EP2585055A1 - Use of stimulators and activators of soluble guanylate cyclase for treating sickle-cell anemia and conserving blood substitutes - Google Patents

Use of stimulators and activators of soluble guanylate cyclase for treating sickle-cell anemia and conserving blood substitutes

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Publication number
EP2585055A1
EP2585055A1 EP11726444.0A EP11726444A EP2585055A1 EP 2585055 A1 EP2585055 A1 EP 2585055A1 EP 11726444 A EP11726444 A EP 11726444A EP 2585055 A1 EP2585055 A1 EP 2585055A1
Authority
EP
European Patent Office
Prior art keywords
treatment
compounds
prophylaxis
formulas
cell anemia
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP11726444.0A
Other languages
German (de)
French (fr)
Inventor
Johannes-Peter Stasch
Eva-Maria Becker
Hubert TRÜBEL
Herbert Himmel
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Adverio Pharma GmbH
Original Assignee
Bayer Intellectual Property GmbH
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Filing date
Publication date
Application filed by Bayer Intellectual Property GmbH filed Critical Bayer Intellectual Property GmbH
Priority to EP11726444.0A priority Critical patent/EP2585055A1/en
Priority to EP13179993.4A priority patent/EP2687210A1/en
Publication of EP2585055A1 publication Critical patent/EP2585055A1/en
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid, pantothenic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid, pantothenic acid
    • A61K31/198—Alpha-aminoacids, e.g. alanine, edetic acids [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • 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
    • A61K31/425—Thiazoles
    • A61K31/426—1,3-Thiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • 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
    • A61K31/425—Thiazoles
    • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/53—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with three nitrogens as the only ring hetero atoms, e.g. chlorazanil, melamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics

Definitions

  • the present invention relates to the novel use of soluble guanylate cyclase stimulators and / or activators for the treatment of sickle cell anemia and the preservation of blood substitutes. Furthermore, the present invention relates to the use of soluble guanylate cyclase stimulators and / or activators in combination with PDE5 inhibitors for the treatment of sickle cell anemia and preservation of blood substitutes.
  • PH Pulmonary hypertension
  • Hb free hemoglobin
  • sGC soluble guanylate cyclase
  • transgenic mice exclusive expression of human sickle cell hemoglobin
  • PH various disease symptoms
  • sGC activators and stimulators for the treatment of sickle cell anemia and the associated PH as well as other pathologies (e.g., end-organ damage affecting the brain, kidney or heart).
  • an sGC activator and stimulator could in the future, the previously observed side effects [Weiskopf, Anesthesia & Analgesia, 110: 3; 659-661, 2010], which are due to reduced NO availability, mitigate and thus make a clinical application possible.
  • sGC activators and stimulators the soluble guanylate cyclase is directly stimulated, so that the missing NO effect is replaced.
  • sGC stimulators are not affected by free Hb.
  • sGC stimulators and sGC activators under acute and especially chronic conditions should show positive effects on blood pressure, oxygen saturation and the resulting plasma and tissue cGMP levels under experimental conditions.
  • These experimental conditions consist on the one hand of healthy animals, animals under external free Hb application or genetically modified animals such.
  • Sickle cell mice transgenic mice that exclusively express human sickle cell hemoglobin beta.
  • PDE5 inhibitors such as sildenafil, vardenaf il and Tadalafil repeatedly pointed to an increased occurrence of back pain. That's why it could be of extraordinary interest, o.g. sGC stimulators and activators can already be differentiated at the preclinical level of PDE 5 inhibitors.
  • the object of the present invention is to provide substances for the treatment of safeniaemia and preservation of blood substitutes.
  • the solution to this problem is to find the following sGC stimulators and / or sGC activators alone or in combination and the combination of sGC stimulators and / or sGC activators with PDE5 inhibitors.
  • cGMP cyclic guanosine monophosphate
  • NO nitric oxide
  • GTP guanosine triphosphate
  • the soluble guanylate cyclases consist of two subunits and most likely contain one heme per heterodimer that is part of the regulatory center. This is central to the activation mechanism. NO can bind to the iron atom of the heme and thus significantly increase the activity of the enzyme. On the other hand, heme-free preparations can not be stimulated by NO. Also, carbon monoxide (CO) is able to bind to the central iron atom of the heme, with stimulation by CO being significantly less than by NO.
  • CO carbon monoxide
  • guanylate cyclase plays a crucial role in various physiological processes, in particular in the relaxation and proliferation of smooth muscle cells, platelet aggregation and adhesion, neuronal signaling and diseases based on a disturbance of the above operations.
  • the NO / cGMP system may be suppressed, which may, for example, lead to hypertension, platelet activation, increased cell proliferation, endothelial dysfunction, arteriosclerosis, angina pectoris, heart failure, myocardial infarction, thrombosis, stroke and sexual dysfunction.
  • a NO-independent treatment option for such diseases which is aimed at influencing the cGMP pathway in organisms, is a promising approach on account of the expected high efficiency and low side effects.
  • the present invention is the use of soluble guanylate cyclase stimulators and / or activators for the treatment of sickle cell disease and preservation of blood substitutes.
  • pyrrolo [2,3-d] pyrimidin-6-one (24), 4-amino-5,5-dimethyl-2- [3- (2,4,6-trifluorobenzyl) imidazo [l, 5-a] pyridine -l-yl]] -5,7-dihydro-6H-pyrrolo [2,3-d] pyrimidin-6-one (25), 4-amino-2- [3- (2-cyclopentylethyl) imidazo [l, 5-a] pyridin-1-yl] -5,5-dimethyl-5,7-dihydro-6H-pyrrolo [2,3-d] pyrimidin-6-one (26) disclosed in WO 2010/065275. -6-
  • Another aspect of the invention is the combination of soluble guanylate cyclase stimulators and / or activators with PDE5 inhibitors for use in the treatment of sickle cell anemia and preservation of blood substitutes.
  • PDE5 inhibitors are suitable for combination for use in the treatment of sickle cell anemia and preservation of blood substitutes:
  • Tadalafil ((6R, 12aR) -2,3,6,7,12,12a - hexahydro - 2 - methyl - 6 - (3,4 - methylene - dioxyphenyl) pyrazino (1 ', 2': 1, 6) pyrido (3,4-b) indole-1, 4-dione), vardenafil (2- (2-ethoxy-5- (4-ethyl-piperazin-1-yl-1-sulfonyl) -phenyl) -5-methyl-7-propyl 3H-imidazo (5,1-f) (l, 2,4) triazin-4-one), sildenafil (3- [2-ethoxy-5- (4-methylpiperazin-1-yl) sulfonyl-phenyl] - 7-methyl-9-propyl-1,2,4,7,8-tetrazabicyclo [4.3.0] nona-3,8,10-trien-5-one),
  • the present invention is a process for the treatment and / or prophylaxis of sickle cell anemia and preservation of blood substitutes using a therapeutically effective amount of one or more of the compound of the invention according to the formulas (1) to (26).
  • Another object of the present invention is the use of one or more of the compounds of the invention according to the formulas (1) to (26) for the manufacture of a medicament for the treatment and / or prophylaxis of sickle cell anemia and preservation of blood substitutes.
  • Another object of the present invention is a pharmaceutical composition containing one or more compounds of the invention of formulas (1) to (26) and one or more other active ingredients, in particular for the treatment and / or prophylaxis of sickle cell anemia and preservation of blood substitutes.
  • Another object of the present invention is the use of one or more compounds of the formulas (1) to (26) in combination with one or more of the following PDE5 inhibitors: Tadalafil ((6R, 12aR) -2,3,6,7 , 12,12a - hexahydro - 2 - methyl - 6 - (3,4 - methylene - dioxyphenyl) pyrazino (l ', 2': l, 6) pyrido (3,4 - b) indole - l, 4 - dione) , Vardenafil (2- (2-ethoxy-5- (4-ethyl-piperazin-1-yl-1-sulfonyl) -phenyl) -5
  • Another object of the present invention is the use of one or more Verbidn 11, of formulas (3) to (7) according to claim 1 in combination with vardenafil and / or sildenafil for the treatment and / or prophylaxis of sickle cell anemia and preservation of blood substitutes.
  • Another object of the present invention is the use of one or more of the compounds of formulas (1) to (26) in combination with the abovementioned PDE5 inhibitors for the treatment and / or prophylaxis of traumatized patients who receive an artificial blood substitute.
  • Another object of the present invention is a method for the treatment and / or prophylaxis of sickle cell anemia and preservation of blood substitutes using a therapeutically effective amount of one or more compounds of the invention according to the formulas (1) to (26) in combination with the above-mentioned PDE5 inhibitors ,
  • Another object of the present invention is the use of one or more of the compounds of the invention according to the formulas (1) to (26) in combination with the above-mentioned PDE5 inhibitors for the manufacture of a medicament for the treatment and / or prophylaxis of sickle cell anemia and preservation of blood substitutes.
  • Another object of the present invention is a pharmaceutical composition containing one or more of the compounds of the invention formulas (1) to (26) in combination with the above-mentioned PDE5 inhibitors and one or more other active ingredients, in particular for the treatment and / or prophylaxis of sickle cell disease and preservation of blood substitutes.
  • Another object of the present invention is the use of one or more compounds of formulas (1) to (26) for the treatment and / or prophylaxis of sickle cell anemia wherein the application is by inhalation.
  • Another object of the present invention Use of one or more compounds of formulas (1) to (26) in combination with the above-mentioned PDE5 inhibitors for the treatment and / or prophylaxis of sickle cell anemia wherein the application is by inhalation.
  • NO and heme independent sGC activators were identified with the compound of formula (5) as the prototype of this class. Common characteristics of these substances are that in combination with NO they exert only an additive effect on the enzyme activation, and that the activation of the oxidized or heme-free enzyme is markedly stronger compared to the heme-containing enzyme [Evgenov et al., Ibid .; JP Stasch et al., Br. J. Pharmacol. 136 (2002), 773; JP Stasch et al., J. Clin. luvest. 116 (2006), 2552].
  • the compounds of the invention show an unpredictable, valuable pharmacological and pharmacokinetic activity spectrum.
  • the sGC stimulators and / or activators result in vasorelaxation and / or platelet aggregation inhibition and / or lowering blood pressure and / or increasing coronary blood flow and microcirculation in general to lead. They are therefore suitable for the treatment and / or prophylaxis of diseases, preferably of cardiovascular diseases, in humans and animals.
  • the compounds of the invention are therefore also suitable for the treatment of cardiovascular diseases such as for the treatment of hypertension, primary and / or secondary prevention and treatment of heart failure, for the treatment of stable and unstable angina pectoris, pulmonary hypertension, peripheral and cardial vascular diseases, arrhythmias Treatment of thromboembolic disorders and ischaemias such as myocardial infarction, stroke, transitory and ischemic attacks, peripheral circulatory disorders, prevention of restenosis such as after thrombolytic therapy, percutaneous transluminal angioplasty (PTA), percutaneous transluminal coronary angioplasty (PTCA) and bypass and for the treatment of arteriosclerosis, asthmatic disorders , Diseases of the genitourinary system such as prostatic hypertrophy, erectile dysfunction, female sexual dysfunction and incontinence are used.
  • cardiovascular diseases such as for the treatment of hypertension, primary and / or secondary prevention and treatment of heart failure
  • arrhythmias Treatment of thromboembolic disorders and ischaemias such as myocardi
  • the compounds of the invention may be used to treat primary and secondary Raynaud's phenomenon, microcirculatory disorders, claudication, peripheral and autonomic neuropathies, diabetic microangiopathies, diabetic nephropathy, diabetic retinopathy, diabetic ulcers on the extremities, CREST syndrome, erythematosis, onychomycosis, tinnitus, Dizziness, sudden hearing loss and rheumatic diseases.
  • the compounds according to the invention are suitable for the treatment of respiratory distress syndromes and chronic obstructive pulmonary diseases (COPD), of acute and chronic renal insufficiency and for the promotion of wound healing. Furthermore, the compounds according to the invention are also suitable for regulating cerebral blood flow and are effective agents for combating migraine. They are also suitable for the prophylaxis and control of the consequences of cerebral infarct events (Apoplexia cerebri) such as stroke, cerebral ischaemias and craniocerebral trauma , Likewise, the compounds of the invention can be used to combat pain.
  • COPD chronic obstructive pulmonary diseases
  • the compounds according to the invention can also be used for the treatment and / or prevention of micro- and macrovascular damage (vasculitis), reperfusion damage, arterial and venous thrombosis, edema, cancer (skin cancer, liposarcoma, carcinomas of the gastrointestinal tract, liver, pancreas , Lung, kidney, ureter, prostate and genital tract), diseases of the central nervous system and neurodegenerative disorders (stroke, Alzheimer's disease, Parkinson's disease, dementia, epilepsy, depression, multiple sclerosis), inflammatory diseases, immune diseases (Morbus Crohn's disease, ulcerative colitis, lupus erythematosus, rheumatoid arthritis, asthma), kidney disease (glomerulonephritis), thyroid disease (hyperthyroidism), diseases of the pancreas (pancreatitis), liver fibrosis, skin diseases (psoriasis, acne, eczema, atopic dermatitis, dermatitis,
  • Another object of the present invention is the use of the compounds of the invention for the treatment and / or prophylaxis of diseases, preferably of thromboembolic diseases and / or thromboembolic complications.
  • thromboembolic disorders include in particular diseases such as heart attack with ST segment elevation (STEMI) and without ST segment elevation (non-STEMI), stable angina pectoris, unstable angina pectoris, reocclusions and restenosis following coronary interventions such as angioplasty, stent or aortocoronary bypass, peripheral arterial occlusive disease, pulmonary embolism, deep venous thrombosis and renal vein thrombosis, transient ischemic attacks, and thrombotic and thromboembolic stroke and pulmonary hypertension.
  • diseases such as heart attack with ST segment elevation (STEMI) and without ST segment elevation (non-STEMI)
  • stable angina pectoris such as unstable angina pectoris, reocclusions and restenosis following coronary interventions such as angioplasty, stent or aortocoronary bypass, peripheral arterial occlusive disease, pulmonary embolism, deep venous thrombosis and renal vein thrombosis, transient
  • the substances are therefore also useful in the prevention and treatment of cardiogenic thromboembolism, such as brain ischemia, stroke and systemic thromboembolism and ischaemia, in patients with acute, intermittent or persistent cardiac arrhythmias, such as atrial fibrillation, and those who undergo cardioversion, further in patients with valvular disease or with intravascular bodies such.
  • cardiogenic thromboembolism such as brain ischemia, stroke and systemic thromboembolism and ischaemia
  • cardiac arrhythmias such as atrial fibrillation
  • intravascular bodies such as atrial fibrillation
  • Artificial heart valves, catheters, intra-aortic balloon counterpulsation and pacemaker probes are suitable for the treatment of disseminated intravascular coagulation (DIC).
  • DIC disseminated intravascular coagulation
  • Thromboembolic complications also occur in microangiopathic hemolytic anemias, sickle cell anemia, thalassemia, inherited forms of spherocytosis, elliptocytosis and ovalcytosis, malaria, Moskowitch syndrome, hemolytic uraemia syndrome, extracorporeal blood circulation, such as.
  • hemofiltration, ventricular assist devices and artificial heart, and heart valve prostheses, blood transfusions, cardiopulmonary bypass and organ transplantation eg lung, heart, kidney, liver
  • the compounds according to the invention are also particularly suitable for primary and / or secondary prevention and treatment of cardiac insufficiency.
  • cardiac insufficiency also encompasses more specific or related forms of disease such as right heart failure, left heart failure, global insufficiency, ischemic cardiomyopathy, dilated cardiomyopathy, congenital heart defects, valvular heart failure, valvular heart failure, mitral valve stenosis, mitral valve insufficiency, aortic valve stenosis, aortic valve insufficiency, tricuspid stenosis, tricuspid insufficiency, pulmonary valve stenosis, Pulmonary valvular insufficiency, combined heart valve defects, myocarditis, chronic myocarditis, acute myocarditis, viral myocarditis, diabetic heart failure, alcoholic cardiomyopathy, cardiac storage disorders, diastolic heart failure, and systolic heart failure.
  • Another object of the present invention is the use of the compounds of the invention for the treatment and / or prophylaxis of diseases
  • the present invention is also suitable for the treatment of traumatized patients who receive an artificial blood substitute [Weisskopf 2010].
  • Another object of the present invention is the use of the compounds of the invention for the manufacture of a medicament for the treatment and / or prophylaxis of diseases, in particular the aforementioned diseases.
  • Another object of the present invention is a method for the treatment and / or prophylaxis of diseases, in particular the aforementioned diseases, using a therapeutically effective amount of a compound of the invention.
  • Another object of the present invention is the use of the compounds of the invention for the manufacture of a medicament for the treatment and / or prophylaxis of sickle cell anemia and preservation of blood substitutes.
  • Another object of the present invention is a method for the treatment and / or prophylaxis of sickle cell anemia and preservation of blood substitutes, using a therapeutically effective amount of a compound of the invention.
  • the compounds of the invention may be used alone or as needed in combination with other agents.
  • Another object of the present invention are pharmaceutical compositions containing a compound of the invention and one or more other active ingredients, in particular for the treatment and / or prophylaxis of the aforementioned diseases.
  • suitable combination active ingredients are lipid metabolism-altering active substances, antidiabetic drugs, blood pressure depressants, circulation-promoting and / or antithrombotic agents, antioxidants, aldosterone and mineralocorticoid receptor antagonists, vasopressin receptor antagonists, organic nitrates and NO.
  • IP receptor agonists positive inotropic compounds, ACE inhibitors, cGMP and cAMP modulating compounds, neutrophilic ecstasis inhibitors, signal transduction cascade inhibiting compounds, heart energy modulating compounds, chemokine receptor antagonists, p38 kinase inhibitors, NPY agonists, orexin agonists, anorectics, PAF-AH Inhibitors, antiphlogistics (COX inhibitors, LTB 4 receptor antagonists, LTB 4 synthesis inhibitors), analgesics (aspirin), antidepressants and other psychotropic drugs.
  • COX inhibitors antiphlogistics
  • LTB 4 receptor antagonists LTB 4 synthesis inhibitors
  • analgesics aspirin
  • antidepressants other psychotropic drugs.
  • the present invention relates, in particular, to combinations with at least one of the compounds according to the invention having at least one active ingredient which alters the lipid metabolism, an antidiabetic agent, a hypotensive agent and / or an antithrombotic agent.
  • the compounds of the invention may preferably be with one or more
  • the fat metabolism-altering active substances by way of example and preferably from the group of HMG-CoA reductase inhibitors from the class of statins, such as, for example and preferably, lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, rosuvastatin,
  • CETP inhibitors such as, by way of example and by way of preference, torcetrapib, JTT-705 or CETP vaccine (Avant), PPAR- ⁇ and / or PPAR-8 agonists such as, by way of example and preferably, pioglitazone or rosiglitazone and / or GW-501516, RXR modulators , FXR modulators, LXR modulators, thyroid hormones and / or thyroid mimetics such as, by way of example and by way of preference, D-thyroxine or 3,5,3'-triiodothyronine (T3), ATP citrate lyase inhibitors,
  • Lp (a) antagonists cannabinoid receptor 1 antagonists such as by way of example and preferably rimonabant or SR-147778, leptin receptor agonists, bombesin receptor agonists, histamine receptor agonists, agonists of the niacin receptor, for example and preferably niacin, Acipimox, A mecanical or Radecol, as well as the antioxidants / free-radical scavengers as exemplified and preferably probucol, AGI-1067, BO-653 or AEOL-
  • Insulin and insulin derivatives are preferably insulin and insulin derivatives as well as orally active hypoglycemic Understood agents.
  • Insulin and insulin derivatives here include both insulins of animal, human or biotechnological origin as well as mixtures thereof.
  • the orally active hypoglycemic agents preferably include sulphonylureas, biguanides, meglitinide derivatives, glucosidase inhibitors and PPAR-gamma agonists, and by way of example and preferably those from the group of sulphonylureas, such as by way of example and preferably tolbutamide, glibenclamide, glimepiride, glipizide or gliclazide, biguanides , such as by way of example and with preference metformin, meglitinide derivatives, such as by way of example and preferably repaglinide or nateglinide, glucosidase inhibitors, such as by way of example and preferably miglitol or acarbose, oxadiazolidinones, thiazolidinediones, GLP 1 receptor agonists, glucagon antagonists, insulin sensitizers, CCK 1 receptor
  • hypotensive agents by way of example and preferably from the group of calcium antagonists such as, by way of example and by way of preference, nifedipine, amlodipine, verapamil or diltiazem, angiotensin AII antagonists such as, for example and preferably, losartan, valsartan, candesartan, embusartan or telmisartan, ACE inhibitors , as exemplified and preferably, enalapril, captopril, ramipril, delapril, fosinopril, quinopril, perindopril or trandopril, beta-receptor blockers such
  • Sympathetic tone lowering agents such as, for example and preferably, reserpine, clonidine or alpha-methyl-dopa, or in combination with a potassium channel agonist, such as, for example and preferably, minoxidil, diazoxide, dihydralazine or hydralazine;
  • Antithrombotic agents by way of example and preferably from the group of platelet aggregation inhibitors, such as by way of example and preferably aspirin, clopidogrel, ticlopidine, cilostazol or dipyridamole, or the anticoagulants such as thrombin inhibitors, such as by way of example and preferably ximelagatran, melagatran, bivalirudin or Clexane, a GPIIb / IIIa antagonist such as, by way of example and preferably, tirofiban or abciximab, a factor Xa inhibitor such as, by way of example and by way of preference, rivaroxaban (BAY 59-7939), DU-176b, apixaban , Otamixaban, Fidexaban, Razaxaban, Fondaparinux, Idraparinux, PMD-3112, YM-150, KFA-1982, EMD-503982, MCM-17, MLN-10
  • SSR-126512 or SSR-128428 with heparin or a low molecular weight (LMW) heparin derivative or with a vitamin K antagonist, such as by way of example and preferably coumarin,
  • Aldosterone and mineralocorticoid receptor antagonists such as by way of example and preferably spironolactone or eplerenone;
  • Vasopressin receptor antagonists such as by way of example and preferably Conivaptan, Tolvaptan, Lixivaptan or SR-121463;
  • Organic nitrates and NO donors such as, for example, sodium nitroprusside, nitroglycerin, isosorbide mononitrate, isosorbide dinitrate, molsidomine or SIN-1, or in combination with inhaled NO;
  • IP receptor agonists such as, by way of example, and iloprost, treprostinil, beraprost, and NS-304
  • Positive inotropic compounds such as, by way of example and by way of illustration, cardiac glycosides (digoxin), beta-adrenergic and dopaminergic agonists such as isoproterenol, epinephrine, norepinephrine, dopamine or dobutamine;
  • Calcium sensitizers such as by way of example and preferably levosimendan
  • cGMP cyclic guanosine monophosphate
  • cAMP cyclic adenosine monophosphate
  • PDE phosphodiesterases
  • sildenafil sildenafil
  • Vardenafil tadalafil
  • PDE 3 inhibitors such as milrinone
  • Natriuretic peptides such as “atrial natriuretic peptide” (ANP, anaritide), "B-type natriuretic peptide” or “brain natriuretic peptide” (BNP, nesiritide), "C-type natriuretic peptide” (CNP) and urodilatin; NO-independent, but heme-dependent guanylate cyclase stimulators, in particular the compounds described in WO 00/06568, WO 00/06569, WO 02/42301 and WO 03/095451;
  • Guanylate cyclase NO- and heme-independent activators in particular the compounds described in WO 01/19355, WO 01/19776, WO 01/19778, WO 01/19780, WO 02/070462 and WO 02/070510;
  • HNE human neutrophilic ecstasy
  • the signal transduction cascade inhibiting compounds such as tyrosine kinase inhibitors and multikinase inhibitors, in particular sorafenib, imatinib, gefitinib and
  • Compounds influencing the energy metabolism of the heart such as, for example, estimoxir, dichloroacetate, ranolazine and trimetazidine.
  • Particularly preferred in the context of the present invention are combinations containing at least one of the compounds according to the invention and one or more further active ingredients selected from the group consisting of HMG-CoA reductase inhibitors (statins), diuretics, antidiabetics, beta-receptor blockers, organic nitrates and NO donors, ACE inhibitors, angiotensin AII antagonists, aldosterone and mineralocorticoid receptor antagonists, vasopressin receptor antagonists, platelet aggregation inhibitors and anticoagulants, and their use for the treatment and / or prevention of the aforementioned disorders.
  • HMG-CoA reductase inhibitors statins
  • diuretics antidiabetics
  • beta-receptor blockers organic nitrates and NO donors
  • ACE inhibitors angiotensin AI
  • the compounds according to the invention can act systemically and / or locally.
  • they 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.
  • the compounds according to the invention can be administered in suitable administration forms.
  • the compounds of the invention in crystalline and / or amorphized and / or dissolved form such as tablets (uncoated or coated tablets, for example with enteric or delayed-dissolving or insoluble coatings which control the release of the compound of the invention), orally disintegrating tablets or films / wafers, films / lyophilisates, capsules (e.g. Hard or soft gelatin capsules), dragees, granules, pellets, powders, emulsions, suspensions, aerosols or solutions.
  • tablets uncoated or coated tablets, for example with enteric or delayed-dissolving or insoluble coatings which control the release of the compound of the invention
  • orally disintegrating tablets or films / wafers, films / lyophilisates capsules (e.g. Hard or soft gelatin capsules), dragees, granules, pellets, powders, emulsions, suspensions, aerosols or solutions.
  • Parenteral administration can be accomplished by bypassing a resorption step (e.g., intravenously, intraarterially, intracardially, intraspinal, or intralumbar) or by resorting to absorption (e.g., intramuscularly, subcutaneously, intracutaneously, percutaneously, or intraperitoneally).
  • a resorption step e.g., intravenously, intraarterially, intracardially, intraspinal, or intralumbar
  • absorption e.g., intramuscularly, subcutaneously, intracutaneously, percutaneously, or intraperitoneally.
  • parenteral administration are suitable as application forms u.a. Injection and infusion preparations in the form of solutions, suspensions, emulsions, lyophilisates or sterile powders.
  • the oral application is preferred.
  • Inhalation medicines including powder inhalers, nebulizers
  • nasal drops solutions, sprays
  • lingual, sublingual or buccal tablets films / wafers or capsules
  • suppositories ear or ophthalmic preparations
  • vaginal capsules aqueous suspensions (lotions, shake mixtures)
  • lipophilic suspensions ointments
  • creams transdermal therapeutic systems (such as patches)
  • milk Pastes, foams, scattering powders, implants or stents.
  • the compounds according to the invention can be converted into the stated administration forms. This can be done in a conventional manner by mixing with inert, non-toxic, pharmaceutically suitable excipients.
  • These adjuvants include, among others. Carriers (for example microcrystalline cellulose, lactose, mannitol), solvents (for example liquid polyethylene glycols), emulsifiers and dispersants or wetting agents (for example sodium dodecyl sulfate, polyoxysorbitanoleate), binders (for example polyvinylpyrrolidone), synthetic and natural polymers (for example albumin), stabilizers ( For example, antioxidants such as ascorbic acid), dyes (eg, inorganic pigments such as iron oxides) and flavor and / or odoriferous.
  • Carriers for example microcrystalline cellulose, lactose, mannitol
  • solvents for example liquid polyethylene glycols
  • emulsifiers and dispersants or wetting agents for example sodium dodecy
  • compositions containing at least one inventive compound preferably together with one or more inert non-toxic, pharmaceutically suitable excipient, as well as their use for the treatment of sickle cell anemia and preservation of blood substitutes.
  • parenterally administered amounts of about 5 to 100 mg per 24 hours to achieve effective results.
  • the amount is about 5 to 250 mg per 24 hours.
  • Method 1 Device Type MS: Waters ZQ; Device Type HPLC: Agilent 1100 Series; UV DAD; Column: Thermo Hypersil GOLD 3 ⁇ 20 mm x 4 mm; Eluent A: 1 l of water + 0.5 ml of 50% formic acid, eluent B: 1 l of acetonitrile + 0.5 ml of 50% strength formic acid; Gradient: 0.0 min 100% A -> 3.0 min 10% A -> 4.0 min 10% A -> 4.1 min 100% A (flow 2.5 ml / min); Oven: 55 ° C; Flow: 2 ml / min; UV detection: 210 nm.
  • Method 2 Instrument: Waters ACQUITY SQD UPLC System; Column: Waters Acquity UPLC HSS T3 1.8 ⁇ 50 x 1 mm; Eluent A: 1 l of water + 0.25 ml of 99% formic acid, eluent B: 1 l of acetonitrile + 0.25 ml of 99% formic acid; Gradient: 0.0 min 90% A -> 1.2 min 5% A -> 2.0 min 5% A Furnace: 50 ° C; Flow: 0.40 ml / min; UV detection: 210 - 400 nm.
  • the diazonium salt thus prepared was added in portions to a 0 ° C cold solution of 12.81 g (85.45 mmol) of sodium iodide in acetone (329 ml) and the mixture stirred for 30 min at RT.
  • the reaction mixture was added to ice water (1.8 L) and extracted twice with ethyl acetate (487 mL each).
  • the collected organic phases were washed with saturated aqueous sodium chloride solution (244 ml), dried, filtered and concentrated. This gave 12.1 g (86% purity, 60% of theory) of the desired compound as a brown solid.
  • the crude product was reacted without further purification.
  • the aqueous phase was separated and extracted twice more with ethyl acetate (200 ml each time).
  • the combined organic phases were washed twice with 10% aqueous sodium chloride solution (100 ml each), dried and concentrated in vacuo.
  • the crude product was reacted without further purification.
  • Variant B Preparation starting from Example 10A: 39.23 g (85.75 mmol) of the compound from Example 10A were initially charged in DMF (800 ml) and then 4 g of palladium (10% on carbon) were added. It was hydrogenated with stirring overnight at normal hydrogen pressure. The mixture was filtered through kieselguhr and washed with a little DMF and then with a little methanol and concentrated to dryness. The residue was combined with ethyl acetate and stirred vigorously, the precipitate was filtered off with suction, washed with ethyl acetate and diisopropyl ether and dried over Sicapent under high vacuum.
  • 29 ⁇ M enzyme solution [0-10 nM soluble guanylate cyclase (prepared according to Hönicka et al., J. Mol. Med. 77, 14-23 (1999)) in 50 mM TEA, 2 mM MgCl 2 , 0.1%. BSA (fraction V), 0.005%> Brij ®, pH 7.5] placed in a microplate and 1 ⁇ of the substance to be tested is added (as a serially diluted solution in DMSO). The mixture is incubated for 10 min at room temperature.
  • the enzyme reaction is started by adding 20 straten ⁇ sub- [1.25 mM guanosine 5'-triphosphate (Fa. Sigma) in 50 mM TEA, 2 mM MgCl 2, 0.1%> BSA (fraction V), 0.005%) Brij ®, pH 7.5] and continuously measured luminometrically.
  • the degree of stimulation by the substance to be tested can be determined relative to the signal of the non-stimulated reaction.
  • the cellular activity of the compounds of the invention is measured on a recombinant guanylate cyclase reporter cell line as described in F. Wunder et al., Anal. Biochem. 339, 104-112 (2005). l.c vascular relaxatory effect in vitro
  • aorta Rabbits are stunned and bled by a stroke of the neck.
  • the aorta is harvested, detached from adherent tissue, divided into 1.5 mm wide rings and placed individually under bias in 5 ml organ baths with 37 ° C warm, carbogen-gassed Krebs-Henseleit solution of the following composition (in each case mM): Sodium chloride: 119; Potassium chloride: 4.8; Calcium chloride dihydrate: 1; Magnesium sulfate heptahydrate: 1 .4; Potassium dihydrogen phosphate: 1 .2; Sodium hydrogencarbonate: 25; Glucose: 10.
  • the force of contraction is detected with Statham UC2 cells, amplified and digitized via A / D converter (DAS-1802 HC, Keithley Instruments Munich) and registered in parallel on a chart recorder.
  • a / D converter DAS-1802 HC, Keithley Instruments Munich
  • phenylephrine is added cumulatively to the bath in increasing concentration.
  • the substance to be examined is added in each subsequent passage in increasing doses and the height of the contraction is compared with the height of the contraction achieved in the last predistortion. This is used to calculate the concentration required to reduce the level of the control value by 50% (IC 50 value).
  • the standard application volume is 5 ⁇ , the DMSO content in the bath solution corresponds to 0.1%. 2.
  • a commercially available telemetry system from DATA SCIENCES INTERNATIONAL DSI, USA is used for the blood pressure measurement on awake rats described below.
  • the system consists of 3 main components: - Implantable transmitters (Physiotel® telemetry transmitters)
  • Data acquisition computer are connected.
  • the telemetry system allows a continuous recording of blood pressure heart rate and body movement on awake animals in their habitual habitat.
  • the experimental animals are kept individually in macroion cages type 3 after transmitter implantation. You have free access to standard food and water.
  • the day - night rhythm in the experimental laboratory is changed by room lighting at 6:00 in the morning and at 19:00 in the evening.
  • the TAI 1 PA - C40 telemetry transmitters are surgically implanted at least 14 days before the first attempt under aseptic conditions.
  • the animals so instrumented are repeatedly used after healing of the wound and ingrowth of the implant.
  • the fasting animals are anaesthetized with pentobabital (Nembutal, Sanofi: 50 mg / kg ip) and shaved and disinfected on the ventral side.
  • pentobabital Nembutal, Sanofi: 50 mg / kg ip
  • the system's liquid-filled measuring catheter above the bifurcation is inserted cranially into the descending aorta and secured with tissue adhesive (VetBonD TM, 3M).
  • the transmitter housing is fixed intraperitoneally to the abdominal wall musculature and the wound is closed in layers.
  • an antibiotic is administered for infection prophylaxis (Tardomyocel COMP Bayer 1 ml / kg sc)
  • a solvent-treated group of animals is used as a control.
  • Experimental procedure The existing telemetry measuring device is configured for 24 animals. Each trial is registered under a trial number (VYear month day).
  • the instrumented rats living in the plant each have their own receiving antenna (1010 receivers, DSI).
  • the implanted transmitters can be activated externally via a built-in magnetic switch. They will be put on the air during the trial run.
  • the emitted signals can be recorded online by a data acquisition system (Dataquest TM A.R.T. for Windows, DSI) and processed accordingly. The storage of the data takes place in each case in a folder opened for this purpose which carries the test number.
  • SBP Systolic blood pressure
  • ACT Activity - Activity
  • the measured value acquisition is repeated computer-controlled in 5-minute intervals.
  • the absolute value of the source data is corrected in the diagram with the currently measured barometric pressure (Ambient Pressure Reference Monitor, APR-1) and in individual data stored. Further technical details can be found in the extensive documentation of the manufacturer (DSI).
  • test substances will take place at 9 o'clock on the day of the experiment. Following the application, the parameters described above are measured for 24 hours.
  • the collected individual data are sorted with the analysis software (DATAQUEST TM A.RT. TM ANALYSIS).
  • the blank value is assumed here 2 hours before application, so that the selected data set covers the period from 7:00 am on the day of the experiment to 9:00 am on the following day.
  • the data is smoothed over a presettable time by averaging (15 minutes average) and transferred as a text file to a disk.
  • the presorted and compressed measured values are transferred to Excel templates and displayed in tabular form.
  • the filing of the collected data takes place per experiment day in a separate folder that bears the test number. Results and test reports are sorted in folders and sorted by paper.
  • the blood pressure and the heart rat By introducing a PE50 arterial catheter into the left femoral artery, the blood pressure and the heart rat (Transducer Ref. 5203660: Fa. Braun CH).
  • the substance is administered as a bolus or continuous infusion via a venous catheter in the left femoral vein.
  • the right external iliac artery is ligated under sterile conditions.
  • anesthetic conditions e.g., inhalation anesthesia, isoflurane, enflurane
  • the femoral artery must be additionally ligated in order to produce a less perfusion.
  • the test animals are treated orally, intragastrically (gastric tube, feed or drinking water intake), intraperitoneally, intravenously, intraarterially, intramuscularly, by inhalation or subcutaneously with the test substances.
  • extracellular hemoglobin previously obtained from donor animals, is applied to the animals.
  • test substances are administered acute or once or several times daily for up to 5 weeks, enterally or parenterally daily, or are administered over subcutaneously implanted osmotic minipumps (e.g., Alzet pumps). Microperfusion and temperature of the lower extremities are documented during the trial.
  • the rats are under anesthesia, a temperature-sensitive laser Doppler probe (Periflux) glued to the paws and thus measured micro perfusion and skin temperature.
  • samples such as blood (interim diagnosis) and other body fluids or organs are taken to perform further in vitro tests or blood pressure and heart rate are measured via a catheter in the carotid artery for documentation of hemodynamics.
  • the animals are killed painlessly.
  • mice eg, eNOS knock out mice, wild-type mice C-57 B16 or ApoE knock out mice, sickle cell mice, .
  • mice were caged and trained in cages with an impeller 4-5 weeks before the start of the experiment. Two weeks before the start of the experiment, the movements of the mice in the impeller are recorded by a photocell using a computer and various running parameters such as the daily running distance, the individual distances covered, but also their temporal distribution over the day.
  • the animals are randomized according to their natural running behavior in groups (8-12 animals) (control group, sham group and one or more substance groups).
  • test animals are administered orally, intragastrically (gastric tube, feed or drinking water intake), intraperitoneally, intravenously, intraarterially, intramuscularly, by inhalation or subcutaneously with the Treated test substances.
  • the test substances are administered once or several times daily or repeatedly enterally or parenterally daily for up to 5 weeks or there is a continuous application via subcutaneously implanted osmotic minipumps.
  • the running behavior of the animals is observed and recorded over 5-8 weeks.
  • the animals are killed painlessly.
  • samples such as blood and other body fluids or organs are taken to carry out further in vitro investigations (S. Vogelsberger New animal models for the indication Claudicatio Intermittens (Paperback), Publisher: WB Laufersweiler Verlag (March 2006), ISBN 10: 383595007X , ISBN-13: 978-3835950078).
  • hemodynamics in anesthetized dogs are 25-35 kg healthy or heart failure Mongrel dogs ® (Marshall BioResources, Marshall Farms Inc; Clyde NY, USA) both sexes used.
  • the anesthesia is induced by slow intravenous administration of 25 mg / kg of sodium thiopental (Trapanal ®) and 0:15 mg / kg Alcuronium chloride (Alloferin ®) and during the experiment maintained by a continuous infusion of 12:04 mg / kg * h fentanyl (Fentanyl ®) , 0.25 mg / kg * h Droperidol (Dihydrobenzperidol ® ) and 15 ⁇ g / kg / h Alcuronium Chloride (Alloferin ® ).
  • the animals are ventilated via the ventilator with a constant breathing volume, so that an end-tidal CO 2 concentration of about 5% is achieved. Ventilation takes place with room air, enriched with approx. 30% oxygen (normoxie).
  • a catheter filled with fluid is implanted in the femoral artery to measure blood pressure.
  • a 2-lumiger Swan-Ganz catheter is ® jugularis on the V. into the pulmonary artery washed in (distal lumen for measuring the pulmonary arterial pressure, proximal lumen for measuring the central venous pressure).
  • a temperature sensor at the tip of the catheter determines the continuous cardiac output (CCO).
  • the blood flow is measured on different vascular beds such as the coronary artery, the carotid artery or the femoral artery by attaching flow probes (Transonic Flowprobe) to the respective vessels.
  • the hnksventrikuläre pressure is measured after the introduction of a Mikro-Tip catheter (Millar Instruments ®) through the carotid artery into the left ventricle and, derived from the dP / dt as a measure of contractility.
  • Substances are administered iv via the femoral vein or intraduodenally as a cumulative dose-response curve (bolus or continuous infusion).
  • the hemodynamic signals are recorded and evaluated by means of pressure transducers / amplifiers and PONEMAH ® as data acquisition software.
  • a pacemaker is implanted into the dogs under sterile conditions. After induction of anesthesia with pentobarbital-Na (15 to 30 mg kg -1 iv) followed by intubation and subsequent ventilation (room air, Sulla 808, Dräger® , Germany) Anesthesia maintained by continuous infusion of pentobarbital (1-5 mg kg "1 h " 1 ) and fentanyl (10-40 ⁇ g kg "1 h " 1 ). A pacing lead (Setrox S60 ®, Biotronik, Germany) is implanted placed over an insertion of the left jugular vein and the right ventricle.
  • the cable is connected to the pacemaker ( Logos® , Biotronik, Germany), which is positioned in a small subcutaneous pocket between the shoulder blades. Only 7 days after the procedure, ventricular pacing to achieve heart failure is started at a rate of 220 beats / min for a period of 10-28 days.
  • the pacemaker logos® , Biotronik, Germany
  • Hemodynamic parameters were continuously measured by intravascular catheters connected by Combitrans® transducers to Gould® transducers (series 6600) connected to the PoNeMah® acquisition and analysis system.
  • Ventilation takes place with room air enriched with approx. 40% oxygen (Normoxie).
  • catheters are inserted into the femoral artery for blood pressure measurement and a Swan- Ganz® catheter is inserted through the jugular vein into the pulmonary artery (distal lumen for measurement of pulmonary artery pressure, proximal lumen for measurement of central venous pressure).
  • a temperature sensor at the tip of the catheter determines the continuous cardiac output (CCO).
  • CO continuous cardiac output
  • the hemodynamic signals are recorded and evaluated by means of pressure transducers / amplifiers and PONEMAH ® as data acquisition software. 2.g.) Inhalation of sGC simulators and activators
  • Acute pulmonary hypertension was induced by a thromboxane A2 analogue infusion (0.12-0.14 ⁇ g / kg / min 9,11 -dideoxy-9a, 11 ⁇ -epoxymethanoprostaglandin F2a (U-46619, Sigma, Germany)) or monocrotaline administration in rats of the Substances were carried out using the Nebutec® or Aeroneb® Pro Vernebier system or after solid nebulization (Pauluhn), which were switched into the inspiratory limb of the respiration. The substances were used as solids or solutions depending on the molecular structure. The hemodynamic signals are recorded and evaluated by means of pressure transducers / amplifiers and PONEMAH ® or CardioMems ® as data acquisition software.
  • Hot Plate Type Socrel DS 37, Ugo Basile Co.
  • mice e.g., NMRI jaws, sickle cell mice.
  • the temperature of the hot plate is 50 ° C, the maximum duration of measurement per animal 90 seconds (cut off time).
  • a hot plate pre-value is determined for each animal. Criteria for latency (in sec.) To the pain reaction are paw shaking, paw licking or jumping. After administration of the test substance for about 8 days, the second test result is carried out under the same conditions.

Abstract

The invention relates to novel uses of stimulators and activators of soluble guanylate cyclase for treating sickle-cell anemia and for conserving blood substitutes. The invention also relates to the use of stimulators and activators of soluble guanylate cyclase combined with PDE 5 inhibitors for treating sickle-cell anemia and conserving blood substitutes.

Description

VERWENDUNG VON STIMULATOREN UND AKTIVATOREN  USE OF STIMULATORS AND ACTIVATORS
DER LÖSLICHEN GUANYLATZYKLASE BEHANDLUNG VON SICHELZELLANÄMIE UND KONSERVIERUNG VON BLUTERSATZSTOFFEN  THE SOLUBLE GUANYLATZYKLASE TREATMENT OF SICKLE LAMINA AND PRESERVATION OF BLOODS
Die vorliegende Erfindung betrifft die neue Verwendung von Stimulatoren und/oder Aktivatoren der löslichen Guanylatzyklase zur Behandlung von Sichelzellanämie und Konservierung von Blutersatzstoffen. Des Weiteren betrifft die vorliegende Erfindung die Verwendung von Stimulatoren und/oder Aktivatoren der löslichen Guanylatzyklase in Kombination mit PDE5 Inhibitoren zur Behandlung von Sichelzellanämie und Konservierung von Blutersatzstoffen. The present invention relates to the novel use of soluble guanylate cyclase stimulators and / or activators for the treatment of sickle cell anemia and the preservation of blood substitutes. Furthermore, the present invention relates to the use of soluble guanylate cyclase stimulators and / or activators in combination with PDE5 inhibitors for the treatment of sickle cell anemia and preservation of blood substitutes.
In den letzten Jahrzehnten mehrt sich das Wissen bezüglich der Rolle von NO in der Blutfluss- Regulation und der kardiovaskulären Hömostase. Pulmonare Hypertension (PH) ist eine häufige Komplikation bei der Sichelzellerkrankung, der eine Störung in der NO-Signalkaskade in Kombination mit erhöhtem oxidativem Stress zu Grunde liegt. PH ist dabei eine Komplikation bei Sichelzellanämie und ein starker Prädiktor für Mortalität. Bei Sichelzellpatienten findet sich eine Resistenz gegenüber NO, hevorgerufen zum einen durch das unverzügliche Abfangen von gebildetem NO durch freies Hämoglobin (Hb), welches beim Zerfall der Erythrocyten freigestzt wird, zum anderen aber auch durch eine erhöhte Oxidation und Degradation der löslichen Guanylatzyklase (sGC), die durch NO als endogenen Liganden aktiviert wird. Zur Klärung der weiteren Pathophysiologie könnten insbesondere transgene Mäuse (exklusive Expression von humanem Sichelzellhämoglobin), die verschiedene Krankheitserscheinungen aufweisen (u.a. PH), hilfreich sein (Blood. 2007;109: 3088-3098). Hier beanspruchen wir die Anwendung von sGC-Aktivatoren und -Stimulatoren für die Behandlung von Sichelzellanämie und der damit verbundenen PH sowie anderen Krankheitserscheinungen (z.B. Endorganschäden, die Hirn, Niere oder Herz betreffen). In recent decades, knowledge of the role of NO in blood flow regulation and cardiovascular hyperstasis increases. Pulmonary hypertension (PH) is a common complication of sickle cell disease, due to a disorder in the NO signaling cascade combined with increased oxidative stress. PH is a complication of sickle cell disease and a strong predictor of mortality. Resistance to NO is found in sickle cell patients, caused by the immediate capture of NO by free hemoglobin (Hb), which is released when erythrocytes decay, but also by increased oxidation and degradation of soluble guanylate cyclase (sGC). which is activated by NO as endogenous ligand. To clarify the further pathophysiology, in particular transgenic mice (exclusive expression of human sickle cell hemoglobin), which exhibit various disease symptoms (inter alia PH), could be helpful (Blood., 2007; 109: 3088-3098). Here we claim the use of sGC activators and stimulators for the treatment of sickle cell anemia and the associated PH as well as other pathologies (e.g., end-organ damage affecting the brain, kidney or heart).
Daneben treten die gleichen o.g. pathophysiologischen Mechanismen in Kraft, wenn Bluttansfusionen (z.B. durch Lagerung etc. mit erhöhter freier Hb Konzentration) bei Patienten mit einer Transfusions-Indikation appliziert werden. In addition, the same o.g. pathophysiological mechanisms when blood transfusions (e.g., by storage, etc., with increased free Hb concentration) are administered to patients with a transfusion indication.
Weiterhin könnte die Kombination eines sGC-Aktivators und -Stimulators mit einem künstlichen Hb-basierten Sauerstoffträgers zukünftig die bisher beobachteten Nebenwirkungen [Weiskopf, Anaesthesia & Analgesia, 110:3; 659-661, 2010], die durch eine verminderte NO-Verfügbarkeit bedingt sind, mitigieren und somit eine klinische Anwendung möglich machen. Durch sGC-Aktivatoren und Stimulatoren wird die lösliche Guanylatzyklase direkt stimuliert, sodass die fehlende NO-Wirkung ersetzt wird. sGC Stimulatoren werden nicht von freiem Hb beeinflusst. Durch sGC Aktivatoren ist es möglich, auch oxidierte Formen der löslichen Guanylatzyklase unabhängig von NO direkt zu stimulieren. Diese oxidierte Form könnte sich in Geweben, die oxidativem Stress ausgesetzt sind, in höheren Konzentrationen anreichern, so dass es durch den Einsatz von sGC Aktivatoren auch zu einer gerichteten Behandlung von Geweben, die unter oxidativem Stress stehen, kommen sollte. Furthermore, the combination of an sGC activator and stimulator with an artificial Hb-based oxygen carrier could in the future, the previously observed side effects [Weiskopf, Anesthesia & Analgesia, 110: 3; 659-661, 2010], which are due to reduced NO availability, mitigate and thus make a clinical application possible. Through sGC activators and stimulators, the soluble guanylate cyclase is directly stimulated, so that the missing NO effect is replaced. sGC stimulators are not affected by free Hb. By using sGC activators, it is also possible to directly stimulate oxidized forms of soluble guanylate cyclase independently of NO. This oxidized form could accumulate in tissues exposed to oxidative stress in higher concentrations, so that the use of sGC activators should also result in the directed treatment of tissues under oxidative stress.
Deswegen sollten sGC Stimulatoren und sGC Aktivatoren unter akuten und insbesondere unter chronischem Einsatz positive Effekte auf den Blutdruck, die Sauerstoffsättigung und die resultierenden Plasma und Gewebs cGMP Spiegel unter experimentelle Bedingungen zeigen. Diese experimentellen Bedingungen bestehen zum einen aus gesunden Tieren, Tieren unter externer freier Hb Applikation oder auch gentechnisch veränderte Tiere wie z.B. Sichelzellmäuse (transgene Mäuse, die exklusiv humanes Sichelzell Hämoglobin beta exprimieren). Hierbei werden die Experimente mit sGC Stimulatoren und Aktivatoren „head to head" gegen den PDE5 Inhibitor Sildenafil durchgeführt, da Sildenafil kürzlich in der klinischen Erprobung (Phase II) bei Patienten mit Sichelzellanämie gescheitert ist. Es führte zu einer Erhöhung der Schmerzkrisen bei den Patienten, sodass die Studie vorzeitig abgebrochen wurde. Daher ist es zwingend notwendig, die Frage zu klären, ob es durch die Erhöhung von cGMP (im Falle von sGC Stimulatoren und Aktivatoren über eine Stimulation der sGC, im Falle von Sildenafil über eine Inhibition des Abbaus des gebildeten cGMP durch Hemmung der Phosphodiesterase 5) zu Stande kommt, oder ob dies eine Eigenschaft von Sildenafil oder allgemein der PDE5 Inhibitoren ist. Das bisherige klinische Programm mit sGC Stimulatoren und Aktivatoren zeigt bisher keine Hinweise, auf eine erhöhte Inzidenz von Schmerzepisoden bei den behandelten Probanden. Im Gegensatz dazu, wird bei PDE5 Inhibitoren wie Sildenafil, Vardenafil und Tadalafil immer wieder auf ein erhöhtes Vorkommen von Rückenschmerz hingewiesen. Deswegen könnte es von aussergewöhnlichem Interesse sein, o.g. sGC Stimulatoren und Aktivatoren bereits auf präklinischer Ebene von PDE 5 Inhibitoren differenziert werden können. Therefore, sGC stimulators and sGC activators under acute and especially chronic conditions should show positive effects on blood pressure, oxygen saturation and the resulting plasma and tissue cGMP levels under experimental conditions. These experimental conditions consist on the one hand of healthy animals, animals under external free Hb application or genetically modified animals such. Sickle cell mice (transgenic mice that exclusively express human sickle cell hemoglobin beta). Here, the experiments with sGC stimulators and activators "head to head" against the PDE5 inhibitor sildenafil are being carried out, as sildenafil has recently failed in clinical trials (phase II) in patients with sickle cell anemia, leading to an increase in pain crises in patients, Therefore, it is imperative to clarify the question whether it is due to the increase of cGMP (in the case of sGC stimulators and activators via stimulation of sGC, in the case of sildenafil via an inhibition of the degradation of the formed cGMP by inhibition of phosphodiesterase 5), or whether this is a property of sildenafil or PDE5 inhibitors in general The current clinical program with sGC stimulators and activators has so far shown no evidence of an increased incidence of pain episodes in treated subjects. In contrast, in PDE5 inhibitors such as sildenafil, vardenaf il and Tadalafil repeatedly pointed to an increased occurrence of back pain. That's why it could be of extraordinary interest, o.g. sGC stimulators and activators can already be differentiated at the preclinical level of PDE 5 inhibitors.
Basierend auf diesem Stand der Technik ist die Aufgabe der vorliegenden Erfindund die Bereitstellung von Substanzen zur Behandlung von Sichezellanämie und Konservierung von Blutersatzstoffen. Die Lösung dieser Aufgabe ist das Auffinden der im folgenden genannten sGC Stimulatoren und/oder sGC Aktivatoren alleine oder in Kombination sowie die Kombination von sGC Stimulatoren und/oder sGC Aktivatoren mit PDE5 Inhibitoren. Based on this prior art, the object of the present invention is to provide substances for the treatment of safeniaemia and preservation of blood substitutes. The solution to this problem is to find the following sGC stimulators and / or sGC activators alone or in combination and the combination of sGC stimulators and / or sGC activators with PDE5 inhibitors.
Eines der wichtigsten zellulären Übertragungssysteme in Säugerzellen ist das cyclische Guanosin- monophosphat (cGMP). Zusammen mit Stickstoffmonoxid (NO), das aus dem Endothel freigesetzt wird und hormonelle und mechanische Signale überträgt, bildet es das NO/cGMP-System. Die Guanylatcyclasen katalysieren die Biosynthese von cGMP aus Guanosintriphosphat (GTP). Die bisher bekannten Vertreter dieser Familie lassen sich sowohl nach strukturellen Merkmalen als auch nach der Art der Liganden in zwei Gruppen aufteilen: Die partikulären, durch natriuretische Peptide stimulierbaren Guanylatcyclasen und die löslichen, durch NO stimulierbaren Guanylatcyclasen. Die löslichen Guanylatcyclasen bestehen aus zwei Untereinheiten und enthalten höchstwahrscheinlich ein Häm pro Heterodimer, das ein Teil des regulatorischen Zentrums ist. Dieses hat eine zentrale Bedeutung für den Aktivierungsmechanismus. NO kann an das Eisenatom des Häms binden und so die Aktivität des Enzyms deutlich erhöhen. Hämfreie Präparationen lassen sich hingegen nicht durch NO stimulieren. Auch Kohlenmonoxid (CO) ist in der Lage, an das Eisen-Zentralatom des Häms zu binden, wobei die Stimulierung durch CO deutlich geringer ist als die durch NO. Durch die Bildung von cGMP und der daraus resultierenden Regulation von Phosphodiesterasen, Ionenkanälen und Proteinkinasen spielt die Guanylatcyclase eine entscheidende Rolle bei unterschiedlichen physiologischen Prozessen, insbesondere bei der Relaxation und Proliferation glatter Muskelzellen, der Plättchenaggregation und -adhäsion, der neuronalen Signalübertragung sowie bei Erkrankungen, welche auf einer Störung der vorstehend genannten Vorgänge beruhen. Unter patho- physiologischen Bedingungen kann das NO/cGMP-System supprimiert sein, was zum Beispiel zu Bluthochdruck, einer Plättchenaktivierung, einer vermehrten Zellproliferation, endothelialer Dysfunktion, Arteriosklerose, Angina pectoris, Herzinsuffizienz, Myokardinfarkt, Thrombosen, Schlaganfall und sexueller Dysfunktion führen kann. One of the most important cellular transmission systems in mammalian cells is cyclic guanosine monophosphate (cGMP). Together with nitric oxide (NO), which is released from the endothelium and transmits hormonal and mechanical signals, it forms the NO / cGMP system. The guanylate cyclases catalyze the biosynthesis of cGMP from guanosine triphosphate (GTP). The Previously known members of this family can be divided into two groups according to both structural features and the nature of the ligands: the particulate guanylate cyclases stimulable by natriuretic peptides and the soluble, NO-stimulable guanylate cyclases. The soluble guanylate cyclases consist of two subunits and most likely contain one heme per heterodimer that is part of the regulatory center. This is central to the activation mechanism. NO can bind to the iron atom of the heme and thus significantly increase the activity of the enzyme. On the other hand, heme-free preparations can not be stimulated by NO. Also, carbon monoxide (CO) is able to bind to the central iron atom of the heme, with stimulation by CO being significantly less than by NO. Through the formation of cGMP and the resulting regulation of phosphodiesterases, ion channels and protein kinases, guanylate cyclase plays a crucial role in various physiological processes, in particular in the relaxation and proliferation of smooth muscle cells, platelet aggregation and adhesion, neuronal signaling and diseases based on a disturbance of the above operations. Under pathophysiological conditions, the NO / cGMP system may be suppressed, which may, for example, lead to hypertension, platelet activation, increased cell proliferation, endothelial dysfunction, arteriosclerosis, angina pectoris, heart failure, myocardial infarction, thrombosis, stroke and sexual dysfunction.
Eine auf die Beeinflussung des cGMP-Signalweges in Organismen abzielende NO-unabhängige Behandlungsmöglichkeit für derartige Erkrankungen ist aufgrund der zu erwartenden hohen Effizienz und geringen Nebenwirkungen ein vielversprechender Ansatz. A NO-independent treatment option for such diseases, which is aimed at influencing the cGMP pathway in organisms, is a promising approach on account of the expected high efficiency and low side effects.
Zur therapeutischen Stimulation der löslichen Guanylatcyclase wurden bisher ausschließlich Verbindungen wie organische Nitrate verwendet, deren Wirkung auf NO beruht. Dieses wird durch Biokonversion gebildet und aktiviert die lösliche Guanylatcyclase durch Angriff am Eisen-Zentralatom des Häms. Neben den Nebenwirkungen gehört die Toleranzentwicklung zu den entscheidenden Nachteilen dieser Behandlungsweise. For therapeutic stimulation of soluble guanylate cyclase, only compounds such as organic nitrates have been used, whose action is based on NO. This is formed by bioconversion and activates the soluble guanylate cyclase by attack on the central iron atom of the heme. In addition to the side effects, tolerance development is one of the decisive disadvantages of this treatment.
In den letzten Jahren wurden einige Substanzen beschrieben, die die lösliche Guanylatcyclase direkt, d.h. ohne vorherige Freisetzung von NO stimulieren, wie beispielsweise 3-(5'-Hydroxymethyl-2'- furyl)-l-benzylindazol [YC-1 ; Wu et al., Blood 84 (1994), 4226; Mülsch et al., Brit. J. Pharmacol. 120 (1997), 681], Fettsäuren [Goldberg et al., J. Biol. Chem. 252 (1977), 1279], Diphenyl- iodonium-hexafluorophosphat [Pettibone et al., Eur. J. Pharmacol. 116 (1985), 307], Iso- liquiritigenin [Yu et al., Brit. J. Pharmacol. 114 (1995), 1587] sowie verschiedene substituierte Pyrazol-Derivate (WO 98/16223) und hier als sGC Stimulatoren im Sinne dieser Erfindung zu verstehen sind. In recent years, some substances have been described which stimulate soluble guanylate cyclase directly, ie without prior release of NO, such as 3- (5'-hydroxymethyl-2'-furyl) -l-benzylindazole [YC-1; Wu et al., Blood 84 (1994), 4226; Mülsch et al., Brit. J. Pharmacol. 120 (1997), 681], fatty acids [Goldberg et al., J. Biol. Chem. 252 (1977), 1279], diphenyliodonium hexafluorophosphate [Pettibone et al., Eur. J. Pharmacol. 116 (1985), 307], iso-liquiritigenin [Yu et al., Brit. J. Pharmacol. 114 (1995), 1587] and various substituted ones Pyrazole derivatives (WO 98/16223) and are here to be understood as sGC stimulators in the context of this invention.
Gegenstand der vorliegen Erfindung ist die Verwendung von Stimulatoren und/oder Aktivatoren der löslichen Guanylatzyklase zur Behandlung von Sichelzellanmämie und Konservierung von Blutersatzstoffen. The present invention is the use of soluble guanylate cyclase stimulators and / or activators for the treatment of sickle cell disease and preservation of blood substitutes.
Besonders bevorzugt sind die Verbindungen (l)-(26) wie im folgenden dargsetellt: Particular preference is given to the compounds (I) - (26) as shown below:
• 2- [ 1 -(2-Fluorbenzyl)- 1 H-pyrazolo[3 ,4-b]pyridin-3 -yl] -5-(4-mo^holinyl)-4,6-pyrimidindiamin (1), offenbart als Beispiel 16 in WO 00/06569, • 2- [1- (2-fluorobenzyl) -1 H -pyrazolo [3,4-b] pyridin-3-yl] -5- (4-monothinyl) -4,6-pyrimidinediamine (1) as Example 16 in WO 00/06569,
• 2- [ 1 -(2-Fluorobenzyl)- 1 H-pyrazolo[3 ,4-b]pyridin-3 -yl] -5-(4-pyridinyl)-4-pyrimidinamin (2), offenbart als Beispiel 1 in WO 02/42301 , 2- [1- (2-fluorobenzyl) -1 H -pyrazolo [3,4-b] pyridin-3-yl] -5- (4-pyridinyl) -4-pyrimidinamine (2), disclosed as Example 1 in WO 02/42301,
• Methyl-4,6-diamino-2- [ 1 -(2-fluorobenzyl)- 1 H-pyrazolo[3 ,4-b]pyridin-3 -yl] -5-pyrimidinyl- (methyl)carbamat (3), offenbart als Beispiel 8 in WO 03/095451, Methyl 4,6-diamino-2- [1- (2-fluorobenzyl) -1 H -pyrazolo [3,4-b] pyridin-3-yl] -5-pyrimidinyl (methyl) carbamate (3), disclosed as Example 8 in WO 03/095451,
• Methyl-4,6-diamino-2- [ 1 -(2-fluorobenzyl)- 1 H-pyrazolo[3 ,4-b]pyridin-3 -yl] -5-pyrimidinyl- carbamat (4), offenbart als Beispiel 5 in WO 03/095451 · 4-({(4-carboxybutyl)[2-(2- {[4-(2-phenylethyl)benzyl]oxy}phenyl)ethyl]amino} methyl) carbonsäure (5), offenbart als Beispiel 8a in WO 01/019780, Methyl 4,6-diamino-2- [1- (2-fluorobenzyl) -1 H -pyrazolo [3,4-b] pyridin-3-yl] -5-pyrimidinyl carbamate (4), disclosed as an example 5 in WO 03/095451 * 4 - ({(4-carboxybutyl) [2- (2- {[4- (2-phenylethyl) benzyl] oxy} phenyl) ethyl] amino} methyl) carboxylic acid (5), disclosed as Example 8a in WO 01/019780,
• Methyl- {4,6-diamino-2-[5-fluor-l-(2-fluorbenzyl)-lH-pyrazolo[3,4-b]pyridin-3-yl]pyrirm 5-yl}carbamat (6), Methyl {4,6-diamino-2- [5-fluoro-1- (2-fluorobenzyl) -1H-pyrazolo [3,4-b] pyridin-3-yl] pyrrmine-5-yl} carbamate (6) .
• Methyl- {4,6-diamino-2-[5-fluor-l-(2-fluorbenzyl)-lH-pyrazolo[3,4-b]pyridin-3-yl]pyrirm 5-yl}methylcarbamat (7), Methyl {4,6-diamino-2- [5-fluoro-1- (2-fluorobenzyl) -1H-pyrazolo [3,4-b] pyridin-3-yl] -pyramido-5-yl} methylcarbamate (7) .
• Methyl- {4,6-diamino-2-[5-fluor-l-(2-fluorbenzyl)-lH-pyrazolo[3,4-b]pyridin-3-yl]pyrimi 5-yl} (2,2,2-trifluorethyl)carbamat (8), Methyl {4,6-diamino-2- [5-fluoro-1- (2-fluorobenzyl) -1H-pyrazolo [3,4-b] pyridin-3-yl] pyrimi5-yl} (2,2 , 2-trifluoroethyl) carbamate (8),
• 5-Chlor-2-(5-chlorothiophen-2-sulfonylamino-N-(4-(morpholin-4-sulfonyl)-phenyl)-benzamid als Natriumsalz (9), offenbart in WO00/02851, · 2-(4-Chloro-phenylsulfonylamino)-4,5-dimethoxy-N-(4-(thiomorpholin-4-sulfonyl)-phenyl)- benzamid (10), offenbart in WO00/02851, • 5-Chloro-2- (5-chlorothiophene-2-sulfonylamino-N- (4- (morpholine-4-sulfonyl) -phenyl) -benzamide as the sodium salt (9), disclosed in WO00 / 02851, 2- (4 Chloro-phenylsulfonylamino) -4,5-dimethoxy-N- (4- (thiomorpholine-4-sulfonyl) -phenyl) -benzamide (10) disclosed in WO00 / 02851,
• 1 - {6-[5-Chloro-2-( {4-trans-4-}trifluoromethyl)cyclohexyl]benzyl} oxy)phenyl] pyridin-2-yl} - 5-(trifluoromethyl)-lH-pyrazol-4-carbonsäure (11), offenbart in WO 2009/032249, 1 - {6- [5-chloro-2- ({4-trans-4-trifluoromethyl) cyclohexyl] benzyl} oxy) phenyl] pyridin-2-yl} - 5- (trifluoromethyl) -1H-pyrazole-4-carboxylic acid (11) disclosed in WO 2009/032249,
• l-[6-(2-(2-Methyl-4-(4-trifluoromethoxyphenyl)benzyloxy)-phenyl)pyridin-2-yl]-5- trifluoromethyl-pyrazol-4-carbonsäure (12), offenbart in WO 2009/071504, • 1- [6- (2- (2-Methyl-4- (4-trifluoromethoxyphenyl) benzyloxy) -phenyl) pyridin-2-yl] -5-trifluoromethyl-pyrazole-4-carboxylic acid (12) disclosed in WO 2009 / 071504,
• 1 [6-(3,4-dichlorophenyl)-2-pyridinyl-5-(trifluoromethyl)-lH-pyrazole-4-cabonsäure (13), offenbart in WO 2009/068652, 1 [6- (3,4-dichlorophenyl) -2-pyridinyl-5- (trifluoromethyl) -1H-pyrazole-4-carboxylic acid (13) disclosed in WO 2009/068652]
• 1 -( {2- [3 -Chlor-5-(trifluoromethyl)phenyl] -5-methyl- 1 ,3 -thiazol-4-yl} methyl)- 1 H-pyrazole- 4-carbonsäure (14), 4-( {2- [3 -(Trifluoromethyl)phenyl] - 1 ,3 -thiazol-4-yl} methyl)benzoesäure (15) and 1 -( {2- [2-Fluoro-3 -(trifluoromethyl)phenyl] -5-methyl- 1 ,3 -thiazol-4-yl} methyl)- 1 H- pyrazol-4-carbonsäure (16) offenbart in WO 2009/123316, · 4-Amino-2-[5-chlor-3(3,3,3-triflou^ropyl)-lH-indazol-lyl]-5,5-dimethyl-5,7-dihydro-6H- pyrrolo[2,3-d]pyrimidin-6-on (17), 4-Amino-2[5-chlor-3-(2,3,6-trifluorbenzyl)-lH-indazol- lyl]-5,5-dimethyl-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-on (18), 4-Amino-5,5- dimethyl-2-[3-(2,3,6-trifluorbenzyl)lH-thieno[3,4-c]pyrazol-l-yl]-5,7-dihydro-6H- pyrrolo[2,3-d]pyrimidin-6-on (19), 4-Amino-5,5-dimethyl-2-[3-(2,3,6-trifluorbenzyl)lH- thieno[2,3-d]pyrazol-l-yl]-5,5-dimethyl-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-on (20),• 1 - ({2- [3-Chloro-5- (trifluoromethyl) phenyl] -5-methyl-1,3-thiazol-4-yl} methyl) -1 H -pyrazole-4-carboxylic acid (14), 4 - ({2- [3- (trifluoromethyl) phenyl] -1,3-thiazol-4-yl} methyl) benzoic acid (15) and 1- ({2- [2-fluoro-3- (trifluoromethyl) phenyl] - 5-methyl-1,3-thiazol-4-yl} methyl) -1H-pyrazole-4-carboxylic acid (16) disclosed in WO 2009/123316, 4-amino-2- [5-chloro-3 (3 , 3,3-trifluorophenyl) -1H-indazolyl] -5,5-dimethyl-5,7-dihydro-6H-pyrrolo [2,3-d] pyrimidin-6-one (17), 4- Amino-2 [5-chloro-3- (2,3,6-trifluorobenzyl) -1H-indazolyl] -5,5-dimethyl-5,7-dihydro-6H-pyrrolo [2,3-d] pyrimidine -6-one (18), 4-amino-5,5-dimethyl-2- [3- (2,3,6-trifluorobenzyl) -1H-thieno [3,4-c] pyrazol-1-yl] -5 , 7-dihydro-6H-pyrrolo [2,3-d] pyrimidin-6-one (19), 4-amino-5,5-dimethyl-2- [3- (2,3,6-trifluorobenzyl) lH- thieno [2,3-d] pyrazol-1-yl] -5,5-dimethyl-5,7-dihydro-6H-pyrrolo [2,3-d] pyrimidin-6-one (20),
4-Amino-5,5-dimethyl-2-[7-(2,3,6-trifluorobenzyl)irmdazo[l,5-b]pyridazin-5-yl]-5,7- dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-on (21), 4-Amino-2-[6-chlor-3-(2,3,6- trifluorobenzyl)imidazo[l,5-a]pyridin-l-yl] ]-5,5-dimethyl-5,7-dihydro-6H-pyrrolo[2,3- d]pyrimidin-6-on (22), 4-Amino-2-[6-fluor-3-(2,3,6-trifluorobenzyl)imidazo[l ,5-a]pyridin- 1-yl] ]-5,5-dimethyl-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-on (23), 4-Amino-2-[6-fluor-4-Amino-5,5-dimethyl-2- [7- (2,3,6-trifluorobenzyl) irmdazo [l, 5-b] pyridazin-5-yl] -5,7-dihydro-6H-pyrrolo [2 , 3-d] pyrimidin-6-one (21), 4-amino-2- [6-chloro-3- (2,3,6-trifluorobenzyl) imidazo [l, 5-a] pyridin-1-yl] ] -5,5-dimethyl-5,7-dihydro-6H-pyrrolo [2,3-d] pyrimidin-6-one (22), 4-amino-2- [6-fluoro-3- (2,3 , 6-trifluorobenzyl) imidazo [l, 5-a] pyridin-1-yl]] -5,5-dimethyl-5,7-dihydro-6H-pyrrolo [2,3-d] pyrimidin-6-one (23 ), 4-amino-2- [6-fluoro
3-(2,3,6-trifluorobenzyl)6-fluoroim 3- (2,3,6-Trifluorobenzyl) 6-fluoroim
pyrrolo[2,3-d]pyrimidin-6-on (24), 4-Amino-5,5-dimethyl-2-[3-(2,4,6- trifluorobenzyl)imidazo[l,5-a]pyridin-l-yl] ]-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-on (25), 4-Amino-2-[3-(2-cyclopentylethyl)imidazo[l,5-a]pyridin-l-yl]-5,5-dimethyl-5,7- dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-on (26), offenbart in WO 2010/065275. -6- pyrrolo [2,3-d] pyrimidin-6-one (24), 4-amino-5,5-dimethyl-2- [3- (2,4,6-trifluorobenzyl) imidazo [l, 5-a] pyridine -l-yl]] -5,7-dihydro-6H-pyrrolo [2,3-d] pyrimidin-6-one (25), 4-amino-2- [3- (2-cyclopentylethyl) imidazo [l, 5-a] pyridin-1-yl] -5,5-dimethyl-5,7-dihydro-6H-pyrrolo [2,3-d] pyrimidin-6-one (26) disclosed in WO 2010/065275. -6-
Ġ Ĩ22) Ġ Ĩ22)
Ġ Ĩ25) Ġ Ĩ25)
(26). (26).
Verbindungen der Formeln (1), (2), (3), (4), (6)-(8) und (17)-(26) sind bekannt als Stimulatoren der löslichen Guanylatzyklase. Besonders bevorzugt sind die Verbindungen der Formeln (3), (4), (6) und (7). Compounds of formulas (1), (2), (3), (4), (6) - (8) and (17) - (26) are known as soluble guanylate cyclase stimulators. Particularly preferred are the compounds of the formulas (3), (4), (6) and (7).
Verbindungen der Formeln (5) und (9)-(16) sind bekannt als Aktivatoren der löslichen Guanylatzyklase. Besonders bevorzugt ist die Verbindung der Formel (5). Compounds of formulas (5) and (9) - (16) are known as soluble guanylate cyclase activators. Particularly preferred is the compound of formula (5).
Ein weiterer Gegestand der Erfindung ist die Kombination von Stimulatoren und/oder Aktivatoren der löslichen Guanylatcyclase mit PDE5 Inhibitoren zur Verwendung zur Behandlung von Sichelzellanämie und Konservierung von Blutersatzstoffen. Another aspect of the invention is the combination of soluble guanylate cyclase stimulators and / or activators with PDE5 inhibitors for use in the treatment of sickle cell anemia and preservation of blood substitutes.
Die im folgenden aufgelisteten PDE5 Inhibitoren eignen sich für die Kombination zur Verwendung zur Behandlung von Sichelzellanämie und Konservierung von Blutersatzstoffen: The following PDE5 inhibitors are suitable for combination for use in the treatment of sickle cell anemia and preservation of blood substitutes:
Tadalafil ((6R,12aR) -2,3,6,7,12,12a - Hexahydro - 2 - methyl - 6 - (3,4-methylene -dioxyphenyl) pyrazino(l ',2': 1 ,6) pyrido(3,4-b)indole- 1 ,4-dione), Vardenafil (2-(2-Ethoxy-5-(4-ethylpiperazin- 1 -yl- l-sulfonyl)phenyl)-5-methyl-7-propyl-3H-imidazo (5,1-f) (l,2,4)triazin-4-one), Sildenafil (3-[2- ethoxy-5-(4-methylpiperazin-l-yl)sulfonyl-phenyl]- 7- methy 1- 9- propy 1-2,4,7,8- tetrazabicyclo [4.3.0]nona -3,8,10-trien-5-one), Udenafil 5-[2-propyloxy-5-(l-methyl-2- pyrrolidinylethylarmdosulfonyl)phenyl]-methy Tadalafil ((6R, 12aR) -2,3,6,7,12,12a - hexahydro - 2 - methyl - 6 - (3,4 - methylene - dioxyphenyl) pyrazino (1 ', 2': 1, 6) pyrido (3,4-b) indole-1, 4-dione), vardenafil (2- (2-ethoxy-5- (4-ethyl-piperazin-1-yl-1-sulfonyl) -phenyl) -5-methyl-7-propyl 3H-imidazo (5,1-f) (l, 2,4) triazin-4-one), sildenafil (3- [2-ethoxy-5- (4-methylpiperazin-1-yl) sulfonyl-phenyl] - 7-methyl-9-propyl-1,2,4,7,8-tetrazabicyclo [4.3.0] nona-3,8,10-trien-5-one), uddenafil 5- [2-propyloxy-5- ( 1-methyl-2-pyrrolidinylethylarmedosulfonyl) phenyl] methyl
7-one, Dasantafiil 7-(3 -Bromo-4-methoxybenzyl)- 1 -ethyl-8- [[( 1 ,2)-2-hydroxycyclopentyl] amino] -3 - (2-hydroxyethyl)-3,7-dihydro-l-purine-2,6-dione, Avanafil 4- {[(3-chloro-4- methoxyphenyl)methyl]amino}-2-[(2S)-2-(hydroxymethyl)pyrrolidin-l-yl]-N-(pyrimidin-2- ylmethyl)pyrimidine-5-carboxamide, Mirodenafil, Lodenafil, UK 369.003, UK 371.800, SLx 2101 of Surface Logix, LAS 34 79Triazolo[l,2-]xanthine,6-methyl-4-propyl-2-[2-propoxy-5-(4- methylpiperazino)sulfonyl]phenyl oder Salze, Hydrate oder Hydrate der Salze davon. Besonders bevorzugt sind Kobinationen von Verbindungen der Formeln (3), (4), (6), (7) und /oder (5) mit Vardenafil und/oder Sildenafil. 7-one, Dasantafiil 7- (3-bromo-4-methoxybenzyl) -1-ethyl-8- [[(1, 2) -2-hydroxycyclopentyl] amino] -3- (2-hydroxyethyl) -3,7- dihydro-l-purine-2,6-diones, avanafil 4- {[(3-chloro-4-methoxyphenyl) -methyl] -amino} -2 - [(2S) -2- (hydroxymethyl) -pyrrolidin-1-yl] - N- (pyrimidin-2-ylmethyl) pyrimidines-5-carboxamides, mirodenafil, lodenafil, UK 369,003, UK 371,800, SLx 2101 of Surface Logix, LAS 34, 79Triazolo [1,2] xanthines, 6-methyl-4-propyl 2- [2-propoxy-5- (4-methylpiperazino) sulfonyl] phenyl or salts, hydrates or hydrates of the salts thereof. Particular preference is given to combinations of compounds of the formulas (3), (4), (6), (7) and / or (5) with vardenafil and / or sildenafil.
Gegenstand der vorliegenden Erfindung ist ein Verfahren zur Behandlung und/oder Prophylaxe von Sichelzellanämie und Konservierung von Blutersatzstoffen unter Verwendung einer therapeutisch wirksamen Menge einer oder mehrerer der erfindungsgemäßen Verbindung gemäß der Formeln (1) bis (26). The present invention is a process for the treatment and / or prophylaxis of sickle cell anemia and preservation of blood substitutes using a therapeutically effective amount of one or more of the compound of the invention according to the formulas (1) to (26).
Ein weiterer Gegenstand der vorliegenden Erfindung ist die Verwendung von einer oder mehrerer der erfindungsgemäßen Verbindungen gemäß der Formeln (1) bis (26) zur Herstellung eines Arzneimittels zur Behandlung und/oder Prophylaxe von Sichelzellanämie und Konservierung von Blutersatzstoffen. Another object of the present invention is the use of one or more of the compounds of the invention according to the formulas (1) to (26) for the manufacture of a medicament for the treatment and / or prophylaxis of sickle cell anemia and preservation of blood substitutes.
Ein weiterer Gegenstand der vorliegenden Erfindung ist ein Arzneimittel, enthaltend eine oder mehrere erfindungsgemäße Verbindung der Formeln (1) bis (26) und einen oder mehrere weitere Wirkstoffe, insbesondere zur Behandlung und/oder Prophylaxe von Sichelzellanämie und Konservierung von Blutersatzstoffen. Ein weiterer Gegenstand der vorliegenden Erfindung ist die Verwendung von einer oder mehrerer Verbindungen der der Formeln (1) bis (26) in Kombination mit einem oder mehreren der folgenden PDE5 Inhibitoren: Tadalafil ((6R,12aR) -2,3,6,7,12,12a - Hexahydro - 2 - methyl - 6 - (3,4- methylene -dioxyphenyl) pyrazino(l',2':l,6) pyrido(3,4-b)indole-l,4-dione), Vardenafil (2-(2- Ethoxy-5-(4-ethylpiperazin- 1 -yl- 1 -sulfonyl)phenyl)-5-methyl-7-propyl-3H-imidazo (5, 1 -f) (l,2,4)triazin-4-one), Sildenafil (3-[2-ethoxy-5-(4-methylpiperazin-l-yl)sulfonyl-phenyl]- 7- methy 1- 9- propy 1-2,4,7,8- tetrazabicyclo [4.3.0]nona -3,8,10-trien-5-one), Udenafil 5-[2-propyloxy-5-(l- methyl-2-pyrrolidinylethylamidosulfonyl)phenyl]-methyl-3-propyl-l,6-dihydro-7H-pyrazolo(4,3- d)pyrimidine-7-one, Dasantafil 7-(3-Bromo-4-methoxybenzyl)-l-ethyl-8-[[(l,2)-2- hydroxycyclopentyl] amino] -3 -(2-hydroxyethyl)-3 ,7-dihydro- 1 -purine-2,6-dione, Avanafil 4- { [(3 - chloro-4-methoxyphenyl)methyl]amino}-2-[(2S)-2-(hydroxymethyl)pyrrolidin-l-yl]-N-(pyrimidin-2- ylmethyl)pyrimidine-5-carboxamide, Mirodenafil, Lodenafil, UK 369.003, UK 371.800, SLx 2101 of Surface Logix, LAS 34 79Triazolo[l,2-]xanthine,6-methyl-4-propyl-2-[2-propoxy-5-(4- methylpiperazino)sulfonyl]phenyl oder Salze, Hydrate oder Hydrate der Salze davon zur Behandlung und/oder Prophylaxe von Sichelzellanämie und Konservierung von Blutersatzstoffen. Ein weiterer Gegenstand der vorliegenden Erfindung ist die Verwendung von einer oder mehrerer Verbidnungen der Formeln (3) bis (7) gemäß Anspruch 1 in Kombination mit Vardenafil und/oder Sildenafil zur Behandlung und/oder Prophylaxe von Sichelzellanämie und Konservierung von Blutersatzstoffen. Ein weiterer Gegenstand der vorliegenden Erfindung ist die Verwendung von einer oder mehrerer der Verbindungen der Formeln (1) bis (26) in Kombination mit den oben genannten PDE5 Inhibitoren zur Behandlung und/oder Prophylaxe von traumatisierten Patienten, welche einen künstlichen Blutersatzstoff erhalten. Ein weiterer Gegenstand der vorliegenden Erfindung ist ein Verfahren zur Behandlung und/oder Prophylaxe von Sichelzellanämie und Konservierung von Blutersatzstoffen unter Verwendung einer therapeutisch wirksamen Menge einer oder mehrerer erfindungsgemäßen Verbindung gemäß der Formeln (1) bis (26) in Kombination mit den oben genannten PDE5 Inhibitoren. Another object of the present invention is a pharmaceutical composition containing one or more compounds of the invention of formulas (1) to (26) and one or more other active ingredients, in particular for the treatment and / or prophylaxis of sickle cell anemia and preservation of blood substitutes. Another object of the present invention is the use of one or more compounds of the formulas (1) to (26) in combination with one or more of the following PDE5 inhibitors: Tadalafil ((6R, 12aR) -2,3,6,7 , 12,12a - hexahydro - 2 - methyl - 6 - (3,4 - methylene - dioxyphenyl) pyrazino (l ', 2': l, 6) pyrido (3,4 - b) indole - l, 4 - dione) , Vardenafil (2- (2-ethoxy-5- (4-ethyl-piperazin-1-yl-1-sulfonyl) -phenyl) -5-methyl-7-propyl-3H-imidazo (5, 1-f) (1, 2 , 4) triazin-4-one), sildenafil (3- [2-ethoxy-5- (4-methylpiperazin-1-yl) sulfonyl-phenyl] -7-methyl-9-propyl 1-2,4,7 , 8-tetrazabicyclo [4.3.0] nona-3,8,10-trien-5-one), Udenafil 5- [2-propyloxy-5- (1-methyl-2-pyrrolidinylethylamidosulphonyl) phenyl] -methyl-3- propyl-l, 6-dihydro-7H-pyrazolo (4,3-d) pyrimidines-7-one, dasantafil 7- (3-bromo-4-methoxybenzyl) -l-ethyl-8 - [[(l, 2) -2-hydroxycyclopentyl] amino] -3- (2-hydroxyethyl) -3,7-dihydro-1-purine-2,6-diones, avanafil 4- {[(3-chloro-4-methoxyphenyl) methyl] amino} -2 - [(2S) -2- (hydro xymethyl) pyrrolidin-1-yl] -N- (pyrimidin-2-ylmethyl) pyrimidines-5-carboxamides, mirodenafil, lodenafil, UK 369,003, UK 371,800, SLx 2101 of Surface Logix, LAS 34, 79Triazolo [1,2-xanthine , 6-methyl-4-propyl-2- [2-propoxy-5- (4-methylpiperazino) sulfonyl] phenyl or salts, hydrates or hydrates of the salts thereof for the treatment and / or prophylaxis of sickle cell anemia and preservation of blood substitutes. Another object of the present invention is the use of one or more Verbidnungen of formulas (3) to (7) according to claim 1 in combination with vardenafil and / or sildenafil for the treatment and / or prophylaxis of sickle cell anemia and preservation of blood substitutes. Another object of the present invention is the use of one or more of the compounds of formulas (1) to (26) in combination with the abovementioned PDE5 inhibitors for the treatment and / or prophylaxis of traumatized patients who receive an artificial blood substitute. Another object of the present invention is a method for the treatment and / or prophylaxis of sickle cell anemia and preservation of blood substitutes using a therapeutically effective amount of one or more compounds of the invention according to the formulas (1) to (26) in combination with the above-mentioned PDE5 inhibitors ,
Ein weiterer Gegenstand der vorliegenden Erfindung ist die Verwendung einer oder mehrerer der erfindungsgemäßen Verbindungen gemäß der Formeln (1) bis (26) in Kombination mit den oben genannten PDE5 Inhibitoren zur Herstellung eines Arzneimittels zur Behandlung und/oder Prophylaxe von Sichelzellanämie und Konservierung von Blutersatzstoffen. Another object of the present invention is the use of one or more of the compounds of the invention according to the formulas (1) to (26) in combination with the above-mentioned PDE5 inhibitors for the manufacture of a medicament for the treatment and / or prophylaxis of sickle cell anemia and preservation of blood substitutes.
Ein weiterer Gegenstand der vorliegenden Erfindung ist ein Arzneimittel, enthaltend eine oder mehrere der erfindungsgemäßen Verbindungen der Formeln (1) bis (26) in Kombination mit dem oben genannten PDE5 Inhibitoren und einen oder mehrere weitere Wirkstoffe, insbesondere zur Behandlung und/oder Prophylaxe von Sichelzellanämie und Konservierung von Blutersatzstoffen. Another object of the present invention is a pharmaceutical composition containing one or more of the compounds of the invention formulas (1) to (26) in combination with the above-mentioned PDE5 inhibitors and one or more other active ingredients, in particular for the treatment and / or prophylaxis of sickle cell disease and preservation of blood substitutes.
Ein weiterer Gegenstand der vorliegenden Erfindung ist die Verwendung von einer oder mehrerer Verbindungen der Formeln (1) bis (26) zur Behandlung und/oder Prophylaxe von Sichelzellanämie wobei die Anwendung inhalativ erfolgt. Ein weiterer Gegenstand der vorliegenden Erfindung Verwendung von einer oder mehrerer Verbindungen der Formeln (1) bis (26) in Kombination mit den oben genannten PDE5 Inhibitoren zur Behandlung und/oder Prophylaxe von Sichelzellanämie wobei die Anwendung inhalativ erfolgt. Another object of the present invention is the use of one or more compounds of formulas (1) to (26) for the treatment and / or prophylaxis of sickle cell anemia wherein the application is by inhalation. Another object of the present invention Use of one or more compounds of formulas (1) to (26) in combination with the above-mentioned PDE5 inhibitors for the treatment and / or prophylaxis of sickle cell anemia wherein the application is by inhalation.
Darüber hinaus wurden NO- und Häm-unabhängige sGC-Aktivatoren, mit der Verbindung der Formel (5) als Prototyp dieser Klasse, identifiziert. Gemeinsame Charakteristika dieser Substanzen sind, dass sie in Kombination mit NO nur einen additiven Effekt auf die Enzymaktivierung ausüben, und dass die Aktivierung des oxidierten oder hämfreien Enzyms im Vergleich zum hämhaltigen Enzym deutlich stärker ist [Evgenov et al., ibid.; J.P. Stasch et al., Br. J. Pharmacol. 136 (2002), 773; J.P. Stasch et al., J. Clin. luvest. 116 (2006), 2552]. Spektroskopische Untersuchungen lassen erkennen, dass die Verbindung der Formel (5) die oxidierte Hämgruppe verdrängt, die durch Schwächung der Eisen-Histidin-Bindung nur schwach an der sGC gebunden ist. Auch wurde gezeigt, dass das charakteristische sGC-Hämbindungsmotiv Tyr-x-Ser-x-Arg sowohl für die Interaktion der negativ geladenen Propionsäuren der Hämgruppe als auch für die Wirkung von die Verbindung der Formel (5) zwingend erforderlich ist. Vor diesem Hintergrund wird angenommen, dass die Bindungsstelle von die Verbindung der Formel (5) an der sGC identisch zur Bindungsstelle der Hämgruppe ist [J.P. Stasch et al., J. Clin. Invest. 116 (2006), 2552]. Die in der vorliegenden Erfindung beschriebenen Verbindungen sind nun ebenfalls in der Lage, die Häm-freie Form der löslichen Guanylatcyclase zu aktivieren. Dies wird auch dadurch belegt, dass diese neuartigen Aktivatoren einerseits am Häm-haltigen Enzym keine synergistische Wirkung mit NO zeigen und andererseits sich ihre Wirkung nicht durch den Häm-abhängigen Inhibitor der löslichen Guanylatcyclase, lH-l,2,4-Oxadiazolo[4,3-a]chinoxalin-l-on (ODQ), blockieren lässt, sondern durch diesen sogar potenziert wird [vgl. O.V. Evgenov et al., Nature Rev. Drug Disc. 5 (2006), 755; J.P. Stasch et al., J. Clin. Invest. 116 (2006), 2552] und hier als sGC Aktivatoren im Sinne dieser Erfindung zu verstehen sind. Dazu sollen insbesondere folgende sGC Aktivatorklassen zählen gemäß den Verbindungen der Formeln (5) und (9)-(16). In addition, NO and heme independent sGC activators were identified with the compound of formula (5) as the prototype of this class. Common characteristics of these substances are that in combination with NO they exert only an additive effect on the enzyme activation, and that the activation of the oxidized or heme-free enzyme is markedly stronger compared to the heme-containing enzyme [Evgenov et al., Ibid .; JP Stasch et al., Br. J. Pharmacol. 136 (2002), 773; JP Stasch et al., J. Clin. luvest. 116 (2006), 2552]. Spectroscopic studies indicate that the compound of formula (5) displaces the oxidized heme group, which is weakly bound to the sGC by weakening the iron-histidine bond. It was also shown that the characteristic sGC binding motif Tyr-x-Ser-x-Arg for both the interaction of the negatively charged propionic acids of the heme group and for the effect of the compound of the Formula (5) is mandatory. Against this background, it is considered that the binding site of the compound of the formula (5) at the sGC is identical to the binding site of the heme group [JP Stasch et al., J. Clin. Invest. 116 (2006), 2552]. The compounds described in the present invention are now also able to activate the heme-free form of soluble guanylate cyclase. This is also demonstrated by the fact that these novel activators on the one hand on the heme-containing enzyme show no synergistic effect with NO and on the other hand their effect is not due to the heme-dependent inhibitor of soluble guanylate cyclase, lH-l, 2,4-oxadiazolo [4, 3-a] quinoxaline-1-one (ODQ), but is even potentiated by it [cf. OV Evgenov et al., Nature Rev. Drug Disc. 5 (2006), 755; JP Stasch et al., J. Clin. Invest. 116 (2006), 2552] and are to be understood here as sGC activators within the meaning of this invention. In particular, the following sGC activator classes should be included according to the compounds of the formulas (5) and (9) - (16).
Die erfindungsgemäßen Verbindungen zeigen ein nicht vorhersehbares, wertvolles pharmakologisches und pharmakokinetisches Wirkspektrum. Neben der Verwendung zur Behandlung von Sichelzellanämie und Konservierung von Blutersatzstoffen führen die sGC Stimulatoren und/oder Aktivatoren zu einer Ge fäßre laxation und/o der zu einer Thrombozytenaggregationshemmung und/oder zu einer Blutdrucksenkung und/oder zu einer Steigerung des koronaren Blutflusses sowie der Mikrozirkulation generell führen. Sie eigenen sich daher zur Behandlung und/oder Prophylaxe von Krankheiten, vorzugsweise von kardiovaskulären Erkrankungen, bei Menschen und Tieren. The compounds of the invention show an unpredictable, valuable pharmacological and pharmacokinetic activity spectrum. In addition to being used to treat sickle cell anemia and to preserve blood substitutes, the sGC stimulators and / or activators result in vasorelaxation and / or platelet aggregation inhibition and / or lowering blood pressure and / or increasing coronary blood flow and microcirculation in general to lead. They are therefore suitable for the treatment and / or prophylaxis of diseases, preferably of cardiovascular diseases, in humans and animals.
Die erfindungsgemäßen Verbindungen eignen sich daher auch zur Behandlung von kardiovaskulären Erkrankungen wie beispielsweise zur Behandlung des Bluthochdrucks, der primären und/oder sekundären Prävention sowie Behandlung der Herzinsuffizienz, zur Behandlung stabiler und instabiler Angina pectoris, pulmonaler Hypertonie, peripheren und kardialen Gefäßerkrankungen, Arrhythmien, zur Behandlung von thromboembolischen Erkrankungen und Ischämien wie Myokardinfarkt, Hirnschlag, transistorischen und ischämischen Attacken, peripheren Durchblutungsstörungen, zur Verhinderung von Restenosen wie nach Thrombolysetherapien, percutantransluminalen Angioplastien (PTA), percutan-transluminalen Koronarangioplastien (PTCA) und Bypass sowie zur Behandlung von Arteriosklerose, asthmatischen Erkrankungen, Krankheiten des Urogenitalsystems wie beispielsweise Prostatahypertrophie, erektile Dysfunktion, weibliche sexuelle Dysfunktion und Inkontinenz eingesetzt werden. Außerdem können die erfindungsgemäßen Verbindungen zur Behandlung von primärem und sekundärem Raynaud-Phänomen, von Mikrozirkulationsstörungen, Claudicatio, peripheren und autonomen Neuropathien, diabetischen Mikroangiopathien, diabetischer Nephropathie, diabetischer Retinopathie, diabetischen Geschwüren an den Extremitäten, CREST-Syndrom, Erythematose, Onychomykose, Tinnitus, Schwindelanfälle, Hörsturz sowie von rheumatischen Erkrankungen verwendet werden. The compounds of the invention are therefore also suitable for the treatment of cardiovascular diseases such as for the treatment of hypertension, primary and / or secondary prevention and treatment of heart failure, for the treatment of stable and unstable angina pectoris, pulmonary hypertension, peripheral and cardial vascular diseases, arrhythmias Treatment of thromboembolic disorders and ischaemias such as myocardial infarction, stroke, transitory and ischemic attacks, peripheral circulatory disorders, prevention of restenosis such as after thrombolytic therapy, percutaneous transluminal angioplasty (PTA), percutaneous transluminal coronary angioplasty (PTCA) and bypass and for the treatment of arteriosclerosis, asthmatic disorders , Diseases of the genitourinary system such as prostatic hypertrophy, erectile dysfunction, female sexual dysfunction and incontinence are used. In addition, the compounds of the invention may be used to treat primary and secondary Raynaud's phenomenon, microcirculatory disorders, claudication, peripheral and autonomic neuropathies, diabetic microangiopathies, diabetic nephropathy, diabetic retinopathy, diabetic ulcers on the extremities, CREST syndrome, erythematosis, onychomycosis, tinnitus, Dizziness, sudden hearing loss and rheumatic diseases.
Ferner eignen sich die erfindungsgemäßen Verbindungen zur Behandlung von Respiratory Distress- Syndromen und chronisch-obstruktiven Atemwegserkrankungen (COPD), von akuter und chronischer Niereninsuffizienz sowie zur Förderung der Wundheilung. Weiterhin eignen sich die erfindungsgemäßen Verbindungen auch zur Regulation der cerebralen Durchblutung und stellen wirkungsvolle Mittel zur Bekämpfung von Migräne dar. Auch eignen sie sich zur Prophylaxe und Bekämpfung der Folgen cerebraler Infarktgeschehen (Apoplexia cerebri) wie Schlaganfall, cerebraler Ischämien und des Schädel-Hirn-Traumas. Ebenso können die erfindungsgemäßen Verbindungen zur Bekämpfung von Schmerzzuständen eingesetzt werden. Darüber hinaus können die erfindungsgemäßen Verbindungen auch zur Behandlung und/oder Prävention von mikro- und makrovaskulären Schädigungen (Vasculitis), Reperfusionsschäden, arteriellen sowie venösen Thrombosen, Ödemen, Krebserkrankungen (Hautkrebs, Liposarcome, Karzinome des Magen-Darm-Traktes, der Leber, Bauchspeicheldrüse, Lunge, Niere, Harnleiter, Prostata und des Genitaltraktes), von Erkrankungen des Zentralen Nervensystems und neurodegenerativen Störungen (Schlaganfall, Alzheimer'sche Krankheit, Parkinson'sche Krankheit, Demenz, Epilepsie, Depressionen, Multiple Sklerose), von Entzündungserkrankungen, Immunerkrankungen (Morbus Crohn, Colitis ulcerosa, Lupus erythematodes, rheumatoide Arthritis, Asthma), Nierenerkrankungen (Glomerulonephritis), Schilddrüsenerkrankungen (Hyperthyreose), Erkrankungen der Bauchspeicheldrüse (Pankreatitis), Leberfibrose, Hauterkrankungen (Psoriasis, Akne, Ekzeme, Neurodermitis, Dermatitis, Keratitis, Narbenbildung, Warzenbildung, Frostbeulen), viralen Erkrankungen (HPV, HCMV, HIV), Kachexie, Osteoporose, Gicht, Inkontinenz sowie zur Wundheilung und Angiogenese eingesetzt werden. Furthermore, the compounds according to the invention are suitable for the treatment of respiratory distress syndromes and chronic obstructive pulmonary diseases (COPD), of acute and chronic renal insufficiency and for the promotion of wound healing. Furthermore, the compounds according to the invention are also suitable for regulating cerebral blood flow and are effective agents for combating migraine. They are also suitable for the prophylaxis and control of the consequences of cerebral infarct events (Apoplexia cerebri) such as stroke, cerebral ischaemias and craniocerebral trauma , Likewise, the compounds of the invention can be used to combat pain. In addition, the compounds according to the invention can also be used for the treatment and / or prevention of micro- and macrovascular damage (vasculitis), reperfusion damage, arterial and venous thrombosis, edema, cancer (skin cancer, liposarcoma, carcinomas of the gastrointestinal tract, liver, pancreas , Lung, kidney, ureter, prostate and genital tract), diseases of the central nervous system and neurodegenerative disorders (stroke, Alzheimer's disease, Parkinson's disease, dementia, epilepsy, depression, multiple sclerosis), inflammatory diseases, immune diseases (Morbus Crohn's disease, ulcerative colitis, lupus erythematosus, rheumatoid arthritis, asthma), kidney disease (glomerulonephritis), thyroid disease (hyperthyroidism), diseases of the pancreas (pancreatitis), liver fibrosis, skin diseases (psoriasis, acne, eczema, atopic dermatitis, dermatitis, keratitis, scarring, warts , Fros tuberculosis), viral diseases (HPV, HCMV, HIV), cachexia, osteoporosis, gout, incontinence as well as wound healing and angiogenesis.
Weiterer Gegenstand der vorliegenden Erfindung ist der Einsatz der erfindungsgemäßen Verbindungen zur Behandlung und/oder Prophylaxe von Erkrankungen, vorzugsweise von thromboembo- lischen Erkrankungen und/oder thromboembolischen Komplikationen. Another object of the present invention is the use of the compounds of the invention for the treatment and / or prophylaxis of diseases, preferably of thromboembolic diseases and / or thromboembolic complications.
Zu den„thromboembolischen Erkrankungen" im Sinne der vorliegenden Erfindung zählen insbesondere Erkrankungen wie Herzinfarkt mit ST-Segment-Erhöhung (STEMI) und ohne ST- Segment-Erhöhung (non-STEMI), stabile Angina Pectoris, instabile Angina Pectoris, Reokklusionen und Restenosen nach Koronarinterventionen wie Angioplastie, Stentimplantation oder aortokoronarem Bypass, periphere arterielle Verschlusskrankheiten, Lungenembolien, tiefe venöse Thrombosen und Nierenvenenthrombosen, transitorische ischämische Attacken sowie thrombotischer und thromboembolischer Hirnschlag und pulmonale Hypertonie. Die Substanzen eignen sich daher auch zur Prävention und Behandlung von kardiogenen Thrombo- embolien, wie beispielsweise Hirn-Ischämien, Schlaganfall (Stroke) und systemischen Thromboembolien und Ischämien, bei Patienten mit akuten, intermittierenden oder persistierenden Herzarrhythmien, wie beispielsweise Vorhofflimmern, und solchen, die sich einer Kardioversion unterziehen, ferner bei Patienten mit Herzklappen-Erkrankungen oder mit intravasalen Körpern, wie z. B. künstlichen Herzklappen, Kathetern, intraaortale Ballongegenpulsation und Schrittmachersonden. Darüber hinaus sind die erfindungsgemäßen Verbindungen zur Behandlung der disseminierten intravasalen Gerinnung (DIC) geeignet. For the purposes of the present invention, "thromboembolic disorders" include in particular diseases such as heart attack with ST segment elevation (STEMI) and without ST segment elevation (non-STEMI), stable angina pectoris, unstable angina pectoris, reocclusions and restenosis following coronary interventions such as angioplasty, stent or aortocoronary bypass, peripheral arterial occlusive disease, pulmonary embolism, deep venous thrombosis and renal vein thrombosis, transient ischemic attacks, and thrombotic and thromboembolic stroke and pulmonary hypertension. The substances are therefore also useful in the prevention and treatment of cardiogenic thromboembolism, such as brain ischemia, stroke and systemic thromboembolism and ischaemia, in patients with acute, intermittent or persistent cardiac arrhythmias, such as atrial fibrillation, and those who undergo cardioversion, further in patients with valvular disease or with intravascular bodies such. Artificial heart valves, catheters, intra-aortic balloon counterpulsation and pacemaker probes. In addition, the compounds of the invention are suitable for the treatment of disseminated intravascular coagulation (DIC).
Thromboembolische Komplikationen treten ferner auf bei mikroangiopathischen hämolytischen Anämien, Sichelzellanämie, Thalassämie, vererbten Formen von Spherozytose, Eliptozytose und Ovalzytose, Malaria, Moskowitch Syndrom, hämolytisches Uraemie Syndrom, extrakorporalen Blutkreisläufen, wie z. B. Hämodialyse, Hämofiltration, ventricular assist devices und Kunstherz, sowie Herzklappenprothesen, Bluttransfusionen, beim Kardiopulmonalen Bypass und Organtransplantationen (z.B. Lunge, Herz, Niere, Leber), Hämolyse auf Grund von intravasculären Devices oder durch Hämodialyse sowie bei der Anwendung von künstlichen Hb-basierten Sauerstoffträgern. Thromboembolic complications also occur in microangiopathic hemolytic anemias, sickle cell anemia, thalassemia, inherited forms of spherocytosis, elliptocytosis and ovalcytosis, malaria, Moskowitch syndrome, hemolytic uraemia syndrome, extracorporeal blood circulation, such as. As hemodialysis, hemofiltration, ventricular assist devices and artificial heart, and heart valve prostheses, blood transfusions, cardiopulmonary bypass and organ transplantation (eg lung, heart, kidney, liver), hemolysis due to intravascular devices or by hemodialysis and in the application of artificial Hb based oxygen carriers.
Besonders eignen sich die erfindungsgemäßen Verbindungen auch zur primären und/oder sekundären Prävention sowie Behandlung der Herzinsuffizienz. The compounds according to the invention are also particularly suitable for primary and / or secondary prevention and treatment of cardiac insufficiency.
Im Sinne der vorliegenden Erfindung umfasst der Begriff Herzinsuffizienz auch spezifischere oder verwandte Krankheitsformen wie Rechtsherzinsuffizienz, Linksherzinsuffizienz, Globalinsuffizienz, ischämische Kardiomyopathie, dilatative Kardiomyopathie, angeborene Herzfehler, Herzklappenfehler, Herzinsuffizienz bei Herzklappenfehlern, Mitralklappenstenose, Mitralklappeninsuffizienz, Aortenklappenstenose, Aortenklappeninsuffizienz, Trikuspidalstenose, Trikuspidalinsuffizienz, Pulmonalklappenstenose, Pulmonalklappeninsuffizienz, kombinierte Herzklappenfehler, Herzmuskelentzündung (Myokarditis), chronische Myokarditis, akute Myokarditis, virale Myokarditis, diabetische Herzinsuffizienz, alkoholtoxische Kardiomyopathie, kardiale Speichererkrankungen, diastolische Herzinsuffizienz sowie systolische Herzinsuffizienz. Weiterer Gegenstand der vorliegenden Erfindung ist die Verwendung der erfindungsgemäßen Verbindungen zur Behandlung und/oder Prophylaxe von Erkrankungen, insbesondere der zuvor genannten Erkrankungen. For the purposes of the present invention, the term cardiac insufficiency also encompasses more specific or related forms of disease such as right heart failure, left heart failure, global insufficiency, ischemic cardiomyopathy, dilated cardiomyopathy, congenital heart defects, valvular heart failure, valvular heart failure, mitral valve stenosis, mitral valve insufficiency, aortic valve stenosis, aortic valve insufficiency, tricuspid stenosis, tricuspid insufficiency, pulmonary valve stenosis, Pulmonary valvular insufficiency, combined heart valve defects, myocarditis, chronic myocarditis, acute myocarditis, viral myocarditis, diabetic heart failure, alcoholic cardiomyopathy, cardiac storage disorders, diastolic heart failure, and systolic heart failure. Another object of the present invention is the use of the compounds of the invention for the treatment and / or prophylaxis of diseases, in particular the aforementioned diseases.
Desweiteren bietet sich die vorliegende Erfindung auch für die Behandlung von traumatisierten Patienten, welche einen künstlichen Blutersatzstoff erhalten [Weisskopf 2010] an. Furthermore, the present invention is also suitable for the treatment of traumatized patients who receive an artificial blood substitute [Weisskopf 2010].
Weiterer Gegenstand der vorliegenden Erfindung ist die Verwendung der erfindungsgemäßen Verbindungen zur Herstellung eines Arzneimittels zur Behandlung und/oder Prophylaxe von Erkrankungen, insbesondere der zuvor genannten Erkrankungen. Another object of the present invention is the use of the compounds of the invention for the manufacture of a medicament for the treatment and / or prophylaxis of diseases, in particular the aforementioned diseases.
Weiterer Gegenstand der vorliegenden Erfindung ist ein Verfahren zur Behandlung und/oder Prophylaxe von Erkrankungen, insbesondere der zuvor genannten Erkrankungen, unter Verwendung einer therapeutisch wirksamen Menge einer erfindungsgemäßen Verbindung. Another object of the present invention is a method for the treatment and / or prophylaxis of diseases, in particular the aforementioned diseases, using a therapeutically effective amount of a compound of the invention.
Weiterer Gegenstand der vorliegenden Erfindung ist die Verwendung der erfindungsgemäßen Verbindungen zur Herstellung eines Arzneimittels zur Behandlung und/oder Prophylaxe von Sichelzellanämie und Konservierung von Blutersatzstoffen. Weiterer Gegenstand der vorliegenden Erfindung ist ein Verfahren zur Behandlung und/oder Prophylaxe von Sichelzellanämie und Konservierung von Blutersatzstoffen, unter Verwendung einer therapeutisch wirksamen Menge einer erfindungsgemäßen Verbindung. Another object of the present invention is the use of the compounds of the invention for the manufacture of a medicament for the treatment and / or prophylaxis of sickle cell anemia and preservation of blood substitutes. Another object of the present invention is a method for the treatment and / or prophylaxis of sickle cell anemia and preservation of blood substitutes, using a therapeutically effective amount of a compound of the invention.
Die erfindungsgemäßen Verbindungen können allein oder bei Bedarf in Kombination mit anderen Wirkstoffen eingesetzt werden. Weiterer Gegenstand der vorliegenden Erfindung sind Arzneimittel, enthaltend eine erfindungsgemäße Verbindung und einen oder mehrere weitere Wirkstoffe, insbesondere zur Behandlung und/oder Prophylaxe der zuvor genannten Erkrankungen. Als geeignete Kombinationswirkstoffe seien beispielhaft und vorzugsweise genannt: den Fettstoffwechsel verändernde Wirkstoffe, Antidiabetika, Blutdruck-Senker, durchblutungsfördernd und/oder antithrombotisch wirkende Mittel sowie Antioxidantien, Aldosteron- und Mineralokortikoid-Rezeptor-Antagonisten, Vasopressin- Rezeptor-Antagonisten, organische Nitrate und NO-Donatoren, IP- Rezeptor Agonisten, positiv inotrop wirksamen Verbindungen, ACE Inhibitoren, cGMP und cAMP modulierende Verbindungen, Inhibitoren der neutrophilen Ekstase, die Signaltransduktionskaskade inhibierende Verbindungen, den Energiestoffwechsel des Herzens modulierende Verbindungen, Chemokin-Rezeptor- Antagonisten, p38-Kinase-Inhibitoren, NPY-Agonisten, Orexin-Agonisten, Anorektika, PAF-AH- Inhibitoren, Antiphlogistika (COX-Inhibitoren, LTB4-Rezeptor- Antagonisten, LTB4-Synthese Hemmer), Analgetika (Aspirin), Antidepressiva und andere Psychopharmaka. The compounds of the invention may be used alone or as needed in combination with other agents. Another object of the present invention are pharmaceutical compositions containing a compound of the invention and one or more other active ingredients, in particular for the treatment and / or prophylaxis of the aforementioned diseases. Examples of suitable combination active ingredients are lipid metabolism-altering active substances, antidiabetic drugs, blood pressure depressants, circulation-promoting and / or antithrombotic agents, antioxidants, aldosterone and mineralocorticoid receptor antagonists, vasopressin receptor antagonists, organic nitrates and NO. Donors, IP receptor agonists, positive inotropic compounds, ACE inhibitors, cGMP and cAMP modulating compounds, neutrophilic ecstasis inhibitors, signal transduction cascade inhibiting compounds, heart energy modulating compounds, chemokine receptor antagonists, p38 kinase inhibitors, NPY agonists, orexin agonists, anorectics, PAF-AH Inhibitors, antiphlogistics (COX inhibitors, LTB 4 receptor antagonists, LTB 4 synthesis inhibitors), analgesics (aspirin), antidepressants and other psychotropic drugs.
Gegenstand der vorliegenden Erfindung sind insbesondere Kombinationen mit mindestens einer der erfindungsgemäßen Verbindungen mit mindestens einem den Fettstoffwechsel verändernden Wirk- Stoff, einem Antidiabetikum, einem blutdrucksenkenden Wirkstoff und/oder einem antithrombotisch wirkenden Mittel. The present invention relates, in particular, to combinations with at least one of the compounds according to the invention having at least one active ingredient which alters the lipid metabolism, an antidiabetic agent, a hypotensive agent and / or an antithrombotic agent.
Die erfindungsgemäßen Verbindungen können vorzugsweise mit einem oder mehreren The compounds of the invention may preferably be with one or more
• den Fettstoffwechsel verändernden Wirkstoffen, beispielhaft und vorzugsweise aus der Gruppe der HMG-CoA-Reduktase-Inhibitoren aus der Klasse der Statine, wie beispielhaft und vorzugsweise Lovastatin, Simvastatin, Pravastatin, Fluvastatin, Atorvastatin, Rosuvastatin,The fat metabolism-altering active substances, by way of example and preferably from the group of HMG-CoA reductase inhibitors from the class of statins, such as, for example and preferably, lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, rosuvastatin,
Cerivastatin oder Pitavastatin, Inhibitoren der HMG-CoA-Reduktase-Expression, Squalensynthese-Inhibitoren wie beispielhaft und vorzugsweise BMS-188494 oder TAK-475, ACAT-Inhibitoren wie beispielhaft und vorzugsweise Melinamide, Pactimibe, Eflucimibe oder SMP-797, LDL-Rezeptor-Induktoren, Cholesterin-Absorptionshemmer wie beispielhaft und vorzugsweise Ezetimibe, Tiqueside oder Pamaqueside, polymeren Gallensäureadsorber, wie beispielhaft und vorzugsweise Cholestyramin, Colestipol, Colesolvam, CholestaGel oder Colestimide, Gallensäure-Reabsorptionshemmer, wie beispielhaft und vorzugsweise ASBT (= IBAT)-Inhibitoren wie z.B. AZD-7806, S-8921, AK-105, BARI-1741, SC-435 oder SC-635,, MTP-Inhibitoren wie beispielhaft und vorzugsweise Implitapide oder JTT-130, Lipase- Inhibitoren wie beispielhaft und vorzugsweise Orlistat, LpL-Aktivatoren, Fibrate, Niacin,Cerivastatin or pitavastatin, inhibitors of HMG-CoA reductase expression, squalene synthesis inhibitors such as, by way of example and not limitation, BMS-188494 or TAK-475, ACAT inhibitors such as, and by way of example, melinamide, pactimibe, eflucimibe or SMP-797, LDL receptor Inducers, cholesterol absorption inhibitors such as, by way of example and by way of preference, ezetimibe, tiqueside or pamaqueside, polymeric bile acid adsorbents, such as by way of example and preferably cholestyramine, colestipol, colesolvam, cholesta gel or colestimide, bile acid reabsorption inhibitors such as, for example and preferably, ASBT (= IBAT) inhibitors such as AZD-7806, S-8921, AK-105, BARI-1741, SC-435 or SC-635 ,, MTP inhibitors such as, by way of example, and preferably, implitapide or JTT-130, lipase inhibitors such as, and preferably, orlistat, LpL activators , Fibrates, niacin,
CETP-Inhibitoren, wie beispielhaft und vorzugsweise Torcetrapib, JTT-705 oder CETP Vaccine (Avant), PPAR-γ- und/oder PPAR-8-Agonisten, wie beispielhaft und vorzugsweise Pioglitazone oder Rosiglitazone und/oder GW-501516, RXR-Modulatoren, FXR-Modulatoren, LXR-Modulatoren, Thyroidhormone und/oder Thyroidmimetika wie beispielhaft und vorzugsweise D-Thyroxin oder 3,5,3'-Triiodothyronin (T3), ATP-Citrat-Lyase-Inhibitoren,CETP inhibitors such as, by way of example and by way of preference, torcetrapib, JTT-705 or CETP vaccine (Avant), PPAR-γ and / or PPAR-8 agonists such as, by way of example and preferably, pioglitazone or rosiglitazone and / or GW-501516, RXR modulators , FXR modulators, LXR modulators, thyroid hormones and / or thyroid mimetics such as, by way of example and by way of preference, D-thyroxine or 3,5,3'-triiodothyronine (T3), ATP citrate lyase inhibitors,
Lp(a)-Antagonisten, Cannabinoid-Rezeptor 1 -Antagonisten, wie beispielhaft und vorzugsweise Rimonabant oder SR- 147778, Leptin-Rezeptor-Agonisten, Bombesin-Rezeptor-Agonisten, Histamin-Rezeptor-Agonisten, Agonisten des Niacin-Rezeptors, wie beispielhaft und vorzugsweise Niacin, Acipimox, Acifran oder Radecol, sowie der Antioxidantien / Radikalfänger wie beispielhaft und vorzugsweise Probucol, AGI-1067, BO-653 oder AEOL-Lp (a) antagonists, cannabinoid receptor 1 antagonists such as by way of example and preferably rimonabant or SR-147778, leptin receptor agonists, bombesin receptor agonists, histamine receptor agonists, agonists of the niacin receptor, for example and preferably niacin, Acipimox, Acifran or Radecol, as well as the antioxidants / free-radical scavengers as exemplified and preferably probucol, AGI-1067, BO-653 or AEOL-
10150, 10150
• Antidiabetika, die in der Roten Liste 2009/1, Kapitel 12 genannt sind. Unter Antidiabetika werden vorzugsweise Insulin und Insulinderivate sowie oral wirksame hypoglykämische Wirkstoffe verstanden. Insulin und Insulinderivate umfasst hierbei sowohl Insuline tierischen, menschlichen oder biotechnologischen Ursprungs als auch Gemische hieraus. Die oral wirksamen hypoglykämischen Wirkstoffe umfassen vorzugsweise Sulphonylharnstoffe, Biguanide, Meglitinid-Derivate, Glukosidase-Inhibitoren und PPAR-gamma-Agonisten sowie beispielhaft und vorzugsweise jenen aus der Gruppe der Sulphonylharnstoffe, wie beispielhaft und vorzugsweise Tolbutamid, Glibenclamid, Glimepirid, Glipizid oder Gliclazid, Biguanide, wie beispielhaft und vorzugsweise Metformin, Meglitinid-Derivate, wie beispielhaft und vorzugsweise Repaglinid oder Nateglinid, Glukosidase-Inhibitoren, wie beispielhaft und vorzugsweise Miglitol oder Acarbose, Oxadiazolidinone, Thiazolidindione, GLP 1 -Rezeptor- Agonisten, Glukagon- Antagonisten, Insulin- Sensitizer, CCK 1-Rezeptor-• Anti-diabetic medicines mentioned in the Red List 2009/1, Chapter 12. Among antidiabetics are preferably insulin and insulin derivatives as well as orally active hypoglycemic Understood agents. Insulin and insulin derivatives here include both insulins of animal, human or biotechnological origin as well as mixtures thereof. The orally active hypoglycemic agents preferably include sulphonylureas, biguanides, meglitinide derivatives, glucosidase inhibitors and PPAR-gamma agonists, and by way of example and preferably those from the group of sulphonylureas, such as by way of example and preferably tolbutamide, glibenclamide, glimepiride, glipizide or gliclazide, biguanides , such as by way of example and with preference metformin, meglitinide derivatives, such as by way of example and preferably repaglinide or nateglinide, glucosidase inhibitors, such as by way of example and preferably miglitol or acarbose, oxadiazolidinones, thiazolidinediones, GLP 1 receptor agonists, glucagon antagonists, insulin sensitizers, CCK 1 receptor
Agonisten, Leptin-Rezeptor-Agonisten, Inhibitoren von Leberenzymen, die an der Stimulation der Glukoneogenese und/oder Glykogenolyse beteiligt sind, Modulatoren der Glukoseaufnahme sowie der Kaliumkanalöffner, wie z.B. denjenigen, die in WO 97/26265 und WO 99/03861 offenbart sind; · den Blutdruck senkenden Wirkstoffen, beispielhaft und vorzugsweise aus der Gruppe der Calcium- Antagonisten wie beispielhaft und vorzugsweise Nifedipin, Amlodipin, Verapamil oder Diltiazem, Angiotensin AII-Antagonisten, wie beispielhaft und vorzugsweise Losartan, Valsartan, Candesartan, Embusartan oder Telmisartan, ACE-Inhibitoren, wie beispielhaft und vorzugsweise Enalapril, Captopril, Ramipril, Delapril, Fosinopril, Quinopril, Perindopril oder Trandopril, beta-Rezeptoren-Blocker, wie beispielhaft und vorzugsweise Propranolol, Atenolol,Agonists, leptin receptor agonists, inhibitors of liver enzymes involved in the stimulation of gluconeogenesis and / or glycogenolysis, modulators of glucose uptake, and potassium channel openers, e.g. those disclosed in WO 97/26265 and WO 99/03861; The hypotensive agents, by way of example and preferably from the group of calcium antagonists such as, by way of example and by way of preference, nifedipine, amlodipine, verapamil or diltiazem, angiotensin AII antagonists such as, for example and preferably, losartan, valsartan, candesartan, embusartan or telmisartan, ACE inhibitors , as exemplified and preferably, enalapril, captopril, ramipril, delapril, fosinopril, quinopril, perindopril or trandopril, beta-receptor blockers such as, by way of example and by way of preference, propranolol, atenolol,
Timolol, Pindolol, Alprenolol, Oxprenolol, Penbutolol, Bupranolol, Metipranolol, Nadolol, Mepindolol, Carazalol, Sotalol, Metoprolol, Betaxolol, Celiprolol, Bisoprolol, Carteolol, Esmolol, Labetalol, Carvedilol, Adaprolol, Landiolol, Nebivolol, Epanolol oder Bucindolol, alpha-Rezeptoren-Blocker, wie beispielhaft und vorzugsweise Prazosin, ECE-Inhibitoren, Rhokinase Inhibitoren und der Vasopeptidase-Inhibitoren, sowie der Diuretika, wie beispielhaft und vorzugsweise einem Schleifendiuretikum wie Furosemid, Bumetanid oder Torsemid, oder einem Thiazid- oder Thiazid-ähnlichen Diuretikum wie Chlorthiazid oder Hydrochlorthiazid oder AI Antagonisten wie Rolofylline, Tonopofylline und SLV-320; Timolol, pindolol, alprenolol, oxprenolol, penbutolol, bupranolol, metipranolol, nadolol, mepindolol, carazalol, sotalol, metoprolol, betaxolol, celiprolol, bisoprolol, carteolol, esmolol, labetalol, carvedilol, adaprolol, landiolol, nebivolol, epanolol or bucindolol, alpha- Receptor blockers such as by way of example and preferably prazosin, ECE inhibitors, rhokinase inhibitors and the vasopeptidase inhibitors, and diuretics, such as by way of example and preferably a loop diuretic such as furosemide, bumetanide or torsemide, or a thiazide or thiazide-like diuretic such as chlorothiazide or hydrochlorothiazide or AI antagonists such as Rolofylline, Tonopofylline and SLV-320;
• den Sympathikustonus senkende Mittel wie beispielhaft und vorzugsweise Reserpin, Clonidin oder alpha-Methyl-Dopa, oder in Kombination mit einem Kaliumkanal- Agonisten, wie beispielhaft und vorzugsweise Minoxidil, Diazoxid, Dihydralazin oder Hydralazin, Sympathetic tone lowering agents such as, for example and preferably, reserpine, clonidine or alpha-methyl-dopa, or in combination with a potassium channel agonist, such as, for example and preferably, minoxidil, diazoxide, dihydralazine or hydralazine;
• antithrombotisch wirkenden Mitteln, beispielhaft und vorzugsweise aus der Gruppe der Thrombozytenaggregationshemmer, wie beispielhaft und vorzugsweise Aspirin, Clopidogrel, Ticlopidin, Cilostazol oder Dipyridamol, oder der Antikoagulantien wie Thrombin-Inhibitoren, wie beispielhaft und vorzugsweise Ximelagatran, Melagatran, Bivalirudin oder Clexane, einem GPIIb/IIIa-Antagonisten, wie beispielhaft und vorzugsweise Tirofiban oder Abciximab, einem Faktor Xa-Inhibitor, wie beispielhaft und vorzugsweise Rivaroxaban (BAY 59-7939), DU- 176b, Apixaban, Otamixaban, Fidexaban, Razaxaban, Fondaparinux, Idraparinux, PMD-3112, YM-150, KFA-1982, EMD-503982, MCM-17, MLN-1021 , DX 9065a, DPC 906, JTV 803,Antithrombotic agents, by way of example and preferably from the group of platelet aggregation inhibitors, such as by way of example and preferably aspirin, clopidogrel, ticlopidine, cilostazol or dipyridamole, or the anticoagulants such as thrombin inhibitors, such as by way of example and preferably ximelagatran, melagatran, bivalirudin or Clexane, a GPIIb / IIIa antagonist such as, by way of example and preferably, tirofiban or abciximab, a factor Xa inhibitor such as, by way of example and by way of preference, rivaroxaban (BAY 59-7939), DU-176b, apixaban , Otamixaban, Fidexaban, Razaxaban, Fondaparinux, Idraparinux, PMD-3112, YM-150, KFA-1982, EMD-503982, MCM-17, MLN-1021, DX 9065a, DPC 906, JTV 803,
SSR-126512 oder SSR-128428, mit Heparin oder einem low molecular weight (LMW)- Heparin-Derivat oder mit einem Vitamin K-Antagonisten, wie beispielhaft und vorzugsweise Coumarin, SSR-126512 or SSR-128428, with heparin or a low molecular weight (LMW) heparin derivative or with a vitamin K antagonist, such as by way of example and preferably coumarin,
• Aldosteron- und Mineralokorticoid-Rezeptor-Antagonisten, wie beispielhaft und vorzugsweise Spironolacton oder Eplerenon; Aldosterone and mineralocorticoid receptor antagonists, such as by way of example and preferably spironolactone or eplerenone;
• Vasopressin-Rezeptor- Antagonisten, wie beispielhaft und vorzugsweise Conivaptan, Tolvaptan, Lixivaptan oder SR-121463,; Vasopressin receptor antagonists, such as by way of example and preferably Conivaptan, Tolvaptan, Lixivaptan or SR-121463;
• organischen Nitraten und NO-D onatoren wie b eispielhaft und vorzugweis e Natriumnitroprussid, Nitroglycerin, Isosorbidmononitrat, Isosorbiddinitrat, Molsidomin oder SIN- 1 , oder in Kombination mit inhalativem NO; Organic nitrates and NO donors such as, for example, sodium nitroprusside, nitroglycerin, isosorbide mononitrate, isosorbide dinitrate, molsidomine or SIN-1, or in combination with inhaled NO;
• IP-Rezeptor Agonisten wie wie beispielhaft und vorzugweise Iloprost, Treprostinil, Beraprost und NS-304 IP receptor agonists such as, by way of example, and iloprost, treprostinil, beraprost, and NS-304
• positiv-inotrop wirksamen Verbindungen, wie beispielhaft und vorzugsweise Herzglycosiden (Digoxin), beta-adrenergen und dopaminergen Agonisten wie Isoproterenol, Adrenalin, Noradrenalin, Dopamin oder Dobutamin,; Positive inotropic compounds, such as, by way of example and by way of illustration, cardiac glycosides (digoxin), beta-adrenergic and dopaminergic agonists such as isoproterenol, epinephrine, norepinephrine, dopamine or dobutamine;
• Calcium-Sensitizern, wie beispielhaft und vorzugsweise Levosimendan; Calcium sensitizers, such as by way of example and preferably levosimendan;
• Verbindungen, die den Abbau von cyclischem Guanosinmonophosphat (cGMP) und/oder cyclischem Adenosinmonophosphat (cAMP) inhibieren, wie beispielsweise Inhibitoren der Phosphodiesterasen (PDE) 1, 2, 3, 4 und/oder 5, insbesondere PDE 5 -Inhibitoren wie Sildenafil, Vardenafil und Tadalafil sowie PDE 3 -Inhibitoren wie Milrinone; Compounds which inhibit the degradation of cyclic guanosine monophosphate (cGMP) and / or cyclic adenosine monophosphate (cAMP), for example inhibitors of phosphodiesterases (PDE) 1, 2, 3, 4 and / or 5, in particular PDE 5 inhibitors such as sildenafil, Vardenafil and tadalafil and PDE 3 inhibitors such as milrinone;
• natriuretischen Peptiden, wie z.B. "atrial natriuretic peptide" (ANP, Anaritide), "B-type natriuretic peptide" oder "brain natriuretic peptide" (BNP, Nesiritide), "C-type natriuretic peptide" (CNP) sowie Urodilatin; • NO-unabhängigen, jedoch Häm-abhängigen Stimulatoren der Guanylatcyclase, wie insbesondere den in WO 00/06568, WO 00/06569, WO 02/42301 und WO 03/095451 beschriebenen Verbindungen; Natriuretic peptides such as "atrial natriuretic peptide" (ANP, anaritide), "B-type natriuretic peptide" or "brain natriuretic peptide" (BNP, nesiritide), "C-type natriuretic peptide" (CNP) and urodilatin; NO-independent, but heme-dependent guanylate cyclase stimulators, in particular the compounds described in WO 00/06568, WO 00/06569, WO 02/42301 and WO 03/095451;
• NO- und Häm-unabhängigen Aktivatoren der Guanylatcyclase, wie insbesondere den in WO 01/19355, WO 01/19776, WO 01/19778, WO 01/19780, WO 02/070462 und WO 02/070510 beschriebenen Verbindungen; Guanylate cyclase NO- and heme-independent activators, in particular the compounds described in WO 01/19355, WO 01/19776, WO 01/19778, WO 01/19780, WO 02/070462 and WO 02/070510;
• Inhibitoren der humanen neutrophilen Ekstase (HNE), wie beispielsweise Sivelestat und DX- 890 (Reltran); • inhibitors of human neutrophilic ecstasy (HNE), such as Sivelestat and DX-890 (Reltran);
• die Signaltransduktionskaskade inhibierenden Verbindungen, wie beispielsweise Tyrosinkinase- Inhibitoren und Multikinase Inhibitoren, insbesondere Sorafenib, Imatinib, Gefitinib undThe signal transduction cascade inhibiting compounds, such as tyrosine kinase inhibitors and multikinase inhibitors, in particular sorafenib, imatinib, gefitinib and
Erlotinib; und/oder erlotinib; and or
• den Energiestoffwechsel des Herzens beeinflussenden Verbindungen, wie beispielweise Eto- moxir, Dichloracetat, Ranolazine und Trimetazidine kombiniert werden. Besonders bevorzugt im Rahmen der vorliegenden Erfindung sind Kombinationen enthaltend mindestens eine der erfindungsgemäßen Verbindungen sowie einen oder mehrere weitere Wirkstoffe ausgewählt aus der Gruppe bestehend aus HMG-CoA-Reduktase-Inhibitoren (Statine), Diuretika, Antidiabetika, beta-Rezeptoren-Blocker, organische Nitrate und NO-Donatoren, ACE-Inhibitoren, Angiotensin AII-Antagonisten, Aldosteron- und Mineralokortikoid-Rezeptor-Antagonisten, Vasopressin-Rezeptor-Antagonisten, Thrombozytenaggregationshemmer und Antikoagulantien, sowie deren Verwendung zur Behandlung und/oder Prävention der zuvor genannten Erkrankungen. • Compounds influencing the energy metabolism of the heart, such as, for example, estimoxir, dichloroacetate, ranolazine and trimetazidine. Particularly preferred in the context of the present invention are combinations containing at least one of the compounds according to the invention and one or more further active ingredients selected from the group consisting of HMG-CoA reductase inhibitors (statins), diuretics, antidiabetics, beta-receptor blockers, organic nitrates and NO donors, ACE inhibitors, angiotensin AII antagonists, aldosterone and mineralocorticoid receptor antagonists, vasopressin receptor antagonists, platelet aggregation inhibitors and anticoagulants, and their use for the treatment and / or prevention of the aforementioned disorders.
Die erfindungsgemäßen Verbindungen können systemisch und/oder lokal wirken. Zu diesem Zweck können sie auf geeignete Weise appliziert werden, wie z.B. oral, parenteral, pulmonal, nasal, sublingual, lingual, buccal, rectal, dermal, transdermal, conjunctival, otisch oder als Implantat bzw. Stent. The compounds according to the invention can act systemically and / or locally. For this purpose, they 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.
Für diese Applikationswege können die erfindungsgemäßen Verbindungen in geeigneten Applikationsformen verabreicht werden. For these administration routes, the compounds according to the invention can be administered in suitable administration forms.
Für die orale Applikation eignen sich nach dem Stand der Technik funktionierende schnell und/oder modifiziert die erfindungsgemäßen Verbindungen abgebende Applikationsformen, die die erfindungsgemäßen Verbindungen in kristalliner und/ oder amorphisierter und/oder gelöster Form enthalten, wie z.B. Tabletten (nicht überzogene oder überzogene Tabletten, beispielsweise mit magensaftresistenten oder sich verzögert auflösenden oder unlöslichen Überzügen, die die Freisetzung der erfindungsgemäßen Verbindung kontrollieren), in der Mundhöhle schnell zerfallende Tabletten oder Filme/Oblaten, Filme/Lyophylisate, Kapseln (beispielsweise Hart- oder Weichgelatinekapseln), Dragees, Granulate, Pellets, Pulver, Emulsionen, Suspensionen, Aerosole oder Lösungen. For the oral administration are according to the prior art functioning rapidly and / or modified compounds of the invention donating application forms, the compounds of the invention in crystalline and / or amorphized and / or dissolved form such as tablets (uncoated or coated tablets, for example with enteric or delayed-dissolving or insoluble coatings which control the release of the compound of the invention), orally disintegrating tablets or films / wafers, films / lyophilisates, capsules (e.g. Hard or soft gelatin capsules), dragees, granules, pellets, powders, emulsions, suspensions, aerosols or solutions.
Die parenterale Applikation kann unter Umgehung eines Resorptionsschrittes geschehen (z.B. intravenös, intraarteriell, intrakardial, intraspinal oder intralumbal) oder unter Einschaltung einer Resorption (z.B. intramuskulär, subcutan, intracutan, percutan oder intraperitoneal). Für die parenterale Applikation eignen sich als Applikationsformen u.a. Injektions- und Infusionszubereitungen in Form von Lösungen, Suspensionen, Emulsionen, Lyophilisaten oder sterilen Pulvern. Parenteral administration can be accomplished by bypassing a resorption step (e.g., intravenously, intraarterially, intracardially, intraspinal, or intralumbar) or by resorting to absorption (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.
Bevorzugt ist die orale Applikation. The oral application is preferred.
Für die sonstigen Applikationswege eignen sich z.B. Inhalationsarzneiformen (u.a. Pulverinhalatoren, Nebulizer), Nasentropfen, -lösungen, -sprays; lingual, sublingual oder buccal zu applizierende Tabletten, Filme/Oblaten oder Kapseln, Suppositorien, Ohren- oder Augenpräparationen, Vaginalkapseln, wässrige Suspensionen (Lotionen, Schüttelmixturen), lipophile Suspensionen, Salben, Cremes, transdermale therapeutische Systeme (wie beispielsweise Pflaster), Milch, Pasten, Schäume, Streupuder, Implantate oder Stents. For the other routes of administration are suitable, for example Inhalation medicines (including powder inhalers, nebulizers), nasal drops, solutions, sprays; lingual, sublingual or buccal tablets, films / wafers or capsules, suppositories, ear or ophthalmic preparations, vaginal capsules, aqueous suspensions (lotions, shake mixtures), lipophilic suspensions, ointments, creams, transdermal therapeutic systems (such as patches), milk, Pastes, foams, scattering powders, implants or stents.
Ebenfalls bevorzugt ist die inhalative Applikation. Die erfindungsgemäßen Verbindungen können in die angeführten Applikationsformen überführt werden. Dies kann in an sich bekannter Weise durch Mischen mit inerten, nichttoxischen, pharmazeutisch geeigneten Hilfsstoffen geschehen. Zu diesen Hilfsstoffen zählen u.a. Trägerstoffe (beispielsweise mikrokristalline Cellulose, Laktose, Mannitol), Lösungsmittel (z.B. flüssige Poly- ethylenglycole), Emulgatoren und Dispergier- oder Netzmittel (beispielsweise Natriumdodecylsulfat, Polyoxysorbitanoleat), Bindemittel (beispielsweise Polyvinylpyrrolidon), synthetische und natürliche Polymere (beispielsweise Albumin), Stabilisatoren (z.B. Antioxidantien wie beispielsweise Ascorbinsäure), Farbstoffe (z.B. anorganische Pigmente wie beispielsweise Eisenoxide) und Geschmacks- und / oder Geruchskorrigentien. Also preferred is the inhalation application. The compounds according to the invention can be converted into the stated administration forms. This can be done in a conventional manner by mixing with inert, non-toxic, pharmaceutically suitable excipients. These adjuvants include, among others. Carriers (for example microcrystalline cellulose, lactose, mannitol), solvents (for example liquid polyethylene glycols), emulsifiers and dispersants or wetting agents (for example sodium dodecyl sulfate, polyoxysorbitanoleate), binders (for example polyvinylpyrrolidone), synthetic and natural polymers (for example albumin), stabilizers ( For example, antioxidants such as ascorbic acid), dyes (eg, inorganic pigments such as iron oxides) and flavor and / or odoriferous.
Weiterer Gegenstand der vorliegenden Erfindung sind Arzneimittel, die mindestens eine erfin- dungsgemäße Verbindung, vorzugsweise zusammen mit einem oder mehreren inerten nichttoxischen, pharmazeutisch geeigneten Hilfsstoff enthalten, sowie deren Verwendung zur Behandlung von Sichelzellanämie und Konservierung von Blutersatzstoffen. Im Allgemeinen hat es sich als vorteilhaft erwiesen, bei parenteraler Applikation Mengen von etwa 5 bis 100 mg je 24 Stunden zur Erzielung wirksamer Ergebnisse zu verabreichen. Bei oraler Applikation beträgt die Menge etwa 5 bis 250 mg je 24 Stunden. Another object of the present invention are pharmaceutical compositions containing at least one inventive compound, preferably together with one or more inert non-toxic, pharmaceutically suitable excipient, as well as their use for the treatment of sickle cell anemia and preservation of blood substitutes. In general, it has been found advantageous to administer parenterally administered amounts of about 5 to 100 mg per 24 hours to achieve effective results. When administered orally, the amount is about 5 to 250 mg per 24 hours.
Trotzdem kann es gegebenenfalls erforderlich sein, von den genannten Mengen abzuweichen, und zwar in Abhängigkeit von Körpergewicht, Applikationsweg, individuellem Verhalten gegenüber dem Wirkstoff, Art der Zubereitung und Zeitpunkt bzw. Intervall, zu welchem die Applikation erfolgt. Nevertheless, it may be necessary to deviate from the stated amounts, depending on body weight, route of administration, individual behavior towards the active ingredient, type of preparation and time or interval at which the application is carried out.
Die Prozentangaben in den folgenden Tests und Beispielen sind, sofern nicht anders angegeben, Gewichtsprozente; Teile sind Gewichtsteile. Lösungsmittelverhältnisse, Verdünnungsverhältnisse und Konzentrationsangaben von flüssig/flüssig-Lösungen beziehen sich jeweils auf das Volumen. Die Angabe "w/v" bedeutet "weight/volume" (Gewicht/Volumen). So bedeutet beispielsweise "10% w/v": 100 ml Lösung oder Suspension enthalten 10 g Substanz. The percentages in the following tests and examples are by weight unless otherwise indicated; Parts are parts by weight. Solvent ratios, dilution ratios and concentration data of liquid / liquid solutions are based on volume. The term "w / v" means "weight / volume". Thus, for example, "10% w / v" means: 100 ml of solution or suspension contains 10 g of substance.
Experimenteller Teil Experimental part
A. Beispiele A. Examples
Abkürzungen und Akronyme: Abbreviations and acronyms:
aq. wässrige Lösung aq. aqueous solution
ber. Berechnet Calculated
DCI direkte chemische Ionisation (bei MS)  DCI direct chemical ionization (in MS)
DMF Dimethylformamid  DMF dimethylformamide
DMSO Dimethylsulfoxid  DMSO dimethyl sulfoxide
d. Th. der Theorie (bei Ausbeute) d. Th. Of theory (at yield)
eq. Äquivalent(e) eq. Equivalent (s)
ESI Elektrospray-Ionisation (bei MS)  ESI electrospray ionization (in MS)
Et Ethyl  Et ethyl
gef. Gefunden gef. Found
h Stunde(n) h hour (s)
HPLC Hochdruck-, Hochleistungsflüssigchromatographie HPLC high pressure, high performance liquid chromatography
HRMS hochaufgelöste Massenspektrometrie HRMS high-resolution mass spectrometry
konz. konzentriert conc. concentrated
LC/MS Flüssigchromatographie-gekoppelte Massenspektrometrie LC / MS liquid chromatography-coupled mass spectrometry
LiHMDS Lithiumhexamethyldisilazid LiHMDS lithium hexamethyldisilazide
Me Methyl  Me methyl
min Minute(n) min minute (s)
MS Massenspektrometrie NMR Kernresonanzspektrometrie MS mass spectrometry NMR nuclear magnetic resonance spectrometry
Pd2dba3 Tris-(dibenzylidenaceton)-dipalladium Pd 2 dba 3 tris (dibenzylideneacetone) dipalladium
Ph Phenyl  Ph phenyl
RT Raumtemperatur  RT room temperature
Rt Retentionszeit (bei HPLC) R t retention time (by HPLC)
THF Tetrahydrofuran  THF tetrahydrofuran
UV Ultraviolett-Spektrometrie  UV ultraviolet spectrometry
v/v Volumen zu Volumen- Verhältnis (einer Lösung)v / v volume to volume ratio (of a solution)
XPHOS Dicyclohexyl-(2',4',6'-triisopropylbiphenyl-2-yl)-phosphin XPHOS dicyclohexyl- (2 ', 4', 6'-triisopropylbiphenyl-2-yl) -phosphine
LC/MS-Methoden: LC / MS methods:
Methode 1 : Gerätetyp MS: Waters ZQ; Gerätetyp HPLC: Agilent 1100 Series; UV DAD; Säule: Thermo Hypersil GOLD 3 μ 20 mm x 4 mm; Eluent A: 1 1 Wasser + 0.5 ml 50%-ige Ameisensäure, Eluent B: 1 1 Acetonitril + 0.5 ml 50%>-ige Ameisensäure; Gradient: 0.0 min 100% A -»■ 3.0 min 10% A -> 4.0 min 10% A -> 4.1 min 100% A (Fluss 2.5 ml/min); Ofen: 55°C; Fluss: 2 ml/min; UV-Detektion: 210 nm. Method 1: Device Type MS: Waters ZQ; Device Type HPLC: Agilent 1100 Series; UV DAD; Column: Thermo Hypersil GOLD 3 μ 20 mm x 4 mm; Eluent A: 1 l of water + 0.5 ml of 50% formic acid, eluent B: 1 l of acetonitrile + 0.5 ml of 50% strength formic acid; Gradient: 0.0 min 100% A -> 3.0 min 10% A -> 4.0 min 10% A -> 4.1 min 100% A (flow 2.5 ml / min); Oven: 55 ° C; Flow: 2 ml / min; UV detection: 210 nm.
Methode 2: Instrument: Waters ACQUITY SQD UPLC System; Säule: Waters Acquity UPLC HSS T3 1.8 μ 50 x 1 mm; Eluent A: 1 1 Wasser + 0.25 ml 99%>ige Ameisensäure , Eluent B : 1 1 Acetonitril + 0.25 ml 99%>ige Ameisensäure; Gradient: 0.0 min 90% A -> 1.2 min 5% A -> 2.0 min 5% A Ofen: 50 °C; Fluss: 0.40 ml/min; UV-Detektion: 210 - 400 nm. Method 2: Instrument: Waters ACQUITY SQD UPLC System; Column: Waters Acquity UPLC HSS T3 1.8 μ 50 x 1 mm; Eluent A: 1 l of water + 0.25 ml of 99% formic acid, eluent B: 1 l of acetonitrile + 0.25 ml of 99% formic acid; Gradient: 0.0 min 90% A -> 1.2 min 5% A -> 2.0 min 5% A Furnace: 50 ° C; Flow: 0.40 ml / min; UV detection: 210 - 400 nm.
Ausgangsverbindungen und Intermediate: Starting compounds and intermediates:
Beispiel 1A Example 1A
2,6-Dichlor-5-fluornicotinamid 2,6-dichloro-5-fluoronicotinamide
Eine Suspension aus 25 g (130.90 mmol) 2,6-Dichlor-5-fluor-3-cyanopyridin in konz. Schwefelsäure (125 ml) wurde 1 h bei 60-65°C gerührt. Nach Abkühlen auf RT wurde der Kolbeninhalt auf Eiswasser gegossen und dreimal mit Essigsäureethylester (je 100 ml) extrahiert. Die vereinigten organischen Phasen wurden mit Wasser (100 ml) und anschliessend mit gesättigter wässriger Natriumhydrogencarbonat-Lösung (100 ml) gewaschen, getrocknet und am Rotations Verdampfer eingeengt. Das erhaltene Material wurde am Hochvakuum getrocknet. A suspension of 25 g (130.90 mmol) of 2,6-dichloro-5-fluoro-3-cyanopyridine in conc. Sulfuric acid (125 ml) was stirred at 60-65 ° C for 1 h. After cooling to RT, the contents of the flask were poured into ice-water and extracted three times with ethyl acetate (100 ml each time). The combined organic phases were washed with water (100 ml) and then with saturated aqueous sodium bicarbonate solution (100 ml), dried and concentrated on a rotary evaporator. The resulting material was dried under high vacuum.
Ausbeute: 24.5 g (90 % d. Theorie) Yield: 24.5 g (90% of theory)
'H-NMR (400 MHz, DMSO-de): δ = 7.95 (br s, 1H), 8.11 (br s, 1H), 8.24 (d, 1H). Beispiel 2A 'H-NMR (400 MHz, DMSO-de): δ = 7.95 (brs, 1H), 8.11 (brs, 1H), 8.24 (d, 1H). Example 2A
2-Chlor-5-fluornicotinamid 2-chloro-5-fluoronicotinamide
Einer Suspension von 21.9 g (335.35 mmol) Zink in Methanol (207 ml) wurden bei RT 44 g (210.58 mmol) 2,6-Dichlor-5-fluornicotinamid zugegeben. Danach wurde mit Essigsäure (18.5 ml) versetzt und unter Rühren für 24 h zum Rückfluss erhitzt. Danach wurde der Kolbeninhalt vom Zink dekantiert und Essigsäureethylester (414 ml) sowie gesättigte wässrige Natriumhydrogencarbonat- Lösung (414 ml) zugegeben und intensiv ausgerührt. Anschliessend wurde über Kieselgur abgesaugt und dreimal mit Essigsäureethylester (je 517 ml) nachgewaschen. Die organische Phase wurde abgetrennt und die wässrige Phase mit Essigsäureethylester (258 ml) gewaschen. Die vereinigten organischen Phasen wurden einmal mit gesättigter wässriger Natriumhydrogencarbonat-Lösung (414 ml) gewaschen, getrocknet und im Vakuum eingeengt. Die so erhaltenen Kristalle wurden mit Dichlormethan (388 ml) versetzt und 20 min. ausgerührt. Es wurde erneut abgesaugt und mit Diethylether nachgewaschen und trockengesaugt. To a suspension of 21.9 g (335.35 mmol) of zinc in methanol (207 ml) was added at RT 44 g (210.58 mmol) of 2,6-dichloro-5-fluoronicotinamide. Acetic acid (18.5 ml) was then added and the mixture was heated to reflux with stirring for 24 h. The contents of the flask were then decanted from the zinc and ethyl acetate (414 ml) and saturated aqueous sodium bicarbonate solution (414 ml) were added and the mixture was thoroughly stirred. The mixture was then filtered off with suction through kieselguhr and washed three times with ethyl acetate (517 ml each time). The organic phase was separated and the aqueous phase washed with ethyl acetate (258 ml). The combined organic phases were washed once with saturated aqueous sodium bicarbonate solution (414 ml), dried and concentrated in vacuo. The crystals thus obtained were washed with Dichloromethane (388 ml) and 20 min. stirred. It was again filtered off with suction and washed with diethyl ether and sucked dry.
Ausbeute: 20.2 g (53 % d. Theorie) Yield: 20.2 g (53% of theory)
'H-NMR (400 MHz, DMSO-de): δ = 7.87 (br s, 1H), 7.99 (dd, 1H), 8.10 (br s, 1H), 8.52 (d, 1H). Beispiel 3A 'H-NMR (400 MHz, DMSO-de): δ = 7.87 (brs, 1H), 7.99 (dd, 1H), 8.10 (brs, 1H), 8.52 (d, 1H). Example 3A
2-Chlor-5-fluornicotinonitril 2-chloro-5-fluornicotinonitril
Eine Suspension von 46.2 g (264.66 mmol) 2-Chlor-5-fluornicotinamid in Dichlormethan (783 ml) wurde mit 81.2 ml (582.25 mmol) Triethylamin versetzt und auf 0°C gekühlt. Unter Rühren wurden dann 41.12 ml (291.13 mmol) Trifluoressigsäureanhydrid langsam zugetropft und 1.5 h bei 0°C nachgerührt. Die Reaktionslösung wurde danach zweimal mit gesättigter wässriger Natrium- hydrogencarbonat-Lösung (je 391 ml) gewaschen, getrocknet und im Vakuum eingeengt. A suspension of 46.2 g (264.66 mmol) of 2-chloro-5-fluoronicotinamide in dichloromethane (783 ml) was admixed with 81.2 ml (582.25 mmol) of triethylamine and cooled to 0 ° C. 41.12 ml (291.13 mmol) of trifluoroacetic anhydride were then slowly added dropwise with stirring and stirred at 0 ° C. for 1.5 h. The reaction solution was then washed twice with saturated aqueous sodium bicarbonate solution (each 391 ml), dried and concentrated in vacuo.
Ausbeute: 42.1 g (90 % d. Theorie). Yield: 42.1 g (90% of theory).
'H-NMR (400 MHz, DMSO-de): δ = 8.66 (dd, 1H), 8.82 (d, 1H). Beispiel 4A 'H-NMR (400 MHz, DMSO-de): δ = 8.66 (dd, 1H), 8.82 (d, 1H). Example 4A
5-Fluor-lH-pyrazolo[3,4-b]pyridin-3-amin 5-fluoro-lH-pyrazolo [3,4-b] pyridin-3-amine
Eine Suspension aus 38.5 g (245.93 mmol) 2-Chlor-5-fluornicotinonitril wurde in 1 ,2-Ethandiol (380 ml) vorgelegt und danach mit Hydrazinhydrat (1 19.6 ml, 2.459 mol) versetzt. Es wurde unter Rühren für 4 h zum Rückfluss erhitzt. Beim Abkühlen fiel das Produkt aus. Die gelben Kristalle wurden mit Wasser (380 ml) versetzt und 10 min. bei RT ausgerührt. Anschliessend wurde die Suspension über eine Fritte abgesaugt, mit Wasser (200 ml) und mit -10°C-kaltem THF (200 ml) nachgewaschen. Der Rückstand wurde im Hochvakuum über Phosphorpentoxid getrocknet. Ausbeute: 22.8 g (61 % d. Theorie) A suspension of 38.5 g (245.93 mmol) of 2-chloro-5-fluoronicotinonitrile was initially charged in 1,2-ethanediol (380 ml), followed by addition of hydrazine hydrate (1.196 ml, 2.459 mol). It was heated to reflux with stirring for 4 h. Upon cooling, the product precipitated. The yellow crystals were added with water (380 ml) and stirred for 10 min. stirred at RT. The suspension was then filtered off with suction through a frit, washed with water (200 ml) and with -10 ° C. cold THF (200 ml). The residue was dried under high vacuum over phosphorus pentoxide. Yield: 22.8 g (61% of theory)
H-NMR (400 MHz, DMSO-de): δ = 5.54 (s, 2H), 7.96 (dd, 1H), 8.38 (m, 1H), 12.07(m, 1H). H-NMR (400 MHz, DMSO-de): δ = 5.54 (s, 2H), 7.96 (dd, 1H), 8.38 (m, 1H), 12.07 (m, 1H).
Beispiel 5A Example 5A
5-Fluor-3-iod-lH-pyrazolo[3,4-b]pyridin 5-fluoro-3-iodo-lH-pyrazolo [3,4-b] pyridine
In THF (329 ml) wurden 10 g (65.75 mmol) 5-Fluor-lH-pyrazolo[3,4-b]pyridin-3-amin vorgelegt und auf 0°C abgekühlt. Anschliessend wurden 16.65 ml (131.46 mmol) Bortrifluorid-Diethylether- Komplex langsam zugesetzt. Die Reaktionsmischung wurde weiter auf -10°C abgekühlt. Danach wurde eine Lösung von 10.01 g (85.45 mmol) Isopentylnitrit in THF (24.39 ml) langsam zugefügt und weitere 30 min nachgerührt. Die Mischung wurde mit kaltem Diethylether (329 ml) verdünnt und der entstandene Feststoff abfiltriert. Das so hergestellte Diazoniumsalz wurde portionsweise in eine 0°C kalte Lösung von 12.81 g (85.45 mmol) Natriumiodid in Aceton (329 ml) gegeben und die Mischung 30 min bei RT nachgerührt. Die Reaktionsmischung wurde auf Eiswasser (1.8 1) gegeben und zweimal mit Essigsäureethylester (je 487 ml) extrahiert. Die gesammelten organischen Phasen wurden mit gesättigter wässriger Natriumchorid-Lösung (244 ml) gewaschen, getrocknet, filtriert und eingeengt. Man erhielt 12.1 g (86%-ige Reinheit, 60 % d. Th.) der gewünschten Verbindung als braunen Feststoff. Das Rohprodukt wurde ohne weitere Reinigung umgesetzt. 10 g (65.75 mmol) of 5-fluoro-1H-pyrazolo [3,4-b] pyridin-3-amine were initially charged in THF (329 ml) and cooled to 0 ° C. Subsequently, 16.65 ml (131.46 mmol) of boron trifluoride-diethyl ether complex were slowly added. The reaction mixture was further cooled to -10 ° C. Thereafter, a solution of 10.01 g (85.45 mmol) of isopentyl nitrite in THF (24.39 ml) was slowly added and stirred for a further 30 min. The mixture was diluted with cold diethyl ether (329 ml) and the resulting solid filtered off. The diazonium salt thus prepared was added in portions to a 0 ° C cold solution of 12.81 g (85.45 mmol) of sodium iodide in acetone (329 ml) and the mixture stirred for 30 min at RT. The reaction mixture was added to ice water (1.8 L) and extracted twice with ethyl acetate (487 mL each). The collected organic phases were washed with saturated aqueous sodium chloride solution (244 ml), dried, filtered and concentrated. This gave 12.1 g (86% purity, 60% of theory) of the desired compound as a brown solid. The crude product was reacted without further purification.
LC-MS (Methode 1): Rt = 1.68 min; MS (ESIpos): m/z = 264 (M+H)+ LC-MS (Method 1): R t = 1.68 min; MS (ESIpos): m / z = 264 (M + H) +
Beispiel 6A 5-Fluor-l-(2-fluorbenzyl)-3-iod-lH-pyrazolo[3,4-b]pyridin Example 6A 5-Fluoro-1- (2-fluorobenzyl) -3-iodo-1H-pyrazolo [3,4-b] pyridine
In DMF (2538 ml) wurden 141 g (462.1 1 mmol) der Verbindung aus Beispiel 5A vorgelegt und anschließend 96.09 g (508.32 mmol) 2-Fluorbenzylbromid sowie 165.62 g (508.32 mmol) Cäsiumcarbonat zugefügt. Die Mischung wurde zwei Stunden bei RT gerührt. Anschließend wurde die Reaktionsmischung auf gesättigte wässrige Natriumchlorid-Lösung (13670 ml) gegeben und zweimal mit Essigester (5858 ml) extrahiert. Die gesammelten organischen Phasen wurden mit gesättigter wässriger Natriumchlorid-Lösung (3905 ml) gewaschen, getrocknet, filtriert und eingeengt. Der Rückstand wurde an Kieselgel (Laufmittel: Petrolether/Essigsäureethylester 97:3) chromatographiert und die Produktfraktionen eingeengt. Der erhaltene Feststoff wurde in Dichlormethan gelöst und einmal mit gesättigter wässriger Natriumthiosulfatlösung (500 ml) und anschliessend mit gesättigter wässriger Natriumchlorid-Lösung (500 ml) gewaschen. Es wurde zur Trockene eingeengt und der Rückstand mit Diethylether aufgeschlämmt, abgesaugt und am Hochvakuum getrocknet. Man erhielt 106.6 g (62 % d. Th.) der gewünschten Verbindung. 141 g (462.1 l mmol) of the compound from Example 5A were initially charged in DMF (2538 ml), and then 96.09 g (508.32 mmol) of 2-fluorobenzyl bromide and 165.62 g (508.32 mmol) of cesium carbonate were added. The mixture was stirred at RT for two hours. Subsequently, the reaction mixture was added to saturated aqueous sodium chloride solution (13670 ml) and extracted twice with ethyl acetate (5858 ml). The collected organic phases were washed with saturated aqueous sodium chloride solution (3905 ml), dried, filtered and concentrated. The residue was chromatographed on silica gel (eluent: petroleum ether / ethyl acetate 97: 3) and the product fractions were concentrated. The resulting solid was dissolved in dichloromethane and washed once with saturated aqueous sodium thiosulfate solution (500 ml) and then with saturated aqueous sodium chloride solution (500 ml). It was concentrated to dryness and the residue was slurried with diethyl ether, filtered off with suction and dried under high vacuum. 106.6 g (62% of theory) of the desired compound were obtained.
LC-MS (Methode 1): Rt = 2.57 min LC-MS (Method 1): R t = 2.57 min
MS (ESIpos): m/z = 372 (M+H)+ 'H-NMR (400 MHz, DMSO-de): δ = 5.73 (s, 2H), 7.13 - 7.26 (m, 3H), 7.33 - 7.41 (m, 1H), 7.94 (dd, 1H), 8.69 - 8.73 (m, 1H). MS (ESIpos): m / z = 372 (M + H) + 'H-NMR (400 MHz, DMSO-de): δ = 5.73 (s, 2H), 7.13 - 7.26 (m, 3H), 7.33 - 7.41 (m, 1H), 7.94 (dd, 1H), 8.69-8.73 (m, 1H).
Beispiel 7A Example 7A
2-[5-Fluor-l-(2-fluorbenzyl)-lH-pyrazolo[3,4-b]pyridin-3-yl]-5-nitropyrimidin-4,6-diami 2- [5-fluoro-l- (2-fluorobenzyl) -lH-pyrazolo [3,4-b] pyridin-3-yl] -5-nitro-4,6-diamino
In 1,4-Dioxan (86 ml) wurden 860 mg (2.32 mmol) der Verbindung aus Beispiel 6A unter Argon vorgelegt und die Reaktionsmischung 10 min mit Argon gespült. Danach wurden 3.51 ml (6.95 mmol) Hexabutyldizinn sowie 483 mg (2.55 mmol) 2-Chlor-5-nitropyrimidin-4,6-diamin (Herstellung erfolgte analog zu: Helvetica Chimica Acta (1951), 34, 835-40) zugefügt. Anschließend wurden 860 mg (0.744 mmol) Tetrakis(triphenylphosphin)-palladium(0) zugegeben und die Reaktionsmischung über Nacht zum Rückfluss erhitzt. Danach wurde auf RT abgekühlt, mit Wasser versetzt und zweimal mit Essigsäureethylester extrahiert. Die gesammelten organischen Phasen wurden über Natriumsulfat getrocknet, filtriert und eingeengt. Der Rückstand wurde in Essigsäureethylester ausgerührt, der Feststoff abfiltriert und im Hochvakuum getrocknet. Man erhielt 355 mg (62%-ige Reinheit, 24% d. Th.) der gewünschten Verbindung. Das Rohprodukt wurde ohne weitere Reinigung umgesetzt. In 1,4-dioxane (86 ml) 860 mg (2.32 mmol) of the compound from Example 6A were placed under argon and the reaction mixture was purged with argon for 10 min. Thereafter, 3.51 ml (6.95 mmol) of hexabutylditin and 483 mg (2.55 mmol) of 2-chloro-5-nitropyrimidine-4,6-diamine (preparation was carried out analogously to: Helvetica Chimica Acta (1951), 34, 835-40) were added. Subsequently, 860 mg (0.744 mmol) of tetrakis (triphenylphosphine) palladium (0) were added and the reaction mixture was heated to reflux overnight. It was then cooled to RT, treated with water and extracted twice with ethyl acetate. The collected organic phases were dried over sodium sulfate, filtered and concentrated. The residue was stirred in ethyl acetate, the solid was filtered off and dried under high vacuum. 355 mg (62% purity, 24% of theory) of the desired compound were obtained. The crude product was reacted without further purification.
LC-MS (Methode 2): Rt = 1.03 min LC-MS (Method 2): R t = 1.03 min
MS (ESIpos): m/z = 399 (M+H)+ Beispiel 8A MS (ESIpos): m / z = 399 (M + H) + Example 8A
5-Fluor-l-(2-fluorbenzyl)-lH-pyrazolo[3,4-b]pyridin-3-carbonitril 5-fluoro-l- (2-fluorobenzyl) -lH-pyrazolo [3,4-b] pyridine-3-carbonitrile
Eine Suspension aus 16.03 g (43.19 mmol) 5-Fluor-l-(2-fluorbenzyl)-3-iod-lH-pyrazolo[3,4- b]pyridin (Beispiel 6A) und 4.25 g (47.51 mmol) Kupfercyanid wurden in DMSO ( 120 ml) vorgelegt und 2 h bei 150°C gerührt. Nach Abkühlen wurde der Kolbeninhalt auf ca. 40°C abgekühlt, auf eine Lösung aus konz. Ammoniakwasser (90 ml) und Wasser (500 ml) gegossen, mit Essigsäureethylester (200 ml) versetzt und kurz ausgerührt. Die wässrige Phase wurde abgetrennt und noch zweimal mit Essigsäureethylester (je 200 ml) extrahiert. Die vereinigten organischen Phasen wurden zweimal mit 10%-iger wässriger Natriumchlorid-Lösung (je 100 ml) gewaschen, getrocknet und im Vakuum eingeengt. Das Rohprodukt wurde ohne weitere Reinigung umgesetzt. A suspension of 16.03 g (43.19 mmol) of 5-fluoro-1- (2-fluorobenzyl) -3-iodo-1H-pyrazolo [3,4-b] pyridine (Example 6A) and 4.25 g (47.51 mmol) of copper cyanide were added DMSO (120 ml) and stirred at 150 ° C for 2 h. After cooling, the flask contents were cooled to about 40 ° C, to a solution of conc. Ammonia water (90 ml) and water (500 ml) were poured, mixed with ethyl acetate (200 ml) and stirred briefly. The aqueous phase was separated and extracted twice more with ethyl acetate (200 ml each time). The combined organic phases were washed twice with 10% aqueous sodium chloride solution (100 ml each), dried and concentrated in vacuo. The crude product was reacted without further purification.
Ausbeute: 11.1 g (91 % d. Theorie) Yield: 11.1 g (91% of theory)
Tl-NMR (400 MHz, DMSO-de): δ = 5.87 (s, 2H), 7.17 - 7.42 (m, 4H), 8.52 (dd, 1H), 8.87 (dd, 1H). Beispiel 9A Tl NMR (400 MHz, DMSO-de): δ = 5.87 (s, 2H), 7.17-7.42 (m, 4H), 8.52 (dd, 1H), 8.87 (dd, 1H). Example 9A
5-Fluor-l-(2-fluorbenzyl)-lH-pyrazolo[3,4-b]pyridin-3-carboxiniidaniid-Acetat 5-fluoro-l- (2-fluorobenzyl) -lH-pyrazolo [3,4-b] pyridin-3-acetate carboxiniidaniid
CHgCOOHCH g COOH
Zu 2.22 g (41.07 mmol) Natriummethanolat in Methanol (270 ml) wurden 11.1 g (41.07 mmol) 5- Fluor-l-(2-fluorbenzyl)-lH-pyrazolo[3,4-b]pyridin-3-carbonitril (Beispiel 8A) gegeben und 2 h bei RT gerührt. Danach wurden 2.64 g (49.29 mmol) Ammoniumchlorid und Essigsäure (9.17 ml) zugegeben und über Nacht zum Rückfluss erhitzt. Danach wurde bis zur Trockene eingeengt und der Rückstand in Wasser (100 ml) und Essigsäureethylester (100 ml) aufgenommen und mit 2N Ntronlauge auf pH 10 gestellt. Es wurde für ca. 1 h bei RT intensiv gerührt. Die erhaltene Suspension wurde abgesaugt und mit Essigsäureethylester (100 ml), Wasser ( 100 ml) und nochmals Essigsäureethylester (100 ml) nachgewaschen. Der Rückstand wurde über Phosphorpentoxid im Hochvakuum getrocknet. To 2.22 g (41.07 mmol) of sodium methoxide in methanol (270 ml) were added 11.1 g (41.07 mmol) of 5-fluoro-1- (2-fluorobenzyl) -1H-pyrazolo [3,4-b] pyridine-3-carbonitrile (Example 8A) and stirred for 2 h at RT. Thereafter, 2.64 g (49.29 mmol) of ammonium chloride and acetic acid (9.17 ml) were added and heated to reflux overnight. It was then concentrated to dryness and the residue was taken up in water (100 ml) and ethyl acetate (100 ml) and adjusted to pH 10 with 2N sodium hydroxide solution. It was stirred vigorously for about 1 h at RT. The resulting suspension was filtered off with suction and washed with ethyl acetate (100 ml), water (100 ml) and again ethyl acetate (100 ml). The residue was dried over phosphorus pentoxide in a high vacuum.
Ausbeute: 9.6 g (78 % d. Th.) Yield: 9.6 g (78% of theory)
MS (ESIpos): m/z = 288 (M+H)+ 'H-NMR (400 MHz, DMSO-de): δ = 1.85 (s, 3H), 5.80 (s, 2H), 7.14 - 7.25 (m, 3H), 7.36 (m, 1H), 8.42 (dd, 1H), 8.72 (dd, 1H). MS (ESIpos): m / z = 288 (M + H) + H-NMR (400 MHz, DMSO-de): δ = 1.85 (s, 3H), 5.80 (s, 2H), 7.14 - 7.25 (m , 3H), 7.36 (m, 1H), 8.42 (dd, 1H), 8.72 (dd, 1H).
Beispiel 10A Example 10A
2-[5-Fluor-l-(2-fluorbenzyl)-lH-pyrazolo[3,4-b]pyridin-3-yl]-5-[(E)-phenyldiazenyl]pyrirmdin-4,6 diamin 2- [5-Fluoro-1- (2-fluorobenzyl) -1H-pyrazolo [3,4-b] pyridin-3-yl] -5 - [(E) -phenyldiazenyl] pyrimidine-4,6-diamine
Zu Wasser (40 ml) und konz. Salzsäure (7.07 ml) wurden unter Rühren 3.85 g (41.34 mmol) Anilin gegeben und auf 0°C gekühlt. Anschliessend wurde eine Lösung von 2.85 g (41.34 mmol) Natriumnitrit in Wasser (21 ml) zwischen 0°C und 5°C zugetropft und 15 min bei 0°C nachgerührt. Danach wurde bei 0°C eine Lösung aus 4.28 g (52.25 mmol) Natriumacetat in Wasser (19 ml) zügig zugetropft und dann unter gutem Rühren eine Lösung aus 2.73 g (41.34 mmol) Malonsäuredinitril in Ethanol (10 ml) zugetropft. Nach 2 h bei 0°C wurde der entstandene Niederschlag abgesaugt und dreimal mit Wasser (je 50 ml) und mit Petrolether (50 ml) gewaschen. Der noch feuchte Rückstand wurde in DMF (46 ml) gelöst und in eine Lösung aus 9.5 g (33.07 mmol) 5-Fluor-l-(2-fluorbenzyl)- lH-pyrazolo[3,4-b]pyridin-3-carboximidamid-Ac etat (B eisp ie l 9A) in D MF (46 ml) und Triethylamin (5.76 ml) bei genau 85°C zugetropft. Anschließend wurde 4 h bei 100°C nachgerührt und über Nacht auf RT abkühlen lassen. Die Mischung wurde auf Wasser (480 ml) gegossen und 1 h bei RT ausgerührt. Nach Absaugen des Niederschlags wurde dieser zweimal mit Wasser (je 100 ml) und zweimal mit Methanol (je 50 ml) nachgewaschen, und anschliessend im Hochvakuum getrocknet. To water (40 ml) and conc. Hydrochloric acid (7.07 ml) was added with stirring 3.85 g (41.34 mmol) of aniline and cooled to 0 ° C. Subsequently, a solution of 2.85 g (41.34 mmol) of sodium nitrite in water (21 ml) was added dropwise between 0 ° C and 5 ° C and stirred at 0 ° C for 15 min. Thereafter, a solution of 4.28 g (52.25 mmol) of sodium acetate in water (19 ml) was rapidly added dropwise at 0 ° C and then with good stirring, a solution of 2.73 g (41.34 mmol) of malononitrile in ethanol (10 ml) was added dropwise. After 2 h at 0 ° C, the resulting precipitate was filtered off with suction and washed three times with water (50 ml each) and with petroleum ether (50 ml). The still moist residue was dissolved in DMF (46 ml) and poured into a solution of 9.5 g (33.07 mmol) of 5-fluoro-1- (2-fluorobenzyl) -1H-pyrazolo [3,4-b] pyridine-3-carboximidamide -Ca etate (Example 9A) in D MF (46 ml) and triethylamine (5.76 ml) were added dropwise at exactly 85 ° C. The mixture was then stirred for 4 h at 100 ° C and allowed to cool to RT overnight. The mixture was poured onto water (480 ml) and stirred at RT for 1 h. After filtering off the precipitate, it was washed twice with water (100 ml each time) and twice with methanol (50 ml each time), and then dried under high vacuum.
Ausbeute: 9.6 g (59 % d. Theorie) LC-MS (Methode 2): Rt = 1.21 min MS (ESIpos): m/z = 458 (M+H)+ Beispiel IIA 2-[5-Fluor-l-(2-fluorbenzyl)-lH-pyrazolo[3,4-b]pyridin-3-yl]pyrimidin-4,5,6-triami Yield: 9.6 g (59% of theory) LC-MS (Method 2): R t = 1.21 min MS (ESIpos): m / z = 458 (M + H) + Example IIA 2- [5-fluoro-1 - (2-fluorobenzyl) -lH-pyrazolo [3,4-b] pyridin-3-yl] pyrimidine-4,5,6-Triami
Variante A: Darstellung ausgehend von Beispiel 7A: Variant A: Representation starting from Example 7A:
In Pyridin (30 mL) wurden 378 mg (0.949 mmol) der Verbindung aus Beispiel 7A vorgelegt und anschließend 143 mg (0.135 mmol) Palladium (10%ig auf Kohle) zugefügt. Die Mischung wurde über Nacht bei RT unter Wasserstoff-Normaldruck hydriert. Anschließend wurde die Suspension durch Kieselgur filtriert und der Filterkuchen mit Ethanol nachgewaschen. Das Filtrat wurde eingeengt und lieferte 233 mg (81 %ig, 51% d. Th.) der gewünschten Verbindung, die ohne weitere Reinigung umgesetzt wurden. In pyridine (30 ml), 378 mg (0.949 mmol) of the compound from Example 7A were initially charged and then 143 mg (0.135 mmol) of palladium (10% on carbon) were added. The mixture was hydrogenated overnight at RT under normal hydrogen pressure. The suspension was then filtered through kieselguhr and the filter cake was washed with ethanol. The filtrate was concentrated to afford 233 mg (81%, 51% of theory) of the desired compound, which were reacted without further purification.
Variante B: Darstellung ausgehend von Beispiel 10A: In DMF (800 ml) wurden 39.23 g (85.75 mmol) der Verbindung aus Beispiel 10A vorgelegt und anschliessend 4 g Palladium (10%ig auf Kohle) zugefügt. Es wurde unter Rühren über Nacht bei Wasserstoff-Normaldruck hydriert. Der Ansatz wurde über Kieselgur filtriert und mit wenig DMF und danach mit wenig Methanol nachgewaschen und zur Trockene eingeengt. Der Rückstand wurde mit Essigsäureethylester versetzt und kräftig gerührt, der Niederschlag abgesaugt, mit Essigsäureethylester und Diisopropylether nachgewaschen und über Sicapent im Hochvakuum getrocknet. Variant B: Preparation starting from Example 10A: 39.23 g (85.75 mmol) of the compound from Example 10A were initially charged in DMF (800 ml) and then 4 g of palladium (10% on carbon) were added. It was hydrogenated with stirring overnight at normal hydrogen pressure. The mixture was filtered through kieselguhr and washed with a little DMF and then with a little methanol and concentrated to dryness. The residue was combined with ethyl acetate and stirred vigorously, the precipitate was filtered off with suction, washed with ethyl acetate and diisopropyl ether and dried over Sicapent under high vacuum.
Ausbeute: 31.7 g (100 % d. Theorie) Yield: 31.7 g (100% of theory)
LC-MS (Methode 2): Rt = 0.81 min LC-MS (Method 2): Rt = 0.81 min
MS (ESIpos): m/z = 369 Ausführungsbeispiele : Beispiel 1 MS (ESIpos): m / z = 369 Exemplary embodiments: Example 1
Methyl- {4,6-diamino-2-[5-fluor-l-(2-fluorbenzyl)-lH-pyrazolo[3,4-b]pyridin-3-yl]pyrimidin-5- yljcarbamat Methyl {4,6-diamino-2- [5-fluoro-1- (2-fluorobenzyl) -1H-pyrazolo [3,4-b] pyridin-3-yl] pyrimidin-5-yl carbamate
In Pyridin (600 ml) wurden 31.75 g (86.20 mmol) der Verbindung aus Beispiel 1 1A unter Argon vorgelegt und auf 0°C abgekühlt. Anschließend wurde eine Lösung von 6.66 ml (86.20 mmol) Chlorameisensäuremethylester in Dichlormethan (10 ml) zugetropft und die Mischung lh bei 0°C gerührt. Danach wurde die Reaktionsmischung auf RT gebracht, im Vakuum eingeengt und mehrfach mit Toluol kodestilliert. Der Rückstand wurde mit Wasser/Ethanol verrührt und danach über eine Fritte abgesaugt und nachfolgend mit Ethanol und Essigsäureethylester gewaschen. Anschliessend wurde der Rückstand abermals mit Diethylether verrührt, abgesaugt und anschliessend im Hochvakuum getrocknet. In pyridine (600 ml) 31.75 g (86.20 mmol) of the compound from Example 1 1A were placed under argon and cooled to 0 ° C. Subsequently, a solution of 6.66 ml (86.20 mmol) of methyl chloroformate in dichloromethane (10 ml) was added dropwise and the mixture lh stirred at 0 ° C. Thereafter, the reaction mixture was brought to RT, concentrated in vacuo and codistilled several times with toluene. The residue was stirred with water / ethanol and then filtered with suction through a frit and subsequently washed with ethanol and ethyl acetate. The residue was then stirred again with diethyl ether, filtered off with suction and then dried under high vacuum.
Ausbeute: 24.24 g (65 % d. Theorie) LC-MS (Methode 2): Rt = 0.79 min Yield: 24.24 g (65% of theory) LC-MS (Method 2): R t = 0.79 min
MS (ESIpos): m/z = 427 (M+H)+ MS (ESIpos): m / z = 427 (M + H) +
'H-NMR (400 MHz, DMSO-de): δ = 3.62 (br. s, 3H), 5.79 (s, 2H), 6.22 (br. s, 4H), 7.10 - 7.19 (m, 2H), 7.19 - 7.26 (m, 1H), 7.32 - 7.40 (m, 1H), 7.67 (br. s, 0.2H), 7.99 (br. s, 0.8H), 8.66 (m, 1H), 8.89 (d, 1H). Beispiel 2 'H-NMR (400 MHz, DMSO-de): δ = 3.62 (br.s, 3H), 5.79 (s, 2H), 6.22 (br.s, 4H), 7.10-7.19 (m, 2H), 7.19 - 7.26 (m, 1H), 7.32 - 7.40 (m, 1H), 7.67 (br s, 0.2H), 7.99 (br s, 0.8H), 8.66 (m, 1H), 8.89 (d, 1H) , Example 2
Methyl- {4,6-diamino-2-[5-fluor-l-(2-fluorbenzy^ Methyl {4,6-diamino-2- [5-fluoro-1- (2-fluoro-benzene]
yl} methylcarbamat yl} methylcarbamate
200 mg (0.469 mmol) Methyl- {4,6-diamino-2-[5-fluor-l-(2-fluorbenzyl)-lH-pyrazolo[3,4-b]pyridin- 3-yl]pyrimidin-5-yl}carbamat (Beispiel 1) wurden in THF (5ml) bei 0°C vorgelegt. Danach wurden 0.704 ml (0.704 mmol) Lithiumhexamethyldisilazan-Lösung (IM in THF) zugegeben und 20 min bei dieser Temperatur gerührt. Anschliessend wurden 43.8 μΐ (0.704 mmol) lodmethan zugegeben und auf RT erwärmt. Nach lh bei dieser Temperatur wurde mit Wasser abgebrochen (1 ml), eingeengt und der Rückstand mittels präparativer RP-HPLC (Wasser (+0.05 % Ameisensäure)-Acetonitril Gradient) getrennt. 200 mg (0.469 mmol) of methyl {4,6-diamino-2- [5-fluoro-1- (2-fluorobenzyl) -1H-pyrazolo [3,4-b] pyridin-3-yl] pyrimidine-5 yl} carbamate (Example 1) were initially charged in THF (5 ml) at 0 ° C. Thereafter, 0.704 ml (0.704 mmol) of lithium hexamethyldisilazane solution (III in THF) were added and the mixture was stirred at this temperature for 20 minutes. Subsequently, 43.8 μΐ (0.704 mmol) of iodomethane were added and warmed to RT. After lh at this temperature was quenched with water (1 ml), concentrated and the residue by preparative RP-HPLC (water (+0.05% formic acid) acetonitrile gradient) separated.
Ausbeute: 90 mg (44 % d. Theorie) Yield: 90 mg (44% of theory)
LC-MS (Methode 2): Rt = 0.85 min LC-MS (Method 2): R t = 0.85 min
MS (ESIpos): m/z = 441 (M+H)+ 'H-NMR (400 MHz, DMSO-de): δ = 3.00 (s, 3H), 3.53 (s, 2.2H), 3.66 (s, 0.8H), 5.81 (s, 2H), 6.57 (br. s, 4H), 7.13 (m, 2H), 7.22 (m, 1H), 7.35 (m, 1H), 8.67 (m, 1H), 8.87 (dd, 1H). MS (ESIpos): m / z = 441 (M + H) + H-NMR (400 MHz, DMSO-de): δ = 3.00 (s, 3H), 3.53 (s, 2.2H), 3.66 (s, 0.8H), 5.81 (s, 2H), 6.57 (br s, 4H), 7.13 (m, 2H), 7.22 (m, 1H), 7.35 (m, 1H), 8.67 (m, 1H), 8.87 ( dd, 1H).
Beispiel 3 Example 3
Methyl- {4,6-diamino-2-[5-fluor-l-(2-fluorbenzyl)-lH-pyrazolo[3,4-b]pyridin-3-yl]pyrimidin-5- yl} (2,2,2-trifluorethyl)carbamat Methyl {4,6-diamino-2- [5-fluoro-1- (2-fluorobenzyl) -1H-pyrazolo [3,4- b] pyridin-3-yl] pyrimidin-5-yl} (2,2 , 2-trifluoroethyl) carbamate
Es wurden 3.470 g (8.138 mmol) der Verbindung aus Beispiel 1 in 35 ml THF suspendiert, bei 0°C mit 358 mg (8.952 mmol) Natriumhydrid (60%-ige Suspension in Mineralöl) versetzt und 90 Min. bei 0°C gerührt, wobei sich eine Lösung bildete. Es wurden 2.519 g (8.952 mmol) 2,2,2- Trifluorethyltrichlormethansulfonat addiert und das Gemisch für 48 h bei RT gerührt. Anschliessend wurde mit Wasser verrührt und am Rotationsverdampfer eingeengt. Der Rückstand wurde in Essigsäureethylester aufgenommen, die organische Phase zweimal mit Wasser gewaschen und über Natriumsulfat getrocknet. Es wurden 5.005 g der Zielverbindung erhalten (79 % d. Th., Reinheit nach HPLC 65%). 250 mg des Rückstands wurden mittels präparativer HPLC gereinigt (Eluent: Methanol/Wasser, Gradient 30:70 -> 90: 10). 3,470 g (8,138 mmol) of the compound from Example 1 were suspended in 35 ml of THF, and 358 mg (8,952 mmol) of sodium hydride (60% suspension in mineral oil) were added at 0 ° C. and the mixture was stirred at 0 ° C. for 90 min , where a solution formed. 2,519 g (8,952 mmol) of 2,2,2-trifluoroethyltrichloromethanesulfonate were added and the mixture was stirred at RT for 48 h. The mixture was then stirred with water and concentrated on a rotary evaporator. The residue was taken up in ethyl acetate, the organic phase washed twice with water and dried over sodium sulfate. 5,005 g of the target compound were obtained (79% of theory, purity according to HPLC 65%). 250 mg of the residue were purified by preparative HPLC (eluent: methanol / water, gradient 30:70 → 90:10).
LC-MS (Methode 2): Rt = 0.97 min; MS (EIpos): m/z = 509 [M+H]+. LC-MS (Method 2): R t = 0.97 min; MS (EIpos): m / z = 509 [M + H] + .
'H-NMR (400 MHz, DMSO-de): δ [ppm] = 3.63 (s, 3H), 4.06-4.15 (m, 2H), 5.80 (s, 2H), 6.46 (s br, 4H) 7.11-7.15 (m, 2H), 7.20-7.25 (m, 1H), 7.33-7.38 (m, 1H), 8.66 (dd, 1H), 8.91 (dd, 1H). 'H-NMR (400 MHz, DMSO-de): δ [ppm] = 3.63 (s, 3H), 4.06-4.15 (m, 2H), 5.80 (s, 2H), 6.46 (s br, 4H) 7.11- 7.15 (m, 2H), 7.20-7.25 (m, 1H), 7.33-7.38 (m, 1H), 8.66 (dd, 1H), 8.91 (dd, 1H).
B) Bewertung der physiologischen Wirksamkeit B) Assessment of physiological efficacy
Die Eignung der erfindungsgemäßen Verbindungen zur Behandlung von Erkrankungen mit erhöhtem Hb Konzentrationen wie kann in folgenden Assaysystemen gezeigt werden: 1.) In vitro Assays l.a.) sGC Enzym Assay: Stimulation der rekombinanten löslichen Guanylatcyclase (sGC) in vitro The suitability of the compounds according to the invention for the treatment of diseases with elevated Hb concentrations as can be demonstrated in the following assay systems: 1.) in vitro assays, l.a.) sGC enzyme assay: stimulation of the recombinant soluble guanylate cyclase (sGC) in vitro
Die Untersuchungen zur Stimulation der rekombinanten löslichen Guanylatcyclase (sGC) durch die erfindungsgemäßen Verbindungen mit und ohne Natriumnitroprussid sowie mit und ohne den Häm- abhängigen sGC-Inhibitor lH-l,2,4-Oxadiazolo[4,3-a]chinoxalin-l-on (ODQ) werden nach der in folgender Literaturstelle im Detail beschriebenen Methode durchgeführt: M. Hoenicka, E.M. Becker, H. Apeler, T. Sirichoke, H. Schroeder, R. Gerzer und J.-P. Stasch, "Purified soluble guanylyl cyclase expressed in a baculovirus/Sf9 System: Stimulation by YC-1 , nitric oxide, and carbon oxide", J. Mol. Med. 77 (1999), 14-23. Die Häm-freie Guanylatcyclase wird durch Zugabe von Tween 20 zum Probenpuffer (0.5% in der Endkonzentration) erhalten. Die Aktivierung der sGC durch eine Prüfsubstanz wird als x-fache Stimulation der Basalaktivität angegeben. Lösliche Guanylatcyclase (sGC) setzt unter Stimulation GTP zu cGMP und Pyrophosphat (PPi) um. PPi wird mit Hilfe des nachfolgend beschriebenen Tests nachgewiesen. Das im Test entstehende Signal nimmt mit fortschreitender Umsetzung zu und dient als Maß für die sGC-Enzymaktivität unter der gegebenen Stimulation. The studies on the stimulation of recombinant soluble guanylate cyclase (sGC) by the compounds according to the invention with and without sodium nitroprusside and with and without the heme-dependent sGC inhibitor 1 -H, 2,4-oxadiazolo [4,3-a] quinoxaline-1 on (ODQ) are carried out according to the method described in detail in the following reference: M. Hoenicka, EM Becker, H. Apeler, T. Sirichoke, H. Schroeder, R. Gerzer and J.-P. Stasch, "Purified soluble guanylyl cyclase expressed in a baculovirus / Sf9 system: stimulation by YC-1, nitric oxide, and carbon oxide", J. Mol. Med. 77 (1999), 14-23. The heme-free guanylate cyclase is obtained by adding Tween 20 to the sample buffer (0.5% in final concentration). Activation of sGC by a test substance is reported as x-fold stimulation of basal activity. Soluble guanylate cyclase (sGC) converts GTP into cGMP and pyrophosphate (PPi) upon stimulation. PPi is detected using the test described below. The signal generated in the test increases as the reaction progresses and serves as a measure of the sGC enzyme activity under the given stimulation.
Zur Durchführung des Tests werden 29 μΐ Enzymlösung [0-10 nM lösliche Guanylatcyclase (hergestellt nach Hönicka et al., J. Mol. Med. 77, 14-23 (1999)) in 50 mM TEA, 2 mM MgCl2, 0.1% BSA (Fraktion V), 0.005%> Brij®, pH 7.5] in eine Mikroplatte vorgelegt und 1 μΐ der zu testenden Substanz (als seriell verdünnte Lösung in DMSO) hinzugegeben. Der Ansatz wird 10 min bei Raum- temperatur inkubiert. Anschließend werden 20 μΐ Detektionsmix [1.2 nM Firefly-Luciferase (Photinus pyralis-Luciferase, Fa. Promega), 29 μΜ Dehydro-Luziferin (hergestellt nach Bitler & McElroy, Arch. Biochem. Biophys. 72, 358 (1957)), 122 μΜ Luziferin (Fa. Promega), 153 μΜ ATP (Fa. Sigma) und 0.4 mM DTT (Fa. Sigma) in 50 mM TEA, 2 mM MgCl2, 0.1% BSA (Fraktion V), 0.005%) Brij®, pH 7.5] zugegeben. Die Enzymreaktion wird durch Zugabe von 20 μΐ Sub- stratlösung [1.25 mM Guanosin-5'-triphosphat (Fa. Sigma) in 50 mM TEA, 2 mM MgCl2, 0.1%> BSA (Fraktion V), 0.005%) Brij®, pH 7.5] gestartet und kontinuierlich luminometrisch vermessen. Das Maß der Stimulation durch die zu testende Substanz kann relativ zum Signal der nicht stimulierten Reaktion bestimmt werden. To perform the assay, 29 μM enzyme solution [0-10 nM soluble guanylate cyclase (prepared according to Hönicka et al., J. Mol. Med. 77, 14-23 (1999)) in 50 mM TEA, 2 mM MgCl 2 , 0.1%. BSA (fraction V), 0.005%> Brij ®, pH 7.5] placed in a microplate and 1 μΐ of the substance to be tested is added (as a serially diluted solution in DMSO). The mixture is incubated for 10 min at room temperature. Subsequently, 20 μΐ detection mix [1.2 nM firefly luciferase (Photinus pyralis luciferase, Promega), 29 μΜ dehydro-luciferin (prepared according to Bitler & McElroy, Arch. Biochem., Biophys., 72, 358 (1957)), 122 μΜ luciferin (Fa. Promega), 153 μΜ ATP (Sigma Fa.) (Fa. Sigma) and 0.4 mM DTT in 50 mM TEA, 2 mM MgCl 2, 0.1% BSA (fraction V), 0.005%) Brij ®, pH 7.5 ] admitted. The enzyme reaction is started by adding 20 stratlösung μΐ sub- [1.25 mM guanosine 5'-triphosphate (Fa. Sigma) in 50 mM TEA, 2 mM MgCl 2, 0.1%> BSA (fraction V), 0.005%) Brij ®, pH 7.5] and continuously measured luminometrically. The degree of stimulation by the substance to be tested can be determined relative to the signal of the non-stimulated reaction.
Durch Zugabe von 25 μΜ lH-l,2,4-Oxadiazolo[4,3-a]chinoxalin-l-on (ODQ) zur Enzymlösung und anschließende 30-minütige Inkubation wird die Aktivierung der Häm- freien Guanylatcyclase untersucht und mit der Stimulation des nativen Enzyms verglichen. Addition of 25 μl of lH-l, 2,4-oxadiazolo [4,3-a] quinoxaline-1-one (ODQ) to the enzyme solution followed by a 30-minute incubation is used to study activation of heme-free guanylate cyclase and stimulation of the native enzyme.
1. b. Wirkung an rekombinanter Guanylatcyclase-Reporterzelllinie 1. b. Effect on recombinant guanylate cyclase reporter cell line
Die zelluläre Wirkung der erfindungsgemäßen Verbindungen wird an einer rekombinanten Guanylat- cyclase-Reporterzelllinie, wie in F. Wunder et al., Anal. Biochem. 339, 104-112 (2005) beschrieben, bestimmt. l.c Gefäß relaxierende Wirkung in vitro The cellular activity of the compounds of the invention is measured on a recombinant guanylate cyclase reporter cell line as described in F. Wunder et al., Anal. Biochem. 339, 104-112 (2005). l.c vascular relaxatory effect in vitro
Kaninchen werden durch Nackenschlag betäubt und entblutet. Die Aorta wird entnommen, von anhaftendem Gewebe befreit, in 1.5 mm breite Ringe geteilt und einzeln unter einer Vorspannung in 5 ml-Organbäder mit 37°C warmer, Carbogen-begaster Krebs-Henseleit-Lösung folgender Zusammensetzung gebracht (jeweils mM): Natriumchlorid: 119; Kaliumchlorid: 4.8; Calciumchlorid- Dihydrat: 1 ; Magnesiumsulfat-Heptahydrat: 1 .4 ; Kaliumdihydrogenphosphat: 1 .2 ; Natriumhydrogencarbonat: 25; Glucose: 10. Die Kontraktionskraft wird mit Statham UC2-Zellen erfasst, verstärkt und über A/D-Wandler (DAS- 1802 HC, Keithley Instruments München) digitalisiert sowie parallel auf Linienschreiber registriert. Zur Erzeugung einer Kontraktion wird Phenylephrin dem Bad kumulativ in ansteigender Konzentration zugesetzt. Nach mehreren Kontroll- zyklen wird die zu untersuchende Substanz in jedem weiteren Durchgang in jeweils steigender Dosierung zugesetzt und die Höhe der Kontraktion mit der Höhe der im letzten Vordurchgang erreichten Kontraktion verglichen. Daraus wird die Konzentration errechnet, die erforderlich ist, um die Höhe des Kontrollwertes um 50% zu reduzieren (IC50-Wert). Das Standardapplikationsvolumen beträgt 5 μΐ, der DMSO-Anteil in der Badlösung entspricht 0.1%. 2.) in vivo Messung Rabbits are stunned and bled by a stroke of the neck. The aorta is harvested, detached from adherent tissue, divided into 1.5 mm wide rings and placed individually under bias in 5 ml organ baths with 37 ° C warm, carbogen-gassed Krebs-Henseleit solution of the following composition (in each case mM): Sodium chloride: 119; Potassium chloride: 4.8; Calcium chloride dihydrate: 1; Magnesium sulfate heptahydrate: 1 .4; Potassium dihydrogen phosphate: 1 .2; Sodium hydrogencarbonate: 25; Glucose: 10. The force of contraction is detected with Statham UC2 cells, amplified and digitized via A / D converter (DAS-1802 HC, Keithley Instruments Munich) and registered in parallel on a chart recorder. To create a contraction, phenylephrine is added cumulatively to the bath in increasing concentration. After several control cycles, the substance to be examined is added in each subsequent passage in increasing doses and the height of the contraction is compared with the height of the contraction achieved in the last predistortion. This is used to calculate the concentration required to reduce the level of the control value by 50% (IC 50 value). The standard application volume is 5 μΐ, the DMSO content in the bath solution corresponds to 0.1%. 2.) in vivo measurement
2. a). Radiotelemetrische Blutdruckmessung an wachen, spontan hypertensiven Ratten 2. a). Radiotelemetric blood pressure measurement on awake, spontaneously hypertensive rats
Für die im Folgenden beschriebene Blutdruckmessung an wachen Ratten wird ein im Handel erhältliches Telemetriesystem der Firma DATA SCIENCES INTERNATIONAL DSI, USA eingesetzt. Das System besteht aus 3 Hauptkomponenten: - Implantierbare Sender (Physiotel® Telemetrietransmitter) A commercially available telemetry system from DATA SCIENCES INTERNATIONAL DSI, USA is used for the blood pressure measurement on awake rats described below. The system consists of 3 main components: - Implantable transmitters (Physiotel® telemetry transmitters)
- Empfänger (Physiotel® Receiver), die über einen Multiplexer (DSI Data Exchange Matrix ) mit einem - Receivers (Physiotel® receivers) connected via a multiplexer (DSI Data Exchange Matrix) with a
- Datenakquisitionscomputer verbunden sind. Data acquisition computer are connected.
Die Telemetrieanlage ermöglicht eine kontinuierliche Erfassung von Blutdruck Herzfrequenz und Körperbewegung an wachen Tieren in ihrem gewohnten Lebensraum. The telemetry system allows a continuous recording of blood pressure heart rate and body movement on awake animals in their habitual habitat.
Tiermaterial animal material
Die Untersuchungen werden an ausgewachsenen weiblichen spontan hypertensiven Ratten (SHR Okamoto) mit einem Körpergewicht von >200 g durchgeführt. SHR/NCrl von Okamoto Kyoto School of Medicine, 1963 wurden aus männlichen Wistar Kyoto Ratten mit stark erhöhtem Blutdruck und weiblichen mit leicht erhöhtem Blutdruck gekreuzt und in der Fl 3 an die U.S. National Institutes of Health abgegeben. The investigations are carried out on adult female spontaneously hypertensive rats (SHR Okamoto) with a body weight of> 200 g. SHR / NCrl from Okamoto Kyoto School of Medicine, 1963 were crossed out of male Wistar Kyoto rats with high blood pressure and female with slightly elevated blood pressure, and in Fl 3 with U.S. Patent No. 4,806,014. National Institutes of Health.
Die Versuchstiere werden nach Senderimplantation einzeln in Makroion - Käfigen Typ 3 gehalten. Sie haben freien Zugang zu Standardfutter und Wasser. The experimental animals are kept individually in macroion cages type 3 after transmitter implantation. You have free access to standard food and water.
Der Tag - Nacht - Rhythmus im Versuchslabor wird per Raumbeleuchtung um 6:00 Uhr morgens und um 19:00 Uhr abends gewechselt. The day - night rhythm in the experimental laboratory is changed by room lighting at 6:00 in the morning and at 19:00 in the evening.
Senderimplantation transmitter implantation
Die eingesetzten Telemetriesender TAI 1 PA - C40 werden den Versuchstieren mindestens 14 Tage vor dem ersten Versuchs eins atz unter aseptischen Bedingungen chirurgisch implantiert. Die so instrumentierten Tiere sind nach Abheilen der Wunde und Einwachsen des Implantats wiederholt einsetzbar. The TAI 1 PA - C40 telemetry transmitters are surgically implanted at least 14 days before the first attempt under aseptic conditions. The animals so instrumented are repeatedly used after healing of the wound and ingrowth of the implant.
Zur Implantation werden die nüchternen Tiere mit Pentobabital (Nembutal, Sanofi: 50mg/kg i.p. ) narkotisiert und an der Bauchseite weiträumig rasiert und desinfiziert. Nach Eröffnung des Bauchraumes entlang der Linea alba wird der flüssigkeitsgefüllte Meßkatheter des Systems oberhalb der Bifurcation nach cranial in die Aorta descendens eingesetzt und mit Gewebekleber (VetBonD TM, 3M) befestigt. Das Sendergehäuse wird intraperitoneal an der Bauchwandmuskulatur fixiert und die Wunde wird schichtweise verschlossen. Postoperativ wird zur Infektionsprophylaxe ein Antibiotikum verabreicht (Tardomyocel COMP Bayer 1ml/kg s.c.) For implantation, the fasting animals are anaesthetized with pentobabital (Nembutal, Sanofi: 50 mg / kg ip) and shaved and disinfected on the ventral side. After opening the abdominal cavity along the alba line, the system's liquid-filled measuring catheter above the bifurcation is inserted cranially into the descending aorta and secured with tissue adhesive (VetBonD ™, 3M). The transmitter housing is fixed intraperitoneally to the abdominal wall musculature and the wound is closed in layers. Postoperatively, an antibiotic is administered for infection prophylaxis (Tardomyocel COMP Bayer 1 ml / kg sc)
Substanzen und Lösungen Substances and solutions
Wenn nicht anders beschrieben werden die zu untersuchenden Substanzen jeweils einer Gruppe von Tieren (n = 6 ) per Schlundsonde oral verabreicht. Entsprechend einem Applikationsvolumen von 5 ml/kg Körpergewicht werden die Testsubstanzen in geeigneten Lösungsmittelgemischen gelöst oder in 0.5 %-iger Tylose suspendiert. Unless otherwise described, the substances to be tested are each administered orally to a group of animals (n = 6) by gavage. According to an application volume of 5 ml / kg body weight, the test substances are dissolved in suitable solvent mixtures or suspended in 0.5% Tylose.
Eine Lösungsmittel- behandelte Gruppe von Tieren wird als Kontrolle eingesetzt. Versuchsablauf Die vorhandene Telemetrie - Meßeinrichtung ist für 24 Tiere konfiguriert. Jeder Versuch wird unter einer Versuchsnummer registiert (VJahr Monat Tag). A solvent-treated group of animals is used as a control. Experimental procedure The existing telemetry measuring device is configured for 24 animals. Each trial is registered under a trial number (VYear month day).
Den in der Anlage lebenden instrumentierten Ratten ist jeweils eine eigene Empfangsantenne zugeordnet (1010 Receiver, DSI ). The instrumented rats living in the plant each have their own receiving antenna (1010 receivers, DSI).
Die implantierten Sender sind über einen eingebauten Magnetschalter von außen aktivierbar. Sie werden bei Versuchsvorlauf auf Sendung geschaltet. Die ausgestrahlten Signale können durch ein Datenakquisitionssystem (Dataquest TM A.R.T. for WINDOWS, DSI ) online erfasst und entsprechend aufgearbeitet werden. Die Ablage der Daten erfolgt jeweils in einem hierfür eröffneten Ordner der die Versuchsnummer trägt. The implanted transmitters can be activated externally via a built-in magnetic switch. They will be put on the air during the trial run. The emitted signals can be recorded online by a data acquisition system (Dataquest ™ A.R.T. for Windows, DSI) and processed accordingly. The storage of the data takes place in each case in a folder opened for this purpose which carries the test number.
Im Standardablauf werden über je 10 Sekunden Dauer gemessen: - Systolischer Blutdruck (SBP) In the standard procedure, duration is measured for 10 seconds each: - Systolic blood pressure (SBP)
- Diastolischer Blutdruck (DBP) - Diastolic blood pressure (DBP)
- Arterieller Mitteldruck (MAP) - Mean Arterial Pressure (MAP)
- Herzfrequenz (HR) - heart rate (HR)
- Aktivität (ACT). Die Messwerterfassung wird rechnergesteuert in 5 Minuten Abständen wiederholt. Die als Absolutwert erhobenen Quelldaten werden im Diagramm mit dem aktuell gemessenen Barometerdruck (Ambient Pressure Reference Monitor; APR-1) korrigiert und in Einzeldaten abgelegt. Weitere technische Details sind der umfangreichen Dokumentation der Herstellerfirma (DSI) zu entnehmen. - Activity (ACT). The measured value acquisition is repeated computer-controlled in 5-minute intervals. The absolute value of the source data is corrected in the diagram with the currently measured barometric pressure (Ambient Pressure Reference Monitor, APR-1) and in individual data stored. Further technical details can be found in the extensive documentation of the manufacturer (DSI).
Wenn nicht anders beschrieben erfolgt die Verabreichung der Prüfsubstanzen am Versuchstag um 9.00 Uhr. Im Anschluss an die Applikation werden die oben beschriebenen Parameter 24 Stunden gemessen. Unless otherwise stated, the administration of the test substances will take place at 9 o'clock on the day of the experiment. Following the application, the parameters described above are measured for 24 hours.
Auswertung evaluation
Nach Versuchsende werden die erhobenen Einzeldaten mit der Analysis-Software (DATAQUEST TM A. R.T. TM ANALYSIS) sortiert. Als Leerwert werden hier 2 Stunden vor Applikation angenommen, so dass der selektierte Datensatz den Zeitraum von 7:00 Uhr am Versuchstag bis 9:00 Uhr am Folgetag umfasst. After the end of the test, the collected individual data are sorted with the analysis software (DATAQUEST TM A.RT. TM ANALYSIS). The blank value is assumed here 2 hours before application, so that the selected data set covers the period from 7:00 am on the day of the experiment to 9:00 am on the following day.
Die Daten werden über eine voreinstellbare Zeit durch Mittelwertbestimmung geglättet (15 Minuten Average) und als Textdatei auf einen Datenträger übertragen. Die so vorsortierten und komprimierten Messwerte werden in Excel- Vorlagen übertragen und tabellarisch dargestellt. Die Ablage der erhobenen Daten erfolgt pro Versuchstag in einem eigenen Ordner, der die Versuchsnummer trägt. Ergebnisse und Versuchsprotokolle werden in Papierform nach Nummern sortiert in Ordnern abgelegt. The data is smoothed over a presettable time by averaging (15 minutes average) and transferred as a text file to a disk. The presorted and compressed measured values are transferred to Excel templates and displayed in tabular form. The filing of the collected data takes place per experiment day in a separate folder that bears the test number. Results and test reports are sorted in folders and sorted by paper.
Literatur literature
Klaus Witte, Kai Hu, Johanna Swiatek, Claudia Müssig, Georg Ertl and Bj örn Lemmer: Experimental heart failure in rats: effects on cardio vascular circadian rhythms and on myocardial ß- adrenergic signaling. Cardiovasc Res 47 (2): 203-405, 2000; Kozo Okamoto: Spontaneous hypertension in rats. Int Rev Exp Pathol 7: 227- 270, 1969; Maarten van den Buuse: Circadian Rhythms of Blood Pressure, Heart Rate, and Locomotor Activity in Spontaneously Hypertensive Rats as Measured With Radio-Telemetry. Physiology & Behavior 55(4): 783-787, 1994 Klaus Witte, Kai Hu, Johanna Swiatek, Claudia Muessig, Georg Ertl and Bjorn Lemmer: Experimental heart failure in rats: effects on cardio vascular circadian rhythms and on myocardial ß-adrenergic signaling. Cardiovasc Res 47 (2): 203-405, 2000; Kozo Okamoto: Spontaneous hypertension in rats. Int Rev. Exp Pathol 7: 227-270, 1969; Maarten van den Buuse: Circadian Rhythms of Blood Pressure, Heart Rate, and Locomotor Activity in Spontaneously Hypertensive Rats as Measured with Radio Telemetry. Physiology & Behavior 55 (4): 783-787, 1994
2.b.) Messung von Blutfluss und Blutdruck in Ratten 250 - 350 g schwere Wistar Ratten (Hsd Cpb:Wu) bzw. 330 - 520 g schwere ZDF Ratten (ZDF/Crl- Lepr fa/fa) werden mittels 2.5% Isofluran im Sauerstoff - Lachgasgemisch (40:60) narkotisiert. Zur Bestimmung des Blutflusses in der A. carotis, A. femoralis wird die narkotisierte Ratte in Rückenlage gebracht und anschließend die linkseitige A. carotis und rechtsseitige A. femoralis vorsichtig freipräpariert. Der Blutfluss wird durch das Anbringen von Flusssonden (Transonic Flowprobe) an den Gefäßen gemessen. Durch das Einbringen eines PE50-Arterien-Katheter in die linksseitige A. femoralis wird der Blutdruck und die Herzratte (Transducer Ref. 5203660: Fa.Braun CH) bestimmt. Die Substanzgabe erfolgt als Bolus bzw. Dauerinfusion über einen Venen-Katheder in der linksseitigen V. femoralis. 2.b.) Measurement of blood flow and blood pressure in rats 250 to 350 g Wistar rats (Hsd Cpb: Wu) or ZDF rats weighing 330 to 520 g (ZDF / Crl-Lepr fa / fa) are treated with 2.5% isoflurane in rats Oxygen - nitrous oxide mixture (40:60) anesthetized. To determine the blood flow in the carotid artery, femoral artery, the anesthetized rat is placed supine and then the left carotid artery and the right femoral artery are carefully dissected free. Blood flow is measured by attaching flow probes (Transonic Flowprobe) to the vessels. By introducing a PE50 arterial catheter into the left femoral artery, the blood pressure and the heart rat (Transducer Ref. 5203660: Fa. Braun CH). The substance is administered as a bolus or continuous infusion via a venous catheter in the left femoral vein.
2.c.) Prüfung von durchblutungsfördernden Substanzen (Mikrozirkulation) 2.c.) Testing of circulation-promoting substances (microcirculation)
Zur Erzeugung einer Minderperfusion wird bei Ratten (z.B. ZDF Ratten) in Narkose (z.B. Inhalationsnarkose Isofluran, Enfluran) unter sterilen Bedingungen die rechte A. iliaca externa ligiert. Je nach Kollateralisierungsgrad der Tiere muß zur Erzeugung einer Minderperfusion zusätzlich noch die A. femoralis ligiert werden. Im Anschluß an die Operation oder auch präventiv werden die Versuchstiere oral, intragastral (Magensonde, Futter- oder Trinkwasseraufnahme), intraperitoneal, intravenös, intraarteriell, intramuskulär, inhalativ oder subcutan mit den Prüfsubstanzen behandelt. Alternativ wird bei den Tieren extrazelluläres Hämoglobin appliziert, das zuvor aus Spendertieren gewonnen wurde. Die Prüfsubstanzen werden akut oder für bis zu 5 Wochen einmal oder mehrfach enteral oder parenteral täglich appliziert oder es erfolgt eine Dauerapplikation über subkutan implantierte osmotische Minipumpen (z.B. Alzet Pumpen). Die Mikroperfusion und Temperatur der unteren Extremitäten werden während dem Versuch dokumentiert. Dabei wird den Ratten unter Narkose eine temperatursensitive Laserdopplersonde (Periflux) auf die Pfoten geklebt und somit Microperfusion und Hauttemperatur gemessen. Je nach Versuchsplan werden Proben wie Blut (Interimsdiagnostik) und sonstige Körperflüssigkeiten oder Organe entnommen, um in vitro weitere Untersuchungen durchzuführen oder es wird Zur Dokumentation der Hämodynamik über einen Katheter in der A. carotis Blutdruck und Herzfrequenz gemessen. Am Ende des Experiments werden die Tiere schmerzlos getötet. To induce low perfusion in rats (e.g., ZDF rats) under anesthetic conditions (e.g., inhalation anesthesia, isoflurane, enflurane), the right external iliac artery is ligated under sterile conditions. Depending on the degree of collateralization of the animals, the femoral artery must be additionally ligated in order to produce a less perfusion. Following the operation or also preventively, the test animals are treated orally, intragastrically (gastric tube, feed or drinking water intake), intraperitoneally, intravenously, intraarterially, intramuscularly, by inhalation or subcutaneously with the test substances. Alternatively, extracellular hemoglobin, previously obtained from donor animals, is applied to the animals. The test substances are administered acute or once or several times daily for up to 5 weeks, enterally or parenterally daily, or are administered over subcutaneously implanted osmotic minipumps (e.g., Alzet pumps). Microperfusion and temperature of the lower extremities are documented during the trial. The rats are under anesthesia, a temperature-sensitive laser Doppler probe (Periflux) glued to the paws and thus measured micro perfusion and skin temperature. Depending on the experimental plan, samples such as blood (interim diagnosis) and other body fluids or organs are taken to perform further in vitro tests or blood pressure and heart rate are measured via a catheter in the carotid artery for documentation of hemodynamics. At the end of the experiment, the animals are killed painlessly.
2.d.) Prüfung von durchblutungsfördernden Substanzen (Motorische Funktion) im Laufradversuch 2.d.) Testing of circulation-promoting substances (motor function) in the impeller test
Zur Bestimmung der motorischen Funktion wird das Laufverhalten von Mäusen (z.B. eNOS knock out Mäuse, Wildtyp Mäuse C-57 B16 oder ApoE knock out Mäuse, Sichelzellmäuse, ...) in Laufrädern untersucht. Um die Mäuse an die freiwillige Benutzung des Laufrades zu gewöhnen, werden 4-5 Wochen vor Beginn des Versuches die Tiere in Käfigen mit Laufrad vereinzelt und trainiert. 2 Wochen vor Start des Experimentes werden die Bewegungen der Mäuse im Laufrad durch eine Photozelle mittels Computer aufgezeichnet und verschiedene Laufparameter wie z.B. die tägliche Laufstrecke, die zurückgelegten Einzelstrecken, aber auch Ihre zeitliche Verteilung über den Tag bestimmt. Die Tiere werden nach ihrem natürlichen Laufverhalten in Gruppen (8-12 Tiere) randomisiert (Kontrollgruppe, Sham Gruppe und ein bis mehrere Substanzgruppen). Im Anschluß daran werden die Versuchstiere oral, intragastral (Magensonde, Futter- oder Trinkwasseraufnahme), intraperitoneal, intravenös, intraarteriell, intramuskulär, inhalativ oder subcutan mit den Prüfsubstanzen behandelt. Die Prüfsubstanzen werden für bis zu 5 Wochen einmal oder mehrfach enteral oder parenteral täglich appliziert oder es erfolgt eine Dauerapplikation über subkutan implantierte osmotische Minipumpen. Das Laufverhalten der Tiere wird über 5-8 Wochen beobachtet und aufgezeichnet. Am Ende des Experiments werden die Tiere schmerzlos getötet. Je nach Versuchsplan werden Proben wie Blut und sonstige Körperflüssigkeiten oder Organe entnommen, um in vitro weitere Untersuchungen durchzuführen (S. Vogelsberger Neue Tiermodelle für die Indikation Claudicatio Intermittens (Taschenbuch), Verlag: WB Laufersweiler Verlag (März 2006), ISBN-10: 383595007X, ISBN-13: 978-3835950078). To determine the motor function, the running behavior of mice (eg, eNOS knock out mice, wild-type mice C-57 B16 or ApoE knock out mice, sickle cell mice, ...) was investigated in wheels. In order to accustom the mice to the voluntary use of the wheel, the animals are caged and trained in cages with an impeller 4-5 weeks before the start of the experiment. Two weeks before the start of the experiment, the movements of the mice in the impeller are recorded by a photocell using a computer and various running parameters such as the daily running distance, the individual distances covered, but also their temporal distribution over the day. The animals are randomized according to their natural running behavior in groups (8-12 animals) (control group, sham group and one or more substance groups). Thereafter, the test animals are administered orally, intragastrically (gastric tube, feed or drinking water intake), intraperitoneally, intravenously, intraarterially, intramuscularly, by inhalation or subcutaneously with the Treated test substances. The test substances are administered once or several times daily or repeatedly enterally or parenterally daily for up to 5 weeks or there is a continuous application via subcutaneously implanted osmotic minipumps. The running behavior of the animals is observed and recorded over 5-8 weeks. At the end of the experiment, the animals are killed painlessly. Depending on the experimental design, samples such as blood and other body fluids or organs are taken to carry out further in vitro investigations (S. Vogelsberger New animal models for the indication Claudicatio Intermittens (Paperback), Publisher: WB Laufersweiler Verlag (March 2006), ISBN 10: 383595007X , ISBN-13: 978-3835950078).
2.e.) Hämodynamik im narkotisierten Hund Es werden 25-35 kg schwere gesunde oder herzinsuffiziente Mongrel® Hunde (Marshall BioResources, Marshall Farms Inc; Clyde NY; USA) beiderlei Geschlechts verwendet. Die Narkose wird eingeleitet durch langsame i.V. Gabe von 25 mg/kg Natrium Thiopental (Trapanal®) und 0.15 mg/kg Alcuronium Chlorid (Alloferin®) und während des Experimentes aufrechterhalten mittels einer Dauerinfusion aus 0.04 mg/kg*h Fentanyl (Fentanyl®), 0.25 mg/kg*h Droperidol (Dihydrobenzperidol®) und 15 μg/kg/h Alcuronium Chloride (Alloferin®). Nach der Intubation werden die Tiere über die Beatmungsmaschine mit konstanten Atemvolumen beatmet, so dass eine endtidale CO2 Konzentration von etwa 5% erreicht wird. Die Beatmung erfolgt mit Raumluft, angereichert mit ca. 30% Sauerstoff (Normoxie). Zur Messung der hämodynamischen Parameter wird ein mit Flüssigkeit gefüllter Katheter in die A. femoralis zur Messung des Blutdruckes implantiert. Ein 2-lumiger Swan-Ganz® Katheter wird über die V. jugularis in die Pulmonalarterie eingeschwemmt (distales Lumen zur Messung des pulmonalarteriellen Druckes, proximales Lumen zur Messung des zentralen Venendruckes). Mittels Temperaturfühler an der Spitze des Katheters wird der kontinuierliche Cardiac Output (CCO) bestimmt. Der Blutfluss wird an unterschiedlichen Gefäßbetten wie z.B. der Koronararterie, der A. Carotis oder der Femoralarterie durch das Anbringen von Flusssonden (Transonic Flowprobe) an den jeweiligen Gefäßen gemessen. Der hnksventrikuläre Druck wird nach Einführung eines Mikro-Tip-Katheters (Miliar® Instruments) über die A. carotis in den linken Ventrikel gemessen und davon das dP/dt als Maß für die Kontraktilität abgeleitet. Substanzen werden i.v. über die V. femoralis oder intraduodenal als kumulative Dosis- Wirkungskurve (Bolus oder Dauerinfusion) appliziert. Die hämodynamischen Signale werden mittels Druckaufnehmern / Verstärkern und PONEMAH® als Datenerfassungssoftware aufgezeichnet und ausgewertet. 2.e.) hemodynamics in anesthetized dogs are 25-35 kg healthy or heart failure Mongrel dogs ® (Marshall BioResources, Marshall Farms Inc; Clyde NY, USA) both sexes used. The anesthesia is induced by slow intravenous administration of 25 mg / kg of sodium thiopental (Trapanal ®) and 0:15 mg / kg Alcuronium chloride (Alloferin ®) and during the experiment maintained by a continuous infusion of 12:04 mg / kg * h fentanyl (Fentanyl ®) , 0.25 mg / kg * h Droperidol (Dihydrobenzperidol ® ) and 15 μg / kg / h Alcuronium Chloride (Alloferin ® ). After intubation, the animals are ventilated via the ventilator with a constant breathing volume, so that an end-tidal CO 2 concentration of about 5% is achieved. Ventilation takes place with room air, enriched with approx. 30% oxygen (normoxie). To measure hemodynamic parameters, a catheter filled with fluid is implanted in the femoral artery to measure blood pressure. A 2-lumiger Swan-Ganz catheter is ® jugularis on the V. into the pulmonary artery washed in (distal lumen for measuring the pulmonary arterial pressure, proximal lumen for measuring the central venous pressure). A temperature sensor at the tip of the catheter determines the continuous cardiac output (CCO). The blood flow is measured on different vascular beds such as the coronary artery, the carotid artery or the femoral artery by attaching flow probes (Transonic Flowprobe) to the respective vessels. The hnksventrikuläre pressure is measured after the introduction of a Mikro-Tip catheter (Millar Instruments ®) through the carotid artery into the left ventricle and, derived from the dP / dt as a measure of contractility. Substances are administered iv via the femoral vein or intraduodenally as a cumulative dose-response curve (bolus or continuous infusion). The hemodynamic signals are recorded and evaluated by means of pressure transducers / amplifiers and PONEMAH ® as data acquisition software.
Zur Induktion von Herzinsuffizienz wird den Hunden unter sterilen Bedingungen ein Schrittmacher implantiert. Nach Induktion der Narkose mittels Pentobarbital-Na (15 to 30 mg kg"1 i.v.) gefolgt von einer Intubation und anschliessender Ventilation (room air; Sulla 808, Dräger®, Germany) wird die Narkose durch kontinuierliche Infusion von Pentobarbital (1-5 mg kg"1 h"1) und Fentanyl (10-40 μg kg"1 h"1) aufrechterhalten. Ein Schrittmacherkabel (Setrox S60®, Biotronik, Germany) wird implantiert über eine Insertion der linken Vena Jugularis und im rechten Ventrikel plaziert. Das Kabel wird mit dem Schrittmacher(Logos®, Biotronik, Germany) verbunden, der in einer kleinen subkutanen Tasche zwischen den Schulterblättern positioniert wird. Erst 7 Tage nach dem Eingriff wird das ventrikuläre Pacing zur Erzielung einer Herzinsuffiziens bei einer Frequenz von 220 Schlägen/minüber einen Zeitraum von 10-28 Tagen gestartet. To induce heart failure, a pacemaker is implanted into the dogs under sterile conditions. After induction of anesthesia with pentobarbital-Na (15 to 30 mg kg -1 iv) followed by intubation and subsequent ventilation (room air, Sulla 808, Dräger® , Germany) Anesthesia maintained by continuous infusion of pentobarbital (1-5 mg kg "1 h " 1 ) and fentanyl (10-40 μg kg "1 h " 1 ). A pacing lead (Setrox S60 ®, Biotronik, Germany) is implanted placed over an insertion of the left jugular vein and the right ventricle. The cable is connected to the pacemaker ( Logos® , Biotronik, Germany), which is positioned in a small subcutaneous pocket between the shoulder blades. Only 7 days after the procedure, ventricular pacing to achieve heart failure is started at a rate of 220 beats / min for a period of 10-28 days.
2.f.) Hämodynamik im narkotisierten Ferkel 2.f.) hemodynamics in anesthetized piglets
Hemodynamic parameters were continuously measured by intravascular catheters connected by Combitrans® transducers to Gould® transducers (series 6600) connected to the PoNeMah® acquisition and analysis System. Hemodynamic parameters were continuously measured by intravascular catheters connected by Combitrans® transducers to Gould® transducers (series 6600) connected to the PoNeMah® acquisition and analysis system.
Es werden 2-6 kg schwere gesunde Göttinger Minipigs® Ellegaard (Ellegaard, Denmark) beiderlei Geschlechts verwendet. Die Narkose erfolgt durch die Gabe von 7.5 mg/ml Ketamin, 1.125 mg/ml Midazolam (Infusionsrate 6 - 25 ml/h) und 0.6 mg/h Pancuroniumbromid (Pancuronium®, Organon, Oss, Netherland). Nach der Intubation werden die Tiere über die Beatmungsmaschine mit konstanten Atemvolumen beatmet (10-12 ml/kg, 35 Atemzüge/min Avea, Viasys Healthcare, USA), so dass eine endtidale CO2 Konzentration von etwa 5% erreicht wird. Die Beatmung erfolgt mit Raumluft, angereichert mit ca. 40% Sauerstoff (Normoxie). Zur Messung der hämodynamischen Parameter werden Katheter in die A. femoralis zur Messung des Blutdruckes und ein Swan-Ganz® Katheter über die V. jugularis in die Pulmonalarterie eingeschwemmt (distales Lumen zur Messung des pulmonalarteriellen Druckes, proximales Lumen zur Messung des zentralen Venendruckes). Mittels Temperaturfühler an der Spitze des Katheters wird der kontinuierliche Cardiac Output (CCO) bestimmt. Die hämodynamischen Signale werden mittels Druckaufnehmern / Verstärkern und PONEMAH® als Datenerfassungssoftware aufgezeichnet und ausgewertet. 2.g.) Inhalation von sGC Simulatoren und Aktivatoren It can be used 2-6 kg healthy Göttingen minipigs ® Ellegaard (Ellegaard, Denmark) of both sexes. Anesthesia is achieved by administration of 7.5 mg / ml ketamine, 1250 mg / ml midazolam (infusion rate 6 - 25 ml / h) and 0.6 mg / h pancuronium bromide (Pancuronium®, Organon, Oss, Netherland). After intubation, the animals are ventilated via the ventilator with constant respiratory volume (10-12 ml / kg, 35 breaths / min Avea, Viasys Healthcare, USA), so that an end-tidal CO 2 concentration of about 5% is achieved. Ventilation takes place with room air enriched with approx. 40% oxygen (Normoxie). To measure hemodynamic parameters, catheters are inserted into the femoral artery for blood pressure measurement and a Swan- Ganz® catheter is inserted through the jugular vein into the pulmonary artery (distal lumen for measurement of pulmonary artery pressure, proximal lumen for measurement of central venous pressure). A temperature sensor at the tip of the catheter determines the continuous cardiac output (CCO). The hemodynamic signals are recorded and evaluated by means of pressure transducers / amplifiers and PONEMAH ® as data acquisition software. 2.g.) Inhalation of sGC simulators and activators
Die Experimente wurden in narkotisierten Göttinger Minischweinen (2.5-4 kg, Narkose: 7.5 mg/ml Ketamin, 1.125 mg/ml Midazolam (Infusionsrate 6 - 25 ml/h) and 0.6 mg/h Pancuroniumbromid (Pancuronium®, Organon, Oss, Niederlande), narkotisierten Ratten und wachen telemetrisch instrumentierten Hunden durchgeführt. Die narkotisierten Tiere wurden nach Intubation künstlich mit Raumluft, angereichert mit 40% Sauerstoff beatmet ( Schweine: 10-12 ml/kg, 35 breaths/min using Avea, Viasys Healthcare, USA). Akute Pulmonale Hypertonie wurde durch eine Thromboxan A2 Analoga Infusion (0.12 - 0.14 μg/kg/min 9, 11 -dideoxy-9a, 11 α-epoxymethanoprostaglandin F2a (U-46619, Sigma, Germany)) oder Monocrotalingabe bei ratten induziert. Die Vernebelung der Substanzen erfolgte unter Verwendung des Nebutec® oder Aeroneb® Pro Vernebier Systems oder nach Feststoffverneblung (Pauluhn), die in den Inspirationsschenkel der Beatmung geschaltet wurden. Die Substanzen wurden als Feststoffe oder Lösungen in Abhängigkeit zur Molekularstruktur eingesetzt. Die hämodynamischen Signale werden mittels Druckaufnehmern / Verstärkern und PONEMAH® oder CardioMems® als Datenerfassungssoftware aufgezeichnet und ausgewertet. The experiments were carried out in anesthetized Göttingen mini-pigs (2.5-4 kg, narcosis: 7.5 mg / ml ketamine, 1.125 mg / ml midazolam (infusion rate 6-25 ml / h) and 0.6 mg / h pancuronium bromide (Pancuronium®, Organon, Oss, The Netherlands The anesthetized animals were artificially ventilated after intubation with room air enriched with 40% oxygen (pigs: 10-12 ml / kg, 35 breaths / min using Avea, Viasys Healthcare, USA). Acute pulmonary hypertension was induced by a thromboxane A2 analogue infusion (0.12-0.14 μg / kg / min 9,11 -dideoxy-9a, 11α-epoxymethanoprostaglandin F2a (U-46619, Sigma, Germany)) or monocrotaline administration in rats of the Substances were carried out using the Nebutec® or Aeroneb® Pro Vernebier system or after solid nebulization (Pauluhn), which were switched into the inspiratory limb of the respiration. The substances were used as solids or solutions depending on the molecular structure. The hemodynamic signals are recorded and evaluated by means of pressure transducers / amplifiers and PONEMAH ® or CardioMems ® as data acquisition software.
2.h.) Testung der Schmerzschwelle/Latenzzeit im Hot Plate Test 2.h.) Testing the pain threshold / latency time in the hot plate test
Hot Plate: Typ Socrel DS 37, Firma Ugo Basile.Die Messung wird an Mäusen (z.B. NMRI Maüse, Sichelzellmäuse) durchgefürt. Die Temperatur der Hot Plate beträgt 50°C , die maximale Dauer der messung je Tier 90 Sekunden (cut off Zeit). Unmittelbar vor der Applikation der Testsubstanz (Prüfsubstanzen z.B. über Futter, Trinkwasser, po Gabe, Alzet Pumpen über mehrere Tage) wird für jedes Tier ein Hot Plate Vorwert bestimmt. Kriterien für die Latenzzeit (in sec.) bis zur Schmerzreaktion sind Pfotenschütteln, Pfotenlecken oder Springen. Nach ca. 8-tägiger Verabreichung der Testsubstanz wird unter denselben Bedingungen das 2. Messergebnis durchgeführt. Hot Plate: Type Socrel DS 37, Ugo Basile Co. The measurement is performed on mice (e.g., NMRI jaws, sickle cell mice). The temperature of the hot plate is 50 ° C, the maximum duration of measurement per animal 90 seconds (cut off time). Immediately prior to administration of the test substance (test substances, for example via feed, drinking water, po dosing, Alzet pumps over several days), a hot plate pre-value is determined for each animal. Criteria for latency (in sec.) To the pain reaction are paw shaking, paw licking or jumping. After administration of the test substance for about 8 days, the second test result is carried out under the same conditions.

Claims

Ansprüche claims
Verwendung von Stimulatoren und Aktivatoren der löslichen Guanylatzyklase zur Behandlung von Sichelzellanämie und Konservierung von Blutersatzstoffen. Use of soluble guanylate cyclase stimulators and activators for the treatment of sickle cell anemia and preservation of blood substitutes.
Verwendung von Stimulatoren und Aktivatoren der löslichen Guanylatzyklase der Formeln (1) bis (26) Use of soluble guanylate cyclase stimulators and activators of the formulas (1) to (26)
( 3 ) ( 4 ) -50- (3) (4) -50-
-51 - -51 -
10 (16) 10 (16)
(19) (19)
(25) -56- (25) -56-
- 57 - - 57 -
zur Behandlung und/oder Prophylaxe von Sichelzellanämie und Konservierung von Blutersatzstoffen. for the treatment and / or prophylaxis of sickle cell anemia and preservation of blood substitutes.
4. Verwendung von einer oder mehrerer Verbindungen der Formeln (1) bis (26) gemäß Anspruch 1 zur Behandlung und/oder Prophylaxe von traumatisierten Patienten, welche einen künstlichen Blutersatzstoff erhalten. 4. Use of one or more compounds of the formulas (1) to (26) according to claim 1 for the treatment and / or prophylaxis of traumatized patients who receive an artificial blood substitute.
5. Verfahren zur Behandlung und/oder Prophylaxe von Sichelzellanämie und Konservierung von Blutersatzstoffen unter Verwendung einer therapeutisch wirksamen Menge einer oder mehrerer der erfindungsgemäßen Verbindung gemäß der Formeln (1) bis (26) gemäß Anspruch 1. 6. Verwendung von einer oder mehrerer der erfindungsgemäßen Verbindungen gemäß der Formeln (1) bis (26) gemäß Anspruch 1 zur Herstellung eines Arzneimittels zur Behandlung und/oder Prophylaxe von Sichelzellanämie und Konservierung von Blutersatzstoffen. 5. A method for the treatment and / or prophylaxis of sickle cell anemia and preservation of blood substitutes using a therapeutically effective amount of one or more of the compound of the invention according to the formulas (1) to (26) according to claim 1. 6. Use of one or more of the inventive Compounds according to formulas (1) to (26) according to claim 1 for the manufacture of a medicament for the treatment and / or prophylaxis of sickle cell anemia and preservation of blood substitutes.
7. Arzneimittel, enthaltend eine oder mehrere erfindungsgemäße Verbindung der Formeln (1) bis (26) gemäß Anspruch 1 und einen oder mehrere weitere Wirkstoffe, insbesondere zur Behandlung und/oder Prophylaxe von Sichelzellanämie und Konservierung von7. A pharmaceutical composition comprising one or more compounds of the invention of the formulas (1) to (26) according to claim 1 and one or more further active compounds, in particular for the treatment and / or prophylaxis of sickle cell anemia and preservation of
Blutersatzstoffen. Blood substitutes.
8. Verwendung von einer oder mehrerer Verbindungen der der Formeln (1 ) bis (26) gemäß Anspruch 1 in Kombination mit einem oder mehreren der folgenden PDE5 Inhibitoren: 8. Use of one or more compounds of the formulas (1) to (26) according to claim 1 in combination with one or more of the following PDE5 inhibitors:
Tadalafil ((6R,12aR) -2,3,6,7,12,12a - Hexahydro - 2 - methyl - 6 - (3,4-methylene - dioxyphenyl) pyrazino(l',2':l,6) pyrido(3,4-b)indole-l,4-dione), Vardenafil (2-(2-Ethoxy-5-(4- ethylpiperazin- 1 -yl- 1 -sulfonyl)phenyl)-5-methyl-7-propyl-3H-imidazo (5, 1 -f) (1 ,2,4)triazin-4- one), Sildenafil (3-[2-ethoxy-5-(4-methylpiperazin-l-yl)sulfonyl-phenyl]- 7- methy 1- 9- propy 1- 2,4,7,8- tetrazabicyclo [4.3.0]nona -3,8,10-trien-5-one), Udenafil 5-[2-propyloxy-5-(l-methyl-2- pyrrolidinylethylamidosulfonyl)phenyl]-methyl-3-propyl-l,6-dihydro-7H-pyrazolo(4,3- d)pyrimidine-7-one, Dasantafil 7-(3-Bromo-4-methoxybenzyl)-l-ethyl-8-[[(l,2)-2- hydroxycyclopentyl] amino] -3 -(2-hydroxyethyl)-3 ,7-dihydro- 1 -purine-2,6-dione, Avanafil 4- { [(3 - chloro-4-methoxyphenyl)methyl]amino}-2-[(2S)-2-(hydroxymethyl)pyrrolidin-l-yl]-N- (pyrimidin-2-ylmethyl)pyrimidine-5-carboxamide, Mirodenafil, Lodenafil, UK 369.003, UK 371.800, SLx 2101 of Surface Logix, LAS 34779Triazolo[l,2-]xanthine,6-methyl-4-propyl-2-[2- propoxy-5-(4-methylpiperazino)sulfonyl]phenyl oder Salze, Hydrate oder Hydrate der Salze davon zur Behandlung und/oder Prophylaxe von Sichelzellanämie und Konservierung von Blutersatzstoffen. Tadalafil ((6R, 12aR) -2,3,6,7,12,12a - hexahydro - 2 - methyl - 6 - (3,4 - methylenedioxyphenyl) pyrazino (1 ', 2': 1, 6) pyrido (3,4-b) indole-1,4-dione), vardenafil (2- (2-ethoxy-5- (4-ethyl-piperazin-1-yl-1-sulfonyl) -phenyl) -5-methyl-7-propyl 3H-imidazo (5, 1-f) (1, 2,4) triazine-4-one), sildenafil (3- [2-ethoxy-5- (4-methylpiperazin-1-yl) sulfonyl-phenyl] - 7-methyl-9-propyl-2,4,7,8-tetrazabicyclo [4.3.0] nona-3,8,10-trien-5-one), uddenafil 5- [2-propyloxy-5- (5-one) 1-methyl-2-pyrrolidinylethyl-amidosulfonyl) -phenyl] -methyl-3-propyl-1,6-dihydro-7H-pyrazolo (4,3-d) -pyrimidines-7-one, dasantafil 7- (3-bromo-4-methoxybenzyl ) -l-ethyl-8 - [[(l, 2) -2-hydroxycyclopentyl] amino] -3- (2-hydroxyethyl) -3,7-dihydro-1-purine-2,6-dione, avanafil 4- {[(3-chloro-4-methoxyphenyl) methyl] amino} -2 - [(2S) -2- (hydroxymethyl) pyrrolidin-1-yl] -N- (pyrimidin-2-ylmethyl) pyrimidines-5-carboxamides, Mirodenafil, Lodenafil, UK 369.003, UK 371.800, SLx 2101 of Surface Logix, LAS 34779Triazolo [l, 2-] xanthines, 6-methyl-4-propyl-2- [2- propoxy-5- (4-methylpiperazino) sulfonyl] phenyl or salts, hydrates or hydrates of the salts of which for the treatment and / or prophylaxis of sickle cell anemia and preservation of blood substitutes.
9. Verwendung von einer oder mehrerer Verbidnungen der Formeln (3) bis (7) gemäß Anspruch 1 in Kombination mit Vardenafil und/oder Sildenafil gemäß Anspruch 8 zur Behandlung und/oder Prophylaxe von Sichelzellanämie und Konservierung von Blutersatzstoffen. 9. Use of one or more Verbidnungen of formulas (3) to (7) according to claim 1 in combination with vardenafil and / or sildenafil according to claim 8 for the treatment and / or prophylaxis of sickle cell anemia and preservation of blood substitutes.
10. Verwendung von einer oder mehrerer der Verbindungen der Formeln (1 ) bis (26) gemäß Anspruch 1 in Kombination mit PDE5 Inhibitoren gemäß Anspruch 8 zur Behandlung und/oder Prophylaxe von traumatisierten Patienten, welche einen künstlichen Blutersatzstoff erhalten. 10. Use of one or more of the compounds of the formulas (1) to (26) according to claim 1 in combination with PDE5 inhibitors according to claim 8 for the treatment and / or prophylaxis of traumatized patients who receive an artificial blood substitute.
11. Verfahren zur Behandlung und/oder Prophylaxe von Sichelzellanämie und Konservierung von Blutersatzstoffen unter Verwendung einer therapeutisch wirksamen Menge einer oder mehrerer erfindungsgemäßen Verbindung gemäß der Formeln (1) bis (26) gemäß Anspruch 1 in Kombination mit PDE5 Inhibitoren gemäß Anspruch 8. 11. A method for the treatment and / or prophylaxis of sickle cell anemia and preservation of blood substitutes using a therapeutically effective amount of one or more compounds of the invention according to the formulas (1) to (26) according to claim 1 in combination with PDE5 inhibitors according to claim 8.
12. Verwendung einer oder mehrerer der erfindungsgemäßen Verbindungen gemäß der Formeln (1) bis (26) gemäß Anspruch 1 in Kombination mit PDE5 Inhibitoren gemäß Anspruch 8 zur Herstellung eines Arzneimittels zur Behandlung und/oder Prophylaxe von Sichelzellanämie und Konservierung von Blutersatzstoffen. 12. Use of one or more of the compounds according to the invention according to the formulas (1) to (26) according to claim 1 in combination with PDE5 inhibitors according to claim 8 for the manufacture of a medicament for the treatment and / or prophylaxis of sickle cell anemia and preservation of blood substitutes.
13. Arzneimittel, enthaltend eine oder mehrere der erfindungsgemäßen Verbindungen der Formeln (1) bis (26) gemäß Anspruch 1 in Kombination mit PDE5 Inhibitoren gemäß Anspruch 8 und einen oder mehrere weitere Wirkstoffe, insbesondere zur Behandlung und/oder Prophylaxe von Sichelzellanämie und Konservierung von Blutersatzstoffen. 13. A pharmaceutical composition containing one or more of the compounds of the formulas (1) to (26) according to claim 1 in combination with PDE5 inhibitors according to claim 8 and one or more further active compounds, in particular for the treatment and / or prophylaxis of sickle cell anemia and preservation of blood substitutes.
14. Verwendung von einer oder mehrerer Verbindungen der Formeln (1) bis (26) gemäß Anspruch 14. Use of one or more compounds of the formulas (1) to (26) according to claim
1 zur Behandlung und/oder Prophylaxe von Sichelzellanämie wobei die Anwendung inhalativ erfolgt. 1 for the treatment and / or prophylaxis of sickle cell anemia wherein the application is by inhalation.
15. Verwendung von einer oder mehrerer Verbindungen der Formeln (1) bis (26) gemäß Anspruch 1 in Kombination mit PDE5 Inhibitoren gemäß Anspruch 8 zur Behandlung und/oder15. Use of one or more compounds of the formulas (1) to (26) according to claim 1 in combination with PDE5 inhibitors according to claim 8 for the treatment and / or
Prophylaxe von Sichelzellanämie wobei die Anwendung inhalativ erfolgt. Prophylaxis of sickle cell anemia using inhalatively.
EP11726444.0A 2010-06-25 2011-06-21 Use of stimulators and activators of soluble guanylate cyclase for treating sickle-cell anemia and conserving blood substitutes Withdrawn EP2585055A1 (en)

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