Patent Grant
8802849
This is the National Phase of PCT/HU2008/000016, filed Feb. 19, 2008.
The present invention relates to novel tricyclic benzo[4,5]thieno-[2,3-d]pyrimidine-4-yl-amin derivatives (I), their pharmaceutically acceptable salts, to the process for producing the compounds and their use as a pharmaceutically active agent and as pharmaceutical compositions for prophylaxis and/or treatment of proliferative diseases such as cancer.
The novel compound have formula (I)
wherein:
A: methylene (CH2), or methylidene (CH),
R1: hydrogen, phenyl, mono-, di-, tri-, tetra-, or pentasubstitued phenyl, five or six membered ring with one or more heteroatom, saturated or unsaturated heterocycle, for example pyrazole, imidazole, isoxazole, furane, pyrrole, tiophene, thiasole, isothiazole, tiasole, pirane, piridine, pirimidine, dioxane, morpholine, thiomorpholine, pyridazine, pyrazine, piperazine,
R2: hydrogen, substituted or unsubstituted alkyl or cycloalkyl.
The epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) is the archetypal member of a receptor tyrosine kinase family comprised of four closely related proteins called EGFR, HER2 (human EGF-related receptor), HER3 and HER4) All of these transmembrane receptors contain an intrinsic kinase activity that modifies tyrosine residues on the receptor itself as well as on downstream signaling molecules. This kinase activity is stimulated when members of the EGF family of growth factors bind to the receptor. EGFR signal transduction occurs via a multi-stage process initiated in normal cells by interactions between the receptor and ligand. Ligand binding to the extracellular domain induces receptors to dimerize. Dimerization can occur between two molecules of the same receptor (homodimerization) or between different members of EGFR family (heterodimerization) and activates the intracellular protein kinase domains of each receptor to cross-phosphorylate tyrosine residues of the other EGFR molecule. Preclinical studies have demonstrated a strong correlation between elevated levels of EGFR expression and tumorigenesis. In some cases, alterations in EGFR levels alone are sufficient to induce cancer. For example, cultured cells transfected with appropriate vectors acquire transformed behavior in culture when EGFR is overexpressed. Additionally, elevated levels of EGFRs have been detected in many different types of cultured human tumor cell lines including squamous-cell carcinoma (SCC) of the skin, oral cavity, esophagus and lung. There is evidence to suggest that similar alterations in EGFR expression may occur during human tumorigenesis or tumor progression as increased EGFR expression is frequently observed in breast, bladder, cervical, kidney, ovarian, lung and various SCCs.
Frequency of elevated EGFR expression in different types of epithelial tumors:
Inhibitors of the EGFR-PTK are therefore accepted to have great therapeutic potential in the treatment of malignant and nonmalignant epithelial diseases (many type of cancer), and proliferative disorders of the epidermis such as psoriasis. [1,2,3] 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.
In recent years, a number of different classes of compounds have been reported as tyrosine kinase inhibitors and reviewed in several articles [4-7].
The present invention is relating to compounds of the formula (I):
wherein:
A: methylene (CH2), or methylidene (CH),
R1: hydrogen, phenyl, mono-, di-, tri-, tetra-, or pentasubstitued phenyl, five or six membered ring with one or more heteroatom, saturated or unsaturated heterocycle, for example pyrazole, imidazole, isoxazole, furane, pyrrole, tiophene, thiasole, isothiazole, tiasole, pirane, piridine, pirimidine, dioxane, morpholine, thiomorpholine, pyridazine, pyrazine, piperazine.
R2: hydrogen, substituted or unsubstituted alkyl or cycloalkyl.
The following compounds are representative of the compounds of formula (I):
The subject of the invention as well the use of compounds according to the above compounds or the compounds in form of pharmaceutical compositions for prophylaxis and/or treatment of proliferative diseases such as cancer.
The invention is relates to the process for producing compounds of the formula (I) and their pharmaceutically acceptable salts where R1, R2 and A are as mentioned above. According to the process cyclohexanone is condensed with cyan-acetic acid ethylester and elementary sulfur in equimolar proportions in watery and alcoholic solution under heating then the produced amine derivative of the formula (IV) is transformed to a thieno-pyrimidine compound of the formula (Va) by heating with formamide in a ring closing reaction;
or the compound of formula (IV) is brought into reaction with R3-carbonyl chloride where R3 is substituted or unsubstituted alkyl or cyclopropyl group in presence of a base and transformed to an acyl derivative of formula (VI) where R3 is as mentioned above then the received compound is heated with aqua ammonia and the compound of closed ring of formula (Vb) where R3 is as mentioned above is produced in this way; or
LC-MS analyses were performed by Waters chromatograph/ZMD mass spectrometer equipped with Waters 996 DAD UV detector Waters 2700 autosampler and Waters 600 controller.
Supelco Discovery RP-AmideC16 column was used in gradient mode at 3 milliliter/min flow rate.
Initial solvent: 10% AcCN/90% Water/0.05% HCOOH. Solvent B: 100% AcCN
Gradient: 0% B till 30 sec, 0-80% between 30-120 sec, 80% till 240 sec, 80-0% between 240-260 sec, 0% till 360 sec.
Injection: 5 μg
Solvents were purchased from Riedel-deHaën Company (Acetonitrile G Chromasolv (34998)
Formic acid extra pure (27001) Distilled water was purified by Mili-Q Academic equipment.
Details of mass spectrometry: Ionization: ES+/ES−, Source block temp: 120° C.
Desolvation temp: 350° C. Desolvation Gas: 400 L/min Cone Gas: 100 L/min
Capillary: 3000 V Cone: 25 V
Extractor: 3 V Rf, Lens: 0.2 V, Scan: 120 to 1000 m/z in 1 sec, Inter-scan delay: 0.1 s
NMR
300 MHz H1-NMR analyses were performed by Bruker AC-300 equipment at 25° C. DMSO-d6 was generally used as solvent while the exceptions are given.
General Synthetic Methods:
A process of making a compound of the formula (I) below can be made by the following general scheme:
1 mol cyclohexanone and 1 mol cyano-acetic acid ethyl ester were solved in 800 milliliter ethyl alcohol, 1 mol sulphur and 35 milliliter distilled water were added to the solution. The mixture was cooled down between to 3-5° C. and 35 milliliter triethyl-amine was added to it. The reaction mixture was stirred for 2 hours at reflux temperature then it was allowed to cool down to room temperature. The precipitated solid was wiltered off, washed with water and n-hexane, then dried.
Yield: 77%.
A solution of 10 millimol IV in 15 milliliter formamide was stirred at 100° C. for 3 hours then cooled down to room temperature. The reaction mixture was poured onto crushed ice with continuous stirring then collected by filtration. The product was washed with water and n-hexane, then dried.
Yield: 65%
A suspension of 10 millimol IV in 10 milliliter 25% aqueous NH3 was stirred at 90° C. for 24 hours in closed vial. The reaction mixture was cooled down to room temperature, and poured onto crushed ice with continuous stirring then collected by filtration. The product was washed with water and n-hexane, then dried.
Yield: 88%
The solution of 1 millimol V in 5 mL POCl3 was stirred at 100° C. for 3 hours. The reaction mixture was cooled down to room temperature, and poured onto crushed ice with continuous stirring. The solid was filtered off, washed with n-hexane and dried over P2O5. This product and 2 millimol amine reagent were solved in 2.5 milliliter propan-2-ol, and the solution was stirred at 100° C. for 6 hours in closed vial. The reaction mixture was poured onto crushed ice with continuous stirring, the pH was adjusted with 1N aqueous HCl to pH=7. The solid was filtered off, washed with water and n-hexane then dried.
Yields: 66-85° A
Compound 1-15 were prepared according to this method.
Physicochemical properties of compounds 1-15.
1H-NMR δ [ppm] in DMSO-d6
11 millimol cyclopropanecarbonyl chloride was added dropwise to the solution of 10 millimol IV in 25 milliliter pyridine at 5° C. The reaction mixture was stirred at this temperature for 1 hour, poured onto icecold 1N aqueous HCl, the precipitated solid was filtered off, washed with water and n-hexane, then dried.
Yield: 80%
8.00 g MnO2 (Activated, Fluka) was added to the suspension of 10 millimol VI in 60 milliliter toluene, and the suspension was stirred at reflux temperature for 5 days. The reaction mixture cooled down to 60° C., filtrated, and evaporated to give the desired compound VII as solid crystal.
Yield: 72%
A suspension of 10 millimol VII in 10 milliliter 25% aqueous NH3 was stirred at 90° C. for 24 hours in closed vial. The reaction mixture was cooled down to room temperature, and poured onto crushed ice with continuous stirring then collected by filtration. The product was washed with water and n-hexane, then dried.
Yield: 75%
The solution of 1 millimol VIII in 5 milliliter POCl3 was stirred at 100° C. for 3 hours. The reaction mixture was cooled down to room temperature, and poured onto crushed ice with continuous stirring. The solid was filtered off, washed with n-hexane and dried over P2O5. This product and 2 millimol amine reagent were solved in 2.5 milliliter propan-2-ol, and the solution was stirred at 100° C. for 6 hours in closed vial. The reaction mixture was poured onto crushed ice with continuous stirring, the pH was adjusted with 1N aqueous HCl to pH=7. The solid was filtered off, washed with water and n-hexane then dried.
Yields: 70-88%
The solution of 10 millimol VII in 50 milliliter methanesulfonic acid was stirred at room temperature for 48 hours. The reaction mixture was poured onto icecold 25% aqueous NH3 solution, the soliw was filtered off, washed with water and n-hexane, then dried.
Yield: 62%
The solution of 10 millimol IX in 15 milliliter milliliter formamide was stirred at 100° C. for 3 hours then cooled down to room temperature. The reaction mixture was poured onto crushed ice with continuous stirring then collected by filtration. The product was washed with water and n-hexane, then dried.
Yield: 72%
The solution of 1 millimol X in 5 milliliter POCl3 was stirred at 100° C. for 3 hours. The reaction mixture was cooled down to room temperature, and poured onto crushed ice with continuous stirring. The solid was filtered off, washed with n-hexane and dried over P2O5. This product and 2 millimol amine reagent were solved in 2.5 milliliter propan-2-ol, and the solution was stirred at 100° C. for 6 hours in closed vial. The reaction mixture was poured onto crushed ice, the pH was adjusted with 1N aqueous HCl to pH=7. The solid was filtered off, washed with water and n-hexane then dried.
Yields: 70-88%
Compound 21-23 were prepared according to this method.
1H-NMR δ [ppm] in DMSO-d6
General description of the kinase assays: the kinase activity was assayed in 96-well microtiter plates at a final compound concentration of 10 μM in a total volume of 50 μl. Compounds were dissolved in 100% DMSO to prepare a 10 mM stock solution and than diluted with buffer to reach a 10 μM (or the required) final concentration. The kinase concentration was used to yield 10% ATP turn over. The ATP concentration was used at the KmATP and 12.5 μCi/ml adenosine 5′[γ-33P]triphosphate. The substrate concentration was used at 5 fold excess over the Km for the substrate. The reaction was incubated for 1 hour at room temperature and stopped by addition of 10 μl 50 mM EDTA. The assay was transferred to a 96 well MAPH filter plate (Millipore). The radioactivity was counted on a Microbeta microplate counter.
Inhibitory activity of prepared compounds at 10 μM concentration (percent of inhibition):
Ten compounds were found to inhibit EGFR-PTK with higher than 96% inhibitory activity at 10 μM concentration. Most active compounds were tested on EGFR-PTK assay to determine their IC50 values.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/HU2008/000016 | 2/19/2008 | WO | 00 | 10/4/2010 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2009/104026 | 8/27/2009 | WO | A |
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| 03057149 | Jul 2003 | WO |
| 2004032716 | Apr 2004 | WO |
| 2006044524 | Apr 2006 | WO |
| 2006136402 | Dec 2006 | WO |
| 2007059905 | May 2007 | WO |
| Entry |
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| Number | Date | Country | |
|---|---|---|---|
| 20110015214 A1 | Jan 2011 | US |
