N1-(3,4-dichlorophenyl)maleamide derivatives and the use thereof in the treatment of inflammatory disorders

Abstract

Provided herein are compounds of Formula I which are thymic stromal lymphopoietin (TSLP) or thymic stromal lymphopoietin receptor (TSLPR) inhibitory compounds, capable of disrupting the protein-protein interaction between TSLP and TSLPR. Compounds of Formula I and compositions thereof modulate activity or inhibit TSLP and TSLPR binding, and are useful for downregulating atopy-relevant pro-inflammatory cytokines. In particular, compounds of Formula I as described herein are useful in the treatment of skin disease, allergic asthma, allergic rhinitis, drug allergies, and food allergies. Furthermore, the compounds described herein are absorbed by the skin. (I)

Description

[0001] N1-(3,4-DICHLOROPHENYL)MALEAMIDE DERIVATIVES AND THE USE THEREOF IN THE TREATMENT OF INFLAMMATORY DISORDERS

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 632,836 filed 11 April 2024 entitled "SMALL MOLECULE INHIBITORS FOR THE TREATMENT OF INFLAMMATORY DISORDERS".

[0004] TECHNICAL FIELD

[0005] This invention relates to the field of thymic stromal lymphopoietin receptor (TSLPR) complex inhibitor compounds. In particular, the TSLPR inhibitor compounds are described herein for use in the treatment of TSLP-mediated diseases.

[0006] BACKGROUND

[0007] Epithelium-derived thymic stromal lymphopoietin (TSLP) is a master regulator of type 2 helper T (Th2) cell-driven inflammation. As such, TSLP is distinctly increased in skin and lung tissue of atopic dermatitis (AD) patients and asthmatics, respectively [1-3]. AD is the most common inflammatory skin disease worldwide with currently up to 25% of children and 1-3% of adults being affected [4]. Besides genetic predispositions, environmental and immunological factors contribute to AD manifestation and pathogenesis rendering it a multifactorial disease. Defects in the epidermal barrier of human skin favor sensitization and strong inflammatory responses to external stimuli ultimately triggering a systemic response [5]. These skin barrier defects may be inherited. Prominent examples are mutations in the filaggrin gene, which have been identified in 10-50% of AD patients and are a major predisposing factor for AD [6].

[0008] Notably, < 50% of AD patients with moderate-to-severe phenotype undergo the atopic march meaning concomitant sensitization to indoor and later outdoor allergens, progressing to asthma and allergic rhinitis at later time points [7, 8]. Although the underlying mechanism remains largely unknown, the contribution of TSLP has been firmly established [8-12]. TSLP is an IL-7-like cytokine that exerts its biological activities by binding to its receptor (i.e. thymic stromal lymphopoietin receptor (TSLPR)), the TSLP-TSLPR heterodimer then recruits the IL-7 receptor a to form the active receptor complex [5, 6]. This receptor complex is expressed by a wide range of immune cells including dendritic cells (DCs), macrophages, and T cells, but also epithelial cells and neurons [5]. TSLP binding activates the TSLPR complex, which in turn activates protein tyrosine kinases and transcription factors including STATS, STATS, STAT6, and GATA3, which promote the expression of inflammatory cytokines such as IL-4 and IL-13 [7, 8]. Moreover, TSLP activates DCs that subsequently prime human CD4+ T cells into Th2 cytokine-producing cells in local lymph nodes [9-11]. TSLP signaling in CD4+ T cells is also required for memory formation after Th2 sensitization

[0012] and the activation of group 2 innate lymphoid cells, which further drive the pathogenesis of inflammatory skin diseases

[0013] . Recent data also indicate that TSLP triggers transcriptional changes in Th2 cells skewing them towards pathogenic phenotypes that produce greater amounts of pro-inflammatory cytokines than conventional Th2 cells [8].

[0009] Two isoforms of human TSLP have been identified: short and long TSLP. The short isoform (sfTSLP; 63 - 60 amino acids) is constitutively expressed, critical for epithelial homeostasis, and does not bind to the TSLPR. In contrast, IfTSLP (long isoform, 159 amino acids) is inducible, can be massively upregulated in atopic diseases, and promotes inflammation rendering both, IfTSLP and TSLPR, putative therapeutic targets [14, 15]. Accordingly, the inhibition or knockout of TSLP signaling has shown great therapeutic potential

[0016] .

[0010] The knowledge about the central role of TSLP in atopic diseases has sparked interest in therapeutically targeting TSLP. In fact, the anti-TSLP antibody tezepelumab (AstraZeneca AB™) is now being approved for the treatment of severe asthma

[0017] .

[0011] However, small molecule TSLP inhibitors, which would allow a topical, non-invasive application, have not yet been developed as disrupting protein-protein interactions with small molecules is challenging. Nonetheless, in addition to lower costs of small molecules versus biologies, a topical small molecule therapeutic would be of valuable since the TSLPR complex drives the initial sensitization for allergic asthma locally in the skin [8, 18, 19]. SUMMARY

[0012] The present invention is based in part, on the surprising discovery that compounds of Formula I as described herein, have thymic stromal lymphopoietin receptor (TSLPR) complex formation inhibitory activity. The compounds of Formula I represent potent and safe small molecule TSLPR inhibitors, suitable for topical administration and which provide the opportunity not only to expand the therapeutic and preventive options in atopic diseases, provide inhibitors that target TSLP / TSLPR / IL4 / IL13 related diseases and / or and inflammatory diseases. The compounds may be useful in the treatment of one or more of the following: skin disease; asthma; allergies; chronic obstructive pulmonary disease (COPD); chronic rhinosinusitis; atopic kerato-conjunctivitis; non-celiac gluten sensitivity; eosinophilic granulomatosis with polyangiitis (EGPA); and eosinophilic esophagitis (EoE). Alternatively, the compounds may be useful in the treatment of one or more of the following: skin disease; allergic asthma; allergic rhinitis; drug allergies; and food allergies. A skin disease may be selected from one or more of the following: atopic dermatitis; urticaria; skin granulomas; nonthrombocytopenic palpable purpura; skin infarcts; and livedo reticulari. Allergies may be selected from one or more of the following: allergic asthma; allergic rhinitis; drug allergies; and food allergies.

[0013] Also described herein is a human-based atopic diseases drug discovery platform that combines a bioengineered atopic-like skin disease model and 3D bronchial epithelial model on a microfluidic multi-organ chip. This platform enabled us to study the preclinical efficacy and safety of our compounds in a complex human-like environment. This has great translational potential due to the avoidance of species-related differences, which are prominent and challenging in the TSLP context.

[0014] In a first embodiment, there is provided a compound, the compound having the structure of

[0015] Formula I , wherein, Ri is selected from: H;

[0016] or a pharmaceutically acceptable salts, solvates, hydrates, hydrated salts, or optical isomers, racemates, diastereoisomers or enantiomers thereof.

[0017] In a further embodiment, there is provided a compound, the compound having the structure of Formula I: wherein, Ri is selected from: H; or a pharmaceutically acceptable salts, solvates, hydrates, hydrated salts, or optical isomers, racemates, diastereoisomers or enantiomers thereof, for use in inhibiting thymic stromal lymphopoietin receptor (TSLPR) complex formation.

[0018] In a further embodiment, there is provided a pharmaceutical composition, the pharmaceutical composition comprises a compound described herein and a pharmaceutically acceptable carrier.

[0019] In a further embodiment, there is provided a use of a pharmaceutical composition comprising a compound as described herein and a pharmaceutically acceptable carrier, for one or more of the following: (a) treating one or more of the following: skin disease; asthma; allergies; chronic obstructive pulmonary disease (COPD); chronic rhinosinusitis; atopic kerato-conjunctivitis; non-celiac gluten sensitivity; eosinophilic granulomatosis with polyangiitis (EGPA); and eosinophilic esophagitis (EoE), wherein the pharmaceutical composition comprises a compound s and a pharmaceutically acceptable carrier; (b) modulating cytokine expression; and (c) modulating TSLPR activity or inhibiting TSLPR. in a further embodiment, there is provided use of a compound as described herein in the manufacture of a medicament for one or more of the following: (a) treating one or more of the following: skin disease; asthma; allergies; COPD; chronic rhinosinusitis; atopic keratoconjunctivitis; non-celiac gluten sensitivity; EGPA; and EoE, wherein the pharmaceutical composition comprises a compound of any one of claims 1-4 and a pharmaceutically acceptable carrier; (b) modulating cytokine expression; and (c) modulating TSLPR activity or inhibiting TSLPR.

[0020] In a further embodiment, there is provided a use of a compound described herein for one or more of the following: (a) treating one or more of the following: skin disease; asthma; allergies; COPD; chronic rhinosinusitis; atopic kerato-conjunctivitis; non-celiac gluten sensitivity; EGPA; and EoE; (b) modulating cytokine expression; and (c) modulating TSLPR activity or inhibiting TSLPR. In a further embodiment, there is provided a method of treating one or more of the following: skin disease; asthma; allergies; COPD; chronic rhinosinusitis; atopic keratoconjunctivitis; non-celiac gluten sensitivity; EGPA; and EoE, the method comprising administering a compound as described herein to a patient in need thereof.

[0021] In a further embodiment, there is provided a method of modulating cytokine expression, the method comprising administering a compound described herein to a patient in need thereof.

[0022] In a further embodiment, there is provided a method of modulating TSLPR activity or inhibiting TSLPR, the method comprising administering a compound described herein to a patient in need thereof.

[0023] In a further embodiment, there is provided a compound as described herein, for inhibiting TSLPR complex formation modulates cytokine expression.

[0024] In a further embodiment, there is provided a compound as described herein, for modulating cytokine expression modulates IL4 or IL 13 expression.

[0025] In a further embodiment, there is provided a compound as described herein, for treating one or more of the following: skin disease; allergic asthma; allergic rhinitis; drug allergies; and food allergies.

[0026] In a further embodiment, there is provided a compound as described herein, for treating one or more of the following: skin disease; asthma; allergies; chronic obstructive pulmonary disease (COPD); chronic rhinosinusitis; atopic kerato-conjunctivitis; non-celiac gluten sensitivity; eosinophilic granulomatosis with polyangiitis (EGPA); and eosinophilic esophagitis (EoE). R1may be selected from may be selected from: H R1may be selected from:

[0027] Alternatively, R1may be selected from: H or a pharmaceutically acceptable salts, solvates, hydrates, hydrated salts, or optical isomers, racemates, diastereoisomers or enantiomers thereof, when used in inhibiting thymic stromal lymphopoietin receptor (TSLPR) complex formation. R1may be selected from: H; P and R1may be selected from: H ; and R1may be selected from: and R1may be selected from: H ; and I . R1may be selected from: H and . R1may be selected from: H; ; and . R1may be selected from: H; ; and R1may be selected from: H; and R1maybe selected from: H, P- . R1may be selected from: H;

[0028]

[0029] The skin disease may be selected from one or more of the following: atopic dermatitis; urticaria; skin granulomas; nonthrombocytopenic palpable purpura; skin infarcts; and livedo reticulari.

[0030] The allergies may be selected from one or more of the following: allergic asthma; allergic rhinitis; drug allergies; and food allergies. The allergy may be a skin allergy.

[0031] The modulating of cytokine expression may be modulating of IL4 or IL13.

[0032] The compounds or pharmaceutical compositions may be for the treatment of: an inflammatory skin disease; an atopic disease; a skin disease; an allergic asthma; an allergic rhinitis; and a food allergy.

[0033] The compounds or pharmaceutical compositions may be for the treatment of an atopic dermatitis or any IgE related condition. The compounds or pharmaceutical compositions may be for the treatment of: an atopic dermatitis; atopic keratoconjunctivitis; eosinophilic esophagitis (EoE); and non-celiac gluten sensitivity.

[0034] The compounds or pharmaceutical compositions may be for the treatment of chronic obstructive pulmonary disease; chronic rhinosinusitis with nasal polyps; chronic spontaneous urticaria; and eosinophilic esophagitis (EoE); and severe asthma. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIGURE 1 shows the identification of both TSLPR and TSLP inhibitors, a) Schematic representation of TSLP, TSLPR and IL-7Ra forming a ternary complex; binding interfaces are termed as site I, II and III. b) Overview of the virtual screening of potential small-molecule TSLP inhibitors and compound selection. A library of compounds from Specs and VitasM™ was screened using 3D pharmacophore models, c, d) In vitro screening of twelve potential compounds in TSLP-activated HuT78 cells. Cells were treated with different concentrations of putative TSLP / TSLP receptor inhibitors for 36 hours. IL-4 and IL-13 expression were detected with ELISA. Mean ± sem., n=3. e, f) Inhibition of Th2 cytokine secretion from human primary CD4+T cells. TSLP activated human primary CD4+T cells from three donors were treated with different concentrations of compound 7,13 and 14 for 36 hours followed by quantification of IL-13 and IL-4 secretion via ELISA. Mean ± sem., n=3. g, h, i) Cell viability. Primary human keratinocytes, dermal fibroblasts, and HuT78 cells were treated with different concentrations of compound C7, C13 and C14. After 24 hours, cell viability was determined by MTT assay. Mean ± sem., n=3. j) Proposed binding modes: The proposed binding mode ofTSOPR- targeting C7 resembles the part of TSLP in site I. The nitrogen atoms present in in the imidazole ring mimic the interaction formed by Argl53 of TSLP with Vall93 of TSLPR thus anchoring the molecule in the interdomain elbow region of TSLPR. Additionally, the dichlorophenyl moiety forms hydrophobic interactions with Leu39, Tyrl43, Tyrl94 and Vail 14 in our docking experiments thus suggesting further stabilization of the surmised binding conformation. The proposed binding mode of TSLP-targeting C13 is characterized by charge interactions and a hydrogen bonding network of the carboxylate moiety with Arg150 and Argl53 of TSLP, while the methylphenyl ring is interacting with Val67 and Leul47. In addition, there is a pi-cation interaction with Argl53

[0036] FIGURE 2 shows the development of initial novel TSLP-TSLPR inhibitors, a) Design of new TSLP inhibitors incorporating features of potent compounds from initial screening campaign and hits from the NCI-DTP library, b) Compounds synthesized to optimize TSLPR inhibitory activity, c, d) Inhibition of IL-13 and IL-4 secretion from Hut78 cells determined by ELISA, were synthesized. TSLP activated HuT78 cells were treated with different concentrations of 16 newly synthesized TSLP inhibitors for 36 hours. Mean ± se., n=4. e, f) TSLP-activated human primary CD4+T cells were treated with 20 μM of four promising compounds. Mean ± s.e. n=3. *p<0.05, **p<0.01, ***p<0.001. g) Crystal structure of lead compound BP79, confirming the alkene substituents are in the cis-orientation and an intramolecular hydrogen bond between the aniline nitrogen atom and distal carbonyl oxygen confers rigidity to the overall structure.

[0037] FIGURE 3 shows the functional validation of BP79. a, b) TSLP-activated primary CD4+T cells were treated with different concentrations of the TSLP inhibitors BP75 and BP79 for 36 hours, respectively. IL-13 and IL-4 secretion was quantified by ELISA. Mean ± s.e. n=6. c, d, e) Cytotoxicity of BP79 in primary human keratinocytes (n=4), fibroblasts (n=5), and CD4+ T cells (n=5) determined by MTT assay. Mean ± s.e. f) TSLP mediated Signaling inhibition. TSLP activates STAT6 in CD4+T cells and enhance Th2 cytokine secretion and activates STATS in keratinocytes and regulate filaggrin expression, g) Inhibition of TSLP-mediated T cell hyperproliferation. Primary CD4+T cells were treated with TSLP inhibitors (or DMSO) and stimulated with TSLP for 1, 2 and 5 days. Cell proliferation was determined using a cell counter. Mean ± s.e. *p<0.05, **p<0.01, ***p<0.001. g) Primary human CD4+ T cells and h) keratinocytes were treated with BP79 and stimulated with TSLP. BP79 inhibited STAT6 and STATS activation in primary CD4+T cells and primary keratinocytes, respectively.

[0038] FIGURE 4 shows target engagement and specificity of BP79. a) Proximity ligation assay (PLA): TSLP binds to TSLPR and forms a ternary complex with IL-7Ra at the cell surface initiating intracellular signaling. PLA was performed to verify the inhibition of TSLP- mediated ternary complex formation via BP79. Primary keratinocyte cells were treated with the TSLP inhibitors and stimulated with TSLP. Cells were fixed and the interaction between

[0039] TSLPR and IL-7Ra was observed with PLA assay. BP79 inhibited the TSLP:TSLPR:IL- 7Ra ternary complex formation, b) In vitro thermal shift assay. BP79 thermally stabilized TSLPR up to 45°C. c) To determine if BP79 decreases the cytokine expression through directbinding of upstream kinases such as JAK1 / 2 or other kinase targets, BP79 was screened against 97 kinase targets using the KINOMEscan™ platform (radio-ligand displacement assay performed by Eurofins Discovery Services). FIGURE 5 shows a) Skin permeation of BP79 was determined using a Franz cell setup followed by MS analysis showing BP79 permeation after 8 and 24 hours, respectively, b) Expression of filaggrin and TSLP in normal (FLG+) and atopic-like (FLG-) skin models as determined by immunofluorescence staining, c) Efficacy testing of BP79 in normal (FLG+) and atopic-like (FLG-) skin models in the presence or absence of activated CD4+ cells emulating atopic-like disease conditions in a human-based setup. BP79 or the DMSO vehicle control were topically applied over three consecutive days. Cytokine expression was then quantified using MSD U-PLEX Cytokine Array Assay Kit™.

[0040] FIGURE 6 shows development and utilization of an atopic diseases-organ chip for preclinical BP79 testing, a, b) Normal or atopic-like skin (disease) models were co-cultivated with 3D normal bronchial epithelial models on a microfluidic two-organ chip. Activated CD4+ T cells were added to the circuitto closely emulate the disease conditions. Following chip and model preparation, BP79 and the reference drug tacrolimus were topically applied once / day over four days onto the skin (disease) model. Chip experiment was terminated after 4 days followed by comprehensive chip and model characterization, c) H&E staining of cocultivated skin (normal and diseased) and lung tissue models w / or w / o topical application of BP79 or tacrolimus, d) Culture media was collected from the chip followed by cytokine expression analysis using ELISA. Topical application of BP79 significantly reduced IL-13, IL- 4, TSLP and periostin. Also, mode of action-specific effects was reflected by this analysis showing distinct differences in the treatment responses to BP79 and tacrolimus, e, f) Except for LDH and glucose concentration fluctuations due to media changes, LDH and glucose levels indicate stable co-culture conditions, g) Filaggrin expression in untreated (control), BP79- treated, and tacrolimus-treated atopic-like skin disease models, h) TSLP expression and CD4+T cell infiltration in healthy, untreated and BP79 & tacrolimus-treated atopic-like skin disease models. BP79 treatment significantly reduced TSLP expression and CD4+T cell migration compared to untreated disease models, i) Volcano plot showing the differential expression between treated and untreated diseased skin. Top 20 gene names are indicated on the plot. Genes significant at FDR < 0.01 with at least 2-fold difference between conditions are indicated by color, j) Treated diseased skin is more similar to untreated healthy skin than to treated healthy skin: A, Spearman correlation between averaged expression values for the three groups: healthy skin, untreated; diseased skin, untreated; diseased skin, treated. B, number of DEGs at FDR < 0.01 and absolute log2 FC > 1 (at least two-fold change), k) Selected results of gene set enrichments. The curves are receiver-operator characteristic (ROC) curves showing enrichment of a gene set for a given contrast. X axis corresponds to the ordered list of genes; Y axis correspond to the fraction of the given gene set present in the percentile of the list indicated by the X. (A) and (B) show gene set enrichments in the comparison of diseased skin, untreated and diseased skin, treated; (C) and (D) show gene set enrichment in the contrast treated vs untreated diseased skin, co-cultured with lung tissue. (A), protein synthesis (tmod gene set collection, ID DC.M5.9); (B), ribosome (KEGG pathways gene set, ID hsa03010); (C), aminoacyl tRNA synthesis (KEGG pathways gene set, ID hsa00970); (D), sodium proton exchangers (REACTOME pathways gene set, ID R-HSA- 425986). All gene set enrichments are significant at FDR < 0.001. *p<0.05, **p<0.01, ***p<0.001.

[0041] FIGURE 7 shows further development Biological screen of 26 BP79 analogs, a) IL-13 expression in HuT78 cells after treatment with 1μM of inhibitor for 36 hours, b) IL-4 expression in HuT78 cells after treatment with 1μM of inhibitor for 36 hours. Bars represent mean percent cytokine expression relative to the DMSO control ± S.E.M. between experiments. N = 2 independent experiments performed in technical duplicates. Nontreated (NT), PMA-ionomycin stimulated (PI), PMA-ionomycin and TSLP stimulated (PI + TSLP), a PMA-ionomycin + TSLP stimulated 0.4% DMSO vehicle control (DMSO) were included.

[0042] FIGURE 8 shows a dose-dependent screen of eight BP79 analogs in CD4+ T-cells, a) IL-13 expression in primary CD4+ T-cells after treatment with lllnM, 333nM, and lOOOnM of inhibitor for 36 hours, b) IL-4 expression primary CD4+ T-cells after treatment with lllnM, 333nM, and lOOOnM of inhibitor for 36 hours. Bars represent mean percent cytokine expression relative to the DMSO control ± S.E.M., N = 3 independent experiments conducted in technical duplicates. Non-treated (NT), CD3 / CD28 stimulated (CD3 / CD28), CD3 / CD28 and TSLP stimulated (CD3 / CD28 + TSLP), and vehicle (CD3 / CD28 + TSLP + DMSO) controls were included. FIGURE 9 shows Optimization of TSLP mediated Th2 cytokines secretion assay condition.

[0043] Bars represent the mean ± s.d., n=3. *p<0.05, **p<0.01, ***p<0.001.

[0044] FIGURE 10 shows Optimization of TSLP mediated Th2 cytokines secretion assay condition.

[0045] Bars represent the mean ± s.d., n=2.

[0046] DETAILED DESCRIPTION

[0047] The following detailed description will be better understood when read in conjunction with the appended figures. For the purpose of illustrating the invention, the figures demonstrate embodiments of the present invention. However, the invention is not limited to the precise arrangements, examples, and instrumentalities shown.

[0048] Any terms not directly defined herein shall be understood to have the meanings commonly associated with them as understood within the art of the invention.

[0049] "Inhibition of thymic stromal lymphopoietin receptor (TSLPR) complex formation" as used herein is meant to encompass interference with the ability of TSLPR to interact with long form thymic stromal lymphopoietin (IfTSLP) and / or interleukin 7 receptor a-chain (IL- 7Ra). Particularly, such that there may be a reduction in Th2 cytokine-producing cells, and / or a subsequent reduction in IL-13 and / or IL-4.

[0050] As used herein "skin diseases" may include inflammatory skin disease, eczema, atopic dermatitis, skin granulomas, nonthrombocytopenic palpable purpura, urticarial, skin infarcts, and livedo reticulari.

[0051] As used herein "allergies" may include allergic asthma, allergic rhinitis, drug allergies, and food allergies.

[0052] As used herein "atopic diseases" may include any disease related to the overproduction of immunoglobulin E (IgE) and is often an exaggerated response to an allergen. Atopic diseases include, but are not limited to atopic dermatitis, asthma, and allergic rhinitis, food allergies, drug allergies, allergic conjunctivitis, urticaria, angioedema, eosinophilic esophagitis (EoE), non-celiac gluten sensitivity, and anaphylaxis. The compounds described herein as defined by Formula I, may be useful for the treatment of one or more of the following: skin disease; allergic asthma; allergic rhinitis; and food allergies. Alternatively, the compounds described herein as defined by Formula I, may be useful for the treatment of one or more of the following: skin disease; asthma; allergies; chronic obstructive pulmonary disease (COPD); chronic rhinosinusitis; atopic keratoconjunctivitis; non-celiac gluten sensitivity; eosinophilic granulomatosis with polyangiitis (EGPA); and eosinophilic esophagitis (EoE). Alternatively, the compounds described herein as defined by Formula I, may be useful for the treatment of one or more of the following: atopic dermatitis; skin granulomas; nonthrombocytopenic palpable purpura; urticaria; skin infarcts; and livedo reticulari. Alternatively, the compounds described herein as defined by Formula I, may be useful for the treatment of one or more of the following: allergic asthma; allergic rhinitis; drug allergies; and food allergies. Alternatively, the compounds described herein as defined by Formula I, may be useful for the treatment of one or more of the following: a IL-13 mediated disease; a IL-4 mediated disease; a IgE mediated disease; a TSLPR mediated disease; or a Th2 mediated disease.

[0053] Those skilled in the art will appreciate that the point of covalent attachment of the moiety to the compounds as described herein may be, for example, and without limitation, cleaved under specified conditions. Specified conditions may include, for example, and without limitation, in vivo enzymatic or non-enzymatic means. Cleavage of the moiety may occur, for example, and without limitation, spontaneously, or it may be catalyzed, induced by another agent, or a change in a physical parameter or environmental parameter, for example, an enzyme, light, acid, temperature or pH. The moiety may be, for example, and without limitation, a protecting group that acts to mask a functional group, a group that acts as a substrate for one or more active or passive transport mechanisms, or a group that acts to impart or enhance a property of the compound, for example, solubility, bioavailability or localization.

[0054] Compounds as described herein may be in the free form or in the form of a salt thereof. In some embodiment, compounds as described herein may be in the form of a pharmaceutically acceptable salt, which are known in the art (Berge S. M. et al., / . Pharm. Sci. (1977) 66(1):1- 19). Pharmaceutically acceptable salt as used herein includes, for example, salts that have the desired pharmacological activity of the parent compound (salts which retain the biological effectiveness and / or properties of the parent compound and which are not biologically and / or otherwise undesirable). Compounds as described herein having one or more functional groups capable of forming a salt may be, for example, formed as a pharmaceutically acceptable salt. Compounds containing one or more basic functional groups may be capable of forming a pharmaceutically acceptable salt with, for example, a pharmaceutically acceptable organic or inorganic acid. Pharmaceutically acceptable salts may be derived from, for example, and without limitation, acetic acid, adipic acid, alginic acid, aspartic acid, ascorbic acid, benzoic acid, benzenesulfonic acid, butyric acid, cinnamic acid, citric acid, camphoric acid, camphorsulfonic acid, cyclopentanepropionic acid, diethylacetic acid, digluconic acid, dodecylsulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, glucoheptanoic acid, gluconic acid, glycerophosphoric acid, glycolic acid, hemisulfonic acid, heptanoic acid, hexanoic acid, hydrochloric acid, hydrobromic acid, hydriodic acid, 2- hydroxyethanesulfonic acid, isonicotinic acid, lactic acid, malic acid, maleic acid, malonic acid, mandelic acid, methanesulfonic acid, 2-napthalenesulfonic acid, naphthalenedisulphonic acid, p-toluenesulfonic acid, nicotinic acid, nitric acid, oxalic acid, pamoic acid, pectinic acid, 3-phenylpropionic acid, phosphoric acid, picric acid, pimelic acid, pivalic acid, propionic acid, pyruvic acid, salicylic acid, succinic acid, sulfuric acid, sulfamic acid, tartaric acid, thiocyanic acid or undecanoic acid. Compounds containing one or more acidic functional groups may be capable of forming pharmaceutically acceptable salts with a pharmaceutically acceptable base, for example, and without limitation, inorganic bases based on alkaline metals or alkaline earth metals or organic bases such as primary amine compounds, secondary amine compounds, tertiary amine compounds, quaternary amine compounds, substituted amines, naturally occurring substituted amines, cyclic amines or basic ion-exchange resins. Pharmaceutically acceptable salts may be derived from, for example, and without limitation, a hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation such as ammonium, sodium, potassium, lithium, calcium, magnesium, iron, zinc, copper, manganese or aluminum, ammonia, benzathine, meglumine, methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, isopropylamine, tripropylamine, tributylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, glucamine, methylglucamine, theobromine, purines, piperazine, piperidine, procaine, N-ethylpiperidine, theobromine, tetramethylammonium compounds, tetraethylammonium compounds, pyridine, N,N-dimethylaniline, N-methylpiperidine, morpholine, N-methylmorpholine, N-ethylmorpholine, dicyclohexylamine, dibenzylamine, N,N-dibenzylphenethylamine, 1-ephenamine, N,N'-dibenzylethylenediamine or polyamine resins. In some embodiments, compounds as described herein may contain both acidic and basic groups and may be in the form of inner salts or zwitterions, for example, and without limitation, betaines. Salts as described herein may be prepared by conventional processes known to a person skilled in the art, for example, and without limitation, by reacting the free form with an organic acid or inorganic acid or base, or by anion exchange or cation exchange from other salts. Those skilled in the art will appreciate that preparation of salts may occur in situ during isolation and purification of the compounds or preparation of salts may occur by separately reacting an isolated and purified compound.

[0055] In some embodiments, compounds and all different forms thereof (e.g. free forms, salts, polymorphs, isomeric forms) as described herein may be in the solvent addition form, for example, solvates. Solvates contain either stoichiometric or non-stoichiometric amounts of a solvent in physical association the compound or salt thereof. The solvent may be, for example, and without limitation, a pharmaceutically acceptable solvent. For example, hydrates are formed when the solvent is water or alcoholates are formed when the solvent is an alcohol.

[0056] In some embodiments, compounds and all different forms thereof (e.g. free forms, salts, solvates, isomeric forms) as described herein may include crystalline and amorphous forms, for example, polymorphs, pseudopolymorphs, conformational polymorphs, amorphous forms, or a combination thereof. Polymorphs include different crystal packing arrangements of the same elemental composition of a compound. Polymorphs usually have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optical and electrical properties, stability and / or solubility. Those skilled in the art will appreciate that various factors including recrystallization solvent, rate of crystallization and storage temperature may cause a single crystal form to dominate. In some embodiments, compounds and all different forms thereof (e.g. free forms, salts, solvates, polymorphs) as described herein include isomers such as geometrical isomers, optical isomers based on asymmetric carbon, stereoisomers, tautomers, individual enantiomers, individual diastereomers, racemates, diastereomeric mixtures and combinations thereof, and are not limited by the description of the formula illustrated for the sake of convenience.

[0057] In some embodiments, pharmaceutical compositions as described herein may comprise a salt of such a compound, preferably a pharmaceutically or physiologically acceptable salt. Pharmaceutical preparations will typically comprise one or more carriers, excipients or diluents acceptable for the mode of administration of the preparation, be it by injection, inhalation, topical administration, lavage, or other modes suitable for the selected treatment. Suitable carriers, excipients or diluents (used interchangeably herein) are those known in the art for use in such modes of administration.

[0058] Suitable pharmaceutical compositions may be formulated by means known in the art and their mode of administration and dose determined by the skilled practitioner. For parenteral administration, a compound may be dissolved in sterile water or saline or a pharmaceutically acceptable vehicle used for administration of non-water soluble compounds such as those used for vitamin K. For enteral administration, the compound may be administered in a tablet, capsule or dissolved in liquid form. The tablet or capsule may be enteric coated, or in a formulation for sustained release. Many suitable formulations are known, including, polymeric or protein microparticles encapsulating a compound to be released, ointments, pastes, gels, hydrogels, or solutions which can be used topically or locally to administer a compound. A sustained release patch or implant may be employed to provide release over a prolonged period of time. Many techniques known to one of skill in the art are described in Remington: the Science & Practice of Pharmacy by Alfonso Gennaro, 20thed., Lippencott Williams & Wilkins, (2000). Formulations for parenteral administration may, for example, contain excipients, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes. Biocompatible, biodegradable lactide polymer, lactide / glycolide copolymer, or poly oxy ethylene-poly oxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for modulatory compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauiyl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.

[0059] Compounds or pharmaceutical compositions as described herein or for use as described herein may be administered by means of a medical device or appliance such as an implant, graft, prosthesis, stent, etc. Also, implants may be devised which are intended to contain and release such compounds or compositions. An example would be an implant made of a polymeric material adapted to release the compound over a period of time.

[0060] An "effective amount" of a pharmaceutical composition as described herein includes a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as reduced tumor size, increased life span or increased life expectancy. A therapeutically effective amount of a compound may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the compound to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as smaller tumors, increased life span, increased life expectancy or prevention of the progression of prostate cancer to an androgen-independent form. Typically, a prophylactic dose is used in subjects prior to or at an earlier stage of disease, so that a prophylactically effective amount may be less than a therapeutically effective amount.

[0061] It is to be noted that dosage values may vary with the severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected by medical practitioners. The amount of active compound(s) in the composition may vary according to factors such as the disease state, age, sex, and weight of the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It may be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.

[0062] In general, compounds as described herein should be used without causing substantial toxicity. Toxicity of the compounds as described herein can be determined using standard techniques, for example, by testing in cell cultures or experimental animals and determining the therapeutic index, i.e., the ratio between the LD50 (the dose lethal to 50% of the population) and the LD100 (the dose lethal to 100% of the population). In some circumstances however, such as in severe disease conditions, it may be appropriate to administer substantial excesses of the compositions. Some compounds as described herein may be toxic at some concentrations. Titration studies may be used to determine toxic and non-toxic concentrations. Toxicity may be evaluated by examining a particular compound’s or composition’s specificity across cell lines using PC3 cells as a negative control that do not express AR. Animal studies may be used to provide an indication if the compound has any effects on other tissues. Systemic therapy that targets the AR will not likely cause major problems to other tissues since anti-androgens and androgen insensitivity syndrome are not fatal.

[0063] Compounds as described herein may be administered to a subject. As used herein, a "subject" may be a human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc. The subject may be suspected of having or at risk for having a disease that would benefit from inhibition of thymic stromal lymphopoietin receptor (TSLPR) complex formation, inhibition of cytokine expression, or for the inhibition of IL4 and / or IL13 expression. Thymic stromal lymphopoietin (TSLP) is involved in the pathogenesis of atopic diseases. Its pathophysiological relevance has sparked great interest in therapeutically targeting TSLP. Yet, so far, there are no small-molecule TSLP inhibitors due to the challenging nature of disrupting the protein-protein interaction between TSLP and the thymic stromal lymphopoietin receptor (TSLPR).

[0064] Here, we report the development of small-molecule TSLP receptor inhibitors using structure-based virtual screening and docking of >1,000,000 (i.e. 1,524,680) compounds followed by iterative chemical synthesis. BP79 emerged as the initial lead compound that effectively abrogates TSLP-triggered cytokine release at low micromolar concentrations. Topical application of BP79 yields efficient skin absorption followed by significant downregulation of atopy-relevant proinflammatoiy cytokines in immunocompetent skin disease models.

[0065] For further analysis, a human atopic disease drug discovery platform was developed using microfluidic multi-organ chips. Here, topical application of BP79 onto atopic-like skin disease models, that were co-cultivated with lung models in the presence of Th2 cells effectively suppressed immune cell infiltration and key atopy mediators including IL-13, IL- 4, TSLP, and periostin, while upregulating skin barrier proteins such as filaggrin. RNA-Seq analysis corroborated these findings and also indicated protective downstream effects on the lung epithelium.

[0066] To the best of our knowledge, this represents the first report of a potent and safe small molecule TSLPR inhibitor, which has the potential to expand the therapeutic and preventive options in atopic diseases. Further, it serves as a starting point for further developments in targeting TSLP as a central player of inflammation.

[0067] Although our understanding the pathological mechanisms of atopic diseases has improved, comparably little progress has been made in the context of treating and / or preventing atopic diseases.

[0068] Glucocorticoids and calcineurin-inhibitors are still considered first-line medications, although they are associated with dose-limiting side-effects such as skin atrophy for glucocorticoids and increased risk of malignancies for calcineurin-inhibitors [36, 37]. Furthermore, many moderate-to-severe cases do not respond well to the available treatment options leaving these patients with a high disease burden. In Canada, for example, 19% of children affected by atopic dermatitis (AD) need to try > 15 different treatments to achieve sufficient disease control

[0038] .

[0069] Recently, several new drugs such as JAK inhibitors have been approved, which expands treatment options. Similarly, biologies such as the anti-IL-4 / IL-13 antibody dupilumab are now available for the treatment of moderate-to-severe AD. While clinical data show favorable effectiveness and a good safety profile, there are still a certain number of therapyresistant patients. Also, biologies are generally associated with high therapy costs of ~$30,000 US per year

[0039] . Thus the development of safe and effective small molecule drugs especially for moderate-to-severe atopic dermatitis is urgently needed

[0038] , which holds particularly true for children who are most frequently affected.

[0070] TSLP has been recognized as a central driver of atopic diseases and, thus, has received increasing attention as a therapeutic target. Consequentially, anti-TSLP antibodies such as tezepelumab have been developed, acknowledging the key role of TSLP in the pathogenesis of atopic diseases. Interestingly, while being approved for severe asthma, disappointing results were obtained for AD with clinical studies showing limited effectiveness in dampening the inflammation and itch

[0040] .

[0071] While biologies such as tezepelumab have very high target specificity, and, thus fewer side effects, they are accompanied by high costs and complex storage and shipping conditions. Furthermore, topical drug administration offers distinct advantages (i.e. localized drug delivery and improved patient acceptance) over the systemic administration of antibodies. Also, primary TSLP-mediated sensitization for allergic asthma happens in the skin in the context of AD

[0041] . Thus, a topical application of TSLP inhibitors on infant skin early on could potentially even avert the onset of disease progression and, thus, the "atopic march".

[0072] In order to close current therapeutic gaps, we leveraged in silica approaches, rational drug design, and the application of state-of-the-art chemical biology tools to develop small molecule TSLP inhibitors. Four out of twelve top virtual screening hits were biologically active and were found to significantly reduce the release of TSLP-induced Th2 cytokines in a dose-dependent manner (FIGURE 1 - i.e. C2, C6, C7, and C13). This represents a promising result considering that there are currently no other small molecules known to target this protein-protein interaction interface. For C7, the proposed binding to a lipophilic pocket (Leu39, Vai 114, Tyrl43 and Tyrl94) in TSLPR might explain its in vitro activity over other virtual hits, while for C13 the combination of hydrogen bonding and the charge interaction between the carboxylate moiety and Arg150 and 153 seem essential for TSLP binding (FIGURE lj).

[0073] Building onto the structural features of C7 and C13, novel TSLP inhibitors were synthesized and tested for their efficacy to inhibit TSLP-mediated effects. BP79 emerged as our lead candidate due to strong inhibitory effects in the micromolar range in relevant cell types including primary, activated CD4+ T cells while showing good cytocompatibility (FIGURE 2).

[0074] Its target engagement was confirmed by a range of methods including proximity ligation (PLA) and thermal shift assays (FIGURE 3). We further demonstrate that BP79 is not a broad-spectrum kinase inhibitor, and importantly lacked activity against the JAKs, which are canonical mediators of TSLP-induced STAT signaling and inflammatory cytokine expression (FIGURE 4). A PLA assay confirmed that BP79 disrupts the ternary receptor complex formation between TSLP / TSLPR / IL- 7Ra, thus inhibiting downstream signaling. In CD4+ T cells, this receptor complex activates STAT6 and, thus enhances Th2 cytokine secretion from CD4+T cells

[0042] . In keratinocytes, STAT3 is predominantly activated which downregulates filaggrin and upregulates TSLP expression

[0013] . BP79 was able to efficiently block STAT6 and STAT3 phosphorylation, respectively thus preventing TSLP-dependent downstream signaling events. Moreover, we found that BP79 treatment abrogated TSLP-mediated proliferation of CD4+T cells (FIGURES 3 and 4).

[0075] Aiming to study the pharmacological efficacy and safety in a complex disease model, we opted to develop a human-based atopic drug discovery platform leveraging an organ-on-chip technology (FIGURE 6). This decision was driven by the fact that conducting these studies in animal models proved difficult. While murine and human TSLP operate in a similar manner [6, 20], they only share 43% and 35% homology, respectively, for TSLPR, with no cross-reactivity between the species

[0043] . Therefore, to the best of our knowledge, there is no animal model available to accurately mimic human TSLP-mediated Th2 inflammation in atopic diseases. Our TSLP inhibitors have been specifically designed to block human TSLPR, rendering currently available mouse models unsuitable. Additionally, atopic diseases do not spontaneously develop in most rodents and, thus, are usually highly artificially induced, with uncertain fidelity to the human situation

[0044] .

[0076] 3D skin models and excised human skin provide a more realistic representation of the skin barrier and enables the evaluation of permeability and efficacy of BP79 in a more clinically relevant context. Furthermore, complex human-based organ-on-a-chip setups have emerged as suitable alternatives to address this translational gap

[0045] . Since TSLP is involved in the progression of atopic diseases and also plays a critical role in the manifestation of allergic asthma, we developed an immunocompetent two-organ-on-chip setup as a drug discovery platform that has broad application potential for preclinical testing of anti-inflammatory drugs beyond small molecule TSLP inhibitors.

[0077] The atopic diseases-on-chip platform reliably demonstrated the anti-inflammatory effects of BP79 and differentiated its mode of action from tacrolimus, a calcineurin inhibitor. The latter predominantly dampens inflammation through inhibitory effects on T cell proliferation by blocking IL-2 production

[0046] (FIGURE 6).

[0078] To gain more insights particularly on the effects of BP79 on the co-cultivated lung tissue, RNA-Seq was performed. Interestingly, pathway enrichment analysis of treated vs. untreated lung models showed enrichment in the KEGG and REACTOME aminoacyl tRNA biosynthesis pathway, which plays a critical role in immune regulations, chemo-attraction for leukocytes, and contribute to the maturation, activation, and recruitment of immune cells (FIGURE 6j)

[0047] . Other enriched REACTOME pathways included sodium proton exchangers (p=0.0048), which regulate the water content of lung fluids. Some studies suggest that dysregulated airway hydration may promote airway inflammation 48. For instance, sodium proton exchanger regulatory factor 1, another enriched pathway, was shown to facilitate allergic airway inflammation in mice accompanied by increased immune cell infiltration, goblet cell hyperproliferation, increased Th2 cytokine expression

[0049] . Taken together, our study reports the first potent small molecule TSLP inhibitors that exert potent anti-inflammatory effects in the low micromolar range and has a great potential to broaden the spectrum of topically applicable drugs for the treatment of atopic dermatitis and, maybe even the prevention of the progression of atopic diseases to other epithelial. Furthermore, we present a complex, human-based drug discovery platform for atopic diseases which may close the current gap of human-based, translational preclinical models in the area of atopic diseases.

[0079] MATERIALS & METHODS

[0080] Cell culture

[0081] HuT78 cells were sourced from the American Type Culture Collection (ATCC). Primary human peripheral blood mononuclear cells (PBMCs) and CD4+T cells were isolated from healthy blood donor buffy coats (CREB approval 2019.023). Primary human dermal fibroblasts (FBs) and keratinocytes (KCs) were isolated from juvenile foreskin according to standard procedures (CREB approval #H19-03096). Written informed consents were obtained from the donors. Normal human lung fibroblasts were purchased from CELL applications™ (Cat# 506-05a). Normal human bronchial epithelial cells (NHBEs) were purchased from EPITHELIX™ (Cat# EP51AB).

[0082] PBMCs were isolated from buffy coat using Lymphoprep™ density gradient centrifugation (STEMCELL™, Catalog # 07851) and CD4 T cells were separated from PBMCs using EasySep™ human CD4+ T cell isolation kit (STEMCELL™, Cat# 17912) following the manufacturer’s instructions. HuT78, PBMCs and CD4+ T cells were cultured in RPMI-1640 Medium (Sigma™, R8758) supplemented with 10% (v / v) of FBS (Sigma™, F1051) and 1% (v / v) of Penicillin- Streptomycin (Sigma™, P4333); media were change every three days. Dermal FBs and Normal human lung FBs were cultured in DMEM - high glucose (Sigma™, D6429) supplemented with 10% (v / v) of fetal bovine serum (FBS) (Sigma™, F1051) and 1% (v / v) of Penicillin-Streptomycin (Sigma™, P4333), medium change was performed every two days. KCs were cultured with EpiLife™ medium (Gibco™, MEPI500CA) supplemented with human keratinocyte growth supplement (Gibco™, s-001- 5). NHBEs were cultured with PhneumaCult-Ex™ medium (STEMCELL™, Cat# 05008). Media were changed every other day, unless otherwise stated. All cells were maintained at 37 °C in a humidified 5% CO2 incubator.

[0083] Reagents and antibodies

[0084] Potential TSLP small molecule inhibitors were obtained from VitasM™, Specs™ and NCI-DTP libraries. Uncoated Human IL-13, IL-4, and TSLP ELISA Kits were obtained from Invitrogen™ (Cat# 88-7439-88, # 88-7046-88, # 88-7497-88), Periostin ELISA kit was purchased from R&D Systems™ (Cat# DY3548B) and the U-PLEX human cytokine array assay kit from Meso Scale Discovery, Inc.™ (Cat# K15067L-1). BCA protein assay kit was obtained from G Bioscience™ (Cat# 786-570). JAK1 / 2 inhibitor-Ruxolitinib (INCB018424) was purchased from Selleckchem™. JAK2 inhibitor (AZD1480), Stat3 / Stat5 inhibitor (SH-4-54) and Stat6 inhibitor (AS1517499) were purchased from Cayman Chemicals™. StatS inhibitor (573108), human recombinant TSLP (Cat# SRP4896), phorbol-12-myristate-13-acetate (PMA, Cat# P1585), ionomycin (Cat# 10634), human anti-CD4 antibody (Cat# MABF419), Thiazolyl Blue Tetrazolium Bromide (MTT, Cat# M2128), Proximity Ligation Assay Kit™ (Cat# DU092103- 1KT) were purchased from Sigma Aldrich™. GAPDH (Cat#2118S), p-STAT5 (Y694) (Cat#9359S), STATS (Cat#25656S), p-STAT3 (Y705) (Cat#9131S), p-STAT6 (Y641) (Cat# 56554S), STAT6 (Cat#5397S), SOD1 (Cat#2770S), P-Jak2 (Cat#3776S), Periostin (Cat#91771S), TSLP (Cat# 97630S) were obtained from Cell Signaling Technology™. Jakl (Cat# MAS-32780, p-Jakl (Cat# PAS-104554), Jak2 (Cat# 702434), IL-7Ra (Cat#14-1278- 82) antibodies were obtained from Invitrogen™. Filaggrin (Cat# ab814688) antibody was obtained from Abeam™. ImmunoCult™ Human CD3 / CD28 T Cell Activator was purchased from STEMCELL™ (Cat # 10991). PureLink™ RNA Mini Kit was obtained from Invitrogen™ (Cat# 12183018A). iScript cDNA Synthesis Kit™ and SsoAdvanced Universal SYBR Green Supermix™ were purchased from Bio-Rad™ (Cat# 1708891 and # 1725270).

[0085] In silica screening of library compounds

[0086] Protein preparation: All modeling studies were performed using the crystal structure of human TSLP in complex with TSLPR and IL-7Ra (PDB 21 code: 5jll 23). Since we were interested in designing ligands to disrupt the TSLP: TLSPR interface, we prepared chain A and B of this crystal structure by removing crystal waters and by assigning protonation states using the "ProtonateSD"

[0050] application implemented in MOE v2019.0102

[0051] .

[0087] Cavity detection and binding site analysis: After removing TSLP [PDB 5] 11 23, chain A) and TSLPR [PDB 5] 11 23, chain B), respectively, we performed a binding site analysis using "Site finder", a cavity detection program based on 52, implemented in MOE v2019.0102

[0051] . The analysis was focused on site I

[0020] at the interface between TSLP and TSLPR. Since the interaction of Argl50, Argl53 of TSLP with Asp92 of TSLPR is known to be crucial [6, 20], only solutions including this interaction were considered.

[0088] 3D pharmacophore-based virtual screening and database preparation

[0089] 3D pharmacophores were developed using LigandScout 4.4™. As no crystal structure with co-crystallized small molecule nor any small molecule TSLP inhibitors were available, key interactions were manually placed at the interface for the side of TSLP or TSLPR, respectively. Both 3D pharmacophore models were used to screen 1,524,680 commercially available compounds from Specs™ [Delft, Netherlands) and VitasM™ (Hong Kong, China). Before screening, both databases were standardized, assigned correct charges at pH 7.4 and counter ions were removed using an in-house standardization workflow using KNIME 53™ and RDKit™

[0051] . LigandScout’s™ [22, 24] idbgen™ program was used to pre- calculate conformational models for all molecules. Virtual hits were filtered limiting the number of rotatable bonds, molecular weight, and matching features to the developed pharmacophore. All compounds with less than seven rotational bonds and greater than 260 Daltons molecular weight with at least five matching chemical features were selected for docking studies.

[0090] Molecular docking

[0091] To check whether virtual hits from 3D pharmacophore screening are actually able to fulfill the desired interaction pattern, a molecular docking step was included in the virtual screening workflow. Virtual hits were docked using GOLD v5.2™

[0054] with standard parameters and GoldScore 55™ as a scoring function. Binding site residues were defined as all protein residues in 10A distance from the key residues Asp92 or Argl53 for TSLPR and TSLP, respectively. Resulting docking poses were minimized in their respective binding pockets using LigandScout’s™ [22, 24] MMFF94 [56, 57] implementation.

[0092] For NCI-DTP compounds, chemical structures were imported into Maestro™ (Schrodinger™) and prepared for docking using the ligprep function. TSLPR from PDB 5J11 was prepared for docking using standard workflows, which included preprocessing, removing water molecules and energy minimization. A docking grid was set as a cube with 10 A sides centered around TSLPR Asp92. Compounds were docked with extra precision and docking scores were ranked. Top scoring compounds were acquired from the NCI; however, some compounds were not available at the time of these experiments.

[0093] General Chemical Methods

[0094] Detailed schemes, chemical procedures and analytical data are included below. Reagents were sourced from Millipore Sigma™ (Burlington, MA, USA), Combi-blocks™ (San Diego, CA, USA) or AK Scientific™ (Union City, CA, USA) and used without modification. Anhydrous solvents were sourced from Sigma Millipore and used without further modifications. Compounds were purified using automated flash chromatography (Biotage Isolera™, Uppsala, Sweden) with ACS grade solvents (hexanes, ethyl acetate, methanol, and dichloromethane) from Fisher Scientific™ (Waltham, MA, USA), or preparative HPLC (Agilent™, Infinity II 1290™) in gradients of water and acetonitrile (Millipore Sigma™). LCMS data were collected using an Agilent™ (1260 Infinity II HPLC™ equipped with an Infinity Lab LC / MSD mass detector). NMR data were collected using a 400 MHz Bruker Ascend™. Compounds were dissolved in DMSO before testing, which was also used as vehicle controls throughout the assessment of compound activity.

[0095] Enzyme-linked immunosorbent assay (ELISA)

[0096] HuT78 and primary CD4+T cells (0.5x106cells / ml) were activated with 100 ng / mL PMA, 1 pg / mL ionomycin, and 50 ng / mL TSLP for 36 hours. TSLP inhibitors were added in selected wells. After 36 hours, the culture media were collected, and the levels of IL-4 and IL-13 were determined using human IL-4 and IL-13 ELISA kits according to the manufacturer’s instructions. MTT assay

[0097] HuT78 (0.5x106cells / ml), human primary CD4+T (0.5x106cells / ml), keratinocytes (0.5x105cells / well) and fibroblast (0.5x105cells / well) cells were treated with a range of different TSLP inhibitor concentrations for 24 hours. Subsequently, MTT (5 mg / ml) reagent was added for 4 hours. Acidified isopropanol or dimethyl sulfoxide (DMSO) were added to solubilize the formazan crystals, and the absorbance was measured at 570 nm using a microplate reader (BioTek Instruments™, USA). Cell viability was determined as a percentage of the vehicle control group treated with a medium containing 0.2% DMSO.

[0098] Immunoblotting

[0099] Western blot analysis was performed in HuT78, primary CD4+ T cells, and keratinocytes treated with TSLP inhibitors to determine the target binding and inhibition of TSLP mediated downstream signaling pathway. Following treatment with TSLP for 20 min in CD4+T cells and HuT78 cells and for 24 hours in keratinocytes, the cells were lysed with RIPA buffer containing a protease and phosphatase inhibitor cocktail. Total protein concentration was determined with a BCA assay kit. Twelve micrograms of total protein were separated on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred onto polyvinylidene fluoride (PVDF) membranes. The levels of indicated proteins were blotted by incubating with primary antibodies (1:1,000) overnight at 4 °C, followed by incubation with secondary antibodies (1:5,000) for I h at room temperature (RT). Immunoreactive proteins were visualized using an Infrared western blot imaging system (Odessy CLx™).

[0100] Proximity Ligation Assay (PLA)

[0101] Proximity ligation assay was performed to determine the protein-inhibitor binding efficacy. Primary keratinocytes were cultured in coverslips overnight. Next day, cells were treated with 10 μM TSLP inhibitors and equal volume of DMSO as a vehicle control and incubated for 1 hour at 37°C, 5% CO2. Cells were then stimulated with 50 ng / ml TSLP for 30 mins and subsequently fixed in 4% formaldehyde. Samples were processed using a DuoLink PLA™ kit with Anti-Mouse PLUS and Anti- Goat MINUS™, and Red PLA detection reagent, and primary antibodies for TSLPR and IL-7Ra, according to the manufacturer’s protocol. Red fluorescence was detected using the Texas Red™ filter. 25 to 40 images were captured per group using EVOS M5000™ fluorescence microscope (Thermo Scientific™, USA). Data analysis was performed using Image)™ software according to a previously published protocol

[0058] .

[0102] In vitro thermal shift assay (TSA)

[0103] The assay was performed as described previously

[0010] . Briefly, recombinant TSLPR protein was dissolved in 25 mM HEPES (pH=7,4), 150 mM NaCl and ImM DTT at a final concentration of 0.5 pg / ml. TSLPR protein was incubated with 20 μM BP79, along with DMSO control, in 50 pL reaction buffer at 25 °C for 10 min, and then heated at various temperatures for 3 min. Samples were centrifuges at 14000 rpm for 10 minutes at 4°C. 30 pl of supernatants were collected carefully from the top and mix with 4x Laemmli sample buffer. TSLPR protein levels were measured by Western blotting.

[0104] Kinase panel screening

[0105] BP79 was tested at 10 μM in the KinomeScan™ (97 kinases) radio ligand displacement assay from Eurofins Discovery Services™ in duplicate. Values displayed in FIGURE 4C are the average of the duplicates from a single experiment.

[0106] Generation of the 3D epithelial models

[0107] A skin disease models that closely emulates atopic dermatitis in vitro was prepared according to previously established procedures (1). In brief, the filaggrin gene (FLG) was knocked down in keratinocytes using a siRNA (Invitrogen™, Cat# 87663809) with HiPerFect transfection reagent (Qiagen™, Cat# 301705). After 24h, dermal Fbs (2.1 x 104cells / well), FBS (Sigma™, F1051) (21pL / well) and type I bovine collagen (Advanced BioMatrix™, Cat# 5005) were brought to neutral pH and poured into transwell insert for 24-well plates (Corning, Cat# 353504) with a growth area of 0.33 cm2(Costar™, Cat# 3422). After incubation for 1 hour at room temperature and another hour at 37°C, 200 pL of EpiLife TM culture medium were added and the system was transferred to a 37°C incubator with 5% (v / v) CO2 and 95% (v / v) humidity for another 2 hours. Subsequently, 3 x 105cells / insert normal and FLG knockdown KCs (were added on top of the collagen matrix. After 24h, the model was lifted to the air-liquid interface and the medium was changed to the keratinocyte differentiation medium (KDM). The skin models were cultured for 11 days (at 37°C, with 5% C02 and 95% humidity). Medium change was performed every other day.

[0108] Bronchial epithelial models were generated as described elsewhere (2). Briefly, normal human lung Fibroblasts (1.9 x 104cells / well) and FBS (Sigma™, F1051) (9.5 pL / well) were embedded in type I bovine collagen (Advanced BioMatrix™, Cat# 5005) mimicking the bronchial epithelial stroma. The mixture was brought to neutral pH and decanted (total volume per well is 79.2 pL) into 3D transwell permeable supports insert for 24-well plates (Corning™, Cat# 353504) with a growth area of 0.33 cm2 (Costar™, Cat# 3422). After incubation for 2 hours at 37°C, 200 pL of PneumaCult-Ex™ medium (STEMCELL™, Cat# 05008) were added and the system was transferred to a 37°C incubator with 5% (v / v) CO2 and 95% (v / v) humidity for another 2 hours. After this incubation, NHBEs in PneumaCult- Ex™ medium (STEMCELL™, Cat# 05008) (2.65 x 105cells / insert) were added on top of the collagen matrix. After 24h, the model was lifted to the air-liquid interface and the medium was changed to PneumaCult™ - ALI medium (STEMCELL™, Cat# 05001). The bronchial epithelial models were cultured for 18 days (at 37°C, with 5% CO2 and 95% humidity). Medium change was performed every other day.

[0109] Skin Permeation Testing

[0110] Skin permeation of BP79 was evaluated using the Franz cell setup. The acceptor compartments of the Franz cells were filled with phosphate buffered saline (PBS; pH 7.4, 32°C). Punched disks of excised human skin (CREB approval # H19-03096) were mounted onto the Franz diffusion cells (static type, volume 12mL, diameter 15mm), with the stratum corneum exposed to air and the dermis in contact with the PBS. Following 30 min equilibration time, 100 pL of 20 μM BP79 or vehicle control was applied topically on the skin. The Franz cells were sealed with Parafilm™ to prevent evaporation of the test solution. To determine the amount of permeated BP79, buffer solution from the acceptor medium was sampled after 2, 4, 8, and 24 hours, respectively and stored at -20°C. For LC / MS analysis, the samples were extracted twice using 10 mb of ethyl acetate. The extracts were combined and evaporated to dryness under nitrogen at 45°C and reconstituted in 100 pL acetonitrile. Subsequently, BP79 detection was performed using the UHPLC / MS / MS system consisted of an Agilent 1290 Infinity Binary Pump™, a 1290 Infinity Sampler™, a 1290 Infinity Thermostat™, and a 1290 Infinity Thermostatted Column Compartment™ (Agilent™, Mississauga, Ontario, Canada) connected to an AB SCIEX QTRAP™ 5500 hybrid linear ion trap triple quadrupole mass spectrometer equipped with a Turbo Spray source (AB SCIEX™, Concord, Ontario, Canada).

[0111] Chromatographic separation was performed using an InfinityLab Poroshell 120™ EC-C18, 3x50mm, 1.9pm particle size (Agilent™, Santa Clara, CA, USA) with column temperature of 30°C. The mobile phase consisted of 0.1% formic acid in deionized water as solvent A and 0.1% formic acid in acetonitrile as solvent B with the flow rate of 450 pL / min. The EC gradient was started with 90% of solvent A at initial condition (t = 0 min) and decrease to 10% in 4 min (t = 4 min), then held for 1.5 min (t= 5.5 min). The gradient return to the initial condition of 90% solvent A and equilibrate for 1.4min (t = 7 min) before the next injection. The injection volume was 5 pL. The mass spectrometer was operated in positive ionization mode with the following conditions: Curtain gas 30 units, Collision gas (CAD) high, lonspray 5500 V, temperature 450 °C, ion source gas 1, 40 units, ion source gas 2, 60 units. Nitrogen gas was used for curtain gas, collision gas, ion source gas 2 (vaporizing gas), and zero air was used for ion source gas 1 (nebulizing gas). Declustering potential 100, Entrance potential 10, Resolution QI Unit, Resolution Q3 Unit, and dwell time was 150 msec. The MRM transitions were m / z 259.0 -> 214.0 with collision energy (CE) 21, collision cell exit potential (CXP) 4 and m / z 259.0 -> 242.1.0 with CE 17, CXP 6. Data were acquired using the Analyst 1.5.2.™ software on a MicrosoftWindows XP Professional™ operating platform.

[0112] Generation of 3D immunocompetent skin models supplemented with activated CD4+ T cells

[0113] Normal (FLG+) and filaggrin-deficient (FLG-) skin models were generated using the methods described above. Primary human CD4+T cells were activated using ImmunoCult™ Human CD3 / CD28 T Cell Activator for 24 hours. At day 12, 0.3 x 106 activated CD4+ T cells were applied underneath the dermis directly onto the cell culture insert membrane of the skin models and cultured for 2 more days. Skin models were treated with 20 μM TSLP inhibitor-BP79 three times topically, models treated with vehicle control (DMSO) and medium only served as control. After end of the treatment, culture media were collected for cytokine array assay. Cytokine array assay

[0114] Culture media were collected from the 3D immunocompetent skin tissue models. Cytokines including TSLP, IL-2, IL-4, IL-5, IL-9, IL-10, IL-13, IL-22, TNF-a, and IFN-y, levels were quantified by a multiplex assay U-PLEX platform with electro-chemiluminescent enzyme- linked immunosorbent assays [Meso Scale Discovery™ (MSD), USA]. The assay was performed according to the manufacturer’s instructions, and data were acquired by MESO QuickPlex SQ 120™ plate reader.

[0115] Organ-on-chip-based co-cultivation of skin (disease) and bronchial epithelial model

[0116] HUMIMIC Chip3plus were purchased from TissUse™ (Berlin, Germany). Prior to chip connection, the HUMIMIC™ starter control unit (TissUse™, Berlin, Germany) was configured to 30 bpm (air flow of 1.5 L / min) with +500 mbar pressure-out and -500 mbar vacuum-out, mimicking recirculation mode of the capillaries [3]. With this set-up, HUMIMIC™ Chip3plus was first placed in a 37°C incubator for 24 hours. Subsequently, the chips were connected to the HUMIMIC™ starter and were perfused for 24 hours. Lastly, the media in the chip were changed into an 800 pL medium mix (1:1:1 of KDM, PneumaCult™ - ALI medium, and complete RPMI medium, hydrocortisone was depleted from KDM and PneumaCult™ - ALI medium as glucocorticoids suppress T cell function [4]). The chip was then perfused for 24 hours. After the preparation, 5 x 105activated CD4+ T cells in fresh 800 pL medium mix were applied to the chip. The FLG knockdown skin model and bronchial epithelial model were transferred into the chip, avoiding the formation of air bubbles and start perfusion. The day of transfer is considered day 1 of the OOC cultivation. 10 pL of a 20 μM solution of the anti TSLP compounds (BP79) were topically applied onto the skin model on day 1 - four hours since the starting of perfusion. On day 2, the OOC system was replenished with fresh 5 x 105activated CD4+ T cells in 800 pL medium mix. BP79 was applied to the skin model after four hours of replenishment. After 24 hours (on day 3), BP79 was applied to the skin model again. After another 24 hours (day 4), final BP79 application was performed and both skin and bronchial epithelial models were harvested in 4 hours. Here, the culture medium was collected, the skin models were cry-frozen and bronchial epithelial models were paraffin embedded for further characterizations.

[0117] H&E Staining Sections of skin models (6 pm) and bronchial epithelial models (4 pm) were obtained using a cryotome or microtome for histological analyses. Hematoxylin (Thermo Scientific™, Cat# 6765007) and eosin (Thermo Scientific™, Cat# 6766007) (H&E) staining was performed according to standard staining procedures.

[0118] Immunofluorescence assay

[0119] Tissue sections were fixed using 4% formaldehyde for 10 min at room temperature. Then, tissues were permeabilized with 0.5% TritonX-100™ in PBS for 10 mins at room temperature; washed with PBS containing 0.0025% BSA and 0.025% Tween 20™ and blocked with normal goat serum (1:20 in PBS). The sections were incubated overnight at 4°C with primary antibodies (in PBS, 0.0025% BSA, 0.025% Tween 20™). Subsequently, the sections were incubated for an additional 1 h at room temperature with secondary antibodies (1:400 in PBS, 0.0025% BSA, 0.025% Tween 20™) and counter stained with DAPI mounting medium. Slides were then imaged using EVOS M5000™ fluorescence microscope.

[0120] LDH and glucose assay

[0121] Lactate dehydrogenase (LDH) and glucose concentrations of the medium in the chip were monitored. Here, medium was collected at day 2 and day 4 and then the LDH concentration was determined using the cytotoxicity detection kit PLUS™ (Roche™, Cat# 04744934001) following the manufacturer’s instructions. Glucose consumption in the OOC system was measured using the glucose LiquiColor™ (Stanbio™, Cat# 1070-125) following the manufacturer’s instructions.

[0122] Real-time quantitative polymerase chain reaction (qPCR)

[0123] For gene expression analysis, total RNA was extracted from the skin and lung models. Briefly, models were lysed in PureLink RNA Mini Kit™ lysis buffer and then homogenized for 30 s at 25 Hz using a TissueLyzer™ (Qiagen™, Germany). RNA was isolated according to the manufacturer’s protocol. Total RNA was quantified using Nanodrop 2000™ (Thermo Scientific™, USA). cDNA was synthesized, using iScriptTM™ cDNA synthesis kit. qPCR was performed with SsoAdvanced Universal SYBR Green Supermix™ using a StepOnePlus™ Real- Time PGR System (Applied Biosystems™, USA). The following Primer sequences were used. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as house-keeping gene. TABLE 1: Primer sequences for qPCR

[0124] RNA-Seq processing and analysis

[0125] The reads were aligned to the human genome, v. GRCh38, p7 using the STAR aligner, v. 2.7.8. a

[0057] . For quality control, we used FastQC™

[0058] and MultiQC™

[0059] . Read counts were collected using featureCounts™

[0060] . Features with fewer than a total of 10 counts or fewer than 5 samples with at least 3 counts were eliminated.

[0126] For differential gene expression analysis, we used the R package DESeq2

[0061] , v. 1.30.1. Gene set enrichment testing was done with the CERNO algorithm implemented in the tmod package

[0062] , v. 0.50.7. with gene sets sourced from the MSigDB™ using the msigdbr package

[0063] , v. 7.4.1. P-values were corrected for multiple testing using the Benjamini-Hochberg method

[0064] .

[0127] Statistical analysis

[0128] Each experiment was performed at least three times, and the data were shown as the mean ± standard error. Data were analyzed using Sigma Plot™ version 10.0. Unpaired student’s t- test was used with P < 0.05 considered statistically significant.

[0129] Chemical Synthesis of BP75 and BP79 and analogs thereof General Procedure A: Ring-opening condensation 3,4-dichloroaniline (or 4-phenoxyaniline, 1 equiv.) and maleic anhydride were dissolved in toluene. Triethylamine (1.1 equiv.) was added to the mixture and stirred for 24h at 70°C. The product was purified by flash chromatography using a mixture of DCM and methanol.

[0130] Scheme 1: Synthesis of BP75

[0131] Intermediate 1. (Z)-4-oxo-4-((4-phenoxyphenyl)amino)but-2-enoic acid was prepared as outlined in General Procedure A on a 2.807mmol scale.

[0132] Intermediate 2. (Z)-4-oxo-4-((4-phenoxyphenyl)amino)but-2-enoic acid (4.014mmol, 1 equiv.) was dissolved in DCM. Thionyl chloride (80.280mmol, 20 equiv.) was added dropwise to the solution and the reaction was stirred for 16h at room temperature. 3-chloro- l-(4- phenoxyphenyl)pyrrolidine-2, 5-dione was purified by flash chromatography using a mixture of hexane and ethyl acetate.1H NMR (400 MHz, CDCI3) δ 7.47 - 7.34 (m, 3H), 7.17 (t, J = 7.6 Hz, 1H), 7.12 - 6.95 (m, 5H), 4.73 (dd, J = 8.7, 4.1 Hz, 1H), 3.40 (dd, J = 18.9, 8.7 Hz, 1H), 3.02 (dd, J = 18.9, 4.1 Hz, 1H). 13C NMR (101 MHz, CDCh) δ 171.93, 158.16, 156.21, 132.41, 130.36, 130.03, 124.12, 120.77, 119.54, 118.95, 116.51, 49.03, 39.33. (752.3 mg, 62% yield).

[0133] BP75. 3-chloro-l-(4-phenoxyphenyl)pyrrolidine-2, 5-dione (2.025mmol, 1 equiv.) and 2- imidazolidinethione (4.050mmol, 2 equiv.) were dissolved in DMF and stirred for 6h at 70°C. The reaction was diluted with water and the product was extracted by ethyl acetate. 3-((4,5- dihydro-lH-imidazol-2-yl)thio)-l-(4-phenoxyphenyl)pyrrolidine-2, 5-dione was purified by flash chromatography using a mixture of hexane and ethyl acetate.1H NMR (400 MHz, DMSO) δ 10.26 (s, 1H), 7.45 - 7.36 (m, 2H), 7.35 - 7.26 (m, 3H), 7.16 (t, J = 7.4 Hz, 1H), 7.05 - 6.98 (m, 2H), 6.75 - 6.68 (m, 1H), 4.88 (dd, J = 10.0, 3.5 Hz, 1H), 4.17 (td, J = 8.2, 1.9 Hz, 2H), 3.63 (td, J = 8.5, 2.3 Hz, 2H), 3.24 (dd, J = 16.9, 3.6 Hz, 1H), 3.02 (dd, J = 17.0, 10.0 Hz, 1H).13C NMR (101 MHz, DMSO) δ 168.59, 167.30,160.94, 157.54, 156.86, 140.63, 130.67, 124.10, 119.31, 114.44, 113.97, 109.51, 61.18, 51.85, 41.77. HPLC-MS tR7.84 minutes, 91.2% purity, [C19H17N3O3S + H] = 369.1, found 368.7 (325.8mg, 40% yield).

[0134] Scheme 1: Synthesis of BP79

[0135] Intermediate 3. (Z)-4-((3,4-dichlorophenyl)amino)-4-oxobut-2-enoic acid was prepared as outlined in General Procedure A on a 1.020 mmol scale (221. Omg, 90% yield).1H NMR (400 MHz, Acetone) δ 7.98 )d, J=2.3 Hz, 1H), 7.42 (dd, J=8.8, 2.4 Hz, 1H), 7.35 (d, J=8.7 Hz, 1H), 6.20 (d, / =13.2 Hz, 1H), 6.07-5.98 (m, 1H).

[0136] Intermediate 4. (Z)-4-((3,4-dichlorophenyl)amino)-4-oxobut-2-enoic acid (6.031 mmol, 1 equiv.) was dissolved in DCM. Thionyl chloride (206.522 mmol, 34 equiv.) was added dropwise to the solution and the reaction was stirred for 16h at room temperature. 3-chloro- l-(3,4-dichlorophenyl)pyrrolidine-2, 5-dione was purified by flash chromatography using a mixture of hexanes and ethyl acetate.1H NMR (400 MHz, CDCI3) δ 7.49 (d, / =1.2 Hz, 1H), 7.49 - 7.39 (m, 1H), 7.23 - 7.11 (m, 1H), 4.72 (dd, J=8.8, 1.3 Hz, 1H), 3.42 (dd, J=19.0, 1.3 Hz, 1H), 3.02 (dd, J = 19.0, 1.2 Hz, 1H). (1546.5mg, 92% yield).

[0137] BP79. 3-chloro-l-(3,4-dichlorophenyl)pyrrolidine-2, 5-dione (1.175mmol, 1 equiv.) was added to a solution of 3 mL ACN and 1.5 mL ammonium hydroxide (30-33%) and stirred for 24h at room temperature. The crude precipitate was isolated by vacuum filtration. N1-(3,4- dichlorophenyljmaleamide was purified by flash chromatography using a mixture of hexane and ethyl acetate. NMR (400 MHz, DMSO) δ 11.30(s, 1H), 8.01 (d, J=2.4 Hz, 1H), 7.95 (s, 1H), 7.57 (d, / =8.7 Hz, 1H), 7.49-7.42 (m, 2H), 6.28 (s, 2H).13C NMR 101 (MHz, DMSO) δ 167.08, 164.38, 139.44, 132.69, 132.04, 131.50, 131.19, 125.33, 120.90, 119.77.

[0138] Chemical Synthesis and Characterization of BP79 Analogs

[0139] General Procedure B: Amide-coupling Reactions

[0140] Intermediate 3 (260.08g / mol, 1 equiv.) was placed in a round-bottom flask and sealed with a rubber septum. The flask was vented with N2. Anhydrous DMF (0.5mL) was injected into the flask and the solution was stirred. DIPEA (2 equiv.) was injected into the flask and stirred. The amine (1 equiv.) was dissolved in anhydrous DMF (0.3mL) and injected into the flask. HATU

[0141] (1.1 equiv.) was dissolved in warmed anhydrous DMF (0.8mL) and injected into the flask. The reaction was stirred for 16h at room temperature. The reaction was poured into water and extracted by ethyl acetate. The organic layer was washed with saturated sodium bicarbonate, water, and brine. The organic layer was dried by MgSCH and gravity filtered. The products were purified by automated flash chromatography using a mixture of hexane and ethyl acetate.

[0142]

[0143] Scheme A.8: Synthesis of BP252.

[0144] BP252. N1-benzyl-N4-(3,4-dichlorophenyl)maleamide was prepared as outlined in General

[0145] Procedure B using benzylamine on a 0.327mmol scale. The reaction mixture was poured into water (10mL). The crude product was extracted in ethyl acetate (10mL x 3). The organic layers were combined and washed with water (30mL x 3), brine (30mL), and dried by MgS04. The crude product was gravity filtered and purified by flash chromatography on a 10g silica column using a hexane:ethyl acetate gradient at a flow rate of 20mL / min. The gradient began at 30% ethyl acetate and ended at 60% ethyl acetate (22.7mg, 18% yield). 1H NMR (400 MHz, DMSO) δ 11.12 (s, 1H), 8.98 (t, / = 6.0 Hz, 1H), 8.03 (d, / = 2.5 Hz, 1H),

[0146] 7.62 - 7.45 (m, 2H), 7.35 - 7.22 (m, 5H), 6.35 (d, / = 1.9 Hz, 2H), 4.35 (dj = 5.9 Hz, 2H).13C

[0147] NMR (101 MHz, DMSO) δ 165.01, 164.62, 139.47, 139.24, 132.88, 131.49, 131.18, 128.77,

[0148] 127.94, 127.41, 125.30, 120.90, 119.80, 42.73. HPLC-MS tR= 7.060min, 99.8% purity,

[0149] [C17H14CI2N2O2 + H] = 349.0, found 349.0.

[0150] Scheme A.9: Synthesis of BP253.

[0151] BP253. N1-cyclopentyl- N4-(3,4-dichlorophenyl)maleamide was prepared as outlined in

[0152] General Procedure B using cyclopentylamine on a 0.779mmol scale. The reaction mixture was poured into water (10mL). The crude product was extracted in ethyl acetate (10mL x

[0153] 3). The organic layers were combined and washed with water (30mL x 3), brine (30mL), and dried by MgSO4. The crude product was gravity filtered and initially purified by flash chromatography on a 10g silica column using a hexane:ethyl acetate gradient at a flow rate of 20mL / min. The gradient began at 40% ethyl acetate and ended at 80% ethyl acetate. The product was further purified by reverse phase high performance liquid chromatography using an Agilent Prep 100A C18 column (30 x 50mm, 5μm particle size) with a flow rate of

[0154] 20mL / min. A water:acetonitrile gradient began with 95% and ended with 5% water was used to isolate the final product (111.16mg, 44% yield).1H NMR (400 MHz, DMSO) δ 11.49

[0155] (s, 1H), 8.56 (d, / = 7.3 Hz, 1H), 8.00 (dj = 2.4 Hz, 1H), 7.61 - 7.42 (m, 2H), 6.27 (dj = 2.1

[0156] Hz, 2H), 4.05 (q, / = 6.6 Hz, 1H), 1.82 (dqj = 12.9, 6.3 Hz, 2H), 1.64 (t, / = 7.7 Hz, 2H), 1.56 -

[0157] 1.36 (m, 4H).13C NMR (101 MHz, DMSO) δ 164.01, 163.96, 138.98, 132.65, 131.02, 130.81, 130.72, 124.80, 120.38, 119.31, 50.46, 32.09, 23.47. HPLC-MS tR= 9.522min, 99.5% purity,

[0158] [C15H16CI2N2O2 + H] = 327.0, found 327.0.

[0159] Scheme A.10: Synthesis of BP254

[0160] BP254. Methyl (4-((3,4-dichlorophenyl)amino)-4-oxobut-2-enoyl)glycinate was prepared as outlined in General Procedure B using methyl glycinate hydrochloride on a 1.083mmol scale. The reaction mixture was poured into water (10mL). The crude product was extracted in ethyl acetate (10mL x 3). The organic layers were combined and washed with water (30mL x 3), brine (30mL), and dried by MgS04. The crude product was gravity filtered and purified by flash chromatography on a 10g silica column using a hexane: ethyl acetate gradient at a flow rate of 20mL / min. The gradient began at 25% ethyl acetate and ended at 100% ethyl acetate (72.8mg, 20% yield).1H NMR (400 MHz, DMSO) δ 10.92 (s,

[0161] IH), 8.96 (tj = 5.9 Hz, IH), 8.01 (d, / = 2.4 Hz, IH), 7.57 (dj = 8.8 Hz, IH), 7.47 (ddj = 8.7,

[0162] 2.4 Hz, IH), 3.95 (d, / = 5.9 Hz, 2H), 3.64 (s, 3H).13C NMR (101 MHz, DMSO) δ 169.98,

[0163] 164.69, 164.31, 138.92, 133.22, 131.00, 130.69, 129.52, 124.87, 120.47, 119.37, 51.79,

[0164] 40.64. HPLC-MS tR= 7.987min, 88.9% purity, [C13H12CI2N2O4- H] = 329.0, found 329.0.

[0165] Scheme A.12: Synthesis of BP256.

[0166] BP256. N1-(3,4-dichlorophenyl)-N4-(4-hydroxyphenethyl)maleamide was prepared as outlined in General Procedure B using 4-(2-aminoethyl)phenol on a 0.495mmol scale. The reaction mixture was poured into water (10mL). The crude product was extracted in ethyl acetate (10mL x 3). The organic layers were combined and washed with NaHCO3(10mL), water (10mL), brine (10mL), and dried by MgS04. The crude product was gravity filtered and purified by flash chromatography on a 10g silica column using a hexane:ethyl acetate gradient at a flow rate of 20mL / min. The gradient began at 25% ethyl acetate and ended at

[0167] 100% ethyl acetate (39.4mg, 21% yield). NMR (400 MHz, DMSO) δ 11.36 (s, 1H), 9.15 (s,

[0168] 1H), 8.60 (d, / = 5.9 Hz, 1H), 8.01 (dj = 2.3 Hz, 1H), 7.57 (dj = 8.8 Hz, 1H), 7.48 (ddj = 8.8,

[0169] 2.5 Hz, 1H), 7.01 (d, / = 8.3 Hz, 2H), 6.70 - 6.63 (m, 2H), 6.33 - 6.22 (m, 2H), 3.31 - 3.26 (m,

[0170] 2H), 2.64 (t, / = 7.4 Hz, 2H).13C NMR (101 MHz, DMSO) δ 164.97, 164.47, 156.16, 139.43,

[0171] 132.97, 131.51, 131.35, 131.19, 129.95, 129.73, 125.32, 120.89, 119.81, 115.59, 41.22,

[0172] 34.44. HPLC-MS tR= 8.687min, 97.3% purity, [C18H16CI2N2O3 - H] = 377.1, found 377.0.

[0173]

[0174] Scheme A.13: Synthesis of BP257.

[0175] BP257. N1-(3,4-dichlorophenyl)-.N4-(l-(4-methoxyphenethyl))maleamide was prepared as outlined in General Procedure B using l-(4-methoxyphenyl)ethan-l-amine on a

[0176] 0.603mmol scale. The reaction mixture was poured into water (10mL). The crude product was extracted in ethyl acetate (10mL x 3). The organic layers were combined and washed with NaHCO3(10mL), water (10mL), brine (10mL), and dried by MgSO4. The crude product was gravity filtered and purified by flash chromatography on a 10g silica column using a hexane:ethyl acetate gradient at a flow rate of 20mL / min. The gradient began at 20% ethyl acetate and ended at 80% ethyl acetate (76.8mg, 34% yield).1H NMR (400 MHz, DMSO) δ

[0177] 11.20 (s, 1H), 8.90 (d, / = 8.1 Hz, 1H), 8.00 (dj = 2.4 Hz, 1H), 7.57 (d, / = 8.8 Hz, 1H), 7.46

[0178] (ddj = 8.7, 2.4 Hz, 1H), 7.25 (dj = 8.3 Hz, 2H), 6.87 (d, / = 8.3 Hz, 2H), 6.37 - 6.25 (m, 2H),

[0179] 4.93 (p, / = 7.1 Hz, 1H), 3.72 (s, 3H), 1.36 (d, / = 7.0 Hz, 3H).13C NMR (101 MHz, DMSO) δ

[0180] 164.24, 163.38, 158.15, 138.98, 135.84, 132.81, 131.00, 130.70, 130.43, 127.28, 124.79,

[0181] 120.41, 119.31, 113.63, 55.05, 47.42, 22.16. HPLC-MS tR= 9.395min, 97.5% purity,

[0182] [C19H18CI2N2O3 - H] = 391.1, found 391.0.

[0183]

[0184] BP258. N1-(3,4-dichlorophenyl)-Al4-((3-trifluoromethyl)benzyl)maleamide was prepared as outlined in General Procedure B using 3 -trifluoromethyl benzylamine on a 0.684mmol scale. The reaction mixture was poured into water (10mL). The crude product was extracted in ethyl acetate (10mL x 3). The organic layers were combined and washed with NaHCO3(10mL), water (10mL), brine (10mL), and dried by MgSO4. The crude product was gravity filtered and purified by flash chromatography on a 10g silica column using a hexane:ethyl acetate gradient at a flow rate of 20mL / min. The gradient began at 30% ethyl acetate and ended at 60% ethyl acetate (74.4mg, 26% yield).1H NMR (400 MHz, DMSO) δ

[0185] 10.89 (s, 1H), 8.96 (t, J = 6.0 Hz, 1H), 8.07 (dj = 2.4 Hz, 1H), 7.77 (s, 1H), 7.64 - 7.52 (m,

[0186] 4H), 7.47 (dd, / = 8.8, 2.4 Hz, 1H), 6.37 (qj = 12.1 Hz, 2H), 4.44 (d, / = 5.9 Hz, 2H).13C NMR

[0187] (101 MHz, DMSO) δ 165.15, 163.81, 140.56, 139.00, 131.96, 131.47, 131.06, 130.92, 130.64,

[0188] 129.21, 124.87, 120.39, 119.25, 41.68. HPLC-MS tR= 9.743min, 98.6% purity,

[0189] [C18H13CI2F3N2O3 - H] = 415.0, found 415.0.

[0190] Scheme A.15: Synthesis of BP259.

[0191] BP259. N1-(3,4-dichlorophenyl)-.N4-((3-methyl)benzyl)maleamide was prepared as outlined in General Procedure B using 3 -trifluoromethyl benzylamine on a 0.641mmol scale. The reaction mixture was poured into water (10mL). The crude product was extracted in ethyl acetate (10mL x 3). The organic layers were combined and washed with NaHCO3(10mL), water (10mL), brine (10mL), and dried by MgSO4. The crude product was gravity filtered and purified by flash chromatography on a 10g silica column using a hexane:ethyl acetate gradient at a flow rate of 20mL / min. The gradient began at 30% ethyl acetate and ended at 60% ethyl acetate (32.0mg, 14% yield).1H NMR (400 MHz, DMSO) δ

[0192] 11.12 (s, 1H), 8.93 (t, J = 5.9 Hz, 1H), 8.05 (dj = 2.4 Hz, 1H), 7.61 - 7.54 (m, 1H), 7.48 (ddj

[0193] = 8.8, 2.4 Hz, 1H), 7.19 (ddj = 17.8, 10.3 Hz, 2H), 7.07 (t, J = 9.0 Hz, 2H), 6.35 (s, 2H), 4.31

[0194] (dj = 5.8 Hz, 2H), 2.28 (s, 3H).13C NMR (101 MHz, DMSO) δ 164.54, 164.11, 139.01,

[0195] 138.67, 137.39, 132.07, 131.01, 130.96, 130.71, 128.18, 128.08, 127.53, 124.54, 120.39,

[0196] 119.30, 42.17, 20.98. HPLC-MS tR= 9.672min, 98.3% purity, [C18H16CI2N2O2 - H] = 361.1, found 361.0.

[0197] Scheme A.16: Synthesis of BP260.

[0198] BP260. N1-(3,4-dichlorophenyl)-N4-((4-tetrahydropyranyl)methyl)maleamide was prepared as outlined in General Procedure B using 4-aminomethyltetrahydropyran on a

[0199] 0.733mmol scale. The reaction mixture was poured into water (10mL). The crude product was extracted in ethyl acetate (10mL x 3). The organic layers were combined and washed with NaHCO3(10mL), water (10mL), brine (10mL), and dried by MgSO4. The crude product was gravity filtered and purified by flash chromatography on a 10g silica column using a hexane:ethyl acetate gradient at a flow rate of 20mL / min. The gradient began at 50% ethyl acetate and ended at 100% ethyl acetate (93.1mg, 36% yield).1H NMR (400 MHz, CDCI3) δ

[0200] 12.42 (s, 1H), 7.93 (dj = 2.4 Hz, 1H), 7.51 (ddj = 8.7, 2.4 Hz, 1H), 7.37 (dj = 8.7 Hz, 1H),

[0201] 6.93 (s, 1H), 6.27 (d, / = 13.5 Hz, 1H), 6.16 (d, / = 13.5 Hz, 1H), 4.03 - 3.94 (m, 2H), 3.38 (tdj

[0202] = 11.7, 2.0 Hz, 2H), 3.28 (t, / = 6.5 Hz, 2H), 1.84 (ddt, / = 11.6, 8.1, 4.2 Hz, 1H), 1.65 (d, / =

[0203] 12.9 Hz, 2H), 1.36 (qd, / = 12.2, 4.5 Hz, 2H).13C NMR (101 MHz, DMSO) δ 165.19, 164.49,

[0204] 139.44, 132.66, 131.52, 131.50, 131.19, 125.30, 120.86, 119.77, 67.18, 44.93, 35.10, 30.86.

[0205] HPLC-MS tR= 8.754, 97.5% purity, [C16H18CI2N2O3+ H] = 357.1, found 357.0.

[0206]

[0207] Scheme A.17: Synthesis of BP261.

[0208] BP261. Methyl 3-((4-((3,4-dichlorophenyl)amino)-4-oxobut-2-enamido)methyl)benzoate was prepared as outlined in General Procedure B using methyl 3-(aminomethyl)benzoate on a 0.742mmol scale. The reaction mixture was poured into water (10mL). The crude product was extracted in ethyl acetate (10mL x 3). The organic layers were combined and washed with NaHCO3(10mL), water (10mL), brine (10mL), and dried by MgSO4. The crude product was gravity filtered and purified by flash chromatography on a 10g silica column using a hexane:ethyl acetate gradient at a flow rate of 20mL / min. The gradient began at

[0209] 30% ethyl acetate and ended at 60% ethyl acetate (94.2mg, 31% yield).1H NMR (400 MHz, DMSO) δ 10.99 (s, 1H), 9.02 (t, / = 6.0 Hz, 1H), 8.02 (dj = 2.4 Hz, 1H), 7.92 (s, 1H), 7.85 (dtj

[0210] = 7.7, 1.5 Hz, 1H), 7.58 (dd, / = 12.0, 8.3 Hz, 2H), 7.48 (ddd, / = 7.8, 4.6, 2.2 Hz, 2H), 6.40 -

[0211] 6.32 (m, 2H), 4.42 (d, / = 5.9 Hz, 2H), 3.84 (s, 3H).13C NMR (101 MHz, DMSO) δ 166.18,

[0212] 164.74, 164.08, 139.68, 138.98, 132.46, 132.09, 131.03, 130.90, 130.68, 129.72, 128.77,

[0213] 128.12, 127.78, 124.85, 120.44, 119.32, 52.13, 41.89. HPLC-MS tR= 8.754, 97.8% purity,

[0214] [C19H16CI2N2O4 + H] = 407.1, found 407.0.

[0215] Scheme A.18: Synthesis of BP262.

[0216] BP262. Intermediate 3 (260.07g / mol, 0.605mmol, 1 equiv.) was placed in a round-bottom flask and sealed with a rubber septum. The flask was vented with N2 gas. Pyridine (ImL) was injected into the flask and the solution was stirred. 4-Methoxy aniline (0.605mmol, 1 equiv.) was resuspended in pyridine (0.5mL) and injected to the flask. Phosphoryl chloride

[0217] (0.666mmol, 1.1 equiv.) was injected into the flask in a dropwise manner and the reaction was allowed to stir for 16h at room temperature. The reaction was quenched in cold water

[0218] (10mL) and extracted by ethyl acetate (10mL x 3). The organic layers were combined and washed with NaHCO3(10mL) and brine (10mL). The crude product was dried by MgSO4and gravity filtered. The product was initially purified by automated flash chromatography using a hexane:ethyl acetate gradient beginning at 25% ethyl acetate and ending at 100% ethyl acetate. The product was further purified by reverse phase high performance liquid chromatography using an Agilent Prep 100A C18 column (30 x 50mm, 5μm particle size) with a flow rate of 20mL / min. A water:acetonitrile gradient began with 95% and ended with 5% water was used to isolate the final product (5.3mg, 2% yield).1H NMR (400 MHz, DMSO) δ 11.27 (s, 1H), 10.68 (s, 1H), 8.16 (s, 1H), 7.63 (dd, / = 24.3, 8.9 Hz, 4H), 7.23 (d, / = 3.9 Hz, 2H), 6.92 (d, / = 8.6 Hz, 2H), 3.73 (s, 3H).13C NMR (101 MHz, DMSO) δ 164.38,

[0219] 162.61, 155.54, 139.03, 131.97, 131.84, 131.21, 131.00, 130.72, 124.83, 120.95, 120.43,

[0220] 119.36, 113.92, 55.18. HPLC-MS tR= 9.715min, 99.2% purity, [C17H14CI2N2O3- H] = 363.0, found 363.1.

[0221] Scheme A.19: Synthesis of BP263.

[0222] BP263. Intermediate 3 (260.07g / mol, 0.484mmol, 1 equiv.) was placed in a round-bottom flask and sealed with a rubber septum. The flask was vented with N2 gas. Pyridine (LOmL) was injected into the flask and the solution was stirred. 2-Methylthio aniline (0.484mmol, 1 equiv.) was resuspended in pyridine (0.5mL) and injected into the flask. Phosphoryl chloride (0.532mmol, 1.1 equiv.) was injected into the flask in a dropwise manner and the reaction was allowed to stir for 16h at room temperature. The reaction was quenched in cold water (10mL) and extracted by ethyl acetate (10mL x 4). The organic layers were combined and washed with brine (15mL x 2). The crude product was dried by MgSO4and gravity filtered. The product was initially purified by automated flash chromatography using a hexane:ethyl acetate gradient beginning at 20% ethyl acetate and ending at 80% ethyl acetate. The product was further purified by reverse phase high performance liquid chromatography using an Agilent Prep 100A C18 column (30 x 50mm, 5μm particle size) with a flow rate of 20mL / min. A water:acetonitrile gradient began with 95% and ended with 5% water was used to isolate the final product (2.4mg, 1% yield).1H NMR (400 MHz,

[0223] MeOD) δ 7.98 (s, 1H), 7.70 (s, 1H), 7.54 - 7.40 (m, 3H), 7.24 (dd, / = 5.9, 3.5 Hz, 2H), 6.56 (d,

[0224] J = 12.6 Hz, 1H), 6.45 (dj = 12.6 Hz, 1H), 2.43 (s, 3H).13C NMR (101 MHz, DMSO) δ 163.00,

[0225] 162.87, 139.30, 134.82, 134.26, 134.02, 131.60, 131.32, 127.28, 126.75, 125.88, 125.72,

[0226] 121.07, 119.96, 15.63. HPLC-MS tR= 10.149, 98.4% purity, [C17H14CI2N2O2S + H] = 381.0, found 381.0.

[0227] Scheme A.20: Synthesis of BP264.

[0228] BP264. N1-([l,l'-biphenyl]-3-ylmethyl)-N4-(3,4-dichlorophenyl)maleamide was prepared as outlined in General Procedure B using [l,l'-biphenyl]-3-ylmethanamine on a 0.512mmol scale. The reaction was poured into water (10mL) and brine (ImL). The crude product was extracted by ethyl acetate (10mL x 3). The organic layers were combined and washed with NaHCO3(10mL), water (10mL), brine (10mL), and dried by MgSO4. The crude product was gravity filtered and initially purified by flash chromatography on a 10g silica column using a hexane:ethyl acetate gradient at a flow rate of 20mL / min. The hexane:ethyl acetate gradient began at 20% ethyl acetate and ended at 80% ethyl acetate. The product was further purified by high performance liquid chromatography using an Agilent Prep 100A

[0229] C18 column (30 x 50mm, 5μm particle size) with a flow rate of 20mL / min. A water:acetonitrile gradient began with 95% and ended with 5% water was used to isolate the final product (lO.Omg, 5% yield).1H NMR (400 MHz, Acetone) δ 12.75 (s, 1H), 8.81 (s,

[0230] 1H), 8.11 (s, 1H), 7.70 - 7.35 (m, 11H), 6.50 (dj = 13.3 Hz, 1H), 6.27 (dj = 13.3 Hz, 1H),

[0231] 4.65 (d, / = 4.8 Hz, 2H).13C NMR (101 MHz, Acetone) δ 166.44, 163.51, 142.16, 141.59,

[0232] 139.99, 139.67, 136.54, 136.28, 132.75, 131.51, 131.40, 129.98, 129.72, 128.30, 127.75,

[0233] 127.67, 127.30, 126.75, 121.68, 120.14. HPLC-MS tR= 10.836, 97.5% purity, [C23H18CI2N2O2

[0234] - H] = 423.1, found 423.0.

[0235] Scheme A.22: Synthesis of BP266.

[0236] BP266. Tert-butyl 4-(4-((3,4-dichlorophenyl)amino)-4-oxobut-2-enamido)piperidine-l- carboxylate was prepared as outlined in General Procedure B using tert-butyl 4- aminopiperidine-l-carboxylate on a 0.393mmol scale. The reaction mixture was poured into water (10mL) and brine (ImL). The crude product was extracted in ethyl acetate

[0237] (10mL x 6). The organic layers were combined and washed with NaHCO3(15mL), brine

[0238] (15mL), and dried by MgSO4. The crude product was gravity filtered and purified by flash chromatography on a 10g silica column using a hexane:ethyl acetate gradient at a flow rate of 20mL / min. The gradient began at 20% ethyl acetate and ended at 80% ethyl acetate

[0239] (71.2mg, 41% yield). NMR (400 MHz, CDCI3) δ 12.58 (s, 1H), 7.89 (dj = 2.4 Hz, 1H), 7.49

[0240] (ddj = 8.7, 2.4 Hz, 1H), 7.33 (dj = 8.7 Hz, 1H), 6.24 - 6.14 (m, 2H), 4.04 (s, 2H), 3.99 - 3.93

[0241] (m, 1H), 2.90 (t, / = 12.7 Hz, 2H), 1.92 (dd, / = 13.0, 3.8 Hz, 2H), 1.43 (s, 9H), 1.42 - 1.37 (m,

[0242] 2H).13C NMR (101 MHz, CDCI3) δ 164.73, 162.59, 154.73, 137.70, 136.51, 132.65, 130.48,

[0243] 130.44, 127.55, 121.76, 119.49, 80.02, 47.50, 38.68, 31.45, 28.44. HPLC-MS tR= 10.214min,

[0244] 98.6% purity, [C20H25CI2N3O4 - H] = 440.1, found 440.1.

[0245] BP267. Tert-butyl 4-(4-((3,4-dichlorophenyl)amino)-4-oxobut-2-enamido)piperidine-l- carboxylate (442.34g / mol, 0.115mmol, 1 equiv.) was dissolved in dichloromethane (ImL) in a round-bottom flask. Trifluoroacetic acid (6.230mmol, 57 equiv.) was added dropwise to the stirred solution and the reaction was stirred for 2h. The reaction was concentrated under reduced pressure. N1-(3,4-dichlorophenyl)- N4-(piperidin-4-yl)maleamide 2,2,2- trifluoroacetate was resuspended in dichloromethane and solvent was evaporated under reduced pressure, repeated a total of 6 times (25.6mg, 65% yield).1H NMR (400 MHz,

[0246] MeOD) δ 7.96 (dj = 2.2 Hz, 1H), 7.50 - 7.39 (m, 2H), 6.33 (dj = 1.8 Hz, 2H), 4.07 - 4.01 (m,

[0247] 1H), 3.43 (dtj = 13.3, 4.2 Hz, 2H), 3.18 - 3.08 (m, 2H), 2.17 (ddj = 14.2, 4.1 Hz, 2H), 1.80

[0248] (ddt, / = 14.4, 10.6, 5.5 Hz, 2H).13C NMR (101 MHz, MeOD) 5 166.61, 164.58, 138.78,

[0249] 132.63, 132.57, 132.37, 130.85, 127.41, 121.83, 119.92, 78.70, 44.79, 43.08, 38.21, 28.29.

[0250] HPLC-MS tR= 7.118min, 98.9% purity, [C15H17CI2N3O2- H] = 340.1, found 340.0.

[0251] Scheme A.24: Synthesis of BP268.

[0252] BP268. N1-(3,4-dichlorophenyl)-N4-(l-phenylpropyl)maleamide was prepared as outlined in General Procedure B using 1-phenylpropan-l-amine on a 0.327mmol scale. The reaction mixture was poured into water (10mL) and brine (ImL). The crude product was extracted in ethyl acetate (10mL x 3). The organic layers were combined and washed with NaHCO3 (10mL), water (10mL), brine (10mL), and dried by MgSO4. The crude product was gravity filtered and purified by flash chromatography on a 10g silica column using a hexane: ethyl acetate gradient at a flow rate of 20mL / min. The gradient began at 20% ethyl acetate and ended at 60% ethyl acetate (22.7mg, 18% yield). NMR (400 MHz, MeOD) δ 7.95 (d, / =

[0253] 2.1 Hz, 1H), 7.46 - 7.43 (m, 2H), 7.35 - 7.30 (m, 4H), 7.24 (tq, / = 5.7, 2.7 Hz, 1H), 6.39 - 6.26

[0254] (m, 2H), 4.84 (t, / = 7.4 Hz, 1H), 1.84 (pt, / = 10.3, 5.4 Hz, 2H), 0.93 (t, / = 7.3 Hz, 3H).13C

[0255] NMR (101 MHz, MeOD) 5 166.50, 165.59, 143.48, 139.56, 134.08, 133.38, 132.62, 131.58,

[0256] 129.52, 128.28, 127.79, 122.60, 120.68, 56.88, 30.38, 11.26. HPLC-MS tR= 11.060min,

[0257] 95.1% purity, [C19H18CI2N2O2- H] = 375.1, found 375.1.

[0258] Scheme A.25: Synthesis of BP269.

[0259] BP269. N1-(3,4-dichlorophenyl)-N4-(2-(dimethylamino)ethyl)maleamide was prepared as outlined in General Procedure B using N1,Mimethylethane- 1,2-diamine on a 0.427mmol scale. The organic layers were combined and washed with brine (15mL x 2). The reaction mixture was poured into water (10mL) and brine (ImL). The crude product was extracted in ethyl acetate (10mL x 3). The organic layers were combined and washed with NaHCO3

[0260] (10mL), water (10mL), brine (10mL), and dried by MgS04. The crude product gravity filtered and was purified by high performance liquid chromatography using an Agilent Prep

[0261] 100A C18 column (30 x 50mm, 5μm particle size) with a flow rate of 20mL / min. A water:acetonitrile gradient began with 95% and ended with 5% water was used to isolate the final product (5.1mg, 3.6% yield). NMR (400 MHz, DMSO) δ 9.67 (s, 1H), 8.83 (s, 1H),

[0262] 8.09 (s, 1H), 7.68 - 7.49 (m, 2H), 7.05 (dj = 15.2 Hz, 1H), 6.97 (dj = 15.0 Hz, 1H), 3.53 (d, /

[0263] = 6.3 Hz, 2H), 3.19 (t, J = 6.1 Hz, 2H), 2.81 (s, 6H).13C NMR (101 MHz, DMSO) δ 163.26,

[0264] 162.72, 138.88, 134.66, 132.06, 131.08, 130.79, 125.26, 120.50, 119.40, 57.88, 45.04, 36.94.

[0265] HPLC-MS tR= 7.426min, 99.1% purity, [C14H17CI2N3O2 + H] = 330.1, found 330.0.

[0266] Scheme A.26: Synthesis of BP270.

[0267] BP270. N4-(3,4-dichlorophenyl)-N4-((S)-l-phenyethyl)maleamide was prepared as outlined in General Procedure B using (S)-l-phenylethan-l-amine on a 0.246mmol scale.

[0268] The reaction mixture was poured into water (10mL) and brine (ImL). The crude product was extracted in ethyl acetate (10mL x 3). The organic layers were combined and washed with NaHCO3(10mL), water (10mL), brine (10mL), and dried by MgSO4. The crude product was gravity filtered and purified by flash chromatography on a 10g silica column using a hexane:ethyl acetate gradient at a flow rate of 20mL / min. The gradient began at 20% ethyl acetate and ended at 70% ethyl acetate (34.8mg, 39% yield).1H NMR (400 MHz, Acetone) δ

[0269] 12.81 (s, 1H), 8.75 (s, 1H), 8.08 (dj = 2.3 Hz, 1H), 7.50 (s, 2H), 7.42 (dj = 7.6 Hz, 2H), 7.37

[0270] - 7.31 (m, 2H), 7.28 - 7.23 (m, 1H), 6.45 (ddj = 13.4, 2.3 Hz, 1H), 6.23 (dd, / = 13.3, 2.4 Hz,

[0271] 1H), 5.19 (q, / = 7.5 Hz, 1H), 1.53 (dd, / = 7.0, 2.2 Hz, 3H).13C NMR (101 MHz, DMSO) δ

[0272] 164.25, 163.48, 143.95, 138.97, 132.73, 130.98, 130.69, 130.38, 128.26, 126.77, 126.08,

[0273] 124.77, 120.39, 119.30, 48.01, 22.22. HPLC-MS tR=10.608min, 96.4% purity,

[0274] [C18H16CI2N2O2 - H] = 361.1, found 361.0.

[0275] Scheme A.27: Synthesis of BP271.

[0276] BP271. N4-(3,4-dichlorophenyl)-N4-(( / ?)-l-phenyethyl)maleamide was prepared as outlined in General Procedure B using (R)-l-phenylethan-l-amine on a 0.246mmol scale.

[0277] The reaction mixture was poured into water (10mL) and brine (ImL). The crude product was extracted in ethyl acetate (10mL x 3). The organic layers were combined and washed with NaHCO3(10mL), water (10mL), brine (10mL), and dried by MgSO4. The crude product was gravity filtered and purified by flash chromatography on a 10g silica column using a hexane:ethyl acetate gradient at a flow rate of 20mL / min. The gradient began at 20% ethyl acetate and ended at 70% ethyl acetate (31.4mg, 31% yield).1H NMR (400 MHz, Acetone) δ

[0278] 12.82 (s, 1H), 8.77 (s, 1H), 8.08 (dj = 1.9 Hz, 1H), 7.50 (dj = 2.4 Hz, 2H), 7.42 (d, / = 7.3 Hz,

[0279] 2H), 7.35 (tj = 7.5 Hz, 2H), 7.26 (t, J = 7.3 Hz, 1H), 6.46 (dj = 13.4 Hz, 1H), 6.23 (dj = 13.4

[0280] Hz, 1H), 5.20 (pj = 7.2 Hz, 1H), 1.53 (d, / = 7.0 Hz, 3H).13C NMR (101 MHz, DMSO) δ

[0281] 164.25, 163.49, 143.96, 138.98, 132.76, 131.00, 130.69, 130.40, 128.27, 126.78, 126.08,

[0282] 124.78, 120.40, 119.30, 48.03, 22.22. HPLC-MS tR=10.310min, 99.1% purity,

[0283] [C18H16CI2N2O2+ H] = 363.1, found 363.1.

[0284] Scheme A.28: Synthesis of BP272.

[0285] BP272. N1-(3,4-dichlorophenyl)-.N4-(4-methoxybenzyl)maleamide was prepared as outlined in General Procedure B using 4-methoxy benzylamine on a 0.246mmol scale. The reaction mixture was poured into water (10mL) and brine (ImL). The crude product was extracted in ethyl acetate (10mL x 3). The organic layers were combined and washed with NaHCO3(10mL), water (10mL), brine (10mL), and dried by MgSO4. The crude product was gravity filtered and purified initially by flash chromatography on a 10g silica column using a hexane:ethyl acetate gradient at a flow rate of 20mL / min. The gradient began at 15% ethyl acetate and ended at 70% ethyl acetate. The product was further purified by high performance liquid chromatography using an Agilent Prep 100A C18 column (30 x 50mm,

[0286] 5μm particle size) with a flow rate of 20mL / min. A water:acetonitrile gradient began with

[0287] 95% and ended with 5% water was used to isolate the final product (17.0mg, 14% yield). 1H NMR (400 MHz, Acetone) δ 12.88 (s, 1H), 8.71 (s, 1H), 8.11 (s, 1H), 7.52 (s, 2H), 7.29 (d, /

[0288] = 8.6 Hz, 2H), 6.93 - 6.84 (m, 2H), 6.46 (d, / = 13.4 Hz, 1H), 6.24 (d, / = 13.3 Hz, 1H), 4.48 (d,

[0289] J = 3.5 Hz, 2H), 3.77 (s, 3H).13C NMR (101 MHz, DMSO) δ 164.37, 164.15, 158.33, 139.00,

[0290] 132.47, 131.00, 130.71, 130.65, 130.63, 128.85, 124.80, 120.41, 119.32, 113.70, 55.06,

[0291] 41.72. HPLC-MS tR=10.310min, 99.5% purity, [C18H16CI2N2O3- H] = 377.1, found 377.0.

[0292] Scheme A.29: Synthesis of BP273.

[0293] C

[0294] BP273. N1-(3,4-dichlorophenyl)-N4-(l-(3-methoxyphenyl)ethyl)maleamide was prepared as outlined in General Procedure B using (3-methoxyphenyl)ethan-l-amine on a

[0295] 0.340mmol scale. The reaction mixture was poured into water (10mL) and brine (ImL). The crude product was extracted in ethyl acetate (10mL x 3). The organic layers were combined and washed with NaHCO3(10mL), water (10mL), brine (10mL), and dried by MgSO4. The crude product was gravity filtered and purified by flash chromatography on a

[0296] 10g silica column using a hexane:ethyl acetate gradient at a flow rate of 20mL / min. The gradient began at 20% ethyl acetate and ended at 70% ethyl acetate. (46.2mg, 35% yield). 1H NMR (400 MHz, DMSO) δ 11.10 (s, 1H), 8.92 (dj = 8.1 Hz, 1H), 8.01 (dj = 2.4 Hz, 1H),

[0297] 7.57 (d, / = 8.8 Hz, 1H), 7.46 (dd, / = 8.8, 2.4 Hz, 1H), 7.23 (t, / = 7.8 Hz, 1H), 6.94 - 6.85 (m,

[0298] 2H), 6.80 (dd, / = 8.2, 2.6 Hz, 1H), 6.33 (d, / = 3.0 Hz, 2H), 4.95 (p, / = 7.1 Hz, 1H), 3.74 (s,

[0299] 3H), 1.37 (d, / = 7.0 Hz, 3H).13C NMR (101 MHz, DMSO) δ 164.20, 163.55, 159.30, 145.67,

[0300] 138.97, 132.45, 131.00, 130.69, 129.32, 124.81, 120.41, 119.31, 118.27, 112.07, 111.93,

[0301] 54.98, 48.02, 22.33. HPLC-MS tR=9.498min, 95.1% purity, [C19H18CI2N2O3+ H] = 393.1, found 393.1.

[0302] Scheme A.30: Synthesis of BP274.

[0303] BP274. N1-(3,4-dichlorophenyl)-N4-(3,4-dimethylbenzyl)maleamide was prepared as outlined in General Procedure B using 3,4-dimethylbenzylamine on a 0.313mmol scale. The reaction mixture was poured into water (10mL) and brine (ImL). The crude product was extracted in ethyl acetate (10mL x 3). The organic layers were combined and washed with NaHCO3(10mL), water (10mL), brine (10mL), and dried by MgSO4. The crude product was gravity filtered and purified initially by flash chromatography on a 10g silica column using a hexane:ethyl acetate gradient at a flow rate of 20mL / min. The gradient began at 15% ethyl acetate and ended at 60% ethyl acetate. The product was further purified by high performance liquid chromatography using an Agilent Prep 100A C18 column (30 x 50mm,

[0304] 5μm particle size) with a flow rate of 20mL / min. A water:acetonitrile gradient began with

[0305] 95% and ended with 5% water was used to isolate the final product (38.0mg, 32% yield). 1H NMR (400 MHz, Acetone) 5 12.90 (s, 1H), 8.67 (s, 1H), 8.12 (t, J = 1.5 Hz, 1H), 7.56 - 7.49

[0306] (m, 2H), 7.13 (s, IH), 7.08 (d, / = 2.0 Hz, 2H), 6.47 (d, / = 13.3 Hz, IH), 6.24 (d, / = 13.3 Hz,

[0307] IH), 4.47 (d, / = 5.8 Hz, 2H), 2.22 (s, 3H), 2.21 (s, 3H).13C NMR (101 MHz, DMSO) δ 164.45,

[0308] 164.09, 139.00, 135.97, 134.68, 132.20, 131.02, 130.91, 130.70, 129.32, 128.71, 124.91,

[0309] 124.81, 120.40, 119.30, 42.02, 40.15, 39.94, 39.73, 39.52, 39.31, 39.10, 38.89, 19.35, 18.99.

[0310] HPLC-MS tR= 9.903min, 95.6% purity, [C19H18CI2N2O2+ H] = 377.1, found 377.1.

[0311] Scheme A.31: Synthesis of BP275.

[0312] BP275. N1-(3,4-dichlorophenyl)-.N4-(l-(p-tolyl)ethyl)maleamide was prepared as outlined in General Procedure B using l-(p-tolyl)ethan-l-amine on a 0.340mmol scale. The reaction mixture was poured into water (10mL) and brine (ImL). The crude product was extracted in ethyl acetate (10mL x 3). The organic layers were combined and washed with NaHCO3

[0313] (10mL), water (10mL), brine (10mL), and dried by MgSO4. The crude product was gravity filtered and purified by flash chromatography on a 10g silica column using a hexane: ethyl acetate gradient at a flow rate of 20mL / min. The gradient began at 10% ethyl acetate and ended at 50% ethyl acetate (49.1mg, 39% yield). NMR (400 MHz, DMSO) δ 11.18 (s, 1H),

[0314] 8.91 (d, / = 8.0 Hz, 1H), 8.00 (dj = 2.4 Hz, 1H), 7.57 (dj = 8.8 Hz, 1H), 7.46 (ddj = 8.8, 2.4

[0315] Hz, 1H), 7.21 (d, / = 7.8 Hz, 2H), 7.12 (dj = 7.7 Hz, 2H), 6.34 (d, / = 12.3 Hz, 1H), 6.30 (d, / =

[0316] 12.4 Hz, 1H), 4.94 (p, / = 7.2 Hz, 1H), 2.26 (s, 3H), 1.36 (d, / = 7.0 Hz, 3H).13C NMR (101

[0317] MHz, DMSO) δ 164.21, 163.43, 140.89, 138.96, 135.83, 132.82, 130.99, 130.68, 130.40,

[0318] 128.78, 126.01, 124.78, 120.40, 119.30, 47.76, 22.18, 20.60. HPLC-MS tR= 10.174min,

[0319] 95.3% purity, [C19H18CI2N2O2- H] = 375.1, found 375.1.

[0320] Scheme A.32: Synthesis of BP276.

[0321] BP276. N1-(3,4-dichlorophenyl)- N4-(2-phenylpropan-2-yl)maleamide was prepared as outlined in General Procedure B using 2-phenylpropan-2-amine on a 0.412mmol scale. The reaction mixture was poured into water (10mL) and brine (ImL). The crude product was extracted in ethyl acetate (10mL x 3). The organic layers were combined and washed with NaHCO3(10mL), water (10mL), brine (10mL), and dried by MgSO4. The crude product was gravity filtered and purified initially by flash chromatography on a 10g silica column using a hexane:ethyl acetate gradient at a flow rate of 20mL / min. The gradient began at 15% ethyl acetate and ended at 80% ethyl acetate (13.8mg, 9% yield).1H NMR (400 MHz, CDCI3)

[0322] 8 11.98 (s, 1H), 7.82 (dj = 2.3 Hz, 1H), 7.51 - 7.45 (m, 1H), 7.41 - 7.27 (m, 7H), 6.16 (qj =

[0323] 13.4 Hz, 2H), 1.76 (s, 6H).13C NMR (101 MHz, CDCI3) δ 164.67, 162.66, 145.70, 137.66,

[0324] 135.91, 132.52, 131.42, 130.31, 128.55, 127.07, 124.72, 121.82, 119.57, 77.36, 77.04, 76.72,

[0325] 57.03, 28.78.13C NMR (101 MHz, CDCI3) δ 164.67, 162.66, 145.70, 137.66, 135.91, 132.52,

[0326] 131.42, 130.31, 128.55, 127.07, 124.72, 121.82, 119.57, 77.36, 77.04, 76.72, 57.03, 28.78.

[0327] HPLC-MS tR= 9.791min, 95.2% purity, [C19H18CI2N2O2 - H] = 375.1, found 375.1.

[0328] Scheme A.33: Synthesis of BP277. F3C.

[0329] H

[0330] Cl N

[0331] Cl o N

[0332] H

[0333] BP277. N1-(3,4-dichlorophenyl)- N4-(l-(3-(trifluoromethyl)phenyl)ethyl)maleamide was prepared as outlined in General Procedure B using l-(3-(trifluoromethyl)phenyl)ethan-l- amine on a 0.581mmol scale. The reaction mixture was poured into water (10mL) and brine (ImL). The crude product was extracted in ethyl acetate (10mL x 3). The organic layers were combined and washed with NaHCO3(10mL), water (10mL), brine (10mL), and dried by MgSO4. The crude product was gravity filtered and purified initially by flash chromatography on a 10g silica column using a hexane:ethyl acetate gradient at a flow rate of 20mL / min. The gradient began at 10% ethyl acetate and ended at 60% ethyl acetate

[0334] (101.7mg, 41% yield). NMR (400 MHz, DMSO) δ 10.89 (s, 1H), 8.96 (dj = 7.8 Hz, 1H),

[0335] 8.03 (d, / = 2.4 Hz, 1H), 7.75 (s, 1H), 7.65 (dj = 7.4 Hz, 1H), 7.62 - 7.53 (m, 3H), 7.46 (dd, / =

[0336] 8.7, 2.4 Hz, 1H), 6.35 (s, 2H), 5.06 (pj = 7.2 Hz, 1H), 1.40 (d, / = 7.0 Hz, 3H).13C NMR (101

[0337] MHz, DMSO) δ 163.99, 163.96, 145.70, 138.98, 131.48, 131.38, 131.02, 130.63, 130.34,

[0338] 129.27, 124.82, 123.51, 123.47, 122.67, 122.63, 120.37, 119.24, 47.76, 40.15, 39.94, 39.73,

[0339] 39.52, 39.31, 39.10, 38.89, 22.22. HPLC-MS tR= 9.716min, 96.9% purity, [C19H15CI2F3N2O2 -

[0340] H] = 429.1, found 429.0. Scheme A.34: Synthesis of BP287.

[0341] BP287. N1-(4-methylsulfonyl)benzyl- N4-(3,4-dichlorophenyl)maleamide was prepared as outlined in General Procedure B using 4-methylsulfonyl benzylamine on a 0.576mmol scale. The reaction mixture was poured into water (10mL) and brine (ImL). The crude product was extracted in ethyl acetate (10mL x 3). The organic layers were combined and washed with NaHCO3(10mL), water (10mL), brine (10mL), and dried by MgSO4. The crude product was gravity filtered and purified initially by flash chromatography on a 10g silica column using a hexane:ethyl acetate gradient at a flow rate of 20mL / min. The gradient began at 25% ethyl acetate and ended at 100% ethyl acetate (72.5mg, 29% yield).1H NMR

[0342] (400 MHz, CDCI3) δ 12.03 (s, 1H), 8.11 (s, 1H), 7.88 (ddj = 5.7, 2.8 Hz, 3H), 7.55 (dj = 8.0

[0343] Hz, 2H), 7.48 (ddj = 8.8, 2.4 Hz, 1H), 7.36 (dj = 8.7 Hz, 1H), 6.19 (s, 2H), 5.22 (pj = 7.1 Hz,

[0344] 1H), 3.05 (s, 3H), 1.58 (dj = 7.0 Hz, 3H).13C NMR (101 MHz, CDCI3) δ 164.82, 162.78,

[0345] 149.19, 139.57, 137.64, 136.04, 132.76, 130.95, 130.59, 127.97, 127.46, 121.93, 119.65,

[0346] 77.48, 77.16, 76.84, 49.43, 44.61, 38.80, 21.73. HPLC-MS tR= 8.789min, 93.5% purity,

[0347] [C19H18CI2N2O4S - H] = 439.0, found 439.0.

[0348] Crystallography

[0349] A single colourless blade crystals of BP79 recrystallised from a mixture of methanol and DCM by slow evaporation. A suitable crystal with dimensions 0.12 x 0.09 x 0.03 mm3was selected and mounted on a mylar loop in oil on a Bruker APEX II™ area detector diffractometer

[0068] .

[0350] The crystal was kept at a steady T = 115(2) K during data collection. The structure was solved with the XT™ 2018 / 2

[0066] solution program using Intrinsic Phasing methods and by using Olex2™

[0065] as the graphical interface. The model was refined with XL™

[0067] using full matrix least squares minimisation on F2.

[0351] Crystal Data

[0352] C10H8C12N2O2, Mr = 259.08, monoclinic, P21 / c(No. 14), a = 3.69390(10) A, b = 22.9196(7) A, c = 12.1562(4) A, b = 93.042(2)°, a = g = 90°, V = 1027.73(5) A3, T = 115(2) K, Z = 4, Z' = 1, m(MoKa) = 0.615, 11708 reflections measured, 3073 unique (Rint = 0.0359) which were used in all calculations. The final wRz was 0.0969 (all data) and R1 was 0.0404 (I>2 s(I)). EXAMPLES

[0353] EXAMPLE 1: Identification of hit compounds from chemical libraries

[0354] The ternary signaling complex of TSLP with its receptor forms in two steps: First, TSLP interacts with TSLPR to form a binary complex through distinct interactions and electrostatic complementarity. TSLP displays a positively charged surface and interacts with the negatively charged interdomain elbow region of TSLPR to form site I. Formation of site I is essential for the second step, in which the TSLP:TSLPR complex recruits IL-7Ra, thus, forming the signaling complex [6, 20]. TSLP then establishes a T-shaped ternary complex through two extensive interaction interfaces namely site I (TSLP:TSLPR) and site II (TSLP: IL-7Ra). TSLP bridges the two receptors and site III (TSLPR:IL-7Ra) is formed involving the interaction between the membrane proximal residues of the two receptors [6, 20]. We therefore, exploited structural information from the crystal structure of the ternary complex (PDB

[0021] code:5Jll [6]) and developed a 3D pharmacophore

[0022] for virtual screening based on the interaction of TSLP with TSLPR (site I) aiming to identify small molecules that hinder TSLPR complex formation (FIGURES la, b).

[0355] Pocket detection was applied to identify druggable binding sites on the surface of the complex. Cavity analysis indicated that the TSLP:TSLPR interface contains more druggable pockets than site II or site III. A thorough analysis of the crystal structure revealed key electrostatic complimentary interactions between Arg150, Argl53 of TSLP with Asp92 of TSLPR [6, 20]. Available mutational and fragment-based inhibitor identification studies also reported the integral role of these key residues thus solidifying our rationale to proceed with site I [6, 20, 23]. Careful consideration of all information from cavity detection, mutation data, and docking studies enabled us to develop 3D pharmacophore models using LigandScout 4.4™ [22, 24] that specifically represent binding hotspots at the TSLP:TSLPR interface (FIGURE la). Two 3D pharmacophores, one representing ligand interaction patterns for TSLPR and the other targeting TSLP, were developed and used for virtual screening to identify potential small molecule binders inhibiting TSLP:TSLPR complex formation. A library of 1,524,680 purchasable compounds from Specs™ (Delft, Netherlands) and VitasM™ (Hong Kong, China) was screened using these two 3D pharmacophores. A total of 3,838 and 56,309 virtual hits were obtained, respectively (FIGURE lb). Virtual hits were filtered by molecular weight (>250 Da), rotatable bonds (<7), and a minimum of 5 matching chemical features from the 3D pharmacophore model for further enrichment. The filtering resulted in the remaining 2,864 and 1,196 molecules as potential inhibitors for TSLPR and TSLP, respectively. To assess whether virtual hits fulfill the previously defined interaction patterns represented by the 3D pharmacophore, molecular docking experiments were performed by docking them into the prepared protein structure (PDB

[0021] code: 5J11 [6]) using GOLD v5.2™

[0025] . The resulting poses were compared with the 3D pharmacophore alignment from the virtual screening and similar conformations were prioritized. Subsequently, the selected molecules were screened focusing on the pre-defined key interactions and their fit in the cavity.

[0356] Eventually, twelve compounds of diverse chemical structures were selected for initial biological testing in the human TSLPR-expressing T cell line HuT78 26. First, the optimal experimental setup was determined showing 36 h as the ideal time for TSLP-mediated activation (FIGURE 9). We further investigated the suitability of two activation approaches, PMA-Ionomycin and CD3 / CD28, the latter mimicking dendritic cell-mediated activation. Although both activation methods resulted in similar cytokine release patterns, PMA- Ionomycin was selected for screening as it triggered higher cytokine levels overall (FIGURE 10).

[0357] Of the twelve tested compounds, C2, C6, C7 and C13 (FIGURES lc, d) significantly reduced TSLP-triggered IL-4 and IL-13 release in a concentration-dependent manner. C2, C6 and C7 have highly similar chemical structures, with subtle differences in their substitution patterns around their benzene ring, with the 3,4-dichlorobenzene functionality of C7 demonstrating the highest activity. Thus, C7 and C13 were further evaluated in human primary CD4+T cells yielding equally strong inhibitory effects at 20 μM concentrations (FIGURES le, f) while inducing no major cytotoxicity in primary human keratinocytes and fibroblasts (FIGURES 1g, h, i). The proposed binding mode of C7, designed to bind TSLPR, resembles the part of TSLP in site I. The nitrogen atoms present in the imidazole ring mimic the interaction formed by Argl53 of TSLP with Vall93 of TSLPR thus anchoring the molecule in the interdomain elbow region of TSLPR. Additionally, the dichlorophenyl moiety forms hydrophobic interactions with Leu39, Tyrl43, Tyrl94 and Vail 14 in our docking experiments thus suggesting further stabilization of the surmised binding conformation (FIGURE lj). The proposed binding mode of C13, designed to bind TSLP, is characterized by charge interactions and a hydrogen bonding network of the carboxylate moiety with Arg150 and Argl53 of TSLP, while the methylphenyl ring is interacting with Val67 and Leul47. In addition, there is a pi-cation interaction with Argl53 (FIGURE lj).

[0358] To initiate structure activity relationship (SAR) studies with these top inhibitor scaffolds, we utilized the National Cancer Institute™ (USA) - Discovery Therapeutics Program™ (NCI-DTP) library to search for chemical analogues of C7 and C13. Substructure and similarity searches were performed using InstantJChem™ (ChemAxon™), identifying 811 chemical analogues. These compounds were prepared for docking into the TSLP-TSLPR interaction interface using GLIDE™ (Schrodinger™). Eighty-four top scoring structural analogues of C7 and C13 were selected and tested in vitro using HuT78 cell line (data not shown). Some of these compounds incorporated features of both C7 and C13 and retained some of their IL-4 and IL-13 inhibitory activity. None of the compounds from the NCI-DTP library outperformed C7 and C13, however, these data were useful in guiding design efforts focused on improving the activity of these validated hit compounds.

[0359] EXAMPLE 2: Hit-to-Lead Development of novel TSLP Inhibitors

[0360] Using inspiration from C7, C13 and the NCI-DTP compounds, a series of analogues were produced incorporating a diverse range of chemical functionalities. Specific efforts were made to explore the features outlined in FIGURES 2a, b, modifying substituents on the aniline, rigidifying the core, exploring the impact of heterocycle substitutions, and incorporating ring systems with hydrogen bonding capacity. Notably, some of the most active NCI-DTP compounds did not possess a terminal aromatic ring system and instead possessed a carbamoylsulfanyl moiety (i.e., NSC13363 and NSC13350). Sixteen diverse compounds were synthesized and again screened for their efficacy to block TSLP-induced IL4 and IL13 release in Hut78 cells. From these sixteen compounds, BP79 most effectively blocked IL-13 (80% inhibition at 20 μM) and IL-4 (60% inhibition at 20 μM) release in a concentration-dependent manner (FIGURES 2 c, d). Combining features of C7 and C13 (i.e., BP86) led to a decrease in potency compared to both C7 and C13, supporting that these compounds are not likely to act as binders of both TSLP and TSLPR. Replacement of the sulfur atom with other heteroatoms appears to be reasonably tolerated, yet the most profound discovery was that the terminal dihydroimidazole or aromatic moiety was not required for potent inhibitory activity, as observed with BP79 (FIGURE 2g). This series also contained BP84, which was previously reported as an inhibitor of the TSLP signaling pathway, however in our hands this compound did not affect TSLP-induced expression of IL4 and IL 13 in a statistically significant fashion.

[0361] Together with moderately active (BP96) and non-active compounds (BP75, BP80), BP79 was assessed in human primary CD4+T cells to investigate inhibition of TSLP-mediated IL4 and IL13 expression (FIGURES 2 e, f). Again, BP79 demonstrated potent inhibition of IL4 and IL13 expression, BP96 gave a moderate level of inhibition, and the inactive compounds showed no inhibition of cytokine expression. This was further supported in dose-response BP79 inhibited TSLP-induced IL4 / IL13 expression in primary CD4+T cells with even stronger inhibitory effects (> 80% inhibition) at 10 μM and 20 μM concentrations (FIGURES 3 a, b). No significant cytotoxicity was observed at 20μM in primary human skin cells and T cells (viability > 80%) (FIGURES 3 c-f) indicating that BP79 is well tolerated by primary cells. As an additional positive effect, the TSLP-induced T cell hyperproliferation was abolished following BP 79 treatment (FIGURE 3 f).

[0362] Next, we assessed the impact of BP79 on TSLP signaling. TSLP activates JAK1 and JAK2 and subsequently phosphorylates downstream signaling proteins such as STAT3, STATS and STAT6. To identify the secondary messengers that stimulate Th2 cytokine secretion in primary human CD4+ T cells, TSLP activated T cells were treated with inhibitors of JAK1 / 2 (ruxolinitib

[0027] and AZD1870

[0028] ), STAT3 / 5 (SH4-54)

[0029] , STATS (573108)

[0030] , and STAT6 (AS1517499)

[0031] inhibitors showing that targeting the JAK-STAT pathway impairs IL-4 and IL-13 secretion (data not shown). In particular, the inhibition of JAK1 / 2, and STAT6 effectively downregulated Th2 cytokine secretion. Additionally, the STAT3 / 5 dual inhibitor suppressed IL-4 and IL-13 secretion. However, STATS inhibition by 573108 only modestly decreased Th2 cytokine secretion, primarily at rather high concentrations (100 μM). In keratinocytes, TSLP activates STAT3, thus, downregulating the skin barrier protein filaggrin while upregulating TSLP expression [13, 32].

[0363] Interestingly, BP79 treatment blocked STAT3 and STAT6 phosphorylation in keratinocytes and CD4+ T cells, respectively (FIGURES 3 g, h) suggesting that its inhibitory effect on IL-4 and IL-13 release indeed results from an inhibition of TSLP signaling.

[0364] EXAMPLE 3: BP79 abolishes TSLPR complex formation without broad-spectrum kinase inhibitory effects

[0365] To verify the interaction of BP79 with TSLPR, we next investigated if BP79 blocks TSLP- mediated ternary complex formation using a proximity ligation assay (PLA). To visualize TSLPR / IL-7Ra co-localization in human primary keratinocytes, TSLPR and IL-7Ra-specific antibodies were used to visualize receptor complex formation following TSLP stimulation. Strikingly, BP79 treatment strongly inhibited TSLP-mediated receptor complex formation showing > 10-fold inhibition compared to the untreated control (FIGURE 4a). The nonactive compound BP75 did not prevent the complex formation (data not shown).

[0366] To further confirm the binding between BP79 and TSLPR, a western blot-based in vitro thermal shift assay on recombinant TSLPR was performed in the presence of BP79. While the TSLPR band was hardly detectable at 37°C, pre-treatment with 20 μM BP79 stabilized the receptor up to 45°C (FIGURE 4b) indicative of a direct interaction between BP79 and TSLPR.

[0367] Finally, to evaluate if BP79 decreases the cytokine expression through direct binding of upstream kinases such as JAK1 / 2 or other kinase targets, BP79 was screened against 97 kinase targets using the KINOMEscan™ platform (radioligand displacement assay performed by Eurofins Discovery Services™). BP79 demonstrated moderate binding of some kinases at 10 μM. Notably, MAPKAPK2 and MARK3 showed > 50% indicative of additional kinase targets. There were limited inhibitory effects against JAK2, JAK3 and TYK2, which are part of the canonical JAK-STAT pathway that regulates STAT activation and inflammatory cytokine expression. While BP79 does not appear to be a broad-spectrum kinase inhibitor, these data cannot entirely rule out possible polypharmacologic effects associated with this compound (FIGURE 4c).

[0368] EXAMPLE 4: BP79 efficiently penetrates human skin and inhibits TSLP-mediated inflammation in an atopic-like skin disease model

[0369] Efficient skin absorption is a prerequisite for topical application and is governed by the physicochemical properties of the compounds. As such, we have defined key criteria for the initial in silico work such as a molecular weight < 800 Da and moderate lipophilicity (logP 1- 3). As BP79 fulfills these criteria (Mw = 259.01 Da and calculated logP = 1.79 (MarvinSketch 23.1™, ChemAxon™)), it was not surprising that we indeed observed efficient skin absorption indicated by the detection of permeated BP79 after 8 h and even more pronounced 24 h (FIGURE 5a).

[0370] We next utilized a previously established atopic dermatitis-like 3D skin disease model to test BP79 effects in tissue models of high clinical biomimicry. This skin disease model is based on filaggrin gene knockdown (FIGURE 5b), while the inflammatory phenotype is induced by adding IL4 and IL13

[0033] . Previous studies have verified its atopic-like characteristics including impaired skin barrier function, high TSLP expression, facilitated T cell migration, and impaired skin surface pH regulation [33-35]. In line with previous studies, TSLP expression significantly increased in the disease models and the addition of activated CD4+ T cells further enhanced TSLP secretion and an array of other, atopy-relevant markers such as IL5, IL2, IL4, IL9, IL13, and IFNγ (FIGURES 5b, c) compared to healthy skin samples. Importantly, the topical application of BP79 highly and significantly reduced the expression of these markers plus IL22 and TNFa indicative of strong anti- inflammatory effects.

[0371] EXAMPLE 5: BP79 exerts potent anti-inflammatory effects in a complex, human-based atopic diseases- on-a-chip model: A novel drug discovery platform

[0372] Due to limited functional cross-reactivity between human and murine TSLP and distinct inter-species related differences, further preclinical testing of BP79 in an in vivo model proved extremely difficult.

[0373] To overcome this limitation, we developed a microfluidic two-organ chip setup to study the efficacy and safety of BP79 in a complex, human-based setup. This platform contained an atopic-like skin disease model that was co-cultivated with a 3D bronchial epithelial tissue model over four days on a dynamic organ chip platform that ensured media circulation and flow from the skin to the healthy lung tissue (FIGURES 6 a, b). Activated CD4+ T cells were added to the circuit due to their role as direct TSLP effector cells.

[0374] Initially, we focused on model establishment and validation. Importantly, no histological changes were observed in the bronchial epithelial models after co-cultivation with normal skin models. However, the atopic-like skin model showed a significantly thickened epidermal layer and strong parakeratosis and exerted detrimental effects on the bronchial epithelial models that was characterized by impaired tissue cohesiveness and pronounced cell shedding (FIGURE 6c). The culture media of the atopic diseases chip contained significantly higher levels of classic Th2- derived cytokines like IL4, IL13, as well as TSLP and periostin (FIGURE 6d). Except for fluctuations due to media changes, LDH and glucose levels remained constant indicative of a homeostatic setup (FIGURES 6e, f).

[0375] Next, BP79 was topically applied onto the atopic-like skin disease models once every 24 h over 4 days. As a consequence, a distinct downregulation of key inflammation markers such as secreted TSLP, periostin, and IL-13 to levels observed in healthy chip setups were observed (FIGURE 6d). BP79 treatment also restored the impaired skin barrier, as exemplified by increased filaggrin expression, and decreased TSLP expression within the skin tissue (FIGURES 6g, h). At the same time, T cell infiltration into the skin models was efficiently blocked which occurred at high levels in the untreated atopic conditions (FIGURE 6h).

[0376] To determine the ability of our atopic diseases chip to reflect drug-specific effects, we included the clinically approved calcineurin inhibitor tacrolimus as a reference. While tacrolimus also significantly suppressed IL-4 and IL-13 secretion, it did not modulate the expression or secretion of periostin and TSLP. Similarly, it did neither prevent T cell migration into the disease model nor increased filaggrin expression (FIGURE 6 d, g, h).

[0377] For the bronchial epithelial models, increased TSLP expression was noted after cocultivation with untreated skin disease models which was less pronounced after BP79 treatment. Similarly, a more pronounced CD4+ T cell infiltration was observed in bronchial models co-cultivated with the untreated disease models which was reduced after BP79 treatment although less pronounced compared to skin (data not shown).

[0378] To gain additional insights on the effect of BP79 treatment on both skin and lung tissue, both tissues (treated and untreated) were subjected to transcriptomic analysis with RNASeq. Subsequently, we compared treated versus untreated skin and lung samples. For atopic-like and normal skin models, we observed a large number of differentially expressed genes, indicating a strong treatment effect. At FDR < 0.01, there were 1080 genes with at least 2- fold difference in treated versus untreated diseased skin models (FIGURE 6i) substantiating the presence of the disease phenotype, and the anti-inflammatory effect of BP79. On gene level, the topical application of BP79 onto diseased skin significantly changed protein synthesis and cell metabolism (data not shown) as exemplified by, for example, the enriched TMOD-gene set related to protein synthesis and the KEGG-Ribosome pathway (FIGURE 6k).

[0379] Further, we found that the transcription profiles of treated diseased skin were more similar to healthy skin than untreated diseased skin. For example, at FDR < 0.01 and absolute log2 FC > 2, there were 1080 DEGs between treated and untreated diseased skin, 809 DEGs between treated diseased skin and healthy skin, and as much as 3331 DEGs between untreated diseased skin and healthy skin. Furthermore, the Spearman correlation coefficients of the average gene expression were highest when comparing healthy skin with treated diseased skin (FIGURE 6j).

[0380] Interestingly, beneficial effects on the lung tissue were noted following BP79 administration onto the skin model. Pathway enrichment analysis for treated vs. untreated lungs showed enrichment in the KEGG and REACTOME aminoacyl tRNA biosynthesis pathway. Another enriched REACTOME pathway was sodium proton exchangers (p=0.0048) (FIGURE 6k).

[0381] EXAMPLE 6: Further development of lead inhibitor, BP79

[0382] Preliminary screen of BP79 analogues (TABLE 2) demonstrated several inhibitors showing equivalent inhibition of IL13 and IL-4 expression in HuT78 cells after treatment with 1μM of inhibitor for 36 hours compared to BP79 as shown in FIGURES 7a) and b). FIGURE 7 shows a further biological screen of twenty-six (26) BP79 analogs, a) IL-13 expression in HuT78 cells after treatment with 1μM of inhibitor for 36 hours, b) IL-4 expression in HuT78 cells after treatment with 1μM of inhibitor for 36 hours. Non-treated (NT), PMA- ionomycin stimulated (PI), PI and TSLP stimulated (PI + TSLP), a PMA-ionomycin + TSLP stimulated 0.4% DMSO vehicle control (DMSO) were included as controls. Although some of the BP79 analogs were able to inhibit IL-13 as well as BP79, there were also some that did not inhibit as well (i.e. BP243; BP261; BP262; BP264; BP266; BP269; BP273; BP276; and BP277). However, all of the BP79 analogs showed very similar IL-4 inhibition. Eight (8) of the BP79 analogs were chosen to test dose dependent inhibition of both IL-13 and IL- 4 (FIGURE 8) and compared against BP79. The does dependent assays involved CD4+ T- cells after treatment with lllnM, 333nM, and lOOOnM of inhibitor for 36 hours and compared cytokine expression relative to DMSO controls.

[0383] TABLE 2: BP79 analogues

[0384] Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. The word "comprising" is used herein as an open-ended term, substantially equivalent to the phrase "including, but not limited to", and the word "comprises" has a corresponding meaning. As used herein, the singular forms "a”, "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a thing" includes more than one such thing. Citation of references herein is not an admission that such references are prior art to an embodiment of the present invention. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples and drawings.

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Claims

CLAIMS:

1. A compound, the compound having the structure of Formula I:wherein,Ri is selected from: H;or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, wherein Ri is selected from: H;3. The compound of claim 1 or 2, wherein Ri is selected from: H;4. The compound of claim 1, 2, or 3, wherein Ri is selected from:; and5. A compound, the compound having the structure of Formula I:Formula I,wherein,Ri is selected from:; or a pharmaceutically acceptable salt thereof, for use in inhibiting thymic stromal lymphopoietin receptor (TSLPR) complex formation.

6. The compound of claim 5, wherein Ri is selected from: H;;7. The compound of claim 5 or 6, wherein Ri is selected from: H; and I8. The compound of claim 5, 6, or 7, wherein Ri is selected from:

9. The compound of any one of claims 5-8, wherein inhibiting TSLPR complex formation modulates cytokine expression.

10. The compound of claim 9, wherein modulating cytokine expression modulates IL4 or IL13 expression.

11. A compound of any one of claims 1-10, for treating one or more of the following: skin disease; allergic asthma; allergic rhinitis; drug allergies; and food allergies.

12. A compound of any one of claims 1-10, for treating one or more of the following: skin disease; asthma; allergies; chronic obstructive pulmonary disease (COPD); chronic rhinosinusitis; atopic kerato-conjunctivitis; non-celiac gluten sensitivity; eosinophilic granulomatosis with polyangiitis (EGPA); and eosinophilic esophagitis (EoE).

13. The compound of claim 11 or 12, wherein the skin disease is selected from one or more of the following: atopic dermatitis; urticaria; skin granulomas; nonthrombocytopenic palpable purpura; skin infarcts; and livedo reticulari.

14. The compound of claim 12, wherein the allergies are selected from one or more of the following: allergic asthma; allergic rhinitis; drug allergies; and food allergies.

15. A pharmaceutical composition, the pharmaceutical composition comprises a compound of any one of claims 1-10 and a pharmaceutically acceptable carrier.

16. The pharmaceutical composition of claim 15, wherein the pharmaceutical composition is for one or more of the following:(a) treating one or more of the following: skin disease; asthma; allergies; COPD; chronic rhinosinusitis; atopic kerato-conjunctivitis; non-celiac gluten sensitivity; EGPA; and EoE;(b) modulating cytokine expression; and(c) modulating TSLPR activity or inhibiting TSLPR.

17. The pharmaceutical composition of claim 16, wherein modulating cytokine expression is modulating of IL4 or IL13.

18. Use of a pharmaceutical composition comprising a compound of any one of claims 1- 10 and a pharmaceutically acceptable carrier, for one or more of the following:(a) treating one or more of the following: skin disease; asthma; allergies; COPD; chronic rhinosinusitis; atopic kerato-conjunctivitis; non-celiac gluten sensitivity; EGPA; and EoE;(b) modulating cytokine expression; and(c) modulating TSLPR activity or inhibiting TSLPR.

19. The use of claim 18, wherein the modulating of cytokine expression is modulating ofIL4 or IL13.

20. Use of a compound of any one of claims 1-10 in the manufacture of a medicament for one or more of the following:(a) treating one or more of the following: skin disease; asthma; allergies; COPD; chronic rhinosinusitis; atopic kerato-conjunctivitis; non-celiac gluten sensitivity; EGPA; and EoE;(b) modulating cytokine expression; and(c) modulating TSLPR activity or inhibiting TSLPR.

21. Use of a compound of any one of claims 1-10 for one or more of the following:(a) treating one or more of the following: skin disease; asthma; allergies; COPD; chronic rhinosinusitis; atopic kerato-conjunctivitis; non-celiac gluten sensitivity; EGPA; and EoE;(b) modulating cytokine expression; and(c) modulating TSLPR activity or inhibiting TSLPR.

22. The use of claim 20 or 21, wherein modulating cytokine expression is modulating of IL4 or IL13.

23. A method of treating one or more of the following: skin disease; asthma; allergies; COPD; chronic rhinosinusitis; atopic kerato-conjunctivitis; non-celiac gluten sensitivity;EGPA; and EoE, the method comprising administering a compound of any one of claims 1- 10 to a patient in need thereof.

24. A method of modulating cytokine expression, the method comprising administering a compound of any one of claims 1-10 to a patient in need thereof.

25. A method of modulating TSLPR activity or inhibiting TSLPR, the method comprising administering a compound of any one of claims 1-10 to a patient in need thereof.

26. The method of claim 24, wherein modulating cytokine expression is modulating of IL4 or IL13.

27. The method of claim 25, wherein the modulating TSLPR activity or inhibiting TSLPR is for the treatment TSLPR-mediated inflammatory condition.

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