Transcript 1. dia - mono.eik.bme.hu

Synthesis of thiophene and α-terthiophene derivatives with antiproliferative
activity
Zsolt Székelyhidia*, János Patób, László Őrfia,b,c, Frigyes Wáczeka, Péter Bánhegyia, Gyöngyi Bökönyid, Edit Z. Szabód, Edit Várkondia, Ákos Papa, 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:
The uncontrolled growth of tumour cells has been linked in many cases to inappropriate signal transduction. Targeting the signal transduction process in order to control tumour growth has
become an attractive area of drug research. Kinases are the most important signalling enzymes and most of the signal transduction therapy based drug research is aimed to inhibit the false or
overexpressed signal of certain pathologically relevant kinases1. Thousands of compounds around several scaffolds have been developed as potential kinase inhibitory drugs.
A few terthiophenes are known as PKC inhibitors2,3 and nitrile derivatives of thiophene inhibit EGF-RTK (epidermal growth factor receptor tyrosine kinase)4,5. Both kinases are involved in cell
proliferation, thus we have planned and synthesised a series of new thiophene and terthiophene derivatives as well as some of known structurally related PKC inhibitors (4a, 10, 11, 14), and
studied them in cell proliferation test on EGFR overexpressing tumour cell line (A431).
EGFR (epidermal growth factor receptor) is a transmembrane protein composed of three domains: the extracellular ligand-binding domain, a transmembrane region and an intracellular protein
kinase domain. A number of different ligands, including EGF, neuregulins, heparinbinding EGF, β-cellulin and transforming growth factor-α have shown the capability to bind to the extracellular
domain of EGFR. The binding of the ligand to EGFR induces the formation of active dimers that are able to turn on a transduction signal through the cell. EGFR activation lies in the multiple
processes involved in cell proliferation, adhesion, migration, development of angiogenesis, and inhibition of apoptosis.4
Biological test
Fig.1
+
Cl
Cl
S
1
2
S
S
S
O
O
O
O
b
S
S
a
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.
Cut-off limit for “effective” compounds was set for differences expressed as (T/C6h - T/C48) > 80%. IC50 values
were generated from IC50 graphs.
4a
3
Reagents and conditions: (a) AlCl3, DCM, 0 0C; (b) Lawesson`s reagent
We have tried the synthetic route described in the literature to prepare α-terthiophene. According to this method
succinyl chloride 1 was reacted with thiophene 2 in the presence of aluminium chloride in dichloromethane at 0 0C to
obtain the dithiophene-1,4-diketone 3 in 80% yield. Thionation using Lawesson`s reagent yielded -terthiophene 4a
in 90%.2
Unfortunately, the yield of the first step was much less than expected and the product was contaminated. Moreover,
this route was not suitable to obtain asymmetric terthiophene analogs. For this reason the following synthetic route
was developed. Thiophene-2-carboxaldehyde 5 paraformaldehyde and a secondary amines in ethanol produced
Mannich–bases 6, 7 and 8. The Mannich-bases were reacted with thiophene-carboxaldehyde and NaCN in abs. DMF to
obtain dithiophene-diketones 3, 9. Thionation using phosphorus pentasulfide yielded terthiophene 4a and 4b.
Fig.2
Product
a
+
R
(HCHO) n
S
4a
4b
6a
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21*
22*
23
24
25
26
27
28
S
S
O
5
O
b
O
O
S
3
6a R= Me2N
7 R= morpholine
8 R= piperidine
c
b'
S
S
S
4a
O
c
S
O
S
S
S
S
T/C6h (%)
50 μM
90.70
99.30
94.90
97.80
99.90
100.00
93.70
91.40
100.00
95.60
100.00
100.00
95.90
94.30
95.40
92.80
94.80
0.00
0.20
75.00
99.90
97.50
97.20
61.00
97.30
9
4b
T/C48h (%)
50 μM
2.80
77.30
76.00
7.50
78.60
96.70
15.80
33.80
0.20
37.10
60.10
56.00
11.70
36.50
14.10
27.20
90.60
0.00
0.00
0.00
62.20
3.40
43.20
0.00
62.00
(T/C6h - T/C48h)
[%]
87.90
22.00
18.90
90.30
21.30
3.30
77.90
57.60
99.80
58.50
39.90
44.00
84.20
57.80
81.30
65.60
4.20
0.00
0.20
75.00
37.70
94.10
54.00
61.00
35.30
s.d.
avarage
0.03
7.43
1.40
1.06
5.30
16.46
0.76
3.25
0.29
5.10
2.75
9.12
0.66
1.25
1.81
1.00
5.03
0.72
1.14
3.42
4.32
1.05
5.40
5.21
11.06
IC50
[μM]
11.66
16.06
4.33
16.25
32.42
15.87
Reagents and conditions: (a) ethanol, secondary amine, 12 h, reflux temperature; (b) thiophene-2-carboxaldehyde; (b’) thiophene-3-carboxaldehyde;
NaCN, abs. DMF, 12 h, room temperature; (c) P2S5, NaHCO3, THF/hexane=3/1, 12 h, room temperature
Compound 4a
Compound 7
125
Compound 12
125
103,4
100
-Terthiophene 4a was lithiated with LDA in abs. THF under argon at –78 degree then the lithium compound was
quenched with DMF to give -terthiophenedialdehyde 10. Reduction of the aldehyde functions with lithium
aluminiumhydride yields alcohol 11.2 Amino-guanidine derivatives (12, 21-26) were synthesised from the Mannichbases, (di) thiophene-diketones and α-terthiophene-dialdehyde with amino-guanidine in ethanol. Condensation of αterthiophene-dialdehyde 10 with malonitrile in pyridine yields a tetracyano derivative 13. 2-Cyano--terthiophene 14
was prepared from α-terthiophene and chlorosulfonyl isocyanate in dichloromethane followed by reaction with DMF.
-Terthiophene-5- carboxylic acid 15 was obtained from the lithiated terthiophene quenched by carbon dioxyde7.
Fig.3
102,0
98,8
100
2
y = 0,0709x - 5,6038x + 105,65
R2 = 0,9971
75
125
94,5
99,7
98,8
98,7
74,5
71,8
75
55,8
50
49
2
y = 0,0394x - 3,8587x + 101,9
2
R = 0,9956
66,3
50
25
y = 0,152x2 - 9,349x + 87,579
R2 = 0,9685
0,01
-28
25
0,1
8,6
4,33 μM
1
0,2
10
100
2,8
0
0,01
0,1
10 11,66 μM
1
-25
100
7,5
0
0,01
16,06 μ
M
0,1
1
10
100
-104
Compound 18
Compound 16
Compound 25
125
125
125
110,8
100
S
O
S
S
4a
OH
y = 0,0401x2 - 3,79x + 100,89
2
R = 0,9976
50
11
S
0
0,01
c
O
10
H
e
H2N
f
S
S
S
S
97,5
100
96,3
93,8
75
66,5
87,1
y = -0,0136x2 - 1,0146x + 98,77
R2 = 0,9992
75
50
50
25
25
y = 0,0359x2 - 3,7328x + 100,38
2
R = 0,9633
66,9
1
10
14,1
100
0
0,01
32,42 μ
M
0,1
1
10
100
0
0,01
3,4
0,1
1
10
15,87 μM
100
NH2
N
N
H
0,1
16,25 μM
H
12
N
H
S
NH
Discussion
N
14
O
S
N
HN
d
S
S
S
S
N
N
OH
15
S
11,7
S
S
95,6
100,0
25
a
S
75
S
b
S
100
87,3
HO
S
97,9
100,5
N
N
13
Reagents and conditions: (a) LDA, abs. THF, -78 0C, 2 h; DMF, 12 h, room temperature; (b) LAH, abs. THF,
4 h, room temperature; (c) aminoguanidine, EtOH, reflux temperature, 12 h; (d) malonitrile, pyridine, 100 0C, 2 h;
chlorosulfonyl isocyanate, abs.DCM; DMF, 1 h, room temperature; (f) LDA, abs. THF, -78 0C, 2 h; CO2, 12 h, room temperature
(e)
The Mannich-bases 6a and 6b were excellent reagent to prepare diketone, hydrazone and amino-guanidine
derivatives. 6a was reacted with aryl-aldehydes and NaCN in abs. DMF produced diketone derivatives 3, 16-20.
21-26 were synthesised from the Mannich-bases and (di) thiophene-diketones with amino-guanidine in ethanol.
The hydrazones 27 and 28 were obtained from 6a and aryl-hydrazins in EtOH at reflux temperature.
We have synthesised and characterised a series of novel thiophene and α-terthiophene derivatives and they were tested in
cell proliferation assay on EGFR overexpressing tumour cell line (A431) and six of them were found to be active. One
compound (12) had excellent antiproliferative activity and two amino-guanidine derivatives 21, 22 had cytotoxic effect.
Among the known PKC inhibitors (dialdehyde 10, nitrile 14 and dihydroxyl 11 derivatives) only the parent α-terthiophene 4a
and its dialdehyde 10 showed antiproliferative effect. Out of the 25 synthesised thiophene and α-terthiophene analogs six
had better than 80% proliferation inhibitory activity without cytotoxic effect.
The biological test results showed that the disubstituted analogs had stronger inhibitory activity than the monosubstituted
ones and basic moieties enhanced their activity. Terthiophene-diaminoguanidine derivative 12 has 99.80% inhibitory activity
against A431 cell culture, while unsubstituted α-terthiophene 4a inhibited the proliferation with 87.90%. An interesting
observation was that the asymmetric terthiophene 4b has lower inhibitory effect than symmetric terthiophene 4a.
Current efforts in our laboratory are directed toward the synthesis of new terthiophene derivatives to increase the water
solubility of the molecule by coupling with hydrophilic moieties, retaining the biological activity. Another important viewpoint
is the selectivity, so we are going to test the active compounds in other cell lines.
Fig.4
O
S
O
a
S
O
S
R
N
N
O
6a
6b
3 R= 2-thienyl
16 R= 4-CF3-phenyl
17 R= 3-CF3-phenyl
18 R= 4-F-phenyl
19 R= 3-OMe-phenyl
20 R= 3,4-di-OMe-phenyl
3, 16-20
b
c
b
b
R
N
R
N
NH
R
N
S
N
NH 2
N
25 R= 2-thiophene
26 R= 3-thiophene
N
HN
HN
27 R= 3-Br
28 R= 4-F
21-24
Janet Dancey; Edward A. Sausville. Nature Reviews. April 2003, vol 2, 296-313
2. Darrick S.H.L.; Kim; Curtis L. Ashendel; Qin Zhou; Ching-te Chang; Eung-Seok Lee; Ching-jerChang. Bioorg.Med.Chem.Lett.
1998, 8, 2695-2698.
N
H
4. Aviv Gazit; Pnina Yaish; Chaim Gilon; Alexander Levitzki. J.Med.Chem. 1989, 32, 2344-2352.
NH
5. Valerie G. Brunton; Martin J. Lear; David J. Robins; Sharon Williamson; Paul Workman. Anti-Cancer Drug Des. 1994, 9,
291-309.
N
NH 2
•
3. Wei-Chu Xu; Curtis L. Ashendel; Ching-te Chang; Ching-jer Chang. Bioorg.Med.Chem.Lett. 1999, 9, 2279-2282.
S
HN
References
6. X. Pivot. CancerFutures. March 2002, vol 1, 90-93.
NH 2
21 R= 2-thienyl
22 R= 4-CF3-phenyl
23 R= 3-OMe-phenyl
24 R= 3,4-di-OMe-phenyl
7. Jacques Kagan; Sudershan K. Arora; Ayse Üstünol. J.Org.Chem. 1983, 48, 4076-4078.
8. Oliver MH.; Harrison NK.; Bishop JE.; Cole PJ.; Laurent GJ. J. Cell. Sci. 1989, 92, 513-518.
9. Faith M. Uckum; Sharon Pendergrass; Danielle Maher; Dan Zhu; Lisa Tuel-Ahlgren; Chen Mao; T.K. Venkatachalam.
Bioorg.Med.Chem.Lett. 1999, 9, 3411-3416
Reagents and conditions: (a) aryl-aldehyde, NaCN, abs. DMF, 12 h, room temperature; (b) aminoguanidine, EtOH, reflux temperature, 12 h (c) phenyl-hydrazine,
EtOH/HCl, reflux temperature, 6 h
We gratefully acknowledge the contributions of the following colleagues who support our work: János Pató, Richárd Schwáb, Edit Szabó, Ildikó Szlágyi, István Varga