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  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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  • A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
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  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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  • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
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  • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
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  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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Definitions

• the present invention relates to LATS (large tumor suppressor kinase) inhibitors.
• the present invention further relates to 6-6 fused bicyclic heteroaryl compounds and
• compositions comprising such compounds.
• the present invention also relates to ex-vivo use of such compounds to produce cellular material for cell therapy/transplantation.
• the present invention further relates to methods of generating an expanded population of cells, such as an expanded population of ocular cells for example comprising limbal stem cells (LSCs) or corneal endothelial cells (CECs) involving the use of a LATS inhibitor, as well as the population of cells such as ocular cells comprising for example limbal stem cells (LSCs) or corneal endothelial cells (CECs) and preparations, uses and methods of therapy comprising said cells.
• LSCs limbal stem cells
• CECs corneal endothelial cells
• the present invention also relates to 6-6 fused bicyclic heteroaryl compounds, compositions comprising such compounds, and their use in in promoting wound healing, particularly for treatment of burns, acute and chronic skin ulcers, including vascular, diabetic and pressure ulcers, such as venous leg ulcers, diabetic foot ulcers, pressure ulcers.
• the present invention additionally relates to 6-6 fused bicyclic heteroaryl compounds, compositions comprising such compounds, and their use in liver regeneration and liver regrowth as well as in the prevention of damage and in the maintenance or improvement of function of organs ex-vivo, with or without perfusion devices.
• Organ regeneration and/or healing is an issue crucial to treat many serious health issues.
• corneal blindness is the third leading cause of blindness worldwide.
• Approximately half of all the cornea transplants worldwide are performed for treatment of corneal endothelial dysfunction.
• the cornea is a transparent tissue comprising different layers: corneal epithelium, Bowman's membrane, stroma, Descemet's Membrane and endothelium.
• the corneal endothelium also comprises a monolayer of human corneal endothelial cells and helps maintain corneal transparency via its barrier and ionic pump functions. It plays a crucial role in maintaining the balance of fluid, nutrients and salts between the corneal stroma and the aqueous humor.
• endothelial cell density must be maintained, however endothelial cell density can be significantly decreased as a result of trauma, disease or endothelial dystrophies. The density of the cells also decreases with aging.
• Human corneal endothelium has a limited propensity to proliferate in vivo. If the density of cells falls too low, the barrier function may be compromised. Loss of endothelial barrier function results in corneal edema and loss of visual acuity. The clinical condition of bullous keratopathy may be one resulting complication.
• corneal transplantation is one of the most common forms of organ transplantation, the availability of donor corneas required is extremely limited.
• a 2012-2013 global survey quantified the considerable shortage of corneal graft tissue, finding that only one cornea is available for every 70 needed (Gain at el., (2016) Global Survey of Corneal Transplantation and Eye Banking. JAMA Ophthalmol. 134:167-173).
• the corneal epithelium also needs to be maintained in the eye.
• the corneal epithelium is composed of a layer of basal cells and multiple layers of a non-keratinized, stratified, squamous epithelium. It is essential in maintaining the clarity and the regular refractive surface of the cornea. It acts as a transparent, renewable protective layer over the corneal stroma and is replenished by a stem cell population located in the limbus.
• limbal stem cell deficiency a condition in which limbal stem cells are diseased or absent, a decrease in the number of healthy limbal stem cells results in a decreased capacity for corneal epithelium renewal.
• Limbal stem cell deficiency may arise as a result of injuries from chemical or thermal burns, ultraviolet and ionizing radiation, or even as a result of contact lens wear; genetic disorders like aniridia, and immune disorders such as Stevens Johnson syndrome and ocular cicatricial pemphigoid.
• Loss of limbal stem cells can be partial or total; and may be unilateral or bilateral. Symptoms of limbal stem cell deficiency include pain, photophobia, non healing painful corneal epithelial defects, corneal neovascularization, replacement of the corneal epithelium by conjunctival epithelium, loss of corneal transparency and decreased vision that can eventually lead to blindness.
• Holoclar is an ex vivo expanded preparation of autologous human corneal epithelial cells containing stem cells.
• a biopsy of healthy limbal tissue is taken from the patient, expanded ex vivo and frozen until surgery.
• the thawed cells are grown on a membrane comprising fibrin, and then surgically implanted onto the eye of the patient.
• the therapy is intended for use in adults with moderate to severe limbal stem cell deficiency due to physical or chemical ocular burns.
• GRWR graft-to-recipient weight ratio
• New therapeutic approaches to promote cell proliferation are thus greatly needed for conditions affecting a range of organs throughout the body, such as the eye, liver and skin.
• the present invention relates to compounds, salts thereof, and compositions thereof, wherein the compounds are LATS (large tumor suppressor kinase) inhibitors. These compounds have use in therapies for the conditions and purposes detailed above.
• LATS large tumor suppressor kinase
• the present invention relates to a compound of Formula A2 or a subformulae thereof, or a salt, or stereoisomer thereof,
• the compound is according to Formula I or Formula II, or subformulae thereof, or a salt thereof:
• the invention in another aspect, relates to a pharmaceutical composition
• a pharmaceutical composition comprising a therapeutically effective amount of a compound according to the definition of Formula A2 or subformulae thereof, or a pharmaceutically acceptable salt thereof, or subformulae thereof and one or more pharmaceutically acceptable carriers.
• the invention relates to a combination, in particular a pharmaceutical combination, comprising a therapeutically effective amount of the compound according to the definition of Formula A2 or subformulae thereof, or a pharmaceutically acceptable salt thereof, and one or more therapeutically active agent.
• the invention relates to compounds and compositions that may be used in therapy.
• the present invention relates to a method of LATS inhibition in a cell or cell population using a compound of Formula A1 or subformulae thereof or a salt thereof, a stereoisomer thereof:
• the salt is a pharmaceutically acceptable salt.
• the compound, salt thereof is selected from 3-(pyridin-4-yl)-N-(1 -(trifluoromethyl)cyclopropyl)-2,6- naphthyridin-1 -amine; N-(1 -methylcyclopropyl)-7-(pyridin-4-yl)isoquinolin-5-amine; 2- (pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)pyrido[3,4-d]pyrimidine.
• the compound, or a salt thereof is selected from N-(tert-butyl)-2-(pyridin-4-yl)-1 ,7- naphthyridin-4-amine; and N-methyl-2-(pyridin-4-yl)-N-[(2S)-1 , 1 , 1 -trifluoropropan-2- yl]pyrido[3,4-d]pyrimidin-4-amine.
• the present invention relates to a method of LATS inhibition in an ocular cell population using a compound of Formula A1 or subformulae thereof or a salt thereof, or a stereoisomer thereof.
• the present invention relates to a method of LATS inhibition in a cell population comprising limbal stem cells using a compound of Formula A1 or subformulae thereof or a salt thereof, or a stereoisomer thereof.
• the present invention relates to a method of LATS inhibition in a cell population comprising corneal endothelial cells using a compound of Formula A1 or subformulae thereof or a salt thereof, or a stereoisomer thereof.
• the method of LATS inhibition in a cell population is performed ex vivo.
• said compound is present in a concentration of 0.5 to 100 micromolar, preferably 0.5 to 25 micromolar, more preferably 1 to 20 micromolar, particularly preferably of about 3 to 10 micromolar.
• the compound is present for 12 to 16 days, particularly preferably the compound is present for 14 days.
• the compound is present for one to two weeks and subsequently the cells are cultured for a period in growth medium without supplementation with said compound, preferably wherein the period is one to two weeks.
• the LATS inhibitor inhibits LATS1 or LATS2, or LATS1 and LATS2. In a more preferred embodiment the LATS inhibitor inhibits LATS1 and LATS2. In another preferred embodiment of the method of LATS inhibition in a cell population comprising limbal stem cells said method further comprises genetically modifying said limbal stem cells. In another preferred embodiment of the method of LATS inhibition in a cell population comprising corneal endothelial cells said method further comprises genetically modifying said corneal endothelial cells. Preferably said genetically modifying comprises introducing into said cell a gene editing system which specifically targets a gene associated with facilitating a host versus graft immune response.
• the method of LATS inhibition in a cell population comprises the further step after generation of an expanded population of cells of rinsing those cells to substantially remove the compound according to the invention.
• the invention relates to an expanded cell population comprising limbal stem cells obtainable by the method of LATS inhibition in a cell population comprising limbal stem cells according to the invention.
• the invention relates to a an expanded cell population comprising limbal stem cells obtained by the method of LATS inhibition in a cell population comprising limbal stem cells according to the invention.
• the invention relates to a population of corneal endothelial cells obtainable by the method of LATS inhibition in a cell population comprising corneal endothelial cells according to the invention.
• the invention relates to a population of corneal endothelial cells obtained by the method of LATS inhibition according to the invention.
• the invention relates to an ocular cell delivery preparation, comprising a cell population obtainable or obtained by the method of LATS inhibition according to the invention and a composition suitable for ocular delivery which is a localising agent.
• the localising agent is GelMa (which is methacrylamide modified gelatin, and is also known as gelatin methacrylate).
• the localising agent is fibrin or fibrin glue.
• the cell delivery preparation of limbal stem cells has greater than 20% limbal stem cells.
• the cell delivery preparation of limbal stem cells has greater than 20% p63alpha positive cells.
• the cell population obtainable or obtained by the method of LATS inhibition according to the invention or cell delivery preparation according to the invention has only trace levels of the compound according to the invention.
• corneal endothelial cells are present in the cell delivery preparation at a density greater than 500 cells per mm 2 (area).
• the cell population obtainable or obtained by the method of LATS inhibition according to the invention or cell delivery preparation according to the invention has only trace levels of the compound according to the invention.
• the invention in another aspect relates to a method of culturing cells comprising culturing a population of cells in the presence of a LATS inhibitor.
• the cells can be a cell population as described and/or as provided herein.
• the cells are ocular cells or liver cells.
• the cells are ocular cells.
• the invention relates to a method of culturing cells comprising culturing a population of cells comprising limbal stem cells in the presence of a LATS inhibitor.
• the invention relates to a method of culturing cells comprising culturing a population of cells comprising corneal endothelial cells in the presence of a LATS inhibitor.
• the invention relates to a method of culturing cells comprising culturing a cell population comprising limbal stem cells, wherein the LATS inhibitor is a compound of Formula A1 or subformulae thereof or salt thereof according to the invention.
• the invention relates to a method of culturing cells comprising culturing a population comprising corneal endothelial cells, wherein the LATS inhibitor is a compound of Formula A1 or subformulae thereof or salt thereof according to the invention.
• the salt is a pharmaceutically acceptable salt.
• said compound is present in a concentration of 0.5 to 100 micromolar, preferably 0.5 to 25 micromolar, more preferably 1 to 20 micromolar, particularly preferably of about 3 to 10 micromolar.
• the compound is present for 12 to 16 days, particularly preferably the compound is present for 14 days.
• the compound is present for one to two weeks and subsequently the cells are cultured for a period in growth medium without supplementation with said compound, preferably wherein the period is one to two weeks.
• the LATS inhibitor inhibits LATS1 or LATS2, or LATS1 and LATS2. In a more a preferred embodiment the LATS inhibitor inhibits LATS1 and LATS2.
• said method further comprises genetically modifying cells.
• said genetically modifying comprises introducing into said cell a gene editing system which specifically targets a gene associated with facilitating a host versus graft immune response.
• the cells are ocular cells.
• said method further comprises genetically modifying limbal stem cells.
• said method further comprises genetically modifying corneal endothelial cells.
• the method of culturing cells comprises the further step after generation of an expanded population of cells of rinsing those cells to substantially remove the compound according to the invention.
• the invention relates to an expanded cell population obtainable by the method of culturing cells according to the invention. In another aspect the invention relates to an expanded cell population obtained by the method of culturing cells according to the invention. Preferably the cells are ocular cells. In one aspect the invention relates to an expanded cell population comprising limbal stem cells obtainable by the method of culturing cells comprising limbal stem cells according to the invention. In another aspect the invention relates to an expanded cell population comprising limbal stem cells obtained by the method of culturing cells comprising limbal stem cells according to the invention. In one aspect the invention relates to a population of corneal endothelial cells obtainable by the method of culturing cells comprising corneal endothelial cells according to the invention.
• the invention relates to a population of corneal endothelial cells obtained by the method of culturing cells comprising corneal endothelial cells according to the invention.
• an ocular cell delivery preparation comprising a cell population obtainable or obtained by the method of culturing cells according to the invention and a composition suitable for ocular delivery which is a localising agent.
• the localising agent is GelMa.
• the localising agent is fibrin or fibrin glue.
• the cell delivery preparation of limbal stem cells has greater than 20% limbal stem cells.
• the cell delivery preparation of limbal stem cells has greater than 20% p63alpha positive cells.
• corneal endothelial cells are present in the cell delivery preparation of corneal endothelial cells, at a density greater than 500 cells per mm 2 (area).
• the cell population obtainable or obtained by the method of culturing cells according to the invention or cell delivery preparation according to the invention has only trace levels of the compound according to the invention.
• the invention in another aspect relates to a method of cell population expansion comprising the step of a) culturing a seeding population of cells in the presence of a LATS inhibitor to generate an expanded population of cells.
• the method of cell population expansion is performed ex vivo.
• the cells are ocular cells or liver cells.
• the cells are ocular cells.
• the invention relates to a method of cell population expansion comprising the step of a) culturing a seeding population of cells comprising limbal stem cells in the presence of a LATS inhibitor to generate an expanded population of cells comprising limbal stem cells.
• the LATS inhibitor is a compound of Formula A1 or subformulae thereof or salt thereof, according to the invention.
• the invention in a futher aspect relates to a method of cell population expansion comprising the step of a) culturing a seeding population of cells comprising limbal stem cells in the presence of a compound of Formula A1 or subformulae thereof, or a salt thereof to generate an expanded population of cells comprising limbal stem cells.
• said compound is selected from Formula A2 or subformulae thereof or a salt thereof.
• the invention relates to a method of cell population expansion comprising the step of a) culturing a seeding population of cells comprising corneal endothelial cells in the presence of a LATS inhibitor to generate an expanded population of cells comprising corneal endothelial cells.
• the LATS inhibitor is a compound of Formula A1 or subformulae thereof or salt thereof, according to the invention.
• the invention relates to a method of cell population expansion comprising the step of a) culturing a seeding population of cells comprising corneal endothelial cells in the presence of a compound of Formula A1 or subformulae thereof, or a pharmaceutically acceptable salt thereof to generate an expanded population of cells comprising corneal endothelial cells.
• said compound is selected from Formula A2 or subformulae thereof.
• the salt is a pharmaceutically acceptable salt.
• said compound is selected from the group of compounds consisting of N-methyl-2-(pyridin-4-yl)-N-(1 , 1 ,1 -trifluoropropan-2-yl)pyrido[3,4-d]pyrimidin-4- amine; 2-methyl-1 -(2-methyl-2-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4- yl)amino)propoxy)propan-2-ol; 2,4-dimethyl-4-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4- yl)amino)pentan-2-ol; N-(tert-butyl)-2-(pyridin-4-yl)-1 ,7-naphthyridin-4-amine; 2-(pyridin-4-yl)- N-(1 -(trifluoromethyl)cyclobutyl)pyrido[3,4-d]
• said compound or a salt thereof is selected from N-(tert-butyl)-2-(pyridin-4- yl)-1 ,7-naphthyridin-4-amine and (S)-N-methyl-2-(pyridin-4-yl)-N-(1 , 1 ,1 -trifluoropropan-2- yl)pyrido[3,4-d]pyrimidin-4-amine.
• said compound is present in a concentration of 0.5 to 100 micromolar, preferably 0.5 to 25 micromolar, more preferably 1 to 20 micromolar, particularly preferably of about 3 to 10 micromolar.
• the compound is present for 12 to 16 days, particularly preferably the compound is present for 14 days.
• the compound is present for one to two weeks and subsequently step b) is performed wherein the cells are cultured for a period in growth medium without supplementation with said compound, preferably wherein the period is one to two weeks.
• the compounds according to Formula A1 or subformulae thereof produce greater than 30 fold expansion of the seeded amount of cells.
• the compounds according to Formula A1 and subformulae thereof produce 100 fold to 2200 fold, preferably 600 fold to 2200 fold expansion of the seeded amount of cells.
• the method according to the invention produces a cell population with greater than 20% limbal stem cells.
• the method according to the invention produces a cell population with greater than 50% limbal stem cells.
• the method of cell population expansion relating to limbal stem cells according to the invention produces a cell population with greater than 20% expressing p63alpha.
• the method of cell population expansion relating to limbal stem cells according to the invention produces a cell population with greater than 50% expressing p63alpha.
• the compounds according to Formula A1 or subformulae thereof produce greater than 10 fold expansion of the seeded amount of cells.
• the compounds according to Formula A1 or subformulae thereof produce 15 fold to 600 fold, preferably 20 fold to 550 fold expansion of the seeded amount of cells.
• the LATS inhibitor inhibits
• said method of cell population expansion further comprises use of a gene editing system.
• said method comprises use of a gene editing system which specifically targets a gene associated with facilitating a host versus graft immune response.
• the cells are ocular cells or liver cells.
• the cells are ocular cells.
• said method of cell population expansion further comprises genetically modifying limbal stem cells, preferably wherein said genetically modifying comprises introducing into said cell a gene editing system which specifically targets a gene associated with facilitating a host versus graft immune response.
• said method further comprises genetically modifying corneal endothelial cells, preferably wherein said genetically modifying comprises introducing into said cell a gene editing system which specifically targets a gene associated with facilitating a host versus graft immune response.
• the method of cell population expansion further comprises step c) rinsing the expanded population of cells to substantially remove the compound according to the invention.
• the invention relates to a kit comprising a LATS inhibitor, growth medium and instructions for cell population expansion.
• the invention relates to a cell population obtainable by the method of cell population expansion according to the invention.
• the invention relates to a cell population obtained by the method of cell population expansion according to the invention.
• the invention relates to an ocular cell population obtainable by the method of cell population expansion according to the invention.
• the invention relates to an ocular cell population obtained by the method of cell population expansion according to the invention.
• the invention relates to a cell population comprising limbal stem cells obtainable by the method of cell population expansion relating to limbal stem cells according to the invention.
• the invention relates to a cell population comprising limbal stem cells obtained by the method of cell population expansion relating to limbal stem cells according to the invention.
• the invention relates to a cell population comprising corneal endothelial cells obtainable by the method of cell population expansion relating to corneal endothelial cells according to the invention.
• the invention relates to a cell population comprising corneal endothelial cells obtained by the method of cell population expansion relating to corneal endothelial cells according to the invention.
• an ocular cell delivery preparation comprising a cell population obtainable or obtained by the method of cell population expansion according to the invention and a composition suitable for ocular delivery which is a localising agent.
• the localising agent is GelMa. In another specific embodiment the localising agent is fibrin or fibrin glue.
• the cell delivery preparation of limbal stem cells has greater than 20% limbal stem cells. Also preferably the cell delivery preparation of limbal stem cells has greater than 20% p63alpha positive cells.
• corneal endothelial cells are present in the cell delivery preparation of corneal endothelial cells, at a density greater than 500 cells per mm 2 (area).
• the cell population obtainable or obtained by the method of cell population expansion or cell delivery preparation according to the invention has only trace levels of the compound according to the invention.
• the method further comprises use of a gene editing system.
• the gene editing system is used for genetically modifying cells.
• genetically modifying comprises reducing or eliminating the expression and/or function of a gene associated with facilitating a host versus graft immune response.
• the gene editing system specifically targets a gene associated with facilitating a host versus graft immune response.
• said gene editing system is selected from the group consisting of a CRISPR gene editing system, a TALEN gene editing system, a zinc finger nuclease gene editing system, a meganuclease gene editing system, AAV vector driven homologous recombination and lentiviral vectors-based genome editing technologies.
• the invention relates to an isolated cell population, wherein greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of cells are limbal stem cells.
• greater than 70% are limbal stem cells.
• Particularly preferably greater than 90% are limbal stem cells.
• the cells have been gene edited.
• the invention in another aspect relates to an isolated cell population, wherein greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of cells are p63alpha expressing cells.
• greater than 70% are p63alpha positive.
• Particularly preferably greater than 90% are p63alpha positive.
• the cells have been gene edited.
• the invention relates to a cell population comprising limbal stem cells or the cell population according to the invention, wherein one or more of said cells comprises a non-naturally occurring insertion or deletion of one or more nucleic acid residues of a gene associated with facilitating a host vs graft immune response, wherein insertion and/or deletion results in reduced or eliminated expression or function of said gene.
• said gene is selected from the group consisting of B2M, HLA-A, HLA-B and HLA-C.
• the cells have genetically modified levels of B2M expression.
• the invention relates to a cell population comprising corneal endothelial cells or the cell population according to the invention, wherein one or more of said cells comprises a non-naturally occurring insertion or deletion of one or more nucleic acid residues of a gene associated with facilitating a host vs graft immune response, wherein insertion and/or deletion results in reduced or eliminated expression or function of said gene.
• said gene is selected from the group consisting of B2M, HLA-A, HLA-B and HLA-C.
• the cells have genetically modified levels of B2M expression.
• the invention relates to a cell population comprising limbal stem cells or corneal endothelial cells which have been gene edited. In a further aspect the invention relates to a cell population comprising limbal stem cells which have been gene edited.
• the gene editing was performed by CRISPR.
• the B2M gene was edited.
• the invention relates to a growth promoting agent of cells comprising a LATS inhibitor.
• the invention relates to a growth promoting agent of ocular cells comprising a LATS inhibitor.
• the invention relates to a growth promoting agent of limbal stem cells comprising a LATS inhibitor.
• the LATS inhibitor is a compound of Formula A1 or subformulae thereof or salt thereof.
• the invention relates to a growth promoting agent of limbal stem cells comprising a compound of Formula A1 or subformulae thereof or salt theroef.
• the invention relates to a growth promoting agent of corneal endothelial cells comprising a LATS inhibitor.
• the LATS inhibitor is a compound of Formula A1 or subformulae thereof or a salt thereof.
• the invention relates to a growth promoting agent of corneal endothelial cells comprising a compound of Formula A1 or subformulae thereof or a salt thereof.
• the invention relates to a pharmaceutical composition
• a pharmaceutical composition comprising a compound of Formula A2 or subformulae thereof, or a pharmaceutically acceptable salt, or
• the composition further comprises a preservation or cryopreservation solution.
• the invention relates to a cell proliferation medium comprising a LATS inhibitor and a growth medium.
• the LATS inhibitor is a compound of Formula A1 or subformulae thereof according to the invention.
• the invention relates to a cell proliferation medium comprising a compound of Formula A1 or subformulae thereof according to the invention and a growth medium.
• the cell proliferation medium additionally comprises cells as provided herein.
• the cell proliferation medium additionally comprises ocular cells.
• the cell proliferation medium additionally comprises limbal stem cells.
• the limbal stem cells are in suspension.
• the cell proliferation medium comprises corneal endothelial cells.
• the corneal endothelial cells are in suspension.
• the invention relates to a cell preparation comprising a LATS inhibitor and cells of a cell population as described and/or provided herein.
• a cell preparation comprising a LATS inhibitor and ocular cells.
• the invention relates to a cell preparation comprising a LATS inhibitor and limbal stem cells.
• a cell preparation comprising a LATS inhibitor and corneal endothelial cells.
• the LATS inhibitor is a compound of Formula A1 or subformulae thereof according to the invention.
• the invention relates to a cell preparation comprising a compound of Formula A1 according to the invention and limbal stem cells.
• the invention relates to a cell preparation comprising a compound of Formula A1 according to the invention and corneal endothelial cells.
• the cell preparation further comprises a growth medium.
• the cell preparation further comprises a preservation or
• the invention relates to an ocular cell delivery preparation, comprising a cell preparation according to the invention and a composition suitable for ocular delivery which is a localising agent.
• the localising agent is GelMa.
• the localising agent is fibrin or fibrin glue.
• the invention relates to an ocular cell delivery preparation, comprising a cell preparation according to the invention and a composition suitable for ocular delivery which is a localising agent.
• the localising agent is GelMa.
• the localising agent is fibrin or fibrin glue.
• the cell preparation according to the invention has only trace levels of the compound according to the invention.
• greater than 20% of the cells in the cell delivery preparation are limbal stem cells.
• greater than 20% of the cells in the cell delivery preparation are p63alpha expressing cells.
• the invention relates to an ocular cell delivery preparation, comprising a cell preparation according to the invention and a composition suitable for ocular delivery which is a localising agent.
• a composition suitable for ocular delivery which is a localising agent.
• the localising agent is GelMa.
• the corneal endothelial cells are in suspension.
• the corneal endothelial cells are present in the cell delivery preparation at a density greater than
• the corneal endothelial cells are present at a density of 1000 to 3500 cells/mm 2 (area), more preferably 2000 to about 3000 cells/mm 2
• the cell preparation according to the invention has only trace levels of the compound according to the invention.
• the growth medium in the methods or cell preparation according to the invention is selected from the group consisting of Dulbecco's Modified Eagle's Medium (DMEM) supplemented with Fetal Bovine Serum (FBS), human endothelial Serum Free (SF) Medium with human serum, X-VIV015 medium and DMEM/F12 which is optionally supplemented with calcium chloride; preferably X-VIV015 medium.
• DMEM Dulbecco's Modified Eagle's Medium
• FBS Fetal Bovine Serum
• SF human endothelial Serum Free
• X-VIV015 medium X-VIV015 medium
• DMEM/F12 which is optionally supplemented with calcium chloride
• the preservation or cryopreservation solution according to the invention comprises a solution which is Optisol or PBS (phosphate buffered saline) and the cryopreservation solution additionally comprises glycerol, dimethyl sulfoxide, propylene glycol or
• the invention relates to a kit comprising a composition suitable for ocular delivery and a LATS inhibitor.
• the LATS inhibitor is a compound of Formula A2 or subformulae thereof according to the invention.
• the invention relates to a kit comprising a composition suitable for ocular delivery and compound of Formula A2 or subformulae thereof according to the invention.
• the kit has instructions for use.
• the composition suitable for ocular delivery is a localising agent or topical eye drops.
• the composition suitable for ocular delivery is a localising agent.
• the kit further comprises limbal stem cells.
• the composition suitable for ocular delivery is a localising agent which is GelMa.
• the composition suitable for ocular delivery is a localising agent which is fibrin or fibrin glue.
• greater than 20% of the cells in the kit are limbal stem cells.
• the LATS inhibitor is a compound of Formula A2 or subformulae thereof according to the invention.
• the kit has instructions for use.
• kits greater than 20% of the cells in the kit are p63alpha expressing cells.
• the kit comprises corneal endothelial cells.
• the composition suitable for ocular delivery of corneal endothelial cells is a localising agent which is GelMa.
• corneal endothelial cells are present in a monolayer.
• the corneal endothelial cells are present at a density greater than 500 cells per mm 2 (area).
• the corneal endothelial cells are present at a density of 1000 to 3500 cells/mm 2 (area), more preferably 2000 to about 3000 cells/mm 2 (area).
• the composition suitable for ocular delivery is a localising agent which is a biomatrix.
• the composition suitable for ocular delivery according to the invention is a localising agent selected from the group consisting of fibrin, collagen, gelatin, cellulose, amniotic membrane, fibrin glue, polyethylene (glycol) diacrylate (PEGDA), GelMA, localising agents comprising a polymer, cross-linked polymer, or hydrogel comprising one or more of hyaluronic acid, polyethylene glycol, polypropylene glycol, polyethylene oxide, polypropylene oxide, poloxamer, polyvinyl alcohol, polyacrylic acid, polymethacrylic acid, polyvinyl pyrrolidone, poly(lactide-co-glycolide), alginate, gelatin, collagen, fibrinogen, cellulose, methylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, hydroxypropyl-guar, gellan
• composition suitable for ocular delivery is a localising agent which is GelMa, fibrin or fibrin glue.
• composition suitable for ocular delivery is a localising agent which is GelMa.
• composition suitable for ocular delivery is a localising agent which is GelMa.
• the composition suitable for ocular delivery is a localising agent which is fibrin or fibrin glue.
• the localising agent is fibrin glue.
• Fibrin glues are known in the art, including, for example, TISSEEL VH Fibrin sealant (Baxter AG, Vienna, Austria) (Panda et al., 2009, Indian J Ophthalmol. Sep-Oct; 57(5): 371-379).
• fibrin glue is used for the delivery of limbal stem cells.
• GelMa is used for the delivery of corneal endothelial cells.
• limbal stem cells are present in combination with the localising agent.
• greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of cells are limbal stem cells.
• Preferably greater than 20% are limbal stem cells. More preferably greater than 50% are limbal stem cells.
• greater than 70% are limbal stem cells.
• Particularly preferably greater than 90% are limbal stem cells.
• greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of cells are p63alpha expressing cells.
• greater than 70% are p63alpha positive cells.
• Particularly preferably greater than 90% are p63alpha positive cells.
• corneal endothelial cells are present in combination with the localising agent.
• the corneal endothelial cells are in a monolayer. More preferably the corneal endothelial cells are present at a density greater than 500 cells per mm 2 (area). Particularly preferably the corneal endothelial cells are present at a density of 1000 to 3500 cells/mm 2 (area), more particularly preferably 2000 to about 3000 cells/mm 2 (area).
• the LATS inhibitor inhibits LATS1 or LATS2, or LATS1 and LATS2. In a more particularly preferred embodiments according to the invention the LATS inhibitor inhibits LATS1 and LATS2.
• the compound is selected from the group consisting of 2-methyl-1 -(2-methyl-2-((2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4- yl)amino)propoxy)propan-2-ol; N-(tert-butyl)-2-(pyridin-4-yl)-1 ,7-naphthyridin-4-amine; N- methyl-2-(pyridin-4-yl)-N-(1 ,1 ,1 -trifluoropropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine; (S)-N- methyl-2-(pyridin-4-yl)-N-(1 ,1 ,1 -trifluoropropan-2-yl)pyrido
• said compound or a salt thereof is selected from N-(tert-butyl)-2-(pyridin-4- yl)-1 ,7-naphthyridin-4-amine and (S)-N-methyl-2-(pyridin-4-yl)-N-(1 ,1 , 1-trifluoropropan-2- yl)pyrido[3,4-d]pyrimidin-4-amine.
• said compound or a salt thereof is N-(tert- butyl)-2-(pyridin-4-yl)-1 ,7-naphthyridin-4-amine.
• the compound according to the invention is present in a concentration of 0.5 to 100 micromolar, preferably 0.5 to 25 micromolar, more preferably 1 to 20 micromolar, particularly preferably of about 3 to 10 micromolar.
• the invention relates in one aspect to a method of transplanting a population of cells to a subject, said method comprising administering the population of cells obtainable or obtained by the method of cell population expansion or method of culturing cells or method of LATS inhibition according to the invention.
• the invention further relates to a method of transplanting a population of ocular cells onto the eye of a subject, said method comprising administering the population of cells obtainable or obtained by the method of cell population expansion or method of culturing cells or method of LATS inhibition according to the invention, wherein the cells are ocular cells.
• ocular cells are limbal stem cells or corneal endothelial cells.
• the invention relates in another aspect to a method of transplanting a population of ocular cells onto the cornea of a subject, said method comprising administering the cell delivery preparation according to the invention.
• the invention relates in another aspect to a method of transplanting a population of cells comprising limbal stem cells onto the cornea of a subject, said method comprising administering the population of cells comprising limbal stem cells obtainable or obtained by the method of cell population expansion or method of culturing cells or method of LATS inhibition according to the invention.
• the invention relates in another aspect to a method of transplanting a population of cells comprising limbal stem cells onto the cornea of a subject, said method comprising administering the cell delivery preparation according to the invention.
• the invention relates in another aspect to a method of transplanting a cell population comprising limbal stem cells onto the cornea of a subject, said method comprising expanding a cell population comprising limbal stem cells by culturing said population with cell proliferation medium comprising a LATS inhibitor according to the invention, preferably rinsing the expanded cell population to substantially remove the LATS inhibitor, and administering said cells onto the cornea of said subject.
• said cell population is combined with a biomatrix prior to said administration.
• said cell population is combined with a biomatrix which is GelMA prior to said administration.
• said cell population is combined with fibrin glue prior to said administration.
• said cell population is combined with a carrier which is a contact lens.
• the cell population comprising limbal stem cells is combined with a biomatrix which is GelMA and the GelMA is polymerized on a carrier which is a contact lens.
• the cell population comprising limbal stem cells is combined with fibrin glue and a contact lens.
• the invention relates in one aspect to a method of transplanting a population of corneal endothelial cells onto the cornea of a subject, said method comprising administering the population of corneal endothelial cells obtainable or obtained by the method of cell population expansion or method of culturing cells or method of LATS inhibition according to the invention.
• the invention relates in another aspect to a method of transplanting a population of corneal endothelial cells onto the cornea of a subject, said method comprising administering the cell delivery preparation according to the invention.
• the invention relates in another aspect to a method of transplanting a population of cells comprising corneal endothelial cells onto the cornea of a subject, said method comprising expanding a population of cells comprising corneal endothelial cells by culturing said population with cell proliferation medium comprising a LATS inhibitor according to the invention, rinsing the expanded population of cells to substantially remove the LATS inhibitor, and administering said cells onto the cornea of said subject.
• said cells are combined with a biomatrix prior to said administration.
• said cells are combined with a biomatrix which is GelMA prior to said administration.
• said corneal endothelial cells are combined with a biomatrix which is bioprinted onto the ocular surface.
• said corneal endothelial cells are combined with a biomatrix which is GelMA and bioprinted onto the ocular surface by polymerising the GelMA by a light triggered reaction.
• the invention relates in another aspect to a method of transplanting a population of cells to the eye of a subject, comprising combining the cells with a biomatrix to form a cell/biomatrix mixture, injecting the mixture into the eye of the subject or applying the mixture onto the surface of the eye of the subject, and bioprinting the cells in or on the eye by guiding and fixing the cells, such as on the cornea, using a light source, such as an Ultraviolet A or white light source.
• a light source such as an Ultraviolet A or white light source.
• the light source produces light of a wavelength that is at least 350 nm. In certain embodiments, the light source produces light in the 350 nm to 420 nm range.
• an LED light source can be used to produce a light having a wavelength of 365 nm or 405 nm, or any other wavelength above 350 nm, or a mercury lamp with a bandpass filter can be used to produce a light having a wavelength of 350 nm to 700 nm, for example a wavelength of 365 nm or 405 nm.
• the light source produces visible, white light having a wavelength, for example, in the 400 nm to 700 nm range.
• the cells are ocular cells, such as corneal cells, for example corneal endothelial cells.
• the invention relates in another aspect to a method of transplanting a population of corneal endothelial cells to the eye of a subject, comprising culturing a population of corneal endothelial cells in a cell proliferation medium that comprises a LATS inhibitor, combining the corneal endothelial cells with a biomatrix to form a cell/biomatrix mixture, injecting the mixture into the eye of the subject, and bioprinting the cells in the eye by guiding and fixing the cells on the cornea using a light source, such a UVA or LED or visible light source.
• a light source such as a UVA or LED or visible light source.
• the invention relates in a further aspect to a method of prophylaxis or treatment of an ocular disease or disorder using a LATS inhibitor.
• the LATS inhibitor is a compound of Formula A1 or subformulae thereof according to the invention.
• the invention relates in yet a further aspect to a method of prophylaxis or treatment of an ocular disease or disorder using a compound according to Formula A2 or subformulae thereof according to the invention.
• the method of prophylaxis or treatment of an ocular disease or disorder further comprises the method of LATS inhibition in a cell population or the method of cell population expansion according to the invention, wherein said cells are ocular cells.
• the method of prophylaxis or treatment of an ocular disease or disorder comprises administering to a subject in need thereof of a therapeutically effective amount of a cell population obtainable or obtained by the method of cell population expansion according to the invention, wherein said cells are ocular cells.
• the method of prophylaxis or treatment of an ocular disease or disorder comprises administering to a subject in need thereof of a therapeutically effective amount of the cell delivery preparation according to the invention, wherein said cells are ocular cells.
• the method of prophylaxis or treatment of an ocular disease or disorder the method comprises the steps of the method of transplanting a population of cells comprising ocular cells to the eye of a subject according to the invention.
• the ocular cells are limbal stem cells or corneal endothelial cells.
• the method of prophylaxis or treatment of an ocular disease or disorder comprises the steps of the method of transplanting a population of cells comprising limbal stem cells onto the cornea of a subject according to the invention.
• the method of prophylaxis or treatment of an ocular disease or disorder comprises the steps of the method of transplanting a population of corneal endothelial cells onto the cornea of a subject according to the invention.
• the cell population obtainable or obtained by the method of cell population expansion according to the invention or cell delivery preparation according to the invention is administered simultaneously or sequentially with an agent or agents selected from the group consisting of dexamethasone, cyclosporine, tobramycin, and cefazolin.
• the invention relates to a YAP (yes associated protein) modulator for use in a method of transplanting a population of cells to a subject, which comprises administering to a subject in need thereof of a therapeutically effective amount of a cell population obtainable or obtained by the method of cell population expansion according to the invention or cell delivery preparation according to the invention.
• said YAP modulator is a LATS inhibitor.
• the cells are ocular cells.
• the invention relates to a YAP (yes associated protein) modulator for use in a method of transplanting a population of cells comprising limbal stem cells onto the cornea of a subject, which comprises administering to a subject in need thereof of a therapeutically effective amount of a cell population obtainable or obtained by the method of cell population expansion according to the invention or cell delivery preparation according to the invention.
• YAP yes associated protein
• said YAP modulator is a LATS inhibitor.
• the invention in another aspect relates to a YAP modulator for use in a method of treating limbal stem cell deficiency, comprising administering to a subject in need thereof of a therapeutically effective amount of a cell population obtainable or obtained by the method of cell population expansion according to the invention or cell delivery preparation according to the invention.
• said YAP modulator is a LATS inhibitor.
• the invention relates to a YAP (yes associated protein) modulator for use in a method of transplanting a population of corneal endothelial cells onto the cornea of a subject, which comprises administering to a subject in need thereof of a therapeutically effective amount of a cell population obtainable or obtained by the method of cell population expansion according to the invention or cell delivery preparation according to the invention.
• said YAP modulator is a LATS inhibitor.
• the invention in another aspect relates to a YAP modulator for use in a method of treating corneal endothelial dysfunction, comprising administering to a subject in need thereof of a therapeutically effective amount of a cell population obtainable or obtained by the method of cell population expansion according to the invention or cell delivery preparation according to the invention.
• said YAP modulator is a LATS inhibitor.
• the present invention relates to a method of treatment of a disease or disorder comprising administering to a subject in need thereof a cell population, wherein the population has been grown in the presence of an agent capable of inhibiting the activity of LATS1 and LATS2 kinases; thereby inducing YAP translocation and driving downstream gene expression for cell proliferation.
• the agent is a compound of Formula A1 or subformulae thereof, or a pharmaceutically acceptable salt thereof.
• the cells are ocular cells.
• the invention relates to a compound according to Formula A2 or subformulae thereof, or a pharmaceutically acceptable salt thereof according to the invention for use in therapy or as a medicament.
• the compound is for use in an ocular disease or disorder.
• the invention relates to a LATS inhibitor for use in an ocular disease or disorder, preferably wherein the LATS inhibitor is a compound.
• the LATS inhibitor is a compound.
• the compound is a compound of Formula A1 or subformulae thereof, or a pharmaceutically acceptable salt thereof according to the invention.
• the invention relates to the use of a compound of Formula A2 or subformulae thereof, or a pharmaceutically acceptable salt thereof according to the invention in the manufacture of a medicament.
• the invention relates to the use of a compound of Formula A1 or subformulae thereof, or a pharmaceutically acceptable salt thereof according to the invention in the manufacture of a medicament to treat an ocular disease or disorder.
• the use further comprises the method of LATS inhibition in a cell population or the method of cell population expansion according to the invention.
• the use comprises administering to a subject in need thereof a therapeutically effective amount of a cell population obtainable or obtained by the method of cell population expansion according to the invention.
• the use comprises administering to a subject in need thereof of a therapeutically effective amount of the cell delivery preparation according to the invention.
• the use comprises the steps of the method of transplanting a population of cells comprising ocular cells onto the cornea of a subject according to the invention.
• the use comprises the steps of the method of transplanting a population of cells comprising limbal stem cells onto the cornea of a subject according to the invention.
• the use comprises the steps of the method of transplanting a population of cells comprising corneal endothelial cells onto the cornea of a subject according to the invention.
• the cell population obtainable or obtained by the method of cell population expansion according to the invention or cell delivery preparation according to the invention is administered simultaneously or sequentially with an agent or agents selected from the group consisting of dexamethasone, cyclosporine, tobramycin, and cefazolin.
• the ocular disease or disorder is associated with limbal stem cell deficiency.
• the ocular disease or disorder is limbal stem cell deficiency.
• the ocular disease or disorder is limbal stem cell deficiency which arises due to an injury or disorder selected from the group consisting of chemical burns, thermal burns, radiation injury, aniridia,
• sclerocornea multiple endocrine neoplasia, Stevens Johnson syndrome, ocular cicatricial pemphigoid, collagen vascular diseases; chronic non-auto-immune inflammatory disorders arising from contact lens use, dry eye disease, rosacea, staph marginal, keratitis (including bacterial, fungal & viral keratitis), pterygia or neoplasm, limbal stem cell deficiency arising after multiple eye surgeries, excision of pterygia or neoplasm or cryotherapy; and limbal stem cell deficiency arising as a result of medication toxicity from a medication selected from the group consisting of preservatives (thimerosal, benzalkonium), topical anesthetics, pilocarpine, beta blockers, mitomycin, 5-fluorouracil, silver nitrate, and oral medications causing Stevens Johnson syndrome.
• the ocular disease or disorder is limbal
• the ocular disease or disorder is associated with decreased corneal endothelial cell density.
• the ocular disease or disorder is corneal endothelial dysfunction.
• the ocular disease or disorder is corneal endothelial dysfunction which is selected from the group consisting of Fuchs endothelial corneal dystrophy, bullous keratopathy (including pseudophakic bullous keratopathy and aphakic bullous keratopathy), corneal transplant failure, posterior polymorphous corneal dystrophy, congenital hereditary endothelial dystrophy, X-linked endothelial corneal dystrophy, aniridia, and corneal endothelitis.
• the ocular disease or disorder is selected from the group consisting of Fuchs endothelial corneal dystrophy, bullous keratopathy (including pseudophakic bullous keratopathy and aphakic bullous keratopathy) and corneal transplant failure.
• the invention further relates to methods of promoting wound healing, particularly for treating or ameliorating the symptoms of burns, acute and chronic skin ulcers, comprising administering to a subject in need thereof an effective amount of a LATS inhibitor.
• the invention relates to a method of promoting wound healing comprising administering a therapeutically effective amount of a compound of Formula A1 or subformulae thereof, or a pharmaceutically acceptable salt, or a stereoisomer thereof.
• the invention relates to compounds and compositions that may be used in promoting wound healing. In another aspect, the invention relates to compounds and compositions that may be used for the manufacture of a medicament for promoting wound healing.
• the present invention also relates to a method of promoting wound healing, particularly for treating or ameliorating the symptoms of burns, acute skin ulcers, and chronic skin ulcers, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of Formula A1 or subformulae thereof, and optionally with a second therapeutic agent that is another compound of the invention or one other type of therapeutic agent.
• the present invention also relates to a method of promoting ocular wound healing comprising administering to an eye of a subject a therapeutically effective amount of a compound of the invention.
• the ocular wound is a corneal wound.
• the ocular wound is an injury or surgical wound.
• the invention relates to compounds and compositions that may be used in liver regeneration and liver regrowth.
• the invention relates to a method of promoting liver regeneration and liver regrowth comprising administering a therapeutically effective amount of a compound of Formula A1 or subformulae thereof, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.
• the invention relates to compounds and compositions that may be used for the manufacture of a medicament for liver regeneration and liver regrowth.
• the present invention also relates to a method for liver regeneration and liver regrowth, particularly for treatment of insufficient liver regrowth following transplantation of marginal grafts; for supporting enhanced regrowth of the remnant liver mass following extensive hepatectomy; for regeneration of patients' of livers following acute liver failure from viral hepatitis, drug -induced liver injury, autoimmune hepatitis, ischemic- and congestive liver disease; and for treatment of patients with chronic liver injury and underlying liver fibrosis, from non-alcoholic steatohepatitis, alcoholic steatohepatitis, chronic viral hepatitis B and C, hemochromatosis, alpha-1 anti-trypsin deficiency, Wilson's disease and drug-induced liver fibrosis to enhance both regenerative capacity and accelerate fibrosis resolution, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the present invention and optionally with a second therapeutic agent that is another compound of the invention or one other type of therapeutic agent.
• the invention relates to a method of generating cellular material for cell therapy and/or transplantation comprising the ex-vivo use of a compound of Formula A1 or subformulae thereof, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.
• the cellular material may comprise ocular, liver or skin cells.
• the invention relates to a method of promoting liver regeneration and liver regrowth comprising the ex-vivo use of a compound of Formula A1 or subformulae thereof, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.
• the present invention also relates to an ex-vivo method for liver cell population expansion, comprising use of a compound of the present invention or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.
• Figure 1 LATS inhibitors (compound ex. 49 and ex. 133) induce YAP dephosphorylation in LSCs within one hour of treatment as shown by Western blot.
• Figure 2 Immunolabelling of p63-alpha in limbal stem cell cultures indicates that the LSC population can be expanded when it is maintained in medium comprising the LATS inhibitors (compound ex. 49 and ex. 133).
• Figure 2A In the presence of growth medium and DMSO, only a few isolated cells attach to the culture dish and survive up to 6 days. Most cells expressed the human nuclear marker, but few expressed p63alpha.
• Figures 2B and 2C In contrast, in the presence of LATS inhibitors: compound example no. 49 and example no.
• FIG. 3 LATS1 and LATS2 knockdown by siRNA activates LSC proliferation in culture as shown by percentage of EdU positive cells.
• FIG. 4 Immunolabelling of LSC markers DeltaN-p63 alpha/beta/gamma, ABCG2 and C/EBP delta indicates that LSCs maintained in a culture medium containing the LATS inhibitors (compound ex. 49 and ex. 133) express typical markers of LSCs. Results indicate that cells cultured in DMSO do not typically express markers such as DeltaN-p63 alpha/beta/gamma, ABCG2 and C/EBP delta, normally expressed by LSCs. In contrast, cells cultured in the presence of LATS inhibitors express markers such as DeltaN-p63 alpha/beta/gamma, ABCG2 and C/EBP delta, normally expressed by LSCs.
• FIG. 5 Immunolabelling of undifferentiated LSC marker p63alpha and corneal epithelium cell marker keratin 12 shows that LSC populations expanded using a culture medium comprising the LATS inhibitors (Fig 5A: compound ex. 49, Fig 5B: compound ex. 47, Fig 5C: compound ex. 12, Fig 5D: compound ex. 261 ) can differentiate into corneal epithelium cells when transferred to conditions that enable differentiation. Shown are views where the transition from the p63alpha-positive cell identity to the keratin-12 identity are occurring.
• FIG. 6 LSCs were labeled using a fluorescent protein in order to confirm that LSCs attached to a contact lens using GelMA polymerization can be delivered to the surface of the rabbit eye ex vivo. Arrows show site of attachment of LSCs.
• Figure 7 Fig 7A: Transplanted eye, keratin-12 staining; Fig. 7B: Transplanted eye, keratin- 19 staining; Fig. 7C: non-transplanted control eye, keratin-12 staining; Fig 7D: non- transplanted control eye, keratin- 19 staining.
• Fig. 7A a keratin-12-positive corneal epithelium
• Fig. 7B Arrow is pointing to absence of keratin 19 staining.
• Fig. 7C Arrow is pointing to absence of keratin-12 staining. Instead, signs of conjunctivalization were observed, as showed by the presence of keratin-19 staining.
• Fig. 7D Arrow is pointing to areas of positive keratin-19 staining.
• FIG. 8 Fig. 8A: Rabbit eye transplanted with human LSCs; Fig. 8B: Control rabbit eye, non-transplanted with human LSCs.
• Fig. 8A Rabbit eye transplanted with human LSCs;
• Fig. 8B Control rabbit eye, non-transplanted with human LSCs.
• Figure 9 Microscopic images of LSCs delivered via TISSEEL to collagen coated 24 well plate show repopulation of the cells to cover the culture surface within 2 weeks.
• FIG. 10 CellTracker Green CMFDA labeled LSCs were delivered to human cornea ex vivo and covered with protective contact lens.
• Figure 11 Red and green dye labeled HEK-293 cells were bioprinted into a Yin-Yang pattern on top of a rabbit cornea ex vivo (shown as dark and light gray pattern).
• Figure 12 Reducing immune rejection by CRISPR/Cas9-mediated deletion of the beta-2- microglobulin (B2M) gene in LSCs: FACS analyses show that CRISPR-mediated deletion of B2M and subsequent elimination of HLA A, B and C occurred in 21 percent of the LSCs.
• Figure 13 The population of B2M-negative/HLA A,B,C-negative LSCs was expanded using compound example no. 48a to produce a cell preparation where 97 percent of the cells do not express HLA A, B, C.
• Figure 14 LATS inhibitors (compounds ex. 133 and ex. 49) induce translocation of YAP into the nucleus in Corneal Endothelial Cells (CECs).
• CECs Corneal Endothelial Cells
• Figure 15 LATS inhibitors (compound ex. 133 and ex. 49) induce YAP dephosphorylation in CECs within one hour of treatment. As shown in Fig. 15a by Western blot; Fig. 15b: graph showing phosphorylated YAP levels normalized to beta-actin; and Fig.15c: graph showing phosphorylated YAP levels normalized to total YAP.
• Figure 16 CECs grown in presence or absence of LATS inhibitors.
• An Incucyte system (Essen Biosciences) was used to measure CEC confluence by real-time quantitative live-cell analysis over a time course.
• Compound ex. 49 black squares
• ex. 133 light grey squares
• CEC proliferation was minimal in the vehicle (DMSO, dark grey squares).
• FIG. 17 LATS1 and LATS2 knockdown by siRNA activates corneal endothelial cell proliferation in culture as shown by percentage of EdU positive cells.
• Figure 18 Zonula Occludens-1 (ZO-1 ) immunolabelling indicates that CECs proliferated in the presence of the LATS inhibitor, compound ex. 49, (Fig. 18b) form tight junctions, an endothelial structure and retain a normal cell size and morphology characteristic of functional CECs. CECs proliferated in the presence of the vehicle alone (DMSO) show signs of polymegatism characteristic of dysfunctional CECs (Fig. 18a).
• DMSO vehicle alone
• FIG 19 Quantitative RT-PCR analysis indicates that a corneal endothelial cell population expanded with the LATS inhibitor, compound ex. 49, express genes normally expressed by corneal endothelial cells in vivo, including Collagen 8a2, AQP1 , SLc4A1 1 . The cells do not express markers of other epithelia present in the eye, including RPE65 (a marker of retinal pigmented epithelium) and CD31 (a marker of vascular epithelium).
• Figure 20 Immunohistochemical analysis indicates that a corneal endothelial cell population expanded with the LATS inhibitor, compound ex. 49, express genes normally expressed by corneal endothelial cells in vivo, including Na/K ATPase (Fig. 20a) and Collagen 8a2 (Fig. 20b).
• Figure 21 FACS analysis of the corneal endothelium cell population expanded in the presence of the LATS inhibitor, compound ex. 47, and CECs cultured in the absence of a LATS inhibitor.
• the cell population expanded in the presence of the LATS inhibitor expresses low levels of CD73 (Fig. 21 a, grey line), CD44 (Fig. 21 b, grey line), CD166 (Fig. 21 c, grey line) and CD105 (Fig. 21 d, grey line); while the cell population cultured without the LATS inhibitor expresses high levels of CD44, CD73, CD105 and CD166 (black lines).
• Figure 22 Bubble depression method depicting method 1 as described further under the "bio-printing section", in which a biomatrix is applied to an eye to deliver a cell preparation according to the invention.
• Fig. 22a Inject bolus of biomatrix;
• Fig. 22b Inject bubble beneath biomatrix to spread over cornea;
• Fig. 22c Cure biomatrix with UV or blue light source;
• Fig. 22d Remove bubble and replace with balanced salt solution.
• Figure 23 Subtractive method using femtosecond (FS) laser, depicting method 2 as described further under the "bio-printing section", in which a biomatrix is applied to an eye to deliver a cell preparation according to the invention.
• Fig. 23a Remove dysfunctional endothelium with FS;
• Fig. 23b Inject biomatrix in anterior chamber;
• Fig. 23c Cure biomatrix with UV or blue light source;
• Fig. 23d Detach unwanted biomatrix with FS;
• Fig. 23e Remove detached biomatrix with forceps.
• Figure 24 Dye mask method, depicting method 3 as described further under the "bio- printing section", in which a biomatrix is applied to an eye to deliver a cell preparation according to the invention.
• Fig. 24a Stain the endothelium with dye (e.g. Typan Blue);
• Fig. 24b Peel the dysfunctional endothelium;
• Fig. 24c Inject dyed biomatrix;
• Fig. 24d Cure biomatrix with UV or blue light source;
• dye e.g. Typan Blue
• Fig. 24b Peel the dysfunctional endothelium
• Fig. 24c Inject dyed biomatrix
• Fig. 24d Cure biomatrix with UV or blue light source
• Figure 25 Dry dispense method, depicting method 4 as described further under the "bio- printing section", in which a biomatrix is applied to an eye to deliver a cell preparation according to the invention.
• Fig. 25a Drain anterior chamber;
• Fig. 25b Dispense biomatrix to posterior cornea with soft tip / brush cannula;
• Fig. 25c Cure biomatrix with UV or blue light source;
• Fig. 25d Refill eye with balanced salt solution.
• Figure 26 Schematic showing bioprinting device as described further in Example C1 1 .
• Figure 27 Results showing cells can be bioprinted on the posterior side of the cornea by using a handheld device that projects 365 nm UVA light through the cornea. After unpolymerized and unattached material was rinsed, a circular pattern of fluorescent protein- labelled cells was retained on the posterior side of the cornea.
• Figure 28 CECs bioprinted on the posterior side of the cornea can rebuild a corneal endothelium in a rabbit model of corneal endothelium dystrophy. Results indicated that in experimental rabbits, the corneal endothelium structure can be detected using ZO-1 immunohistochemistry (Fig. 28A). In the right eye of a rabbit where the corneal endothelium was surgically removed and no CEC was bioprinted, the ZO-1 staining is absent, indicating an absence of normal corneal endothelium structure (Fig. 28B). In the right eye of a rabbit where the corneal endothelium was surgically removed and CEC were bioprinted, the ZO-1 staining is present, indicating that a corneal endothelium structure has been rebuilt (Fig.
• Fig. 28D and Fig. 28E show that human nuclear antigen immunostaining is absent in eyes that did not receive any human CECs.
• human nuclear antigen-positive cells cover the imaged field in eyes where human CECs were bioprinted (Fig.28F), indicating that the ZO-1 -labeled corneal endothelium structure shown in Fig. 28C is composed of the human CECs bioprinted on the posterior side of the rabbit cornea.
• Figure 29 Red-fluorescent-protein labeled HEK-293 cells were bioprinted into constructs of different letters on the posterior side of human cornea ex vivo.
• Figure 30 Vector map shows the design of the AAV2 vector used to express the CRISPR system in LSCs and CECs. ( Figure 30 discloses SEQ ID NO: 28).
• Figure 31 FACS analysis of AAV-mediated expression of the CRISPR system enabled deletion of B2M and subsequent elimination of HLA A, B and C in LSCs.
• Figure 32 FACS analysis of AAV-mediated expression of the CRISPR system enabled deletion of B2M and subsequent elimination of HLA A, B and C in CECs.
• Figure 33A is a western blot of pYAP in lysate of human HaCaT cells that were untreated or treated by 40 pM each of siRNA against MST1/2 or LATS1/2; actin was used as control.
• Figure 33B is a western blot of pYAP in lysate of human HaCaT cells that were untreated or treated by 9 ⁇ of Example 133; actin was used as control.
• Figure 33C is a graph of the relative inhibitory activity against LATS1 versus concentration of Example 133 ranging from ⁇ 10 "4 to 1 ⁇ .
• the calculated IC 50 of Example 133 against LATS1 was 1 .3 nM.
• Figure 34 is a bar graph of relative Cyr61/Gapdh expression levels versus concentration of Example 133 at 0, 0.2 and 2 mg/mL.
• Figure 35A shows micrographs of mouse skin treated topically with vehicle or Example 133.
• Figure 35B is a scatter plot comparing the percentage of Ki67+ cells in mouse skin treated with vehicle or Example 133.
• LATS is the abbreviated name of the large tumor suppressor kinase.
• LATS as used herein refers to LATS1 and/or LATS2.
• LATS1 as used herein refers to the large tumor suppressor kinase 1 and LATS2 refers to the large tumor suppressor kinase 2.
• LATS1 and LATS2 both have serine/threonine protein kinase activity.
• LATS1 and LATS2 have been given the
• LATS1 Human Genome Organisation (HUGO) Gene Nomenclature Committee identifiers: HGNC ID 6514 and HGNC ID 6515 respectively.
• LATS1 is sometimes also referred to in the art as WARTS or wts
• LATS2 is sometimes referred to in the art as KPM.
• Representative LATS sequences include, but are not limited to, the protein sequences available from the National Center for Biotechnology Information protein database with the accession numbers NP_004681 .1 (LATS1 ) and NP_001257448.1 (LATS1 ) and NP_055387.2 (LATS 2), as shown below.
• LATS1 NP_004681 .1 (Serine/threonine-protein kinase LATS1 isoform 1 , homo sapiens)(SEQ ID NO: 1 :)
• LATS1 serine/threonine-protein kinase LATS1 isoform 2 [Homo sapiens]
• LATS 2 NP_055387.2 serine/threonine-protein kinase LATS2 [Homo sapiens]. ((SEQ ID NO: 3:)
• LATS is thought to negatively regulate YAP1 activity.
• ⁇ 1 refers to the yes-associated protein 1 , also known as YAP or YAP65, which is a protein that acts as a transcriptional regulator of genes involved in cell proliferation.
• LATS kinases are serine/threonine protein kinases that have been shown to directly phosphorylate YAP which results in its cytoplasmic retention and inactivation. Without phosphorylation by LATS, YAP translocates into the nucleus, forming a complex with a DNA binding protein, TEAD, and results in downstream gene expression.
• Barry ER & Camargo FD The Hippo superhighway: signaling crossroads converging on the Hippo/Yap pathway in stem cells and development.
• the Hippo/YAP pathway is involved in numerous cell types and tissues in mammalian systems, including various cancers.
• the Hippo pathway is evidently involved in the intestine, stomach and esophagus, pancreas, salivary gland, skin, mammary gland, ovary, prostate, brain and nervous system, bone, chrondrocytes, adipose cells, myocytes, T lymphocytes, B lymphocytes, myeloid cells, kidney, and lung. See Nishio et al., 2017, Genes to Cells 22:6-31 .
• Compounds of Formula A1 or subformulae thereof, in free form or in salt form are potent inhibitors of LATS1 and/or LATS2.
• the compounds of Formula A2 or subformulae thereof, in free form or in salt form are potent inhibitors of LATS1 and LATS2.
• the inhibition efficacy of the compounds against LATS1 were assayed by the LATS1 Biochemical HTRF Assay as described in Example A1 below.
• the inhibition efficacy of the compounds of the invention against LATS1 (LATS1 IC 50 in micromolar) in this assay are reported in Table 1A. It should be noted that compounds with IC 50 greater than 1 micromolar are considered inactive in this assay.
• LATS2 Biochemical Caliper Assay The inhibition efficacy of selected compounds against LATS2 were assayed by the LATS2 Biochemical Caliper Assay as described in Example A3 below.
• the inhibition efficacy of the compounds of the invention against LATS2 (LATS2 IC 50 in micromolar) in this assay are also reported in Table 1 A. It should be noted that compounds with IC 50 greater than 1 micromolar are considered inactive in this assay.
• the invention therefore relates to a compound of Formula A2:
• X 1 is CH or N
• ring A is unsubstituted or substituted by 1 to 2 substituents independently selected from halogen, cyano, C 1-6 alkyl,
• R° is hydroxyl or C ⁇ alkoxy
• R 1 is hydrogen or C 1-6 alkyl
• R 2 is selected from
• R 6 is selected from hydrogen, C 1-6 alkyl that is unsubstituted or substituted by R° or -C(0)R°;
• (xii) 6-membered heterocycloalkyi comprising, as ring members, 1 to 2 heteroatoms independently selected from N, O and S and that is unsubstituted or substituted by 1 to 2 substituents independently selected from hydroxyl, halogen, C 1-6 alkyl, C ! -eialkylamino, and
• (xv) 9- or 10-membered fused bicyclic heteroaryl comprising, as ring member, 1 to 2 heteroatoms independently selected from N and O;
• R 1 and R 2 can be taken together with the nitrogen atom to which both are bound to form a 4- to 6-membered heterocycloalkyi that can include, as ring members, 1 to 2 additional heteroatoms independently selected from N, O, and S, wherein the 4- to 6-membered heterocycloalkyl formed by R 1 and R 2 taken together with the nitrogen atom to which both are bound is unsubstituted or substituted by 1 to 3 substituents independently selected from halogen, C 1-6 alkyl, C ⁇ haloalkyl, and R°; R 3 is selected from hydrogen, halogen and C 1-6 alkyl; and
• R 5 is selected from hydrogen, halogen and -NH-(3- to 8-membered heteroalkyl), wherein the 3- to 8-membered heteroC 3 . 8 alkyl of the -NH-(3- to 8-membered heteroalkyl) comprises 1 to 2 oxygen atoms as chain members and is unsubstituted or substituted by R°;
• R 2 is not C 2 - 4 alkyl that is substituted with a substituent selected from -NH 2, or f-butyl-carbamoyl-amino and that is optionally further substituted with unsubstituted phenyl; and
• compounds of the present invention refers to compounds of Formula A2 or subformulae thereof, or salts thereof, as well as all stereoisomers (including diastereoisomers and enantiomers), rotamers, tautomers and isotopically labeled compounds (including deuterium substitutions), as well as inherently formed moieties.
• Embodiment 1 A compound of Formula A2 or a salt thereof, as described above.
• Embodiment 2. A compound of Formula A2 according to embodiment 1 , or a salt thereof, wherein
• ring A is unsubstituted or substituted by 1 to 2 substituents independently selected from halogen, cyano, C 1 -6 alkyl, C ⁇ haloalkyl, -NH 2 and C 3 . 6 cycloalkyl.
• Embodiment 3 A compound of Formula A2, or a salt thereof, according to embodiment 1 , wherei from , which are each unsubstituted or substituted by 1 to 2 substituents independently selected from cyano, halogen, C 1-6 alkyl, C ⁇ haloalkyl, N H 2 , and C 3 . 6 cycloalkyl;
• Embodiment 4 A compound of Formula A2, or a salt thereof, according to any one of embodiments 1 to 3, wherein ring A is selected from , and N' ⁇ 0 , which are each unsubstituted or substituted by a substituent selected from halogen , cyano, C 1 -6 alkyl, 6halo H 2 ;
• Embodiment 5 A compound of Formula A2 , or a salt thereof, according to any one of embodiments 1 to 3 wherein ring A is selected from
• Embodiment 6 A compound of Formula A2 , or a salt thereof, according to any one of embodiments 1 to 5, wherein ring A is selected from
• Embodiment 7 A compound of Formula A2 , or a salt thereof, according to any one of embodiments 1 to 6, wherein ring A is selected from and Embodiment 8.
• Embodiment 10 A compound of Formula A2 or a salt thereof, according to any one of
• Embodiment 1 1 A compound of For a salt thereof, according to any one of embodiments 1 to 7, wherein ring A '
• Embodiment 12 A compound of For a salt thereof, according to any one of embodiments 1 to 7, wherein ring A is Embodiment 13.
• Embodiment 14 A compound of Formula A2 or a salt thereof, according to any one of
• Embodiment 16 A compound of Formula A2, or a salt thereof, according to any one of
• Embodiment 17 A compound of Form or a salt thereof, according to any one of embodiments 1 to 4, wherein ring A is
• Embodiment 18 A compound of Form a salt thereof, according to any one of
• Embodiment 18A A compound of Formula A2, or a salt thereof, according to any one of
• Embodiment 19 A compound of Formula A2, or a salt thereof, according to any one of embo from hich are each unsubstituted or substituted by
• substituents independently selected from cyano, halogen, C 1-6 alkyl, C 1-6 haloalkyl, NH 2 , and C 3 . 6 cycloalkyl; , which are each unsubstituted or substituted by C 1-6 alkyl.
• Embodiment 20 A compound of Formula A2, or a salt thereof, according to any one of embodiments 1 to 19, wherein R 1 is selected from hydrogen, methyl and ethyl.
• Embodiment 21 A compound of Formula A2, or a salt thereof, according to any one of embodiments 1 to 20, wherein R 1 is methyl.
• Embodiment 22 A compound of Formula A2, or a salt thereof, according to any one of embodiments 1 to 20, wherein R 1 is hydrogen.
• Embodiment 23 A compound of Formula A2, or a salt thereof, according to any one of embodiments 1 to 22, wherein
• R 2 is selected from
• R 6 is selected from hydrogen, C 1-6 alkyl that is unsubstituted or substituted by R° or -C(0)R°;
• (c) 4-membered heterocycloalkyi comprising, as ring member, a heteroatom selected from N and O and that is unsubstituted or substituted by 1 to 2 substituents independently selected from C ⁇ haloalkyl, R°, C ⁇ ealkylamino, -C(0)R°, and C 1-6 alkyl that is unsubstituted or substituted by R° or -C(0)R°.
• Embodiment 24 A compound of Formula A2, or a salt thereof, according to any one of embodiments 1 to 22, wherein
• R 2 is selected from
• R 6 is selected from hydrogen, C 1-6 alkyl that is unsubstituted or substituted by R° or -C(0)R°;
• Embodiment 25 A compound of Formula A2, or a salt thereof, according to any one of embodiments 1 to 23, wherein
• R 2 is selected from
• R 6 is selected from hydrogen, C 1-6 alkyl that is unsubstituted or substituted by hydroxyl or -C(0)H;
• Embodiment 26 A compound of Formula A2, or a salt thereof, according to any one of embodiments 1 to 25, wherein
• R 2 is selected from
• R 6 is selected from hydrogen, and C 1-6 alkyl that is unsubstituted or substituted by hydroxyl;
• Embodiment 28. A compound of Formula A2, or a salt thereof, according to any one of embodiments 1 to 27, wherein R 2 is C ⁇ alkyl that is unsubstituted or substituted by hydroxyl.
• Embodiment 29 A compound of Formula A2, or a salt thereof, according to any one of embodiments 1 to 27, wherein R 2 is C 1 -6 alkyl that is unsubstituted or substituted by
• Embodiment 30 A compound of Formula A2, or a salt thereof, according to any one of embodiments 1 to 27, wherein R 2 is C 1 -6 alkyl that is unsubstituted or substituted by -0-Ci_ 6 alkyl-OH.
• Embodiment 31 A compound of Formula A2, or a salt thereof, according to any one of embodiments 1 to 26, wherein R 2 is C 3 . 6 cycloalkyl that is unsubstituted or substituted by a substituent selected from C ⁇ haloalkyl, Ci.ealkylamino, R°, and C 1 -6 alkyl that is unsubstituted or substituted by hydroxyl.
• Embodiment 32 A compound of Formula A2, or a salt thereof, according to any one of embodiments 1 to 26 and 31 , wherein R 2 is unsubstituted C 3 . 6 cycloalkyl.
• Embodiment 33 A compound of Formula A2, or a salt thereof, according to any one of embodiments 1 to 26 and 31 , wherein R 2 is C 3 . 6 cycloalkyl that is substituted by C 1 -6 alkyl or C ⁇ haloalkyl.
• Embodiment 34 A compound of Formula A2, or a salt thereof, according to any one of embodiments 1 to 25, wherein R 2 is selected from isopropyl, s-butyl, f-butyl, 2-methyl-but-2- -trimethylpentan-2-yl,
• Embodiment 34A A compound of Formula A2, or a salt thereof, according to any one of
• Embodiment 35 A compound of Formula A2, or a salt thereof, according to any one of embodiments 1 to 23 and 25 to 27, wherein R 2 is selected from n-propyl, isopropyl, f-butyl,
• Embodiment 36 A compound of Formula A2, or a salt thereof, according to any one of ments 2 is selected from Embodiment 37.
• Embodiment 38. A compound of Formula A2, or a salt thereof, according to any one of 7, 30, and 34 to 36 wherein R 2 is ⁇ "" OH 0 r
• Embodiment 39 A compound of Formula A2, or a salt thereof, according to any one of embodiments 1 to 27, 31 , and 34 to 36 wherein R 2 is * ⁇ OH .
• Embodiment 40 A compound of Formula A2, or a salt thereof, according to any one of 1 , 34 and 35 wherein R 2 is selected from
• Embodiment 41 A compound of Formula A2, or a salt thereof, according to any one of embodiments 1 to 26, 31 , 33 to 35 and 40, wherein R 2 is selected from * V 0 r
• Embodiment 42 A compound of Formula A2, or a salt thereof, according to any one of embodime 26, 31 , 33 to 35 and 40, wherein R 2 is or Embodiment 43.
• Embodiment 45 A compound of Formula A2, or a salt thereof, according to any one of embodiments 1 to 23, 25 to 30, 35, and 44 wherein R 2 is n-propyl.
• Embodiment 46 A compound of Formula A2, or a salt thereof, according to any one of embodiments 1 to 30, 35, and 44, wherein R 2 is isopropyl.
• Embodiment 47 A compound of Formula A2, or a salt thereof, according to any one of embodiments 1 to 30, 35, and 44, wherein R 2 is f-butyl.
• Embodiment 48 A compound of Formula A2, or a salt thereof, according to any one of embodiments 1 to 19, wherein
• X 1 is CH
• R 1 and R 2 taken together with the nitrogen atom to which both are bound to form a 4- to 6- membered heterocycloalkyl that can include, as ring members, 1 to 2 additional heteroatoms independently selected from N, O, and S, wherein the 4- to 6-membered heterocycloalkyl formed by R 1 and R 2 taken together with the nitrogen atom to which both are bound is unsubstituted or substituted by 1 to 3 substituents independently selected from halogen, C 1-6 alkyl, C ⁇ haloalkyl, and R°.
• Embodiment 49 A compound of Formula A2, or a salt thereof, according to any one of embodiments 1 to 19, and 48 wherein
• X 1 is CH
• R 1 and R 2 are taken together with the nitrogen atom to which both are bound to form a 5- or 6-membered heterocycloalkyl that can include, as ring member, 1 to 2 additional heteroatom selected from N, O and S, wherein the 5- or 6-membered heterocycloalkyl formed by R 1 and R 2 taken together with the nitrogen atom to which both are bound is unsubstituted or substituted by 1 to 3 substituents independently selected from hydroxyl, C 1-4 alkyl and C ⁇ haloalkyl.
• Embodiment 50 A compound of Formula A2, or a salt thereof, according to any one of embodiments 1 to 19, 48 and 49 wherein
• X 1 is CH
• R 1 and R 2 are taken together with the nitrogen atom to which both are bound to form a 6- membered heterocycloalkyl that can include, as ring member, an additional heteroatom selected from N and O, wherein the 6-membered heterocycloalkyl formed by R 1 and R 2 taken together with the nitrogen atom to which both are bound is unsubstituted or substituted by 1 to 3 substituents independently selected from hydroxyl, C 1-6 alkyl and C ⁇ haloalkyl.
• Embodiment 51 A compound of Formula A2, or a salt thereof, according to any one of embodiments 1 to 19, and 48 to 50 wherein
• X 1 is CH
• R 1 and R 2 are taken together with the nitrogen atom to which both are bound to form a 6- membered heterocycloalkyl selected from piperidinyl, piperazinyl and morpholinyl, wherein the piperidinyl, piperazinyl or morpholinyl is unsubstituted or substituted by 1 to 3 substituents independently selected from hydroxyl and C 1-6 alkyl.
• Embodiment 52 A compound of Formula A2, or a salt thereof, according to any one of embodiments 1 to 51 , wherein R 3 is selected from hydrogen, chloro and methyl.
• Embodiment 53 A compound of Formula A2, or a salt thereof, according to any one of embodiments 1 to 52, wherein R 3 is hydrogen.
• Embodiment 53A A compound of Formula A2, or a salt thereof, according to any one of embodiments 1 to 52, wherein R 3 is chloro.
• Embodiment 53B A compound of Formula A2, or a salt thereof, according to any one of embodiments 1 to 52, wherein R 3 is methyl.
• Embodiment 54 A compound of the Formula A2, or a salt thereof, according to any one of embodiments 1 to 53, wherein R 5 is selected from hydrogen and chloro.
• Embodiment 55 A compound of Formula A2, or a salt thereof, according to any one of embodiments 1 to 54, wherein R 5 is hydrogen.
• Embodiment 56 A compound of Formula A2, or a salt thereof, according to embodiment 1 , wherein the compound is of Formula A3:
• X 1 is CH or N
• Ring A is , each of which is unsubstituted or
• R 1 is hydrogen or unsubstituted C 1-6 alkyl
• R 6 is selected from hydrogen and C 1-6 alkyl that is unsubstituted or substituted by hydroxyl; or
• Embodiment 57 A compound of Formula A2, or a salt thereof, according to embodiment 56
• ring A is F 3 C or
• Embodiment 58 A compound of Formula A2, or a salt thereof, according to embodiment 56 or embodiment 57, wherein R 2 is
• Embodiment 59 A compound of Formula A2, or a salt thereof, according to embodiment 56 or embodiment 57, wherein R 2 is n-propyl or tert-butyl that is unsubstituted or substituted by trifluromethyl.
• Embodiment 60 A compound of the Formula A2, or a salt thereof, according to
• embodiment 1 selected from: N-(2-cyclopropylpropan-2-yl)-2-(pyridin-4-yl)pyrido[3,4- d]pyrimidin-4-amine; N,N-diethyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine; N-ethyl-N- (propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine; 2-(pyridin-4-yl)-N-(1 ,1 ,1 - trifluoropropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine; N-methyl-N-(propan-2-yl)-2-(pyridin-4- yl)pyrido[3,4-d]pyrimidin-4-amine; N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-
• Embodiment 60A A compound of the Formula A2, or a salt thereof, according to embodiment 1 , selected from: N-methyl-2-(pyridin-4-yl)-N-(1 , 1 ,1 -trifluoropropan-2- yl)pyrido[3,4-d]pyrimidin-4-amine; 2-methyl-1 -(2-methyl-2- ⁇ [2-(pyridin-4-yl)pyrido[3,4- d]pyrimidin-4-yl]amino ⁇ propoxy)propan-2-ol; 2,4-dimethyl-4- ⁇ [2-(pyridin-4-yl)pyrido[3,4- d]pyrimidin-4-yl]amino ⁇ pentan-2-ol; N-tert-butyl-2-(pyrimidin-4-yl)-1 ,7-naphthyridin-4-amine; 2-(pyridin-4-yl)-N-[1 -(trifluoromethyl)cyclobuty
• Embodiment 60B A compound of the Formula A2, or a salt thereof, according to embodiment 1 , selected from: N-(tert-butyl)-2-(pyridin-4-yl)-1 ,7-naphthyridin-4-amine; and N-methyl-2-(pyridin-4-yl)-N-[(2S)-1 , 1 ,1 -trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine.
• Embodiment 60C A compound of the Formula A2, or a salt thereof, according to embodiment 1 , wherein the compound is N-(tert-butyl)-2-(pyridin-4-yl)-1 ,7-naphthyridin-4- amine.
• Embodiment 61 A compound of Formula I, or a salt thereof
• R° is hydroxyl or C ⁇ alkoxy
• R 1 is hydrogen or C 1-6 alkyl
• R 2 is selected from
• R 6 is selected from hydrogen, C 1-6 alkyl that is unsubstituted or substituted by R° or -C(0)R°;
• (xii) 6-membered heterocycloalkyl comprising, as ring members, 1 to 2 heteroatoms independently selected from N, O and S and that is unsubstituted or substituted by 1 to 2 substituents independently selected from hydroxyl, halogen, C 1 -6 alkyl,
• (xv) 9- or 10-membered fused bicyclic heteroaryl comprising, as ring member, 1 to 2 heteroatoms independently selected from N and O;
• R 3 is selected from hydrogen, halogen and C 1 -6 alkyl
• R 5 is selected from hydrogen, halogen and -NH-(3- to 8-membered heteroalkyl), wherein the 3- to 8-membered heteroC 3 . 8 alkyl of the -NH-(3- to 8-membered heteroalkyl) comprises 1 to 2 oxygen atoms as chain members and is unsubstituted or substituted by R°;
• R 1 , R 3 and R 5 are H; then R 2 is not C 4 alkyl that is
• Embodiment 62 A compound of Formula I according to embodiment 61 , or a salt thereof, wherein
• * represents the point of attachment of ring A to the remainder of the molecule and ring A is unsubstituted or substituted by 1 to 2 substituents independently selected from halogen, cyano, C 1-6 alkyl, C ⁇ haloalkyl, -NH 2 and C 3 . 6 cycloalkyl.
• Embodiment 63 A compound of Formula I, or a salt thereof, according to embodiment 61 or embodiment 62, wherein ring A is selected from , V H ⁇ N _ ⁇ 3 ⁇ 4 , which are each unsubstituted or substituted by 1 to 2 substituents independently selected from cyano, halogen, C 1-6 alkyl, C ⁇ haloalkyl, NH 2 , and C . 6 cycloalkyl;
• Embodiment 64 A compound of Formula I, or a salt thereof, according to any one of embodiments 61 to 63, wherein ring A is selected from and N- 0 that are each unsubstituted or substituted by a substituent selected from halogen, cyano,
• Embodiment 65 A compound of Formula I, or a salt thereof, according to any one of embodiments 61 to 63 wherein rin A is selected from
• Embodiment 66 A compound of Formula I, or a salt thereof, according to any one of embodiments 61 to 65 wherein rin A is selected from
• Embodiment 67 A compound of Formula I, or a salt thereof, according to any one of embodiments 61 to 66 wherein ring A is selected from
• Embodiment 68 A compound of Formula I or a salt thereof, according to any one of embodiments 61 to 67, wherein ring A '
• Embodiment 69 A compound of Formula I or a salt thereof, according to any one of embodiments 61 to 67, wherein ring A is
• Embodiment 70 A compound of Formula I or a salt thereof, according to any one of embodiments 61 to 67, wherein ring A is
• Embodiment 71 A compound of Formula I, or a salt thereof, according to any one of embodiments 61 to 67, wherein ring A is
• Embodiment 72 A compound of Formul thereof, according to any one of embodiments 61 to 67, wherein ring A is
• Embodiment 73 A compound of Formula I, or a salt thereof, according to any one of embodiments 61 to 65, wherein ring A is CI Embodiment 74. A compound of Formula I or a salt thereof, according to any one of
• Embodiment 75 A compound of Formula I or a salt thereof, according to any one of
• Embodiment 76 A compound of Formula I, or a salt thereof, according to any one of
• Embodiment 78 A compound of Formul thereof, according to any one of
• Embodiment 78A A compound of Formula I, or a salt thereof, according to any one of
• Embodiment 79 A compound of Formula I, or a salt thereof, according to any one of embodiments 61 to 63, wherein ring A is selected , SsL
• Embodiment 80 A compound of Formula I, or a salt thereof, according to any one of embodiments 61 to 79, wherein R 1 is selected from hydrogen, methyl and ethyl.
• Embodiment 81 A compound of Formula I, or a salt thereof, according to any one of embodiments 61 to 80, wherein R 1 is methyl.
• Embodiment 82 A compound of Formula I, or a salt thereof, according to any one of embodiments 61 to 80, wherein R 1 is hydrogen.
• Embodiment 83 A compound of Formula I, or a salt thereof, according to any one of embodiments 61 to 82, wherein
• R 2 is selected from
• R 6 is selected from hydrogen, C 1-6 alkyl that is unsubstituted or substituted by R° or -C(0)R°;
• (c) 4-membered heterocycloalkyi comprising, as ring member, a heteroatom selected from N and O and that and that is unsubstituted or substituted by 1 to 2 substituents independently selected from C ⁇ haloalkyl, R°, C ⁇ ealkylamino, -C(0)R°, and C 1-6 alkyl that is unsubstituted or substituted by R° or -C(0)R°.
• Embodiment 84 A compound of Formula I, or a salt thereof, according to any one of embodiments 61 to 82, wherein
• R 2 is selected from
• R 6 is selected from hydrogen, C 1-6 alkyl that is unsubstituted or substituted by R° or -C(0)R°;
• C 3 . 6 cycloalkyl that is unsubstituted or substituted by 1 to 2 substituents independently selected from C ⁇ haloalkyl, R°, C ⁇ ealkylamino, -C(0)R°, and C 1-6 alkyl that is unsubstituted or substituted by R° or -C(0)R°; and
• 4-membered heterocycloalkyi comprising, as ring member, a heteroatom selected from N and O and that and that is unsubstituted or substituted by 1 to 2 substituents independently selected from C ⁇ haloalkyl, R°, C ⁇ ealkylamino, -C(0)R°, and C 1-6 alkyl that is unsubstituted or substituted by R° or -C(0)R°.
• Embodiment 85 A compound of Formula I, or a salt thereof, according to any one of embodiments 61 to 83, wherein
• R 2 is selected from
• R 6 is selected from hydrogen, C 1-6 alkyl that is unsubstituted or substituted by hydroxyl or -C(0)H;
• Embodiment 86 A compound of Formula I, or a salt thereof, according to any one of embodiments 61 to 85, wherein
• R 2 is selected from (a) C 1-8 alkyl that is unsubstituted or substituted by 1 to 2 substituents independently selected from
• R 6 is selected from hydrogen, and C 1-6 alkyl that is unsubstituted or substituted by hydroxyl;
• Embodiment 87 A compound of Formula I, or a salt thereof, according to any one of embodiments 61 to 86, wherein R 2 is C 1-6 alkyl that is unsubstituted or substituted by 1 to 2 substituent independently selected from C ⁇ haloalkyl and -OR 6 , wherein R 6 is selected from hydrogen, and C 1-6 alkyl that is unsubstituted or substituted by hydroxyl.
• Embodiment 88 A compound of Formula I, or a salt thereof, according to any one of embodiments 61 to 87, wherein R 2 is C 1-6 alkyl that is unsubstituted or substituted by hydroxyl.
• Embodiment 89 A compound of Formula I, or a salt thereof, according to any one of embodiments 61 to 87, wherein R 2 is C 1-6 alkyl that is unsubstituted or substituted by C- ⁇ 6 haloalkyl.
• Embodiment 90 A compound of Formula I, or a salt thereof, according to any one of embodiments 61 to 87, wherein R 2 is C 1-6 alkyl that is unsubstituted or substituted by -O- 6 alkyl-OH.
• Embodiment 91 A compound of Formula I, or a salt thereof, according to any one of embodiments 61 to 86, wherein R 2 is C 3 . 6 cycloalkyl that is unsubstituted or substituted by a substituent selected from C ⁇ haloalkyl, Ci.ealkylamino, R°, and C 1-6 alkyl that is unsubstituted or substituted by hydroxyl.
• Embodiment 92 A compound of Formula I, or a salt thereof, according to any one of embodiments 61 to 86 and 91 , wherein R 2 is unsubstituted C 3 . 6 cycloalkyl.
• Embodiment 93 A compound of the Formula I, or a salt thereof, according to any one of embodiments 61 to 86 and 91 , wherein R 2 is C 3 . 6 cycloalkyl that is substituted by C 1-6 alkyl or C ⁇ haloalkyl.
• Embodiment 94 A compound of the Formula I, or a salt thereof, according to any one of embodiments 61 to 85, wherein R 2 is selected from n-propyl, isopropyl, s-butyl, f-butyl, 2-
• Embodiment 94A A compound of the Formula I, or a salt thereof, any one of according to embodiments 61 to 85, wherein R 2 is selected from
• Embodiment 95 A compound of Formula I, or a salt thereof, according to any one of embodiments 61 to 83 and 85 to 87, wherein R 2 is selected from
• Embodiment 96 A compound of Formula I, or a salt thereof, according to any one of
• Embodiment 97 A compound of Formula I, or a salt thereof, according to any one of embodiments 61 to 87, 89, and 94 to 96, wherein R 2 iisc *' c r
• Embodiment 98 A compound of Formula I, or a salt thereof, according to any one of embodiments 61 to 87, 90, and 94 to 96 wherein R 2 is " ⁇ 0 ⁇ ⁇ .
• Embodiment 99 A compound of Formula I, or a salt thereof, according to any one of
• Embodiment 100 A compound of Formula I, or a salt thereof, according to any one of e F Embodiment 101 .
• Embodiment 103 A compound of Formula I, or a salt thereof, according to any one of embodiments 61 to 86, 91 to 95, 100 and 102, wherein R 2 is
• Embodiment 104 A compound of Formula I, or a salt thereof, according to any one of embodiments 61 to 83, 85 to 90, and 95, wherein R 2 is selected from n-propyl, isopropyl and f-butyl.
• Embodiment 105 A compound of Formula I, or a salt thereof, according to any one of embodiments 61 to 83, 85 to 90, 95, and 104, wherein R 2 is n-propyl.
• Embodiment 106 A compound of the Formula I, or a salt thereof, according to any one of embodiments 61 to 90, 95, and 104, wherein R 2 is isopropyl.
• Embodiment 107 A compound of Formula I, or a salt thereof, according to any one of embodiments 61 to 90, 95, and 104, wherein R 2 is f-butyl.
• Embodiment 108. A compound of Formula I, or a salt thereof, according to any one of embodiments 61 to 107, wherein R 3 is selected from hydrogen, chloro and methyl.
• Embodiment 109 A compound of Formula I, or a salt thereof, according to any one of embodiments 61 to 108, wherein R 3 is hydrogen.
• Embodiment 109A A compound of Formula I, or a salt thereof, according to any one of embodiments 61 to 108, wherein R 3 is chloro.
• Embodiment 109B A compound of Formula I, or a salt thereof, according to any one of embodiments 61 to 108, wherein R 3 is methyl.
• Embodiment 1 10. A compound of the Formula I, or a salt thereof, according to any one of embodiments 61 to 109, wherein R 5 is selected from hydrogen, and chloro. Embodiment 1 1 1 . A compound of Formula I, or a salt thereof, according to any one of embodiments 61 to 1 10, wherein R 5 is hydrogen.
• Embodiment 1 A compound of Formula I, or a salt thereof, according to embodiment 61 , wherein the compound is of Formula V:
• Ring A is ich is unsubstituted or
• R 1 is hydrogen or unsubstituted C 1-6 alkyl
• R 6 is selected from hydrogen and C 1-6 alkyl that is unsubstituted or substituted by hydroxyl; or
• Embodiment 1 13 A compound of Formula I, or a salt thereof, according to embodiment 1 12, wherein ring A is selected from
• Embodiment 1 A compound of Formula I, or a salt thereof, according to embodiment
• Embodiment 1 A compound of Formula I, or a salt thereof, according to embodiment 1 12
• Embodiment 1 16 A compound of Formula I, or a salt thereof, according to any one of
• Embodiment 1 16A A compound of Formula I, or a salt thereof, according to any one of
• R 2 is selected from
• Embodiment 1 A compound of Formula I, or a salt thereof, according to any one of
• Embodiment 120 A compound of the Formula I , or a salt thereof, according to embodiment 61 , selected from: N-(2-cyclopropylpropan-2-yl)-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4- amine; N,N-diethyl-2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine; N-ethyl-N-(propan-2-yl)-2- (pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-amine; 2-(pyridin-4-yl)-N-(1 ,1 , 1-trifluoropropan-2- yl)pyrido[3,4-d]pyrimidin-4-amine; N-methyl-N-(propan-2-yl)-2-(pyridin-4-yl)pyrido[3,4- d]pyrimidin-4-amine; N-(propan-2
• Embodiment 120A A compound of the Formula I , or a salt thereof, according to embodiment 61 , selected from: N-methyl-2-(pyridin-4-yl)-N-(1 , 1 , 1-trifluoropropan-2- yl)pyrido[3,4-d]pyrimidin-4-amine; 2-methyl-1 -(2-methyl-2- ⁇ [2-(pyridin-4-yl)pyrido[3,4- d]pyrimidin-4-yl]amino ⁇ propoxy)propan-2-ol; 2,4-dimethyl-4- ⁇ [2-(pyridin-4-yl)pyrido[3,4- d]pyrimidin-4-yl]amino ⁇ pentan-2-ol; 2-(pyridin-4-yl)-N-[1 - (trifluoromethyl)cyclobutyl]pyrido[3,4-d]pyrimidin-4-amine; N-propyl-2-(pyridin-4- yl)
• Embodiment 120B A compound of the Formula I , or a salt thereof, according to embodiment 61 , wherein the compound is N-methyl-2-(pyridin-4-yl)-N-[(2S)-1 ,1 ,1 - trifluoropropan-2-yl]pyrido[3,4-d]pyrimidin-4-amine.
• Embodiment 121 A compound of Formula II, or a salt thereof,
• R° is hydroxyl or C ⁇ alkoxy
• R 1 is hydrogen or C 1-6 alkyl
• R 2 is selected from
• R 6 is selected from hydrogen, C 1-6 alkyl that is unsubstituted or substituted by R° or -C(0)R°;
• (xii) 6-membered heterocycloalkyi comprising, as ring members, 1 to 2 heteroatoms independently selected from N, O and S and that is unsubstituted or substituted by 1 to 2 substituents independently selected from hydroxyl, halogen, C 1 -6 alkyl, C ! -eialkylamino, and
• (xv) 9- or 10-membered fused bicyclic heteroaryl comprising, as ring member, 1 to 2 heteroatoms independently selected from N and O;
• R 1 and R 2 can be taken together with the nitrogen atom to which both are bound to form a 4- to 6-membered heterocycloalkyi that can include, as ring members, 1 to 2 additional heteroatoms independently selected from N , O, and S, wherein the 4- to 6-membered heterocycloalkyi formed by R 1 and R 2 taken together with the nitrogen atom to which both are bound is unsubstituted or substituted by 1 to 3 substituents independently selected from halogen, C 1 -6 alkyl, C ⁇ haloalkyl, and R°;
• R 3 is selected from hydrogen, halogen and C 1 -6 alkyl
• R 5 is selected from hydrogen, halogen and -NH-(3- to 8-membered heteroalkyl), wherein the 3- to 8-membered heteroC 3 .
• 8 alkyl of the -NH-(3- to 8-membered heteroalkyl) comprises 1 to 2 oxygen atoms as chain members and is unsubstituted or substituted by R°.
• Embodiment 122 A compound of formula II according to embodiment 121 , or a salt thereof, wherein
• Embodiment 123 A compound of Formula II, or a salt thereof, according to embodiment 121 or embodiment 122, wherein ring A is selected
• Embodiment 124 A compound of Formula II, or a salt thereof, according to any one of embodiments 121 to 123 wherein rin A is selected from which are each unsubstituted or substituted by a substituent selected from halogen, cyano, C 1-6 alkyl, and -NH 2 ;
• Embodiment 125 A com ound of Formula II, or a salt thereof, according to any one of
• Embodiment 126 A compound of Formula II, or a salt thereof, according to any one of embodiments 121 to 125 wherein ring A is selected from
• Embodiment 127 A compound of Formula II, or a salt thereof according to any one of
• Embodiment 129 A compound of Formula II or a salt thereof, according to any one of embodiments 121 to 127, wherein ring A '
• Embodiment 130 A compound of Formula II or a salt thereof, according to any one of embodiments 121 to 127, wherein ring A is
• Embodiment 131 A compound of Formula thereof, according to any one of embodiments 121 to 127, wherein ring A is
• Embodiment 132 A compound of Formula thereof, according to any one of embodiments 121 to 127, wherein ring A '
• Embodiment 133 A compound of Formula II, or a salt thereof, according to any one of
• Embodiment 134 A compound of Formula II or a salt thereof, according to any one of embodiments 121 to 125, wherein ring A ' Embodiment 135.
• Embodiment 136 A compound of Formula II , or a salt thereof, according to any one of
• Embodiment 137 A compound of Formula II , or a salt thereof, according to any one of embodiments 121 to 124, wherein ring A is Embodiment 138.
• Embodiment 139 A compound of Formula II , or a salt thereof, according to any one of embodiments 121 to 123, wherein is selected from _ " N -H t and , which are each unsubstituted or substituted by
• Embodiment 140 A compound of Formula II , or a salt thereof, according to any one of embodiments 121 to 139, wherein R 1 is selected from hydrogen, methyl and ethyl.
• Embodiment 141 A compound of Formula II, or a salt thereof, according to any one of embodiments 121 to 140, wherein R 1 is methyl.
• Embodiment 142 A compound of Formula II, or a salt thereof, according to any one of embodiments 121 to 140, wherein R 1 is hydrogen.
• Embodiment 143 A compound of Formula II, or a salt thereof, according to any one of embodiments 121 to 142, wherein
• R 2 is selected from
• R 6 is selected from hydrogen, C 1-6 alkyl that is unsubstituted or substituted by R° or -C(0)R°;
• R 2 is selected from
• R 6 is selected from hydrogen, C 1-6 alkyl that is unsubstituted or substituted by R° or -C(0)R°;
• (c) 4-membered heterocycloalkyi comprising, as ring member, a heteroatom selected from N and O and that is unsubstituted or substituted by 1 to 2 substituents independently selected from C 1-6 haloalkyl, R°, Ci.ealkylamino, -C(0)R°, and C 1-6 alkyl that is unsubstituted or substituted by R° or -C(0)R°.
• Embodiment 145 A compound of Formula II, or a salt thereof, according to any one of embodiments 121 to 143, wherein
• R 2 is selected from (a) C 1-8 alkyl that is unsubstituted or substituted by 1 to 2 substituents independently selected from
• R 6 is selected from hydrogen, C 1-6 alkyl that is unsubstituted or substituted by hydroxyl or -C(0)H;
• R 7a is hydrogen or C 1-6 alkyl
• R 7b is selected from hydrogen, -C(0)-C 1-6 alkoxy, and C 1-6 alkyl that is unsubstituted or substituted by
• Embodiment 146 A compound of Formula II, or a salt thereof, according to any one of embodiments 121 to 145, wherein
• R 2 is selected from
• R 6 is selected from hydrogen, and C 1-6 alkyl that is unsubstituted or substituted by hydroxyl;
• Embodiment 147 A compound of Formula II, or a salt thereof, according to any one of embodiments 121 to 146, wherein R 2 is C 1-6 alkyl that is unsubstituted or substituted by 1 to 2 substituent independently selected from C ⁇ haloalkyl and -OR 6 , wherein R 6 is selected from hydrogen, and C 1-6 alkyl that is unsubstituted or substituted by hydroxyl.
• Embodiment 148. A compound of Formula II, or a salt thereof, according to any one of embodiments 121 to 147, wherein R 2 is C 1-6 alkyl that is unsubstituted or substituted by hydroxy I.
• Embodiment 149 A compound of Formula II, or a salt thereof, according to any one of embodiments 121 to 147, wherein R 2 is C 1-6 alkyl that is unsubstituted or substituted by C- ⁇ 6 haloalkyl.
• Embodiment 150 A compound of Formula II, or a salt thereof, according to any one of embodiments 121 to 147, wherein R 2 is C 1-6 alkyl that is unsubstituted or substituted by -O- d. 6 alkyl-OH.
• Embodiment 151 A compound of the Formula II, or a salt thereof, according to any one of embodiments 121 to 146, wherein R 2 is C 3 . 6 cycloalkyl that is unsubstituted or substituted by a substituent selected from C ⁇ haloalkyl, Ci.ealkylamino, R°, and C 1-6 alkyl that is unsubstituted or substituted by hydroxyl.
• Embodiment 152 A compound of Formula II, or a salt thereof, according to any one of embodiments 121 to 146 and 151 , wherein R 2 is unsubstituted C 3 . 6 cycloalkyl.
• Embodiment 153 A compound of Formula II, or a salt thereof, according to any one of embodiments 121 to 146 and 151 , wherein R 2 is C 3 . 6 cycloalkyl that is substituted by 6 alkyl or C ⁇ haloalkyl.
• Embodiment 154 A compound of Formula II, or a salt thereof, according to any one of embodiments 121 to 145, wherein R 2 is selected from n-propyl, isopropyl, s-butyl, f-butyl, 2- methyl-but-2-yl, 2,4,4-trimethylpentan-2-yl,
• Embodiment 155 A compound of Formula II , or a salt thereof, according to any one of embodiments 121 to 143 and 145 to 147, wherein R 2 is selected from n-propyl, isopropyl, t-
• Embodiment 156 A compound of Formula II , or a salt thereof, according to any one of
• Embodiment 157 A compound of Formula II , or a salt thereof, according to any one of embodiments 121 to 147, 149, and 154 to 156, wherein R 2 is * X C F 3 .
• Embodiment 158 A compound of Formula II , or a salt thereof, according to any one of 0r
• Embodiment 159 A compound of Formula II , or a salt thereof, according to any one of embodiments 121 to 147, 151 , and 154 to 156, wherein R 2 is *" ⁇ ⁇ ) ⁇ .
• Embodiment 160 A compound of Formula II, or a salt thereof, according to any one of
• Embodiment 161 A compound of Formula II, or a salt thereof, according to any one of
• Embodiment 162 A compound of Formula II, or a salt thereof, according to any one of embodiments 121 to 136, 151 , 153 to 155 and 160, wherein
• Embodiment 163 A compound of Formula II, or a salt thereof, accord to any one of embodiments 121 to 136, 151 , 152, 154, 155, and 160, wherein R 2 is Embodiment 164.
• Embodiment 165 A compound of Formula II, or a salt thereof, according to any one of embodiments 121 to 143, 145 to 150, 155, and 164, wherein R 2 is n-propyl.
• Embodiment 166 A compound of Formula II, or a salt thereof, according to any one of embodiments 121 to 150, 155, and 164, wherein R 2 is isopropyl.
• Embodiment 167 A compound of Formula II, or a salt thereof, according to any one of embodiments 121 to 150, 155, and 164, wherein R 2 is f-butyl.
• Embodiment 168. A compound of Formula II, or a salt thereof, according to any one of embodiments 121 to 139, wherein R 1 and R 2 taken together with the nitrogen atom to which both are bound to form a 4- to 6-membered heterocycloalkyi that can include, as ring members, 1 to 2 additional heteroatoms independently selected from N, O, and S, wherein the 4- to 6-membered heterocycloalkyi formed by R 1 and R 2 taken together with the nitrogen atom to which both are bound is unsubstituted or substituted by 1 to 3 substituents independently selected from halogen, C 1-6 alkyl, C ⁇ haloalkyl, and R°.
• Embodiment 169 A compound of Formula II, or a salt thereof, according to any one of embodiments 121 to 139, and 168, wherein R 1 and R 2 are taken together with the nitrogen atom to which both are bound to form a 5- or 6-membered heterocycloalkyi that can include, as ring member, 1 to 2 additional heteroatom selected from N, O and S, wherein the 5- or 6- membered heterocycloalkyi formed by R 1 and R 2 taken together with the nitrogen atom to which both are bound is unsubstituted or substituted by 1 to 3 substituents independently selected from hydroxyl, C 1-4 alkyl and C ⁇ haloalkyl.
• Embodiment 170 A compound of Formula II, or a salt thereof, according to any one of embodiments 121 to 139, 168, and 169, wherein R 1 and R 2 are taken together with the nitrogen atom to which both are bound to form a 6-membered heterocycloalkyi that can include, as ring member, an additional heteroatom selected from N and O, wherein the 6- membered heterocycloalkyi formed by R 1 and R 2 taken together with the nitrogen atom to which both are bound is unsubstituted or substituted by 1 to 3 substituents independently selected from hydroxyl, C 1-6 alkyl and C ⁇ haloalkyl.
• Embodiment 171 Embodiment 171 .
• Embodiment 172 A compound of Formula II, or a salt thereof, according to any one of embodiments 121 to 171 , wherein R 3 is selected from hydrogen, chloro and methyl.
• Embodiment 173. A compound of Formula II, or a salt thereof, according to any one of embodiments 121 to 172, wherein R 3 is hydrogen.
• Embodiment 174 A compound of the Formula II, or a salt thereof, according to any one of embodiments 121 to 173, wherein R 5 is selected from hydrogen, and chloro.
• Embodiment 175. A compound of Formula II, or a salt thereof, according to any one of embodiments 121 to 174, wherein R 5 is hydrogen.
• Embodiment 176. A compound of the Formula II, or a salt thereof, according to embodiment 121 , wherein the compound is of Formula VI:
• R 1 is hydrogen or unsubstituted C 1-6 alkyl
• R 6 is selected from hydrogen, C 1-6 alkyl that is unsubstituted or substituted by R°; or
• R 1 and R 2 may be taken together with the nitrogen to which they are bound to form a 6- membered heterocycloalkyl, which is unsubstituted or substituted by 1 to 3 substituted selected from C 1-6 alkyl and hydroxyl.
• Embodiment 177 A com ound of the Formula II, or a salt thereof, according to embodiment
• Embodiment 179 A compound of the Formula II, or a salt thereof, according to any one of embodiments 176 to 178, wherein R 2 is tert-butyl.
• Embodiment 180 A compound of the Formula II, or a salt thereof, according to
• embodiment 121 selected from: N-(4-methoxy-2-methylbutan-2-yl)-2-(pyridin-4-yl)-1 ,7- naphthyridin-4-amine; N-[2-methyl-1 -(propan-2-yloxy)propan-2-yl]-2-(pyridin-4-yl)-1 ,7- naphthyridin-4-amine; N-[(2S)-butan-2-yl]-2-(pyridin-4-yl)-1 ,7-naphthyridin-4-amine; N-[(2R)- butan-2-yl]-2-(pyridin-4-yl)-1 ,7-naphthyridin-4-amine; N-(1 -methoxy-2-methylpropan-2-yl)-2- (pyridin-4-yl)-1 ,7-naphthyridin-4-amine; N-methyl-N-(propan-2-yl)-2-(pyridin-4
• Embodiment 180a A compound of the Formula II, or a salt thereof, according to embodiment 121 , wherein the compound is N-(tert-butyl)-2-(pyridin-4-yl)-1 ,7-naphthyridin-4- amine.
• Embodiment 181 A compound of Formula A1 , or a salt thereof,
• X 1 and X 2 are each independently CH or N;
• R° is hydroxyl or C ⁇ alkoxy
• R 1 is hydrogen or C 1-6 alkyl
• R 2 is selected from
• R 6 is selected from hydrogen, C 1-6 alkyl that is unsubstituted or substituted by R° or -C(0)R°;
• (xii) 6-membered heterocycloalkyi comprising, as ring members, 1 to 2 heteroatoms independently selected from N, O and S and that is unsubstituted or substituted by 1 to 2 substituents independently selected from hydroxyl, halogen, C 1-6 alkyl, C ! -eialkylamino, and
• (xv) 9- or 10-membered fused bicyclic heteroaryl comprising, as ring member, 1 to 2 heteroatoms independently selected from N and O;
• R 1 and R 2 can be taken together with the nitrogen atom to which both are bound to form a 4- to 6-membered heterocycloalkyl that can include, as ring members, 1 to 2 additional heteroatoms independently selected from N , O, and S, wherein the 4- to 6-membered heterocycloalkyl formed by R 1 and R 2 taken together with the nitrogen atom to which both are bound is unsubstituted or substituted by 1 to 3 substituents independently selected from halogen, C 1 -6 alkyl, C ⁇ haloalkyl, and R°;
• R 3 is selected from hydrogen, halogen and C 1 -6 alkyl
• R 5 is selected from hydrogen, halogen and -NH-(3- to 8-membered heteroalkyl), wherein the 3- to 8-membered heteroC 3 .
• 8 alkyl of the -NH-(3- to 8-membered heteroalkyl) comprises 1 to 2 oxygen atoms as chain members and is unsubstituted or substituted by R°.
• Embodiment 182 A compound of Formula A1 or a salt thereof, for use in ocular diseases or disorders according to embodiment 181 , wherein the compound is of the formula selected from Formulae I to IV:
• Embodiment 183 A compound of Formula A1 or a salt thereof, for use in ocular diseases or disorders according to embodiment 181 , wherein the compound is selected from 3-(pyridin- 4-yl)-N-(1 -(trifluoromethyl)cyclopropyl)-2,6-naphthyridin-1 -amine; N-(1 -methylcyclopropyl)-7- (pyridin-4-yl)isoquinolin-5-amine; 2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1 - yl)pyrido[3,4-d]pyrimidine; N-(tert-butyl)-2-(pyridin-4-yl)-1 ,7-naphthyridin-4-amine; and N- methyl-2-(pyridin-4-yl)-N-[(2S)-1 ,1 , 1-trifluoropropan-2-yl]pyrido[3,4
• Embodiment 184 A compound of the Formula A1 or a salt thereof, for use in ocular diseases or disorders according to embodiment 181 , wherein the compound is according to any one of embodiments 1 to 180.
• Embodiment 185 Use of a compound of the Formula A1 , or a salt thereof,
• X 1 and X 2 are each independently CH or N;
• R° is hydroxyl or C ⁇ alkoxy
• R 1 is hydrogen or C 1-6 alkyl
• R 2 is selected from
• R 6 is selected from hydrogen, C 1-6 alkyl that is unsubstituted or substituted by R° or -C(0)R°;
• (xii) 6-membered heterocycloalkyi comprising, as ring members, 1 to 2 heteroatoms independently selected from N, O and S and that is unsubstituted or substituted by 1 to 2 substituents independently selected from hydroxyl, halogen, C 1 -6 alkyl,
• (xv) 9- or 10-membered fused bicyclic heteroaryl comprising, as ring member, 1 to 2 heteroatoms independently selected from N and O;
• R 1 and R 2 can be taken together with the nitrogen atom to which both are bound to form a 4- to 6-membered heterocycloalkyi that can include, as ring members, 1 to 2 additional heteroatoms independently selected from N , O, and S, wherein the 4- to 6-membered heterocycloalkyi formed by R 1 and R 2 taken together with the nitrogen atom to which both are bound is unsubstituted or substituted by 1 to 3 substituents independently selected from halogen, C 1 -6 alkyl, C ⁇ haloalkyl, and R°;
• R 3 is selected from hydrogen, halogen and C 1 -6 alkyl
• R 5 is selected from hydrogen, halogen and -NH-(3- to 8-membered heteroalkyl), wherein the 3- to 8-membered heteroC 3 .
• 8 alkyl of the -NH-(3- to 8-membered heteroalkyl) comprises 1 to 2 oxygen atoms as chain members and is unsubstituted or substituted by R°.
• Embodiment 186. Use of a compound of the Formula A1 , or a salt thereof,
• X 1 and X 2 are each independently CH or N ;
• ring A is unsubstituted or substituted by 1 to 2 substituents independently selected from halogen, cyano, C 1 -6 alkyl, C 1 -6 haloalkyl, -NH 2 , C ⁇ ealkylamino, di-(C 1 -6 alkyl)amino, C 3 . 6 cycloalkyl, and phenylsulfonyl;
• R° is hydroxyl or C 1 -6 alkoxy
• R 1 is hydrogen or C h alky!
• R 2 is selected from
• R 6 is selected from hydrogen, C 1 -6 alkyl that is unsubstituted or substituted by R° or -C(0)R°;
• (xii) 6-membered heterocycloalkyi comprising, as ring members, 1 to 2 heteroatoms independently selected from N, O and S and that is unsubstituted or substituted by 1 to 2 substituents independently selected from hydroxyl, halogen, C 1 -6 alkyl, C ! -eialkylamino, and
• (xv) 9- or 10-membered fused bicyclic heteroaryl comprising, as ring member, 1 to 2 heteroatoms independently selected from N and O;
• R 1 and R 2 can be taken together with the nitrogen atom to which both are bound to form a 4- to 6-membered heterocycloalkyi that can include, as ring members, 1 to 2 additional heteroatoms independently selected from N , O, and S, wherein the 4- to 6-membered heterocycloalkyl formed by R 1 and R 2 taken together with the nitrogen atom to which both are bound is unsubstituted or substituted by 1 to 3 substituents independently selected from halogen, C 1-6 alkyl, C ⁇ haloalkyl, and R°;
• R 3 is selected from hydrogen, halogen and C 1-6 alkyl
• R 5 is selected from hydrogen, halogen and -NH-(3- to 8-membered heteroalkyl), wherein the 3- to 8-membered heteroC 3 .
• 8 alkyl of the -NH-(3- to 8-membered heteroalkyl) comprises 1 to 2 oxygen atoms as chain members and is unsubstituted or substituted by R°.
• Embodiment 187 Use of a compound of the Formula A1 or a salt thereof, according to embodiment 185 or 186, wherein the compound is of the formula selected from Formulae I to IV:
• Embodiment 188 Use of a compound of Formula A1 or a salt thereof, according to embodiment 185 or 186, wherein the compound is selected from 3-(pyridin-4-yl)-N-(1- (trifluoromethyl)cyclopropyl)-2,6-naphthyridin-1 -amine; N-(1 -methylcyclopropyl)-7-(pyridin-4- yl)isoquinolin-5-amine; and 2-(pyridin-4-yl)-4-(3-(trifluoromethyl)piperazin-1-yl)pyrido[3,4- d]pyrimidine.
• Embodiment 188A Use of a compound of the Formula Al, or a salt thereof, according to embodiment 185 or 186, wherein the compound is selected from: N-methyl-2-(pyridin-4-yl)- N-(1 ,1 ,1 -trifluoropropan-2-yl)pyrido[3,4-d]pyrimidin-4-amine; 2-methyl-1 -(2-methyl-2- ⁇ [2- (pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino ⁇ propoxy)propan-2-ol; 2,4-dimethyl-4- ⁇ [2- (pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]arTiino ⁇ pentan-2-ol; N-tert-butyl-2-(pyrimidin-4-yl)-1 ,7- naphthyridin-4-amine; 2-(pyridin-4-yl)-N-[1 -(tri
• Embodiment 188B Use of a compound of the Formula Al, or a salt thereof, according to embodiment 185 or 186, wherein the compound is selected from: N-(tert-butyl)-2-(pyridin-4- yl)-1 ,7-naphthyridin-4-amine; and N-methyl-2-(pyridin-4-yl)-N-[(2S)-1 , 1 ,1 -trifluoropropan-2- yl]pyrido[3,4-d]pyrimidin-4-amine.
• Embodiment 188C Use of a compound of the Formula Al, or a salt thereof, according to embodiment 185 or 186, wherein the compound is selected from: N-(tert-butyl)-2-(pyridin-4- yl)-1 ,7-naphthyridin-4-amine; and N-methyl-2-(pyridin-4-yl)-N-[(2S)-1 , 1 ,1 -tri
• Embodiment 189 Use of a compound of Formula A1 or a salt thereof, according to embodiment 185 or 186, wherein the compound is according to any one of embodiments 1 to 180.
• Embodiment 190 A method of treatment of an ocular disease or disorder comprising administering to a subject in need thereof a cell population, wherein the cell population has been grown in the presence of a compound of Formula A1 , or a salt thereof,
• X 1 and X 2 are each independently CH or N;
• R° is hydroxyl or C ⁇ alkoxy
• R 1 is hydrogen or C 1-6 alkyl
• R 2 is selected from
• R 6 is selected from hydrogen, C 1 -6 alkyl that is unsubstituted or
• heteroatoms independently selected from N and O;
• heteroatoms independently selected from N and O;
• R 1 and R 2 can be taken together with the nitrogen atom to which both are bound to form a 4- to 6-membered heterocycloalkyl that can include, as ring members, 1 to 2 additional heteroatoms independently selected from N, O, and S, wherein the 4- to 6- membered heterocycloalkyl formed by R 1 and R 2 taken together with the nitrogen atom to which both are bound is unsubstituted or substituted by 1 to 3 substituents independently selected from halogen, C 1-6 alkyl, C ⁇ haloalkyl, and R°;
• R 3 is selected from hydrogen, halogen and C 1-6 alkyl
• -NH-(3- to 8-membered heteroalkyl) is selected from hydrogen, halogen and -NH-(3- to 8-membered heteroalkyl), wherein the 3- to 8-membered heteroC 3 .
• 8 alkyl of the -NH-(3- to 8-membered heteroalkyl) comprises 1 to 2 oxygen atoms as chain members and is unsubstituted or substituted by R°.
• Embodiment 190A A method of treatment of an ocular disease or disorder comprising administering to a subject in need thereof a limbal stem cell population, wherein said population has been grown in the presence of a compound of Formula A1 , or a salt thereof,
• X 1 and X 2 are each independently CH or N;
• R° is hydroxyl or C ⁇ alkoxy
• R 1 is hydrogen or C 1-6 alkyl
• R 2 is selected from
• R 6 is selected from hydrogen, C 1-6 alkyl that is unsubstituted or substituted by R° or -C(0)R°;
• phenyl that is unsubstituted or substituted by 1 to 2 substituents independently selected from halogen, C 1-6 alkyl and R°; (d) C 3 . 6 cycloalkyl that is unsubstituted or substituted by 1 to 2 substituents independently selected from C ⁇ haloalkyl, R°, C ⁇ ealkylamino, di- ⁇ ! -ealky amino, -C(0)R°, and 6alkyl that is unsubstituted or substituted by R° or -C(0)R°; and
• R 1 and R 2 can be taken together with the nitrogen atom to which both are bound to form a 4- to 6-membered heterocycloalkyl that can include, as ring members, 1 to 2 additional heteroatoms independently selected from N, O, and S, wherein the 4- to 6- membered heterocycloalkyl formed by R 1 and R 2 taken together with the nitrogen atom to which both are bound is unsubstituted or substituted by 1 to 3 substituents independently selected from halogen, C 1 -6 alkyl, C ⁇ haloalkyl, and R°;
• R 3 is selected from hydrogen, halogen and C 1 -6 alkyl
• R 5 is selected from hydrogen, halogen and -NH-(3- to 8-membered heteroalkyl), wherein the 3- to 8-membered heteroC 3 .
• 8 alkyl of the -NH-(3- to 8-membered heteroalkyl) comprises 1 to 2 oxygen atoms as chain members and is unsubstituted or substituted by R°.
• Embodiment 191 A method of treatment of an ocular disease or disorder comprising administering to a subject in need thereof a corneal endothelial cell population, wherein the population has been grown in the resence of a compound of Formula A1 , or a salt thereof,
• X 1 and X 2 are each independently CH or N ;
• R° is hydroxyl or C ⁇ alkoxy
• R 1 is hydrogen or C 1-6 alkyl
• R 2 is selected from
• heteroatoms independently selected from N and O;
• heteroatoms independently selected from N and O;

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