Patent Application
20080039447
245 mg (1 mmol) of 2-chloro-4-2-propynylaminopyrimidine is dissolved in 2 ml of acetonitrile, and a suspension of 4-(difluoromethylthio)-aniline hydrochloride [produced from 352 mg (2 mmol) of 4-(difluoromethylthio)-aniline, 1 ml of acetonitrile and 0.5 ml of aqueous HCl (4M in dioxane)] is added at room temperature. Then, the reaction mixture is refluxed overnight under N2 atmosphere. After cooling, the mixture is filtered, the remaining solid phase is washed with H2O and dried. A yield of 328 mg (85%) of the product can be expected
1.55 g (4.9 mmol) of compound 20 is dissolved in 5.5 ml of epibromohydrin, and 1.38 g of K2CO3 and 65 mg of tetrabutylammonium bromide are added to it. The reaction mixture is stirred under nitrogen atmosphere at 100° C. for 1 hour. After ethyl acetate is added, the resulting precipitate is collected and recrystallized from ethanol. The product yield is 1.15 g (62%) as a white powder.
Substance 40 is produced analogously to Example 2.
0.2 ml of a 0.5 M 4-phenylpiperazine solution in DMPU is added to a solution of 19 mg (0.05 mmol) of substance 51 in N,N′-dimethylpropylurea (DMPU). The reaction mixture is kept for 18 hours at a temperature of 80° C. After cooling, 3.5 ml of tertiary butyl methyl ether is added, and the organic phase is extracted 5 times with 1.5 ml of H2O and then evaporated in a vacuum. The remaining residue is chromatographed on 1.7 g (15 μM) of Lichrosphere Si60 (gradient: dichloromethane/hexane 1:1 to DCM and then dichloromethane/methanol 99:1 to 93:7). A product yield of 17 mg (64%) is achieved.
Similarly produced are also the following compounds:
The following compounds were similarly produced in the described examples.
The following compounds were produced analogously to the described synthesis processes according to Diagram 1 or 2:
All NMR spectra are measured in the indicated solvent or in DMSO.
Diastereomere1/2(ca. 1:3)
Diastereomere1/2(ca. 6:1)
Diastereomere1/2(ca. 1:1)
Diastereomere1/2(ca. 1:1)
Diastereomere1/2(ca. 1:1)
Diastereomere1/2(ca. 1:1)
Diastereomere3/4(ca. 1:1)
Diastereomer 1
Diastereomer 2
(+)-Enantiomer
(−)-Enantiomer
Diastereomer 1
Diastereomer 2
Diastereomer 1
Diastereomer 2
Compounds Nos. 159, 160, 161, 163, 167, 168, 170, 174, 175, 191, 192, 203 and 204 that are identified with *) can be produced by the process variants that are described under Example No. 292.
202 mg (0.60 mmol) of the compound of Example No. 122 is mixed with 1 ml of water and 0.2 g (1.2 mmol) of bromine and stirred at room temperature. After 24 hours, 0.2 g (1.2 mmol) of bromine is added again, and it is stirred for another 24 hours at room temperature. The solvent is evaporated by means of underpressure, and the remaining residue is purified by chromatography (Flashmaster II, DCM/MeOH 7:3). 17 mg (0.04 mmol, 7%) of the product is obtained as a white solid.
Analogously to the process for the production of intermediate products that is described under Example 6.0, the following compounds were also produced:
Analogously to production example 1, the following compounds were also produced:
According to the production variants below, the following compounds are also synthesized:
30 mg (0.0678 mmol) of compound No. 278 is dissolved in 1 ml of methanol/tetrahydrofuran 1:1. After adding ≈10 mg of sodium borohydride, stirring is continued for 2 hours. Then, it is quenched with ≈3-4 drops of glacial acetic acid while being cooled, and it is concentrated by evaporation. Below, the crude product is taken up with a little water, suctioned off, rewashed with acetonitrile and dried in a vacuum at 60° C. Yield: 21 mg (70% of theory) of the desired compound.
The production of the oxime ether is carried out according to the following general reaction diagram:
R8 and R9 have the meanings that are indicated in general formula I.
50 mg (0.12 mmol) of compound No. 283, 34 mg of hydroxylammonium chloride and 150 mg of pulverized KOH are refluxed for 2 hours in 2 ml of ethanol. Then, it is poured onto ice water and acidified with glacial acetic acid, extracted 3 times with dichloromethane/isopropanol 4:1, dried with magnesium sulfate and concentrated by evaporation. The residue is suspended with acetonitrile, suctioned off and dried at 60° C. Yield: 28 mg (54% of theory) of the desired compound.
Mass
ESI:
MH+ 429 (29%)
371 (61%)
289 (91%)
Similarly produced were also the following compounds:
50 mg (0.12 mmol) of compound No. 283 and 7.5 mg (0.132 mmol) of cyclopropylamine are dissolved in 2 ml of 1,2-dichloroethane. After 9.1 mg (0.144 mmol) of sodium cyanoborohydride is added, it is allowed to stir for 12 more hours. Then, it is diluted with dichloromethane/isopropanol 4:1, washed 2× with water, dried with magnesium sulfate and concentrated by evaporation. The residue is chromatographed on silica gel with dichloromethane/methanol 95:5. Yield: 18 mg (33% of theory) of the desired compound.
Produced similarly are also compounds Nos. 159, 160, 161, 163, 167, 168, 170, 174, 175, 191, 192, 203 and 204.
Produced similarly to Example 1 are also the following two compounds:
0.2 mmol of sulfonic acid fluoride is introduced into the reactor of a synthesizer. 1.0 ml of solvent, preferably 2-butanol, is added. 0.2 ml (0.2 mmol) of DMAP—dissolved in a solvent, for example DMSO or 2-butanol—and 0.2 ml (0.2 mmol) of the amine, dissolved in 2-butanol, are added in succession via a pipette. The reaction mixture is then stirred for 20 hours at 80° C. After the reaction is completed, the crude product is pipetted off, and the reactor is rewashed with 1.0 ml of THF. The solution of the crude product is then concentrated by evaporation and purified by HPLC.
The compounds below were produced:
The production of the pyrimidine-sulfonic acid fluorides is carried out analogously to the production of the sulfonic acid amides.
Similarly produced to the above-described examples were also the following para-compounds:
The diastereomer mixture was separated in the two corresponding racemates (A and B) by means of HPLC. Conditions:
Below, racemates A and B in each case were separated by means of chiral HPLC.
Production of the intermediate stages preferably used for the synthesis of the compounds of general formula I according to the invention.
11.1 g (66 mmol) of 2,4-dichloro-5-fluoropyrimidine is dissolved in 60 ml of acetonitrile, and 10.2 ml (73 mmol) of triethylamine and 6.0 ml (86 mmol) of propynylamine are added. The reaction mixture is stirred overnight at room temperature and then poured into water. The mixture is extracted by means of ethyl acetate, the combined organic phases are dried on MgSO2, and the solvent is evaporated by means of underpressure. After the remaining material is recrystallized with diisopropyl ether/hexane, the yield is 10.6 g (87% of theory) of the product.
The 4-(diaminocyclohexyl) derivatives that are described below are synthesized via reductive aminations of the described keto derivative with use of triacetoxy borohydride (Abdel-Magid, Carson, Harris, Maryanoff, Sha, J. Org. Chem. 1996, 61, 3849). The keto derivative is obtained by TPAP oxidation (Griffith, Ley, Aldrichimica Acta 1990, 23, 13) of the corresponding alcohol.
Similarly produced are also the following intermediate compounds:
3.19 g (14 mmol) of 5-bromo-2,4-dichloropyrimidine is mixed with 8.06 g (63 mmol) of 4,4,4-trifluorobutanol, and 0.74 ml (8.4 mmol) of trifluoromethanesulfonic acid is slowly added to it. The reaction mixture is stirred overnight at room temperature and then poured into water. The mixture is extracted by means of ethyl acetate, the combined organic phases are dried on MgSO2, and the solvent is evaporated by means of underpressure. The product is always contaminated with varying amounts of 2,4-bisalkoxypyrimidine. The remaining material is therefore purified by means of gradient chromatography with silica gel as a carrier medium (eluant: hexane and hexane/ethyl acetate at a 9:1 ratio). This process results in a yield of 1.70 g (38%) and also yields 1.93 g (34%) of 5-bromo-2,4-bis-(4,4,4-trifluorobutoxy)pyrimidine (starting compound).
Similarly produced are also the following compounds:
Analogously to process examples 1 and 2, the following intermediate products are also produced:
Diastereomer1/2
Diastereomer 1
Diastereomer 2
Diastereomer 1
Diastereomer1 + 2(ca. 1:1)
Diastereomer1 + 2(ca. 1:1)
Diastereomer3 + 4(ca. 1:1)
Diastereomer1 + 2(ca. 1:1)
4.5 g (20 mmol) of 2-bromobutyraldehyde diethyl acetyl (Pfaltz-Bauer Company) and 5.2 g (80 mmol) of sodium azide are stirred for 5 days in 15 ml of DMF at 100° C. Then, it is poured onto cold dilute sodium bicarbonate solution, extracted 3× with ether, the organic phase is dried with magnesium sulfate and concentrated by evaporation: raw yield 1.87 g (50% of theory).
936 mg of the crude product is dissolved in 50 ml of methanol, mixed with palladium on carbon (10%) and stirred for 12 hours under H2 atmosphere. After the catalyst is filtered off and after concentration by evaporation, 457 mg (57% of theory) of the desired amine remains.
148 mg (0.5 mmol) of intermediate product compound 1.18 is dissolved in 1 ml of glacial acetic acid. At room temperature, 0.5 ml of 1N hydrochloric acid is added, and it is stirred for 12 hours. For working-up, it is poured onto ice water and carefully neutralized with pulverized sodium bicarbonate. Then, it is extracted 3× with ethyl acetate, the organic phase is dried with magnesium sulfate and concentrated by evaporation, crude product 104 mg (83% of theory) of the aldehyde of compound 4.0. The crude product can be used without further purification.
100 mg (0.356 mmol) of compound 6.0 and 126 mg of N-methylmorpholine-N-oxide are dissolved in 5 ml of dichloromethane and stirred for 10 minutes with pulverized molecular sieve (4 A). Then, 6 mg of tetrapropylammonium perruthenate is added, and it is stirred for 4 more hours at room temperature. After concentration by evaporation, it is chromatographed on silica gel (hexane/ethyl acetate 4:1>2:1). Yield: 75 mg (76% of theory) of the ketone of compound 5.0.
265 mg (1 mmol) of compound 4.2 is dissolved in 20 ml of tetrahydrofuran. While being cooled in an ice bath, 5 equivalents of methylmagnesium bromide (3 molar solution in ether) is added in portions. Then, it is stirred for 3 more hours at room temperature and then quenched with water while being cooled. Then, it is mixed with ammonium chloride solution, extracted 3× with ethyl acetate, the organic phase is dried with magnesium sulfate and concentrated by evaporation. Flash chromatography (hexane/ethyl acetate 2:1) yields 213 mg (76% of theory) of the alcohol of compound 6.0.
ESI:MH+ 282 (100%) 276 (5%)
Similarly produced are also the following intermediate products:
Subjects of this invention are thus also compounds of general formula Ia
in which
D stands for halogen, and X, R1, and R2 have the meanings that are indicated in general formula (I).
Those intermediate products of general formula Ia, in which D stands for chlorine and X, R1 and R2 have the meanings that are indicated in the general formula, are especially valuable.
Another subject of this invention are also those compounds that fall under industrial property right DE 4029650, whose action is in the fungicide range and which are not described as CDK inhibitors, however, and also their use for treating cancer is not described.
The invention thus relates in addition to pharmaceutical agents that comprise a compound of general formula I in which
R2, R3, R4, R7 and R8 have the meanings that are indicated in general formula I, as well as isomers, diastereomers, enantiomers and salts thereof.
The agents according to the invention can also be used for treating cancer, auto-immune diseases, cardiovascular diseases, chemotherapy agent-induced alopecia and mucositis, infectious diseases, nephrological diseases, chronic and acute neurodegenerative diseases and viral infections, whereby cancer is defined as solid tumors and leukemia; auto-immune diseases are defined as psoriasis, alopecia and multiple sclerosis; cardiovascular diseases are defined as stenoses, arterioscleroses and restenoses; infectious diseases are defined as diseases that are caused by unicellular parasites; nephrological diseases are defined as glomerulonephritis; chronic neurodegenerative diseases are defined as Huntington's disease, amyotrophic lateral sclerosis, Parkinson's disease, AIDS dementia and Alzheimer's disease; acute neurodegenerative diseases are defined as ischemias of the brain and neurotraumas; and viral infections are defined as cytomegalic infections, herpes, hepatitis B or C, and HIV diseases.
The following examples describe the biological action of the compounds according to the invention without limiting the invention to these examples.
Recombinant CDK2- and CycE-GST-fusion proteins, purified from baculovirus-infected insect cells (Sf9), were obtained by Dr. Dieter Marmé, Klinik für Tumorbiologie [Clinic for Tumor Biology], Freiburg. Histone IIIS, which was used as a kinase substrate, was purchased by the Sigma Company.
CDK2/CycE (50 ng/measuring point) was incubated for 15 minutes at 22° C. in the presence of various concentrations of test substances (0 μm, as well as within the range of 0.01-100 μm) in assay buffer [50 mmol of tris/HCl pH 8.0, 10 mmol of MgCl2, 0.1 mmol of Na ortho-vanadate, 1.0 mmol of dithiothreitol, 0.5 μm. of adenosine triphosphate (ATP), 10 μg/measuring point of histone IIIS, 0.2 μCi/measuring point of 33P-gamma ATP, 0.05% NP40, 12.5% dimethyl sulfoxide]. The reaction was stopped by adding EDTA solution (250 mmol, pH 8.0, 14 μl/measuring point).
From each reaction batch, 10 μl was applied to P30 filter strips (Wallac Company), and non-incorporated 33P-ATP was removed by subjecting the filter strips to three washing cycles for 10 minutes each in 0.5% phosphoric acid. After the filter strips were dried for one hour at 70° C., the filter strips were covered with scintillator strips (MeltiLex™ A, Wallac Company) and baked for one hour at 90° C. The amount of incorporated 33P (substrate phosphorylation) was determined by scintillation measurement in a gamma-radiation measuring device (Wallac).
Cultivated human tumor cells (as indicated) were flattened out at a density of 5000 cells/measuring point in a 96-hole multititer plate in 200 μl of the corresponding growth medium. After 24 hours, the cells of one plate (zero-point plate) were colored with crystal violet (see below), while the medium of the other plates was replaced by fresh culture medium (200 μl), to which the test substances were added at various concentrations (0 μm, as well as in the range of 0.01-30 μm; the final concentration of the solvent dimethyl sulfoxide was 0.5%). The cells were incubated for 4 days in the presence of test substances. The cell proliferation was determined by coloring the cells with crystal violet: the cells were fixed by adding 20 μl/measuring point of a 11% glutaric aldehyde solution for 15 minutes at room temperature. After three washing cycles of the fixed cells with water, the plates were dried at room temperature. The cells were colored by adding 100 μl/measuring point of a 0.1% crystal violet solution (pH was set at 3 by adding acetic acid). After three washing cycles of the colored cells with water, the plates were dried at room temperature. The dye was dissolved by adding 100 μl/measuring point of a 10% acetic acid solution. The extinction was determined by photometry at a wavelength of 595 nm. The change of cell growth, in percent, was calculated by standardization of the measured values to the extinction values of the zero-point plate (=0%) and the extinction of the untreated (0 μm) cells (=100%).
The results of Examples 1 and 2 are cited in the following tables.
To prove the superiority of the compounds according to the invention compared to the known compounds, the compounds according to the invention were compared to known reference compounds and structurally-similar known compounds in the enzyme test. The result is cited in the following table:
Nr. 48
Nr. 9
Nr. 11
Olomoucine
Roscovitine
Kenpaullone
Alsterpaullone
Purvalanol A
It can be seen from the results of the table that both in the enzyme test and in the cell test, the compounds according to the invention have significantly higher activities in the enzyme and in the MCF-7 cells than the compounds that are known from the prior art. The compounds according to the invention are thus far superior to the known compounds.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10127581.1 | May 2001 | DE | national |
| 10212098.6 | Mar 2002 | DE | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10156759 | May 2002 | US |
| Child | 11819307 | US | |
| Parent | 10842419 | May 2004 | US |
| Child | 10156759 | US |
