EP2670408A1 - (5s,8s)-3-(4'-chlor-3'-fluor-4-methlybiphenyl-3-yl)-4-hydroxy-8-methoxy-1-azaspiro[4.5]dec-3-en-2-one (compound a) for treatment - Google Patents

Abstract

The invention relates to (5s,8s)-3-(4'-chlor-3'-fluor-4-methlybiphenyl-3-yl)-4-hydroxy-8-methoxy-1-azaspiro[4.5]dec-3-en-2-one for therapeutic purposes, pharmaceutical agents, and the use thereof in treatment, in particular for the prophylaxis and treatment of tumor diseases.

Description

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

 The present invention relates to (5s, 8s) -3- (4'-chloro-3'-fluoro-4-methylbiphenyi-3-yl) -4-hydroxy-8-methoxy-1-azaspiro [4.5] decyl 3-en-2-one (= compound A) for therapeutic purposes,

Pharmaceutical compositions 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, Harw ood., 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.

 There are two human ACC isoforms known. ACC1 and ACC2 encoded by two different genes (LuTFI ABU-ELHEIGA et al, 1995, Jane WIDMER, et al., 1996). ACC I is expressed in lipogenic tissue (liver, adipose tissue), localized 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. This controls fatty acid oxidation by inhibiting camitine palmityl transferase I. the enzyme that facilitates entry of long-chain fatty acids into the mitochondria for β-oxidation (Milgraum LZ et al., 1 97. 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 activation by citrate. a feedback inhibition by long-chain fatty acids, reversible phosphorylation and / or inactivation or modulation of the

Enzyme production by altered gene expression.

ACC I 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 the

Fatty acid oxidation in oxidative tissues, has been upregulated by 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, Miigraum, et al, 1997, Yahagi, et al., 2005 ). This phenotype most likely contributes to the development and progression of tumors that are 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.

PCT Patent Application PCT / EPP99 / 01787, published as WO 99/48869 corresponding to European Patent EP 1 066 258 B1, relates to novel arylphenyl-substituted cyclic ketoenols, a majority of the processes for their preparation and their use as pesticides and herbicides.

Pharmaceutical properties of 3-acylpyrrolidine-2,4-diones are described above (Suzuki, S. et al., Chem. Pharm. Bull., J., 5,120 (1967)). Furthermore, N-phenylpyrrolidine-2,4-diones were synthesized by R. Schmierer and H. Mildenberger (Liebigs Ann. Chem. 1985, 1095). Biological activity of these compounds has not been described.

 EP-A-0 262 399 and GB-A-2 266 888 disclose similarly structured compounds (3-arylpyrrolidine-2,4-diones), of which, however, no herbicidal, insecticidal or acaricidal activity has become known , Unsubstituted, bicyclic 3-arylpyrrolidine-2,4-dione derivatives are known (EP-A-355 599, EP-A-415 21 1 and JP-A-12-053.) With herbicidal, insecticidal or acaricidal activity 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 I H-Aiylpyrrolidin-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 95/26 954, 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 102 00505 9892, 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 09/1 15262. WO 10/052 161. WO 10/063378,

WO 10/063670, WO 10/063380, WO 10/066780 and WO 10/102758.

In addition, ketal-substituted 1H-arylpyrrolidine-2,4-diones from WO 99/16748 and (spiro) - Aryl-pyrrolidinediones of JP-A-14 205 984 and Ito M. et al. Bioscience. Biotechnology and Biochemistry 67, 1230-1238, (2003). In addition, WO 06/02441 1 discloses herbicidal compositions containing ketoenols.

It is known that certain substituted A 1 -dihydrofuran-2-one derivatives have herbicidal properties (cf., DE-A-4 014 420). The synthesis of used as starting compounds

Tetronic acid derivatives (such as 3- (2-methyl-phenyl) -4-hydroxy-5- (4-fluoro-phenyl) -Δ-dihydrofuranone (2)) are also described in DE-A-4 014 420. Similarly structured compounds without indication of insecticidal and / or acaricidal activity are known from the publication Campbell et al. J.

Chem. Soc, Perkin Trans. 1, 1985. (8) 1567-76. Farther

Derivatives having herbicidal, acaricidal and insecticidal properties are known from: EP-A-528 156, EP-A-647 637. WO 95/26 954. WO 96/20 196, WO 96/25 395.

 WO 96/35 664, WO 97/01 535, WO 97/02 243, WO 97/36 868, WO 98/05 638, WO 98/06 72, WO 99/16748, WO 98/25 928, WO 99/43 649. WO 99/48 869, WO 99/55 673. 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-A ³ -dihydrothiphen-on derivatives are known from (WO 95/26 345, 96/25 395,

WO 97/01 535, WO 97/02 243, WO 97/36 868, WO 98/05638, WO 98/25928, WO 99/16748, WO 99/43649, WO 99/48869, WO 99/55673. WO 01 / I 7972. WO 01/23354, WO 01/74770, WO 03/013249, WO 04/080 962, WO 04/1 1 1 042. WO 05/092897, WO 06/029799 and

WO 07/096058. Certain phenyl-pyrone derivatives unsubstituted in the phenyl ring have already been disclosed (compare AM Chirazi, T. Kappe and E. Ziegler, Arch. Pharm. 309, 558 (1976) and K.H. Boitze and K. Heidenbluth, Chem. Ber. 9J. 2849). Phenyl-pyrone derivatives having herbicidal, acaricidal and insecticidal properties which are substituted in the phenyl ring are described in EP-A-588 137, WO 96/25 395, WO 96/35 664, WO 97/01 535, WO 97/02 243, WO 97 / 16,436, 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 04/1 1 1 042, WO 05/092897, WO 06/029799 and

WO 07/096058. Certain 5-phenyl-1,3-thiazine derivatives which are unsubstituted in the phenyl ring have already been disclosed (see E. Ziegler and E. Steiner, Monatsh., 95, 147 (1964), R. Ketcham, T. Kappe and E. Ziegler, J. Heterocycl Chem, J, 0. 223 (1973)). Substituted in the phenyl ring

Herbicidal, acaricidal and insecticidal derivatives are described in WO 94/14785,

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 04/1 1 1042, WO 05/092897, WO 06/029799 and WO 07/096058.

It is known that certain substituted 2-arylcyclopentanediones. herbicides, insecticides and acaricidal properties (see, e.g., U.S. Patent Nos. 4,283,348; 4,338,122; 4,436,666; 4,526,723;

4 551 547; 4,632,698; WO 96/01 798; WO 96/03 366. WO 97/14667 and WO 98/39281, WO 99/43649, WO99 / 48869, WO 99/55673, WO 01/17972, WO 01/74770, WO 03/062244, WO 04/080962 , WO04 / 114042, WO05 / 092897, WO06 / 029799, WO07 / 080066, WO07 / 096058, WO09 / 019005, WO09 / 019015, WO09 / 049851, WO10 / 069834, WO10 / 000773, WO10 / 057880, WO 1 0/0 1 94. WO 10/089210, WO 10/102848 and WO 101/3232. 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-eno-ones from Edwards et al. J. Chem. Soc. S, (1967), 405-9. An insecticidal or acaricidal activity is not described. Also, 2- (2,4,6-trimethylphenyl) -l, 3-indanedione is from J. Economic Entomology. 66, (1973), 584 and the published patent application 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 (US 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, and 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 04/1 1 1 042, WO 05/092897, WO 06/029799, WO 07/096058, WO 08/071405,

WO 08/1 10307, WO 08/110308, WO 09/074314, WO 08/145336, WO 09/015887, WO 09/0743, WO 10/046194, WO 10/081755, and WO 10/08921 1.

 It is known that certain substituted 4-aryl-pyrazolidine-3,5-diones have acaricidal, insecticidal and herbicidal properties (see for example WO 92/16510, EP-A-508 126, WO 96/1 1 574,

 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 04/1 1 1 042,

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-hydroxypropyltetrahydropyridones with acaricidal, insecticidal and herbicidal properties are known (JP 1 1 152273). 4-Hydroxyl et rahydropy ridone were also known 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, 4-phenyl-6- (2-phenethyl) -5,6-dihydropyron is known from the synthesis of kawalactone derivatives (Kappe et al., Arch. Pharm. 309, 558-564 (1976)). 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 having Applications in crop protection are in WO 01/98288 and

WO 07/09658.

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

WO 2005/0891 18 and WO2007 / 039286 generically disclose nitrogen-containing bicyclic

Structures for therapy, wherein 5-biphenyl-substituted cyclic ketoenols are not specifically mentioned. 4-phenyl-substituted [1.2] oxazine-3,5-diones are described as herbicides for the first time in WO 01/17972. Furthermore, 4-acyl-substituted [1.2] oxazine-3,5-diones have been described as pesticides, but above all as herbicides and growth regulators, for example in EP-A-39 48 89; WO 92/07837, US 5,728,831 and as herbicides and pesticides in WO 03/048138.

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. Concurrent with the present application of priority, the subject of which is the therapeutic use of compound A alone, i.a. filed a PCT application, the subject has the therapeutic use of numerous cyclic ketoenols and the priority of a German application with the application number DE 102010008644.4 claims. Compound A is Example I-1118 in the PCT application, but was in the

DE 102010008644.4 not yet included.

The structurally closest state of the art could be the example I-I-16 of WO99 / 48869, which differs from compound A only by a missing fluorine atom on the outer phenyl ring of the biphenyl. The therapeutic use of Example 11-16 of WO99 / 48869 is also an article in DE 102010008644.4 and the PCT application claiming the priority of DE 102010008644.4 (Example 1-2).

However, the structurally closest state of the art could also be the example I-I-a-31 of WO03 / 059065, 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 102010008644.4 and the PCT application claiming the priority of DE 102010008644.4 (Example 1-8 1).

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 for the prophylaxis and therapy of

Tumors are suitable and advantages over known in the art

Have structures. It was now überras

especially suitable for the treatment of diseases. It was unpredictable whether and which of those known as insecticides or herbicides

 Structures solves the task of the invention particularly well, namely a structure that can be used particularly well in the treatment of human diseases.

Compound A has comparable enzyme inhibition data as Example I-I-a-16 of

WO99 / 48869, which could be considered structurally closest prior art. Surprisingly, however, the compound A is characterized by a broader therapeutic window in the M C F 7 - X eno ra 11 - M odel I 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-3 1 of WO03 / 059065, which could also be considered as the structurally closest prior art.

From the large group of known as insecticides, fungicides or herbicides cyclic

Ketoenol surprisingly has highlighted Compound A by better enzyme inhibition and / or better in vivo efficacy at tolerated doses.

Also considered to be covered by the present invention is the use of the

Physiologically acceptable salts of Compound A. Physiologically acceptable salts of Compound A also include salts of common bases such as, by way of example and by way of example, alkali metal salts (e.g., sodium and potassium salts), alkaline earth salts (e.g., calcium and magnesium salts), and ammonium salts derived from ammonia or organic

Amines having I to 16 ( ^' atoms, such as, by way of example and by way of preference, ethylamine, diethylamine, triethylamine, ethyldiisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dicyclohexylamine,

Dimctln amino ethanol. Prokain, 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 may be applied in a suitable manner, e.g. oral, parenteral, pulmonary, nasal, sublingual, lingual, buccal, rectal, dermal, transdermal, conjunctival, otic or as an implant or stent. For these routes of administration, compound A can be administered in suitable administration forms.

For the oral administration are according to the state of the art functioning fast and / or modified the compound A donating application forms containing the compound A in crystalline and / or amorphised and / or dissolved form, such as. Tablets (uncoated or coated tablets, for example with enteric or delayed-release or insoluble coatings which control the release of the compound of the invention), orally disintegrating tablets or films / wafers, films / lyophilisates, capsules in the oral cavity

(For example, hard or soft gelatin capsules), dragees, granules, pellets, powder. Emulsions. Suspensions, aerosols or solutions.

 Parenteral administration can be done by bypassing a resorption step (e.g.

intravenously, intraarterially, intracardially, intraspinally or intralumbarally) or with the involvement of resorption (e.g., 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 suitable, for example Inhalation medicines (i.a.

Powder inhalers. Nebulizer), nose drops, solutions. sprays; lingual, sublingual or buccal tablets to be applied, films / wafers or capsules, suppositories, ear or

Eye preparations, vaginal capsules, aqueous suspensions (lotions, shaking 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 happen. These excipients include, inter alia, excipients (for example

microcrystalline cellulose, lactose, mannitol), solvents (eg liquid polyethylene glycols), emulsifiers and dispersing or wetting agents (for example sodium dodecylsuifate, polyoxy sorbitan oleate), binders (for example polyvinylpyrrolidone), synthetic and natural polymers (for example albumin), stabilizers (eg antioxidants such as for example

Ascorbic acid), dyes (e.g., inorganic pigments such as iron oxides), and flavor and / or smell corrigents.

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, moisturizers, lubricants, adsorbents and adsorbents. Diluents, solvents, cosolvents, emulsifiers, solubilizers, flavoring agents, colorants,

Preservatives, stabilizers, wetting agents, salts for changing the osmotic pressure or buffers are used.

Mention should be made of Remington ^'s Pharmaceutical Science, 15th ed. Mack Publishing Company, Fast Pennsylvania (1980). 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 are present.

For the purposes of the invention, auxiliaries may be, for example, salts, saccharides (mono-, di-, tri- and oligo- and / or polysaccharides), proteins, amino acids, peptides, fats, waxes. oils

Hydrocarbons and derivatives thereof, wherein the excipients may be of natural origin or may be obtained synthetically or partially synthetically. 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 the compound A for the prophylaxis and therapy of human diseases, in particular of tumor diseases.

In particular, compound A can be used to inhibit or reduce cell proliferation and / or cell division and / or to induce apoptosis.

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 prostatic hyperplasia (BPH),

 solid tumors and

 hematological tumors.

As solid tumors according to the invention, for example, tumors of the breast,

Respiratory tract, the brain, the reproductive system, the gastrointestinal tract, the genitourinary tract, the eye, the liver, the skin, the head and neck, the thyroid gland, the parathyroid gland, the bone and the connective tissue and metastases of these tumors.

For example, hematological tumors are treatable

 multiple myeloma,

 Lymphomas or

 Leukemias.

For example, treatable as breast tumors are:

 Breast carcinomas 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 - II - inflammatory breast cancer.

For example, tumors of the respiratory tract are treatable

 non-small cell lung cancers and

 small cell bronchial carcinomas.

For example, tumors of the brain are treatable

 gliomas,

 glioblastomas,

 astrocytomas,

 Meningiomas and

 Medulloblastomas.

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

 prostate cancers,

 Malignant testicular tumors and

 Penis cancer.

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

 endometrial

 cervical cancer

 ovarian cancer

 Vaginalkarzimome

 Vulvarkarzinome

For example, tumors of the gastrointestinal tract are treatable:

 Colorectal carcinomas

 anal cancers

 gastric cancers

 pancreatic cancer

 Ösophagukarzinome

 Gallbladder carcinomas

 Small intestine cancer

 Salivary gland carcinomas

 Neuroendocrine tumors

Gastrointestinal stromal tumors For example, tumors of the urogenital tract are treatable:

Hamblasenkarzinome

 Renal cell carcinoma

 Carcinomas of the renal pelvis and the urinary tract

For example, tumors of the eye are treatable:

 retinoblastoma

 Intraocular melanomas

Treatable as tumors of the liver, for example:

 Hepatocellular carcinomas

 Cholangiocellular carcinomas

Tumors of the skin are treatable, for example:

 Malignant melanomas

 Basal cell carcinomas

 squamous

 Kaposi's sarcoma

 Merkel cell carcinoma

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

 laryngeal cancer

 Carcinomas of the pharynx and oral cavity

For example, sarcomas are treatable:

 Soft tissue sarcomas

 osteosarcomas

For example, lymphomas are treatable:

- Non-Hodgkin's lymphoma

 Hodgkin's lymphoma

 Cutaneous lymphoma

 Lymphomas of the central nervous system

AIDS-associated lymphomas Treatable as leukemias, for example:

 Acute myeloid leukemias

 Chronic myeloid leukemias

 Acute lymphocytic leukemia

- Chronic lymphocytic leukemia

 Hairy cell leukemia

Advantageously, the compound A can be used 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

 Pank easka rzi nomen. Renal cell carcinoma, hepatocellular carcinoma, malignant melanoma and other skin tumors, non-small cell lung carcinoma, endometrial carcinoma, colorectal carcinoma, gastric carcinoma and prostate carcinoma. The compound A can be used with particular advantage for the prophylaxis and / or therapy of:

 Breast cancers, in particular of hormone receptor negative and hormone receptor positives, as well as pancreatic carcinomas, non-small cell lung carcinomas, endometrial carcinomas, colorectal carcinomas, gastric carcinomas and prostate carcinomas.

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.

A further subject of the present application is compound A for the prophylaxis and / or therapy of breast carcinomas, pancreatic carcinomas, renal cell carcinomas, hepatocellular carcinomas, malignant melanomas and other skin tumors, non-small cells,

Bronchial carcinoma, endometrial carcinoma, colorectal carcinoma, gastric carcinoma or prostate cancer.

An advantageous object of the present application is compound A for the prophylaxis and / or therapy of breast cancers, in particular of hormone receptor negative and hormone receptor positives, as well as pancreatic carcinomas, non-small cell lung carcinomas, endometrial carcinomas, colorectal carcinomas, gastric carcinomas and prostate carcinomas. 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 treatment of breast, pancreatic, renal cell carcinoma, hepatocellular carcinoma, malignant melanoma and other skin tumors, non-small cells, bronchial carcinoma, endometrial carcinoma, colorectal carcinoma, gastric carcinoma or prostate cancer.

An advantageous subject 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 cancers, in particular of hormone receptor negative and hormone receptor positives, as well as

 Pancreatic carcinoma, non-small cell lung carcinoma, endometrial carcinoma,

Colorectal carcinomas, gastric carcinomas and prostate cancers.

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, gastric carcinoma or prostate cancer.

An advantageous subject of the present application is the use of the compound A for the prophylaxis and / or therapy of breast cancers, in particular of hormone receptor negative and hormone receptor positives. as well as pancreatic carcinomas, non-small cells

 Bronchial carcinoma, endometrial carcinoma, colorectal carcinoma, gastric carcinoma and prostate cancer.

The present application further relates to pharmaceutical formulations in the form of tablets containing compound A for the prophylaxis and / or therapy of breast carcinomas, pancreatic carcinomas, renal cell carcinomas, hepatocellular carcinomas, malignant melanomas and other skin tumors, non-small cell carcinomas, bronchial carcinomas, Endometrial carcinoma, colorectal carcinoma, gastric carcinoma or prostate cancer. An advantageous subject of the present application are pharmaceutical formulations in the form of tablets containing compound A for the prophylaxis and / or therapy of

Breast cancers, in particular of hormone receptor negative and hormone receptor positives, as well as pancreatic carcinomas, non-small cell lung carcinomas, endometrial carcinomas, colorectal carcinomas, gastric carcinomas and prostate carcinomas.

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 may be associated with known anti-hyperproliferative. cytostatic or cytotoxic substances used to treat 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:

Afmitor. Aldesleukin, alendronic acid, alfaferone, alitretinoin. Allopurinol. Aloprim. Aloxi, altretamine, aminoglutethimide. Amifostine, Amrubicin, Amsacrine, Anastrozoi, Anzmet, Aranesp, Arglabin. Arsenic trioxide, Aromasin. 5-azacytidine, azathioprine, BCG or tice-BCG, bestatin. Beta methasone acetate, betamethasone sodium phosphate, bexarotene, bleomycin sulfate, broxuridine, bortezomib. Busuifan, calcitonin. Campath, capecitabine, carboplatin. Casodex, cefeson,

Celmoleukin. Cerubidin, chlorambucil, cisplatin, cladribine, clodronic acid, cyclophosphamide, cytarabine. Dacarbazine, dactinomycin. DaunoXome, Decadron. Decadron phosphate. Delicious roe. Denileukin Diftitox, Depomedrol, Deslorelin, Dexrazoxane, Diethylstilbestrol, Diflucan, Docetaxel, Doxifluridine, Doxorubicin, Dronabinol, DW-166HC, Eligard, Elitek, Ellence, Einend, Epirubicin, Epoetin-alfa, Epogen, Eptaplatin. Ergamisol, Estrace, Estradiol, Estramustine Sodium Phosphate, Ethinyl Estradiol. Ethyol, etidronic acid, etopophos. Etoposide. Fadrozole, farston, filgrastim, finasteride, fligrastim, floxuridine, fluconazole, fludarabine, 5-fluorodeoxyuridine monophosphate, 5-fluorouracil (5-FU), fluoxymesterone, II utain id. Formestan, Fosteabin. Fotemustine. fulvestrant, Gammagard, Gemcitabine, Gemtuzumab, Gleevec, Giiadel, Goserelin. G ra ni be ron - H vdroch 1 o rid. Histrelin, Hycamtin, Hydrocorton, erythro-hydroxynonyladincin. Hydroxyurea, ibritumomab tiuxetan, idarubicin. Ifosfamide, interferon-alpha, interferon-alpha-2, interferon-alpha-2a,

Interferon-alpha-2β, Interferon-alpha-n 1. Interferon-alpha-n3, Intercron-beta. Interferon gamma-la, Interleukin-2, Intron A, Iressa. Irinotecan. Kytril, lapatinib, lentinan sulfate, letrozole, leucovorin, leuprolide. Lcuprolid acetate. Levamisole. Levofolic acid calcium salt, levothroid, levoxyl, lomustine, lonidamine, marinol, mechlorethamine, mccobalamin. Medroxyprogcristone acetate, metastol acetate. Melphalan, Menest. 6-mercaptopurine. Mesna, methotrexate, metvix, miltefosine, minocycline, mitomycin C, mitotane, mitoxantrone, mod renal, myocet, ncdaplatin. Neulasta, Neumega, Neupogen. Nilutamide. Nolvadex, NSC-631570, OCT-43, Octreotide. Ondansetron hydrochloride, Orapred. Oxaliplatin, Paclitaxel, Pcdiapred, Pcgaspargasc. Pegasys. Pcntostatin, Picibanil. Pilocarpine hydrochloride. Pirarubicin. Plicamycin. Porfimer sodium, Prcdnimustin.

Prednisolone, Prednisone, Premarin, Procarbazine, Procrit. Raltitrexed, RDEA1 19, Rebif. Rhenium 186 etidronate, rituximab, Roferon-A. Ronuirtid. Salagen. Sandostatin, Sargramostim, Seniustin. Sizofirane, sobuzoxan, solu-medrol, streptozocin, strontium-89-chloride, tamoxifen,

Tamsulosin. Tasonermin. Tastolacton. Taxoters, Teceleukin. Temozolomide. Teniposide. Testosterone propionate. Test red. Thioguanine. Thiotepa, thyrotropin. Ti ludronic acid. Topotecan. Torem ifen. Tositumomab, Tastuzumab, Teosulfan, Tretinoin, Trexall, Trimetlu Imekunin. Trimetrexate, triptorelin acetate. Triptorelin pamoate. UFT, uridine. Valrubicin. Vesnarinone. Vinblastine. Vincristine. Vindesine. Vinorelbine. Virulizin. Zinecard, Zinostatin Stimalamer, Zofran; ABI-007, Acolbifen, Actimmun. Affinitak, aminopterin. Arzoxifen, Asoprisnil. Atamestane. Atrasentan. BAY 43-9006 (sorafenib), Avastin, CCI-779, CDC-501, celebrex, cetuximab, crisnatol, cyproterone acetate, decitabine. DN-101, doxorubicin MTC. dSLIM, dutasteride. Edotecarin. Eflornitliin. Exatecan, fenretinide, histamine dihydrochloride, histrelin-hydrogei-implant, holmium-166-DOTMP, ibandronic acid. Interferon-gamma. Intron PEG, ixabepilone. Keyhole limpet hemocyanin, L-

651582, lanreotide, lasofoxifene, Libra. Lonafarnib, Miproxifen. Minodronate. MS-209, iiposomal MTP-PE. M.X-6, Nafarelin. Nemorubicin. Neovastat, Nolatrexed. Oblimersen. Onko-TCS, Osidem. Pac I i taxel - polymatamate, pamidronate disodium, PN-401, QS-21, quazepam, R-1 49. Raloxides, ranpirnas, 13 -is -retetinic acid, satraplatin, seocalcitol, T-1 38067. Tarceva , Taxoprexin, thymosin-alpha. Tiazofurin, tipifarnib, tirapazamine, TLK-286, toremi fen. TransMID-107R, Valspodar. Vapreotide. Vatalanib. Verteporfin. Vinflunine. Z-100th 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 conclusive: Aminoglutethimide, L-asparaginase, azathioprine, Bleomycin, busiilfan, carboplatin, carmustine, chlorambucil, cisplatin, colaspase, cyclophosphamide, cytarabine. Dacarbazine.

Dactinomycin, daunorubicin, diethylstilbestrol, 2 ', 2'-difluorodoxycytidine, docetaxel, doxorubicin (adriamycin), epirubicin, epothilone and its derivatives. erythro Hydroxynonyladenin. Ethinyl estradiol, etoposide, fludarabine phosphate, 5-fluorodeoxyuridine, 5-fluorodeoxyuridine monophosphate, 5-fluorouracil, fluoxymcesterone. Flutamide. Hexamethylmelamine, hydroxyurea, hydroxyprogesterone caproate, idarubicin. Ifosfamide, interferon, irinotecan. Leucovorin, Lomustin.

Mechlorethainin, medroxyprogesterone acetate. Megestrol acetate, melphalan. 6-mercaptopurine, mesna, methotrexate, mitomycin C, mitotane, mitoxantrone, paclitaxel. Pentostatin, .V-phosphonoacetyl-L-aspartate (PALA). Plicamycin. Prednisolone. Prednisone. Procarbazine, raloxifene, semustin, streptozocin, tamoxifen, teniposide. Tcstosteron propionate. Thioguanine, thiotepa, topotecan. Trimethylmelamine, uridine, vinblastine. Vincristine. Vindcsin and Vinorelbin.

Promisingly, compound A can also be combined with biological therapeutics such as antibodies (e.g., Avastin, Rituxan, Erbitux, Herceptin) and recombinant proteins. Compound A may also be used in combination with other antiangiogenic agents

Therapies, such as Avastin, axitinib. Regorafenib, Recentin. Sorafenib or sunitinib. Combinations with inhibitors of the proteasome and mTOR as well as antihormones and steroidal metabolic enzyme inhibitors are beneficial because of their favorable

Side effects profile particularly suitable. In general, the combination of compound A with other cytostatic or cytotoxic agents has the following aims:

• 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 to use the chemotherapeutic agents used in lower doses than in monotherapy;

 • the possibility of a more tolerable therapy with fewer side effects compared to

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.

EXPERIMENTAL PART 1. COMPARATIVE EXAMPLES Table V shows Example I-1-a-16 of WO99 / 488691 and Example I-1-a-3 1 of

WO03 / 059065, which Applicant regards as the closest prior art.

Tab. V

Method 1 (UPLC-MS)

 Instrument: Waters Acquity UPLC-MS SQD 3001; Column: Acquity UPLC BEH C18 1.7 50x2. 1 mm; Eluent A: water + 0.1% formic acid, eluent B: acetonitrile; Gradient: 0-1.6 min 1-99% B, 1 .6-

2.0 minutes 99% B; Flow 0.8 ml / min; Temperature: 60 ° C; Injection: 2 μΐ; DAD scan: 210-400 nM.

Method 2 (UPLC-MS):

Instrument: Waters Acquity UPLC-MS ZQ4000; Column: Acquity UPLC BEH C 18 1.7 50x2.1mm; 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 μΐ; DAD scan: 210-400 nM.

Method 3 (U PLC-MS):

 Instrument: Waters Acquity UPLC-MS SQD 3001; Column: Acquity UPLC BEH C 18 1.7 50x2.1mm; Eluent A: water + 0.1% formic acid, eluent B: acetonitrile; Gradient: 0-1.6 min 1-99% B, 1 .6-

2.0 minutes 99% B; Flow 0.8 ml / min; Temperature: 60 ° C; Injection: 2 μΐ; DAD scan: 210-400 um.

Preparation of Comparative Example V.I.

a) Interniediate

Intermedia! V.l.l

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

5.00 g (19.18 mmol) of (4'-chloro-4-methylbiphenyl-3-acetic acid (EP 2029531 A1 and US Pat

2009/298828 Al) 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. Drying in a fine vacuum gave 5.4 g (100% of theory) of the title compound as a brownish oil. Ή NMR (300MHz, CDCl ₃ ): δ [ppm] = 2.36 (s, 3H), 4.22 (s, 2H), 7.29 (d, 1H), 7.35-7.55 (m, 6H).

Intermediate V.l.2

 Mcthyl-cis- 1 -! I (4'-chloro-4-methylbiphcyl-3- \ 1 (acetyl) amino J -4-methoxycyclohexanecarboxylate

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 DM AP and 8.4 ml (60.5 mmol) of triethylamine were added at room temperature under nitrogen dissolved in 1 ml of dichlorometliane. Subsequently, a solution of 6.75 g (24.2 mmol) of the intermediate V. I was added dropwise. I in 60 ml dichlorometlian added. The resulting reaction mixture was stirred at room temperature overnight. to

 Work-up was diluted with dichloromethane and the organic phase was washed with water. saturated sodium bicarbonate solution and aqueous, 5% citric acid. To

Drying over sodium sulfate 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 (300MHz, DMSO-d ₆ ): δ [ppm] = 1.3 1 - 1.47 (m, 2H), 1.60 - 1.73 (m, 2H), 1.75 - 1.86 (m, 2H), 2.00 - 2.1 1 (in, 2H), 2.27 (s, 3H), 3.09 - 3.20 (m, 1H), 3.21 (s, 3H), 3.5 1 (s, 3H), 3.58 (s, 2H), 7.23 (d, 1H) , 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, = 1.38 min; MS (ESIpos): m / z = 430 | M + H | , b) final product V.I.

(5s, 8s) -3- (4'-chloro-4-methylbiphenyl-3-yl) -4-hydroxy-8-methoxy-l-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 Ν, Ν-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 liter of ice-water, adjusted to pH 3 with 1N aqueous hydrogen chloride solution, stirred for 3 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.

Ή-NM (300MHz, DMSO-d ₆ ): δ [ppm j = 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, = 1. 19 minutes; MS (ESIpos): m / z = 398 [M + Hf.

Comparative Example V.2

 Comparative Example V.2 is Example I-1 -a-3 1 of WO03 / 059065.

 The therapeutic use of Example I-I-A-31 of WO03 / 059065 is also an article in DE 1020 1 0008644.4 and the PCT application which has the priority of DE 1020 1 0008644.4 in

Claiming (Example 1 -8 1).

2. Connection A

Preparation of the compound A

a) Intermediate

 Intermediate A.l

(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 under argon were added 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). 20 h at room temperature. Then, toluene and water were added, adjusted with concentrated aqueous hydrogen chloride solution, a pH of 1-2, stirred for 10 minutes, the phases separated, extracted twice with toluene, dried the combined organic phases over sodium sulfate. filtered and narrowed. 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. Ή-NMR (300MHz, 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, = 1.31 min; MS (ESIneg): m / z = 277 | M H | , Intermediate A.2

Methyl cis- 1 - {[(4'-chloro-3'-fluoro-4-methyl-1-butyl-3-yl) -acetyl] -amino} -4-methoxycyclohexanecarboxylate

10.0 g (35.9 mmol) of the intermediate A.I 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. This gave 10.8 g (100% of theory) (4'-chloro-3'-fluoro-4-methylbiphenyl-3-yl) -acetyl chloride. 10.6 g (35.7 mmol) of (4'-chloro-3'-fluoro-4-methylbiphenyi-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 admixed with saturated, aqueous sodium bicarbonate solution. The phases were separated and the aqueous phase extracted twice with ethyl acetate. The combined organic phases were over

Dried sodium sulfate, filtered and concentrated. 8.50 g were obtained

methoxycyclohexancarboxylat. 8.02 g (42.8 mmol) of metin 1-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 added with water, extracted with dichloromethane. The combined organic phases were washed with IN 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.

Ή-NMR (300MHz, 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 - 2.1 1 (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 (i.e. , 1H), 7.61 - 7.72 (m, 2H), 8.30 (s, 1H). LC-MS (Method 2): R, = 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-ene-2 on

To 15.7 g (35.0 mmol) of the intermediate A.2 in 60 ml of N, N-Dimethyiformamid were under Nitrogen 4.32 g (38.5 mmol) of potassium tert-butoxide given. The reaction mixture was stirred 20

Minutes at room temperature. Then the reaction mixture was added to ice water, added dropwise 160 ml of 1 N aqueous hydrochloric acid solution, 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.

Ή-NMR (300MHz, 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 + HJ

3. Assays

Human ACCI enzyme assay

 The ACC l inhibition data were collected with two different assays (A I and B l)

Assay AI (= (A1))

 The inhibitory activity of the substances of this invention against acetyl-CoA carboxylase 1 (ACC 1) was measured in the ACC 1 assay described in the following paragraphs.

The basic principle of the assay is the measurement of the adenosine diphosphate (ADP) formed as a co-product by means of an HTRF "-based, homogeneous homo-resolved fluorescence assay (HTRF).

As an enzyme, ( ^' -terminal FLAG -tagged recombinant human ACC 1 (GenBank Accession No. NM 1 8834, amino acids 39- end) expressed in baculovirus-transfected insect cells (Hi) and purified by affinity chromatography on anti-FLAG M2 Affinity .. gel (sigma-Aldrich) was used Alternatively, commercial information available ( ^'-terminal ffis-getagtes ACC I (BPS Biosciencc San Diego, CA. catalog No. 50200. amino acids 39 -. be used end) For the assay, 50. In a 1 × 10 fold concentrated solution of the test substance in DMSO is pipetted into a black low-volume 384-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany) 2 μl of a solution of ACC 1 in assay buffer [50 mM HEPES / NaOH pH 7.5 , 1 2mM sodium bicarbonate, 2mM MgC ^' h, 2mM potassium citrate, 0.005% (w / v) bovine

Serum albiimine (BSA)] was added and the mixture was incubated for 1 min to allow pre-binding of the substances to the enzyme prior to the enzyme reaction. Then it would become

Enzyme reaction started by addition of 3 μl of a solution of adenosine triphosphate (ATP, 83.5 μM => estrogen conc. Ion in 5 μl assay volume is 50 μl, Amersham Pharmacia Biotech # 27-2056-01) and Acctyl-CoA ( 33.4 μ M => final concentration in 5 μΐ assay volume is 20 μΜ, Roche Biosciencc # 10101893001) in assay buffer and the resulting mixture is incubated for 20 min at 22 ° C for the reaction time. The concentration of the ACC I was adjusted to the respective activity of the enzyme and adjusted so that the assay worked in the linear range. ^'T ypical concentrations ranged from 2.5 ng / ul.

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

The resulting mixture was incubated for one hour at 22 ° C to allow binding of the europium cryptate-labeled anti-A D P antibody to the ADP formed by the enzyme reaction 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, e.g. a Rubystar or Pherastar (both BMG Labtcchnologics. Offenburg,

Germany), which measured fluorescence emissions at 620 tim and 665 nm after excitation at 350 μm. The ratio of 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 Europiuni cryptate-labeled anti-A DP antibody less ADP). The data were normalized (enzyme reaction without inhibitor = 0% inhibition, all other assay components but no enzyme = 100% inhibition). Typically, the test substances were incubated on the same microtiter plates at 10 different concentrations in the range of 20 LI to I nM (20 μΜ, 6.7 μΜ, 2.2 μΜ, 0.74 μΜ, 0.25 μΜ, 82 ηΜ, 27 ηΜ, 9.2 ηΜ, The dilution series were assayed at the level of 100X concentrated solution by serial 1: 3 dilutions) in duplicate for each concentration prior to assay, and IC50 values were calculated with a 4-parameter fit for an in-house software was used. Assay Bl (= (B 1))

 The hACC I inhibitory activity of the substances of this invention was measured in the hACC 1 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 of Promega. In this case, the adenosine tri-phosphate (ATP) which is not consumed in the enzyme reaction is first quantitatively converted into cA MP by means of an adenylate cyclase ("ADP-GLO reagent"), then the adenylate cyclase is stopped and then (.K i nase Detecti on Reagent "), the ADP formed is then converted into ATP and converted into a glow luminescence signal in a luciferase-based reaction. Recombinant C-terminal FLAG-tagged human ACC 1 (acetyl-coenzyme A carboxylase alpha transcript variant 1) (Gen Bank 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 100-fold concentrated solution of the test substance in DMSO was converted to a white low volume (Greiner Bio-One, Frickenhausen, Germany) pipetted. 2.5 μΐ of a solution of hACC I in assay buffer [50 mM HEPES / NaOH pH 7.5, 2 mM MgCl ₂ , 2 mM potassium citrate. 12 mM NaHCCK 2 mM di-thio-threitol (DTT), 0.005% (w / v) bovine serum albumin (BSA) | and the mixture was incubated for 15 minutes to allow pre-binding of the substances to the enzyme prior to the enzyme reaction. Then the

Enzyme reaction started by the addition of 2.5 μΙ of a solution of adenosine tri-phosphate (ATP, 100 M => 50 ° C) and acetyl-CoA (20 μM

=> Final concentration in 5 μΐ assay volume is 10 μΜ) in assay buffer and the resulting

Mixture for the reaction time of 45 min at 22 ° C incubated. The concentration of hACC I 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 / μΐ. The reaction was stopped by adding 2.5 μΐ of the "ADP-GLO reagent" (diluted 1: 1.5-fold) and the

resulting mixture I h incubated at 22 ° C to completely convert the unreacted ATP in cAMP. Subsequently, 2.5 μ! of the "Kinase Detection Reagent" (1.2 times more concentrated than indicated by the manufacturer), the resulting mixture incubated for 1 h at 22 ° C and then the luminescence measured with a suitable meter (Viewlux or Topcount from Perkin-Elmer or Pherastar from BMG Labtechnologies) The amount of light emitted was taken as the amount of ADP formed and thus for the enzyme activity of hACC I. The data were normalized (enzyme reaction without inhibitor = 0% inhibition, all others

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 μΜ to 1 nM (20 μΜ, 6.7 μΜ, 2.2 μΜ, 0.74 μΜ, 0.25 μΜ, 82 ηΜ, 27 ηΜ, 9.2 ηΜ, 3.1 nM and I nM, the serial dilutions were assayed at the level of the I OX-fold concentrated solution by serial 1: 3 dilutions) in duplicate for each concentration prior to assay and IC50 values were calculated using a 4-parameter fit using an inhousc software. Human ACC2 Ecoassay

The A C C 2 - 1 nh i bi t i on sdatcn were collected with two different assays (A2 and B2) Assay A2 (= (A2))

 The inhibitory activity of the substances of this invention against acetyl-CoA carboxylase 2 (ACC2) was measured in the ACC2 assay described in the following paragraphs.

The basic principle of the assay is the measurement of the co-product adenosine diphosphate (ADP) using an HTRF ^! -based competitive immunoassays (HTRF = Hpmogeneous Time Resolved Fluorescence).

The commercially available ( ^' -terminal His-tagged ACC2 from BPS Bioscience (San Diego, CA, catalog No. 50201) was used as the enzyme. End amino acids 39, expressed in baculovirus-infected Sf9 insect cells and purified by Ni-NTA affinity chromatography. used.

For the assay, 50 μl of a 100X concentrated solution of the test substance in DMSO was pipetted into a black low-volume 384well tube (Greiner Bio-One, Frickenhausen, Germany), 2 μl of a solution of ACC2 in assay buffer [50 mM HEPES / NaOH pH 7.5, 12 mM sodium in bicarbonate. 2mM MgCL, 2mM potassium citrate. 0.005% (w / v) bovine

Serum Albumin (BSA) | was added and the mixture incubated for 15 min to allow pre-binding of the substances to the enzyme prior to the enzyme reaction. Then the

Enzyme reaction is started by adding 3 ul of a solution of adenosine triphosphate (ATP, 83.5} iM = ^• final concentration in 5 μΐ assay volume is 50 μΜ, Amersham Pharmacia Biotech # 27- 2056-01) and Acctyl-CoA (33.4 μ M => Final concentration in 5 μΐ assay volume is 20 μΜ, Roche Bioscience # 10101893001) in assay buffer and the resulting mixture for the reaction time of 20 min at 22 ° C incubated. 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 / μΙ. The reaction urde stopped by successive additions of 2.5 microliters of a solution of d2-labeled ADP (HTRF ^® Trans Screener ™ ADP kit, Cis biointemational. Marcoulc. France) (in the EDTA-containing HTRF ^® Trans Screener ™ ADP detection buffer in HTRF ^® Trans Screener ™ 50mM HEPES pH 7.0, 60mM EDTA, 0.1% (w / v) BSA, 0.02% Natnumazide, 400mM potassium fluoride) and 2.5μl of a solution of europium cryptate-labeled anti-ADP Kit. ADP antibody (HTRF ^® Transscreener ™ ADP Kit) in HTRF * Transscreener ™ 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 ADP formed by the enzyme reaction 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 measuring instrument, eg a Rubystar or Pherastar (both BMG Labtechnologies, Offenburg,

Germany), which measured fluorescence emissions at 620 μm 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 in the range of 20 μΜ to 1 nM (20 μΜ, 6.7 μΜ, 2.2 μΜ, 0.74 μΜ, 0.25 μΜ, 82 ηΜ, 27 ηΜ, 9 , 2 ηΜ, 3, 1 ηΜ and 1 nM, the serial dilutions were assayed at the level of 100X concentrated solution by serial 1: 3 dilutions) in duplicate for each concentration before the assay and IC50 values were calculated using a 4-parameter -Fit, for which an inhouse 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, Gen Bank Accession No. NP_001084) (amino acids 27-end), expressed in baculovirus-infected insect cells (Hi5) and purified by anti-FLAG affinity chromatography.

For the assay, 50 μl of a 100 × 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 μ! of a solution of hACC2 in assay buffer [50 mM HEPES / NaOH pH 7.5. 2m M MgCl. 2 mM potassium citrate. 12 mM NaHCO ₃ , 2 mM thio-threitol (DTT). 0.005% (w / v) bovine serum albumin (BSA) | and the mixture incubated for 15 min to allow pre-binding of the substances to the enzyme prior to the enzyin reaction. Then the

Enzyme reaction started by adding 2.5 μΐ of a solution of adenosine tri-phosphate (ATP, 100 μΜ => final concentration in 5 μΐ assay volume is 50 μΜ) and acetyl-CoA (20 μΜ

=> Final concentration in 5 μΐ assay volume is 10 μΜ) in assay buffer and the resulting

Mixture for the reaction time of 45 min at 22 ° C incubated. 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 / μί. The reaction was stopped by adding 2.5 μΐ of the "ADP-GLO reagent" (diluted 1: 1.5 fold) and the resulting mixture incubated at 22 ° C for 1 h to completely exchange the unreacted ATP in cAMP Subsequently, 2.5 μΐ 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 hACC 2 enzyme activity. The data were normalized (enzyme reaction without inhibitor = 0% inhibition, all other assay components but no enzyme = 100% inhibition). Typically, the test substance was incubated on the same microtiter plates at 10 different concentrations ranging from 20 μΜ to 1 nM (20 μΜ, 6.7 μΜ, 2.2 μΜ, 0.74 μΜ, 0.25 μΜ, 82 μη, 27 μη, 9.2 μη, 3.1 μM and 1 μM , the serial dilutions were assayed at the level of 100X concentrated solution by serial 1: 3 dilutions) in duplicates for each concentration before assay and IC50 values were calculated using a 4-parameter fit using in-house software has been. Non-human ACCase assay

The assay was incubated at room temperature in a clear 384-wel! microtiter plate

carried out. It determines the inorganic phosphate released from ATP in the ACCase reaction.

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

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 AMMOKOBDAT in 4 M HCl. The solution was allowed to stand for 30 minutes. After filtration through a 0.45 μιη

 Pol tetrafluoroethylene (PTFE) filters were added to 0.1 part of Triton X-100 (1.5%) in distilled water.

ACCase enzyme was extracted from oat seedlings harvested 9 days after seeding and partially purified by 0-40% ammonium sulfate precipitation followed by ionic Australian Q-Sepharose chromatography.

Fashion-of-action attempt

Before determining the efficacy in the MCF-7 model, part of the test substances were tested in a "Mode of action" experiment based on the short-term application of a test substance capable of ACC I and / or ACC2 in the living

Organism to inhibit after oral administration, leading to a reduction of Malonvl-CoA in the tumor. For this purpose, 2 million human MCF-7 breast cancer cells were experimentally subcutaneously injected into female nude mice (NM RI-nude (nu / nu) mice, Taconic M & B A / S, the day before application of a pellet for the release of estrogen over a period of at least 60 days) injected. 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.). Ce! L assays

In accordance with the invention, the substances were tested in cell-based assays, that is, the ability of the substances to tumor cell proliferation after 96 hours

Inhibit substance incubation. Cell stability was tested using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega). The cells were at a density of 2000-5000

Cells / well (depending on the cell line) seeded in ΙΟΟμΙ growth medium on 96well microtiter plates. For every cell line examined. Cells were seeded on a separate plate to determine luminescence at t = 0 hours and t = 96 hours. After an incubation at 37 ° C., the luminescence values for the t = 0 samples were determined. 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 the

Luminescences between substance-treated and control values were used to determine the percent growth inhibition.

The substances were investigated in the following cell lines, which exemplified the indicated

Indications represented:

Cell line source indication

 MCF7 ATCC hormone receptor positive breast cancer

 PC3 ATCC prostate cancer

 You 145 NCI prostate cancer

 FCC 1 ATCC endometrial carcinoma nom

 K M 1 2 NCI colorectal carcinoma

 HEC 1A ATCC endometrial carcinoma

 DNA-G CLS pancreatic carcinoma

 BxPC3 ATCC pancreatic carcinoma

 H460 ATCC non-small cell Bonchia! karzi nom

 CA L- 1 20 ATCC hormone receptor negative breast carcinoma

 BT-20 ATCC hormone receptor negative breast cancer

 SNU16 ATCC gastric carcinoma

LNCaP ATCC prostate 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 daily orally a defined dose over a period of 1, 2 or 3 weeks the test substance and were daily observed for mortality and body weight. The highest administrable dose was defined as MTD, during the treatment phase and in the day

 Follow-up time no animal died and it did not drop more than 10%

Body weight compared to the starting weight came.

To determine the antitumoral efficacy, various xenograft models were then used in which the test substances were given at their MTD and at lower dosages. In addition to various 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 was administered subcutaneously to these mice on the day before the tumors 1 in the plating on the release of estrogen (17β-oestradiol 0.36 mg, release over 60 days). 2 x tumor cells (suspended in Medium + Matrigel) 1: 1, final 0, 1 ml) were then injected subcutaneously into the flank of each animal on the next day. If the tumors 20-25 mm ²

Had reached the tumor area, the mice were randomized into the therapy groups and started the therapy. Therapy was then measured 2-3 times weekly

Tumor area and body weight continued until the average tumor size in the

Control group, which had received only the vehicle of the test substance, or in one of

Treatment groups had reached 120mm ² . At this time, the experiment was stopped in all groups and the prepared tumors were weighed.

The T / C value calculated as the primary success parameter was calculated either on the basis of the effect on the tumor weight or on the tumor area: Mean value of the tumor area / area in the treatment group divided by the average of the tumor weights / areas in the vehicle group. Analysis of ACC1 expression in tumor and normal tissues

The ACC I expression was determined by means of a microrray. For this, the RNA was isolated from different tumor tissues and the corresponding normal weights. Methodologically, Trizol RNA extraction reagent (Invitrogen) was used and purification was performed using the RNeasy Mini Kit (Qiagen). In addition, DNase I (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" of

 Affymetrix and then the array on an Affymetrix GeneChip 3000 scanner (Affymetrix) read. Evaluation and quality control were performed using the Expressionist Pro 4.0 Refiner (Gene Data) 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

These data show that Compound A and Comparative Example V.I 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 example from the cell assays. Tab. 2

4.3 Maximum Tolerable Dose (MTD)

Comparative Example V.I.

Comparative Example V.1 was applied to female nude mice (NMRI nu / nu):

Dose: s. Tab. 3

 Type of application: per os

 Vehicle: PEG400 / ethanol / solutol HS15 (70/5/25, v / v / v)

(Solutol HS 15: polyoxyethylene ester of

Application volume: 10ml / kg

Scheme: s. Tab. 3

The treatment phase was followed by an observation period of 7 days. The resulting death and the effect on body weight are summarized in Table 3.

Tab. 3

 Substance dose (mg / kg) regimen body weight (%) deaths

V. l 10 14 days. Twice daily minus 1 2 1 of 3

V. 1 20 14 days, twice daily minus 29 2 of 3

V. l 30 14 days, 2 times a day minus 1 1 2 of 3

Vehicle 2 1 day. once a day plus 1 5 0 out of 5

V. l 15 21 days, once a day minus 1 1 out of 5

V. l 20 21 days, once a day minus 2 1 out of 5 V. l. 25 21 days, once a day minus 3 1 out of 5

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

 As a result, the MTD is less than 15 mg / kg for a 2-day regimen once daily.

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 to female nude mice (NMR! Nu / nu):

Dose: 7.5mg / kg

Type of application: per os

 Vehicle: PEG400 / ethanol / Solutol HS 15 70/5/25, v / v / v)

 Application volume: 10ml / 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 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).

 FIG. 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 10mg / kg 2qd or 12.5mg / kg 2qd by day 43

further treated. 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.I, if any, is very small.

Connection A

Compound A was administered to female nude mice (NMR! Nu / nu):

Dose: s. Tab. 4

Type of application: per os

Vehicle: PEG400 / ethanol / Solutol HS 15 70/5/25, v / v / v) Application volume: 10ml / 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

As a result, the MTD for a 2-day treatment at I daily dosing is below 40 mg / kg and above 30 mg / kg.

Compound A was subsequently applied in the MCF-7 breast cancer xenograft model to female nude mice (N M R! Nii / nu):

 Dose: s. Tab. 5

 Type of application: per os

 Vehicle: PEG400 / ethanol / Solutol HS 15 70/5/25, v / v / v)

 Application volume: 10ml / kg

 Scheme: 30 days, once a day (qd)

 Mice per dose group: 10-13

Tab. 5

 Substance dose (mg / kg) T / C T / C

 (based on (based on tumor areac) tumor weight)

Vehicle 1.00 1.00

Compound A 20 0.56 (ns) 0.42 (p = 0.003)

Compound A 25 0.51 (p <0.05) 0.37 (p <0.001)

Compound A 30 0.49 (p <0.05) 0.35 (p <0.001)

Compound 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 tumor area-based T / C values since there was no normal distribution of the measurements. Subsequently, all therapy groups were compared to the vehicle group by means of the Dürrns 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 value of 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 with I daily dosing.

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

Pankreaskarziomen. 5. Formulation

Tablets containing Compound A a) Preparation of a Pharmaceutical Formulation by Direct Tabletting

 Tablets were prepared according to the composition of Table 6 containing the compound by direct tabletting.

Tab. 6

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

For the production of 50 tablets wound in a Fantaschale

 3.351 g mannitol, spray dried

 2.004 g of cellulose, microcrystalline

 0.499 g of N a-C rosca sleeve 1 ose and

 3.992 g Exemplified compound 1-1 18

 premixed by careful trituration. The mixture was poured into a 100ml screwcap; transferred and homogenized for 10 minutes in a Turbula mixer. After adding 0.149 g of magnesium stearate, it was again mixed for 1 min in the Turbula mixer.

The resulting molding compound was pressed by means of an eccentric tablet press (Kersch EK 2) into biconvex tablets with a diameter of 8 mm and a radius of curvature of 1 2 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 Mass Disintegration time

Beginning 8 IN 198.7 mg 1: 28 min

 Mid 95 N 196.8 mg 1: 28 min.

 End 97N 199.0 mg 1: 32 min.

Mean 9 IN 198.2 mg 1: 29 min. c) in vitro dissolution

 In vitro release of Compound A from the prepared tablets was carried out 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 Figure 4 show the results.

Tab. 7

 * SDS = sodium lauryl sulfate (added because solubility at pH 1 and pH 6.8 is not sufficient) 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:

ACC 1 expression in tumor and corresponding normal tissue

 1: healthy breast tissue (2 samples)

 2: breast tumor tissue (26 samples)

 3: healthy colon tissue (30 samples)

 4: colon tumor tissue (7 1 samples)

 5: healthy lung tissue (27 samples)

 6: lung tumor tissue (40 samples)

 7: healthy pancreatic tissue (22 samples)

 8: Pancreatic tumor tissue (19 samples)

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

 Therapeutic efficacy of Compound A in hormone-dependent MCF-7

Breast cancer xenograft model.

 Release curve of compound A from tablets

Claims

claims

1. Use of (5s, 8s) -3- (4'-chloro-3'-fluoro-4-methyl-biphenyl-3-yl) -44-hydroxy-8-methoxy-1-azaspiro [4.5] dec-3-yl en-2-on for the manufacture of a drug.

2. Use according to claim I for the preparation of a medicament for prophylaxis

 and / or therapy of tumor diseases.

3. Use according to claim 2 for the manufacture of a medicament for prophylaxis

 and / or therapy of breast cancers, pancreatic carcinomas, non-small cells

Bronchial carcinoma, endometrial carcinoma, colorectal carcinoma,

 Gastric carcinoma and prostate cancer.

4. Use of (5s, 8s) -3- (4'-chloro-3'-fluoro-4-methylbiphenyl-3-yl) -4-hydroxy-8-methoxy-1-azaspiro [4.5] dec-3-3 en-2-on for the prophylaxis and / or therapy of

 Diseases of humans or other mammals.

5. Use according to claim 4 for the prophylaxis and / or therapy of

 Tumor diseases.

6. Use according to claim 5 for the prophylaxis and / or therapy of breast, pancreatic, non-small cell lung, endometrial, colorectal, gastric and prostatic carcinoma.

7. (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 use as a medicinal product.

8. (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 tumor diseases.

9. (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 breast, pancreatic, non-small cell lung, endometrial, colorectal, gastric and prostatic carcinoma.

10. 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-one for the prophylaxis and / or treatment of breast, pancreatic, non-small cell lung, endometrial, colorectal, gastric and prostate cancers.

1 1. (5s, 8s) -3- (4'-chloro-3'-fluoro-4-methylbiphenyl-3-yl) -4-hydroxy-8-methoxy-1-azaspiro [4.5] dec-3-ene -2-on in combination with another active stool