Classifications

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  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
  • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
  • A61K31/4412—Non condensed pyridines; Hydrogenated derivatives thereof having oxo groups directly attached to the heterocyclic ring
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
  • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
  • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
• • 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
• • 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
• • 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
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
  • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
  • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
  • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D213/62—Oxygen or sulfur atoms
  • C07D213/63—One oxygen atom
  • C07D213/65—One oxygen atom attached in position 3 or 5
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D261/00—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings
  • C07D261/02—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings
  • C07D261/06—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings having two or more double bonds between ring members or between ring members and non-ring members
  • C07D261/10—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings having two or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D261/18—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D333/00—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom
  • C07D333/02—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings
  • C07D333/04—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom
  • C07D333/26—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D333/38—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
  • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
  • C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
  • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
  • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

• the invention relates to substituted benzamide derivatives, particularly to compounds that selectively modulate the cardiac sarcomere, and specifically to compounds, pharmaceutical formulations and methods of treatment for systolic heart failure, including congestive heart failure.
• the "sarcomere” is an elegantly organized cellular structure found in cardiac and skeletal muscle made up of interdigitating thin and thick filaments; it comprises nearly 60% of cardiac cell volume.
• the thick filaments are composed of "myosin,” the protein responsible for transducing chemical energy (ATP hydrolysis) into force and directed movement. Myosin and its functionally related cousins are called motor proteins.
• the thin filaments are composed of a complex of proteins. One of these proteins, "actin” (a filamentous polymer) is the substrate upon which myosin pulls
• Myosin is the most extensively studied of all the motor proteins. Of the thirteen distinct classes of myosin in human cells, the myosin-ll class is responsible for contraction of skeletal, cardiac, and smooth muscle. This class of myosin is significantly different in amino acid composition and in overall structure from myosin in the other twelve distinct classes (Goodson HV and Spudich JA. (1993) Proc. Natl. Acad. Sci. USA 90:659-663). Myosin-ll consists of two globular head domains linked together by a long alpha-helical coiled-coiled tail that assembles with other myosin-lls to form the core of the sarcomere's thick filament.
• the globular heads have a catalytic domain where the actin binding and ATP functions of myosin take place.
• actin binding and ATP functions of myosin take place.
• the release of phosphate leads to a change in structural conformation of the catalytic domain that in turn alters the orientation of the light-chain binding lever arm domain that extends from the globular head; this movement is termed the powerstroke.
• This change in orientation of the myosin head in relationship to actin causes the thick filament of which it is a part to move with respect to the thin actin filament to which it is bound (Spudich JA. (2001 ) Nat Rev Mol Cell Biol. 2(5):387-92).
• actin filament also Ca 2+ modulated
• Mammalian heart muscle consists of two forms of cardiac myosin, alpha and beta, and they are well characterized (Robbins, supra).
• the beta form is the predominant form (> 90 percent) in adult human cardiac muscle. Both have been observed to be regulated in human heart failure conditions at both transcriptional and translational levels (Miyata supra), with the alpha form being down-regulated in heart failure.
• the components of the cardiac sarcomere present targets for the treatment of heart failure, for example by increasing contractility or facilitating complete relaxation to modulate systolic and diastolic function, respectively.
• CHF Congestive heart failure
• systolic dysfunction an impairment of cardiac contractility (with a consequent reduction in the amount of blood ejected with each heartbeat).
• systolic dysfunction with compensatory dilation of the ventricular cavities results in the most common form of heart failure, "dilated cardiomyopathy,” which is often considered to be one in the same as CHF.
• the counterpoint to systolic dysfunction is diastolic dysfunction, an impairment of the ability to fill the ventricles with blood, which can also result in heart failure even with preserved left ventricular function.
• Congestive heart failure is ultimately associated with improper function of the cardiac myocyte itself, involving a decrease in its ability to contract and relax.
• systolic and/or diastolic dysfunction such as atherosclerosis, hypertension, viral infection, valvular dysfunction, and genetic disorders.
• Patients with these conditions typically present with the same classical symptoms: shortness of breath, edema and overwhelming fatigue.
• dilated cardiomyopathy the cause of their heart dysfunction is ischemic heart disease due to coronary atherosclerosis.
• Acute congestive heart failure also known as acute "decompensated” heart failure
• a new myocardial infarction, discontinuation of medications, and dietary indiscretions may all lead to accumulation of edema fluid and metabolic insufficiency even in the resting state.
• a therapeutic agent that increases heart function during such an acute episode could assist in relieving this metabolic insufficiency and speeding the removal of edema, facilitating the return to the more stable "compensated" congestive heart failure state.
• Patients with very advanced congestive heart failure particularly those at the end stage of the disease also could benefit from a therapeutic agent that increases heart function, for example, for stabilization while waiting for a heart transplant.
• Other potential benefits could be provided to patients coming off a bypass pump, for example, by administration of an agent that assists the stopped or slowed heart in resuming normal function.
• Patients who have diastolic dysfunction could benefit from a therapeutic agent that modulates relaxation.
• Inotropes are drugs that increase the contractile ability of the heart. As a group, all current inotropes have failed to meet the gold standard for heart failure therapy, i.e., to prolong patient survival. In addition, current agents are poorly selective for cardiac tissue, in part leading to recognized adverse effects that limit their use. Despite this fact, intravenous inotropes continue to be widely used in acute heart failure (e.g., to allow for reinstitution of oral medications or to bridge patients to heart transplantation) whereas in chronic heart failure, orally given digoxin is used as an inotrope to relieve patient symptoms, improve the quality of life, and reduce hospital admissions.
• the present invention provides compounds, pharmaceutical compositions and methods for the treatment of heart disease including CHF, particularly systolic heart failure.
• the compositions are selective modulators of the cardiac sarcomere, for example potentiating cardiac myosin.
• R 1 is alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl;
• R 2 is acetyl, acetylamino, acetylene, alkoxycarbonyl, carboxamido, cyano, halo, heteroaryl, hydrogen, nitrile, nitro, and trifluoromethyl;
• R 3 is aryl, substituted aryl, heteroaryl, and substituted heteroaryl; including single stereoisomers, mixtures of stereoisomers, and the pharmaceutically acceptable salts thereof.
• the compounds of Formula I are useful as active agents in practice of the methods of treatment and in manufacture of the pharmaceutical formulations of the invention, and as intermediates in the synthesis of such active agents.
• the invention relates to compounds represented by
• R 1 is alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl;
• R 2 is acetyl, acetylamino, acetylene, alkoxycarbonyl, carboxamido, cyano, halo, heteroaryl, hydrogen, nitrile, and trifluoromethyl;
• R 3 is aryl, substituted aryl, heteroaryl, and substituted heteroaryl; or a single stereoisomer, or mixture of stereoisomers, or a pharmaceutically acceptable salt thereof.
• R 1 is optionally substituted aryl or heteroaryl, preferably phenyl, substituted phenyl or pyridinyl (especially 3-chlorophenyl, 4-fluorophenyl, 4- hydroxyphenyl, 3-methylphenyl, pyridin-2-yl, pyridin-3-yl or pyridin-4-yl);
• R 2 is acetyl, acetylene, alkoxycarbonyl, cyano, halo, heteroaryl, hydrogen or
• the invention relates to compounds represented by
• R 1 is alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl;
• R 2 is nitro
• R 3 is aryl, substituted aryl, heteroaryl or substituted heteroaryl; provided that: when R 1 is pyridin-3-yl, R 3 is not 3-bromophenyl, 3-fluorophenyl, 4-fluorophenyl, furan-2-yl, 4-methoxyphenyl or 1 -methyl- 1 H-pyrazol-3-yl; when R 1 is 4-hydroxyophenyl, R 3 is not furan-2-yl; when R 1 is 4-fluorophenyl, R 3 is not furan-2-yl or pyridin-4-yl; and when R 3 is pyridin-4-yl, R 1 is not phenyl, 3-chlorophenyl, 4-hydroxyphenyl or 3- methylphenyl; or a single stereoisomer, or mixture of stereoisomers, or a pharmaceutically acceptable salt thereof.
• R 1 is optionally substituted aryl or heteroaryl, preferably phenyl, substituted phenyl or pyridinyl (especially 3-chlorophenyl, 4-fluorophenyl, 4-hydroxyphenyl, 3-methylphenyl, pyridin-2-yl or pyridin-3-yl); and
• R 3 is optionally substituted aryl or optionally substituted heteroaryl (especially phenyl, 4-fluorophenyl, 4-cyanophenyl, 4-methoxyphenyl, 4-methylphenyl, isoxazol-5-yl, thiophene-2-yl or 5-methyl-thiophene-2-yl).
• the invention relates to a pharmaceutical formulation including a pharmaceutically acceptable excipient, and to a method of treatment for
• R 1 is alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl;
• R 2 is acetyl, acetylamino, acetylene, alkoxycarbonyl, carboxamido, cyano, halo, heteroaryl, hydrogen, nitrile, nitro or trifluoromethyl;
• R 3 is aryl, substituted aryl, heteroaryl or substituted heteroaryl; or a single stereoisomer, or mixture of stereoisomers, or a pharmaceutically acceptable salt thereof.
• Preferred in this aspect are the above-described preferred compounds, isomers and salts, and those where:
• R 1 is substituted phenyl (especially 4-fluorophenyl) and R 2 is nitro;
• R 1 is heteroaryl (especially pyridin-3-yl) and R 2 is fluoro;
• R 1 is heteroaryl (especially pyridin-3-yl) and R 2 is nitro, particularly where R 3 is phenyl, 4-fluorophenyl, 4-cyanophenyl, 4-methoxyphenyl or 4-methylphenyl.
• the present invention provides methods of screening for compounds that will bind to myosin (particularly myosin III, ⁇ cardiac myosin or ⁇ cardiac myosin), for example compounds that will displace or compete with the binding of the compounds of the invention.
• the methods comprise combining a labeled compound of the invention, myosin, and at least one candidate agent and determining the binding of the candidate agent to myosin.
• the invention provides methods of screening for modulators of the activity of myosin.
• the methods comprise combining a compound of the invention, myosin, and at least one candidate agent and determining the effect of the candidate agent on the activity of myosin.
• DIEA N,N-diisopropylethylamine
• Ph phenyl
• substituted alkyl includes optionally substituted cycloalkyl groups, which in turn are defined as including optionally substituted alkyl groups, potentially ad infinitum
• substitution or substitution patterns e.g., substituted alkyl includes optionally substituted cycloalkyl groups, which in turn are defined as including optionally substituted alkyl groups, potentially ad infinitum
• Alkyl is intended to include linear, branched, or cyclic hydrocarbon structures and combinations thereof.
• Lower alkyl refers to alkyl groups of from 1 to 5 carbon atoms. Examples of lower alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, s-and t-butyl and the like.
• Preferred alkyl groups are those of C 20 or below. More preferred alkyl groups are those of C13 or below. Still more preferred alkyl groups are
• Cycloalkyl is a subset of alkyl and includes cyclic hydrocarbon groups of from 3 to 13 carbon atoms. Examples of cycloalkyl groups include c-propyl, c- butyl, c-pentyl, norbornyl, adamantyl and the like.
• alkyl refers to alkanyl, alkenyl and alkynyl residues; it is intended to include cyclohexylmethyl, vinyl, allyl, isoprenyl and the like.
• Alkylene is another subset of alkyl, referring to the same residues as alkyl, but having two points of attachment.
• alkylene examples include ethylene (-CH 2 CH 2 -), propylene (-CH 2 CH 2 CH 2 -), dimethylpropylene (-CH 2 C(CH 3 ) 2 CH 2 -) and cyclohexylpropylene (-CH 2 CH 2 CH(C 6 H ⁇ 3 )-).
• alkyl residue having a specific number of carbons all geometric isomers having that number of carbons are intended to be encompassed; thus, for example, "butyl” is meant to include n-butyl, sec-butyl, isobutyl and t-butyl; "propyl” includes n-propyl and isopropyl.
• alkoxy refers to the group -O-alkyl, preferably including from 1 to 8 carbon atoms of a straight, branched, cyclic configuration and combinations thereof attached to the parent structure through an oxygen. Examples include methoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy, cyclohexyloxy and the like. Lower-alkoxy refers to groups containing one to four carbons.
• substituted alkoxy refers to the group -O-(substituted alkyl).
• One preferred substituted alkoxy group is "polyalkoxy" or -O-(optionally substituted alkylene)-(optionally substituted alkoxy), and includes groups such as -OCH 2 CH 2 OCH 3 , and glycol ethers such as polyethyleneglycol and -O(CH 2 CH 2 O) x CH 3 , where x is an integer of about 2-20, preferably about 2-10, and more preferably about 2-5.
• Another preferred substituted alkoxy group is hydroxyalkoxy or -OCH 2 (CH 2 ) y OH, where y is an integer of about 1-10, preferably about 1-4.
• Acyl refers to groups of from 1 to 10 carbon atoms of a straight, branched, cyclic configuration, saturated, unsaturated and aromatic and combinations thereof, attached to the parent structure through a carbonyl functionality. One or more carbons in the acyl residue may be replaced by nitrogen, oxygen or sulfur as long as the point of attachment to the parent remains at the carbonyl. Examples include acetyl, benzoyl, propionyl, isobutyryl, t-butoxycarbonyl, benzyloxycarbonyl and the like. "Lower- acyl” refers to groups containing 1 to 4 carbons and "acyloxy” refers to the group O-acyl.
• amino refers to the group -NH 2 .
• substituted amino refers to the group -NHR or -NRR where each R is independently selected from the group: optionally substituted alkyl, optionally substituted alkoxy, optionally substituted
• Aryl and “heteroaryl” mean a 5-, 6- or 7-membered aromatic or heteroaromatic ring containing 0-4 heteroatoms selected from O, N or S; a bicyclic 9- or
• the aromatic 6- to 14-membered aromatic carbocyclic rings include, e.g., phenyl, naphthalene, indane, tetralin, and fluorene and the 5- to 10-membered aromatic heterocyclic rings include, e.g., imidazole, oxazole, isoxazole, oxadiazole, pyridine, indole, thiophene, benzopyranone, thiazole, furan, benzimidazole, quinoline, isoquinoline, quinoxaline, pyrimidine, pyrazine, tetrazole and pyrazole.
• alkoxy refers to the group -O-aralkyl.
• heterooaralkoxy refers to the group -O-heteroaralkyl;
• aryloxy refers to -O-aryl; and
• heteroaryloxy refers to the group -O-heteroaryl.
• Alkyl refers to a residue in which an aryl moiety is attached to the parent structure via an alkyl residue. Examples include benzyl, phenethyl, phenylvinyl, phenylallyl and the like.
• Heteroaralkyl refers to a residue in which a heteroaryl moiety is attached to the parent structure via an alkyl residue. Examples include furanylmethyl, pyridinylmethyl, pyrimidinylethyl and the like.
• ATPase refers to an enzyme that hydrolyzes ATP.
• ATPases include proteins comprising molecular motors such as the myosins.
• Halogen or "halo” refers to fluorine, chlorine, bromine or iodine.
• Dihaloaryl, dihaloalkyl, trihaloaryl etc. refer to aryl and alkyl substituted with a plurality of halogens, but not necessarily a plurality of the same halogen; thus 4-chloro-3-fluorophenyl is within the scope of dihaloaryl.
• Heterocycle means a cycloalkyl or aryl residue in which one to four of the carbons is replaced by a heteroatom such as oxygen, nitrogen or sulfur.
• heterocycles that fall within the scope of the invention include imidazoline, pyrrolidine, pyrazole, pyrrole, indole, quinoline, isoquinoline, tetrahydroisoquinoline, benzofuran, benzodioxan, benzodioxole (commonly referred to as methylenedioxyphenyl, when
• N-heterocyclyl refers to a nitrogen-containing heterocycle as a substituent residue.
• heterocyclyl encompasses heteroaryl, which is a subset of heterocyclyl.
• N-heterocyclyl residues include 4-morpholinyl, 4- thiomorpholinyl, 1-piperidinyl, 1-pyrrolidinyl, 3-thiazolidinyl, piperazinyl and 4-(3,4- dihydrobenzoxazinyl).
• substituted heterocyclyl include 4-methyl-1- piperazinyl and 4-benzyl-1-piperidinyl.
• “Isomers” are different compounds that have the same molecular formula.
• “Stereoisomers” are isomers that differ only in the way the atoms are arranged in space.
• “Enantiomers” are a pair of stereoisomers that are non-superimposable mirror images of each other. A 1 :1 mixture of a pair of enantiomers is a “racemic” mixture. The term “(. ⁇ .)” is used to designate a racemic mixture where appropriate.
• “Diastereoisomers” are stereoisomers that have at least two asymmetric atoms, but which are not mirror-images of each other. The absolute stereochemistry is specified according to the Cahn-lngold-Prelog R-S system.
• stereochemistry at each chiral carbon may be specified by either R or S.
• Resolved compounds whose absolute configuration is unknown can be designated (+) or (-) depending on the direction (dextro- or levorotatory) which they rotate plane polarized light at the wavelength of the sodium D line.
• Certain of the compounds described herein contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)-.
• the present invention is meant to include all such possible isomers, including racemic mixtures, optically pure forms and intermediate mixtures.
• Optically active (R)- and (S)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques.
• the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.
• pharmaceutically acceptable carrier or “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
• compositions S7155P1 WO - 12 -
• pharmaceutically acceptable salt refers to salts that retain the biological effectiveness and properties of the compounds of this invention and, which are not biologically or otherwise undesirable. In many cases, the compounds of this invention are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto. Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids.
• Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.
• Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.
• Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.
• Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like; particularly preferred are the ammonium, potassium, sodium, calcium and magnesium salts.
• Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, specifically such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.
• Substituted- alkyl, aryl, heteroaryl and heterocyclyl refer respectively to alkyl, aryl, heteroaryl and heterocyclyl wherein one or more (up to about 5, preferably up to about 3) hydrogen atoms are replaced by a substituent independently selected from the group: optionally substituted alkyl (e.g., fluoroalkyl), optionally substituted alkoxy, alkylenedioxy (e.g.
• optionally substituted amino e.g., alkylamino and dialkylamino
• optionally substituted amidino optionally substituted aryl (e.g., phenyl), optionally substituted aralkyl (e.g., benzyl), optionally substituted aryloxy (e.g., phenoxy), optionally substituted aralkoxy (e.g., benzyloxy), carboxy (-COOH), carboalkoxy (i.e.,
• sulfanyl refers to the groups: -S-(optionally substituted alkyl),
• sulfinyl refers to the groups: -S(O)-H, -S(O)-(optionally substituted alkyl), -S(O)-(optionally substituted amino), -S(O)-(optionally substituted aryl), -S(O)-(optionally substituted heteroaryl), and -S(O)-(optionally substituted heterocyclyl).
• sulfonyl refers to the groups: -S(O 2 )-H, -S(O 2 )-(optionally substituted alkyl), -S(O 2 )-(optionally substituted amino), -S(O 2 )-(optionally substituted aryl), -S(O 2 )-(optionally substituted heteroaryl), -S(O 2 )-(optionally substituted heterocyclyl) ,-S(O 2 )-(optionally substituted alkoxy), -S(O 2 )-optionally substituted aryloxy), -S(O 2 )-(optionally substituted heteroaryloxy), and -S(O 2 )-(optionally substituted heterocyclyloxy).
• therapeutically effective amount refers to that amount of a compound of Formula I that is sufficient to effect treatment, as defined below, when administered to a mammal in need of such treatment.
• the therapeutically effective amount will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the particular compound of Formula I chosen, the dosing regimen to be followed, timing of administration, the manner of administration and the like, all of which can readily be determined by one of ordinary skill in the art.
• treatment means any treatment of a disease in a mammal, including: a) preventing the disease, that is, causing the clinical symptoms of the disease not to develop; b) inhibiting the disease, that is, slowing or arresting the development of clinical symptoms; and/or c) relieving the disease, that is, causing the regression of clinical symptoms.
• the present invention is directed to the compounds represented by
• Formula I which are selective modulators of the cardiac sarcomere (e.g., by stimulating or otherwise potentiating the activity of cardiac myosin), as follows:
• R 1 is selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl;
• R 2 is selected from the group: acetyl, acetylamino, acetylene, alkoxycarbonyl, carboxamido, cyano, halo, heteroaryl, hydrogen, nitrile, nitro, and trifluoromethyl; and
• R 3 is selected from the group consisting of aryl, substituted aryl, heteroaryl, and substituted heteroaryl; including single stereoisomers, mixtures of stereoisomers, and the pharmaceutically acceptable salts thereof.
• the compounds of Formula I are useful as active agents in practice of the methods of treatment and in manufacture of the pharmaceutical formulations of the invention, and as intermediates in the synthesis of such active agents.
• Formula IA i.e., the compound according to Formula I where R 1 is pyridin-3-yl, R 2 is trifluoromethyl and R 3 is 4-fluorophenyl, can be named 4-fluoro- ⁇ /-[3-(pyridin-3-yloxy)-5-trifluoromethyl- phenyl]-benzamide.
• R 1 is pyridin-3-yl
• R 2 is trifluoromethyl
• R 3 is 4-fluorophenyl
• Formula IB i.e., the compound according to Formula I where R 1 is 4-fluorophenyl, R 2 is nitro and R 3 is isoxalyl-5-yl, can be named isoxazole-5-carboxylic acid [3-(4-fluoro-phenoxy)-5-nitro- phenyl-amide.
• reaction times and conditions are intended to be approximate, e.g., taking place at about atmospheric pressure within a temperature range of about -10°C to about 110°C over a period of about 1 to about 24 hours; reactions left to run overnight average a period of about 16 hours.
• solvent each mean a solvent inert under the conditions of the reaction being described in conjunction therewith [including, for example, benzene, toluene, acetonitrile, tetrahydrofuran (“THF”), dimethylformamide (“DMF”), chloroform, methylene chloride (or dichloromethane), diethyl ether, methanol, pyridine and the like].
• solvents used in the reactions of the present invention are inert organic solvents.
• q.s means adding a quantity sufficient to achieve a stated function, e.g., to bring a solution to the desired volume (i.e., 100%).
• Isolation and purification of the compounds and intermediates described herein can be effected, if desired, by any suitable separation or purification procedure such as, for example, filtration, extraction, crystallization, column chromatography, thin-layer chromatography or thick-layer chromatography, or a combination of these procedures.
• suitable separation and isolation procedures can be had by reference to the examples hereinbelow. However, other equivalent separation or isolation procedures can, of course, also be used.
• the (R)- and (S)-isomers may be resolved by methods known to those skilled in the art, for example by formation of diastereoisomeric salts or complexes which may be separated, for example, by crystallisation; via formation of
• S7155P1 WO - 17 - diastereoisomeric derivatives which may be separated, for example, by crystallisation, gas-liquid or liquid chromatography; selective reaction of one enantiomer with an enantiomer-specific reagent, for example enzymatic oxidation or reduction, followed by separation of the modified and unmodified enantiomers; or gas-liquid or liquid chromatography in a chiral environment, for example on a chiral support, such as silica with a bound chiral ligand or in the presence of a chiral solvent.
• a compound of Formula I can be dissolved in a lower alkanol and placed on a Chiralpak AD (205 x 20 mm) column (Chiral Technologies, Inc.) conditioned for 60 min at 70% EtOAc in Hexane.
• a further step may be required to liberate the desired enantiomeric form.
• specific enantiomer may be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one enantiomer to the other by asymmetric transformation.
• Reaction Scheme 1 illustrates synthesis of the compounds of Formula I, starting from a substituted nitro-benzene, which is condensed with an R alcohol, optionally derivatized to introduce the R 2 substituent, reduced to the corresponding amino-benzene and acidified to afford Formula I.
• nitro-benzenes of Formula 101 e.g., 3-fluoro-5-iodo-nitro-benzene and 3,5-difluoro-nitro-benzene
• R4alcohols of Formula 102 e.g., 4-fluoro-phenol and pyridin-3-ol
• Other reactants are likewise commercially available or may be readily prepared by those skilled in the art using commonly employed synthetic methodology.
• S7155P1 WO - 19 alcohol of Formula 102 are contacted in a solvent (e.g., DMF) in the presence of a base (e.g., K 2 CO 3 ).
• a solvent e.g., DMF
• a base e.g., K 2 CO 3
• the solution is stirred for 1-96 hours at room temperature to 150°C to afford the corresponding alkoxy-, aryloxy- or heteroaryloxy-nitro-benzene of Formula 103, which is conventionally isolated and purified.
• the compound of Formula 103 can be carried forward to Reaction Scheme 1 , Step 2 or directly to Reaction Scheme 1 , Step 3.
• R 2 is acetylene
• a compound of Formula 103 wherein Y is I or Br
• palladium e.g., PdOAc
• copper e.g., Cul
• a base e.g., diethylamine
• trimethylsilylacetylene of Formula 104 followed by removal of the trimethylsilyl with a fluoride source (e.g., tetrabutylammonium fluoride "TBAF").
• a fluoride source e.g., tetrabutylammonium fluoride "TBAF"
• the reaction takes place over 1-48 hours at room temperature to 150°C, or for 1-30 min. with microwave irradiation, to afford the corresponding acetylene-substituted nitro-benzene of Formula 105.
• this reaction can be run on a compound of Formula I where R 2 is I or Br (e.g., where a compound of Formula 103 is carried forward directly to Reaction
• R 2 is heteroaryl
• a compound of Formula 103 wherein Y is I or Br is contacted with a heteroaryl organometalic (e.g., an organozinc, an organoboronic acid, or an organotin) of Formula 104 in the presence of a palladium catalyst.
• the reaction takes place over 1-48 hours at room temperature to 150°C, or for 1-30 min. with microwave irradiation, to afford the corresponding heteroaryl-substituted nitro-benzene of Formula 105.
• this reaction can be run on a compound of Formula I where R 2 is I or Br (e.g., where a compound of Formula 103 is carried forward directly to Reaction Scheme 1 , Step 3).
• the R 2 -substituted nitro-benzene of Formula 105 can be conventionally isolated and purified, or carried forward without isolation and purification.
• Compounds prepared by the above-described process of the invention can be identified, e.g., by the presence of a detectable amount of Formula 106 or 107. While it is well known that pharmaceuticals must meet pharmacopoeia standards before approval and/or marketing, and that synthetic reagents (such as 4-fluoro-benzoic acid) and precursors (such as Formula 106) should not exceed the limits prescribed by pharmacopoeia standards, final compounds prepared by a process of the present invention may have minor, but detectable, amounts of such materials present, for example at levels in the range of 95% purity with no single impurity greater than 1%. These levels can be detected, e.g., by emission spectroscopy. It is important to monitor the purity of pharmaceutical compounds for the presence of such materials, which presence is additionally disclosed as a method of detecting use of a synthetic process of the invention.
• a substituted amino-benzene compound of Formula 106 is condensed with an aryl- or heteroaryl-acid or acid chloride of Formula 107 to afford the corresponding compound of Formula I.
• a racemic mixture of isomers of a compound of Formula I is placed on a chromatography column and separated into (R)- and (S)- enantiomers.
• a compound of Formula I is contacted with a pharmaceutically acceptable acid to form the corresponding acid addition salt.
• a pharmaceutically acceptable acid addition salt of Formula I is contacted with a base to form the corresponding free base of Formula I.
• R 1 is optionally substituted aryl or heteroaryl. o Especially where optionally substituted aryl is phenyl or substituted phenyl, and/or where heteroaryl is pyridinyl.
• R 1 is pyridin-3-yl and R 2 is fluoro.
• R 1 is 4-fluorophenyl and R 2 is nitro.
• R 1 is pyridin-2-yl, pyridin-3-yl or 4-fluorophenyl.
• R 2 is acetyl, acetylene, an alkyl ester, cyano, halo, optionally substituted heteroaryl, hydrogen, nitro (subject to the provisos identified above and in the claims) or trifluoromethyl. o Especially where halo is fluoro; where the alkyl ester is ethoxycarbonyl; and/or where heteroaryl is oxazolyl, pyrazinyl, pyridinyl, pyrimidinyl, tetrazolyl or thiazolyl, optionally substituted with lower alkoxy or oxo.
• heteroaryl is oxazol-2-yl, pyrazin-2-yl, pyridin-1-yl (particularly 3-methoxy-2-oxo-2H-
• R 2 is acetylene, cyano, ethoxycarbonyl, fluoro, hydrogen, nitro, oxazol-2-yl, pyridin-2-yl, pyrimidin-2-yl, thiazol-2-yl or trifluoromethyl.
• R 3 is optionally substituted aryl or optionally substituted heteroaryl. o Especially when R 3 is optionally substituted aryl, where aryl is phenyl, optionally substituted with lower alkoxy, lower alkyl, halo (particularly bromo, chloro or fluoro), hydroxy, hydroxy-lower alkyl or nitro.
• R 1 is pyridin-3-yl.
• R 1 is optionally substituted aryl or heteroaryl.
• optionally substituted aryl is phenyl or substituted phenyl, and/or where heteroaryl is pyridinyl.
• S7155P1 WO 23 More preferably pyridin-2-yl or pyridin-3-yl. o More preferably phenyl optionally substituted with lower alkyl, halo or hydroxy.
• R 1 is pyridin-2-yl, pyridin-3-yl or 4-fluorophenyl.
• R 2 is acetyl, acetylene, an alkyl ester, cyano, halo, optionally substituted heteroaryl, hydrogen, nitro or trifluoromethyl.
• halo is fluoro; where the alkyl ester is ethoxycarbonyl; and/or where heteroaryl is oxazolyl, pyrazinyl, pyridinyl, pyrimidinyl, tetrazolyl or thiazolyl, optionally substituted with lower alkoxy or oxo.
• heteroaryl is oxazol-2-yl, pyrazin-2-yl, pyridin-1-yl (particularly 3-methoxy-2-oxo-2H-pyridin-1 -yl), pyridin-2-yl, pyrimidin-2-yl, tetrazol-2-yl (particularly 1 -methoxymethoxy-1 H-tetrazol-5-yl, 2-methoxymethoxy-2H-tetrazol-5-yl) or thiazol-2-yl.
• R 2 is acetylene, cyano, ethoxycarbonyl, fluoro, hydrogen, nitro, oxazol — 2-yl, pyridin-2-yl, pyrimidin-2-yl, thiazol-2-yl or trifluoromethyl. ecially where R 1 is optionally substituted aryl or heteroaryl.
• pyridin-2-yl or pyridin-3-yl Preferably pyridin-2-yl or pyridin-3-yl.
• phenyl optionally substituted with lower alkyl, halo or hydroxy.
• o More preferably 3-chlorophenyl, 4-fluorophenyl, 4-hydroxyphenyl and 3-methylphenyl.
• R 1 is pyridin-2-yl, pyridin-3-yl or 4-fluorophenyl.
• R 2 is acetyl, acetylene, an alkyl ester, cyano, halo, optionally substituted heteroaryl, hydrogen, nitro or trifluoromethyl.
• halo is fluoro; where the alkyl ester is ethoxycarbonyl; and/or where heteroaryl is oxazolyl, pyrazinyl, pyridinyl, pyrimidinyl, tetrazolyl or thiazolyl, optionally substituted with lower alkoxy or oxo.
• heteroaryl is oxazol-2-yl, pyrazin-2-yl, pyridin-1-yl (particularly 3-methoxy-2-oxo-2H-pyridin-1-yl), pyridin-2-yl, pyrimidin-2-yl, tetrazol-2-yl (particularly 1 -methoxymethoxy-1 H-tetrazol-5-yl, 2-methoxymethoxy-2/-/-tetrazol-5-yl) or thiazol-2-yl.
• R 2 is acetylene, cyano, ethoxycarbonyl, fluoro, hydrogen, nitro, oxazol- 2-yl, pyridin-2-yl, pyrimidin-2-yl, thiazol-2-yl or trifluoromethyl.
• R 2 is acetyl, acetylene, an alkyl ester, cyano, halo, optionally substituted heteroaryl, hydrogen, nitro or trifluoromethyl.
• halo is fluoro; where the alkyl ester is ethoxycarbonyl; and/or where heteroaryl is oxazolyl, pyrazinyl, pyridinyl, pyrimidinyl, tetrazolyl or thiazolyl, optionally substituted with lower alkoxy or oxo.
• heteroaryl is oxazol-2-yl, pyrazin-2-yl, pyridin-1-yl (particularly 3-methoxy-2- oxo-2H-pyridin-1-yl), pyridin-2-yl, pyrimidin-2-yl, tetrazol-2-yl (particularly 1 -methoxymethoxy-1 H-tetrazol-5-yl, 2-methoxymethoxy-2f- -tetrazol-5-yl) or thiazol-2-yl.
• R 2 is acetylene, cyano, ethoxycarbonyl, fluoro, hydrogen, nitro, oxazol-2-yl, pyridin-2-yl, pyrimidin-2-yl, thiazol-2-yl or trifluoromethyl.
• R 1 is heteroaryl and R 3 is substituted phenyl. o Especially where R 1 is pyridin-3-yl.
• R 2 is acetylene, cyano, ethoxy-carbonyl, fluoro, hydrogen, nitro, oxazol-2-yl, pyridin-2-yl, pyrazin-2-yl, thiazol-2-yl or trifluoromethyl.
• R 2 is nitro or fluoro.
• R 3 is 4-fluorophenyl
• compositions of matter where R 2 is cyano, ethoxycarbonyl, hydrogen, fluoro, hydrogen, pyridin-2-yl, pyrazin-2-yl, or trifluoromethyl. o Especially where R 3 is 4-fluorophenyl.
• R 1 is pyridin-3-yl.
• R 1 is substituted phenyl and R 2 is nitro. o Especially where R 1 is 4-fluorophenyl.
• R 1 is heteroaryl and R 2 is fluoro. o Especially where R is pyridin-3-yl.
• R 1 is heteroaryl and R 2 is nitro. o Especially where R 1 is pyridin-3-yl.
• R 3 is optionally substituted aryl.
• R 3 is phenyl, 4-fluorophenyl, 4-cyanophenyl, 4-methoxyphenyl or 4-methylphenyl.
• R 3 is optionally substituted aryl, optionally substituted heteroaryl or optionally substituted heteroarylamino
• the above-described groups and sub-groups are individually preferred and can be combined to describe further preferred aspects of the invention.
• the compounds of the present invention are selective for and modulate the cardiac sarcomere, and are useful to bind to and/or potentiate the activity of cardiac myosin, increasing the rate at which myosin hydrolyzes ATP.
• modulate means either increasing or decreasing myosin activity
• potentiate means to increase activity. It has also been determined in testing representative compounds of the invention, that their administration can also increase the contractile force in cardiac muscle fiber.
• the compounds, pharmaceutical formulations and methods of the invention are used to treat heart disease, including but not limited to: acute (or decompensated) congestive heart failure, and chronic congestive heart failure; particularly diseases associated with systolic heart dysfunction.
• Additional therapeutic utilities include administration to stabilize heart function in patients awaiting a heart transplant, and to assist a stopped or slowed heart in resuming normal function following use of a bypass pump.
• ATP hydrolysis is employed by myosin in the sarcomere to produce force.
• an increase in ATP hydrolysis would correspond to an increase in the force or velocity of muscle contraction.
• myosin ATPase activity is stimulated >100 fold.
• ATP hydrolysis not only measures myosin enzymatic activity but. also its interaction with the actin filament.
• a compound that modulates the cardiac sarcomere can be identified by an increase or decrease in the rate of ATP hydrolysis by myosin, preferably exhibiting a 1.4 fold increase at concentrations less than 10 ⁇ M (more preferably, less than 1 ⁇ M).
• Preferred assays for such activity will employ myosin from a human source, although myosin from other organisms can also be used. Systems that model the regulatory role of calcium in myosin binding are also preferred.
• a biochemically functional sarcomere preparation can be used to determine in vitro ATPase activity, for example, as described in U.S. Serial Number 09/539,164, filed March 29, 2000.
• S7155P1 WO - 28 - sarcomere including calcium sensitivity of ATPase hydrolysis, can be reconstituted by combining its purified individual components (particularly including its regulatory components and myosin).
• Another functional preparation is the in vitro motility assay. It can be performed by adding test compound to a myosin-bound slide and observing the velocity of actin filaments sliding over the myosin covered glass surface (Kron SJ. (1991 ) Methods Enzymol. 196:399-416).
• the in vitro rate of ATP hydrolysis correlates to myosin potentiating activity, which can be determined by monitoring the production of either ADP or phosphate, for example as described in Serial No. 09/314,464, filed May 18, 1999.
• ADP production can also be monitored by coupling the ADP production to NADH oxidation (using the enzymes pyruvate kinase and lactate dehydrogenase) and monitoring the NADH level either by absorbance or fluorescence (Greengard, P., Nature 178 (Part 4534): 632-634 (1956); Mol Pharmacol 1970 Jan;6(1):31-40).
• Phosphate production can be monitored using purine nucleoside phosphorylase to couple phosphate production to the cleavage of a purine analog, which results in either a change in absorbance (Proc Natl Acad Sci U S A 1992 Jun 1 ;89(11 ):4884-7) or fluorescence (Biochem J 1990 Mar 1 ;266(2):611-4). While a single measurement can be employed, it is preferred to take multiple measurements of the same sample at different times in order to determine the absolute rate of the protein activity; such measurements have higher specificity particularly in the presence of test compounds that have similar absorbance or fluorescence properties with those of the enzymatic readout.
• Test compounds can be assayed in a highly parallel fashion using multiwell plates by placing the compounds either individually in wells or testing them in mixtures. Assay components including the target protein complex, coupling enzymes and substrates, and ATP can then be added to the wells and the absorbance or fluorescence of each well of the plate can be measured with a plate reader.
• a preferred method uses a 384 well plate format and a 25 ⁇ L reaction volume.
• a pyruvate kinase/lactate dehydrogenase coupled enzyme system (Huang TG and hackney DD. (1994) J Biol Chem 269(23): 16493-16501 ) is used to measure the rate of ATP hydrolysis in each well.
• the assay components are added in buffers and reagents. Since the methods outlined herein allow kinetic measurements, incubation periods are optimized to give adequate
• the assay is done in real time giving the kinetics of ATP hydrolysis, which increases the signal to noise ratio of the assay.
• Modulation of cardiac muscle fiber contractile force can be measured using detergent permeabilized cardiac fibers (also referred to as skinned cardiac fibers), for example, as described by Haikala H, et al (1995) J Cardiovasc Pharmacol 25(5):794-801. Skinned cardiac fibers retain their intrinsic sarcomeric organization, but do not retain all aspects of cellular calcium cycling, this model offers two advantages: first, the cellular membrane is not a barrier to compound penetration, and second, calcium concentration is controlled. Therefore, any increase in contractile force is a direct measure of the test compound's effect on sarcomeric proteins. Tension measurements are made by mounting one end of the muscle fiber to a stationary post and the other end to a transducer that can measure force.
• the force transducer After stretching the fiber to remove slack, the force transducer records increased tension as the fiber begins to contract. This measurement is called the isometric tension, since the fiber is not allowed to shorten.
• Activation of the permeabilized muscle fiber is accomplished by placing it in a buffered calcium solution, followed by addition of test compound or control. When tested in this manner, compounds of the invention caused an increase in force at calcium concentrations associated with physiologic contractile activity, but very little augmentation of force in relaxing buffer at low calcium concentrations or in the absence of calcium (the EGTA data point).
• Selectivity for the cardiac sarcomere and cardiac myosin can be determined by substituting non-cardiac sarcomere components and myosin in one or more of the above-described assays and comparing the results obtained against those obtained using the cardiac equivalents.
• S7155P1 WO 30 then be assessed in whole organ models, such as such as the Isolated Heart (Langendorff) model of cardiac function, in vivo using echocardiography or invasive hemodynamic measures, and in animal-based heart failure models, such as the Rat Left Coronary Artery Occlusion model. Ultimately, activity for treating heart disease is demonstrated in blinded, placebo-controlled, human clinical trials.
• whole organ models such as the Isolated Heart (Langendorff) model of cardiac function
• echocardiography or invasive hemodynamic measures such as the Rat Left Coronary Artery Occlusion model.
• Rat Left Coronary Artery Occlusion model such as the Rat Left Coronary Artery Occlusion model.
• the compounds of Formula I are administered at a therapeutically effective dosage, e.g., a dosage sufficient to provide treatment for the disease states previously described.
• a daily dose is from about 0.05 to 100 mg/kg of body weight, preferably about 0.10 to 10.0 mg/kg of body weight, and most preferably about 0.15 to 1.0 mg/kg of body weight.
• the dosage range would be about 3.5 to 7000 mg per day, preferably about 7.0 to 700.0 mg per day, and most preferably about 10.0 to 100.0 mg per day.
• the amount of active compound administered will, of course, be dependent on the subject and disease state being treated, the severity of the affliction, the manner and schedule of administration and the judgment of the prescribing physician; for example, a likely dose range for oral administration would be about 70 to 700 mg per day, whereas for intravenous administration a likely dose range would be about 700 to 7000 mg per day, the active agents being selected for longer or shorter plasma half-lives, respectively.
• Administration of the compounds of the invention or the pharmaceutically acceptable salts thereof can be via any of the accepted modes of administration for agents that serve similar utilities including, but not limited to, orally, subcutaneously, intravenously, intranasally, topically, transdermally, intraperitoneally, intramuscularly, intrapulmonarilly, vaginally, rectally, or intraocularly. Oral and parenteral administration are customary in treating the indications that are the subject of the present invention.
• Pharmaceutically acceptable compositions include solid, semi-solid, liquid and aerosol dosage forms, such as, e.g., tablets, capsules, powders, liquids, suspensions, suppositories, aerosols or the like.
• the compounds can also be administered in sustained or controlled release dosage forms, including depot injections, osmotic pumps, pills, transdermal (including electrotransport) patches, and the like, for prolonged and/or timed, pulsed administration at a predetermined rate.
• sustained or controlled release dosage forms including depot injections, osmotic pumps, pills, transdermal (including electrotransport) patches, and the like, for prolonged and/or timed, pulsed administration at a predetermined rate.
• compositions are provided in unit dosage forms suitable for single administration of a precise dose.
• the compounds can be administered either alone or more typically in combination with a conventional pharmaceutical carrier, excipient or the like (e.g., mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, sodium crosscarmellose, glucose, gelatin, sucrose, magnesium carbonate, and the like).
• a conventional pharmaceutical carrier e.g., mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, sodium crosscarmellose, glucose, gelatin, sucrose, magnesium carbonate, and the like.
• the pharmaceutical composition can also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents and the like (e.g., sodium acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate, and the like).
• the pharmaceutical formulation will contain about 0.005% to 95%, preferably about 0.5% to 50% by weight of a compound of the invention.
• Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania.
• the compounds of the invention can be co-administered with, and the pharmaceutical compositions can include, other medicinal agents, pharmaceutical agents, adjuvants, and the like.
• suitable additional active agents include, for example: therapies that retard the progression of heart failure by down- regulating neurohormonal stimulation of the heart and attempt to prevent cardiac remodeling (e.g., ACE inhibitors or ⁇ -blockers); therapies that improve cardiac function by stimulating cardiac contractility (e.g., positive inotropic agents, such as the ⁇ - adrenergic agonist dobutamine or the phosphodiesterase inhibitor milrinone); and therapies that reduce cardiac preload (e.g., diuretics, such as furosemide).
• therapies that retard the progression of heart failure by down- regulating neurohormonal stimulation of the heart and attempt to prevent cardiac remodeling e.g., ACE inhibitors or ⁇ -blockers
• therapies that improve cardiac function by stimulating cardiac contractility e.g., positive inotropic agents, such as the ⁇ - adrenergic agonist dobut
• the compositions will take the form of a pill or tablet and thus the composition will contain, along with the active ingredient, a diluent such as lactose, sucrose, dicalcium phosphate, or the like; a lubricant such as magnesium stearate or the like; and a binder such as starch, gum acacia, polyvinylpyrrolidine, gelatin, cellulose, cellulose derivatives or the like.
• a powder, marume, solution or suspension e.g., in propylene carbonate, vegetable oils or triglycerides
• a gelatin capsule e.g., in propylene carbonate, vegetable oils or triglycerides
• Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, etc. an active compound as defined above and optional pharmaceutical adjuvants in a carrier (e.g., water, saline, aqueous dextrose, glycerol, glycols, ethanol or the like) to form a solution or suspension.
• a carrier e.g., water, saline, aqueous dextrose, glycerol, glycols, ethanol or the like
• injectables can be prepared in conventional forms, either as liquid solutions or suspensions, as emulsions, or in solid forms suitable for dissolution or suspension in liquid prior to injection.
• the percentage of active compound contained in such parenteral compositions is highly dependent on the specific nature thereof, as well as the activity of the compound and the needs of the subject.
• compositions will comprise 0.2-2% of the active agent in solution.
• Formulations of the active compound or a salt may also be administered to the respiratory tract as an aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose. In such a case, the particles of the formulation have diameters of less than 50 microns, preferably less than 10 microns.
• myosin is bound to a support and a compound of the invention is added to the assay.
• the compound of the invention can be bound to the support and the myosin added.
• Classes of compounds among which novel binding agents may be sought include specific antibodies, non-natural binding agents identified in screens of chemical libraries, peptide analogs, etc. Of particular interest are screening assays for candidate agents that have a low toxicity for human cells.
• assays may be used for this purpose, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc.) and the like. See, e.g., U.S. Patent No. 6,495,337, incorporated herein by reference.
• Dose responses are measured using a calcium-buffered, pyruvate kinase and lactate dehydrogenase-coupled ATPase assay containing the following reagents (concentrations expressed are final assay concentrations): Potassium PIPES (12 mM), MgCI 2 (2 mM), ATP (1 mM), DTT (1 mM), BSA (0.1 mg/ml), NADH (0.5 mM), PEP (1.5 mM), pyruvate kinase (4 U/ml), lactate dehydrogenase (8 U/ml), and antifoam (90 ppm). The pH is adjusted to 6.80 at 22°C by addition of potassium hydroxide. Calcium levels
• S7155P1 WO • 36 - are controlled by a buffering system containing 0.6 mM EGTA and varying concentrations of calcium, to achieve a free calcium concentration of IxlO "4 M to 1x10 " ° M.
• bovine cardiac myosin subfragment-1 typically 0.5 ⁇ M
• bovine cardiac actin 14 ⁇ M
• bovine cardiac tropomyosin typically 3 ⁇ M
• bovine cardiac troponin typically 3-8 ⁇ M
• concentrations of tropomyosin and troponin are determined empirically, by titration to achieve maximal difference in ATPase activity when measured in the presence of 1 mM EGTA versus that measured in the presence of 0.2 mM CaCI 2 .
• concentration of myosin in the assay is also determined empirically, by titration to achieve a desired rate of ATP hydrolysis. This varies between protein preparations, due to variations in the fraction of active molecules in each preparation.
• Compound dose responses are typically measured at the calcium concentration corresponding to 50% of maximal ATPase activity (pCa 5 o), so a preliminary experiment is performed to test the response of the ATPase activity to free calcium concentrations in the range of IxlO "4 M to 1x10 ⁇ 8 M. Subsequently, the assay mixture is adjusted to the pCaso (typically 3x10 "7 M).
• Assays are performed by first preparing a dilution series of test compound, each with an assay mixture containing potassium Pipes, MgCI 2 , BSA, DTT, pyruvate kinase, lactate dehydrogenase, myosin subfragment-1 , antifoam, EGTA, CaCI 2 , and water.
• the assay is started by adding an equal volume of solution containing potassium Pipes, MgCI 2 , BSA, DTT, ATP, NADH, PEP, actin, tropomyosin, troponin, antifoam, and water. ATP hydrolysis is monitored by absorbance at 340 nm.
• the AC1.4 is defined as the concentration at which ATPase activity is 1.4-fold higher than the bottom of the dose curve.
• Preferred compounds of the invention have an AC1.4 less than 10 ⁇ M; and more preferably, less than 1 ⁇ M.
• the heart is perfused with modified Krebs buffer supplemented with 3.3% collagenase (169 ⁇ /mg activity, Class II, Worthington Biochemical Corp., Freehold, NJ) and 25 ⁇ M final calcium concentration until the heart becomes sufficiently blanched and soft.
• the heart is removed from the cannulae, the atria and vessels discarded and the ventricles are cut into small pieces.
• the myocytes are dispersed by gentle agitation of the ventricular tissue in fresh collagenase containing Krebs prior to being gently forced through a 200 ⁇ m nylon mesh in a 50 cc tube. The resulting myocytes are resuspended in modified Krebs solution containing 25 ⁇ m calcium.
• Myocytes are made calcium tolerant by addition of a calcium solution (100 mM stock) at 10 minute intervals until 100 ⁇ M calcium is achieved. After 30 minutes the supernatant is discarded and 30 - 50 ml of Tyrode buffer (137 mM NaCL, 3.7 mM KCL, 0.5 mM MgCL, 11 mM glucose, 4 mM Hepes, and 1.2 mM CaCI 2 , pH 7.4) is added to cells. Cells are kept for 60 min at 37°C prior to initiating experiments and used within 5 hrs of isolation.
• Tyrode buffer 137 mM NaCL, 3.7 mM KCL, 0.5 mM MgCL, 11 mM glucose, 4 mM Hepes, and 1.2 mM CaCI 2 , pH 7.4
• Tyrode buffer containing myocytes are placed in perfusion chambers (series 20 RC-27NE; Warner Instruments) complete with heating platforms. Myocytes are allowed to attach, the chambers heated to 37°C, and the cells then perfused with 37°C Tyrode buffer. Myocytes are field stimulated at 1 Hz in with platinum electrodes (20% above threshold). Only cells that have clear striations, and are quiescent prior to pacing are used for contractility experiments. To determine basal contractility, myocytes are imaged through a 40x objective and using a variable frame
• 6B FCHOCARDIOGRAPHY Animals are anesthetized with isoflurane and maintained within a surgical plane throughout the procedure. Core body temperature is maintained at 37°C by using a heating pad. Once anesthetized, animals are shaven and hair remover is applied to remove all traces of fur from the chest area. The chest area is further prepped with 70% ETOH and ultrasound gel is applied. Using
• 6C BOLUS AND INFUSION EFFICACY
• rats are treated as described above.
• Five pre-dose M-Mode images are taken at 30 second intervals prior to bolus injection or infusion of compounds.
• M-mode images are taken at 30 second intervals up to 10 minutes and every minute or at five minute intervals thereafter.
• Bolus injection or infusion is via the tail vein.
• Infusion parameters are determined from pharmacokinetic profiles of specific compounds.
• 6D RESULTS Compounds of the present invention show activity when tested by this method.
• Sham animals are treated the same, except that the suture is not tied. The incision is closed in three layers. The rat is ventilated until able to ventilate on its own. The rats are extubated and allowed to recover on a heating pad. Animals receive buprenorphine (0.01-0.05 mg/kg SQ) for post operative analgesia. Once awake, they are returned to their cage. Animals are monitored daily for signs of infection or distress. Infected or moribund animals are euthanized. Animals are weighed once a week. [00111] 7C. FFFiCACY ANALYSIS Six weeks after surgery, rats are scanned for signs of myocardial infarction using ultrasound as described above.
• mice Only those animals with decreased fractional shortening compared to sham rats are utilized in efficacy experiments. In all experiments, there are four groups: sham + vehicle, sham + compound, LCO + vehicle, and LCO + compound. At 7-12 weeks post LCO, rats receive a bolus injection or are infused with test compound. As in Example 6, five pre-dose M-Mode images are taken at 30 second intervals prior to bolus injection or infusion of compound. After injection, M-mode images are taken at 30 second intervals up to 10 minutes, and thereafter every minute or at five minute intervals. Fractional shortening is determined from the M-mode images. Comparisons between the pre-dose fractional shortening and post compound treatment are performed by ANOVA and a post-hoc Student - Newman - Keuls with the StatView statistical program (SAS Institute). A p value ⁇ 0.05 is considered significant.

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