AU2015221570B2 - Treatment of pluripotent cells - Google Patents

Abstract

The present invention is directed to methods to treat pluripotent cells, whereby the pluripotent cells can be efficiently expanded in culture and differentiated by treating the pluripotent cells with an inhibitor of GSK-3B enzyme activity.

Description

TREATMENT OF PLURIPOTENT CELLS FIELD OF THE INVENTION

The present application is a divisional application of Australian Application No. 2009239447, which is incorporated in its entirety herein by reference.

The present invention is directed to methods to treat pluripotent cells, whereby the pluripotent cells can be efficiently expanded in culture and differentiated by treating the pluripotent cells with an inhibitor of GSK-3B enzyme activity.

BACKGROUND

Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

Advances in cell-replacement therapy for Type I diabetes mellitus and a shortage of transplantable islets of Langerhans have focused interest on developing sources of insulin-producing cells, or β cells, appropriate for engraftment. One approach is the generation of functional β cells from pluripotent cells, such as, for example, embryonic stem cells.

In vertebrate embryonic development, a pluripotent cell gives rise to a group of cells comprising three germ layers (ectoderm, mesoderm, and endoderm) in a process known as gastrulation. Tissues such as, for example, thyroid, thymus, pancreas, gut, and liver, will develop from the endoderm, via an intermediate stage. The intermediate stage in this process is the formation of definitive endoderm. Definitive endoderm cells express a number of markers, such as, HNF-3 beta, GATA-4, Mixll, CXCR4 and SOX-17.

Formation of the pancreas arises from the differentiation of definitive endoderm into pancreatic endoderm. Cells of the pancreatic endoderm express the pancreatic-duodenal homeobox gene, PDX-1. In the absence of PDX-1, the pancreas fails to develop beyond the formation of ventral and dorsal buds. Thus, PDX-1 expression marks a critical step in pancreatic organogenesis. The mature pancreas contains, among other cell types, exocrine tissue and endocrine tissue. Exocrine and endocrine tissues arise from the differentiation of pancreatic endoderm.

The generation of a sufficient amount of cellular material for transplantation requires a source of the cellular material that can be efficiently expanded in culture, and efficiently differentiated into the tissue of interest, for example, functional β cells.

Current methods to culture humaarem^onic stem ceUf am^mspiexvfttey. feguiro tfeo use of exogenous feeiom, or chemically defined media, in order for the ceils to proliferate without loosing their pluripOfeacy. Furthermoredifeentiafiou often results in a decreese m the cells to expmd in culture.

In one example, Cheon ei a! (BioReprod DO 1:! 0.1095/hiolreprod, 1051)46870, October 19,2005) disclose a feeder-free, serum-free culture system in which embryonic stem ceils are maintained in unconditioned scrum replacement (SR) medium supplemented with difcwt growth feefom capable of triggering embryonic stem cell self-renewal.

In another example, US20050235440 discloses a defined media useful in culturing stem cells, including undifferentiated primate primordial stem colls, in solution, the media is substantially isotonic as compared to the idem cells being cultured. In a given culture, the particular medium comprises a base medium and au amoun t of each of hPGF, insu lin, and ascorbic acid necessary to support substantially undiSerentiaied growth of the primordial stem cells.

In anot her example, W020050B0845 discloses a method for maintenance of an: undifferentiated stem cell, said, method comprising exposing a stem cell to a member of the transforming growth. factor-beta (TOFP) family of proteins, a member of the fibroblast growth, factor (FGF) ''fMnfl:y^of;pmtp:kfδ·j'.iόϊ' nicotinamide (MIC): M an amount sufficient to maintain the cell In mt undifierenti ated state for a sufficient amount of time to achieve » desired result.

Inhibitors of glycogen synthase kinase-3 (GSK-3) are known ’to pffiotote proilferatfoh and expansion of adult stem cells. In one example, Tateisbi ei ai. (Biochemical and Biophysical Research Communications |2§&7) 352:635) show that mhibi tibn of GSK-3 enhances growth and survi val of human cardiac stem ceils (hC'SCs) mcoveted lfom the neonatal, or adult human heart and having mesenchymal features.

For example, RuhfsOn *».// ffi'sAR 1-R*% 62-"-o25 2, (21)07)) stales “'Wrt signaling stimulates islet p cell proliferation. h> another example, W0200?0i64B$ *hat Edition, of GiSll-3 inhibitors to the cultiire of noo-'embrwoic stem cells, including multipotent adult progenitor cells, leads to the raahttenauce of a piuripoteat phenotype during expansion and results in a more robust dillomutiatidu response.

In another example, US2006030042 uses a method of inhibiting GSK-3, either by addhion of Wot oc a small molecule inhibitor of GSK-3 ettoyme activity, to maintam use of a feeder cel! layer, . to another :gXi8^|^;Wd2^&^36473 reports the addition of aGSK~3B inhibitor, to stabilise piuripoteut cells through transcriptional activation of c-ntvc and stabilization of c-rnye protein.

In another example, W02006100400 reports the use of a stem cell culture medium containing a GSK-3 inhibitor and a gp 130 agonist to maintain a sdf-renewmg population 0Fplari.potsat:.ste;Cdl^/ifteludtttg:mo«se:!or'h.iK«a«. embryonic stem cells.

In anotbef Sxaotple, Sato ei ai. (Nature Medicine (2004) 10:53-63) show that inhibition of GSK~3 with a specific pharmacological compound can maintain the imdiifcentfated phenotype of embryonic stem ceils ,and>suidain expmssidnof piuripoten t state-specific transcription factors such as 0ct-3/95, Rex-f, and.Nan.bg,

In another example, Maurer etal (Journal of Proieorne Research. (2007) 6:1.198-1208} show that adult, neuronal stem cells treated mth a GSK-3: inhibitor show enhanced neuronal differentiation, specifically by promoting:transcription of p-eatenm target genes and decreasing apoptosis.

In another example, Gregory e/ at (Annals of the Mew Suth: Academy of Sciences (2005) 1049:97-106) report that inhibitors of GSK-38

In another example, Feng et al (Biochemical aid Biophysical Research Comm uneations (2004) 324:1333-1339) show that henaatopototiebiftotontlatipn from embryonic stem cells is associated with down-regulation of the Wnt/0-cutenin pathway, where Wnt is a natural inhibitor of GSK3.

[0020] Therefore, there still remains a significant need to develop methods for treating pluripotent stem cell such that they can be expanded to address the current clinical needs, while retaining the potential to differentiate into pancreatic endocrine cells, pancreatic hormone expressing cells, or pancreatic hormone secreting cells.

SUMMARY

[0020a] According to one aspect, the present invention provides a method to expand and differentiate human pluripotent cells, comprising the steps of: a. culturing human pluripotent cells, and b. treating the human pluripotent cells with an inhibitor of glycogen synthase kinase 3β (GSK-3B) enzyme activity, wherein the inhibitor is 3-[1-(3-hydroxy-propyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione.

[0020b] Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

[0021] The present invention provides a method to expand and differentiate pluripotent cells by treating the pluripotent cells with an inhibitor of GSK-3B enzyme activity.

[0022] In one embodiment, the present invention provides a method to expand and differentiate pluripotent cells, comprising the steps of: a. culturing pluripotent cells, and b. treating the pluripotent cells with an inhibitor of GSK-3B enzyme activity.

[0023] In one embodiment, the pluripotent cells are differentiated into cells expressing markers characteristic of the definitive endoderm lineage.

[0024] The pluripotent cells may be human embryonic stem cells, or they may be cells expressing pluripotency markers derived from human embryonic stem cells, according to the methods disclosed in 60/913475.

[0025] In one embodiment, the inhibitor of GSK-3B enzyme activity is a compound of the Formula (I):

Formula (I)

In one embodiment, the inhibitor of GSfC~3B enzyme activity is a compound of the Formula (11):

Formula (II)

In one embodiment,, the inhibitor of GS!C~3B enzyme activity is a coni|>ot3rix| of the Formula (11 f);

Formula (III) brief description m the figures

Figure! shows the effect of a range of concentrations of the compound JHJ 17.189731 on cel! number, as determined by the number of nuclei observed (Panel A) and Sox··! 7 expression, a& determined by intensi ty of immnoofluoreseent staining (Panel B). Results were obtained bom cells of the human embryonic stem eel 1 line HI (white bars), or cells of file human embryonic stem cell line 389 (black bmh using the IM Cell Analyzer 1000 (QEIfealthcam).

Figure 2 shows the effect of nfhageeimone^ compound JNJ 1716379b on cell number, as determined by the number of nuclei observed (Panel A) and Sox-! 7 expression, as determined by intensity of imnmnoduorascent staining (Panel B), Results were obtained bum cells of the human embryonic stem cell line HI (white fears), or oolls of the human ampoule stem ceil line 119 (hlaefe ham), using the (IK Cell Analyzer 1000 (GE Healthcare).

Figured shows the effect of a range of coneentmtions of fe com pound JMJ 17223375 on cell number, as determined by die unmfeer of nuclG observed (Panei A) and Sox-17 expression:, as determined bystaining (Panel B). Results were obtained from cells of five human embryonic stem cell line HI (white bars), or cells of the human. embryonic stem cell line H9 (Mack bars), using the IN Cell Analyser 1000 (GEMealthcate),

Ffgnr e 4 shows the ef fee t o f a range of concentrations of tire eonipouiid JMJ I % 157698 on cel l n umber, as; determin ed by the number of n uclei ©hsetwed (Panel A) and Sox-l? expression, as determined by intensity of im.mun0fhmre$eeat staining (Panel B), Results were obtained from cells of tbe human embryonic stem coll line Hi (white bars), or cells of the human, embryonic stem cell lino H9 (black bars), using the IN Cell Analyzer 1606 (GE Healthcare),

Figure 5 shows the effect ofa range of concentrations«of the compound JMJ 26158615: on cell number, as determined: by the number of nuclei observed (Panel A) and S6x~17 expression, as determined by inteasity of immmioBuorescent staining (Panel B), Results were obtained tfom cells of the bbman embryonic stem: cell line HI (white bars), or cells of ibe hntaaa embryonic stent cell. line 119 (black bars), using the IN Cell Analyzer 1666 (OE 'Healthcare),

Figure 6 shows the effect of a range of concentrations of the compound JNJ 2648319? on eel! number, as determined by the number of nuclei observed (Panel A) and Sox-l 7 expression,, as determined by intensity of imjnunofluoreseeta staining (Panel B). Results were obtained from cells of the, human embryonic stem cell line HI (white bars), or cells of the: human embryonic stem cell line H9 (Mack bars), using the IN Ceil Analyzer 1606 (GE Healthcare), figure 7 shows the effect of a range of eoncentnitions of the contpoond 3M3 26483249 On cell number, as deiettnined by the number of nuclei Observed (Panel A) and Sox-17 expression, as determined by intensity of Immunollnoreseent staining (Panel B). Results were obtained fmm cells of the human embryonic stem,cell line H I (white bum), or cells of the human embryonic stem: cell line H9 (black bars), using the IN Ceil Analyzer 1600 (GE Healthcare),

Figur e 8 shows the effect: of a mage of concentrations of the. compound/JNJ 10220007 on cell number, as determined by the number of nuclei observed (Panel A:) and $ox»i7 expressions as determined by: intensity of immraofiuoreseont staining (Pane! B). Results Wemohtained from, cells of the human embryonic stem ceil lineSi (white bars), or cells of tite human embryonic stem cell line I 19 (black bars),:: using: 1¾¾ IM Cei! Analyzer 1000 (GE Healthcare);

Figure 9 shows the expression of C/XCR4 on. the surface Of eeiJs, as determined by immnnofluoresconf staining and flow· cytometric analysis, on cells treated with the compounds shown, according to the metlwds described in Example 8.

Figure 10 shows tire expression. of CXCR4 (Panel. A), HNF-3 beta (Panel B),:and Sox-U (Panel: C’|, as detormiaed: by real-time 'FCR, in cells treated with the compounds Shown, according to the methods described I» Example 8.

Figure fl shows t he effect of a range of con centration s of the compounds shown on cell number, as determined by the number of nuclei observed (PMciA) and Pdx-1 expression, as determined by intensity of inrmunoflitorescent staining (Panel B), using the M OH Analyzer 1000 (GEllealtheare), Ceils were treated according to the methods described in Example 9.

Figure 12 shows the effect of a range of concentrations of the compounds shown on Pdx-1 expression (White bars) and H/NFA) (hlaelv bars), as determined by maritinte PCR* Cells were treated according to the methods described in Example 9:,

Figure 13 shows the effect of a range of eeneentmtions of the compounds shown on cell number, as demrmlned by the n umber of nuclei observed (Panel A) and insulin expression, as determined by intensity of immunofluoreseent staining (Bane! B), using the IN Cell, Analyzer 1000 (01: Bealthcare), Cells were treated aoeording to: the methods described in Example 18:

Figure 14 shows effect of a range of concentrations of the compounds shown on Pdx-1 : expression: (white bars) and. insulin (black bars), as deiemuned by real-time PCR, Cells were treated according to the .methods described in Example t.0l

Figure ISshowsthe pfifcet of a range of concenimtions of the compounds shown on ceil number* as: determined by the number of nuclei observed (Panel A) and rimslin expression, as determined by intensity of in orron» 0 imrescen i stainIng (Panel B), using the IN Cell Analyzer 1000 (OB Healthcare). Cells were treated according to the methods described in Example 1:11

.&ETAILE& BESCRlPTICiN

For clarity of diselosisre, andinof by wayof limitation, the detailed description of the myentiotx is: dtyiried.mto the fb IIowing subsections: that describe,or: illustrate certain feaiiims^ ombbdlMents, or applications of the present invention;

Definitions

Stem cells die undifferentiated ceils.defined by their ability at the single: cell level to both, sdfAonsw and. dtttoailnie to produce progeny cells, including self renewing progenitors,: «on-renewing progenitors, and termhtally dil&enfiated cells. Stem ceils are; also characterized by their ability to difiemnilate in vitro Into fenetionaieelis ofyuribiis' ce ll lineages from multiple germ layers (endederm, mesodenn and ectoderm), as well as to gibe rise m tissues df multiple gerih laycm following transplantation and to contribute substantially to most, if not all, tissues following injection imo blastocysts.

Stem cells are clsssifted by iheirdevdepmental potential as: fl) totipotent, meaning able to give rise to ail cell types-; (2) ρΐηπροΐοηί, meaning able to give rise to all embryonic ceil types: (3) multlpotent, meaning able to give rise to a subset of cell lineages, but all withi n a particular tissue, organ, or physiological system (tor example, hematopoietic stem cells (BSC:) can produce progeny dial include HSC (seif- renewal), blood cell restricted obgopotent progenitors and all edl types and elements (e.g,., platelets) that are norma! components of theblood); (4) oligopoifenh meaning able to give rise to a more restricted subset of ceil lineages than muitipoteni stein cells; and (5) unipoieut, meaning able togive rise to a single cell lineage (e.g., spermatOgehic stem cells).

Differentiation is the process by which an unspecialized ("uncommitted") or less specialized cell acquires the features of a specialized eel! such as, for example, a nerve cell or a muscle cell. A differentiated of diffefentiat»EiP“indttc«d:eefl is ode that has tahen ott a mom specialized ("committed") position within, the lineage of a4e:iL-''19ie:'tdmi. 4<e0t^|Riile(d%'wh'hp applied to the process of ditfefeniiation, refers to a ceil that has proceeded so the differentiation. pathway to a point where, under iiormai cirenmstattees, it will continue to differentiate into a specific cell type or subset of cell types, and. cannot, under normal circumstances, dlffemniiate imo a different cell type Off evert to a less differentiated cell type. Detoi fferent iatiem refers to the process by which a cel 1 reverts to a less specialized (or committed) position within the lineage of a cell. As used herein, the lineage of a cell defines the heredity of the cell, i,e„ which cells it came bom and what cells it can give rise to, The lineage of a cell places the ceil within a hereditary scheme of development and differentiation. A lineage-speciftc marker refers to a characteristic spectfteaily associated with the phenotype of cells of a lineage of interest and can he used to assess the diiferenpation of an nneornmitted ceM to the lineage of interest. "β-eeli lineage’' refer to ceils with: positive gene expression for the transcription factor FD3C-1 and at least one of the following transcription factors: NGN-3, Nkx2,2, Nksfet, NburOD. M~l, BNF-a beta, MA.FA, Pax4, and Pdxb. Cells expressing markers characteristic of the f) cell lineage include p celis:, ‘‘Cells expressing markers eharaeteristie Of the de^nitive endoderih lineage’’ as used herein refer to cells expressing at least one Of the following markets; SOX-17, GATA~4, BNF-3 beta, G:SC,; Cor 1., Hodaf FGffo Braehyury, Mixffike homeobox protein, PGF4 :€D4E, eomesodermin (HOMES), DKK4, FGF17, Q&amp;f CXCR4, «it, CD99, or :0ΤΧ2, Cells expressing markers characteristic of the definitive endoderm lineage : include primitive streak precursor cells, primitive streak:cells, moseddoderm cells and definitive endoderm cells. '“Gelts ekpmssifig:markets eharaeteristie ofritopanefeatic ehdpderm lineage*’ as used herein: refer to ©ells expressing at least one of the following markers:; POX-!., HNF- 1 beta* PTF~ I alpha, HhiFfo, or HBfo Cells expressing markers chameterisfe of the pancreatic ehdodemi lineage include pancreatic endoderm cells, “Cells expresshtg markers chameteristie of the pancreatic endocrine lineagC: as used herein refer to cells expressing at least one of the fohowing markers: KGN-3, KeuroD, l.sleM, PDX-l, NKM.1, Pax-4, Ngo-3. orPTIM alpha, Cells expressing markers eltaraeterisdc of the pancreatic endocrine lineage include pancreatic endocrine cells* pancreatic hormone expressing ceils, and pancreatie ftopnone secreting cells, and cells of the (Feel I lineage; “Definitive endodenn'’ as used herein refers to cells which bear the characteristics of ceils arising from the epiblast during gastrulation and which formffee gastrointestinal tract and its derivative^ Definitive endodOrm cells express the following markers: E1NF-3 beta, GATA-4, SQ7C-I7, Cerberus, OTX2, gooseeokl, C-Kit, CD99, andMixIl. “Extraembryonic eadodfeem” as used herein refers ®a population of cells expressing at least one bfthpfoKowhig'iaiAem-SO^^, AFP, and SPARC. "’’Markers” as used herein, are nucioiC'Mdor^l^pe|>ti&amp;"'iiioiee«ks''that'arediffereittial!^ expressed hi a cell of interest, in th is eonfokcdtfforentiai exptAssion meaas'.aa fobfigi&amp;hd level lor a. positive marker and: a decreased level for a negative marker. The detectable level of the marker nucleic acid or polypeptide is sufficiently higher dr lower in the cells of interest compared to other cells, such that the cell of interest can he identified and distinguished fmm other ceils using any of a variety of methods known In the art. “Mesendoderm cell” as used herein refers to a cell expressing at least one of the following martet CD4«, eomesodermin (HOMES), SOXG7, DKK4,X1NP~3 beta, GSC, FGF.17, GATA-6. “Panereatie endocrine celP, or '‘panematle hormone expressing cell'11 as used herein refers to a ccdl capable of expressing at least ope of the following hprmpnbs: insulin, glucagon:, somatostatin, and: pancreatic polypeptide. “Paiscreafle ferWOTe secretiiTg cell” as used hereijs refers to a cell capafefc of secietirig at least oae^fthes&amp;l'I^WijSg 'hormones: insulin, glucagon, somatostatin;. sod pancreatic polypeptide.

‘Tre-pnifiiiiye streak! cell” as used herei n refers to a cel l expressing;, at least one: of the .followliig;parkers: Nodal,; or FGFB

Primitive streak eelP as used herein refers to a cell expressing at least one of the following marker!: ptofeitu, or FOF4,

In one eoibodinKot,: the preserdinventiOn provides a differentiation of piuripotent eel is comprising treating the piuripotent cells with an inhibitor of GSK.»3Ee»ieyrne activity.

In one embodiment, the present invention provides a method to expand and differentiate plartpotent cells, eon^risiog the sieps of c. Gulturing piuripotent CClISs and d. Treating rhv pinripoteut ceiiswithian inhibitor of G&amp;K-dB enzyme activity.

In one embodiment, the p totipotent ceils are differentiated into cells expressing markers: characteristic of the definiti ve Mdodeofi lineage.

Markers characteristic of the:defimdve endodemt lineage are selected from:the .group consisting Of SOXJT, GATA4, HnfiTbeia, GSC, Cer!, Nodal, FGF8, Bmchyury, Mix-like homeobox pOJteitr, :FG:F4 €D4gveomeaodemiitt (EOMES), DKK4, FGFi 7, GATAh, CX!CR4, CP49v;abd;Ci'fX2. Goniemp fated in the present invention is a cell, derived fttkh feat 4?xpfes#§!s at feast one of the markers clmractenatic of ; thc definitive eudoderrn. iincagc. I n one aspect of the present invention , a ceil, expressing piatkors oltaxaetertstie of the dofiwfive endOderm lipeagoisw primitive streak precursor edl, In an; alternate aspect, a ceil expressing markers characteristic: of the: definitive ebdoderm. lineage is a otesendoderm eelh In an altenmto aspect, a cell expressing markers characteristic of the definitive endoderto lineage is a definitive ehiioderm cell

The pluripotent cells; may be troated^th·^Inhibitor of GSK-3II enzyme activity: for atoutbne to about 72 tours, Aitemativejydthe plufipoietlt ceils may be treated with the inhibitor of GSK-3B enzyme activity for about 12 to about 4¾ hours. Alternatively, the piuripotent cells may be treated with the inhibitor of GSK-3B enzyme activity tor about 4:S bouts.

Is one embodimeab the inhibitor of GSK-3S enzyme activity is used at a. concentration pf about 10DhM: to: about ΙΟΟμΜ. Aheraatiyeiy, the inhibite*':dfOSR~|Pet^yt^e activity is used at a. conOOntatioh of about1 IpMto about lOuM. Ahemaiiveiyytbe inhibitor of GSK-3B enzyme activity is used at a concentration of about I0uM,

Compounds s«ifohie for use in the methods of the 'jpi^sent layenilha

In orsc embodiment , the inhibitor of GSKto S enzyme activi ty is a compound' of the Formula (I):

Formula (1) whercur.

Ri is phenyl, substituted phenyl wherein the phenyl substituents are selected brim the group consisting of Cusilliyl:, halogen, nitro, trifinoromethyl and nitrile, or pyrimidinyl;

Ms: is phenyl, substituted phenyl wherein the phenyl substituents are selected from the group consisting ofCojaltCyl, halogen, nitre, triiluorometftyi and nitrile, or pyrimidinyl which is Optionally T>^4albyl Substituted, and. at least oneof Mi and to is pyriotidinyi; to is hydrogen,: 2~(trimethyIsilyl)ethoxynieihyL Cs^aikoxyearbonyi, aryleayearbonyi, arylGirsalkyloxyCarbonyl, arylCi.saikyl, substituted. arylCfosMkyl wherein,the Ope or more ar^I: subsiltiteiiti» ^re indep^ndfisotly selected from the group; consisting .of'Cfrvsal'kyf .'.C{„ssilko^ halogen, amioo, Cosalkyiamioo. and di€ ^salkytainiiKg phihaliitiiddCr^alky!, :ar£imoCtoa&amp;yl. diamiftoCr^aikyh suocimmidoCfuoSdkyk tn-sSlkylcarbonyl, arylvarbonyl, di,5alkyfearhonyiCi.:safkyi and aryloxycatimnyiCumikyi; A is /vinyiene, ethyuyiene m

R$. the group consistiog of hydrogeu, Chatted, phenyl and pheBylCi,.sai;%l; q is 0-9; X is selected from the group consisting of hydrogen, hydroxy*: vinyl, substituted viny l wherein one or more vinyl substituents are each selected .feat, the group consisting of fluaHue, bromine, chlorine and Iodine, ethyrry! f sMf>Stituted ethyny! wherein the ethynyl substituents are selected fern the group consisting Of fluorine, bromine chlorine and iodine,: Cusalkyli substituted^ or more alkyl substituents arc each group consisting of Ci g5ajfc0xyt. tribaloalkyh phthaifeidb^M'-totUo, Csecyeloallyi, €i..5:alkosy, substitutedtOri^toyi^berdm· tile alkyl substituents are selected from the group consisiingoi phtiutiimido and amino* phthalintiddoky, phenoxy, Substituted phenoxy wherein the one or more phenyl substituents are eaeh sglected from the group consisting of ^s^aJkyk halogen .and C^alkoky, phenyl, substituted phenyl wherein, the one or more phenyl substituents are each, selected from the group: consisting of Ci^aikyl,: halogen and Cfr aikoxy, aryiCipalkyi, substituted arylOusalkyl wherein the one or more aryl subsuiuenw arepacb selected from the group consisting of Cesalkyi, halogen aud€tm8ikoxy,ns'yloxy€5.salkylainiito, G{mdkylami«0, difejalfcyianuno, nitrile, oxime, bcnxyloxyimmo, <1 Ksnlkyl0xyirm.no, phthallmido, suceimmido, Ciieaikyiearbonyloxy, phenyidarbohyloxy, substituted phenyicarboOyioxy wherein the one or more phenyl substituents arc each selected from the group consisting of Cmalkyl, halogen andCuydkoxy, phenylCusalkylcarlxmyloxy wherein the oneor more-phenyl siibstimeMs are each selected from tile group consistiug of Cs.solkyls. hatogen and Ciyaikoxy, ambmearhonyloxy, Cgsalkylafl^nc^arbORyl^y, diCgmlkyfeownoearbanyioxy, Cutelkoxyeaibonyloxy, substituted Cusalknxyesfbonyloxy Wherein; the one or more alkyl the group consiidihg: of methyl, ethyl, isopropyl and hexyl, phenoxyeatbonyldxy, substituted phenoxyearbonyloxy wherein the end- brMobtfchb^ each: selected, from

tM group eonxisfing of Cesulkyl, Ci.galkqxy add halogen, Ch-salkyltfrio, substituted Ci-salkylthio wherein the alkyl substituents ^selected from the. gmup consisting of hydroxy and phihalimido. Gi^alfeylsnllbuyl, pheoylsultbuyi:subsliluted plteuyisu!.fdpyl wherein tbe one or more phenyl substituents are each selected front the group consisting Of bromine, fluorine, chloride,; Cusalkoxy and trifluoromethyi; with the proviso that if A is

q is 0 and X is H, then R= niay hot be 2- (nirncifryLxilyi )cthoxymcthy 1; and pharmaceutically acceptable salts thereof

An example of the is substituted phenyl ami Rv is pyrimidm-3-yl.

Aw exao^le of the iav«tio» inclndes a conupoond of Formula (I) wheroinRj is 4~flnOfophenyl

An example of the invention ineludes a compound of Formula (I) wherein Ey is hydrogtet, arylGosaikyk or Mibstituted ary!Ci.,salkyf

An example o f the invention includes a com pound of Formula (I) wherein ,R3 is hyd rogen or phenyiC:,ndkyl

An example o f the invention includes a compound of Form ula (1) wherein A is ethynyiene and q is 0-5.

An example of the invention includes a compound of Formula (1) wherein X is sucdnimido, hydroxy, methyl, phenyl, €·. salkylsuifowyk Cfr scycloalkyi,

Cusafkyfcarbtmyloxy, Cj.saikexy, phenylcarbonyioxy, C^alkykmmo, diCgsalkykmmo or nitri le.

Compounds of Formula .(1) ar© disclosed in commonly assigned United States Patent Number 63214,830, the: complete disclosure of which is herein incorporated by reference.

An example of the Invention includes a compound of Formula (I) wherein the compound is selected from the group consisting eft

Compound Napte 1 §(4)<(4-fl«oi»piiei^iH(3H4-pyri5y'i}iiftida^ies 2 ddd-fluorophcnyl)»· i 43ithenylprppyl)fe44^yTidyl)imldazale, 3 5"(4~fluorophenyl)- i A3“phenylpropyl)NA4-pw'idyi)imida2ole:. 4 4'-f4-f1.'uO'roph'e»yI)-2~lodQ"i-{3-;ph!e»ylj>«5pyj)"iS-{4-pyridyi)itaid'^soIes 5 4-(4-fkoropitenyl)~2d|44iydtO>;yimtyn-flyylVl~(3”phenyitiropyl)~5~{4~ pyridyilimidaeoie, 6 4-(4~tluoroph.enyl:}~544~pyridyl)~!~[2~(tri:me:tby!silyl)eth0xymethyl]·' imidazole, 7 S~(4~finorophenylN4~(4'^yridyl)~I-[2-(trimethyisil.yiiethoxyrnethyi]·' imidazole, 8 5<4»fl«oropheny l)“2~iodo^ld4-pyridyi)~ l«[2> (trimethyisilyl)etltoxymethyl]“imldazole, 9 5^'4“fluoro|)hettyl)-4“(^“py^dy^)“2*(M«i.eth'ykilyi)ethiayi-l-P'· (trimetliylsilyl)eihoxyt»etltyl]"imidazole, 10 2“(2"ChloroviByl)"5“(4»fluorophenyl)"4~(4»pyridyi)»imidazoles

Compound Name ΐ I 3-{4-Saoroptayi)-4< 1 p> r i dy 1)-- i 4.2«(trimethyisilyi lethox vrn cih y! j - imi daxoJe-2-carbox a! dehyde, 12 242,2~dibromoeihy!eae".! -yIj-5~(4~floo.rophenY!)N~(4~pyridy!)~ 1 -[2- (trimeibyNily!)etliosyajetliy!]-imidaz0!e~2“CarboxaIdehycl:e, :13 5(4:)-(4-fla0TOpliesy i)-2<3~hydioxy-3~pbe«yl~propyi:i-:1 ~y!}-4(5 )-(4-. pyridyi)iiTOdazob, 14 544~flaoropbeByI)N44-pyridyl)-'NP»(trimethyIsilyl}ethoxymetfeyIj-2'· oximinoimidazole, 15 5-(4~.H'tioroplie%' !)-4-(4~pyddyl)~2~lmidaz0l£ oxime, 16 2-(5-chiOTOpeniyn~ X ~y i}~4-(4-fIuoropheny!)~ 1 -(3 -phenyipi^yl)~5~(4-pyrtdyl)iraida2ole, 17 4-{4-flu0rophejiyl>“2-(4-N-pheftyicarbamoyloxyb«tya-I -yl) 1 -(3-pbeny1propyl}-5-(4~pyridyl)iBiidaeoles 17 2-{4-eMorobisty:o-1 -yl>4-(4~flu0ioplienyi> J-(3.-phenytpropyl>5-(4~ pyridyDuYndazolC; and IS 2~(4-diffietf^!aniioobutyTi-.l-yl)-4~(4-f]uorophefiyl)-l~(3~pheny!propyi}- 5 44~pyridyi)imidazofc,

An example of the invention includes a compound of Formula (I) -wherein the compound b Compound 5 of the formula:

Compound 5.

In one embodiment, the inhibitor of GSK--3B enzyme activity Is a compound of the Formula (II):

Formula .(0)

Wherein.; R is .selected from the group consisting of R«, -Ci.a#lkyi-R«, <Wdkenyi-R8s, -€^aikynyl~R;i and cyan»; IG is selected the group consisting of CYCloalkyl, hetemcyclyb·aryl and hetaroaryt; R! is selected from the group consisting of hydmgen. HCi-§aikyI-R\ ~C"2ike0y!-1¾" ¥ -C^I1^yriyi-R5,-'-C(0)-(CM)aikyi-R9s -C(0}^iyl-RN, C{0)^0-(G5.s)aikyhR5; -CfOVO-aryi-R8, ^0>NH(Cj^1kyl~R\ ~C(0)~Ne(aryl-Rs)s -CiOmC^alkvi-R^, -heteroeyciyldC, -aryi-R6 and -heieroaryi-R0; wherein heterocydyl and heteroaryl are attached to the azamdole nitroge» atom, in the one position via a heterocyelyl or heteroaryl ring carbon atom;

Rs i s 1 to 2 substituen ts independently selected from the group consisting of hydrogen, -Ο-(0>_s)a'Ikyis <)-(C^)alkyl·Oe^O^Cw)alkyl·ςKC^t)alkyl> -0-fC:.v)alk>!-NH;. <)~(C:r.s)al.kyRNItfCi-salkyi), -0-(Cy>aIkyl-NiX;;.*aIky().-, -fHC^jalkyl~S-(C:s..s)a!fey 1, -04C\*)alfcyi-$0>-(Cjulkyi, -0-(0^)alkv I -SOrNH2, -0-(C:..daikyl-S0rNH({;; ,dky!}. -0~iCrdalkyI-$OrNf€u,a!ky:p, -0-0(0)1 h ~0-C(0HCut)aikyl? -G-€(0)-NI-!2, -0-,C(0)-NB(C^aikyi}5 -0-C<0)-N(C^aIkyi)2, ~0-(C 1 -s}aIky |~€(0)B, ~0-(Ct.g}a.Ikyl~C(0)~(Ci...s)aikyi, -0~(Cr^)ai.kyl-C02H, -0-(Cj..2)al.ky ί-€(0)-0-(€ j..da!ky!. -0-(CuXlaiky i-C({>)-NH;>, -0“(C^fs)alkyl“C(0)“dH:(C^.¾aLkyi), -0-(€r..8)aLkyl-CX0)-N(Cv;gilkyi)2, "C(0)H. -qOMCodalikyl -CO.H, -C(O)-0-i€na}alkyl ~C(OVMI-0, -Ο NH >-NH2, - Ct 0)- N H(C usa iky 1), -€(0}-N(C u*a I iky 1.)2, -SH, -S-iCi ,}<dkyi, -S-(Ct .:js)al'ky l-S~(C }.:S)aiky k -S~(C1.g}al.ky!-0-(Cr.da).ky), -S -(Ci ...s)al:kyI-0»(C 1 .s)aikyl -OH, -S^C^siaikyi-O^Ci^kyi-NH^, -S-(Ci.s)alkyI-0-fC2s)alkyl-NHCCrealkyl), -S-CCi^dkyi^HCi^^lkyl-^CC^alkyl^. -S-(CKa)alkyl-Ne(Ci...saikyi}, -SCh-fC^ndkyl -SOa-NHs, -S02-NH{Cs.^alky|), -SO?-N(Gt.saikyik, -N-RX cyaao, (halo)s j, hydroxy, nitro, oxo, -cycloalkyl-Rv -heteroeyelyl-Rf -aryi-R^ and -heteroaryl-Ri5;

10' is 1. to 4 substituents attached to a carbon or nitrogen atom independently selected from the group consisting of'hydrogen, -Cj^alkyi, -C.?,salkenyl5 -Cs-satkynyk -G(0)H, -t:XO)-(Cus)aikyL -CO-H, -C(0>04Cu8)aikyl, ~e(0)~NB2, -CX Ni l )-NH2, -CCOVNBCCusalkyl), -C(.0)-NtCj.<;)8ikyi)2s -SOr(Cps)aikyi ~S02~NH2, -SOa-NWC,.3alkyl), -S02-N<CVsaiky%, -(C^alkyl-M-RX ~Ct;\.N}aikyRihafo)re, -(Cf^)aikyROH, -aryl-RX -(Ci.s)aIkyl-aryRR* and -(Ci.<i)alkyl"heteroaryhR^; with the proviso that, when R6, is attached to a carbon atom, R* is further selected from the group consisting of -CVsalkoxy, -(Cn^aikoxy-Cfcato)!-^ -SB, ~S~(€l^s)alkyL ~N~R', eyano, halo, hydroxy, ftitro* oxo and dieteroaryl-RlY R' is 2 substituents independently selected 1¾¾¾ the group consisting of hydrogen, «Cj-.salkyl»~fb-.i«')ikenyls -G^alkyayi, -(Cj^jalkyi-OH. -(Ci,s)alk:yl~0hCh4)alkyI, -(C).paskyi-Nil.. -(Cj^ky 1-NH(Cvsalkyl), IC^Mkyi-NfCi.«alky 1}2, -(C,^aikyi^-(C,^)aikyit C(0)B, -C (OHC: .ydkyL ip^(Ct^)aiky), -C(Q)-NH2j ~€(0)"-ΝΙ;Ι(€s..sulkyf), C(0)~N(C)..8aikyi)1, -SOHC^ikyi -SCN-NB,, -SO(€.t.salky 1), -SOe-NK'i uukylR ·0{Ν}··ΝΗ;. -cydoalkyl-R3, -(Ci.8)alkyl-'heterocycly!'-R:>, -aryl-RX -(Gi.s)alkyl~aryi~Rs and RCh .s)alkyi4ieteroaryl~R.y R* Is 1 to 4 substituents attached to a carbon ot nitrogen ..atom independently selected from the group consisting of hydrogen, vCsyalkyl, -.(Cj^aikyl-Chalojio. and -(C{^)alkyi~OH; with the proviso that,-whim R* is attached to a carbon atom, Rvis further selected from the group consisting of -tlsalkoxy, -NHj, ".NfiCC^alky!),· eyano, halo» R€i,Xta!koxyRhal0)1..3; hydroxy and. niiro; E;>' is I to 2 substituents independently selected from the group consisting of hydrogen, -Cj-saikoxy, -MR, -NH(C;.$afkyl}, -NfCj^alkylij, eyano, (halo)ro, hydroxy and niiro; R~ Is one substituent attached to a carbon, or nitrogen, atom selected from the group consisting of hydrogen, -CrodkybRy -C^alkcnybR .y -C^toibynylR\ ~C(0)Hs ~C(OHCh..s)ai:kyb:Rs5 ~G(DbNH2! ^Q^fi^aikyl-R9), ~C(G>Ni C; .„aIkyER\ R:(OhNH(arybR\ -CfO)-cyeioai:kyhRs, -C(0)--hcterocyelyhRs, C(0}-aryl-R3, -CCO)-heteroaryl-Rs, -0.11!. -0(05-0-(0: οΙΚυΙ-ΙΓ, ~C(0}~G-aryi~Rs5 -SOrCCosialkyl-R^, 4SOraVyl-R'\ -eyeloaikyl-RX -aryi-R0 and ~(Cs»s}a1kyl-N-R?; with .‘y . the proviso that, when. R" is attached, to a carbon atom, R“ is further selected from the group consisting of -Ci.aalko'.y-R*, ~N-R;, eyano, halogen, hydroxy, nitro, oxo, -heteroeycl.yl-R6 and -hetcroaryi-R*; IV is 1 to 3 substituents attached to a carbon atom independemiy selected from the group consisting of hydrogen, 4V.gdkyt~R!!J, -(h.galfcenyl-R:*3, -Cj^alkynyl-Rkt, -Cusalkoxy-R^, ~C(0)B, -'€(0)->(C(^alkyl-E3’, -C(0)-NH?., ~C(0 }--N ΗiCj^alkyl-R^), -C(Q)-N(€^aikyI^2, • yCOdd* ^{(^^O^Gi^alkyl-R;', “CiCGj-O-aryl·-^, -SOriGi^ky^i^ ^Q^-aryleR*»· “bhR'-, cy&amp;oo, halogen, hydroxy, nhro, -cycloaikyi-E'',, AeterocyclyRRy -aryl-R* and dteteroatyi-R'·; R4 is .1 to 4 substituents attphedtda carbon mm itid^eMejitly seiected from the group ^6®ϊ^ί^4ί«|::θ#^χ^ίο^ί,'Λ€ϊ4^3^·“^,ν» ~C(C»H, -C(Di»(e}.«)aIky RRr -€(0)-», -G(0|-NHfC 1^¾ ♦C7OVN(C}a‘aikyl-R?)2S;^<0)-cy^a&amp;yl»R^:^CO)*Mei^yielyi-Rxi,“C{0)HaJ3?I«'R!i» ’•CCOl-h.^to'ajyi^^, -CfFiBENBa*. -COsH» , ~G((i)~CRaryl~R , -Sit ·8··(€5.χ)3ϋ<νΙ·Ε;'\ SOr{Ci,,)aikyi-R'>. -SOr-aryi-iG, -SOr-Nit, -SOrN0(Ci.#lkyhR9),^^ -N~R/, eyano, halogen, hydroxy, nitro, -eyeloafkyl-E*, -beierocyciy 1 ~Rt -aiyRR4 and -heieroaryh-R4'; RKi.is 1 to 2.·substituents rodependentiy selected from tho group consisting of hydrogen, -biBfGnsalfcyf), rK(€,^ai%i^* cyano, (halo'te, hydroxy, oir.ro and oxo; and, t and t are independently selected from the poop consisting of 0, S, (ϊ^ΟΗ) aod f H,H); with, the proviso that one of Ϋ and Z is O and the sthor is selected from the group consisting of 0S S3 (!d30H) and (H JI >; and pharmaceutically acceptable salts thereof

Embodim en ts of the present in ventton iaelnde eompoiinds of Formula (11) wherein, R is selected tent the group consisting ofRe^-Gi^aitei-R;,, ^^afkenyERs, -G^dkynyl-Ra and cyano.

Embodiments of the present invention include compounds of Formula (H) wherein, R» is group consisting of hetemGyclyk aryl and hereroaryl.

In one embodiment, Rs is selected from the group consisting of dihydro-pyranyi, pheny l, naphthyl, thienyl, pyk^:iyl^.i^ida^iyli.^^ffis6;^is:ipi^rldiayi¥ aaaindelyi, ipdaroiyl, bcnxoinryk benzothienyl, dibenzofuryl and dibenrothie?ryl

Embodiments of the present invention include compounds of Formula (ii) wherein, R! is selected from the group consisting of hydrogen, -Ci^alkyidf5, -Q,4alkenyRR·, ~C-2_4&amp;ikyny1.-R'\ -C(0)-(Ci-4)a&amp;y!~R^ -C(0)-aryl-R^ ~C{0)-0-(-Ci4>a.lkyl-R9, -C(0)-O-aryUR*s -C(i>) NHiC :..,u!ky 1 · R'-G(0)-NH(8ryI~Rs)3-C<0)-N{Ct4aik5?i-R% -SOHC^n^alkyl-R*, *pyetoalkyl“R*y dieterocvclyl-Ry -aryl-R® and. dieteroaryi-Rs wherein, heterocyelyl and heteroatyi. are attached to the axaindole nitrogen atom, in the one position via a heierocyolyl or heteroatyi ring carbon atom.

In one· embodiment, B.:! is selected from the group consisting of hydrogen, HCj^sikyl-Ry -aryi-R” and dreteroaryi-Kf; wherein heteroaryi is attached to the azaiudole nitrogen atom inthe one position via a heteroaryi ring carbon atom.

In one embodiment, Rs is'selected from the group consisting of hydrogen, -Cj^aikyRR^ and. ~naph thyl-R'·’.

Embodiments of the present invention include compounds of Formula (II) wherein, JS? is I to 2 substituents independently selected from the group consisting of hydrogen, ~ CMC > 4)a! ky!, ~0~(€ i Mai hyl -OH, ~0~(€ s .uialky! ~G-(C i Ma Iky I, -0~(€?.4}aiky!MiM ~0-(Ci.4)alkyl-NH{C{..4allcyl), -<MCi.4)aUiyl-N{C| .4« Iky 1.)2, frMCtMalkyiMRCMiaikyi, "0“(C?-4.hdkyi”S0?,-siCr4)alhyh -0-(.0 .M^lkyi-SOsfNB;;, fr)~(C:r4)al.kyRS02-Ni:i:(Cj.4allfy!}, -<MCMMxyl-SO;rN(<'Ma!ky!h. -C)~C(O)H, ··O· C(())· i C ·..fla 1 kyI, -0-C(0>N%, ••O-f.YOV-NΗ{€η#Μ> -0-€{O)~N(C.Ma!ky%, frMQMa!kyU:\0)H:! -0-(!CM>al{syi-CiPHCi^alkyl, -0-<Cw)a!kyIXt>2Bs -0-(Cj4:)aikyhC(0}^0-'CCj.4)aikyis~0-{Ci4)alkyK'{0}"TsiH29 ~0-(C 1.4)aiky l~€(0)~N H(Cj^aikyi}, -<KCi4)alkyM^O)-N(CM.aIkyi>ri-C(0)H, -€(0)-(€54}alkyl5 -GCfeH, ~C(0)-0,(€H)a!kyi, -0(0)-^¾ ^€(ΝΟ>ΝΒ2,. -C(0)"NIi(Cj,4alkyt), -OObMC 5 MkyiK -SI1 -S-(C, ,)a)ky). -S-f'C^E...r)alkyi-S--(C^..,riiilkyh.-S~(Ci^,r)alkyi-·D“(C.^....dmlkyi, ~S~(C4 -4;}aiky! -0-(0,Malkyl-OH, -S<Cn4)aikyI-0<C}.4)alkyi-NH2, -S-CCw)alkyr-0-(Cii4>aa£yb:NH(CMalkyl), -S-CC,4)alky]-CKCi4¥kyl“N(C,4alkyl)2, -S-(Ci4)alkyI"NH(Ci4aIkyl),~S02-{Ci.4.)alkyl, -S02~N%,-S02-NH(CMalkyf), *S02~N(Cj.4alkyl}2, -N-Ry cyano, (halo) {..3., hydroxy, nitre, oxo, -eycloalkyl-R6, “heteroeyclyl-R4’, -aryl-R^ and. -hetcroaryl-R0, fe :#%· t to 2: substituents independently selected front .the group consisting of »N~R7, hydimy and -hetemaryl-R*

In one embodiment, R^is 1 to 2 substituents Independently selected from the group “N~R:, hydroxy* -imidazoiyi-R*, -triazolyl-R" and

Embodi ments of the present indention· include compounds of Formula (II) wherein , R* is 1 to 4 substituents attached to a carbon or nitrogen atom independents selected from the group consisting of hydrogen* -OMafkyl, 4ib,<jalkonyL -CboniRynyi, -0(O)1:L X®HCi4^1kyI, -COdf 0(0)-0-(0 M.l&amp;lkyl, -CCOfNFk,-C(NH)-N% -sorNH,, -S0j--Nif(Cn4aikyi), -S03~N(e*#lkyih, ^Cw)alM'N^^^)a}ky!.-(lial0)Wi -(Cu4)aibyi~0H, -aryl-Ri5, -(Cj^ialJcybarybR^ sod with the proviso that, when R* is attached to a carbon atom,R* is further selected from the group consisting of -C^aBcoxy, -SH, -S4€w)alkyi· -N-R7, oyuno, halo, by droxy, inifro,, 6ix&amp; and -heteroaryl-R*.

In one embodiment, R ' is hydrogen..

Embodiments Of the present in vention melode compounds of Formula (II) wherein, R. Is 2 substifnemsi independentiy selected from the group consisting of hydmgen, -Chalky!, df2,4a;Sbsbyi, -C^kl^y^yi -(€|μ)&amp;Ι% l-Ofl -(Ci.^alkyl-O-fCri^ialkyl, ~(0],4)ulkyi-NI-i2, ^<C;e,4)iaifeyI.^:e:(Cr^alkyi'^ -(€ ^)alkyi-S-(€s .to&amp;ikyl ~CK>)H, -C(0)~(0j:.0¾Ikyl, - C(0)-0^;0*,4:}alkyI, -C(0)-H(0:o4SSkyC)2, -SO^COiOaikyl -Sa2~Mfr, -S&amp;rNH<CMafky&amp; -SOrbi(0i.4^kyl:b, -CiN)-Nfl2, -cycloalkyRRfts -(CuOalkyi-heterocyeiyRR 4 -aryfriO,: -(0 H)a Iky 1-ary i ~R: and -«b^lalkyi -hetemary i-JO,

In one embodiment R7 is 2 substituents independently selected from the group: consisting of of hydrogen, S0m, -CfOl-CCi^lalkyl, -0(0)-0-(0. M)alkyl5.-SO-NH,, -S0aM(Ci,4alk>-i) and rSOrNCC^aikyi),.

Embodiments of the present invention include eomponnds of Formula (ii) wherein, R* is I to 4 snhstituents attached to.a carbon or nitipgen atom independently selected horn the group consisting ,of hydrogen, .^Μ^%1,^0ί:4)«ϊ%ΚΜο)ο3 and dCo^aikyl-Oi·!; with the proviso that, when R® is attached to a carbon atom, R* k. further selected from. the group consisting of aifcoxy, -Nl-R, ~NH(€lui&amp;lky 1),..-N(€i.4kSkylE,eyano,;halo, ^Ci4JaI^oxy^|tato^..3v%dmxy and nitro. in one embodiment, R" Is hydrogen.

Embodiments of the present invention, include compounds of Fort»»lai(H) wherein,: Rs is I to S sohstituents mdependently selected, from the group consisting of hydrogen, -C;;oa!hoxy, ~bl|:i2, -MidRRatdkyl}, "N{C},4a&amp;yl)2:, eyano:, (fk.{p)ms hydroxy and nitro.

In one embodiment, R'5 is hydrogen,

Enibodimcirtif of the present invention inelnde compounds of Itomtula (II) wherein, Rzk one substituent attached to a carbon or nitmgen atom selected from the group consisting of hydrogen, ~€t,.#ikyi-R', -Cb^alkenyoR^, '•Ca^alkynyl-R5, -€(0)1:1, -(XOMCVRdkybR9, -C(0}-NH5, -e(Q}-NB(€n#ikyl-R^ -Gpi-ISiifli.mikyl-R^, -€{0>heior^^oiyi-<R%;:uCfO)^8s^tR\· -€(<>)-hMemaryl-R*, -CCER -€{0)-0~iCi ^aihyl-R^-eCOEP-aryi-R55, -SOr•cSOraryER8, ~ey chalky RR*, -aryRRf and «φstalky!-N-RR with * * .* *>k * ·>-· the pmviso thag when RA is attached to a carbon atom, R* is\fdtttier'^lectMfl»m'ihd' group consisting of-Cv^alkoxy-R*, -N-R?, eyano, halogen, hydroxy, niro, oxo, -heterocyclyi-R6 and -hefetoa:ryi~R‘\

In one embodiment, Rf is one substituent attached to a. cati5d^:Op.iittOgdp::aiOib,Mec(ld from the group consisting of hydrogen,:-Ci^alkyl-R', “Ci^aikenyl-R^^-Cs^alkynyl-#,: -COdrh -C(0)~0-(Cs4)fdky!~Rs, -cypibaikyl-R6; ~aryt~R* and -CCihalkyl-N-R; with the •s proviso that, when. Rhs attached: to a nitrogen atom, a qaateruium salt is not tonned; and, with the proviso that, when. Rzis; attached, to a carbon atom, E? is further selected from the group consisting'.#-C?s -ISER*, eyano, halogen, hsdeoxy, nitro, pxo, -heteroeyelyi-R6 and· dteteroarybiR1';

Ip one : embodiment, Rf is one sobstimeni attached to a carbon or nitrogen.·atom selected, from tire #0«# consisting of hydrogen, -Gn#alky and -aryhR *; with the: proviso t hat, when f£ is attached to a nitrogen: atom, a quatennirm salt is not ippned; and, with the proviso that when R': is attached to a carbon atom, R is Rather selected from the group consisting of-N~RY halogen, hydroxy and -heteroaryl-R6,

Embodiments of the presen t invention: include eomponnds: of Formula III) wherein, Rs is 1. to 3 sobatitoents attached to a e&amp;fboh atony tndependendy selected Ifoat the group consi sting of hydrogen, -Cn^aikoxy-R:1^ -0(0)0,

•-G(0bN(Ce4alRyi»R%, di^-cycidalkyl-R^, -C^'hPtetocyefyRR^p-CiOI-atyl-R^ -C(0)~heteioaryRR:^-€(^H)~NH&amp; CQsEt, -G(0)-0RCM)a]kyi~R* ~C(O)~0-atyi-Rs, -SOy-CGi.^alkyi-RY ~SGa-aryi*R\ -hi~R;, -fCMtalkyl-N-Ri evano, halogen, hydroxy., nitro, -cycIoalkybRY -heteroeycM-RY ~aryl~E* and -heteroaryl-RY

In one embodiment, .Rf is one substituent attached to a carbon atom selected from, the group consisting: of hydrogen, .,-C.j,4alkyl-RH!, -CY^alkenyl-R^, '-Co.^aikynyl-R.^, "CKaalfcoxyd^V^ClOiEk -COyK, -NBCCh^alkyl), -NfCUaifeyl)*, cyano, halogen, hydroxy and nitre.

In one embodiment, R* is one xobsti tuent attached to a carbon atom selected from the gronp consisting of hydrogen, -G,u4alkyRR/iY -MBs, -NH(Gnua!kyl), dd(C j,4&amp;!kyi}2, halogen and hydroxy .

Embodiments of the present invention indnde: compounds of Formula (li) wherein, R."5 is I to 4 substituents attached to a carbon atom iTidependehtly selected front the gronp consisting of hyd rogen, -G^aiky i-R'Y -Ca^sSkenyl-R’Y -Gs^aikynyt-E1^ ~C) ,;alkaxy-RK>, RSpi-CCj^kikyl-R9, -C(0)-NH3, -CiORbiHiCMalkyl-R9},

~Cf0)~N(CK4a&amp;yl4R%v ~€CO)~helerocyclyl~RY <l{0)-aryi4RY -Gp;Rheroroary!»R^~£pHENH2, -COA -C(0)-0~(C stalky l-A -GfOVO-aryt-R, -Sid, -SQ-iG^lalkyl-R^ -$0raryrf, -SOrN% ~SCI|“HH(C:i.^RyRR^.^02^Oj^aIkyl~R52> -N~R\ eyano, halogen, hydroxy, nitro, royeloalkyl-RY and -hetetoatylA.

In 4 substituents attached to a carbon atom independently selected fern the gmop consisting of hydrogen, »€ ^aIkyWl5 % -C’^alkenyldl e, ~C;>.,jalkynyl-Rii!, -€= ,aikoxy~RK!, -€(0)B, -Me(Cyaifel^ -N'(C, ,a)ky)},, cyan®, Miogen, hydroxy, «itr®, -eycioalkyi, -hetcrocyelyh -aryl and -hcteroaryl

In one embodiment, R ! is I to 4 substituents attached to a carbon atom independently selected from the group conatsbug of hydrogen, CMa!kyf~R.ivk C^alkoxy-E.^, -NB2s, -NH((’s .ubkyi), -Ν(Οι .«alkylfe, halogen and hydroxy.

In one embodiment, R* is 1 to 4 substituents attached to a carbon atom independently selected from the gronp consisting of hydmgeh, Ch^alkoxy-E’^, -¾¾ -NliCCi ^alkyl), -N« i faikyi}>, chlorine, fluorine and hydroxy.

Embodiments of the pr«^eittinyentioftdn.eiadc:oompda&amp;'df Formula (II) wherein, R.fe Is I to 2 sobstitiiiits independently selectedfrom the group eonslsting of hydmgen, -Nflr, -NI:I(Ct4alkyi^ -N(C: jaikylh, cyan®, <hak>)s λ» hydroxy, oitro and oxo.

Iri One embodiment, Rki is 1 to 2 kuhsirtoents independently selected from the group consisting of hydrogen and (bakth/e

In one embodiment, Rf<: is 1. to 2 substituents independently selected from the group consisting of hydrogen and (floerois.

Embodiments Of the present invention include compounds of Formula (II) wherein., Y and Z are independently seieded horn the group consisting of 0, S, (FI,OB) and (0,1¾ with, the proviso that one of ¥ and Z is 0 and the other is selected .from the group consisting of Qy S, (Ej,OH) and (B,B).

In one embodiment, ¥ and Z.&amp;m. indepmiteitiy-aeleeted. ft^m'lhfc'group conaisting of 0 and U1B}· with the proviso that one of ¥ and Z is 0, and the other is selected hoot the group consisting of 0 and (FkH).

In one :emhodimen t, Y and: Z are Independently selected.· from 0,

Compounds of Formula. (II) are disclosed in commonly assigned. United States Patent Nuniber 7,125,878, the complete disclosure of'which is herein incorporated by reference.

An example of the invention includes a compound of 'Formula (If) wherein the compound is selected from the group consisting, of;

Compound. Name I. 3-(:2~eld.oraplmnyl}4dldii~bydroxypm|yyl}U.^~|>ynxsIo(2,3~b)p)i'idt.n--3~ yl j-1 i:FpyiToie»2,5~dio«e, 2 3»(2-cMorophenyl)"4“[ 1 -P-<dimcthylamitto)propyl]- if/~pymdo[2s3·" ^]ρνΓΐ4Ιηο»3~γ1]~Ι27~ρ2ίΤ01ε~2,5~4.Ιοηθ, 3 3~[I-(3-:hydroxypropyl)~l.i7~pyrrolo[2:,3~0]pyridin~3-ylj-4-'(l~ « aphthateny 1)- iH~pym>le~2 ,5-dione, 4 3 -{Ϊ -[3*(dimethy3amtno)propyl]~ lffrpyrrolo[2,3~b]pyridin-3-yl]-4~( I -uaphtlialcnyl)-1 l:frpyTxo!e-25S-dioue, 5 3.>{5->chlomhen^o[^|thim>-3-yl}-4“P~P»hydro*ypix>pyI)-1.//~pyTtQloP,3'-i 0jpyridi.fte:-3-)d.j”Lf?T-pym>le--2,.5--diofte, 6 3-[l -(3 -hydroxy propy l)-1 H-pyno io [2,3-0 Jpynd.m-3 ~yl ]-4~( I H~md axol-3-yl)- li-Fpyrmle-2,3-dione, 7 3-(I”eihyi-l/i”pyTOdo[2,3-0]pyrid,in-3”yf)-4“[i-(3f-h.ydroxypropyl)”f/·/-pynolo[2;3~0]pyridl:n-3-yl]-li/-pyrmIe“255-dlone, 8 3~[ 1 -(3-hydroxypropyI)~l //-pyrroio[2!3~0jpyridin-3-y !.]-4-{2~ mefhoxypfeenyl}» I /frpyn'ole-2,S-diono, 9 3»[! "(3-hy droxypmpy I)» I .fr-pyrrolop ,3 -Ojpyrid i.n-3-y 1]-4-(3 -methoxypheny 1)-li/-pyrrole-2,5~dione. 10 3~(2-chlorcf~4~lk$orophenyi}-4"[ 1 ~(3~hydroxypropyi>! //~pyrrok>[23-d]pyndi»e-3«yi}-1 #-pyrroie-2* $ -diomy 11 3-[l-{34tydfoxypiOpyr)".l.//-pyn<oio[2s3"5]pyridin~3*'yI]-4"[2- (inft«oroBietlwl)plie:Hyl]~i/l~pyrrole~2f5~dioB«s 12 3~[ i -(3"hydro*ypTOpy!)·' 1 //“pynOio:[2»3~i>]pyridiB“3-y !]-4"(2~pyridiayi}“ 1 //-pyn:ole-2 ,5 ~db«e, .13 3~[3~cliioro-5-(Pil2uomrB.eihyI}~2~pyridi:nyrj~4~[.l-(3~h.ydroxypropyl)-I./i~ pynBio[2?3”<&amp;]py!idin--3-yl]-3i/--pyrn>ie--2,5~dione, 14 3'-[i“(3“dydxDxypn)py!}4//-'pyrre3o[23-6;]pyridi3i~3~yi]-4«(2”thieiiyl}·· 1 //-pyrro\e~2,5 -dioncx lilpyridine-S-yil-i^-pyiToi^jS-diOine» i 6 3-[ 1 ~(3~by droxypropy!}-1 i?-pyra2ol~3 - v S] -4-( 1 -(3 -hydro xypropy f)-1 H~ pyOTf o{23~5]pyridin~3~yI]-1 ii^py rfQle-2, S-dionev i? 3«[l-C3-hydiroxypfropyl )3i^-pytroio[2s3-^]pyri4m~3^yl}<4“( 1 tf-imidazol·- 2~y!)~ l//-pyrrote~2,5-diotte, .18 3~[ I-(3-hydroxypmpyl}~l.i2-'imidaxo)-'4-ylj-4~[ i -(d'-liydroxypropyl)·' 1i?" pym>lo[23'^y^dk-3ryl^1i¥-pyiTOk-23'^o»'P» 19 3~[l“(2-%draxyeihyi)"i/i3mida2oM»yl]-4"[l-(3~hydroxyp!X>pyl)”l/^ pyTToio[23-^]pyridffl~3~y!j-'l/f~py.rrole~2J~dioBe:i 20 3-[ 1 ~[3~(diBieliiy!.amlB0)pi'opyi.]“i /2-mdaz»l-3~yI]~4~[ l -(2-aaphthaleay!)-1 /3“pyirt)lo[2s3 --Olpyridm-d -yl]»1 /f-pyrrob-S J-dione,, 21 3-dld3-hydmxypr0py!}”li/»mda2oi~3“yl]'-4»[i~(2“ni5phihaleayl)»l/3-pyrrolo [2,3~6]:pyri di π-3-yl ]- 1 l2-pym>Ie-2,5-dione, 22 3"[(£?}"2~(4~ili30fop1isnyl}eihenyi]~4~[l''(3-'hydfoxypropyf}~ll/~ p¥mjio[2,3-4]|>yridio -3-y IJ- 1 //-pyrrote-2,5 -dimo> 23 3~|3,4“di'hydro~2//~pyrao:-6--yl}~4-[ J -{3~hydroxyprofry))- I/f~pyrroto[2,3” ft]pyridine“3-yl]-llif“pyitole^2,SH!kme, 24 ^-[i-CSdiydro^ypTOpylidfH-pynOiop^-dlpyridiivS-ylJ-fSJ’^-l/i" •pyn'ob]-2»5-di0«e, 23 3424)e«zo&amp;taayi>4-[1^3diydmxypTOpyi)-"IS'pyf'K}l:0[2,3-'&amp;3pyrIdia~3~ yi]-!//-pyrrolS“2p~dio»es 26 3-[ 1 -(3-hydrDxy.pmpyi).-1 //-pyrmio[23"^]pyridii>3-'yl]-4“(.l -methyl-1H~ pym^l~3~y!)-I/2~pyrrol:e~2,5~dioiie:i 27 ^^-^^^^“[UCS-hydrDxypropy^ 1 //«pyiroloP^-^lpyrid^-S-yll^jS-' dioxo~12f”pym>Ie“3~carbomt5'de, 28 3-d:3)drizo[i&amp;^thiPn-4~yi-4~[ 1 -O-hydroxyptopyl)- i//-pyrmlo^S- l>]pyridine-3~yl f~ I/Z-py rroie-2 .5-dkme, 29 3»(4«-di6enzoferaiiyl)~4~[i-(3“hydrGxypn)py!)”l//~pym)io[23”d]pyndirs-3~yl]~ l//-pyrro.le~2,5-di0iie, 30 3~(2~hydTOxyph.e«y!}''4-[l~(3''meihoxypfopyi}-lff'pyi!r0to[2,3v'd]py^dm·' 3-yi] - |//-pyrmie~2»S>4i0Be, 31 3”C3,4-dimedioxypi}.onyi>4-fl “p-methoxypropy!}- i //-.pyrro)o[2,3-6]pyridine“3-y!,]-1 //-pytroie-2,5-diode, 32 3“(3,4-dihydroxypte«yl)“4“| 1 ~(34>ydroxypropyl)“J//~pym;4oi253'· &amp;]py rid i ne-3-y 1]~ 1 ^-pyrrok-2,5-dioim 33 3“(2»methoxyphenyI)'-4»[i~(2“HaplPhalenyl)-'l/i~pyrrolo[2J“d]pyn.din~3“ ylj- 1.//-pyTO>le-2s§.-dione, 34 [3~[3~[2s5~dibydro^-”(2~metimypheiwl)~23~^ioxo-llf--pyn-of~3~yl]--lif~ pyra^o{23-£]pyridtP* i -yl.]propyl]-earbamic acid 2-snethylpropyl eaten 35 3~[1-(3-amiftCipropyl)”!./3~pyo'o!o[23”ii]pyi'^ift~3"yi]“4'-(2“ methoxyphe«yI)“1.//“pyn:ole*2»5^toBe> 36 A^p-[3"[2!5"di!iydro~4~f2~mcthoxyphenyl)-2s5~dioxo-113-pyrF0l--3~y1j~ ]JY-p}^Tolop,3~^]pyridm.-l -yljpmpylj-acetamide, 37 A^|3-[3-[2J5-dihydTO~4-i2-metho^yphetty!:)-'2s5~dioso~l&amp;-pyriol"'3~yl]~ l/ApyFrolo[23-b]pyridm-1 -vljpropyll-sei&amp;mide, 38 332-fnethoxypheiiyl)-4-[ I -p-ii/AtetrazoF 1 -yi)propyl]-l/3-pyrrota[2,3·' 6]pyridiiie~3-yl|- lH~py rrole-2,5 -dione, 39 3 -(2-medioxypheoy!)~4~[ 1 -p-C2/:/-tetraxo!”2-y!)propyi] -1 i;/~pyrrolo [2,3-AJpyridme-S-yil-lii-pye'oie^jS-dione, 40 3-[]~(3~dydroxy-propyB-H3”pyrrob[2,3“b]py«dro--3~yl]~4”py:tazir!~2--yl-pyrnsfe-d^-dione, 41 3-(2.4xlbietiK>xy“pyriQiidiii”5“yl3“4"[l--(34iydrcvxy-propyi)--lH-pyrrolo[23~b]pytidi.a-3-yij-pyrrole~2,S-di0ec, 42 4~{3~[4-(2,4-diffseih0xy-pyriiiiddm~5~yf}~2,5~dioxo-2,5~dibydro-.iI-i-pym>i”3-yl]“pynOl0p3”i>]pyi'ldin·- i -yl). -butymnitrile, 43 4- p-[4-( I-methyl--1 H-pyr^o!-3-yl)-2i5*<iiP5(o>2s5-diliyidro-l H-pyrroi-3-yl]-pyr«ib[2;,3“bjpyridi«-l-y!}-butyronitrile, aid 44 3-(2e4-dinieth0xy~pyrimidia~5~yi)-4“{.!-pheiieihyi-.l B~pym)lo[2,3“ blpyrklifK^-S-yil-pyrroic-d^S-dioae,

Att example of the invention includes a compound' of Formula (II) wherein the compound is selected from the group consisting of:

Compound 11 Compound 26 Compound 40

Compound 41 Compound 42 Compound 43

Compound 44 la one embodiment, the inhibitor of OSK-3B enzyme activity is a compound of the Formula (HI):

Formula (III) wherein A andE are independently selected from the group consisting ofrhydrogeo subsdtuted tearboin atom and a nitrogen atom; wherein

Is independemjy selected 'from the group eOMMirig of l/Mndole, /. ipyriditse and 1 /Aindazoie; Z Is selected from 0; aiierHMiyely, Z Is seleemd fom dihydm; wherem each hydrogen atom Is attached fry a smgle frOfrd; R.;. and R$ are independently selected from CY^alkyl, C;> galkcnyl and (frsaikyoyi optionally substituted with oxo; feis selected from the group consisting of-Gesulkyl··, A^alkenyfr, AIAsalkynyk dHCrsialkyMA, (wfrereiu any ofr tfre foregoing alkyl, alkenyl and alkyoyl linking groups are straigfrt carbon chains optionally sobstdirted with onuto lour substituents independently selected: from the group consisting of Cosalkyl, Cj^alkoxy, €.ualboxy(€m)alkyl, carboxyl, earboxyi(Cs^)aikyi -C^saikyi-C(0)0”(C:i.^)aikyh amino (substituted with a substituent indupemtoily selected from the group, consisting of hydrogen and •ChalkyI), aminO(CM)alkyi (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen ami CMalfcyl), halogen, (hab)io(Cs-sMkyi5 (hafofofrCubaikoxy, hydroxy, hydroxy(Cux)aikyl and oxo; and, wherein any of the foregoing alkyl, , alkenyl and aikynyl linking groups are optionally .substituted with one to two substilueuts independently selected from the group consisting ofheterocydyl, axyfrheteroaryk heteroeyclyI(€.ubalkyl, aryl(€fos)alkyls heieroary it C\ .s)alkyi, spiracyebaiky! and xpfroheieroeydyi (wherein any of the foregoing eyeloalkyi, heteroeyelyl, .aryl and heteroary! substituents: are optionally substituted with one to font substituents independently selected from the group consisting of Ci-saikyh €t.ydkoxy, Ci^aikoxy(C«)ailk.y..h carboxyl, carboxyl(Ci.sfraikyi, amino (substituted with a substituent Independently selected, from the group consisting of hydrogen and Chalky!}, afflino(€i.g)alkyl (wherein amino is 'substituted with a .substituent independently -selected from- the group consisting of hydrogen and Cmalfcyi), halogen, (halo)i..5(Ci.s)alkyl, fhalefr/frCbsialkoxy, hydroxy and hydroxyCCubalkyl: and, wherein any of the foregoing lieteroeycly l substituents are optionally substituted with oxo)). cycloaikyl., heteroeyelyi., aryl, heteroaryl (wherein eyebalkyi, beieroeyclyk aryl and heteroary! are optionally substituted with one to tour substituents independently selected from the group consisting of Croalkyl, Cgsalfcoxy, €;i,salkoxy(Cfrs)aIkyl, carboxy l, carhoxyi(GK*)aikyl, amino (substituted with a.-substituent independently selected from the group consisting of hydrogen and Chalky!}. amlno(€^)a!ky! (wherein amino is safesti.tuted.with a substituent independently selected: from the group consisting of hydrogen and C>Maikyi), halogen, (haloij^CCi^alky'fe (halofe.3(Ci.u)aikoxyy hydroxy and hydroxy(Cs.s)alkyf; and, wherein heteroeyeiy! is optionally substituted with oxo), -(0-(CBfo^W0-, -0-(CH2)m>-0-(CH2)r^0-? -(0-(0-12)} -6)a -0~(CB v) I,,-N fMOb); ·*-<Κ -0-(€Η2)ηβ-0·4Ο·Ι2)η«-ΝΚ6-, frC)--(CB>}ifoow'S-N ••0-tCHv)!,.-S-{CH2)jw-0··, -OCH^^OlCHdi^SvN^ -NR6~NR?~, ~NR$~(C1;fr)s..6-NS.r, -NfV(€02)KrNR7~(CH2)i.$-NRs-, -NR6.-C(0)-5 ••CiOt NIfr,·, “C(0)HCIfr)GW“NRs“(CH2)o,6“C(0)», ~QfraM2)o6»N%fr€^

~S03~ (wherein &amp;<>, R? and Bs arei»depeti<Je«% .selected fromthe group consisting of hydrops, Chalky!, C^alkoN v{Cufralkyf Ca;i'boxyI(Cj..s:}ajkyf ammOiCogjalky! (ydretelh amino issifostimted with «Substituent independently selected thorn the group consisting of hydrogen and Ci.**lky!>, hydEJxy(Cps}ai%l> &amp;ad hetcroary1(Cj.-?s)aikyI (xvherein the feregoidg hefotoeyelyl, aryl ahdfoetecoaryl subsatueuts are Optionally stfostituted with .one to fear substituents independent ly selected, from the group consisting Of ffr^alkyl, Cs...8a|koxy, C ^«alkoky(C^fi)aikyl5: carboxyl, earboxyl(ChyJaikyf, amino: (substituted with a. substituent independently selected from the group consisting of hydrogen: and"Chalky 1),: amind(Ch,-s)alkyl (wherein, amino is substituted with a snbstitiieni independently selected from the group consisting of hydrogen and Chalky i), halogen, (ha{.o)^(Cns)aUcyi* (halon.,.dC^s)al.koxys:hydroxy and hydroxy(Ci^)alky!; and, wherein heterocyClyl is optionally subxti mied witltOxo}); with the proviso that if A. and E are selected, from a .hydrogen sofestidhed carbon. aioniythen, R2 is selected from the group consisting of ~€3.xalfcynyhi: -OfrC^aikyhO-, -OfrCs-sfalkcnyi-O-v -OfrCi3:,sla%uyhO-, <:(0^(Cu.a)ail<yl"€(0)-:(wherein any of the foregoing alkyl, alkenyland alkynytJinking groups; are straight: carbon chains optionally substituted with one to foursUhstituents independently selected from the group consisting of .Chalky h £waikoxyXuAoxyCfeixs)^kyh carboxyl, parboxylfCfrklaikyi, -€(0)0~(Ciy)aiky!, -€i^alkyi-C(0)0~(Ci.s)aikyi, amino (substituted with a substituent independently selected from the group consisting of hydrogen and CMaikyi),::aani»o(Ci^)alkyl fwhefoin amino is substituted with a substituent independently selected from the group consisting of hydrogen and %^alkyi|t halogen, (halofio(Gi-s)aikyk (halofr4(Ci.<i)alkoxy, hydroxy, :hydmxy(Cnss)aikyl. and oxo; and> wherein any of the foregoing alkyl, alkenyl and aikyayl linking groups are optionally stibstituted with one to two substituents independently .^I^t^iffopthe group consisting of heterocyclyk aryl, heteroaryl, hete.roeyolyl(Ci -s)al kyl, arylCCj^alkyl, heteroaryl(Ci,s)alkyl, spirocyofoaikyi and spiroheterocydyi (wherein any of the foregoing cyoloalkyf heterocyclyh aryl and heteroaryl substituents are optionally substituted with one to four stfostituents independently selected from the group consisting of Chalky!* Cusaikoxy, Cs..salkoxy(C^)alkyl, carboxyl* ca:rboxyl(Ci.„s)alkyl> amino (substituted with, a substituent independently selected. from tit e group consisting of hydrogen, and Ci^alkyl}, ammo(Ci,s)aikyl. (wherein amino is substituted with a substituent independently, selected from the group consist ing of hydrogen and C1.-4a.ikyi), halogen* <ba 1 o>i .¾(Cj -s)atky 1 ^ (halo)i.3(€s.s;)alkoxy, hydroxy and bydmxy(C;..j5}aik.yi; and, wherein any of the foregoing heterocyclyf substituents are optionally substituted with oxo}), eycloalkyl (wherein cycloalkyl, is optionally substituted with one to four substituents independently selected from the group consisting of Chalky!» Ci^alkoxy, €osalkoxy(Ci.2)aikyi carboxyl, earboxyI{C {. )alkyI, amino (substituted with a substituent independently selected from, the group consisting of hydrogen and Chalky!), atnino(€5.g)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and Chalky!), halogen., (halajh^Cstalky], (haloRoCCifoaikoxy, hydroxy and hydroxy(Ci^)alkyl)« '(O-CCHcltwiio-O-, ~0"(CB2)^^r0»(CH^)s,4~0-, -0-(01:¾}i 4~O-(0I-b)i .$~0t-((,.I‘l2).i~{ϋ-((_,Η> ).:.(>)l -i"bl Ιΐχ-, "0'gt.H;:!)i.*-0~(C.H2}s^-N.Rs“> “(O-iCH /fi xOco-S-’, ^(Ή2)1:^^03£>ί.^.,^€^)ι;^(Η€Η2)ι.^8-, -NR6-biR7~,-NRHCliOirs-MR.,-, -NRrCCHOrx^lO-CCHif^sdSRr.-NR^CiCOV, -C(0)-NR^f -c:iOi-(a^-NRr(ce2W( n>κ-ttMcmuraouviι,), -c tonau, ~nr7~? ~NRg-€(0}-NR7-, -NRrC(N10i \Rr, -0-((¾),,5-Nll6-(GH2)nrS-j: -S-CCBfo^-NRs-iCBB^-O-, -SyOiOb ..-NR^-fCi 0 )= :-S- and. -NR6-(CI^)i,.rS-(ChO}t.is-NR7- (wherein R^.-R? and -R«:are independently selected from the group consisting of hydrogen. Chalky}, €.^alkoxy(€;}^)alkyl, carboxyl(Ci ..s)a!ky|, amino(Ci.8)alkyl (wherein amino is substituted with a subslimeat independently selected from, the group consisting of hydrogen and. Ci-aalky!}* hydrdxy(Cj-s)alky!, :heteK>cyclyl(Cos)alkyh afyl(Cfog)aikyl and heiereatyl(Cj.s)alkyl (wherein, the foregoing heieroeydyl, aryl and beteroaryl substituents axe optionally substituted with one to four subslituents independently selected from the group consisting of Chalky!, Ci-saikoxy, Ch.^alkoxy(C.fos)alkyk carboxyl, carboxyl(€^*)aIky!, amino (substituted with a substituent independently selected from the group consisting of hydrogen and Ch.ndkyS}, am.mo(Ci-.s;)aikyl (wherein amino is substituted, with a substituent independently selected from the group consisting of hydrogen and CMalkyj), habge:riv(frala}ie(Cn.s)d&amp;y;l (haIo)i.gCi.g)aifc<>xy, hydroxy and 'hydmxy(Cfi,jj|aikyl; and, wherein hetcroeyclyl is optionally si^siiiptsd/wjlfe oxo|; and, wherein. %is selected from the group eonMsilng of carboxy i(C },K iallcyi, atm«©^€|^)a)kyt (wherein amino is sa.bsiito.ted with a subxtituentindependemly sdetiteti fern tile group consisting of hj<d3rpx^0i-«>aili^l, .hefesssocjycjyti^i^^ aryfrXms.laikyl and il^t^ftijaryK.'CL^alk^i^^heir^m.foregoing heterocyelyfr aryl and hctefoaryl stibstituents are optionally substituted witboneto four stfostituents independently selected frotnthe: group consisting of C.-saikyi. G.Kgalkosys :C^;Salkoxy(Ci^:}aikyl5 carboxyl, €£ufo0xyi((f^.s)al%!y:aifoao (Substituted with a substituent :lad0pendently selected from, foe group consisting of hydrogen and. Ci^aikyl), aaiino(C5.g)aIkyI (wherein. amino is substituted with a substituent independently selected·front the gitstip oonsistittg of liydtiogen and C^#lkyi),halogen, (halofro^ ihalo)· .frO foalkoxy, hydroxy and frydroxy(€ns}aikyh and* wherein. heforocycfyj is Optionally: substituted with oxofi; and,

Er and R y are independently selected: from the group consisting of hydrogen, Ό^alkyh. Cb-salkonyf £m*alkynyl ("wherein, alkyl, alkenyl and alkynyl are optionally substituted with a substituent selected from the group consisting of Ci*salkOxyf alkoxy(Ci*$}alkyl, carboxyl,: carboxyKCr,s)aihyi:, amino (substituted with a substituent independently seiectedifroni the group consisting: of hydrogen and C}4dkyi),tamin^Qt^kyIi<yi^er^h amino is substituted with, a substituent independently selected frotu the gfoirp consisting of hydrogen and Csualkyl), (hafo)to> fhafo}s,.;i(€es)alkyl, (hafo'fofrCh^lalkoxy, hydroxy, hydroxy(G|,s}aikyl and οχο}, C'j,salkoxys:CreXifexycarbonyl, (hafolilslGi^aikoxy, C| .^alkylthio, atylf freforoaryl. .(iyhdl'pfo. aryl and heteroaryf are Optionally stsbstittifod with a substituent seiccted from the group consisting of Ci^alkyh Ci-fsalkoxy, :a!'koxy(Cii,s)alkyi, carboxyl, cad>oxy:i(£'i.^)aikyl, amino (substituted with a substituent independently selected, from, the group consistragnf hydrogen, and Ci^alkyl), amlnoCCtblalkyl: (wherein amino is substituted with. a.substituent independently selected froth the group consisting:of hydrogen and C ^alky l), halogen, {ba|0l)|,:dCfo8)aikyl, .(halo)j..3(C},«)al.koxy, hydroxy and:hydmxy(€iy}alkyi), amino (substituted with a substituent iadependestly selected from the group consisting of hydrogen, htM Chalky!), cyano, halogen, hytifoxy and nitro; and pharmaceutically acceptable salts thereof

In one embodiment, a compound, of Formula (111) is a compound selected- from the group consisting of:

Formula (Ilia) Formula (illb.)

Formula (Hie) Formula (Hid)

Formula (Ole) Formula (Olf)

Formula (Illg). Formula (Hlh)

Formula (fifi) Formula (Hlj)

Formula (Iilk; Formula (Dll)

Formula (Him) Formula (I I hr) wherein all other variables are as previously defined; and, pharmaceutically acceptable salts thereof, in one embodiment, a. compound of Formula (HI) Is a compound selected from the group consisting, of;

Formula ( Ola) Formula (111b)

Formula (III!) Formula {Oii.)

Formula {113 j > wherem all other variables. are as. prowowsly defined; and, pharmaceolidally acceptable salts thereof..

Compounds of Formula (III) are disclosed In commonly assigned United States Patent Mrmber b*F2h~$2% tbe cootpleie disclosure of wbicb is :E#6^V;mc0^rat0d\%-#^lbfe«co-

Atr exampleOf the inventta includes a compound of Formula0ΪΙ>^ therein the compound is sMeeied fiom the group consMag of:

Compound Name 1 -6,7,9» ί 0,12, i 3,15,16^ctahydro»2 3/3-3,26.: ί 7,22 -dimetheno -5 /7 dlpynd»[2,3~έ::3’ f2’~q Jpym>fo|3,4- n:} f 1,4,7,1 0,1.9] trioxadkzacye io hemeoame-23,2 5(24/9)-dicme, 2 1.0,11,13,14,16,17, l:9,20,22,23-decahydro~9,4:24,29“dirr5eihesiO" ΪΗ- dipyridop3"ff^322t”r|pyrroio[3,4~ if][l ,4,7,10,13522jleiraoxadlazacyctotefeaiioame-.l ,3(2ff)~dione, 3 10,11,13,14,16,1729,2CK22,23,2S,26-dodecahydm~9,4;27,32--dlmsiheno~ 1 i-^dipyrido[2,3”f :322S"W]pyTK>!o[3,4» ij[l ',4,7,10,13,16,25 Ipentaox.adiaxacyclohepiacosffle-'l ,3(2/i)~dione, 4 6,7,9,1(1,12,13 diexahydro~20//~5,23:14,19~dimeflieno-5^ diheozo[6,a]pyrrolo[3,4-4d[ 1,4,7,16]dioa.8diazacycloociadecme~ 20,22(21iifl-dione, 5 6,7,9,10,12,13,15,16~ocialiydm~23/C5s26;l 7,22-dime&amp;euQ~5H~ di.be.nzo|>Cy |pyrrolo[3,4~n] [ 1,4,7,10,19jtrioxMia«acycIohe»@icosine'-23,25(24if)~dione, 6 10,11., 13,14,16,17,19,20,22,23 -decahydro-9,4:24,29-dimetiseao> IH-dlbenxo[oy]pyrTOio[3,4~y][L4,7,1.O,13522]tetrao)iadiaxaeyci0teltaooaiue~ l,3(2/7}“dbne, 7 10,ll,l3iI4,16,17,!9,20,22,23,25,2(Cd6deeabydm4),4;27,;>2brmetb.an:o- l/2~dib<aixo[y,>vjpyrro[o[3,4~ ^[1,4,7,10,13, l.i>,25 !pcataoxad:iazacyckiliepiacoa0ie:»vi,3(2i7)~dioae, 8 1 2dwd.n.>6./'/,.l §77-5,22; 13,18:7,114rlnteiheoopyrido[23~/)pyrmlo[3,4'· mj[l ,9]beiizodia2acycbkep(adedne~l 9,21(20/7>dlone, 9 12 ~ h y dro - 6/Λ19//-5,2.2 :1 3·, 1 S-dimetheno-?,11 -nitri tepyrido[2s3" j]pyn'Oio[3>49?i][ 1 ^Jbenzodiazacvcioheptadecme"! 9^2 i(20/f J-dioac, 10 6!Jf7s9>i0»i^l3-hexahydiO-20.W"5»2:3::1.4iS.l9“di-m$th^o-3.M“pyrido[2,3” 6]pynTdo[3 A~n ] [4,7 51,10]be:n:zodioxadiazacyclo0clad.ecisie“20,22(21 /7)-diorte, 1.1 6,7,9,10,12» 13,15,16~oeiah}3ro~233/~5,26:l 7,22~dlmeihe»0~5//~pyrid42,3~· a]pyiToSo[3,4--£/][4,7,l 0,1,1 Slhenzotrioxadiazae'ycioheneicosine-2 3,2 5 (24M}~diQm. 12 11-ethyl-6,7,10,11,12 J 3,15,. 16-GGtahydro-23 /7-5,26:17,2 2~dimeihe.n0-5i3,966dil)eaz£)[^,y]pyn'0l£):[3,4- xj[l,7,4,10,19]dioxstriazacyclohe0dcosme-23s25(24i;i}-ds0n4i, 13 6,7,10,11,12,13,15,16-ociahydrQ-11 -meth:yl~23//-S,26:17,22-diTOetheno-5/7,977~d:ibeD2of2,y]pyrrolo[3,4~ «JCl^AlQJ-^'jdioxatriasacyckdK'udcoNine-ZS^S^di^-dliofte, 14 6,7,10,11,12,13, i S, 16-oeiahydro- 11 <j ~me%!eihy!)~23//-3,26:17,22-dlffietheftO~5.i9f9H~dftefia0[fc,:yjpynOlop,4~ m,7A 1049jdioxairia?acyolohpnp!cosine-23,25{ 24/f)-dioae, 15 7,8,9,10,1 i ,12,13,14,15, ib-deeahydro-B, i 1 ,'14-tfimethyl-6/733/7·· 5v26fl.7,22~d5metliettediben.zo[i?,/]pyrr0lol3,4·' y][] ,4,7,1OJ 3]pe«iaazacycl0li0S£ic0si«e--23,25(24:3/)-diotte, 16 6,7,10,11,12,13,15,1 b-octahydfo-1Ϊ -methy1-23/^-5,26-methe«b-17,22-ttMlo-5/3f,9./i“diteJzo[^JpytfoIo[3,4- /?][ 1,7,4,10,19jdi0xatriazacyel0heneio0sme“23,25(24i7)“dloae, 17 11'ethyl-6,7,10,11,1.2,.13,15* 1 6-octahydro-23/7-5,26-meihe»^17;22~iiitrtl.c>- 5i7,9i7-dibenz0[pyjpynrol0[3,4~ ,7,4,10,19]dioxatrlazacyclohe0eico,sinc-23,25{24//)“dione, 18 1 l~ethyl~6,7,l0,11,12, .13-,15,10-ottafaydro-23/i-S,26:17,22-dimelheno- 5/is9/Mip)^ido[23>l::3iJ22g]pyrro!o[3,4" n}[l J,4s10J9jdioxain.CTcyciol)^Beicosiii,e-2335(241;/}-dio»e; d3pyrMo[2!3--i:3',2,~£/]pyn»io[3,4- .«•][13A10,19]dloxaihja4iazacyclohen.ekosiae“23i25(24//)“dio'ni5i, 20 7,8,94 0,.11,1243,1445, Ϊ Mecahydro-(6i/,23//-5^0:17,22^ dimetb.eriodipy.d.doC2,3~r3,>2,-i]pyrmla[3,4- p] [ 1, ?, 13 jtriazacy cloheftci cosia c-23,25{24//}-cHonc. 21 Ϊ l-ethyI-73,9,10,114243J4J5,:i6-decahy4ro~6//33H”5,26:lt dim.e&amp;sa6dlpyndo[2j3“«:3*,2,“if'jpywolo[3'!,4^ </][] ,7,0]iriaKacyeIoheireicodfie-23,25(24/7)--dione, 22 6,7,8,9,10,11,.12,13,..14,15-deeahydro-22/f--552S: 16,21 -din^tteo-5»-dipyri do[2,3 ~m :3 ',23? JpyfroloP >4~p][ 1,6,12] its azacycloei easmcs-22,24(2 3//) -dioae, 23 10~ethyi-6,7,8,94 0,11,12,13,.14,.1 S~deca!iydtt>~22:«~5,23; 16,21 -dimeihono-523-dipyrido [2,3-m: 3224φρΒχ4ο[ 3 A-p] [ 1,6,12]triazscydodcosffie-22,24(23.fl>dione, 24 7,8,9,1546,1.7,18~hi':piahydro-6//,25/7-5,28'.19,24-dlniiotb£:no-10,14“ niinlodipyrido[23-/> :3',2!~A]py.rs'o!o[3,4-e] [1,10] diaaaey elotrieosm®-25»27(26/fj~dioBe, 25 7,8,9,10,.11,13,14,15,!.6-fto«ahy*o~6.ffs:23«~5,26:1.7,22-di:rdethenodi.pyrido[2,3 -~h: 3 ',2'"l?]pyrrob[3,4»6] [ 1,1 OJdiazacyclofaeneicoske» 12,2 3,2 5(3460-tritrne, 26 7,8,9,11,1243,14~6eptahydro4>/£217i~5^4:15,20Hiimetheiiodipyrido[2,3" 6:3,,2'-6]pym>lo[3,4-e][l,.iOjdia2a.cyclon0nadeci«e-lO,21,23(22f/)-trione, 27 6,7,8,9,.10,1.1512713,. 14,! 5nieeahydto~7 J4-dihydroxy-(7$f 148)-22H~ 5,25:16,21 -dlmeiheao-5^dipyrido[23~^:3,!2'--^]pym)lo[3,4~ c] [ i, 10 jdiazacy cloeicostoe-22 3(23l?>cl<me,. 28 6,7,9'JO,IWte.eihettG-SitA^ipiyridpiS^-^:3‘,2'“«]py«ro1o[3i44'][1i,4j7>'ii63dioxaifii^cycioop<adecme-2€ff22{2).ic0“ dlone, 29 6,73 0,11,12,134 5,16-octahydro·· 11 “(2~methoxyethy!)"2366-5,26-metheno-17,22~RMriio~577,9ff-dibeozo[A,yjpyrrolo[3,4~ n][ 1,7,4,1049]dioxaiidazacycloheneic6sine-23i,25(24/'/ Cdione, 30 6,7,10,114 2,13,15.4 6-ectahydro-J i-(2-h>'droxyethyl)-23//-5s26:.17,2.2“ di:ffietbeao-3i7,9//»di3en^p,^]pyrrolo(354- »]P ,7,4,10,19]dioxaln.azacycbhe«sicosii5e-23,25(24.fif)-diotie, and 31 6,7,9,1042,1344,15,16,17~decabydm~14~meihyl-24665,274.8,23-dlTnatlienQ~537~dibenm[/a'!pyn'olo;[3,4~ t>j[l ,4,7,11,2O]diax.al'nazacyciodoc0sine^24,26(25.ii7}~dlo:ne,

An example of the Inxeniion includes a confound: of Formula (Hi) wherein the compound is selected from the group consisting of;

Compound 1 Compound 2 Compound 5

Compound 6

Other examples of the invention, include a compound selected from the group consisting of:

Compound Name la To be provided 2a 3~[HM(2~h ydroxyctJrjdtoeihyiao^inojproiyyij-llJ-indaz^l-d-yij- 4-[ 1 -(3-pyridinyi)-1 B-mdoi-S-ylj'JH-pytTole-^S-dione, 3 a 3,3-diehia.ro-ISj·-p-chloi'0-4^[(3f4, 12U2a~telrahydm~ JTi- [1,4]thiaxiBoP>4 -e j [ 1.4]hcnxodiaxepin~l J C6B)~ y i )c arbo n y Ij ρ it any I ] -ben zamt de,. 4a 3-p ~(2-hydroxy-ethyl Kl B-ftt.dol-3-yJ.]-4-{ j.-pyridip~3-yl- IH^ndoi- 3-yl)-pyrro!c-2,5-dionc, 5 a 3-(2-methoxy-pheny 1)-4-(1 -pyiidin-3'-yH B-mdoi“3-yl)-pym>lc- 2,3-drone, 6a (>~|[2-[[4-(2.4-<Jielilor0plienyi.)-5-(4~:nK4hy1-.lfi-imidaxel~2-yl)-2- pyrimidinylj&amp;minolethyiJaniinoj-S-pyridineearbonihile, 7a 3~(5~chloro~l -methyl-1 H-indoi-3-yl)-4-[l~(3-imlda2ol-i -yl- prapyl}-1 H”indazo!-3-yl]-pyrrole-2,5"^it)^es

Ha 3-{5-«b.1.oro-1 -mg$feyMH4ijdol-3idM*{ i -<3-[.1·,243 jiriazol- l.-yl- propyl)-! H-ied.azoJ-3~yi]~pyrmie~2,:3~di:ones 9a 3-[l-(3~iiydt®xy~propyi}-iO~pyny>lo|t233-blpyridk^3~yi|~4~(l- methyl-l B-pytazo! - 3-y l>pym>le-2s5-dione, 10» To be provided. 11» 3-[Ί -(3~bydroxy~3~iTieihy irbidy i)-lB~iodazol-3~yl]~4~il-pyddi.a-3- yi.-1B-mdol.-3·-yl)»pyn'oie-2,5-dtobe, 13a 3-[H2»hydtOKycihyl> 1 H-indazoi-3 -y1M>{ * i>yrimidin-5-yI- 1H- i!idoI-3-y!)-pynole~2s5~dione5 i 3a 3~[ 1 ~(2 -hydroxy-ethyl}-1 B-rado!-3 ~yij~4~{ 1 -pyrorodm-S -y I- III- ind0l-3~y I)~p:ym>.i e~2,3 -dion % 14a (112)-8,9,10,13,14, IS-hsmhydro^b: 17,2:1- di(methend)pym>lof 3,4-h] [1,15 ,7]dioxazacyclotricosim> 22,240 B,23K)-dme, 15» 3-(S-eidoro-.l.~pyi:idi«~3~y!~l]:l~mdoI~3~yl)~4-[l-(3~hydimy- propy 1)-1H-iad azol »3-y I j-pyra) le - 2,5-dioHe, 16a 3"(2-meiho:xy-phenyl)~4-{ 1 -(3-meihoxy-propyl)- i H~pyrrDlo[3,2« c]pyrldm-3-ylj-pyiro! e~2,5 -dioxie, 17a 3~['l~13“hydt«!Xy-propYl)~lH4adazo.l-3~yi’j-4-[I-(fetraliydro-pyi^«- 4»y!)»lH~iadol"3'*yI]“pyrroie”2,5'-diooei 18a 2-{3-[4-(5“CMon>‘l~methyl-1B-mdol-3-yi)“2,5-d.ioxo-2,5-dihydtO“ 1 Id-pyrrol-3-y l]-iodazoi~ 1-yi)-N-(2-hydroxy-ethyl.)-acetam.k!es. 19a 4~(3<hloroi>.he«yl)-6-(3~dlmethy!ami.TOi?ropyI}~5i6~<1ihy4rO"4B'· 2,4j6“tria2a"cyc!0pe«ta[c]fluoriiie-1,3 -diene,· '20a I4~eihyi-6,7,9, .10J3,MS 1 Sa6~oeiahydro~ 12B,2314-5,26:1732- dimeihenodibaftz0.[.k,q]pyrrolo'[3i,4- «I[13JJOJ9]d:ioxatriaMcyclotefieic0sme--23?25(24HHioBe, 2 la: !4-feenzy}.~6,?,9,1CM3,14,15,1 .(«>ctahydf0-12M523«^,2O:l?32- di(«iethenQ)dlben2:0[kiq]|)yrr0l0[3i4” «] [ 1,4,7,10,19]dsoxadkza.cye leheni eokne-23,25(24H)-diones :22a 3-(l-|24'2~(2~hydroxy~ethoxy)”ethox'y5”elfcyi}~.].H4adioi'‘3-yl);-44'l·” (3-hydroxy-eihyl)-1 H-indol-S-ylj-pyiToie-SjS-dione, 23a 6,7,8,9,10,1 U2,13-octahydro~B,l i-dimeihyi-5,23;l4,l9- diaKtheii0-2Oll~dlbe:n:zo[k,q]pyrrola[3s4-· nj[[l ,4,7,1 Olieiraaxacyd0oeiade0lne-2O,22(21 .Hjh'dlone, 24a. 7,8,9,10,12,13,16,17,1 SdO-deeadydro-S, 17-dims%l-15Η,26Β~ 539:20,25-diTnetheit©-6H-.dihensotk,qjpytT0lo[3·,4-»}[ 1,4,7,10,1.9,2 2]dl0xaieSiaaxaeycloieS:rac0sirie~26,28(27H)~di0ne, 25a 1.4-( )2-6.in4.m.ed.iyl)~6,7i9,1.O,1.3,.14,lSJf00ctahydr0-]213,23l-l·· 5,26:17,22-di(metheno)dibei5ro[k,q]pyiTol:G[3s4- nj[.1,4,7,10,19jdioxatdazacyd0heoic0s.me-23,25(24H)-dioBe, 26a 14-(2-lhieayi(oofh> 1 jA7,9,; 0.15,14, i 5J.6-oc&amp;hydro-128I,23Il- 5,26:3.7,2 3-dii4octhcaK^)dibeaz0(ksq |ρ;/0Χ)Ι.ο[3,4-«][! ,4,7, Ϊ O,19]diox.atriaxacyci0ha6cosiae-23,25(24H)-d]o00, 27a 1.4-(1 -naphthytaethyl)-4?,9,10,13,14,15,16-octohydro-l 2H,23H- 5,26:17,22-di(metheBo)dibe»2o[k,q]pyrroIa[3s4" n}[ 1,4,7,10,19 Jdioxat ri azacyc tahenic«sp;e-23,25(24H)~tKone, 28 a 14-(pyndin-4~ylBjelhy 1)-6,7,9,10,1 3,1.4,15, i 6~ociahydm~ 1.2B,23 H- 5>26::17,22-di(metheji0')dibeiizo[k,qjpymilo[3,4-' ^[l^JilOj.OJdio^.airiaxacryci.ohmjcostec-.I.'^^j 54B)-dion.e, 29a 3»[l-(2~|2~[2-(l,2,3s44eirah:ydro"BapM!ialea~!~y!aiTMxio}~i;th.osy]~ ethoxy) -eibyl)- lH-indoi--3-y!]-4~{ |:~[2-(1,2,3,4-iefraliydfo-oap hifo&amp;l en~ I -y I ami«p)-ethylj- J B-mdol-S-yl] -pyrrol e-2,5-dione, 30a 3-[1.~(3"diraothykBimo-phenyl)~l H-mdol-3-y!J>4-i 1 ~(2~hydroxy~ ethyl}-] hl”iik1a:zol“3-yl]-pyrrob~2,5-dioBe.. 3 la 3-[5«chloro-l-(6“dtoeihylamiaO“pyridm-3--yl>]B“indoI-3“yi]-4-[l- (2~hydroxy-ei!iyi)-] ll-itk1a.EoI-3~yl]~pymd.e~2,5~diooes add 32a 5~(5~chlom~3-|4-[.1-(2~hydiOxy~eihy!)-lI-l-hidazO:l~3~yl]-2,5~dioxo- 2,3-dthydrO”iH-pyrrol“3-yI)-mdol-!-yl)"meGtmic acid .methyl ester.

Other examples of the invention include a compound selected from the group oons.ist.mg of:

Compound, la Compound -2a- Compound 3a

Compound % Compound :5 a Compound 6a

Compound 7a Compound 8a Compound 9a

Compound 10a Compound 11a Compound 1:2a

'.Compound 13a Compound 14a Compound 15a

Compound Ida Compound !?a Compound 18a

Compound 19a Compound 20a Compound 21a.

Cofcipoand 22a Compound 23a Compound 24a

Compound 25a Compound 26a Compound 2?a

Compotmd 2¾ Compound 2¾ Gorapoy a d 3 Oa

Compound 3 la Compound 32a

Cellssuitable fortreatment according to the methods of'.the presentInvention

Plunpoient cells, suitable for nse In the, present invention express at least one of tile following pforipotency markets selected from the: group consisting of: ABCG2,;cnpto, Eoxp3, CdnSexm43:, Comi:exin45, Octfo SOX-2, Nanog, hTEfoi; :Um~h XEP42, SSEA-3, SSEA:-4, Tml fofo and Tral-83. in one embodiment,: foe plnripotenf cells arc embryonic steft/cells.. In an alternate enfoodiTnenty the plnopotent eellx are cells eapmasisg pkrlpotency markers derived from embryorsfo stern ceils, In one embodiment, the embryonic stem cells are Homan. I'soIatioii,-exi>a»sIoii anti culture of trama» embryonic stem eells

Charmiertutkm inhuman em&amp;ryomc stem celis: Bi«M« enforyotnc stem cells may express one or more o:f tire stage-specific embryonic antigens (SSE&amp;); 3 and 4, and markers detectable using antibodies designated T:ra~l~60 andiTra4-8i fThdsmm etaL· iSeience .282:! ί 45> 1998). Diffe^t^dosi of human embrypiiie stem cells/4 vitro results •is the loss ofESEA~4< Tra- 1-60,and IVa-l-gl expressionlifpresent! and increased expression of SS;EA~1. lltfoifemfoated human embryonic stem cells typically have-alkaline phosphatase activity, which can be detected by fixing the cells with: 4% parafoomldehyde, and then developing with. Vector Red as a substrate, as described by the hianniietarer (Vector Laboratories, Burlingame Calif } Undifferentiated piuripoieoi stem cells also typically express 0et-4 and TERT, an detected, by RT4CR>

Another desirable phenotype of propagated human embryonic stem ceils ip a potential to differenti ate into cells of a 11 three germinal layers: endodermj mesoderm, and ectoderm tissues, Pluripotency of human emhiyonie stein cells can be confirmed, forexamplfe by inieptiug eeSIs into SOD mice, fixing the teratomas that form using 4% paraformaldehyde, and then examining them histologically tor evidence of cell types from the three germ layers Alternatively, piuripoteney may be determined by the creation of embryoid bodies and assessing the embryoid bodies: for the presence of markers associated with. the three germinal layers.

Propagated human embryonic stem cel! lines may be karyotyped using a standard Q~ banding technique and compared m published karyotypes of the corresponding primate species, K is desirable to obtain cells that ha ve a "normal karyotype", which means that the cells are eupioid, wherein all human chromosomes are present and not noticeably altered.

Sources ofkmmn emltrwmc stem mils: Types of human embryonic stem cells that may be used include established lines of human embryonic cells derived from tissue formed after gestation, including pre-embryonic tissue (such as, for example, a blastocyst}, .embryonic tissue, or fetal fissue taken any time during gestation,, typically hut not necessarily before appmximately 10-42 weeks gestation, blonffirmbng examples are established lines of human embryonic stem eelisor human embryonic gem eeils, suck as, for example the human embryonic stem cell lines Ml* H?s and H9 (WiCell),. Also contemp feted is use of the compositions; of this disclosure during the initial establishment or stabilization of such cells, in which case the source ceiSswwtuld'be primary pi uri potent ceils taken directly from thwspufce tissues. Also suitable are eel;!k: taken from, a piuripotent stem cell population already enimted in the absenee of leeder cells. Also suitable are mutant human embryonic stem cell lines; such as, for example, SCO l:v: (BtesaGen, Athens, GA),:

In one embodiment, hhimait enibryohie stem cells are prepared as described by Thomson etal (O.S. Fat. No. 5,843,780; Sefende: 282; 1143, 1998; Curr. Top. Dev. Biol 38:133 ft, 199B; Proc, Natl Acad, Sri. I S. A 92:7844, 1995).

Culture of human embryonic- stem imihe In one embodiment, human embryonic idem, cells are cultured iu a culture system that is essemially free of feeder ceils, but nonetheless supports proliferation of hum.® embryonic stem cells without undergoing substantial differentiation. The growth of human embryonic stem ceils in feeder'tree culture without differentiation is supported using a medium conditioned'.by culturing previously with another cell type. Alternatively, the growth of human embryonic stem cells in fcederTree culture without difrerentiation is supported using a chemically defined medium.

In an alternate embodiment, human embryonic stem cells are initially cultured layer of feeder cells thatsupport the human embryonic stem cells in various ways. The human embryonicare theu transferred fo a culture system that id essentially free of feeder cells, but nonetheless supports proliferation of human embryonic stem cells without undergoing substantial differentiation.

Examples of conditioned media suitable for use in the present invention are disclosed in 11820029072117, iiSbb42048, W020050I4799. and Xu etal (StemCells 22: 972-980, 2004), ..An example of a chemically defined medium suitable lor use in the present iBvertiion may be found in US2fi07O0IG01 1..

Suitable culture media may be made from foe following components, such as, for example, Dolbeceo’s modified Eaple’s medium. (DMEM), Gihco # 11965.-#2pK^dcktut. Dulbcceo's modified Eagle's medium (K.0 DMEM), Gibe» #10329-4)] 8: Ham's FI2/50%: DMF.M basal, medium; 200 mM L-glutamltte, Oibeo F15030-02?; uon~essefttiai amino acid solution, Gibco 11140-050; β- mereaptoefoanol, Sigma // 1V17522; human reeombiuaut basic fiferoblast growth factor (bEGEj, Oibeo # 13256-020.

In one embodiment, the human embryonic::hfofofohiis.^-::p-iafod· onto a suitable culture substrate that is treated prior to treatment according to the methods of the present invention. In one embodiment, the treatmeht is an exforeelfolar matrix component, such as, for example, tbose denyed from basement memfoane or that may form part of adhesion molecule receptor-ligand couplings. In one embodiment, a foe suitable culture substrate is Mbhigelif} (Beefoh Plekensoah Mairigel® is a sbiuble pfophtatiou from Eugelbreth-lfolm-Swarm: tumor ceils that gels;at toom temperature to form a reconstituted basement membrane.

Other extracellular matrix components and component mixtures are suitable as an alternative. This may include laminin,: fibroueetin, pmteogiyean, cntactin, heparan snlfote, and the like, alone m.,in'.various combinations.

The human embryonic .stem bells arc plated, onto foe substrate in. a suitable distribution and in foe presence of a medium that promotes cell survival, ptopagaiion,;and:reteniion:pf the desirablecharaeierisiies, All these characteristics benefit from careful attention to the Seedlhg distribution and bah readily be determined by one of ski ll in the art. isolation, expansion and coltere of ceils expressing pieripotesey markers that are derived from human emlmyenle stom ceils

In one embodiment, cells expressing pforipotm«y,:marfe^>^4pay^:^mfo»*mft embryonic stem, eelis by a method composing foe steps ofi a, Gn Inuring hump. embryonic £tem call s , b, Di £ferenti atin g foe Innhau fohbryonic item eel is info cells expressing inarkers elmraeteristie of defeiiive eadodetm sells* and c, Removing the cells, and subsequently culturingIhemxnsdef hypoxic conditions, 0». a tissue cukum subsimfothat is not predicated with a protein or an extracellular matrix prior to culmring the cells.

In one embodiment, cells expressing glfoipofohey niarkers are derived Rom hutnan. embryonic stem ceils by a nmfood comprising the: steps of: a. Culturing human embryonic stem cells, and b. Removing the cells, and subsequently culturing them under hypoxic conditions, on a tissue culture pbstrate that is pot prefocated with a protein or an extraeellukr matrix.

Cell culture tipilef hypoxic conditions 0» a tissue culture substrate that Is not pro-treated with a protein.or an extracellular matrix: in one embodiment, the colls are cultured under hypoxic conditions, on a tissue culture substrate that is not coated with ah extraeclloiaf matrix for about 1 to about 2d days. In an alternate emk)dimont, the ceils are enltured under hypoxic conditions, on a tissue culture substrate that is not coated with an, cxtraeeliular matrix for about 5 to about 211 days. In an alternate embodiment, the cells arc cultured under hypoxic conditions, on a tissue enltufo substfate that is not coated with au extracellular matrix for about. 15 days. in one embodiment, the hypoxic condition, is about 1% 0.> to about 20% (>>. la an alternate embodiment, the hypoxic condition is about 2% 0¾ to about 10% ().?. ia an alternate embodiment, the hypoxic condition is about 3% O,·

The cells may be eukured, under hypoxic conditions on a t(i^oo:'Od:Iihn^::$ubstr^::tetis hot pre-treated with a protein or an extracellular matrix, ia medipm cOntaiaing serara, setivin A, and a Wnr ligand . Alternatively, foe medium may also contain IGF-1.

The culture medium may have a serum concentration in the range of abouf 3% to about '5%.· In an alternate embodhacut, the senua conceutratidn uiay be about 3%. . Aetiyin A may be used at aeoneetUTaiion from about Ipg/ml to about ifrOpgdnf. Jo an alternate embodiment, the concentration nray be about IpgAul to about Ipg/ml. In another alternate embodiment, the concentration may be. about Ipg/nd to about ICKIug/ml. In another alternate embodiment, the concentration may be about 50ng/rni to about iOOng/rnl In another alternate embodiment, the concentration may be about lOOng/ml t he Wni ligand may be selected from the group consisting of Wni-1, Wnt-3a, Wnt-Sa and In one ^bodiment, the Wnt ligand is Wht-l·. in an alternate embodiment, tM Writ ligand is Wnt-3a.

The W ut ligand m ay be used at a coheenimtion of about tug/ral to about l(100ng/mL In an alternate embodiment, the Writ ligand may be used a? a concentration of about I0ng/mi to about lOOug/ntl,.; itl one enibodiment, the eoneentraiion of the Wni ligand is about: ;2frng/mb:

IGF-1 may he used.at a eouceutrafron of about Ing/mlto about lQ0ng/mi. in an alternate embodiment, the: ICS F--1 may be used at . a concentration of about lOng/ml to about lOOng/tdf In,one: embodiment, tbeiebheedtrafron oflpF-l is about 50ng/mL

The,cells eupmssing plnripoteucy markers, derived by the methods of the present invention are capable of expansion in culture under hypoxic conditions, on tissue culture substrate that is not pre-ireated with a: protein or an. extracellular matrix-.

The cells expressing pl uripoteticy nuufceiS derived by the methods of the present invention express at least one of the following ploripotency: markers selected bom the group consisting of: ABGPS^eripio, Fokl33, Counexin43, €onn.exind5, Ootd, S©y~2, Nanog, and Tra 1-81.

Further differentlafin» of cefls expressing markers charaeteristk of the definitive etiiloderm lineage

Fills exposing uprfcers characteristic of the definitive erfooderm lineage may be differentiated ibto cells expressing markets diaraeteristie of the pancreatic endoderm lineage by any method in the art.

For example, cells expressing markers characteristic of die definitive endoderm lineage may be differentiated into cells expressing markers eharaefetlsfie of the pancreatic endoderm 1 Ineage according to the methods disclosed in D" Amour er a/. Nature Biotechnology 24,1391- 1401 (2006).

For example, cells expressing markers characteristic of the definitive endoderm Imeage are itrther differentiated into cells expressing markers characteristic of the pancreatic endoderm. lineage, by treating the cells expressing markers characteristic of the definitive endoderm lineage with a fibroblast growth factor and KAADroyciopanime, then removing the medium containing die fibroblast growth factor and KAAp-eyeiopamine and subsequently culturing the cells in medium containing retinoic acid, a fibroblast growth factor and IfAAPAwelopaminc, Aa example of this method is disclosed in D5 Anipo! et at. Nature Biotechnology, 24·;:. 1392-1401, (2006).

Markers characteristic ofthe pancreatic endoderm lineage are selected front the group consisting of Fdxl. HNF-lbeta, FTFla^ HNF-6S HB9 and PROXL Suitable for use in the present invention is a cell that expresses at least one of the market's ehataetorisiie of the pancreatic ehdodenn lineage. In one aspect of the present invention, a coll expressing markers characteristic ofthe pancreatic endoderm lineage is a pas^^ic:bPdod«^i bdtt.

Further differed flatten, of cells expressing markers ehaeaeierisf ie of fie paucreaff e endoderm lineage

Cells expressing markers characteristic of the pancreatic endoderm lineage may be differentiated into ceils expressing markers characteristic ofthe pancreatic endocrine lineage by any method in the art.

For example, cells expressing markers characteristic of the pancreatic endoderm lineage may be differentiated into cells expressing markers characteristic: of die pancreatic endocrine lineage according to -the methods disclosed in O 'A mour ei at,'Nature Biotechnology 24, 1392 - 1401 (2006),

Mafirem eharacteristicof the pancreatic endocrine lineage are selected from the group consisting of HC3M-3, Neurol), Islet-!, Pdx~!.s NKIX6J, Pa:x~4s Ngn~3:i and PTF-! alpha,. In one etnbodirp&amp;ht^-at |yap¥^.aaic;^i4o<^n3e:^l1 Is capable of expressing at least one of :'^eWl0mnp;^onnos^;riBSttIin, glucagon, somatostatin, and pancreatic polypeptide. Suitable lor use id the present invention is a ce ll that expresses at least one of the markers characteristic of the pancreatic endocrine lineage, In one aspect of the presentindention, a cell expressing .markers characteristic of the pancreatic, endocrine lineage is a pancreatic endocrine cell. The pancreatic endocrine cCO may he a pancreatic hormone expressing cell, Alternatively, the pancreatic endocrine cell may: he a pancreatic hormone secreting cell.

In one aspect of the present invention, the pancreatic endocrine cell is a cell expressing markers characteristic of die β cell lineage. A cell expressing markers characteristic of the β cell lineage expresses Pdx! and at least one of the following transcription fectors: •M W3,lMkx2i:2, N:kx6.15:TIeuroD, Isl-I. HNF-3 heta, MAFA, Bax4, and Faxh, In one aspect of the1 present irreentipfo a cell expressing: markers characteristic of the: β cel! lineage is a β cell.

Deteeiloti Of cells expressing markers characteristic of tire definitive emloderm linage

Formation of cells: Expressing niarkcrs characteristic of the delditree eadoderin lineage may be deteretmed by testing for the presence of the markers before and afier following a particular protoeoL Pluripotent Steffi cells typically: do not express such markers. Thus, di®f«*idatiop:0f pluripptiatl'ceife is delectcd when cells begin: to express therm

The efficiency of differentiation .may be determined by exposing a treated cell population to an agent (such as an antibody) that specifically recoginzes a protein marker expressed By cells expressing markers eharaetedstie oftk©'id^M^vei^oden5Siin^agd>:

Methods for assessing -expression of protein undmteleic acidtiB^^,ittcalteil or isolated colls ate '.standard in the ait These include quantitative reverse tran^enptase polymerase chain reaction (lO^pCE), Northern,blots* in site hybndimtioo: (¾¾ e.g., Cumem Protocols m Molecular Biology (A us «feel et ri/,, eds, 2001 impplemeni)),: and immunoassays Such' a$ initpaaofijd^^ of sectioned material. Western

Wotting, .aad^'tnarkwAftt ar&amp;aeeesslfele in intact ceils, flow cytometry analysis (FACS) (see., e,g., Barlow and Lane, .-Using Antibodies: A laboratory Manual, Mew York; Cold Sp fing H arbor t,aboratory FressXI99b):T

Exanrples of antibodies useiid lor delecting certain protein markem are listed in. TaMe ΪΑ, It should, be noted that alternate antibodies directed to the same markers that arc recognised, by the antibodies listed in TaMo I A are available, or can -be readily developed. Such alternate antibodies can: also be employed lor assessing expression of markers In the cells isolated iu accordance with thepresent in vention.

For example, characteristics of pluripotent stern ceils: are well known to those skilled in. the art, and additional characteristics of pluripotent stem, cells CGhtinno to be identified. Hlnripotent stem cell markers include, for example, the expression of one or more of the Jbllowing; ABCG2, eripto, :FoxD3,Connexia43, ConnexrndS, Oet4, SoxS,^Manog, hTEET, If ΓΡ -1,2PP42, SSEAM, SSEAM-, Tral »60, Tra!-81.

Alter treating pluripotent stem, cells with the methods of thp:present invention, the differentiated .cells may he purified by exposing: a treated cell population to an agent (such us an antibody) that specifically tceogtiizes a protein-marker, such as (U£GE4, expressed by cells expressing, markers characteristic of the definitive endoderm lineage.

Detection of cells expressing markers characteristic of the pancreatic emiiKlcrm linage • Markers characteristic of the pancreatic endoderm. lineage are: well known to those skilled in: the art,-arid-additional markers characteristic of the pancreatic endoderm lineage continue tothC: identified. These markers; can be used to confirm: that the cells treated in : accordance with the present invention have differentiated to acquire the properties characteristic#the pancreatic endoderm lineage, Pancreafioendedenn lineage .specific markers include the -expression# one or more transcription factors such as. ter examp le. Hlxb9, FTP- la, PDX-1, ΠΝί'# 1 INF-1 beta.

The efficiency of differentiation may be determined by exposing a treated cell population: to ah: agedt.;(sneh as an antibody) that specifically reedgitipes a protein ma#er expressed by cells expressing markers characteristic of the pancmatieendoderm lineage.

Methods for; assessing: expression of protein anffnacieichcid markers in cultured or isolated eells are standard in the art. These, inehtde qoanitaive reyerse transcriptase polymerase chain reaction (RT-PCR), Northern blots, m si(u hybridization (see. e.g., CurrentProtocols in Molecular Biology (Ausubel et&amp;l., Ms, 2001 supplement}}; and immunoassays sued m immunobistoehcmieal analysts of sectioned ntateriai. Western: biottiogi apd Ihr raarkers that are accessible inJptact eelisv flow cytometry analysis (EACH) (see. e.g,. Harlow and Lane, Using Antibodies: A Laboratory Manual, Hew York: Col:d Spring Harbor Laboratory Press (1998)).

Examples of antibodies useful for detecting certain protein markers are listed in Table IA. it should denoted that alternate antibodies directed to the same markers that am: .recognizedby·the;antibodies listed in TabielA are available,or eMbefeadily developed. Such alternate antibodies:can: also be employed for assessing expression,of markers in thecelis isolated in.accordance with the present invention.

Betectiosr of ceils expressing markers characteristic of the pancreatic endoerloe linage

Markers characteristic of cells of the pancreatic endocrine lineage are well, known to those skilled in the art,: and additional markers: characteristic of the pancreaiie endocrine: lineage continue to be identified. These markers can be used to confirm that the cells treated in accordance with the present invention have differentiated to acquire the properties characteristic of the pancreatic endocrine lineage. Pancreatic endocrine lineage specific markers include the expression;.#1 Ofeor more transcription factors such as, for example, NGN-3. MeurdD, IsleteL

Markers characteristic of ceils of the β cel! Encage ate well knowo to those skilled in the art, and additional' markers characteristic of the 0 ceil lineage continue to be identified. These markers can be used to confirm that the colls treated in accordance with the present invention have differentiated to acquire the properties characteristic of the fbeell lineage, β celtlineage specific characteristics include the expression of otic or-more transcription factors such: as, for example, Pdxl (pancreatic andduodenaf homeobox gene-1), HkxS.2, NkxbJ, Isli, Paxfo Pax4, NetuoD, finflb, ffcfi-fo ΒοίΤΕχ^η and MafA, among others. These transcription factors are wellestablished in: the ait for Identification, of endocrine cells, See,e.g:,, Edlund (Nature Reviews foenetics 3: S24T>33 (2002)).

The efficiency! of differentiation may be determided by exposing a treated cell population to an agent (such as an antibody) drat specifically rccopiises a proiem nwrkcr expressed by cells expresstng markers eharactefistic of the pancreatic endocrine lineage, AlSentaiiyely ,. f^e0ctcacy:.Qfidiffei!ddfi^foft may be determined by exposing; a treated cell population· to ah agent (such as an antibody ) that specifically recognises a protein marker expressed by cells expressing markers characteristic of the | cell lineage.

Methods for assessing expression of protein and mieleie acid markers in cultured or isolated cells are standard in the art, These include quantitative reverse transcriptase polymerase chain reaction (RT-PCR), Northern b!o%in situ- hybridization (see, e.g., •Current Protocols in Molecular Biology' (Ausubdl el «/,, eds, 2001 supplement)), and immunoassays such as tmmnnqhistochemlcai analysis of sectioned material, Westefn blotting, and for markers that are accessible in intact cells, flow cytometry analysis (FAC'S » t ace, e.g,, Barlow and Lane, Using Antibodies; A Laboratory Manual, Hew York; Cold Spring Harbor Laboratory Press (1998)),

Examples of antibodies useful for detecting certain protein markers are listed in Table M, It should be opted that alternate antibodies directed fo the same markers that are recognized by the antibodies listed in Table IA are availabie, or can be readily developed. Snob alternate antibodies can also be employed for assessing expression of markers in foe cells isolated in accordance with the present mvention,

The presentinvention is farther illustrated, bnt not limned by, the following examples.

Human Embrworstp Stem Cell Ctdterb

Stem cells are"iuad-ilfm»iiktad.;ceUs defined by their ability at the single cell level to both self-rencw and differentiate to produce pfogeny cells, including seiCrenewing ptdgefiitom»«©i3^^^Mg:pmgemtoiSi,Md terminally differentiated cells, Stem cells are also characterised by their ability to differentiate in vitro into functional cells of various cell lineages from Multiple germ !ayepr(endoderrn, mesoderm and ectoderm), as well as to give rise; to tissues of multiple germ layera following transplantation and to contribute substantially to most, if ndt ail, tissoes following :ln|ection into blastocysts.

The hitman: embryonic stem cell lines HI,: id? and H9:were:obtained from WiCell Eesearch Institute, bic,, (Madison, WI) and cultured according to instrnefiotss provided by the source institute,: Briefly, cells wereeuituredipn mouse embiyomc ftbmblasf (MEF) feeder cells iuiESsell medium consisting of DMEM2F12 (Invltmgen/QlBOO) supplemented with 21¾¾ Imoekout serum repiaeomeot, 10(1 nM MEM nonessential amino acids, OJ mM beta mremaptoefhanoi 2mM L-glntamine with 4ng/ml human basic fibroblast growth lac^r^bF6F)|^lifr0ml».vib?e^ea/0IBCD^ MEFeelis, derived from El3 to 13.5 mouse embryos, were purchased from dharles River, MEF ceils were expanded in DMEivt medium .supplemented with 1-0% FBS (Hyelone), 2mM glutamine, and 100 mM MEM nonessentiai amino acids, Sob-confluent MEF cell cultures were treated with Hfog/mi mitomycin € (Sigma, Si. Louis, MO) for 3h to arrest cell division, then Uyp.siuized and plated at 2x 10' /cm" on 0.13¾ bovine gelatin-coated dishes. MEF eel Is from passage two through four were used as feeder layers, Human embryonic stem cells plated on MEF ceil feeder layers were cultured at 3?°G in an atmosphere of 5% CO# within a humidified tissue culture incubator. When confiuentfapproximaieiy 5~7 days after plating), Ituman embryonic stem cells were treated with Intg/ml eollagenase type IV (Inviimgen/CIBCO) for 5-10 min and then getiiiy scraped off the surl^co uMng a 5-mi pipette. Cells were spun at 900 rpm for 5 min, and the pellet was resuspended and re-plated at a 1:3 to 1:4 ratio of cells in fresh culture medium.

In parallel:, HI, H7S and 8¾ human embryonicceils were also seededon plates •Coated wtE- a'1:30diiuidon of growth factor reduced MATEIGELJ^ (BD Bidseienees) and cultured in MEF-eondlgoned media:supplemented with 8ng/ml bFGF, The cells, editored oh MATEIGEE* were routinely passaged with. coliagenasoIV (ihyiirogea/GIBGC))» Diapasc (BD Bioscieuces) or Liberals enzyme (Source). Some of the: human embryonic: stem eel! cultures were incubated under hypoxic conditions (approximately 3¾ (¾).

Example 2

Pefivatidn anti Clulafe of Ceils Expressing Plurfpoieney Markers, Perwed Irom

Homan Embry oak Stem Cells

Cells from the human embryonic stem cell lines H i and M9 various passages (Passage 30-54) were: editored under hypoxic condifibp (approximately 3%£E) for at least three passages. The cells: were cultured lit MEF-CM supplemented with 8 ug/mi of bFGF sad plated on M ATIilGEL coated plates according to Example 1.

Cells were their treated with I3MEM/FO medium suppjomented with 0.5¾ FBS, 20 ng/tel WNT-3a (Catalog# i324-WM*002s R&amp;£> Systems, MN), and 160 ftg/ml ActivM-A (R&amp;P Systems, MN) fof twb days followed by treatment with DMEM/E 1:2 media supplemented with 2% FBS and: 100 nghnl Aeiivin--A (AA) for an additional 3 to 4 days. This protocol resulted in si|prMcant hpre^iation of definitive endoderrn markers.:

The cells were then treated with TrypEETS4i Express solution (Invihugen, CAj for 5 mips. Released eells were tbSuspended in BMEM~E12 + 2% FBS medium, recovered by cenmiEsgafion., and counted using s hemoeytometer. The released cells were seeded at 1000“ 10,000 cellstem4 on tissue culture polystyrene (TCPS) treated flasks and cultured in DMEM-F12 t 250 FIS T 100 ng/ml activin-A * 20 ng/rn! WNT-3A under hypoxic conditions (approximately 3% ()?) at 37 *£·:m standard tb&amp;iteeldture ineufeamr. The TCPS flaks were not coated with MATR1GEL or other exterceliular matrix proteins, 'fhe media was changed daily . In some o»ifur^The;mdiiawas.:Furffe'aapplemoated''Witfr 10-50 hg/mi of IGF-l·(insulin growth faetoAl from E&amp;D Systems, MN) or IX ITS (Insulin, transferrin, and selenium Bonrinvitrogen, €»). In some ofthe culture conditions tile basal mfedia (DM-Hii 12% FBS) was foriliersupplementecl with CM uiM mercaptoethanof (invitrogeu, €&amp;.) and sOnAis&amp;ential amino adds (1¾ NB&amp;Airom 'imdtroge.0vC.AK

Following S to 1 $ days of otdtnrmg, dlst&amp;cf pell colonies appeared surrotthded fey a .large number of enlarged cells that appear to fee in senescence. At approximafely 50 .to .60% conflnenCy, the cultures wdte passaged fey exposure to TiypLEW Express solution for 5 mins at room temperature. The released: cells were resuspended in DMEIVfeFi.2 + 2% 'FBS medium, recovered by centri&amp;gation, and seeded at 1.0,000 celis/emd on tissue treatedilasks in BMEM-F12 + 2%FBS + 100 ng/rataetmm A + 20 ng/mi: V^H'F^SA +/- 50 ng/'ml of 1GF~I> This media, will be (briber re&amp;tred io as the “growth ipedi'd* '.

Example 3

Berivaion of Cells EXpressintg Elnrlppteaey Markers Iron? a Single CeO Suspension of Human Embryonic Stem Cells

Ceils ftom'tSerhnm^/^biyiQBife -stem cell lines Η ! P33 and FS9 P4§ were cultured under hypoxic condition (approximately 3% O;·) for at least three passages. The cells were cultured in MBF-CM ^plcmented with B og/ml of feFGF and plated on MATRlOEL coated plates according to Example L At approximately 40% coofiueney, the cultures were exposed to IwpLE m Express solution (invitrogen, CA) lor 5 mins. Released cells were resuspended in BMBM~F!2 + 2% FBE medium, recovered by centrifugation, and counted using a herooeytometer; The released ceils were seeded "af 1000::'to 10,000 eeils/cnr on tissue eulture/pplystyrene (TCPS) treated flasks and:cultured: In DM-P.12 + 2% FBS x l00: hg/ml aettvin-A 4 20 ng/mi WMT-3A + 50 ng/nt.l of IpF-l + OJ mM mereaptoethanol (Invitrogeu, CA) and non-essential amino acids (;X, felEAA from lofeitrogem GA) under hypoxie coudifions (approximately 3% 0¾) at 37 *C in standard tissue culture iucufeator. The TGES Basks were not coated with MATfeRoEL (mother extarcelluiar matrix proteins. The media v^rohbn^l:tjN:!ly2 The Erst passage cells are referred to as P L

Example 4 for Expansion of Cells Expressing Flnripotone^ Markers Derived from Htmtan Embryonic Stem Cells·

Cells expressing plaripoteitey markers derived from human ombtypmc stem cells have been: soccessfully oiftorod: m lheyfoilowing media coBrpositions for at least 2-3(1 passages:

1. 0M-F12 -1-.¾% FBS:s· H)0 ng-nu ΛΛ -t- 20 ng/ml WNT-3A 2. DM-F12 + 2% FBS a 100 ng-nri AA. + 20 ng/ml WNl-M-P 50 ng/ml IGF-1 3. DM-F12 4 2% FBS: e- i 00 ng-ml AA :- 20 ng/ml WN133A-H lOngml 1(11--( 4. DM-F.12 -r2% FBS - 50 ng/ml AA -:- 20 ng/ml. WNT-3A f 50 ng/ml KJF-j 5. DM-FI2 -I- 2% FBS + 50 dg/ml AA t 10 ng/rni WNT-3A + 50 ng/ml IGF-! 6. GM-F12 r 2% FBS + 50 ng/ml A A t 20 ng/m! WNT-3A f 10 ng/ml IGF-! 7. DM-FI2 : 2% FBS a 100 ng/m! AA -:- 10 ng/ml WNT-3A : Ϊ0 ng/mi IGF-! 8. HEScGRG defined media (Gbemicon, GA)

Tire basal component of the ahoye: listed media may be replaced wi&amp; similar media such asiRFMI, DMEM:, CRM 1.,, Knockout and F12.

Exam pid 4

Effects of Iiiislbitbrs of GSK-3fl Enzyme Activity on the Viability of Cells ExpressMg Piarlpotency Markers

Derivation add maintenance of cells ekpm%iBg plaripoteiicy makers was dondneted as Iras been described in Ixainple 2. Celia were grown in £$vtEM:F]2 supplemented with 2% FCS (iBvifrogen), 10:0 Bg/ml Aetivin A, 20 ng/ml WnW3as and 50 .ng/ml IGF(R&amp;D Biosystemsb Cells were seeded at a density of 10,000 ceils/cnr on Falcon polystyrene .flask.*· and grown in r»m>kwr culture M 5% €0>, jew oxygen. After reaching 60- 70% confluence, cells were passed by washing the monolayer withPpS and inenbadag with TrypLE {invitregen} fbr 3-5 minutes to allow deiactonent and single cel! dispersal

Screening was conducted using test compounds front a proprietary library of srnal 1 molecules: selected for their ability to inhibit GSf%3:B emsyree activity, Compounds from this library wore made available as IniM stocks, ma 96-well plate formatin ADmM HBFES, 30%; DMSO. For assay, cells expressing pluripotency markers were washed, counted, and plated in normal culture medium a t a seeding density of20,000 cells per well in 96-wcll Clear-bottom, dark-well plates (Gostar), This seeding density was previously determined to yield optimal monolayer formation in overnight culture. On the following day, culture medium was removed, ceil monolayers were rinsed three times with PBS, and test compounds were added to the wells in SOn! aliquots, each diluted into assay medium at a final assay concentration of CM day 2 of the assay, medium was removed from each well and replaced with: a fresh aliquot of test compounds diluted into assay medium. Assay medium on days f and 2 of culture consisted ofBMEM:F12 suppiemeuted with ft.5% FCS and JOOngmd Activiu. A., On. days 3 and 4 of culture, medium; was removed from each well and replaced With OMBM;P12 supplemented with. 2% FCS and .lOOngtod Activln A (no test compound); Oft day 4 of assay, ISulof MI'S (Fromega} was added to each well and plates were .incubated afd?^ for 1.5 to: 4' hours priori» reading optical density at 490 am on a SpectraMax iMoleeular Devices) instrument Statistical measures consisting pfmsah, standard deviation, and coefficient of variation were eaicidatedVfOP«^h.dupIicide'-def:, Tbxieity was calculated: for each test well, relative to a'posuive eontrol (wells treated witlt Actlvin A and WntSa on. days 1 and 2 of culture).

Table ΟM a compilation of all :screening resul ts, Gelis cx:pfds§ing.pl«rippfonoy:'mark#§' were plated initially ns a coniueni monolayer in this assay; hence, tire results are representative of a toxicity measure over the four-day culture period:. Results are expressed as percentage viability of control, and demonstrato variable toxicity for some compounds at the lO ttM screening concentration used, A larger proportion of the compounds have minimal or no measurable toxicity ip this cell-based assay. A small: panel of select compounds was repeat tested over a iiarrow: dose titmtion range, again using cells expressing pluripotebcy Markers m a similar assay as described above. Table III is a summary of these resuitei: demohsiteting variable dose titration effects for a range of toxie and .non-toxic compounds.

Effects of inhibitors of GSK~3p Eniyme Activity on the Biffereatistion and Proliferation of Human Embryonic Stem Cells BeiermSued nslog &amp; High Content

Screening Assay M8mtonane^.»fhoman^^!Ota«,^mtcelMH$:fec)'^:Coa4uctodasJs^^4'1n: Example 1, Colonies of ceils were maintained in an undifferentiated, pi.unpotent state with passage on avenge every four days, Passage was perfotened by exposing cell eidtures to a solution of eollagenase; fl tng/mi; Sigma-Aldrich) for 10 to 30 minutes at .37°C followed by gentle sefoping with a pipette tip to recover cell clusters. Clusters were allowed to sediment by gravity, followed by washing to remove residual eollagenase.

Cell clusters were split at: ail 3 ratio for routine maintenance culture or a I ;1; ratio for immediate assay , The Imman emhryoole stem eell lines used were ntamiaioed at passage : numbers less than passage: SO and, routinely evaluated: for normal karyotypic phetTOtype and absence of mycoplasma contamination.

Cell dusters used in foe assay Were evenly resuspended in normal eulMfemedinm and plated onto MATRlGlL-coated 96-well Packard. V1EWPLATES (PerkinElntef) in volumes of lOdpl/welb hfEF eondhfoaed medinm supplemented with tog/ml bEGF was used; for initial plating and recovery. Daily feeding was conducted % aspirating spent vulture medium from each well and replacing with, on equal volume of fresh medium. Plates were Maintained at 3?e€, 5¾¾ €(¾ id a humidified box throughout the deration pf assay.

Screening was conducted «sing test compounds fmm a proprietary library of small .molecules selected for their ability to inhibit GSK-3B enzyme activity, Compounds from tins library wmc made available as IrnM stocks, in a 96~wcll plate format in SOrnM IIEPES, 30% DMSO, Screening compounds· were tested in triplicate or duplicate sets. Primary screening assays were Initiated by aspirating culture medium from each well followed by three washes hr1 PBS to remove residual growth factors and serum. Test: volumes of:8t) to I ΟΟμΙ 'peti.weE· were: added back containing 'DMEMrFi 2 base medium (Invitrogen): supplemented with 0.5%: FCS (ByCbne) and IO0ng/ml activin A (R&amp;D Biosysteres) plus iOpM test compound. Positive control wells contained the same base tuediumssubstitntiug l,O%0ng/ml :Wnt3a (R&amp;D Biosystems>fer tlte test compound, Negative control wells contained base tnediam with 0.5% PCS and activin A alone (AA only) or alternatively, 0,555 FGS without aotivi»:^:-0rAV»t3ll|ttbii*caiment), Wells were aspirated and hid again with identical :SoIntiom b« day 2 of assay. On. days 3 and 4, all assay wells: were aspirated and convened to DMEM;F12 supplemented with 2¾. PCS and l.O0ng/rni activin.A. (without test:compound, or WntSa); parallel: negative coufroI wells were maintained in DMEM’F 12 base medium, with 255 ECS anti activin A (AA only) or alternatively, 2% ECS without activin A (no treatment).

At the end of culture, cells In 96~weil plates were fixed with 4% paraformaldehyde at room temperature for 20 minutes, washed three times with PBS, and then perrneain lined with 0.5% Triton X-100 tor 20 minutes at roont temperature, Alternatively, ceils were fixed with ice cbid 70% ethanol overnight at -20%', washed three times with PBS, and then penneabiIked with Triton X-100 for 5 minutes at 4°C. Alter fixing and permeahilming, cells were washed again three times with PBS and then blocked with 4% chicken serxun (Invitrogen) th PBS for 30 minutes at rootir temperature:. Primary ahfihddles (goat: anti-hurnan. Sdxl? and goat ah.d-humah::pN:E--3betat R<&amp;D Systems) were diluted 1:100 in 4% chicken serum and added to ceils for one hour at room temperature. Alexa Fluor 4ht conjugated secondary antibody (chicken:anti-goat Ig©;; Molecular Probes) was dinted 1:200 in PBS and added after washing the cells three times with PBS. To eoiniterstain rmeleg 5 rnM DraqS (Alexis Biochemicals) was added for five minutes at room temperature, ©ells were washed once with PBS and left in 100 mi/well PBS for imaging.

Ceils were imaged using an IN Cell Analyzer 1000 (GEIfealthcare) nitlkipg the 5l0O8bs diehroic for cells stained'with Dra.q5 and Alexa Fluor 4¾ Exposure times were: c^mizecl ;using; a positive: control wd!h; and wells with secondary only for untreated negative controls. Twelve fields per well were obtained to compensate for nay' cell loss during the traatnlent and: staining procedures, 'tota! celSin ontbers and total cell intensity ibriox-1? and BNP-3heta were measured using tbs HM Ceil Developer Toolbox ..1 ,·;6·COE Healthcain} software. %gnfoniation for the nuclei was determined based on grey-scale levels (baseline range 100-300) and nuclear sis?. Averages and standard deviations were ealculatedior replicateSv Total protein expression was reported as total i ntensi ty or Inlegrated inteMty, defined as total ftnoreseence of the cell ilrnes afoa of the cell. Background was eliminated based on acceptance eriteri a of grey-seale ranges between 300 to 300(} and form factors greater than or equal to 0.4. Total Intensity data were normalized by dividing tbe total intensities for each well by the average total intensity for the Wntla/Activin A positivecontrol, fformalined data was calculated lor averages and standard deviation lor each replicate set. summary of all screening results. Table ¥ is a list of hits iiom this screening. Strong bits are defined as greater than or equal to 120% of control 'values; moderate hits are defined as foiling within the interval of 60-120% of control 'values. A significant number of compounds induce both a proliferative response in this assay. In parallel* a significantnumber of conipdnnds induce diiforehtiahon in this assay, as tnensured by the protein expression Of Sox 17 and Hnfo3b transcription fitetors.

Example 6

Effects of InhililtPrs of GSK»3p Enzyme Activity on the Proliferation of Human Inforyeme Stem Ceils Defermlneil using a Plate Meatier Assay

Maintenance of human embryonic s tem cells (B9 or H i lines) was conducted as described in Example 1. Colonics of cells were maintained in an undifforenfiated, piuripoteni state with passage on average every four days. Passage was performed by exposing cell cultures to a solution of collagenase (1 mg/ml; Sigrna-Aldrich) for 10 to 30 minutes at 37¾ followed by gentle scraping with. a. pipette tip to recover cell clnstem. Clusters wem allowed to sediment and washed to remove residua] collagenase. Cell clusters were split at a ratio of! :3 monolayer area for routine cul ture or a 1:1 ratio for irnmedi#e assay. The human embryonls stem cell hnes usedfeifei® examples were maintained at passage numbers less than 50 and routinely evaluated for normal karyotypic phenotype as well as absence of ntycoplasrn conmminabon.

Cell clusters used in assay were evenly resuspended in normal culture medium and plated id1^'M^^KiEL^hted;^vwdil. Packard VIEWPLATES (FerkipElmer} in volumes of i0Oul/welL MEF conditioned medium supplemented with 8og/ml BBSP) was used lor initial plating^andreeoyety. Daily feeding conducted by. medi um from each, well and replaeing with M· equal vol time of fresh medium, Plates were maintained at;3:7^C in a humidified box, 5% CO? thmugbpuf the duration of assay.

Primary screening: assays were initiated by aspij^d.ng.c»itwm-^^ispi:fno«i. each well followed hy three washes in PBS to remove residual growth: factors and serum. Test volumes of SDDQOpi per well were added back: containing: DMEM:F13 base medium ^|«:yi^geJt).suppi«m^ted!'Wl'di,0..5^VFiCS (ByCfene) and lOOng/ml aetivin A (R&amp;D Bidsystems) and IfoiM test compound. Positive control wells contained the same medium substituting j0A10ng/mi WntSa: (Ib&amp;D Biosystems). Negative control wells contained base meditifn with. 0,3¾¾ PC’S without;aetivin .A or Wnt3a, Screening compounds were tested in triplicate, Wells: were aspirated and fed again with identical solutions oh day 2 of the assay. Oft days 3 and 4, all assay wells were aspirated and converted; $>-pMEM:F J2 knppfemente4vwii:h:''2%'.FCS and 100og/ml aetivin A with the exception of negative control wells, which were maintsmedin DMEM'FO base medium with 2% PC S.

On day 4 of assay , I S-20 nl of MTS (Promega| was ad ded to each, well and plates were Incubated: at 37°C for U to 4 hours, Denxliometrlc readings at CJD490 were determined using a Molecular Devices spectrophotometer plate reader. Average readings for replicate sets were calculated along with, standard deviation and coefficient of variation. Experimental Wells were compared to the Aetivin A/Wht3a positive control to calculate a percent control value as a measure of prplifemtion.

Table VI Is a representative summary of alf screening msolts, Table VII is a list of bits from this screening, Stronghlts. are defined: as; greater than or equa I to 120% of control values; moderate .hits Ore defined as falling within the Interval of 00-120% of control values, A significant number Of compounds kduce.^p.fol.tT2^^0;tesp6ase. in this assay.

Example 7

Effects of<5SK«3p Enzyme Inhibitors on the PifferostNation and Tholiferatlon of Ilnman Embryonic Stem Cells : Bose Tifmtinn of Lead Compoands

It was important to confirm the activity of hits identified from primary' screening, and fttrther analyze the range of activity by dose titration. New samples of a selective subset of primary screening hits were obtained as dry powders, sohfoiiized to make fresh stock reagents, and diluted into secondary confirmation assays to evaluate effects on biiman embryonic stem cells.

Cohort? of two human embryonic stem cells (HI and H9) was conducted as described in Example t. Cioionies of ceils were maintained in an undifferentiated, piurt potent state on Matrigei1^ flnvitmgebfoeoated: polystyrene plastic, using:a '1:30 dilution of Matrigel1*In 0MEM:FI2 to coat the surface, Cells Were split by epyfolbc passageevery four days on average. Passage was performed by exposing cell monolayers tosolution of collagebase (1 mphf;;: Sigma-Aidrich)dor '1:0 to 60 minutes at 37~C followed by gentle scraping with,.a pipette tip to recover cell clusters. Clusters were Allowed to sediment by gravity, then washed to; remove residual coliagenase. Cell: clusters were split at a 1:3 ratio for maintenance cnlturc or a 1:1 ratio for subsequent assay. The human embryonic stem cell lines were maintained at less than passage 50 and routinely evaluated for normal karyotypic phenotype and absenceof mycoplasma contamination,

PrefnirfflkM· of ceils for ussimcCeil dusters of the HI or HO: human embryonic Stem eel! lines used in the assay were evenly resuspended in culturefo.edium:ahd plated onto |yfatrigel:iM-coated 96«well Packard VIEWPLATES:(l3erklnEi.nreri in volumes of ΙΟΟμΙ/well. MEF conditioned medium supplemented with bng/ml bFGF was used for initial plating and expansion. X>ai!y feeding was conducted by aspirating spent culture 'medi^avfew'wh well and replacing with: an equal volume of imsh niedtom. Cultures were allowed to expand ode to three days after pMIng prior to inflating assay. Plates were maintainod at 3?°0, :5% €0¾ m a hqrnldi&amp;d box for the duration of assay .

Preparation of compotmdsmd assay medium; A subset of hits resulting from primary screening was used for follow-up study and subsequent secondary assays. Twenty compounds available as dry powdeto wereisoinbilized as itoM stocks in DMSO and stored; dessicatsd at ~2Φ€. until use, Immediately prior to assays compound stocks were dilated 1:1000 to ntakedOuM: test, compound. inDMEMT 12 base .medium (luvitrogen} supplemented with 0,5¾¾ PCS iByCIonet and lOOug/ml Aetivin A (Rt&amp;D BiosystemsX This was further diluted;'two-lbld in wpcs id make a seven pdfpi d||idi%Pwve,f6f e^-ch compound, also in DMBlrirf 12 base mediuto with 0.5% PCS and 1.00n.g?'ml Activin A.

Semndary screening assay: Assay was initiated by aspirating culture: medium from cell monolayers in, each well followed by three washes: in PBS to remove residual growth factors add serum.. Test volumes of 1 0%1 per well Were added back containing medium with 0.5% PCS and difterent concentrations of inhibitor compounds with 100ng/mi Activin A, without Wnt3a. Positive control wells contained the same base medium with 0,5% PCS and with 20ng/mi Wnt3a (RAD Biosysiems) In the absence of test compound, Negative control wells contained the same base medium with 0.5% PCS, in the absence of Activin Af Wotda, or test compound. Assay wells were aspirated and fed again with identical concentrations oftest compound or control solutions on day 2 of assay. On days 3 and 4, all assay wells were aspirated and fed with DMBM;P12 supplemented with 2% PCS and IO0ng/nd Aetivm A in the absence of both test compound or Wnt3a. Parallel negative control wells were maintained on days 3 and 4 in ΠΜΡ:Μ;Ρ 12 base mednun with 2% PCS.

Assayemiuatim; At the end of culture, cells i»:96~weii plates were washed twice with. PBS then, fixed with 4% psrafomsaidehyde at mom temperature fbr 20 minutes- washed three times more with PBS, and then permeabilked with 0,5% Triton X-100 for 20 minutes at mom temperature. After fixing and. penueabilixingi ceils were washed again three times with PBS and then blocked with4%:dhieken serum. (Invitrogeh) in PBS for 30 minutes at. room temperature. Primary antibodies (goat anti-human Sox ! / ; R&amp;i) Systems) were diluted 1-:-100 in-4% chicken serum and added to the ceils for one hour at mom. temperature, Al^c4;:FUtof40e0®^¥gittedi.i^»!daiy antibody (chickeu anO-goat lg€h Molecular l*rOfees) was diluted 1:200 in PBS and added to each well after washing the cells three times: with PBS. To eounterstain nuclei* 2pg/nilifoechst 33343 (luvitrogen): was added. ^ten.':t»mute8-M::room-tent^ralnre;. Cells were washed, once with KiSaod lefoin Hit)gl/weil PBS tor imaging.

Cells wore imaged using an IK'Cell Analyzer 1000 (GBIlealfhcare) utilizing Ole 51 OOSbs diehrotc for cells stained withEoeehst: 33342 .and Alexa Fluer:4&amp;8. Exposute times were optimized using positive control wells and:wells stained with secondary: antibody alone as ah,nntmated negative control, Imagesfitsoi 1.5 fields per well were: acquired to compensate for :auy cell loss during the treatment and staining procedures.

Measurements: for total cell number and total SoxT7 intensity were obtained for each well using IK Cell Developer 'toolbox 1.7 (G£ Healthcare) software, Segmentation for the nuclei was doterttuned based on grey-scale lewis (baseline range 100-300) and nuclear size, Awmges and standard deviariohs wem ebleidaied for each replicate data set. Total Sox 17 protein expression was reported as total intensity or Integrated intensi ty, defined as total fiuoreseen.ee of the cell times area of the cell. Background was eiminated based on acceptance criteria of grey-scate ranges b^ween 300 ίο 30OO and form factors greater than or equal to 0.4, Total intensity data were normalized by dividingthe^ tcdaf mtensities for each well by the average Iota! intensity for the Wnt3aMetivln A positive control. Nororaltxed data were calculated for averages and standard deviations for each replicate set

Results

Results are shown for eight G:Sfo-3:B: enzyme inhibitors where activity was confirmed: add potency was determined by titration: in this secondary assay. Data: presented show compound effects on cell number and Sox 17 intensity where respective data points were averaged from a duplicate set and mined lor each parameter from identical fields and wells. In this example, Sox! 7 expression is indicative of definiti ve endoderm differssntiati.^a... Results for cel! number and Sox 17 intensity, respectively:, using the HI human embryonic stem cell line are shown in Tables VIII anti DC, Rosiilis lor the H9 Jmhian embryonic stem cell Imouce Shown in Tables X and XI, Positive control values; were nonmlfeed to L0OO tor cell number end Sox i? Intensity. Negative eontrol vaiues: were lessTfeau bJRR lor ceil number and les$~lhan 0.065 iprSoM? Intensity with, both eel 11 lues, A graphic: ppritaya!: of these data, comparing both human embryonic stem cel! lines and including a: dose titration of each compound, is: provided in Figures I. in S. Cell number is pibsetvted in panel A; Sox. 17 intensity is shown in panel B, Those data confirm that each compound can promote IBS: cell proliferation and defrmdve endoderm differeatiatioft arid, identify ah optimal range of activity.

Examples

Effects of GSfiriSpEnxymC Inhibitors on the Expression of Additional Markers Associated with DeiMMvc Endoderna

It was Important to deoionstrate that lead compounds could also induce other markers indicati ve of definitive endoderm differentiation, in addition to the transcription factor S0xl7< A select· subset of bits was tested tor their ability to promote expressiori of 'CXCRd, o^tf«oo::«oe^ioC:piOfcm, and I:INF»3 beta, afranaeription factor also associated with definitive endoderm differeMiaiiOn,

Preparations^ oeUs jot assay: Cell duSters: from, the Hit human embryonisstem eel! line used in. the assay were evenly resuspended in. culture medioor and plated onto MAIRlGILlMmdafed (f :30: diiution) 6~wel| plates (Coming) in volumes of 2 nb/wcli. MEF conditioned medium supplemented with 8ng/ml feFGF was used tor initial, plating and. expansion, Gaily feeding was conducted by aspirating spent; culture medium from each well and,replacing· wi th, an equal volume of fresh medium. Chiitures were allowed,to expand one to three days after plating prior to initiating assay. Elates were maintained: at .37% 5% €02 for the duration of assay ,

Preparatimpfeompounds and assay medium: A subset of seven hits resulting from primary sefeeriing: was used for fbllow^up study and suFvmmnfsecondary assays,: Meat compounds were lixed as lOroM stocks in DMSO and stored dessieated at ~20°(2 and! use. Immediately prior to compound stocks were dl&amp;ted to a final concentration ranging between IpM sndduM in 0Ι9ΙΕΜ:Ε12 base medhnn fln.vitregen) dapfifeaiMfe4^i^:'!Eb:S%-.FCS. (I iyCioderaod IbOiigdrsi .Activity A. (R$£D; Biosysiems), T^e assay was Jttitiated by aspirating culture medium from cob monolayers in each well followed by three washes in PBS to remove residual growth factors and serum. Test volumes of 2ml perwell were added back containing medium with 0.5% PCS and different eoneenmatioM of inlnbitor compounds with I00ng/mi Aetivtu A, without Wat3a, Positive control weds contained the same base medium and b.S% PCS with IbOngbnl Aetivin A and 2flngdni Wnt3a(RM> Biosyslems) in the absence of test compound, Negative control wells contained base medium with 0,5% PCS, in the absence of Aeti vin A, Wut3a, or test compound. Assay weljs were aspirated and fed again With identical concentrations of test compound or control solutions on day 2 of assay , On days 3 and 4, all assay wells were aspirated and; fed with BMEM:P12 supplemented with: 2% PCS and lOClng/m! Activity A in the absence of both tost compound or Wni3a. Parallel negative control wells were maintained on days 3 and 4 in Dfdl;M:F12 base medium with 2% PCS. AMaP evaluMian; At the cud of culture, cell monolayers were washed with PBS and harvested from culture plates by incubating 5 minutes with 1rypld2*M Express solution (Invitrogen, CA). Ceils were resuspended in MEP conditioned otedinm. and ^lit into two equal samples, One setof samples was further stained with various fluorescent labeled antibodies and sulgected to flow cytometric (EACSV analysis. A second parallel set of samples was subjected to quantitative PCR.

Cells for FACS analysis were washed into PBS and blocked for 15 minutes at 4c>C in 0. 125% human gamma-globulin (Sigma eat# 0-438# diluted in PBS and BD FACS staining buffer. AKquoia of cells {approximately 105 cells each) were stained fer 30 minutes at 4*C with antibodies direrely conjugated to a fluorescent tag and having specificity lor CD9 PF. (BD#5553?2k CD99 PE te&amp;liagdM11(.:09904), or CXCR-4 AFC (R&amp;D Bystcnts cat# ΡΛΒ17 3 A). After a series of washes in BB FACS staining buffer. ceils were stained: wife 7-AAD (BD# 559925) to assess viability and analyzed on a BD FACS Array in$irament(BD BiQseienc8s)^C0iiee{ing at least 10,000 events. Mouse IgChkisotype control antibodies for both PP. and A RC were used to gate percent positive cells, ••Celts teqi^^itattve PCR vvem-f^^sed^i-'llNA extraction, puriEeation, and eDNA synthesis. RNA samples were purified by binding to a silica-gel membrace {Rueasy Μάη Kit, Qiagen, CA) in the gmseneedfan ethanol-containing, high-salt buffer followed by washing to mmove cqntannnants. The RNA was further purified using a TURBO DMA-.f^fe.t.fAjOsbbttv-'fec.)» and high-quality RI4A was eluted in water. Yield and purity were assessed by A2b0 and A2M rbadings on a speotroptotbineter. cD A A copies were made tom purified: ENA using an Applied Biosysietnaj Inc, (AB1, CA) high: capacity cDNA archive kit.

Unless otherwise stated, all reagents lot real-time FOR ampli&amp;atbn and quantitation were purchased from AM, Real-time PCR reactions were performed using the ARi PRISM 7900 Sequence Detection System, XAQMAAI UNIVERSA1. PCR MASTER. MIX (ABI, CA) was used with 20 ng of reverse: transcribed RNA iit a total reaction volume of 20 μΐ, Each ePNAstuppie was run ,in, duplicate to correct for pipetting errors. Primers and RAM-labeled TA:QMAN probes were used at:concentrations of200 PM.

The level of expression for each target gene was normalized using a human giyccra!ilehyde-3~phosphate dehydrogenase (G APDH) endogenous control, previously developed by ABL Primer and probe sets are: listed as follows; CXCR4 (HS00237052¾ GAPDfl C43108S4E), HNF3h (H^^7^)/SOX0Xp^Kvpai?£l4:S(^25,::fc;^d and reverse: part #4304971),

After an initial incubation at 50°C for 2 nim Mlowed by 95°€ for 10 :m%: samples were eyeled 40 times in two stages, a denaturaiioa step at 95*C for 15 sec ©flowed by an atmeaiing/estension step at 60rtC for 1 min. Data analysis was canied out using GENEAMP 7000 Sequence Defection System software, For each pnmer/probe set, a Gt value was determined as the cycle number at which the Rnoreseence intensity reached a specific value in the middle of the exponential region of amplification. Relative gene expression levels were ealetdated rising the comparative €imethod. Briefly, lot each cDNA. sample, the endogenous: control Ct value was subtracted front the gene of interest Ct to give the delta Gt value iACt). The normalized amount of target was calculated .as.:2-amifitficafron to be 1.00$». effide»fey;, Final #k were expressed relative to a calibrator sample.

Eeswlts

Figure 9 displays the FACS analysis of percent positive ceils expressing CXCR4 surface receptor after treatment with venous GSE3 inhibitors. Two ePneetiiratiOns of each conipouad, ranging between ,-ΙμΜ and -%¾ are shown relative to an untreated population of cells (negative control) or cells treated with Activin A and Wnt3 (positive control). Figure if panels», fc, trod c show real-time P€R data for CXCR4, Sox 17, and HMFSbeta, which are also considered to be markers of definitive endoderm. Both FAGS and real-time PGR analysis demonstratea significant increase In each of these markers observed in differentiated eelis relative to untreated control cells. Expression levels of these definitive endoderm. markets were bifidvalent in some cases to the positive control, demonstrating; that a OSK3inhibitor can substitute for Wnt3a at this stage of /ddfereuilatiou.

Example 9

Effects of CISK~3p Enzyme Inhibitors on the Formation of Pancreatic Endoderm

It was important to demonstrate that treatment with GSK3p inhibitors during induction of definitive endoderm did not prevent the subsequent diflercndation of other cell types, such, as pancreatic bndodeAh, for example, A select subset of hits was tested %rthelf ability to promote expression of PD30 and FiM'b, key transcription factors associated with pancreatic endodemu

Maintenance of human embryonic stenvoeltsfBl and. H9 lines) was conducted as described in Example 1. Colonies of cells were: maintalned in ao undifferentiated, pluripoteat state with passage on average every foui days. Passage was performed by exposing cell eultoms in a- solution of eollagenase (1 utgmd; Sigma-Aidrich) for 1:0; to 30 minutes at 3?°C, fol lowed by gentle scraping with a. pipette tip to recover cell clusters. Clusters were allowed to sediment by gravity, followed by washing to remove residual eollagen&amp;se, Cell dusters were split at a i;3 ratio for routine maintenance culture or a 1 ; i ratio for subsequent assay. The human embryonic stem ceil lines used were maintained at less than passage 50 and routindy evaluated for normal karyotypic phenotype and absence of 'ipyd^iaxsma^c^fehamatioh, C&amp;Ppmpamtimpppsmy: Cell clusters of the H i human embryonis stem cell line used in the assay were evenly resuspended in unhung medium and plated onto MATRiOEl/^-coated .(1:30 dilution) 24-well plates (black well; Arctic White) in volumes of i ml/weik MEF eondihoned medium supplemental with Sngdnl bfGF was used for initial plating and expansion. In a second experiment, clusters of h.ES cells from the B9 line were plated in 9fowcil plates on mou^embiyOnicfoederipilEF) layers, po^kmsly inaetlvuted by treating with tnitomyein C fSigma Chemical Co). Culture medium for hES cells on M1F monolayers consisted of Dkl EM'Fl 2 with 20% fCnockout Serum Repiaeer (Invitmgeul supptemetited with rninimai esseniiahamino acids (Invttmgen), L-ghuamine, and 2«mercapioethahol. Daily feeding was conducted by aspirating spent culture medium ..froth;each, well and replacing with an. equal, volume of fresh medfunp Cifilures were allowed to expand one to three days after plating prior to initiating: assay. Plates were maintained at SAC, 5% €(¾. for the duration· of assay,

Prep&amp;miim pfeompoumis ami. mmy nmMmv A subset of eight hits resulting 'from primary screening was Used for folfowmp study and subsequent secondary assays. Neat compounds were Mlubtlined as ICImM stocks in. DhlSO and stored, dessicated at ™20CC until use. Immediately prior to assay, compoundstocks were diluted to a final concentration ranging between. ΙμΜ and 5μΜ in base medium with additives.

In this assay, GSO inhibitors were included only on days 1 and 2 of the defrnitive endoderm differentiation step, substituting for Wnfoa, Bmbryonfostem eel! cultures on MATRIGEL*'* were initiated as described in luamptes 2 and I above by aspirating culture medium from cel! monolayers in each well followed by three washes lh SPSS ip remove residual growth factors m&amp;mmm. For differentiation to-definitive dndodorm, tost volumes (0.5 ml per well for 2fe-wellpistes,: 100 μί per well for 9(5-weIL '.pfetos) were added: containing i)MESvl:FI2 ffiediom with ) 0.5% FCS and.'different eonegniraiions ciPi^biil.fefcompoutJds with i 00 ng/mf Acdviu A,: without;"Wn.t3a> .P0sidm'eontmiwA'e0ntst»ed:tit.e same base medium: with 0J% FCS and with. iC)()ng/Pit Acmdn Λ and 20ng/ml Wnt3a (R&amp;O Biosysiems) in the absence of test compound. 'Negative eohtfof wells contained the same base medium with 0.5% PCS, ip the absence of Aefivln A, Whi3a, or test compound, Assay wells were aspirated and fed. again witllMentlcai conc« compound or control solutions Op day 2 of assay* On days 3 pod 4, all assay wells were; aspirated::and fed widi0&amp;fEM'Fj2 supplemented with .2% FCS.and lObngfml .Activin A lathe absence, of both, test compound or 'WintSjii Parallel negative: control weds were maintained, on days 3 and 4 in DMBM:Fi2 base mdftatn. with 2% PCS. for differentiation. to pancreatic endodenjp cells were treated for three daySi leediPg: daily with OMEM;Pl;2 base medium. containing 2% FCS with. 0,2S;uM RAAD eyelopantine CEMD Blosciences) and 20 ng/ml FCrFf (R&amp;D Biosystems}. Cells were then treated for aft additional four days,:feeding daily with BMEM:F12 containing 3% B27 Onvitogen), 0.25 uM KAAD eydopamtt®, 2 μΜ Retinoic Acid (RA; Sigmaw^idrieh) and 20 Pgfrnl FGF7, Parallel negative control wells were maintained throughout in DMEMrFl 2 base medium with 2% PCS. (stage 2) or 1 % B27 (stage 3} and without any other addmyM,

Parallel cultures of 139 human embryonic cells were grown on MFF feeder layers, and ^ffc^fia^ad-.to.padeieatic endodenm Definitive endoderm diffefentiMion was achieved by culturing the cells in medium consistingof RPMI-1640 (Invitrogen) eOnfainlng no serum on. day 1 and 0*2% FCS on days 2 and 3 ai:o«tV^thiififerent.<^PCMtmtlons:oF' inhibitor compounds and 100 ng-rni Actiyip A. Positive control wells contained the Same base medi urn (wi th or without serum) wi th ! 00ng/ml Act Ivin A. and 20ug/ml Wai3 a (RMD Biosystems) In the absence of test compound; N egative Control' wel ls con tained, the sameibase medium: with or withoht scrum, in the absence of Activin A, Wnt3av or test Qompound:. Assay wells were aspirated and. fed again with identical concentrations of test compound or control solutions on day 2 of assay. On day 3, all assay wells were aspirated and fed wdi RPMI~ 1640 supplemented with. 2% PCS and lOOngftnl Aetivln A in the absence of both test compound and Wn8ti.: Parallel negative control wells were maintained on day 3 in RPM1-1649 base medintd widt'2% PCS. Cells were; differentiated into pancreatic endoderm by treating the ceils lor four days, foeding daily with RPMI-164Q: base medium containing 2% PCS with 0,25 roM RAAB eydopmnioe (BMP Bioseienees) and 50 ng/mlECMIO ($&amp;.P Biosystems):. Subsequently; colls wore treated for three days duration; feeding daily with APM14640 containing 1% B27 (hwitfogenk 0.25: rhM KA AP cyclopamine, 2 ®Μ RetinoiC: Acid (RA; Sigma-Aldrich) and 50 ng/ml FGFT0, Parallel negative control wells were maintained throughout in RPMI-1640 base tuediu.rn/w|th 2% FCS;(stage 2) or 1% B27fstage 3) and without any other additives, dsxqy evaluation: At the end the differendation, celts were examined us described in Example 8 for gene expression by real4h«e PCR, For high contest Bumeseenee staining? cells in 96-w.di plates were washed twice with PBS then fixed with 4¾% paraformaldehyde at room temperature for 20 minutes;. with PBS, and then peromabihxed with 0.5% Triton X-100 for 2© minutes at room temperature. After fixing and petmeahiiidng, cells were washed again three times with PBS and blocked with 4% chicken aennn (ΙηνΐίΑ^εη)in PBS for 30 minutes at room temperature. Primary antibody (goat aniFhuman Pdxi; Santa Cruz) was diluted 1:100 in 4% chiokon serum and added to eglls for two hours at room temperature, Alexa Fluor 488 cotgugaied secondary autibody (ehickeu anti-goat IgG; Molecular Probes) was diluted 1:200 in PBS and added to each well after washing the: cells three times with PBS. To eouuferstain nuclei, 2gg/ml Boeehst 33342 (lnvitogen) was: added for ten minutes at room temperature. Cells were washed bnee with PBS and left in 100 unwell PBS for imaging.

Cells were imaged using an IN Ceil Analyzer 1000 CCB Heaithcare) utilizing the 51008bs diehrme for cells stained with Hoechst 33342 and Alcxa Fluor 488. Exposure times were optimized using positive control wells and wells stained with secondary antibody alone, images Bom 15 fields per well were acquired to compensate for any cell loss during the treatment and staining procedures. Measurements tor total cel l number an d total Pdx i intensity were obtained for each well using IN Cel Developer Toolbox 1,7 (GE Healthcare) software. Segmentation for tire nuclei was determined baaed on grey»seale levels (baseline range 100-300) and nuclear size. Averages and standard deviations were calculated for each. re|>Iicate data set. Total Bdx i protein expression was reported as total intensity or integratecl intensity, defined as total B.uoroseenee: of the cell. . iim:es: area of the celt Background was eliminated based on acceptance criteria of grey-seal o ranges between 396 to 3000. Total intensity data were normalized by dividing the total intensities for eaeh; well by the average total, intensity for the Wnt3a/Aeiivin, A: positive control. Normalized data were calculated for averages and standard deviations for each replicate set.

Cells for quantitative PCE were lysed in R I T buifer (Qiagcn) and then processed for RNA extraction, purification, and cONA synibosis. RNA samples were purified by binding to a silica-gel membrane (Rneasy Miui Rit, Qiagen, CA) in the presence of an ethanol-containing, high-salt buffer followed % washing ro mmove eontaminants. The EN A was further purified using a TURBO DN A-fioe kit (Ambion, loe,), and high-quality RNA was then eluted in water. Yield and purity were assessed by A260 and A281) readings on a spectrophotometer. eBNA copies were made from purified RNA Using an Applied Biosystenm, Ine, (A Bl, CAfdugh eapacity cDNA arehive kit

Unless otherwise stated, all reagents for real-tiroe FCE. amplification and quantitation were purchased from ABi. Real-time BCR reactions were performed using the ABI PRISM 7900 Sequence Detection System, TAQMAN UN.IYP.RSA1. PCR MASTER: MIX was used with. 20 ng of isveme tjpnseribedRNA In a total reaction volume of 20 μΐ Each eDNA sample wus mn in duplicate to correct for pipetting errors. Primers and FAM-labeled TAQMAN probes were used m eouceniratiops of 200 uM. The level of expression for each targip gene was normalized using a human g!yeeraidehyde~3-plrosphate dehydrogenase (GAPDH) endogenous control previously developed by ABI, Primer andprobe sets are iisfed as follows: PDX1 (Hs0Q2308.30jhtI), GARDE (431R884B), and HNF6 (lds0l)413554 ml).

After an initial inenbatioo at fib €· for 2 rnirt followed by 95®C for 10 min, samples were cycled a denatufodon step at 95°C for 15 see followed bv ah aooealfog^xteoafoii siep at 00*0 for I pars. Data analysts was carried out using GENEAMPQ70O# Sequence DeteetloB System software» For eaehpitaor/pmbo seb value was defenained m the oyele mifober at which the fluorescence ihtensity reached a specific value: in the middle of the exponential region of amplification, Relative gene expression levels were calculated using the: eomparafive <3t melhod. Briefly, for each cDISI A sample, the ehdogbMpsfooMml.CtvdiyiO -was suhtfoeied frofe the geuo of tntereA Ct fo give the delta Ct value (ACt), The normalized amount of target was Calculated as 2* Δ€ί> assuming amplification to he 100% efficiency. Final data were expressed relative fo a calibrator sample,

Results

Results are shown for eigM ClSfo-dp enzyme inhibitor. Data presented in Figure XI from high content analysis show effects on cell number (panel A) and Pdxl intensity (pane!: B;) for the H I HIS cell line, -wheto foapectiyg::#%pofoP wofoldvd&amp;ged from a duplicate sample set and mined for each parameter from identical fields and wells. Data presented In Figure 12 frotn real-time PCR show effects of these spall molecule inhibitors on induced expmssion of two transcription factors, Pdxl and HNP6. in these examples, Pdx l aad HMFb expression are indi cative of pancreatic endodertn differentiation, GSRdp inhibitor compounds in these assays can substitute for WnOa during early stages of Cell lineage commifinent; resulting cells sustain a capacity to form pancreatic endoderm daring later sequential stages of differentiation.

Example 10

Effects of GSKsip EuzpBC Inhibitors on the Formation of Pauerentk Endocrine

Cells

It was important to demonsfrafe that treatment with; <3$K3 inhibitors during induction of definitive endpclerm did not prevent ; the subsequent differentiatipn of other cell types, sack as paiacreatic«adocriae cells» or insulin pmduciag cells, for example. A select subset of hits was tested for their ability to pfoinote o^ptosaiob of pancreatic hormones. €dlprepamti<mfi>r assay: Pancreatic endoderm ceils obtained according to the methods described so Example9fcultored on 96~wellplaies and 24-we!i plates) weto subsequently subjected to agents that cause the cells fodtffofoMiateinto panefoatte homtOtie expressing cells.

Assay for cultures Of the Hi human embryonic· stem cell iineorrMATEl@EL:i?ii was initlated as desersbed in Examples 7-9 above by ^pirating culture medium %rm cell Monolayers in each well followed by three washes in PBS to remove' residual growth factors and serum. For differentietionfodefinilive endoderm, test volumes (0.5 Ml per well for 24-well plates, 100 μΐ per wellfor 96-well plates) were added containing; medium wi th 0. 5% PCS; and. different concentrations of inhibitor compounds with 100 ngfod Activin A., without Wnida. Positive·"control wdlscontumed the same base medium and 0.5% FCS with lOQng/mI Acti vin A add.20ng/naf Wnt3.a;{R&amp;l> the absence of test compound, Negative control wells contained the same base medium with 0,5% ECS, in the absence of Activin A, W«t3a, or test compound. Assay wells were aspirated and fed again with identical concentrations of test compound or control solutions on day 2 of assay. On days 3,4, and 5, all assay wells were aspirated and fed with 0MpM:Fl2 supplemented with 2% FGS and lOOngdni Activin A in the absence of both test compound or Wnt3a, Parallel negative days 3, 4, and 5 in DMEM:FI2 base medium with 2% PCS. For differentiation to pancreatic endoderm, cells were treated for three days, ieedtug da.0y with DMEMtF !2 base medium eesdaining 2% FCE with 0.25 μΜ KAAD cyetopaMoc FEMD Btoseienees) and 20 ng/ntl FGF7 (RA.0 Btosystems). Cells were subsequently treated for four days, feeding daily with DMEM:F12 eoutainlng 1 % B27 flnvhmgeh), 0.25 μΜ fCAAD cycSopamihe, 2 μΜ Retinoic Acid (E.A; Sigrna-Aldrseh) and 20 ngfml FOF7. Parallel negative control wells dnfoig stages 2 and 3 were maintained tbroughout in 0MEM:F12 base medium with 2% ECS or 1% B27 and without any other additives. After formatfon of pancreatic endoderm, • edils : Iklldrior six days dumtion, feeding daily with DMIMtFl 2 base medium containing 1% B27 with i μΜ DAFTfgamma secreiase inhibitor· ΕΜ0

Bioscienees)and 50 ng/ral Bkeudin 4 (Siipna-A-ldrleh):, Cells were then .'treated for another three days duration, feeding daily: with DMEM;F12 base medium containing1% 82.7, SO ng/fel Exendin 4;S SO ngdal IGF (R&amp;D Biosystefes):Md: SO n:g/ml I IGF (R.&amp;D .Biosystems). Parallel negative control wefe feiMg^wtfe .01&amp;EM;F 1.2 base medium wife ISO 827 and without any other additives, -dlswaj' evaimtwm At the end: of culture» eel!&amp;, were treated as in. Examples 7 and 8 above for evaluation analysis dr real-time P€R.,

For high, conteutTIuoreseenee staining,·. cells: in OS-well plates were washed twice with: PBS then fixed with 4% paraformaldehyde at room temperature for 20 minutes, v&amp;fched three times foote with PBS, andithen pertneahilized with 0.3% Triton Χ-Ι00 for20 minutasai: mom temperature. After fixing and permeahllizing, ceils were washed again three times with PBS and,blocked wife 4% ducfcen,serum (iuvltrogen) in PBS; for 30 minnies at. room tempemtare. Primary antibody (guinea pig anti^wineinsuSin,: cross-reactive with human, insulin; DalfoCyfomation) diluted ! :500 in 4% goat serutnand added to cells for one hour at. mom tefopemture. Celia were washed three times with PBS andthenatainefi wife Alexa Fluor 4SS ©anj:ugated. secondary antibody (goat anti-guinea pig IgG; Molecular Probes) diluted 1:100 in 4% goat scrum. To eoonterstain nuclei, 2pgftnl Boechsi 33342 (tefirogen) was added for ten minutes at mom temperature, Ceils were washed once wife PBS and left in 100 glAveil PBS for imaging.

Cells were Imaged using an IN Cell Analyxer 1000 (BE Healthcare) utilising the SlOOSbs diehrote: for cells, stained wife. Booehsi 33342 ajfo:AIexa Fluor 4S8. Exposure times were, optimised nsihgipositivc eontrol wells and wells stained with: secondary antibody alone. Images ;feom 15 fields per well were aegutred: fo; eompensate for any eel! loss during the: :treaimefe:8ad.§feim«g'p«>e®dam,·. Measurement for iota! .cell number and total insulin intensity wereobtained for each well using IN Cell Developer Toolbox 1.7 (GE Healthcare) software. Segmentation for the nuclei was detenniued based on grey-seale levels (baseline range 100-300) and unclear siM. Averages and standard deviations .weie cale«Iated,for::each replicate: data set. Total insulin protein; expression was; reported as total inteasiiy or integra&amp;diimenMty, defined: as total flnoreseen.ee of fee ee 11 times area of the ©ell Background was eliminated based on acceptance catena of pey-seale rmigos between 300 to 3000. Total intensity data, were nonnahxedby dividing the total by the average total intensity for the Wot3uAeiiviu A positive control. Normalized data were calculated lor averages and standard deviations for each triplicate set.

Cells for quantitative PC St wets; lysed in R.S.T holier (Qiagenj and then processed tor RNA extractiorg puriiomtioTg and cDNA synthesis. RNA samples were pari tied by binding to a sflica-gei membrane (feeasy Mini Kig Qiagen,· CA)-in the presence of an eth anol-eontai oirsg, high -sal t buffer to S lowed by washing to nmiove con taminan ts. The RNA was farther pnribed nsing a TURBC DNA-tbse kit (Arnblon, INC), and high» quality ENA was elated in water, Yield and purity were assessed by A200 and A280 readings on &amp; spectrophotometen cDNA copies were made from purified RNA using an Applied Biosysienug Inc. (ABI, CA) high capacity cDNA archive kh.

Unless otherwise stated, all reagents for real-time P€R amplification and quantitation were purchased from ABI. Real-time PCR foacdons Were peffonafod using the ABi PRISM® 7900 Sequence Selection System. TASMAN® UNIVERSAL PCR MASTER M1 XT i A 81, € A) was used: V with 20 ng Of reverse transcribed.. RNA tn a total reaction volume of 20 ui Each cDNA. samp le was. run In. d uplicate to correct for pipetting, errors, Printers anti EAM-Iabeled TA:QMAN@probes were used: at concentrations Of 200 oM, The level of expression for each target gene was normalized, using a, human glyeersidehyde-S-phosplmte dehydrogenase fUAPDH) endogenous eontroi previously developed by ABI, Primer and probe sets are listed,as follows: PDX1 Tes(102:fo830JsI)yinsulin psO03S57?3), and OAPDB (43J0884E).

After an Initial incubation at 50*® for 2 min followed by 95*® for 10 min, samples were cycled 40 tunes in two stages, a denaturation step at 95*C for 15 sec followed by an aRneaiingfoxtensfon step at 60*0 for 1 min. Data analysis was carried out using OiNEAMP®70O0 Sequence Detection. System, software. For each primebprobe set, a Ct value was determined as the cycle number at which the fluorescence intensity reached a. specific value in the middieof the exponential region of amplification. Relative gene expression levels were calculated using the comparative Ci method, Bfiefly, for each cDNk sample, the endogenous control β value was subtracted from the geneof imprest Gj to: give the delta G( value (ACt). The normalized amount of target was calculated as 2' M i, assuming ampti.fication to be: .1.1)()% efficiency, mhdive to a calibrator sample.

Results ..Eesuitsvare shown foreight GSK-3B enzyme inhibitors. Dailipreseofed in Figure: 1.3 from: high content analysts show compound, effects oh cell number (phel A) and insulin :intensity (panel B) fe the HI hES cell line where respective data points were averaged from a triplicate set and mined for each parameter from identical fields and wells. Data presented in Eignre 14 from realtime PCR show compound effects torBdxi and insulin, fh these examples, Pdxl and insulin: expression are indicati ve of pancreatic endoderm. ;^^endPio»-;aadi;g^ierati©p. of hormonal positive cells, Selcetiye GSfeSp ihhihitor compounds in these assays can substitute for WuPa during early stages of cell lineage commitment and can induce and sustain pancreatic beta cell formation during later sepeniial stages of differohtiatiotg as evident from both, insulin iftrmnnostaining and reafrtime PCR.

Example II stive Effects of GSK-3£ Enzyme Inhibitors on theFormation ofIfeoereaUr Endocrine

Cells

If was important to dentonstrate that treatmeatmeth C5Sfv3|3 inhibitors could improve pancreatic beta cell differentiation if added, during inultiple phases: of cell fate commitment; A select subset:of hits was: tested bysequential timed addition to enhance insulin, expression associated widr pancreatic:hormonal positive cells.

Preparation $-$$IJ0hrm$ay: CeU^pepamtkmf&amp;r assay·: Pancreatic endoderm cells obtained according to the methods described in Example 9 and Ifeieulturod on 9h~ weliplsies)were subsequently subjected to agerus that cause the cells todtfferenfiato into pancreatic hormone expressing/cells.

Assay fer cultures of the Η1 human embryonic stem cel! line on M ATRlGELiM was initiated as described in Examples 7~9 above by aspirating; culture medium bom cell monolayer 1» bach well followed by three washes in PBS to remove residual growth factors arid serum. For differentiation to definitive endodetm,, teat volumes (100 μΐ per well for Jfewell plates) were added containing,medium·with 0,5%·'FOB and.different concentrations of Inhibitor compounds with 100 ng/ml Activin A, without Wnf3a.

Positive: ebntrol veils contained the same base medium and 0.5% :FGS: with SOCtog ml Activin A and 20ng/ml Wnfda (R&amp;P Biosystems) in the absence of test eoMpound. Negative control wells contained the same base medium with 0.5% FCS,in theiabsenee of Acfiyin A, Wnt3a, or test compound:. Assay wells were aspirated p:d .fed again with identical concentrations of test compound or control solutions on day 2 of assay. On days 3,: 4» and :5, ail assay wells were aspirated and hid. with DMEMfF 12 supplemented with 2% PCS and lOtog/ml Activin A in the absence of both test compound or Wnt3a,

Parallel negative conttol wells were maintained on days 3,4, and 5 in DMEM:FI2 base medinth with 2% FCS, For differentiation to pancmatlc endoderm, cells were treated for three dap, feeding daily with DMEM:F1 2 base medium containing 2% PCS with 0.25 μΜ EAAO cyeiojj^ine^^^ ng/«tl FGF7 (EM> Biosysterm).

Cells wereAnfesequmily treated for lour days, feeding daily with DMEM:FI.2 containing 1% B27 flnvitrogen), 0.25 μΜ KAAD cyelopaifene, 2 μΜ Retinoic Acid (EA; Sigma." Aldrieb) arid 20 ngferl FGF7. Paral lel negative control wells were maintained throughout in DMEM:FI2 base medium with 2% ICS of! % B27 and without any other additives. After formation feather for six days duration, feeding alfernafing days with DMEM;Fi2 base medium containing l% B27 with 1 μΜ DAFT (gamma secreiase inhibitor: HMD Bioseiences) and 50 ng/ml Exendin d (Sigma-· Aidrieh)and I μΜ TGPbeta Rl hdtifeitor II(AEKi5inl|tife>ri.iMD Bioscieno^s)., During this six day period, GSK3j3 inhibitors were added back to rbspecii ve wells, using the same concentration as previous treatment at the initiation of differentiation. Ceils were then treated tor another three days duration, feeding alfematkig days with DMEM:F12 ,h®se medium containing 154 B27, 50 ng/ml Exendin 4, 50 ug/ml IGF (R&amp;D Biosystems) and 50 ng/hri HQF(R&amp;X> BfosystefosFand 1 pMTGFheta Ri inhibitor II (ALK5 inhibitor EM© BiosdeneeS), During this three day period, GSK30 inMbitoris wore added back ho respective wells, using ifee same concentration as previous treatment at the initiation of dirierentiatiou, Parallel sets of positive oo»aoFweIfe'^^'tr^^::M':the presence or absence of 20ng/ml Wnt3a. Parallel negative eoniroi wells were maintained tiimughotti in DMEM:FI2 basemedlum with 1% B27 and without any other additives. ;4p# evaluation: At the end of euitore, ceils were treated as in Examples JO above for evaluation by high content analysis.

For high cmvient fluorescence staining, ceils in 96-weii plates were washed twice with PBS then fixed wdfhdAa paraformaldehyde at room temperature for 20 mi mrtes, washed three times more with PBS, and then pcrmeahilired with 0:534 Triton. X-I0O for 20 minutes at morn temperamre, After fixing arfo penrieabiliaing,: cells were washed again three times with PBS and blocked with: 4% chicken: serum (luvitrogen) in PBS for 30 minutes at rootn temperatare. Primary antibody (guinea pig anrifowine insulin, cross-reacti ve with human, insulin; DakoCyiomation) was diluted 1:500 in 454 goat serum and added to cells for one hour at room temperature. Fells were washed three times with PBS and then stained with Alex a. Fluor 488 cohlugated. secondary antibody (goat anti-guinea pig IgG; Molecular Probes) diluted 1:100 in. 454 goat serum. To eounterstain nuclei 2gg/inl Boechst 33342 (luvitrogen) was addedfor ten minutes at room temperature* Cells were washed once with PBS arid iefHn 100 pl/weli PBfofor imaging.

Cells were imaged using an 114 Ceil Analyser 1.000 (GE Healthcare) utilmrig the 5I008hs dichroic for cells stained with. Hoechst 33342 and Alexa Fluor 480, Exposure times were optimized using1positive control wells and wells stained with secondary antibody alone. Images: from 15 fields per well were acquired to compensate: for any cell toss during the: treatmefri and stahnng procedures. Measurements for total cell numher and total insulin intensity' were oh tamed for each well using IN Cell Developer Toolbox 1.7 (GE Healthcare) software. Segmentation for the nuclei was determined, based, on grey-scale levels (baseline range 100-300) and nuclear size. Averages arid standard deviations were calculated for each replicate data set: Total insulin preteift expression was reported: as total intensity or integraied hrtensityr defined as total fluorescence of die cell times area όί the cel l Background seas cHmln:«ted based on acceptance cri teria of grey-scale ranges between 300 to 3000. Total intensity data were normalized by: dividing the total intensities tor each well fey the average total:: mOtosity te positi ve control.: Normalised data: wore calculated for averages and standard1 deviations tor each triplicate, set,

MemiM

Results are shown for eight 6SK-3B enzyme Inhibitors. Data presented In Figure 15 fiord; high eontenf analysis show compound {panel intensity (panel B) for the H I kES cell line, where respective data points were averaged front: a trip! i cate set and mined: for each pafam eter from identical fields and we I Is, Ip this exatupSeNnsulm expression is indicative of dil&amp;'entiation to hormonal positive pancreatic cel Is. Select! ve GS113 p inhibitor compounds in these assays can substitute for Wnt3a dufifigeafiy stages of cell lineage compntmimt and, when added at later stages of difierentiation, appear to promote enhanced insulin gxjMmsioft relative to a positive control sample,

Pufelieations cited throughout this tlocoment are hereby incorporated fey reference in their entirety. Although the various aspects of the Invention have beehi;ikstm!N^b^y0.by' reference to examples and preferred embodiments, it will be appreciated that the scope of the invention Is defined not by the foregoing: description, but by· the following claims properly construed under prinelpiesof patent law.

Table ΙΑ: List of primary antibodies used for FACS and immunostamminganalysis.

Table lb: List of secondary conjugated' antibodies used, for FACS and i ramluKsstam minganai ym&amp;.

Table II; Effects of Inhibitors of GSK-3B Enzyme Activity on the Viability of Cells Expressing FI uripotency Markers.

Table II I: Effects of inlhMtors of GSK-3B Enzyme Activity on the Viability of Cells Expressing Plnopoteucy Markers,

Table IV: Effects of Inhibitors- of GSK.-3B Eozyme &amp;n ^&amp;d0em^tkuonymd proliferation of lusmaa enferyome steep. cells.

Table V, Eifects el Inhibitor®· of 0SK-3B E nxyt.no Activity on the different? abort and' proliferation. of human srnbryemc stem cells.

Table VI: Effects of Inhibitors of GSK--3B Ensyme Activity oh the proliferation of 'toman embryonic stem cells.

Table VII: Effects of Inhibitors of GSK-TB Enzyme Activity oh the proliferation ofbianatrembryohk stem cells.

Table VIII: Doee-DEPEND ANT Effects of inhibitors of GSK.-3B Enzyme Aclivsiy on the proliferation of CELLS OF THE human' embryonic stem cell LIKE HL

Table IX: Dose-DEPE N DANT Effects of KhibHots- of GSK-3B Enzyme Activity cm the DIFFERENTIATION of CELLS OF THE human embryonic stem celFUNB HL

Table X: Dusc-DEFENDA.NT Effects of InlMbitim. of GSK-3B Enzyme Activity '00 the proliferatba of CELLS OF THE Luma»' embryonic stem cell

UHL ILL

Table XI: Dose-’DEFEMDA.MT Effects ofKhibHors ofGSK~3B Enzyme Activity bathe DIFFERENTIATION of CELLS OF THE human embryonic stem ceil LINE H9.

Claims (10)

1. THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:-

   1. A method to expand and differentiate pluripotent cells, comprising the steps of: a. culturing pluripotent cells, and b. treating the pluripotent cells with an inhibitor of glycogen synthase kinase 3β (GSK-3B) enzyme activity, wherein the inhibitor is 3-[1-(3-hydroxy-propyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione.
2. 2. The method of claim 1, wherein the pluripotent cells are embryonic stem cells.
3. 3. The method of claim 1, wherein the human pluripotent cells are cells expressing pluripotency markers of embryonic stem cells.
4. 4. The method of claim 3, wherein the cells expressing pluripotency markers express at least one of ATP-binding cassette, sub-family G, member 2 (ABCG2), crypto, forkhead box D3 (FoxD3), Connexin43, Connexin45, POU domain transcription factor Oct4, sex determining region Y-box 2 (SOX-2), Nanog, human telomerase reverse transcriptase (hTERT), undifferentiated embryonic cell transcription factor 1 (UTF-1), zinc finger protein 42 (ZFP42), stage-specific embryonic antigen 3 (SSEA-3), stage-specific embryonic antigen 4 (SSEA-4), tumor-related antigen-1-60 (Tra1-60), and tumor-related antigen 1 81 (Tra1-81).
5. 5. The method of any one of claims 1 to 4, wherein the pluripotent cells are differentiated into cells expressing markers characteristic of the definitive endoderm lineage.
6. 6. The method of any one of claims 1 to 5, wherein the pluripotent cells are treated with the inhibitor of GSK-3B enzyme activity for about one to about 72 hours.
7. 7. The method of and one of claims 1 to 5, wherein the pluripotent cells are treated with the inhibitor of GSK-3B enzyme activity for about 12 to about 48 hours.
8. 8. The method of any one of claims 1 to 5, wherein the pluripotent cells are treated with the inhibitor of GSK-3B enzyme activity for about 48 hours.
9. 9. The method of any one of claims 1 to 8, wherein the inhibitor of GSK-3B enzyme activity is used at a concentration of about 100nM to about 100μΜ. 10 The method of any one of claims 1 to 8, wherein the inhibitor of GSK-3B enzyme activity is used at a concentration of about 1 μΜ to about 10μΜ.
10. 11. The method of any one of claims 1 to 8, wherein the inhibitor of GSK-3B enzyme activity is used at a concentration of about 10μΜ.