Feeder-based and feeder-free stem cell culture systems for stratified epithelial stem cells and related methods of use

Abstract

The present invention relates to a culture medium system useful for the isolation and epigenetically stable propagation of normal stem cells and cancer stem cells derived from epithelial cancers in cultures derived from stratified epithelial tissues. In certain embodiments, the culture system is a feeder-free system. TIFF2022551869000029.tif99140

Description

[Technical Field]

[0001] Priority claims This application claims the benefit of priority to U.S. Provisional Application No. 62 / 913,226, filed October 10, 2020, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Background of the Invention Stratified epithelium differs from simple epithelium in that it is multilayered. Thus, it is often found where the body lining must withstand mechanical or chemical stimuli, allowing layers to be abraded and lost without exposing the underlying epithelium. Cells flatten as the layers become more apical, but in the most basal layers, cells may be squamous or cuboidal.

[0003] Stratified epithelium includes columnar, cuboidal, and squamous types. Squamous epithelium lines surfaces such as the skin and alveoli, and is found to allow simple passive diffusion, which is also found in alveolar epithelium. Specialized squamous epithelium also forms the lining of cavities, for example, in blood vessels as endothelium, in the pericardium as mesothelium, and in other body cavities.

[0004] Cuboidal epithelial cells have a cube-like shape and appear square in cross section. The cell nucleus is large, spherical, and located in the center of the cell. Cuboidal epithelium is commonly found in secretory tissues, such as exocrine glands, or absorptive tissues, such as the pancreas and the lining of renal tubules, and is also found in the ducts of glands. The germinal epithelium covering the ovaries in women and lining the walls of the seminiferous tubules of the testes are also cuboidal. Cuboidal cells provide protection and, depending on their location and specialization, may actively or passively pump materials into and out of the lumen. Simple cuboidal epithelium typically differentiates to form the secretory and ductal portions of glands. Stratified cuboidal epithelium protects areas such as the ducts of sweat, mammary, and salivary glands.

[0005] Columnar epithelial cells are elongated and cylindrical, with a height at least four times greater than their width. The nucleus is elongated and usually located near the base of the cell. Columnar epithelium forms the lining of the stomach and intestine. Cells may possess microvilli to maximize surface area for absorption, and these microvilli may form a brush border. Some cells have cilia to move mucus in the function of mucociliary clearance. Other ciliated cells are found in the fallopian tubes, uterus, and central canal of the spinal cord. Some columnar cells are specialized for sensory reception, for example, in the nose, ears, and taste buds. Hair cells of the inner ear have stereocilia similar to microvilli. Goblet cells are modified columnar cells and are found between columnar epithelial cells in the duodenum. They secrete mucus, which acts as a lubricant. Simple, non-ciliated columnar epithelium tends to exhibit absorptive functions. Stratified columnar epithelium, although rare, is found in the lobar ducts of salivary glands, eyes, pharynx, and genitalia. It consists of a layer of cells overlying at least one other layer of epithelial cells that can be squamous, cuboidal, or columnar.

[0006] The isolation and long-term expansion of primary cells, specifically stem / progenitor cell populations, is a fundamental and important basic technology in various biological fields, including developmental biology and stem cell biology, as well as in medical science. Cells of stratified epithelial tissues are highly regenerative and are responsible for many human cancers and inflammatory / autoimmune diseases, but the cloning of epithelial stem cells has been limited due to the difficulty of maintaining these cells in an immature state. However, the inability to maintain the immaturity of stem cell populations in vitro limits the long-term expansion of various types of human epithelial stem cells.

[0007] For example, the majority of human cancers originate from epithelial tissues. Since the concept of cancer stem cells ("CSCs") was introduced in the late 1990s, it has become accepted as the underlying mechanism for tumor initiation, growth, and ultimately, drug resistance. These stem cells have influenced all approaches to cancer research and treatment because they help explain the mechanistic progression of cancer from more benign to more aggressive forms. Although the majority of anticancer drugs kill the majority of tumor cells, they ultimately fail to induce a sustained clinical response because they are unable to eliminate critical CSCs that are resistant to existing cancer treatments, including targeted drugs, chemotherapy, and radiation therapy. Surviving CSCs generate new tumors and metastases, leading to disease recurrence. Recurrent tumors become more malignant, spread rapidly, and become resistant to radiation therapy and previously used drugs, resulting in a poor prognosis for cancer patients.

[0008] A complicating factor is that many tumors are thought to contain heterogeneous populations of CSCs, which exhibit a range of tumor-promoting activities and drug sensitivities. Therefore, the special survival of CSCs or subsets of CSCs from heterogeneous CSC populations may explain many treatment failures and highlight new directions for enhancing cancer therapy. To develop truly effective treatments that can produce durable clinical responses, it is crucial to develop drugs that can target and kill CSCs. CSCs have only recently begun to be accurately identified thanks to technological advances that facilitate the identification, isolation, and characterization of distinct tumor cell subpopulations that differ in their ability to form and perpetuate tumors. Therefore, there is a need for methods and reagents for the isolation and stable passage and expansion of stratified epithelial CSCs, which are useful in drug screening.

[0009] It is an object of the present invention to provide systems and reagents for the rapid isolation / cloning of stratified epithelial stem cells, particularly from small biopsies, under conditions that preserve epigenetic memory and faithfully preserve the in vivo characteristics of the stem cells as they were present in the tissue biopsy during repeated expansion and passaging in culture, so that patient-specific diagnostic and treatment strategies (e.g., inflammatory diseases and metaplasia / tumor) or for regenerative medicine are scalable, efficient, and ultimately sufficiently affordable to be performed on a patient-by-patient basis. Summary of the Invention

[0010] In one aspect, the present invention provides a method for producing a medicament for the treatment of a medicament comprising the steps of: (1) culturing dissociated epithelial cells from a stratified epithelial tissue sample to form stem cell colonies; (a) ROCK (Rho kinase) inhibitor, (c) mitogenic growth factor, (d) insulin or IGF, (e) TrkA inhibitor (GW441756), and (h) Oct4 activator; comprising at least one of a VEGF inhibitor, a tyrosine kinase inhibitor, and / or an FGF10 or FGF10 agonist; optionally, further comprising a TGFβ signaling pathway inhibitor (e.g., a TGFβ inhibitor or a TGFβ receptor inhibitor); optionally, further comprising a bone morphogenetic protein (BMP) antagonist; Optionally, culturing the dissociated cells and cell colonies in a medium further comprising a Wnt agonist; cells derived from the tissue sample are optionally in fluid or direct contact with division-inactive feeder cells, but preferably cultured in the absence of feeder cells; cells from the tissue sample are optionally in contact with an extracellular matrix (e.g., basement membrane matrix) or other biomatrix or synthetic matrix; (2) isolating a single stem cell from the cell colony; and (3) individually culturing the isolated single stem cells from step (2) in a medium to form a culture of purified stem cell clones in contact with (optionally) feeder cells and / or a basement membrane matrix, each of the stem cell clones representing a clonal expansion of epithelial stem cells present in the stratified epithelial tissue sample, thereby isolating stratified epithelial stem cells. The present invention provides a method for isolating stem cells from epithelial tissue, preferably stratified epithelial tissue, such as normal or diseased tissue, comprising:

[0011] In certain preferred embodiments, the culture medium comprises a VEGF inhibitor, preferably a VEGF inhibitor that is a small molecule tyrosine kinase inhibitor. In certain preferred embodiments, the culture medium comprises a VEGF inhibitor and lacks FGF10 or an FGF10 agonist. In certain preferred embodiments, the culture medium comprises both a VEGF receptor kinase inhibitor and a tyrosine kinase inhibitor, which may be the same compound or different compounds. In certain preferred embodiments, the culture medium comprises a VEGF receptor kinase inhibitor and a pan-ABL1 kinase inhibitor, which may be the same compound or different compounds.

[0012] In feeder-free embodiments of the invention, the medium further comprises (i) a SYK inhibitor, (j) an LPA receptor antagonist, (k) a GSK3 inhibitor, and (l) a CK2 inhibitor.

[0013] In certain embodiments, the epithelial tissue is derived from a patient with a disease, disorder, or abnormal condition, and is affected by the disease, disorder, or abnormal condition. In certain embodiments, the stratified epithelial stem cells are adult stratified epithelial stem cells. In certain embodiments, the stratified epithelial stem cells are fetal stratified epithelial stem cells.

[0014] In certain embodiments, the medium optionally further comprises nicotinamide and / or comprises a Notch agonist.

[0015] In certain other embodiments, the medium specifically lacks one or both of nicotinamide and / or contains a Notch agonist.

[0016] In certain embodiments, in step (1), the (epithelial) cells are dissociated from the tissue through enzymatic digestion with enzymes, such as collagenase, protease, dispase, pronase, elastase, hyaluronidase, accutase, or trypsin.

[0017] In certain embodiments, in step (1), the (epithelial) cells are dissociated from the tissue through dissolution of the extracellular matrix surrounding the (epithelial) cells.

[0018] In certain embodiments involving fluid-mediated contact with feeder cells, the mitotically inactivated cells are mitotically inactivated fibroblasts, preferably human or mouse fibroblasts, e.g., 3T3-J2 cells. Mitotic inactivation can be achieved by administration of mitomycin C or other chemical-based mitotic inhibitors, gamma irradiation, X-ray irradiation, and / or UV light irradiation.

[0019] In certain embodiments involving contact with an extracellular matrix, the extracellular matrix is ​​a basement membrane matrix, e.g., a laminin-containing basement membrane matrix (e.g., MATRIGEL™ basement membrane matrix (BD Biosciences)), preferably growth factor-reduced. In other embodiments, the biopolymer is selected from the group consisting of collagen, chitosan; fibronectin, fibrin, and mixtures thereof.

[0020] In certain embodiments, the basement membrane matrix does not support three-dimensional growth or does not form the three-dimensional matrix necessary to support three-dimensional growth.

[0021] In certain embodiments, the medium further comprises serum, preferably FBS (more preferably non-heat-inactivated FBS), for example, at a concentration of 5% to 15%, e.g., 10% FBS.

[0022] In certain embodiments, ROCK inhibitors include Rho kinase inhibitor VI (Y-27632, (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide)), fasudil, or HA1077 (5-(1,4-diazepan-1-ylsulfonyl)isoquinoline), or HI 152 ((S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride).

[0023] In certain embodiments, the BMP antagonist includes noggin, DAN, DAN-like proteins containing a DAN cystine-knot domain (e.g., Cerberus and Gremlin), chordin, chordin-like proteins containing a chordin domain, follistatin, follistatin-related proteins containing a follistatin domain, sclerostin / SOST, decorin, or a-2 macroglobulin. In certain preferred embodiments, the BMP antagonist is noggin.

[0024] In certain embodiments, the medium contains a Wnt agonist, such as R-spondin1, R-spondin2, R-spondin3, R-spondin4, an R-spondin mimetic, a Wnt family protein (e.g., Wnt-3a, Wnt-5, Wnt-6a), Norrin, or a GSK inhibitor (e.g., CHIR99021).

[0025] In certain embodiments, the mitogenic growth factors include EGF, keratinocyte growth factor (KGF), TGFa, BDNF, HGF, and / or FGF (eg, FGF7 or FGF10).

[0026] In certain embodiments, TGFβ receptor inhibitors include SB431542 (4-(4-(5-benzo[1,3]dioxol-5-yl)-4-(pyridin-2-yl)-1H-imidazol-2-yl)benzamide), A83-01, SB-505124, SB-525334, LY 364947, SD-208, or SJN 2511.

[0027] In certain embodiments, the TGFβ (signaling) inhibitor binds to and reduces the activity of one or more serine / threonine protein kinases selected from the group consisting of ALK5, ALK4, TGFβ receptor kinase 1, and ALK7.

[0028] In certain embodiments, the TGFβ (signaling) inhibitor is added at a concentration of 1 nM to 100 μM, 10 nM to 100 μM, 100 nM to 10 μM, or approximately 1 μM.

[0029] In certain embodiments, the VEGF inhibitor is aflibercept, pegaptanib, tivozanib, 3-(4-bromo-2,6-difluoro-benzyloxy)-5-[3-(4-pyrrolidin-1-yl-butyl)-ureido]-isothiazole-4-carboxylic acid amide hydrochloride, axitinib, N-(4-bromo-2-fluorophenyl)-6-methoxy-7-[(1-methylpiperidin-4-yl-)methoxy]quinazolin-4-amine, an inhibitor of VEGF-R2 and VEGF-R1, axitinib, N,2-dimethyl-6-(2-(1-methyl-1H-imidazol-2-yl)thieno[3,2-b]pyridine- and 7-benzyloxy)benzo[b]thiophene-3-carboxamide, a tyrosine kinase inhibitor of RET / PTC oncogenic kinase, N-(4-bromo-2-fluorophenyl)-6-methoxy-7-[(1-methylpiperidin-4-yl)methoxy]quinazolin-4-amine, a pan-VEGF-R kinase inhibitor; a protein kinase inhibitor, a multitargeted human epidermal growth factor receptor (HER)1 / 2 and vascular endothelial growth factor receptor (VEGFR)1 / 2 receptor family tyrosine kinase inhibitor, cediranib, sorafenib, vatalanib, glufanide disodium, a VEGFR2-selective monoclonal antibody, angiozyme, an siRNA-based VEGFR1 inhibitor, 5-((7-benzyloxyquinazolin-4-yl)amino)-4-fluoro-2-methylphenol hydrochloride, derivatives thereof, and combinations thereof.

[0030] In certain preferred embodiments, the VEGF inhibitor is a VEGF receptor inhibitor, more preferably a VEGF receptor kinase inhibitor, such as tivozanib (AV-951), AZD2932, midostaurin (pkc412), BAW2881 (NVP-BAW2881), nintedanib (BIBF 1120), SU5402, SU1498, BFH772, sorafenib, sunitinib, dovitinib (TKI258), semaxanib (SU5416), hypericin, vatalanib, ZM306416, AAL993, SU4312, DMXAA, or foretinib.

[0031] In certain embodiments, the medium contains a tyrosine kinase inhibitor, such as nilotinib, ponatinib, and dasatinib (but does not contain bosutinib or imatinib). In certain preferred embodiments, the tyrosine kinase inhibitor is a pan-ABL1 kinase inhibitor, such as ponatinib or dasatinib.

[0032] In certain embodiments, the media contains both a VEGF receptor kinase inhibitor and a tyrosine kinase inhibitor, which may be the same compound or different compounds, for example, a combination of ponatinib and tivozanib.

[0033] In certain embodiments, the TrkA inhibitor is selected from BMS-754807, GW441756, PF-06273340, Sitravatinib (MGCD516), ANA-12, GNF-5837, Belizatinib (TSR-011), Larotrectinib (LOXO-101) Sulfate, Lestaurtinib, Entrectinib (RXDX-101), GNF 5837, and AG-879. Preferably, the TrkA inhibitor is selective for TrkA over TrkB or TrkC, e.g., GW441756 and Sitravatinib (MGCD516). Preferably, the TrkA inhibitor is a potent and selective TrkA inhibitor, e.g., GW441756, having an IC50 of 10 nM or less, with the IC50 for inhibition of c-Raf1 and CDK2 being at least 100-fold greater than the IC50 for inhibition of TrkA.

[0034] In certain embodiments, the Oct4 activator is capable of activating a reporter gene driven by the Oct4 promoter, such as a luciferase gene under the transcriptional control of the Oct4 promoter, and more preferably, a reporter gene driven by both the Oct4 promoter and the Nanog promoter. Furthermore, when added to the reprogramming mixture together with the reprogramming factor tetrad (Oct4, Sox2, c-Myc, and Klf4), the Oct4 activator enhances iPSC reprogramming efficiency and accelerates the reprogramming process. Exemplary Oct4 activators are taught, for example, in U.S. Patent Application No. 20150191701 and Li et al. (2012) "Identification of Oct4-activating compounds that enhance reprogramming efficiency" PNAS 109(51):20853-8.

[0035] In certain embodiments, the Oct4 activator has the formula: TIFF0007815111000001.tif35128, wherein: X 1is C(R 12 ) or N; X 2 is C(R 4 ) or N; X 3 is C(R 5 ) or N; R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are independently selected from hydrogen, halogen, —CN, —NO2, —NH2, —CF3, —CCl3, —OH, —SH, —SO3H, —C(O)OH, —C(O)NH2, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R 2 and R 3 may be optionally linked to form a substituted or unsubstituted heterocycloalkyl or a substituted or unsubstituted heteroaryl.

[0036] Specifically, in feeder-free embodiments, the medium may also contain a SYK (spleen tyrosine kinase) inhibitor. Representative SYK inhibitors may be selected from the group consisting of entospletinib (GS-9973), fostamatinib (R788), R406, cerdulatinib (PRT062070), and TAK-659.

[0037] Specifically, in feeder-free embodiments, the medium may also contain an LPA receptor antagonist, e.g., an antagonist that inhibits inositol phosphate production induced by LPA1 and LPA3, each with a Ki of 1000 μM or less, and is a substantially weaker inhibitor of LPA2, LPA4, LPA5, and LPA6, i.e., each with a Ki of 5000 μM or less. Ki16198 is a preferred LPA receptor antagonist and is the methyl ester of Ki16425.

[0038] Specifically, in feeder-free embodiments, the medium may also contain a GSK3 inhibitor. Exemplary GSK3 inhibitors include CHIR-99021 (CT99021) HCl, SB216763, CHIR-98014, TWS119, tideglusib, SB415286, CHIR-99021 (CT99021), AZD2858, AZD1080, AR-A014418, TDZD-8, LY2090314, BIO-acetoxime, IM-12, 1-azakempaullone, indirubin, and 6-BIO.

[0039] Specifically, in embodiments that are feeder-free, the medium may also include a CK2 inhibitor, such as CX-4945 (silmitasertib), CX-8184, DMAT, ellagic acid, or TTP22.

[0040] In another aspect, the present invention provides single cell clones of epithelial stem cells, or in vitro cultures thereof, such as those comprising the medium of the present invention, wherein the epithelial stem cells substantially lack expression of markers associated with differentiated cell types of the epithelial tissue from which they were derived.

[0041] In another aspect, the present invention provides single cell clones of non-embryonic epithelial stem cells or in vitro cultures thereof, such as those comprising the medium of the present invention, wherein the non-embryonic epithelial stem cells have an immature, undifferentiated morphology characterized by small, round cell shape with a high nucleus / cytoplasm ratio.

[0042] In a related aspect, the present invention also provides libraries or collections of single-cell clones of the present invention, or in vitro cultures thereof (e.g., comprising the media of the present invention). In certain embodiments, the library or collection may contain single-cell clones derived from the same tissue / organ type. In certain embodiments, the library or collection may contain single-cell clones isolated from the same type of tissue / organ type but derived from different members of the population. In certain embodiments, one or more (preferably each) members of the population are homozygous at at least one tissue-typing locus (e.g., HLA-A, HLA-B, and HLA-D). In certain embodiments, at least one tissue-typing locus (e.g., an HLA locus described above) is modified in cloned stem cells, e.g., via TALEN technology or CRISPR technology (see below), to generate a universal donor cell line (e.g., hepatocytes) lacking tissue antigens encoded by the tissue-typing loci (e.g., HLA-A, HLA-B, and HLA-D, etc.). See Torikai et al. (Blood, 122(8):1341-1349, 2013, incorporated herein by reference). In certain embodiments, populations can be defined by ethnicity, age, sex, disease status, or common characteristics of the population. A library or collection can have at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 180, 200, 250, 300, or more members.

[0043] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (1) isolating epithelial stem cells from tissue corresponding to tissue affected by a disease, disorder, or abnormal condition of a subject using any of the methods of the present invention; (2) optionally, altering the expression of at least one gene in the epithelial stem cells to generate altered epithelial stem cells; (3) Reintroducing the isolated or altered epithelial stem cells or their clonal expansion into the subject. wherein at least one adverse effect or symptom of the disease, disorder, or abnormal condition in the subject is alleviated.

[0044] In certain embodiments, the expression of at least one gene in an epithelial stem cell is genetically, recombinantly, and / or epigenetically altered to generate an altered epithelial stem cell.

[0045] In certain embodiments, the tissue from which the epithelial stem cells are isolated is derived from a healthy adult or fetal (ie, non-embryonic) subject.

[0046] In certain embodiments, the tissue from which the epithelial stem cells are isolated is derived from a subject, hi certain embodiments, the tissue from which the epithelial stem cells are isolated is diseased tissue affected by a disease, disorder, or abnormal condition.

[0047] In certain embodiments, the tissue from which the epithelial stem cells are isolated is adjacent to diseased tissue affected by a disease, disorder, or abnormal condition.

[0048] In certain embodiments, at least one gene is underexpressed in tissue affected by the disease, disorder, or abnormal condition of the subject, and expression of the at least one gene is enhanced in the altered epithelial stem cells.

[0049] In certain embodiments, at least one gene is overexpressed in a tissue affected by the disease, disorder, or abnormal condition of the subject, and expression of the at least one gene is reduced in the altered epithelial stem cells.

[0050] In certain embodiments, step (2) is accomplished by introducing exogenous DNA or RNA into the epithelial stem cells.

[0051] In yet another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (1) isolating epithelial stem cells from a subject using any of the methods of the present invention; (2) generating cell lines of epithelial stem cells via single-cell clonal expansion; (3) contacting test cells derived from the cell line with a plurality of candidate compounds; and (4) identifying one or more compounds that produce a predetermined phenotypic change in the test cell; The present invention provides a method for screening a compound, comprising:

[0052] The use of the words "a" or "an," when used in conjunction with the word "comprising" in the claims and / or this specification, can mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more." The word "about" means + or -5% of the specified number.

[0053] [The present invention 1001] A defined culture medium for isolating stratified epithelial stem cells and stably maintaining the epigenetics of the stratified epithelial stem cells over multiple passages, comprising: basal medium; and ROCK (Rho kinase) inhibitors, mitogenic growth factors, insulin or IGF, TrkA inhibitors, and Oct4 activators, respectively Including, at least one of a VEGF inhibitor, a tyrosine kinase inhibitor, and / or an FGF10 or FGF10 agonist Including, TGFβ signaling pathway inhibitors and / or bone morphogenetic protein (BMP) antagonists and optionally further comprising A defined culture medium that supports epigenetically stable growth and proliferation of stem cells of stratified epithelial tissue origin in culture. [The present invention 1002] Allows for the passage of cells in a feeder-free manner, further including SYK inhibitors, LPA receptor antagonists, GSK3 inhibitors, and CK2 inhibitors; 1001 defined culture medium of the present invention. [The present invention 1003] The defined culture medium of invention 1001 or 1002, wherein the epithelial stem cells are in contact with an extracellular matrix or other biomatrix. [The present invention 1004] 4. The defined culture medium of any one of claims 1001 to 1003, wherein the stem cells are isolated from a tissue sample taken from normal epithelial tissue. [The present invention 1005] Stem cells, Tissue samples taken from diseased epithelial tissue, e.g., from inflammatory or autoimmune patients The defined culture medium of any one of 1001 to 1004 of the present invention, isolated from [The present invention 1006] 1004. The defined culture medium of any of claims 1001 to 1003, wherein the stem cells are isolated from a tissue sample taken from a tumor. [The present invention 1007] A clonal stratified epithelial stem cell isolated using any of the defined culture media of the present invention. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0054] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0055] [Figure 1] Representative images of cloned human epithelial stem cells derived from tumors of the epidermis, upper airway, distal airway, bladder, esophagus, and ovary. Human epithelial tissues were digested and plated onto irradiated 3T3-J2 feeders in the presence of SQM medium. [Figure 2]Figure 2A: Single-cell-derived lineages of human bladder stem cells were seeded in an air-liquid interface system. Single bladder stem cells can differentiate into all cell types present in the bladder epithelium, including basal cells, transitional epithelium, and basal cells. Figure 2B: 1,000 single bladder epithelial stem cells were seeded on irradiated 3T3-J2 feeders in the presence of SQM medium and allowed to form over 500 colonies. Colony-forming ability remained unchanged after seven passages, approximately 100 days of culture, and 200 cell divisions. Figure 2C: CNV, BAF (B allele frequency), and LRR (log R ratio) profiles of lineages from P1 to P7 demonstrated genomic stability during passage. [Figure 3A] Representative images of human distal airway stem cells at passages 5 and 25. Lineage CNV, BAF (B allele frequency), and LRR (log R ratio) profiles of human DASCs at P5–P25 demonstrated genomic stability during passage. [Figure 3B] Single-cell derived lineages of human DASCs were seeded into ALI cultures and differentiated into club cells (CC10), type I pneumocytes (AQP4), and type II pneumocytes (SEPTB). [Figure 3C] The generation of DASCs by the methods of the present invention results in a high degree of clonogenicity (clonal potential), which is maintained over multiple passages (compare passages 5 and 25), as well as the observed genetic and epigenetic stability of these stem cell clones. [Figure 4] Figure 4A: Single-cell-derived lineages of human upper airway stem cells were seeded at the air-liquid interface for in vitro differentiation. Single cells differentiated into ciliated (tubulin) and goblet (MUC5AC) cells. Figure 4B: Left, representative image of human upper airway stem cell lineages growing on irradiated 3T3-J2 feeders in the presence of SQM medium. Right, lineage-derived cells were transplanted into NSG mice to form upper airway epithelium containing ciliated and goblet cells. [Figure 5] Stem cells of a single cell-derived lineage of human skin were seeded into an air-liquid interface differentiation system and induced to differentiate into squamous epithelium resembling human skin. [Figure 6]Representative images of cloned human epithelial stem cells derived from tumors of the epidermis, upper airway, distal airway, bladder, esophagus, and ovary. Human epithelial tissue was digested and plated in the presence of SGM-63+ medium without mouse feeder support. [Figure 7] Single-cell-derived lineage stem cells of human upper airway epithelium were transplanted into NSG mice to generate structures resembling normal human upper airway epithelium based on histology and immunostaining using markers specific for ciliated cells (tubulin), goblet cells (MUC5AC), and club cells (CC10). [Figure 8] Figure 8A-E: Representative images of cloned human epithelial stem cells derived from skin using the B1 media system. Human skin tissue was digested and plated onto irradiated 3T3-J2 feeders in the presence of specialized media. A. Brightfield image of human dermal epithelial stem cells. B. Stem cell colonies stained positively with anti-p63 antibody. C. Stem cell colonies stained positively with anti-Krt5 antibody. D. Stem cell colonies stained positively with anti-Ki67 antibody, indicating that the cells were highly proliferative. E. Single human dermal stem cells were sorted into individual wells of a 384-well cell culture dish. Based on rhodamine staining, more than 60% of the cells were clonogenic. [Figure 9] Figure 9A-E: Representative images of cloned human epithelial stem cells derived from the bladder. Human bladder tissue was digested and plated on irradiated 3T3-J2 feeders in the presence of specialized medium. A. Brightfield image of human bladder epithelial stem cells. B. Stem cell colonies stained positively with anti-p63 antibody. C. Stem cell colonies stained positively with anti-Krt5 antibody. D. Stem cell colonies stained positively with anti-Ki67 antibody, indicating that the cells were highly proliferative. E. Single human bladder stem cells were sorted into individual wells of a 384-well cell culture dish. Based on rhodamine staining, more than 60% of the cells are clonogenic. [Figure 10]Figure 10A-E: Representative images of cloned human epithelial stem cells derived from salivary glands. Human salivary gland tissue was digested and plated on irradiated 3T3-J2 feeders in the presence of specialized medium. A. Brightfield image of human salivary gland epithelial stem cells. B. Stem cell colonies stained positively with anti-p63 antibody. C. Stem cell colonies stained positively with anti-Krt5 antibody. D. Stem cell colonies stained positively with anti-Ki67 antibody, indicating that the cells were highly proliferative. E. Single human salivary gland stem cells were sorted into individual wells of a 384-well cell culture dish. Based on rhodamine staining, more than 60% of the cells were clonogenic. [Figure 11] Figure 11A-D: Representative images of cloned human epithelial stem cells derived from the airway. Human airway tissue was digested and plated on irradiated 3T3-J2 feeders in the presence of specialized medium. A. Brightfield image of human airway epithelial stem cells. B. Stem cell colonies stained positively with anti-p63 antibody. C. Stem cell colonies stained positively with anti-Krt5 antibody. D. Single human airway stem cells were sorted into each well of a 384-well cell culture dish. More than 70% of the cells are clonogenic based on rhodamine staining. [Figure 12] Figures 12A-B: Single-cell-derived lineages of human upper or distal airway stem cells were induced to differentiate in an air-liquid interface system. 12A. Single-cell-derived lineages of human upper airway stem cells were differentiated into ciliated cells (tubulin) and goblet cells (MUC5AC). 12B. Single-cell-derived lineages of human DASCs were seeded into ALI cultures and differentiated into club cells (CC10), type I pneumocytes (AQP4), and type II pneumocytes (SEPTB). DETAILED DESCRIPTION OF THE INVENTION

[0056] DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS 1. Overview The invention described herein relates to a method for isolating and / or maintaining in culture non-embryonic (e.g., adult or fetal) epithelial stem cells derived from the stratified epithelium of an organ. Epithelial stem cells so isolated from various tissues or organs can self-renew or proliferate indefinitely in vitro, are pluripotent, and can differentiate into the various differentiated cell types normally found in the tissue or organ from which they were isolated. Cultures (including in vitro cultures) containing such isolated epithelial stem cells are also within the scope of the invention.

[0057] Furthermore, isolated epithelial stem cells can be propagated through clonal expansion of a single isolated stem cell to generate clones (e.g., in vitro cultures) in which at least about 40%, 70%, or 90% or more of the cells can be further passaged as clones of single-cell origin. Thus, stem cells isolated using the methods of the present invention can be uniquely manipulated in vitro through standard molecular biology techniques, such as the introduction of exogenous genetic material via infection or transfection.

[0058] As used herein, "epithelial stem cells" include adult stem cells isolated from adult tissues or organs, and fetal stem cells isolated from prenatal tissues or organs.

[0059] In related embodiments, the methods of the invention described herein isolate fetal stem cells from fetal or prenatal tissues or organs. In certain embodiments, when fetal tissues or organs are the source of the stem cells, particularly when the fetus is a human fetus, the methods of the invention do not destroy the fetus or impair its normal development. In other embodiments, the source of fetal tissue is obtained from aborted fetuses, dead fetuses, macerated fetal material, or cells, tissues, or organs excised therefrom.

[0060] The methods of the present invention are applicable to any animal stratified epithelial tissue that contains epithelial stem cells, including tissue from humans, non-human mammals, non-human primates, rodents (including, but not limited to, mice, rats, polecats, hamsters, guinea pigs, and rabbits), livestock (including, but not limited to, pigs, cattle, sheep, goats, horses, and camels), birds, reptiles, fish, pet or other companion animals (e.g., cats, dogs, birds), or other vertebrates.

[0061] The classification of stratified epithelium is based on the shape of the cells in the superficial layer. For example, if the superficial layer consists of flat cells, it is part of stratified squamous epithelium. Stratified epithelium is divided into three different types:

[0062] "Stratified non-keratinizing squamous epithelium" The shape of the cells of stratified non-keratinizing squamous epithelium changes from the base to the free surface and is divided into four sections: · Basal layer: trabecular dark cells with round nuclei; · Parabasal layer: polygonal dark cells arranged in stratified tissue; ·Stratum spinosum: Polyhedral polygonal cells connected by desmosomes; Superficial layer: Flat cells that decompose and desquamate in the outermost layer It can be divided into:

[0063] This type of epithelium is found in the mucous membranes of the oral cavity and esophagus, as well as in the vagina and eye (corneal epithelium).

[0064] "Stratified keratinized squamous epithelium" The outermost cell layer of the epithelium consists of flat cells without nuclei, which are transformed into scales. They are called the stratum corneum, and their purpose is to provide mechanical protection for the underlying tissue from dehydration. Stratified keratinized squamous epithelium is divided into five sections: ·Basal layer; ·Spinous layer; · Granular layer: flat cells containing keratohyalin granules; ·Transparent layer: conversion area; ·Stratum corneum It can be divided into:

[0065] "Transitional epithelium (urothelium)" The urothelium consists of a basal layer, several intermediate cell layers, and an umbrella cell layer. Umbrella cells (superficial cells) are large and often have two nuclei. A very dense network of cytoplasm, the crusta, is located below the apical membrane. The cell membrane consists primarily of a firm plaque containing uroplakin (a transmembrane protein). Transitional epithelium is found primarily in the efferent urinary tract, i.e., the renal pelvis, ureter, bladder, and initial part of the urethra.

[0066] "Pseudostratified epithelium" An important feature of this epithelium type is that the cells are in contact with the basement membrane, but not all of them reach the free surface. Cells that reach the free surface belong to the columnar type. Cells that do not reach the free surface are in the basal layer and have round nuclei. The term pseudostratified comes from the appearance of this epithelium. The cell nuclei appear at different heights, giving the false impression of multiple cell layers. Non-ciliated pseudostratified epithelium is found, for example, in the epididymal ducts and vas deferens, while ciliated pseudostratified epithelium with kinocilia is found in the respiratory tract (nasal cavity and bronchi).

[0067] In certain embodiments, the epithelial tissue is isolated from a healthy or normal individual.

[0068] In certain embodiments, the epithelial tissue is isolated from diseased tissue (eg, tissue affected by a disease), disordered tissue (eg, tissue affected by a disorder), or tissue having any other abnormal condition.

[0069] As used herein, the term "disease" includes an abnormal or medical condition that affects the body of an organism and is generally associated with specific symptoms and signs. Diseases can be caused by external factors (such as infectious diseases, including papillomavirus infections or sexually transmitted diseases) or by internal dysfunction (such as autoimmune diseases or cancer). In a broad sense, "disease" can also include conditions that cause pain, disability, suffering, social problems, or death in the affected individual, or similar problems in those who come into contact with that individual. In this broader sense, it can include damage, disability, disorders, syndromes, infections, isolated symptoms, abnormal behavior, and atypical variations in structure and function, although in other contexts and for other purposes, these may be considered distinct categories. In certain preferred embodiments, stem cells are isolated from tumor biopsies.

[0070] In some embodiments, epithelial tissue is isolated from an individual with disease, disorder or other abnormal condition, but the epithelial tissue itself may not be affected by disease, disorder or abnormal condition.For example, epithelial tissue may be isolated from a patient with inflammatory bowel disease or gastric cancer from the healthy part of the intestine (in the case of IBD) or stomach (in the case of tumor) that has not yet been affected by inflammatory condition or cancer.In some embodiments, epithelial tissue may be proximal or distal to disease, disorder or abnormal tissue.

[0071] In certain embodiments, the epithelial tissue is isolated from an individual who is predisposed to or at high risk for developing a disease, disorder, or other abnormal condition, e.g., based on the individual's genetic makeup, family history, lifestyle choices (e.g., smoking, diet, exercise habits), previous viral infections, etc., but who has not yet developed the disease, disorder, or other abnormal condition or exhibits detectable symptoms of the disease, disorder, or other abnormal condition.

[0072] Another aspect of the invention provides an epithelial stem cell isolated by any of the methods of the invention, or an in vitro culture thereof.

[0073] In yet another aspect, the present invention further provides a single cell clone of an isolated epithelial stem cell, or an in vitro culture thereof, wherein at least about 40%, 50%, 60%, 70%, or about 80% of the cells in the single cell clone are capable of expanding to give rise to the single cell clone when isolated as a single cell.

[0074] Each single cell clone may be cultured for at least about 10, 100, 10 3 pieces, 10 4 pieces, 10 5 pieces, 10 6 It may contain one or more cells.

[0075] In a related aspect, the present invention provides single cell clones of isolated epithelial stem cells or in vitro cultures thereof, wherein the epithelial stem cells, when isolated as single cells, are capable of self-renewal for about 50, 70, 100, 150, 200, 250, 300, 350, or about 400 generations or more.

[0076] In certain embodiments, the in vitro culture comprises a medium of the present invention (e.g., a modified medium of the present invention as described below). See the sections below describing the media of the present invention. Each of the media described therein is incorporated herein by reference. In certain embodiments, epithelial stem cells can differentiate into differentiated cell types of the epithelial tissue from which they were originally biopsied, or, in the case of cancer stem cells, into the tumor of that tissue origin. For example, isolated epithelial stem cells of the present invention can differentiate into one or more cell types normally found in the epithelial tissue from which they were derived.

[0077] In certain embodiments, epithelial stem cells can differentiate into organized structures that resemble structures or substructures found in the tissue from which they were derived, for example, isolated hepatic stem cells of the present invention can differentiate into liver tissue-like structures that resemble liver epithelium, and isolated gastrointestinal stem cells of the present invention can differentiate into GI tissue-like structures that resemble gastrointestinal epithelium.

[0078] In certain embodiments, epithelial stem cells have an immature, undifferentiated morphology characterized by a small, round cell shape with a high nuclear / cytoplasmic ratio.

[0079] A further aspect of the present invention is a method for producing a pharmaceutical composition comprising the steps of: (1) using any of the methods of the present invention to isolate non-embryonic (e.g., adult) stem cells from regenerative tissue corresponding to tissue affected by a disease, disorder, or abnormal condition in a subject; (2) altering the expression of at least one gene in the epithelial stem cells to produce altered epithelial stem cells; (3) Reintroducing the altered epithelial stem cells or clonal expansions, or tissue grafts derived from the cultures, into the subject. The present invention provides a method of treating a subject having a disease, disorder, or abnormal condition in need thereof, comprising alleviating at least one adverse effect or symptom of the disease, disorder, or abnormal condition in the subject, or as a means of regenerating / replacing damaged regenerative tissue, comprising administering to the subject a therapeutically effective amount of ...

[0080] For example, step (2) of the method can be achieved by introducing exogenous DNA or RNA into isolated epithelial stem cells, which increases or decreases the expression of a target gene in the epithelial stem cells. Any art-recognized molecular biology technique can be used to alter gene expression in cells, for example, in vitro or ex vivo. Such methods can include, but are not limited to, transfection or infection with a viral or non-viral vector that may encode a coding sequence for a protein or functional fragment thereof that is dysfunctional or missing in the target cell, or may encode RNA (antisense RNA, siRNA, miRNA, shRNA, ribozyme, etc.) that disrupts the function of the target gene.

[0081] In certain embodiments, the tissue from which the epithelial stem cells are isolated is derived from a healthy subject, preferably one that is HLA-type matched to the subject in need of treatment.

[0082] In certain embodiments, the tissue from which the epithelial stem cells are isolated is derived from the subject, and the isolated epithelial stem cells are autologous to the subject.

[0083] In certain embodiments, the tissue from which the epithelial stem cells are isolated is a diseased tissue affected by a disease, disorder, or abnormal condition.

[0084] In certain embodiments, the tissue from which the epithelial stem cells are isolated is adjacent to diseased tissue affected by a disease, disorder, or abnormal condition.

[0085] In certain embodiments, at least one gene is underexpressed in tissue affected by the disease, disorder, or abnormal condition of the subject, and expression of the at least one gene is enhanced in the altered epithelial stem cells.

[0086] In certain embodiments, at least one gene is overexpressed in a tissue affected by the disease, disorder, or abnormal condition of the subject, and expression of the at least one gene is reduced in the altered epithelial stem cells.

[0087] In another aspect, the present invention also provides methods for screening for agents or conditions that alter the "phenotype" of cells, such as differentiation, epigenetics, survival, etc., of stem cells of regenerative tissues, whether normal or derived from a cancer / disease state. In an exemplary embodiment, the method comprises: (1) using any of the methods of the present invention to isolate epithelial stem cells (including cancer stem cells) from a regenerative tissue of a subject; (2) generating one or more stem cell lines from the epithelial stem cells via single-cell clonal expansion; (3) contacting test cells derived from the cell line with one or more candidate compounds; and (4) identifying compounds that cause a predetermined phenotypic change in the test cells; This screening method of the present invention can be used for target identification and validation. For example, a potential target gene in epithelial stem cells isolated from a patient in need of treatment may function abnormally (either overexpressed or underexpressed) to cause a phenotype associated with a disease, disorder, or abnormal condition. Clonal expansion of epithelial stem cells isolated using the method of the present invention can be subjected to the screening method of the present invention to test an array of potential compounds (such as small molecule compounds) to identify one or more compounds that can correct, alleviate, or reverse the phenotype.

[0088] In another embodiment, epithelial stem cells can be isolated from the regenerative tissue of a patient in need of treatment, such as regenerative tissue affected by a disease, disorder, or abnormal condition. Clonal expansion of epithelial stem cells isolated using the methods of the present invention can be subjected to the screening methods of the present invention to test an array of potential compounds (such as small molecule compounds or RNA-based antagonists, such as siRNA libraries) to identify one or more compounds that can correct, alleviate, or reverse the phenotype. Target genes affected by effective compounds can be further identified, for example, by microarray, RNA-Seq, or PCR-based expression profile analysis.

[0089] Epithelial stem cells isolated using the methods of the present invention and their clonal expansion may further be useful for toxicological screening or research, such that toxicological analysis and testing can be personalized for individual patients who will receive certain medications or medical interventions.

[0090] Epithelial stem cells isolated using the methods of the present invention and their clonal expansion may also be useful for regenerative medicine, where either autologous stem cells or stem cells isolated from HLA-matched healthy donors can be induced to differentiate in vitro, ex vivo, or in vivo into regenerative tissues or organs to treat existing conditions or prevent / delay the onset of such conditions. Such stem cells may also be genetically manipulated prior to induced differentiation.

[0091] 2. Methods for Obtaining and / or Cultivating Stem Cells One aspect of the present invention relates to a method for isolating epithelial stem cells from epithelial tissue, as generally described above.

[0092] To illustrate, one step of the method involves culturing dissociated epithelial cells derived from epithelial tissue in contact with (optionally) a first population of division-inactive feeder cells and / or an extracellular matrix, e.g., a basement membrane matrix, to form epithelial cell clones.

[0093] In certain embodiments, (epithelial) cells are dissociated from the tissue through enzymatic digestion with enzymes including, but not limited to, one or more of collagenase, protease, dispase, pronase, elastase, hyaluronidase, accutase, and / or trypsin.

[0094] These enzymes or functional equivalents are well known in the art and, in almost all cases, are commercially available.

[0095] In other embodiments, (epithelial) cells can be dissociated from tissue samples through dissolution of the extracellular matrix surrounding the (epithelial) cells. One suitable reagent for this embodiment of the invention includes a non-enzymatic proprietary solution sold by BD Biosciences (San Jose, CA) as BD™ Cell Recovery Solution (BD Catalog No. 354253), which allows for the recovery of cells cultured in BD MATRIGEL™ Basement Membrane Matrix for subsequent biochemical analysis.

[0096] In certain embodiments, the feeder cells may include certain lethally irradiated fibroblasts, such as mouse 3T3-J2 cells. The feeder cells can form a feeder cell layer on top of the basement membrane matrix.

[0097] Suitable 3T3-J2 cell clones are well known in the art (see, e.g., Todaro and Green, "Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines," Cell Biol. 17:299-313, 1963) and are readily publicly available. For example, Waisman Biomanufacturing (Madison, Wisconsin) sells irradiated 3T3-J2 feeder cells that are produced and tested according to cGMP guidelines. These cells are initially obtained by the vendor from Dr. Howard Green's laboratory under a Material Transfer Agreement and are of sufficient quality to support, for example, skin gene therapy and wound healing clinical trials. The vendor also states that each vial of 3T3 cells contains a minimum of 3 x 10 cells manufactured in a fully compliant clean room. 6 The bank contains 1000 cells and is certified mycoplasma-free and low-endotoxin. Additionally, the cell bank has been fully tested for adventitious agents, including mouse viruses. These cells have been screened for keratinocyte culture support and are mitomycin C-free.

[0098] The method of the present invention provides for the use of feeder cells, such as the mouse 3T3-J2 clone of fibroblasts. Generally, without being limited to a particular phenotype, feeder cell layers are often used to support the culture of stem cells and / or inhibit differentiation. A feeder cell layer is generally a monolayer of cells that is co-cultured with the cells of interest and provides a suitable surface for their growth. The feeder cell layer provides an environment in which the cells of interest can grow. Feeder cells are often mitotically inactivated (e.g., by (lethal) irradiation or treatment with mitomycin C) to prevent their proliferation.

[0099] In some embodiments, the feeder cells are appropriately screened and are GMP-grade human feeder cells, e.g., sufficient to support the clinical-grade stem cells of the present invention. For GMP-grade human feeder cells grown in a medium containing GMP-quality FBS, see Crook et al. (Cell Stem Cell 1(5):490-494, 2007, incorporated by reference).

[0100] In some embodiments, feeder cells can be labeled with the marker that stem cells lack, so that stem cells can be easily distinguished and isolated from feeder cells.For example, feeder cells can be modified to express fluorescent markers, such as GFP or other similar fluorescent markers.Fluorescently labeled feeder cells can be separated from stem cells, for example, by FACS sorting.

[0101] Any of a number of physical separation methods known in the art can be used to separate the stem cells of the present invention from feeder cells.Other than FACS, such physical methods can include various immunoaffinity methods based on specific expressed markers.For example, the stem cells of the present invention can be isolated based on the specific stem cell markers they express and by using antibodies specific to these markers.

[0102] In one embodiment, the stem cells of the present invention can be isolated by FACS, for example, using an antibody against one of these markers. Fluorescence-activated cell sorting (FACS) can be used to detect markers characteristic of a particular cell type or lineage. As will be apparent to those skilled in the art, this can be achieved through a fluorescently labeled antibody or a fluorescently labeled secondary antibody that has binding specificity for the primary antibody. Examples of suitable fluorescent labels include, but are not limited to, FITC, Alexa Fluor® 488, GFP, CFSE, CFDA-SE, DyLight 488, PE, PerCP, PE-Alexa Fluor® 700, PE-Cy5 (TRI-COLOIT), PE-Cy5.5, PI, PE-Alexa Fluor* 750, and PE-Cy7. This list of fluorescent markers is provided for illustrative purposes only and is not intended to be limiting.

[0103] For example, it will be clear to those skilled in the art that FACS analysis using stem cell specific antibody will provide purified stem cell population.However, in some embodiments, it may be desirable to further purify cell population by performing additional FACS analysis using one or more other identifiable markers, such as those that select other than feeder.

[0104] For some competitive methods, the presence of feeder cells can complicate the cell passage in these competitive methods, so the use of feeder cells is considered undesirable.For example, cells must be separated from feeder cells at each passage, and new feeder cells are required at each passage.In addition, the use of feeder cells can cause feeder cells to be mixed with desired cells.

[0105] However, the use of a feeder layer is not necessarily a disadvantage of the present invention, since the isolated stem cells of the present invention can be, and indeed preferably are, passaged as single cells, and thus the potential risk of feeder contamination during passaging is minimized, if not eliminated.

[0106] In certain embodiments, the basement membrane matrix is ​​a laminin-containing basement membrane matrix (eg, MATRIGEL™ basement membrane matrix (BD Biosciences)), preferably growth factor reduced.

[0107] In certain embodiments, basement membrane matrix does not support three-dimensional growth or does not form the three-dimensional matrix necessary to support three-dimensional growth. Thus, when seeding basement membrane matrix, it is generally not required that the basement membrane matrix be deposited on the support in a particular shape or form, e.g., forming a dome shape or form and maintaining such shape or form after solidification, to support three-dimensional growth. In certain embodiments, the basement membrane matrix is ​​distributed or spread evenly over a flat surface or support structure (such as a flat-bottom tissue culture dish or well).

[0108] In certain embodiments, the basement membrane matrix is ​​first thawed, diluted to an appropriate concentration (e.g., 10%) with cold (e.g., about 0-4°C) feeder cell growth medium, plated, and allowed to solidify on a flat surface by warming to 37°C in a tissue culture incubator with an appropriate CO2 content (e.g., about 5%). Lethally irradiated feeder cells are then plated onto the solidified basement membrane matrix at an appropriate density so that the plated feeder cells form a subconfluent or confluent feeder cell layer on the basement membrane matrix overnight. The feeder cells are preferably cultured in a feeder cell medium, such as a medium (e.g., 3T3-J2 growth medium) containing basal tissue culture medium with high glucose (e.g., about 4.5 g / L), no L-glutamine, and no sodium pyruvate (e.g., DMEM (Invitrogen catalog no. 11960; high glucose (4.5 g / L), no L-glutamine, no sodium pyruvate), 10% calf serum (not heat-inactivated), one or more antibiotics (e.g., 1% penicillin-streptomycin), and L-glutamine (e.g., about 1.5 mM, or 1-2 mM, or 0.5-5 mM, or 0.2-10 mM, or 0.1-20 mM).

[0109] According to the method of the present invention, epithelial cell colonies become detectable after dissociated cells derived from the tissue of origin are cultured in the stem cell medium of the present invention for several days (e.g., 3 to 4 days or about 10 days).

[0110] In certain embodiments, single cells can be isolated from these epithelial cell colonies, for example, by enzymatic digestion. Suitable enzymes for this purpose include trypsin, such as warmed 0.25% trypsin (Invitrogen catalog number 25200056). In certain embodiments, the enzymatic digestion is substantially complete, such that essentially all cells in the epithelial cell clone are dissociated from other cells and become single cells. In certain embodiments, the method includes culturing the isolated single cells in modified growth medium (preferably after washing and resuspending the single cells) in contact with a second population of lethally irradiated feeder cells and a second basement membrane matrix in modified growth medium. Optionally, the isolated single cells may be passed through a cell strainer of appropriate size (e.g., 40 microns) before plating the single cells onto the feeder cells and basement membrane matrix.

[0111] In certain embodiments, the modified growth medium is changed periodically (e.g., every day, every 2 days, every 3 days, or every 4 days, etc.) until single cell clones or clonal expansions of the isolated single stem cells are formed.

[0112] In certain embodiments, single colonies of stem cells can be isolated, for example, using cloning rings. The isolated stem cell clones can be expanded to develop lineage cell lines, i.e., cell lines derived from a single stem cell.

[0113] In certain embodiments, a single stem cell can be isolated from the clonal expansion of a single stem cell and passaged again as a single stem cell.

[0114] 3. Culture Medium The present invention provides various cell culture media for isolating, culturing, and / or differentiating stem cells of the present invention, including basal media supplemented with numerous factors to create stem cell culture media for regenerative tissue stem cells. Factors that can be added to basal media or modified media are first described below. Then, to illustrate specific, non-limiting aspects of the present invention, several exemplary basal and modified media of the present invention are described in further detail.

[0115] Rock (Rho kinase) inhibitors Without wishing to be bound by any particular theory, the addition of a Rock inhibitor may prevent anoikis, particularly when culturing single stem cells. Rock inhibitors include (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632, Sigma-Aldrich), 5-(1,4-diazepan-1-ylsulfonyl)isoquinoline (fasudil or HA1077, Cayman Chemical), (1S,)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride (H1 152, Tocris). Bioscience), and N-(6-fluoro-1H-indazol-5-yl)-2-methyl-6-oxo-4-(4-(trifluoromethyl)phenyl)-1,4,5,6-tetrahydropyridine-3-carboxamide (GSK429286A, Stemgent). In certain embodiments, the final concentration for Y27632 is about 1-5 μM or 2.5 μM. A Rho kinase inhibitor, such as Y-21632, can be added to the culture medium every 1, 2, 3, 4, 5, 6, or 7 days for the first 7 days of stem cell culture.

[0116] Wnt agonists The Wnt signaling pathway is defined by a series of events that occur when a Wnt protein ligand binds to a cell surface receptor that is a member of the Frizzled receptor family. This results in the activation of the Dishevelled (Dsh) family of proteins, which inhibit a complex of proteins containing axin, GSK-3, and the protein APC, to degrade intracellular β-catenin. The resulting concentrated nuclear β-catenin enhances transcription by the TCF / LEF family of transcription factors. As used herein, "Wnt agonist" includes agents that directly or indirectly activate TCF / LEF-mediated transcription in cells, such as by modulating the activity of any of the proteins / genes in the Wnt signaling cascade (e.g., by enhancing the activity of a positive regulator of the Wnt signaling pathway or inhibiting the activity of a negative regulator of the Wnt signaling pathway).

[0117] The Wnt agonist is selected from true Wnt agonists that bind to and activate members of the Frizzled receptor family, including all Wnt family proteins, inhibitors of intracellular beta-catenin degradation, and activators of TCF / LEF. The Wnt agonist can stimulate Wnt activity in cells by at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70%, at least about 90%, at least about 100%, at least about 2-fold, 3-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1000-fold or more, compared to the level of Wnt activity in the absence of the Wnt agonist. As known to those skilled in the art, Wnt activity can be determined by measuring Wnt transcriptional activity, for example, using Tcf luciferase reporter constructs in pTOPFLASH and pFOPFLASH (see Korinek et al., Science 275:1784-1787, 1997, incorporated herein by reference).

[0118] Representative Wnt agonists may include secreted glycoproteins, including Wnt-1 / Int-1, Wnt-2 / Irp (Int-1 related protein), Wnt-2b / 13, Wnt-3 / Int-4, Wnt-3a (R&D systems), Wnt-4, Wnt-5a, Wnt-5b, Wnt-6 (Kirikoshi et al, Biochem. Biophys. Res. Com., 283:798-805, 2001), Wnt-7a (R&D systems), Wnt-7b, Wnt-8a / 8d, Wnt-8b, Wnt-9a / 14, Wnt-9b / 14b / 15, Wnt-10a, Wnt-10b / 12, Wnt-11, and Wnt-16. A summary of human Wnt proteins is provided in "The Wnt Family of Secreted Proteins," R&D Systems Catalog, 2004 (incorporated herein by reference).

[0119] Additionally, Wnt agonists include secreted proteins of the R-spondin family, which are involved in activating and regulating the Wnt signaling pathway and include at least four members: R-spondin 1 (NU206, Nuvelo, San Carlos, CA), R-spondin 2 (R&D systems), R-spondin 3, and R-spondin 4. Wnt agonists also include Norrin (R&D systems), a secreted regulatory protein (also known as Norrie Disease Protein or NDP) that functions similarly to Wnt proteins by binding with high affinity to the Frizzled 4 receptor and inducing activation of the Wnt signaling pathway (Kestutis Planutis et al., BMC Cell Biol. 8:12, 2007).

[0120] Wnt agonists include those having the following structure, as described in Liu et al. (Angew Chem. Int. Ed. Engl. 44 13):1987-1990, 2005), which is incorporated herein by reference: Further included is a small molecule agonist of the Wnt signaling pathway, an aminopyrimidine derivative (N4-[(2H-1,3-benzodioxol-5-yl)methyl)-6-(3-methoxyphenyl)pyrimidine-2,4-diamine) of TIFF0007815111000002.tif39128.

[0121] GSK inhibitors include small interfering RNA (siRNA, Cell Signaling), lithium (Sigma), Kenpaullone (Biomol International, Leost et al., Eur. J. Biochem. 267: 5983-5994, 2000), 6-bromoindirubin-30-acetoxime (Meyer et al., Chem. Biol. 10: 1255-1266, 2003), SB 216763, and SB 415286 (Sigma-Aldrich), as well as FRAT family members and FRAT-derived peptides that prevent the interaction of GSK-3 with axin. A summary is provided by Meijer et al. (Trends in Pharmacological Sciences 25: 471-480, 2004, incorporated herein by reference). Methods and assays for determining the level of GSK-3 inhibition are known in the art and may include, for example, methods and assays such as those described in Liao et al. (Endocrinology 145(6):2941-2949, 2004, incorporated herein by reference).

[0122] In certain embodiments, the Wnt agonist is selected from one or more of a Wnt family member, R-spondins 1-4 (such as R-spondin 1), Norrin, Wnt3a, Wnt-6, and a GSK inhibitor.

[0123] In certain embodiments, the Wnt agonist comprises or consists of R-spondin 1. R-spondin 1 can be added to the culture medium of the present invention at a concentration of at least about 50 ng / mL, at least about 75 ng / mL, at least about 100 ng / mL, at least about 125 ng / mL, at least about 150 ng / mL, at least about 175 ng / mL, at least about 200 ng / mL, at least about 300 ng / mL, or at least about 500 ng / mL. In certain embodiments, R-spondin 1 is about 125 ng / mL.

[0124] In certain embodiments, any of the specific protein-based Wnt agonists mentioned herein, such as R-spondin1 through R-spondin4, any Wnt family member, etc., may be replaced with a natural, synthetic, or recombinantly produced homolog or fragment thereof that retains at least about 80%, 85%, 90%, 95%, or 99% of the respective Wnt agonist activity, and / or has at least about 60%, 70%, 80%, 90%, 95%, 97%, or 99% amino acid sequence identity, as measured by art-recognized sequence alignment software based on either global alignment techniques (e.g., the Needleman-Wunsch algorithm) or local alignment techniques (e.g., the Smith-Waterman algorithm). The sequences of representative Wnt agonists mentioned herein are represented in SEQ ID NOs. 10-17.

[0125] During the culture of the stem cells of the present invention, a Wnt family member can be added to the culture medium every day, every two days, every three days, every four days, every five days, or more, while changing the culture medium every day, every two days, every three days, every four days, every five days, or more.

[0126] In certain embodiments, the Wnt agonist is selected from the group consisting of R-spondin, Wnt-3a, and Wnt-6, or a combination thereof. In certain embodiments, R-spondin and Wnt-3a are used together as Wnt agonists. In certain embodiments, the R-spondin concentration is about 125 ng / mL, and the Wnt3a concentration is about 100 ng / mL.

[0127] mitogenic growth factors Mitogenic growth factors suitable for the present invention may include the family of growth factors including epidermal growth factor (EGF) (Peprotech), transforming growth factor alpha (TGFa, Peprotech), basic fibroblast growth factor (bFGF, Peprotech), brain-derived neurotrophic factor (BDNF, R&D Systems), and keratinocyte growth factor (KGF, Peprotech).

[0128] EGF is a potent mitogen for a variety of cultured ectodermal and mesodermal cells and exerts profound effects on the differentiation of certain cells in vivo and in vitro, as well as some fibroblasts in cell culture. The EGF precursor exists as a membrane-bound molecule that is proteolytically cleaved to generate a 53-amino acid peptide hormone that stimulates cells. EGF can be added to the culture medium of the present invention at a concentration of 1 to 500 ng / mL. In certain embodiments, the final EGF concentration in the medium is at least about 1, 2, 5, 10, 20, 25, 30, 40, 45, or 50 ng / mL, but not more than about 500, 450, 400, 350, 300, 250, 200, 150, 100, 50, 30, or 20 ng / mL. In certain embodiments, the final EGF concentration is about 1-50 ng / mL, or about 2-50 ng / mL, or about 5-30 ng / mL, or about 5-20 ng / mL, or about 10 ng / mL.

[0129] The same concentration can be used for FGFs such as FGF10 or FGF7. When multiple types of FGFs, such as FGF7 and FGF10, are used, the above-mentioned FGF concentrations can refer to the total concentration of all FGFs used in the medium.

[0130] In certain embodiments, any of the specific mitogenic growth factors mentioned herein, such as, for example, EGF, TGFα, bFGF, BDNF, KGF, etc., may be replaced with a natural, synthetic, or recombinantly produced homolog or fragment thereof that retains at least about 80%, 85%, 90%, 95%, 99% of the respective mitogenic growth factor activity and / or has at least about 60%, 70%, 80%, 90%, 95%, 97%, 99% amino acid sequence identity as measured by art-recognized sequence alignment software based on either global alignment techniques (e.g., the Needleman-Wunsch algorithm) or local alignment techniques (e.g., the Smith-Waterman algorithm).

[0131] The sequences of representative mitogenic growth factors referred to herein are represented in SEQ ID NOs. 18-27.

[0132] During the culture of the stem cells of the present invention, mitogenic growth factors can be added to the medium, for example, every day, every two days, with the medium being changed daily.

[0133] Any member of the bFGF family can be used. In certain embodiments, FGF7 and / or FGF10 are used. FGF7 is also known as KGF (keratinocyte growth factor). In certain embodiments, a combination of mitogenic growth factors such as EGF and KGF or EGF and BDNF is added to the culture medium of the present invention. In certain embodiments, a combination of mitogenic growth factors such as EGF and KGF or EGF and FGF10 is added to the culture medium of the present invention.

[0134] BMP inhibitors Bone morphogenetic proteins (BMPs) bind as dimeric ligands to a receptor complex consisting of two distinct receptor serine / threonine kinases, type I and type II receptors. The type II receptor phosphorylates the type I receptor, leading to activation of this receptor kinase. The type I receptor then phosphorylates specific receptor substrates (such as SMADs), resulting in a signaling pathway that leads to transcriptional activation.

[0135] As used herein, BMP inhibitors include agents that inhibit BMP signaling through its receptor.In one embodiment, BMP inhibitors bind to BMP molecules to form complexes, for example, by preventing or inhibiting the binding of BMP molecules to BMP receptors, thereby neutralizing BMP activity.Examples of such BMP inhibitors can include antibodies specific to BMP ligands or their antigen-binding portions.Other examples of such BMP inhibitors include dominant-negative mutants of BMP receptors, such as soluble BMP receptors, that bind to BMP ligands and prevent the ligands from binding to natural BMP receptors on cell surfaces.

[0136] Alternatively, BMP inhibitors can include drugs that function as antagonists or inverse agonists.This type of inhibitor binds to BMP receptor and prevents BMP from binding to the receptor.An example of such a drug is an antibody that specifically binds to BMP receptor and prevents BMP from binding to antibody-bound BMP receptor.

[0137] In certain embodiments, BMP inhibitors inhibit the BMP-dependent activity in cells by at most 90%, at most 80%, at most 70%, at most 50%, at most 30%, at most 10%, or about 0% (near complete inhibition), compared to the level of BMP activity in the absence of inhibitor.As known to those skilled in the art, BMP activity can be determined by measuring the transcriptional activity of BMP, for example, as exemplified in Zilberberg et al. ("A rapid and sensitive bioassay to measure bone morphogenetic protein activity," BMC Cell Biology 8:41,2007, incorporated herein by reference).

[0138] Several classes of natural BMP binding proteins are known, including Noggin (Peprotech), Chordin and Chordin-like proteins containing Chordin domains (R&D systems), Follistatin and follistatin-related proteins containing Follistatin domains (R&D systems), DAN and DAN-cystine knot domain-containing DAN-like proteins (e.g., Cerberus and Gremlin) (R&D systems), Sclerostin / SOST (R&D systems), Decorin (R&D systems), and α2 macroglobulin (R&D systems), or as described in US 8,383,349. Exemplary BMP inhibitors for use in the methods of the present invention are selected from Noggin, DAN, and DAN-like proteins containing Cerberus and Gremlin (R&D systems). These diffusible proteins can bind to BMP ligands with varying degrees of affinity and inhibit BMP from accessing signaling receptors.

[0139] Any of the above BMP inhibitors may be added, alone or in combination, to the culture medium of the present invention when desired.

[0140] In certain embodiments, the BMP inhibitor is Noggin, which may be added to the respective culture medium at a concentration of at least about 10 ng / mL, or at least about 20 ng / mL, or at least about 50 ng / mL, or at least about 100 ng / mL (e.g., 100 ng / mL).

[0141] In certain embodiments, any of the specific BMP inhibitors mentioned herein, such as noggin, chordin, follistatin, DAN, cerberus, gremlin, sclerostin / SOST, decorin, and α2 macroglobulin, may be replaced with a natural, synthetic, or recombinantly produced homolog or fragment thereof that retains at least about 80%, 85%, 90%, 95%, 99% of the respective BMP inhibitory activity and / or has at least about 60%, 70%, 80%, 90%, 95%, 97%, 99% amino acid sequence identity as measured by art-recognized sequence alignment software based on either global alignment techniques (e.g., the Needleman-Wunsch algorithm) or local alignment techniques (e.g., the Smith-Waterman algorithm).

[0142] The sequences of representative BMP inhibitors referred to herein are represented in SEQ ID NOs. 1-9.

[0143] During the culture of the stem cells of the present invention, the BMP inhibitor can be added to the culture medium every day, every two days, every three days, or every four days, while changing the culture medium every day, every two days, every three days, or every four days, as appropriate.

[0144] VEGF inhibitors In certain embodiments, the VEGF inhibitor is aflibercept, pegaptanib, tivozanib, 3-(4-bromo-2,6-difluoro-benzyloxy)-5-[3-(4-pyrrolidin-1-yl-butyl)-ureido]-isothiazole-4-carboxylic acid amide hydrochloride, axitinib, N-(4-bromo-2-fluorophenyl)-6-methoxy-7-[(1-methylpiperidin-4-yl-)methoxy]quinazolin-4-amine, an inhibitor of VEGF-R2 and VEGF-R1, axitinib, N,2-dimethyl-6-(2-(1-methyl-1H-imidazol-2-yl)thieno[3,2-b]pyridin-7-yloxy)benzo[b]thiophene-3-carboxamide, a tyrosine kinase inhibitor of the RET / PTC oncogenic kinase. and a combination thereof.

[0145] In certain preferred embodiments, the VEGF inhibitor is a VEGF receptor inhibitor, more preferably a VEGF receptor kinase inhibitor such as tivozanib (AV-951), AZD2932, midostaurin (pkc412), BAW2881 (NVP-BAW2881), nintedanib (BIBF 1120), SU5402, SU1498, BFH772, sorafenib, sunitinib, dovitinib (TKI258), semaxanib (SU5416), hypericin, vatalanib, ZM306416, AAL993, SU4312, DMXAA, or foretinib.

[0146] In certain embodiments, the VEGF receptor inhibitor is afatinib, imatinib, dacomitinib, dasatinib, ponatinib, KD-019, bosutinib, lapatinib ditosylate, AZD9291, neratinib, poziotinib, S-222611, suramin hexasodium, AL-6802, BGB-102, PB357, pyrotinib, nib), sunitinib, sorafenib tosylate, pazopanib, regorafenib, apatinib, axitinib, carbozantinib, lenvatinib, nintedanib, vandetanib, tivozanib, anlotinib, midostaurin, muparfostat, BMS-690514, ENMD-2076 , golvatinib, lucitanib, motesanib, necuparinib, RAF265, famitinib, telatinib, X82, ALNVSP, altiratinib, ABT348, MGCD516, OB318, ODM203, HHGV678, LY-3012207, CS2164, ilorasertib, radotinib, bafetinib, NRCAN-019, ABL001, metatinib tromethamine, rebastinib tosylate, or a multi-tyrosine kinase inhibitor such as VX-15.

[0147] tyrosine kinase inhibitors In certain embodiments, the medium comprises a tyrosine kinase inhibitor, such as nilotinib, ponatinib, dasatinib, gefitinib, erlotinib, sunitinib, or cabozantinib. In certain preferred embodiments, the tyrosine kinase inhibitor is a pan-ABL1 kinase inhibitor, such as ponatinib or dasatinib.

[0148] In certain embodiments, the media contains both a VEGF receptor kinase inhibitor and a tyrosine kinase inhibitor, which may be the same compound or different compounds, for example, a combination of ponatinib and tivozanib.

[0149] TGFβ or TGFβ receptor inhibitors TGF-β signaling is involved in many cellular functions, including cell growth, cell fate, and apoptosis. Signaling typically begins with the binding of a TGF-β superfamily ligand to a type II receptor, which recruits and phosphorylates a type I receptor. The type I receptor then phosphorylates SMADs, which function as nuclear transcription factors and regulate target gene expression. Alternatively, TGF-β signaling can activate the MAP kinase signaling pathway, for example, via p38 MAP kinase.

[0150] TGFβ superfamily ligands include bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), anti-Mullerian hormone (AMH), activin, nodal, and TGFβ.

[0151] TGFβ inhibitors, as used herein, include agents that reduce the activity of the TGFβ signaling pathway. There are many different ways of disrupting the TGFβ signaling pathway known in the art, any of which can be used in the present invention. For example, TGFβ signaling can be disrupted by inhibiting TGFβ expression using small interfering RNA strategies; inhibiting furin (a TGFβ-activating protease); inhibiting the pathway with physiological inhibitors, such as inhibiting BMPs with noggin, DAN, or DAN-like proteins; neutralizing TGFβ with monoclonal antibodies; inhibiting TGFβ receptor kinase 1 (also known as activin receptor-like kinase, ALK5), ALK4, ALK6, ALK7, or other TGFβ-related receptor kinases with small molecule inhibitors; inhibiting Smad2 and Smad3 signaling by overexpressing the physiological inhibitor Smad7 or by using thioredoxin as a Smad anchor, rendering the Smads inactivatable (Fuchs, Inhibition of TGFβ Signaling for the Treatment of Tumor Metastasis and Fibrotic Diseases. Current Signal Transduction Therapy 6(1):29-43(15), 2011).

[0152] For example, a TGFβ inhibitor may target a serine / threonine protein kinase selected from TGFβ receptor kinase 1, ALK4, ALK5, ALK7, or p38. ALK4, ALK5, and ALK7 are all closely related receptors of the TGFβ superfamily. ALK4 has a GI number of 91; ALK5 (also known as TGFβ receptor kinase 1) has a GI number of 7046; and ALK7 has a GI number of 658. An inhibitor of any of these kinases reduces the enzymatic activity of one or more of these kinases. Inhibition of ALK and p38 kinase has previously been shown to be associated with B-cell lymphoma (Bakkebo et al., "TGF-β-induced growth inhibition in B-cell lymphoma correlates with Smad 1 / 5 signaling and constitutively active p38MAPK," BMC Immunol. 11:57, 2010).

[0153] In certain embodiments, a TGFβ inhibitor can bind to and inhibit the activity of a Smad protein, such as R-SMAD or SMAD1-5 (ie, SMAD1, SMAD2, SMAD3, SMAD4, or SMAD5).

[0154] In certain embodiments, the TGFβ inhibitor may bind to and reduce the activity of a Ser / Thr protein kinase selected from TGFβ receptor kinase 1, ALK4, ALK5, ALK7, or p38.

[0155] In certain embodiments, the media of the present invention comprises an inhibitor of ALK5.

[0156] In certain embodiments, the TGFβ inhibitor or TGFβ receptor inhibitor does not include a BMP antagonist (ie, is an agent other than a BMP antagonist).

[0157] Various methods are known for determining whether a substance is a TGFβ inhibitor.For example, a cell assay can be used in which cells are stably transfected with a reporter construct containing the human PAI-1 promoter or Smad binding site that drives a luciferase reporter gene.The inhibition of luciferase activity compared to a control group can be used as a measure of the activity of a compound (De Gouville et al., Br.J.Pharmacol.145(2):166-177,2005, incorporated herein by reference).Another example is the ALPHASCREEN® phosphosensor assay for measuring kinase activity (Drew et al., J.Biomol.Screen.16(2):164-173,2011, incorporated herein by reference).

[0158] The TGFβ inhibitor useful for the present invention can be a protein, peptide, small molecule, small interfering RNA, antisense oligonucleotide, aptamer, antibody or antigen-binding portion thereof. The inhibitor can be naturally occurring or synthetic. Examples of small molecule TGFβ inhibitors that can be used in the context of the present invention include, but are not limited to, the small molecule inhibitors listed in Table 1 below.

[0159] Table 1: Small molecule TGF inhibitors targeting receptor kinases TIFF0007815111000003.tif142134

[0160] One or more of the inhibitors listed in Table 1 above, or a combination thereof, can be used as a TGFβ inhibitor in the present invention. In certain embodiments, the combination can include SB-525334 and SD-208 and A83-01; SD-208 and A83-01; or SD-208 and A83-01.

[0161] Those skilled in the art will recognize that there are numerous other small molecule inhibitors that are primarily designed to target other kinases but also inhibit TGFβ receptor kinase at high concentrations. For example, SB-203580 is a p38 MAP kinase inhibitor that can inhibit ALK5 at high concentrations (e.g., approximately 10 μM or higher). Such inhibitors that inhibit the TGFβ signaling pathway can also be used in the present invention. In certain embodiments, A83-01 can be added to the culture medium at a concentration of 10 nM to 10 μM, or 20 nM to 5 μM, or 50 nM to 1 μM. In certain embodiments, A83-01 can be added to the culture medium at approximately 500 nM. In certain embodiments, A83-01 can be added to the culture medium at a concentration of 350 to 650 nM, 450 to 550 nM, or approximately 500 nM. In certain embodiments, A83-01 can be added to the culture medium at a concentration of 25-75 nM, 40-60 nM, or about 50 nM.

[0162] SB-431542 can be added to the culture medium at a concentration of 80 nM to 80 μM, 100 nM to 40 μM, 500 nM to 10 μM, or 1 to 5 μM. For example, SB-431542 can be added to the culture medium at about 2 μM.

[0163] SB-505124 can be added to the culture medium at a concentration of 40 nM to 40 μM, 80 nM to 20 μM, or 200 nM to 1 μM. For example, SB-505124 can be added to the culture medium at about 500 nM.

[0164] SB-525334 can be added to the culture medium at a concentration of 10 nM to 10 μM, or 20 nM to 5 μM, or 50 nM to 1 μM. For example, SB-525334 can be added to the culture medium at about 100 nM.

[0165] LY 364947 can be added to the culture medium at a concentration of 40 nM to 40 μM, or 80 nM to 20 μM, or 200 nM to 1 μM. For example, LY 364947 can be added to the culture medium at about 500 nM.

[0166] SD-208 can be added to the culture medium at a concentration of 40 nM to 40 μM, or 80 nM to 20 μM, or 200 nM to 1 μM. For example, SD-208 can be added to the culture medium at about 500 nM.

[0167] S JN 2511 can be added to the culture medium at a concentration of 20 nM to 20 μM, or 40 nM to 10 μM, or 100 nM to 1 μM. For example, A83-01 can be added to the culture medium at approximately 200 nM.

[0168] p38 inhibitors "p38 inhibitors" may include inhibitors that directly or indirectly negatively regulate p38 signaling, such as agents that bind to and reduce the activity of at least one p38 isoform. p38 protein kinase (see GI No. 1432) is part of the mitogen-activated protein kinase (MAPK) family. MAPKs are serine / threonine-specific protein kinases that respond to extracellular stimuli, such as environmental stress and inflammatory cytokines, and regulate various cellular activities, such as gene expression, differentiation, division, proliferation, and cell survival / apoptosis. p38 MAPK exists as α, β, β2, γ, and δ isoforms.

[0169] Various methods are known to determine whether a substance is a p38 inhibitor, such as the detection of phosphorylation at Thrl80 / Tyrl82 with a phospho-specific antibody, which provides a well-established measure of cellular p38 activation or inhibition; biochemical recombinant kinase assays; tumor necrosis factor alpha (TNFα) secretion assays; and the DiscoverRx high-throughput screening platform for p38 inhibitors. Several p38 activity assay kits also exist (e.g., Millipore, Sigma-Aldrich).

[0170] In certain embodiments, high concentrations (e.g., greater than 100 nM, greater than 1 μM, greater than 10 μM, or greater than 100 μM) of p38 inhibitors can have the effect of inhibiting TGFβ. In other embodiments, p38 inhibitors do not inhibit TGFβ signaling.

[0171] Various p38 inhibitors are known in the art (see, e.g., Table 1). In some embodiments, the inhibitor that directly or indirectly negatively regulates p38 signaling is selected from the group consisting of SB-202190, SB-203580, VX-702, VX-745, PD-169316, RO-4402257, and BIRB-796.

[0172] In certain embodiments, the medium contains both (a) an inhibitor that binds to and reduces the activity of one or more kinases from the group consisting of ALK4, ALK5, and ALK7; and (b) an inhibitor that binds to and reduces the activity of p38.

[0173] In certain embodiments, the medium comprises an inhibitor that binds to and reduces the activity of ALK5 and an inhibitor that binds to and reduces the activity of p38.

[0174] In one embodiment, the inhibitor binds to its target (e.g., TGFβ and / or p38) and reduces its activity by more than 10%, more than 30%, more than 60%, more than 80%, more than 90%, more than 95%, or more than 99% compared to a control, as assessed by a cellular assay. Examples of cellular assays for measuring target inhibition are well known in the art, as described above.

[0175] TGFβ and / or p38 inhibitors may have IC50 values ​​of 2000 nM or less; less than 1000 nM; less than 100 nM; less than 50 nM; less than 30 nM; less than 20 nM, or less than 10 mM. The IC50 value refers to the effectiveness of an inhibitor in inhibiting the biological or biochemical function of its target. IC50 indicates the amount of a specific inhibitor required to inhibit a kinase by 50%. The IC50 value can be calculated according to the assay method described above. TGFβ and / or p38 inhibitors can exist in various forms, including natural or modified substrates, enzymes, receptors, small organic molecules, such as small natural or synthetic organic molecules up to 2000 Da, preferably less than 800 Da, peptidomimetics, inorganic molecules, peptides, polypeptides, antisense oligonucleotide aptamers, and structural or functional mimetics thereof, including small molecules.

[0176] In certain embodiments, the inhibitor of TGFβ and / or p38 may be an aptamer. As used herein, the term "aptamer" refers to a strand of oligonucleotide (DNA or RNA) that can adopt a highly specific three-dimensional conformation. Aptamers are designed to have high binding affinity and specificity for certain target molecules, including extracellular and intracellular proteins. Aptamers can be generated, for example, using the SELEX process (see, for example, Tuerk and Gold, Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA Polymerase. Science 249:505-510, 1990, incorporated herein by reference).

[0177] In certain embodiments, the inhibitor of TGFβ and / or p38 can be a small synthetic molecule having a molecular weight of 50-800 Da, 80-700 Da, 100-600 Da, or 150-500 Da.

[0178] In certain embodiments, inhibitors of TGFβ and / or p38 include pyridinylimidazoles or 2,4-disubstituted teridines or quinazolines, such as, for example: Contains TIFF0007815111000004.tif36128.

[0179] Specific examples of inhibitors of TGFβ and / or p38 that may be used in accordance with the present invention include, but are not limited to, SB-202190, SB-203580, SB-206718, SB-227931, VX-702, VX-745, PD-169316, RO-4402257, BIRB-796, A83-01 SB-431542, SB-505124, SB-525334, LY 364947, SD-208, SJ 2511 (see Table 2).

[0180] For example, SB-202190 can be added to the culture medium at a concentration of 50 nM to 100 μM, or 100 nM to 50 μM, or 1 μM to 50 μM. For example, SB-202190 can be added to the culture medium at approximately 10 μM.

[0181] SB-203580 can be added to the culture medium at a concentration of 50 nM to 100 μM, or 100 nM to 50 μM, or 1 μM to 50 μM. For example, SB-203580 can be added to the culture medium at approximately 10 μM.

[0182] VX-702 can be added to the culture medium at a concentration of 50 nM to 100 μM, or 100 nM to 50 μM, or 1 μM to 25 μM. For example, VX-702 can be added to the culture medium at approximately 5 μM.

[0183] VX-745 can be added to the culture medium at a concentration of 10 nM to 50 μM, or 50 nM to 50 μM, or 250 nM to 10 μM. For example, VX-745 can be added to the culture medium at approximately 1 μM.

[0184] PD-169316 can be added to the culture medium at a concentration of 100 nM to 200 μM, or 200 nM to 100 μM, or 1 μM to 50 μM. For example, PD-169316 can be added to the culture medium at approximately 20 μM.

[0185] RO-4402257 can be added to the culture medium at a concentration of 10 nM to 50 μM, or 50 nM to 50 μM, or 500 nM to 10 μM. For example, RO-4402257 can be added to the culture medium at approximately 1 μM.

[0186] BIRB-796 can be added to the culture medium at a concentration of 10 nM to 50 μM, or 50 nM to 50 μM, or 500 nM to 10 μM. For example, BIRB-796 can be added to the culture medium at approximately 1 μM.

[0187] For applicable concentrations for other factors in Table 2, see Table 1 and associated text above.

[0188] Table 2: Exemplary TGFβ and / or p38 inhibitors TIFF0007815111000005.tif226134TIFF0007815111000006.tif98128

[0189] Thus, in some embodiments, inhibitors that directly or indirectly negatively regulate TGFβ and / or p38 signaling are added to the culture medium at a concentration of 1 nM to 100 μM, 10 nM to 100 μM, 100 nM to 10 μM, or about 1 μM. For example, the total concentration of one or more inhibitors is 10 nM to 100 μM, 100 nM to 10 μM, or about 1 μM.

[0190] Oct4 activator The Oct4 activator is a drug that can activate a reporter gene driven by the Oct4 promoter, such as a luciferase gene under the transcriptional control of the Oct4 promoter, and more preferably, can activate both a reporter gene driven by the Oct4 promoter and a reporter gene driven by the Nanog promoter. Furthermore, when added to the reprogramming mixture together with the reprogramming factor tetrad (Oct4, Sox2, c-Myc, and Klf4), the Oct4 activator enhances iPSC reprogramming efficiency and accelerates the reprogramming process. Exemplary Oct4 activators are taught, for example, in U.S. Patent Application No. 20150191701 and Li et al. (2012) "Identification of Oct4-activating compounds that enhance reprogramming efficiency", PNAS 109(51):20853-8.

[0191] In certain embodiments, the Oct4 activator has the following formula: TIFF0007815111000007.tif35128, wherein: X 1 is C(R 12 ) or N; X 2 is C(R 4 ) or N; X 3 is C(R 5 ) or N; R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12are independently selected from hydrogen, halogen, —CN, —NO2, —NH2, —CF3, —CCl3, —OH, —SH, —SO3H, —C(O)OH, —C(O)NH2, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R 2 and R 3 may together form a substituted or unsubstituted heterocycloalkyl or a substituted or unsubstituted heteroaryl.

[0192] In certain preferred embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are independently selected from hydrogen, halogen, —CN, —NO, —NH, —CF, —CCl, —OH, —SH, —SOH, —C(O)OH, —C(O)NH, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, or substituted or unsubstituted heterocycloalkyl.

[0193] In certain preferred embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are independently selected from hydrogen, halogen, —CN, —NO 2 , —NH 2 , —CF 3 , —CCl 3 , —OH, —SH, —SO 3 H, —C(O)OH, —C(O)NH 2 , substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl.

[0194] In certain preferred embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 is hydrogen, halogen, -CN, -NO2, -NH2, -CF3, -CCl3, -OH, -SH, -SO3H, -C(O)OH, -C(O)NH2, substituted or unsubstituted C1 to C 10 and independently selected from alkyl, substituted or unsubstituted 2- to 10-membered heteroalkyl, or substituted or unsubstituted 3- to 8-membered heterocycloalkyl.

[0195] In certain preferred embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 is hydrogen, halogen, -CN, -NO2, -NH2, -CF3, -CCl3, -OH, -SH, -SO3H, -C(O)OH, -C(O)NH2, substituted or unsubstituted C1 to C 10 alkyl, or substituted or unsubstituted 2- to 10-membered heteroalkyl.

[0196] In certain preferred embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12are independently selected from hydrogen, halogen, —CN, —NO 2 , —NH 2 , —CF 3 , —CCl 3 , —OH, —SH, —SO 3 H, —C(O)OH, —C(O)NH 2 , unsubstituted alkyl, unsubstituted heteroalkyl, or substituted heterocycloalkyl.

[0197] In certain preferred embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are independently selected from hydrogen, halogen, —CN, —NO 2 , —NH 2 , —CF 3 , —CCl 3 , —OH, —SH, —SO 3 H, —C(O)OH, —C(O)NH 2 , unsubstituted alkyl, or unsubstituted heteroalkyl.

[0198] In certain preferred embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 is hydrogen, halogen, -CN, -NO2, -NH2, -CF3, -CCl3, -OH, -SH, -SO3H, -C(O)OH, -C(O)NH2, unsubstituted C1 to C 10 and independently selected from alkyl, unsubstituted 2- to 10-membered heteroalkyl, or unsubstituted 3- to 8-membered heterocycloalkyl.

[0199] In certain preferred embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R9 , R 10 , R 11 , and R 12 is hydrogen, halogen, -CN, -NO2, -NH2, -CF3, -CCl3, -OH, -SH, -SO3H, -C(O)OH, -C(O)NH2, unsubstituted C1 to C 10 alkyl, or unsubstituted 2-10 membered heteroalkyl.

[0200] In certain preferred embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 is hydrogen, halogen, unsubstituted C1-C 10 alkyl, or unsubstituted 2-10 membered heteroalkyl.

[0201] In certain preferred embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are independently selected from hydrogen, halogen, —N(CH 3 ) 2 , unsubstituted C 1 -C 5 alkyl, or unsubstituted C 1 -C 5 alkoxy.

[0202] In certain preferred embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R12 are independently selected from hydrogen, halogen, —N(CH 3 ) 2 , unsubstituted C 1 -C 5 alkyl, methoxy, ethoxy, or propoxy.

[0203] In certain embodiments, the Oct4 activator is: TIFF0007815111000008.tif142129.

[0204] In certain embodiments, the Oct4 activator has the structure: OAC1 with TIFF0007815111000009.tif29128.

[0205] TrkA inhibitors Representative examples of TrkA inhibitors include BMS-754807, GW441756, PF-06273340, sitravatinib (MGCD516), ANA-12, GNF-5837, belizatinib (TSR-011), larotrectinib (LOXO-101) sulfate, lestaurtinib, entrectinib (RXDX-101), GNF5837, and AG-879.

[0206] Preferably, the TrkA inhibitor is selective for TrkA over TrkB or TrkC, such as GW441756 and sitravatinib (MGCD516).

[0207] Preferably, the TrkA inhibitor is a potent and selective TrkA inhibitor, e.g., GW441756, having an IC50 of 10 nM or less, with the IC50 for inhibition of c-Raf1 and CDK2 being at least 100-fold greater than the IC50 for inhibition of TrkA.

[0208] Other representative Trk inhibitors can be those found in U.S. Patent No. 9,187,489 and International Publication No. WO 2013 / 183578, both of which are incorporated by reference in their entireties. Exemplary Trk inhibitors include PLX7486 and DS-6051.

[0209] Non-limiting examples of Trk inhibitors can be found in U.S. Publication No. 2015 / 0306086 and International Publication No. WO 2013 / 074518, both of which are incorporated by reference in their entireties. Exemplary Trk inhibitors include TSR-011.

[0210] Further examples of Trk inhibitors can be found in U.S. Patent No. 8,637,516, International Publication No. WO 2012 / 034091, U.S. Patent No. 9,102,671, International Publication No. WO 2012 / 116217, U.S. Publication No. 2010 / 0297115, International Publication No. WO 2009 / 053442, U.S. Patent No. 8,642,035, International Publication No. WO 2009092049, U.S. Patent No. 8,691,221, International Publication No. WO2006131952 (all of which are incorporated by reference in their entirety). Exemplary Trk inhibitors include GNF-4256, described in Cancer Chemother. Pharmacol. 75(1):131-141, 2015; and GNF-5837 (N-[3-[[2,3-dihydro-2-oxo-3-(1H-pyrrol-2-ylmethylene)-1H-indol-6-yl]amino]-4-methylphenyl]-N'-[2-fluoro-5-(trifluoromethyl)phenyl]-urea), described in ACS Med. Chem. Lett. 3(2):140-145, 2012 (each incorporated herein by reference in its entirety).

[0211] Additional examples of Trk inhibitors include those disclosed in U.S. Publication No. 2010 / 0152219, U.S. Patent No. 8,114,989, and International Publication No. WO 2006 / 123113, all of which are incorporated by reference in their entireties. Exemplary Trk inhibitors include: AZ623 described in Cancer 117(6):1321-1391,2011; AZD6918, described in Cancer Biol. Ther. 16(3):477-483, 2015; AZ64, described in Cancer Chemother.Pharmacol.70:477-486,2012; AZ-23 ((S)-5-chloro-N2-(1-(5-fluoropyridin-2-yl)ethyl)-N4-(5-isopropoxy-1H-pyrazol-3-yl)pyrimidine-2,4-diamine), described in Mol. Cancer Ther. 8:1818-1827, 2009; and AZD7451 (each incorporated by reference in its entirety).

[0212] Trk inhibitors can include those described in U.S. Patent Nos. 7,615,383; 7,384,632; 6,153,189; 6,027,927; 6,025,166; 5,910,574; 5,877,016; and 5,844,092 (each incorporated by reference in its entirety).

[0213] Further examples of Trk inhibitors include: CEP-751, described in Int. J. Cancer 72:672-679, 1997; CT327, described in Acta Derm.Venereol.95:542-548,2015; Compounds described in International Publication No. WO 2012 / 034095; Compounds described in U.S. Patent No. 8,673,347 and International Publication No. WO 2007 / 022999; Compounds described in U.S. Patent No. 8,338,417; Compounds described in International Publication No. WO 2016 / 027754; Compounds described in U.S. Patent No. 9,242,977; compounds described in U.S. Publication No. 2016 / 0000783; Sunitinib (N-(2-diethylaminoethyl)-5-[(Z)-(5-fluoro-2-oxo-1H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide), described in PLoS One 9:e95628,2014; Compounds described in International Publication No. WO 2011 / 133637; Compounds described in U.S. Patent No. 8,637,256; Compounds described in Expert. Opin. Ther. Pat. 24(7):731-744, 2014; Compounds described in Expert Opin. Ther. Pat. 19(3):305-319, 2009; (R)-2-phenylpyrrolidine-substituted imadizopyridazines, for example, (4-((5-chloro-4-(methylamino)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-3-methoxyphenyl)(morpholino)methanone, described in ACS Med. Chem. Lett. 6(5):562-567, 2015; GTx-186 and others described in PLoS One 8(12):e83380,2013; K252a ((9S-(9α,10β,12α))-2,3,9,10,11,12-hexahydro-10-hydroxy-10-(methoxycarbonyl)-9-methyl-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocin-1-one), described in Mol. Cell Biochem. 339(1-2):201-213, 2010; 4-aminopyrazolylpyrimidines, for example, AZ-23 (((S)-5-chloro-N2-(1-(5-fluoropyridin-2-yl)ethyl)-N4-(5-isopropoxy-1H-pyrazol-3-yl)pyrimidine-2,4-diamine) described in J. Med. Chem. 51(15):4672-4684, 2008); PHA-739358 (danusertib), described in Mol. Cancer Ther. 6:3158, 2007; Go 6976 (5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-12-propanenitrile), described in J. Neurochem. 72:919-924, 1999; GW441756 ((3Z)-3-[(1-methylindol-3-yl)methylidene]-1H-pyrrolo[3,2-b]pyridin-2-one) described in IJAE 115:117,2010; Milciclib (PHA-848125AC), described in J. Carcinog. 12:22, 2013; AG-879 ((2E)-3-[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-2-cyano-2-propenethioamide); Altiratinib (N-(4-((2-(cyclopropanecarboxamido)pyridin-4-yl)oxy)-2,5-difluorophenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide); Cabozantinib (N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide); Lestaurtinib ((5S,6S,8R)-6-hydroxy-6-(hydroxymethyl)-5-methyl-7,8,14,15-tetrahydro-5H-16-oxa-4b,8a,14-triaza-5,8-methanodibenzo[b,h]cycloocta[jkl]cyclopenta[e]-as-indacen-13(6H)-one); Dovatinib (4-amino-5-fluoro-3-[6-(4-methylpiperazin-1-yl)-1H-benzimidazol-2-yl]quinolin-2(1H)-one mono-2-hydroxypropanoate hydrate); Sitravatinib (N-(3-fluoro-4-((2-(5-(((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide); ONO-5390556; Regorafenib (4-[4-({[4-chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)-3-fluorophenoxy]-N-methylpyridine-2-carboxamide hydrate); VSR-902A (all of the above references are incorporated herein by reference in their entirety).

[0214] In some embodiments, the Trk inhibitor is (6R)-9-Fluoro-2,11,15,19,20,23-hexaazapentacyclo[15.5.2.1 7,11 .0 2,6 .0 20,24 ]pentacosa-1(23),7,9,17(24),18,21-hexaene-16,25-dione; (6R)-12-Oxa-2,16,20,21,24,26-hexaazapentacyclo[16.5.2.1 7,11 .0 2,6 .0 21,25 ]hexacosa-1(24),7(26),8,10,18(25),19,22-heptaen-17-one; (6R)-9-Fluoro-13-oxa-2,11,17,21,22,25-hexaazapentacyclo[17.5.2.0 2,6 .0 7,12 .0 22,26 ]hexacosa-1(25),7,9,11,19(26),20,23-heptaen-18-one; (6R)-9-Fluoro-15-hydroxy-13-oxa-2,11,17,21,22,25-hexaazapentacyclo[17.5.2.0 2,6 .0 7,12 .0 22,26]hexacosa-1(25),7,9,11,19(26),20,23-heptaen-18-one; (6R,13S)-9-Fluoro-13-hydroxy-2,11,15,19,20,23-hexaazapentacyclo-[15.5.2.1 7,11 .0 2,6 .0 20,24 ]pentacosa-1(23),7,9,17(24),18,21-hexaene-16,25-dione; (6R,15R)-9-Fluoro-15-hydroxy-13-oxa-2,11,17,21,22,25-hexaazapentacyclo-[17.5.2.0 2,6 .0 7,12 .0 22,26 ]hexacosa-1(25),7,9,11,19(26),20,23-heptaen-18-one; (6R,13R)-9-Fluoro-13-hydroxy-2,11,15,19,20,23-hexaazapentacyclo-[15.5.2.1 7,11 .0 2,6 .0 20,24 ]pentacosa-1(23),7,9,17(24),18,21-hexaene-16,25-dione; (6R)-9-Fluoro-13-oxa-2,11,16,20,21,24-hexaazapentacyclo[16.5.2.0 2,6 .0 7,12 .0 21,25 ]pentacosa-1(24),7,9,11,18(25),19,22-heptaen-17-one; (6R)-9-Fluoro-13-oxa-2,11,18,22,23,26-hexaazapentacyclo[18.5.2.0 2,6 .0 7,12 .0 23,27 ]heptacosa-1(26),7,9,11,20(27),21,24-heptaen-19-one; (6R)-9-Fluoro-2,11,16,20,21,24-hexaazapentacyclo[16.5.2.1 7,11 .0 2,6 .0 21,25]Hexacosa-1(24),7,9,18(25),19,22-hexaene-17,26-dione; (6R)-9-Fluoro-2,11,13,16,20,21,24-heptaazapentacyclo[16.5.2.0 2,6 .0 7,12 .0 21,25 ]pentacosa-1(24),7,9,11,18(25),19,22-heptaen-17-one; (6R)-9-Fluoro-2,11,13,17,21,22,25-heptaazapentacyclo[17.5.2.0 2,6 .0 7,12 .0 22,26 ]hexacosa-1(25),7,9,11,19(26),20,23-heptaen-18-one; (6R)-9-Fluoro-13,16-dioxa-2,11,20,21,24-pentaazapentacyclo[16.5.2.0 2,6 .0 7,12 .0 21,25 ]-pentacosa-1(24),7,9,11,18(25),19,22-heptaen-17-one; (6R)-9-Fluoro-14-oxa-2,11,18,19,22-pentaazapentacyclo[14.5.2.1 7,11 .0 2,6 .0 19,23 ]tetracosa-1(22),7,9,16(23),17,20-hexaene-15,24-dione; (6R)-9-Fluoro-13,16-dioxa-2,11,17,21,22,25-hexaazapentacyclo[17.5.2.0 2,6 .0 7,12 .0 22,26 ]hexacosa-1(25),7,9,11,19(26),20,23-heptaen-18-one; (6R,13R)-9,13-difluoro-2,11,15,19,20,23-hexaazapentacyclo[15.5.2.1 7,11 .0 2,6 .0 20,24 ]pentacosa-1(23),7,9,17(24),18,21-hexaene-16,25-dione; (6R)-9-Fluoro-17-methyl-13-oxa-2,11,17,21,22,25-hexaazapentacyclo[17.5.2.0 2,6 .0 7,12 .0 22,26 ]hexacosa-1(25),7,9,11,19(26),20,23-heptaen-18-one; (6R)-9,15,15-trifluoro-13-oxa-2,11,17,21,22,25-hexaazapentacyclo[17.5.2.0 2,6 .0 7,12 .0 22,26 ]hexacosa-1(25),7,9,11,19(26),20,23-heptaen-18-one; (6R)-9-Fluoro-13-oxa-2,17,21,22,25-pentaazapentacyclo[17.5.2.0 2,6 .0 7,12 .0 22,26 ]hexacosa-1(25),7,9,11,19(26),20,23-heptaen-18-one; (6R)-9-Fluoro-13-oxa-2,16,20,21,24-pentaazapentacyclo[16.5.2.0 2,6 .0 7,12 .0 21,25 ]pentacosa-1(24),7,9,11,18(25),19,22-heptaene; 1-[(6R)-9-fluoro-13-oxa-2,16,20,21,24-pentaazapentacyclo[16.5.2.0 2,6 .0 7,12 .0 21,25 ]pentacosa-1(24),7,9,11,18(25),19,22-heptaen-16-yl]ethan-1-one; 1-[(6R)-9-fluoro-13-oxa-2,16,20,21,24-pentaazapentacyclo[16.5.2.0 2,6 .0 7,12 .0 21,25 ]pentacosa-1(24),7,9,11,18(25),19,22-heptaen-16-yl]-2-hydroxyethan-1-one; (6R)-9-Fluoro-13-oxa-2,17,21,22,25-pentaazapentacyclo[17.5.2.0 2,6 .0 7,12 .0 22,26 ]hexacosa-1(25),7,9,11,19(26),20,23-heptaene; (6R)-9-Fluoro-16-methanesulfonyl-13-oxa-2,16,20,21,24-pentaazapentacyclo[16.5.2.0 2,6 .0 7,12 .0 21,25 ]pentacosa-1(24),7,9,11,18(25),19,22-heptaene; 2-[(6R)-9-fluoro-13-oxa-2,16,20,21,24-pentaazapentacyclo[16.5.2.0 2,6 .0 7,12 .0 21,25 ]pentacosa-1(24),7,9,11,18(25),19,22-heptaen-16-yl]acetic acid; (6R)-9-Fluoro-17-methanesulfonyl-13-oxa-2,17,21,22,25-pentaazapentacyclo[17.5.2.0 2,6 .0 7,12 .0 22,26 ]hexacosa-1(25),7,9,11,19(26),20,23-heptaene; (6R)-N-ethyl-9-fluoro-13-oxa-2,17,21,22,25-pentaazapentacyclo[17.5.2.0 2,6 .0 7,12 .0 22,26 ]Hexacosa-1(25),7,9,11,19(26),20,23-heptaene-17-carboxamide; (6R)-N-ethyl-9-fluoro-13-oxa-2,16,20,21,24-pentaazapentacyclo-[16.5.2.0 2,6 .0 7,12 .0 21,25 ]Pentacosa-1(24),7,9,11,18(25),19,22-heptaene-16-carboxamide; (6S)-9-Fluoro-4,13-dioxa-2,11,17,21,22,25-hexaazapentacyclo[17.5.2.0 2,6 .0 7,12 .0 22,26 ]Hexacosa-1(25),7(12),8,10,19(26),20,23-heptaene-3,18-dione; (6S)-9-Fluoro-4,13-dioxa-2,11,16,20,21,24-hexaazapentacyclo[16.5.2.0 2,6 .0 7,12 .0 21,25 ]Pentacosa-1(24),7(12),8,10,18(25),19,22-heptaene-3,17-dione; (6R)-9-Fluoro-2,11,16,20,21,24-hexaazapentacyclo[16.5.2.0 2,6 .0 7,12 .0 21,25 ]pentacosa-1(24),7,9,11,18(25),19,22-heptaen-17-one; (6R)-9-Fluoro-15-methyl-2,11,16,20,21,24-hexaazapentacyclo[16.5.2.0 2,6 .0 7,12 .0 21,25 ]pentacosa-1(24),7,9,11,18(25),19,22-heptaen-17-one; (6R,13R)-9-Fluoro-13-methyl-2,11,15,19,20,23-hexaazapentacyclo[15.5.2.1 7,11 .0 2,6 .0 20,24 ]pentacosa-1(23),7,9,17(24),18,21-hexaene-16,25-dione; (6R,13S)-9-Fluoro-13-methyl-2,11,15,19,20,23-hexaazapentacyclo[15.5.2.1 7,11 .0 2,6 .0 20,24 ]pentacosa-1(23),7,9,17(24),18,21-hexaene-16,25-dione; (6R)-9-Fluoro-15,15-dimethyl-13-oxa-2,11,17,21,22,25-hexaazapentacyclo[17.5.2.0 2,6 .0 7,12 .0 22,26 ]hexacosa-1(25),7,9,11,19(26),20,23-heptaen-18-one; (6R)-9-Fluoro-15,15-dimethyl-2,11,16,20,21,24-hexaazapentacyclo[16.5.2.0 2,6 .0 7,12 .0 21,25 ]pentacosa-1(24),7,9,11,18(25),19,22-heptaen-17-one; (6R)-9-Fluoro-13-oxa-2,11,16,17,21,25,26,29-octaazahexacyclo[21.5.2.0 2,6 .0 7,12 .0 16,20 .0 26,30 ]triaconta-1(29),7,9,11,17,19,23(30),24,27-nonaen-22-one; (6R)-9-Fluoro-13-oxa-2,11,19,21,25,26,29-heptaazahexacyclo[21.5.2.0 2,6 .0 7,12 .0 15,20 .0 26,30 ]triaconta-1(29),7,9,11,15(20),16,18,23(30),24,27-decaen-22-one; (6R)-9-Fluoro-13,13-dimethyl-2,11,15,19,20,23-hexaazapentacyclo[15.5.2.1 7,11 .0 2,6 .0 20,24 ]pentacosa-1(23),7,9,17(24),18,21-hexaene-16,25-dione; (4R,6R,15S)-9-Fluoro-4,15-dihydroxy-13-oxa-2,17,21,22,25-pentaazapentacyclo[17.5.2.0 2,6 .0 7,12 .0 22,26]hexacosa-1(25),7(12),8,10,19(26),20,23-heptaen-18-one; (4R,6S,15S)-9-Fluoro-4,15-dihydroxy-13-oxa-2,17,21,22,25-pentaazapentacyclo[17.5.2.0 2,6 .0 7,12 .0 22,26 ]hexacosa-1(25),7(12),8,10,19(26),20,23-heptaen-18-one; (4R,6R)-9-Fluoro-4-hydroxy-13-oxa-2,17,21,22,25-pentaazapentacyclo[17.5.2.0 2,6 .0 7,12 .0 22,26 ]hexacosa-1(25),7(12),8,10,19(26),20,23-heptaen-18-one; (4R,6S)-9-Fluoro-4-hydroxy-13-oxa-2,17,21,22,25-pentaazapentacyclo[17.5.2.0 2,6 .0 7,12 .0 22,26 ]hexacosa-1(25),7(12),8,10,19(26),20,23-heptaen-18-one; (4R,6R)-9-Fluoro-4-hydroxy-13-oxa-2,16,20,21,24-pentaazapentacyclo[16.5.2.0 2,6 .0 7,12 .0 21,25 ]pentacosa-1(24),7,9,11,18(25),19,22-heptaen-17-one; (4R,6S)-9-Fluoro-4-hydroxy-13-oxa-2,16,20,21,24-pentaazapentacyclo[16.5.2.0 2,6 .0 7,12 .0 21,25 ]pentacosa-1(24),7,9,11,18(25),19,22-heptaen-17-one; (4R,6R,15R)-9-Fluoro-4,15-dihydroxy-13-oxa-2,17,21,22,25-pentaazapentacyclo[17.5.2.0 2,6 .07,12 .0 22,26 ]hexacosa-1(25),7(12),8,10,19(26),20,23-heptaen-18-one; (4R,6S,15R)-9-Fluoro-4,15-dihydroxy-13-oxa-2,17,21,22,25-pentaazapentacyclo[17.5.2.0 2,6 .0 7,12 .0 22,26 ]hexacosa-1(25),7(12),8,10,19(26),20,23-heptaen-18-one; and (15S)-4,4,9-trifluoro-15-hydroxy-13-oxa-2,17,21,22,25-pentaazapentacyclo[17.5.2.0 2,6 .0 7,12 .0 22,26 ]hexacosa-1(25),7(12),8,10,19(26),20,23-heptaen-18-one, or It is selected from the group consisting of pharmaceutically acceptable salts thereof.

[0215] In some embodiments, the Trk inhibitor is (R)-N-tert-butyl-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-(pyridin-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-(3-methylpyridin-2-yl)pyrazolo[1,5-a]-pyrimidine-3-carboxamide; (R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-(2-morpholinoethyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-((5-methylfuran-2-yl)methyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-(1-methyl-1H-pyrazol-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-((trans)-4-hydroxycyclohexyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-(1-hydroxy-2-methylpropan-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-(2-methyl-1-morpholinopropan-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-methylpyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-1-(5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carbonyl)piperidine-4-carboxylic acid; (R)-2-(1-(5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carbonyl)piperidin-4-yl)acetic acid; (R)-N-Cyclopropyl-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-N-Cyclobutyl-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; N-((2S)-bicyclo[2.2.1]heptan-2-yl)-5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-(1-(hydroxymethyl)cyclopropyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-(2-hydroxy-2-methylpropyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)((S)-3-hydroxypyrrolidin-1-yl)methanone; (5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)((R)-3-hydroxypyrrolidin-1-yl)methanone; (R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-(tetrahydro-2H-pyran-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-((1-methyl-1H-imidazol-4-yl)methyl)pyrazole[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-((1-methyl-1H-pyrazol-4-yl)methyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-(2-(1-methyl-1H-imidazol-5-yl)ethyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-(2-(2-oxoimidazolidin-1-yl)ethyl)pyrazole[1,5-a]pyrimidine-3-carboxamide; (R)—N-(2-(1H-imidazol-4-yl)ethyl)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazole[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-((R)-2,3-dihydroxypropyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N,N-dimethylpyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)—N-(2-(1H-imidazol-1-yl)ethyl)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-((S)-2,3-dihydroxypropyl)pyrazzolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-(5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)(3-hydroxyazetidin-1-yl)methanone; (R)-(5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)(3-hydroxy-3-methylazetidin-1-yl)methanone; trans-4-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)cyclohexanecarboxylic acid; 5-((R)-2-(5-fluoro-2-methoxyphenyl)pyrrolidin-1-yl)-N-((trans)-4-hydroxycyclohexyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(3-fluorophenyl)pyrrolidin-1-yl)-N-((trans)-4-hydroxycyclohexyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-N-tert-butyl-5-(2-(3-fluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-N-cyclopropyl-5-(2-(3-fluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)—N-(2-cyanopropan-2-yl)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)—N-(cyanomethyl)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-(1-fluoro-2-methylpropan-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; N-cyclopropyl-5-((2R,4R)-2-(3-fluorophenyl)-4-hydroxypyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; N-tert-butyl-5-((2R,4R)-2-(3-fluorophenyl)-4-hydroxypyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((2R,4R)-2-(3-fluorophenyl)-4-hydroxypyrrolidin-1-yl)-N-methylpyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-(1-(methylsulfonyl)piperidin-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-(1-sulfamoylpiperidin-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-(2-(methylsulfonamido)ethyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-(2-sulfamoylethyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-N-cyclopropyl-5-(2-(5-fluoro-2-methoxyphenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoro-2-methoxyphenyl)pyrrolidin-1-yl)-N-(2-hydroxy-2-methylpropyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-(4-hydroxy-4-methylcyclohexyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (diastereomer 1); 5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-(4-hydroxy-4-methylcyclohexyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (diastereomer 2); (R)-N-Cyclopropyl-5-(2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-N-tert-butyl-5-(2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoro-2-methoxyphenyl)pyrrolidin-1-yl)-N-(2-morpholinoethyl)-pyrazolo[1,5-a]pyrimidine-3-carboxamide; N-((S)-2,3-dihydroxypropyl)-5-((R)-2-(5-fluoro-2-methoxyphenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; N-((R)-2,3-dihydroxypropyl)-5-((R)-2-(5-fluoro-2-methoxyphenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-(2-methyl-1-(methylsulfonamido)propan-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-N-(2-amino-2-methylpropyl)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-N-tert-butyl-5-(4,4-difluoro-2-(3-fluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-(1,3-dihydroxy-2-methylpropan-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-((3S,4R)-3-fluoropiperidin-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; N-((S)-2,3-dihydroxypropyl)-5-((R)-2-(5-fluoro-2-(trifluoromethyl)phenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; N-((R)-2,3-dihydroxypropyl)-5-((R)-2-(5-fluoro-2-(trifluoromethyl)phenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoro-2-(trifluoromethyl)phenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-methoxypyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(5-(2,5-difluorophenyl)-2,2-dimethylpyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-N-Cyclopropyl-5-(5-(2,5-difluorophenyl)-2,2-dimethylpyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)—N-(2-cyanopropan-2-yl)-5-(2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-(1-(methylsulfonyl)piperidin-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)—N-(1-fluoro-2-methylpropan-2-yl)-5-(2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-(tetrahydro-2H-pyran-4yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-methoxypyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(3-fluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; ((R)-5-(2-(3-fluorophenyl)pyrrolidin-1-yl)-N-methoxypyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(3-fluoro-5-(2-morpholinoethoxy)phenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-N-cyclopropyl-5-(2-(3-fluoro-5-(2-methoxyethoxy)phenyl)pyrrolidin-1-yl)pyrazole[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(3-fluoro-5-(2-methoxyethoxy)phenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-N-Cyclopropyl-5-(2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-N-tert-butyl-5-(2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)-N-(1-fluoro-2-methylpropan-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)-N-methoxypyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-1-(5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)-cyclopropanecarboxylic acid; (R)-N-cyclopropyl-5-(2-(3-fluoro-5-(2-morpholinoethoxy)phenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoro-2-(2-morpholinoethoxy)phenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-N-cyclopropyl-5-(2-(5-fluoro-2-(2-morpholinoethoxy)phenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-((S)-2,3-dihydroxypropoxy)pyrazolo[1-,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoro-2-(2-methoxyethoxy)phenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-N-cyclopropyl-5-(2-(5-fluoro-2-(2-methoxyethoxy)phenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-(1-methylcyclopropyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-(5-(2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)(3-hydroxy-3-methylazetidin-1-yl)methanone; (R)-5-(2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-isopropylpyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-(5-(2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)(pyrrolidin-1-yl)methanone; (R)—N-(5-fluoropyridin-2-yl)-5-(2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-(5-(2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)(3-methoxyazetidin-1-yl)methanone; N-(3-chloro-2-fluoropropyl)-5-((R)-2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-(1-(trifluoromethyl)cyclopropyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-((trans)-4-hydroxycyclohexyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-((cis)-4-hydroxycyclohexyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-N-Cyclobutyl-5-(2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-(1-methylcyclobutyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-((1S,2S)-2-hydroxycyclopentyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-((1S,2R)-2-hydroxycyclopentyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-((1S,3S)-3-hydroxycyclopentyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-N-(cyclopropylmethyl)-5-(2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-(1-(hydroxymethyl)cyclopropyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-(5-(2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)(3-hydroxyazetidin-1-yl)methanone; 5-((R)-2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-((S)-2-hydroxypropyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-((R)-2-hydroxypropyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-(2-hydroxy-2-methylpropyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-(2-hydroxyethyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; N-(1-cyclopropylethyl)-5-((R)-2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-methylpyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-((R)-1-hydroxypropan-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-((S)-1-hydroxypropan-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-(1-methoxypropan-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-(2-hydroxy-3-methoxypropyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-((trans)-2-hydroxycyclopentyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-((S)-1-hydroxy-3-methylbutan-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-((R)-1-hydroxy-3-methylbutan-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; N-((R)-1-cyclopropylethyl)-5-((R)-2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; N-((S)-1-cyclopropylethyl)-5-((R)-2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-(3-hydroxy-2,2-dimethyl-propyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-Azetidin-1-yl(5-(2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)methanone; (R)-(5-(2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)(3-(hydroxymethyl)azetidin-1-yl)methanone; (5-((R)-2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)((S)-3-hydroxypyrrolidin-1-yl)methanone; 5-((R)-2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-((R)-1,1,1-trifluoropropan-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-((S)-1,1,1-trifluoropropan-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-(2,2,2-trifluoroethyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-(1-hydroxy-2-methylpropan-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-((1R,2R)-2-hydroxycyclopentyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)—N-(2,2-difluoroethyl)-5-(2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-((1R,2S)-2-hydroxycyclopentyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-((1R,2R)-2-hydroxycyclohexyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-(5-(2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)(piperidin-1-yl)methanone; 5-((R)-2-(5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-((2R,3S,4S)-3-(hydroxymethyl)bicyclo[2.2.1]heptan-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-(5-(2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)(3-hydroxyazetidin-1-yl)methanone; 5-((R)-2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)-N-((trans)-4-hydroxycyclohexyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-tert-butyl 3-(5-(2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)propylcarbamate; (R)—N-(3-aminopropyl)-5-(2-(5-fluoro-2-oxo-1,2-dihydropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; N-((S)-2,3-dihydroxypropyl)-5-((R)-2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; N-((S)-3-chloro-2-hydroxypropyl)-5-((R)-2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; N-((R)-3-chloro-2-hydroxypropyl)-5-((R)-2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)—N-(2-chloroethoxy)-5-(2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-(5-(2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)(3-hydroxy-3-methylazetidin-1-yl)methanone; (R)-5-(2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)-N-(3-hydroxypropyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; N-(2,3-dihydroxypropyl)-5-((R)-2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; N-((R)-2,3-dihydroxypropyl)-5-((R)-2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)-N-(4-hydroxybutyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)—N-(2-tert-butoxyethoxy)-5-(2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)-N-methylpyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)-N-((1S,3S)-3-hydroxycyclopentyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)-N-(2-hydroxyethyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)-N-((S)-2-hydroxypropyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)-N-((R)-2-hydroxypropyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)-N-(2-hydroxy-2-methylpropyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)—N-(1,3-dihydroxypropan-2-yl)-5-(2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)-N-(6-oxo-1,6-dihydropyridin-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; R)-5-(2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)-N-(1-(methylsulfonyl)piperidin-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-N-(2-chloroethyl)-5-(2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)—N-(2-bromoethoxy)-5-(2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-(2-hydroxyethyl)pyrazolo[1,5a]pyrimidine-3-carboxamide; 5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-(2-hydroxypropyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-(2-hydroxypropyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-(3-hydroxy-2,2-dimethylpropyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-((1S,3S)-3-hydroxycyclopentyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-(2-(4-hydroxypiperidin-1-yl)ethyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-(2-(4-methylpiperazin-1-yl)ethyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-(2-methoxyethyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-(1,3-dihydroxypropan-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-((2S,3R)-1,3-dihydroxybutan-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-((2S,3S)-1,3-dihydroxybutan-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-((2R,3S)-1,3-dihydroxybutan-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-((S)-1-hydroxypropan-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-((S)-1-hydroxybutan-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-((S)-1-hydroxy-3-methylbutan-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-N-((S)-1-hydroxy-3,3-dimethylbutan-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; N-cyclopropyl-5-(2-(5-fluoro-2-methylpyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; N-cyclopropyl-5-(2-(2-ethyl-5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo-[1,5-a]pyrimidine-3-carboxamide; (R)-N-tert-butyl-5-(2-(5-fluoro-2-methylpyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoro-2-methylpyridin-3-yl)pyrrolidin-1-yl)-N-isopropylpyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-N-Cyclobutyl-5-(2-(5-fluoro-2-methylpyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoro-2-methylpyridin-3-yl)pyrrolidin-1-yl)-N-methylpyrazolo-[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoro-2-methylpyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoro-2-methylpyridin-3-yl)pyrrolidin-1-yl)-N-(2-hydroxyethyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoro-2-methylpyridin-3-yl)pyrrolidin-1-yl)-N-((R)-2-hydroxypropyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoro-2-methylpyridin-3-yl)pyrrolidin-1-yl)-N-(1-methylcyclopropyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoro-2-methylpyridin-3-yl)pyrrolidin-1-yl)-N-(2-methoxyethyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-(5-(2-(5-fluoro-2-methylpyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)(3-hydroxyazetidin-1-yl)methanone; (R)-5-(2-(5-fluoro-2-methylpyridin-3-yl)pyrrolidin-1-yl)-N-(1-(hydroxymethyl)cyclopropyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(5-fluoro-2-methylpyridin-3-yl)pyrrolidin-1-yl)-N-((trans)-4-hydroxycyclohexyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(5-fluoro-2-methylpyridin-3-yl)pyrrolidin-1-yl)-N-((cis)-4-hydroxycyclohexyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(5-fluoro-2-methylpyridin-3-yl)pyrrolidin-1-yl)-N-((1S,3S)-3-hydroxycyclopentyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(5-fluoro-2-methylpyridin-3-yl)pyrrolidin-1-yl)-N-((1R,2R)-2-hydroxycyclopentyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(5-fluoro-2-methylpyridin-3-yl)pyrrolidin-1-yl)-N-((R)-quinuclidin-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(2-ethyl-5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-((trans)-4-hydroxycyclohexyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(2-ethyl-5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-((1S,3S)-3-hydroxycyclopentyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(2-ethyl-5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-(2-hydroxy-2-methylpropyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-N-tert-butyl-5-(2-(5-fluoro-2-oxo-1,2-dihydropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)—N-(2-chloroethyl)-5-(2-(5-fluoro-2-oxo-1,2-dihydropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; N-cyclopropyl-5-((2R)-2-(2-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-5-fluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((2R)-2-(2-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-5-fluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; N-cyclopropyl-5-((2R)-2-(3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-5-fluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((2R)-2-(3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-5-fluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; N-cyclopropyl-5-((2R)-2-(3-(2,3-dihydroxypropoxy)-5-fluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((2R)-2-(3-(2,3-dihydroxypropoxy)-5-fluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; N-cyclopropyl-5-((2R)-2-(2-(2,3-dihydroxypropoxy)-5-fluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((2R)-2-(2-(2,3-dihydroxypropoxy)-5-fluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((2R,5S)-2-(5-fluoropyridin-3-yl)-5-(hydroxymethyl)pyrrolidin-1-yl)-N-((R)-1,1,1-trifluoropropan-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((2R,5S)-2-(5-fluoropyridin-3-yl)-5-(hydroxymethyl)pyrrolidin-1-yl)-N-((S)-1,1,1-trifluoropropan-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((2R,5S)-2-(5-fluoropyridin-3-yl)-5-(hydroxymethyl)pyrrolidin-1-yl)-N-(1-methylcyclopropyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((2R,5S)-2-(5-fluoropyridin-3-yl)-5-(hydroxymethyl)pyrrolidin-1-yl)-N-isopropyl-pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((2R,4S)-2-(3-fluorophenyl)-4-hydroxypyrrolidin-1-yl)-N-((S)-1,1,1-trifluoropropan-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((2R,4S)-2-(3-fluorophenyl)-4-hydroxypyrrolidin-1-yl)-N-isopropylpyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((2R,4S)-2-(3-fluorophenyl)-4-hydroxypyrrolidin-1-yl)-N-methylpyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((2S,5R)-5-(5-fluoropyridin-3-yl)-2-(hydroxymethyl)-2-methylpyrrolidin-1-yl)-N-isopropylpyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((2S,5R)-5-(5-fluoropyridin-3-yl)-2-(hydroxymethyl)-2-methylpyrrolidin-1-yl)-N-((S)-1,1,1-trifluoropropan-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-(5-(2-(2-amino-5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)(azetidin-1-yl)methanone; (R)-tert-butyl 3-(5-(2-(2-chloro-5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamido)propylcarbamate; (R)-N-(3-aminopropyl)-5-(2-(2-chloro-5-fluoropyridin-3-yl)pyrrolidinyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)—N-(2-tert-butoxyethoxy)-5-(2-(2-chloro-5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(2-chloro-5-fluoropyridin-3-yl)pyrrolidin-1-yl)-N-(2-hydroxyethoxy)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-N-tert-butyl-5-(2-(5-fluoro-1-methyl-2-oxo-1,2-dihydropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoro-1-methyl-2-oxo-1,2-dihydropyridin-3-yl)pyrrolidin-1-yl)-N-isopropylpyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-N-Cyclopropyl-5-(2-(5-fluoro-1-methyl-2-oxo-1,2-dihydropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoro-1-methyl-2-oxo-1,2-dihydropyridin-3-yl)pyrrolidin-1-yl)-N-(6-methylpyridin-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-N-Cyclobutyl-5-(2-(5-fluoro-1-methyl-2-oxo-1,2-dihydropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoro-1-methyl-2-oxo-1,2-dihydropyridin-3-yl)pyrrolidin-1-yl)-N-(pyridin-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)—N-(cyclopropylmethyl)-5-(2-(5-fluoro-1-methyl-2-oxo-1,2-dihydropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(5-fluoro-1-methyl-2-oxo-1,2-dihydropyridin-3-yl)pyrrolidin-1-yl)-N-((S)-1-hydroxy-3,3-dimethylbutan-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(5-fluoro-1-methyl-2-oxo-1,2-dihydropyridin-3-yl)pyrrolidin-1-yl)-N-((1R,2R)-2-hydroxycyclohexyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; N-((R)-1-cyclopropylethyl)-5-((R)-2-(5-fluoro-1-methyl-2-oxo-1,2-dihydropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; N-((S)-1-cyclopropylethyl)-5-((R)-2-(5-fluoro-1-methyl-2-oxo-1,2-dihydropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoro-1-methyl-2-oxo-1,2-dihydropyridin-3-yl)pyrrolidin-1-yl)-N-(1-methylcyclopropyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; 5-((R)-2-(5-fluoro-1-methyl-2-oxo-1,2-dihydropyridin-3-yl)pyrrolidin-1-yl)-N-((trans)-4-hydroxycyclohexyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoro-1-methyl-2-oxo-1,2-dihydropyridin-3-yl)pyrrolidin-1-yl)-N-(5-fluoropyridin-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)-N-(3-methyl-1H-pyrazol-5-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)-5-(2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)-N-(1-methyl-1H-pyrazol-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)—N-(3-cyclopropyl-1H-pyrazol-5-yl)-5-(2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; (R)—N-(3-ethyl-1H-pyrazol-5-yl)-5-(2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide; and (R)-5-(2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)-N-(1-isopropyl-1H-pyrazol-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide, or a pharmaceutically acceptable salt thereof.

[0216] In some embodiments, the Trk inhibitor is 5-fluoro-2-[[(1S)-1-(5-fluoro-2-pyridyl)ethyl]amino]-6-[(5-isopropoxy-1H-pyrazol-3-yl)amino]pyridine-3-carbonitrile; ((2E)-3-[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-2-cyano-2-propenethioamide); 2,2-Dichloro-N-[3-[(7-chloroquinolin-4-yl)amino]propyl]-N-methylacetamide; N-[3-[[2,3-dihydro-2-oxo-3-(1H-pyrrol-2-ylmethylene)-1H-indol-6-yl]amino]-4-methylphenyl]-N'-[2-fluoro-5-(trifluoromethyl)phenyl]-urea; (S)-5-chloro-N2-(1-(5-fluoropyridin-2-yl)ethyl)-N4-(5-isopropoxy-1H-pyrazol-3-yl)pyrimidine-(S)—N-(1-(5-fluoropyrimidin-2-yl)ethyl)-3-(5-isopropoxy-1H-pyrazol-3-yl)-3H-imidazo[4,5-b]pyridin-5-amine,4-diamine; 5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-12-propanenitrile; 1,3-dihydro-3-[(1-methyl-1H-indol-3-yl)methylene]-2H-pyrrolo[3,2-b]pyridin-2-one, or It is selected from the group consisting of pharmaceutically acceptable salts thereof.

[0217] In some embodiments, the Trk inhibitor is (2R)-2-({4-[(5-cyclopropyl-1H-pyrazol-3-yl)amino]-5-fluoropyrimidin-2-yl}-amino)-2-(4-fluorophenyl)ethanol; 5-Bromo-N 4 -(3-cyclopropyl-1H-pyrazol-5-yl)-N 2 -[(1S)-1-(4-fluorophenyl)ethyl]pyrimidine-2,4-diamine; (2R)-2-({5-chloro-4-[(3-cyclopropyl-1H-pyrazol-5-yl)amino]pyrimidin-2-yl}-amino)-2-(4-fluorophenyl)ethanol; (2R)-2-({5-chloro-4-[(3-isopropoxy-1H-pyrazol-5-yl)amino]pyrimidin-2-yl}amino)-2-(4-fluorophenyl)ethanol; (3S)-3-({5-chloro-4-[(5-cyclopropyl-1H-pyrazol-3-yl)amino]pyrimidin-2-yl}-amino)-3-(4-fluorophenyl)-N-methylpropanamide; 2-({5-chloro-2-{[(1S)-1-(4-fluorophenyl)ethyl]amino}-6-[(5-isopropoxy-1H-pyrazol-3-yl)amino]-pyrimidin-4-yl}amino)propane-1,3-diol; 2-[(5-chloro-6-[(3-cyclopropyl-1H-pyrazol-5-yl)amino]-2-{[(1S)-1-(4-fluorophenyl)ethyl]amino]pyrimidin-4-yl)amino}propane-1,3-diol; 5-Chloro-N 4 -(5-cyclopropyl-1H-pyrazol-3-yl)-N 2 -[(1S)-(4-fluoro-phenyl)-ethyl]-6-(4-methyl-piperazin-1-yl)-pyrimidine-2,4-diamine; (2R)-2-({4-[(5-cyclopropyl-1H-pyrazol-3-yl)amino]-7-fluoroquinazolin-2-yl}amino)-2-(4-fluorophenyl)ethanol; and 2-[(5-chloro-6-[(5-cyclopropyl-1H-pyrazol-3-yl)amino]-2-{[(1R)-1-(4-fluorophenyl)-2-hydroxyethyl]amino}pyrimidin-4-yl)amino]propane-1,3-diol, or The compound is selected from the group consisting of acceptable salts thereof.

[0218] In some embodiments, the Trk inhibitor is 1-(3-tert-butyl-1-phenyl-1H-pyrazol-5-yl)-3-(trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)urea; 1-(3-tert-butyl-1-p-tolyl-1H-pyrazol-5-yl)-3-(trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)urea hydrochloride; trans-1-(4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; trans-1-(4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-isopropyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)urea; 1-(1,3-dimethyl-1H-pyrazol-5-yl)-3-(trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)urea; 1-(3-tert-butyl-1-(pyridin-3-yl)-1H-pyrazol-5-yl)-3-(trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)urea; 1-(3-tert-butyl-1-(4-fluorophenyl)-1H-pyrazol-5-yl)-3-(trans-1-(2-methoxy-ethyl)-4-phenylpyrrolidin-3-yl)urea; 1-(3-cyclopropyl-1-phenyl-1H-pyrazol-5-yl)-3-(trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)urea; 1-(1,3-diphenyl-1H-pyrazol-5-yl)-3-(trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)urea; 1-(trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)-3-(3-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-(3-isopropyl-1-phenyl-1H-pyrazol-5-yl)-3-(trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)urea; 1-(trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)-3-(1-methyl-3-phenyl-1H-pyrazol-5-yl)urea; 1-(trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-(trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)-3-(1-phenyl-1H-pyrazol-5-yl)urea; 1-(3-tert-butyl-1-(2-fluorophenyl)-1H-pyrazol-5-yl)-3-(trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)urea; 1-(3-tert-butyl-1-(3-fluorophenyl)-1H-pyrazol-5-yl)-3-(trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)urea; 1-(3,4-dimethyl-1-phenyl-1H-pyrazol-5-yl)-3-(trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)urea; 1-(trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)-3-(2-(pyridin-3-yl)-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-(trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)-3-(1-methyl-1H-pyrazol-5-yl)urea; 1-(trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)thiourea; 1-(2-(3-fluorophenyl)-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)-3-(trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)urea; 1-(2-(4-fluorophenyl)-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)-3-(trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)urea; 1-(3-cyclopentyl-1-phenyl-1H-pyrazol-5-yl)-3-(trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)urea; 1-(1-ethyl-3-phenyl-1H-pyrazol-5-yl)-3-(trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)urea; 1-(trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)-3-(2-phenyl-4,5,6,7-tetrahydro-2H-indazol-3-yl)urea; 1-(trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)-3-(2-methyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-(1,3-dimethyl-4-phenyl-1H-pyrazol-5-yl)-3-(trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)urea; 1-(3-tert-butyl-1-o-tolyl-1H-pyrazol-5-yl)-3-(trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)urea; 1-(3-tert-butyl-1-m-tolyl-1H-pyrazol-5-yl)-3-(trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)urea; 1-(trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)-3-(1-methyl-4-phenyl-1H-pyrazol-5-yl)urea; 1-(4-cyano-3-methyl-1-phenyl-1H-pyrazol-5-yl)-3-(trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)urea; 1-(trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)-3-(2-(1-methyl-1H-pyrazol-4-yl)-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-(3-tert-butyl-1-(tetrahyro-2H-pyran-4-yl)-1H-pyrazol-5-yl)-3-(trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)urea; 1-(trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)-3-(2-(pyridin-2-yl)-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-(6,6-dimethyl-2-phenyl-2,4,5,6-tetrahydroxychloro(clo)penta[c]pyrazol-3-yl)-3-(trans-1-(2-methoxyethyl)-4-phenyl-pyrrolidin-3-yl)urea; 1-(7,7-dimethyl-2-phenyl-4,5,6,7-tetrahydro-2H-indazol-3-yl)-3-(trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)urea; 1-(trans-1-(2-methoxyethyl)-4-(pyridin-4-yl)pyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; trans-1-(4-(3-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; trans-1-(-4-(3-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-isopropyl-1-phenyl-1H-pyrazol-5-yl)urea; trans-1-(4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-isopropyl-1-phenyl-1H-pyrazol-5-yl)urea; trans-1-(4-(3-chlorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-isopropyl-1-phenyl-1H-pyrazol-5-yl)urea; trans-1-(4-(2-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-isopropyl-1-phenyl-1H-pyrazol-5-yl)urea; trans-1-(3-isopropyl-1-phenyl-1H-pyrazol-5-yl)-3-(1-(2-methoxyethyl)-4-(thiophen-2-yl)pyrrolidin-3-yl)urea; 1-((3,4-trans)-4-(2,4-dimethylthiazol-5-yl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-(trans-1-(2-methoxyethyl)-4-(oxazol-5-yl)pyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-(trans-4-(isoxazol-5-yl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-((3,4-trans)-1-(2-methoxyethyl)-4-(3-methoxyphenyl)pyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-(1-(2-methoxyethyl)-4-(thiazol-2-yl)pyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-((3S,4R)-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-(1,3-diphenyl-1H-pyrazol-5-yl)-3-((3S,4R)-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)urea; 1-((3S,4R)-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)-3-(1-methyl-3-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)-3-(1-phenyl-3-(trifluoromethyl)-1H-pyrazol-5-yl)urea; 1-(1,4-dimethyl-3-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)urea; 1-(3-cyclopropyl-1-methyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1-methyl-3-(pyridin-2-yl)-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1-methyl-3-(pyridin-3-yl)-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1,1'-dimethyl-1H,1'H-3,4'-bipyrazol-5-yl)urea; 1-(3-(3-cyanophenyl)-1-methyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(3-(4-cyanophenyl)-1-methyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(imidazo[1,2-a]pyridin-5-yl)-1-methyl-1H-pyrazol-5-yl)urea; 1-(4-chloro-1,3-diphenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-bromo-1,3-diphenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-3-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1,3-dimethyl-4-phenyl-1H-pyrazol-5-yl)urea; 1-(4-cyano-3-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-1-methyl-3-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-bromo-1-methyl-3-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-cyano-3-(cyanomethyl)-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(3-(2-cyanopropan-2-yl)-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-ethyl-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1'-methyl-1-phenyl-1H,1'H-3,4'-bipyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-3-(oxetan-3-ylmethoxy)-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-3-((3-methyloxetan-3-yl)methoxy)-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(((S)-2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(((R)-2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-(3,4-dimethyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)urea; tert-Butyl 3-(3-((3S,4R)-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)ureido)-2-phenyl-4,6-dihydropyrrolo[3,4-c]pyrazole-5(2H)-carboxylate; 1-(3-isopropyl-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)urea; 1-((3S,4R)-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)-3-(2-phenyl-4,6-dihydro-2H-furo[3,4-c]pyrazol-3-yl)urea; 1-((3S,4R)-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)-3-(2-phenyl-4,6-dihydro-2H-thieno[3,4-c]pyrazol-3-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3,4-dimethyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-isopropyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(2-phenyl-4,6-dihydro-2H-furo[3,4-c]pyrazol-3-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(2-phenyl-4,6-dihydro-2H-thieno[3,4-c]pyrazol-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(2-phenyl-4,6-dihydro-2H-furo[3,4-c]pyrazol-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(2-phenyl-4,6-dihydro-2H-thieno[3,4-c]pyrazol-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3,4-dimethyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1-methyl-3-phenyl-1H-pyrazol-5-yl)urea; 1-(3-(1-hydroxy-2-methylpropan-2-yl)-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)urea; 1-((3S,4R)-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)-3-(5-oxido-2-phenyl-4,6-dihydro-2H-thieno[3,4-c]pyrazol-3-yl)urea; 1-((3S,4R)-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)-3-(1-methyl-3-(pyridin-4-yl)-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1-methyl-3-(pyridin-4-yl)-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1-methyl-3-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1-methyl-3-(thiophen-2-yl)-1H-pyrazol-5-yl)urea; 1-((3S,4R)-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)-3-(3-(methoxymethyl)-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)-3-(3-(methoxymethyl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1-methyl-3-p-tolyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1-methyl-3-m-tolyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1-methyl-3-o-tolyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(3-methoxyphenyl)-1-methyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(2-methoxyphenyl)-1-methyl-1H-pyrazol-5-yl)urea; 1-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-5-yl)-3-((3S,4R)-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)urea; 1-((3S,4R)-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)-3-(3-(4-methoxyphenyl)-1-methyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-1-(2-methoxyethyl)-4-(3-(trifluoromethyl)phenyl)pyrrolidin-3-yl)-3-(1-methyl-3-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1-methyl-3-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(2,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1-methyl-3-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(4fluorophenyl)-1-methyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(3-fluorophenyl)-1-methyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(2-fluorophenyl)-1-methyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-(3-(1-hydroxy-2-methylpropan-2-yl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(1-hydroxy-2-methylpropan-2-yl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-(3-(4-chlorophenyl)-1-methyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4 difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(2,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-5-yl)urea; Methyl 4-(5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-1-methyl-1H-pyrazol-3-yl)benzoate; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1-(2-hydroxyethyl)-3-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(methoxymethyl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(methoxymethyl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1-methyl-3-(4-(methylthio)phenyl)-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1,3-diphenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(3-methoxypropyl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-(3,4-dimethyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(4-(2-methoxyethoxy)phenyl)-1-methyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methoxy-3-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(hydroxymethyl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(2-hydroxyethyl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(2-methoxyethyl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-(3-(benzyloxy)-1-methyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(2-methoxyethoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; trans-1-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-methoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-(3-(cyanomethoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluoro-phenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(4-methoxybenzyloxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-methoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(2-fluoroethoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(2-hydroxy-2-methylpropoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-ethoxy-1-phenyl-1H-pyrazol-5-yl)urea; 1-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(2-hydroxyethoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-(2-cyclohexyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)-3-(2-(pyridin-4-yl)-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1-methyl-3-(5-methylpyrazin-2-yl)-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1,4-dimethyl-3-(5-methylpyrazin-2-yl)-1H-pyrazol-5-yl)urea; Ethyl 5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-1-phenyl-1H-pyrazole-4-carboxylate; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1-methyl-3-(pyrazin-2-yl)-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-methoxy-1-methyl-4-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-ethoxy-1-methyl-4-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)urea dihydrochloride; 1-(5-acetyl-2-phenyl-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-3-((3S,4R)-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-(hydroxymethyl)-3-(methoxymethyl)-1-phenyl-1H-pyrazol-5-yl)urea; 4-(5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-1-methyl-1H-pyrazol-3-yl)benzoic acid; 4-(5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-1-methyl-1H-pyrazol-3-yl)benzamide; 4-(5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-1-methyl-1H-pyrazol-3-yl)-N-methylbenzamide; 4-(5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-1-methyl-1H-pyrazol-3-yl)-N,N-dimethylbenzamide; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(4-(hydroxymethyl)phenyl)-1-methyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1-methyl-3-(4-(methylsulfonyl)phenyl)-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-fluoro-3-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-fluoro-1-methyl-3-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-fluoro-1,3-diphenyl-1H-pyrazol-5-yl)urea; 2-Methoxyethyl 4-(5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-1-methyl-1H-pyrazol-3-yl)benzoate; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(5,5-dioxido-2-phenyl-4,6-dihydro-2H-thieno[3,4-c]pyrazol-3-yl)urea; 1-(5,5-dioxido-2-phenyl-4,6-dihydro-2H-thieno[3,4-c]pyrazol-3-yl)-3-((3S,4R)-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(5,5-dioxido-2-phenyl-4,6-dihydro-2H-thieno[3,4-c]pyrazol-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-3-(2-(methylsulfonyl)ethoxy)-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-(hydroxymethyl)-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-((R)-2,3-dihydroxypropoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-((S)-2,3-dihydroxypropoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(2-hydroxyethoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea hydrochloride; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-((S)-2-hydroxypropoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea hydrochloride; 1-((3R,4S)-4-hydroxy-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-((3R,4S)-4-fluoro-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-(trans-4-phenyl-1-(2-(trifluoromethoxy)ethyl)pyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-(trans-1-(2-(methylthio)ethyl)-4-phenylpyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-((3S,4R)-1-((S)-2-methoxypropyl)-4-phenylpyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-((3,4-trans)-4-phenyl-1-(4,4,4-trifluorobutyl)pyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-((3S,4R)-1-(cyanomethyl)-4-(3,4-difluorophenyl)pyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-((3S,4R)-1-(cyanomethyl)-4-(3,4-difluorophenyl)pyrrolidin-3-yl)-3-(3-(2-methoxyethoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3,4-trans)-1-(cyanomethyl)-4-phenylpyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-((3S,4R)-1-(cyanomethyl)-4-phenylpyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 2-((3R,4S)-3-phenyl-4-(3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)ureido)pyrrolidin-1-yl)acetamide; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-hydroxyethyl)pyrrolidin-3-yl)-3-(3,4-dimethyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((trans)-1-(3,3,4,4,4-pentafluorobutyl)-4-phenylpyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-((trans)-1-ethyl-4-phenylpyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-((trans)-4-phenyl-1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-(3,4-dimethyl-1-phenyl-1H-pyrazol-5-yl)-3-((trans)-4-phenyl-1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl)urea; 1-(3-(2-methoxyethoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((trans)-4-phenyl-1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl)urea; 1-((trans)-4-phenyl-1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-(3,4-dimethyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-phenyl-1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl)urea; 1-(3-(2-methoxyethoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-phenyl-1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3-fluorophenyl)-1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl)-3(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-(3,4-dimethyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3-fluorophenyl)-1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3-fluorophenyl)-1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl)-3(3-(2-methoxyethoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3-fluorophenyl)-1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl)-3(1-methyl-3-phenyl-1H-pyrazol-5-yl)urea; 1-((3R,4S)-4-(3-fluorophenyl)-1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3R,3S)-4-(3-fluorophenyl)-1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl)urea; 1-((trans)-1-(1,3-difluoropropan-2-yl)-4-phenylpyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; (trans)-tert-butyl 3-(3-methoxyphenyl)-4-(3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)ureido)pyrrolidine-1-carboxylate; 1-((trans)-4-(3-chlorophenyl)-1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl)-3(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)-3-((trans)-4-(pyridin-2-yl)-1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl)urea; 1-((trans)-4-(4-fluorophenyl)-1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl)-3(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-((trans)-4-(4-chlorophenyl)-1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl)-3(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-((trans)-4-(2-chlorophenyl)-1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl)-3(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)-3-((trans)-4-(pyridin-3-yl)-1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl)urea; 1-((trans)-4-(2-fluorophenyl)-1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl)-3(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-((trans)-4-(4-fluorophenyl)-1-(2,2-difluoroethyl)pyrrolidin-3-yl)-3-(2phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(1H-pyrazol-4-yl)pyrrolidin-3-yl)-3-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(1H-pyrazol-4-yl)pyrrolidin-3-yl)-3-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(1H-pyrazol-3-yl)pyrrolidin-3-yl)-3-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(3-methyl-1H-pyrazol-4-yl)pyrrolidin-3-yl)-3-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(3-(trifluoromethyl)-1H-pyrazol-4-yl)pyrrolidin-3-yl)-3-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(1H-pyrazol-4-yl)pyrrolidin-3yl)-3-(3-((S)-2-hydroxypropoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(1H-pyrazol-4-yl)pyrrolidin-3yl)-3-(3-((R)-2,3-dihydroxypropoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,5-difluoro-phenyl)-1-(1H-pyrazol-4-yl)pyrrolidin-3-yl)-3-(3,4-dimethyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(1H-pyrazol-4-yl)pyrrolidin-3-yl)-3-(3,4-dimethyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(1H-pyrazol-4-yl)pyrrolidin-3-yl)-3-(3-(3-methoxypropyl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(1H-pyrazol-4-yl)pyrrolidin-3-yl)-3-(3-(2-methoxyethoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(1H-pyrazol-4-yl)pyrrolidin-3-yl)-3-(3-(2-hydroxy-2-methylpropoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-1-(1-methyl-1H-pyrazol-5-yl)-4-phenylpyrrolidin-3-yl)urea; 1-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3R,4S)-1-(1-methyl-1H-pyrazol-5-yl)-4-phenylpyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(1-methyl-1H-pyrazol-5-yl)pyrrolidin-3-yl)-3-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3R,4S)-4-(3,5-difluorophenyl)-1-(1-methyl-1H-pyrazol-5-yl)pyrrolidin-3-yl)-3-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-phenylpyrrolidin-3-yl)-3-(3-ethoxy-4methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyphenyl)pyrrolidin-3-yl)-3-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-1-(2-fluorophenyl)-4-phenylpyrrolidin-3-yl)urea; 1-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-1-(4-fluorophenyl)-4-phenylpyrrolidin-3-yl)urea; 1-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-1-(2-methylphenyl)-4-phenylpyrrolidin-3-yl)urea; 1-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-1-(2-methoxyphenyl)-4-phenylpyrrolidin-3-yl)urea; 1-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-1-(2-chlorophenyl)-4-phenylpyrrolidin-3-yl)urea; 1-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-phenyl-1-(2-(trifluoromethoxy)phenyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-1-(2,6-difluorophenyl)-4-phenylpyrrolidin-3-yl)-3-(3-ethoxy-4methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-1-(2-methoxypyridin-4-yl)-4-phenylpyrrolidin-3-yl)urea; 1-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-1-(2-methoxypyridin-3-yl)-4-phenylpyrrolidin-3-yl)urea; 1-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-1-(2-ethoxypyridin-3-yl)-4-phenylpyrrolidin-3-yl)urea; 1-(1',4-dimethyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)-3-((3S,4R)-1-(2-methoxypyridin-3-yl)-4-phenylpyrrolidin-3-yl)urea; 1-((3S,4R)-1-(2-methoxypyridin-3-yl)-4-phenylpyrrolidin-3-yl)-3-(4-methyl-1,3-diphenyl-1H-pyrazol-5-yl)urea; 1-(4-bromo-1'-methyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)-3-((3S,4R)-1-(2-methoxypyridin-3-yl)-4-phenylpyrrolidin-3-yl)urea; 1-(4-bromo-1,3-diphenyl-1H-pyrazol-5-yl)-3-((3S,4R)-1-(2-methoxypyridin-3-yl)-4-phenylpyrrolidin-3-yl)urea; 1-((3S,4R)-1-((1,2,3-thiadiazol-4-yl)methyl)-4-(3,4-difluorophenyl)pyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-((3S,4R)-1-((1,2,3-thiadiazol-4-yl)methyl)-4-(3,4-difluorophenyl)pyrrolidin-3-yl)-3-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-1-((1,2,3-thiadiazol-4-yl)methyl)-4-(3,4-difluorophenyl)pyrrolidin-3-yl)-3-(3-(cyanomethoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-1-((1,2,3-thiadiazol-4-yl)methyl)-4-(3,4-difluorophenyl)pyrrolidin-3-yl)-3-(1',4-dimethyl-1-phenyl-1H,11'H-3,4'-bipyrazol-5-yl)urea; 1-((3S,4R)-1-((1,2,3-thiadiazol-4-yl)methyl)-4-(3,4-difluorophenyl)pyrrolidin-3-yl)-3-(4-methyl-3-(1-methyl-1H-imidazol-4-yl)-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-((1-methyl-1H-1,2,3-triazol-4-yl)methyl)pyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(1,3-dimethoxypropan-2-yl)pyrrolidin-3-yl)-3-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(1-methoxypropan-2-yl)pyrrolidin-3-yl)-3-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((trans)-4-(4-fluorophenyl)-1-(2-(methylamino)ethyl)pyrrolidin-3-yl)-3(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-((trans)-1-((1H-imidazol-2-yl)methyl)-4-phenylpyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; Methyl 3-methoxy-2-((trans)-3-phenyl-4-(3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)ureido)pyrrolidin-1-yl)propanoate; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(1-methoxypropan-2-yl)pyrrolidin-3-yl)-3-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(1-hydroxy-3-methoxypropan-2-yl)pyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(3-hydroxy-1-methoxy-3-methylbutan-2-yl)pyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 2-((3R,4S)-3-(3,4-difluorophenyl)-4-(3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)ureido)pyrrolidin-1-yl)-3-methoxypropanoic acid hydrochloride; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(1-hydroxy-3-methoxypropan-2-yl)pyrrolidin-3-yl)-3-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-(4-chloro-1'-methyl-1-phenyl-1H,1'H-3,4'-bipyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(1-hydroxy-3-methoxypropan-2-yl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3yl)-3-(3-methoxy-1-phenyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)urea; 1-(3-(2-fluoroethoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3R,4S)-4-phenyl-1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl)urea; 1-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3R,4S)-4-phenyl-1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl)urea; 1-(3-(cyanomethoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3R,4S)-4-phenyl-1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl)urea; 1-(1',4-dimethyl-1-phenyl-1H,1'H-3,4'-bipyrazol-5-yl)-3-((3R,4S)-4-phenyl-1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-1'-methyl-1-phenyl-1H,1'H-3,4'-bipyrazol-5-yl)-3-((3R,4S)-4-phenyl-1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl)urea; 1-(3-(((S)-2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3R,4S)-4-phenyl-1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl)urea; 1-(3-((R)-2,3-dihydroxypropoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3R,4S)-4-phenyl-1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl)urea; (R,S)1-((2α,3β,4α)-2-methyl-4-phenyl-1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; (R,S)-1-((3(3,4α,5α)-5-methyl-4-phenyl-1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-((S)-1,1,1-trifluoro-3-hydroxypropan-2-yl)pyrrolidin-3-yl)-3-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-((S)-1,1,1-trifluoro-3-methoxypropan-2-yl)pyrrolidin-3-yl)-3-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-((S)-1,1,1-trifluoro-3-methoxypropan-2-yl)pyrrolidin-3-yl)-3-(3-((S)-2-hydroxypropoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-(4-chloro-1'-methyl-1-phenyl-1H,1'H-3,4'-bipyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-((R)-1,1,1-trifluoro-3-methoxypropan-2-yl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-((R)-1,1,1-trifluoro-3-methoxypropan-2-yl)pyrrolidin-3-yl)-3-(3-((S)-2-hydroxypropoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-methyl-4-(methylthio)-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3yl)-3-(3-(3-methoxypropyl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-(3-(1,1-difluoro-2-hydroxyethyl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(3-(1,1-difluoro-2-hydroxyethyl)-4-methyl-1-phenyl-1H-pyrazol-5yl)-3-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(3-(1,1-difluoro-2-hydroxyethyl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(3-(1,1-difluoro-2-hydroxyethyl)-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(2-hydroxyethyl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(2hydroxyethyl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(2-hydroxy-2-methylpropyl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-((S)-2-hydroxypropyl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-((R)-2-hydroxypropyl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; Ethyl 5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-4-methyl-1-phenyl-1H-pyrazole-3-carboxylate; 5-(3-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-N,4-dimethyl-1-phenyl-1H-pyrazole-3-carboxamide; 1-(trans-4-(3-chloro-4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1',4-dimethyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)urea; 1-(trans-4-(4-chloro-3-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1',4-dimethyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)urea; 1-(trans-4-(3-chloro-5-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1',4-dimethyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)urea; 1-(trans-4-(3-chlorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1',4-dimethyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-3-(5-methyl-1,3,4-oxadiazol-2-yl)-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-3-(3-methyl-1,2,4-oxadiazol-5-yl)-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-1-phenyl-3-(3-(trifluoromethyl)-1,2,4-oxadiazol-5-yl)-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-1-phenyl-3-(3-(trifluoromethyl)-1,2,4-oxadiazol-5-yl)-1H-pyrazol-5-yl)urea; 5-(3-(trans-4-(3-chloro-4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-N,4-dimethyl-1-phenyl-1H-pyrazole-3-carboxamide; 5-(3-(trans-4-(4-chloro-3-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-N,4-dimethyl-1-phenyl-1H-pyrazole-3-carboxamide; 1-(trans-4-(4-chloro-3-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-3-(1-methyl-2-oxo-1,2-dihydropyridin-4-yl)-1-phenyl-1H-pyrazol-5-yl)urea; 1-(trans-4-(3-chloro-4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-3-(1-methyl-2-oxo-1,2-dihydropyridin-4-yl)-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1',4,5'-trimethyl-1-phenyl-1H,1'H-[3,3'-bipyrazol]-5-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1',4,5'-trimethyl-1-phenyl-1H,1'H-[3,3'-bipyrazol]-5-yl)urea; 1-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1',4,5'-trimethyl-1-phenyl-1H,1'H-[3,3'-bipyrazol]-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(2',4,5'-trimethyl-1-phenyl-1H,2'H-[3,3'-bipyrazol]-5-yl)urea; 1-(4-cyclopropyl-1'-methyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-isopropyl-1'-methyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-ethyl-1'-methyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1-(4-fluorophenyl)-1',4-dimethyl-1H,1'H-[3,4'-bipyrazol]-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1-(3-fluorophenyl)-1',4-dimethyl-1H,1'H-[3,4'-bipyrazol]-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1-(2-fluorophenyl)-1',4-dimethyl-1H,1'H-[3,4'-bipyrazol]-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1-(3-chlorophenyl)-1',4-dimethyl-1H,1'H-[3,4'-bipyrazol]-5-yl)urea; 1-(1-(3-chloro-4-fluorophenyl)-1',4-dimethyl-1H,1'H-[3,4'-bipyrazol]-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(1-(3-chloro-2-fluorophenyl)-1',4-dimethyl-1H,1'H-[3,4'-bipyrazol]-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1-(4-fluorophenyl)-1',4-dimethyl-1H,1'H-[3,4'-bipyrazol]-5-yl)urea; 1-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1-(3-fluorophenyl)-1',4-dimethyl-1H,1'H-[3,4'-bipyrazol]-5-yl)urea; 1-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1-(2-fluorophenyl)-1',4-dimethyl-1H,1'H-[3,4'-bipyrazol]-5-yl)urea; 1-((3S,4R)-4-(3,4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1-(3-chlorophenyl)-1',4-dimethyl-1H,1'H-[3,4'-bipyrazol]-5-yl)urea; 1-(1-(3-chloro-4-fluorophenyl)-1',4-dimethyl-1H,1'H-[3,4'-bipyrazol]-5-yl)-3-((3S,4R)-4-(4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(1-(3-chloro-2-fluorophenyl)-1',4-dimethyl-1H,1'H-[3,4'-bipyrazol]-5-yl)-3-((3S,4R)-4-(4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(2,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1',4-dimethyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)urea; 1-((3S,4R)-4-(3-cyanophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1',4-dimethyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)urea; 1-((3S,4R)-4-(4-cyanophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1',4-dimethyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)urea; 1-(1',4-dimethyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)-3-((3S,4R)-1-(2-methoxyethyl)-4-(p-tolyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-1,3-diphenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-1,3-diphenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4,5-trifluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-1,3-diphenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-1,3-diphenyl-1H-pyrazol-5-yl)urea; 1-(4-bromo-3-methyl-1-phenyl-1H-pyrazol-5-yl)-3-trans-1-(2-methoxyethyl)4-(1-methyl-1H-pyrazol-4-yl)pyrrolidin-3-yl)urea; 1-(1',4-dimethyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)-3-(trans-1-(2-methoxyethyl)-4-(1-methyl-1H-pyrazol-4-yl)pyrrolidin-3-yl)urea; 1-(1',4-dimethyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)-3-((trans-1-(2-methoxyethyl)-4-(1,2,3-thiadiazol-4-yl)pyrrolidin-3-yl)urea; 1-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-(trans-1-(2-methoxyethyl)-4-(3-(trifluoromethyl)phenyl)pyrrolidin-3-yl)urea; 1-(3,4-dimethyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-1-(2-methoxyethyl)-4-(3-(trifluoromethyl)phenyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(5-fluoropyridin-3-yl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-((3R,4S)-4-(5-fluoropyridin-3-yl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-(3-(2-fluoroethoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(5fluoropyridin-3-yl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(3-(2-fluoroethoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3R,4S)-4-(5fluoropyridin-3-yl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(trans-4-(5-fluoropyridin-3-yl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(2-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 1-(trans-4-(5-chloropyridin-3-yl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-(trans-4-(5-chloropyridin-3-yl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3,4-dimethyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-(trans-4-(5-fluoropyridin-3-yl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-1,3-diphenyl-1H-pyrazol-5-yl)urea; 1-(1',4-dimethyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)-3-(trans-4-(5-fluoropyridin-2-yl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(1',4-dimethyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)-3-((3S,4R)-4-(3-fluoropyridin-4-yl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-3-(1-methyl-1H-1,2,4-triazol-3-yl)-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1'-(2-methoxyethyl)-4-methyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)urea; 1-(3-cyano-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1'-(2-hydroxyethyl)-4-methyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-3-(2-methyl-2H-1,2,3-triazol-4-yl)-1-phenyl-1H-pyrazol-5-yl)urea; 1-(3-bromo-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(5-methyl-6-oxo-2-phenyl-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(5-methyl-6-oxo-2-phenyl-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-3-((5-methyl-1,3,4-oxadiazol-2-yl)methoxy)-1-phenyl-1H-pyrazol-5-yl)urea; 1-(4-chloro-3-ethoxy-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-ethoxy-4-fluoro-1-phenyl-1H-pyrazol-5-yl)urea; 1-(4-bromo-3-(2-hydroxy-2-methylpropoxy)-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-3-(2-hydroxy-2-methylpropoxy)-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-((S)-2-hydroxybutoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; Ethyl 2-((5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-4-methyl-1-phenyl-1H-pyrazol-3-yl)oxy)acetate; 1-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(2hydroxy-2-methylpropoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(2-hydroxy-2-methylpropoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-((R)-2-hydroxypropoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-((R)-2-hydroxypropoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-((R)-2-hydroxypropoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-1-phenyl-3-((R)-3,3,3-trifluoro-2-hydroxypropoxy)-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-((S)-2-hydroxypropoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-1-phenyl-3-((S)-3,3,3-trifluoro-2-hydroxypropoxy)-1H-pyrazol-5-yl)urea; 1-(4-chloro-3-(2-hydroxy-2-methylpropoxy)-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-3-(2-hydroxy-2-methylpropoxy)-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-3-((R)-2-hydroxypropoxy)-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-3-((R)-2-hydroxypropoxy)-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-3-((R)-2-hydroxypropoxy)-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-bromo-3-((R)-2-hydroxypropoxy)-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-bromo-3-((R)-2-hydroxypropoxy)-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-bromo-3-((R)-2-hydroxypropoxy)-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-((R)-2-hydroxybutoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-((R)-2-hydroxybutoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-((R)-2-hydroxybutoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; Ethyl 4-bromo-5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-1-phenyl-1H-pyrazole-3-carboxylate; 1-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1'-(2-methoxyethyl)-4-methyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)urea; 1-((3S,4R)-4-(3-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1'-(2-methoxyethyl)-4-methyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)urea; 1-((3S,4R)-4-(3-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-3-(2-methyl-2H-1,2,3-triazol-4-yl)-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-3-(2-methyl-2H-1,2,3-triazol-4-yl)-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-3-(2-morpholinoethoxy)-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(2-(1,3-dioxoisoindolin-2-yl)ethoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; tert-butyl 4-(2-((5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-4-methyl-1-phenyl-1H-pyrazol-3-yl)oxy)ethyl)piperazine-1-carboxylate; trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)-3-(2-phenyl-2H-indazol-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(2-phenyl-2H-indazol-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-3-((1-methyl-1H-1,2,4-triazol-3-yl)methoxy)-1-phenyl-1H-pyrazol-5yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(((S)-2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-ethoxy-4-methyl-1-(pyrazin-2-yl)-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(2-(pyridazin-4-yl)-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)urea; 5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-4-methyl-1-phenyl-1H-pyrazol-3-yldimethylcarbamate; 5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-4-methyl-1-phenyl-1H-pyrazol-3-ylmorpholine-4-carboxylate; 1-(3-(((S)-2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-1-(2-methoxyethyl)-4-(3,4,5-trifluorophenyl)pyrrolidin-3-yl)urea; 1-(3-(((R)-2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-1-(2-methoxyethyl)-4-(3,4,5-trifluorophenyl)pyrrolidin-3-yl)urea; 1-(3-((S)-2-(tert-butyldimethylsilyloxy)propoxy)-4-methyl-1-phenyl-1-pyrazol-5-yl)-3-((3S,4R)-1-(2-methoxyethyl)-4-(3,4,5-trifluorophenyl)pyrrolidin-3-yl)urea; 1-(3-(2-hydroxy-2-methylpropoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-1-(2-methoxyethyl)-4-(3,4,5-trifluorophenyl)pyrrolidin-3-yl)urea; 1-(3-((S)-2-(tert-butyldimethylsilyloxy)-3-methoxypropoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-3-(methoxy-methyl)-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-bromo-3-(methoxy-methyl)-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-3-(methoxy-methyl)-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-bromo-3-(methoxy-methyl)-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-3-(methoxy-methyl)-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(4fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-bromo-3-(methoxy-methyl)-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-3-(1,1-difluoro-2-hydroxyethyl)-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-3-(1,1-difluoro-2-hydroxyethyl)-1-phenyl-1H-pyrazol-5yl)-3-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-3-(1,1-difluoro-2-hydroxyethyl)-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-3-((S)-2-hydroxypropyl)-1-phenyl-1H-pyrazol-5-yl)-3((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-3-((R)-2-hydroxypropyl)-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-bromo-3-((R)-2-hydroxypropyl)-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-3-(2-hydroxy-2-methylpropyl)-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-3-(3-methyl-1,2,4-oxadiazol-5-yl)-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-bromo-3-(2-cyanopropan-2-yl)-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-3-(2-cyanopropan-2-yl)-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-bromo-1'-methyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)-3-((3S,4R)-4(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-bromo-1'-methyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)-3-((3S,4R)-4(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-bromo-1'-methyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)-3-((3-4-(4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-1'-methyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)-3-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-1'-methyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-1'-methyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)-3-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-1'-methyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)-3-((3S,4R)-4-phenyl-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-1,3-diphenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-bromo-1,3-diphenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-bromo-1,3-diphenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(4-fluorophenyl)-1(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-3-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-bromo-3-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-1'-methyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)-3-((3S,4R)-4-(5-fluoropyridin-3-yl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-fluoro-1'-methyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)-3-((3S,4R)-4-(5-fluoropyridin-3-yl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-bromo-3-methyl-1-phenyl-1H-pyrazol-5-yl)-3-trans-4-(5-fluoropyridin-3-yl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-bromo-3-methyl-1-phenyl-1H-pyrazol-5-yl)-3-trans-4-(5-fluoropyridin-3-yl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-bromo-1,3-diphenyl-1H-pyrazol-5-yl)-3-(trans-4-(5-fluoropyridin-3-yl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-1,3-diphenyl-1H-pyrazol-5-yl)-3-(trans-4-(5-fluoropyridin-3-yl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-bromo-3-methyl-1-phenyl-1H-pyrazol-5-yl)-3-(trans-4-(5-fluoropyridin-2-yl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-fluoro-1'-methyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-fluoro-1'-methyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)urea; 1-(4-bromo-1'-methyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)-3-((3S,4R)-4(5-fluoropyridin-3-yl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(1',4-dimethyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)-3-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(1',4-dimethyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)-3-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(1',4-dimethyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)-3-((3S,4R)-4-(3,4,5-trifluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(1',4-dimethyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(1',4-dimethyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)-3-((3S,4R)-4-(fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1'-(4-methoxybenzyl)-4-methyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1'-(4-methoxybenzyl)-4-methyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)urea trifluoroacetate; 2-(4-chloro-5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-1-phenyl-1H-pyrazol-3-yl)ethyl acetate; 1-(4-chloro-3-(2-hydroxyethyl)-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(cis-3-hydroxycyclobutyl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(trans-3-hydroxycyclobutyl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-(4-chloro-3-(cis-3-hydroxycyclobutyl)-1-phenyl-1H-pyrazol-5-yl)3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-3-((1r,3S)-3-hydroxycyclobutyl)-1-phenyl-1H-pyrazol-5yl)-3-(trans-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)3-(3-(cis-3-hydroxycyclobutyl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(trans-3-hydroxycyclobutyl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(cis-3-hydroxycyclobutyl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(trans-3-hydroxycyclobutyl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-4-methyl-1-phenyl-1H-pyrazole-3-carboxylic acid; 5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-N,4-dimethyl-1-phenyl-1H-pyrazole-3-carboxamide; 5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-N,N,4-trimethyl-1-phenyl-1H-pyrazole-3-carboxamide; 5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-N-ethyl-4-methyl-1-phenyl-1H-pyrazole-3-carboxamide; 5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-N-isopropyl-4-methyl-1-phenyl-1H-pyrazole-3-carboxamide; 5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-4-methyl-1-phenyl-1H-pyrazole-3-carboxamide; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-ethoxy-4-(hydroxymethyl)-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3-chloro-4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1',4-dimethyl-1-phenyl-1H,1'H-3,4'-bipyrazol-5-yl)urea; 1-((3S,4R)-4-(4-chloro-3-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1',4-dimethyl-1-phenyl-1H,1'H-3,4'-bipyrazol-5-yl)urea; 1-((3S,4R)-4-(3-chloro-5-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1',4-dimethyl-1-phenyl-1H,1'H-3,4'-bipyrazol-5-yl)urea; 2-((3R,4S)-3-(3,4-difluorophenyl)-4-(3-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)ureido)pyrrolidin-1-yl)acetate; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(3,3,3-trifluoro-2-hydroxypropyl)pyrrolidin-3-yl)-3-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-hydroxypropyl)pyrrolidin-3-yl)-3-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-1-(2-cyanoethyl)-4-(3,4-difluorophenyl)pyrrolidin-3-yl)-3-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 2-((3R,4S)-3-(3,4-difluorophenyl)-4-(3-(3-ethoxy-4-methyl-1-phenyl-1H-pyrazol-5-yl)ureido)pyrrolidin-1-yl)-N-methylacetamide; 1-(1-cyclohexyl-3,4-dimethyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(3-hydroxy-2-(hydroxymethyl)propoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-1-phenyl-3-(2,2,2-trifluoroethoxy)-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-1-phenyl-3-(2,2,2-trifluoroethoxy)-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-1-phenyl-3-(2,2,2-trifluoroethoxy)-1H-pyrazol-5-yl)urea; 1-(3-(2,2-difluoroethoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-1-phenyl-3-(2,2,2-trifluoroethoxy)-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-1-phenyl-3-(pyridin-2-yl)-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4 difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-1-phenyl-3-(pyridin-4-yl)-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4 difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-1-phenyl-3-(pyridin-3-yl)-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4 difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-bromo-1-phenyl-3-(pyridin-3-yl)-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-bromo-1-phenyl-3-(pyridin-2-yl)-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-3-phenyl-1-(pyridin-3-yl)-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4 difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-1-phenyl-3-(pyridin-3-yl)-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-1-phenyl-3-(pyridin-4-yl)-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-1-phenyl-3-(pyridin-2-yl)-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(5-fluoropyridin-3-yl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(5-fluoropyridin-3-yl)-4-methyl-1-(pyridin-3-yl)-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1',4-dimethyl-1-phenyl-1H,1'H-[3,3'-bipyrazol]-5-yl)urea; 1-(1',4-dimethyl-1-phenyl-1H,1'H-[3,3'-bipyrazol]-5-yl)-3-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1',4-dimethyl-1-phenyl-1H,1'H-[3,3'-bipyrazol]-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(2',4-dimethyl-1-phenyl-1H,2'H-[3,3'-bipyrazol]-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1-(5-fluoropyridin-3-yl)-1',4-dimethyl-1H,1'H-[3,4'-bipyrazol]-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1',4-dimethyl-1-(5-methylpyridin-3-yl)-1H,1'H-[3,4'-bipyrazol]-5-yl)urea; 1-(1-(5-chloropyridin-3-yl)-1',4-dimethyl-1H,1'H-[3,4'-bipyrazol]-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-1-phenyl-1'-(2,2,2-trifluoro-1-(2,2,2-trifluoroethoxy)ethyl)-1H,1'H-[3,4'-bipyrazol]-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-1-phenyl-1'-(2,2,2-trifluoroethyl)-1H,1'H-[3,4'-bipyrazol]-5-yl)urea; 1-(1'-(cyclopropylmethyl)-4-methyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(1'-(cyclopropanecarbonyl)-4-methyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-1'-(methylsulfonyl)-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1'-isopropyl-4-methyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-1-phenyl-3-(pyrimidin-5-yl)-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-3-(1-methyl-2-oxo-1,2-dihydropyridin-4-yl)-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-3-(1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1',4,5'-trimethyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1',3',4-trimethyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)urea; 1-(1'-cyclopropyl-4-methyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-3-(2-methylthiazol-5-yl)-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-3-(2-methylpyrimidin-5-yl)-1-phenyl-1H-pyrazol-5-yl)urea; 1-(3-(2-aminopyrimidin-5-yl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(2,4-dimethylthiazol-5-yl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(2,6-dimethylpyridin-4-yl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-(3-(6-aminopyridin-3-yl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(3-bromo-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-3-(6-oxo-1-(2,2,2-trifluoroethyl)-1,6-dihydropyridin-3-yl)-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1'-isopropyl-4-methyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)urea; 1-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-3-(1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-1-phenyl-1H-pyrazol-5-yl)urea; 1-(3-bromo-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3R,4S)-4-phenyl-1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl)urea; 1-(4-methyl-3-(1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-1-phenyl-1H-pyrazol-5-yl)-3-((3R,4S)-4-phenyl-1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl)urea; 1-(3-(2-aminopyrimidin-5-yl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3((3R,4S)-4-phenyl-1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl)urea bis(2,2,2-trifluoroacetate); 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1'-ethyl-4-methyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)urea; 1-(1'-ethyl-4-methyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)-3-((3S,4R)-4(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-4-methyl-1-phenyl-1H-pyrazol-3-yl trifluoromethanesulfonate; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(2-methoxypyrimidin-5-yl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(2-(dimethylamino)pyrimidin-5-yl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(2-methoxypyrimidin-5-yl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-(3-(2-(dimethylamino)pyrimidin-5-yl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(1'-ethyl-4-methyl-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)-3-((3S,4R)-4(3-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-3-(1-methyl-2-oxo-1,2-dihydropyridin-4-yl)-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-3-(1-methyl-2-oxo-1,2-dihydropyridin-4-yl)-1-phenyl-1H-pyrazol-5-yl)urea; 1-(3-cyclopropyl-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(3-cyclopropyl-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(1-isopropyl-6-oxo-1,6-dihydropyridin-3-yl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(1isopropyl-6-oxo-1,6-dihydropyridin-3-yl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-((S)-2-hydroxypropoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea dihydrochloride; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-1-phenyl-3-(2-(piperazin-1-yl)ethoxy)-1H-pyrazol-5-yl)urea trihydrochloride; 1-(3-(benzyloxy)-4-chloro-1-methyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 2-((5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-4-methyl-1-phenyl-1H-pyrazol-3-yl)oxy)acetic acid; 2-((5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-4-methyl-1-phenyl-1H-pyrazol-3-yl)oxy)-N-ethylacetamide; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-ethyl-3-(2-hydroxy-2-methylpropoxy)-1-phenyl-1H-pyrazol-5-yl)urea; 1-(3-(2-aminoethoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; N-(2-((5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-4-methyl-1-phenyl-1H-pyrazol-3-yl)oxy)ethyl)methanesulfonamide; N-(2-((5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-4-methyl-1-phenyl-1H-pyrazol-3-yl)oxy)ethyl)acetamide; 1-(3-(2-(4-acetylpiperazin-1-yl)ethoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 2-((5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-4-methyl-1-phenyl-1H-pyrazol-3-yl)oxy)acetamide; N-(5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-3-ethoxy-1-phenyl-1H-pyrazol-4-yl)-2,2,2-trifluoroacetamide; 1-(4-amino-3-ethoxy-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-ethoxy-4-(2-hydroxyethyl)-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-3-(2-(4-methylpiperazin-1-yl)ethoxy)-1-phenyl-1H-pyrazol-5-yl)urea trihydrochloride; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-3-(2-morpholino-2-oxoethoxy)-1-phenyl-1H-pyrazol-5-yl)urea; 4-bromo-5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-1-phenyl-1H-pyrazole-3-carboxylic acid; 4-bromo-5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-N-methyl-1-phenyl-1H-pyrazole-3-carboxamide; 4-bromo-5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-N-methoxy-1-phenyl-1H-pyrazole-3-carboxamide; 1-(4-chloro-1'-(2-methoxyethyl)-1-phenyl-1H,1'H-[3,4'-bipyrazol]-5-yl)-3((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-((S)-2,3-dihydroxypropoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-((R)-2,3-dihydroxypropoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-(3-((R)-2,3-dihydroxypropoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-1-(2-methoxyethyl)-4-(3,4,5-trifluorophenyl)pyrrolidin-3-yl)urea; 1-(3-((S)-2,3-dihydroxypropoxy)-4-methyl-1-phenyl-1H-pyrazol-5yl)-3-((3S,4R)-1-(2-methoxyethyl)-4-(3,4,5-trifluorophenyl)pyrrolidin-3-yl)urea; 1-(3-((S)-2-hydroxypropoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-1-(2-methoxyethyl)-4-(3,4,5-trifluorophenyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-((S)-2-hydroxy-3-methoxypropoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-((R)-2-hydroxy-3-methoxypropoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)urea; 1-(3-((S)-2-hydroxy-3-methoxypropoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)3-((3S,4R)-1-(2-methoxyethyl)-4-(3,4,5-trifluorophenyl)pyrrolidin-3-yl)urea; 1-(3-((R)-2-hydroxy-3-methoxypropoxy)-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-1-(2-methoxyethyl)-4-(3,4,5-trifluorophenyl)pyrrolidin-3-yl)urea; 1-(4-bromo-1,1'-dimethyl-1H,1'H-[3,4'-bipyrazol]-5-yl)-3-((3S,4R)4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-1,1'-dimethyl-1H,1'H-[3,4'-bipyrazol]-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-1-phenyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; tert-butyl 4-(4-chloro-5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-1phenyl-1H-pyrazol-3-yl)piperidine-1-carboxylate; 1-(4-chloro-1-phenyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-5-yl)-3-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-3-(3,5-dimethylisoxazol-4-yl)-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; (R)-tert-butyl 2-(4-chloro-5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-1phenyl-1H-pyrazol-3-yl)pyrrolidine-1-carboxylate; (S)-tert-butyl 2-(4-chloro-5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-1phenyl-1H-pyrazol-3-yl)pyrrolidine-1-carboxylate; 1-(4-bromo-1-phenyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; tert-butyl 4-(4-bromo-5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-1phenyl-1H-pyrazol-3-yl)piperidine-1-carboxylate; 1-(4-bromo-1-phenyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-5-yl)-3-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-bromo-3-(3,5-dimethylisoxazol-4-yl)-1-phenyl-1H-pyrazol-5-yl)-3-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; (R)-tert-butyl 2-(4-bromo-5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-1-phenyl-1H-pyrazol-3-yl)pyrrolidine-1-carboxylate; tert-butyl 4-((4-bromo-5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-1phenyl-1H-pyrazol-3-yl)methoxy)piperidine-1-carboxylate; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(l-phenyl-3-(piperidin-4-yl)-1H-pyrazol-5-yl)urea dihydrochloride; 1-(4-chloro-1-phenyl-3-(piperidin-4-yl)-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea dihydrochloride; 1-(4-bromo-1-phenyl-3-(piperidin-4-yl)-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea dihydrochloride; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-1-phenyl-3-((R)-pyrrolidin-2-yl)-1H-pyrazol-5-yl)urea dihydrochloride; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-1-phenyl-3-((S)-pyrrolidin-2-yl)-1H-pyrazol-5-yl)urea dihydrochloride; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-chloro-1-phenyl-3-((R)-pyrrolidin-2-yl)-1H-pyrazol-5-yl)urea dihydrochloride; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-1-phenyl-3-((S)-pyrrolidin-2-yl)-1H-pyrazol-5-yl)urea dihydrochloride; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-chloro-1-phenyl-3-((S)-pyrrolidin-2-yl)-1H-pyrazol-5-yl)urea dihydrochloride; 1-(4-bromo-1-phenyl-3-((R)-pyrrolidin-2-yl)-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea dihydrochloride; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1-phenyl-3-((piperidin-4-yloxy)methyl)-1H-pyrazol-5-yl)urea dihydrochloride; 1-(4-chloro-1-phenyl-3-((piperidin-4-yloxy)methyl)-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea dihydrochloride; 1-(4-bromo-1-phenyl-3-((piperidin-4-yloxy)methyl)-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea dihydrochloride; 1-(4-bromo-3-(1-(methylsulfonyl)piperidin-4-yl)-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(3-(1-acetylpiperidin-4-yl)-4-bromo-1-phenyl-1H-pyrazol-5-yl)-3((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-chloro-1-phenyl-3-(1-(trifluoromethylsulfonyl)piperidin-4-yl)-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea hydrochloride; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-3-((R)-1-(methylsulfonyl)pyrrolidin-2-yl)-1-phenyl-1H-pyrazol-5-yl)urea; 1-(3-((R)-1-acetylpyrrolidin-2-yl)-4-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-3-((R)-1-methylpyrrolidin-2-yl)-1-phenyl-1H-pyrazol-5-yl)urea dihydrochloride; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-3-((S)-1-methylpyrrolidin-2-yl)-1-phenyl-1H-pyrazol-5-yl)urea dihydrochloride; 1-(4-bromo-3-((1-(methylsulfonyl)piperidin-4-yloxy)methyl)-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(3-((1-acetylpiperidin-4-yloxy)methyl)-4-bromo-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-(4-isopropyl-5-oxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)-4-methyl-1phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-3-(4-methyl-5-oxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-1-phenyl-3-(pyrazin-2-yloxy)-1H-pyrazol-5-yl)urea; 1-((3S,4R)-1-(2-methoxyethyl)-4-(3,4,5-trifluorophenyl)pyrrolidin-3-yl)-3-(4-methyl-3-(1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-1-phenyl-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-methoxy-1-phenyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-methoxy-1-phenyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-methoxy-1-phenyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-methoxy-1-phenyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)urea; 1-((trans)-4-(4-chloro-3-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-5-oxo-2-phenyl-2,5-dihydro-1H-pyrazol-3-yl)urea; 1-((trans)-4-(3-chloro-4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-5-oxo-2-phenyl-2,5-dihydro-1H-pyrazol-3-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-5-oxo-2-phenyl-2,5-dihydro-1H-pyrazol-3-yl)urea; 1-((3S,4R)-1-(2-methoxyethyl)-4-(3,4,5-trifluorophenyl)pyrrolidin-3-yl)-3-(4-methyl-5-oxo-2-phenyl-2,5-dihydro-1H-pyrazol-3-yl)urea; 1-((3S,4R)-4-(3-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-5-oxo-2-phenyl-2,5-dihydro-1H-pyrazol-3-yl)urea; 1-((trans)-4-(3-chloro-5-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-5-oxo-2-phenyl-2,5-dihydro-1H-pyrazol-3-yl)urea; 1-(4-cyano-3-methoxy-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-((3S,4R)-4-(3-chloro-5-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-5-oxo-2-phenyl-2,5-dihydro-1H-pyrazol-3-yl)urea; 1-(4-cyano-1-phenyl-3-(trifluoromethyl)-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 1-(4-cyano-5-oxo-2-phenyl-2,5-dihydro-1H-pyrazol-3-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)urea; 5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-3-methoxy-1-phenyl-1H-pyrazole-4-carboxamide; 5-(3-((3S,4R)-4-(3,4-difluoro-phenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-3-methyl-1-phenyl-1H-pyrazole-4-carboxamide; 5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-3-ethyl-1-phenyl-1H-pyrazole-4-carboxamide; 5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-1-phenyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide; 5-(3-((trans)-4-(3-chloro-4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3yl)ureido)-3-methyl-1-phenyl-1H-pyrazole-4-carboxamide; 5-(3-((trans)-4-(4-chloro-3-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3yl)ureido)-3-methyl-1-phenyl-1H-pyrazole-4-carboxamide; 5-(3-((trans)-4-(3-chloro-5-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3yl)ureido)-3-methyl-1-phenyl-1H-pyrazole-4-carboxamide; 5-(3-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-3-methyl-1-phenyl-1H-pyrazole-4-carboxamide; 5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-1-phenyl-1H-pyrazole-4-carboxamide; 5-(3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)ureido)-1-phenyl-1H-pyrazole-4-carboxamide; 1-(4-bromo-3-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)guanidine dihydrochloride; 1-(4-bromo-3-methyl-1-phenyl-1H-pyrazol-5-yl)-3-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)thiourea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-3-(1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-1-phenyl-1H-pyrazol-5-yl)thiourea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1',4-dimethyl-1-phenyl-1H,1'H-3,4'-bipyrazol-5-yl)thiourea; 1-((3S,4R)-4-(4-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1',4 dimethyl-1-phenyl-1H,1'H-3,4'-bipyrazol-5-yl)thiourea; trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)urea; trans-1-(2-methoxyethyl)-4-phenylpyrrolidin-3-yl)-3-(pyrazolo[1,5-a]pyridin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(pyrazolo[1,5-a]pyridin-3-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(5-methyl-3-phenyl-1-(pyrazin-2-yl)-1H-pyrazol-4-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1,5-dimethyl-3-phenyl-1H-pyrazol-4-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1,5-dimethyl-3-phenyl-1H-pyrazol-4-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1-isopropyl-5-methyl-3-phenyl-1H-pyrazol-4-yl)urea; 1-((3S,4R)-4-(3,5-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1-isopropyl-5-methyl-3-phenyl-1H-pyrazol-4-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(5-methyl-3-phenyl-1-(2,2,2-trifluoroethyl)-1H-pyrazol-4-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1-ethyl-5-methyl-3-phenyl-1H-pyrazol-4-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1-ethyl-3-methyl-5-phenyl-1H-pyrazol-4-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1-methyl-5-phenyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(1-methyl-3-phenyl-5-(trifluoromethyl)-1H-pyrazol-4-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(3-methyl-1-phenyl-1H-pyrazol-4-yl)urea; 1-((3S,4R)-4-(3,4-difluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(l-phenyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)urea; or an acceptable salt thereof.

[0219] In some embodiments, the Trk inhibitor is 5-Chloro-N 4 -(5-cyclopropyl-1H-pyrazol-3-yl)-N 2 -(1-phenylethyl)pyrimidine-2,4-diamine; 5-Bromo-N 4 -(3-ethyl-1H-pyrazol-5-yl)-N 2 -(1-phenylethyl)pyrimidine-2,4-diamine; N 4 -(3-tert-butyl-1H-pyrazol-5-yl)-5-chloro-N 2 -(1-phenylethyl)pyrimidine-2,4-diamine; N 4 -(3-cyclopropyl-1H-pyrazol-5-yl)-N 2 -(1-phenylethyl)-5-(trifluoromethyl)pyrimidine-2,4-diamine; 5-Bromo-N 4 -(3-cyclopropyl-1H-pyrazol-5-yl)-N 2 -[(1S)-1-(4-fluorophenyl)ethyl]pyrimidine-2,4-diamine; 5-Bromo-N 4 -(3-cyclopropyl-1H-pyrazol-5-yl)-N2 -[(1S)-1-phenylpropyl]pyrimidine-2,4-diamine; 5-Bromo-N 4 -(3-cyclopropyl-1H-pyrazol-5-yl)-N 2 -[(1S)-1-(4-nitrophenyl)ethyl]pyrimidine-2,4-diamine; (2R)-2-({5-bromo-4-[(3-cyclopropyl-1H-pyrazol-5-yl)amino]pyrimidin-2yl}amino)-2-phenylethanol; 5-Bromo-N 4 -(5-cyclopropyl-1H-pyrazol-3-yl)-N 2 -(1-phenylethyl)pyrimidine-2,4-diamine; 5-Chloro-N 4 -(5-cyclopropyl-1H-pyrazol-3-yl)-N 2 -(1-phenylpropyl)pyrimidine-2,4-diamine is selected from the group consisting of:

[0220] Syk inhibitors Specifically, in feeder-free embodiments, the medium may also contain a SYK (spleen tyrosine kinase) inhibitor. Representative SYK inhibitors may be selected from the group consisting of entospletinib (GS-9973), fostamatinib (R788), R406, celdulatinib (PRT062070), and TAK-659.

[0221] In some embodiments, the Syk inhibitor has the following structure: Entospletinib with TIFF0007815111000010.tif49128.

[0222] The chemical name of entospletinib is 6-(1H-indazol-6-yl)-N-(4-morpholinophenyl)imidazo[1,2-a]pyrazin-8-amine. Entospletinib, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, can be prepared according to the procedures described in U.S. Patent Nos. 8,748,607 and 8,450,321 and U.S. Patent Application Publication No. 2015 / 0038505.

[0223] In some embodiments, the Syk inhibitor has the formula: TIFF0007815111000011.tif44128 or an ester, stereoisomer, or tautomer thereof, In the above formula, R1 is TIFF0007815111000012.tif75128, where TIFF0007815111000013.tif2128 shows the point of attachment to the rest of the compound in formula, R2 is H or 2-hydroxyethoxy; R3 is H or methyl; R4 is H or methyl.

[0224] In one embodiment, each of R2, R3, and R4 is H, and R1 is as defined above. In one embodiment, R2 is H, R3 is methyl, R4 is H, and R1 is as defined above. In one embodiment, R2 is H, R3 is H, R4 is methyl, and R1 is as defined above. In one embodiment, R2 is 2-hydroxyethoxy, R3 is methyl, R4 is H, and R1 is as defined above. In one embodiment, R2 is 2-hydroxyethoxy, R3 is methyl, R4 is H, and R1 is as defined above. In one embodiment, R2 is 2-hydroxyethoxy, R3 is H, R4 is methyl, and R1 is as defined above.

[0225] In one embodiment, the SYK inhibitor is: TIFF0007815111000014.tif227160TIFF0007815111000015.tif168153 Suitable Syk inhibitors are described in U.S. Patent No. 9,290,050.

[0226] Syk inhibitors utilized in the present invention include compounds disclosed in U.S. Patent Nos. 9,290,050 and 6,432,963 and U.S. Patent Application Publication No. US2004 / 0029902 A1, each of which is incorporated herein by reference in its entirety. Exemplary Syk inhibitors from these references include: 2-(2-aminoethylamino)-4-(3-methylanilino)pyrimidine-5-carboxamide; 2-(2-aminoethylamino)-4-(3-trifluoromethylanilino)pyrimidine-5-carboxamide; 2-(4-aminobutylamino)-4-(3-trifluoromethylanilino)pyrimidine-5-carboxamide; 2-(2-aminoethylamino)-4-(3-bromoanilino)pyrimidine-5-carboxamide; 2-(2-aminoethylamino)-4-(3-nitroanilino)pyrimidine-5-carboxamide; 2-(2-aminoethylamino)-4-(3,5-dimethylanilino)pyrimidine-5-carboxamide; 2-(2-aminoethylamino)-4-(2-naphthylamino)pyrimidine-5-carboxamide; 2-(cis-2-aminocyclohexylamino)-4-(3-methylanilino)pyrimidine-5-carboxamide; 2-(cis-2-aminocyclohexylamino)-4-(3-bromo-anilino)pyrimidine-5-carboxamide; 2-(cis-2-aminocyclohexylamino)-4-(3,5-dichloroanilino)pyrimidine-5-carboxamide and 2-(cis-2-aminocyclohexylamino)-4-(3,4,5-trimethoxyanilino)pyrimidine-5-carboxamide; N2,N4-[(2,2-dimethyl-4H-benzo[1,4]oxazin-3-one)-6-yl]-5-fluoro-2,4-pyrimidinediamine; N4-(3,4-dichlorophenyl)-5-fluoro-N2-(indazolin-6-yl)-2,4-pyrimidinediamine; N4-(3,4-ethylenedioxyphenyl)-5-fluoro-N2-(1-methyl-indazolin-5-yl)-2,4-pyrimidinediamine; N2,N4-bis(3-hydroxyphenyl)-5-fluoro-2,4-pyrimidinediamine; N2,N4-bis(3,4-ethylenedioxyphenyl)-5-fluoro-2,4-pyrimidinediamine; N4-(1,4-benzoxazin-6-yl)-5-fluoro-N2-[3-(N-methylamino)carbonylmethyleneoxyphenyl]-2,4-pyrimidinediamine; N2,N4-bis(3-aminophenyl)-5-fluoro-2,4-pyrimidinediamine; N4-(3,4-ethylenedioxyphenyl)-5-fluoro-N2-[3-(N-methylamino)-carbonylmethyleneoxyphenyl]-2,4-pyrimidinediamine; 5-Fluoro-N4-(3-hydroxyphenyl)-N2-[3-(N-methylamino)carbonylmethyleneoxyphenyl]-2,4-pyrimidinediamine; N4-(3-hydroxyphenyl)-5-trifluoromethyl-N2-[3-(N-methylamino)carbonylmethyleneoxyphenyl]-2,4-pyrimidinediamine; 5-Fluoro-N4-[(1H)-indol-6-yl]-N2-[3-(N-methylamino)carbonylmethyleneoxyphenyl]-2,4-pyrimidinediamine; 5-Fluoro-N4-(3-hydroxyphenyl)-N2-[3-(N-methylamino)carbonylmethyleneoxyphenyl]-2,4-pyrimidinediamine; 5-Fluoro-N2-(3-methylaminocarbonylmethyleneoxyphenyl)-N4-[2-H-pyrido[3,2-b]-1,4-oxazin-3(4H)-one-6-yl]-2,4-pyrimidinediamine; N4-(3,4-ethylenedioxyphenyl)-5-fluoro-N2-[3-(2-hydroxyethylamino)carbonylmethyleneoxyphenyl]-2,4-pyrimidinediamine; 5-Fluoro-N4-(3-hydroxyphenyl)-N2-[3-(N-methylamino)carbonylmethyleneoxyphenyl]-2,4-pyrimidine-diamine; N2,N4-bis(indol-6-yl)-5-fluoro-2,4-pyrimidinediamine; 5-Fluoro-N2-[2-(2-hydroxy-1,1-dimethylethylamino)carbonylbenzofuran-5-yl]-N4-(3-hydroxyphenyl)-2,4-pyrimidinediamine; N2-[3-(N2,3-dihydroxypropylamino)carbonylmethyleneoxyphenyl]-N4-(3,4-ethylenedioxyphenyl)-5-fluoro-2,4-pyrimidinediamine; N2-(3,5-dimethoxyphenyl)-N4-(3,4-ethylenedioxyphenyl)-5-fluoro-2,4-pyrimidinediamine; N4-(3,4-ethylenedioxyphenyl)-5-fluoro-N2-[3-(1,3-oxazol-5-yl)phenyl]-2,4-pyrimidinediamine; N4-(3,4-ethylenedioxyphenyl)-5-fluoro-N2-[3-(N-methylamino)-carbonylmethyleneoxyphenyl]-2,4-pyrimidinediamine; 5-Fluoro-N2-(3-hydroxyphenyl)-N4-[4-(3-phenyl-1,2-4-oxadiazol-5-yl)methyleneoxyphenyl]-2,4-pyrimidinediamine; N4-(3,4-ethylenedioxyphenyl)-5-fluoro-N2-(indazolin-6-yl)-2,4-pyrimidinediamine; 5-Fluoro-N4-(3-hydroxyphenyl)-N2-(indazolin-6-yl)-2,4-pyrimidinediamine; N4-(3,4-ethylenedioxyphenyl)-5-fluoro-N2-(1-methyl-indazolin-5-yl)-2,4-pyrimidinediamine; 5-Fluoro-N4-(3-hydroxyphenyl)-N2-(1-methy-indazolin-5-yl)-2,4-pyrimidinediamine; N4-(3,4-ethylenedioxyphenyl)-5-fluoro-N2-[4-(3-phenyl-1,2,4-oxadiazol-5-yl)methyleneoxyphenyl]-2,4-pyrimidinediamine; N4-(3,5-dimethyl-4-hydroxyphenyl)-5-fluoro-N2-[3-[2-(N-morpholino)ethyleneoxy]phenyl]-2,4-pyrimidinediamine; N4-(3,5-dimethyl-4-hydroxyphenyl)-5-fluoro-N2-[3-[2-(N-morpholino)ethyloxy]phenyl]-2,4-pyrimidine-diamine; N4-(3-chloro-4-hydroxy-5-methylphenyl)-5-fluoro-N2-[3-[2-(N-morpholino)ethyloxy]phenyl]-2,4-pyrimidinediamine; N2-(3-tert-butylcarbonylaminophenyl)-N4-(3-hydroxyphenyl)-5-fluoro-2,4-pyrimidinediamine; N4-(3-tert-butylphenyl)-N2-[3-(N-methylamino)carbonylmethyleneoxyphenyl]-5-fluoro-2,4-pyrimidine-diamine; N4-(3-tert-butylphenyl)-N2-[3-(N2,3-dihydroxypropylamino)carbonylmethyleneoxyphenyl]-5-fluoro-2,4-pyrimidinediamine; N2-[3-(N2,3-dihydroxypropylamino)carbonylmethyleneoxyphenyl]-5-fluoro-N4-(3-isopropylphenyl)-2,4-pyrimidinediamine; N4-[4-(cyanomethyleneoxy)phenyl]-5-fluoro-N2-(3-hydroxyphenyl)-2,4-pyrimidinediamine; N4-(3,5-dimethyl-4-hydroxyphenyl)-5-fluoro-N2-[3-(N-piperazino)carbonylmethyleneoxyphenyl]-2,4-pyrimidinediamine; N4-(3,5-dimethyl-4-hydroxyphenyl)-5-fluoro-N2-[3-[2-(N-piperazino)ethoxy]phenyl]-2,4-pyrimidine-diamine bishydrochloride; N4-(3,4-ethylenedioxyphenyl)-5-fluoro-N2-[4-(2-hydroxyethyloxy)phenyl]-2,4-pyrimidinediamine; N4-(1,4-benzoxazin-3-one-6-yl)-5-fluoro-N2-(3-hydroxyphenyl)-2,4-pyrimidinediamine; (+ / -)-5-Fluoro-N2-[(N-methylacetamido-2)-3-phenoxy]-N4-(2-methyl-1,4-benzoxazin-6-yl)-2,4-pyrimidinediamine; N2-(1,4-benzoxazin-3-one-6-yl)-5-fluoro-N4-(3-hydroxyphenyl)-2,4-pyrimidinediamine; N4-(3-chloro-4-trifluoromethoxyphenyl)-5-fluoro-N2-[3-(N-methylamino)carbonylmethyleneoxyphenyl]-2,4-pyrimidinediamine; 5-Fluoro-N4-(3-hydroxy-4-methylphenyl)-N2-[3-[(N-methylamino)carbonylmethyleneoxy]phenyl]-2,4-pyrimidinediamine; 5-Fluoro-N4-(3-hydroxyphenyl)-N2-[4-methyl-3-[(N-methylamino)carbonylmethyleneoxy]phenyl]-2,4-pyrimidinediamine; 5-Fluoro-N4-(3-hydroxy-4-methoxyphenyl)-N2-[3-(N-methylamino)carbonylmethyleneoxyphenyl]-2,4-pyrimidinediamine; N4-(3-chloro-4-methylphenyl)-5-fluoro-N2-[3-(N-methylamino)-carbonylmethyleneoxyphenyl]-2,4-pyrimidinediamine; N4-(3-chloro-4-methoxyphenyl)-5-fluoro-N2-[3-[(N-methylamino)carbonylmethyleneoxy]phenyl]-2,4-pyrimidinediamine; 5-Fluoro-N4-1(1H)-indol-5-yl]-N2-[3-[(N-methylamino)carbonylmethyleneoxy]phenyl]-2,4-pyrimidine-diamine; 5-Fluoro-N4-(3-hydroxyphenyl)-N2-[1-(methoxycarbonyl)methyl-indazolin-5-yl]-2,4-pyrimidinediamine; 5-Fluoro-N4-(3-hydroxyphenyl)-N2-[1-(3-hydroxypropyl)indazolin-6-yl]-2,4-pyrimidinediamine; N4-(3,4-ethylenedioxyphenyl)-5-fluoro-N2-[1-(3-hydroxypropyl)indazolin-5-yl]-2,4-pyrimidinediamine; 5-Fluoro-N4-(3-hydroxyphenyl)-N2-[1-(3-hydroxypropyl)indazolin-5-yl]-2,4-pyrimidinediamine; 5-Fluoro-N2-[1-(3-hydroxypropyl)indazolin-5-yl]-N4-(4-isopropoxyphenyl)-2,4-pyrimidinediamine; N4-(3,4-ethylenedioxyphenyl)-5-fluoro-N2-[1-[2(N-methylaminocarbonyl)ethyl]-indazolin-5-yl]-2,4-pyrimidinediamine; 5-Fluoro-N4-(4-isopropoxyphenyl)-N2-[1-[2(N-methylaminocarbonyl)ethyl]-indazolin-5-yl]-2,4-pyrimidinediamine; N4-[(2,2-dimethyl-4H-benzo[1,4]oxazin-3-one)-6-yl]-5-fluoro-N2-[3-(methylaminocarbonylmethylene-oxy)phenyl]-2,4-pyrimidinediamine; N4-[(2,2-dimethyl-4H-benzo[1,4]oxazin-3-one)-6-yl]-5-fluoro-N2-(1-methylindazolin-5-yl)-2,4-pyrimidinediamine; N4-[(2,2-difluoro-4H-benzo[1,4]oxazin-3-one)-6-yl]-5-fluoro-N2-[3-(methylaminocarbonylmethyleneoxy)phenyl]-2,4-pyrimidinediamine; N4-1(2,2-dimethyl-4H-5-pyridol-1,4]oxazin-3-one)-6-yl]-5-fluoro-N2-[3-(methylaminocarbonylmethyleneoxy)phenyl]-2,4-pyrimidinediamine; 5-Fluoro-N2-(3-methylaminocarbonylmethyleneoxyphenyl)-N4-[2H-pyrido[3,2-b]-1,4-oxazin-3(4H)-one-6-yl]-2,4-pyrimidinediamine; N4-(4-amino-3,4-dihydro-2H-1-benzopyran-6-yl)-5-fluoro-N2-[3-(N-methylamino)carbonylmethyleneoxyphenyl]-2,4-pyrimidinediamine; N4-(3-chloro-4-hydroxy-5-methylphenyl)-5-fluoro-N2-[3-[2-(N-piperazino)ethoxy]phenyl]-2,4-pyrimidinediamine; and N4-(3-methylcarbonyloximephenyl)-5-fluoro-N2-[3-(N-methylamino)carbonylmethyleneoxyphenyl]-2,4-pyrimidinediamine; or their salts These include, but are not limited to:

[0227] LPA receptor antagonist Specifically, in feeder-free embodiments, the medium may also contain an LPA receptor antagonist, e.g., an antagonist that inhibits inositol phosphate production induced by LPA1 and LPA3, each with a Ki of 1000 μM or less, and is a substantially weaker inhibitor of LPA2, LPA4, LPA5, and LPA6, i.e., each with a Ki of 5000 μM or less. Ki16198 is a preferred LPA receptor antagonist and is the methyl ester of Ki16425.

[0228] Other LPA receptor antagonists include H2L5765834, H2L5186303, Ki 16425, Ro 6842262, and C LPA5 4.

[0229] In one aspect, provided herein is an LPA receptor inhibitor, or a salt, solvate, polymorph, prodrug, metabolite, N-oxide, stereoisomer, or isomer thereof, having a structure as set forth in US20170042915A1, for example, one of the following structures: TIFF0007815111000016.tif100142TIFF0007815111000017.tif210133TIFF0007815111000018.tif59128

[0230] GSK3 inhibitors Specifically, in feeder-free embodiments, the medium may also contain a GSK3 inhibitor. Exemplary GSK3 inhibitors include CHIR-99021 (CT99021) HCl, SB216763, CHIR-98014, TWS119, tideglusib, SB415286, CHIR-99021 (CT99021), AZD2858, AZD1080, AR-A014418, TDZD-8, LY2090314, BIO-acetoxime, IM-12, 1-azakempaullone, indirubin, and 6-BIO.

[0231] Suitable GSK-3 inhibitors, methods for their synthesis, and assays for GSK inhibition are also described, for example, in WO 03 / 004472, WO 03 / 055492, WO 03 / 082853, WO 2004 / 018455, WO 2004 / 037791, 06 / 001754, WO 07 / 040436, WO 07 / 040438, WO 07 / 040439, WO 07 / 040440, WO08 / 002244, WO08 / 002245, and Coghlan et al. Chemistry & Biology 2000, 7(10):793-803. GSK-3 inhibitors are also reviewed, for example, in Cohen et al. Nature Reviews Drug Discovery 2004, 3:479-487; Kramer et al. International Journal of Alzheimer's Disease Volume 2012, Article ID 381029, 32 pages; and Eldar-Finkelman et al., Front Mol Neurosci. 2011; 4:32. In one embodiment, the GSK-3 inhibitor is lithium, for example, a lithium salt such as lithium carbonate, lithium citrate, lithium chloride, lithium orotate, lithium bromide, or lithium chloride. In another embodiment, the GSK-3 inhibitor is 3-(2,4-dichlorophenyl)-4-(l-methyl-lH-indol-3yl)-lH-pyrrole-2,5-dione (SB216763) or 3-(3-chloro-4-hydroxyphenylamino)-4-(2 nitrophenyl)-lH-pyrrole-2,5-dione (SB-415286), the structures of which are shown as the following formulas: TIFF0007815111000019.tif37128 (named SB216763 and SB-415286 in the literature).

[0232] Additional GSK3 inhibitors include 6-BIO, hymenialdisine, dibromocantharelline, CT98014, CT98023, CT99021, TWS119, AR-A014418, AZD-1080, kenpaullone, alsterpaullone, cazpaullone, aloisine A, manzamine A, palinurine, tricantine, TDZD-8, NP00111, P031115, P031112 (tideglusib), HMK-32, and L803-mts, the chemical structures and syntheses of which are described in Eldar-Finkelman et al., Front Mol Neurosci.2011;4:32 or where reference is indicated.

[0233] CK2 inhibitors Specifically, in embodiments that are feeder-free, the medium may also include a CK2 inhibitor, such as CX-4945 (silmitasertib), CX-8184, DMAT, ellagic acid, or TTP22.

[0234] Further exemplary CK2 inhibitors are those taught in PCT Publication WO 2017 / 070137 A1, for example, those having the formula: TIFF0007815111000020.tif36128, including enantiomers, diastereomers, tautomers, acceptable salts, prodrugs, hydrates, or solvates thereof; In the above formula, R4 is 1 to 3 R e C replaced with 1-4 Alkyl, C 3-6 cycloalkyl and 1 to 3 R e heterocyclyl substituted with; Two R7 groups, together with the nitrogen atom to which they are both attached, form a 4- to 7-membered monocyclic or 7- to 12-membered bicyclic heterocycle substituted with 1 to 4 R, which is bounded by carbon atoms and NR8a and 1 to 3 additional heteroatoms selected from the group consisting of S(O)2, O, and S(O)2; R8, in each occurrence, is H, F, CI, Br, 1 to 4 R e C replaced with 1-4 Alkyl, ═O (ketone), 1 to 5 R e C replaced with 2-4 Alkenyl, -(CHR g ) r OR b , -CHR g ) r S(0) p R c , -(CHR g ) r C(=O)(CHR g ) r R d , -(CHR g ) r NR a R a , -(CHR g ) r C(=O)NR a R a , -(CHR g ) r C(=O)NR a S(O) p R c , -(CHR g ) r NR a (CR g R g ) r C(=O)R d , -(CHR g ) r NR a C(=O)OR b , -(CHR g ) r OC(=O)(CHR g ) r R d , -(CHR g ) r OC(=O)(CHR g ) r C(=O)OR d , -(CHR g ) r OC(=O)(CHRg ) r C(=O)NR a R a , -(CHR g ) r OC(=O)(CHR g ) r NR a C(=O)R b , -(CHR g ) r OC(=O)(CHR g ) r NR a R a , -(CHR g ) r NR a C(=O)NR a R a , -(CHR g ) r C(=O)(CH2) r OR b , -(CHR g ) r C(=O)(CHR g ) r OC(=O)R b , -(CHR g ) r S(0)2NR a R a , -(CHR g ) r NR a S(0) p NR a R a , -(CHR g ) r NR a S(0) p R c , -OPO3H, 1 to 5 R e -(CHR g ) r -C 3-6 Cycloalkyl, 1 to 4 R e -(CHR g ) r -aryl, and 1 to 4 R e -(CHR g ) r -heterocyclyl; R8a is H, 1 to 5 R e C replaced with 1-4 Alkyl, 1 to 5 R e C replaced with 2-4 Alkenyl, -(CHR g ) r OR b , -(CHR g ) r S(0) p R c , -(CHRg)rC(=O)(CHRg)rRd, -CHRg)rNRaRa, -(CHRg)rC(=O)NRaRa, -(CHR g ) r C(=O)NR a S(O) p R c , -(CHR g ) r NR a (CR g R g ) r C(=O)Rd, -(CHR g ) r NHC(=O)OR b , -(CHR g ) r OC(=O)(CHR g ) r R d , -(CHR g ) r OC(=O)(CHR g ) r C(=O)OR d , -(CHR g ) r OC(=O)(CHR g ) r C(=O)NR a R a , -(CHR g ) r OC(=O)(CHR g ) r NR a C(=O)R b , -(CHR g ) r OC(=O)(CHR g ) r NR a R a , -(CHRg ) r NR a (CHR g ) r C(=O)NR a R a , -(CHR g ) r C(=O)OR b , -(CHR g ) r C(=O)(CHR g ) r OC(=O)R b , -(CHR g ) r S(O)NR a R a , -(CHR g ) r NR a S(O) p NR a R a , -(CHR g rNR a S(O) p R c , -OPO3H, 1 to 5 R e -(CHR g ) r -C 3-6 Cycloalkyl, 1 to 4 R e -(CHR g ) r -aryl, and 1 to 4 R e -(CHR g ) r -heterocyclyl; R a is a compound consisting of H, CN, and 1 to 5 R e C replaced with 1-6 Alkyl, 1 to 5 R e C replaced with 2-6 Alkenyl, 1 to 5 R e C replaced with 2-6 Alkynyl, 1 to 5 R e substituted with -(CH2) r -C3-io carbocyclyl and 1 to 5 R e substituted with -(CH2) r-heterocyclyl; or R a and R a consists of 1 to 5 R atoms, along with the nitrogen atom to which they are both attached. e forming a heterocycle substituted with; R b Each occurrence of H and 1 to 5 R e C replaced with 1-6 Alkyl, 1 to 5 R e C replaced with 2-6 Alkenyl, 1 to 5 R e C replaced with 2-6 Alkynyl, 1 to 5 R e substituted with -(CH2) r -C3-io carbocyclyl and 1 to 5 R e substituted with -(CH2) r -heterocyclyl; R c is 1 to 5 R in each occurrence. e Ci-6 alkyl substituted with 1 to 5 R e C replaced with 2-6 Alkenyl, 1 to 5 R e C replaced with 2-6 Alkynyl, C 3-6 independently selected from the group consisting of carbocyclyl, and heterocyclyl; R d is, in each occurrence, H, OH, 1 to 5 R e C replaced with 1-6 Alkyl, 1 to 5 R e C replaced with 2-6 Alkenyl, 1 to 5 R e C replaced with 2-6 Alkynyl, 1 to 5 R e substituted with -(CH2) r -C3-io carbocyclyl and 1 to 5 R e substituted with -(CH2) r -heterocyclyl; R e is a string of H, N3, and 1 to 5 R in each occurrence. f C replaced with1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, -(CH2) r -C 3-6 Cycloalkyl, (CH2) r -Heterocyclyl, F, CI, Br, -(CH2) r CN, NO2, =O, -OPO3H, -OSi(C 1-4 alkyl)3, (CH2) r O.C. 1-5 Alkyl, -(CH2) r O(CH2) r O.C. 1-5 Alkyl, -(CH2) r OH, -(CH2) r S(O)2C 1-5 Alkyl, -(CH2) r S(O)2R f , -(CH2) r NHS(O)2C 1-5 Alkyl, -S(O)2NH2, -SH, -(CH2) r NR f R f , -(CH2) r NHC(=O)OR f , -(CH2) r NHC(=O)R f , -(CH2) r NHC(=NH)NR f R f , -(CH2) r C(=O)(CH2) r R f , and -(CH2) r C(=O)OR f independently selected from the group consisting of: R f is, in each occurrence, H, -CH2OH, -(CH2) r O.C. 1-5 Alkyl, (optionally substituted with F, CI, OH, NH2) C 1-5 C optionally substituted with alkyl, NH 3-6 Cycloalkyl, -(CH2) r S(O)PC 1-4 Alkyl, -NHC(=O)C 1-4Alkyl, -C(=O)NH2, -C(=O)OC 1-4 Alkyl, -C(=O)C 1-4 Alkyl, -(CH2) r -phenyl, optionally substituted with alkyl -(CH2) r -heterocyclyl, and CN, or R f and R f C, along with the nitrogen atom to which they are both attached. 1-4 forming a heterocycle which may be substituted with alkyl; R g is H, F, OH, and C in each occurrence 1-5 independently selected from the group consisting of alkyl; p, at each occurrence, is independently selected from the group consisting of 0, 1, and 2; and r is independently selected from the group consisting of 0, 1, 2, 3, 4, and 5 in each occurrence.

[0235] Notch agonists The culture medium of the present invention may additionally contain a Notch agonist. Notch signaling has been shown to play an important role in both cell fate determination and cell survival and proliferation. Notch receptor proteins can interact with a number of surface-bound or secreted ligands, including, but not limited to, Jagged-1, Jagged-2, Delta-1, or Delta-like-1, Delta-like-3, and Delta-like-4. Upon ligand binding, Notch receptors are activated by sequential cleavage events involving members of the ADAM protease family, as well as by intramembrane cleavage regulated by the gamma-secretase presinilin. This results in the translocation of the intracellular domain of Notch to the nucleus, where it transcriptionally activates downstream genes.

[0236] "Notch agonist," as used herein, includes molecules that stimulate Notch activity in cells by at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70%, at least about 90%, at least about 100%, at least about 3-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, or more, compared to the level of Notch activity in the absence of the Notch agonist. As is known in the art, Notch activity can be determined by measuring the transcriptional activity of Notch, for example, by the 4xwtCBF1-luciferase reporter construct described by Hsieh et al. (Mol. Cell. Biol. 16:952-959, 1996, incorporated herein by reference).

[0237] In certain embodiments, the Notch agonist is selected from Jagged-1, Delta-1, and Delta-like-4, or active fragments or derivatives thereof. In certain embodiments, the Notch agonist is the DSL peptide (Dontu et al., Breast Cancer Res., 6:R605-R615, 2004) having the amino acid sequence CDDYYYGFGCNKFCRPR (SEQ ID NO:36). The DSL peptide (ANA spec) may be used at a concentration of 10 μM to 100 nM, or at least 10 μM, but not more than 100 nM. In certain embodiments, the final concentration of Jagged-1 is about 0.1 to 10 μM; or about 0.2 to 5 μM; or about 0.5 to 2 μM; or about 1 μM.

[0238] In certain embodiments, any of the specific Notch agonists mentioned herein, such as Jagged-1, Jagged-2, Delta-1, and Delta-like-4, may be replaced with a natural, synthetic, or recombinantly produced homolog or fragment thereof that retains at least about 80%, 85%, 90%, 95%, 99% of the respective Wnt agonist activity, and / or has at least about 60%, 70%, 80%, 90%, 95%, 97%, 99% amino acid sequence identity as measured by art-recognized sequence alignment software based on either global alignment techniques (e.g., the Needleman-Wunsch algorithm) or local alignment techniques (e.g., the Smith-Waterman algorithm).

[0239] Representative Notch agonist sequences referred to herein are represented in SEQ ID NOs. 28-35.

[0240] Notch agonists can be added to the culture medium every 1, 2, 3, or 4 days for the first 1-2 weeks of stem cell culture.

[0241] Nicotinamide The culture medium of the present invention may be supplemented with nicotinamide or its analogs, precursors, or mimetics, such as methylnicotinamide, benzamide, pyrazinamide, thymine, or niacin. Nicotinamide may be added to the culture medium at a final concentration of 1 to 100 mM, 5 to 50 mM, or preferably 5 to 20 mM. For example, nicotinamide may be added to the culture medium at a final concentration of approximately 10 mM. Similar concentrations of nicotinamide analogs, precursors, or mimetics may also be used, alone or in combination.

[0242] Extracellular matrix (ECM) Extracellular matrix (ECM), used interchangeably herein with "basement membrane matrix," is secreted by connective tissue cells and comprises a variety of polysaccharides, water, elastin, and proteins, which may include proteoglycans, collagen, entactin (nidogen), fibronectin, fibrinogen, fibrillin, laminin, and hyaluronic acid. The ECM can provide a suitable substrate and microenvironment useful for the selection and culture of stem cells of the present invention.

[0243] In certain embodiments, the stem cells of the present invention are attached to or in contact with ECM.Various types of ECM are known in the art and can contain different compositions, including different types of proteoglycans and / or different combinations of proteoglycans.ECM can be provided by culturing ECM-producing cells, such as certain fibroblasts.Examples of extracellular matrix-producing cells include chondrocytes, which mainly produce collagen and proteoglycans; fibroblasts, which mainly produce type IV collagen, laminin, interstitial procollagen, and fibronectin; and colonic myofibroblasts, which mainly produce collagen (type I, type III, and type V), chondroitin sulfate proteoglycans, hyaluronic acid, fibronectin, and tenascin-C.

[0244] In certain embodiments, at least some of the ECM is produced by a murine 3T3-J2 clone that can be grown on MATRIGEL™ basement membrane matrix (BD Biosciences) as a feeder cell layer.

[0245] Alternatively, ECM can be commercially available. Examples of commercially available extracellular matrices are extracellular matrix proteins (Invitrogen) and MATRIGEL™ basement membrane matrix (BD Biosciences). The use of ECM to culture stem cells can enhance the long-term survival of stem cells and / or the continued existence of undifferentiated stem cells. Alternatives can be scaffolds such as fibrin matrix or fibrin gel, or glycerol-treated allografts that have been initially depleted of cells.

[0246] In certain embodiments, the ECM for use in the methods of the present invention comprises at least two distinct glycoproteins, such as two different types of collagen, or collagen and laminin. The ECM may be a synthetic hydrogel extracellular matrix or a naturally occurring ECM. In certain embodiments, the ECM is provided by MATRIGEL™ basement membrane matrix (BD Biosciences), which comprises laminin, entactin, and collagen IV.

[0247] Culture medium The cell culture medium used in the methods of the present invention can include any cell culture medium, such as a culture medium buffered to about pH 7.4 (e.g., about pH 7.2 to 7.6) with a carbonate-based buffer. Many commercially available tissue culture media may be suitable for the methods of the present invention, including, but not limited to, Dulbecco's Modified Eagle's Medium (DMEM, e.g., DMEM without L-glutamine and containing high glucose), Minimum Essential Medium (MEM), Knockout-DMEM (KO-DMEM), Glasgow Minimum Essential Medium (G-MEM), Basal Eagle's Medium (BME), DMEM / Ham's F12, Advanced DMEM / Ham's F12, Iscove's Modified Dulbecco's Medium, and Minimal Essential Medium (MEM), Ham's F-10, Ham's F-12, Medium 199, and RPMI 1640 medium.

[0248] Cells may be cultured in an atmosphere containing 5-10% CO2 (e.g., at least about 5%, 10% or less CO2, or about 5% CO2). In certain embodiments, the cell culture medium is DMEM / F12 (e.g., a 3:1 mixture) or RPMI 1640 supplemented with L-glutamine, insulin, penicillin / streptomycin, and / or transferrin. In certain embodiments, Advanced DMEM / F12 or Advanced RPMI, which are optimized for serum-free culture and already contain insulin, are used. Advanced DMEM / F12 or Advanced RPMI medium may be further supplemented with L-glutamine and penicillin / streptomycin. In certain embodiments, the cell culture medium is supplemented with one or more purified, natural, semi-synthetic, and / or synthetic factors described herein. In certain embodiments, the cell culture medium is supplemented with about 10% fetal bovine serum (FBS) that is not heat-inactivated prior to use. For example, additional supplements such as B-27® serum-free supplement (Invitrogen), N-acetylcysteine ​​(Sigma), and / or N2 serum-free supplement (Invitrogen), or Neurobasal (Gibco), TeSR (StemGent), etc. may be added to the culture medium.

[0249] In certain embodiments, the medium may contain one or more antibiotics (such as penicillin / streptomycin) to prevent contamination. In certain embodiments, the medium may have an endotoxin content of less than 0.1 endotoxin units / mL, or may have an endotoxin content of less than 0.05 endotoxin units / mL. Methods for determining the endotoxin content of culture medium are known in the art.

[0250] The cell culture medium according to the present invention allows the survival and / or proliferation and / or differentiation of epithelial stem cells on an extracellular matrix. As used herein, the term "cell culture medium" is synonymous with "culture medium," "culture medium," or "cell culture medium."

[0251] The modified (growth) medium of the present invention comprises, in a basal medium, (a) a ROCK (Rho kinase) inhibitor, (b) a Wnt agonist, (c) a mitogenic growth factor, (d) a TGFβ signaling pathway inhibitor such as a TGFβ inhibitor or a TGFβ receptor inhibitor, and (e) insulin or IGF; the medium optionally further comprises a bone morphogenetic protein (BMP) antagonist.

[0252] Thus, in one aspect, the present invention provides a basal medium (basal medium) comprising insulin or an insulin-like growth factor; T3 (3,3',5-triiodo-L-tyrosine); hydrocortisone; adenine; EGF; and 10% fetal bovine serum (not heat-inactivated) in a DMEM:F12 3:1 medium supplemented with L-glutamine.

[0253] In certain embodiments, the basal medium comprises about 5 μg / mL insulin; about 2×10 M T3 (3,3′,5-triiodo-L-tyrosine); about 400 ng / mL hydrocortisone; about 24.3 μg / mL adenine; about 10 ng / mL EGF; and 10% fetal bovine serum (not heat inactivated) in DMEM:F12 3:1 medium supplemented with about 1.35 mM L-glutamine.

[0254] In certain embodiments, the concentration for each of the media components mentioned in the immediately preceding paragraph is independently 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95% higher or lower than the respective stated value, or 2-fold, 3-fold, 5-fold, 10-fold, 20-fold higher than the respective stated value. For example, in an exemplary media, the insulin concentration may be 6 μg / mL (20% higher than the stated 5 μg / mL), the EGF concentration may be 5 ng / mL (50% lower than the stated 10 ng / mL), and each remaining component has the same concentration as the stated concentration.

[0255] In a related aspect, the invention provides a basal medium containing cholera enterotoxin. In another embodiment, the basal medium does not contain cholera enterotoxin.

[0256] The basal medium may further contain one or more antibiotics such as penicillin / streptomycin and / or gentamicin.

[0257] Basal medium can be used to create modified growth medium (or simply, modified medium) by adding one or more of the factors listed above.

[0258] 4. Protein sequences of representative culture medium factors Some representative (non-limiting) protein factors used in the media and methods of the present invention are provided below. For each listed factor, numerous homologs or functional equivalents are known in the art and can be easily retrieved from public databases, such as GenBank, EMBL, and / or NCBI RefSeq, to ​​name a few. Additional proteins or their peptide fragments or the polynucleotides encoding them, for example, functional homologs from human or non-human mammals, can be easily retrieved from public sources, for example, through sequence-based searches such as NCBI BLASTp or BLASTn, or both.

[0259] TIFF0007815111000021.tif251146TIFF0007815111000022.tif240146TIFF00078151110 00023.tif240146TIFF0007815111000024.tif234146TIFF0007815111000025.tif195146

[0260] 5. How to differentiate stem cells Isolated stem cells (e.g., epithelial stem cells) can be induced to differentiate into differentiated cells normally present in the tissue or organ from which they were derived or isolated. Other tissues include the fallopian tubes, endometrium (uterus), male ductus efferentus, male epididymis, male vas deferens, male ejaculatory duct, male bulbourethral gland, and seminal vesicles. Differentiated cells may express markers characteristic of differentiated cells and can be easily distinguished from stem cells that do not express such differentiated cell markers.

[0261] 6. Markers Generally, for all of the markers described below, gene expression can be measured at the RNA level. Additionally, expression of certain markers can also be detected by protein expression, for example, using antibodies specific for the protein encoded by the marker gene.

[0262] 7. How to use In a further aspect, the present invention provides for the use of stem cells of the present invention isolated from various cultures in drug discovery screens, toxicity assays, animal-based disease models, or medicine, such as regenerative medicine.

[0263] Genetic manipulation of cloned stem cells For example, stem cells isolated by the methods of the present invention are suitable for many types of genetic manipulation, including the introduction of exogenous genetic material capable of modulating the expression of one or more target genes of interest. Such types of gene therapy can be used, for example, in methods for repairing damaged or diseased tissues. Briefly, appropriate vectors, including adenoviral, lentiviral, or retroviral gene delivery vehicles (see below), can be used to deliver genetic information such as DNA and / or RNA to any of the stem cells of the present invention. Those skilled in the art can replace or repair specific genes targeted by gene therapy. For example, to replace a non-functional gene, a normal gene can be inserted into a non-specific location in the genome of the affected cell. In another example, an abnormal gene sequence can be replaced with a normal gene sequence through homologous recombination. Alternatively, selective reversion may restore a gene to its normal function. Another example is the alteration of the regulation (the degree to which a gene is turned on or off) of a specific gene. Preferably, stem cells are treated ex vivo using a gene therapy approach and then transferred into a mammal, preferably a human in need of treatment.

[0264] Art-recognized methods for genetic manipulation, including transfection and infection with various types of nucleic acid constructs (e.g., with viral vectors), can be applied to stem cells so isolated.

[0265] For example, heterologous nucleic acids (e.g., DNA) can be introduced into the stem cells of the present invention using chemical materials or biological vectors (viruses), by physical processes (e.g., electroporation, sonoporation, optical transfection, protoplast fusion, impalefection, hydrodynamic delivery, nanoparticles, magnetofection). Chemical-based transfection can be based on calcium phosphate, cyclodextrins, polymers (e.g., cationic polymers such as DEAE-dextran or polyethyleneimine), highly branched organic compounds such as dendrimers, liposomes (e.g., cationic liposomes, lipofection such as lipofection using lipofectamine), or nanoparticles (with or without chemical or viral functionality).

[0266] The nucleic acid construct comprises a nucleic acid molecule of interest and is generally capable of expressing the nucleic acid molecule of interest in a cell into which it is introduced.

[0267] In certain embodiments, the nucleic acid construct is an expression vector in which a nucleic acid molecule encoding a gene product such as a polypeptide, or a nucleic acid that antagonizes expression of a polypeptide (e.g., an siRNA, miRNA, shRNA, antisense sequence, aptamer, ribozyme, etc.), is operably linked to a promoter capable of expressing the nucleic acid molecule in a target cell (e.g., an isolated stem cell).

[0268] The term "expression vector" generally refers to a nucleic acid molecule capable of achieving expression of a gene / nucleic acid molecule it contains in a cell compatible with such sequence. These expression vectors typically contain at least a suitable promoter sequence and, optionally, a transcription termination signal. The nucleic acid or DNA or nucleotide sequence encoding the polypeptide is incorporated into a DNA / nucleic acid construct capable of introduction into and expression in an in vitro cell culture as identified in the methods of the present invention.

[0269] DNA constructs prepared for introduction into specific cells typically contain a replication system recognized by the cell, a DNA segment intended to encode the desired polypeptide, and transcriptional and translational initiation and termination control sequences operably linked to the polypeptide-encoding segment. A DNA segment is "operably linked" when it is placed into a functional relationship with another DNA segment. For example, a promoter or enhancer is operably linked to a coding sequence if it stimulates the transcription of that sequence. DNA for a signal sequence is operably linked to DNA encoding a polypeptide if it is expressed as a preprotein involved in the secretion of the polypeptide. Generally, operably linked DNA sequences are contiguous, and in reading phase, in the case of a signal sequence. However, enhancers need not be contiguous with the coding sequence whose transcription they control. Linking is accomplished by ligation at convenient restriction sites or by adapters or linkers inserted instead.

[0270] The selection of an appropriate promoter sequence generally depends on the host cell selected for expression of the DNA segment. Examples of suitable promoter sequences include eukaryotic promoters well known in the art (see, e.g., Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Third Edition, 2001). Transcriptional control sequences typically include a heterologous enhancer or promoter recognized by the cell. Suitable promoters include the CMV promoter. Expression vectors may contain a replication system and utilize transcriptional and translational control sequences along with an insertion site for a polypeptide-encoding segment. Examples of viable combinations of cell lines and expression vectors are described in Sambrook and Russell (2001, supra) and Metzger et al. (1988) Nature 334:31-36.

[0271] Some aspects of the present invention relate to the use of a nucleic acid construct or expression vector comprising a nucleotide sequence as defined above, wherein the vector is a vector suitable for gene therapy. Anderson(Nature 392:25-30,1998);Walther and Stein(Drugs 60:249-71,2000);Kay et al.(Nat.Med.7:33-40,2001);Russell(J.Gen.Virol.81:2573-604,2000);Amado and Chen(Science 285:674-6,1999);Federico(Curr.Opin.Biotechnol.10:448-53,1999);Vigna and Naldini(J.Gene Med.2:308-16,2000);Marin et al.(Mol.Med.Today 3:396-403,1997);Peng and Suitable vectors for gene therapy are known in the art, such as those described in Russell (Curr. Opin. Biotechnol. 10:454-7, 1999); Sommerfeld (J. Gen. Virol. 80:3049-64, 1999); Reiser (Gene Ther. 7:910-3, 2000); and the references cited therein (all incorporated by reference). Examples include integrating and non-integrating vectors, such as those based on retroviruses, adenoviruses (AdV), adeno-associated viruses (AAV), lentiviruses, poxviruses, alphaviruses, and herpesviruses.

[0272] Particularly suitable gene therapy vectors include adenoviral (Ad) and adeno-associated viral (AAV) vectors. These vectors infect a wide variety of dividing and non-dividing cell types. Furthermore, adenoviral vectors are capable of high-level transgene expression. However, due to the episomal nature of adenoviral and AAV vectors after cell entry, these viral vectors are best suited for therapeutic applications requiring only transient expression of the transgene, as described above (Russell, J. Gen. Virol. 81:2573-2604, 2000; Goncalves, Virol J. 2(1):43, 2005). Preferred adenoviral vectors have been modified to reduce host responses, as outlined by Russell (2000, supra). The safety and efficacy of AAV gene transfer has been extensively studied in humans, with promising results in the liver, muscle, CNS, and retina (Manno et al., Nat. Medicine 2006; Stroes et al., ATYB 2008; Kaplitt, Feigin, Lancet 2009; Maguire, Simonelli et al. NEJM 2008; Bainbridge et al., NEJM 2008).

[0273] AAV2 is the most well-characterized serotype for gene transfer research in both humans and experimental models.AAV2 exhibits natural tropism for skeletal muscle, neurons, vascular smooth muscle cells, and hepatocytes.Other examples of adeno-associated virus-based non-integrating vectors include AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and pseudotyped AAV.The use of non-human serotypes such as AAV8 and AAV9 may be useful to overcome these immunological responses in subjects, and clinical trials have just begun (ClinicalTrials.gov identification number: NCT00979238). For gene transfer into hepatocytes, adenovirus serotype 5, or AAV serotypes 2, 7, or 8 have been shown to be effective vectors and are therefore preferred Ad or AAV serotypes (Gao, Molecular Therapy 13:77-87, 2006).

[0274] An exemplary retroviral vector for application in the present invention is a lentivirus-based expression construct. Lentivirus vectors have the unique ability to infect non-dividing cells (Amado and Chen, Science 285:674-676, 1999). The construction and use of lentivirus-based expression constructs are described in U.S. Patent Nos. 6,165,782, 6,207,455, 6,218,181, 6,277,633, and 6,323,031, as well as Federico (Curr. Opin. Biotechnol. 10:448-53, 1999) and Vigna et al. (J. Gene Med. 2:308-16, 2000). Generally, gene therapy vectors are expression vectors in the sense that they contain a nucleotide sequence encoding the gene product (e.g., polypeptide) of the present invention to be expressed, and the nucleotide sequence is operably linked to an appropriate control sequence as described above. Such control sequences will at least include a promoter sequence. Suitable promoters for expression of a nucleotide sequence encoding a polypeptide from a gene therapy vector include, for example, the cytomegalovirus (CMV) intermediate early promoter, viral long terminal repeat promoters (LTRs) such as those derived from murine Moloney leukemia virus (MMLV), Rous sarcoma virus, or HTLV-1, the simian virus 40 (SV40) early promoter, and the herpes simplex virus thymidine kinase promoter. Additional suitable promoters are described below.

[0275] Several inducible promoter systems have been described that can be induced by administration of organic or inorganic small molecule compounds. Such inducible promoters include those regulated by heavy metals, such as the metallothionein promoter (Brinster et al., Nature 296:39-42, 1982; Mayo et al., Cell 29:99-108, 1982), those regulated by RU-486 (a progesterone antagonist) (Wang et al., Proc. Natl. Acad. Sci. USA 91:8180-8184, 1994), those regulated by steroids (Mader and White, Proc. Natl. Acad. Sci. USA 90:5603-5607, 1993), and those regulated by tetracycline (Gossen and Bujard, Proc. Natl. Acad. Sci. USA 89:5547-5551, 1992; U.S. Patent No. 5,464,758; Furth et al., U.S. Patent No. 5,464,758). al, Proc. Natl. Acad. Sci. USA 91:9302-9306, 1994; Howe et al, J. Biol. Chem. 270:14168-14174, 1995; Resnitzky et al, Mol. Cell. Biol. 14:1669-1679, 1994; Shockett et al, Proc. Natl. Acad. Sci. USA 92:6522-6526, 1995), and the tTAER system, which is based on a multi-chimeric transactivator composed of the tetR polypeptide as the activation domain of VP16 and the ligand-binding domain of the estrogen receptor (Yee et al, 2002, US 6,432,705).

[0276] Suitable promoters for nucleotide sequences encoding small RNAs for knockdown of specific genes by RNA interference (see below) include polymerase III promoters in addition to the polymerase II promoters mentioned above. RNA polymerase III (pol III) is responsible for the synthesis of a variety of small nuclear and cytoplasmic non-coding RNAs, including 5S, U6, adenovirus VA1, vault, telomerase RNA, and tRNA. The promoter structures of many genes encoding these RNAs have been determined, and RNA pol III promoters have been found to fall into three types of structures (for reviews, see Geiduschek and Tocchini-Valentini, Annu. Rev. Biochem. 57:873-914, 1988; Willis, Eur. J. Biochem. 212:1-11, 1993; Hernandez, J. Biol. Chem. 276:26733-36, 2001). Particularly suitable for siRNA expression are type 3 RNA pol III promoters, in which transcription is driven by cis-acting elements found only in the 5'-flanking region, i.e., upstream of the transcription start site. Upstream sequence elements include the traditional TATA box (Mattaj et al., Cell 55:435-442, 1988), proximal sequence element, and distal sequence element (DSE; Gupta and Reddy, Nucleic Acids Res. 19:2073-2075, 1991).

[0277] Examples of genes under the control of type 3 pol III promoters are the U6 small nuclear RNA (U6 snRNA) gene, the 7SK gene, the Y gene, the MRP gene, the HI gene, and the telomerase RNA gene (see, e.g., Myslinski et al., Nucl. Acids Res. 21:2502-09, 2001).

[0278] The gene therapy vector may optionally contain one or more additional nucleotide sequences encoding a second or additional polypeptide. The second or additional polypeptide may be a (selectable) marker polypeptide that allows for identification, selection, and / or screening of cells containing the expression construct. Suitable marker proteins for this purpose include, for example, the fluorescent protein GFP, and the selectable marker genes HSV thymidine kinase (for selection with HAT medium), bacterial hygromycin B phosphotransferase (for selection with hygromycin B), Tn5 aminoglycoside phosphotransferase (for selection with G418), and dihydrofolate reductase (DHFR) (for selection with methotrexate), CD20, and the low-affinity nerve growth factor gene. The sources for obtaining these marker genes and methods for their use are provided in Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York, 2001.

[0279] Alternatively, the second or additional nucleotide sequence may encode a polypeptide derived from the transgenic cells that provides a safety mechanism that allows the subject to be cured if deemed necessary. Such a nucleotide sequence, often referred to as a suicide gene, encodes a polypeptide capable of converting a prodrug into a toxic substance capable of killing the transgenic cells in which the polypeptide is expressed. Suitable examples of such suicide genes include, for example, the Escherichia coli (E. coli) cytosine deaminase gene or one of the thymidine kinase genes derived from herpes simplex virus, cytomegalovirus, and varicella-zoster virus; in this case, ganciclovir may be used as a prodrug to kill IL-10 transgenic cells in the subject (see, e.g., Clair et al., Antimicrob. Agents Chemother. 31:844-849, 1987).

[0280] To knock down the expression of a specific polypeptide, gene therapy vectors or other expression constructs are used to express the desired nucleotide sequence, preferably encoding an RNAi agent, i.e., an RNA molecule capable of RNA interference or a part of an RNA molecule capable of RNA interference. Such RNA molecules are called siRNA (for example, small interfering RNA, including short hairpin RNA). The desired nucleotide sequence comprises antisense-coded DNA encoding antisense RNA for a region of target gene mRNA, and / or sense-coded DNA encoding sense RNA for the same region of target gene mRNA. In the DNA construct of the present invention, antisense-coded DNA and sense-coded DNA are operably linked to one or more promoters as defined herein above, which can express antisense RNA and sense RNA, respectively. "siRNA" includes small interfering RNA, which is a short double-stranded RNA that is not toxic in mammalian cells (Elbashir et al., Nature 411:494-98, 2001; Caplen et al., Proc. Natl. Acad. Sci. USA 98:9742-47, 2001). The length of siRNA is not necessarily limited to 21-23 nucleotides. As long as it does not exhibit toxicity, the length of siRNA is not particularly limited. "siRNA" can be, for example, at least about 15, 18, or 21 nucleotides to 25, 30, 35, or 49 nucleotides in length. Alternatively, the double-stranded RNA portion of the final transcription product of the expressed siRNA can be, for example, at least about 15, 18, or 21 nucleotides to 25, 30, 35, or 49 nucleotides in length.

[0281] "Antisense RNA" preferably refers to an RNA strand that has a sequence complementary to a target gene mRNA and is thought to induce RNAi by binding to the target gene mRNA.

[0282] A "sense RNA" is complementary to an antisense RNA and has a sequence that anneals to its complementary antisense RNA to form an siRNA.

[0283] In this context, the term "target gene" includes the gene whose expression is to be silenced by the siRNA expressed by the system of the present invention, and can be selected arbitrarily.For example, as the target gene, a gene whose sequence is known but whose function is not yet elucidated, and a gene whose expression is thought to cause a disease, are preferably selected.As long as the partial sequence of the mRNA of the gene, which has at least 15 nucleotides or more in length that can be bound to one strand of siRNA (antisense RNA strand), has been determined, the genome sequence of the target gene may not be completely elucidated.Therefore, even if the full-length sequence has not been determined, some sequences (preferably at least 15 nucleotides) of the gene, expressed sequence tags (ESTs), and parts of mRNA can be selected as "target genes".

[0284] The double-stranded RNA portion of an siRNA in which two RNA strands are paired is not limited to being perfectly paired, and may contain unpaired portions due to mismatches (corresponding nucleotides are not complementary) or bulges (corresponding complementary nucleotides are missing in one strand). Unpaired portions may be present to the extent that they do not interfere with siRNA formation. As used herein, a "bulge" includes one to two unpaired nucleotides, and the double-stranded RNA region of an siRNA in which two RNA strands are paired preferably contains one to seven, and more preferably one to five, bulges.

[0285] As used herein, the term "mismatch" refers to a mismatch that may be present in the double-stranded RNA region of an siRNA in which two RNA strands are paired. In certain mismatches, one nucleotide is guanine and the other is uracil. Such mismatches may be due to, but are not limited to, a C to T mutation, a G to A mutation, or a mixture thereof in the DNA encoding the sense RNA. Furthermore, in the present invention, the double-stranded RNA region of an siRNA in which two RNA strands are paired may contain both bulges and mismatches, preferably 1 to 7, more preferably 1 to 5, in total. Such mismatches (e.g., mismatches or bulges) suppress the recombination between the antisense-encoding DNA and the sense-encoding DNA described below, stabilizing the siRNA expression system described below. Furthermore, although it is difficult to sequence stem-loop DNA that does not contain a mismatch in the double-stranded RNA region of an siRNA in which two RNA strands are paired, introducing mismatches or bulges as described above makes sequencing possible. Furthermore, siRNAs containing mismatches or bulges in the paired double-stranded RNA region have the advantage of being stable in E. coli or animal cells.

[0286] The terminal structure of siRNA may be blunt or sticky (overhanging) as long as the siRNA is capable of silencing target gene expression through the RNAi effect. The sticky (overhanging) end structure is not limited to 3' overhangs but can also include 5' overhangs, as long as they are capable of inducing the RNAi effect. Furthermore, the number of overhanging nucleotides is not limited to the previously reported 2 or 3, but can be any number as long as the overhang is capable of inducing the RNAi effect. For example, the overhang may consist of 1 to 8 nucleotides, preferably 2 to 4 nucleotides. Herein, the total length of an siRNA having a sticky end structure is expressed as the sum of the length of the paired double-stranded portion and the length of the pair, including the overhanging single strands at both ends. For example, in the case of a 19-bp double-stranded RNA portion with 4-nucleotide overhangs at both ends, the total length is expressed as 23 bp. Furthermore, because this overhanging sequence has low specificity for the target gene, it is not necessarily complementary (antisense) or identical (sense) to the target gene sequence. Furthermore, as long as the siRNA can maintain its gene silencing effect on the target gene, the siRNA may contain, for example, a low-molecular-weight RNA (which may be a natural RNA molecule such as tRNA, rRNA, or viral RNA, or an artificial RNA molecule) in the protruding portion at one end.

[0287] Furthermore, the terminal structure of "siRNA" is necessarily a cutoff structure at both ends as described above, and may have a stem-loop structure in which one end of the double-stranded RNA is connected by a linker RNA ("shRNA"). The length of the double-stranded RNA region (stem-loop portion) may be, for example, at least 15, 18, or 21 nucleotides to 25, 30, 35, or 49 nucleotides. Alternatively, the length of the double-stranded RNA region that is the final transcription product of the expressed siRNA may be, for example, at least 15, 18, or 21 nucleotides to 25, 30, 35, or 49 nucleotides.

[0288] Furthermore, the length of the linker is not particularly limited, as long as it has a length that does not interfere with the pairing of the stem portion.For example, the linker portion can have a cloverleaf tRNA structure to ensure stable pairing of the stem portion and to prevent recombination between the DNA encoding that portion.Even if the linker has a length that interferes with the pairing of the stem portion, it is possible to construct the linker portion with an intron, for example, so that the intron is excised during the processing of precursor RNA into mature RNA, thereby allowing the pairing of the stem portion.In the case of stem-loop siRNA, either end (head or tail) of the RNA without loop structure can have a low molecular weight RNA.As mentioned above, this low molecular weight RNA can be a natural RNA molecule such as tRNA, rRNA, snRNA, or viral RNA, or an artificial RNA molecule.

[0289] The DNA construct of the present invention contains a promoter as defined above to express antisense and sense RNAs from the antisense and sense coding DNAs, respectively. In principle, the number and location of promoters within the construct can be freely selected, as long as they are capable of expressing the antisense and sense coding DNAs. As a simple example of the DNA construct of the present invention, a tandem expression system can be formed in which a promoter is located upstream of the antisense and sense coding DNAs. This tandem expression system can produce siRNAs with the above-mentioned cutoff structures at both ends. In a stem-loop siRNA expression system (stem expression system), the antisense and sense coding DNAs are arranged in opposite directions and connected via a linker DNA to construct a single unit. A promoter is linked to one side of this unit to construct the stem-loop siRNA expression system. The length and sequence of the linker DNA are not particularly limited herein, and the linker DNA can have any length and sequence, as long as it is not a termination sequence and does not interfere with the pairing of the stem portion during the production of mature RNA as described above. As an example, DNA encoding the aforementioned tRNA or the like can be used as linker DNA.

[0290] In both the tandem expression system and the stem-loop expression system, the 5' end has a sequence that can promote transcription from the promoter. More specifically, in the case of tandem siRNA, the efficiency of siRNA production can be improved by adding a sequence that can promote transcription from the promoter to the 5' end of the antisense and sense coding DNA. In the case of stem-loop siRNA, such a sequence can be added to the 5' end of the above-mentioned unit. The transcript from such a sequence can be used in a state attached to siRNA as long as it does not interfere with target gene silencing by siRNA. If this condition interferes with gene silencing, it is preferable to trim the transcript using a trimming means (e.g., a ribozyme known in the art). It will be clear to those skilled in the art that antisense and sense RNAs can be expressed in the same vector or in different vectors. To avoid adding excessive sequences downstream of the sense and antisense RNAs, it is preferable to place a transcription terminator at the 3' end of each strand (the strand encoding the antisense and sense RNAs). A terminator can be a sequence of four or more consecutive adenine (A) nucleotides.

[0291] Genome editing Genome editing can be used to change the genomic sequence of the cloned stem cells of the present invention, for example, cloned cancer (or other disease) stem cells, by introducing heterologous transgenes or inhibiting the expression of target endogenous genes.Such genetically modified stem cells can be used for regenerative medicine (see below) or wound healing.Therefore, in certain embodiments, the regenerative medicine (see below) method of the present invention comprises the use of the stem cells of the present invention whose genomic sequence is modified by genome editing.

[0292] Genome editing can be carried out using art-recognized technologies such as ZFN / TALEN technology or CRISPR technology (see review by Gaj et al., Trends in Biotech. 31(7):397-405, 2013, the entire text and all references cited therein are incorporated herein by reference). Such technology allows virtually any gene to be manipulated in a wide range of cell types and organisms by inducing DNA double-strand (DSB) breaks that stimulate error-prone non-homologous end joining (NHEJ) or homology-directed repair (HDR) at specific genomic locations, thereby enabling a wide range of gene modifications.

[0293] Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are chimeric nucleases composed of a programmable sequence-specific DNA binding module linked to a nonspecific DNA cleavage domain. They are artificial restriction enzymes (REs) created by fusing the zinc finger or TAL effector DNA binding domain with a DNA cleavage domain. Zinc finger (ZFs) or transcription activator-like effectors (TALEs) can be engineered to bind to any desired target DNA sequence and fused to the DNA cleavage domain of the RE, thereby creating engineered restriction enzymes (ZFNs or TALENs) specific to the desired target DNA sequence. When ZFNs / TALENs are introduced into cells, they can be used for in situ genome editing. Indeed, the versatility of ZFNs and TALENs can be extended to effector domains other than nucleases, such as transcriptional activators and repressors, recombinases, transposases, DNA and histone methyltransferases, and histone acetyltransferases, to affect genome structure and function.

[0294] The Cys2-His2 zinc finger domain is one of the most common types of DNA-binding motifs found in eukaryotes and represents the second most frequently encoded protein domain in the human genome. Individual zinc fingers contain approximately 30 amino acids in a conserved ββα configuration. Key to the application of zinc finger proteins for specific DNA recognition was the development of non-natural arrays containing more than three zinc finger domains. This advance was facilitated by the structure-based discovery of highly conserved linker sequences, which enabled the construction of synthetic zinc finger proteins that recognize DNA sequences 9–18 bp long. This design proved to be the optimal strategy for constructing zinc finger proteins that recognize specific continuous DNA sequences in complex genomes. Suitable zinc fingers can be obtained by a modular assembly approach (e.g., by selection of large combinatorial libraries or using preselected libraries of zinc finger modules generated by rational design). Zinc finger domains that recognize nearly all 64 possible nucleotide triplets have been developed, and preselected zinc finger modules can be linked in tandem to target DNA sequences containing a set of these DNA triplets. Alternatively, selection-based approaches such as oligomerized pool engineering (OPEN), which take into account environment-dependent interactions between neighboring fingers, can be used to select new zinc finger arrays from randomized libraries. A combination of the two approaches can also be used.

[0295] Engineered zinc fingers are commercially available. Sangamo Biosciences (Richmond, CA, USA) has developed a proprietary platform for zinc finger construction (CompoZr) in collaboration with Sigma-Aldrich (St. Louis, MO, USA) that allows researchers to bypass zinc finger construction and validation altogether, and thousands of proteins are already available. Broadly, zinc finger protein technology allows for the targeting of virtually any sequence.

[0296] TAL effectors are proteins secreted by the plant pathogenic bacterium Xanthomonas that contain a DNA-binding domain containing a repeat of a highly conserved 33–34 amino acid sequence, with the exception of the 12th and 13th amino acids. These two positions are highly variable (Repeat Variable Diresidues, or RVDs) and show a strong correlation with specific nucleotide recognition. This simple relationship between amino acid sequence and DNA recognition has enabled the engineering of specific DNA-binding domains by selecting combinations of repeat segments containing appropriate RVDs. Similar to zinc fingers, modular TALE repeats are linked together to recognize contiguous DNA sequences. Numerous effector domains, including nucleases, transcriptional activators, and site-specific recombinases, have been made available for fusion with TALE repeats for targeted gene modification. Rapid assembly of custom TALE arrays can be achieved by using strategies including "Golden Gate" molecular cloning, high-throughput solid-phase assembly, and ligation-independent cloning techniques, all of which can be used in the present invention for genome editing of cloned stem cells.

[0297] TALE repeats can be easily assembled using numerous tools available in the art, such as a library of TALENs targeting 18,740 human protein-coding genes (Kim et al., Nat. Biotechnol. 31, 251-258, 2013). Custom-designed TALE arrays are also commercially available, for example, through Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, KY, USA), and Life Technologies (Grand Island, NY, USA).

[0298] Nonspecific DNA cleavage domains derived from the ends of REs, such as FokI endonuclease (or FokI cleavage domain variants, such as Sharkey, with mutations designed to improve cleavage specificity and / or activity), can be used to construct hybrid nucleases that are active in yeast assays (and also active in plant and animal cells). To improve ZFN activity, transient low-temperature culture conditions can be used to increase nuclease expression levels; co-delivery of site-specific nucleases with DNA end-processing enzymes, as well as the use of fluorescent surrogate reporter vectors that allow enrichment of cells modified by ZFNs and TALENs, can also be used. The specificity of ZFN-mediated genome editing can also be improved by the use of zinc finger nickases (ZF nickases), which exploit the observation that induction of nicked DNA stimulates HDR without activating the error-prone NHEJ repair pathway.

[0299] The simple relationship between the amino acid sequence of a TALE binding domain and DNA recognition allows for designable proteins. A publicly available software program (DNAWorks) can be used to calculate appropriate oligonucleotides for assembly in two-step PCR. Numerous modular assembly schemes for generating modified TALE constructs have also been reported and are known in the art. Both methods offer a systematic approach to modifying DNA binding domains that is conceptually similar to the modular assembly method for generating zinc finger DNA recognition domains.

[0300] After the TALEN gene is assembled, it is introduced into target cells by a vector using any art-recognized method (such as electroporation or transfection using cationic lipid-based reagents, or various viral vectors such as plasmid vectors, adenovirus vectors, AAV vectors, and integrase-deficient lentivirus vectors (IDLV)). Alternatively, TALENs can be delivered to cells as mRNA, which eliminates the possibility of genomic integration of TALEN-expressed proteins. This can also dramatically increase the level of homology-directed repair (HDR) during gene editing and the success of gene transfection. Finally, direct delivery of purified ZFN / TALEN proteins into cells can also be used. This approach does not involve the risk of insertional mutagenesis and results in fewer off-target effects than delivery systems that rely on expression from nucleic acids, making it ideal for use in studies requiring precise genome modification in cells such as the stem cells of the present invention.

[0301] TALENs can be used to edit genomes by inducing double-strand breaks (DSBs), to which cells respond through repair mechanisms. Non-homologous end joining (NHEJ) reconnects DNA from either side of a double-strand break where there is little or no sequence overlap for annealing. A simple heteroduplex break assay can be performed to detect differences between two PCR-amplified alleles. Cleavage products can be visualized in simple agarose gel or slab gel systems. Alternatively, DNA can be introduced into the genome through NHEJ in the presence of an exogenous double-stranded DNA fragment.

[0302] Homologous recombination repair can also introduce foreign DNA at DSBs because the transfected double-stranded sequence is used as a template for repair enzymes. TALENs have been used to generate knockout C. elegans, rats, and zebrafish, and to generate stably modified human embryonic stem cells and induced pluripotent stem cell (iPSC) clones.

[0303] For stem cell-based therapy, ZFNs and TALENs can correct the underlying cause of disease through precise genome modification, thus permanently eliminating symptoms. For example, ZFN-induced HDR can be used to directly correct disease-causing mutations associated with X-linked severe combined immunodeficiency (SCJD), hemophilia B, sickle cell disease, α1-antitrypsin deficiency, and many other genetic diseases by either repairing defective target genes or knocking out target genes. Furthermore, these site-specific nucleases can also be used to safely insert therapeutic transgenes into the stem cells of the present invention at specific "safe harbor" locations within the human genome. Such technology can be used in combination with the stem cells of the present invention in gene therapy, including autologous stem cell transplantation-based treatments, in which one or more genes of cloned (diseased or normal) stem cells are manipulated to increase or reduce / eliminate target gene expression.

[0304] Alternatively, the CRISPR / Cas system may be used to efficiently induce targeted genetic modifications in stem cells of the present invention. The CRISPR / Cas (CRISPR-related) system, or "Clustered Regulatory Interspaced Short Palindromic Repeats," is a genetic locus that contains multiple short direct repeats and provides adaptive immunity to bacteria and archaea. The CRISPR system relies on crRNA and tracrRNA for sequence-specific silencing of invading foreign DNA. The term "tracrRNA" refers to a non-coding RNA that promotes crRNA processing and is a trans-activating chimeric RNA required to activate RNA-guided Cas9 cleavage. CRISPR RNA or crRNA base-pairs with tracrRNA to form a two-RNA structure that guides Cas9 endonuclease to the complementary DNA site for cleavage.

[0305] There are three types of CRISPR / Cas systems: in Type II systems, Cas9 serves as an RNA-guided DNA endonuclease that cleaves DNA upon crRNA-tracrRNA target recognition. In bacteria, CRISPR systems provide adaptive immunity against invading foreign DNA through RNA-guided DNA cleavage. By redesigning the crRNA, CRISPR / Cas systems can be retargeted to cleave virtually any DNA sequence. In fact, it has been shown that CRISPR / Cas systems can be directly transferred into human cells by co-delivering a plasmid expressing the Cas9 endonuclease and the necessary crRNA components. These programmable RNA-guided DNA endonucleases have demonstrated multiple gene disruption capabilities and target-specific integration in iPS cells and therefore can be used in the stem cells of the present invention as well.

[0306] Cancer stem cells The methods and reagents of the present invention also enable the culture and isolation of cancer-derived cancer stem cells (CSCs) derived from epithelial tissue samples / biopsies or from other stratified regenerative tissues, which CSCs can be used in a number of applications that were previously impossible or impractical, in part due to the inability to obtain such CSCs as single-cell clones in large quantities.

[0307] For example, a CSC library established from a patient using the method of the present invention allows comparison between sensitive and resistant clones from the same patient for directed drug discovery efforts. Certain genes may be up-regulated or down-regulated in resistant clones compared to sensitive clones. Inhibitors of up-regulated genes can be further validated as drug target genes, for example, by testing the ability to down-regulate target genes in resistant clones and determining the effect on drug resistance. Conversely, restoring or overexpressing down-regulated genes in resistant clones can also overcome drug resistance.

[0308] Thus, in one aspect, the present invention provides a drug discovery method using CSCs isolated using the method and medium of the present invention to identify genes that are up-regulated or down-regulated in drug-resistant CSC clones, the method comprising: (1) obtaining a large number of cell clones from cancer tissue (e.g., from a cancer patient) using the methods of the present invention; (2) contacting the large number of cell clones with one or more chemical compounds (e.g., anticancer drugs) under conditions in which a small percentage (e.g., at most 1%, 0.5%, 0.2%, 0.1%, 0.05%, 0.01%, or less) of drug-resistant clones survive; (3) comparing the gene expression profile of the drug-resistant clones with that of sensitive clones (e.g., one or more cell clones randomly selected prior to step (2) that are suspected to be sensitive to drug treatment), thereby identifying genes that are up- or down-regulated in the surviving drug-resistant clones. Includes:

[0309] In certain embodiments, the method further comprises inhibiting the expression of up-regulated genes in surviving drug-resistant clones.For example, the up-regulated genes can be commonly up-regulated in two or more surviving drug-resistant clones, which are derived from the same or different tumor types, and from the same or different patients.In certain embodiments, the up-regulated genes can be specific to the patient from which CSCs are isolated.This can be useful in designing personalized medicine or treatment plans for patients.

[0310] In certain embodiments, the method further comprises restoring or increasing the expression of down-regulated genes in surviving drug-resistant clones.For example, the down-regulated genes can be commonly down-regulated in two or more remaining drug-resistant clones from the same type of tumor or different types of tumor, from the same patient, or from different patients.In certain embodiments, the down-regulated genes can be specific for the patient from which CSCs are isolated.This can also be useful in designing personalized medicine or treatment plans for patients.

[0311] In a related aspect, the present invention provides a drug discovery method using CSCs isolated using the methods and media of the present invention to identify candidate compounds that inhibit the growth of or promote the death of drug-resistant CSCs, the method comprising: (1) obtaining a large number of cell clones from cancer tissue (e.g., from a cancer patient) using the methods of the present invention; (2) contacting the large number of cell clones with one or more compounds (e.g., anticancer drugs) under conditions such that a small percentage of drug-resistant clones remains (e.g., at most 1%, 0.5%, 0.2%, 0.1%, 0.05%, 0.01%, or less); (3) contacting the remaining drug-resistant clones with a number of candidate compounds; and (4) Identifying one or more candidate compounds that inhibit the growth of or promote the de...

Claims

1. A defined culture medium for isolating stratified epithelial stem cells and stably maintaining the epigenetics of the stratified epithelial stem cells over multiple passages, comprising: a basal medium; and Direct inhibitors of ROCK (Rho kinase), epidermal growth factor (EGF), insulin or IGF, TrkA inhibitors, ponatinib, and OAC1 Including, At least one of VEGF receptor inhibitor, FGF10 or FGF10 agonist Including, Selective TGFβ receptor inhibitors and / or noggin and optionally further comprising A defined culture medium that supports epigenetically stable growth and proliferation of stem cells of stratified epithelial tissue origin in culture.

2. Allows for the passage of cells in a feeder-free manner, further including SYK inhibitors, LPA receptor antagonists, GSK3 inhibitors, and CK2 inhibitors; 10. The defined culture medium of claim 1.

3. 3. The defined culture medium of claim 1 or 2, wherein the epithelial stem cells are in contact with an extracellular matrix or other biomatrix.

4. 4. The defined culture medium of any one of claims 1 to 3, wherein the stem cells are isolated from a tissue sample taken from normal epithelial tissue.

5. 5. The defined culture medium of any one of claims 1 to 4, wherein the stem cells are isolated from a tissue sample taken from the diseased epithelial tissue.

6. 4. The defined culture medium of any one of claims 1 to 3, wherein the stem cells are isolated from a tissue sample taken from a tumor.

7. 6. The defined culture medium of claim 5, wherein the stem cells are isolated from a tissue sample derived from an inflammatory or autoimmune patient.

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