IUSM-Purdue TREAT-AD Milestone 2 Target Enabling Package: LYN (LYN proto-oncogene, Src family tyrosine kinase)

IUSM-Purdue TREAT-AD Center Milestone 2 Target Enabling Package LYN (LYN proto-oncogene, Src family tyrosine kinase) Gene Symbol/Name NCBI Gene ID UniProt ID Ensembl ID LYN (LYN proto-oncogene, Src family tyrosine kinase) 4067 P07948 ENSG00000162711 Alternatives JTK8 Corresponding Author Pathum M. Weerawarna Contributing Authors Avi L. Benitah, Timothy I. Richardson Collaborating Authors Supervision Brent Clayton, Jeff Dage, Kun Huang, Bruce Lamb, Andrew Mesecar, Alan Palkowitz, Timothy I. Richardson Date Approved by Admin Core October 15, 2025 Document version Version 1.0 Document version date November 6, 2025 Citation https://doi.org/10.5281/zenodo.17545570 Sections of this work have been and will be submitted for peer review and prospective publication Affiliations Indiana University School of Medicine , Purdue University Funding U54AG065181 1. ABSTRACT The TaRget Enablement to Accelerate Therapy Development for Alzheimer’s Disease (TREAT-AD) centers were established to provide high-quality research tools and technologies to validate and advance the next generation of drug targets for Alzheimer’s disease (AD). Data, methods, and experimental resources are being openly disseminated through the AD Knowledge Portal to accelerate the discovery and characterization of AD-relevant therapeutic targets. These resources are compiled as Target Enabling Packages (TEPs) that provide open access to validated reagents, assays, and chemical tools. Here, the IUSM-Purdue TREAT-AD Center presents a Milestone 2 (M2) TEP that evaluates the experimental feasibility of initiating a drug discovery program targeting Lyn kinase by establishing and assessing initial in vitro assays and tool molecules. Lyn is a Src-family nonreceptor tyrosine kinase that serves as a critical regulator of immune signaling through the phosphorylation of both immunoreceptor tyrosine-based activation (ITAM) and inhibitory (ITIM) motifs. Aberrant Lyn activation has been implicated in autoimmune diseases, hematologic malignancies, and neurodegeneration, including AD, where Lyn expression and autophosphorylation are elevated in microglia surrounding amyloid plaques. To enable pharmacological studies of Lyn function in AD models, we pursued a ligand-based optimization strategy starting from imatinib, based on the observation that imatinib inhibits Lyn kinase but lacks the potency and selectivity required for a suitable probe molecule. Given that imatinib acts as a type II kinase inhibitor and the advantages of this binding mode for kinase selectivity, we reasoned that rational modifications could enhance both potency and selectivity within the Src-family of kinases and provide new chemotype chemically distinct from imatinib. Through systematic exploration of medium-length phenoxymethyl and phenyl amide substitutions, we identified a series of potent and selective type II Lyn inhibitors (e.g., TAD-0411662, TAD-0411836, TAD0411837), some exhibiting single-digit nanomolar Ki values and >500-fold selectivity over Hck, the closest SFK homolog. Kinome-wide profiling confirmed high specificity toward tyrosine kinases with minimal offtarget activity, establishing their suitability as in vitro pharmacological probes, while also highlighting physicochemical properties that currently limit their use in in vivo animal studies that required brain penetration. These newly developed compounds represent best in class Lyn-selective type II inhibitors within the SFKs and provide pharmacological tools and associated data for dissecting Lyn-dependent signaling pathways in AD and other diseases. Future directions for optimizing physicochemical properties for sufficient brain exposure in vivo are outlined. 2. BACKGROUND Lyn kinase, a member of the Src family (SFK) of nonreceptor tyrosine kinases, plays a pivotal role in transducing signals downstream of immune receptors, primarily through the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) and immunoreceptor tyrosine-based inhibitory motifs (ITIMs).1-3 Although the ITAM phosphorylating function of Lyn is considered redundant, as other SFK members, such as Fyn and Blk, are capable of compensating for this activity in its absence, the ITIM phosphorylating function of Lyn is unique.4,5 This dual functionality enables Lyn to modulate cellular activation thresholds and function as a master regulator of immune and nonimmune signaling, particularly in hematopoietic, nervous system, epithelial, and endocrine cells.6 Structurally, Lyn shares the conserved domain architecture characteristic of SFKs, consisting of an N-terminal SH4 domain followed by a unique region, SH3 and SH2 regulatory domains, and a catalytic SH1 domain.3 Several other nonreceptor tyrosine kinase families, including Tec, FAK, Syk, JAK, and Abl, also exhibit modular architectures similar to those of SFKs.7 Among these, the Abl family is the most closely related to SFKs from both structural and regulatory perspectives. Like SFKs, Abl kinases maintain tight control over their enzymatic activity through phosphorylation-dependent activation and inhibition, as well as through the formation of closed inactive conformations mediated by intramolecular SH3 and SH2 domain interactions. As a result, SFKs often appear in the off-target kinase panels of most early Abl inhibitor development campaigns.8,9 Dysregulation of Lyn kinase, either through overexpression or constitutive activation, has been implicated in various pathological conditions, including autoimmune diseases, hematological malignancies, solid tumors, and neurodegenerative disorders such as AD.10-15 The expression of Lyn in microglial increases in response to amyloid beta (Aβ), a primary pathological hallmark of AD in humans and a phenotype replicated in murine amyloidosis models of AD.16 Increased Lyn expression has also been found in human and mouse microglia compared to other SFKs.17 Furthermore, post-mortem AD patient brains have also shown elevated Lyn levels in microglia.10 Interestingly, the broad-spectrum Src kinase inhibitor PP1 (4-amino-5-(4-methylphenyl)-7-(tbutyl)pyrazolo[3,4-d]pyrimidine),18 which mainly targets Lyn, was shown to inhibit Aβ-triggered neurotoxin production and improve neuronal survival.10 Additionally, independent studies have documented increased Lyn activation in mouse microglia exposed to Aβ oligomers, akin to the microglia from the post-mortem AD patient brains.19,20 In a detailed study by Gwon et al., the role of Lyn in AD was explored, revealing its significant involvement in Aβ-induced neurotoxicity and tau hyperphosphorylation via FcγRIIb2 phosphorylation.21 The study observed a rapid increase in Lyn activity upon exposure to oligomeric Aβ1-42 in mouse neuronal cells. They also found that Lyn activation was inhibited in FcγRIIb2 knockout neurons, suggesting that FcγRIIb2-Aβ1-42 interactions mediated the activation of Lyn. An analysis of AD patient brain tissue revealed a three-fold increase in Lyn's autophosphorylation in the hippocampus compared to non-AD controls. Further experimentations confirmed the direct phosphorylation of the FcγRIIb2 ITIM at Tyr273 by Lyn upon exposure to oligomeric Aβ1-42. Interestingly, the knockdown of Lyn expression significantly suppressed Aβ1-42-induced cell death in neuroblastoma and hippocampal cell lines, underscoring Lyn's role in countering Aβ1-42-induced neurotoxicity. Additionally, phosphorylated FcγRIIb2 by Lyn was found to recruit SHIP2 to FcγRIIb2, leading to tauhyperphosphorylation. Lastly, a novel Lyn inhibitor (KICG2576) was shown to lessen Aβ-FcγRIIb2-mediated neuronal cell death and rescue Aβ-induced memory impairment in mice. Overall, this study establishes the role of Lyn in AD in the context of Aβ-triggered neurotoxicity and tau hyperphosphorylation, which are the main pathological features of AD. Despite increasing evidence supporting the role of Lyn kinase in various disease conditions, selective inhibitors targeting of Lyn remains an unmet need. A primary hurdle arises from the high degree of structural homology within the SFKs, which include Fyn, Hck, Lck, Blk, c-Src, Fgr, and Yes.1 These kinases frequently co-participate in immune receptor signaling cascades, particularly Fyn, Hck, and Lck, making it essential for small-molecule inhibitors targeting Lyn as a therapeutic strategy to be highly selective for Lyn over other SFKs.22-24 This selectivity is critical to attributing pharmacological phenotypic outcomes to the function of Lyn. While some existing SFK inhibitors that target Lyn exhibit broad kinome-wide selectivity, they often lack intra-family specificity and inhibit a broad spectrum of SFKs, limiting their utility as Lynselective chemical probes. One rational strategy to achieve kinase selectivity is to exploit inactive conformational states of the catalytic domain.25 In contrast to type I inhibitors that bind the ATP-competitive active (DFG-in/αC-in) conformation, type II inhibitors engage a DFG-out inactive conformation, often with αC-in positioning.26 This mode of binding mainly leverages structural differences in allosteric pockets that are less conserved across kinases, thereby offering enhanced selectivity. In addition, kinases differ in their intrinsic conformational equilibria and their propensity to sample inactive states, providing a unique opportunity to design inhibitors that selectively stabilize these less populated conformations, as recently demonstrated by extensive solution-phase NMR structure elucidation studies.27 While recent work has demonstrated the feasibility of developing type I1/2 inhibitors that preferentially bind the DFG-in/αC-out inactive conformation (second known inactive conformation) to achieve SRC-B subfamily selectivity, which includes Lyn, there are no reports in the literature exploring the type II inhibitor binding mode to develop potent Lyn inhibitors with selectivity within the SFKs.28 Preliminary data from our group and others suggest that certain type II inhibitors, such as imatinib , masitinib and bafetinib, can potently inhibit Lyn kinase with modest selectivity over other SFK members.29,30 Given the characteristic biphasic three-step binding mechanism of type II inhibitors31,32, which involves an initial conformational selection followed by physical binding and an induced fit step that involves residues distal to the traditional ATP binding site, we hypothesized that existing type II inhibitors could be rationally modified to improve Src family selectivity while retaining or enhancing Lyn potency. To this end, as a proof of concept, we conducted a systematic ligand-based optimization of imatinib, a known type II kinase inhibitor, to develop a series of novel type II inhibitors with enhanced Lyn selectivity within the SFKs. This effort led to the identification of several potent derivatives that exhibit significant improvements in intrinsic selectivity across the SFK family, representing some of the most selective chemical probes for Lyn reported to date. Herein, we report the design rationale, synthesis, and biological evaluation of these compounds with the objective of catalyzing and supporting further investigation into Lyn as a target for the treatment of AD. 3. RESULTS AND DISCUSSION 3.1 Inhibitor Design Rationale Imatinib, also known commercially as “Gleevec” or “Glivec,” the marketed mesylate salt of imatinib, is a potent and selective inhibitor of Abl (Bcr-Abl) and was first reported by Zimmermann et al. in 1997.33 It became the first FDA-approved kinase inhibitor for treating chronic myelogenous leukemia (CML). The medicinal chemistry effort that led to the development of imatinib began with the identification of lead compound (1) from a screening campaign for protein kinase C (PKC) (serine/threonine kinase) inhibitors. Compound 1 features a phenylaminopyrimidine scaffold substituted at the 3ʹ-position with a 3-pyridyl group (Figure 1).34 Hit-to-lead optimization of this scaffold led to compound 2, in which the 3-position of the phenyl ring is substituted with a benzamide moiety, resulting in a dual inhibitor of PKC-α and plateletderived growth factor receptor (PDGF-R) with low micromolar inhibitory activity.35 Interestingly, introduction of a so called “flag-methyl group” at the 6-position of the bridge phenyl ring of compound 2 completely diminished PKCα inhibitory activity and improved PDGFR inhibitory activity to a submicromolar level (IC50 = 0.1 µM), resulting in the PDGFR-selective inhibitor 3. Figure 1. Evolution of the clinically approved kinase inhibitor imatinib from a PKC hit (Compound 1). Initial SAR studies around 1 led to the identification of the PKCα/PDGF-R dual inhibitor 2. Introduction of a “flag-methyl” substitution on Compound 2 diminished PKCα activity and enhanced PDGF-R potency, yielding the PDGF-R selective inhibitor 3. Subsequent SAR efforts explored two major structural classes, ultimately leading to imatinib through incorporation of a solubilizing N-methylpiperazine moiety. (Highlighted IC50 values reflect activity against key kinase targets, demonstrating increasing selectivity for PDGF-R and BCR-Abl, along with nanomolar potency against Lyn kinase and moderate selectivity over Hck). Subsequently, the optimization of this compound as a potent and selective inhibitor of v-Abl was reported, culminating in the discovery of imatinib.33 The SAR studies explored 3-pyridyl, 4-pyridyl, and 3-indolyl substitutions at the 3ʹ position of the aminopyrimidine ring, along with various substitutions at the 3ʹ position of the phenyl ring, classifying the resulting molecules into two structural series (classes A and B), in the presence or absence of the “flag-methyl group” at the 6-position of the bridge phenyl ring (Figure 1). Within structural class B, compounds bearing a 3ʹ-pyridyl group on the aminopyrimidine ring and various benzamide derivatives at the 3-position of the phenyl ring (as shown in Figure 1) exhibited potent v-Abl inhibition with submicromolar IC50 values. Among these, the initial lead compound (compound 3), which showed submicromolar PDGF-R inhibitory activity, also inhibited v-Abl with an IC50 of 0.4 µM, functioning as a dual inhibitor of PDGF-R and v-Abl. However, these early compounds suffered from poor aqueous solubility and low oral bioavailability.33,34 To address these limitations, a highly polar Nmethylpiperazine moiety was introduced at the 4-position of the benzamide ring, connected via a spacer to the phenyl core, resulting in the chemical structure of imatinib. This modification not only improved aqueous solubility and oral bioavailability but also enhanced potency, reducing the v-Abl IC50 from 0.4 µM to 38 nM and the PDGF-R IC50 from 0.1 µM to 50 nM, paving the way for the first clinically approved kinase inhibitor. In addition, it displayed weak inhibitory activity against the SFK member c-Src, with an IC50 value of 15.7 µM. Our in-house screening of imatinib against the SFKs revealed that it potently inhibits Lyn kinase in vitro, with a Ki value of 119 nM and a 13-fold selectivity over Hck, the closest relative of Lyn in the SFK phylogenetic tree (Figure 1). Figure 2. (A) Schematic representation of the three-step, biphasic type II kinase inhibitor binding mechanism. Binding proceeds via a conformational selection (CS) step from the active DFG-in to the inactive DFG-out conformation, followed by physical binding to form the initial encounter complex. This is subsequently followed by an induced fit (IF) transition, yielding the final inhibitor-bound complex. (B) Multiple sequence alignment of representative Src family kinases (Src (P12931), Fyn (P06241), Hck (P08631), and Lyn (P12931)) from two structural groups (SRC-A and SRC-B), highlighting key regions relevant to type II inhibitor binding that are distal to the ATP-binding pocket: the linker region (SH2-KD), p-loop, and activation loop. Residue differences are depicted in color. For years, the remarkable selectivity of imatinib for Abl over c-Src remained unexplained in terms of direct binding interactions observed in X-ray crystal structures, as the key interacting residues are identical, and both enzymes adopt the DFG-out inactive conformation. It was only after a decade that the precise binding mechanism of imatinib was elucidated through extensive NMR studies and pre-steady-state, nonequilibrium stopped-flow kinetics experiments that monitored intrinsic fluorescence quenching upon inhibitor binding.7,27,32 The mechanism involves a three-step process31,36 comprising an initial conformational selection step, during which the kinase exists in a pre-existing equilibrium between DFGin and DFG-out conformations (Figure 2A). This is followed by selective binding of the inhibitor to the DFGout state and subsequently by an induced-fit step characterized by a slow conformational rearrangement of the activation loop and changes in the p-loop. According to this mechanism, the experimentally observed selectivity arises primarily from the slow activation loop rearrangement (induced fit step) and the conformational equilibrium between DFG-in and DFG-out states (conformational selection), which are governed by structural features involving less conserved residues distal to the traditional ATP binding pocket, rather than by direct interactions with residues in the ATP binding pocket (Figure 2B). Given the empirical evidence of moderate selectivity of imatinib for Lyn over Hck observed in our in-house validation, and the unique three-step biphasic binding mechanism, which involves less conserved residues both between and within kinase families, located distal to the traditional ATP binding pocket, we hypothesized that systematic modifications to the imatinib scaffold could yield a potent type II inhibitor with improved selectivity within the SFKs. Careful evaluation of the original imatinib SAR studies described above indicates that it was limited to various substituted benzamides and their corresponding heterocycles at the 3-position of the phenyl ring. As a result, the tail region, which interacts with the allosteric pocket and can influence activation loop dynamics, was restricted to relatively short substitutions. The incorporation of the N-methylpiperazine moiety that gave rise to imatinib with an extended tail region, was primarily a strategy to improve the solubility and oral bioavailability of the original series.34 In contrast, we employed a ligand-based approach that modified the tail group for selectivity. We synthesized a series of derivatives bearing tail groups with medium-length, a region of chemical space that was not explored in the original imatinib SAR studies, to investigate the impact of these modifications on Lyn inhibitory activity and selectivity within the SFKs. Figure 3. (A) Overview of tail region structural modifications explored in this study. Initial modifications involved the introduction of a phenoxymethyl group with substitutions at the para (p), meta (m), and ortho (o) positions, yielding compounds 4-28 and probing chemical space not previously explored in the original imatinib SAR. Compounds 2937 incorporate various para substituted phenyl groups in the tail region, revisiting some reported analogs while also introducing new derivatives. Compounds 38-40 featuring ortho-additions (Y = H, F, Cl) to assess its impact on potency and selectivity. (B) General synthetic route used for the preparation of compounds 4-40. As shown in Figure 3A, we used phenoxymethyl groups bearing different substituents at the para (p), meta (m), and ortho (o) positions on the phenyl ring next to the amide carbonyl group to systematically evaluate the effects of these modifications on Lyn inhibitory activity and selectivity within the SFKs. The selection of the phenoxymethyl group as the tail moiety was based on two criteria: (a) it positions the terminal phenyl ring two atoms distal to the amide carbonyl, in contrast to the directly attached phenyl ring in the original imatinib SAR, thereby yielding a tail group of intermediate length, while preserving a molecular architecture distinct from the original imatinib scaffold; and (b) the commercial availability of a broad range of substituted phenoxyacetic acid derivatives enabled the efficient synthesis of a small, focused compound library via amide coupling with a common amine scaffold, affording final analogs in good to excellent yields (Figure 3B). We also synthesized a small set of derivatives bearing phenyl groups with different substituents at the para position, directly attached to the amide carbonyl group, similar to the original imatinib SAR, to revisit some of the reported chemical space and explore new analogs for evaluating their inhibitory activity against Lyn kinase. Within the original SAR studies, the “flag-methyl group” at the 6-position of the bridge phenyl ring diminished PKC inhibitory activity while increasing PDGF-R inhibitory activity, indicating that these two enzymes are sensitive to this substitution.33 However, v-Abl inhibitory activity was not affected by the introduction of the “flag-methyl group,” as evidenced by equipotent inhibition of v-Abl by both compounds 2 and 3 (IC50 = 0.4 µM). This prompted us to synthesize compounds lacking the substitution at this position as well as two analogs bearing Cl and F substituents, respectively, to evaluate the impact of substitution at this position on Lyn inhibitory activity and selectivity. These compounds were synthesized similarly to the phenoxymethyl derivatives from commercially available starting materials using an amide coupling reaction, as shown in Figure 3B. 3.2 Evaluation of in vitro Lyn Inhibitory Activity and Selectivity Having synthesized novel Lyn kinase inhibitors, we next determined their inhibitory activity and selectivity within the SFKs using the HotSpot kinase assay, a radiometric, filtration-based method widely regarded as the 'gold standard' for detecting kinase activity due to its high sensitivity and reliability. This assay utilizes γ-32P-ATP as the phosphate donor, enabling the direct measurement of phosphorylated substrate formation.37 All compounds were assayed against full-length Lyn kinase and Hck, with Hck selected as the initial reference kinase to assess intrinsic selectivity within the SFK family. Since Hck is the closest SFK family member to Lyn, according to phylogenetic analysis, we hypothesized that achieving selective Table 1. Evaluation of Lyn/Hck Inhibition by tail group phenoxymethyl-substituted analogs (4-28) p-substitution (R2, R3 = H) Compound R1 Lyn Ki (nM) Hck Ki (μM) Lyn/Hck 4 (TAD-0411476) F 330 ± 57 29.4 ± 4.4 88 5 (TAD-0411477) Cl 275 ± 197 75 273 6 (TAD-0411478) Br 123 ± 21 47.3 ± 9.6 382 7 (TAD-0411659) I 159 ± 12 8.6 ± 3.9 54 8 (TAD-0411669) CH 3 377 ± 43 18.7 ± 5.3 50 9 (TAD-0411670) CF3 115 ± 26 19.9 ± 1.2 174 10 (TAD-0411479) OCH3 305 ± 88 34.5 ± 4.5 111 11 (TAD-0411671) OCF3 149 ± 59 38.6 255 12 (TAD-0422249) NH2 1.7 (μM) 15.0 ± 3.6 9 13 (TAD-0411658) NO 2 60 (μM) inactive NA 14 (TAD-0411657) CN 658 ± 105 75 114 m-substitution (R1, R3 = H) Compound R2 Lyn Ki (nM) Hck Ki (μM) Lyn/Hck 15 (TAD-0411664) F 152 ± 34 4.2 ± 1.0 28 16 (TAD-0411665) Cl 254 ± 29 14.6 ± 1.4 57 17 (TAD-0411666) Br 377 ± 40 10.3 ± 3.3 27 18 (TAD-0411668) CH 3 456 ± 37 13.5 ± 1.1 30 19 (TAD-0411737) OCH3 6.8 ± 2.5 (μM) 34.9 ± 7.1 5 20 (TAD-0411836) NO2 732 ± 2.5 14.2 ± 4.3 19 o-substitution (R1, R2 = H) Compound R3 Lyn Ki (nM) Hck Ki (μM) Lyn/Hck 21 (TAD-0411661) F 92.5 ± 2.8 2.5 27 22 (TAD-0411662) Cl 8.7 ± 3.9 0.57 ± 0.03 65 23 (TAD-0411663) Br 30.4 ± 9.2 1.0 33 24 (TAD-0411667) CH3 34.6 ± 0.7 1.0 29 25 (TAD-0411832) OCH3 47.6 ± 31.9 3.1 ± 0.3 65 26 (TAD-0411828) NO2 47.7 ± 11.1 6.5 ± 0.7 136 o,p-substitution (R2 = H) Compound R1, R3 Lyn Ki (nM) Hck Ki (μM) Lyn/Hck 27 (TAD-0411834) Cl, CH3 86 ± 23 43 ± 10 500 28 (TAD-0411734) Cl, Cl 67 ± 8 3.9 ± 0.3 58 IC50 values were determined using the HotSpot kinase assay (10 µM ATP) with full-length protein and converted to Ki values using the Cheng-Prusoff equation38 to facilitate direct comparison (𝐾𝐾𝑚𝑚 ATP values: Lyn = 15 μM, Hck = 30 µM). Values are reported as the average of at least two independent experiments ± SEM. Inhibitor constants are given in nanomolar (nM) for Lyn and micromolar (µM) for Hck. inhibition of Lyn over Hck would translate to improved overall selectivity within the SFKs. Table 1 and 2 summarizes the apparent Ki values calculated from the IC50 values, along with the Lyn over Hck selectivity profiles of the compounds. 3.2.1 Effect of Tail Group Phenoxymethyl Substitution on Lyn Inhibitor Activity and Selectivity The first-generation of inhibitors we synthesized, bearing para-substituted phenoxy methyl tail groups (compounds 4–14), displayed submicromolar Lyn inhibitory activity, with some compounds showing comparable potency to imatinib, such as compound 6 (Br substitution) and 9 (CF3 substitution). Strong electron-withdrawing groups (EWGs), both in terms of resonance and inductive effects, such as nitro and nitrile (compounds 13 and 14), as well as strong electron-donating groups (EDGs) bearing hydrogen bond donors, such as amines (compound 12) at the para position, tend to negatively impact Lyn inhibitory activity. Interestingly, despite this observation, halogens, along with other EWGs and EDGs lacking hydrogen bond donors, were generally well tolerated at this position regardless of their size, affording compounds 4–11 with good Lyn inhibitory activity in the submicromolar range (< 400 nM) and improved Lyn over Hck selectivity compared to imatinib. Notably, some compounds achieved excellent selectivity of over 300-fold, as observed for 6 (Br substitution). Overall, these findings suggest that substitution at the para position with halogens, EWGs, or EDGs lacking hydrogen bond donors are well tolerated, with minimal steric constraints, and serves as a key driver of selectivity, either by modulating polar interactions within the DFG-out extended binding pocket or by differentially influencing the activation loop conformational change associated with the induced-fit step of the binding mechanism between Lyn and Hck. Compounds 15–20 represent novel Lyn inhibitors featuring meta-substituted phenoxymethyl tail groups. Similar to the first-generation, the presence of strong EWGs, both by resonance and inductive effects, such as nitro (compound 20), weakened Lyn inhibitory activity, while halogen substitutions (F, Cl, and Br) provided submicromolar Lyn inhibitory activities comparable to para-substituted analogs, along with moderate improvements in Lyn over Hck selectivity (25to 60-fold) compared to imatinib. Interestingly, unlike in the case of para-substitution, methoxy-substitution at the meta-position resulted in reduced Lyn inhibitory activity. Overall, although halogen substitutions at the meta-position yielded potent Lyn inhibitors, substitution at the meta-position was not a significant modulator of selectivity, in contrast to the more pronounced selectivity observed with para-substitution. Inhibitors bearing ortho-substituted phenoxymethyl tail groups were the most successful among all the derivatives, breaking the submicromolar barrier and reaching low nanomolar Lyn inhibitory potency. All compounds, regardless of the substitution size or electronic properties, exhibited improved Lyn inhibitory activity and selectivity, indicating that a wide range of substitutions are well tolerated at this position. In particular, chlorine substitution at the ortho-position (compound 22) yielded single-digit nanomolar potency, with a Ki value of 8.7 nM and 65-fold selectivity for Lyn over Hck. Although the Lyn over Hck selectivity was not as high as that observed for the para-substituted analogs, it was nonetheless improved and comparable to that of the meta-substituted series. groups, we identified a series of novel analogs that exhibited low nanomolar Lyn inhibitory activity with significantly improved selectivity over closely related family members such as Hck and Fyn. Biochemical characterization against a broader kinome panel consisting of selected members from different kinase groups revealed that the lead compounds maintained high specificity for tyrosine kinases, while also inhibiting PDGF-R and c-Kit, similar to imatinib, as two of the main off-targets. Evaluation of physicochemical and ADME properties highlighted aqueous solubility concerns, along with high metabolic clearance in mouse microsomes, pinpointing issues associated with the phenoxymethyl moiety and its metabolism, factors that require further optimization for in vivo probe development. However, the excellent MDCK permeability observed for the lead compounds suggests that they can still be used as in vitro chemical probes for dissecting Lyn-specific signaling pathways in cellular models. 5. METHODS 5.1 General Procedures Unless otherwise noted, all of the reagents used in the synthesis were obtained from commercial sources and used as received. All solvents were purified by passage through a solvent column composed of activated alumina and stored under an argon or nitrogen atmosphere. All of the syntheses were carried out using flame-dried glassware. Normal phase and reverse phase chromatography were performed on a Teledyne-ISCO NextGen 300 instrument using prepacked silica gel or C18-functionalized silica gel columns available from Teledyne-ISCO, or on a Teledyne-ISCO Combiflash using prepacked silica gel columns available from Agela or Welch. 1H NMR and 13C NMR spectra were recorded in DMSO-d6 or CDCl3 as solvents on a Bruker AVANCE III 500 spectrometer or 400 at 26 °C. Chemical shifts are reported in parts per million (ppm, δ) and referenced to DMSO-d6 (2.50 ppm for 1H NMR and 39.52 ppm for 13C NMR) or CDCl3 (7.26 ppm for 1H NMR and 77.2 ppm for 13C NMR). Coupling constants (J) are reported in Hz, and spin multiplicities are described as s (singlet), br (broad singlet), d (doublet), t (triplet), q (quartet), and m (multiplet). High-resolution mass spectra (HRMS) were measured with an Agilent 6210 LC-TOF (ESI,APCI, APPI) mass spectrometer. Purities of the final compounds were greater than 95%, as determined by reverse-phase HPLC analysis on an Agilent 1260 analytical HPLC system by following method: gradient table C; column Shimadzu Nexcol C18 (5 μm, 50 mm x 3.0 mm); mobile phase A, 1% formic acid in H2O; mobile phase B, 1% formic acid in MeCN; flow rate, 0.5 mL/min; detection wavelength, 214 nM; column temperature, 40 °C. General Procedure A (Amide Coupling): To a stirred solution of commercially available carboxylic acids (1.5 eq) in 6 mL DCM were added CDI, (1.5 eq), and DIPEA (3 eq). The solution was allowed to stir at rt for 1 hour. To the solution was then added 1 eq of the commercially available amine, 4-amino-2-[4-(3-pyridyl)- 2-pyrimidinylamino]toluene. The solution was allowed to stir at rt for 24 hours. The solution was then evaporated in vacuo and the resulting residue was purified by flash chromatography (9:1 DCM:MeOH). 5.2 Synthesis and Spectral Data for Compounds 4-40 Compound 4 (TAD-0411476) N-{3-[4-(3-pyridyl)-2-pyrimidinylamino]-4-tolyl}(pfluorophenoxy)acetamide N H N N N N H O O F Compound 4 was synthesized according to general procedure A. (1H NMR: (500 MHz, DMSO) δ 10.04 (s, 1H), 9.29 – 9.23 (m, 1H), 8.96 (s, 1H), 8.69 (dd, J = 4.7, 1.7 Hz, 1H), 8.51 (d, J = 5.1 Hz, 1H), 8.46 (dt, J = 8.1, 1.9 Hz, 1H), 7.95 (d, J = 2.2 Hz, 1H), 7.50 (ddd, J = 7.9, 4.7, 0.9 Hz, 1H), 7.43 (d, J = 5.2 Hz, 1H), 7.34 (dd, J = 8.2, 2.2 Hz, 1H), 7.22 – 7.12 (m, 3H), 7.06 – 7.00 (m, 2H), 4.68 (s, 2H), 2.21 (s, 3H). LRMS m/z (ESI+): 430.25 [M+H]+) Compound 5 (TAD-0411477) N-{3-[4-(3-pyridyl)-2-pyrimidinylamino]-4-tolyl}(pchlorophenoxy)acetamide N H N N N N H O O Cl Compound 5 was synthesized according to general procedure A. (1H NMR: 400 MHz, DMSO δ 10.05 (s, 1H), 9.25 (d, J = 2.3 Hz, 1H), 8.94 (s, 1H), 8.68 (dd, J = 4.8, 1.7 Hz, 1H), 8.50 (d, J = 5.1 Hz, 1H), 8.45 (dt, J = 8.1, 2.1 Hz, 1H), 7.94 (d, J = 2.2 Hz, 1H), 7.48 (dd, J = 8.0, 4.8 Hz, 1H), 7.43 (d, J = 5.1 Hz, 1H), 7.39 – 7.29 (m, 3H), 7.18 (d, J = 8.3 Hz, 1H), 7.05 – 7.00 (m, 2H), 4.70 (s, 2H), 2.20 (s, 3H). LRMS m/z (ESI+): 446.15 [M+H]+). Compound 6 (TAD-0411478) N-{3-[4-(3-pyridyl)-2-pyrimidinylamino]-4-tolyl}(pbromophenoxy)acetamide N H N N N N H O O Br Compound 6 was synthesized according to general procedure A. (1H NMR: (500 MHz, DMSO) δ 10.07 (s, 1H), 9.26 (d, J = 2.3 Hz, 1H), 8.96 (s, 1H), 8.69 (dd, J = 4.8, 1.7 Hz, 1H), 8.51 (d, J = 5.1 Hz, 1H), 8.46 (dt, J = 8.0, 2.0 Hz, 1H), 7.95 (d, J = 2.3 Hz, 1H), 7.51 – 7.45 (m, 3H), 7.43 (d, J = 5.2 Hz, 1H), 7.33 (dd, J = 8.2, 2.2 Hz, 1H), 7.18 (d, J = 8.3 Hz, 1H), 7.02 – 6.95 (m, 2H), 4.71 (s, 2H), 2.21 (s, 3H). LRMS m/z (ESI+): 490.30 [M+H]+) Compound 7 (TAD-0411659) N-{3-[4-(3-pyridyl)-2-pyrimidinylamino]-4-tolyl}(piodophenoxy)acetamide N H N N N N H O O I Compound 7 was synthesized according to general procedure A. (1H NMR: 400 MHz, DMSO δ 10.06 (s, 1H), 9.26 (d, J = 2.3 Hz, 1H), 8.95 (s, 1H), 8.70 (dd, J = 4.8, 1.7 Hz, 1H), 8.52 (d, J = 5.1 Hz, 1H), 8.46 (dt, J = 8.0, 2.0 Hz, 1H), 7.96 (d, J = 2.2 Hz, 1H), 7.67 – 7.60 (m, 2H), 7.48 (dd, J = 8.1, 4.7 Hz, 1H), 7.44 (d, J = 5.1 Hz, 1H), 7.33 (dd, J = 8.1, 2.2 Hz, 1H), 7.19 (d, J = 8.3 Hz, 1H), 6.90 – 6.83 (m, 2H), 4.71 (s, 2H), 2.22 (s, 3H). LRMS m/z (ESI+): 538.20 [M+H]+). Compound 8 (TAD-0411669) N-{3-[4-(3-pyridyl)-2-pyrimidinylamino]-4-tolyl}(p-tolyloxy)acetamide N H N N N N H O O Compound 8 was synthesized according to general procedure A. (1H NMR: (500 MHz, DMSO) δ 10.01 (s, 1H), 9.26 (d, J = 2.3 Hz, 1H), 8.95 (s, 1H), 8.69 (dd, J = 4.8, 1.7 Hz, 1H), 8.51 (d, J = 5.2 Hz, 1H), 8.45 (dt, J = 8.0, 2.0 Hz, 1H), 7.96 (d, J = 2.2 Hz, 1H), 7.48 (dd, J = 8.0, 4.8 Hz, 1H), 7.43 (d, J = 5.1 Hz, 1H), 7.34 (dd, J = 8.2, 2.2 Hz, 1H), 7.18 (d, J = 8.3 Hz, 1H), 7.11 (d, J = 8.3 Hz, 2H), 6.92 – 6.86 (m, 2H), 4.64 (s, 2H), 2.23 (s, 3H), 2.21 (s, 3H). LRMS m/z (ESI+): 426.20 [M+H]+). Compound 9 (TAD-0411670) N-{3-[4-(3-pyridyl)-2-pyrimidinylamino]-4-tolyl}[p- (trifluoromethyl)phenoxy]acetamide N H N N N N H O O CF3 Compound 9 was synthesized according to general procedure A. (1H NMR: (500 MHz, DMSO) δ 10.13 (s, 1H), 9.26 (dd, J = 2.4, 0.9 Hz, 1H), 8.96 (s, 1H), 8.68 (dd, J = 4.9, 1.7 Hz, 1H), 8.51 (d, J = 5.1 Hz, 1H), 8.46 (dt, J = 8.0, 2.0 Hz, 1H), 7.96 (d, J = 2.2 Hz, 1H), 7.69 (d, J = 8.8 Hz, 2H), 7.47 (ddd, J = 8.1, 4.8, 0.9 Hz, 1H), 7.43 (d, J = 5.2 Hz, 1H), 7.33 (dd, J = 8.2, 2.2 Hz, 1H), 7.19 (dd, J = 8.7, 2.3 Hz, 3H), 4.83 (s, 2H), 2.21 (s, 3H). LRMS m/z (ESI+): 480.35 [M+H]+) Compound 10 (TAD-0411479) N-{3-[4-(3-pyridyl)-2-pyrimidinylamino]-4-tolyl}(pmethoxyphenoxy)acetamide N H N N N N H O O OCH3 Compound 10 was synthesized according to general procedure A. (1H NMR: (400 MHz, DMSO) δ 9.97 (s, 1H), 9.25 (d, J = 2.2 Hz, 1H), 8.94 (s, 1H), 8.68 (dd, J = 4.9, 1.7 Hz, 1H), 8.50 (d, J = 5.1 Hz, 1H), 8.45 (dt, J = 8.0, 2.0 Hz, 1H), 7.95 (d, J = 2.2 Hz, 1H), 7.49 (dd, J = 8.0, 4.7 Hz, 1H), 7.42 (d, J = 5.1 Hz, 1H), 7.34 (dd, J = 8.3, 2.2 Hz, 1H), 7.17 (d, J = 8.3 Hz, 1H), 6.98 – 6.92 (m, 2H), 6.91 – 6.84 (m, 2H), 4.61 (s, 2H), 2.20 (s, 3H). LRMS m/z (ESI+): 442.35 [M+H]+). Compound 11 (TAD-0411671) N-{3-[4-(3-pyridyl)-2-pyrimidinylamino]-4-tolyl}(ptrifluoromethoxyphenoxy)acetamide N H N N N N H O O OCF3 Compound 11 was synthesized according to general procedure A. (1H NMR: (500 MHz, DMSO) δ 10.08 (s, 1H), 9.26 (dd, J = 2.4, 0.8 Hz, 1H), 8.96 (s, 1H), 8.69 (dd, J = 4.7, 1.7 Hz, 1H), 8.51 (d, J = 5.1 Hz, 1H), 8.46 (dt, J = 8.0, 2.0 Hz, 1H), 7.96 (d, J = 2.2 Hz, 1H), 7.49 (ddd, J = 8.1, 4.9, 0.9 Hz, 1H), 7.43 (d, J = 5.2 Hz, 1H), 7.36 – 7.29 (m, 3H), 7.19 (d, J = 8.3 Hz, 1H), 7.13 – 7.06 (m, 2H), 4.75 (s, 2H), 2.21 (s, 3H). LRMS m/z (ESI+): 496.30 [M+H]+) Compound 15 (TAD-0411664) N-{3-[4-(3-pyridyl)-2-pyrimidinylamino]-4-tolyl}(mfluorophenoxy)acetamide N H N N N N H O O F Compound 15 was synthesized according to general procedure A. (1H NMR: 400 MHz, DMSO δ 10.05 (s, 1H), 9.27 (d, J = 2.3 Hz, 1H), 8.96 (s, 1H), 8.69 (dd, J = 4.8, 1.7 Hz, 1H), 8.52 (d, J = 5.2 Hz, 1H), 8.46 (dt, J = 8.0, 2.0 Hz, 1H), 7.96 (d, J = 2.2 Hz, 1H), 7.49 (dd, J = 8.0, 4.8 Hz, 1H), 7.44 (d, J = 5.1 Hz, 1H), 7.39 – 7.29 (m, 2H), 7.19 (d, J = 8.3 Hz, 1H), 6.95 – 6.74 (m, 3H), 4.74 (s, 2H), 2.22 (s, 3H). LRMS m/z (ESI+): 430.40 [M+H]+). Compound 16 (TAD-0411665) N-{3-[4-(3-pyridyl)-2-pyrimidinylamino]-4-tolyl}(mchlorophenoxy)acetamide N H N N N N H O O Cl Compound 16 was synthesized according to general procedure A. (1H NMR: 400 MHz, DMSO δ 10.05 (s, 1H), 9.27 (d, J = 2.3 Hz, 1H), 8.96 (s, 1H), 8.70 (dd, J = 4.8, 1.6 Hz, 1H), 8.52 (d, J = 5.1 Hz, 1H), 8.47 (dt, J = 8.0, 2.0 Hz, 1H), 7.96 (d, J = 2.2 Hz, 1H), 7.50 (dd, J = 8.0, 4.8 Hz, 1H), 7.44 (d, J = 5.1 Hz, 1H), 7.35 (ddd, J = 8.2, 5.2, 3.0 Hz, 2H), 7.20 (d, J = 8.3 Hz, 1H), 7.12 (t, J = 2.2 Hz, 1H), 7.02 (ddd, J = 17.9, 8.1, 2.2 Hz, 2H), 4.75 (s, 2H), 2.22 (s, 3H).LRMS m/z (ESI+): 446.30 [M+H]+). Compound 17 (TAD-0411666) N-{3-[4-(3-pyridyl)-2-pyrimidinylamino]-4-tolyl}(mbromophenoxy)acetamide N H N N N N H O O Br Compound 17 was synthesized according to general procedure A. (1H NMR: (500 MHz, DMSO) δ 10.05 (s, 1H), 9.26 (d, J = 2.3 Hz, 1H), 8.95 (s, 1H), 8.69 (dd, J = 4.8, 1.6 Hz, 1H), 8.51 (d, J = 5.1 Hz, 1H), 8.46 (dt, J = 8.1, 2.0 Hz, 1H), 7.96 (d, J = 2.2 Hz, 1H), 7.48 (dd, J = 8.1, 4.8 Hz, 1H), 7.43 (d, J = 5.2 Hz, 1H), 7.34 (dd, J = 8.1, 2.2 Hz, 1H), 7.30 – 7.22 (m, 2H), 7.22 – 7.15 (m, 2H), 7.06 – 7.00 (m, 1H), 4.74 (s, 2H), 2.21 (s, 3H). LRMS m/z (ESI+): 490.35 [M+H]+). Compound 18 (TAD-0411668) N-{3-[4-(3-pyridyl)-2-pyrimidinylamino]-4-tolyl}(m-tolyloxy)acetamide N H N N N N H O O Compound 18 was synthesized according to general procedure A. (1H NMR: (500 MHz, DMSO) δ 10.01 (s, 1H), 9.26 (d, J = 2.2 Hz, 1H), 8.95 (s, 1H), 8.69 (dd, J = 4.8, 1.6 Hz, 1H), 8.51 (d, J = 5.1 Hz, 1H), 8.46 (dt, J = 8.0, 2.0 Hz, 1H), 7.96 (d, J = 2.2 Hz, 1H), 7.49 (dd, J = 8.0, 4.8 Hz, 1H), 7.43 (d, J = 5.1 Hz, 1H), 7.34 (dd, J = 8.2, 2.2 Hz, 1H), 7.18 (dt, J = 7.8, 3.7 Hz, 2H), 6.84 (t, J = 2.0 Hz, 1H), 6.79 (dd, J = 8.4, 2.4 Hz, 2H), 4.66 (s, 2H), 2.28 (s, 3H), 2.21 (s, 3H). LRMS m/z (ESI+): 426.20 [M+H]+). Compound 19 TAD-0411737 N-{3-[4-(3-pyridyl)-2-pyrimidinylamino]-4-tolyl}(mmethoxyphenoxy)acetamide N H N N N N H O O OCH3 Compound 19 was synthesized according to general procedure A. (1H NMR: 400 MHz, DMSO δ 10.03 (s, 1H), 9.26 (d, J = 2.2 Hz, 1H), 8.96 (s, 1H), 8.69 (dd, J = 4.8, 1.6 Hz, 1H), 8.52 (d, J = 5.1 Hz, 1H), 8.46 (dt, J = 8.0, 2.0 Hz, 1H), 7.96 (d, J = 2.1 Hz, 1H), 7.49 (dd, J = 8.0, 4.8 Hz, 1H), 7.44 (d, J = 5.2 Hz, 1H), 7.35 (dd, J = 8.3, 2.2 Hz, 1H), 7.21 (td, J = 8.4, 3.9 Hz, 2H), 6.61 – 6.55 (m, 3H), 4.68 (s, 2H), 3.74 (s, 3H), 2.21 (s, 3H). LRMS m/z (ESI+): 442.20 [M+H]+). Compound 20 (TAD-0411836) (N-(4-methyl-3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)-2-(3nitrophenoxy)acetamide) N H N N N N H O O NO2 Compound 20 was synthesized according to general procedure A. (1H NMR: (500 MHz, DMSO) δ 10.14 (s, 1H), 9.26 (d, J = 2.3 Hz, 1H), 8.97 (s, 1H), 8.68 (dd, J = 4.8, 1.7 Hz, 1H), 8.51 (d, J = 5.1 Hz, 1H), 8.46 (dt, J = 8.0, 2.0 Hz, 1H), 7.95 (d, J = 2.2 Hz, 1H), 7.89 – 7.82 (m, 2H), 7.62 (t, J = 8.2 Hz, 1H), 7.52 – 7.47 (m, 2H), 7.44 (d, J = 5.1 Hz, 1H), 7.34 (dd, J = 8.2, 2.2 Hz, 1H), 7.20 (d, J = 8.3 Hz, 1H), 4.88 (s, 2H), 2.21 (s, 3H). LRMS m/z (ESI+): 457.15 [M+H]+) Compound 21 (TAD-0411661) N-{3-[4-(3-pyridyl)-2-pyrimidinylamino]-4-tolyl}(ofluorophenoxy)acetamide N H N N N N H O O F Compound 21 was synthesized according to general procedure A. (1H NMR: 400 MHz, DMSO δ 10.13 (s, 1H), 9.26 (d, J = 2.3 Hz, 1H), 8.96 (s, 1H), 8.70 (dd, J = 4.8, 1.7 Hz, 1H), 8.52 (d, J = 5.2 Hz, 1H), 8.47 (dt, J = 8.1, 2.0 Hz, 1H), 7.95 (d, J = 2.2 Hz, 1H), 7.49 (dd, J = 8.0, 4.8 Hz, 1H), 7.44 (d, J = 5.1 Hz, 1H), 7.32 (dd, J = 8.2, 2.2 Hz, 1H), 7.29 – 7.22 (m, 1H), 7.19 (d, J = 8.3 Hz, 1H), 7.16 – 7.10 (m, 2H), 6.98 (ddd, J = 11.5, 6.4, 3.7 Hz, 1H), 4.81 (s, 2H), 2.22 (s, 3H). LRMS m/z (ESI+): 430.25 [M+H]+). Compound 22 (TAD-0411662) N-{3-[4-(3-pyridyl)-2-pyrimidinylamino]-4-tolyl}(ochlorophenoxy)acetamide N H N N N N H O O Cl Compound 22 was synthesized according to general procedure A. (1H NMR: (500 MHz, DMSO) δ 10.11 (s, 1H), 9.25 (d, J = 2.3 Hz, 1H), 8.95 (s, 1H), 8.69 (dd, J = 4.8, 1.7 Hz, 1H), 8.51 (d, J = 5.1 Hz, 1H), 8.46 (dt, J = 8.0, 2.0 Hz, 1H), 7.95 (d, J = 2.2 Hz, 1H), 7.50 – 7.41 (m, 3H), 7.33 – 7.26 (m, 2H), 7.19 (d, J = 8.3 Hz, 1H), 7.09 (dd, J = 8.3, 1.4 Hz, 1H), 6.99 (td, J = 7.6, 1.4 Hz, 1H), 4.83 (s, 2H), 2.21 (s, 3H). LRMS m/z (ESI+): 446.25 [M+H]+). Compound 23 (TAD-0411663) N-{3-[4-(3-pyridyl)-2-pyrimidinylamino]-4-tolyl}(obromophenoxy)acetamide N H N N N N H O O Br Compound 23 was synthesized according to general procedure A. (1H NMR: 400 MHz, DMSO δ 10.05 (s, 1H), 9.26 (d, J = 2.3 Hz, 1H), 8.96 (s, 1H), 8.70 (dd, J = 4.7, 1.6 Hz, 1H), 8.52 (d, J = 5.1 Hz, 1H), 8.47 (dt, J = 8.0, 2.0 Hz, 1H), 7.95 (d, J = 2.4 Hz, 1H), 7.62 (dd, J = 7.9, 1.6 Hz, 1H), 7.49 (dd, J = 8.0, 4.8 Hz, 1H), 7.44 (d, J = 5.2 Hz, 1H), 7.38 – 7.31 (m, 2H), 7.20 (d, J = 8.3 Hz, 1H), 7.11 – 7.05 (m, 1H), 6.93 (td, J = 7.6, 1.3 Hz, 1H), 4.83 (s, 2H), 2.22 (s, 3H).LRMS m/z (ESI+): 490.20 [M+H]+). Compound 24 (TAD-0411667) N-{3-[4-(3-pyridyl)-2-pyrimidinylamino]-4-tolyl}(o-tolyloxy)acetamide N H N N N N H O O Compound 24 was synthesized according to general procedure A. (1H NMR: (500 MHz, DMSO) δ 10.01 (s, 1H), 9.25 (d, J = 2.3 Hz, 1H), 8.95 (s, 1H), 8.69 (dd, J = 4.8, 1.7 Hz, 1H), 8.51 (d, J = 5.2 Hz, 1H), 8.46 (dt, J = 8.1, 2.0 Hz, 1H), 7.95 (d, J = 2.2 Hz, 1H), 7.48 (dd, J = 8.0, 4.8 Hz, 1H), 7.43 (d, J = 5.2 Hz, 1H), 7.32 (dd, J = 8.2, 2.2 Hz, 1H), 7.21 – 7.10 (m, 3H), 6.87 (t, J = 7.8 Hz, 2H), 4.71 (s, 2H), 2.24 (s, 3H), 2.21 (s, 3H). LRMS m/z (ESI+): 426.25 [M+H]+). Compound 25 (TAD-0411832) N-{3-[4-(3-pyridyl)-2-pyrimidinylamino]-4-tolyl}(omethoxyphenoxy)acetamide N H N N N N H O O O Compound 25 was synthesized according to general procedure A. (1H NMR: (500 MHz, DMSO) δ 10.01 (s, 1H), 9.26 (d, J = 2.2 Hz, 1H), 8.96 (s, 1H), 8.69 (dd, J = 4.8, 1.7 Hz, 1H), 8.51 (d, J = 5.2 Hz, 1H), 8.46 (dt, J = 8.0, 2.0 Hz, 1H), 7.96 (d, J = 2.4 Hz, 1H), 7.49 (dd, J = 8.0, 4.8 Hz, 1H), 7.43 (d, J = 5.2 Hz, 1H), 7.33 (dd, J = 8.1, 2.3 Hz, 1H), 7.19 (d, J = 8.3 Hz, 1H), 7.04 – 6.93 (m, 3H), 6.88 (td, J = 7.6, 1.6 Hz, 1H), 4.68 (s, 2H), 3.80 (s, 3H), 2.21 (s, 3H). LRMS m/z (ESI+): 442.20 [M+H]+) Compound 26 (TAD-0411828) N-{3-[4-(3-pyridyl)-2-pyrimidinylamino]-4-tolyl}(onitrophenoxy)acetamide N H N N N N H O O O2N Compound 26 was synthesized according to general procedure A. (1H NMR: (500 MHz, DMSO) δ 10.04 (s, 1H), 9.25 (dd, J = 2.3, 0.8 Hz, 1H), 8.96 (s, 1H), 8.69 (dd, J = 4.9, 1.7 Hz, 1H), 8.51 (d, J = 5.1 Hz, 1H), 8.46 (dt, J = 8.1, 2.0 Hz, 1H), 7.97 – 7.90 (m, 2H), 7.66 (ddd, J = 8.9, 7.4, 1.7 Hz, 1H), 7.49 (ddd, J = 8.1, 4.8, 0.9 Hz, 1H), 7.44 (d, J = 5.1 Hz, 1H), 7.31 (ddd, J = 16.3, 8.4, 1.7 Hz, 2H), 7.20 (d, J = 8.3 Hz, 1H), 7.16 (td, J = 7.7, 7.3, 1.1 Hz, 1H), 4.95 (s, 2H), 2.21 (s, 3H). LRMS m/z (ESI+): 457.55 [M+H]+) Compound 27 (TAD-0411834) N-{3-[4-(3-pyridyl)-2-pyrimidinylamino]-4-tolyl}(4-chloro-2tolyloxy)acetamide N H N N N N H O O Cl Compound 27 was synthesized according to general procedure A. (1H NMR: (500 MHz, DMSO) δ 10.04 (s, 1H), 9.26 (d, J = 2.3 Hz, 1H), 8.96 (s, 1H), 8.69 (dd, J = 4.8, 1.7 Hz, 1H), 8.51 (d, J = 5.1 Hz, 1H), 8.46 (dt, J = 8.0, 2.0 Hz, 1H), 7.93 (d, J = 2.3 Hz, 1H), 7.48 (dd, J = 8.0, 4.8 Hz, 1H), 7.43 (d, J = 5.2 Hz, 1H), 7.31 (dd, J = 8.2, 2.2 Hz, 1H), 7.25 (d, J = 2.7 Hz, 1H), 7.22 – 7.17 (m, 2H), 6.90 (d, J = 8.7 Hz, 1H), 4.74 (s, 2H), 2.23 (s, 3H), 2.21 (s, 3H). LRMS m/z (ESI+): 460.15 [M+H]+) Compound 28 (TAD-0411734) N-{3-[4-(3-pyridyl)-2-pyrimidinylamino]-4-tolyl}(2,4dichlorophenoxy)acetamide N H N N N N H O O Cl Cl Compound 28 was synthesized according to general procedure A. (1H NMR: 400 MHz, DMSO δ 10.12 (s, 1H), 9.26 (d, J = 2.3 Hz, 1H), 8.95 (s, 1H), 8.70 (dd, J = 4.8, 1.6 Hz, 1H), 8.52 (d, J = 5.1 Hz, 1H), 8.47 (dt, J = 8.1, 2.0 Hz, 1H), 7.94 (d, J = 2.2 Hz, 1H), 7.61 (d, J = 2.6 Hz, 1H), 7.49 (dd, J = 8.0, 4.8 Hz, 1H), 7.44 (d, J = 5.2 Hz, 1H), 7.38 (dd, J = 8.9, 2.6 Hz, 1H), 7.30 (dd, J = 8.2, 2.2 Hz, 1H), 7.19 (d, J = 8.3 Hz, 1H), 7.13 (d, J = 9.0 Hz, 1H), 4.87 (s, 2H), 2.22 (s, 3H). LRMS m/z (ESI+): 480.20 [M+H]+). Compound 29 (TAD-0411728) N-{3-[4-(3-pyridyl)-2-pyrimidinylamino]-4-tolyl}p-fluorobenzamide N H N N N N H O F Compound 29 was synthesized according to general procedure A. (1H NMR: 400 MHz, DMSO δ 10.23 (s, 1H), 9.29 (d, J = 2.2 Hz, 1H), 8.98 (s, 1H), 8.70 (dd, J = 4.8, 1.7 Hz, 1H), 8.53 (d, J = 5.1 Hz, 1H), 8.49 (dt, J = 8.1, 2.0 Hz, 1H), 8.08 (d, J = 2.2 Hz, 1H), 8.07 – 8.02 (m, 2H), 7.53 (dd, J = 8.0, 4.8 Hz, 1H), 7.48 (dd, J = 8.2, 2.2 Hz, 1H), 7.44 (d, J = 5.1 Hz, 1H), 7.41 – 7.34 (m, 2H), 7.23 (d, J = 8.2 Hz, 1H), 2.24 (s, 3H). LRMS m/z (ESI+): 400.20 [M+H]+). Compound 30 (TAD-0411729) N-{3-[4-(3-pyridyl)-2-pyrimidinylamino]-4-tolyl}p-chlorobenzamide N H N N N N H O Cl Compound 30 was synthesized according to general procedure A. (1H NMR: 400 MHz, DMSO δ 10.28 (s, 1H), 9.29 (d, J = 2.2 Hz, 1H), 8.99 (s, 1H), 8.70 (d, J = 4.6 Hz, 1H), 8.53 (d, J = 5.2 Hz, 1H), 8.49 (d, J = 8.3 Hz, 1H), 8.09 (s, 1H), 8.00 (d, J = 8.5 Hz, 2H), 7.62 (d, J = 8.5 Hz, 2H), 7.53 (dd, J = 8.0, 4.8 Hz, 1H), 7.48 (d, J = 8.3 Hz, 1H), 7.45 (d, J = 5.1 Hz, 1H), 7.23 (d, J = 8.3 Hz, 1H), 2.24 (s, 3H).LRMS m/z (ESI+): 416.25 [M+H]+). Compound 31 (TAD-0411730) N-{3-[4-(3-pyridyl)-2-pyrimidinylamino]-4-tolyl}p-bromobenzamide N H N N N N H O Br Compound 31 was synthesized according to general procedure A. (1H NMR: 400 MHz, DMSO δ 10.28 (s, 1H), 9.29 (d, J = 2.3 Hz, 1H), 8.98 (s, 1H), 8.70 (dd, J = 4.8, 1.7 Hz, 1H), 8.53 (d, J = 5.1 Hz, 1H), 8.51 – 8.47 (m, 1H), 8.09 (d, J = 2.1 Hz, 1H), 7.92 (d, J = 8.5 Hz, 2H), 7.76 (d, J = 8.5 Hz, 2H), 7.53 (dd, J = 8.0, 4.8 Hz, FUNDING SOURCES This work was primarily supported by NIA grant U54AG065181 (Palkowitz, Lamb, Richardson) to the IUSM Purdue TREAT-AD Center. KEY WORDS Lyn, Src, Alzheimer’s disease, Type II Kinase Inhibitors DATA AVAILABILITY Datasets will be made available via the AD Knowledge Portal. REFERENCES 1. Brown MT, Cooper JA. Regulation, substrates and functions of src. Biochim Biophys Acta. Jun 7 1996;1287(2-3):121-49. doi:10.1016/0304-419x(96)00003-0 2. Mkaddem SB, Murua A, Flament H, et al. Lyn and Fyn function as molecular switches that control immunoreceptors to direct homeostasis or inflammation. Nat Commun. Aug 15 2017;8(1):246. doi:10.1038/s41467-017-00294-0 3. Weerawarna PM, Richardson TI. 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