SIEBENEICHER HOLGER (DE)
POOK ELISABETH (DE)
ROTTMANN ANTJE (DE)
NAGEL JENS (DE)
FISCHER OLIVER (DE)
| WO2020011921A1 | 2020-01-16 | |||
| WO2019081343A1 | 2019-05-02 | |||
| WO2011017142A1 | 2011-02-10 | |||
| WO2020011921A1 | 2020-01-16 | |||
| WO2012112363A1 | 2012-08-23 |
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| CLAIMS 1. A compound of general formula (I): wherein: X represents a nitrogen or a carbon atom; Y represents a nitrogen or a carbon atom, • wherein the unit X-Y represents either a N-C group or a C=N group respectively; R1 and R2are linked to each other to form a five-membered heterocyclic ring containing from 2 to 3 nitrogen atoms, substituted with R5; and R3 represents oxo or is absent; or R1 represents oxo; and R2 and R3 are linked to each other to form a five-membered heterocyclic ring containing from 2 to 3 nitrogen atoms, substituted with R5; R4 represents fluoro, chloro, or methyl; R5 represents hydrogen, C1-C4-alkyl, or cyclopropyl; and stereoisomers, N-oxides, hydrates, solvates, and salts thereof, and mixtures of same. 2. A compound of general formula (II): wherein: the unit L1 - L2 represents a group selected from: R5 represents hydrogen, C1-C4-alkyl, or cyclopropyl; and stereoisomers, N-oxides, hydrates, solvates, and salts thereof, and mixtures of same. 3. A compound of general formula (III): wherein: R5 represents hydrogen, C1-C4-alkyl, or cyclopropyl; and stereoisomers, N-oxides, hydrates, solvates, and salts thereof, and mixtures of same. 4. A compound of general formula (IV): wherein: R5 represents hydrogen or C1-C4-alkyl; and stereoisomers, N-oxides, hydrates, solvates, and salts thereof, and mixtures of same. 5. A compound of general formula (V): wherein: R5 represents hydrogen or C1-C4-alkyl; and stereoisomers, N-oxides, hydrates, solvates, and salts thereof, and mixtures of same. 6. A compound of general formula (VI): wherein: R5 represents hydrogen or C1-C4-alkyl; and stereoisomers, N-oxides, hydrates, solvates, and salts thereof, and mixtures of same. 7. A compound of general formula (VII): wherein: R5 represents hydrogen or C1-C4-alkyl; and stereoisomers, N-oxides, hydrates, solvates, and salts thereof, and mixtures of same. 8. A compound of general formula (VIII): wherein: R5 represents hydrogen or C1-C4-alkyl; and stereoisomers, N-oxides, hydrates, solvates, and salts thereof, and mixtures of same. 9. The compound according to any one of claims 1 or 2 which is: 2-[3-tert-butyl-8-(morpholin-4-yl)-5-oxopyrido[2,3-e][1,2,4]triazolo[4,3-c]pyrimidin-6(5H)-yl]- N-(5-fluoropyridin-2-yl)acetamide; 2-[2-tert-butyl-8-(morpholin-4-yl)-5-oxoimidazo[1,2-c]pyrido[2,3-e]pyrimidin-6(5H)-yl]-N-(5- fluoropyridin-2-yl)acetamide; 2-[(2±)-2-tert-butyl-8-(morpholin-4-yl)-5-oxo-2,3-dihydroimidazo[1,2-c]pyrido[2,3- e]pyrimidin-6(5H)-yl]-N-(5-fluoropyridin-2-yl)acetamide; 2-[(3±)-3-tert-butyl-8-(morpholin-4-yl)-2,5-dioxo-2,3-dihydroimidazo[1,2-c]pyrido[2,3- e]pyrimidin-6(5H)-yl]-N-(5-fluoropyridin-2-yl)acetamide; 2-[3-cyclopropyl-8-(morpholin-4-yl)-5-oxopyrido[2,3-e][1,2,4]triazolo[4,3-c]pyrimidin-6(5H)- yl]-N-(5-fluoropyridin-2-yl)acetamide; or a stereoisomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same. 10. The compound according to claim 1 which is: 2-[3-tert-butyl-8-(morpholin-4-yl)pyrido[2,3-d][1,2,4]triazolo[4,3-b]pyridazin-6-yl]-N-(5- fluoropyridin-2-yl)acetamide; 2-[3-tert-butyl-8-(morpholin-4-yl)-5-oxopyrido[3,2-d][1,2,4]triazolo[4,3-a]pyrimidin-10(5H)- yl]-N-(5-fluoropyridin-2-yl)acetamide; or a stereoisomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same. 11. A compound of general formula (I) according to any one of claims 1 to 10 for use in the treatment or prophylaxis of a disease. 12. A pharmaceutical composition comprising a compound of general formula (I) according to any one of claims 1 to 10 and one or more pharmaceutically acceptable excipients. 13. Use of a compound of general formula (I) according to any one of claims 1 to 10 for the treatment or prophylaxis of a disease. 14. Use of a compound of general formula (I) according to any one of claims 1 to 10 for the preparation of a medicament for the treatment or prophylaxis of a disease. 15. Use according to any one of claims 13 or 14, wherein the disease is a neurogenic disorder, such as a gynecological disorder, urinary tract disease state, respiratory disorder or a pain- associated disease or disorder. 16. Use according to claim 15, wherein the disease is endometriosis, moderate to severe pain associated with endometriosis in women of reproductive age; overactive bladder (OAB) with symptoms of urge urinary incontinence, urgency, urinary frequency and nocturia; interstitial cystitis and/ or bladder pain syndrome; refractory and/ or unexplained chronic cough; neuropathic pain associated with diabetic peripheral neuropathy (NP-DPN); cancer and/ or chemotherapy related neuropathic pain; postherpetic neuralgia and postsurgical and/ or posttraumatic neuropathic pain; irritable bowel syndrome with diarrhea (IBS D); Prurigo nodularis; chronic pruritus of unknown origin; itch; heart failure and/ or central and obstructive sleep apnea. |
R 5 represents hydrogen, C 1 -C 4 -alkyl, or cyclopropyl; and stereoisomers, N-oxides, hydrates, solvates, and salts thereof, and mixtures of same. In a further embodiment, the present invention relates to compounds of general formula (III) wherein: R 5 represents hydrogen, C 1 -C 4 -alkyl, or cyclopropyl; and stereoisomers, N-oxides, hydrates, solvates, and salts thereof, and mixtures of same.
In a further embodiment, the present invention relates to compounds of general formula (IV) wherein: R 5 represents hydrogen or C 1 -C 4 -alkyl; and stereoisomers, N-oxides, hydrates, solvates, and salts thereof, and mixtures of same. In a further embodiment, the present invention relates to compounds of general formula (V) wherein: R 5 represents hydrogen or C 1 -C 4 -alkyl; and stereoisomers, N-oxides, hydrates, solvates, and salts thereof, and mixtures of same. In a further embodiment, the present invention relates to compounds of general formula (VI) wherein: R 5 represents hydrogen or C 1 -C 4 -alkyl; and stereoisomers, N-oxides, hydrates, solvates, and salts thereof, and mixtures of same. In a further embodiment, the present invention relates to compounds of general formula (VII) wherein: R 5 represents hydrogen or C 1 -C 4 -alkyl; and stereoisomers, N-oxides, hydrates, solvates, and salts thereof, and mixtures of same. In a further embodiment, the present invention relates to compounds of general formula (VIII) wherein: R 5 represents hydrogen or C 1 -C 4 -alkyl; and stereoisomers, N-oxides, hydrates, solvates, and salts thereof, and mixtures of same. The present invention covers any sub-combination within any embodiment or aspect of the present invention of compounds of general formula (I), supra. The present invention covers methods of preparing compounds of the present invention of general formula (I), said methods comprising the steps as described in the Experimental Section herein. The compounds according to the invention of general formula (I) are prepared according to the following schemes 1 to 6. The schemes and procedures described below illustrate synthetic routes to the compounds of general formulae (III), (IV), (V), (VI), (VII), and (VIII) of the invention and are not intended to be limiting. It is clear to the person skilled in the art that the order of transformations as exemplified in schemes 1 to 6 can be modified in various ways. The order of transformations exemplified in these schemes is therefore not intended to be limiting. Appropriate protecting groups and their introduction and cleavage are well-known to the person skilled in the art. Specific examples are described in the subsequent paragraphs. The starting materials are either commercially available or can be prepared according to procedures available from the public domain, as understandable to the person skilled in the art. Specific examples are described in the Experimental Section. Six routes for the preparation of compounds of general formulae (III) – (VIII) are described in schemes 1 to 6. Synthesis of compounds of general formula (I) of the present invention Compounds of general formula (III) can be synthesised according to the route depicted in Scheme 1. Starting from 5-bromo-3-fluoro-pyridine-2-carbonitrile, pyridyl nitrile esters of formula 2 can be obtained by SN reaction using an appropriate glycine ester, for example glycine tert-butyl ester in presence of an appropiate base, such as DIPEA in an appropriate solvent like DMA at temperatures between 70°C and 140°C. Nitrile hydrolysis to obtain the corresponding amide of formula 3 can be achieved by oxidative alkaline hydrolysis using hydrogen peroxide and an appropriate aqueous base, like for example aqueous Na 2 CO 3 solution in an appropriate solvent like acetone at temperatures between -30°C and +80°C. Cyclization to dioxopyridopyrimidine of formula 4 may be accomplished by reaction of intermediate 3 with an appropriate activated carbonyl reagent, like for example carbonyl diimidazole in an appropriate solvent, like DMA at temperatures ranging from r. t. to 150°C. Introduction of an amine, like for example morpholine, is possible by nucleophilic substitution of bromo precursor of formula 5 with the respective amine in an appropriate solvent like for example DMSO under elevated temperatures ranging from 80°C to 150°C. Dioxo compound 5 can be converted to chloro oxo compound 6 using an appropriate chlorinating reagent, like for example phosphoroxichloride under basic conditions using for example DIPEA in an appropriate solvent like acetonitrile at temperatures ranging from r. t. to reflux. Hydrazide compound 7 may be obtained by reaction of chloro precursor 6 with the appropriate hydrazide in an appropriate solvent like for example dioxane at elevated temperatures ranging from 40 °C to 80°C. Simulataneous cyclization and ester deprotection to triazole acid 8 is possible by treatment with an appropriate acid, for example HCl in an appropriate solvent like dioxane at temperatures ranging from r. t. to reflux. Target compounds of general formula (I) are accessible using acid precursor 8, 5-fluoropyridin-2-amine as amine component and an appropriate acid activating agent like HATU in the presence of base, like for example N-methyl morpholine in an appropriate solvent like DMF at temperatures between -30°C and + 60°C. Scheme 1 Compounds of general formula (IV) can be synthesised according to the route depicted in Scheme 2. Commercially available 5-bromopyridine-2,3-dicarboxylic acid can be converted to the corresponding anhydride of formula 2 using an appropriate activating agent, like for example HATU, EDC or thionyl chloride, optionally in presence of a base like DIPEA in an appropriate solvent like DMF at temperatures ranging from -30°C to 60°C. Anhydride 2 may be converted to mono methyl ester 3 using methanol in an appropriate solvent, for example dioxane at temperatures ranging from r. t. to 60°C. Introduction of the tert butyl ester side chain may be possible by Friedel Crafts acylation of intermediate 3 with 3-tert-butoxy-3-oxo-propanoic acid in presence of Lewis acid, like for example MgCl2 and an activating agent, like for example CDI to afford intermediate of general formula 4. Reaction with hydrazine leads to cyclized oxo pyridazine of formula 5. Oxo pyridazine 5 can be converted to chloro compound 6 using an appropriate chlorinating reagent, like for example phosphoroxichloride under basic conditions using for example DIPEA in an appropriate solvent like acetonitrile at temperatures ranging from r. t. to reflux. Tricyclic compound of general formula 7 may be obtained by reaction of chloro precursor 6 with the appropriate hydrazide in an appropriate solvent like for example dioxane at elevated temperatures ranging from 40°C to 80°C. Introduction of an amine, like for example morpholine, is possible by nucleophilic substitution of bromo precursor of general formula 7 with the respective amine in an appropriate solvent like for example DMSO under elevated temperatures ranging from 80°C to 150°C. Deprotection of the tert butyl ester in compounds of general formula 8 may be possible by treatment with acid, for example HCl in dioxane or with TFA oprionally in an appropriate solvent like dichloromethane at temperatures ranging from - 30°C to 60°C. Target compounds of general formula (IV) are accessible using acid precursor 9, 5-fluoropyridin-2-amine as amine component and an appropriate acid activating agent like HATU in the presence of base, like for example N-methyl morpholine in an appropriate solvent like DMF at temperatures between -30°C and + 60°C.
Scheme 2 Compounds of general formula (V) can be synthesised according to the route depicted in Scheme 3. Starting from 5-bromo-3-fluoro-pyridine-2-carbonitrile, pyridyl nitrile esters of formula 2 can be obtained by SN reaction using an appropriate glycine ester, for example glycine tert-butyl ester in presence of an appropiate base, such as DIPEA in an appropriate solvent like DMA at temperatures between 70°C and 140°C. Reaction with an isocyanate source, for example trichloroacetyl isocyanate, leads to bicyclic intermediate 3. Further condensation of amidine 3 with a substituted alpha-bromo carbonyl compound leads to tricyclic intermediate of general formula 4. Introduction of an amine, like for example morpholine, is possible by nucleophilic substitution of bromo precursor of formula 5 with the respective amine in an appropriate solvent like for example DMSO under elevated temperatures ranging from 80°C to 150°C. Ester deprotection to acid intermediate of general formula 6 is possible by treatment with an appropriate acid, for example HCl in an appropriate solvent like dioxane at temperatures ranging from r. t. to reflux. Target compounds of general formula (V) are accessible using acid precursor 6, 5-fluoropyridin-2-amine as amine component and an appropriate acid activating agent like HATU in the presence of base, like for example N-methyl morpholine in an appropriate solvent like DMF at temperatures between -30°C and + 60°C.
Scheme 3 Compounds of general formula (VI) can be synthesised according to the route depicted in Scheme 4. Starting from methyl 5-bromo-3-fluoro-pyridine-2-carboxylate, pyridyl glycine esters of formula 2 can be obtained by SN reaction using an appropriate glycine ester, for example glycine tert-butyl ester in presence of an appropiate base, such as DIPEA in an appropriate solvent like DMA at temperatures between 70°C and 140°C. Reaction with an isothiocyanate source, for example benzoyl isothiocyanate, leads to bicyclic oxo thio pyrimidine 3. Intermediate 3 can be converted with hydrazine to intermediate 4. Further condensation of intermediate 4 with a substituted alpha-bromo carbonyl compound leads to tricyclic intermediate of general formula 4. Introduction of an amine, like for example morpholine, is possible by nucleophilic substitution of bromo precursor of formula 5 with the respective amine in an appropriate solvent like for example DMSO under elevated temperatures ranging from 80°C to 150°C. Ester deprotection to acid intermediate of general formula 7 is possible by treatment with an appropriate acid, for example HCl in an appropriate solvent like dioxane at temperatures ranging from r. t. to reflux. Target compounds of general formula (VI) are accessible using acid precursor 7, 5-fluoropyridin-2- amine as amine component and an appropriate acid activating agent like HATU in the presence of base, like for example N-methyl morpholine in an appropriate solvent like DMF at temperatures between -30°C and + 60°C. Scheme 4 Compounds of general formula (VII) can be synthesised according to the route depicted in Scheme 5. Commercially available 5-bromo-3-fluoropicolinonitrile 1 can be converted in a SNAr reaction using an appropriate glycine ester, for example glycine tert-butyl ester in presence of an appropiate base, such as DIPEA in an appropriate solvent like DMA at temperatures between 70°C and 140°C. The pyridyl glycine esters of formula 3 can be obtained in a transition metal catalysed coupling reaction with nucleophiles like amines, preferably morpholine, or alkohols by using for example a Pd- or Cu catalyst in presence of an appropriate ligand like a mono- or bidentate phosphine ligang and an appropriate base like potassium tert-butylate in an aprotic solvent such as DMF at temperatures ranging from 80°C to 150°C.. Cyclization to aminopyrimidinone intermediates of formula 4 may be performed by reaction with trichloroacctyl isocyanate that can subsequently be condensed with 2-halogen substituted carboxylic acids of formula 5 to receive pyrazolones of formula 6. To perform this conversion the carboxylic acids of formula 5 have to be activated using for example a chlorinating reagent such as oxalyl chloride in presence of a catalytic amount of DM and a base such as DIPEA in an appropriate aprotic solvent such as DMA. Deprotection of the tert butyl ester in compounds of general formula 6 may be possible by treatment with acid, for example HCl in dioxane or with TFA oprionally in an appropriate solvent like dichloromethane at temperatures ranging from -30°C to 60°C. Target compounds of general formula (VII) are accessible using acid precursor 7, 5-fluoropyridin-2- amine as amine component and an appropriate acid activating agent like HATU in the presence of base, like for example N-methyl morpholine in an appropriate solvent like DMF at temperatures between -30°C and +60°C.
Scheme 5 Compounds of general formula (VIII) can be synthesised according to the route depicted in Scheme 6. According to the methods described in Scheme 5 intermediates of formula 3 can be derived in a two step sequence from commercially available 5-bromo-3-fluoropicolinonitrile 1. The nitriles of formula 3 may be converted with optionally substituted 1,2-diamino ethanes of formula 4 in the presence of a base like triethylamine and an aprotic solvent such as DMA at temperatures ranging from 80°C to 180°C.The resulting imidazoline intermediates of formula 5 can be cyclized to the pyrimidinones of formula 6 with an appropriate activated carbonyl reagent, like for example carbonyl diimidazole in an appropriate solvent, like DMA at temperatures ranging from r. t. to 150°C. Deprotection of the tert butyl ester in compounds of general formula 6 may be possible by treatment with acid, for example HCl in dioxane or with TFA oprionally in an appropriate solvent like dichloromethane at temperatures ranging from -30°C to 60°C. Target compounds of general formula (VIII) are accessible using acid precursor 7, 5-fluoropyridin-2-amine as amine component and an appropriate acid activating agent like HATU in the presence of base, like for example N-methyl morpholine in an appropriate solvent like DMF at temperatures between -30°C and +60°C. Scheme 6 formula (I) of the present invention demonstrate a valuable pharmacological spectrum of action and pharmacokinetic profile if supported by data, both of which could not have been predicted. Compounds of the present invention have surprisingly been found to effectively and selectively inhibit P2X3 and it is possible therefore that said compounds be used for the treatment or prophylaxis of diseases, preferably neurogenic disorders in humans and animals. Other substituents than morpholine contained in compounds of general formula (I) of the present invention have also been found to have comparable inhibitory potency against P2X3, for example a dimethylamino substituent or a cyclopropyl moiety. Synthesis of such compounds is possible by related routes by either reacting the described halo-pyridine with the desired functionalized substituent (like for example dimethylamine under Hartwig-Buchwald or SN conditions or cyclopropylboronic acid under Suzuki conditions) or by directly using the appropriately substituted pyridyl building block at the beginning of the reaction sequence. Method for treatment The present invention also relates to a method for using a compound of formula (I) and compositions thereof, to treat mammalian neurogenic diseases and disorders. This method comprises administering to a mammal in need thereof, including a human, an amount of a compound of formula (I) or composition thereof, which is effective to treat the disease or disorder. Neurogenic diseases and disorders include but are not limited to genitourinary, gastrointestinal, respiratory diseases, cardiovascular disease associated with autonomic imbalance caused by increased chemoreceptor sensitivity, and pain as such as well as pain-related diseases, conditions and disorders, gynecological diseases, urinary tract disease states, pain-related diseases and disorders, pain-associated diseases and disorders, neurological diseases and disorders, neurodegenerative diseases and disorders and dermatological diseases and disorders. Gynecological diseases include but are not limited to dysmenorrhea (primary and secondary dysmenorrhea), dyspareunia, endometriosis, and adenomyosis; endometriosis-associated pain; endometriosis-associated symptoms, wherein said symptoms are in particular dysmenorrhea, dyspareunia, dysuria, or dyschezia; endometriosis-associated proliferation; pelvic hyper- sensitivity. Preference is given to endometriosis and to moderate to severe pain associated with endometriosis in women of reproductive age. Urinary tract disease states include, but are not limited to those associated with the bladder outlet obstruction; urinary incontinence conditions such as reduced bladder capacity, increased frequency of micturition, urge incontinence, stress incontinence, or bladder hyperreactivity; benign prostatic hypertrophy; prostatic hyperplasia; prostatitis; detrusor hyperreflexia; overactive urinary bladder and symptoms related to overactive urinary bladder wherein said symptoms are in particular increased urinary frequency, nocturia, urinary urgency or urge incontinence; pelvic hypersensitivity; urethritis; prostatitis; prostatodynia; cystitis, in particular Interstitial cystitis; idiopathic bladder hypersensitivity and bladder pain syndrome,. Preference is given to overactive bladder (OAB) with symptoms of urge urinary incontinence, urgency, urinary frequency and nocturia. Furthermore, preference is given to interstitial cystitis and bladder pain syndrome as well. Neurological diseases and disorders include, but are not limited to epilepsy, partial and generalized seizures. Respiratory diseases and disorders include, but are not limited to asthma, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, acute cough, chronic cough including chronic idiopathic and refractory and/ or unexplained chronic cough, bronchospasm and interstitial lung disease (including idiopathic pulmonary fibrosis). Preference is given to refractory and/ or unexplained chronic cough. Cardiovascular diseases and disorders include, but are not limited to those associated with nerve fiber sensitization, and/or other pathological conditions associated with autonomic imbalance caused by increased chemoreceptor sensitivity, in particular for the treatment of breathing disorders, Cheyne Stokes respiration, central and obstructive sleep apnea, cardiovascular disease, hypertension, resistant hypertension, and heart failure, which are related to increased activity of P2X3 receptors. Gastrointestinal dieases and disorders include, but are not limited to irritable bowel syndrome (IBS), epigastric pain syndrome (functional dyspepsia syndrome), functional abdominal bloating with distension, inflammatory bowel disease (IBD), biliary colic and other biliary disorders, renal colic, diarrhea-dominant IBS; gastroesophageal reflux, gastrointestinal distension, ulcerative colitis (Crohn’s disease). Preference is given to irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). Furthermore, irritable bowel syndrome with diarrhea (IBS D) is preferred. Neurodegenerative diseases and disorders include, but are not limited to Alzheimer's disease, Multiple Sclerosis, Parkinson’s disease, Brain ischemia and traumatic brain injury. Dermatological diseases and disorders include, but are not limited to Prurigo nodularis, chronic pruritus of unknown origin, pruritus due to kidney or liver disease, Brachioradial pruritus, Rosacea, Chronic hand eczema, Dyshidrotic eczema, Atopic dermatitis and Psoriasis. Pain-related diseases and disorders include, but are not limited to acute, chronic, inflammatory and neuropathic pain syndromes. Pain-related diseases and disorders include, but are not limited to pain syndromes selected from the group consisting of acute, chronic, inflammatory and neuropathic pain, preferably inflammatory pain, low back pain, surgical pain, postsurgical neuropathic pain, posttraumatic neuropathic pain, visceral pain, dental pain, periodontitis, premenstrual pain, endometriosis- associated pain, pain associated with fibrotic diseases, central pain, pain due to burning mouth syndrome, pain due to burns, pain due to migraine, cluster headaches, pain due to nerve injury, pain due to neuritis, neuralgias, pain due to poisoning, pain due to ischemic injury, pain due to interstitial cystitis, cancer-related neuropathic pain, chemotherapy-related neuropathic pain, pain due to viral, parasitic or bacterial infections, pain due to traumatic nerve-injury, pain due to post- traumatic injuries (including fractures and sport injuries), pain due to trigeminal neuralgia, pain associated with small fiber neuropathy, pain associated with diabetic neuropathy, postherpetic neuralgia, chronic lower back pain, neck pain phantom limb pain, pelvic pain syndrome, chronic pelvic pain, neuroma pain, complex regional pain syndrome, bladder pain syndrome, pain associated with gastrointestinal distension, chronic arthritic pain and related neuralgias, and pain associated with cancer, Morphine-resistant pain, pain associated with chemotherapy, HIV and HIV treatment-induced neuropathy; and pain associated with diseases or disorders selected from the group consisting of hyperalgesia, allodynia, functional bowel disorders (such as irritable bowel syndrome) and arthritis (such as osteoarthritis, rheumatoid arthritis and ankylosing spondylitis). Pain-associated diseases and disorders include, but are not limited to hyperalgesia, allodynia, functional bowel disorders (such as irritable bowel syndrome), gout, arthritis (such as osteoarthritis, rheumatoid arthritis and ankylosing spondylitis), burning mouth syndrome, burns, migraine or cluster headaches, nerve injury, traumatic nerve injury, post-traumatic injuries (including fractures and sport injuries), neuritis, neuralgias, poisoning, ischemic injury, interstitial cystitis, cancer, trigeminal neuralgia, small fiber neuropathy, diabetic neuropathy, chronic arthritis and related neuralgias, HIV and HIV treatment-induced neuropathy, pruritus; impaired wound healing and disease of the skeleton like degeneration of the joints. Preference is given to peripheral neuropathic pain. In particular, preference is given to neuropathic pain associated with diabetic peripheral neuropathy (NP-DPN). Furthermore, preference is given to cancer and/ or chemotherapy related neuropathic pain, to postherpetic neuralgia and to postsurgical and/ or posttraumatic neuropathic pain. The present invention relates to a method for using the compounds of the present and compositions thereof to treat inflammation, in particular neurogenic inflammation. The term "inflammation" is also understood to include any inflammatory disease, disorder or condition per se, any condition that has an inflammatory component associated with it, and/or any condition characterized by inflammation as a symptom, including, inter alia, acute, chronic, ulcerative, specific, allergic, infection by pathogens, immune reactions due to hypersensitivity, entering foreign bodies, physical injury, and necrotic inflammation, and other forms of inflammation known to those skilled in the art. The term thus also includes, for the purposes of this invention, inflammatory pain, pain generally and/or fever. The present invention relates to a method for using the compounds of the present invention and compositions thereof to treat fibromyalgia, myofascial disorders, viral infections (e.g. influenza, common cold, herpes zoster, hepatitis C and AIDS), bacterial infections, fungal infections, surgical or dental procedures, arthritis, osteoarthritis, juvenile arthritis, rheumatoid arthritis, juvenile onset rheumatoid arthritis, rheumatic fever, ankylosing spondylitis, Hodgkin's disease, systemic lupus erythematosus, vasculitis, pancreatitis, nephritis, bursitis, conjunctivitis, iritis, scleritis, uveitis, wound healing, dermatitis, eczema, stroke, autoimmune diseases, allergic disorders, rhinitis, ulcers, mild to moderately active ulcerative colitis, familial adenomatous polyposis, coronary heart disease, sarcoidosis and any other disease with an inflammatory component. The present invention relates to a method for using the compounds of the present invention and compositions thereof to treat mammalian, including human disorders and diseases which are not linked to inflammatory mechanisms, such as in the reduction of bone loss in a subject. Diseases that may be mentioned in this regard include osteoporosis, osteoarthritis, Paget's disease and/or periodontal diseases. The inventive compounds can therefore be used in medicaments for treatment and/or prophylaxis of the above-mentioned diseases. These diseases and/ or disorders have been well characterized in humans, but also exist with a similar etiology in other mammals and can be treated by administering pharmaceutical compositions of the present invention. The term “treating” or “treatment” as used in the present text is used conventionally, e.g., the management or care of a subject for the purpose of combating, alleviating, reducing, relieving, improving the condition of a disease or disorder, such as neurogenic disorders or diseases. The compounds of the present invention can be used in therapy and prevention, i.e. prophylaxis, of neurogenic diseases, conditions and disorders. In accordance with a further aspect, the present invention covers pharmaceutical compositions, in particular a medicament, comprising a compound of general formula (I), as described supra, or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, a salt thereof, particularly a pharmaceutically acceptable salt, or a mixture of same, and one or more excipients), in particular one or more pharmaceutically acceptable excipient(s). Conventional procedures for preparing such pharmaceutical compositions in appropriate dosage forms can be utilized. The present invention furthermore covers pharmaceutical compositions, in particular medicaments, which comprise at least one compound according to the invention, conventionally together with one or more pharmaceutically suitable excipients, and to their use for the above mentioned purposes. It is possible for the compounds according to the invention to have systemic and/or local activity. For this purpose, they can be administered in a suitable manner, such as, for example, via the oral, parenteral, pulmonary, nasal, sublingual, lingual, buccal, rectal, vaginal, dermal, transdermal, conjunctival, otic route or as an implant or stent. For these administration routes, it is possible for the compounds according to the invention to be administered in suitable administration forms. For oral administration, it is possible to formulate the compounds according to the invention to dosage forms known in the art that deliver the compounds of the invention rapidly and/or in a modified manner, such as, for example, tablets (uncoated or coated tablets, for example with enteric or controlled release coatings that dissolve with a delay or are insoluble), orally- disintegrating tablets, films/wafers, films/lyophylisates, capsules (for example hard or soft gelatine capsules), sugar-coated tablets, granules, pellets, powders, emulsions, suspensions, aerosols or solutions. It is possible to incorporate the compounds according to the invention in crystalline and/or amorphised and/or dissolved form into said dosage forms. Parenteral administration can be effected with avoidance of an absorption step (for example intravenous, intraarterial, intracardial, intraspinal or intralumbal) or with inclusion of absorption (for example intramuscular, subcutaneous, intracutaneous, percutaneous or intraperitoneal). Administration forms which are suitable for parenteral administration are, inter alia, preparations for injection and infusion in the form of solutions, suspensions, emulsions, lyophylisates or sterile powders. Examples which are suitable for other administration routes are pharmaceutical forms for inhalation [inter alia powder inhalers, nebulizers], nasal drops, nasal solutions, nasal sprays; tablets/films/wafers/capsules for lingual, sublingual or buccal administration; suppositories; eye drops, eye ointments, eye baths, ocular inserts, ear drops, ear sprays, ear powders, ear-rinses, ear tampons; vaginal capsules, aqueous suspensions (lotions, mixturae agitandae), lipophilic suspensions, emulsions, ointments, creams, transdermal therapeutic systems (such as, for example, patches), milk, pastes, foams, dusting powders, implants or stents. The compounds according to the invention can be incorporated into the stated administration forms. This can be effected in a manner known per se by mixing with pharmaceutically suitable excipients. Pharmaceutically suitable excipients include, inter alia, • fillers and carriers (for example cellulose, microcrystalline cellulose (such as, for example, Avicel ® ), lactose, mannitol, starch, calcium phosphate (such as, for example, Di-Cafos ® )), • ointment bases (for example petroleum jelly, paraffins, triglycerides, waxes, wool wax, wool wax alcohols, lanolin, hydrophilic ointment, polyethylene glycols), • bases for suppositories (for example polyethylene glycols, cacao butter, hard fat), • solvents (for example water, ethanol, isopropanol, glycerol, propylene glycol, medium chain-length triglycerides fatty oils, liquid polyethylene glycols, paraffins), • surfactants, emulsifiers, dispersants or wetters (for example sodium dodecyl sulfate), lecithin, phospholipids, fatty alcohols (such as, for example, Lanette ® ), sorbitan fatty acid esters (such as, for example, Span ® ), polyoxyethylene sorbitan fatty acid esters (such as, for example, Tween ® ), polyoxyethylene fatty acid glycerides (such as, for example, Cremophor ® ), polyoxethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, glycerol fatty acid esters, poloxamers (such as, for example, Pluronic ® ), • buffers, acids and bases (for example phosphates, carbonates, citric acid, acetic acid, hydrochloric acid, sodium hydroxide solution, ammonium carbonate, trometamol, triethanolamine), • isotonicity agents (for example glucose, sodium chloride), • adsorbents (for example highly-disperse silicas), • viscosity-increasing agents, gel formers, thickeners and/or binders (for example polyvinylpyrrolidone, methylcellulose, hydroxypropylmethylcellulose, hydroxypropyl- cellulose, carboxymethylcellulose-sodium, starch, carbomers, polyacrylic acids (such as, for example, Carbopol ® ); alginates, gelatine), • disintegrants (for example modified starch, carboxymethylcellulose-sodium, sodium starch glycolate (such as, for example, Explotab ® ), cross- linked polyvinylpyrrolidone, croscarmellose-sodium (such as, for example, AcDiSol ® )), • flow regulators, lubricants, glidants and mould release agents (for example magnesium stearate, stearic acid, talc, highly-disperse silicas (such as, for example, Aerosil ® )), • coating materials (for example sugar, shellac) and film formers for films or diffusion membranes which dissolve rapidly or in a modified manner (for example polyvinylpyrrolidones (such as, for example, Kollidon ® ), polyvinyl alcohol, hydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose, hydroxypropyl- methylcellulose phthalate, cellulose acetate, cellulose acetate phthalate, polyacrylates, polymethacrylates such as, for example, Eudragit ® )), • capsule materials (for example gelatine, hydroxypropylmethylcellulose), • synthetic polymers (for example polylactides, polyglycolides, polyacrylates, polymethacrylates (such as, for example, Eudragit ® ), polyvinylpyrrolidones (such as, for example, Kollidon ® ), polyvinyl alcohols, polyvinyl acetates, polyethylene oxides, polyethylene glycols and their copolymers and blockcopolymers), • plasticizers (for example polyethylene glycols, propylene glycol, glycerol, triacetine, triacetyl citrate, dibutyl phthalate), • penetration enhancers, • stabilisers (for example antioxidants such as, for example, ascorbic acid, ascorbyl palmitate, sodium ascorbate, butylhydroxyanisole, butylhydroxytoluene, propyl gallate), • preservatives (for example parabens, sorbic acid, thiomersal, benzalkonium chloride, chlorhexidine acetate, sodium benzoate), • colourants (for example inorganic pigments such as, for example, iron oxides, titanium dioxide), • flavourings, sweeteners, flavour- and/or odour-masking agents. The present invention furthermore relates to a pharmaceutical composition which comprise at least one compound according to the invention, conventionally together with one or more pharmaceutically suitable excipient(s), and to their use according to the present invention. In accordance with another aspect, the present invention covers pharmaceutical combinations, in particular medicaments, comprising at least one compound of general formula (I) of the present invention and at least one or more further active ingredients, in particular for the treatment and/or prophylaxis of neurogenic disorders, in particular of genitourinary, gastrointestinal, respiratory, cardiovascular disease associated with autonomic imbalance caused by increased chemoreceptor sensitivity, and pain-related diseases, conditions and disorders. Particularly, the present invention covers a pharmaceutical combination, which comprises: • one or more first active ingredients, in particular compounds of general formula (I) as defined supra, and • one or more further active ingredients, suitable for the treatment of neurogenic disorders, genitourinary, gastrointestinal, respiratory, cardiovascular disease associated with autonomic imbalance caused by increased chemoreceptor sensitivity, and pain-related diseases, conditions and disorders. The term “combination” in the present invention is used as known to persons skilled in the art, it being possible for said combination to be a fixed combination, a non-fixed combination or a kit-of-parts. A “fixed combination” in the present invention is used as known to persons skilled in the art and is defined as a combination wherein, for example, a first active ingredient, such as one or more compounds of general formula (I) of the present invention, and a further active ingredient are present together in one unit dosage or in one single entity. One example of a “fixed combination” is a pharmaceutical composition wherein a first active ingredient and a further active ingredient are present in admixture for simultaneous administration, such as in a formulation. Another example of a “fixed combination” is a pharmaceutical combination wherein a first active ingredient and a further active ingredient are present in one unit without being in admixture. A non-fixed combination or “kit-of-parts” in the present invention is used as known to persons skilled in the art and is defined as a combination wherein a first active ingredient and a further active ingredient are present in more than one unit. One example of a non-fixed combination or kit-of-parts is a combination wherein the first active ingredient and the further active ingredient are present separately. It is possible for the components of the non-fixed combination or kit-of- parts to be administered separately, sequentially, simultaneously, concurrently or chronologically staggered. The compounds of the present invention can be administered as the sole pharmaceutical agent or in combination with one or more other pharmaceutically active ingredients where the combination causes no unacceptable adverse effects. The present invention also covers such pharmaceutical combinations. For example, the compounds of the present invention can be combined with known hormonal therapeutic agents. The compounds of the present invention can be combined with therapeutic agents or active ingredients, that are already approved or that are still under development for the treatment and/ or prophylaxis of diseases, which are related to or mediated by P2X3 receptor. Such therapeutic agents or active ingredients are for example, but not limited, to 5-(2,4-Diamino-pyrimidin-5- yloxy)-4-isopropyl-2-methoxy-benzenesulfonamide (Gefapixant/ MK-7264/AF-219), (5-(5-iodo- 2-isopropyl-4-methoxy-phenoxy)-pyrimidine-2,4-diamine (AF-353), 5-[2-isopropyl-4-methoxy-5- (methylsulfonyl)phenoxy]pyrimidine-2,4-diamine (AF-130), 2-[[4-amino-5-(5-iodo-4-methoxy-2- propan-2-ylphenoxy)-pyrimidin-2-yl]amino]propane-1,3-diol (AF-906), and (S)-methyl 2-((2-(2,6- difluoro-4-(methylcarbamoyl)-phenyl)-5-methyl-1H-benzo[d]imi dazol-1-yl)methyl)morpholine-4- carboxylate (BLU-5937/ NEO 5937). The compounds of the present invention can be combined with therapeutic agents or active ingredients, that are already approved or that are still under development for the treatment and/ or prophylaxis of diseases, which are related to other targets like NK1 inhibitors, for example 2- (R)-(4-Fluoro-2-methyl-phenyl)-4-(S)-((8aS)-6-oxohexahydro-p yrrolo[1,2-a]-pyrazin-2-yl)- piperidine-1-carboxylic acid [1-(R)-(3,5-bis-trifluoromethyl-phenyl)-ethyl]-methylamide (Orvepitant), 3-[(3aR,4R,5S,7aS)-5-{(1R)-1-[3,5-bis(trifluoromethyl)phenyl ]ethoxy}-4-(4- fluorophenyl)octahydro-2H-isoindol-2-yl]cyclopent-2-en-1-one (Serlopitant), 5-[((2S,3R)-2- 5- ([(2R,3S)-2-((R)-1-[3,5-bis(trifluoromethyl)phenyl]ethoxy)-3 -(4-fluorophenyl)- morpholino]methyl)-1H-1,2,4-triazol-3(2H)-one (Aprepitant), [2-[1-[[3,5-bis(trifluoromethyl)- phenyl]methyl]-5-pyridin-4-yltriazol-4-yl]pyridin-3-yl]-(2-c hlorophenyl)methanone (Tradipitant), (2R,4S)-4-(4-Acetylpiperazin-1-yl)-N--2-(4-fluoro-2-methylph enyl)-N-methylpiperidine-1- carboxamide (Casopitant), 2-[3,5-bis(trifluoromethyl)phenyl]-N,2-dimethyl-N-[4-(2-meth yl- phenyl)-6-(4-methyl-piperazin-1-yl)pyridin-3-yl]propanamide (Netupitant), [3-[[(2R,3S)-2-[(1R)- 1-[3,5-bis(trifluoro-methyl)phenyl]ethoxy]-3-(4-fluorophenyl )morpholin-4-yl]methyl]-5-oxo-4H- 1,2,4-triazol-1-yl]phosphonic acid (Fosaprepitant), (5S,8S)-8-[[(1R)-1-[3,5-bis(trifluoromethyl)- phenyl]ethoxy]methyl]-8-phenyl-1,9-diazaspiro[4.5]decan-2-on e (Rolapitant), (2S)-N-[(1R)-1- [3,5-bis(trifluoromethyl)phenyl]ethyl]-2-(4-fluoro-2-methylp henyl)-N-methylpiperazine-1- carboxamide (Vestipitant), 2-[1-[2-[(2R)-4-[2-[3,5-bis(trifluoromethyl)phenyl]acetyl]-2 -(3,4- dichlorophenyl)morpholin-2-yl]ethyl]piperidin-4-yl]-2-methyl propanamide (Burapitant), [4-[5-[[2- [3,5-bis(trifluoromethyl)phenyl]-2-methylpropanoyl]-methylam ino]-4-(2-methylphenyl)pyridin-2- yl]-1-methylpiperazin-1-ium-1-yl]methyl hydrogen phosphate (Fosnetupitant), or NK1/ NK3 inhibiotrs, for example N-[6-[(7S,9alphaS)-7-(hydroxymethyl)-3,4,6,7,9,9alpha-hexahy dro-1H- pyrazino[2,1-c][1,4]oxazin-8-yl]-4-(4-fluoro-2-methylphenyl) pyridin-3-yl]-2-[3,5-bis(trifluoro- methyl)phenyl]-N,2-dimethylpropanamide (Elinzanetant), or nicotinic Acetylcholine modulators, for example N-(2-((3-pyridinyl)Methyl)-1-azabicyclo[2.2.2]oct-3-yl)benzo furan-2-carboxamide (Bradanicline/ ATA-101). The compounds of the present invention can be administered as the sole pharmaceutical agent or in combination with one or more other pharmaceutically active ingredients where the combination causes no unacceptable adverse effects. The present invention also covers such pharmaceutical combinations. For example, the compounds of the present invention can be combined with known indication agents. For example, the compounds of the present invention can be combined with known hormonal therapeutic agents. In particular, the compounds of the present invention can be administered in combination or as comedication with Selective Progesterone Receptor Modulators (SPRMs) or hormonal contraceptives. SPRMs and hormonal contraceptives can be administered via oral, subcutan, transdermal, intrauterine or intravaginal route, for example as Combined Oral Contraceptives (COCs), or Progestin-Only-Pills (POPs) or hormone-containing devices like implants, patches or intravaginal rings. COCs include but are not limited to birth control pills or a birth control method that includes a combination of an estrogen (estradiol) and a progestogen (progestin). The estrogenic part is in most of the COCs ethinyl estradiol. Some COCs contain estradiol or estradiol valerate. Said COCs contain the progestins norethynodrel, norethindrone, norethindrone acetate, ethynodiol acetate, norgestrel, levonorgestrel, norgestimate, desogestrel, gestodene, drospirenone, dienogest, or nomegestrol acetate. Birth control pills include for example but are not limited to Yasmin, Yaz, both containing ethinyl estradiol and drospirenone; Microgynon or Miranova containing levonorgestrel and ethinyl estradiol; Marvelon containing ethinyl estradiol and desogestrel; Valette containing ethinyl estradiol and dienogest; Belara and Enriqa containing ethinyl estradiol and chlormadinonacetate; Qlaira containing estradiol valerate and dienogest as active ingredients; and Zoely containing estradiol and normegestrol. POPs are contraceptive pills that contain only synthetic progestogens (progestins) and do not contain estrogen. They are colloquially known as mini pills. POPs include but are not limited to Cerazette containing desogestrel; Microlut containing levonorgestrel and Micronor containing norethindrone. Other Progeston-Only forms are intrauterine devices (IUDs), for example Mirena containing levonorgestrel or injectables, for example Depo-Provera containing medroxyprogesterone acetate, or implants, for example Implanon containing etonogestrel. Other hormone-containing devices with contraceptive effect which are suitable for a combination with the compounds of the present invention are vaginal rings like Nuvaring containing ethinyl estradiol and etonogestrel or transdermal systems like a contraceptive patch, for example Ortho- Evra or Apleek (Lisvy) containing ethinyl estradiol and gestodene. A preferred embodiment of the present invention is the administration of a compound of general formula (I) in combination with a COC or a POP or other Progestin-Only forms as well as vaginal rings or contraceptive patches as mentioned above. The compounds of the present invention can be combined with therapeutic agents or active ingredients, that are already approved or that are still under development for the treatment and/ or prophylaxis of diseases which are related to or mediated by P2X3 receptor. For the treatment and/ or prophylaxis of urinary tract diseases, the compounds of the present invention can be administered in combination or as comedication with any substance that can be applied as therapeutic agent in the following indications: Urinary tract disease states associated with the bladder outlet obstruction; urinary incontinence conditions such as reduced bladder capacity, increased frequency of micturition, urge incontinence, stress incontinence, or bladder hyperreactivity; benign prostatic hypertrophy; prostatic hyperplasia; prostatitis; detrusor hyperreflexia; overactive bladder and symptoms related to overactive bladder wherein said symptoms are in particular increased urinary frequency, nocturia, urinary urgency or urge incontinence; pelvic hypersensitivity; urethritis; prostatitis; prostatodynia; cystitis, in particular interstitial cystitis; idiopathic bladder hypersensitivity. For the treatment and/ or prophylaxis of overactive bladder and symptoms related to overactive bladder, the compounds of the present invention can be administered in combination or as comedication, independently or in addition to behavioral therapy like diet, lifestyle or bladder training, with anticholinergics like oxybutynin, tolterodine, propiverine, solifenacin, darifenacin, trospium, fesoterdine; ß-3 agonists like mirabegron; neurotoxins like onabutolinumtoxin A; or antidepressants like imipramine, duloxetine. For the treatment and/ or prophylaxis of interstitial cystitis, the compounds of the present invention can be administered in combination or as comedication, independently or in addition to behavioral therapy like diet, lifestyle or bladder training, with pentosans like elmiron; NSAIDS (Non-Steroidal Antiinflammatory Drugs), either unselective NSAIDS like ibuprofen, diclofenac, aspirin, naproxen, ketoprofen, indomethacin; as well as Cox-2 selective NSAIDS like Parecoxib, Etoricoxib, Celecoxib; antidepressants like amitriptyline, imipramine; or antihistamines like loratadine. For the treatment and/ or prophylaxis of gynaecological diseases, the compounds of the present invention can be administered in combination or as comedication with any substance that can be applied as therapeutic agent in the following indications: dysmenorrhea, including primary and secondary dysmenorrhea; dyspareunia; endometriosis; endometriosis-associated pain; endometriosis-associated symptoms, wherein said symptoms are in particular dysmenorrhea, dyspareunia, dysuria, or dyschezia. For the treatment and/ or prophylaxis of dysmenorrhea, including primary and secondary dysmenorrhea; dyspareunia; endometriosis and endometriosis-associated pain, the compounds of the present invention can be administered in combination or as comedication with pain medicaments, in particular NSAIDS like ibuprofen, diclofenac, aspirin, naproxen, ketoprofen, indomethacin; as well as Cox-2 selective NSAIDS like Parecoxib, Etoricoxib, Celecoxib; or in combination with ovulation inhibiting treatment, in particular COCs as mentioned above or contraceptive patches like Ortho-Evra or Apleek (Lisvy); or with progestogenes like dienogest (Visanne); or with GnRH analogous, in particular GnRH agonists and antagonists, for example leuprorelin, nafarelin, goserelin, cetrorelix, abarelix, ganirelix, degarelix; or with androgens: danazol. For the treatment and/ or prophylaxis of endometriosis and endometriosis-associated pain, the compounds of the present invention can be administered in combination or as comedication with GnRH antagonists like Elagolix, Linzagolix, or Relugolix. For the treatment and/ or prophylaxis of endometriosis and endometriosis-associated pain, the compounds of the present invention can be administered in combination or as comedication with Selective Progesterone Receptor Modulators (SPRMs) or Progesterone antagonists like Vilaprisan, Ulipristal acetate, Telapristone, or Mifepristone. For the treatment and/ or prophylaxis of diseases which are associated with pain, or pain syndromes, the compounds of the present invention can be administered in combination or as comedication with any substance that can be applied as therapeutic agent in the following indications: pain-associated diseases or disorders like hyperalgesia, allodynia, functional bowel disorders (such as irritable bowel syndrome) and arthritis (such as osteoarthritis, rheumatoid arthritis and ankylosing spondylitis), burning mouth syndrome, burns, migraine or cluster headache, nerve injury, traumatic nerve injury, post-traumatic injuries (including fractures and sport injuries), neuritis, neuralgia, poisoning, ischemic injury, interstitial cystitis, trigeminal neuralgia, small fiber neuropathy, diabetic neuropathy, chronic arthritis and related neuralgias, HIV and HIV treatment- induced neuropathy. The compounds of the present invention can be combined with other pharmacological agents and compounds that are intended to treat inflammatory diseases, inflammatory pain or general pain conditions. In addition to well-known medicaments which are already approved and on the market, the compounds of the present invention can be administered in combination with inhibitors of PTGES (prostaglandin E synthase), with inhibitors of IRAK4 (interleukin-1 receptor-associated kinase 4) and with antagonists of the prostanoid EP4 receptor (prostaglandin E2 receptor 4). In particular, the compounds of the present invention can be administered in combination with pharmacological endometriosis agents, intended to treat inflammatory diseases, inflammatory pain or general pain conditions and/or interfering with endometriotic proliferation and endometriosis associated symptoms, namely with inhibitors of Aldo-keto-reductase1C3 (AKR1C3) and with functional blocking antibodies of the prolactin receptor. For the treatment and/ or prophylaxis of chronic cough and symptoms related to chronic cough, the compounds of the present invention can be administered in combination or as comedication with cough suppressants like dextromethorphan, benzonatate, codeine or hydrocodone; with inhalative agents to treat eosinophilic bronchitis, COPD or asthma like budesonide, beclomethasone, fluticasone, theophylline, ipatropiumbromid, montelukast or salbutamol; with drugs like proton pump inhibitors which are used to treat acid reflux, for example omeprazole, esomeprazole, lansoprazole, ranitidine, famotidine, cimetidine; and promotility agents such as metoclopramide; with nasal or topical glucocorticoids like fluticasone or mometasone or triamcinolone; or with oral antihistamines like loratadine, fexofenadine or cetirizine. The compounds of the present invention can be combined with other pharmacological agents and compounds that are intended for the treatment, prevention or management of cancer. In particular, the compounds of the present invention can be administered in combination with 131I-chTNT, abarelix, abiraterone, aclarubicin, ado-trastuzumab emtansine, afatinib, aflibercept, aldesleukin, alemtuzumab, Alendronic acid, alitretinoin, altretamine, amifostine, aminoglutethimide, Hexyl aminolevulinate,amrubicin, amsacrine, anastrozole, ancestim, anethole dithiolethione, angiotensin II, antithrombin III, aprepitant, arcitumomab, arglabin, arsenic trioxide, asparaginase, axitinib, azacitidine, basiliximab, belotecan, bendamustine, belinostat, bevacizumab, bexarotene, bicalutamide, bisantrene, bleomycin, bortezomib, buserelin, bosutinib, brentuximab vedotin, busulfan, cabazitaxel, cabozantinib, calcium folinate, calcium levofolinate, capecitabine, capromab, carboplatin, carfilzomib, carmofur, carmustine, catumaxomab, celecoxib, celmoleukin, ceritinib, cetuximab, chlorambucil, chlormadinone, chlormethine, cidofovir, cinacalcet, cisplatin, cladribine, clodronic acid, clofarabine, copanlisib, crisantaspase, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, darbepoetin alfa, dabrafenib, dasatinib, daunorubicin, decitabine, degarelix, denileukin diftitox, denosumab, depreotide, deslorelin, dexrazoxane, dibrospidium chloride, dianhydrogalactitol, diclofenac, docetaxel, dolasetron, doxifluridine, doxorubicin, doxorubicin + estrone, dronabinol, eculizumab, edrecolomab, elliptinium acetate, eltrombopag, endostatin, enocitabine, enzalutamide, epirubicin, epitiostanol, epoetin alfa, epoetin beta, epoetin zeta, eptaplatin, eribulin, erlotinib, esomeprazole, estradiol, estramustine, etoposide, everolimus, exemestane, fadrozole, fentanyl, filgrastim, fluoxymesterone, floxuridine, fludarabine, fluorouracil, flutamide, folinic acid, formestane, fosaprepitant, fotemustine, fulvestrant, gadobutrol, gadoteridol, gadoteric acid meglumine, gadoversetamide, gadoxetic acid, gallium nitrate, ganirelix, gefitinib, gemcitabine, gemtuzumab, Glucarpidase, glutoxim, GM-CSF, goserelin, granisetron, granulocyte colony stimulating factor, histamine dihydrochloride, histrelin, hydroxycarbamide, I- 125 seeds, lansoprazole, ibandronic acid, ibritumomab tiuxetan, ibrutinib, idarubicin, ifosfamide, imatinib, imiquimod, improsulfan, indisetron, incadronic acid, ingenol mebutate, interferon alfa, interferon beta, interferon gamma, iobitridol, iobenguane (123I), iomeprol, ipilimumab, irinotecan, Itraconazole, ixabepilone, lanreotide, lapatinib, Iasocholine, lenalidomide, lenograstim, lentinan, letrozole, leuprorelin, levamisole, levonorgestrel, levothyroxine sodium, lisuride, lobaplatin, lomustine, lonidamine, masoprocol, medroxyprogesterone, megestrol, melarsoprol, melphalan, mepitiostane, mercaptopurine, mesna, methadone, methotrexate, methoxsalen, methylaminolevulinate, methylprednisolone, methyltestosterone, metirosine, mifamurtide, miltefosine, miriplatin, mitobronitol, mitoguazone, mitolactol, mitomycin, mitotane, mitoxantrone, mogamulizumab, molgramostim, mopidamol, morphine hydrochloride, morphine sulfate, nabilone, nabiximols, nafarelin, naloxone + pentazocine, naltrexone, nartograstim, nedaplatin, nelarabine, neridronic acid, nivolumabpentetreotide, nilotinib, nilutamide, nimorazole, nimotuzumab, nimustine, nitracrine, nivolumab, obinutuzumab, octreotide, ofatumumab, omacetaxine mepesuccinate, omeprazole, ondansetron, oprelvekin, orgotein, orilotimod, oxaliplatin, oxycodone, oxymetholone, ozogamicine, p53 gene therapy, paclitaxel, palifermin, palladium-103 seed, palonosetron, pamidronic acid, panitumumab, pantoprazole, pazopanib, pegaspargase, PEG-epoetin beta (methoxy PEG-epoetin beta), pembrolizumab, pegfilgrastim, peginterferon alfa-2b, pemetrexed, pentazocine, pentostatin, peplomycin, Perflubutane, perfosfamide, Pertuzumab, picibanil, pilocarpine, pirarubicin, pixantrone, plerixafor, plicamycin, poliglusam, polyestradiol phosphate, polyvinylpyrrolidone + sodium hyaluronate, polysaccharide-K, pomalidomide, ponatinib, porfimer sodium, pralatrexate, prednimustine, prednisone, procarbazine, procodazole, propranolol, quinagolide, rabeprazole, racotumomab, radium-223 chloride, radotinib, raloxifene, raltitrexed, ramosetron, ramucirumab, ranimustine, rasburicase, razoxane, refametinib , regorafenib, risedronic acid, rhenium-186 etidronate, rituximab, romidepsin, romiplostim, romurtide, roniciclib , samarium (153Sm) lexidronam, sargramostim, satumomab, secretin, sipuleucel-T, sizofiran, sobuzoxane, sodium glycididazole, sorafenib, stanozolol, streptozocin, sunitinib, talaporfin, tamibarotene, tamoxifen, tapentadol, tasonermin, teceleukin, technetium (99mTc) nofetumomab merpentan, 99mTc- HYNIC-[Tyr3]-octreotide, tegafur, tegafur + gimeracil + oteracil, temoporfin, temozolomide, temsirolimus, teniposide, testosterone, tetrofosmin, thalidomide, thiotepa, thymalfasin, thyrotropin alfa, tioguanine, tocilizumab, topotecan, toremifene, tositumomab, trabectedin, tramadol, trastuzumab, trastuzumab emtansine, treosulfan, tretinoin, trifluridine + tipiracil, trilostane, triptorelin, trametinib, trofosfamide, thrombopoietin, tryptophan, ubenimex, valatinib , valrubicin, vandetanib, vapreotide, vemurafenib, vinblastine, vincristine, vindesine, vinflunine, vinorelbine, vismodegib, vorinostat, vorozole, yttrium-90 glass microspheres, zinostatin, zinostatin stimalamer, zoledronic acid, zorubicin. Furthermore, the compounds of the present invention can be combined with active ingredients, which are well known for the treatment of cancer-related pain and chronic pain. Such combinations include, but are not limited to NSAIDS (either unselective NSAIDS like ibuprofen, diclofenac, aspirin, naproxen, ketoprofen and indomethacin; and Cox-2 selective NSAIDS like Parecoxib, Etoricoxib and Celecoxib), step II opiods like codeine phosphate, dextropropoxyphene, dihydro¬codeine,Tramadol), step III opiods like morphine, fentanyl, buprenorphine, oxymorphone, oxycodone and hydromorphone; and other medications used for the treatment of cancer pain like steroids as Dexamethasone and methylprednisolone; bisphosphonates like Etidronate, Clodronate, Alendronate, Risedronate, and Zoledronate; tricyclic antidepressants like Amitriptyline, Clomipramine, Desipramine, Imipramine and Doxepin; class I antiarrhythmics like mexiletine and lidocaine; anticonvulsants like carbamazepine, Gabapentin, oxcarbazepine, phenytoin, pregabalin, topiramate, alprazolam, diazepam, flurazepam, pentobarbital and phenobarbital. Based upon standard laboratory techniques known to evaluate compounds useful for the treatment of neurogenic, by standard toxicity tests and by standard pharmacological assays for the determination of treatment of the conditions identified above in mammals, and by comparison of these results with the results of known active ingredients or medicaments that are used to treat these conditions, the effective dosage of the compounds of the present invention can readily be determined for treatment of each desired indication. The amount of the active ingredient to be administered in the treatment of one of these conditions can vary widely according to such considerations as the particular compound and dosage unit employed, the mode of administration, the period of treatment, the age and sex of the patient treated, and the nature and extent of the condition treated. The total amount of the active ingredient to be administered will generally range from about 0.001 mg/kg to about 200 mg/kg body weight per day, and preferably from about 0.01 mg/kg to about 50 mg/kg body weight per day. Clinically useful dosing schedules will range from one to three times a day dosing to once every four weeks dosing. In addition, it is possible for "drug holidays", in which a patient is not dosed with a drug for a certain period of time, to be beneficial to the overall balance between pharmacological effect and tolerability. It is possible for a unit dosage to contain from about 0.5 mg to about 400 mg of active ingredient and can be administered one or more times per day or less than once a day. The average daily dosage for administration by injection, including intravenous, intramuscular, subcutaneous and parenteral injections, and use of infusion techniques will preferably be from 0.01 to 200 mg/kg of total body weight. The average daily rectal dosage regimen will preferably be from 0.01 to 200 mg/kg of total body weight. The average daily vaginal dosage regimen will preferably be from 0.01 to 200 mg/kg of total body weight. The average daily topical dosage regimen will preferably be from 0.1 to 200 mg administered between one to four times daily. The transdermal concentration will preferably be that required to maintain a daily dose of from 0.01 to 200 mg/kg. The average daily inhalation dosage regimen will preferably be from 0.01 to 100 mg/kg of total body weight. Of course the specific initial and continuing dosage regimen for each patient will vary according to the nature and severity of the condition as determined by the attending diagnostician, the activity of the specific compound employed, the age and general condition of the patient, time of administration, route of administration, rate of excretion of the drug, drug combinations, and the like. The desired mode of treatment and number of doses of a compound of the present invention or a pharmaceutically acceptable salt or ester or composition thereof can be ascertained by those skilled in the art using conventional treatment tests. Methods of testing for a particular pharmacological or pharmaceutical property are well known to persons skilled in the art. The example testing experiments described herein serve to illustrate the present invention and the invention is not limited to the examples given. EXPERIMENTAL SECTION Abbreviations: The following table lists the abbreviations used in this paragraph and in the Examples section as far as they are not explained within the text body. Other abbreviations have their meanings customary per se to the skilled person.
Other abbreviations have their meanings customary per se to the skilled person. The various aspects of the invention described in this application are illustrated by the following examples which are not meant to limit the invention in any way. The example testing experiments described herein serve to illustrate the present invention and the invention is not limited to the examples given.
EXPERIMENTAL SECTION - GENERAL PART All reagents, for which the synthesis is not described in the experimental part, are either commercially available, or are known compounds or may be formed from known compounds by known methods by a person skilled in the art. The compounds and intermediates produced according to the methods of the invention may require purification. Purification of organic compounds is well known to the person skilled in the art and there may be several ways of purifying the same compound. In some cases, no purification may be necessary. In some cases, the compounds may be purified by crystallization. In some cases, impurities may be stirred out using a suitable solvent. In some cases, the compounds may be purified by chromatography, particularly flash column chromatography, using for example prepacked silica gel cartridges, e.g. Biotage SNAP cartidges KP-Sil ® or KP- NH ® in combination with a Biotage autopurifier system (SP4 ® or Isolera Four ® ) and eluents such as gradients of hexane/ethyl acetate or DCM/methanol. In some cases, the compounds may be purified by preparative HPLC using for example a Waters autopurifier equipped with a diode array detector and/or on-line electrospray ionization mass spectrometer in combination with a suitable prepacked reverse phase column and eluents such as gradients of water and acetonitrile which may contain additives such as trifluoroacetic acid, formic acid or aqueous ammonia. In some cases, purification methods as described above can provide those compounds of the present invention which possess a sufficiently basic or acidic functionality in the form of a salt, such as, in the case of a compound of the present invention which is sufficiently basic, a trifluoroacetate or formate salt for example, or, in the case of a compound of the present invention which is sufficiently acidic, an ammonium salt for example. A salt of this type can either be transformed into its free base or free acid form, respectively, by various methods known to the person skilled in the art, or be used as salts in subsequent biological assays. It is to be understood that the specific form (e.g. salt, free base etc.) of a compound of the present invention as isolated and as described herein is not necessarily the only form in which said compound can be applied to a biological assay in order to quantify the specific biological activity. Furthermore, the intermediates and examples according to the invention may be present as rotational isomers, in particular in NMR studies. In cases where the presence of rotamers are clearly visible by NMR, it is stated in the experimental section. Purity figures are generally based on corresponding peak integrations in the LC/MS chromatogram, but may additionally also have been determined with the aid of the 1 H NMR spectrum. In solvent-containing or contaminated batches, the formal yield may be ">100%"; in these cases the yield is not corrected for solvent or purity. The multiplicities of proton signals in 1 H NMR spectra reported in the paragraphs which follow represent the signal form observed in each case and do not take account of any higher-order signal phenomena. In general, the stated chemical shift refers to the centre of the signal in question. In the case of broad multiplets, an interval is given. Signals obscured by solvent or water were either tentatively assigned or have not been listed. Significantly broadened signals – caused, for example, by rapid rotation of molecular moieties or because of exchanging protons – were likewise assigned tentatively (often referred to as a broad multiplet or broad singlet or broad doublet) or are not listed. The 1 H NMR data of selected synthesis intermediates and working examples are stated in the form of 1 H NMR peak lists. For each signal peak, first the δ[ppm] = value in ppm and then the signal intensity in round brackets are listed. The δ[ppm] = value/signal intensity number pairs for different signal peaks are listed with separation from one another by commas. The peak list for an example therefore takes the following form: δ[ppm] = 1 (intensity 1 ), δ[ppm] = 2 (intensity 2 ), ..., δ[ppm] = i (intensity i ), ... , δ[ppm] = n (intensity n ). The intensity of sharp signals correlates with the height of the signals in a printed example of an NMR spectrum in cm and shows the true ratios of the signal intensities in comparison with other signals. In the case of broad signals, several peaks or the middle of the signal and the relative intensity thereof may be shown in comparison to the most intense signal in the spectrum. The lists of the 1 H NMR peaks are similar to the conventional 1 H NMR printouts and thus usually contain all peaks listed in a conventional NMR interpretation. In addition, like conventional 1 H NMR printouts, they may show solvent signals, signals of stereoisomers of the target compounds which are likewise provided by the invention, and/or peaks of impurities. The peaks of stereoisomers of the target compounds and/or peaks of impurities usually have a lower intensity on average than the peaks of the target compounds (for example with a purity of > 90%). Such stereoisomers and/or impurities may be typical of the particular preparation process. Their peaks can thus help in identifying reproduction of our preparation process with reference to "by-product fingerprints". An expert calculating the peaks of the target compounds by known methods (MestreC, ACD simulation, or using empirically evaluated expected values) can, if required, isolate the peaks of the target compounds, optionally using additional intensity filters. This isolation would be similar to the peak picking in question in conventional 1 H NMR interpretation. A detailed description of the presentation of NMR data in the form of peak lists can be found in the publication "Citation of NMR Peaklist Data within Patent Applications" (cf. Research Disclosure Database Number 605005, 2014, 1 August 2014 or http://www.researchdisclosure.com/searching-disclosures). In the peak picking routine described in Research Disclosure Database Number 605005, the parameter "MinimumHeight" can be set between 1% and 4%. Depending on the type of chemical structure and/or depending on the concentration of the compound to be analysed, it may be advisable to set the parameters "MinimumHeight" to values of < 1%. Analysis methods: LC-MS, Analytical Method A: Instrument: Waters Acquity UPLCMS SingleQuad; Column: Acquity UPLC BEH C181.7 µm, 50x2.1mm; eluent A: water + 0.1 vol % formic acid (99%), eluent B: acetonitrile; gradient: 0-1.6 min 1-99% B, 1.6-2.0 min 99% B; flow 0.8 ml/min; temperature: 60 °C; DAD scan: 210-400 nm. Purification Methods: Biotage Isolera TM chromatography system (http://www.biotage.com/product-area/flash- purification) using pre-packed silica and pre-packed modified silica cartridges. Preparative HPLC, Method A: Instrument: pump: Labomatic HD-5000 or HD-3000, head HDK 280, lowpressure gradient module ND-B1000; manual injection valve: Rheodyne 3725i038; detector: Knauer Azura UVD 2.15; collector: Labomatic Labocol Vario-4000; column: Chromatorex RP C-18 10 µm, 125x30mm; eluent A: water + 0.1 vol % formic acid (99%), eluent B: acetonitrile; gradient A: 0 - 15 min 1 – 25% B; flow: 60 mL/min; gradient B: 0 - 15 min 10 – 50% B; flow: 60 mL/min; gradient C: 0 - 15 min 15 – 55% B; flow: 60 mL/min; gradient D: 0 - 15 min 30 – 70% B; flow: 60 mL/min; gradient E: 0 - 15 min 40 – 80% B; flow: 60 mL/min; gradient F: 0 - 15 min 65 – 100% B; flow: 60 mL/min; temperature: 25 °C; solution: max.250 mg / 2ml dimethyl sulfoxide; injection: 1 x 2 ml; Detection: UV 254 nm; Software: SCPA PrepCon5. EXPERIMENTAL SECTION - INTERMEDIATES Chemical naming of the examples and intermediates was performed using ACD software by ACD/LABS (Batch version 12.01.) or Marvin software by ChemAxon (Batch version 4.1.7 or higher). Reaction times are either specified explicitly in the protocols of the experimental section, or reactions were run until completion. Chemical reactions were monitored and their completion was judged using methods well known to the person skilled in the art, such as thin layer chromatography, e.g. on plates coated with silica gel, or by LC-MS methods. Intermediate 1 tert-butyl N-(5-bromo-2-carbamoylpyridin-3-yl)glycinate To a solution of 5-bromo-3-fluoropyridine-2-carboxamide (3.00 g, 13.7 mmol; CAS-RN:[669066- 90-4]) and N,N-diisopropylethylamine (6.0 ml, 34 mmol; CAS-RN:[7087-68-5]) in dry N,N- dimethylacetamide (30 ml) was added tert-butyl glycinate hydrogen chloride (1/1) (2.76 g, 16.4 mmol; CAS-RN:[27532-96-3]). The reaction mixture was stirred for 20 h at 80°C. The reaction was allowed to cool to room temperature and then the solution was partitioned between ethyl acetate and water. The organic layer was washed twice with brine, dried with a water repellant filter and concentrated. The organic layer was washed twice with brine, dried with a water repellant filter and concentrated. The residue was triturated with 10 ml MtBE and the suspension was added to 150 ml hexane were added. The precipitate thus obtained was collected by filtration, washed with hexane and dried to afford 3.28 g (71% yield, 98% purity) of the title compound as a white solid. 1 H NMR (500 MHz, DMSO-d6) δ [ppm]: 1.43 (s, 9H), 4.04 (d, 2H), 7.32 (d, 1H), 7.55 (br d, 1H), 7.87 (d, 1H), 8.03 (br d, 1H), 8.82 (t, 1H). LC-MS (Method A): R t = 1.26 min; MS (ESIpos): m/z = 330 [M+H] + Intermediate 2 tert-butyl (7-bromo-2,4-dioxo-3,4-dihydropyrido[3,2-d]pyrimidin-1(2H)-y l)acetate To a stirred solution of tert-butyl N-(5-bromo-2-carbamoylpyridin-3-yl)glycinate (1.77 g, 5.36 mmol, intermediate 1) in N,N-dimethylformamide (25 ml) was added sodium hydride (60% dispersion in mineral oil) (429 mg, 60 % purity, 10.7 mmol; CAS-RN:[7646-69-7]) at 4°C. The mixture was stirred for 1 h while cooled by an ice-bath. Then carbonyldiimidazole (3.48 g, 21.4 mmol; CAS-RN:[530-62-1]) was added in portions, the ice-bath was removed and the reaction mixture was stirred for 2 h at room temperature. The reaction was carefully quenched with water and the aqueous phase was extracted with ethyl acetate. The combined organics were washed with brine, dried with a water repellant filter and the solvent was removed under reduced pressure. The obtained residue was triturated with methyl tert-butyl ether (60 ml), the precipitate was collected by vacuum filtration and dried to afford 856 mg (44% yield, 95% purity) of the title compound. 1 H NMR (400 MHz, CHLOROFORM-d) δ [ppm]: 1.49 (s, 9H), 4.73 (s, 2H), 7.52 (d, 1H), 7.72 (s, 1H), 8.67 (d, 1H). LC-MS (Method A): R t = 0.97 min; MS (ESIpos): m/z = 356 [M+H] + Intermediate 3 tert-butyl [7-(morpholin-4-yl)-2,4-dioxo-3,4-dihydropyrido[3,2-d]pyrimi din-1(2H)-yl]acetate To a solution of tert-butyl (7-bromo-2,4-dioxo-3,4-dihydropyrido[3,2-d]pyrimidin-1(2H)-y l)acetate (400 mg, 1.12 mmol, intermediate 2) in dimethyl sulfoxide (4.0 ml) was added morpholine (970 µl, 11 mmol; CAS-RN:[110-91-8]). The reaction mixture was heated at 110°C for 4 h. The mixture was allowed to cool to ambient temperature and was poured into water (20 mL). The precipitate thus obtained was collected by vacuum filtration, washed with water and acetonitrile. The solid was dried in a vacuum drying cabinet (16 h; 40°C; 10 mbar) to afford 242 mg (58% yield, 98% purity) of the title compound. 1 H NMR (400 MHz, DMSO-d6) δ [ppm]: 1.41 (s, 9H), 3.41-3.46 (m, 4H), 3.72-3.77 (m, 4H), 4.82 (s, 2H), 6.86 (d, 1H), 8.31 (d, 1H), 11.55 (br s, 1H). LC-MS (Method A): R t = 0.86 min; MS (ESIneg): m/z = 361 [M-H]- Intermediate 4 tert-butyl 2-(4-chloro-7-morpholino-2-oxo-pyrido[3,2-d]pyrimidin-1-yl)a cetate and tert-butyl 2-(2-chloro-7-morpholino-4-oxo-pyrido[3,2-d]pyrimidin-1-yl)a cetate Tert-butyl [7-(morpholin-4-yl)-2,4-dioxo-3,4-dihydropyrido[3,2-d]pyrimi din-1(2H)-yl]acetate (50.0 mg, 138 µmol, intermediate 3) was dissolved in acetonitrile (1.0 mL) and N,N- diisopropylethylamine (77 µl, 830 µmol; CAS-RN:[10025-87-3]), then phosphoroxychloride (77 µl, 830 µmol; CAS-RN:[10025-87-3]) was added. The reaction mixture was heated at 50°C for 30 minutes. The mixture was concentrated under reduced pressure and the residue was co- evaporated three times with dioxane (5 mL). The crude mixture containing tert-butyl 2-(4-chloro- 7-morpholino-2-oxo-pyrido[3,2-d]pyrimidin-1-yl)acetate and tert-butyl 2-(2-chloro-7-morpholino- 4-oxo-pyrido[3,2-d]pyrimidin-1-yl)acetate was used in the next step without purification. Intermediate 5 tert-butyl 2-[4-[2-(2,2-dimethylpropanoyl)hydrazino]-7-morpholino-2-oxo -pyrido[3,2-d]pyrimidin- 1-yl]acetate and tert-butyl 2-[2-[2-(2,2-dimethylpropanoyl)hydrazino]-7-morpholino-4-oxo -pyrido[3,2-d]pyrimidin- 1-yl]acetate ; To a crude mixture of tert-butyl 2-(4-chloro-7-morpholino-2-oxo-pyrido[3,2-d]pyrimidin-1- yl)acetate and tert-butyl 2-(2-chloro-7-morpholino-4-oxo-pyrido[3,2-d]pyrimidin-1-yl)a cetate (110 mg, 289 µmol, intermediate 4) in dry dioxane (3.0 mL) was added 2,2- dimethylpropanehydrazide (101 mg, 867 µmol; CAS-RN:[42826-42-6]). The reaction mixture was heated at 80°C for 1 h. The reaction mixture was allowed to cool to room temperature and the solution was added to a saturated sodium bicarbonate solution (10 mL). The aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with brine, dried with a water repellant filter and evaporated to dryness. The crude mixture composed of tert-butyl 2-[4-[2-(2,2-dimethylpropanoyl)hydrazino]-7-morpholino-2-oxo -pyrido[3,2-d]pyrimidin-1-yl]- acetate and tert-butyl 2-[2-[2-(2,2-dimethylpropanoyl)hydrazino]-7-morpholino-4-oxo -pyrido[3,2- d]pyrimidin-1-yl]acetate was used in the next step without purification (85 mg, 64%). LC-MS (Method A): R t = 1.07 min; MS (ESIneg): m/z = 459 [M-H]-. Intermediate 6 [3-tert-butyl-8-(morpholin-4-yl)-5-oxopyrido[2,3-e][1,2,4]tr iazolo[4,3-c]pyrimidin-6(5H)-yl]acetic acid The crude product composed of tert-butyl {4-[2-(2,2-dimethylpropanoyl)hydrazinyl]-7- (morpholin-4-yl)-2-oxopyrido[3,2-d]pyrimidin-1(2H)-yl}acetat e and tert-butyl 2-[2-[2-(2,2- dimethylpropanoyl)hydrazino]-7-morpholino-4-oxo-pyrido[3,2-d ]pyrimidin-1-yl]acetate (85.0 mg, 185 µmol, intermediate 5) was dissolved in a solution of hydrochloric acid in dioxane (4M) (460 µl, 4.0 M, 1.8 mmol; CAS-RN:[7647-01-0]) and the reaction mixture was heated at 80°C for 4 h. After this time the reaction was cooled to room temperature and the solvent was removed under reduced pressure. The obtained residue was dissolved in 2 ml dimethyl sulfoxide /water (1:1), filtered via syringe filter and purified by HPLC (Method A, gradient C) to afford 21.0 mg (29% yield, 97% purity) of the title compound and 14.5 mg (20% yield, 92% purity) of [3-tert-butyl-8- (morpholin-4-yl)-5-oxopyrido[3,2-d][1,2,4]triazolo[4,3-a]pyr imidin-10(5H)-yl]acetic acid. 1 H NMR (400 MHz, DMSO-d6) δ [ppm]: 1.41 (s, 9H), 3.43-3.51 (m, 4H), 3.73-3.82 (m, 4H), 5.08 (s, 2H), 7.22 (d, 1H), 8.48 (d, 1H), 13.32 (br s, 1H). LC-MS (Method A): R t = 0.84 min; MS (ESIpos): m/z = 387 [M+H] + Intermediate 7 [3-tert-butyl-8-(morpholin-4-yl)-5-oxopyrido[3,2-d][1,2,4]tr iazolo[4,3-a]pyrimidin-10(5H)-yl]acetic acid The title compound resulted as a side product under the synthesis described at intermediate 6. LC-MS (Method A): R t = 0.78 min; MS (ESIpos): m/z = 387 [M+H] + Intermediate 8 5-bromo-2-{[methoxy]carbonyl}pyridine-3-carboxylic acid 3-Bromo-5H,7H-furo[3,4-b]pyridine-5,7-dione (27.1 g, 119 mmol; CAS-RN:[98278-78-5]) was added portion-wise over 5 minutes into cooled to 0°C methanol (180 mL). The reaction mixture was stirred for 30 min at 0°C and then allowed to warm to room temperature and stirred for 16 h. The solvent was removed in vacuo. The residue was recrystallized from toluene (200 mL). The precipitate was filtered, washed with cooled toluene and dried in vacuo to afford 18.4 g (60% yield) of the title compound. Intermediate 9 methyl 5-bromo-3-(3-tert-butoxy-3-oxopropanoyl)pyridine-2-carboxyla te To 5-bromo-2-(methoxycarbonyl)nicotinic acid (18.4 g, 70.8 mmol, intermediate 8) in acetonitrile (285 mL) in reaction flask #1, CDI (12.6 g, 77.9 mmol) was added portion-wise and the mixture was heated to 60°C for 3 h. After that period the reaction mass (RM#1) was allowed to cool to room temperature. To a mixture of potassium 3-tert-butoxy-3-oxopropanoate (29.5 g, 149 mmol) and acetonitrile (285 mL) in reaction flask #2, cooled to 5-10 °C, triethylamine (20.7 mL, 149 mmol) was added dropwise over 5 min followed by the addition of magnesium chloride (16.9 g, 177 mmol). The reaction mass (RM#2) was stirred for 3 h at room temperature, then cooled to 0°C and RM#1 was added portionwise over 5-15 min. The reaction mixture was stirred for 16 h at room temperature. The solvent was removed in vacuo. The residue was diluted with aqueous saturated sodium carbonate solution (10% aq. sol., 285 mL) and dichloromethane (285 mL). The obtained mixture was filtrated through cotton; the filtrate layers were separated. The aqueous layer was extracted with dichloromethane (3 × 145 mL). The combined organic layer was washed with citric acid (15% aq. sol., 425 mL), sodium chloride (5% aq. sol., 2 × 425 mL), dried over sodium sulfate, filtered and evaporated under reduced pressure to afford 20.1 g (79% yield) of the title compound. Intermediate 10 tert-butyl (3-bromo-8-oxo-7,8-dihydropyrido[2,3-d]pyridazin-5-yl)acetat e A solution of methyl 5-bromo-3-(3-tert-butoxy-3-oxopropanoyl)pyridine-2-carboxyla te (13.4 g, 37.3 mmol, intermediate 9) in methanol (100 mL) was cooled to -5-0 °C. Then a solution of hydrazine hydrate (1.96 g, 39.2 mmol) in methanol (60 mL) was added dropwise over 10 min. The reaction mixture was slowly allowed to reach room temperature and stirred for 24 hours. The precipitate formed was filtered, washed with methanol (25 mL) and dried in vacuo to afford 5.94 g (47% yield) of the title compound. Intermediate 11 tert-butyl [3-(morpholin-4-yl)-8-oxo-7,8-dihydropyrido[2,3-d]pyridazin- 5-yl]acetate tert-Butyl (3-bromo-8-oxo-7,8-dihydropyrido[2,3-d]pyridazin-5-yl)acetat e (450 mg, 1.32 mmol, intermediate 10) (6.64 g, 19.5 mmol) was dissolved in dimethyl sulfoxide (34 mL). Then morpholine (17.0 g, 195 mmol) was added. The reaction mixture was heated to 130°C for 16 h. After that period, the reaction mass was diluted with cold water (200 mL). The precipitate formed was filtered, washed with water twice and dried in vacuo to afford 5.66 g (84% yield) of the title compound. Intermediate 12 tert-butyl [8-chloro-3-(morpholin-4-yl)pyrido[2,3-d]pyridazin-5-yl]acet ate POCl3 (124 mL, 1310 mmol) was cooled to 0°C and N,N-diisopropylethylamine (8.55 mL, 49.0 mmol) was added dropwise. Then tert-butyl [3-(morpholin-4-yl)-8-oxo-7,8-dihydropyrido[2,3- d]pyridazin-5-yl]acetate (5.66 g, 16.3 mmol, intermediate 11) was added portionwise. The reaction mixture was heated to 50°C for 3 hours. After that period, the reaction mixture was evaporated in vacuo. The residue was diluted with dichloromethane (300 mL) and poured into aqueous saturated sodium bicarbonate solution (600 mL). After pH has been confirmed to be ≥7, the layers were separated. The aqueous layer was extracted with dichloromethane (300 mL). The combined organic layer was washed with sodium chloride (5% aq. sol., 2 × 600 mL), dried over sodium sulfate, filtered and evaporated under reduced pressure to afford 5.63 g (95% yield) of the title compound, that was used in the next step without further purification. Intermediate 13 tert-butyl {8-[2-(2,2-dimethylpropanoyl)hydrazino]-3-(morpholin-4-yl)py rido[2,3-d]pyridazin-5- yl}acetate tert-Butyl {4-[2-(2,2-dimethylpropanoyl)hydrazinyl]-7-(morpholin-4-yl)- 2-oxopyrido[3,2-d]-pyrimi- din-1(2H)-yl}acetate (5.50 g, 15.1 mmol, intermediate 12) and 2,2-dimethylpropanehydrazide (5.25 g, 45.2 mmol) were mixed in dioxane (140 mL). The reaction mixture was heated to 100 °C (an oil bath) for 16 hours. The solvent was removed in vacuo. The residue was dissolved in dichloromethane (250 mL), washed with ammonium chloride (aq. sat., 250 mL). Then aqueous layer was extracted with dichloromethane (250 mL). The combined organic layer was washed with ammonium chloride (aq. sat., 500 mL), aqueous sodium chloride solution (5% aq. sol., 2 × 500 mL), dried over sodium sulfate, filtered and evaporated under reduced pressure. The residue was purified by column chromatography (chloroform/methanol gradient from 49:1 to 24:1; TLC (14:1); Rf=0.3) to give 3.59 g of the title compound (56% yield). Intermediate 14 [3-tert-butyl-8-(morpholin-4-yl)pyrido[2,3-d][1,2,4]triazolo [4,3-b]pyridazin-6-yl]acetic acid {8-[2-(2,2-dimethylpropanoyl)hydrazino]-3-(morpholin-4-yl)py rido[2,3-d]pyridazin-5-yl}acetate (2.59 g, 6.07 mmol, intermediate 13) was dissolved in dichloromethane (80 mL). Then 4 M HCl·dioxane (155 mL, 303 mmol) was added. The reaction mixture was heated to 45 °C (an oil bath) for 16 hours. After that period, the solvents were removed in vacuo to afford the title compound (quantitative yield). Intermediate 15 tert-butyl N-(2-cyano-5-cyclopropylpyridin-3-yl)glycinate To a stirred solution of 5-bromo-3-fluoropyridine-2-carbonitrile (4.00 g, 19.9 mmol; CAS- RN:[886373-28-0])in anhydrous N,N-dimethylacetamide was added tert-butyl glycinate hydrochloride (1/1) (3.67 g, 21.9 mmol; CAS-RN:[27532-96-3]) and N,N-diisopropylethylamine (8.7 ml, 50 mmol; CAS-RN:[7087-68-5]). The reaction mixture was stirred at 80°C for 16 h. After this time the mixture was allowed to cool to ambient temperature, water (100 ml) was added and the aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with brine (twice with 30 ml), dried with a water-repellant filter and concentrated to afford 4.21 g (66% yield, 98% purity) of the title compound. 1 H NMR (400 MHz, CHLOROFORM-d) δ [ppm]: 1.51 (s, 9H), 3.86 (d, 2H), 5.30 (br s, 1H), 7.08 (d, 1H), 8.06 (d, 1H) LC-MS (Method A): Rt = 1.27 min; MS (ESIpos): m/z = 312 [M+H] + Intermediate 16 tert-butyl N-[2-cyano-5-(morpholin-4-yl)pyridin-3-yl]glycinate To a solution of tert-butyl N-(5-bromo-2-cyanopyridin-3-yl)glycinate (400 mg, 1.28 mmol, intermediate 15) in dimethyl sulfoxide (5.0 ml) was added morpholine (1.1 ml, 13 mmol; CAS- RN:[110-91-8]). The reaction mixture was heated at 110°C for 16 h. The reaction mixture was allowed to cool to ambient temperature and was added to 30 ml water. The precipitate thus obtained was collected by vacuum filtration, washed with water and hexane and dried to afford 344 mg (76% yield, 90% purity) of the title compound as a light brown solid. 1 H NMR (400 MHz, CHLOROFORM-d) δ [ppm]: 1.49 (s, 9H), 3.26-3.30 (m, 4H), 3.83-3.87 (m, 6H), 5.10 (br t, 1H), 6.10 (d, 1H), 7.73 (d, 1H). LC-MS (Method A): Rt = 1.07 min; MS (ESIpos): m/z = 319 [M+H] + Intermediate 17 tert-butyl N-{2-[(4±)-4-tert-butyl-4,5-dihydro-1H-imidazol-2-yl]-5-(mo rpholin-4-yl)pyridin-3- yl}glycinate To a solution of tert-butyl N-[2-cyano-5-(morpholin-4-yl)pyridin-3-yl]glycinate (500 mg, 1.57 mmol, intermediate 16) in N,N-dimethylacetamide (10 mL) is added (±)-3,3-dimethybutane-1,2- diamine (549 mg, 4.72 mmol, CAS-RN:[157750-90-8]) and triethylamine (1.32 mL, 9.47 mmol) (561 mg, 5.55 mmol). The reaction mixture is heated at 150°C for 16 h. Aller cooling down to room temperature, the mixture is diluted with ethyl acetate and the organic layer is washed with water, brine and dried over anhydrous sodium sulfate and evaporated to dryness to afford the title compound that is used in the next step without further purification. MS (ESIpos): m/z = 418 [M+H] + . Intermediate 18 tert-butyl [(2±)-2-tert-butyl-8-(morpholin-4-yl)-5-oxo-2,3-dihydroimid azo[1,2-c]pyrido[2,3- e]pyrimidin-6(5H)-yl]acetate To a solution of tert-butyl N-{2-[(4±)-4-tert-butyl-4,5-dihydro-1H-imidazol-2-yl]-5-(mo rpholin-4- yl)pyridin-3-yl}glycinate (350 mg, 0.84 mmol, intermediate 17) in NMP (9.2 mL), is added carbonyldiimidazole (381 mg, 2.35 mmol) and 4-dimethylaminopyridine (42.0 mg, 0.34 mmol). The reaction mixture is heated at 150°C for 16 h. The reaction mixture is poured into water, extractcd with ethyl acetate, dried over anhydrous sodium sulfate. The solvent is evaporated to dryness and the crude is purified via flash chromatography to afford the title compound. MS (ESIpos): m/z = 444 [M+H] + . Intermediate 19 [(2±)-2-tert-butyl-8-(morpholin-4-yl)-5-oxo-2,3-dihydroimid azo[1,2-c]pyrido[2,3-e]pyrimidin- 6(5H)-yl]acetic acid
To tert-butyl [(2±)-2-tert-butyl-8-(morpholin-4-yl)-5-oxo-2,3-dihydroimid azo[1,2-c]pyrido[2,3- e]pyrimidin-6(5H)-yl]acetate (42.1 mg, 95 µmol, intermediate 18) is added a solution of hydrochloric acid (4M in dioxane) (886 µL, 4.0 M, 3.54 mmol; CAS-RN:[7647-01-0]). The reaction mixture is heated at 80°C for 2 h. After this time the reaction is allowed to cool to room temperature and the solvent is evaporated under reduced pressure. The residue is triturated with methyl tert-butyl ether (2.5 ml) and the solid is collected by vacuum filtration and dried to afford the title compound. MS (ESIneg): m/z = 386 [M-H]-. Intermediate 20 tert-butyl (4-amino-7-bromo-2-oxopyrido[3,2-d]pyrimidin-1(2H)-yl)acetat e tert-Butyl N-(5-bromo-2-cyanopyridin-3-yl)glycinate (500 mg, 1.60 mmol, intermediate 15) was dissolved in tetrahydrofuran (12 ml) and the solution was cooled by an ice-bath. Then trichloroacetyl isocyanate (1.0 ml, 1.6 M, 1.6 mmol; CAS-RN:[3019-71-4]) was added dropwise and the reaction mixture was stirred for 1 h at room temperature until the carbonyl intermediate was formed. The reaction was quenched at 4°C with excess of methanol und the solvents were removed under reduced pressure. The residue was taken up in 2 ml ammonia in methanol (14 ml, 7.0 M, 95 mmol; CAS-RN:[7664-41-7]) and the resulting mixture was stirred at room temperature for 64 h. The obtained precipitate was collected by vacuum filtration, washed with cold methanol and dried to afford 315 mg (54% yield, 98% purity) of the title compound as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ [ppm]: 1.41 (s, 9H), 4.76 (s, 2H), 8.14 (s, 1H), 8.20 (d, 1H), 8.23 (br s, 1H), 8.52 (d, 1H) LC-MS (Method A): Rt = 0.95 min; MS (ESIpos): m/z = 355 [M+H] + Intermediate 21 tert-butyl [7-bromo-4-(2-bromo-3,3-dimethylbutanamido)-2-oxopyrido[3,2- d]pyrimidin-1(2H)- yl]acetate To a solution of tert-butyl (4-amino-7-bromo-2-oxopyrido[3,2-d]pyrimidin-1(2H)-yl)acetat e (300 mg, 845 µmol, intermediate 20) in pyridine (5.0 ml) was added 2-bromo-3,3-dimethylbutanoic acid (198 mg, 1.01 mmol; CAS-RN:[50364-40-4]) and 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (648 mg, 3.38 mmol; CAS-RN:[25952-53-8]). The reaction mixture was stirred for 16 h at room temperature. To the mixture were added further 0,5 eq.2- bromo-3,3-dimethylbutanoic acid (99 mg, 0.50 mmol; CAS-RN:[50364-40-4]) and 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (648 mg, 3.38 mmol; CAS-RN:[25952- 53-8]) and the reaction was stirred for further 24 h at room temperature. Then the mixture was poured into 20 ml aqueous hydrochloric acid (2M) and extracted with ethyl acetate. The combined organic layers were washed with brine, dried with a water repellent filter and concentrated. The crude material was purified by Biotage Isolera™ chromatography (SNAP KP- Sil – 25 g, eluting with hexane-ethyl acetate, 19:1 to 9:11) to afford 274 mg (44% yield, 72% purity) of the title compound as a white solid. 1 H NMR (400 MHz, CHLOROFORM-d) δ [ppm]: 1.17-1.33 (m, 9H), 1.49 (s, 9H), 4.77 (d, 1H), 4.87 (br d, 1H), 5.30 (s, 1H), 7.55 (d, 1H), 8.51 (br s, 1H), 10.01 (br s, 1H). LC-MS (Method A): R t = 1.39 min; MS (ESIpos): m/z = 531 [M+H] + Intermediate 22 tert-butyl (8-bromo-3-tert-butyl-2,5-dioxo-2,3-dihydroimidazo[1,2-c]pyr ido[2,3-e]pyrimidin- 6(5H)-yl)acetate To a stirred solution of tert-butyl [7-bromo-4-(2-bromo-3,3-dimethylbutanamido)-2- oxopyrido[3,2-d]pyrimidin-1(2H)-yl]acetate (200 mg, 376 µmol, intermediate 21) in anhydrous N,N-dimethylformamide (3.0 mL) was added N,N-diisopropylethylamine (160 µl, 940 µmol; CAS- RN:[7087-68-5]). The reaction mixture was set under nitrogen atmosphere and heated at 120°C for 2 hours. The reaction was cooled to room temperature, water was added and the aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with brine, dried with a water repellant filter and concentrated under reduced pressure. The residue was taken up in 2 ml acetonitrile/water (7:3), filtered via syringe filter and purified by HPLC (Method 1, gradient D). The solvent of the product fractions was evaporated under reduced pressure. The precipitate thus obtained was collected by vacuum filtration, washed with hexane and dried to afford 148 mg (86% yield, 99% purity) of the title compound as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ [ppm]: 0.99 (s, 9H), 1.41 (s, 9H), 4.32 (s, 1H), 4.80-4.87 (d, 1H), 4.87-4.95 (d, 1H), 8.43 (d, 1H), 8.77 (d, 1H). LC-MS (Method A): R t = 1.21 min; MS (ESIpos): m/z = 451 [M+H] + Intermediate 23 tert-butyl [(3±)-3-tert-butyl-8-(morpholin-4-yl)-2,5-dioxo-2,3-dihydro imidazo[1,2-c]pyrido[2,3- e]pyrimidin-6(5H)-yl]acetate
To a solution of tert-butyl (8-bromo-3-tert-butyl-2,5-dioxo-2,3-dihydroimidazo[1,2-c]pyr ido[2,3- e]pyrimidin-6(5H)-yl)acetate (100 mg, 222 µmol, intermediate 22) in dimethyl sulfoxide (750 µl) was added morpholine (190 µl, 2.2 mmol; CAS-RN:[110-91-8]). The reaction mixture was heated for 1 h at 100°C. Upon completion the mixture was allowed to cool to ambient temperature and poured into water (30 ml). The obtained precipitate was collected by vacuum filtration, washed with water and dried to afford 98.0 mg (95% yield, 98% purity) of the title compound as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ [ppm]: 0.98 (s, 9H), 1.40 (s, 9H), 3.50-3.60 (m, 4H), 3.76 (t, 4H), 4.24 (s, 1H), 4.88 (s, 2H), 6.93 (d, 1H), 8.46 (d, 1H). LC-MS (Method A): R t = 1.10 min; MS (ESIpos): m/z = 459 [M+H] + Intermediate 24 [(3±)-3-tert-butyl-8-(morpholin-4-yl)-2,5-dioxo-2,3-dihydro imidazo[1,2-c]pyrido[2,3-e]pyrimidin- 6(5H)-yl]acetic acid To a solution of tert-butyl [3-tert-butyl-8-(morpholin-4-yl)-2,5-dioxo-2,3-dihydroimidaz o[1,2- c]pyrido[2,3-e]pyrimidin-6(5H)-yl]acetate (95.0 mg, 208 µmol, intermediate 23) in dichloromethane (1.4 ml) was added trifluoroacetic acid (160 µl, 2.1 mmol; CAS-RN:[76-05-1]). The reaction mixture was stirred for 16 hours at room temperature. The volatiles were evaporated and the oily residue was triturated with ethyl acetate. The obtained solid was collected by filtration, washed with methyl tert-butyl ether and dried to afford 80.0 mg (94% yield, 98% purity) of the title compound as a yellow powder. 1 H NMR (400 MHz, DMSO-d6) δ [ppm]: 0.98 (s, 9H), 3.55-3.64 (m, 4H), 3.73-3.79 (m, 4H), 4.30 (s, 1H), 4.89 (s, 2H), 7.00 (d, 1H), 8.49 (d, 1H), 13.21 (br s, 1H). LC-MS (Method A): Rt = 0.74 min; MS (ESIpos): m/z = 402 [M+H] + Intermediate 25 tert-butyl [4-amino-7-(morpholin-4-yl)-2-oxopyrido[3,2-d]pyrimidin-1(2H )-yl]acetate A mixture of tert-butyl (4-amino-7-bromo-2-oxopyrido[3,2-d]pyrimidin-1(2H)-yl)acetat e (1.67 g, 4.7 mmol, intermediate 20) and morpholine (4.10 g, 47 mmol) in dimethyl sulfoxide (10 mL) was stirred at 110°C for 4 h. Upon completion, the mixture was cooled to room temperature and poured into water. The precipitated solid was collected, washed with water, acetonitrile and dried to afford 1.20 g (70% yield) of the title compound. Intermediate 26 tert-butyl [2-tert-butyl-8-(morpholin-4-yl)-5-oxoimidazo[1,2-c]pyrido[2 ,3-e]pyrimidin-6(5H)- yl]acetate A mixture of tert-butyl [4-amino-7-(morpholin-4-yl)-2-oxopyrido[3,2-d]pyrimidin-1(2H )-yl]acetate (1.22 g, 3.4 mmol, intermediate 25), 1-bromo-3,3-dimethylbutan-2-one (0.67 g, 3.7 mmol) and N,N-diisopropylethylamine (0.53 g, 4.0 mmol) in N,N-dimethylformamide was stirred at room temperature for 16 h. Upon completion, the mixture was poured into water and extracted with ethyl acetate. The organic layer was washed with water, dried and evaporated to dryness to afford 1.2 g (80% yield) of the title compound, that was used in the next step without further purification Intermediate 27 [2-tert-butyl-8-(morpholin-4-yl)-5-oxoimidazo[1,2-c]pyrido[2 ,3-e]pyrimidin-6(5H)-yl]acetic acid A flask was charged with tert-butyl [2-tert-butyl-8-(morpholin-4-yl)-5-oxoimidazo[1,2- c]pyrido[2,3-e]pyrimidin-6(5H)-yl]acetate (1.2 g, 2.6 mmol, intermediate 26), followed by addition of saturated ethereal hydrogen chloride (30 mL). The mixture was stirred at room temperature overnight. The solid was collected, washed with acetone and dried to afford 1.10 g (92% yield) of the title compound. Intermediate 28 tert-butyl {4-[2-(cyclopropanecarbonyl)hydrazinyl]-7-(morpholin-4-yl)-2 -oxopyrido[3,2- d]pyrimidin-1(2H)-yl}acetate To a solution of tert-butyl [4-chloro-7-(morpholin-4-yl)-2-oxopyrido[3,2-d]pyrimidin-1(2 H)- yl]acetate (55.0 mg, 144 µmol, intermediate 4) in dry dioxane (1.5 ml) was added cyclopropanecarbohydrazide (43.4 mg, 433 µmol; CAS-RN:[6952-93-8]). The reaction mixture was heated at 80°C for 30 minutes. The reaction mixture was allowed to cool to room temperature and the solution was added to 10 ml saturated sodium bicarbonate solution. The aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with brine, dried with a water repellant filter and evaporated to dryness. The crude product was used with further purification in the next reaction. Intermediate 29 [3-cyclopropyl-8-(morpholin-4-yl)-5-oxopyrido[2,3-e][1,2,4]t riazolo[4,3-c]pyrimidin-6(5H)- yl]acetic acid The crude product tert-butyl {4-[2-(cyclopropanecarbonyl)hydrazinyl]-7-(morpholin-4-yl)-2 - oxopyrido[3,2-d]pyrimidin-1(2H)-yl}acetate (110 mg, 247 µmol, intermediate 28) was dissolved in a solution of hydrochloric acid in dioxane (4M) (1mL) and the reaction mixture was heated at 80°C for 16 h. After this time the reaction was cooled to room temperature and the solvent was removed under reduced pressure. The residue was diluted with 2 ml dimethyl sulfoxide / water (1:1) and purified by preparative HPLC (Method A, gradient C). The product fractions were pooled and concentrated in vacuo to afford 45.0 mg (37% yield, 76% purity) of the title compound. 1 H NMR (400 MHz, DMSO-d6) δ [ppm]: 1.07-1.15 (m, 4H), 2.78-2.88 (m, 1H), 3.39-3.46 (m, 4H), 3.75-3.80 (m, 4H), 5.02 (s, 2H), 7.19 (d, 1H), 8.40 (d, 1H)(acidic proton missing) LC-MS (Method A): Rt = 0.68 min; MS (ESIpos): m/z = 371 [M+H] +
EXPERIMENTAL SECTION - EXAMPLES Example 1 2-[3-tert-butyl-8-(morpholin-4-yl)-5-oxopyrido[2,3-e][1,2,4] triazolo[4,3-c]pyrimidin-6(5H)-yl]-N- (5-fluoropyridin-2-yl)acetamide To a suspension of [3-tert-butyl-8-(morpholin-4-yl)-5-oxopyrido[2,3-e][1,2,4]tr iazolo[4,3-c]- pyrimidin-6(5H)-yl]acetic acid (20.0 mg, 51.8 µmol, intermediate 6) in pyridine (720 µl) was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (39.7 mg, 207 µmol; CAS- RN:[25952-53-8]) and 5-fluoropyridin-2-amine (8.70 mg, 77.6 µmol; CAS-RN:[21717-96-4]). The reaction mixture was stirred for 1 h at room temperature. The reaction mixture was diluted with acetonitrile/water (7:3)(2 mL), filtered via syringe filter and purified by HPLC (Method A, gradient C) to afford 7.10 mg (28% yield, 99% purity) of the title compound as a white powder. 1 H NMR (400 MHz, DMSO-d6) δ [ppm]: 1.42 (s, 9H), 3.41-3.48 (m, 4H), 3.71-3.81 (m, 4H), 5.26 (s, 2H), 7.23 (d, 1H), 7.75 (td, 1H), 8.02 (br dd, 1H), 8.37 (d, 1H), 8.48 (d, 1H), 11.06 (s, 1H). LC-MS (Method A): R t = 1.01 min; MS (ESIpos): m/z = 481 [M+H] + Example 2 2-[3-tert-butyl-8-(morpholin-4-yl)-5-oxopyrido[3,2-d][1,2,4] triazolo[4,3-a]pyrimidin-10(5H)-yl]-N- (5-fluoropyridin-2-yl)acetamide To a suspension of [3-tert-butyl-8-(morpholin-4-yl)-5-oxopyrido[3,2-d][1,2,4]tr iazolo[4,3- a]pyrimidin-10(5H)-yl]acetic acid (13.9 mg, 35.0 µmol, intermediate 7) in pyridine (500 µl) is added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (27.5 mg, 144 µmol; CAS- RN:[25952-53-8]) and 5-fluoropyridin-2-amine (6.04 mg, 53.9 µmol; CAS-RN:[21717-96-4]). The reaction mixture is stirred for 1 h at room temperature. The reaction mixture is diluted with acetonitrile/water (7:3)(2 mL), filtered via syringe filter and purified by HPLC to afford the title compound. MS (ESIpos): m/z = 481 [M+H]- Example 3 2-[3-tert-butyl-8-(morpholin-4-yl)pyrido[2,3-d][1,2,4]triazo lo[4,3-b]pyridazin-6-yl]-N-(5- fluoropyridin-2-yl)acetamide [3-tert-Butyl-8-(morpholin-4-yl)pyrido[2,3-d][1,2,4]triazolo [4,3-b]pyridazin-6-yl]acetic acid (2.25 g, 6.07 mmol, intermediate Fehler! Verweisquelle konnte nicht gefunden werden.) and 5- fluoropyridin-2-amine (1.02 g, 9.10 mmol) were mixed in N,N-dimethylformamide (70 mL) and N,N-diisopropylethylamine (4.71 g, 36.4 mmol) was added. The mixture was stirred at r.t. for 10 min and HATU (3.46 g, 9.10 mmol) was added portion-wise over 1 min. The reaction mixture was stirred at r.t. for 16 hours, diluted with sodium chloride (5% aq. sol., 285 mL) and extracted with dichloromethane (2 × 360 mL). The combined organic layer was washed with sodium chloride (5% aq. sol., 5 × 720 mL), dried over sodium sulfate, filtered and evaporated under reduced pressure. The residue was purified by column chromatography (chloroform/methanol gradient from 24:1 to 14:1; TLC (9:1); Rf=0.25) to afford 611 mg (22% yield) of the title compound. 1H NMR (400 MHz, CHLOROFORM-d) δ [ppm]: 1.50 (s, 9H), 3.85 (d, 2H), 5.24 (br s, 1H), 6.70 (d, 1H), 6.86 (dd, 1H), 7.26 (d, 1H) Example 4 2-[(2±)-2-tert-butyl-8-(morpholin-4-yl)-5-oxo-2,3-dihydroim idazo[1,2-c]pyrido[2,3-e]pyrimidin- 6(5H)-yl]-N-(5-fluoropyridin-2-yl)acetamide
To a suspension of [(2±)-2-tert-butyl-8-(morpholin-4-yl)-5-oxo-2,3-dihydroimid azo[1,2- c]pyrido[2,3-e]pyrimidin-6(5H)-yl]acetic acid (8.0 mg, 20.6 µmol, intermediate 19) in pyridine (500 µL) is added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (15.9 mg, 83 µmol; CAS-RN:[25952-53-8]) and 5-fluoropyridin-2-amine (3.47 mg, 36.0 µmol; CAS- RN:[21717-96-4]). The reaction mixture is stirred for 1 h at room temperature. The reaction mixture is diluted with acetonitrile/water (7:3)(2 mL), filtered via syringe filter and purified by HPLC to afford the title compound. MS (ESIpos): m/z = 482 [M+H] + . Example 5 2-[(3±)-3-tert-butyl-8-(morpholin-4-yl)-2,5-dioxo-2,3-dihyd roimidazo[1,2-c]pyrido[2,3- e]pyrimidin-6(5H)-yl]-N-(5-fluoropyridin-2-yl)acetamide [(3±)-3-tert-Butyl-8-(morpholin-4-yl)-2,5-dioxo-2,3-dihydro imidazo[1,2-c]pyrido[2,3-e]pyrimidin- 6(5H)-yl]acetic acid (78.0 mg, 194 µmol, intermediate 24) was dissolved in pyridine (1.5 ml), then 5-fluoropyridin-2-amine (32.7 mg, 291 µmol; CAS-RN:[21717-96-4]) and 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (149 mg, 777 µmol; CAS-RN:[25952- 53-8]) were added. The reaction mixture was stirred for 1 hours at room temperature. The reaction was quenched with 20 ml hydrochloric acid (1M) and extracted with ethyl acetate. The combined organic phases were washed with brine and concentrated. The obtained solid was suspended in methyl tert-butyl ether (15 ml), stirred vigorously for 10 minutes. The solid was collected by vacuum filtration, washed with methyl tert-butyl ether and dried to afford 72.1 mg (73% yield, 97% purity) of the title compound 1 H NMR (400 MHz, DMSO-d6) δ [ppm]: 0.98 (s, 9H), 3.49-3.59 (m, 4H), 3.73 (t, 4H), 4.23 (s, 1H), 4.98-5.04 (m, 1H), 5.05-5.13 (m, 1H), 7.01 (d, 1H), 7.36-7.41 (m, 1H), 8.04 (br dd, 1H), 8.36 (d, 1H), 8.46 (d, 1H), 8.54-8.60 (m, 1H). LC-MS (Method A): Rt = 0.96 min; MS (ESIneg): m/z = 494 [M-H]- Example 6 2-[2-tert-butyl-8-(morpholin-4-yl)-5-oxoimidazo[1,2-c]pyrido [2,3-e]pyrimidin-6(5H)-yl]-N-(5- fluoropyridin-2-yl)acetamide To an ice cooled solution of [2-tert-butyl-8-(morpholin-4-yl)-5-oxoimidazo[1,2-c]pyrido[2 ,3- e]pyrimidin-6(5H)-yl]acetic acid (1.05 g, 2.3 mmol, intermediate 27), 5-fluoropyridin-2-amine (0.282 g, 2.5 mmol), N-methyl morpholine (1.16 g, 11.5 mmol) in N,N-dimethylformamide (7 mL) was added HATU (1.17 g, 3.1 mmol). The reaction mixture was warmed to room temperature and stirred for 16 h. Upon completion, the mixture was poured into water and extracted with ethyl acetate. The organic layer was washed with water, brine, dried and evaporated in vacuo and the residue was crystallized with methyl tert-butyl ether to afford 690 mg (63% yield) of the title compound. 1 H NMR (400 MHz, DMSO-d6) δ [ppm]: 1.33 (s, 9H), 3.34-3.38 (m, 4H), 3.71-3.80 (m, 4H), 5.24 (s, 2H), 7.23 (d, 1H), 7.51 (s, 1H), 7.75 (td, 1H), 8.02 (br dd, 1H), 8.38 (dd, 2H), 11.07 (s, 1H). Example 7 2-[3-cyclopropyl-8-(morpholin-4-yl)-5-oxopyrido[2,3-e][1,2,4 ]triazolo[4,3-c]pyrimidin-6(5H)-yl]- N-(5-fluoropyridin-2-yl)acetamide
To a suspension of [3-cyclopropyl-8-(morpholin-4-yl)-5-oxopyrido[2,3-e][1,2,4]t riazolo[4,3- c]pyrimidin-6(5H)-yl]acetic acid (20.0 mg, 54.0 µmol, intermediate 29) in pyridine (750 µl) was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (41.4 mg, 216 µmol; CAS- RN:[25952-53-8]) and 5-fluoropyridin-2-amine (9.08 mg, 81.0 µmol; CAS-RN:[21717-96-4]). The reaction mixture was stirred for 1 hour at room temperature. The residue was taken up in 2 ml acetonitrile/water (7:3), filtered via syringe filter and purified by preparative HPLC (Method A, gradient C). The product fractions were pooled and evaporated to dryness. The obtained solid was triturated with 2 ml methyl tert-butyl ether and collected by vacuum filtration to afford 5.9 mg (21% yield, 88% purity) of the title compound as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ [ppm]: 1.07-1.14 (m, 4H), 2.80-2.90 (m, 1H), 3.37-3.43 (m, 4H), 3.70-3.78 (m, 4H), 5.18 (s, 2H), 7.21 (d, 1H), 7.70-7.80 (m, 1H), 8.05 (br dd, 1H), 8.38 (dd, 2H), 11.07 (s, 1H). LC-MS (Method A): R t = 0.91 min; MS (ESIneg): m/z = 463 [M-H]-
EXPERIMENTAL SECTION – BIOLOGICAL ASSAYS Examples were tested in selected biological assays one or more times. When tested more than once, data are reported as either average values or as median values, wherein • the average value, also referred to as the arithmetic mean value, represents the sum of the values obtained divided by the number of times tested, and • the median value represents the middle number of the group of values when ranked in ascending or descending order. If the number of values in the data set is odd, the median is the middle value. If the number of values in the data set is even, the median is the arithmetic mean of the two middle values. Examples were synthesized one or more times. When synthesized more than once, data from biological assays represent average values or median values calculated utilizing data sets obtained from testing of one or more synthetic batch. The in vitro activity of the compounds of the present invention can be demonstrated in the following assays: 1) Intracellular calcium measurement to assess antagonist activity at human P2X3 receptors (hP2X3 CHO) The determination of antagonistic activity at the P2X3 receptor of the compounds of the invention was performed by use of a recombinant cell line. These cell line derives originally from the Chinese hamster ovary (CHO) cell line (Tjio J. H.; Puck T. T., 1958, J. Exp. Med. 108: 259– 271). The cell line is stably transfected with the human P2X3 receptor and a calcium-sensitive photoprotein, mitochondrial photina, which, after reconstitution with the cofactor coelenterazine, emits light in dependence of calcium binding [Bovolenta S, Foti M, Lohmer S, Corazza S., J Biomol Screen. 2007 Aug;12(5):694-704]. The strength of the photina luminescence signal corresponds to the level of receptor activation upon agonist binding. An inhibitor would decrease the signal depending on its potency and concentration. Bioluminescence was detected using a suitable luminometer [Milligan G, Marshall F, Rees S, Trends in Pharmacological Sciences 17, 235-237 (1996)]. Test procedure: On the day before the assay, the cells are plated out in culture medium (DMEM/F12 (PAN, P04- 41451), 10% FCS) in 384-well microtiter plates and kept in a cell incubator (96% humidity, 5% v/v CO 2 , 30°C). On the day of the assay medium is replaced by 2 mM Ca-tyrode buffer containing 5 µg/ml coelenterazine. Plates are incubated for 3 hours at 37°C (96% humidity, 5% v/v CO 2 ). After incubation the test substances in various concentrations are placed for 10 minutes in the wells of the microtiter plate before the agonist α,β-methylene-ATP at EC 50 concentration is added. The resulting light signal is measured immediately in the luminometer. 2) and 3) Intracellular calcium measurement to compare antagonist activity at human P2X3 receptors (hP2X31321N1) and at human P2X2/3 receptors (hP2X231321N1) The comparison of antagonistic activity at the P2X3 versus the P2X23 receptor of the compounds of the invention were performed by use of recombinant cell lines. These cell lines derived originally from the human astrocytoma cell line 1312N1 (Macintyre EH, Pontén J, Vatter AE. Acta Pathol Microbiol Scand A. 1972;80(2):267-83). The cell lines are stably transfected with the human P2X3 receptor forming homotrimeric P2X3 receptors or are co-transfected with P2X2 and P2X3 forming heterotrimeric P2X23 receptors. Stimulation of the receptors with the agonist ATP leads to a conformational change of the receptors and influx of extracellular calcium ions through the open ion channel. The cytoplasmatic calcium transient is detected via the calcium sensitive dye Fluo8. The strength of Fluo8 fluorescence signal corresponds to the level of receptor activation. An inhibitor would decrease the signal depending on its potency and concentration. Fluorescence was measured by use of a suitable fluorescence reader. Test procedure: On the day before the assay, the cells were plated in culture medium (DMEM high glucose, 10% FCS, 1% MEM non-essential amino acids , 4mM Glutamax) in 384-well poly-D-lysine coated microtiter plates and kept in a cell incubator (96% humidity, 5% v/v CO 2 , 37°C). On the day of the assay medium was exchanged by Fluo8 containing buffer and incubated for 60 minutes. Test compounds were added at various concentrations and plates were incubated for 10 minutes. In the fluorescence reader a 3 seconds baseline measurement was performed and the agonist ATP was applied at EC 50 concentration of the respective receptor during constant fluorescence measurement for 120 seconds. Results: