EP1768654A2 - Inhibiteurs selectifs des corticoide-synthases humaines - Google Patents

Inhibiteurs selectifs des corticoide-synthases humaines

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Publication number
EP1768654A2
EP1768654A2 EP05769814A EP05769814A EP1768654A2 EP 1768654 A2 EP1768654 A2 EP 1768654A2 EP 05769814 A EP05769814 A EP 05769814A EP 05769814 A EP05769814 A EP 05769814A EP 1768654 A2 EP1768654 A2 EP 1768654A2
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European Patent Office
Prior art keywords
hydrogen
alkyl
hydroxy
radicals
pyridine
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EP05769814A
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German (de)
English (en)
Inventor
Rolf Hartmann
Sarah Ulmschneider
Ursula MÜLLER-VIERA
Rita Bernhardt
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POMBIOTECH GmbH
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Universitaet des Saarlandes
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    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
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    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
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    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
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    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/06—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom containing only hydrogen and carbon atoms in addition to the ring nitrogen atom
    • C07D213/16—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom containing only hydrogen and carbon atoms in addition to the ring nitrogen atom containing only one pyridine ring
    • C—CHEMISTRY; METALLURGY
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/26—Radicals substituted by halogen atoms or nitro radicals
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    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/28—Radicals substituted by singly-bound oxygen or sulphur atoms
    • C07D213/30—Oxygen atoms
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    • C07D217/00—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems
    • C07D217/12—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with radicals, substituted by hetero atoms, attached to carbon atoms of the nitrogen-containing ring
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    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D233/64—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms, e.g. histidine
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    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/26—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms

Definitions

  • the invention relates to compounds for the selective inhibition of the human corticoids synthases CYPIIIIB and CYP1 1B2, their preparation and use for the treatment of hypercortisolism and diabetes mellitus or cardiac insufficiency and myocardial fibrosis.
  • corticosteroids Human adrenal glands are divided into two areas, the adrenal medulla and the adrenal cortex. The latter secretes a number of hormones known as corticosteroids that fall into two categories. Glucocorticoids (especially hydrocortisone or cortisol) act primarily on carbohydrate and glucose metabolism, secondarily they can retard wound healing by interfering with the inflammatory process and the formation of fibrous tissue. The second category, mineral corticoids, are involved primarily in the retention of sodium and the excretion of potassium. The most important and effective mineral corticoid is aldosterone.
  • hydrocortisone or cortisol act primarily on carbohydrate and glucose metabolism, secondarily they can retard wound healing by interfering with the inflammatory process and the formation of fibrous tissue.
  • mineral corticoids are involved primarily in the retention of sodium and the excretion of potassium. The most important and effective mineral corticoid is aldosterone.
  • glucocorticoid biosynthesis is u. a. controlled by adrenocorticotropin (ACTH).
  • ACTH adrenocorticotropin
  • CYPIIBl Steroid II ⁇ hydroxylase
  • Hypercortisolism in particular Cushing's syndrome, as well as a special form of diabetes mellitus characterized by an extreme morning increase in cortisol plasma levels.
  • cortisol Elevated cortisol levels are also associated with neurodegenerative disorders.
  • Aldosterone secretion is regulated by a variety of signals: the
  • Plasma concentrations of sodium and potassium and the multi-step renin-angiotensin-aldosterone system renin is secreted by the kidneys in response to low blood pressure releasing angiotensin I from a precursor peptide. Angiotensin I will turn to
  • Vasoconstrictor is. In addition, it acts as a hormone to stimulate the release of aldosterone (Weber, KT. & Brilla, CG., Circulation 83: 1849-1865
  • CYP11B2 aldosterone synthase
  • mitochondrial cytochrome P450 enzyme catalyzes the formation of the most potent mineral corticoid aldosterone from its steroidal substrate 11-deoxycorticosterone (Kawamoto, T. et al., Proc. Natl. Acad USA 89: 1458-1462 (1992)).
  • Excessive plasma aldosterone concentrations are associated with and contribute to the progression of these diseases, such as congestive heart failure and congestive heart failure, myocardial fibrosis, ventricular arrhythmia, cardiac fibroblast stimulation, cardiac hypertrophy, renal perfusion and hypertension (Brilla, CG, Herz 25: 299-306 (2000)).
  • RAAS renin-angiotensin system
  • its pathophysiological activation occurs in particular in patients with chronic heart failure or renal underperfusion or renal artery stenoses (Young, M., Funder, JW, Trends Endocrinol, Metab., 11: 224 -226 (2000)).
  • both elevated plasma aldosterone and angiotensin II levels as well as cardially locally-secreted aldosterone induce fibrotic structural changes of the myocardium, as a result of which the formation of myocardial fibrosis leads to further reduction Cardiac function (Brilla, CG, Cardiovasc., Res. 47: 1-3 (2000); Lijnen, P. & Petrov, VJ Mol. Cell. Cardiol. 32: 865-879 (2000)).
  • Fibrotic structural changes are characterized by the formation of tissue characterized by an abnormally high amount of fibrotic material (mainly collagen strands). Such fibroses are in some situation, e.g. wound healing, useful, but may be harmful, i.a. if they affect the function of internal organs. In myocardial fibrosis, the heart muscle is traversed by fibrotic strands that make the muscle stiff and inflexible, thereby impairing its function. Since even in patients with mild heart failure mortality is 10-20%, it is urgently necessary to intervene here with a suitable drug therapy. Despite long-term treatment with digitalis glycosides, diuretics, ACE inhibitors or AT II antagonists, plasma aldosterone levels remain elevated in patients and the medication has no effect on fibrotic structural changes.
  • fibrotic material mainly collagen strands
  • Mineralcorticoid antagonists in particular aldosterone blocking agents, are already the subject of numerous patents or patent applications.
  • the steroidal mineralocorticoid receptor antagonist spironolactone (17-hydroxy-7-alpha-mercapto-3-oxo-17 ⁇ -pregn-4-ene-21-carboxylic acid ⁇ -lactone acetate; Aldactone ®) aldosterone Receptors competitively blocked to aldosterone, thus preventing the receptor-mediated aldosterone effect.
  • US 2002/0013303, US 6,150,347 and US 6,608,047 describe the dosage of spironolactone for the therapy or prevention of cardiovascular diseases and myocardial fibrosis while maintaining the normal electrolyte and water balance of the patient.
  • Mespirenone (15,16-methylene-17-spirolactones) and its derivatives have been considered as promising alternatives to spironolactone because they have only a low percentage of the spironolactone antiandrogenic activity (Losert, W. et al., Drug Res. 36: 1583-1600 (nickisch, K. et al., J Med Chem 30 (8): 1403-1409 (1987); Nickisch, K. et al., J. Med. Chem. 34: 2464-2468 (1991); Agarwal , MK, Lazar, G., Renal Physiol., Biochem., 14: 217-223 (1991)).
  • Mespirenone blocks aldosterone biosynthesis as part of a complete mineral corticoid biosynthesis inhibition (Weindel, K. et al., Arzneiffenfor ⁇ tion 41 (9): 946-949 (1991)). However, like spironolactone, mespirenone inhibits aldosterone biosynthesis only in very high concentrations.
  • WO 01/34132 describes methods for the treatment, prevention or blocking of pathogenic changes due to vascular injury (restenosis) in mammals by the administration of an aldosterone antagonist, namely eplerenone (an aldosterone receptor antagonist) or related structures which are partially epoxysteroidal and all derived from 20-spiroxanes.
  • WO 96/40255 US 2002/0123485, US 2003/0220312 and US 2003/0220310 describe therapeutic methods for the treatment of cardiovascular diseases, myocardial fibrosis or cardiac hypertrophy by using a combination therapy of an angiotensin II antagonist and an epoxy-steroidal aldosterone receptor Antagonists such as Eplerone or Epoxymexrenone.
  • Selective aldosterone synthase inhibitors may also be a promising class of drugs that, after myocardial infarction, promote the healing of compromised myocardial tissue with reduced scarring, thereby reducing the incidence of serious complications.
  • WO 01/76574 describes a pharmaceutical composition comprising an inhibitor of aldosterone formation or one of its pharmaceutically acceptable salts, optionally in combination with other active substances.
  • WO 01/76574 relates to the use of non-steroidal inhibitors of aldosterone formation which were commercially available at that time, in particular to the (+) - enantiomer of fadrozole, a 4- (5,6,7,8-tetrahydroimidazo (1, 5) a) pyridin-5-yl) benzonitrile, and its synergistic effect with angiotensin II receptor antagonists.
  • Anastrozole (Arimidex ®) and Exemestane (Coromasin ®) are other non ⁇ steroidal aromatase inhibitors. Their field of application is the treatment of breast cancer by inhibiting aromatase, which converts androstenedione and testosterone into estrogen.
  • the human steroid II ⁇ hydroxylase CYPIIIB1 shows greater than 93% homology to human CYPl 1B2 (Kawamoto, T. et al., Proc Natl Acad, See, USA 89: 1458-1462 (1992); Taymans, SE et al al., J. Clin. Endocrinol. Metab. 83: 1033-1036 (1998)).
  • strong inhibitors of aldosterone synthase must not affect steroid IL ⁇ hydroxylase and must therefore be tested for their selectivity.
  • nonsteroidal inhibitors of aldosterone synthase should preferably be used as therapeutics, since fewer side effects on the endocrine system are to be expected. This has been pointed out in previous studies, as well as the fact that the development of selective CYP11B2 inhibitors that do not affect CYPIIBl is hampered by the high similarity of the two enzymes (Ehmer, P. et al., J. Steroid Biochem 81: 173-179 (2002); Hartmann, R. et al., Eur. J. Med. Chem. 38: 363-366 (2003)).
  • the inhibitors should also interfere as little as possible with other P450 (CYP) enzymes.
  • CYP P450
  • the only drug known today that affects corticoid synthesis in humans is the aromatase (estrogen synthase, CYP19) inhibitor fadrozole, which is used in breast cancer therapy. It may also affect aldosterone and cortisone levels, but only at ten times the therapeutic dose (Demers, LM et al., J. Clin Endocrinol, Metabol 70: 1162-1166 (1990)).
  • Schizosaccharomyces pombe cells stably expressing human CYPl 1B2 and for subsequent selection of selectivity with V79MZ cells stably expressing either CYPl 1B2 or CYPIIBI (Ehmer, et al.
  • Inhibitor of human CYPl 1B2 (and strong aromatase inhibitor) and four others identified as non-selective but more potent than CYPIIBl inhibitors (A: CYP11B2 inhibitor; B 1 D: non-selective CYPIII inhibitors):
  • Structures were strong CYPIIBl inhibitors and therefore should not be considered for immediate use as selective CYP11B2 inhibitors.
  • R 1 , R 2, R 4, R 5 independently of one another, hydrogen, C 4 alkyl, C 2-4 alkenyl, C 3 - 6 cycloalkyl, hydroxy, Ci -4 alkoxy, Ci -4 hydroxyalkyl, halo, nitro or optionally substituted amino, and one of R 1 , R 2 , R 4 , R 5 is hydrogen; R 3 is a 4-imidazolyl radical; R 6 is hydrogen; R 7 is hydrogen or Ci- 4 alkyl; R 8
  • Ci- 4 alkyl hydroxy, or Ci -4 alkoxy
  • R 9 is hydrogen or Ci_ 4 alkyl
  • R 1 , R 2 , R 4 and R 5 are hydrogen or one to three of R 1 , R 2 , R 4 , R 5 independently halogen, hydroxy, NH 2, halo-Ci- 6 alkyl, CI_ 6 - alkyl, Ci 6 alkoxy, or HO- (Ci- 6) alkyl;
  • R 3 is a 4-imidazolyl radical;
  • R 6 and R 7 are hydrogen; one of R 6 , R 7 , R 8 and R 9 is a cyclic radical selected from phenyl, naphthyl, tetrahydronaphthyl, C 3-7 cycloalkyl and C 5-7 cycloalkenyl, or a methyl radical containing such a cyclic radical and optionally one or two Ci -6 -Al ky I residues carries two of the radicals R 6,
  • Undanylidenemethyl pyridine (CAS132819-71-7), Z-2- (1-undanylidenemethyl) -r-methylpyrene and Z-2- (undanylidenemethyl) benzothiazole.
  • Reaction conditions (a) NaBH 4 , MeOH / CH 2 Cl 2 , 15 min at 0 ° C., 1 h at RT; (b) PPh 3 HBr, benzene, 12h, reflux; (c) EtONa, 4 (5) -imidazole carboxaldehyde, N 2 , 12h, reflux; (d) Isomer separation by flash column chromatography.
  • the invention thus provides
  • R 1 and R 2 are independently selected from H, halo, CN, hydroxy, nitro, alkyl, alkoxy, alkylcarbonyl, alkylcarbonyloxy, alkylsulfinyl and alkylsulfonyl (wherein the alkyl radicals are straight, branched or cyclic, saturated or unsaturated and having 1 to 3 radicals R 12 may be substituted), aryl and Heteroaryl radicals and their partially or completely saturated equivalents, which may be substituted by 1 to 3 radicals R 12 , aryloxy and heteroaryloxy radicals, where aryl and heteroaryl have the abovementioned meaning, -COOR 11 , -SO 3 R 11 , -CHO, - CHNR 11 , -N (R n ) 2 , -NHCOR 11 and - NHS (O) 2 R 11 ;
  • R 3 is selected from nitrogen-containing monocyclic or bicyclic heteroaryl radicals and their partially or fully saturated equivalents, which may be substituted with 1 to 3 R 12 radicals and have at least one nitrogen atom not bonded to the methylidene C atom and unsubstituted;
  • R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 10 are independently selected from H, halo, CN, hydroxy, nitro, lower alkyl, lower alkoxy, lower alkylcarbonyl, lower alkylcarbonyloxy, lower alkylcarbonylamino, lower alkylsulfonylamino, lower alkylthio, lower alkylsulfinyl and lower alkylsulfonyl (wherein the lower alkyl groups may be straight, branched or cyclic, saturated or unsaturated and substituted with 1 to 3 R 12's ), -N (R n ) 2 , -COOR 11 and -SO 3 R 11 , or
  • R 8 or R 9 with R 6 or R 7 and / or with R 8 or R 9 of the adjacent C atom form one or two double bonds
  • R 8 (and R 9 ) with R 6 (and R 7 ) or with R 8 (and R 9 ) of the adjacent C atom and the associated carbon atoms form a saturated or unsaturated fused aryl or heteroaryl ring
  • the atoms of the fused aryl or heteroaryl ring can be substituted by 1-3 radicals R 12 , or R 4 and R 10 together form a methylene, ethylene or ethylidene bridge, it being possible for the atoms of the bridge to be substituted by one or two radicals R 12 , or a ring atom in the ortho position of the heteroaryl radical of R 3 directly or via a methylene radical or methylidene bridge forms a bond with R 6 and / or R 7 , wherein the bridging atom may be substituted with one or two R 12 radicals;
  • R 11 is
  • Lower alkylsulfonyl hydroxy-lower alkyl, hydroxy-lower alkoxy, hydroxy-lower alkylcarbonyl, hydroxy-lower alkylcarbonyloxy, hydroxy-lower alkylcarbonylamino, hydroxy-lower alkylthio, hydroxy-lower alkylsilinyl, hydroxy-lower alkylsulfonyl, mono- and bis (hydroxy-lower alkyl) amino and mono- and polyhalogenated Lower alkyl (wherein the lower alkyl groups may be straight chain, branched or cyclic, saturated or unsaturated); n is an integer from 1 to 3; or a pharmaceutically acceptable salt thereof for the treatment of hypercortisolism, diabetes mellitus, heart failure and
  • n 1, R 1 , R 2 and R 4 - R 10 is hydrogen, then R 3 is not 4-imidazolyl or 4-pyridyl;
  • n 1, R 1 and R 4 - R 10 is hydrogen and R 2 is F, Cl, Br or CN, then R 3 is not 4-imidazolyl;
  • R 3 is not 4-imidazolyl, 4-pyridyl, 4-methyl-3-pyridyl or 3-nitroimidazo [1, 2] a] pyrid-2-yl;
  • n 1 or 2;
  • three of the radicals R 1, R 2, R 4 and R 5 are independently hydrogen, Ci -4 alkyl, C 2-4 alkenyl, C 3-7 cycloalkyl, hydroxy, Ci -4 alkoxy, hydroxy-Ci - 4 -alkyl, halogen, trifluoromethyl, nitro or optionally substituted amino and the fourth radical of R 1 , R 2 , R 4 and R 5 is hydrogen, R 6 is hydrogen, R 7 is hydrogen or Ci -4 -Al ky I is , R, Ci -4 -Al ky I, hydroxy, or Ci -4 alkoxy 8 is hydrogen, R 9 and R 10 are independently hydrogen or Ci -4 alkyl, then R3 is not 4-imidazolyl;
  • n 1 or 2
  • three of the radicals R 1, R 2, R 4 and R 5 are independently hydrogen, hydroxy, amino, halo Ci -6 alkyl, Ci -6 alkyl, Ci -6 alkoxy or hydroxy-Ci- 6 are alkyl and the fourth radical selected from R 1, R 2, R 4 and R 5 is hydrogen, one of R 6 , R 7 , R 8 and R 9 is C 3-7 -cycloalkyl, C 5-7 -cycloalkenyl, C 3-7 -
  • Cycloalkylmethyl or C 3-7 -Cycloalkenylmethyl is, the methyl group may be substituted with one or two Ci -6 -Al ky I residues, two of the radicals R 6, R 7, R 8 and R 9 are independently hydrogen, hydroxy, C - 6 alkyl, halo-Ci- 6 -alkyl, Ci -6 -alkoxy or hydroxy-Ci -6- alkyl, and the remaining radicals R 6 , R 7 , R 8 and R 9
  • R 10 is hydrogen or C 6 -alkyl, then R 3 is not 4-
  • R 4 - R 10 is hydrogen, then R 3 is not 4-pyridyl;
  • n 2
  • R 1 is hydrogen
  • R 2 is hydroxy
  • Ci -4 -alkoxy or Ci -4 Al -Al is kylcarbonyloxy
  • R 4 - R 9 is hydrogen
  • R 10 is hydrogen or Ci -4 alkyl
  • R 3 is not 4-
  • n 1, R 1 , R 2 , R 4 , R 5 , R 8 - R 10 is hydrogen, R 6 and R 7 are both hydrogen or both methyl, then R 3 is not 2-pyridyl;
  • R 3 is not 4-methyl-3-pyridyl or its pharmaceutically acceptable salts
  • Alkyl radicals and “alkoxy radicals” in the context of the invention may be straight-chain, branched or cyclic and be saturated or (partially) unsaturated. Preferred alkyl radicals and alkoxy radicals are saturated or have one or more double and / or triple bonds.
  • straight-chain or branched alkyl radicals those with 1 to 10 C atoms, in particular those with 1 to 6 C atoms, are particularly preferred.
  • cyclic alkyl radicals mono- or bicyclic alkyl radicals having 3 to 15 C atoms, in particular monocyclic alkyl radicals having 3 to 8 C atoms, are particularly preferred.
  • “Lower alkyl radicals” and “lower alkoxy radicals” for the purposes of the invention are straight-chain, branched or cyclic saturated lower alkyl radicals and lower alkoxy radicals or those having a double or triple bond. In the case of the straight-chain ones, those having 1 to 6 C atoms, in particular having 1 to 3 C atoms, are particularly preferred. In the cyclic ones, those having 3 to 8 C atoms are particularly preferred.
  • “Aryls” for the purposes of the present invention include mono-, bi- and tricyclic aryl radicals having 3 to 18 ring atoms, which may optionally be fused with one or more saturated rings.
  • anthracenyl dihydronaphthyl, fluorenyl, hydrindanyl, indanyl, indenyl, naphthyl, naphthenyl, phenanthrenyl, phenyl and tetralinyl.
  • 'heteroaryl radicals' are mono- or bicyclic heteroaryl radicals having 3 to 12 ring atoms, which preferably have 1 to 5 heteroatoms selected from nitrogen, oxygen and sulfur and which may be fused with one or more saturated rings.
  • the preferred nitrogen-containing monocyclic and bicyclic heteroaryls include benzimidazolyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinolyl, quinoxalinyl, cinnolinyl, dihydroindolyl, dihydroisoindolyl, dihydropyranyl, dithiazolyl, homopiperidinyl, imidazolidinyl, imidazolinyl, imidazolyl, indazolyl, indolyl, isoquinolyl, isoindolyl, isothiazolidinyl, isothiazolyl, Isoxazolidinyl, isoxazolyl, morpholinyl, oxadiazolyl, oxazolidinyl, oxazolyl, phthalazinyl, piperazinyl, piperidyl, pteridinyl, purinyl, pyrazolidin
  • mono- or bicyclic heteroaryl radicals having from 5 to 10 ring atoms, which preferably have from 1 to 3 nitrogen atoms, very particular preference to isoquinolyl, imidazolyl, pyridyl and pyrimidyl.
  • Anelliere aryl or heteroaryl rings in the context of the present invention include such monocyclic rings having 5 to 7 ring atoms, which are fused via two adjacent ring atoms with the adjacent ring. They can be saturated or unsaturated.
  • the fused heteroaryl rings comprise 1 to 3 heteroatoms, preferably nitrogen, sulfur or oxygen atoms, more preferably oxygen atoms.
  • Preferred fused aryl rings are cyclohexyl, cyclohexenyl, cyclopentyl, cyclopentenyl and benzyl
  • preferred heteroaryl rings are furanoyl, dihydropyranyl, pyranyl, pyrrolyl, imidazolyl, pyridyl and pyrimidyl.
  • “Pharmaceutically acceptable salts” for the purposes of the present invention thereby include salts of the compounds with organic acids (such as lactic acid, acetic acid, amino acid, oxalic acid, etc.), inorganic acids (such as HCl, HBr, phosphoric acid, etc.) and, if the compounds have acid substituents , also with organic or inorganic bases. Preferred are salts with oxalic acid and HCl. Preferred compounds of embodiment (1) of the invention are those having the formulas (Ia) to (Ig), the compounds of the formula (Ia), (Ib), (Ic) and (Id) being particularly preferred:
  • a preferred embodiment of the compounds (Ia), (Ib) and (Ic) are the compounds of the following formula (Ih):
  • R 1 is H, halogen, CN, O-alkyl, O-alkenyl, O-alkynyl, alkyl, alkenyl or alkynyl, n is 1-3 and Het is a heteroaromatic with 5-10 ring atoms with 1-3 nitrogen atoms, and their pharmaceutically acceptable salts.
  • a particularly preferred embodiment of the compounds (Ia), (Ib) and (Ic) are the compounds of the following formula (Ii):
  • R 1 is H, halogen, CN, O-alkyl, O-alkenyl, O-alkynyl, alkyl, alkenyl or alkynyl, n is 1 or 2 and the double bonds have E or Z configuration, and pharmaceutically acceptable salts thereof.
  • R 1 or R 2 are independently selected from hydrogen , Halogen, CN, hydroxy, Ci-io-alkyl and Ci-I 0 -Al koxyresten, wherein the alkyl radicals or alkoxy radicals are straight-chain and saturated and may be substituted by 1 to 3 radicals R 12 ; and or
  • R 3 is selected from nitrogen-containing monocyclic heteroaryl radicals having 5 to 10 ring atoms and 1 to 3 nitrogen atoms, in particular selected from isoquinolyl, imidazolyl, oxazolyl, pyrazinyl, pyrazolyl, pyridyl, pyrimidyl, pyrrolyl, thiazolyl, triazinyl and triazoyl; and or
  • R 4, R 5, R 6, R 7, R 8, R 9, R CN, hydroxy and Ci 10 are independently selected from H, halogen, Ci -6 alkyl and koxyresten -6 -alkyl, which may be substituted by 1 to 3 radicals R 12 ; and or
  • R 12 is selected from H, halogen, hydroxyl, CN, Ci- 3 alkyl and Ci -3 -alkoxy; and or
  • Particularly preferred compounds of this type are those of the formulas (I) and (Ia) to (Ig), in particular compounds of the formulas (Ia) to (Ic) in which
  • R 1 or R 2 is hydrogen
  • the other of the substituents R 1 or R 2 is selected from H, fluorine, chlorine,
  • R 3 is selected from isoquinolyl, pyridyl, imidazolyl and pyrimidyl; and (iv) R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 are H.
  • R 1 or R 2 is selected from H, fluoro, chloro, CN, hydroxy, Ci -3 alkyl, and Ci -3 alkoxy;
  • R 3 is selected from pyridyl, imidazolyl, isoquinolyl and pyrimidyl;
  • R 5, R 6, R 7, R 8, R 9 and R 12 are H; and
  • V is a double bond.
  • the compounds of formula (I) may be in E and Z configuration depending on the position of substituents R 3 and R 10 .
  • the present invention encompasses both the mixture of isomers and the isolated E and Z compounds.
  • the compounds (I) have chiral centers (eg, the C atoms substituted with R 6 / R 7 and R 8 / R 9 ). Again, both the mixtures of the stereoisomers and the isolated individual compounds of the invention are included.
  • Preferred compounds of the formula (I) are the following compounds: E, Z-3- (1,2,3,4-tetrahydronaphth-1-ylidenemethyl) -pyridine, E, Z-3- (6-fluoro-1, 2,3,4-tetrahydronaphth-1-ylidenemethyl) -pyridine, E, Z-3- (6-chloro-l, 2,3,4-tetrahydronaphth-1-ylidene-methyl) -pyridine, E, Z-3 ( 6-methoxy-l, 2,3,4-tetrahydronaphth-l-ylidenemethyl) pyridine, E, Z-3- (7-fluoro-1, 2,3,4-tetrahydronaphth-1-ylidenemethyl) -pyridine, E, Z-3- (7-chloro-l, 2,3,4-tetrahydronaphth-l -ylidenemethyl) pyridine, E, Z-3- (7-methoxy-1,2,
  • the chemical compounds according to the invention can be synthesized in the process according to embodiment (3) by reducing the compound (II) to the corresponding alcohol and an adjoining Wittig reaction (cf., Examples 1-3).
  • the process preferably takes place according to the following general synthesis scheme:
  • Reaction conditions (a) NaBH 4 , MeOH / CH 2 Cl 2 , 15 min at 0 ° C., 1 h at RT; (b) PPh 3 «HBr, benzene, 12 h reflux; (c) heterocyclic carbonyl compound (III), base (preferably K 2 CO 3 with 18-crown-6, or EtONa), 12 h reflux.
  • the key step of the synthesis is a Wittig reaction using various heterocyclic carbonyl compounds, preferably aldehydes, and suitable salts, in particular phosphonium salts, of the bicyclic component.
  • a suitable reducing agent preferably NaBH 4
  • alcohol intermediates are formed. These are converted into their phosphonium salts.
  • a suitable base in particular K 2 CO 3 , for example in dry CH 2 Cl 2
  • a suitable phase transfer catalyst preferably 18-crown-6.
  • suitable base NaOEt for example in ethanol, without addition of a phase transfer catalyst is preferred.
  • the mixture of E and Z isomers obtained after the Wittig reaction can be used as a mixture or separated into its isomers.
  • the separation is carried out by crystallization or chromatographic methods, preferably by
  • the isomers can be converted into their stable salts, preferably in HCl or oxalic acid salts.
  • these are preferably stable hydrochlorides or oxalates, for the inventive use according to embodiment (1), these are preferably pharmaceutically acceptable salts.
  • the synthesis according to the invention can be used for the preparation of the E and Z isomers of the compounds according to the invention.
  • the yields can be increased drastically by the synthesis of the invention compared to previously known methods (up to 90%), in particular, the proportion of Z-isomer in the product can be significantly increased.
  • a preferred application of the synthesis is therefore the preparation of the Z-isomers of the compounds according to embodiment (3) of the invention.
  • the compounds of structure (Id) can be prepared in a four-step synthesis starting from acenaphthene or a suitable derivative thereof, optionally followed by purification eg by chromatographic separation (see also the reaction scheme below): nitration of the acenaphene (Chen, M. et al , Ranliao Gongye 38: 21-23 (2001)) and subsequent hydrogenation (Friedman, OM et al., J. Am. Chem. Soc. 71: 3010-3013 (1949)) provides, inter alia, 3-aminoacenaphthene.
  • the mixture of the resulting bromine compounds is isolated and reacted directly in a Suzuki coupling with 3-pyridineboronic acid to give the desired product.
  • the desired product, the acenaphthene derivative 50 is subsequently isolated, for example, by means of flash column chromatography.
  • the oil thus prepared can be converted to increase the stability in the corresponding hydrochloride.
  • Reaction conditions (a) HNO 3, in acetic anhydride for 20 hours at 1O 0 C; (b) H 2 , Pt / C (5%), in THF; (c) 1. NaNO 2, HBr, 0 ° C, 2. CuBr in HBr / toluene, O salt 0 C, the addition of the diazonium, 10 min at 0 ° C, 2 hours at 100 0 C; (d) Na 2 CO 3 solution, 3-pyridineboronic acid, in methanol, tetrakis (triphenylphosphine) palladium, N 2 , reflux, 12 h.
  • the test of the compounds according to the invention for use in accordance with embodiment (1) is carried out in in vitro test systems, preferably on more than one in wt test system.
  • the first step of these tests according to the invention comprises testing with nonspecific bovine adrenal CYPIIB from mitochondria for action of the test substances (Hartmann, R. et al., J. Med. Chem. 38: 2103-2111 (1995)).
  • the second step involves testing with human CYPIIB enzymes, preferably human CYPIIBI and CYP11B2.
  • human enzymes can either be expressed recombinantly, in particular in Schizosaccharomyces pombe or V79 cells, or in a tested human cell line, in particular the adrenocortical tumor cell line NCI-H295R (see example 5).
  • substances for use according to the invention according to (1) which have an effect on human CYPIIB enzymes, since there is no or only a very slight correlation between test data with bovine and human enzymes (compare Example 9).
  • fission yeast and V79MZh cells recombinantly expressing CYPIIB1 and CYP11B2 and NCI-H295R cells are suitable for the identification of novel therapeutically active compounds according to embodiment (1) for humans.
  • the compounds 41b, 42b, 44b, 45a, 45b, 48b, 49b are particularly suitable of the imidazole derivatives, which in comparison to the non-selective CYP inhibitor ketoconazole (78%) has a high inhibitory activity in the range of 90% (Table 4).
  • the Z isomers are particularly suitable for use according to (5), Z-4- (5-chloro-1-indanylidenemethyl) imidazole is most preferably 48 b ( Ex. 9); the latter is a highly potent CYP11B2 inhibitor (IC 50 : 4 nM), which has a five-fold selectivity in comparison with CYPIIBI (IC 50 : 20 nM).
  • a screening test can be used in recombinant S. pombe, especially CYPIIB2-expressing S. pombe Pl (Ex. 5A).
  • S. pombe especially CYPIIB2-expressing S. pombe Pl (Ex. 5A).
  • CYPIIB2-expressing S. pombe Pl Ex. 5A
  • For a further investigation on the use according to use (5) are then selected especially those compounds which show a higher inhibitory effect than the reference fadrozole.
  • compounds can be tested for their use according to (5) in V79 MZh cells (hamster lung fibroblasts) expressing either CYPIII Bl or CYPl 1B2 for their activity and selectivity (Ex. 5B).
  • V79 MZh cells hamster lung fibroblasts
  • CYPIII Bl CYPIII Bl
  • CYPl 1B2 CYPl 1B2
  • Different inhibition profiles are found: inhibitors which are either selective for CYPIIB1 or for CYP1B2, and inhibitors which can inhibit both CYPIIB enzymes.
  • the imidazole derivatives 41b, 42b, 44b, 45a, 45b, 46a, 48a and 49a and the compounds 6a, 8a, IQa, 13b are particularly suitable for the selective inhibition of CYP11B2 the imidazole derivatives 48b and 49b and many others of the compounds presented here (see Examples 6-9).
  • the inhibition of CYP19 by the test compounds can be performed in vitro using human placental microsomes and [I ⁇ , 2 ⁇ - 3 H] testosterone as substrate (modified according to: Thompson, EA Jr. & Siterii, PK, J. Biol. Chem. 249: 5364-5372 (1974)) (Example 4).
  • the inhibition of CYP 17 by the test substances can be determined in vitro with microsomes from E. coli recombinantly expressing CYP17 and progesterone as substrate (Example 4).
  • the NCI-H295R cell line is commercially available and is often used as a model for the human adrenal cortex.
  • the cells were first isolated in 1980 (Gazdar, AF et al., Cancer Res. 50: 5488-5496 (1990)) and contain 5 steroidogenic CYP450 enzymes, including 17-alpha-hydroxylase, CYPIIIBl and CYP11B2. Because all steroidogenic CYP enzymes found in the adrenal cortex are expressed in this cell line, it is an important tool in estimating the selectivity of inhibitors in vitro.
  • NCI-H295R is human cells, but also that in V79MZhllBl or V79MZhllB2 only one target enzyme is recombinantly expressed in an otherwise completely CYP-enzyme-free system, while NCI-H295R represents a much more complex model.
  • NCI-H295R represents a much more complex model.
  • Compound 50 was tested in V79 cells for inhibition of CYPIII and CYPl 1B2.
  • Compound 50 inhibits human aldosterone synthase in the deep nanomolar range and additionally shows only a very weak inhibition of the human CYPIIB.
  • the substance is not only highly potent but also very selective.
  • the substances of formula (I) which are suitable for use in accordance with embodiment (5) can be used to develop a drug which can improve the quality of life of patients with cardiac insufficiency or myocardial fibrosis and decisively reduce mortality.
  • the substances of formula (I) suitable for use in accordance with embodiment (5) may further serve for the development of a drug which can improve the quality of life of patients with hypercortisolism or diabetes mellitus and decisively reduce mortality.
  • the results of the present invention clearly show that it is possible for the
  • Target enzyme CYPIIBI to develop inhibitors that are highly active, but which have little effect on CYPl 1B2, which has a high structural and functional homology to CYPIIBl.
  • the compounds of the invention are as individual compounds and in combination with other active ingredients and excipients z.
  • active ingredients and excipients z for example, for the inhibition of human and mammalian P450 oxygenases, especially for the inhibition of human or mammalian aldosterone synthase, especially for the inhibition of human aldosterone synthase CYPl 1B2 with low impairment of human CYPIIBl and vice versa for the inhibition of CYPIIBl, while low impaired CYP11B2 is suitable in vitro and in vivo.
  • CYP1B2-selective compounds can be used in the preparation of medicaments for the treatment of heart failure, (myo) cardiac fibrosis, (congestive heart failure), hypertension, and primary hyperaldosteronism in humans and mammals.
  • the CYPIIBl-selective compounds can be used in the preparation of medicaments for the therapy of hypercortisolism and diabetes mellitus.
  • These medicaments or the pharmaceutical compositions according to embodiment (4) of the invention may contain, in addition to the compounds according to the invention, further active ingredients as well as suitable excipients and carriers. Suitable excipients and carriers are determined by the person skilled in the art depending on the field of application and the form of application.
  • the invention further includes a method or use of the compound of the invention for the prevention, slowing down or treatment of any of the following diseases or conditions: diabetes mellitus, hypercortisolism, hypertension, congestive heart failure, renal failure, especially chronic renal failure, restenosis, atherosclerosis, nephropathy , Coronary heart disease, increased formation of collagen, fibrosis, each associated with or not associated with the onset of hypertension, by administration of a pharmaceutical preparation according to the invention.
  • diseases or conditions diabetes mellitus, hypercortisolism, hypertension, congestive heart failure, renal failure, especially chronic renal failure, restenosis, atherosclerosis, nephropathy , Coronary heart disease, increased formation of collagen, fibrosis, each associated with or not associated with the onset of hypertension, by administration of a pharmaceutical preparation according to the invention.
  • this method is useful for preventing, slowing the course or therapy of myocardial fibrosis, congestive heart failure or congestive heart failure and comprises administering to the subject or an active dose of an aldosterone synthase inhibitor or a pharmaceutically acceptable salt thereof according to the invention affected mammal.
  • this method is useful for preventing, slowing the course or therapy of stress-dependent refractory diabetes mellitus or hypercortisolism and comprises administering an effective dose of a steroid hydroxylase inhibitor of the invention, particularly steroid IL ⁇ hydroxylase inhibitor, or a pharmaceutical acceptable salt thereof to the affected human or the affected mammal.
  • a steroid hydroxylase inhibitor of the invention particularly steroid IL ⁇ hydroxylase inhibitor, or a pharmaceutical acceptable salt thereof to the affected human or the affected mammal.
  • Reaction conditions (a) NaBH 4 , MeOH / CH 2 Cl 2 , 15 min at 0 ° C., 1 h at RT; (b) PPh 3 «HBr, benzene, 12 h reflux; (c) heterocyclic carbonyl compound, K 2 CO 3 and 18-crown-6 in CH 2 Cl 2 , 12 h reflux.
  • 3- (2-fluorophenyl) propanoic acid (Houghton, RP et al., J.Chem.Soc.Perkin.Trans. 1: 925-931 (1984)) was synthesized as a starting material in two steps : Knoevenagel reaction of malonic acid with 2-fluorobenzaldehyde (Rabjohn, M., Org. Synth. Collective 327-329 (1963)) followed by catalytic reduction of the produced 3- (2-fluorophenyl) acrylic acid (24Jy) (Luo, J. Chem K et al., J.
  • Reaction conditions (a) pyridine, piperidine, 1) for 15 min at 6O 0 C, 2) 45 minutes at 85 0 C, 3) for 3 h at HO 0 C; (b) PtO 2 H 2 O, MeOH, 12h at RT; (c) oxalyl chloride, DMF, 12h RT; (d) AICI 3 , O 0 C, 3.5h reflux, then 12h at RT.
  • 1,3-Thiazole-5-carbaldehyde (27i) was prepared in two steps (Dondoni, A. et al., Synthesis 11: 998-1001 (1987)):
  • the alcohol thus obtained was then converted directly into the phosphonium salt.
  • 40 mmol of the alcohol and 13.7 g of triphenylphosphonium bromide (40 mmol) (Hercouet, A. & Le Corre, M., Synth., Comm., 157-158 (1988)) were suspended in 50 ml of benzene and refluxed for 12 hours under a nitrogen atmosphere , The precipitate was filtered off and dried. The solid was suspended in dry diethyl ether and stirred for 10 minutes. The phosphonium salt was filtered off and washed with diethyl ether.
  • the free base was either dissolved in acetone and an excess of oxalic acid in acetone to afford the oxalate or it was dissolved in dry diethyl ether and an excess of HCl in diethyl ether added to the hydrochloride receive.
  • IR c ⁇ V 1 v max 3022, 2934, 2841, 2427, 1609, 1548, 1455, 1016, 902, 815, 760, 749.
  • IR c ⁇ V 1 v max 3056, 3019, 2953, 2278, 1621, 1569, 1460, 1351, 860, 818, 789, 756.
  • Anal. (Ci 6 H 15 N-HCl) C; H; N. 3-rfz ') -3,4-Dihvdronaphthalin-lf2H') -ylidenennethyllPyridin-hvdrochlorid (3BO cleaning.
  • FCC EtOAc: hexane, 1: 1). Yield 18%, white solid, mp 206 0 C..
  • IR cnrT 1 v max 3046, 2936, 1524, 1458, 813, 757.
  • 1 H NMR 400 MHz, DMSO-d 6 ) ⁇ 1.63-1.69 (m, 2H, H-3).
  • IR cnrT 1 v max 3017, 2399, 1637, 1591, 1484, 1240, 936, 859.
  • Anal. (C i 5 Hi 2 NF -HCl-0.5 H 2 O) C; N; H: lime 5.21, found 4.59.3-r (Z) - (5-fluoro-2- t 3-dihydro-1H-indan-1 ylidene) methyllpyridine hydrochloride (5b1)
  • IR cm '1 v max 3050, 3014, 2395, 1552, 1244, 1224, 933, 858, 813, 705.
  • IR OTT 1 v ma ⁇ 2361, 1583, 1511, 1247, 1209, 833, 805.
  • Anal. (C i H 5 H 12 NF-HCl-0.5 H 2 O) C H, N.
  • IR cnrT 1 v max 3003, 2955, 2630, 1619, 1590, 1499, 1199, 1189, 1072, 875, 815, 790, 750.
  • Anal. (Ci 5 H 12 NCI-HCl-0.3 H 2 O) C; H; N. 4-r (E) - (5-Chloro-2- t 3-dihydro-1H-indan-1-ylidenes) methyl-1-pyridine hydrochloride (8a1) Purification: FCC (EtOAc: hexane, 1: 1) Yield 29%. yellow solid, mp 213 ° C.
  • IR cnrT 1 v max 2844, 2361, 1632, 1602, 1545, 1489, 1309, 1256, 1227, 1109, 1021, 832, 797.
  • Anal. (Ci 6 Hi 5 ON-HCl-H 2 O) C; N; H: lime. 6.22, found 5.34.
  • IR cnrT 1 v max 2386, 1602, 1586, 1543, 1502, 1236, 1124, 1035, 899, 848, 833, 801.
  • Anal. (Ci 7 H 17 ON-HCI) C; H; N. 3-r (Z) - (6-methoxy-3,4-dihydronaphthalene-1 (2H) -ylidene) -ethylpyridine-hydrochloride (13b ' ).
  • IR cnrT 1 v max 3044, 1943, 2843, 2429, 1626, 1504, 1258, 1234, 1189, 1178, 1032, 881, 853, 831, 809.
  • Anal. (Ci 7 Hi 7 ON HCl 0.3 H 2 O) C; H; N.
  • IR c ⁇ V 1 v max 3041, 2935, 2823, 1631, 1595, 1565, 1494, 1253, 1139, 820, 797.
  • Anal. (Ci 7 H 17 ON-HCl-0.6 H 2 O) C; H; N. 3-r (E) - (6-Methoxy-2- t 3-dihydro-1H-indan-1-ylidenes) -ethylpyridine hydrochloride (15a 1 ).
  • IR cnrT 1 Vm a x 3051, 3001, 2736, 1604, 1508, 1497, 1222, 1200, 1020, 893, 806.
  • Anal. (CI 6 H 15 ON HCl 0.8 H 2 O) C; H; N. 4-r (Z) - (6-methoxy-2- t 3-dihydro-1H-indan-1-ylidenes) -ethylpyridine hydrochloride (161.0) Purification: FCC (EtOAc: hexane, 1: 1). Yield 13%. yellow solid, mp 207 0 C.
  • 1 H NMR 500 MHz, DMSO-d 6) ⁇ 3.03 to 3.05 (m, 2H, H-2),.
  • IR cnrT 1 v ma ⁇ 2931, 2835, 2419, 1714, 1603, 1586, 1550, 1514, 1466, 1454, 1254, 1215, 1139, 1028, 1016, 871, 855, 833, 800.
  • IR cnrT 1 v max 3027, 2930, 2360, 1627, 1597, 1571, 1503, 1358, 1257, 1218, 1192, 1141, 1025, 872, 844, 786.
  • Anal. (Ci 8 H 19 O 2 N HCI 1, H 2 O) C; H; N.
  • IR cm '1 v max 3032, 2984, 2921, 2880, 2595, 1632, 1590, 1552, 1475, 1247, 1092, 1045, 825, 806.
  • Anal. (Ci 7 H 17 ON HCl 0.2 H 2 O) C; H; N. 3 - f (E) -r5- (benzyloxy) -2,3-dihydro-1H-indan-1-ylidenemethylpyridine hydrochloride (20Al, Prepared from (20J)) Purification: FCC (EtOAc: hexane, 1.:. 3) 13% yield, yellow solid, mp 209 0 C.
  • IR cm- 1 v max 3013, 2408, 1635, 1552, 938, 885, 825, 784.
  • Anal. (Ci 6 H 15 N-HCl-0.3 H 2 O) H; N; C: lime. 74.56, found 75.52.
  • IR OTT 1 v max 2917, 2460, 1596, 1510, 1204, 880, 813.
  • IR cnrT 1 v ma 3003, 2839, 2363, 2083, 1605, 1584, 1481, 1468. 1455, 1303, 1067, 894, 794.
  • Anal. (Ci 6 H 15 ON-HCI-1, 2H 2 O) C; H; N.
  • IR cnrT 1 v max 3087, 2924, 2377, 1635, 1548, 1201, 1179, 1073, 919, 864, 825, 801.
  • Anal. C14 H13 ONS-HCl) C; H; N. 5-rrzi-r5-methoxy-2,3-dihydro-lH-indan-l-ylidene ') methyl-l, 3-thiazol-hvdrochloride (. 27b1 Prepared from (Z7J) cleaning.
  • FCC EtOAc: hexane Yield 9%, white solid, mp 211 ° C.
  • IR cnrT 1 v max 2940, 2361, 1598, 1549, 1492, 1318, 1298, 1253, 1110, 1032, 822, 798, 782, 770.
  • Anal. (Ci 4 Hi 3 ONS-HCl-0.4 H 2 O) C; H; N.
  • IR cnrT 1 v max 2930, 2852, 1563, 1340, 1250, 979, 813.
  • IR cnrT 1 v max 2929, 2360, 2341, 1574, 1244, 836, 759.
  • IR cnrT 1 v max 3042, 3007, 2955, 2423, 1540, 1442, 809, 767, 722.
  • Anal. (Ci 9 Hi 3 N-HCl) C; H; N. 4- (9H-Fluoren-9-ylidenemethyl) pyridine hydrochloride (34).
  • IR OTT 1 v m a ⁇ 3050, 3004, 2950, 1627, 1585, 1498, 1481, 807, 775, 727. Anal.
  • IR cm- 1 v max 3029, 2961, 2925, 2360, 1730, 1547, 1475, 1250, 1084, 1019, 933, 859, 825, 816.
  • Anal. (Ci 6 H 15 N-HCl-0.6 H 2 O) C; H; N.
  • Reaction conditions (a) AICI 3 , benzene, 3h reflux; (b) triflic anhydride, dry pyridine, 15 min at 0-5 0 C, then for 2 h at RT; (c) Zn, PPh3, KCN, Ni (PPh 3) 2 CI 2, MeCN, 2 h at 6O 0 C.
  • Reaction conditions (a) AlCl 3, 40-50 0 C; (b) HgCl 2 ZZn, H 2 O, toluene, 24h reflux, addition of HCl every 6h; (c) PPA, 40 min at 7O 0 C.
  • the resulting alcohol was converted to the phosphonium salt.
  • 40 mmol of the alcohol and 13.7 g of triphenylphosphonium bromide (40 mmol) were suspended in 25 ml of benzene and refluxed under nitrogen for 12 h.
  • the precipitate was filtered off and dried, then taken up in dry diethyl ether and stirred for 10 min.
  • the phosphonium salt was finally filtered off and washed with acetone.
  • a sodium ethoxide solution was prepared.
  • 2.1 g of imidazole-4 (5) -carbaldehyde (22 mmol) was added.
  • the free base was either dissolved in acetone and an excess of oxalic acid in acetone to afford the oxalate or it was dissolved in dry diethyl ether and an excess of HCl in diethyl ether added to the hydrochloride receive.
  • cnrT 1 v ma x 3055, 3020, 2960, 2920, 2840, 1643, 1601, 1460, 985, 757.
  • Anal. (C 13 H 12 N 2 -C 2 H 2 O 4 ) C; H; N. 5-rfZ1-2,3-dihydro-1H-ind-1-ylidene-methyl-1H-imidazolium oxalate (42r /).
  • n 1, 2 Reaction conditions: (a) NaBH 4 , MeOH / CH 2 Cl 2 , 15 min at 0 ° C., 1 h at RT; (b) PPh 3 «HBr, benzene, 12 h reflux; (c) EtONa, 4 (5) -imidazole carboxaldehyde, N 2 , 12 h reflux; (d) Isomer separation by flash column chromatography.
  • reaction mixture was then poured into water and extracted several times with dichloromethane. The combined organic phases were dried over MgSO 4 and the solvent removed in vacuo. After purification, 2.5 mmol of the sulfonamide (42ia / 42ib, 48ia / 48ib) was taken up in a few ml of dioxane and 75 ml of 4N HCl added. The mixture was refluxed overnight with stirring. Upon cooling to room temperature, the hydrochloride precipitated and could be filtered off and washed with dry diethyl ether (quantitative yield based on the sulfonic acid amide).
  • IR (Powder) cnrT 1 v max 3124, 2923, 2361, 1465, 1386, 1174, 1080, 962, 724.
  • IR cnrT 1 v max 3381, 3165, 3082, 2989, 2822, 2362, 2686, 2651, 1519, 1267, 1142, 841, 815, 748.
  • Anal. (Ci 3 Hi 2 N 2 -HCI-0.5 H 2 O) C, H, N.
  • IR OTT 1 v ma ⁇ 2971, 2901, 1612, 1578, 1550, 1449, 1329, 1261, 1065, 1048, 805.
  • Example 5 Enzyme assay systems for testing compounds for inhibition of CYP enzymes in vitro
  • CYP17 expressed recombinantly in E. coli
  • Human placental CYP19 Human placental CYP19
  • bovine adrenal CYPIII Hartmann, RW et al., J. Med. Chem. 38: 2103-2111 (1995)
  • E. coli strain pJL17 / 0R in which the human CYP17 and the rats NADPH-P450 reductase were coexpressed, was prepared according to the method of Ehmer et al. and stored (Ehmer, PB et al., J. Steroid Biochem. Mol. Biol. 75: 57-63 (2000)).
  • phosphate buffer 0.05 M, pH 7.4, 1 mM MgCl 2 , 0.1 mM EDTA and 0.1 mM DTT.
  • the bacteria were spun down and resuspended in 10 ml of ice-cold TES buffer (0.1 M Tris-acetate, pH 7.8, 0.5 mM EDTA, 0.5 M sucrose). 4 mg lysozyme in 10 ml ice-cold water was added to give a final concentration of 0.2 mg / ml. This was followed by a thirty minute incubation with continuous shaking on ice. The spheroplasts were recovered by a new centrifugation step at 12,000 g for 10 min and resuspended in 3 ml of ice-cold phosphate buffer (composition thus, plus 0.5 mM PMSF).
  • the cells were disrupted on ice with an ultrasonic wand.
  • the whole cells and cell debris were spun down at 3,000 g for 7 min.
  • the supernatant was again added at 50,000 g for 20 min 4 0 C centrifuged.
  • the membrane pellet which was resuspended in 2 ml of phosphate buffer (composition see above) with 20% glycerol with the aid of an Ultra-Turrax® stick, was deposited.
  • the protein concentration was determined by the method of Lowry et al. determined (Lowry, OH et al., J. Biol. Chem. 193: 265-275 (1951)). Aliquots having an approximate protein concentration of 5 mg / ml were stored until use at -70 0 C.
  • Placenta (St. Joseph's Hospital, Saarmaschinen-Dudweiler, Germany) according to the method of Thompson and Siiteri (Thompson, E.A. & Siiteri, P.K., J. Biol. Chem. 249: 5364-5372 (1974)).
  • the isolated microsomes were suspended in a minimal volume of phosphate buffer (0.05 M, pH 7.4, 20% glycerol).
  • DTT (10 mM) and EDTA (1 mM) were added to protect the enzyme from degradation reactions.
  • the protein concentration was determined according to Lowry et al. determined (Lowry, O.H. et al., J. Biol. Chem. 193: 265-275 (1951)) and should be about 35 mg / ml after work-up.
  • Sucrose buffer (0.25 M sucrose, 0.05 M Tris, pH 7.4). After removing the attached fatty tissue, the adrenal medulla was carefully separated from the adrenal cortex with a pair of scissors. The pieces of adrenal cortices were roughly minced with scissors, washed with Tris-sucrose buffer, weighed and finely minced in the above buffer (2 ml per g of tissue) with a hand blender. Thereafter, the tissue was homogenized with an Ultraturrax rod. To separate coarse cell debris and cell nuclei, the homogenate was centrifuged twice at 900g and 4 ° C for 15 min. The supernatant was then centrifuged for 35 min at 11000 g to recover the mitochondria fraction.
  • the precipitate was resuspended in Tris-sucrose buffer and centrifuged again at 11000 g for 35 min. This washing step was carried out a total of twice. After the last centrifugation, the pellet was resuspended in Tris-sucrose buffer containing 0.001M EDTA was contained, resuspended and frozen at -70 0 C.
  • the mitochondrial suspension Prior to use of the enzyme in the CYPIIB inhibition assay, the mitochondrial suspension was adjusted to a protein concentration of 5 mg / ml with 18- Hydroxylase buffer diluted (0.05M Tris, 1.2mM MgCl 2 , 6.0mM KCl, 140mM NaCl, 2.5mM CaCl 2 ) (Ayub, M. & Levell, MJ, J. Steroid Biochem. 32: 515-524 (1989)). Protein determination was carried out according to Lowry (Lowry, OH et al., J. Biol. Chem. 193: 265-275 (1951)). D) Determination of the percent inhibition of CYP17
  • a solution of 6.25 nmol of progesterone (in 5 ⁇ l of MeOH) was dissolved in 140 ⁇ l of phosphate buffer (0.05 M, pH 7.4, 1 mM MgCl 2 , 0.1 mM EDTA and 0.1 mM DTT) and combined with 50 ul NADPH regenerating system (phosphate buffer with 10 mM IMADP ® , 100 mM glucose-6-phosphate and 2.5 units of glucose-6-phosphate dehydrogenase) and inhibitor (in 5 ul DMSO) at 37 0 C for 5 min preincubated. Control incubations were performed in parallel with 5 ⁇ l of DMSO without inhibitor.
  • the reaction was started by adding 50 ⁇ l of a 1 to 5 diluted membrane suspension in phosphate buffer (0.8-1 mg protein per ml). After mixing the batch was incubated at 37 0 C for 30 min. The reaction was stopped by addition of 50 ⁇ l IN HCl.
  • the steroids were extracted with 1 ml EtOAc. After a centrifugation step (5 min at 2500 g), 900 ⁇ l of the organic phase were transferred to an Eppendorf vessel with 250 ⁇ l of the incubation buffer and 50 ⁇ l of 1 N HCl and shaken again. After centrifugation, 800 ⁇ l of the organic phase was taken, placed in a new vessel and evaporated to dryness. The samples were dissolved in 50 ⁇ l of a water-methanol mixture (1: 1) and analyzed by HPLC. The substrate turnover was calculated from the ratio of the areas of the product peaks (17 ⁇ -hydroxyprogesterone and 16 ⁇ -hydroxyprogesterone) to that of the substrate peak. The activity of the inhibitors was calculated from the reduced substrate conversion after addition of inhibitors according to the following formula:
  • the assay was performed approximately analogously to that of Foster et al. A detailed description can be found in Hartmann and Batzl 1986 (Foster, AB et al., J Med Chem 26: 50-54 (1983); Graves, PE & Salhanick, HA, Endocrinology 105: 52 - 57 (1979); Hartmann, RW & Batzl, C, J. Med. Chem. 29: 1362-1369 (1986)). The enzyme activity was monitored by measuring the 3 H 2 O formed during the aromatization from [l ⁇ - 3 H] androstenedione.
  • Each reaction vessel contained 15 nM radiolabeled [LSS 3 H] androstenedione (equivalent to 0.08 uCi) and 485 nM unlabelled androstenedione, 2 mM IMADP ®, 20 mM glucose-6-phosphate, 0.4 units glucose-6-phosphate dehydrogenase and inhibitor (0-100 ⁇ M) in phosphate buffer (0.05 M, pH 7.4).
  • the compounds to be tested were dissolved in DMSO and diluted with buffer to the desired concentration. The final DMSO concentration of the control and inhibitor incubation was about 2%.
  • Each jar was preincubated for 5 min in a water bath at 30 0 C. Addition of the microsomal protein (0.1 mg) started the reaction.
  • the total volume of each batch was 200 ⁇ l.
  • 200 .mu.l ice-cold 1 mM HgCl 2 solution the reaction was stopped after 14 min.
  • 200 ⁇ l of a 2% aqueous suspension of dextran-coated charcoal (DCC) was added to absorb the steroids and the vessels were shaken for 20 minutes. Thereafter, the activated carbon was centrifuged off at 1,500 g for 5 min.
  • the radioactive water ( 3 H 2 O) in the supernatant was determined by scintillation measurement by means of a LKB-Wallac ⁇ -counter.
  • the IC 50 values were calculated by a semilogarithmic plot of percent inhibitor inhibition inhibition. From this, the molar concentration at which 50% inhibition occurs was read.
  • Corticosterone 200 ⁇ M was mixed with inhibitor (1 ⁇ M) and mitochondrial enzyme (0.5 mg / 0.5 ml) with the addition of a regenerating system consisting of NADP + (ImM), glucose-6-phosphate (7 mM) and glucose-6-phosphate dehydrogenase (1 IU / 0.5 ml).
  • a regenerating system consisting of NADP + (ImM), glucose-6-phosphate (7 mM) and glucose-6-phosphate dehydrogenase (1 IU / 0.5 ml).
  • ImM NADP +
  • glucose-6-phosphate 7 mM
  • glucose-6-phosphate dehydrogenase 1 IU / 0.5 ml
  • the nulls were spiked with 250 ⁇ L of HCl prior to pipetting the enzyme.
  • the steroids were separated by shaking with 1 ml of ethyl acetate (10 min). After centrifugation (15,000 g, 10 min), 900 ⁇ l of the organic phase were shaken with 250 ⁇ l of 1 N NaOH (10 min) and 800 ⁇ l of supernatant were again washed with 250 ⁇ l of buffer.
  • the steroids were taken up in 20 ⁇ l of distilled methanol and 10 ⁇ l of the methanolic solution were separated by HPLC (stationary phase: Nucleosil 120-5 C18 column with a 1 cm long 7 ⁇ m precolumn; 50% methanol in water, flow: 1.1 ml / min, detection: UV detector).
  • HPLC stationary phase: Nucleosil 120-5 C18 column with a 1 cm long 7 ⁇ m precolumn; 50% methanol in water, flow: 1.1 ml / min, detection: UV detector.
  • 18-OH corticosterone (retention time: 10 minutes) and corticosterone (retention time: 21 minutes) were separated.
  • the height of the 18-OH corticosterone peak was used for the evaluation.
  • the percent inhibition of 18-hydroxylation of corticosterone by the inhibitors was based on the zero values on the mean values of the control incubations.
  • Each inhibitor was tested at least twice for its 18-hydroxylase inhibitory activity at a concentration of 1 ⁇ M.
  • the determination of the amounts of 18-OH corticosterone formed in the examination of the incubation time and the substrate saturation was carried out by means of a calibration line.
  • Example 6 Biological Assay Systems for Testing Compounds for Selective Inhibition of Human CYPIIB1 and CYP11B2 in vitro
  • a split yeast suspension (S.pombe PEI) with a cell density of 3-10 7 cells / ml was prepared from a freshly grown cuticle using fresh EMMG (pH 7.4), modified according to Ehmer et al. (Ehmer, PB et al., J. Steroid, Biochem., Mol. Biol. 81, 173-179 (2002)). 492.5 ⁇ l of this cell suspension were mixed with 5 ⁇ l of inhibitor solution (50 ⁇ M of the compound to be tested in ethanol or DMSO) and incubated at 32 ° C. for 15 min. Controls were added with 5 ⁇ l of ethanol.
  • the enzyme reaction was started by adding 2.5 ⁇ l of 11-deoxycorticosterone (20 ⁇ M, containing 1.25 nCi [4- 14 C] II deoxycorticosterone, in ethanol), then shaking horizontally at 32 ° C for 6 h.
  • the assay was stopped by extraction of the sample with 500 ⁇ l EtOAc. After centrifugation (10,000 g, 2 min), the EtOAc phase was removed and evaporated to dryness. The residue was taken up in 10 ⁇ l of chloroform. The conversion of the substrate to corticosterone was analyzed by HPTLC (see below).
  • Equation 2 The percentage inhibition caused by an inhibitor at the concentration used was calculated according to Equation 2. Equation 2:
  • V79 MZhIlBl and V79 MZhllB2 which recombinantly express the human aldosterone synthase or steroid 11-.beta.-hydroxylase and according to Denner et al. were prepared (Denner, K. et al, Pharmacogenetics 5:. 89-96 (1995)) was treated in a CO 2 incubator at 37 0 C and in water vapor saturated atmosphere with 5% CO 2 in cell culture dishes with 60 or 90 mm diameter cultured. Both cell lines were cultured in DMEM + containing 10% FCS and the antibiotics penicillin and streptomycin (1%) to protect against bacterial contamination.
  • the cells were passaged every 2-3 days after treatment with trypsin / EDTA, since the doubling density was 1 to 2 days, depending on the number of cells.
  • the cells were passaged a maximum of 12 - 15 times to exclude possible cell changes. If needed further, freshly thawed cells were used.
  • FCS Fetal Calf Serum
  • the pH of the medium was adjusted to 7 ', 2-7', 3.
  • FCS was added after sterile filtration.
  • V79 MZh HBl and V79 MZh 11B2 cells (8-10 5 cells per well) were grown on 24-well cell culture plates with 1.9 cm 2 culture area per well (Nunc. Roskilde, Denmark) until confluency. Prior to testing, the existing DMEM culture medium was removed and 450 ⁇ l of fresh DMEM with inhibitor added in at least three different concentrations to each well to determine the IC 50 value. After preincubation (60 min, 37 ° C), the reaction was terminated by addition of 50 ⁇ l DMEM with 2.5 ⁇ l solution of the substrate 11-deoxycorticosterone (20 ⁇ M, containing 1.25 nCi [4- 14 C] II-deoxycorticosterone) Ethanol) started.
  • V79 MZh HBl cells were incubated for 120 min, the V79 MZh 11B2 cells for 40 min. Controls without inhibitor were treated in the same way.
  • the enzyme reactions were stopped by extraction of the supernatant with 500 ⁇ l EtOAc.
  • the samples were centrifuged (10000 g, 2 min), the solvent was removed and evaporated. The residue was taken up in 10 ⁇ l of chloroform and analyzed by HPTLC (see below).
  • the conversion for V79 MZhIlBl was calculated in accordance with Equation 1 (Ex. 5A), where:
  • PSL B PSL for cortisol and corticosterone
  • Equation 3 Equation 3:
  • PSLD OC PSL for background 11-deoxycorticosterone (DOC) PSL HG PSL The percentage inhibition caused by an inhibitor in the particular concentration used was calculated according to Equation 2 (Example 5A).
  • the IC 50 value is defined as the concentration of the inhibitor at which the enzyme is inhibited to 50%. It was calculated by determining the percent inhibition at at least 3 different inhibitor concentrations, all of which must be within the linear range of the sigmoid IC 50 curve (log C /% inhibition).
  • imaging plates (BAS MS2340, for 14 C samples, Raytest, Straubenhardt, Germany) were exposed for 48 h to the HPTLC plates.
  • the imaging plates were scanned with the phosphoimager system Fuji FLA 3000 (Raytest, Straubenhardt, Germany) and the steroids quantified.
  • Example 7 Inhibition of adrenal CYPIIB enzymes in vitro by rdihydronaphthalene or dihydroindan-1 (2H) -ylidenemethyl-3-pyridines [dihydronaphthalene or dihydroindan-1 (2H) -ylidenemethyl] -3-pyridines were prepared as in Examples 5 and 6 described as inhibitors tested. The results of the tests are summarized in Tab. Tab. 1: [Dihydronaphthalene or dihydroindan-1 (2H) -ylidenemethyl] -3-pyridines; Inhibition of adrenal CYPIIB enzymes, CYP17 and CYP19 in vitro
  • Fadrozole 68 9.7 1.0 7 0.0295 a Mean of 4 determinations, standard deviation ⁇ 10%.
  • S. pomöe cells expressing human CYP11B2; Substrate deoxycorticosterone, 100 nM; Inhibitor, 500 nM.
  • c mean of 4 determinations, standard deviation ⁇ 20%.
  • e Haster fibroblasts expressing human CYP11B2; Substrate deoxycorticosterone, 100 0 nM. f E.
  • Example 8 Inhibition of adrenal CYPIIB enzymes in vitro by rdihydronaphthalene or dihydroindan-1 (2H) -ylidene-methyl-4-pyridines [dihydronaphthalene or dihydroindan-1 (2H) -ylidenemethyl] -4-pyridines were prepared as in Examples 5 and 6 described as inhibitors tested. The results of the tests are summarized in Tab.
  • Fadrozole 68 9.7 1.0 7 0.0295 a Mean of 4 determinations, standard deviation ⁇ 10%.
  • S. pomöe cells expressing human CYP11B2; Substrate deoxycorticosterone, 100 nM; Inhibitor, 500 nM.
  • c mean of 4 determinations, standard deviation ⁇ 20%.
  • Haster fibroblasts expressing human CYPIIBl; Substrate deoxycorticosterone, 100 nM.
  • Hector fibroblasts expressing human CYP11B2; Substrate deoxycorticosterone, 100 nM. f E. coli expressing the human CYP17; 5 mg / ml protein; Substrate progesterone, 2.5 ⁇ M; Inhibitor, 2.5 ⁇ M. 9 mean of 4 determinations, standard deviation ⁇ 5%; h human placental CYP19, 1 mg / ml protein; Substrate testosterone, 2.5 ⁇ M; (nd not determined)
  • Example 9 Inhibition of adrenal CYPIIB enzymes, CYP 17 and CYP 19 in vitro by further dihydroindan-1 (2H) -ylidene-methyll-heterocycles
  • Fadrozole 68 9.7 1.0 7 0.0295 a Mean of 4 determinations, standard deviation ⁇ 10%.
  • S. pomöe cells expressing human CYP11B2; Substrate deoxycorticosterone, 100 nM; Inhibitor, 500 nM.
  • c mean of 4 determinations, standard deviation ⁇ 20%.
  • e Haster fibroblasts expressing human CYP11B2; Substrate deoxycorticosterone, 100 nM. f E.
  • Example 10 Inhibition of adrenal CYPIIB enzymes and CYP17 and CYP19 in vitro by rDihydronaphthalene or dihydroindan-1 (2H) -ylidenemethyl-4-imidazoles
  • f Haster fibroblasts expressing human CYP11B2; Substrate deoxycorticosterone, 100 nM. (nd not determined)
  • Example 11 Inhibition of CYP enzymes in vitro by the reference compounds ketoconazole and fadrozole
  • Ketocoanzol or fadrozole were tested as inhibitors as described in Examples 5 and 6. The results of the tests are summarized in Tab. 6.
  • a percent inhibition of CYPIIBl in NCI-H295R, inhibitor concentration 2.5 ⁇ M (for IC 50 - determination at least 3 different concentrations); Preincubation 1 h, substrate: [ 3 H] -deoxycortisol (RSS, 500 nM); Incubation time: 48 h; Extraction with dichloromethane; Determination of cortisol after HPLC separation (methanol-water 50:50, RP18) b: percent inhibition of CYP11B2 in NCI-H295R, inhibitor concentration 2.5 ⁇ M (for IC 50 - determination at least 3 different concentrations); Stimulation with K + -containing saline [20 mM K + ] preincubation 1 h, substrate: [ 3 H] -corticosterone (B, 500 nM); Incubation time: 24 h; Extraction with dichloromethane; Determination of [ 3 H] -18-hydroxycorticosterone and [ 3 H] aldosterone after
  • To prepare a mixture of labeled and unlabeled substance 38 ⁇ l of unlabelled deoxycortisol (0.5 mM in ethanol) and 41.6 ⁇ l of [1,2- 3 H (N)] deoxycortisol (1 mCi / ml, 52 Ci / mmol NEN-Perkin-Elmer) in ethanol with 120.4 ⁇ l of ethanol.
  • the corticosterone substrate solution (final concentration in the assay 500 nM) consisted of 38.4 [1.2- 3 H (N)] -corticosterone (1 mCi / ml, 76.5 Ci / mmol NEN-Perkin-Elmer) in ethanol, 39.0 ⁇ l of unlabeled corticosterone solution (0.5 mM in ethanol) and 122.6 ⁇ l of ethanol.
  • Deoxycorticosterone which was also used at a final concentration of 500 nM, was composed of 18 ⁇ l of [ 14 C] -labeled deoxycorticosterone (60.0 mCi / mmol, 0.5 nCi / ⁇ l) in ethanol in admixture with 54 ⁇ l unlabeled substance (0.5 mM in ethanol) and 228 ⁇ l of ethanol.
  • the 24-well plate was then kept for 2 incubator at 37 0 C and 5% CO 2 in the CO.
  • the incubation period was 3 hours using deoxycorticosterone as substrate, 24 hours for corticosterone and 48 hours for deoxycortisol.
  • Test stop After the incubation times, the contents of the wells were removed as quantitatively as possible after a brief swirl and inactivated by mixing with 1000 ⁇ l of dichloromethane in a 2 ml Eppendorf tube. After shaking for 10 minutes, centrifugation was carried out for phase separation, and the upper organic phase was transferred to a 1.5 ml Eppendorf tube.
  • the residue was taken up in 10 ⁇ l of chloroform and applied in the middle of the concentration zone of an HPTLC plate.
  • the steroids were separated by double development with a flow agent composed of chloroform, methanol and water in a ratio of 300: 20: 1.
  • the separation was carried out by HPLC on a RP18 column with the eluent methanol: water 1: 1 and a flow rate of 0.25 ml / min, the detection was carried out using a Berthold Radiomonitor 509.
  • the exposed film was scanned in the Phosphoimager FLA 3000 after two days.
  • Equation 4 the conversion for the substrate deoxycortisol was calculated after HPLC separation: Equation 4: g / p _ ⁇ Jortisone ⁇ Jortison y, ir ⁇ ajortison + Ajortisol + ⁇ RSSl
  • Equation 5 For the substrate corticosterone, Equation 5 was: Equation 5:
  • Example 5A The percentage inhibition caused by an inhibitor in the particular concentration used was calculated according to Equation 2 (Example 5A). The determination of the IC 50 value was carried out as described in Example 5B.
  • Compound 50 was tested in V79 cells for inhibition of CYPIII and CYPl 1B2.
  • Compound 50 inhibits human aldosterone synthase in the deep nanomolar range and additionally shows only a very weak inhibition of the human CYPIIB.
  • the substance is not only highly potent but also very selective.
  • substances of the class of substituted in the 3-position 1,2-Dihydroacenaphthylenes are new lead structures that can lead to even more potent and simultaneously highly selective CYP11B2 inhibitors.
  • Deoxycorticosterone 100 nM.
  • c hamster fibroblasts expressing human CYP11B2 Substrate deoxycorticosterone, 100 nM.

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Abstract

L'invention concerne des composés utilisés pour l'inhibition sélective des corticoïde-synthases humaines CYP11B1 et CYP11B2, leur production et leur utilisation dans le traitement de l'hypercortisolisme, du diabète sucré ou de l'insuffisance cardiaque et de la fibrose myocardique.
EP05769814A 2004-07-21 2005-07-21 Inhibiteurs selectifs des corticoide-synthases humaines Withdrawn EP1768654A2 (fr)

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DE102004035322A1 (de) 2006-02-16
JP2008506753A (ja) 2008-03-06
WO2006008316A3 (fr) 2006-07-27
WO2006008316A2 (fr) 2006-01-26
US20090105278A1 (en) 2009-04-23

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