Stem cell culture method

Abstract

The present invention relates to compounds for stabilizing cells, and methods of using the same, which prevent cell differentiation and promote cell survival, including but not limited to when the cells are isolated or when the cells exist outside of their normal culture medium or outside of a tissue environment. The present invention relates to two small molecule compounds, each of which acts by a slightly different mechanism but is useful as a preventative and therapeutic compound for a number of different disease indications, including, but not limited to, cancer, tissue injury, and stroke.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 61 / 200,808, filed December 3, 2008, which is incorporated herein in its entirety for all purposes. [Background technology]

[0002] 2. Background of the Invention Embryonic stem cells (ESCs) are pluripotent cells with the capacity to self-renew indefinitely and differentiate into all cell types of the body (Thomson, JA et al., Science 282 (5391):1145-1147 (1998); Thomson, JA and Odorico, JS, Trends Biotechnol 18 (2):53-57 (2000). This capability offers hope that ESCs may one day be used to replace lost and damaged cells, as well as to provide therapeutic approaches beyond the reach of conventional drugs. However, to fully realize the clinical potential of hESCs, robust culture conditions that are chemically defined, feeder-free and animal-derived product-free must be established. Although several chemically defined media have been reported (Yao, S. et al., Proc Natl Acad Sci USA 103 (18):6907-6912 (2006); Lu, J. et al., Proc Natl Acad Sci USA 103 (15):5688-5693 (2006); Ludwig, TE et al., Nat Biotechnol 24 (2):185-187 (2006)), most of them are still unsatisfactory because the cells in them perform suboptimally. Especially under these conditions, they undergo extensive cell death when the cells are passaged to single cells by trypsin.Numerous signaling pathways that mediate hESC self-renewal are known, including FGF, TGF-β, Wnt, etc. (James, D. et al., Development 132 (6):1273-1282 (2005)(Non-Patent Document 6); Xu, RH et al., Nat Methods 2 (3):185-190 (2005)(Non-Patent Document 7); Beattie, GM et al., Stem Cells 23 (4):489-495 (2005)(Non-Patent Document 8); Greber B., Lehrach, H., and Adjaye, J., Stem Cells 25 (2):455-464 (2007)(Non-Patent Document 9); Sato, N. et al., Nat Med 10 (1):55-63 (2004)(Non-Patent Document 10)). However, none of them appear to play a survival factor role in this process, and the molecular mechanism remains unclear. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Thomson, JA et al., Science 282 (5391):1145-1147 (1998) [Non-Patent Document 2] Thomson, JA and Odorico, JS, Trends Biotechnol 18 (2):53-57 (2000) [Non-Patent Document 3] Yao, S. et al., Proc Natl Acad Sci USA 103 (18):6907-6912 (2006) [Non-Patent Document 4] Lu, J. et al., Proc Natl Acad Sci USA 103 (15): 5688-5693 (2006) [Non-Patent Document 5] Ludwig, TE et al., Nat Biotechnol 24 (2):185-187 (2006) [Non-Patent Document 6] James, D. et al., Development 132 (6):1273-1282 (2005) [Non-Patent Document 7] Xu, RH et al., Nat Methods 2 (3):185-190 (2005) [Non-Patent Document 8] Beattie, GM et al., Stem Cells 23 (4):489-495 (2005) [Non-Patent Document 9] Greber B., Lehrach, H., and Adjaye, J., Stem Cells 25 (2):455-464 (2007) [Non-Patent Document 10] Sato, N. et al., Nat Med 10 (1):55-63 (2004)( Summary of the Invention

[0004] BRIEF SUMMARY OF THE INVETION The present invention provides a compound having the formula: TIFF0007621405000001.tif32128In formula, Ring A is a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; Ring B is a substituted or unsubstituted heterocycloalkyl, or a substituted or unsubstituted heteroaryl; L 1 -C(O)-NR 2 - or -C(O)-NR 2 - and; L 2 is a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene; and R 1and R 2 is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0005] In some embodiments, ring A is substituted or unsubstituted aryl.

[0006] In some embodiments, ring A is substituted or unsubstituted phenyl.

[0007] In certain embodiments, Ring B is a substituted or unsubstituted heterocycloalkyl, or a substituted or unsubstituted heteroaryl.

[0008] In some embodiments, Ring B is a substituted or unsubstituted heteroaryl.

[0009] In certain embodiments, Ring B is substituted or unsubstituted pyrazolyl, substituted or unsubstituted furanyl, substituted or unsubstituted imidazolyl, substituted or unsubstituted isoxazolyl, substituted or unsubstituted oxadiazolyl, substituted or unsubstituted oxazolyl, substituted or unsubstituted pyrrolyl, substituted or unsubstituted pyridyl, substituted or unsubstituted pyrimidyl, substituted or unsubstituted pyridazinyl, substituted or unsubstituted thiazolyl, substituted or unsubstituted triazolyl, substituted or unsubstituted thienyl, substituted or unsubstituted dihydrothieno-pyrazolyl, substituted or unsubstituted thianaphthalene, substituted or unsubstituted phenyl, substituted or unsubstituted phenyl, substituted or unsubstituted phenyl, substituted or unsubstituted phenyl, substituted or unsubstituted phenyl substituted or unsubstituted benzoyl, substituted or unsubstituted carbazolyl, substituted or unsubstituted benzothienyl, substituted or unsubstituted benzofuranyl, substituted or unsubstituted indolyl, substituted or unsubstituted quinolinyl, substituted or unsubstituted benzotriazolyl, substituted or unsubstituted benzothiazolyl, substituted or unsubstituted benzoxazolyl, substituted or unsubstituted benzimidazolyl, substituted or unsubstituted isoquinolinyl, substituted or unsubstituted isoindolyl, substituted or unsubstituted acridinyl, substituted or unsubstituted benzoisazolyl, or substituted or unsubstituted dimethylhydantoin.

[0010] In one embodiment, L 2 is a substituted or unsubstituted C1-C 10 It is an alkyl.

[0011] In one embodiment, L 2 is unsubstituted C1-C 10 It is an alkyl.

[0012] In one embodiment, L 2 is methylene.

[0013] In some embodiments, ring A is substituted or unsubstituted aryl; ring B is substituted or unsubstituted heteroaryl; R 1 is hydrogen; and L 2 is unsubstituted C1-C 10 It is an alkyl.

[0014] In one embodiment, R2 is hydrogen.

[0015] In one embodiment, R 1 is hydrogen or unsubstituted C1-C 10 It is an alkyl.

[0016] In one embodiment, R 1 is hydrogen.

[0017] In some embodiments, the compound has the formula: TIFF0007621405000002.tif32135, where y is an integer from 0 to 3; z is an integer from 0 to 5; and X is -N=, -CH=, or -CR=. 5 = R 3 , R 4 , and R 5 are independently CN, S(O)nR 6 , N.R. 7 R 8 , C(O)R 9 , N.R. 10 -C(O)R 11 , N.R. 12 -C(O)-OR 13 , -C(O)NR 14 R 15 , -NR 16 S(O)2R 17 , -OR 18 , -S(O)NR 19 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, where n is an integer from 0 to 2, and where z is greater than 1, then two R 3 the moieties may be linked together to form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and R 6 , R 7 , R 8 , R 9 , R 10 , R11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , and R 19 is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0018] In one embodiment, L 2 is a substituted or unsubstituted C1-C 10 It is an alkyl.

[0019] In one embodiment, L 2 is unsubstituted C1-C 10 It is an alkyl.

[0020] In one embodiment, L 2 is methylene.

[0021] In some embodiments, X is -N= or -CH=.

[0022] In some embodiments, z is 2 and two R 3 The moieties are linked together to form a substituted or unsubstituted heterocycloalkyl.

[0023] In some embodiments, z is 1.

[0024] In certain embodiments, y is 0 or 1.

[0025] In one embodiment, R 3 -OR 18 and R 18 is hydrogen or unsubstituted C1-C 10 It is an alkyl.

[0026] In one embodiment, L 2is methylene; X is -N= or -CH=; R 1 is hydrogen; and y and z are 0.

[0027] In some embodiments, the compound has the formula: TIFF0007621405000003.tif143128

[0028] The present invention also provides a compound having the formula: TIFF0007621405000004.tif30128In formula, Ring D is a substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl; L 3 is -C(O)NH- or -S(O)2-NH-; R 20 is substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R 21 -NR 22 R 23 -OR 24 and; R 22 and R 23 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or joined together to form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl; R 24is a substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, or is linked together to form a substituted or unsubstituted cycloalkyl of a substituted or unsubstituted heterocycloalkyl.

[0029] In some embodiments, Ring D is substituted or unsubstituted phenyl.

[0030] In one embodiment, R 20 is substituted or unsubstituted alkyl or substituted or unsubstituted cycloalkyl.

[0031] In one embodiment, R 20 is a substituted or unsubstituted C 1 -C 10 alkyl, or substituted or unsubstituted 3- to 7-membered cycloalkyl.

[0032] In one embodiment, R 20 is a substituted or unsubstituted C 1 -C 5 alkyl, or substituted or unsubstituted 3- to 6-membered cycloalkyl.

[0033] In one embodiment, R 20 is unsubstituted C 1 -C 5 alkyl, or unsubstituted 3- to 6-membered cycloalkyl.

[0034] In one embodiment, R 22 is hydrogen; and R 23 is a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl.

[0035] In one embodiment, R 22 is hydrogen; and R 23is a substituted or unsubstituted substituted or unsubstituted aryl.

[0036] In one embodiment, R 22 and R 23 are linked together to form a substituted or unsubstituted heterocycloalkyl, or a substituted or unsubstituted heteroaryl.

[0037] In one embodiment, R 22 and R 23 are linked together to form a substituted or unsubstituted pyrrolyl, substituted or unsubstituted isoindolinyl, substituted or unsubstituted piperidinyl, or substituted or unsubstituted tetrahydroquinolinyl.

[0038] In some embodiments, the compound has the formula: TIFF0007621405000005.tif52128, where w is an integer from 0 to 1; q is an integer from 0 to 7; R 25 , R 26 , R 27 , and R 28 are independently -CN, -NR 29 R 30 , -C(O)R 31 , -NR 32 -C(O)R 33 , -NR 34 -C(O)-OR 35 , -C(O)NR 36 R 37 , -NR 38 S(O)2R 39 , -OR 40 , -S(O)NR 41 , -S(O) v NR 42 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, where v is an integer from 0 to 2; R 29 , R 30 , R 31 , R 32 , R33 , R 34 , R 35 , R 36 , R 37 , R 38 , R 39 , R 40 , R 41 , and R 42 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, where R 25 and R 26 , or R 26 and R 27 may be linked to form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0039] In one embodiment, R 28 -OR 40 where R 40 is hydrogen or unsubstituted C1-C 10 It is an alkyl.

[0040] In one embodiment, R 40 is hydrogen or unsubstituted C1-C5 alkyl.

[0041] In some embodiments, the compound has the formula: TIFF0007621405000006.tif46128

[0042] In one embodiment, R 20 is unsubstituted C1~C 10 It is an alkyl.

[0043] In one embodiment, R 28 -OR 40 where R 40 is hydrogen or unsubstituted C1-C 10C1-C substituted with alkyl or substituted or unsubstituted C3-C6 cycloalkyl 10 It is an alkyl.

[0044] In certain embodiments, q is 1.

[0045] In some embodiments, the compound has the formula: TIFF0007621405000007.tif43128

[0046] In some embodiments, the compound has the formula: TIFF0007621405000008.tif232121TIFF0007621405000009.tif36128

[0047] The present invention also provides a method for stabilizing an isolated cell in vitro, in some embodiments, the method comprising contacting an animal cell with a compound of Formula I or III in an amount sufficient to stabilize the cell.

[0048] In some embodiments, the method further comprises altering the state or environment of the cell in the presence of a compound, wherein the altering in the absence of the compound results in a change in the cellular programming of the cell. In some embodiments, the altering comprises at least one of thawing the cell and dissociating the cell from other cells.

[0049] In some embodiments, the cells are adherent. In some embodiments, the cells are in suspension.

[0050] In some embodiments, the method further comprises determining a trait of the cell.

[0051] In some embodiments, the method includes isolating the cells from the animal. In some embodiments, the animal is a human. In some embodiments, the animal is a non-human animal.

[0052] In some embodiments, the compound is a compound of formula I. In some embodiments, the compound is a compound of formula III.

[0053] The present invention also provides a method of ameliorating a condition in an animal, in some embodiments, the method comprises administering to an animal in need thereof a compound of Formula I or III in an amount sufficient to ameliorate the condition.

[0054] In some embodiments, the condition is selected from the group consisting of tissue injury, stroke, and cancer. In some embodiments, the tissue is selected from the group consisting of pancreas, liver, intestine, lung, and kidney.

[0055] In some embodiments, the condition comprises at least partial rejection of a transplanted tissue or organ, hi some embodiments, the transplant comprises transplantation of bone marrow, umbilical cord blood, purified hematopoietic stem or progenitor cells, cardiac cells, neural cells, pancreatic beta cells, or hepatic cells.

[0056] In some embodiments, the compound is a compound of formula I. In some embodiments, the compound is a compound of formula III.

[0057] The present invention also provides a method for maintaining cell survival, in one embodiment, the method comprises producing isolated stem cells, progenitor cells or differentiated cells, and inducing stabilization of E-cadherin in the isolated cells, thereby maintaining cell survival.

[0058] In some embodiments, the inducing step comprises contacting the isolated stem cells with a compound of formula I in an amount sufficient to increase survival of the isolated stem cells by at least 2-fold compared to the absence of the compound.

[0059] In some embodiments, the inducing step comprises culturing the isolated stem cells on a surface, wherein a molecule comprising an E-cadherin ectodomain is tethered to the surface.

[0060] The invention also provides a population of isolated cells comprising a molecule that stabilizes E-cadherin in the isolated cells in an amount sufficient to enhance survival of the isolated cells by at least two-fold compared to the absence of the molecule.

[0061] In some embodiments, the molecule comprises a compound of formula I.

[0062] In some embodiments, the cell is selected from the group consisting of a stem cell, an induced stem cell, a pluripotent stem cell, a progenitor cell, a differentiated cell, a beta cell, and a fibroblast cell.

[0063] The invention also provides a population of isolated cells comprising a compound of Formula I or III in an amount sufficient to enhance survival of the isolated cells by at least 2-fold compared to the absence of the compound.

[0064] In certain embodiments, the cell is selected from the group consisting of a stem cell, an induced stem cell, a pluripotent stem cell, a progenitor cell, a differentiated cell, a beta cell, and a fibroblast cell.

[0065] The present invention also provides a method for maintaining stem cell survival. In one embodiment, the method includes producing isolated cells; and maintaining cell survival by activating protein kinase C (PKC) in the isolated cells.

[0066] In one embodiment, the activating step comprises contacting the isolated cells with phorbol 12-myristate 13-acetate (PMA) in an amount sufficient to increase survival of the cells compared to survival in the absence of PMA.

[0067] The present invention also provides an isolated population of stem cells comprising a protein kinase C activator in an amount sufficient to enhance survival of the isolated stem cells by at least two-fold compared to survival in the absence of the PKC activator.

[0068] definition The abbreviations used herein have their conventional meaning within the chemical and biological arts.

[0069] The term "stabilizing a cell" means substantially reducing or eliminating the response of a cell to a change in condition or environment to which the cell is exposed. In this context, "substantially reducing" means that the response is at least 50% less than the response that would occur in the absence of the stabilizing component (e.g., a compound of the invention).

[0070] The term "changing the state or environment of a cell" refers to changing the temperature, medium (e.g., carbon source, salt concentration, growth factor), dissociating the cell into isolated cells, thawing the cell, or changing an element of the environment surrounding the cell. As described herein, changing the state or environment of a cell often changes the trait or programming of the cell. For example, when isolated, stem cells and some other cells undergo differentiation and / or death in response to certain changes, such as separation, thawing, etc. Thus, changing the state can reduce or eliminate cell viability, while the viability of the stabilized cells described herein is not substantially reduced under similar changes of state. Changes in cell programming can also be monitored as the cell's response to a particular stimulus characteristic of a cell type and / or by the expression of one or a set of characteristic genes or gene products. As a non-limiting example, human pluripotent stem cells are known to express at least some, and optionally all, of the following markers: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49 / 6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, Rex1, and Nanog. Such expression may change as stem cells lose pluripotency or otherwise differentiate. Stabilized human pluripotent stem cells will maintain their characteristic expression pattern even after the state is changed.

[0071] An "isolated" cell is one that has been substantially separated or purified away from other cells of an organism.

[0072] The term "dissociating" a cell refers to the process of separating the cell from other cells or a surface (e.g., a culture plate surface). For example, cells can be dissociated from an animal or tissue by mechanical or enzymatic methods. Alternatively, cells that aggregate in vitro can be dissociated from each other. In yet another alternative, adherent cells are dissociated from a culture plate or other surface. Thus, dissociation can include severing the extracellular matrix (ECM) and the interaction of the cells with the matrix (e.g., the surface of the culture), or severing the ECM between the cells.

[0073] "Determining the trait of a cell" means evaluating the quality or characteristics of a cell, such as gene expression or gene expression patterns characteristic of a cell type, the response of a cell to a stimulus or environment, the ability to differentiate or dedifferentiate, the presence or absence of a particular morphology, etc.

[0074] Where chemical substituents are specified by their conventional chemical formula and written from left to right, they also include those substituents that are chemically equivalent to those that result from writing the structure from right to left. For example, -CH2O- is the same as -OCH2-.

[0075] The term “alkyl,” by itself or as part of another substituent, means, unless otherwise stated, straight (i.e., unbranched) or branched chain, or combinations thereof, which may be fully saturated, mono- or polyunsaturated, and which can include di- and polyvalent radicals, and has the specified number of carbon atoms (i.e., C1-C2). 10means 1 to 10 carbons). Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, e.g., n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Unsaturated alkyl groups are groups that have one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. Preferred alkyl groups are C 1-6 It is an alkyl group.

[0076] The term "alkylene" by itself or as part of another substituent means a divalent radical derived from alkyl, including, but not limited to, -CH2CH2CH2CH2-. Typically, alkyl (or alkylene) groups have from 1 to 24 carbon atoms, with groups having 10 or fewer carbon atoms being exemplified in the present invention. A "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, generally having 8 or fewer carbon atoms. Preferred alkylene groups are those having a C 1-6 It is an alkylene group.

[0077] The term "heteroalkyl," by itself or in combination with another term, means, unless otherwise stated, a stable linear or branched chain or cyclic hydrocarbon radical, or combinations thereof, consisting of at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si and S, where the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. The heteroatoms O, N, P, and S and Si may be located at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -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, -CH=CH-N(CH3)-CH3, O-CH3, -O-CH2-CH3, and -CN. Up to two heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. Similarly, the term "heteroalkylene" by itself or as part of another substituent means a divalent radical derived from heteroalkyl, including, but not limited to, -CH-CH-S-CH-CH- and -CH-S-CH-CH-NH-CH-. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). Furthermore, for alkylene and heteroalkylene linking groups, no directionality of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O)R'- denotes both -C(O)R' and -R'C(O)-.As stated above, heteroalkyl groups, as used herein, include groups that are attached to the remainder of the molecule through a heteroatom, such as -C(O)R', -C(O)NR', ​​-NR'R'', -OR', -SR', and / or -SOR'. When "heteroalkyl" is designated followed by a specific heteroalkyl group, such as -NR'R'', it will be understood that the terms heteroalkyl and -NR'R'' are not overlapping or mutually exclusive. Rather, the specific heteroalkyl group is designated to add clarity. Thus, the term "heteroalkyl" should not be construed herein to exclude specific heteroalkyl groups, such as -NR'R'', etc. Preferred heteroalkyl groups are C. 1-6 It is a heteroalkyl group.

[0078] As used herein, the term "heteroalkylene" refers to a heteroalkyl group that links at least two other groups, as defined above. The two moieties linked to the heteroalkylene may be linked to the same atom or different atoms of the heteroalkylene. Preferred heteroalkylene groups are 1-6 It is a heteroalkylene group.

[0079] The terms "cycloalkyl" and "heterocycloalkyl", by themselves or in combination with other terms, refer to cyclic versions of "alkyl" and "heteroalkyl", respectively, unless otherwise stated. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 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. "Cycloalkylene" and "heterocycloalkylene" refer to the divalent radicals derived from cycloalkyl and heterocycloalkyl, respectively. Cycloalkyl and heterocycloalkyl groups are defined as C 3-8 Cycloalkyl groups and C 3-8 Heterocycloalkyl group, or C 5-8 Cycloalkyl groups and C 5-8 It may be a heterocycloalkyl group.

[0080] The terms "halo" or "halogen", by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl" are meant to include monohaloalkyl and polyhaloalkyl. For example, the term "halo(C1-C4)alkyl" is meant to include, but is not limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0081] The term "aryl" refers to a polyunsaturated, aromatic, or hydrocarbon substituent, which may be a single ring or multiple rings (preferably from one to three rings) that are fused together or linked by a covalent bond, unless otherwise stated. The term "heteroaryl" refers to an aryl group (or ring) containing from 1 to 4 heteroatoms selected from N, O, and S, where the nitrogen and sulfur atoms may be oxidized and the nitrogen atom may be quaternized. Heteroaryl groups can be attached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples 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-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, Examples include 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. Substituents for each of the above aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. "Arylene" and "heteroarylene" refer to divalent radicals derived from aryl and heteroaryl, respectively. The aryl groups of the present invention preferably have 5 to 12 ring members, more preferably 6 to 10 ring members. The heteroaryl groups of the present invention preferably have from 5 to 12 ring members, more preferably from 5 to 10 ring members.

[0082] For brevity, the term "aryl" when used in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above. Thus, the term "arylalkyl" is meant to include radicals in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl, etc.), including alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced, for example, by an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, etc.).

[0083] As used herein, the term "oxo" refers to an oxygen that is double bonded to a carbon atom.

[0084] As used herein, the term "alkylsulfonyl" refers to a moiety having the formula -S(O2)-R', where R' is an alkyl group as defined above. R' may have a specified number of carbons (e.g., "C1-C4 alkylsulfonyl").

[0085] Each of the above terms (e.g., "alkyl," "heteroalkyl," "aryl" and "heteroaryl") are meant to include both substituted and unsubstituted forms of the radical being described. Exemplary substituents for each type of radical are provided below.

[0086] Substituents for the alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) include, but are not limited to, -OR', ═O, ═NR', ═N-OR', -NR'R'', -SR', -halogen, -SiR'R''R''', -OC(O)R', -C(O)R', -COR', -CONR'R'', -OC(O )NR'R'', -NR''C(O)R', -NR'-C(O)NR''R''', -NR''C(O)2R', -NR-C(NR'R''R''')=NR'''', -NR-C(NR'R'')=NR''', -S(O)R', -S(O)2R', -S(O)2NR'R'', -NRSO2R', -CN and -NO2 in a number ranging from 0 to (2m'+1), where m' is the total number of carbon atoms in such radical. Each of R', R'', R''' and R'''' preferably independently represents hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1 to 3 halogens), substituted or unsubstituted alkyl, alkoxy or thioalkoxy group, or arylalkyl group. When a compound of the invention contains two or more R groups, each of the R groups is independently selected, as are, for example, each of the R', R'', R''', and R'''' groups when two or more of these groups are present. When R' and R'' are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example, -NR'R'' is meant to include, but not be limited to, 1-pyrrolidinyl and 4-morpholinyl.From the above discussion of substituents, one of skill in the art will understand that the term "alkyl" is meant to include groups that contain carbon atoms bonded to groups other than hydrogen groups, such as haloalkyl (e.g., -CF and -CHCF) and acyls (e.g., -C(O)CH, -C(O)CF, -C(O)CHOCH, etc.).

[0087] Similar to the substituents described for the alkyl radicals, the substituents for the aryl and heteroaryl groups are varied and include, for example, halogen, -OR', -NR'R'', -SR', -halogen, -SiR'R''R''', -OC(O)R', -C(O)R', -COR', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR''R''', - and wherein R′, R″, R′″ and R″″ are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. When a compound of the invention contains two or more R groups, each of the R groups is independently selected, as is, for example, each of the R', R'', R''', and R'''' groups when two or more of these groups are present.

[0088] Two substituents on adjacent atoms in an aryl or heteroaryl ring are represented by the formula -TC(O)-(CRR'), where T and U are independently -NR-, -O-, -CRR'- or a single bond, and q is an integer from 0 to 3. qAlternatively, two substituents on adjacent atoms in an aryl or heteroaryl ring can be formed of the formula -A-(CH2) (wherein A and B are independently -CRR'-, -O-, -NR-, -S-, -S(O)-, -S(O)2-, -S(O)2NR'- or a single bond, and r is an integer from 1 to 4. r Optionally, one of the single bonds of the new ring thus formed may be replaced with a double bond. Alternatively, two substituents on adjacent atoms in the aryl or heteroaryl ring may be replaced with a substituent of the formula -(CRR') where s and d are independently integers from 0 to 3, and X' is -O-, -NR'-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR'-. s -X'-(CR''R''') d The substituents R, R', R'', and R''' are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.

[0089] As used herein, the term "heteroatom" or "ring heteroatom" is intended to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).

[0090] As used herein, "substituent" means a group selected from the following moieties: (A) -OH, -NH2, -SH, -CN, -CF3, -NO2, oxo, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and (B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, each of which is substituted with at least one substituent selected from the following: (i) oxo, -OH, -NH2, -SH, -CN, -CF3, -NO2, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, substituted with at least one substituent selected from: (a) oxo, -OH, -NH2, -SH, -CN, -CF3, -NO2, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl substituted with at least one substituent selected from oxo, -OH, -NH2, -SH, -CN, -CF3, -NO2, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, and unsubstituted heteroaryl.

[0091] As used herein, a "size-limited substituent" or "size-limited substituent" refers to a group selected from any of the substituents described above for "substituents," where each of the substituted or unsubstituted alkyl groups is a substituted or unsubstituted C-C 20 each of the substituted or unsubstituted heteroalkyls is a substituted or unsubstituted 2-20 membered heteroalkyl, each of the substituted or unsubstituted cycloalkyls is a substituted or unsubstituted C4-C8 cycloalkyl, and each of the substituted or unsubstituted heterocycloalkyls is a substituted or unsubstituted 4-8 membered heterocycloalkyl.

[0092] As used herein, a "lower substituted moiety" or "lower substituent" means a group selected from all of the substituted moieties described above with respect to "substituents," where each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2-8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C5-C7 cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 5-7 membered heterocycloalkyl.

[0093] The term "pharmaceutically acceptable salts" is intended to include salts of active compounds prepared using relatively non-toxic acids or bases, depending on the specific substituents found on the compounds described herein. In cases where the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium salts, potassium salts, calcium salts, ammonium salts, organic amino salts, or magnesium salts, or similar salts. In cases where the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharma- ceutically acceptable acid addition salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid, or phosphorous acid, and the like, as well as salts derived from relatively non-toxic organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and the like. Also included are salts of amino acids, such as arginates, and salts of organic acids, such as glucuronic acid or galacturonic acid (see, e.g., Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.

[0094] Therefore, the compounds of the present invention may exist as salts together with pharma- ceutically acceptable salts.The present invention includes such salts.Examples of such salts include hydrochloride, hydrobromide, sulfate, methanesulfonate, nitrate, maleate, acetate, citrate, fumarate, tartrate (e.g. (+)-tartrate, (-)-tartrate, or mixtures thereof, including racemic mixtures), succinate, benzoate, and salts containing amino acids such as glutamic acid.These salts may be prepared by methods known to those skilled in the art.

[0095] The neutral forms of the compounds are preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents.

[0096] In addition to salt forms, the present invention also provides compounds in the form of prodrugs.Prodrugs of the compounds described herein are compounds that undergo chemical changes easily under physiological conditions to produce the compounds of the present invention.Furthermore, prodrugs can also be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment.For example, prodrugs can be placed in a transdermal patch reservoir with suitable enzymes or chemical reagents to be slowly converted to the compounds of the present invention.

[0097] Certain compounds of the present invention can exist in unsolvated form or in solvated form, including hydrated form.In general, solvated form is equivalent to unsolvated form and is included within the scope of the present invention.Certain compounds of the present invention can exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent in terms of the applications contemplated by the present invention and are intended to be within the scope of the present invention.

[0098] Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, tautomers, geometric isomers and individual isomers are encompassed within the scope of the present invention. Compounds of the present invention do not include those known in the art to be so unstable that they cannot be synthesized and / or isolated.

[0099] The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may contain unnatural proportions of atomic isotopes, such as tritium ( 3 H), iodine-125( 125 I) or carbon-14( 14 C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are encompassed within the scope of the present invention. [The present invention 1001] The following formula: Compound with TIFF0007621405000010.tif32128: During the ceremony, Ring A is substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; Ring B is a substituted or unsubstituted heterocycloalkyl, or a substituted or unsubstituted heteroaryl; L 1 -C(O)-NR 2 -OR-NR 2 -C(O)-; L 2 is a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene; and R 1 and R 2 is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. [The present invention 1002] 1001. A compound of the present invention, wherein ring A is substituted or unsubstituted aryl. [The present invention 1003] 1001. A compound of the present invention, wherein ring A is substituted or unsubstituted phenyl. [The present invention 1004] The compound of the present invention 1001, wherein Ring B is a substituted or unsubstituted heterocycloalkyl, or a substituted or unsubstituted heteroaryl. [The present invention 1005] 1001. A compound of the present invention, wherein ring B is a substituted or unsubstituted heteroaryl. [The present invention 1006] Ring B is substituted or unsubstituted pyrazolyl, substituted or unsubstituted furanyl, substituted or unsubstituted imidazolyl, substituted or unsubstituted isoxazolyl, substituted or unsubstituted oxadiazolyl, substituted or unsubstituted oxazolyl, substituted or unsubstituted pyrrolyl, substituted or unsubstituted pyridyl, substituted or unsubstituted pyrimidyl, substituted or unsubstituted pyridazinyl, substituted or unsubstituted thiazolyl, substituted or unsubstituted triazolyl, substituted or unsubstituted thienyl, substituted or unsubstituted dihydrothieno-pyrazolyl, substituted or unsubstituted thianaphthenyl, substituted or unsubstituted aryl, substituted or unsubstituted aryl- ... The compound of the present invention is substituted or unsubstituted carbazolyl, substituted or unsubstituted benzothienyl, substituted or unsubstituted benzofuranyl, substituted or unsubstituted indolyl, substituted or unsubstituted quinolinyl, substituted or unsubstituted benzotriazolyl, substituted or unsubstituted benzothiazolyl, substituted or unsubstituted benzoxazolyl, substituted or unsubstituted benzimidazolyl, substituted or unsubstituted isoquinolinyl, substituted or unsubstituted isoindolyl, substituted or unsubstituted acridinyl, substituted or unsubstituted benzoisazolyl, or substituted or unsubstituted dimethylhydantoin. [The present invention 1007] L 2 is substituted or unsubstituted C1-C 10 The compound of the present invention 1001, which is alkyl. [The present invention 1008] L 2Unsubstituted C1-C 10 The compound of the present invention 1001, which is alkyl. [The present invention 1009] L 2 The compound of the present invention, wherein is methylene. [The present invention 1010] Ring A is substituted or unsubstituted aryl; Ring B is substituted or unsubstituted heteroaryl; R 1 is hydrogen; and L 2 Unsubstituted C1-C 10 is alkyl, 1001 compounds of the present invention. [The present invention 1011] R 2 The compound of the present invention 1001, wherein is hydrogen. [The present invention 1012] R 1 is hydrogen or unsubstituted C1-C 10 The compound of the present invention 1001, which is alkyl. [The present invention 1013] R 1 The compound of the present invention 1001, wherein is hydrogen. [The present invention 1014] The following formula: Compound of the present invention 1001 having TIFF0007621405000011.tif32135: During the ceremony, y is an integer from 0 to 3; z is an integer from 0 to 5; X is -N=, -CH=, or -CR 5 = and; R 3 , R 4 , and R 5 However, independently, -CN, -S(O) n R 6 , -NR 7 R 8 , -C(O)R 9 , -NR 10 -C(O)R 11 , -NR 12 -C(O)-OR 13 , -C(O)NR14 R 15 , -NR 16 S(O)2R 17 , -OR 18 , -S(O)NR 19 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, where n is an integer from 0 to 2, and where z is greater than 1, then two R 3 the moieties may be linked together to form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , and R 19 is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. [The present invention 1015] L 2 is substituted or unsubstituted C1-C 10 The compound of the present invention 1014, which is alkyl. [The present invention 1016] L 2 Unsubstituted C1-C 10 The compound of the present invention 1014, which is alkyl. [The present invention 1017] L 2 The compound of the present invention 1014, wherein is methylene. [The present invention 1018] The compound of the present invention 1014, wherein X is -N= or -CH=. [The present invention 1019] z is 2 and two R 3 The compound of the invention 1014, wherein the moieties are linked together to form a substituted or unsubstituted heterocycloalkyl. [The present invention 1020] The compound of the present invention 1014, wherein z is 1. [The present invention 1021] The compound of the present invention 1014, wherein y is 0 or 1. [The present invention 1022] R 3 -OR 18 and R 18 is hydrogen or unsubstituted C1-C 10 The compound of the present invention 1014, which is alkyl. [The present invention 1023] L 2 is methylene; X is -N= or -CH=; R 1 is hydrogen; and y and z are 0; The compound of the present invention 1014. [The present invention 1024] The following formula: Compound 1001 of the present invention having TIFF0007621405000012.tif142128. [The present invention 1025] The following formula: Compound with TIFF0007621405000013.tif30128: During the ceremony, Ring D is substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl; L 3 is -C(O)-NH- or -S(O)2-NH-; R 20is substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R 21 But -NR 22 R 23 -OR 24 and; R 22 and R 23 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or joined together to form a substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted heteroaryl; and R 24 are substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or are joined together to form a substituted or unsubstituted cycloalkyl of a substituted or unsubstituted heterocycloalkyl. [The present invention 1026] 1025 compounds of the present invention, wherein Ring D is substituted or unsubstituted phenyl. [The present invention 1027] R 20 is substituted or unsubstituted alkyl or substituted or unsubstituted cycloalkyl. [The present invention 1028] R 20 is substituted or unsubstituted C1-C 10 The compound of the present invention 1025, which is alkyl, or substituted or unsubstituted 3- to 7-membered cycloalkyl. [The present invention 1029] R 20is a substituted or unsubstituted C1-C5 alkyl, or a substituted or unsubstituted 3- to 6-membered cycloalkyl. [The present invention 1030] R 20 is unsubstituted C1-C5 alkyl, or unsubstituted 3-6 membered cycloalkyl. [The present invention 1031] R 22 is hydrogen; R 23 is substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; 1025 compounds of the present invention. [The present invention 1032] R 22 is hydrogen; and R 23 is a substituted or unsubstituted substituted or unsubstituted aryl; 1025 compounds of the present invention. [The present invention 1033] R 22 and R 23 are linked together to form a substituted or unsubstituted heterocycloalkyl or a substituted or unsubstituted heteroaryl. [The present invention 1034] R 22 and R 23 are linked together to form a substituted or unsubstituted pyrrolyl, a substituted or unsubstituted isoindolinyl, a substituted or unsubstituted piperidinyl, or a substituted or unsubstituted tetrahydroquinolinyl. [The present invention 1035] The following formula: Compound of the present invention 1025 having TIFF0007621405000014.tif52128: During the ceremony, w is an integer from 0 to 1; q is an integer from 0 to 7; R 25 , R 26 , R27 , and R 28 independently, -CN, -NR 29 R 30 , -C(O)R 31 , -NR 32 -C(O)R 33 , -NR 34 -C(O)-OR 35 , -C(O)NR 36 R 37 , -NR 38 S(O)2R 39 , -OR 40 , -S(O)NR 41 , -S(O) v NR 42 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, where v is an integer from 0 to 2; R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , R 35 , R 36 , R 37 , R 38 , R 39 , R 40 , R 41 , and R 42 is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R 25 and R 26 , or R 26 and R 27 may be linked to form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. [The present invention 1036] R 28 -OR40 where R 40 is hydrogen or unsubstituted C1-C 10 The compound of the present invention 1035, which is alkyl. [The present invention 1037] R 40 is hydrogen or unsubstituted C1-C5 alkyl. [The present invention 1038] The following formula: Compound 1035 of the present invention having TIFF0007621405000015.tif46128. [The present invention 1039] R 20 Unsubstituted C1~C 10 The compound of the present invention 1038, which is alkyl. [The present invention 1040] R 28 -OR 40 where R 40 is hydrogen or unsubstituted C1-C 10 C1-C substituted with alkyl or substituted or unsubstituted C3-C6 cycloalkyl 10 The compound of the present invention 1038, which is alkyl. [The present invention 1041] The compound of the present invention 1038, wherein q is 1. [The present invention 1042] The following formula: Compound 1038 of the present invention having TIFF0007621405000016.tif43128. [The present invention 1043] The following formula: Compound of the present invention 1025 having TIFF0007621405000017.tif157128TIFF0007621405000018.tif111128. [The present invention 1044] contacting an animal cell with a compound of formula I or III (e.g., any of the compounds of any of inventions 1001-1043) in an amount sufficient to stabilize the cell; 23. A method for stabilizing isolated cells in vitro, comprising: [The present invention 1045] The method of claim 1044, further comprising altering the state or environment of said cell in the presence of said compound, said altering in the absence of said compound resulting in a change in cellular programming of said cell. [The present invention 1046] The method of claim 1045, wherein said altering step comprises at least one of thawing said cells and dissociating said cells from other cells. [The present invention 1047] The method of claim 1044, wherein the cells are adherent. [The present invention 1048] The method of claim 1044, wherein the cells are in suspension. [The present invention 1049] The method of claim 1044, further comprising the step of determining a trait of said cell. [The present invention 1050] The method of claim 1044, further comprising the step of isolating said cells from the animal. [The present invention 1051] The method of claim 1050, wherein the animal is a human. [The present invention 1052] The method of claim 1050, wherein said animal is a non-human animal. [The present invention 1053] The method of claim 1044, wherein said compound is a compound of formula I. [The present invention 1054] The method of claim 1044, wherein said compound is a compound of formula III. [The present invention 1055] 1. A method of ameliorating a condition in an animal, comprising: administering to an animal in need thereof a compound of formula I or III (e.g., any of the compounds of any of inventions 1001-1043) in an amount sufficient to ameliorate the condition. A method comprising: [The present invention 1056] The method of claim 1055, wherein said condition is selected from the group consisting of tissue injury, stroke, and cancer. [The present invention 1057] The method of claim 1056, wherein said tissue is selected from the group consisting of pancreas, liver, intestine, lung, and kidney. [The present invention 1058] The method of claim 1055, wherein said condition comprises at least partial rejection of a transplanted tissue or organ. [The present invention 1059] The method of claim 1058, wherein said transplantation comprises transplantation of bone marrow, umbilical cord blood, purified hematopoietic stem or progenitor cells, cardiac cells, neural cells, pancreatic beta cells, or hepatic cells. [The present invention 1060] The method of claim 10, wherein said compound is a compound of formula I. [The present invention 1061] The method of claim 1055, wherein said compound is a compound of formula III. [The present invention 1062] 1. A method for maintaining cell viability, comprising: Producing isolated stem, progenitor or differentiated cells; and Inducing stabilization of E-cadherin in the isolated cells, thereby maintaining cell survival. A method comprising: [The present invention 1063] The method of claim 1062, wherein the inducing step comprises contacting the isolated stem cells with a compound of formula I (e.g., any of compounds of claims 1001-1024) in an amount sufficient to enhance survival of the isolated stem cells by at least 2-fold compared to the absence of the compound. [The present invention 1064] The method of claim 1062, wherein said inducing step comprises culturing said isolated stem cells on a surface, wherein a molecule comprising an E-cadherin ectodomain is tethered to said surface. [The present invention 1065] A population of isolated cells comprising a molecule that stabilizes E-cadherin in the isolated cells in an amount sufficient to enhance survival of the isolated cells by at least two-fold compared to the absence of the molecule. [The present invention 1066] The population of cells of invention 1065, wherein the molecule comprises a compound of formula I (eg, any of compounds of inventions 1001-1024). [The present invention 1067] The population of cells of the present invention 1065, wherein said cells are selected from the group consisting of stem cells, induced stem cells, pluripotent stem cells, progenitor cells, differentiated cells, beta cells, and fibroblasts. [The present invention 1068] A population of isolated cells comprising a compound of formula I or III (e.g., any of compounds of the present invention 1001-1043) in an amount sufficient to enhance survival of the isolated cells by at least 2-fold compared to the absence of the compound. [The present invention 1069] The population of claim 1068, wherein said cells are selected from the group consisting of stem cells, induced stem cells, pluripotent stem cells, progenitor cells, differentiated cells, beta cells, and fibroblasts. [The present invention 1070] 1. A method for maintaining stem cell survival, comprising: Producing the isolated cells; and maintaining cell survival by activating protein kinase C (PKC) in the isolated cells; A method comprising: [The present invention 1071] The method of claim 1070, wherein the activating step comprises contacting the isolated cells with phorbol 12-myristate 13-acetate (PMA) in an amount sufficient to improve survival of the isolated cells compared to survival in the absence of PMA. [The present invention 1072] A population of isolated stem cells comprising a protein kinase C activator in an amount sufficient to enhance survival of the isolated stem cells by at least 2-fold compared to survival in the absence of the PKC activator. [The present invention 1073] Each substituent is independently C 1-10 Alkyl, C 1-10and R is selected from the group consisting of heteroalkyl, -OR', =O, =NR', =N-OR', -NR'R'', -SR', -halogen, -SiR'R''R''', -OC(O)R', -C(O)R', -COR', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR''R''', -NR''C(O)2R', -NR-C(NR'R''R''')=NR'''', -NR-C(NR'R'')=NR''', -S(O)R', -S(O)2R', -S(O)2NR'R'', -NRSO2R', -CN, -NO2, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, wherein each of R', R'', R''', and R'''' is independently selected from hydrogen, C 1-10 Alkyl group, C 1-10 The compound of the present invention 1001 is selected from the group consisting of a heteroalkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, and an arylalkyl group. [The present invention 1074] Ring A is 1 to 5 R 3 cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which may be optionally substituted with a group; Ring B is 1 to 5 R 4 heterocycloalkyl or heteroaryl, each of which may be optionally substituted with a group; L 1 -C(O)-NR 2 -OR-NR 2 -C(O)-; L 2 But, bond, C 1-10 Alkylene, or C 1-10 is heteroalkylene; R 1 and R 2 are independently hydrogen, C 1-10 Alkyl, C 1-10 Heteroalkyl, C 3-8 Cycloalkyl, C 3-8 heterocycloalkyl, aryl, or heteroaryl; R 3 and R 4each independently represents -CN, -S(O) n R 6 , -NR 7 R 8 , -C(O)R 9 , -NR 10 -C(O)R 11 , -NR 12 -C(O)-OR 13 , -C(O)NR 14 R 15 , -NR 16 S(O)2R 17 , -OR 18 , -S(O)NR 19 , C 1-10 Alkyl, C 1-10 heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, where n is an integer from 0 to 2, and two R 3 the moieties may be linked together to form a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; and R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , and R 19 are independently hydrogen, C 1-10 Alkyl, C 1-10 heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; 1001 compounds of the present invention. [The present invention 1075] Each substituent is independently C 1-10 Alkyl, C 1-10and R is selected from the group consisting of heteroalkyl, -OR', =O, =NR', =N-OR', -NR'R'', -SR', -halogen, -SiR'R''R''', -OC(O)R', -C(O)R', -COR', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR''R''', -NR''C(O)2R', -NR-C(NR'R''R''')=NR'''', -NR-C(NR'R'')=NR''', -S(O)R', -S(O)2R', -S(O)2NR'R'', -NRSO2R', -CN, -NO2, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, wherein each of R', R'', R''', and R'''' is independently selected from hydrogen, C 1-10 Alkyl group, C 1-10 selected from the group consisting of heteroalkyl groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, and arylalkyl groups; 1025 compounds of the present invention. [The present invention 1076] Ring D is aryl or heteroaryl, each optionally substituted with 1 to 5 R groups; L 3 is -C(O)NH- or -S(O)NH-; R 20 each of which may be substituted with 1 to 5 R groups; 1-10 Alkyl, C 1-10 heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R 21 But -NR 22 R 23 -OR 24 and; R 22 and R 23 are independently hydrogen, C 1-10 Alkyl, C 1-10 heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or linked together to form a heterocycloalkyl or heteroaryl, each of which may be substituted with 1-5 R groups; R24 each of which may be substituted with 1 to 5 R groups; 1-10 Alkyl, C 1-10 heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl; and Each R group is independently C 1-10 Alkyl, C 1-10 and R, -R, -R', -SR, -halogen, -SiR'R''R''', -OC(O)R', -C(O)R', -COR', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR''R''', -NR''C(O)R', -NR-C(NR'R''R''')=NR'''', -NR-C(NR'R'')=NR''', -S(O)R', -S(O)R', -S(O)NR'R'', -NRSO2R', -CN, -NO2, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, wherein each of R', R'', R''', and R'''' is independently selected from hydrogen, C 1-10 Alkyl group, C 1-10 selected from the group consisting of heteroalkyl groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, and arylalkyl groups; 1025 compounds of the present invention. [Brief description of the drawings]

[0100] [Figure 1A] Newly synthesized small molecules dramatically enhance the survival of hESCs after single cell dissociation without compromising their overall long-term self-renewal and developmental potential. The chemical structures of Thiazovivin / Tzv and Pyrintegrin / Ptn are shown. [Figure 1B] A newly synthesized small molecule dramatically enhances the survival of hESCs after single cell dissociation without compromising their overall long-term self-renewal and developmental potential. ALP staining of hESC colonies grown from dissociated single cells seeded at low density and treated as described. [Figure 1C]A novel synthetic small molecule dramatically enhances the survival of hESCs after single cell dissociation without compromising their overall long-term self-renewal and developmental potential. Ratio of ALP-positive colonies to total initially seeded cells. [Figure 1D] A novel synthetic small molecule dramatically enhances the survival of hESCs after single cell dissociation without compromising their overall long-term self-renewal and developmental potential. Immunostaining of hESCs maintained long-term in medium containing Ptn or Tzv is shown. [Figure 1E] A novel synthetic small molecule dramatically enhances the survival of hESCs after single cell dissociation without compromising their overall long-term self-renewal and developmental potential. Sections of 5-week teratomas formed from long-term expanded hESCs maintained in medium containing Tzv (i, ii) or Ptn (iii, iv). Neuroepithelium (ectoderm), cartilage (mesoderm), and simple epithelium (endoderm) (i); neuroepithelium (ectoderm), simple epithelium and liver epithelium (endoderm) (ii); neuroepithelium (ectoderm), cartilage (mesoderm), and renal tubular epithelium (endoderm) (iii); neuroepithelium (ectoderm), skeletal muscle (mesoderm), and renal tubular epithelium (endoderm) (iv). [Figure 1F] A novel synthetic small molecule dramatically enhances the survival of hESCs after single cell dissociation without compromising their overall long-term self-renewal and developmental potential. (G-banding analysis of hESCs after more than 20 passages grown in the presence of compounds Ptn or Tzv. Unless otherwise stated, all above hESCs were cultured in chemically defined medium and feeder-free on Matrigel-coated plates. [Figure 2A] After cell dissociation, Tzv stabilizes E-cadherin and protects hESCs from death in suspension culture. Cell death analysis of dissociated hESCs grown on Matrigel or in suspension with or without treatment with Ptn or Tzv. [Figure 2B] After cell dissociation, Tzv stabilizes E-cadherin and protects hESCs from death in suspension culture. Phase contrast images of hESCs grown on non-coated plates treated with the indicated molecules. [Figure 2C]After cell dissociation, Tzv stabilizes E-cadherin and protects hESCs from death in suspension culture. Western blot analysis of E-cadherin in hESCs transfected with specific siRNA against E-cadherin or GFP. [Figure 2D] After cell dissociation, Tzv stabilizes E-cadherin and protects hESCs from death in suspension culture. Cell death analysis by TUNEL staining of dissociated hESCs transfected with specific siRNA against E-cadherin or GFP in the presence of Tzv. [Figure 2E] After cell dissociation, Tzv stabilizes E-cadherin and protects hESCs from death in suspension culture.ALP staining of dissociated hESCs transfected with specific siRNA against E-cadherin or GFP in the presence of Tzv. [Figure 2F] After cell dissociation, Tzv stabilizes E-cadherin and protects hESCs from death in suspension culture. Western blot analysis of full-length E-cadherin in hESCs before and after trypsinization. [Figure 2G] After cell dissociation, Tzv stabilizes E-cadherin and protects hESCs from death in suspension culture. Time-course Western blot analysis of full-length E-cadherin expression in hESCs treated with DMSO, Tzv, or Ptn for the indicated times after trypsin dissociation. [Figure 2H] After cell dissociation, Tzv stabilizes E-cadherin and protects hESCs from death in suspension culture. Flow cytometric analysis of the surface abundance of E-cadherin on hESCs after trypsinization in the presence of Tzv. DMSO was used as a control. [Figure 2I] After cell dissociation, Tzv stabilizes E-cadherin and protects hESCs from death in suspension culture. Semiquantitative RT-PCR of E-cadherin in hESCs treated or not with Tzv. [Figure 2J] After cell dissociation, Tzv stabilizes E-cadherin and protects hESCs from death in suspension culture. Endocytosis analysis of E-cadherin in the presence or absence of Tzv. [Figure 2K] After cell dissociation, Tzv stabilizes E-cadherin and protects hESCs from death in suspension culture. Cell survival analysis of hESCs grown on plates coated with BSA or different concentrations of E-cad-Fc chimera. [Figure 3A] Ptn and Tzv protect hESCs from cell death in adhesion medium after dissociation by maintaining and reactivating integrin activity. Growth curves of hESCs grown on Matrigel with different time courses of Ptn and Tzv treatment. Group 1, Ptn treatment only for the first 24 hours; Group 2, Ptn treatment continuously throughout the culture period; Group 3, Tzv treatment only for the first 24 hours; Group 4, Tzv treatment continuously throughout the culture period. For each condition, dissociated cells were plated at 10 × 104 cells per well of a 6-well plate. [Figure 3B] Ptn and Tzv protect hESCs from cell death in adhesion medium after dissociation by maintaining and reactivating integrin activity. Phase contrast images of hESCs seeded on different matrices and treated with the indicated compounds 12 h later. [Figure 3C] Ptn and Tzv protect hESCs from cell death in adhesion medium after dissociation by maintaining and reactivating integrin activity. Dissociated hESCs were plated on Matrigel-coated plates and allowed to adhere for 3 hours in the presence of compounds or with integrin β1-blocking antibodies as indicated. Percentage of adhesion was calculated as described in Materials and Methods. [Figure 3D] Ptn and Tzv protect hESCs from cell death in adhesion medium after dissociation by maintaining and reactivating integrin activity Western blot analysis of integrin β1 expressed by hESCs before and after trypsinization. [Figure 3E] Ptn and Tzv protect hESCs from cell death in adhesion medium after dissociation by maintaining and reactivating integrin activity. Time-course Western blot analysis of integrin expression in hESCs after trypsin dissociation and treatment with DMSO, Tzv, or Ptn for the indicated times. [Figure 3F] Ptn and Tzv protect hESCs from cell death in adhesion medium after dissociation by maintaining and reactivating integrin activity. Flow cytometric analysis of active conformation of β1 integrin in trypsin-dissociated hESCs after treatment with Tzv or Ptn. [Figure 3G] Ptn and Tzv protect hESCs from cell death in adhesion medium after dissociation by maintaining and reactivating integrin activity. Immunostaining analysis of active conformation of β1 integrin in trypsin-dissociated hESCs after treatment with Tzv or Ptn. [Figure 3H] Ptn and Tzv protect hESCs from cell death in adhesion medium after dissociation by maintaining and reactivating integrin activity. Cell adhesion of hESCs treated or not with the β1-activating antibody, TS2 / 16. [Figure 3I] Ptn and Tzv protect hESCs from cell death in adhesion medium after dissociation by maintaining and reactivating integrin activity. ALP staining of hESCs treated or not with the β1-activating antibody, TS2 / 16. [Figure 3J] Ptn and Tzv protect hESCs from cell death in adhesion medium after dissociation by maintaining and reactivating integrin activity. Cell adhesion of hESCs treated with Tzv or Ptn with or without PKC inhibitors. [Figure 3K] Ptn and Tzv protect hESCs from cell death in adhesion medium after dissociation by maintaining and reactivating integrin activity. Immunostaining of β1 integrin in the active conformation in hESCs treated or not with PMA (10 nM). [Figure 3L] Ptn and Tzv protect hESCs from cell death in adhesion medium after dissociation by maintaining and reactivating integrin activity.Cell adhesion of hESCs treated with the indicated compounds. [Figure 3M]Ptn and Tzv protect hESCs from cell death in adhesion medium after dissociation by maintaining and reactivating integrin activity ALP staining of hESCs treated with the indicated compounds. [Figure 4A] PI-3K and ERK mediated growth factor receptors are the major survival and anti-differentiation signaling pathways, respectively, emanating from the hESC niche. Cell death analysis of dissociated hESCs plated on Matrigel and treated as indicated. [Figure 4B] PI-3K and ERK mediated growth factor receptors are the major survival and anti-differentiation signaling pathways, respectively, emanating from the hESC niche. Western blot showing the phosphorylation status of different growth factor receptors in hESCs treated with Ptn for 2 hours. DMSO was used as a control. [Figure 4C] PI-3K and ERK mediated growth factor receptors are the major survival and anti-differentiation signaling pathways, respectively, emanating from the hESC niche. Cell death analysis of dissociated hESCs in suspension treated with the indicated conditions. [Figure 4D] PI-3K and ERK mediated growth factor receptors are the major survival and anti-differentiation signaling pathways, respectively, emanating from the hESC niche. Immunoprecipitation showing the interaction of E-cadherin with EGFR1 and Erb2. [Figure 4E] PI-3K and ERK, via growth factor receptors, are the major survival and anti-differentiation signaling pathways, respectively, emanating from the hESC niche. Western blot showing AKT phosphorylation status in hESCs treated with Ptn for the indicated time periods. [Figure 4F] PI-3K and ERK, via growth factor receptors, are the major survival and anti-differentiation signaling pathways, respectively, emanating from the hESC niche. Western blot showing AKT and ERK phosphorylation status in the presence of Ptn or in the presence of integrin β1 blocking antibody together with Ptn. [Figure 4G]PI-3K and ERK, via growth factor receptors, are the major survival and differentiation inhibitory signaling pathways, respectively, emanating from the hESC niche. Western blot showing AKT phosphorylation status in hESCs treated with Ptn or with the indicated receptor inhibitors. [Figure 4H] PI-3K and ERK via growth factor receptors are the major survival and differentiation inhibitory signaling pathways, respectively, emanating from the hESC niche. Cell death analysis of hESCs treated with Ptn for 24 hours or with Ptn together with PI-3K or MEK inhibitors for 24 hours. [Figure 4I] PI-3K and ERK mediated growth factor receptors are the major survival and anti-differentiation signaling pathways, respectively, emanating from the hESC niche. Percentage of SSEA4 negative cells following treatment with MEK inhibitors. [Figure 5A] FIG. 1 represents compounds of the present invention including thiazovivin and its derivatives. [Figure 5B] FIG. 1 represents compounds of the present invention including thiazovivin and its derivatives. [Figure 6A] FIG. 1 represents compounds of the present invention including pyrin tegrin and its derivatives. [Figure 6B] FIG. 1 represents compounds of the present invention including pyrin tegrin and its derivatives. [Figure 6C] FIG. 1 represents compounds of the present invention including pyrin tegrin and its derivatives. [Figure 6D] FIG. 1 represents compounds of the present invention including pyrin tegrin and its derivatives. [Figure 6E] FIG. 1 represents compounds of the present invention including pyrin tegrin and its derivatives. [Figure 6F] FIG. 1 represents compounds of the present invention including pyrin tegrin and its derivatives. [Figure 6G] FIG. 1 represents compounds of the present invention including pyrin tegrin and its derivatives. [Figure 6H]FIG. 1 represents compounds of the present invention including pyrin tegrin and its derivatives. [Figure 6I] FIG. 1 represents compounds of the present invention including pyrin tegrin and its derivatives. [Figure 6J] FIG. 1 represents compounds of the present invention including pyrin tegrin and its derivatives. [Figure 6K] FIG. 1 represents compounds of the present invention including pyrin tegrin and its derivatives. [Figure 6L] FIG. 1 represents compounds of the present invention including pyrin tegrin and its derivatives. [Figure 6M] FIG. 1 represents compounds of the present invention including pyrin tegrin and its derivatives. [Figure 6N] FIG. 1 represents compounds of the present invention including pyrin tegrin and its derivatives. [Figure 6O] FIG. 1 represents compounds of the present invention including pyrin tegrin and its derivatives. [Figure 6P] FIG. 1 represents compounds of the present invention including pyrin tegrin and its derivatives. [Figure 6Q] FIG. 1 represents compounds of the present invention including pyrin tegrin and its derivatives. [Figure 6R] FIG. 1 represents compounds of the present invention including pyrin tegrin and its derivatives. [Figure 6S] FIG. 1 represents compounds of the present invention including pyrin tegrin and its derivatives. [Figure 6T] FIG. 1 represents compounds of the present invention including pyrin tegrin and its derivatives. [Figure 6U] FIG. 1 represents compounds of the present invention including pyrin tegrin and its derivatives. [Figure 6V] FIG. 1 represents compounds of the present invention including pyrin tegrin and its derivatives. [Figure 6W] FIG. 1 represents compounds of the present invention including pyrin tegrin and its derivatives. [Figure 6X] FIG. 1 represents compounds of the present invention including pyrin tegrin and its derivatives. [Figure 6Y]FIG. 1 represents compounds of the present invention including pyrin tegrin and its derivatives. [Figure 6Z] FIG. 1 represents compounds of the present invention including pyrin tegrin and its derivatives. [Figure 6AA] FIG. 1 represents compounds of the present invention including pyrin tegrin and its derivatives. [Figure 6AB] FIG. 1 represents compounds of the present invention including pyrin tegrin and its derivatives. [Figure 6AC] FIG. 1 represents compounds of the present invention including pyrin tegrin and its derivatives. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0101] Detailed Description I. Introduction The present invention provides novel compounds and methods of their use. Two small molecule compounds are provided that prevent cell differentiation and promote cell survival, including but not limited to when cells are isolated or when cells are outside of their normal medium or tissue environment. Although the compounds act by somewhat different mechanisms, both are useful as preventative and therapeutic compounds for a number of different disease indications, including but not limited to cancer, tissue injury, and stroke.

[0102] II. Compounds that promote cell survival and / or inhibit differentiation In one aspect, a compound is provided that promotes cell survival and / or inhibits differentiation. In one embodiment, the compound has the formula: In formula (I), ring A is substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. Ring B is substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted heteroaryl.

[0103] L 1 -C(O)-NR 2 -OR-NR2 -C(O)-. 2 is a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene.

[0104] R 1 and R 2 is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0105] In some embodiments, ring A is a substituted or unsubstituted aryl. Ring A may be a substituted or unsubstituted phenyl.

[0106] In another embodiment, Ring B is a substituted or unsubstituted heterocycloalkyl or a substituted or unsubstituted heteroaryl. Ring B may be a substituted or unsubstituted heteroaryl. Ring B is substituted or unsubstituted pyrazolyl, substituted or unsubstituted furanyl, substituted or unsubstituted imidazolyl, substituted or unsubstituted isoxazolyl, substituted or unsubstituted oxadiazolyl, substituted or unsubstituted oxazolyl, substituted or unsubstituted pyrrolyl, substituted or unsubstituted pyridyl, substituted or unsubstituted pyrimidyl, substituted or unsubstituted pyridazinyl, substituted or unsubstituted thiazolyl, substituted or unsubstituted triazolyl, substituted or unsubstituted thienyl, substituted or unsubstituted dihydrothieno-pyrazolyl, substituted or unsubstituted thianaphthenyl, substituted or unsubstituted cycloalkyl ... or unsubstituted carbazolyl, substituted or unsubstituted benzothienyl, substituted or unsubstituted benzofuranyl, substituted or unsubstituted indolyl, substituted or unsubstituted quinolinyl, substituted or unsubstituted benzotriazolyl, substituted or unsubstituted benzothiazolyl, substituted or unsubstituted benzoxazolyl, substituted or unsubstituted benzimidazolyl, substituted or unsubstituted isoquinolinyl, substituted or unsubstituted isoindolyl, substituted or unsubstituted acridinyl, substituted or unsubstituted benzoisazolyl, or substituted or unsubstituted dimethylhydantoin.

[0107] L 2 is a substituted or unsubstituted C1-C 10 In some embodiments, L 2 is unsubstituted C1-C 10 It is an alkyl group. 2 can also be substituted or unsubstituted methylene (eg, unsubstituted methylene).

[0108] R 2 R may be hydrogen. 1 is hydrogen or unsubstituted C1-C 10 In some embodiments, R 1 is simply hydrogen.

[0109] In certain embodiments of formula (I), ring A is a substituted or unsubstituted aryl, ring B is a substituted or unsubstituted heteroaryl, R 1 is hydrogen and L 2 is unsubstituted C1-C 10 It is an alkyl.

[0110] In another embodiment, the compound that promotes cell survival and / or inhibition of differentiation has the formula: TIFF0007621405000020.tif32135In formula (II), y is an integer from 0 to 3, and z is an integer from 0 to 5. X is -N=, -CH=, or -CR=. 5 = R 1 and L 2 is as described in formula (I) above.

[0111] R 3 , R 4 , and R 5 are independently -CN, -S(O) n R 6 , -NR 7 R 8 , -C(O)R 9 , -NR 10 -C(O)R 11 , -NR 12 -C(O)-OR 13 , -C(O)NR 14 R 15 , -NR 16 S(O)2R 17 , -OR 18 , -S(O)NR 19 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, where n is an integer from 0 to 2, and where z is greater than 1, then two R 3 The moieties may be linked together to form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0112] R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , and R 19 is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0113] In one embodiment, L 2 is a substituted or unsubstituted C1-C 10 It is an alkyl group. 2 is unsubstituted C1-C 10 Alternatively, L 2 is substituted or unsubstituted methylene (eg, unsubstituted methylene).

[0114] In other embodiments, X is -N= or -CH=. The symbol z may be 2. In yet other embodiments, two R 3 The moieties are linked together to form a substituted or unsubstituted heterocycloalkyl. The symbol z can be 1. The symbol y can be 0 or 1. R 3 -OR 18 R 18 is hydrogen or unsubstituted C1-C 10 It may be alkyl.

[0115] In one embodiment, L 2 is substituted or unsubstituted methylene (e.g., unsubstituted methylene), X is -N= or -CH=, R 1 is hydrogen, and y and z are 0.

[0116] In another embodiment, the compound has the formula: TIFF0007621405000021.tif155162

[0117] In yet another embodiment, the compound of formula I is a compound in which ring A is selected from 1 to 5 R 3 ring B is a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group, each of which may be substituted with 1 to 5 R 4 is a heterocycloalkyl or heteroaryl, each of which may be substituted by a group; L 1 -C(O)-NR 2 -OR-NR 2 -C(O)-;L 2 is bond, C 1-10 Alkylene, or C 1-10 Heteroalkylene; R 1 and R 2 are independently hydrogen, C 1-10 Alkyl, C 1-10 Heteroalkyl, C 3-8 Cycloalkyl, C 3-8 heterocycloalkyl, aryl, or heteroaryl; R 3 and R 4 each independently represents -CN, -S(O) n R 6 , -NR 7 R 8 , -C(O)R 9 , -NR 10 -C(O)R 11 , -NR 12 -C(O)-OR 13 , -C(O)NR 14 R 15 , -NR 16 S(O)2R 17 , -OR 18 , -S(O)NR 19 , C 1-10 Alkyl, C 1-10 heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, where n is an integer from 0 to 2, and two R 3The moieties may be linked together to form a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; and R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , and R 19 are independently hydrogen, C 1-10 Alkyl, C 1-10 It is heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.In yet other embodiments, the compound of formula I is a compound other than thiazovivin.

[0118] In other embodiments, the compound that promotes cell survival and / or inhibition of differentiation has the formula: In formula (III), ring D is a substituted or unsubstituted aryl or a substituted or unsubstituted heteroaryl. 3 is -C(O)NH- or -S(O)NH-.

[0119] R 20 is substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. R 21 -NR 22 R 23 -OR 24 It is.

[0120] R 22 and R 23are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, or are joined together to form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl.

[0121] R 24 is a substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, or is linked together to form a substituted or unsubstituted cycloalkyl of a substituted or unsubstituted heterocycloalkyl.

[0122] In another embodiment, L 3 is a bond, -O-, -C(O)NH- or -S(O)2NH-, R 20 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, and rings D and R 21 is as defined above. In certain other embodiments, L 3 is a bond, -O- or -S(O)2NH-, and rings D, R 20 , and R 21 is as defined above, and L 3 If -S(O)2NH-, then R 20 is hydrogen. 3 is a bond or -O-, and rings D and R 20 , and R 21 is as defined above.

[0123] In some embodiments, Ring D is substituted or unsubstituted phenyl.

[0124] In other embodiments, R 20 is substituted or unsubstituted alkyl or substituted or unsubstituted cycloalkyl. 20 is a substituted or unsubstituted C1-C 10 R may be an alkyl group or a substituted or unsubstituted 3- to 7-membered cycloalkyl group. 20 may be a substituted or unsubstituted C1-C5 alkyl, or a substituted or unsubstituted 3- to 6-membered cycloalkyl. 20 is unsubstituted C1-C5 alkyl, or unsubstituted 3- to 6-membered cycloalkyl.

[0125] In yet another embodiment, R 22 is hydrogen and R 23 is substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. 22 is hydrogen and R 23 is a substituted or unsubstituted aryl; or R 22 and R 23 are linked together to form a substituted or unsubstituted heterocycloalkyl or a substituted or unsubstituted heteroaryl. R 22 and R 23 may be linked together to form a substituted or unsubstituted pyrrolyl, a substituted or unsubstituted isoindolinyl, a substituted or unsubstituted piperidinyl, or a substituted or unsubstituted tetrahydroquinolinyl.

[0126] In some embodiments, the compound has the formula: In formula (IV), w is an integer from 0 to 1, and q is an integer from 0 to 7. 20 is as defined above in the definition of the compound of formula (III). 25 , R 26 , R 27 , and R 28are independently -CN, -NR 29 R 30 , -C(O)R 31 , -NR 32 -C(O)R 33 , -NR 34 -C(O)-OR 35 , -C(O)NR 36 R 37 , -NR 38 S(O)2R 39 , -OR 40 , -S(O)NR 41 , -S(O) v NR 42 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, where v is an integer from 0 to 2.

[0127] R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , R 35 , R 36 , R 37 , R 38 , R 39 , R 40 , R 41 , and R 42 is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0128] R 25 and R 26 , or R 26 and R 27 may be linked to form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0129] In one embodiment, R 28 -OR 40 R 40 is hydrogen or unsubstituted C1-C 10 alkyl, and R 40 may be hydrogen, or unsubstituted C1-C5 alkyl.

[0130] The compound may have the formula: TIFF0007621405000024.tif47135In formula (V), R 20 , R 28 and q are as defined above in the definition of formula (IV). 20 is unsubstituted C1-C 10 It is an alkyl group. 28 -OR 40 R 40 is hydrogen or unsubstituted C1-C 10 C1-C substituted with alkyl or substituted or unsubstituted C3-C6 cycloalkyl 10 The symbol q may be 1.

[0131] In another embodiment, the compound has the formula: TIFF0007621405000025.tif45149In formula (VI), R 20 , R 28 and q are as defined above in the definition of formula (IV) or formula (VI).

[0132] In another embodiment, the compound has the formula: TIFF0007621405000026.tif125128TIFF0007621405000027.tif164163

[0133] In another embodiment, the compound of formula III is a compound in which ring D is aryl or heteroaryl, each of which may be substituted with 1-5 R groups; 3 is -C(O)NH- or -S(O)NH-; R 20each of which may be substituted with 1 to 5 R groups; 1-10 Alkyl, C 1-10 R is heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; 21 But -NR 22 R 23 -OR 24 ;R 22 and R 23 are independently hydrogen, C 1-10 Alkyl, C 1-10 heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or joined together to form a heterocycloalkyl or heteroaryl, each of which may be substituted with 1 to 5 R groups; R 24 each of which may be substituted with 1 to 5 R groups; 1-10 Alkyl, C 1-10 heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; and each R group is independently C 1-10 Alkyl, C 1-10 and R is selected from the group consisting of heteroalkyl, -OR', =O, =NR', =N-OR', -NR'R'', -SR', -halogen, -SiR'R''R''', -OC(O)R', -C(O)R', -COR', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR''R''', -NR''C(O)2R', -NR-C(NR'R''R''')=NR'''', -NR-C(NR'R'')=NR''', -S(O)R', -S(O)2R', -S(O)2NR'R'', -NRSO2R', -CN, -NO2, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, wherein each of R', R'', R''', and R'''' is independently selected from hydrogen, C 1-10 Alkyl group, C 1-10 The compound is selected from the group consisting of a heteroalkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, and an arylalkyl group.

[0134] In some embodiments, each of the above-mentioned substituents in the compounds of formulas (I)-(VI) is substituted with at least one substituent. More specifically, in some embodiments, each of the above-mentioned substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, and / or substituted heteroalkylene in the compounds of formulas (I)-(VI) is substituted with at least one substituent. In other embodiments, at least one or all of these groups are substituted with at least one size-limited substituent. Alternatively, at least one or all of these groups are substituted with at least one lower substituent.

[0135] In other embodiments of the compounds of Formulae (I)-(VI), each of the substituted or unsubstituted alkyl is a substituted or unsubstituted C-C 20 each of the substituted or unsubstituted heteroalkyl groups is a substituted or unsubstituted 2-20 membered heteroalkyl group; each of the substituted or unsubstituted cycloalkyl groups is a substituted or unsubstituted C3-C8 cycloalkyl group; each of the substituted or unsubstituted heterocycloalkyl groups is a substituted or unsubstituted 3-8 membered heterocycloalkyl group; each of the substituted or unsubstituted alkylene groups is a substituted or unsubstituted C1-C 20 Each of the alkylene and / or substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2- to 20-membered heteroalkylene.

[0136] In certain embodiments, each of the substituted or unsubstituted alkyls is a substituted or unsubstituted C1-C8 alkyl, each of the substituted or unsubstituted heteroalkyls is a substituted or unsubstituted 2-8 membered heteroalkyl, each of the substituted or unsubstituted cycloalkyls is a substituted or unsubstituted C5-C7 cycloalkyl, each of the substituted or unsubstituted heterocycloalkyls is a substituted or unsubstituted 5-7 membered heterocycloalkyl, and / or each of the substituted or unsubstituted alkylenes is a substituted or unsubstituted C1-C8 alkylene, and / or each of the substituted or unsubstituted heteroalkylenes is a substituted or unsubstituted 2-8 membered heteroalkylene.

[0137] In certain other embodiments, the compounds of Formulae (I)-(VI) are 1-10 Alkyl, C 1-10 and each of R', R'', R''', and R'' is independently selected from the group consisting of heteroalkyl, -OR', =O, =NR', =N-OR', -NR'R'', -SR', -halogen, -SiR'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', -NR-C(NR'R''R''')=NR'''', -NR-C(NR'R'')=NR''', -S(O)R', -S(O)2R', -S(O)2NR'R'', -NRSO2R', -CN, -NO2, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, where each of R', R'', R''', and R'''' is independently selected from the group consisting of hydrogen, C 1-10 Alkyl, C 1-10 It is a heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or arylalkyl group.

[0138] III.How to use The compounds of the present invention are useful for a wide variety of purposes.For example, the compounds promote survival in situations where cells (e.g., for isolated cells) would otherwise undergo apoptosis or die.In some embodiments, cells are stabilized for at least a certain period of time, for example, 10 minutes, 30 minutes, or 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 24 hours, 48 ​​hours, or 96 hours.Furthermore, the compounds are useful for maintaining the status quo of cell differentiation in situations where cells would otherwise differentiate or otherwise change cell programming.These effects lead to a number of uses of the compounds, either in vitro or in vivo.

[0139] A. In Vivo Applications The compounds of the present invention are useful for reducing tissue damage, and therefore can be administered to treat, alleviate, or prevent tissue damage. In some embodiments, the compounds of the present invention are administered to individuals who have or are at risk of having tissue damage in internal organs. Internal organs include, but are not limited to, the brain, pancreas, liver, intestine, lung, kidney, or heart, and are injured, for example, by burns or cuts. For example, in some embodiments, the compounds of the present invention are effective in reducing infarct size in ischemic reperfusion injury. Thus, the compounds of the present invention can be administered to individuals who may have, have, or have had a stroke. Similarly, the compounds of the present invention can be administered to individuals who may have, have, or have had a heart attack or heart damage.

[0140] The inventors have found that the compounds of the present invention can prevent cell death, for example in epithelial cells. For example, the inventors have spread primary human pancreatic islet / beta cells plated as single cells onto tissue culture plates coated with matrigel or laminin. In normal cell culture medium for beta cells without Tzv, substantial cell death was observed as a result. However, when Tzv (1-2 mM) was added to the medium, cell death was inhibited. Similar effects were observed in other epithelial early cells, such as neural cells. Thus, in some embodiments, the compounds of the present invention are administered to an individual in need of pancreatic beta cells and / or pancreatic islet cells, where administration of the compound results in an increase in the number of beta cells or islet cells in the individual.

[0141] In addition, the compounds of the present invention (e.g., compounds of formula I or III) are effective in increasing blood flow and inhibiting inflammatory responses. For example, compounds of formula I enhance monocyte adhesion and migration across endothelial cell monolayers, thereby reducing inflammatory responses (data not shown). Thus, in some embodiments, the compounds of the present invention are administered to individuals who need increased blood flow and / or reduced inflammation (e.g., have cerebral ischemia). Individuals who need reduced inflammation include individuals who have inflammatory diseases or diseases mediated by inflammatory conditions. Examples of inflammatory diseases include, but are not limited to, chronic obstructive pulmonary disease, osteoarthritis, tendonitis or bursitis, gouty arthritis, polymyalgia rheumatica, fibromyalgia, pelvic inflammatory disease, and arthritis, including rheumatoid arthritis.

[0142] In some embodiments, the compounds of the present invention are used to treat or alleviate cancer. In some cases, the compounds of the invention are useful in treating cancer, e.g., cell types, glioma, mesothelioma, melanoma, lymphoma, leukemia, adenocarcinoma, breast cancer, ovarian cancer, cervical cancer, glioblastoma, leukemia, lymphoma, prostate cancer, and Burkitt's lymphoma, head and neck cancer, colon cancer, colorectal cancer, non-small cell lung cancer, small cell lung cancer, esophageal cancer, gastric cancer, pancreatic cancer, hepatobiliary cancer, gallbladder cancer, small intestine cancer, rectal cancer, kidney cancer, bladder cancer, prostate cancer, penile cancer, urethral cancer, testicular cancer, cervical cancer, vaginal cancer, uterine cancer, ovarian cancer, thyroid cancer, parathyroid cancer, adrenal cancer, pancreatic endocrine cancer, carcinoid tumors, bone cancer, skin cancer, retinoblastoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma (for further examples of cancers, see Cancer: Principles and Practice (De Vita, VT et al., eds, 1997) are administered to treat

[0143] Cancer cell metastasis is a process that typically involves epithelial-mesenchymal transition / EMT (e.g., epithelial cells to fibroblastic cells). The inventors have found that the compounds of the present invention (i.e., Tvz and Ptn) can induce MET (the reverse of EMT) and inhibit EMT, and have shown that the compounds are effective in reducing or preventing cancer metastasis. Thus, in some embodiments, the compounds of the present invention are administered to individuals experiencing or at risk of cancer metastasis. For example, the inventors have found that the compounds of formula I and III inhibit the metastasis of epithelial cancers, including but not limited to breast cancer and hepatocellular carcinoma.

[0144] In certain embodiments, the compounds of the invention are administered to individuals suffering from or at risk of suffering from hypertension and / or atherosclerosis.

[0145] The compounds of formula I and III (i.e., Tvz and Ptn) are effective in promoting axonal regeneration and functional recovery of injured central nervous system. For example, the inventors have found that Tvz can promote neurite outgrowth from primary neural cells from mice. Tzv (3 μM) was tested using mouse P1 cortical explants and found to result in axonal outgrowth. Tzv was added to the medium 20 minutes after plating, and DMSO was used as a control. The explants were observed in the medium for 4 days. Tzv showed a dramatic effect on promoting axonal outgrowth, which was more pronounced than the first division. Thus, in some embodiments, the compounds of the present invention are administered to individuals with central nervous system injuries or those who require or would otherwise benefit from axonal regeneration.

[0146] In certain embodiments, the compounds of the invention are administered to individuals suffering from diabetes, insulin resistance, or otherwise in need of enhanced beta cell survival or at risk of losing beta cell function.

[0147] The compounds of the present invention also find use in improving negative symptoms and other organ, cell or tissue transplantation.As described herein, the compounds of the present invention are effective in stabilizing and maintaining the contextual programming of cells.Therefore, in some embodiments, the compounds of the present invention are administered to an individual during or after cell, tissue or organ transplantation.Examples of transplantation include, but are not limited to, transplantation of bone marrow, umbilical cord blood, purified hematopoietic stem / progenitor cells, cardiac cells, neural cells, pancreatic β cells, and hepatic cells.

[0148] B. In Vitro Applications The compounds of the present invention are effective in stabilizing cells exposed to a wide variety of conditions. Many animal cells lose viability, undergo apoptosis, and / or change programming when separated (when in suspension or adherent) (e.g., stem cells often die or differentiate when separated). The compounds of the present invention, when mixed with such cells, are effective in preventing the response of such cells to changes in the environment. In some embodiments, cells are separated from animals and contacted with a sufficient amount of the compounds of the present invention to prevent the loss of cellular capacity and / or change in cellular programming. In some embodiments, such separated cells are useful for diagnosis because they retain traits that the cells would otherwise lose due to the separation process and the cell's response to the separation itself. Examples of traits that are retained include gene expression patterns, cellular responsiveness to stimuli, ligands, or drugs, and cellular capacity.

[0149] The stability of a cell population can be monitored, for example, by monitoring the expression of gene products. For example, certain gene products are tissue or cell type specific and can be monitored before and after a change in condition or environment (e.g., changing cell culture medium, thawing cells, separating cells from other cells) to determine whether the change affects cell programming. In some embodiments, cells immediately before being exposed to a change in condition or environment, or relatively soon after the change (e.g., within one minute, five minutes, an hour, etc., depending on the context), are contacted with a sufficient amount of a compound of the present invention so that one or more cell expression markers remain substantially identical. "Substantially identical" is understood in the art, depending on the context. In some embodiments, "substantially identical" means that the expression of a gene product associated with a particular cell type does not change by more than about 10%, 20%, or 30% (e.g., compared to the expression before the treatment) after a particular treatment on the cells.

[0150] In one embodiment, the present invention provides a method for promoting survival and differentiation inhibition in ex vivo stem cells.For example, the present inventors have found that the compound of formula I or III (i.e., Tzv and Ptn) is effective in promoting survival and differentiation inhibition in human embryonic stem cells, mouse embryonic stem cells, multiple neural stem cells, skin stem cells, mesenchymal stem cells, hematopoietic stem cells, stromal stem cells and epithelial stem cells by contacting the cells with the compound of formula I or III immediately after cell separation.

[0151] Thus, the invention provides cell populations and / or tissues that are contacted with a compound of the invention (e.g., a compound of Formula I or III) in an amount sufficient to stabilize the cells, e.g., prevent or reduce the response of the cells to a change in condition (e.g., separation from the tissue, thawing the cells). In some embodiments, for example, the cells or tissues that are contacted with the compound of the invention are in a frozen or liquid state. In some embodiments, the cells / tissues are thawed from a frozen state while contacted with a compound of the invention in an amount sufficient to prevent or reduce cell damage or differentiation.

[0152] In some embodiments, the compound of the present invention is contacted with a population of stem cells, progenitor cells, or differentiated cells. Exemplary stem cells include pluripotent stem cells, embryonic stem cells, and induced pluripotent stem cells (iPS cells). Exemplary stem cells further include human embryonic stem cells, mouse embryonic stem cells, multiple neural stem cells, skin stem cells, mesenchymal stem cells, hematopoietic stem cells, stromal stem cells, and epithelial stem cells. Any type of progenitor cell can be used, including but not limited to endodermal progenitor cells, mesodermal progenitor cells (e.g., muscle progenitor cells, bone progenitor cells, blood progenitor cells), and ectodermal progenitor cells (e.g., epithelial tissue progenitor cells and neural progenitor cells). A wide variety of differentiated cells are also known. Examples of differentiated cells include but are not limited to fibroblasts, cardiac cells, neural cells, pancreatic beta cells, hepatic cells, epithelial cells, and intestinal cells. The cells described herein can be human or non-human cells. In some embodiments, the cells are human cells. In certain embodiments, the cells are mouse, dog, bovine, pig, rat, or non-human primate cells.

[0153] The ability to maintain cell viability and cell programming allows for improved methods of drug screening and diagnosis. For example, in some embodiments, cells are screened for response in the presence of at least one compound of the present invention (e.g., a compound of formula I or III) that maintains cell viability, and are further contacted with at least one of a plurality of agents (e.g., a compound library) and monitored for response. Various screening methods are known. This method has been found to be particularly advantageous for use with cells that would otherwise be less viable under the conditions of the screening method (e.g., where it is convenient to use isolated cells, cells in suspension, adherent cells, etc.). The cells may be, for example, stem cells, progenitor cells, or differentiated cells as described herein. The cellular response may be any desired response. Some responses in cell-based screening assays include, but are not limited to, gene expression (e.g., based on expression of a reporter gene or quantified by PCR or other detection techniques), cell viability or loss thereof, induction of apoptosis, etc.

[0154] The agent used in the screening method may be, for example, a small organic compound (e.g., molecular weight less than 10,000 daltons, e.g., less than 8000, 6000, 4000, 2000 daltons), lipid, carbohydrate, polypeptide, antibody, nucleic acid (e.g., oligonucleotide, DNA, RNA, ribozyme, short inhibitory RNA (siRNA), microRNA (miRNA), etc.).

[0155] In some embodiments, the assay is designed to screen large combinatorial libraries by automating the assay steps, which are typically performed in parallel (e.g., in a microtiter format or microwell plate in a robotic assay), and providing compounds from any convenient source. Combinatorial libraries can be completely random or can contain members that contain a core structure based on one or more promising lead compounds. Combinatorial libraries can be completely synthetic or contain some or all members from natural sources, such as bacteria, fungi, plants, insects, and vertebrates (e.g., Xenopus (frog) or Anguilla (eel)), and invertebrates (e.g., Strongylocentrotus (sea urchin) or mollusks). See also Boldi, Combinatorial Synthesis of Natural Product Based Libraries, 2006, CRC Press.

[0156] In one embodiment, a high throughput screening method involves providing a combinatorial chemical or peptide library that contains a large number of potential therapeutic compounds (potential modulator or ligand compounds). Such "combinatorial chemical or peptide libraries" are then screened using one or more assays, as described herein, to identify library members (particular chemical species or subclasses) that exhibit a desired characteristic activity. Compounds thus identified can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics.

[0157] A combinatorial chemical library is a diverse collection of compounds made by chemical or biological synthesis by combining chemical "building blocks" such as multiple reagents. For example, a linear combinatorial chemical library, such as a polypeptide library, is formed by combining a set of chemical building blocks (amino acids) in every possible way to obtain a compound of a particular length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized by such combinatorial mixing of chemical building blocks.

[0158] The preparation and screening of combinatorial compound libraries are known to those skilled in the art. For example, see U.S. Patent Nos. 5,663,046; 5,958,792; 6,185,506; 6,541,211; and 6,721,665, the disclosures of which are also incorporated herein by reference. Such combinatorial compound libraries include, but are not limited to, peptide libraries (see, for example, U.S. Patent No. 5,010,175; Furka, Int. J. Pept. Prot. Res. 37:487-493 (1991); Houghton, et al., Nature 354:84-88 (1991); Combinatorial Peptide Library Protocols, Cabilly, ed., 1997, Humana Press). Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to, peptoids (e.g., PCT Publication WO 91 / 19735), coded peptides (e.g., PCT Publication WO 93 / 20242), random bio-oligomers (e.g., PCT Publication WO 92 / 00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines, and dipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913(1993)), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), non-peptidic peptidomimetics with glucose scaffolds (Hirschmann et al., J. Amer. Chem. Soc. 114:6568 (1992)), and the like. 114:9217-9218(1992)), analogous organic synthesis of small molecule compound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661(1994), Combinatorial Libraries: Synthesis, Screening and Application Potential, Cortese, ed., 1995, Walter De Gruyter Inc, and Obrecht and Villalgordo, Solid-Supported Combinatorial and Parallel Synthesis of Small-Molecular-Weight Compound Libraries, 1998, Elsevier Science Ltd.), oligocarbamates (Cho et al., Science 261:1303(1993)), and / or peptidyl phosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleic acid libraries (see Ausubel, Sambrook, and Russell, all supra, and U.S. Pat. Nos. 6,955,879, 6,841,347, 6,830,890, 6,828,098, 6,573,098, and 6,399,334), peptide nucleic acid libraries (see, e.g., U.S. Pat. Nos. 5,539,083, 5,864,010, and 6,756,199), antibody libraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314 (1996) and PCT / US96 / 10287), carbohydrate libraries (see, e.g., Liang et al., Science, 274:1520-1522 (1996), U.S. Patent No. 5,593,853, and Solid Support Oligosaccharide Synthesis and Combinatorial Carbohydrate Libraries, Seeberger, ed., 2004, John Wiley & Sons (E-book)), organic small molecule libraries (e.g., benzodiazepines, see Baum C&EN, January 18, page 33 (1993) and U.S. Pat. No. 5,288,514; isoprenoids, see U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, see U.S. Pat. No. 5,549,974; pyrrolidines, see U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, see U.S. Pat. No. 5,506,337, etc.). See also Combinatorial Library Design and Evaluation: Principles, Software Tools, and Applications in Drug Discovery, Ghose et al., eds., 2001, Marcel Dekker; Molecular Diversity and Combinatorial Chemistry: Libraries and Drug Discovery, Chaiken and Janda, eds., 1996, Oxford Univ Pr.; and Combinatorial Library Methods and Protocols, English ed., 2002, Humana Press.

[0159] Equipment for preparing combinatorial libraries is commercially available (see, e.g., Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburn, MA, Applied Biosystems, Foster City, CA, Millipore, Bedford, MA, and Caliper Life Sciences, Hopkinton, MA).

[0160] In some embodiments, screening assays can be conveniently performed in multi-well plates (e.g., 96-well, 384-well, etc.), where each agent to be screened is tested separately in one well. In some embodiments, two or more candidate agents are tested in one reaction mixture.

[0161] C. A different target for a similar effect As illustrated in the examples below, the inventors have become aware of the role of several gene products in the cellular response to the compounds of the invention, which has led to the discovery that cells can also be stabilized by manipulating the gene products as described below.

[0162] 1. E-cadherin The present inventors have found that increasing E-cadherin expression enhances stem cell survival.Thus, the present invention provides a method for stabilizing and / or increasing the expression of E-cadherin in cells, thereby stabilizing cells against changes in conditions that may otherwise be detrimental to cell viability.Stabilizing E-cadherin can include, for example, contacting cells with a compound that increases the expression of E-cadherin or protects E-cadherin from proteolytic cleavage in some manner.

[0163] In some embodiments, the present invention provides a method of culturing stem cells (including, but not limited to, human or mouse embryonic stem cells) in a container in which the surface is coated with a protein comprising at least the ectodomain of E-cadherin, optionally linked to another component such as a fusion protein, thereby stabilizing the cells (e.g., maintaining or increasing the viability of the cells and / or maintaining cellular programming). An ectodomain is a portion of a membrane protein that extends into the extracellular space. In some embodiments, an ectodomain is a portion of a protein that initiates contact with a surface and results in signal transduction. The ectodomain of E-cadherin is described, for example, in Ito et al., Oncogene 18(50):7080-90(1999). In some embodiments, at least the ectodomain of E-cadherin is fused to a dimerizing polypeptide sequence, thereby allowing for stabilized dimerization of the ectodomain. By "dimerizing polypeptide" is meant an amino acid sequence that forms a homodimer, thereby allowing two polypeptides to form a dimer. An example of a dimerizing polypeptide includes, but is not limited to, an IgGFc fragment. In one embodiment, stem cells (including, but not limited to, human or mouse embryonic stem cells, pluripotent stem cells, iPS cells) are dissociated from one another and cultured in a vessel having a surface coated with a protein that includes at least the ectodomain of E-cadherin, optionally linked to another moiety such as a fusion protein, thereby improving cell viability and stabilizing the cells compared to the viability of similarly treated cells cultured in a vessel that is not coated with the polypeptide.

[0164] 2. Protein Kinase C The present invention also provides stabilization of cells by contacting the cells with a protein kinase C activator. As described herein, dissociated hESCs were treated with a protein kinase C activator and grown in the presence of a matrigel matrix, resulting in significantly improved cell attachment and colony formation, thereby improving cell viability. Thus, the present invention provides improved cell viability by culturing cells in the presence of a protein kinase C activator, thereby improving cell viability, proliferation and / or adhesion. In some embodiments, a protein kinase C activator is contacted with a population of stem cells, progenitor cells, or differentiated cells in an amount sufficient to improve cell viability and / or survival time and / or adhesion. Exemplary stem cells include pluripotent cells, embryonic stem cells, induced pluripotent stem cells (iPS cells), or others described herein. Examples of protein kinase C activators include, but are not limited to, phorbol esters (e.g., phorbol 12-myristate 13-acetate (PMA) or the phorbol esters described in U.S. Patent Application Publication No. 20080226589) or peptide agonists (e.g., as described in U.S. Patent No. 6,165,977).

[0165] 3. Integrin β1 The present invention also provides stabilization of cells by contacting the cells with an integrin β1 activator. As described herein, dissociated hESCs were treated with an integrin β1 activator and the cells were grown on lamins, resulting in significantly improved cell adhesion and colony formation, thereby improving cell viability. Thus, the present invention provides improved cell viability by culturing cells in the presence of an integrin β1 activator, thereby improving cell viability, proliferation and / or adhesion. In some embodiments, an integrin β1 activator is contacted with a population of stem cells, progenitor cells, or differentiated cells in an amount sufficient to improve cell viability and / or survival time and / or adhesion. Examples of stem cells include pluripotent cells, embryonic stem cells, induced pluripotent stem cells (iPS cells), or others described herein. Examples of integrin β1 activators include, but are not limited to, integrin β1 activating antibodies, such as TS2 / 16 (commercially available from Thermo Scientific, Rockfield, Ill., etc.).

[0166] IV. Cell populations As described herein, the invention provides cells in a mixture (e.g., cell culture) with one or more compounds described herein (e.g., compounds of formula I or III, including but not limited to Tzv and Pt-, or protein kinase C activators, or integrin β1 activators). In some embodiments, the compound is present in the mixture at a concentration sufficient to maintain viability or cellular programming in response to a change in the cellular environment or condition (e.g., thawing). For example, in some embodiments, the compound is present at a concentration of at least 0.1 M, such as at least 1, 10, 100, 1000, 10000, or 100000 nM, such as 0.1 nM to 100000 nM, such as 1 nM to 10000 nM, such as 10 nM to 10000 nM, such as 1 to 10 μM. In some embodiments, the mixture is present in an artificial container (e.g., a test tube, a petri dish, etc.). Thus, in some embodiments, the cell is an isolated cell (not part of an animal). In some embodiments, the cells are adherent cells or cells in suspension. In some embodiments, the cells are separated or dissociated from tissue samples (e.g., biopsy tissue) from animals (human or non-human), placed in containers, and contacted with one or more compounds (e.g., compounds of formula I or III) described herein. The cells can then be cultured and optionally stimulated to differentiate into specific cell types or cell lineages, or a recombinant expression cassette can be introduced into the cells, and optionally returned to the same or different animals.

[0167] V. Cell Culture The cells can be cultured by any method known in the art. The cells can be cultured in suspension or as adherent cells, as appropriate.

[0168] In some embodiments, cells (e.g., stem cells) are cultured in contact with feeder cells. Examples of feeder cells include, but are not limited to, fibroblasts, such as mouse embryonic fibroblasts (MEFs). Methods for culturing cells on feeder cells are known in the art.

[0169] In some embodiments, cells are cultured in the absence of feeder cells. For example, cells can be directly attached to a solid culture surface (e.g., a culture plate) via, for example, a molecular tether. Examples of molecular tethers include, but are not limited to, matrigel, extracellular matrix (ECM), ECM analogs, laminin, fibronectin, or collagen. However, those skilled in the art will recognize that this is a non-limiting list and other molecules can be used to attach cells to a solid surface. Methods for initially attaching tethers to a solid surface are known in the art.

[0170] VI. Formulations and Methods of Administration Formulations (including, for example, but not limited to, compounds of the present invention suitable for administration) include aqueous and non-aqueous solutions, isotonic sterile solutions containing antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic, aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. In carrying out the present invention, compositions can be administered, for example, orally, nasally, topically, intravenously, intraperitoneally, or intrathecally. Formulations of compounds can be provided in unit dose or multi-dose sealed containers, such as ampoules and vials. Solutions and suspensions can be prepared from sterile powders, granules, and tablets of the above-mentioned types. Modifiers can also be administered as part of prepared foods or drugs.

[0171] The dose administered to a patient in the context of the present invention should be sufficient to induce a beneficial response in the subject over time. The optimal dose level for any patient will be determined by a variety of factors, including the potency of the particular modulator used, the patient's age, weight, physical activity, and diet, possible combinations with other drugs, and the severity of the disease or condition being addressed. The size of the dose will also be determined by the existence, nature, and extent of any adverse side effects associated with the administration of a particular compound or vector to a particular subject.

[0172] When determining the effective amount of active ingredient to administer, the physician may evaluate the circulating plasma concentration of the compound or agent, the toxicity of the compound or agent, and the production of antibodies to the compound or agent. In general, the equivalent dose of the compound or agent is about 1 ng / kg to 10 mg / kg for a typical subject. EXAMPLES

[0173] VII. Examples The following examples are offered to illustrate, but not to limit, the claimed invention.

[0174] Example 1: To improve the chemically defined medium conditions and to clarify the molecular mechanism of hESC death after single cell dissociation, we performed a high-throughput trait screen of 50,000 synthetic compounds to identify small molecules that promote hESC survival after trypsin dissociation. This screen identified two groups of compounds that significantly increased cell survival after dissociation and also maintained hESC colony morphology as well as alkaline phosphatase (ALP) expression. Further compound optimization and activity analysis led to the discovery of two lead molecules, a 2,4-disubstituted thiazole (named thiazovivin / Tzv) and a 2,4-disubstituted pyrimidine (named pyrintegrin / Ptn) (Figure 1A), for further functional and mechanistic characterization.

[0175] (Table 1) Activity data TIFF0007621405000028.tif113164TIFF0007621405000029.tif220164TIFF0007621405000030.tif241164TIFF0007621405000031.tif92164 1 Ratio of ALP-positive colonies to the total number of cells initially plated.

[0176] Compounds Tzv or Ptn enhance the survival of single hESCs by more than 20-fold after enzymatic dissociation on Matrigel-coated plates (Fig. 1B,C). hESCs were serially passaged for more than 20 generations in chemically defined media containing Tzv or Ptn. Under these conditions, cells uniformly maintained hESC-characteristic morphology, expression of typical pluripotency markers, and normal karyotype (Fig. 1D,E). When these cells were injected into nude mice, complex teratomas consisting of all three primary germ layer tissues developed (Fig. 1F). These results, confirmed in several independent hESC lines, collectively and convincingly demonstrated that both compounds can substantially promote hESC survival without compromising self-renewal and full developmental potential.

[0177] hESCs are known to have difficulty in forming embryoid bodies (EBs) in suspension culture after single cell dissociation due to a large number of cell deaths. Therefore, we further tested whether Tzv or Ptn could promote the survival of dissociated hESCs in suspension. Interestingly, Tzv significantly improved the survival of hESCs in both adherent and suspension cultures. In contrast, Ptn promoted the survival of hESCs only in adherent cultures (e.g., on Matrigel-coated plates) and had no effect in suspension cultures (Fig. 2A). These findings suggest that at least two different mechanisms are involved in these two types of cell death under ECM / Matrigel or suspension conditions, and that Tzv and Ptn function in different ways. hESCs formed favorable cell aggregates and could differentiate into various lineages when cultured in suspension in the presence of Tzv (Fig. 2B) (data not shown). Because cell aggregation is most often mediated by cell-cell adhesion, and E-cadherin is both the primary cell-cell adhesion molecule and is highly expressed in hESCs (Eastham, AM et al., Cancer Res 67(23):11254-11262(2007)), we tested the effect of a specific E-cadherin-blocking antibody on the formation of multicellular aggregates. Culturing cells in the presence of the antibody significantly inhibited Tzv treatment-induced cell survival and the formation of large, compact aggregates, indicating that functional E-cadherin engagement is required for Tzv-induced cell survival and the assembly of multicellular aggregates (Figure 2B). Furthermore, knockdown of E-cadherin by specific siRNA in hESCs dramatically reduced Tzv treatment-induced cell survival and markedly reduced the number of ALP-positive colonies (Figure 2C, D, E). These results suggest that Tzv enhances hESC survival in suspension, possibly acting through E-cadherin-mediated cell-cell adhesion.

[0178] Next, we investigated E-cadherin expression in hESCs after trypsin dissociation. We found that most of the full-length E-cadherin was cleaved after trypsin dissociation (Figure 2F). This finding was consistent with a report that the extracellular domain of E-cadherin has an intracellular proteolytic cleavage site near the transmembrane domain (Damsky, CH et al., Cell 34(2):455-466(1983)). In cells that were not treated with Tzv, newly synthesized full-length E-cadherin appeared 1 hour after enzyme treatment and disappeared 4 hours later, suggesting that newly synthesized E-cadherin in dissociated hESCs is unstable. However, E-cadherin expression was significantly increased in Tzv-treated cells (Figure 2G). Furthermore, flow cytometry analysis revealed that cell surface E-cadherin in hESCs was significantly increased by Tzv (Figure 2H). Thus, Tzv may affect cell adhesion by regulating the cell surface levels of E-cadherin. Semiquantitative RT-PCR revealed similar amounts of E-cadherin transcripts in mock control and Tzv-treated cells (Fig. 2I), suggesting that the difference in E-cadherin protein amount was not due to changes in transcript abundance. Tzv likely exerts its effect by stabilizing E-cadherin on the cell surface. Finally, endocytosis assay revealed that the internalization of E-cadherin was significantly blocked by Tzv. These results indicate that Tzv regulates E-cadherin activity by inhibiting the endocytosis of E-cadherin (Fig. 2J).

[0179] Cell-cell dissociation with trypsin leads to rapid cleavage and subsequent destabilization of E-cadherin. We hypothesized that E-cadherin stability may be mediated by homophilic interactions between cells. Thus, homophilic ligation of E-cadherin with recombinant ligands may stabilize E-cadherin and affect hESC survival. To test this hypothesis, we coated plates with a dimeric E-cadherin-Fc chimeric protein containing the ectodomain of E-cadherin fused to an IgGFc fragment (Ecad-Fc). Remarkably, dissociated hESCs attached to the coated surface and their viability was significantly increased in a dose-dependent manner (Figure 2K), confirming that E-cadherin-mediated cell-cell adhesion is a key regulator of hESC survival.

[0180] Both Tzv and Ptn show dramatic effects on the survival of hESCs grown on Matrigel-coated plates. Such survival-promoting effects are unlikely to be due to effects on cell proliferation, but may be largely due to the increased cell adhesion ability following the cell dissociation and seeding process (Figure 3A, B). Indeed, dissociated hESCs treated with Tzv or Ptn showed dramatically increased adhesion to Matrigel or laminin. In contrast, the adhesion of hESCs to gelatin or polylysine, which does not involve integrins (Figure 3B, and data not shown), was not affected by Ptn or Tzv treatment. The main component of Matrigel is laminin, and it has been reported that the laminin receptor β1 integrin is highly expressed in hESCs (Xu, C. et al., Nat Biotechnol 19(10):971-974 (2001)). To test whether Ptn or Tzv act through β1 integrin, we pretreated cells with a blocking antibody against β1 integrin and observed that the increased cell adhesion induced by compound treatment was completely abolished, suggesting that Tzv and Ptn mediate cell adhesion to ECM substrates through β1 integrin (Figure 3C).

[0181] To gain insight into the mechanism of β1 integrin regulation by Tzv and Ptn, we examined whether the compound effects were due to changes in integrin expression. In contrast to E-cadherin, β1 integrin was not cleaved by trypsin. Western blot analysis revealed that the expression of β1 integrin was increased, making the compound effects unlikely. Thus, Tzv and Ptn likely affect cell adhesion by modulating integrin activity (Figure 3D, E). To examine the effect of compound treatment on β1 integrin activity, we used the monoclonal antibody HUTS-21, which specifically binds to the activated form of β1 integrin (Luque, A. et al., J Biol Chem 271 (19):11067-11075(1996)). Notably, compound treatment increased the level of HUTS-21 binding (Figure 3F, G). Collectively, these results suggest that Tzv and Ptn increase cell adhesion by regulating integrin activity from the inside-out.

[0182] If both compounds enhanced cell adhesion by converting integrins to their active conformation, then treating cells with integrin-activating antibodies that lock integrins in their active conformation should have a similar effect. Indeed, when dissociated hESCs were plated on laminin in the presence of TS2 / 16, an activating antibody against β1 integrin (van de Wiel-van Kemenade, E. et al., J Cell Biol 117 (2):461-470 (1992)), cell adhesion was significantly increased and cells formed more colonies compared to controls (Figure 3H, I). These results suggest that the increased adhesion that occurs when cells are treated with these compounds involves a mechanism that induces integrin activation.

[0183] To further investigate the molecular mechanism of integrin activity regulation by Tzv and Ptn, we examined the effect of several pathway inhibitors. We found that bisindolylmaleimide I, a specific inhibitor of PKC, could antagonize the increase in cell adhesion induced by Ptn, but had no effect on cell adhesion induced by Tzv, suggesting that PKC may mediate the action of Ptn, but not Tzv (Fig. 3J). To further confirm the role of PKC in hESC survival, dissociated hESCs were treated with phorbol 12-myristate 13-acetate (PMA), a PKC agonist. PMA treatment caused integrin activation and a substantial increase in cell adhesion and colony formation (Fig. 3K, L, M).

[0184] The fate of stem cells is influenced by their niche, which consists of growth factors, cell-ECM interactions, and cell-cell interactions. The fact that hESC survival is highly dependent on cell-cell interactions and / or cell-ECM interactions has revealed the importance of hESC's in vitro niche, which has not been recognized before. More importantly, cell-intrinsic protein expression (e.g., E-cadherin and integrins) and regulatory mechanisms (e.g., protein stabilization and activation) are not only responsive to niche components, but are also essential niche components, suggesting that stem cells have the intrinsic ability to form their own niche in the absence of other exogenous factors or cell types, but that these exogenous factors or cell types can participate in and enhance the self-regulatory niche mechanism of cells.

[0185] The interaction between the physical / structural environment and growth factors plays a crucial role in controlling cell fate (Comoglio, PM, Boccaccio, C., and Trusolino, L., Curr Opin Cell Biol 15 (5): 565-571 (2003)). To examine whether growth factors are involved in integrin-mediated hESC survival, we treated dissociated hESCs with individual highly specific growth factor receptor inhibitors together with Tzv or Ptn. We found that chemical inhibition of FGFR, IGFR, EGFR1, or Erb2 significantly reduced the pro-survival effect induced by Tzv or Ptn treatment (Figure 4A). In addition, Ptn significantly increased phosphorylation of growth factor receptors, suggesting that the engagement of growth factor receptors is required for integrin-mediated cell survival (Figure 4B). Similarly, inhibition of FGFR, IGFR, EGFR1, or Erb2 significantly abolished Tzv-induced hESC survival in suspension culture. Furthermore, Tzv induced E-cadherin binding to EGFR1 and ERB2, indicating a critical role for growth factor receptors in E-cadherin-mediated cell survival (Figure 4C,D).

[0186] Phosphatidylinositol-3-kinase (PI-3K) signaling and MAPK / ERK are key regulators of hESC self-renewal (Armstrong, L. et al., Hum Mol Genet 15 (11): 1894-1913 (2006); Paling, NR et al., J Biol Chem 279 (46):48063-48070 (2004); Pyle, AD, Lock, LF, and Donovan, PJ, Nat Biotechnol 24 (3):344-350 (2006); Li, J. et al., Differentiation 75 (4):299-307 (2007)). Phosphorylation of ERK and AKT, downstream effectors of PI-3K, was increased upon treatment of dissociated hESCs with Ptn, and this increase was abolished by integrin-blocking antibodies (Fig. 4E, F). Furthermore, activation of AKT and ERK by Ptn was blocked by inhibitors of FGFR, IGFR, EGFR, or Erb2 (Fig. 4G and data not shown). Chemical inhibition of PI-3K action significantly antagonized the survival effect induced by Ptn (Fig. 4H). Inhibition of ERK induced differentiation of hESCs, although it had no dramatic effect on Ptn-induced survival (Fig. 4I). These results indicated that activation of PI-3K is the major survival signaling, and activation of ERK is the differentiation-suppressing signaling generated by the niche through activation of growth factor receptors.

[0187] In summary, we have identified two novel synthetic small molecules with distinct mechanisms of action that greatly enhance hESC survival after single cell dissociation through high-throughput trait screening. The newly identified biological tools (e.g., defined recombinant Ecad-Fc for hESC attachment in adherent culture; activating antibodies for enhancing cell survival and attachment) through such chemical and mechanistic characterization will enable more robust hESC culture and significantly facilitate hESC applications, such as gene targeting or drug discovery. More importantly, the thorough mechanistic characterization uncovered a previously unrecognized niche mechanism required to sustain hESC survival and proliferation. Such a niche consists of E-cadherin-mediated hESC-hESC interactions, integrin-mediated cell-ECM interactions, and growth factors. Previous studies have pointed towards a critical role of growth factors for hESC self-renewal. However, full activation of growth factor signaling requires not only the presence of growth factors and receptors, but also interactions with a specific microenvironment. When this physical / structural environment is disrupted, growth factors alone are insufficient for ESC self-renewal.

[0188] Recently, it was reported that differentiated fibroblasts generated from hESCs in self-renewal medium create an in vitro niche for hESCs (Bendall, SC et al., Nature 448(7157):1015-1021 (2007)). Such differentiated cells are very rarely observed in long-term cultures under our and others' chemically defined medium conditions, suggesting that such artificial niches may be created by differences in the medium. However, our study reveals unique cell-autonomous (i.e., cell-cell interactions) and cell-independent (i.e., cell-ECM and -growth factor) niche mechanisms that likely play an important role in controlling the in vivo fate of adult stem cells in the survival and self-renewal of hESCs.

[0189] Cell-cell dissociation with trypsin not only resulted in the destabilization of E-cadherin but also in the inactivation of integrins, indicating that the signaling that maintains integrin activity is sensitive to enzymatic treatment. Feeder cell-conditioned medium (serum rich in growth factors) offered little protection against cell death after single cell dissociation. Furthermore, the fact that high-density cell seeding also induces increased cell adhesion / survival suggests that the signaling required to maintain integrin activity is not due to secreted factors but rather to physical cell-cell interactions. Tzv inhibits the endocytosis of E-cadherin, thereby protecting cells from death in suspension. Similarly, by inhibiting endocytosis, Tzv may stabilize signaling from the cell surface, thereby maintaining integrin activity. On the other hand, Ptn may activate PKC by mimicking downstream signaling by physical cell-cell interactions. Future identification of Ptn targets may provide new insights into the mechanisms by which cell-cell adhesion controls cell-ECM interactions. Our study also illustrates the potential and advancement of high-throughput compound screening in stem cell research. Further development and application of such chemical approaches in stem cells will undoubtedly result in the identification of additional novel small molecules and gaining insight into the mechanisms for precise control of cell fate in vitro and in vivo.

[0190] method cell culture Human ESC lines, H1, HUES7, and HUES9, were cultured on irradiated MEF feeder cells in DMEM-F12 supplemented with 2 mM L-glutamine, 1x non-essential amino acids, 20% serum replacement (Invitrogen), and 10 ng / ml basic fibroblast growth factor (Invitrogen). Chemically defined, feeder-free hESC culture medium has been described previously (Yao, S. et al., Proc Natl Acad Sci USA 103 (18):6907-6912 (2006)). Briefly, hESCs were grown on Matrigel-coated tissue culture plates in N2B27-CDM (DMEM-F12 supplemented with 1x N2 supplement, 1x B27 supplement, 2mM L-glutamine, 0.11mM 2-mercaptoethanol, 1x non-essential amino acids, and 0.5mg / ml BSA (fraction V) and 20ng / ml bFGF) and 20ng / mL bFGF. Human ESCs were passaged every 5-6 days with 0.05% trypsin.

[0191] For clonal survival assays, single hESCs were diluted to clonal density and plated onto 96-well Matrigel-coated plates. For low-density survival assays, 500 cells were plated onto 96-well Matrigel-coated plates. To visualize hESC colonies, cultures were fixed in 4% paraformaldehyde in PBS for 5 min, washed once with PBS, and stained for alkaline phosphatase activity according to the manufacturer's instructions. ALP-positive colonies were counted using an inverted microscope.

[0192] reagent ALP detection kit and integrin antibody were from Chemicon. AG825 (Erb2 inhibitor), AG1478 (EGFR inhibitor), and PPP (IGFR1 inhibitor) were purchased from Calbiochem. An antibody raised against the intracytoplasmic tail of E-cadherin (Transduction Laboratories, Lexington, KY) was used for immunoprecipitation. TS2 / 16 antibody was from Pierce. Antibodies against the extracellular domain of the E-cadherin molecule were from Zymed (Carlsbad). Antibodies against extracellular signal-regulated kinases / MAPKs, EGFR1, ERB2, GADPH, and phosphorylated AKT were from Cell Signaling. Mouse monoclonal anti-phosphorylated tyrosine (4G-10 clone) was from Upstate Biotechnology. Ptn and Tzv were added to the medium at 2 μM.

[0193] High-Throughput Compound Screening Trypsinizable hESC lines HUES7 or HUES9 were used for screening. hESCs were cultured in chemically defined medium on Matrigel-coated plates as described above. Cells were then harvested using trypsin. hESCs were plated at 4,000 cells per well on Matrigel-coated 384-well plates. After one hour for cells to settle, compounds from a library of 50,000 distinct heterocycles were added to each well (final concentration 2 μM). After an additional 6 days of culture, with medium and compounds changed on day 3, cells were stained for ALP expression and examined for compact colony morphology.

[0194] Immunostaining analysis Immunostaining was performed as previously described (Yao, S. et al., Proc Natl Acad Sci USA 103 (18):6907-6912 (2006)). Briefly, cells were fixed with 4% paraformaldehyde for 15 min at room temperature (RT). Cells were then incubated in blocking buffer for 1 h at RT. Primary antibody incubation was performed overnight at 4°C. The following commercial antibodies were used at a concentration of 1:100 in blocking buffer: anti-SSEA4, anti-Oct4 (Chemicon), anti-Nanog (Chemicon). Staining was visualized using FITC-, cy3-, or cy5-conjugated secondary antibodies (Jackson ImmunoResearch).

[0195] Teratoma formation and karyotyping Teratoma formation experiments were performed by injecting 3-5 million hESCs (maintained in the presence of compounds Tvz or Ptn) under the kidney capsule of nude mice. After 4-5 weeks, all mice developed teratomas that were excised and analyzed immunohistologically by Scripps Research Institute Research Histology Service and Animal Resources. Compound-treated cells were karyotyped by standard G-banding in the Cytogenetics Laboratory at Children's Hospital Oakland. No chromosomal abnormalities were detected in 10 randomly selected nuclei.

[0196] TUNEL assay Differently treated hESCs were dissociated with trypsin and fixed with 4% paraformaldehyde. Staining was performed according to the manufacturer's instructions (MBL Laboratories, Watertown, MA). After staining, samples were analyzed by flow cytometry using a FACS Calibur flow cytometer (BD).

[0197] Flow cytometry analysis To assess the expression of E-cadherin, activated integrins and SSEA4, dissociated cells (3 × 10 5 ) were washed with PBS and resuspended in PBS containing 2% goat serum. Cells were then incubated with the appropriate antibody for 1 h at 4°C, washed with blocking solution, and labeled with FITC-conjugated secondary antibody for 30 min at 4°C. Cells were then washed and analyzed on a FACS Calibur flow cytometer.

[0198] Cell adhesion assay Cell adhesion assays were performed in 96-well microtiter plates coated with Matrigel. After trypsinization, hESCs were resuspended in chemically defined medium containing the desired compound. Cells were then added to microtiter wells and incubated at 37°C for 3 hours. Unbound and loosely bound cells were removed by shaking and washing, and the remaining cells were then immediately fixed. Wells were washed three times with 200 μl H2O and attached cells were stained with crystal violet (Sigma). The absorbance of each well at 570 nm was then measured. For experiments with blocking antibodies, cells were preincubated with the antibody on ice for 30 minutes and adhesion assays were performed in the presence of the antibody. Assays for each sample were performed in triplicate independently.

[0199] Endocytosis assay hESCs were incubated on ice with 1.5 mg / ml sulfosuccinimidyl 2-(biotinamido)ethyl-dithioproprionic acid (sulfo-NHS-SS-biotin) (Pierce Chemical Co.), washed, and the reaction was stopped. Endocytosis of E-cadherin is mediated by Ca 2+The biotinylated proteins were induced by depletion of β-terminal ...

[0200] Example 2: Synthesis of N-benzyl-2-(pyrimidin-4-ylamino)thiazole-4-carboxamide (thiazovivin) chemical synthesis Using the chemical synthesis examples below and chemical synthesis methods generally known in the art, one of ordinary skill in the art can produce the compounds disclosed herein (e.g., compounds of Formulas (I)-(VI)).

[0201] All commercially obtained compounds were used without further purification. NMR spectra were recorded on a Bruker (400 MHz) instrument. Chemical shifts (δ) were measured in ppm and coupling constants (J) were reported in Hz. LCMS was performed using a reversed-phase liquid chromatography-mass spectrometry Agilent 1100 LCMS system with an API-ES ionization source. High pressure liquid chromatography was performed using a C18 column with a linear gradient of 10% to 90% solution A (acetonitrile containing 0.035% trifluoroacetic acid) in solution B (water containing 0.05% trifluoroacetic acid) for 7.5 min, followed by 90% A for 2.5 min.

[0202] Synthesis of N-benzyl-2-(pyrimidin-4-ylamino)thiazole-4-carboxamide (thiazovivin) Benzylamine was added to 4-formyl-3,5-dimethoxyphenoxymethyl-functionalized polystyrene resin (PAL) via reductive amination to give PAL-benzylamine resin. See Ding, S.; Grey, NS Wu, X.; Ding, Q.; Schultz, PGJ Am. Chem. Soc. 2002, 124, 1594-1596. A reaction flask containing PAL-benzylamine resin (200 mg, 0.2 mmol), 2-bromothiazole-4-carboxylic acid (83 mg, 0.4 mmol), bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP-Cl) (153 mg, 0.6 mmol), and diisopropylethylamine (0.17 mL, 1 mmol) in DMF (3 mL) was shaken at room temperature for 24 h. The resin was washed with methanol, dichloromethane, and dried under vacuum to give PAL resin-N-benzyl-2-bromothiazole-4-carboxamide, which was added to a flame-dried reaction vial followed by 4-aminopyrimidine (95 mg, 1 mmol), Pd2(dba)3 (46 mg, 0.05 mmol), Xantphos (87 mg, 0.15 mmol), and NaO t Bu (192 mg, 2 mmol) was added. The vial was securely capped, degassed, and then backfilled with argon and anhydrous dioxane (1.5 mL). The reaction was shaken at 90 °C for 24 h. The resin was washed with sodium diethyldithiocarbamate solution (0.05 M in DMF), methanol, and dichloromethane, and dried under vacuum. The resin was then cleaved with a cleavage cocktail of TFA:CH2Cl2:H2O (45:55:5) (2 mL) for 2 h. The resin was filtered, and the filtrate was collected and dried under vacuum to give the crude product, which was then purified by HPLC to give the title compound (30 mg, 48%). TIFF0007621405000032.tif21128N-Benzyl-2-(pyrimidin-4-ylamino)thiazole-4-carboxamide C 15 H 13 Calculated accurate mass of N5OS: 311.1, measured value: LCMS m / z = 334.1 (M+Na +). TIFF0007621405000033.tif21155

[0203] Example 3: Synthesis of thiazovivin derivatives TIFF0007621405000034.tif26151

[0204] Amines R suitable for functionalizing polystyrene resin (PAL) with 4-formyl-3,5-dimethoxyphenoxymethyl through reductive amination 1 PAL-benzylamine resin was obtained by pre-addition of NH2. A mixture of PAL-benzylamine resin (200 mg, 0.2 mmol, 1.0 equiv), 2-bromothiazole carboxylic acid 1 (0.4 mmol, 2.0 equiv), bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP-Cl) (0.6 mmol, 3.0 equiv), and diisopropylethylamine (1 mmol, 5.0 equiv) in anhydrous DMF (3 mL) was shaken at ambient temperature for 24 h. The resin was washed with methanol, dichloromethane, and dried under vacuum, which was added to a flame-dried reaction vial and then the corresponding R 2 NH2 (1 mmol, 5.0 equiv.), Pd2(dba)3 (0.05 mmol), Xantphos (0.15 mmol), and NaO t Bu (2 mmol, 10.0 equiv) was added. The vial was securely capped, degassed, and then backfilled with argon and anhydrous dioxane (1.5 mL). The reaction was shaken at 90 °C for 24 h. The resin was washed with sodium diethyldithiocarbamate solution (0.05 M in DMF), methanol, and dichloromethane, and dried under vacuum. The resin was then cleaved with a cleavage cocktail of TFA:CHCl:HO=45:55:5 (2 mL) for 2 h. The resin was filtered, and the filtrate was collected and dried under vacuum to give the crude product, which was then purified by HPLC to give the desired title compound 3. TIFF0007621405000035.tif75162TIFF0007621405000036.tif230162TIFF0007621405000037.tif129162

[0205] Example 4: Synthesis of N-(cyclopropylmethyl)-4-(4-(6-hydroxy-3,4-dihydroquinolin-1(2H)-yl)pyrimidin-2-ylamino)benzenesulfonamide (pyrintegrin) A reaction flask containing 2,4-dichloropyrimidine (372 mg, 2.5 mmol), 6-methoxy-1,2,3,4-tetrahydroquinoline (489 mg, 3 mmol), and diisopropylethylamine (0.52 mL, 3 mmol) in n-butanol (10 mL) was warmed at 40° C. overnight. The solvent was evaporated and the residue was purified by flash column chromatography to give 2-chloro-4-(6-methoxy-3,4-dihydroquinolin-1(2H)-yl)pyrimidine (551 mg, 80%). This intermediate (250 mg, 0.91 mmol) was dissolved in dichloromethane and treated with BBr3 (1 M in dichloromethane) (1 mL, 1 mmol) at −78° C. The reaction mixture was allowed to slowly warm to room temperature, stirred for 1 h, poured into water, and extracted with dichloromethane. The combined organic layers were dried over anhydrous Na2SO4 and concentrated. The residue was purified by flash column chromatography to give 2-chloro-4-(6-hydroxy-3,4-dihydroquinolin-1(2H)-yl)pyrimidine (154 mg, 65%). To a stirred solution of 2-chloro-4-(6-hydroxy-3,4-dihydroquinolin-1(2H)-yl)pyrimidine (29 mg, 0.11 mmol) and 4-amino-N-(cyclopropylmethyl)benzenesulfonamide (27 mg, 0.12 mmol) in DMF (0.5 mL) was added p-toluenesulfonic acid (2M in dioxane) (55 μL, 0.11 mmol). The reaction mixture was stirred at 90° C. overnight and then purified by HPLC to give the title compound (27 mg, 56%). TIFF0007621405000038.tif26128N-(cyclopropylmethyl)-4-(4-(6-hydroxy-3,4-dihydroquinolin-1(2H)-yl)pyrimidin-2-ylamino)benzenesulfonamide C 23 H 25 Calculated accurate mass of N5O3S: 451.2, measured value LCMS m / z = 452.3 (M+H + ). TIFF0007621405000039.tif29159

[0206] Example 5: Synthesis of pyrin tegrin derivatives TIFF0007621405000040.tif21152 2,4-Dichloropyrimidine 4 (1.0 equiv.) in n-butanol, R 1 A mixture of NH2 (1.2 equiv.) and diisopropylethylamine (1.2 equiv.) was heated at 80 °C overnight. The solvent was evaporated and the residue was purified by flash column chromatography to give intermediate 10 in excellent yield (>80%), which was dissolved in R 2 Treated with NH2 (1.2 equiv.) and p-toluenesulfonic acid (2 M in dioxane) (1.2 equiv.) was added. The reaction mixture was stirred at 90 °C overnight and then directly purified by preparative HPLC to give pyrithinegrin derivative 11 in excellent yield. TIFF0007621405000041.tif26157TIFF0007621405000042.tif241157TIFF00076214050 00043.tif244157TIFF0007621405000044.tif229157TIFF0007621405000045.tif102157

[0207] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes will be suggested to those skilled in the art in light of them, which are within the spirit and scope of this application and the appended claims. All references, patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

Claims

1. 1. A method for modulating integrin activity to enhance cell survival and / or proliferation, comprising the steps of: (a) obtaining isolated cells, including stem cells; and (b) contacting the separated cells with a compound sufficient to enhance survival and / or proliferation of the cells compared to the absence of the compound; Here, the compound has the following formula (II): or a racemate, diastereomer, tautomer, or geometric isomer thereof, or a pharma- ceutically acceptable salt thereof: (In the formula, L 2 is unsubstituted C 1 -C 6 is alkylene; y is an integer from 0 to 1; z is an integer from 0 to 2; X is -N=, or -CH=; R 1 is hydrogen or unsubstituted C 1 -C 6 is alkyl; R 3 -C(O)NR 14 R 15 , -OR 18 or substituted or unsubstituted C 1 -C 6 alkyl, and the substituted C 1 -C 6 The alkyl substituents are halogens, where z is 2, and two R 3 The moieties are linked together and are unsubstituted C having at least one heteroatom selected from the group consisting of O, N, P, Si, and S. 3 -C 8 Forming a heterocycloalkyl; R 4 -OR 18 and R 14 , R 15 , and R 18 are independently hydrogen or unsubstituted C 1 -C 6 is alkyl, The alkyl and alkylene groups are saturated and linear or branched or combinations thereof.

2. The method of claim 1 , wherein the method modulates β1 integrin activity.

3. The isolated cells are (a) in adhesion medium; or (b) in suspension culture; or (c) are single dissociated cells; or (d) The cells are aggregated. The method according to claim 1 or 2.

4. The method according to any one of claims 1 to 3, wherein the step of contacting further comprises the step of subculturing the cells in a medium containing the compound.

5. 5. The method of claim 4, wherein the subculturing step comprises subculturing the cells for at least 20 generations.

6. The medium is (a) a chemically defined medium; and / or (b) feeder cell-free or non-feeder cell conditioned medium; and / or (c) Media free of animal-derived products The method according to claim 4 or 5,

7. The contacted cells are (a) maintaining uniformity; and / or (b) maintain pluripotency; and / or (c) express pluripotency markers; and / or (d) maintaining a normal karyotype; and / or (e) capable of self-renewal; and / or (f) reduced cell death; and / or (g) capable of forming teratomas; and / or (h) decreased cell differentiation; and / or (i) capable of differentiating into the three primary germ layers; and / or (j) capable of differentiation into specific cell lineages; and / or (k) capable of forming embryoid bodies following single cell dissociation; and / or (l) After single cell dissociation, cell adhesion ability is increased. The method according to any one of claims 1 to 6.

8. The compound is 8. The method of any one of claims 1 to 7, wherein the compound is selected from the group consisting of: or a racemate, diastereomer, tautomer, or geometric isomer thereof, or a pharma- ceutically acceptable salt thereof.

9. The compound is selected from the group consisting of: or a racemate, diastereomer, tautomer, or geometric isomer thereof, or a pharma- ceutically acceptable salt thereof.

10. 10. A composition comprising an isolated cell obtained using the method according to any one of claims 1 to 9, and the compound of formula (II), or a racemate, diastereomer, tautomer, or geometric isomer thereof, or a pharma- ceutically acceptable salt thereof.

11. A method for modulating integrin activity to enhance cell survival and / or proliferation, comprising the steps of: (a) obtaining isolated cells, including stem cells; and (b) contacting the separated cells with a compound sufficient to enhance survival and / or proliferation of the cells compared to the absence of the compound; wherein the compound is or a racemate, diastereomer, tautomer, or geometric isomer thereof, or a pharma- ceutically acceptable salt thereof.

12. A composition comprising an isolated cell obtained using the method of claim 11, and a compound of claim 11, or a racemate, diastereomer, tautomer, or geometric isomer thereof, or a pharma- ceutically acceptable salt thereof.

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