Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
  • C07D487/04—Ortho-condensed systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure

Definitions

• MLKs Mixed lineage kinases
• MAPKKKs MAPK-kinase kinases
• SAPK/JNK stress activated protein kinase/c-Jun N-terminal kinase
• MKKs p38 kinase pathways
• SAPK/JNK and p38 kinase are specific MAPKs that, like other eukaryotic MAPKs, are activated in response to a multitude of stimuli including exposure to inflammatory cytokines, hormones, and growth factors as well as cellular stresses such as heat shock, inhibition of protein glycosylation, and exposure to ultraviolet irradiation. Fanger et al, Curr. Opin. Genet. Dev.; 7(1): 67-74 (1997).
• MLK1-MLK4 Three subfamilies of MLKs have been previously identified and grouped on the basis of domain arrangements and sequence homology within their catalytic domains.
• the MLKs (MLK1-MLK4) contain an amino-terminal SRC homology domain (SH3), followed sequentially by a catalytic kinase domain, a leucine zipper region, and a Cdc42/Rac-interactive binding domain (CRIB Motif).
• SH3 SRC homology domain
• Cdc42/Rac-interactive binding domain Cdc42/Rac-interactive binding domain
• MLK1-MLK4 share approximately 75% homology within their catalytic domains and approximately 65% homology from the SH3 domain to the CRIB motif. However, the carboxy terminus of each of these kinases diverge, indicating these regions may serve different regulatory functions. Nature Reviews; 3: 663-664 (2002).
• the duel leucine zipper bearing kinases represent the second family of MLKs and are characterized by a kinase domain followed by two leucine zipper motifs, separated by a 31 amino acid spacer.
• the catalytic domains of the two DLKs (DLK and leucine zipper kinase (LZK)) share approximately 87%o sequence homology but again diverge in their carboxy terminus.
• the final subfamily of MLK is represented by zipper sterile ⁇ otmotif kinase (ZAK). ZAK shares homology with the other MLKs tlirough the leucine zipper domain, but again diverges from the others at the carboxy terminus. Nature Reviews: 3: 663-664 (2002).
• MLKs While all of the MLKs have been shown to activate the c-Jun N-terminal kinase pathway, some have also been shown to activate the p38 kinase pathway as well.
• MAPKKKs have been identified which activate the JNK and p38 kinase pathways, including MEK kinase; apoptosis inducing kinase 1 (ASK1); and transforming growth factor beta (TGF ⁇ )-activated kinase (TAK1). Nature Reviews: 3: 663-664 (2002).
• Congestive heart failure is a complex disorder with several etiologies including hypertension, myocardial injury, and hemodynamic overload.
• One of the adaptive responses of the heart to these stresses is hypertrophy of the cardiac myocyte, characterized by altered gene transcriptional regulation, increased protein synthesis, and increased organization of the myo fibril. This hypertrophy, in turn, may lead to remodeling of the heart and subsequent failure.
• SAPK/JNK stress activated protein kinase
• MLK-7 a novel MAPKKK, designated as MLK-7, was identified from a database mining effort of a cDNA library from human failed heart tissue.
• the cDNA encodes for a 55 kDa. protein with serine/threonine kinase activity when expressed and purified from insect cells.
• MLK-7 activates the SAPK/JNKl pathway in rat neonatal cardiac myocytes and modulates fetal expression of marker genes for cardiac hypertrophy.
• MLK-7 increased expression of atrial natruiretic factor (ANF) and decreased expression of ⁇ myosin heavy chain ( ⁇ MHC) mRNAs in rat neonatal cardiac myocytes.
• MLK-7 also increased protein synthesis in cardiac myocytes as evidenced by increased
• the present invention is directed to the discovery that dihydropyrrolopyrazole- derivative compounds of the present invention, as defined below, are antagonists of the mitogen activated protein kinasase kinase kinase, MLK-7. Accordingly, the present invention provides a compound of the formula:
• Rl represents hydrogen, halo, or (Cl-C4)alkyl
• R2 represents:
• R 3 represents independently at each occurrence amino, hydroxy, (Cj-C4)alkyl
• R4 and R ⁇ represent independently at each occurrence amino, hydroxy, (C_ - C4)alkyl, or (C i -C4)alkoxy;
• R6 and R 7 represent independently at each occurrence amino or (Cl-C4)alkyl;
• R8 represents independently at each occurrence amino, (Ci -C4)alkyl, or (Ci - C4)alkoxy;
• R9 and RlO represent independently at each occurrence (Cl-C4)alkyl;
• R! 1 represents independently at each occurrence (Cl-C4)alkyl or a substituent selected from the group consisting of: (a) -(CH 2 ) n -X-Y
• Y and Y' represent independently at each occurrence amino, hydroxy, (C ⁇ -C4)alkyl, (C ⁇ -C4)alkoxy, (C ⁇ -C4)alkoxycarbonyl, NH-(C ⁇ - C4)alkylamine, or N,N-(C ⁇ -C4)dialkylamine, provided that where X or X' represents S, then Y or Y" is not amino or hydroxy;
• Rl2 and R--* 3 represent independently at each occurrence hydrogen or (Ci - C4)alkyl, or Rl2 and R- ⁇ together with the nitrogen atom to which they are attached form a piperidino, pyrrolidino, morpholino or a methylpiperazino group; R!4 represents independently at each occurrence hydroxy, amino, or (Ci -
• Rl5 and R1" each represent independently at each occurrence hydrogen or (Ci - C4)alkyl, or a pharmaceutically acceptable salt thereof.
• the present invention provides a method of treating or preventing congestive heart failure, comprising administering to a patient in need thereof an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof.
• the present invention provides pharmaceutical compositions of compounds of Formula I, including the pharmaceutically acceptable salts and hydrates thereof, comprising a compound of Formula I in combination with a pharmaceutically acceptable carrier, diluent or excipient. This invention also encompasses novel intermediates, and processes for the synthesis of the compounds of Formula I.
• the present invention provides the use of a compound of
• Formula I for the manufacture of a medicament for treating or preventing congestive heart failure.
• the present invention provides compounds with affinity for the mitogen activated protein kinase kinase kinase designated as MLK-7, which could be used to antagonize or partially antagonize kinase activity and therby influence physiological functions related to kinase levels and/or kinase activity, h this regard, such ligands are believed to be useful in treating or preventing physioligcal disorders susceptible to MLK7 modulation, particularly MLK-7 antagonism.
• methods for the treatment or prevention of physiolgical disorders susceptible to MAPKKK modulation, particularly MLK-7 antagonism constitute an important embodiment of the present invention.
• the present invention provides compounds useful as MLK-7 modulators.
• the present invention provides compounds useful as MLK-7 antagonists.
• compounds of Formula I are believed to be novel and, thus, to constitute yet another important embodiment of the present invention, hi addition, compounds of the present invention may also exert antagonist activity at other MAPKKKs as well as inhibiting the action of other serine/threonine kinases such as the TGF ⁇ receptors, Type I and Type II.
• prodrug refers to a compound of Formula I which has been structurally modified such that in vivo the prodrug is converted, for example, by hydrolytic, oxidative, reductive, or enzymatic cleavage, into the parent molecule ("drug") as given by Formula I.
• prodrugs may be, for example, metabolically labile ester derivatives of the parent compound where said parent molecule bears a carboxylic acid group. Conventional procedures for the selection and preparation of suitable prodrugs are well known to one of ordinary skill in the art.
• Typical pharmaceutically acceptable salts include those salts prepared by reaction of the compounds of the present invention with a pharmaceutically acceptable mineral or organic acid or an organic or inorganic base. Such salts are known as acid addition and base addition salts. It is further understood by the skilled reader that salt forms of pharmaceutical compounds are commonly used because they are often more readily crystallized, or more readily purified, than are the free bases.
• Acids commonly employed to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as -toluenesulfonic, methanesulfonic acid, oxalic acid, /? -bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like.
• inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like
• organic acids such as -toluenesulfonic, methanesulfonic acid, oxalic acid, /? -bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like.
• salts examples include the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, bromide, iodide, hydroiodide, dihydroiodide, acetate, propionate, decanoate, caprylate, acrylate, formate, hydrochloride, dihydrochloride, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-l,4-dioate, hexyne-l,6-dioate, benzoate, chlorobenzoate, methylbenzoate, hydroxybenzoate, methoxybenzoate, phthalate, xylenesulfonate, phenylacetate,
• Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like.
• bases useful in preparing the salts of this invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate, and the like.
• the particular counterion forming a part of any salt of this invention is usually not of a critical nature, so long as the salt as a whole is pharmacologically acceptable and as long as the counterion does not contribute undesired qualities to the salt as a whole. It is further understood that such salts may exist as a hydrate.
• stereoisomer refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures which are not interchangeable. The three-dimensional structures are called configurations.
• enantiomer refers to two stereoisomers whose molecules are nonsuperimposable mirror images of one another.
• chiral center refers to a carbon atom to which four different groups are attached.
• diastereomers refers to stereoisomers which are not enantiomers.
• two diastereomers which have a different configuration at only one chiral center are referred to herein as “epimers”.
• racemate racemic mixture
• racemic modification refer to a mixture of equal parts of enantiomers.
• the compounds of the present invention may have one or more chiral centers and may, therefore, exist in a variety of stereoisomeric configurations. As a consequence of these chiral centers the compounds of the present invention may occur as racemates, mixtures of enantiomers, and as individual enantiomers as well as diastereomers and mixtures of diastereomers. All such racemates, enantiomers, and diastereomers are within the scope of the present invention. Enantiomers of the compounds provided by the present invention can be resolved, for example, by one of ordinary skill in the art using standard techniques such as those described by J. Jacques, et al, "Enantiomers, Racemates, and Resolutions", John Wiley and Sons, Inc., 1981.
• R and S where used herein are as commonly used in organic chemistry to denote specific configuration of a chiral center.
• the term “R” (rectus) refers to that configuration of a chiral center with a clockwise relationship of group priorities (highest to second lowest) when viewed along the bond toward the lowest priority group.
• the term “S” (sinister) refers to that configuration of a chiral center with a counterclockwise relationship of group priorities (highest to second lowest) when viewed along the bond toward the lowest priority group.
• the priority of groups is based upon their atomic number (in order of decreasing atomic number). A partial list of priorities and a discussion of stereochemistry is contained in "Nomenclature of Organic Compounds: Principles and Practice", (J.H. Fletcher, et al, eds., 1974) at pages 103-120.
• the specific stereoisomers and enantiomers of compounds of Formula I can be prepared by one of ordinary skill in the art utilizing well known techniques and processes, such as those disclosed by Eliel and Wilen, "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., 1994, Chapter 7; Separation of Stereoisomers, Resolution, Raceniization; and by Collet and Wilen, "Enantiomers, Racemates, and Resolutions", John Wiley & Sons, Inc., 1981.
• specific stereoisomers and enantiomers can be prepared by stereospecific syntheses using enantiomerically and geometrically pure, or enantiomerically or geometrically enriched starting materials.
• the specific stereoisomers and enantiomers can be resolved and recovered by techniques such as chromatography on chiral stationary phases, enzymatic resolution or fractional recrystallization of addition salts formed by reagents used for that purpose.
• Pg refers to a suitable oxygen or nitrogen protecting group.
• Suitable oxygen or nitrogen protecting groups refers to those groups intended to protect or block the oxygen or nitrogen group against undesirable reactions during synthetic procedures. Whether the term “Pg", where used herein, represents an oxygen protecting group or a nitrogen protecting group will be readily apparent to the ordinarily skilled artisan. The suitability of the oxygen or nitrogen protecting group used will depend upon the conditions that will be employed in subsequent reaction steps wherein protection is required, and is well within the knowledge of one of ordinary skill in the art.
• nitrogen protecting groups are disclosed in Greene, "Protective Groups In Organic Synthesis," (John Wiley & Sons, New York (1981)).
• Suitable nitrogen protecting groups comprise acyl groups such as formyl, acetyl, propionyl, pivaloyl, t- butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o- nitrophenoxyacetyl, .alpha.-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4- nitrobenzoyl, and the like; sulfonyl groups such as benzenesulfonyl, p-toluenesulfonyl and the like; carbamate forming groups such as benzyloxycarbonyl, p- chlorobenzyloxycarbonyl, p-
• oxygen protecting groups are also disclosed in Greene (supra). Suitable oxygen protecting groups comprise alkyl groups such as methyl, ethyl, and the like; silyl groups such as t-butyldimethylsilyl, t- butyldiphenylsilyl, triisopropylsilyl, and the like, with t-butyldimethylsilyl being preferred. Other commonly used oxygen protecting groups include benzyl, 4-nitrophenyl methyl, benzoyl, and the like.
• (C ⁇ -C 4 )alkyl refers to a straight or branched, monovalent, saturated aliphatic chain of 1 to 4 carbon atoms and includes, but is not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and the like.
• (C ⁇ -C 6 )alkyl refers to a straight or branched, monovalent, saturated aliphatic chain of 1 to 6 carbon atoms and includes, but is not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, n-hexyl, and the like. It is understood that the term “(C ⁇ -C 4 )alkyl” is included within the definition of "(C ⁇ -C 6 )alkyl".
• (CrC ⁇ o)alkyl refers to a straight or branched, monovalent, saturated aliphatic chain of 1 to 10 carbon atoms and includes, but is not limited to methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tertiary butyl, pentyl, isopentyl, hexyl, 2,3-dimethyl-2 -butyl, heptyl, 2,2-dimethyl-3-pentyl, 2-methyl-2-hexyl, octyl, 4-methyl-3 -heptyl and the like. It is understood that the terms "(C ⁇ -C 4 )alkyl" and "(C ⁇ -C 6 )alkyl” are included within the definition of "(C ⁇ -C ]0 )alkyl".
• Me methyl, ethyl, propyl, isopropyl, butyl and tert-butyl respectively.
• (C ⁇ -C 4 )alkoxy refers to an oxygen atom bearing a straight or branched, monovalent, saturated aliphatic chain of 1 to 4 carbon atoms and includes, but is not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, and the like.
• (C ⁇ -C 6 )alkoxy refers to an oxygen atom bearing a straight or branched, monovalent, saturated aliphatic chain of 1 to 6 carbon atoms and includes, but is not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, n- pentoxy, n-hexoxy, and the like. It is understood that the term “(C ⁇ -C 4 )alkoxy” is included within the definition of "(C ⁇ -C 6 )alkoxy”.
• amino(C ⁇ -C 4 )alkyl refers to a straight or branched, monovalent, saturated aliphatic chain of 1 to 4 carbon atoms bearing an amino group attached to one of the carbon atoms.
• hydroxy(C ⁇ -C )alkyl refers to a straight or branched, monovalent, saturated aliphatic chain of 1 to 4 carbon atoms bearing a hydroxyl group attached to one of the carbon atoms.
• hydroxy(d- C 6 )alkyl refers to a straight or branched, monovalent, saturated aliphatic chain of 1 to 6 carbon atoms bearing a hydroxyl group attached to one of the carbon atoms. It is understood that the term “hydroxy(C ⁇ -C 4 )alkyl” is included within the definition of "hydroxy(C ⁇ -C 6 )alkyl".
• hydroxy(C ⁇ -C 4 )alkoxy refers to an oxygen atom bearing a straight or branched, monovalent, saturated aliphatic chain of 1 to 4 carbon atoms with a hydroxyl group attached to one of the carbon atoms.
• hydroxy(C 1 -C 6 )alkoxy refers to an oxygen atom bearing a straight or branched, monovalent, saturated aliphatic chain of 1 to 6 carbon atoms with a hydroxyl group attached to one of the carbon atoms. It is understood that the term “hydroxy(C ⁇ - C 4 )alkoxy” is included within the definition of "hydroxy(C ⁇ -C 6 )alkoxy”.
• (C ⁇ -C 4 )alkyl-(C ⁇ -C 4 )alkoxy refers to a straight or branched, monovalent, saturated aliphatic chain of 1 to 4 carbon atoms which has a ( - C )alkoxy group attached to the aliphatic chain.
• (C ⁇ -C 4 )alkoxy-(C ⁇ -C 4 )alkoxy refers to an oxygen atom bearing straight or branched, monovalent, saturated aliphatic chain of 1 to 4 carbon atoms which has a (C ⁇ -C 4 )alkoxy group attached to the aliphatic chain.
• (C ⁇ -C )alkyl-COR4 refers to a straight or branched, monovalent, saturated aliphatic chain of 1 to 4 carbon atoms which has a COR ⁇ group attached to the aliphatic chain (R ⁇ being as defined elsewhere herein).
• (C ⁇ -C )alkoxy-COR5" refers to an oxygen atom bearing a straight or branched, monovalent, saturated aliphatic chain of 1 to 4 carbon atoms which has a COR ⁇ group attached to the aliphatic chain (R- ⁇ being as defined elsewhere herein).
• amino(C ⁇ -C 4 )alkyl-COR4" refers to an amino(C ⁇ -
• hydroxy(C -C 4 )alkyl-COR4" refers to a hydroxy(C ⁇ -C 4 )alkyl group which has a COR ⁇ group attached to the aliphatic chain (R4 being as defined elsewhere herein).
• halo refers to a chlorine, bromine, iodine or fluorine atom, unless otherwise specified herein.
• halo(C ⁇ -C )alkyl refers to a straight or branched, monovalent, saturated aliphatic chain of 1 to 4 carbon atoms bearing one or more halo groups attached to one or more of the carbon atoms.
• halo(C ⁇ -C 6 )alkyl refers to a straight or branched, monovalent, saturated aliphatic chain of 1 to 6 carbon atoms bearing one or more halo groups attached to one or more of the carbon atoms. It is understood that the term “halo(C ⁇ -C 4 )alkyl” is included within the definition of "halo(C ⁇ -C 6 )alkyl".
• halo(C]-C 4 )alkoxy refers to an oxygen atom bearing a straight or branched, monovalent, saturated aliphatic chain of 1 to 4 carbon atoms bearing one or more halo groups attached to one or more of the carbon atoms.
• halo(C ⁇ -C 6 )alkoxy refers to an oxygen atom bearing a straight or branched, monovalent, saturated aliphatic chain of 1 to 6 carbon atoms bearing one or more halo groups attached to one or more of the carbon atoms. It is understood that the term “halo(C ⁇ -C )alkoxy” is included within the definition of "halo(C ⁇ -C 6 )alkoxy".
• halophenyl(C ⁇ -C4)alkyl refers to a straight or branched, monovalent, saturated aliphatic chain of 1 to 4 carbon atoms bearing phenyl group wich is further substituted with a halo moiety.
• halophenyl(C ⁇ -C4)alkyl refers to a straight or branched, monovalent, saturated aliphatic chain of 1 to 4 carbon atoms bearing phenyl group wich is further substituted with a halo moiety.
• C4)alkyl include (4-chlorophenyl)methyl, (3-chlrorophenyl)methyl, (4- chlorophenyl)ethyl, (3-chlrorophenyl)ethyl, and the like
• (C -C 6 )alkenyl refers to a straight or branched, monovalent, unsaturated aliphatic chain having from two to six carbon atoms and having a double bond.
• Typical (C -C 6 )alkenyl groups include ethenyl (also known as vinyl), 1- methylethenyl, 1 -methyl- 1-propenyl, 1-butenyl, 1-hexenyl, 2-methyl-2-propenyl, 1- propenyl, 2-propenyl, 2-butenyl, 2-pentenyl, and the like.
• (C -C 6 )alkynyl refers to a straight or branched, monovalent, unsaturated aliphatic chain having from two to six carbon atoms and having a triple bond.
• aryl refers to a monovalent carbocyclic group containing one or more fused or non-fused phenyl rings and includes, for example, phenyl, 1- or 2-naphthyl, 1 ,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, and the like.
• aryl (C ⁇ -C 6 )alkyl refers to a straight or branched, monovalent, saturated aliphatic chain of 1 to 6 carbon atoms which has an aryl group attached to the aliphatic chain
• aryl (C ⁇ -C 4 )alkyl refers to a straight or branched, monovalent, saturated aliphatic chain of 1 to 4 carbon atoms which has an aryl group attached to the aliphatic chain. It is understood that the term “aryl (C ⁇ -C 4 )alkyl” is included within the definition of “aryl (C ⁇ -C 6 )alkyT. Examples of “aryl (C ⁇ -C 6 )alkyl” include:
• aryl(C ⁇ -C 6 )alkoxy refers to an oxygen atom bearing a straight or branched, monovalent, saturated aliphatic chain of 1 to 6 carbon atoms wherein said aliphatic chain, in turn, bears an aryl group.
• (C 3 -C ⁇ o)cycloalkyl refers to a saturated hydrocarbon ring structure composed of one or more fused or unfused rings containing from three to ten carbon atoms.
• Typical (C 3 -C ⁇ o)cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, adamantanyl, and the like.
• “(C 3 - C 7 )cycloalkyl” refers to a saturated hydrocarbon ring structure composed of one or more fused or unfused rings containing from three to seven carbon atoms. It is understood that the definition of "(C 3 -C 7 )cycloalkyl" is included within the definition of "(C 3 - C ⁇ o)cycloalkyl".
• (C ⁇ -C 4 )alkyl-(C 3 -C 7 )cycloalkyT refers to a straight or branched, monovalent, saturated aliphatic chain of 1 to 4 carbon atoms which has a (C -C 7 )cycloalkyl attached to the aliphatic chain. Included within the term “(C ⁇ -C )alkyl- (C 3 -C 7 )cycloalkyl” are the following:
• (C 3 -C 7 )cycloalkoxy refers to an oxygen atom bearing a saturated hydrocarbon ring structure composed of one or more fused or unfused rings containing from three to seven carbon atoms.
• ( - ) alkoxycarbonyl refers to a carbonyl group having a (C ⁇ -C 4 )alkyl group attached to the carbonyl carbon through an oxygen atom. Examples of this group include t-butoxycarbonyl, methoxycarbonyl, ethoxycarbonyl and the like.
• (Ci -C4)alkyl-(C ⁇ -C4)alkoxycarbonyl refers to a straight or branched, monovalent, saturated aliphatic chain of 1 to 4 carbon atoms bearing a carbonyl group having a (C ⁇ -C )alkyl group attached to the carbonyl carbon through an oxygen atom. Examples include methoxycarbonyl methyl, ethoxycarbonyl methyl, 2- methoxycarbonyl ethyl, and the like.
• (C ⁇ -C4)alkylcarbonyl refers to a carbonyl group bearing a a straight or branched, monovalent, saturated aliphatic chain of 1 to 4 carbon atoms attached to the carbonyl carbon.
• heterocycle refers to a saturated or unsaturated, five- or six-membered ring, which contains one to four heteroatoms selected from the group consisting of oxygen, sulfur, and nitrogen. It is understood that the remaining atoms are carbon and that the heterocycle may be attached at any point which provides for a stable structure.
• heterocycle groups include thiopheneyl, furanyl, tetrahydrofuryl, imidazolyl, pyrazolyl, pyrrolyl, thiazolyl, thiazolidinyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, thiadiazolyl, oxadiazolyl, tetrazolyl, pyridyl, pyridinyl, pyrimidyl, pyrazinyl, pyridazinyl, triazinyl, imidazolyl, dihydropyrimidyl, tetrahydropyrimdyl, pyrrolidinyl, piperidinyl, piperazinyl, pyrazolidinyl, pyrimidinyl, imidazohdimyl, morpholinyl, pyranyl, thiomorpholinyl, dioxo-thiomorpholinyl, and the like
• benzofused heterocyclic ring refers to a saturated or unsaturated, five- or six-membered ring, which contains one to four heteroatoms selected from the group consisting of oxygen, sulfur, and nitrogen, and which is fused to a phenyl group. It is understood that the remaining atoms are carbon and that the benzofused heterocycle may be attached at any point on either the heterocyclic or phenyl ring which provides for a stable structure.
• benzofused heterocyclic rings include benzoxazole, benzoimidazole, benzofuran, benzothiophene, benzo[l,3]-dioxolyl, benzothiazole, 2,2-dioxy-2,3-dihydro- lH-2 ⁇ 6 -benzo[c]thiophene, azaindole, and indole.
• NH(C 3 -C 7 )cycloalkyl refers to an amino group substituted with a saturated hydrocarbon ring structure composed of one or more fused or unfused rings containing from three to seven carbon atoms.
• N,N-(C ⁇ -C )dialkylamine refers to a nitrogen atom substituted with two independently selected straight or branched, monovalent, saturated aliphatic chains of 1 to 4 carbon atoms. Included within the term "N,N-(C ⁇ -C )dialkylamine”
• C 6 )dialkylamine are -N(CH 3 ) 2 , -N(CH 2 CH 3 ) 2 , -N(CH 2 CH 2 CH 3 ) 2 , -N(CH 2 CH 2 CH 2 CH 3 ) 2 , and the like.
• NH-(C ⁇ -C 4 ) alkylamine refers to a nitrogen atom substituted with a straight or branched, monovalent, saturated aliphatic chains of 1 to 4 carbon atoms.
• NH-(C ⁇ -C 4 ) alkylamine Included within the term "NH-(C ⁇ -C 4 ) alkylamine" are -NH(CH 3 ), -NH(CH 2 CH 3 ), - NH(CH 2 CH 2 CH 3 ), -NH(CH 2 CH 2 CH 2 CH 3 ), and the like.
• the designation " ' ' ⁇ ⁇ " refers to a bond that protrudes backward out of the plane of the page.
• mitogen activated protein kinase kinase kinase modulator or “MAPKKK modulator” refers to a ligand which binds to any one of the mitogen activated protein kinase kinase kinases and either agonizes, antagonizes, partially agonizes, or partially antagonizes the kinase 's activity.
• mitogen activated protein kinase kinase kinase antagonist or “MAPKKK antagonist” refers to a ligand which binds to any one of the mitogen activated protein kinase kinase kinases and either antagonizes, or partially antagonizes the kinase 's activity.
• mixed lineage kinase-7 antagonist or “MLK-7 antagonist” refers to an antagonist of the specific MAPKKK designated as “mixed lineage kinase-7" or “MLK-7” and is included within the meanings of "mitogen activated protein kinase kinase modulator” or “mitogen activated protein kinase kinase kinase antagonist”.
• mixed lineage kinase-7 refers to the mitogen activated protein kinase kinase kinase, subtype 7, as described by Bloem et al. , J. Mol. Cell Cardiol.; 33: 1739-1750, (2001), of the larger class of mitogen activated protein kinase kinase kinases, which functions as a serine/threonine kinase to activate cellular signaling pathways.
• CHF congestive heart failure
• congestive heart disease refers to a disease state of the cardiovascular system whereby the heart is unable to efficiently pump an adequate volume of blood to meet the requirements of the body's tissues and organ systems.
• CHF is charachterized by left ventricular failure (systolic dysfunction) and fluid accumulation in the lungs, with the underlying cause being attributed to one or more heart or cardiovascular disease states including coronary artery disease, myocardial infarction, hypertension, diabetes, myocardial injury, hemodynamic overload, valvular heart disease, and cardiomyopathy.
• diastolic congestive heart failure refers to a state of CHF characterized by impairment in the ability of the heart to properly relax and fill with blood.
• systolic congestive heart failure refers to a state of CHF characterized by impairment in the ability of the heart to properly contract and eject blood. It is understood that the terms “diastolic congestive heart failure” and “systolic congestive heart failure” are included within the term “congestive heart failure" or "CHF”.
• physiological disorders may present as a “chronic” condition, or an “acute” episode.
• chronic where used herein, means a condition of slow progress and long continuance.
• a chronic condition is treated when it is diagnosed and treatment continued throughout the course of the disease.
• acute means an exacerbated event or attack, of short course, followed by a period of remission.
• the treatment of physiological disorders contemplates both acute events and chronic conditions. In an acute event, compound is administered at the onset of symptoms and discontinued when the symptoms disappear. As described above, a chronic condition is treated throughout the course of the disease.
• the term "patient” refers to a mammal, such a mouse, gerbil, guinea pig, rat, dog or human. It is understood, however, that the preferred patient is a human.
• the terms “treating”, “treatment”, or “to treat” each mean to alleviate symptoms, eliminate the causation of resultant symptoms either on a temporary or permanent basis, and to prevent, slow the appearance, or reverse the progression or severity of resultant symptoms of the named disorder.
• the methods of this invention encompass both therapeutic and prophylactic administration.
• the term "effective amount” refers to the amount or dose of the compound, upon single or multiple dose administration to the patient, which provides the desired effect in the patient under diagnosis or treatment.
• An effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by the use of known techniques and by observing results obtained under analogous circumstances. In determining the effective amount or dose of compound administered, a number of factors are considered by the attending diagnostician, including, but not limited to: the species of mammal; its size, age, and general health; the degree of involvement or the severity of the disease involved; the response of the individual patient; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.
• a typical daily dose will contain from about 0.01 mg/kg to about 100 mg/kg of each compound used in the present method of treatment.
• daily doses will be about 0.05 mg/kg to about 50 mg/kg, more preferably from about 0.1 mg/kg to about 25 mg/kg.
• Oral administration is a preferred route of administering the compounds employed in the present invention whether administered alone, or as a combination of compounds capable of acting as a MAPKKK modulator. Oral administration, however, is not the only route, nor even the only preferred route.
• Other preferred routes of administration include transdermal, percutaneous, pulmonary, intravenous, intramuscular, intranasal, buccal, sublingual, or intrarectal routes.
• one of the compounds may be administered by one route, such as oral, and the other may be administered by the transdermal, percutaneous, pulmonary, intravenous, intramuscular, intranasal, buccal, sublingual, or intrarectal route, as particular circumstances require.
• the route of administration may be varied in any way, limited by the physical properties of the compounds and the convenience of the patient and the caregiver.
• the compounds employed in the present invention may be administered as pharmceutical compositions and, therefore, pharmaceutical compositions incorporating compounds of Formula I are important embodiments of the present invention.
• Such compositions may take any physical form that is pharmaceutically acceptable, but orally administered pharmaceutical compositions are particularly preferred.
• Such pharmaceutical compositions contain, as an active ingredient, an effective amount of a compound of Formula I, including the pharmaceutically acceptable salts and hydrates thereof, which effective amount is related to the daily dose of the compound to be administered.
• Each dosage unit may contain the daily dose of a given compound, or may contain a fraction of the daily dose, such as one-half or one-third of the dose.
• the amount of each compound to be contained in each dosage unit depends on the identity of the particular compound chosen for the therapy, and other factors such as the indication for which it is given.
• the pharmaceutical compositions of the present invention may be formulated so as to provide quick, sustained, or delayed release of the active ingredient after administration to the patient by employing well known procedures. The following discussion provides typical procedures for preparing pharmaceutical compositions incorporating the compounds of the present invention. However, the following is in no way intended to limit the scope of the pharmaceutical compositons provided by the present invention.
• compositions are preferably formulated in a unit dosage form, each dosage containing from about 1 to about 500 mg of each compound individually or in a single unit dosage form, more preferably about 5 to about 300 mg (for example 25 mg).
• unit dosage form refers to a physically discrete unit suitable as unitary dosages for a patient, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical carrier, diluent, or excipient.
• compositions contain from about 0.5%) to about 50% of the compounds in total, depending on the desired doses and the type of composition to be used.
• the amount of the compound is best defined as the "effective amount", that is, the amount of each compound which provides the desired dose to the patient in need of such treatment.
• the activity of the compounds employed in the present invention do not depend on the nature of the composition, hence, the compositions are chosen and formulated solely for convenience and economy.
• Capsules are prepared by mixing the compound with a suitable diluent and filling the proper amount of the mixture in capsules.
• suitable diluents include inert powdered substances such as starches, powdered cellulose especially crystalline and microcrystalline cellulose, sugars such as fructose, mannitol and sucrose, grain flours, and similar edible powders.
• Tablets are prepared by direct compression, by wet granulation, or by dry granulation. Their formulations usually incorporate diluents, binders, lubricants and disintegrators as well as the compound. Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts such as sodium chloride and powdered sugar. Powdered cellulose derivatives are also useful. Typical tablet binders are substances such as starch, gelatin and sugars such as lactose, fructose, glucose and the like.
• Natural and synthetic gums are also convenient, including acacia, alginates, methylcellulose, polyvinylpyrrolidine and the like. Polyethylene glycol, ethylcellulose and waxes can also serve as binders. Tablets are often coated with sugar as a flavor and sealant.
• the compounds may also be formulated as chewable tablets, by using large amounts of pleasant-tasting substances such as mannitol in the formulation, as is now well-established practice.
• Instantly dissolving tablet-like formulations are also now frequently used to assure that the patient consumes the dosage form, and to avoid the difficulty in swallowing solid objects that bothers some patients.
• a lubricant is often necessary in a tablet formulation to prevent the tablet and punches from sticking in the die.
• the lubricant is chosen from such slippery solids as talc, magnesium and calcium stearate, stearic acid and hydrogenated vegetable oils.
• Tablet disintegrators are substances which swell when wetted to break up the tablet and release the compound. They include starches, clays, celluloses, algins and gums. More particularly, corn and potato starches, methylcellulose, agar, bentonite, wood cellulose, powdered natural sponge, cation-exchange resins, alginic acid, guar gum, citrus pulp and carboxymethylcellulose, for example, may be used, as well as sodium lauryl sulfate.
• Enteric formulations are often used to protect an active ingredient from the strongly acid contents of the stomach. Such formulations are created by coating a solid dosage form with a film of a polymer which is insoluble in acid environments, and soluble in basic environments. Exemplary films are cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose acetate succinate.
• Cocoa butter is a traditional suppository base, which may be modified by addition of waxes to raise its melting point slightly.
• Water-miscible suppository bases comprising, particularly, polyethylene glycols of various molecular weights are in wide use, also.
• Transdermal patches have become popular recently. Typically they comprise a resinous composition in which the drugs will dissolve, or partially dissolve, which is held in contact with the skin by a film which protects the composition. Many patents have appeared in the field recently. Other, more complicated patch compositions are also in use, particularly those having a membrane pierced with innumerable pores through which the drugs are pumped by osmotic action. It is understood by one of ordinary skill in the art that the formulation procedures as described above can be readily applied to a method of treating physiological disorders susceptible to MAPKKK modulation or MLK-7 modulation , and particularly congestive heart failure.
• Rl represents hydrogen or methyl
• R2 represents: (a) phenyl; (b) phenyl optionally substituted one to three times with a substituent independently selected from the group consisting of:
• R2 represents:
• benzimidazole benzofuran, benzothiophene, benzo[l,3]-dioxolyl, benzothiazole, 2,2-dioxy-2,3-dihydro-lH-2 ⁇ 6 -benzo[c]thiophene, and indole optionally substituted one or two times with a substituent independently selected from the group consisting of: (i) amino, or (ii) (C ⁇ -C 4 )alkyl or (g) cyclohexyl;
• benzimidazole benzofuran, benzothiophene, benzo[ 1 ,3]-dioxolyl, benzothiazole, 2,2-dioxy-2,3-dihydro-lH-2 ⁇ -benzo[c]thiophene, and indole optionally substituted one or two times with a substituent independently selected from the group consisting of: (i) amino, or (ii) methyl; or (g) cyclohexyl;
• R13 independently represent hydrogen or methyl or R12 and R13 together with the nitrogen to which they are attached form a piperidino, pyrrolidino, morpholino or a methylpiperazino group;
• benzimidazole benzofuran, benzothiophene, benzo[l,3]-dioxolyl, benzothiazole, 2,2-dioxy-2,3-dihydro-lH-2 ⁇ 6 -benzo[c]thiophene, and indole optionally substituted one or two times with a substituent independently selected from the group consisting of:
• R2 represents phenyl or phenyl optionally substituted one to three times with a substituent independently selected from the group consisting of:
• R2 represents phenyl or phenyl optionally substituted one to three times with a substituent independently selected from the group consisting of:
• Y represents amino, (C ⁇ -C4)alkyl, (C ⁇
• X' represents -S-, -CH 2 -, -NH-, or -CO-, and Y' represents
• Rl 1 represents a group selected from the following:
• R13 independently represent hydrogen or methyl or R12 and R13 together with the nitrogen to which they are attached form a piperidino, pyrrolidino, morpholino or a methylpiperazino group;
• (M) R2 represents phenyl, 4-(N-acetylamino)phenyl, 2-hydroxyphenyl, 3- hydroxyphenyl, 4-hydroxyphenyl, 2-aminophenyl, 3-aminophenyl, 4- aminophenyl, 3-chlorophenyl, 4-chlorophenyl, 3-nitrophenyl, 4- nitrophenyl, 3-(methylsulfonylamino)phenyl, 4- (methylsulfonylamino)phenyl, 2-ethoxyphenyl, 3-ethoxyphenyl, 4- ethoxyphenyl, 4-methoxyphenyl, 4-fluoro-3 -methylphenyl, 4-Fluoro-2- methylphenyl, 4-bromophenyl, 4-ethylsulfanylphenyl, 4- methylsulfanylphenyl, 4-cyanophenyl, 4-acetylphenyl, 2- carboxamidophenyl, 4-(2-carboxy-
• R2 represents thiopheneyl, furanyl, imidazolyl, pyrazolyl, pyrrolyl, thiazolyl, triazolyl, pyridinyl, pyrimidyl, pyrazinyl, pyridiazinyl, piperidinyl, piperazinyl, pyrimidinyl, dioxo-thiomo ⁇ holinyl; or thiophenyl, furanyl, imidazolyl, pyrazolyl, pyrrolyl, thiazolyl, triazolyl, pyridinyl, pyrimidyl, pyrazinyl, pyridiazinyl, piperidinyl, piperazinyl, pyrimidinyl, or dioxo-thiomo ⁇ holinyl optionally substituted one to three times with a substituent independently selected from the group consisting of:
• R2 represents thiopheneyl, furanyl, imidazolyl, pyrazolyl, pyrrolyl, thiazolyl, triazolyl, pyridinyl, pyrimidyl, pyrazinyl, pyridiazinyl, piperidinyl, piperazinyl, pyrimidinyl, dioxo-thiomo ⁇ holinyl; or thiophenyl, furanyl, imidazolyl, pyrazolyl, pyrrolyl, thiazolyl, triazolyl, pyridinyl, pyrimidyl, pyrazinyl, pyridiazinyl, piperidinyl, piperazinyl, pyrimidinyl, or dioxo-thiomo ⁇ holinyl optionally substituted one to three times with a substituent independently selected from the group consisting of:
• R2 represents thiopheneyl, furanyl, imidazolyl, pyrazolyl, pyrrolyl, thiazolyl, triazolyl, pyridinyl, pyrimidyl, pyrazinyl, pyridiazinyl, piperidinyl, piperazinyl, pyrimidinyl, dioxo-thiomo ⁇ holinyl; or thiophenyl, furanyl, imidazolyl, pyrazolyl, pyrrolyl, thiazolyl, triazolyl, pyridinyl, pyrimidyl, pyrazinyl, pyridiazinyl, piperidinyl, piperazinyl, pyrimidinyl, or dioxo-thiomo ⁇ holinyl optionally substituted one to three times with a substituent independently selected from the group consisting of:
• R2 represents thiophen-2-yl, thiophen-3-yl, pyridin-4-yl, pyridin-3-yl, furan-3-yl, furan-2-yl, thiazol-2-yl, pyrazin-2-yl, pyridin-2-yl, lH-pyrrol-
• R2 represents benzimidazole, benzofuran, benzothiophene, benzo[ 1,3]- dioxolyl, benzothiazole, 2,2-dioxy-2,3-dihydro-lH-2 ⁇ 6 -benzo[c]thiophene, indole; or benzoimidazole, benzofuran, benzothiophene, benzo[l,3]- dioxolyl, benzothiazole, 2,2-dioxy-2,3-dihydro-lH-2 ⁇ 6 -benzo[c]thiophene, or indole optionally substituted one or two times with a substituent independently selected from the group consisting of:
• R2 represents lH-Indol-5-yl, Benzo[l,3]dioxol-5-yl, Benzo[b]tbiophen-2- yl, Benzofuran-2-yl, 4-Benzo[b]thiophen-3-yl, lH-Indol-2-yl, 2,2-Dioxy- 2,3-dihydro-lH-2 ⁇ 6 -benzo[c]thiophen-5-yl, lH-benzoimdazol-2-yl, or 2- amino benzothiazol-6-yl,; or (U) R2 represents cyclohexyl.
• the compounds of the present invention may be purified by methods known to one skilled in the art. These methods include crystallization, precipitation, normal phase silica gel chromatography and reverse-phase high-performace chromatography that is mass-guided and/or UV-guided. The purity of compounds is determined by LC/MS analysis.
• the salt composition is determined by HPLC anaysis.
• the HPLC system consists of a Shimadzu SCL-IOA controller, SIL-10A auto injector, LCIOAS pump, and a SPD- 10A UN detector (Kyoto, Japan).
• the Prevail Organic Acid column (25 cm x 4.6 mm ID.) is obtained from Alltech Associates Inc.(Deerfield, IL).
• the HPLC operating conditions consist of a mobile phase comprising 0.2M KH2PO4 (no pH adjustment).
• the mobile phase flow rate is set at 1.5 mL/minute with sample injections of 10 ⁇ L.
• the UN detector is set at 195 nm and run time is 3 minutes. TFA is quantitated by linear regression of peak area response versus standards prepared at known TFA concentrations.
• Scheme 1 depicts the synthesis of the dihydro-4H-pyrrolo[l,2- bjpyrazole core structure, substituted by heteroaromatics, preferably pyridine (with or without substitution) and quinoline.
• the quinoline may be substituted as well with a variety of aromatic or heterocyclic rings, specifically at the 7-position of the quinoline.
• a quinoline of Formula 2 can be prepared by reaction of an aniline with a vinyl ketone in the presence of an appropriate acid catalyst such as cone. ⁇ SO 4 in a suitable solvent such as dioxane.
• step B is sequentially treated with a strong base such as potassium hexamethyldisilazide, lithium hexamethyl-disilazide, or lithium diisopropyl amide at -78 °C in a suitable solvent such as tetrahydrofuran, and treated with a picolonic acid ester of Formula 3, such as methyl picolinate, which may be additionally substituted on the pyridine ring, to yield a methyl ketone of Formula 4.
• a strong base such as potassium hexamethyldisilazide, lithium hexamethyl-disilazide, or lithium diisopropyl amide at -78 °C in a suitable solvent such as tetrahydrofuran
• a picolonic acid ester of Formula 3 such as methyl picolinate, which may be additionally substituted on the pyridine ring, to yield a methyl ketone of Formula 4.
• step C Condensation of the methyl ketone (step C) with 1-aminopyrrolidinone hydrochloride (Formula 5) provides an imine of Formula 6 under basic conditions at room temperature (pyridine/ethanol or sodium ethoxide in ethanol). The imine undergoes cyclization by treatment with base such as sodium hydride or cesium carbonate under standard conditions (step D) to yield the 5,6- dihydro-4H-pyrrolo[l,2- bjpyrazole (Formula 7) with appropriate pyridyl and quinolinyl substitution.
• base such as sodium hydride or cesium carbonate
• H 2 SO 4 (14.4 mL, 270 mmol) is added to a solution of 3-bromoaniline (Aldrich, 30.0 g, 174 mmol) in 1,4-dioxane (1 L) at RT.
• the mixture is heated to reflux and treated with methyl vinyl ketone (Aldrich, 19.5 mL, 270 mmol) in 1,4-dioxane (50 mL) dropwise over 3 h. Heating is continued for 1 h after the addition, followed by removal of the solvent in vacuo.
• the residue is dissolved in water (100 mL), neutralized with Na 2 CO 3 and extracted with CH C1 .
• Scheme 2 depicts an alternative route for the preparation of the dihydropyrrolopyrazole central structure, beginning with step E.
• step E ethyl picolinylacetate (Formula 8) is reacted with 1-aminopyrrolidione hydrochloride (Formula 9) to yield an imine.
• the picolinate may be substituted at the 6-position of the nitrogen heterocycle.
• the reaction is conducted in a suitable solvent such as ethanol, N- methylpyrrolidinone or pyridine, with pyridine being the preferred solvent.
• the reaction is carried out at temperatures of about 60 °C to ambient for 4-24 hours.
• the products can be isolated and purified by techniques described above.
• Step F depicts the cyclization of a compound of Formula 10 to give an optionally substituted compound of Formula 11.
• the appropriate compound of Formula 10 is reacted with a suitable base that can form the anion of the hydrazone, cesium carbonate being the preferred base in a suitable solvent, preferably N,N-dimethylformamide, at 100 °C.
• a suitable solvent preferably N,N-dimethylformamide, at 100 °C.
• the ethyl ester of the pendant carboxylate on the resultant dihydropyrrolopyrazole Formula 11 is removed as in Step G by ethanolic base hydrolysis with gentle heating under standard conditions.
• the acid may be converted to a halide of Formula 13, preferably a bromide, as in Step H with N- halosuccinimide in the presence of a weak base such as sodium bicarbonate in a suitable solvent such as DMF, at room temperature.
• a weak base such as sodium bicarbonate
• a suitable solvent such as DMF
• Step I depicts the transformation of the halide of Formula 13 to a boronic acid of Formula 14, by treatment of the halide with a strong base such as n-butyllithium at -78 °C, followed by quenching of the intermediate carbanions with a source of boron, such as triisopropyl borate.
• the boronic acid may be liberated from the boronic ester upon workup with aqueous ammonium chloride. This transformation is well known and appreciated in the art (Larock, R. C, Comprehensive Organic Transformations, 2 nd Ed., copyright 1999, John Wiley & Sons, pp 741-742).
• the boronic acid or boronic ester of Formula 14 may subsequently be used as a leaving group in combination with a substituted aryl- or heteroarylhalide in the presence of a suitable palladium catalyst, preferably tetrakis(triphenylphosphine)palladium (0), and a suitable base such as potassium carbonate to further give compounds of Formula (I) (Suzuki reaction see: Miyaura, N.; Yanagi, T.; Suzuki, A. The Palladium-Catalyzed Cross Coupling Reaction of Phenylboronic Acid with Haloarenes in the Presence of Bases. Synth. Commun., 1981, 513-518).
• a suitable palladium catalyst preferably tetrakis(triphenylphosphine)palladium (0)
• a suitable base such as potassium carbonate
• the organic phase is separated and the aqueous phase is extracted 3 times with 30 mL portions of CHC1 3 .
• the combined organic extracts are washed with saturated brine, dried (Na 2 SO 4 ), filtered, and evaporated to a solid mass.
• the crude solid is purified by MPLC on silica gel using a linear gradient of 50% EtOAc/Hexanes to 80% EtOAc/Hexanes over 50 min.
• the chromatographed product is crystallized from Et O/Hexanes to yield the title compound, 0.528 g (77%), as white solid.
• Scheme 3 depicts the preparation of the compounds of Formula I tlirough a route which differs from Scheme 1.
• a 4-haloheterocycle of Formula 17a, 17b is prepared in two steps from a precursor 4-hydroxyheterocycle by conversion first to a triflate as in Step J.
• the triflate (Formula 16) is prepared by reaction of the 4- hydroxyquinoline with trifluoromethanesulfonic anhydride in the presence of a polar, basic solvent such as pyridine at 0 °C.
• halide salt such as potassium iodide (Step K) or lithium bromide (Step L)
• Step K a halide salt
• Step L lithium bromide
• a polar solvent such as dimethylformamide or acetonitrile
• temperatures ranging from RT to 100 °C The haloheterocycle can be coupled to a pyrrol opyrazole boronic acid of Formula 14 (Step M) in the presence of a palladium catalyst, preferably tris(dibenzylidineacetone)dipalladium (0) (Pd (dba) 3 ), with a suitable base such as potassium carbonate.
• a palladium catalyst preferably tris(dibenzylidineacetone)dipalladium (0) (Pd (dba) 3
• suitable base such as potassium carbonate.
• An additional ligand for the palladium such as triphenylphosphine, may be used. All of the reagents are combined in a suitable solvent, typically dioxane, and stirred at reflux temperature. All products (Formula 18) can be isolated and purified by silica gel chromatography (MPLC), reverse phase HPLC, or trituration of solid as described above.
• MPLC silica gel chromatography
• HPLC reverse phase HPLC
• R' and R" alkyl, aryl, heterocycle, or halogen or together form an aromatic or heterocyclic ring
• Step N A representative transformation is seen in Step N, in which a metal- nucleophile, such as trialkylstannyls, or boranes with a suitable base such as potassium carbonate, sodium alkoxides (sodium methoxide, or sodium ethoxide) or potassium alkoxides (potassium methoxide, or potassium ethoxide) can be used with a palladium catalyst, previously described, preferably tris(dibenzylidineacetone)dipalladium (0) (Pd (dba) 3 ).
• An additional ligand for the palladium such as triphenylphosphine, may be used.
• the stannyl or boronic acid reagents may be aromatic with one or more substituents or variously substituted heteroaromatics. Protecting groups may be required on the O, N, or S-containing functional groups.
• organozinc reagents may also be employed to effect the net displacement of the halide at the C-7 position by an alkyl group. All of the reagents are combined in a suitable solvent, typically dioxane, isopropyl alcohol, tetrahydrofuran, toluene or ethylene glycol dimethyl ether, stirred at temperatures from room temperature to reflux.
• the coupling of arylhalides with metallo-aromatics and metallo-alkyls by similar methods has been reviewed in the literature (Stanforth, S. P. Tetrahedron, 1998, 54, 263-303).
• the product of Formula 20 may require an additional synthetic step to remove a group such as t-butoxycarbonyl or trimethylsilyl, by treatment with trifluoroacetic acid or tetra-n-butylammonium fluoride, respectively.
• Such deprotection conditions are well known in the art. (Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic Synthesis (3 rd ed.); Wiley: New York, 1999.) All products can be isolated and purified by silica gel chromatography (MPLC), reverse phase HPLC, or trituration of solid as described above.
• R2 aryl, heterocycle, cycloalkyl
• the reaction mixture is cooled, diluted with 2 mL water and 4 mL EtOAc, and agitated. The water layer is removed, 2 mL water is added, and the extraction procedure repeated. The organic layer is eluted over 1 g silica gel cartridge with 25 mL MeOH containing one drop concentrated NH 4 OH. The MeOH solution is evaporated under reduced pressure. The residue is dissolved in 20 mL EtOH and evaporated again. Purified product is isolated as a free base by trituration with DMSO and drying at high vacuum.
• the product TFA salt (53.2 mg, 43% yield) is obtained by freeze drying appropriate fractions (based on LC/MS analysis) from reverse phase HPLC using Waters Symmetry C18 column with a gradient of 10 to 70% B in A, where A is water containing 0.1% TFA and B is CH 3 CN containing 0.1% TFA.
• MS ES+ m/e 395 (M+l of free base)
• Example 55 Using the method of Example 55, changing only the procedure to isolate the crude zwitterionic product from the aqueous layer by precipitation with dilute HCl when there is an acidic functionality situated on the aryl ring of the boronic acid, the following compounds are made.
• Scheme 5 demonstrates an alternative means of attaching various aryl rings and heterocycles at the C-7 position of the 7-bromoquinoline of Formula 19.
• Step O illustrates that the bromide can be converted to a boronic ester of Formula 21 that can then be coupled to aryl-, heterocyclic- or alkylbromides in metal-catalyzed reactions to provide C- 7 substituted quinoline compounds of Formula 20.
• the conversion of the heteroaryl bromide (Formula 19) is accomplwashed by treatment with bis(pinacolato)diboron and KOAc in a polar, high boiling solvent such as DMSO.
• a palladium catalyst preferably l,l'-bis(diphenylphosphino)ferrocene palladium (II) chloride complex with dichloromethane (PdCl (dppf)
• PdCl (dppf) dichloromethane
• the boronate (Formula 21) is coupled (step P) to an aryl- or heterocyclic halide with a suitable base such as potassium carbonate, sodium alkoxides (sodium methoxide, or sodium ethoxide) or potassium alkoxides (potassium methoxide, or potassium ethoxide) in combination with a palladium catalyst, as previously described, preferably tris(dibenzylidineacetone)dipalladium (0) (Pd 2 (dba) 3 ).
• a palladium catalyst preferably tris(dibenzylidineacetone)dipalladium (0) (Pd 2 (dba) 3 ).
• An additional ligand for the palladium such as triphenylphosphine, may be used.
• reagents are combined in a suitable solvent, typically dioxane, isopropyl alcohol, tetrahydrofuran, toluene or ethylene glycol dimethyl ether, stirred at temperatures from room temperature to reflux.
• a suitable solvent typically dioxane, isopropyl alcohol, tetrahydrofuran, toluene or ethylene glycol dimethyl ether, stirred at temperatures from room temperature to reflux.
• the product of Formula 20 may require an additional deprotection step to remove a group such as t-butoxycarbonyl or trimethylsilyl, by treatment with trifluoroacetic acid or tetra- n-butylammonium fluoride, respectively. All products can be isolated and purified by silica gel chromatography (MPLC), reverse phase HPLC, or trituration of solid as described above.
• Purified product (29.2 mg, 32%) is obtained by freeze drying appropriate fractions (based on MS analysis) from reverse phase HPLC using Waters Symmetry CI 8 column with a gradient of 10 to 70% B in A, where A is water containing 0.1% TFA and B is CH 3 CN containing 0.1 % > TFA.
• Example 73 Using the method of Example 73, changing only the workup procedure to isolate the crude product from the aqueous layer by precipitation or evaporation after treatment with dilute HCl when there is an acidic functionality situated on the aryl ring of the aryl bromide coupling partner, the following compounds are prepared:
• Step Q depicts the modification of the 7-position of the quinoline by attachment of a nitrogen heterocycle, either aromatic or alkyl, to form a carbon-nitrogen bond to provide compounds of Formula 22.
• This transformation requires a base, preferably potassium tert-butoxide, a high boiling polar solvent such as dimethylsulfoxide, and a metal catalyst, preferably copper (0).
• the reaction is conducted at high temperatures, up to 200 °C, and following workup, the products are purified by reverse phase HPLC. Copper-catalyzed nucleophihc substitutions of aryl halides have been reviewed by Lindley (See: Lindley, J. Tetrahedron 1984, 40, 1433-1456).
• Step Q can also be accomplished by palladium catalysis, preferably tris(dibenzylideneacetone) dipalladium (0) (Pd 2 (dba) ) ) with heterocyclicamines such as piperidine, piperazine, morpholine, or thiomorpholine.
• An alkoxide base for example, sodium tert-butoxide, as well as 18-crown-6 and 2-(di-t-butylphosphino)biphenyl (BINAP) are also preferred for the transformation.
• the preferred solvent is tetrahydrofuran.
• the palladium-catalyzed method has been described by Wolfe and Buchwald (Wolfe, J. P.; Buchwald, S. L. J. Org. Chem. 1997, 62, 6066-6068).
• Purified product 36.3 mg (37%) is obtained by freeze drying appropriate fractions (based on MS analysis) from reverse phase HPLC using Waters Symmetry C18 column with a gradient of 10 to 70% B in A, where A is water containing 0.1% TFA and B is CH 3 CN containing 0.1 %> TFA.
• Tris(dibenzylideneacetone) dipalladium (0) (0.014 g, 0.015 mmol) and 2-(di-t-butylphosphino)biphenyl (0.009 g, 0.03 mmol) are added, followed by degassing and flushing with nitrogen.
• the mixture is stirred at RT for 8 h under nitrogen.
• the crude mixture is diluted with EtOAc (20 mL) and extracted with saturated NaHCO 3 .
• the organic phase is separated and washed with saturated brine, dried (Na 2 SO 4 ), filtered, and evaporated to a solid mass.
• Primary amides maybe prepared from the carboxylic acids as well, via an intermediate acid chloride that is obtained by treatment with either oxalyl chloride or thionyl chloride.
• the intermediate acid chloride is reacted with a source of ammonia, such as gaseous NH 3 or ammonium hydroxide, to yield the amide.
• a source of ammonia such as gaseous NH 3 or ammonium hydroxide
• substituted secondary and tertiary amides are obtained from the carboxylic acids by reaction with a coupling reagent such as HBTU in the presence of an amine base such as diisopropylethylamine, in a suitable solvent, preferably dimethylformamide, or dichloromethane, or tetrahydrofuran.
• a coupling reagent such as HBTU
• an amine base such as diisopropylethylamine
• suitable solvent preferably dimethylformamide, or dichloromethane, or tetrahydrofuran.
• the alkylation of a phenolic "X" takes advantage of the reactivity of the functional group as a nucleophile through treatment with an electrophile possessing a good leaving group (for example, triflate, iodide, bromide, or chloride).
• a base such as potassium carbonate, sodium hydride, or an anhydrous alkoxide can be used to deprotonate the phenol and increase its nucleophilicity, in a suitable polar aprotic solvent, for example tetrahydrofuran, dimethylformamide, dioxane. These reactions are performed at temperatures ranging from ambient to reflux.
• the cap is positioned, and mixture heated in oil bath at 60 °C for 16 h. Most of the solvent is removed under vacuum, and the residue is triturated with 15 mL of saturated K 2 CO 3 solution, giving 127 mg of the crude titled compound as a free base.
• the purified product 169 mg, (80%) is obtained by freeze drying appropriate fractions (based on MS analysis) from reverse phase HPLC using Waters Symmetry C18 column with a gradient of 10 to 70% B in A, where A is water containing 0.1% TFA and B is CH 3 CN containing 0.1% TFA.
• GST glutathione-s-transferase
• the in vitro kinase reaction is run with recombinant MLK-7 at 1 nm and various concentrations of test compound in a reaction mixture containing 2 mM DTT, 30 uM ATP, 5 mM MgC12, 5 uM MBP, 4% DMSO, 5 uCi 33P in 44 mM HEPES,(pH 7.4).
• the reaction is carried out for 2 hours at room temperature and then stopped by the addition of phosphoric acid to 5.5%.
• the phosphorylated MBP is collected on a filter and radioactivity associated with the membrane measured using a scintillation counter.
• Inhibitors are identified by the ability to reduce the transfer of radioactive phosphate from ATP to MBP compared to controls run without test compound.
• IC50 values defined as the concentration of test compound required to decrease the transfer of radioactive phosphate from ATP to MBP by 50%, are then determined. Ki values (which refers to the dissociation constant of an enzyme-antagonist complex and serves as an index of ligand binding) for each respective test compound can also be calculated by application of the Cheng-Prusoff equation as described in Cheng et al, Relationship Between The Inhibition Constant (Ki) and The Concentration of Inhibitor Which Causes 50% inhibition (IC50) of an Enzymatic Reaction, Biochem. Pharmacol., 22: 3099-31088; (1973).
• Representative compounds of the present invention have an IC50 in the MLK-7 kinase assay of ⁇ 10,000nM Table I (see infra.) provides IC50 data from the afore mentioned MLK-7 kinase assay for a representative sample of the exemplified compounds of the present invention.
• Mammalian expression vectors containing either MLK-7 or J ⁇ K cD ⁇ A are co- transfected into Cos cells DMEM supplemented with 5% fetal calf serum and 0.1 mg.ml ampicillin.
• the cDNA is expressed for 24 hours at which time media is aspirated and replaced with media containing the selected concentration of test compound.
• cells are lysed and analysed for phospho-JNK (pJNK) levels using the pJNK luminex assay according to the manufacturers instruction (BioRad).
• pJNK phospho-JNK
• MLK-7 inhibitors are evaluated based on the ability to reduce pJNK levels in the Cos cells compared to control samples incubated in the absence of test compound.
• Test compounds may also be evaluated for the ability to inhibit phospho-p38 formation in cardiac myocytes.
• Primary cardiac myocytes are collected by trypsin digestion of neo-natal rat heart tissue. The cells incubated in Dulbecco's modified Eagles' medium DMEM/F12 (1:1 v/v) supplemented with 2 g/L bovine serum albumin, 3 mM MEM sodium pyruvic acid, 15 mM HEPES, 100 ⁇ g/mL ampicillin, 1 ⁇ g/mL transferrin, 10 ng/mL sodium selenite, and 1 ⁇ g/mL insulin are infected with recombinant adenovirus expressing MLK-7.
• MLK-7 inhibitors are evaluated based on the ability to reduce phospho-p38 levels in cardiac myocytes compared to control samples incubated in the absence of test compound.
• In vitro and cell based kinase assay protocols for MLK-7 similar to those described above, can be readily designed by the ordinarily skilled artisan. Bloem et al, J. Mol.
• United States Patent No. 6,146,832 provides a detailed description of the isolation of the cDNA encoding MLK-7 (designated therein as CSAPK 2), expression of recombinant MLK-7 (CSAPK2) protein in bacterial cells and expression of recombinant MLK-7.

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