|Publication number||USRE39608 E1|
|Application number||US 10/935,683|
|Publication date||May 1, 2007|
|Filing date||Nov 23, 1999|
|Priority date||Nov 27, 1998|
|Also published as||CA2352554A1, CA2352554C, CN1184208C, CN1332731A, DE59908600D1, EP1133477A1, EP1133477B1, US6448271, WO2000032579A1|
|Publication number||10935683, 935683, PCT/1999/9004, PCT/EP/1999/009004, PCT/EP/1999/09004, PCT/EP/99/009004, PCT/EP/99/09004, PCT/EP1999/009004, PCT/EP1999/09004, PCT/EP1999009004, PCT/EP199909004, PCT/EP99/009004, PCT/EP99/09004, PCT/EP99009004, PCT/EP9909004, US RE39608 E1, US RE39608E1, US-E1-RE39608, USRE39608 E1, USRE39608E1|
|Inventors||Wilfried Lubisch, Michael Kock, Thomas Höger, Sabine Schult, Roland Grandel, Reinhold Müller|
|Original Assignee||Abbott Gmbh & Co. Kg|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (11), Non-Patent Citations (6), Referenced by (30), Classifications (45), Legal Events (3)|
|External Links: USPTO, USPTO Assignment, Espacenet|
This application is a 371 of PCT/EP99/09004 filed Nov. 23, 1999.
The present invention relates to novel benzimidazoles, their preparation and their use as inhibitors of the enzyme poly(ADP-ribose)polymerase or PARP (EC 220.127.116.11) for the preparation of drugs.
Poly(ADP-ribose)polymerase (PARP) or, as it is also known, poly(ADP-ribose)synthase (PARS) is a regulatory enzyme which is found in cell nuclei (K. Ikai et al., J. Histochem. Cytochem. 31 (1983), 1261-1264). It is assumed that PARP plays a role in repairing DNA breaks (M. S. Satoh et al., Nature 356 (1992), 356-358). Damage to or breaks in the DNA strands activate the enzyme PARP which, if it has been activated, catalyses the transfer of ADP-ribose from NAD (S. Shaw, Adv. Radiat.Biol. 11 (1984), 1-69). Nicotinamide is liberated from NAD. Nicotinamide is converted back into NAD with consumption of the energy carrier ATP by other enzymes. Overactivation of PARP would accordingly result in an unphysiologically high consumption of ATP, and this leads to cell damage and cell death in extreme cases.
It is known that radicals such as the superoxide anion, NO and the hydrogen peroxide can lead to DNA damage in cells and hence activate PARP. The formation of large amounts of radicals is observed in a number of pathophysiological conditions, and it is assumed that this accumulation of radicals leads or contributes to the observed cell or organ damage. These include, for example, ischemic conditions of organs, as in stroke, myocardial infarct (C. Thiemermann et al., Proc. Natl. Acad. Sci USA 94 (1997), 679-683) or ischemia of the kidneys, as well as reperfusion damage as occurs, for example, following the lysis of myocardial infarct (see above: C. Thiermermann et al.). The inhibition of the enzyme PARP might accordingly be a means for preventing or reducing this damage at least in part. PARP inhibitors might therefore constitute a new therapeutic principle for treating a number of disorders.
The enzyme PARP influences the repair of DNA damage and could thus also play a role in therapy of cancer diseases, since the higher action potential against tumor tissue was observed in combination with cytostatic substances (G. Chen et al. Cancer Chemo. Pharmacol. 22 (1988), 303).
Nonlimiting examples of tumors are leukemia, glioblastomas, lymphomas, melanomas, carcinomas of the breast and cervical carcimonas. It was also found that PARP inhibitors can have an immunosuppressive effect (D. Weltin et al. Int. J. Immunopharmacol. 17 (1995), 265-271).
It was also discovered that PARP is involved in immunological diseases or disorders in which the immune system plays an important role, for example rheumatoid arthritis and septic shock, and that PARP inhibitors can have an advantageous effect on the course of the disorder (H. Kröger et al. Inflammation 20 (1996), 203-215; W. Ehrlich et al. Rheumatol. Int. 15 (1995), 171-172; C. Szabo et al., Proc. Natl. Acad. Sci. USA 95 (1998), 3867-3872; S. Cuzzocrea et al. Eur. J. Pharmacol. 342 (1998), 67-76).
For the purposes of this invention, PARP is also understood as meaning isoenzymes of the PARP enzyme described above.
Furthermore, the PARP inhibitor 3-aminobenzamide exhibited protective effects in a model for circulatory shock (S. Cuzzocrea et al., Br. J. Pharmacol. 121 (1997), 1065-1074).
PARP is also involved in diabetes mellitus (V. Burkhart et al., Nature Medicine (1999), 5314-19).
Benzimidazoles have been widely described.
The synthesis of 2-phenylbenzimidaz-4-ylamides which also carry a substituted alkyl chain on the amide radical and which are said to have a cytotoxic effect is mentioned in J. Med. Chem. 33 (1990), 814-819. WO 97/04771 mentions 4-benzimidazolamides which inhibit PARS. In particular, derivatives which carry a phenyl ring in the 2-position, where the phenyl ring may furthermore be substituted by simple substituents such as nitro, methoxy or CF3, are described there as being effective. Although some of these substances exhibit good inhibition of the enzyme PARP, the derivatives described there have the disadvantage that they have little or no solubility in aqueous solutions and hence cannot be applied as an aqueous solution.
Benzimidazoles which carry a piperidine ring in the 2-position have also been described. Thus, in J. Het. Chem. 24 (1987), 31, derivatives have been prepared as antihistamine drugs. In J. Het. Chem. 32 (1995), 707 and J. Het. Chem. 26 (1989), 541, analogous compounds having the same use have been described. 2-Piperidinylbenzimidazoles are mentioned in EP 818454 as antihistamine drugs and in WO 9736554 as agents against hepatitis. Derivatives are likewise mentioned in CA 80, 146143, Fr. 2103639 and in Khim. Ceterotsikl. Soedin 1 (1974), 104.
However, the importance of substituents on the phenylaromatics in the benzimidazole fragment has not been investigated. Furthermore, those benzimidazoles which carry a 4- to 8-membered heterocycle, in particular a piperidine ring, in the 2-position have not been described to date as being PARP inhibitors.
The present application describes the surprising finding that the introduction of a carboxamide radical on the benzimidazole aromatic gives benzimidazoles which constitute novel and highly effective PARP inhibitors, provided that they are substituted in the 2-position by a saturated heterocycle.
In a number of treatments, such as for stroke, the active compounds are applied intravenously as an infusion solution. For this purpose, it is necessary to have substances, in this case PARP inhibitors, which have sufficient water solubility at or about physiological pH (i.e. pH of 5-8), so that an infusion solution can be prepared. However, many of the PARP inhibitors described, in particular the more effective PARP inhibitors, have the disadvantage that they exhibit only little or no water solubility at the pH values and are therefore not suitable for intravenous application. Such active compounds can be applied only with excipients which are intended to impart water solubility (cf. WO 97/04771). These excipients, for example polyethylene glycol and dimethyl sulfoxide, frequently cause side effects or are even not tolerated. No highly effective PARP inhibitors having sufficient water solubility have been described to date.
It was surprisingly found that benzimidazoles which carry a piperidine ring on the imidazole ring are highly effective inhibitors and, owing to the incorporating of the aliphatic amine radical, permit salt formation with acids, resulting in substantially improved water solubility and hence permitting the preparation of an infusion solution.
The present invention describes novel benzimidazole derivatives of the formula I which have advantages over the compounds described above and constitute potent PARP inhibitors and at the same time have sufficient water solubility. When compounds of the formula I are referred to, they are understood as meaning the compounds of the formulae Ia and Ib. The present invention relates to substituted benzimidazoles of the formula I:
The compounds of the formula I where R1 is hydrogen are preferred.
The compounds of the formula I where R2 is hydrogen are preferred.
The compounds of the formula I where R4 is hydrogen are preferred.
The compounds of the formula I where R3 is hydrogen are preferred.
The compounds of the formula I where R3 is hydrogen, C1-C6-alkyl, benzyl or phenethyl are preferred.
The compounds of the formula I where R1, R2 and R4 are each hydrogen and A is piperidine which is bonded at the 4-position on the benzimidazole and R3 is hydrogen, C1-C6-alkyl, benzyl or phenethyl and is bonded in the 1-position on the piperidine ring are particularly preferred.
The respective meanings of R5 to R10 are independent of one another in R1 to R4.
The preferred meanings of NR8R9, NR24R25 and NR32R33, as cyclic amine, are piperidine, pyrrolidine, piperazine and homopiperazine. In the case of piperazine and homopiperazine, the ring may preferably furthermore carry a radical of branched or straight-chain C1-C6-alkyl, C3-C7-cycloalkyl-C1-C4-alkyl, CO—R7 or phenyl.
The preferred meaning of A is piperidine, pyrrolidine, piperazine, morpholine or homopiperazine.
The compounds of the formula I where A is piperazine or piperidine are particularly preferred.
The compounds of the formula I may be used in the form of racemates, enantiomerically pure compounds or diastereomers. If enantiomerically pure compounds are desired, these can be obtained, for example, by carrying out a classical resolution of the racemate with the compounds of the formula I or their intermediates used in a suitable optically active base or acid.
The saturated or monounsaturated cyclic structures A may be present as cis-isomers, trans-isomers or mixtures thereof.
The present invention also relates to compounds which are mesomers or tautomers of compounds of the formula I.
The present invention furthermore relates to the physiologically tolerated salts of the compound I, which can be obtained by reacting compounds I with a suitable acid or base. Suitable acids and bases are listed, for example, in Fortschritte der Arzneimittelforschung, 1966, Birkhäuser Verlag, Vol. 10, pages 224-285. These include, for example, hydrochloric acid, citric acid, tartaric acid, lactic acid, phosphoric acid, methanesulfonic acid, acetic acid, formic acid, maleic acid, fumaric acid, etc., and sodium hydroxide, lithium hydroxide, potassium hydroxide and Tris.
Prodrugs are understood as meaning those compounds which are metabolized in vivo to give compounds of the formula I. Typical prodrugs are phosphates, carbamates of amino acids, esters and others.
The preparation of the novel benzimidazoles I can be carried out by various routes which are shown in synthesis scheme 1.
The benzimidazole I or VII is obtained by condensation of the aldehyde V with phenylenediamines V1, the procedure preferably being carried out in polar solvents, such as ethanol or dimethylformamide, and with the addition of acids, such as acetic acid, at elevated temperatures, as a rule from 80 to 120° C. It is advantageous for the reaction to add weak oxidizing agents, such as copper(II) salts, which are added as aqueous solution.
If, in the benzimidazole VII, R is NH2, novel compounds I are formed directly in the condensation. Otherwise, if R is O-alkyl these esters can be reacted with ammonia, if required at elevated temperatures and superatmospheric pressure, to give the amide I. Alternatively, the esters VII can be reacted with hydrazine in polar solvents, such as the alcohols butanol and ethanol or dimethylformamide, at elevated temperatures, preferably from 80 to 130° C., the result being hydrazide VII (R=NHNH2) which can then be reduced under reductive conditions, for example with Raney nickel in alcohols under reflux, to give the amide I.
The radical R1 on the benzimidazole radical in I (R1=H) is introduced under conventional alkylating conditions. Benzimidazoles I are alkylated with R1—L, where L is a leaving group, using a base at from 25 to 150° C., but mainly at elevated temperatures such as from 60 to 130° C., the novel product I where R1≈hydrogen being obtained. The procedure is carried out in solvents, for example dimethylformamide, dimethylsulfoxide, alcohols, e.g. ethanol, ketones, e.g. methyl ethyl ketone or acetone, aliphatic ethers, e.g. tetrahydrofuran, and hydrocarbons, e.g. toluene, it also being possible to use mixtures. Suitable bases are, for example, alcoholates, e.g. sodium ethanolate and potassium tert-butanolate, carbonates, e.g. potassium carbonate, hydrides, e.g. sodium hydride, and hydroxides, e.g. sodium hydroxide and potassium hydroxide.
Various crown ethers, such as 18-crown-6, may also be added in catalytic amounts. Phase transfer conditions may also be employed (for methods, cf. R. C. Larock, Comprehensive Organic Transformations, 1989, page 445 et seq.). The leaving group L used may be a halide, e.g. bromide, chloride or iodide, or, for example, a tolysate or mesylate.
Alternatively to the aldehydes V shown in Scheme 1 it is also possible to use benzoic acids, such as IX (cf. Scheme 2), or benzonitriles, such as XIII (cf. Scheme 3), instead of the benzaldehyde. The preparation of these derivatives is carried out analogously to the preparation of the substituted benzaldehydes V. Starting from IX, the condensation to give VII is carried out in two stages. First, the benzoic acid XI is reacted with the aniline VI with a peptide-like coupling to give the amide XII. The conditions used here are the conventional ones which are listed, for example, in Houben-weyl, Methoden der Organischen Chemie, 4th Edition, E5, Chapter V, or C. R. Larock, Comprehensive Organic Transformations, VCH Publisher, 1989, page 972 et seq. Cyclization to the benzimidazole is then effected at elevated temperatures, for example from 60 to 180° C., with or without solvents, such as dimethylformamide, with the addition of acids, such as acetic acid, or directly in acetic acid itself.
The reaction of the phenylenediamine VI with a benzonitrile XIII is likewise effected under conventional conditions. It is possible to employ solvents, such as dimethylformamide, with the addition of acids at elevated temperatures, such as from 60 to 200° C. However, it is also possible to use the conventional methods for the preparation of amides from benzonitriles, as described in J. Amer. Chem. Soc. (1957), 427 and J. Org. Chem. (1987), 1017.
The substituted benzimidazoles I contained in the present invention are inhibitors of the enzyme poly(ADP-ribose) polymerase or PARP (EC 18.104.22.168).
The inhibitory effect of the substituted benzimidazoles I was determined by an enzyme test already known in the literature, the Ki value being determined as a measure of activity. The benzimidazoles I were measured in this way for an inhibitory effect of the enzyme poly(ADP-ribose) polymerase or PARP (EC 22.214.171.124).
The substituted benzimidazoles of the formula I are inhibitors of poly(ADP-ribose) polymerase (PARP) or, as it is also referred to, poly(ADP-ribose)synthase (PARS) and can therefore be used for the treatment and prophylaxis of disorders which are associated with increased activity of these enzymes.
The compounds of the formula I can be used for preparing drugs for the treatment of damage following ischemias and for prophylaxis where ischemias of various organs are expected.
The present benzimidazoles of the formula I can then be used for the treatment and prophylaxis of neurodegenerative disorders which occur after ischemia, trauma (craniocerebral trauma), massive bleeding, subarachnoid hemorrhages and stroke, and of neurodegenerative disorders such as multi-infarct dementia. Alzheimer's disease and Huntington's disease and of epilepsies, in particular of generalized epileptic attacks, for example petit mal and tonoclonic attacks and partial epileptic attacks such as temporal lobe, and complex partial attacks, and furthermore for the treatment and prophylaxis of cardiac damage following myocardial ischemias and damage to the kidneys following renal ischemias, for example acute renal insufficiency, acute renal failure, damage which is caused by drug therapy such as, for example, during ciclosporin therapy or damage which occurs during or after a kidney transplantation. Furthermore, the compounds of the formula I can be used for the treatment of acute myocardial infarction and damage which occurs during and after its lysis under treatment with drugs (for example with TPA, reteplase or streptokinase or mechanically with a laser or Rotablator) and of microinfarcts such as, for example, during and after replacement of the heart valve, aneurysm resections and heart transplantations. The present benzimidazoles I can also be used for the treatment of a revascularization of critically narrowed coronary arteries, for example in PCTA and bypass operations, and critically narrowed peripheral arteries, for example arteries of the leg. Moreover, the benzimidazoles I may be useful in the chemotherapy of tumors and their metastasis and for the treatment of inflammations and rheumatic disorders, for example rheumatoid arthritis. In addition, the compounds of the formula I can be used to treat diabetes mellitus or to treat sepsis and multiorgan failure such as, for example, during septic shock and adult respiratory distress syndrome (ARDS, shock lung).
The novel drug formulations contain a therapeutically effective amount of the compounds I in addition to the conventional drug excipients.
For local external application, for example in the form of powders, ointments or sprays, the active compounds may be present in the conventional concentrations. As a rule, the active compounds are present in an amount of from 0.001 to 1, preferably from 0.001 to 0.1, % by weight.
In the case of internal use, the preparations are administered in single doses. From 0.1 to 100 mg per kg of body weight are administered in a single dose. The formulation can be administered daily in one or more doses, depending on the type and severity of the disorders.
Depending on the desired method of application, the novel drug formulations contain the conventional carriers and diluents in addition to the active compound. For local external application, pharmaceutical excipients such as ethanol, isopropanol, oxethylated castor oil, oxethylated hydrogenated castor oil, polyacrylic acid, polyethylene glycol, polyethylene glycol stearate, ethoxylated fatty alcohols, liquid paraffin, vaseline and lanolin, may be used. For internal use, for example, lactose, propylene glycol, ethanol, starch, talc and polyvinylpyrrolidone are suitable.
Antioxidants, such as tocopherol and butylated hydroxyanisole, and butylated hydroxytoluene, flavor-improving additives, stabilizers, emulsifiers and lubricants may furthermore be present.
The substances contained in the formulation in addition to the active compound, and the substances used in the preparation of pharmaceutical formulations, are toxicologically safe and are compatible with the respective active compound. The preparation of the drug formulations is carried out in a conventional manner, for example by mixing the active compound with other conventional carriers and diluents.
The drug formulations can be administered by various methods of application, for example perorally, parenterally, such as intravenously by infusion, subcutaneously, intraperitoneally and topically. Thus, the formulations such as tablets, emulsions, infusion and injection solutions, pastes, ointments, gels, creams, lotions, powders and sprays are possible.
In addition to the substances stated in the examples, the following compounds are particularly preferred and can be synthesized according to said preparation methods:
a) N-(2-Amino-3-ethoxycarbonyl)-1-(tert-butoxycarbonyl) piperidine-4-carboxanilide
5.5 g (24 mmol) of 1-(tert-butoxycarbonyl)piperidine-4-carboxylicacid and 4.3 g (24 mmol) of ethyl 2,3-diaminobenzoate were dissolved with 6.0 g (60 mmol) of triethylamine and 3.2 g (24 mmol) of 1-hydroxybenzotriazole in 100 ml of anhydrous tetrahydrofuran. At 0° C., 4.6 g (24 mmol) of N′-(3-dimethylaminopropyl)-N-ethylcarbodiimide were then added and the whole was stirred for 1 hour. Stirring was then continued for 24 hours at room temperature. The reaction mixture was evaporated down under reduced pressure and the residue obtained was partitioned between ethyl acetate and aqueous sodium bicarbonate solution. The ethyl acetate phase was also washed with 5% strength aqueous citricacid solution, dried and evaporated down under reduced pressure. 8.4 g of the product were obtained.
b) Ethyl 2-(1-(tert-butoxycarbonyl)piperidin-4-yl) benzimidazole-4-carboxylate
8.1 g of the intermediate 1a in 100 ml of concentrated acetic acid were refluxed for 30 minutes. The whole was then evaporated down under reduced pressure and the residue was partitioned between ethyl acetate and water. The ethyl acetate phase was also washed with aqueous sodium bicarbonate solution and water then evaporated down under reduced pressure. 4.6 g of the product were obtained.
c) 2-Piperidin-4-ylbenzimidazole-4-carboxylate×2 HCl
3.7 g (9.9 mmol) of the intermediate 1b were added to 50 ml of a 4M solution of hydrogen chloride in dioxane and stirred for 1 hour at room temperature. Thereafter, the batch was diluted with a large amount of ether and the resulting precipitate was filtered off with suction. 3.2 g of the product was obtained.
2.7 g (7.8 mmol) of the intermediate 1c and 2.7 g (54 mmol) of hydrazine in 30 ml of n-butanol were refluxed for 15 hours. Thereafter, the whole was evaporated down under reduced pressure and the residue obtained was partitioned between ethyl acetate and aqueous sodium bicarbonate solution. The organic phase was separated off, dried and evaporated down under reduced pressure. 0.9 g of the product was obtained.
e) 2-Piperidin-4-ylbenzimidazole-4-carboxamide×2 HCl
About 2.4 g of Raney nickel in 20 ml of water were added to 0.8 g (3.1 mmol) of the intermediate 1d in 20 ml of dimethylformamide, and the whole was heated to 100° C. for 8 hours. The reaction mixture was then filtered. The residue was taken up in ethanol and a crude product was precipitated by adding ether. The precipitate was dissolved in isopropanol, and a solution of hydrogen chloride in isopropanol was added. The resulting precipitate was filtered off with suction. 0.52 g of the product was obtained.
1H-NMR (D6-DMSO). δ=1.8-2.3 (4H), 2.8-3.5 (5H), 7.2 (1H), 7.7 (1H), 7.8 (1H), 8.5 (broad) and 9.2 (broad) ppm.
The example was prepared analogously to Example 1.
1H-NMR (D6-DMSO). δ=1.7 (1H), 1.9-2.2 (4H), 2.75 (1H), 3.8 (1H), 7.2 (1H), 7.6 (1H), 7.8 (1H) and 9.3 (broad) ppm.
a) Methyl 2-(N-acetylpiperidin-4-yl)benzimidazole-4-carboxylate
3.3 g (19.9 mmol) of methyl 2,3-diaminobenzoate were dissolved in 100 ml of methanol, and a solution of 4.0 g (25.8 mmol) of N-acetylpiperidine-4-carbaldehyde in 100 ml of methanol was added dropwise at room temperature. The whole was stirred for about 10 minutes at room temperature. Thereafter, 5.2 g (25.8 mmol) of copper(II) acetate, which was dissolved in 100 ml of water, were added dropwise and the whole was refluxed for 30 minutes. After cooling, 25 ml of concentrated hydrochloricacid were added carefully and the whole was again refluxed. 7.15 g (29.8 mmol) of sodium sulfide nonahydrate, dissolved in 100 ml of water, were then added dropwise and the whole was boiled for a further 10 minutes. After cooling, the reaction solution was evaporated down under reduced pressure. The residue obtained was dispersed in water and filtered. The filtrate was rendered alkaline with aqueous sodium bicarbonate solution and was extracted several times with ethyl acetate. The combined organic phases were washed with water, dried and evaporated down under reduced pressure. 4.5 g of the product were obtained.
4.3 g (14.9 mmol) of the intermediate 3a were refluxed with 3.7 g (74.3 mmol) of hydrazine hydrate in 100 ml of ethanol for 2.5 hours. The whole was then evaporated down under reduced pressure, the crude product obtained being used directly in the following reaction step.
5 g Raney nickel were added to a mixture of 100 ml of dimethylformamide and 50 ml of water. The residue from reaction step 3b, dissolved with water, was then carefully added dropwise at room temperature so that the gas evolution observed could be controlled. The whole was then heated to 100° C. for 2 hours. After cooling, filtration was carried out and the filtrate was evaporated down under reduced pressure. The residue obtained was taken up in a little methylene chloride and the product was precipitated by carefully adding ether. 3.2 g of the product were obtained.
1H-NMR (D6-DMSO). δ=1.8-2.3 (4H), 2.8-3.5 (5H), 7.2 (1H), 7.7 (1H), 7.8 (1H), 8.5 (broad) and 9.2 (broad) ppm.
0.25 g (1 mmol) of the product from Example 2, 59 mg (1 mmol) of n-propanal and 125 μl (2 mmol) of acetic acid were dissolved in 25 ml of ethanol. Thereafter, 64 mg (1 mmol) of sodium cyanoborohydride were added at room temperature and the whole was stirred for 16 hours. The reaction solution was evaporated down under reduced pressure and the residue was partitioned between methylene chloride and aqueous sodium bicarbonate solution. The organic phase was washed with water, separated off, dried and evaporated down under reduced pressure. The residue obtained was purified chromatographically using the mobile phase 4/1 ethyl acetate/methanol. 0.07 g of the product being obtained.
1H-NMR (D6-DMSO). δ=0.9 (3H), 1.5 (2H), 1.9 (2H), 2.3 (2H), 2.9 (2H), 3.3 (1H), 7.25 (1H), 7.6 (1H), 7.8 (1H), 9.3 (1H) and 12.8 (1H) ppm.
1.3 g (3.8 mmol) of the product from Example 6 were dissolved in 20 ml of isopropanol, and 50 ml of isopropanolic hydrochloride solution were added. The whole was stirred for 1 hour at room temperature. The resulting precipitate was filtered off with suction, 1.1 g of the product being obtained.
1H-NMR (D6-DMSO). δ=1.95-2.3 (3H), 2.45 (1H), 3.2 (1H), 3.5 (1H), 3.9 (1H), 7.6 (1H) and 7.95 (2H) ppm.
a) Ethyl 2-amino-3-(N—(O-tert-butoxycarbonyl)piperidin-3-yl)amido-benzoate
4 g (17.4 mmol) of N—(O-tert-butoxycarbonyl) piperidine-3-carboxylicacid and 4.8 ml (34.9 mmol) of triethylamine were dissolved in 100 ml of anhydrous tetrahydrofuran. 1.7 ml (17.4 mmol) of ethyl chloroformate, dissolved in 10 ml of anhydrous tetrahydrofuran, were then added dropwise at −10° C. The whole was stirred for 1 hour at 0° C. Thereafter, 2.9 g (17.4 mmol) of methyl 2,3-diaminobenzoate were added, once again at −10° C., and the whole was stirred for 12 hours at room temperature. The reaction solution was evaporated down under reduced pressure and the residue obtained was partitioned between ethyl acetate and water. The organic phase was also washed with aqueous sodium bicarbonate solution and water, dried and evaporated down under reduced pressure. 5.5 g of the product were obtained.
b) Methyl 2-(N—(O-tert-butoxycarbonyl)piperidin-3-yl) benzimidazole-4-carboxylate
5.4 g (14.3 mmol) of the product from 6a in 100 ml of acetic acid were refluxed for 75 minutes. After cooling, the whole was evaporated down under reduced pressure and the resulting residue was purified chromatographically using the mobile phase 1/1 ethyl acetate/heptane. 2.7 g of the product were obtained.
c) 2-(N—(O-tert-Butoxycarbonyl)piperidin-3-yl benzimidazole-4-carbohydrazide
2.3 g (6.4 mmol) of the product from 6b were refluxed with 1.6 g (32 mmol) of hydrazine hydrate in 20 ml of ethanol for 2.5 hours. After cooling, the whole was evaporated down under reduced pressure. The residue was treated with water, the resulting precipitate being filtered off with suction and dried. 1.6 g of the product were obtained.
d) 2-(N-O-tert-Butoxycarbonyl)piperidin-3-yl) benzimidazole-4-carboxamide
1.6 g of the product from 6c were reacted analogously to the method from 3c. 1.3 g of the product were obtained.
1H-NMR (D6-DMSO). δ=1.4 (1H), 1.5 (1H), 2.9 (1H), 3.1 (1H), 3.9 (1H), 4.2 (1H), 7.3 (1H), 7.7 (1H), 7.8 (1H), 9.1 (broad) and 13 (broad) ppm.
The substances mentioned in the following examples were prepared in analogy to Examples 1 to 6:
1H-NMR (D6-DMSO); δ=1.6-1.8(3H), 2.1(2H), 2.3(1H), 2.8(1H), 3.1(1H), 3.2(1H), 3.5(2H), 7.2-7.4(6H), 7.6(2H), 7.8(2H) and 9.2 (broad) ppm.
1H-NMR (D2O); δ=2.1(2H), 2.3(1H), 2.5(1H), 3.1(3H), 3.2(1H), 3.5(1H), 4.0(2H), 7.7(1H) and 8.0(2H) ppm.
1H-NMR (D6-DMSO); δ=2.5(4H), 3.3(4H), 7.2(1H), 7.6-7.7(2H), 7.8(1H) and 9.3(1H) ppm.
1H-NMR (D6-DMSO); δ=0.9(3H), 1.5(2H), 1.9(2H), 2.0 (4H), 2.3(2H), 2.9(3H), 7.2(1H), 7.6(2H), 7.8(1H) and 9.3 (broad) ppm.
1H-NMR (D6-DMSO): δ=2.0-2.5(6H), 2.8(2H), 3.1(1H), 3.2-3.4(3H), 3.7(1H), 3.8-4.0(2H), 7.3-7.5(5H), 7.7(1H) and 8.0(2H) ppm.
1H-NMR (CF3COOD): δ=1.9(1H), 2.6(1H), 3.8(1H), 3.9-4.2(4H), 4.3(1H), 4.8(1H) and 7.5-8.2(8H) ppm.
1H-NMR (D2O): δ=2.3(2H), 2.6(2H), 3.3(2H), 3.8(3H), 4.5(2H) and 7.5-8.0(8H) ppm.
1H-NMR (D6-DMSO): δ=1.4(2H), 1.6-2.0(6H), 2.0-2.4 (7H), 2.7-3.0(6H), 7.2(1H), 7.7(2H), 7.8(1H) and 9.4 (broad) ppm.
1H-NMR (D6-DMSO): δ=0.9(3H), 1.2-1.5(6H), 1.7-2.1 (6H), 2.3(2H), 2.8-3.0(4H), 7.3(1H), 7.6-7.8(3H), 9.4(1H) and 12.8 (broad) ppm.
1H-NMR (D6DMSO): δ=0.9(6H), 1.8-2.1(10H), 2.9 (2H), 7.2(1H), 7.6(2H), 7.8(1H), 9.2(1H) and 12.5 (broad) ppm.
1H-NMR (D6-DMSO): δ=0.9(3H), 1.3(2H), 1.7(2H), 2.2-2.4(4H), 3.0-3.2(4H), 3.4-3.6(3H), 7.5(1H), 7.8-8.0 (2H), 8.0(1H), 8.7 (broad) and 10.9 (broad) ppm.
1H-NMR (D6-DMSO): δ=0.9(6H), 1.7(3H), 2.2-2.4(4H), 3.1(4H), 3.3(1H), 3.7(2H), 7.5(1H), 7.8-8.0(3H), 8.7 (broad) and 10.5 (broad) ppm.
1H-NMR (D6-DMSO): δ=2.5 (3H), 2.9 (3H), 3.3-3.8 (5H), 3.9 (1H), 5.0 (1H), 7.4 (1H), 7.7 (1H), 7.8 (1H), 7.9 (1H) and 8.6 (broad) ppm.
1.83 g (3.67 mmol) of the product from Example 23 were introduced into 250 ml of methanol with 1 g of 10% palladium on carbon and hydrogenated with about 165 ml of hydrogen. The catalyst was filtered off with suction, and the filtrate was concentrated. The residue was dissolved in 20 ml of isopropanol, and 50 ml of isopropanolic hydrochloricacid solution were added. The resulting precipitate was filtered off with suction to obtain 1.1 g of the product.
1H-NMR (D6-DMSO): δ=3.2-3.7(5H), 4.0(1H), 5.2(1H), 7.4(1H), 7.8(1H), 7.9(1H) and 10.2 (broad) ppm.
1H-NMR (D6-DMSO): δ=1.25(6H), 2.3(4H), 3. 1(1H), 3.4-3.6(4H), 3.7(1H), 7.5(1H), 7.7-8.0(3H), 8.7(1H) and 10.7 (broad) ppm.
1H-NMR (D6-DMSO): δ=2.95-3.7 (7H), 3.8-4.9 (4H), 7.1-7.55 (8H), 7.65 (2H), 7.85 (2H), 7.94 (1H), 8.7 (broad) and 12.2 (broad) ppm.
1H-NMR (D6-DMSO): δ=1.7(2H), 1.8-2.0(6H), 2.1(4H), 2.5-2.7(2H), 2.8-3.0(4H), 3.5(4H), 7.2-7.5(11H), 7.7(1H), 8.6(1H), 9.5(1H), 12.3 (broad) ppm.
A 96-well microtiter plate (Falcon) was coated with histones (type II-AS; SIGMA H7755). In addition, histones were dissolved in carbonate buffer (0.05 M NaHCO3; pH 9.4) to a concentration of 50 μg/ml. The individual wells of the microtiter plate were incubated overnight, each with 100 μl of the histone solution. Thereafter, the histone solution was removed and the individual wells were incubated with 200 μl of a 1% strength BSA (bovine serum albumin) solution in carbonate buffer for 2 hours at room temperature. Washing was then carried out three times with wash buffer (0.05% Tween10 in PBS). For the enzyme reaction, 50 μl of the enzyme reaction solution per well (5 μl of reaction buffer (1M Tris-HCl pH 8.0, 100 mM MgCl2, 10 mM DTT), 0.5 μl of PARP (c=0.22 μg/μl), 4 R1 activated DNA (SIGMA D-4522, 1 mg/ml in water), 40.5 μl of H2O were preincubated with 10 μl of an inhibitor solution for 10 minutes. The enzyme reaction was started by adding 40 μl of a substrate solution (4 μl of reaction buffer (see above), 8 μl of NAD solution (100 μm in H2O), 28 μl of H2O). The reaction time was 20 minutes at room temperature. The reaction was stopped by washing three times with wash buffer (see above). This was followed by incubation for one hour at room temperature with a specific anti-poly-ADP-ribose antibody. The antibodies used were monoclonal anti-poly(ADP-ribose) antibodies “10H” (Biomol SA-276).
The antibodies were used in a 1:5000 dilution in antibody buffer (1% BSA and PBS; 0.05% Tween20). Washing three times with wash buffer was followed by incubation for an hour at room temperature with the secondary antibody. Here, an anti-mouse-IgG coupled with peroxidase (Boehringer Mannheim) was used for the monoclonal antibody and an anti-rabbit-IgG coupled with peroxidase (SIGMA A-6154) was used for the rabbit antibody, each in a 1:10,000 dilution in an antibody buffer. After washing three times with wash buffer, the color reaction was carried out using a 100 μl/well of color reagent (SIGMA, TMB ready-mix, T8540) for about 15 minutes at room temperature. The color reaction was stopped by a 100 μl of 2M H2SO4. Measurement was then carried out immediately (450 against 620 nm; ELISA “Easy Reader” EAR340AT plate reader, SLT-Lab instruments, Austria). The Ki can be determined in a conventional manner from the inhibition curves at various substrate concentrations.
A compound to be measured was dissolved directly in a specified volume of water and the resulting solution was brought to a pH of from 5 to 6 with a sodium acetate solution so that the concentration of the active compound to be tested was reached. If the test substance was not present as a water-soluble salt, it was dissolved in a very small amount of dimethyl sulfoxide and then diluted with water (final concentration of dimethyl sulfoxide≦1%), after which the pH was adjusted here too. Here, Example 1 according to the invention gave a solubility of >0.5%.
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|U.S. Classification||514/322, 546/187, 544/370, 544/364, 514/316, 514/211.08, 514/394, 548/306.1, 540/575, 514/254.06, 546/199, 514/253.09|
|International Classification||A61P25/00, C07D, C07D235/30, C07D403/04, A61P, A61K, A61P25/14, A61P9/10, A61P13/12, A61P25/08, A61P35/00, A61K31/496, A61P3/12, C07D401/14, A61K31/4184, A61P25/16, A61P25/28, A61P43/00, A61P29/00, A61P9/00, A61P31/04, C07D403/02, A61P17/02, C07D401/04, A61K31/454|
|Cooperative Classification||C07D235/30, C07D401/04, C07D403/04, C07D401/14|
|European Classification||C07D235/30, C07D401/04, C07D403/04, C07D401/14|
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