Transcript 5,6,7,8-Tetrahydro-benzo[4,5]thieno[2,3-d]

5,6,7,8-Tetrahydro-benzo[4,5]thieno[2,3-d]pyrimidin-4-yl-amine Derivatives as
Potent and Selective, ATP Side Directed Inhibitors of the EGF-Receptor Protein
Tyrosine Kinase
Péter Bánhegyia*, László Őrfia,b,c, Frigyes Wáczeka, Zsolt Székelyhidia, Gyöngyi Bökönyid, Edit Z. Szabód, Edit Várkondia, Richárd Schwába and György Kéria,b,d
aCooperative
Research Centre, Semmelweis University, Rippl-Rónai u. 37., 1062 Budapest, Hungary
bVichem Chemie Ltd., Hermann u. 15., 1022 Budapest, Hungary
cDept. of Pharmaceutical Chemistry, Semmelweis University, Hőgyes u. 9., 1092 Budapest, Hungary
dPeptide Biochemistry Research Group, Hung. Acad. Sci - Semmelweis Univ., Puskin u. 9., 1088 Budapest, Hungary
*Tel.: +36-1-3010614; Fax: +36-1-3010613; [email protected]
Introduction
Protein tyrosine kinases (PTK) play a fundamental role in signal transduction pathways. 4 Deregulated PTK activity has been observed in many proliferative diseases (e.g. Cancer, psoriasis,
restenosis, etc.).1 Tyrosine kinases are therefore attractive targets for the design of new therapeutic agents. The PTK`s can be divived into subgroups which have similar structural organization
and amino acid sequence similarity within their kinase domains.2 The family of the epidermal growth factor receptor (EGF-R) PTK belongs to the largel class of the transmembrane growth
factor receptor PTK`s. This EGF-R family contains four members, the EGF-R kinase (c-erb B-1 gene product), the p185erb B2 (c-erb B-2 gene product), and the recently identified c-erb B-3 and c-erb
B-4 gene products. EGF-R and its ligands (EGF, TGF-α) have been implicated in numerous tumors of epithelial origin (e.g. squamous cell carcinoma: breast, ovarian, NSC lung cancer; etc.)1,3 and
proliferative disorders of the epidermis such as psoriasis.4 Inhibitors of the EGF-R PTK could therefore have great therapeutic potential in the treatment of malignant and nonmalignant
epithelial deseases. Due to the involvement of tyrosine kinases in many signal transduction pathways, it will be important to develop inhibitors with high selectivity at the enzyme level.
Peter Traxler et all published5 the 4-(phenylamino)pyrrolopirimidin derivates as ATP site directed inhibitors of the
EGF-R PTK. Aromatization of the tetramethylene ring attached to the pyrrole ring (X=(CH2)4 -> (CH)4) incrased the
potency.
The benzo[4,5]thieno[3,2-d]pyrimidin-4-yl-phenyl derivates were described by D. Hollis Showalter et all6 as efective
EGF-R PTK inhibitors. (Fig.1)
A couple of 5,6,7,8-tetrahydro-benzo[4,5]thieno [2,3-d]pyrimidin-4-ylamine compounds of general structure 8 and 9
were designed and preparated starting out from the structures of the most active tricyclic EGF-R PTK inhibitor
molecules 1 and 2. Present poster is aiming the publication of the synthesis and biological data of these molecules.
X
R
1a
1b
1c
1d
1e
1f
1g
1h
2a
2b
2c
N R
HN
S
N
N
X
N
N
N
H
2a,b,c
1a,b,c,d,e,f,g,h
R
-(CH2)4-(CH2)4-(CH2)4-(CH2)4-(CH2)4-(CH2)4-(CH2)4-(CH)4-
EGF-R IC50 (µM)
0.31
0.82
0.029
0.046
0.42
0.86
0.36
0.006
191
538
1.8
H
3-Me
3-Cl
3-Br
3-OH
3-OMe
3-CF3
3-Cl
Bn
CH(Me)Ph
3-BrPh
Fig.1
Synthetic procedures
Compounds of general structure 8 and 9 can be obtained from common starting material 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile (3), which was preparated from cyclohexanone, malonitrile and sulphur by Gewald
reaction. Ring-closed derivates 4 and 5 were preparated by heating of 3 in formic acid or in cyclopropanecarbonic
acid. The pyrimidone intermediers 4, 5 were refluxed in phosphoroxychloride yielding active imidoyl chloride
derivates 6, 7 which were reacted with the correspoding aniline resulting the desired compound families. Fig2
Cl
O
O
d
N
S
+
c
N
S
N
e
N
N
S
6
4
N
S
+
Cl
O
b
S
d
N
S
S
e
N
N
S
7
5
R
HN
N
N
Compound
T/C6h [%]
(at 50M)
T/C48h [%]
(at 50M)
T/C6h -T/C48h
[%]
Toxic
Antiproliferative
IC50 [M]
8a
89.267
22.641
66.626
No
Yes
20.21
8b
82.73
34.51
48.22
No
No
8c
95.117
79.198
15.919
No
No
8d
91.69
34.74
56.95
No
Yes
8e
92.198
77.757
14.441
No
No
8f
84.8
26.3
58.5
No
Yes
8g
75.185
46.039
29.146
No
No
8h
86.114
21.597
64.517
No
Yes
9a
81.136
66.631
14.505
No
No
9b
94.319
81.139
13.18
No
No
9c
94.724
71.252
23.472
No
No
9d
84.03
74.85
9.18
No
No
9e
92.176
52.882
39.294
No
No
9f
75.185
46.039
29.146
No
No
9g
94.967
35.245
59.722
No
Yes
N
9a-g
Reagents and conditions:
(a) Gewald reaction (Gewald, K.; Schinke, E.; Böttcher, H. Chem. Ber. 1966, 99, 94 )
(b) Stirred in Cyclopropanecarboxylic acid, at 120 oC, for 2 hours
(c) Refluxed in 80 % Formic acid, for 2 hours
(d) Refluxed in phosphoroxychlorid for 1 hour
(e) Stirred with the correspoding aniline in Ethanol
O
N
S
N
R
N
S
42.33
For assaying tyrosine kinase function a modified protocol of Sigma (PTK101) was used. 96-well plates were coated
with Poly(Glu,Tyr) substrate in PBS at 37˚C overnight, then washed with PBS containing 0,05% Tween once. Then
EGFR and ATP in reaction buffer were added and incubation at 37˚C for 30 minutes followed. Plates were washed 5
times and HRP-conjugated anti-phosphotyrosine antibody was added at a dilution of 1:1000. After 30 minutes of
incubation at 37˚C, plates were washed 5x and OPD was added along with H2O2. Reaction was stopped with H2SO4
and plates were read by ELISA reader at 490 nm.
N
8a
b
c
d
e
f
g
h
R
Cl
NO2
NH2
CN
OCH3
CF3
OH
Br
R
9a Cl
b NO2
c NH2
d CN
e OCH3
CF3
f
g OH
N
O
N
Reference16
EGF-R PTK IC50 = 0.78 uM
Br
HN
Fig.3
N
N
H
Waters Alliance LC system, equipped with Micromass Qvadrupole MS detector was used for the purity, chemical
identity analysis. The chemical identities of molecules were also checked by 1H NMR spectra, which were made on
Bruker 300 MHz NMR spectrometer in DMSO-d6.
Compound
Remaining activity
at 10 uM
8a
40%
8b
100%
8c
64%
8d
44%
8e
100%
8f
75%
8g
44%
8h
41%
9a
71%
9b
100%
9c
100%
9d
85%
9e
90%
9f
95%
9g
99%
Reference1
10%
Reference2
62%
Br
HN
HN
25.98
Materials – All compounds were dissolved in DMSO and diluted in reaction buffer (500 mM Hepes pH 7.4, 200 mM
MgCl2, 1 mM MnCl2, 2 mM Na3VO4) for tyrosine kinase assay. Single concentration of compounds (10 μM) containing
1% DMSO were tested in duplicates.
For tyrosine kinase assay, 50 ng recombinant EGFR enzyme (ProQinase) and 30 μg Poly(Glu,Tyr) substrate (Sigma)
was used. Phosphorylated substrate was detected by HRP-conjugated anti-phosphotyrosine antibody and OPD
(Orthophenildiamine) (Sigma). and the reference EGFR inhibitor was used as positive control.
The following 5,6,7,8-tetrahydro-benzo[4,5]thieno [2,3-d]pyrimidin-4-ylamine compounds of general structure 8, 9
were designed and preparated. Fig3
R
38.34
Statistics – Results are expressed as OD of the percentage of Treated / Control wells (T/C%).
Fig.2
HN
19.22
8a-h
NH2
3
Human A431 epidermoid carcinoma cells were cultured in DMEM (Dulbecco’s Mod Eagle Medium) supplemented with
10% FCS (foetal calf serum), 200 mM L-glutamine, 10000 U/ml penicillin and 10 mg/ml streptomycin (Gibco Life Sci)
at 37˚C and 5% CO2. Cells were seeded into 96-well plates and incubated for 16 hours before serial dilutions of
compounds were added. Cells were treated for 6 and 48 hours. Cells used for 6 hour and 48 hour treatment were
seeded at 4x104 and 1x104 per well respectively. Antiproliferative efficacy of the compounds was analysed with
Methylene blue test8.
All compounds were dissolved in DMSO and diluted in cell culture medium for the-proliferation tests in final
concentrations of 50, 10, 2, 0.4, 0.08 μM and tested in duplicates. Cycloheximide, a well established inducer of
apoptosis was used as positive control.
Antiproliferative effect was first expressed as a percentage of the optical density (OD) of treated (T) and control (C)
wells after both 6 and 48 hours (T/C*100). Because new protein synthesis is required for apoptosis in immortalized
cell lines, compounds that induce programmed cell death will show significantly less antiproliferative activity after 6h
than after 48h. In the optimal case an apoptosis inducing compound will cause 100% viability after 6h and 0% after
48h. Therefore, analysing T/C48 versus T/C6h will correlate with the apoptosis inducing “specificity” of a compound.
Kinase assay:
N
N
a
Proliferative assay:
R
HN
N
Biological tests
N
Reference25
EGF-R PTK IC50 = 0.046 uM
Fig.4
References
1. Aaronson, S. A. Growth Factors and Cancer. Science, 1991, 254, 1146-1152
2. Hanks, S. K., Quinn, A., M., Hunter, T. The protein kinase family: conserved features and phylogeny at the catalytic
domains. Science, 1988, 241 42-52
3. Ulrich, A., Schleissinger, J. Signal Transduction by Receptors with Tyrosine KinaseActivity. Cell 1990, 61, 203-212
4. Elder, J. T.; Fisher, G. J.’ Lindquist, P. B.; Bennett, G. L.; Pittelkow, M. R.; Coffey, R. J.; Ellingsworth, L; Derynck, R.;
Voorhees, J. J. Overexpression of transforming growth factor α in psoriatic epidermis. Science 1989, 243, 811-814
5. Peter M. Traxler, Pascal Furet, Helmut Mett, Elisabeth Buchdunger, Thomas Meyer, Nicholas Lydon> J. Med. Chem,
1996, 39, 2285-2292
Discussion
We have synthesised and characterised two series of novel 5,6,7,8-tetrahydro-benzo[4,5]thieno[2,3-d]pyrimidin-4-yl-amine
derivates, and they were tested in cell proliferation assay on EGFR overexpressing tumour cell line (A431) and against EGF-R
PTK inhibition. Five of them were found to be active on the proliferation assay (8a, 8d, 8f, 8h, 9g) and four on the EGF kinase
assay (8a, 8d, 8g, 8h). We found the cyclopropyl moiety at 2 position (general formula 9) ruind the inhibitory activity of the
compounds in both assays. Fig4
Current efforts in our laboratory are directed toward the synthesis of new 5,6,7,8-tetrahydro-benzo[4,5]thieno[2,3d]pyrimidin-4-yl-amine derivatives (e.g. the unsaturated ring system, another R1 groups, etc.) to increase the biological
activity. Another important viewpoint is the selectivity, so we are going to test the active compounds in other cell lines.
6. H. D. Hollis Showalter et all. J. Med. Chem, 1999, 42, 5464-5474
We gratefully acknowledge the contributions of the following colleagues who support our work: Ildikó Szilágyi, István Varga, Ferenc Jaczina