AU2017202571B2

AU2017202571B2 - Treatment of pluripotent cells

Info

Publication number: AU2017202571B2
Application number: AU2017202571A
Authority: AU
Inventors: Janet E. Davis; Jiajian Liu
Original assignee: Janssen Biotech Inc
Current assignee: Janssen Biotech Inc
Priority date: 2008-04-24
Filing date: 2017-04-19
Publication date: 2018-06-14
Anticipated expiration: 2029-04-22
Also published as: AU2017202571A1; AU2015221570A1; AU2015221570B2

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

2017202571 19 Apr 2017

ABSTRACT

2017202571 19 Apr 2017 [0001] [0001a] [0001b] [0002] [0003] [0004]

TREATMENT OF PLURIPOTENT CELLS

FIELD OF THE INVENTION

The present application is a divisional application of Australian Application No. 2015221570, which is incorporated in its entirety herein by reference.

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

- 1 of PDX-l, the pancreas fails to develop beyond the formation of ventral and dorsal buds. Thus, PDX-l 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.

[0005] 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. -y

2017202571 19 Apr 2017

- 1a2017202571 19 Apr 2017

10006]

10007] |0008| [0009|

10010] |0011|

Current methods to culture human embryonic stem cells are complex; they require the use of exogenous factors, or chemically defined media in order for the cells to proliferate without loosing their pluripotency. Furthermore differentiation of embryonic stem cells often results in a decrease in the cells to expand in culture.

In one example, Cheon et al (BioRcprod DO1;10.1095/biolrcprod. 105.046870, October 19, 2005) disclose a feeder-free, serum-free culture system in which embryonic stem cells are maintained in unconditioned serum replacement (SR) medium supplemented with different growth factors capable of triggering embryonic stem cell self-renewal.

In another example, US20050233446 discloses a defined media useful in culturing stem cells, including undifferentiated primate primordial stem cells. In solution, the media is substantially isotonic as compared to the stem cells being cultured. In a given culture, the particular medium comprises a base medium and an amount of each of bFGF, insulin, and ascorbic acid necessary to support substantially undifferentiated growth of the primordial stem cells.

In another example, W02005086845 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 (TGFP) family of proteins, a member of the fibroblast growth factor (FGF) family of proteins, or nicotinamide (NIC) in an amount sufficient to maintain the cell in an undifferentiated state for a sufficient amount of time to achieve a desired result.

Inhibitors of glycogen synthase kinase-3 (GSK-3) are known to promote proliferation and expansion of adult stem cells. In one example, Tatcishi et al. (Biochemical and Biophysical Research Communications (2007) 352: 635) show that inhibition of GSK-3 enhances growth and survival of human cardiac stem cells (hCSCs) recovered from the neonatal or adult human heart and having mesenchymal features.

For example, Rulifson et al (PNAS 144, 6247-6252, (2007)) states “Wnt signaling stimulates islet β cell proliferation.

. ? _

2017202571 19 Apr 2017 |00121

10013] |0014]

10015]

100161

100171 [00181 |0019]

In another example, W020070I6485 reports that addition of GSK-3 inhibitors to the culture of non-embryorric stem cells, including multipotent adult progenitor cells, leads to the maintenance of a pluripotent phenotype during expansion and results in a more robust differentiation response.

In another example, US2006030042 uses a method of inhibiting GSK-3, either by addition of Wnt or a small molecule inhibitor of GSK-3 enzyme activity, to maintain embryonic stem cells without the use of a feeder cell layer.

In another example, W02006026473 reports the addition of a GSK-3B inhibitor, to stabiiize pluripotent cells through transcriptional activation of c-myc and Stabilization of c-myc protein.

In another example, W02006100490 reports the use of a stem cell culture medium containing a GSK-3 inhibitor and a gp.130 agonist to maintain a self-renewing population of pluripotent stem cells, including mouse or human embryonic stem cells.

In another example, Sato etal. (Nature Medicine (2004) 10:55-63) show that inhibition of GSK-3 with a specific pharmacological compound can maintain the undifferentiated phenotype of embryonic stem cells and sustain expression of pluripotent state-specific transcription factors such as Oct-3/4, Rex-I, and Nanog.

In another example, Maurer etal (Journal of Proteome Research (2007) 6:1198-1208) show that adult, neuronal stem cells treated with a GSK-3 inhibitor show enhanced neuronal differentiation, specifically by promoting transcription of β-catenin target genes and decreasing apoptosis.

In another example, Gregory et at (Annals of the New York Academy of Sciences (2005) 1049:97-106) report that inhibitors ofGSK-3B enhance in vitro osteogenesis.

In another example, Feng et ai (Biochemical and Biophysical Research CommuncationS (2004) 324:1333-1339) show that hematopoietic differentiation from embryonic stem cells is associated with down-regulation of the Wnt/p-catenin pathway, where Wnt is a natural inhibitor of GSK3.

-3-42017202571 20 Apr 2017 [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.

[0020a] 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.

SUMMARY [0020b] According to a first aspect, the present invention provides a method for differentiating pluripotent stem cells into definitive endoderm cells comprising treating the pluripotent stem cells with medium supplemented with a compound of formula (III)

wherein the compound is selected from the group consisting of:

a. 6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)-2pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile;

b. 3-[1-(3-hydroxy-3-methyl-butyl)-1H-indazol-3-yl]-4-(1-pyridin-3-yl-1H-indol-3-yl)-pyrrole2,5-dione;

c. 3-[1-[3-[(2-hydroxyethyl)methylamino]propyl]-1H-indazol-3-yl]-4-[1-(3-pyridinyl)-1 H-indol3-yl]-1 H-pyrrole-2,5-dione;

d. 14-ethyl-6,7,9,10,13,14,15,16-octahydro-12H,23H-5,26:17,22dimethenodibenzo[k,q]pyrrolo[3,4-n][1,4,7,10,19]dioxatriazacycloheneicosine-23,25(24H)dione;

e. 14-(2-thienylmethyl)-6,7,9,10,13,14,15,16-octahydro-12H,23H-5,26:17,22di(metheno)dibenzo[k,q]pyrrolo[3,4-n][1,4,7,10,19]dioxatriazacyclohenicosine-23,25(24H)dione; and

f. 14-(1-naphthylmethyl)-6,7,9,10,13,14,15,16-octahydro-12H,231-1-5,26:17,22di(metheno)dibenzo[k,q]pyrrolo[3,4-n][1,4,7,10,19]dioxatriazacyclohenicosine-23,25(24H)dione.

2017202571 20 Apr 2017

- 4a [0020c] According to a second aspect, the present invention provides a method of differentiating pluripotent stem cells into pancreatic endoderm or pancreatic endocrine comprising differentiating pluripotent stem cells into definitive endoderm cells using the method of the first aspect and differentiating the definitive endoderm cells into pancreatic endoderm or pancreatic endocrine cells.

[0020d] According to a third aspect, the present invention provides definitive endoderm cells when produced by the method of the first aspect.

[0020e] According to a fourth aspect, the present invention provides pancreatic endoderm or pancreatic endocrine when produced by the method of the second aspect.

[0020q 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):

-4b2017202571 20 Apr 2017

Formula (I)

2017202571 19 Apr 2017 |00261

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

-5|0027) In one embodiment, the inhibitor of GSK.-3B enzyme activity isa compound of the

Formula (III ):

2017202571 19 Apr 2017

BRIEF DESCRIPTION OF THE FIGURES

10028] Figure 1 shows the effect of a range of concentrations of the compound JNJ 17189731 on cell number, as determined by the number of nuclei observed (Panel A) and Sox-17 expression, as determined by intensity of immunofluoresccnt staining (Panel B). Results were obtained from cells of the human embryonic stem cell line Η1 (white bars), or cells of the human embryonic stem cell line H9 (black bars), using the ΓΝ Cell Analyzer 1000 (GE Healthcare).

100291 Figure 2 shows the effect of a range of concentrations of the compound JNJ 17163796 on cell number, as determined by the number of nuclei observed (Panel A) and Sox-17 expression, as determined by intensity of immunofluoresccnt staining (Panel B). Results were obtained from cells of the human embryonic stem cell line Η1 (white bars), or cells of the human embryonic stem cell line H9 (black bars), using the IN Cell Analyzer 1000 (GE Healthcare).

|0030| Figure 3 shows the effect of a range of concentrations of the compound JNJ 17223375 on cell number, as determined by the number of nuclei observed (Panel A) and Sox-17

-62017202571 19 Apr 2017 |003lJ |0032| [0033] |0034| expression, as determined by intensity of immunofluorescent staining (Panel B). Results were obtained from cells of the human embryonic stem cell line Hl (white bars), or cells of the human embryonic stem cell line H9 (black bars), using the IN Cell Analyzer 1000 (GE Healthcare).

Figure 4 shows the effect of a range of concentrations of the compound JNJ 18157698 on cell number, as determined by the number of nuclei observed (Panel A) and Sox-17 expression, as determined by intensity of immunofluorescent staining (Panel B). Results were obtained from cells of the human embryonic stem cell line Hl (white bars), or cells of the human embryonic stem cell line H9 (black bars), using the IN Cell Analyzer 1000 (GE Healthcare),

Figure 5 shows the effect of a range of concentrations of the compound JNJ 26158015 on cell number, as determined by the number of nuclei observed (Panel A) and Sox-17 expression, as determined by intensity of immunofluorescent staining (Panel B), Results were obtained from cells of the human embryonic stem cell line Η1 (white bars), or cells of the human embryonic stem cell line H9 (black bars), using the IN Cell Analyzer 1000 (GE Healthcare),

Figure 6 shows the effect of a range of concentrations of the compound JNJ 26483197 on cell number, as determined by the number of nuclei observed (Panel A) and Sox-17 expression, as determined by intensity of immunofluorescent staining (Panel B). Results were obtained from cells of the human embryonic stem cell line H] (white bars), or cells of the human embryonic stem cell line H9 (black bars), using the IN Cell Analyzer 1000 (GE Healthcare),

Figure 7 shows the effect of a range of concentrations of the compound JNJ 26483249 on cell number, as determined by the number of nuclei observed (Panel A) and Sox-17 expression, as determined by intensity of immunofluorescent staining (Panel B), Results were obtained from cells of the human embryonic stem cell line Hl (white bars), or cells of the human embryonic stem cell line H9 (black bars), using the IN Cell Analyzer 1000 (GE Healthcare).

-72017202571 19 Apr 2017

1003?!

10036| |00371 |0038| |00391

10040] [0041|

Figure 8 shows the effect of a range of concentrations of the compound JNJ 10220067 on cell number, as determined by the number of nuclei observed (Panel A) and Sox-17 expression, as determined by intensity of immunofluorcscent staining (Panel B). Results were obtained from cells of the human embryonic stem cell line Hl (white bars), or cells of the human embryonic stem cell line H9 (black bars), using the IN Cell Analyzer 1000 (GE Healthcare),

Figure 9 shows the expression of CXCR4 on the surface of cells, as determined by immunofluorescent staining and flow cytometric analysis, on cells heated with the compounds shown, according to the methods described in Example 8.

Figure 10 shows the expression ofCXCR4 (Panel A), HNF-3 beta (Panel B), and Sox-17 (Panel C), as determined by real-time PCR, in cells treated with the compounds shown, according to the methods described in Example 8.

Figure 11 shows the effect of a range of concentrations of the compounds shown on cell number, as determined by the number of nuclei observed (Panel A) and Pdx-1 expression, as determined by intensity of immunofluorescent staining (Panel B), using the IN Cell Analyzer 1000 (GE Healthcare). Cells 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 Pdx1 expression (white bars) and HNF-6 (black bars), as determined by real-time PCR. Cells were treated according to the methods described in Example 9.

Figure 13 shows the effect of a range of concentrations of the compounds shown on cell number, as determined by the number of nuclei observed (Panel A) and insulin expression, as determined by intensity of immunofluorescent staining (Panel B), using the IN Cell Analyzer 1000 (GE Healthcare), Cells were treated according to the methods described in Example 10.

Figure 14 shows effect of a range of concentrations of the compounds shown on Pdx-1 expression (white bars) and insulin (black bars), as determined by real-time PCR. Cells were treated according to the methods described in Example 10.

-8Figure 15 shows the effect of a range of concentrations of the compounds shown on cell number, as determined by the number of nuclei observed (Panel A) and insulin expression, as determined by intensity of immunofluorescent staining (Panel B), using the IN Cell Analyzer 1000 (GE Healthcare). Ceils were treated according to the methods described in Example 11.

2017202571 19 Apr 2017 |00421

I0043I |0044|

DETAILED DESCRIPTION

For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the following subsections that describe or illustrate certain features, embodiments, or applications of the present invention.

Definitions

Stem cells are undifferentiated cells defined by their ability at the single cell level to both self-rencw and differentiate to produce progeny cells, including self-renewing progenitors, non-renewing progenitors, and terminally differentiated cells. Stem cells arc also characterized by their ability to differentiate in vitro into functional cells of various cell lineages from multiple germ layers (endoderm, mesoderm and ectoderm), as well as to give rise to tissues of multiple germ layers following transplantation and to contribute substantially to most, if not all, tissues following injection into blastocysts.

Stem cells are classified by their developmental potential as: (1) totipotent, meaning able to give rise to all embryonic and extraembryonic cell types; (2) pluripotent, meaning able to give rise to all embryonic cell types; (3) multipotent, meaning able to give rise to a subset of cell lineages, but all within a particular tissue, organ, or physiological system (for example, hematopoietic stem cells (HSC) can produce progeny that include HSC (self- renewal), blood cell restricted oligopotent progenitors and all cell types and elements (e.g., platelets) that are normal components of the blood); (4) oligopotent, meaning able to give rise to a more restricted subset of cell lineages than multipotent stem cells; and (5) unipotent, meaning able to give rise to a single cell lineage (e.g., spermatogenic stem cells).

-92017202571 19 Apr 2017

100451 [00461 |0047| [00481

Differentiation is the process by which an unspecialized (uncommitted) or less specialized cell acquires the features of a specialized cell such as, for example, a nerve cel! or a muscle cell. A differentiated or differentiation-induced cell is one that has taken on a more specialized {committed) position within the lineage of a cell. The term “committed”, when applied to the process of differentiation, refers to a cell that has proceeded in the differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types, and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type. De-differentiation refers to the process by which a cell 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 from and what cells it can give rise to. The lineage of a cell places the cell within a hereditary scheme of development and differentiation. A lineage-specific marker refers to a characteristic specifically associated with the phenotype of cells of a lineage of interest and can be used to assess the differentiation of an uncommitted cell to the lineage of interest.

β-cell lineage refer to cells with positive gene expression for the transcription factor PDX-1 and at least one of the following transcription factors: NGN-3, Nkx2.2< Nkx6.1, NeuroD, Isl-1, HNF-3 beta, MAFA, Pax4, and Pax6. Cells expressing markers characteristic of the β cell lineage include β cells.

“Cells expressing markers characteristic of the definitive endoderm lineage” as used herein refer to cells expressing at least one of the following markers: SOX-17, GATA-4, HNF-3 beta, GSC, Cerl, Nodal, FGF8, Brachyury, Mix-like homeobox protein, FGF4 CD48, eomesodermin (EOMES), DKK4, FGF17, GATA-6, CXCR4, C-Kit, CD99, or OTX2. Cells expressing markers characteristic of the definitive endoderm lineage include primitive streak precursor cells, primitive streak cells, mesendoderm cells and definitive endoderm ceils.

“Cells expressing markers characteristic of the pancreatic endoderm lineage” as used herein refer to cells expressing at least one of the following markers: PDX-1, HNF- 102017202571 19 Apr 2017

10049) |0050)

100511 [0052| [0053) |0054|

Ibeta, PTF-1 alpha, HNF-6, or HB9. Cells expressing markers characteristic of the pancreatic endoderm lineage include pancreatic endoderm cells.

“Cells.expressing markers characteristic of the pancreatic endocrine lineage” as used herein refer to cells expressing at least one of the following markers: NGN-3, NeuroD, Islet-1, PDX-1, NKX6.1, Pax-4, Ngn-3, or PTF-t alpha. Cells expressing markers characteristic of the pancreatic endocrine lineage include pancreatic endocrine cells, pancreatic hormone expressing cells, and pancreatic hormone secreting cells, and cells of the β-celJ lineage.

“Definitive endoderm” as used herein refers to cells which bear the characteristics of cells arising from the epiblast during gastrulation and which form the gastrointestinal tract and its derivatives. Definitive endoderm cells express the following markers: HNF3 beta, GATA-4, SOX-17, Cerberus, OTX2, goosecoid, C-K.it, CD99, and Mixl 1, “Extraembryonic endoderm as used herein refers to a population of cells expressing at least one of the following markers: SOX-7, AFP, and SPARC.

Markers as used herein, are nucleic acid or polypeptide molecules that are differentially expressed in a cell of interest. In this context, differential expression means an increased level for a positive marker and a decreased level for a negative marker. The detectable level of the marker nucleic acid or polypeptide is sufficiently higher or lower in the cells of interest compared to other cells, such that the cell of interest can be identified and distinguished from other cells using any of a variety of methods known in the art, “Mescndodcrm cell” as used herein refers to a cell expressing at least one of the following markers: CD48, eomesodermin (EOMES), SOX-17, DKK4, HNF-3 beta,

GSC, FGF17, GATA-6.

“Pancreatic endocrine cell”, or “pancreatic hormone expressing cell” as used herein refers to a cell capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin, and pancreatic polypeptide.

- 11 2017202571 19 Apr 2017

100551 |0056| [00571 |00581 |0059[

100601

100611 “Pancreatic hormone secreting cell” as used herein refers to a cell capable of secreting at least one of the following hormones: insulin, glucagon, somatostatin, and pancreatic polypeptide.

“Pre-primitive streak cell” as used herein refers to a cell expressing at least one of the following markers: Nodal, or FGF8 “Primitive streak cell as used herein refers to a cell expressing at least one of the following markers: Brachyury, Mix-like homeobox protein, or FGF4.

In one embodiment, the present invention provides a method for the expansion and differentiation of pluripotent cells comprising treating the pluripotent cells with an inhibitor of GSK-3B enzyme activity.

In one embodiment, the present invention provides a method to expand and differentiate pluripotent cells, comprising the steps of:

c. Culturing pluripotent cells, and

d. Treating the pluripotent cells with an inhibitor of GSK-3B enzyme activity.

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

Markers characteristic of the definitive endoderm lineage are selected from the group consisting of SOX17, GATA4, Hnf-3beta, GSC, Cerl, Nodal, FGF8, Brachyury, Mixlike homeobox protein, FGF4 CD48, eomesodermin (EOMES), DKK4, FGF17, GATA6, CXCR4, C-Kit, CD99, and OTX2. Contemplated in the present invention is a cell, derived from a pluripotent cell that expresses at least one of the markers characteristic of the definitive endoderm lineage. In one aspect of the present invention, a cell expressing markers characteristic of the definitive endoderm lineage is a primitive streak precursor ceil. In an alternate aspect, a cell expressing markers characteristic of the definitive endoderm lineage is a mesendoderm cell. In an alternate aspect, a cell expressing markers characteristic of the definitive endoderm lineage is a definitive endoderm cell.

- 122017202571 19 Apr 2017

100621

10063]

10064|

The pluripotent cells may be treated with the inhibitor of GSK.-3B enzyme activity for about one to about 72 hours. Alternatively, the pluripotent cells may be treated with the inhibitor of GSK.-3B enzyme activity for about 12 to about 48 hours. Alternatively., the pluripotent cells may be treated with the inhibitor of GSK-3B enzyme activity for about hours.

In one embodiment, the inhibitor of GSK-3B enzyme activity is used at a concentration of about lOOnM to about ΙΟΟμΜ. Alternatively, the inhibitor of GSK-3B enzyme activity is used at a concentration of about 1 μΜ to about 10μΜ. Alternatively, the inhibitor of GSK.-3B enzyme activity is used at a concentration of about 10μΜ,

Compounds suitable for use in the methods of the present invention

In one embodiment, the inhibitor of GSK.-3B enzyme activity is a compound of the Formula fi):

Formula (J) [0065) |00661

10067)

I0068J wherein;

Ri is phenyl, substituted phenyl wherein the phenyl substituents are selected from the group consisting of Cj^alkyl, halogen, nitro, trifluoromethyl and nitrile, of pyrimidinyl;

R₂ is phenyl, substituted phenyl wherein the phenyl substituents are selected from the group consisting of Cj^alkyl, halogen, nitro, triffuoromethyl and nitrile, or pyrimidinyl which is optionally Chalky 1 substituted, and at least one of Ri and R₂ is pyrimidinyl;

Ri is hydrogen, 2-(trimethyl.silyl)etlioxymethyl, Ci.₅alkoxycarbonyl, aryloxycarbonyl, aryICi.salkyloxycarbonyl, arylCi^alkyl, substituted arylCi^alkyl wherein the one or more

- 13 aryl substituents are independently selected from the group consisting of Ci/alkyl,

C|/alkoxy, halogen, amino, Cι/alkylamino, and diCi/alkylamino, phthalimidoC i.salkyl, aminoCi/alkyl, di ami noC,/alkyl, succinimidoCi/alkyl, Ci/alkylcarbonyl, arylcarbonyl,

Ci/alkylcarbonylCi/alkyl and aryloxycarbonylCi/alkyl,

2017202571 19 Apr 2017 |0069| EL, is /OR₅

10070) |0071|

A is vinylene, ethynylene or

R₅ is .selected from the group consisting of hydrogen, Cj/alkyl, phenyl and phenylC r/alkyl;

[0072| q is 0-9;

[0073] X is selected from the group consisting of hydrogen, hydroxy, vinyl, substituted vinyl wherein one or more vinyl substituents are each selected from the group consisting of fluorine, bromine, chlorine and iodine, ethynyl, substituted ethynyl wherein the ethynyl substituents are selected from the group consisting of fluorine, bromine chlorine and iodine, Cj/alkyl, substituted Cj/alkyl wherein the one or more alkyl substituents are each selected from the group consisting of C i/alkoxy, trihaloalkyl, phthalimido and amino, Cri/cycloalkyl, C ι/alkoxy, substituted C|/alkoxy wherein the alkyl substituents are selected from the group consisting of phthalimido and amino, phthalfrnidooxy, phenoxy, substituted phenoxy wherein the one or more phenyl substituents are each selected from the group consisting of Cj/alkyl, halogen and Cj/alkoxy, phenyl, substituted phenyl wherein the one or more phenyl substituents are each selected from the group consisting of Cj/alkyl, halogen and Cj/alkoxy, arylCi/alkyl, substituted arylCj/alkyl wherein the one or more atyl substituents are each selected from the group consisting of Cj/alkyl, halogen and Ci/alkoxy, aryloxyCi/alkylamino, Ct/alkylamino, diC|/alkylamino, nitrile, oxime, benxyloxyimino, C i/alkyloxyimino, phthalimido, succinimido, Ci/alkylcarbonytoxy, phenylcarbonyloxy, substituted phenylcarbonyloxy wherein the one or more phenyl substituents are each selected from the group consisting of Cj/alkyl,

- 142017202571 19 Apr 2017

10074|

10075] |0076]

10077|

10078] [00791 |0074] |0075] |0076]

10077|

10078] [00791 halogen and Ci-jalkoxy, phenylCj-salkylcarbonyloxy wherein the one or more phenyl substituents are each selected from the group consisting of Ci-salkyl, halogen and

Cj.salkoxy, aminocarbonyloxy, Cj.salkylaminocarbonylcixy, diC|.5alkylaminocarbonyloxy, Ci-jalkoxycarbonyloxy, substituted Cj^alkoxycarbonyloxy wherein the one or more alkyl substituents are each selected from the group consisting of methyl, ethyl, isopropyl and hexyl, phenoxycarbonyloxy, substituted phenoxycarbonyloxy wherein the one or more phenyl substituents are each selected from the group consisting ofCi-salkyl, Ci-jalkoxy and halogen, Chalky Ithio, substituted ChalkyIthio wherein the alkyl substituents are selected from the group consisting of hydroxy and phthalimido, Cualkylsulfonyl, phenylsulfonyl, substituted phenylsulfonyl wherein the one or more phenyl substituents are each selected from the group consisting of bromine, fluorine, chloride, Cj.galkoxy and trifluoromethyl; with the proviso that if A

Ν' is , q is 0 and X is H, then Ri may not be 2-(trimethylsilyl)cthoxymethyl; and pharmaceutically acceptable salts thereof.

An example of the invention includes a compound of Formula (I) wherein R[ is substituted phenyl and R? is pyrimidin-3-yh

An example of the invention includes a compound of Formula (I) wherein R| is

4-fluorophenyl.

An example of the invention includes a compound of Formula (1) wherein Ri is hydrogen, arylCualkyl, or substituted arylCi-jalkyl.

An example of the invention includes a compound of Formula (I) wherein R < is hydrogen or phenylCi-jalkyl.

An example of the invention includes a compound of Formula (I) wherein A is ethynylene and q is 0-5.

An example of the invention includes a compound of Formula (1) wherein X is succinimide, hydroxy, methyl, phenyl, Ci->alkylsulfonyl, C?-scycloalkyl,

- 152017202571 19 Apr 2017

Ci-salkylcarbonyloxy, Ci-jalkoxy, phenylcarbonyloxy, Ci-jalkylamino, diCj^alkylamino or nitrile.

10080) Compounds of Formula (1) are disclosed in commonly assigned United States Patent

Number 6,214,830, the complete disclosure of which is herein incorporated hy reference.

[0081) An example of the invention includes a compound of Formula (I) wherein the compound is selected from the group consisting of;

Compound Name

5{4)-(4-fluorophenyl)-4(5>(4-pyridy [)imidazole,

4-(4-fIuoropheny1)-l-(3-phenylpropyl)-5-(4-pyridyl)iniidazole,

5-(4-fluorophenyl)-1 -(3-pheny Ipropy l)-4-(4-pyridyl)imidazole,

4-(4-fluorophenyl)-2-iodo-1 -(3-phenyIpropy l}-5-(4-pyridyl)imidazolc.

4-{4-fluorophenyl)-2-{4-bydroxybutyn-1 -yl)-1 -(3-pheny Ipropy I )-5-(4pyridyl)imidazole, ή 4-(4-fluorOpheny])-5-(4-pyridyl)-l-[2-(trimethylsilyl)ethoxymethyl]i midazole,

5-(4-fluorophenyl)-4-(4-pyridyl)-U[2-(trimethylsilyl)ethoxymethyl]imidazole,

5-(4-fluoropheny l)-2-iodo-4-(4-pyridyl)-1 -[2(tri methy lsilyl)ethoxyme thy l]-imidazo le,

5-(4-fluorophenyl)-4-(4-pyridyl)-2-(trimcthylsilyr)ethmyl-1 -[2(trimethylsilyl)ethoxymethy]]-imidazolc₍

2-{2-chlorovinyl)-5-(4-fluorophenyl)-4-(4-pyrid_]yl)-imidazole,

- 162017202571 19 Apr 2017

Compound	Name
11	5-(4-ftuorophenyl )-4-(4-pyridyl)-l-[2-(trimethyl sily l)ethoxymethy 1] - imidazoIc-2-cafboxaldehyde,
12	2-[2,2-dibromoethyIene-l-yl]-5-(4-fIuorophenyl}-4-(4-pyridyl)-l-[2- (trimethylsilyl)ethoxymethyl]-tmidazole-2-carboxaldehyde,
13	5{4)-(4-fluorophenyl)-2-(3-hydroxy-3-phenyl-propyn-1 -y 1)-4(5 )-(4- pyridyl)imidazole,
14	5-(4-fluorophenyl )-4-( 4-pyri dyl)-1 -[2-( trimethy Ssily 1 )ethoxymethy 1J-2- oximino imidazole,
15	5-(4-fluorophenyl)-4-(4-pyridyl)-2-imidazole oxime.
16	2-(5-chIoropentyn-l“yl)-4-(4-fluarophenyJ)-l-(3-phenylpropy])-5“(4- pyridyl)imidazole,
17	4-(4-fluorophenyl)-2-(4-N-phenylcarbamoytoxybutyn-l -yl)l -(3- phenylpropyl)-5-(4-pyridyl)imidazole.
17	2-(4-chtorobutyn-l-yl)-4-(4-fluorophenyi)-l-(3-phenylpropyl)-5-(4- pyridyl)imidazole, and
18	2-i4-dimethylaminobutyn-1 -yl)-4-(4- fluorophenyl)-1 -(3-phenylpropyl)- 5-(4-pyridy])imidazole.

|00821 An example of the invention includes a compound of Formula (1) wherein the compound is Compound 5 of the Formula:

- 172017202571 19 Apr 2017

|0083] 1 π one embodiment, the inhibitor of GSK-3B enzyme activity is a compound of the

Formula (II):

[0084] Wherein:

100851 R is selected from the group consisting of Ra, -Ci-8alkyl-R._n, -Cj-salkenyl-Ru, -Cj-salkynyl-Ra and cyano;

[0086] Ra is selected from the group consisting of cycloalkyl, heterocyclyl, aryl and heteroaryl;

- 182017202571 19 Apr 2017

10087) R¹ is selected from the group consisting of hydrogen, -Cj-salkyl-R⁵, -C2-?alkenyl-R\ -C2_salkynyl-R⁵, -C(O)-(C,:S)alkyl-R⁹, -C(O)-aryl-R^s, -C(O)-O-(Ch8)alkyl-R⁹, -C(O)-O-aryl-R^s, -C(O)-NH(C^alkyl-R⁹), -C(O)-NH(aryl-R⁸), -C(O)-N(Cj.8alkyl-R⁹)₂, -SO2“(Ci.s)alkyl-R⁹, -SOj-aryl-R^s, -cycloa!kyI-R^fi, -hcterocyclyl-R⁰, -aryl-R^fi and -hetcroaryl-R⁶; wherein heterocyclyl and heteroaryl are attached to the azaindole nitrogen atom in the one position via a heterocyclyl or heteroaryl ring carbon atom;

)0088) R⁵ is 1 to 2 substituents independently selected from the group consisting of hydrogen, -O-(C,.₈)alkyl, -O-(C,.₈)aLkyl-OH, -0-((2,.aialkyl-O-fC^alkyl, -O-(Ci.₈)alkyl-NH₂, -O-(Ci.₈)alkyl-NH(Ci-_Salkyl), -O-(C,.₈)alkyl-N(Ci^alkyl)₂, -O-(C,.₈)alkyl-S-(Ci-₈)alkyl, -O-iC^alkyl-SOHCi^alkyl, -O-(Ci.₈)alkyt-SO₂-NH_2i

-O-(C,.₈)alkyl-SO₃-NHCCi.salkyl), -O-(Cus)alkyl-SO₂-N(C,.₈alkyl)₂, -O-C(O)H, -O-C(O)-(C|-₈)alkyl, -O-C(O)-NH₂, -O-C(O)-NH(Cualkyf)„ -O-C(O)-N(C, _salkyl)₂, -O-(C].₈)alkyl-C(O)H, -O-(C_!-₈)a]kyI-C(O)-(Ci._B)alkyl, -O-(C,.₈)alkyI-CO₂H, -O-(Ci^)alkyl-C(O)-O-(C,.₈)alkyl, -O-(C ,.₈)alkyl-C(O)-NH₂,

-O-(C₁__s)alkyl-C(0)-NH(C₁._salkyl), -O-iCL.^alkyl-CfOJ-NfCt-salkylh, -C(O)H, -C(O)-(C,.s)alkyl, -CO₂H, -C(O)-O-(C,.₈)alkyl, -C(0)-NH₂, -C(NH)-NH_Z, -C(O)-NH(Ci_₈alkyl), -C(O)-N(C|.₈alkyl)₂, -SH, -S-(C,.₈)alkyl, -S-{C|._s)alkyl-S-(Ci,_R)alkyl, -S-(C^)alkyl-O-(C,,₈)alkyl,

-S-iCj-sJalkyl-O-iCuiJalkyl-OH’-S-(C₁.₈)alkyt-O-(Ci,₈)alkyl-NH₂, -S-(Ci.₈)alkyl-O-(Ci^)alkyl-NH(Ci.salkyl), -S-tChsjalkyl-O-CCi^alkyl-NfChsalkylE -S-(C|.s)alkyl-NH(Ci-salkyl), -SO₂-(C,.₈)alkyl, -SO₂-NH₂, -SO₂-NH(Ci-₈alkyl), -SO₂-N(C|-₈alkyl)₂, -N-R⁷, cyano, (halo)ro, hydroxy, nitro, Oxo, -cycloalkyl-R⁶, -heterocyclyl-R⁶, -ary l-R⁶ and -heteroaryl-R⁶;

10089) R⁶ is 1 to 4 substituents attached to a carbon or nitrogen atom independently selected from the group consisting of hydrogen, -Ci_₈alkyl, -C^salkenyl. -C₂.₈alkynyl, -C(O)H, -(2(0)-((2 i-s)a!kyl, -CO₂H, -C(O)-0-(C_1B)alkyl, -C(0)-NH₂, -<2(ΝΗ)-ΝΗ₂,

-C(O>NH(C,.«alkyl), -C(O)-N(C,__R)alkyl)₂, -SO₂-(C,.₈)alkyl, -SO₂-NH₂, -SOrNH(C,.₈alkyl)_s -SO.₂-N(C,.₈alkyI)₂, -(Chalky 1-N-R⁷, -(Cf.B)alkyl-(haJo)i.j, -(C|-g)a!kyl-OH, -aryl-R⁸, -(Cj.8)alkyl-aryl-R⁸ and -(Ci._B)atkyl-heteroaryl-R^s; with the proviso that, when R^fi is attached to a carbon atom, R⁶ is further selected from the group

- 192017202571 19 Apr 2017 consisting of -C^alkoxy, -(Ci^)alkoxy-(halo)|.j, -SH, -S-(Ci-s)alkyl, -N-R⁷, cyano, halo, hydroxy, nitro, oxo and -heteroaryl-R⁸;

100901 R⁷ is 2 substituents independently selected from the group consisting of hydrogen,

-Chalky!, -CXalkcnyl, -CXalkynyl, -(Ci.s)alkyl-OH, -(Ci.s)alkyl-O-(Ci.s)alkyl, -(C|.s)alkyl-NH2,-(C|^)aikyl-NH(Ci__salkyl), -(C^alkyl-NiCEsalkylh, -(C^)alkyl-S-(C,._s)alkyl, -C(O)H, -C(O)-(C,^)alkyI, -C(O)-O-(C^)alkyl, -C(O)-NH₂, -C(O)-NH(Cu_Salkyl), -C(O)-N(Ci._salkyl)₂, -SOHCrsJalkyl, -SO₂-NH₂, -SOz-NH(Ci-salkyl), -SO₂-N(Cf_₈alky[)₂, -C(N)-NH_2l -cycloalkyl-R⁸, -{Ci-sjalkyl-heterocyclyl-R⁸, -aryl-R⁸, -(Cufialkyl-aryl-R⁸ and -(Ci-g)aikyl-heteroaryl-R⁸;

100911 R^s is 1 to 4 substituents attached to a carbon or nitrogen atom independently selected from the group consisting of hydrogen, -Ci-galkyl, -(C].s)alkyl-(halo)i_3 and -{C|.s)alkyl-OH; with the proviso that, when R^a is attached to a carbon atom, R⁸ is further selected from the group consisting of -Cj.salkoxy, -NH.₂, -NH(Cj,8alkyl), -N(C|.salkyl)₂, cyano, halo, -(C_{t 8})aikoxy-(halo)i.₂, hydroxy and nitro;

[00921 is I to 2 substituents independently selected from the group consisting of hydrogen,

-Ci-₈alkoxy, -NH₂, -NHfCi-salkyl), -N(Ci-₈alkyl)₂, cyano, (haloJi.j, hydroxy and nitro;

100931 R⁷ is one substituent attached to a carbon or nitrogen atom selected from the group consisting of hydrogen, -Ci-galkyl-R³, -C2_salkenyl-R⁵, -C2.₈alkynyl-R⁵, -C(0)H, -C(O)-(C|4i)alkyl-R⁹, -C(O)-NH2, -CO-NHfCXalkyl-R⁹), -C(O)-N(C,.aalkyl-R⁹)2, -C(O)-NH( aryl-R⁷), -C(0)-cycloalkyl-R^s, -C(O)-heterocyclyl-R\ -C(O)-aryl-R\ -CfOI-heteroaryl-R⁷, -CO2H, -QOJ-O-CCu^alkyt-R⁹, -C(O)-O-aryI-R⁸, -SO2-(C,._s)alkyl-R⁹, -SO2-aryl-R'^s, -cycloalkyl-R⁶, -aryl-R⁶ and -(C,.s)a1kyl-N-R⁷; with the proviso that, when R² is attached to a carbon atom, R² is further selected from the group consisting of -Ci-salkoxy-R⁵, -N-R⁷, cyano, halogen, hydroxy, nitro, oxo, -heterocyclyl-R⁶ and -heteroaryl-R⁶;

[0094] R³ is 1 to 3 substituents attached to a carbon atom independently selected from the group consisting of hydrogen, -Ci-galkyl-R¹⁹, -CEgalkenyl-R¹, -C2-salkynyl-R¹⁰, -Crsalkoxy-R¹⁰, -C(O)H, -C(O)-(Cw)aIkyl-R⁹, -C(O)-NH₂, -C(O)-NH(C|-₈alkyl-R⁹),

- 20-GOVNOAsalkyl-R⁹);!, -C(0)-cycIoalkyl-R^s, -C(O)-heterocyclyl-R⁸, -C(O)-aryl-R^s,

-C(O)-heteroaryl-R^K, -C(NH)-NH₂, -CO₂H, -C(O)-O-(C_M)alkyl-R⁹, -C(O)-O-aryl-R\

-SO₂-(C|.a)alkyl-R⁹, -SO₂-aiyl-R^a, -N-R⁷, cyano. halogen, hydroxy, nitro. -cycloalkyl-R^s,

-heterocyclyl-R⁸, -aryl-R⁸ and -heteraaryl-R⁸;

2017202571 19 Apr 2017 |0095] |0096| [00971

I0098I (01001 |0101I [01021

R⁴ is I to 4 substituents attached to a carbon atom independently selected from the group consisting of hydrogen, -CYalkyl-R¹⁰, -C2_nalkenyl-R¹⁰, -CYalkynyl-R¹⁰, -Ci^alkoxy-R¹⁰, -C(O)H, -C(O)-(CM)alkyl-R⁹, -C(O)-NH2, -C(O)-NH(Cl.salkyI-R⁹), -C(O)-N(Ci.salkyl-R⁹)2, -C(O)-cycIoalkyl-R⁸, -C(O)-heterocyclyl-R⁸, -C(O)-aryl-R⁸, -C(O)-heteroaryl-R⁸, -C(NH)-NH2, -CO2H, -CiOj-O-tCuOalkyl-R⁹, -C(O)-O-aryl-R^s, -SH, -S-(C|Aalky!-R^l<!, -SCMC^alkyf-R⁹, -SO2-aryl-R^s, -SO2-NH2, -SO?-NH(Cj.salkyl-R⁹), -SO2-N(Ci.salkyl-R^yb, -N-R⁷, cyano, halogen, hydroxy, nitro, -cycloalkyl-R⁸, -heterocyclyi-R⁸, -aryl-R^s and -heteroaryl-R⁸;

R¹⁰ is 1 to 2 substituents independently selected from the group consisting of hydrogen, -NH₂, -NH(C| _salkyi), -NfC[._salkyt)₂, cyano, (halo)u, hydroxy, nitro and oxo; and,

Y and Z are independently selected from the group consisting of O, S, (H,OH) and (H,H); with the proviso that one of Y and Z is O and the other is selected from the group consisting of O, S, (H,OH) and (H,H); and pharmaceutically acceptable salts thereof.

Embodiments of the present invention include compounds of Formula (II) wherein. R is selected from the group consisting of R_a, -C_MalkyI-R_a, -CYalkenyl-R,, -C2„4alkynyl-R_aand cyano.

Embodiments of the present invention include compounds of Formula (II) wherein, R_a is selected from the group consisting of heterocyclyl, aryl and heteroaryl.

In one embodiment, R_a is selected from the group consisting of dihydro-pyranyl, phenyl, naphthyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, azaindolyl, indazolyl, benzofuryl, benzothienyl, dibenzofury I and dibenzothienyl.

Embodiments of the present invention include compounds of Formula (11) wherein, R' is selected from the group consisting of hydrogen, -Ci.^alkyl-R⁵, -C^alkenyl-R⁵,

- 21 2017202571 19 Apr 2017

-C^alkynyl-R⁵, -C(O)-(Chalkyl-R', -C(O)-aryl-R^s, -C(O)-O-(C,^)alkyl-R^!>1

-C(O)-O-aryl-R\ -C(O)-NH(Cj ^alkyl-R⁹), -C(O)-NH(aryl-R⁸), -C(O)-N(CMa1kyl-R⁹)2, “SOi-(CM)alkyl-R⁹s -SO₂-aryl-R^s, -cycloalkyl-R⁶, -heterocyclyl-R⁶, -aryl-R⁸ and

-heteroaryl-R⁶; wherein heterocyclyl and hetcroaryl are attached to the azaindole nitrogen atom in the one position via a heterocyclyl or hcteroaryl ring carbon atom.

101031 In one embodiment. R^l is selected from the group consisting of hydrogen, -Ci^alkyl-R⁵, -aryl-R⁶ and -heteroaryl-R⁶; wherein heteroaryl is attached to the azaindole nitrogen atom in the one position via a heteroaryl ring carbon atom.

|0104| In one embodiment, R¹ is selected from the group consisting of hydrogen, -Ci₄alkyl-R⁵and -napbthyl-R^B.

10105) Embodiments of the present invention include compounds of Formula (11) wherein, R⁵ is I to 2 substituents independently selected from the group consisting of hydrogen, -O-(C_M)alkyl, -O-(C_M)alkyl-OH, -O-(Cj^)alkyl-O-(Cw)afkyl, -O-(C|.₄)alkyl-NH_z, -O-(C_M)alkyl-NH{C_Malkyl)₅ -0-(C,.4)alkyl-N(C_l4alkyl)_z, ,O-(C|.₄)alkyl-S-(Ci^)alkyl, -O-(CM)alkyl-S0r(Ci.₄)alkyl, -O-(Ci-fialky I-SO_Z-NH_Z}

-O-(CL-4)alkyl-S0₂-NH(CMalkyl), -O-(Ci.₄)alkyl-SO₂-N(CL.₄alkyl)_z, -O-C(O)H, -O-C(O)-(C|.₄)alkyl, -O-C(O)-NH_Z, -O-C(O)-NH(C_Matkyl)„ -O-C(O)-N(C_Malkyl)₂, -O-(CiMalkyl-C(O)H, -0-(C_(J,)alkyl-C(O)-(C_M)alkyl, -O-(C_M)alkyl-CO₂H, -O-(C_M)alkyl-C(O)-O-(C'_M)alkyl, O-(C |.₄)alkyl-C(O)-NH₂,

-O-(C,.₄)aIkyl-C(O)-NH(CMalkyl)_j -O-iCwlalkyl-CiOJ-NiCMalkylh, -C(O)H. -C(O)-(C_M)alkyl, -CO_ZH, -C(O)-O-iC_M)alkyl, -C(O)-NH₂, -C(NH)-NH_Z, -C(O)-NH(C,toaIkyl), -C(O)-N(C|-₄aIkyl)_z, -SH, -S-(C).₄)aIkyl, -S-(Ci.₄)alkyl-S-(Ci.₄)alkyl, -S-(CM)alkyl-O-(C_|j4)alkyl,

-S-(C_M)alkyl-O-(C_M)alkyl-OH, -S-(C_M)alkyl-O-(C_M)alkyl-NH₂, -S-(Ci.4)alkyl-O-(C_M)alkyl-NH(Ci.₄alkyl), -S-(C_M)alkyl-O-(Ci.₄)alkyl-N(CMalkyl)_z, -S-(C_M)alkyl-NH(C_MaJkyl), -SO_z-(C_M)alkyl, -SO_Z-NH_Z, iSO₂-NH(Ci-₄alkyl), -SO₂-N(C|j(alkyl)₂, -N-R⁷, cyano, (halo)j.3, hydroxy, nitro, oxo, -cycloalkyl-R⁶, -heterocycly!-R⁶, -aryl-R⁶ and -heteroaryl-R⁶.

- 222017202571 19 Apr 2017 |01061 In one embodiment, R⁵ is 1 to 2 substituents independently selected from the group consisting of hydrogen, -O-(C_M)alkyl, -N-R, hydroxy and -heteroaryl-R⁶.

101071 In one embodiment, R⁵ is 1 to 2 substituents independently selected from the group consisting of hydrogen, -O-(CM)alkyl, -N-R⁷, hydroxy, -imidazolyl-R⁶, -triazolyl-R⁶ and -tetrazolyl-R⁶.

|0108| Embodiments of the present invention include compounds of Formula (II) wherein, R⁶ is to 4 substituents attached to a carbon or nitrogen atom independently selected from the group consisting of hydrogen, -Ci^alkyl, -C₂ualkenyl, -C₂^alkynyl, -C(O)H, -C(O)-(C_M)alkyl, -CO₂H, -C(O)-O-(C]^)alkyl, -C(O)-NH₂, -C(NH)-NH₂, -C(O)-NH(C_Malkyl), -C(O)-N(C_M)alkyl)₂, -SO₂-(C_w)atkyl, -SO₂-NH₂, -SO₂-NH(C_Ma[kyl), -SO₂-NfC_Malkyl)₂, -(CA)alkyl-N-R⁷, -(C_M)alkyl-(halo)u_}-(CiuOalkyl-OH, -aryl-R⁸, -(CM)alkyl-ary 1-R^S and -(CM)alkyl-heteroaryl-R⁸; with the proviso that, when R⁶ is attached to a carbon atom, R⁶ is further selected from the group consisting of-Ci ^alkoxy, -(Cs^)alkoxy-(halo)i„3, -SH, -S-(Ci^)alkyl, -N-R⁷, cyano, halo, hydroxy, nitro, oxo and -heteroaryl-R^s,

10109) In one embodiment, R⁶ is hydrogen.

101101 Embodiments of the present invention include compounds of Formula (II) wherein, R⁷ is substituents independently selected from the group consisting of hydrogen, -CMalkyl, -C_Malkenyl, -C^alkynyl, -(C_M)alkyl-OH, -(C_M)alkykO-(C_M)alkyl, -(C_M)alkyl-NH₂, -(Cw)alkyl-NH(CMalliyl), -(C_M)alkyl-N(Cwalkyl)_2j -(C_w)alkyl-S-(CM)alkyl, -C(O)H, -C(O)-(C_M)alkyl, -C(O)-O-(C,.₄)alkyl_a -C(O)-NH_2> -C(O)-NH(C,.₄alkyl),

-C(O)-N(C_Malkyl)₂, -SO₂-<C|.₄)alkyl, -SO₂-NH₂, -SO₂-NH(C|.₄alkyl)_!

-SO₂-N(Ci ialkyl)₂, -C(N)-NH_2i -cycloalkyl-R^s, -(CuJalkyl-hcterocyclyl-R³, -aryl-R³, -{Ci-4)alkyl-aryl-R^s and -(Ci-₄)alkyl-heteroaryI-R^s.

101111 In one embodiment R⁷ is 2 substituents independently selected from the group consisting of of hydrogen, -C,_₄alkyl, -C(O)H, -C(O)-(C_M)aIkyl, -C(O)-O-(C_M)alkyl, -SO₂-NH_2l-SO₂-NH(Ci.₄alkyl) and -SO₂-N(Ct-₄aIkyl)₂.

- 23 2017202571 19 Apr 2017

101121 |0113) |01141 [0115] [0116| [0117]

Embodiments of the present invention include compounds of Formula (II) wherein, R⁸ is L to 4 substituents attached to a carbon or nitrogen atom independently selected from the group consisting of hydrogen, -C|.iialkyl, -(Ci/alkyl-Oialo/ and -(Ci_4)alkyl-OH: with the proviso that, when R^s is attached to a carbon atom, R^s is further selected from the group consisting of -Cm alkoxy, -NH₂, -NlbCi.jalkyl), -N(CMalkyl)₂, cyano, halo, -(Ci_i)alkoxy“(haIo)i-3, hydroxy and nitro,

In one embodiment, R⁸ is hydrogen.

Embodiments of the present invention include compounds of Formula (II) wherein, R⁹ is 1 to 2 substituents independently selected from the group «consisting of hydrogen, -Ci„ialkoxy, -NH₂, -NH(C_MaIkyI), -NfCualkyOi, cyano, (halo)u, hydroxy and nitro.

In one embodiment, R⁹ is hydrogen.

Embodiments of the present invention include compounds of Formula (II) wherein, R is one substituent attached to a carbon or nitrogen atom selected from the group consisting of hydrogen, -Chalky l-R⁵, -Ci^alkenyl-R⁵, -Cj.^alkynyl-R⁵, -C(O)H„

-C(OHCM)alky!-R^y, -C(0)-NH2, -C(O)-NH(Cualkyl-R⁹), -C(O)-N(Ci-,aIkyl-R⁹)2, -C(O)-NH(aryl-R⁸), -C(0)-cycl0alkyl-R^§, -C(O)-heterocyclyl-R⁸, -C(O)-aryl-R⁸, -C(O)-hetcroaryl-R^s, -CO2H, -C(0)-O-(CM)alkyl-R⁹, -C(O)-O-aryl-R⁸, -SO2-(C_M)alkyl-R⁹, -SO2-aryl-R⁸, -cycloalkyl-R⁶, -aryl-R⁶ and -(CM)alkyl-N-R⁷; with the proviso that, when R² is attached to a carbon atom, R² is further selected from the group consisting of -C Malkoxy-R^J, -N-R⁷, cyano, halogen, hydroxy, nitro, oxo, -heterocyclyl-R⁶ and -heteroaryl-R⁶.

In one embodiment, R is one substituent attached to a carbon or nitrogen atom selected from the group consisting of hydrogen, -Cmalkyl-R⁵, -C2.4alkenyl-R⁵, -C24alkynyl-R^s, -CO2H, -C(O)-O-(CM)alkyl-R‘^J, -cycloalkyhR⁶, -aryl-R^e and -(CM)alkyl-N-R⁷; with the proviso that, when R² is attached to a nitrogen atom, a quaternium salt is not formed; and, with the proviso that, when R² is attached to a carbon atom, R² is further selected from the group consisting of -Ci^alkoxy-R⁵, -N-R⁷, cyano, halogen, hydroxy, nitro, oxo, -heterocyclyl-R⁶ and -hcteroaryl-R⁶.

- 242017202571 19 Apr 2017

101181 In one embodiment, R² is one substituent attached to a carbon or nitrogen atom selected from the group consisting of hydrogen, -C_Malkyl-R⁵ and -aryl-R⁶; with the proviso that, when R² is attached to a nitrogen atom, a quatemium salt is not formed; and, with the proviso that when R² is attached to a carbon atom, R² is further selected from the group consisting of -N-R⁷, halogen, hydroxy and -heteroaryl-R⁶.

[0119| Embodiments of the present invention include compounds of Formula (II) wherein, R³ is to 3 substituents attached to a carbon atom independently selected from the group consisting of hydrogen, -Crralkyl-R¹⁰, -C₂.₄alkenyl-R^!0, -C2^alkynyl-R¹⁰,

-C^alkoxy-R¹⁹, -C(O)H, -CiOEfCiffialkyl-R⁹, -C(O)-NH₂, -C(O)-NH (Chalky I-R^

-C(O)-N(C _Malkyl-R⁹)₂,-C(O)-cycIoalkyl-R^s, -C(O)-heterocyclyl-R^s, -C(O)-aryl-R-)

-C(O)-Iicteroaryl-R^s, -C(NH)-NH₂, -CO₂H, -C(O)-O-(Ci^)alkyl-R^s, -C(O)-O-aryl-R⁸,

-SO₂-(Ci„g)alkyl-R^S, -SO₂-aryl-R⁸, -N-R⁷, -(CM)alkyl-N-R⁷, cyano, halogen, hydroxy, i S S & S nitro, -cycloalkyl-R , -heterocycIyl-R , -aryl-R and-heteroaryl-R .

[0120) In one embodiment, R'¹ is one substituent attached to a carbon atom selected from the group consisting of hydrogen, -Ci^alkyl-R¹⁰, -Cj^alkenyl-R¹⁰, -C?^alkynyl-R¹⁰, -Cr4alkoxy-R^l(J, -C(O)H, -CO2H, -NH₂, -NH(C_Malkyl), -N(C_Malkyl)₂, cyano, halogen, hydroxy and nitro.

101211 In one embodiment, R³ is one substituent attached to a carbon atom selected from the group consisting of hydrogen, -Cj^alkyl-R¹⁰, -NH₂, -NH(C|^alkyl), -N(CMalkyl)₂, halogen and hydroxy.

[01221 Embodiments of the present invention include compounds of Formula (II) wherein, R⁴ is to 4 substituents attached to a carbon atom independently selected from the group consisting of hydrogen, -Cj_4alkyl-R^IU, -C2^alkenyl-R^lcl, -Cz^alkynyl-R¹⁰, -CMalkoxy-R¹⁰, -C(O)H, -C(O)-(C, A)alkyl-R\ -C(O)-NH2, -C(O)-NH(Ch4alkyl-R^i>), -C(O)-N(Ciaalkyl-R⁹)2, -C(O)-cycloalkyl-R^s, -C(O)-heterocyclyl-R^s, -C(O)-aryl-R⁸, -C(O)-heteroaryl-R⁸, -C(NH)-NH2, -CO2H, -C(Oj-O-(CM)aIkyI-R^s, -C(O)-O-aryl-R^s, -SH, -S-(CM)alkyl-Rⁱ⁰, -SO2-(C .ffialkyl-R⁹, -SO2-aryl-R⁸, -SO_rNH_?,

-SO₂-NH(Ci_4alkyt-R⁹),-SO2-N(CMalkyl-R⁹)2, -N-R⁷, cyano, halogen, hydroxy, nitro, -cycloalkyl-R⁸, -heterocyclyl-R⁸, -aryl-R^s and -heteroaryl-R⁹,

-252017202571 19 Apr 2017 |01231 1 n one embodiment, R⁴ is 1 to 4 substituents attached to a carbon atom independently selected from the group consisting of hydrogen, -C, ^alkvl-R¹⁰, -CT^alkenyl-R¹,

-C_Malkynyl-R^lc, -CMalkoxy-R^w, -C(O)H. -COZH, -NH_Z, -NH(C_Malkyl), -N(C_walkyl)₂, cyano, halogen, hydroxy, nitro, -cycloalkyl, -heterocyclyl, -aryl and -heteroaryl.

10124) In one embodiment, R⁴ is 1 to 4 substituents attached to a carbon atom independently selected from the group consisting of hydrogen, Ci_4alkyl-R^lu, Ci_4alkoxy-R¹⁰, -NH2, -NH(C|^alkyl), -N(Ci-4alkyl)₂, halogen and hydroxy, |0125) In one embodiment, R⁴ is 1 to 4 substituents attached to a carbon atom independently selected from the group consisting of hydrogen, CMalkyl-R¹⁰, Cj^alkoxy-R¹⁰, -NH₂, -NH(Ci^alkyl), -N(Cnalkyt)₂, chlorine, fluorine and hydroxy, [0126] Embodiments of the present invention include compounds of Formula (Il) wherein, R¹⁰ is 1 to 2 substituents independently selected from the group consisting of hydrogen, -NH₂, -NH(C iJtalkyl), -N(CM^lkylfe, cyano, (halo)j.3, hydroxy, nitro and oxo,

10127J In one embodiment, R¹⁰ is 1 to 2 substituents independently selected from the group consisting of hydrogen and (halo)u.

[0128| In one embodiment, R¹⁰ is 1 to 2 substituents independently selected from the group consisting of hydrogen and (fluoro)j.

[01291 Embodiments ofthe present invention include compounds of Formula (II) wherein, Y and Z are independently selected from the group consisting of O, S, (H,OH) and (H,H); with the proviso that one of Y and Z is O and the other is selected from the group consisting of O, S, (H,OH) and (H,H).

|0130| In one embodiment, Y and Z are independently selected from the group consisting of O and with the proviso that one of Y and Z is O, and the other is selected from the group consisting of O and

10131] In one embodiment, Y and Z are independently selected from O.

- 262017202571 19 Apr 2017 [0132| Compounds of Formula (Π) are disclosed in commonly assigned United States Patent

Number 7,125,878, the complete disclosure of which is herein incorporated by reference.

|0133| An example of the invent ion includes a compound of Formula (11) wherein the compound is selected from the group consisting of:

Compound Name

3“(2-chlorophenyl)-4-[]-(3-hydroxyprapyl)-l//-pyrrofo[2,3-/>]pyridin-3yl]-l //-pyrrole-2,5-dione,

3-(2-chloropheny l)-4-[l -[3-(dimethylamino )propy t ]-l//-pyrrolo[2,36]pyridine-3-yl]-l//-pyrrole-2,5-dione,

3-[l-(3-hydroxypropyl)-l//-pyrrolo[2,3-Z>]pyridin-3-yl]-4-(1naphthaieny 1)-1 //-pyrro le-2,5-dione,

3-[ 1 -[3-(dimethylamino)propyl]-l //-pyrrolo[2,3-b]pyridin-3-yl]-4-( I naphthalenyl)-1 //-pyrrole-2,5-dione,

3-(5-chlorobcnzo[0]thien-3-yl)-4-[l-(3-hydroxypropyl)-l//-pynOlo[2.3h]pyrid ine-3-y 1 ] -1 //-pyrrole-2,5 -dione,

3-[ 1 -{3-hydroxypropyl)-l //-pyrrolo[2,3-/j]pyridin-3-yl]-4-( 1 //-indazol-3y 1)-1//-pyrrole-2,5-dione,

3-{l-ethyl-l/7-pyrrolo[2,3-Z?]pyridin-3-yl)-4-[l-(3-hydroxypropyl)-l//pyrrolo[2,3-Z>]pyridin-3-yl]-1 //-pyrrole-2,5-dione,

3-[l-(3-hydiOxypropyl)-l//-pyiTO]o[2,3-5]pyridin-3-yl]-4-(2mcthoxyphenyl)-l//-pyrrole-2,5-dione,

3-[ l-(3-hydroxypropyl)-l//-pyrrolo[2,3-d]pyridin-3-yl]-4-(3methoxyphenyl)-l//-pyrrole-2,5-dione,

- 272017202571 19 Apr 2017

3-(2-chloro-4-fluorophenyl)-4-[l-(3-hydroxyprapyl)-l/7-pyrrolo[2,36]pyridine-3-yl]-1 //-pyrrole-2,5-dione,

3-[l-(3-hydroxypropyl)-l//-pynolo[2₁3-Z?]pyridin-3-yl]-4-[2<trifluoroinetliyr)phenyl]-l/i-pyiTOlc-2,5-dione,

3-[l-{3-hydroxypropyl)-l//-pyiro]o[2,3-0]pyridin-3-yl]-4-(2-pyridiny])1 //-pyrro le-2,5-dione,

3-[3-chloi'o-5-(trifluoromethyl)-2-pyridirLyI]-4-[l-(3-hydroxypropyl)-l// pyrroIo[2,3-0]pyridln-3-yl]-l //-pyrrole-2,5 -dione,

3-[ 1 -(3-hydroxypropyl)-1 //-pyiTolo[2,3-i]pyridin-3-yl]-4-(2-thienyl)I //-pyrrole-2,5-dione,

3-(2,5-dich]oro-3-thienyl)-4-[l-(3-hydiOxypropyl)-l//-pynOlo[2.3Z>]pyridine-3-yl]-l //-pyrrole-2,5-dione,

3-[l-(3-hydroxypropyl)-1 //-pyrazo!-3-ylJ-4-[ 1 -(3-hydroxypropyl)-1//pyrrolo[2,3-Z?]pyridin-3-yl]-1 //-pyrrole-2,5-dione,

3-[l-(3-hydroxypropyl)-l//-pyrrolo[2,3-/)]pyridin-3-yl]-4-(]//-imidazol2-yl)-l //-pyrrole-2,5-dione,

3-[l-(3-hydroxypropyl)-17/-imidazol-4-yt]-4-[t-(3-hydroxypropyl)-17/pyrrolo[2,3-6]pyridm-3-yl]-l//-pyrrole-2,5-dione,

3-[ 1 -(2-hydroxyethyl)-1 //-imidazol-4-yl]-4-[ 1 -(3-hydroxypropyl)-1Hpyrrolo [2,3 -ύ] pyndin-3-y 1]-1 //-pyrro le-2,5 -dione,

3-[l-[3-(dirficthylamino)propyl]-l//-indazol-3-yl]-4-[]-(2-naphthalenyl)· l//-pynOlo[2,3-0]pyridin-3-y[]-l//-pyrrole-2,5-dione,

3-[ 1 -(3-hydroxypropyl)-l i/-indazol-3-yl]-4-[l -(2-naphthalenyl)-1//pyrrolo[2,3-Z)]pyridin-3-yl]-17/-pyrrale-2,5-dione,

-282017202571 19 Apr 2017

3-[(£)-2-(4-fluorophenyl)ethenyl]-4-[l-(3-hydraxypiOpyl)-l//pyrrolo[2,3-Z)]pyridin-3-yl]-I//-pyrrale-2,5-dione,

3-(3,4-dihydro-2//-pyran-6-yl)-4-[ 1 -(3-hydroxypropyl)- l//-pyrrolo[2,30]pyridi nc-3-y I ] -1 //-pyrro le-2,5-dion e,

4-[ l-(3-liydiO.\ypiOpyl)“l//-pynolo[2,3-0]pyridin-3-yl]-[3,3'-bi-l Hpyrrote]-2,5-dione,

3-(2-benzofuranyl)-4-|4-(3-hydroxypropy[)-l//-pyrrolo[2,3-0]pyridm-3yl]-1 //-pyrro le-2,5-di one,

3-(1 -(3-hydroxypropyl)-1 //-pyrrolo[2,3-Z>]pyridin-3-yl]-4-( 1 -methyl-1// pyrazol-3-yl)-] //-pyrrole-2,5-dione,

2,5-dihydro-4-[l-(3-hydroxypropyl)-1//-pyrrolo[2,3-/>]pyridin-3-yl]-2,5dioxo-l//-pyrrole-3-carbonitrile,

3-dibenzo[/?,z/]thicn-4-yl-4-[] -(3-hydroxypropyl)-l//-pyrroio[2,3Z>]pyridine-3-yl]-l//-pyrrole-2,5-dione,

3-(4-dibenzofuranyl )-4-[ 1 -(3-hydroxypropyl)- I //-pyrro lo[2,3-/j]pyrid in3-yl]-l//-pyrrole-2,5-dione,

3-(2-hydroxyphenyl)-4-[ 1-(3-methoxypropyi)-l //-pyrrolo [2,3-/j]pyridin· 3-yl]-l//-pyrrole-2,5-dione,

3-(3,4-dimethoxypheny])-4-[l-(3-methoxypropyl)-l//-pyiTolo[2,3ifipyridi nc-3-y 1] -1 //-pyrro le-2,5 -dione,

3-(3,4-dihydroxypheny I )-4-( I -(3-hydroxypropyl)-1 //-pyrrolo[2,3&]pyridine-3-yl]-1 //-pyrrole-2,5-dione,

3-(2-methoxyphenyl)-4-[ I -(2-naphthalenyl)-1 //-pyrroIo[2,3-0]pyridin-3 yl]-1 //-pyrrole-2,5 -dion e,

- 292017202571 19 Apr 2017 [3-[3-[2,5-dihydro-4-(2-methoxyphenyl)-2,5-dioxo-l//-pyrrol-3-yl]-17/pyrrola[2,3-Z}]pyndin-l-yl]propyl]-carbamic acid 2-methylpropyl ester,

3-[ 1 -(3-aminopropyl)- l/7-pyrralo[2,3-&]pyridin-3-yl]-4-(2mcthoxyphenyl)-17/-pynoIe-2,5-dione, ?7-[3-[3-[2,5-dihydro-4-{2-methoxypheny 1)-2,5-dioxo-1 //-pynol-3-yl]1 7/-pyrrolo[2,3-/j]pyridin-1 -yljpropy 1] -acetamide,

V-[3-[3-[2,5-dihydrori-(2-methoxyplieny 1)-2,5-dioxo-l/7-pytTOl-3-yl]1 Ti-pyrrolo [2,3-ft]pyr id in-1-y Ijpropy 1]-su 1 famide,

3-(2-methoxypheny])-4-[]-[3-(l//-tetrazol-l-yL)propyl]-17/-pyrrolo[2,36]pyridine-3-yl]-177-pyrrole-2,5-dione,

3-(2-metboxyphcny!)-4-[l -[3-(2//-tetrazol-2-yl)propyl]-l//-pynolo[2,3i)]pyridine-3-ylj-l/7-pyrrole-2,5-dione,

3-[ 1 -(3-hydroxy-propyl)-l H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-ylpyrroIe-2,5-dione,

3-(2,4-dimethoxy-pyrimidin-5-yl)-4-[ I -(3-hydroxy-propyl)-l HpynOlo[2,3-bjpyridin-3-yl]-pyiTole-2,5-dione,

4- [3-[4-(2,4-dimethoxy-pyrimidin-5-yI)-2,5-dioxo-2,5-dihydro-l HpyriOl-3-yl]-pyrrolo[2,3 -bjpyridin-1 -ylj -butyronitrile,

4-{3-[4-( 1 -methyl-lH-pyrazol-3-yl)-2,5-dioxo-2,5-dihydro-l H-pyrrol-3yl]-pyrTolo[2,3-b]pyridin-] -yl j -butyronitrile, and

3-(2,4-dimethoxy-pyrimidin-5-yl)-4-( 1 -phenethyl-] H-pyrrolo[2,3b]pyridine-3-yl)-pyrrole-2,5-dione.

|0134| An example of the invention includes a compound of Formula (11) wherein the compound is selected from the group consisting of:

-302017202571 19 Apr 2017

Compound 44

10135J In one embodiment, the inhibitor of GSK.-3B enzyme activity is a compound of the

Formula (III);

-31 2017202571 19 Apr 2017

|0136) wherein

10137)

A and E arc independently selected from the group consisting of a hydrogen substituted //^x

carbon atom and a nitrogen atom; wherein N is independently selected from the group consisting of 1/7-indote, ]/7-pyrrolo[2,3-b]pyridine, l//-pyrazolo[3,4ftjpyridine and 1 /7-indazole;

|0138) Z is selected from O; alternatively, Z is selected from dihydro; wherein each hydrogen atom is attached by a single bond;

10139) Ri and Rs are independently selected from C[.«alkyl, Cz-salkenyl and C₂__Ralkynyl optionally substituted with oxo;

|0140) R-2 is selected from the group consisting of -Cualkyl-, -CYsalkenyl-, -CAsalkynyl-,

-O-(C|._s)alkyl-O-, -O-iQAalkenyl-O-, -O-(C₂__R)alkynyl-O-. -C(O)-(C_hS)alkyl-C(O)(wherein any of the foregoing alkyl, alkenyl and alkynyl linking groups are straight carbon chains optionally substituted with one to four substituents independently selected

-322017202571 19 Apr 2017 from the group consisting ofCi.«alkyl, Ci-galkoxy, C|^aIkoxy(Ci-s)alkyl, carboxyl, carboxy 1(Ci__s)alkyl, -C(O)O-(C|._s)alkyl, -Ci._ftalkyl-C(O)O-(Ci._s)alkyl, amino (substituted with a substituent independently selected from the group consisting of hydrogen and Cualkyl), aniino(Cj.s)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and Ci^allcyl), halogen, (ha!o)].₃(CL^)alkyl, (halo)i.₃(C^alkoxy, hydroxy, hydroxy(C|._s)alkyl and oxo; and, wherein any of the foregoing alkyl, alkenyl and alkynyl linking groups are optionally substituted with one to two substituents independently selected from the group consisting of heterocyclyl, aryl, heteroaiyl, heterocyclyl(Ci__a)alkyl, atyl(Ci.g)alkyl, hetciOaryl(Cj-s)alkyl, spirocycloalkyl and spiroheteroeyclyl (wherein any of the foregoing cycloalkyi, heterocyclyl, aryl and heteroaryl substituents are optionally substituted with one to four substituents independently selected from the group consisting of C,.«alkyl, Ct-salkoxy, C|.,salkoxy(Ci.«)alkyl, carboxyl, carboxyl(C|.s)alkyl, amino (substituted with a substituent independently selected from the group consisting of hydrogen and Ci^alkyl), amino(Ci-g)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and Cj^alkyl ), halogen, (halo)[.₃(Ci-g)alkyI, (halo)[.₃{Ci.g)alkoxy, hydroxy and hydroxy(Ci-s)alkyl; and, wherein any of the foregoing heterocyclyl substituents are optionally substituted with oxo)), cycloalkyi, heterocyclyl, aryl, heteroaryl (wherein cycloalkyi, heterocyclyl, aryl and heteroaryl are optionally substituted with one to four substituents independently selected from the group consisting gf Ci__BaIkyl,C|.«alkoxy, C μ «alkoxy (C|.s)alkyl, carboxyl, carboxy l(C|.s)alkyl₁ amino (substituted with a substituent independently selected from the group consisting of hydro gen and Chalky!), amino(Ci_g)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and Chalky]), halogen, (halo)].₃(C|_s)alkyl, (halo)].₃(C[_g)alkoxy, hydroxy and hydraxy{C|.s)alkyl; and, wherein heterocyclyl is optionally substituted with oxo), -(0-(CH₂)i^)o5'0’, -O-(CH₂)j.₆-O-(CH₂)_w-O-, -(HCH₂)_w-0-(CH₂)_w.O4CH₂)_u.0., -(0-(CH2),^)o.₅-NRi-, -O-(CH₂)i._G-NR<₅-(CH₂),^O-,-O-(CH₂},-₆-O-(CH₂)_l._s-NR_i-, -(O-(CHi)t^)₀.₅-S-. -0<CH₂),.₆-S-(CH₂)^-0-, -O-(CH₂)_w-O-(CH₂)_w-S-, -NR,,-, -NRe-NR?-, -NR_g-{CH₂)i._g-NR₇-, -NR_€-(CH₂)i.₆-NR₇-(CH₂)_r6-NRR-, -NR^-CfO)-, -C(O)-NR<r, -C(O)-(CH₂WNR_G-(CH₃)₀.₆-C(O)-,

-33 2017202571 19 Apr 2017

-NR₆-(CH₂)₀.₆-C(O)-(CH₂)₁.₆-C(Oh(CH₂)c.₆-NR7-,-NR₆-C(O)-NR₇-, -NR6-C(NR₇)-NR_S-, -O-(CH₂)i_₆-NRHCH₂WS-, -S-iCHjJj.e-NRACH,)-S-(CH₂),.₆-NR₆-(CH₂)^-S-, -NR6-(CH₂)|.6-S-(CH₂)i4i-NR7- and -SO₂- (wherein Re, R? and Rk are independently selected from the group consisting of hydrogen, Ct-salkyl, C|.salkoxy(C|.s)alkyl, carboxyl(Cj.8)alkyl, amino(Ci_s)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and Cj.4a.lkyl ), hydroxy(Ci_is)alkyl, heterocyclyl(C't^)alkyl, aryl(C].»)alkyI and heteroaryl(Ci__K)alkyl (wherein the foregoing heterocyclyl, aryl and heteroaryl substituents are optionally substituted with one to four substituents independently selected from the group consisting ofCrsalkyl, Ci^alkoxy, Ci.Ralkoxy(Cj_8)alkyl, carboxyl, carboxyl(Ci.g)alkyl, amino (substituted with a substituent independently selected from the group consisting of hydrogen and Ci^alkyl), amino(Ci-8)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and Chalky I), halogen, (halo)i_i(Cj.is)aJkyl, (halo)iACi-s)alkoxy, hydroxy and hydroxy(C)^)alkyl; and, wherein heterocyclyl is optionally substituted with oxo)); with the proviso that, if A and E are selected from a hydrogen substituted carbon atom, then R₂ is selected from the group consisting of -CAalkynyl-, -O-(Ci-8)alkyl-O-, -O-(C₂._s)alkenyl-O-,

-O-(C₂_s)alkynyl-O-, -C(O)-(C|,s)alkyl-C(O)- (wherein any of the foregoing alkyl, alkenyl and alkynyl linking groups are straight carbon chains optionally substituted with one to four substituents independently selected from the group consisting of Ct-galkyl, C^alkoxy, Ci.salkoxy(Ci-s)alkyl, carboxyl, carboxylfCi.sjalkyt, -C(O)O-(C|.B)alkyl,

-Ci salkyl-C(O.)O-(C| s)alkyl, amino (substituted with a substituent independently selected from the group consisting of hydrogen and Chalky I), amino(Cfrs)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting ofhydrogen and Chalky 1), halogen, (halo)].i(Cns)alkyl, (halo)].₂(C|_s)alkoxy, hydroxy, hydroxy(Ci.fi)alkyl and oxo; and, wherein any of the foregoing alkyl, alkenyl and alkynyl linking groups are optionally substituted with one to two substituents independently selected from the group consisting of heterocyclyl, aryl, heteroaryl, heterocyclyl(C|-s)alkyl, aryl(Ci.s)alkyl, heteroaryl(Ci.g)alkyl, spirocycioalkyl and spiroheterocyclyl (wherein any of the foregoing cycloalkyl, heterocyclyl, aryl and hetcroaryl substituents are optionally substituted with one to four substituents

-342017202571 19 Apr 2017 independently selected from the group consisting of Ci/alkyl, Ci/alkoxy,

C|,_salkoxy(Ci_f!)alkyl, carboxyl, carboxy I (C|._s)alkyl, amino (substituted with a substituent independently selected from the group consisting of hydrogen and C|/alkyi), amino(Ci/)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and Ci/alkyl), halogen, (halo)i/(C| .gjalkyl, (halo)i/(Ci/)alkoxy, hydroxy and hydroxy(C].g)alkyl; and, wherein any of tire foregoing heterocyclyl substituents are optionally substituted with oxo)), cycloalkyl (wherein cycloalkyl is optionally substituted with one to four substituents independently selected from the group consisting ofCj-galkyl, Ci/alkoxy, Ci/aIkoxy(’Ci/)alkyl, carboxyl, carboxyl(Ci/)alkyl, amino (substituted with a substituent independently selected from the group consisting of hydrogen and Ci/alkyl), amino(Ci/)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and Cj/alkyl), halogen, (halo)i/(Ci/)alkyl, (halo)i/(C|.s)alkoxy, hydroxy and hydroxy(Ci__g)alkyl),-(O-(CH₂)i/)_t/-O-, -0-(CH₂)_I/r0-(CH2),._ii-O-, -O-(CH₂)₁.₆-O-(CH₂)₁/-O-(CH₂)i.₆-O-, -(O-CCtkh.eh.s-NRi-,

-O-(CH₂)^-NR_i-(CH₂)₁/_rO-₎-0-(CH₂)_J/-O-(CH₂)i/-NR₆-, -(O-(CH₂),/)_Q/-S-, -0-((^)^-8-(013^/-0-, -O-(CH₂)i/-O-(CH₂)i/-S-, -NRs-W, -NR(i-(CH₃),/-NR₇-, -NRHCHzKe-NRriCHs^-NRs-, -NR₉-C(O)-₅ -C(O)-NR_y-,

-C(O)-(CH₂)₀.₆’NR_ii-(CH₂)0.₆-C(O)-, -NR₆-(CH₂F._fi-C(O)-(CH₂),.₆-C(O)-(CH₂WNR₇-, ,NR^C(0)-NR₇-, -NR_fi-C(NR₇)-NRs-, -O-(CH₂)i__s-NR₆-(CH₂)_]/-S-, -STCHOiz-NRi-tCH^M-O-, -S-(CH₂)|.₆-NRf,-(CH₂)i/-S- and

-NR6-(CH₂)i/-S-(CH₂)i/-NR₇- (wherein Rs, R₇ and Rs are independently selected from the group consisting of hydrogen, Ct__salkyl, G/alkoxy(Ci/)alkyl, carboxy l(Ci_₈)alkyl, amino(CL-s)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and Cj/alkyl), hy droxy (C//)alkyl, heterocyclyl(Ci-s)alkyl, aryl(Ci/)alkyl and heteroaryl(Ci-s)aIkyt (wherein the foregoing heterocyclyl, aryl and heteroaryl substituents are optionally substituted with one to four substituents independently selected from the group consisting of Ci^alkyl, C i/alkoxy, C|_salkoxy(Ci_s)alkyl, carboxyl, carboxyl(Ci_g)alkyl, amino (substituted with a substituent independently selected from tire group consisting of hydrogen and C|/alkyl), amino(Ci/)alkyl (wherein amino is substituted with a substituent independently selected

-352017202571 19 Apr 2017 from the group consisting of hydrogen and C].₄alkyl), halogen, (halo)i^(C i-sjalkyl, (halo)_!.3(C[_f_t)alkoxy₃ hydroxy and hydroxy(Cp_S)alkyJ; and, wherein heterocyclyl is optionally substituted with oxo); and, wherein Rg is selected from the group consisting of Ci-salkyl, C|.salkoxy(Ci,s)alkyl, carboxyl(Ci,s)alkyl, amino(C).R)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and Cj.₄alkyl), hydroxy(Ci,g)atkyl, hetcrocyclyl(Ci-s)ajkyt, aiyl(C[^)alky 1 and heteroaryl(Ci_8)alkyl (wherein the foregoing heterocyclyl, aryl and heteroaryi substituents are optionally substituted with one to four substituents independently selected from the group consisting of Ci-gaLkyl, Ci_₈alkoxy, Ci._salkoxy(Cb₈)alkyl, carboxyl, carboxyI(Cj.:))alkyl, amino (substituted with a substituent independently selected from the group consisting of hydrogen, and C i_₄alkyl), amino(C|_s)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and Chalky 1), halogen, (halo)i-j(Ci^)alkyl, (halo)]-jt(C|^)alkoxy, hydroxy and hydroxy(Cjji)alky!; and, wherein heterocyclyl is optionally substituted with oxo)); and, [0141] R| and Ri are independently selected from the group consisting of hydrogen, Cj.galkyl, Cz-salkenyl, Cj.palkynyl (wherein alkyl, aLkenyl and alkynyl are optionally substituted with a substituent selected from the group consisting of C’i_₈alkoxy, alkoxy(C i_s)alkyl, carboxyl, carboxyl(Ci.B)alkyl, amino (substituted with a substituent independently selected from the group consisting of hydrogen and C|.₄alkyl), amino(C| .slalky 1 (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and Cualkyl), (halo)i_3, (haloh-aCCi-sMkyl, (halofiXCrslalkoxy, hydroxy, hydroxy(C|.s)alkyl and oxo). C i.«alkoxy, Ci^alkoxycarbonyl, (lialo)).«(C).«)alkoxy. Crsalkylthio, aryl, heteroaryi (wherein aryl and heteroaryi are optionally substituted with a substituent selected from the group consisting of Chalky!, Cigalkoxy, alkoxy(Ci_g)alkyl, carboxyl, carboxyl(Ci-s)alkyl, amino (substituted with a substituent independently selected from the group consisting of hydrogen and C|.₄alkyl), amino(C|.s)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and Cualkyl), halogen, (halo)i-.i(C i Aalkyl, (halo)j j(C!_s)aIkoxy, hydroxy and hydroxy(Ci.g)alkyl), amino (substituted with a substituent independently selected from the group consisting of hydrogen and Chalky!), cyano, halogen, hydroxy and nitro; and pharmaceutically acceptable salts thereof.

-36|01421

In one embodiment, a compound of Formula (III) is a compound selected from the group

2017202571 19 Apr 2017 consisting of:

Formula (Ule)

Formula (Illf)

-372017202571 19 Apr 2017

Formula (Illk)

Formula (Illi)

-382017202571 19 Apr 2017

10143] wherein all other variables are as previously defined; and, pharmaceutically acceptable salts thereof.

[0144| In one embodiment, a compound of Formula (Π1) is a compound selected from the group consisting of:

-392017202571 19 Apr 2017

Η

Formula (lllj) [0145] wherein all other variables are as previously defined; and, pharmaceutically acceptable salts thereof.

|0146| Compounds of Formula (Ill) are disclosed hr commonly assigned United States Patent Number 6,828,327, the complete disclosure of which is herein incorporated by reference

10147) An example ofthe invention includes a compound of Formula (ΠΊ) wherein the compound is selected from the group consisting of:

-402017202571 19 Apr 2017

Compound Name

Ί 6,7,9,10,12,13,15,16-octahydm-23//-5^26:17,22-dimetheno-5/7dipyrido[2,3-£:3',2'-ij]pyrroIo[3,4“

n][1,4,7,10,19]trioxadjazacyclohenicosinc-23,25(24//)-dione,

10,11, J 3,14,16,17,19,20,22,23-decahydro-9,4:24,29-dimetheno-) Hdipyrido [2,3-tt: 3 ',2'-f]pyrro lo[3,41,4,7,10,13,22]tetraoxadiazacyclotetracosine-1,3(2//)-dione,

10,11,13,14,16,17,19,20,22,23_s25,26-dodecahydro-9,4:27,32-dimethenol/7-dipyrida[2,3-<y:3',2'-«/|pyrrolo[3,4r] [ 1,4,7,10,13,16,25 Jpentaoxadiazacy cloheptacosine-1,3 (2//)-dione,

6,7,9,10,12, l3-hcxahydiO-20//-5,23:I4,19-dimetheno-5//dibsnzo[A,«]pyiTolo[3,4-A^r] [1,4,7,16]dioxadiazacyclooctadecine20,22(2 l//)-dione,

6,7,9,10,12,13,15,16-octahydro-23//-5,26:17,22-dimetheno-5/7dibenzo[4,g]pyrrolo[3,4'rt][l,4,7,10,19]trioxadiazacycloheneicosine23,25(24//)-dione,

10,ll,13,14,I6,17,19,20^2,23-decahydro-9,4:2429-dimetheno-l//dibenzo[«, i]pyrrolo[3,4-</][I,4,7,10,13,22]tetraoxadiazacyclotetracosinel,3(2//)-dione,

10,11,13,14,16,17,19,20,22,23₁25,26-dodecahydro-9,4;27,32-dimetheno1//-d ibenzo[i/,H^j]pyn:oIa [ 3,4r] [1,4,7,10,13,16,25]pentaoxadiazacycloheptacosine-1,3(2//)-dione,

12-hydro-6//, 19//-5,22:13,18:7,1 l-trimethenopyrido[2,37]pyrrolo[3,4m][l ,9]benzodiazacycloheptadecine-l 9,21 (20//)-dione,

-41 2017202571 19 Apr 2017

12-hydro-6//,l 9//-5,22:13,18-dimetheno-7,11 -nitrilopyrido[2,3/]pyiTolo[3,4-ni][l,9]benzodiazacyctoheptadecine-19,21(20//)-dione,

6,7,9,10,12, l3-hexahydro-20//-5,23:I4,19-dimetheno-5//-pyrido[2,3£]pyrrolo[3,4-/i][4,7, l,10]benzodioxadiazacyclooctadccine-20,22(21//)dione,

6,7,9,10,12,13,15,) 6-octahydro-23//-5,26;l 7,22-dimetheno-5//-pyrido[2,3 njpyrrolo [3,4-c/][4,7,10,1,13]benzotrioxadiazacycioficnei cosine23, 25(24/7)-dione,

11 -ethyl-6,7,10,11,12,13,15,16-octahydro-23//-5,26:17,22-dimetheno5//,9//-dibenzo[A*,^]pyrrolo[3,4«][ 1,7,4,10,19] dioxatri azacycloheneicosinc-23,25(24//)-dione,

6,7,10,1 lj 12,.13,15,16-octahydro-l I-methy1-23/7-5,26:17,22-dirnetherio5//,9//-dibenzo[A',^]pyrrolo[3,4n][ 1,7,4,10,19]dioxatriazacycloheneicosine-23,25(24//)-dione,

6,7,10,11,12,13,15,16-octahydro-l 1 -(1 -methy lethy1)-23//-5,26:17,22dimetheno-5//,9//'dibenzo[A,7]pyiTolo[3,4n] [ 1,7,4,10,19] dioxatri azacyc loheneicosine-23,25(24Z/)-d tone,

7,8,9,10,11,12,13,14,15, l6-decahydro-8,11,14-trimethy 1-6//, 23//5,26:17,22-dimethenodibenzo[/j,f]pyiTolo[3,4g][l ,4,7,10,13]pentaazacyctoheneicosine-23,25(24//)-dione,

6,7,10,11,12,13,15,16-octahydro-l l-mcthyI-23//-5,26-metheno-17,22mtrilo-5//.9//-dibenzo[/i,i/]pyrro[o[3,4n][l ,7,4,10,19]dioxatriazacycIohcneicosine-23,25(24//)-dione,

11-ethyl-6,7,10,1I,12,13,I5,16-oetahydro-23//-5,26-metheno-17,22-nitrilo 5//,.9//-dibenzo[£,i/]pyna]o]3,4n][l,7,4,10,19]dioxatriazacycloheneicosine-23,25(24//)-dione,

- 42 2017202571 19 Apr 2017

11-ethyl-6,7,10,11,12,13,15,16-oetahydro-23//-5,26:17,22-dimetheno5 H, 9/i-dipyrido[2,3-U3' ,2 '-G'pyrro lo [3,4n] [ 1,7,4,10,19]dioxatriazacycloheneicosine-23,25(247/)-dione,

6,7,9,10,12,13,15,16-octahydro-237/-5,26:17,22-dihictheno-57/dipyrido[2,3-7:3',2'-gJpyrrolo[3,4n] [ 1,7,4,10,19]dioxathiadiazacyclohenci cosine-23,25(247/)-dione,

7,8,9,10,11,12,13,14,15,16-decahydro-(67/,237/-5,26:17,22dimethenodipyrido[2,3-rt:3',2'-f]pyriO]o[3,4<7 ] [ 1,7,13] triazacyc loheneicosine-23,25(247/)-dicme,

11 -ethy 1-7,8,9,10,11,12,13,14,15,16-decahydro-677,237/-5,26:17,22dimethenodipyrido[2,3-«:3',2'-i]pyiTolo[3,4g][ 1,7,13]triazacycloheneicosine-23,25(247/)-dione,

6,7,8,9,10,11,12,13,14,15-dccahydro-227/-5,25:16,21 -dimetheno-57/dipyrido[2,3-wi ;3',2'-y]pyrrolo[3,4-p][ 1,6,12]triazacycloeicosine22,24(23/7)-dione,

10-ethy 1-6,7,8,9,10,11,12,13,14,15 -decahy dro-22/7-5.25:16,21 -dimetheno57/-dipyrido[2,3-7«:3’,2'-s]pyn^,olo[3,4’/?][ 1,6,12]triazacycloeicosine22,24(237/)-dione,

7,8,9,15,16,17,18-heptahydro-67/,2577-5,28:19,24-dimetheno-10,14nitrilodipyrido[2,3-6:3',2'-7]pyrrolo[3,4-e][ 1,1 Ojdiazacyclotricosine25,27(267/)-dione,

7,8,9,10,11,13,14,15,16-nonahydro-6//,23//-5,26:17,22dimethenodipyrido[2,3-6:3^l,2'-6]pyn^,olo[3,4-c][],10]diazacycloheneic0sinc 12,23,25 (247/)- trione,

7,8,9,11,12,13,14-heptahydro-6/7,2]/7-5,24:15,20-dimetliertc)dipyrido[2,36:3',2'-6]pyrrolo[3,4-t'][l,10]dia2acyclononadecine-10,2l,23(22T/)-trione,

-43 2017202571 19 Apr 2017

6,7,8,9,10,11,12,13,14,15-decahydro-7,14-dihydroxy-(7A147f)-22/75,25:16,21-dimetlieno-5/7-dipyrido[2,3-Z):3^,,2'-/j]pyrrolo[3,4e][1,1 0]diazacyclocicosine-22,24(23/7)-dione,

6,7,9,10,12,13-hcxahydro-2077-5,23:14,19-dimetheno-5/7-dipyrido[2,3h :3',2'-«]pyiTolo[3,4-E][ 1,4,7,16]dioxadiazacyclooctadecine-20,22(21/7)dione,

6,7,10,11,12,13,15,16-octahydro-l l-(2-methoxyethyl)-23/7-5_I26-metheno 17,22-nitrilo-5//,9//-dibenzo[£,(y]pyrrolo[3,4n][ 1,7,4,10,19]dioxatriazacycIoheneicosine-23,25(24/7)-dione,

6,7,10,11,12,13,15,16-octahydro-l I-(2-hydroxyethy1)-23/7-5,26:17,22dimctheno-5/7,9/7-dibenzo[A\^]pyrrolo[3₁4n] [I,7,4,10,19]dioxatriazacycIohencicosine-23,25(24/7)-dione, and

6,7,9,10,12,13,14,15,16,17-dccahy dro-14-mcthy1-2477-5,27 :18,23dimetheno-5/7-dibenzo[/,r]pyn'olo[3,4o] [ 1,4,7,11,20] dioxatriazacycIodocosine-24,26(25/7)-dione.

|0148] An example of the invention includes a compound of Formula (III) wherein the compound is selected from the group consisting of:

Compound 1 Compound 2 Compound 5

-442017202571 19 Apr 2017

|Θ149| Other examples of the invention include a compound selected from die group consisting of:

Compound Name la To be provided

2a 3-[l-[3-[(2-hydroxyethy])methylamino]propyl]-lH-indazol-3-yl]4-[l-(3-pyridinyl)-lH-indot-3-yl]-lH-pyrrole-2,5-dione,

3a 3,5-dichloro-N-[3-chloro-4-[(3,4,32,12a-tetraliydro-IH[ 1,4]thiazino[3,4-c] [ 1₅4]benzodiazcpin-l I (6H)yl)carbonyl]phcnyl]-benzamide,

4a 3 -[ 1 -(2-hydroxy -ethyl)-1 H-i ndo l-3-yI]-4-( 1 -pyridin-3-yl-1 H-in dol 3-yl)-pyrrole-2,5-dione,

5a 3-{2-methoxy-pheny 1)-4-(1 -pyridin-3-yl-I H-indol-3-yl)-pyrrole2,5-dione,

6a 6-[[2-[[4-(2,4-dichloropheny l)-5-(4-methy 1-1 H-imidazol-2-yl )-2pyrimidinyl]amino]ethyl]amino]-3-pyr]dinccarbonitrile,

-452017202571 19 Apr 2017

7a 3 -(5-ch loro-1 -methyl-1 H-indo l-3-yl)-4-[ 1 -(3-imidazol-l -y lpropyl)-lH-indazol-3-yl]-pyrrole-2,5-dione,

8a 3-(5-chloro- 1-methyl-I H-indol-3-yl)-4-[l-(3-[ 1,2,3 ]triazol-l-ylpropyl)-1 H-indazoI-3-yl]-pyrroIc-2,5-dione,

9a 3-[l-(3-hydiOxy-propyl)-IH-pynolo[2,3-b]pyridin-3-yl]-4-( 1methy 1-1H -pyrazol-3 -y 1 )-pyrro le-2,5-dione,

10a To be provided lla 3-[ 1 -(3-hydroxy-3-methyl-buty I)-1 H-indazol-3-yl]-4-( 1 -pyridin-3 y 1-1 H-indol-3-yl)-pyrrote-2,5-dione,

12a 3-[I-(24iydroxy-ethyl)-lH-tndazol-3-yI]-4-(l-pyrimidin-5-yl-lHtindol-3-yl)-pyrrole-2,5-dione,

13a 3 -[ I -(2-hydroxy-ethyl)-1H -in dol -3-yl]-4-( 1 -pyriraiding -yl-1Hindol-3-yl)-pyrrole-2,5-dione,

14a (] lZ)-8,9,10,13,14,l5-hexahydro-2,6:17,2Idi(metheno)pyrrolo[3,4-h][I,15,7]dioxazacyclotricosine22,24( I H,23H)-dione,

15a 3 -{5-chloro-1 -pyridin-3 -y 1-1 H-indol -3-y 1 )-4-[ 1 -(3 -hydroxypropyl)- 1 H-indazol-3-yl]-pyrroIe-2,5-dione,

16a 3-(2-methoxy-phenyl)-4-[l-(3-methoxy-propyl)-lH-pyrrolo[3,2c]pyridin-3-yl]-pyrroIe-2,5-dione,

17a 3-[l-(3-hydroxy-propyl)-lH-indazor-3-yl]-4-[ l-(tetrahydro-pyran·

-y I)-1 H-indo I -3-y I ]-pyrro le-2,5-dione,

18a 2-{3-[4-(5-chlora-1-methyl-1 H-indol-3-yl)-2,5-dioxo-2,5-dihydro

H-pyrrol-3-yl]-indazol-1 -yl} -N-(2-hydroxy-ethyl)-acetamide,

-464-(3-ch loro-phenyl )-6-(3-di methy lamino-propy I )-5,6-dihydro-4H2,4,0-triaza-cyclopenta[c]fluQiine-1,3-dione,

14-cthyl-6,7,9,10,13,14,15,16-octahydro-12H,23 H-5,26:17,22dimethenodibenzo[k,q]pyrrolof3,4n J [ 1,4,7,10,19]dioxatriazacyclohen ei cos ine-23,25 (24H)-dione,

14-benzyl-6,7,9,10,13,14,15,16-octahydro-12H.23H-5.26:17,22di(metheno)dibenzo[k,q]pyrralo[3,4n][l,4,7,10,19]dioxatriazacyclohenicosine-23,25(24H)-dione,

3-( l-{2-[2-(2-hydroxy-ethoxy)-ethoxy]-ethyl}-1 H-indol-3-yl)-4-[l (2-hydroxy-ethyl)-lH-indol-3-yI]-pyrrole-2,5-dione,

6,7,8,9,10,11,12,13-octahydro-8,1 I -dimethyl-5,23:14,19dimetheno-20H-dibenzo[k,q]pyrroIo[3,4n][ 1,4,7,10]tetraazacyclooctadeeine-20,22(21 H)-dione,

7,8,9,10,12,13,16,17,18,19-decahydro-8,17-dimethyl-15H,26H5.29:20,25-dirricthcno-6H-dibcrLzofk,q]pyrrolo[3,4n][l ,4,7,10,19,22]dioxatetraazacyclotetracosme-26,28(27H)-dione,

J 4-(2-fury lmethyl )-6.7,9,10,13,14,15,16-octahydro- 12H,23H5,26:17,22-di(metheno)dibenzo[k,q]pyrrolo[3,4n][l,4,7,10,19]dioxatriazacyclohenicosine-23,25(24H)-dione,

14-(2-thienylmethyl )-6,7,9,10,13,14,15,16-octahydro-12H.23H5 /26:17,22-di(metheno)dibenzo[k,q]pyrroto[3,4n][ 1,4,7,10,19]dioxatriazaeyclohenicosine-23,25(24H)-dione,

-472017202571 19 Apr 2017

27a 14-( 1 -naphthylmethy 1)-6,7,9,10 J 3,14,15,16-octahydro-i 2H,23H5,26:17,22-dt(metlieno)dibenzo[k,q]pyrrolo[3,4n][1,4,7,10,19]dioxatriazacyclohenicosine-23,25(24H)-dione,

28a 14-(pyridin-4-y lmethyI)-6,7,9,10,13,14,15,16-oetahydro-12H,23H5,26:17,22-di(metheno)djbenzo[k,q]pyrrolo[3,4n ][ 1,4,7,10,19]dioxatriazacyclohenicosinc-23,25(24H)-dione,

29a 3-[ I -(2-{2-[2-(1,2,3,4-tctrahydro-naphthalcn-l-ylamino)-ethoxy]ethoxy}-cthyl)-lH-indol-3-yl]-4-{ l-[2-(I,2,3,4-tetrahydronaphthalen-]-ylamino)-ethyl]- lH-indol-3-ylf-pyrrole-2,5-dionc,

30a 3-[l-(3-dimethylamino-phenyl)-lH-indol-3-yl]-4-[l-(2-bydroxye thy!1 H-indazol-3-y I] -pyrrole-2,5-dione,

31a 3-[5-cbloro-]-(6-dimethylamino-pyridin-3-yl)-]H-indol-3-y]]-4-[l(2-hydroxy-ethyl)-lH-indazol-3-yI]-pyrrole-2,5-dione, and

32a 5-i5-chloro-3-{4-[l-(2-hydroxy-ethyl}-lH-indazol-3-yi]-2,5-dioxo2,5-dihydro-IH-pyrrol-3-ylf-indol-l-yl)-nicotinic acid methyl ester.

10150| Other examples of the invention include a compound selected from the group consisting of:

-482017202571 19 Apr 2017

Compound 4a

Compound 5a

Compound 6a

Compound 7a

Compound 8a

Compound 9a

-492017202571 19 Apr 2017

Compound 16a

Compound 17a

Compound 18a

-502017202571 19 Apr 2017

Compound 19a

Compound 22a

Compound 23a

Compound 25 a

Compound 26a

-51 2017202571 19 Apr 2017

Compound 3 J a

Compound 32a

Cells suitable for treatment according to the methods of the present invention [0151] Pluripotent cells, suitable for use in the present invention express at least one of the following pluripotency markers selected from the group consisting of: ABCG2, cripto, FoxD3, Connexin43, Connexin45, Oct4, SOX-2, Nanog, hTERT, UTF-1, ZFP42, SSEA3, SSEA-4, Tral-60, and TraI-81.

[0152] 1 n one embodiment, the pluripotent cells are embryonic stem cells. In an alternate embodiment, the pluripotent cells are cells expressing pluripotency markers derived from embryonic stem cells. In one embodiment, the embryonic stem cells are human.

-52Isolation, expansion and culture of human embryonic stem cells

2017202571 19 Apr 2017 [0153)

10154] [0155] [01561

Characterization of human embryonic stem cells: Human embryonic stem cells may express one or more of the stage-specific embryonic antigens (SSEA) 3 and 4, and markers detectable using antibodies designated Tra-1-60 and Tra-1-81 (Thomson etal., Science 282:1145, 1998). Differentiation of human embryonic stem cells in vitro results in the loss of SSEAA, Tra- ] -60, and Tra-1 -81 expression (if present) and increased expression of SSEA-1. Undifferentiated human embryonic stem cells typically have alkaline phosphatase activity, which can be detected by fixing the cells with 4% paraformaldehyde, and then developing with Vector Red as a substrate, as described by the manufacturer (Vector Laboratories, Burlingame Calif.) Undifferentiated pluripotent stem cells also typically express Oct-4 and TERT, as detected by RT-PCR.

Another desirable phenotype of propagated human embryonic stem cells is a potential to differentiate into cells of all three germinal layers: endoderm, mesoderm, and ectoderm tissues. Pluripotency of human embryonic stem cells can be confirmed, for example, by injecting cells into SCID mice, fixing the teratomas that form using 4% paraformaldehyde, and then examining them histologically for evidence of cell types from the three germ layers. Alternatively, pluripotency may be determined by the creation of embryoid bodies and assessing the embryoid bodies forthe presence of markers associated with the three germinal layers.

Propagated human embryonic stem cell lines may be karyotyped using a standard Gbanding technique and compared to published karyotypes of the corresponding primate species. It is desirable to obtain cells that have a normal karyotype, which means that the cells are euploid, wherein al] human chromosomes are present and not noticeably altered.

Sources of human embryonic stem cells: Types of human embryonic stem cells that may he 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 tissue taken any time during gestation, typically but not necessarily before approximately 10-12 weeks gestation. Non-limiting examples are

-53 2017202571 19 Apr 2017 established lines of human embryonic stem cells or human embryonic germ cells, such as, for example the human embryonic stem cell lines Hl, H7, and H9 (WiCell). Also contemplated is use of the compositions of this disclosure during the initial establishment or stabilization of such cells, in which case the source cells would be primary pluripotent cells taken directly from the source tissues. Also suitable arc cells taken from a pluripotent stem cell population already cultured in the absence of feeder cells. Also suitable are mutant human embryonic stem cell lines, such as, for example, BGOI v (BresaGen, Athens, GA).

10157) In one embodiment. Human embryonic stem cells are prepared as described by Thomson etai. (U.S. Pat. No. 5,843,780, Science 282:1145, 1998; Curr. Top, Dev. Biol. 38:133 ff„ 1998; Proc. Natl. Acad. Sci. H.S.A. 92:7844, 1995).

|015Sj Culture of human embryonic stem cells: In one embodiment, human embryonic stem cells are cultured in a culture system that is essentially free of Feeder cells, but nonetheless supports proliferation of human embryonic stem cells without undergoing substantial differentiation. The growth of human embryonic stem cells in feeder-free 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 feeder-free culture without differentiation is supported using a chemically defined medium.

[0159) In an alternate embodiment, human embryonic stem cells are initially cultured layer of feeder cells that support the human embryonic stem Cells in various ways. The human embryonic are then transferred to a culture system that is essentially free of feeder cells, but nonetheless supports proliferation of human embryonic stem cells without undergoing substantial differentiation.

[01601 Examples of conditioned media suitable for use in the present invention are disclosed in US20020072117, US6642048, W02005014799, and Xu et al (Stem Cells 22: 972-980, 2004).

-542017202571 19 Apr 2017

101611 An example of a chemically defined medium suitable for use in the present invention may be found in US2007001001 L |0162| Suitable culture media may be made from the following components, such as, for example, Dulbecco's modified Eagle's medium (DMEM), Gibco # 11965-092; Knockout Dulbecco's modified Eagle's medium (KO DMEM), Gibco # 10829-018; Ham's F12/50% DMEM basal medium; 200 mM L-glutamine, Gibco # 15039-027; non-essential amino acid solution, Gibco 11140-050; β- mercaptoetbanol, Sigma # M7522; human recombinant basic fibroblast growth factor (bFGE), Gibco # 13256-029.

|01631 1 n one embodiment, the human embryonic stem cells are plated onto a suitable culture substrate that is treated prior to treatment according to the methods of the present invention, tn one embodiment, the treatment is an extracellular matrix component, such as, for example, those derived from basement membrane or that may form part of adhesion molecule receptor-ligand couplings. In one embodiment, a the suitable culture substrate is Matrigel® (Becton Dickenson), Matrigel® is a soluble preparation from Engelbreth-Holm-Swarm tumor cells that gels at room temperature to form a reconstituted basement membrane,

101641 Other extracellular matrix components and component mixtures are suitable as an alternative. This may include laminin, fibronectin, proteoglycan, entactin, heparan sulfate, and the like, alone or in various combinations.

10165| The human embryonic stem cells are plated onto the substrate in a suitable distribution and in the presence of a medium that promotes cell survival, propagation, and retention of the desirable characteristics. All these characteristics benefit from careful attention to the seeding distribution and can readily be determined by one of skill in the art.

Isolation, expansion and culture ol cells expressing pluripotency markers that are derived from human embryonic stem cells [0166] In one embodiment, cells expressing pluripotency markers are derived from human embryonic stem cells by a method comprising the steps of:

-552017202571 19 Apr 2017 |0167| |0168)

10169] |0170|

a. Culturing human embryonic stem cells,

b. Differentiating the human embryonic stem cells into cells expressing markers characteristic of definitive endoderm cells, and

c. Removing the cells, and subsequently culturing them under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix prior to culturing the cells.

In one embodiment, cells expressing pturipotency markers are derived from human embryonic stem cells by a method 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 substrate that is not pre-treated with a protein or an extracellular matrix.

Cell culture under hypoxic conditions on a tissue culture substrate that is not pretreated with a protein or an extracellular matrix

In one embodiment, the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not coated with an extracellular matrix for about 1 to about 20 days. In an alternate embodiment, the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not coated with an extracellular matrix for about 5 to about 20 days. In an alternate embodiment, the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not coated with an extracellular matrix for about 15 days.

In one embodiment, the hypoxic condition is about 1% Cf to about 20% Cf. In an alternate embodiment, the hypoxic condition is about 2% Cf to about 10% Cf. In an alternate embodiment, the hypoxic condition is about 3% Cf.

The cells may be cultured, under hypoxic conditions on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing serum, activin A, and a Wnt ligand. Alternatively, the medium may also contain IGF-1,

-562017202571 19 Apr 2017 |0171| |0172|

10173)

10174J |01751

10176) [01771

The culture medium may have a serum concentration in the range of about 2% to about

5%. In an alternate embodiment, the serum concentration may be about 2%.

Activin A may be used at a concentration from about 1 pg/ml to about 1 00pg/ml. In an alternate embodiment, the concentration may be about 1 pg/ml to about 1 pg/ml. In another alternate embodiment, the concentration may be about 1 pg/ml to about [OOng/ml In another alternate embodiment, the concentration may be about 50ng/ml to about 1 OOng/ml. In another alternate embodiment, the concentration may be about 1 OOng/ml.

The Wnt ligand may be selected from the group consisting of Wnt-1, Wnt-3a, Wnt-5a and Wnt-7a. In one embodiment, the Wnt ligand is Wnt-1. In an alternate embodiment, the Wnt ligand is Wnt-3a.

The Wnt ligand may be used at a concentration of about 1 ng/ml to about lOOOng/ml. In an alternate embodiment, the Wnt ligand may be used at a concentration of about IQng/ml to about 1 OOng/ml. In one embodiment, the concentration of the Wnt ligand is about 20ng/ml.

1GF-1 may be used at a concentration of about 1 ng/ml to about 1 OOng/ml. In an alternate embodiment, the IGF-lmay be used at a concentration of about lOng/ml to about lOOng/ml. In one embodiment, the concentration of IGF-I is about 50ng/ml.

The cells expressing pluripotency 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-treated with a protein or an extracellular matrix.

The cells expressing pluripotency markers derived by the methods of the present invention express at least one of the following pluripotency markers selected from the group consisting of: ABCG2, cripto, FoxD3. Connexin43, Cortnexin45, Oct4, SOX-2, Nanog, hTERT, UTF-1, ZFP42, SSEA-3, SSEA-4, Tral-60, and Tral-81.

Further differentiation of cells expressing markers characteristic of the definitive endoderm lineage

-572017202571 19 Apr 2017

101781 Cells expressing markers characteristic of the definitive endoderm lineage may he differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage by any method in the art.

10179] For example, cells expressing markers characteristic of the definitive endoderm lineage may be differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage according to the methods disclosed in D’Amour etal, Nature Biotechnology 24, 1392 - 1401 (2006).

101801 For example, cells expressing markers characteristic of the definitive endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage, by treating the cells expressing markers characteristic of the definitive endoderm Uncage with a fibroblast growth factor and KAAD-cyclopamine, then removing the medium containing the fibroblast growth factor and KAAD-cyclopamine and subsequently culturing the cells in medium containing retinoic acid, a fibroblast growth factor and KAAD-cyclopamine. An example of this method is disclosed in D’ Amour et al, Nature Biotechnology, 24: 1392-1401, (2006).

|(I1811 Markers characteristic of the pancreatic endoderm lineage are selected from the group consisting of Pdxl, HNF-lbeta, PTFla, HNF-6, HB9 and PROX1, Suitable for use in the present invention is a cell that expresses at least one of the markers characteristic of the pancreatic endoderm lineage. In one aspect of the present invention, a cell expressing markers characteristic of the pancreatic endoderm lineage is a pancreatic endoderm cell.

Further differentiation of cells expressing markers characteristic of the pancreatic endoderm lineage

10182J Cells expressing markers characteristic of the pancreatic endoderm lineage may be differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage by any method in the art.

[0183] For example, cells expressing markers characteristic of the pancreatic endoderm lineage may be differentiated into cells expressing markers characteristic of the pancreatic

-582017202571 19 Apr 2017 endocrine lineage according to the methods disclosed in D¹ Amour at ai. Nature Biotechnology 24, 1392 - 1401 (2006).

|0184| Markers characteristic of the pancreatic endocrine lineage are selected from the group consisting of NGN-3, NeuroD, Islet-1, Pdx-1, NKX6.1, Pax-4, Ngn-3, and PTF-1 alpha. In one embodiment, a pancreatic endocrine cell is capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin, and pancreatic polypeptide. Suitable for use in the present invention is a cell that expresses at least one of the markers characteristic of the pancreatic endocrine lineage. In one aspect of the present invention, a cell expressing markers characteristic of the pancreatic endocrine lineage is a pancreatic endocrine cell. The pancreatic endocrine cell may be a pancreatic hormone expressing cell. Alternatively, the pancreatic endocrine cell may be a pancreatic hormone secreting cell.

101851 In one aspect of the present invention, the pancreatic endocrine cell is a cell expressing markers characteristic of the β cell lineage. A cell expressing markers characteristic of the β cell lineage expresses Pdxl and at least one of the following transcription factors: NGN-3, Nkx2.2, Nkx6.1, NeuroD. Isl-1, HNF-3 beta. MAFA, Pax4, and Pax6. In one aspect of the present invention, a cell expressing markers characteristic of the β cell lineage is a β cell.

Detection of cells expressing markers characteristic of the definitive endoderm linage |0186| Formation of cells expressing markers characteristic of the definitive endoderm lineage may be determined by testing for the presence of the markers before and after following a particular protocol. Pluripotent stem cells typically do not express such markers. Thus, differentiation of pluripotent cells is detected when cells begin to express them.

|0187| The efficiency of differentiation may be determined by exposing a treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker expressed by cells expressing markers characteristic of the definitive endoderm lineage.

-592017202571 19 Apr 2017 [0188| Methods for assessing expression of protein and nucleic acid markers in cultured or isolated cells are standard in the art. These include quantitative reverse transcriptase polymerase chain reaction (RT-PCR), Northern blots, in situ hybridization (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds, 2001 supplement)), and immunoassays such as immunohistochemical analysis of sectioned material, Western blotting, and for markers that are accessible in intact cells, flow cytometry analysis (FACS) (see, e.g., Harlow and Lane, Using Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press (1998)).

10189] Examples of antibodies useful for detecting certain protein markers are listed in Table IA. It should be noted that alternate antibodies directed to the same markers that are recognized by the antibodies listed in Table IA are available, or can be readily developed. Such alternate antibodies can also be employed for assessing expression of markers in the cells isolated in accordance with the present invention.

|0190] For example, characteristics of pluripotent stem cells are well known to those skilled in the art, and additional characteristics of pluripotent stem cells continue to be identified. Pluripotent stem cell markers include, for example, the expression of one or more of the following: ABCG2, cripto, FoxD3, Connexin43, Connexin45, Oct4, Sox2, Nanog, hTERT, UTF-l, ZFP42, SSEA-3, SSEA-4, Tral-60, Tral-81.

|01911 After treating pluripotent stem cells with the methods of the present invention, the differentiated cells may be purified by exposing a treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker, such as CXCR4, expressed by cells expressing markers characteristic of the definitive endoderm lineage.

Detection of cells expressing markers characteristic of the pancreatic endoderm linage [01921 Markers characteristic of the pancreatic endoderm lineage are well known to those skilled in the art, and additional markers characteristic of the pancreatic endoderm 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

-602017202571 19 Apr 2017 characteristic of the pancreatic endoderm lineage. Pancreatic endoderm lineage specific markers include the expression of One or more transcription factors such as, for example,

Hlxh9, PTF-la, PDX-I, HNF-6, HNF-lbeta.

10193J The efficiency of differentiation may be determined by exposing a treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker expressed by cells expressing markers characteristic of the pancreatic endoderm lineage.

[0194| Methods for assessing expression of protein and nucleic acid markers in cultured or isolated cells are standard in the art. These include quantitative reverse transcriptase polymerase chain reaction (RT-PC'R), Northern bLots, ift situ hybridization (see, e.g., Current Protocols in Molecular Biology (Ausubel et a/., eds. 2001 supplement)), and immunoassays such as immunohistochemical analysis of sectioned material, Western blotting, and for markers that are accessible in intact cells, flow cytometry analysis (FACS) (see, e.g., Harlow and Lane, Using Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press (1998)).

[0195] Examples of antibodies useful for detecting certain protein markers are listed in Table IA. It should be noted that alternate antibodies directed to the same markers that are recognized by the antibodies listed in Table IA are available, or can be readily developed. Such alternate antibodies can also be employed for assessing expression of markers in the cells isolated in accordance with the present invention.

Detection of cells expressing markers characteristic of the pancreatic endocrine linage |0196| Markers characteristic of cells of the pancreatic endocrine lineage are well known to those skilled in the art, and additional markers characteristic of the pancreatic 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 of one or more transcription factors such as, for example, NGN-3, NeuroD, lslet-1.

-61 2017202571 19 Apr 2017 [01971 [0198|

101991 [02001

Markers characteristic of cells of the β cell lineage are well known to those skilled in the art, and additional markers characteristic of the β cell 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 β-cell lineage, β cell lineage specific characteristics include the expression of one or more transcription factors such as, for example, Pdxl (pancreatic and duodenal homeobox gene-1), Nkx2.2, Nkx6.1, Isll, Pax6, Pax4, NeuroD, Hnflb, Hnf-6, Hnf-3beta, and MafA, among others. These transcription factors are well established in the art for identification of endocrine cells. See, e.g., Edlund (Nature Reviews Genetics 3: 524-632 (2002)).

The efficiency of differentiation may be determined by exposing a treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker expressed by cells expressing markers characteristic of the pancreatic endocrine lineage. Alternatively, the efficiency of differentiation may be determined by exposing a treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker expressed by cells expressing markers characteristic of the β cell lineage.

Methods for assessing expression of protein and nucleic acid markers in cultured or isolated cells are standard in the art. These include quantitative reverse transcriptase polymerase chain reaction (RT-PCR), Northern blots, in situ hybridization (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 2001 supplement)), and immunoassays such as immunohistochemical analysis of sectioned material, Western blotting, and for markers that are accessible in intact cells, flow cytometry analysis (FACS) (see, e.g., Harlow and Lane, Using Antibodies; A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press (1998)).

Examples of antibodies useful for detecting certain protein markers are listed in Table IA. It should be noted that alternate antibodies directed to the same markers that are recognized by the antibodies listed in Table IA are available, or can be readily developed. Such alternate antibodies can also be employed for assessing expression of markers in the cells isolated in accordance with the present invention.

The present invention is further illustrated, but not limited by, the following examples.

10201J

-622017202571 19 Apr 2017

Example t

Human Embryonic Stem Cell Culture

Stem cells arc undifferentiated cells defined by their ability at the single cell level to both seif-renew and differentiate to produce progeny cells, including self-renewing progenitors, non-renewing progenitors, and terminally differentiated cells. Stem cells are also characterized by their ability to differentiate in vitro into functional cells of various cell lineages from multiple germ layers (endoderm, mesoderm and ectoderm), as well as to give rise to tissues of multiple germ layers following transplantation and to contribute substantially to most, if not all, tissues following injection into blastocysts.

)0202) The human embryonic stem cell lines H1, H7 and H9 were obtained from WiCell

Research Institute, Inc.j (Madison, WI) and cultured according to instructions provided by the source institute. Briefly, cells were cultured on mouse embryonic fibroblast (MEF) feeder cells in ES cell medium consisting of DMEM/F12 (Invitrogen/GIBCO) supplemented with 20% knockout serum replacement, 100 nM MEM noncssential amino acids, 0.5 mM beta-mercaptoethanol, 2mM L-glutamine with 4ng/ml human basic Fibroblast growth factor (bFGF) (all from Invitrogen/GIBCO), MEF cells, derived from El3 to 13.5 mouse embryos, were purchased from Charles River, MEF cells were expanded in DMEM medium supplemented with 10% FBS (Hyclone), 2mM glutamine, and 100 mM MEM nonessential amino acids. Sub-confluent MEF cell cultures were treated with 10ug/ml mitomycin C (Sigma, St, Louis, MO) for 3h to arrest cell division, then trypsinized and plated at 2x 10⁴/cm² on 0,1 % bovine gelatin-coated dishes. MEF cells from passage two through four were used as feeder layers. Human embryonic stem cells plated on MEF cell feeder layers were cultured at 37°C in an atmosphere of 5% CO?/ within a humidified tissue culture incubator. When confluent (approximately 5-7 days after plating), human embryonic stem cells were treated with Img/ml collagenase type IV (Invitrogen/GIBCO) for 5-10 min and then gently scraped off the surface using a

5-ml 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.

-63 In parallel, Hl, H7, and H9 human embryonic stem ceils were also seeded on plates coated with a 1:30 dilution of growth factor reduced MATRIGEL™ (BD Biosciences) and cultured in MEF-conditioned media supplemented with & ng/ml bFGF. The cells cultured on MATRIGEL™ were routinely passaged with collagenase IV (Invitrogen/GIBCO), Dispase (BD Bioscienccs) or Liberase enzyme (Source). Some of the human embryonic stem cell cultures were incubated under hypoxic conditions (approximately 3% O2).

2017202571 19 Apr 2017

102031 |0204|

102051 |0206]

Example 2

Derivation and Culture of Cells Expressing Pluripotency Markers, Derived from Human Embryonic Stem Cells

Cells from the human embryonic stem cell lines Hl and H9 various passages (Passage 30-54) were cultured under hypoxic conditionsi(approximatcly 3% Cf ) for at least three passages. The cells were cultured in MEF-CM supplemented with 8 ng/ml of bFGF and plated on MATRIGEL coated plates according to Example 1,

Cells were then treated with DMEM/FI2 medium supplemented with 0.5% FBS, 20 ng/ml WNT-3a (Catalog# 1324-WN-002, R&D Systems, MN), and 100 ng/ml Activin-A (R&D Systems, MN) for two days followed by treatment with DMEM/F12 media supplemented with 2% FBS and 100 ng/ml Activin-A (AA) for an additional 3 to 4 days. This protocol resulted in significant upregulation of definitive endoderm markers.

The cells were then treated with TrypLE™Express solution (Invitrogen, CA) for 5 mins. Released cells were resuspended in DMEM-F12 + 2% FBS medium, recovered by centrifugation, and counted using a hcmocytometer. The released cells were seeded at 1000-10,000 cclls/cm²on tissue culture polystyrene (TCPS) treated flasks and cultured in DMEM-F 12 + 2% FBS + 100 ng/ml activin-A + 20 ng/ml WNT-3A under hypoxic conditions (approximately 3% Cf) at 37 °C in standard tissue culture incubator. The TCPS flaks were not coated with MATRIGEL or other extarcellular matrix proteins. The media was changed daily. In some cultures, the media was further supplemented with 10-50 ng/ml of IGF-1 (insulin growth factor-f from R&D Systems, MN) or IX ITS

-642017202571 19 Apr 2017 (Insulin, transferrin, and selenium from Invitrogen, Ca). In some of the culture conditions the basal media (DM-F12 + 2% FBS) was further supplemented with 0,1 mM mcrcaptoethanol (Invitrogen, CA) and non-essential amino acids (IX, NEAA from

Invitrogen, CA),

10207] Following 5 to 15 days of culturing, distinct cell colonies appeared surrounded by a large number of enlarged cells that appear to be in senescence. At approximately 50 to 60% confluency, the cultures were passaged by exposure to TrypLE™ Express solution for 5 mins at room temperature. The released cells were resuspended in DMEM-F12 + 2% FBS medium, recovered by centrifugation, and seeded at 10,000 cells/cm² on tissue culture polystyrene (TCPS) treated flasks in DMEM-F12 + 2%FBS + 100 ng/ml activinA + 20 ng/ml WNT-3A +/- 50 ng/ml of IGF-I. This media will be further referred to as the “growth media”.

Example 3

Derivation of Ceils Expressing Pluripotency Markers from a Single Ceil Suspension of Human Embryonic Stem Cells

10208] Cells from the human embryonic stem cell tines Hl P33 and H9 P45 were cultured under hypoxic conditions (approximately 3% O₂) for at least three passages. The cells were cultured in MEF-CM supplemented with 8 ng/ml of bFGF and plated on MATRIGEL coated plates according to Example I. At approximately 60% confluency, the cultures were exposed to TrypLE ™ Express solution (Invitrogen, CA) for 5 mins. Released cells were resuspended in DMEM-FI2 + 2% FBS medium, recovered by centrifugation, and counted using a hemocytometer, The released cells were seeded at 1000 to 10,000 cells/cm² on tissue culture polystyrene (TCPS) treated flasks and cultured in DM-F12 + 2% FBS + 100 ng/ml activin-A + 20 ng/ml WNT-3A + 50 ng/ml of IGF-I + 0.1 mM mercaptoethanol (Invitrogen, CA) and non-essential amino acids (IX, NEAA from Invitrogen, CA) under hypoxic conditions (approximately 3% O₂) at 37 °C in standard tissue culture incubator. The TCPS flasks were not coated with MATRIGEL or other extarcellular matrix proteins. The media was changed daily. The first passage cells are referred to as P1.

-652017202571 19 Apr 2017

Example 4

Various Growth Media Useful for Expansion of Cells Expressing Pluripotency Markers Derived from Human Embryonic Stem Cells

10209) Cells expressing pluripotency markers derived from human embryonic stem cells have been successfully cultured in the following media compositions for at least 2-30 passages:

1. DM-F12 + 2% FBS + 100 ng/ml AA + 20 ng/ml WNT-3A

2. DM-F12 + 2% FBS + 100 ng/ml AA + 20 ng/ml WNT-3A + 50 ng/ml 1GF-1

3. DM-F12 + 2% FBS + 100 ng/ml AA + 20 ng/ml WNT-3 A + 10 ng/ml IGF-1

4. DM-F12 + 2% FBS + 50 ng/ml AA + 20 ng/ml WNT-3A + 50 ng/ml IGF-I

5. DM-F12 + 2% FBS + 50 ng/ml AA + 10 ng/ml WNT-3A + 50 ng/ml IGF-I

6. DM-F 12 + 2% FBS + 50 ng/ml AA + 20 ng/ml WNT-3A + 10 ng/ml IGF-I

7. DM-F 12 + 2% FBS + 100 ng/ml AA + 10 ng/ml WNT-3 A + 10 ng/ml IGF-I

8. HEScGRO defined media (Cbemicon, CA)

The basal component of the above listed media may be replaced with similar media such as, RPMI, DMEM, CRML, Knockout ™DMEM, and FI2.

Example 4

Effects of Inhibitors of GSK-3p Enzyme Activity on the Viability of Cells Expressing Pluripotency Markers [0210) Derivation and maintenance of cells expressing pluripotency makers was conducted as has been described in Example 2. Cells were grown in DMEM:F12 supplemented with 2% FCS (Invitrogen), 100 ng/ml Activin A, 20 ng/ml Wnt-3a, and 50 ng/ml IGF(R&D Biosystems). Cells were seeded at a density of 10,000 cells/cm“ on Falcon polystyrene

-662017202571 19 Apr 2017 flasks and grown in monolayer culture at 37^CC’, 5% CO₂, low oxygen. After reaching 6070% confluence, cells were passed by washing the monolayer with PBS and incubating with TrypLE (Invitrogen) for 3-5 minutes to allow detachment and single cell dispersal.

[02111 Screening was conducted using test compounds from a proprietary library of small molecules selected for their ability to inhibit GSK-3B enzyme activity. Compounds from this library were made available as 1 mM stocks, in a 96-well plate format in 50mM HEPES, 30% DMSO. For assay, cells expressing pluripotency markers were washed, counted, and plated in normal culture medium at a seeding density of 20,000 cells per well in 96-well clear-bottom, dark-well plates (Costar). This seeding density was previously determined to yield optimal monolayer formation in overnight culture. On the following day, culture medium was removed, cell monolayers were rinsed three times with PBS, and test compounds were added to the wells in 80μ1 aliquots, each diluted into assay medium at a final assay concentration of ΙΟμΜ. On 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 1 and 2 of culture consisted of DMEM :F 12 supplemented with 0.5% FCS and 1 OOng/ml Activin A. On days 3 and 4 of culture, medium was removed from each well and replaced with DMEM:F12 supplemented with 2% FCS and 1 OOng/ml Activin A (no test compound). On day 4 of assay, 15μΙ of MTS (Promega) was added to each well and plates were incubated af 37°C for 1.5 to 4 hours prior to reading optical density at 490 nm on a SpectraMax (Molecular Devices) instrument. Statistical measures con sisting of mean, standard deviation , and coefficient of variation were calculated for each duplicate set. Toxicity was calculated for each test well relative to a positive control (wells treated with Activin A and Wnt3a on days 1 and 2 of culture).

102121 Table II is a compilation of all screening results. Cells expressing pluripotency markers were plated initially as a confluent monolayer in this assay; hence, the results are representative of a toxicity measure over the four-day culture period. Results are expressed as percentage viability of control, and demonstrate variable toxicity for some compounds at the ΙΟμΜ screening concentration used. A larger proportion of the compounds have minimal or no measurable toxicity in this cell-based assay.

-672017202571 19 Apr 2017 [02131 A small panel of select compounds was repeat tested over a narrow dose titration range, again using cells expressing pluripotency markers in a simitar assay as described above.

Table 111 is a summary of these results, demonstrating variable dose titration effects for a range of toxic and non-toxic compounds.

Example 5

Effects of Inhibitors of GSK-3fi Enzyme Activity on the Differentiation and Proliferation of Human Embryonic Stem Cells Determined using a High Content

Screening Assay [0214| Maintenance of human embryonic stem cells (H9 Line) was conducted as described in Example 1. Colonies of cells were maintained in ail undifferentiated, pluripotent state with passage on average every four days. Passage was performed by exposing cell cultures to a solution of collagenase (1 mg/ml; Sigma-Aldrich) for 10 to 30 minutes at 37°C followed by gentle scraping with a pipette tip to recover cell clusters. Clusters were allowed to sediment by gravity, followed by washing to remove residual collagenase.

Cell clusters were split at a 1:3 ratio for routine maintenance culture or a 1:1 ratio for immediate assay. The human embryonic stem cell lines used were maintained at passage numbers less than passage 50 and routinely evaluated for normal karyotypic phenotype and absence of mycoplasma contamination.

|02l5| Cell clusters used in the assay were evenly resuspended in normal culture medium and plated onto MATRIGEL-coated 96-well Packard VIEWPLATES (PerkinElmer) in volumes of ΙΟΟμΙ/welL MEF conditioned medium supplemented with 8ng/ml bFGF was used for initial plating and recovery. Daily feeding was conducted by aspirating spent culture medium from each well and replacing with an equal volume of fresh medium. Plates were maintained at 37°C, 5% CO? in a humidified box throughout the duration of assay.

|0216] Screening was conducted using test compounds from a proprietary library of small molecules selected for their ability to inhibit GSK-3B enzyme activity. Compounds from this library were made available as 1 mM stocks, in a 96-well plate format in 50mM

-682017202571 19 Apr 2017

HEPES, 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 in PBS to remove residual growth factors and serum. Test volumes of 80 to 100μ1 per well were added back containing DMEM:F12 base medium (tnvitrogen) supplemented with 0.5% FCS (HyClone) and lOOng/ml activin A (R&D Biosystems) plus 10μΜ test compound. Positive control wells contained the same base medium, substituting 10-20ng/ml Wnt3a (R&D Biosystems) for the test compound. Negative control wells contained base medium with 0.5% FCS and activin A atone (AA only) or alternatively, 0.5% FCS without activin A or Wnt3a (no treatment). Wells were aspirated and fed again with identical solutions on day 2 of assay. On days 3 and 4, all assay wells were aspirated and converted to DMEM:F12 supplemented with 2% FCS and lOOng/ml activin A (without test compound or Wnt3a); parallel negative control wells were maintained in DMEM:F12 base medium with 2% FCS and activin A (AA only) or alternatively, 2% FCS without activin A (no treatment).

|0217| At the end of culture, cells in 96-well plates were fixed with 4% paraformaldehyde at room temperature for 20 minutes, washed three times with PBS, and then permcabilized with 0.5%i Triton X-100 for 20 minutes at room temperature. Alternatively, cells were fixed with ice cold 70% ethanol overnight at -20°C, washed three times with PBS, and then permcabilized with Triton X-100 for 5 minutes at 4°C. After fixing and permeabilizing, cells were washed again three times with PBS and then blocked with 4% chicken serum (Invitragen) in PBS for 30 minutes at room temperature. Primary antibodies (goat anti-human Sox 17 and goat anti-human HNF-3beta; R&D Systems) were diluted 1:100 in 4% chicken serum and added to cells for one hour at room temperature. Alexa Fluor 488 conjugated secondary antibody (chicken anti-goat IgG; Molecular Probes) was diluted 1:200 in PBS and added after washing the cells three times with PBS. To counterstain nuclei, 5 mM Draq5 (Alexis Biochemicals) was added for five minutes at room temperature. Cells were washed once with PBS and left in 100 ml/wcll PBS for imaging.

[0218| Cells were imaged using an IN Cell Analyzer 1000 (GE Healthcare) utilizing the 51008bs dichroic for cells stained with Draq5 and Alexa Fluor 488. Exposure times were

-692017202571 19 Apr 2017 optimized using a positive control wells and wells with secondary only for untreated negative controls. Twelve fields per well were obtained to compensate for any cell loss during the treatment and staining procedures. Total cell numbers and total cell intensity for Sox-17 and HNF-3beta were measured using the IN Cell Developer Toolbox 1.6 (GE Healthcare) software. Segmentation for the nuclei was determined based on grey-scale levels {baseline range 100-300) and nuclear size. Averages and standard deviations were calculated for replicates. Total protein expression was reported as total intensity or integrated intensity, defined as total fluorescence of the cell times area of the cell. Background was eliminated based on acceptance criteria of grey-scale ranges between 300 to 3000 and form factors greater than or equal to 0.4. Total intensity data were normalized by dividing the total intensities for each well by the average total intensity for the Wnt3a/Activin A positive control. Normalized data was calculated for averages and standard deviation for each replicate set, [0219) Table IV is a representative summary of all screening results. Table V is a list of hits from this screening. Strong hits are defined as greater than or equal to 120% of control values; moderate hits are defined as falling 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 significant number of compounds induce differentiation in this assay, as measured by the protein expression of Sox 17 and Hnf-3b transcription factors.

Example 6

Effects of Inhibitors of GSK-3P Enzyme Activity on the Proliferation of Human Embryonic Stem Cells Determined using a Plate Reader Assay |0220| Maintenance of human embryonic stem Cells (H9 or Η1 lines) was conducted as described in Example 1. Colonics of cells were maintained in an undifferentiated, pluripotent state with passage on average every four days. Passage was performed by exposing cell cultures to a solution of collagenase (1 mg/ml; Sigma-Aldrich) for 10 to 30 minutes at 37°C followed by gentle scraping with a pipette tip to recover cell clusters. Clusters were allowed to sediment and washed to remove residual collagenase. Cell clusters were split at a ratio of 1:3 monolayer area for routine culture or a 1:1 ratio for

- 702017202571 19 Apr 2017 [0221| |0222[ |02231 immediate assay. The human embryonis stem cell· tines used for these examples were maintained at passage numbers less than 50 and routinely evaluated for normal karyotypic phenotype as well as absence of mycoplasm contamination.

Cell clusters used in assay were evenly resuspended in norma] culture medium and plated into MATRIGEL-coatcd 96-well Packard VIEWRLATES (PerkinElmer) in volumes of I ΟΟμΙ/well. MEF conditioned medium supplemented with 8ng/ml bFGF) was used tor initial plating and recovery. Daily feeding was conducted by aspirating spent culture medium from each well and replacing with an equal volume of fresh medium. Plates were maintained at 37°C in a humidified box, 5% CO? throughout the duration of assay.

Primary screening assays were initiated by aspirating culture medium from each well followed by three washes in PBS to remove residual growth factors and serum. Test volumes of 80-100 ul per well were added back containing DMEM :F 12 base medium (Invitrogen) supplemented with 0.5% FCS (HyCJone) and lOOng/ml activin A (R&D Biosystems) and 10μΜ test compound. Positive control wells contained the same medium substituting 10-20ng/ml Wnt3a (R&D Biosystems). Negative control wells contained base medium with 0.5% FCS without activin A or Wnt3a. Screening compounds were tested in triplicate. Wells were aspirated and fed again with identical solutions on day 2 of the assay. On days 3 and 4, all assay wells were aspirated and converted to DMEM:F12 supplemented with 2% FCS and lOOng/ml activin A with the exception of negative control wells which were maintained in DMEM:FI2 base medium with 2% FCS.

On day 4 of assay, 15-20μΙ of MTS (Promega) was added to each well and plates were incubated at 37^QC for 1.5 to 4 hours. Densitometric readings at OD490 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 Activin A/Wnt3a positive control to calculate a percent control value as a measure of proliferation.

-71 2017202571 19 Apr 2017 |02241

I0225I [0226)

10227)

Table VI is a representative summary of all screening resuits. Table VH is a list of hits from this screening. Strong hi ts are defined as greater than or equal to 120% of control values; moderate hits are defined as falling within the interval of 60-120% of control values, A significant number of compounds induce a proliferative response in this assay.

Example 7

Effects of GSK-3p Enzyme Inhibitors on the Differentiation and Proliferation of Human Embryonic Stem Cells: Dose Titration of Lead Compounds

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

Culture of two human embryonic stem cells (Η 1 and H9) was conducted as described in Example I. Colonies of cells were maintained in an undifferentiated, pluripotent state or Matrigel™ (Invitrogen)-coated polystyrene plastic, using a 1:30 dilution of Matrigel™ in DMEM:F12 to coat the surface. Cells were split by enzymatic passage every four days on average. Passage was performed by exposing cell monolayers to a solution of collagenase (1 mg/ml; Sigma-Aldrich) for 10 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 collagenase. Celt clusters were split at a 1:3 ratio for maintenance culture 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 absence of mycoplasma contamination.

Preparation of cells for assay: Cell clusters of the Hl or H9 human embryonic stem cell lines used in the assay were evenly resuspended in culture medium and plated onto Matrigel™-coated 96-well Packard VIEWPLATES (PerkinElmer) in volumes of 1 ΟΟμΙ'/well. MEF conditioned medium supplemented with 8ng/ml bFGF was used for initial plating and expansion. Daily feeding was conducted by aspirating spent culture

-722017202571 19 Apr 2017 medium From each wel! and replacing with an equal volume of fresh medium. Cultures were allowed to expand one to three days after plating prior to initiating assay. Plates were maintained at 37°C, 5% CO₂ in a humidified box for the duration of assay.

[0228] Preparation of compounds and 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 powders were solubilized as lOmM stocks in DMSO and stored dessicated at -20°C until use. Immediately prior to assay, compound stocks were diluted 1:1000 to make 10μΜ test compound in DMEM:F12 base medium (Invitrogen) supplemented with 0.5% FCS (HyClone) and lOOng/ml Activin A (R&D Biosystcms). This was further diluted two-fold in series to make a seven point dilution curve for each compound, also in DMEM:F12 base medium with 0.5% FCS and lOOng/ml Activin A.

10229) Secondary 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 and serum. Test volumes of 100μΙ per well were added back, containing medium with 0.5% FCS and different concentrations of inhibitor compounds with lOOng/ml Activin A, without Wnt3a. Positive control wells contained the same base medium with 0.5% FCS and with 20ng/ml Wnt3a (R&D Biosystems) in the absence of test compound. Negative control wells contained the same base medium with 0.5% FCS, in the absence of Activin A, Wnt3a, 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 and 4, all assay wells were aspirated and fed with DMEM:F12 supplemented with 2% FCS and lOOng/ml Activin A in the absence of both test compound or Wnt3a. Parallel negative control wells were maintained on days 3 and 4 in DMEM:F12 base medium with 2% FCS.

|0230] Assay evaluation: At the end of culture, cells in 96-well plates were washed twice with PBS then fixed with 4% paraformaldehyde at room temperature for 20 minutes, washed three times more with PBS, and then permeabilized with 0.5% Triton X-I00 for 20 minutes at room temperature. After fixing and permeabilizing, cells were washed again three times with PBS and then blocked with 4% chicken serum (Invitrogen) in PBS for

- 73 2017202571 19 Apr 2017 minutes at room temperature. Primary antibodies (goat anti-human Sox!7; R&D Systems) were diluted 1:100 in 4% chicken serum and added to the cells for one hour at room temperature. Alexa Fluor 488 conjugated secondary antibody (chicken 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 counterstain nuclei, 2gg/ml Hocchst 33342 (Invitrogen) was added for ten minutes at room temperature. Cells were washed once with PBS and left in 100 μΙ/well PBS for imaging.

|0231) Cells were imaged using an IN Cell Analyzer 1000 (GE Healthcare) utilizing the 51008bs dichrolc for cells stained with Hoechst 33342 and Alexa Fluor 488. Exposure times were optimized using positive control wells and wells stained with secondary antibody alone as an untreated negative control. Images from 15 fields per well¹ were acquired to compensate for any cell loss during the treatment and staining procedures.

Measurements for total cell number and total Sox-17 intensity were obtained 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 and standard deviations were calculated for each replicate data set. Total Soxl7 protein expression was reported as total intensity or integrated intensity, defined as total fluorescence of the cell times area of the cell. Background was eliminated based on acceptance criteria of grey-scale ranges between 300 to 3000 and form factors greater than or equal to 0.4, Total intensity data were normalized hy dividing the total intensities for each well by the average total intensity for the Wnt3a/Activin A positive control. Normalized data were calculated for averages and standard deviations for each replicate set.

Results

10232) Results arc shown for eight GSK.-3B enzyme inhibitors where activity was confirmed and 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 for each parameter from identical fields and wells. In this example, Sox 17 expression is indicative of definitive endoderm

- 742017202571 19 Apr 2017 differentiation. Results for cell number and Sox 17 intensity, respectively, using the Hl human embryonic stem cell line are shown in Tables VIII and IX. Results for the H9 human embryonic stem cell line are shown in Tables X and XI. Positive control values were normalized to 1.000 for cell number and Sox 17 intensity. Negative control values were less-than 0.388 for cell number and less-than 0.065 for Soxl7 intensity with both cell lines. A graphic portrayal of these data, comparing both human embryonic stem cell lines and including a dose titration of each compound, is provided in Figures 1 to 8. Cell number is presented in panel A; Sox 17 intensity is shown in panel B. These data confirm that each compound can promote hES cell proliferation and definitive endoderm differen tiation and identify an optimal range of activity.

Examples

Effects of GSK-3 β Enzyme Inhibitors on the Expression of Additional Markers Associated with Definitive Endoderm [0233| It was important to demonstrate that lead compounds could also induce other markers indicative of definitive endoderm differentiation, in addition to the transcription factor Sox 17. A select subset of hits was tested for their ability to promote expression of CXCR4, a surface receptor protein, and HNF-3 beta, a transcription factor also associated with definitive endoderm differentiation.

[02341 Preparation of cells for assay: Cell clusters from the Hl human embryonis stem cell line used in the assay were evenly resuspended in culture medium and plated onto MATRrGEL™-coated (1:30 dilution) 6-wcll plates (Coming) in volumes of 2 mEwell. MEF conditioned medium supplemented with 8ng/ml bFGF was used for initial plating and expansion. Daily feeding was conducted by aspirating spent culture medium from each well and replacing with an equal volume of fresh medium. Cultures were allowed to expand one to three days after plating prior to initiating assay. Plates were maintained at 37°C, 5% CO₂ for the duration of assay,

10235] Preparation of compounds and assay medium: A subset of seven hits resulting from primary screening was used for follow-up study and subsequent secondary assays. Neat

-752017202571 19 Apr 2017 compounds were solubilized as LOmM stocks in DMSO and stored dessicated at -20°C until use. Immediately prior to assay, compound stocks were diluted to a final concentration ranging between ΙμΜ and 5μΜ in DMEM:Fl2 base medium (Invitrogen) supplemented with 0.5% FCS (HyCIone) and lOOng/ml Activin A (R&D Biosystems).

10236] Assay: The assay was initiated by aspirating culture medium from cell monolayers in each well followed by three washes in PBS to remove residual growth factors and serum. Test volumes of 2ml per well were added back containing medium with 0.5% FCS and different concentrations of inhibitor compounds with lOOng/ml Activin A, without Wnt3a. Positive control wells contained the same base medium and 0.5% FCS with lOOng/ml Activin A and 20ng/ml Wnt3a (R&D Biosystems) in the absence of test compound. Negative control wells contained base medium with 0.5% FCS, in the absence of Activin A, Wnt3a, 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 and 4, all assay wells were aspirated and fed with DMEM:FI2 supplemented with 2% FCS and lOOng/ml Activin A in the absence of both test compound or Wnt3a. Parallel negative control wells were maintained on days 3 and 4 in DMEM:F12 base medium with 2% FCS,

10237] Assay evaluation: At the end of culture, cell monolayers were washed with PBS and harvested from culture plates by incubating 5 minutes with TrypLE™ Express solution (Invitrogen, CA). Cells were resuspended in MEF conditioned medium and split into two equal samples. One set of samples was further stained with various fluorescent labeled antibodies and subjected to flow cytometric (FACS) analysis. A second parallel set of samples was subjected to quantitative PCR.

|0238) Cells for FACS analysis were washed into PBS and blocked for 15 minutes at 4°C in 0. 1*25% human gamma-globulin (Sigma cat# G-4386) diluted in PBS and BD FACS staining buffer, Aliquots of cells (approximately 10⁵ cells each) were stained for 30 minutes at 4“C with antibodies directly conjugated to a fluorescent tag and having specificity for CD9 PE (BD#555372), CD99 PE (CaItag#MHCD9904), or CXCR-4 APC (R&D Systems cat# FAB 173 A). After a series of washes in BD FACS staining buffer,

- 762017202571 19 Apr 2017 cells were stained with 7-AAD (BD# 559925) to assess viability and analyzed on a BD

FACS Array instrument (BD Biosciences), collecting at least 10,000 events. Mouse lgG|k isotype control antibodies for both PE and APC were used to gate percent positive cells.

10239] Cells For quantitative PCR were processed for RNA extraction, purification, and cDNA synthesis. RNA samples were purified by binding to a silica-gel membrane (Rneasy Mini Kit, Qiagen, CA) in the presence of an ethanol-containing, high-salt buffer followed by washing to remove contaminants. The RNA was further purified using a TURBO DNAfree kit (Ambion, Inc.), and high-quality RNA was eluted in water. Yield and purity were assessed by A260 and A280 readings on a spectrophotometer. cDNA copies were made from purified RNA using an Applied Biosystems, Inc. (ABI. CA) high capacity cDNA archive kit.

|0240| Unless otherwise staled, all reagents for real-time PCR amplification and quantitation were purchased from ABI. Real-time PCR reactions were performed using the ABI PRISM 7900 Sequence Detection System. TAQMAN UNIVERSAL PCR MASTER MIX (ABI, C A) was used with 20 ng of reverse transcribed RNA in a total reaction volume of 20 μΐ. Each cDNA sample was run in duplicate to correct for pipetting errors. Primers and FAM-labeled TAQMAN probes were used at concentrations of200 nM.

The level of expression for each target gene was normalized using a human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) endogenous control previously developed by ABI, Primer and probe sets are listed as follows: CXCR4 (Hs00237052), GAPDH (4310884E), HNF3b (Hs00232764), SOX 17 (probe part # 450025, forward and reverse part # 4304971).

10241] After an initial incubation at 50°C for 2 min followed by 95°C for 10 min, samples were cycled 40 times in two stages, a denaturation step at 95°C for 15 see followed by an annealing/extension step at 60^sC for 1 min. Data analysis was carried out using GENE AMP 7000 Sequence Detection System software. For each primer/probe set, a Ct value was determined as the cycle number at which the fluorescence intensity reached a specific value in the middle of the exponential region of amplification. Relative gene

-77expression levels were calculated using the comparative Ct method. Briefly, for each cDNA sample, the endogenous control Ct value was subtracted from the gene of interest

Ct to give the delta Ct value (ACt). The normalized amount of target was calculated as 2ACt, assuming amplification to he 100% efficiency. Final data were expressed relative to a calibrator sample.

2017202571 19 Apr 2017

Results [0242) Figure 9 displays the FACS analysis of percent positive cells expressing CXCR4 surface receptor after treatment with various GSK3 inhibitors. Two concentrations of each compound, ranging between 1 uM and 5μΜ, are shown relative to an untreated population of cells (negative control) or cells treated with Activin A and Wnt3 (positive control). Figure 10 panels a, b, anti c show real-time PCR data for CXCR4, Sox 17, and HNF3beta, which are also considered to be markers of definitive endoderm. Both FACS and real-time PCR analysis demonstrate a significant increase in each of these markers observed in differentiated cells relative to untreated control cells. Expression levels of these definitive endoderm markers were equivalent in some cases to the positive control, demonstrating that a GSK3 inhibitor can substitute for Wnt3a at this stage of differentiation.

Example 9

Effects of GSK-33 Enzyme inhibitors on the Formation of Pancreatic Endoderm

10243| It was important to demonstrate that treatment with GSK.3P inhibitors during induction of definitive endoderm did not prevent the subsequent differentiation of oilier cell types, such as pancreatic endoderm, for example. A select subset of hits was tested for their ability to promote expression of PDX1 and HNF6, key transcription factors associated with pancreatic endoderm.

[»2441 Maintenance of human embryonic stem cells (Hl and H9 lines) was conducted as described in Example 1. Colonies of cells were maintained in an undifferentiated, pluripotent state with passage on average every four days. Passage was performed by

-782017202571 19 Apr 2017 exposing cell cultures to a solution of collagenase (1 mg/ml; Sigma-Aldrich) for 10 to 30 minutes at 37°C, followed by gentle scraping with a pipette tip to recover cell clusters. Clusters were allowed to sediment by gravity, followed by washing to remove residual collagenase. Cell clusters were split at a 1:3 ratio for routine maintenance culture or a 1:1 ratio for subsequent assay. The human embryonic stem cell lines used were maintained at less than passage 50 and routinely evaluated for normal karyotypic phenotype and absence of mycoplasma contamination.

[0245) Cell preparation of assay: Cell clusters of the Η1 human embryonis stem cell line used in the assay were evenly resuspended in culture medium and plated onto MATR1GEL™coated (1:30 dilution) 24-welJ plates (black well; Arctic White) in volumes of 1 ml/well. MEF conditioned medium supplemented with 8ng/ml bFGF was used for initial plating and expansion, in a second experiment, clusters of hES celts from the H9 line were plated in 96-well plates on mouse embryonic feeder (MEF) layers, previously inactivated by treating with mitomycin C (Sigma Chemical Co), Culture medium for hES cells on MEF monolayers consisted of DMEM:FI2 with 20% Knockout Serum Replacer (Invitrogen) supplemented with minimal essential amino acids (Invitrogen), L-glutamine, and 2-mercaptoethanol. Daily feeding was conducted by aspirating spent culture medium from each well and replacing with an equal volume of fresh medium. Cultures were allowed to expand one to three days after plating prior to initiating assay. Plates were maintained at 37°C, 5% CO₂ for the duration of assay.

10246) Preparation of compounds and assay medium: A subset of eight hits resulting from primary screening was used for follow-up study and subsequent secondary assays. Neat compounds were solubilized as lOmM stocks in DMSO and stored dessicated at 20°C until use. Immediately prior to assay, compound stocks were diluted to a final concentration ranging between 1 liM and 5μΜ in base medium with additives, [0247) Assay: In this assay, GSK3 inhibitors were included only on days 1 and 2 of the definitive endoderm differentiation step, substituting for Wnt3a. Embryonic stem cell cultures on MATRIGEL™ were initiated as described in Examples 7 and 8 above by aspirating culture medium from cell monolayers in each welt followed by three washes in

- 792017202571 19 Apr 2017

PBS to remove residual growth factors and serum. For differentiation to definitive endoderm, test volumes (0.5 ml per well for 24-well plates, 100 μΙ per well for 96-well plates) were added containing DMEM:F12 medium with ) 0.5% FCS and different concentrations of inhibitor compounds with 100 ng/ml Activin A, without Wnt3a.

Positive control wells contained the same base medium with 0.5% FCS and with 1 OOng/ml Activin A and 20ng/ml Wnt3a (R&D Biosystems) in the absence of test compound. Negative control wells contained the same base medium with 0.5% FCS, in the absence of Activin A, Wnt3a, 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 and 4, all assay wells were aspirated and fed with DMEM:F12 supplemented with 2% FCS and lOOng/ml Activin A in the absence of both test compound or Wnt3a. Parallel negative control wells were maintained on days 3 and 4 in DMEM:F12 base medium with 2% FCS. For differentiation to pancreatic endoderm, cells were treated for three days, feeding daily with DMEM:F12 base medium containing 2% FCS with 0.25 μΜ KAAD cyclopamine (EMD Biosciences) and 20 ng/ml FGF7 (R&D Biosystems). Cells were then treated for an additional four days, feeding daily with DMEM:F12 containing 1% B27 (lnvitrogen), 0.25 μΜ KAAD cyclopamine, 2 μΜ Retinoic Acid (RA; Sigma-AIdrich) and 20 ng/ml’FGF7, Parallel negative control wells were maintained throughout in DMEM:F12 base medium with 2% FCS (stage 2) or 1% B27 (stage 3) and without any other additives, |0248| Parallel cultures of H9 human embryonic cells were grown on MEF feeder layers, and differentiated to pancreatic endoderm. Definitive endoderm differentiation was achieved by culturing the cells in medium consisting of RPMI-1640 (lnvitrogen) containing no serum on day 1 and 0.2% FCS on days 2 and 3 along with different concentrations of inhibitor compounds and 100 ng/ml Activin A. Positive control wells contained the same base medium (with or without serum) with 1 OOng/ml Activin A and 20ng/ml Wnt3a (R&D Biosystems) in the absence of test compound. Negative control wells contained the same base medium with or without serum, in the absence of Activin A, Wnt3a, 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 day 3, all assay welts were

- 802017202571 19 Apr 2017 aspirated and fed with RPMI-1640 supplemented with 2% FCS and lOOng/ml Activin A in the absence of both test compound and Wnt3a. Parallel negative control wells were maintained on day 3 in RPMI-1640 base medium with 2% FCS. Cells were differentiated into pancreatic endoderm by treating the cells for four days, feeding daily with RPMI-1640 base medium containing 2% FCS with 0.25 mM KAAD cyelopaminc (EMD Biosciences) and 50 ng/ml FGF 10 (R&D Biosystems). Subsequently, cells were treated for three days duration, feeding daily with RPMI-1640 containing 1% B27 (Invitrogen), 0.25 mM KAAD cyclopamine, 2 mM Retinoic Acid (RA; Sigma-Aldrich) and 50 ng/ml FGF10. Parallel negative control welts were maintained throughout in RPMI-1640 base medium with 2% FCS (stage 2) or 1% B27 (stage 3) and without any other additives.

10249| Assay evaluation: At the end the differentiation, cells were examined as described in Example 8 for gene expression by real-time PCR. For high content fluorescence staining, cells in 96-well plates were washed twice with PBS then fixed with 4% paraformaldehyde at room temperature for 20 minutes, washed three times more with PBS, and then permeabilized with 0.5% Triton X-l 00 for 20 minutes at room temperature. After fixing and permeabilizing, cells were washed again three times with PBS and blocked with 4% chicken serum (Invitrogen) in PBS for 30 minutes at room temperature. Primary antibody (goat anti-human Pdxl; Santa Cruz) was diluted 1:100 in 4% chicken serum and added to cells for two hours at room temperature. Alexa Fluor 488 conjugated secondary antibody (chicken 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 counterstain nuclei, 2gg/ml Hoechst 33342 (Invitrogen) was added for ten minutes at room temperature. Cells were washed once with PBS and left in 100 μΙ/well PBS for imaging.

10250| Cells were imaged using an IN Cell Analyzer 1000 (GE Healthcare) utilizing the 51008bs diehroic for cells stained with Hoechst 33342 and Alexa Fluor 488. Exposure times were optimized using positive 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 treatment and staining procedures. Measurements for total cell number and total Pdx 1

- 81 2017202571 19 Apr 2017 intensity were obtained 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 and standard deviations were calculated for each replicate data set. Total Pdx 1 protein expression was reported as total intensity or integrated intensity, defined as total fluorescence of the cell times area of the cell. Background was eliminated based on acceptance criteria of grey-scalc ranges between 300 to 3000. Total intensity data were normalized by dividing the total intensities for each well by the average total intensity for the Wnt3a/Activin A positive control. Normalized data were calculated for averages and standard deviations for each replicate set.

|02511 Cells for quantitative PCR were lysed in RLT buffer (Qiagen) and then processed for RNA extraction, purification, and cDNA synthesis. RNA samples were purified by binding to a silica-gel membrane (Rneasy Mini Kit, Qiagen, CA) in the presence of an ethanol-containing, high-salt buffer followed by washing to remove contaminants. The RNA was further purified using a TURBO DNA-free kit (Ambion, inc,), and high-quality RNA was then eluted in water. Yield and purity were assessed by A260 and A280 readings on a spectrophotometer. cDNA copies were made from purified RNA using an Applied Biosystems, Inc, (AB1, C'A) high capacity cDNA archive kit.

10252) Unless otherwise stated, all reagents for real-time PCR amplification and quantitation were purchased from AB1. Real-time PCR reactions were performed using the AB1 PRISM 7900 Sequence Detection System. TAQMAN UNIVERSAL PCR MASTER MIX was used with 20 ng of reverse transcribed RNA in a total reaction volume of 20 μΙ. Each cDNA sample was run in duplicate to correct for pipetting errors. Primers and FAM-labcled TAQMAN probes were used at concentrations of 200 nM. The level of expression for each target gene was normalized using a human glyceraldehyde-3phosphate dehydrogenase (GAPDH) endogenous control previously developed by AB1. Primer and probe sets are listed as follows: PDX1 (Hs00236830__ml), GAPDH (4310884E), and HNF6 (Hs00413554_ml).

- 822017202571 19 Apr 2017

10253) [02541

I0255J

After an initial incubation at 50^QC for 2 min followed by 95^CC for 10 min, samples were cycled 40 times in two stages, a denaturation step at 95 °C for 15 sec followed by an annealing/extension step at 60°C for 1 min. Data analysis was carried out using GENEAMP0700G Sequence Detection System software, For each primcr/probe set, a Ct value was determined as the cycle number at which the fluorescence intensity reached a specific value in tire middle of the exponential region of amplification. Relative gene expression levels were calculated using the comparative Ct method. Briefly, for each cDNA sample, the endogenous control Ct value was subtracted from the gene of interest Ct to give the delta Ct value (ACt). The normalized amount of target was calculated as 2ACt, assuming amplification to be ] 00% efficiency. Final data were expressed relative to a calibrator sample.

Results

Results are shown for eight GSK.-3p enzyme inhibitors. Data presented in Figure 11 from high content analysis show effects on cell number (panel A) and Pdxl intensity (panel B) for the Hl hES cell line, where respective data points were averaged from a duplicate sample set and mined for each parameter from identical fields and wells. Data presented in Figure 12 from real-time PCR show effects of these small molecule inhibitors on induced expression of two transcription factors, Pdxl and HNF6. In these examples, Pdxl and HNF6 expression are indicative ofpanereatic endoderm differentiation. GSK33 inhibitor compounds in these assays can substitute for Wnt3a during early stages of cell lineage commitment; resulting cells sustain a capacity to form pancreatic endoderm during later sequential stages of differentiation.

Example 10

Effects of GSK-30 Enzyme Inhibitors on the Formation of Pancreatic Endocrine Cells

It was important to demonstrate that treatment with GSK3 inhibitors during induction of definitive endoderm did not prevent the subsequent differentiation of other cell types.

- 83 2017202571 19 Apr 2017 such as pancreatic endocrine cells, or insulin producing celts, for example. A select subset of hits was tested for their ability to promote expression of pancreatic hormones.

10256J Cell preparation far assay: Pancreatic endoderm cells obtained according to the methods described in Example 9 (cultured on 96-wellplates and 24-wcll plates) were subsequently subjected to agents that cause the cells to differentiate into pancreatic hormone expressing cells.

|0257] Assay for cultures of the Η1 human embryonic stem cell line on MATRIGEL™ was initiated as described in Examples 7-9 above by aspirating culture medium from cell monolayers in each well followed by three washes in PBS to remove residual growth factors and serum. For differentiation to definitive endoderm, test volumes (0.5 ml per well for 24-well plates, 100 μΙ per well for 96-well plates) were added containing medium with 0.5% FCS and different concentrations of inhibitor compounds with 100 ng/ml Activin A, without Wnt3a. Positive control wells contained the same base medium and 0.5% FCS with lOOng/ml Activin A and 20ng/mI Wnt3a (R&D Biosystems) in the absence of test compound. Negative control wells contained the same base medium with 0.5% FCS, in the absence of Activin A, Wnt3a, 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 DMEM:FI2 supplemented with 2% FCS and lOOng/ml Activin A in the absence of both test compound or Wnt3a. Parallel negative control welts were maintained on days 3, 4, and 5 in DMEM:F12 base medium with 2% FCS. For differentiation to pancreatic endoderm, cells were treated for three days, feeding daily with DMEM:F12 base medium containing 2% FCS with 0.25 μΜ KAAD cyclopamine (EMD Biosciences) and 20 ng/ml FGF7 (R&D Biosystems). Cells were subsequently treated for four days, feeding daily with DMEM:F 12 containing 1% B27 (Invitrogen), 0.25 μΜ KAAD cyclopamine, 2 μΜ Retinoic Acid (RA; Sigma-Aldrich) and 20 ng/ml FGF7. Parallel negative control wells during stages 2 and 3 were maintained throughout in DMEM:F12 base medium with 2% FCS or 1 % B27 and without any other additives. After formation of pancreatic endoderm, cells were treated further for six days duration, feeding daily with DMEM:F12 base medium containing 1% B27 with 1 μΜ DAPT (gamma secretase inhibitor: EMD

- 842017202571 19 Apr 2017

Biosciences) and 50 ng/ml Exendin 4 (Sigma-Aldrich). Cells were then treated for another three days duration, feeding daily with DMEM:FI2 base medium containing 1%

B27, 50 ng/ml Exendin 4, 50 ng/ml IGF (R&D Biosystems) and 50 ng/ml HGF (R&D

Biosystems). Parallel negative control wells were maintained throughout in DMEM;F12 base medium with 1% B27 and without any other additives.

10258J Assay evaluation: At the end of culture, cells were treated as in Examples 7 and 8 above for evaluation by high content analysis or real-time,PCR.

|02591 For high content fluorescence staining, cells in 96-well plates were washed twice with PBS then fixed with 4% paraformaldehyde at room temperature for 20 minutes, washed three times more with PBS, and then permeabilized with 0.5% Triton X-100 for 20 minutes at room temperature. After fixing and permeabilizing, cells were washed again three times with PBS and blocked with 4% chicken serum (Invitrogen) in PBS for 30 minutes at room temperature. Primary antibody (guinea pig anti-swine insulin, crossreactive with human insulin; DakoCytomation) was diluted 1:500 in 4% goat serum and added to cells for one hour at room temperature. Cells were washed three times with PBS and then stained with Alexa Fluor 488 conjugated secondary antibody (goat antiguinea pig IgG; Molecular Probes) diluted 1:100 in 4% goat serum, To counterstain nuclei, 2,ug/ml Hoechst 33342 (Invitrogen) was added for ten minutes at room temperature. Cells were washed once with PBS and left in 100 μΙ/well PBS for imaging.

10260) Cells were imaged using an IN Cell Analyzer 1000 (GE Healthcare) utilizing the 51008bs dichroic for cells stained with Hoechst 33342 and Alexa Fluor 488. Exposure times were optimized using positive control wells and wells stained with secondary antibody alone. Images from 15 fields per well were acquired to compensate for any cell loss during the treatment and staining procedures. Measurements for total cell number and total insulin intensity were obtained 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 and standard deviations were calculated for each replicate data set. Total insulin protein expression was reported as total intensity or integrated intensity, defined as total fluorescence of the cell times area

-852017202571 19 Apr 2017 of the cell. Background was eliminated based on acceptance criteria of grey-scale ranges between 300 to 3000. Total intensity data were normalized by dividing the total intensities for each well by the average total intensity for the Wnt3a/Activin A positive control, Normalized data were calculated for averages and standard deviations for each triplicate set.

[02611 Cells for quantitative PCR were lysed in RLT buffcr(Qiagen) and then processed for RNA extraction, purification, and cDNA synthesis. RNA samples were purified by binding to a silica-gel membrane (Rneasy Mini Kit, Qiagen, CA) in the presence of an ethanol-containing, high-salt buffer followed by washing to remove contaminants. The RNA was further purified using a TURBO DNA-free kit (Ambion, INC), and highquality RNA was eluted in water. Yield and purity were assessed by A260 and A280 readings on a spectrophotometer. cDNA copies were made from purified RNA using an Applied Biosystems, Inc. (ABl, CA) high capacity cDNA archive kit.

[0262) Unless otherwise stated, all reagents for real-time PCR amplification and quantitation were purchased from ABL Real-time PCR reactions were performed using the ABl PRISM® 7900 Sequence Detection System. TAQMAN® UNIVERSAL PCR MASTER ΜIX® (ABl, CA) was used with 20 ng of reverse transcribed RNA in a total reaction volume of 20 μΙ. Each cDNA sample was run in duplicate to conect for pipetting errors. Primers and FAM-labcled TAQMAN®probes were used at concentrations of 200 nM. The level of expression for each target gene was normalized using a human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) endogenous control previously developed by ABl. Primer and probe sets are listed as follows: PDX1 (Hs00236830_m1), Insulin (Hs00355773), and GAPDH (4310884E).

102631 After an initial incubation at 50^cC for 2 min followed by 95°C for 10 min, samples were cycled 40 times in two stages, a denaturation step at 95°C for 15 sec followed by an anneal ing/ex tension step at 60°C for 1 min. Data analysis was carried out using GENEAMP®7000 Sequence Detection System software. For each primer/probe set, a Ci value was determined as the cycle number at which the fluorescence intensity reached a specific value in the middle of the exponential region of amplification, Relative gene

- 86expression levels were calculated using the comparative Ct method. Briefly, for each cDNA sample, the endogenous control C_t value was subtracted from the gene of interest

C_t to give the delta Ct value (AC_L). The normalized amount of target was calculated as 2' ^Λ°, assuming amplification to be 100% efficiency. Final data were expressed relative to a calibrator sample.

2017202571 19 Apr 2017

Results [0264| Results are shown for eight GSK-3B enzyme inhibitors. Data presented in Figure 13 from high content analysis show compound effects on cell number (panel A) and insulin intensity (panel B) forthe Hl 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 Figure 14 from real-time PCR show compound effects for Pdxl and insulin In these examples, Pdxl and insulin expression are indicative of pancreatic endoderm differentiation and generation of hormonal positive cells. Selective GSK3p inhibitor compounds in these assays can substitute for Wnt3a during early stages of cell lineage commitment and can induce and sustain pancreatic beta cell formation during later sequential stages of differentiation, as evident from both insulin immunostaining and real-time PCR.

Example 11

Additive Effects of GSK-3P Enzyme Inhibitors on the Formation of Pancreatic Endocrine Cells

It was important to demonstrate that treatment with GSKJp inhibitors could improve pancreatic beta cell differentiation if added during multiple phases of cell fate commitment. A select subset of hits was tested by sequential timed addition to enhance insulin expression associated with pancreatic hormonal positive cells.

Preparation ofcells far assay: Cell preparation far assay: Pancreatic endoderm cells obtained according to the methods described in Example 9 and Id (cultured on 96- 872017202571 19 Apr 2017 wellplates) were subsequently subjected to agents that cause the cells to differentiate into pancreatic hormone expressing cells.

Assay for cultures of the Hl human embryonic stem cell line on MATRIGEL™ was initiated as described in Examples 7-9 above by aspirating culture medium from cell monolayers in each well followed by three washes in PBS to remove residual growth factors and serum. For differentiation to definitive endoderm, test volumes ¢100 μΙ per well for 96-well plates) were added containing medium with 0,5% FCS and different concentrations of inhibitor compounds with 100 ng/ml Activin A, without Wnt3a.

Positive control wells contained the same base medium and 0.5% FCS with 1 OOng/ml Activin A and 20ng/ml Wnt3a (R&D Biosystems) in the absence of test compound. Negative control wells contained the same base medium with 0.5% FCS, in the absence of Activin A, Wnt3a, 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 DMEM:FI2 supplemented with 2% FCS and 1 OOng/ml Activin A in the absence of both test compound or Wnt3a.

Parallel negative control wells were maintained on days 3, 4, and 5 in DMEM:FI2 base medium with 2% FCS. For differentiation to pancreatic endoderm, cells were treated for three days, feeding daily with DMEM:F12 base medium containing 2% FCS with 0.25 μΜ KAAD cyclopamine (EMD Biosciences) and 20 ng/ml FGF7 (R&D Biosystems). Cells were subsequently treated for four days, feeding daily with DMEM:F12 containing 1% B27 (Invitrogen), 0.25 μΜ KAAD cyclopamine, 2 μΜ Retinoic Acid (RA; SigmaAldrich) and 20 ng/ml FGF7, Parallel negative control wells were maintained throughout in DMEM:F12 base medium with 2% FCS or 1% B27 and without any other additives. After formation of pancreatic endoderm, cells were treated further for six days duration, feeding alternating days with DMEM:F 12 base medium containing 1% B27 with I μΜ DAPT (gamma sccrctase inhibitor; EMD Biosciences) and 50 ng/ml Excndin 4 (SigmaAldrich) and 1 μΜ TGFbeta RI inhibitor II (ALK5 inhibitor; EMD Biosciences). During this six day period, GSK3P inhibitors were added back to respective wells, using the same concentration as previous treatment at the initiation of differentiation. Cells were then treated for another three days duration, feeding alternating days with DMEM :F 12

-882017202571 19 Apr 2017 base medium containing 1% B27, 50 ng/ml Exendin 4, 50 ng/ml IGF (R&D Biosystems) and 50 ng/ml HGF (R&D Biosystems), and 1 μΜ TGFbeta Rl inhibitor II (ALK5 inhibitor; EMD Biosciences). During this three day period, GSK3P inhibitors were added back to respective wells, using the same concentration as previous treatment at the initiation of differentiation. Parallel sets of positive control wells were treated in the presence or absence of 20ng/ml Wnt3a. Parallel negative control wells were maintained throughout in DMEM:F12 base medium with 1% B27 and without any other additives.

10265] Assay evaluation: At the end of culture, cells were treated as in Examples 10 above for evaluation by high content analysis,

10266] For high content fluorescence staining, cells in 96-well plates were washed twice with

PBS then fixed with 4% paraformaldehyde at room temperature for 20 minutes, washed three times more with PBS, and then permeabilized with 0.5% Triton X-100 for 20 minutes at room temperature. After fixing and permeabilizing, cells were washed again three times with PBS and blocked with 4% chicken scrum (Invitrogen) in PBS for 30 minutes at room temperature. Primary antibody (guinea pig anti-swine insulin, crossreactive with human insulin; DakoCytomation) was diluted 1:500 in 4% goat scrum and added to cells for one hour at room temperature. Cells were washed three times with PBS and then stained with Alexa Fluor 488 conjugated secondary antibody (goat anti guinea pig IgG; Molecular Probes) diluted 1:100 in 4% goat serum. To counterstain nuclei, 2pg/ml Hoechst 33342 (Invitrogen) was added for ten minutes at room temperature. Cells were washed once with PBS and left in 100 ul/well PBS for imaging.

10267] Cells were imaged using an IN Cell Analyzer 1000 (GE Healthcare) utilizing the 51008bs dichroic for cells stained with Hoechst 33342 and Alexa Fluor 488. Exposure times were optimized using positive control wells and wells stained with secondaiy antibody alone. Images from 15 fields per well were acquired to compensate for any cell loss during the treatment and staining procedures. Measurements for total cell number and total insulin intensity were obtained 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 and standard deviations were

- 892017202571 19 Apr 2017 calculated for each replicate data set. Total insulin protein expression was reported as total intensity or integrated intensity, defined as total fluorescence of the cell times area of the cell. Background was eliminated based on acceptance criteria of grey-scale ranges between 300 to 3000. Total intensity data were normalized by dividing the total intensities for each well by the average total intensity for the Wnt3a/Activin A positive control, Normalized data were calculated for averages and standard deviations for each triplicate set.

Results

1112681 Results are shown for eight GSK.-3B enzyme inhibitors. Data presented in Figure 15 from high content analysis show compound effects on cell number (panel A) and insulin intensity (panel B) for the Hl hES cell line, where respective data points were averaged from a triplicate set and mined for each parameter from identical fields and wells. In this example, insulin expression is indicative of differentiation to hormonal positive pancreatic cells. Selective GSK.3P inhibitor compounds in these assays can substitute for Wnt3a during early stages of cell lineage commitment and, when added at later stages of differentiation, appear to promote enhanced insulin expression relative to a positive control sample.

[0269] Publications cited throughout this document are hereby incorporated by reference in their entirety. Although the various aspects of the invention have been illustrated above 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 principles of patent law.

-902017202571 19 Apr 2017

Table IA: List of primary antibodies used for FACS and immunostaimninganalysis.

Antibody	Supplier	I so type	Clone
SSEA-1	Chemicon (CA)	Mouse IgM	MC-480
SSEA-3	Chemicon (CA)	Mouse lgG3	MC-631
SSEA-4	Chemicon (CA)	Rat IgM	MC-813-70
TRA 1-60	Chemicon (CA)	Mouse IgM	TRA 1-60
TRA 1-81	Chemicon (CA)	Mouse IgM	TRA 1-81
TRA 1-85	Chemicon (CA)	Mouse IgGl	TRA 1-85
AP	R&D Systems	Mouse IgGl	B4-78
HNF3p	R&D Systems	Goat IgG
PDX1	Santa Cruz Biotechnology, INC	Goat tgG	A-l 7
GATA4	R&D Systems	Goat IgG
Sox 17	R&D Systems	Goat IgG
CD 9	BD	Mouse IgGl	M-L 13

-91 Table lb: List of secondary conjugated antibodies used for FACS and immunostainininganalysis.

2017202571 19 Apr 2017

Secondary Conjugated Antibody	Supplier	Dilution
Goat Anti-Mouse IgG APC conjugated	Jackson ImmunoResearch (PA)	1:200
Goat Anti-Mouse IgG PE conjugated	Jackson ImmunoResearch (PA)	1:200
Donkey anti-rabbit PE or- APC conjugated	Jackson ImmunoResearch (PA)	1:200
Donkey anti-goat PE or — APC conjugated	Jackson ImmunoResearch (PA)	1:200
Goat anti-mouse IgM PE	SouthemBiotech (AL)	1:200
Goat anti-Rat IgM PE	SouthemBiotech (AL)	1:200
Goat anti-mouse IgG3 PE	SouthemBiotech (AL)	1:200

-92Table II: Effects of Inhibitors of GSK.-3B Enzyme Activity on the Viability of Cells

Expressing Pluripotency Markers.

2017202571 19 Apr 2017

	Raw data (duplicate)	Average	S.D.	% cv	% Control
JNJ5226780	0.785 0.790	0.788	0.00382	0.48	94.0
JNJ10179026	0.148 0.152	0.150	0.00247	1.65	4.8
JNJ17154215	0.427 0.462	0.444	0.02496	5.62	46.0
JNJ17205955	0.643 0.638	0.641	0.00368	0.57	73.5
JNJ180125	0.762 0.762	0.762	0.00007	0.01	90.4
JNJ18157546	0.850 0.824	0.837	0.01824	2.18	101.0
JNJ19370026	0.911 0.884	0.898	0.01881	2.10	109.5
JNJ19567340	0.723 0.743	0.733	0.01421	1.94	86.4
JNJ7830433	0.161 0.169	0.165	0.00559	3.39	6.9
JNJ10179130	0.767 0.789	0.778	0.01556	2.00	92.6
JNJ17154215	0.512 0.555	0.533	0.03048	5.72	58.4
JNJ17205955	0.282 0.293	0.288	0.00792	2.75	24.1
JNJ18014061	0.764 0,723	0.743	0.02892	3.89	87.9
JNJ18157698	0.853 0.858	0.855	0.00382	0.45	103.5
JNJ19376240	0.832 0.837	0.834	0.00361	0.43	100.6
JNJ195674O5	0.726 0.725	0.725	0.00042	0.06	85.3
JNJ8706646	0.132 0.137	0.134	0.00368	2.74	2.6
JNJ10182562	0.797 0.793	0.795	0.00346	0.44	95.1
JNJ17157659	0.776 0.787	0.782	0.00792	1.01	93.2
JNJ17205994	0.164 0:148	0.156	0.01131	7.24	5.6
JNJ18014074	0.475 0.383	0.429	0.06548	15.26	43.8
JNJ18157698	0.823 0.774	Q.79B	0.03444	4.31	95.8
JNJ19386042	0.781 0.729	0.755	0.03649	4.83	89.5
JNJ19573541	0.143 0.149	0.146	0.00396	2.72	4.2
JNJ8710481	0.716 0.716	0.716	0.00014	0.02	84.1
JNJ10182562	0.804 0.802	0.803	0.00148	0.18	96.2
JNJ17163042	0.900 0.877	0.888	0.01626	1.83	108.2
JNJ17226703	0.824 0.799	0.812	0.01725	2.13	97.4
JNJ18018338	0.744 0.819	0.781	0.05261	6.73	93.2
JNJ18157711	0.952 0.966	0.959	0.00933	0.97	118.1
JNJ19410833	0.952 0.919	0.935	0.02277	2.43	114.8
JNJ19574867	0.776 0.777	0.777	0.00042	0.05	92.5
JNJ8710481	0.691 0.617	0.654	0.05254	8.03	75,4
JNJ10184655	0.162 0.134	0.148	0.02022	13.66	4.5
JNJ10166565	0.791 0.608	0.700	0.12947	18.50	81.8
JNJ17982133	0.153 0.129	0.141	0.01676	11.87	3.5
JNJ18018351	0.731 0.585	0.658	0.10317	15.68	75.9
DMSO	0.789 0.700	0.744	0.06279	8.44	88Ό
JNJ19410859	0.909 0.675	0.792	0.16546	20.88	94.7
JNJ19574880	0.164 0.151	0.157	0.00926	5.89	5.8
JNJ10148307	0.706 0.672	0.689	0.02404	3.49	83.9

-93 2017202571 19 Apr 2017

JNJ10222784	0.641	0.601	0.621	0.02878	4.63	73.7
JNJ17174664	0.882	0.748	0.815	0.09504	11.66	102.5
JNJ17989049	0.822	0.802	0.812	0.01400	1.72	102.1
JNJ18047991	0.777	0,764	0.771	0.00919	1.19	95.9
DMSO	0,798	0.771	0.735,	0.01916	2.44	98.0
JNJ19410872	0.791	0.789	0.790	0.00134	0.17	98.7
JNJ20948798	0.628	0.640	0.634	0.00806	1,27	75.6
JNJ10164830	0.149	0.135	0.142	0.00969	6.81	2.7
JNJ10222927	0.803	0.782	0.792	0.01492	1.88	99.1
JNJ17187027	0.125	0.129	0.127	0.00318	2.51	0.4
JNJ17994873	0.315	0.542	0.428	0.15995	37.34	45.2
JNJ18055726	0.820	0.748	0.784	0.05091	6.49	97.9
JNJ18157711	0.154	0.165	0.160	0.00806	5.05	5.3
JNJ19558929	0.737	0.730	0.734	0.00481	0.66	90.4
JNJ21192730	0,659	0.647	0.653	0.00813	1,25	78.5
JNJ10164895	0.165	0.154	0.159	0.00785	4.93	5.2
JNJ10231273	0.637	0.554	0.595	0.05876	9.87	69.9
JNJ17187053	0.684	0.588	0.636	0.06809	10.71	76.0
JNJ17994899	0.750	0.624	0.687	0.08945	13.02	83.5
JNJ18077800	0.678	0.618	0.648	0.04285	6.61	77.8
JNJ19363357	0.777	0.667	0.722	0.07757	10.74	88.7
DMSO	0.799	0.649	0.724	0.10564	14.59	89.0
JNJ21194667	0.648	0.625	0.636	0.01662	2.61	76.0
JNJ10172058	0.601	0.620	0.611	0.01308	2.14	72.2
JNJ10259847	0.695	0.702	0.698	0.00552	0.79	85.2
JNJ17193774	0.568	0.709	0.639	0.09956	15.59	76.4
JNJ17994912	0.623	0.765	0.694	0.10041	14.46	84.6
JNJ18157074	0.758	0.762	0.760	0.00297	0.39	94.3
JNJ19369233	0.487	0.434	0.461	0,03769	8.18	49.9
JNJ19567314	0,690	0.686	0.688	0.00262	0.38	83.7
JNJ21196227	0.535	0.550	0.543	0.01089	2.01	62.1
JNJ10178727	0.743	0.638	0.691	0.07446	10.78	84.1
JNJ10259847	0.694	0.603	0.649	0.06449	9.94	77.8
JNJ17200976	0,160	0,186	0.173	0.01824	10.56	7.2
JNJ17994925	0.662	0.566	0.614	0.06788	11.05	72.7
JNJ18157087	0.600	0.514	0.557	0.06102	10.96	64.2
JNJ19369246	0,685	0.524	0.605	0.11427	18.90	71.3
JNJ19567327	0.731	0.525	0.628	0.14552	23.18	74.7
JNJ24843611	0.715	0.596	0.655	0.08436	12.87	78.8
JNJ24843611	0.592	0.572	0.582	0.01393	2.39	70.0
JNJ25758785	0.614	0.611	0.613	0.00177	0.29	74.6
JNJ26064571	0.766	0.849	0.807	0.05869	7.27	104,3
JNJ26130403	0.830	0.813	0.822	0.01195	1.45	106.5
JNJ26170794	0.727	0.730	0.728	0.00198	0.27	92.2
JNJ26241774	0.713	0.836	0.774	0.08733	11,28	99.3
JNJ26367991	0.726	0.719	0.722	0.00523	0.72	91.3
JNJ26483310	0.646	0.681	0.663	0,02510	3.78	82.4

-942017202571 19 Apr 2017

JNJ243261B5	0.651	0.649
JNJ25758850	0.642	0.622
JNJ26067626	0.843	0.672
JNJ26134771	0.734	0.815
JNJ26J70820	0,823	0.743
JNJ26241917	0.871	0.874
JNJ26714220	0.652	0.642
JNJ26483223	0.617	0.633
JNJ24843572	0.657	0.555
JNJ25758863	0.684	0.809
JNJ26067652	0.901	0.735
JNJ26150202	0.791	0.768
JNJ26170833	0.948	0,764
JNJ2G2432O4	0.821	0.608
JNJ26399906	0.745	0.685
JNJ26483236	0.624	0.618
JNJ24843585	0.652	0.624
JNJ25873419	0.773	0.662
JNJ26069901	0.856	0.834
JNJ26153647	0.828	0.800
JNJ26177086	0.821	0.841
JNJ26247143	0.816	0.787
JNJ26399906	0.744	0.737
JNJ26483249	0.699	0.679
JNJ25753520	0.186	0.208
JNJ25887537	0.665	0.699
JNJ26077883	0.810	0.683
JNJ26158015	0.141	0.162
DMSO	0.784	0.605
JNJ26248729	0.726	0.590
JNJ26399945	0.635	0.620
JNJ26483249	0.697	0.695
JNJ25753403	0.154	0.153
JNJ25900641	0.616	0.645
JNJ22791671	0.909	0.830
JNJ26158054	0.150	0.150
JNJ26177762	0.981	1.056
JNJ26261105	0.166	0.189
JNJ26399971	0.718	0.451
JNJ26483262	0.652	0.647
JNJ25757173	0.503	0.529
JNJ25900654	0,603	0.609
JNJ26116922	0.856	0.793
JNJ26893438	0.883	0.848
JNJ26184457	0.779	0.784
JNJ26361712	0.892	0.914
JNJ263999S4	0.544	0.537

0.650	0.00120	0.19	80.3
0.632	0.01407	2.23	77.5
0.758	0.12099	15.97	96.7
0.774	0.05728	7.40	99.3
0.783	0.05699	7.28	100.6
0.872	0.00219	0.25	114.2
0.647	0.00721	1.12	79.8
0.625	0.01174	1.88	76.5
0.656	0.00134	0.20	81.2
0.746	0.08803	11.80	95.0
0.818	0.11731	14.34	106.0
0.779	0.01591	2.04	100.1
0.856	0.12982	15.17	111.7
0.714	0.15033	21.05	90.1
0.715	0.04243	5.94	90.2
0.621	0.00417	0.67	76.0
0.638	0.01916	3.00	78.5
0.718	0.07792	10.86	90.6
0.845	0.01570	1.86	110.1
0.814	0.02008	2.47	105.4
0.831	0.01421	1.71	108.0
0.802	0.02072	2.58	103.5
0.741	0.00453	0.61	94.1
0.689	0.01464	2.12	86.3
0.197	0.01541	7.83	11.3
0.682	0.02432	3.57	85.2
0.746	0.09030	12.10	95.0
0.151	0.01506	9.95	4.3
0.695	0.12671	18.25	87.1
0.658	0.09624	14.63	81.5
0.628	0.01068	1.70	76.9
0.696	0.00113	0.16	87.3
0.154	0.00042	0.28	4.5
0.630	0.02072	3.29	82.1
0.869	0.05614	6.46	121.0
0.150	0.00028	0.19	3.9
1.018	0.05303	5.21	145.3
0.177	0.01626	9.19	8.3
0.584	0.18887	32.34	74.6
0.649	0.00389	0.60	85.2
0.516	0.01860	3.61	63.5
0.606	0.00424	0.70	78.1
0.824	0.04419	5.36	113.7
0.866	0.02503	2.89	120.5
0.781	0.00368	0.47	106.7
0.903	0.01591	1.76	126.6
0.540	0.00460	0.85	67.5

-952017202571 19 Apr 2017

JNJ265119O1	0.532	0.682	0.607	0.10543	17.37	78.3
JNJ25757173	0.665	0.645	0.655	0.01400	2.14	86.1
JNJ25900706	0.676	0.677	0.677	0.00035	0.05	89.7
JNJ26120601	0.935	0.807	0.871	0.09115	10.47	121.3
JNJ26158093	0.916	0.859	0.887	0.03981	4.49	124.0
JNJ26219050	0.907	0.891	0.899	0.01124	1.25	125.9
JNJ26361725	0.909	0.896	0.902	0.00919	1.02	126.4
JNJ26399997	0.682	0.797	0.740	0.08118	10.98	99.9
JNJ26511927	0.679	0.644	0.661	0.02510	3.80	87.2
JNJ25757238	0.300	0.223	0.261	0.05452	20.88	22.0
JNJ26047723	0.183	0.175	0.179	0.00573	3.20	8.6
JNJ26120614	0.741	0.728	0.734	0.00884	1.20	99.1
JNJ26158106	0,935	0,906	0.921	0.02051	2.23	129.4
JNJ26219063	0.131	0.128	0.129	0.00212	1.64	0.5
JNJ26366730	0.138	0.137	0.138	0,00092	0.67	1.9
JNJ26400049	0.241	0.227	0.234	0.01032	4.41	17.6
JNJ26941226	0.604	0.639	0.622	0.02475	3.98	80.7
JNJ257587O7	0.247	0.182	0.215	0.04617	21.52	14.4
JNJ26054912	0.659	0.634	0.647	0.01718	2.66	84.8
JNJ26128726	0.758	0.575	0.667	0.12961	19.44	88.1
JNJ26161343	0.166	0.170	0.168	0.00276	1.64	6.9
JNJ26220454	0.651	0.559	0.605	0.06541	10.81	78.0
JNJ26367991	0.803	0.694	0.748	0.07693	10.28	101.3
JNJ26483197	0.823	0.634	0.728	0.13378	18.37	98.1
JNJ26511953	0.624	0.618	0.621	0.00431	0.69	80.6
RWJ351001	0.639	0.603	0.621	0.02553	4.11	73.6
RWJ382867	0.143	0.149	0.146	0.00403	2.76	2.9
RWJ682205	0.817	0.818	0.818	0.00071	0.09	102.8
RWJ665862	0,742	0.752	0.747	0.00679	0.91	92.2
RWJ670804	0.856	0.905	0.881	0.03479	3.95	112.1
RWJ673829	0.650	0.576	0.613	0.05268	8.59	72.4
RWJ675260	0.768	0.724	0.746	0.03097	4.15	92.2
RWJ675946	0.556	0.549	0.553	0.00537	0.97	63.4
RWJ351958	0.227	0.242	0.235	0.01103	4.70	16.1
RWJ395477	0.634	0.663	0.649	0.02044	3.15	77.7
RWJ447228	0.141	0.128	0.135	0.00919	6.83	1.3
RWJ666167	0.847	0.832	0.840	0.01110	1.32	106.0
RWJ670908	0.803	0.845	0.824	0.02998	3.64	103.7
RWJ67383O	0.860	0.860	0.860	0.00035	0.04	109.1
RWJ675261	0.528	0.497	0.513	0.02227	4.34	57.5
RWJ675948	0,683	0.688	0.686	0.00332	0.48	83.1
RWJ447228	0.611	0.628	0.620	0.01202	1.94	73.3
RWJ414342	0.719	0.749	0.734	0.02143	2.92	90.3
RWJ5537Q9	0.916	0.838	0.877	0.05487	6.26	111,6
RWJ666168	0.771	0.740	0.755	0.02178	2.88	93.5
RWJ670984	0.820	0.852	0.836	0.02305	2.76	105.5
RWJ674239	0.971	0.913	0.942	0.04137	4.39	121.2

-962017202571 19 Apr 2017

RWJ67543O	0.839	0.743	0.791	0.06746	8.53	98.8
RWJ676061	0.562	0.527	0.544	0.02440	4.48	62.2
RWJ35219O	0.678	0.661	0.670	0.01195	1.78	80.8
RWJ414984	0.722	0.713	0.717	0.00658	0.92	87.9
RWJ65978O	0,802	0.801	0.802	0.00106	0.13	100.4
RWJ666205	0.854	0.857	0.855	0.00205	0.24	108.4
RWJ671232	0,767	0.798	0.782	0.02157	2.76	97.5
RWJ57424O	0.789	0.776	0.782	0.00870	1.11	97.5
RWJ675266	0,720	0.709	0.714	0.00764	1.07	87.4
RWJ676Q85	0.641	0.618	0.630	0.01619	2.57	74.9
RWJ352244	0.603	0.584	0.593	0.01372	2.31	69.4
RWJ425264	0.135	0.158	0.146	0.01633	11.18	3.0
RWJ66244O	0.792	0.572	0.682	0.15563	22.83	82.6
RWJ666213	0.752	0.593	0.673	0,11292	16.79	81.2
RWJ672667	0.805	0.598	0.702	0.14644	20.87	85.5
RWJ674241	0.599	0.504	0.552	0.06682	12.11	63.2
RWJ675366	0.714	0.593	0.654	0.08549	13.08	78.4
RWJ676137	0,699	0.698	0.698	0.00099	0.14	85.0
RWJ352628	0.690	0.674	0.682	0.01131	1.66	83-3
RWJ4252S8	0.616	0.634	0.625	0,01301	2:08	74.8
RWJ66386O	0.809	0.817	0.813	0.00552	0.68	103.0
RWJ667045	0.128	0,133	0.131	0,00361	2.76	0.7
RWJ672932	0.821	0.811	0.816	0.00721	0.88	103.4
RWJ674320	0.456	0.474	0.465	0.01223	2.63	50.8
RWJ675369	0.762	0.766	0.764	0.00304	0.40	95.7
RWJ676139	0.680	0.663	0.671	0.01195	1.78	81.8
RWJ353258	0.615	0.635	0.625	0.01400	2.24	74.8
RWJ355923	0,681	0.698	0.689	0.01266	1.84	84.5
RWJ664545	0.830	0.807	0.818	0.01584	1.94	103.8
RWJ667046	0.869	0.849	0.859	0.01442	1.68	109.9
RWJ672934	0.821	0.841	0.831	0.01428	1.72	105.7
RWJ674817	0.819	0.840	0.830	0.01485	1.79	105.5
RWJ675430	0.795	0.793	0.794	0.00078	0.10	100.1
RWJ676431	0.640	0.636	0.638	0.00283	0.44	76.7
RWJ355131	0.610	0.628	0.619	0.01266	2.05	73.9
RWJ425271	0.143	0.144	0.144	0.00035	0.25	2.6
RWJ353709	0.804	0.903	0.853	0.07000	8.20	109.0
RWJ667069	0.918	0.854	0.886	0.04483	5.06	113.9
RWJ673313	0.105	1.080	0.593	0.68971	116.37	70.0
RWJ674855	0.877	0.860	0.868	0.01209	1.39	111.3
RWJ675578	0.808	0.695	0.751	0.07941	10,57	93.8
RWJ676432	0.720	0.697	0.709	0.01648	2.33	87.3
RWJ355923	0.636	0.621	0.629	0.01054	1.68	75.4
RWJ425348	0.640	0.634	0.637	0.00474	0.74	76.6
RWJ665436	0.833	0.833	0.833	0.00000	0.00	106.0
RWJ6691S2	0.887	0.846	0.866	0.02934	3.39	111,0
RWJ673515	0.845	0.877	0.861	0.02326	2.70	110.2

-972017202571 19 Apr 2017

RWJ674855	0.794	0.784
RWJ675605	0.770	0.786
RWJ67657	0.629	0.659
RWJ356205	0.584	0.558
RWJ445224	0.707	0.679
RWJ665588	0.727	0.578
RWJ669327	0.742	0.629
DMSO	0.653	0.507
RWJ675104	0.722	0.568
RWJ675881	0.643	0.581
RWJB76639	0.608	0.590
JNJ26511966	0,597	0.610
JNJ26511979	0.687	0.668
JNJ26512005	0.840	0.832
JNJ26533065	0.831	0.822
JNJ26533091	0.863	0.856
JNJ265331O4	0,886	0.802
JNJ26533156	0.753	0.687
JNJ26714181	0.455	0.463
JNJ26714194	0,668	0.678
JNJ26714207	0.181	0.171
JNJ26714220	0.832	0.842
JNJ26875563	0.795	0.802
JNJ22791671	0.157	0.140
JNJ26893438	0.153	0.153
JNJ26941226	0.168	0.154
JNJ28572128	0.670	0.641
RWJ67694	0.706	0.679
RWJ676940	0,788	0.666
RWJ677545	0.879	0.785
RWJ678986	0.168	0.176
RWJ680665	0.946	0.848
RWJ680667	0.187	0.202
RWJ680668	0.906	0.688
RWJ680669	0.715	0.674
RWJ680858	0.695	0.700
RWJ680858	0.665	0.631
RWJ680879	0,590	0.613
RWJ680885	0,681	0.687
RWJ680991	0.829	0.821
RWJ680992	0.822	0.790
RWJ680993	0.671	0.684
RWJ681140	0.686	0.668
RWJ681142	0.212	0.197
RWJB81146	0.666	0.666
RWJ681945	0.736	0.656
RWJ68498	0.726	0.610

0.789	0.00686	0.87	99.4
0.778	0.01138	1.46	97.8
0.644	0.02128	3.30	77.7
0.571	0.01817	3.18	66.8
0.693	0.01987	2.87	85.0
0.652	0.10536	16.15	78.9
0.685	0.07969	11.63	83.8
0.580	0.10310	17.78	65.0
0.645	0.10904	16.90	77.9
0.612	0.04384	7.16	72.9
0.599	0.01245	2.08	70.9
0.603	0.00926	1.54	71.2
0.677	0.01336	1.97	82.4
0.836	0.00594	0.71	106.1
0.826	0.00587	0.71	104.7
0.860	0.00509	0.59	109.7
0.844	0.05954	7.05	107.3
0.720	0.04660	6.47	88.8
0.459	0.00587	1.28	49.6
0.673	0.00764	1.13	81.7
0.176	0.00658	3.74	7.2
0.837	0,00658	0.79	106.3
0.798	0.00445	0.56	100.5
0.148	0.01202	8.11	3.0
0.153	0.00035	0.23	3.7
0.161	0.00969	6.02	4.9
0.655	0.02079	3.17	79.1
0.693	0.01888	2.73	84.7
0.727	0.08627	11.86	89.8
0.832	0.06640	7.98	105.6
0.172	0.00537	3.13	6.6
0.897	0.06972	7.77	115.3
0.194	0.01089	5.61	9.9
0.797	0.15394	19.31	100.3
0.694	0,02850	4.10	84.9
0.697	0.00339	0.49	85.3
0.648	0,02369	3.66	78.0
0.601	0.01655	2.75	71.0
0,684	0.00382	0.56	83.3
0.825	0.00530	0.64	104.5
0.806	0.02270	2.82	101.6
0.677	0.00912	1.35	82.3
0.677	0.01266	1.87	82.3
0.204	0.01047	5.12	11.5
0.666	0.00007	0,01	80.7
0.696	0.05643	8.11	85.1
0.668	0.08217	12.30	81.0

-982017202571 19 Apr 2017

JNJ28850601	0.303	0.310	0.306	0,00488	1.59	26.7
DMSO	0.786	0.659	0.722	0.09001	12.46	89.1
DMSO	0.673	0.649	0.661	0.01676	2.53	79.9
DMSO	0.701	0.686	0.693	0.01D11	1.46	84.8

-99Table III: Effects oflnhibitors oFGSK-3B Enzyme Activity on the Viability of Cells

Expressing Pluripotency Markers,

2017202571 19 Apr 2017

	cmpd cone (uM)	Raw data (duplicate)	Average	S.D.	%cv	% Control
EXPRES 01 medium		0.6379 0.6180	0.6280	0.0141	2.2	74.6
no treatment		0.7412 0.7038	0.7225	0.0264	3.7	88.7
AAonly		0.7674 0.8047	0.7861	0.0264	3.4	98.3
AA + Wnt3a		0.7754 0,8200	0.7977	0.0315	4.0	100.0
JNJ26512005	10	0.1412 0.1515	0.1464	0.0073	5.0	2.4
JNJ26512005	5	0.1501 0.1444	0.1473	0.0040	2.7	2.5
JNJ26512005	2.5	0.1541 0.4254	0.2898	0,1918	66.2	23.9
JNJ26533065	10	0.1272 0.1282	0.1277	0.0007	0.6	-0.4
JNJ26533065	5	0.5862 0.5880	0.5871	0.0013	0.2	68.4
JNJ26533065	2.5	0.7613 0.7603	0.7608	0.0007	0.1	94.5
JNJ26533156	10	0.1481 0.15S2	0.1537	0.0078	5.1	3.5
JNJ26533156	5	0.1479 0.1639	0:1559	0.0113	7.3	3.8
JNJ26533156	2.5	0.2861 0.2477	0.2669	0.0272	10.2	20.4
JNJ26714194	10	0.2092 0.2426	0.2259	0.0236	10.5	14.3
JNJ26714194	5	0.6815 0.7128	0.6972	0.0221	3.2	84.9
JNJ26714194	2.5	0.7389 0.7870	0.7630	0.0340	4.5	94.8
JNJ26150202	10	0.1381 0.1398	0,1390	0.0012	0.9	1.3
JNJ26150202	5	0.7826 0.7578	0.7702	0.0175	2.3	95.9
JNJ26150202	2.5	0.8231 0.7742	0.7987	0.0346	4.3	100.1
JNJ26158015	10	0.1352 0.1326	0.1339	0.0018	1.4	0.5
JNJ26158015	5	0.2632 0.2604	0.2618	0.0020	0.8	19.7
JNJ26158015	2.5	0.4160 0.5314	0.4737	0.0816	17.2	51.4
RWJ670804	10	0.4447 0.4791	0.4619	0.0243	5.3	49.7
RWJ670804	5	0.6902 0.6884	0.6893	0.0013	0.2	83.8
RWJ670804	2.5	0.7476 0.7483	0.7480	0.0005	0.1	92.5
JNJ26170833	10	0.6790 0.6704	0.6747	0.0061	0.9	81.6
JNJ26170833	5	0.7833 0.7924	0.7879	0.0064	0,8	98.5
JNJ26170833	2.5	0.8155 0.8389	0.8272	0.0165	2.0	104.4
JNJ26177086	10	0.6533 0.6884	0,6709	0.0248	3.7	81.0
JNJ26177086	5	0.7697 0.7738	0.7718	0.0029	0.4	96.1
JNJ26177086	2.5	0.8119 0.8219	0.8169	0.0071	0.9	102.9
JNJ26177762	10	0.1242 0.1323	0.1283	0.0057	4.5	-0.4
JNJ26177762	5	0.1263 0,1303	0.1283	0.0028	2.2	-0.3
JNJ26177762	2.5	0.8480 0.7725	0.8103	0.0534	6.6	101.9
RWJ673515	10	0.1695 0.1890	0,1793	0.0138	7.7	7.3
RWJ673515	5	0.7217 0.7435	0.7326	0.0154	2.1	90.2
RWJ673515	2.5	0.7812 0.7221	0.7517	0.0418	5.6	93.1
EXPRES Olmedium		0.6294 0.6363	0.6329	0.0049	0.8	70.3
no treatment		0.7156 0.7356	0.7256	0.0141	1.9	83.3
AA only		0.8732 0.9046	0.8889	0.0222	2,5	106.0
AA + Wnt3a		0.8415 0.8500	0.8458	0.0060	0.7	100.0

- 1002017202571 19 Apr 2017

JNJ19370026	10	0.1403,0.1493	0.1448	0.0064	4.4	2.3
JNJ19370026	5	0.4434 0.3878	0.4156	0.0393	9.5	40.1
JNJ19370026	2.5	0.7734 0.8038	0.7886	0.0215	2.7	92.0
JNJ26483197	10	0.2993 0.3026	0.3010	0.0023	0.8	24.1
JNJ26483197	5	0.7023 0.6299	0.6661	0.0512	7,7	75.0
JNJ26483197	2.5	0.7835 0.8043	0.7939	0.0147	1.9'	92.a
RWJ675605	10	0.7205 0.7369	0,7287	0.0116	1.6	83.7
RWJ575605	5	0.7769 0.8272	0.8021	0.0356	4.4	93.9
RWJ675605	2.5	0.8214 0.8640	0.8427	0.0301	3.6	99.6
RWJ67543Q	10	0.6275 0.5980	0.6128	0.0209	3.4	67.5
RWJ67543O	5	0.7159 0.7222	0.7191	0.0045	0.6	82.3
RWJ675430	2.5	0.9245 0.9403	0.9324	0.0112	1.2	112.1
RWJ675948	10	0.7220 0.6670	0.6945	0.0389	5.6	78,9
RWJ675948	5	0.7526 0.7486	0.7506	0.0028	0.4	86.7
RWJ675948	2.5	0.7557 0.7390	0.7474	0.0118	1.6	86,3
JNJ26483249	10	0.8214 0.8636	0.8425	0.0298	3.5	99.5
JNJ264B3249	5	0.7996 0.7873	0.7935	0.0087	1.1	92.7
JNJ26483249	2.5	0.8669 0.8195	0:8432	0.0335	4.0	99.6
RWJ67657	10	0.6195 0.5908	0.6052	0.0203	3.4	66.5
RWJ67657	5	0.8047 0.8319	0.8183	0.0192	2.4	96.2
RWJ67657	2.5	0.8041 0.7900	0.7971	0.0100	1.3	93.2
RWJ676639	10	0.1261 0.1520	0.1391	0.0183	13.2	1.5
RWJ676639	5	0.1303 0.1263	0.1283	0.0028	2.2	0.0
RWJ676639	2.5	0.4482 0.4051	0.4267	0.0305	7.1	41.6

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Table VI: Effects of lnliibitors of GSK-3B Enzyme Activity on the proliferation of human embryonic stem cells.

JNJ number	Raw Data	Average	S.D.	%cv	% Control
conditioned medium	1.1348 1.0099 1.1092	1,0846	0,0660	6,1	116,5
no treatment	0.9344 0.5977 0.8454	0.7925	0.1745	22.0	85.2
AA/DMSO	0.3878 0.2434 0.2252	0.2855	0.0891	31.2	30.7
AA/Wnt3a/DM5O	0.6098 1.0804 0.7635	0.8179	0.2403	25.8	100.0
RWJ351001	0.3418 0.4276 0.5751	0.4482	0.1180	26.3	48.2
RWJ351958	0.1362 0,1531 0,1532	0.1475	0.0098	6.6	15.8
RWJ352190	1.3764 1.2753 1.3208	1.3242	0.0506	3.8	142.3
RWJ3S2244	0.6923 0.5994 0.6134	0.6350	0.0501	7.9	68.2
RWJ352628	1.7896 1.4721 2.1908	1.8175	0.3602	19.8	195.3
RWJ353250	1.7591 1.6274 1.6518	1.6794	0.0701	4.2	180.4
RWJ3S5131	0.3702 0.3193 0.3368	0.3421	0.0259	7.6	36.8
RWJ355923	0.5876 0.6384 0,9154	0.7138	0.1764	24.7	76.7
RWJ3562Q5	0.3074 0.2328 0.2920	0.2774	0.0394	14.2	29.8
RWJ382867	0.1311 0.1245 0.1288	0.1281	0.0034	2.6	13.8
RWJ395477	0.1270 0.2778 0.1916	0.1988	0.0757	38.1	21.4
RWJ414342	0.2166 0.3062 0.2915	0.2714	0.0481	17.7	29.2
RWJ414934	0.4362 0.3728 0.2481	0.3524	0.0957	27.2	37.9
RWJ425Z64	0.1560 0.1481 0.1359	0.1467	0.0101	6.9	15.8
RWJ425268	0.2932 0.3883 0.6258	0.4358	0.1713	39.3	46.8
RWJ425271	0.1362 0.1479 0.1298	0.1380	0.0092	6.7	14.8
RWJ425348	0.2198 0.2159 0.2300	0.2219	0.0073	3.3	23.8
RWJ445224	0.7624 0.2705 0.2478	0.4269	0.2908	68.1	45.9
RWJ447228	0.1239 0.1233 0.1269	0.1247	0.0019	1.5	13.4
RWJ553709	0.1277 0.1254 0.6980	0.3170	0.3299	104.1	34.1
RWJ659780	0.2665 0.3215 0.2605	0.2828	0.0336	11.9	30.4
RWJ66244Q	0.2395 0.3235 0.1333	0.2321	0.0953	41.1	24.9
RWJ6638G0	0.2646 0.1873 0.1293	0.1937	0.0679	35.0	20.8
RWJW4545	0.3590 0.2790 0.1515	0.2632	0.1047	39.8	28.3
RW J665436	0.4690 0.5805 0.3349	0.4615	0.1230	26.6	49.6
JNJ number	Raw Data	Average	S.D.	%cv	% Control
conditioned medium	1.1525 1.1269 1.1140	1.1311	0.0196	1,7	71.0
no treatment	1.2057 1.2358 1.3132	1.2516	0,0555	4.4	78.6
AA/DMSO	0.2622 0.2073 0.2830	0.2508	0.0391	15.6	15.8
AA/Wnt3a/DMSO	1.3943 1.7976 1.8000	1.5922	0.2136	13.4	100.0
RWJ665588	0.1930 0.2223 0,2167	0.2107	0.0156	7.4	13.2
RWJ665862	0.1757 0.1813 0,1835	0.1802	0.0040	2.2	11.3
RWJ666167	0.1473 0.1880 0.1732	0.1695	0.0206	12.2	10.6
RWJ666168	0.1330 0.1362 0.1867	0.1520	0.0301	19.8	9.5
RWJ666205	0.8191 0.5493 0.6526	0.6737	0.1361	20.2	42.3
RW J666213	0.4008 0.2779 0.3869	0.3552	0.0673	18.9	22.3
RWJ667045	0.1220 0.1248 0.1251	0.1240	0.0017	1.4	7.8
RWJ667046	0.2883 0.3308 0.5503	0.3898	0.1406	36.1	24.5
RWJG670G9	0.2835 0.4024 0.5698	0.4186	0.1438	34.4	26.3
RWJGG9t32	0.3704 0.6073 0.5280	0.5019	0.1206	24.0	31.5
RWJ6G9327	0.2266 0.1815 0.2289	0.2123	0.0267	12.6	13.3

- 1092017202571 19 Apr 2017

RWJ670804	1.0820 1.1862 1.1076	1,1253	0.0543	4.8	70,7
RWJ670908	0.3590 0.5457 0,6123	0.5057	0.1313	26Ό	31.8
RWJ670934	0.2198 0.3564 0.3202	0.2988	0.0708	23.7	18.8
RWJ671232	0.2928 0.2920 0.3659	0.3169	0.0424	13.4	19.Θ
RWJ672667	0.3349 0.3013 0.3507	0.3290	0.0252	7.7	20.7
RWJ672332	0.1852 0.1924 0.2349	0.2042	0.0269	13.2	12.8
RWJ672934	0.2170 0.3003 0.1877	0.2350	0.0584	24.9	14.8
RWJ673313	0.3094 0.2515 0.1881	0.2497	0.0607	24.3	15.7
RWJ673515	1.8452 1.7710 1.5591	1.7251	0,1485	8.6	108.3
RWJ673829	0.7305 0.7067 0.6250	0.6874	0.0553	8.0	43.2
RWJ673830	0.2113 0.1800 0.1547	0.1820	0.0284	15.6	11.4
RWJ674233	1.5225 1.5912 0.1081	1.0739	0.8371	78.0	67.4
RWJ674240	0.4006 1.2807 0.1162	0.5992	0.6071	101.3	37.6
RWJ674241	0.1972 0.1839 0.1162	0.1658	0.0434	26.2	10.4
RWJ674320	0.1351 0.1318 0.1169	0.1279	0,0097	7.6	8.0
JNJ number	Raw Data	Average	S.D.	%CV	% Control
conditioned medium	1.0568 1.0604	1.0586	0.0025	0.2	71.9
no treatment	1.1544 0.9576	1.0560	0.1392	13.2	71.7
AA only * DMSO	0.6329 0.8434	0.7382	0.1488	20.2	47.1
AA + Wnt3a + DMSO	1.2704 1.8669	1.4229	0.2960	20.8	100.0
RWJ674817 ¹	0.5617 0.2098	0.3858	0.2488	64.5	19.9
RWJ674855	0.6850 0.5853	0.6352	0.0705	11.1	39.2
RWJ674355	0.7496 0.9187	0.8342	0.1196	14.3	54.5
RWJ675104	0.2320 0.2124	0.2222	0.0139	6.2	7.3
RWJ675260	0.8079 1.4391	1.1235	0.4463	39.7	76.9
RWJ6752G1	0.8310 0.7318	0.7814	0.0701	9.0	50.5
RWJ675Z66	1.0646 1.1384	1.1015	0.0522	4.7	75.2
RWJ675366	0.6344 1.0400	0.8372	0.2868	34.3	54.8
no cells	0.1335 0.2070	0.1703	0.0520	30.5	3.3
RWJ67S369	0.8643 0.4060	0.6352	0.3241	51.0	39.2
RWJ67543Q	1,7922 1.8533	1.8228	0.0432	2.4	130.9
RWJ67557B	0.1914 0.2371	0.2143	0.0323	15.1	6,7
RWJ675605	1.8401 1.7563	1.7982	0.0593	3.3	129.0
RWJ675881	1.0301 1.0356	1.0329	0.0039	0.4	69.9
RWJ675946	0.1306 0.1338	0.1322	0.0023	1.7	0.3
RWJ675948	1.7143 1.6506	1.6825	0.0450	2.7	120.0
RWJ6760E1	0.4170 0.4956	0.4563	0.0556	12.2	25.4
RWJ676085	0.1772 0.2348	0.2060	0.0407	19.8	6.0
RWJ676137	1.0231 1.2392	1.1312	0.1528	13.5	77.5
RWJ676139	1.9718 2.0997	2,0358	0,0904	4.4	147.3
RWJ676431	1.5168 1.6872	1.6020	0.1205	7.5	113.8
RWJG76432	1.6935 1.9710	1.8323	0,1962	10.7	131.6
RWJ67657	1.2655 1.1829	1.2242	0.0584	4.8	84.7
RWJ676639	1.3481 1.3168	1.3325	0.0221	1.7	93.0
JNJ26511966	0,6444 0.7239	0.6842	0,0562	8.2	43.0
JNJ2E511979	0.2046 0.3076	0.2561	0.0728	28.4	9.9
JNJ26512005	1.3627 1.0693	1.2160	0.2075	17.1	84.0
JN J26533065	0.8722 0.9660	0.9191	0.0663	7.2	61.1
JNJ26533091	1.0332 0.4554	0.7443	0.4086	54.9	47.6
JNJ26533104	0.8775 0.7347	0.8061	0.1010	12.5	52.4

- 1102017202571 19 Apr 2017

JNJ2653315G	1.7865 1.2008	1.4937	0.4142	27.7	105.5
JNJ26714181	0.2396 0.1584	0.1990	0.0574	28.9	5.5
JNJZ6714194	0.8122 1.0827	0.9475	0.1913	20.2	63.3
JNJ26714207	0.1342 0.1363	0.1353	0.0015	1.1	0.6
JNJ26714220	1.0462 0.5838	0.8150	0.3270	40.1	53.1
JNJ26875563	0.4586 0.2903	0.3745	0.1190	31.8	19.0
JNJ22791671	0.1277 0.1402	0.134Q	0.0088	6.6	0.5
JNJ26893438	0.1258 0.1324	0.1291	0.0047	3,6	0.1
JNJZ6941226	0.1219 0.1216	0.1218	0.0002	0.2	-0.5
JNJ28572128	0.4223 0.4721	0.4472	0.0352	7.9	24.7
JNJ2S850601	0.1514 0.1396	0.1455	0.0083	5.7	1.4
JNJ number	Raw Data	Average	S.D.	%CV	% Control
conditioned medium	0.7423 0.7081	0.7252	0.0242	3,3	87.7
no treatment	0.4936 0.5689	0.5313	0.0532	10.0	59.8
AA only + DMSO	0.1433 0.1939	0.1686	0.0358	21.2	7.6
AA + Wnt3a + DMSO	0,6808 0.9406	0.8107	0.1837	22.7	100,0
JNJ17994873	0.2447 0.1331	0.1889	0.0789	41.8	10.6
JNJ17994899	0.1537 0.1302	0.1420	0.0166	11.7	3.8
no Cells	0.1163 0.1147	0.1155	0.0011	1.0	0Ό
JNJ17994912	0.2994 0.2592	0,2793	0.0284	10.2	23.6
JNJ17994925	0,1353 0.2121	0.1737	0.0543	31.3	8.4
JNJ180125	0.1267 0.1419	0.1343	0.0107	8.0	2.7
JNJ18014061	0.1376 0.1676	0.1526	0.0212	13.9	5.3
JNJ18014074	0,1134 0.1103	0.1119	0.0022	2.0	-0.5
JNJ1S018338	0.1318 0.1478	0.1398	0.0113	8.1	3.5
JNJ18018351	0.2569 0.2124	0.2347	0.0315	13.4	17.1
JNJ18047991	0.2674 0.2636	0.2655	0.0027	1.0	21.6
JNJ1805572G	0.4357 0.3467	0.3912	0.0629	16.1	39.7
JNJ1B077800	0.1265 0.1588	0.1427	0.0228	16.0	3.9
JNJ18157074	0.1662 0.2521	0.2092	0.0607	29.0	13,5
JNJ18157087	0,1596 0.1566	0.1531	0.0021	1.3	6.1
JNJ18157646	0.2725 0.1636	0.2181	0.0770	35.3	14.8
JNJ18157711	1.2256 1.0636	1.1446	0.1146	10.0	148.0
JNJ18157711	0.1134 0.1070	0.1102	0.0045	4.1	-0.8
JNJ19363357	0.1469 0.1495	0.1482	0.0018	1.2	4.7
JNJ19369233	0.1169 0.1122	0.1146	0.0033	2.9	-0.1
JNJ19369248	0.1595 0.1422	0,1509	0.0122	8.1	5.1
JNJ19370026	1.0484 1.0749	1.0617	0.0187	1.8	136.1
JNJ19376240	0.3012 0.2347	0.2680	0.0470	17,5	21.9
JNJ19386042	0.1267 0.1510	0.1389	0.0172	12.4	3.4
JNJ19410833	1.1902 1.1487	ΐ,1'695	0.0293	2.5	151.6
JNJ19410859	0.6400 0.7076	0.6738	0,0478	7,1	80.3
JNJ19410872	0.1701 0.1752	0.1727	0.0036	2.1	8.2
JNJ19558929	0.3435 0.3488	0.3462	0.0037	1,1	33.2
JNJ19567314	0.4032 0.3548	0.3790	0,0342	9.0	37.9
JNJ19587327	0.1602 0.1502	0.1552	0.0071	4.6	5.7
JNJ19567340	0.1604 0.2079	0.1842	0.0336	18.2	9.9
JNJ19567405	0.1646 0.1592	0.1619	0.0038	2.4	6.7
JNJ19573541	0,1779 0.2273	0.2026	0.0349	17.2	12.5
JNJ19574867	0.1225 0.1443	0.1334	0.0154	11.6	2.6

- Ill 2017202571 19 Apr 2017

JNJ19S74BS0	0,1300 0.1291	0.1296	0.0006	0.5	2.0
JNJ20&4&73&	0.1263 0.1336	0.1300	0.0052	4.0	2.1
JNJ21192730	0.2778 0.1326	0.2052	0.1027	50.0	12.9
JNJ21194667	0.2569 0.1219	0.1894	0.0955	50.4	10.6
JNJ21196227	0.1640 0.1158	0.1399	0,0341	24.4	3.5
JNJ24843611	1.1486 0.8970	1.0228	0.1779	17.4	130.5
JNJ24B43611	0.1358 0.1201	0.1280	0.0111	8.7	1.8
JNJ24326135	0.1257 0.1257	0.1257	0.0000	0.0	1.5
JNJ24B43S72	0.4676 0.4803	0.4740	0.0090	1.9	51.6
JNJ number	Raw Data	Average	S.O.	%cv	% Control
conditioned medium	0.6935 0.7803	0,7369	0.0614	8.3	104.8
no treatment	0.4735 0.6069	0.5402	0.0943	17.5	71.5
AA only + DMSO	0.1428 0.1656	0.1542	0.0161	10.5	6.3
AA + Wnt3a + DMSO	0.5702 0.8468	0.7085	0.1956	27.6	100.0
JNJ24S435B5	0.1599 0.2380	0.1990	0.0552	27.8	13.8
JNJ25753520	0,1287 0.1244	0,1266	0.0030	2.4	1.6
no cells	0.1241 0.1100	0.1171	0.0100	8.5	0.0
JNJ25753403	0.1235 0.1152	0.1194	0.0059	4.9	0.4
JNJ25757173	0.1199 0.1278	0.1239	0,0056	4.5	1.1
JNJ257S7173	0.1174 0.1162	0.1168	0.0008	0.7	-0,1
JNJ25757238	1.1100 0.9464	1.0282	0.1157	11.3	154,1
JNJ25758707	0.1247 0.1115	0.1181	0.0093	7.9	0.2
JNJ257ES785	0.2640 0.1688	0.2164	0.0673	31.1	16.8
JNJ257S8350	0.2313 0.1307	0.1810	0.0711	39.3	10.8
JNJ25758863	0.8639 0.9218	0.8929	0.0409	4.6	131.2
JNJ25873419	0.2540 0.2320	0.2430	0.0156	6.4	21.3
JNJ2S88T537	0.1809 0.3077	0.2443	0.0897	36.7	21.5
JNJ259DO641	0,1892 0.1872	0.1882	0.0014	0.8	12.0
JNJ2590O654	0.1967 0.2492	0.2230	0.0371	16.7	17.9
JNJ25900706	0.3346 0.1619	0.2483	0.1221	49.2	22.2
JNJ26047723	0.1106 0.1138	0.1122	0.0023	2.0	-0.8
JKJ26054912	0.1224 0.1445	0.1335	0,0156	11.7	2.8
JNJ26064S71	0.1312 0.1270	0.1291	0.0030	2.3	2.0
JNJ26067626	0.1653 0.2114	0.1884	0.0326	17.3	12.0
JNJ260676S2	0.1732 0.1467	0.1600	0,0187	11.7	7.2
JNJ26069901	0.1618 0.2754	02186	0.0803	36.7	17.2
JNJ26077883	1.0006 0.9631	0.9819	0.0265	2.7	146.2
JNJ26116922	0.6472 0.4319	0.5396	0.1522	28.2	71.4
JNJ26120601	0.1539 0.1469	0.1504	0.0049	3.3	5.6
JNJZ612O614	0.1127 0.1309	0.1218	0.0129	10.6	0.6
JNJ26128726	0.6887 0.5860	0.6374	0.0726	11.4	88.0
JNJ2G1304Q3	0.1141 0.1094	0.1118	0,0033	3.0	-O’;9
JWJ26134771	0.2774 0.1690	0.2232	0.0767	34.3	17.9
JNJ26150202	0.9482 1.1150	1.0316	0.1179	11.4	154.6
JN J26153647	0.7687 0.6804	0.7246	0.0624	8.6	102.7
JNJ2615B015	0.7125 0.3347	0.5236	02671	51.0	68.7
JNJ26158054	0.1446 0.1221	0.1334	0.0159	11.9	2.7
JNJ26158093	1.0968 1.3108	1.2038	0.1513	12.6	183.8
JNJ26158108	0.3167 0.3415	0.3291	0.0175	5.3	35.8
JNJ26161343	0.1261 0.1144	0.1203	0.0083	6.9	0.5

-1122017202571 19 Apr 2017

JNJ26170794	0.2223 0.2930	0.2577	0.0500	19.4	23.8
JNJ26170S20	0.1265 0.1236	0.1251	0,0021	1.6	1.3
JNJZ6170833	1.1940 0.9431	1.0686	0.1774	16.6	160.9
JNJZ61770B6	1.0689 0.6879	0.8784	0.2694	30.7	128.7
JNJ26177762	1.0444 0.7603	0.9024	0.2009	22.3	132.8
JNJ261B4457	0.1443 0.1209	0.1326	0.0165	12.5	2.6
JNJZ6219050	0.1152 0.1309	0.1231	0.0111	9.0	1.0
JNJ number	Raw Data	Average	S.D.	%CV	% Control
conditioned medium	0.7590 0.7451	0.7521	0.0098	1.3	98.0
no treatment	0.5687 0.4490	0.5089	0.0846	16.6	60.4
AA only + DMSO	0.1988 0.1522	0.1755	0,0330	18.8	8.9
AA + Wnt3a + DMSO	0.6837 0.8460	0,7649	0.1148	15.0	100.0
JNJZ6219063	0.1911 0.1101	0.1506	0.0573	38.0	5.0
JNJ26220454	0.2772 0.1151	0.1962	0.1146	58.4	12.1
no celts	0.1278 0.1084	0.1181	0.0137	11.6	0.0
JNJ26241774	0.1443 0.2120	0.1782	0.0479	26.9	9.3
JNJ26241917	0.4413 0.2238	0.3326	0.1538	46.2	33.2
JNJ26243204	0.1098 0.1085	0.1092	0.0009	0.8	-1.4
JNJ26247143	0.1389 0.2147	0.1768	0.0536	30.3	9.1
JNJ2624S729	0.1852 0.1342	0.1597	0.0361	22.6	6.4
JNJ26261105	0,1114 0.1295	0.1205	0.0128	10.6	0.4
JNJ26361712	0.5375 0.6158	0.5767	0.0554	9.6	70.9
JNJ26361725	0.1259 0.1441	0.1350	0.0129	9.5	2.6
JNJ263E6730	0.1206 0.1312	0,1259	0.0075	6.0	1,2
JNJ26367991	0.2269 0.2857	0.2563	0.0416	16.2	21.4
JNJ2E3C7931	0.1140 0.1079	0.1110	0.0043	3,9	-1.1
JNJ26399906	0.9589 0.8868	0.9229	0.0510	5.5	124.4
JNJ2G399906	1.0442 0.9622	1.0032	0.0580	5.8	136.8
JNJ26339945	0.1961 0.1735	0.1848	0.0160	8.6	10.3
JNJ26399971	0.5732 0.5216	0.5474	0.0365	6.7	66.4
JNJ2E399984	0.1273 0.1217	0.1245	0.0040	3.2	1.0
JKJ26399997	0.5932 0.6671	0.6302	0.0523	8.3	79.2
JNJ26400049	0.1444 0.1368	0.1406	0.0054	3.8	3.5
JNJ264E3197	1.0786 1.0891	1.0839	0.0074	0.7	149.3
JNJ264B3310	0.5418 0.2338	0.3878	0.2178	56.2	41.7
JNJ26483223	0.1268 0.2052	0,1660	0.0554	33.4	7.4
JNJ264S3236	0.1169 0.1184	0.1177	0.0011	0.9	-0.1
JNJ26483249	0.8618 1.0400	0.9509	0.1260	13.3	128.8
JNJ26483249	0.8430 1.0187	0.9309	0.1242	13.3	125.7
JNJ264S3262	0.3659 0.3168	0.3414	0.0347	10.2	34.5
JNJ26511901	0.9184 0.8116	0.8650	0.0755	8.7	115.5
JNJ26511927	0.2384 0.3156	0.2770	0,0546	19.7	24.6
JNJ26511953	0.2297 0.1469	0.1883	0.0585	31.1	10.9
RWJB7694	0.1955 0.1256	0.1606	0.0494	30.8	6.6
RWJ676940	0.1658 0.1704	0.1681	0.0033	1.9	7.7
RWJ677545	0.1399 0.1303	0.1351	0.0068	5.0	2.6
RWJ678936	0.1234 0.1236	0.1235	0.0001	0.1	0.8
RWJ680665	0.1397 0.2147	0.1772	0.0530	29.9	9,1
RWJ6B0667	0.1218 0.1310	0,1264	0.0065	5.1	1.3
RWJ6fiOG68	0.1456 0.1981	0.1719	0.0371	21.6	8.3

- 113 2017202571 19 Apr 2017

RWJ6B06G9	0.5412 0.1898	0.3655	0.2485	68.0	38.2
RWJ68085B	0.1996 0.1245	0.1621	0,0531	32.8	6.8
RWJeaoBso	0.1418 0.2014	0.1716	0.0421	24.6	8.3
RWJ6B0S79	0,1106 0.1197	0.1152	0.0064	5.6	-0,5
RWJ680885	0.1159 0.1272	0.1216	0.0080	6.6	0.5
JNJ number	Raw Data	Average	S.D.	%cv	% Control
conditioned medium	0.8077 0.7210	0.7644	0.0613	8.0	74.7
no treatment + DMSO	0.4638 0,4073	0,4356	0,0400	9,2	36.7
AA/Wnt3a	0.8466 0.9935	0.9830	0.2592	26.4	100.0
JNJ10222784	0.8095 0.9055	0.8575	0.0679	7.9	85.5
JNJ10222927	0.3519 0.4708	0.4114	0,0841	20.4	33.9
JNJ1 0231273	0.1609 0.1275	0.1442	0.0236	16.4	3.1
JNJ10259847	0.5020 0.2733	0.3877	0.1617	41.7	31.2
JNJ10259847	0.3413 0.4146	0.3780	0.0518	13.7	30.1
JNJ17154215	0.1176 0.1174	0.1175	0.0001	0.1	0.0
JNJ17154215	0.1148 0.1410	0.1279	0,0185	14.5	1,2
JNJ17157659	0.2394 0.2450	0.2422	0,0040	1.6	14.4
JNJ17163042	0.3672 0.3098	0.3385	0.0406	12.0	25.5
JNJ10166565	0.2722 0.1593	0.2158	0.0798	37Ό	11.3
JNJ17174664	0.5079 0,4349	0.4714	0.0516	11.0	40.9
JNJ17187027	0,1076 0.1168	0.1122	0.0065	5.8	-0.6
JNJ17187053	0.2569 0.2151	0.2360	0.0296	12.5	13.7
JNJ17193774	0.2846 0.4376	0.3611	0.1082	30.0	28,1
JNJ17200976	0.1168 Q.1136	0.1152	0.0023	2.0	-0.3
JNJ17205955	0.1168 0.1152	0.1160	0.0011	1.0	-0.2
JNJ17205955	0.1137 0.1195	0.1166	0.0041	3.5	-0.1
JNJ17205994	0.1154 0.1152	0.1153	0.0001	0.1	-0.3
JNJ17226703	0.2188 0.2353	0.2271	0.0117	5.1	12.6
JNJ17982133	0,4588 0.2521	0,3555	0.1462	41.1	27.5
JNJ17989049	0.3081 0.1961	0.2521	0.0792	31.4	15.5
JNJ number	Raw Data	Average	S.D.	%cv	% Control
conditioned medium	0.7914 1.1189	0.9552	0,2316	24.2	93.3
no treatment	0.4215 0.5259	0.4737	0.0738	15.6	39.8
no cells	0.1152 0.1160	0.1156	0.0006	0.5	0.0
AA/Wnt3a	0.7168 0.8836	1.0151	0.2016	19.9	100Ό
RWJ680991	0.2882 0.2308	0.2844	0,0499	17.6	18.8
RWJ680992	0.3049 0.2845	0.3127	0.0282	9.0	21.9
RWJ6B0993	0.5403 0.2570	0.3855	0.1332	34.6	30.0
RWJ681140	0.7323 0.3034	0.4388	0,2041	46.5	35,9
RWJ681142	0.1185 0.1216	0.1199	0.0018	1,5	0.5
RWJ681146	0.2496 0.2683	0,2302	0.0376	16.3	12.7
RWJ631945	0.1548 0.1356	0,1513	0.0134	8.8	4.0
RWJ6819A	0.1555 0.1450	0.1581	0.0161	10.2	4.7
RWJ682205	0.2347 0.1920	0.3785	0.2589	68.4	29.2
RWJ447228	0.1842 0.2093	0.3793	0.2585	68.2	29.3
RWJ675430	0.7223 0.8707	0.4291	0.2452	57.2	34.8
RWJ355923	0.6268 0.3192	0.3354	0.1667	49.7	24.4

- 1142017202571 19 Apr 2017

Table VII: Effects of Inhibitors of GSK-3B Enzyme Activity on the proliferation of human embryonic stem cells,

List Strong Hits >=120% control

JNJ Number	% Control Value
RWJ352628	195.3
JNJ26158093	183.8
RWJ35325B	180.4
JNJ26170833	160.9
JNJ2G150202	154.6
JNJ257S7238	154.1
JNJ19410833	151.6
JNJ264S3197	149.3
JNJ18157711	148.0
RWJ676139	147.3
JNJ26077883	146.2
RWJ352190	142.3
JNJ2639990G	136.8
JNJ19370026	136.1
JNJ26177762	132.8
RWJG7643Z	131.6
JNJ257588&3	131.2
RWJ67543Q	130.9
JNJ24843G11	130.5
RWJ675605	129.0
JNJ264S3249	128.8
JNJ26177086	128.7
JNJ2G483249	125.7
JNJ2639990G	124.4
RWJ675948	120,0

List Moderate Hits
60-120% control
JNJ Number	% Control Value
JNJ26511901	115.5
RWJG76431	113,8
RWJB73515	108.3
JNJ26533156	105.5
JNJ26153647	102.7
RWJ676639	93.0
JNJ26128726	88Ό
JNJ10222784	85.5
RWJ67657	84.7
JNJ26512005	84Ό
JNJ19410859	80.3
JNJ26399997	79.2
RWJ676137	77.5
RWJG752G0	76.9
RWJ355923	76.7
RWJ675266	75.2
JNJ26116922	71.4
JNJ2G3G1712	70.9
RWJG70804	70.7
RWJ675B81	69.9
JNJ26153015	68.7
RWJ352244	68.2
RWJS74239	67.4
JNJ2G399971	66.4
JNJ2B714134	63.3
JNJ26533065	61.1

- 115 Table VIII: Dose-DEPENDANT Effects of Inhibitors of GSK-3B Enzyme

Activity on the proliferation of CELLS OF THE human embryonic stem cell

LINE Hl.

JNJ 18157638

2017202571 19 Apr 2017

CencelitlatipU

JNJ17189731

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Cell number ! SD

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0.600

0,034

0.294

0.076

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0-826 0.,030

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1.106 0.056

0.BS9 0,213

0.6S0 0.079

JN12&150702

Cell number I SD

0.436 0.690

0.7ΕΘ 0.490

1001 0129

1.150 0.043

0639 0 240

0617 ..D232 iio·? ήί.'ϊχί

JNJ2t533065

Cell nunilrerl SD

U.UJ1 ,0,027 ......fiiO'......‘;CL354.

tt.*¹ ’“I r·'' “•f-l·

16S3 :0.170

.....‘ΐ.|$.......0.101

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1.049 .0.063

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0.742

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10.127

20.020

JNJ26433219

JNJ17225e?1

JNJI7229453

Cell nmiihor SD

06B 0.074 ¢173.......0.207 ' ίί420......o.32E ¢850........0 736

0.910 D.0S1

0,860 0,131

0.742 0.051

JNJ26170B33

Cell number SD

0.129 0.170

D.530 0.030

1174 GDIS

113 0.057

IBS 0 041

1,-153 0.102

9M 'doss

JHJ28533156

Cell liiinibei j SD

3S7 Π 0?3 1213 __ 0177 1,.706 0.142

Cell number | SO

Cell number | SD

0.290 0.307 /0,458 /',U3^3' □ 000 oosg □ CEO □.067

0002 ' Uli

0640

0,739

0605

0,705

0.774

0.104 aim

0.036

0,034

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0.932

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0.D25

0,038

0.005

JNJ261770BS

JHJZ6177762

Cell number SD

0,412 0.0Θ1

1.128 0,026

1.031 0.217

0.914 0 100

0601 0.136

0.785 0.121

0559 0 060

JNJ2C714194

Cell number SD ,,,0.052.......U.OcZ

Ϊ'.ΪΒΙ “^J,B.J34 T ?53 ' Otcti Liia'.......n.W

1741 0.031

1.041 0 007

Cell number [ Sil

996 '0.908 1 005 1.200 1 111 0.859 '0612 :0.246 :0.179

6.066 :0.085 =0.300 iO.DS4 io.059

JNJ3026562

Cell immbei | SD

053 □ 024 .0.905. ,0.036

Tiff

Liei'. ';'O062 ί?3Ϊ'-'^Ϋθΐ52

1.216 0.007

IOi ,0065

- 116Activity on the DIFFERENTIATION of CELLS OF THE human embryonic stem cell LINE Hl.

jHjiaisreaa

2017202571 19 Apr 2017

Table IX: Dose-DEPENDANT Effects of Inhibitors of GSK.-3B Enzyme

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0.341 0.105

JHJ2S15TO15

Snx17 intensity | SD

W·

0200 0.732 0324 0+3) bis

0+01 + C20 0157 0.065 6.113 □42Z 'ΟΌ+Γ

S4«i7'lh»^SrSD oooor 0.010

0+35 0.049

1336 aisg

.......Ϊ2'-·ϊ3.........0.030

0.997

731 o+a

JHJ2S512O05

0CS5 0.172

0Γ27

Snx17 Intensity SO

0+0Q

DOOQ

0270'

0+70

0970*

0.742

0.533

0.000

0382

6.434

0.021

0.043

0.643

SoitiT lritjanfeHB l' SO aos itust .......Tig% <$£@6 '’'W'.......® mjaassiar “Qffii

0.W

0.497 ' 0.993 1 061 0.937

863

Mrtnasffl

Soit171nt6n«1te[~SB

w

1.0EB

1»

CO43 'aion am?· '0,015

Saxff InfeieBy I SO

C0E4 0.063

Sajtff JnmgHvI SBlies w

Lttj.......:ο:ΰιΰ’

ϊ.-ite 'o,D3i i ii»' +.006

0.031 '+033 0.043 :0 0=6

0,739 +,074

JHJ17725871

Scx17 Imfcrtajty | SP

SanK HKaTeajij SB +W

TffiS......Ϊ0.002

'.38 - <063 /+071 [ o ni

..0.167

70.032

0.767

0.692 +651

JHJ177a45g

0.076 0 14.4 0.065 0 136

062

JHJ2615O2A2

Sax17 iMtfeasity | SO

0.491

0.150

0,800

0310

0+67

0.516 a/ta [□.681 +224 :+,201„,gM, .pi® ’tSW

SoirtT intauHte so

„.....mjlJM

14+15 '+,174

+.233

1055

0.569

0.655

0.451

046 0,124 0 118 0.443 +jwi ^169^:

1.?¥3' 3 282 :+8^: ''+062 trass' +.176

O.E%B ^; 0.015

0.267 :0230

Snx17 InlgnUty | SO

S^inriftiid^rSP

028.1 :0350:

0.46.0 +.183

1.019' 2.132

0960.'......++06 i'.'oso'

SSsF^i+Sb:

-100255331¾

Spxt7lnteii8lty I SD .0+02.^., +H}1 ' .........0<ϊ i:azs· 0.078 i .172 0.01 =

1.107 016$

1.060 0.125

0.402 '+005

586

0,786

0,700

O.TBZ :0229 + 041 + 185 +,051 +,132 '0 061

JNJ2&177OOT

SnifIT Interwjtyl SD '0 059· 0 036 + ‘31 jS® _ άζ» ιά« +W j&lis

.......+¾.......W

.....0.000.. 0-001

o.g ' O+ffl”

587 0.655 0.753 n’mc

W2617776?

0.035

0.034

0.023 : 0.001 '0+98 6.043

0.330

0,916 vast +3iS

S714194 §2ϊί g’-ipBiwiafa j W

Sitx17 Intensity I SD

0.701

Τ’!

o ?2a ...... 1.+59

+.136 i.Sfis' ......iasa

0.307 0.146 0 + 19 +.003

70186 + +Γ

......nits»......'..SOT'250· O'77 rise.........“lafifif

1ΛΪ2

SUXHT lHtetliity j 50

0,313

1.140

0.998 +'oce +036 + 006

- n+fiL TwiT *20 i.tzi 1.241 1.23, 1.034 , :0+46 +0,070 [0038 ..0.010-7 To+ai

0014

+.008

- 117Activity on the proliferation of CELLS OF THE human embryonic stem cell

LINE H9.

JNJ1S15769S

2017202571 19 Apr 2017

Table X: Dosc-DEPENDANT Effects of Inhibitors of GSK-3B Enzyme i Ctneenftatlbn

JHJ17153796.

JhSL

ί.........f.....

........3^'

1.25 /'''0.636' 0 313 0.156

Cuncentraflon

IiiMI s'

25

0.625

0313

Soil cflntraBbrT

JH*L

£.

3i

LSS

......’WT ~

.......BW'

ICencerTtrirtlon

B>2L tfl.

:.......

p.3',3 ΐ 0.156

CeUtlurnlier I 5D

0,131 0.209 aur.......b.141 /0.140 0 112

0.307' '' o.isa' π Via

o.os3 acts

0.3® 0.B01

JHJ2S1W15

Cell number SD

0:001

0023 ' o eai toil oi®y D-W.

0.000 bi;?4i

0.223

0.4B1

o.ioa

0.L33

JHJ13OTI076

Cellntnnbcr .| SD

0.033 O,£OO

0.032 OCH lissa.........OJ '

.................0032

Sir

0.330 ,,,,,,,,¾¾..

jifcaesiaiiB

Celliwrobgi 1 SO ,0,097.........O....

' nS? inn ,0:0)1 fegsi

0.090. ''ftW

8.8»”' ®

j.$4' 0(148

O.T® 0009

Cell number SD

JHJ17189731 Cell number | 5ΪΓ ___-ttIL,

Έΐ®;......

USS. :iW7

3.86-7 0.070 ole® 0.109

JNJ17223375

number	I SD	Cell number	I SD
0.123	e loe	0.770	0.077
0,954	:0 146	0.456	o.on
0.796	0 101	0 384	0 247
0 54)	;0.Q51	0,325	;O.oq:
0332	‘0/049		0.009
0.206	10,005	0 172	0.071
0.142	10,039	0.138	0,048

J HJ 76483197

Call number | SD

0.785 10.192

1.067 ' ϊ .369

1.477

0.099

0.540

0.206

JHJ2615O2D2

Cell bum her

JHJ2S483249

Cell number

SD :0336 :6:025 io 1.47 Ϊ0 108 :0094 :0069 ,0.134 0 087

SO

0462 .:0.094

0.433 .0050 £21 :0.229

O&3........00.030

JK& /Q,tg2 S,t§2 0 025 +03057.......j0M

JHJ26573065

Sell number ttOqtp .......af

0.2CB' +01320

0388-..........0'.0t9

0.304 :0.113

12E7 ills

□.ί® 'aose

0X47 0.067

J »426170833

Cell numhei~Γ$5~

0CC2 :0.001,

1.325 0015

i.a&

.kWi ______

3®, Jy²

.................ii

JHJ26S33156

Cell number

JHJ17275B71

Cell Biimher | S0~

JNJ172J445B

Cell number

SD

377 0,336+ οϊέκ 0.222 0.2® 0,200 0 174 _^{:D 040}|'.1081 iooie 0035 :0,096 IDS®,

.....icosT

0.000 0 052 ' 0003 ' 0.1DS 0 169 0.119 0.067

0.000

O.OlB 0303 0.003

0.041 :0,02b ' 0-110

JHjg81T7O»___

Cell ΰϊιι~Μΐ>βϊ~ Τ$ΪΓ

JNJ26177762

Cell number SD

1Λ17

D.733 t&c

JNJ26711194

10,043	1.022	0.422
:0 030	1.201	0 1Π9
:□ .122	,.197	0.D6B
.ί.ρ.θ’β	1039	0,213
:0,072	0,086	n mi
'0,000	0.437	LkUbb
•0 015	0.276	0 043

0.042

0.862

0.098

0.012

Cell number I SP

JNJ3026S82

Cell number I SO

W	IqM	O&5
0.2&f	io.®	0.914-
0 086	:0033	0.267
□.063	: 0.004	0.210-

₄G,EW4, _t	..j®®® «Ma ..J.ER·	0 0,7	:0.001 ^J“‘i0.TO
0 033	:0.037
cSsK	ifl.OTG	0044	:0.028
W*S#T	4&,0ffi1	0.100	:0.033
03i7	ta	0.057	0-032
0,132	'0,014	0 070	0.043

- 118Activity oil the DIFFERENTIATION of CELLS OF THE human embryonic stem cell LINE H9.

JH.H8157698

2017202571 19 Apr 2017

Table XI: Dose-DEPENDANT Effects of Inhibitors of GSK-3B Enzyme

Conceftitation atUiijante?

awayttam

JNJi72233?5

IgM] SowtT IhtetBriW | 5D 3t»17 Intensity [ SB

o.isz

:........0213.....

:......ΉΜ.....

' 1 <5,.....

75·

S : Con ceiiitrirthHi a 157 ; orb : ' 0 625......

25 : 2.5

i.........A...............

: ID ' ' |g»l

0:315 0355 u tai 0,105 0.121

JHJ26158015

o.oeg

0.141 o 153

0.311 0453 ' 1.012 OJ9B6 ' 0.430 0.002

W8 *t(.0C5

0.076'

DW

0423

0.HS

JKJ2HE31S7 :5^17 Intensity f SD SoxlZ Intensity] 50

0:354 0.044

5-1 01.:62

0.505.......jr.2

D.943 0.419

0.659 0.23B

0.019 0C19 ttiioi acai

JHJ1B37M26

Sw17 lctetW~r^P

SoxIT IHffeiwIly | SD~ Sexi? Intinirily I SD~ ; 0,093 εοοϊσ' Τι® ftLtiia

1017,

0:158 :3.432 0.111'

3,022 0,005

001254*3249

0.149 0399 0.673 1 110 0.857 0,134 ''□129

J141211150202

0.063 bibs o'.IS?

01W2

0.012

0.0b?

0.037

SaxlZ Intensity SO

..0116

0.459 “otSt Ti 14 0.140 :0,047 .cozi w

TIDES .10079 ;ooee :0,110

JHJ17225B71

4ox11 Intensity I so Sum? IntsTijfty | SD i}.135 3 051¹ ' ':Ef:O ^:0.01S

'.O2^; 2 0.134

Εζ.2Ε2 :0.,03

0,209 00«

0.,£4 :,023

JHJ261703»

Sax171ntawt}tvl SO

Snx17 Intensity | SD

096 0.025

........b.'nb......:'o,'b3b

l.'jy&t :0.013 '02MT.....:0J»2

0297' Π 235

353 0.030

0694 0.123

JHJ1722445B

132 0146 Γ 196 0129 0177 0174 0200

DOES

0,0(6

0084'

0.029

0,030

0070

0022

Sexi/ Intensity [ SO

0.039 0 070 0137' 0.075 0 053 0,038 boob

0.D10 :0.027 :0.049 0.017 :0.005 ;ο,οΰι a'.roo

JNJ26T77O8S SohT?1 «itsre^ty l SO* _ - ....

~SnxT7~l'niansitvlSD*

0:001

0009 ioSSb..........o'®

0.202

D.2S9

0.300.

O.DCD inee io,tob 'flW |Β®

Π234 10,076

E:342 :0 028 ow aiao Uss.......3.C43 aity.......fcrai oa, raffi'

p.ppl abba up® aza¹ □.'crzS' ' b.izS 0087 □.000

0.002

0.019

0-®5

0.000:

11759 :0.051

054 il.W

b.o'73......:o.oes ' 0.053 ...... 0.775 ' 0.2 VJ :0.001 ^! 0.003

- 119- 120 -

Claims

CLAIMS:

1. A method for differentiating pluripotent stem cells into definitive endoderm cells comprising treating the pluripotent stem cells with medium supplemented with a compound of formula (III)

2017202571 20 Apr 2017 wherein the compound is selected from the group consisting of:

a. 6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)-2pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile;

b. 3-[1-(3-hydroxy-3-methyl-butyl)-1H-indazol-3-yl]-4-(1-pyridin-3-yl-1H-indol-3-yl)-pyrrole2,5-dione;

c. 3-[1-[3-[(2-hydroxyethyl)methylamino]propyl]-1H-indazol-3-yl]-4-[1-(3-pyridinyl)-1 H-indol3-yl]-1 H-pyrrole-2,5-dione;

d. 14-ethyl-6,7,9,10,13,14,15,16-octahydro-12H,23H-5,26:17,22dimethenodibenzo[k,q]pyrrolo[3,4-n][1,4,7,10,19]dioxatriazacycloheneicosine-23,25(24H)dione;

e. 14-(2-thienylmethyl)-6,7,9,10,13,14,15,16-octahydro-12H,23H-5,26:17,22di(metheno)dibenzo[k,q]pyrrolo[3,4-n][1,4,7,10,19]dioxatriazacyclohenicosine-23,25(24H)dione; and

f. 14-(1-naphthylmethyl)-6,7,9,10,13,14,15,16-octahydro-12H,23H-5,26:17,22di(metheno)dibenzo[k,q]pyrrolo[3,4-n][1,4,7,10,19]dioxatriazacyclohenicosine-23,25(24H)dione.
2. The method of claim 1, wherein the method further comprises culturing pluripotent stem cells prior to treating the cells with the compound of formula (III).
3. The method of claim 1 or claim 2, wherein the pluripotent stem cells are treated with the compound of formula (III) for 12 to 48 hours.
4. The method of claim 1 or claim 2, wherein the pluripotent stem cells are treated with the compound of formula (III) for 1 to 72 hours.

- 121 2017202571 20 Apr 2017
5. The method of any one of claims 1 to 4, wherein the compound of formula (III) is used at a concentration of 100 nM to 100 μΜ.
6. The method of any one of claims 1 to 4, wherein the compound of formula (III) is used at a concentration of 1 μΜ to 10 pM.
7. The method of any one of claims 1 to 6, wherein the pluripotent stem cells are human stem cells.
8. The method of claim 7, wherein the human stem cells are human embryonic stem cells.
9. The method of any one of claims 1 to 8, wherein the compound of formula (III) is 6-((2[[4-(2,4-dichlorophenyl)-5-(4-methyl-1 H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3pyridine-carbonitrile.
10. The method of any one of claims 1 to 8, wherein the compound of formula (III) is 3-(1-(3[(2-hydroxyethyl)methylamino]propyl]-1 H-indazol-3-yl]-4-[1 -(3-py rid i ny I)-1 H-i n dol-3-y I]-1 hlpyrrole^,5-dione.
11. The method of any one of claims 1 to 8, wherein the compound of formula (III) is 3-(1-(3hydroxy-3-methyl-butyl)-1 H-indazol-3-yl]-4-(1 -pyridin-3-yl-1 H-indol-3-yl)-pyrrole-2,5-dione.
12. The method of any one of claims 1 to 8, wherein the compound of formula (III) is 14ethyl-6,7,9,10,13,14,15,16-octahydro-12H,23H-5,26:17,22-dimethenodibenzo[k,q]pyrrolo[3,4n][1,4,7,10,19]dioxatriazacycloheneicosine-23,25(24H)-dione.
13. The method of any one of claims 1 to 8, wherein the compound of formula (III) is 14-(2thienylmethyl)-6,7,9,10,13,14,15,16-octahydro-12H,23H-5,26:17,22di(metheno)dibenzo[k,q]pyrrolo[3,4-n][1,4,7,10,19]dioxatriazacyclohenicosine-23,25(24H)dione.
14. The method of any one of claims 1 to 8, wherein the compound of formula (III) is 14-(1naphthylmethyl)-6,7,9,10,13,14,15,16-octahydro-12H,23H-5,26:17,22di(metheno)dibenzo[k,q]pyrrolo[3,4-n][1,4,7,10,19]dioxatriazacyclohenicosine-23,25(24H)dione.

- 122 2017202571 20 Apr 2017
15. The method of any one of claims 1 to 14, wherein the medium is further supplemented with activin A.
16. The method of claim 15, wherein the medium is supplemented with 1 pg/ml to about 100pg/ml of activin A.
17. The method of claim 15, wherein the medium is further supplemented with serum.
18. The method of claim 17, wherein the medium is supplemented with 2% to 5% of serum.
19. The method of any one of claims 1 to 8, wherein the compound of formula (III) is an inhibitor of glycogen synthase kinase 3β enzyme activity.
20. A method of differentiating pluripotent stem cells into pancreatic endoderm or pancreatic endocrine comprising differentiating pluripotent stem cells into definitive endoderm cells using the method of any one of claims 1 to 19 and differentiating the definitive endoderm cells into pancreatic endoderm or pancreatic endocrine cells.
21. The method of claim 20, wherein the method comprises differentiating pluripotent stem cells into pancreatic endocrine and wherein the method further comprises differentiating pancreatic endoderm cells into pancreatic endocrine cells.
22. Definitive endoderm cells when produced by the method of any one of claims 1 to 19.
23. Pancreatic endoderm or pancreatic endocrine when produced by the method of claim 20 or claim 21.

1/20

2017202571 19 Apr 2017

Figure IA

1.5 φ > o 1.0

S 'T u £ ti in c 2 o o . . φ α o 0,0 O'

Cell number min iu.

0.157 0.313 0.625 1.25 2.5 5 10

Concentration of JNJ17189731 [μΜ]

Figure IB

Sox17 Expression ms * « c <2 8 o

Ο Q. 0 £

1.5

1.0

0.5

0.0 nnim di

0.157 0.313 0.625 1.25 2.5 5 10

Concentration of JNJ17189731 [μΜ]

2/20

2017202571 19 Apr 2017

Figure 2A

Cell number

Φ > 2 ·- Γ « £ 0 o φ α o φ

>

(0

J

0.157 0.313 0.625 1.25 2.5 5 10

Concentration of JNJ17163796 [μΜ]

Figure 2B *^w £ o Φ Q. X.

Φ > 2

Sox11 Expression .11.

ί

0.157 0.313 0.625 1.25 2.5 5 10

Concentration of JNJ17163796 [μΜ]

3/20

2017202571 19 Apr 2017

Figure 3A

Cell number ^21.0 ο α o 0.0

QC

Figure 3B

0.157 0.313 0.625 1.25 2.5 5

Concentration of JNJ17223375 [μΜ]

Sox17 Expression >£$ ¹⁰

2*0 0.5 ο α o

0.0

γΕί E-ι ri E-i - r, r ι 1 1 _

0.157 0.313 0.625 1.25 2.5 5 10

Concentration of JNJ17223375 [μΜ]

4/20

Figure 4A

Cell number

2017202571 19 Apr 2017 * ·-1.5

1-0 * « c 0.5

- 0 0 nn

Φ 0. 0 U.U £

it ~L PI

0.157 0.313 0.625 1.25 2.5 5

Concentration of JNJ18157698 [μΜ]

Figure 4B

Φ >

m * = ί 0 o ®Q,0 £

1.5

1.0

0.5

0.0

Sox17 Expression

5 Η ή γΞπ ι_

0.157 0.313 0.625 1.25 2.5

Concentration of JNJ18157698 [μΜ]

5/20

2017202571 19 Apr 2017

Figure 5A

Figure 5B

Sox17 Expression o

* u Φ > p

2 5 £

12 ο o o a υ £

1.5

1.0

0.5

0.0

14 a i j

0.157 0.313 0.625 1.25 2.5

Concentration of JNJ26158015 [μΜ]

6/20

2017202571 19 Apr 2017

Figure 6A

Cell number

5».

> .t £

Hi po

2.0

1.5

1.0

0.5

0.0 fb fl 1 r!J J

0.157 0.313 0.625 1.25 2.5 5 10

Concentration of JNJ26483197 [μΜ]

Figure 6B * Φ = >2 ra £ +> ^w E o o ⁰ΐαϋ X

Sox'! 7 Expression

1.5 -, 1.0 0.5- 0.0 fk- 1 □lj 1

0.157 0.313 0.625 1.25 2.5 5 10

Concentration of JNJ26483197 [μΜ]

7/20

2017202571 19 Apr 2017

Figure 7 A

Cell number s„_¹·⁵ > 5 S¹'° • o o0.5

Φ Q. 0 * 0.0 l El LA ί·

0.157 0.313 0,625 1.25 2.5 5

Concentration of JNJ26483249 [μΜ]

Figure 7B

Sox17 Expression ** fl) _ Φ > 0 >5$ * tt C 12 ο o Φ Q, o Οΐ

1.5 1.0 0.5 0.0 rn *

I 1¾ A

I I

0.157 0.313 0.625 1.25 2.5 5

Concentration of JNJ26483249 [μΜ]

8/20

2017202571 19 Apr 2017

Figure 8A

S.J·⁵ >S^°

5 ο O0.5 Φ Q. 0

1C 0.0

Cell number

0.157 0.313 0.625 1.25 2,5 5

Concentration of JNJ 10220067 [μΜ]

Figure 8B

Sox17 Expression o

* ¢Φ > 0 t « c

JS ο o

0 QO

1.5

1.0

0,5

0,0

-E- r? I* -Ϊ- ή L Π

0.157 0.313 0.625 1.25 2,5 5 10

Concentration of JNJ10220067 [μΜ]

9/20

2017202571 19 Apr 2017

Figure 9

CXCR4 Expression

10/20

2017202571 19 Apr 2017

Figure 10-A

CXCR4 mRNA level

11/20

2017202571 19 Apr 2017

Figure 10-B

12/20

2017202571 19 Apr 2017

Figure 10-C

Soxi 1 mRNA level

5μΜ

13/20

2017202571 19 Apr 2017

Figure 11-A

Oonlroi

ΙμΜ' 3μΜ ΙμΜ 3μΜ ΙμΜ 3μΜ ΙμΜ 3μΜ 1μΜ 3μΜ ΙμΜ' ϊμΜ ΙμΜ

JNJ1W5 ¹ 0

1D2OT JI

14/20

2017202571 19 Apr 2017

Figure 11-B

Pdx1 expression

15/20

Ο (Μ

Lh

Ph

Ο

Figure 12

IT) (Μ

Ο (Μ

Ο (Μ

-2.5

S2.0 »15 « ΙΛ “05 ο

OmiLMhJ

NegaSve tefte 3μΜ ΙμΜ 3μΜ ΙμΜ 3μΜ ί ΙμΜ 3μΜ' ΙμΜ 3μΜ 1μΜ 3μΜ ΙμΜ 3μΜ 1μΜ ra <υ

LT

Cffliifol

058015 «3137 «2Μ

3μΜ >163756

16/20

2017202571 19 Apr 2017

Figure 13-A

Cell number

17/20

2017202571 19 Apr 2017

Figure 13-B

Insulin expression

2 2.5 j 2.0 £ 1.5 S 1,0 oQ.5 ζο.ο >

+- ω

cc a

>

ra co

a)

Li I.____Ll .1 ii

Φ >

<0

O

CL

3.

1 I i ϊ co τ co -3.

1 μΜ

Control JNJ17189731

JNJ18i57698! ' JNJ17223375 «19? JNJW?

JNJ17163796

MM

18/20

JW17163796 «53249

19/20

20/20

2017202571 19 Apr 2017

Figure 15-B

Insulin expression