MXPA06008062A - 2, 4 - diaminopyrimidines and their use for inducing cardiomyogenesis - Google Patents

Abstract

The present invention provides compounds of formula (I) useful for inducing cardiomyogenesis in mammalian cells, particularly embryonic stem cells, in vitro and in vivo.

Description

2,4-DlAMINOPIRIDINAS AND ITS USE FOR INDUCTION OF CARDIOMYOGENESIS CROSS REFERENCE WITH RELATED APPLICATIONS The present application claims the priority of the North American Provisional Patent Application No. 60 / 537,144, filed on January 16, 2004, the teachings of which are incorporated in their entirety to the present invention as a reference, Background of the Invention. Heart diseases are a problem worldwide, they comprise many different diseases and conditions. Cardiomyopathy, for example, is a disease of the heart muscle where the heart loses its ability to pump blood, and in some cases, the heart rhythm is disturbed, leading to irregular heartbeat or arrhythmias. Cardiomyopathy affects ten out of every one thousand American citizens of all ages and is a major reason for heart transplantation. The condition tends to be progressive and sometimes gets worse very quickly. Understanding the development and function of the heart muscle can facilitate the use of stem cells. Stem cells are multipotent cells with the ability to self-renew and differentiate into specialized cells in response to appropriate signals. See, for example, the publication by Spradling et al., Nature, 414: 98-104 (2001). Most tissues have progenitor / endogenous stem cells that at the time of injury to the organ can proliferate and differentiate at the damaged site. However, the heart of an adult is composed mainly of cells differentiated in post-mitotic and terminal form. Although a subpopulation of myocardial cells has recently been identified as a cardiac stem cell, its limited availability hinders therapeutic applications. See for example the publication of Beltrami and associates, Cell, 114: 763-776 (2003). Stem cells derived from other tissues, such as bone marrow, have been shown to be able to repair cardiac damage in animal models, but inefficient differentiation and possible fusion with somatic cells limit their use in cardiac repair. See for example the publication of Ferrari and associates, Science, 279: 1528-30 (1998). Embryonic pluripotent stem (ES) cells represent a possible unlimited source of functional cardiomyocytes. Such cardiomyocytes could probably facilitate the therapeutic application of ES cells in cardiac diseases, as well as provide important tools to test the molecular mechanism of cardiomyocyte differentiation and heart development. However, to date the in vitro differentiation of ES cells into cardiomyocytes comprises a poorly defined, inefficient, and relatively non-selective process. See for example the publication of Boheler and associates., Circ. Res. 91: 189-201 (2002). Therefore, the art recognizes the need for compositions and methods to induce and direct the differentiation of ES cells into cardiomyocytes. There is a particular need for small cells that can induce differentiation in vivo and in vitro of ES cells in cells of a myocardial lineage. The present invention satisfies these and other needs. Summary of the Invention The present invention provides compositions and novel methods for inducing and directing the differentiation of ES cells into cells of a myocardial lineage. One embodiment of the present invention provides compounds of Formula I that have the following structure: In Formula I, R1 is a functional group including, but not limited to, hydrogen, C? -4alkyl, C3-8cycloalkyl and C0-2alkylaryl, substituted with 0-2 R1a groups which are independently selected, and are functional group, including but not limited to, halogen, C. 4alkyl, C1-4alkoxy, -OH, -N (R1b, R1b), -SO2N (R1b, R1), -C (O) N (R1b, R1b). heterocycloalkyl and -O-aryl, or when the R1a groups are on adjacent ring atoms, are optionally taken together to form a functional group including, but not limited to, O- (CH2) 1-2-O-, - OC (CH3) 2CH2- and - (CH2) 3- -, or R1 is optionally taken together with the nitrogen to which it adheres to form a heterocycle, optionally substituted with C? -4alkyl, C-8cycloalkyl, C -? - 4alkylhydroxy and C0-2alkylaryl; each R1b group is independently selected and is a functional group including, but not limited to, hydrogen and C-i. 4alkyl. In Formula I, R2 is a functional group including, but not limited to, C? -4alkyl, C3-8cycloalkyl, and C0-2alkyl or substituted with groups 0-2 R2a. The R2a group is independently selected and is a functional group including, but not limited to, halogen, C1-4alkyl, C4-4alkoxy, -N (R2, R2b), -SO2N (R2, R2b), -C ( O) N (R2, R2b) and -O-aryl, or when the R2a groups are on adjacent ring atoms, are optionally taken together to form a functional group including, but not limited to, -O- (CH2) ? -2-O-, -OC (CH3) 2CH2- and - (CH2) 3-4-; and each group R2b is independently selected and is a functional group including, but not limited to, hydrogen, and C-i. alkyl, R3, in Formula I, is usually hydrogen, or R3 is optionally in conjunction with R2 and the nitrogen to which both are attached to form a heterocycle, optionally substituted with, for example, C1-4alkyl or C0-2alkylaryl. The compounds of the present invention include all pharmaceutically acceptable salts, isomers, solvates, hydrates and prodrugs thereof. In another embodiment, the present invention provides methods for inducing cardiomyogenesis. The mammalian cells are contacted with a compound of Formula I or II, while the mammalian cell differentiates into a cell of a myocardial lineage. The contact step can be in vivo or in vitro. By virtue of their ability to induce cardiomyogenesis, the compounds of Formula I are useful for treating heart muscle conditions such as cardiomyopathy and arrhythmia, and for repairing damage to heart muscle tissue resulting from a heart attack for example. Another embodiment of the present invention provides methods for treating heart muscle diseases by contacting a mammalian cell with a compound of Formula I, wherein the mammalian cell differentiates into a cell of a myocardial lineage. The mammalian cell can also be contacted with other compounds or proteins favorable for cardiomyogenesis. If the mammalian cell is contacted with a compound of Formula I or II in vitro, the differentiated cells are administered to an individual with a treatable condition, thereby treating the condition. In some embodiments, the mammalian cell adheres to a solid support (e.g., a three-dimensional matrix or a flat surface) or is injected to damaged sites of the myocardium. In some embodiments, the mammalian cell is contacted with a compound of Formula I or II in vivo. If the mammalian cells are contacted with a compound of Formula I or II in vivo, the step of contacting can be by oral, intravenous, subcutaneous or intraperitoneal administration of the compound to the mammal. In some embodiments, the differentiation of the mammalian cell into a cell of a myocardial lineage is detected. In some embodiments, the differentiation of a mammalian cell into a myocardioblast cell is detected through the detection of the expression of a cardiomyogenesis marker gene, for example, atrial natriuretic factor ("AND"). In other embodiments, the differentiation of the cell of a mammal in a cell of a myocardial lineage is detected through the detection of the expression of a specific transcription factor of the cardiac muscle cell (for example MEF2 or Nkx2.5 or the homodomain transcription factor HOP). In other embodiments, the differentiation of the mammalian cell into a cell of a myocardial lineage is detected through the detection of expression of a specific gene of the cardiac muscle (e.g., myosin light chain 2V or eHAND). In yet other embodiments, differentiation of a mammalian cell from a myocardial lineage cell is detected by detecting the expression of a specific cardiac gene such as GATA-4 or by the expression of a gene involved in the contraction capacity of the muscle. cardiac muscle such as the sarcomeric myosin heavy chain (MHC). In additional modalities, differentiation can be detected by observing palpitation of the heart muscle using standard techniques well known to those skilled in the art. In some embodiments, the mammalian cell is a stem cell (e.g., an embryonic stem cell or an embryonic carcinoma cell.) In some embodiments, the stem cell is isolated from a mouse (e.g., an undifferentiated R1 embryonic stem cell. of murine or a P19 carcinoma cell) or of a primate (for example a human) In some embodiments of the above methods, the compound administered to mammalian cells is cardiogenol A, B, C or D, or a composition that it comprises one or more of cardiogenol A, B, C or D. From the detailed description set forth below, other embodiments and advantages of the present invention may be appreciated Brief Description of the Figures Figure 1. A high performance test of cardiomyogenesis using a promoter-ANF reporter assay This figure shows the data obtained using a stable P19 clone harboring a reporter plasmid of ANF promoter that ex Luciferase prey The graph shows a 5 to 7 fold increase in the luciferase signal from this P19 clone after several days under standard cardiomyogenesis differentiation conditions for P19 cells (EB formation and treatment with 1% DMSO (see the publication of Sherjank IS, Trends Cardiovasc Med. 9: 139-143 (1999)). Figure 2. Immunostaining of cardiac muscle markers in ESCs cells (A - E) and P19CL6 cells (F) treated with 0.25 μM cardiogenol C: (A) and (F) Heavy Chain of Miosin (green); (B) GATA-4 (red); (C) MEF2 (red); (D) Nkx2.5 (red); and (E) Heavy Chain of Miosin (green) and MEF2 (red). The nuclei of the cell were stained with DAPI (blue). The cells were fixed with 4% paraformaldehyde (Sigma) for 20 minutes. Cell staining was carried out in PBS (Gibco) with 0.3% Triton X-100 and 6% horse serum. Primary antibodies were used in the following dilutions: myosin heavy chain MF20 mouse monoclonal antibody (MHC) (Hybridoma Bank of Developing Studies 1: 200), polyclonal rabbit anti-GATA-4 antibody (Santa Cruz Biotech, 1 : 300), rabbit anti-MEF2 antibody (Santa Cruz Biotech, 1: 100) and goat anti-Nkx2.5 antibody (Santa Cruz Biotech, 1: 100). Secondary antibodies were anti-mouse conjugated by Cy2 (1: 300), or anti-mouse or anti-goat antibodies conjugated with Cy3 (Jackson ImmunoResearch, 1: 500). The nuclei of the cells were stained with DAPI (Roche). Images were taken with a Nikon Eclipes TE2000 microscope with 200-fold magnification. Images with double or triple labels were assembled in Metamorph. Figure 3. Immunostaining of ESCs without cardiogenol C treatment (control). (TO). MHC (green) and Nuclei (Blue). (B) GATA-4- (Red) and Nuclei (Blue). Compare with figures 2A and 2E, respectively. Figure 4. This figure shows a list of additional compounds of the present invention that can be used to induce cardiomyogenesis in mammalian cells. Detailed Description of the Invention I. Introduction. The present invention provides compounds, compositions and methods for differentiating cells of a mammal in cells of a myocardial lineage. More particularly, the present invention provides compounds of Formula I and II which are useful for differentiating cells from a mammal in cells of a myocardial lineage. In some embodiments, a composition comprising the compound of Formula I and II. In other embodiments, methods are provided for inducing cardiomyogenesis in mammalian cells. The myogenesis can be induced in vivo or in vitro according to the methods of the present invention. II. Definitions Unless defined otherwise, all technical and scientific terms used in the present invention generally have the same meaning as commonly understood by one skilled in the art to which the present invention pertains. Generally, the nomenclature used in the present invention and in the laboratory procedures for organic and analytical chemistry are those commonly known and used in the art. The term "alkyl" by itself or as part of another substituent, means, unless otherwise specified, a straight or branched chain or cyclic hydrocarbon radical, or combination thereof, which may be completely saturated, mono or polyunsaturated and may include di and multivalent radicals, having the designated carbon atom number (for example Cn-C10 means from one to ten carbons). Examples of saturated hydrocarbon radicals include groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl) methyl, cyclopropylmethyl, homologs and isomers, for example , n-pentyl, n-hexyl, n-heptyl, n-octyl and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2- (butadienyl), 2,4-pentadienyl, 3- (1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl and homologues and higher isomers. The term "alkyl" unless otherwise noted also means that it includes the alkyl derivatives defined in more detail below as "heteroalkyl". Alkyl groups that are limited to hydrocarbon groups are referred to as "homoalkyl". The term "alkylene" by itself or as part of another substituent means a divalent radical derived from an alkane, as exemplified by CH2CH2CH2CH2-, and further includes those groups which are described below as "heteroalkylene".

Normally, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, wherein groups having 10 or fewer carbon atoms are preferred in the present invention. A "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group that generally has eight or fewer carbon atoms. The terms "alkoxy", "alkylamino" and "alkylthio" (or thioalkoxy) are used in their conventional sense and refer to alkyl groups attached to the rest of the molecule through an oxygen atom, an amino group or an atom of sulfur, respectively. The term "heteroalkyl", by itself or in combination with another term, means unless stated otherwise, a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof consisting of a manifested number of carbon atoms and one to three heteroatoms selected from the group consisting of O, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom (s) O, N and S can be placed at any interior position of the heteroalkyl group. The heteroatom can be placed in any position of the heteroalkyl group, including the position in which the alkyl group adheres to the heteroalkyl group. rest of the molecule. Examples include -CH2-CH2-O-CH3, -CH2-CH2O-CH2-CH2- (CH3) 2, -CH2-CH2-NH-CH3, -CH2-CH2-N (CH3) -CH3j-CH2-S -CH2-CH3, -CH2-CH2-S (O) -CH3, -CH2-CH2-S (O) 2 -CH3, -CH = CH-O-CH3, -Si (CH3) 3, CH2-CH = N-OCH3, and -CH = CH-N (CH3) -CH3. Up to two heteroatoms can be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si (CH3) 3. In a similar way, the term "heteroalkylene" by itself or as part of other substituents means a divalent radical derived from heteroalkyl, as exemplified by -CH2-CH2-S-CH2CH2- and -CH2-S-CH2-CH2-NH-CH2 -. For heteroalkylene groups, the heteroatoms may also occupy either or both of the chain terms (eg, alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further, for linking groups of alkylene and heteroalkylene, the orientation of the linking group is not implied.

The terms "cycloalkyl" and "heterocycloalkyl", by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of "alkyl" and "heteroalkyl", respectively. In addition, for heterocycloalkyl, a heteroatom can occupy the position in which the heterocycle adheres to the rest of the molecule. Examples of cycloalkyl include cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include 1- (1, 2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl , tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. The terms "halo" or "halogen", by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine or iodine atom. In addition, the terms "haloalkyl", mean that they include monohaloalkyl and polyhaloalkyl. For example, the term "halo (C1-C) alkyl" is intended to include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like. The term "aryl" means, unless otherwise stated, a polyunsaturated, normally aromatic hydrocarbon substituent, which may be a single ring or multiple rings (up to three rings) that are fused together or covalently linked. The term "heteroaryl" refers to aryl groups (or rings) containing from zero to four heteroatoms selected from N, O, and S wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atoms are optionally quaternized. A heteroaryl group can be attached to the rest of the molecule through a heteroatom. Examples without limitation of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazoyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3- furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl,. purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl and 6-quinolyl. The substituents for each of the aryl and heteroaryl ring systems noted above are selected from the group of acceptable substituents described below. For brevity, the term "aryl" when used in combination with other terms (eg, aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above. Thus, the term "arylalkyl" means that it includes the radicals in which an aryl group is attached to an alkyl group (eg, benzyl, phenethyl, pyridylmethyl and the like) including alkyl groups in which a carbon atom ( for example, a methylene group) has been replaced, for example, by an oxygen atom (for example phenoxymethyl, 2-pyridyloxymethyl, 3- (1-naphthyloxy) propyl, and the like). Each of the foregoing terms (eg, "alkyl", "heteroalkyl", "aryl" and "heteroaryl") are intended to include both substituted and unsubstituted forms of the indicated radical. Preferred substituents of each type of radical are given below. Substituents for the alkyl and heteroaryl radicals (including the groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be a variety of groups selected from: -OR \ = O, = NR \ = N-OR ', -NR'R'0 -SR0 -halogen, -SR'R "R" \ -OC (O) R \ -C (O) R \ -CO2R \ -CONR'R ", -OC (O) NR'R", -NR "C (O) R ', -NR'-C (O) NR" R' ", -NR" C (O) 2R ', -NH-C (NH2) = NH, NR'C (NH2) = NH, -NH-C (NH2) = NR \ - S (O) R \ -S (O) 2R0 -S (O) 2NR'R ", -CN and -NO2 in a number which fluctuates from zero to (2m '+ 1), where m' is the total number of carbon atoms in said radical R ', R "and R"' each independently refer to hydrogen, (C? -C8 ) unsubstituted alkyl and heteroalkyl, unsubstituted aryl, aryl substituted with 1 to 3 halogens, unsubstituted alkyl, alkoxy or thioalkoxy groups, or aryl- (C -? - C) alkyl groups When R 'and R "adhere to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5, 6 or 7 membered ring. For example, -NR'R "means that it includes 1-pyrrolidinyl and 4-morpholinyl. From the foregoing description of the substituents, one skilled in the art will understand that the term" alkyl "means that it includes groups such as haloalkyl (eg. example, -CF3 and -CH2CF3) and acyl (e.g., -C (O) CH3, -C (O) CF3, -C (O) CH2OCH3, and the like). Similarly, substituents for the aryl groups and heteroaryl are varied and are selected from: -halogen, -OR \ -OC (O) R ', - NR'R'0 -SR', -R ', -CN, -NO2, -CO2R', -CONR'R ", -C (O) R ', -OC (O) NR'R", -NR "C (O) R \ -NR" C (O) 2R', -NR'-C (O) NR "R "', -NH-C (NH2) = NH, NR'C (NH2) = NH, -NH-C (NH2) = NR ', -S (O) R', -S (O) 2R ', -S (O) 2NR'R ", -N3, - CH (Ph) 2, perfluoro (C, -C4) alkoxy, and perfluoro (C -? - C) alkyl, in a number ranging from zero to a total number of open valencies in the aromatic ring system; R ', R "and R'" are independently selected from hydrogen, (C ^ Cs) alkyl and heteroalkyl, aryl and unsubstituted heteroaryl (unsubstituted aryl) - (C-C4) alkyl, and (unsubstituted aryl) oxy- (C1-C4) alkyl Two of the substituents on the adjacent atoms of the aryl or heteroaryl ring can be optionally replaced with a substituent of the formula -TC (O) - (CH2) qU-, wherein T and U are independently -NH-, -O-, -CH2- or a bond, and q is an integer from 0 to 2. Alternatively, two of the substituents on the adjacent atoms of the aryl or heteroaryl ring can be optionally replaced with a substituent of the formula -A- (CH2), -B-, wherein A and B are independently -CH2-, -O-, -NH-, -S-, -S ( O) -, -S (O) 2-, -S (O) 2NR'- or a single bond, and r is an integer from 1 to 3. One of the single bonds of the newly formed ring can be optionally replaced with a double link Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring can be optionally replaced with a substituent of the formula - (CH2) S- X- (CH2) t-, where syt are integers independently from 0 to 3 , and X is -O-, -NR'-, -S-, -S (O) -, -S (O) 2-, or -S (O) 2NRA The substituent R 'in -NR'- and - S (O) 2NR'- is selected from hydrogen or (C -? - C6) unsubstituted alkyl.

The terms "halo" or "halogen" as used in the present invention refer to substituents Cl, Br, F or I. The term "haloalkyl" and the like, refer to aliphatic carbon radicals having at least one atom of hydrogen replaced by an atom of Cl, Br, F or I, which include mixtures of the different halo atoms. Trihaloalkyl includes trifluoromethyl and the like, for example in the form of preferred radicals. The terms "alkoxy", "alkylamino" and "alkylthio" (or thioalkoxy) are used in their conventional sense, and refer to the alkyl groups attached to the rest of the molecule through an oxygen atom, an amino group, or a sulfur atom, respectively. As used in the present invention, the term "heteroatom" means that it includes oxygen (O), nitrogen (N) and sulfur (S). The term "pharmaceutically acceptable salts" means that they include salts of the active compounds that are prepared with relatively non-toxic acids or bases, depending on the particular substituents found in the compounds described herein. When the compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of said compounds with a sufficient amount of the desired base, either pure or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When the compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of said compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbon, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydroiodic, or phosphorous acids and the like, as well as the salts derived therefrom. of relatively non-toxic organic acids acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-toluenesulfonic, citric, tartaric, methanesulfonic, and the like. They also include salts of amino acids such as arginate, and the like, and salts of organic acids such as glucuronic or galactunoric acids and the like (see, for example, the publication by Berge et al., "Pharmaceutical Salts" "Journal of Pharmaceutical Science, 66: 1-19 (1977).) Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted to their base or acid addition salts.The neutral forms of the compounds can be regenerated by contacting salt with a base or acid and isolating the parent compound in the conventional manner The origin form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, although otherwise the salts are equivalent to the form of origin of the compound for the purposes of the present invention In addition, of the salt forms, the present invention n provides compounds that are in a prodrug form. The prodrugs of the compounds described in the present invention are the compounds that readily pass through chemical changes under physiological conditions to provide the compounds of the present invention. In addition, prodrugs can be converted to the compounds of the present invention through chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir with an appropriate enzyme or chemical reagent.

Certain compounds of the present invention may exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, solvated forms are equivalent to unsolvated forms and are projected to be within the scope of the present invention. Certain compounds of the present invention can exist in multiple crystals or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are projected to be within the scope thereof. Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers and individual isomers are all projected to be within the scope of the present invention. The compounds of the present invention may also contain unnatural proportions of atomic isotopes in one or more of the atoms constituting said compounds. For example, the compounds can be radiolabelled with radioactive isotopes, such as tritium (3H), iodine-125 (125l), or carbon-14 (14C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are projected to be within the scope of the present invention. The term "cardiomyogenesis", as used in the present invention, refers to the differentiation of progenitor cells or precursors into cardiac muscle cells (e.g. cardiomyocytes) and the growth of cardiac muscle tissue. The progenitor or precursor cells can be pluripotent stem cells such as, for example, embryonic stem cells. The progenitor or precursor cells can be cells previously consigned to a myocardial lineage (eg, pre-cardiomyocyte cells) or cells that are not previously consigned (eg, multipotent adult stem cells). A "stem cell" as used in the present invention refers to any pluripotent cell or multipotent cell or progenitor cell or self-renewing precursor cell that has the ability to differentiate into multiple cell types. Stem cells suitable for use in the methods of the present invention, include those that have the ability to differentiate into myocardial lineage cells, e.g., cardiomyocytes. Suitable stem cells for use in the methods of the present invention include, for example, embryonic stem cells ("ESCs") and embryonic carcinoma cells ("EC"). Pluripotent embryonic stem cells have the ability to differentiate into all types of tissue, including neuronal cells, muscle cells, blood cells, etc. See for example the publication of Spradling and associates (2001). The term "differentiate" or "differentiation" as used in the present invention, refers to the process by which progenitor or precursor cells (e.g., stem cells) differentiate into specific cell types, e.g., cardiomyocytes. A differentiated cell can be identified through a number of characteristics that are unique or distinctive with respect to that of the particular cell type. For example, differentiated cells can be identified by their patterns of gene expression and protein expression. Normally, cells of a myocardial lineage express genes such as, for example, sarcomeric myosin heavy chain, myosin light chain 2V, eHAND, and ANF. See for example the publication of Small and Associates., Cell, 110: 725-735 (2002); Shin and associates., Cell, 110: 725-35 (2002). Also normally expressed by myocardial lineage cells are specific transcription factors of cardiac muscle cells such as MEF2, Nkx2.5 or the homodomain transcription factor HOP. See, for example, the Edmondson and Associates publication, Development, 1251-1263 (1994); Lin et al., Science, 276: 1404-1407 (1997). Additional transcription factors that are involved in the differentiation of cardiomyocytes include, for example, GATA4 (see for example, the publication of Grepin and associates, Development, 124: 2387-95 (1997)). One skilled in the art will recognize that other cardiac muscle specific genes can be used to monitor and determine differentiation. A "cardiomyocyte marker gene" is a gene that is expressed only by developing cardiomyocytes or only in rare form through other cell types, so that the marker gene is useful for the determination of whether a cell is a cardiomyocyte. An example of a cardiomyocyte marker gene is ANF, a polypeptide hormone that is synthesized mainly in cardiac myocytes and is a downstream object of several transcription factors of cardiomyogenesis. A "solid support", as used in the present invention in relation to induction of cardiomyogenesis, refers to a three-dimensional matrix or a planar surface on which stem cells can be cultured. The solid support can be derived from naturally occurring substances (for example based on proteins) or synthetic substances. For example, matrices based on naturally occurring substances may be composed of autologous bone fragments or commercially available bone substitutes as described, for example in the publication by Clokie et al., J. Craniofac. Surg. 13 (1): 111-21 (2002 and Isaksson, Swed, Dent J. Suppl 84: 1-46 (1992).) Suitable synthetic matrices are described, for example, in U.S. Patent Nos. 5,041,138, 5,512,474, and 6,425,222. For example, biodegradable man-made polymers such as polyglycolic acid, polyorthoester, or polyanhydride can be used for solid support.Calcium carbonate, aragonite, and porous ceramics (e.g., dense hydroxyapatite ceramics) are also suitable for use in the solid support Polymers such as polypropylene, polyethylene glycol, and polystyrene can also be used in solid support Cells grown and differentiated into a solid support that is a three-dimensional matrix normally grow on all surfaces of the matrix, for example, internal and The cells grown and differentiated into a solid support that is normally flat grows in a monolayer.The term "solid support" is also used In this context, the "solid support" refers to a polymeric support, such as a bead, that can be partially soluble in a suitable solvent or in the context of the preparation of compounds of Formula I. completely insoluble, and used to bind, for example, a reagent or a reagent of the reaction. Suitable solid supports include, but are not limited to, PAL resin, Wang resin and polystyrene resin. The term "culture" as used in the present invention refers to the maintenance of cells under conditions in which they can proliferate, differentiate and prevent senescence. For example, in the present invention, cultured embryonic stem cells proliferate and differentiate into cells of a myocardial cell lineage. The cells can be grown in a growth medium containing suitable growth factors, for example, a cocktail of growth factors containing proteins that facilitate or increase the development of cardiomyocytes. lll. Compounds of the Present Invention and Methods for its Preparation. A. The Compounds of Formula 1 In one aspect, the present invention provides compounds of Formula I having the following structure: In Formula I, R1 is a functional group including, but not limited to, hydrogen, C? -4alkyl, C3_8cycloalkyl and C0-2alkyl, substituted with groups 0-2R1a which are independently selected, and are functional groups, including but not limited to, halogen, C-? 4alkyl, C 1-4 alkoxy, -OH, -N (R 1b, R 1b), -SO 2 N (R 1b, R 1b), -C (O) N (R b, R1b). heterocycloalkyl and -O-aryl, or when the R1a groups are on adjacent ring atoms, are optionally taken together to form a functional group including, but not limited to, O- (CH2)? -2-O-, - OC (CH3) 2CH2- and - (CH2) 3-4-, or R1 is optionally taken together with the nitrogen to which it adheres to form a heterocycle, optionally substituted with C? -alkyl, C3. β-cycloalkyl, C 1 -alkylhydroxy and Co-alkylaryl; each R 1b group is independently selected and is a functional group including, but not limited to, hydrogen and C 4 alkyl. In Formula I, R2 is a functional group that includes but is not limited to, C1-alkyl, C-8-chloroalkyl, and C0-2-alkylaryl, substituted with groups 0-2 R 2a. The R group is independently selected and is a functional group including, but not limited to, halogen, C-a-alkyl, C-4alkoxy, -N (R2b, R2b), -SO2N (R2b, R2b), -C ( O) N (R2, R2b) and -O-aryl, or when the R2a groups are on adjacent ring atoms, are optionally taken together to form a functional group including, but not limited to, -O- (CH2) ? -2-O-, -OC (CH3) 2CH2- and - (CH2) 3--; and each group R 2b is independently selected and is a functional group that includes, but is not limited to, hydrogen, and C ?. alkyl, R3, in Formula I, is usually hydrogen, or R3 is optionally taken together with R2 and the nitrogen to which both are attached to form a heterocycle, optionally substituted with, for example, C-i. 4alkyl or Co-2alkylaryl. The compounds of the present invention include all pharmaceutically acceptable salts, isomers, solvates, hydrates and prodrugs thereof. In one embodiment, R1 is a functional group that includes, but is not limited to, the following: In a preferred embodiment, R1 is In one embodiment, R is a functional group that includes, but is not limited to, the following: In certain preferred embodiments, R3 is hydrogen. However, in other embodiments, R2 and R3 and the nitrogen to which both are attached form a heterocycle. Examples of suitable heterocycles include, but are not limited to, the following: In a preferred embodiment, the compounds of the present invention have the following general structure: In formula II, above, R2 is as defined above with respect to Formula I. In preferred embodiments, R2 of Formula I and II include, but are not limited to, the following: Preferred compounds of the present invention include, but are not limited to, the following (which are referred to in the present invention as Cardiogenol A, B, C and D, respectively): Other preferred compounds of the present invention include, but are not limited to, the compounds of examples set forth in Figure 4. The compounds of Formula I and II can be easily classified with respect to their ability to induce cardiomyogenesis using methods of in vitro and in vivo classification set forth below, and in particular, in the examples. B. Preparation of Compounds.

The compounds of the present invention can be prepared either by solid phase synthesis or solution phase. I. Solid Phase Synthesis. Methods directed to solid phase synthesis of the compounds of Formula I and II are described in Example 1, as well as in the publication by Ding et al., J. Am. Chem. Soc. 124 (8): 1594 (2002), Ding et al., J. Am. Chem. Soc, 124 (49): 14520-14521 (2002) and in US Patent Application No. 10 / 687,220 filed October 15, 2003, Application for US Patent No. 60/328, 763, filed October 12, 2001, U.S. Patent Application No. 60 / 331,835, filed November 20, 2001, U.S. Patent Application No. 60 / 346,480, filed January 7, 2002, Patent Application No. 60 / 348,089, filed January 10, 2002, and US Patent Application No. 10 / 270,030, filed October 12, 2002 (containing Legal File No. 21288-000340), whose teachings are incorporated to the present invention as reference. In general, 2-ethanolamine is coupled to the polystyrene resin (4-formyl-3,5-dimethoxyphenoxy) methyl (PAL resin) by reductive amination. The resin-PAL (1 g, 1.1 mmol) in DMF (4 mL) and ethanolamine (5.5 mmol), acetic acid (0.65 mL, 1.13 mmol) and sodium triacetoxyborohydride (720 mg, 3.4 mmol) are subsequently added to the mixture. solution. The mixture is stirred gently at room temperature for approximately 12 hours. Subsequently the resulting resin is washed, for example, with DMF (10 L, 3 times), methanol (10 mL, 3 times) and dichloromethane (10 mL, 3 times). Subsequently, the resin bound with aniline is reacted with 2,4-dichloropyrimidine (2.2 mmole) and diisopropylethylamine (0.5 mL, 3 mmole) in 1-butanol (5 mL) at a temperature of 80 ° C for 12 hours. Subsequently the resulting resin is washed as described above. The PAL resin bound with pyrimidine (100 mg, 0.1 mmole) is mixed, for example, with different aromatic amines (1.0 mmole) in 1 ml of butanol. The reaction mixture is heated to a temperature of 120 ° C for about 12 hours to produce the desired products. The resulting resin is then washed as described above and dissociated with CH2Cl: TFA: Me2S: H2O / 45: 5: 5 (v / v / v / v, 0.5 mL) at room temperature for about 2 hours. The solution is collected and dried in vacuo to produce the desired crude product. Subsequently, the crude products are easily purified, using, for example, RP-HPCL for preparation with H2O (with 0.1% TFA) and MeCN as solvents. Those skilled in the art will appreciate that similar solid phase synthesis techniques can be used to prepare the other compounds of Formula I and II. 2. Synthesis of the Solution Phase. The compounds of the present invention can be prepared by solution phase synthesis as set forth in Example 1. Typically, the solution phase synthesis of the compounds of Formula I comprises first replacing a 2,4-dihaloheteroaryl (such as a 2,4-dichloropurine) with a suitable substituent (such as a hydroxyethylamino group) under suitable reaction conditions known to one skilled in the art. This is followed by substitution with a suitable second substituent (such as a suitably substituted aniline (e.g., (4-phenyl amino), 4-phenoxyaniline, 4-methoxyaniline, 4-amino-trans-stilnene, etc.) under suitable reaction conditions known to those skilled in the art The compounds of the present invention can be purified using standard methods (such as RP-HPLC preparation) known to those skilled in the art. Compositions to Induce Cardiomyogenesis.

The compositions of the present invention can be used to induce cardiomyogenesis in mammalian cells. Generally speaking, a mammalian cell is contacted with a compound of Formula I, wherein the mammalian cell differentiates into a cell of a myocardial lineage. The mammalian cell can be contacted with a compound of Formula I (or a composition thereof) either in vivo or in vitro. For example, cardiogenol C can be administered directly to the injured or impaired heart muscle intravenously or by direct administration during surgery. A. In vivo Induction of Cardiomyogenesis The compounds of Formula I, as well as compositions thereof, can conveniently be used to induce cardiomyogenesis in vivo. The compounds and compositions of the present invention are administered to an individual mammal, for example a mammal such as a human, in an amount effective to induce the differentiation of mammalian cells into cells of a myocardial lineage. By virtue of their ability to induce cardiomyogenesis, the compounds of Formula I are useful for the repair of damaged myocardium in acute heart diseases and for treating conditions such as cardiomyopathy. In a preferred embodiment, the compounds and compositions of the present invention are used to generate cardiomyocytes for the purpose of studying cardiac muscle tissue development. In another preferred embodiment, the compounds and compositions of the present invention are used during the treatment of a subject in need of repair or augmentation of damaged or ened cardiac muscle tissue. In another embodiment, the compositions of the present invention are used to treat a subject who wishes the augmentation or improvement of cardiac muscle tissue that is not damaged or ened. Such subjects may include, for example, those at risk of heart disease or disease. One skilled in the art will appreciate that the compositions of the present invention can be used alone or in combination with other compounds and therapeutic regimens to induce cardiomyogenesis. For example, a compound of Formula I may be administered together with purified or synthesized growth factors and other agents or combinations thereof that increase the development of cardiac muscle tissue. An effective amount of the composition will be administered by the existence, nature and degrees of any adverse side effects that accompany the administration of the composition, the LD50 of the composition; and the side effects of the composition in various concentrations. Typically, the amount of composition administered will range from about 0.01 to about 20 mg per kg, more usually from about 0.05 to about 15 mg per kg, even more usually from about 0.1 to about 10 mg per body weight. The compositions can be administered, for example, by intravenous, oral, intraperitoneal, or subcutaneous infusion. Oral administration is the preferred method of administration. Compound formulations may be presented in unit doses or in sealed multi-dose containers such as ampoules and flasks. The compositions of the present invention are usually formulated with a pharmaceutically acceptable carrier prior to administration to an individual or subject. Pharmaceutically acceptable carriers are determined, in part, through the particular composition being administered (e.g. Cardiogenol C) as well as through the particular method for administering the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see for example, Remington's Pharmaceutical Sciences, 17th Edition, 1989). Formulations suitable for oral administration may consist of (a) liquid solutions, such as an effective amount of the compound of Formula I suspended in diluents such as water, saline or PEG 400.; (b) capsules, scented pads or tablets each containing a predetermined amount of the active ingredient in the form of liquids, solids, granules and gelatin; (c) suspensions in a suitable liquid; and (d) suitable emulsions. The tablet forms may include one or more of the following: lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, acid stearic, and other excipients, colorants, fillers, binders, diluents, buffering agents, wetting agents, preservatives, flavoring agents, inks, disintegrating agents and pharmaceutically combattable vehicles. The forms of lozenges may comprise the active ingredient in a flavor, for example, sucrose, as well as lozenges comprising the active ingredient in an inert base, such as gelatin and glycerin emulsions or sucrose and acacia, gels and the like, which contain, in addition to the active ingredient, carriers known in the art.

The compositions of the present invention may be in formulations suitable for other routes of administration such as, for example, intravenous, intraperitoneal, or subcutaneous infusion. The formulations include, for example, aqueous and non-aqueous solutions, sterile isotonic injection solutions which may contain anti-oxidants, bacteriostatic buffers and melted solutions which convert the isotonic formulation with the blood of the projected container, and sterile aqueous and non-aqueous suspensions which may include suspending agents, solubilizers, thickening agents, preservatives and stabilizers. Injection solutions and suspensions can be prepared from sterile powders, granules and tablets. The dose administered to a patient within the context of the present invention should be sufficient to effect a beneficial therapeutic response for the patient over time. For example, if the compositions of the present invention are administered to treat or prevent cardiomyopathy, the dose administered to the patient should be sufficient to prevent, slow or reverse the decreased ability of the heart muscle to contract rhythmically. The dose will be determined through the effectiveness of the particular composition employed and the condition of the patient, as well as the body weight and surface area of the patient to be treated. The size of the dose will also be determined by the existence, nature and degree of any adverse side effects that accompany the administration of a particular composition in a particular patient. B. In vitro induction of cardiomyogenesis. The compositions of the present invention can be conveniently used to induce cardiomyogenesis in vitro. The mammalian cells are contacted with the compositions, wherein the mammalian cells differentiate into cells of a myocardial lineage. 1. Suitable cells. Cells that will be differentiated into cells of a myocardial lineage can be derived from any suitable mammal. For example, the cells can be obtained from rodents such as, for example, mice, rats, guinea pigs and rabbits; non-rodent mammals, such as, for example, dogs, cats, pigs, sheep, horses, cows and goats; primates such as, for example, chimpanzees and humans. The cells that will be differentiated can be primary cells or they can be cells that are kept in culture. If the cells are kept in culture, they are usually contacted with the compounds / compositions of the present invention between the 12th and 15th passage in the culture. Techniques and methods for establishing a primary cell culture for use in the methods of the present invention are known to those skilled in the art (see for example the Humason Publication, ANIMAL TISSUE TECHNIQUES, 4th edition, WH Freeman and Company ( 1979), and Ricciardelli et al., In Vitro Cell Dev. Biol., 25: 1016 (1989)). Human mesenchymal stem cells (MSCs) can be obtained by isolating pluripotent mesenchymal stem cells from other cells in the bone marrow or other MSC source. Bone marrow cells can be obtained from the iliac crest, femoral crest, tibias, spines, ribs or other medullary spaces. Other sources of human mesenchymal stem cells include embryonic albumin sac, placenta, umbilical cord, fetal and adolescent skin, blood, adipose tissue and muscle satellite cells, typically cells from a tissue specimen containing mesenchymal stem cells. they are grown in a growth medium containing growth factors that (1) stimulate mesenchymal stem cell growth without differentiation, (2) allow selective adhesion only of mesenchymal stem cells to a substrate surface. for an adequate amount of time, the non-adherent matter is removed from the surface of the substrate, thus providing an expanded population of mesenchymal stem cells. Therefore, homogeneous MSC populations are obtained by positive selection of adherent marrow and periosteal cells that are free of associated markers with either hematopoietic cells or differentiated mesenchymal cells. Preferably, the mammalian cells contacted through the compounds of the present invention are stem cells, particularly embryonic stem cells (ESCs). Methods for the isolation of human and animal ESCs are well known in the art. See for example the Brook FA Publication, Proc. Nati Acad. Sci. USA, 94: 5709-12 (1997); Grounds and associates, J. Histochem. and Cytochem., 50: 589-610 (2002); Reubinoff, Nat. Biotech. 18: 399-404 (2000). Mammalian embryonic stem cells include, for example, murine R1 cells and human embryonic stem cells. 2. General Cultivation Methods. Mammalian cells (e.g., ESCs) can be contacted with a compound of Formula I alone, in combination with other compounds of Formula I, either together in a single mixture or in sequences, or in the presence of other factors of growth. Those skilled in the art will appreciate that the amount of the compounds, e.g., the amount of any cardiogenol A, B, C, or D, and growth factors can be adjusted to facilitate the induction of differentiation in particular cell types. . For example, the amount of a cardiogenol contacted with the cell typically ranges from about 0.01 μM (52 ng / ml) to about 10 μM (2.6 μg / ml), more usually from about 0.02 μM to about 5 μM, even more usually from about 0.05 μM to about 1 μM, still more typically from about 0.075 μM to about 0.5 μM, and most typically about 1 μM. This aspect of the present invention depends on routine techniques in the field of cell culture. Suitable cell culture methods and conditions can be determined by those skilled in the art using known methodology (see for example Freshney and Associates Publication, CULTURE OF ANIMAL CELLS, 3rd edition, 1994). In general, the cell culture environment includes consideration of factors such as the substrate for cell growth, cell density and cellular contract, gas phase, medium and temperature. Incubation of cells is generally carried out under conditions known to be optimal for cell growth. Such conditions may include, for example, a temperature of about 37 ° C and a humidified atmosphere containing about 5% CO2. The duration of incubation can vary widely, depending on the desired results. In general, the incubation preferably continues until the cells are expressed properly. Proliferation is conveniently determined using 3H thymidine incorporation or BrdU labeling. Plastic plates, flasks or roller bottles can be used to grow cells according to the methods of the present invention. Suitable culture containers include, for example, multiple deposit plates, Petri dishes, tissue culture tubes, bottles, roller bottles and the like. The cells are grown in optimal densities that are determined empirically based on the cell type. The cells are usually passed 12 to 15 times and discarded after 15 passes. Cultured cells normally grow in an incubator that provides a suitable temperature, for example, the body temperature of the animal from which the cells were obtained, taking into account regional variations in temperature. Generally, a temperature of 37 ° C is the preferred temperature for cell culture. Most incubators are modified at approximately atmospheric conditions. The important constituents of the gas phase are oxygen and carbon dioxide. Normally, atmospheric oxygen tensions are used for cell cultures. Culture containers are normally ventilated in the incubator atmosphere to allow gas exchange using gas permeable lids or by avoiding the seal of culture containers. Carbon dioxide plays an important role in pH stabilization, along with the regulator in the cell medium and is usually in a concentration of 1 to 10% in the incubator. The preferred CO2 concentration is usually 5%. The defined cell media is available in packaging, premixed powders or previously sterilized solutions. Examples of commonly used media include MEM-a, DME, RPMI 1640, DMEM, Iscove's complete medium or McCoy's medium (see, for example, Gibco BRL / Life Technologies Catalog and Reference Guide; Sigma Catalog). Normally, MEM-a or DMEM are used in the methods of the present invention. The defined cell culture media is often supplemented with 5 to 20% serum, usually heat deactivated serum, for example, human, horse, calf and fetal bovine serum. Normally, 10% fetal bovine serum is used in the methods of the present invention. The culture medium is normally buffered to maintain the cells at a pH of preferably from about 7.2 to about 7.4. Other supplements for the medium typically include, for example, antibiotics, amino acids and sugars and growth factors. C. Detection of cardiomyogenesis. After administration of the compositions of the present invention in vivo or in vitro, the induction of cardiomyogenesis can be detected through a number of different methods, including but not limited to: detection of expression of cardiomyocyte-specific proteins, detection of expression of cardiac muscle cell-specific transcription factors, detection of expression of essential proteins for cardiac muscle function and detection of palpitation of cardiac muscle cells. Specific examples of cardiomyocyte specific proteins and cardiac muscle cell-specific transcription factors are described in the present invention. 1. Detection of cardiomyocyte-specific proteins. Expression of cardiac muscle cell differentiation can be detected by measuring the specific protein level of cardiac muscle cell or mRNA. The level of cardiac muscle cell-specific proteins can be conveniently measured using immunoassays, such as immunohistochemical staining, Western blotting, ELISA and the like, with an antibody that selectively binds the particular cardiomyocyte specific proteins or a fragment from the same. Detection of the protein using protein-specific antibodies in immunoassays is known to those skilled in the art (see for example, Harlow &Lane Publication, Antibodies: A Laboratory Manual (1988), Coligan, Current Protocols in Immunology ( 1991); Goding, Monoclonai Antibodies: Principies and Practice 2nd edition, 1986); and Kohier & Milstein, Nature 256: 495-497 (1975). To measure the mRNA, amplification is preferred, for example, PCR, LCR, or hybridization assays, eg, Northern hybridization, RNAse protection and spot spotting. The level of protein or mRNA is selected, for example, using directly or indirectly labeled detection agents, for example, nucleic acids labeled in fluorescent or radioactive form, labeled antibodies in radioactive or enzymatic form. These assays are well known to those skilled in the art and are described, for example, in Ausubel and Associates Publication, ed. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (2001).

Normally, cardiac muscle cell protein ANF expression is used to detect differentiated cardiomyocytes. ANF is a polypeptide hormone that is synthesized mainly in cardiac myocytes and is a downstream target of several cardiomyogenesis transcription factors; it is considered a specific cardiomyocyte "marker" gene (Boer, Exp. Cell Res., 207: 421-29 (1993).) Activation of the ANF gene can be measured, for example, by inserting the ANF promoter region into an upstream plasmid reporter of a readily detectable protein or an enzyme whose activity can be easily detected, such as luciferase.An increase in the level of expression of the reporter gene under these circumstances indicates a differentiation of cardiomyocytes a) Immunohistochemical detection. For direct immunohistochemical staining of cells to detect, for example, cardiomyocyte-specific genes, the cells are seeded in 96-well assay plates at a suitable density and treated with an appropriate amount of a compound of the formula I (e.g. cardiogenol A), either alone or with other growth factors for a suitable time. The cells are subsequently fixed in a 10% formalin solution. The fixed cells are again washed and stained with a specific reagent for the protein of interest (eg, an antibody specific for the protein, or if an enzyme reporter gene is used, a reagent whose detection capability, for example, by methods fluorometric change the presence of the reporter gene enzyme) using methods known to those skilled in the art (see for example Harlow &Lane Publication, 1988, supra; Coligan, 1991, supra; Goding, 1986, supra; and Kohier; &Milstein, 1975, supra). The photographic images of the cells are taken and counted manually from the images of the positive cells that express the specific gene of cardiomyocyte. 2. Detection of specific transcription factors of cardiac muscle cell. The expression of cardiac muscle cell-specific transcription factors can be detected using reporter gene assays. A variety of reporter gene assays are known to a person skilled in the art. See for example, the New and Associated Publication, Phytother. Res., 17: 439-48 (2003); Schenborn and associates, Mol. Biotechnol. 13: 29-44 (1999). Reporter genes such as, for example, chloramphenicol acetyltransferase, firefly luciferase, bacterial luciferase or β-galactosidase can be used in the reporter gene assays. The reporter construct is usually transfected temporarily or stably in a cell. The promoter region of the relevant gene is normally amplified by suitable PCR primers. The resulting PCR product is inserted into a suitable cloning vector, amplified and sequenced. The resulting plasmid is digested with suitable restriction enzymes and the resulting fragment is inserted into a vector comprising a reporter gene. (a) Transfected cells temporarily. For reporter gene assays with transfected cells in a temporary manner, the cells are normally plated in a plate of six reservoirs at a density of approximately 30,000 cells / reservoir in 2 ml of growth medium and incubated overnight or for an adequate time . The plasmid DNA is transfected into the cells using a suitable transfection reagent. After 8 hours, the transfected cells are plated in 96-well assay plates (e.g. Corning) and treated with an appropriate amount of a compound of the formula I (e.g., cardiogenol A). The cells are incubated for 4 days, subsequently the activity of the reporter gene in the cells is assayed using methods known to those skilled in the art. b) Stably transfected cells.

For reporter gene assays with stably transfected cells, the cells are normally plated in a plate of six reservoirs at a density of approximately 30,000 cells / reservoir in 2 ml of growth medium incubated overnight or for a suitable time. An appropriate amount of the reporter plasmid and a vector comprising a selectable marker (e.g., an antibiotic resistance gene) are transfected together into the cells using a suitable transfection reagent. After a suitable incubation time, the cells are seeded in a 10 cm culture dish and an appropriate amount of antibiotic is added to the culture medium. Fresh antibiotic is added at appropriate intervals. Antibiotic resistant colonies are pooled to produce the transfected cells. Stable way The transfected cells are plated in 96-well assay plates (e.g. Corning) and treated with an appropriate amount of the compound of formula I (e.g., cardiogenol A). The cells are incubated for 4 days, then the activity of the reporter gene in the cells is assayed using methods known to those skilled in the art. 3. Administration of differentiated cardiomyocytes. Differentiated cardiomyocytes can be administered to a subject through any means known to those skilled in the art. In one embodiment of the present invention, differentiated cardiomyocytes on an intact solid support (eg, a three-dimensional matrix or a planar surface) can be administered to the subject, for example, by surgical implant. Alternatively, differentiated cardiomyocytes can be separated from the matrix, for example, by treatment with a protease, prior to administration to a subject, for example, intravenously, subcutaneously, or intraperitoneally. In some embodiments of the present invention, the embryonic stem cells are extracted and subsequently contacted with a matrix for proliferation and differentiation in cells of a myocardial lineage. The cells can be extracted from the subject to be treated, for example, autologous (thus avoiding the rejection of the implant by immunology) or can be extracted from a second subject, for example heterologous. In any case, the administration of cells can be combined with an adequate immunosuppressive treatment. Differentiated cardiomyocytes according to the methods of the present invention can be administered to a subject through means known in the art. Suitable means of administration include, for example, intravenous, subcutaneous, intraperitoneal and surgical implant administration. Cardiomyocytes can be injected directly into cardiac muscle or applied topically, for example, during heart surgery. The cells may be in formulations suitable for administration, such as, for example, sterile isotonic aqueous and non-aqueous injection solutions, which may contain antioxidants, buffers, bacteriostats and melted solutions which render the formulation isotonic with the blood of the projected receptor. , and aqueous and non-aqueous sterile suspensions which may include suspending agents, solubilizers, thickening agents, stabilizers and preservatives. The injection solutions and suspensions can be prepared from sterile powders, granules and tablets. For surgical implantation, differentiated cells are usually left on an intact solid support, for example, a three-dimensional matrix or flat surface. The matrix or flat surface is surgically implanted at the appropriate site in a subject. For example, a patient in need of a replacement of a part of the cardiac muscle tissue may have differentiated cells on an intact solid support implanted surgically. In determining the effective amount of cells that will be administered in the treatment or prophylaxis of conditions due to impaired or impaired cardiac muscle cells, the specialist evaluates cellular toxicity, transplant reactions, progress of disease and production of anti-cell antibodies. For administration, differentiated cardiomyocytes according to the methods of the present invention can be administered in an amount effective to provide heart muscle cells to the subject, taking into account the side effects of cardiomyocytes in various concentrations, as applied to the mass and general health of the patient. Administration can be achieved through a single dose or divided doses. EXAMPLES The following examples are offered to illustrate, but not limit the present invention. 1. Example 1: Synthesis and characterization of cardiogenol A, B, C, and D. All the chemicals used for the synthesis were purchased from Aldrich. 2,4-Dichloropyrimidine (200 mg, 1.34 mmol) was dissolved in 5 ml of ethanol and 282 μL (1.62 mmol) of di-isopropylethylamine (DIEA) and 90 mg (1.47 mmol) of ethanolamine was added to the solution. Subsequently, the reaction mixture was heated at a temperature of 50 ° C for 2 hours to produce 2-chloro-4- (1-hydroxyethylamino) -pyrimidine (80% yield). 20 mg (0.12 mmol) of 2-chloro-4- (1-hydroxyethylamino) -pyrimidine were dissolved in 1-butanol (1 ml) and 42.4 mg (0.23 mmol) of 4- (phenylamino) -aniline were added. The reaction mixture was heated at a temperature of 200 ° C for 15 minutes in a microwave reactor to produce cardiogenol A (85% yield). Similarly, the use of 42.7 mg (0.23 mmol) of 4-phenoxyaniline, 28.4 mg (0.23 mmol) of 4-methoxyaniline or 45.0 mg (0.23 mmol) of 4-amino-trans-stilbene will produce cardiogenol B (80% production ), C (90% production) or D (75% production), respectively. The compounds were purified by preparative HPLC using H2O (with 0.1% TFA) and MeCN as solvents with a linear gradient of 5% to 90% MeCN in 10 minutes. The desired peaks were collected and dried by freezing to produce final products. Cardiogenol A: 1 H NMR (400 MHz, DMSO): d (ppm) 3. 44 (m, 2H), 3.57 (m, 2H), 6.20 (d, 1H, H = 7.2), 6.83 (t, 1H, J = 7.2), 7.08 (m, 5H), 7.23 (t, 2H, J = 8.3), 7.34 (d, 2H, J = 7.7), 7.67 (d, 1H, J = 6.6), 8.25 (s, 1H), 10.18 (s, 1H). High resolution mass spectrometry (MALDI-FTMS): Calculated [MH +] (C? 8H20N5O) 322.1662, found 322.1660. Cardiogenol B: 1 H NMR (400 MHz, DMSO): d (ppm) 3. 45 (m, 2H), 3.56 (m, 2H), 6.24 (d, 1H, J = 7.2), 7.03 (m, 5H), 7.14 (t, 1H, J = 7.4), 7.40 (t, 2H, J = 7.5), 7.55 (d, 2H, J = 8.6), 7.73 (d, 1H, J = 7.0), 8.97 (s, 1H), 10.30 (s, 1H). High resolution mass spectrometry (MALDI-FTMS): Calculated [MH +] (C- | 8H1gN4O2) 323.1502, found 323.1498. Cardiogenol C: 1 H NMR (400 MHz, DMSO): d (ppm) 3. 42 (m, 2H), 3.55 (m, 2H), 3.72 (s, 3H), 6.21 (d, 1H, J = 7.2), 6.97 (d, 2H, J = 8.9), 7.41 (d, 2H, J = 8.4), 7.66 (d, 1H, J = 7.1), 8.93 (s, 1H), 10.07 (s, 1H). High resolution mass spectrometry (MALDI-FTMS): Calculated [MH +] (C13H? 7N4O2) 261.1346, found 261.1342. Cardiogenol D: 1H NMR (400 MHz, DMSO): d (ppm) 3.48 (m, 2H), 3.62 (m, 2H), 6.26 (d, 1H, J = 7.2), 7.27 (m, 3H), 7.38 ( t, 2H, J = 7.5), 7.61 (m, 6H), 7.77 (d, 1H, J = 7.0), 9.00 (s, 1H), 10.35 (s, 1H). High resolution mass spectrometry (MALDI-FTMS): Calculated [MH +] (C20H21N4O) 333.1710, found 333.1711. In addition to the above solution phase synthesis methods, the compounds of the present invention can also be made using solid phase synthesis methods as indicated below: Generally, 2-ethanolamine was coupled to polystyrene resin (4- formyl-3,5-dimethoxyphenoxy) methyl (PAL resin) by reductive amination. The resin-PAL (1 g, 1.1 mmol) was suspended in DMF (4 ml) and subsequently ethanolamine (5.5 mmol), acetic acid (0.65 ml, 1.13 mmol) and sodium triacetoborohydride (720 mg, 3.4) were added to the solution. mmol). The mixture was stirred gently at room temperature for 12 hours. The resulting resin was subsequently washed with DMF (10 ml three times), methanol (10 ml, three times), and dichloromethane (10 ml, three times). The resin bound by aniline was subsequently reacted with 2,4-dichloropyrimidine (2.2 mm) and diisopropylethylamine (05 ml, 3 mmol) in 1-butanol (5 ml) at a temperature of 80 ° C for 2 hours. Subsequently the resulting resin was washed as described above. The PAL resin bound with pyrimidine (100 mg, 0.1 mmol) was mixed with different aromatic amines (1.0 mmol) in 1 ml of butanol. The reaction mixture was heated at a temperature of 120 ° C for 2 hours to produce the desired products. The resulting mixture was subsequently washed as described above and dissociated with CH2Cl2: TFA: Me2S: H2O / 45: 45: 5: 5 (v / v / v / v, 0.5 ml) at room temperature for 2 hours. The solution was collected and dried in vacuo to yield the desired crude product. The crude products can be further purified using RP-HPLC preparation, using H2O (with 0.1% TFA) and MeCN as solvents. 2. Example 2: Cell culture and high performance classification for induction molecules of cardiomyogenesis. Cells of embryonic carcinoma (s) P19 (from ATCC) were grown in MEM-alpha with 7.5% newborn calf serum and 2.5% FBS (Gibco) at a temperature of 37 ° C in 5% CO2. P19CL6 cells (a gift from Dr. Michael Schneider and Dr. Nakamura Teruya) were grown in MEM-alpha with 10% FBS (from Gibco) at a temperature of 37 ° C in 5% CO2. A fragment (~700 bps) containing a rat ANF promoter region was amplified using PCR primers (5'-ccgacgcgtgaaacatcacattggttgcctt and 5'-ccgctcgagcactctctggtttctctctc) and subsequently subcloned into the plasmid of the luciferase reporter PGL3-BV using sites of restriction Mlul and Xhol. A stable P19 clone harboring the reporter plasmid produced a 5- to 7-fold increase (FIG. 1) in the luciferase signal under the standard cardiomyogenesis differentiation conditions for P19 cells (EB formation and treatment with 1% DMSO (see Publication Skerjank IS, Trends Cardiovasc Med., 9: 139-143 (1999)) This cell line was used to classify a heterocycle library of 100,000 compounds in a monolayer format according to the following method: 103 cells were plated in each Deposit of plates of 384 deposits with 100 μL of induction medium (MEM-alpha with 5% FBS); 500 nL of a 1 mM compound solution was subsequently added in each tank. After treatment with the compound for 3 days, the medium was changed without additional compounds added. Luciferase activity was measured after 7 days of compound treatment using the Bright-Glo luciferase assay kit (Promega). It was identified with approximately 80 compounds that activated more than four times the luciferase activity in the absence of EBs. MHC is one of the essential motor proteins responsible for the contraction capacity of the heart muscle and was used as a test for secondary differentiation. 35 of the 80 compounds identified in the classification assay described above also included heavy chain expression of induced sarcomeric myocin (MHC) in P19CL6 cells. The P19CL6 cell line is a subclone of the P19 EC cells with the greatest potential for cardiomyogenesis. See the Publication of Habara-Ohkubo, Cell Struct. Funct. 21: 101-110 (1996). The MHC expression was determined in P19CL6 cells by immunostaining cells with anti-MHC antibody (MF20) (Figure 2F). 3. Example 3: Identification of cardiogens A, B, C, and D in the form of compounds with induction activity of cardiomyogenesis.

Among the 35 compounds identified in the previous example, four diaminopyrimidines, cardiogenol A-D (Table 1), were the most potent in the induction of MHC expression. Table 1 Compound Name EC50 Activity Toxicity EC50 Optimal Cardiogenol D A ° "1 μM ++++ 2.5 μM The optimal activities of the cardiogenols in Table 1 are indicated by a series of "+" signs, as follows: + +: 10-25% cells are positive for MHC after 7 days; + + +: 25-40% cells are positive for MHC after 7 days; + + + +: 40-55% cells are positive for MHC after 7 days. To confirm that these compounds are general induction agents of cardiomyogenesis, their effects were analyzed in undifferentiated R1 mouse ESCs. Mouse ESCs R1 can be maintained in a pluripotent state with the addition of the leukemia inhibitory factor (LIF) in the culture medium. The embryonic stem cell line R1 was cultured in tissue culture dishes coated with gelatin with DMEM Elimination with replacement of serum ES 15%, 1 mM L-glutamine (from Gibco), existence of non-essential amino acids at 1% , existence of 1% nucleosides, 0.1 mM of beta-mercaptomethanol (from Specialty Media) and 1000 units / ml of leukemia inhibition factor (LIF, from Chemicon). For differentiation, R1 cells were plated in a monolayer (10,000 cells / reservoir) in plates of 384 deposits or 96 gelatin-coated deposits with 100 μL of DMEM with 10% FBS and 0.25 μM of compounds. The LIF was not present during the differentiation. After 7 days in culture (3 days with compounds and subsequently, after changing the medium without added additional compound, another 4 days), the presence of pulsating heart muscle was visualized under a microscope. In addition to the MHC expression (Figure 2A), the cardiac specific gene, GATA-4, was detected by immunofluorescent staining using anti-GATA-4 antibody (Figure 2B). GATA-4 is a restricted transcription factor for developing heart and its overexpression increases cardiomyogenesis in P19 cells (Grepin and associates, Development, 124: 2387-95 (1997); Chadron et al., Cell and Dev. Biol. , 10: 85-91 (1999), Gag et al, Nature, 424: 443-447 (2003) As shown in Figure 3, neither MHC nor GATA-4 are expressed in undifferentiated R1 mouse ESCs. It was also observed that treatment with the compound decreased cell proliferation without significant cell death, indicating that this process is not simply a selection of cardiac precursor cells with cell death in other lineages 4. Example 4: Cardiogenol C is a potent inducer of cardiomyogenesis in embryonic stem cells Cardiogenol C has an aniline of p-metonym substituted in the position C2 of pyrimidine and is very potent with EC50 at 0.1 μM to induce the differentiation of positive cardiomyocytes MHC of ESCs. l C showed significant cellular toxicity only at concentrations greater than 25 μM, after treating R1 cells with 0.25 μM of compound for 3 days and further culturing in a medium without compound for 4 days, more than 50% of cells stained positive for MHC and more than 90% of the cells are positive for GATA-4, consistent with the previous observation that GATA-4 is expressed earlier than MHC. See Publication of Boheler and associates, Circ. Res., 91: 189-201 (2002). In addition, there were many areas of palpitation in R1 cells treated with cardiogenol C, demonstrating that these MHC positive cells can form functional heart muscle. These results indicate that the majority of the cell population was induced by cardiogenol C to differentiate into cardiac lineage (in the absence of aggregation and EB formation). This is in contrast to the standard current method for inducing cardiomyogenesis in ESCs by aggregation and formation of EBs, which results in only 5% of the cell population that forms cardiomyocytes. See the Boheler and Associates Publication (2002). 5. Example 5: Detection of cardiac muscle cell-specific transcription factors in differentiated ESCs using cardiogenol C. To further characterize the activity of cardiogenol C, the expression of cardiac muscle cell-specific transcription factors MEF2 and Nkx2.5 was examined (Figure 2C and D). Members of the MEF2 family are essential for the development of muscles. See for example, Edmondson and Associates Publication, Development, 1251-1263 (1994); Lin and associates, Science, 276: 1404-1407 (1997). Nkx2.5 together with GATA-4 regulates the expression of multiple cardiac muscle-specific genes (eg, myosin light chain 2V, atrial natriuretic factor, eHAND, and homeodomain transcription factor HOP). See for example, Publication of Small and Associates, Cell, 110: 725-735 (2002); Shin and associates, Cell, 110: 725-35 (2002). In addition, the targeted disruption of the Nkx2.5 gene is lethal to the embryo and results in the arrest of cardiac development. See for example, Lyons and Associates Publication, Genes Dev., 9: 1654-66 (1995). Approximately 90% of the cells treated with cardiogenol C stained positive for MEF2 and Nkx2.5, confirming in addition that ESCs differentiate in cardiac muscle by cardiogenol C. All publications and patent applications mentioned in this specification are incorporated as reference , as if each publication or individual patent application were specifically and individually indicated to be incorporated as a reference. Although the above invention has been described with certain details by way of illustration and example for purposes of clarity of understanding, those skilled in the art will appreciate in light of the teachings of the present invention, that certain changes and modifications can be made to the same without departing from the spirit or scope of the appended claims.

Claims (51)

1. R E I V I N D I C A C I O N S 1. A compound of formula I having the following structure:

   wherein: R1 is a member selected from the group consisting of hydrogen, C1-4alkyl, C3.8cycloalkyl and C0-2alkylaryl, substituted with 0-2 R1a groups which are independently selected from the group consisting of halogen, C1-4alkyl, C1 -4alcoxy, -OH, -N (R1b, R1b), -SO2N (R1b, R1b), heterocycloalkyl and -O-aryl or when said R1a groups are in adjacent ring atoms, are optionally taken together to form a functional group including, but not limited to, O- (CH2) 1-2-O-, -OC (CH3) 2CH2- and - (CH2) 3-4-, or R1 is optionally taken together with the nitrogen to which it adheres to form a heterocycle, optionally substituted with C1. alkyl, C3-cycloalkyl, C1-4alkylhydroxy and C0-2alkylaryl; each group R1b is a member that is independently selected from the group consisting of hydrogen and C? -alkyl.

   R2 is a member selected from the group consisting of C -? - alkyl, C3-8cycloalkyl and C0-2alkylaryl, substituted with groups 0-2 groups R2a which are independently selected from the group consisting of halogen, C-t. 4alkyl, C-, - 4alcoxy, -N (R2b, R2b), -SO2N (R2b, R2b), -C (O) N (R2b, R2b) and -O-aryl, or when the R2a groups are in atoms of adjacent rings are optionally taken together to form a member selected from the group consisting of -O- (CH2) 1.2-O-, -OC (CH3) 2CH2- and - (CH2) 3-4-; and each group R2 is a member that is independently selected from the group consisting of hydrogen, and C -4alkyl; and R3 is hydrogen, or R2 is optionally taken in conjunction with R3 and the nitrogen to which both are attached form a heterocycle, optionally substituted with, for example, C4-4alkyl or C0-2alkylaryl.
2. 2. The compound according to claim 1, characterized in that R1 is a member selected from the group consisting of:
3. 3. The compound according to claim 2, characterized in that R1 is:
4. 4. The compound according to claim 1, characterized in that R2 is a member selected from the group consisting of:
5. 5. The compound according to claim 1, characterized in that R3 is hydrogen.
6. 6. The compound according to claim 1, characterized in that R2 and R3 and the nitrogen to which both are adhered form a heterocycle.
7. The compound according to claim 6, characterized in that the heterocycle is a member selected from the group consisting of:
8. 8. The compound according to claim 1, characterized in that the compound has the following structure:
9. The compound according to claim 1 or 8, characterized in that R2 is a member selected from the group consisting of:
10. 10. The compound according to claim 1, characterized in that the compound is a member selected from the group consisting of:
11. 11. A pharmaceutical composition comprising a compound according to claim 1 or claim 10 and a pharmaceutically acceptable carrier.
12. 12. A method for inducing myocardiogenesis, wherein the method comprises: contacting a mammalian cell with a compound according to claim 1, characterized in that the mammalian cell differentiates into a myocardial lineage cell.
13. The method according to claim 12, characterized in that the compound according to claim 1 is a pharmaceutically acceptable carrier.
14. 14. The method according to claim 12, characterized in that the mammalian cell is in a mammal.
15. 15. The method according to claim 14, characterized in that the contact step is by oral administration of the compound with the mammal.
16. 16. The method according to claim 14, characterized in that the step of contacting is by intravenous administration of the compound to the mammal.
17. 17. The method according to claim 14, characterized in that the step of contacting is by subcutaneous administration of the compound to the mammal.
18. 18. The method of compliance with the claim

    14, characterized in that the step of contacting is by intraperitoneal administration of the compound to the mammal.
19. 19. The method according to claim 12, characterized in that it further comprises detecting the differentiation of the mammalian cell in a cell of a myocardial lineage.
20. 20. The method of compliance with the claim

    19, characterized in that the differentiation of the mammalian cell in a cell of a myocardial lineage is detected, detecting the expression of a cardiomyocyte marker gene.
21. 21. The method according to the claim

    20, characterized in that the cardiomyocyte marker gene encodes the atrial natriuretic factor.
22. 22. The method of compliance with the claim

    19, characterized in that the differentiation of the mammalian cell in a cell of a myocardial lineage is detected, detecting the expression of a specific transcription factor of the cardiac muscle cell.
23. 23. The method according to the claim

    22, characterized in that the cardiac muscle specific transcription factor is selected from the group consisting of MEF2 and Nkx2.5.
24. 24. The method according to claim 19, characterized in that the differentiation of the mammalian cell into a cell of a myocardial lineage is detected, detecting the expression of a motor protein involved in the contraction of cardiac muscle.
25. 25. The method according to claim 24, characterized in that the motor protein is a motor protein of the heavy chain of sarcomeric myocin.
26. 26. The method according to claim 19, characterized in that the differentiation of the mammalian cell in a cardiac lineage cell is detected, detecting the expression of a specific cardiac gene.
27. 27. The method according to claim 26, characterized in that the cardiac specific gene is GATA-4.
28. 28. The method according to claim 12, characterized in that the mammalian cell is an embryonic stem cell.
29. 29. The method of compliance with the claim

    28, characterized in that the embryonic stem cell is isolated from a mouse.
30. 30. The method of compliance with the claim

    29, characterized in that the embryonic stem cell is an embryonic stem cell R1.
31. 31. The method according to claim 12, characterized in that the mammalian cell is an embryonic carcinoma cell.
32. 32. The method according to claim 31, characterized in that the carcinoma cell is isolated from a mouse.
33. 33. The method according to claim 32, characterized in that the mouse carcinoma cell is an embryonic carcinoma cell P19.
34. 34. The method according to claim 12, characterized in that the mammalian cell is an embryonic primate stem cell.
35. 35. The method of compliance with the claim

    12, characterized in that the mammalian cell is an embryonic human stem cell.
36. 36. The method according to claim 12, characterized in that the mammalian cell is contacted in addition with a cardiomyogenesis enhancing protein.
37. 37. The method according to claim 36, characterized in that the cardiomyogenesis enhancing protein is a growth factor involved in cardiomyogenesis.
38. 38. The method according to claim 12, characterized in that the mammalian cell adheres to a solid support.
39. 39. The method according to claim 38, characterized in that the solid support is a three-dimensional matrix.
40. 40. The method according to claim 38, characterized in that the solid support is a flat surface.
41. 41. A method for inducing cardiomyogenesis, wherein the method comprises: contacting a mammalian cell with a compound according to claim 1, whereby the mammalian cell differentiates into a cell of myocardial lineage.
42. 42. The method according to claim 41, characterized in that the mammalian cell is in a mammal.
43. 43. The method according to claim 41, characterized in that the contact step is by oral administration of the compound with the mammal.
44. 44. The method according to claim 41, characterized in that the step of contacting is by intravenous administration of the compound to the mammal.
45. 45. The method according to the claim

    41, characterized in that the step of contacting is by subcutaneous administration of the compound to the mammal.
46. 46. The method according to claim 41, characterized in that the step of contacting is by intraperitoneal administration of the compound to the mammal.
47. 47. A method for treating a condition of the heart muscle, wherein the method comprises: contacting a mammalian cell with a compound according to claim 1, characterized in that the mammalian cell differentiates into a cell of a myocardial lineage.
48. 48. The method of compliance with the claim

    47, characterized in that the heart muscle condition is associated with damaged myocardium.
49. 49. The method of compliance with the claim

    48, characterized in that the heart muscle condition is cardiomyopathy.
50. 50. The method according to claim 47, characterized in that it further comprises administering the cell of a myocardial lineage to an individual with a condition, thereby treating the condition.
51. 51. The method according to claim 50, characterized in that the administration is by surgical implant.

    R E S U M N N The present invention provides compounds of formula (I), useful for inducing cardiomyogenesis in mammalian cells, particularly embryonic, invitro and in vivo stem cells.