DE102011011040A1 - (5s, 8s) -3- (4'-chloro-3'-fluoro-4-methylbiphenyl-3-yl) -4-hydroxy-8-methoxy-1-azaspiro [4.5] dec-3-en-2- on (compound A) for therapy - Google Patents

Abstract

The invention relates to (5s, 8s) -3- (4'-chloro-3'-fluoro-4-methylbiphenyl-3-yl) -4-hydroxy-8-methoxy-1-azaspiro [4.5] dec-3-ene -2-one for therapeutic purposes, pharmaceutical agents and their use in therapy, in particular for the prophylaxis and therapy of tumor diseases.

Description

The present invention relates to (5s, 8s) -3- (4'-chloro-3'-fluoro-4-methylbiphenyl-3-yl) -4-hydroxy-8-methoxy-1-azaspiro [4.5] dec-3 en-2-one (= compound A) for therapeutic purposes, pharmaceutical agents containing compound A and their use in therapy, in particular for the prophylaxis and / or therapy of tumor diseases.

Acetyl-CoA carboxylases (ACCs) play a key role in cellular fatty acid homeostasis. ACCs are biotin-containing enzymes that catalyze the carboxylation of acetyl-CoA to malonyl-CoA in an ATP-dependent manner ( Kim, 1997 ; Harwood, 2005 ; Tong, 2005 ). This reaction, which runs as two half reactions, a biotin carboxylase (BC) reaction and a carboxyl transferase (CT) reaction, is the first preliminary step in fatty acid biosynthesis and is the rate-limiting step for the pathway. Two human ACC isoforms are known, ACC1 and ACC2, which are encoded by two different genes ( LuTFI ABU-ELHEIGA et al, 1995 . Jane WIDMER, et al. 1996 ). ACC1 is expressed in lipogenic tissue (liver, adipose tissue), located in the cytosol, and fills the malonyl-CoA pool, which serves as a C2 unit donor for the de novo synthesis of long-chain fatty acids by FASN and subsequent chain extension. ACC2 is mainly expressed in oxidative tissues (liver, heart, skeletal muscle) ( Bianchi et al., 1990 ; Kim, 1997), associated with the mitochondria, and regulates a second pool of malonyl-CoA. It controls fatty acid oxidation by inhibiting carnitine palmitate transferase I, the enzyme that facilitates the entry of long-chain fatty acids into the mitochondria for β-oxidation ( Milgraum LZ, et al., 1997 Widmer J. et al., 1996). Both enzymes show a very large sequence homology and are similarly regulated by a combination of transcriptional, translational and prosttranslational mechanisms. In humans as well as in animals, ACC activity is strictly controlled by a variety of dietary, hormonal and other physiological mechanisms such as forward allosteric citrate activation, long chain fatty acid feedback inhibition, reversible phosphorylation and / or inactivation or modulation enzyme production by altered gene expression.

ACC1 knockout mice are embryonic lethal ( Swinnen, et al., 2006 . Abu-Elheiga, et al. 2005 ). ACC2 knockout mice show reduced malonyl-CoA levels in skeletal and cardiac muscle, increased fatty acid oxidation in muscle, decreased liver fat levels, decreased levels of total body fat, increased levels of UCP3 in skeletal muscle (as a sign of increased energy expenditure), decreased body weight, decreased levels of plasma free fatty acids, decreased plasma glucose levels, decreased levels of glycogen in tissues and are protected from diet-induced diabetes and obesity ( Abu-Elheiga et al., 2001, 2003 ; Oh et al., 2005 ).

In addition to involvement in fatty acid synthesis in lipogenic tissues and fatty acid oxidation in oxidative tissues, up-regulation of ACC and increased lipogenesis has been observed in many tumor cells ( Swinnen, et al., 2004 . Heemers, et al., 2000 . Swinnen, et al., 2002 . Rossi, et al., 2003 . Milgraum, et al., 1997 . Yahagi, et al., 2005 ). This phenotype most likely contributes to the development and progression of tumors, but the regulatory mechanisms for this have yet to be clarified.

EP0454782 and US5759837 protect the use of fatty acid synthesis inhibitors to inhibit tumor cell growth. Cyclic ketoenols are not revealed.

A number of substances have been discovered that are capable of inhibiting plants and / or insect ACC.

The PCT patent application PCT / EPP99 / 01787 , published as WO 99/48869 that the European patent EP 1 066 258 B1 refers to new arylphenyl-substituted cyclic ketoenols, to a majority of the processes for their preparation and their use as pesticides and herbicides.

Of 3-acyl-pyrrolidine-2,4-diones pharmaceutical properties are described above ( Suzuki, S. et al. Chem. Pharm. Bull. 15 1120 (1967) ). Furthermore, N-phenylpyrrolidine-2,4-diones of R. Schmierer and H. Mildenberger (Liebigs Ann. Chem. 1985, 1095) synthesized. Biological activity of these compounds has not been described.

In EP-A-0 262 399 and GB-A-2 266 888 similarly structured compounds (3-aryl-pyrrolidine-2,4-diones) are disclosed, but of which no herbicidal, insecticidal or acaricidal activity has become known. With herbicidal, insecticidal or acaricidal action are unsubstituted, bicyclic 3-aryl-pyrrolidine 2,4-dione derivatives ( EP-A-355 599 . EP-A-415 211 and JP-A-12-053 670 ) and substituted monocyclic 3-arylpyrrolidine-2,4-dione derivatives ( EP-A-377 893 and EP-A-442,077 ).

Also known are polycyclic 3-arylpyrrolidine-2,4-dione derivatives ( EP-A-442 073 ) and 1H-arylpyrrolidinone-dione derivatives ( EP-A-456 063 . EP-A-521 334 . EP-A-596 298 . EP-A-613,884 . EP-A-613,885 . WO 95/01 971 . WO 95/26954 . WO 95/20 572 . EP-A-0 668 267 . WO 96/25 395 . WO 96/35 664 . WO 97/01 535 . WO 97/02 243 . WO 97/36868 . WO 97/43275 . WO 98/05638 . WO 98/06721 . WO 98/25928 . WO 99/24437 . WO 99/43649 . WO 99/48869 . WO 99/55673 . WO 01/17972 . WO 01/23354 . WO 01/74770 . WO 03/013249 . WO 03/062244 . WO 2004/007448 . WO 2004/024 688 . WO 04/065366 . WO 04/080962 . WO 04/111042 . WO 05/044791 . WO 05/044796 . WO 05/048710 . WO 05/049569 . WO 05/066125 . WO 05/092897 . WO 06/000355 . WO 06/029799 . WO 06/056281 . WO 06/056282 . WO 06/089633 . WO 07/048545 . DEA 10 2005 059 892 . WO 07/073856 . WO 07/096058 . WO 07/121868 . WO 07/140881 . WO 08/067873 . WO 08/067910 . WO 08/067911 . WO 08/138551 . WO 09/015801 . WO 09/039975 . WO 09/049851 . WO 09/115262 . WO10 / 052161 . WO 10/063378 . WO 10/063670 . WO10 / 063380 . WO10 / 066780 and WO10 / 102758 ,

In addition, ketal-substituted 1-H-arylpyrrolidine-2,4-diones are made WO 99/16748 and (spiro) ketal-substituted N-alkoxy-alkoxy-substituted arylpyrrolidinediones JP-A-14 205 984 and Ito M. et al. Bioscience, Biotechnology and Biochemistry 67, 1230-1238, (2003) known. Besides, are off WO 06/024411 Herbicidal agents containing ketoenols known.

It is known that certain substituted Δ ³ -dihydrofuran-2-one derivatives have herbicidal properties (cf. DE-A-4 014 420 ). The synthesis of the tetronic acid derivatives used as starting compounds (such. B. 3- (2-methyl-phenyl) -4-hydroxy-5- (4-fluorophenyl) -Δ ³ -di-hydrofuranon- (2)) is also in DE-A-4 014 420 described. Similarly structured compounds without indication of an insecticidal and / or acaricidal activity are from the publication Campbell et al., J. Chem. Soc., Perkin Trans. 1, 1985, (8) 1567-76 known. Furthermore, 3-aryl-Δ ³ -dihydrofuranone derivatives having herbicidal, acaricidal and insecticidal properties are known from: EP-A-528 156 . EP-A-647 637 . WO 95/26954 . WO 96/20 196 . WO 96/25 395 . WO 96/35 664 . WO 97/01 535 . WO 97/02 243 . WO 97/36868 . WO 98/05 638 . WO 98/06 721 . WO 99/16748 . WO 98/25 928 . WO 99/43 649 . WO 99/48869 . WO 99/55673 . WO 01/23354 . WO 01/74 770 . WO 01/17972 . WO 04/024 688 . WO 04/080 962 . WO 04/111 042 . WO 05/092 897 . WO 06/000355 . WO 06/029 799 . WO 07/048545 . WO 07/073856 . WO 07/096058 . WO 07/121868 . WO 07/140881 . WO 08/067911 . WO 08/083950 . WO 09/015801 . WO09 / 039975 and PCT / EP2010 / 003020 ,

Also 3-aryl-Δ ³ -dihydrothiphen-on derivatives are known from ( WO 95/26 345 . 96/25 395 . WO 97/01 535 . WO 97/02 243 . WO 97/36868 . WO 98/05638 . WO 98/25928 . WO 99/16748 . WO 99/43649 . WO 99/48869 . WO 99/55673 . WO 01/17972 . WO 01/23354 . WO 01/74770 . WO 03/013249 . WO 04/080 962 . WO 04/111 042 . WO 05/092897 . WO 06/029799 and WO 07/096058 ,

Certain phenyl-pyrone derivatives unsubstituted in the phenyl ring have already become known (cf. AM Chirazi, T. Kappe and E. Ziegler, Arch. Pharm. 309, 558 (1976) and K.-H. Boltze and K. Heidenbluth, Chem. Ber. 91, 2849 ). In the phenyl ring substituted phenyl-pyrone derivatives having herbicidal, acaricidal and insecticidal properties are in EP-A-588,137 . WO 96/25 395 . WO 96/35 664 . WO 97/01 535 . WO 97/02 243 . WO 97/16436 . WO 97/19941 . WO 97/36868 . WO 98/05638 . WO 99/43649 . WO 99/48869 . WO 99/55673 . WO 01/17972 . WO 01/74770 . WO 03/013249 . WO 04/080 962 . WO 04/111 042 . WO 05/092897 . WO 06/029799 and WO 07/096058 described.

Certain, unsubstituted in the phenyl ring 5-phenyl-1,3-thiazine derivatives have already become known (see. E. Ziegler and E. Steiner, Monatsh. 95, 147 (1964) . R. Ketcham, T. Kappe and E. Ziegler, J. Heterocycl. Chem. 10, 223 (1973) ). In the phenyl ring substituted 5-phenyl-1,3-thiazine derivatives with herbicidal, acaricidal and insecticidal activity are in WO94 / 14785 . WO 96/25 395 . WO 96/35 664 . WO 97/01 535 . WO 97/02 243 . WO 97/02 243 . WO 97/36868 . WO 99/43649 . WO 99/48869 . WO 99/55673 . WO 01/17972 . WO 01/74770 . WO 03/013249 . WO 04/080 962 . WO 04/111 042 . WO 05/092897 . WO 06/029799 and WO 07/096058 described.

It is known that certain substituted 2-arylcyclopentanediones have herbicidal, insecticidal and acaricidal properties (cf., for example, ( U.S. 4,283,348 ; 4,338,122 ; 4,436,666 ; 4 526 723 ; 4 551 547 ; 4,632,698 ; WO 96/01 798 ; WO 96/03366 . WO 97/14667 such as WO 98/39281 . WO 99/43649 . WO99 / 48869 . WO 99/55673 . WO 01/17972 . WO 01/74770 . WO 03/062244 . WO 04/080962 . WO04 / 111042 . WO05 / 092897 . WO06 / 029799 . WO07 / 080066 . WO07 / 096058 WO09 / 019005 . WO09 / 019015 . WO09 / 049851 . WO 10/069834 . WO10 / 000773 . WO10 / 057880 . WO10 / 081894 . WO10 / 089210 . WO10 / 102848 and WO10 / 133232 , In addition, similarly substituted compounds are known; 3-hydroxy-5,5-dimethyl-2-phenylcyclopent-2-en-1-one from the publication Micklefield et al., Tetrahedron, (1992), 7519-26 and the natural product Involutin (-) - cis-5- (3,4-dihydroxyphenyl) -3,4-dihydroxy-2- (4-hydroxyphenyl) -cyclopent-2-en-one from the publication Edwards et al., J. Chem. Soc. S, (1967), 405-9 , An insecticidal or acaricidal action is not described. Also, 2- (2,4,6-trimethylphenyl) -1,3-indanedione is from the publication J. Economic Entomology, 66, (1973), 584 and the publication DE-A 2 361 084 with indication of herbicidal and acaricidal effects.

It is known that certain substituted 2-arylcyclohexanediones possess herbicidal, insecticidal and acaricidal properties ( U.S. 4,175,135 . 4 256 657 . 4 256 658 . 4 256 659 . 4,257,858 . 4,283,348 . 4,303,669 . 4,351,666 . 4 409 153 . 4,436,666 . 4 526 723 . 4 613 617 . 4,659,372 . DE-A 2 813 341 , such as Wheeler, TN, J. Org. Chem. 44, 4906 (1979) ) WO 99/43649 . WO 99/48869 . WO 99/55673 . WO 01/17972 . WO 01/74770 . WO 03/013249 . WO 04/080 962 . WO 04/111 042 . WO 05/092897 . WO 06/029799 . WO 07/096058 . WO 08/071405 . WO 08/110307 . WO 08/110308 . WO 09/074314 . WO 08/145336 . WO 09/015887 . WO09 / 074314 . WO10 / 046194 . WO10 / 081755 , and WO10 / 089211 ,

It is known that certain substituted 4-aryl-pyrazolidine-3,5-diones have acaricidal, insecticidal and herbicidal properties (cf. WO 92/16510 . EP-A-508 126 . WO 96/11 574 . WO 96/21 652 . WO 99/47525 . WO 01/17351 . WO 01/17352 . WO 01/17353 . WO 01/17972 . WO 01/17973 . WO 03/028 466 . WO 03/062 244 . WO 04/080 962 . WO 04/111 042 . WO 05/005428 . WO 05/016873 . WO 05/092897 . WO 06/029799 and WO 07/096058 ,

It is known that certain tetrahydropyridones have herbicidal properties ( JP 0832530 ). In addition, special 4-hydroxytetrahydropyridones with acaricidal, insecticidal and herbicidal properties are known (US Pat. JP 11152273 ). Also known were 4-hydroxytetrahydropyridones as pesticides and herbicides in WO 01/79204 and WO 07/096058 , 4-hydroxy-quinolones are disclosed in WO 03/01045 ,

It is known that certain 5,6-dihydropyrone derivatives have antiviral properties as protease inhibitors ( WO 95/14012 ). Furthermore, the 4-phenyl-6- (2-phenethyl) -5,6-dihydropyron from the synthesis of kawalactone derivatives is known ( Cap et al., Arch. Pharm. 309, 558-564 (1976). , In addition, 5,6-dihydropyrone derivatives are known as intermediates ( White, JD, Brenner, JB, Deinsdale, MJ, J. Amer. Chem. Soc. 93, 281-282 (1971) , 3-phenyl-5,6-dihydropyrone derivatives with applications in crop protection are in WO 01/98288 and WO 07/09658 described.

4'-biphenyl-substituted tetronic acid derivatives are disclosed in WO 2008/022725 for the therapy of viral diseases.

WO 2005/089118 and WO2007 / 039286 disclose generic nitrogen-containing bicyclic structures for therapy, with 5'-biphenyl-substituted cyclic ketoenols not being specifically named.

4-phenyl-substituted [1.2] oxazine-3,5-diones are the first herbicides in WO 01/17972 described. Furthermore, 4-acyl-substituted [1.2] oxazine-3,5-diones have been described as pesticides but above all as herbicides and growth regulators z. In EP-A-39 48 89 ; WO 92/07837 . US 5,728,831 and as herbicides and pesticides in WO 03/048138 ,

The compound A is specifically disclosed in WO2008 / 067910 (Table 1, page 26, line 4). WO2008 / 067910 does not disclose the suitability of Compound A for therapeutic purposes.

The present application establishes the priority for the therapeutic use of Compound A. Concurrently with the present priority application, the subject of which is the therapeutic use of Compound A only, a PCT application has been submitted, inter alia, which has the therapeutic use of numerous cyclic ketoenols and which claims the priority of a German application with the application number DE 10 2010 008 644.4. Compound A is Example 1-118 in the PCT application, but was in the DE 10 2010 008 644.4 not included yet.

The structurally closest prior art could be Example I-1-a-16 of the WO99 / 48869 form, which differs from the compound A only by a lack of fluorine atom on the outer phenyl ring of the biphenyl. The therapeutic use of Example I-1-a-16 of WO99 / 48869 is also one Item in DE 10 2010 008 644.4 and the PCT application claiming the priority of DE 10 2010 008 644.4 (Example 1-2).

However, the structurally closest prior art could also Example I-1-a-31 of the WO03 / 059065 form, which differs from the compound A by the replacement of the fluorine atom on the outer phenyl ring of the biphenyl by a chlorine atom. The therapeutic use of Example I-1-a-31 of WO03 / 059065 is also an article in DE 10 2010 008 644.4 and the PCT application claiming the priority of DE 10 2010 008 644.4 (Example 1-81).

Based on this prior art, the object of the present invention is to provide a particularly effective structure for the therapy of diseases.

In particular, the structure according to the invention should be suitable for the prophylaxis and therapy of tumor diseases and have advantages over structures known in the prior art.

besonders gut für die Therapie von Erkrankungen geeignet ist.It has now surprisingly been found that the compound A

especially suitable for the treatment of diseases.

It was unpredictable whether and which of the structures known as insecticides or herbicides particularly well achieves the object according to the invention, namely a structure which can be used particularly well in the treatment of human diseases.

Compound A has comparable enzyme inhibition data as Example I-1-a-16 of WO99 / 48869 which could be considered structurally closest prior art. Surprisingly, however, compound A is characterized by a broader therapeutic window in the MCF7 xenograft model than this prior art, which has no effectiveness in this model at tolerated doses.

Compound A has better enzyme inhibition data than Example I-1-a-31 of WO03 / 059065 , which could also be considered as the structurally closest prior art.

From the large group of cyclic ketoenols known as insecticides, fungicides or herbicides, compound A has surprisingly been highlighted by better enzyme inhibition and / or better in vivo efficacy at tolerated doses.

Also to be regarded as covered by the present invention is the use of the physiologically acceptable salts of the compound A.

Physiologically acceptable salts of compound A also include salts of conventional bases such as, by way of example and by way of example, alkali metal salts (eg sodium and potassium salts), alkaline earth salts (eg calcium and magnesium salts) and ammonium salts derived from ammonia or organic amines with 1 to 16 carbon atoms, such as, by way of example and by way of preference, ethylamine, diethylamine, triethylamine, ethyldiisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, dimethylaminoethanol, procaine, dibenzylamine, N-methylmorpholine, arginine, lysine, ethylenediamine and N-methylpiperidine.

Another object of the present invention are pharmaceutical compositions containing compound A and at least one or more other active ingredients, in particular for the prophylaxis and / or therapy of tumor diseases.

Compound A can act systemically and / or locally. For this purpose, it can be applied in a suitable manner, such as. As oral, parenteral, pulmonary, nasal, sublingual, lingual, buccal, rectal, dermal, transdermal, conjunctival, otic or implant or stent.

For these routes of administration, compound A can be administered in suitable administration forms.

For oral administration are according to the prior art functioning fast and / or modified compound A donating application forms containing the compound A in crystalline and / or amorphized and / or dissolved form, such as. As tablets (uncoated or coated tablets, for example, with enteric or delayed-dissolving or insoluble coatings that control the release of the compound of the invention), in the oral cavity rapidly disintegrating tablets or films / wafers, films / lyophilisates, capsules (for example, hard or soft gelatin capsules), dragees, granules, pellets, powders, emulsions, suspensions, aerosols or solutions. Parenteral administration can be carried out bypassing a resorption step (eg intravenously, intraarterially, intracardially, intraspinally or intralumbarly) or using absorption (for example intramuscularly, subcutaneously, intracutaneously, percutaneously or intraperitoneally). For parenteral administration are suitable as application forms u. a. Injection and infusion preparations in the form of solutions, suspensions, emulsions, lyophilisates or sterile powders.

For the other routes of administration are z. Inhalation medicaments (including powder inhalers, nebulizers), nasal drops, solutions, sprays; lingual, sublingual or buccal tablets, films / wafers or capsules, suppositories, ear or ophthalmic preparations, vaginal capsules, aqueous suspensions (lotions, shake mixtures), lipophilic suspensions, ointments, creams, transdermal therapeutic systems (such as patches), milk, Pastes, foams, scattering powders, implants or stents.

The compound A can be converted into the mentioned application forms. This can be done in a conventional manner by mixing with inert, non-toxic, pharmaceutically suitable excipients. These adjuvants include u. a. Excipients (for example microcrystalline cellulose, lactose, mannitol), solvents (for example liquid polyethylene glycols), emulsifiers and dispersing or wetting agents (for example sodium dodecyl sulfate, polyoxysorbitanoleate), binders (for example polyvinylpyrrolidone), synthetic and natural polymers (for example albumin), stabilizers (For example, antioxidants such as ascorbic acid), dyes (eg, inorganic pigments such as iron oxides) and flavor and / or odoriferous.

Another object of the present invention are pharmaceutical compositions containing the compound A, usually together with one or more inert, non-toxic, pharmaceutically suitable excipients, and their use for the purposes mentioned above.

The formulation of compound A into pharmaceutical preparations is carried out in a manner known per se, by converting the active substance (s) into the desired administration form with the auxiliaries customary in galenicals.

As auxiliaries may, for example, vehicles, fillers, disintegrants, binders, humectants, lubricants, adsorbents and agents, diluents, solvents, cosolvents, emulsifiers, solubilizers, flavoring agents, colorants, preservatives, stabilizers, wetting agents, salts for changing the osmotic pressure or buffers are used. It is up Remington's Pharmaceutical Science, 15th ed Mack Publishing Company, East Pennsylvania (1980) to point.

The pharmaceutical formulations can
in solid form, for example as tablets, dragees, pills, suppositories, capsules, transdermal systems or
in semi-solid form, for example as ointments, creams, gels, suppositories, emulsions or
in liquid form, for example as solutions, tinctures, suspensions or emulsions.

Auxiliaries for the purposes of the invention may be, for example, salts, saccharides (mono-, di-, tri-, oligo-, and / or polysaccharides), proteins, amino acids, peptides, fats, waxes, oils, hydrocarbons and derivatives thereof, the auxiliaries may be of natural origin or synthetically or partially synthetically obtained.

For oral or oral administration, in particular tablets, dragees, capsules, pills, powders, granules, lozenges, suspensions, emulsions or solutions come into question. For parenteral administration in particular suspensions, emulsions and above all solutions in question.

The present invention relates to the use of compound A for the prophylaxis and therapy of human diseases, in particular of tumor diseases. The compound A can be used in particular to inhibit or reduce cell proliferation and / or cell division and / or to induce apoptosis.

• – Psoriasis,
• – Keloide und andere Hyperplasien, die die Haut betreffen,
• – gutartige Prostathyperplasien (BPH),
• – solide Tumore und
• – hämatologische Tumore.

The compound A is particularly suitable for the prophylaxis and / or treatment of hyperproliferative diseases such as

• - psoriasis,
• - keloids and other skin-related hyperplasias
• - benign prostate hyperplasia (BPH),
• - solid tumors and
• - hematological tumors.

As solid tumors according to the invention, for example, tumors of the breast, the respiratory tract, the brain, the reproductive organs, the gastrointestinal tract, the genitourinary tract, the eye, the liver, the skin, the head and neck, the thyroid gland, the parathyroid gland, are treatable Bone and connective tissue and metastases of these tumors.

• – multiple Myelome,
• – Lymphome oder
• – Leukämien.

For example, hematological tumors are treatable

• - multiple myeloma,
• - Lymphomas or
• - leukemia.

• – Mammakarzinome mit positivem Hormonrezeptorstatus
• – Mammakarzinome mit negativem Hormonrezeptorstatus
• – Her-2 positive Mammakarzinome
• – Hormonrezeptor- und Her-2 negative Mammakarzinome
• – BRCA-assoziierte Mammakarzinome
• – entzündliches Mammakarzinom.

For example, treatable as breast tumors are:

• - Breast cancer with positive hormone receptor status
• - Breast cancer with negative hormone receptor status
• - Her-2 positive breast cancers
• - Hormone receptor and Her-2 negative breast cancers
• - BRCA-associated breast cancer
• - inflammatory breast cancer.

• – nicht-kleinzellige Bronchialkarzinome und
• – kleinzellige Bronchialkarzinome.

For example, tumors of the respiratory tract are treatable

• - non-small cell lung carcinomas and
• - small cell bronchial carcinomas.

• – Gliome,
• – Glioblastome,
• – Astrozytome,
• – Meningiome und
• – Medulloblastome.

For example, tumors of the brain are treatable

• - glioma,
• - glioblastomas,
• - astrocytomas,
• - Meningiomas and
• - Medulloblastomas.

• – Prostatakarzinome,
• – Maligne Hodentumore und
• – Peniskarzinome.

For example, tumors of the male reproductive organs are treatable:

• - prostate carcinomas,
• - Malignant testicular tumors and
• - Penile carcinomas.

• – Endometriumkarzinome
• – Zervixkarzinome
• – Ovarialkarzinome
• – Vaginalkarzimome
• – Vulvarkarzinome

For example, tumors of the female reproductive organs are treatable:

• - endometrial carcinomas
• - cervical carcinomas
• - ovarian carcinomas
• - vaginal carcinomas
• - Vulvar carcinomas

• – Kolorektale Karzinome
• – Analkarzinome
• – Magenkarzinome
• – Pankreaskarzinome
• – Ösophagukarzinome
• – Gallenblasenkarzinome
• – Dünndarmkarzinome
• – Speicheldrüsenkarzinome
• – Neuroendokrine Tumore
• – Gastrointestinale Stromatumore

For example, tumors of the gastrointestinal tract are treatable:

• - Colorectal carcinomas
• - Anal carcinomas
• - gastric carcinoma
• - Pancreatic carcinomas
• - esophageal carcinomas
• - Gallbladder carcinomas
• - Small intestinal carcinomas
• - Salivary gland carcinomas
• - Neuroendocrine tumors
• - Gastrointestinal stromal tumors

• – Harnblasenkarzinome
• – Nierenzellkarzinome
• – Karzinome des Nierenbeckens und der ableitenden Harnwege

For example, tumors of the urogenital tract are treatable:

• - bladder carcinomas
• - renal cell carcinomas
• - carcinomas of the renal pelvis and the urinary tract

• – Retinoblastome
• – Intraokulare Melanome

For example, tumors of the eye are treatable:

• - Retinoblastomas
• - Intraocular melanomas

• – Hepatozelluläre Karzinome
• – Cholangiozelluläre Karzinome

Treatable as tumors of the liver, for example:

• - Hepatocellular carcinomas
• - Cholangiocellular carcinomas

• – Maligne Melanome
• – Basaliome
• – Spinaliome
• – Kaposi-Sarkome
• – Merkelzellkarzinome

For example, tumors of the skin are treatable:

• - Malignant melanomas
• - basaliomas
• - Spinaliomas
• - Kaposi's sarcoma
• - Merkel cell carcinomas

• – Larynxkarzinome
• – Karzinome des Pharynx und der Mundhöhle

Tumors of the head and neck, for example, are treatable:

• - Laryngeal carcinomas
• - Carcinoma of the pharynx and oral cavity

• – Weichteilsarkome
• – Osteosarkome

For example, sarcomas are treatable:

• - soft tissue sarcoma
• - Osteosarcomas

• – Non-Hodgkin-Lymphome
• – Hodgkin-Lymphome
• – Kutane Lymphome
• – Lymphome des zentralen Nervensystems
• – AIDS-assoziierte Lymphome

For example, lymphomas are treatable:

• - Non-Hodgkin's lymphoma
• - Hodgkin lymphoma
• - cutaneous lymphomas
• - Lymphomas of the central nervous system
• - AIDS-associated lymphomas

• – Akute myeloische Leukämien
• – Chronische myeloische Leukämien
• – Akute lymphatische Leukämien
• – Chronische lymphatische Leukämien
• – Haarzellleukämien

Treatable as leukemias, for example:

• - Acute myeloid leukemias
• - Chronic myeloid leukemia
• - Acute lymphocytic leukemia
• - Chronic lymphocytic leukemia
• - hair cell leukemia

The compound A can be used with particular advantage for the prophylaxis and / or therapy of:
Breast cancers, in particular of hormone receptor negative, hormone receptor positives or BRCA-associated breast carcinomas, as well as
Pancreatic carcinoma, renal cell carcinoma, hepatocellular carcinoma, malignant melanoma and other skin tumors, non-small cell lung carcinoma, endometrial carcinoma, colorectal carcinoma and prostate carcinoma.

These diseases are well characterized in humans but also exist in other mammals.

Another object of the present application is the compound A for use as a medicament, in particular for the prophylaxis and / or therapy of tumor diseases.

Another object of the present application is the compound A for the prophylaxis and / or treatment of breast carcinoma, pancreatic carcinoma, renal cell carcinoma, hepatocellular carcinoma, malignant melanoma and other skin tumors, non-small cell, bronchial carcinoma, endometrial carcinoma, colorectal carcinoma or prostate cancer.

Another object of the invention is the use of the compound A for the preparation of a medicament.

Another object of the present application is the use of the compound for the manufacture of a medicament for the prophylaxis and / or therapy of tumor diseases.

Another object of the present application is the use of the compound A for the manufacture of a medicament for the prophylaxis and / or therapy of breast, pancreatic, renal cell carcinoma, hepatocellular carcinoma, malignant melanoma and other skin tumors, non-small cell, bronchial carcinoma, endometrial carcinoma, colorectal carcinoma or prostate carcinoma ,

Another object of the present application is the use of the compound for the prophylaxis and / or therapy of tumor diseases.

Another object of the present application is the use of compound A for the prophylaxis and / or treatment of breast, pancreatic, renal cell carcinoma, hepatocellular carcinoma, malignant melanoma and other skin tumors, non-small cells, bronchial carcinoma, endometrial carcinoma, colorectal carcinoma or prostate cancer.

A further subject of the present application are pharmaceutical formulations in the form of tablets containing compound A for the prophylaxis and / or treatment of breast, pancreatic, renal cell carcinoma, hepatocellular carcinoma, malignant melanoma and other skin tumors, non-small cells, bronchial carcinoma, endometrial carcinoma, colorectal carcinoma or prostate carcinoma ,

Another object of the invention is the use of compound A for the treatment of diseases associated with proliferative processes.

Compound A can be used alone or as needed in combination with one or more other pharmacologically active substances, as long as this combination does not lead to undesirable and unacceptable side effects. Another object of the present invention are therefore pharmaceutical compositions containing compound A and one or more other active ingredients, in particular for the prophylaxis and / or therapy of the aforementioned diseases.

For example, compound A can be combined with known anti-hyperproliferative, cytostatic or cytotoxic substances for the treatment of cancers. The combination of compound A with other substances commonly used for cancer therapy or else with radiotherapy is particularly indicated.

Examples of suitable combination active ingredients are:
Afinitor, aldesleukin, alendronic acid, alfaferone, alitretinoin, allopurinol, aloprim, aloxi, altretamine, aminoglutethimide, amifostine, amrubicin, amsacrine, anastrozole, anzmet, aranesp, arglabine, arsenic trioxide, aromasine, 5-azacytidine, azathioprine, BCG or tice-BCG, Bestatin, betamethasone acetate, betamethasone sodium phosphate, bexarotene, bleomycin sulfate, broxuridine, bortezomib, busulfan, calcitonin, campath, capecitabine, carboplatin, casodex, cefesone, celmoleukin, cerubidine, chlorambucil, cisplatin, cladribine, clodronic acid, cyclophosphamide, cytarabine, Dacarbazine, Dactinomycin, DaunoXome, Decadron, Decadron Phosphate, Delestrogen, Denileukin Diftitox, Depomedrol, Deslorelin, Dexrazoxane, Diethylstilbestrol, Diflucan, Docetaxel, Doxifluridine, Doxorubicin, Dronabinol, DW-166HC, Eligard, Elitek, Ellence, Emend, Epirubicin, Epoetin -Alfa, Epogen, Eptaplatin, Ergamisol, Estrace, Estradiol, Estramustine Sodium Phosphate, Ethinylestradiol, Ethyol, Etidronic Acid, Etopophos, Etoposide, Fadrozole, Farston, Filgrastim, Finasteride, Fligrastim, Floxuridine, Fluconazole, Fludarabine, 5-Fluorodeoxyuridine Monophosphate, 5-Fluorouracil (5 -FU), fluoxymesterone, flutamide, formestane, fosteabin, fotemustine, fulvestrant, gammagard, gemcitabine, gemtuzumab, gleevec, gliadel, goserelin, granisetron hydrochloride, histrelin, hycamtin, hydrocorton, erythro-hydroxynonyladenine, hydroxyurea, ibritumomab tiuxetan, idarubicin, ifosfamide , Interferon alpha, Interferon alpha 2, Interferon alpha 2α, Interferon alpha 2β, Interferon alpha n1, Interferon alpha n3, Interferon beta, Interferon gamma 1a, Interleukin 2, Intron A, Iressa, Irinotecan, Kytril, Lapatinib, Lentinan Sulfate, Letrozole, Leucovorin, Leuprolide, Leuprolide Acetate, Levamisole, Levofolic Acid Calcium Salt, Levothroid, Levoxyl, Lomustine, Lonidamine, Marinol, Mechlorethamine, Mecobalamin, Medroxypro gesterone acetate, megestrol acetate, melphalan, menest, 6-mercaptopurine, mesna, methotrexate, metvix, miltefosine, minocycline, mitomycin C, mitotane, mitoxantrone, modrenal, myocet, nedaplatin, neulasta, neumega, neupogen, nilutamide, nolvadex, nsc -631570, OCT-43, octreotide, ondansetron hydrochloride, Orapred, oxaliplatin, paclitaxel, pediapred, pegaspargase, pegasys, pentostatin, picibanil, pilocarpine hydrochloride, pirarubicin, plicamycin, porfimer sodium, prednimustine, prednisolone, prednisone, premarin, procarbazine , Procrit, Raltitrexed, RDEA119, Rebif, Rhenium-186-Etidronate, Rituximab, Roferon-A, Romurtide, Salagen, Sandostatin, Sargramostim, Semustin, Sizofiran, Sobuzoxan, Solu-Medrol, Streptozocin, Strontium-89-Chloride, Synthroid, Tamoxifen , Tamsulosin, Tasonermin, Tastolactone, Taxoter, Teceleukin, Temozolomide, Teniposide, Testosterone Propionate, Testred, Thioguanine, Thiotepa, Thyrotropin, Tiludronic Acid, Topotecan, Toremifene, Tositumomab, Tastuzumab, Teosulfan, Tretinoin, Trexall, Trimethyl melamine, trimetrexate, triptorelin acetate, triptorelin pamoate, UFT, uridine, valrubicin, vesnarinone, vinbiastine, vincristine, vindesine, vinorelbine, virulizine, zinecard, zinostatin zestran, zofran; ABI-007, Acolbifen, Actimmun, Affinitak, Aminopterin, Arzoxifen, Asoprisnil, Atamestan, Atrasentan, BAY 43-9006 (Sorafenib), Avastin, CCI-779, CDC-501, Celebrex, Cetuximab, Crisnatol, cyproterone acetate, decitabine, DN-101, doxorubicin MTC, dSLIM, dutasteride, edotecarin, eflornithine, exatecan, fenretinide, histamine dihydrochloride, histrelin hydrogel implant, holmium-166-DOTMP, ibandronic acid, interferon-gamma, intron-PEG, ixabepilone, keyhole limpet Hemocyanin, L-651582, Lanreotide, Lasofoxifen, Libra, Lonafarnib, Miproxifen, Minodronate, MS-209, Liposomal MTP-PE, MX-6, Nafarelin, Nemorubicin, Neovastat, Nolatrexed, Oblimersen, Onko-TCS, Osidem, Paclitaxel Polyglutamate, pamidronate disodium, PN-401, QS-21, quazepam, R-1549, raloxifene, ranpirnas, 13-cis-retinoic acid, satra-platinum, seocalcitol, T-138067, tarceva, taxoprexin, thymosin-alpha-1, Tiazofurin, tipifarnib, tirapazamine, TLK-286, toremifene, TransMID-107R, valspodar, vapreotide, vatalanib, verteporfin, vinflunine, Z-100, zoledronic acid, and combinations thereof.

In a preferred embodiment, the compound A can be combined with anti-hyperproliferative agents, which may be by way of example-without this enumeration being exhaustive:
Aminoglutethimide, L-asparaginase, azathioprine, 5-azacytidine, bleomycin, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, colaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, diethylstilbestrol, 2 ', 2'-difluorodoxycytidine, docetaxel, doxorubicin ( Adriamycin), epirubicin, epothilone and its derivatives, erythro-hydroxynonyladenine, ethinyl estradiol, etoposide, fludarabine phosphate, 5-fluorodeoxyuridine, 5-fluorodeoxyuridine monophosphate, 5-fluorouracil, fluoxymesterone, flutamide, hexamethylmelamine, hydroxyurea, hydroxyprogesterone caproate, idarubicin, Ifosfamide, interferon, irinotecan, leucovorin, lomustine, mechlorethamine, medroxyprogesterone acetate, megestrol acetate, melphalan, 6-mercaptopurine, mesna, methotrexate, mitomycin C, mitotane, mitoxantrone, paclitaxel, pentostatin, N-phosphonoacetyl-L-aspartate (PALA ), Plicamycin, prednisolone, prednisone, procarbazine, raloxifene, semustine, streptozocin, tamoxifen, teniposide, testosterone propionate, thioguanine, thiotepa, to potecan, trimethylmelamine, uridine, vinblastine, vincristine, vindesine and vinorelbine.

Promisingly, compound A can also be combined with biological therapeutics such as antibodies (eg, Avastin, Rituxan, Erbitux, Herceptin) and recombinant proteins.

Compound A may also provide positive effects in combination with other anti-angiogenic therapies, such as Avastin, axitinib, regorafenib, recentin, sorafenib, or sunitinib. Combinations with proteasome and mTOR inhibitors as well as antihormones and steroidal metabolic enzyme inhibitors are particularly suitable because of their favorable side effect profile.

• • eine verbesserte Wirksamkeit bei der Verlangsamung des Wachstums eines Tumors, bei der Reduktion seiner Größe oder sogar bei seiner völligen Eliminierung im Vergleich zu einer Behandlung mit einem einzelnen Wirkstoff;
• • die Möglichkeit, die verwendeten Chemotherapeutika in geringerer Dosierung als bei der Monotherapie einzusetzen;
• • die Möglichkeit einer verträglicheren Therapie mit weniger Nebeneffekten im Vergleich zur Einzelgabe;
• • die Möglichkeit zur Behandlung eines breiteren Spektrums von Tumorerkrankungen;
• • das Erreichen einer höheren Ansprechrate auf die Therapie;
• • eine längere Überlebenszeit der Patienten im Vergleich zur heutigen Standardtherapie.

In general, the following objectives can be pursued with the combination of compound A with other cytostatic or cytotoxic agents:

• • improved efficacy in slowing down the growth of a tumor, reducing its size or even eliminating it completely compared to treatment with a single drug;
• • the possibility of using the chemotherapeutic agents used in lower doses than in monotherapy;
• • the possibility of a more tolerable therapy with fewer side effects compared to a single dose;
• • the ability to treat a wider range of tumors;
• • achieving a higher response rate to therapy;
• • longer patient survival compared to today's standard therapy.

In addition, compound A may also be used in conjunction with radiotherapy and / or surgical. Intervention be used.

Experimental part

1. Comparative Examples

Table V shows Example I-1-a-16 of WO99 / 488691 and Example I-1-a-31 of WO03 / 059065 which Applicant regards as the closest prior art.

Tab. V

LC-MS and HPLC methods:

Method 1 (UPLC-MS)

• Instrument: Waters Acquity UPLC-MS SQD 3001; Column: Acquity UPLC BEH C18 1.7 50 × 2.1 mm; Eluent A: water + 0.1% formic acid, eluent B: acetonitrile; Gradient: 0-1.6 min 1-99% B, 1.6-2.0 min 99% B; Flow 0.8 ml / min; Temperature: 60 ° C; Injection: 2 μl; DAD scan: 210-400 nM.

Method 2 (UPLC-MS):

• Instrument: Waters Acquity UPLC-MS ZQ4000; Column: Acquity UPLC BEH C18 1.7 50 × 2.1 mm; Eluent A: water + 0.05% formic acid, eluent B: acetonitrile + 0.05% formic acid; Gradient: 0-1.6 min 1-99% B, 1.6-2.0 min 99% B; Flow 0.8 ml / min; Temperature: 60 ° C; Injection: 2 μl; DAD scan: 210-400 nM.

Method 3 (UPLC-MS):

• Instrument: Waters Acquity UPLC-MS SQD 3001; Column: Acquity UPLC BEH C18 1.7 50 × 2.1 mm; Eluent A: water + 0.1% formic acid, eluent B: acetonitrile; Gradient: 0-1.6 min 1-99% B, 1.6-2.0 min 99% B; Flow 0.8 ml / min; Temperature: 60 ° C; Injection: 2 μl; DAD scan: 210-400 nm.

Preparation of Comparative Example V.1.

a) Intermediate

Intermediate V.1.1

(4'-chloro-4-methylbiphenyl-3-yl) acetyl chloride

5.00 g (19.18 mmol) of (4'-chloro-4-methylbiphenyl-3-yl) acetic acid ( EP 2029531 A1 and US 2009/298828 A1 ) were dissolved in 36.51 g (306.84 mmol) of thionyl chloride. The reaction mixture was stirred for four hours at 80 ° C and then evaporated in vacuo. After drying in a fine vacuum, 5.4 g (100% of theory) of the title compound were obtained as a brownish oil.
¹ H-NMR (300 MHz, CDCl ₃ ): δ [ppm] = 2.36 (s, 3H), 4.22 (s, 2H), 7.29 (d, 1H), 7.35-7.55 (m, 6H).

Intermediate V.1.2

Methyl-cis-1 - {[(4'-chloro-4-methylbiphenyl-3-yl) acetyl] amino} -4-methoxycyclohexancarboxylat

5.41 g (24.2 mmol) of methyl cis-1-amino-4-methoxycyclohexanecarboxylate hydrochloride ( EP 1791816 A1 and WO 2006/29799 A1 ), 14.8 mg (1.21 mmol) of DMAP and 8.4 ml (60.5 mmol) of triethylamine were dissolved at room temperature under nitrogen in 118 ml of dichloromethane. Subsequently, a solution of 6.75 g (24.2 mmol) of the intermediate V.1.1 in 60 ml of dichloromethane was added dropwise. The resulting reaction mixture was stirred at room temperature overnight. For working up, the mixture was diluted with dichloromethane and the organic phase was washed with aqueous, saturated sodium bicarbonate solution and aqueous 5% citric acid. After drying over sodium sulfate, it was filtered and concentrated. This gave 10.9 g of the title compound, which were further purified by chromatography on silica gel (mobile phase: hexane-ethyl acetate gradient).
¹ H-NMR (300 MHz, DMSO-d ₆ ): δ [ppm] = 1.31-1.47 (m, 2H), 1.60-1.73 (m, 2H), 1.75-1.86 (m, 2H), 2.00-2.11 ( m, 2H), 2.27 (s, 3H), 3.09-3.20 (m, 1H), 3.21 (s, 3H), 3.51 (s, 3H), 3.58 (s, 2H), 7.23 (d, 114), 7.43 (dd, 1H), 7.46-7.54 (m, 3H), 7.61-7.68 (m, 2H), 8.31 (s, 1H).
LC-MS (Method 1): R _t = 1.38 min; MS (ESIpos): m / z = 430 [M + H] ⁺ .

b) Final product V.1

(5s, 8s) -3- (4'-chloro-4-methylbiphenyl-3-yl) -4-hydroxy-8-methoxy-1-azaspiro [4.5] dec-3-en-2-one

To 8.87 g (20.6 mmol) of the intermediate V.1.2 in 103 ml of N, N-dimethylformamide were added at room temperature under nitrogen 4.63 g (41.3 mmol) of potassium tert-butoxide. The reaction mixture was stirred at 80 ° C for 60 minutes. For workup, the cooled reaction mixture was poured onto 1 l of ice water, adjusted to pH 3 with 1 N aqueous hydrogen chloride solution, stirred for three hours, the precipitate was filtered off with suction, washed with water and dried. The crude product was further purified by stirring in diethyl ether overnight, filtering and drying. 7.75 g (94% of theory) of the title compound were obtained.
¹ H-NMR (300 MHz, DMSO-d ₆ ): δ [ppm] = 1.39-1.62 (m, 4H), 1.84-2.05 (m, 4H), 2.18 (s, 3H), 3.07-3.20 (m, 1H), 3.26 (s, 3H), 7.30 (d, 1H), 7.34 (d, 1H), 7.45-7.53 (m, 3H), 7.62-7.68 (m, 2H), 8.18 (br, s, 1H) , 10.82 (brs s, 1H).
LC-MS (Method 3): R _t = 1.19 min; MS (ESIpos): m / z = 398 [M + H] ⁺ .

Comparative Example V.2

Comparative Example V.2 is Example I-1-a-31 of WO03 / 059065.

The therapeutic use of Example I-1-a-31 of WO03 / 059065 is also an object in DE 10 2010 008 644.4 and the PCT application claiming the priority of DE10 2010 008 644.4 (Example 1-81).

2. Connection A

Preparation of the compound A

a) Intermediate

Intermediate A.1

(4'-chloro-3'-fluoro-4-methylbiphenyl-3-yl) acetic acid

To a solution of 40.0 g (175 mmol) of (5-bromo-2-methylphenyl) acetic acid ( EP 1791816 and WO 2006/29799 ) in a mixture of 437 ml (437 mmol) of degassed IN aqueous sodium hydroxide solution, 160 ml of degassed water and 160 ml of degassed tetrahydrofuran were added under argon 33.5 g (192 mmol) of (4-chloro-3-fluorophenyl) boronic acid. The mixture was stirred for 10 minutes, treated with 507 mg (1.75 mmol) of tri-tert-butylphosphonium tetrafluoroborate and 532 mg (1.75 mmol) of palladium (II) acetylacetonate and stirred for 20 h at room temperature. Then toluene and water were added, adjusted to pH 1-2 with concentrated aqueous hydrogen chloride solution, stirred for 10 minutes, the phases separated, extracted twice with toluene, the combined organic phases dried over sodium sulfate, filtered and concentrated one. Of the The residue was stirred in 300 ml of a 6/1 mixture of n-hexane / tert-butyl methyl ether for 30 minutes, filtered off with suction, washed with n-hexane and dried in vacuo. 38.0 g (78% of theory) of the title compound were obtained.
¹ H-NMR (300 MHz, DMSO-d ₆ ): δ [ppm] = 2.27 (s, 3H), 3.67 (s, 2H), 7.27 (d, 1H), 7.49-7.59 (m, 3H), 7.61 -7.75 (m, 2H), 12.4 (s, 1H).
LC-MS (method 1): R _t = 1.31 min; MS (ES Ineg): m / z = 277 [M - H] ^- .

Intermediate A.2

Methyl-cis-1 - {[(4'-chloro-3'-fluoro-4-methylbiphenyl-3-yl) acetyl] amino} -4-methoxycyclohexancarboxylat

10.0 g (35.9 mmol) of the intermediate A.1 were dissolved in 14.9 ml (205 mmol) of thionyl chloride. The reaction mixture was stirred for 1 h at 90 ° C and then concentrated. 10.8 g (100% of theory) of (4'-chloro-3'-fluoro-4-methylbiphenyl-3-yl) -acetyl chloride were obtained. 10.6 g (35.7 mmol) of (4'-chloro-3'-fluoro-4-methylbiphenyl-3-yl) -acetyl chloride were dissolved in 120 ml of acetonitrile. 12.0 g (53.7 mmol) of methyl cis-1-amino-4-methoxycyclohexanecarboxylate hydrochloride (described in EP 1791816 and WO 2006/29799 ) were taken up in ethyl acetate and treated with saturated aqueous sodium bicarbonate solution. The phases were separated and the aqueous phase extracted twice with ethyl acetate. The combined organic phases were dried over sodium sulfate, filtered and concentrated. 8.50 g of methyl cis-1-amino-4-methoxycyclohexanecarboxylate were obtained. 8.02 g (42.8 mmol) of methyl cis-1-amino-4-methoxycyclohexanecarboxylate in 120 ml of acetonitrile were admixed with 17.3 g (125 mmol) of potassium carbonate. Under ice-cooling, the solution of the acid chloride was added dropwise and stirred overnight at room temperature. It was then concentrated, the residue was combined with water, extracted with dichloromethane, the combined organic phases were washed with 1N aqueous hydrogen chloride solution and saturated aqueous sodium bicarbonate solution, dried over sodium sulfate, filtered and concentrated. This gave 15.7 g (98% of theory) of the title compound, which were reacted without further purification.
¹ H-NMR (300 MHz, DMSO-d ₆ ): δ [ppm] = 1.30-1.47 (m, 2H), 1.60-1.74 (m, 2H), 1.75-1.85 (m, 2H), 1.99-2.11 ( m, 2H), 2.28 (s, 3H), 3.09-3.20 (m, 1H), 3.21 (s, 3H), 3.52 (s, 3H), 3.58 (s, 2H), 7.24 (d, 1H), 7.46 - 7.55 (m, 2H), 7.57 (d, 1H), 7.61-7.72 (m, 2H), 8.30 (s, 1H).
LC-MS (Method 2): R _t = 1.36 min; MS (ESIpos): m / z = 448 [M + H] ⁺ .

b) End product compound A

(5s, 8s) -3- (4'-chloro-3'-fluoro-4-methylbiphenyl-3-yl) -4-hydroxy-8-methoxy-1-azaspiro [4.5] dec-3-en-2- on

To 15.7 g (35.0 mmol) of the intermediate A.2 in 60 ml of N, N-dimethylformamide were added under nitrogen 4.32 g (38.5 mmol) of potassium tert-butoxide. The reaction mixture was stirred for 20 minutes at room temperature. Subsequently, the reaction mixture was poured into ice-water, 160 ml of 1N aqueous solution was added dropwise Hydrogen chloride solution was added, stirred for 30 minutes, filtered off with suction, washed with water and dried the precipitate. This gave 14.2 g (97% of theory) of the title compound.
¹ H-NMR (300 MHz, DMSO-d ₆ ): δ [ppm] = 1.40-1.62 (m, 4H), 1.85-2.04 (m, 4H), 2.19 (s, 3H), 3.07-3.20 (m, 1H), 3.27 (s, 3H), 7.31 (d, 1H), 7.39 (d, 1H), 7.48-7.57 (m, 2H), 7.60-7.73 (m, 2H), 8.20 (s, 1H), 10.82 (s, 1H).
LC-MS (Method 1): R _t = 1.22 min; MS (ESIpos): m / z = 416 [M + H] ⁺ .

3. Assays

Human ACC1 enzyme assay

The ACC1 inhibition data were collected with two different assays (A1 and B1)

Assay A1 (= (A1))

The inhibitory activity of the substances of this invention against acetyl-CoA carboxylase 1 (ACC1) was measured in the ACC1 assay described in the following paragraphs. Basic principle of the assay is to measure the Adenosindiphoshats formed as co-product (ADP) using a HTRF ^® -based competitive immunoassay (HTRF = Homogeneous Time Resolved Fluorescence).

As an enzyme, C-terminal FLAG-tagged recombinant human ACC1 (GenBank Accession # NM_198834, amino acids 39- end) expressed in baculovirus transfected insect cells (Hi5) and purified by affinity chromatography on Anti- ^FLAG® M2 affinity gel (Sigma Aldrich). Alternatively, commercially available C-terminal His-tagged ACC1 from BPS Bioscience (San Diego, CA, catalog number 50200. amino acids 39- end) can be used. For the assay, 50 μl of a 100X concentrated solution of the test substance in DMSO were pipetted into a black low-volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 μl of a solution of ACC1 in assay buffer [50 mM HEPES / NaOH pH 7.5, 12mM sodium bicarbonate, 2mM MgCl ₂ , 2mM potassium citrate, 0.005% (w / v) bovine serum albumin (BSA)] and the mixture incubated for 15 min to pre-bind the substances to the enzyme prior to the enzyme reaction to enable. Then, the enzyme reaction was started by adding 3 μl of a solution of adenosine triphosphate (ATP, 83.5 μM = final concentration in 5 μl of assay volume is 50 μM, Amersham Pharmacia Biotech # 27-2056-01) and acetyl-CoA (33.4 μM => final concentration in 5 μl assay volume is 20 μM, Roche Bioscience # 10101893001) in assay buffer and the resulting mixture incubated for 20 min at 22 ° C for the reaction time. The concentration of the ACC1 was adjusted to the respective activity of the enzyme and adjusted so that the assay worked in the linear range. Typical concentrations were in the range of 2.5 ng / μl.

The reaction was quenched by successive additions of 2.5 microliters of a solution of d2-labeled ADP (HTRF ^® Trans Screener ^TM ADP kit, Cis biointernational, Marcoule, France) (in the EDTA-containing HTRF ^® Trans Screener ^TM ADP detection buffer in HTRF ^® Transscreener ^™ ADP Kit containing 50mM HEPES pH 7.0, 60mM EDTA, 0.1% (w / v) BSA, 0.02% sodium azide, 400mM potassium fluoride) and 2.5μl of a solution of europium cryptate labeled anti-ADP antibody (HTRF ^® Trans Screener ^TM ADP Kit) Trans Screener in HTRF ^{® TM} ADP detection buffer.

The resulting mixture was incubated for one hour at 22 ° C to allow binding of the europium cryptate-labeled anti-ADP antibody to the enzyme-produced ADP and the d2-labeled ADP. Subsequently, the amount of the complex of d2-labeled ADP and europium-cryptate-labeled anti-ADP antibody was determined by measuring the resonance energy transfer from the europium cryptate to the d2. For this purpose, in an HTRF meter, z. B. a Rubystar or Pherastar (both BMG Labtechnologies, Offenburg, Germany), the fluorescence emissions measured at 620 nm and 665 nm after excitation at 350 nm. The ratio of the emissions at 665 nm and at 622 nm was used as a measure of the amount of the complex of d2-labeled ADP and europium cryptate-labeled anti-ADP antibody and thus indirectly as a measure of the amount of unlabelled formed in the enzyme reaction ADP taken (higher ratio of emissions at 665 nm and at 622 nm a more complex of d2-labeled ADP and europium-cryptate-labeled anti-ADP antibody ⇔ less ADP). The data were normalized (enzyme reaction without inhibitor = 0% inhibition, all other assay components but no enzyme = 100% inhibition). Usually, the test substances were incubated on the same microtiter plates at 10 different concentrations ranging from 20 μM to 1 nM (20 μM, 6.7 μM, 2.2 μM, 0.74 μM, 0.25 μM, 82 nM, 27 nM, 9 , 2 nM, 3.1 nM and 1 nM, serial dilutions were assayed at 100-fold concentrated solution level by serial 1: 3 dilutions) in duplicate for each concentration prior to assay and IC50 values were calculated using a 4-parameter -Fit, for which an in-house software was used.

Assay B1 (= (B1))

The hACC1 inhibitory activity of the substances of this invention was measured in the hACC1 assay described in the following paragraphs.

Essentially, the enzyme activity is measured by quantifying the adenosine di-phosphate (ADP) formed as a by-product of the enzyme reactions using the ADP-Glo ^™ detection system from Promega. In this case, the adenosine tri-phosphate (ATP) not consumed in the enzyme reaction is first quantitatively converted into cAMP by means of an adenylate cyclase ("ADP-GLO reagent"), the adenylate cyclase is then stopped and then ("kinase detection reagent") is then converted the formed ADP into ATP and converted this into a glow luminescence signal in a luciferase-based reaction.

The enzyme used was recombinant C-terminal FLAG-tagged human ACC1 (acetyl-coenzyme A carboxylase alpha transcript variant 1) (GenBank Accession No. NM_198834) (amino acids 39- end) expressed in baculovirus-infected insect cells (Hi5) and purified by anti-FLAG affinity chromatography.

For the assay, 50 μl of a 100X concentrated solution of the test substance in DMSO were pipetted into a white low-volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2.5 μl of a solution of hACC1 in assay buffer [50 mM HEPES / NaOH pH 7.5, 2mM MgCl ₂ , 2mM potassium citrate, 12mM NaHCO ₃ , 2mM thio-threitol (DTT), 0.005% (w / v) bovine serum albumin (BSA)] and the mixture for 15 min incubated to allow a Vorbindung the substances to the enzyme prior to the enzyme reaction. Then the enzyme reaction was started by adding 2.5 μl of a solution of adenosine tri-phosphate (ATP, 100 μM => final concentration in 5 μl assay volume is 50 μM) and acetyl-CoA (20 μM => final concentration in 5 μl assay volume is 10 μM) in assay buffer and the resulting mixture is incubated for the 45 min reaction time at 22 ° C. The concentration of hACC1 was adjusted to the respective activity of the enzyme and adjusted so that the assay worked in the linear range. Typical concentrations were in the range of 1.75 ng / μl. The reaction was stopped by adding 2.5 μl of the "ADP-GLO reagent" (diluted 1: 1.5 fold) and the resulting mixture incubated for 1 h at 22 ° C to fully add the unreacted ATP into cAMP convict. Subsequently, 2.5 μl of the "Kinase Detection Reagent" (1.2 times more concentrated than indicated by the manufacturer) was added, the resulting mixture incubated for 1 h at 22 ° C and then the luminescence with a suitable instrument (Viewlux or Topcount from Perkin-Elmer or pherastar from BMG Labtechnologies). The amount of light emitted was taken as a measure of the amount of ADP formed and thus of the enzyme activity of hACC1. The data were normalized (enzyme reaction without inhibitor = 0% inhibition, all other assay component but no enzyme = 100% inhibition). Typically, the test substance was incubated on the same microtiter plates at 10 different concentrations ranging from 20 μM to 1 nM (20 μM, 6.7 μM, 2.2 μM, 0.74 μM, 0.25 μM, 82 nM, 27 nM, 9.2 nM, 3.1 nM and 1 nM, serial dilutions were assayed at 100-fold concentrated solution level by serial 1: 3 dilutions) in duplicate for each concentration prior to assay, and IC ₅₀ values were calculated using a 4-parameter fit using in-house software.

Human ACC2 enzyme assay

ACC2 inhibition data were collected using two different assays (A2 and B2)

Assay A2 (= (A2))

The inhibitory activity of the substances of this invention on acetyl-CoA carboxylase 2 (ACC2) was measured in the ACC2 assay described in the following paragraphs. Basic principle of the assay is to measure the Adenosindiphoshats formed as co-product (ADP) using a HTRF ^® -based competitive immunoassay (HTRF = Homogeneous Time Resolved Fluorescence).

As an enzyme, commercially available C-terminal His-tagged ACC2 from BPS Bioscience (San Diego, CA, Catalog No. 50201. Amino Acids 39-end, expressed in baculovirus-infected Sf9 insect cells and purified by Ni-NTA affinity chromatography) was used.

For the assay, 50 μl of a 100X concentrated solution of the test substance in DMSO were pipetted into a black low-volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 μl of a solution of ACC2 in assay buffer [50 mM HEPES / NaOH pH 7.5, 12mM sodium bicarbonate, 2mM MgCl ₂ , 2mM potassium citrate, 0.005% (w / v) bovine serum albumin (BSA)] and the mixture for 15 min incubated to allow a Vorbindung the substances to the enzyme prior to the enzyme reaction. Then, the enzyme reaction was started by adding 3 μl of a solution of adenosine triphosphate (ATP, 83.5 μM = final concentration in 5 μl of assay volume is 50 μM, Amersham Pharmacia Biotech # 27-2056-01) and acetyl-CoA (33.4 μM => final concentration in 5 μl assay volume is 20 μM, Roche Bioscience # 10101893001) in assay buffer and the resulting mixture incubated for 20 min at 22 ° C for the reaction time. The concentration of the ACC2 was adjusted to the respective activity of the enzyme and adjusted so that the assay worked in the linear range. Typical concentrations were in the range of 0.6 ng / μl.

The reaction was quenched by successive additions of 2.5 microliters of a solution of d2-labeled ADP (HTRF ^® Trans Screener ^TM ADP kit, Cis biointernational, Marcoule, France) (in the EDTA-containing HTRF ^® Trans Screener ^TM ADP detection buffer in HTRF ^® Transscreener ^™ ADP Kit containing 50mM HEPES pH 7.0, 60mM EDTA, 0.1% (w / v) BSA, 0.02% sodium azide, 400mM potassium fluoride) and 2.5μl of a solution of europium cryptate labeled anti-ADP antibody (HTRF ^® Trans Screener ^TM ADP Kit) Trans Screener in HTRF ^{® TM} ADP detection buffer.

The resulting mixture was incubated for one hour at 22 ° C to allow binding of the europium cryptate-labeled anti-ADP antibody to the enzyme-produced ADP and the d2-labeled ADP. Subsequently, the amount of the complex of d2-labeled ADP and europium-cryptate-labeled anti-ADP antibody was determined by measuring the resonance energy transfer from the europium cryptate to the d2. For this purpose, in an HTRF meter, z. B. a Rubystar or Pherastar (both BMG Labtechnologies, Offenburg, Germany), the fluorescence emissions measured at 620 nm and 665 nm after excitation at 350 nm. The ratio of the emissions at 665 nm and at 622 nm was used as a measure of the amount of the complex of d2-labeled ADP and europium cryptate-labeled anti-ADP antibody and thus indirectly as a measure of the amount of unlabelled formed in the enzyme reaction ADP taken (higher ratio of emissions at 665 nm and at 622 nm »more complex of d2-labeled ADP and europium-cryptate-labeled anti-ADP antibody ⇔ less ADP). The data were normalized (enzyme reaction without inhibitor = 0% inhibition, all other assay components but no enzyme = 100% inhibition). Usually, the test substances were incubated on the same microtiter plates at 10 different concentrations ranging from 20 μM to 1 nM (20 μM, 6.7 μM, 2.2 μM, 0.74 μM, 0.25 μM, 82 nM, 27 nM, 9 , 2 nM, 3.1 nM and 1 nM, the serial dilutions were assayed at 100-fold concentrated solution level by serial 1: 3 dilutions) in duplicate for each concentration and IC ₅₀ values were calculated with a 4- Parameter Fit, for which an in-house software was used.

Assay B2 (= (B2))

The hACC2 inhibitory activity of the substances of this invention was measured in the hACC2 assay described in the following paragraphs.

Essentially, the enzyme activity is measured by quantifying the adenosine di-phosphate (ADP) formed as a by-product of the enzyme reactions using the ADP-Glo ^™ detection system from Promega. In this case, the adenosine tri-phosphate (ATP) not consumed in the enzyme reaction is first quantitatively converted into cAMP by means of an adenylate cyclase ("ADP-GLO reagent"), the adenylate cyclase is then stopped and then ("kinase detection reagent") is then converted the formed ADP into ATP and converted this into a glow luminescence signal in a luciferase-based reaction.

The enzyme used was recombinant C-terminal FLAG-tagged human ACC2 (acetyl-coenzyme A carboxylase 2, GenBank Accession No. NP_001084) (amino acid 27-end) expressed in baculovirus-infected insect cells (Hi5) and purified by anti-FLAG affinity chromatography.

For the assay, 50 μl of a 100X concentrated solution of the test substance in DMSO were pipetted into a white low-volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2.5 μl of a solution of hACC2 in assay buffer [50 mM HEPES / NaOH pH 7.5, 2mM MgCl ₂ , 2mM potassium citrate, 12mM NaHCO ₃ , 2mM thio-threitol (DTT), 0.005% (w / v) bovine serum albumin (BSA)] and the mixture for 15 min incubated to allow a Vorbindung the substances to the enzyme prior to the enzyme reaction. Then the enzyme reaction was started by adding 2.5 μl of a solution of adenosine tri-phosphate (ATP, 100 μM => final concentration in 5 μl assay volume is 50 μM) and acetyl-CoA (20 μM => final concentration in 5 μl assay volume is 10 μM) in assay buffer and the resulting mixture is incubated for the 45 min reaction time at 22 ° C. The concentration of hACC2 was adjusted to the respective activity of the enzyme and adjusted so that the assay worked in the linear range. Typical concentrations were in the range of 2 ng / μl. The reaction was stopped by adding 2.5 μl of the "ADP-GLO" Reagent "(1: diluted 1.5 times) and the resulting mixture for 1 h at 22 ° C to completely convert the unreacted ATP in cAMP. Subsequently, 2.5 μl of the "Kinase Detection Reagent" (1.2 times more concentrated than indicated by the manufacturer) was added, the resulting mixture incubated for 1 h at 22 ° C and then the luminescence with a suitable instrument (Viewlux or Topcount from Perkin-Elmer or pherastar from BMG Labtechnologies). The amount of light emitted was taken as a measure of the amount of ADP formed and thus of the enzyme activity of hACC2. The data were normalized (enzyme reaction without inhibitor = 0% inhibition, all other assay component but no enzyme = 100% inhibition). Typically, the test substance was incubated on the same microtiter plates at 10 different concentrations ranging from 20 μM to 1 nM (20 μM, 6.7 μM, 2.2 μM, 0.74 μM, 0.25 μM, 82 nM, 27 nM, 9.2 nM, 3.1 nM and 1 nM, serial dilutions were assayed at 100-fold concentrated solution level by serial 1: 3 dilutions) in duplicate for each concentration prior to assay, and IC ₅₀ values were calculated using a 4-parameter fit using in-house software.

Non-human ACCase assay

The assay was performed at room temperature in a transparent 384-well microtiter plate. It determines the inorganic phosphate released from ATP in the ACCase reaction.

The assay contained 50 mM Tris-HCl pH 8.3, 50 mM KCl, 2.5 mM MgCl ₂ , 0.5 mM ATP, 0.8 mM dithiothreitol (DTT), 30 mM NaHCO ₃ , 0.1 mM acetyl-CoA, 0.04% bovine serum albumin and 0.4 μg partially purified ACCase enzyme in a final volume of 40 μl. After 45 minutes of incubation, the reaction was stopped with 150 μl of malachite green solution and the absorbance was read out at 620 after 30 minutes.

The malachite green (MG) solution was prepared by mixing 3 parts of 0.6 mM MG-HCl solution in distilled water with 1 part of 8.5 mM ammonium molybdate in 4 M HCl. The solution was allowed to stand for 30 minutes. After filtration through a 0.45 μm polytetrafluoroethylene (PTFE) filter, 0.1 part of Triton X-100 (1.5%) in distilled water was added.

ACCase enzyme was extracted from oat seedlings harvested 9 days after seeding and partially purified by 0-40% ammonium sulfate precipitation followed by ion exchange chromatography on Q-sepharose.

Mode-of-action test

Before determining the efficacy in the MCF-7 model, part of the test substances were tested in a mode of action experiment. The principle of this experiment is that the short-term application of a test substance which is able to inhibit ACC1 and / or ACC2 in the living organism after oral administration leads to a reduction of malonyl-CoA in the tumor. Experimentally, 2 million human MCF-7 breast cancer cells were injected subcutaneously into female nude mice (NMRI-nude (nu / nu) mice, Taconic M & B A / S, 1 day before administration of a pellet for the release of estrogen over a period of at least 60 days) , Once the tumor had reached an area of about 60-70 mm ² , the oral administration of the test substance took place over a period of 1-3 days, then the intratumoral content of malonyl-CoA was determined at defined times and compared with the vehicle control. The method is described in Anal Chem. 2008 Aug 1; 80 (15): 5736-42. Epub 2008 Jul 9.).

Cell assays

In accordance with the invention, the substances were tested in cell-based assays, which means the ability of the substances to inhibit tumor cell proliferation after a 96-hour substance incubation. Cell viability was tested using the CellTiter-Glo Luminescent Cell Viability Assay ^® (Promega). The cells were seeded at a density of 2000-5000 cells / well (depending on the cell line) in 100 μl growth medium on 96 well microtiter plates. For each cell line examined, cells were seeded on a separate plate to determine luminescence at t = 0 hours and t = 96 hours. After overnight incubation at 37 ° C, the luminescence values were determined for the t = 0 samples. The dose plates for the t = 96 hour time points were treated with substances diluted in growth medium. The cells were then incubated for 96 hours at 37 ° C, then the luminescence values were determined for the t = 96-hour samples. For data analysis, the t = 0 values were subtracted from the t = 96 hours values for treated and untreated samples. The percentage differences in luminescence between substance-treated and control values were used to determine the percent growth inhibition.

The substances were investigated in the following cell lines, which exemplify the indicated indications: cell line source indication MCF7 ATCC Hormone receptor positive breast cancer PC3 ATCC prostate cancer Du145 NCI prostate cancer ECC 1 ATCC endometrial cancer KM12 NCI Colorectal carcinoma HEC1A ATCC endometrial cancer

Xenograft model

Xenograft models were used in immunosuppressed mice to determine antitumoral efficacy in the living organism.

First, the maximum tolerable dose (MTD) was determined according to the following protocol: female nude mice (NMRI-nude (nu / nu) mice, Taconic M & B A / S) received a daily oral dose of 1, 2 or 3 weeks the test substance and were daily observed for mortality and body weight. MTD was defined as the highest dose that could be administered, during which no animal died during the treatment phase and during the 7-day follow-up period and there was no more than 10% decrease in body weight compared to baseline.

To determine the antitumoral efficacy then various xenograft models were used in which the test substances were given at their MTD and in lower dosages. Among several other models, the breast cancer model with hormone-dependent human MCF-7 cells in female nude mice (NMRI-nude (nu / nu) mice, Taconic M & B A / S) was primarily used. For this purpose, a pellet for the release of estrogen (17β-oestradiol 0.36 mg, release over 60 days) was applied subcutaneously to these mice the day before the tumor cell implantation. Two million tumor cells (suspended in medium + Matrigel) 1: 1, final 0.1 ml) were injected subcutaneously into the flank of each animal on the next day. When the tumors reached 20-25 mm ² tumor area, mice were randomized into therapy groups and therapy started. Therapy was then continued with 2-3 times weekly measurement of tumor area and body weight until the average tumor size in the control group, which had received only the vehicle of the test substance or in one of the treatment groups, reached 120 mm ² . At this time, the experiment was stopped in all groups and the prepared tumors were weighed. As a primary parameter of success, the T / C value was calculated either by the effect on the tumor weight or on the tumor area: mean of the tumor weights / areas in the treatment group divided by the mean of the tumor weights / areas in the vehicle group.

Analysis of ACC1 expression in tumor and normal tissues

The ACC1 expression was determined by means of a microrray. For this, the RNA was isolated from different tumor tissues and the corresponding normal tissues. Methodically, Trizol RNA extraction reagent (Invitrogen) was used and purification was performed using the RNeasy Mini Kit (Qiagen). In addition, DNaseI (Qiagen) digestion was performed to eliminate genomic DNA. For quality control, total RNA analysis was performed using an RNA LabChip on an Agilent Bioanalyzer 2100 Platform (Agilent Technologies) and RNA concentration determined using the Peqlab NanoDrop System. For hybridization, the "one-cycle eukaryotic target labeling assay" from Affymetrix was used and the array was then read on an Affymetrix GeneChip 3000 scanner (Affymetrix). Evaluation and quality control were performed using the Expressionist Pro 4.0 Refiner (GeneData) software.

4. Results:

4.1. enzyme assay

Table 1 summarizes the results with respect to compound A and the comparative examples from the enzyme assays. Tab. 1 Example no ACC1 (= A1) IC50 [μmol / l] ACC2 (= A2) IC50 [μmol / l] ACC1 (= B1) IC50 [μmol / l] ACC2 (= B2) IC50 [μmol / l] V.1 12:28 12:37 0084 0822 V.2 0327 1414 0428 2.61 A 0129 0690 0102 1:38

These data show that Compound A and Comparative Example V.1 inhibit the enzymes to a comparable extent while Comparative Example V.2 is inferior.

4.2 Cellassays

Table 2 summarizes the results with respect to compound A and the comparative examples from the cell assays. Tab. 2 Example no MCF7 IC50 [μmol / l] PC3 IC50 [μmol / l] Du145 IC50 [μmol / l] ECC1 IC50 [μmol / l] KM12 IC50 [μmol / l] HEC-1A IC50 [μmol / l] V.1 0057 V.2 0270 A 0037 0025 0039 0221 0275 1.76

4.3 Maximum Tolerable Dose (MTD)

Comparative Example V.1

Comparative Example V.1 was administered to female nude mice (NMRI nu / nu): Dose: s. Tab. 3 administration: per os Vehicle: PEG400 / ethanol / Solutol HS15 (70/5/25, v / v / v) (Solutol HS15: polyoxyethylene ester of 12-hydroxystearic acid) Application volume: 10 ml / kg scheme: s. Tab. 3

The treatment phase was followed by an observation period of 7 days. The resulting deaths and the effect on body weight are summarized in Table 3. Tab. 3 substance Dose (mg / kg) scheme Body weight (%) mortality V.1 10 14 days, twice a day minus 12 1 of 3 V.1 20 14 days, twice a day minus 29 2 of 3 V.1 30 14 days, twice a day minus 11 2 of 3 vehicle 21 days, once a day plus 15 0 out of 5 V.1 15 21 days, once a day minus 1 1 out of 5 V.1 20 21 days, once a day minus 2 1 out of 5 V.1 25 21 days, once a day minus 3 1 out of 5

As a result, the MTD for a 14-day treatment with twice daily administration is below 10 mg / kg.

As a result, the MTD is less than 15 mg / kg when given once a day for 21 days.

As deaths and body weight loss occurred in all groups, the MTD could not be determined.

Comparative Example V.1 was subsequently applied in the MCF-7 breast cancer xenograft model female nude mice (NMRI nu / nu): Dose: 7.5 mg / kg administration: per os Vehicle: PEG400 / ethanol / Solutol HS 15 70/5/25, v / v / v) Application volume: 10 ml / kg scheme: 27 days, twice a day (2qd) mice: 10

This dose was relatively well tolerated by day 37 of the experiment (no body weight loss, 1 in 10 animals died). A therapeutic efficacy defined as a T / C value of <= 0.5 could not be observed in this dose group compared to the vehicle control (T / C value based on tumor area = 0.86).

2 shows the tumor area as a function of the number of days after implantation of the tumor cells.

The remaining animals were treated with 10 mg / kg 2qd or 12.5mg / kg 2qd until day 43. However, these two higher dosing regimens were poorly tolerated (1 in 4 and 4 in 5 deaths, respectively).

In summary, the therapeutic window of Comparative Example V.1, if any, is very small.

Connection A

Compound A was administered to female nude mice (NMRI nu / nu): Dose: s. Tab. 4 administration: per os Vehicle: PEG400 / ethanol / Solutol HS 15 70/5/25, v / v / v) Application volume: 10 ml / kg scheme: s. Tab. 4

The treatment phase was followed by an observation period of 7 days. The deaths that occurred and the effect on body weight are summarized in Table 4. Tab. 4 substance Dose (mg / kg) scheme Body weight (%) mortality vehicle 21 days, once a day plus 5 0 out of 5 Connection A 20 21 days, once a day plus 2 0 out of 5 Connection A 30 21 days, once a day plus 1 0 out of 5 Connection A 40 21 days, once a day minus 3 1 out of 5 Connection A 50 21 days, once a day minus 29 5 out of 5 Connection A 60 21 days, once a day minus 24 5 out of 5

As a result, the MTD for a 21-day treatment is less than 40 mg / kg once daily and above 30 mg / kg when given once daily.

Compound A was subsequently applied in the MCF-7 breast cancer xenograft model to female nude mice (NMRI nu / nu): Dose: s. Tab. 5 administration: per os Vehicle: PEG400 / ethanol / Solutol HS 15 70/5/25, v / v / v) Application volume: 10 ml / kg scheme: 30 days, once a day (qd) Mice per dose group: 10-13 Tab. 5 substance Dose (mg / kg) T / C (based on tumor area T / C (based on tumor weight) vehicle 1:00 1:00 Connection A 20 0.56 (ns) 0.42 (p = 0.003) Connection A 25 0.51 (p <0.05) 0.37 (p <0.001) Connection A 30 0.49 (p <0.05) 0.35 (p <0.001) Connection A 35 0.44 (p <0.05) 0.31 (p <0.001)

For the statistical evaluation of the experiment, the non-parametric ANOVA test was used for the T / C values based on the tumor area since there was no normal distribution of the measured values. Subsequently, all therapy groups were compared to the vehicle group by Dunns Posttest. The resulting p-values are shown in the table (ns: not significant = p> 0.05). Since there was a normal distribution for the T / C values based on tumor weight, the ANOVA test for parametric values was used for analysis. Subsequently, all therapy groups were compared to the vehicle group by means of the Bonferroni posttest. The resulting p-values are shown in Table 5.

Since in this experiment all dose groups used reached the target T / C (based on tumor weight) of <0.5, a statistical evaluation of the experiment was performed, the results of which are also shown in Table 5. Compound A statistically significantly inhibited tumor growth (based on both tumor weight and tumor area) from a dose of 25 mg / kg given once daily. Based on the tumor weight, this effect was already statistically significant from a dose of 20 mg / kg given once daily.

3 shows the tumor area as a function of the number of days after implantation of the tumor cells.

Thus, the comparison between Comparative Example V.1 and Compound A shows that no antitumor efficacy is observed for Comparative Example V.1 even at a poorly tolerated dose, while biologically significant and significant antitumoral effects for Compound A are within a tolerable dose range from 20 mg / kg qd to 35 mg / kg qd.

ACC1 expression in tumor and normal tissue

The expression of ACC1 in tumor and corresponding normal tissue was determined by microarray ( 1 ). The expression of ACC1 was significantly upregulated compared to normal breast cancer, colorectal carcinoma, bronchial carcinoma and pancreatic carcinoma.

5. Formulation

Tablets containing compound A.

a) Preparation of a Pharmaceutical Formulation by Direct Tableting

Tablets were prepared according to the composition of Table 6 containing the compound A by means of direct tabletting. Tab. 6 starting materials Mass / tablet [mg] Connection A 80.0 Mannitol, spray dried 67.0 Cellulose, microcrystalline 40.0 Na croscarmellose 10.0 magnesium stearate 3.0 total 200.0

The pharmaceutical formulation can be prepared by any suitable method, in particular powder mixing and direct tabletting in any desired scale.

For the preparation of 50 tablets were in a Fantaschale 3.351 g Mannitol, spray dried 2.004 g Cellulose, microcrystalline 0.499 g Na croscarmellose and 3.992 g Example Compound 1-118 premixed by careful trituration. The mixture was transferred to a 100 ml screw-cap jar and homogenized for 10 minutes in a Turbula mixer. After addition of 0.149 g of magnesium stearate was mixed again for 1 min in the Turbula mixer.

The resulting molding compound was compressed by means of an eccentric tablet press (Korsch EK 2) into biconvex tablets with a diameter of 8 mm and a radius of curvature of 12 mm.

b) Breaking strength

The breaking strength (by means of Schleuniger's breaking strength tester), mass and disintegration time in water at 37 ° C. (using the apparatus described in monograph 2.9.1 European Pharmacopoeia) of the tablets obtained were tested at the beginning, middle and end of the tableting process. breaking strength Dimensions disintegration time Beginning 81 N 198.7 mg 1:28 min center 95 N 196.8 mg 1:28 min. The End 97 N 199.0 mg 1:32 min. medium 91 N 198.2 mg 1:29 min.

c) In vitro dissolution

In vitro release of Compound A from the prepared tablets was determined by Apparatus 2 (paddle method) according to USP. The release test was carried out in each case in 900 ml of different media at 37 ° C. and with a stirring speed of 75 revolutions per minute. Each determination was carried out in triplicate. The content was determined by HPLC. Table 7 and 4 show the results. Tab. 7 medium % released after 15 minutes 30 min 45 min 60 min 90 min 0.1N HCl + 1% SDS * (pH 1) 20.5% 32.1% 37.1% 41.2% 45.3% USP phosphate buffer pH = 6.8 + 1% SDS * 43.2% 55.6% 62.0% 65.7% 70.1% USP phosphate buffer pH = 8.0 80.1% 87.5% 89.6% 90.4% 91.2% * SDS = sodium lauryl sulphate (added since solubility at pH 1 and pH 6.8 is insufficient)

d) Short term stability of the pharmaceutical formulation

The produced tablets were subjected to a short term stability test for 1 month at 25 ° C / 60% relative humidity and at 40 ° C / 75% relative humidity. The tablets were stable under both conditions in terms of content and degradation products, analyzed by HPLC.

Characters:

1 : ACC1 expression in tumor and corresponding normal tissue

2 : Therapeutic efficacy of Comparative Example V.1 in hormone-dependent MCF-7 breast cancer xenograft model.

3 : Therapeutic efficacy of Compound A in hormone-dependent MCF-7 breast cancer xenograft model.

4 : Release curve of compound A from tablets

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• EP 0454782 [0005]
• US 5759837 [0005]
• EP 99/01787 [0007]
• WO 99/48869 [0007, 0010, 0012, 0013, 0014, 0015, 0016, 0017, 0026, 0032]
• EP 1066258 B1 [0007]
• EP 0262399 A [0009]
• GB 2266888 A [0009]
• EP 355599A [0009]
• EP 415211 A [0009]
• JP 12-053670 A [0009]
• EP 377893A [0009]
• EP 442077 A [0009]
• EP 442073A [0010]
• EP 456063A [0010]
• EP 521334A [0010]
• EP 596298A [0010]
• EP 613884A [0010]
• EP 613885 A [0010]
• WO 95/01971 [0010]
• WO 95/26954 [0010, 0012]
• WO 95/20572 [0010]
• EP 0668267A [0010]
• WO 96/25395 [0010, 0012, 0013, 0014, 0015]
• WO 96/35664 [0010, 0012, 0014, 0015]
• WO 97/01535 [0010, 0012, 0013, 0014, 0015]
• WO 97/02243 [0010, 0012, 0013, 0014, 0015, 0015]
• WO 97/36868 [0010, 0012, 0013, 0014, 0015]
• WO 97/43275 [0010]
• WO 98/05638 [0010, 0012, 0013, 0014]
• WO 98/06721 [0010, 0012]
• WO 98/25928 [0010, 0012, 0013]
• WO 99/24437 [0010]
• WO 99/43649 [0010, 0012, 0013, 0014, 0015, 0016, 0017]
• WO 99/55673 [0010, 0012, 0013, 0014, 0015, 0016, 0017]
• WO 01/17972 [0010, 0012, 0013, 0014, 0015, 0016, 0017, 0018, 0023]
• WO 01/23354 [0010, 0012, 0013]
• WO 01/74770 [0010, 0012, 0013, 0014, 0015, 0016, 0017]
• WO 03/013249 [0010, 0013, 0014, 0015, 0017]
• WO 03/062244 [0010, 0016, 0018]
• WO 2004/007448 [0010]
• WO 2004/024688 [0010]
• WO 04/065366 [0010]
• WO 04/080962 [0010, 0012, 0013, 0014, 0015, 0016, 0017, 0018]
• WO 04/111042 [0010, 0012, 0013, 0014, 0015, 0016, 0017, 0018]
• WO 05/044791 [0010]
• WO 05/044796 [0010]
• WO 05/048710 [0010]
• WO 05/049569 [0010]
• WO 05/066125 [0010]
• WO 05/092897 [0010, 0012, 0013, 0014, 0015, 0016, 0017, 0018]
• WO 06/000355 [0010, 0012]
• WO 06/029799 [0010, 0012, 0013, 0014, 0015, 0016, 0017, 0018]
• WO 06/056281 [0010]
• WO 06/056282 [0010]
• WO 06/089633 [0010]
• WO 07/048545 [0010, 0012]
• DE 102005059892 [0010]
• WO 07/073856 [0010, 0012]
• WO 07/096058 [0010, 0012, 0013, 0014, 0015, 0016, 0017, 0018, 0019]
• WO 07/121868 [0010, 0012]
• WO 07/140881 [0010, 0012]
• WO 08/067873 [0010]
• WO 08/067910 [0010]
• WO 08/067911 [0010, 0012]
• WO 08/138551 [0010]
• WO 09/015801 [0010, 0012]
• WO 09/039975 [0010, 0012]
• WO 09/049851 [0010, 0016]
• WO 09/115262 [0010]
• WO 10/052161 [0010]
• WO 10/063378 [0010]
• WO 10/063670 [0010]
• WO 10/063380 [0010]
• WO 10/066780 [0010]
• WO 10/102758 [0010]
• WO 99/16748 [0011, 0012, 0013]
• JP 14205984 A [0011]
• WO 06/024411 [0011]
• DE 4014420 A [0012, 0012]
• EP 528156 A [0012]
• EP 647637A [0012]
• WO 96/20196 [0012]
• WO 04/024688 [0012]
• WO 08/083950 [0012]
• EP 2010/003020 [0012]
• WO 95/26345 [0013]
• EP 588137A [0014]
• WO 97/16436 [0014]
• WO 97/19941 [0014]
• WO 94/14785 [0015]
• US 4283348 [0016, 0017]
• US 4338122 [0016]
• US 4436666 [0016, 0017]
• US 4526723 [0016, 0017]
• US 4551547 [0016]
• US 4632698 [0016]
• WO 96/01798 [0016]
• WO 96/03366 [0016]
• WO 97/14667 [0016]
• WO 98/39281 [0016]
• WO 07/080066 [0016]
• WO 09/019005 [0016]
• WO 09/019015 [0016]
• WO 10/069834 [0016]
• WO 10/000773 [0016]
• WO 10/057880 [0016]
• WO 10/081894 [0016]
• WO 10/089210 [0016]
• WO 10/102848 [0016]
• WO 10/133232 [0016]
• DE 2361084 A [0016]
• US 4175135 [0017]
• US 4256657 [0017]
• US 4256658 [0017]
• US 4256659 [0017]
• US 4257858 [0017]
• US 4,303,669 [0017]
• US 4351666 [0017]
• US 4409153 [0017]
• US 4613617 [0017]
• US 4659372 [0017]
• DE 2813341 A [0017]
• WO 08/071405 [0017]
• WO 08/110307 [0017]
• WO 08/110308 [0017]
• WO 09/074314 [0017, 0017]
• WO 08/145336 [0017]
• WO 09/015887 [0017]
• WO 10/046194 [0017]
• WO 10/081755 [0017]
• WO 10/089211 [0017]
• WO 92/16510 [0018]
• EP 508126 A [0018]
• WO 96/11574 [0018]
• WO 96/21652 [0018]
• WO 99/47525 [0018]
• WO 01/17351 [0018]
• WO 01/17352 [0018]
• WO 01/17353 [0018]
• WO 01/17973 [0018]
• WO 03/028466 [0018]
• WO 05/005428 [0018]
• WO 05/016873 [0018]
• JP 0832530 [0019]
• JP 11152273 [0019]
• WO 01/79204 [0019]
• WO 03/01045 [0019]
• WO 95/14012 [0020]
• WO 01/98288 [0020]
• WO 07/09658 [0020]
• WO 2008/022725 [0021]
• WO 2005/089118 [0022]
• WO 2007/039286 [0022]
• EP 394889A [0023]
• WO 92/07837 [0023]
• US 5728831 [0023]
• WO 03/048138 [0023]
• WO 2008/067910 [0024, 0024]
• DE 102010008644 [0025, 0026, 0027, 0090]
• WO 03/059065 [0027, 0033, 0086, 0090]
• WO 99/488691 [0086]
• EP 2029531 A1 [0087]
• US 2009/298828 A1 [0087]
• EP 1791816 A1 [0088]
• WO 2006/29799 A1 [0088]
• EP 1791816 [0091, 0092]
• WO 2006/29799 [0091, 0092]

Cited non-patent literature

• Kim, 1997 [0002]
• Harwood, 2005 [0002]
• Tong, 2005 [0002]
• LuTFI ABU-ELHEIGA et al., 1995 [0002]
• Jane WIDMER, et al. 1996 [0002]
• Bianchi et al., 1990 [0002]
• Milgraum LZ, et al., 1997 [0002]
• Swinnen, et al., 2006 [0003]
• Abu-Elheiga, et al. 2005 [0003]
• Abu-Elheiga et al., 2001, 2003 [0003]
• Oh et al., 2005 [0003]
• Swinnen, et al., 2004 [0004]
• Heemers, et al., 2000 [0004]
• Swinnen, et al., 2002 [0004]
• Rossi, et al., 2003 [0004]
• Milgraum, et al., 1997 [0004]
• Yahagi, et al., 2005 [0004]
• Suzuki, S. et al. Chem. Pharm. Bull. 15 1120 (1967) [0008]
• R. Schmierer and H. Mildenberger (Liebigs Ann. Chem. 1985, 1095) [0008]
• Ito M. et al. Bioscience, Biotechnology and Biochemistry 67, 1230-1238, (2003) [0011]
• Campbell et al., J. Chem. Soc., Perkin Trans. 1, 1985, (8) 1567-76 [0012]
• AM Chirazi, T. Kappe and E. Ziegler, Arch. Pharm. 309, 558 (1976) [0014]
• K.-H. Boltze and K. Heidenbluth, Chem. Ber. 91, 2849 [0014]
• E. Ziegler and E. Steiner, Monatsh. 95, 147 (1964) [0015]
• R. Ketcham, T. Kappe and E. Ziegler, J. Heterocycl. Chem. 10, 223 (1973) [0015]
• Micklefield et al., Tetrahedron, (1992), 7519-26 [0016]
• Edwards et al., J. Chem. Soc. S, (1967), 405-9 [0016]
• J. Economic Entomology, 66, (1973), 584 [0016]
• Wheeler, TN, J. Org. Chem. 44, 4906 (1979) [0017]
• Cap et al., Arch. Pharm. 309, 558-564 (1976) [0020]
• White, JD, Brenner, JB, Deinsdale, MJ, J. Amer. Chem. Soc. 93, 281-282 (1971) [0020]
• Remington's Pharmaceutical Science, 15th ed Mack Publishing Company, East Pennsylvania (1980). [0045]

Claims (11)

Use of (5s, 8s) -3- (4'-chloro-3'-fluoro-4-methylbiphenyl-3-yl) -4-hydroxy-8-methoxy-1-azaspiro [4.5] dec-3-ene 2-on for the manufacture of a drug. Use according to claim 1 for the preparation of a medicament for the prophylaxis and / or therapy of tumor diseases. Use according to claim 2 for the manufacture of a medicament for the prophylaxis and / or therapy of breast, pancreatic, renal cell carcinoma, hepatocellular carcinoma, malignant melanoma and other skin tumors, non-small cell, bronchial carcinoma, endometrial carcinoma, colorectal carcinoma or prostate carcinoma. Use of (5s, 8s) -3- (4'-chloro-3'-fluoro-4-methylbiphenyl-3-yl) -4-hydroxy-8-methoxy-1-azaspiro [4.5] dec-3-ene 2-on for the prophylaxis and / or treatment of human or other mammalian diseases. Use according to claim 4 for the prophylaxis and / or therapy of tumor diseases. Use according to claim 5 for the prophylaxis and / or therapy of breast cancers, pancreatic carcinomas, renal cell carcinomas, hepatocellular carcinomas, malignant melanomas and other skin tumors, non-small cells, bronchial carcinomas, endometrial carcinomas, colorectal carcinomas or prostate carcinomas. (5s, 8s) -3- (4'-chloro-3'-fluoro-4-methylbiphenyl-3-yl) -4-hydroxy-8-methoxy-1-azaspiro [4.5] dec-3-en-2- on for use as a medicine. (5s, 8s) -3- (4'-chloro-3'-fluoro-4-methylbiphenyl-3-yl) -4-hydroxy-8-methoxy-1-azaspiro [4.5] dec-3-en-2- on for the prophylaxis and / or therapy of tumor diseases. (5s, 8s) -3- (4'-chloro-3'-fluoro-4-methylbiphenyl-3-yl) -4-hydroxy-8-methoxy-1-azaspiro [4.5] dec-3-en-2- on for the prophylaxis and / or treatment of breast cancers, pancreatic carcinomas, renal cell carcinomas, hepatocellular carcinomas, malignant melanomas and other skin tumors, non-small cell carcinomas, bronchial carcinomas, endometrial carcinomas, colorectal carcinomas or prostate cancers. Pharmaceutical formulation in the form of a tablet containing (5s, 8s) -3- (4'-chloro-3'-fluoro-4-methylbiphenyl-3-yl) -4-hydroxy-8-methoxy-1-azaspiro [4.5] dec -3-en-2-on for the prophylaxis and / or treatment of breast cancer, pancreatic carcinoma, renal cell carcinoma, hepatocellular carcinoma, malignant. Melanoma and other skin tumors, non-small cells, bronchial carcinoma, endometrial carcinoma, colorectal carcinoma or prostate cancer. (5s, 8s) -3- (4'-chloro-3'-fluoro-4-methylbiphenyl-3-yl) -4-hydroxy-8-methoxy-1-azaspiro [4.5] dec-3-en-2- on in combination with another active ingredient

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