Autophagy modulators and methods of use

Abstract

Disclosed herein are aspects of a compound having a structure according to Formula I. The compound may have a structure according to Formula II. In some aspects, the compound may be useful for modulating autophagy. Also disclosed are aspects of a method for making the compound, and aspects of a method for using the compound. The compounds may be useful for treating a disease or condition, such as a disease or condition where modulating autophagy may be beneficial.

Description

[0001] 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 AUTOPHAGY MODULATORS AND METHODS OF USE CROSS REFERENCE TO RELATED APPLICATION This application claims the benefit of the earlier filing date of U.S. provisional patent application No.63 / 707,961, filed October 16, 2024, which is incorporated herein by reference in its entirety. ACKNOWLEDGMENT OF GOVERNMENT SUPPORT This invention was made with government support under grant number R01 NS114413 awarded by the National Institutes of Health. The government has certain rights in the invention. FIELD This disclosure relates to novel autophagy modulating compounds and compositions and to methods of treating disease. BACKGROUND Macroautophagy (hereafter autophagy) is a conserved, eukaryotic, catabolic pathway that maintains homeostasis by degrading and recycling intracellular components. Cellular cargo is sequestered within the double-layered membrane of an autophagosome that fuses with a lysosome to form the autolysosome, where the cargo is degraded by enzymes contained within the acidic lysosome lumen. The resulting products are then recycled and used for other cellular processes. Autophagy has been implicated in a wide array of processes including aging, cancer, and neurodegeneration. In the past decade, modulation of the autophagy pathway has emerged as a promising therapeutic strategy for a wide range of diseases, including a rare genetic disease called Niemann-Pick Type C Disease (NPC). SUMMARY Disclosed herein are aspects of a compound according to Formula I 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 A or a pharmaceutically acceptable salt thereof. With respect to 10 membered heteroaryl. R1 is selected from C1-6alkyl, C6-10aryl, C6- membered heteroaryl, 6-10 membered heteroaryl-C alkyl, C 1-6 3-C1-6alkyl, 5- or 6-membered heterocyclyl or 5- or 6-membered heterocyclyl-C1-6alkyl, -NR5R6, - alkyl-NR5R6, or -NRa-alkyl-NR5R6, where R1is optionally substituted with one or more groups independently selected from -NR7R8, -alkyl-NR7R8, -C(O)R7, -C(O)NR7R8, -SO2R7, C6-10aryl-C1-6alkyl, 6-10 membered heteroaryl-C1-6alkyl, C3-6cycloalkyl-C1-6alkyl, or 5- or 6- membered heterocyclyl-C1-6alkyl, and each Raindependently is H or C1-6alkyl. R2and R3are independently selected from H, C1-6alkyl, C6-10aryl, C6-10aryl-C1-6alkyl, 6-10 membered heteroaryl, 6-10 membered heteroaryl-C1-6alkyl, C3-6cycloalkyl, C3-6cycloalkyl-C1-6alkyl, 5- or 6-membered heterocyclyl or 5- or 6-membered heterocyclyl-C1-6alkyl , or R2 and R3, together with the N atom to which they are attached form a 5- or 6- membered heterocyclyl, where R2and R3are independently optionally substituted with one or more substituents selected from C1-6alkyl, -SO2C1-6alkyl, -C(O)R7, or C(O)NR7R8, such as C1-6alkyl, -SO2C1-6alkyl, or -C(O)C1-6alkyl. R4 is H or C1-6alkyl. R5 and R6 are independently selected from H, -C(O)R7, -C(O)NR7R8, -SO2R7, C1-6alkyl, C6-10aryl, C6-10aryl-C1-6alkyl, 6-10 membered heteroaryl, 6-10 membered heteroaryl- C1-6alkyl, C3-6cycloalkyl, C3-6cycloalkyl-C1-6alkyl, 5- or 6-membered heterocyclyl, or 5- or 6- membered heterocyclyl-C1-6alkyl, or R5and R6together form a 5- or 6-membered optionally substituted heterocyclyl, where R5 and R6 are independently optionally substituted with halo or C1-6alkyl. R7 and R8 are independently selected from C1-6alkyl, C6-10aryl, C6-10aryl-C1-6alkyl, 6- 10 membered heteroaryl, 6-10 membered heteroaryl-C1-6alkyl, C3-6cycloalkyl, C3-6cycloalkyl- C1-6alkyl, 5- or 6-membered heterocyclyl, or 5- or 6-membered heterocyclyl-C1-6alkyl, where R7and R8are independently optionally substituted with one or more groups independently selected from -NR5R6, -alkyl-NR5R6, -C(O)R5, -C(O)NR5R6, -SO2R5, C6-10aryl, C6-10aryl-C1-6alkyl, 6-10 membered heteroaryl, 6-10 membered heteroaryl-C1-6alkyl, C3-6cycloalkyl, C1-6cycloalkyl-C1-6alkyl, 5- or 6-membered heterocyclyl, and 5- or 6-membered heterocyclyl-C1-6alkyl. 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 Additionally, with respect to Formula I, the compound is not, or is other than, . and / or a or a a compound disclosed herein. The foregoing and other objects, features, and advantages of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures. BRIEF DESCRIPTION OF THE DRAWINGS FIG.1A provides representative eGFP-LC3 HTS results (compounds at 20µM) where the x and y-axis represent each duplicate of plate 13 in the library with an R2of 0.8 (left). The red circles represent hits where the normalized average puncta / cell z-score was above 2.199, and the blue triangles represent cytotoxic compounds that had a nuclear count z-score below - 1.799. Data are normalized to the average DMSO value. And on the right statistical parameters are provided that were used to evaluate HTS quality and identify hits, including representative data from plate 13. Data for other plates are provided in FIG.7. FIG.1B provides digital images illustrating eGFP-LC3 HeLa cells after a 4-hour treatment with DMSO, CQ (20 µM), 1 (5 µM), and 2 (5 µM), where blue is the nucleus stained with Hoechst 33342, the green cytosol is diffusely located LC3-I, and the green puncta are indicative of LC3-II located in autophagosome membranes. FIG.1C is a graph illustrating the autophagy modulators from the eGFP-LC3 HTS that were evaluated in the filipin assay for 24 hours in I1061T NPC1 fibroblasts in duplicate and their activity assessed using z-score, where 1 and 2 (10 µM, red circles) had z-scores comparable to 2HPβCD (1 mM, green circles). Filipin intensity is measured as the pit integrated intensity, which is the total pixel intensity over all the pit areas in the image. FIG.1D provides the structure of hit compound 1. FIG.1E provides the structure of hit compound 2. 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 FIG.1F is a graph of filipin intensity versus compound, illustrating the evaluation of 1 and 2 (5 and 10 µM), Rap (4 µM), and 2HPβCD (1 mM) in filipin assay. Data are presented as biological duplicates. Significance was determined using Ordinary ANOVA followed by Dunnett’s multiple comparison test using GraphPad Prism 9.0, where ns = not significant (P>0.05) and **** is P≤0.0001. FIG.2A is a graph illustrating average puncta per cell count following treatment with compounds 1 (5 and 10 µM) and 2 (5 and 10 µM) compared to CQ (20 µM), Rap (4 µM), and 2HPβCD (1 mM) in the eGFP-LC3 HeLa assay. Data are presented as biological duplicates. FIG.2B is a graph illustrating resynthesized 2 (EC50 =14.33 µM) in an eGFP-LC3 assay at multiple doses (0.63-80 µM) compared to CQ (20 µM) and DMSO. FIG.2C is a graph illustrating resynthesized 2 (20 and 40 µM), DMSO, 2HPβCD (1 mM), and 2HPβCD (300 µM) in a filipin assay in I1061T NPC1 fibroblasts. Filipin intensity is measured as the pit integrated intensity, which is the total pixel intensity over all the pit areas in the image. FIG.2D provides representative images from mCherry-GFP-LC3 dual reporter HeLa assay after 24 hours compound treatment following treatment with DMSO, CQ (20 µM), 1 (10 µM) and 2 (20 µM). FIG.2E is a graph illustrating the quantitative effect of 1 (5-80 µM) on average number of autolysosomes vs. autophagosomes compared to DMSO and CQ (20 µM) in dual reporter assay. FIG.2F is a graph illustrating the quantitative effect of 2 (5-80 µM) on average number of autolysosomes versus autophagosomes compared to DMSO and CQ (20 µM) in dual reporter assay. FIG.3 is a table providing the activity of compound 2 analogues in eGFP-LC3 assay and filipin assay. The EC50for eGFP-LC3 assay was calculated from an 8-point dose (0.63- 80 μM) treatment after 4 hours and was measured as the average puncta / cell. eGFP-LC3 data are presented as mean ± SEM of three independent experiments each with duplicate biological replicates. The EC50 for filipin intensity was calculated using an 8-point dose (0.63-80 μM) treatment after 24 hours and was measured as the pit integrated intensity. Filipin data are presented as mean ± SEM of two independent experiments each with duplicate biological replicates. 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 FIG.4A is a graph illustrating puncta / cell counts in DQ-BSA assay following compound treatment (24 hours) in I1061T NPC1 fibroblasts. The effects of 1 (10 µM), 2 (20 µM), 2HPβCD (1 mM), CQ (20 µM), and BafA1 (100 nM) were evaluated. Values are normalized to DMSO and data are presented as mean ± SEM of two independent experiments each with duplicate biological replicates. FIG.4B is a graph illustrating puncta / cell counts in DQ-BSA assay following treatment (6 hours) in HeLa cells after treatment with 1 (10, 20 µM), 2 (20, 40 µM), 2HPβCD (1 mM), CQ (20 µM), and BafA1 (100 nM). Values are normalized to DMSO and data are presented as mean ± SEM of two independent experiments each with duplicate biological replicates. FIG.4C is a graph illustrating the viability of I1061T NPC1 fibroblasts following treatment with DMSO, 1 and 2 (10-80 µM), 2HPβCD (1 mM), CQ (40 µM), BafA1 (100 nM) in the CellTiter-Glo 2.0 assay after 24 hours. Data are presented as mean ± SEM of three independent experiments each with duplicate biological replicates. FIG.4D provides representative P-p70S6K and p70S6K immunoblots after 24 hours compound treatment in I1061T NPC1 fibroblasts with DMSO, Rap (1 µM), 2HPβCD (1 mM), CBZ (100 µM), and compounds 2 (5 µM) and 1 (5 µM). FIG.4E provides representative P-p70S6K and p70S6K immunoblots following treatment of I1061T NPC1 fibroblasts with resynthesized compounds 1 (20 µM) and 2 (80 µM), CQ (20 µM), and BafA1 (0.2 µM). FIG.4F provides a representative NPC1 immunoblot of I1061T NPC1 fibroblasts following treatment with DMSO, 1 (10 and 20 µM), 2 (10 and 20 µM), and 2HPβCD (1 mM) (top) and a graph illustrating quantified NPC1 immunoblot presented as mean ± SEM of three independent experiments normalized to β-actin (bottom). FIG.4G is a graph illustrating results from a filipin assay in NPC1 null HeLa cells following treatment with DMSO, 1 (40 µM), 2 (80 µM), and 2HPβCD (0.3 and 1 mM). Data are presented as mean ± SEM of three independent experiments each with duplicate biological replicates. Significance was determined using Ordinary one-way ANOVA followed by Dunnett’s multiple comparison test using GraphPad Prism 9.0, where ns = not significant (P>0.05), *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001. FIG.5A is a table providing the name of the affected protein, followed by the gene name, and the ratio represents protein expression after 24-hour treatment with 2 (80 µM) in I1061T NPC1 fibroblasts divided by the protein expression after treatment with the vehicle, 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 DMSO. The green represents a decrease in protein expression, and the p-value is a measure of significance where ns = not significant (P>0.05), *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001. FIG.5B provides representative immunoblot images evaluating levels of pro-LGMN and LGMN with β-actin control after 24-hour treatment with DMSO, 2 (80 µM), CQ (20 µM), and BafA1 (100 nM) in I1061T NPC1 fibroblasts (top), and a graph illustrating the proteins quantified by densitometry using ImageJ from three independent experiments and are presented as mean ± SEM (bottom). FIG.5C provides representative immunoblot images evaluating levels of CTSZ with β-actin control after 24-hour treatment with DMSO, 2 (80 µM), CQ (20 µM), and BafA1 (100 nM) in I1061T NPC1 fibroblasts (top), and a graph illustrating the proteins quantified by densitometry using ImageJ from three independent experiments and are presented as mean ± SEM (bottom). FIG.5D provides representative immunoblot images evaluating levels of pro-CTSB and CTSB with β-actin control after 24-hour treatment with DMSO, 2 (80 µM), CQ (20 µM), and BafA1 (100 nM) in I1061T NPC1 fibroblasts (top), and a graph illustrating the proteins quantified by densitometry using ImageJ from three independent experiments and are presented as mean ± SEM (bottom). FIG.5E provides representative immunoblot images evaluating levels of pro-CTSD and CTSD with β-actin control after 24-hour treatment with DMSO, 2 (80 µM), CQ (20 µM), and BafA1 (100 nM) in I1061T NPC1 fibroblasts (top), and a graph illustrating the proteins quantified by densitometry using ImageJ from three independent experiments and are presented as mean ± SEM (bottom). FIG.5F provides representative immunoblot images evaluating levels of glycosylated LAMP1 and non-glycosylated LAMP1 with β-actin control after 24-hour treatment with DMSO, 2 (80 µM), CQ (20 µM), and BafA1 (100 nM) in I1061T NPC1 fibroblasts (top), and a graph illustrating the proteins quantified by densitometry using ImageJ from three independent experiments and are presented as mean ± SEM (bottom). FIG.5G provides a relationship map illustrating the relationship of significantly altered proteins, identified via mass spectrometry. The proteins were entered into WebGestalt for relationship mapping, and genes involved in protein catabolic processes are highlighted in red. Protein changes detected in this dataset are represented by larger circles, and the smaller circles represent the top-ranking neighbors assigned by WebGestalt. 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 FIG 5H is a table illustrating genes that were not detected to have significant changes but were deemed “top ranking neighbors”. FIG.6A provides a graph illustrating immunoblot analysis of proteasomal and autophagic markers p21, where I1061T fibroblasts were treated with CQ (20 μM), MG132 (10 μM), Rap (200 nM), PIK-III (10 μM), or compound 2 for 24 hours prior to lysis and analysis. FIG.6B provides a graph illustrating immunoblot analysis of proteasomal and autophagic markers p27, where I1061T fibroblasts were treated with CQ (20 μM), MG132 (10 μM), Rap (200 nM), PIK-III (10 μM), or compound 2 for 24 hours prior to lysis and analysis. FIG.6C provides a graph illustrating immunoblot analysis of proteasomal and autophagic markers p62, where I1061T fibroblasts were treated with CQ (20 μM), MG132 (10 μM), Rap (200 nM), PIK-III (10 μM), or compound 2 for 24 hours prior to lysis and analysis. FIG.6D provides a graph illustrating immunoblot analysis of proteasomal and autophagic markers LC3, where I1061T fibroblasts were treated with CQ (20 μM), MG132 (10 μM), Rap (200 nM), PIK-III (10 μM), or compound 2 for 24 hours prior to lysis and analysis. FIG.6E is a Western blot of a glycan digestion assay performed with HeLa cells treated for 24 hours with DMSO, HPβCD (1 mM), BafA1 (0.1 μM), or compound 2 (20 μM). Lysates were subjected to no treatment (NT), Endo H treatment (E), or PNGase treatment (P) for 3 hours prior to western blot analysis. FIG.6F is a graph illustrating quantified immunoblots for NT and E samples. Data are presented as mean ± SEM of three independent experiments. Significance was determined by unpaired two-tailed t-test comparing control (DMSO) E-Sen band intensity with the E-Sen band intensity for each of the compounds using GraphPad Prism 9.0, where ns = not significant (P>0.05), *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001. FIG.6G is a Western blot of a glycan digestion assay performed with I1061T NPC1 fibroblasts treated for 24 hours with DMSO, HPβCD (1 mM), BafA1 (0.1 μM), or compound 2 (20 μM). Lysates were subjected to no treatment (NT), Endo H treatment (E), or PNGase treatment (P) for 3 hours prior to western blot analysis. FIG.6H is a graph illustrating the quantified immunoblots for E samples with bands at 180 kDa (E-Res) and 140 kDa (E-Sen). Data are presented as mean ± SEM of three independent experiments. Significance was determined by unpaired two-tailed t-test 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 comparing control (DMSO) E-Sen band intensity with the E-Sen band intensity for each of the compounds using GraphPad Prism 9.0, where ns = not significant (P>0.05), *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001. FIG.7 provides graphs illustrating evaluation of positive control in eGFP-LC3 assay. Left provides eGFP-LC3 results after compound treatment (4 hours) with CQ (10, 20, 40 μM) and vehicle (DMSO). Right provides results for CQ evaluated in dose to assess EC50(14.95 μM) where all data are presented as mean ± SEM of three independent experiments each with duplicate biological replicates. Significance was determined using Ordinary ANOVA followed by Dunnett’s multiple comparison test using GraphPad Prism 9.0, where ns = not significant (P>0.05) and **** is P≤0.0001. FIG.8 provides eGFP-LC3 HTS results (20 µM) from 32 plates (001-3 to 032-3) where the x and y-axis represent each duplicate and their corresponding R2is in the upper left-hand corner of each graph. Red dots represent potential autophagy activators and aqua dots represent cytotoxic compounds. Some compounds in the library precipitated out of solution, such as in plate 002-3, skewing the R2values for these plates. FIG.9 provides graphs of activity of resynthesized 1 (EC50=10.97 µM) in eGFP-LC3 assay at multiple doses (0.63-80 µM) compared to the sample average of CQ (20 µM) and DMSO (left), and activity of resynthesized 1 (20 and 40 µM), DMSO, and 2HPβCD (1 mM) in filipin assay in I1061T NPC1 fibroblasts (right). Filipin intensity is measured as the pit integrated intensity, which is the total pixel intensity over all the pit areas in the image. All data are presented as mean ± SEM of three independent experiments each with duplicate biological replicates. Significance was determined using Ordinary ANOVA followed by Dunnett’s multiple comparison test using GraphPad Prism 9.0, where ns = not significant (P>0.05), *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001. FIG.10 is a graph illustrating eGFP-LC3 assay dose response curves (0.63-80 µM) for resynthesized hit compound 2 and first- and second-generation analogues after 4-hour compound treatment. Data are presented as mean ± SEM of three independent experiments each with duplicate biological replicates. Data points were fitted using the Dose-response- Special, X is log (concentration) function in GraphPad Prism 9.0. FIG.11 is a graph of filipin assay dose response curves (0.63-80 µM) for resynthesized hit compound 2 and second-generation analogues after 24 hours compound treatment. Data are presented as mean ± SEM of two independent experiments each with 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 duplicate biological replicates. Data points were fitted using the Dose-response- Special, X is log (concentration) function in GraphPad Prism 9.0. FIG.12 provides graphs illustrating immunoblot quantification of protease precursors after compound treatment (24 hours) in I1061T NPC1 fibroblasts. Left provides quantified pro-LGMN results from FIG.5A after treatment with DMSO, 2 (80 µM), CQ (10 µM), and BafA1 (100 nM), and right provides quantified pro-CTSB results from FIG.5C after treatment with DMSO, 2 (80 µM), CQ (10 µM), and BafA1 (100 nM). All data are presented as mean ± SEM of three independent experiments. Data were normalized first to the β-actin loading control for each sample and then to DMSO per each independent experiment. Significance was determined using Ordinary ANOVA followed by Dunnett’s multiple comparison test using GraphPad Prism 9.0, where ns = not significant (P>0.05), *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001. FIG.13A is a graph illustrating quantified data for glycan digestion experiments performed in WT fibroblasts following treatment for 24 hours with DMSO and compound 2 (20 µM). Data are presented as the mean ±SEM of three independent experiments. Significance was determined by paired two-tailed t-test using GraphPad Prism 9.0, where ns = not significant (P>0.05), *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 FIG.13B provides immunoblots for the three independent glycan digestion experiments in WT fibroblasts in (A) following treatment for 24 hours with DMSO, HPβCD (1 mM), or compound 2 (20 µM). Lysates were subjected to no treatment (NT), EndoH treatment (E), or PNGase treatment (P) for 3 hours prior to western blot analysis. FIG.13C provides immunoblots for the additional two independent glycan digestion experiments in HeLa cells. following treatment for 24 hours with DMSO, HPβCD (1 mM), compound 2 (20 µM), or BafA1 (100 nM). Lysates were subjected to no treatment (NT), EndoH treatment (E), or PNGase treatment (P) for 3 hours prior to western blot analysis. FIG.13D provides immunoblots for the additional two independent glycan digestion experiments in I1061T NPC1 fibroblasts following treatment for 24 hours with DMSO, HPβCD (1 mM), compound 2 (20 µM), or BafA1 (100 nM). Lysates were subjected to no treatment (NT), EndoH treatment (E), or PNGase treatment (P) for 3 hours prior to western blot analysis. FIG.14 is a table of activity data for additional exemplary compounds. 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 DETAILED DESCRIPTION I. Terms and Definitions Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features of the disclosure are apparent from the following detailed description and the claims. The disclosure of numerical ranges should be understood as referring to each discrete point within the range, inclusive of endpoints, unless otherwise noted. Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, percentages, temperatures, times, and so forth, as used in the specification or claims, are to be understood as being modified by the term “about.” Unless context indicated otherwise, “about” refers to plus or minus 5% of a reference value. For example, “about” 100 refers to 95 to 105. Accordingly, unless otherwise implicitly or explicitly indicated, or unless the context is properly understood by a person of ordinary skill in the art to have a more definitive construction, the numerical parameters set forth are approximations that may depend on the desired properties sought and / or limits of detection under standard test conditions / methods as known to those of ordinary skill in the art. When directly and explicitly distinguishing aspects from discussed prior art, the aspect numbers are not approximates unless the word “about” is recited. Although there are alternatives for various components, parameters, operating conditions, etc. set forth herein, that does not mean that those alternatives are necessarily equivalent and / or perform equally well. Nor does it mean that the alternatives are listed in a preferred order unless stated otherwise. Abbreviations 2HPβCD:(2-hydroxypropyl)-β-cyclodextrin; BafA1: Bafilomycin A1 CBZ: Carbamazepine CQ: Chloroquine ER-phagy: Endoplasmic Reticulum Autophagy 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 ER: Endoplasmic Reticulum ERAD: Endoplasmic Reticulum-associated Degradation GFP: Green Fluorescent Protein HTS: High-Throughput Screen LAMP-1: Lysosome-Associated Membrane Protein 1 LC3: Light Chain 3 LE / Lys: Late Endosomes / Lysosomes MEF: Mouse Embryonic Fibroblasts MoA: Mechanisms of Action MTOR: Mechanistic Target of Rapamycin NPC: Niemann-Pick Type C RT: Room Temperature TMT: Tandem Mass Tagging The following terms and expressions used herein have the indicated meanings. Terms used herein may be preceded and / or followed by a single dash, “-”, or a double dash, “=“, to indicate the bond order of the bond between the named and its parent single dash indicates a single bond and a double dash indicates a double bond. In the absence of a single or double dash it is understood that a single bond is formed between the substituent and its parent moiety; further, substituents are intended to be read “left to right” unless a dash indicates otherwise. For example, C1-C6alkoxycarbonyloxy and -OC(O)C1-C6alkyl indicate the same functionality; similarly arylalkyl and –alkylaryl indicate the same functionality. “Alkenyl” means a straight or branched chain hydrocarbon containing from 2 to 10 carbons, such as from 2 to 8 carbons, or from 2 to 6 carbons, unless otherwise specified, and containing at least one carbon-carbon double bond. Representative examples of alkenyl include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4- pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, 3-decenyl, and 3,7-dimethylocta-2,6- dienyl. Alkenylene is defined identically to “alkylene” except for containing a carbon-carbon double bond. “Alkoxy” means an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, and hexyloxy. 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 “Alkyl” means a straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms, such as from 1 to 8 carbons, from 1 to 6 carbons, or from 1 to 4 carbons, unless otherwise specified. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl. When an “alkyl” group is a linking group between two other moieties (optionally referred to as an alkylene group defined herein), then it may also be a straight or branched chain; examples include, but are not limited to -CH2-, -CH2CH2-, -CH2CH2CHC(CH3)-, and -CH2CH(CH2CH3)CH2-. "Alkylene" refers to a bidentate moiety obtained by removing two hydrogen atoms from an alkane. An "alkylene" is positioned between two other chemical groups and serves to connect them. An example of a straight chain alkylene group is –(CH2)n–, but an alkylene group also can be branched. An alkyl, e.g., methyl, or alkylene, e.g., -CH2CH2-, group can be substituted, for example, independently, with one or more of halo, trifluoromethyl, trifluoromethoxy, hydroxy, alkoxy, nitro, cyano, alkylamino, and amino groups, for example. “Alkynyl” means a straight or branched chain hydrocarbon group containing from 3 to 6 carbon atoms and containing at least one carbon-carbon triple bond. Representative examples of alkynyl include, but are not limited, to acetylenyl, 1-propynyl, 2-propynyl, 3- butynyl, 2-pentynyl, and 1-butynyl. “Alkynylene is defined identically to “alkylene” except for containing a carbon-carbon triple bond. “Amino” means a group of formula –NRaRbwherein Raand Rbare independently selected from hydrogen and C1-C6alkyl. Acetylamino means a -NHC(=O)CH3group. “Aryl” means a phenyl (i.e., monocyclic aryl), or a bicyclic ring system containing at least one phenyl ring or an aromatic bicyclic ring containing only carbon atoms in the aromatic bicyclic ring system. The bicyclic aryl can be azulenyl, naphthyl, or a phenyl fused to a monocyclic cycloalkyl, a monocyclic cycloalkenyl, or a monocyclic heterocyclyl. The bicyclic aryl is attached to the parent molecular moiety through any carbon atom contained within the aromatic portion of the bicyclic system, or any carbon atom with the napthyl or azulenyl ring. The fused monocyclic cycloalkyl or monocyclic heterocyclyl portions of the bicyclic aryl are optionally substituted with one or two oxo (=O) and / or thia (=S) groups. Representative examples of the bicyclic aryls include, but are not limited to, azulenyl, naphthyl, dihydroinden-1-yl, dihydroinden-2-yl, dihydroinden-3-yl, dihydroinden-4-yl, 2,3- dihydroindol-4-yl, 2,3-dihydroindol-5-yl, 2,3-dihydroindol-6-yl, 2,3-dihydroindol-7-yl, inden-1-yl, inden-2-yl, inden-3-yl, inden-4-yl, dihydronaphthalen-2-yl, dihydronaphthalen-3- 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 yl, dihydronaphthalen-4-yl, dihydronaphthalen-1-yl, 5,6,7,8-tetrahydronaphthalen-1-yl, 5,6,7,8-tetrahydronaphthalen-2-yl, 2,3-dihydrobenzofuran-4-yl, 2,3-dihydrobenzofuran-5-yl, 2,3-dihydrobenzofuran-6-yl, 2,3-dihydrobenzofuran-7-yl, benzo[d][1,3]dioxol-4-yl, benzo[d][1,3]dioxol-5-yl, 2H-chromen-2-on-5-yl, 2H-chromen-2-on-6-yl, 2H-chromen-2-on- 7-yl, 2H-chromen-2-on-8-yl, isoindoline-1,3-dion-4-yl, isoindoline-1,3-dion-5-yl, inden-1- on-4-yl, inden-1-on-5-yl, inden-1-on-6-yl, inden-1-on-7-yl, 2,3-dihydrobenzo[b][1,4]dioxan- 5-yl, 2,3-dihydrobenzo[b][1,4]dioxan-6-yl, 2H-benzo[b][1,4]oxazin3(4H)-on-5-yl, 2H- benzo[b][1,4]oxazin3(4H)-on-6-yl, 2H-benzo[b][1,4]oxazin3(4H)-on-7-yl, 2H- benzo[b][1,4]oxazin3(4H)-on-8-yl, benzo[d]oxazin-2(3H)-on-5-yl, benzo[d]oxazin-2(3H)- on-6-yl, benzo[d]oxazin-2(3H)-on-7-yl, benzo[d]oxazin-2(3H)-on-8-yl, quinazolin-4(3H)-on- 5-yl, quinazolin-4(3H)-on-6-yl, quinazolin-4(3H)-on-7-yl, quinazolin-4(3H)-on-8-yl, quinoxalin-2(1H)-on-5-yl, quinoxalin-2(1H)-on-6-yl, quinoxalin-2(1H)-on-7-yl, quinoxalin- 2(1H)-on-8-yl, benzo[d]thiazol-2(3H)-on-4-yl, benzo[d]thiazol-2(3H)-on-5-yl, benzo[d]thiazol-2(3H)-on-6-yl, and, benzo[d]thiazol-2(3H)-on-7-yl. In certain aspects, the bicyclic aryl is (i) naphthyl or (ii) a phenyl ring fused to either a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, or a 5 or 6 membered monocyclic heterocyclyl, wherein the fused cycloalkyl, cycloalkenyl, and heterocyclyl groups are optionally substituted with one or two groups which are independently oxo or thia. In certain aspects the aryl may be substituted by one or more halo, alkyl, haloalkyl, or alkoxy groups. In certain aspects of the disclosure, the aryl group is phenyl or substituted phenyl. “Arylalkyl” means an aryl group attached to the parent molecular moiety by an alkylene group. “Cycloalkyl” means a monocyclic or a bicyclic cycloalkyl ring containing from 3 to 8 carbon atoms, such as from 3 to 6 carbons, where such groups can be saturated or unsaturated, but not aromatic. In certain aspects, cycloalkyl groups are fully saturated. In certain aspects, the cycloalkyl may be substituted by one or more halo, alkyl, haloalkyl, or alkoxy groups. In certain aspects, the cycloalkyl is cyclopentyl, cyclohexyl, or cycloheptyl. “Cycloalkylalkyl” means a cycloalkyl group attached to the parent molecular moiety by an alkylene group. “Halo” or “halogen” means -Cl, -Br, -I or -F. In certain aspects, “halo” or “halogen” refers to -Cl or -F. “Haloalkyl” means at least one halogen, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein. Representative examples of haloalkyl include, but are not limited to, chloromethyl, 2-fluoroethyl, trifluoromethyl, 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 pentafluoroethyl, and 2-chloro-3-fluoropentyl. In certain aspects, each “haloalkyl” is a fluoroalkyl, for example, a polyfluoroalkyl such as a substantially perfluorinated alkyl. “Heteroaryl” means a monocyclic heteroaryl or a bicyclic ring system containing at least one heteroaromatic ring. The monocyclic heteroaryl can be a 5 or 6 membered ring. The 5 membered ring consists of two double bonds and one, two, three or four nitrogen atoms and optionally one oxygen or sulfur atom. The 6 membered ring consists of three double bonds and one, two, three or four nitrogen atoms. The 5 or 6 membered heteroaryl is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the heteroaryl. Representative examples of monocyclic heteroaryl include, but are not limited to, furyl, imidazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, oxazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrazolyl, pyrrolyl, tetrazolyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, and triazinyl. The bicyclic heteroaryl consists of a monocyclic heteroaryl fused to a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocyclyl, or a monocyclic heteroaryl. The fused cycloalkyl or heterocyclyl portion of the bicyclic heteroaryl group is optionally substituted with one or two groups which are independently oxo or thia. When the bicyclic heteroaryl contains a fused cycloalkyl, cycloalkenyl, or heterocyclyl ring, then the bicyclic heteroaryl group is connected to the parent molecular moiety through any carbon or nitrogen atom contained within the monocyclic heteroaryl portion of the bicyclic ring system. When the bicyclic heteroaryl is a monocyclic heteroaryl fused to a phenyl ring, then the bicyclic heteroaryl group is connected to the parent molecular moiety through any carbon atom or nitrogen atom within the bicyclic ring system. Representative examples of bicyclic heteroaryl include, but are not limited to, benzimidazolyl, benzofuranyl, benzothienyl, benzoxadiazolyl, benzoxathiadiazolyl, benzothiazolyl, cinnolinyl, 5,6- dihydroquinolin-2-yl, 5,6-dihydroisoquinolin-1-yl, furopyridinyl, indazolyl, indolyl, isoquinolinyl, naphthyridinyl, quinolinyl, purinyl, 5,6,7,8-tetrahydroquinolin-2-yl, 5,6,7,8- tetrahydroquinolin-3-yl, 5,6,7,8-tetrahydroquinolin-4-yl, 5,6,7,8-tetrahydroisoquinolin-1-yl, thienopyridinyl, 4,5,6,7-tetrahydrobenzo[c][1,2,5]oxadiazolyl, and 6,7- dihydrobenzo[c][1,2,5]oxadiazol-4(5H)-onyl. In certain aspects, the fused bicyclic heteroaryl is a 5 or 6 membered monocyclic heteroaryl ring fused to either a phenyl ring, a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, a 5 or 6 membered monocyclic heterocyclyl, or a 5 or 6 membered monocyclic heteroaryl, wherein the fused cycloalkyl, cycloalkenyl, and heterocyclyl groups are optionally substituted with one or two groups which are independently oxo or thia. In certain aspects, the heteroaryl may be substituted by one or more halo, alkyl, haloalkyl, or alkoxy groups. In certain aspects, the 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 heteroaryl group is furyl, imidazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyrazolyl, pyrrolyl, thiazolyl, thienyl, triazolyl, benzimidazolyl, benzofuranyl, indazolyl, indolyl, or quinolinyl. “Heteroarylalkyl” means a heteroaryl group attached to the parent molecular moiety by an alkylene group. “Heterocyclyl” means a monocyclic heterocycle. The monocyclic heterocycle is a 3, 4, 5, 6 or 7 membered ring containing at least one heteroatom independently selected from O, N, and S where the ring is saturated or unsaturated, but not aromatic. The 3 or 4 membered ring contains 1 heteroatom selected from O, N and S. The 5 membered ring can contain zero or one double bonds and one, two or three heteroatoms selected from O, N and S. The 6 or 7 membered ring contains zero, one or two double bonds and one, two, three, or four heteroatoms selected from O, N and S. The monocyclic heterocycle is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the monocyclic heterocycle. Representative examples of monocyclic heterocycle include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl,1,1-dioxidothiomorpholinyl (thiomorpholine sulfone), thiopyranyl, and trithianyl. In certain aspects, the heterocyclyl may be substituted by one or more halo, alkyl, haloalkyl, or alkoxy groups. In certain aspects, the heterocyclyl is pyrrolidinyl, piperidinyl, piperazinyl, or morpholinyl. “Heterocyclylalkyl” means a heterocyclyl group attached to the parent molecular moiety by an alkylene group. “Saturated” means the referenced chemical structure does not contain any multiple carbon-carbon bonds. For example, a saturated cycloalkyl group as defined herein includes cyclohexyl, cyclopropyl, and the like. “Unsaturated” means the referenced chemical structure contains at least one multiple carbon-carbon bond, but is not aromatic. For example, an unsaturated cycloalkyl group as defined herein includes cyclohexenyl, cyclopentenyl, cyclohexadienyl, and the like. "Pharmaceutically acceptable salts" refers to salts or zwitterionic forms of the present compounds. Salts of the present compounds can be prepared during the final isolation and purification of the compounds or separately by reacting the compound with an acid having a suitable cation or a base having a suitable anion. The pharmaceutically acceptable salts of the 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 present compounds can be acid addition salts formed with pharmaceutically acceptable acids. Examples of acids which can be employed to form pharmaceutically acceptable salts include inorganic acids such as nitric, boric, hydrochloric, hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic, maleic, succinic, tartaric, and citric. Nonlimiting examples of salts of compounds of the disclosure include, but are not limited to, the hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, 2-hydroxyethansulfonate, phosphate, hydrogen phosphate, acetate, adipate, alginate, aspartate, benzoate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerolphosphate, hemisulfate, heptanoate, hexanoate, formate, succinate, fumarate, maleate, ascorbate, isethionate, salicylate, methanesulfonate, mesitylenesulfonate, naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylproprionate, picrate, pivalate, propionate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, paratoluenesulfonate, undecanoate, lactate, citrate, tartrate, gluconate, methanesulfonate, ethanedisulfonate, benzene sulphonate, and p-toluenesulfonate salts. In addition, available amino groups present in the compounds of the disclosure can be quaternized with methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; and benzyl and phenethyl bromides. In light of the foregoing, any reference to compounds of the present disclosure appearing herein is intended to include the present compounds as well as pharmaceutically acceptable salts thereof. “Modulating” or “modulate” refers to the treating, prevention, suppression, enhancement or induction of a function, condition or disorder. For example, the compounds of the disclosure may be effective modulators of neurodegenerative diseases or conditions. In an aspect, the neurodegenerative disease or condition is Niemann-Pick Type C Disease (NPC). “Treating” or “treatment” covers the treatment of a disease or disorder described herein, in a subject, preferably a human, and includes: i. inhibiting a disease or disorder, i.e., arresting its development; ii. relieving a disease or disorder, i.e., causing regression of the disorder; iii. slowing progression of the disorder; and / or iv. inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 “Subject” refers to a warm blooded animal such as a mammal, preferably a human, or a human child, which is afflicted with, or has the potential to be afflicted with one or more diseases and disorders described herein. II. Introduction NPC is an autosomal recessive neurodegenerative disease characterized by an accumulation of unesterified cholesterol in various tissues, including the brain, liver, and spleen. This accumulation is caused by a mutation in the NPC1 gene in 95% of cases, or in the NPC2 gene in 5% of cases, where the NPC1 and NPC2 proteins coordinate cholesterol efflux from late endosomes / lysosomes (LE / Lys). The most common mutation in NPC1 is an I1061T missense mutation that occurs in 20% of patients, where the endogenous I1061T mutant misfolds in the endoplasmic reticulum (ER) and is degraded by two pathways that function complementarily: selective endoplasmic reticulum autophagy (ER-phagy) and MARCH6-dependent endoplasmic reticulum-associated degradation (ERAD). However, overexpression of I1061T mutant NPC1 enables the misfolded proteins that escape degradation to still be able to clear unesterified cholesterol. This observation led the inventors to hypothesize that a late-stage autophagy inhibitor could potentially prevent NPC1 protein degradation through the autophagy pathway, thus making the mutant NPC1 available for cholesterol transport and thereby ameliorating NPC disease. To further support autophagy inhibition as a strategy to treat NPC, it has been observed that upon autophagy induction in NPC fibroblasts, a significant increase in unesterified cholesterol levels is observed, suggesting that autophagy contributes towards lipid storage. Because the goal is to mitigate lipid storage defects, an autophagy inhibitor could potentially be a viable option to treat NPC disease. There are also studies that support an alternative hypothesis – that an autophagy inducer could ameliorate NPC disease. This is supported by the observation that NPC1- knockout mouse embryonic fibroblasts (MEFs), NPC1 knockdown human embryonic stem cell (hESC)-derived neurons, NPC1 human dermal fibroblasts heterozygous for P237S splice mutation and I1061T missense mutation, and the brain of NPC1-deficient mice (C57BLKS / J spm and BALB / c npc1nihmutations) all had an accumulation of autophagosomes. This suggests that stalling the autophagy machinery in both NPC1-knockout / downs and cells with disease-relevant mutations is detrimental and that the induction of autophagic flux could resolve this issue. 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 One of the most studied autophagy inducers, Rapamycin (Rap), targets the mechanistic target of rapamycin (mTOR) which inadvertently affects other processes like cell growth and metabolism. Carbamazepine (CBZ) uses an mTOR-independent pathway to induce autophagy by lowering inositol levels, while also restoring autophagy defects in NPC1 induced pluripotent stem cell (iPSC)-derived hepatic and neuronal cells from fibroblasts of NPC patients with various mutations including I1061T. A possible treatment could be the combination of an autophagy inducer like Rap with a cholesterol clearance enhancer, like 2- hyroxypropyl-β-cyclodextrin (2HPβCD), to bypass the misfolded NPC1 protein and foster autophagosome-lysosome fusion and the clearance of cholesterol. The mechanism of 2HPβCD has not been fully elucidated, but it is known that it enters the cell through endocytosis, activates AMP-activated protein kinase (AMPK), and uses lysosome-associated membrane protein 1 (LAMP-1) to clear cholesterol in the absence of a functional NPC1 protein.2HPβCD is not FDA-approved, but it is in clinical trials as a treatment for NPC disease. However, its inability to cross the blood-brain barrier requires the delivery of the drug through uncomfortable, highly concentrated, intrathecal injections, highlighting the need for an orally available, small-molecule therapeutic option. Autophagy inhibitors face similar specificity challenges as inducers, where chloroquine (CQ) and its analogue, hydroxychloroquine (HCQ), are the only FDA-approved drugs used to inhibit autophagy, an effect that they cause through deacidification of the lysosome and disruption of autophagosome-lysosome fusion in late-stage autophagy. Bafilomycin A1 (BafA1) is another lysosomotropic agent that deacidifies the lysosome; however, it produces this effect through inhibition of the Vacuolar type H+-ATPase (V- ATPase) proton pump, which prevents autophagosome-lysosome fusion, and inhibits late- stage autophagy. Because both autophagy inducers and late-stage inhibitors have potential as NPC therapeutics, the goal of the present disclosure was to discover novel, mTOR-independent autophagy modulators that also enhance cholesterol clearance in NPC patient-derived fibroblasts without cytotoxicity. This was accomplished by employing a phenotypic high- throughput screen (HTS) to identify autophagy modulators followed by filipin staining in I1061T NPC1 fibroblasts to monitor unesterified cholesterol clearance after treatment with the identified autophagy modulators. The activity of two autophagy modulators in cells was investigated to gain insight into the mechanisms of action (MoA) of these molecules. Both compounds cleared unesterified cholesterol from NPC patient-derived fibroblasts and this 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 activity was dependent on the presence of mutant NPC1 protein. Furthermore, an increase in the expression levels of both NPC1 and LAMP1 was observed, which could be contributing to enhanced cholesterol clearance by compound 2. Through Tandem Mass Tagging (TMT) and mass spectrometry, it was determined that compound 2 significantly decreased levels of various lysosomal proteases, a phenomenon that has been shown to offer neuroprotection in NPC models, which could also contribute to amelioration of the NPC phenotype. III. Compounds Disclosed herein are aspects of a compound that acts as an autophagy modulator. In some aspects, the compound has a structure according to Formula I A or a pharmaceutically acceptable salt thereof. With respect to 10 membered heteroaryl, such as a 6-9 membered heteroaryl, that heteroatoms selected from N, O or S, such as 1, 2, or 3 heteroatoms selected from N, O or S. In some aspects, ring A is a monocyclic heteroaryl, but in other aspects, ring A is a bicyclic heteroaryl, and may be a thienopyrimidine ring. . 10aryl-C1-6alkyl, 6-10 membered heteroaryl, 6-10 membered heteroaryl-C1-6alkyl, C3-6cycloalkyl, C3-6cycloalkyl-C1-6alkyl, 5- or 6- membered heterocyclyl or 5- or 6-membered heterocyclyl-C1-6alkyl, and a group of formula - NR5R6, -alkyl-NR5R6, or, -NRa-alkyl-NR5R6, wherein the C1-6alkyl, C6-10aryl, C6-10aryl-C1-6alkyl, 6-10 membered heteroaryl, 6-10 membered heteroaryl-C1-6alkyl, C3-6cycloalkyl, C3-6cycloalkyl-C1-6alkyl, 5- or 6-membered heterocyclyl, or 5- or 6-membered heterocyclyl-C1-6alkyl, is optionally substituted with one or more groups independently selected from -NR7R8, 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 -alkyl-NR7R8, -C(O)R7, -C(O)NR7R8, -SO2R7, C6-10aryl-C1-6alkyl, 6-10 membered heteroaryl-C1-6alkyl, C3-6cycloalkyl-C1-6alkyl, and 5- or 6-membered heterocyclyl-C1-6alkyl. In some aspects, R1 is -NRa-C1-6alkyl-NR5R6, -NRa-phenyl, or 5- or 6-membered nitrogen-containing heterocyclyl, such as piperidinyl, piperazinyl, pyrrolidinyl, or morpholinyl. In some aspects, R1 is -NRa-C1-6alkyl-NR5R6, or -NRa-phenyl. In any aspects, R1may be unsubstituted or substituted with 1, 2, 3, or more substituents selected from C1-6alkyl, or halo. Rais H or C1-6alkyl. In some aspects, Rais H. R2and R3are independently selected from H, C1-6alkyl, C6-10aryl, C6-10aryl-C1-6alkyl, 6-10 membered heteroaryl, 6-10 membered heteroaryl-C1-6alkyl, C3-6cycloalkyl, C3-6cycloalkyl-C1-6alkyl, 5- or 6-membered heterocyclyl or 5- or 6-membered heterocyclyl-C1-6alkyl , or R2 and R3, together with the N atom to which they are attached form a 5- or 6- membered heterocyclyl, wherein the C1-6alkyl, C6-10aryl, C6-10aryl-C1-6alkyl, 6-10 membered heteroaryl, 6-10 membered heteroaryl-C1-6alkyl, C3-6cycloalkyl, C3-6cycloalkyl-C1-6alkyl, 5- or 6-membered heterocyclyl and 5- or 6-membered heterocyclyl-C1-6alkyl are optionally substituted with one or more groups independently selected from -NR7R8, -alkyl-NR7R8, - C(O)R7, -C(O)NR7R8, -SO2R7, C6-10aryl, C6-10aryl-C1-6alkyl, 6-10 membered heteroaryl, 6-10 membered heteroaryl-C1-6alkyl, C3-6cycloalkyl, C3-6cycloalkyl-C1-6alkyl, 5- or 6-membered heterocyclyl or 5- or 6-membered heterocyclyl-C1-6alkyl. In some aspects, R2 and R3 are independently H, C1-6alkyl, C3-6cycloalkyl, C3-6cycloalkyl-C1-6alkyl-, or 5- or 6-membered heterocyclyl, or R2and R3together with the nitrogen to which they are attached form a 5- or 6-membered heterocyclyl, such as, for example, piperazinyl, piperidinyl, morpholinyl, or pyrrolidinyl. In some aspects, R2and / or R3, or a heterocyclyl formed therefrom, are unsubstituted, but in other aspects, R2 and / or R3, or a heterocyclyl formed therefrom, are substituted with 1, 2, 3, or more substituents, such as 1 or 2 substituents, selected from C1-6alkyl, -SO2C1-6alkyl, -C(O)R7, or C(O)NR7R8, such as C1-6alkyl, -SO2C1-6alkyl, or -C(O)C1-6alkyl. In some aspects, R2 and R3 independently are H, or C1-6alkyl, and in certain aspects, R2and R3independently are C1-6alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl. In other aspects, R2and R3together with the nitrogen to which they are attached form a 5- or 6-membered heterocyclyl, such as, for example, piperazinyl, piperidinyl, morpholinyl, or pyrrolidinyl. The 5- or 6-membered heterocyclyl may be unsubstituted or it may be substituted with 1, 2, 3, or more substituents, such as, 1, or 2 substituents, or 1 substituent, 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 selected from C1-6alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec- butyl, or tert-butyl. R4 is H or alkyl, such as H or C1-6alkyl. In some aspects, R4 is H, but in other aspects, R4is methyl. R5 and R6 are independently selected from H, -C(O)R7, -C(O)NR7R8, -SO2R7, C1-6alkyl, C6-10aryl, C6-10aryl-C1-6alkyl, 6-10 membered heteroaryl, 6-10 membered heteroaryl- C1-6alkyl, C3-6cycloalkyl, C3-6cycloalkyl-C1-6alkyl, 5- or 6-membered heterocyclyl, or 5- or 6- membered heterocyclyl-C1-6alkyl, or R5and R6together form a 5- or 6-membered optionally substituted heterocyclyl (for example, piperazinyl, piperidinyl, morpholinyl, or pyrrolidinyl), where R5and R6are independently optionally substituted with halo or C1-6alkyl. In some aspects, R5 and R6 are independently H, C1-6alkyl (such as, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl), or R5and R6together form a 5- or 6-membered optionally substituted heterocyclyl. In some aspects, R5and R6are independently C1-6alkyl, such as methyl, ethyl, n- propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl. In other aspects, R5and R6together form a 5- or 6-membered optionally substituted heterocyclyl, such as morpholinyl, N-(C1-6alkyl)piperazinyl such as N-methylpiperazinyl, piperidinyl, or pyrrolidinyl. R7 and R8 are independently selected from C1-6alkyl, C6-10aryl, C6-10aryl-C1-6alkyl, 6- 10 membered heteroaryl, 6-10 membered heteroaryl-C1-6alkyl, C3-6cycloalkyl, C3-6cycloalkyl- C1-6alkyl, 5- or 6-membered heterocyclyl, or 5- or 6-membered heterocyclyl-C1-6alkyl, wherein the C1-6alkyl, C6-10aryl, C6-10aryl-C1-6alkyl, 6-10 membered heteroaryl, 6-10 membered heteroaryl-C1-6alkyl, C3-6cycloalkyl, C3-6cycloalkyl-C1-6alkyl, 5- or 6-membered heterocyclyl and 5- or 6-membered heterocyclyl-C1-6alkyl are optionally substituted with one or more groups independently selected from -NR5R6, -alkyl-NR5R6, -C(O)R5, -C(O)NR5R6, - SO2R5, C6-10aryl, C6-10aryl-C1-6alkyl, 6-10 membered heteroaryl, 6-10 membered heteroaryl- C1-6alkyl, C3-6cycloalkyl, C1-6cycloalkyl-C1-6alkyl, 5- or 6-membered heterocyclyl, and 5- or 6-membered heterocyclyl-C1-6alkyl. In some aspects, R7 and R8 are independently selected from C1-6alkyl. In some aspects, the compound of formula I does not include 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 . a With respect to Formula II, R1, R2, defined for Formula I. In some aspects, the compound has a structure according to Formula III or a pharmaceutically acceptable salt thereof. With respect to Formula III, R2, as previously defined for Formula I. L is –(CRa2)n-, such as –(CH2)n-, where n is from 1 to 6, such as from 2 to 6, from 3 to 6, or 3, 4, 5, or 6, such as 3, or 4, and in some aspects, n is 3. In some aspects, the compound has a structure according to Formula IV or a pharmaceutically acceptable salt thereof. With respect to Formula IV, R1 and defined for Formula I, and R9 is H, C1-6alkyl, -SO2C1-6alkyl, -C(O)R7, or C(O)NR7R8, such as C1-6alkyl, -SO2C1-6alkyl, or - C(O)C1-6alkyl. In some aspects, R9 is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 sec-butyl, tert-butyl, -SO2methyl, -SO2ethyl, -SO2n-propyl, -SO2isopropyl, -SO2n-butyl, - SO2isobutyl, -SO2sec-butyl, -SO2tert-butyl, -C(O)methyl, -C(O)ethyl, -C(O)n-propyl, - C(O)isopropyl, -C(O)n-butyl, -C(O)isobutyl, -C(O)sec-butyl, or -C(O)tert-butyl. And in certain aspects, R9is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert- butyl. In some aspects, the compound has a structure according to Formula V or a pharmaceutically acceptable salt thereof. With respect to Formula V, Ra, defined for Formula III, and R9is as previously defined for Formula IV. In any aspects of Formulas I-V, R1 may , , In any aspects of Formulas I-V, the -NR2R3 moiety may , , 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 Exemplary compounds according to Formula I include, but are not limited to: , , , 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 or 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 In an alternative aspect, the compound has a structure according to Formula VI or a pharmaceutically acceptable salt thereof With respect to Formula VI, R1for Formula I. IV. Pharmaceutical Compositions and Methods of Administration In other aspects, the disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of a compound having structural Formula I, or pharmaceutically acceptable salts thereof as described herein, and one or more pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants, excipients, or carriers. The pharmaceutical composition can be used, for example, for treating a disease or condition where modulation of autophagy would provide a benefit. In an aspect, the disease or condition is selected from liver disease, diabetes, kidney disease, heart disease, inflammatory bowel disease, neurodegenerative disease, and cancer. In an aspect, the disease or condition is a neurodegenerative disease or disorder. In an aspect, the neurodegenerative disease or condition is selected from Alzheimer’s disease, Parkinson’s disease, Huntington's disease, Amyotrophic lateral sclerosis, or Niemann-Pick Type C. In an aspect, the neurodegenerative disease or disorder is Niemann-Pick Type C. In an aspect, the disease or condition is cancer. In an aspect, the cancer is selected from lung cancer, pancreatic ductal adenocarcinoma, melanoma, breast cancer, ovarian cancer and colorectal cancer. In certain aspects, the disclosure provides a pharmaceutical composition comprising the compounds of the disclosure together with one or more pharmaceutically acceptable excipients or vehicles, and optionally other therapeutic and / or prophylactic ingredients. Such excipients include liquids such as water, saline, glycerol, polyethylene glycol, hyaluronic acid, ethanol, and the like. The term “pharmaceutically acceptable vehicle” refers to a diluent, adjuvant, excipient or carrier with which a compound of the disclosure is administered. The terms “effective amount” or “pharmaceutically effective amount” refer to a nontoxic but sufficient amount of the agent to provide the desired biological result. That result can be reduction 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An appropriate “effective” amount in any individual case can be determined by one of ordinary skill in the art using routine experimentation. “Pharmaceutically acceptable carriers” for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington’s Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990). For example, sterile saline and phosphate-buffered saline at physiological pH can be used. Preservatives, stabilizers, dyes and even flavoring agents can be provided in the pharmaceutical composition. For example, sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid can be added as preservatives. Id. at 1449. In addition, antioxidants and suspending agents can be used. Id. Suitable excipients for non-liquid formulations are also known to those of skill in the art. A thorough discussion of pharmaceutically acceptable excipients and salts is available in Remington’s Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990). Additionally, auxiliary substances, such as wetting or emulsifying agents, biological buffering substances, surfactants, and the like, can be present in such vehicles. A biological buffer can be any solution which is pharmacologically acceptable and which provides the formulation with the desired pH, i.e., a pH in the physiologically acceptable range. Examples of buffer solutions include saline, phosphate buffered saline, Tris buffered saline, Hank’s buffered saline, and the like. Depending on the intended mode of administration, the pharmaceutical compositions can be in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, suspensions, creams, ointments, lotions or the like, preferably in unit dosage form suitable for single administration of a precise dosage. The compositions will include an effective amount of the selected drug in combination with a pharmaceutically acceptable carrier and, in addition, can include other pharmaceutical agents, adjuvants, diluents, buffers, and the like. In general, the compositions of the disclosure will be administered in a therapeutically effective amount by any of the accepted modes of administration. Suitable dosage ranges depend upon numerous factors such as the severity of the disease to be treated, the age and relative health of the subject, the potency of the compound used, the route and form of administration, the indication towards which the administration is directed, and the preferences and experience of the medical practitioner involved. One of ordinary skill in the 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 art of treating such diseases will be able, without undue experimentation and in reliance upon personal knowledge and the disclosure of this application, to ascertain a therapeutically effective amount of the compositions of the disclosure for a given disease. Thus, the compositions of the disclosure can be administered as pharmaceutical formulations including those suitable for oral (including buccal and sub-lingual), rectal, nasal, topical, pulmonary, vaginal or parenteral (including intramuscular, intra-arterial, intrathecal, subcutaneous and intravenous) administration or in a form suitable for administration by inhalation or insufflation. The preferred manner of administration is intravenous or oral using a convenient daily dosage regimen which can be adjusted according to the degree of affliction. For solid compositions, conventional nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like. Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, and the like, an active compound as described herein and optional pharmaceutical adjuvants in an excipient, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension. If desired, the pharmaceutical composition to be administered can also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and the like. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington’s Pharmaceutical Sciences, referenced above. In yet another aspect is the use of permeation enhancer excipients including polymers such as: polycations (chitosan and its quaternary ammonium derivatives, poly-L-arginine, aminated gelatin); polyanions (N-carboxymethyl chitosan, poly-acrylic acid); and, thiolated polymers (carboxymethyl cellulose-cysteine, polycarbophil-cysteine, chitosan- thiobutylamidine, chitosan-thioglycolic acid, chitosan-glutathione conjugates). For oral administration, the composition will generally take the form of a tablet, capsule, a softgel capsule or can be an aqueous or nonaqueous solution, suspension or syrup. Tablets and capsules are preferred oral administration forms. Tablets and capsules for oral use can include one or more commonly used carriers such as lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. Typically, the compositions of the disclosure can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, starch, sucrose, glucose, methyl callulose, 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents, and coloring agents can also be incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like. When liquid suspensions are used, the active agent can be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like and with emulsifying and suspending agents. If desired, flavoring, coloring and / or sweetening agents can be added as well. Other optional components for incorporation into an oral formulation herein include, but are not limited to, preservatives, suspending agents, thickening agents, and the like. Parenteral formulations can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solubilization or suspension in liquid prior to injection, or as emulsions. Preferably, sterile injectable suspensions are formulated according to techniques known in the art using suitable carriers, dispersing or wetting agents and suspending agents. The sterile injectable formulation can also be a sterile injectable solution or a suspension in a nontoxic parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that can be employed are water, Ringer’s solution and isotonic sodium chloride solution. In addition, sterile, fixed oils, fatty esters or polyols are conventionally employed as solvents or suspending media. In addition, parenteral administration can involve the use of a slow release or sustained release system such that a constant level of dosage is maintained. Parenteral administration includes intraarticular, intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, and include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Administration via certain parenteral routes can involve introducing the formulations of the disclosure into the body of a patient through a needle or a catheter, propelled by a sterile syringe or some other mechanical device such as 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 an continuous infusion system. A formulation provided by the disclosure can be administered using a syringe, injector, pump, or any other device recognized in the art for parenteral administration. Preferably, sterile injectable suspensions are formulated according to techniques known in the art using suitable carriers, dispersing or wetting agents and suspending agents. The sterile injectable formulation can also be a sterile injectable solution or a suspension in a nontoxic parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that can be employed are water, Ringer’s solution and isotonic sodium chloride solution. In addition, sterile, fixed oils, fatty esters or polyols are conventionally employed as solvents or suspending media. In addition, parenteral administration can involve the use of a slow release or sustained release system such that a constant level of dosage is maintained. Preparations according to the disclosure for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. Such dosage forms can also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. They can be sterilized by, for example, filtration through a bacteria retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured using sterile water, or some other sterile injectable medium, immediately before use. Sterile injectable solutions are prepared by incorporating one or more of the compounds of the disclosure in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Thus, for example, a parenteral composition suitable for administration by injection is prepared by stirring 1.5% by weight of active ingredient in 10% by volume propylene glycol and water. The solution is made isotonic with sodium chloride and sterilized. Alternatively, the pharmaceutical compositions of the disclosure can be administered in the form of suppositories for rectal administration. These can be prepared by mixing the 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 agent with a suitable nonirritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols. The pharmaceutical compositions of the disclosure can also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, propellants such as fluorocarbons or nitrogen, and / or other conventional solubilizing or dispersing agents. Preferred formulations for topical drug delivery are ointments and creams. Ointments are semisolid preparations which are typically based on petrolatum or other petroleum derivatives. Creams containing the selected active agent, are, as known in the art, viscous liquid or semisolid emulsions, either oil-in-water or water-in-oil. Cream bases are water- washable, and contain an oil phase, an emulsifier and an aqueous phase. The oil phase, also sometimes called the “internal” phase, is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol; the aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant. The emulsifier in a cream formulation is generally a nonionic, anionic, cationic or amphoteric surfactant. The specific ointment or cream base to be used, as will be appreciated by those skilled in the art, is one that will provide for optimum drug delivery. As with other carriers or vehicles, an ointment base should be inert, stable, nonirritating and nonsensitizing. Formulations for buccal administration include tablets, lozenges, gels and the like. Alternatively, buccal administration can be effected using a transmucosal delivery system as known to those skilled in the art. The compounds of the disclosure can also be delivered through the skin or muscosal tissue using conventional transdermal drug delivery systems, i.e., transdermal “patches” wherein the agent is typically contained within a laminated structure that serves as a drug delivery device to be affixed to the body surface. In such a structure, the drug composition is typically contained in a layer, or “reservoir,” underlying an upper backing layer. The laminated device can contain a single reservoir, or it can contain multiple reservoirs. In one aspect, the reservoir comprises a polymeric matrix of a pharmaceutically acceptable contact adhesive material that serves to affix the system to the skin during drug delivery. Examples of suitable skin contact adhesive materials include, but are not limited to, polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates, polyurethanes, and the like. Alternatively, the drug-containing reservoir and skin contact 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 adhesive are present as separate and distinct layers, with the adhesive underlying the reservoir which, in this case, can be either a polymeric matrix as described above, or it can be a liquid or gel reservoir, or can take some other form. The backing layer in these laminates, which serves as the upper surface of the device, functions as the primary structural element of the laminated structure and provides the device with much of its flexibility. The material selected for the backing layer should be substantially impermeable to the active agent and any other materials that are present. The compositions of the disclosure can be formulated for aerosol administration, particularly to the respiratory tract and including intranasal administration. The compound will generally have a small particle size, for example, of the order of 5 microns or less. Such a particle size can be obtained by means known in the art, for example by micronization. The active ingredient is provided in a pressurized pack with a suitable propellant such as a chlorofluorocarbon (CFC) for example dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane, carbon dioxide or other suitable gas. The aerosol can conveniently also contain a surfactant such as lecithin. The dose of drug can be controlled by a metered valve. Alternatively, the active ingredients can be provided in a form of a dry powder, for example a powder mix of the compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidine (PVP). The powder carrier will form a gel in the nasal cavity. The powder composition can be presented in unit dose form, for example, in capsules or cartridges of e.g., gelatin or blister packs from which the powder can be administered by means of an inhaler. A pharmaceutically or therapeutically effective amount of the composition will be delivered to the subject. The precise effective amount will vary from subject to subject and will depend upon the species, age, the subject’s size and health, the nature and extent of the condition being treated, recommendations of the treating physician, and the therapeutics or combination of therapeutics selected for administration. Thus, the effective amount for a given situation can be determined by routine experimentation. For purposes of the disclosure, generally a therapeutic amount will be in the range of about 0.01 mg / kg to about 250 mg / kg body weight, more preferably about 0.1 mg / kg to about 10 mg / kg, in at least one dose. In larger mammals the indicated daily dosage can be from about 1 mg to 300 mg, one or more times per day, more preferably in the range of about 10 mg to 200 mg. The subject can be administered as many doses as is required to reduce and / or alleviate the signs, symptoms, or causes of the disorder in question, or bring about any other desired alteration of a biological 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 system. When desired, formulations can be prepared with enteric coatings adapted for sustained or controlled release administration of the active ingredient. The foregoing may be better understood by reference to the following methods, results and discussion which are presented for purposes of illustration and are not intended to limit the scope of the disclosure. V. Results & Discussion Upon autophagy induction, ATG3, ATG7, and the ATG5-ATG12-ATG16L1 complex conjugates Light Chain 3 (LC3)-I, which is diffusely located throughout the cytosol, to phosphatidylethanolamine (PE) and generates membrane-associated LC3-II. LC3-II is anchored into the developing autophagosome membrane and promotes expansion and closure of the nascent autophagosome, and it is considered a marker of autophagosome formation. When tagged with a fluorophore, like green fluorescent protein (GFP), LC3-I and LC3-II can be visualized through fluorescence microscopy and used to assess autophagy induction by measuring autophagosome formation through the production of LC3-II. LC3-I appears like a green cytoplasmic pool, and LC3-II appear like green distinct dots, or puncta, enabling efficient quantification. HeLa cells stably expressing eGFP-tagged LC3 were used to identify small molecules that induce autophagy and cause the localization change of eGFP-LC3 from the cytosol to the autophagosome membrane to form puncta. An important aspect of this assay to take into consideration is that an autophagy inducer and late-stage inhibitor would have the same phenotype, as an inducer would increase the formation of autophagosomes, and a late-stage inhibitor would cause the accumulation of autophagosomes due to inhibition of autophagosome maturation. Both possibilities result in an increase of puncta, and thus additional evaluation is necessary to discern the true nature of newly identified autophagy modulators. The late-stage autophagy inhibitor CQ caused a robust accumulation of autophagosomes, as indicated by a significant increase in puncta per cell, and was thus selected as the positive control for the eGFP-LC3 assay, and the vehicle, DMSO, was selected as the negative control (FIG.7).10,000 small molecules from ChemDiv were screened in duplicate for the ability to increase puncta levels, and compounds with a z-score of greater than 2.199 in both duplicates were considered hits. Compounds with a z-score of less than -1.799 for cell count were deemed cytotoxic and excluded from subsequent evaluation (FIG.1A). With a seeding density of 3,000 cells / well, 4 hour compound treatment, and 10x magnification, the eGFP-LC3 assay consistently yielded plates with a Z-factor (Z’) 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 of 0.4-0.6 (FIG.1A, Eq.1) and Percent Coefficient of Variation (%CV) of less than 20% (FIG.1A, Eq.2), highlighting the reproducibility of the assay and the reliability of identified hits (FIG.1A). From the 10,000 compounds screened in the eGFP-LC3 assay, the hits were selected and prioritized based on z-score (FIG.1A, Eq.3), where 312 were able to increase autophagosome / puncta levels (FIG.8). The 312 autophagy modulators were then tested in homozygous I1061T NPC1 patient-derived skin fibroblasts to determine if any of these compounds could clear accumulated unesterified cholesterol. Cholesterol levels were quantified using filipin dye, a naturally fluorescent polyene macrolide antibiotic that binds to unesterified / free cholesterol, but not esterified sterols, and is the current initial method for the diagnosis of NPC disease. Interestingly, of all of the autophagy modulators, only two compounds out of the 312 were identified that significantly decreased filipin intensity in patient-derived cells and increased LC3-II puncta levels (FIG.1C), termed compounds 1 and 2 (FIGS.1D and 1E). The current investigational NPC treatment, 2HPβCD, also decreased filipin intensity, but the autophagy inducer, rapamycin, did not (FIG.1F). These results indicate that not all autophagy modulators enhance cholesterol efflux, indicating that the mechanism of action must be critical for the induction of cholesterol clearance. In preparation for additional mechanistic studies with 1 and 2, convergent synthetic routes were developed with multiple branching points to facilitate analogue generation and to expediently access to each of the hit compounds (Schemes 1 and 2 in the Examples). Activity of the prioritized compounds, 1 and 2, was evaluated in the eGFP-LC3 assay along with CQ and Rap, which increased puncta count, and 2HPβCD, which did not increase puncta count (FIG.2A). The inability of 2HPβCD to significantly increase puncta count highlights that not all molecules that enhance cholesterol efflux also modulate autophagy, complementing the earlier observation that not all autophagy modulators decrease unesterified cholesterol levels. The two resynthesized hits were then evaluated in the eGFP-LC3 assay, and it was determined that 1 and 2 had similar potencies (EC50 of 10.97 and 14.33 µM, respectively) (FIGS.2B and 9 left). The resynthesized compounds were also tested in the filipin assay at multiple doses where 1 and 2 decreased filipin intensity, as did 2HPβCD (FIGS.2C and 9 right). To discern whether the newly discovered compounds, 1 and 2, were autophagy activators or late-stage inhibitors, the dual reporter assay was conducted. The dual reporter assay relies on the pH-sensitive GFP fluorophore and pH-insensitive mCherry red 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 fluorophore to differentiate autophagy activators from inhibitors in HeLa cells stably expressing tandem mCherry and eGFP tags on LC3 (mCherry-eGFP-LC3). Once the autophagosome (AP) fuses with the lysosome to form the autolysosome (AL), the pH- sensitive GFP is quenched and only mCherry fluoresces, causing AL to appear red in images. The co-localization of GFP and mCherry in autophagosomes causes AP to appear yellow in images. Quantitatively, an autophagy inducer would increase the number of AP and increase or maintain levels of AL, whereas a late-stage inhibitor would significantly increase the number of AP but decrease the number of AL as compared to DMSO due to the inhibition of autophagic flux. After 24-hour treatment with CQ in the dual reporter assay, yellow puncta were visualized, as expected, whereas 1 produced a mix of red and yellow puncta, consistent with an autophagy activator, and 2 produced a similar phenotype to CQ, suggesting it is a late- stage inhibitor and / or affects lysosome acidity (FIG.2D). Quantitatively, DMSO-treated cells exhibited a ratio of 2:2 AP:AL and CQ treatment caused an increase in the number of AP and decrease in the number of AL to give a ratio of 6:1 AP:AL. (FIGS.2E and 2F). Across several concentrations (5-80 µM), compound 1 had an approximate ratio of 4:2.4 AP:AL, which means the levels of AP and AL increased compared to DMSO treatment, indicative of an autophagy inducer (FIG.2E). Compound 2 (40 µM) elicited a ratio of 4.5:1 AP:AL, which means the levels of AP increased, and the levels of AL decreased compared to DMSO treatment, similar to CQ, and consistent with an autophagy late-stage inhibitor (FIG.2F). However, at a lower concentration (10 µM), 2 had an AP:AL ratio of 3.8:2.5, which is more similar to the profile of an autophagy activator. Therefore, this compound may be activating autophagy at concentrations close to its EC50 and that administration at higher concentrations could negatively impact autophagic flux by creating an abundance of autophagosomes in excess of the lysosomes available for fusion. In this example, quantified data for FIGS.2B, 2C, 2E, and 2F are presented as mean ± SEM of three independent experiments each with duplicate biological replicates. Significance was determined using Ordinary ANOVA followed by Dunnett’s multiple comparison test using GraphPad Prism 9.0, where ns = not significant (P>0.05), *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001. Next, a collection of compound 2 analogues was prepared using the optimized synthetic route to further explore the structure-activity relationships (SAR) of this scaffold and to identify which moieties are required to maintain activity to inform future optimization efforts in preparation for in vivo efficacy studies. This initial set of analogues modified the 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 piperazine (Ra) position and the amide (Rb) position (FIGS.3, 10 and 11). Reducing the lipophilicity of the amino substituent at Rbresulted in a significant reduction or complete loss of autophagy activation activity. Modification of Ra from ethyl to methyl piperazine reduced activity by 2-fold, and modification from a cyclic piperazine to diethyl amine completely abolished activity. It was decided to further explore modifications to the Ra position and maintaining the basic amine at this position was determined to be useful for activity. The propyl-substituted piperazine (AA2123) displayed slightly improved activity in the autophagy assay and similar activity to compound 2 in the filipin assay. Modification of this position to incorporate a larger, branched sec-butyl group (AA2124) increased activity in both assays, indicating that larger substituents at this position are well-tolerated and may improve interactions of the compound with its target. To test if 1 or 2 affect lysosomal protease activity, the Dye Quenched-Bovine Serum Albumin (DQ-BSA) assay was implemented. DQ-BSA is labeled with a self-quenching dye that only fluoresces once it is endocytosed and digested, which creates isolated fluorophores that are no longer quenched and become brightly fluorescent, indicative of proteolytic activity. Because I1061T NPC1 fibroblasts have a defective NPC1 protein located in late endosomes and lysosomes, this can affect the proteolytic activity of enzymes found in the lysosome which would impact DQ-BSA processing. Proteolytic processing of DQ-BSA by 1 and 2 was evaluated in NPC patient-derived fibroblasts to determine how they impact this process in the context of I1061T NPC1 (FIG.4A). CQ decreased proteolytic activity in I1061T NPC1 fibroblasts, whereas 2HPβCD and BafA1 increased proteolytic activity, and compounds 1 and 2 elicited a 3-fold increase in proteolytic activity after 24 hours as compared to DMSO. When performed in HeLa cells with a wild-type (WT) NPC1 protein for 6 hours, a method typically used for evaluating inhibition of lysosome acidification or function by late-stage autophagy inhibitors, CQ and BafA1 drastically decreased proteolytic activity, as expected (FIG.4B).1 and 2 did not significantly affect DQ-BSA processing in HeLa cells at concentrations near the EC50 in the eGFP-LC3 and filipin assays; however, at higher concentrations, 1 and 2 did decrease DQ-BSA processing, but not the same degree as BafA1 and CQ. This means 1 and 2 significantly enhance proteolytic activity in the presence of I1061T NPC1 compared to the vehicle control, which indicates potential restoration of lysosome function in these cells. To gain additional insight into the mechanisms of 1 and 2, the CellTiter-Glo viability assay was conducted, and neither one of the hit compounds displayed significant cytotoxicity after 24 hours in I1061T NPC1 patient-derived fibroblasts across multiple concentrations (10- 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 80 µM) (FIG.4C). The effect of 1 and 2 on mTOR activity was also evaluated in I1061T NPC1 fibroblasts after 24-hour treatment to determine if the hit compounds were inducing autophagosome accumulation in an mTOR-dependent or mTOR-independent manner. The serine / threonine protein kinase, mTOR, contributes to the regulation of multiple cellular processes, including cell growth, protein translation, and metabolic processes. Inhibition of mTOR prevents phosphorylation of p70S6 Kinase (p70S6K), a substrate of mTOR that has a role in translation, and thus compounds that inhibit the activity of this kinase will decrease phosphorylation of p70S6K. Because mTOR has many functions in the cell, its inhibition could have undesirable effects. The well-established mTOR inhibitor Rap prevented substrate phosphorylation, but neither the controls, mTOR-independent CBZ and HPβCD, nor the hit compounds, 1 and 2, decreased formation of P-p70S6K, suggesting they do not inhibit the function of mTOR (FIGS.4D and 4E). Next, the effects of 1 and 2 on I1061T NPC1 protein expression levels were evaluated after 24-hour treatment, and both compounds increased NPC1 protein levels (FIG.4F). An increase in NPC1 expression levels could improve unesterified cholesterol clearance by providing excess protein to facilitate cholesterol efflux from the lysosome and late endosome. To test if the decrease in unesterified cholesterol is dependent on the NPC1 protein, 1 and 2 were evaluated in the filipin assay using NPC1 null HeLa cells to assess cholesterol clearance in the absence of NPC1 protein (FIG.4G). 2HPβCD retained the ability to clear unesterified cholesterol, whereas 1 and 2 were unable to clear unesterified cholesterol. This suggests that the activity of compounds 1 and 2 is dependent on the presence of NPC1 protein. Next, tandem mass tagging and mass spectrometry were used to quantify changes in protein expression levels following 24-hour compound treatment in I1061T NPC1 fibroblasts. Compound 1 had a minimal impact on protein expression levels in these cells compared to DMSO treatment, so additional experiments will be performed to determine the mechanism of this compound. Compound 2 significantly decreased various protease levels, including Legumain (LGMN) and members of the cathepsin family such as Cathepsin Z (CTSZ), Cathepsin B (CTSB), and Cathepsin D (CTSD) (FIG.5A). Changes in protease expression levels induced by compound 2 treatment were validated using western blotting and tested alongside the late-stage inhibitors CQ and BafA1 (FIGS.5B-5F). For FIGS.5B- 5F, data were normalized first to the β-actin loading control for each sample and then to DMSO per each independent experiment. Significance was determined using Ordinary ANOVA followed by Dunnett’s multiple comparison test using GraphPad Prism 9.0, where 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 ns = not significant (P>0.05), *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001. Compound 2 significantly decreased expression of the cysteine endopeptidase LGMN, but to a lesser extent than CQ and BafA1 (FIG.5B), and pro-LGMN, the precursor to mature LGMN that can be differentially activated to a carboxypeptidase or an endopeptidase, was not significantly affected by compound 2 treatment (FIG.12 left). Compound 2 also decreased cathepsin levels, which are primarily found in the endosomal / lysosomal system and are the most abundant lysosomal proteases (FIGS.5C-5E). Compound 2 decreased CTSZ levels, but to a lesser extent than CQ and BafA1 (FIG.5C), and decreased CTSB levels to a comparable degree as CQ and BafA1 (FIG.5D) but did not have a significant effect on pro-CTSB levels (FIG.12 right). Compound 2 also decreased CTSD levels, whereas CQ decreased CTSD levels slightly more and BafA1 did not significantly decrease CTSD levels (FIG.5E). Finally, the effect of compound 2 on LAMP1 expression and glycosylation was also tested as LAMP1 binds cholesterol and aids in lysosomal cholesterol export, and the state of LAMP1 glycosylation may have a role in NPC disease. It was observed that compound 2 increased levels of glycosylated and non-glycosylated LAMP1 to the greatest degree, whereas treatment with BafA1 had no significant effect (FIG.5F). It is possible that excess LAMP1 could also be contributing to enhanced cholesterol efflux from the LE / Lys induced by compound 2 through an NPC1-dependent mechanism. The observations that CQ, BafA1, and compound 2 affect cell viability, proteolytic activity, and protein expression levels with different trends further supports the hypothesis that compound 2 has a different mechanism than CQ and BafA1 through modulation of a unique target that could provide an alternative therapeutic strategy with improved selectivity and therapeutic window. Next the WEB-based Gene SeT AnaLysis Toolkit (WebGestalt) was used to identify affected pathways based on the acquired proteomic data (data not shown), and it was discovered that the observed expression changes in these proteases, as well as some of the other affected proteins, were related through the action of FBXO6 (F-box only protein 6), an SKP1-CUL1-F-box protein (SCF) E3 ligase that is involved in the ERAD pathway for misfolded luminal proteins by promoting their ubiquitination and degradation, with particular affinity for high mannose N-linked glycoproteins (FIGS.5G and 5H). Therefore, it is possible that direct or indirect inhibition of FBXO6 activity could not only result in decreased expression levels of lysosomal proteases, but it could also allow misfolded NPC1 protein to escape degradation and remain available to facilitate unesterified cholesterol clearance. These observed changes in protein expression trends after treatment with 2 were especially intriguing considering previous experiments using an NPC1-deficient mouse 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 model revealed increased amounts of CTS Z, B, and D. Significant decreases in expression levels of these cathepsins were observed following treatment of I1061T NPC1 fibroblasts with 2. Furthermore, CTSZ and LGMN expression levels were also significantly elevated in late-stage NPC disease in the Npc1- / -mouse model. It is possible that 2 is also able to ameliorate the impact of an NPC1 deficit by reducing the levels of cathepsins and LGMN, which could reduce neuronal toxicity. This effect could also be useful in other diseases like cancer, as overexpressed and hyper-secreted CTSD is observed in ovarian, breast, lung, endometrial, and prostate cancer, as well as melanoma and malignant glioma. High levels of LGMN have also been associated with peritoneal metastasis, and lowering these levels could be beneficial in cancer. Furthermore, FBXO6-promoted degradation of certain protein targets has been implicated in ovarian cancer development and inhibition of ER stress-induced apoptosis. Therefore, while compound 2 shows promise in models of NPC disease, it is also possible that this compound could be beneficial in certain cancer types by decreasing protease expression levels and preventing ubiquitination and degradation of key tumor suppressors. To assess the effect of compound 2 on proteasomal degradation, the levels of several different markers for proteasomal (p21, p27) and autophagic degradation (p62, LC3 I / II) were measured following 24-hour treatment with autophagy modulators or MG132 as an inhibitor of the proteasome (FIGS.6A-6D). As expected, MG132 treatment caused an accumulation of proteins that are primarily degraded by the proteasome, p21 (FIG.6A) and p27 (FIG.6B) but did not cause an accumulation of p62 (FIG.6C), which is primarily degraded by autophagy. Autophagy inhibitors, CQ and PIK-III, did cause a significant accumulation of p62. By contrast, compound 2 did not cause an accumulation of p21, p27, or p62, indicating that this compound does not broadly inhibit proteasomal activity and providing further support that it is not inhibiting autophagy. To determine how compound 2 impacts NPC1 expression and trafficking, glycan digestion experiments were performed. Glycans are added to NPC1 in the endoplasmic reticulum (ER), and the glycosylated protein is trafficked to the Golgi where the glycans are modified and become resistant to endoglycosidase H (Endo H) digestion but remain sensitive to digestion by peptide-N-glycosidase F (PNGase F). Because I1061T NPC1 is recognized as misfolded in the ER and sent for degradation before reaching the Golgi, its glycans remain sensitive to Endo H digestion. In both HeLa (FIGS.6E and 6F) and WT fibroblasts (FIGS. 13A and 13B), a significant change in NPC1 expression levels was not observed following treatment with 2. In contrast, treatment with compound 2 caused a significant increase in the 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 amount of Endo-H sensitive (E-Sen) I1061T NPC1 (FIGS.6G and 6H), indicating that the compound does not restore glycosylation of mutant NPC1 and that the primary effect is likely prevention of the degradation of the E-sen NPC1. In fact, it has been previously shown that increasing the cellular population of E-sen, mutant NPC1 results in late endosomal localization of the mutant protein, likely due to the ability of a proportion of this protein to fold correctly and escape ERAD quality control checkpoints, which promotes clearance of cholesterol from lysosomes despite the trafficking defect. A total of 312 autophagy modulators were identified, but only two of these modulators significantly reduced unesterified cholesterol accumulation in NPC patient- derived fibroblasts. The two modulators were unable to clear cholesterol in NPC1 null cells, indicating a requirement for NPC1 for the observed activity. Although compound 1 had a profile consistent with an autophagy inducer, there were not as many significant changes in protein expression levels, so additional experiments are necessary to elucidate the mechanism of this compound. Compound 2 induced significant reduction in the expression of several lysosomal proteases and an increase in the expression levels of glycosylated LAMP1 and I1061T NPC1 proteins, and pathway analysis revealed an E3 ligase, FBXO6, as a potential target for this compound. The mechanism of this compound is distinct from 2HPβCD, because 2HPβCD does not increase I1061T NPC1 protein expression levels and its activity is not dependent on the presence of NPC1 protein. Additional mechanistic studies were performed for compound 2 to evaluate the putative role of FBXO6 in the mechanism of this small molecule, and compound 2 may be preventing proteasomal degradation of mutant NPC1 without broadly inhibiting proteosome activity, which results in the observed enhanced cholesterol clearance. While the inability to remove misfolded mutant NPC1 protein through proteasomal degradation could increase ER stress, it appears that compound 2 may serve as a small-molecule chaperone that increases the proportion of properly folded mutant NPC1 and ameliorates cytotoxicity through upregulation of the autophagy pathway to a level that is predominantly cytoprotective. In addition, compound 2 significantly decreases lysosomal protease levels which may prevent autophagy-mediated degradation of NPC1 and exert a neuroprotective effect in NPC disease. VI. Exemplary Aspects The following numbered paragraphs illustrate exemplary aspects of the disclosed technology. Paragraph 1. A compound of Formula I 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 or a pharmaceutically acceptable A membered heteroaryl; C1-6alkyl, C6-10aryl, C6-10aryl-C1-6alkyl, 6-10 membered heteroaryl, 6-10 C1-6alkyl, C3-6cycloalkyl, C3-6cycloalkyl-C1-6alkyl, 5- or 6- membered heterocyclyl or 5- or 6-membered heterocyclyl-C1-6alkyl, -NR5R6, -alkyl-NR5R6, or -NRa-alkyl-NR5R6, where R1is optionally substituted with one or more groups independently selected from -NR7R8, -alkyl-NR7R8, -C(O)R7, -C(O)NR7R8, -SO2R7, C6-10aryl-C1-6alkyl, 6-10 membered heteroaryl-C1-6alkyl, C3-6cycloalkyl-C1-6alkyl, or 5- or 6- membered heterocyclyl-C1-6alkyl; each Raindependently is H or C1-6alkyl; R2 and R3 are independently selected from H, C1-6alkyl, C6-10aryl, C6-10aryl-C1-6alkyl, 6-10 membered heteroaryl, 6-10 membered heteroaryl-C1-6alkyl, C3-6cycloalkyl, C3-6cycloalkyl-C1-6alkyl, 5- or 6-membered heterocyclyl or 5- or 6-membered heterocyclyl-C1-6alkyl , or R2and R3, together with the N atom to which they are attached form a 5- or 6- membered heterocyclyl, where R2 and R3 are independently optionally substituted with one or more substituents selected from C1-6alkyl, -SO2C1-6alkyl, -C(O)R7, or C(O)NR7R8, such as C1-6alkyl, -SO2C1-6alkyl, or -C(O)C1-6alkyl; R4is H or C1-6alkyl; R5 and R6 are independently selected from H, -C(O)R7, -C(O)NR7R8, -SO2R7, C1-6alkyl, C6-10aryl, C6-10aryl-C1-6alkyl, 6-10 membered heteroaryl, 6-10 membered heteroaryl- C1-6alkyl, C3-6cycloalkyl, C3-6cycloalkyl-C1-6alkyl, 5- or 6-membered heterocyclyl, or 5- or 6- membered heterocyclyl-C1-6alkyl, or R5and R6together form a 5- or 6-membered optionally substituted heterocyclyl, where R5 and R6 are independently optionally substituted with halo or C1-6alkyl; R7 and R8 are independently selected from C1-6alkyl, C6-10aryl, C6-10aryl-C1-6alkyl, 6- 10 membered heteroaryl, 6-10 membered heteroaryl-C1-6alkyl, C3-6cycloalkyl, C3-6cycloalkyl- C1-6alkyl, 5- or 6-membered heterocyclyl, or 5- or 6-membered heterocyclyl-C1-6alkyl, where R7and R8are independently optionally substituted with one or more groups independently 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 selected from -NR5R6, -alkyl-NR5R6, -C(O)R5, -C(O)NR5R6, -SO2R5, C6-10aryl, C6-10aryl-C1-6alkyl, 6-10 membered heteroaryl, 6-10 membered heteroaryl-C1-6alkyl, C3-6cycloalkyl, C1-6cycloalkyl-C1-6alkyl, 5- or 6-membered heterocyclyl, and 5- or 6-membered heterocyclyl-C1-6alkyl; and wherein the compound is not . NR5R6, - NRa-phenyl, or 5- or 6-membered nitrogen-containing heterocyclyl, and is optionally substituted. Paragraph 3. The compound of paragraph 1 or paragraph 2, wherein R1is -NRa-C1-6alkyl-NR5R6, or -NRa-phenyl and is optionally substituted. Paragraph 4. The compound of any one of paragraphs 1-3, wherein R1is unsubstituted. Paragraph 5. The compound of any one of paragraphs 1-3, wherein R1is substituted with 1, 2, or 3 substituents selected from C1-6alkyl, or halo. Paragraph 6. The compound of any one of paragraphs 1-5, wherein each Rais H. Paragraph 7. The compound of any one of paragraphs 1-6, wherein R2and R3are independently H, C1-6alkyl, C3-6cycloalkyl, C3-6cycloalkyl-C1-6alkyl-, or 5- or 6-membered heterocyclyl, and where R2and R3are independently optionally substituted. Paragraph 8. The compound of paragraph 7, wherein R2 and R3 are independently C1-6alkyl. Paragraph 9. The compound of any one of paragraphs 1-6, wherein R2 and R3 together with the nitrogen to which they are attached form a 5- or 6-membered optionally substituted heterocyclyl. Paragraph 10. The compound of paragraph 9, wherein R2and R3together with the nitrogen to which they are attached form an optionally substituted piperazinyl, piperidinyl, morpholinyl, or pyrrolidinyl ring. Paragraph 11. The compound of paragraph 10, wherein R2 and R3 together with the nitrogen to which they are attached form an optionally substituted piperazinyl ring. 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 Paragraph 12. The compound of any one of paragraphs 9-11, wherein the 5- or 6- membered heterocyclyl formed by R2and R3is substituted with 1, 2, or 3 substituents selected from C1-6alkyl or -SO2C1-6alkyl. Paragraph 13. The compound of paragraph 12, wherein the 5- or 6-membered heterocyclyl is piperazinyl substituted with C1-6alkyl or -SO2C1-6alkyl. Paragraph 14. The compound of paragraph 12 or paragraph 13, wherein the substituents are selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl. Paragraph 15. The compound of any one of paragraphs 9-14, wherein R2 and R3 together with the atom to which they are attached , , R4is C1-6alkyl. Paragraph 17. The compound of paragraph 16, wherein R4 is methyl. Paragraph 18. The compound of any one of paragraphs 1-17, wherein the compound has a structure according to Formula II or a pharmaceutically acceptable Paragraph 19. The compound of any one of paragraphs 1-18, wherein the compound has a structure according to Formula IV 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 or a pharmaceutically acceptable R9 is H, C1-6alkyl, -SO2C1-6alkyl, - C(O)R7, or C(O)NR7R8, such as C1-- or -C(O)C1-6alkyl. Paragraph 20. The compound of any one of paragraphs 1-18, wherein the compound has a structure according to Formula III or a pharmaceutically –(CRa2 n ) - and n is from 1 to 6. Paragraph 21. The compound of any one of paragraphs 1-20, wherein R5 and R6 are independently H or C1-6alkyl. Paragraph 22. The compound of paragraph 21, wherein R5and R6are independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl. Paragraph 23. The compound of any one of paragraphs 1-20, wherein R5and R6together form a 5- or 6-membered optionally substituted heterocyclyl. Paragraph 24. The compound of any one of paragraphs 1-23, wherein R7and R8are independently selected from C1-6alkyl. Paragraph 25. The compound of any one of paragraphs 20-23, wherein the compound has a structure according to Formula V or a pharmaceutically acceptable is H, C1-6alkyl, -SO2C1-6alkyl, - C(O)R7, or C(O)NR7R8, such as C1-6alkyl, -SO2C1-6alkyl, or -C(O)C1-6alkyl. 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 Paragraph 26. The compound of paragraph 25, wherein R9 is C1-6alkyl, -SO2C1-6alkyl, or -C(O)C1-6alkyl. Paragraph 27. The compound of paragraph 25, wherein R9 is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl. Paragraph 28. The compound of any one of paragraphs 1-19, wherein R1 is or from , 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 , according to Formula VI 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 or a pharmaceutically Paragraph 31. A . one of paragraphs 1-31 and a pharmaceutically acceptable diluent or excipient. Paragraph 33. A method of treating a disease or condition where modulation of autophagy would provide a benefit comprising treating a subject with an effective amount of a compound according to any one of paragraphs 1-31. Paragraph 34. A method, comprising administering to the subject having a disease or condition, an effective amount of a compound according to any one of paragraphs 1-31, or a pharmaceutical composition thereof. Paragraph 35. The method of paragraph 33 or paragraph 34, wherein the disease or condition is selected from liver disease, diabetes, kidney disease, heart disease, inflammatory bowel disease, neurodegenerative disease, and cancer. Paragraph 36. The method of paragraph 35, wherein the disease or condition is a neurodegenerative disease or disorder. Paragraph 37. The method of paragraph 36, wherein the neurodegenerative disease or condition is selected from Alzheimer’s disease, Parkinson’s disease, Huntington's disease, Amyotrophic lateral sclerosis, or Niemann-Pick Type C. Paragraph 38. The method of paragraph 36, wherein the neurodegenerative disease or disorder is Niemann-Pick Type C. 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 Paragraph 39. The method of paragraph 34, wherein the disease or condition is cancer. Paragraph 40. The method of paragraph 39, wherein the cancer is selected from lung cancer, pancreatic ductal adenocarcinoma, melanoma, breast cancer, ovarian cancer and colorectal cancer. Paragraph 41. A method of modulating autophagy in a cell, comprising contacting the cell with an effective amount of a compound of any one of paragraphs 1-31. Paragraph 42. A use of a compound according to any one of paragraphs 1-31 in the preparation of a medicament for the treatment of liver disease, diabetes, kidney disease, heart disease, inflammatory bowel disease, neurodegenerative disease, or cancer. Paragraph 43. A use of a compound according to any one of paragraphs 1-31 in the preparation of a medicament to modulate autophagy. Paragraph 44. A compound according to any one of paragraphs 1-31 for use in a method of administration to a subject in need thereof. Paragraph 45. The compound of paragraph 44, wherein the subject has liver disease, diabetes, kidney disease, heart disease, inflammatory bowel disease, neurodegenerative disease, or cancer. Paragraph 46. A compound according to any one of paragraphs 1-31 for use in a method of modulating autophagy. VII. Examples Materials and Methods Control Compounds: DMSO (Corning, #25-950-CQC), CQ (Sigma-Aldrich, #C6628), Rap (LC Laboratories, #R5000), BafA1 (LC Laboratories, #B-1080), CBZ (Sigma- Aldrich, C4024) were purchased from commercial vendors.2HPβCD was provided by Roquette (KleptoseHPB). eGFP-LC3 Assay HeLa cells stably expressing the green fluorescent protein on LC3 were a gift from Ramnik Xavier from Massachusetts General Hospital.50 µL of eGFP-LC3 HeLa cells were plated at a cell density of 3,000 cells / well in a black 384-well plate (Corning, #3764) in DMEM (Corning, 15-013-CV) supplemented with 10% FBS (Sigma-Aldrich, #12306C), 1x pen-strep (Corning, #30-002-Cl), and 1 % L-glutamine (Corning, #25-005-Cl), and incubated overnight at 37 °C and 5 % CO2. After 24 hours, a Biomek NXPliquid handler (Beckman- 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 Coulter) was used to transfer compounds from a compound plate into the assay plate, where compounds had a final concentration of 20 µM, the control, CQ (20 µM), and the other tested compounds included Rap (4 µM), and 2HPβCD (1 mM). The assay plate was incubated at 37 °C and 5 % CO2for 4 hours, and then the MultiFlo FX (BioTek, #MFXPW) was used to aspirate the media and add 25 µL of 4% paraformaldehyde (PFA) (Electron Microscopy Sciences, #15710) to each well to fix the cells. The cells incubated in the PFA for 12 minutes at room temperature (RT) in the dark, after which the solution from each well was aspirated and 25 µL of PBS was added and then aspirated to clean the wells. Hoechst 33342 nuclear stain (Thermo, #H3570) was diluted in PBS and 25 µL dispensed into each well for a final concentration of 2 µg / mL and incubated in the dark at room temperature for 10 minutes. The Hoechst solution was aspirated, 25 µL of PBS added to each well, the plate was sealed using the PlateMax semi-automatic plate sealer (Axygen) and imaged at 10x magnification on the ImageXpress Micro (IXM) XLS automated fluorescent microscope (Molecular Devices) using the DAPI and FITC filters. The images were analyzed using MetaXpress software where an acceptable Z’ (Z factor) was between 0.4-0.6 for cell-based assays, a % CV of less than 20 %, and a z-score greater than 2.199 in two replicates was used to identify hits in the HTS.312 autophagy modulators were identified from the ChemDiv library to use in subsequent experiments. Prioritized hits were then synthesized and validated in the eGFP- LC3 experiments. Filipin Assay I1061T NPC1 patient-derived fibroblasts were plated at 35,000 cells / ml in a black 384-well assay plate (PerkinElmer, #6057300) at 50 µL / well using DMEM (Corning, 15-013- CV) supplemented with 20% FBS (Sigma-Aldrich, #12306C), 1x pen-strep (Corning, #30- 002-Cl), and 1% L-glutamine (Corning, #25-005-Cl) and incubated at 37 °C and 5 % CO2for 24 hours. Cells were then treated with test compounds (10 µM), Rap (4 µM), and 2HPβCD (1 mM) using the Biomek NXPliquid handler and incubated for an additional 24 hours at 37 °C and 5% CO2. Media was aspirated from each well using the MultiFlo FX, and replaced with 25 µL of 4% PFA for 1 hour at RT. After 1 hour, the PFA solution was aspirated, and the fibroblasts rinsed three times with PBS containing 50 mM NH4Cl. Cells were then stained with 25 µL of 0.05 mg / mL filipin (Sigma-Aldrich, #F9765) where the filipin was dissolved in PBS that was mixed with 10% FBS (Sigma-Aldrich, #12306C). The assay plate was then left at RT in a drawer covered in foil for 2 hours. To conclude the assay, cells were rinsed three 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 times with PBS without ammonium chloride, 25 µL of PBS dispensed into each well, the plate sealed using the PlateMax semi-automatic plate sealer (Axygen) and imaged immediately using the DAPI filter at 20x magnification with the IXM XLS automated fluorescent microscope (Molecular Devices). The images were analyzed using MetaXpress software and quantified as pit integrated intensity, the total pixel intensity divided by all the pit areas in the image, where wells with a z-score of less than -1.25 were considered hits. Filipin Assay in NPC1 null HeLa cells NPC1 null HeLa cells, a gift from Judith Storch, Rutgers, were plated at 38,000 cell / ml in a black 384-well plate (PerkinElmer, #6057300) in warmed media (same media as eGFP-LC3 assay), and all remaining steps for the assay and analysis were performed as with the I1061T NPC1 fibroblast cell line. mCherry-GFP-LC3 (Dual Reporter) Assay HeLa cells stably expressing mCherry-GFP-LC3 were a gift from Ramnik Xavier at Massachusetts General Hospital. The dual reporter assay was performed following the same protocol as the eGFP-LC3 assay, except the cells underwent 24 hours compound treatment and the plates were imaged using 3 filters: DAPI for Hoechst, FITC for GFP, and Texas Red for mCherry. CellProfiler 3.1.9, rather than MetaXpress, was used to analyze the images by determining the number of autophagosomes and autolysosomes to gain insight into autophagic flux. Dye Quenched-Bovine Serum Albumin (DQ-BSA) in HeLa cells HeLa cells were plated in a black 384-well plate (Corning, #3764) at 2,500 cells / well in 40 µL of warmed media (same media as eGFP-LC3 assay) and incubated overnight at 37 °C and 5% CO2. The next day, DQ-BSA stock of 1 mg / mL (Invitrogen, #D12051) was diluted in warmed media, and 10 µL dispensed into each well for a final concentration of 10 µg / mL. The plate was incubated at 37 °C and 5% CO2 for 1 hour, and then the media removed using the MultiFlo FX. The wells were washed with 1x PBS twice and replaced with 40 µL of media. DMSO, 2HPβCD (1 mM), CQ (20 µM), BafA1 (100 nM), 1 (10, 20 µM), 2 (20,40 µM) were pin-transferred into the assay plate using the Biomek NXPliquid handler (Beckman-Coulter) and incubated at 37 °C and 5% CO2 for 6 hours. Hoechst 33342 was diluted in warmed media and 10 µL dispensed into each well using the Multi Flo FX the 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 last 30 minutes of incubation for a final concentration of 2 µg / mL. The contents of each well were then aspirated using the MultiFlo FX and replaced with 40 µL of 1x HBSS. The plate was sealed using the PlateMax semi-automatic plate sealer (Axygen) and imaged at 20x magnification on the IXM XLS automated fluorescent microscope (Molecular Devices) using the DAPI and Texas Red filters. The images were analyzed using MetaXpress software. Dye Quenched-Bovine Serum Albumin (DQ-BSA) in I1061T NPC1 fibroblasts I1061T NPC1 patient-derived fibroblasts (28,000 cells / mL) were plated in a 384-well black plate (Corning, #3764) using the same media as the filipin assay and incubated overnight at 37 °C and 5% CO2. The next day, DQ-BSA was added as with the DQ-BSA in HeLa cells and then removed using the MultiFlo FX. The wells were washed twice with PBS and replaced with 40 µL of warmed media. DMSO, 2HPβCD (1 mM), CQ (20 µM), BafA1 (100 nM), 1 (10 µM), 2 (20 µM) were pin-transferred into the assay plate using the Biomek NXPliquid handler (Beckman-Coulter) and incubated at 37 °C and 5% CO2for 24 hours. Hoechst 33342 (2 µg / mL) was then used to stain the nuclei and replaced with 40 µL of 1x HBSS. The plate was sealed and imaged as described for the HeLa cells. Cytotoxicity Assay I1061T NPC1 patient-derived fibroblasts were plated in a white 384-well plate (Corning, #3765) at 28,000 cells / mL with 50 µL / well and incubated at RT for 1 hour and then placed in the 37 °C incubator in a container containing a water reservoir for 24 hours. Cells were then compound treated with DMSO, 2HPβCD (1 mM), CQ (20 µM), BafA1 (100 nM), 1 (10 - 80 µM), and 2 (10 - 80 µM) and incubated in 37 °C and 5% CO2for 24 hours. The next day, the assay plate and CellTiter-Glo 2.0 reagent (Promega, #G9242) were both incubated at RT for 30 minutes, and then 25 µL of CellTiter-Glo 2.0 reagent to each well for a total volume of 75 µL / well. The assay plate was covered in foil and placed on the orbital shaker for 10 minutes at 150 rpm. The luminescence reading was taken on the SpectraMax i3x (Molecular Devices) using the Softmax Pro 6.5.1 software where the integration time was set to 500 ms and the read height set to 6.50 mm from the plate. 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 Immunoblotting NPC1 Immunoblots I1061T NPC1 patient-derived fibroblasts were plated in a 24-well plate (Corning, #353047) at a seeding density of 75,000 cells / well in the same media as filipin and left in room temp for 1 hour to allow cells to adhere to plate bottom, and then placed into the 37 °C incubator for 24 hours. DMSO, 2HPβCD (1 mM), 1 (10, 20 µM), and 2 (10, 20 µM) were added to wells by hand and incubated at 37 °C and 5% CO2 for 24 hours. Cells were then lysed using NP-40 Lysis Buffer made in a 15 mL tube comprised of 10 mL of 1xTris- Buffered Saline (TBS) (Corning, #46-012-CM), 1 Pierce protease and phosphatase inhibitor tablet (Thermo, #A32959), and 1 % IGEPAL (Sigma-Aldrich, #56741). Lysate was centrifuged (Thermo Scientific, Sorvall Legend Micro 21R) at 18,000 g for 30 minutes at 4 °C to remove debris, the supernatant was separated by 10% SDS-PAGE (120 V, 1.5 h) and protein was transferred onto PVDF membrane (EMD Millipore, #IPFL00010) at 25 V for 1 hour. The membrane was blocked with 5% Blotting-grade Blocker (BioRad, #1706404) in TBS (Corning, #46-012-CM) supplemented with Tween-20 (VWR, #500-018-3) (TBS-T) for 1 hour at room temperature and then incubated overnight with primary antibody for NPC1 (1:1000 ) (Abcam, EPR5209) and β-actin (1:2000) (Cell Signaling Technology, 8457) in 5% Blotting-grade Blocker in TBS-T. The blots were washed thrice with TBS-T and incubated with HRP-conjugated secondary antibody in 5% blotting-grade blocker for 1 hour at room temperature. Membranes were washed again with TBS-T and proteins visualized using anti- rabbit IgG (Cell Signaling Technology, #7074S) with SuperSignal West Pico Plus Stable Peroxide Solution (Thermo, #1863097) and luminol / enhancer solution (Thermo, #1863096) using the c Series Capture Software on the Azure Imaging System. P70S6K and Phospho-P70S6K Immunoblots Same as NPC1 immunoblot protocol, except 1% BSA (Sigma-Aldrich, 9048-46-8) was used for the blocking step for the phospho-P70S6K antibody. Additionally, after visualizing the phosphorylated p70S6K (1:500) (Cell Signaling Technology, 9205S) and β- actin (1:2000) on the Azure system, the membrane was stripped using stripping buffer (Thermo Scientific, #21059) and re-probed with the non-phosphorylated p70S6K antibody (1:1000) (Cell Signaling Technology, #2708S). 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 LGMN, CTS Z, B, D, and LAMP1 Immunoblots I1061T NPC1 fibroblasts were plated at a density of 132,000 cells / well in a 6-well plate (Corning, # 353046) in the same media as the filipin assay and grown for 24 hours. Cells were treated with DMSO, 2 (80 µM), CQ (10 µM), and BafA1 (0.1 µM) and incubated for 24 hours. The rest of the blot is the same as for NPC1 immunoblot, except the primary antibodies included LGMN (1:1000) (Cell Signaling Technology, 93627S), CTSZ (1:750) (ThermoFisher Scientific, PA5-62072), CTSB (1:750) (Cell Signaling Technology, #31718S), CTSD (1:750) (Cell Signaling Technology, 2284S), and LAMP1 (1:750) (Cell Signaling Technology, #3243) and that 1% BSA (Sigma-Aldrich, 9048-46-8) was used as the blocking buffer for CTSZ and CTSD. Membranes were stripped with stripping buffer (Thermo Scientific, #21059) and re-probed with β-actin (Cell Signaling Technology, 8457). LC3 Immunoblots I1061T NPC1 fibroblasts were plated at a density of 125,000 cells / well in a 12-well plate (Corning, #353043) in the same media as the filipin assay and grown for 24 hours. Cells were treated with DMSO (0.1% v), CQ (20 μM), 2HPβCD (1 mM), BafA1 (0.2 μM), and 2 (40μM and 20μM) and incubated for 24 hours. The rest of the protocol is the same as for NPC1 immunoblots, except the LC3B (1:1000) (Cell Signaling Technology, 2775S). Membranes were stripped with stripping buffer (Thermo Scientific, #21059) and re-probed with β-actin (1:2000) (Cell Signaling Technology, 8457). Tandem Mass Tag and Mass Spectrometry Analysis Sample Prep I1061T NPC1 patient-derived fibroblasts were cultured in the same media as filipin assay at 1,500,000 cells / ml in six 10 cm tissue culture dishes (Corning, 353003) and grown overnight (22-24 h) in 37 °C and 5% CO2. Three plates were then treated with DMSO and three plates with either 1 (40 µM) or 2 (80 µM) for 24 hours. Cells were then harvested, rinsed with 1x PBS (Corning, 21-040-CM) thrice, and flash frozen in a 1.5 mL microcentrifuge tube. Cells were resuspended in 70 µL Lysis Buffer (10% SDS in 100mM TEAB, 1 mM protease inhibitor, and phosphatase inhibitor [10 mM Na4O7P2, 1 mM PMSF, 1 mM Sodium Orthovanadate, 1 mM β-Glycerophosphate disodium salt, 1 mM Sodium Fluoride]) and sonicated (QSonica Sonicators, model CL-18) for 3 sonication cycles where 1 cycle consisted of 10 sec (1 sec on and 1 sec off). After sonication, samples were centrifuged 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 (Eppendorf, Centrifuge 5424 R) at 14,000 rpm for 10 minutes at 20 °C and the supernatant transferred to a new microcentrifuge tube. Bicinchoninic acid (BCA) Assay (Thermo Scientific, 23225) was performed in a 96- well plate (Fisher Scientific, 12565501) and analyzed on a Molecular Devices, VERSA max tunable microplate reader to determine protein concentration in accordance with the manual. 75 µg protein after treatment with 1, and 100 µg protein after treatment with 2, was transferred into a new microcentrifuge tube, where proteins were reduced with DL- Dithiothreitol (Sigma-Aldrich, D0632) and alkylated with iodoacetamide (Sigma, I1149). The sample was then trypsin digested (Thermo Scientific, 90058) using a micro S-trap spin column (Protifi, C02-mini / micro-80) following provided protocol with minor modification. After loading the acidified protein mixture onto the S-Trap, captured proteins were rinsed 7 times with s-trap buffer, and 1:50 trypsin to protein ratio was used for overnight digestion. After elution of digested peptides, samples were dried down in vacuo (Labconco Acid- resistant CentriVap Concentrator). Samples were then labeled using the TMTsixplex Isobaric Reagent Set (Thermo Scientific, 90061) in accordance with the provided protocols. Briefly, 0.8 mg of TMT reagent was resuspended in 41 µL MeCN and added to each of the six samples. The mixture incubated at RT for 1 hour and then quenched with 8 µL 5% hydroxylamine for 15 minutes. The 6 samples (three DMSO replicates, and three compound- treated replicates) were combined into 1 microcentrifuge tube and dried down in vacuo. A series of MeCN solutions with percentages ranging from 1 to 80% MeCN were prepared. The protein sample was then resuspended via vortexing in 800 µL of 1% MeCN solution and fractionated manually into twelve 1.5 mL microcentrifuge tubes using Oasis HLB cartridge (Waters, WAT094225), Nitrogen gas, and the series of MeCN solutions. After fractionation, samples were dried down in vacuo and stored in -20 °C. Samples were then resuspended in 100 µL of 0.1% formic acid (FA) to yield an estimated final concentration of 0.5 µg / µL in each fraction. The samples were then vortexed 40 times with pulsing, centrifuged at 14,000 g for 10 minutes, and 50 µL supernatant transferred to LCMS vials (Thermo Scientific, C4011-13). LC-MS Data Acquisition Approximately 250 ng of protein from each of the 12 samples was then injected into an Agilent 1260 Infinity nanoLC system (Agilent Technologies, Santa Clara, CA) coupled with a Q Exactive mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Samples 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 were loaded onto a Thermo NanoViper trap column (75 µm x 20 mm, 3 µm C18, 100 Å) (Thermo Fisher Scientific, Bremen, Germany) and washed with solvent A (0.1% FA in water) for 10 minutes at 2 μL / min flowrate and then loaded onto an Agilent Zorbax 300SB- C18 column (0.075 × 150 mm, 3.5 μm 300 Å) at 5% B (0.1% FA in MeCN). Separation was carried out using a 60-min gradient going from 5 to 60% B with a flowrate of 0.25 μL / min. The system was then increased from 60-90% B in 0.1 minutes, then maintained at 90% B for 10 minutes prior to a 15-min re-equilibration segment at 5% B prior to the next run. Mass spectra were collected using data-dependent acquisition (DDA) with a capillary temperature of 250 °C and spray voltage of 1.5 kV. Full MS scans were collected at a mass resolution of 70,000 with a scan range of 375–1600 m / z. Automatic gain control (AGC) target was set at 1 × 106 for a maximum injection time (IT) of 100 ms. The top ten most intense peaks were selected for MS / MS analysis, with an isolation width of 1.5 m / z. MS / MS spectra were acquired at a resolution of 17,500, ACG target 1 × 105, maximum IT of 50 ms. The first fixed mass was set at 100 m / z. Parent ions were fragmented at a normalized collision energy (NCE) of 27%. Dynamic exclusion was set for 20 s. Parent ions with charges of 1 and larger than 6 were excluded. Data Analysis Raw files were analyzed using Thermo Proteome Discoverer 2.3.0.523 (Thermo Fisher Scientific, Bremen, Germany). Using the Sequest HT search engine, data were searched against the UniProt Homo Sapien database (42,368 gene sequence; downloaded June 12, 2019) with 10 ppm precursor mass tolerance. A maximum missed cleavage of 2 by trypsin was set for amino acid sequence between 6 and 144 residues in length. Fragment masses were searched with a tolerance of ± 0.02 Da. Dynamic modifications included Oxidation (+15.995 Da; M), TMT6plex (+229.163 Da; S, T), Deaminated (+0.984 Da; N, Q, R), and Acetylation (+42.011 Da; N-terminus). Carbamidomethylation (+57.021 Da; C) and TMT6plex (+229.163 Da; K, N-terminus) were set as static modifications. Both peptides and PSMs were set to a target false discovery rate (FDR) of ≤ 0.05 for peptide-spectrum matches with moderate confidence and ≤ 0.01 for matches with high confidence. All protein hits contain two or more peptide matches. Quantification was performed using relative abundance of TMT6plex reporter ions intensities, based on signal to noise values. Protein ratios were calculated based on group protein abundances. For statistical analysis, t-test was performed based on the abundances of individual proteins. 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 Glycan Digestion Assays I1061T NPC1 patient-derived fibroblasts were plated in a 6-well tissue culture treated plate (Corning, #353046) at a density of 200,000 cells / mL with 2 mL of the cell suspension in each well. The cells were left outside of the incubator in the dark at 25 °C for 30 minutes to allow the cells to settle and begin adhering to the bottom of the plate before transferring to the incubator for 24 hours. The following day, the cells were treated with DMSO (2 μL), HPβCD (1 mM), BafA1 (100 nM), or 2 (20 μM) and left for 24 hours in the cell incubator. After 24 hours, the cells were lysed with 100 μL of M-PER Mammalian Protein Extraction Reagent (Thermo, #78501) supplemented with a Pierce protease and phosphatase inhibitor tablet (Thermo, #A32959). Lysate was centrifuged at 18,000 g for 30 minutes at 4 °C to remove debris and the supernatant was transferred to a clean microcentrifuge tube. Protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo, #23225). For the PNGase F, Endo H, and NT (not treated) samples, a 10 μL reaction volume was made by combining 10 μg of glycoprotein, 1 μL of 10x Glycoprotein Denaturing Buffer (NEB, #B1704SVIAL) and ultrapure H2O if necessary to bring total volume to 10 μL. Samples were denatured for 10 mins at 100 °C, cooled on ice for 5 minutes, then briefly spun down. For the PNGase F reaction, 2 μL 10x GlycoBuffer 2 (NEB, #B3704SVIAL), 2 μL 10% NP-40 (NEB, #B2704SVIAL), and 6 μL H2O were added to the reaction vial to make a final volume of 20 μL. The samples were briefly spun down, then 0.5 μL of PNGaseF stock (NEB, #P0704SVIAL) was added to the reaction and incubated at 37 °C for 3 hours. For the Endo H and NT samples, 2 μL of 10x GlycoBuffer 3 (NEB, # B1720SVIAL), 0.75 μL Endo H (NEB, # P0702SVIAL), and ultrapure H2O were added to the reaction vial to bring total volume to 20 μL. Endo H was not added to the NT reaction vial. These samples were incubated at 37 °C for 3 hours. Reactions were stopped by adding 7.7 μL of 4x LDS Sample Buffer (Thermo, #NP0007) and taken onto immunoblotting. The samples were run on a NuPAGE 4 to 12% Bis-Tris Gel (Thermo Fisher Scientific, #NP0335BOX) following manufacturer's instructions for gel running buffer and transfer buffer. For experiments with HeLa cells, cells were plated in a 12-well tissue culture treated plate (Corning, #353043) at a density of 120,000 cells / mL with 1 mL of the cell suspension in each well. The cells were treated with DMSO (2 μL), HPβCD (1 mM), BafA1 (0.2 μM), or 2 (20 μM) and lysed after 24 hours with 75 μL of M- PER Mammalian Protein Extraction Reagent (Thermo, #78501) supplemented with a Pierce protease and phosphatase inhibitor tablet (Thermo, #A32959). For the PNGase F, Endo H, and NT reactions, 5 μg of glycoprotein was used. For experiments with WT NPC1 fibroblasts, cells were plated in a 6-well tissue culture plate (Corning, #353046) at a density 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 of 200,000 cells / mL with 2 mL of the cell suspension in each well. The cells were treated with DMSO (2 μL), HPβCD (1 mM), BafA1 (0.2 μM), or 2 (20 μM) and lysed after 24 hours with 100 μL of M-PER Mammalian Protein Extraction Reagent (Thermo, #78501) supplemented with a Pierce protease and phosphatase inhibitor tablet (Thermo, #A32959). For the PNGase F, Endo H, and NT reactions, 10 μg of glycoprotein was used. Synthesis Compounds of the invention may be prepared as shown in Schemes 1-4. Scheme 1 Synthetic Route to Access Hit Compound 1

[0002] 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 Scheme 2 Synthetic Route to Access Hit Compound 2

[0003] 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 Scheme 3 Synthesis of Substituents for Compound 2 Analogs O Me O Me HO OO S O S R1

[0004] 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 Scheme 4 Synthetic Route to Access Biotinylated Hit Compound 2 (Biotin-2) General Synthetic Methods All chemicals were purchased from Sigma-Aldrich, Alfa-Aesar, Acros Organics, TCI America, or Oakwood Chemicals and were used without further purification unless otherwise noted. Reaction progress was monitored by TLC (Merck KGaA Silica gel 60 F254 glass backed plates). Reaction progress and mass spectrometry data were collected by LCMS (Agilent 1260 Series automated chromatographic system outfitted with a Thermo Scientific Accucore column (2.1 x 50mm, 2.6 µm particle size)) utilizing a gradient elution mobile phase of 25% MeCN:H2O to 95% MeCN:H2O over 2 minutes, then holding at 95% MeCN:H2O for 2 minutes (0.200 mL / min flow rate, 30 °C column compartment, detection 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 modes: wavelengths of 254 and 280 nm and an Agilent 6120 quadrupole MS). NMR data were collected on a Bruker AV 500 MHz spectrometer outfitted with a Bruker 5mm1H19F / BBO S2 Z-gradient probe, and data were processed with Mestrenova (Mestrelab Research). Data were recorded at ambient temperature and are reported as chemical shift (ppm) relative to solvent peak (1H NMR: CDCl3 = 7.26 ppm, MeOD = 3.31 ppm, DMSO = 2.50 ppm;13C NMR: CDCl3= 77.16 ppm, MeOD = 49.00 ppm, DMSO = 39.52 ppm). Multiplicity is reported as follows: s = singlet, d = doublet, t = triplet, q = quartet, p = quintet / pentet, dd = doublet of doublets, dt = doublet of triplets, td = triplet of doublets, ddd = doublet of doublet of doublets, ddt = doublet of doublet of triplets, dddd = doublet of doublet of doublet of doublets. High-resolution mass spectrometry data were collected on a Waters Synapt G2-Si ESI MS, a Micromass 70-VSE equipped with an EI / CI source, or a Waters Q- TOF Ultima ESI and were collected at the University of Illinois at Urbana-Champaign School of Chemical Sciences. IR data were collected on a ThermoScientific Nicolet IS5 spectrophotometer outfitted with a ThermoFisher Scientific iD5 ATR. Microwave reactions were performed in a Biotage Initiator + microwave reactor and reaction mixtures were purified on a Biotage Isolera One automated chromatography system with normal-phased silica gel columns and C18-reversed phase silica gel columns. Synthetic Methods for Hit Compounds N-(2-cyclohexyl nicotinamide (1) To a flame dried flask was added 6-(3-phenylpiperazin-1-yl)nicotinic acid (33.3 mg, 0.118 mmol) in anhydrous DMF (2.0 mL).1-Hydroxybenzotriazole hydrate (HOBt) (39.7 mg, 0.235 mmol, 20 wt.% water) was then added into the reaction flask. The mixture was stirred for 10 minutes until all solids were dissolved, and N-(2-aminoethyl)-N-methylcyclohexanamine (7) (18.4 mg, 0.118 mmol) was then added as a solution in anhydrous DMF (0.5 mL). The resulting mixture was stirring at room temperature for 10 minutes, and DIPEA (122.6 µl, 0.705 mmol) followed by 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (27.1 mg, 0.235 mmol) was slowly added into the reaction mixture. The resulting mixture was allowed to stir at room temperature for 14 hours. Once the reaction reached completion, the solution was purified via normal-phase silica gel column chromatography (CH2Cl2:MeOH / 9:1) to 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 yield the product as pale yellow oil (14.6 mg, 25% over 2 steps). IR (neat): 3304, 2925, 2850, 1632, 1599, 1489, 1404, 1326, 1251, 1169, 700 cm-1.1H NMR (500 MHz, CDCl3) δ 8.57 (s, 1H), 7.93 (d, J = 8.8 Hz, 1H), 7.44 (d, J = 7.3 Hz, 2H), 7.35 (t, J = 7.3 Hz, 2H), 7.31 – 7.27 (m, 1H), 7.01 (s, 1H), 6.62 (d, J = 8.9 Hz, 1H), 4.34 (d, J = 11.0 Hz, 2H), 3.83 (d, J = 8.7 Hz, 1H), 3.45 (d, J = 5.2 Hz, 2H), 3.22 (d, J = 9.2 Hz, 1H), 3.04 (p, J = 12.0 Hz, 2H), 2.87 (t, J = 11.6 Hz, 1H), 2.66 (t, J = 5.2 Hz, 2H), 2.40 (s, 1H), 2.28 (s, 3H), 1.78 (d, J = 7.0 Hz, 4H), 1.62 (d, J = 12.3 Hz, 1H), 1.27 – 1.18 (m, 6H), 1.07 (s, 1H).13C NMR (125 MHz, CDCl3) δ 165.9, 160.2, 147.3, 141.6, 136.9, 128.6, 127.9, 127.1, 119.2, 105.7, 63.2, 60.3, 52.5, 51.5, 46.0, 45.0, 37.2, 36.8, 28.6, 26.2, 25.9. HRMS (ES+) m / z calculated for C25H36N5O+[M+H]+: 422.2920; found: 422.2928. Ethyl 2-bromo-2-phenylacetate To charged with argon was added 2- bromo-2-phenylacetic acid (2.15 g, 10.0 mmol) as a suspension in anhydrous ethanol (20.0 mL). The mixture was cooled to 0 °C and thionyl chloride (1.46 mL, 20.0 mmol) was then added to the reaction mixture dropwise. The reaction mixture was allowed to warm to room temperature and stir for 3 hours. The resulting mixture was diluted with water and the organic phase was separated. The aqueous phase was extracted with EtOAc (3x) and the combined organic extracts were washed with brine, dried over Na2SO4, and concentrated in vacuo to afford the crude product as a pale yellow oil (2.38 g, 98%), which was used in the next step without further purification.1H NMR (500 MHz, CDCl3) δ 7.53 (dd, J = 25.8, 6.4 Hz, 2H), 7.42 – 7.29 (m, 3H), 5.36 (s, 1H), 4.24 (qq, J = 10.8, 7.2 Hz, 2H), 1.28 (t, J = 7.1 Hz, 3H).13C NMR (125 MHz, CDCl3) δ 168.3, 135.9, 129.3, 128.8, 128.7, 128.0, 62.5, 59.2, 46.9, 14.0. 3-phenylpiperazin-2-one (3) To a ethyl 2-bromophenylacetate (1.35 g, 5.54 mmol) in 0.5M anhydrous ethanol was added ethylenediamine (0.74 mL, 11.1 mmol) dropwise. After the addition was completed, sodium ethoxide (754.0 mg, 11.1 mmol) was added and the reaction was heated to 65 °C for 18 hours. The resulting mixture was concentrated in vacuo to afford the crude product. The crude product was purified by normal- 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 phase silica gel column chromatography (CH2C2: MeOH / 9:1) to yield the desired product as a white solid (734.5 mg, 75%).1H NMR (500 MHz, CDCl3) δ 7.42 (d, J = 7.2 Hz, 2H), 7.36 (t, J = 7.4 Hz, 2H), 7.30 (t, J = 7.2 Hz, 1H), 6.61 (s, 1H), 4.58 (s, 1H), 3.58 – 3.49 (m, 1H), 3.42 – 3.34 (m, 1H), 3.16 (dt, J = 12.2, 4.2 Hz, 1H), 3.07 (ddd, J = 12.6, 8.6, 4.2 Hz, 1H), 1.88 (s, 1H).13C NMR (125 MHz, CDCl3) δ 170.8, 139.3, 128.6, 128.4, 128.0, 64.0, 43.3, 41.1. 2-phenylpiperazine (4) To a 2-one (3) (352.4 mg, 2.0 mmol), in 12 mL of anhydrous THF was mg, 9.0 mmol) in small portions at 0 °C. The mixture was slowly warmed up to room temperature and then heated to 75 °C for 3 hours. The resulting mixture was slowly quenched with water followed by 10% NaOH to quench excess LiAH4. The mixture was filtered, and the filter cake was extracted with EtOAc (3x). The combined organic filtrate and extracts were dried over Na2SO4 and evaporated to give crude product that was purified by normal-phase silica gel column chromatography (CH2Cl2:MeOH:Et3N / 9:1:0.1) to yield the desired product as white solid (232.6 mg, 72%).1H NMR (500 MHz, CDCl3) δ 7.33 (d, J = 7.4 Hz, 2H), 7.27 (t, J = 7.3 Hz, 2H), 7.21 (t, J = 7.0 Hz, 1H), 3.71 (d, J = 10.1 Hz, 1H), 3.04 (d, J = 11.9 Hz, 1H), 2.94 (dt, J = 23.0, 12.0 Hz, 3H), 2.82 (dd, J = 16.2, 8.2 Hz, 1H), 2.66 (t, J = 11.1 Hz, 1H), 2.22 (s, 2H).13C NMR (125 MHz, CDCl3) δ 142.6, 128.4, 127.4, 126.9, 61.9, 54.1, 47.6, 45.9. Methyl 6-chloronicotinate (5) To a acid (1.575 g, 10.0 mmol) in 0.25M anhydrous methanol under argon was added thionyl chloride (1.46 mL.20.0 mmol) dropwise. The reaction mixture was allowed to stir at room temperature for 3 hours. The resulting mixture was diluted with water and the organic phase was separated. The aqueous phase was extracted with EtOAc (3x), and the combined organic extracts were washed with brine, dried with Na2SO4, and concentrated in vacuo to afford the crude product that was purified by normal-phase silica gel column chromatography (Hex:EtOAc / 8:2) to yield the desired product as white solid (1.669 g, 97%).1H NMR (500 MHz, MeOD) δ 8.90 (s, 1H), 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 8.31 (d, J = 8.2 Hz, 1H), 7.56 (d, J = 8.3 Hz, 1H), 3.94 (s, 3H).13C NMR (125 MHz, MeOD) δ 164.7, 155.1, 150.5, 139.8, 125.4, 124.3, 51.7. Methyl 6-(3- dried microwave vial flask charged with an argon (4) (200.0 mg, 1.23 mmol), methyl 6-chloronicotinate (S5) (211.5 mg, 1.23 mmol), and anhydrous DMF (0.1 M). The resulting mixture was heated in a microwave reactor to 110 °C for 3 hours. After the reaction reached completion, the mixture was concentrated in vacuo and purified by normal-phase silica gel column chromatography (CH2Cl2:MeOH / 9:1) to yield the desired product as a yellow oil (185.8 mg, 51%).1H NMR (500 MHz, CDCl3) δ 8.78 (s, 1H), 8.00 (d, J = 8.6 Hz, 1H), 7.44 (d, J = 7.4 Hz, 2H), 7.35 (t, J = 7.3 Hz, 2H), 7.30 (t, J = 7.2 Hz, 1H), 6.58 (d, J = 9.1 Hz, 1H), 4.39 (t, J = 10.8 Hz, 2H), 3.84 (d, J = 8.0 Hz, 4H), 3.21 (d, J = 11.0 Hz, 1H), 3.09 (t, J = 13.0 Hz, 1H), 3.00 (t, J = 11.0 Hz, 1H), 2.93 – 2.85 (m, 1H), 2.11 (d, J = 33.6 Hz, 1H).13C NMR (125 MHz, CDCl3) δ 166.5, 160.7, 151.1, 141.5, 138.5, 128.6, 128.0, 127.1, 114.7, 105.2, 60.4, 52.2, 51.7, 46.0, 44.9. (3-phenylpiperazin-1-yl) reaction flask was added methyl 6-(3-phenylpiperazin-1-yl)nicotinate (70.0 mg, 0.235 mmol) as a solution in anhydrous THF (2.0 mL) and lithium hydroxide (11.3 mg, 0.471 mmol) as a solution in water (1.0 mL). The resulting mixture was stirring at room temperature overnight. After the reaction reached completion, the solvent was evaporated to yield the crude product as white solid (65.3 mg, 98%). The crude product was carried forward without further purification. 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 2-(2-(cyclohexyl(methyl)amino)ethyl)isoindoline-1,3-dione To a flame-dried round bottom flask was added a solution of N-methylcyclohexylamine (766.0 mg, 6.77 mmol) in 1.0 mL of anhydrous MeCN. K2CO3 (180.7 mg, 1.30 mmol) followed by (N-2-bromo-ethyl)- phthalimide (254.1 mg, 1,0 mmol) was then added into the mixture. The reaction mixture was heated at reflux (90 °C) under nitrogen atmosphere for 2 days. The reaction mixture was cooled to room temperature and filtered through a pad of celite and concentrated in vacuo. The crude product was then purified by normal-phase silica gel column chromatography (CH2Cl2 / MeOH 9:1) to yield the alkylated product (149.2 mg, 52%)1H NMR (500 MHz, CDCl3) δ 7.82 (dd, J = 5.2, 3.0 Hz, 2H), 7.68 (dd, J = 5.3, 3.0 Hz, 2H), 3.75 (t, J = 6.8 Hz, 2H), 2.69 (t, J = 6.8 Hz, 2H), 2.33 (s, 4H), 1.68 (dd, J = 22.7, 11.2 Hz, 4H), 1.56 (d, J = 12.9 Hz, 1H), 1.32 – 0.95 (m, 6H).13C NMR (125 MHz, CDCl3) δ 168.5, 133.8, 132.2, 123.1, 63.0, 50.7, 38.2, 36.5, 28.7, 26.3, 25.9. N-(2-Aminoethyl)-N- a round-bottom reaction flask was added a solution of 2-(2-(cyclohexyl(methyl)amino)ethyl)isoindoline-1,3-dione (150.0 mg, 0.52 mmol) in 0.1 M of anhydrous MeOH. Hydrazine solution (26.0 µL, 0.52 mmol) was then added dropwise, and the resulting mixture was heated at 65 °C for 30 minutes. The reaction mixture was then concentrated in vacuo, diluted with CH2Cl2, and filtered over a pad of celite. The filtrate was concentrated to provide the desired product as a colorless oil (71.1 mg, 87%). The crude product was carried forward without further purification. N-(3-(butyl(ethyl)amino) 1-yl)thieno[2,3 d]pyrimidine-6-carboxamide (2) To a flame dried flask was added 5-methyl-4-(4- methylpiperazin-1-yl)thieno[2,3-d]pyrimidine-6-carboxylic acid (11) (0.45 g, 1.54 mmol) in anhydrous CH2Cl2 (14 mL).1-Hydroxybenzotriazole hydrate (HOBt) (0.31 g, 2.31 mmol, 20 wt.% water) was then added into the reaction flask followed by 1-Ethyl-3-(3- 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 dimethylaminopropyl)carbodiimide (EDC) (0.36 g, 2.31 mmol). The mixture was stirred at room temperature for 20min. DIPEA (0.54 ml, 3.08 mmol) was then added dropwise followed by N-butyl-N-ethylpropane-1,3-diamine (8) (0.29 g, 1.85 mmol) as a solution in anhydrous CH2Cl2(1.4 mL). The resulting mixture was allowed to stir at room for 29 hours. The resulting mixture was diluted with water and organic phase was separated. The aqueous phase was extracted with CH2Cl2(3 x) and the combined organic extracts were washed with brine, dried with Na2SO4 and concentrated in vacuo to afford the crude product that was purified by silica gel column chromatography (CH2Cl2:MeOH:TEA / 9:1:0.1) to yield the product as a yellow oil (0.40 g, 60% over two steps).1H NMR (500 MHz, MeOD) δ 8.51 (s, 1H), 3.58 (s, 4H), 3.42 (t, J = 6.7 Hz, 2H), 2.72 (s, 3H), 2.65 – 2.57 (m, 8H), 2.53 – 2.47 (m, 2H), 2.35 (s, 3H), 1.84 – 1.76 (m, 2H), 1.51 – 1.43 (m, 2H), 1.36 – 1.26 (m, 3H), 1.07 (t, J = 7.1 Hz, 3H), 0.92 (t, J = 7.3 Hz, 3H).13C NMR (125 MHz, MeOD) δ 167.23, 163.68, 163.25, 152.81, 132.46, 129.02, 120.39, 54.07, 52.83, 50.77, 49.63, 44.71, 38.50, 28.15, 25.53, 20.43, 15.16, 12.97, 9.96. HRMS (ES+) m / z calculated for C22H37N6OS+[M+H]+: 433.2750; found: 433.2747. 2-(3-(butyl(ethyl)amino) a flame-dried round bottom flask was added a solution of N-ethylbutan-1-amine (3.06ml mL, 22.38 mmol) in 0.33M of anhydrous MeCN, then, K2CO3 (3.09 g, 22.38 mmol) was added. The reaction was stirred at room temperature for 20 minutes. (N-3-bromo-propyl)-phthalimide (2 g, 77.46 mmol) was then added into the mixture. The reaction mixture was heated at reflux (75 °C) under a nitrogen atmosphere for 17 hours. The reaction mixture was cooled to room temperature, filtered through a pad of celite, and concentrated in vacuo. The crude residue was then purified by normal-phase silica gel column chromatography (CH2Cl2:MeOH / 9:1) to afford the product as a yellow oil (1.25 g, 78%).1H NMR (500 MHz, CDCl3) δ 7.80 (s, 2H), 7.67 (s, 2H), 3.69 (t, J = 7.0 Hz, 2H), 2.47 (s, , 2.36 (t, J = 6.8 Hz, 2H), 1.84 – 1.74 (m, 2H), 1.34 (d, J = 6.6 Hz, 2H), 1.30 – 1.20 (m, 2H), 0.95 (t, J = 6.3 Hz, 3H), 0.86 (t, J = 7.0 Hz, 3H).13C NMR (125 MHz, CDCl3) δ 168.4, 133.8, 132.2, 123.1, 53.1, 51.0, 47.3, 36.6, 29.1, 26.1, 20.7, 14.1, 11.6. 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 N-butyl-N-ethylpropane-1,3- flask was added a solution of 2-(3-(butyl(ethyl)amino) mg, 1.387 mmol) in 0.1 M of anhydrous MeOH. Hydrazine solution (275 µL, 5.548 mmol) was then added dropwise and the resulting mixture was heated at 65 °C for 4 hours. The reaction mixture was then concentrated in vacuo, diluted with CH2Cl2, and filtered over a pad of celite. The filtrate was concentrated to provide the desired product as a colorless oil (182.9 mg, 72%). The crude product was carried forward without further purification. Diethyl 5-amino-3- To a flame dried flask was added ethyl acetoacetate (3.83 mL, 30.0 mmol), sulfur (962.1 mg, 30.0 mmol), and anhydrous ethanol (75.0 mL, 0.4 M). Diethylamine (3.10 mL, 15.0 mmol) was added followed by addition of ethyl cyanoacetate (3.18 mL, 30.0 mmol) into the reaction mixture dropwise. The resulting mixture was then stirred at 65 °C for 3 hours. After cooling to room temperature, the mixture was directly poured into ice / H2O mixture containing a few drops of conc. HCl solution. The formed solid was collected by filtration to yield the desired product as brown solid (6020.9 mg, 78%) without further purification.1H NMR (500 MHz, DMSO-d6) δ 7.90 (s, 2H), 4.20 (q, J = 7.0 Hz, 2H), 4.14 (q, J = 7.1 Hz, 2H), 2.58 (s, 3H), 1.26 (t, J = 7.1 Hz, 3H), 1.21 (t, J = 7.1 Hz, 3H).13C NMR (125 MHz, DMSO-d6) δ 167.2, 165.2, 162.5, 147.9, 106.7, 106.6, 60.3, 59.9, 16.2, 14.8, 14.7. Ethyl 4-hydroxy-5-methylthieno carboxylate (9) To a microwave vial was added diethyl 5-amino-3-methylthiophene-2,4-dicarboxylate (1286.5 mg, 5 mmol), formamide (10.0 mL, 0.5 M), and acetic acid (0.33 mL, 15 M). The resulting mixture was heated to 150 °C for 40 hours. Water (62.5 mL, 0.08 M) was added after the mixture was 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 cooled to 70 °C. The suspension was then cooled to room temperature with stirring. The formed solid was filtered, washed with water, and condensed in vacuo to yield the desired product as brown solid (1004.0 mg, 84%) without further purification.1H NMR (500 MHz, DMSO-d6) δ 8.18 (d, J = 2.1 Hz, 1H), 4.28 (q, J = 7.0 Hz, 2H), 2.79 (s, 3H), 1.28 (t, J = 7.0 Hz, 3H).13C NMR (125 MHz, DMSO-d6) δ 166.2, 162.3, 158.8, 148.8, 143.8, 124.3, 121.9, 61.6, 15.3, 14.6. Ethyl 4-chloro-5-methylthieno carboxylate (10) To a flame-dried microwave vial was added ethyl 4- [2,3-d]pyrimidine-6-carboxylate (9) (100.0 mg, 0.42 mmol) in anhydrous toluene (2.1 mL). Phosphoryl chloride (0.047 mL, 0.50 mmol) followed by DIPEA (0.058 mL, 0.34 mmol). The mixture was heated to 105 °C for 16 hours. The resulting mixture was cooled to room temperature, diluted with CH2Cl2, and then neutralized with 6M NaOH solution. The organic phase was separated and condensed in vacuo to yield the desired product as pale-yellow solid (105.1 mg, 98%) without further purification.1H NMR (500 MHz, CDCl3) δ 8.84 (s, 1H), 4.41 (q, J = 7.1 Hz, 2H), 3.03 (s, 3H), 1.41 (t, J = 7.1 Hz, 3H).13C NMR (125 MHz, CDCl3) δ 169.2, 162.1, 157.3, 154.2, 138.9, 129.0, 128.5, 62.1, 15.8, 14.2. HRMS (ESI) m / z calculated for C10H10ClN2O2S+[M+H]+: 257.0152; found: 257.0144. Ethyl 5-methyl-4-(4- d]pyrimidine-6-carboxylate (11) To a flame dried microwave vial was added ethyl 4-chloro-5-methylthieno[2,3-d]pyrimidine-6- carboxylate (10) (150.0 mg, 0.584 mmol), methylpiperazine (64.8 µl, 0.584 mmol), and 0.5 M anhydrous ethanol. Triethylamine (0.24 mL, 1.753 mmol) was added into the mixture dropwise (3.0 eq.), and the resulting mixture was heated in a microwave reactor for 1 hour at 65 °C. The mixture was then cooled down to room temperature, diluted with water, and 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 extracted with EtOAc (3x). The organic phase was combined and concentrated in vacuo to yield the desired product as a brown oil (153.7 mg, 82%) without further purification.1H NMR (500 MHz, CDCl3) δ 8.55 (s, 1H), 4.35 (q, J = 7.1 Hz, 2H), 3.55 (s, 4H), 2.78 (s, 3H), 2.57 (s, 4H), 2.34 (s, 3H), 1.38 (t, J = 7.1 Hz, 3H).13C NMR (125 MHz, CDCl 3) 163.4, 162.8, 153.9, 139.1, 124.0, 120.3, 61.4, 54.5, 50.2, 46.1, 16.7, 14.3. 5-methyl-4-(4-methylpiperazin-1- 6-carboxylic acid To a glass vial was added ethyl 5-methyl-4-(4- yl)thieno[2,3-d]pyrimidine-6- carboxylate (11) (46.3 mg, 0.145 mmol), lithium hydroxide (6.9 mg, 0.289 mmol), and 0.1 M THF / H2O (2:1) The reaction was stirred at room temperature for 2 hours then the solvent was evaporated to yield the desired product as pale yellow solid (33.0 mg, 78%). The crude product was carried on to the next step without further purification. Synthetic Methods for Compound 2 Analogues tert-butyl 4-(methylsulfonyl)piperazine-1-carboxylate (S1) To a flame-dried flask was added tert-butyl piperazine-1-carboxylate (100.0 mg, 0.54mmol), K2CO3 (149 mg, 1.08 mmol), and anhydrous acetonitrile (5.4 ml, 0.1M). The mixture was stirred at room temperature for 20 minutes, then, methanesulfonyl chloride (0.06 ml, 0.81 mmol) was added dropwise. The reaction was heated at 70 °C for one hour. The resulting mixture was diluted with water and organic phase was separated. The aqueous phase was extracted with CH2Cl2 (3 x) and the combined organic extracts were washed with brine, dried with Na2SO4 and concentrated in vacuo to be purified by silica gel column chromatograph (CH2Cl2:MeOH / 9:1) to afford the product as a white power (0.14 g, 100%).1H NMR (500 MHz, CDCl3) δ 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 3.37 (t, J = 7.25 Hz 4H), 3.01 (t, J = 5.07 Hz, 4H), 2.63 (s, 3H), 1.30 (s, 9H).13C NMR (125 MHz, CDCl3) δ 153.6, 79.7, 77.1, 76.9, 76.6, 45.1, 42.8, 33.9, 27.8. tert-butyl 4-propylpiperazine-1- To a flame-dried flask was added tert- butyl piperazine-1-carboxylate (100.0 mg, , K2CO3 (149 mg, 1.08 mmol), and anhydrous acetonitrile (5.4 ml, 0.1M). The mixture was stirred at room temperature for 20 minutes, then, 1-bromopropane (0.07 ml, 0.81 mmol) was added dropwise. The reaction was heated at 70 °C for 15 hour. The resulting mixture was diluted with water and organic phase was separated. The aqueous phase was extracted with CH2Cl2 (3 x) and the combined organic extracts were washed with brine, dried with Na2SO4and concentrated in vacuo to be purified by silica gel column chromatograph (CH2Cl2:MeOH / 9:1) to afford the product as a yellow oil (0.12 g, 100%).1H NMR (500 MHz, CDCl3) δ 3.42 (t, J = 5.21 Hz, 4H), 2.37 (t, J = 5.09 Hz, 4H), 2.29 (m, 2H), 1.49 (m, 2H), 1.43 (s, 3H), 0.88 (t, J = 7.37 Hz, 3H).13C NMR (125 MHz, CDCl3) δ 154.7, 79.6, 77.3, 77.0, 76.8, 60.6, 52.9, 43.9, 43.0, 28.4, 19.8, 11.9. tert-butyl 4-(sec-butyl)piperazine-1- (S3) To a flame-dried flask was added tert-butyl piperazine-1-carboxylate (100.0 mg, 0.54mmol), K2CO3(224 mg, 1.62 mmol), and anhydrous acetonitrile (5.4 ml, 0.1M). The mixture was stirred at room temperature for 20 minutes, then, 2-bromobutane (0.12 ml, 1.08 mmol) was added dropwise. The reaction was heated at 70 °C for 2 days. The resulting mixture was diluted with water and organic phase was separated. The aqueous phase was extracted with CH2Cl2(3 x) and the combined organic extracts were washed with brine, dried with Na2SO4 and concentrated in vacuo to be purified by silica gel column chromatograph (CH2Cl2:MeOH / 9:1) to afford the product as a yellow oil (0.12 g, 87%).1H NMR (500 MHz, CDCl3) δ 3.38 (m, 4H), 2.44 (m, 2H), 2.39 (m, 2H), 1.53 (m, 1H), 1.43 (s, 9H), 1.25 (m, 1H), 0.93 (d, J = 6.57 Hz, 3H), 0.87 (t, J = 7.39 Hz, 3H). 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 202513C NMR (125 MHz, CDCl3) δ 154.6, 79.3, 77.2, 76.9, 76.7, 60.8, 48.0, 44.3, 43.4, 28.3, 26.0, 13.7, 11.2. Ethyl 4-(4-ethylpiperazin-1-yl)-5- pyrimidine-6-carboxylate (11A) To a flame dried flask was added ethyl [2,3-d]pyrimidine-6-carboxylate (10) (150.0 mg, 0.584 mmol), ethylpiperazine (74.2 µl, 0.584 mmol), and 0.5 M anhydrous ethanol. Triethylamine (0.24 ml, 1.753 mmol) was added into the mixture dropwise (3.0 eq.), and the resulting mixture was warmed up to 65 °C and stirred at elevated temperature for one hour. The mixture was then cooled down to room temperature, diluted with water, and extracted with EtOAc (3x). The organic phase was combined and concentrated in vacuo to yield the desired product as brown solids (64.8 mg, 81%).1H NMR (500 MHz, CDCl3) δ 8.52 (s, 1H), 4.34 (q, J = 7.1 Hz, 2H), 3.54 (s, 4H), 2.76 (s, 3H), 2.58 (s, 4H), 2.46 (dd, J = 14.1, 7.0 Hz, 2H), 1.36 (t, J = 7.1 Hz, 3H), 1.09 (t, J = 7.1 Hz, 3H).13C NMR (125 MHz, CDCl3) δ 169.0, 163.3, 162.8, 153.9, 139.1, 123.9, 120.2, 61.4, 52.3, 52.3, 50.2, 16.7, 14.3, 11.8. Ethyl 4-(diethylamino)-5- 6-carboxylate (11B) To a flame dried flask was added ethyl 4-chloro-5-methylthieno[2,3-d]pyrimidine-6-carboxylate (10) (150.0 mg, 0.584 mmol), diethylamine (60.4 µl, 0.584 mmol), and 0.5 M anhydrous ethanol. Triethylamine (0.24 ml, 1.753 mmol) was added into the mixture dropwise (3.0 eq.), and the resulting mixture was warmed up to 65 °C and stirred at elevated temperature for one hour. The mixture was then cooled down to room temperature, diluted with water, and extracted with EtOAc (3x). The organic phase was combined and concentrated in vacuo to yield the desired product as brown solids (148.8 mg, 96%).1H NMR (500 MHz, CDCl3) δ 8.53 (s, 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 1H), 4.37 (q, J = 7.1 Hz, 2H), 3.55 (q, J = 7.1 Hz, 4H), 2.78 (s, 3H), 1.39 (t, J = 7.1 Hz, 3H), 1.17 (t, J = 7.1 Hz, 6H).13C NMR (125 MHz, CDCl3) δ 168.7, 163.2, 162.9, 153.5, 139.7, 123.2, 120.6, 61.3, 44.8, 16.7, 14.4, 12.6. Ethyl 5-methyl-4-(4- d]pyrimidine-6-carboxylate (11C) To a flame dried flask was added [2,3-d]pyrimidine-6- carboxylate (10) (38 mg, 0.15 mmol), 1-propylpiperazine (S1) (56 mg, 0.44 mmol), and 0.5 M anhydrous ethanol. Triethylamine (0.07ml, 0.60 mmol) was added into the mixture dropwise, and the resulting mixture was warmed up to 65 °C and stirred at elevated temperature for 19 hours. The reaction mixture was then cooled down to room temperature and diluted with water and organic phase was separated. The aqueous phase was extracted with CH2Cl2 (3 x) and the combined organic extracts were washed with brine, dried with Na2SO4 and concentrated in vacuo to be purified by silica gel column chromatograph (CH2Cl2:MeOH / 9:1) to afford product as a yellow oil (47.94 mg, 89%).1H NMR (500 MHz, CDCl3) δ 8.56 (s, 1H), 4.37 (q, J = 7.12 Hz, 2H), 3.59 (s, 4H), 2.79 (s, 3H), 2.61 (s, 4H), 2.40 (s, 2H), 1.56 (q, J = 8.20 Hz, 2H), 1.40 (t, J =7.12 Hz, 3H), 0.92 (t, J = 7.37 Hz, 3H).13C NMR (125 MHz, CDCl3) δ 169.0, 163.1, 162.7, 153.8, 139.0, 120.1, 77.2, 76.9, 76.7, 61.3, 60.3, 52.4, 49.9, 19.6, 16.5, 14.2, 11.7. Ethyl 4-(4-(sec-butyl)piperazin- [2,3-d]pyrimidine-6-carboxylate (11D) To a flame dried flask was added ethyl 4-chloro-5-methylthieno[2,3-d]pyrimidine-6- carboxylate (10) (36 mg, 0.15 mmol), 1-(sec-butyl)piperazine (S3) (58 mg, 0.41 mmol), and 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 0.5 M anhydrous ethanol. Triethylamine (0.07ml, 0.60 mmol) was added into the mixture dropwise, and the resulting mixture was warmed up to 65 °C and stirred at elevated temperature for 19 hours. The reaction mixture was then cooled down to room temperature and diluted with water and organic phase was separated. The aqueous phase was extracted with CH2Cl2 (3 x) and the combined organic extracts were washed with brine, dried with Na2SO4and concentrated in vacuo to be purified by silica gel column chromatograph (CH2Cl2:MeOH / 9:1) to afford product as a yellow oil (45.15 mg, 88%).1H NMR (500 MHz, CDCl3) δ 8.54 (s, 1H), 4.36 (q, J = 7.15 Hz, 2H), 3.54 (t, J = 4.54 Hz, , 2.78 (s, 4H), 2.58 (t, J = 5.04 Hz, 4H), 2.35 (m, 2H), 1.51 (m, 2H), 1.38 (t, J = 7.12 Hz, , 0.90 (t, J =7.38 Hz, 3H).13C NMR (125 MHz, CDCl3) δ 169.1, 162.8, 153.9, 139.1, 120.2, 77.3, 77.0, 76.8, 61.4, 50.6, 47.7, 29.7, 25.7, 16.7, 14.3, 13.8, 11.2. Ethyl 5-methyl-4-(4- thieno[2,3-d]pyrimidine-6- carboxylate (11E) To a flame dried flask was added ethyl 4-chloro-5-methylthieno[2,3- d]pyrimidine-6-carboxylate (10) (36 mg, 0.15 mmol), 1-(methylsulfonyl)piperazine (S1) (68 mg, 0.41 mmol), and 0.5 M anhydrous ethanol. Triethylamine (0.07ml, 0.60 mmol) was added into the mixture dropwise, and the resulting mixture was warmed up to 65 °C and stirred at elevated temperature for 19 hours. The reaction mixture was then cooled down to room temperature and diluted with water and organic phase was separated. The aqueous phase was extracted with CH2Cl2 (3 x) and the combined organic extracts were washed with brine, dried with Na2SO4and concentrated in vacuo to be purified by silica gel column chromatograph (CH2Cl2:MeOH / 9:1) to afford product as a white-yellow powder (47.51 mg, 88%).1H NMR (500 MHz, CDCl3) δ 8.60 (s, 1H), 4.38 (q, J = 7.12 Hz, 2H), 3.68 (t, J = 4.85 Hz, 4H), 3.38 (t, J = 4.91 Hz, 4H), 2.82 (s, 3H), 2.81 (s, 4H), 1.40 (t, J = 7.11 Hz, 3H).13C NMR (125 MHz, CDCl3) δ 168.7, 163.0, 162.6, 153.4, 138.3, 125.0, 120.4, 61.6, 50.0, 45.2, 34.8, 16.5, 14.3. 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 Ethyl 5-methyl-4-(pyrrolidine-1- 6-carboxylate (11F) To a flame dried flask was added ethyl 4- [2,3-d]pyrimidine-6-carboxylate (10) (50 mg, 0.20 mmol), pyrrolidine (0.02 ml, 0.20 mmol), and 0.5 M anhydrous ethanol. Triethylamine (0.08ml, 0.59 mmol) was added into the mixture dropwise (3.0 eq.), and the resulting mixture was warmed up to 65 °C and stirred at elevated temperature for 4 hours. The mixture was then cooled down to room temperature, diluted with water, and extracted with CH2Cl2 (3x). The organic phase was combined and concentrated in vacuo to yield the desired product as yellow oil (56.96 mg, 100%).1H NMR (500 MHz, CDCl3) δ 8.36 (s, 1H), 4.32 (q, J = 7.11, 2H), 3.66 (m, 4H), 2.71 (s, 3H), 1.88 (m, 4H), 1.35 (t, J = 7.11, 3H).13C NMR (125 MHz, CDCl3) δ 168.5, 162.9, 160.4, 153.5, 139.9, 121.7, 118.4, 61.1, 51.0, 25.4, 18.6, 14.3. 5-methyl-4-(4-propylpiperazin-1- 6-carboxylic acid To a reaction vial was added ethyl 5-methyl-4-(4-propylpiperazin-1-yl)thieno[2,3-d]pyrimidine-6- carboxylate (11D) (25 mg, 0.07 mmol), lithium hydroxide (2 mg, 0.09 mmol), 0.15 M anhydrous THF and 0.30 M water. The reaction was left at room temperature for 20 hours then the solvent was evaporated to yield the desired product as pale-yellow solids, and the crude was carried on the next step without further purification. 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 4-(4-(sec-butyl)piperazin-1-yl)-5- pyrimidine-6-carboxylic acid To a reaction vial was added ethyl 4-(4- 1-yl)-5-methylthieno[2,3- d]pyrimidine-6-carboxylate (11C) (25 mg, 0.07 mmol), lithium hydroxide (2 mg, 0.09 mmol), 0.15 M anhydrous THF and 0.30 M water. The reaction was left at room temperature for 20 hours then the solvent was evaporated to yield the desired product as pale-yellow solids, and the crude was carried on the next step without further purification. 5-methyl-4-(4-(methylsulfonyl) [2,3-d]pyrimidine-6-carboxylic acid To a reaction vial was added ethyl 5-methyl-4-(4-(methylsulfonyl)piperazin-1-yl)thieno[2,3- d]pyrimidine-6-carboxylate (11E) (25 mg, 0.07 mmol), lithium hydroxide (2 mg, 0.09 mmol), 0.15 M anhydrous THF and 0.30 M water. The reaction was left at room temperature for 20 hours then the solvent was evaporated to yield the desired product as white-yellow solids, and the crude was carried on the next step without further purification. 5-methyl-4-(pyrrolidine-1-yl) 6-carboxylic acid To a reaction vial was added ethyl 5-methyl-4-(pyrrolidine-1-yl)thieno[2,3-d]pyrimidine-6-carboxylate (11F) (56 mg, 0.19 mmol), lithium hydroxide (5.51 mg, 0.23 mmol), 0.15 M anhydrous THF and 0.30 M water. The reaction was left at room temperature for 20 hours then the solvent was 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 evaporated to yield the desired product as white-yellow solid, and the crude was carried on the next step without further purification. 4-(4-ethylpiperazin-1-yl)-5- 6-carboxylic acid To a reaction vial was added ethyl 4-(4- -5-methylthieno[2,3-d]pyrimidine-6- carboxylate (11A) (64.8 mg, 0.194 mmol), lithium hydroxide (9.3 mg, 0.388 mmol), 0.15 M anhydrous THF and 0.30 M water (0.30 M). The reaction was left at room temperature for 2 hours then the solvent was evaporated to yield the desired product as pale yellow solids, and the crude was carried on the next step without further purification. 4-(Diethylamino)-5-methylthieno 6-carboxylic acid To a reaction vial was added ethyl 4-(diethylamino)-5-methylthieno[2,3-d]pyrimidine-6-carboxylate (11B) (148.8 mg, 0.561 mmol), lithium hydroxide (26.9 mg, 1.122 mmol), 0.15 M anhydrous THF and 0.30 M water (0.30 M). The reaction was left at room temperature for 2 hours then the solvent was evaporated to yield the desired product as a pale yellow solid, and the crude was carried on the next step without further purification. 2-(3-Morpholinopropyl) of morpholine (0.49 ml, 5.56 mmol) in 11.3 ml of MeCN, was added K2CO3 (1.55 g, 11.19 mmol) and (N-3-bromo- 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 propyl)-phthalimide (1.0 g, 3.73 mmol). The reaction mixture was heated at refluxed (75 °C) under nitrogen atmosphere for 18 hr. The reaction mixture was cooled to room temperature and filtered through a pad of celite and concentrated in vacuo. The crude was then purified by column chromatography (CH2Cl2:MeOH / 9:1) to afford the product as a yellow oil (0.82 g, 80%)1H NMR (500 MHz, CDCl3) δ δ 7.79 (m, 2H), 7.67 (m, 2H), 3.73 (t, J = 6.90 Hz, 3H), 3.48 (t, J = 4.74 Hz, 3H), 2.37 (t, J = 7.69 Hz, 3H), 2.32 (m, 4H), 1.82 (t, J = 6.86 Hz, 3H).13C NMR (125 MHz, CDCl3) δ 168.4, 133.8, 132.3, 123.1, 66.8, 56.4, 53.6, 36.6, 24.7. 2-(3-(4-methylpiperazin-1-yl) To a solution of N- methylpiperizine (0.166 ml, 1.50 mmol) in 1.5 mL of MeCN, was added K2CO3(207.0 mg, 1.50 mmol) and (N-3-bromo-propyl)-phthalimide (134.0 mg, 0.50 mmol). The reaction mixture was heated at refluxed (90 °C) under nitrogen atmosphere for 2 days. The reaction mixture was cooled to room temperature and filtered through a pad of celite and concentrated in vacuo. The crude was then purified by column chromatography (Hex / EtOAc 9:1) to yield the alkylated product (107.8 mg, 75%).1H NMR (500 MHz, CDCl3) δ 7.77 – 7.70 (m, 2H), 7.65 – 7.58 (m, 2H), 3.65 (t, J = 6.8 Hz, 2H), 3.31 (s, 2H), 2.63 – 2.16 (m, 8H), 2.12 (s, 3H), 1.79 – 1.71 (m, 2H).13C NMR (125 MHz, CDCl3) δ 168.4, 133.8, 132.2, 123.1, 55.8, 54.6, 52.5, 45.5, 36.5, 25.1. 2-(3-(Pyrrolidine-1-yl)propyl) a solution of pyrrolidine (0.123 ml, 1.50 mmol) in 1.5 mL of MeCN, was added K2CO3 (207.0 mg, 1.50 mmol) and (N-3-bromo- propyl)-phthalimide (134.0 mg, 0.50 mmol). The reaction mixture was heated at refluxed (90 °C) under nitrogen atmosphere for 2 days. The reaction mixture was cooled to room temperature and filtered through a pad of celite and concentrated in vacuo. The crude was 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 then purified by column chromatography (Hex / EtOAc 9:1) to yield the alkylated product (38.1 mg, 30%).1H NMR (500 MHz, CDCl3) δ 7.80 (d, J = 2.5 Hz, 2H), 7.67 (d, J = 2.7 Hz, 2H), 3.72 (t, J = 6.6 Hz, 2H), 2.52 (t, J = 7.3 Hz, 2H), 2.47 (s, 4H), 1.92 – 1.84 (m, 2H), 1.66 (s, 4H).13C NMR (125 MHz, CDCl3) δ 168.4, 133.8, 132.2, 123.1, 53.9, 53.7, 36.5, 27.5, 23.4. 2-(3-(diethylamino)propyl) of diethylamine (0.155 ml, 1.50 mmol) in 1.5 mL of was mg, 1.50 mmol) and (N-3-bromo- propyl)-phthalimide (134.0 mg, 0.50 mmol). The reaction mixture was heated at refluxed (90 °C) under nitrogen atmosphere for 2 days. The reaction mixture was cooled to room temperature and filtered through a pad of celite and concentrated in vacuo. The crude was then purified by column chromatography (Hex / EtOAc 9:1) to yield the alkylated product (93.5 mg, 72%).1H NMR (500 MHz, CDCl3) δ 7.77 (dd, J = 4.8, 3.1 Hz, 2H), 7.64 (dd, J = 4.8, 3.0 Hz, 2H), 3.66 (t, J = 7.2 Hz, 2H), 2.45 (q, J = 6.8 Hz, 6H), 1.82 – 1.73 (m, 2H), 0.92 (t, J = 7.1 Hz, 6H).13C NMR (125 MHz, CDCl3) δ 168.3, 133.8, 132.2, 123.1, 50.3, 46.6, 36.5, 26.0, 11.5. 2-(3-(dibutylamino)propyl) of dibutylamine (0.253 ml, 1.50 mmol) in 1.5 mL of MeCN, was added K2CO3(207.0 mg, 1.50 mmol) and (N-3-bromo- propyl)-phthalimide (134.0 mg, 0.50 mmol). The reaction mixture was heated at refluxed (90 °C) under nitrogen atmosphere for 2 days. The reaction mixture was cooled to room temperature and filtered through a pad of celite and concentrated in vacuo. The crude was then purified by column chromatography (Hex / EtOAc 9:1) to yield the alkylated product 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 (132.0 mg, 85%).1H NMR (500 MHz, CDCl3) δ 7.76 (d, J = 1.6 Hz, 2H), 7.64 (d, J = 2.6 Hz, 2H), 3.64 (t, J = 7.2 Hz, 2H), 2.42 (t, J = 6.9 Hz, 2H), 2.31 (t, J = 7.2 Hz, 4H), 1.79 – 1.70 (m, 2H), 1.30 (dd, J = 14.2, 7.4 Hz, 4H), 1.21 (dt, J = 13.1, 6.7 Hz, 5H), 0.82 (t, J = 7.1 Hz, 6H).13C NMR (125 MHz, CDCl3) δ 168.3, 133.8, 132.2, 123.0, 53.6, 51.6, 36.6, 29.1, 26.2, 20.6, 14.1. 3-(4-methylpiperazin-1-yl) of 2-(3-(4-methylpiperazin-1- yl)propyl)isoindoline-1,3-dione mg, in 0.1 M of anhydrous EtOH was added hydrazine solution (74.8 µL, 1.494 mmol), and the resulting mixture was heated at 65 °C for 30 minutes. The reaction mixture was then concentrated in vacuo, diluted with DCM, and filtered over a pad of celite. The filtrate was concentrated to provide the desired product as colorless oil (31.8 mg, 54%). The crude was carried forward for the next step without further purification. 3-(pyrrolidine-1-yl)propan-1- of 2-(3-(pyrrolidine-1- yl)propyl)isoindoline-1,3-dione (38.1 mg, 0.147 mmol) in 0.1 M of anhydrous EtOH was added hydrazine solution (29.5 µL, 0.590 mmol), and the resulting mixture was heated at 65 °C for 30 minutes. The reaction mixture was then concentrated in vacuo, diluted with DCM, and filtered over a pad of celite. The filtrate was concentrated to provide the desired product as colorless oil (13.76 mg, 73%). The crude was carried forward for the next step without further purification. 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 N,N-diethylpropane-1,3-diamine To a solution of 2-(3-(diethylamino)propyl)isoindoline- 1,3-dione (93.5 mg, 0.359 mmol) in 0.1 M of anhydrous EtOH was added hydrazine solution (71.92 µL, 1.437 mmol), and the resulting mixture was heated at 65 °C for 30 minutes. The reaction mixture was then concentrated in vacuo, diluted with DCM, and filtered over a pad of celite. The filtrate was concentrated to provide the desired product as colorless oil (44.4 mg, 95%). The crude was carried forward for the next step without further purification. 3-morpholinopropan-1-amine To morpholinopropyl)isoindoline-1,3- dione (0.26 g, 0.95 mmol) in 0.1 M of anhydrous EtOH was added hydrazine solution (0.18 ml, 3.8 mmol), and the resulting mixture was heated at 65 °C for 17 hr. The reaction mixture was then concentrated in vacuo, diluted with CH2Cl2, and filtered over a pad of celite. The filtrate was concentrated to provide the desired product as colorless oil (0.101 g, 74%). The crude was carried forward for the next step without further purification. N,N-dibutylpropane-1,3- (dibutylamino)propyl)isoindoline- 1,3-dione (132.0 mg, 0.417 mmol) in 0.1 M of anhydrous EtOH was added hydrazine solution (83.5 µL, 1.669 mmol), and the resulting mixture was heated at 65 °C for 30 minutes. The reaction mixture was then concentrated in vacuo, diluted with CH2Cl2, and filtered over a pad of celite. The filtrate was concentrated to provide the desired product as colorless oil (66.0 mg, 85%). The crude was carried forward for the next step without further purification. 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 N-(3-(dibutylamino) 1-yl)thieno[2,3- d]pyrimidine-6- added 5-methyl-4-(4- methylpiperazin-1-yl)thieno[2,3-d]pyrimidine-6-carboxylic acid (60.0 mg, 0.205 mmol) in DMF (2.0 ml), HOBt (59.0 mg, 0.308 mmol) was then added into the reaction flask. Until all the solids have been dissolved, N1,N1-dibutylpropane-1,3-diamine (57.4 mg, 0.308 mmol) was added as a solution in DMF (0.2 ml). The resulting mixture was stirring at room temperature for 10 minutes before the slowly addition of DIPEA (107.0 µl, 0.615 mmol), EDC (35.5 mg, 0.308 mmol) was finally added into the reaction mixture, and the resulting mixture was allowed to stir at room for 14 hours. Once the reaction went completion, the solution was directly purified via reverse column chromatography (ACN / H2O 1:1) to yield the product (16.1 mg, 17%). IR (neat) ʋmax = 2955, 1632, 1540, 1497, 1498, 1444, 1289, 1142, 985, 800 cm-1.1H NMR (500 MHz, MeOD) δ 8.50 (s, 1H), 3.57 (s, 3H), 3.42 (t, J = 6.7 Hz, 2H), 2.71 (s, 3H), 2.61 (d, J = 10.5 Hz, 3H), 2.59 (s, 2H), 2.50 – 2.46 (m, 4H), 2.35 (s, 3H), 1.83 – 1.76 (m, 2H), 1.46 (dd, J = 15.4, 8.0 Hz, 4H), 1.32 (dd, J = 14.9, 7.4 Hz, 6H), 0.92 (t, J = 7.3 Hz, 6H).13C NMR (125 MHz, MeOD) δ 167.2, 163.6, 163.2, 152.8, 132.4, 129.1, 120.4, 54.1, 53.5, 51.5, 49.6, 44.7, 38.6, 28.3, 25.6, 20.4, 15.2, 13.0. HRMS (ES+) m / z calculated for C24H41N6OS+[M+H]+: 461.3063; found: 461.3060. 5-methyl-4-(4- 1-yl)propyl)thieno[2,3- d]pyrimidine-6-carboxamide (2B) To a flame dried flask was added 5-methyl-4-(4- methylpiperazin-1-yl)thieno[2,3-d]pyrimidine-6-carboxylic acid (40.0 mg, 0.137 mmol) in DMF (1.4 ml), HOBt (105 mg, 0.274 mmol) was then added into the reaction flask. Until all the solids have been dissolved, 3-(4-methylpiperazin-1-yl)propan-1-amine (21.69 mg, 0.137 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 mmol) was added as a solution in DMF (0.2 ml). The resulting mixture was stirring at room temperature for 10 minutes before the slowly addition of DIPEA (143.0 µl, 0.822 mmol), EDC (31.6 mg, 0.274 mmol) was finally added into the reaction mixture, and the resulting mixture was allowed to stir at room for 14 hours. Once the reaction went completion, the solution was directly purified via reverse column chromatography (ACN / H2O 1:1) to yield the product (13.6 mg, 23%). IR (neat) ʋmax= 2936, 1633, 1538, 1444, 1357, 1286, 1144, 985, 797 cm-1.1H NMR (500 MHz, MeOD) δ 8.50 (s, 1H), 3.57 (s, 4H), 3.43 (t, J = 6.7 Hz, 2H), 2.71 (s, 4H), 2.68 – 2.40 (m, 12H), 2.35 (s, 4H), 2.27 (s, 3H), 1.87 – 1.79 (m, 2H).13C NMR (125 MHz, MeOD) δ 167.2, 163.6, 163.2, 152.8, 132.5, 129.0, 120.4, 55.8, 54.3, 54.1, 52.3, 49.6, 44.7, 44.6, 38.4, 25.8, 15.2. HRMS (ES+) m / z calculated for C21H34N7OS+[M+H]+: 432.2546; found: 432.2547. N-(3-(diethylamino)propyl)- 1-yl)thieno[2,3- d]pyrimidine-6-carboxamide (2C) To a flame dried flask was added 5-methyl-4-(4- methylpiperazin-1-yl)thieno[2,3-d]pyrimidine-6-carboxylic acid (11) (60.0 mg, 0.205 mmol) in DMF (2.0 ml), HOBt (59.0 mg, 0.308 mmol) was then added into the reaction flask. Until all the solids have been dissolved, N1,N1-diethylpropane-1,3-diamine (40.1 mg, 0.308 mmol) was added as a solution in DMF (0.2 ml). The resulting mixture was stirring at room temperature for 10 minutes before the slowly addition of DIPEA (107.0 µl, 0.615 mmol), EDC (35.5 mg, 0.308 mmol) was finally added into the reaction mixture, and the resulting mixture was allowed to stir at room for 14 hours. Once the reaction went completion, the solution was directly purified via reverse column chromatography (can / H2O 1:1) to yield the product (10.8 mg, 13%). IR (neat) ʋmax= 2968, 1640, 1540, 1498, 1289, 1142, 1070, 985 cm-1.1H NMR (500 MHz, MeOD) δ 8.51 (s, 1H), 3.58 (s, 4H), 3.45 (t, J = 6.6 Hz, 2H), 2.86 – 2.77 (m, 6H), 2.72 (s, 3H), 2.62 (s, 4H), 2.36 (s, 3H), 1.93 – 1.83 (m, 2H), 1.16 (t, J = 7.1 Hz, 6H).13C NMR (125 MHz, MeOD) δ 167.3, 163.9, 163.2, 152.9, 132.8, 128.8, 120.4, 54.1, 49.9, 49.6, 46.6, 44.7, 37.9, 25.0, 15.2, 9.2. HRMS (ES+) m / z calculated for C20H33N6OS+[M+H]+: 405.2437; found: 405.2438. 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 N-(3-(dimethylamino) 1-yl)thieno[2,3- d]pyrimidine-6-carboxamide was added 5-methyl-4-(4- methylpiperazin-1-yl)thieno[2,3-d]pyrimidine-6-carboxylic acid (60.0 mg, 0.205 mmol) in DMF (2.0 ml), HOBt (59.0 mg, 0.308 mmol) was then added into the reaction flask. Until all the solids have been dissolved, N1,N1-dimethylpropane-1,3-diamine (0.162 ml, 0.308 mmol) was added as a solution in DMF (0.2 ml). The resulting mixture was stirring at room temperature for 10 minutes before the slow addition of DIPEA (107.0 µl, 0.615 mmol), EDC (35.5 mg, 0.308 mmol) was finally added into the reaction mixture, and the resulting mixture was allowed to stir at room for 14 hours. Once the reaction went completion, the solution was directly purified via reverse column chromatography (ACN / H2O 1:1) to yield the product (10.8 mg, 14%). IR (neat) ʋmax = 2931, 1633, 1541, 1497, 1445, 1366, 1290, 1261, 985, 668 cm-1.1H NMR (500 MHz, MeOD) δ 8.50 (s, 1H), 3.57 (s, 4H), 3.42 (t, J = 6.9 Hz, 2H), 2.71 (s, 3H), 2.63 (s, 4H), 2.46 – 2.41 (m, 2H), 2.35 (s, 3H), 2.27 (s, 6H), 1.86 – 1.78 (m, 2H).13C NMR (125 MHz, MeOD) δ 167.3, 163.7, 163.2, 152.8, 132.4, 129.2, 120.4, 57.0, 54.1, 49.6, 44.7, 44.1, 38.3, 26.6, 15.1. HRMS (ES+) m / z calculated for C18H29N6OS+[M+H]+: 377.2124; found: 377.2125. 5-methyl-4-(4- yl)propyl)thieno[2,3- d]pyrimidine-6-carboxamide (2E) To a flame dried flask was added 5-methyl-4-(4- methylpiperazin-1-yl)thieno[2,3-d]pyrimidine-6-carboxylic acid (60.0 mg, 0.205 mmol) in DMF (2.0 ml), HOBt (59.0 mg, 0.308 mmol) was then added into the reaction flask. Until all the solids have been dissolved, 3-(pyrrolidin-1-yl)propan-1-amine (39.4 mg, 0.308 mmol) 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 was added as a solution in DMF (0.2 ml). The resulting mixture was stirring at room temperature for 10 minutes before the slow addition of DIPEA (107.0 µl, 0.615 mmol), EDC (35.5 mg, 0.308 mmol) was finally added into the reaction mixture, and the resulting mixture was allowed to stir at room for 14 hours. Once the reaction went completion, the solution was directly purified via reverse column chromatography (ACN / H2O 1:1) to yield the product (4.95 mg, 6%). IR (neat) ʋmax= 2931, 1634, 1539, 1515, 1444, 1289, 1261, 1142, 985 cm-1.1H NMR (500 MHz, MeOD) δ 8.50 (s, 1H), 3.58 (s, 4H), 3.44 (t, J = 6.4 Hz, 2H), 2.72 (s, 3H), 2.62 (s, 10H), 2.35 (s, 3H), 1.91 – 1.81 (m, 6H).13C NMR (125 MHz, MeOD) δ 167.3, 163.7, 163.2, 152.8, 132.5, 129.1, 120.4, 54.1, 53.8, 53.7, 49.6, 44.7, 38.3, 27.8, 22.8, 15.2. HRMS (ES+) m / z calculated for C20H31N6OS+[M+H]+: 403.2280; found: 403.2277. N-(3-(butyl(ethyl)amino) methylthieno[2,3- d]pyrimidine-6-carboxamide (2F) To a flame dried flask was added 4-(4-ethylpiperazin-1- yl)-5-methylthieno[2,3-d]pyrimidine-6-carboxylic acid (42.8 mg, 0.14 mmol) in DMF (1.4 ml), HOBt (110.0 mg, 0.28 mmol) was then added into the reaction flask. Until all the solids have been dissolved, N-butyl-N-ethylpropane-1,3-diamine (8) (33.2 mg, 0.21 mmol) was added as a solution in DMF (0.2 ml). The resulting mixture was stirring at room temperature for 10 minutes before the slow addition of DIPEA (97.5 µl, 0.42 mmol), EDC (32.3 mg, 0.28 mmol) was finally added into the reaction mixture, and the resulting mixture was allowed to stir at room for 14 hours. Once the reaction went completion, the solution was directly purified via reverse column chromatography (ACN / H2O 1:1) to yield the product (9.08 mg, 15%). IR (neat) ʋmax = 2931, 1640, 1541, 1499, 1445, 1384, 1286, 1090, 975 cm-1.1H NMR (500 MHz, MeOD) δ 8.62 (s, 1H), 3.81 (s, 3H), 3.51 (t, J = 6.6 Hz, 2H), 3.35 (d, J = 12.1 Hz, 3H), 3.28 (d, J = 7.4 Hz, 2H), 3.27 – 3.24 (m, 2H), 3.19 – 3.15 (m, 4H), 2.79 (s, 3H), 2.06 (dd, J = 15.1, 7.4 Hz, 2H), 1.75 – 1.69 (m, 2H), 1.44 (dd, J = 15.0, 7.5 Hz, 2H), 1.36 (dt, J = 11.7, 7.3 Hz, 8H), 1.01 (t, J = 7.4 Hz, 3H).13C NMR (125 MHz, CDCl3) δ 167.7, 164.1, 162.6, 153.0, 132.8, 129.3, 120.8, 52.1, 52.0, 50.6, 49.8, 36.7, 25.5, 23.9, 19.5, 15.0, 12.5, 8.5, 7.7. HRMS (ES+) m / z calculated for C23H39N6OS+[M+H]+: 447.2906; found: 447.2898. 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 N-(3-(butyl(ethyl)amino) [2,3-d]pyrimidine-6- carboxamide (2G) To a -5-methylthieno[2,3- d]pyrimidine-6-carboxylic acid (37.1 mg, 0.14 mmol) in DMF (1.4 ml), HOBt (110.0 mg, 0.28 mmol) was then added into the reaction flask. Until all the solids have been dissolved, N-butyl-N-ethylpropane-1,3-diamine (8) (33.2 mg, 0.21 mmol) was added as a solution in DMF (0.2 ml). The resulting mixture was stirring at room temperature for 10 minutes before the slow addition of DIPEA (97.5 µl, 0.42 mmol), EDC (32.3 mg, 0.28 mmol) was finally added into the reaction mixture, and the resulting mixture was allowed to stir at room for 14 hours. Once the reaction went completion, the solution was directly purified via reverse column chromatography (ACN / H2O 1:1) to yield the product (10.2 mg, 18%). IR (neat) ʋmax = 2967, 1633, 1542, 1497, 1452, 1383, 1287, 1032, 790 cm-1.1H NMR (500 MHz, MeOD) δ 8.44 (s, 1H), 3.61 (q, J = 7.0 Hz, 4H), 3.47 (t, J = 6.6 Hz, 2H), 3.04 (dd, J = 15.6, 8.3 Hz, 4H), 2.97 – 2.91 (m, 2H), 2.71 (s, 3H), 2.01 – 1.93 (m, 2H), 1.68 – 1.59 (m, 2H), 1.39 (dt, J = 14.8, 7.5 Hz, 2H), 1.24 (d, J = 7.2 Hz, 3H), 1.18 (d, J = 7.1 Hz, 6H), 0.98 (t, J = 7.4 Hz, 3H).13C NMR (125 MHz, MeOD) δ 167.1, 164.2, 163.0, 152.6, 133.7, 127.6, 120.5, 52.3, 50.1, 44.7, 37.2, 26.4, 24.5, 19.8, 15.4, 12.7, 11.4, 8.4. HRMS (ES+) m / z calculated for C21H36N5OS+[M+H]+: 406.2641; found: 406.2636. N-(3-chlorophenyl)-5-methyl- thieno[2,3-d]pyrimidine-6- carboxamide (2H) To a flame dried flask containing CH2Cl2(2.1 ml), 5-methyl-4-(4- methylpiperazin-1-yl)thieno[2,3-d]pyrimidine-6-carboxylic acid (60 mg, 0.21 mmol), HOBt (43 mg, 0.32 mmol), and EDC (50 mg, 0.32 mmol) were added. The reaction was stirred at room temperature for 20min, then, DIPEA (0.07 ml, 0.42 mmol) followed by 3-chloroaniline 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 (0.03ml, 0.27mmol) The resulting mixture was allowed to stir at room for 4 days. The resulting mixture was diluted with water and organic phase was separated. The aqueous phase was extracted with CH2Cl2 (3 x) and the combined organic extracts were washed with brine, dried with Na2SO4and concentrated in vacuo to afford the crude product that was purified by silica gel column chromatography (CH2Cl2:MeOH:TEA / 9:1:0.1) to yield the product as a light-yellow oil (13.62 mg, 17% over two steps). IR (neat) ʋmax= 2922, 2850,1650,1591, 1536, 1246, 828, 727,1H NMR (500 MHz, CDCl3) δ 8.58 (s, 1H), 7.93 (s, 1H), 7.77 (t, J = 2.03 Hz, 1H), 7.49 (d, J = 8.25 Hz, 1H), 7.29 (t, J = 8.07 Hz, 1H), 7.15 (ddd, J = 7.97, 1.96, 0.91 Hz, 1H), 3.71 (s, 4H), 2.81 (s, 3H), 2.77 (s, 2H), 2.48 (s, 2H).13C NMR (125 MHz, CDCl3) δ 167.9, 162.9, 160.9, 153.8, 138.6, 134.8, 130.1, 125.0, 120.7, 120.2, 118.3, 54.0, 49.4, 45.4, 29.7, 16.5. HRMS (ES+) m / z calculated for C19H21ClN5OS+[M+H]+: 402.1150; found: 402.1149. 5-methyl-4-(4- thieno[2,3-d]pyrimidine-6- carboxamide (2I) To a flame dried flask containing CH2Cl2(0.7 ml), 5-methyl-4-(4- methylpiperazin-1-yl)thieno[2,3-d]pyrimidine-6-carboxylic acid (35 mg, 0.12 mmol), HOBt (24 mg, 0.18 mmol), and EDC (21 mg, 0.18 mmol) were added. The reaction was stirred at room temperature for 20min, then, DIPEA (0.04 ml, 0.24 mmol).3-morpholinopropan-1- amine (26 mg, 0.18 mmol) was added as a solution in CH2Cl2(0.3 ml). The resulting mixture was allowed to stir at room for 18 hours. The resulting mixture was diluted with water and organic phase was separated. The aqueous phase was extracted with CH2Cl2(3 x) and the combined organic extracts were washed with brine, dried with Na2SO4 and concentrated in vacuo to afford the crude product that was purified by silica gel column chromatography (CH2Cl2:MeOH:TEA / 9:1:0.1) to yield the product as a light-yellow oil (34.86 mg, 70% over two steps). IR (neat) ʋmax = 2931, 2850, 1633, 1537, 1515, 1496, 1439, 1287, 1261, 1114, 971, 605.1H NMR (500 MHz, CDCl3) δ 8.54 (s, 1H), 7.75 (s, 1H), 3.72 (t, J = 4.66 Hz, 2H), 3.53 (m, 3H), 2.75 (s, 3H), 2.58 (m, 4H), 2.53 (t, J = 6.00 Hz, 3H), 2.49 (s, 4H), 2.34 (s, 3H), 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 1.79 (p, J = 6.07 Hz, 2H).13C NMR (125 MHz, CDCl3) δ 167.3, 163.2, 162.8, 153.4, 134.2, 127.3, 120.8, 66.7, 58.5, 54.4, 53.9, 50.1, 45.9, 45.8, 40.6, 24.0, 16.4, 8.6. N-(3-(butyl(ethyl)amino) yl)-5-methylthieno[2,3- d]pyrimidine-6- or a flask containing CH2Cl2 (0.7 ml), 4-(4-(sec-butyl)piperazin-1-yl)-5-methylthieno[2,3-d]pyrimidine-6-carboxylic acid (22 mg, 0.07 mmol), HOBt (13.5 mg, 0.1 mmol), and EDC (15.5 mg, 0.1 mmol) were added. The reaction was stirred at room temperature for 20min, then, DIPEA (0.02 ml, 0.14 mmol). N-butyl-N-ethylpropane-1,3-diamine (8) (12.7 mg, 0.08 mmol) was added as a solution in CH2Cl2(0.3 ml). The resulting mixture was allowed to stir at room for 18 hours. The resulting mixture was diluted with water and organic phase was separated. The aqueous phase was extracted with CH2Cl2(3 x) and the combined organic extracts were washed with brine, dried with Na2SO4 and concentrated in vacuo to afford the crude product that was purified by silica gel column chromatography (CH2Cl2:MeOH:TEA / 9:1:0.1) to yield the product as a yellow oil (11.3 mg, 34% over two steps). IR (neat) ʋmax = 2958, 2929, 1633, 1536, 1496, 1437, 1255, 983.1H NMR (500 MHz, CDCl3) δ 8.58 (s, 1H), 8.54 (s, 1H), 3.56 (m, 2H), 3.52 (s, 4H), 2.77 (s, 3H), 2.66 (m, 8H), 2.49 (m, 3H), 1.78 (s, 2H), 1.58 (td, J = 14.07, 6.58 Hz, 1H), 1.46 (m, 2H), 1.29 (m, 4H), 1.07 (t, J = 7.14 Hz, 1H), 1.00 (d, J = 6.51 Hz, 3H), 0.91 (t, J = 7.41Hz, 3H), 0.85 (t, J = 7.35 Hz, 3H).13C NMR (125 MHz, CDCl3) δ 167.5, 163.2, 153.3, 133.9, 120.7, 70.6, 60.9, 52.9, 52.9, 50.8, 26.2, 24.4, 20.7, 16.6, 14.0, 11.3. HRMS (ES+) m / z calculated for C23H39N6OS+[M+H]+: 475.3214; found: 475.3213. 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 N-(3-(butyl(ethyl)amino)propyl)-5-methyl-4-(4-propylpiperazin-1-yl)thieno[2,3- d]pyrimidine-6-carboxamide (2K, or AA2123) To a flame dried flask containing CH2Cl2(0.7 ml added 5-methyl-4-(4-propylpiperazin-1-yl)thieno[2,3-d]pyrimidine-6-carboxylic acid (22 mg, 0.07 mmol), HOBt (13.5 mg, 0.1 mmol), and EDC (15.5 mg, 0.1 mmol) were added. The reaction was stirred at room temperature for 20min, then, DIPEA (0.02 ml, 0.14 mmol). N-butyl-N-ethylpropane-1,3-diamine (8) (12.7 mg, 0.08 mmol) was added as a solution in CH2Cl2 (0.3 ml). The resulting mixture was allowed to stir at room for 18 hours. The resulting mixture was diluted with water and organic phase was separated. The aqueous phase was extracted with CH2Cl2 (3 x) and the combined organic extracts were washed with brine, dried with Na2SO4and concentrated in vacuo to afford the crude product that was purified by silica gel column chromatography (CH2Cl2:MeOH:TEA / 9:1:0.1) to yield the product as a light-yellow oil (13.56 mg, 45% over two steps). IR (neat) ʋmax= 2929, 2870,1632,1536, 1496, 1435, 984.1H NMR (500 MHz, CDCl3) δ 8.58 (s, 1H), 8.55 (s, 1H), 3.57 (m, 2H), 3.53 (s, 4H), 2.66 (s, 3H), 2.60 (m, 5H), 2.51 (s, 2H), 2.36 (m, 2H), 1.79 (s, 2H), 1.54 (m, 3H), 1.48 (m, 2H), 1.26 (m, 3H), 1.07 (m, 3H), 0.92 (t, J = 7.36 Hz, 3H), 0.85 (t, J = 7.35 Hz, 3H).13C NMR (125 MHz, CDCl3) δ 183.7, 167.3, 163.1, 162.8, 153.2, 133.8, 120.7, 77.2, 77.0, 76.9, 76.7, 70.4, 60.5, 52.9, 52.7, 50.2, 47.3, 29.6, 24.3, 20.6, 19.8, 16.4, 13.8, 11.8, 10.7. HRMS (ES+) m / z calculated for C24H41N6OS+[M+H]+: 461.3057; found: 461.3054. N-(3-(butyl(ethyl)amino) piperazin-1- yl)thieno[2,3-d]pyrimidine-6-carboxamide (2L) To a flame dried flask containing CH2Cl2 (0.7 ml), 5-methyl-4-(4-(methylsulfonyl)piperazin-1-yl)thieno[2,3-d]pyrimidine-6-carboxylic acid (23 mg, 0.07 mmol), HOBt (13.5 mg, 0.1 mmol), and EDC (15.5 mg, 0.1 mmol) were added. The reaction was stirred at room temperature for 20 minutes, then, DIPEA (0.02 ml, 0.14 mmol). N-butyl-N-ethylpropane-1,3-diamine (8) (12.7 mg, 0.08 mmol) was added as a solution in CH2Cl2(0.3 ml). The resulting mixture was allowed to stir at room for 18 hours. The resulting mixture was diluted with water and organic phase was separated. The aqueous phase was extracted with CH2Cl2(3 x) and the combined organic extracts were washed with 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 brine, dried with Na2SO4 and concentrated in vacuo to afford the crude product that was purified by silica gel column chromatography (CH2Cl2:MeOH:TEA / 9:1:0.1) to yield the product (18.16 mg, 56% over two steps). IR (neat) ʋmax = 2927, 2847,1634,1536,1496,1340, 1323, .1H NMR (500 MHz, CDCl3) δ 8.78 (s, 1H), 8.60 (s, 1H), 3.63 (t, J = 3.75 Hz, 4H), 3.56 (q, J = 5.39 Hz, 3H), 3.38 (t, J = 4.86 Hz, 4H), 2.82 (s, 2H), 2.79 (s, 3H), 2.62 (m, 4H), 2.48 (t, J = 7.96 Hz, 3H), 1.76 (m, 2H), 1.43 (m, 2H), 1.25 (h, J = 7.34 Hz, 3H), 1.05 (t, J = 7.13 Hz, 3H), 0.84 (t, J = 7.35 Hz, 3H).13C NMR (125 MHz, CDCl3) δ 167.7, 162.9, 162.5, 153.2, 133.3, 121.1, 70.6, 53.3, 50.0, 47.5, 45.2, 41.4, 34.8, 28.4, 24.5, 20.8, 16.2, 14.0, 11.1. HRMS (ES+) m / z calculated for C22H37N6O3S2+[M+H]+: 497.2363; found: 497.2362. N-(3-(butyl(ethyl)amino) yl)thieno[2,3-d]pyrimidine- 6-carboxamide (2M) To a flame dried flask containing CH2Cl2(0.7 ml), 5-methyl-4- (pyrrolidine-1-yl)thieno[2,3-d]pyrimidine-6-carboxylic acid (27 mg, 0.10 mmol), HOBt (20.23 mg, 0.15 mmol), and EDC (23.00 mg, 0.15 mmol) were added. The reaction was stirred at room temperature for 20 minutes, then, DIPEA (0.03 ml, 0.20 mmol). N-butyl-N- ethylpropane-1,3-diamine (8) (16.0 mg, 0.10 mmol) was added as a solution in CH2Cl2(0.3 ml). The resulting mixture was allowed to stir at room temperature for 18 hours. The resulting mixture was diluted with water and organic phase was separated. The aqueous phase was extracted with CH2Cl2 (3 x) and the combined organic extracts were washed with brine, dried with Na2SO4and concentrated in vacuo to afford the crude product that was purified by silica gel column chromatography. (CH2Cl2:MeOH:TEA / 9:1:0.1) to yield the product as a yellow oil (22.38 mg, 56% over two steps). IR (neat) ʋmax= 2958, 2871,1633, 1536, 1496, 1446, 726.1H NMR (500 MHz, CDCl3) δ 8.37 (s, 1H), 8.12 (s, 1H), 3.69 (m, 4H), 3.59 (q, J = 5.67 Hz, 2H), 2.88 (s, 4H), 2.74 (s, 3H), 1.99 (s, 2H), 1.90 (m, 4H), 1.60 (s, 2H), 1.32 (q, J = 7.44 Hz, 2H), 1.23 (m, 4H), 0.90 (t, J = 7.34 Hz, 3H).13C NMR (125 MHz, CDCl3) δ 167.4, 163.7, 160.4, 153.0, 134.4, 126.7, 118.9, 52.2, 51.1, 47.3, 38.8, 26.5, 25.6, 24.2, 20.5, 18.8, 13.9, 9.7. HRMS (ES+) m / z calculated for C21H34N5OS+[M+H]+: 404.248; found: 404.270. 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 Synthetic Methods for Biotin-2 Probe N-ethyl-4-((triisopropylsilyl) a flamed fried flask, 4- (ethylamino)butan-1-ol (0.78 7.8 ml of CH2Cl2at 0℃, then, triethylamine (TEA, 1.56 mmol, 0.22 ml) was added. The reaction was stirred at 0℃ for 20min. Subsequently, triisopropylsilyl chloride (TIPS, 0.17 mmol, 0.25 mL) was added. The mixture was stirred at room temperature for 5 hours. The reaction mixture was diluted with water and organic phase was separated. The aqueous phase was extracted with CH2Cl2(3 x) and the combined organic extracts were washed with brine, dried with Na2SO4 and concentrated in vacuo. The crude product was carried on the next step without further purification.1H NMR (500 MHz, CDCl3) δ 3.65 (t, J = 6.21 Hz, 2H), 3.46 (s, 1H), 2.66 (m, 4H), 1.60 (m, 2H), 1.54 (m, 2H), 1.13 (t, J = 7.19 Hz, 1H), 1.00 (s, 9H), 1.00 (s, 9H).13C NMR (125 MHz, CDCl3) δ 63.1, 49.0, 43.6, 30.6, 25.6, 18.1, 17.9, 17.8, 17.7, 14.2, 12.4, 12.1, 11.9, 11.7, 11.2. Me OTIPS2-(3-(ethyl(4-( 1,3-dione To a flamed dried flask, N-ethyl-4-((triisopropylsilyl)oxy)butan-1-amine (12) (1.21 mmol, 0.33 g) was added in 3.6 ml of can, K2CO3 (3.3 mmol, 0.46 g) was added to the reaction. The mixture was allowed to stir at room temperature for 20 minutes. Then, (N-3-bromo-propyl)- phthalimide (1.1 mmol, 0.29 g) was added. The reaction mixture was heated 65 ℃ for 18 hours. The reaction was cooled at room temperature and diluted with water and organic phase was separated. The aqueous phase was extracted with EtOAc (3 x) and the combined organic extracts were washed with brine, dried with Na2SO4and concentrated in vacuo to be purified by silica gel column chromatography (CH2Cl2::MeOH / 9:1) to afford the product as a light- yellow oil (0.43 g, 88%).1H NMR (500 MHz, CDCl3) δ 7.67 (dd, J = 5.42, 3.04 Hz, 2H), 7.56 (dd, J = 5.43, 3.03 Hz, 2H), 3.57 (t, J = 7.27 Hz, 2H), 3.53 (m, 2H), 2.41 (m, 4H), 2.34 (m, 2H), 1.72 (p, J = 7.25 Hz, 2H), 1.39 (m, 2H), 0.89 (m, 25H).13C NMR (125 MHz, 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 CDCl3) δ 168.0, 133.7, 132.0, 122.9, 63.1, 52.9, 50.8, 47.0, 36.3, 30.8, 25.9, 22.9, 17.9, 11.8, 11.2. N1-ethyl-N1-(4-( diamine (13) To a solution of 2- (3-(ethyl(4-((triisopropylsilyl) 1,3-dione (0.22 mmol, 100 mg) in 0.5 M EtOH was added hydrazine (0..88 mmol, 0.04 ml), and the resulting mixture was heated at 65℃ for 18 hr. The reaction mixture was then concentrated in vacuo, diluted with DCM, and filtered over a pad of celite. The filtrate was concentrated to provide the desired product as colorless oil. The crude product was carried forward for the next step without further purification.1H NMR (500 MHz, MeOD) δ 3.75 (t, J = 5.86 Hz, 2H), 2.68 (t, J = 7.06 Hz, 2H), 2.57 (q, 3H), 2.51 (m, 3H), 1.65 (m, 2H), 1.57 (m, 4H), 1.09 (s, 18H).13C NMR (125 MHz, MeOD) δ 63.0, 52.9, 50.8, 39.7, 30.7, 28.5, 22.5, 17.1, 11.8, 10.1. N-(3-(ethyl(4-( 4-(4- methylpiperazin-1-yl)thieno[2,3-d]pyrimidine-6-carboxamide (14) To a flame dried flask containing CH2Cl2 (0.7 ml), 5-methyl-4-(4-methylpiperazin-1-yl)thieno[2,3-d]pyrimidine-6- carboxylic acid (10) (27 mg, 0.09 mmol), HOBt (18.91 mg, 0.14 mmol), and EDC (21.73 mg, 0.14 mmol) were added. The reaction was stirred at room temperature for 20min. Then, DIPEA (0.18 mmol, 0.03 ml) was added. N1-ethyl-N1-(4- ((triisopropylsilyl)oxy)butyl)propane-1,3-diamine (13) (0.1 mmol, 33 mg) was added in 0.5 ml CH2Cl2. The reaction mixture was allowed to stir at room temperature for 17 hr. The resulting mixture was diluted with water and organic phase was separated. The aqueous phase was extracted with CH2Cl2(3 x) and the combined organic extracts were washed with brine, dried with Na2SO4 and concentrated in vacuo to afford the crude product that was purified by 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 silica gel column chromatography CH2Cl2:MeOH:TEA / 9:1:0.1) to yield the product as a yellow oil (33.08 mg, 59% over two steps).1H NMR (500 MHz, CDCl3) δ 8.62 (s, 1H), 8.54 (s, 1H), 3.59 (t, J = 6.13 Hz, 4H), 3.52 (m, 8H), 2.76 (s, 4H), 2.58 (m, 14H), 2.48 (t, J = 7.66 Hz, 3H), 2.33 (s, 4H), 1.71 (m, 4H), 1.52 (m, 4H), 1.45 (m, 4H), 1.03 (m, 8H), 0.98 (m, 33H).13C NMR (125 MHz, CDCl3) δ 167.3, 163.2, 162.6, 153.3, 134.1, 127.8, 120.9, 63.0, 54.6, 53.6, 53.4, 50.3, 47.5, 46.1, 41.5, 31.0, 24.7, 22.8, 18.0, 16.4, 11.9, 11.3. N-(3-(ethyl(4- 1- yl)thieno[2,3-d]pyrimidine-6-carboxamide To a flamed dried reaction vessel, N-(3- (ethyl(4-((triisopropylsilyl)oxy)butyl)amino)propyl)-5-methyl-4-(4-methylpiperazin-1- yl)thieno[2,3-d]pyrimidine-6-carboxamide (0.03 mmol, 16 mg) was added in 0.05M MeOH at 0 ℃, subsequently HCl (0.12 mmol, 4 µL) was added dropwise. The reaction was stirred at 0 ℃ for 2 hours, then the solvent was evaporated to yield the desired product as yellow oil, and the crude product was carried on the next step without further purification.1H NMR (500 MHz, MeOD) δ 8.81 (s, 1H), 4.58 (d, J = 13.98 Hz, 2H), 3.87 (s, 2H), 3.68 (d, J = 11.10 Hz, 2H), 3.63 (q, J = 4.84 Hz, 3H), 3.53 (d, J = 6.39 Hz, 2H), 3.37 (s, 2H), 3.28 (m, 3H), 3.21 (m, 3H), 2.98 (s, 3H), 2.79 (s, 3H), 2.11 (s, 2H), 1.85 (s, 2H), 1.64 (t, J = 6.66 Hz, 3H), 1.36 (t, J = 6.71 Hz, 3H).13C NMR (125 MHz, MeOD) δ 162.8, 161.8, 158.3, 148.2, 132.5, 131.5, 119.2, 60.6, 52.5, 52.3, 50.0, 42.3, 40.6, 36.9, 29.0, 23.8, 20.6, 15.9, 7.8. 4-(ethyl(3-(5- 6 carboxamido)propyl)amino)butyl pent-4-ynoate (15) To a flamed died reaction flask, N- 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 (3-(ethyl(4-hydroxybutyl)amino)propyl)-5-methyl-4-(4-methylpiperazin-1-yl)thieno[2,3- d]pyrimidine-6-carboxamide (0.02 mmol, 11 mg) was added in 0.1M CH2Cl2, then, triethylamine (0.026 mmol, 4 µL) was added dropwise. The reaction was stirred at 0 ℃ for 20 minutes, subsequently, hex-5-ynoyl chloride (0.022 mmol, 3 µL) was added dropwise. The reaction was stirred at 0 ℃ for 30 minutes, then the solvent was evaporated to yield the desired product as yellow oil, and the crude product was carried on to the next step without further purification. 4- propyl)amino)butyl-4-(1-(12-oxo-17-((3aS,6aR)-2-oxohexahydro-1H-thieno[2,3- d]imidazol-5-yl)-3,6,9-trioxa-13-azaheptadecyl)-1H-1,2,3-triazol-4-yl)butanoate (Biotin- 2) To a flame dried reaction flask, 4-(ethyl(3-(5-methyl-4-(4-methylpiperazin-1- yl)thieno[2,3-d]pyrimidine-6 carboxamido)propyl)amino)butyl pent-4-ynoate (15) (0.014 mmol, 7.6 mg) was added in 0.01 M THF:H2O. Then, Azide-PEG3-biotin conjugate (0.017 mmol, 7.5 mg, was added followed by 20% CuSO4·5H2O and sodium ascorbate (0.028 mmol, 5.5 mg). The reaction heated at 90℃ for 2 hr. The reaction mixture was concentrated in vacuo. The crude product was purified by neutral alumina column chromatography (CH2Cl2:MeOH:NH4OH / 9:1:1) to afford the product as a white powder (10.61 mg, 77%).1H NMR (500 MHz, MeOD) δ 8.63 (s, 1H), 7.83 (s, 1H), 4.55 (t, J = 5.08 Hz, 2H), 4.50 1H), 4.31 (m, 1H), 4.13 (m, 4H), 3.88 (t, J = 5.05 Hz, 2H), 3.61 (s, 6H), 3.59 (s, 5H), 3.53 (t, J = 5.66 Hz, 4H), 3.21 (m, 5H), 2.98 (s, 3H), 2.92 (dd, J = 9.92, 6.73 Hz, 1H), 2.80 (s, 3H), 2.71 (m, 3H), 2.39 (t, J = 7.28 Hz, 2H), 2.21 (t, J = 7.38 Hz, 2H), 2.10 (t, J = 8.05 Hz, 2H), 1.95 (p, J = 10.45 Hz, 2H), 1.85 (s, 3H), 1.74 (m, 4H), 1.61 (m, 5H), 1.43 (m, 4H), 1.36 (m, 3H), 1.31 (m, 6H).13C NMR (125 MHz, MeOD) δ 174.7, 173.4, 167.8, 164.0, 162.5, 153.0, 132.4, 129.9, 122.9, 120.9, 70.1, 70.0, 70.0, 70.0, 69.2, 69.0, 63.1, 61.9, 60.2, 55.6, 52.4, 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 51.8, 49.9, 49.8, 42.3, 39.7, 38.9, 36.7, 35.3, 32.8, 31.6, 29.3, 28.3, 28.1, 25.4, 24.4, 24.2, 23.8, 20.4, 15.1, 7.8. HRMS (ES+) m / z calculated for C46H74N12O8S2+[M+H]+: 987.8272; found: 987.609. tert-butyl 4-isobutylpiperazine-1- a dried reaction vial containing a solution of 1-BOC-piperazine (1.0 eq) (0.2 M) was added K2CO3 (3.0 eq). The reaction mixture stirred for 20 minutes at room temperature. To the reaction mixture was added 1-bromo-2-methylpropane (3 eq) dropwise. The reaction mixture was heated to 80 °C overnight. Upon completion, the solvent was then removed under reduced pressure. The crude residue was dissolved in dichloromethane and washed with distilled water. The aqueous phase was extracted 3x with dichloromethane. The organic phase was dried over sodium sulfate and concentrated under vacuum to yield the desired product as a tan oil (0.610 g, 94%). The crude product was carried forward without further purification.1H NMR (600 MHz, CDCl3) δ 3.41 (t, J = 5.2 Hz, 4H), 2.32 (t, J = 5.1 Hz, 4H), 2.07 (d, J = 7.4 Hz, 2H), 1.81 – 1.73 (m, 1H), 1.45 (s, 9H), 0.89 (d, J = 6.6 Hz, 6H). IR (neat): 2954, 2931, 2869, 2808, 2782, 1694, 1457 cm-11-isobutylpiperazine: To a dried a solution of tert-butyl 4- isobutylpiperazine-1-carboxylate (1.0 eq) in dichloromethane (0.2 M) was added trifluoroacetic acid (8.0 eq). The reaction mixture stirred at room temperature for two hours. Upon completion, the solvent was then removed under reduced pressure. Additional volatiles were removed under nitrogen overnight to yield the desired product as a pale orange oil. The crude product was carried forward without further purification. 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 20251H NMR (600 MHz, CDCl3) δ 3.87 (d, J = 6.7 Hz, 3H), 3.76 (s, 2H), 3.65 (s, 3H), 3.49 (s, 1H), 3.22 (s, 1H), 2.99 (d, J = 7.4 Hz, 2H), 2.09 (dt, J = 13.7, 6.9 Hz, 2H), 1.00 (d, J = 6.6 Hz, 6H). ethyl 4-(4-isobutylpiperazin-1- d]pyrimidine-6-carboxylate: To a dried reaction vial containing a (4.0 eq) in ethanol (0.5 M) was added triethylamine (3.0 eq). The reaction mixture stirred for 20 minutes at room temperature. To the reaction mixture was added ethyl 4-chloro-5-methylthieno[2,3- d]pyrimidine-6-carboxylate (1.0 eq). The reaction mixture was heated to 65 °C for one hour. Upon completion, the solvent was then removed under reduced pressure. The crude residue was dissolved in dichloromethane. The reaction was quenched with distilled water. The aqueous phase was extracted 3x with dichloromethane. One additional wash of the organic phase with 0.1 M HCl was performed. The organic phase was dried over sodium sulfate and concentrated under vacuum to yield the desired product as a tan oil (0.217 g, 102%). The crude product was carried forward without further purification.1H NMR (600 MHz, CDCl3) δ 8.63 (s, 1H), 4.39 (dq, J = 14.2, 7.1 Hz, 3H), 4.02 (d, J = 19.7 Hz, 4H), 3.63 (s, 2H), 2.87 (d, J = 7.1 Hz, 2H), 2.81 (s, 3H), 2.15 (dq, J = 13.6, 6.8 Hz, 1H), 1.34 (t, J = 7.3 Hz, 3H), 1.07 (d, J = 6.7 Hz, 5H). IR (neat): 2919, 2850, 2360, 2340, 1676, 1583, 1540, 1499, 1447 cm-1N-(3-(butyl(ethyl)amino) -5-methylthieno[2,3- d]pyrimidine-6-carboxamide: To a dried reaction vial containing a solution of ethyl 4-(4- 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 isobutylpiperazin-1-yl)-5-methylthieno[2,3-d]pyrimidine-6-carboxylate (1.0 eq) in THF:H2O (1:2; 0.15 M: 0.30 M) was added LiOH (1.2 eq) slowly. The reaction mixture stirred overnight at room temperature. Upon completion, the solvent was then removed under reduced pressure. The crude residue was dissolved in dichloromethane (0.1 M) and transferred to a dried reaction vial equipped with a stir bar. To the reaction solution were added EDC (1.5 eq) and HOBt (1.5 eq). The reaction mixture stirred for 20 minutes at room temperature. To the reaction mixture were added N1-butyl-N1-ethylpropane-1,3-diamine (1.2 eq) and DIPEA (2.0 eq). The reaction mixture stirred for 24-48 hours at room temperature. The reaction was quenched with distilled water. The aqueous phase was extracted 3x with dichloromethane. The organic phase was dried over sodium sulfate and concentrated under vacuum. The crude product was purified by silica gel column chromatography (CH2Cl2:MeOH:TEA / 9:1:0.1) to yield the product as a tan oil (18.0 mg, 6.3% over two steps).1H NMR (600 MHz, CDCl3) δ 8.53 (s, 1H), 7.93 (s, 1H), 3.62 (q, J = 5.9 Hz, 2H), 3.58 – 3.51 (m, 3H), 3.07 (q, J = 9.0 Hz, 4H), 2.98 – 2.90 (m, 2H), 2.78 (s, 3H), 2.57 (s, 3H), 2.15 (q, J = 6.7 Hz, 4H), 1.81 (s, 1H), 1.68 (p, J = 7.6 Hz, 2H), 1.41 – 1.35 (m, 3H), 1.32 (t, J = 7.4 Hz, 3H), 1.24 (s, 3H), 0.94 (t, J = 7.4 Hz, 3H), 0.91 (d, J = 6.6 Hz, 6H).).13C NMR (151 MHz, CDCl3) δ 168.02, 163.85, 163.30, 162.60, 154.13, 153.44, 133.29, 120.80, 77.37, 77.16, 76.95, 66.82, 53.12, 52.39, 51.41, 50.34, 46.92, 37.46, 29.83, 25.35, 23.84, 20.98, 20.28, 16.79, 13.74, 8.73. HRMS (ESI): C8H13N2O2+, Calculated: [M+H]+, 475.3214; Found: [M+H]+, 475.3231. IR (neat): 3273, 2956, 2924, 2872, 2851, 2811, 2782, 2361, 2338, 1674, 1642, 1540, 1515, 1500, 1442 cm-1tert-butyl 4-isopropylpiperazine-1- To a dried reaction vial containing a solution of BOC-piperazine (1.0 eq) in acetonitrile (0.2 M) was added K2CO3(3.0 eq). The reaction mixture stirred for 20 minutes at room temperature. To the reaction mixture was added the desired alkyl bromide (3 eq) dropwise. The reaction mixture was heated to 80 °C 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 overnight. Upon completion, the solvent was then removed under reduced pressure. The crude residue was dissolved in dichloromethane and washed with distilled water. The aqueous phase was extracted 3x with dichloromethane. The organic phase was dried over sodium sulfate and concentrated under vacuum to yield the desired product as a tan oil (0.666 g, 109%). The crude product was carried forward without further purification.1H NMR (600 MHz, CDCl3) δ 3.36 (t, J = 5.1 Hz, 4H), 2.62 (p, J = 6.5 Hz, 1H), 2.38 (t, J = 5.1 Hz, 4H), 1.39 (s, 9H), 0.97 (d, J = 6.6 Hz, 6H). IR (neat): 2968, 2931, 2810, 1695, 1454, 1419 cm-11-isopropylpiperazine: To a dried reaction vial containing a solution of tert-butyl 4- isopropylpiperazine-1-carboxylate (1.0 eq) in dichloromethane (0.2 M) was added trifluoroacetic acid (8.0 eq). The reaction mixture stirred at room temperature for two hours. Upon completion, the solvent was then removed under reduced pressure. Additional volatiles were removed under nitrogen overnight to yield the desired product as a slightly yellow oil. The crude product was carried forward without further purification.1H NMR (600 MHz, CDCl3) δ 3.59 (s, 3H), 3.53 (s, 2H), 3.52 – 3.44 (m, 3H), 1.37 (d, J = 6.7 Hz, 6H). ethyl 4-(4-isopropylpiperazin-1- d]pyrimidine-6-carboxylate: To a dried reaction vial containing a solution of 1-isopropylpiperazine (4.0 eq) in ethanol (0.5 M) was added triethylamine (3.0 eq). The reaction mixture stirred for 20 minutes at room temperature. To the reaction mixture was added ethyl 4-chloro-5-methylthieno[2,3- d]pyrimidine-6-carboxylate (1.0 eq). The reaction mixture was heated to 65 °C for one hour. Upon completion, the solvent was then removed under reduced pressure. The crude residue was dissolved in dichloromethane. The reaction was quenched with distilled water. The 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 aqueous phase was extracted 3x with dichloromethane. One additional wash of the organic phase with 0.1 M HCl was performed. The organic phase was dried over sodium sulfate and concentrated under vacuum to yield the desired product as a tan oil (0.211 g, 103 / %). The crude product was carried forward without further purification.1H NMR (600 MHz, CDCl3) δ 8.56 (s, 1H), 4.33 (q, J = 7.1 Hz, 2H), 4.04 (d, J = 14.3 Hz, 2H), 3.92 (t, J = 13.4 Hz, 2H), 3.43 (p, J = 6.7 Hz, 1H), 3.38 (d, J = 11.8 Hz, 2H), 3.07 (qd, J = 7.3, 4.5 Hz, 2H), 2.96 (d, J = 12.7 Hz, 2H), 2.74 (s, 3H), 1.35 (t, J = 7.2 Hz, 3H), 1.31 (d, J = 6.7 Hz, 6H), 1.27 (t, J = 7.3 Hz, 3H). IR (neat): 2985, 2920, 2850, 2361, 2339, 1675, 1540, 1499, 1444 cm-1N-(3-(butyl(ethyl)amino) yl)-5-methylthieno[2,3- d]pyrimidine-6-carboxamide: To a dried reaction vial containing a solution of ethyl 4-(4- isopropylpiperazin-1-yl)-5-methylthieno[2,3-d]pyrimidine-6-carboxylate (1.0 eq) in THF:H2O (1:2; 0.15 M: 0.30 M) was added LiOH (1.2 eq) slowly. The reaction mixture stirred overnight at room temperature. Upon completion, the solvent was then removed under reduced pressure. The crude residue was dissolved in dichloromethane (0.1 M) and transferred to a dried reaction vial equipped with a stir bar. To the reaction solution were added EDC (1.5 eq) and HOBt (1.5 eq). The reaction mixture stirred for 20 minutes at room temperature. To the reaction mixture were added N1-butyl-N1-ethylpropane-1,3-diamine (1.2 eq) and DIPEA (2.0 eq). The reaction mixture stirred for 24-48 hours at room temperature. The reaction was quenched with distilled water. The aqueous phase was extracted 3x with dichloromethane. The organic phase was dried over sodium sulfate and concentrated under vacuum. The crude product was purified by silica gel column chromatography (CH2Cl2:MeOH:TEA / 9:1:0.1) to yield the product as a tan oil (22.2 mg, 8% over two steps).1H NMR (600 MHz, CDCl3) δ 8.57 (s, 1H), 8.18 (s, 1H), 4.02 (s, 3H), 3.66 (q, J = 5.9 Hz, 3H), 3.14 (t, J = 6.9 Hz, 5H), 3.01 (s, 3H), 2.82 (s, 3H), 2.22 (p, J = 6.5 Hz, 2H), 1.74 (s, 2H), 1.41 (dt, J = 18.9, 7.4 Hz, 6H), 1.35 (s, 4H), 1.27 (s, 3H), 0.99 (t, J = 7.4 Hz, 3H), 0.92 – 0.80 (m, 2H). 4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 202513C NMR (151 MHz, CDCl3) δ 168.51, 163.73, 153.26, 120.89, 77.37, 77.16, 76.95, 51.13, 49.89, 47.33, 46.86, 37.08, 29.84, 24.93, 23.71, 20.23, 17.33, 16.73, 13.72, 8.53. HRMS (ESI): C8H13N2O2+, Calculated: [M+H]+, 461.3057; Found: [M+H]+, 461.3068 IR (neat): 3379, 3246, 2962, 2923, 2874, 2851, 2361, 2340, 1638, 1540, 1500, 1443 cm-1In view of the many possible aspects to which the principles of the disclosure may be applied, it should be recognized that the illustrated aspects are only preferred examples of the disclosure and should not be taken as limiting the scope of the disclosure. Rather, the scope of the disclosure is defined by the following claims. We therefore claim as the disclosure all that comes within the scope and spirit of these claims.

Claims

4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 We claim:

1. A compound of Formula Ior a pharmaceutically acceptable A membered heteroaryl; C1-6alkyl, C6-10aryl, C6-10aryl-C1-6alkyl, 6-10 membered heteroaryl,6-10 membered heteroaryl-C1-6alkyl, C3-6cycloalkyl, C3-6cycloalkyl-C1-6alkyl, 5- or 6- membered heterocyclyl or 5- or 6-membered heterocyclyl-C1-6alkyl, -NR5R6, -alkyl-NR5R6, or -NRa-alkyl-NR5R6, where R1 is optionally substituted with one or more groups independently selected from -NR7R8, -alkyl-NR7R8, -C(O)R7, -C(O)NR7R8, -SO2R7, C6-10aryl-C1-6alkyl, 6-10 membered heteroaryl-C1-6alkyl, C3-6cycloalkyl-C1-6alkyl, or 5- or 6- membered heterocyclyl-C1-6alkyl; each Raindependently is H or C1-6alkyl; R2and R3are independently selected from H, C1-6alkyl, C6-10aryl, C6-10aryl-C1-6alkyl, 6-10 membered heteroaryl, 6-10 membered heteroaryl-C1-6alkyl, C3-6cycloalkyl, C3-6cycloalkyl-C1-6alkyl, 5- or 6-membered heterocyclyl or 5- or 6-membered heterocyclyl-C1-6alkyl , or R2 and R3, together with the N atom to which they are attached form a 5- or 6- membered heterocyclyl, where R2and R3are independently optionally substituted with one or more substituents selected from C1-6alkyl, -SO2C1-6alkyl, -C(O)R7, or C(O)NR7R8, such as C1-6alkyl, -SO2C1-6alkyl, or -C(O)C1-6alkyl; R4 is H or C1-6alkyl; R5and R6are independently selected from H, -C(O)R7, -C(O)NR7R8, -SO2R7, C1-6alkyl, C6-10aryl, C6-10aryl-C1-6alkyl, 6-10 membered heteroaryl, 6-10 membered heteroaryl- C1-6alkyl, C3-6cycloalkyl, C3-6cycloalkyl-C1-6alkyl, 5- or 6-membered heterocyclyl, or 5- or 6- membered heterocyclyl-C1-6alkyl, or R5 and R6 together form a 5- or 6-membered optionally substituted heterocyclyl, where R5and R6are independently optionally substituted with halo or C1-6alkyl;4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 R7 and R8 are independently selected from C1-6alkyl, C6-10aryl, C6-10aryl-C1-6alkyl, 6- 10 membered heteroaryl, 6-10 membered heteroaryl-C1-6alkyl, C3-6cycloalkyl, C3-6cycloalkyl- C1-6alkyl, 5- or 6-membered heterocyclyl, or 5- or 6-membered heterocyclyl-C1-6alkyl, where R7and R8are independently optionally substituted with one or more groups independently selected from -NR5R6, -alkyl-NR5R6, -C(O)R5, -C(O)NR5R6, -SO2R5, C6-10aryl, C6-10aryl-C1-6alkyl, 6-10 membered heteroaryl, 6-10 membered heteroaryl-C1-6alkyl, C3-6cycloalkyl, C1-6cycloalkyl-C1-6alkyl, 5- or 6-membered heterocyclyl, and 5- or 6-membered heterocyclyl-C1-6alkyl; and wherein the compound is not .

2. The compound of claim 1, wherein R1is -NRa-C1-6alkyl-NR5R6, -NRa-phenyl, or 5- or 6-membered nitrogen-containing heterocyclyl, and is optionally substituted.

3. The compound of claim 1 or claim 2, wherein R1is -NRa-C1-6alkyl-NR5R6, or -NRa-phenyl and is optionally substituted.

4. The compound of any one of claims 1-3, wherein R1is unsubstituted.

5. The compound of any one of claims 1-3, wherein R1 is substituted with 1, 2, or 3 substituents selected from C1-6alkyl, or halo.

6. The compound of any one of claims 1-5, wherein each Rais H.

7. The compound of any one of claims 1-6, wherein R2and R3are independently H, C1-6alkyl, C3-6cycloalkyl, C3-6cycloalkyl-C1-6alkyl-, or 5- or 6-membered heterocyclyl, and where R2and R3are independently optionally substituted.

8. The compound of claim 7, wherein R2and R3are independently C1-6alkyl.4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 9. The compound of any one of claims 1-6, wherein R2and R3together with the nitrogen to which they are attached form a 5- or 6-membered optionally substituted heterocyclyl.

10. The compound of claim 9, wherein R2and R3together with the nitrogen to which they are attached form an optionally substituted piperazinyl, piperidinyl, morpholinyl, or pyrrolidinyl ring.

11. The compound of claim 10, wherein R2and R3together with the nitrogen to which they are attached form an optionally substituted piperazinyl ring.

12. The compound of any one of claims 9-11, wherein the 5- or 6-membered heterocyclyl formed by R2and R3is substituted with 1, 2, or 3 substituents selected from C1-6alkyl or -SO2C1-6alkyl.

13. The compound of claim 12, wherein the 5- or 6-membered heterocyclyl is piperazinyl substituted with C1-6alkyl or -SO2C1-6alkyl.

14. The compound of claim 12 or claim 13, wherein the substituents are selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl.

15. The compound of any one of claims 9-14, wherein R2 and R3 together with the or4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 16. The compound of any one of claims 1-15, wherein R4 is C1-6alkyl.

17. The compound of claim 16, wherein R4 is methyl.

18. The compound of any one of claims 1-17, wherein the compound has a structure according to Formula IIor a pharmaceutically acceptable 19. The compound of any one of claims 1-18, wherein the compound has a structure according to Formula IVor a pharmaceutically acceptable salt thereof, wherein R9 is H, C1-6alkyl, -SO2C1-6alkyl, - C(O)R7, or C(O)NR7R8, such as C1-6alkyl, -SO2C1-6alkyl, or -C(O)C1-6alkyl.

20. The compound of any one of claims 1-18, wherein the compound has a structure according to Formula III or a pharmaceutically–(CRa2)n- and n is from 1 to 6.4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 21. The compound of any one of claims 1-20, wherein R5 and R6 are independently H or C1-6alkyl.

22. The compound of claim 21, wherein R5and R6are independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl.

23. The compound of any one of claims 1-20, wherein R5 and R6 together form a 5- or 6-membered optionally substituted heterocyclyl.

24. The compound of any one of claims 1-23, wherein R7and R8are independently selected from C1-6alkyl.

25. The compound of any one of claims 20-23, wherein the compound has a structure according to Formula Vis H, C1-6alkyl, -SO2C1-6alkyl, - C(O)R7, or C(O)NR7R8, such as C1-6alkyl, -SO2C1-6alkyl, or -C(O)C1-6alkyl.

26. The compound of claim 25, wherein R9 is C1-6alkyl, -SO2C1-6alkyl, or - C(O)C1-6alkyl.

27. The compound of claim 25, wherein R9 is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl.

28. The compound of any one of claims 1-19, wherein R1 is ,4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 or,4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 or30. The compound of claim 1, wherein the compound has a structure according to Formula VI4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 or a pharmaceutically acceptable salt thereof.

31. A compound having a structure .

32. A composition, comprising a compound according to any one of claims 1-31 and a pharmaceutically acceptable diluent or excipient.

33. A method of treating a disease or condition where modulation of autophagy would provide a benefit comprising treating a subject with an effective amount of a compound according to any one of claims 1-31.

34. A method, comprising administering to the subject having a disease or condition, an effective amount of a compound according to any one of claims 1-31, or a pharmaceutical composition thereof.

35. The method of claim 33 or claim 34, wherein the disease or condition is selected from liver disease, diabetes, kidney disease, heart disease, inflammatory bowel disease, neurodegenerative disease, and cancer.

36. The method of claim 35, wherein the disease or condition is a neurodegenerative disease or disorder.

37. The method of claim 36, wherein the neurodegenerative disease or condition is selected from Alzheimer’s disease, Parkinson’s disease, Huntington's disease, Amyotrophic lateral sclerosis, or Niemann-Pick Type C.4239-114921-02 10 / 16 / 25 E-188-2025-0-PC-01 FILED ELECTRONICALLY ON OCTOBER 16, 2025 38. The method of claim 36, wherein the neurodegenerative disease or disorder is Niemann-Pick Type C.

39. The method of claim 34, wherein the disease or condition is cancer.

40. The method of claim 39, wherein the cancer is selected from lung cancer, pancreatic ductal adenocarcinoma, melanoma, breast cancer, ovarian cancer and colorectal cancer.

41. A method of modulating autophagy in a cell, comprising contacting the cell with an effective amount of a compound of any one of claims 1-31.

42. A use of a compound according to any one of claims 1-31 in the preparation of a medicament for the treatment of liver disease, diabetes, kidney disease, heart disease, inflammatory bowel disease, neurodegenerative disease, or cancer.

43. A use of a compound according to any one of claims 1-31 in the preparation of a medicament to modulate autophagy.

44. A compound according to any one of claims 1-31 for use in a method of administration to a subject in need thereof.

45. The compound of claim 44, wherein the subject has liver disease, diabetes, kidney disease, heart disease, inflammatory bowel disease, neurodegenerative disease, or cancer.

46. A compound according to any one of claims 1-31 for use in a method of modulating autophagy.

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