Patent Application
20160237045
The invention relates to substituted phenylalanine derivatives and to processes for preparation thereof, and to the use thereof for production of medicaments for treatment and/or prophylaxis of diseases, especially of cardiovascular disorders and/or severe perioperative blood loss.
Blood coagulation is a protective mechanism of the organism which helps to “seal” defects in the wall of the blood vessels quickly and reliably. Thus, loss of blood can be avoided or kept to a minimum. Haemostasis after injury of the blood vessels is effected mainly by the coagulation system in which an enzymatic cascade of complex reactions of plasma proteins is triggered. Numerous blood coagulation factors are involved in this process, each of which factors converts, on activation, the respectively next inactive precursor into its active form. At the end of the cascade comes the conversion of soluble fibrinogen into insoluble fibrin, resulting in the formation of a blood clot. In blood coagulation, traditionally the intrinsic and the extrinsic system, which end in a final joint reaction path, are distinguished. Here, factors Xa and IIa (thrombin) play key roles: Factor Xa bundles the signals of the two coagulation paths since it is formed both via factor VIIa/tissue factor (extrinsic path) and via the tenase complex (intrinsic path) by conversion of factor X. The activated serine protease Xa cleaves prothrombin to thrombin which, via a series of reactions, transduces the impulses from the cascade to the coagulation state of the blood.
In the more recent past, the traditional theory of two separate regions of the coagulation cascade (extrinsic and intrinsic path) has been modified owing to new findings: In these models, coagulation is initiated by binding of activated factor VIIa to tissue factor (TF). The resulting complex activates factor X, which in turn leads to generation of thrombin with subsequent production of fibrin and platelet activation (via PAR-1) as injury-sealing end products of haemostasis. Compared to the subsequent amplification/propagation phase, the thrombin production rate is low and as a result of the occurrence of TFPI as inhibitor of the TF-FVIIa-FX complex is limited in time.
A central component of the transition from initiation to amplification and propagation of coagulation is factor XIa. In positive feedback loops, thrombin activates, in addition to factor V and factor VIII, also factor XI to factor XIa, whereby factor IX is converted into factor IXa, thus, via the factor IXa/factor Villa complex generated in this manner, rapidly producing relatively large amounts of factor Xa. This triggers the production of large amounts of thrombin, leading to strong thrombus growth and stabilizing the thrombus.
The formation of a thrombus or blood clot is counter-regulated by fibrinolysis. Activation of plasminogen by tissue plasminogen activator (tPA) results in formation of the active serine protease, plasmin, which cleaves polymerized fibrin and thus degrades the thrombus. This process is referred to as fibrinolysis—with plasmin as key enzyme.
Uncontrolled activation of the coagulation system or defects in the inhibition of the activation processes may cause formation of local thromboses or embolisms in vessels (arteries, veins, lymph vessels) or heart chambers. This may lead to serious thrombotic or thromboembolic disorders. In addition, systemic hypercoagulability may lead to consumption coagulopathy in the context of a disseminated intravasal coagulation.
In the course of many cardiovascular and metabolic disorders, there is an increased tendency for coagulation and platelet activation owing to systemic factors such as hyperlipidaemia, diabetes or smoking, owing to changes in blood flow with stasis, for example in atrial fibrillation, or owing to pathological changes in vessel walls, for example endothelial dysfunctions or atherosclerosis. This unwanted and excessive haemostasis may, by formation of fibrin- and platelet-rich thrombi, lead to thromboembolic disorders and thrombotic complications with life-threatening conditions.
Thromboembolic disorders are the most frequent cause of morbidity and mortality in most industrialized countries [Heart Disease: A Textbook of Cardiovascular Medicine, Eugene Braunwald, 5th edition, 1997, W.B. Saunders Company, Philadelphia].
The anticoagulants known from the prior art, for example substances for inhibiting or preventing blood coagulation, have various, frequently grave disadvantages. Accordingly, in practice, efficient treatment methods or the prophylaxis of thrombotic/thromboembolic disorders are found to be very difficult and unsatisfactory.
In the therapy and prophylaxis of thromboembolic disorders, use is made, firstly, of heparin which is administered parenterally or subcutaneously. Because of more favourable pharmacokinetic properties, preference is these days increasingly given to low-molecular-weight heparin; however, the known disadvantages described hereinbelow encountered in heparin therapy cannot be avoided either in this manner. Thus, heparin is orally ineffective and has only a comparatively short half-life. In addition, there is a high risk of bleeding, there may in particular be cerebral haemorrhages and bleeding in the gastrointestinal tract, and there may be thrombopaenia, alopecia medicomentosa or osteoporosis [Pschyrembel, Klinisches Wörterbuch [clinical dictionary], 257th edition, 1994, Walter de Gruyter Verlag, page 610, keyword “Heparin”; Rompp Lexikon Chemie, Version 1.5, 1998, Georg Thieme Verlag Stuttgart, keyword “Heparin”]. Low-molecular-weight heparins do have a lower probability of leading to the development of heparin-induced thrombocytopaenia; however, they can also only be administered subcutaneously. This also applies to fondaparinux, a synthetically produced selective factor Xa inhibitor having a long half-life.
A second class of anticoagulants are the vitamin K antagonists. These include, for example, 1,3-indanediones and in particular compounds such as warfarin, phenprocoumon, dicumarol and other coumarin derivatives which non-selectively inhibit the synthesis of various products of certain vitamin K-dependent coagulation factors in the liver. Owing to the mechanism of action, the onset of action is only very slow (latency to the onset of action 36 to 48 hours). The compounds can be administered orally; however, owing to the high risk of bleeding and the narrow therapeutic index complicated individual adjustment and monitoring of the patient are required [J. Hirsh, J. Dalen, D. R. Anderson et al., “Oral anticoagulants: Mechanism of action, clinical effectiveness, and optimal therapeutic range” Chest 2001, 119, 8S-21S; J. Ansell, J. Hirsh, J. Dalen et al., “Managing oral anticoagulant therapy” Chest 2001, 119, 22S-38S; P. S. Wells, A. M. Holbrook N. R. Crowther et al., “Interactions of warfarin with drugs and food” Ann. Intern. Med. 1994, 121, 676-683]. In addition, other side-effects such as gastrointestinal problems, hair loss and skin necroses have been described.
More recent approaches for oral anticoagulants are in various phases of clinical evaluation or in clinical use, but they have also shown disadvantages, for example highly variable bioavailability, liver damage and bleeding complications.
For antithrombotic medicaments, the therapeutic width is of central importance: The interval between the therapeutically active dose for coagulation inhibition and the dose where bleeding may occur should be as large as possible so that maximum therapeutic activity is achieved at a minimum risk profile.
In various in vivo models with, for example, antibodies as factor XIa inhibitors, but also in factor XIa knock-out models, the antithrombotic effect with small/no prolongation of bleeding time or extension of blood volume was confirmed. In clinical studies, elevated factor XIa concentrations were associated with an increased event rate. However, factor XI deficiency (haemophilia C), in contrast to factor VIIIa or factor IXa (haemophilia A and B, respectively), did not lead to spontaneous bleeding and was only noticed during surgical interventions and traumata. Instead, protection against certain thromboembolic events was found.
In the event of hyperfibrinolytic states, there is inadequate wound closure, which causes severe, sometimes life-threatening, bleeding. This bleeding can be stopped by the inhibition of fibrinolysis with antifibrinolytics, by which plasmin activity is reduced. Corresponding effects with the plasminogen inhibitor tranexamic acid have been shown in various clinical studies.
It is therefore an object of the present invention to provide novel compounds for treatment and/or prophylaxis of cardiovascular disorders and/or severe perioperative blood loss in man and animals, said compounds having a wide therapeutic range.
WO89/11852 describes, inter alia, substituted phenylalanine derivatives for treatment of pancreatitis, and WO 2007/07016 describes substituted thiophene derivatives as factor XIa inhibitors.
The invention provides compounds of the formula
in which
R1 represents a group of the formula
where # is the point of attachment to the nitrogen atom,
R5 represents 5-membered heteroaryl,
Compounds according to the invention are the compounds of the formula (I) and the salts, solvates and solvates of the salts thereof, and also the compounds encompassed by formula (I) and specified hereinafter as working example(s), and the salts, solvates and solvates of the salts thereof, to the extent that the compounds encompassed by formula (I) and specified hereinafter are not already salts, solvates and solvates of the salts.
The compounds of the invention may, depending on their structure, exist in different stereoisomeric forms, i.e. in the form of configurational isomers or else, if appropriate, of conformational isomers (enantiomers and/or diastereomers, including those in the case of atropisomers). The present invention therefore encompasses the enantiomers and diastereomers, and the respective mixtures thereof. The stereoisomerically uniform constituents can be isolated from such mixtures of enantiomers and/or diastereomers in a known manner; chromatography processes are preferably used for this, especially HPLC chromatography on an achiral or chiral phase.
If the compounds of the invention can occur in tautomeric forms, the present invention encompasses all the tautomeric forms.
The present invention also encompasses all suitable isotopic variants of the compounds of the invention. An isotopic variant of a compound of the invention is understood here to mean a compound in which at least one atom within the compound of the invention has been exchanged for another atom of the same atomic number, but with a different atomic mass from the atomic mass which usually or predominantly occurs in nature. Examples of isotopes which can be incorporated into a compound of the invention are those of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine, chlorine, bromine and iodine, such as 2H (deuterium), 3H (tritium), 13C, 14C, 15N, 17O, 18O, 32P, 33P, 33S, 34S, 35S, 36S, 18F, 36Cl, 82Br, 123I, 124I, 129I, an 131I. Particular isotopic variants of a compound of the invention, especially those in which one or more radioactive isotopes have been incorporated, may be beneficial, for example, for the examination of the mechanism of action or of the active ingredient distribution in the body; due to comparatively easy preparability and detectability, especially compounds labelled with 3H or 14C isotopes are suitable for this purpose. In addition, the incorporation of isotopes, for example of deuterium, may lead to particular therapeutic benefits as a consequence of greater metabolic stability of the compound, for example an extension of the half-life in the body or a reduction in the active dose required; such modifications of the compounds of the invention may therefore in some cases also constitute a preferred embodiment of the present invention. Isotopic variants of the compounds of the invention can be prepared by the processes known to those skilled in the art, for example by the methods described further down and the procedures described in the working examples, by using corresponding isotopic modifications of the respective reagents and/or starting compounds.
Preferred salts in the context of the present invention are physiologically acceptable salts of the compounds according to the invention. However, the invention also encompasses salts which themselves are unsuitable for pharmaceutical applications but which can be used, for example, for the isolation or purification of the compounds according to the invention.
Physiologically acceptable salts of the compounds according to the invention include acid addition salts of mineral acids, carboxylic acids and sulphonic acids, for example salts of hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, methanesulphonic acid, ethanesulphonic acid, toluenesulphonic acid, benzenesulphonic acid, naphthalenedisulphonic acid, acetic acid, trifluoroacetic acid, propionic acid, lactic acid, tartaric acid, malic acid, citric acid, fumaric acid, maleic acid and benzoic acid.
Physiologically acceptable salts of the compounds according to the invention also include salts of conventional bases, by way of example and with preference alkali metal salts (e.g. sodium and potassium salts), alkaline earth metal salts (e.g. calcium and magnesium salts) and ammonium salts derived from ammonia or organic amines having 1 to 16 carbon atoms, by way of example and with preference ethylamine, diethylamine, triethylamine, ethyldiisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, dimethylaminoethanol, procaine, dibenzylamine, N-methylmorpholine, arginine, lysine, ethylenediamine, N-methylpiperidine and choline.
Solvates in the context of the invention are described as those forms of the compounds according to the invention which form a complex in the solid or liquid state by coordination with solvent molecules. Hydrates are a specific form of the solvates in which the coordination is with water.
The present invention additionally also encompasses prodrugs of the compounds of the invention. The term “prodrugs” encompasses compounds which for their part may be biologically active or inactive but are converted during their residence time in the body into compounds according to the invention (for example by metabolism or hydrolysis).
The two ways (A) and (B) of representing a 1,4-disubstituted cyclohexyl derivative shown below are equivalent to one another and identical, and in both cases describe a trans-1,4-disubstituted cyclohexyl derivative.
This applies especially to the structural element of tranexamamide, for example N-[(trans-4-{[(tert-butoxycarbonyl)amino]methyl}cyclohexyl)carbonyl and trans-4-(aminomethyl)-cyclohexyl]carbonyl}. In the present invention, representation (A) is used.
The three ways (C), (D) and (E) of representing tautomers of a triazole derivative shown below are equivalent to one another and identical and in all cases describe a 1,4-disubstituted triazole derivative.
This applies especially to the following structural elements: 1H-1,2,4-triazol-3-yl, 1H-1,2,4-triazol-5-yl, 4H-1,2,4-triazol-3-yl and 4H-1,2,4-triazol-5-yl. Y1 and Y2 here are different substituents.
The two ways (F) and (G) of representing tautomers of a tetrazole derivative shown below are equivalent to one another and identical and in all cases describe a tetrazole derivative.
This applies especially to the following structural elements: 1H-tetrazol-5-yl and 2H-tetrazol-5-yl. Y3 here is the remainder of the compound.
The compounds according to the invention of the formula
and all L-phenylalanine intermediates are described as the (S) configuration at the stereocentre marked with an * in the above formula, since L-phenylalanine derivatives are introduced into the synthesis as central units. In the preparation of the compounds according to the invention, the coupling of the L-phenylalanine intermediates with the amine H2N—R1 can result in partial epimerization at the stereocentre marked with an *. Thus, a mixture of the compounds according to the invention of (S) enantiomer and (R) enantiomer can arise. The main component is the (S) enantiomer depicted in each case. The mixtures of (S) enantiomer and (R) enantiomer can be separated into their enantiomers by methods known to those skilled in the art, for example by chromatography on a chiral phase.
The enantiomers can be separated either directly after the coupling of the L-phenylalanine intermediates with the amine H2N—R1 or at a later synthesis intermediate, or else the inventive compounds can be separated themselves. Preference is given to the separation of the enantiomers directly after the coupling of the L-phenylalanine intermediates with the amine H2N—R1.
In the context of the present invention, the term “treatment” or “treating” includes inhibition, retardation, checking, alleviating, attenuating, restricting, reducing, suppressing, repelling or healing of a disease, a condition, a disorder, an injury or a health problem, or the development, the course or the progression of such states and/or the symptoms of such states. The term “therapy” is used here synonymously with the term “treatment”.
The terms “prevention”, “prophylaxis” and “preclusion” are used synonymously in the context of the present invention and refer to the avoidance or reduction of the risk of contracting, experiencing, suffering from or having a disease, a condition, a disorder, an injury or a health problem, or a development or advancement of such states and/or the symptoms of such states.
The treatment or prevention of a disease, a condition, a disorder, an injury or a health problem may be partial or complete.
In the context of the present invention, unless specified otherwise, the substituents are defined as follows:
Alkyl represents a straight-chain or branched alkyl radical having 1 to 4 carbon atoms, preferably 1 to 3 carbon atoms, by way of example and with preference methyl, ethyl, n-propyl, isopropyl, 2-methylprop-1-yl, n-butyl and tert-butyl.
Alkoxy represents a straight-chain or branched alkoxy radical having 1 to 4 carbon atoms, preferably 1 to 3 carbon atoms, by way of example and with preference methoxy, ethoxy, n-propoxy, isopropoxy, 2-methylprop-1-oxy, n-butoxy and tert-butoxy.
Alkylamino represents an amino group having one or two independently selected, identical or different, straight-chain or branched alkyl radicals each having 1 to 3 carbon atoms, for example and with preference methylamino, ethylamino, n-propylamino, isopropylamino, N,N-dimethylamino, N,N-diethylamino, N-ethyl-N-methylamino, N-methyl-N-n-propylamino, N-isopropyl-N-n-propylamino and N,N-diisopropylamino. C1-C3-Alkylamino represents, for example, a monoalkylamino radical having 1 to 3 carbon atoms or a dialkylamino radical having 1 to 3 carbon atoms in each alkyl radical.
Alkoxycarbonyl represents a straight-chain or branched alkoxy radical attached via a carbonyl group and having 1 to 4 carbon atoms, preferably 1 to 3 carbon atoms, for example and with preference methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, isopropoxycarbonyl, n-butoxycarbonyl and tert-butoxycarbonyl.
Alkylaminocarbonyl represents an amino group having one or two independently selected, identical or different, straight-chain or branched alkyl substituents each having 1 to 3 carbon atoms, attached via a carbonyl group, for example and with preference methylaminocarbonyl, ethylaminocarbonyl, n-propylaminocarbonyl, isopropylaminocarbonyl, N,N-dimethylaminocarbonyl, N,N-diethylaminocarbonyl, N-ethyl-N-methylaminocarbonyl, N-methyl-N-n-propylaminocarbonyl, N-isopropyl-N-n-propylaminocarbonyl and N,N-diisopropylaminocarbonyl. C1-C3-Alkylaminocarbonyl represents, for example, a monoalkylaminocarbonyl radical having 1 to 3 carbon atoms or a dialkylaminocarbonyl radical having 1 to 3 carbon atoms in each alkyl substituent.
Cycloalkyl represents a monocyclic cycloalkyl group having 3 to 6 carbon atoms, preferred examples of cycloalkyl being cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
Heterocyclyl attached via a nitrogen atom in the definition of the R4 radical represents a saturated or partly unsaturated monocyclic radical attached via a nitrogen atom and having 5 or 6 ring atoms and up to 3 heteroatoms and/or hetero groups, preferably 1 or 2 heteroatoms and/or hetero groups, from the group consisting of S, O, N, SO and SO2, where a nitrogen atom may also form an N-oxide, for example and with preference pyrrolidinyl, morpholinyl, thiomorpholinyl, piperidinyl and piperazinyl, particularly preferably morpholinyl and piperazinyl.
4- to 8-membered heterocyclyl attached via a carbon atom in the definition of the radicals R10 and R12 represents a saturated or partially unsaturated monocyclic or bicyclic radical which is attached via a carbon atom and which has 4 to 8 ring atoms, preferably 5 or 6 ring atoms, and up to 3 heteroatoms and/or hetero groups, preferably 1 or 2 heteroatoms and/or hetero groups from the series S, O, N, SO and SO2, where a nitrogen atom may also form an N-oxide, for example and with preference azetidinyl, pyrrolidinyl, piperidinyl, tetrahydropranyl, 3-azabicyclo[3.1.0]hex-6-yl, 8-azabicyclo[3.2.1]oct-3-yl and azepanyl, particularly preferably piperidinyl.
4- to 7-membered heterocycle in the definition of the R10 and R11 radicals and the radicals R12 and R13 represents a saturated or partially unsaturated, monocyclic or bicyclic radical having 4 to 7 ring atoms, preferably 5 or 6 ring atoms, and up to 3 heteroatoms and/or hetero groups, preferably 1 or 2 heteroatoms and/or hetero groups, from the group of S, O, N, SO and SO2, where one nitrogen atom may also form an N-oxide, for example and with preference azetidinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, piperidinyl, piperazinyl, 3-azabicyclo[3.1.0]hex-6-yl, 8-azabicyclo[3.2.1]oct-3-yl and azepanyl, particularly preferably morpholinyl, piperidinyl and piperazinyl.
5-membered heteroaryl in the definition of the R5 radical is an aromatic monocyclic radical having 5 ring atoms and up to 4 heteroatoms and/or hetero groups from the group of S, O, N, SO and SO2, where one nitrogen atom may also form an N-oxide, for example and with preference thienyl, furyl, pyrrolyl, thiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, pyrazolyl, imidazolyl, triazolyl and tetrazolyl, more preferably triazolyl and tetrazolyl, most preferably tetrazolyl.
5-membered heterocycle in the definition of the R7 and R8 radicals represents a saturated, partially unsaturated or aromatic monocyclic radical having 5 ring atoms and up to 2 heteroatoms and/or hetero groups from the group of S, O, N, SO and SO2, where one nitrogen atom may also form an N-oxide. This 5-membered heterocycle together with the phenyl ring to which it is attached represents, for example and with preference, 2,3-dihydro-1-benzothiophen-5-yl, 1,3-dihydro-2-benzothiophen-5-yl, 2,3-dihydro-1-benzofuran-5-yl, 1,3-dihydro-2-benzofuran-5-yl, indolin-5-yl, isoindolin-5-yl, 2,3-dihydro-1H-indazol-5-yl, 2,3-dihydro-1H-benzimidazol-5-yl, 1,3-dihydro-2,1-benzoxazol-5-yl, 2,3-dihydro-1,3-benzoxazol-5-yl, 1,3-dihydro-2, 1-benzothiazol-5-yl, 2,3-dihydro-1,3-benzothiazol-5-yl, 1H-benzimidazol-5-yl, 1H-indazol-5-yl, 1,2-benzoxazol-5-yl, indol-5-yl, isoindol-5-yl, benzofuran-5-yl, benzothiophen-5-yl, 2,3-dihydro-1-benzothiophen-6-yl, 1,3-dihydro-2-benzothiophen-6-yl, 2,3-dihydro-1-benzofuran-6-yl, 1,3-dihydro-2-benzofuran-6-yl, indolin-6-yl, isoindolin-6-yl, 2,3-dihydro-1H-indazol-6-yl, 2,3-dihydro-1H-benzimidazol-6-yl, 1,3-dihydro-2,1-benzoxazol-6-yl, 2,3-dihydro-1,3-benzoxazol-6-yl, 1,3-dihydro-2, 1-benzothiazol-6-yl, 2,3-dihydro-1,3-benzothiazol-6-yl, 1H-benzimidazol-6-yl, 1H-indazol-6-yl, 1,2-benzoxazol-6-yl, indol-6-yl, isoindol-6-yl, benzofuran-6-yl and benzothiophen-6-yl, particularly preferably 2,3-dihydro-1H-indazol-6-yl and 1H-benzimidazol-6-yl.
5- to 7-membered heterocyclyl in the definition of the radical R15 represents a saturated or partially unsaturated, monocyclic or bicyclic radical having 5 to 7 ring atoms, preferably 5 or 6 ring atoms, and up to 3 heteroatoms and/or hetero groups, preferably 1 or 2 heteroatoms and/or hetero groups, from the group consisting of S, O, N, SO and SO2, where one nitrogen atom may also form an N-oxide, for example and with preference pyrrolidinyl, morpholinyl, thiomorpholinyl, piperidinyl, piperazinyl, tetrahydropyranyl, 3-azabicyclo[3.1.0]hex-6-yl, 8-azabicyclo[3.2.1]oct-3-yl and azepanyl, particularly preferably piperidinyl and tetrahydropyranyl.
In the formulae of the group which may represent R1, the end point of the line marked by # in each case does not represent a carbon atom or a CH2 group, but is part of the bond to the atom to which R1 is attached.
Preference is given to compounds of the formula (I) in which
R1 represents a group of the formula
Preference is also given to compounds of the formula (I) in which
R1 represents a group of the formula
R2 represents hydrogen
R3′ represents hydrogen,
R3b represents hydrogen,
R4 represents amino, hydroxymethyl, —S(O)2NR10R11, —C(O)NR12R13 or —NR14(CO)R15,
and the salts thereof, the solvates thereof and the solvates of the salts thereof.
Preference is also given to compounds of the formula (I) in which
R3 represents a group of the formula
R2 represents hydrogen
R3a represents hydrogen,
R3b represents hydrogen,
R4 represents amino, hydroxymethyl or —C(O)NR12R13,
and the salts thereof, the solvates thereof and the solvates of the salts thereof.
Preference is also given to compounds of the formula (I) in which
R1 represents a group of the formula
R2 represents hydrogen
R3a represents hydrogen,
R3b represents hydrogen,
R4 represents amino, hydroxymethyl or —C(O)NR12R13,
and the salts thereof, the solvates thereof and the solvates of the salts thereof.
Preference is also given to compounds of the formula (I) in which
R1 represents a group of the formula
Preference is also given to compounds of the formula (I) in which R2 represents hydrogen.
Preference is also given to compounds of the formula (I) in which R3a represents hydrogen.
Preference is also given to compounds of the formula (I) in which R3b represents hydrogen.
Preference is also given to compounds of the formula (I) in which
Preference is also given to compounds of the formula (I) in which
R4 represents amino, hydroxymethyl or —C(O)NR12R13,
Irrespective of the particular combinations of the radicals specified, the individual radical definitions specified in the particular combinations or preferred combinations of radicals are also replaced as desired by radical definitions from other combinations.
Very particular preference is given to combinations of two or more of the abovementioned preferred ranges.
The invention further provides a process for preparing the compounds of the formula (I), or the salts thereof, solvates thereof or the solvates of the salts thereof, wherein the compounds of the formula
in which
R1, R2, R3a, R3b and R4 have the meaning given above, are reacted with an acid.
The reaction is generally effected in inert solvents, preferably within a temperature range from room temperature to 60° C. at standard pressure.
Inert solvents are, for example, halogenated hydrocarbons such as dichloromethane, trichloromethane, tetrachloromethane or 1,2-dichloroethane, or ethers such as tetrahydrofuran or dioxane, preference being given to dioxane.
Acids are, for example, trifluoroacetic acid or hydrogen chloride in dioxane, preference being given to hydrogen chloride in dioxane.
The compounds of the formula (II) are known or can be prepared by
[A] reacting compounds of the formula
in which
R1 and R2 have the meaning given above, and
X1 represents bromine or iodine,
with compounds of the formula
in which
R3a, R3b and R4 have the meaning given above and
Q1 represents B(OH)2, a boronic ester, preferably pinacol boronate, or —BF3−K+, under Suzuki coupling conditions,
or
[B] reacting compounds of the formula
in which
R1 and R2 have the meaning given above, and
Q2 represents B(OH)2, a boronic ester, preferably pinacol boronate, or —BF3−K+, with compounds of the formula
in which
R1a, R3b and R4 have the meaning given above and
X2 represents bromine or iodine,
under Suzuki coupling conditions,
or
[C] reacting compounds of the formula
in which
R2, R3a, R3b and R4 have the meaning given above,
with compounds of the formula
H2N—R1 (VIII),
in which
R1 has the meaning given above,
in the presence of a dehydrating reagent.
The reaction in process [A] is generally effected in inert solvents, in the presence of a catalyst, optionally in the presence of an additional reagent, optionally in a microwave, preferably within a temperature range from room temperature to 150° C. at standard pressure to 3 bar.
Catalysts are, for example, palladium catalysts customary for Suzuki reaction conditions, preference being given to catalysts such as dichlorobis(triphenylphosphine)palladium, tetrakistriphenylphosphinepalladium(0), palladium(II) acetate/triscyclohexylphosphine, tris(dibenzylideneacetone)dipalladium, bis(diphenylphosphaneferroc enyl)palladium (II) chloride, 1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene(1,4-naphtho quinone)palladium dimer, allyl(chloro)(1,3-dimesityl-1,3-dihydro-2H-imidazol-2-ylidene)palladium, palladium(II) acetate/dicyclohexyl(2′,4′,6′-triisopropylbiphenyl-2-yl)phosphine, [1, 1-bis(diphenylphosphino)ferrocene]palladium(II) chloride monodichloromethane adduct or XPhos precatalyst [(2′-aminobiphenyl-2-yl)(chloro)palladium dicyclohexyl(2′,4′,6′-triisopropylbiphenyl-2-yl)phosphane (1:1)], preference being given to tetrakistriphenylphosphinepalladium(0), [1,1-bis-(diphenylphosphino)ferrocene]palladium(II) chloride monodichloromethane adduct or XPhos precatalyst [(2′-aminobiphenyl-2-yl)(chloro)palladium dicyclohexyl(2′,4′,6′-triisopropylbiphenyl-2-yl)phosphane (1:1)].
Additional reagents are, for example, potassium acetate, caesium carbonate, potassium carbonate or sodium carbonate, potassium tert-butoxide, caesium fluoride or potassium phosphate, which may be present in aqueous solution; preferred additional reagents are those such as potassium acetate or a mixture of potassium acetate and sodium carbonate.
Inert solvents are, for example, ethers such as dioxane, tetrahydrofuran or 1,2-dimethoxyethane, hydrocarbons such as benzene, xylene or toluene, or carboxamides such as dimethylformamide or dimethylacetamide, alkyl sulphoxides such as dimethyl sulphoxide, or N-methylpyrrolidone or acetonitrile, or mixtures of the solvents with alcohols such as methanol or ethanol and/or water, preference being given to toluene, dimethylformamide or dimethyl sulphoxide.
The compounds of the formula (IV) are known, can be synthesized from the corresponding starting compounds by known processes or can be prepared analogously to the processes described in the Examples section.
The reaction in process [B] is effected as described for process [A].
The compounds of the formula (VI) are known, can be synthesized from the corresponding starting compounds by known processes or can be prepared analogously to the processes described in the Examples section.
The reaction in process [C] is generally effected in inert solvents, optionally in the presence of a base, preferably within a temperature range from 0° C. to the reflux of the solvents at standard pressure.
Suitable dehydrating agents here are, for example, carbodiimides such as N,N′-diethyl-, N,N′-dipropyl-, N,N′-diisopropyl-, N,N′-dicyclohexylcarbodiimide, N-(3-dimethylaminoisopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) (optionally in the presence of pentafluorophenol (PFP)), N-cyclohexylcarbodiimide-N′-propyloxymethyl-polystyrene (PS-carbodiimide) or carbonyl compounds such as carbonyldiimidazole, or 1,2-oxazolium compounds such as 2-ethyl-5-phenyl-1,2-oxazolium 3-sulphate or 2-tert-butyl-5-methylisoxazolium perchlorate, or acylamino compounds such as 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, or propanephosphonic anhydride, or isobutyl chloroformate, or bis-(2-oxo-3-oxazolidinyl)phosphoryl chloride or benzotriazolyloxytri(dimethylamino)phosphonium hexafluorophosphate, or O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU), 2-(2-oxo-1-(2H)-pyri dyl)-1, 1,3,3-tetramethyluronium tetrafluoroborate (TPTU), (benzotriazol-1-yloxy)bisdimethylaminomethylium fluoroborate (TBTU) or O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU), or 1-hydroxybenzotriazole (HOBt), or benzotriazol-1-yloxytris(dimethyl amino)phosphonium hexafluorophosphate (BOP), or ethyl cyano(hydroxyimino)acetate (Oxyma), or (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholinocarbenium hexafluorophosphate (COMU), or N-[(dimethylamino) (3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)methylidene]-N-methylmethanaminium hexafluorophosphate, or 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide (T3P), or mixtures of these, with preference being given to N-[(dimethylamino)(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)methylidene]-N-methylmethanaminium hexafluorophosphate or 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide (T3P).
Bases are, for example, alkali metal carbonates such as sodium carbonate or potassium carbonate, or sodium bicarbonate or potassium bicarbonate, or organic bases such as trialkylamines, for example triethylamine, N-methylmorpholine, N-methylpiperidine, 4-dimethylaminopyridine or diisopropylethylamine; preference is given to diisopropylethylamine
Inert solvents are, for example, halogenated hydrocarbons such as dichloromethane or trichloromethane, hydrocarbons such as benzene, or other solvents such as nitromethane, tetrahydrofuran, dioxane, dimethylformamide, dimethyl sulphoxide, acetonitrile or pyridine, or mixtures of the solvents, preference being given to tetrahydrofuran or dimethylformamide or a mixture of dimethylformamide and pyridine.
The compounds of the formula (VIII) are known, can be synthesized from the corresponding starting compounds by known processes or can be prepared analogously to the processes described in the Examples section.
The compounds of the formula (III) are known or can be prepared by reacting compounds of the formula
in which
R2 has the meaning given above and
X1 represents bromine or iodine,
with compounds of the formula (VIII) in the presence of a dehydrating reagent.
The reaction is carried out as described for process [C].
The compounds of the formula (IX) are known, can be synthesized from the corresponding starting compounds by known processes or can be prepared analogously to the processes described in the Examples section.
The compounds of the formula (V) are known or can be prepared by reacting compounds of the formula (III) with 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi-1,3,2-dioxaborolane.
The reaction is generally effected in inert solvents, in the presence of a catalyst, optionally in the presence of an additional reagent, optionally in a microwave, preferably within a temperature range from room temperature to 150° C. at standard pressure to 3 bar. Hydrolysis in an acidic medium affords the corresponding boronic acids. Workup with potassium dihydrogenfluoride solution (KHF2 solution) affords the corresponding trifluoroborates.
Catalysts are, for example, palladium catalysts customary for the borylation of aryl halides, preference being given to catalysts such as dichlorobis(triphenylphosphine)palladium, tetrakistriphenylphosphinepalladium(0), palladium(II) acetate/triscyclohexylphosphine, tris(dibenzylideneacetone)dipalladium, bis(diphenylphosphineferrocenyl)palladium(II) chloride, 1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene(1,4-naphthoquinone)palladium dimer, allyl(chloro)(1,3-dimesityl-1,3-dihydro-2H-imidazol-2-ylidene)palladium, palladium(II) acetate/dicyclohexyl(2′,4′,6′-triisopropylbiphenyl-2-yl)phosphine, [1,1-bis(diphenylphosphino)ferrocene]palladium(II) chloride monodichloromethane adduct or XPhos precatalyst [(2′-aminobiphenyl-2-yl)(chloro)palladium dicyclohexyl(2′,4′,6′-triisopropylbiphenyl-2-yl)phosphane (1:1)], preference being given to tetrakistriphenylphosphinepalladium(0) and [1,1-bis(diphenylphosphino)ferrocene]palladium(II) chloride.
Additional reagents are, for example, potassium acetate, caesium carbonate, potassium carbonate or sodium carbonate, potassium tert-butoxide or sodium tert-butoxide, caesium fluoride, potassium phosphate or potassium phenoxide, preference being given to potassium acetate.
Inert solvents are, for example, ethers such as dioxane, tetrahydrofuran or 1,2-dimethoxyethane, hydrocarbons such as benzene, xylene or toluene, or carboxamides such as dimethylformamide or dimethylacetamide, alkyl sulphoxides such as dimethyl sulphoxide, or N-methylpyrrolidone or acetonitrile, preference being given to dioxane, dimethylformamide or dimethyl sulphoxide.
Literature: K. L. Billingslay, T. E. Barde, S. L Buchwald, Angew. Chem. 2007, 119, 5455 or T. Graening, Nachrichten aus der Chemie, January 2009, 57, 34.
The compounds of the formula (VII) are known or can be prepared by reacting compounds of the formula (IX) with compounds of the formula (IV) under Suzuki coupling conditions.
The reaction is carried out as described for process [A].
The preparation of the starting compounds and of the compounds of the formula (I) can be illustrated by the synthesis scheme below.
The compounds according to the invention have an unforeseeable useful pharmacological activity spectrum and good pharmacokinetic behaviour. They are compounds that influence the proteolytic activity of the serine proteases FXIa and kallikrein, and possibly plasmin. The inventive compounds inhibit the enzymatic cleavage of substrates that assume a major role in the activation of the blood coagulation cascade and platelet aggregation. If the inventive compounds inhibit plasmin activity, the result is inhibition of fibrinolysis.
They are therefore suitable for use as medicaments for treatment and/or prophylaxis of diseases in man and animals.
The present invention further provides for the use of the compounds according to the invention for the treatment and/or prophylaxis of disorders, in particular cardiovascular disorders, preferably thrombotic or thromboembolic disorders and/or thrombotic or thromboembolic complications.
“Thromboembolic disorders” in the sense of the present invention include in particular disorders such as acute coronary syndrome (ACS), ST-segment elevation myocardial infarction (STEMI) and non-ST-segment elevation myocardial infarction (non-STEMI), stable angina pectoris, unstable angina pectoris, reocclusions and restenoses after coronary interventions such as angioplasty, stent implantation or aortocoronary bypass, peripheral arterial occlusion diseases, pulmonary embolisms, venous thromboses, especially in deep leg veins and renal veins, transitory ischaemic attacks and also thrombotic and thromboembolic stroke.
The inventive compounds are therefore also suitable for the prevention and treatment of cardiogenic thromboembolisms, for example brain ischaemias, stroke and systemic thromboembolisms and ischaemias, in patients with acute, intermittent or persistent cardial arrhythmias, for example atrial fibrillation, and those undergoing cardioversion, and also in patients with heart valve disorders or with artificial heart valves.
In addition, the inventive compounds are suitable for the treatment and prevention of disseminated intravascular coagulation (DIC) which may occur in connection with sepsis inter alia, but also owing to surgical interventions, neoplastic disorders, burns or other injuries and may lead to severe organ damage through microthromboses.
Thromboembolic complications are also encountered in microangiopathic haemolytic anaemias, extracorporeal circulatory systems, such as haemodialysis, and also prosthetic heart valves.
In addition, the inventive compounds are also used for influencing wound healing, for the prophylaxis and/or treatment of atherosclerotic vascular disorders and inflammatory disorders, such as rheumatic disorders of the locomotive system, coronary heart diseases, of heart failure, of hypertension, of inflammatory disorders, for example asthma, inflammatory pulmonary disorders, glomerulonephritis and inflammatory intestinal disorders, for example Crohn's disease or ulcerative colitis or acute renal failure, and additionally likewise for the prophylaxis and/or treatment of dementia disorders, for example Alzheimer's disease. In addition, the inventive compounds can be used for inhibiting tumour growth and the formation of metastases, for microangiopathies, age-related macular degeneration, diabetic retinopathy, diabetic nephropathy and other microvascular disorders, and also for the prevention and treatment of thromboembolic complications, for example venous thromboembolisms, for tumour patients, especially those undergoing major surgery or chemo- or radiotherapy.
In addition, the inventive compounds are also suitable for the prophylaxis and/or treatment of pulmonary hypertension.
The term “pulmonary hypertension” includes certain forms of pulmonary hypertension, as determined, for example, by the World Health Organization (WHO). Examples include pulmonary arterial hypertension, pulmonary hypertension associated with disorders of the left heart, pulmonary hypertension associated with pulmonary disorders and/or hypoxia and pulmonary hypertension owing to chronic thromboembolisms (CTEPH).
“Pulmonary arterial hypertension” includes idiopathic pulmonary arterial hypertension (IPAH, formerly also referred to as primary pulmonary hypertension), familial pulmonary arterial hypertension (FPAH) and associated pulmonary-arterial hypertension (APAH), which is associated with collagenoses, congenital systemic-pulmonary shunt vitia, portal hypertension, HIV infections, the ingestion of certain drugs and medicaments, with other disorders (thyroid disorders, glycogen storage disorders, Morbus Gaucher, hereditary teleangiectasia, haemoglobinopathies, myeloproliferative disorders, splenectomy), with disorders having a significant venous/capillary contribution, such as pulmonary-venoocclusive disorder and pulmonary-capillary haemangiomatosis, and also persisting pulmonary hypertension of neonatants.
Pulmonary hypertension associated with disorders of the left heart includes a diseased left atrium or ventricle and mitral or aorta valve defects.
Pulmonary hypertension associated with pulmonary disorders and/or hypoxia includes chronic obstructive pulmonary disorders, interstitial pulmonary disorder, sleep apnoea syndrome, alveolar hypoventilation, chronic high-altitude sickness and inherent defects.
Pulmonary hypertension owing to chronic thromboembolisms (CTEPH) comprises the thromboembolic occlusion of proximal pulmonary arteries, the thromboembolic occlusion of distal pulmonary arteries and non-thrombotic pulmonary embolisms (tumour, parasites, foreign bodies).
The present invention further provides for the use of the inventive compounds for production of medicaments for the treatment and/or prophylaxis of pulmonary hypertension associated with sarcoidosis, histiocytosis X and lymphangiomatosis.
In addition, the inventive substances may also be useful for the treatment of pulmonary and hepatic fibroses.
In addition, the inventive compounds may also be suitable for treatment and/or prophylaxis of disseminated intravascular coagulation in the context of an infectious disease, and/or of systemic inflammatory syndrome (SIRS), septic organ dysfunction, septic organ failure and multiorgan failure, acute respiratory distress syndrome (ARDS), acute lung injury (ALI), septic shock and/or septic organ failure.
In the course of an infection, there may be a generalized activation of the coagulation system (disseminated intravascular coagulation or consumption coagulopathy, hereinbelow referred to as “DIC”) with microthrombosis in various organs and secondary haemorrhagic complications. Moreover, there may be endothelial damage with increased permeability of the vessels and seeping of fluid and proteins into the extravasal lumen. As the infection progresses, there may be failure of an organ (for example kidney failure, liver failure, respiratory failure, central-nervous deficits and cardiovascular failure) or multiorgan failure.
In the case of DIC, there is a massive activation of the coagulation system at the surface of damaged endothelial cells, the surfaces of foreign bodies or injured extravascular tissue. As a consequence, there is coagulation in small vessels of various organs with hypoxia and subsequent organ dysfunction. This can be prevented by the inventive compounds. A secondary effect is the consumption of coagulation factors (for example factor X, prothrombin and fibrinogen) and platelets, which reduces the coagulability of the blood and may result in heavy bleeding.
In addition, the inventive compounds are also useful for the prophylaxis and/or treatment of hyperfibrinolysis. The prophylaxis and/or treatment may reduce or eliminate severe perioperative blood loss. Severe bleeding occurs in major operations, for example coronary artery bypass surgery, transplants or hysterectomy, and in the event of trauma, in the event of haemorrhagic shock or in the event of postpartum haemorrhage. In the aforementioned indications, there may be perioperative use of extracorporeal circulation systems or filter systems, for example heart and lung machines, haemofiltration, haemodialysis, extracorporeal membrane oxygenation or a ventricular support system, for example artificial heart. This additionally requires anticoagulation, for which the inventive compounds can also be used.
The inventive compounds are also suitable for anticoagulation during kidney replacement procedures, for example in the case of continuous veno-venous haemofiltration or intermittent haemodialysis.
In addition, the compounds according to the invention can also be used for preventing coagulation ex vivo, for example for preserving blood and plasma products, for cleaning/pretreating catheters and other medical auxiliaries and instruments, for coating synthetic surfaces of medical auxiliaries and instruments used in vivo or ex vivo or for biological samples which could contain factor XIa.
The present invention further provides for the use of the compounds according to the invention for the treatment and/or prophylaxis of disorders, especially the disorders mentioned above.
The present invention further provides for the use of the compounds according to the invention for production of a medicament for the treatment and/or prophylaxis of disorders, especially the disorders mentioned above.
The present invention further provides a method for the treatment and/or prophylaxis of disorders, especially the disorders mentioned above, using a therapeutically effective amount of a compound according to the invention.
The present invention further provides the compounds according to the invention for use in a method for the treatment and/or prophylaxis of disorders, especially the disorders mentioned above, using a therapeutically effective amount of a compound according to the invention.
The present invention further provides medicaments comprising a compound according to the invention and one or more further active compounds.
The present invention further provides a method for preventing the coagulation of blood in vitro, especially in banked blood or biological samples which could contain factor XIa, which is characterized in that an anticoagulatory amount of the inventive compound is added.
The present invention further provides medicaments comprising a compound according to the invention and one or more further active compounds, in particular for the treatment and/or prophylaxis of the disorders mentioned above. Preferred examples of active compounds suitable for combinations include:
“Combinations” for the purpose of the invention mean not only dosage forms which contain all the components (so-called fixed combinations) and combination packs which contain the components separate from one another, but also components which are administered simultaneously or sequentially, provided that they are used for prophylaxis and/or treatment of the same disease. It is likewise possible to combine two or more active ingredients with one another, meaning that they are thus each in two-component or multicomponent combinations.
The compounds of the invention can act systemically and/or locally. For this purpose, they can be administered in a suitable manner, for example by the oral, parenteral, pulmonal, nasal, sublingual, lingual, buccal, rectal, dermal, transdermal, conjunctival or otic route, or as an implant or stent.
The compounds of the invention can be administered in administration forms suitable for these administration routes.
Suitable administration forms for oral administration are those which function according to the prior art and deliver the inventive compounds rapidly and/or in modified fashion, and which contain the inventive compounds in crystalline and/or amorphized and/or dissolved form, for example tablets (uncoated or coated tablets, for example having enteric coatings or coatings which are insoluble or dissolve with a delay, which control the release of the compound according to the invention), tablets which disintegrate rapidly in the mouth, or films/wafers, films/lyophilisates, capsules (for example hard or soft gelatin capsules), sugar-coated tablets, granules, pellets, powders, emulsions, suspensions, aerosols or solutions.
Parenteral administration can be accomplished with avoidance of a resorption step (for example by an intravenous, intraarterial, intracardiac, intraspinal or intralumbar route) or with inclusion of a resorption (for example by an intramuscular, subcutaneous, intracutaneous, percutaneous or intraperitoneal route). Administration forms suitable for parenteral administration include preparations for injection and infusion in the form of solutions, suspensions, emulsions, lyophilizates or sterile powders.
Parenteral administration is preferred.
Suitable administration forms for the other administration routes are, for example, pharmaceutical forms for inhalation (including powder inhalers, nebulizers), nasal drops, solutions or sprays; tablets for lingual, sublingual or buccal administration, films/wafers or capsules, suppositories, preparations for the ears or eyes, vaginal capsules, aqueous suspensions (lotions, shaking mixtures), lipophilic suspensions, ointments, creams, transdermal therapeutic systems (for example patches), milk, pastes, foams, dusting powders, implants or stents.
The compounds of the invention can be converted to the administration forms mentioned. This can be accomplished in a manner known per se by mixing with inert, nontoxic, pharmaceutically suitable excipients. These excipients include carriers (for example microcrystalline cellulose, lactose, mannitol), solvents (e.g. liquid polyethylene glycols), emulsifiers and dispersing or wetting agents (for example sodium dodecylsulphate, polyoxysorbitan oleate), binders (for example polyvinylpyrrolidone), synthetic and natural polymers (for example albumin), stabilizers (e.g. antioxidants, for example ascorbic acid), colourants (e.g. inorganic pigments, for example iron oxides) and flavour and/or odour correctants.
The present invention further provides medicaments comprising at least one inventive compound, preferably together with one or more inert nontoxic pharmaceutically suitable excipients, and the use thereof for the purposes mentioned above.
In the case of parenteral administration, it has generally been found to be advantageous to administer amounts of about 5 to 250 mg every 24 hours to achieve effective results. In the case of oral administration, the amount is about 5 to 500 mg every 24 hours.
In spite of this, it may be necessary, if appropriate, to deviate from the amounts specified, specifically depending on body weight, administration route, individual behaviour towards the active ingredient, type of formulation, and time or interval of administration.
Unless stated otherwise, the percentages in the tests and examples which follow are percentages by weight; parts are parts by weight. Solvent ratios, dilution ratios and concentration data for the liquid/liquid solutions are based in each case on volume. “w/v” means “weight/volume”. For example, “10% w/v” means: 100 ml of solution or suspension comprise 10 g of substance.
HPLC and LC/MS methods:
Method 1 (LC-MS):
Instrument: Waters ACQUITY SQD UPLC system; column: Waters Acquity UPLC HSS T3 1.8 g 50 mm×1 mm; mobile phase A: 1 l of water+0.25 ml of 99% strength formic acid, mobile phase B: 1 l of acetonitrile+0.25 ml of 99% strength formic acid; gradient: 0.0 min 90% A→1.2 min 5% A→2.0 min 5% A; oven: 50° C.; flow rate: 0.40 ml/min; UV detection: 210-400 nm.
Method 2 (LC-MS):
Instrument: Micromass Quattro Premier with Waters UPLC Acquity; column: Thermo Hypersil GOLD 1.9μ 50 mm×1 mm; mobile phase A: 1 l of water+0.5 ml of 50% strength formic acid, mobile phase B: 1 l of acetonitrile+0.5 ml of 50% strength formic acid; gradient: 0.0 min 97% A→0.5 min 97% A→3.2 min 5% A→4.0 min 5% A; oven: 50° C.; flow rate: 0.3 ml/min; UV detection: 210 nm.
Method 3 (LC-MS):
Instrument: Waters ACQUITY SQD UPLC system; column: Waters Acquity UPLC HSS T3 1.8μ 30 mm×2 mm; mobile phase A: 1 l of water+0.25 ml of 99% strength formic acid, mobile phase B: 1 l of acetonitrile+0.25 ml of 99% strength formic acid; gradient: 0.0 min 90% A→1.2 min 5% A→2.0 min 5% A oven: 50° C.; flow rate: 0.60 ml/min; UV detection: 208-400 nm.
Method 4 (LC-MS):
Instrument: Waters Acquity UPLC-MS SQD 3001; column: Acquity UPLC BEH C18 1.7μ 50 mm×2.1 mm; mobile phase A: water+0.1% formic acid, mobile phase B: acetonitrile; gradient: 0-1.6 min 1-99% B, 1.6-2.0 min 99% B; flow rate: 0.8 ml/min; temperature: 60° C.; injection: 2 μl; DAD scan: 210-400 nm; ELSD.
Method 5 (LC-MS):
Instrument: Waters Acquity UPLC-MS SQD 3001; column: Acquity UPLC BEH C18 1.7μ 50 mm×2.1 mm; mobile phase A: water+0.2% ammonia, mobile phase B: acetonitrile; gradient: 0-1.6 min 1-99% B, 1.6-2.0 min 99% B; flow rate: 0.8 ml/min; temperature: 60° C.; injection: 2 μl; DAD scan: 210-400 nm; ELSD.
Method 6 (HPLC):
System: Labomatic HD-3000 HPLC gradient pump, Labomatic Labocol Vario-2000 fraction collector; column: Chromatorex C-18 125 mm×30 mm, mobile phase A: 0.1% formic acid in water, mobile phase B: acetonitrile, gradient: A 95%/B 5%→A 55%/B 45%; flow rate: 150 ml/min; UV detection: 254 nm.
Method 7 (HPLC):
System: Labomatic HD-3000 HPLC gradient pump, Labomatic Labocol Vario-2000 fraction collector; column: Chromatorex C-18 125 mm×30 mm, mobile phase A: 0.1% formic acid in water, mobile phase B: acetonitrile; gradient: A 90%/B 10%→A 50%/B 50%; flow rate: 150 ml/min; UV detection: 254 nm.
Method 8 (HPLC):
System: Labomatic HD-3000 HPLC gradient pump, Labomatic Labocol Vario-2000 fraction collector; column: Chromatorex C-18 125 mm×30 mm, mobile phase A: 0.1% formic acid in water, mobile phase B: acetonitrile; gradient: A 85%/B 15%→A 45%/B 55%; flow rate: 150 ml/min; UV detection: 254 nm.
Method 9 (HPLC):
System: Labomatic HD-3000 HPLC gradient pump, Labomatic Labocol Vario-2000 fraction collector; column: Chromatorex C-18 125 mm×30 mm, mobile phase A: 0.1% formic acid in water, mobile phase B: acetonitrile; gradient: A 80%/B 20%→A 40%/B 60%; flow rate: 150 ml/min; UV detection: 254 nm.
Method 10 (HPLC):
Instrument: Waters autopurification system SQD; column: Waters XBridge C18 5 g 100 mm×30 mm; mobile phase A: water+0.1% formic acid (99%), mobile phase B: acetonitrile; gradient: 0-8.0 min 1-100% B, 8.0-10.0 min 100% B; flow rate 50.0 ml/min; temperature: RT; injection: 2500 μl; DAD scan: 210-400 nm.
Method 11 (HPLC):
Instrument: Waters autopurification system SQD; column: Waters XBridge C18 5 g 100 mm×30 mm; mobile phase A: water+0.2% ammonia (32%), mobile phase B: acetonitrile; gradient: 0-8.0 min 1-100% B, 8.0-10.0 min 100% B; flow rate 50.0 ml/min; temperature: RT; injection: 2500 μl; DAD scan: 210-400 nm.
Method 12 (LC-MS):
MS instrument: Waters (Micromass) QM; HPLC instrument: Agilent 1100 series; column: Agient ZORBAX Extend-C18 3.0 mm×50 mm 3.5 micron; mobile phase A: 1 l of water+0.01 mol of ammonium carbonate, mobile phase B: 1 l of acetonitrile; gradient: 0.0 min 98% A→0.2 min 98% A→3.0 min 5% A→4.5 min 5% A; oven: 40° C.; flow rate: 1.75 ml/min; UV detection: 210 nm.
Method 13 (LC-MS):
Instrument: Waters ACQUITY SQD UPLC system; column: Waters Acquity UPLC HSS T3 1.8μ 50 mm×1 mm; mobile phase A: 1 l of water+0.25 ml of 99% strength formic acid, mobile phase B: 1 l of acetonitrile+0.25 ml of 99% strength formic acid; gradient: 0.0 min 95% A→6.0 min 5% A→7.5 min 5% A; oven: 50° C.; flow rate: 0.35 ml/min; UV detection: 210-400 nm.
Method 14 (LC-MS):
MS instrument: Waters (Micromass) Quattro Micro; HPLC instrument: Agilent 1100 series; column: YMC-Triart C18 3μ 50 mm×3 mm; mobile phase A: 1 l of water+0.01 mol of ammonium carbonate, mobile phase B: 1 l of acetonitrile; gradient: 0.0 min 100% A→2.75 min 5% A→4.5 min 5% A; oven: 40° C.; flow rate: 1.25 ml/min; UV detection: 210 nm.
Method 15 (HPLC):
Column: Reprosil C18; 10 μm; 125 mm×30 mm; mobile phase A: water+0.01% trifluoroacetic acid, mobile phase B: methanol; gradient: 0.0-5.0 min 20% B→6.50 min 40% B→17.00 min 100% B→19.5-23.0 min 40% B; flow rate: 50 ml/min.
Microwave:
The microwave reactor used was an instrument of the Biotage™ Initiator type.
When compounds according to the invention are purified by preparative HPLC by the above-described methods in which the eluents contain additives, for example trifluoroacetic acid, formic acid or ammonia, the compounds according to the invention may be obtained in salt form, for example as trifluoroacetate, formate or ammonium salt, if the compounds according to the invention contain a sufficiently basic or acidic functionality. Such a salt can be converted to the corresponding free base or acid by various methods known to the person skilled in the art. Weaker salts can be converted to the corresponding chlorides by addition of a little hydrochloride.
In the case of the synthesis intermediates and working examples of the invention described hereinafter, any compound specified in the form of a salt of the corresponding base or acid is generally a salt of unknown exact stoichiometric composition, as obtained by the respective preparation and/or purification process. Unless specified in more detail, additions to names and structural formulae, such as “hydrochloride”, “trifluoroacetate”, “sodium salt” or “x HCl”, “x CF3COOH”, “x Na+” should not therefore be understood in a stoichiometric sense in the case of such salts, but have merely descriptive character with regard to the salt-forming components present therein.
This applies correspondingly if synthesis intermediates or working examples or salts thereof were obtained in the form of solvates, for example hydrates, of unknown stoichiometric composition (if they are of a defined type) by the preparation and/or purification processes described.
If the starting compounds and examples contain an L-phenylalanine derivative as the central unit, the corresponding stereocentre is described as the (S) configuration. In the absence of further information, there was no check in individual cases as to whether partial epimerization of the stereocentre took place in the coupling of the L-phenylalanine intermediates with the amine H2N—R1. Thus, a mixture of the inventive compounds of (S) enantiomer and (R) enantiomer may be present. The main component is the (S) enantiomer depicted in each case.
N,N-Diisopropylethylamine (381 ml, 2186 mmol) was added to a solution of methyl 4-bromo-L-phenylalaninate (250 g, 874 mmol) and trans-4-{[(tert-butoxycarbonyl)amino]methyl}cyclohexanecarboxylic acid (225 g, 874 mmol) in ethyl acetate (5012 ml). A 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide solution (50% in dimethylformamide, 766 ml, 1312 mmol) was added dropwise, and the suspension was then stirred at RT for 3 h. The reaction mixture was then stirred into water and extracted three times with ethyl acetate. The organic phase was washed with saturated aqueous sodium hydrogencarbonate solution, saturated aqueous ammonium chloride solution, and saturated aqueous sodium chloride solution. The solution was dried over sodium sulphate and the solvent was removed. This gave 420 g (97% of theory) of the title compound.
1H-NMR (400 MHz, DMSO-d6): δ=0.68-0.92 (m, 2H), 1.04-1.32 (m, 4H), 1.37 (s, 9H), 1.48-1.73 (m, 4H), 2.03 (m, 1H), 2.74 (m, 2H), 2.78-2.90 (m, 1H), 2.94-3.05 (m, 1H), 4.36-4.50 (m, 1H), 6.72-6.85 (m, 1H), 7.17 (d, 2H), 7.46 (d, 2H), 8.15 (d, 1H).
LC-MS (Method 1): Rt=1.14 min; MS (ESIpos): m/z=497 [M+H]+.
A solution of methyl 4-bromo-N-[(trans-4-{[(tert-butoxycarbonyl)amino]methyl}-cyclohexyl)carbonyl]-L-phenylalaninate in tetrahydrofuran (3000 ml) was admixed with a solution of lithium hydroxide (72 g, 3015 mmol) in water (600 ml). The suspension was stirred at RT for 16 h. The reaction mixture was acidified with 1N hydrochloric acid solution and admixed with ethyl acetate. The organic phase was washed with saturated aqueous sodium chloride solution and dried over sodium sulphate, and the solvent was removed. This gave 284 g (97% of theory) of the title compound.
1H-NMR (400 MHz, DMSO-d6): δ=0.71-0.90 (m, 2H), 1.22 (d, 4H), 1.37 (s, 9H), 1.45-1.73 (m, 5H), 2.03 (m, 1H), 2.67-2.88 (m, 3H), 2.95-3.09 (m, 1H), 4.38 (m, 1H), 6.77 (s, 1H), 7.17 (d, 2H), 7.46 (d, 2H), 7.99 (d, 1H), 12.65 (br. s, 1H).
LC-MS (Method 1): Rt=1.03 min; MS (ESIneg): m/z=481 [M−H]−.
N,N-Diisopropylethylamine (9.6 ml, 55 mmol) was added to a solution of 4-bromo-N-[(trans-4-{[(tert-butoxycarbonyl)amino]methyl}cyclohexyl)carbonyl]-L-phenylalanine (11 mg, 22 mmol) and 4-(1H-tetrazol-5-yl)aniline (4 g, 24 mmol) in DMF (161 ml). At 0° C., a 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide solution (50% in DMF, 16.9 g, 27 mmol) was added dropwise, and the suspension was then stirred at RT for 16 h. The reaction mixture was stirred into ethyl acetate (13 000 ml) and extracted three times with water (1570 ml each time). The organic phase was dried with sodium sulphate and the solvent was removed. The crude product was stirred with acetonitrile and filtered off with suction. This gave 11.4 g (78% of theory) of the title compound.
1H-NMR (400 MHz, DMSO-d6): δ=0.67-0.90 (m, 2H), 1.24 (m, 4H), 1.37 (s, 9H), 1.51-1.74 (m, 4H), 2.02-2.17 (m, 1H), 2.71-2.79 (m, 2H), 2.79-2.89 (m, 1H), 2.99-3.06 (m, 1H), 3.06-3.16 (m, 1H), 3.51-3.67 (m, 1H), 4.55-4.74 (m, 1H), 6.01-6.02 (m, 1H), 6.69-6.84 (m, 1H), 7.21-7.32 (m, 2H), 7.43-7.55 (m, 2H), 7.64-7.76 (m, 2H), 7.88-7.99 (m, 2H), 8.03-8.14 (m, 1H), 10.25 (s, 1H).
LC-MS (Method 1): Rt=1.07 min; MS (ESIneg): m/z=624 [M−H]−.
0.6 ml (1.2 mmol) of a 2M sodium carbonate solution in water was added to a solution of 250 mg (0.40 mmol) of 4-bromo-N-alpha-[(trans-4-{[(tert-butoxycarbonyl)amino]methyl}cyclohexyl)carbonyl]-N-[4-(2H-tetrazol-5-yl)phenyl]-L-phenylalaninamide and 99 mg (0.60 mmol) of 2-(dihydroxyboranyl)benzoic acid in 2 ml DMF, and the mixture was degassed with argon for 5 min. 29.2 mg (0.04 mmol) of 1,1′-bis(diphenylphosphino)ferrocenepalladium(II) chloride were added and the mixture was stirred at 120° C. in a preheated oil bath for 30 min. Another 29.2 mg (0.04 mmol) of 1,1′-bis(diphenylphosphino)ferrocenepalladium(II) chloride were added and the mixture was stirred at 120° C. in a preheated oil bath for 16 h. The reaction solution was separated by preparative HPLC (mobile phase: gradient of acetonitrile/water with 0.1% trifluoroacetic acid). The product-containing fractions were combined and concentrated on a rotary evaporator. The residue was dried under high vacuum. 61 mg (22% of theory, 94% purity) of the title compound were obtained.
1H NMR (400 MHz, DMSO-d6) δ=0.68-0.92 (m, 2H), 1.04-1.42 (m, 12H), 1.68 (m, 4H), 1.96-2.19 (m, 1H), 2.75 (t, 2H), 2.82-2.99 (m, 1H), 2.99-3.15 (m, 1H), 4.53-4.85 (m, 1H), 6.67-6.86 (m, 1H), 7.15-7.22 (m, 1H), 7.23-7.37 (m, 4H), 7.39-7.73 (m, 3H), 7.82 (t, 2H), 7.92-8.04 (m, 2H), 8.09-8.22 (m, 1H), 10.42 (s, 1H), 12.36-13.03 (m, 1H), 16.55 (br. s, 1H).
LC-MS (Method 1): Rt=0.99 min; MS (ESIneg): m/z=666 [M−H]−.
100 mg (0.16 mmol) of 4-bromo-N-alpha-[(trans-4-{[(tert-butoxycarbonyl)amino]methyl}cyclohexyl)carbonyl]-N-[4-(2H-tetrazol-5-yl)phenyl]-L-phenylalaninamide, 87 mg (0.32 mmol) of 2-(morpholin-4-ylsulphonyl)phenylboronic acid and 18 mg (0.02 mmol) of tetrakis(triphenylphosphine)palladium(0) were stirred in 1.5 ml of 1,2-dimethoxyethane and 0.5 ml of ethanol. After the addition of 1.2 ml of 2N sodium carbonate solution, the mixture was stirred at 100° C. for 4 h and at RT for 7 days. The salts in the reaction mixture were filtered off. The filtrate was separated by preparative HPLC (mobile phase: gradient of acetonitrile/water with 0.1% trifluoroacetic acid). This gave 86 mg of a mixture of the title compound and the partially deprotected title compound, which was used directly in the next stage.
LC-MS (Method 1): Rt=1.06 min; MS (ESIneg): m/z=772 [M−H]−.
0.36 ml (0.72 mmol) of a 2M sodium carbonate solution in water was added to a solution of 150 mg (0.24 mmol) of 4-bromo-N-alpha-[(trans-4-{[(tert-butoxycarbonyl)amino]methyl}cyclohexyl)carbonyl]-N-[4-(2H-tetrazol-5-yl)phenyl]-L-phenylalaninamide and 84 mg (0.36 mmol) of [2-(morpholin-4-ylcarbonyl)phenyl]boronic acid in 2.5 ml DMF, and the mixture was degassed with argon for 5 min. 17.5 mg (0.02 mmol) of 1,1′-bis(diphenylphosphino)ferrocenepalladium(II) chloride were added and the mixture was stirred at 120° C. in a preheated oil bath for 30 min. The reaction solution was separated by means of preparative HPLC (eluent: methanol/water gradient, 0.01% trifluoroacetic acid). The product-containing fractions were combined and concentrated on a rotary evaporator. The residue was dried under high vacuum. 99 mg (52% of theory, 93% purity) of the title compound were obtained.
1H NMR (400 MHz, DMSO-d6) δ=0.68-0.91 (m, 2H), 1.36 (m, 14H), 1.54-1.80 (m, 4H), 2.02-2.16 (m, 1H), 2.75 (m, 2H), 2.82-3.00 (m, 2H), 3.02-3.25 (m, 3H), 3.26-3.63 (m, 3H), 4.63-4.78 (m, 1H), 6.70-6.87 (m, 1H), 7.26-7.56 (m, 9H), 7.83 (d, 2H), 7.99 (d, 2H), 8.11-8.23 (m, 1H), 10.40-10.56 (m, 1H).
LC-MS (Method 1): Rt=1.28 min; MS (ESIneg): m/z=929 [M−H]−.
0.06 ml (0.35 mmol) of N,N-diisopropylamine and 66 mg (0.18 mmol) of N-[(dimethylamino)(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)methylidene]-N-methylmethanaminium hexafluorophosphate were added to a solution of 78 mg (0.12 mmol) of 4′-[(2S)-2-{[(trans-4-{[(tert-butoxycarbonyl)amino]methyl}cyclohexyl)carbonyl]amino}-3-oxo-3-{[4-(2H-tetrazol-5-yl)phenyl]amino}propyl]biphenyl-2-carboxylic acid and 0.02 ml (0.23 mmol) of diethylamine in 1 ml of DMF, and the mixture was stirred at RT for 16 h. The contents of the flask was separated by preparative HPLC (mobile phase: gradient of acetonitrile/water with 0.1% trifluoroacetic acid). The product-containing fractions were combined, concentrated on a rotary evaporator, and the residue was dried under high vacuum. 31 mg (34% of theory, 93% purity) of the title compound were obtained.
1H NMR (400 MHz, DMSO-d6) δ=0.63 (m, 2H), 0.73-0.91 (m, 4H), 1.13-1.31 (m, 3H), 1.36 (s, 9H), 1.47-1.79 (m, 4H), 2.03-2.16 (m, 1H), 2.57-2.68 (m, 2H), 2.69-3.11 (m, 6H), 3.40 (m, 2H), 4.60-4.76 (m, 1H), 6.72-6.87 (m, 1H), 7.20-7.49 (m, 7H), 7.82 (d, 2H), 7.99 (d, 2H), 8.07-8.26 (m, 1H), 10.44 (s, 1H).
LC-MS (Method 1): Rt=1.06 min; MS (ESIpos): m/z=723 [M+H]+.
0.06 ml (0.35 mmol) of N,N-diisopropylamine and 67 mg (0.18 mmol) of N-[(dimethylamino)(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)methylidene]-N-methylmethanaminium hexafluorophosphate were added to a solution of 78 mg (0.12 mmol) of 4′-[(2S)-2-{[(trans-4-{[(tert-butoxycarbonyl)amino]methyl}cyclohexyl)carbonyl]amino}-3-oxo-3-{[4-(2H-tetrazol-5-yl)phenyl]amino}propyl]biphenyl-2-carboxylic acid and 0.02 ml (0.23 mmol) of piperidine in 1 ml of DMF, and the mixture was stirred at RT for 16 h. The contents of the flask was separated by preparative HPLC (mobile phase: gradient of acetonitrile/water with 0.1% trifluoroacetic acid). The product-containing fractions were combined, concentrated on a rotary evaporator, and the residue was dried under high vacuum. This gave 34 mg (86% of theory) of the title compound.
1H NMR (400 MHz, DMSO-d6) δ=0.50-0.65 (m, 1H), 0.72-0.93 (m, 2H), 0.99-1.40 (m, 18H), 1.48-1.79 (m, 4H), 2.00-2.18 (m, 1H), 2.75 (m, 4H), 3.01-3.16 (m, 1H), 3.18-3.28 (m, 1H), 3.34-3.41 (m, 2H), 4.57-4.75 (m, 1H), 6.71-6.88 (m, 1H), 7.16-7.57 (m, 8H), 7.83 (m, 2H), 7.99 (m, 2H), 8.17 (d, 1H), 10.47 (s, 1H).
LC-MS (Method 1): Rt=1.08 min; MS (ESIpos): m/z=735 [M+H]f.
0.05 ml (0.31 mmol) of N,N-diisopropylamine and 60 mg (0.16 mmol) of N-[(dimethylamino)(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)methylidene]-N-methylmethanaminium hexafluorophosphate were added to a solution of 70 mg (0.11 mmol) of 4′-[(2S)-2-{[(trans-4-{[(tert-butoxycarbonyl)amino]methyl}cyclohexyl)carbonyl]amino}-3-oxo-3-{[4-(2H-tetrazol-5-yl)phenyl]amino}propyl]biphenyl-2-carboxylic acid and 39 mg (0.21 mmol) of tert-butyl piperazine-1-carboxylate in 1 ml of DMF, and the mixture was stirred at RT for 16 h. The contents of the flask was separated by preparative HPLC (mobile phase: gradient of methanol/water with 0.1% trifluoroacetic acid). The product-containing fractions were combined, concentrated on a rotary evaporator, and the residue was dried under high vacuum. This gave 37 mg (88% of theory) of the title compound.
1H NMR (400 MHz, DMSO-d6) S=0.69-0.91 (m, 2H), 1.04 (d, 4H), 1.09-1.28 (m, 3H), 1.36 (d, 18H), 1.53-1.78 (m, 4H), 2.00-2.17 (m, 1H), 2.59-2.79 (m, 3H), 2.86-2.98 (m, 3H), 3.00-3.15 (m, 2H), 4.57-4.76 (mmol), 1H), 6.68-6.86 (m, 1H), 7.20-7.55 (m, 9H), 7.76-7.88 (m, 2H), 8.00 (d, 2H), 8.04-8.16 (m, 1H), 10.47 (s, 1H).
LC-MS (Method 1): Rt=1.12 min; MS (ESIneg): m/z=834 [M−H]−.
55 μl (0.31 mmol) of N,N-diisopropylethylamine and 59.8 mg (0.16 mmol) of N-[(dimethylamino)(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)methylidene]-N-methylmethanaminium hexafluorophosphate were added to a solution of 70 mg (0.11 mmol) of 4′-[(2S)-2-{[(trans-4-{[(tert-butoxycarbonyl)amino]methyl}cyclohexyl)carbonyl]amino}-3-oxo-3-{[4-(2H-tetrazol-5-yl)phenyl]amino}propyl]biphenyl-2-carboxylic acid and 42.0 mg (0.21 mmol) of tert-butyl 4-aminopiperidine-1-carboxylate in 1 ml DMF. The mixture was stirred at RT for 16 h. The contents of the flask was separated by preparative HPLC (Method 15). The product-containing fractions were combined and concentrated on a rotary evaporator. The residue was dried under high vacuum. 39 mg (38% of theory, 86% purity) of the title compound were obtained.
LC-MS (Method 1): Rt=1.10 min; MS (ESIpos): m/z=850 [M+H]+.
55 μl (0.31 mmol) of N,N-diisopropylethylamine and 59.8 mg (0.16 mmol) of N-[(dimethylamino)(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)methylidene]-N-methylmethanaminium hexafluorophosphate were added to a solution of 70 mg (0.11 mmol) of 4′-[(2S)-2-{[(trans-4-{[(tert-butoxycarbonyl)amino]methyl}cyclohexyl)carbonyl]amino}-3-oxo-3-{[4-(2H-tetrazol-5-yl)phenyl]amino}propyl]biphenyl-2-carboxylic acid and 14.2 mg (0.21 mmol) of methanamine hydrochloride in 1 ml DMF. The mixture was stirred at RT for 16 h. The contents of the flask was separated by preparative HPLC (Method 15). The product-containing fractions were combined and concentrated on a rotary evaporator. The residue was dried under high vacuum. This gave 28 mg (40% of theory) of the title compound.
LC-MS (Method 1): Rt=0.96 min; MS (ESIpos): m/z=681 [M+H]+.
3.6 ml (7.2 mmol) of a 2M sodium carbonate solution in water was added to a solution of 1500 mg (2.4 mmol) of 4-bromo-N-alpha-[(trans-4-{[(tert-butoxycarbonyl)amino]methyl}cyclohexyl)carbonyl]-N-[4-(2H-tetrazol-5-yl)phenyl]-L-phenylalaninamide and 786.8 mg (3.6 mmol) of 2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline in 25 ml DMF, and the mixture was degassed with argon for 5 min. 175 mg (0.24 mmol) of 1,1′-bis(diphenylphosphino)ferrocenepalladium(II) chloride were added and the mixture was stirred at 120° C. in a preheated oil bath overnight. The mixture was filtered through kieselguhr and washed through with water. The filtrate was diluted with ethyl acetate and water and extracted. The aqueous phase was acidified with 10% strength citric acid solution and once more washed with ethyl acetate. The combined organic phases were dried over magnesium sulphate and concentrated under reduced pressure. The residue was stirred with ethyl acetate and precipitated with cyclohexane. The solid was filtered off and dried under high vacuum. This gave 1040 mg (67% of theory) of the title compound.
LC-MS (Method 1): Rt=1.05 min; MS (ESIpos): m/z=639 [M+H]+.
93.3 μl (0.54 mmol) of N,N-diisopropylethylamine and 128.1 mg (0.34 mmol) of N-[(dimethylamino)(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)methylidene]-N-methylmethanaminium hexafluorophosphate were added to a solution of 100 mg (0.15 mmol) of tert-butyl [(trans-4-{[(2S)-3-(2′-aminobiphenyl-4-yl)-1-oxo-1-{[4-(2H-tetrazol-5-yl)phenyl]amino}propan-2-yl]carbamoyl}cyclohexyl)methyl]carbamate and 97 mg (0.34 mmol) of N-(tert-butoxycarbonyl)glycylglycylglycine in 2 ml of ethyl acetate. 0.5 ml of DMF was added to improve solubility, and the mixture was stirred at RT for 16 h. No conversion was observed; the mixture was stirred at 60° C. for 1 h and 1.2 eq of (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate were then added and the mixture was stirred at RT. No product was formed, and therefore 1 more eq. each of N-[(dimethylamino)(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)methylidene]-N-methylmethanaminium hexafluorophosphate, N,N-diisopropylethylamine and N-(tert-butoxycarbonyl)glycylglycylglycine were added and the mixture was stirred at RT overnight. The contents of the flask was diluted with water and ethyl acetate and the organic phase was washed twice with water, dried over magnesium sulphate and concentrated under reduced pressure. This gave 61 mg (43% of theory) of the title compound.
LC-MS (Method 1): Rt=1.01 min; MS (ESIpos): m/z=910 [M+H]+.
66.7 μl (0.38 mmol) of N,N-diisopropylethylamine, 109.4 μl (0.18 mmol) of a 50% strength solution of 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide in DMF and 1 ml of DMF were added to a solution of 100 mg (0.15 mmol) of tert-butyl [(trans-4-{[(2S)-3-(2′-aminobiphenyl-4-yl)-1-oxo-1-{[4-(2H-tetrazol-5-yl)phenyl]amino}propan-2-yl]carbamoyl}cyclohexyl)methyl]carbamate and 34.7 mg (0.34 mmol) of (dimethylamino)acetic acid in 2.5 ml of ethyl acetate, and the mixture was stirred at RT for 4 h. There was no reaction. 69.8 mg (0.18 mmol) of N-[(dimethylamino)(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)methylidene]-N-methylmethanaminium hexafluorophosphate were added and the mixture was stirred at RT overnight. This gave a mixture of starting material and product. Addition of a further 1 eq. of (dimethylamino)acetic acid, 1.2 eq. of (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate and heating of the reaction mixture to 50° C. gave no improvement. The mixture was concentrated and the residue was taken up in DMF and water and separated by preparative HPLC. 57 mg (39% of theory, 75% purity) of the title compound were obtained.
LC-MS (Method 1): Rt=0.80 min; MS (ESIpos): m/z=724 [M+H]+.
140 μl (0.80 mmol) of N,N-diisopropylethylamine and 143.4 mg (0.28 mmol) of (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate were added to a solution of 150 mg (0.23 mmol) of tert-butyl [(trans-4-{[(2S)-3-(2′-aminobiphenyl-4-yl)-1-oxo-1-{[4-(2H-tetrazol-5-yl)phenyl]amino}propan-2-yl]carbamoyl}cyclohexyl)methyl]carbamate and 65.8 mg (0.51 mmol) of tetrahydro-2H-pyran-4-carboxylic acid in 2 ml of ethyl acetate. The mixture was stirred at RT for 16 h. There was still starting material present. 1 eq. each of tetrahydro-2H-pyran-4-carboxylic acid and N,N-diisopropylethylamine were added, and the mixture was stirred at 50° C. for 2 h. 1.2 eq of N-[(dimethylamino)(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)methylidene]-N-methylmethanaminium hexafluorophosphate were added, and the mixture was stirred overnight. The contents of the flask were concentrated, taken up in a little DMF and separated by preparative HPLC (mobile phase: gradient of acetonitrile/water with 0.1% trifluoroacetic acid). The product-containing fractions were combined and concentrated on a rotary evaporator. The residue was dried under high vacuum. 88 mg (45% of theory, 88% purity) of the title compound were obtained.
LC-MS (Method 1): Rt=0.99 min; MS (ESIpos): m/z=751 [M+H]+.
93.3 μl (0.54 mmol) of N,N-diisopropylethylamine and 200.5 μl (0.34 mmol) of a 50% strength solution of 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide in ethyl acetate were added to a solution of 100 mg (0.15 mmol) of tert-butyl [(trans-4-{[(2S)-3-(2′-aminobiphenyl-4-yl)-1-oxo-1-{[4-(2H-tetrazol-5-yl)phenyl]amino}propan-2-yl]carbamoyl}cyclohexyl)methyl]carbamate and 77.2 mg (0.34 mmol) of 1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid in 2 ml of ethyl acetate. 1 ml of DMF was added to improve solubility, and the mixture was stirred at RT for 16 h. There was still starting material present. 1 eq. each of a 50% strength solution of 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide in ethyl acetate and N,N-diisopropylethylamine were added, and the mixture was stirred first at 50° C. for 2 h and then at RT overnight. 1 eq. of 1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid was added, and the mixture was stirred at RT for a further night. An improvement of the starting material:product ratio was observed. 1.2 eq of N-[(dimethylamino)(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)methylidene]-N-methylmethanaminium hexafluorophosphate were added, and the mixture was stirred overnight. The contents of the flask were concentrated, taken up in a little DMF and separated by preparative HPLC (mobile phase: gradient of acetonitrile/water with 0.1% trifluoroacetic acid). The product-containing fractions were combined and concentrated on a rotary evaporator. The residue was dried under high vacuum. 35 mg (25% of theory, 94% purity) of the title compound were obtained.
LC-MS (Method 1): Rt=1.14 min; MS (ESIneg): m/z=850 [M+H]+.
0.36 ml (0.72 mmol) of a 2M sodium carbonate solution in water was added to a solution of 150 mg (0.24 mmol) of 4-bromo-N-alpha-[(trans-4-{[(tert-butoxycarbonyl)amino]methyl}cyclohexyl)carbonyl]-N-[4-(2H-tetrazol-5-yl)phenyl]-L-phenylalaninamide and 64.3 mg (0.36 mmol) of (2-acetamidophenyl)boronic acid in 2.5 ml DMF, and the mixture was degassed with argon for 5 min. 17.52 mg (0.024 mmol) of 1,1′-bis(diphenylphosphino)ferrocenepalladium(II) chloride were added and the mixture was stirred at 120° C. in a preheated oil bath for 2 h. The mixture was filtered through kieselguhr and washed through with ethyl acetate. The filtrate was diluted with ethyl acetate and water, acidified with 10% strength citric acid and once more washed with ethyl acetate. The organic phase of the filtrate was dried over magnesium sulphate and concentrated under reduced pressure. The residue was suspended in ethyl acetate, and the solid obtained was filtered off and dried under high vacuum. This gave 73 mg (42% of theory) of the title compound.
LC-MS (Method 1): Rt=0.98 min; MS (ESIpos): m/z=681 [M+H]+.
0.36 ml (0.72 mmol) of a 2M sodium carbonate solution in water was added to a solution of 150 mg (0.24 mmol) of 4-bromo-N-alpha-[(trans-4-{[(tert-butoxycarbonyl)amino]methyl}cyclohexyl)carbonyl]-N-[4-(2H-tetrazol-5-yl)phenyl]-L-phenylalaninamide and 54.6 mg (0.36 mmol) of [2-(hydroxymethyl)phenyl]boronic acid in 1.5 ml DMF, and the mixture was degassed with argon for 5 min. 17.52 mg (0.024 mmol) of 1,1′-bis(diphenylphosphino)ferrocenepalladium(II) chloride were added and the mixture was stirred at 75° C. for 6 h. The mixture was filtered through kieselguhr and washed through with ethyl acetate. The filtrate was diluted with ethyl acetate and water, acidified with 10% strength citric acid and once more washed with ethyl acetate. The organic phase of the filtrate was dried over magnesium sulphate and concentrated under reduced pressure. The residue was dissolved in a little isopropanol and stirred with diethyl ether. The solid obtained was filtered off and dried under high vacuum. This gave 75 mg (46% of theory) of the title compound.
LC-MS (Method 1): Rt=1.00 min; MS (ESIpos): m/z=654 [M+H]+.
4-Bromo-N-[(trans-4-{[(tert-butoxycarbonyl)amino]methyl}cyclohexyl)carbonyl]-L-phenylalanine (1500 mg, 3.1 mmol) and methyl 3-[5-(4-aminophenyl)-1H-1,2,4-triazol-3-yl]-2,2,3,3-tetrafluoropropanoate (1185 mg, 3.7 mmol) were dissolved in 10 ml of pyridine and admixed with 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide (50% in DMF, 7.2 ml, 12.4 mmol) and stirred at 85° C. for 5 h. Water was added, and the pyridine was removed under reduced pressure. Dilute ammonium chloride solution was added to the residue, and the mixture was extracted three times with ethyl acetate. The combined organic phases were washed with water and saturated aqueous sodium chloride solution, and dried over sodium sulphate and under reduced pressure. The residue was purified chromatographically (silica gel, mobile phase: dichloromethane/methanol=10/1). This gave 1675 mg (63% of theory) of the title compound.
1H NMR (400 MHz, DMSO-d6): δ=ppm 0.82 (m, 2H), 1.05-1.30 (m, 3H), 1.36 (s, 9H), 1.53-1.60 (m, 1H), 1.68 (m, 3H), 2.03-2.14 (m, 1H), 2.70-2.78 (m, 2H), 2.80-2.91 (m, 1H), 2.97-3.09 (m, 1H), 3.95 (s, 2H), 4.57-4.72 (m, 1H), 6.72-6.82 (m, 1H), 7.26 (d, 2H), 7.48 (d, 2H), 7.73-7.81 (m, 2H), 7.96 (d, 2H), 8.15 (d, 1H), 10.44 (s, 1H), 15.19 (br. s, 1H).
LC-MS (Method 1): Rt=1.23 min; MS (ESIpos): m/z=785.4 [M+H]+.
2,2,3,3-Tetrafluoro-3-[5-(4-nitrophenyl)-1H-1,2,4-triazol-3-yl]propanoic acid (30.3 g, 90.8 mmol) was dissolved in methanol (500 ml) and admixed with concentrated sulphuric acid (3 ml). The mixture was stirred at 65° C. for 22 h. Concentrated sulphuric acid (5 ml) was then added, and the mixture was stirred once again at 65° C. for 22 h. Sodium hydrogencarbonate was added at RT to pH=7, the mixture was filtered and the solvent was removed under reduced pressure. The residue was stirred in petroleum ether and diethyl ether and then filtered. This gave 31.6 g (77% of theory) of the title compound.
LC-MS (Method 1): Rt=0.96 min; MS (ESIpos): m/z=349.1 [M+H]+.
Methyl 2,2,3,3-tetrafluoro-3-[5-(4-nitrophenyl)-1H-1,2,4-triazol-3-yl]propanoate (24.0 g, 68.9 mmol) was initially charged in THF (370 ml) and admixed with palladium/charcoal (10%, 50% water-moist) under an argon atmosphere. Hydrogenation was effected with hydrogen (1 bar) at RT for 18 h. The mixture was filtered through kieselguhr and washed with dichloromethane/methanol 9:1. The filtrate was concentrated and the residue was dried under reduced pressure. This gave 21.7 g (99% of theory) of the title compound.
LC-MS (Method 1): Rt=0.78 min; MS (ESIpos): m/z=319.1 [M+H]+.
Methyl 3-{5-[4-({4-bromo-N-[(trans-4-{[(tert-butoxycarbonyl)amino]methyl}cyclohexyl)-carbonyl]-L-phenylalanyl}amino)phenyl]-4H-1,2,4-triazol-3-yl}-2,2,3,3-tetrafluoropropanoate (100 mg, 0.13 mmol) and (2-carbamoylphenyl)boric acid (25 mg, 0.15 mmol) were dissolved in 1 ml dimethylformamide, aqueous sodium carbonate solution (2M, 0.13 ml, 0.26 mmol) was added and the mixture was degassed. After addition of 9 mg (0.01 mmol) of 1,1′-bis(diphenylphosphino)ferrocenepalladium(II) chloride, the reaction mixture was stirred at 75° C. for 18 h. More (2-carbamoylphenyl)boric acid (16 mg, 0.1 mmol), aqueous sodium carbonate solution (2M, 0.13 ml, 0.26 mmol) and 9 mg (0.01 mmol) of 1,1′-bis(diphenylphosphino)ferrocenepalladium(II) chloride were added, and the reaction mixture was stirred at 75° C. for 3 h. The reaction solution was filtered through a Millipore syringe filter and purified via preparative HPLC (mobile phase: acetonitrile/water with 0.1% trifluoroacetic acid (gradient)). This gave 38.6 mg (30% of theory) of the title compound.
LC-MS (Method 1): Rt=1.03 min; MS (ESIpos): m/z=810.4 [M+H]f.
4-Bromo-N-alpha-[(trans-4-{[(tert-butoxycarbonyl)amino]methyl}cyclohexyl)carbonyl]-N-[4-(1H-tetrazol-5-yl)phenyl]-L-phenylalaninamide (150 mg, 0.24 mmol) and tert-butyl 4-[2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl]piperazine-1-carboxylate (116 mg, 0.29 mmol) were dissolved in 1.8 ml of dimethylformamide, aqueous sodium carbonate solution (2M, 0.24 ml, 0.47 mmol) was added and the mixture was degassed. After addition of 18 mg (0.02 mmol) of 1,1′-bis(diphenylphosphino)ferrocenepalladium(II) chloride, the reaction mixture was stirred at 120° C. for 0.5 h. The reaction solution was filtered through a Millipore syringe filter and purified via preparative HPLC (mobile phase: acetonitrile/water with 0.1% trifluoroacetic acid (gradient)). This gave 79 mg (35% of theory) of the title compound.
LC-MS (Method 1): Rt=0.9 min; MS (ESIpos): m/z=822.4 [M+H]+.
Methyl 3-{5-[4-({4-bromo-N-[(trans-4-{[(tert-butoxycarbonyl)amino]methyl}cyclohexyl)-carbonyl]-L-phenylalanyl}amino)phenyl]-4H-1,2,4-triazol-3-yl}-2,2,3,3-tetrafluoropropanoate (100 mg, 0.13 mmol) and (2-isopropoxyphenyl)boric acid (27 mg, 0.15 mmol) were dissolved in 1 ml dimethylformamide, aqueous sodium carbonate solution (2M, 0.13 ml, 0.26 mmol) was added and the mixture was degassed. After addition of 9 mg (0.01 mmol) of 1,1′-bis(diphenylphosphino)ferrocenepalladium(II) chloride, the reaction mixture was stirred at 75° C. for 18 h. More (2-carbamoylphenyl)boric acid (80 mg, 0.1 mmol), aqueous sodium carbonate solution (2M, 0.13 ml, 0.26 mmol) and 9 mg (0.01 mmol) of 1,1′-bis(diphenylphosphino)ferrocenepalladium(II) chloride were added, and the reaction mixture was stirred at 75° C. for 3 h. The reaction solution was filtered through a Millipore syringe filter and purified via preparative HPLC (mobile phase: acetonitrile/water with 0.1% trifluoroacetic acid (gradient)). This gave 59 mg (51% of theory) of the title compound.
LC-MS (Method 1): Rt=1.26 min; MS (ESIpos): m/z=825.2 [M+H]+.
0.41 ml (1.62 mmol) of 4M hydrogen chloride in 1,4-dioxane was added to a solution of 82 mg (0.11 mmol) of tert-butyl [(trans-4-{[(2S)-3-[2′-(morpholin-4-ylsulphonyl)biphenyl-4-yl]-1-oxo-1-{[4-(2H-tetrazol-5-yl)phenyl]amino}propan-2-yl]carbamoyl}cyclohexyl)methyl]carbamate in 3 ml of 1,4-dioxane, and the mixture was stirred at RT overnight. The reaction mixture was concentrated; the residue was taken up in methanol and separated twice by preparative HPLC (mobile phase: gradient of acetonitrile/water with 0.1% trifluoroacetic acid). The product-containing fractions were combined and admixed with 0.2 ml of 4M hydrogen chloride in 1,4-dioxane and the mixture was concentrated on a rotary evaporator. The residue was dried under high vacuum. This gave 10 mg (11% of theory, 87% pure) of the title compound.
LC-MS (Method 1): Rt=0.77 min; MS (ESIneg): m/z=672 [M−H−HCl]−.
1.00 ml (4.00 mmol) of 4M hydrogen chloride in 1,4-dioxane was added to a solution of 82 mg (0.11 mmol) of tert-butyl [(trans-4-{[(2S)-3-[2′-(morpholin-4-ylcarbonyl)biphenyl-4-yl]-1-oxo-1-{[4-(2H-tetrazol-5-yl)phenyl]amino}propan-2-yl]carbamoyl}cyclohexyl)methyl]carbamate in 5 ml of tetrahydrofuran, and the mixture was stirred at RT for 16 h. The solvent was removed on a rotary evaporator and the residue was dried under high vacuum. The residue was triturated with acetonitrile, filtered, washed with acetonitrile and dried under high vacuum. 57 mg (71% of theory, 93% purity) of the title compound were obtained.
1H NMR (400 MHz, DMSO-d6) δ=0.92 (m, 2H), 1.11-1.35 (m, 2H), 1.38-1.56 (m, 1H), 1.76 (m, 5H), 2.08-2.22 (m, 2H), 2.83-3.02 (m, 2H), 3.13 (m, 4H), 3.31-3.54 (m, 4H), 4.64-4.81 (m, 1H), 7.26-7.56 (m, 8H), 7.67-8.07 (m, 8H), 8.18-8.31 (m, 1H), 10.64 (d, 1H).
LC-MS (Method 1): Rt=0.71 min; MS (ESIneg): m/z=635 [M−H−HCl]−.
1.00 ml (4.00 mmol) of 4M hydrogen chloride in 1,4-dioxane was added to a solution of 28 mg (0.04 mmol) of tert-butyl [(trans-4-{[(2S)-3-[2′-(diethylcarbamoyl)biphenyl-4-yl]-1-oxo-1-{[4-(2H-tetrazol-5-yl)phenyl]amino}propan-2-yl]carbamoyl}cyclohexyl)methyl]carbamate in 1 ml of tetrahydrofuran, and the mixture was stirred at RT for 16 h. The solvent was removed on a rotary evaporator, and the residue was stirred in acetonitrile and filtered. The solid formed was washed with acetonitrile and diethyl ether and dried under high vacuum. 22 mg (77% of theory, 90% purity) of the title compound were obtained.
1H NMR (400 MHz, DMSO-d6) δ=0.64 (m, 3H), 0.74-1.00 (m, 4H), 1.09-1.35 (m, 2H), 1.75 (m, 5H), 2.02-2.24 (m, 1H), 2.63 (m, 2H), 2.76-3.15 (m, 4H), 3.33-3.51 (m, 1H), 4.62-4.76 (m, 1H), 7.16-7.53 (m, 8H), 7.85 (m, 5H), 8.03 (d, 2H), 8.16-8.35 (m, 1H), 10.59 (s, 1H).
LC-MS (Method 1): Rt=0.75 min; MS (ESIneg): m/z=621 [M−H−HCl]−.
1.00 ml (4.00 mmol) of 4M hydrogen chloride in 1,4-dioxane was added to a solution of 30 mg (0.04 mmol) of tert-butyl [(trans-4-{[(2S)-1-oxo-3-[2′-(piperidin-1-ylcarbonyl)biphenyl-4-yl]-1-{[4-(2H-tetrazol-5-yl)phenyl]amino}propan-2-yl]carbamoyl}cyclohexyl)methyl]carbamate in 1 ml of tetrahydrofuran, and the mixture was stirred at RT for 16 h. The solvent was removed on a rotary evaporator, and the residue was stirred in acetonitrile and filtered. The solid formed was washed with acetonitrile and diethyl ether and dried under high vacuum. 23 mg (78% of theory, 91% purity) of the title compound were obtained.
1H NMR (400 MHz, DMSO-d6) δ=0.44-0.62 (m, 1H), 0.79-1.85 (m, 14H), 2.04-2.22 (m, 1H), 2.59-2.70 (m, 2H), 2.78-3.15 (m, 3H), 3.20-3.40 (m, 3H), 4.56-4.74 (m, 1H), 7.22-7.54 (m, 8H), 7.65-7.91 (m, 5H), 8.01 (s, 2H), 8.15-8.36 (m, 1H), 10.58 (s, 1H).
LC-MS (Method 1): Rt=0.76 min; MS (ESIneg): m/z=633 [M−H−HCl]−.
1.50 ml (6.00 mmol) of 4M hydrogen chloride in 1,4-dioxane was added to a solution of 37 mg (0.04 mmol) of tert-butyl 4-({4′-[(2S)-2-{[(trans-4-{[(tert-butoxycarbonyl)amino]methyl}cyclohexyl)carbonyl]amino}-3-oxo-3-{[4-(2H-tetrazol-5-yl)phenyl]amino}propyl]biphenyl-2-yl}carbonyl)piperazine-1-carboxylate in 2 ml of 1,4-dioxane, and the mixture was stirred at RT for 16 h. The solvent was removed on a rotary evaporator, and the residue was stirred in acetonitrile and filtered. The solid formed was washed with acetonitrile and diethyl ether and dried under high vacuum. 31 mg (90% of theory, 91% purity) of the title compound were obtained.
1H NMR (400 MHz, DMSO-d6) δ=0.81-1.01 (m, 2H), 1.13-1.37 (m, 2H), 1.40-1.55 (m, 1H), 1.60-1.70 (m, 1H), 1.70-1.88 (m, 4H), 2.13-2.26 (m, 1H), 2.58-2.70 (m, 3H), 2.74-3.04 (m, 2H), 3.04-3.23 (m, 3H), 3.52-3.65 (m, 1H), 3.66-3.79 (m, 1H), 4.58-4.81 (m, 1H), 7.14-7.62 (m, 8H), 7.74-7.91 (m, 5H), 7.97-8.08 (m, 2H), 8.20-8.48 (m, 1H), 8.95-9.20 (m, 1H), 10.59-10.81 (m, 1H), 16.78 (br. s, 1H).
LC-MS (Method 1): Rt=0.58 min; MS (ESIneg): m/z=634 [M−H−HCl]−.
1.50 ml (6.00 mmol) of 4M hydrogen chloride in 1,4-dioxane was added to a solution of 39 mg (0.05 mmol) of tert-butyl 4-[({4′-[(2S)-2-{[(trans-4-{[(tert-butoxycarbonyl)amino]methyl}cyclohexyl)carbonyl]amino}-3-oxo-3-{[4-(2H-tetrazol-5-yl)phenyl]amino}propyl]biphenyl-2-yl}carbonyl)amino]piperidine-1-carboxylate in 2 ml of 1,4-dioxane, and the mixture was stirred at RT for 16 h. The solvent was removed on a rotary evaporator, and the residue was stirred in acetonitrile and filtered. The solid formed was washed with acetonitrile and diethyl ether and dried under high vacuum. 30 mg (82% of theory, 90% purity) of the title compound were obtained.
1H NMR (400 MHz, DMSO-d6) δ=0.84-1.02 (m, 2H), 1.13-1.35 (m, 2H), 1.40-1.67 (m, 4H), 1.76 (m, 5H), 2.11-2.23 (m, 1H), 2.59-2.69 (m, 2H), 2.83-3.00 (m, 3H), 3.02-3.16 (m, 3H), 3.76-3.97 (m, 1H), 4.58-4.81 (m, 1H), 7.24-7.55 (m, 8H), 7.87 (m, 5H), 8.04 (d, 2H), 8.22-8.38 (m, 2H), 8.54-8.69 (m, 1H), 8.74-8.90 (m, 1H), 10.59-10.83 (m, 1H), 16.84 (br. s, 1H).
LC-MS (Method 1): Rt=0.58 min; MS (ESIneg): m/z=648 [M−H−HCl]−.
1.50 ml (6.00 mmol) of 4M hydrogen chloride in 1,4-dioxane was added to a solution of 29 mg (0.04 mmol) of tert-butyl [(trans-4-{[(2S)-3-[2′-(methylcarbamoyl)biphenyl-4-yl]-1-oxo-1-{[4-(2H-tetrazol-5-yl)phenyl]amino}propan-2-yl]carbamoyl}cyclohexyl)methyl]carbamate in 2 ml of 1,4-dioxane, and the mixture was stirred at RT for 16 h. The solvent was removed on a rotary evaporator, and the residue was stirred in acetonitrile and filtered. The solid formed was washed with acetonitrile and diethyl ether and dried under high vacuum. This gave 19 mg (70% of theory) of the title compound.
1H NMR (400 MHz, DMSO-d6) δ=0.81-0.98 (m, 2H), 1.24 (m, 2H), 1.43-1.55 (m, 1H), 1.59-1.66 (m, 1H), 1.76 (m, 3H), 2.07-2.22 (m, 1H), 2.63 (t, 2H), 2.88-3.01 (m, 1H), 3.09 (m, 1H), 3.57 (s, 3H), 4.72 (d, 1H), 7.20-7.53 (m, 8H), 7.85 (m, 5H), 7.97-8.08 (m, 3H), 8.29 (d, 1H), 10.62 (s, 1H).
LC-MS (Method 1): Rt=0.67 min; MS (ESIneg): m/z=579 [M−H−HCl]−.
2 ml (6 mmol) of 4M hydrogen chloride in 1,4-dioxane was added to a solution of 60 mg (0.09 mmol) of tert-butyl [(trans-4-{[(2S)-3-(2′-aminobiphenyl-4-yl)-1-oxo-1-{[4-(2H-tetrazol-5-yl)phenyl]amino}propan-2-yl]carbamoyl}cyclohexyl)methyl]carbamate in 3 ml of tetrahydrofuran, and the mixture was stirred at RT for 3 h. The solid formed was filtered, washed with tetrahydrofuran and acetonitrile and dried under high vacuum. 50 mg (83% of theory, 94% purity) of the title compound were obtained.
1H NMR (400 MHz, DMSO-d6) δ=0.80-1.02 (m, 2H), 1.10-1.36 (m, 2H), 1.41-1.66 (m, 2H), 1.70-1.88 (m, 3H), 2.13-2.23 (m, 1H), 2.63 (br. s., 2H), 2.92-3.03 (m, 1H), 3.09-3.24 (m, 1H), 4.65-4.80 (m, 1H), 7.22-7.53 (m, 8H), 7.87 (m, 5H), 8.05 (d, 2H), 8.36 (d, 1H), 10.68 (s, 1H).
LC-MS (Method 1): Rt=0.73 min; MS (ESIneg): m/z=537 [M−H−HCl]−.
0.5 ml (2 mmol) of 4M hydrogen chloride in 1,4-dioxane was added to a solution of 60 mg (0.06 mmol) of N-(tert-butoxycarbonyl)glycylglycyl-N-{4′-[(2S)-2-{[(trans-4-{[(tert-butoxycarbonyl)amino]methyl}cyclohexyl)carbonyl]amino}-3-oxo-3-{[4-(2H-tetrazol-5-yl)phenyl]amino}propyl]biphenyl-2-yl}glycinamide in 1 ml of tetrahydrofuran, and the mixture was stirred at RT for 16 h. A further 0.25 ml (1 mmol) of 4M hydrogen chloride in 1,4-dioxane was added, and the mixture was stirred at RT for 16 h. The solid formed was filtered, washed with tetrahydrofuran and acetonitrile and dried under high vacuum. 45 mg (83% of theory, 93% purity) of the title compound were obtained.
1H NMR (400 MHz, DMSO-d6) δ=0.91 (m, 2H), 1.08-1.32 (m, 2H), 1.42-1.86 (m, 6H), 2.12-2.24 (m, 1H), 2.56-2.71 (m, 2H), 2.98 (m, 1H), 3.15 (m, 1H), 3.54-3.66 (m, 2H), 3.70-3.87 (m, 4H), 4.64-4.79 (m, 1H), 7.15-7.47 (m, 7H), 7.56 (d, 1H), 7.75-8.27 (m, 10H), 8.29-8.44 (m, 2H), 8.75 (br. s., 1H), 9.22 (s, 1H), 10.74 (br. s., 1H).
LC-MS (Method 1): Rt=0.53 min; MS (ESIneg): m/z=708 [M−H−HCl]−.
0.5 ml (2.00 mmol) of 4M hydrogen chloride in 1,4-dioxane was added to a solution of 50 mg (0.05 mmol) of tert-butyl [(trans-4-{[(2S)-3-(2′-{[(dimethylamino)acetyl]amino}biphenyl-4-yl)-1-oxo-1-{[4-(2H-tetrazol-5-yl)phenyl]amino}propan-2-yl]carbamoyl}cyclohexyl)methyl]carbamate in 1.5 ml of tetrahydrofuran, and the mixture was stirred at RT for 16 h. A further 0.25 ml (1.00 mmol) of 4M hydrogen chloride in 1,4-dioxane was added, and the mixture was stirred at RT for 16 h. The solid formed was washed with tetrahydrofuran and dried under high vacuum. This gave 35.5 mg (96% of theory) of the title compound.
1H NMR (400 MHz, DMSO-d6) δ=0.92 (d, 2H), 1.16-1.33 (m, 2H), 1.42-1.84 (m, 8H), 2.17 (t, 1H), 2.58-2.67 (m, 2H), 2.70 (br. s., 6H), 2.95 (dd, 2H), 3.12 (dd, 1H), 3.60 (t, 3H), 3.95 (s, 2H), 4.71 (m, 1H), 7.20-7.51 (m, 9H), 7.88 (m, 5H), 8.05 (d, 2H), 8.36 (d, 1H), 9.83 (br. s, 1H), 10.22 (d, 1H), 10.72 (d, 1H).
LC-MS (Method 1): Rt=0.55 min; MS (ESIneg): m/z=622 [M−H−HCl]−.
1.0 ml (4 mmol) of 4M hydrogen chloride in 1,4-dioxane was added to a solution of 85 mg (0.1 mmol) of tert-butyl [(trans-4-{[(2S)-1-oxo-3-{2′-[(tetrahydro-2H-pyran-4-ylcarbonyl)amino]biphenyl-4-yl}-1-{[4-(2H-tetrazol-5-yl)phenyl]amino}propan-2-yl]-carbamoyl}cyclohexyl)methyl]carbamate in 4 ml of tetrahydrofuran, and the mixture was stirred at RT for 16 h. A further 0.8 ml (1 mmol) of 4M hydrogen chloride in 1,4-dioxane was added, and the mixture was stirred at RT for 16 h. The solid formed was filtered off and washed with tetrahydrofuran, acetonitrile, diethyl ether and ethyl acetate and dried under high vacuum. 70 mg (88% of theory, 90% purity) of the title compound were obtained.
1H NMR (400 MHz, DMSO-d6) δ=0.81-1.00 (m, 2H), 1.12-1.34 (m, 2H), 1.55 (m, 5H), 1.76 (m, 6H), 2.10-2.21 (m, 1H), 2.38-2.46 (m, 1H), 2.57-2.69 (m, 2H), 2.85-2.98 (m, 1H), 3.05-3.16 (m, 1H), 3.20-3.29 (m, 2H), 3.60 (t, 3H), 3.74-3.92 (m, 2H), 4.63-4.78 (m, 1H), 7.18-7.48 (m, 8H), 7.66-7.90 (m, 5H), 8.02 (d, 2H), 8.25 (d, 1H), 9.20 (s, 1H), 10.53 (s, 1H). 27
LC-MS (Method 1): Rt=0.70 min; MS (ESIneg): m/z=649 [M−H−HCl]−.
0.6 ml (2.33 mmol) of 4M hydrogen chloride in dioxane was added to a solution of 35 mg (39 μmol) of tert-butyl 4-({4′-[(2S)-2-{[(trans-4-{[(tert-butoxycarbonyl)amino]methyl}cyclohexyl)carbonyl]amino}-3-oxo-3-{[4-(2H-tetrazol-5-yl)phenyl]amino}propyl]biphenyl-2-yl}carbamoyl)piperidine-1-carboxylate in 1.6 ml of THF. After 4 h, a further 15 eq. of 4M hydrogen chloride in dioxane were added, and the mixture was stirred at RT for 16 h. The precipitated solid was filtered off with suction, washed with dioxane and acetonitrile, then dried under high vacuum. This gave 28 mg (98% of theory) of the title compound.
LC-MS (Method 1): Rt=0.58 min; MS (ESIpos): m/z=650 [M+H−HCl]+.
1.5 ml (6.03 mmol) of 4M hydrogen chloride in dioxane were added to a solution of 72 mg (100 μmol) of tert-butyl [(trans-4-{[(2S)-3-(2′-acetamidobiphenyl-4-yl)-1-oxo-1-{[4-(2H-tetrazol-5-yl)phenyl]amino}propan-2-yl]carbamoyl}cyclohexyl)methyl]carbamate in 3 ml of THF. After 4 h at RT, conversion was incomplete. A total of 45 eq. of 4M hydrogen chloride in dioxane were added over a period of 2 days, until the product ratio improved no further. The precipitated solid was filtered off with suction and washed repeatedly with acetonitrile, acetonitrile/dichloromethane and with acetonitrile/dichloromethane/tetrahydrofuran. The product was then dried under high vacuum. This gave 50 mg (72% of theory) of the title compound.
LC-MS (Method 1): Rt=0.64 min; MS (ESIpos): m/z=581 [M+H−HCl]+.
1H NMR (400 MHz, DMSO-d6) δ ppm 0.83-0.98 (m, 2H), 1.09-1.33 (m, 2H), 1.50 (d, 1H), 1.63 (d, 1H), 1.71-1.80 (m, 4H), 1.86 (s, 3H), 2.16 (br. s., 1H), 2.64 (t, 2H), 2.89-2.99 (m, 1H), 3.12 (dd, 1H), 4.65-4.79 (m, 1H), 7.22-7.35 (m, 5H), 7.36-7.46 (m, 3H), 7.80 (br. s., 2H), 7.85 (d, 2H), 8.03 (d, 2H), 8.28 (d, 1H), 9.21 (s, 1H), 10.59 (br. s., 1H).
0.84 ml (3.35 mmol) of 4M hydrogen chloride in dioxane were added to a solution of 73 mg (112 μmol) of tert-butyl [(trans-4-{[(2S)-3-[2′-(hydroxymethyl)biphenyl-4-yl]-1-oxo-1-{[4-(2H-tetrazol-5-yl)phenyl]amino}propan-2-yl]carbamoyl}cyclohexyl)methyl]carbamate in 3 ml of THF. The mixture was stirred at RT overnight. The precipitated solid was filtered off with suction and washed repeatedly with tetrahydrofuran and acetonitrile/diethyl ether. The product was then dried under high vacuum. 45 mg (57% of theory, 89% purity) of the title compound were obtained.
LC-MS (Method 1): Rt=0.68 min; MS (ESIpos): m/z=554 [M+H−HCl]+.
1H NMR (400 MHz, DMSO-d6) δ ppm 0.93 (d, 2H), 1.10-1.34 (m, 2H), 1.48 (br. s., 1H), 1.58 (d, 1H), 1.70-1.81 (m, 3H), 2.16 (t, 1H), 2.63 (t, 2H), 2.91-3.03 (m, 1H), 3.14 (dd, 1H), 4.37 (s, 2H), 4.71-4.81 (m, 1H), 7.17 (d, 1H), 7.29 (d, 2H), 7.37 (d, 2H), 7.56 (d, 1H), 7.84 (d, 4H), 8.03 (d, 2H), 8.28 (d, 1H), 10.56 (br. s., 1H).
119 μl (0.48 mmol) of 4M hydrogen chloride in dioxane were added to a solution of 38.6 mg (48 μmol) of 3-[5-(4-{[(2S)-2-{[(trans-4-{[(tert-butoxycarbonyl)amino]methyl}cyclohexyl)carbonyl]amino}-3-(2′-carbamoylbiphenyl-4-yl)propanoyl]amino}phenyl)-1H-1,2,4-triazol-3-yl]-2,2,3,3-tetrafluoropropanoic acid in 1.9 ml of dioxane. The mixture was then stirred at RT for 18 h. Acetonitrile was added and the solid obtained was filtered, washed with acetonitrile and dried under high vacuum. 31 mg (81% of theory) of the title compound were obtained.
1H NMR (400 MHz, DMSO-d6): δ=ppm 0.80-1.02 (m, 2H), 1.14-1.37 (m, 2H), 1.43-1.54 (m, 1H), 1.57-1.67 (m, 1H), 1.76 (m, 3H), 2.10-2.23 (m, 1H), 2.63 (m, 2H), 2.85-3.00 (m, 1H), 3.03-3.18 (m, 1H), 4.60-4.78 (m, 1H), 7.22 (s, 1H), 7.27-7.50 (m, 8H), 7.61 (s, 1H), 7.81 (m, 4H), 7.99 (d, 2H), 8.25 (d, 1H), 10.51 (s, 1H), 15.14 (br. s, 1H).
LC-MS (Method 1): Rt=0.63 min; MS (ESIpos): m/z=710.1 [M+H−HCl]+.
119 μl (0.48 mmol) of 4M hydrogen chloride in dioxane were added to a solution of 78 mg (95 μmol) of tert-butyl [(trans-4-{[(2S)-1-oxo-3-[2′-(piperazin-1-ylmethyl)biphenyl-4-yl]-{[4-(1H-tetrazol-5-yl)phenyl]amino}propan-2-yl]carbamoyl}cyclohexyl)methyl]carbamate in 2 ml of dioxane. The mixture was then stirred at RT for 18 h. The solid obtained was filtered off, washed with acetonitrile and dried under high vacuum. 51 mg (81% of theory) of the title compound were obtained.
1H NMR (400 MHz, DMSO-d6): δ=ppm 0.69-1.01 (m, 4H), 1.11-1.37 (m, 4H), 1.43-1.81 (m, 8H), 2.10-2.24 (m, 1H), 2.63 (d, 2H), 2.90-3.02 (m, 2H), 3.08-3.21 (m, 2H), 4.64-4.84 (m, 1H), 7.28 (m, 4H), 7.42 (m, 4H), 7.87 (m, 6H), 8.04 (d, 2H), 8.31 (d, 1H), 9.04 (br. s, 1H), 9.58 (br. s, 1H), 10.65 (d, 1H), 12.03 (br. s, 1H).
LC-MS (Method 1): Rt=0.58 min; MS (ESIpos): m/z=622.4 [M+H−HCl]+.
179 μl (0.71 mmol) of 4M hydrogen chloride in dioxane were added to a solution of 59 mg (72 μmol) of 3-[5-(4-{[(2S)-2-{[(trans-4-{[(tert-butoxycarbonyl)amino]methyl}cyclohexyl)carbonyl]amino}-3-(2′-isopropoxybiphenyl-4-yl)propanoyl]amino}phenyl)-1H-1,2,4-triazol-3-yl]-2,2,3,3-tetrafluoropropanoic acid in 2 ml of dioxane. The mixture was then stirred at RT for 18 h. Acetonitrile was added and the solid obtained was filtered, washed with acetonitrile and dried under high vacuum. 18 mg (32% of theory) of the title compound were obtained.
1H NMR (400 MHz, DMSO-d6): δ=ppm 0.93 (m, 2H), 1.16 (d, 6H), 1.18-1.35 (m, 2H), 1.41-1.54 (m, 1H), 1.58-1.67 (m, 1H), 1.68-1.82 (m, 3H), 2.16 (t, 1H), 2.64 (t, 2H), 2.85-2.99 (m, 1H), 3.03-3.14 (m, 1H), 4.45-4.56 (m, 1H), 4.65-4.77 (m, 1H), 6.99 (t, 1H), 7.08 (d, 1H), 7.19-7.37 (m, 3H), 7.42 (d, 2H), 7.81 (m, 4H), 7.98 (d, 2H), 8.24 (d, 1H), 10.51 (s, 1H), 15.15 (br. s, 1H).
LC-MS (Method 1): Rt=0.79 min; MS (ESIpos): m/z=725.3 [M+H−HCl]+.
The suitability of the compounds according to the invention for treating thromboembolic or hyperfibrinolytic disorders can be demonstrated in the following assay systems:
a) Test Descriptions (In Vitro)
a.1) Measurement of FXIa Inhibition
The factor XIa inhibition of the substances according to the invention is determined using a biochemical test system which utilizes the reaction of a peptidic factor XIa substrate to determine the enzymatic activity of human factor XIa. Here, factor XIa cleaves from the peptic factor XIa substrate the C-terminal aminomethylcoumarin (AMC), the fluorescence of which is measured. The determinations are carried out in microtitre plates.
Test substances are dissolved in dimethyl sulphoxide and serially diluted in dimethyl sulphoxide (3000 μM to 0.0078 μM; resulting final concentrations in the test: 50 μM to 0.00013 μM). In each case 1 μl of the diluted substance solutions is placed into the wells of white microtitre plates from Greiner (384 wells). Subsequently, the following are added successively: 20 μl of assay buffer (50 mmol/l Tris buffer pH 7.4; 100 mmol/l sodium chloride; 5 mmol/l calcium chloride; 0.1% bovine serum albumin) and 20 μl of factor XIa from Kordia (0.45 nM in assay buffer). After 15 min of incubation, the enzyme reaction is started by addition of 20 μl of the factor XIa substrate Boc-Glu(OBzl)-Ala-Arg-AMC dissolved in assay buffer (10 μM in assay buffer) from Bachem, the mixture is incubated at room temperature (22° C.) for 30 min and fluorescence is then measured (excitation: 360 nm, emission: 460 nm). The measured emissions of the test batches with test substance are compared to those of control batches without test substance (only dimethyl sulphoxide instead of test substance in dimethyl sulphoxide), and IC50 values are calculated from the concentration/activity relationships. Activity data from this test are listed in Table A below:
a.2) Determination of the Selectivity
To demonstrate the selectivity of the substances with respect to FXIa inhibition, the test substances are examined for their inhibition of other human serin 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 are dissolved (50 mmol/l of Tris buffer [C,C,C-tris(hydroxymethyl)aminomethane], 100 mmol/l of sodium chloride, 0.1% BSA [bovine serum albumin], 5 mmol/l of calcium chloride, pH 7.4) and incubated for 15 min with test substance in various concentrations in dimethyl sulphoxide and also with dimethyl sulphoxide without test substance. The enzymatic reaction is then started by addition of the appropriate substrates (5 μmol/l of Boc-Ile-Glu-Gly-Arg-AMC from Bachem for factor Xa and trypsin, 50 μmol/l of MeOSuc-Ala-Phe-Lys-AMC from Bachem for plasmin). After an incubation time of 30 min at 22° C., fluorescence is measured (excitation: 360 nm, emission: 460 nm). The measured emissions of the test mixtures with test substance are compared to the control mixtures without test substance (only dimethyl sulphoxide instead of test substance in dimethyl sulphoxide) and IC50 values are calculated from the concentration/activity relationships.
a.3) Thrombin Generation Assay (Thrombogram)
The effect of the test substances on the thrombogram (thrombin generation assay according to Hemker) is determined in vitro in human plasma (Octaplas R from Octapharma).
In the thrombin generation assay according to Hemker, the activity of thrombin in coagulating plasma is determined by measuring the fluorescent cleavage products of the substrate I-1140 (Z-Gly-Gly-Arg-AMC, Bachem). The reactions are carried out in the presence of varying concentrations of test substance or the corresponding solvent. To start the reaction, reagents from Thrombinoscope (30 pM or 0.1 pM recombinant tissue factor, 24 μM phospholipids in HEPES) are used. In addition, a thrombin calibrator from Thrombinoscope is used whose amidolytic activity is required for calculating the thrombin activity in a sample containing an unknown amount of thrombin. The test is carried out according to the manufacturer's instructions (Thrombinoscope BV): 4 μl of test substance or of the solvent, 76 μl of plasma and 20 μl of PPP reagent or thrombin calibrator are incubated at 37° C. for 5 min. After addition of 20 μl of 2.5 mM thrombin substrate in 20 mM HEPES, 60 mg/ml of BSA, 102 mM of calcium chloride, the thrombin generation is measured every 20 s over a period of 120 min. Measurement is carried out using a fluorometer (Fluoroskan Ascent) from Thermo Electron fitted with a 390/460 nm filter pair and a dispenser.
Using the Thrombinoscope software, the thrombogram is calculated and represented graphically. The following parameters are calculated: lag time, time to peak, peak, ETP (endogenous thrombin potential) and start tail.
a.4) Determination of Anticoagulatory Activity
The anticoagulatory activity of the test substances is determined in vitro in human and animal plasma (for example mouse, rat, rabbit, pig and dog plasma). To this end, blood is drawn off in a mixing ratio of sodium citrate/blood of 1:9 using a 0.11 molar sodium citrate solution as receiver. Immediately after the blood has been drawn off, it is mixed thoroughly and centrifuged at about 4000 g for 15 minutes. The supernatant is pipetted off.
The prothrombin time (PT, synonyms: thromboplastin time, quick test) is determined in the presence of varying concentrations of test substance or the corresponding solvent using a commercial test kit (Neoplastin® from Boehringer Mannheim or Hemoliance® RecombiPlastin from Instrumentation Laboratory). The test compounds are incubated with the plasma at 37° C. for 3 minutes. Coagulation is then started by addition of thromboplastin, and the time when coagulation occurs is determined. The concentration of test substance which effects a doubling of the prothrombin time is determined.
The activated partial thromboplastin time (aPTT) is determined in the presence of varying concentrations of test substance or the corresponding solvent using a commercial test kit (C.K. Prest from Diagnostica Stago). The test compounds are incubated with the plasma and the PTT reagent (cephalin, kaolin) at 37° C. for 3 minutes. Coagulation is then started by addition of a 25 mM aqueous calcium chloride solution, and the time when coagulation occurs is determined. The concentration of test substance which brings about a 1.5-fold extension of the aPTT is determined. Activity data from this test are listed in Table B below:
a.5) Determination of Fibrinolytic Activity
Antifibrinolytic activity in vitro is assessed in human, platelet-free plasma. Tissue factor (TF) (1 pM) and tissue plasminogen activator (tPA) (40 nM) are pipetted into plasma together with 12.5 mM aqueous calcium chloride solution and substance. On occurrence of clotting, the subsequent clot lysis is determined photometrically over a period of 30 minutes.
b) Determination of Antithrombotic Activity (In Vivo)
b.1) Arterial Thrombosis Model (Iron(II) Chloride-Induced Thrombosis) in Combination with Ear Bleeding Time in Rabbits
The antithrombotic activity of the FXIa inhibitors is tested in an arterial thrombosis model. Thrombus formation is triggered here by causing chemical injury to a region in the carotid artery in rabbits. Simultaneously, the ear bleeding time is determined.
Male rabbits (Crl:KBL (NZW)BR, Charles River) receiving a normal diet and having a body weight of 2.2-2.5 kg are anaesthetized by intramuscular administration of xylazine and ketamine (Rompun, Bayer, 5 mg/kg and Ketavet, Pharmacia & Upjohn GmbH, 40 mg/kg body weight). Anaesthesia is furthermore maintained by intravenous administration of the same preparations (bolus: continuous infusion) via the right auricular vein.
The right carotid artery is exposed and the vessel injury is then caused by wrapping a piece of filter paper (10 mm×10 mm) on a Parafilm® strip (25 mm×12 mm) around the carotid artery without disturbing the blood flow. The filter paper contains 100 μL of a 13% strength solution of iron(II) chloride (Sigma) in water. After 5 min, the filter paper is removed and the vessel is rinsed twice with aqueous 0.9% strength sodium chloride solution. 30 min after the injury the injured region of the carotid artery is extracted surgically and any thrombotic material is removed and weighed.
The test substances are administered either intravenously to the anaesthetized animals via the femoral vein or orally to the awake animals via gavage, in each case 5 min and 2 h, respectively, before the injury.
Ear bleeding time is determined 2 min after injury to the carotid artery. To this end, the left ear is shaved and a defined 3-mm-long incision (blade Art. Number 10-150-10, Martin, Tuttlingen, Germany) is made parallel to the longitudinal axis of the ear. Care is taken here not to damage any visible vessels. Any blood that extravasates is taken up in 15 second intervals using accurately weighed filter paper pieces, without touching the wound directly. Bleeding time is calculated as the time from making the incision to the point in time where no more blood can be detected on the filter paper. The volume of the extravasated blood is calculated after weighing of the filter paper pieces.
c) Determination of Fibrinolytic Activity (In Vivo)
c. 1) Hyper-Fibrinolytic Rats
The determination of antifibrinolytic activity in vivo is conducted in hyperfibrinolytic rats. After anaesthetization and catheterization of the animals, hyperfibrinolysis is triggered by infusion of tissue plasminogen activator (tPA) (8 mg/kg/h). 10 minutes after commencement of tPA infusion, the substances are administered as an i.v. bolus. After a further 15 minutes, tPA infusion is ended and a transsection of the tail is conducted. Subaqual bleeding (in physiological saline at 37° C.) is observed over 30 minutes and the bleed time is determined.
The substances according to the invention can, for example, be converted to pharmaceutical preparations as follows:
Tablet:
Composition:
100 mg of the compound of Example 1, 50 mg of lactose (monohydrate), 50 mg of maize starch, 10 mg of polyvinylpyrrolidone (PVP) and 2 mg of magnesium stearate.
Tablet weight 212 mg. Diameter 8 mm, radius of curvature 12 mm.
Production:
The mixture of the compound of Example 1, lactose and starch is granulated with a 5% strength solution (m/m) of the PVP in water. After drying, the granules are mixed with the magnesium stearate for 5 min. This mixture is compressed in a conventional tabletting press (see above for format of the tablet).
Oral Suspension:
Composition:
1000 mg of the compound of Example 1, 1000 mg of ethanol (96%), 400 mg of Rhodigel and 99 g of water.
10 ml of oral suspension correspond to a single dose of 100 mg of the compound of the invention.
Production:
The Rhodigel is suspended in ethanol, and the compound of Example 1 is added to the suspension. The water is added while stirring. The mixture is stirred for about 6 h until swelling of the Rhodigel is complete.
Solution for Oral Administration:
Composition: 500 mg of the compound of the invention, 2.5 g of polysorbate and 97 g of polyethylene glycol 400. 20 g of oral solution correspond to a single dose of 100 mg of the compound of the invention.
Production:
The compound of the invention is suspended in the mixture of polyethylene glycol and polysorbate with stirring. The stirring operation is continued until dissolution of the compound of the invention is complete.
i.v. Solution:
The compound of the invention is dissolved in a concentration below the saturation solubility in a physiologically acceptable solvent (e.g. isotonic saline solution, glucose solution 5% and/or polyethylene glycol 400/water 30% m/m). The solution is subjected to sterile filtration and dispensed into sterile and pyrogen-free injection vessels.
| Number | Date | Country | Kind |
|---|---|---|---|
| 13186059.5 | Sep 2013 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2014/070323 | 9/24/2014 | WO | 00 |
