Methods of activating FOXN1

Abstract

The present disclosure provides methods of promoting thymus regeneration, immune reconstitution, activating FOXN1, promoting skin wound healing, and / or augmenting hair growth in a subject in need thereof.

Description

METHODS OF ACTIVATING FOXN1 BACKGROUND Field

[0001] The present disclosure provides methods of promoting thymus regeneration, immune reconstitution, activating FOXN1, promoting skin wound healing, and / or augmenting hair growth in a subject in need thereof. Background

[0002] The thymus is a specialized primary lymphoid organ of the immune system that facilitates maturation of T cells, which play a central role in the adaptive immune response. Although the thymus constantly undergoes acute stresses and regeneration cycles, it undergoes age-related involution over time, and is very sensitive to insults, e.g., infections, stress, and cytoreductive therapies. Thymus dysfunction resulting from any of these factors can lead to immunodeficiency and a general increase in mortality.

[0003] FOXN1 is a transcription factor that regulates thymic epithelial cell development and thymus organogenesis. FOXN1 deficiency is known to disrupt thymic architecture and impair thymic T cell development, while administration of recombinant FOXN1 protein to mice increases thymic epithelial cell development. Thus, compounds that promote FOXN1 expression, i.e., FOXN1 activators, may be useful in promoting thymus regeneration.

[0004] Therefore, there is a need in the art for methods of promoting thymus regeneration, immune reconstitution, FOXN1 activating, promoting skin wound healing, and / or augmenting hair growth. BRIEF SUMMARY

[0005] The present disclosure provides a method for promoting thymus regeneration in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a Compound of the Disclosure.

[0006] In some aspects, the subject is a human.

[0007] In some aspects, the thymus of the subject is damaged by physiological or external factors including stress, infection, glucocorticoids, cytoreductive therapies, aging, or a combination thereof.

[0008] In some aspects, the subject is in need of thymus regeneration due to hematopoietic stem cell transplantation, immune deficiency due to HIV, HIV therapy, autoimmune disease, inborn errors of thymic development, administration of a vaccine, age-related immune senescence, administration of exogenous glucocorticoids, bacterial or viral infections, cancer-related treatments such as radiation, immunotherapy, and cytoreductive treatments, zinc deficiency, thymus involution, or a combination thereof.

[0009] The present disclosure also provides a method of promoting immune reconstitution in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a Compound of the Disclosure.

[0010] In some aspects, the subject is a human.

[0011] In some aspects, the thymus of the subject is damaged by physiological or environmental factors including stress, infection, glucocorticoids, cytoreductive therapies, aging, or a combination thereof.

[0012] In some aspects, the subject is in need of immune reconstitution due to hematopoietic stem cell transplantation, immune deficiency due to HIV, HIV therapy, autoimmune disease, inborn errors of thymic development, administration of a vaccine, age-related immune senescence, administration of exogenous glucocorticoids, bacterial or viral infections, cancer-related treatments such as radiation, immunotherapy, and cytoreductive treatments, zinc deficiency, thymus involution, or a combination thereof.

[0013] The present disclosure also provides a method of activating FOXN1 in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a Compound of the Disclosure.

[0014] In some aspects, the thymus of the subject is damaged by physiological or environmental factors including stress, infection, glucocorticoids, cytoreductive therapies, aging, or a combination thereof.

[0015] In some aspects, the subject is in need of FOXN1 activation due to hematopoietic stem cell transplantation, immune deficiency due to HIV, HIV therapy, autoimmune disease, inborn errors of thymic development, administration of a vaccine, age-related immune senescence, administration of exogenous glucocorticoids, bacterial or viral infections, cancer-related treatments such as radiation, immunotherapy, and cytoreductivetreatments, zinc deficiency, thymus involution, diabetes-related ulcers, foot ulcers, chronic wounds, alopecia, or a combination thereof.

[0016] In some aspects, the subject is in need of FOXN1 activation to treat or prevent immune deficiency due to HIV, autoimmune disease, age-related immune senescence, bacterial or viral infections, zinc deficiency, thymus involution, diabetes-related ulcers, foot ulcers, chronic wounds, alopecia, or a combination thereof.

[0017] In some aspects, the subject is in need of FOXN1 activation to promote skin wound healing.

[0018] In some aspects, the subject has a diabetes-related ulcer, a foot ulcer, a chronic wound, or a combination thereof.

[0019] In some aspects, the subject is in need of FOXN1 activation to augment hair growth.

[0020] In some aspects, the subject has alopecia.

[0021] In some aspects, the subject is a human.

[0022] The present disclosure also provides a method of enhancing immune function or promoting thymic regeneration in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a Compound of the Disclosure, wherein the subject is in need of immune function enhancement or thymic regeneration due to administration of CAR-T cells or other adoptive T-cell therapies, recovery from sepsis or critical illness, idiopathic T-cell lymphopenia, immunosuppression following solid organ transplantation, or a combination thereof. In some aspects, the subject is at risk of or recovering from infection due to impaired T-cell function. In some aspects, the subject is an elderly individual, an immunocompromised patient, a recipient of immunosuppressive therapy, or a combination thereof.

[0023] The present disclosure also provides a method of promoting immune tolerance or reducing transplant-related complications in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a Compound of the Disclosure, wherein the subject is receiving a hematopoietic stem cell transplant or solid organ transplant. In some aspects, the method reduces the risk of graft-versus-host disease (GVHD). In some aspects, the method enhances immune reconstitution.

[0024] The present disclosure also provides a method of treating or supporting recovery from a thymic epithelial tumor in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a Compound of the Disclosure, wherein the subject has a thymoma or thymic carcinoma.

[0025] The present disclosure also provides a method of promoting immune surveillance or reducing cancer risk in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a Compound of the Disclosure, wherein the subject has an age-related decline in immune function, a personal or familial history of cancer, or a genetic predisposition to immune dysfunction.

[0026] The present disclosure also provides a method of augmenting hair growth or restoring hair loss in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a Compound of the Disclosure, wherein the hair loss is chemotherapy-induced, radiation-induced, or idiopathic in origin.

[0027] The present disclosure also provides a method of promoting skin repair or regeneration in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a Compound of the Disclosure, wherein the subject has a radiation-induced skin injury, a surgical wound, or age-related skin atrophy.

[0028] The present disclosure also provides a method of promoting dermal regeneration or treating skin aging in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a Compound of the Disclosure.

[0029] The present disclosure also provides a method of preconditioning a non-human donor organism for xenotransplantation of a composite organ comprising thymic tissue, comprising administering to the donor a therapeutically effective amount of a Compound of the Disclosure, wherein the method improves thymic tissue function, promotes immune system development, or enhances compatibility of the graft with the recipient immune system.

[0030] Additional embodiments and advantages of the disclosure will be set forth, in part, in the description that follows, and will become apparent from the description, or can be learned by practice of the disclosure. The embodiments and advantages of the disclosure will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing summary and the following detailed description are exemplary and explanatory only, and are not restrictive of the invention as claimed.DETAILED DESCRIPTION I. Definitions

[0031] As used herein, the terms “treat,” “treating,” “treatment,” and the like refer to eliminating, reducing, or ameliorating a disease or condition, and / or symptoms associated therewith. Although not precluded, treating a disease or condition does not require that the disease, condition, or symptoms associated therewith be completely eliminated. The term “treat” and synonyms contemplate administering a therapeutically effective amount of a Compound of the Disclosure to a subject in need of such treatment. The treatment can be symptomatically orientated, for example, to suppress symptoms. It can be effected over a short period, a medium term, or as a long-term treatment, for example within the context of a maintenance therapy.

[0032] The term “disease” or “condition” denotes disturbances and / or anomalies that as a rule are regarded as being pathological conditions or functions, and that can manifest themselves in the form of particular signs, symptoms, and / or malfunctions.

[0033] The term “therapeutically effective amount” or “effective dose” as used herein refers to an amount of the active ingredient(s) that is(are) sufficient, when administered by a method of the disclosure, to efficaciously deliver the active ingredient(s) for the treatment of the disease or condition of interest to a subject in need thereof.

[0034] The use of the terms “a,” “an,” “the,” and similar referents in the context of describing the disclosure (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated. Recitation of ranges of values herein merely are intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended to better illustrate the disclosure and is not a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0035] The term “about,” as used herein, includes the recited number ± 10%. Thus, “about 10” means 9 to 11.II. Compounds of the Disclosure

[0036] The present disclosure provides small molecules recited in Table 1 below, and the pharmaceutically acceptable salts and solvates, e.g., hydrates, thereof. These compounds, and the salts and solvates thereof, are collectively referred to herein as “Compounds of the Disclosure” or individually as a “Compound of the Disclosure.” Compounds of the Disclosure are FOXN1 activators. FOXN1 activators are useful in promoting thymus regeneration and / or immune reconstitution in a subject in need thereof, and / or in treating diseases or conditions wherein activation of FOXN1 provides a therapeutic benefit to a subject. Table 1III. Therapeutic Methods of the Disclosure

[0037] The present disclosure provides a method of promoting thymus regeneration in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a Compound of the Disclosure or a pharmaceutical composition thereof.

[0038] In some aspects, the Compound of the Disclosure is selected from any of the compounds listed in Table 1.

[0039] In some aspects, the Compound of the Disclosure is selected from the group consisting of:

[0040] In some aspects, the Compound of the Disclosure is selected from the group consisting of:.

[0041] In some aspects, the Compound of the Disclosure is selected from the group consisting of:

[0042] In some aspects, the subject is a human.

[0043] In some aspects, the thymus of the subject is damaged by stress, infection, glucocorticoids, cytoreductive therapies, aging, or a combination thereof.

[0044] In some aspects, the subject is in need of thymus regeneration due to hematopoietic stem cell transplantation, immune deficiency due to HIV, HIV therapy, autoimmune disease, inborn errors of thymic development, administration of a vaccine, age-related immune senescence, administration of exogenous glucocorticoids, bacterial or viral infections, cancer-related treatments such as radiation, immunotherapy, and cytoreductive treatments, zinc deficiency, thymus involution, or a combination thereof.

[0045] The present disclosure also provides a method of promoting immune reconstitution in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a Compound of the Disclosure or a pharmaceutical composition thereof.

[0046] In some aspects, the Compound of the Disclosure is selected from any of the compounds listed in Table 1.

[0047] In some aspects, the Compound of the Disclosure is selected from the group consisting of:

[0048] In some aspects, the Compound of the Disclosure is selected from the group consisting of:.

[0049] In some aspects, the Compound of the Disclosure is selected from the group consisting of:

[0050] In some aspects, the subject is a human.

[0051] In some aspects, the thymus of the subject is damaged by stress, infection, glucocorticoids, cytoreductive therapies, aging, or a combination thereof.

[0052] In some aspects, the subject is in need of immune reconstitution due to hematopoietic stem cell transplantation, immune deficiency due to HIV, HIV therapy, autoimmune disease, inborn errors of thymic development, administration of a vaccine, age-related immune senescence, administration of exogenous glucocorticoids, bacterial or viral infections, cancer-related treatments such as radiation, immunotherapy, and cytoreductive treatments, zinc deficiency, thymus involution, or a combination thereof.

[0053] The present disclosure provides a method of activating FOXN1 in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a Compound of the Disclosure or a pharmaceutical composition thereof.

[0054] In some aspects, the Compound of the Disclosure is selected from any of the compounds listed in Table 1.

[0055] In some aspects, the Compound of the Disclosure is selected from the group consisting of:

[0056] In some aspects, the Compound of the Disclosure is selected from the group consisting of:.

[0057] In some aspects, the Compound of the Disclosure is selected from the group consisting of:

[0058] In some aspects, the subject is a human.

[0059] In some aspects, the subject is in need of activating FOXN1 because the thymus of the subject is damaged by stress, infection, glucocorticoids, cytoreductive therapies, aging, or a combination thereof.

[0060] In some aspects, the subject is in need of FOXN1 activation due to hematopoietic stem cell transplantation, immune deficiency due to HIV, HIV therapy, autoimmune disease, inborn errors of thymic development, administration of a vaccine, age-related immune senescence, administration of exogenous glucocorticoids, bacterial or viral infections, cancer-related treatments such as radiation, immunotherapy, and cytoreductive treatments, zinc deficiency, thymus involution, diabetes-related ulcers, foot ulcers, chronic wounds, alopecia, or a combination thereof.

[0061] In some aspects, the subject is in need of FOXN1 activation to treat or prevent immune deficiency due to HIV, autoimmune disease, age-related immune senescence, bacterial or viral infections, zinc deficiency, thymus involution, diabetes-related ulcers, foot ulcers, chronic wounds, alopecia, or a combination thereof.

[0062] In some aspects, the subject is in need of FOXN1 activation to promote skin wound healing in the subject.

[0063] In some aspects, the subject has a diabetes-related ulcer, a foot ulcer, a chronic wound, or a combination thereof.

[0064] In some aspects, the subject is in need of FOXN1 activation to augment hair growth in the subject.

[0065] In some aspects, the subject has alopecia.

[0066] In some aspects, the subject is a human.

[0067] The present disclosure also provides a method of enhancing immune function or promoting thymic regeneration in a subject in need thereof, comprising administering tothe subject a therapeutically effective amount of a Compound of the Disclosure or a pharmaceutical composition thereof, wherein the subject is in need of immune function enhancement or promoting thymic regeneration due to administration of CAR-T cells or other adoptive T-cell therapies, recovery from sepsis or critical illness, idiopathic T-cell lymphopenia, immunosuppression following solid organ transplantation, or a combination thereof. In some aspects, the Compound of the Disclosure is selected from any one of the compounds listed in Table 1. In some aspects, the subject is at risk of or recovering from infection due to impaired T-cell function. In some aspects, the subject is an elderly individual, an immunocompromised patient, a recipient of immunosuppressive therapy, or a combination thereof.

[0068] The present disclosure also provides a method of promoting immune tolerance or reducing transplant-related complications in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a Compound of the Disclosure or a pharmaceutical composition thereof, wherein the subject is receiving a hematopoietic stem cell transplant or solid organ transplant. In some aspects, the Compound of the Disclosure is selected from any one of the compounds listed in Table 1. In some aspects, the method reduces the risk of graft-versus-host disease (GVHD). In some aspects, the method enhances immune reconstitution.

[0069] The present disclosure also provides a method of treating or supporting recovery from a thymic epithelial tumor in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a Compound of the Disclosure or a pharmaceutical composition thereof, wherein the subject has a thymoma or thymic carcinoma. In some aspects, the Compound of the Disclosure is selected from any one of the compounds listed in Table 1.

[0070] The present disclosure also provides a method of promoting immune surveillance or reducing cancer risk in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a Compound of the Disclosure or a pharmaceutical composition thereof, wherein the subject has an age-related decline in immune function, a personal or familial history of cancer, or a genetic predisposition to immune dysfunction. In some aspects, the Compound of the Disclosure is selected from any one of the compounds listed in Table 1.

[0071] The present disclosure also provides a method of augmenting hair growth or restoring hair loss in a subject in need thereof, comprising administering to the subject atherapeutically effective amount of a Compound of the Disclosure or a pharmaceutical composition thereof, wherein the hair loss is chemotherapy-induced, radiation-induced, or idiopathic in origin. In some aspects, the Compound of the Disclosure is selected from any one of the compounds listed in Table 1.

[0072] The present disclosure also provides a method of promoting skin repair or regeneration in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a Compound of the Disclosure or a pharmaceutical composition thereof, wherein the subject has a radiation-induced skin injury, a surgical wound, or age-related skin atrophy. In some aspects, the Compound of the Disclosure is selected from any one of the compounds listed in Table 1.

[0073] The present disclosure also provides a method of promoting dermal regeneration or treating skin aging in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a Compound of the Disclosure or a pharmaceutical composition thereof. In some aspects, the Compound of the Disclosure is selected from any one of the compounds listed in Table 1.

[0074] The present disclosure also provides a method of preconditioning a non-human donor organism for xenotransplantation of a composite organ comprising thymic tissue, comprising administering to the donor a therapeutically effective amount of a Compound of the Disclosure or a pharmaceutical composition thereof, wherein the method improves thymic tissue function, promotes immune system development, or enhances compatibility of the graft with the recipient immune system. In some aspects, the Compound of the Disclosure is selected from any one of the compounds listed in Table 1.

[0075] The present disclosure provides compounds that are FOXN1 activators for promoting thymus regeneration and / or immune reconstitution, and / or for the treatment of diseases and conditions wherein activation of FOXN1 has a beneficial effect. These compounds typically have EC50 (the drug concentration that results in 50% FOXN1 activation) values of less than 100 μM, e.g., less than 50 μM, less than 25 μM, and less than 5 μM, or less than about 1 µM.

[0076] In some aspects, Compounds of the Disclosure are administered in admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice. Pharmaceutical compositions for use in accordance with the present disclosure are formulated in a conventional manner using one or morephysiologically acceptable carriers comprising excipients and / or auxiliaries that facilitate processing of Compounds of the Disclosure.

[0077] Compounds of the Disclosure can be readily combined with pharmaceutically acceptable carriers. Standard pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 19th ed.1995. Such carriers enable the active agents to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained by adding the Compound of the Disclosure to a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.

[0078] Suitable excipients include fillers such as saccharides (for example, lactose, sucrose, mannitol, or sorbitol), cellulose preparations, calcium phosphates (for example, tricalcium phosphate or calcium hydrogen phosphate), as well as binders such as starch paste (using, for example, maize starch, wheat starch, rice starch, or potato starch), gelatin, tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or polyvinyl pyrrolidone. If desired, one or more disintegrating agents can be added, such as the above-mentioned starches and also carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate. Buffers and pH modifiers can also be added to stabilize the pharmaceutical composition.

[0079] Auxiliaries are typically flow-regulating agents and lubricants such as, for example, silica, talc, stearic acid or salts thereof (e.g., magnesium stearate or calcium stearate), and polyethylene glycol. Dragee cores are provided with suitable coatings that are resistant to gastric juices. For this purpose, concentrated saccharide solutions can be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, polyethylene glycol, and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. In order to produce coatings resistant to gastric juices, solutions of suitable cellulose preparations such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate can be used. Dye stuffs or pigments can be added to the tablets or dragee coatings, for example, for identification or in order to characterize combinations of active compound doses.

[0080] Compounds of the Disclosure can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampules or in multidose containers, with an added preservative. The compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing, and / or dispersing agents.

[0081] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active agent in water-soluble form. Additionally, suspensions of a Compound of the Disclosure can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils or synthetic fatty acid esters. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension. Optionally, the suspension also can contain suitable stabilizers or agents that increase the solubility of the compounds and allow for the preparation of highly concentrated solutions. Alternatively, a composition of the present disclosure can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0082] In particular, the Compounds of the Disclosure can be administered orally, buccally, or sublingually in the form of tablets containing excipients, such as starch or lactose, or in capsules or ovules, either alone or in admixture with excipients, or in the form of elixirs or suspensions containing flavoring or coloring agents. Such liquid preparations can be prepared with pharmaceutically acceptable additives, such as suspending agents. Compounds of the Disclosure also can be injected parenterally, for example, intravenously, intramuscularly, subcutaneously, or intracoronarily. For parenteral administration, the Compounds of the Disclosure are typically used in the form of a sterile aqueous solution which can contain other substances, for example, salts or monosaccharides, such as mannitol or glucose, to make the solution isotonic with blood.EXAMPLES EXAMPLE 1 Synthesis of 2-(6-(3,5-dimethylisoxazol-4-yl)-2-methyl-1H-pyrrolo[3,2-c]pyridin-4-yl)phenol (Compound 88)

[0083] Step 1: To a suspension of 2,6-dichloropyridin-4-amine (20.0 g, 1 eq, 123 mmol) and potassium acetate (48.2 g, 4 eq, 491 mmol) in acetic acid (100 mL), ICl (19.9 g, 6.15 mL, 1 eq, 123 mmol) in acetic acid (100 mL) was added dropwise over 30 minutes. The mixture was heated to 40°C and stirred for 24 hours. The mixture was cooled to rt and water (200 mL) was added. The precipitate formed was collected by filtration, washed with water (50 mL), and dried in vacuo to afford 2,6-dichloro-3-iodopyridin-4-amine (36.2 g, 0.12 mol, 96%, 94% purity) as a pale orange solid.

[0084] 1H NMR in DMSO-d6 was consistent with product structure at >95% purity.1H NMR (400 MHz, DMSO) δ 6.87 (s, 2H), 6.56 (s, 1H).

[0085] The product was analysed by LCMS (Cortecs C18+, 90 Å, 30 x 2.1 mm, 2.7 μm, 3 min method, 0.1% formic acid, 2-100% MeCN / water): m / z 288.8 / 290.8 (M+H)+(ES+) at 1.24 min, 93.8% purity at 254 nm.

[0086] Step 2: To a solution of 2,6-dichloro-3-iodopyridin-4-amine (4 x 1 g, 1 eq, 3.46 mmol) in DMF (10.0 mL) in four microwave vials, prop-1-yne (693 mg, 17.3 mL, 1.00 molar, 5 eq, 17.3 mmol), CuI (65.9 mg, 0.1 eq, 0.35 mmol) and bis-(triphenylphosphino)- palladous chloride (243 mg, 0.1 eq, 0.35 mmol) were added. The mixture was degassed with N2(bubbling) for 5 minutes and was stirred at 60°C for 2 days. The mixture was cooled to rt, diluted with EtOAc (100 mL) and washed with sat. aq. NH4Cl (50 mL). The organiclayer was further washed with water (100 mL) and brine (2 x 100 mL). The organic layer was dried over Na2SO4, filtered and concentrated in vacuo.

[0087] The crude product was purified by chromatography on silica gel (80 g cartridge, 0- 50% EtOAc / isohexane) to afford 2,6-dichloro-3-(prop-1-yn-1-yl)pyridin-4-amine (2.47 g, 8.6 mmol, 62%, 70% purity) as a tan solid.

[0088] 1H NMR in DMSO-d6 was consistent with product structure at 70% purity.1H NMR (400 MHz, DMSO) δ 6.89 (s, 2H), 6.61 (s, 1H), 2.13 (s, 3H).

[0089] The product was analysed by LCMS (Cortecs C18+, 90 Å, 30 x 2.1 mm, 2.7 μm, 3 min method, 0.1% formic acid, 2-100% MeCN / water): m / z 201.0 / 203.0 (M+H)+(ES+) at 1.26 min, 70.9% purity 260 nm + / - 90 nm.

[0090] Step 3: To a solution of 2,6-dichloro-3-(prop-1-yn-1-yl)pyridin-4-amine (2.47 g, 70% wt, 1 eq, 8.60 mmol) in DMF (40.0 mL), potassium tert-butoxide (1.93 g, 2 eq, 17.2 mmol) was added. The mixture was stirred at 80°C for 2 hours. The mixture was cooled to rt and water (100 mL) was added. The precipitate formed was collected by filtration, washed with water (10 mL) and dried in vacuo. The crude product was purified by chromatography on silica gel (40 g cartridge, 0-100% EtOAc / isohexane) to afford 4,6- dichloro-2-methyl-1H-pyrrolo[3,2-c]pyridine (1.45 g, 6.9 mmol, 80%, 95% purity) as a tan solid.

[0091] 1H NMR in DMSO-d6 was consistent with product structure at >95% purity.1H NMR (400 MHz, DMSO) δ 11.95 (s, 1H), 7.39 (d, J = 0.9 Hz, 1H), 6.32 – 6.26 (m, 1H), 2.41 (d, J = 1.0 Hz, 3H).

[0092] The product was analysed by LCMS (Cortecs C18+, 90 Å, 30 x 2.1 mm, 2.7 μm, 3 min method, 0.1% formic acid, 2-100% MeCN / water): m / z 201.0 / 203.0 (M+H)+(ES+) at 1.32 min, 96.0% purity 260 nm + / - 90 nm.

[0093] Step 4: To a solution of 4,6-dichloro-1,2-dimethyl-1H-pyrrolo[3,2-c]pyridine (85.0 mg, 1 eq, 395 μmol) in 2-Methyltetrahydrofuran (1.20 mL), potassium carbonate (164 mg, 296 μL, 4.00 molar, 3 eq, 1.19 mmol) and 2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2- yl)phenol (87.0 mg, 82.8 μL, 1 eq, 395 μmol) were added. The mixture was degassed (N2 bubbling) for 5 minutes and Pd(dppf)Cl2 (28.9 mg, 0.1 eq, 39.5 μmol) was added. The mixture was further degassed (N2bubbling) for 5 minutes and was stirred at 80°C for 3 hours. The mixture was cooled to rt and concentrated in vacuo. The crude product was purified by chromatography on silica gel (12 g cartridge, 0-100% EtOAc / isohexane) toafford 2-(6-chloro-1,2-dimethyl-1H-pyrrolo[3,2-c]pyridin-4-yl)phenol (75.0 mg, 0.26 mmol, 66%, 95% purity) as a pale yellow solid.

[0094] 1H NMR in DMSO-d6 was consistent with product structure at >95% purity.1H NMR (500 MHz, DMSO) δ 12.60 (s, 1H), 7.99 (dd, J = 7.8, 1.6 Hz, 1H), 7.72 (d, J = 0.9 Hz, 1H), 7.33 (td, J = 7.6, 1.6 Hz, 1H), 7.02 – 6.95 (m, 2H), 6.80 (s, 1H), 3.75 (s, 3H), 2.48 (d, J = 1.0 Hz, 3H).

[0095] The product was analysed by LCMS (Cortecs C18+, 90 Å, 30 x 2.1 mm, 2.7 μm, 3 min method, 0.1% formic acid, 2-100% MeCN / water): m / z 273.0 / 275.0 (M+H)+(ES+) at 1.84 min, 98.7% purity 254 nm.

[0096] Step 5: To a solution of 2-(6-chloro-2-methyl-1H-pyrrolo[3,2-c]pyridin-4- yl)phenol (70.0 mg, 1 eq, 271 μmol) in 1,4-dioxane (2.00 mL), 3,5-dimethyl-4-(4,4,5,5- tetramethyl-1,3,2-dioxaborolan-2-yl)isoxazole (90.5 mg, 1.5 eq, 406 μmol) and potassium carbonate (112 mg, 541 μL, 1.50 molar, 3 eq, 812 μmol) were added. The mixture was degassed (N2bubbling) for 5 minutes and Pd-118 (17.6 mg, 0.1 eq, 27.1 μmol) was added. The mixture was further degassed (N2 bubbling) for 5 minutes and was stirred at 90°C for 30 minutes. The mixture was cooled to rt and concentrated in vacuo. The crude product was purified by chromatography on silica gel (12 g cartridge, 0-100% EtOAc / isohexane) to afford the desired product with low purity. The product was further purified by preparative HPLC: the residue was dissolved in 1.8 mL with DMSO, filtered and purified by reversed phase preparative HPLC (Waters 2767 Sample Manager, Waters 2545 Binary Gradient Module, Waters Systems Fluidics Organiser, Waters 515 ACD pump, Waters 515 Makeup pump, Waters 2998 Photodiode Array Detector, Waters QDa) on a Waters X- Select CSH C18 ODB prep column, 130Å, 5 µm, 30 mm X 100 mm, flow rate 40 mL min- 1 eluting with a 0.1% formic acid in water-MeCN gradient over 8.5 mins using UV across all wavelengths with PDA as well as a QDA and ELS detector. At-column dilution pump gives 2 mL min-1 ACN over the entire method, which is included in the following MeCN percentages. Gradient information: 0.0-0.5 min, 17.5% MeCN; 0.5-5.5 min, ramped from 17.5% MeCN to 47.5% MeCN; 5.5-5.6 min, ramped from 47.5% MeCN to 100% MeCN; 5.6-8.5 min, held at 100% MeCN. The clean fractions were evaporated in a Genevac to afford 2-(6-(3,5-dimethylisoxazol-4-yl)-2-methyl-1H-pyrrolo[3,2-c]pyridin-4-yl)phenol (21.0 mg, 62 μmol, 23%, 95% purity) as a pale yellow solid.

[0097] 1H NMR in DMSO-d6 was consistent with product structure at >95% purity.1H NMR (400 MHz, DMSO) δ 14.81 (s, 1H), 11.94 (s, 1H), 8.23 – 8.16 (m, 1H), 7.46 (s, 1H),7.35 – 7.26 (m, 1H), 7.03 – 6.92 (m, 2H), 6.86 (s, 1H), 2.53 (s, 3H), 2.35 (s, 3H). (3H, s under the DMSO peak, see HSQC).

[0098] The product was analysed by LCMS (Cortecs C18+, 90 Å, 30 x 2.1 mm, 2.7 μm, 3 min method, 0.1% formic acid, 2-100% MeCN / water): m / z 320.2 (M+H)+(ES+) at 0.90 min, 99.6% purity 254 nm. EXAMPLE 2 Synthesis of N-(4-chlorobenzyl)-3-ethyl-[1,2,4]triazolo[4,3-b]pyridazin-6-amine (Compound 67)

[0099] Step 1: A suspension of (Z)-3-chloro-6-hydrazineylidene-1,6-dihydropyridazine (1.00 g, 1 eq, 6.92 mmol) in propionic acid (10.2 g, 10.4 mL, 20 eq, 138 mmol) was stirred at 100°C for 2 hours. The mixture was cooled to rt and concentrated in vacuo. The residue was dissolved in EtOAc (20 mL) and washed with sat. aq. NaHCO3 solution (50 mL). The aqueous layer was further extracted with EtOAc (3 x 20 mL). The organic layers were combined, washed with brine (50 mL), dried over Na2SO4and concentrated in vacuo to afford 6-chloro-3-ethyl-[1,2,4]triazolo[4,3-b]pyridazine (0.74 g, 3.8 mmol, 56%, 95% purity) as a pale brown solid.

[0100] 1H NMR in DMSO-d6was consistent with product structure at >95% purity.1H NMR (400 MHz, DMSO) δ 8.41 (d, J = 9.6 Hz, 1H), 7.46 (d, J = 9.6 Hz, 1H), 3.08 (q, J = 7.5 Hz, 2H), 1.37 (t, J = 7.5 Hz, 3H).

[0101] The product was analysed by LCMS (Cortecs C18+, 90 Å, 30 x 2.1 mm, 2.7 μm, 3 min method, 0.1% formic acid, 2-100% MeCN / water): m / z 183.0 / 185.0 (M+H)+(ES+) at 0.81 min, 98.4% purity 260 nm + / - 90 nm.

[0102] Step 2: To a solution of 6-chloro-3-ethyl-[1,2,4]triazolo[4,3-b]pyridazine (50.0 mg, 1 eq, 274 μmol) in ethanol (2.00 mL), (4-chlorophenyl)methanamine (58.2 mg, 50.0 μL, 1.5 eq, 411 μmol) was added and the mixture was stirred at 80°C for 48 hours. The mixture was cooled to rt and concentrated in vacuo. The crude product was purified by chromatography on silica gel (12 g cartridge, 0-5% MeOH / DCM) to afford N-(4-chlorobenzyl)-3-ethyl-[1,2,4]triazolo[4,3-b]pyridazin-6-amine (40 mg, 0.13 mmol, 48%, 95% purity) as a pale yellow solid.

[0103] 1H NMR in DMSO-d6 was consistent with product structure at >95% purity.1H NMR (400 MHz, DMSO) δ 7.91 – 7.84 (m, 2H), 7.47 – 7.36 (m, 4H), 6.80 (d, J = 9.8 Hz, 1H), 4.43 (d, J = 5.7 Hz, 2H), 2.89 (q, J = 7.6 Hz, 2H), 1.24 (t, J = 7.5 Hz, 3H).

[0104] The product was analysed by LCMS (Cortecs C18+, 90 Å, 30 x 2.1 mm, 2.7 μm, 3 min method, 0.1% formic acid, 2-100% MeCN / water): m / z 288.1 / 290.0 (M+H)+(ES+) at 1.32 min, 100.0% purity 254 nm. EXAMPLE 3 Synthesis of N-(2-hydroxyphenyl)-6-(isobutyl(methyl)amino)nicotinamide (Compound 57)

[0105] Step 1: To a solution of methyl 6-chloronicotinate (300 mg, 1 eq, 1.75 mmol) in DMF (5.00 mL), DIPEA (452 mg, 609 μL, 2 eq, 3.50 mmol) and methyl(2- methylpropyl)amine (229 mg, 315 μL, 1.5 eq, 2.62 mmol) were added. The mixture heated to 80°C and stirred for 18 hours. The mixture was cooled to rt and water (20 mL) was added. The mixture was extracted with EtOAc (3 x 10 mL). The organic layers were combined, washed with brine (2 x 50 mL) dried over Na2SO4, filtered and concentrated in vacuo. The crude product was purified by chromatography on silica gel (12 g cartridge, 0- 50% EtOAc / isohexane) to afford methyl 6-(isobutyl(methyl)amino)nicotinate (343 mg, 1.5 mmol, 84%, 95% purity) as a pale yellow solid.

[0106] 1H NMR in DMSO-d6was consistent with product structure at >95% purity.1H NMR (400 MHz, DMSO) δ 8.61 (dd, J = 2.5, 0.7 Hz, 1H), 7.90 (dd, J = 9.1, 2.4 Hz, 1H), 6.70 – 6.63 (m, 1H), 3.77 (s, 3H), 3.42 (d, J = 7.5 Hz, 2H), 3.07 (s, 3H), 2.03 (hept, J = 6.9 Hz, 1H), 0.86 (d, J = 6.6 Hz, 6H).

[0107] The product was analysed by LCMS (Cortecs C18+, 90 Å, 30 x 2.1 mm, 2.7 μm, 3 min method, 0.1% formic acid, 2-100% MeCN / water): m / z 223.2 (M+H)+(ES+) at 1.04 min, 100.0% purity 254 nm.

[0108] Step 2: To a solution of methyl 6-(isobutyl(methyl)amino)nicotinate (0.34 g, 95% Wt, 1 eq, 1.5 mmol) in THF (3.00 mL) / MeOH (1.00 mL) / H2O (1.00 mL), LiOH, H2O (67 mg, 1.1 eq, 1.6 mmol) was added and the mixture was stirred at 50°C for 3 hours. The mixture was concentrated in vacuo to afford 6-(isobutyl(methyl)amino)nicotinic acid, Lithium (0.32 g, 1.4 mmol, 97%, 95% purity) as a tan solid.

[0109] 1H NMR in DMSO-d6 was consistent with product structure at >95% purity.1H NMR (400 MHz, DMSO) δ 8.51 (d, J = 2.3 Hz, 1H), 7.86 (dd, J = 8.7, 2.2 Hz, 1H), 6.43 (d, J = 8.8 Hz, 1H), 3.33 (d, J = 7.4 Hz, 2H), 2.99 (s, 3H), 2.00 (hept, J = 6.9 Hz, 1H), 0.85 (d, J = 6.7 Hz, 6H).

[0110] The product was analysed by LCMS (Cortecs C18+, 90 Å, 30 x 2.1 mm, 2.7 μm, 3 min method, 0.1% formic acid, 2-100% MeCN / water): m / z 209.2 (M+H)+(ES+) at 0.61 min, 98.9% purity 260 nm + / - 90 nm.

[0111] Step 3: To a solution of 6-(isobutyl(methyl)amino)nicotinic acid, Lithium (70.0 mg, 1 eq, 325 μmol) in DMF (1.00 mL), 2-aminophenol (106 mg, 3 eq, 976 μmol), DIPEA (126 mg, 170 μL, 3 eq, 976 μmol) and HATU (148 mg, 1.2 eq, 390 μmol) were successively added. The mixture was stirred at rt for 6 hours. The mixture was diluted with water (10 mL). The mixture was extracted with EtOAc (3 x 10 mL). The organic layers were combined, washed with brine (2 x 50 mL) dried over Na2SO4, filtered and concentrated in vacuo. The crude product was purified by chromatography on silica gel (12 g cartridge, 0- 100% EtOAc / isohexane) to afford the desired product with low purity. The residue was purified again by chromatography on RP Flash C18 (12 g cartridge, 10-70% (0.1% formic acid in MeCN) / (0.1% formic acid in water)) to afford N-(2-hydroxyphenyl)-6- (isobutyl(methyl)amino)nicotinamide (10.0 mg, 32 μmol, 9.8%, 95% purity) as a pale yellow solid.

[0112] 1H NMR in DMSO-d6 was consistent with product structure at >95% purity.1H NMR (400 MHz, DMSO) δ 9.91 (s, 1H), 9.40 (s, 1H), 8.70 (d, J = 2.5 Hz, 1H), 8.02 (dd, J = 9.0, 2.5 Hz, 1H), 7.66 – 7.57 (m, 1H), 7.05 – 6.97 (m, 1H), 6.90 (dd, J = 8.1, 1.5 Hz, 1H), 6.81 (td, J = 7.6, 1.5 Hz, 1H), 6.69 (d, J = 9.1 Hz, 1H), 3.43 (d, J = 7.4 Hz, 2H), 3.08 (s, 3H), 2.05 (hept, J = 7.0 Hz, 1H), 0.87 (d, J = 6.7 Hz, 6H).

[0113] The product was analysed by LCMS (Cortecs C18+, 90 Å, 30 x 2.1 mm, 2.7 μm, 3 min method, 0.1% formic acid, 2-100% MeCN / water): m / z 300.2 (M+H)+(ES+) at 0.97 min, 100.0% purity 254 nm.EXAMPLE 4 Synthesis of N-((1-isobutyl-1H-imidazol-5-yl)methyl)-3-(1-methyl-6-oxo-1,6-dihydropyridazin- 3-yl)benzamide (Compound 154)

[0114] Step 1: To a solution of methyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2- yl)benzoate (1.0 g, 1.5 eq, 4.0 mmol) in ACN (8.00 mL), 6-bromo-2-methylpyridazin- 3(2H)-one (0.50 g, 1 eq, 2.6 mmol), K2CO3(1.1 g, 2.0 mL, 4.00 molar, 3 eq, 7.9 mmol) and Pd(dppf)Cl2 (0.19 g, 0.1 eq, 0.26 mmol) were added. The mixture was degassed (Vacuum / N2), heated to 80°C and stirred for 1 hour. The mixture was cooled to rt and water (10 mL) was added. The solid was collected by filtration, washed with water (10 mL) and dried in vacuo. The crude product was purified by chromatography on silica gel (24 g cartridge, 0-100% EtOAc / isohexane) to afford methyl 4-(1-methyl-6-oxo-1,6- dihydropyridazin-3-yl)benzoate (0.61 g, 2.4 mmol, 90%, 95% purity) as a white solid.

[0115] 1H NMR in DMSO-d6was consistent with product structure at >95% purity.1H NMR (400 MHz, DMSO) δ 8.13 (d, J = 9.8 Hz, 1H), 8.10 – 8.00 (m, 4H), 7.09 (d, J = 9.7 Hz, 1H), 3.88 (s, 3H), 3.77 (s, 3H).

[0116] The product was analysed by LCMS (Cortecs C18+, 90 Å, 30 x 2.1 mm, 2.7 μm, 3 min method, 0.1% formic acid, 2-100% MeCN / water): m / z 245.0 (M+H)+(ES+) at 1.13 min, 100.0% purity 254 nm.

[0117] Step 2: To a solution of methyl 4-(1-methyl-6-oxo-1,6-dihydropyridazin-3- yl)benzoate (0.34 g, 1 eq, 1.4 mmol) in THF (3.00 mL) / MeOH (1.00 mL) / H2O (1.00 mL), LiOH, H2O (64 mg, 1.1 eq, 1.5 mmol) was added. The mixture was stirred at 45°C for 1 hour. The mixture was concentrated in vacuo to afford 4-(1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)benzoic acid, lithium (0.32 g, 1.3 mmol, 92%, 95% purity) as a white solid.

[0118] 1H NMR in DMSO-d6 was consistent with product structure at >95% purity.1H NMR (400 MHz, DMSO) δ 8.05 (d, J = 9.7 Hz, 1H), 7.97 – 7.89 (m, 2H), 7.81 – 7.74 (m, 2H), 7.02 (d, J = 9.7 Hz, 1H), 3.74 (s, 3H).

[0119] The product was analysed by LCMS (Cortecs C18+, 90 Å, 30 x 2.1 mm, 2.7 μm, 3 min method, 0.1% formic acid, 2-100% MeCN / water): m / z 231.0 (M+H)+(ES+) at 0.85 min, 98.0% purity 260 nm + / - 90 nm.

[0120] Step 3: To a solution of 4-(1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)benzoic acid, lithium (100 mg, 1 eq, 422 μmol) in DMF (2.00 mL), (1-isobutyl-1H-imidazol-5- yl)methanamine dihydrochloride (124 mg, 1.3 eq, 548 μmol), DIPEA (272 mg, 367 μL, 5 eq, 2.11 mmol) and HATU (240 mg, 1.5 eq, 632 μmol) were successively added. The mixture was stirred at rt for 2 hours. The mixture was diluted with water (10 mL) and extracted with EtOAc (3 x 10 mL). The organic layers were combined, washed with brine (2 x 50 mL) dried over Na2SO4, filtered and concentrated in vacuo. The crude product was purified by chromatography on silica gel (12 g cartridge, 0-100% EtOAc / isohexane) to afford N-((1-isobutyl-1H-imidazol-5-yl)methyl)-3-(1-methyl-6-oxo-1,6-dihydropyridazin- 3-yl)benzamide (133 mg, 0.35 mmol, 82%, 95% purity) as a pale yellow solid.

[0121] 1H NMR in DMSO-d6 was consistent with product structure at >95% purity.1H NMR (400 MHz, DMSO) δ 8.96 (t, J = 5.6 Hz, 1H), 8.12 (d, J = 9.8 Hz, 1H), 7.98 (d, J = 1.0 Hz, 4H), 7.56 (d, J = 1.2 Hz, 1H), 7.07 (d, J = 9.7 Hz, 1H), 6.84 (d, J = 1.1 Hz, 1H), 4.49 (d, J = 5.5 Hz, 2H), 3.79 (d, J = 7.4 Hz, 2H), 3.76 (s, 3H), 1.95 (dq, J = 13.6, 6.8 Hz, 1H), 0.82 (d, J = 6.7 Hz, 6H).

[0122] The product was analysed by LCMS (Cortecs C18+, 90 Å, 30 x 2.1 mm, 2.7 μm, 3 min method, 0.1% formic acid, 2-100% MeCN / water): m / z 366.2 (M+H)+(ES+) at 0.70 min, 100.0% purity 254 nm.EXAMPLE 5 Synthesis of 4-chloro-5-((1-(2-methoxyethyl)-1H-indol-5-yl)amino)-2-methylpyridazin-3(2H)- one (Compound 41)

[0123] Step 1: To a solution of 1H-indol-5-amine (1.00 g, 1 eq, 7.57 mmol) in dry DMF (30.0 mL) at 0°C, NaH (363 mg, 60% Wt, 1.2 eq, 9.08 mmol) was added. The mixture was stirred for 30 minutes at the same temperature and 2-bromoethylmethyl ether (1.26 g, 860 μL, 1.2 eq, 9.08 mmol) was added dropwise over 5 minutes. The mixture was warmed to rt and was stirred for 18 hours. Water (50 mL) was cautiously added and the mixture was extracted with EtOAc (3 x 50 mL). The organic layers were combined, washed with brine (2 x 100 mL) dried over Na2SO4, filtered and concentrated in vacuo. The crude product was purified by chromatography on silica gel (40 g cartridge, 0-100% 3 / 1 EtOAc / EtOH in isohexane) to afford 1-(2-methoxyethyl)-1H-indol-5-amine (1.03 g, 5.1 mmol, 68%, 95% purity) as a pale yellow solid.

[0124] 1H NMR in DMSO-d6was consistent with product structure at >95% purity.1H NMR (400 MHz, DMSO) δ 7.18 – 7.09 (m, 2H), 6.66 (d, J = 2.1 Hz, 1H), 6.51 (dd, J = 8.6, 2.1 Hz, 1H), 6.10 (dd, J = 3.0, 0.8 Hz, 1H), 4.45 (s, 2H), 4.18 (t, J = 5.5 Hz, 2H), 3.59 (t, J = 5.4 Hz, 2H), 3.20 (s, 3H).

[0125] The product was analysed by LCMS (Cortecs C18+, 90 Å, 30 x 2.1 mm, 2.7 μm, 3 min method, 0.1% formic acid, 2-100% MeCN / water): m / z 191.2 (M+H)+(ES+) at 0.14 min, 95.8% purity 254 nm.

[0126] Step 2: To a solution of 4,5-dichloro-2-methylpyridazin-3(2H)-one (100 mg, 1 eq, 559 μmol) in DMSO (1.00 mL), DIPEA (144 mg, 195 μL, 2 eq, 1.12 mmol) and 1-(2- methoxyethyl)-1H-indol-5-amine (128 mg, 1.2 eq, 670 μmol) were added. The mixture was stirred at 120°C under microwave irradiation for 18 hours. The mixture was cooled to rt and EtOAc (10 mL) was added. The mixture was washed with sat. aq. NaHCO3solution (10 mL). The aqueous layer was further extracted with EtOAc (10 mL) and the organic layers were combined, washed with brine (20 mL), dried over Na2SO4, filtered and concentrated in vacuo. The crude product was purified by chromatography on silica gel (12g cartridge, 0-100% EtOAc / isohexane) to afford the desired product with low purity. The crude product was purified by chromatography on RP Flash C18 (12 g cartridge, 10-60% (0.1% formic acid in MeCN) / (0.1% formic acid in water)) to afford 4-chloro-5-((1-(2- methoxyethyl)-1H-indol-5-yl)amino)-2-methylpyridazin-3(2H)-one (79.0 mg, 0.23 mmol, 40%, 95% purity) as a tan solid.

[0127] 1H NMR in DMSO-d6 was consistent with product structure at >95% purity.1H NMR (500 MHz, DMSO) δ 8.62 (s, 1H), 7.54 (d, J = 8.7 Hz, 1H), 7.46 – 7.37 (m, 3H), 7.02 (dd, J = 8.6, 2.1 Hz, 1H), 6.43 (dd, J = 3.1, 0.8 Hz, 1H), 4.34 (t, J = 5.3 Hz, 2H), 3.66 (t, J = 5.3 Hz, 2H), 3.59 (s, 3H), 3.23 (s, 3H).

[0128] The product was analysed by LCMS (Cortecs C18+, 90 Å, 30 x 2.1 mm, 2.7 μm, 3 min method, 0.1% formic acid, 2-100% MeCN / water): m / z 333.0 / 335.0 (M+H)+(ES+) at 1.23 min, 98.8% purity 254 nm. EXAMPLE 6 Synthesis of 2-(((5-chlorothiophen-2-yl)methyl)(methyl)amino)-4H-pyrido[1,2-a]pyrimidin-4- one (Compound 34)

[0129] To a solution of 2-chloro-4H-pyrido[1,2-a]pyrimidin-4-one (35.0 mg, 1 eq, 194 μmol) in EtOH (1.00 mL), DIPEA (50.1 mg, 67.5 μL, 2 eq, 388 μmol) and 1-(5- chlorothiophen-2-yl)-N-methylmethanamine (37.6 mg, 1.2 eq, 233 μmol) were added. The mixture was heated to 80°C and stirred for 18 hours. The mixture was cooled to rt and concentrated in vacuo. The crude product was purified by chromatography on silica gel (12 g cartridge, 0-100% EtOAc / isohexane) to afford 2-(((5-chlorothiophen-2- yl)methyl)(methyl)amino)-4H-pyrido[1,2-a]pyrimidin-4-one (46.0 mg, 0.14 mmol, 74%, 95% purity) as a white solid.

[0130] 1H NMR in DMSO-d6 was consistent with product structure at >95% purity.1H NMR (400 MHz, DMSO) δ 8.82 – 8.75 (m, 1H), 7.88 – 7.79 (m, 1H), 7.43 – 7.35 (m, 1H), 7.09 (td, J = 6.9, 1.4 Hz, 1H), 7.02 – 6.93 (m, 2H), 5.48 (s, 1H), 4.90 (s, 2H), 3.01 (s, 3H).

[0131] The product was analysed by LCMS (Cortecs C18+, 90 Å, 30 x 2.1 mm, 2.7 μm, 3 min method, 0.1% formic acid, 2-100% MeCN / water): m / z 306.0 / 308.0 (M+H)+(ES+) at 1.55 min, 100.0% purity 254 nm. EXAMPLE 7 Synthesis of N-(4,7-dimethyl-2-oxo-2H-chromen-6-yl)-4-methylbenzenesulfonamide (Compound 19)

[0132] To a solution of 6-amino-4,7-dimethyl-2H-chromen-2-one (0.10 g, 1 eq, 0.53 mmol) in DCM (2.00 mL), DIPEA (0.14 g, 0.18 mL, 2 eq, 1.1 mmol) and 4- methylbenzenesulfonyl chloride (0.10 g, 1 eq, 0.53 mmol) were added. The mixture was stirred at rt for 2 hours. DMF (1.00 mL) was added to help with solubility and the mixture was stirred at rt for 18 hours. Sat. aq. NH4Cl (5 mL) was added and the layer were separated. The aqueous layer was extracted with DCM (2 x 10 mL). The organic layers were combined, dried over Na2SO4, filtered and concentrated in vacuo. The crude product was purified by chromatography on silica gel (12 g cartridge, 0-100% EtOAc / isohexane) to afford the desired product with low purity. The residue was purified by chromatography on silica gel (12 g cartridge, 0-50% EtOAc / isohexane) to afford N-(4,7-dimethyl-2-oxo-2H- chromen-6-yl)-4-methylbenzenesulfonamide (22.0 mg, 61 μmol, 12%, 95% purity) as a pale yellow solid.

[0133] 1H NMR in DMSO-d6 was consistent with product structure at >95% purity.1H NMR (400 MHz, DMSO) δ 9.68 (s, 1H), 7.58 – 7.50 (m, 2H), 7.37 (d, J = 8.1 Hz, 2H), 7.22 (d, J = 0.8 Hz, 1H), 7.15 (s, 1H), 6.34 (d, J = 1.4 Hz, 1H), 2.37 (s, 3H), 2.20 (d, J = 1.3 Hz, 3H), 2.06 (s, 3H).

[0134] The product was analysed by LCMS (Cortecs C18+, 90 Å, 30 x 2.1 mm, 2.7 μm, 3 min method, 0.1% formic acid, 2-100% MeCN / water): m / z 344.0 (M+H)+(ES+) at 1.39 min, 97.5% purity 254 nm.EXAMPLE 8 Synthesis of 1-methyl-6-(piperidin-1-ylsulfonyl)benzo[cd]indol-2(1H)-one (Compound 14)

[0135] Step 1: To a solution of benzo[cd]indol-2(1H)-one (2.00 g, 1 eq, 11.8 mmol) in DMF (20.0 mL), K2CO3(3.27 g, 2 eq, 23.6 mmol) and MeI (2.18 g, 961 μL, 1.3 eq, 15.4 mmol) were added. The mixture was stirred at 45°C for 18 hours. Partial conversion observed by LCMS. MeI (3.36 g, 1.48 mL, 2 eq, 23.6 mmol) was added and stirring at 45°C was continued for 24 hours. The mixture was cooled to rt and water (100 mL) was added. The mixture was extracted with EtOAc (3 x 50 mL). The organic layers were combined, washed with brine (2 x 100 mL), dried over Na2SO4 and concentrated in vacuo. The crude product was purified by chromatography on silica gel (40 g cartridge, 0-50% EtOAc / isohexane) to afford 1-methylbenzo[cd]indol-2(1H)-one (2.01 g, 10 mmol, 88%, 95% purity) as a pale yellow solid.

[0136] 1H NMR in DMSO-d6was consistent with product structure at >95% purity.1H NMR (400 MHz, DMSO) δ 8.21 – 8.14 (m, 1H), 8.07 – 8.03 (m, 1H), 7.84 – 7.75 (m, 1H), 7.66 – 7.62 (m, 1H), 7.59 – 7.52 (m, 1H), 7.16 (d, J = 7.0 Hz, 1H), 3.38 (s, 3H).

[0137] The product was analysed by LCMS (Cortecs C18+, 90 Å, 30 x 2.1 mm, 2.7 μm, 3 min method, 0.1% formic acid, 2-100% MeCN / water): m / z 184.2 (M+H)+(ES+) at 1.21 min, 80.3% purity 260 nm + / - 90 nm.

[0138] Step 2: To a solution of 1-methylbenzo[cd]indol-2(1H)-one (1.00 g, 1 eq, 5.46 mmol) in Chloroform (30.0 mL), chlorosulfonic acid (1.91 g, 1.10 mL, 3 eq, 16.4 mmol) was added dropwise over 5 minutes. The mixture was heated to 50°C for 3 hours. The mixture was cooled to rt and was poured onto ice-water (100 mL). The mixture was extracted with DCM (2 x 50 mL). The organic layers were combined, dried over Na2SO4, filtered and concentrated in vacuo to afford 1-methyl-2-oxo-1,2-dihydrobenzo[cd]indole- 6-sulfonyl chloride (0.494 g, 1.6 mmol, 29%, 90% purity) as a yellow solid.

[0139] 1H NMR in DMSO-d6was consistent with product structure at 90% purity.1H NMR (400 MHz, DMSO) δ 8.73 (dd, J = 8.4, 0.7 Hz, 1H), 8.01 (dd, J = 7.0, 0.7 Hz, 1H), 7.84 (d, J = 7.3 Hz, 1H), 7.78 (dd, J = 8.4, 7.0 Hz, 1H), 7.05 (d, J = 7.4 Hz, 1H), 3.36 (s, 3H).

[0140] The product was analysed by LCMS (Cortecs C18+, 90 Å, 30 x 2.1 mm, 2.7 μm, 3 min method, 0.1% formic acid, 2-100% MeCN / water): m / z 282.0 / 284.0 (M+H)+ (ES+) at 1.49 min, 67.1% purity 254 nm.

[0141] Step 3: To a solution of 1-methyl-2-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonyl chloride (0.10 g, 90% Wt, 1 eq, 0.32 mmol) in dry DCM (2.00 mL), N-ethyl-N- isopropylpropan-2-amine (0.12 g, 3 eq, 0.96 mmol), piperidine (41 mg, 47 μL, 1.5 eq, 0.48 mmol) and N,N-dimethylpyridin-4-amine (3.9 mg, 0.1 eq, 32 μmol) were added successively. The mixture was stirred at rt for 18 hours. Sat. aq. NH4Cl (5 mL) was added and the layer were separated. The aqueous layer was extracted with DCM (2 x 10 mL). The organic layers were combined, dried over Na2SO4, filtered and concentrated in vacuo. The crude product was purified by chromatography on silica gel (12 g cartridge, 0-100% EtOAc / isohexane) to afford 1-methyl-6-(piperidin-1-ylsulfonyl)benzo[cd]indol-2(1H)-one (90.0 mg, 0.26 mmol, 81%, 95% purity) as a pale yellow solid.

[0142] 1H NMR in DMSO-d6was consistent with product structure at >95% purity.1H NMR (400 MHz, DMSO) δ 8.66 (d, J = 8.4 Hz, 1H), 8.17 (d, J = 6.9 Hz, 1H), 8.09 (d, J = 7.6 Hz, 1H), 7.96 (dd, J = 8.5, 7.0 Hz, 1H), 7.32 (d, J = 7.6 Hz, 1H), 3.40 (s, 3H), 3.02 – 2.94 (m, 4H), 1.55 – 1.45 (m, 4H), 1.37 – 1.27 (m, 2H).

[0143] The product was analysed by LCMS (Cortecs C18+, 90 Å, 30 x 2.1 mm, 2.7 μm, 3 min method, 0.1% formic acid, 2-100% MeCN / water): m / z 331.0 (M+H)+(ES+) at 1.45 min, 97.4% purity 254 nm.EXAMPLE 9 Synthesis of N-(2-methyl-1,2,3,4-tetrahydroquinolin-4-yl)-N-phenylacetamide (Compound 6)

[0144] Step 1: 4-chloroquinoline (2.00 g, 1 eq, 12.2 mmol) was added to a solution of sodium methoxide (5.4 M in MeOH) (6.60 g, 22.6 mL, 5.40 molar, 10 eq, 122 mmol) in MeOH (25.0 mL). The mixture was heated to 70°C and was stirred for 5 hours. The mixture was concentrated in vacuo, diluted with DCM (50 mL) and washed with water (50 mL). The aqueous layer was extracted with DCM (2 x 25 mL). The organic layers were combined, dried over Na2SO4, filtered and concentrated in vacuo to afford 4- methoxyquinoline (2.03 g, 12 mmol, 99%, 95% purity) as pale yellow oil.

[0145] 1H NMR in DMSO-d6 was consistent with product structure at >95% purity.1H NMR (400 MHz, DMSO) δ 8.74 (d, J = 5.2 Hz, 1H), 8.14 (dd, J = 8.3, 1.5 Hz, 1H), 7.95 (ddd, J = 8.5, 1.2, 0.6 Hz, 1H), 7.74 (ddd, J = 8.4, 6.8, 1.5 Hz, 1H), 7.56 (ddd, J = 8.2, 6.9, 1.3 Hz, 1H), 7.03 (d, J = 5.2 Hz, 1H), 4.04 (s, 3H).

[0146] The product was analysed by LCMS (Cortecs C18+, 90 Å, 30 x 2.1 mm, 2.7 μm, 3 min method, 0.1% formic acid, 2-100% MeCN / water): m / z 160.2 (M+H)+(ES+) at 0.21 min, 100.0% purity 260 nm + / - 90 nm.

[0147] Step 2: To a solution of 4-methoxyquinoline (1.00 g, 1 eq, 6.28 mmol) in dry THF (30.0 mL) at -78°C, methylmagnesium chloride (3 M in THF) (940 mg, 4.19 mL, 3.00 molar, 2 eq, 12.6 mmol) was added dropwise over 10 minutes. The mixture was stirred for 30 minutes and benzyl chloroformate (2.14 g, 1.79 mL, 2 eq, 12.6 mmol) was added dropwise over 15 minutes. The mixture was slowly warmed to rt and was stirred for 18 bours. Methanol (20 mL) was slowly added followed by the addition of 1 M HCl solution(20 mL). The mixture was concentrated in vacuo and the residue was extracted with DCM (2 x 50 mL). The organic layers were combined, washed with brine (50 mL), dried over Na2SO4, filtered and concentrated in vacuo. The crude product was purified by chromatography on silica gel (40 g cartridge, 0-50% EtOAc / isohexane) to afford benzyl 2- methyl-4-oxo-3,4-dihydroquinoline-1(2H)-carboxylate (1.13 g, 3.6 mmol, 58%, 95% purity) as a pale yellow oil.

[0148] 1H NMR in DMSO-d6was consistent with product structure at >95% purity.1H NMR (400 MHz, DMSO) δ 7.85 (ddd, J = 8.4, 3.7, 1.4 Hz, 2H), 7.60 (ddd, J = 8.7, 7.2, 1.8 Hz, 1H), 7.48 – 7.28 (m, 5H), 7.21 (ddd, J = 8.0, 7.2, 1.1 Hz, 1H), 5.33 – 5.21 (m, 2H), 5.14 – 5.01 (m, 1H), 3.20 (dd, J = 17.4, 5.8 Hz, 1H), 2.55 (d, J = 1.9 Hz, 1H), 1.14 (d, J = 6.9 Hz, 3H).

[0149] The product was analysed by LCMS (Cortecs C18+, 90 Å, 30 x 2.1 mm, 2.7 μm, 3 min method, 0.1% formic acid, 2-100% MeCN / water): m / z 296.0 (M+H)+(ES+) at 1.61 min, 100.0% purity 260 nm + / - 90 nm.

[0150] Step 3: To a solution of benzyl 2-methyl-4-oxo-3,4-dihydroquinoline-1(2H)- carboxylate (1.10 g, 1 eq, 3.72 mmol) in DCM (10.0 mL) at 0°C in an ice / sodium chloride bath, triethylamine (3.02 g, 4.15 mL, 8 eq, 29.8 mmol) was added. Titanium tetrachloride (1 M in toluene) (706 mg, 3.72 mL, 1.00 molar, 1 eq, 3.72 mmol) was added dropwise for 10 minutes. Aniline (694 mg, 680 μL, 2 eq, 7.45 mmol) was added and the mixture was stirred over night at room temperature. The reaction was quenched with saturated NaHCO3solution (10 mL) and the mixture was extracted with ethyl acetate (3 x 20 mL). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered and concentrated in vacuo. The crude product was purified by chromatography on silica gel (40 g cartridge, 0-20% EtOAc / isohexane) to afford benzyl (Z)-2-methyl-4-(phenylimino)-3,4- dihydroquinoline-1(2H)-carboxylate (1.08 g, 2.5 mmol, 67%, 86% purity) as a yellow oil.

[0151] 1H NMR in DMSO-d6 was consistent with product structure at 95% purity.1H NMR (400 MHz, DMSO) δ 8.22 (dd, J = 8.0, 1.6 Hz, 1H), 7.77 (dd, J = 8.5, 1.2 Hz, 1H), 7.51 – 7.29 (m, 8H), 7.25 – 7.15 (m, 1H), 7.13 – 7.04 (m, 1H), 6.87 – 6.71 (m, 2H), 5.30 – 5.18 (m, 2H), 4.97 – 4.86 (m, 1H), 2.90 (dd, J = 16.9, 5.5 Hz, 1H), 2.47 – 2.41 (m, 1H), 1.02 (d, J = 6.9 Hz, 3H).

[0152] The product was analysed by LCMS (Cortecs C18+, 90 Å, 30 x 2.1 mm, 2.7 μm, 3 min method, 0.1% formic acid, 2-100% MeCN / water): m / z 371.2 (M+H)+(ES+) at 2.03 min, 86.2% purity 260 nm + / - 90 nm.

[0153] Step 4: To a solution of benzyl (Z)-2-methyl-4-(phenylimino)-3,4- dihydroquinoline-1(2H)-carboxylate (1.00 g, 1 eq, 2.70 mmol) in MeOH (25.0 mL), NaBH4 (2.04 g, 20 eq, 54.0 mmol) was added by portions. The reaction was stirred overnight at rt. The mixture was concentrated to near dryness under vacuum. The residue was diluted with a sat. aq. solution of NaHCO3 (50 mL) and then extracted with ethyl acetate (3 x 150 mL). The combined organic layers were washed with brine (200 mL), dried over Na2SO4, filtered and concentrated in vacuo. The crude product was purified by chromatography on silica gel (40 g cartridge, 0-20% EtOAc / isohexane) to afford benzyl (2S,4S)-2-methyl-4- (phenylamino)-3,4-dihydroquinoline-1(2H)-carboxylate (0.209 g, 0.49 mmol, 18%, 88% purity) as a pale yellow solid and benzyl (2R,4S)-2-methyl-4-(phenylamino)-3,4- dihydroquinoline-1(2H)-carboxylate (0.328 g, 0.74 mmol, 27%, 84% purity) as a pale yellow solid. Trans isomer:

[0154] 1H NMR in DMSO-d6was consistent with product structure at 95% purity.1H NMR (400 MHz, DMSO) δ 7.62 (dd, J = 8.4, 1.2 Hz, 1H), 7.45 – 7.28 (m, 6H), 7.21 (ddd, J = 8.5, 7.3, 1.7 Hz, 1H), 7.10 – 7.01 (m, 3H), 6.73 – 6.66 (m, 2H), 6.53 (tt, J = 7.2, 1.1 Hz, 1H), 5.88 (d, J = 7.8 Hz, 1H), 5.27 – 5.15 (m, 2H), 4.66 (dq, J = 13.9, 7.0 Hz, 2H), 2.16 (ddd, J = 13.7, 8.3, 5.6 Hz, 1H), 1.88 (dt, J = 13.4, 5.5 Hz, 1H), 1.21 (d, J = 6.6 Hz, 3H).

[0155] The product was analysed by LCMS (Cortecs C18+, 90 Å, 30 x 2.1 mm, 2.7 μm, 3 min method, 0.1% formic acid, 2-100% MeCN / water): m / z 395.2 (M+Na)+(ES+) at 1.98 min, 87.9% purity 260 nm + / - 90 nm. Cis isomer:

[0156] 1H NMR in DMSO-d6 was consistent with product structure at 95% purity.1H NMR (400 MHz, DMSO) δ 7.48 – 7.33 (m, 5H), 7.20 (td, J = 7.5, 1.8 Hz, 1H), 7.15 – 6.97 (m, 4H), 6.69 – 6.60 (m, 2H), 6.55 (tt, J = 7.2, 1.1 Hz, 1H), 6.01 (d, J = 7.7 Hz, 1H), 5.30 – 5.13 (m, 2H), 4.51 (dt, J = 9.5, 7.8 Hz, 1H), 4.25 (s, 1H), 2.61 – 2.51 (m, 2H), 1.28 (td, J = 12.2, 9.7 Hz, 1H), 1.16 (d, J = 6.3 Hz, 3H).

[0157] The product was analysed by LCMS (Cortecs C18+, 90 Å, 30 x 2.1 mm, 2.7 μm, 3 min method, 0.1% formic acid, 2-100% MeCN / water): m / z 395.2 (M+Na)+(ES+) at 1.95 min, 84.1% purity 260 nm + / - 90 nm.

[0158] Step 5 (protocol for trans isomer): To a solution of benzyl (2S,4S)-2-methyl-4- (phenylamino)-3,4-dihydroquinoline-1(2H)-carboxylate (0.20 g, 1 eq, 0.54 mmol) in 1,4- dioxane (3.00 mL), DIPEA (0.10 g, 0.14 mL, 1.5 eq, 0.81 mmol) and acetic anhydride (0.55 g, 0.51 mL, 10 eq, 5.4 mmol) were added. The mixture was heated to 90°C and stirred for 18 hours. The mixture was cooled to rt, diluted with EtOAc (10 mL) and washed with sat. aq. NaHCO3 solution (10 mL). The aqueous layer was extracted with EtOAc (2 x 10 mL). The organic layers were combined, dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by chromatography on silica gel (24 g cartridge, 0-50% EtOAc / isohexane) to afford benzyl (2S,4S)-2-methyl-4-(N-phenylacetamido)-3,4- dihydroquinoline-1(2H)-carboxylate (0.189 g, 0.43 mmol, 81%, 95% purity) as a pale yellow solid.

[0159] 1H NMR in DMSO-d6 was consistent with product structure at 95% purity.1H NMR (400 MHz, DMSO) δ 7.58 – 7.51 (m, 1H), 7.47 – 7.40 (m, 1H), 7.41 – 7.24 (m, 8H), 7.23 – 7.11 (m, 2H), 7.00 (s, 2H), 6.04 (t, J = 8.7 Hz, 1H), 5.10 – 4.98 (m, 2H), 4.62 (dtd, J = 11.5, 6.8, 4.6 Hz, 1H), 2.01 – 1.86 (m, 2H), 1.76 (s, 3H), 1.12 (d, J = 6.8 Hz, 3H).

[0160] The product was analysed by LCMS (Cortecs C18+, 90 Å, 30 x 2.1 mm, 2.7 μm, 3 min method, 0.1% formic acid, 2-100% MeCN / water): m / z 437.2 (M+Na)+(ES+) at 1.74 min, 95.7% purity 260 nm + / - 90 nm.

[0161] Step 5 (protocol for cis isomer): To a solution of benzyl (2R,4S)-2-methyl-4- (phenylamino)-3,4-dihydroquinoline-1(2H)-carboxylate (0.32 g, 1 eq, 0.86 mmol) in 1,4- dioxane (3.00 mL), DIPEA (0.17 g, 0.22 mL, 1.5 eq, 1.3 mmol) and acetic anhydride (0.88 g, 0.81 mL, 10 eq, 8.6 mmol) were added. The mixture was heated to 90°C and stirred for 18 hours. The mixture was cooled to rt, diluted with EtOAc (10 mL) and washed with sat. aq. NaHCO3solution (10 mL). The aqueous layer was extracted with EtOAc (2 x 10 mL). The organic layers were combined, dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by chromatography on silica gel (24 g cartridge, 0-100% EtOAc / isohexane) to afford benzyl (2R,4S)-2-methyl-4-(N-phenylacetamido)-3,4- dihydroquinoline-1(2H)-carboxylate (0.094 g, 0.20 mmol, 23%, 86% purity) as a pale yellow solid.

[0162] 1H NMR in DMSO-d6was consistent with product structure at 90% purity.1H NMR (400 MHz, DMSO) δ 7.60 – 7.53 (m, 1H), 7.48 – 7.11 (m, 13H), 5.20 (d, J = 12.5 Hz, 1H), 5.12 (d, J = 12.5 Hz, 1H), 4.39 (dt, J = 16.2, 7.0 Hz, 1H), 4.16 – 4.04 (m, 1H), 2.26 (s, 1H), 1.88 (d, J = 4.2 Hz, 3H), 1.17 (t, J = 7.1 Hz, 1H), 1.06 – 0.99 (m, 3H).

[0163] The product was analysed by LCMS (Cortecs C18+, 90 Å, 30 x 2.1 mm, 2.7 μm, 3 min method, 0.1% formic acid, 2-100% MeCN / water): m / z 437.2 (M+Na)+(ES+) at 1.79 min, 86.4% purity 260 nm + / - 90 nm.

[0164] Step 6 (protocol for trans isomer): To a solution of benzyl (2S,4S)-2-methyl-4-(N- phenylacetamido)-3,4-dihydroquinoline-1(2H)-carboxylate (0.10 g, 1 eq, 0.24 mmol) in ethanol (2.00 mL), ammonium formate (0.15 g, 10 eq, 2.4 mmol) and Pd / C (26 mg, 10% wt, 0.1 eq, 24 μmol) were added. The mixture was heated to 80°C and stirred for 2 hours. The mixture was cooled to rt, filtered in celite, washed with EtOH (10 mL) and the filtrate was concentrated in vacuo. The crude product was purified by chromatography on silica gel (12 g cartridge, 0-5% (0.7 M ammonia / MeOH) / DCM) to afford the desired product with low purity. The residue was purified by chromatography on RP Flash C18 (12 g cartridge, 10-60% (0.1% formic acid in MeCN) / (0.1% formic acid in water)) to afford N- ((2S,4S)-2-methyl-1,2,3,4-tetrahydroquinolin-4-yl)-N-phenylacetamide (40.0 mg, 0.14 mmol, 56%, 95% purity) as a white solid.

[0165] 1H NMR in DMSO-d6 at 363 K was consistent with product structure at >95% purity.1H NMR (400 MHz, DMSO) δ 7.29 (dd, J = 4.8, 2.0 Hz, 3H), 7.21 – 7.15 (m, 1H), 6.97 (dd, J = 6.5, 2.9 Hz, 2H), 6.95 – 6.87 (m, 1H), 6.55 (td, J = 7.4, 1.3 Hz, 1H), 6.44 (dd, J = 8.0, 1.2 Hz, 1H), 6.01 – 5.94 (m, 1H), 5.25 (s, 1H), 2.66 (ddt, J = 9.6, 4.5, 1.9 Hz, 1H), 1.92 (dddd, J = 13.7, 4.7, 3.6, 1.2 Hz, 1H), 1.76 (s, 3H), 1.66 (ddd, J = 13.8, 9.4, 6.5 Hz, 1H), 0.99 (d, J = 6.3 Hz, 3H).

[0166] The product was analysed by LCMS (Cortecs C18+, 90 Å, 30 x 2.1 mm, 2.7 μm, 3 min method, 0.1% formic acid, 2-100% MeCN / water): m / z 281.2 (M+H)+(ES+) at 1.23 min, 96.1% purity 260 nm + / - 90 nm.

[0167] Step 6 (protocol for cis isomer): To a solution of benzyl (2R,4S)-2-methyl-4-(N- phenylacetamido)-3,4-dihydroquinoline-1(2H)-carboxylate (0.10 g, 1 eq, 0.24 mmol) in ethanol (2.00 mL), ammonium formate (0.15 g, 10 eq, 2.4 mmol) and Pd / C (51 mg, 5% wt, 0.1 eq, 24 μmol) were added. The mixture was heated to 80°C and stirred for 2 hours. The mixture was cooled to rt, filtered in celite, washed with EtOH (10 mL) and the filtrate was concentrated in vacuo. The crude product was purified by chromatography on silica gel (12 g cartridge, 0-5% (0.7 M ammonia / MeOH) / DCM) to afford the desired product with low purity. The residue was purified by chromatography on RP Flash C18 (12 g cartridge, 10- 60% (0.1% formic acid in MeCN) / (0.1% formic acid in water)) to afford N-((2R,4S)-2-methyl-1,2,3,4-tetrahydroquinolin-4-yl)-N-phenylacetamide (15.0 mg, 51 μmol, 21%, 95% purity) as a white solid.

[0168] 1H NMR in DMSO-d6 at 363 K was consistent with product structure at >95% purity.1H NMR (400 MHz, DMSO) δ 7.39 – 7.28 (m, 3H), 7.15 – 7.06 (m, 3H), 6.91 (td, J = 7.9, 1.4 Hz, 1H), 6.57 (td, J = 7.4, 1.2 Hz, 1H), 6.46 (dd, J = 8.1, 1.2 Hz, 1H), 5.99 (s, 1H), 5.21 (s, 1H), 3.47 (ddd, J = 12.9, 7.6, 3.6 Hz, 1H), 1.90 (dd, J = 11.8, 5.6 Hz, 1H), 1.83 (s, 3H), 1.25 (d, J = 12.6 Hz, 1H), 1.07 (d, J = 6.2 Hz, 3H).

[0169] The product was analysed by LCMS (Cortecs C18+, 90 Å, 30 x 2.1 mm, 2.7 μm, 3 min method, 0.1% formic acid, 2-100% MeCN / water): m / z 281.2 (M+H)+(ES+) at 1.20 min, 98.5% purity 260 nm + / - 90 nm. EXAMPLE 10 Biological Assays In vitro FOXN1 activation

[0170] Principle: The primary screening assay used hTEC SVERT 24-B cells containing a NanoLuc luciferase reporter vector under the control of a sequence upstream of FOXN1. Therefore, the primary assay aimed to identify modulators of FOXN1 by identifying compounds which alter luciferase expression in a dose dependent manner via Nano-Glo luciferase assay system (Promega). The counter screen assay uses the same cell line with the NanoLuc luciferase reporter vector however with no promoter region and thus aims to identify compounds that promote luciferase expression in a FOXN1 upstream sequence- independent manner.

[0171] The Nano-Glo luciferase assay system (Promega) worked by providing a substrate (furimazine) for luciferase which produced a product with high intensity, glow type luminescence. This product can be detected by a luminometer.

[0172] Experimental process: Compounds were dosed into 384 well plates using an acoustic liquid handler (ECHO650) to provide 10-point, 2-fold dilutions of each compound (plate also includes DMSO and tool compound controls). Cells were then seeded into these plates using an automated liquid handler (multidrop) and incubated at 37°C, 5% CO2for 24 hours. After this incubation, plates were equilibrated to room temperature and the Nano- Glo luciferase detection reagent (Promega) was added to each well. Plates were then incubated for 10 minutes before luminescence was read using a plate reader. Luminescenceread outs for each compound were normalised to DMSO only controls which were then used to fit dose response curves and calculate EC50 values. Table 2A = <25 µM; B = 25-50 µM; C = 51-75 µM; D = 76-99 µM; E = >99 µM

[0173] It is to be understood that the foregoing embodiments and exemplifications are not intended to be limiting in any respect to the scope of the disclosure, and that the claims presented herein are intended to encompass all embodiments and exemplifications whether or not explicitly presented herein

[0174] All patents and publications cited herein are fully incorporated by reference in their entirety.

Claims

WHAT IS CLAIMED IS:

1. A method of promoting thymus regeneration in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound selected from the group consisting of:, , ,or a pharmaceutically acceptable salt or solvate thereof.

2. The method of claim 1, wherein the compound is selected from the group consisting3. The method of claim 1, wherein the compound is selected from the group consisting of:.

4. The method of claim 1, wherein the compound is selected from the group consisting of:.

5. The method of any one of claims 1-4, wherein the subject is a human.

6. The method of any one of claims 1-5, wherein the thymus of the subject is damaged by stress, infection, glucocorticoids, cytoreductive therapies, aging, or a combination thereof.

7. The method of any one of claims 1-6, wherein the subject is in need of thymus regeneration due to hematopoietic stem cell transplantation, immune deficiency due to HIV, HIV therapy, autoimmune disease, inborn errors of thymic development, administration of a vaccine, age-related immune senescence, administration of exogenous glucocorticoids, bacterial or viral infections, radiation, immunotherapy, cytoreductive treatments, zinc deficiency, thymus involution, or a combination thereof.

8. A method of promoting immune reconstitution in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound selected from the group consisting of:or a pharmaceutically acceptable salt or solvate thereof.

9. The method of claim 8, wherein the compound is selected from the group consisting of:,10. The method of claim 8, wherein the compound is selected from the group consisting of:.

11. The method of claim 8, wherein the compound is selected from the group consisting of:.

12. The method of any one of claims 8-11, wherein the subject is a human.

13. The method of any one of claims 8-12, wherein the thymus of the subject is damaged by stress, infection, glucocorticoids, cytoreductive therapies, aging, or a combination thereof.

14. The method of any one of claims 8-13, wherein the subject is in need of immune reconstitution due to hematopoietic stem cell transplantation, immune deficiency due to HIV, HIV therapy, autoimmune disease, inborn errors of thymic development, administration of a vaccine, age-related immune senescence, administration of exogenous glucocorticoids, bacterial or viral infections, radiation, immunotherapy, cytoreductive treatments, zinc deficiency, thymus involution, or a combination thereof.

15. A method of activating FOXN1 in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound selected from the group consisting of:or a pharmaceutically acceptable salt or solvate thereof.

16. The method of claim 15, wherein the compound is selected from the group consisting of:,17. The method of claim 15, wherein the compound is selected from the group consisting of:.

18. The method of claim 15, wherein the compound is selected from the group consisting of:

19. The method of any one of claims 15-18, wherein the subject is in need of activating FOXN1 because the thymus of the subject is damaged by stress, infection, glucocorticoids, cytoreductive therapies, aging, or a combination thereof.

20. The method of any one of claims 15-19, wherein the subject is in need of FOXN1 activation due to hematopoietic stem cell transplantation, immune deficiency due to HIV, HIV therapy, autoimmune disease, inborn errors of thymic development, administration of a vaccine, age-related immune senescence, administration of exogenous glucocorticoids, bacterial or viral infections, radiation, immunotherapy, cytoreductive treatments, zinc deficiency, thymus involution, diabetes-related ulcers, foot ulcers, chronic wounds, alopecia, or a combination thereof.

21. The method of any one of claims 15-20, wherein the subject is in need of FOXN1 activation to promote skin wound healing.

22. The method of claim 21, wherein the subject has a diabetes-related ulcer, a foot ulcer, a chronic wound, or a combination thereof.

23. The method of any one of claims 15-22, wherein the subject is in need of FOXN1 activation to augment hair growth.

24. The method of claim 23, wherein the subject has alopecia.

25. The method of any one of claims 15-24, wherein the subject is a human.

26. A method of enhancing immune function or promoting thymic regeneration in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound selected from the group consisting of:or a pharmaceutically acceptable salt or solvate thereof, wherein the subject is in need of immune function enhancement or thymic regeneration due to administration of CAR-T cells or other adoptive T-cell therapies, recovery from sepsis or critical illness, idiopathic T-cell lymphopenia, immunosuppression following solid organ transplantation, or a combination thereof.

27. The method of claim 26, wherein the subject is at risk of or recovering from infection due to impaired T-cell function.

28. The method of claim 27, wherein the subject is an elderly individual, an immunocompromised patient, a recipient of immunosuppressive therapy, or a combination thereof.

29. A method of promoting immune tolerance or reducing transplant-related complications in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound selected from the group consisting of:or a pharmaceutically acceptable salt or solvate thereof, wherein the subject is receiving a hematopoietic stem cell transplant or solid organ transplant.

30. The method of claim 29, wherein the method reduces the risk of graft-versus-host disease (GVHD).

31. The method of claim 29, wherein the method enhances immune reconstitution.

32. A method of treating or supporting recovery from a thymic epithelial tumor in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound selected from the group consisting of:or a pharmaceutically acceptable salt or solvate thereof, wherein the subject has a thymoma or thymic carcinoma.

33. A method of promoting immune surveillance or reducing cancer risk in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound selected from the group consisting of:or a pharmaceutically acceptable salt or solvate thereof, wherein the subject has an age-related decline in immune function, a personal or familial history of cancer, or a genetic predisposition to immune dysfunction.

34. A method of augmenting hair growth or restoring hair loss in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound selected from the group consisting of:or a pharmaceutically acceptable salt or solvate thereof, wherein the hair loss is chemotherapy-induced, radiation-induced, or idiopathic in origin.

35. A method of promoting skin repair or regeneration in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound selected from the group consisting of:,or a pharmaceutically acceptable salt or solvate thereof, wherein the subject has a radiation-induced skin injury, a surgical wound, or age-related skin atrophy.

36. A method of promoting dermal regeneration or treating skin aging in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound selected from the group consisting of:or a pharmaceutically acceptable salt or solvate thereof.

37. A method of preconditioning a non-human donor organism for xenotransplantation of a composite organ comprising thymic tissue, comprising administering to the donor a therapeutically effective amount of a compound selected from the group consisting of:or a pharmaceutically acceptable salt or solvate thereof, wherein the method improves thymic tissue function, promotes immune system development, or enhances compatibility of the graft with the recipient immune system.

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