Patent Application
20070142401
The invention relates to the use of compounds (hereinafter: “COMPOUND”) or a N-Oxide or a pharmaceutically acceptable salt thereof having an activity on protein kinases PKC alpha, PKC beta, PKC gamma, PKC epsilon, PKC theta, CDK-1, KDR, PKA, Flt-1, Flt-2, Flt-3 or Flt-4, or on a combination of the above enzymes, for the treatment and/or prevention of neurological and vascular disorders related to beta-amyloid generation and/or aggregation such as neurodegenerative diseases like Down's Syndrome, memory and cognitive impairment, dementia, amyloid neuropathies, brain inflammation, nerve and brain trauma, vascular amyloidosis, or cerebral hemorrhage with amyloidosis.
A compound of formula I
wherein
Ra is H; C1-4alkyl; or C1-4alkyl substituted by OH, NH2, NHC1-4alkyl or N(di-C1-4alkyl)2;
Rb is H; or C1-4alkyl;
R is a radical of formula (a), (b), (c), (d), (e) or (f)
wherein
each of R2, R3, R5, R6, R9, R10, R12, R13, R15 and R′15, independently, is H, halogen, C1-4alkyl, CF3, OH, SH, NH2, C1-4alkoxy, C1-4alkylthio, NHC1-4alkyl, N(di-C1-4alkyl)2 or CN;
either E is —N═ and G is —CH═ or E is —CH═ and G is —N═; and
ring A is optionally substituted,
or a salt thereof.
Preferably a compound of formula I wherein the heterocyclic residue as R1, R4, R7, R8, R11, R14 or Y or formed, respectively, by NR16R17 or NR19R20, is a three to eight membered saturated, unsaturated or aromatic heterocyclic ring comprising 1 or 2 heteroatoms, and optionally substituted on one or more ring carbon atoms and/or on a ring nitrogen atom when present.
More preferably a compound of formula I wherein the heterocyclic residue is R1, R4, R7, R8, R11, R14 or Y or formed, respectively, by NR16R17 or NR19R20, is a residue of formula (γ).
wherein
the ring D is a 5, 6 or 7 membered saturated, unsaturated or aromatic ring;
Xb is —N—, —C— or —CH—;
Xc is —N═, —NRf—, —CRf′═ or —CHRf′— wherein Rf is a substituent for a ring nitrogen atom and is selected from C1-6alkyl; acyl; C3-6cycloalkyl; C3-6cycloalkyl-C1-4alkyl; phenyl; phenyl-C1-4alkyl;
a heterocyclic residue; and a residue of formula β
—R21—Y′ (β)
wherein R21 is C1-4alkylene or C2-4alkylene interrupted by O and Y′ is OH, NH2, NH(C1-4alkyl) or N(C1-4alkyl)2; and Rf′ is a substituent for a ring carbon atom and is selected from C1-4alkyl;
C3-cycloalkyl optionally further substituted by C1-4-alkyl;
wherein p is 1, 2 or 3; CF3;
halogen; OH; NH2; —CH2—NH2; —CH2—OH; piperidin-1-yl; and pyrrolidinyl;
the bond between C1 and C2 is either saturated or unsaturated;
each of C1 and C2, independently, is a carbon atom which is optionally substituted by one or two substituents selected among those indicated above for a ring carbon atom; and
the line between C3 and Xb and between C1 and Xb, respectively, represents the number of carbon atoms as required to obtain a 5, 6 or 7 membered ring D.
Even more preferably a compound of formula I, wherein D is a piperazinyl ring optionally C— and/or N-substituted as specified in claim 3.
Yet even more preferably COMPOUND a compound of formula I wherein
Ra is H; CH3; CH2—CH3; or isopropyl,
Rb is H; halogen; C1-6-alkoxy; or C1-6alkyl, and either
I. R is a radical of formula (a)
II. R is a radical of formula (b)
III. R is a residue of formula (c)
IV. R is a radical of formula (d)
V. R is a radical of formula (e)
The compounds of formula I may exist in free form or in salt form, e.g. addition salts with e.g. organic or inorganic acids, for example, hydrochloric acid, acetic acid, trifluoroacetic acid. It will be appreciated that the compounds of formula I may exist in the form of optical isomers, racemates or diastereoisomers. For example, a ring carbon atom bearing a substituent in the position 3 of the piperazinyl residue is asymmetric and may have the D- or
L- configuration. It is to be understood that the present invention embraces all enantiomers and their mixtures. Similar considerations apply in relation to starting materials exhibiting asymmetric carbon atoms as mentioned.
An especially preferred COMPOUND is a compound of formula I, as herein before described,
wherein
when R is of formula (a)
R1 is -(4-methyl-piperazin-1-yl), 1-piperazinyl, 3-methyl-piperazin-1-yl or -(4,7-diaza-spiro[2.5]oct-7-yl)
R2 is 2-Cl or 2-CH3
R3 is 3-CH3, 3-CF3or H
Ra is H or CH3
And when,
R is of formula (b)
R4 is -(4,7-diaza-spiro[2.5]oct-7-yl), 3-methyl-piperazin-1-yl or 4-methyl-3-methyl-piperazin-1-yl
Ra is H or CH3
And when
R is of formula (c)
R14 is -4-methyl-piperazin-1-yl, 3-methyl-piperazin-1-yl, -4,7-diaza-spiro[2.5]oct-7-yl, 1-piperazinyl, 4-methyl-3-methyl-piperazin-yl, 3-methoxyethyl-piperazin-1-yl, 3-ethyl-piperazin-1-yl, 3-benzyl-piperazin-1-yl or 3-CH2F-piperazin-1-yl
R15 is Cl, Br, CF3, F
R16 is CH3, H, CH2—CH3
Ra is H or CH3
Rb is H, CH2—CH2—CH3, F, CH(CH3)2, Cl, OCH3, CH3 or CH2—CH3
And when
R is of formula (d)
R8 is 3-methyl-piperazin-1-yl, 4-benzyl-1-piperazinyl or 1-piperazinyl
Ra is CH3 or H
And when
R is of formula (e)
R9 is -4,7-diaza-spiro[2.5]oct-7-yl, 3-ethyl-piperazin-1-yl, 3-methyl-piperazin-1-yl, 4-methyl-3-methyl-piperazin-1-yl or 3-ethyl-piperazin-1-yl
Ra is H, CH2—CH3 or CH(CH3)2
Rb is CH3, F, CH(CH3)2, OCH3, CH2—CH3 or Cl
most prefered COMPOUND is 3-[2-Chloro-5-(4-methyl-piperazin-1-yl)-3-trifluoromethyl-phenyl]-4-(1H-indol-3-yl)-pyrrole-2,5-dione having the formula
3-(1H-Indol-3-yl)-4-[2-(4-methyl-piperazin-1-yl)-quinazolin-4-yl]-pyrrole-2,5-dione having the formula
Even more preferred, Compound means any of the other definitions of COMPOUND wherein the compound has an activity on PKC alpha, PKC beta, PKC gamma, PKC epsilon, PKC theta, or on a combination of these enzymes.
Compounds of formula I and methods for the preparation of such compounds are in particular generically and specifically disclosed in the patents and patent application WO2003082859, in particular in the compound claims and the final products of the working examples, the subject-matter of the final products, the pharmaceutical preparations and the claims is hereby incorporated into the present application by reference to this publication.
The general terms used hereinbefore and hereinafter preferably have within the context of this disclosure the following meanings, unless otherwise indicated:
Alkyl or alkoxy may be straight or branched. Phenyl-C1-4alkyl is preferably benzyl or phenethyl. In C1-4alkoxy-C1-4alkyl the alkoxy moiety is preferably methoxy or ethoxy and the alkyl moiety preferably methyl or ethyl; a suitable example is e.g. 2-methoxyethyl. Halogen may be F, Cl, Br or I, preferably F, Cl or Br. Halogeno-C1-4alkyl is alkyl wherein one or more H are replaced by halogen, e.g. Cl or F, e.g. CH2Cl, CH2F or CF3
In the radical of formula (a) or (c), R2 or R15 is preferably in para to R1 or R14, respectively. R3 is preferably in meta to R1. In the radical or formula (e), R9 is preferably 4,7-diaza-spiro [2.5] oct-7-yl.
PKC is protein kinase C
CDK is cyclin dependent kinase
PKA is protein kinase A
Salts are especially the pharmaceutically acceptable salts of compounds of formula I.
Such salts are formed, for example, as acid addition salts, preferably with organic or inorganic acids, from compounds of formula I with a basic nitrogen atom, especially the pharmaceutically acceptable salts. Suitable inorganic acids are, for example, halogen acids, such as hydrochloric acid, sulfuric acid, or phosphoric acid. Suitable organic acids are, for example, carboxylic, phosphonic, sulfonic or sulfamic acids, for example acetic acid, propionic acid, octanoic acid, decanoic acid, dodecanoic acid, glycolic acid, lactic acid, fumaric acid, succinic acid, adipic acid, pimelic acid, suberic acid, azelaic acid, malic acid, tartaric acid, citric acid, amino acids, such as glutamic acid or aspartic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, cyclohexanecarboxylic acid, adamantanecarboxylic acid, benzoic acid, salicylic acid, 4-aminosalicylic acid, phthalic acid, phenylacetic acid, mandelic acid, cinnamic acid, methane- or ethane-sulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, 1,5-naphthalene-disulfonic acid, 2-, 3- or 4-methylbenzenesulfonic acid, methylsulfuric acid, ethylsulfuric acid, dodecylsulfuric acid, N-cyclohexylsulfamic acid, N-methyl-, N-ethyl- or N-propyl-sulfamic acid, or other organic protonic acids, such as ascorbic acid.
In the presence of negatively charged radicals, such as carboxy or sulfo, salts may also be formed with bases, e.g. metal or ammonium salts, such as alkali metal or alkaline earth metal salts, for example sodium, potassium, magnesium or calcium salts, or ammonium salts with ammonia or suitable organic amines, such as tertiary monoamines, for example triethylamine or tri(2-hydroxyethyl)amine, or heterocyclic bases, for example N-ethyl-piperidine or N,N′-dimethylpiperazine.
The invention further relates to the use of COMPOUND or a N-Oxide or a pharmaceutically acceptable salt thereof for the manufacture of medicament having an activity on protein kinases PKC alpha, PKC beta, PKC gamma, PKC epsilon, PKC theta, CDK-1, KDR, PKA, Flt-1, Flt-2, Flt-3 or Flt-4, or on a combination of the above enzymes, for the treatment and/or prevention of neurological and vascular disorders related to beta-amyloid generation and/or aggregation such as neurodegenerative diseases like Down's Syndrome, memory and cognitive impairment, dementia, amyloid neuropathies, brain inflammation, nerve and brain trauma, vascular amyloidosis, or cerebral hemorrhage with amyloidosis.
Depending on species, age, individual condition, mode of administration, and the clinical picture in question, effective doses, for example daily doses of about 10-1000 mg, preferably 10-50 mg or 50-200 mg or 200-400 mg, especially 50-100 mg or 300-400 mg, are administered to warm-blooded animals of about 70 kg bodyweight. For adult patients with neurological and vascular disorders related to beta-amyloid generation and/or aggregation, especially neurodegenerative diseases like Down's Syndrome, memory and cognitive impairment, dementia, amyloid neuropathies, brain inflammation, nerve and brain trauma, vascular amyloidosis, or cerebral hemorrhage with amyloidosis.
The invention relates likewise to a process or a method for the treatment of neurological and vascular disorders related to beta-amyloid generation and/or aggregation, especially neurodegenerative diseases like Down's Syndrome, memory and cognitive impairment, dementia, amyloid neuropathies, brain inflammation, nerve and brain trauma, vascular amyloidosis, or cerebral hemorrhage with amyloidosis. The COMPOUNDS thereof can be administered as such or especially in the form of pharmaceutical compositions, prophylactically or therapeutically, preferably in an amount effective against the said diseases, to a warm-blooded animal, for example a human, requiring such treatment. In the case of an individual having a bodyweight of about 70 kg the daily dose administered is from approximately 0.01 g to approximately 5 g, preferably from approximately 0.25 g to approximately 1.5 g, more preferably 0.01 g to 0.05 g, even more preferably 0.025 g to 0.1 g most preferably 0.05 g to 1 g of a compound of the present invention.
The compounds of formula I may be administered by any conventional route, in particular enterally, e.g. orally, e.g. in the form of tablets or capsules, or parenterally, e.g. in the form of injectable solutions or suspensions, topically, e.g. in the form of lotions, gels, ointments or creams, or in a nasal or a suppository form. Pharmaceutical compositions comprising a compound of formula I in free form or in pharmaceutically acceptable salt form in association with at least one pharmaceutical acceptable carrier or diluent may be manufactured in conventional manner by mixing with a pharmaceutically acceptable carrier or diluent. Unit dosage forms for oral administration contain, for example, from about 0.1 mg to about 500 mg of active substance.
Topical administration is e.g. to the skin. A further form of topical administration is to the eye. The compounds of formula I may be administered in free form or in pharmaceutically acceptable salt form e.g. as indicated above. Such salts may be prepared in conventional manner and exhibit the same order of activity as the free compounds.
The invention relates also to a method for administering to a human subject suffering from a neurological and vascular disorders related to beta-amyloid generation and/or aggregation, especially neurodegenerative diseases like Down's Syndrome, memory and cognitive impairment, dementia, amyloid neuropathies, brain inflammation, nerve and brain trauma, vascular amyloidosis, or cerebral hemorrhage with amyloidosis, COMPOUND or a pharmaceutically acceptable salt thereof, which comprises administering a pharmaceutically effective amount of COMPOUND or a pharmaceutically acceptable salt thereof to the human subject, preferably once daily for a period exceeding 3 months. The invention relates especially to such method wherein a daily dose of 200 to 800 mg, or 10 mg to 200 mg especially 400-600 mg or 10-100 mg, preferably 400 mg or 10-50 mg, of COMPOUND is administered.
The invention also relates in a combination which comprises (a) COMPOUND or a pharmaceutically acceptable salt thereof and (b) a therapeutic agent for the treatment of neurological and vascular disorders related to beta-amyloid generation and/or aggregation, most preferably a combination wherein the combination partners are present in synergistically effective amounts.
The effective dosage of each of the combination partners employed in the combination may vary depending on a variety of factors including the particular combination of the pharmaceutical compound partners, the route of administration, the severity of the disease, the renal and hepatic functions of the patient. The molar ratio (a)/(b) of the combination partners is about 0.1 to 10, most preferably 0.3 to 3 and the unit dosage form contains 20 to 200 mg, most preferably 50 to 150 mg of 3-[2-Chloro-5-(4-methyl-piperazin-1-yl)-3-trifluoromethyl-phenyl]-4-(1H-indol-3-yl)-pyrrole-2,5-dione of the formula I.
Cell Culture
HEK/APPswe cells are plated in microtiter plates precoated with 10 μg/ml poly-D-lysine at 12′000 cells/well in 100 μl/well DMEM medium supplemented with 10% FCS, 0.25 mg/ml G418 sulfate, 1% penicillin streptomycin. The following day, supernatant is replaced with 90 μl/well of fresh medium and 10 μl/well of compound diluted in culture medium are added. Two types of control wells are used: cell culture medium without cells plus 10 μl/well of all compound dilutions (background signals) and cell culture medium from untreated cells (positive control). 24 hours later after compound addition, conditioned medium is collected and Aβ levels determined by a specific sandwich ELISA.
Aβ40 and Aβ42 Detection by Sandwich ELISA
For the sandwich ELISA, the maxisorp microtiterplates are coated overnight at 4° C. with 100 μl/well of the monoclonal antibody 25H 10 diluted 1:1000 in PB for Aβ40 detection or monoclonal antibody B10E7 diluted 1:2750 for detection of Aβ42. Wells are then emptied, washed three times with 350 μl PBS and blocking is performed for 2 hours at room temperature with 200 μl/well of 2% BSA, 0.05% Tween20 in PBS. After washing the wells as described above, 10 μl of the conditioned media samples to be tested are added to wells containing 90 μl of medium and 0.18 μg/ml of biotinylated monoclonal β1 antibody and incubated overnight at 4° C. Wells were washed as described above and 100 μl/well of alkaline phosphatase coupled to streptavidin diluted 1:5′000 in medium are added. After 1 hour incubation at room temperature wells are washed as described above and alkaline phosphatase activity is determined by adding 100 μl/well of diethanolamine buffer, pH 9.8 (100 mM diethanolamine, 1 mM MgCl2, pH adjusted to 9.8 with 2 M HCl) containing the chemiluminescent CSPD substrate (25 mM stock solution diluted 1:416) and the enhancer Emerald II (diluted 1:10). After 15 minutes incubation at room temperature in the dark, plates are measured on the luminometer (Analyst AD; LJL Biosystems, USA Aβ40). Values are given as % reduction of A
MTS Assay
To determine cytotoxicity, cells are tested by the MTS colorimetric kit performed essentially according to the manufacturer's specifications (Promega, #G5430X). After collecting the conditioned medium for the sandwich ELISA, the rest of the conditioned medium is removed completely and replaced with 100 μl/well culture medium containing one fifth of MTS solution prepared as recommended in the kit. After 3 hours incubation at 37° C., absorbance is read at an OD of 490 nm with a reference wavelength set to 630 nm. Values are given as % metabolic rate (n=6). The 0% value is calculated from wells which had no cells, 100% from wells with an untreated cell layer
This compound has the following activities in cell-free enzyme assays:
The compound of Example 2 demonstrates a clear reduction of Aβ secretion in the medium of HEK/APPswe cell cultures at concentrations below 1 microM, without having any effect on cellular viability.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0325032.1 | Oct 2003 | GB | national |
| 0325176.6 | Oct 2003 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP04/12082 | 10/26/2004 | WO | 11/1/2006 |
