Tropomyosin receptor kinase a (TRKA) inhibitors and uses thereof

Abstract

Provided herein are compounds of Formula I-III for use in modulating tropomyosin receptor kinase A (TrkA) activity. In particular, the compounds as described herein may have TrkA inhibitory activity. Furthermore, the compounds as described herein may also have multi-kinase inhibitory activity of Janus kinase 2 (JAK2) and / or Proto-Oncogene Tyrosine-Protein Kinase Src (SRC). The compounds as described herein may useful for the treatment of cancer, osteoarthritis (OA), rheumatoid arthritis (RA), inflammation, acute pain, and chronic pain.

Description

[0001] TROPOMYOSIN RECEPTOR KINASE A (TRKA) INHIBITORS AND USES THEREOF

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application Serial No.

[0004] 63 / 777,154 filed 25 March 2025 entitled " TRKA-TARGETED INHIBITORS AND METHODS OF USE".

[0005] TECHNICAL FIELD

[0006] This invention relates to the field of small molecule inhibitors of tropomyosin receptor kinase A (TrkA) and use of such compounds for the treatment of TrkA-mediated diseases. In particular, the TrkA inhibitor compounds described herein for use in the treatment of TrkA-mediated diseases.

[0007] BACKGROUND

[0008] Within the Signal Transducer and Activator of Transcription (STAT) protein family, STAT3 has been established as a major oncogenic signalling node, whose dysregulation can promote the development and progression of many types of cancer1. Cancers that exhibit overexpression and / or persistently active STAT3 include breast, colon, prostate, pancreatic, lung, brain, and renal cancer, as well as head and neck squamous cell carcinoma, melanoma, glioma, lymphoma, and leukaemia2. Canonically, the STAT3 signalling cascade entails cytokine or growth factor stimulation, which leads to the activation of receptor complexes that recruit and phosphorylate STAT3. Phosphorylated STAT3 monomers then form homodimers and translocate to the nucleus where they promote the expression of pro-tumorigenic and anti-apoptotic gene signatures3. Over the past few decades a much more complex picture of STAT3 signalling has emerged, where different activation patterns, dimerization and oligomerization partners, dimer orientations, activators, co-activators, and deactivators have been identified4-6.

[0009] Accordingly, the STAT3 signalling cascade is found at the center of many cellular processes, with several upstream activators (cytokines and growth factors) and mediators (kinases and phosphatases) as well as a myriad of downstream gene targets7-9. While these findings often highlight the important role of STAT3 in promoting and maintaining cancerous phenotypes, the broad and far-reaching roles of STAT3 in cellular and oncogenic processesmakes evaluating the specific actions of potential STAT3 inhibitors immensely challenging10.

[0010] STAT3 undergoes multiple post-translational modifications including methylation11, acetylation12and phosphorylation (Tyr705 and Ser727)13, which influence the activity, complexation and subcellular localization of the protein6. Multiple tyrosine and serine kinases act on STAT3 which contribute to its aberrant activation in tumour cells, including EGFR14, JAK1 / 215, Src16, BCR-ABL17and others18. Inhibiting STAT3 phosphorylation using tyrosine kinase inhibitors (TKI), in particular targeting JAK1 and JAK2 has gained recent attention as a promising strategy for disrupting STAT3 signalling cascades19. However, targeting upstream kinases has not been the magic bullet for novel cancer therapies, where limitations including on- and off-target adverse effects as well as innate and acquired resistance have limited their clinical utility and efficacy20'21. In addition to targeting upstream kinases, there are an abundance of small molecules that claim to bind directly to STAT3 to block its activity22. While directly targeting STAT3 is an attractive strategy for drug development efforts, like most transcription factors, STAT3 does not harbour an enzyme active site and is largely devoid of cavities that could be easily targeted with smallmolecule inhibitors23.

[0011] Modern chemical biology methods have helped to better characterize the activity of both classic and novel compounds that are proposed as direct STAT3 binders24”26. It has become evident that the earliest of STAT3 inhibitors (STAT31, i.e. STATTIC, S31-201 and others)27”29were promiscuous and induced non-STAT3 dependent phenotypes; however, these compounds were widely used to probe STAT3’s role in cancer and served as benchmarkers of STAT31 anti-cancer activity. The antithesis of these classic compounds appears to be the recently developed STAT3 protein degraders SD-36 and SD-91, which have been thoroughly evaluated for their ability to bind and inhibit STAT3 in biochemical and cellular settings30'31. There is great promise for these compounds as they move towards advanced preclinical testing stages. Many reported STAT31 claim to bind its Src Homology 2 (SH2) domain, which plays an essential role in regulating STAT3’s protein-protein interactions. Indeed, the SH2 domain has become a preferred site for developing STAT31, with potent and highly selective phosphopeptide binding sequences identified32. As such, targetingSTAT3 with small, drug-like molecules appears to be a possible, yet elusive goal which keeps STAT3 positioned at the very edge of the "druggable” proteome.

[0012] Advancements in protein thermal stability assays have provided powerful new tools for interrogating potential STAT3 binders in biochemical and cellular settings33'34. STAT3 thermal shift assays measure differences in its thermal stability in the presence or absence of a proposed inhibitor, where the formation of a STAT3-STAT31 complex will cause a shift in the melting temperature compared to vehicle controls. A benefit of these techniques is that they can evaluate inhibitor binding without honing on a specific binding cavity, which is a limitation of other established biochemical STAT31 assays, such as the fluorescence polarization assay24. Employing thermal stability assays to the STAT31 field has highlighted that many well-established STAT3 inhibitors lacked the ability to selectively bind to STAT3 in biochemical and cellular settings35'36. These findings have challenged the direct binding capabilities of early proposed STAT31 and, echoing calls from drug discovery and development experts,37have emphasized the importance of robust target engagement validation in early drug discovery projects.

[0013] Establishing a target-engagement focused screening and validation approach can mitigate failures and facilitate progression to more advanced stages of the drug development pipeline38. Due to its central role in cancer cell signalling, many cellular effects can be attributed to direct STAT3 inhibition in cells10. However, these same phenotypes are often possible through indirectly blocking STAT3 activity, by targeting STAT3 activators, mediators or outputs39. In this light, careful interpretation of STAT3 cell-based assay results becomes paramount, necessitating due diligence with respect to target-engagement and vigilance towards potential off-target and polypharmacologic effects, especially towards upstream kinases which moderate STAT3 signalling cascades.

[0014] The present work utilizes a STAT31 discovery screening funnel focused on thermal stability assays and target engagement techniques. Furthermore, a high-throughput screen using STAT3 differential scanning fluorimetiy [DSF] and rigorously evaluated promising compounds in complementary and orthogonal assays. While our top compounds ultimately did not validate as direct STAT3 binders, they demonstrated moderate inhibitionof STAT3 activity in cells that prompted kinome profiling experiments and revealed targeting of kinases linked to STAT3 signalling, including JAK2 and Src. Pyrazine compounds and pyridine compounds for inhibiting JAK kinases are known in the art91'92- However, the most potent inhibitory activity was identified with tropomyosin receptor kinase A (TrkA), a neurotrophic growth factor receptor that is known to form fusion gene products leading to rare forms of cancer40. Adhering to our target engagement-focused drug discovery efforts, the TrkA inhibitory scaffold was repurposed and further optimized to identify and validate lead TrkA inhibitors with potent activity in biochemical and cellbased assays.

[0015] TrkA protein (expressed from the NTRK1 gene) is a receptor tyrosine kinase that has been associated with the perineural invasion of cancer cells41and has garnered more attention for its propensity to form fusion mutants in rare cancers. These chimeric proteins arise from chromosomal rearrangements that fuse the N-terminal domain of a partner gene with the tyrosine kinase domain of Trk receptors (FIGURE 6A), resulting in fusion kinases that are necessary and sufficient for driving malignant transformation42. Larotrectinib85’87and entrectinib88-90are FDA-approved Trk inhibitors for the treatment of cancers bearing Trk fusions (FIGURE 6B). Intrigued by the potential applications for new Trk inhibitors and unexpectedly identifying top hits from our STAT3 screening pipeline as potent TrkA binders, we pivoted our target-engagement focus towards exploring the potential of this scaffold to generate new Trk inhibitors. Aiylpyrazinamines are known for tubulin inhibition and used in the treatment of cancer93and other pyrazines are known as protein kinase inhibitors for use in the treatment of infectious disease, cytomegalovirus and cancer94. TrkA is well known as a therapeutic target for cancer95and pain96.SUMMARY

[0016] The present invention is based in part, on the surprising discovery that compounds of Formula I, Formula II, and Formula III as described herein, have tropomyosin receptor kinase A (TrkA) inhibitory activity. Furthermore, the compounds described herein also surprisingly have multi-kinase inhibitory activity of Janus kinase 2 (JAK2) and / or ProtoOncogene Tyrosine-Protein Kinase Src (SRC). It was also discovered, that compounds of Formula I, Formula II, and Formula III as described herein have anti-cancer activity and potential uses in treating osteoarthritis (OA), rheumatoid arthritis (RA), inflammation, acute pain, and chronic pain.

[0017] Herein, we describe the discovery and validation of 6-aryl-N2-(l-phenylethyl)pyrazine-2,3-diamine inhibitors (Pyrazine Inhibitors, PI) as Trk binders. The series of TrkA inhibiting compounds described herein may be useful for the inhibition of TrkA activity in NTRK1 fusion-positive cancers, but also for treatment of other TrkA-associated cancers and TrkA-associated disorders including, but not limited to, osteoarthritis or rheumatoid arthritis-related pain and inflammation. Overexpression of TrkA, TrkB, and TrkC are found in several forms of cancer, including basal cell carcinoma, lung cancer, and neuroblastoma. The compounds described herein may also be useful in preventing perineural invasion (PNI) of cancer cells and may also prevent associated metastasis. The compounds described herein also modulate one or more of tropomyosin receptor kinase A (TrkA); tropomyosin receptor kinase B (TrkB); tropomyosin receptor kinase C (TrkC); Janus kinase 2 (JAK2); and Proto-Oncogene Tyrosine-Protein Kinase Src (SRC). The compounds described herein also modulate one or more of TrkA; JAK2; and SRC. Furthermore, the intersection of the Trk and JAK2 / SRC pathways are provide an important target for the treatment of pain and inflammation. Additionally, TrkA and its respective receptor-ligand, NGF, play a key role in modulating both acute and chronic pain, particularly in conditions associated with inflammation such as endometriosis, Osteoarthritis (OA) or Rheumatoid Arthritis (RA). Alternatively, JAK2 / SRC-linked inflammatory signaling may contribute to tissue pathology, sensitization and persistence. A single molecule, as described herein, could also avoid the PK / tissue-distribution mismatch and extra kinome complexity ofpairing separate Trk and JAK inhibitors. NGF / TrkA pain signaling coexists with inflammatory cytokine or remodeling pathways providing a novel target opportunity.

[0018] In a first embodiment, there is provided a compound, the compound having the structure of Formula I and Formula II:

[0019]

[0020] methyl; ethyl; and tert-butyl; or a stereoisomer thereof or a pharmaceutically acceptable salt thereof.In a further embodiment, there is provided a compound, the compound having the

[0021] structure of Formula

[0022]

[0023] III: Formula III wherein, R3may be selected

[0024]

[0025] or a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

[0026] In a further embodiment, there is provided a pharmaceutical composition, the pharmaceutical composition comprises a compound described herein and a pharmaceutically acceptable carrier.

[0027] 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 treating one or more Trk associated diseases or disorders.

[0028] In a further embodiment, there is provided use of a compound, as described herein, in the manufacture of a medicament for treating one or more Trk associated diseases or disorders.

[0029] In a further embodiment, there is provided a use of a compound described herein for treating one or more Trk associated diseases or disorders.

[0030] In a further embodiment, there is provided a method of treating one or more Trk associated diseases or disorders.

[0031] A Trk associated disease or disorder may be selected from one or more one of:

[0032] (i) TRK fusion-positive tumours;

[0033] (ii) cancer; osteoarthritis (OA); rheumatoid arthritis (RA); inflammation; acute pain; and chronic pain;

[0034] (in) endometriosis;

[0035] (iv) breast cancer; colon cancer; prostate cancer; pancreatic cancer; lung cancer; brain cancer; renal cancer; and head and neck cancer;(v) squamous cell carcinoma; melanoma; glioma; basal cell carcinoma; neuroblastoma; lymphoma; and leukaemia;

[0036] (vi) HER2+ breast cancer; and triple negative breast cancer (TNBCj;

[0037] (vii) cellular congenital mesoblastic nephroma; secretory breast sarcoma; mammary analog secretory carcinoma; and infantile fibrosarcoma;

[0038] ( iiij undifferentiated sarcomas; gliomas; papillary thyroid cancers; spitzoid neoplasms; inflammatory myofibroblastic tumors; and acute leukemias; and

[0039] [ix] cellular congenital mesoblastic nephroma; secretory breast sarcoma; mammary analog secretory carcinoma; infantile fibrosarcoma; undifferentiated sarcomas; gliomas; papillary thyroid cancers; spitzoid neoplasms; inflammatory myofibroblastic tumors; and acute leukemias.

[0040] In a further embodiment, there is provided a method of treating one or more of the following: cancer; osteoarthritis (OA); rheumatoid arthritis (RAj; inflammation; acute pain; and chronic pain, comprising the administration of a compound described herein to a subject in need thereof.

[0041] In a further embodiment, there is provided a use of a compound or pharmaceutical composition as described herein for the treatment of one or more of the following: cancer; osteoarthritis (OAJ; rheumatoid arthritis (RAJ; inflammation; acute pain; and chronic pain. In a further embodiment, there is provided a use of a compound, as described herein, in the manufacture of a medicament for the treatment of one or more of the following: cancer; osteoarthritis (OAJ; rheumatoid arthritis (RAJ; inflammation; acute pain; and chronic pain. In a further embodiment, there is provided a compound, as described herein, for the treatment of one or more of the following: cancer; osteoarthritis (OAJ; rheumatoid arthritis (RAJ; inflammation; acute pain; and chronic pain.

[0042] In a further embodiment, there is provided a compound as described herein, for modulating TrkA activity.

[0043] The compound may be an S-isomer or an R-isomer. The compound may be an S-isomer. The compound may be an R-isomer. X may be selected from: (RJ -methyl; and (SJ -methyl.The compound may be a solvate or hydrate. The compound may be a polymorph or more than one crystalline form.

[0044] X may be selected from: H; methyl; ethyl; propyl; and tert-butyl. X may be selected from: H; methyl; ethyl; and tert-butyl. X may be selected from: H; methyl; ethyl; and propyl. X may be selected from: H; methyl; and ethyl. X may be selected from: H; and methyl. X may be H. X may be methyl. X may be ethyl. X may be tert-butyl.

[0045]

[0046]

[0047]

[0048] The compound may be selected from one or more of the following:

[0049]

[0050]

[0051]

[0052] The compound may be selected from one or more of the following:

[0053]

[0054] following:

[0055]

[0056] selected from one or more of the following:

[0057]

[0058] selected from one or more of the following:

[0059]

[0060] selected from one or more of the following:

[0061]

[0062] The compound may inhibit tropomyosin receptor kinase A (TrkA). The compound may inhibit Janus kinase 2 (JAK2). The compound may inhibit Proto-Oncogene Tyrosine-Protein Kinase Src (SRC). The compound may be used to target TRK fusion proteins. The compound may be used to treat TRK fusion-positive tumours.

[0063] The compound may be used to treat one or more of the following: cancer; osteoarthritis (OA); rheumatoid arthritis (RA); inflammation; acute pain; and chronic pain. The compound may be used for treating endometriosis. The cancer may be selected from one of more of: breast; colon; prostate; pancreatic; lung; brain; renal; and head and neck. The cancer may be selected from one of more of: squamous cell carcinoma; melanoma; glioma; basal cell carcinoma; neuroblastoma; lymphoma; and leukaemia. The cancer may be selected from one of more of: HER2+ breast cancer; and triple negative breast cancer (TNBC). The cancer may be selected from one of more of: cellular congenital mesoblasticnephroma; secretory breast sarcoma; mammary analog secretory carcinoma; and infantile fibrosarcoma. The cancer may be selected from one of more of: undifferentiated sarcomas; gliomas; papillary thyroid cancers; spitzoid neoplasms; inflammatory myofibroblastic tumors; and acute leukemias. The cancer may be a pediatric cancer. The pediatric cancer may be selected from one of more of: cellular congenital mesoblastic nephroma; secretory breast sarcoma; mammary analog secretory carcinoma; infantile fibrosarcoma; undifferentiated sarcomas; gliomas; papillary thyroid cancers; spitzoid neoplasms; inflammatory myofibroblastic tumors; and acute leukemias.BRIEF DESCRIPTION OF THE DRAWINGS

[0064] FIGURE 1 shows (A) Model of recombinant STAT3127'688utilised in high-throughput singledose STAT3 DSF screen, where domains and residues assigned based on PDB ID = 1BG1. Representative STAT3 DSF melt curve is shown on the right, indicating the positive thermal shift induced by the treatment with gpl3O (Ac-pYLPQTV-NH2). (B) Drug screening pipeline used to uncover PI-8 as a tyrosine kinase inhibitor. (C) Expansion of the selected DSF hit into 10 structural analogues altering the aromatic substituents on the 6-aiyl ring of the 2,3diaminopyrzine core (D) STAT3 Luciferase assay single-dose (20 pM) screen and (E) 4-dose follow-up with compound PI-8. F) Heatmap of PI-8 (25 pM) scanEDGE kinase panel screen against 97 kinases. Top 20 tyrosine kinase hits and corresponding kinase inhibition is shown. (G) Determination of the Ka values of PI-8 for the top 4 kinase hits, ABL1, JAK2, Src and TrkA.

[0065] FIGURE 2 shows the predicted binding modes of PI-8 into (A) TrkA DFG-in (B) TrkA DFG-out using GLIDE (Schrodinger) with proposed H-bonding interactions represented by yellow dashes. (C) Summary of obtained Series I analogues and their inhibitory effects in two TrkA biochemical assays. Screening results for both Eurofins TrkA KINOMEscan™ (Eurofins™, left value or only value%) and BPS Bioscience™ TrkA Assay (BPS, right value%).

[0066] FIGURE 3 shows (A) Summary of obtained Series II analogues and their inhibitory effects in two TrkA biochemical assays. Screening results of compounds tested at 100 nM in both Eurofins TrkA KINOMEscan™ (Eurofins™) and BPS Bioscience TrkA Assay (BPS) are displayed. (B) Binding affinities of top 3 hits for TrkA, TrkB and TrkC as determined using the KINOMEscan™ assay (Discovery X™). Mean values determined between 2 independent experiments are shown, representing the dissociation constants (Ka) towards each member of the Trk kinase family. (C) ICso inhibitory values of top TrkA binders for JAK2 and SRC as determined by the Z'-LYTE Kinase activity assay (ThermoFisher™). (D) Antiproliferative activity of top 10 TrkA binders against KM12 cells harbouring the TPM3-TrkA mutant. (E) Summary of cancer cell toxicity ICso values estimated for top 4 TrkA binders against Larotrectinib (LS174T female, colon cancer and MDA-MB-231 female, breast cancer). Themean IC50 value estimated between 3 independent experiments (N=3) is shown for each treatment.

[0067] FIGURE 4 shows (A) Representative Western blots assessing TPM3-pTrkA depletion in KM12 cells after 24 h treatment with BP compounds. Larotrectinib and BP146 were used as positive and negative control treatments, respectively. TPM3-pTrkA (pTrkA) levels were measured against total level of TPM3-TrkA (TrkA) protein. Densitometry analysis was used to determine the pTrkA / TrkA ratio for each treatment condition normalised to the pTrkA / TrkA ratio of the DMSO-treated samples on each blot. (B) Summary of the densitometry results for each compound, showing pTrkA / TrkA ratios averaged between N=3 independent experiments. (C) Representative western blot of KM12 lysates treated with BP15 at 1 pM in the TrkA CETSA, where SOD1 levels in each sample was used as a loading control. (D) The intensity of the TPM3-TrkA bands from C were normalized to the SOD1 bands in each lane, then compared to the 37 °C control to generate relative TPM3-TrkA protein values, plotted as % of 37 °C control. A Boltzmann sigmoidal curve was fit to estimate the Tagg for each treatment. (E) Representative pTrkA Western blot of HA-TrkA overexpressed MDA-MB-231 cells treated with Larotrectinib and top 3 PI compounds at 0.25 pM or (F) 0.5 pM. Cells were treated for 1 h with respective inhibitors and subsequently stimulated with NGF-p for 30 min.

[0068] FIGURE 5 shows (A) Summary of obtained Series III analogues and their inhibitory effects in the Z’-Lyte TrkA inhibition assay at 2-doses (B) Structure of compound PI-15R including their IC50 values for Src, JAK2 and Trk kinases. JAK2 and Src IC50 values for PI-8R from Figure 3C are repeated here for ease of reference. (C) Dose-response kinase selectivity profile of PI-15R against top hits from 100 screened kinases showing >50% compound activity at 2.5 pM.

[0069] (D) Summary of in vitro PK profiling. Microsomal stability half-life and intrinsic clearance (Clint) values for PI-8 and PI-15R were measured at concentration of 0.1 pM with samples collected every 15 min interval over 1 h. Plasma protein binding (ppb) percentage, and Caco-2 permeability assays used a compound concentration of 10 pM. The unidirectional permeability of PI-8 from chamber A to B was determined at pH 6.5 / 7.4, while the permeability coefficient of PI-15R was measured at pH 7.4 / 7.4. For both compounds measurements were taken at 0 and 60 min at 37°C.FIGURE 6 shows (A) NTRK1 (TrkA) gene rearrangements leading to TrkA fusion kinase in cancer and Apo-TrkA (NTRK1) Tyrosine kinase domain with structural motifs labelled. (B) FDA-approved Trk inhibitors to treat Trk fusion-positive cancers and Entrectinib bound to the active " DFG-in” conformer of TrkA as a Type I inhibitor (PDB ID = 5KVT).

[0070] FIGURE 7 shows DSF results of 2,3-diaminopyrazine hits and estimated STAT3 Tm stabilisation relative to gpl30 positive control.

[0071] FIGURE 8 shows pyrazine hits STAT3 DSF 3-dose counter screen summary. Tmstabilisation relative to DMSO control is estimated for both STAT3127’688(containing SH2 domain) and STAT3127’465(SH2-truncated isoform).

[0072] FIGURE 9 shows (A) Single-dose KM12 lysate CETSA, where lysates were treated with 1000 nM of compounds vs. 0.03 % DMSO control and heated at 51 ° C for 3 min. Chemiluminescent blots of TrkA and SOD1 (imaged separately) are shown where MW labels are shown on the left. TrkA bands could be observed at ~100 kD whereby the mutant fusion TPM3-TrkA bands are seen at ~70 kD. (B) Corresponding TPM3-TrkA densitometry analysis relative to 37 ° C vehicle.

[0073] FIGURE 10 shows docking pose of (A) PI-8, (B) PI-10 and (C) PI-15 into TrkA " DFG-out" active site (PDB ID = 6PMB). Interatomic distances measured between arylsubtituents and the basic Arg 599 residue from the hinge motif is shown with yellow dashes for each ligand. Structures of TrkB (PDB ID = 4AT4) and TrkC (PDB ID = 6KZD) were overlapped with the protein-ligand complex where the corresponding Lys 599 from each protein is represented in sticks.

[0074] FIGURE 11 shows a heatmap of Thermofisher Z’-lyte™ kinase screen probing PI-15R at 2.5 pM against 100 kinases (N=2, single concentration).

[0075] FIGURE 12 shows (A) Diagram of the Z’-lyte TrkA inhibition assay. Primary kinase enzymatic reaction phosphorylates a FRET-pair labelled peptide resulting in 10-40 % of the substrate being phosphorylated. Then a "developing” secondary reaction utilising a site-directed proteolytic enzyme, recognizes and cleaves the unphosphorylated substrate. The Coumarin-Fluorescein FRET pair emission ratio is then used to determine the levels ofphosphorylated peptide substrate, indicating inhibition of the enzymatic reaction. The Emission Ratio will remain low if the FRET-peptide is phosphorylated (i.e., no kinase inhibition) and will be high if the FRET-peptide is non-phosphorylated (i.e., kinase inhibition). (B) Kinome inhibition profile of compound PI-15R against the top 18 kinase targets, as determined from FIGURE 11, tested at three concentrations using the Z’-Lyte activity assay. Mean inhibition values from N=2 independent experiments are presented.

[0076] FIGURE 13 shows TRKA, JAK2, and SRC selectivity for compounds 1, 12, 24, 31, 33, 23, 19, 20, and 6 tested for % inhibition using the Thermofisher SelectScreen™ service.

[0077] FIGURE 14 shows average plasma concentrations for PI-15R (10 mg / kg, SQ, (10%DMSO, 10%Kolliphor, PBS, 40 mM) was administered to three 10-week-old female Sprague-Dawley rats. Blood samples were collected via tail-vein catheter over the course of three hours. Sample analysis proceeded via LC-MS.

[0078] FIGURE 15 shows GLIDE™ docking scores of compounds 1-10 in the active site of respective kinase where enantiomers are denoted with (S) or (R) corresponding to the stereo-conformation of the Methyl substituent in the benzylamine group. (A) JAK2 " DFG-in" (PDB ID = 4AQC), Src " DFG-in” (PDB ID = 4U5J), TrkA " DFG-in” (PDB ID = 5KVT). (B) JAK2 " DFG-out” (PDB ID = 3UGC), Src " DFG-out” (PDB ID = 3EL7), TrkA " DFG-out” (PDB ID = 6PMB). 14 / 20 (70 %) of the ligands observed lower docking scores for TrkA " DFG-out” than JAK2 " DFG-out” indicating better binding.

[0079] FIGURE 16 shows a scatter plot of Series I and II PI-8 analogues docked into the TrkA " DFG-out" conformer (PDB ID= 6PMB) compared to the TrkA " DFG-in" (PDB ID = 5KVT). Where applicable, enantiomers are denoted with (S) or (R) corresponding to the stereoconformation of the Methyl substituent in the benzylamine group.

[0080] DETAILED DESCRIPTION

[0081] The following detailed description will be better understood when read in conjunction with the appended figures. For the purpose of illustrating the invention, the figuresdemonstrate embodiments of the present invention. However, the invention is not limited to the precise arrangements, examples, and instrumentalities shown.

[0082] 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.

[0083] Inhibition of tropomyosin receptor kinase A (TrkA) as used herein is meant to encompass interference with the ability of TrkA to interact with the MAPK pathway to inhibit autophosphorylation or phosphorylation of the MAPK pathway. Particularly, such that these compounds may be useful to inhibit TrkA cancers (i.e. fusion-positive cancers and non-fusion-positive cancers), the treatment of osteoarthritis (OA) or rheumatoid arthritis (RA) related pain and / or inflammation, pain generally, or endometriosis.

[0084] The compounds described herein as defined by Formulas I-III, may be useful for the treatment of one or more of the following: cancer; osteoarthritis (OA); rheumatoid arthritis (RA); inflammation; acute pain; and chronic pain. Alternatively, the compounds described herein as defined by Formulas I-III, may be useful for the treatment of endometriosis. " Tropomyosin receptor kinases” as used herein are transmembrane receptor tyrosine kinases named TrkA, TrkB, and TrkC and encoded by the NTRK1, NTRK2, and NTRK3 genes, respectively. TrkA-C are expressed primarily in neuronal tissues and are activated by neurotrophins causing autophosphorylation and phosphorylation of the Ras / MAPK and the PI3K / Akt pathways to regulate cellular processes. Aberrant regulation of Trk signaling can result in a number of cancers (for example, overexpression in HER2+brest cancer, triple negative breast cancer (TNBC), basal cell carcinoma, lung cancer, and neuroblastoma), cancer metastasis, modulating both acute and chronic pain, arthritis-related pain and inflammation, and pelvic pain in endometriosis.

[0085] “NTRK1 fusion-positive cancers” or " TRK fusion-positive tumours” as used herein refer to cancers characterized by chromosomal translocations of the NTRK1, NTRK2, and NTRK3 genes sometimes referred to as " TRK fusions”. In TRK fusions or TRK fusion proteins neurotrophin tyrosine kinase receptors TRKA, TRKB, and TRKC, are often constitutively activated, which results in unregulated activity of TRK signaling. Some cancers that have TRK fusions, include infantile fibrosarcoma, cellular congenital mesoblastic nephroma,secretory breast sarcoma, and mammary analog secretory carcinoma. TRK fusions have also been found in other pediatric cancers, including undifferentiated sarcomas, gliomas, papillary thyroid cancers, spitzoid neoplasms, inflammatory myofibroblastic tumors, and acute leukemias.

[0086] As used herein, "tropomyosin receptor kinase A” (TrkA) belongs to a family of Growth factor receptor tyrosine kinases (GFR-TK) that control synaptic strength and plasticity in the mammalian nervous system. TrkA is also known as Tyrosine kinase receptor A (Trk-A), high affinity nerve growth factor receptor, neurotrophic tyrosine kinase receptor type 1, Tropomyosin-related kinase A, TRKl-transforming tyrosine kinase protein, Tyrosine kinase receptor, gpl40trk, and pl40-TrkA (EC:2.7.10.1) is a protein that in humans is encoded by the NTRK1 gene. TrkA is known to play a role in the development and the maturation of the central and peripheral nervous systems through regulation of proliferation, differentiation and survival of sympathetic and nervous neurons. TrkA has a high affinity receptor for the neurotrophin, nerve growth factor (NGF), but also binds and can be activated by neurotrophin-3 (NTF3). Binding of NGF to TrkA leads to a ligand-induced homodimerization, subsequent endocytosis and phosphorylation to recruit signaling molecules that are involved in cell growth, survival, differentiation and apoptosis97.

[0087] Janus kinase 2 (JAK2) or Tyrosine-protein kinase JAK2 (EC:2.7.10.2) is a non-receptor tyrosine kinase that is involved in regulating cell growth, cell development, cell differentiation and histone modification. Janus kinase 2 (JAK2) / signal transducer and activator of transcription 3 (STAT3) signaling pathway is known to play a role in cytogenesis and is involved in Osteoarthritis (OA) progression98, in Rheumatoid Arthritis (RA), and is also known to play a role in myeloproliferative neoplasm (MPN)100.

[0088] Proto-Oncogene Tyrosine-Protein Kinase Src (SRC), also known as proto-oncogene c-Src, pp60c-src (p60-Src), or SRC1, is a non-receptor tyrosine kinase, whose activity is known to be increase in several cancer tissues.

[0089] 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 pharmaceuticallyacceptable 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.

[0090] 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.

[0091] 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 appreciatethat various factors including recrystallization solvent, rate of crystallization and storage temperature may cause a single crystal form to dominate.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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 pain or discomfort, 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 cancer or metastasis. 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.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.

[0097] 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 LDso (the dose lethal to 50% of the population) and the LDioo (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. Animal studies may be used to provide an indication if the compound has any effects on other tissues.

[0098] 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 TrkA, JAK2, and or SRC.MATERIALS & METHODS

[0099] Chemistry

[0100] A) Synthetic route to obtain compound 1, a racemic mixture of hits CBK063768 and CBK063720 B) Summary of synthetic procedures used to obtain Series I-III analogues.

[0101] CBK063768 and CBK063720 (compound 1) 2- step reaction scheme used to produce arylpyrazine analogues

[0102] X = H, Me, Et, tBu

[0103] General procedure A: 2-amino-3,5-dibromopyrazine (265 mg, 1.05 mmol) was dissolved in n-Butanol (0.15 M final concentration) and benzylamine A (3 eq ), and N, N-Diisopropylethylamine (5eq.) were added. The mixture was irradiated in a microwave reactor (Biotage Initiator 2.5) heating at 140-180 °C for 3-16 h. Upon completion, the reaction mixture was cooled, concentrated to viscous oil in vacuo, and transferred to a separatory funnel using ethyl acetate (50 mL) The organic phase was acid washed with 10 mL of H2O (acidified with 2M HCI (aq ) to pH=3), followed by brine (2 x 10 mL). The remaining organic phase was then dried (Na2SO4) and concentrated to afford crude mixture. The remaining crude was purified through Silica Gel chromatography using a solvent system of Ethyl Acetate and Hexanes to afford the final product as a sticky red oil.

[0104] General procedure B: 6-bromo-N2-[benzyl]pyrazine-2,3-diamine B (56 mg, 0.19 mmol) was dissolved in toluene / EtOH (2:1, 1.5 mL) and phenylboronic acid C (1.5 eq.) and K2CO32M H2O solution (3 eq.) were added to the mixture.

[0105] Pd(dppf)2Cl2(5 mol %) were then added and the resulting solution was sealed in a MW vial and degassed with N2(2 min). The mixture was then irradiated in the microwave reactor (Biotage Initiator 2.5) at 90°C for 45 min. Upon completion, mixture was then cooled down to room temperature and the organic phase was filtered through celite. The organic filtrate was diluted with EA (10 mL) and washed with brine (3 x 5 mL), dried (Na2SO4), and then concentrated under reduced pressure. The remaining crude was purified through silica gel chromatography or prep HPLC to afford the final product as a sticky oil or crystaline solid.

[0106]

[0107] Compounds 1-39 were obtained via 2-step synthesis starting from 2-Amino-3,5-dibromopyrazine. Firstly, a microwave-assisted, region-selective SNAC reaction introduced a differentially substituted benzylamine ring at the 3-position of the pyrazine ring to furnish a 2,3-diamino-6-bromopyrazine intermediate. Diverse aryl groups were then attached to the 6-position of the 2,3-diaminopyrazine ring using a Suzuki cross-coupling reaction to produce the desired final compounds.

[0108]

[0109] Compound 1 (PI-19-06)

[0110] 6-[3,5-Bis(trifluoromethyl)phenyl]-N2-(1-phenylethyl)pyrazine-2,3-diamine was prepared as outlined in general procedure B on a 0.3 mmol scale and purified through silica gel chromatography using a EtOAc / hexanes eluent system to afford the final product as a yellow oil (82 mg, 49 %).1H NMR (400 MHz, DMSO) 68.33 (s, 2H), 8.05 (s, 1H), 7.84 (s, 1H), 7.40 (d, / = 7.7 Hz, 2H), 7.29 (t, / = 7.5 Hz, 2H), 7.17 (t, / = 7.5 Hz, 1H), 6.95 (d, / = 6.2 Hz, 1H), 6.63 (s, 2H), 5.08 (q, / = 6.7 Hz, 1H), 1.53 (d, / = 6.9 Hz, 3H).13C NMR (101 MHz, CDC13) 6 144.6, 143.5, 142.8, 139.7, 137.6, 131.7 (q, J = 33.1 Hz), 128.6, 127.2, 126.1, 125.9, 125.3 (d, J = 2.9 Hz), 123.5 (q, J = 272.7 Hz), 120.8 (m, J = 3.7 Hz), 51.6, 23.1. HPLC-MS tR7.3 min, 96.0 % purity, m / z calculated for [C20H16F6N4+H] = 427.1, found 427.2

[0111]

[0112] Compound 2 (PI-22-46)

[0113] 6-[3-(Methoxycarbonyl)phenyl]-N2-(1-phenylethyl)pyrazine-2,3-diamine was prepared as outlined in general procedure B on a 0.2 mmol scale and purified through silica gel chromatography using a EtOAc / hexanes eluent system to afford the final product (38 mg, 61 %).1H NMR (400 MHz, DMSO) 68.44 (s, 1H), 8.03 (d, / = 6.8 Hz, 1H), 7.81 (d, / = 5.0 Hz, 2H), 7.48 (q, / = 4.2 Hz, 3H), 7.33 (t, / = 7.6 Hz, 2H), 7.19 (t, / = 7.2 Hz, 1H), 6.79 (d, / = 6.0 Hz,1H), 6.39 (s, 2H), 5.23 (m = 6.7 Hz, 1H), 3.92 (s, 3H), 1.54 (d = 6.0 Hz, 3H).13C NMR (101 MHz, DMSO) 5166.9, 146.5, 144.2, 141.4, 139.1, 135.3, 130.4, 129.3, 129.2, 128.6, 127.7, 126.9, 126.6, 125.7, 52.6, 50.6, 23.7. HPLC-MS tR= 6.7 min, 97.3 % purity, m / z calculated for [C20H20N4O + H] = 349.2, found 349.2

[0114]

[0115] Compound 3 (PI-22-47)

[0116] 6-(3-Nitrophenyl)-N2-(1-phenylethyl)pyrazine-2,3-diamine was prepared as outlined in general procedure B on a 0.2 mmol scale and purified through silica gel chromatography using a EtOAc / hexanes eluent system to afford crude which was further purified using prep-HPLC to obtain the final product as a TFA salt (25 mg, 31 %).1H NMR (400 MHz, DMSO) 68.60 (s, 1H), 8.22 (d = 7.7 Hz, 1H), 8.11 (d = 8.0 Hz, 1H), 7.92 (s, 1H), 7.71 (d = 5.0 Hz, 1H), 7.64 (t = 8.0 Hz, 1H), 7.48 (d = 7.4 Hz, 2H), 7.34 (t = 7.4 Hz, 2H), 7.21 (t = 7.2 Hz, 1H), 5.21 (t = 6.1 Hz, 1H), 4.99 (s, 3H), 1.57 (d = 6.8 Hz, 3H).13C NMR (101 MHz, DMSO) 6148.8, 145.4, 142.9, 142.1, 138.7, 134.3, 131.2, 130.5, 128.8, 127.2, 126.6, 122.6, 119.7, 117.2, 51.5, 23.3. HPLC-MS tR= 6.83 min, >99.9 % purity, m / z calculated for [C18H17N5O2 + H] = 336.1, found 336.2

[0117]

[0118] Compound 4 (PI-19-10)

[0119] 6-(4-Fluorophenyl)-N2-(1-phenylethyl)pyrazine-2,3-diamine was prepared as outlined in general procedure B on a 0.1 mmol scale and purified through silica gel chromatography, using a MeOH / DCM eluent system spiked with 1 mol% EtsN, to afford the final product as a yellow solid (21 mg, 64 %). Over 1 year storage at 4 °C, compound degradation was observed. The solid stock was consequently purified a second time using prep HPLC to yield the final product as a TFA salt for biological testing. 'H NMR (400 MHz, MeOD) 67.74 (q = 4.7 Hz, 2H), 7.47 (d = 7.9 Hz, 3H), 7.37 (t = 7.6 Hz, 2H), 7.26 (t = 7.3 Hz, 1H), 7.11(t, J = 8.7 Hz, 2H), 5.33 (qj = 6.9 Hz, 1H), 1.67 (dj = 6.9 Hz, 3H).13C NMR (101 MHz, MeOD) 5 164.4, 161.9, 144.1, 143.3, 139.3, 137.9, 131.9, 131.9, 128.2, 127.2, 127.1, 126.8, 125.8, 115.1, 114.8, 109.6, 51.7, 21.4. HPLC-MS tR= 6.81 min, 99.7 % purity, m / z calculated for [C18H17FN4 + H] = 309.1, found 309.0

[0120] N X

[0121] H

[0122]

[0123] Compound 5 (PI-19-11)

[0124] 6-(3-Fluorophenyl)-N2-(1-phenylethyl)pyrazine-2,3-diamine was prepared as outlined in general procedure B on a 0.2 mmol scale and purified through silica gel chromatography using a EtOAc / hexanes eluent system to afford the final product as a pale-yellow solid (46 mg, 87 %).1H NMR (400 MHz, DMSO) 57.80 (s, 1H), 7.62 (dj = 7.4 Hz, 1H), 7.51 (dj = 11.0 Hz, 1H), 7.43 (dj = 7.2 Hz, 2H), 7.33 (m = 7.4 Hz, 3H), 7.19 (t = 6.7 Hz, 1H), 7.01 (t = 7.4 Hz, 1H), 6.72 (d = 5.8 Hz, 1H), 6.39 (s, 2H), 5.23 (mJ = 6.6 Hz, 1H), 1.53 (dj = 6.5 Hz, 3H).13C NMR (101 MHz, DMSO) 5164.3, 161.9, 146.5, 144.3, 141.3, 135.1, 130.6 (dj = 8.4 Hz), 127.5 (dj = 220.8 Hz), 126.8, 126.0, 120.7, 113.5 (dj = 21.3 Hz), 111.4 (dj = 22.7 Hz), 50.5, 23.7. HPLC-MS tR = 7.03 min, 97.7 % purity, m / z calculated for [C18H17FN4 + H] = 309.1, found 309.2

[0125] N X

[0126] H

[0127]

[0128] Compound 6 (PI-22-48)

[0129] 6-(2,6-Dimethoxyphenyl)-N2-(1-phenylethyl)pyrazine-2,3-diamine was prepared as outlined in general procedure B on a 0.2 mmol scale and purified through silica gel chromatography using a EtOAc / hexanes eluent system to afford the final product (41 mg, 66 %). 1H NMR (400 MHz, DMSO) 57.38 (dj = 7.3 Hz, 2H), 7.30 (tj = 7.6 Hz, 2H), 7.22 (mJ = 6.9 Hz, 2H), 7.00 (s, 1H), 6.66 (dj = 8.3 Hz, 2H), 6.26 (dj = 7.4 Hz, 1H), 5.98 (s, 2H), 5.18 (tj = 6.9 Hz, 1H), 3.56 (s, 6H), 1.47 (dj = 6.7 Hz, 3H).13C NMR (101 MHz, DMSO) 5 158.8, 145.9, 142.7, 141.7, 133.6, 129.8, 129.3, 128.5, 126.8, 118.1, 105.0, 56.1, 49.2, 22.9.HPLC-MS tR= 6.75 min, 98.3 % purity, m / z calculated for [C20H22N4O2 + H] = 351.2, found 351.2

[0130] N

[0131] H

[0132]

[0133] Compound 7 (PI-22-49)

[0134] N2-(l-Phenylethyl)-6-[3-(propan-2-yloxy)phenyl]pyrazine-2,3-diamine was prepared as outlined in general procedure B on a 0.2 mmol scale and purified through silica gel chromatography using a EtOAc / hexanes eluent system to afford the final product (25 mg, 49 %).1H NMR (400 MHz, DMSO) 6 7.73 (s, 1H), 7.42 (d, J = 7.5 Hz, 2H), 7.30 (t, J = 7.8 Hz, 4H), 7.19 (t, J = 7.7 Hz, 2H), 6.70 (q, J = 10.8 Hz, 2H), 6.28 (s, 2H), 5.24 (m, J = 6.6 Hz, 1H), 4.58 (m, J = 5.5 Hz, 1H), 1.53 (d, J = 6.9 Hz, 3H), 1.28 (q, J = 7.9 Hz, 6H).13C NMR (101 MHz, DMSO) 6 158.1, 146.6, 143.8, 141.3, 140.1, 136.4, 129.8, 128.6, 126.8, 126.4, 125.6, 117.1, 115.0, 111.9, 69.4, 50.4, 23.8, 22.4, 22.3. HPLC-MS tR= 7.10 min, 95.7 % purity, m / z calculated for [C21H24N4O + H] = 349.2, found 349.2

[0135]

[0136] Compound PI-8 (PI-22-40)

[0137] 6-[3-(Dimethylamino)phenyl]-N2-(l-phenylethyl)pyrazine-2,3-diamine was prepared as outlined in general procedure B on a 0.2 mmol scale, purified through silica gel chromatography using a EtOAc / hexanes eluent system, and recrystallised from Ethyl Acetate to afford the final product as a black solid (21 mg, 33 %).1H NMR (400 MHz, MeOD) 67.59 (s, 1H), 7.44 (d, J = 7.5 Hz, 2H), 7.30 (t, J = 7.6 Hz, 2H), 7.18 (m, J = 6.7 Hz, 3H), 7.06 (d, J = 7.6 Hz, 1H), 6.70 (q, J = 3.4 Hz, 1H), 5.29 (q, J = 6.9 Hz, 1H), 2.92 (s, 6H), 1.61 (d, J = 7.0 Hz, 3H).13C NMR (101 MHz, MeOD) 6 151.1, 145.9, 142.6, 141.9, 139.3, 138.6, 128.5, 128.0, 126.1, 125.7, 123.5, 113.9, 112.1, 110.3, 50.6, 40.0, 22.4. HPLC-MS tR= 6.38 min, 95.7 % purity, m / z calculated for [C20H23N5 + H] = 334.2, found 334.2

[0138]

[0139] Cl

[0140] Compound 9 (PI-22-50)

[0141] 6-(3,5-Dichlorophenyl)-N2-(l-phenylethyl)pyrazine-2,3-diamine was prepared as outlined in general procedure B on a 0.2 mmol scale and purified through silica gel chromatography using a EtOAc / hexanes eluent system to afford crude, which was further purified using prep-HPLC to obtain the final product as a TFA salt (20 mg, 24 %).XH NMR (400 MHz, DMSO) 67.88 (s, 1H), 7.74 (s, 2H), 7.64 (d, J = 4.2 Hz, 1H), 7.43 (t, J = 7.6 Hz, 3H), 7.34 (t, J = 7.5 Hz, 2H), 7.21 (t, J = 7.1 Hz, 1H), 5.13 (t, J = 5.9 Hz, 1H), 4.97 (s, 3H), 1.55 (d, J = 6.8 Hz, 3H).13C NMR (101 MHz, DMSO) 5159.9, 145.5, 142.7, 142.1, 140.5, 134.9, 133.7, 128.8, 127.2, 127.1, 126.3, 123.7, 117.5, 51.7, 23.4. HPLC-MS tR= 7.31 min, 99.8 % purity, m / z calculated for [C18H16CI2N4 + H] = 359.1, found 359.1

[0142]

[0143] Compound PI-10 (PI-22-39)

[0144] N2-(l-Phenylethyl)-6-(pyridin-3-yl)pyrazine-2,3-diamine was prepared as outlined in general procedure B on a 0.2 mmol scale, purified through Silica Gel chromatography using 100 % EtOAc followed by an eluent system of MeOH / DCM, and recrystallised from Ethyl Acetate / Hexanes to afford the final product (22 mg, 40 %). 'H NMR (400 MHz, MeOD) 6 8.86 (s, 1H), 8.37 (d = 4.2 Hz, 1H), 8.12 (d = 8.1 Hz, 1H), 7.70 (s, 1H), 7.45 (d = 7.5 Hz, 2H), 7.36 (mJ = 5.8 Hz, 3H), 7.20 (t = 7.2 Hz, 1H), 5.26 (q = 6.9 Hz, 1H), 1.62 (d = 6.9 Hz, 3H).13C NMR (101 MHz, MeOD) 5146.5, 145.7, 145.6, 143.7, 142.0, 134.8, 134.4, 132.9, 128.0, 126.2, 125.7, 124.1, 123.6, 50.9, 22.1. HPLC-MS tR= 5.76 min, 95.9 % purity, m / z calculated for [C17H17N5 + H] = 292.2, found 292.2

[0145]

[0146] Compound 11 (PI-22-51)

[0147] 6-{[l,l'-Biphenyl]-4-yl}-N2-(l-phenylethyl)pyrazine-2,3-diamine was prepared as outlined in general procedure B on a 0.1 mmol scale and purified through silica gel chromatography using a EtOAc / hexanes eluent system to afford crude, which was further purified using prep-HPLC to obtain the final product as a TFA salt (14 mg, 26 %).1H NMR (400 MHz, DMSO) 67.90 (d, / = 7.8 Hz, 2H), 7.80 (s, 1H), 7.70 (s, 5H), 7.48 (d, / = 6.6 Hz, 4H), 7.37 (d, / = 5.7 Hz, 3H), 7.24 (d, / = 6.7 Hz, 1H), 5.33 (s, 1H), 4.89 (s, 3H), 1.58 (d = 6.2 Hz, 3H).

[0148] 13C NMR (101 MHz, DMSO) 6145.2, 143.1, 140.9, 140.0, 140.0, 136.6, 135.7, 129.4, 128.8, 128.0, 127.3, 127.2, 127.0, 126.6, 125.9, 114.5, 51.1, 23.1. HPLC-MS tR= 7.34 min, 95.5 % purity, m / z calculated for [C24H22N4 + H] = 367.2, found 367.2

[0149]

[0150] Compound 12 (PI-22-52)

[0151] 3-{5-Amino-6-[(l-phenylethyl)amino]pyrazin-2-yl}phenol was prepared as outlined in general procedure B on a 0.1 mmol scale and purified through silica gel chromatography using a EtOAc / hexanes eluent system to afford the final product (21 mg, 62 %). *H NMR (400 MHz, DMSO) 69.32 (s, 1H), 7.66 (s, 1H), 7.46 (d, / = 7.0 Hz, 2H), 7.31 (t, / = 6.9 Hz, 2H), 7.25 (s, 1H), 7.19 (m, 2H), 7.12 (t, / = 7.3 Hz, 1H), 6.63 (d, / = 6.8 Hz, 2H), 6.23 (s, 2H), 5.32 (t, / = 6.4 Hz, 1H), 1.53 (d, / = 6.4 Hz, 3H).13C NMR (101 MHz, DMSO) 6157.9, 146.4, 143.7, 141.3, 140.0, 136.7, 129.7, 128.6, 126.9, 126.6, 125.3, 115.9, 114.2, 112.0, 50.0, 23.5. HPLC-MS tR= 6.47 min, 95.67 % purity, m / z calculated for [C18H18N4O + H] = 307.2, found 307.1

[0152]

[0153] Compound 13 (PI-22-53)

[0154] 6-(3,5-Dimethylphenyl)-N2-(l-phenylethyl)pyrazine-2,3-diamine was prepared as outlined in general procedure B on a 0.1 mmol scale and purified through silica gel chromatography using a EtOAc / hexanes eluent system to afford the final product (25 mg, 72 %). 'H NMR (400 MHz, DMSO) 67.67 (s, 1H), 7.44 (dj = 7.4 Hz, 2H), 7.31 (m, 4H), 7.19 (tj = 7.3 Hz, 1H), 6.83 (s, 1H), 6.65 (dj = 6.2 Hz, 1H), 6.22 (s, 2H), 5.18 (tj = 6.7 Hz, 1H), 2.26 (s, 6H), 1.52 (dj = 7.0 Hz, 3H).13C NMR (101 MHz, DMSO) 5146.8, 143.6, 141.4, 138.5, 137.5, 137.0, 128.6, 126.7, 126.5, 125.4, 123.0, 50.7, 23.7, 21.5. HPLC-MS tR= 7.17 min, 97.5 % purity, m / z calculated for [C20H22N4 + H] = 319.2, found 318.2

[0155]

[0156] Compound 14 (PI-19-25)

[0157] 6-Phenyl-N2-(l-phenylethyl)pyrazine-2,3-diamine was prepared as outlined in general procedure B on a 0.1 mmol scale and purified through silica gel chromatography using a EtOAc / hexanes eluent system to afford the final product as a white solid (28 mg, 94 %).1H NMR (400 MHz, DMSO) 67.76 (dj = 7.4 Hz, 2H), 7.73 (s, 1H), 7.44 (dj = 7.5 Hz, 2H), 7.32 (mJ = 3.8 Hz, 4H), 7.20 (q = 7.6 Hz, 2H), 6.65 (d = 6.8 Hz, 1H), 6.25 (s, 2H), 5.28 (mJ = 6.7 Hz, 1H), 1.53 (dj = 7.0 Hz, 3H).13C NMR (101 MHz, MeOD) 6145.8, 142.8, 142.0, 138.6, 138.1, 127.9, 126.7, 126.2, 125.9, 124.9, 123.4, 50.6, 22.0. HPLC-MS tR= 6.97 min, 99.3 % purity, m / z calculated for [C18H18N4 + H] = 291.2, found 291.3

[0158]

[0159] Compound PI-15 (PI-22-41)

[0160] N-(3-{5-amino-6-[(l-phenylethyl)amino]pyrazin-2-yl}phenyl)acetamide was prepared asoutlined in general procedure B on a 0.2 mmol scale, purified through silica gel chromatography using a EtOAc / hexanes eluent system to afford crude, which was further purified using prep-HPLC to obtain the final product as a TFA salt (26 mg, 39 %).1H NMR (400 MHz, DMSO) 610.01 (s, 1H), 8.16 (s, 1H), 7.62 (s, 2H), 7.48 (m, 4H), 7.32 (m, 3H), 7.22 (d, / = 7.2 Hz, 1H), 5.37 (t, / = 6.5 Hz, 1H), 2.09 (s, 3H), 1.58 (d = 6.8 Hz, 3H).13C NMR (101 MHz, DMSO) 6168.8, 159.3, 159.0, 145.0, 143.0, 140.9, 140.1, 137.1, 136.9, 129.3, 128.8, 127.3, 126.9, 120.2, 119.2, 116.3, 114.7, 50.8, 24.5, 22.9. HPLC-MS tR= 6.34 min, 97.7 % purity, m / z calculated for [C20H21N5O + H] = 348.2, found 348.2

[0161] N N

[0162] H

[0163]

[0164] Compound 16 (PI-19-27)

[0165] 6-(Naphthalen-l-yl)-N2-(l-phenylethyl)pyrazine-2,3-diamine was prepared as outlined in general procedure B on a 0.1 mmol scale and purified through silica gel chromatography, using a MeOH / DCM eluent system spiked with 1 mol% DIPEA, to afford the final product as black oil (22 mg, 63 %). Over 1 year storage at 4 °C, compound degradation was observed. The solid stock was consequently purified a second time using prep HPLC to yield the final product as a TFA salt for biological testing.XH NMR (400 MHz, DMSO) 87.92 (d, / = 6.8 Hz, 2H), 7.85 (d, / = 8.5 Hz, 1H), 7.51 (t, / = 7.5 Hz, 2H), 7.46 (t, / = 7.9 Hz, 2H), 7.39 (s, 1H), 7.36 (d, / = 3.6 Hz, 4H), 7.30 (t, / = 4.1 Hz, 1H), 7.21 (t, / = 7.7 Hz, 1H), 5.18 (t, / = 6.6 Hz, 1H), 1.52 (d, / = 6.9 Hz, 3H).13C NMR (101 MHz, DMSO) 6157.8, 145.2, 142.7, 141.0, 139.2, 135.2, 134.0, 131.2, 128.8, 128.5, 127.2, 126.5, 126.5, 126.2, 126.1, 125.8, 113.6, 50.6, 23.4. HPLC-MS tR= 7.01 min, 99.0 % purity, m / z calculated for [C22H20N4 + H] = 341.2, found 341.2

[0166]

[0167] Compound PI-17 (PI-19-29)

[0168] 3-{5-amino-6-[(l-phenylethyl)amino]pyrazin-2-yl}-N, N-dimethylbenzamide was prepared as outlined in general procedure B on a 0.1 mmol scale and purified through silica gel chromatography using a MeOH / DCM eluent to afford crude, which was further purifiedusing prep-HPLC to obtain the final product as a TFA salt (33 mg, 88 %). 'H NMR (400 MHz, DMSO) 67.84 (d = 7.5 Hz, 1H), 7.78 (s, 1H), 7.71 (s, 1H), 7.58 (s, 1H), 7.42 (s, 3H), 7.32 (m, 3H), 7.22 (d = 7.0 Hz, 1H), 5.23 (s, 1H), 3.03 (s, 3H), 2.86 (s, 3H), 1.56 (d = 6.2 Hz, 3H).

[0169] 13C NMR (101 MHz, DMSO) 6170.5, 145.4, 142.9, 141.3, 137.4, 136.7, 136.2, 129.1, 128.7, 127.1, 126.6, 126.5, 125.9, 123.7, 115.8, 51.3, 35.2, 23.3. HPLC-MS tR= 6.34 min, 99.2 % purity, m / z calculated for [C21H23N5O + H] = 362.2, found 362.3

[0170]

[0171] Compound 18 (PI-22-54)

[0172] 6-(2H-l,3-Benzodioxol-5-yl)-N2-(l-phenylethyl)pyrazine-2,3-diamine was prepared as outlined in general procedure B on a 0.1 mmol scale and purified through silica gel chromatography using a EtOAc / hexanes eluent system to afford the final product (29 mg, 80 %).1H NMR (400 MHz, DMSO) 67.65 (s, 1H), 7.42 (d = 7.3 Hz, 2H), 7.30 (mJ = 6.4 Hz, 4H), 7.18 (t = 7.3 Hz, 1H), 6.86 (d = 8.1 Hz, 1H), 6.63 (d = 6.6 Hz, 1H), 6.19 (s, 2H), 6.00 (s, 2H), 5.23 (t = 6.8 Hz, 1H), 1.52 (d = 6.9 Hz, 3H).13C NMR (101 MHz, DMSO) 6148.0, 146.6, 146.6, 143.4, 141.3, 136.6, 133.2, 128.6, 126.8, 126.5, 124.8, 118.4, 108.6, 105.6, 101.2, 50.4, 23.7. HPLC-MS tR= 6.79 min, 96.0 % purity, m / z calculated for [C19H18N4O2 + H] = 335.1, found 335.1

[0173] N N

[0174] H

[0175]

[0176] Compound 19 (PI-22-55)

[0177] N-(4-{5-amino-6-[(l-phenylethyl)amino]pyrazin-2-yl}phenyl)methanesulfonamide was prepared as outlined in general procedure B on a 0.1 mmol scale and purified through silica gel chromatography using a EtOAc / hexanes eluent system to afford the final product (23 mg, 55 %).1H NMR (400 MHz, DMSO) 69.70 (s, 1H), 7.71 (t = 8.1 Hz, 3H), 7.43 (d = 7.6 Hz, 2H), 7.31 (t = 7.5 Hz, 2H), 7.18 (d = 17.7 Hz, 3H), 6.66 (d = 6.7 Hz, 1H), 6.23 (s, 2H), 5.25 (t = 6.8 Hz, 1H), 2.97 (s, 3H), 1.52 (d = 6.9 Hz, 3H).13C NMR (101 MHz, DMSO) 6146.6, 143.6, 141.4, 137.4, 136.2, 134.5, 128.6, 126.8, 126.5, 125.9, 125.0, 120.4, 79.6, 50.3, 23.7. HPLC-MS tR = 6.44 min, 95.9 % purity, m / z calculated for [C19H21N5O2S + H] = 384.1, found 384.1

[0178]

[0179] O' +^O

[0180] Compound 20 (PI-22-56)

[0181] 3-{5-Amino-6-[(l-phenylethyl)amino]pyrazin-2-yl}-N, N-dimethyl-5-nitrobenzamide was prepared as outlined in general procedure B on a 0.1 mmol scale and purified through silica gel chromatography using a MeOH / DCM eluent system to afford the final product (35 mg, 73 %).1H NMR (400 MHz, DMSO) 68.61 (s, 1H), 8.12 (s, 1H), 7.97 (s, 2H), 7.44 (d, / = 7.5 Hz, 2H), 7.30 (t = 7.5 Hz, 2H), 7.18 (d, / = 7.3 Hz, 1H), 6.92 (d = 6.0 Hz, 1H), 6.59 (s, 2H), 5.14 (t = 6.6 Hz, 1H), 3.05 (s, 3H), 2.89 (s, 3H), 1.54 (d = 6.9 Hz, 3H).13C NMR (101 MHz, DMSO) 6168.6, 148.8, 146.5, 144.9, 141.4, 140.6, 138.7, 133.4, 128.7, 128.6, 126.9, 126.8, 126.4, 119.7, 119.6, 51.1, 35.3, 23.8. HPLC-MS tR = 6.59 min, 97.8 % purity, m / z calculated for [C21H22N6O3 + H] = 407.2, found 407.2

[0182]

[0183] °\

[0184] Compound 21 (PI-22-57)

[0185] 6-[4-(Methoxycarbonyl)phenyl]-N2-(l-phenylethyl)pyrazine-2,3-diamine was prepared as outlined in general procedure B on a 0.1 mmol scale and purified through silica gel chromatography using a EtOAc / hexanes eluent system to afford crude, which was further purified using prep-HPLC to obtain the final product as a TFA salt (14 mg, 29 %).XH NMR (400 MHz, DMSO) 67.91 (t, / = 5.6 Hz, 4H), 7.84 (s, 1H), 7.65 (d, / = 4.9 Hz, 1H), 7.46 (d, / = 7.5 Hz, 2H), 7.34 (t, / = 7.5 Hz, 2H), 7.21 (t, / = 7.1 Hz, 1H), 5.27 (t, / = 6.2 Hz, 1H), 4.48 (s, 3H), 3.85 (s, 3H), 1.56 (d = 6.8 Hz, 3H).13C NMR (101 MHz, DMSO) 6166.5, 145.3, 142.9,141.7, 141.3, 135.4, 129.9, 128.9, 128.8, 127.2, 126.6, 125.3, 116.8, 52.5, 51.2, 23.2. HPLC- MS tR = 6.83 min, 99.8 % purity, m / z calculated for [C20H20N4O2 + H] = 349.2, found 349.2

[0186]

[0187] Compound 22 (PI-22-27)

[0188] 4-{5-Amino-6-[(l-phenylethyl)amino]pyrazin-2-yl}benzoic acid hydrochloride. To stirred solution of PI-22-57 (6-[4-(methoxycarbonyl)phenyl]-N2-(l-phenylethyl)pyrazine-2,3-diamine) in THF / H2O (3:1) was added lithium hydroxide (10 eq) and the mixture was heated at 50° C and stirred overnight. Upon reaction completion, the mixture was concentrated in vacuo and the residue was dissolved in H2O (25 mL). Aqueous phase was washed with DCM (5 x 20 mL) and acidified (pH ~ 1) using 2M HC1 (aq.). Upon neutralisation the product was observed as a pale-yellow solid precipitate in the aqueous phase. Solid was filtered and washed with H2O on filter funnel. Resulting product was then transferred through Acetone and dried to yield the final product as a HC1 salt (222 mg, 50 %). iH NMR (400 MHz, DMSO) 6 12.90 (s, 1H), 7.98 (s, 2H), 7.90 (s, 4H), 7.81 (s, 2H), 7.50 (d, J = 7.5 Hz, 2H), 7.32 (t, J = 7.4 Hz, 2H), 7.19 (t, J = 7.2 Hz, 1H), 5.28 (t, J = 6.6 Hz, 1H), 1.57 (d, J = 6.8 Hz, 3H).13C NMR (101 MHz, DMSO) 6 167.5, 145.7, 143.1, 141.7, 141.0, 135.4, 130.1, 130.0, 128.7, 127.1, 126.7, 125.2, 51.4, 23.4. HPLC-MS tR= 6.29 min, 97.7 % purity, m / z calculated for [C19H18N4O2 + H] = 335.1, found 335.2

[0189]

[0190] Compound 23 (PI-22-17)

[0191] N2-benzyl-6-[3-(dimethylamino)phenyl]pyrazine-2,3-diamine was prepared as outlined in general procedure B on a 0.3 mmol scale and purified through silica gel chromatography using a EtOAc / hexanes eluent system to afford the final product as a black solid (56 mg, 63 %). Over 1 year storage at 4 °C, compound degradation was observed. The solid stock wasconsequently purified a second time using prep HPLC to yield the final product as a TFA salt for biological testing. 'H NMR (400 MHz, DMSO) 67.81 (s, 1H), 7.75 (s, 1H), 7.43 (d = 7.3 Hz, 2H), 7.35 (t = 7.3 Hz, 2H), 7.24 (m = 7.6 Hz, 4H), 6.73 (d = 6.5 Hz, 1H), 4.71 (s, 2H), 2.92 (s, 6H).13C NMR (101 MHz, DMSO) 6150.8, 143.7, 141.0, 139.6, 137.7, 137.2, 129.6, 128.8, 128.0, 127.4, 114.9, 114.1, 113.0, 109.9, 44.9, 40.9. HPLC-MS tR= 5.95 min, 97.9 % purity, m / z calculated for [C19H21N5 + H] = 320.2, found 320.2

[0192]

[0193] Compound 24 (PI-22-18)

[0194] N2-(2,2-dimethyl-l-phenylpropyl)-6-[3-(dimethylamino)phenyl]pyrazine-2,3-diamine was prepared as outlined in general procedure B on a 0.2 mmol scale and purified through silica gel chromatography using a EtOAc / hexanes eluent system to afford the final product as a black oil (65 mg, 75 %). 'H NMR (400 MHz, MeOD) 67.58 (s, 1H), 7.44 (d, J = 7.6 Hz, 2H), 7.29 (t, J = 7.5 Hz, 2H), 7.25 (s, 1H), 7.20 (q, J = 7.5 Hz, 2H), 7.09 (d, J = 7.6 Hz, 1H), 6.73 (q, J = 3.5 Hz, 1H), 5.23 (s, 1H), 2.99 (s, 6H), 1.07 (s, 9H).13C NMR (101 MHz, MeOD) 6151.1, 142.7, 142.4, 141.8, 139.3, 138.6, 128.6, 128.5, 127.1, 126.3, 123.6, 114.0, 112.2, 110.3, 63.7, 40.1, 34.3, 26.3. HPLC-MS tR= 6.66 min, 95.9 % purity, m / z calculated for [C23H29N5 + H] = 376.2, found 376.3

[0195]

[0196] Compound PI-8S (PI-22-19)

[0197] 6-[3-(Dimethylamino)phenyl]-N2-[(lS)-l-phenylethyl]pyrazine-2,3-diamine was prepared as outlined in general procedure B on a 0.3 mmol scale and purified through silica gel chromatography using a EtOAc / hexanes eluent system to afford the final product as a black oil (68 mg, 73 %).1H NMR (400 MHz, MeOD) 67.59 (s, 1H), 7.44 (d, J = 7.6 Hz, 2H), 7.30 (t, J = 7.5 Hz, 2H), 7.17 (m, J = 6.5 Hz, 3H), 7.06 (d, J = 7.6 Hz, 1H), 6.70 (q, J = 3.4 Hz, 1H), 5.30 (q, J = 6.9 Hz, 1H), 2.92 (s, 6H), 1.61 (d, J = 7.0 Hz, 3H).13C NMR (101 MHz, MeOD) 6151.0, 145.9, 142.6, 141.9, 139.3, 138.6, 128.6, 128.0, 126.2, 125.7, 123.5, 113.9, 112.1, 110.3,50.6, 40.0, 22.4. HPLC-MS tR = 6.21 min, 98.2 % purity, m / z calculated for [C20H23N5 + H] = 334.2, found 334.1

[0198]

[0199] Compound PI-8R (PI-22-22)

[0200] 6-[3-(Dimethylamino)phenyl]-N2-[(lR)-l-phenylethyl]pyrazine-2,3-diamine was prepared as outlined in general procedure B on a 0.3 mmol scale and purified through silica gel chromatography using a EtOAc / hexanes eluent system to afford the final product as a black oil (52 mg, 50 %).1H NMR (400 MHz, MeOD) 67.59 (s, 1H), 7.44 (d, J = 7.6 Hz, 2H), 7.30 (t, J = 7.5 Hz, 2H), 7.17 (m, J = 6.5 Hz, 3H), 7.06 (d, J = 7.6 Hz, 1H), 6.70 (q, J = 3.4 Hz, 1H), 5.30 (q, J = 6.9 Hz, 1H), 2.92 (s, 6H), 1.61 (d, J = 7.0 Hz, 3H).13C NMR (101 MHz, MeOD) 6151.1, 145.9, 142.6, 141.9, 139.3, 138.6, 128.5, 128.0, 126.1, 125.7, 123.5, 113.9, 112.1, 110.3, 50.6, 40.0, 22.4. HPLC-MS tR = 6.31 min, 96.6 % purity, m / z calculated for [C20H23N5 + H] = 334.2, found 334.1

[0201]

[0202] Compound 27 (PI-22-45)

[0203] 6-[3-(Dimethylamino)phenyl]-N2-(l-phenylpropyl)pyrazine-2,3-diamine was prepared as outlined in general procedure B on a 0.1 mmol scale and purified through silica gel chromatography using a EtOAc / hexanes eluent system to afford crude, which was further purified using prep-HPLC to obtain the final product as a TFA salt (18 mg, 29 %).XH NMR (400 MHz, DMSO) 68.45 (s, 1H), 7.81 (d = 6.4 Hz, 1H), 7.70 (s, 1H), 7.43 (d = 7.6 Hz, 2H), 7.33 (t = 7.5 Hz, 2H), 7.21 (q = 8.2 Hz, 4H), 6.80 (d = 7.8 Hz, 1H), 5.04 (q = 6.9 Hz, 1H), 4.93 (s, 3H), 2.97 (s, 6H), 1.90 (m = 7.0 Hz, 2H), 0.96 (t = 7.2 Hz, 3H).13C NMR (101 MHz, DMSO) 6159.4, 159.0, 150.4, 143.8, 143.6, 140.0, 137.6, 136.7, 129.7, 128.8, 127.33, 127.0, 117.9, 115.0, 114.8, 113.7, 111.7, 110.3, 57.7, 41.3, 29.9, 11.7. HPLC-MS tR= 6.28 min, 99.7 % purity, m / z calculated for [C21H25N5 + H] = 348.2, found 348.2

[0204]

[0205] Compound 28 (PI-22-34)

[0206] N2-benzyl-6-[3-(dimethylamino)phenyl]pyrazine-2,3-diamine was prepared as outlined in general procedure B on a 0.2 mmol scale and purified through silica gel chromatography using a EtOAc / hexanes eluent system to afford the final product as a black oil (51 mg, 99%). 1H NMR (400 MHz, MeOD) 67.92 (s, 1H), 7.60 (s, 1H), 7.22 (t, / = 7.8 Hz, 1H), 7.16 (t, / = 7.8 Hz, 1H), 7.07 (d, / = 7.5 Hz, 1H), 7.01 (d, / = 8.4 Hz, 2H), 6.76 (d, / = 7.9 Hz, 1H), 6.70 (d, / = 8.0 Hz, 1H), 5.26 (q, / = 6.8 Hz, 1H), 3.76 (s, 3H), 2.91 (s, 6H), 1.60 (d, / = 7.0 Hz, 3H).13C NMR (101 MHz, MeOD) 6159.8, 151.1, 147.7, 142.6, 141.9, 139.2, 138.6, 129.0, 128.5, 123.4, 117.9, 113.9, 112.1, 111.7, 111.2, 110.3, 54.2, 50.6, 40.0, 22.3. HPLC-MS tR= 6.00 min, 97.7 % purity, m / z calculated for [C21H25N5O + H] = 364.2, found 365.1

[0207]

[0208] Compound 29 (PI-22-37)

[0209] 6-[3-(Dimethylamino)phenyl]-N2-{l-[3-(trifluoromethyl)phenyl]ethyl}pyrazine-2,3-diamine was prepared as outlined in general procedure B on a 0.1 mmol scale and purified through silica gel chromatography using a EtOAc / hexanes eluent system to afford the final product as a black solid (50 mg, 95 %). 'H NMR (400 MHz, MeOD) 67.74 (s, 1H), 7.70 (d, / = 4.3 Hz, 1H), 7.62 (s, 1H), 7.50 (d, / = 3.8 Hz, 2H), 7.15 (t, / = 7.9 Hz, 1H), 7.09 (s, 1H), 7.01 (d, / = 7.5 Hz, 1H), 6.69 (q, / = 3.3 Hz, 1H), 5.34 (q, / = 7.0 Hz, 1H), 2.89 (s, 6H), 1.63 (d, / = 7.0 Hz, 3H).13C NMR (101 MHz, Methanol-d4) 6151.0, 147.5, 142.7, 141.6, 139.2, 138.4, 130.2 (q, / = 31.8 Hz), 129.4, 128.7, 128.5, 125.8, 123.9, 123.1, 122.6 (dq = 57.1, 4.0 Hz), 114.0, 112.1, 109.9, 50.4, 39.9, 22.3. HPLC-MS tR= 6.41 min, 96.8 % purity, m / z calculated for [C21H22F3N5 + H] = 402.2, found 402.2

[0210]

[0211] Compound 30 (PI-22-36)

[0212] 6-[3-(Dimethylamino)phenyl]-N2-[l-(4-methylphenyl)ethyl]pyrazine-2,3-diamine was prepared as outlined in general procedure B on a 0.2 mmol scale and purified through silica gel chromatography using a EtOAc / hexanes eluent system to afford the final product as a black solid (56 mg, 84 %). Over 1 year storage at 4 °C, compound degradation was observed. The solid stock was consequently purified a second time using prep HPLC to yield the final product as a TFA salt for biological testing.XH NMR (400 MHz, DMSO) 87.84 (d, / = 5.5 Hz, 1H), 7.71 (s, 1H), 7.32 (d, / = 7.6 Hz, 2H), 7.22 (t, / = 9.1 Hz, 3H), 7.12 (d, / = 7.6 Hz, 2H), 6.81 (d, / = 7.4 Hz, 1H), 5.24 (t, / = 6.3 Hz, 1H), 2.96 (s, 6H), 2.25 (s, 3H), 1.55 (d = 6.8 Hz, 3H).13C NMR (101 MHz, DMSO) 5159.9, 159.6, 159.2, 158.9, 150.3, 143.4, 142.0, 139.9, 137.5, 136.7, 136.3, 129.7, 129.4, 126.3, 117.9, 115.0, 113.8, 111.4, 110.4, 51.1, 41.3, 23.3, 21.1. HPLC-MS tR = 6.24 min, 95.5 % purity, m / z calculated for [C21H25N5 + H] = 348.2, found 348.2

[0213]

[0214] Compound 31 (PI-22-59)

[0215] 6-[3-(Dimethylamino)phenyl]-N2-[l-(4-methoxyphenyl)ethyl]pyrazine-2,3-diamine was prepared as outlined in general procedure B on a 0.2 mmol scale and purified through silica gel chromatography using a EtOAc / hexanes eluent system to afford crude, which was further purified using prep-HPLC to obtain the final product as a TFA salt (28 mg, 29 %).XH NMR (400 MHz, DMSO) 68.50 (s, 3H), 7.80 (s, 1H), 7.71 (s, 1H), 7.36 (d, / = 6.6 Hz, 2H), 7.26 (t = 7.5 Hz, 3H), 6.89 (d = 6.6 Hz, 2H), 6.84 (s, 1H), 5.25 (s, 1H), 3.71 (d = 1.9 Hz, 3H), 2.98 (s, 6H), 1.55 (d = 4.7 Hz, 3H).X3C NMR (101 MHz, DMSO) 6159.8, 159.4, 159.0, 158.7, 158.6, 150.2, 143.4, 139.9, 137.5, 136.8, 136.8, 129.8, 127.6, 115.1, 114.2, 114.0, 111.3, 110.5, 55.5, 50.7, 41.4, 23.2. HPLC-MS tR = 6.62 min, 96.1 % purity, m / z calculated for [C21H25N5O + H] = 364.2, found 364.1

[0216]

[0217] Compound 32 (PI-22-69)

[0218] 6-[3-(Dimethylamino)phenyl]-N2-[l-(4-methanesulfonylphenyl)ethyl]pyrazine-2,3-diamine was prepared as outlined in general procedure B on a 0.2 mmol scale and purified through silica gel chromatography using a EtOAc / hexanes eluent system to afford crude, which was further purified using prep-HPLC to obtain the final product as a TFA salt (35 mg, 39 %).1H NMR (400 MHz, DMSO) 67.91 (q, / = 7.2 Hz, 4H), 7.71 (t, / = 5.7 Hz, 3H), 7.22 (t, / = 7.8 Hz, 2H), 7.13 (d, / = 7.8 Hz, 2H), 6.81 (d, / = 7.4 Hz, 1H), 5.34 (t, / = 6.0 Hz, 1H), 3.18 (s, 3H), 2.93 (s, 6H), 1.59 (d = 6.8 Hz, 3H).13C NMR (101 MHz, DMSO) 6159.4, 159.0, 151.3, 150.2, 143.3, 140.2, 139.8, 137.4, 136.7, 129.7, 127.6, 127.2, 115.1, 113.9, 112.5, 110.4, 51.3, 44.0, 41.4, 23.2. HPLC-MS tR = 6.24 min, 99.0 % purity, m / z calculated for [C21H25N5O2S + H] = 412.2, found 412.1

[0219]

[0220] Compound 33 (PI-22-62)

[0221] 6-[3-(Dimethylamino)phenyl]-N2-[(3-methylphenyl)methyl]pyrazine-2,3-diamine was prepared as outlined in general procedure B on a 0.2 mmol scale and purified through silica gel chromatography using a EtOAc / hexanes eluent system to afford the final product (43 mg, 54 %).1H NMR (400 MHz, DMSO) 67.76 (s, 1H), 7.23 (s, 2H), 7.20 (d, / = 5.0 Hz, 2H), 7.16 (t, / = 6.4 Hz, 2H), 7.04 (d = 4.3 Hz, 1H), 6.84 (d, / = 5.1 Hz, 1H), 6.61 (d = 2.7 Hz, 1H), 6.14 (s, 2H), 4.62 (d = 5.2 Hz, 2H), 2.89 (s, 6H), 2.28 (s, 3H).13C NMR (101 MHz, DMSO) 6151.1, 143.7, 142.1, 140.9, 139.1, 137.7, 137.5, 129.3, 128.5, 127.7, 125.5, 125.0, 113.4, 111.7, 109.5, 44.5, 40.7, 21.5. HPLC-MS tR = 6.21 min, 96.1 % purity, m / z calculated for [C20H23N5 + H] = 334.2, found 334.2

[0222]

[0223] Compound 34 (PI-22-65)

[0224] 6-[3-(Dimethylamino)phenyl]-N2-[(3,4-dimethylphenyl)methyl]pyrazine-2,3-diamine was prepared as outlined in general procedure B on a 0.3 mmol scale and purified through silica gel chromatography using a EtOAc / hexanes eluent system to afford the final product (28 mg, 31 %). 1H NMR (400 MHz, DMSO) 67.74 (s, 1H), 7.24 (s, 1H), 7.19 (s, 1H), 7.15 (d = 4.6 Hz, 2H), 7.11 (s, 1H), 7.06 (d = 7.6 Hz, 1H), 6.78 (d = 5.2 Hz, 1H), 6.62 (s, 1H), 6.12 (s, 2H), 4.57 (d = 5.1 Hz, 2H), 2.90 (s, 6H), 2.18 (d = 6.5 Hz, 6H).13C NMR (101 MHz, DMSO) 5 151.1, 143.7, 142.1, 139.1, 138.3, 137.4, 136.2, 134.7, 129.7, 129.3, 129.2, 125.4, 113.4, 111.7, 109.5, 44.3, 40.7, 19.9, 19.5. HPLC-MS tR = 6.41 min, 97.2 % purity, m / z calculated for [C21H25N5 + H] = 348.2, found 348.2

[0225]

[0226] Compound 35 (PI-22-66)

[0227] 6-[3-(Dimethylamino)phenyl]-N2-{[3-(trifluoromethyl)phenyl]methyl}pyrazine-2,3-diamine was prepared as outlined in general procedure B on a 0.1 mmol scale and purified through silica gel chromatography using a EtOAc / hexanes eluent system to afford crude, which was further purified using prep-HPLC to obtain the final product as a TFA salt (20 mg, 28 %).1H NMR (400 MHz, DMSO) 68.32 (s, 3H), 8.15 (s, 1H), 7.80 (d = 6.8 Hz, 2H), 7.72 (s, 1H), 7.60 (m = 7.4 Hz, 2H), 7.26 (d = 12.8 Hz, 3H), 6.81 (s, 1H), 4.83 (s, 2H), 2.91 (s, 6H).13C NMR (101 MHz, DMSO) 5159.8, 159.4, 150.2, 144.2, 140.9, 140.6, 137.5, 136.8, 131.9, 129.8, 129.7, 129.4, 126.1, 124.5, 124.5, 124.2, 124.1, 123.4, 115.1, 113.9, 112.6, 110.4, 44.6, 41.2. HPLC-MS tR = 6.78 min, 98.7 % purity, m / z calculated for [C20H20F3N5 + H] = 388.2, found 388.1

[0228]

[0229] Compound 36 (PI-22-67)

[0230] N2-({[l,l'-biphenyl]-3-yl}methyl)-6-[3-(dimethylamino)phenyl]pyrazine-2,3-diamine was prepared as outlined in general procedure B on a 0.2 mmol scale and purified through silica gel chromatography using a EtOAc / hexanes eluent system to afford the final product (45 mg, 58 %). 1H NMR (400 MHz, DMSO) 67.76 (t, / = 4.3 Hz, 2H), 7.61 (d, / = 6.9 Hz, 2H), 7.52 (s, 1H), 7.43 (d, / = 7.9 Hz, 4H), 7.36 (t, / = 3.5 Hz, 1H), 7.24 (s, 1H), 7.14 (t, / = 5.1 Hz, 2H), 6.94 (s, 1H), 6.61 (t = 3.2 Hz, 1H), 6.15 (s, 2H), 4.73 (s, 2H), 2.84 (d, / = 2.8 Hz, 6H).13C NMR (101 MHz, DMSO) 6151.1, 143.8, 142.1, 141.7, 140.7, 140.5, 139.1, 137.5, 129.4, 129.3, 129.3, 127.9, 127.1, 127.1, 126.6, 125.6, 125.5, 113.5, 111.7, 109.5, 44.7, 40.7. HPLC-MS tR = 6.99 min, 95.1 % purity, m / z calculated for [C25H25N5 + H] = 396.2, found 396.1

[0231]

[0232] Compound 37 (PI-22-72)

[0233] Methyl 3-[({3-amino-6-[3-(dimethylamino)phenyl]pyrazin-2-yl}amino)methyl]benzoate was prepared as outlined in general procedure B on a 0.5 mmol scale and purified through silica gel chromatography using a EtOAc / hexanes eluent system to afford the final product (177 mg, 87 %).XH NMR (400 MHz, DMSO) 68.04 (s, 1H), 7.83 (d, / = 7.3 Hz, 1H), 7.77 (s, 1H), 7.69 (d, / = 7.3 Hz, 1H), 7.47 (t, / = 7.5 Hz, 1H), 7.14 (t, / = 7.6 Hz, 3H), 7.00 (s, 1H), 6.60 (d, / = 6.6 Hz, 1H), 6.13 (s, 2H), 4.72 (d = 4.6 Hz, 2H), 3.84 (s, 3H), 2.86 (s, 6H).13C NMR (101 MHz, DMSO) 6166.8, 151.1, 143.8, 142.0, 141.9, 139.0, 137.6, 132.7, 130.1, 129.3, 129.1, 128.4, 127.9, 125.8, 113.4, 111.7, 109.5, 52.5, 44.3, 40.6. HPLC-MS tR= 6.44 min, 96.4 % purity, m / z calculated for [C21H23N5O2 + H] = 378.2, found 378.1

[0234]

[0235] Compound 38 (PI-22-73)

[0236] 6-[3-(Dimethylamino)phenyl]-N2-[(pyridin-3-yl)methyl]pyrazine-2,3-diamine was prepared as outlined in general procedure B on a 0.2 mmol scale and purified using prep-HPLC to obtain the final product as a TFA salt (30 mg, 39 %). 'H NMR (400 MHz, DMSO) 6 9.61 (s, 3H), 8.93 (s, 1H), 8.76 (s, 1H), 8.43 (d = 8.0 Hz, 1H), 8.30 (d = 10.6 Hz, 1H), 7.88 (d, / = 5.6 Hz, 1H), 7.82 (s, 1H), 7.23 (d = 11.1 Hz, 3H), 6.83 (d = 7.7 Hz, 1H), 4.90 (s, 2H), 2.92 (s, 6H).13C NMR (101 MHz, DMSO) 6159.7, 159.3, 150.1, 144.1, 143.4, 143.2, 142.3, 141.0, 138.5, 137.3, 136.8, 129.8, 126.4, 115.3, 114.0, 113.4, 110.5, 42.3, 41.4. HPLC-MS tR= 1.39 min, 98.5 % purity, m / z calculated for [C18H20N6 + H] = 321.2, found 321.1

[0237]

[0238] Compound 39 (PI-22-74)

[0239] 4-{5-Amino-6-[(l-phenylethyl)amino]pyrazin-2-yl}benzoic acid. To stirred solution of PI-22-72 (151.4 mg, 0.401 mmol) in THF / H2O (3:1), was added lithium hydroxide (10 eq) at room temperature. The resulting mixture was heated up to 50° C and stirred overnight. Upon completion, reaction mixture was concentrated in vacuo. The resulting residue was dissolved in H2O (45 mL) and transferred into a separating funnel. DCM (25 ml) were added and the aqueous phase was neutralised (pH ~ 3.5) using 2M HC1 (aq.). Upon neutralisation product precipitated in the aqueous phase as a light brown solid. Solid was washed with DCM (2 x 15 mL), filtered and washed again with cold H2O (3 x 10 mL). The solid was dissolved in MeOH and combined with the DCM organic phases washes from the work-up. The organic phases were dried (Na2SO4) and concentrated to afford the final product (107 mg, 74 %). 'H NMR (400 MHz, DMSO) 68.01 (s, 1H), 7.81 (d, / = 7.4 Hz, 1H), 7.75 (s, 1H), 7.65 (d, / = 7.4 Hz, 1H), 7.44 (t, / = 7.6 Hz, 1H), 7.13 (t, / = 7.1 Hz, 3H), 6.98 (s, 1H), 6.60 (d, / = 7.0 Hz, 1H), 6.14 (s, 2H), 4.71 (d = 4.7 Hz, 2H), 2.86 (s, 6H).13C NMR (101 MHz, DMSO) 6167.8, 151.1, 143.8, 141.9, 141.7, 139.0, 137.6, 132.2, 131.3, 129.3, 128.9, 128.5, 128.0, 125.7, 113.5, 111.7, 109.5, 44.3, 40.7. HPLC-MS tR= 6.36 min, 96.6 % purity, m / z calculated for [C20H21N5O2 + H] = 364.2, found 364.2

[0240]

[0241] Compound PI-IOS (PI-24-01)

[0242] N2-[(lS)-l-phenylethyl]-6-(pyridin-3-yl)pyrazine-2,3-diamine was prepared as outlined in general procedure B on a 0.16 mmol scale and purified using prep-HPLC to obtain the final product as a light-yellow oil (9.3 mg, 29 %).XH NMR (400 MHz, DMSO-d ) 68.96 (d, / = 2.3 Hz, 1H), 8.40 (dd = 4.8, 1.7 Hz, 1H), 8.08 (dt = 8.0, 2.0 Hz, 1H), 7.82 (s, 1H), 7.43 (d, / = 7.4 Hz, 2H), 7.37 - 7. 1 (m, 3H), 7.18 (td, / = 7.1, 1.3 Hz, 1H), 6.75 (d, / = 6.7 Hz, 1H), 6.40 (s, 2H), 5.26 (p = 7.0 Hz, 1H), 1.53 (d = 6.9 Hz, 3H).13C NMR (101 MHz, DMSO) 6147.96, 146.51, 146.43, 144.35, 141.61, 134.01, 133.97, 131.96, 128.63, 126.86, 126.47, 125.97, 123.86, 50.45, 23.63. HPLC-MS tR = 6.83 min, 98.1 % purity, m / z calculated for [C17H17N5 + H] = 292.2, found 292.2

[0243]

[0244] Compound PI-10R (PI-24-07)

[0245] N2-[(lR)-l-phenylethyl]-6-(pyridin-3-yl)pyrazine-2,3-diamine was prepared as outlined in general procedure B on a 0.16 mmol scale and purified using prep-HPLC to obtain the final product as a Formic Acid salt (11.1 mg, 41 %).XH NMR (400 MHz, DMSO-d ) 68.98 - 8.94 (m, 1H), 8.40 (d, / = 4.6 Hz, 1H), 8.16 (s, 1H), 8.08 (d, / = 8.1 Hz, 1H), 7.82 (s, 1H), 7.43 (d = 7.6 Hz, 2H), 7.32 (q, / = 7.8, 7.2 Hz, 3H), 7.18 (t, / = 7.3 Hz, 1H), 6.75 (d, / = 6.7 Hz, 1H), 6.40 (s, 2H), 5.26 (p, / = 6.9 Hz, 1H), 1.53 (d, / = 7.0 Hz, 3H).X3C NMR (101 MHz, DMSO) 6163.53, 147.95, 146.50, 146.43, 144.35, 141.62, 134.02, 133.98, 131.98, 128.63, 126.86, 126.47, 125.96, 123.86, 50.46, 23.62. HPLC-MS tR = 6.72 min, 99.1 % purity, m / z calculated for [C17H17N5 + H] = 292.2, found 292.2

[0246]

[0247] Compound PI-15S (PI-24-03)

[0248] N-[3-(5-amino-6-{[(lS)-l-phenylethyl]amino}pyrazin-2-yl)phenyl]acetamide was prepared as outlined in general procedure B on a 0.16 mmol scale and purified using prep-HPLC to obtain the final product as a Formic Acid salt (8.3 mg, 26 %). 'H NMR (400 MHz, DMSO-d6) 69.92 (s, 1H), 8.16 (s, 1H), 8.07 (s, 1H), 7.65 (d, J = 3.5 Hz, 1H), 7.47 (q, J = 9.9, 8.8 Hz, 4H), 7.29 (d, J = 7.3 Hz, 2H), 7.24 (q, J = 6.3, 4.8 Hz, 1H), 7.18 (t, J = 7.2 Hz, 1H), 6.66 (d, J = 7.1 Hz, 1H), 6.26 (s, 2H), 5.35 (t, J = 7.1 Hz, 1H), 2.07 (d, J = 3.5 Hz, 3H), 1.54 (d, J = 7.0 Hz, 3H).13C NMR (101 MHz, DMSO) 6 168.69, 163.58, 146.33, 143.79, 141.39, 139.91, 139.01, 136.53, 129.07, 128.60, 126.90, 126.78, 125.26, 119.89, 118.06, 115.87, 49.99, 24.53, 23.38. HPLC-MS tR = 7.35 min, 99.3 % purity, m / z calculated for [C20H21N5O + H] = 348.2, found 348.2

[0249] H N N X

[0250] H

[0251]

[0252] Compound PI-15R (PI-24-08)

[0253] N-[3-(5-amino-6-{[(lR)-l-phenylethyl]amino}pyrazin-2-yl)phenyl]acetamide was prepared as outlined in general procedure B on a 0.16 mmol scale and purified using prep-HPLC to obtain the final product as a Formic Acid salt (8.4 mg, 1 %).XH NMR (400 MHz, DMSO-d6) 69.89 (s, 1H), 8.16 (s, 1H), 8.06 (s, 1H), 7.66 (s, 1H), 7.52 - 7.41 (m, 4H), 7.27 (dt, = 24.8, 7.6 Hz, 3H), 7.18 (t, / = 7.4 Hz, 1H), 6.64 (d, / = 7.3 Hz, 1H), 6.23 (s, 2H), 5.35 (p, / = 6.9 Hz, 1H), 2.08 (s, 3H), 1.54 (d, / = 7.0 Hz, 3H).13C NMR (101 MHz, DMSO) 6 168.69, 163.60, 146.33, 143.79, 141.41, 139.92, 139.03, 136.57, 129.06, 128.60, 126.89, 126.79, 125.28, 119.91, 118.09, 115.91, 50.00, 24.53, 23.36. HPLC-MS tR= 7.41 min, 99.2 % purity, m / z calculated for [C20H21N50 + H] = 348.2, found 348.2

[0254] N^ NH

[0255] N N

[0256] H

[0257]

[0258] Compound PI-17S (PI-24-04)

[0259] 3-(5-amino-6-{[(lS)-l-phenylethyl]amino}pyrazin-2-yl)-N, N-dimethylbenzamide wasprepared as outlined in general procedure B on a 0.16 mmol scale and purified through prep-HPLC to afford the final product as a light-brown oil (18.9 mg, 48 %).XH NMR (400 MHz, DMSO-J6) 67.85 - 7.76 (m, 2H), 7.70 (s, 1H), 7.45 - 7.33 (m, 3H), 7.29 (t, / = 7.6 Hz, 2H), 7.22 - 7.17 (m, 2H), 6.73 (d = 6.4 Hz, 1H), 6.35 (s, 2H), 5.21 (t = 6.9 Hz, 1H), 3.03 (s, 3H), 2.87 (s, 3H), 1.53 (d, / = 7.0 Hz, 3H).13C NMR (101 MHz, DMSO) 6170.84, 146.57, 144.04, 141.46, 138.54, 137.22, 135.92, 128.81, 128.57, 126.78, 126.46, 125.74, 125.54, 125.46, 123.45, 50.62, 35.16, 23.64. HPLC-MS tR = 7.39 min, 95.4 % purity, m / z calculated for [C21H23N5O + H] = 362.2, found 362.2

[0260]

[0261] Compound PI-17R (PI-24-09)

[0262] 3-(5-amino-6-{[(lR)-l-phenylethyl]amino}pyrazin-2-yl)-N, N-dimethylbenzamide as outlined in general procedure B on a 0.16 mmol scale and purified prep-HPLC to afford the final product as an oil (9.5 mg, 33 %). 'H NMR (400 MHz, DMSO-J6) 67.84 - 7.75 (m, 2H), 7.69 (d, / = 1.9 Hz, 1H), 7.43 - 7.25 (m, 5H), 7.19 (dt = 10.2, 5.3 Hz, 2H), 6.70 (d, / = 6.5 Hz, 1H), 6.32 (s, 2H), 5.21 (p = 6.9 Hz, 1H), 3.03 (s, 3H), 2.87 (s, 3H), 1.53 (d = 6.9 Hz, 3H).

[0263] 13C NMR (101 MHz, DMSO) 6170.82, 146.58, 144.03, 141.43, 138.51, 137.20, 135.87, 128.81, 128.57, 126.78, 126.45, 125.73, 125.53, 125.46, 123.43, 50.61, 35.17, 23.66. HPLC-MS tR = 7.39 min, 99.2 % purity, m / z calculated for [C21H23N5O + H] = 362.2, found 362.2

[0264]

[0265] Compound 20S (PI-24-05)

[0266] 3-(5-amino-6-{[(lS)-l-phenylethyl]amino}pyrazin-2-yl)-N, N-dimethyl-5-nitrobenzamide was prepared as outlined in general procedure B on a 0.16 mmol scale and purified through prep-HPLC to afford the final product as a red oil (7.5 mg, 17 %).XH NMR (400 MHz, DMSO-J6) 68.61 (s, 1H), 8.12 (s, 1H), 7.97 (d, / = 3.8 Hz, 2H), 7.45 (d, / = 7.5 Hz, 2H),7.30 (t, J = 7.6 Hz, 2H), 7.18 (t, J = 7.4 Hz, 1H), 6.89 (d, / = 6.2 Hz, 1H), 6.56 (s, 2H), 5.15 (p, / = 7.1 Hz, 1H), 3.05 (s, 3H), 2.89 (s, 3H), 1.55 (d, / = 6.9 Hz, 3H).13C NMR (101 MHz, DMSO) 5 168.56, 148.81, 146.53, 144.92, 141.37, 140.65, 138.70, 133.38, 128.66, 128.60, 126.93, 126.84, 126.42, 119.67, 119.63, 51.07, 35.30, 23.75. HPLC-MS tR= 7.96 min, 98.2 % purity, m / z calculated for [C21H22N6O3 + H] = 407.2, found 407.2

[0267]

[0268] Compound 20R (PI-24-10)

[0269] 3-(5-amino-6-{[(lR)-l-phenylethyl]amino}pyrazin-2-yl)-N, N-dimethyl-5-nitrobenzamide was prepared as outlined in general procedure B on a 0.16 mmol scale and purified through prep-HPLC to afford the final product as a red oil (9.8 mg, 30 %).XH NMR (400 MHz, DMSO-d6) 68.61 (d, / = 2.0 Hz, 1H), 8.13 (s, 1H), 7.97 (d, / = 3.6 Hz, 2H), 7.45 (d, / = 7.6 Hz, 2H), 7.30 (t, / = 7.5 Hz, 2H), 7.18 (t, / = 7.3 Hz, 1H), 6.91 (d, / = 6.2 Hz, 1H), 6.57 (s, 2H), 5.15 (p = 6.8 Hz, 1H), 3.05 (s, 3H), 2.89 (s, 3H), 1.54 (d = 6.9 Hz, 3H).13C NMR (101 MHz, DMSO) 5168.56, 148.81, 146.53, 144.92, 141.37, 140.65, 138.69, 133.38, 128.66, 128.60, 126.92, 126.84, 126.42, 119.67, 119.63, 51.07, 35.30, 23.74. HPLC-MS tR= 7.97 min, 98.9 % purity, m / z calculated for [C21H22N6O3 + H] = 407.2, found 407.2

[0270] Purification and characterization of test compounds

[0271] Commercial reagents and solvents were purchased from chemical suppliers and used without further purification. Essential reagents for this study include the following: 3,5-Dibromopyrazin-2-amine (Combi-Blocks™, San Diego, USA), a-Methylbenzylamine, [1,1'-Bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with Dichloromethane (Sigma Aldrich™, St. Louis, USA). Analytical thin layer chromatography was performed using 60-F-254 plates (E. Merck™, Darmstadt, Germany) and visualized using a UV 254 nm lamp. Flash chromatography was performed using a Biotage™ 'Isolera One’ automated flash chromatography system. Preparative HPLC purification was performed on an Agilent G7169B™ column using an acidic reverse phase gradient of 5 % to 99 % MeCN in H2O (0.1 % TFA) over 12 min.XH andX3C NMR spectra were collected using a Bruker Ascend 400™ MHzspectrometer. Chemical shifts of reported spectra are expressed in parts per million (ppm) and referenced to residual solvent peaks.

[0272] Analytical HPLC-MS was performed using an Agilent™ analytical HPLC system (1260 Infinity II™) fit with the Agilent™ HPLC column Zorbax™ SB-C18 2.1 x 50 mm (1.8pm) and streamlined to a single quadrupole LC / MSD (Agilent InfinityLab™ G6125B). Using a flowrate of 0.500 mL / min, a reverse phase solvent gradient of 10 % to 99 % MeCN in H₂O (0.1 % TFA) was used over 12 min: 0-3 min (10 % MeCN); 3-6 min (10-90 % MeCN); 6-9 min (90-99 % MeCN); 9-12 min (99 % MeCN). Analytical UPLC-MS analysis was performed for some samples, where a Waters H-Class Acquity™ UPLC system was employed using a reverse phase gradient of 5 to 95 % of MeCN in H₂O over 8 min.

[0273] Compound analytical samples were prepared as 600 pM (~0.2 mg / mL) solutions in HPLC-gradient Methanol (Sigma-Aldrich™), where 5 pL (~1 pg) of the sample solution were injected into the instrument per analytical run. UV absorbance at 254 nm (integrating area under the curve) was used to characterize purity of the compounds. Retention times (tn) are reported in minutes and all intermediates and final compounds were assessed to be >95% pure by HPLC-MS analysis. Mass spectra was obtained using the API-ES ion source, acquiring positive polarity signals scanning from 100 to 1000 m / z. Compound solids were prepared at 10 mM in DMSO for biological testing.

[0274] Protein expression and purification

[0275] Recombinant STAT3 and NUDT5 protein expression and purification was performed in collaboration with the Protein Science Facility, Department of Medical Biochemistry and Biophysics (MBB), Karolinska Institute, Stockholm, Sweden, as previously reported.36Phosphopeptides corresponding to the gpl30 sequences Ac-(pY)LPQTV, were purchased from Biomatik™ (Cambridge, Canada) and diluted in DMSO prior to use.

[0276] Differential Scanning Fluorimetry (DSF)

[0277] The DSF assay protocol was adapted according to procedures described previously34'35. The procedure and analysis of the STAT3 DSF high throughput screen is outlined in FIGURE 1.

[0278] Hit validation DSF experiments were conducted in DSF assay buffer (100 mM Tris-HCl, 40 mM NaCl, 10 mM MgCl₂, pH=7.40). Recombinant STAT3127’688or STAT3127’465proteins were incubated at a final concentration of 2 pM together with SYPRO™ Orange at "7.5x” (from astock concentration of "5000x”). Inhibitor solutions were added to the protein well following the addition of SYPRO™ Orange to constitute 20 pL final reaction volume. Experiments were conducted using technical triplicates in a 96-well plate (Applied Biosystems™ MicroAmp™ Fast Optical 96-Well Reaction Plate, 0.1 mL) into StepOnePlus™ Real-Time PCR System, Thermo Fisher Scientific™. Heating was conducted using gradients from 25 to 80 °C, reading fluorescence every 1°C where raw fluorescence was plotted versus temperature to obtain the STAT3 melt curve plots.

[0279] Protein Thermal Shift Assay (PTSA)

[0280] STAT3 PTSA was performed as described36. Protein master mixes of STAT3 and NUDT5 were prepared in 0.2 mL PCR tubes using DSF assay buffer. These contained recombinant STAT3 along with the thermally stable NUDT5 loading control. Master mixes were then aliquoted into PCR tubes (18 pL) and 2 pL of inhibitor solutions (1 mM, 10 % DMSO in buffer) or controls (10 % DMSO in buffer, or buffer alone) were added for a total volume of 20 L. The final concentrations in the reaction mixtures were as follows: STAT3 (3.8 pM), NUDT5 (2.4 μM), inhibitors (100 pM), and DMSO (1 %). Samples were incubated for 8 min at a single elevated temperature (ranging from 37 to 63 °C) using a DNA Engine Dyad Peltier Thermal Cycler™ (Bio-Rad™). Upon completion, the contents were placed on ice for two minutes to quench the reaction, then subsequently transferred into 1.5 mL Eppendorf™ tubes and centrifuged at 20000 g for 20 min at 4 °C. The supernatant containing the soluble protein fraction (10 pL) was then removed and prepared for SDS-PAGE separation by adding 2.5 μL of loading buffer (Tris-Cl / SDS, glycerol, p-mercaptoethanol, bromophenol blue, pH 6.8) and heated at 95 °C for 10 minutes. 12.5 pL total volume of each sample were separated by SDS-PAGE on a 10 % polyacrylamide gel. Protein bands were then stained using silver stain (Biorad™). Band intensities were detected by Coomassie (Brilliant Blue G; Sigma™) and Silver Staining (BioRad™) and subsequently analyzed by densitometry using ImageJ™ (National Institutes of Health). Ratios between STAT and NUDT5 loading control for each lane, were then normalized to the STAT3: NUDT5 ratio at 37 °C which was set to 100 %. Fluorescence Polarization (FP) assay

[0281] A Corning 384-well black flat-bottom plate was loaded in triplicates with serial dilutions of STAT3127-688protein and 10 nM of fluorophore-tagged-gp130 (5-aminofluorescein-GpYLPQTV, 5-FAM-gp130) in buffer containing 50 mM NaCl, 10 mM HEPES, 1 mM DTT at pH 7.5, total reaction volume was 50 pL. After 10 min of incubation, polarized fluorescence was measured using a Synergy Neo2™ reader (λex = 485 nm, λem = 535 nm, Xenon lamp medium intensity, 10 flashes). Fluorescence polarization (mP) was plotted against STAT3 concentration and Sigmoidal dose-response nonlinear regression was fit to the data using GraphPad Prism 10™. The estimated ECso value for STAT3 was used as control on each plate. STAT3 FP experiments with test inhibitors were then carried out in triplicates at using 150 nM STAT3 protein, 10 nM of gp130 probe and inhibitor at corresponding concentration (1 % DMSO, 50 pL total volume).

[0282] Biochemical kinase binding and activity assay

[0283] Kinome profiling and kinase Ka determination studies with synthesized compounds were conducted using the KINOMEscan™ biochemical assay47, outsourced to Eurofins DiscoverX™ Corporation, San Diego, US. Additionally, inhibition of TrkA activity using BPS Bioscience™ TrkA assay kit was assessed according to the supplier’s instructions. Experiments were run in duplicates where LOXO-101 (larotrectinib) was used as a positive control. Enzymatic TrkA activity was normalised corresponding to the values obtained for 1 pM larotrectinib -treated TrkA which was set to 0 %. Kinase enzymatic inhibition assessed using the Z’-lyte™ activity assay61was outsourced to Thermo Fisher Scientific™, Madison, US.

[0284] GLIDE docking experiments

[0285] TrkA molecular docking experiments were performed using Schrödinger, Maestro 12.9 GLIDE™ platform using crystal structures of JAK2 (" DFG-in” pdb: 4AQC, " DFG-out” pdb: 3UGC), Src (" DFG-in” pdb: 4U5J, " DFG-out" pdb: 3EL7), TrkA bound by a Type I (PDB ID = 5KVT) and Type II Trk inhibitor (PDB ID = 6PMB). Solvent water molecules and ligands were removed from the protein crystal structure prior to preparation. Preparation and minimisation were then performed using the default settings of the Protein Preparation Wizard™ (OPLS4 force field, EpiK pH=7.4 ± 2.0). Ligands were prepared using the LigPrep™ function, generating possible ionisation states at pH=7.4 ± 2.0 using the OPLS4 force field parameters. The coordinates of the native ligands, bound into the crystal’s active site, were then used as a centroid for defining a 10 x 10 x 10 A cube using the Receptor Grid Generation™ function. The test ligands were then docked into the defined grid where extraprecision (XP) settings were chosen for each ligand. The top pose for each compound and its corresponding XP score were then summarised for each docking experiment. Predicted protein-ligand complexes for top scoring compounds were then exported as a PyMol™ file for further analysis.

[0286] Cell culture

[0287] STATl-null male U3A human fibrosarcoma cells (provided by George Stark; Cleveland Clinic) were stably transfected with STAT3-dependent luciferase80, and were maintained in Dulbecco's modified Eagle's medium (DMEM) (Thermo Fisher Scientific™) containing 10% fetal bovine serum (FBS). Male KM12 cells were purchased from MD Anderson and subcultured at 37° C (5% CO2) according to MDA CCAC protocol. The media contained 10 % FBS in DMEM (4.5 g / L glucose, glutamine, 6x sodium pyruvate) supplemented with IxNEAA. MDA-MB-231 (HTB-26) cells were obtained from American Type Culture Collection™ (ATCC™), provided by Karla Williams and LS174T cells were obtained from the NCI Developmental Therapeutics Program (DTP) repository, provided by Thomas Velenosi. Both cell lines were maintained in DMEM media supplemented with 10% FBS and detached for seeding through media supplemented with 0.25% trypsin / EDTA. MDA-MB-231 overexpressing HA-TrkA cells were established and maintained in culture as described67.

[0288] STAT3 Luciferase Reporter Assay

[0289] STAT3-dependent gene expression assay was conducted as previously reported81. STAT3-luciferase U3A cells (5 x 103) were plated in white opaque 96-well plates and allowed to adhere overnight. Cells were pretreated with compound for 1 h and then stimulated with Oncostatin M (10ng / mL) for 5 to 6 h to activate STAT3. Luciferase activity was detected using the Bright-Glo Luciferase Assay System™ (Promega™) and quantitated using a Luminoskan Ascent Microplate Reader™ (Labsystems™).

[0290] Cell viability experiments

[0291] Cancer cells were lifted upon reaching 60-80% confluence and seeded in 96-well plates (4500 cells / well) in triplicates using a multichannel pipette and a total volume of 180 pL of cell media. After 24 h, 20 pL of (10 % DMSO, cell media) solutions of test compounds were added to each well. KM12 cells were treated at 11-doses with test inhibitors and LS174Tand MDA-MB-231 cells were treated at 6-doses where for each plate the final DMSO concentration in each well including no cell controls was 1 % DMSO. Following 72 h of compound treatment, lOx resazurin solution (22.2 pL) were added to each well on the plate to form a 222.2 pL of lx resazurin. Following 4 h incubation at 37° C, the plate’s fluorescence (540 / 600) was read on a Synergy 2 BioTek™ plate reader. Five individual fluorescence readings for each well were obtained to cover higher surface area of cells.

[0292] The mean fluorescence (N=5 readings per well) and its standard deviation (SD) were obtained for each well. The mean background fluorescence (media only) was subtracted to calculate the background-corrected, mean fluorescence values for each cell-containing well, where the SD from the "media only” was propagated. The mean DMSO control values were then normalized as 100 % and the ratio between each compound-treated sample and the DMSO control mean was used to estimate the cell viability. KM12 dose-response experiments were repeated as 3 independent biological experiments (N=3), where for each individual experiment, cell viability against compound concentration values were plotted using GraphPad Prism™. The [Inhibitor] vs. response (four parameters, ICso and Bottom parameters > 0) regression model was then fit to each graph to estimate the ECso value observed for the corresponding compound-treatment on that plate. The ECso values estimated between all independent experiment plates were then averaged to calculate the mean ECso across all experiments.

[0293] TPM3-TrkA Western blot

[0294] KM12 cells were subcultured as described previously, where upon reaching 60-80 % confluence, cells were seeded onto 6-well plates (450 000 cells per well). 24 h later, test inhibitors at corresponding concentrations were added to the KM12 cell media from 100% DMSO stock solutions. The final DMSO concentration in each well was 0.1 %. After 24 h incubation with test compounds, the media from each well was discarded and the cells were harvested for downstream western blot analysis. Cells were washed with PBS (ImL) and lifted into 15 mL falcon tubes using media containing 0.25% trypsin / EDTA. After centrifugation at 200 g for 5 min, the cell media was discarded and the remaining cell pellets were resuspended into 750 pL of PBS, supplemented with lx Protease Inhibitor Cocktail™ + lx PhosStop™ (Roche™). The tubes were then centrifuged again at 200 g for 5 min and thePBS supernatant was removed using a P1000 pipette. The remaining cell pellets were flash frozen using EtOH + CO2 (s) for 3 min and placed into the -700C freezer for storage.

[0295] Cell pellets were resuspended in TBS (1 % NP-40, 20 mM Tris-Cl, pH 7,5; 150 mM NaCl) supplied with PIC Mini and PhosStop™ (Roche™; 1 tablet each per 10 mL), where 100 pL per cell aliquot were used. The cell suspensions were then incubated in an ethanol / dry ice bath for 3 minutes. Transferred to a 37° C water bath for 3 mins. This was repeated for a total of three freeze-thaw cycles. Lysates were centrifuged at 20 000 x g for 20 mins at 4° C to remove the cellular debris. Supernatant was collected and protein concentration was determined using Pierce Rapid Gold BCA™ where lysates were diluted in lx PBS (supplied with PIC). Lysates were then prepared for gel electrophoresis in lx SDS-PAGE loading buffer, where individual samples contained 20 pg of total protein. Samples were denatured by heating at 80-90 °C in a water bath for 12 minutes and run by SDS-PAGE on 10 % polyacrylamide gels. Proteins were then transferred onto PVDF membranes using a semidry transfer protocol (Bio rad™ Trans-Blot Turbo Transfer System™) using " High MW” transfer setting. The membranes were washed with lx TBS-T wash buffer (3 x 5 min), blocked with fresh blocking buffer (5% skim milk in TBS-T) for 1 h at room temperature and then washed again with 5 mL lx TBS (3 x 5 min). The strips were then incubated with their respective primary antibody solution at 40C overnight.

[0296] An anti-TrkA antibody (catalogue number P04629, Raybiotech™) was used at a dilution of 1:500 in TBS-T and anti-SODl (catalogue code sc-17767, Santa Cruz™) was used ata 1:5000 dilution as the loading control. Alternatively, anti -actin antibody (catalogue number AC-15, Abeam™) was used at a 1:5000 dilution as the equal loading control. Phospho-TrkA (Tyr490) antibody (9141, Cell Signal™) was used at a 1:500 dilution in 5 % BSA lx TBS-T. Following incubation with primary antibodies, the PVDF membranes were washed with TBS-T (3 x 5 min) and then incubated with respective HRP -tagged secondary antibodies at room temperature for 1 h. Anti-rabbit secondary antibody (NA934-1ML, Cytiva™) was used at 1:3000 dilution in 5 % skim milk TBS-T and anti-mouse secondary antibody (NA931-1ML, Cytiva™) was used at 1:3000 in 5 % skim milk TBS-T. Membranes were then washed again with TBS-T (3 x 5 min) and cut horizontally using a scalpel to separate the loading control bands from the TrkA bands. After removing the TBS-T buffer from the container, 1 ml of HRP substrate (Immobilon Forte Western HRP™ substrate) was added onto each membrane. TheHRP substrate was pipetted up and down to bath the membranes for 1 min and the strips were imaged separately for their chemiluminescence on Azure SAPPHIRE™ biomolecular imager.

[0297] After imaging, the PVDF membranes were first washed twice (2 x 10 min) with Abcam’s mild stripping buffer at room temperature. Washed twice in PBS (2 x 10 min), followed by TBS-T (2 x 5 min). The membranes were then blocked with fresh blocking buffer (5% skim milk in TBS-T) for 1 h at room temperature, washed again with 5 mL lx TBS (3 x 5 min) and incubated with respective primary antibody solution as described above.

[0298] KM12 cell lysate CETSA

[0299] KM12 cell were sub-cultured in T75 flasks and harvested as described above. Pellets were lysed in lx TBS buffer (20 mM Tris-Cl, pH 7,5; 150 mM NaCl) supplied with PIC, and a KM12 lysate master mix was prepared where the total protein concentration was in the range between 2.1- 2.5 pg / pL. Lysate aliquots were then dispensed in 0.2 mL PCR tubes, where 18 pL of lysate and 2 pL of inhibitor solutions (10 pM, 0.1% DMSO in buffer) or controls (0.1% DMSO in buffer, or buffer alone) were added for a total volume of 20 pL. The resulting lysate mixtures were left to incubate at room temperature for 30 min prior to heat treatment. Samples were then heated for 3 min at a single elevated temperature (ranging from 37 to 69°C) using a DNA Engine Dyad Peltier Thermal Cycler (Bio-Rad™). Upon completion, the contents were placed on ice for two minutes to quench the reaction, then subsequently transferred into 1.5 mL Eppendorf™ tubes and centrifuged at 20000 g for 20 min at 4°C. The supernatant containing the soluble protein fraction (10 pL) was then removed and prepared for SDS-PAGE separation by adding 2.5 μL of loading buffer (Tris-Cl / SDS, glycerol, β-mercaptoethanol, bromophenol blue, pH 6.8) and heated at 95°C for 10 minutes. Each sample (12.5 pL total volume) was separated by SDS-PAGE on a 10% polyacrylamide gel and transferred onto PVDF membranes for Western blot analysis as described. Membranes were stained with primary antibodies for TrkA as well as the thermally-durable SOD1 loading control.

[0300] Band intensity for both TPM3-TrkA (~70 kDa) and SOD1 were subsequently analyzed by densitometry using ImageJ™ (National Institutes of Health™). Ratios between TPM3-TrkA and SOD1 loading control for each lane, were then normalized to the TPM3-TrkA: SODl ratioat 37°C which was set to 100%. To derive Taggvalues for PTSA or CETSA aggregation curve experiments, non-linear regression fit was performed using the Boltzmann sigmoidal model and the VC50 estimate was calculated as a Taggusing the formula:

[0301] (Top - Bottom)

[0302] y = Bottom +

[0303]

[0304] HA-TrkA MDA-MB-231 Western Blot

[0305] Cells were lysed in buffer (HEPES 40 mM, pH 7.5; EDTA 1 mM pH 8.0; NaCl 120 mM; 10 mM; NaPPi 50 mM; NaF 50 mM; Na₃VO₄ 1.5 mM; Triton-X100 1% (v / v); sodium lauryl sulfate (SDS) 0.1% (v / v); PMSF 1 mM; protease cocktail inhibitor 1% (v / v); glycerol 10% (v / v)) and then frozen (12 h, - 80 °C). The lysates were recovered by scraping and centrifugation (18 300 g; 10 min; 4 °C). Protein extracts in Laemmli buffer 5X (Tris HCl 63 mM; glycerol 10% (v / v); SDS 2% (w / v); p-mercaptoethanol 5% (v / v); bromophenol blue 0,025% (v / v); pH 6,8) were loaded (40 pg / well) and separated on polyacrylamide gel (7.5%, 180 V constant, lhl5). Proteins were transferred (Tris 25 mmol / L, glycine 192 mmol / L, methanol 15% (v / v), H₂O) onto PVDF membranes (100 V, 1.5 h). The membranes were saturated (1 h, 20 °C, shaking) in TBS- 0.1% (v / v) Tween-20 (TBS-T) with 5% (w / v) BSA. Then they were incubated with the primary antibodies diluted in the saturation buffer (BSA 5% (w / v), 16 h, 4 °C, shaking). The following primary antibodies were used: anti-p-Actin (Sigma-Aldrich™, A2066, rabbit), anti-pTrkA (Tyr674 / 675, Cell Signaling™, 9141, rabbit), anti-HA (Biolegend™, 901513). After washing (TBS-T 0.1% (v / v), 5 x 5 min, 20 °C), the membranes were incubated with the secondary antibodies (HRP-linked Anti-rabbit, Goat, 7074, Cell signaling or HRP-linked Goat anti-mouse IgG, 115-035-174, Jackson Immunoresearch™) diluted in TEST (0.1% (v / v)). After the washing step, the chemiluminescence reaction was carried out (West Pico, ThermoScientific™) and photons were detected in the darkroom using a camera (Fusion FX™ spectra, Vilber™).

[0306] In vitro Pharmacokinetic Profiling Experiments

[0307] ADME profiling studies with synthesized compounds were conducted through the ADME-Tox commercial service, outsourced Eurofins DiscoverX™ Corporation, St. Charles, US. In each experiment and if applicable, the respective reference compounds were tested concurrently with PI-8 and PI-15R, and the data were compared with historical valuesdetermined at Eurofins™. Metabolic stability profiling experiments were performed as previously described82, incubating compounds at a final concentration of 0.1 pM with human liver microsomes (0.1 mg / mL) at 37°C. HPLC-MS / MS sampling was used to monitor test compound concentration at 15 min intervals for 60 min. Protein plasma binding (ppb) was measured using equilibrium dialysis in a 96-well block where HPLC-MS / MS was used for analyte detection following 4 h of incubation in blood plasma at 37°C. Test compound and controls were used at concentration of 10 pM and their respective ppb bound % was determined against phosphate buffer (0.05 M sodium phosphate in 0.07 M NaCl, pH 7.5) as described previously83. Cell permeability was assessed ex vivo assay using a layer of Caco-2 cells grown on collagen-coated polycarbonate membranes was performed at 37°C as previously described84. Test compounds or drug controls were incubated at concentration of 10 pM in the starting, donor chamber. Using HPLC-MS / MS the concentration of the corresponding test compound in both donor (A) and receiver (B) chambers were determined across two separate timepoints. For A-B chamber permeability experiments compound concentrations were assessed at 0 and 60 min with (Caco-2, pH 6.5 / 7.4) conditions being used for compound PI-8 and (Caco-2, pH 7.4 / 7.4J conditions used for compound PI-15R respectively.

[0308] Statistical Analysis

[0309] Statistical analysis was performed using GraphPad Prism™ 10.0.0. For each corresponding CETSA experiment, Taggvalues from two independent experiments were obtained and averaged (mean reported as final Taggestimate), where the standard deviation (SD) between those two Taggnumbers was reported as error value. To compute statistical significance, in KM12 cell experiments, ECso, pTrkA / TrkA ratios and Taggestimates for each corresponding treatment together with their SD values, were subjected to comparison to the DMSO control values using ordinary one-way AN OVA analysis with Dunnett's multiple comparisons test. Refer to supplementary information for n values of repeated measurements. A p-value of <0.05 was considered statistically significant: p<0.05 (*), p<0.01 (**), p<0.001 (***), p<0.0001 (****).EXAMPLES

[0310] EXAMPLE 1: STAT3 DSF High-throughput screen (HTS)

[0311] The STAT3 DSF assay is a thermal stability assay, which uses recombinant STAT3¹²⁷⁻⁶⁸⁸ (coiled-coil to SH2 domain), and tests ligands for their ability to directly interact with STAT3 and impact its melting temperature (Tm) 34. To adapt the existing STAT3 DSF protocol for HTS, a master mix of protein and SYPROTM Orange™ was prepared and added to 384-well PCR plates loaded with test compounds (0.05 pL in DMSO, final concentration of 50 pM). Following room temperature incubation for up to 3 h, plates were heated from 45 to 80 °C (ramp rate 4.8 °C per min) while fluorescence at (ex.465 and em. 580 nm) was acquired 10 times per °C. The known STAT3-binding, gpl30 phosphopeptide sequence (Ac-pYLPQTV-NH2, 500 pM (SEQ ID NO.:1) was used as a positive control which induced a ~6.3 °C shift in Tm compared to the vehicle (DMSO) control (FIGURE 1A). In total, ~ 33 000 distinct chemical entities were screened in the STAT3 DSF assay, consisting of diverse compounds from the primary screening set at the Chemical Biology Consortium of Sweden (CBCS™). Tm values were determined from the inflection point of the S-shape melt curve, produced by plotting fluorescence versus temperature. The peak center of the resulting first derivative were used as readouts of STAT3127-688 melting temperatures (Tm). From the screen, 90 primary hits were identified with ATm values greater than 3-times the standard deviation for the screen. These compounds were then subjected to follow-up confirmation assays in dose-response (10, 50 and 100 pM) and those that showed reproducible and dose-dependent stabilization of STAT3127-688 were considered for further analysis. Among the compounds that demonstrated a dose-dependent stabilization of STAT3127-688, three hits containing a 6-aryl-N2-(1-phenylethyl)pyrazine-2,3-diamine inhibitor (PI) scaffold were identified as moderate stabilizers of STAT3 (ATm 0.9 to 1.4 °C), which motivated an initial round of hit expansion and further binding validation experiments with this scaffold (FIGURES 7 and 8).

[0312] EXAMPLE 2: Preliminary Hit Expansion of the PI Scaffold

[0313] Compound 1 was selected as a top hit and expanded into 10 structural analogues to perform an initial SAR study. Compounds 1-10 were subsequently obtained via 2-step synthesis starting from 2-Amino-3,5-dibromopyrazine, diversifying the moieties appendedto the 5-aryl substituent (Scheme 1). Briefly, a microwave-assisted, region-selective SNAr reaction introduced the benzylamino group at the 3-position of the pyrazine starting material to furnish intermediate 1a. Diverse aryl groups were then added using a Suzuki cross-coupling reaction to furnish the 6-position of the 2,3-diaminopyrazine ring and produce the desired final compounds (FIGURE 1C). Compounds were purified using automated flash chromatography (Biotage Isolera™) and their identities were confirmed using analytical LCMS (Agilent InfinityLab™ G6125B) and 1H and 13C NMR (Bruker Ascend™). As an initial hit expansion, ten 6-aryl-2,3-diaminopyrazine compounds were generated, which were subsequently tested according to the target engagement-focused screening funnel described in FIGURE 1B.

[0314] Scheme 1. Synthesis of Compounds 1-10a

[0315]

[0316] aReagents and conditions (a) DIPEA, n-Butanol, pW, 180 °C, 16 h; 82 % (b) K2CO3, Pd(dppf)2C12, Toluene / EtOH (2:1), 90 °C, 45 min; 24-87 %

[0317] EXAMPLE 3: Hit Validation and Target Confirmation

[0318] DSF validation experiments at 3-doses with analogues 1-10 yielded inconclusive results regarding their ability to bind STAT3127’688and induce thermal stabilisation. Overall, a dose-dependent reduction of the fluorescent signal was observed at higher temperatures when the protein was treated with 8 out of 10 compounds. This produced flattened fluorescent curves, yielding a sub-maximal fluorescent response compared to the vehicle control. Control experiments heating compounds with SYPRO Orange™ alone revealed these ligands did not increase fluorescence in DSF in the absence of STAT3. This suggested that the dose-dependent reduction of the fluorescent signal observed with STAT3-treated compounds is dependent on the presence of the protein. However, this effect was not observed for the gpl30 phosphopeptide positive control, which produced comparablefluorescent intensity to the controls and stabilised STAT3 against thermal stress (data not shown). Notably, the decreased fluorescence at high temperatures, seemed to contribute to positive shifts of STAT3127'688Tm, however the distortion of the melt curves questioned the reliability of these results, preventing accurate conclusions about compound binding. To interrogate whether these compounds bound to STAT3127'688at its SH2 domain, a further truncated STAT3 protein fragment was used which lacked the SH2 domain (STAT3127’465). DSF experiments with STAT3127'465demonstrated no significant stabilisation of the truncated STAT3 isoform (data not shown). However, compounds 1, 4, 5, 7, and PI-8 exhibited variations in the fluorescent curves between the short and long forms of STAT3, where the melt pattern appeared more flattened with STAT3127'688, containing the SH2 domain. We hypothesized the observed reduction in fluorescence with these compounds could be mediated by a potential interaction between the SH2 domain, which warranted further investigation with orthogonal STAT3 SH2 domain binding assays. The STAT3 FP assay was subsequently employed to assess SH2 domain binding of compounds 1-10, however no binding to the SH2 domain was observed (data not shown), indicating that these compounds do not act as STAT3 SH2 domain binders.

[0319] Finally, a single-dose, isothermal STAT3 protein thermal shift assay (PTSA) screen was conducted to determine whether compounds 1-10 could stabilise STAT3 against thermal aggregation in a dye-free setting (data not shown). When the protein was probed with 100 pM of the test compounds and heated at 57 °C for 8 minutes, only the gpl30 phosphopeptide successfully rescued STAT3127'688from thermal aggregation. On the contrary, the compounds that displayed distorted melt curves by DSF appeared to reduce STAT3127’688stability when analyzed by PTSA, resulting in less STAT3127'688signal. While this was not the expected finding, it suggests that STAT3127'688aggregation is increased when these compounds are added, which may explain the fluorescence reduction in the DSF experiments, where compound treatment could facilitate protein aggregation at high temperatures. Thus, our hit validation experiments assessing direct binding to recombinant STAT3 in biochemical settings invalidated the ability of these compounds to bind and stabilize STAT3 against thermal stress and we opted to investigate the impact of these compounds on cellular STAT3 activity using a luciferase reporter assay.Three compounds from the PI series were probed at 20 pM in a luciferase reporter assay, which investigates whether compounds can block STAT3-dependent gene expression in U3A-luciferase reporter cells. Briefly, compound-treated cells (or vehicle controls) were exposed to oncostatin M to activate the STAT3 signaling cascade, resulting in luciferase expression. After 6 hours incubation, the luciferase substrate, luciferin, was added and the produced luminescent output was determined as a measurement of STAT3 transcriptional activity. Pretreatment with compounds 1, PI-8 and PI-10, resulted in modest inhibition of STAT3-dependent luminescence compared to the vehicle-treated control, ~ 40% at 20 pM (FIGURE 1D and 1E). With blockade of STAT3-dependent gene expression in cells, but insufficient evidence that these ligands bind directly to STAT3, we hypothesised that the mechanism of action could be due to off-target inhibition of other proteins that act within the STAT3 signalling cascade. Additionally, previous reports on similar structures have claimed close structural analogues inhibit lactate dehydrogenase43or acted as kinase inhibitors44-46, suggesting that upstream kinase inhibition could be a plausible cause of decreased STAT3-dependent gene expression in the luciferase assay.

[0320] EXAMPLE 4: Kinome Profiling Experiments Revealed Potent Tyrosine Kinase Inhibition

[0321] To screen for potential kinase affinity, PI-8 was probed at 25 pM in the scanEDGE kinase assay (Eurofins™, San Diego), which assessed its competitive binding to 97 kinases in a cell-free setting47. At the relatively high concentration of 25pM, PI-8 exhibited binding to multiple tyrosine kinases. Notable hits (70 - 90 % binding inhibition) included ALK, CSF1R, FAK and KIT, where top kinase hits (>90% inhibition) were identified as ABL1, JAK2, Src and TrkA. Among these, TrkA binding was nearly completely blocked by PI-8, resulting in only 0.1% TrkA signal compared to controls (> 99.9% inhibition) (FIGURE IF).

[0322] Follow-up dose-response experiments with the top 4 kinase hits from this screen were then conducted to determine the dissociation constants (Kd) of PI-8 for those enzymes revealing PI-8 was a nanomolar binder of TrkA with a Kd value of 36 nM, showing selectivity for TrkA over, Src (Kd = 640 nM), JAK2 (Kd = 1100 nM) and ABL1 (Kd = 5300 nM), (FIGURE 1E). As such, compound PI-8 was uncovered as a potent TrkA inhibitor, with good selectivity for TrkA over non-Trk kinases. Notably, the affinity for Src and JAK2kinases likely contributed to the impairment of STAT3-dependent gene expression in the luciferase assay, as both of these kinases are known to phosphorylate STAT3.

[0323] We also investigated if TrkA inhibition contributes to impairing STAT3 activity in the luciferase reporter assay using the clinically approved panTrk inhibitor larotrectinib, (TrkA IC50 = 6.5 nM, TrkB IC50 = 8.1 nM, TrkC IC50 = 10.6 nM) showing known off-target inhibition against TNK2 (IC50 = 576 nM)48. No significant effect on STAT3-dependent gene expression was observed with larotrectinib concentrations as high as 20 pM (data not shown). These data support that the PI-8-mediated inhibition of STAT3 activity was not from targeting TrkA, but likely other upstream kinases such as JAK2 and Src. While TrkA has been linked to STAT3 signalling in previous studies,49-51our experiments clearly demonstrate TrkA does not play a significant role in regulating STAT3 transcriptional activity under these specific conditions.

[0324] Although the data supporting PI-8 inhibited STAT3 activity through indirect inhibition were not the findings we had initially hoped for, we surmised that its high affinity for TrkA over other kinases favorably-positioned this hit for further development as a TrkA-targeted inhibitor. Exploring the novelty of this scaffold as a kinase inhibitory pharmacophore, structure-based searches associated the 2-aminopyrazine heterocycle with structural elements of ligands targeting NEK244, FLT345, and MK246kinases, with reported IC50 values of 0.87, 0.07, and 0.75 pM, respectively. In contrast, compound PI-8, in a Eurofins KINOMEscan™ kinase screen probing PI-8 at 25 pM against 97 kinases showed low activity against FLT3 and MK2 (MAPKAPK2) inhibiting these targets by 17% and 11%, respectively, while showing 90.9% and 93.2% inhibition of ALBI nonphosphorylated and phosphorylated kinases, respectively, 96% inhibition of JAK2, 97.5% inhibition of SRC, and 99.9 % inhibition of TRKA (data not shown). A carboxylic acid derivative featuring a 6-aryl-2,3-diaminopyrazine moiety, akin to compound 2, has demonstrated inhibition of lactate dehydrogenase A (LDHA) within the micromolar range (LDHA IC50 = 4 pM)43.

[0325] While the 2,3-diaminopyrazine core has been reported in inhibitors against various enzymatic targets, its activity as potential TrkA inhibitors has notyetbeen explored.

[0326] Furthermore, aligning with our target engagement-focused drug discovery approach, we shifted our research efforts toward optimizing this scaffold as a TrkA inhibitor and exploring its anti-cancer effects within the context of NTRK1 fusion-positive cancer.EXAMPLE 5: Pivoting to Optimizing the PI Scaffold for TrkA Inhibition

[0327] The TrkA kinase domain consists of two lobes connected by a disordered motif known as the " Hinge”, where its active site harbours a conserved DFG motif, consisting of aspartate (D), phenylalanine (F), and glycine (G). The DFG motif is crucial for its functionality and serves as a regulatory switch (FIGURE 6B). The " DFG-in” conformer, where the D668 residue is orientated inward towards the ATP-binding pocket, signifies an active state that enables optimal substrate binding and catalytic activity. Trk Type I inhibitors (ATP-competitive), including clinically approved larotrectinib and entrectinib, have demonstrated impressive results in the clinic, yet acquired resistance and lack of Trk subtype selectivity present current challenges that motivate further development of TrkA inhibitors52'53. Point mutations in TrkA’s active site (e.g. G595R, G667C or F589L) are known to ablate the binding affinity of Type I Trk inhibitors54. Molecular dynamics simulations have indicated changes in the active site xDFG residues cause Trk proteins to adopt a preference for the " DFG-out” conformation, which increases affinity for Type II Trk inhibitors55. Unlike, Type I Inhibitors which bind the " DFG-in” conformer, Type II inhibitors preferentially bind to the inactive " DFG-out” Trk state. In the latter conformation, a hydrophobic "back pocket” is observed adjacent to the conserved DFG motif, which creates a cavity deeper into the active site. Type II binders are known to occupy this less-homologous site, while also making interactions with the kinase active site, allowing for retained affinity towards Trk mutants and offering also some Trk-subtype selectivity.

[0328] Ensuring TrkA selectivity over other Trk-subtypes (TrkB and TrkC) is a desired inhibitory profile as indiscriminate inhibition of Trks can contribute to adverse side effects associated with pan-Trk inhibition, such as dizziness, weight gain, and paresthesia56. Lack of selectivity is a particular obstacle for type I inhibitors due to the high degree of structural similarity shared between the Trks (TrkA's kinase domain exhibits 86% similarity to TrkB and 83% to TrkC). Larotrectinib indiscriminately blocks the activity of all Trk subtypes as a pan-Trk inhibitor, whereas entrectinib also has similar inhibitory activity across the Trk family (TrkA ICso = 1 nM, TrkB ICso = 3 nM, TrkC ICso = 5 nM) and has additional off-target kinase activity against ROS1 and ALK1 (ICso values 7 and 12 nM, respectively)57.

[0329] Developing Type II and Type III Trk inhibitors has recently gained more traction in the fielddue to their potential to retain affinity towards Trk xDFG mutants and offer higher selectivity towards Trk-subtypes, respectively58'59. These advancements in the field have highlighted how understanding the binding mode of Trk inhibitors, can not only help guide binding optimization efforts but also support the prediction of selectivity, which can influence the efficacy and side effects profile of lead compounds. Since the KINOMEscan™ assay investigates active-site-dependent competitive binding, we hypothesised that PI-8 can retain binding towards multiple kinases by engaging a conserved motif between these hit kinases, such as the hinge or DFG sequences in the active site. Docking experiments into the active conformation of JAK2, Src and TrkA were next conducted with compounds 1-10 in efforts to obtain insights about the proposed binding poses and interactions within these enzymes’ ATP-binding sites.

[0330] EXAMPLE 6: In Silico Molecular Docking with JAK2, Src and TrkA Suggest the PI Series Act as Type II Inhibitors

[0331] GLIDE™ (Schrodinger™, Maestro™) docking was performed using protein crystal structures for TrkA (DFG-in pdb: 5KVT, DFG-out pdb: 6PMB), JAK2 (DFG-in pdb: 4AQC, DFG-out pdb: 3UGC) and Src (DFG-in pdb 4U5J, DFG-out pdb: 3EL7). Compounds 1-10 exhibited lowest docking scores for TrkA, predicting stronger binding affinity for TrkA compared to Src and JAK2 in the DFG-in systems (FIGURE 15). When comparing the GLIDE™ docking scores (i.e. TrkA conformational states DFG-in v. DFG-out) for compounds 1-40 (i.e. R and S enantiomers of 1-22 and 24-32) are shown FIGURE 16. Compound 7, having an isopropyl ether has interesting docking profile where the S enantiomer is more potent predicted binder than the R enantiomer. Also, for compound 13, the 3,5-dimethyl gave a much better TrkA docking score for DFG-in than DFG-out, whereby this may be a sign that the compound is more selective than other compounds. There was a trend in the observed pattern in Ka values during the KINOMEscan™ assay, with PI-8’s Ka values decreasing in the order of JAK2 > Src > TrkA, where PI-8 demonstrated docking scores of -4.9, -7.8, and -9.8, respectively. This result indicates a strong correlation between the results of the kinase binding experiments and the computational docking model and motivated further docking experiments in the active site of TrkA. We next docked compounds 1-10 into the DFG-out conformers of the three kinases to gather insights on how this series of compounds couldbe acting in the active site (Type I or Type II binding mode). For TrkA this was done using a " DFG-in” protein co-crystal structure of TrkA hosting the Type I inhibitor entrectinib and a " DFG-out” conformation of TrkA bound to a Type II inhibitor, respectively60. As expected, entrectinib demonstrated better docking scores in the DFG-in active site compared to the " DFG-out” orientation (-12.6 versus -8.7, respectively). Moreover, compounds 1-10 all showed better GLIDE™ docking scores into the " DFG-out” conformers suggesting this scaffold may engage in a Type II mode (FIGURE 2A and B).

[0332] Seven out of the ten ligands demonstrated lowest docking scores for TrkA. The docking model predicted that compounds 1-10, which feature an altered aryl substituents on the 6-position of the 2,3-diaminopyrazine ring, orient this aromatic group towards the solventfrontin the " DFG-out” conformer. Hydrogen bonding between the diaminopyrazine moiety and residues in the hinge motif of TrkA were observed for all docked ligands. For instance, PI-8’s docking pose suggested the dimethylaniline ring is solvent exposed with the pyrazine heterocycle sitting in a tight hydrophobic cleft, forming hydrogen bonds with residues Met592 and Glu598. The modelling suggested the benzylamino group of compounds 1-10 was situated deeper into the active site forming hydrophobic interactions with gatekeeper Phe589 and the DFG motif, where the DFG-adjacent hydrophobic pocket was seen unoccupied. These modelling insights observed across the series of PI-8 analogues were incorporated into ligand modifications, aimed at exploring the scaffold’s SAR towards TrkA inhibition. The " DFG-out” docking pose of PI-8 was among the top scoring poses from this series and inspired further structural modifications altering the dimethylaniline ring, which was predicted to interact with the hinge motif of TrkA.

[0333] EXAMPLE 7: SAR and Antiproliferative Effects of Top Binders in TPM3-TrkA Fusion Cancer Cells

[0334] Compounds 11-22 were produced via the previously reported 2-step synthesis starting from 2-Amino-3,5-dibromopyrazine, increasing the diversity of substituents appended to the aryl group at the 6-position of the 2,3-diaminopyrazine ring. The a-Me benzylamino moiety remained unchanged as the racemate, mirroring compounds 1-10, encompassing 22 compounds total that modified the 6-aryl ring of the pyrazine scaffold (herein grouped under Series I) (FIGURE 2C). This series of analogues were then investigated for theirTrkA binding affinity using Eurofins’ KINOMEscan biochemical assay47. Additionally, inhibition of TrkA activity using BPS’ TrkA assay kit was assessed. Compounds were tested at two concentrations (100 nM and 10 nM), which were selected to gauge higher or lower binding affinity of the compounds in correspondence to PI-8’s Ka value. Nitrogen substitution on the meta position of the 6-aryl ring, as well as polar substituents, such as -NO2, -N(Me)2 and -CONH2, were shown to improve binding compared to unsubstituted benzene. On the contrary, attaching small polar or non-polar substituents at this position, such as -OH, -CF3, -Cl, -F and -Me did not improve binding. Overall, aryl ring modifications with carbonyl-based functional groups resulted in the strongest binding, where adding polar groups 3-NHCOCHs (PI-15) and 4-C00H (22) yielded the most active compounds in this series.

[0335] Next, we explored modifications on the benzylamino group (Series II), which was predicted to interact with the DFG-motif in TrkA (FIGURE 3A), aiming to investigate the stereochemistry and significance of the a-methyl group as well as substituents on the aryl ring. Compounds 23-39 were obtained via the same synthetic route where a region-selective SNAr reaction introduced substituted benzylamines at the 3-position of the pyrazine ring, and a Suzuki cross-coupling reaction attached the 3-dimethylaminophenyl group at the 6-position of the 2,3-diaminopyrazine ring. Screening this second series of analogues in both the TrkA binding and enzymatic activity assays revealed smaller alkyl substituents, such as -Me were preferred over bulkier groups like -‘Bu and -Et on the a-benzylamine position. Removing the a-methyl group did not improve the inhibitory activity and most notably the -stereoisomer of PI-8 (PI-8R) was much more active than the S-stereoisomer (PI-8S). Overall, adding substituents on the benzylamino aryl ring did not result in an increase of its binding potency, though attaching a 3-OMe group (28) or substituting the meta carbon atom for nitrogen atom to form a 3-pyridyl ring (38) were shown to be tolerated modifications. With high affinity TrkA binders being identified among the two series of synthesized analogues, we next sought to evaluate the Trk-subtype selectivity of 3 of the top performing binders, while also examining inhibitory activity towards JAK2 and SRC.

[0336] The Kd values of compounds PI-8, PI-10, and PI-15, for TrkA, TrkB, and TrkC, respectively, were determined using the KINOMEscan™ assay (Eurofins™). Modest TrkA selectivity wasdetermined for PI-8 over other Trk subtypes (TrkA Kd = 52 nM, TrkB Kd = 150 nM, TrkC Kd = 470 nM). While PI-10 did not show any significant binding preference towards any of the Trk subtypes, top binder PI-15 also displayed TrkA selectivity over TrkB and TrkC with Kd values of 31, 60 and 240 nM, respectively (FIGURE 3B). IC50 values for PI-10, PI-15, and compound PI-8R were determined for JAK2 and SRC using the Z'-LYTE61kinase activity assay (FIGURE 3C). All three compounds demonstrated moderate inhibition of JAK2 with selectivity over SRC, with PI-15 showing highest activity for JAK2 (IC50 = 361 nM) compared to SRC (IC50 = 1560 nM). Compound PI-10 showed reduced JAK2 inhibition (IC50 = 663 nM) but displayed improved selectivity over SRC (IC50 = 4520 nM). In contrast, compound PI-8R exhibited lower selectivity between JAK2 and SRC, with IC50 values of 530 nM and 1110 nM, respectively

[0337] These data suggest that attaching -N(Me)2 and -CONH2 groups at the meta position can introduce some selectivity for TrkA over other Trk subtypes, while also contributing to selectivity for JAK2 over SRC. Conversely, the incorporation of a 3-pyridyl ring provided the highest selectivity for JAK2 over SRC but did not enhance selectivity for TrkA over other Trk subtypes. The docking pose of PI-15 suggested H-bonding between the acetamide aryl group and residue Arg599 from the Hinge motif of TrkA. Arg 599, which is a nonconserved TrkA residue substituted for Lys 599 in TrkB and TrkC’s, is solvent-exposed and predicted to sit adjacent to the 6-aiyl 2,3-diaminopyrazine ring by the computational model (FIGURE 10). While H-bonding between PI-8, PI-10 and Arg599 was not predicted, this TrkA-distinct residue might contribute to the modest TrkA selectivity observed with PI-8 and PI-15 in the TrkA biochemical assay. Overall, these screening efforts helped forge a robust SAR study, where tolerated modifications were identified and analogues with improved binding were discovered. Compounds PI-8, PI-10, PI-15, PI-17, 20, 22, PI-8R, 28 and 38 were revealed as top binders of TrkA in biochemical settings.

[0338] We next sought to investigate whether the observed binding affinity of these compounds would translate to cellular inhibition of TrkA activity in cancer cells. The KM12 (male, colon cancer) cell line harbours a TPM3-TrkA fusion kinase arising from an NTRK1 gene translocation. These cells are sensitive to TrkA inhibition and are routinely used to study TrkA inhibitors62. LS174T female, colon cancer and MDA-MB-231 female, breast cancer which are not known to possess Trk fusions, were treated with top TrkA binders toinvestigate the selectivity of compounds for cell lines possessing the Trk fusions.

[0339] Larotrectinib (a pan-Trk inhibitor) was used as a positive control where it potently blocked KM12 cell viability (IC50 = 3.8 ± 2.3 nM), but showed no significant effect on the viability of LS174T and MD-MB-231 cells, even at concentrations as high as 100 pM over a 72-hour treatment period (FIGURES 3D, E). Compounds with the highest activity from the biochemical screen were assayed alongside larotrectinib and Compound 1 as positive and negative controls, respectively. Top binders PI-8, PI-10, PI-15, PI-17 and PI-8R, effectively inhibited KM12 cell viability displaying EC50 values below 300 nM (FIGURE 3D). PI-8, PI-10, and PI-8R demonstrated similar EC50 values at ~200 nM. Top inhibitors of KM12 cell viability were determined to be PI-17 and PI-15, exhibiting EC50 values of 111 ± 50 nM and 38 ± 15 nM, respectively. Compounds 1, 22 and 38 displayed poor EC50 values of 5.3 ± 1.9 pM, 4.1 ± 0.8 pM and 3.7 ± 0.2 pM, respectively. Compound 1, which was one of the least active TrkA binders expectedly did not display strong activity in the KM12 cell viability assay. Analogues 22, and 38, which feature polar groups - pyridyl and carboxylic acid -exhibited TrkA inhibition at 100 nM in the biochemical assay, showing inhibition percentages of 56%, and 31%, respectively. However, their effects on KM12 cell viability did not correlate with these values, as their EC50 values were in the micromolar range. This discrepancy between TrkA binding and cell viability may be due to differences in membrane permeability as an example. The oncogenic TPM3-TrkA fusion kinase that drives the malignant phenotype in KM12 cells is cytosolic and accordingly, high affinity TrkA binders that exhibit low membrane permeability would not be expected to have strong activity.

[0340] Top compounds from the KM12 cell viability assays, PI-8, PI-10, PI-15, and PI-8R, displayed minimal inhibitory activity against the non-TrkA-driven cancer cell lines.

[0341] Compound PI-8 exerted the highest activity against MDA-MB-231 and LS174T cell viability (IC50 values of 10 pM and 12 pM, respectively), but still demonstrating a 50 to 60-fold selectivity for KM12 cells harboring the TPM3-TrkA fusion mutant. Compound PI-10, which showed even less activity against MDA-MB-231 and LS174T cells (cell viability of 43% and 83% at the maximum tested concentration of 100 pM, respectively) displayed an IC50 value of 186 ± 12 nM against KM12 cells, exhibiting excellent selectivity for the TrkA-driven cancer cell lines. These results suggest that the Pl-compounds exhibit selectivitytoward the TPM3-TrkA mutant KM12 cells which is likely attributed to their more potent activity towards TrkA over other kinases. This is in contrast with larotrectinib's highly specific activity in KM12 cells and lack of effect on LS174T and MDA-MB-231 cell viability. The minimal but measurable effects observed in LS174T and MDA-MB-231 cells with the PI compounds could be due to co-inhibition of JAK2 and SRC, which may also be potentially beneficial in a cancer therapeutic context due to the involvement of JAK2 and SRC in many forms of cancer.

[0342] EXAMPLE 8: Inhibition of TPM3-TrkA Phosphorylation and Target-engagement Confirmation via CETSA

[0343] With top binders exhibiting selective inhibition of KM12 cell viability, we next aimed to investigate the effects of these inhibitors on the phosphorylation levels of TPM3-TrkA. Cells were treated for 24 h with increasing doses of compounds followed by cell lysis and Western blot analysis for phosphorylated TrkA (pTrkA) at Tyr490, a major autophosphorylation site of TPM3-TrkA (FIGURE 4A, B). Compounds PI-8, PI-10, PI-15, and PI-17 were probed against 22 and larotrectinib, which were used as negative and positive controls, respectively.

[0344] Notably, PI-8, PI-15 and PI-17 displayed potent inhibitory activity, showing concentrations as low as 123 nM were sufficient to almost completely suppress the pTrkA signal. While we cannot rule out co-inhibition mechanisms disrupting the survival programs in these cells, this data confirm the potent anti-proliferative effects in the NTRK1 fusion-positive cells are correlated with depletion of TPM3-TrkA phosphorylation. On the other hand, suppression of TPM3-TrkA phosphorylation was observed for all tested analogues except PI-10, which did not display reduced pTrkA signals at concentrations as high as 1.1 pM. The lack of observed pTrkA blockade of PI-10 despite demonstrating antiproliferative effects in KM12 cells and high binding affinity for TrkA in biochemical assays is curious, possibly suggesting that this compound binds to TrkA without inhibiting its autophosphorylation. However, further investigation would be required to confirm this hypothesis. Another possibility could be a synergistic off-target effect between modest inhibition (not statistically significant p = 0.22) of pTrkA and other kinases that may be targeted by PI-10 leading to impaired viability in KM12 cells, or simply the timingdifferences within these two assays may play a role in the discrepancies between TrkA phosphorylation (24 h) and cell viability (72 hours).

[0345] To validate whether these observed inhibition effects are due to the binding of TPM3-TrkA, target-engagement of TPM3-TrkA was evaluated using KM12 cell lysate CETSA. KM12 lysates were spiked with 1 pM of compounds and heated at 51 °C for 3 minutes to screen for their ability to rescue TPM3-TrkA from thermal aggregation (FIGURE 9). Among the tested set, PI-15-treated lysates showed the highest increase in soluble TPM3-TrkA, 49% of vehicle control, motivating follow-up CETSA experiments. Although other compounds demonstrated similar binding affinities to PI-15 with the recombinant TrkA protein in biochemical assays, PI-8 and PI-10 failed to stabilize TPM3-TrkA to the same extent at 1 pM in cellular lysates. It is possible PI-8 and PI-10 may not bind as tightly to TPM3-TrkA as PI-15, thereby limiting their ability to rescue the fusion kinase against thermal denaturation at 51°C. Additionally, the cellular environment contains thousands of proteins beyond the intended binding partner, raising the possibility of off-target interactions. Full melt curve CETSA experiments revealed PI-15 induced a 1.9° C increase of TPM3-TrkA’s Taggwhen probed at 1 pM (FIGURES 4C, D), confirming PI-15 successfully engages the TrkA fusion kinase in KM12 lysates.

[0346] Validating our top TrkA inhibitor's ability to bind and inhibit TPM3-TrkA phosphorylation in the cellular environment, we next sought to assess whether it exhibits similar inhibitory activity towards full-length TrkA which is upregulated in HER2+, as well as triple negative breast cancer (TNBC)63'64. Although TrkA has been shown to be overactive in breast cancer biopsies compared to normal tissues, its expression is notably low in widely used TNBC cell lines such as MDA-MB-23165, thus requiring transfection for deeper investigation. The induced overexpression of TrkA in both MCF7 and MDA-MB-231 cells has been associated with increased cell growth, migration, invasion, and survival properties, where blockade of this pathway with kinase inhibitors mitigate these effects66'67. These reports have offered a model for investigating the inhibition of full-length TrkA and highlighted a potential role for TrkA in promoting an aggressive phenotype in breast cancer. To that end, our top compounds PI-8, PI-10 and PI-15 were tested alongside larotrectinib for their ability to block TrkA phosphorylation in MDA-MB-231 (female, TNBC) cells transfected with HA-TrkA (FIGURE 4E, F). A marked decrease in the pTrkA levels was observed when MDA-MB-231 cells were treated with PI-15 at 250 and 500 nM, suggesting this compound can successfully block TrkA phosphorylation in this TrkA-overexpressing breast cancer cell model.

[0347] EXAMPLE 9: Stereochemistry and Preliminary in vitro Pharmacokinetic (PK) Evaluation

[0348] With several PI-8 analogues exhibiting potent TrkA binding, antiproliferative activity, and inhibition of TrkA phosphorylation in cells, we next sought to characterize whether there were differences in activity between the R- or S- enantiomers of the most active compounds. Thus, we synthesized Series III, to specifically explore the impact of the chirality at the benzylic position using the established synthetic route (Scheme 1), but using enantiopure a-Me-benzylamino starting materials. The R- and S-epimers of the top racemate TrkA binders: PI-8, PI-10, PI-15, PI-17, and compound 20 were produced and assessed fortheir inhibition of TrkA enzymatic activity at 10 and 100 nM using the Z’-Lyte assay (FIGURE 5A).

[0349] Notably, all R- isomers exhibited higher %TrkA inhibition than their S- counterparts, confirming that the R-stereochemistry contributes to enhanced activity, regardless of the 6-aryl ring substituents. At 100 nM, the R- enantiomers demonstrated 74-83% inhibition of TrkA, whereas the isomers displayed only 0-20% inhibition. These data align with the Series II biochemical screen results, suggesting that the R-enantiomers exhibit both improved inhibition and binding of TrkA (FIGURE 3A). While the R-Me enantiomers displayed higher activity than their S-counterparts, the latter retained some level of activity at 10 nM, suggesting the R-orientation is beneficial but not strictly required for targetinhibition at the tested concentrations. Among these, the R-Me enantiomer of PI-15 (PI-15R) emerged as the most potent inhibitor, displaying 63% and 83% inhibition of TrkA at 10 nM and 100 nM, respectively.

[0350] Follow-up profiling experiments with PI-15R provided further insights into its selectivity and potency across a broad panel of kinases including Trk subtypes (FIGURE 5B). Dose-response analysis using the Z’-lyte activity assay demonstrated PI-8R potently inhibited Trk kinases (ICso: TrkA = 23 nM, TrkB = 12 nM, TrkC = 9 nM) at lower concentrations than the racemate (PI-8, Kd = 36-470 nM, KinomeSCAN assay), while maintaining Trk-selectivity (IC50 = 9-23 nM) over JAK2 (530 nM) and Src (1100 nM). The top compound PI-15R exhibited even greater potency, with ICso values of 17 nM for TrkA, 8 nM for TrkB, and 5 nM for TrkC, while maintaining selectivity over JAK2 (ICso = 177 nM) and Src (ICso = 502 nM). Interestingly, both R-Me analogues displayed ~2-fold selectivity for TrkB and TrkC over TrkA in the Z’-Lyte assay, whereas the racemic mixtures showed a preference for TrkA in the KinomeSCAN™ binding assay (FIGURE 3B). Moreover, a broader kinome screen (i.e. Thermofisher Z’-lyte kinase screen) probing PI-15R at 2.5 pM against 100 kinase targets (FIGURES 11, 12) showed that PI-15R exhibited potent, TrkA-selective inhibition at low doses (25 nM). Expectedly, some selectivity was lost at higher concentrations, for example observed notable inhibition of JAK2 (76 %), ROS1 (67 %), and TXK (61 %) at 250 nM PI-15R, and broader activity at 2.5 pM (FIGURE 5C).

[0351] With PI-15R emerging as a potent single-digit nanomolar Trk inhibitor, we next sought to evaluate its in vitro PK properties in comparison to the initial hit, PI-8. In vitro PK profiling experiments assessed metabolic stability, plasma protein binding, and cell permeability of both compounds (FIGURE 5D). PI-15R demonstrated improved metabolic stability (ti / 2 = 105 min) compared to PI-8 (ti / 2 = 40 min). While both compounds exhibited high plasma protein binding, PI-15R showed slightly lower binding (95.7%) than PI-8 (99.6%).

[0352] Additionally, PI-15R displayed greater cell permeability through Caco-2 monolayers (8.0 x 10“6cm / s) compared to PI-8 (1.9 x 10“6cm / s). The higher measured protein binding of PI-8 indicates it may be more prone to non-specific interactions with other proteins in the lysate environment, potentially explaining its lack of TPM3-TrkA stabilization in KM12 cell lysates at 1 pM, whereas PI-15 induced thermal stabilization of TPM3-TrkA at the same concentration. Furthermore, PI-15R’s improved permeability may contribute to its stronger anti-proliferative activity in KM12 cells. These findings highlight PI-15R as a more potent and metabolically stable Trk inhibitor, warranting its distinction as a promising candidate for further translational development.

[0353] EXAMPLE 10: TRKA, JAK2, SRC Selectivity

[0354] TRKA, JAK2, and SRC selectivity was determined for compounds 1, 12, 24, 31, 33, 23, 19, 20, and 6 as a % inhibition at 250 nM and 2500 nM using the Thermofisher SelectScreen™ service (FIGURE 13), where all of the compounds showed greater inhibition of TrkA thanSRC and JAK2 and for compounds 1, 24, 31, 32, and 6 % inhibition of SRC and JAK2 was negative and mostly below 10% (except 2500 nM JAK2). Compounds 12, 33, 19, and 20 showed significant JAK2 inhibition and compounds 19 and 20 also showed significant SRC inhibition. Accordingly compounds 12, 19, and 20 show promise as multi-kinase inhibitors.

[0355] EXAMPLE 11: PI-15R Plasma Concentrations

[0356] Average plasma concentrations for PI-15R (10 mg / kg, SQ, (10%DMSO, 10%Kolliphor, PBS, 40 mM) are shown in FIGURE 14 where the compound was administered to three 10-week-old female Sprague-Dawley rats and blood samples were collected via tail-vein catheter over the course of three hours, with subsequent sample analysis proceeded via Liquid chromatography-mass spectrometry (LC-MS). The concentration in plasma peaks

[0357] ZI

[0358] at 1 hour post injection and declines after 2 hours. These plasma concentrations are useful K °

[0359] in determining the compounds potential as a therapeutic agent and for pharmacokinetic (PK) and pharmacodynamic (PD) modeling.

[0360] EXAMPLE 12: Summary of Compound Activities by Series

[0361] The compounds tested as represented by Formulas I and II are shown below with their respective activities in TABLE 1 (Series I) TABLE 2 (Series II), and TABLE 3 (Series III).

[0362] TABLE 1: TESTED COMPOUNDS - SERIES I

[0363] (EuroFins ScanElect™ is a KINOMEscan biochemical assay / BPS Bioscience TrkA Assay™) Compound Structure TrkA Inhibition (%)

[0364] 100 nM 100 nM i.250 nM (10 nM) ii. 2.5 pM Eurofins BPS TrkA Z’lyte ScanElect™ (Activity) (Activity) (binding)

[0365] PI-15 48% 94%

[0366]

[0367] PI-10 44% 95%

[0368] 22 56% 81%

[0369] ^ ^ JAA I i T [19%)

[0370] z z z z Z Z / / /

[0371] <z^

[0372] 20 H2N. AL 32% 86% i.81%

[0373] \=Z ii.95% HN xC N V^ A° 2 ~z=—. N x

[0374] Z —\

[0375] I

[0376] (A A25OT o °^

[0377] NO ZI \o o=z

[0378] PI-17 41% 67%

[0379] _zz

[0380] PI-8 < ( Z 2 Z z—— 36% 55%

[0381] ^^r\ £ £1 x

[0382] 3 H2NXXN. 28% 55%

[0383] AO2

[0384] 0^

[0385] 19 23% 38% i. 72% ii.93%

[0386]

[0387] H2N N 36%

[0388] 0

[0389] 28%

[0390] ? J?? i i \I, I r >^

[0391] z z z z Z Z z z / / / /

[0392] <z^

[0393] H2N N 2% 38%

[0394] JT T1

[0395] HN^N^^r ^^3pi

[0396] )o=

[0397] °°xx O\

[0398] CO

[0399] b 14%

[0400] \ )o c

[0401] \7\ 7

[0402] _zz

[0403] <zz— 14%

[0404] 4% 10% i. 27% ii. 50% 2% 21%

[0405]

[0406]

[0407] TABLE 2: TESTED COMPOUNDS - SERIES II

[0408] Compound Structure TrkA Inhibition (%)

[0409] 100 nM inhibitor Eurofins BPS TrkA ScanElect™ Activity Z z=

[0410] PI-8R 56% 100%

[0411] '? \ i i i T y z z z zz z- ^z z /

[0412] 28 H2N N 41% 66%

[0413] HIT

[0414] Z—ZZ—— / / /

[0415] ~z—.

[0416] PI-8 36% 55% 23 21% 79% 27 H2NX, N. 24% 73%

[0417] 0^

[0418] 38 31% 50%

[0419]

[0420] 24 H2NX, I\L 11% 58% "l w

[0421] 39 o 7% 51%

[0422] \ X

[0423] z z /

[0424] 37 H2N p^N. 6% 48%

[0425] 0 HIT r ^ITw YY \

[0426] Z—

[0427] >yj MZ —

[0428] \y

[0429] 36 H2N N

[0430] z — 5% 42% \zz-~ '

[0431] £

[0432] 29 11% 44%

[0433] ( z z—

[0434] ( o=

[0435] o

[0436] X

[0437] 32 H2N N 4% 46%

[0438] HN^IT

[0439] PI-8S 10% 40%

[0440]

[0441] 34 6% 35%

[0442] 33 T c

[0443] o H2N N

[0444] O 6% 26%

[0445] ' 'f? \ i \ I i i z z

[0446] M zz / i I z z y z z—

[0447] 35 4% 28%

[0448] p

[0449] Z—

[0450] z—

[0451] z—

[0452] Z—

[0453] 31 7% 17%

[0454] 30 10% 11%

[0455]

[0456] TABLE 3: TESTED COMPOUNDS - SERIES III

[0457] Compound Structure Z-lyte™ assay TrkA Inhibition (%)

[0458] 10 nM 100nM PI-15R H2N^NL 63 83

[0459] H I H QA M o

[0460]

[0461] PI-10R H2NX^N. 51 78 J- L JJ

[0462] PI-20R H2N., I\L 48 78

[0463] J i

[0464] p z z z z?——

[0465] HN N ^r^ A N /

[0466] IJAX'' IT

[0467] NO2

[0468] PI-8R 41 76

[0469] )Z°=— /

[0470] Z —\

[0471] PI-17R IZ 34 74 PI-20S H2(Nzz—., I\L 25 20

[0472] N°2

[0473] PI-IOS H2N N 5 20

[0474] HhAirA^N

[0475] PI-15S 8 12

[0476]

[0477] PI-17S 10 0

[0478] PI-8S 7 0

[0479] M Ji

[0480] z z z z J J——

[0481]

[0482] DISCUSSION

[0483] While many small-molecule Signal Transducer and Activator of Transcription 3 inhibitors

[0484] (STAT31) have been reported in the literature, the lack of clinical progress of such agents as anticancer therapies continues to drive innovation in drug discovery efforts within the STAT31 field. This study combined classic STAT3 inhibition assays with modern chemical biology techniques, such as STAT3 thermal stability assays, to execute a thermal-shift-focused screening funnel that enabled assessment of the binding profiles of test compounds in both biochemical and cellular contexts. Through this STAT3 drug discovery screening platform, we uncovered pyrazine-containing compounds that were rigorously explored and devalidated as direct STAT3 binders. However, our approach led to the serendipitous realization that these compounds acted as potent TrkA-targeted inhibitors, with additional moderate activity towards Src and JAK2 at higher concentrations. While our biochemical STAT3 thermal shift assays were inconclusive for direct STAT3 binding, the cellular luciferase reporter assay showed that these compounds weakly blocked STAT3 transcriptional activity and were pivotal for motivating kinome profiling experiments that identified their potent TrkA inhibitory activity.

[0485] The failure to validate STAT3 binding of hits from the DSF HTS may be due to non-specific binding interactions that are present at elevated temperatures in the DSF assay, which alter SYPRO™ Orange’s binding to decrease the dye’s fluorescent intensity. The HTS assay workflow used the first derivative plot of fluorescence versus temperature df / dT), which failed to detect the decreased fluorescence at high temperatures. As this analysis method only considers the temperature at which fluorescence observes maximal changes, itneglects the shape of the melt curve which can hold important information for STAT3 thermal stability, and possibly confound the determination of hits. By examining the raw fluorescence profiles closer, our subsequent dose-response DSF validation experiments offered a more insightful view, where compounds previously thought to bind STAT3 were, in fact, observed to reduce the dynamic range of fluorescence. Fluorophore-free thermal stability experiments hinted at potential protein aggregation or fluorescence interference effects induced by these compounds, which might have been overlooked with the first derivative method. This raises a general concern with false positive rates in DSF thermal stability methodology, which may experience limitations mediated by non-specific signals, susceptible to interference from small-molecule ligands and which can lower confidence in the obtained results.

[0486] Although our hit compounds did not validate as STAT3 binders, the rigorous pursuit of investigating its target-engagement and mechanism of STAT3 inhibition revealed it is a tyrosine kinase inhibitor displaying high affinity for TrkA. An issue with many proposed STAT31 remains the inconsistent validation of their STAT3-engagementin cells. A comprehensive target engagement-focused strategy, integrating orthogonal and cellular assays, can ensure false positives are diligently dismissed, but can also facilitate the identification of novel chemical probes with potential efficacy in other disease models. The integration of kinome profiling experiments early on in our screening funnel was a key addition, enabling a more critical hit validation approach. Moreover, sampling an extended array of potential binding partners across the proteome also increased the likelihood of opening new avenues for optimizing and repurposing hits. The unexpected discovery of PI-8 as a potent TrkA binder and our pivot from STAT3 to TrkA was a notable shift, yet identifying its function as a kinase inhibitor was greatly facilitated through the use of target engagement-focused validation approaches. This highlights how a target-engagement-focused strategy, receptive to unexpected findings, can reveal compounds with intriguing selectivity profiles and pave the way for further development.

[0487] Exploring the SAR of this scaffold for TrkA inhibition, PI-15 was developed with comparable biochemical activity compared to PI-8, yet with improved cellular activity inhibiting TPM3-TrkA and full-length TrkA phosphorylation as well as potently impairingviability in KM12 NTRK1 fusion-positive cancer cells. PI-15 also engaged the TPM3-TrkA fusion protein in KM12 cell lysate CETSA experiments. Notably, PI-15 retained TrkA selectivity displaying 2-fold and 9-fold selectivity for TrkA inhibition over TrkB and TrkC, respectively. Moderate inhibition of JAK2 and SRC in biochemical settings indicated the top TrkA binders might exhibit co-inhibition of these kinases in the cellular environment, however these compounds demonstrated a high degree of selectivity for TrkA-fusion dependent KM12 cells (IC50 < 0.3 pM] compared to non-TrkA-driven cancer cell lines (IC50 > 10 pM). Although polypharmacologic effects may contribute to the low IC50 values against the NTRK1 fusion-positive cancer cells, the depletion of TPM3-TrkA phosphorylation substantiates TrkA inhibition as a major mechanism of action for the strong anti-proliferative effects of the PI compounds.

[0488] The selectivity, but not absolute specificity of these inhibitors for TrkA-driven cancer cell lines likely results from their activity towards other kinases, such as JAK2, as demonstrated in biochemical assays. Future studies will investigate if this combined inhibition of oncogenic kinases may be beneficial within the context of anti-cancer therapy, especially within the context of disrupting resistance pathways that limit the efficacy of clinically used Trk inhibitors. Even so, it is important to recognize interpreting the specificity of these inhibitors for TrkA-driven cells alongside their biochemical kinome profile remains challenging, as correlating biochemical Ka and IC50 values with their effects on cellular viability, requires navigating the nuanced interplay between target engagement and cellular context, particularly the cell’s dependence on the inhibited pathway.

[0489] Moreover, there were also some curious results in this series of compounds, for example, the deprioritized TrkA hit PI-10 was unable to block TPM3-TrkA phosphorylation in cells, yet impaired viability of KM12 cells with an EC50 of 186 nM. We note again that the difference in incubation time may play a role in this finding, where PI-10 treatment was for 24 hours prior to measuring TPM3-pTrkA levels whereas viability was measured after 72 hours. Additionally, it is possible that moderately decreasing pTrkA levels is sufficient to impair viability in these cells. We also cannot discount that PI-10 may exert its impairment of viability through off-target effects, noting that the replacement of the functionalized 5-phenyl group with the 5-(3-pyridyl)-moiety may lead to change in the kinase selectivity profile of this compound which may lead to more promiscuity within the kinome.

[0490] The / ^-stereochemistry of the a-Methylbenzylamino group in this scaffold was shown to be more active than its S- counterpart indicating the stereochemistry of the a-Methylbenzyl moiety is important for its activity. Compound PI-15R was revealed as a potent TrkA-targeted inhibitor which maintained selectivity across the kinome at 25 nM, with low activity against other oncogenic targets such as JAK2 and ROS1. As a highly potent Trk inhibitor, PI-15R demonstrated strong inhibition of all Trk subtypes while remaining selective for Trks over JAK2 and Src. Additionally, it was predicted as a type II binder using GLIDE docking models. Notably, almost all ligands showed a preference for the Type II conformation of TrkA, JAK2, and SRC in our docking experiments, suggesting this series may act as Type II inhibitors. An X-ray crystal structure of PI-15R bound to the TrkA kinase domain would be valuable to confirm whether this binding hypothesis is valid.

[0491] Binding TrkA in a mode that is distinct from traditional Type I inhibitors, may offer opportunities for improved selectivity and safety profiles compared to currently available therapeutics. Furthermore, the moderate inhibition of JAK2 and SRC observed alongside potent TrkA inhibition is not necessarily a disadvantage. Both JAK2 and SRC are well-established anti-cancer drug targets, and this potential polypharmacologic effect could allow for synergistic outcomes in cancer models sensitive to multi-kinase inhibition involving these targets. In vitro PK profiling experiments outlined PI-8 and the more potent -isomer of PI-15 exhibit favourable metabolic stability, with PI-15R showing improved stability and cell permeability positioning this scaffold favourably for future in vivo PK and efficacy studies.

[0492] Overexpression of Trks are found in several forms of cancer, including basal cell carcinoma, lung cancer, neuroblastoma, and others42. Moreover, while Trk inhibitors are used as targeted cancer therapeutics for Trk fusion-positive cancers, there are likely roles for Trk inhibitors beyond these rare cases, with emerging roles of full length TrkA in breast cancer metastasis67'68. Additionally, TrkA and its respective receptor-ligand, NGF, play a key role in modulating both acute and chronic pain, particularly in conditions associated with inflammation such as Osteoarthritis [OA] or Rheumatoid Arthritis (RA)69-71. As such, thereis growing interest in the role of TrkA in chronic pain and the potential utility of TrkA inhibitors as a promising therapeutic approach for chronic pain management72~76. Smallmolecule TrkA inhibitors, such as the selective inhibitor AR786, have shown promise by attenuating pain responses in osteoarthritis77and inflammatory arthritis in rat models, where it also reduced joint swelling78, suggesting potential anti-inflammatory effects. As such, investigating the analgesic and anti-inflammatory properties of the PI compounds presents an exciting possibility for further research. With both JAK278and Src79being linked as modulators of the inflammatory response in RA, a polypharmacologic strategy targeting these kinases alongside TrkA may offer both analgesic and anti-inflammatory effects in inflammatory chronic pain models.

[0493] Overall, this work has led to the discovery of potent TrkA inhibitors that have been validated for target engagement and show promising activity in TrkA fusion-positive cell lines. This was a direct result of the construction and execution of a target engagement-focused screening funnel, that led us away from direct STAT3 binders and instead to a serendipitous outcome. This work emphasises the importance for heightened scrutiny when targeting proteins that are deemed "undruggable” and brings attention to factors to be mindful of when utilising thermal shift assays as screening tools.

[0494] 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.REFERENCES

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Claims

CLAIMS:

1. A compound, the compound having the structure of Formula I or Formula II:Formula II wherein,O OR2is selected from:andX is selected from: H; methyl; ethyl; and tert-butyl;or a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, wherein X is selected from: (R)-methyl; and (S)-methyl.

3. The compound of claim 1 or 2, wherein Ri is selected from:The compound of claim 1 or 2, wherein Ri is selected from:OThe compound of claim 1 or 2, wherein R2 is selected from:

7. The compound of any one of claims 1-6, wherein the compound is selected from one or more of the following:

8. The compound of any one of claims 1-7, wherein the compound is selected from one or more of the following:

9. The compound of any one of claims 1-8, wherein the compound is selected from one or more of the following:

10. The compound of claim 7, wherein the compound is selected from one or more of the following:

11. The compound of claim 7, wherein the compound is selected from one or more of the following:

12. A compound, the compound having the structure of Formula III:Formula IIIwherein,R3is selected from:or a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

13. The compound of claim 12, wherein the compound is selected from one or more of the following:

14. The compound of any one of claims 1-13, wherein the compound inhibits tropomyosin receptor kinase A (TrkA).

15. The compound of any one of claims 1-14, wherein the compound inhibits Janus kinase 2 (JAK2).

16. The compound of any one of claims 1-15, wherein the compound inhibits ProtoOncogene Tyrosine-Protein Kinase Src (SRC).

17. The compound of any one of claims 1-16, wherein the compound is used to target TRK fusion proteins.

18. The compound of any one of claims 1-16, wherein the compound is used to treat TRK fusion-positive tumours.

19. The compound of any one of claims 1-16, for treating one or more of the following: cancer; osteoarthritis (OA); rheumatoid arthritis (RAJ; inflammation; acute pain; and chronic pain.

20. The compound of any one of claims 1-16, for treating endometriosis.

21. The compound of claim 19, wherein the cancer is selected from one of more of: breast; colon; prostate; pancreatic; lung; brain; renal; and head and neck.

22. The compound of claim 19, wherein the cancer is selected from one of more of: squamous cell carcinoma; melanoma; glioma; basal cell carcinoma; neuroblastoma; lymphoma; and leukaemia.

23. The compound of claim 19, wherein the cancer is selected from one of more of:(i) HER2+ breast cancer; and triple negative breast cancer (TNBC);(ii) cellular congenital mesoblastic nephroma; secretory breast sarcoma; mammary analog secretory carcinoma; and infantile fibrosarcoma; or(iii) undifferentiated sarcomas; gliomas; papillary thyroid cancers; spitzoid neoplasms; inflammatory myofibroblastic tumors; and acute leukemias.

24. A compound of any one of claims 19, wherein the cancer is a pediatric cancer selected from one or more of the following: cellular congenital mesoblastic nephroma; secretory breast sarcoma; mammary analog secretory carcinoma; infantile fibrosarcoma; undifferentiated sarcomas; gliomas; papillary thyroid cancers; spitzoid neoplasms; inflammatory myofibroblastic tumors; and acute leukemias.

25. A pharmaceutical composition, the pharmaceutical composition comprises a compound of any one of claims 1-14 and a pharmaceutically acceptable carrier.

26. The pharmaceutical composition of claim 25, wherein the pharmaceutical composition is for one or more of the following: cancer; osteoarthritis (OA); rheumatoid arthritis (RA); inflammation; acute pain; and chronic pain.

27. Use of a pharmaceutical composition, the pharmaceutical composition comprising a compound of any one of claims 1-14 and a pharmaceutically acceptable carrier, for the treatment of one or more of the following: cancer; osteoarthritis (OA); rheumatoid arthritis (RA); inflammation; acute pain; and chronic pain.

28. Use of a compound of any one of claims 1-14 in the manufacture of a medicament for the treatment of one or more of the following: cancer; osteoarthritis (OA); rheumatoid arthritis (RAj; inflammation; acute pain; and chronic pain.

29. Use of a compound of any one of claims 1-14 for the treatment of one or more of the following: cancer; osteoarthritis (OA); rheumatoid arthritis (RA); inflammation; acute pain; and chronic pain.

30. A method of treating one or more of the following: cancer; osteoarthritis (OA); rheumatoid arthritis (RA); inflammation; acute pain; and chronic pain, comprising the administration of a compound of any one of claims 1-14 to a subject in need thereof.

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