RU2792645C2 - Substituted oxopyridine derivative - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to 5-({6-amino-2-[4-(5-chlor-2-cyanophenyl)-5-methoxy-2-oxopyridine-1(2H)-yl]-3-methylhexanoyl}amino)pyrazolo[1,5-a]pyridine-3-carboxamide of the formula (I) or to one of its salts.

EFFECT: new compound is obtained, which can be used in medicine as an inhibitor of destructive activity of plasma kallikrein to produce drugs for the treatment and/or prevention of diseases, in particular cardiovascular diseases, preferably thrombotic or thromboembolic disorders and edema, as well as ophthalmic disorders.

3 cl, 3 ex

Description

The present invention relates to 5-({6-amino-2-[4-(5-chloro-2-cyanophenyl)-5-methoxy-2-oxopyridin-1(2H)-yl]-3-methylhexanoyl}amino)pyrazolo [1,5-a]pyridine-3-carboxamide, to processes for its preparation, to its use in the treatment and/or prevention of diseases, and to its use in the preparation of medicaments for the treatment and/or prevention of diseases, in particular cardiovascular diseases, preferably thrombotic or thromboembolic disorders and edema, as well as ophthalmic disorders, and its use to inhibit the destructive activity of plasma kallikrein for extracorporeal procedures and analytical analyses.

Blood coagulation is a protective mechanism of the body, which helps to quickly and reliably "solder" defects in the walls of blood vessels. Thus, blood loss can be avoided or minimized. Hemostasis after damage to blood vessels is carried out mainly by the blood coagulation system, in which an enzymatic cascade of complex reactions of plasma proteins is launched. Numerous coagulation factors are involved in this process, each of which, when activated, converts the corresponding next inactive precursor to its active form. At the end of the cascade, soluble fibrinogen is converted to insoluble fibrin, resulting in the formation of a blood clot.

The focus is on the fact that the blood coagulation system can be activated, in particular on negatively charged surfaces, which include not only the surface structures of foreign cells (e.g. bacteria), but also artificial surfaces such as vascular prostheses, stents and extracoporal circulation. At the surface, factor XII (FXII) is initially activated to factor XIIa, which subsequently activates factor XI to factor XIa. In addition, factor XIIa also activates plasma prekallikrein (PPK) to plasma kallikrein (PK), which in the potentiation loop first leads to further activation of factor XII, which generally leads to increased initiation of the coagulation cascade. In addition, PK is an important bradykinin-releasing protease which, inter alia, thus leads to increased endothelial permeability. Other described substrates are prorenin and prourokinase, the activation of which can affect the regulatory processes of the renin-angiotensin system and fibrinolysis. Thus, PK activation is an important link between coagulative and inflammatory processes.

Uncontrolled activation of the coagulation system or inadequate inhibition of activation processes can lead to the formation of local thrombosis or embolism in vessels (arteries, veins, lymphatic vessels) or heart cavities. In addition, systemic hypercoagulability can lead to systemic thrombus formation and finally to consumption coagulopathy in the context of diffuse intravascular coagulation. Thromboembolic complications can also occur in extracorporeal circulatory systems, such as during hemodialysis, and in vascular or prosthetic heart valves and stents.

Plasma kallikrein (PK) is associated with other disorders that are associated with increased vascular permeability or chronic inflammatory disorders such as diabetic retinopathy, macular edema and hereditary angioedema or chronic inflammatory bowel disorders. Diabetic retinopathy is primarily caused by microvascular insufficiency, which leads to thickening of the basement membrane of the vessels and loss of vascularized pericytes, followed by vascular occlusion and retinal ischemia, which, due to the thus induced retinal hypoxia, can lead to increased vascular permeability with subsequent formation of macular edema and, due to all the processes present, to the blindness of the patient. In hereditary angioedema (HAE), reduced production of the physiological kallikrein inhibitor C1-esterase inhibitor causes uncontrolled activation of plasma kallikrein, which leads to inflammation with lightning-fast edema and severe pain. In experimental animal models, there are indications that inhibition of plasma kallikrein suppresses vascular hyperpermeability and therefore may prevent the formation of macular edema and/or diabetic retinopathy, or may reduce the acute symptoms of NAE. Oral plasma kallikrein inhibitors can also be used to prevent NAE.

Kinins formed by plasma kallikrein in particular play a causal role in the progression of chronic inflammatory intestinal disorders (CID). Their pro-inflammatory effect through the activation of bradykinin receptors induces and potentiates the progression of the disease. Studies of patients with Crohn's disease show a correlation between the concentration of kallikrein in the intestinal epithelium and the degree of intestinal inflammation. Activation of the kallikrein-kinin system has also been observed in experimental animal studies. Accordingly, inhibition of bradykinin synthesis by kallikrein inhibitors can also be used to prevent and/or treat chronic inflammatory bowel disorders.

In many disorders, the combination of antithrombotic and anti-inflammatory principles may also be particularly attractive in preventing the mutual enhancement of coagulation and inflammation.

WO 2006/030032, inter alia, describes substituted pyridinones as allosteric modulators of the mGluR2 receptor, and WO 2008/079787 describes substituted pyridin-2-ones and their use as glucokinase activators. In Wo2005/123680, Wo 2014/154794, Wo 2014/160592, Wo 2015/011087, Wo 2015/063093, Wo 2015/120777, Wo 2016/046156, Wo 2016/046157, Wo 2016/046158, Wo 2016/046159, Wo 2016/046159, WO 2016/046164, WO 2016/046166, WO 2016/146606, WO 2017/005725, WO 2017/037051 and WO 2018/041122 describe substituted pyridin-2-ones and their use as factor XIa inhibitors and/or kallikrein inhibitors.

Thus, the object of the present invention is to provide a novel compound for the treatment of cardiovascular diseases, in particular thrombotic or thromboembolic disorders, in humans and animals, and for use in biological samples containing plasma prekallikrein (PPK) or plasma kallikrein (PK) to prevent destructive effects caused by the use of plasma kallikrein physiological and artificial substrates.

Surprisingly, a certain substituted oxopyridine derivative has been found to be a highly potent and selective inhibitor of plasma kallikrein.

The present invention provides the compound 5-({6-amino-2-[4-(5-chloro-2-cyanophenyl)-5-methoxy-2-oxopyridin-1(2H)-yl]-3-methylhexanoyl}amino)pyrazolo [1,5-a]pyridine-3-carboxamide which corresponds to the compound of formula (I)

and its salts, its solvates and solvates of its salts.

In addition, the present invention provides the compound 5-({6-amino-2-[4-(5-chloro-2-cyanophenyl)-5-methoxy-2-oxopyridin-1(2H)-yl]-3-methylhexanoyl} amino)pyrazolo[1,5-a]pyridine-3-carboxamide trifluoroacetate, which corresponds to the compound of formula (Ia)

Preferred is 5-({(2,5)-6-amino-2-[4-(5-chloro-2-cyanophenyl)-5-methoxy-2-oxopyridin-1(2H)-yl]-3- methylhexanoyl}amino)pyrazolo[1,5-a]pyridine-3-carboxamide, which corresponds to the compound of formula (Ib)

and its salts, its solvates and solvates of its salts.

Further preferred is the compound 5-({(2S)-6-amino-2-[4-(5-chloro-2-cyanophenyl)-5-methoxy-2-oxopyridin-1(2H)-yl]-3 -methylhexanoyl}amino)pyrazolo[1,5-a]pyridine-3-carboxamide trifluoroacetate, which corresponds to the compound of formula (Ic)

The compounds according to the present invention may, depending on their structure, exist in various stereoisomeric forms, i.e. in the form of configurational isomers or, if necessary, in the form of conformational isomers (enantiomers and/or diastereomers, including in the case of atropisomers). Thus, the present invention covers enantiomers and diastereomers and their respective mixtures. Stereoisomerically homogeneous components can be isolated from such mixtures of enantiomers and/or diastereomers in a known manner; for this, chromatographic methods are preferably used, especially HPLC chromatography on an achiral or chiral phase.

While the compounds of the present invention may be in tautomeric forms, the present invention encompasses all tautomeric forms.

In the context of the present invention, the term "enantiomerically pure" should be understood to mean that the compound in question, with respect to the absolute configuration of the chiral center, is present in an enantiomeric excess of more than 95%, preferably more than 97%. The enantiomeric excess, ee, is calculated in the context of the present invention by evaluating the corresponding chiral phase HPLC chromatogram using the formula below:

ee = [E ^A (% by area) - E ^B (% by area)] × 100% / [E ^A (% by area) + E ^B (% by area)]

( ^EA : major enantiomer, E ^B : minor enantiomer)

The present invention also includes all suitable isotopic variants of the compounds of the present invention. An isotopic variant of a compound of the present invention, in the context of the present invention, is understood to mean a compound in which at least one atom in the compound of the present invention has been replaced by another atom with the same atomic number but an atomic mass other than the atomic mass that is normally or mostly found in nature. Examples of isotopes that can be included in the compound of the present invention are isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, bromine and iodine, such as ² H (deuterium), ³ H (tritium), ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁷ O, ¹⁸ O and ³⁶ Cl. Particular isotopic variants of the compound of the present invention, especially those in which one or more radioactive isotopes have been included, may be preferred, for example, to study the mechanism of action or distribution of the active compound in the body; due to their relatively easy preparation and determination, compounds labeled with 3 H or 14 C isotopes are particularly suitable for this purpose. In addition, the incorporation of isotopes, eg deuterium, may result in particular therapeutic benefits due to greater metabolic stability of the compound, eg increased half-life in the body or reduced active dose required; therefore, such modifications of the compounds of the present invention may, in some cases, also represent a preferred embodiment of the use of the present invention. Isotopic variants of the compounds of the present invention can be prepared by methods known to those skilled in the art, for example, the methods described below and the procedures described in the Working Examples, through the use of appropriate isotopic modifications of the appropriate reagents and/or starting compounds.

Preferred salts in the context of the present invention are the physiologically acceptable salts of the compounds of the present invention. However, the present invention also includes salts which are not in themselves suitable for pharmaceutical use, but can be used, for example, to isolate or purify the compounds of the present invention.

Physiologically acceptable salts of the compounds of the present invention include acid addition salts of mineral acids, carboxylic acids and sulfonic acids, e.g. salts of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, naphthalenedisulfonic acid , acetic acid, trifluoroacetic acid, propionic acid, lactic acid, tartaric acid, citric acid, malic acid, fumaric acid, maleic acid and benzoic acid.

In the context of the present invention, solvates are those forms of the compounds of the present invention which form a complex in the solid or liquid state by coordination with solvent molecules. Hydrates are a special form of solvates in which coordination occurs with water.

The present invention further also encompasses prodrugs of the compounds of the present invention. The term "prodrugs" embraces compounds which, for their part, may be biologically active or inactive, but are converted during their stay in the body into the compounds of the present invention (eg, by metabolism or hydrolysis).

In the context of the present invention, the term "treatment" or "treat" includes inhibiting, slowing, checking, alleviating, attenuating, limiting, reducing, suppressing, eliminating or curing a disease, condition, disorder, injury or health or developmental problem, the course or progression of such conditions and/or symptoms of such conditions. The term "therapy" is used herein as a synonym for the term "treatment".

The terms "prevention", "prophylaxis" and "prevention" are used interchangeably in the context of the present invention and refer to preventing or reducing the risk of developing, experiencing, suffering from or having a disease, condition, disorder, injury or health problem, or developing or progressing such conditions and/or symptoms of such conditions.

The treatment or prevention of a disease, condition, disorder, injury, or health problem may be partial or complete.

The present invention provides a process for the preparation of compounds of formula (I), (Ia), (Ib) and (Ic), or their salts, their solvates or solvates of their salts. Compounds can be synthesized as illustrated in Synthesis Scheme 1:

Scheme 1:

The compounds of the present invention have an unexpected spectrum of useful pharmacological activity. These are compounds that affect the proteolytic activity of the plasma serine protease kallikrein (PK). The compounds of the present invention inhibit PK-catalyzed enzymatic degradation of substrates, which plays an important role in the activation of blood coagulation, in platelet aggregation, and in inflammatory processes, which in particular include an increase in vascular permeability.

They are therefore suitable for use as medicaments for the treatment and/or prevention of diseases in humans and animals.

The present invention also provides the use of the compounds according to the present invention for the treatment and/or prevention of disorders, in particular cardiovascular diseases, preferably thrombotic or thromboembolic disorders and/or thrombotic or thromboembolic complications, and/or ophthalmic disorders, in particular diabetic retinopathy or macular edema, and /or inflammatory disorders, in particular those associated with excessive plasma kallikrein activity, such as hereditary angioedema (HAE), or chronic inflammatory diseases, especially of the intestine, such as Crohn's disease.

Factor XIIa activates plasma prekallikrein (PPK) to plasma kallikrein (PK) in the context of intrinsic activation, which, inter alia, in the potentiation loop, leads to further activation of factor XII, which generally leads to increased initiation of the surface coagulation cascade. Thus, the PK inhibitory activity of the compound of the present invention reduces coagulation by activating the surface, and thus has an anticoagulant effect.

Accordingly, the compounds of the present invention are useful in the treatment and/or prevention of disorders or complications that may result from clot formation.

In the context of the present invention, "thrombotic or thromboembolic diseases" includes diseases that occur in both the arterial and venous vasculature and which can be treated with the compounds of the present invention, in particular disorders in the coronary arteries of the heart, such as acute coronary syndrome ( ACS), ST-segment elevation myocardial infarction (STEMI) and non-ST-segment elevation (non-STEMI), stable angina, unstable angina, reocclusion and restenosis after coronary interventions such as angioplasty, stent implantation or coronary artery bypass grafting, and thrombotic or thromboembolic disturbances in other vessels leading to peripheral arterial occlusive disorders, pulmonary embolism, venous thromboembolism, venous thrombosis, especially in the deep veins of the legs and kidneys, temporary ischemic attacks, as well as thrombotic stroke and thromboembolic stroke.

Stimulation of the coagulation system can occur for various reasons or related disorders. In the context of surgery, immobility, bedridden, infections, inflammation or cancer or cancer therapy, among others, the coagulation system may be highly activated and there may be thrombotic complications, in particular venous thromboses. The compounds of the present invention are therefore suitable for the prevention of thrombosis in the context of surgical interventions in patients suffering from cancer. Thus, the compounds of the present invention are also suitable for the prevention of thrombosis in patients having an activated coagulation system, for example in the stimulation situations described.

The compounds of the present invention are therefore also suitable for the prevention and treatment of cardiogenic thromboembolisms, e.g. cerebral ischemia, stroke and systemic thromboembolisms and ischemias, in patients with acute, intermittent or persistent cardiac arrhythmias, e.g. atrial fibrillation, and in patients undergoing electrical shock pacing. and in patients with valvular heart disease or artificial heart valves.

In addition, the liquid compounds of the present invention are suitable for the treatment and prevention of generalized thrombohemorrhagic syndrome (DIC), which may occur, in particular in connection with sepsis, but also due to surgery, neoplastic disorders, burns or other injuries, and can lead to serious organ damage due to microthrombosis.

In addition, thromboembolic complications occur in microangiopathic hemolytic anemias and in contact of blood with foreign surfaces in the context of extracorporeal circulation, for example, hemodialysis, ECMO ("extracorporeal membrane oxygenation"), LVAD ("left ventricular assist device") and similar methods, AV fistulas , prostheses of blood vessels and valves of the heart.

Moreover, the compounds of the present invention are useful in the treatment and/or prevention of disorders involving microclot formation or fibrin deposition in cerebral blood vessels that can lead to dementia disorders such as vascular dementia or Alzheimer's disease. Here, the clot may contribute to the disorder both through occlusion and by linking other disease-related factors.

Moreover, the compounds according to the present invention are suitable for the treatment and/or prevention of diseases in which, in addition to the procoagulant component, the pro-inflammatory component also plays an important role. Mutual enhancement of blood coagulation and inflammation in particular can be prevented by the compounds of the present invention, thereby greatly reducing the likelihood of thrombotic complications. In this case, both the factor XIa inhibitory component (by inhibiting thrombin production) and the PK inhibitory component can contribute to the anticoagulant and anti-inflammatory effect (eg, via bradykinin). Thus, among other things, they can be considered for the treatment and/or prevention in the context of atherosclerotic vascular disorders, inflammatory processes in the context of rheumatic disorders of the musculoskeletal system, inflammatory disorders of the lung such as pulmonary fibrosis, inflammatory diseases of the kidneys such as glomerulonephritis, inflammatory bowel disorders such as Crohn's disease or ulcerative colitis, or disorders that may be present in the context of an underlying diabetic disease such as diabetic retinopathy or nephropathy.

Kinins formed by plasma kallikrein in particular play a causal role in the progression of chronic inflammatory intestinal disorders (CID). Their pro-inflammatory effect through the activation of bradykinin receptors induces and potentiates the progression of the disease. Studies of patients with Crohn's disease show a correlation between the concentration of kallikrein in the intestinal epithelium and the degree of intestinal inflammation. Activation of the kallikrein-kinin system has also been observed in experimental animal studies. Accordingly, inhibition of bradykinin synthesis by kallikrein inhibitors can also be used to prevent and/or treat chronic inflammatory bowel disorders.

Moreover, the compounds of the present invention can be used to inhibit tumor growth and metastasis formation, as well as to prevent and/or treat thromboembolic complications, for example, venous thromboembolism in cancer patients, in particular those undergoing major surgery or chemo- or radiation therapy. therapy

In addition, the compounds according to the present invention are also suitable for the treatment and/or prevention of disseminated intravascular coagulation in the context of infectious disease and/or systemic inflammatory syndrome (SIRS), septic organ dysfunction, septic organ failure and multiple organ failure, acute respiratory failure syndrome. (ARDS), acute lung injury (ALI), septic shock and/or septic organ failure.

In the course of infection, general activation of the coagulation system (disseminated intravascular coagulation of blood or consumption coagulopathy, hereinafter referred to as "DIC") may occur with microthrombosis in various organs and secondary hemorrhagic complications. In addition, endothelial injury may occur with increased vascular permeability and diffusion of fluid and proteins into the extravascular space. As infection progresses, organ failure (eg, renal failure, liver failure, respiratory failure, central nervous system deficiency, and cardiovascular failure) or multiple organ failure may occur.

In the case of DIC, there is a massive activation of the coagulation system on the surface of damaged endothelial cells, on the surfaces of foreign bodies, or on sutured extravascular tissues. As a result, coagulation occurs in small vessels of various organs with hypoxia and subsequent organ dysfunction. A secondary effect is the depletion of clotting factors (such as factor X, prothrombin, and fibrinogen) and platelets, which reduces blood clotting and can lead to severe bleeding.

In addition to anticoagulant activity, plasma kallikrein is an important protease that releases bradykinin, which, among other things, leads to increased endothelial permeability. Thus, the compounds can be used for the treatment and/or prevention of disorders involving edema formation, such as ophthalmic disorders, in particular diabetic retinopathy, macular edema or hereditary angioedema.

"Ophthalmic diseases" in the context of the present invention includes in particular disorders such as diabetic retinopathy, diabetic macular edema (DME), macular edema, macular edema associated with retinal vein occlusion, age-related macular degeneration (AMD), including dry (non-exudative) and wet (exudative, neovascular) AMD, choroidal neovascularization (CNV), choroidal neovascular membranes (CNVM), cystoid macular edema (CME), epiretinal membranes (ERM) and macular retinal break, myopia-associated choroidal neovascularization, angioid and vascular streaks , retinal detachment, atrophic and hypertrophic changes in the retinal pigment epithelium, retinal venous occlusion, choroidal retinal venous occlusion, retinitis pigmentosa, Stargardt's disease, premature retinopathy, glaucoma, inflammatory diseases of the eye, such as uveitis, scleritis or endophthalmitis, cataracts, refractive errors, nap For example, myopia, hypermetropia, astigmatism or keratoconus, corneal angiogenesis as a consequence of, for example, keratitis, corneal transplantation or keratoplasty, corneal angiogenesis as a consequence of hypoxia (for example, as a result of prolonged contact lens wear), conjunctival pterygium, subcorneal edema, and edema within the cornea.

The compounds according to the present invention are also suitable for the primary prevention of thrombotic or thromboembolic disorders and/or inflammatory disorders and/or disorders with increased vascular permeability in patients in which gene mutations lead to increased enzyme activity or elevated levels of zymogens, and they are established by appropriate tests / measurements of enzyme activity or zymogen concentrations.

The present invention also provides for the use of the compounds of the present invention for the treatment and/or prevention of disorders, especially the disorders mentioned above.

The present invention also provides for the use of the compounds of the present invention for the preparation of a medicament for the treatment and/or prevention of disorders, especially the disorders mentioned above.

The present invention also provides a method for treating and/or preventing disorders, especially the disorders mentioned above, using a therapeutically effective amount of a compound of the present invention.

The present invention also provides compounds of the present invention for use in a method for the treatment and/or prevention of disorders, especially the disorders mentioned above, using a therapeutically effective amount of a compound of the present invention.

In particular, the present invention provides compounds of the present invention for use in a method for the treatment and/or prevention of thrombotic or thromboembolic disorders using a therapeutically effective amount of a compound of the present invention.

In addition, the present invention provides medicinal products containing the compound according to the present invention and one or more other active compounds.

In addition, the compounds of the present invention can also be used to prevent ex vivo coagulation, for example, to protect organs to be transplanted from damage to an organ caused by clot formation, and to protect a recipient organ from thromboemboli from a transplanted organ, to preserve blood products, and plasma, for cleaning/pretreatment of catheters and other medical auxiliaries and instruments, for coating synthetic surfaces of medical auxiliaries and instruments used in vivo or ex vivo.

The compounds of the present invention can also be used in biological samples containing plasma prekallikrein (PPK) or plasma kallikrein (PK) to prevent the damaging effects caused by the use of plasma kallikrein on physiological and artificial substrates.

The present invention further provides a method for preventing blood coagulation in vitro, particularly in blood banks or biological samples that may contain plasma kallikrein, the method being characterized in that an anticoagulant effective amount of a compound of the present invention is added.

The present invention also relates to medicaments containing a compound according to the present invention and one or more additional active compounds, in particular for the treatment and/or prevention of the disorders mentioned above.

"Combinations" in the context of the present invention means not only dosage forms that contain all components (so-called fixed combinations) and combination packs that contain components separate from each other, but also components that are administered simultaneously or sequentially, provided that they are used to prevent and/or treat the same disease. It is also possible to combine two or more active ingredients with each other, which means that each of them is thus in two-component or multi-component combinations.

The compounds of the present invention may act systemically and/or locally. For this purpose, they can be administered by a suitable route, for example, by oral, parenteral, pulmonary, nasal, sublingual, lingual, buccal, rectal, dermal, transdermal, conjunctival or otic route, or in the form of an implant or stent.

The compounds of the present invention may be administered in administration forms suitable for these routes of administration.

Suitable for oral use are administration forms according to the state of the art which deliver the compound according to the invention rapidly and/or in a modified manner and which contain the compounds according to the invention in crystalline and/or amorphous and/or dissolved form, such as, for example, tablets (uncoated or coated shell, e.g. with gastric resistant or delayed or insoluble shells that control the release of the compound of the invention), rapidly disintegrating tablets or films/wafers, films/lyophilisates, capsules (e.g. hard or soft gelatin capsules ), dragees, granulates, pellets, powders, emulsions, suspensions, aerosols or solutions.

Parenteral administration may bypass the absorption step (eg, intravenously, intraarterially, intracardiac, intraspinally, or intralumbally) or by incorporating the absorption process (eg, intramuscularly, subcutaneously, intradermally, transdermally, or intraperitoneally). Suitable forms of administration for parenteral use are, inter alia, injection and infusion preparations in the form of solutions, suspensions, emulsions, lyophilisates or sterile powders.

For extraocular (topical) administration suitable administration forms operate according to the prior art, which release the active compound rapidly and/or in a modified or controlled manner, and which contain the active compound in a crystalline and/or amorphous and or dissolved form, such as for example , eye drops, sprays and lotions (e.g. solutions, suspensions, vesicular/colloidal systems, emulsions, aerosols), eye drop powders, sprays and lotions (e.g. powdered active compound, mixtures, lyophilisates, precipitated active compound), semi-solid preparations eye products (eg hydrogels, in-situ hydrogels, creams and ointments), eye liners (solid and semi-solid preparations, eg bioadhesives, films/plates, tablets, contact lenses).

Intraocular administration includes, for example, intravitreal, subretinal, subscleral, intrachoroidal, subconjunctival, retrobulbar and subtenon administration. For intraocular administration, suitable administration forms operate according to the prior art, which release the active compound in a rapid and/or modified or controlled manner, and which contain the active compound in crystalline and/or amorphous and/or dissolved form, such as, for example, injections and concentrates for injections (e.g. solutions, suspensions, vesicular/colloidal systems, emulsions), powders for injections (e.g. powdered active compound, mixtures, lyophilisates, precipitated active compound), gels for injections (semi-solid preparations, eg hydrogels, in situ hydrogels) and implants (solid preparations, eg biodegradable and non-biodegradable implants, implantable pumps).

Preference is given to oral administration or, in the case of ophthalmic disorders, extraocular and intraocular administration.

For other routes of administration, for example, dosage forms for inhalation (including powder inhalers, nebulizers), drops, solutions or nasal sprays taken on the tongue, under the tongue or buccal tablets, films / cachets or capsules, suppositories, ear or ophthalmic preparations, vaginal capsules, aqueous suspensions (lotions, boluses), lipophilic suspensions, ointments, creams, transdermal therapeutic systems (eg patches), milks, pastes, foams, powders, implants or stents.

Compounds according to the present invention can be translated into these forms of application. This can be done in a known manner by mixing with inert, non-toxic, pharmaceutically acceptable excipients. Such excipients include, but are not limited to, carriers (eg, microcrystalline cellulose, lactose, mannitol), solvents (eg, liquid polyethylene glycol), emulsifiers, and dispersing or wetting agents (eg, sodium dodecyl sulfate, polyoxysorbitan oleate), binders ( e.g. polyvinylpyrrolidone), synthetic and natural polymers (e.g. albumin), stabilizers (e.g. antioxidants such as ascorbic acid), coloring agents (e.g. inorganic pigments such as iron oxide) and flavor and /or smell.

The present invention further provides medicaments comprising at least one compound of the present invention, preferably together with one or more inert, non-toxic, pharmaceutically acceptable excipients, and uses thereof for the above purposes.

In the case of parenteral administration, it has generally been found that, in order to achieve effective results, it is preferable to administer amounts of from about 5 to 250 mg every 24 hours. In the case of oral administration, the amount is from about 5 to 500 mg every 24 hours.

Despite this, if necessary, it may be necessary to deviate from the indicated amounts, in particular, depending on body weight, route of administration, individual behavior in relation to the active ingredient, type of preparation and time or interval of administration.

Unless otherwise indicated, the percentages in the tests and examples that follow are percentages by weight; parts are mass parts. Solvent ratios, dilution factors and concentration data for liquid/liquid solutions are based in each case on volume. "W/v" means "mass/volume". For example, "10% w/v" means: 100 ml of a solution or suspension contains 10 g of the substance.

A) Examples

Abbreviations:

aq. - water

Boc - tert-butyl hydroxycarbonyl

Br - wide (in NMR)

Brsm - Based on the extracted starting material

D - day(s), doublet (in NMR)

Dd - Doublet of doublets (in NMR)

Ddd - Doublet of doublet of doublet of doublet (in NMR)

DMF - N,N-dimethylformamide

DMSO - Dimethyl Sulfoxide

ESI - Electrospray Ionization (in MS)

h - hour(hours)

HATU - O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate

HPLC - High Pressure Liquid Chromatography

HV - High Vacuum

LC-MS - Mac Spectrometry Related to Liquid Chromatography

M - multiplet (in NMR)

Min - minute(s)

MS - Mac Spectroscopy

NMR - Nuclear Magnetic Resonance Spectroscopy

R _t - Retention time (in HPLC)

S - singlet (in NMR)

T - triplet (in NMR)

THF - tetrahydrofuran

TFA - trifluoroacetic acid

Т3Р - 2,4,6-tripropyl-1,3,5,2,4,6-trioxatrifosphine 2,4,6-trioxide

LC-MS ways:

, 1.8 мкм, 2.1 мм × 75 мм; Элюент A: 1 л воды + 0.01% муравьиной кислоты; Элюент В: 1 л ацетонитрила + 0.01% муравьиной кислоты; градиент: 0.0 мин 10% В → 2.5 мин 95% В → 3.5 мин 95% В; печь: 50°С; поток: 0.90 мл/мин; УФ-обнаружение: 210 нм/ оптимальный путь интеграции 210-300 нм.Method 1: MS Device: Thermo Scientific FT-MS; HPLC device: Thermo Scientific UltiMate 3000; column: Waters Acquity UPLC HSS T3, 100

, 1.8 µm, 2.1 mm × 75 mm; Eluent A: 1 L water + 0.01% formic acid; Eluent B: 1 L acetonitrile + 0.01% formic acid; gradient: 0.0 min 10% B → 2.5 min 95% B → 3.5 min 95% B; oven: 50°C; flow: 0.90 ml/min; UV detection: 210nm / optimal integration path 210-300nm.

, 1.8 мкм; 1 мм × 50 мм; Элюент A: 1 л воды + 0.25 мл 99% муравьиной кислоты, Элюент В: 1 л ацетонитрила + 0.25 мл 99% муравьиной кислоты; градиент: 0.0 мин 90% А → 1.2 мин 5% А → 2.0 мин 5% А; печь: 50°С; поток: 0.40 мл/мин; УФ-обнаружение: 210 нм.Method 2: Device: Waters ACQUITY SQD UPLC System; column: Waters Acquity UPLC HSS T3, 100

, 1.8 µm; 1 mm × 50 mm; Eluent A: 1 L water + 0.25 ml 99% formic acid, Eluent B: 1 L acetonitrile + 0.25 ml 99% formic acid; gradient: 0.0 min 90% A → 1.2 min 5% A → 2.0 min 5% A; oven: 50°C; flow: 0.40 ml/min; UV detection: 210 nm.

When the compounds of the present invention are purified by chromatography in which the eluents contain additives, such as trifluoroacetic acid, formic acid, or ammonia, the compounds of the present invention can be obtained in the form of a salt, such as trifluoroacetate, formate, or ammonium salt, if the compounds of the present invention contain sufficient basic or acidic functionality. Such a salt can be converted to the corresponding free base or acid by a variety of methods known to those skilled in the art.

In the case of the synthesis of intermediates and working examples of the present invention described below, any compound indicated in the form of a salt of the corresponding base or acid is usually a salt of unknown exact stoichiometric composition, obtained using an appropriate method of preparation and/or purification. Unless otherwise stated, additions to names and structural formulas such as "hydrochloride", "trifluoroacetate", "sodium salt" or "x HCl", "x CF3COOH", "x Na+" should therefore not be understood in a stoichiometric sense in the case of such salts, since they are only descriptive in relation to the salt-forming components present in them.

This applies accordingly if synthetic intermediates or working examples or salts thereof have been prepared in the form of solvates, eg hydrates, of unknown stoichiometric composition (if they are of a certain type) by the preparation and/or purification methods described.

Source compounds

Example 1A

(2E)-But-2-en-1-yl[4-(5-chloro-2-cyanophenyl)-5-methoxy-2-oxopyridin-1(2H)-yl]acetate

A mixture of 4-chloro-2-(5-methoxy-2-oxo-1,2-dihydropyridin-4-yl)benzonitrile [CAS-RN 1630193-83-7; WO 2015/063093, page 73f., example 2.1C] (11.3 g, 43.2 mmol), (2E)-but-2-en-1-yl bromoacetate [CAS-RN 93455-19-7; S. M. Weinreb et al., J. Org. Chem. 1984, 49, 5058-5064] (10.0 g, 51.8 mmol) and potassium carbonate (8.95 g, 64.8 mmol) in DMF (90 ml) were heated to 100°C for 45 min. The solvent was then removed in vacuo. Water was added and the mixture was extracted three times with ethyl acetate. The combined organic layers were washed with brine and dried over sodium sulfate. The solvent was evaporated and the residue was purified by column chromatography (silica gel; eluent: cyclohexane-ethyl acetate gradient) to give 10.8 g (92% pure, 62% yield) of the title compound.

LC-MS (Method 2): R _t = 0.92 min; MS (ESIpos): m/z = 373 [M+H] ⁺

¹ H-NMR (400 MHz, DMSO-d ₆ ): δ [ppm] = 8.01-7.97 (m, 1H), 7.75-7.71 (m, 2H), 7.58 (s, 1H), 6.52 (s, 1H) , 5.88-5.78 (m, 1H), 5.65-5.55 (m, 1H), 4.74 (s, 2H), 4.59 (d, 2H), 3.62 (s, 3H), 1.70 (dd, 3H).

Example 2A

2-[4-(5-Chloro-2-cyanophenyl)-5-methoxy-2-oxopyridin-1(2H)-yl]-3-methylpent-4-enoic acid (racemic mixture of diastereomers)

(2E)-But-2-en-1-yl [4-(5-chloro-2-cyanophenyl)-5-methoxy-2-oxopyride in-1(2H)-yl]acetate (10.8 g, 92% pure , 26.7 mmol) was dissolved in THF (150 ml) and cooled to -78°C. Lithium bis(trimethylsilyl)amide (58 ml, 1.0 M in THF, 58 mmol) was added. The reaction mixture was stirred for 30 min at -78°C. Then, chloro(trimethyl)silane (7.4 ml, 58 mmol) was added. It was heated to 60° C. and stirred for 1 hour. Water was added to the reaction mixture and it was acidified with 1 N hydrochloric acid. Extracted three times with dichloromethane. The combined organic layers were extracted three times with 1 N aqueous sodium hydroxide solution. The combined base layers were acidified with 4N aqueous hydrochloric acid and extracted five times with dichloromethane. The combined organic phases were dried over sodium sulfate and the solvent was evaporated to give 6.95 g (70% yield) of the title compound as a a racemic mixture of diastereomers (38:62 ratio).

LC-MS (Method 1): R _t = 1.59 min (minor isomer), 1.62 min (major isomer); MS (ESIpos): m/z = 373 [M+H] ⁺

¹ H-NMR (400 MHz, DMSO-d ₆ ): δ [ppm] = 13.16 (br s, 1H), 7.99 (d, 1H major isomer) and 7.98 (d, 1H minor isomer), 7.76-7.69 (m , 2H), 7.44 (s, 1H major) and 7.38 (s, 1H minor), 6.52 (s, 1H major) and 6.46 (s, 1H minor), 5.95 (ddd, 1H major) and 5.59 (ddd, 1H minor ), 5.20 (d, 1H major) and 5.02 (d, 1H minor), 5.18-5.15 (m, 1H), 5.11 (dd, 1H major) and 4.92 (dd, 1H minor), 3.65 (s, 3H major) and 3.62 (s, 3H minor), 3.26-3.11 (m, 1H), 1.18 (d, 3H minor), and 0.87 (d, 3H major).

Example 3A

2-[4-(5-Chloro-2-cyanophenyl)-5-methoxy-2-oxopyridin-1(2H)-yl]-3-methyl-N-(prop-2-en-1-yl)-pent -4-enamide (racemic mixture of diastereomers)

2-[4-(5-Chloro-2-cyanophenyl)-5-methoxy-2-oxopyridin-1(2H)-yl]-3-methylpent-4-enoic acid (racemic mixture of diastereomers) (4.00 g, 10.7 mmol ) and prop-2-en-1-amine (12 ml, 160 mmol) were dissolved in pyridine (40 ml) and heated to 60°C. A solution of T3P in ethyl acetate (19 ml, 50% pure, 32 mmol) was added dropwise and the mixture was further stirred at 60° C. for 1 hour. Water was added and extracted three times with ethyl acetate. Water was added and three times the combined organic layers were dried over sodium sulfate, extracted with ethyl acetate, and the solvent was removed in vacuo. The residue was purified by column chromatography (silica gel; eluent: cyclohexane-ethyl acetate gradient) to give 2.56 g (58% yield) of the title compound as a mixture of diastereomers.

LC-MS (Method 1): R _t = 1.81 min; MS (ESIpos): m/z = 412 [M+H] ⁺

¹ H-NMR (400 MHz, DMSO-d ₆ ): δ [ppm] = 8.84 (t, 1H minor isomer) and 8.69 (t, 1H major isomer), 7.99 (d, 1H major) and 7.97 (d, 1H minor), 7.76-7.68 (m, 2H), 7.64 (s, 1H major) and 7.62 (s, 1H minor), 6.52 (s, 1H major) and 6.46 (s, 1H minor), 5.89-5.43 (m, 3H), 5.22-4.89 (m, 4H), 3.85-3.60 (m, 2H), 3.65 (s, 3H major) and 3.64 (s, 3H minor), 3.13-2.95 (m, 1H), 1.08 (d, 3H minor) and 0.83 (d, 3H major).

Example 4A

tert-butyl allyl {2-[4-(5-chloro-2-cyanophenyl)-5-methoxy-2-oxopyridin-1(2H)-yl]-3-methylpent-4-enoyl}carbamate (racemic mixture of diastereomers)

2-[4-(5-Chloro-2-cyanophenyl)-5-methoxy-2-oxopyridin-1(2H)-yl]-3-methyl-N-(prop-2-en-1-yl)pent- 4-enamide (racemic mixture of diastereomers) (2.50 g, 6.07 mmol) was dissolved in acetonitrile (120 ml). Di-tert-butyl dicarbonate (2.8 ml, 12 mmol) and 4-dimethylaminopyridine (297 mg, 2.43 mmol) were added and the mixture was heated to 60°C for 1 h. The mixture was then concentrated in vacuo and the residue was directly purified using a column chromatography (silica gel; eluent: cyclohexane-ethyl acetate gradient) to give 3.07 g (99% yield) of the title compound as a mixture of diastereomers.

LC-MS (Method 1): R _t = 2.41 min; MS (ESIpos): m/z = 512 [M+H] ⁺

¹ H-NMR (400 MHz, DMSO-d ₆ ): δ [ppm] = 7.99 (d, 1H major isomer) and 7.97 (d, 1H minor isomer), 7.75-7.63 (m, 2H), 7.42 (s, 1H major) and 7.39 (s, 1H minor), 6.51 (s, 1H major) and 6.46 (s, 1H minor), 6.45 (br. d, 1H major) and 6.36 (br. d, 1H minor), 5.96- 5.58 (m, 2H), 5.22-4.92 (m, 4H), 4.24-4.07 (m, 2H), 3.66 (s, 3H major) and 3.62 (s, 3H minor), 3.35-3.20 (m, 1H), 1.484 (s, 9H minor) and 1.478 (s, 9H major), 1.16 (d, 3H minor) and 0.89 (d, 3H major).

Example 5A

tert-Butyl 3-[4-(5-chloro-2-cyanophenyl)-5-methoxy-2-oxopyridin-1(2H)-yl]-4-methyl-2-oxo-2,3,4,7- tetrahydro-1H-azepine-1-carboxylate (racemic mixture of diastereomers)

Tert-butyl allyl {2-[4-(5-chloro-2-cyanophenyl)-5-methoxy-2-oxopyridin-1(2H)-yl]-3-methylpent-4-enoyl}carbamate (racemic mixture of diastereomers) (3.00 g, 5.86 mmol) was dissolved in dichloromethane (1.0 1) and the solution was degassed (argon atmosphere). Benzylidene[1,3-bis(2,4,6-trimethylphenyl)imidazolidin-2-ylidene]ruthenium dichloride-tricyclohexylphosphane (1/1) [CAS-RN 246047-72-3] (249 mg, 293 µmol) was added and the reaction mixture was stirred at 45° C. for 1.5 h. Then, a saturated aqueous sodium bicarbonate solution was added. The organic layer was separated and the aqueous layer was extracted twice with dichloromethane. The combined organic layers were washed with brine and dried over sodium sulfate. The solvent was evaporated and the residue was purified by column chromatography (silica gel; eluent: cyclohexane-ethyl acetate gradient) to give 2.75 g (97% yield) of the title compound as a mixture of diastereomers.

LC-MS (Method 1): R _t = 2.04 (major isomer) and 2.06 min (minor isomer); MS (ESIneg): m/z = 482 [M-H] ^-

¹ H-NMR (400 MHz, DMSO-d ₆ ): δ [ppm] = 8.01 (m, 1H minor isomer) and 8.00 (d, 1H major isomer), 7.78-7.71 (m, 2H), 7.47 (s, 1H major) and 7.31 (s, 1H, minor), 6.60 (s, 1H minor) and 6.56 (s, 1H major), 6.34 (d, 1H major) and 6.18 (d, 1H minor), 6.03-5.94 (m , 1H), 5.90-5.83 (m, 1H minor) and 5.83-5.77 (m, 1H major), 4.57 (dd, 1H minor) and 4.47 (dd, 1H major), 4.44-4.33 (m, 1H), 3.684 (s, 3H minor) and 3.676 (s, 3H major), 3.48-3.38 (m, 1H minor), 3.07-2.97 (m, 1H minor), 1.46 (s, 9H major) and 1.45 (s, 9H minor) , 1.18 (d, 3H minor) and 0.91 (d, 3H major).

Example 6A

tert-Butyl 3-[4-(5-chloro-2-cyanophenyl)-5-methoxy-2-oxopyridin-1(2H)-yl]-4-methyl-2-oxoazepane-1-carboxylate (racemic mixture of diastereomers)

tert-Butyl 3-[4-(5-chloro-2-cyanophenyl)-5-methoxy-2-oxopyridin-1(2H)-yl]-4-methyl-2-oxo-2,3,4,7- tetrahydro-1H-azepine-1-carboxylate (racemic mixture of diastereomers) (2.70 g, 5.58 mmol) was dissolved in ethyl acetate (250 ml) and palladium (10% on charcoal, 594 mg) was added. The reaction mixture was stirred under a hydrogen atmosphere (atmospheric pressure) for 4 hours. The mixture was filtered through diatomaceous earth and the solvent was removed in vacuo. The residue was purified by column chromatography (silica gel; eluent: cyclohexane-ethyl acetate gradient) to give 0.54 g of the main isomer of the starting material and 1.78 g (66% yield, 82% yield based on the recovered starting material) of the title compound as a mixture of diastereomers.

LC-MS (Method 1): R _t = 2.05 min; MS (ESIneg): m/z = 484 [M-H] ^-

¹ H-NMR (400 MHz, DMSO-d ₆ ): δ [ppm] = 8.00 (d, 1H) and 7.99 (d, 1H), 7.77-7.71 (m, 2H), 7.42 (s, 1H minor isomer) and 7.16 (s, 1H major), 6.56 (s, 1H major) and 6.52 (s, 1H minor), 5.89 (s, 1H major) and 5.86-5.66 (m, 1H minor), 4.23-4.13 (m, 1H), 3.68 (s, 3H major) and 3.66 (s, 3H minor), 3.65-3.55 (m, 1H), 2.64-2.56 (m, 1H) and (2.15-2.03 (m, 1H), 1.97-1.55 (m, 4H), 1.46 (s, 9H), 1.14 (d, 3H major), and 0.86 (d, 3H minor).

Example 7A

6-[(tert-Butoxycarbonyl)amino]-2-[4-(5-chloro-2-cyanophenyl)-5-methoxy-2-oxopyridin-1(2H)-yl]-3-methylhexanoic acid (racemic mixture of diastereomers )

tert-Butyl 3-[4-(5-chloro-2-cyanophenyl)-5-methoxy-2-oxopyridin-1(2H)-yl]-4-methyl-2-oxoazepane-1-carboxylate (racemic mixture of diastereomers) (1.77 g, 3.64 mmol) was dissolved in THF (25 ml) and an aqueous solution of lithium hydroxide (3.6 ml, 2.0 M, 7.2 mmol) was added. The mixture was stirred at 30°C for 1 h, then concentrated in vacuo and the residue was dissolved in water. Washed with ethyl acetate. The aqueous phase was acidified with 1N aqueous hydrochloric acid and extracted twice with ethyl acetate. The combined organic layers were washed with brine and dried over sodium sulfate to give 1.78 g (97% yield) of the title compound as a mixture of diastereomers.

LC-MS (Method 1): R _t = 1.80 min (minor isomer) and 1.84 min (major isomer); MS (ESIpos): m/z = 504 [M+H] ⁺

¹ H-NMR (400 MHz, DMSO-d ₆ ): δ [ppm] = 13.13 (br. s, 1H), 8.01-7.97 (m, 1H), 7.76-7.71 (m, 2H), 7.38 (s, 1H), 6.80 (br. t, 1H major isomer) and 6.70 (br. t, 1H minor isomer), 6.51 (s, 1H), 5.10 (d, 1H minor) and 5.05 (d, 1H major), 3.64 ( s, 3H minor) and 3.63 (s, 3H major), 3.01-2.71 and 2.49-2.37 (m, together 3H), 1.64-1.40 and 1.31-1.13 and 1.02-0.93 (m, together 4H), 1.38 (s, 9H major) and 1.34 (s, 9H minor), 1.05 (d, 3H minor) and 0.72 (d, 3H major).

Example 8A

tert-Butyl 1(2H)-yl]-4-methyl-6-oxohexyl}carbamate (racemic mixture of diastereomers)

6-[(tert-Butoxycarbonyl)amino]-2-[4-(5-chloro-2-cyanophenyl)-5-methoxy-2-oxopyridin-1(2H)-yl]-3-methylhexanoic acid (racemic mixture of diastereomers ) (1.08 g, 2.14 mmol) and 5-aminopyrazolo[1,5-a]pyridine-3-carboxamide trifluoroacetate [CAS-RN 1891071-11-6; WO 2016/046158, page 53 example 1.1C] (746 mg, 2.57 mmol) was dissolved in DMF (4.0 ml). HATU (1.22 g, 3.21 mmol) as a solution in DMF (2.0 ml) and then N,N-diisopropylethylamine (370 μl, 2.1 mmol) were added dropwise. The reaction mixture was stirred for 1.5 h at room temperature. More 5-aminopyrazolo[1,5-a]pyridine-3-carboxamide trifluoroacetate (187 mg, 643 µmol) and N,N-diisopropylethylamine (370 µl, 2.1 mmol) were added. After stirring overnight, more HATU (407 mg, 1.07 mmol) and N,N-diisopropylethylamine (370 μl, 2.1 mmol) were added and after 1 hour the reaction mixture was concentrated in vacuo. The residue was crystallized with water, collected by suction filtration and washed with water and dried in vacuo. The compound was purified by column chromatography (silica gel; eluent: dichloromethane/methanol gradient) to give 1.20 g (84% yield) of the title compound as a mixture of diastereomers.

LC-MS (Method 1): R _t = 1.75 min; MS (ESIpos): m/z = 662 [M+H] ⁺

¹ H-NMR (400 MHz, DMSO-d ₆ ): δ [ppm] = 11.14 (br. s, 1H major isomer) and 11.12 (br. s, 1H minor isomer), 8.73-8.66 (m, 2H), 8.47 (s, 1H major) and 8.46 (s, 1H minor), 8.00 (d, 1H major) and 7.99 (d, 1H minor), 7.77-7.71 (m, 2H), 7.63 (s, 1H), 7.60 ( br. s, 1H), 7.26 (dd, 1H), 6.98 (br. s, 1H), 6.76-6.70 (m, 1H), 6.56 (s, 1H minor) and 6.55 (s, 1H major), 5.57 ( d, 1H major) and 5.56 (d, 1H minor), 3.72 (s, 3H major) and 3.71 (s, 3H minor), 2.96-2.76 (m, 2H), 1.64-0.96 (m, 5H), 1.34 ( s, 9H major) and 1.28 (s, 9H minor), 1.05 (d, 3H major) and 0.78 (d, 3H minor).

Separation of the four stereoisomers can be achieved by chiral chromatography: Column and solid phase: 250 mm x 20 mm, Chiralpak IE, 5 µm; Eluent: ethanol; Flow 15.0 ml/min. Three peaks are obtained (7.0-8.5 min, 11.1 min, 13.9 min). The first peak needs additional chromatography for purification: Column and solid phase: 250 mm × 20 mm, Chiralpak IC, 5 µm; Eluent: ethanol; Flow 15.0 ml/min. Two peaks are obtained (6.29 min, 7.33 min).

Analytical HPLC: column 250 mm × 4.6 mm, Chiralcel OX-H, 5 µm; Eluent: isohexane/ethanol 1:1; Flow 1.0 ml/min; Temperature: 40°C.

Stereoisomer 1: R _t = 10.90 min.

Stereoisomer 2: R _t = 7.50 min.

Stereoisomer 3: R _t = 8.85 min.

Stereoisomer 4: R _t = 9.19 min.

Stereoisomer 1 and 4 and stereoisomer 2 and 3 are each other's enantiomers.

Stereoisomer 1 and 4:

¹ H-NMR (400 MHz, DMSO-d ₆ ): δ [ppm] = 11.14 (br. s, 1H), 8.70-8.67 (m, 2H), 8.47 (s, 1H), 8.00 (d, 1H) , 7.75-7.71 (m, 2H), 7.60 (br. s, 1H), 7.62 (br. s, 1H), 7.25 (dd, 1H), 6.98 (br. s, 1H), 6.76-7.70 (m, 1H), 6.55 (s, 1H), 5.57 (d, 1H), 3.71 (s, 3H), 2.93-2.74 (m, 2H), 1.52-1.29 (m, 3H), 1.34 (s, 9H), 1.26 -0.96 (m, 2H), 1.05 (d, 3H).

Stereoisomer 2 and 3:

¹ H-NMR (400 MHz, DMSO-d ₆ ): δ [ppm] = 11.12 (br. s, 1H), 8.71 (d, 1H), 8.68 (d, 1H), 8.46 (s, 1H), 7.99 (d, 1H), 7.76 (d, 1H), 7.73 (dd, 1H), 7.62 (s, 1H), 7.59 (br. s, 1H), 7.25 (dd, 1H), 6.99 (br. s, 1H ), 6.75 (br. t, 1H), 6.56 (s, 1H), 5.56 (d, 1H), 3.70 (s, 3H), 2.95-2.83 (m, 2H), 2.48-2.39 (m, 1H), 1.66-1.53 (m, 1H), 1.51-1.29 (m, 3H), 1.28 (s, 9H), 0.77 (d, 3H).

Working examples

Example 1

5-({6-Amino-2-[4-(5-chloro-2-cyanophenyl)-5-methoxy-2-oxopyridin-1(2H)-yl]-3-methyl-hexanoyl}amino)pyrazolo[1 ,5-a]pyridine-3-carboxamide trifluoroacetate (racemic mixture of diastereomers)

tert-Butyl 1(2H)-yl]-4-methyl-6-oxohexyl}carbamate (racemic mixture of diastereomers) (1.20 g, 1.81 mmol) was dissolved in dichloromethane and cooled with ice water. Trifluoroacetic acid (2.8 ml, 36 mmol) was added. The mixture was warmed to room temperature and stirred for 1 hour. The mixture was then evaporated. Dichloromethane was added and removed in vacuo. This procedure was repeated once. The residue was purified by column chromatography (silica gel; eluent: dichloromethane/methanol gradient) to give 785 mg (64% yield) of the title compound as a mixture of diastereomers.

LC-MS (Method 2): R _t = 0.64 min (major isomer) 0.68 (minor isomer); MS (ESIpos): m/z=562 [M+H] ⁺

¹ H-NMR (400 MHz, DMSO-d ₆ ): δ [ppm] = 11.17 (s, 1H major isomer) and 11.14 (s, 1H minor isomer), 8.73-8.67 (m, 2H), 8.48 (s, 1H), 8.00 (d, 1H major) and 7.99 (d, 1H minor), 7.76-7.72 (m, 2H), 7.69 (br. s, 4H), 7.65 (s, 1H major) and 7.61 (s, 1H minor), 7.28-7.25 (m, 1H), 6.98 (br. s, 1H), 6.58 (s, 1H minor) and 6.57 (s, 1H major), 5.61-5.55 (m, 1H), 3.72 (s, 3H major) and 3.70 (s, 3H minor), 2.87-2.51 (t, 3H), 1.80-1.09 (t, 4H), 1.08 (d, 3H major) and 0.79 (d, 3H minor).

Example 2

5-({(2S)-6-Amino-2-[4-(5-chloro-2-cyanophenyl)-5-methoxy-2-oxopyridin-1(2H)-yl]-3-methylhexanoyl}amino)pyrazolo [1,5-а]pyridine-3-carboxamide trifluoroacetate (stereoisomer 3)

Following the procedure described in Example 1, the separated carbamate stereoisomer 3 according to Example 8A was separately deprotected.

Stereoisomer 3:

LC-MS (Method 1): R _t = 1.05 min; MS (ESIpos): m/z = 562 [M+H] ⁺

¹ H-NMR (400 MHz, DMSO-d ₆ ): δ [ppm] = 11.15 (s, 1H), 8.72 (d, 1H), 8.69 (d, 1H), 8.48 (s, 1H), 8.00 (d , 1H), 7.76-7.72 (m, 2Н), 7.60 (s, 1H), 7.63 (br s, 4H), 7.27 (dd, 1H), 6.98 (br s, 1H), 6.58 (s, 1H), 5.56 (d, 1H), 3.70 (s, 3H), 2.88-2.72 (m, 2H), (1H under DMSO), 1.81-1.67 (m, 1H), 1.63-1.44 (m, 2H), 1.39-1.28 (m, 1H), 0.79 (d, 3H).

Example 3

5-({(2S)-6-Amino-2-[4-(5-chloro-2-cyanophenyl)-5-methoxy-2-oxopyridin-1(2H)-yl]-3-methylhexanoyl}amino)pyrazolo [1,5-а]pyridine-3-carboxamide trifluoroacetate (stereoisomer 4)

Following the procedure described in Example 1, the separated carbamate stereoisomer 4 according to Example 8A was separately deprotected.

Stereoisomer 4:

LC-MS (Method 1): R _t = 0.99 min; MS (ESIpos): m/z = 562 [M+H] ⁺

¹ H-NMR (400 MHz, DMSO-d ₆ ): δ [ppm] = 11.17 (s, 1H), 8.72-8.67 (m, 2H), 8.48 (s, 1H), 8.03-7.98 (m, 1H) , 7.77-7.72 (m, 2Н), 7.65 (s, 1H), 7.62 (br s, 4H), 7.26 (dd, 1H), 6.98 (br s, 1H), 6.57 (s, 1H), 5.59 (d , 1H), 3.72 (s, 3H), 2.83-2.62 (m, 2H), 2.58-2.51 (m, 1H), 1.70-1.44 (m, 2H), 1.08 (d, 3H), 1.26-1.06 (m , 2H).

C) Evaluation of physiological effectiveness

The suitability of the compounds of the present invention for the treatment of thromboembolic disorders can be demonstrated using the following assay systems:

a) Test descriptions (in vitro)

a.1) Determination of plasma kallikrein activity

To determine the inhibition of plasma kallikrein by the substances of the present invention, a biochemical test system is used that uses the reaction of the peptide substrate of plasma kallikrein to determine the enzymatic activity of human plasma kallikrein. Here, plasma kallikrein cleaves from the plasma peptide substrate a C-terminal aminomethylcoumarin (AMC), the fluorescence of which is measured. The determinations are carried out on microtiter plates.

The test substances were dissolved in dimethyl sulfoxide and serially diluted with dimethyl sulfoxide (from 3000 μM to 0.0078 μM; final concentrations in the test: from 50 μM to 0.00013 μM). In each case, 1 µl of diluted solutions of the substances were added to the wells of Greiner white microtiter plates (384 wells). 20 µl assay buffer (50 mM Tris/HCl pH 7.4; 100 mM sodium chloride solution; 5 mM calcium chloride solution; 0.1% bovine serum albumin) and 20 µl Kordia plasma kallikrein (0.6 nM in assay buffer ) were then added sequentially. After 15 min incubation, the enzyme reaction was started by adding 20 µl of H-Pro-Phe-Arg-AMC substrate dissolved in assay buffer (10 µM in assay buffer) from Bachem, the mixture was incubated at room temperature (22°C) for 30 minutes and then the fluorescence was measured (excitation: 360 nm, emission: 460 nm). The measured emissions of test lots with test substance were compared with emissions of control lots without test substance (dimethyl sulfoxide only instead of test substance in dimethyl sulfoxide) and IC ₅₀ values were calculated from concentration/activity ratios. Activity data from this test are listed in Table A below (some as averages of several independent individual determinations):

a.2) Measurement of FXIa inhibition

Factor XIa inhibition of the substances of the present invention is determined using a biochemical test system that uses the factor XIa peptide substrate reaction to determine the enzymatic activity of human factor XIa. Here, factor XIa cleaves from the factor XIa peptide substrate the C-terminal aminomethylcoumarin (AMC) whose fluorescence is measured. The determinations are carried out on microtiter plates.

The test substances were dissolved in dimethyl sulfoxide and serially diluted with dimethyl sulfoxide (from 3000 μM to 0.0078 μM; final concentrations in the test: from 50 μM to 0.00013 μM). In each case, 1 µl of diluted solutions of the substances were added to the wells of Greiner white microtiter plates (384 wells). 20 µl of assay buffer (50 mM Tris/HCl pH 7.4; 100 mM sodium chloride solution; 5 mM calcium chloride solution; 0.1% bovine serum albumin) and 20 µl of factor XIa from Kordia (0.45 nM in assay buffer ) were then added sequentially. After 15 min incubation, the enzyme reaction was started by adding 20 µl of factor XIa substrate Boc-Glu(OBzl)-Ala-Arg-AMC dissolved in assay buffer (10 µM in assay buffer) from Bachem, the mixture was incubated at room temperature (22°C) for 30 minutes and then the fluorescence was measured (excitation: 360 nm, emission: 460 nm). The measured emissions of test lots with test substance were compared with emissions of control lots without test substance (dimethyl sulfoxide only instead of test substance in dimethyl sulfoxide) and IC ₅₀ values were calculated from concentration/activity ratios. Activity data from this test are listed in Table B below (some as averages of several independent individual determinations):

3) Definition of selectivity

To demonstrate the selectivity of the substances for FXIa inhibition, the test substances were tested for their inhibition of other human serine proteases such as factor Xa, trypsin and plasmin. To determine the enzymatic activity of factor Xa (1.3 nmol/l from Kordia), trypsin (83 mU/ml from Sigma) and plasmin (0.1 μg/ml from Kordia), these enzymes were dissolved (50 mmol/l Tris buffer [ C,C,C-tris(hydroxymethyl)aminomethane], 100 mmol/l NaCl, 0.1% BSA [bovine serum albumin], 5 mmol/l calcium chloride, pH 7.4) and incubated for 15 min with the test substance at various concentrations in dimethyl sulfoxide, as well as with dimethyl sulfoxide without the test substance. The enzymatic reaction was then started by adding appropriate substrates (5 µmol/l Boc-Ile-Glu-Gly-Arg-AMC from Bachem for factor Xa and trypsin, 50 µmol/l MeOSuc-Ala-Phe-Lys-AMC from Bachem for plasmin) . After 30 min incubation at 22° C., fluorescence was measured (excitation: 360 nm, emission: 460 nm). The measured emissions of test mixtures with test substance were compared with emissions of control mixtures without test substance (dimethyl sulfoxide only instead of test substance in dimethyl sulfoxide) and IC ₅₀ values were calculated from concentration/activity ratios.

a.4) Determination of anticoagulant activity

The anticoagulant activity of the test substances was determined in vitro in human plasma and rat plasma. For this, blood was collected at a sodium citrate/blood ratio of 1:9, using a 0.11 molar solution of sodium citrate as a receiver. Immediately after taking the blood, it was thoroughly mixed and centrifuged at about 4000 g for 15 minutes. The supernatant was taken with a pipette.

Activated partial thromboplastin time (APTT) was determined in the presence of various concentrations of the test substance or the appropriate diluent using a commercial test kit (aPTT reagent from Siemens). Test compounds were incubated with plasma and aPTT reagent at 37°C for 3 minutes. Then, coagulation was started by adding 25 mM calcium chloride and the time when coagulation occurred was determined. The concentration of test substance that increases by 50% or doubles APTT was determined.

a.5) Determination of the integrity of the endothelium

The activity of the compounds of the present invention was characterized by an in vitro permeability assay on "human umbilical vein cells" (HUVEC). Using the EOS device (EC IS: Electric Cell-Sub Impedance Sensing; Applied Biophysics Inc; Troy, NY), changes in transendothelial electrical resistance (TEER) can be continuously measured through a monolayer of endothelial cells deposited on gold electrodes. HUVEC were plated on a 96-well sensor electrode plate (96W1 E, Ibidi GmbH, Martinsried, Germany). Hyperpermeability of the formed monolayer of confluent cells is induced by stimulation with kininogen, prekallikrein and factor XII (100 nM each). Compounds according to the present invention are added before adding the substances mentioned above. Typical compound concentrations are from 1×10 ^-10 to 1×10 ^-6 M.

a.6) Determination of in vitro permeability of endothelial cells

On another model of hyperpermeability, the activity of substances was determined by modulation of macromolecular permeability. HUVEC were plated on fibronectin coated Transwell filter membrane (24-well plates, 6.5 mm insert with 0.4 μM polycarbonate membrane; Costar #3413). A filter membrane separates the upper cell culture space from the lower cell culture space, with a confluent layer of endothelial cells at the bottom of the upper cell culture space. 250 μg/ml 40 kDa FITC-dextane (Invitrogen, D1844) was added to the upper chamber medium. Monolayer hyperpermeability was induced by stimulation with kininogen, prekallikrein, and factor XII (100 nM each). Every 30 min, samples of the medium were removed from the lower chamber and the relative fluorescence was determined using a fluorimeter as a parameter of changes in macromolecular permeability as a function of time. Compounds according to the present invention are added before adding the substances mentioned above. Typical compound concentrations are from 1×10 ^-10 to 1×10 ^-6 M.

b) Determination of antithrombotic activity (in vivo)

b.1) Model of Arterial Thrombosis (Iron(II) Chloride Thrombosis) in Combination with Ear Bleeding Time in Rabbits

The antithrombotic activity of FXIa inhibitors was tested in a model of arterial thrombosis. Here, thrombus formation was triggered by causing chemical damage to the area of the carotid artery in rabbits. At the same time, the time of ear bleeding was determined.

Male rabbits (Crl: KBL (NZW) BR, Charles River) fed a normal diet and weighing 2.2-2.5 kg were anesthetized by intramuscular injection of xylazine and ketamine (Rompun, Bayer, 5 mg/kg and Ketavet, Pharmacia & Upjohn GmbH, 40 mg/kg body weight). Anesthesia was maintained, in addition, by intravenous administration of the same drugs (bolus: continuous infusion) through the right atrial vein.

The right carotid artery was exposed and vessel injury was induced by wrapping a piece of filter paper (10 mm x 10 mm) around a Parafilm® strip (25 mm x 12 mm) around the carotid artery without disturbing blood flow. The filter paper contains 100 µl of a 13% iron(II) chloride solution (Sigma) in water. After 5 min, the filter paper was removed and the vessel was washed twice with an aqueous solution of sodium chloride with a concentration of 0.9%. 30 minutes after injury, the injured section of the carotid artery was removed surgically, and any thrombotic material was removed and weighed.

Test substances were administered either intravenously to anesthetized animals via the femoral vein or orally to awake animals via a gastric tube, in each case 5 minutes and 2 hours, respectively, prior to injury.

Ear bleeding time was determined 2 min after injury to the carotid artery. To do this, the left ear was shaved and a 3 mm incision (blade, article no. 10-150-10, Martin, Tuttlingen, Germany) was made parallel to the longitudinal axis of the ear. Care must be taken here not to damage the visible vessels. Any blood that was expelled was collected at 15 second intervals with accurately weighed pieces of filter paper without directly touching the wound. Bleeding time was calculated as the time from the incision to the time when no blood could be detected on the filter paper. The volume of extravasated blood was calculated after weighing the pieces of filter paper.

c) Determination of the effect on hemorrhage/edema formation and/or neovascularization in the eye (in vivo)

c.1) Testing the efficacy of substances in a model of laser-induced choroidal neovascularization

This study serves to investigate the efficacy of the test substance in reducing hemorrhage/edema and/or choroidal neovascularization in a rat model of laser-induced choroidal neovascularization.

For this purpose, pigmented Brown-Norway rats that did not show any signs of ophthalmic disorders were selected and randomized into treatment groups. On day 0, animals were anesthetized with an intraperitoneal injection (15 mg/kg xylazine and 80 mg/kg ketamine). After instillation of a drop of 0.5% solution of tropicamide to dilate the pupils, choroidal neovascularization was initiated at six specific locations around the optic nerve using a 532 nm argon laser photocoagulator (diameter 50-75 μm, intensity 150 mW, duration 100 ms). The test substance and the appropriate vehicle (eg, PBS, isotonic saline) were administered either systemically by the oral or intraperitoneal route, or topically to the eye by repeated administration as eye drops or intravitreal injection. The body weight of all animals was determined before the start of the study, and then daily during the study.

On day 21, angiography was performed using a fluorescent fundus camera (eg, Kowe, HRA). Under anesthesia and after another pupil dilation, sodium fluorescein dye was injected subcutaneously (s.c.) at a concentration of 10%. After 2-10 min. took a picture of the back of the eye. The degree of hemorrhage/edema represented by fluorescein leakage was assessed by two to three blind observations and classified by severity from 0 (no hemorrhage) to 3 (strong staining in excess of the actual lesion).

Animals were sacrificed on day 23, after which the eyes were removed and fixed in 4% paraformaldehyde solution for one hour at room temperature. After one wash, the retina was carefully peeled off and the sclera-choroidal complex was stained with FITC antibody to isolectin B4 and then applied to a microscope slide. The preparations thus obtained were evaluated with a fluorescent microscope (Apotom, Zeiss) at an excitation wavelength of 488 nm. Area or volume of choroidal neovascularization (in µm ² and µm ³ , respectively) was calculated by morphometric analysis using Axiovision 4.6 software.

c.2) Testing the efficacy of substances in an oxygen-induced retinopathy model

Oxygen-induced retinopathy has been shown to be a useful animal model for studying pathological retinal angiogenesis. This model is based on the observation that hyperoxia during early postnatal retinal development causes growth arrest or retardation of normal retinal blood vessels. When animals are returned to normoxic room air after the 7-day hyperoxia phase, this is equivalent to relative hypoxia, since the retina lacks the normal vessels that are necessary to ensure an adequate supply of neural tissue under normoxic conditions. The ischemic situation thus induced results in an abnormal neovascularization that bears some resemblance to pathophysiological neovascularization in ocular diseases such as wet AMD. In addition, induced neovascularization is highly reproducible, quantifiable, and is an important parameter for studying disease mechanisms and possible treatments for various forms of retinal disease.

The aim of this study is to investigate the efficacy of daily systemically administered doses of a test compound on retinal vascular growth in an oxygen-induced retinopathy model. Newborn mice C57Bl/6 and their mothers are subjected to hyperoxia (70% oxygen) on the 7th postnatal day (PD7) for 5 days. From PD12, mice are kept under normoxic conditions (room air, 21% oxygen) up to PD17. From day 12 to day 17, mice are treated daily with test substance or appropriate vehicle. On day 17, all mice are anesthetized with isoflurane and then sacrificed by cervical fracture. The eyes are removed and fixed with 4% formalin. After washing in saline with phosphate buffer, the retina is excised, its flat preparation is obtained, which is stained with an antibody to isolectin B4. Neovascularization is quantified using the Zeiss ApoTome.

C) Working examples of pharmaceutical compositions

The substances of the present invention can be formulated into pharmaceutical preparations as follows:

Tablet:

Compound:

100 mg of the compound of Example 1, 50 mg of lactose (monohydrate), 50 mg of cornstarch, 10 mg of polyvinylpyrrolidone (PVP 25) (from BASF, Germany) and 2 mg of magnesium stearate.

The weight of the tablet is 212 mg. Diameter 8 mm, radius of curvature 12 mm.

Receipt:

A mixture of the compound of Example 1, lactose and starch is granulated with a 5% (w/w) solution of PVP in water. After drying, the granules are mixed with magnesium stearate for 5 minutes. This mixture is compressed in a conventional tablet press (see above for tablet format).

Oral suspension:

Compound:

1000 mg of the compound of Example 1, 1000 mg of ethanol (96%), 400 mg of Rhodigel (xanthan gum) (from FMC, USA) and 99 g of water.

10 ml of oral suspension corresponds to one dose of 100 mg of the compound according to the present invention.

Receipt:

Rhodigel was suspended in ethanol and the compound of the present invention was added to the suspension. Water was added with stirring. The mixture was stirred for about 6 hours until the swelling of the Rhodigel was complete.

Solution or suspension for topical use in the eye (eye drops):

A sterile pharmaceutical formulation for topical administration to the eye can be obtained by reconstituting a lyophilisate of a compound of the present invention in sterile saline. Suitable preservatives for such a solution or suspension are, for example, benzalkonium chloride, thiomersal or phenylmercury nitrate in the concentration range from 0.001 to 1% by weight.

Solution or suspension for topical use in the eye (eye drops):

A sterile pharmaceutical formulation for topical administration to the eye can be obtained by reconstituting a lyophilisate of a compound of the present invention in sterile saline. Suitable preservatives for such a solution or suspension are, for example, benzalkonium chloride, thiomersal or phenylmercury nitrate in the concentration range from 0.001 to 1% by weight.

Claims (8)

1. Compound 5-({6-amino-2-[4-(5-chloro-2-cyanophenyl)-5-methoxy-2-oxopyridin-1(2H)-yl]-3-methylhexanoyl}amino)pyrazolo[ 1,5-a]pyridine-3-carboxamide of formula (I)

or one of its salts. 2. Compound 5-({6-amino-2-[4-(5-chloro-2-cyanophenyl)-5-methoxy-2-oxopyridin-1(2H)-yl]-3-methylhexanoyl}amino)pyrazolo[ 1,5-a]pyridine-3-carboxamide trifluoroacetate according to claim 1 of formula (Ia)

3. Compound 5-({(2S)-6-amino-2-[4-(5-chloro-2-cyanophenyl)-5-methoxy-2-oxopyridin-1(2H)-yl]-3-methylhexanoyl} amino)pyrazolo[1,5-a]pyridine-3-carboxamide according to claim 1 of formula (Ib)

or one of its salts.