WO2025076226A1 - Fused heterocycles for treating neurodegenerative diseases - Google Patents

Abstract

Disclosed herein are aspects of compounds according to Formula I and Formula II. Also disclosed are pharmaceutical compositions comprising the compounds and method for making and using the compounds. The compounds are useful as autophagy modulators. Additionally, the disclosed compounds may be useful as therapeutics for treating and/or preventing neurodegenerative diseases including Alzheimer's disease.

Description

COMPOUNDS, COMPOSITIONS AND METHODS FOR TREATING NEURODEGENERATIVE DISEASES

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of, and priority to, the earlier filing date of U.S. Provisional Application No. 63/542,321, filed October 4, 2023, which is incorporated herein by reference in its entirety.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant numbers RF1 AG076653, R01 AG074248, T32 AG057468, F30 AG081091 and F31 CA265072 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 thereof, as well as to methods of treating neurodegenerative disease using the compounds and compositions.

BACKGROUND

Autophagy, an important cellular homeostasis pathway, has a primary role in the catabolic degradation and recycling of long-lived proteins and organelles. This intracellular process allows for the engulfment of protein aggregates in a double-membrane structure known as an autophagosome, and upon fusion with lysosome, allows for the degradation of the autophagic cargo. A functional autophagic pathway is especially critical in neurons. Neurons are post- mitotic and do not replicate, so the need to remove cellular debris and toxins is paramount in keeping neurons alive. In neurons, autophagosomes originate in axons, and travel towards the soma, along the way fusing to lysosomes to create the autolysosomc that undergoes degradation of its contents. Autophagy has been associated with neurodevelopment, neuronal homeostasis, and neuronal activity and plasticity. Alternatively, dysfunction of autophagy has also been related to many neurological disease states.

Protein misfolding and aggregation are one of the most common hallmarks of a variety of neurological disorders. Because of this, autophagy has been implicated in different disease states such as Huntington’s disease (HD), where aggregation occurs from CAG repeats on the first exon of the huntingtin gene, Parkinson’s disease (PD), where a variety of factors cause accumulation of a-synuclein and polyubuiquinated proteins, and amyotrophic lateral sclerosis (ALS), where genetic mutations lead to accumulation of ubiquitinated cytoplasmic inclusions like the protein TDP-43. In these cases, impairment of autophagy and the closely related lysosomal pathway have been implicated in progression of neurodegeneration associated with the disorders. Recent work has also linked autophagic dysfunction to the progression of Alzheimer’s disease (AD). Given the prevalence of neurodegenerative disorders, there exists a need in the art for new compounds that can be used to treat and/or prevent such diseases.

SUMMARY

In an aspect this disclosure provides a compound of formula I or II, or a pharmaceutically acceptable salt thereof,

wherein Ai, A2, A3 and A4 are independently selected from C1-8alkyl, C6-10aryl, 5- to 10-membered heteroaryl, C3-10cycloalkyl and 4- to 10-membered heterocyclyl, and a group of formula -NR3R4; wherein any alkyl, aryl, heteroaryl, cycloalkyl and heterocyclyl group is optionally substituted with one or more groups independently selected from - C(O)R5, -C(O)NRsR6, -SO2R5, C6-10aryl(C1-6alkyl), 5- to 10-membered heteroaryl(C1-6alkyl), C3-10cycloalkyl(C1-6alkyl), 4- to 10-membered heterocyclyl(Ci.6alkyl), urea (- NHC(O)NHi), sulfonamide (-SCEamino), C 1-6alkyl, halo, amino, hydroxy, or amide groups, or a combination thereof.

Each of L1, and L4 are independently absent or C1-C4 alkylene. L3 is absent or is selected from O, NR5, S, C(O), and C1-C4 alkylene. Li and L5 are independently absent or selected from C1-C4 alkylene and - SO2-. And Z is selected from -C(O)R5, -C(O)NR₈R₉, -SO2R5, hydroxy, C1-C4 alkoxy, -C(O)OR₅, -OC(O)R₅, and -C(O)R_S;

Each of R1, Ri, R3, R4, and R5 are independently selected from H, -C(O)R6, -C(O)NR8R9, -SO2R6, C1-6alkyl, -O(C1-6alkyl), halo, C6-10aryl, C6-10arylalkyl, 5- to 10-membered heteroaryl, 5- to 10-membered heteroaryl(C1-6alkyl), C3-10cycloalkyl, C3-10cycloalkyl(Ci-6alkyl), 4- to 10-membered heterocyclyl, and 4- to 10-membered heterocyclylalkyl.

R6 and R7 arc independently selected from H, C1-10alkyl, C2-10alknyl, C6-10aryl, C6-10aryl(Ci-6alkyl), 5- to 10-membered heteroaryl, 5- to 10-membered heteroaryl(C1-6alkyl), C3-10cycloalkyl, C3-10cycloalkyl(Ci. 6alkyl), 4- to 10-membered heterocyclyl, and 4- to 10-membered heterocyclylalkyl.

R8 and R9 are independently selected from H, C1-10alkyl, C2-10alknyl, C6-10aryl, C6-10aryl(C1-6alkyl), 5- to 10-membered heteroaryl, 5- to 10-membered heteroaryl(Ci-ealkyl), C3-10cycloalkyl, C3-10cycloalkyl(C1- ealkyl), 4- to 10-membered heterocyclyl, and 4- to 10-membered heterocyclylalkyl, or Rs and R9 together with the nitrogen to which they are attached form a 5- to 7 membered heterocyclyl, optionally including 1, 2 or 3 additional heteroatoms selected from N, O or S, and optionally substituted with 1, 2, or 3 substituents selected from C1-6alkyl, -CH2phenyl, or -C(O)OC1-6alkyl.

In another aspect, this disclosure is directed to a composition comprising a compound of formula I or II, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent or excipient.

In another aspect, this disclosure is directed to a method of treating a neurodegenerative disease or condition, comprising administering to a subject in need of treatment an effective amount of a compound of formula I or II, or a pharmaceutically acceptable salt thereof. In one aspect, the neurodegenerative disease or condition is selected from Huntington’s disease (HD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and Alzheimer’s disease. In another aspect, the neurodegenerative disease or condition is Alzheimer’s disease.

In another aspect, this disclosure is directed to a compound of formula III

or a pharmaceutically acceptable salt thereof.

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. 1 A summarizes an eGFP-LC3 puncta formation assay in HeLa cells was performed in high- throughput on 10,000 molecules, revealing 312 molecules that were able to increase the puncta/cells levels significantly (z-score > 2.199 in both duplicate biological replicates). Of those, 27 were found to be overtly cytotoxic (unlabeled dark circles), whereas compounds la and 2a were prioritized.

FIG. IB shows representative images from the eGFP-LC3 assay. Cells were plated at a density of 3,000 cells/well and compound treated for 4 h with DMSO, chloroquine (CQ) (20 pM), la (20 p M), or 2a (150 pM). The late-stage autophagy inhibitor CQ was used as a positive control for puncta accumulation. FIG. 1C shows results where mCherry-GFP-LC3 expressing HeLa cells were plated to a density of 3,000 cells/well and compound-treated for 4 h with DMSO, CQ (20 pM), PI-103 (5 pM), la (20 pM), or 2a (40 pM). The PI3K inhibitor PI- 103 and CQ were used as controls to show the effects of both early- and late-stage inhibition, respectively.

FIG. ID summarizes that SAR from the initial high-throughput screen revealed moieties useful for activity in the assay.

FIG. IE shows representative image of LC3 immunoblotting performed on HeLa cells treated with compounds for 4 h (DMSO, compound la (20 pM), or compound 2a (40 pM)). Co-treatment with autophagy inhibitor Bafilomycin Al (BafAl, 100 nM) was used to confirm activation.

FIG. IF shows quantification of LC3 immunoblots to measure activation and promotion of autophagic flux. Data are presented as mean ± SEM of four independent experiments (unpaired t-test,

FIG. 1G shows an immunoblot for phosphorylated P70S6Kinase (p-PS6Kinase) shows that compounds la (20 pM) and 2a (40 pM)) are activating autophagy in an mTOR-independent manner compared to mTOR inhibitor, Rap (1 pM).

FIG. 2 A shows a synthetic scheme for analogues of scaffold 1. The initial compound la (DS 1040) hit and three additional analogues were prepared using a four-step method (longest linear sequence).

FIG. 2B shows a synthetic scheme for analogues of scaffold 2. The initial compound 2a (RH1096) hit and four additional analogues were prepared using a four-step method (longest linear sequence).

FIG. 2C shows structures of the analogues and corresponding EC50 values in the eGFP-LC3 puncta formation assay.

FIG. 2D shows that twelve-point dose response curves were generated for each analogue in the eGFP-LC3 puncta formation assay (300 pM to 0.146 pM for 1 analogs, 1000 pM to 0.488 pM for 2 analogues). Data arc presented as mean + SEM of three independent experiments, each with duplicate biological replicates. Data points at the highest concentrations were omitted if they were found to be cytotoxic (% viability <40%).

FIG. 2E shows percent viability was measured using nuclear count following treatment with compounds relative to DMSO controls. Data are presented as mean ± SEM of three independent experiments, each with duplicate biological replicates.

FIG. 3A shows mass spectrometry-based identification of eluted proteins. Proteins captured by Biotin-RHl 115 were subjected to digestion followed by mass spectrometry analysis. Eluted proteins identified were compared to DMSO and biotin acid as standards. 13 unique proteins (listed in alphabetical order) were identified to interact with the RH1115 probe and when treated with excess of RH1115, 7 proteins were no longer pulled down (bolded). FIG. 3B shows a number of peptide spectrum matches (PSMs) identified for each protein in pulldown sample with Biotin-RHl 115 but not in the negative control samples. LMNA has a higher number of PSMs, observed over 36 peptides, suggesting that it was identified with high confidence.

FIG. 3C shows an immunoblot for LAMP1 after 24-hour compound treatment with RH1115 (50 pM) in HeLa cells (bottom). Proteins were quantified and represented as a mean ± SEM of three biological replicates, normalized to P-actin (top)

FIG. 3D shows quantified immunoblots of Lamin A/C after 24-hour compound treatment with RH1115 (50 pM) in HeLa cells. Proteins were quantified and represented as a mean ± SEM of three biological replicates, normalized to P-actin.

FIG. 3E shows representative blots from three independent Cellular Thermal Shift assays. A549 cells were treated with RH1115 (100 pM) or DMSO for 24 hours and heated at each temperature in duplicate biological replicates for 3 min.

FIG. 3F shows immunoblot analysis of Lamin A/C capture by streptavidin pulldown protocol in the presence of Bzorin-RHl 115 (50 pM) (+/-), biotin acid (-/-), Biotin-RHl 115 (50 pM) with excess RH1115 (100 pM) following lysate addition to the beads (+/+). Data are presented as three independent experiments. P-actin was used as a loading control.

FIG. 3G shows immunoblot analysis of LAMP1 capture by streptavidin pulldown protocol in the presence of Biotin-RHl 115 (50 pM) (+/-), biotin acid (-/-), Biotin- RH1115 (50 pM) with excess RH1115 (100 pM) following lysate addition to the beads (+/+). Data are presented from three independent experiments. P-actin was used as a loading control.

FIG. 4A shows quantification of the number of i³Neurons per unit area following treatment with DMSO (Control), DS 1040, RH1096, RH1103 and RH1115 as a read out of neuronal viability. Data are presented as mean ± SEM from three independent experiments, 150-200 neurons per treatment per experiment.

FIG. 4B shows the percentage of i³Neurons exhibiting strong perinuclear clustering of lysosomes after compound or DMSO treatment. Data are presented as mean ± SEM of four independent experiments, 100-150 neurons per treatment. (*P < 0.05, **P < 0.01, one-way ANOVA followed by multiple comparisons).

FIG. 4C shows high resolution confocal images of i³Neurons treated with compounds or DMSO and stained for LAMP1 to label lysosomes and Tau to label neurites, showing enlarged and brighter lysosomal vesicles.

FIG. 4D shows quantification of mean intensity of LAMPl-positive vesicles in DMSO and RH1115-treated (15 pM) i³Neurons from high resolution confocal images. Data are presented as mean + SEM of three independent experiments, 20-25 neurons per treatment. FIG. 4E: Quantification of mean size of LAMP 1 -positive vesicles in DMSO and RH115-treated (15 pM) i³Neurons from high resolution confocal images. Data are presented as mean ± SEM of three independent experiments, 20-25 neurons per treatment, **P < 0.01, unpaired t-test.

FIG. 4F shows immunoblotting of EAMP 1 in DIV20-21 i³Neurons treated with 15 pM RH1115 for 72 hours vs. 0.1% DMSO treatment. Tubulin is used as a loading control.

FIG. 4G shows that quantification of EAMP1 immunoblot shows a significant increase in EAMP1 after RH1115 treatment (15 pM) compared to DMSO treatment from four independent experiments, mean ± SEM *P < 0.05.

FIG. 5A shows immunoblotting for EC3 in DIV20-21 i³Neurons treated with RH1115 (15 pM) or DMSO (0.1%) for 72 hours.

FIG. 5B shows that quantification of LC3II/LC3I ratio shows that i³Neurons treated with RH1115 exhibit significantly increased LC3 lipidation in comparison to DMSO treated i³Neurons. Mean ± SEM from four independent experiments, **P < 0.01. FIG. 5C: Confocal images of DIVIO i³Neurons stably expressing LC3-RFP-GFP treated with DMSO, RH1115 (15 pM) for 72 hours, or BafAl (100 nM) for 24 hours prior to live imaging using airyscan. Higher magnification images of region outlined by dashed box are depicted to the right of each image.

FIG. 5D shows quantification of percent of autophagosomes in i³Neurons treated with RH1115 (15 pM) or BafAl (100 nM) compared to DMSO.

FIG. 5E shows quantification of percent of autolysosomes in i³Neurons treated with RH1115 (15 pM) or BafAl (100 nM) compared to DMSO.

FIG. 5F shows quantification of mean size of autolysosomes in i³Neurons treated with RH1115 (15 pM) compared to DMSO.

FIG. 5G shows quantification of intensity of autolysosomes in i³Neurons treated with RH1115 (15 pM) compared to DMSO. In D-G data were collected from three independent experiments, 60-70 cells per treatment, mean + SEM ***p < 0.001.

FIG.6A shows a synthetic method to generate substituted guanidine and amidine reagents.

FIG. 6B shows a synthetic method to generate the propyl substituted aldehyde reagent.

FIG. 6C shows a synthetic route to access Biotiu-RH1115.

FIG. 7A summarized kinetic aqueous solubility that was performed on select compounds (100 pM) as a solution in lx PBS. Optical density calculations were performed at 620 nm. Data are presented as the mean + SEM of five independent experiments.

FIG. 7B summarizes eGFP-EC3 dose response that was performed on Biotin-RH 1115 to ensure activity is retained. Data are presented as mean ± SEM from three independent experiments, each with duplicate biological replicates. FIG. 8 is a table of exemplary compounds according to the disclosure and providing activity data for certain compounds, wherein the indicates an extrapolated value based on curve fitting; curve does not level off at the highest concentration, and “n.d.” indicates that the value could not be determined.

FIG. 9 is a second table of exemplary compounds according to the disclosure and providing activity data, wherein the indicates an extrapolated value based on curve fitting; curve does not level off at the highest concentration, and “n.d.” indicates that the value could not be determined.

FIG. 10 is a table of alternative exemplary compounds according to the disclosure and their activity data, wherein the indicates an extrapolated value based on curve fitting; curve does not level off at the highest concentration.

FIG. 11 shows individual gray scale and merged images at t= 10 from time lapse Airyscan images acquired at 2.5 frames/sec of DIV15-16 Control and JIP3 KO i³Neurons stably expressing LC3-RFP-GFP treated with 0.15% DMSO for 72 hours. White arrowheads point to axonal swellings filled with autophagosomes and autolysosomes in JIP3 KO FNeurons. Scale bar, 5pm.

FIG. 12 shows a portion of straightened representative neurite (top) and corresponding kymographs (bottom) depicting LC3-RFP-GFP (red channel) vesicle density and movement, respectively, in Control and JIP3 KO i³Neurons. Scale bar, 1pm. Arrows point to moving vesicles, and white arrowheads point to stationary vesicles. Control images are shown 2x brighter than JIP3 KO to enhance their visibility.

FIG. 13 shows quantification depicting the motile fraction of autophagosomes (RFP+ and GFP+) and autolysosomes (RFP+ only) in DIV15-16 Control and JIP3 KO i³Neurons. Superplots show mean ± SEM as well as individual data (represented as large and small symbols respectively. Circles, triangles, and squares represent each independent experiment). N=3 independent experiments, Control autolysosomes n=99 neurites, JIP3 KO autolysosome n=69 neurites, Control autophagosome n=47 neurites, JIP3 KO autophagosome n=40 neurites; *£><.05; one-way ANOVA with Sidak’s multiple comparisons.

FIG. 14 shows quantification showing percentage of total autophagic vacuoles that arc autolysosomes in DIV15-16 Control and JIP3 KO i³Neurons. Superplots show mean + SEM as well as individual data (represented as large and small circles, triangles and squares). N=3 independent experiments, Control n=45 vesicles, JIP3 KO n=58 vesicles; ns=not significant; unpaired t-test.

FIG.15 shows individual gray scale and merged snapshots at t=10 from time lapse Airyscan images acquired at 2.5 frames/sec of DIV 15- 16 Control and JIP3 KO i³Neurons stably expressing LC3-RFP-GFP and treated for 72 hours with 15|iM RH1115. Scale bar, 5pm.

FIG. 16 shows quantification of autophagic vacuole density (LC3 vesicle number per 10pm of neurite) outside of swellings. Superplots show mean + SEM as well as individual data (represented as large and small circles, triangles and squares). N=3 independent experiments. Control DMSO n=120 neurites, Control RH1115 n=125 neurites, JIP3 KO DMSO n=141 neurites, JIP3 KO RH1115 n=130 neurites; ****p?<.0001; one-way ANOVA with Sidak’s multiple comparisons.

FIG. 17 shows quantification of autophagic vacuole density within swellings. Superplots show mean ± SEM as well as individual data (represented as large and small symbols). N=3 independent experiments, DMSO n=33 swellings, RH1115 n=9 swellings in JIP3 KO i³Neurons (only found in JIP3 KO genotype) *p<.05; unpaired t-test.

FIG. 18 shows high resolution, stitched, greyscale images of neuronal processes of DIV 11 Control and JIP3 KO i³Neurons stained for endogenous LAMP1 after 72-hour treatment with 0.15% DMSO or 15pM RH1115. Arrows point to LAMP 1 -positive vesicle accumulations. Scale bar, 20pm.

FIG. 19 shows quantification of the axonal lysosome accumulation index (swellings greater than 5pm or greater than 10pm normalized to neurite density). Superplots show normalized mean ± SEM as well as individual data (represented as large and small circles, triangles and squares). N=3 independent experiments, n=14 stitched images per condition. ****p<.0001; one-way ANOVA followed by Sidak’s multiple comparisons test.

FIG. 20 shows a treatment paradigm for A [142 measurement: i³Neurons were plated on DIV0 and differentiated until DIV42/43 when media was changed for a weeklong drug treatment. i³Neurons were treated with 0.15% DMSO, 15pM RH1115 or 5pM BACE1- 1 two times over the week (DIV42/43 and DIV47/48) and the conditioned media was collected when the i³Neurons were lysed on DIV49/50.

FIG. 21 shows that extracellular AP42 was measured by ELISA using the media collected from DIV49/50 Control and JIP3 KO i³Neurons following the treatment paradigm described above. One-way ANOVA followed by Sidak’s multiple comparisons. **p<.01, ***p<0.001, ****p<0.0001, ns=not significant. Different symbol shapes represent 3 independent replicates.

FIG. 22 shows high resolution greyscale images of DIV11 JIP3 KO i³Neuron somas treated with 0.15% DMSO or 15pM RH1115 for 72 hours and stained for endogenous LAMPE Somas are outlined with a white dashed line. Scale bar, 5 pm.

FIG. 23 shows quantification of the percent of DIVIO JIP3 KO i³Neurons with LAMP1 perinuclear localization, plots show mean ± SEM, N=3 independent experiments, DMSO treated n=106, RH1115 n= 123, ***p <.001; unpaired t-test.

FIG. 24 shows quantification of the average LAMPl-vesicle size in DIVIO JIP3 KO i³Neurons, plots show mean ± SEM, N=3, DMSO n=21 somas, RH1115 n=22 somas, **p<.01; unpaired t-test.

FIG. 25 shows quantification of the average endogenous LAMP1 fluorescence intensity in DIVIO JIP3 KO i³Neurons, plots show mean ± SEM, N=3 independent experiments, DMSO n=21 somas, RH1115 n=22 somas, ****p<.0001; unpaired t-test.

FIG. 26 shows confocal images of Control HeLa cells transfected with LAMP1-KBS-GFP and treated with 0.15% DMSO or 15pM RH1115 for 6 hours, 48h post-transfection. Cells were stained with anti- GFP and DAPI, Scale bar, 10pm.

FIG. 27 shows quantification of the percent of cells showing the majority of LAMP1-KBS-GFP in the cell periphery, perinuclear area, or a mixed distribution. Plots show mean + SEM, N=3 independent experiments (represented as circles, triangles, and squares). DMSO n=91 cells, RH1115 n=84 cells; *p<0.05, nd=no discovery; grouped analysis multiple unpaired t-tests. P value of individual t-tests for peripheral and perinuclear localization, ***p<0.001.

FIG. 28 shows confocal images of DIV 10-13 Control and JIP3 KO PNeurons treated with 0.15% DMSO or 15,u M RH1115 for 72 hours and stained for Lysotracker Red for 10 minutes. Scale bar, 5pm. Neuronal cell bodies are outlined with a dashed line. Insets show higher magnification image of area outlined by the dashed box within the image. Scale bar, 1pm.

FIG. 29 shows quantification of the mean lysotracker intensity per soma in DIVIO- 13 Control and JIP3 KO PNeurons, transformed using Log 10 to account for the exponential difference between DMSO and RH1115 fluorescence intensity values. Superplots show mean ± SEM as well as individual data (represented as large and small circles, triangles and squares). N=3 independent experiments, Control DMSO n=47 PNeurons, Control RH1115 n=43 PNeurons, JIP3 KO DMSO n=37 PNeurons, JIP3 KO RH1115 n=55 PNeurons; **p<.01; one way ANOVA followed by Sidak’s multiple comparisons.

FIG. 30 shows confocal images of DIV12-13 Control PNeuron somas pre-loaded with 25pg/mL DQ- Red BSA and treated with 0.15% DMSO or 15pM RH1115. Scale bar, 5pm.

FIG. 31 shows quantification of the mean intensity of DQ-Rcd BSA. Supcrplots show mean ± SEM as well as individual data (represented as large and small circles, triangles and squares). N=3 independent experiments, Control DMSO n=72 PNeurons, Control RH1115 n=71 PNeurons, *p <.05; unpaired t-test.

FIG. 32 shows confocal images of DIV 12-13 JIP3 KO PNeuron somas pre-loaded with 25pg/mL DQ-Red BSA and treated with 0.15% DMSO or 15pM RH1115. Scale bar, 5pm.

FIG. 33 shows quantification of the mean intensity of DQ-Red BSA. Superplots show mean ± SEM as well as individual data (represented as large and small circles, squares, and triangles). N=3 independent experiments, JIP3 KO DMSO n=64 PNeurons, JIP3 KO RH1115 n=63 PNeurons, *p<0.05, unpaired t-test.

FIG. 34 shows an immunoblot showing TMEM55B expression in DIV21 Control and JIP3 KO PNeurons treated with 0.15% DMSO or 15pM RH1115 for 72 hours (Actin[3 loading control).

FIG. 35 shows quantification of TMEM55B levels normalized to the loading control. Plots show mean + SEM N=3 independent experiments (represented as circles, triangles and squares); *p<.05, **p<.01; one-way ANOVA with Sidak’s multiple comparisons.

FIG. 36 shows high resolution, stitched images of DIV8 JIP3/4 DKO PNeurons stably expressing LAMP1-GFP and treated with 0.15% DMSO or 15pM RH1115 for 72 hours. LAMP 1 -positive vesicle accumulations are marked by arrows. Scale bar, 20pm. FIG. 37 shows quantification of the length of LAMP 1 -positive vesicle accumulations in DIV8 JIP3/4 DKO LAMP1-GFP i³Neurons treated with 0.15% DMSO or 15pM RH1115. Superplots show mean ± SEM as well as individual data (represented as large and small circles, squares, and triangles). N=3 (here includes 2 independent experiments, and a technical replicate). DMSO treated n=61 swellings, RH1115 treated n=68 swellings.

FIG. 38 shows quantification of the lysosome accumulation index (swellings greater than 5pm, 10pm and 20pm normalized to neurite density). Superplots show mean + SEM as well as individual data (represented as large and small circles, squares, and triangles). N=3 (here includes 2 independent experiments, and a technical replicate), DMSO treated n=12 stitched images, RH1115 treated n=12 stitched images.

FIG. 39 shows that RH1115 treatment rescues locomotor defects in JIP3 KO larval zebrafish.

Decreased total distance moved and average velocity were displayed in JIP3 homozygous knockout (KO) zebrafish larvae, compared to JIP3 wild type (WT) clutch mates. Larval zebrafish treated with 0.05% DMSO or 0.15 pM RH1115 immediately following fertilization for 6 days were utilized for locomotor assay. At 6 days post-fertilization (dpf), total distance moved (mm) of larval zebrafish were compared. Both locomotor phenotypes were shown to be rescued in JIP3 KO zebrafish treated with 0.15 pM RH1115 for 6 days. Each dot represents an individual 6-dpf larvae; WT DMSO N=45, WT RH1115 N=38, JIP3 KO DMSO N=49, JIP3 KO RH1115 N=37; ****p<0.0001, ns=non-significant, one-way ANOVA with Tukcy’s test for multiple comparisons.

FIG. 40 shows that RH1115 treatment rescues locomotor defects in JIP3 KO larval zebrafish.

Decreased total distance moved and average velocity were displayed in JIP3 homozygous knockout (KO) zebrafish larvae, compared to JIP3 wild type (WT) clutch mates. Larval zebrafish treated with 0.05% DMSO or 0.15 pM RH1115 immediately following fertilization for 6 days were utilized for locomotor assay. At 6 days post-fertilization (dpf), average velocity (mm/s) of larval zebrafish were compared. Both locomotor phenotypes were shown to be rescued in J1P3 KO zebrafish treated with 0.15 pM RH1115 for 6 days. Each dot represents an individual 6-dpf larvae; WT DMSO N=45, WT RH1115 N=38, JIP3 KO DMSO N=49, JIP3 KO RH1115 N=37; ****p<0.0001, ns=non-significant, one-way ANOVA with Tukey’s test for multiple comparisons.

DETAILED DESCRIPTION

I. Definitions

The following explanations of terms are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. As used herein, “comprising” means “including” and the singular forms “a” or “an” or “the” include plural references unless the context clearly dictates otherwise. The term “or” refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise.

Although the steps of some of the disclosed methods are described in a particular, sequential order for convenient presentation, it should be understood that this manner of description encompasses rearrangement, unless a particular ordering is required by specific language set forth below. For example, steps described sequentially may in some cases be rearranged or performed concurrently. Additionally, the description sometimes uses terms like “produce” and “provide” to describe the disclosed methods. These terms are high-level abstractions of the actual steps that are performed. The actual steps that correspond to these terms will vary depending on the particular implementation and are readily discernible by one of ordinary skill in the art.

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 compounds similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and compounds are described below. The compounds, methods, and examples are illustrative only and not intended to be limiting, unless otherwise indicated. Other features of the disclosure are apparent from the following detailed description and the claims.

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.” Accordingly, unless otherwise indicated, implicitly or explicitly, the numerical parameters set forth are approximations that can depend on the desired properties sought and/or limits of detection under standard test conditions/methods. When directly and explicitly distinguishing embodiments from discussed prior art, the embodiment numbers are not approximates unless the word “about” is recited. Furthermore, not all alternatives recited herein are equivalents.

Compound embodiments disclosed herein may contain one or more asymmetric elements such as stereogenic centers, stereogenic axes and the like, e.g., asymmetric carbon atoms, so that the chemical conjugates can exist in different stereoisomeric forms. These compound embodiments can be, for example, racemates or optically active forms. For compound embodiments with two or more asymmetric elements, these compound embodiments can additionally be mixtures of diastereomers. For compound embodiments having asymmetric centers, all optical isomers in pure form and mixtures thereof are encompassed by corresponding generic formulas unless context clearly indicates otherwise or an express statement excluding an isomer is provided. In these situations, the single enantiomers, i.e., optically active forms can be obtained by method known to a person of ordinary skill in the art, such as asymmetric synthesis, synthesis from optically pure precursors, or by resolution of the racemates. Resolution of the racemates can also be accomplished, for example, by conventional methods, such as crystallization in the presence of a resolving agent, or chromatography, using, for example a chiral HPLC column. All isomeric forms are contemplated herein regardless of the methods used to obtain them.

All forms (for example solvates, optical isomers, enantiomeric forms, polymorphs, free compound and salts) of an active agent may be employed either alone or in combination. Stereochemical definitions and conventions used herein generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., New York. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes (+/-) D and L or R and S are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and 1 or (+) and (-) are employed to designate the sign of rotation of plane -polarized light by the compound, with (-) or 1 meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory

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 substituent and its parent moiety; a 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, Cl- C6alkoxycarbonyloxy and -OC(O)C1-C6 alkyl 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, 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

“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.

“Alkyl” means a straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms, such as from 1 to 8 or from 1 to 6 carbon atoms, 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, 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 an alkylene group is -(CH₂)n- An alkyl, e.g., methyl, or alkylene, e.g., — CH₂CH₂ — , group can be substituted, independently, with one or more of halo, tri fluoromethyl, 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 “alkylkene” except for containing a carbon-carbon triple bond.

“Amino” means a group of formula -NR^aR^b wherein R^a and R^b are independently selected from hydrogen and C|-C alkyl. Acetylamino means a -NHC(=O)CH₃ group.

“Aryl” means a phenyl (z.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 phenyl 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 and/or thia groups. Representative examples of the bicyclic aryls include, but are not limited to, azulenyl, naphthyl, dihydroinden-l-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-l-yl, inden-2-yl, inden-3-yl, inden-4-yl, dihydronaphthalen-2-yl, dihydronaphthalen-3-yl, dihydronaphthalen-4-yl, dihydronaphthalen-1- yl, 5,6,7,8-tetrahydronaphthalen-l- 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][l,3]dioxol-4-yl, benzo[d][l,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- l,3-dion-4-yl, isoindoline- 1,3- dion-5-yl, inden- l-on-4-yl, inden-l-on-5-yl, inden- l-on-6-yl, inden-l-on-7- yl, 2,3- dihydrobenzo[b][l,4]dioxan-5-yl, 2,3-dihydrobenzo[b][l,4]dioxan-6-yl, 2H- benzo[b][l,4]oxazin3(4H)-on-5-yl, 2H-benzo[b][l,4]oxazin3(4H)-on-6-yl, 2H-benzo[b][l,4]oxazin3(4H)- on-7-yl, 2H-benzo[b] [l,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(lH)-on-5-yl, quinoxalin- 2(lH)-on-6-yl, quinoxalin- 2(lH)-on-7-yl, quinoxalin-2(lH)-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, 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, 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 hctcroaryl 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-l-yl, thienopyridinyl, 4, 5, 6, 7- tetrahydrobenzo[c][l,2,5]oxadiazolyl, and 6,7- dihydrobenzo[c][l,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 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 the group consisting of 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 the group consisting of O, N and S. The 5 membered ring can contain zero or one double bonds and one, two or three heteroatoms selected from the group consisting of O, N and S. The 6 or 7 membered ring contains zero, one or two double bonds and one, two or three heteroatoms selected from the group consisting of 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,l,l-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, a 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. The pharmaceutically acceptable salts of the 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 quatcrnizcd 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, it is believed that compounds of the disclosure are effective modulators of neurodegenerative diseases or conditions. In certain aspects, the neurodegenerative disease or condition is selected from Huntington’s disease (HD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and Alzheimer’s disease. In an aspect, the neurodegenerative disease or condition is Alzheimer’ s disease. “Neurodegenerative disorder” refers to an abnormality in the nervous system of a subject, such as a mammal, in which neuronal integrity is threatened. Without being bound by theory, neuronal integrity can be threatened when neuronal cells display decreased survival or when the neurons can no longer propagate a signal. In several embodiments, neurodegenerative diseases are associated with ER stress and protein aggregation, such as accumulation, oligomerization, fibrillization or aggregation, of two or more, hetero- or homomeric, proteins or peptides in the intracellular or extracellular neuronal environment. Non-limiting examples of neurodegenerative disorders associated with ER stress and protein aggregation include Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis (ALS).

Alzheimer’ s disease (AD) is a progressive brain disorder that occurs gradually and results in memory loss, behavioral and personality changes, and a decline in mental abilities. These losses are related to the death of brain cells and the breakdown of the connections between them. The course of this disease varies from person to person, as does the rate of decline. On average, AD patients live for 8 to 10 years after they are diagnosed, though the disease can last up to 20 years. AD advances by stages, from early, mild forgetfulness to a severe loss of mental function. At first, AD destroys neurons in parts of the brain that control memory, especially in the hippocampus and related structures. As nerve cells in the hippocampus stop functioning properly, short-term memory fails. AD also attacks the cerebral cortex, particularly the areas responsible for language and reasoning.

Parkinson's disease (PD) is an idiopathic, slowly progressive, degenerative CNS disorder characterized by slow and decreased movement, muscular rigidity, resting tremor, and postural instability. The loss of substantia nigra neurons, which project to the caudate nucleus and putamen, results in the depletion of the neurotransmitter dopamine in these areas.

Amyotrophic lateral sclerosis (ALS) is a progressive, usually fatal, neurodegenerative disease caused by the degeneration of motor neurons. The neurons typically affected are located in the lower motor neurons of the brainstem and spinal cord and upper motor neurons in the cerebral cortex. Within 2 to 5 years after clinical onset, the loss of motor neurons leads to progressive atrophy of skeletal muscles, which results in loss of muscular function resulting in paralysis, speech deficits, and death due to respiratory failure. ALS is also known as Lou Gehrig’s disease.

Huntington's disease is an autosomal dominant neurodegenerative disease resulting from mutation in the Huntington gene. The mutation is an expansion of a trinucleotide repeat (CAG) in exon 1 of the Huntington gene, resulting in a polyglutamine expansion in the Huntington protein. The resulting gain of function is the basis for the pathological, clinical and cellular sequelae of Huntington's disease. The primary neuro-anatomical affect is found in the caudate nucleus and putamen, including medium spiny neurons. Clinically, Huntington's disease is characterized by an involuntary choreiform movement disorder, psychiatric and behavioral chances and dementia. The age of onset is usually between 30-50 years of age, although juvenile and late onset cases of Huntington's disease occur. At the cellular level, Huntington's disease is characterized by protein aggregation in the cytoplasm and nucleus of neurons which comprise ubiquitinated terminal fragments of Huntington. (see, e.g., Bence el al., Science, 292:1552-1555, 2001; Walter el al., Mol. Biol. Cell., 12: 1393-1407, 2001).

Multiple sclerosis is a slowly progressive CNS disease characterized by disseminated patches of demyelination in the brain and spinal cord, resulting in multiple and varied neurological symptoms and signs, usually with remissions and exacerbation. An increased family incidence suggests genetic susceptibility, and women are somewhat more often affected than men. The symptoms of MS include weakness, lack of coordination, paresthesias, speech disturbances, and visual disturbances, most commonly double vision. More specific signs and symptoms depend on the location of the lesions and the severity and destructiveness of the inflammatory and sclerotic processes. Relapsing-remitting multiple sclerosis is a clinical course of MS that is characterized by clearly defined, acute attacks with full or partial recovery and no disease progression between attacks. Secondary-progressive multiple sclerosis is a clinical course of MS that initially is relapsing-remitting, and then becomes progressive at a variable rate, possibly with an occasional relapse and minor remission. Primary progressive multiple sclerosis presents initially in the progressive form. A clinically isolated syndrome is the first neurologic episode, which is caused by inflammation/demyelination at one or more sites in the CNS.

“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.

“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

Alzheimer’s disease was the seventh-leading cause of death in the United states in 2021, and it is estimated that by the year 2060, 13.8 million people are projected to get the disease, thus the need for therapeutics to slow or stop disease progression remain a critical challenge. Alzheimer’s disease is characterized by two major neuronal protein aggregations: 1) A0 protein plaques generated from proteolytic processing of amyloid precursor protein (APP) and 2) neurofibrillary tangles of hyperphosphorylated tau protein. These aggregations lead to degradation of the neuron and eventual neuronal cell death. Studies in human AD and mouse models of the disease reveal the presence of numerous autophagic and lysosomal intermediates in brain tissue, suggestive of defects in fusion and/or clearance of these organelles. This dysfunction may be a result of Beclin-1, a key autophagy regulator, expression is reduced in early AD. Moreover, further works show inhibition of basal autophagy in neurons results in morphologies similar to that of an AD model. Taken together, these factors suggest the need for activation of autophagy in these cases.

Lysosomes are the central organelles in maintaining protein and organelle homeostasis in neurons over their long lifespan. In addition to their longevity, their polarity and extreme size add additional spatial challenges for optimal lysosome biogenesis and functioning in neurons. This necessitates efficient lysosome transport as an important adaptation to meet the metabolic demands of the neuron. Indeed, efficient axonal lysosome transport is coupled to the maturation of these organelles and necessary for clearance of cargo from axons. Multiple studies suggest that autophagosomes, which play an important role in clearing damaged organelles and old proteins from axons, engage with endolysosomes and undergo retrograde transport so that cargo can be degraded in the highly acidic lysosomes in the soma. The transport of lysosomes and their precursor organelles along the axon requires the modulation of microtubule tracks by post-translational modification, microtubule associated proteins (MAPs), molecular motors, and adaptor proteins that regulate the connections between motors and their cargo.

The importance of efficient axonal lysosome transport to maintenance of neuronal health and functioning is exemplified by how disruptions to this process are linked to several neurological diseases. A high abundance of axonal lysosomes within swellings at sites of amyloid plaques have been observed in Alzheimer’s disease. These organelles have been linked to increased Amyloid Precursor Protein (APP) processing and exacerbated plaque development in AD. Indeed, loss of JNK-interacting protein 3 (JIP3), a brain-enriched adaptor and a regulator of axonal lysosome transport, results in a similar focal buildup of axonal lysosomes within swellings as well as increased intraneuronal amyloid 042 ( A 042) levels. Furthermore, haploinsufficicncy of JIP3 in an AD mouse model resulted in worsening of amyloid pathology, supporting a model wherein these stalled axonal organelles lead to increased amyloid processing and pathology development. Thus, restoring efficient axonal lysosome transport and maturation could suppress amyloidogenic processing of APP and in turn, the progression and development of AD pathology.

Currently, Alzheimer’s treatment involves the use of cholinesterase inhibitors or N-methyl D- aspartate inhibitors to simply mitigate the symptoms of the disease, but their adverse side-effects and inability to treat the disease itself render them ineffective as a long-term treatment option.

More recently, clinical trials have sought to treat AD by attempting to clear A0 through modulation of key secretase proteins in the formation pathway. 0-site APP cleavage enzyme (BACE1) is a membranebound aspartyl protease responsible for the initiation of APP cleavage to generate A0 and therefore has become a target for inhibition for the purposes of lowering A0 levels and treating AD. Clinical trials of many of these inhibitors revealed successful binding to BACE1 and clearance of A0, however, these trials were stopped due to limited improvement in cognition of AD patients, notable adverse side effects, and/or an inability to successfully clear tau and phosphorylated tau aggregates. Other efforts have looked to target y-secretase, a complex of presenilin, nicastin, Aphl and Pen2 that performs the final cleavage of APP to generate A0.

Specifically, inhibitors of presenilin in clinical trials was also able to reduce Ap production, but were nonspecific and an eventual rebounding of Ap was noted when doses were reduced. This highlights the need for a novel therapeutic target for AD.

Given the connection that autophagy has to neurodegenerative disease, modulation of the process has become a major area of interest for potential treatments. Rapamycin, a well-established mTOR inhibitor and autophagy inducer, has been shown to reduce aggregates and increase protein degradation in HD and ALS mouse models and in addition, has been shown to delay the onset of behavioral abnormalities that arise from such diseases. Given the importance of mTOR in other pathways, alternative mTOR independent activators of autophagy have shown similar success in clearing aggregation and preventing neuronal toxicity in HD and PD models. One such molecule is trehalose, a non-reducing disaccharide not found in mammals, has been implicated in neuronal survival by clearing tau aggregates in mouse models by returning autophagic flux in HD, PD, and ALS. Autophagy induction has also shown success in AD models as well. The use of rapamycin has shown clearance of Ap aggregates and rescues memory defects in AD mouse models. Other mTOR-independent autophagy inducers have also shown success in Ap and tau clearance as well as neuroprotective effects.

The use of high-throughput phenotypic assays has been instrumental in the discovery of many chemical probes that induce a desired cellular phenotype, and these assays are important in multiple stages of drug discovery to identify, validate, and optimize lead molecules. Screening efforts have taken place to identify autophagy modulators that improve model disease phenotypes. However, in many of these cases, these works lack follow-up studies to identify targets and mechanisms of action for the identified compounds. In order to identify more effective therapeutics, the present inventors utilized a phenotypic assay to identify novel therapeutic targets for neurodegenerative diseases. The workflow used by the inventors allowed them to address both hit identification and target validation. To identify autophagy activators that induce desirable neuronal phenotypes, a robust image-based high-throughput phenotypic screen was implemented followed by target identification and validation studies to successfully identify novel lead compounds and protein targets to support efforts to develop novel therapeutic strategies for neurodegenerative diseases. The inventors also have identified new structure-activity relationships and thus have designed new compounds for treating these diseases. II. Compounds

This disclosure provides a compound of formula I or II, or a pharmaceutically acceptable salt thereof,

wherein

Ai, A2, A3 and A4 are independently selected from C1-8alkyl, C6-10aryl, 5- to 10-membered heteroaryl, C3-10cycloalkyl and 4- to 10-membered heterocyclyl, and a group of formula -NR3R4, wherein the alkyl, aryl, heteroaryl, cycloalkyl and heterocyclyl are optionally substituted with one or more groups independently selected from - C(O)Rs, -C(O)NR5R6, -SO2R5, C6-10aryl(C1-6alkyl), 5- to 10-membered heteroaryl(Ci-6alkyl), C3-10cycloalkyl(C1-6alkyl), 4- to 10-membered heterocyclyhC1-6alkyl), urea (- NHC(0)NH2), sulfonamide (-SO2amino), C 1-6alkyl, halo, amino, hydroxy, or amide (for example, C(O)amino) groups, or a combination thereof;

Li, and L4 are independently absent or C1-C4 alkylene;

L3 is absent or is selected from O, NRs, S, C(O), and C1-C4 alkylene;

L2 and L5 are independently absent or selected from C1-C4 alkylene and -SO2-;

Z is selected from -C(O)Rs, -C(O)NR8R9, -SO2R5, hydroxy, C1-C4 alkoxy, -C(O)ORs, -OC(O)R5, and -C(O)R₅.

R1, R2, R3, R4, and R5 are independently selected from H, -C(O)R6, -C(O)NR8R9, -SO2R6, C1-6alkyl, - O(C1-6alkyl), halo, C6-10aryl, C6-10 arylalkyl, 5- to 10-membered heteroaryl, 5- to 10-membered heteroaryl(Ci. 6alkyl), C3-10cycloalkyl, C3-10cycloalkyl(C 1-6alkyl), 4- to 10-membered heterocyclyl, and 4- to 10-membered heterocyclylalkyl;

R6 and R7 are independently selected from H, C1-10alkyl, C2-10alknyl, C6-10aryl, C6-10aryl(C1-6alkyl), 5- to 10-membered heteroaryl, 5- to 10-membered heteroaryl(Ci-ealkyl), C3-10cycloalkyl, C3-10cycloalkyl(C1- 6alkyl), 4- to 10-mcmbcrcd heterocyclyl, and 4- to 10-mcmbcrcd heterocyclylalkyl; and

R_s and Rg are independently selected from H, Ci-ioalkyl, C2-10alknyl, C6-10aryl, C6-10aryl(Ci-ealkyl), 5- to 10-membered heteroaryl, 5- to 10-membered heteroaryl(C1-6alkyl), C 1-10cycloalkyl, C3-10cycloalkyl(C1- 6alkyl), 4- to 10-membered heterocyclyl, and 4- to 10-membered heterocyclylalkyl, or R₈ and Rg together with the nitrogen to which they are attached form a 5- to 7-membered heterocyclyl, optionally including 1, 2 or 3 additional heteroatoms selected from N, O or S, and optionally substituted with 1, 2, or 3 substituents selected from Ci-salkyl, -CH2phenyl, or -C(O)OC1-6alkyl. In some aspects, L1, L3 and L4 are -CH2-.

In some aspects, L2 is -CH2-. In another aspect, L2 is -CH2CH2-. In a further aspect, L2 is -SO2-.

In an aspect, Ai, A2, A3 and A4 are independently selected from

in certain aspects, n is 1.

In certain aspects, Ai, A2, A3 and A4 are independently selected from

In some aspects, Ri is H, C1-6alkyl, or halo, and in certain aspects, R1 is methyl, H, or propyl.

In some aspects, R2 is H. In other aspects, R2 is alkyl, such as methyl; -Oalkyl, such as methoxy; or halo, such as F or Cl.

In some aspects, the L3-Z moiety is selected from -CH2OH; 1-6alkoxy, such as methoxy, ethoxy, or propoxy; or -C(O)NR8R9, where R5 and R6 are as previously defined. In some aspects, Rs and R6 are independently selected from H or C 1-6alkyl, such as methyl, ethyl, propyl, or isopropyl. In certain aspects, R5 is H and R6 is C1-6alkyl. In other aspects, both R5 and R6 independently are C1-6alkyl. In other aspects, R8 and R9 together with the nitrogen to which they are attached form a 5- to 7 membered heterocyclyl, optionally including 1, 2 or 3 additional heteroatoms selected from N, O or S, and optionally substituted with 1, 2, or 3 substituents selected from C1-6alkyl, -CH2phenyl, or -C(O)OC1-6alkyl.

In some aspects, this disclosure provides a compound of formula I-A:

In another aspect, this disclosure provides a compound of formula I-B:

In some aspects, the compound has a formula I-C

In some aspects, the compound has a formula I-D

In some aspects, the compound has a formula I-E

In any aspects, R4 and R5 may each independently selected from H or C1-6alkyl. And in some aspects, R4 and R5 are each independently selected from C1-6alkyl.

In any aspects, Z may be Ci-salkyl or OH. In certain aspects of Formula I or Formula I-D, Z is C1- 8alkyl.

In any aspects, the -L2A2 moiety may be -(Ci-4alkyl)phenyl, such as -CH2phenyl, or - CH2CH2phenyl, optionally substituted with from 1 to 5 substituents selected from Cwalkyl, halo, hydroxy, or a combination thereof.

In any aspects, the -L2A2 moiety may be -(C1-4alkyl)cyclohexane, such as -CH2cyclohexane, or - CH2CH2cyclohexane.

In any aspects, the -L1A1 moiety may be -(C1-4alkyl)cyclohexane, such as -CH2cyclohexane, or -

CH2CH2cyclohexane.

In any aspects, the -L1A1 moiety may be -(C1-4alkyl)heteroaryl, such as

optionally substituted with from 1 to 5, from 1 to 4, or from 1 to 2, substituents selected from Cwalkyl, C1-4alkoxy, halo, hydroxy, or a combination thereof, where R1 is as previously defined.

In particular aspects, the -L1A1 moiety may be -(Ci-4alkyl)heteroaryl, such as

, which is substituted with halogen (preferably F) or C1-4alkoxy (preferably methoxy). In exemplary aspects, the compound is (8-((5-methoxy-l -methyl- lH-indol-3-yl)methyl)-2-phenethyl-2, 8-diazaspiro[4.5]decan-4- yl)methanol or (2-benzyl-8-(cyclohexylmethyl)-2,8-diazaspiro[4.5]decan-4-yl)methanol.

Representative compounds of the Formula I include, but are not limited to:

or a pharmaceutically acceptable salt thereof.

In another aspect, this disclosure provides compounds according to formula II. In some aspects, such compounds can be of a formula:

In another aspect, the compound can be of a formula:

Exemplary compounds according to Formula II include, but are not limited to

or a pharmaceutically acceptable salt thereof.

Compounds according to the present disclosure exhibit improved metabolic stability, cytotoxicity activity, and other properties that lend to their use as therapeutics in treating neurodegenerative diseases/conditions.

III. Pharmaceutical Composition and Administration

In other aspects, this disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of a compound having structural formula I or II, 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 pain in a subject.

In certain aspects, this 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 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 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-thiobutyl amidine, chitosan- thioglycolic acid, chitosanglutathione 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, 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 betalactose, 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 a 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 arc 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 nonaqueous 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 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 arc ointments and creams. Ointments arc 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 drugcontaining reservoir and skin contact 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 poly vinylpyrrolidine (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.

IV. Dosage

A pharmaceutically or therapeutically effective amount of the compound or a composition thereof 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 0.01 mg/kg to 250 mg/kg body weight, more preferably 0.1 mg/kg to 10 mg/kg, in at least one dose. In larger mammals the indicated daily dosage can be from 1 mg to 300 mg, one or more times per day, more preferably in the range of 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 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. Methods

Methods for making compounds according to aspects of the present disclosure are described herein, with particular details being described in FIGS. 2A, 2B, 6A, 6B, 6C, and the Examples provided herein.

Methods for using the compounds according to the present disclosure also are described. In some aspects, the method comprises administering a compound according to the present disclosure to a subject in order to treat a neurodegenerative disease or condition, including age-related disease, protein misfolding/prion diseases, and/or genetic diseases. In some aspects, the neurodegenerative disease or condition is selected from Huntington’s disease (HD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and Alzheimer’s disease. In some particular aspects, compounds of the present disclosure can be used to treat diseases characterized by altered lysosome function and distribution, such as lysosomal storage diseases.

In some aspects, the method can comprise administering the compound, or a composition thereof, using an administration technique known to those in the art, particularly with the benefit of the present disclosure. For example, administration techniques described above for the compositions can be used to administer the compound, including any composition thereof. Dosages described herein can be used in the method.

In particular aspects, the compounds induce autophagic flux in neurons and possess novel mechanisms of action that enable their use in targeting new cellular targets for the treatment of neurodegenerative disease. In some such aspects, the compounds modulate the activity of Lamin A/C and LAMP1 in cells. VI. Overview of Several Aspects

Disclosed herein are compounds of formula I or II, or a pharmaceutically acceptable salt thereof,

wherein

Ai, Aj, A3 and A4 are independently selected from C1-8 alkyl, C6-10aryl, 5- to 10-membered heteroaryl, Cs-iocycloalkyl and 4- to 10-membered heterocyclyl, and a group of formula -NR3R4, wherein the alkyl, aryl, heteroaryl, cycloalkyl and heterocyclyl are optionally substituted with one or more groups independently selected from - C(O)R5, -C(O)NR5R6, -SO2R5, C6-10aryl(Ci-6alkyl), 5- to 10-membered heteroaryl(Ci-6alkyl), C 1-10cycloalkyl(C1-6alkyl), 4- to 10-membered heterocyclyl(Ci-6alkyl), urea (- NHC(O)NH2), sulfonamide (-SO2amino), C1-6alkyl, halo, amino, hydroxy, or amide groups, or a combination thereof;

L1, and L4 are independently absent or C1-C4 alkylene;

L3 is absent or is selected from O, NR5, S, C(O), and C1-C4 alkylene;

L2 and L5 are independently absent or selected from C1-C4 alkylene and -SO2-;

Z is selected from -C(O)R₅, -C(O)NR₈R9, -SO2R5, hydroxy, C1-C4 alkoxy, -C(O)OR₅, -OC(O)R₅, and -C(O)R5;

Ri, R2, R3, R4, and R5 are independently selected from H, -C(O)R6, -C(O)NR,R_y, -SC2R6, C1-6alkyl, - O(Ci-6alkyl), halo, C6-10aryl. C6-10arylalkyl, 5- to 10-membered heteroaryl, 5- to 10-membered heteroaryl(C1- 6alkyl), C 3-10cycloalkyl, C3-10cycloalkyl(C1-6alkyl), 4- to 10-membered heterocyclyl, and 4- to 10-membered heterocyclylalkyl;

R6 and R7 are independently selected from H, C1-10alkyl, C2-10alknyl, C6-10aryl, C6-10aryl(C1-6alkyl), 5- to 10-membered heteroaryl, 5- to 10-membered heteroaryl(Ci-ealkyl), C3-10cycloalkyl, C3-10cycloalkyl(C1- 6alkyl), 4- to 10-membered heterocyclyl, and 4- to 10-membered heterocyclylalkyl; and

R8 and R9 are independently selected from H, C1-10alkyl, C2-10alknyl, C6-10aryl, C6-10aryl(Ci-ealkyl), 5- to 10-membered heteroaryl, 5- to 10-membered heteroaryl(Ci-ealkyl), C3-10cycloalkyl, C3-10cycloalkyl(C1- 6alkyl), 4- to 10-membered heterocyclyl, and 4- to 10-membered heterocyclylalkyl, or Rs and R9 together with the nitrogen to which they are attached form a 5- to 7 membered heterocyclyl, optionally including 1, 2 or 3 additional heteroatoms selected from N, O or S, and optionally substituted with 1 , 2, or 3 substituents selected from C1-6alkyl, -CHiphenyl, or -C(O)OC1-6alkyl. In any or all of the above aspects, Li, L3 and L4 are -CH2-.

In any or all of the above aspects, Ai, A₂, A₃ and A₄ are independently selected from

, where n is from 1 to 5, such as from 1 to 4, or from 1 to 2, and

in certain aspects, n is 1.

In any or all of the above aspects, Ai, A₂, A₃ and A₄ are independently selected from

In any or all of the above aspects, the compound has a structure according to Formula I.

In any or all of the above aspects, the compound has a structure according to formula I-A or I-B

In any or all of the above aspects, the compound has a structure according to formula I-C, I-D or I-E

In any or all of the above aspects, R4 and R5 are each independently selected from H or C 1-8alkyl.

In any or all of the above aspects, Z is OH.

In any or all of the above aspects, Z is C1-8alkyl.

In any or all of the above aspects, the -L2A2 moiety is -(C1-4alkyl) phenyl, optionally substituted with from 1 to 5 substituents selected from C1-4alkyl, halo, hydroxy, or a combination thereof.

In any or all of the above aspects, the -L2A2 moiety is -CH2phenyl or -CH2CH2phenyl, optionally substituted with from 1 to 5 substituents selected from C1-4alkyl, halo, hydroxy, or a combination thereof.

In any or all of the above aspects, the -L2A2 moiety is -(Ci-4alkyl)cyclohexane.

In any or all of the above aspects, the -L2A2 moiety is -CH2cyclohexane, or -CH2CH2cyclohexane.

In any or all of the above aspects, the -L1A1 moiety is-(Ci-4alkyl)cyclohexane

In any or all of the above aspects, the -L1A1 moiety is -CH2cyclohexane, or -CH2CH2cyclohexane.

In any or all of the above aspects, the -L1 Ai moiety is -(C1-4alkyl)heteroaryl, optionally substituted with from 1 to 5 substituents, such as from 1 to 4 substituents, or from 1 to 2 substituents selected from Ci. ialkyl, C ialkoxy, halo, hydroxy, or a combination thereof, where Ri is as previously defined.

In any or all of the above aspects, the -L1A1 moiety is

optionally substituted with from 1 to 5 substituents, such as from 1 to 4 substituent, or from 1 to 2 substituents selected from C1-4alkyl, C1-4alkoxy , halo, hydroxy, or a combination thereof. In any or all aspects, the -L1A1 moiety is

substituted with a halo atom or a Ci-4alkoxy group.

In any or all of the above aspects, the compound is selected from a compound species disclosed herein, or a pharmaceutically acceptable salt thereof. In any or all of the above aspects, the compound has a structure according to Formula II.

In any or all of the above aspects, the compound has a structure according to Formula

In any or all of the above aspects, the compound is selected from

or a pharmaceutically acceptable salt thereof.

Also disclosed herein is a composition comprising a compound according to any or all of the above compound aspects, and a pharmaceutically acceptable diluent or excipient. Also disclosed herein is a method of treating a neurodegenerative disease or condition comprising administering to a subject in need of treatment an effective amount of a compound according to any or all of the above aspects, or a composition thereof.

In any or all of the above aspects, the neurodegenerative disease or condition is selected from Huntington’s disease (HD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and Alzheimer’s disease.

In any or all of the above aspects, the neurodegenerative disease or condition is Alzheimer’s disease.

Also disclosed is the use of a compound according to any or all of the above compound aspects in the manufacture of a medicament for the treatment of a neurodegenerative disease.

VII. Examples

Example 1

General Synthetic Methods: All chemicals for synthetic methods were purchased from Sigma- Aldrich, Alfa-Aesar, Acros Organics, TCI America, Oakwood Chemicals or Chem Impex and were used without further purification unless otherwise noted. Reaction mixtures were purified on a Biotage Isolera One automated chromatography system with silica gel columns. Microwave reactions utilized the Biotage Initiator-i- microwave reactor. Reactions were monitored by TLC (Silica gel 60 F254 Glass Backed plates) and mass spectrometry using LCMS (Agilent 1260 Series automated chromatographic system outfitted with a Thermo Scientific Accucore column (2.1 x 50mm, 2.6 pm particle size)) and an Agilent 6120 quadrupole MS, utilizing a gradient elution mobile phase of 25% ACN:H2O to 95% ACN:HzO over 3 minutes, then holding at 95% ACN:HzO for 2 minutes (0.200 mL/min flow rate, 30 °C column compartment, detection modes: wavelengths of 254 and 280 nm). NMR data were collected on a Bruker AV 500 MHz spectrometer outfitted with a Bruker 5mm 1H19F/BBO S2 Z-gradient probe, and spectra were processed utilizing Mcstrcnova (Mcstrclab Research). Data were recorded at ambient temperature and arc reported as chemical shift (ppm) relative to solvent peak (’H NMR: CDCh = 7.26 ppm, MeOD = 3.31 ppm, D2O — 4.65 ppm; ¹³C NMR: CDCh = 77.16 ppm, MeOD = 49.00). IR data were collected on a ThermoScientific Nicolet IS5 spectrometer outfitted with a ThermoFisher Scientific iD5 ATR. HRMS data were collected by Dr. Furong Sun at the University of Illinois-Urbana Champaign using Waters Q-TOF Ultima ESI.

Cell Culture: HeLa cells stably expressing eGFP-LC3 and mCherry-GFP-LC3 were a gift from Ramnik Xavier at Massachusetts General Hospital, Boston MA. HeLa and A549 cells were purchased from Sigma Aldrich (Ref. #90321013; #86012804). All cells were cultured in DMEM (Corning, #15-013-CV) with 10% FBS (Sigma- Aldrich #2442), 3.6 mM L-glutamine (Corning #25-005-Cl), and lx penicillinstreptomycin (Corning, #3O-OO2-C1). Cultured cells were maintained in a humidified incubator at 37 °C with 5% CO₂. GFP-LC3 Puncta Formation Assay: Compounds tested in the HCS were a curated ChemDiv library provided by the UlCenter for Drug Discovery. HeLa cells expressing eGFP-LC3 were plated at a density of 3,000 cells/well in a black 384-well plate (Corning, #3764) and incubated for 24 hours at 37 °C. After 24 hours, a Biomek NX^P automated liquid handler (Beckman Coulter) transferred compounds (20 pM), DMSO (Corning, #259-50-CQC), chloroquine (CQ, Sigma- Aldrich, #C6628) (20 pM), PI-103 (LC- Laboratories, P-9099) (5 pM), or Baftlomycin Al (BafAl, LC Laboratories, B-1080) (100 nM). The plate was incubated for 4 hours at 37 °C before the media was aspirated using a MultiFlo FX (BioTek, #MFXPW) and 25 pL of 4% paraformaldehyde (PF A) (Electron Microscopy Sciences, #15710) were added. The plate was incubated in the dark at room temperature (RT) for 12 minutes before PFA was aspirated and washed with 50 pL of lx PBS (Corning, 21-040-CM). Following washing, 25 pL Hoechst 33342 nuclear stain (Thermo Scientific, #H3570) at a concentration of 2 pg/mL and incubated in the dark at room temperature (RT) for 10 minutes. The solution was aspirated and 50 pL of lx PBS was added before the plate was sealed using the PlateMax semi-automatic plate sealer (Axygen). The plate was imaged at lOx magnification using the DAPI and FITC filters on the ImageXpress Micro (IXM) XLS automated florescent microscope (Molecular Devices). Images were analyzed using Meta Xpress software in assays. Hits were selected based on assays with a Z’ (Z factor) above 0.4, a % CV of less than 20 %, and a z-score greater 2.199 in two replicates, yielding 312 molecules that meet these requirements.

Subsequent eGFP-LC3 assays run on synthesized molecules were performed in 12-point dose (150 pM to 0.146 pM for 1 analogues, 1000 pM to 0.488 pM for 2 analogues) to generate EC so values. Overtly cytotoxic concentrations (% viability < 40) were excluded. Data are presented as a mean ± SEM as three independent experiments in duplicate.

Dual Reporter Assay: HeLa cells stably expressing mCherry-GFP-LC3 were grown to a density of 3,000 cells/well. The dual reported assay was performed following the same protocol as the eGFP-LC3 Puncta Formation assay. The plates were imaged using three filters: DAPI for nuclear stain, FITC for GFP- LC3 and Texas Red for mCherry. CellProfiler 3.1.9 was used to analyze these images to determine the number of autophagosomes and autolysosomes.

LC3 Immunoblotting: HeLa cells were plated in at a density of 75,000 cells/well in a 24-well dish and left at RT for 1 hour to adhere to the plate before being placed in the incubator at 37 °C for 24 hours. DMSO, Compound la (20 pM), Compound 2a (80 pM) and BafAl (100 nM) were administered by hand, and the plate was returned to the incubator for 4 hours. Cells were lysed using NP-40 Lysis Buffer containing 10 mL of IX TBS (Corning, 46-012-CM), 1 Pierce protease and phosphate inhibitor tablet (ThermoFisher, #A32959), and 1% IGEPAL (Sigma-Aldrich, #56741). Lysate was centrifuged at 12,000 rpm for 30 min at 4 °C to remove cellular debris. The supernatant was combined with 4X NuPAGE LDS Sample Buffer (ThermoFisher, NP0007) and 10X Bolt Sample Reducing Agent (ThermoFisher, B0009) and separated by 10 % SDS-PAGE (120 V, 1.5 h). Protein was transferred onto PVDF membrane (Millipore- Sigma, #IPFL00010) at 25 V for 1 hour. The membrane was blocked with 5% Blotting-grade Blocker (BioRad, #1706404) in TBS spiked with 0.1% Tween-20 (VWR, #97062) and incubated for 1 hour at RT. Membranes were incubated overnight at 4 °C with the primary antibody for LC3 (1:1000) (Cell Signaling Technologies, #2775S) and 0-Actin (1:2000) (Cell Signaling Technologies, #8457L) in 5% Blotting-grade Blocker in TBS-T. The blots were washed three times with TBS-T and incubated with the Anti-Rabbit IgG, HRP-linked secondary antibody (1:4000) (Cell Signaling Technologies, #7074S) for 1 hour at RT. Membranes were washed with TBS-T and incubated with SuperSignal West Pico PLUS Chemiluminescent Substrate (ThermoFisher, #34580) for 3 minutes at RT. Blots were visualized using the c Series Capture Software on the Azure Imaging System. Images were quantified using ImageJ, and data are presented as mean ± SEM of two independent experiments in duplicate.

P70S6K and Phospho- P70S6K Immunoblotting: Slight modifications were made to the procedure for LC3 immunoblotting. Compound 2a was treated at 40 pM. 1% bovine serum albumin (Sigma-Aldrich, A2153) was used for blocking steps. Phosphorylated p70S6K (1:1000) (Cell Signaling Technologies, 9205S) and P- Actin (1:2000) were visualized and stripped using Restore Western Blot Stripping Buffer (Thermo Fisher, #21059). The blot was re -probed with p70S6K (1: 1000) (Cell Signaling Technology, #2708S). Images were analyzed using ImageJ.

Cell Viability Assay: Following the procedure for dose-dependent eGFP-LC3 puncta formation, nuclear counts were determined using the DAPI filter. Nuclear counts were compared to the average DMSO nuclear count to generate a percent viability. Percent viability data were generated as a mean ± SEM of three independent experiments in duplicate. Exemplary results are provided in FIGS. 8-10.

Kinetic Aqueous Solubility Assay: Compounds were diluted with lx PBS to a concentration of 100 pM in a 96-well clear assay plate (Corning, #3628). Diclofenac was used as the soluble control, while dipyridamole was used as the insoluble control. Optical Density readings at 620 ran were taken on a SpectraMax i3x (Molecular Devices) using the Softmax Pro 6.5.1 software. Data are presented as the mean ± SEM of five independent experiments.

Biotinylated Compound Pulldown Assay: HeLa cells were plated at a density of 3.75 x 10⁶ cells/mL in a T-225 flask (Thermo, #159934) in 50 mL of DMEM and then grown for 3 days to confluency. The cells were resuspended by trypsinization then pelleted and stored at -80 °C until ready for cell lysis. Prior to sonication, the pelleted cells were thawed then washed with lx PBS. Cells were then lysed using Pierce IP Lysis Buffer (Thermo, #87788) via sonication. Lysate was centrifuged at 18,000 g for 30 minutes at 4 °C to remove debris and the supernatant was transfer to a clean microcentrifgure tube. Protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo, #23225). 30 pL of Pierce Streptavidin Magnetic Beads (Thermo, #88816) were washed twice with 200 pL of lx PBS. Biotinylated RH1115 (40 pM), biotin acid (40 pM) and DMSO (2 pL) were incubated with the beads while rotating at 4 °C for 2 hours. 500 pg of lysate was then added to the magnetic beads and diluted to 200 pF using the cell lysis buffer. The lysate was left with the beads and compounds for 16 hours before washing twice with 200 pF of 1 X PBS then eluting with elution buffer (Thermo, #1858606). Samples were dried completely using a speed vacuum before proceeding onto sample clean up using S-Trap (Profiti, #CC>2- micro- 10). The dried sample was solubilized in 25 pF of 5% SDS, 50 mM TEAB. Samples were reduced using 20 mM DTT and heated for 5 minutes at 95 °C. Disulfides were then alkylated using 40 mM iodoacetamide and incubated in the dark for 30 minutes. 12% aqueous phosphoric acid at a 1: 10 ratio to yield a final concentration of 1.2% phosphoric acid. Then, 165 pF of S-Trap protein binding buffer (90% aqueous methanol, 100 mM TEAB, pH 7.1) was added to the acidified solution. The prepared solution was then added to the S-Trap microcolumn and centrifuged to capture protein. The column was washed with 150 pL of S-Trap protein binding buffer. 2 pg of trypsin (Thermo, #90058) in 40 pL of digestion buffer (50 mM TEAB) was added to the top of the column and left for 16 hours at 37 °C to digest. Peptides were eluted 40 pL of 50 mM TEAB then 35 pL of 50% ACN, 0.2% formic acid. Samples were dried on speed vacuum then resuspended in 0.1% formic acid for liquid chromatography-mass spectrometry (LC-MS) data collection and analysis.

Liquid Chromatography-Mass Spectrometry (LC-MS) Data Acquisition and Analysis: Resuspended samples were spun down at 14,000 g for 30 minutes before being transferred to polypropylene vials. 0.5 pL of each sample was 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 were analyzed using the following method. First, digested peptides were loaded onto a Thermo NanoViper trap column (75 pm x 20 mm, 3 pm C18, 100 A) (Thermo Fisher Scientific, Bremen, Germany) and washed for 10 minutes with solvent A (0.1% FA in water) at 2 pL/min flowrate. Peptides were then loaded onto an Agilent Zorbax 300SB-C18 column (0.075 x 150 mm, 3.5 pm 300 A) at 5% B (0.1% FA in McCN). Separation was carried out using a 180-min gradient going from 5 to 30% B with a flowrate of 0.25 pL/min. The system was then increased from 30-60% B and from 60-90% B, with each increase in 10-minute period. The system is 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 E7 kV. Full MS scans were collected at a mass resolution of 70,000 with a scan range of 375-2000 m/z. Automatic gain control (AGC) target was set at 1 x 106 for a maximum injection time (IT) of 100 ms. The top ten most intense 10 peaks were selected for MS/MS analysis, with an isolation window of 1.5 m/z. MS/MS spectra were acquired at a resolution of 35,000, ACG target 1 x 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 seconds. Parent ions with charges of 1 and larger than 6 were excluded. All raw data were deposited on MASSIVE.

Raw files were analyzed using Proteome Discoverer 2.3 (Thermo Fisher Scientific, Waltham, MA) using the Sequest HT search engine against the UniProt Homo Sapien database (42,368 gene sequence; downloaded June 12, 2019). Mass error tolerance was set to 10 ppm for precursors, cleaved by trypsin, allowing a maximum of two missed cleavages, with sequence lengths between 6 and 144 amino acids. Fragment masses were searched with a tolerance of ± 0.02 Da. Dynamic modifications included oxidation (M), deamidation (N, Q), and acetylation (N-terminus). Carbamidomethylation was set as a static modification (C). Both peptides and PSMs were set to a target false discovery rate (FDR) of < 0.01 for matches with high confidence. After database searching, protein list was filtered so that only proteins with at least two high- confidence peptide found in 3 replicates remain.

Pulldown Validation Experiments: HeLa cells were plated at a density of 3.75 x 10⁵ cells/mL in a T-225 flask in 50 mL of DMEM and then grown for 3 days to confluency. The cells were resuspended by trypsinization then pelleted and stored at -80 °C until ready for cell lysis. Prior to sonication, the pelleted cells were thawed then washed with 1 x PBS. Cells were resuspended in Pierce IP Lysis Buffer (Thermo, #87788) supplemented with a Pierce protease and phosphatase inhibitor tablet (Thermo, #A32959) and lysed via sonication. Lysate was centrifuged at 18,000 g for 30 minutes at 4 °C to remove debris and the supernatant was transfer to a clean microcentrifuge tube. Protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo, #23225). 40 uL of Pierce Streptavidin Magnetic Beads (Thermo, #88816) were washed twice with 200 pL of IX PBS. Biotm-RHl 115 (50 pM) and biotin acid (50 pM) were incubated with the beads while rotating at 4 °C for 2 hours. 500 pg of lysate was then added to the magnetic beads and diluted to 200 pL using the cell lysis buffer. The lysate was left with the beads and compounds for 16 hours before washing twice with 200 pL of 1 X PBS then eluting with 20 pL IX SDS loading buffer.

Cellular Thermal Shift Assay: A549 cells were plated at a density of 2.5 x 10⁶ cells in a 10 cm dish in DMEM and grown for 16 hours. Cells were then treated with RH1115 (100 pM) or DMSO (50 pL) and incubated for 24 hours. Media was removed and cells were suspended by trypsinization, pelleted washed with PBS, and resuspended to a density of 1.0 x 10⁷ cells/mL in PBS supplemented with Pierce protease inhibitor tablet. 50 pL of each cell suspension was then dispensed into PCR tubes (Bio-Rad, #TBS0201), heated at 50 - 62 ° C in 3-degree increments using a Bio-Rad T100 thermal cycler for 3 minutes, and immediately lysed by freeze-thaw.

Debris was then removed by centrifugation at 18,000 g for 30 minutes at 4 °C and supernatant was probed by immunoblot as previously described for LC3 for the presence of Lamin A/C (1: 1000) and P-actin. Statistical analysis: Statistical analysis was performed using GraphPad Prism version 9.5.0. Data presented in bar graphs utilize the ordinary one-way ANOVA testing. EC50 values were determined using a nonlinear fit function comparing log(agnostic) vs. response - Variable Slope (four parameters). Error bars are mean ± SEM unless otherwise noted. Significance stars correspond to the following P values: ns: P > 0.05; *: P < 0.05; **: P < 0.01; ***: P < 0.001; **»: P < 0.0001.

Protocol for eGFP-LC3 autophagy activation assay and cytotoxicity: HeLa cells stably expressing eGFP-LC3 were plated at a density of 3,000 cells/well in 40 pL/well of media (DMEM (Corning, Cat#15-013-CV), L-Glutamine (Corning, 25-005-CI), lx Pen/Strep (Corning, 30-002-CI), 10% FBS (Sigma, Cat#F2442-500ML)) and grown for 24 hours.

Compounds were pintool transferred (V&P Scientific, FP3NS100) in duplicate 9-point 2-fold dose (32.3 pM to 126 nM, except for 501-A-032 (16.1 pM to 63.0 nM), and AD3128 and AD3127 (8.05 pM to 31.5 nM)) along with controls (DMSO (Sigma, Cat#472301,0.32%) and chloroquine (Sigma, Cat#C6628- 25G, 64.5 pM)). Cells were grown for an additional 4 hours and then fixed by addition of 8 pL/well of 16% paraformaldehyde (Fisher, Cat#50-980-487, 2.6%) and incubated for 12 minutes. All wells were evacuated and washed once with lx PBS (Corning, Cat#21-040-CM, 50 pL/well), and then nuclei and cells were stained by addition of 25 pL/well of Hoechst 33342 (Thermo, Cat#62249, 2.2 pg/mL) and HCS CellMask Near IR (Thermo, Cat#H32722, 2 ng/mL) in lx PBS and incubated for 25 minutes. All wells were evacuated and 25 pL/well of lx PBS was dispensed, plates were sealed and imaged on the ImageXpress HT.ai automated microscope (Molecular Devices) in confocal mode using a 20X water-immersion objective with DAPI, FITC, Cy7 filter sets. Three sites were imaged for each well, with a z-stack of three z-planes imaged at each site. For each z-stack a 2D projection image was generated using maximum intensity projection. IN Carta image analysis software was used to quantify cell number and mean puncta/cell count for each well.

Exemplary results are provided in FIGS. 8-10.

Calculation of activation: Mean puncta/cell is used to determine activation based on the following formula:

Calculation of viability: Cell count is used to determine compound cytotoxicity:

Normalization of puncta count: Mean puncta/cell is normalized by DMSO controls based on the following formula:

Results and Discussion:

To measure autophagy modulation following compound treatment, the eGFP-LC3 puncta formation assay was used as the primary high-content screen (HCS) with the goal of identifying autophagy modulators with a variety of mechanisms of action to discover new cellular targets that improve disease -relevant phenotypes. Pro-LC3 is cleaved by the cysteine protease ATG4 to produce cytosolic LC3-I and upon autophagy activation, ATG7 and ATG3 catalyze the conjugation of phosphatidylethanolamine (PE) to LC3-I by the ATG12-ATG5-ATG16L1 complex to form LC3-II, which is recruited to the autophagosome membrane, and serves as a biomarker for autophagic flux. Compounds that modulate autophagy cause an increase in LC3-II puncta, either by activating autophagy and increasing the number of autophagosomes formed, or by inhibiting autophagosome-lysosome fusion and causing an accumulation of autophagosomes. Both of these possibilities are identified by quantifying the number of green dots (puncta) per cell in the GFP-LC3 assay as an indication of autophagosome numbers. The anti-malarial drug chloroquine (CQ) was used as a positive control in these experiments as it is a late-stage autophagy inhibitor that significantly increases LC3-II accumulation. The optimized HCS reliably performs with Z’ > 0.5 and % CV values <20%, supporting the robust nature of the screen (Table S I). The HCS was performed in duplicate with a library of 10,000 molecules that were obtained from the commercially available ChemDiv collection at a concentration of 20 pM to discover novel autophagy modulators. Of these, 312 hit molecules were identified based on a z-score of > 2.2 for their ability to significantly increase the puncta per cell counts without cytotoxicity in HeLa cells (FIG. 1A and IB).

The 312 compounds were then obtained as a single plate for validation to differentiate activators from late-stage inhibitors using an mCherry-GFP-LC3 dual reporter assay. Upon fusion of the autophagosome to the lysosome, the GFP fluorescence is quenched due to the low pH, leaving the red color from the mCherry. By contrast, GFP fluorescence is not quenched by late-stage inhibitors that prevent autophagosome-lysosomc fusion or lysosomal acidification, and thus red and green fluorescence overlap, resulting in a yellow color. The controls, late-stage inhibitors CQ and bafilomycin Al (BafAl), cause robust accumulation of autophagosomes, observed by the yellow color, as expected. The 312 compounds were successfully classified as activators and late-stage inhibitors, and there was a focus on two activators, la and 2a, based on structure-activity relationships (SAR) observed in the HCS (FIG. 1C and ID). Compound la and analogues with the same core structure reveal that the presence of the indole moiety at R2 can influence activity, as lack of the indole resulted in compounds with no significant activity. The 4-methylpiperazine at R1 was present in both of the active analogues, so synthetic variation at this position was planned. For compound 2a, the tertiary amine in the R3 position was necessary for activity. Replacement of the tertiary amine with an amide resulted in a complete loss of activity, revealing the effects of the basic amine. Synthetic analogues were envisioned to modify the left side of compound 2a to incorporate other tertiary amines to improve potency in the GFP- LC3 puncta formation assay.

To further confirm the conclusion from the dual reporter assay that la and 2a activate autophagy, LC3-II levels were also quantified using western blotting (FIG. IE). Both compound treatments showed significant increases in the levels of LC3-II relative to the DMSO control, indicating autophagy modulation (FIG. IF). To confirm that the increase in LC3-II is not due to late-stage autophagy inhibition, cells were cotreated with the vATPase inhibitor BafAl. If the compounds were late-stage inhibitors, it would be expected to see no additional increase in LC3- II levels with BafAl co-treatment; however, a significant increase in LC3-II levels was observed following treatment with both compounds and a further increase when BafAl was added to prevent LC3-1I turnover through late-stage autophagy inhibition (FIG. IF). These results support the conclusion from the dual reporter assay that la and 2a are true autophagy activators and are enhancing autophagic flux. Next, it was determined if the hits were activating autophagy independently of mTOR. The nature of mTOR in a variety of pathways means that autophagy induction though mTOR inhibition may not be an ideal target for autophagy activation. One of the most well-known biomarkers for mTOR inhibition is the phosphorylation of the mTOR substrate, p70S6K. Treatment with the compounds shows no impact on the phosphorylation levels of p70S6K, unlike the mTOR inhibitor rapamycin (FIG. 1G). This suggests the compounds activate autophagy through a different mechanism of action.

Two synthetic routes were developed to access la and 2a as well as a variety of analogues (FIG. 2A and 2B). The synthesis of la began with a reaction of amidinopyrazole hydrochloride and methylpiperazine under basic conditions to afford the key amidine. Subsequent analogues were prepared using alternative piperazine and aryl derivatives. Preparation of the indole reagent occurred by the straightforward alkylation of 1 H-indole-3-carbaldehyde with propyl bromide and sodium hydride to afford the alkylated indole in good yield. The synthetic strategy to generate pyrimidoazepine intermediates was adapted from Yang and coworkers (FIG. 2A). The synthesis began with the Lewis acid catalyzed ring expansion of 1- boc-4- piperidone with ethyl diazoacetate and BF3-etherate to access the oxoazepane ester 4 intermediate efficiently (71%). This intermediate then underwent a Pinner-type condensation with 4-methylpiperazine-l- carboximidamide (or relevant amidine for analogues) in the presence of sodium ethoxide base. This step enabled the generation of the pyrimidoazepine intermediates 5a-5c in good yield (38-76%). Trifluoroacetic acid was then use to deprotect the BOC-protecting group followed by reductive amination with the 1-propyl- l /7-indole-3-carhaldehydc, catalytic acetic acid, and sodium triacetoxyborohydride to provide la (DS 1040) in 35% over 2 steps. Two additional analogues were synthesized using pyrrolidine and phenyl moieties in the R1 position (lb, 1c), and 4-piperidinecarboxaldehyde was used in place of the aldehyde to generate analogue Id. To prepare 2a, trimethylphosphonoacetate and l-boc-4-piperidinone underwent a Horner- Wadsworth-Emmons reaction using sodium hydride as a base to provide alkene 6 in excellent yield (95%) (FIG. 2B). The alkene was then subjected to 'V-henzyl- 1 -methoxy -N- ((trimethylsilyl)methyl)methanamine and catalytic TFA, to promote a [3+2] cycloaddition which provided spirocycle 7 in good yield (57% (99% BRSM)). Similar to the first synthetic route, a BOC deprotection and reductive amination using 2- pyridinecarboxaldehyde provided the ester intermediate 8a in 32% over 2 steps. Analogues 8b-8e were formed using different aldehydes in the reductive amination step (36-77% over 2 steps). The final reduction of the ester to the primary alcohol using lithium aluminum hydride (LAH) provided 2a (RH1096) and its analogues in good to excellent yields (64-87%). In total, five analogues were created with different aryl or alkyl moieties in the R3 position.

The newly synthesized molecules were tested in the eGFP-EC3 puncta formation assay in 12-point dose to generate EC50 values for each analogue (300 pM to 0.146 pM for 1 analogs, 1000 pM to 0.488 pM for 2 analogues) (FIG. 2C and 2D). The analogues of DS 1040 containing the indole show similar activity in this assay, highlighting the importance of this heterocycle in the molecule’s activity. By contrast, replacing the indole with a 4-pyridine completely eliminates autophagy activation activity. Variation of the R3 position in the 2 analogues resulted in improved activity compared to RH1096. Replacement of the 2- pyridine ring in RH1096 with benzene (2b, RH1103) or cyclohexane (2c, RH1115) resulted in a 3- and 6- fold increase in potency, respectively. Interestingly, modification of the nitrogen in the pyridine from the 2- to 4- position (2d) results in a 2-fold decrease in potency. Percent viability was also measured to ensure the activation response was not coming from cytotoxicity (FIG. 2E). All compounds were found to not be overtly cytotoxic at their EC50 values, indicating that the observed autophagy activation likely was not due to a cell death response. Active analogues were also confirmed to retain autophagy activation in the mCherry-GFP-EC3 dual reporter assay. Next, aqueous kinetic solubility was measured for molecules DS 1040, RH1096, and RH1115. Even at 100 pM concentration, all three molecules were found to be highly soluble, indicating that these molecules are excellent starting points for probe development. Based on these results, compound RH1115 was carried forward for target identification and validation experiments.

To determine the mechanism of action of the most potent hit, RH1115, a biotin-labeled probe was developed to use in a streptavidin bead pulldown assay. The modified synthetic route incorporated a terminal alkyne-containing precursor that was subjected to a copper-catalyzed azide-alkyne cycloaddition reaction with a biotin-tagged azide linker to link biotin to the molecule through formation of a triazole. The terminal alkyne was added to RH1115 in the late stages of the synthetic route, following the ester reduction. The primary alcohol underwent acylation using hex-5-ynoyl chloride and triethylamine to generate the alkyne in good yield (68%). The cycloaddition was performed using the alkyne and Biotin-PEG3 -Azide with catalytic CuSO4 at 90°C for 2 days to reach completion, which resulted in the desired product with a yield of 36%. The eGFP-LC3 puncta formation assay was performed in dose and revealed that Biotin- RH1115 retained activity and was able to significantly increase the puncta/cell count relative to DMSO with an EC50 of 46.2 pM.

A pulldown experiment using RH1115 was performed to identify the target of this molecule to obtain insight into the mechanism of action. Proteins bound to RH1115 were eluted and prepared for mass spectrometry data acquisition and analysis. Through analysis of the resulting data, 13 proteins were identified as being pulled down by the biotinylated compound exclusively, i.e. they were not pulled down with the biotin acid or DMSO negative controls (FIG. 3A). To confirm binding to the compounds, a competition assay was performed in which Biotzn-RHl 115 and excess RH1115 soluble competitor were incubated with the lysate prior to the pulldown. Proteins that were pulled down in the initial assay but were not pulled down in the competition assay confirm the specific interaction with RH1115. Of the initial 13 proteins pulled down, 7 proteins were no longer identified in the mass spectrometry analysis when treated with excess RH1115, providing putative targets for RH1115 (FIG. 3 A and 3B). Proteins with isoforms that were also pulled down by the controls or proteins that were not expressed in neurons were excluded.

Of the remaining three proteins, validation experiments were performed with Lamin A/C, ubiquitin A-52 residue ribosomal protein fusion (UBA-52), and lysosome-associated membrane glycoprotein 1 (LAMP1). Expression tests were performed for all 3 proteins to evaluate protein levels after 24-hour RH1115 treatment (FIG. 3C, S3A and S3B). Results revealed a significant increase in the ratio of glycosylated/non-glycosylated LAMP1 following treatment with RH1115 (FIG. 3C) compared to control treatment. By contrast, expression levels of Lamin A/C were unchanged following treatment with compound RH1115. (FIG. 3D). The interaction of RH1115 with Lamin A/C was then analzyed using a cellular thermal shift assay (CETSA) to measure changes in melting temperature following compound treatment to assess direct binding of the untagged compound to the protein(s) of interest. Both Lamin A and C were found to be stabilized by RH1115, further validating the direct interaction of Lamin A/C with unmodified RH1115 (FIG. 3E).

Next, the mass spectrometry results were further validated through pulldown and competition studies followed by western blot analysis for Lamin A/C, LAMP1, and UBA-52 (FIG. 3F, 3G). UBA-52 was deemed a nonspecific target/false positive for RH1115 because western blot analysis of the pulldown eluent revealed an interaction with the negative control and the inability of the soluble RH1115 competitor to prevent binding of the /ho/m-RH I 1 15 probe. By contrast, Lamin A/C was highly enriched in the eluent after treatment with Biotin-RHl 115, and the interaction with the probe was almost completely prevented by the soluble RH1115 competitor, indicating a specific interaction between Lamin A/C and RH1115 (FIG. 3F). Glycosylated LAMP1 was also pulled down and concentrated in the eluent after treatment with Biotin- RH1115, and this interaction was efficiently prevented by the addition of soluble RH1115 competitor (FIG. 3G), thus confirming that this protein is also a target of compound RH1115.

Given the potent effect of the compounds on autophagy, and the interest in modulation of autophagy as a therapeutic option for neurodegenerative diseases such as AD, the compounds were next evaluated on human iPSC derived neurons (i³Neurons). DIV 10 i³Neurons treated with DS 1040, RH1096, RH1103, and RH1115 for 72 hours starting at DIV 7, showed no overt cytotoxicity as determined by their cell density at the end of the treatment (FIG. 4A).

Examination of LAMP1 staining in these neurons revealed that the compound treatment resulted in a profound effect on lysosome positioning in these i³Neurons (FIG. 4B and 4C). While LAMP1 vesicles, which include a mixture of late endosomes and degradative lysosomes, are normally heterogeneous in their distribution in the neuronal cell body (soma), including larger perinuclear vesicles as well as several smaller peripherally located vesicles, all the compounds resulted a strong perinuclear clustering of these LAMP1 vesicles (FIG. 4B and 4C). In addition to the change in lysosome distribution, treatment with compound RH1115 resulted in increased intensity and mean size of the LAMP1 vesicles (FIG. 4C-E), suggestive of increased LAMP1 levels on the endo-lysosomes. Indeed, the total levels of LAMP1 protein in lysates from neurons treated with compound RH1115 was also found to be increased by 1.5-fold when compared to control neurons (FIG. 4F and 4G).

Given the effect of RH1115 on autophagic flux in HeLa cells and modulation of lysosome positioning, morphology, and potentially its biogenesis (increased LAMP1 localization and recruitment to the perinuclear region), the compound modulated autophagy in neurons was then examined. It was found that i³Neurons treated with RH1115 did indeed exhibit higher LC3-II/LC3-I ratio (FIG. 5A and 5B) as determined by immunoblotting. To evaluate autophagic flux, the nature and distribution of autophagosomes and autolysosomes in i³Neurons derived from iPSCs generated to stably express LC3-GFP-mCherry was examined. Using airy scan live imaging of i³Neurons treated with DMSO, BafAl, or RH1115, the number and fraction of autophagosomes (GFP and mCherry-positive vesicles) and autolysosomes (only mCherry- positive vesicles) in each of these conditions (FIG. 5C) was determined. It was found that while BafAl massively increased the number of autophagosomes per neuronal cell body (FIG. 5C, 5D, S5B, and S5C), RH1115 did not do the same. In contrast, the number of autophagosomes in DMSO and RH1115-treated i³Neurons is far lower. Consistent with BafAl reducing fusion between autophagosomes and lysosomes, the percentage of autolysosomes in BafAl-treated neurons is far less than in RH1115 or DMSO treated i³Neurons (FIG. 5E). In these neurons, RH1115 does not inhibit autophagic flux (as seen by low numbers of autophagosomes), and in fact likely activates autophagic flux as evidenced by LC3-II/LC3-I ratio. Although a dramatic increase in the fraction of autolysosomes is not evident with RH1115 treatment, when examining the absolute number of these vesicles, the autolysosomes are nearly three times as large as in the DMSO treated condition (FIG. 5F) and exhibit increased total mCherry intensity per vesicle, suggestive of potentially fused and larger autolysosomes. Thus, RH1115, in addition to affecting lysosome positioning and biogenesis, likely also increases autophagic flux.

Disease-modifying therapies for AD have remained a challenge in the field of drug discovery. As cases of AD rise globally, there is a need for alternative therapeutic strategies, and selective modulation of autophagy has emerged as a promising approach for the treatment of neurodegenerative and age-related diseases. Current clinical trials have evaluated BACE1 inhibitors, which prevent and clear Af protein aggregates but fail to improve cognitive function in AD patients and have significant side effects, minimizing their effectiveness. Moreover, some of these inhibitors lack the ability to clear tau aggregates in the somatodendritic compartment of neuronal brain cells, which can lead to the formation of tangles inside of neurons which promotes disease progression and eventual patient death. The drawbacks of BACE1 inhibitors demonstrate how target-based methods for drug discovery rely heavily on the modulation of candidate proteins, and even successful modulation of a promising target may not have the hypothesized impact in disease models. By contrast, phenotypic screening provides an unbiased approach to drug discovery. In recent years, phenotypic strategies for drug discovery have become increasingly popular because they can lead to the discovery of small molecules that function through unique mechanisms of action. Subsequent target identification and validation efforts can provide novel targets to affect diseaserelevant phenotypes, which facilitates the development of highly effective, first-in-class therapeutics. Because autophagy has been implicated in a wide range of neurodegenerative diseases, there is an interest in developing optimized autophagy modulators with novel mechanisms to evaluate their ability to ameliorate autophagy defects observed in AD and to determine how the validated protein targets of these modulators are involved in disease pathology and progression.

Lamin A/C was identified as a potential target of RH1115 using an unbiased proteomics approach (FIG. 3B). Nuclear Lamins arc divided into A and B type ligands depending on the structure and expression pattern. The LMNA gene encodes for multiple isoforms of the A type Lamin proteins, including Lamin A and C, formed through alternative splicing and differ from each other by a modified C-terminus and absence of CAAX box in Lamin C. Subsequent validation experiments revealed a direct interaction between Lamin A/C and Biolin-RHl 115, which was confirmed through a competition experiment with soluble RH1115 (FIG. 3F).

Expression levels of Lamin A/C did not significantly change following compound treatment (FIG. 3D), but stabilization of Lamin A/C was observed in the CETSA, which provided further support that a direct interaction occurs between RH1115 and Lamin A/C. Based on these observations, it is likely that modulation of Lamin A and/or C function or localization, but not expression, is important for the activity of RH1115. Lamin A/C are found in the nuclear envelope, where they contribute to several physiological processes, including the maintenance of cellular structure and stability, chromatin regulation, and telomere protection. Numerous diseases, known as Laminopathies, are caused by mutations in the LMNA gene, and recent work has attempted to clarify the effects of Lamins in neurodegeneration. Lamin abnormalities have been found to be present in both Drosophila and human tauopathy, leading to heterochromatin relaxation, DNA damage, and neuronal cell death. Interestingly, despite the importance of Lamin A/C in most cells types, healthy neurons notably have little to no Lamin A expression, which allows for improved flexibility and plasticity. While Mendez-Lopez and co-workers identified Lamin A and C in both control and AD human hippocampal samples, they observed a significant increase of LMNA mRNA and Lamin A/C protein expression in AD samples characterized as high severity cases. More recently, Gil and coworkers noted significant increases of hippocampal neuron expression of Lamin A and a lack of Lamin C in cases of early and late stage AD, while neurons from healthy elderly patients did not show Lamin A expression, suggesting a possible connection between abnormal Lamin A/C expression and AD progression. Although autophagy activation using rapamycin or its analogue temsirolimus has been shown to ameliorate various Laminopathic disease states, the connection of Lamin A/C to autophagy in neurons and neurodegeneration is much less explored. Future studies will investigate the role of Lamin A/C in the regulation of neuronal autophagy in AD and how the interaction of RH1115 with Lamin proteins may be modulating autophagy to assess the potential of Lamin A/C as a novel target for AD therapeutic development.

LAMP1, well-known for its role in the biogenesis and maintenance of lysosomes, was also identified as a target in the proteomics experiments, and validation experiments confirmed a direct interaction between Biotin-RHl 115 and LAMP1 (FIG. 3G). Lysosomes are a contributor to neuronal protein and organelle homeostasis and the clearance of autophagic cargo, and lysosome function has been found to be altered in AD models. Studies in AD mouse neurons have shown accumulation of lysosome-like organelles in amyloid plaques found at swollen axon sites. Additionally, A0 prevents autophagic flux by disrupting normal lysosome distribution in AD models. Treatment with RH1115 also resulted in the change of LAMP1 distribution in the soma of human iPSC neurons (FIG. 4B and 4C) and increased LAMP1 intensity and vesicle size (FIG. 4C and 4E). Retrograde movement of lysosomes to a perinuclear location has been suggested to facilitate autophagosome-lysosome fusion and autophagy induction by compound treatment or transcription factor overexpression has been shown to increase LAMP1 protein levels and perinuclear clustering of lysosomes. Interestingly, a significant increase in the ratio of glycosylated to nonglycosylated LAMP1 following treatment with RH1115 (FIG. 3C) was noted. Maturation of LAMP1 consists of glycosylation of the protein to form a stable glycoprotein layer that maintains the integrity of the lysosome and may indirectly modulate the fusion of lysosomes with phagosomes, autophagosomes, or the plasma membrane. While decreases in protein glycosylation have been observed in AD models, the results are not consistent across regions of the brain, and the glycosylation of LAMP1 specifically has not been extensively studied. Abnormal LAMP1 glycosylation has also been observed in another neurodegenerative disease, Niemann-Pick type Cl (NPC), which is a lysosomal storage disease that affects cholesterol trafficking due to mutations in the NPC1 gene. Both NPC and AD have common pathological features, including A0 accumulation and neurofibrillary tangles, but one of the most significant similarities is the contribution of polymorphisms in the apolipoprotein E (ApoE) for the progression of both diseases. Along with the increase in glycosylation of LAMP1, direct interaction between RH1115 and LAMP1 was observed (FIG. 3G). Taken together, these results suggest that treatment with RH1115 may rescue dysfunctional LAMP1 and restore autophagic flux by promoting lysosome movement and autophagosome-lysosome fusion in neurons. Changes in glycosylation pattern and the contribution of this increase in glycosylated LAMP1 to the observed phenotype will be further investigated.

In summary, a phenotypic assay was implemented to identify molecules that increase the number of autophagosomes, and it was confirmed that the prioritized molecules are mTOR-independent autophagy activators. Through synthetic optimization, more potent analogues of the initial hits could be prepared and thus develop a biotinylated version of the RH1115 analogue that retained its biological activity and phenotypic properties to enable target identification studies that revealed two protein targets of interest with significant implications in neurodegeneration. Finally, it was determined that this compound alters positioning of lysosomes and increases autophagic flux in human iPSC-derived neurons. Given the highly polarized and unique morphology of neurons, and the link between lysosome transport and maturation in these cells, a small molecule that mobilizes endo-lysosomes in neurons could be especially impactful in neurodegenerative diseases. Future studies will evaluate the effects of these small-molecule autophagy modulators on lysosomal pathology and A0 generation in neuronal models of AD to provide additional insight into the therapeutic potential of the identified protein targets, which may reveal alternative biomarkers for clinical evaluation and enable the development of new therapeutic strategies to treat ncurodcgcncration.

Example 2 Synthetic Methods:

General Procedure A: To a flame dried flask equipped with a stir bar was added crude methyl 2- benzyl-2,8-diazaspiro[4.5]decane-4-carboxylate (1.0 eq), the desired aldehyde (5.0 eq), and trifluoroacetic acid (TFA) (12 eq) in methanol (0.5M). The solution was heated to 50 °C for 6 hours before the addition of sodium borohydride (5.0 eq). The reaction was stirred for 12 hours before being removed from heat. The reaction was quenched with H2O and was concentrated under vacuum. General Procedure B: To a flame dried flask equipped with a stir bar was added palladium on carbon (10%) (0.2 eq) and a slurry was formed with a small volume of methanol. The desired carboxylate was then added as a solution in methanol (0.1M) and the reaction was heated to 35

°C for 6 hours. The reaction was filtered through celite and then concentrated under vacuum. The crude product was then dissolved in dichloromethane (0. IM) and the desired sulfonyl chloride was added (1.1 eq) along with triethylamine (1.2 eq). The reaction was heated to 35 °C for 12 hours before being removed from heat. The reaction was extracted with ethyl acetate three times and the organic layer was subsequently dried with sodium sulfate and concentrated under vacuum.

General Procedure C: To a flame dried flask equipped with a stir bar was added palladium on carbon (10%) (0.2 eq) and a slurry was formed with a small volume of methanol. The desired carboxylate was then added as a solution in methanol (0.1M) and the reaction was heated to 35

°C for 6 hours. The reaction was filtered through celite and then concentrated under vacuum. The crude product was then dissolved in acetonitrile (0.1M) and the alkyl halide was added (1.1 eq) along with K2CO3 (2 eq). The reaction was heated to 60 °C for 12 hours before being removed from heat. The reaction was extracted with ethyl acetate three times and the organic layer was subsequently dried with sodium sulfate and concentrated under vacuum.

General Procedure D: To a flame dried flask equipped with a stir bar was added palladium on carbon (10%) (0.2 eq) and a slurry was formed with a small volume of methanol. The desired carboxylate was then added as a solution in methanol (0.1M) and the reaction was heated to 35 °C for 6 hours. The reaction was filtered through celite and then concentrated under vacuum. The crude product was then dissolved in methanol (0.5M) and the desired aldehyde (5.0 eq), and TFA (12 eq) were added. The reaction was heated to 50 °C for 6 hours before the addition of sodium borohydride (5.0 eq). The reaction was stirred for 12 hours before being removed from heat. The reaction was quenched with H2O and was concentrated under vacuum.

General Procedure E: To a flame dried flask equipped with a stir bar was added LiAlH4 and a slurry was formed using a small amount of THF and cooled to 0 °C. The desired carboxylate was then added dropwise as a solution in THF (0.1M) and the reaction was stirred for 10-15 minutes, after which the reaction was quenched with H2O. The reaction mixture was filtered through celite and concentrated under vacuum.

General Procedure F: To a flame dried flask equipped with a stir bar was added LiAlH4 and a slurry was formed using a small amount of THF. The desired carboxylate was then added dropwise as a solution in THF (0.1M). The reaction was stirred for 2 hours at room temperature, after which the reaction was quenched with H2O. The reaction mixture was filtered through celite and concentrated under vacuum. General Procedure G: To a flame dried flask equipped with a stir bar was added sodium ethoxide (2.0 eq), 1 -(tert-butyl) 4-ethyl 5-oxoazepane-l,4-dicarboxylate (1.0 eq), the appropriate amidine (1.5 eq.) and ethanol (0.15 M). The mixture was heated to 75 °C for 3.5 hours before being quenched with water and extracted with DCM.

General Procedure H: To a reaction vessel equipped with a magnetic stir bar was added a 1 : 1 mixture of TFA:DCM (0.20 M) and the substituted Boc-azepine. The reaction stirred for 1 hour at rt. The mixture was concentrated under vacuum and was used crude in the next step.

General Procedure I: To a flame dried flask equipped with a stir bar was added crude azepine ( 1.0 eq), and desired aldehyde (5.0 eq) in 2% AcOH:DMF (0.05M). The solution was heated to 75° C for 2 hours before addition of sodium triacetoxyborohydride (5.0 eq). The reaction stirred for 16 hours before being removed from the heat. The reaction was quenched with H2O, and the aqueous layer was then washed in triplicate with DCM. The organic layer was concentrated under vacuum.

General Procedure J: To a flame dried flask equipped with a stir bar was added crude methyl 2- benzyl-2,8-diazaspiro[4.5]decane-4-carboxylate (1.0 eq), and desired aldehyde (5.0 eq) in 2% AcOH:DMF (0.05M). The solution was heated to 75° C for 1 hour before addition of sodium triacetoxyborohydride (5.0 eq). The reaction stirred for 3 hours before being removed from the heat and stirred for 16 hours at rt. The reaction was quenched with FEO, and the aqueous layer was then washed in triplicate with EtOAc. The recovered aqueous layer was concentrated under vacuum.

General Procedure K: To a microwave vial equipped with a stir bar was added LiAlH4 (2.0 eq) as a solution in anhydrous THF (0.20 M). Desire carboxylate (1.0 eq) was added dropwise in THF (0.20 M). The reaction vessel was sealed and stirred for 2h at 0° C. EtOAc (2 mL) and H2O (1 mL) were added to quench the reaction. The mixture was passed through a short path column of Celite before being concentrated under vacuum.

General Procedure L:

To a flame dried flask equipped with a stir bar was added crude methyl 2-benzyl-2,8- diazaspiro[4.5]decane-4-carboxylate (1.0 eq), the desired aldehyde (5.0 eq), and trifluoroacetic acid (TFA) (12 eq) in methanol (0.5M). The solution was heated to 50 °C for 6 hours before the addition of sodium borohydride (5.0 eq). The reaction was stirred for 12 hours before being removed from heat. The reaction was quenched with H2O and was extracted 3x with EtOAc and NaHCO s. The organic layer was dried with sodium sulfate and subsequently concentrated under vacuum.

General Procedure M: To a flame dried flask equipped with a stir bar was added palladium on carbon (10%) (0.2 eq) and a slurry was formed with a small volume of methanol. The desired carboxylate was then added as a solution in methanol (0.1M) and the reaction was heated to 35 °C for 6 hours. The reaction was filtered through celite and then concentrated under vacuum. The crude product was then dissolved in dichloromethane (0.1M) and the desired sulfonyl chloride was added (1.1 eq) along with triethylamine (1.2 eq). The reaction was heated to 35 °C for 12 hours before being removed from heat. The reaction was extracted with ethyl acetate 3x and the organic layer was subsequently dried with sodium sulfate and concentrated under vacuum.

General Procedure N: To a flame dried flask equipped with a stir bar was added palladium on carbon (10%) (0.2 eq) and a slurry was formed with a small volume of methanol. The desired carboxylate was then added as a solution in methanol (0.1M) and the reaction was heated to 35 °C for 6 hours. The reaction was filtered through celite and then concentrated under vacuum. The crude product was then dissolved in acetonitrile (0.1M) and the alkyl halide was added (1.2 eq) along with K2CO3 (2 eq). The reaction was heated to 60 °C for 12 hours before being removed from heat. The reaction was extracted with ethyl acetate 3x and the organic layer was subsequently dried with sodium sulfate and concentrated under vacuum.

General Procedure O: To a flame dried flask equipped with a stir bar was added LiAlH4(1.5 eq) and a slurry was formed using a small amount of THF and cooled to 0 °C. The desired carboxylate was then added dropwise as a solution in THF (0.1M) and the reaction was stirred for 5 minutes, after which the reaction was quenched with H2O. The reaction mixture was filtered through celite and concentrated under vacuum.

General Procedure P: To a flame dried flask equipped with a stir bar was added LiAlH4 and a slurry was formed using a small amount of THF. The desired carboxylate was then added dropwise as a solution in THF (0.1M). The reaction was stirred for 2 hours at room temperature, after which the reaction was quenched with H2O. The reaction mixture was filtered through celite and concentrated under vacuum.

General Procedure Q: To a flame dried flask equipped with a stir bar was added the desired carboxylate (1.0 eq) and TFA (12 eq) in dichloromethane (IM). The reaction was stirred for 3 hours at room temperature and was extracted 3x with DCM and NaHCO ;. The organic layer was dried with sodium sulfate and subsequently concentrated under vacuum and used crude in the next step.

General Procedure R: To a flame dried flask equipped with a stir bar was added the desired carboxylate (1.0 eq), the desired aldehyde, (1.5 eq), and glacial acetic acid (1.0 eq) in DCM (0.1 M). The solution was heated to 35 °C and stirred for 1 hour before the addition of sodium triaceto xyborohydride (1.5 eq). The reaction was stirred for 12 hours before being removed from heat. The reaction was quenched with H2O and was extracted 3x with EtOAc and NaHCCh. The organic layer was dried with sodium sulfate and subsequently concentrated under vacuum.

General Procedure S: To a flame dried flask equipped with a stir bar was added NaH (1.2 eq) as a 60% dispersion in mineral oil and was subsequently suspended in ACN (0.4M). The desired aldehyde (1.0 eq) was then added and allowed to react for 30 minutes. The desired alkyl halide (5.0 eq) was added and the reaction was stirred for 1 hour. The reaction was quenched with H2O and was extracted 3x with EtOAc and NaHCCh. The organic layer was dried with sodium sulfate and subsequently concentrated under vacuum.

Tert-butyl 4-(2-methoxy-2-oxoethylidene)piperidine-l-carboxylate: To a flame dried flask equipped with a stir bar was added NaH (1.2 eq) as a 60% dispersion in mineral oil and was subsequently suspended in DMF (0.1M). The solution was cooled to 0 °C and then trimethyl phosphonoacetate (1.5 eq) was added dropwise and allowed to react for 30 minutes. Tert-butyl 4- oxopiperidine- 1 -carboxylate (1 g, 1 eq) was then added and the reaction was allowed to stir at room temperature for 6 hours. Reaction was stirred for 6 hours at room temperature and was subsequently quenched with aqueous NH4CL The reaction was extracted with ethyl acetate three times and the organic layer was subsequently dried with sodium sulfate and concentrated under vacuum. The product was purified using column chromatography (0-20% EtOAc:hexane over 7 minutes) to afford the desired product as a white solid: 1.198 g (94%). 'H NMR (500 MHz, CDCI3) 6 5.44 (s, 1H), 3.40 (s, 3H), 3.23 (t, J = 5.9 Hz, 2H), 3.19 (t, J = 5.9 Hz, 2H), 2.66 (t, J = 6.1 Hz, 2H), 2.01 (t, J = 6.0 Hz, 2H), 1.19 (s, 9H). ¹³C NMR (125 MHz, CDCk) 5 166.15, 157.85, 154.13, 114.56, 79.33, 50.52, 44.28, 36.12, 29.24, 28.08.

8-(tert-butyl) 4-methyl 2-benzyl-2,8-diazaspiro[4.5]decane-4,8-dicarboxylate: To a flame dried flask equipped with a stir bar was added solid Tert-butyl 4-(2-methoxy-2- oxoethylidene)piperidine-l- carboxylatc (1.0 eq) and LiF (3.0 eq). N-(Mcthoxymcthyl)-N- (trimcthylsilyhncthyObcnzylaminc was then added (2.0 eq) and the reaction was heated to 140 °C for 24 hours. The reaction was removed from heat and filtered to remove excess LiF. The product was purified using column chromatography (0-100% DCM:ACN over 10 minutes) to afford the desired product as a yellow oil 2.1986 g (84%) 'H NMR (500 MHz, CDC13) 6 7.27 (d, J = 6.7 Hz, 4H), 7.23 - 7.18 (m, 1H), 3.82 (s, 2H), 3.63 (s, 3H), 3.62 (d, J = 4.2 Hz, 2H), 2.94 (t, J = 8.6 Hz, 1H), 2.83 (d, J = 10.3 Hz, 1H), 2.78 (t, J = 8.8 Hz, 2H), 2.69 (t, J = 8.0 Hz, 1H), 2.31 (d, J = 9.2 Hz, 1H), 1.81 - 1.72 (m, 1H), 1.62 (d, J = 13.2 Hz, 1H), 1.51 (d, J = 12.5 Hz, 1H), 1.41 (s, 9H), 1.35 (d, J = 10.6 Hz, 1H). ¹³C NMR (125 MHz, CDC13) 5 173.23, 154.77, 138.83, 128.42, 128.27, 126.98, 79.37, 61.93, 59.84, 55.27, 53.42, 51.46, 44.03, 41.32, 36.88, 32.48, 28.40.

Methyl 2-benzyl-2,8-diazaspiro[4.5]decane-4-carboxylate: To a flame dried flask equipped with a stir bar was added 8-(tert-butyl) 4-methyl 2-benzyl-2,8-diazaspiro[4.5]decane-4,8- dicarboxylate (2.1986 g, 5.66 mmol) and TFA (12 eq, 6.79 mL) in dichloromethane (IM). Reaction was stirred for 3 hours at room temperature and was subsequently concentrated under vacuum and used crude in the next step.

AD-2034: Following General Procedure A, methyl 2-benzyl-2,8-diazaspiro[4.5]decane-4- carboxylate (416.3 mg, 1.444 mmol) and cyclohexane carboxaldehyde (809.87 mg, 7.22 mmol) were used. The product was purified using column chromatography (0-10% DCM:MeOH over 10 minutes) to afford the desired product as a yellow oil 453.1 mg (82%) 'H NMR (500 MHz, Methanol-t/4) 8 7.35 - 7.28 (m, 4H), 7.27 - 7.22 (m, 1H), 3.69 (s, 3H), 3.66 (d, J = 3.8 Hz, 2H), 3.03 (d, J = 14.5 Hz, 2H), 2.95 - 2.90 (m, 1H), 2.86 - 2.75 (m, 3H), 2.45 (d, J = 9.6 Hz, 4H), 2.38 (s, 1H), 2.06 (td, J = 13.1, 4.1 Hz, 1H), 1.81 - 1.69 (m, 6H), 1.69 - 1.57 (m, 4H), 1.28 (qt, J = 12.5, 3.4 Hz, 2H), 1.18 (qt, J = 12.0, 2.6 Hz, 1H), 0.93 (qd, J = 13.3, 4.0 Hz, 2H). ¹³C NMR (125 MHz, Methanol-^) 8 172.86, 138.07, 128.59, 128.03, 126.97, 64.33, 62.08, 59.65, 54.63, 52.78, 51.25, 50.76, 50.66, 42.73, 34.98, 33.85, 31.16, 30.87, 25.97, 25.49.

AD-1016: Following General Procedure B, AD-2034 (45.8 mg, 0.119 mmol) and benzenesulfonyl chloride (20.98 mg, 0.1188 mmol) were used. The product was purified using column chromatography (0-10% DCM:MeOH over 15 minutes) to afford the desired product as an orange solid: 19.8 mg (38%) 'H NMR (500 MHz, CDCh) 5 7.86 (d, J = 7.3 Hz, 2H), 7.61 (t, J = 7.5 Hz, 1H), 7.55 (t, J = 7.6 Hz, 2H), 3.60 - 3.57 (m, 1H), 3.55 (s, 4H), 3.47 (dd, J = 10.4, 6.4 Hz, 1H), 3.26 (s, 2H), 2.72 (t, J = 7.1 Hz, 1H), 2.06 (d, J = 4.1 Hz, 2H), 1.98 (s, 1H), 1.88 (s, 2H), 1.68 (q, J = 15.7 Hz, 6H), 1.42 (d, J = 13.7 Hz, 3H), 1.25 (s, 1H), 1.15 (dq, J = 24.3, 13.0 Hz, 5H), 0.83 (t, J = 11.6 Hz, 3H). ¹³C NMR (125 MHz, CDCh) 5 171.50, 136.70, 132.79, 129.05, 127.45, 65.58, 55.03, 52.19, 51.83, 51.14, 50.76, 48.28, 44.00, 35.05, 31.89, 30.09, 26.69, 26.08.

AD-1059: Following General Procedure E, AD-1016 (35.95 mg, 0.083 mmol) and LiAlH4 (4.73 mg, 0.1245 mmol) were used. The product was purified using column chromatography (0-10% DCM:MeOH over 13 minutes) to afford the desired product as an orange solid: 12.9 mg (17%). 'H NMR (500 MHz, CDCh) 6 7.84 (d, J = 7.1 Hz, 2H), 7.61 (t, J = 7.4 Hz, 1H), 7.54 (t, J = 7.5 Hz, 2H), 3.68 (dd, J = 11.1, 6.1 Hz, 1H), 3.64 (s, 1H), 3.48 - 3.37 (m, 4H), 3.17 (dd, J = 10.3, 7.1 Hz, 1H), 3.06 (d, J = 10.1 Hz, 1H), 2.96 - 2.85 (m, 2H), 2.36 (s, 2H), 2.23 (s, 2H), 2.12 - 1.98 (m, 2H), 1.93 (s, 1 H), 1.79 (d, J = 12.1 Hz, 2H), 1.71 (d, J = 13.0 Hz, 2H), 1.65 (d, J = 11.9 Hz, 1H), 1.58 (s, 1H), 1.31 (d, J = 15.2 Hz, 2H), 1.27 - 1.22 (m, 3H), 1.22 - 1.08 (m, 3H), 0.91 (q, J = 13.4 Hz, 3H). ¹³C NMR (125 MHz, CDCh) 8 136.51, 132.86, 129.10, 127.32, 70.53, 64.95, 60.57, 55.92, 51.22, 50.94, 49.13, 41.65, 34.22, 33.25, 31.70, 27.15, 26.22, 25.79.

TP-1010: Following General Procedure B, AD- 2034 (107.98 mg, 0.281 mmol) and tosylsulfonyl chloride (58.91 mg, 0.309 mmol) were used. The product was purified using column chromatography (0-10% DCM:MeOH over 15 minutes) to afford the desired product as an orange solid: 70.48 mg (56%) 'H NMR (500 MHz, CDCh) 5 7.72 (d, J = 7.8 Hz, 2H), 7.33 (d, J = 7.9 Hz, 2H), 3.56 (s, 3H), 3.55 - 3.51 (m, 1H), 3.45 (dd, J = 10.4, 6.8 Hz, 1H), 3.23 (t, J = 8.3 Hz, 2H), 2.70 (t, J = 7.3 Hz, 1H), 2.56 (d, J = 20.1 Hz, 2H), 2.43 (s, 3H), 2.05 (d, J = 19.9 Hz, 2H), 1.93 (d, J = 31.3 Hz, 2H), 1.80 (s, 1H), 1.68 (q, J = 16.2 Hz, 6H), 1.48 - 1.32 (m, 3H), 1.24 (d, J = 6.3 Hz, 1H), 1.14 (dq, J = 24.2, 12.5 Hz, 4H), 0.82 (q, J = 11.2 Hz, 2H). ¹³C NMR (125 MHz, CDCh) 8 171.52, 143.54, 133.74, 129.66, 127.55, 65.61, 55.08, 52.27, 51.81, 51.20, 50.77, 48.25, 44.01, 35.09, 31.90, 30.20, 29.71, 26.72, 26.10, 21.57.

AD-1069: Following General Procedure E, TP-1010 (70.48 mg, 0.083 mmol) and LiAlH4 (8.94 mg, 0.2355 mmol) were used. The product was purified using column chromatography (0-10% DCM:MeOH over 13 minutes) to afford the desired product as an orange solid: 33.7 mg (51%). 'H NMR (500 MHz, CDCh) 6 7.70 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 7.9 Hz, 2H), 3.65 (dd, J = 11.8, 6.4 Hz, 1H), 3.42 (dd, J = 10.1, 7.7 Hz, 1H), 3.36 (t, J = 9.4 Hz, 2H), 3.15 (dd, J = 10.1, 6.9 Hz, 1H), 3.02 (d, J = 10.1 Hz, 1H), 2.76 (d, J = 11.5 Hz, 2H), 2.42 (s, 3H), 2.25 (d, J = 6.9 Hz, 2H), 2.11 (s, 2H), 1.97 (p, J = 7.1 Hz, 1H), 1.74 (d, J = 11.5 Hz, 3H), 1 .66 (dd, J = 22.7, 12.3 Hz, 4H), 1 .25 (d, J = 14.9 Hz, 3H), 1 .23 - 1 .05 (m, 4H), 0.86 (q, J = 12.1 Hz, 2H). ¹³C NMR (125 MHz, CDCh) 8 143.64, 133.51, 129.71, 127.43, 70.53, 65.24, 60.61, 55.93, 51.22, 50.91, 49.37, 48.70, 41.84, 34.50, 33.91, 31.78, 29.70, 27.79, 26.41, 25.92, 21.58.

AD-1146: To a flame dried flask equipped with a stir bar was added palladium on carbon (10%) (69.7 mg, 0.66 mmol) and a slurry was formed with a small volume of methanol. The AD-2034 (126.9 mg, 0.328 mmol) was then added as a solution in methanol (0. IM) and the reaction was heated to 35 °C for 6 hours. The reaction was filtered through celite and then concentrated under vacuum. The crude product was then dissolved in tetrahydrofuran (0.1M) and 1-methyl-lH- imidazole-4-sulfonyl chloride was added (65.0 mg, 0.36 mmol) along with 4-dimethyl aminopyridine (DMAP) (48 mg, 0.313 mmol). The reaction was heated to 35 °C for 12 hours before being removed from heat. The reaction was extracted with ethyl acetate three times and the organic layer was subsequently dried with sodium sulfate and concentrated under vacuum. The product was purified using column chromatography (0-10% DCM:MeOH over 10 minutes) to afford the desired product as a yellow solid: 20.7 mg (14%). 'H NMR (500 MHz, CDCh) 8 7.51 (d, J = 10.2 Hz, 2H), 7.30 (d, J = 4.4 Hz, 1H), 3.77 (s, 3H), 3.70 (s, 1H), 3.66 (s, 3H), 3.65 - 3.63 (m, 1H), 3.63 - 3.61 (m, 1H), 3.58 (d, J = 10.8 Hz, 1H), 2.84 - 2.71 (m, 4H), 2.37 (dd, J = 25.3, 8.5 Hz, 2H), 2.26 (d, J = 7.0 Hz, 3H), 2.19 (t, J = 12.3 Hz, 1H), 2.03 - 1.93 (m, 1H), 1.80 (d, J = 13.7 Hz, 1H), 1.76 - 1.62 (m, 8H), 1.54 - 1.44 (m, 3H), 1.38 (t, J = 18.0 Hz, 2H), 1.28 - 1.09 (m, 6H), 0.88 (qt, J = 14.3, 8.1 Hz, 3H). ¹³C NMR (125 MHz, CDCh) 6 171.19, 139.30, 137.77, 128.48, 128.34, 127.09, 124.78, 65.14, 64.78, 59.73, 55.27, 52.89, 51.99, 51.77, 51.20, 50.63, 48.27, 43.64, 43.14, 34.58, 34.11, 34.02, 31.59, 31.41, 29.68, 29.17, 26.45, 26.19, 25.89, 25.74.

AD-1147: Following General Procedure E, AD-1146 (20.7 mg, 0.047 mmol) and LiAlH4 (2.69 mg, 0.081 mmol) were used. The product was purified using column chromatography (0-10% DCM:MeOH over 13 minutes) to afford the desired product as an orange solid: 10.4 mg (54%). 'H NMR (500 MHz, CDCh) 5 7.70 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 7.9 Hz, 2H), 3.65 (dd, J = 11.8, 6.4 Hz, 1H), 3.42 (dd, J = 10.1, 7.7 Hz, 1H), 3.36 (t, J = 9.4 Hz, 2H), 3.15 (dd, J = 10.1 , 6.9 Hz, 1 H), 3.02 (d, J = 10.1 Hz, 1 H), 2.76 (d, J = 1 1.5 Hz, 2H), 2.42 (s, 3H), 2.25 (d, J = 6.9 Hz, 2H), 2.11 (s, 2H), 1.97 (p, J = 7.1 Hz, 1H), 1.74 (d, J = 11.5 Hz, 3H), 1.66 (dd, J = 22.7, 12.3 Hz, 4H), 1.25 (d, J = 14.9 Hz, 3H), 1.23 - 1.05 (m, 4H), 0.86 (q, J = 12.1 Hz, 2H). ¹³C NMR (125 MHz, CDCh) 5 143.64, 133.51, 129.71, 127.43, 70.53, 65.24, 60.61, 55.93, 51.22, 50.91, 49.37, 48.70, 41.84, 34.50, 33.91, 31.78, 29.70, 27.79, 26.41, 25.92, 21.58.

AD-2040: Following General Procedure C, AD-2034 (141.5 mg, 0.368 mmol) and phenethyl iodide (93.9 mg, 0.4058 mmol) were used. The product was purified using column chromatography (0-10% DCM:MeOH over 10 minutes) to afford the desired product as an yellow oil: 87.8 mg (60%). 'H NMR (500 MHz, Methanol-^) 5 7.29 - 7.15 (m, 5H), 3.70 (s, 3H), 3.04 (dd, J = 9.7, 7.5 Hz, 1H), 2.89 (d, J = 15.2 Hz, 2H), 2.87 - 2.66 (m, 7H), 2.45 (d, J = 9.7 Hz, 1H), 2.29 (t, J = 8.8 Hz, 3H), 2.19 (d, J = 7.3 Hz, 1H), 2.00 (td, J = 12.9, 4.2 Hz, 1H), 1.81 - 1.63 (m, 6H), 1.62 - 1.49 (m, 3H), 1.34 - 1.26 (m, 2H), 1.20 (dtt, J = 25.0, 12.6, 3.3 Hz, 2H), 0.92 (qd, J = 12.2, 3.0 Hz, 2H). ¹³C NMR (125 MHz, Methanol-rW) 6 172.81, 139.79, 128.32, 128.09, 125.84, 65.05, 62.55, 57.94, 54.94, 52.84, 51.44, 50.77, 50.75, 42.92, 35.70, 34.48, 34.36, 31.51, 31.48, 26.17, 25.68.

AD-1190: Following General Procedure F, AD-2040 (85.3 mg, 0.214 mmol) and LiAfiD (16.3 mg, 0.428 mmol) were used. The product was purified using column chromatography (0-10% DCM:MeOH on neutral alumina over 8 minutes) to afford the desired product as a yellow oil: 66.7 mg (84%). 'H NMR (500 MHz, Methanol-^) 57.30 - 7.16 (m, 5H), 3.71 (dd, J = 10.7, 5.7 Hz, 1H), 3.54 (dd, J = 10.7, 7.5 Hz, 1H), 3.16 (dd, J = 9.9, 7.2 Hz, 1H), 2.91 (dd, J = 17.7, 11.1 Hz, 3H), 2.86 - 2.76 (m, 4H), 2.56 (dd, J = 26.4, 10.0 Hz, 2H), 2.31 (t, J = 8.8 Hz, 3H), 2.28 - 2.18 (m, 1H), 2.04 (p, J = 7.6 Hz, 1H), 1.91 - 1.82 (m, 1H), 1.82 - 1.67 (m, 6H), 1.67 - 1 .55 (m, 4H), 1.34 - 1 .26 (m, 2H), 1 .26 - 1 .14 (m, 2H), 0.93 (qd, J = 1 1 .8, 2.8 Hz, 2H). ¹³C NMR (125 MHz, Methanol-J4) 8 139.35, 128.30, 128.16, 125.96, 65.06, 63.40, 60.68, 58.24, 56.80, 51.47, 50.89, 49.21, 40.76, 35.98, 34.35, 34.10, 31.49, 30.00, 26.17, 25.67.

AD-2051: Following General Procedure D, AD-2034 (442.5 mg, 1.15 mmol) and cyclohexyl carboxaldehyde (645.4 mg, 0.4058 mmol) were used. The product was purified using column chromatography (0-10% DCM:MeOH over 10 minutes) to afford the desired product as an yellow oil: 156.4 mg (35%). ^JH NMR (500 MHz, Methanol-^) 5 3.70 (s, 3H), 3.09 (dd, J = 25.0, 11.6 Hz, 2H), 3.00 (d, J = 8.7 Hz, 1H), 2.90 - 2.85 (m, 2H), 2.85 - 2.79 (m, 2H), 2.55 (d, J = 7.0 Hz, 4H), 2.50 (d, J = 9.7 Hz, 2H), 2.40 (qd, J = 11.9, 7.1 Hz, 2H), 2.14 - 2.04 (m, 1H), 1.86 - 1.75 (m, 7H), 1.75 - 1.61 (m, 10H), 1.51 (ttt, J = 10.7, 6.9, 3.4 Hz, 1H), 1.35 - 1.25 (m, 5H), 1.24 - 1.14 (m, 3H), 1.01 - 0.86 (m, 5H). ¹³C NMR (125 MHz, Methanol-^) 6 172.64, 64.10, 62.98, 62.47, 55.15, 52.41, 51.12, 50.85, 50.56, 48.51, 42.53, 36.36, 34.56, 33.70, 31.46, 31.36, 31.07, 30.46, 26.38, 25.94, 25.73, 25.46.

AD-2052: Following General Procedure F, AD-2040 (77.3 mg, 0.198 mmol) and LiAIFU (15.02 mg, 0.396 mmol) were used. The product was purified using column chromatography (0-10% DCM:MeOH on neutral alumina over 8 minutes) to afford the desired product as a yellow oil: 39.8 mg (54%). 'H NMR (500 MHz, Methanol-^) 57.30 - 7.16 (m, 5H), 3.71 (dd, J = 10.7, 5.7 Hz, 1H), 3.54 (dd, J = 10.7, 7.5 Hz, 1H), 3.16 (dd, J = 9.9, 7.2 Hz, 1H), 2.91 (dd, J = 17.7, 11.1 Hz, 3H), 2.86 - 2.76 (m, 4H), 2.56 (dd, J = 26.4, 10.0 Hz, 2H), 2.31 (t, J = 8.8 Hz, 3H), 2.28 - 2.18 (m, 1H), 2.04 (p, J = 7.6 Hz, 1H), 1.91 - 1.82 (m, 1H), 1.82 - 1.67 (m, 6H), 1.67 - 1.55 (m, 4H), 1.34 - 1.26 (m, 2H), 1.26 - 1.14 (m, 2H), 0.93 (qd, J = 11.8, 2.8 Hz, 2H). ¹³C NMR (125

MHz, Methanol-^) 8 139.35, 128.30, 128.16, 125.96, 65.06, 63.40, 60.68, 58.24, 56.80, 51.47, 50.89, 49.21, 40.76, 35.98, 34.35, 34.10, 31.49, 30.00, 26.17, 25.67.

AD-2064: Following General Procedure A, methyl 2-benzyl-2,8-diazaspiro[4.5]decane-4- carboxylate (198.1 mg, 0.687mmol) and lH-Indole-3-carbaldehyde (498.62 mg, 3.435 mmol) were used. The product was purified using column chromatography (0-10% DCM:MeOH on neutral alumina over 10 minutes) to afford the desired product as a yellow oil 44 mg (15%). 'H NMR (500 MHz, Methanol-c/7) 87.59 (d, J = 8.0 Hz, 1H), 7.34 (d, J = 8.1 Hz, 1H), 7.31 - 7.26 (m, 4H), 7.24 (dt, J = 6.6, 2.9 Hz, 1H), 7.20 (s, 1H), 7.09 (t, J = 7.6 Hz, 1H), 7.03 (t, J = 6.9 Hz, 1H), 3.74 (s, 2H), 3.60 (s, 2H), 3.59 (s, 3H), 2.87 (t, J = 8.1 Hz, 2H), 2.79 (s, 1H), 2.77 - 2.66 (m, 3H), 2.33 (d, J = 9.5 Hz, 1H), 2.28 - 2.13 (m, 2H), 1.94 (td, J = 12.6, 4.1 Hz, 1H), 1.66 (d, J = 12.7 Hz, 1H), 1.57 - 1.45 (m, 2H). ¹³C NMR (125 MHz, Methanol-^) 8 173.09, 138.13, 136.42, 128.60, 128.01, 127.94, 126.86, 125.16, 121.04, 118.74, 118.13, 110.91, 108.66, 59.73, 54.76, 52.97, 52.22, 50.48, 50.44, 49.68, 43.05, 36.26, 31.93.

AD-2081: Following General Procedure F, AD-2064 (22.0 mg, 0.054 mmol) and LiAlH4 (4.08 mg, 0.108 mmol) were used. The product was purified using column chromatography (0-10% DCM:MeOH on neutral alumina over 8 minutes) to afford the desired product as a white solid: 5 mg (23%). 'II NMR (500 MHz, Methanol-^) 5 7.69 (d, J = 7.9 Hz, 1H), 7.48 (s, 1H), 7.43 (d, J = 8.1 Hz, 1H), 7.37 - 7.30 (m, 4H), 7.29 - 7.25 (m, 1H), 7.19 (t, J = 7.6 Hz, 1H), 7.14 (t, J = 7.5 Hz, 1H), 4.37 (s, 2H), 3.72 (s, 2H), 3.63 (dd, J = 10.9, 6.8 Hz, 1H), 3.53 (dd, J = 10.9, 6.5 Hz, 1H), 3.42 - 3.35 (m, 1H), 3.35 (s, 1H), 3.01 (d, J = 8.0 Hz, 2H), 2.94 (s, 1H), 2.85 (d, J = 9.0 Hz, 1H), 2.57 (d, J = 10.1 Hz, 1H), 2.47 - 2.38 (m, 1H), 2.05 (t, J = 7.2 Hz, 1H), 1.96 (d, J = 12.0 Hz, 2H), 1.82 (d, J = 14.9 Hz, 2H). ¹³C NMR (125 MHz, Methanol-fiW) 5 136.51, 128.82, 128.10, 127.63, 127.33, 127.25, 121.91, 119.81, 117.74, 111.45, 60.46, 59.77, 56.08, 51.42, 49.55, 49.22, 40.25, 34.40, 28.48.

4-methylpiperazine-l-carboximidamide (SI)- To a flame dried flask was added N- methylpiperazine (0.554 mL, 4.992 mmol) as a solution in DMF (0.50M). Then, amidinopyrazole HC1 (731.7 mg, 4.992 mmol) and N,N-diisopropylethylamine (0.869 mL, 4.992 mmol) were added before the solution was stirred at 80 °C for 10 hours. The solution was concentrated under vacuum before crystalizing with ether to generate product as yellow crystals: 614.6 mg (87%). 'H NMR (500 MHz, D2O) 8 3.97 (s, 4H), 3.35 (s, 4H), 2.96 (s, 3H). ¹³C NMR (125 MHz, D₂O) 8 156.64, 52.92, 44.45, 44.41. IR (neat) u_max = 3304, 3117, 2941, 2863, 2853, 2810, 1663, 1648, 1596, 1525, 1450, 1289, 1208, 1151, 1131, 1080, 1057, 996, 806. HRMS (ESI) C₆H₁₅N₄. Calculated: [M+H]⁺, 143.1297 Found: [M+H]⁺, 143.1293.

Pyrrolidine-l-carboximidamide (S2)- To a flame dried microwave vial was added pyrrolidine (0.462 mL, 5.624 mmol) as a solution in DMF (0.67M). Then, amidinopyrazole HC1 (824 mg, 5.624 mmol) and N,N- diisopropylethylamine (0.980 mL, 5.624 mmol) were added, and the solution was heated using microwave irradiation for 35 min at 133 °C. The solution was concentrated under vacuum and taken without purification to the next step as a white powder: 710 mg (quant.). 'H NMR (500 MHz, MeOD) 3 7.06 (s, 1H), 3.48 - 3.39 (m, 4H), 2.09 - 1.97 (m, 4H). ¹³C NMR (125 MHz, MeOD) 6 154.73, 46.87, 24.83. IR (neat) u_max = 3315, 3159, 1651, 1621, 1558, 1480, 1467, 1457, 1364, 1223, 1157, 1064, 1038. HRMS (ESI) C5H12N3.

Calculated: [M+H]⁺, 114.1031 Found: [M+H]⁺, 114.1026.

Benzimidamide (S3)- To a flame dry flask was added a solution of ammonium chloride (210.0 mg, 3.882 mmol) as a solution in toluene (2.0M). Trimethylaluminum was added as a solution in toluene (2.0M) and the resulting solution was stirred for 2 hours. Benzonitrile (0.400 mL, 3.882 mmol) was added dropwise and heated at 83 °C for 16 hours. The reaction was concentrated and poured over a silica slurry in DCM. The slurry was gravity filtered and washed with MeOH before concentrating the pure product as a white powder: 410.5 mg (88%). ^XH NMR (500 MHz, MeOD) 3 7.82 (dd, J = 8.44, 1.33 Hz, 2H), 7.78 - 7.72 (m, 1H), 7.65 - 7.59 (m, 2H), 7.57 (s, 1H), 1.46 - 1.32 (m, 1H). ¹³C NMR (125 MHz, MeOD) 3 167.14, 133.81, 129.06, 128.17, 127.51. IR (neat) nmax = 3160, 1660, 1624, 1346, 1224. HRMS (ESI) C₇H₉N₂. Calculated: [M+H]⁺, 121.0766 Found: [M+H]⁺, 121.0763.

l-propyl-lH-indole-3-carbaldehyde (S4)- To a flame dried flask was added lH-indole-3- carbaldehyde (1.0023 g, 6.889 mmol), 1 -bromopropane (12.50 mL, 13.78 mmol), and sodium hydride (0.3040 g, 8.267 mmol) as a solution in DMF (0.1 M). The reaction was stirred for 40 min before being quenched with HzO, and the aqueous layer was washed in triplicate with EtOAc. The organic layer was concentrated under vacuum. The crude product was purified by column chromatography (0-30% EtOAc:Hexanes over 12 min) to afford the desired product as an orange solid: 1.0285 g (80%). ^XH NMR (500 MHz, MeOD) 8 9.84 (s, 1H), 8.17 (d, J = 8.00 Hz, 1H), 8.09 (s, 1H), 7.51 (d, 2 = 8.21 Hz, 1H), 7.38 - 7.19 (m, 2H), 4.22 (t, J = 7.06 Hz, 2H),

1.91 (h, J = 7.31 Hz, 2H), 0.93 (t, J = 7.42 Hz, 3H). ¹³C NMR (125MHz, MeOD) 8 185.40, 140.83, 137.58, 125.12, 123.59, 122.48, 121.33, 117.57, 110.30, 48.23, 22.75, 10.05. IR (neat) u„_iax = 2967, 2934, 2878, 2793, 2723, 2699, 1644, 1609, 1571, 1529, 1486, 1474, 1465, 1454, 1410, 1394, 1381, 1353, 1320, 1284, 1241, 1190, 1171. HRMS (ESI) C12H14NO. Calculated: [M+H]⁺, 188.1075 Found: [M+H]⁺, 188.1071.

l-(tert-butyl) 4-ethyl 5-oxoazepane-l,4-dicarboxylate (3)- To a flame dried flask equipped with a stir bar was added l-Boc-4-piperidone (2.0000g, 10.04 mmol) as a solution in diethyl ether (0.60M). Then, ethyl diazoacetate (1.58 ml, 15.06 mmol) and BFiOEti (1.24 mL, 10.04 mmol) were added dropwise, and the resulting mixture was stirred at -25° C for 1 hour. The reaction was neutralized with sodium bicarbonate, and the aqueous layer was extracted in triplicate with EtOAc. The combined organic extracts were washed with brine, dried with Na2SC>4, and concentrated under vacuum. The crude product was purified by column chromatography (0-20% EtOAc:Hexanes over 10 min) to afford the desired product as a yellow oil: 2.020 g (71%). Product was shown to exist as the keto- and enol- tautomers leading to a half integrations in the proton NMR and splitting in the carbon NMR. ^XH NMR (500 MHz, CDCh) 6 4.11 (q, J = 7.13 Hz, 2H), 3.44 (s, 1H), 3.23 (t, J = 7.17 Hz, 1H), 2.54 (s, 1H), 2.27 (t, J = 7.47 Hz, 1H), 1.81 (p, J 7.30 Hz, 1H), 1.43 (s, 6H), 1.24 (t, J = 7.16 Hz, 3H). ¹³C NMR (125 MHz, CDCh) 5 173.07, 171.91, 155.27, 79.73, 60.43 (d, J = 16.50 Hz), 46.90 (d, J = 37.97 Hz), 43.31, 33.70 (d, J = 73.69 Hz), 28.36, 23.75 (d, J = 48.41 Hz), 21.79 (d, J = 3.24 Hz), 14.17 (d, J = 4.59 Hz). IR (neat) u_max = 2976, 2931, 1741, 1668, 1469, 1445, 1415, 1366, 1317, 1242, 1199, 1158, 1067, 1024, 897, 860. HRMS (ESI) C14H23NO5. Calculated: [M+Na]⁺, 308.1474 Found: [M+Na]⁺, 308.1464.

Tert-butyl 2-(4-methylpiperazin-l-yl)-4-(»xo-3,4,5,6,8,9-hexahydro-7H-pyrimido[4,5- d]azepine-7- carboxylate (4a)- Following General Procedure A, 1 -(tert-butyl) 4-ethyl 5- oxoazepane- 1,4-dicarboxylate (900.0 mg, 3.154 mmol), 4-methylpiperazine-l-carboximidamide (659 mg, 4.637 mmol), and sodium ethoxide as a 1 M solution in ethanol (3.2 mL) were used. Crude product was purified using column chromatography (0-100% MeOH:DCM for 5 minutes, holding at 100% for 4 minutes) to afford the desired product as an off white solid: 800 mg (70%). 'H NMR (500 MHz, CDC13) 5 3.73 (s, 4H), 3.51 (d, J = 44.71 Hz, 4H), 2.75 (d, J = 29.59 Hz, 4H), 2.47 (t, J = 4.90 Hz, 4H), 2.34 (s, 3H), 1.54 - 1.41 (m, 9H). ¹³C NMR (125 MHz, CDC13) 5 167.87, 165.79, 155.02, 151.55, 111.19, 79.64, 54.54, 46.96, 46.00, 44.26, 43.71, 40.18, 28.48, 24.33. IR (neat) Umax = 2972, 2960, 2930, 2795, 1662, 1616, 1574, 1456, 1414, 1385, 1362, 1329, 1304, 1294, 1237, 1203, 1159, 1107, 1084,, 1040, 1002, 935, 884, 862. HRMS (ESI) CI8H30N5O3. Calculated: [M+H]⁺, 364.2349 Found: [M+H]⁺, 364.2332.

Tert-butyl 4-oxo-2-(pyrrolidin-l-yl)-3,4,5,6,8,9-hexahydro-7H-pyrimido[4,5-d]azepine-7- carboxylate (4b)- Following General Procedure A, 1 -(tert-butyl) 4-ethyl 5-oxoazepane-l,4-dicarboxylate (336.0 mg, 1.178 mmol), pyrrolidine- 1-carboximidamide (200 mg, 1.767 mmol), and sodium ethoxide as a 0.15 M solution in ethanol (7.8 mL) were used. Crude product was purified using column chromatography (0-10% MeOH:DCM for 15 minutes) to afford the desired product as an off-white solid: 150 mg (38%). 'H NMR (500 MHz, CDC13) 5 3.54 (t, J = 6.66 Hz, 6H), 3.45 (s, 2H), 2.74 (d, J = 20.18 Hz, 4H), 1.97 (d, J = 6.42 Hz, 4H), 1.46 (s, 9H). ¹³C NMR (125 MHz, CDC13) 8 167.89, 165.35, 155.03, 150.37, 109.91, 79.53, 46.79 (d, 7 = 99.93 Hz), 46.66, 44.23 (d, J= 90.79 Hz), 40.13, 28.48, 25.37, 24.46 (d, J = 25.93 Hz). IR (neat) Umax = 2971, 1687, 1626, 1584, 1457, 1413, 1390, 1364, 1335, 1290, 1236, 1162, 1108, 1085, 949, 938, 868. HRMS (ESI) C17H27N4O3. Calculated: [M+H]⁺, 335.2083 Found: [M+H]⁺, 335.2078.

Tert-butyl 4-oxo-2-phenyl-3,4,5,6,8,9-hexahydro-7H-pyrimido[4,5-d]azepine-7-carboxylate (4c)-

Following General Procedure A, 1 -(tert-butyl) 4-ethyl 5-oxoazepane-l,4-dicarboxylate (285 mg, 0.9987 mmol), benzimidamine (180 mg, 1.498 mmol) and sodium ethoxide as a 0.15 M solution in ethanol (6.7 mL) were used. Crude product was purified using column chromatography (0-10% MeOH:DCM for 10 minutes) to afford the desired product as an off- white solid: 260 mg (76%). 'H NMR (500 MHz, CDCL) 8 8.23 (d, J = 6.89 Hz, 2H), 7.61 - 7.45 (m, 3H), 3.61 (d, 7 = 34.79 Hz, 4H), 3.00 (d, 7 = 49.63 Hz, 4H), 1.49 (s, 8H), 1.45 - 1.42 (m, 1H). ¹³C NMR (125 MHz, CDC1₃) 8 166.09, 165.11, 154.98, 153.63, 131.99, 131.71, 128.81, 127.60, 122.67, 79.87, 45.68 (d, 7 = 98.61 Hz), 44.00 (d, 7 = 93.44 Hz), 39.70, 28.48, 24.81. f IR (neat) rw = 3359, 3140, 2973, 2929, 1661, 1630, 1542, 1456, 1417, 1366, 1330, 1313, 1282, 1263, 1250, 1234, 1168, 1113, 1087, 1062, 969, 940, 902, 857. HRMS (ESI) CI₉H24N₃O₃. Calculated: [M+H]⁺, 342.1818 Found: [M+H]⁺,342.1813.

2-(4-methylpiperazin-l-yl)-7-((l-propyl-lH-indol-3-yl)methyl)-3,5,6,7,8,9-hexahydro-4H- pyrimido[4,5-d]azepin-4-one (la, DS1040)- Following General Procedure B, tert-butyl 2-(4- methylpiperazin-l-yl)-4-oxo-3,4,5,6,8,9-hexahydro-7H-pyrimido[4,5-d]azepine-7-carboxylate (80.3 mg, 0.2209 mmol) and trifluoroacetic acid (1.1 mL) were added. Following conversion to the free amide, General Procedure C was followed. 1 -propyl- lH-indole-3-carbaldehyde (124.1 mg, 0.6630 mmol), sodium triacetoxyborohydride (234.2 mg, 1.105 mmol) in DMF (4.4 mL) and acetic acid (0.090 mL) were added. Crude product was purified using column chromatography (0-100% MeOH:DCM for 15 minutes) to afford the desired product as an orange solid: 33.7 mg (35%). ¹H NMR (500 MHz, MeOD) 5 7.66 (d, J = 7.97 Hz, 1H), 7.40 (d, J = 8.44 Hz, 1H), 7.32 (s, 1H), 7.18 (ddd, J = 8.22, 7.03, 1.12 Hz, 1H), 7.09 (ddd, J = 7.98, 6.97, 0.98 Hz, 1H), 4.14 (t, J = 7.00 Hz, 2H), 4.09 (s, 2H), 3.62 (s, 4H), 2.95 (s, 2H), 2.87 (s, 4H), 2.80 (s, 2H), 2.47 (s, 4H), 2.31 (s, 3H), 1 .85 (h, J = 7.26 Hz, 2H), 0.90 (t, J = 7.38 Hz, 3H). ¹³C NMR (125 MHz, MeOD) 5 165.79, 152.86, 136.40, 129.36, 128.37, 121.39, 119.17, 118.53, 111.19, 111.05, 109.42, 106.46, 53.91, 53.76, 52.86, 51.42, 44.58, 43.77, 35.12, 29.32, 23.18, 21.64, 10.24. IR (neat) u_max =2929, 2782, 1643, 1568, 1455, 1392, 1356, 1340, 1319, 1302, 1293, 1279, 1263, 1217, 1191, 1167, 1149, 1136, 1113, 1076, 1002, 972, 950, 853. HRMS (ESI) C25H36N6O. Calculated: [M+H]⁺, 435.2872 Found: [M+H]⁺, 435.2857.

7-((l-propyl-lH-indol-3-yl)mcthyl)-2-(pyrrolidin-l-yl)-3,5,6,7,8^-hcxahydro-4H- pyrimido[4,5- d]azepin-4-one (lb)- Following General Procedure B, Tert-butyl 4-oxo-2- (pyrrolidin-l-yl)-3,4,5,6,8,9- hexahydro-7H-pyrimido[4,5-d]azepine-7-carboxylate (150 mg, 0.4500 mmol) and trifluoroacetic acid (2.2 mL) were added. Following conversion to the free amide, General Procedure C was followed. 1 -propyl- 1H- indole-3-carbaldehyde (176.0 mg, 0.9390 mmol), sodium triacetoxyborohydride (497 mg, 2.347 mmol) in DMF (9.0 mL) and acetic acid (0.190 mL) were added. Crude product was purified using column chromatography (0-25% MeOH:DCM for 15 minutes) to afford the desired product as a yellow solid: 62.0 mg (33%). 1H NMR (500 MHz, MeOD) δ 7.71 (d, J = 7.94 Hz, 1H), 7.52 (s, 1H), 7.48 (d, J = 8.30 Hz, 1H), 7.25 (t, J = 7.65 Hz, 1H), 7.17 (t, J = 7.52 Hz, 1H), 4.50 (s, 2H), 4.19 (t, J = 7.02 Hz, 2H), 3.52 - 3.41 (m, 4H), 3.37 (s, 2H), 3.27 (s, 2H), 2.98 (t, J = 5.24 Hz, 2H), 2.91 (s, 2H), 2.06 - 1.93 (m, 4H), 1.88 (h, J = 6.46, 5.59 Hz, 2H), 0.92 (t, 7 = 7.38 Hz, 3H). ¹³C NMR (125 MHz, MeOD) 8 161.52, 151.21, 136.47, 131.12, 128.00, 122.00, 120.03, 118.15, 118.00, 109.91, 107.98, 102.37, 53.29, 52.54, 50.71, 46.46, 41.04, 33.49, 24.80, 23.14, 19.88, 10.22. IR (neat) Umax = 2957, 2875, 1632, 1588, 1456, 1393, 1333, 1262, 1237, 1197, 1175, 1125, 1014, 960, 898, 879. HRMS (ESI) C24H32N5O. Calculated: [M+H]⁺, 406.2607 Found: [M+H]⁺, 406.2592.

2-phenyl-7-((l-propyl-lH-indol-3-yl)methyl)-3,5,6,7,8,9-hexahydro-4H-pyrimido[4,5- d]azepin-4-one (lc)- Following General Procedure B, Tert-butyl 4-oxo-2-phenyl-3,4,5,6,8,9- hexahydro-7H-pyrimido[4,5- d]azepine-7-carboxylate (260 mg, 0.7615 mmol) and trifluoroacetic acid (2.5 mL) were added. Following conversion to the free amide, General Procedure C was followed. 1 -propyl- lH-indole-3-carbaldehyde (250 mg, 1.335 mmol), sodium triacetoxyborohydride (707 mg, 3.336 mmol) in DMF (13 mL) and acetic acid (0.270 mL) were added. Crude product was purified using column chromatography (0-10% MeOH:DCM for 10 minutes) to afford the desired product as a yellow solid: 84.4 mg (31%). 'H NMR (500 MHz, MeOD) 8 7.98 - 7.92 (m, 2H), 7.70 (d, J = 7.89 Hz, 1H), 7.55 (t, J = 7.34 Hz, 1H), 7.49 (t, J = 7.46 Hz, 2H), 7.41 (d, J = 7.16 Hz, 2H), 7.20 (ddd, J = 8.21, 6.93, 1.04 Hz, 1H), 7.12 (ddd, J = 7.98, 7.04, 0.99 Hz, 1H), 4.30 (s, 2H), 4.14 (t, J = 7.04 Hz, 2H), 3.26 - 3.19 (m, 2H), 3.14 (q, J = 5.61 Hz, 4H), 3.01 (dd, J = 6.93, 3.61 Hz, 2H), 1.85 (h, 7 = 7.22 Hz, 2H), 0.90 (t, J = 7.37 Hz, 3H). ¹³C NMR (125 MHz, MeOD) 8 164.07, 163.92, 155.54, 136.42, 132.30, 131.45, 130.19, 128.49, 128.16, 127.34, 121.67, 121.52, 119.57, 118.36, 109.65, 104.61, 52.60, 52.29, 50.72, 47.48, 34.06, 23.15, 21.07, 10.25. IR (neat) u_max = 2930, 1635, 1603, 1542, 1505, 1466, 1398, 1327, 1195, 1128, 1013, 943, 898. HRMS (ESI) C26H29N4O. Calculated: [M+H]⁺, 413.2341 Found: [M+H]⁺, 413.2324.

2-phenyl-7-(pyridin-4-ylmethyl)-3,5,6,7,8,9-hexahydro-4H-pyrimido[4,5-d]azepin-4-one (ld)-

Following General Procedure B, tert-butyl 2-(4-methylpiperazin-l-yl)-4-oxo-3,4,5,6,8,9- hexahydro-7H- pyrimido[4,5-d]azepine-7-carboxylate (75.1 mg, 0.286 mmol) and trifluoroacetic acid (0.82 mL) were added. Following conversion to the free amide, General Procedure C was followed. 4-pyridine carboxyladehyde (0.67 mL, 0.572 mmol), sodium triacetoxy borohydri de (302.5 mg, 1.430 mmol) in DMF (5.72 mL) and acetic acid (60.6 pL) were added. Crude product was purified using column chromatography (0-10% Methanolic ammonia:DCM for 15 minutes, holding at 10% for 10 minutes) to afford the desired product as an yellow solid: 63.9 mg (69%). 'H NMR (500 MHz, MeOD) 8 8.47 (s, 1H), 8.41 (d, J = 5.28 Hz, 1H), 7.46 (d, J = 4.66 Hz, 1H), 7.32 (d, J = 5.03 Hz, 1H), 4.81 (s, 1H), 3.68 (s, 2H), 3.65 (s, 4H), 2.81 (d, J = 5.65 Hz, 2H), 2.74 (d, J = 5.36 Hz, 2H), 2.61 (d, J = 8.33 Hz, 2H), 2.54 (t, J = 4.85 Hz, 2H), 2.50 (s, 4H), 2.33 (s, 3H). ¹³C NMR (125 MHz, MeOD) 8 165.84, 152.59, 151.80, 149.44, 148.66, 148.03, 124.26, 122.67 (d, J = 42.80 Hz), 1 12.10, 75.71, 61.42, 54.91 , 53.95, 52.70, 44.61 , 43.80, 23.31. IR (neat) irnax =2922, 2799, 1626, 1563, 1413, 1399, 1290, 1264 ,1142, 1001, 950 ,803, 790, 576. HRMS (ESI) CI₉H₂₆N₆O Calculated: [M+H]⁺, 355.2246 Found: [M+H]⁺, 355.2236.

Tert-butyl 4-(2-methoxy-2-oxoethylidene)piperidine-l-carboxylate (6)- To a flame dried flask equipped with a stir bar was added NaH (60 % dispersion in mineral oil, 240.9 mg, 6.023 mmol) as a suspension in anhydrous DMF (0.10 M). The resulting mixture was cooled to 0° C before dropwise addition of trimethyl phosphonoacetate (1.20 mL, 7.416 mmol). The reaction stirred for 30 min before addition of 1-tert- Butoxycarbonylpiperidin-4-one (0.9985 g, 5.019 mmol) dissolved in DMF (1 mL). The reaction proceeded for 6 hours before quench with NH |C I . The aqueous layer was extracted in triplicate with EtOAc. The combined organic extracts were washed with brine, dried with NazSO i, and concentrated under vacuum. The crude product was purified by column chromatography (0-100% EtOAc:Hexanes over 15 min) to afford the desired product as a white solid: 1.058 g (95%). 'H NMR (500 MHz, CDC1₃) 8 5.57 (s, 1H), 3.53 (s, 3H), 3.33 (dt, J = 14.87, 5.80 Hz, 4H), 2.78 (t, J = 5.86 Hz, 2H), 2.13 (t, J = 5.83 Hz, 2H), 1.32 (s, 9H). ¹³C NMR (125 MHz, CDCL) 8 166.04, 157.83, 154.03, 114.52, 79.22, 50.46, 44.42, 36.09, 29.20, 28.05. IR (neat) tu = 3014, 2968, 2871, 1680, 1652, 1422, 1885, 1478, 1421, 1385, 1364, 1340, 1314, 1255, 1236, 1213, 1139, 1114, 1009, 992, 980, 965, 864, 791, 767, 744, 726, 690, 634. HRMS (ESI) C13H21NO4. Calculated: [M+H- Boc]⁺, 156.1025 Found: [M+H- Boc]⁺, 156.1029.

8-(tert-butyl)-4-methyl-2-benzyl-2,8-diazaspiro[4.5]decane-4,8-dicarboxylate (7)- To a flame dried flask that was purged with argon was added equipped a magnetic stir bar. The flask was cooled to 0° C before addition of tert-butyl 4-(2-methoxy-2-oxoethylidene)piperidine-l- carboxylate (1.005 g, 3.917 mmol) as a solution in anhydrous toluene (0.40 M) and N- (Methoxymethyl)-N-(trimethylsilylmethyl)-benzylamine (1.3948 mg, 5.875 mmol). The mixture stirred for 15 minutes before dropwise addition of TFA (0.060 mL, 0.783 mmol) as a solution in DCM (1.0 M). The reaction was stirred for 2 hours before being concentrated under vacuum. The crude product was purified by column chromatography (0-100% EtOAc:Hexanes over 20 min) to afford the desired product as a clear oil: 861 .6 mg (57%, 99% BRSM). 'H NMR (500 MHz, CDCk) 8 7.28 (t, J = 7.05 Hz, 4H), 7.23 - 7.18 (m, 1H), 3.82 (s, 2H), 3.63 (s, 3H), 3.62 (d, J = 4.13 Hz, 2H), 2.94 (t, J = 8.53 Hz, 1H), 2.83 (d, J = 12.01 Hz, 1H), 2.78 (t, J = 8.93 Hz, 3H), 2.69 (t, J = 8.00 Hz, 1H), 2.31 (d, 7 = 9.18 Hz, 1H), 1.81 - 1.72 (m, 1H), 1.62 (d, J = 13.36 Hz, 1H), 1.51 (d, J = 13.10 Hz, 1H), 1.41 (s, 9H), 1.34 (t, J = 11.21 Hz, 1H). ¹³C NMR (125 MHz, CDC1₃) 8 173.23, 154.77, 138.83, 128.42, 128.27, 126.98, 79.37, 61.93, 59.84, 55.27, 53.42, 51.46, 44.03, 41.32, 36.88, 32.48, 28.40. IR (neat) u_max = 2948, 1733, 1687, 1495, 1453, 1422, 1364, 1273, 1244, 1093, 1028, 978, 951, 911, 860, 738, 698. HRMS (ESI) C22H32N2O4. Calculated: [M+H]⁺, 389.2440 Found: [M+H]⁺, 389.2444.

Methyl 2-benzyl-2,8-diazaspiro[4.5]decane-4-carboxylate (S5)- To a reaction vessel equipped with a magnetic stir bar was added a 1 : 1 mixture of HCTEtOAc (0.30 M) and 8-(tert-butyl)-4- methyl-2-benzyl- 2,8-diazaspiro[4.5]decane-4,8-dicarhoxylate (427.3 mg, 1 .010 mmol). The reaction stirred for 30 minutes at rt before being quenched with H2O. The aqueous layer was washed in triplicate with EtOAc. The recovered aqueous later was concentrated under vacuum. Product was isolated as a yellow foaming solid (454.1 mg) and used crude in the next step.

Methyl 2-benzyl-8-(pyridin-2-ylmethyl)-2,8-diazaspiro[4.5]decane-4-carboxylate (8a)- Following General Procedure D, 2-benzyl-2,8-diazaspiro[4.5]decane-4-carboxylate (227.7 mg, 0.7871 mmol), pyridine-2-carbaldehyde (421.6 mg, 3.936 mmol), and sodium triacetoxyborohydride (834.2 mg, 3.936 mmol) were used. Crude product was purified using column chromatography (0-10% MethanoLDCM over 10 minutes) to afford the desired product as a brown oil: 65.7 mg (62% over 2 steps). 'H NMR (500 MHz, CDCls) 5 8.59 - 8.47 (m, 1H), 7.64 (t, J = 7.70 Hz, 1H), 7.39 (d, J = 7.64 Hz, 1H), 7.32 (q, J = 7.99, 7.44 Hz, 4H), 7.28 - 7.22 (m, 1H), 7.15 (t, J = 6.40 Hz, 1H), 3.67 (s, 5H), 3.61 (s, 2H), 2.99 (s, 1H), 2.86 (d, J = 9.26 Hz, 1H), 2.80 - 2.67 (m, 4H), 2.30 (d, J = 9.19 Hz, 1H), 2.17 (s, 1H), 2.08 (s, 1H), 1.96 (t, J = 12.73 Hz, 1H), 1.71 (d, J = 13.16 Hz, 1H), 1.56 (s, 2H). ¹³C NMR (125 MHz, MeOD) 8 172.58, 155.82, 148.49, 137.36, 136.64, 128.95, 128.18, 127.46, 123.98, 122.90, 62.60, 61.84, 59.57, 54.40, 52.58, 50.92, 50.83, 50.24, 42.95, 35.48, 20.96. IR (neat) tw = 2947, 2804, 1683, 1580, 1570, 1495, 1475, 1434, 1364, 1260, 1168, 1028, 993, 911, 861, 757, 700. HRMS (ESI) C23H29N3O2. Calculated: [M+H]⁺, 380.2338 Found: [M+H]⁺, 380.2338.

Methyl 2,8-dibenzyl-2,8-diazaspiro[4.5]decane-4-carboxylate (8b)- Following General Procedure D, 2- benzyl-2,8-diazaspiro[4.5]decane-4-carboxylate (113.9 mg, 0.3953 mmol), benzaldehyde (209.6 mg, 1.976 mmol), and sodium triacetoxyborohydride (418.8 mg, 1.976 mmol) were used. Crude product was purified using column chromatography (0-25% MethanoLDCM over 25 minutes) to afford the desired product as a clear oil: 27.0 mg (36% over 2 steps). H NMR (500 MHz, CDCh) 8 7.38 - 7.27 (m, 10H), 3.77 (s, 2H), 3.68 (s, 3H), 3.66 (s, 2H), 3.12 (s, 1H), 2.87 (q, J = 8.88, 6.44 Hz, 3H), 2.80 (t, J = 7.63 Hz, 2H), 2.40 (d, J = 9.62 Hz, 1H), 2.33 (s, 1H), 2.22 (s, 1H), 2.08 (s, 1H), 1.84 (d, J = 13.49 Hz, 1H), 1.71 (s, 1H), 1.63 (s, 1H). ¹³C NMR (125 MHz, CDC1₃) 6 175.82, 172.72, 135.65, 132.84, 130.36, 129.34, 128.71, 128.62, 128.57, 128.00, 61.37, 59.57, 54.29, 52.17, 51.93, 49.90, 49.45, 43.21, 34.35, 30.61, 21.79. IR (neat) umax = 2926, 2852, 2806, 1698, 1644, 1553, 1495, 1454, 1485, 1454, 1435, 1365, 1234, 1193, 1168, 1028, 738, 614, 603, 578, 568. HRMS (ESI) C24H30N2O2. Calculated: [M+H]⁺, 379.2386 Found: [M+H]⁺, 379.2388.

Methyl 2-benzyl-8-(cyclohexylmethyl)-2,8-diazaspiro[4.5]decane-4-carboxylate (8c)- Following General Procedure D, 2-benzyl-2,8-diazaspiro[4.5]decane-4-carboxylate (214.9 mg, 0.7450 mmol), cyclohexane carboxaldehyde (417.8 mg, 3.725 mmol), and sodium triacetoxyborohydride (789.5 mg, 3.725 mmol) were used. Crude product was purified using column chromatography (0-100% MethanokDCM over 15 min, holding at 25% for 5 min) to afford the desired product as a yellow oil: 136.8 mg (77% over 2 steps). *H NMR (500 MHz, CDCh) 8 7.31 (q, J = 7.46 Hz, 4H), 7.28 - 7.22 (m, 1H), 3.82 - 3.72 (m, 2H), 3.71 (s, 3H), 3.44 (s, 1H), 3.14 (t, J = 8.96 Hz, 2H), 2.92 (t, J = 11.36 Hz, 2H), 2.83 (t, J = 7.63 Hz, 1H), 2.65 (s, 1H), 2.57 (d, J = 6.70 Hz, 2H), 2.47 (d, J = 9.69 Hz, 1H), 2.33 (t, J = 12.67 Hz, 1H), 2.00 - 1.91 (m, 5H), 1.81 (d, J = 11.84 Hz, 2H), 1.70 (d, J = 12.87 Hz, 3H), 1.62 (d, J = 12.63 Hz, 1H), 1.25 - 1.07 (m, 3H), 0.97 (q, J = 12.24, 11.05 Hz, 2H). ¹³C NMR (125 MHz, MeOD) 8 172.36, 137.41, 128.73, 128.14, 127.22, 62.96, 61.52, 59.42, 54.35, 52.24, 50.98, 50.65, 50.23, 42.22, 33.39, 32.94, 30.70, 29.42, 25.66, 25.23, 21.65. IR (neat) rmax = 2925, 2852, 1651, 1449, 1362, 1263, 1171, 1028, 945, 602, 568, 559. HRMS (ESI) C24H36N2O2. Calculated: [M+H]⁺, 385.2855 Found: [M+H]⁺, 385.2861.

Methyl 2-benzyl-8-(pyridin-4-ylmethyl)-2,8-diazaspiro[4.5]decane-4-carboxylate (8d)- Following

General Procedure D, 2-benzyl-2,8-diazaspiro[4.5]decane-4-carboxylate (214.9 mg, 0.7450 mmol), 4- pyridine carboxaldehyde (399.1 mg, 3.726 mmol), and sodium triacetoxyborohydride (789.7 mg, 3.726 mrao!) were used. Crude product was purified using column chromatography (0-8% MethanokDCM over 10 min, holding at 8% for 5 min) io afford the desired product as a yellow oil: 137.2 mg (58% over 2 steps). 'H NMR (500 MHz, CDC13) 58.53 (d, J = 5.01 Hz, 1 H), 8.49 (d, J = 4.95 Hz, 2H), 7.31 (dd, J = 14.40, 7.15 Hz, 4H), 7.23 (d, J= 3.94 Hz, 2H), 3.68 (s, 5H), 3.42 (s, 2H), 3.02 (t, J= 8.29 Hz, 1 H), 2.86 (d, J= 9.18 Hz, 1H), 2.77 (dt, J = 24.45, 8.14 Hz, 2H), 2.63 (dd, J = 28.93, 11.31 Hz, 2H), 2.31 (d, J = 9.30 Hz, 1H), 2.08 (t, J = 1 1.37 Hz, 1H), 1.99 (d, J = 9.16 Hz, 1H), 1.90 (td, J = 12.30, 11.75, 3.98 Hz, 1H), 1.72 (d, .7 = 13.19 Hz, 1H), 1.52 (d, J= 5.42 Hz, 2H). ¹³C NMR (125 MHz, MeOD) 5 172.98, 152.51, 148.60, 137.52, 128.76, 128.06, 127.14, 124.48, 121.37, 62.28, 61.92, 61.16, 59.78, 53.04, 51.08, 50.66, 50.35, 43.19, 36.53, 32.37.

IR (neat) Umax = 3028, 2945, 2804, 1602, 1561, 1495, 1435, 1415, 1363, 1322, 1299, 1266, 1169, 1091, 1064, 1040, 1028, 993, 955, 912, 812, 794, 740, 700, 628, 616, 603, 593, 581, 568. HRMS (ESI) C23H29N3O2. Calculated: [M+H]⁺, 380.2338 Found: [M+H]⁺, 380.2344.

Methyl 2-benzyl-8-((3,5-dimethylisoxazol-4-yl)methyl)-2,8-diazaspiro[4.5]decane-4- carboxylate (8e)- Following General Procedure D, 2-benzyl-2,8-diazaspiro[4.5]decane-4- carboxylate (214.9 mg, 0.7450 mmol), 3,5-dimethyl-4-isoxazole carbaldehyde (399.1 mg, 3.726 mmol), and sodium triacetoxyborohydride (789.7 mg, 3.726 mmol) were used. Crude product was purified using column chromatography (0-100% MethanoLDCM over 10 min, holding at 10% for 5 min) to afford the desired product as a yellow oil: 137.2 mg (68% over 2 steps). 'H NMR (500 MHz, CDC1₃) 57.37 - 7.28 (m, 4H), 7.26 (t, J = 7.12 Hz, 1H), 3.77 (s, 2H), 3.66 (s, 3H), 3.17 (s, 2H), 3.14 - 3.07 (m, 1H), 2.99 - 2.90 (m, 1H), 2.88 - 2.81 (m, 1H), 2.76 (t, J = 7.75 Hz, 1H), 2.62 (dd, J = 28.36, 10.62 Hz, 2H), 2.38 (d, J = 9.69 Hz, 1H), 2.29 (s, 3H), 2.20 (s, 3H), 2.00 (s, 1H), 1.94 (d, J = 11.05 Hz, 1H), 1.83 (td, J = 12.42, 11.50, 3.91 Hz, 1H), 1.70 (d, J = 13.40 Hz, 1H), 1.52 - 1.39 (m, 2H). ¹³C NMR (125 MHz, CDC13) 6 173.43, 166.50, 160.39, 138.55, 128.61, 128.30, 127.06, 110.48, 62.49, 59.91, 55.21, 53.41, 51.44, 51.07, 50.58, 50.40, 43.88, 37.24, 32.89, 11.05, 10.28. IR (neat) u_max = 2921, 2804, 1730, 1679, 1640, 1583, 1495, 1452, 1434, 1362, 1298, 1260, 1194, 1069, 1028, 989, 956, 911, 886, 801, 742, 699. HRMS (ESI) C23H31N3O3. Calculated: [M+H]⁺, 398.2444 Found: [M+H]⁺, 398.2452.

(2-benzyl-8-(pyridin-2-ylmethyl)-2,8-diazaspiro[4.5]decan-4-yl)methanol (2a, RH1096)- Following the

General Procedure E, LiAlEU (3.9 mg, 0.1017 mmol) and methyl 2-benzyl-8- (pyridin-2-ylmethyl)-2,8- diazaspiro[4.5]decane-4-carboxylate (19.3 mg, 0.0509 mmol) were used. Crude product was purified using column chromatography (0-100% MethanoLDCM over 10 min) to afford the desired product as a clear oil: 11.4 mg (64%). 'H NMR (500 MHz, CDCh) 6 8.55 (d, J = 4.70 Hz, 1H), 7.64 (t, J = 7.66 Hz, 1H), 7.39 (d, J = 7.80 Hz, 1H), 7.32 (t, 7 = 7.30 Hz, 2H), 7.28 (d, 7 = 7.13 Hz, 3H), 7.15 (t, 1H), 3.74 (s, 2H), 3.63 (d, 7 = 8.43 Hz, 2H), 3.60 (s, 2H), 2.87 - 2.73 (m, 3H), 2.67 (s, 2H), 2.28 (d, 7 = 9.62 Hz, 1H), 2.25 - 2.12 (m, 2H), 1.91 - 1.82 (m, 2H)., 1.79 (d, 7 = 13.66 Hz, 1H), 1.69 (td, 7= 11.67, 10.30, 3.68 Hz, 1H), 1.61 (d, 7 = 13.56 Hz, 1H). ¹³C NMR (125 MHZ, CDCh) 5 158.90, 149.22, 138.35, 136.35, 128.53, 128.45, 127.16, 123.13, 121.94, 64.98, 64.02, 63.64, 60.31, 57.59, 52.10, 51.34, 41.20, 39.14, 32.47. IR (neat) n_max = 3374, 2924, 1645, 1595, 1436, 1366, 1230, 1092, 1028, 743. HRMS (ESI) C22H29N3O. Calculated: [M+H]⁺, 352.2389 Found: [M+H]⁺, 352.2393.

(2,8-dibenzyl-2,8-diazaspiro[4.5]decan-4-yl)methanol (2b)- Following the General Procedure E, LiAlH4 (8.3 mg, 0.2198 mmol) and methyl 2,8-dibenzyl-2,8-diazaspiro[4.5]decane-4- carboxylate (20.7 mg, 0.1099 mmol) were used. Crude product was purified using column chromatography (0-20% MethanohDCM over 20 min) to afford the desired product as a clear oil: 26.1 mg (68%). 'H NMR (500 MHz, CDCh) 5 7.43 - 7.29 (m, 10H), 5.80 (s, 1H), 3.88 (q, 2H), 3.75 - 3.64 (m, 4H), 3.11 (t, 1H), 3.00 (dd, 7 = 10.58, 4.47 Hz, 1H), 2.96 - 2.85 (m, 3H), 2.72 (d, 7 = 10.45 Hz, 1H), 2.44 - 2.29 (m, 2H), 2.07 (t, 7 = 9.57, 7.49 Hz, 1H), 2.00 - 1.90 (m, 2H), 1.81 (d, 7 = 14.04 Hz, 1H), 1.71 (d, 7 = 14.08 Hz, 1H). ¹³C NMR (125 MHz, MeOD) 5 132.72, 129.79, 129.17, 128.26, 128.17, 128.10, 127.79, 127.70, 62.84, 61.98, 60.45, 59.82, 56.27, 50.56, 50.07, 40.91, 35.71, 29.84, 29.34. IR (neat) n_max = 3369, 2921, 2851, 2802, 1557, 1494, 1452, 1378, 1261, 1074, 1028, 991, 913, 798, 740. HRMS (ESI) C23H30N2O. Calculated: [M+H]⁺, 351.2436 Found: [M+H]⁺, 351.2440.

(2-benzyl-8-(cyclohexylmethyl)-2,8-diazaspiro[4.5]decan-4-yl)methanol (2c, RH1115)- Following the

General Procedure E, LiAIH 1 (6.9 mg, 0.1815 mmol) and methyl 2-benzyl-8- (cyclohexylmethyl)-2,8- diazaspiro[4.5]dccane-4-carboxylatc (34.9 mg, 0..0908 mmol) were used. Crude product was purified using column chromatography (0-100% MethanohDCM over 15 min) to afford the desired product as a yellow oil: 18.0 mg (74%). 'H NMR (500 MHz, CDCh) 5 7.43 - 7.27 (m, 5H), 3.96 - 3.79 (m, 2H), 3.78 - 3.61 (m, 2H), 3.19 (s, 2H), 3.08 (s, 1H), 2.88 (s, 2H), 2.71 (d, 7 = 10.02 Hz, 1H), 2.58 (d, 7 = 6.79 Hz, 3H), 2.37 - 2.19 (m, 2H), 2.11 (s, 1H), 2.01 (s, 1H), 1.94 (d, 7 = 14.48 Hz, 1H), 1.83 (d, 7 = 13.31 Hz, 3H), 1.72 (d, 7 = 11.48 Hz, 3H), 1.68 - 1.61 (m, 1H), 1.23 (q, J = 12.58 Hz, 2H), 1.19 - 1.09 (m, 1H), 0.98 (q, / = 12.73, 11.89 Hz, 2H). ¹³C NMR (125 MHz, MeOD) 8 130.18, 129.06, 128.24, 127.56, 63.13, 62.69, 60.38, 59.68, 56.02, 50.88, 50.57, 40.34, 34.18, 33.00, 30.55, 28.33, 25.65, 25.18, 20.90. IR (neat) u_max = 3329, 2919, 2849, 2800, 1574, 1494, 1448, 1378, 1297, 1263, 1119, 1072, 1028, 994, 892, 844, 798, 739, 698, 652. HRMS (ESI) C23H36N2O. Calculated: [M+H]⁺, 357.2906 Found: [M+H]⁺, 357.2904.

(2-benzyl-8-(pyridin-4-ylmethyl)-2,8-diazaspiro[4.5]decan-4-yl)methanol (2d)- Following the General

Procedure E, L1AIH4 (10.0 mg, 0.2635 mmol) and methyl 2-benzyl-8- (cyclohexylmethyl)-2,8- diazaspiro[4.5]decane-4-carboxylate (50.3 mg, 0.1325 mmol) were used. Crude product was purified using column chromatography (0-100% MethanoLDCM over 15 min) to afford the desired product as a yellow oil: 37.3 mg (81%).^ NMR (500 MHz, CDC13) 8 8.52 (d, J = 5.09 Hz, 2H), 7.38 - 7.27 (m, 5H), 7.25 (s, 2H), 6.55 (s, 1H), 3.86 - 3.75 (m, 2H), 3.73 (d, J = 3.62 Hz, 2H), 3.46 (s, 2H), 2.99 (d, J = 4.86 Hz, 2H), 2.90 (d, J = 10.27 Hz, 1H), 2.61 (s, 1H), 2.55 (d, J= 10.25 Hz, 1H), 2.18 - 2.09 (m, 1H), 2.04 (s, 2H), 1.97 (t, / = 4.75 Hz, 1H), 1.81 (t, / = 10.00 Hz, 1H), 1.73 (d, J= 16.20 Hz, 1H), 1.68 (d, 7 = 10.30 Hz, 1H), 1.60 (d, /= 13.51 Hz, 1H). ¹³C NMR (125 MHz, MeOD) 8 148.71, 148.58, 136.45, 129.12, 128.18, 127.50, 124.47, 63.07, 61.26, 60.69, 60.02, 56.54, 51.16, 50.51, 49.31, 40.98, 36.84, 30.97. IR (neat) Umax = 3396, 2913, 2803, 1604, 1560, 1417, 1362, 1221, 1131, 1090, 1028, 992, 813, 790, 744, 700, 663, 636, 611, 574, 567.

HRMS (ESI) C22H29N3O. Calculated: [M+H]⁺, 352.2389 Found: [M+H]⁺, 352.2394.

(2-benzyl-8-((3,5-dimethylisoxazol-4-yl)methyl)-2,8-diazaspiro[4.5]decan-4-yl)methanol (2e)-

Following the General Procedure E, LiAlH4 (9.5 mg, 0.2516 mmol) and methyl 2-benzyl-8- (cyclohexylmethyl)-2,8-diazaspiro[4.5]decane-4-carboxylate (50.0 mg, 0.1258 mmol) were used. Crude product was purified using column chromatography (0-100% MethanoLDCM over 20 min) to afford the desired product as a yellow oil: 40.3 mg (87%). 'H NMR (500 MHz, CDC13) 8 7.49 (d, J = 5.42 Hz, 2H), 7.38 (d, J = 5.92 Hz, 3H), 4.09 (d, J = 13.63 Hz, 1H), 4.01 (d, J = 12.90 Hz, 1H), 3.72 (d, J = 4.65 Hz, 2H), 3.31 (t, 7 = 8.15 Hz, 1H), 3.22 (s, 3H), 3.06 (d, 7 = 11.03 Hz, 1H), 2.89 (d, 7 = 11.05 Hz, 1H), 2.60 (s, 2H),

2.32 (s, 3H), 2.22 (s, 3H), 2.12 (s, 1H), 2.02 (s, 2H), 1.69 (td, J = 33.01, 26.90, 12.77 Hz, 4H). ¹³C NMR (125 MHz, MeOD) 8 167.60, 160.36, 129.46, 128.41, 128.14, 110.02, 62.71, 60.24, 59.71, 56.16, 50.60, 50.12, 49.60, 48.70, 41.28, 36.36, 30.54, 9.62, 8.73. IR (neat) max = 3400, 2926, 2808, 1645, 1421, 1362, 1223, 1092, 1028, 989, 745, 701, 582, 580, 566. HRMS (ESI) C22H31N3O2. Calculated: [M+H]⁺, 370.2495 Found: [M+H]⁺, 370.2497.

(2-benzyl-8-(cyclohexylmethyl)-2,8-diazaspiro[4.5]decan-4-yl)methyl hex-5-ynoate (S6)- To a flame dried flask that was purged with argon was added equipped a magnetic stir bar. The flask was cooled to 0 °C before addition of (2-benzyl-8-(cyclohexylmethyl)-2,8-diazaspiro[4.5]decan- 4-yl)methanol (14.2 mg, 0.0398 mmol) and triethylamine (8.1 mg, 0.0797 mmol) as a solution in DCM (0.01 M). The reaction was stirred for 5 minutes before addition of hex-5-ynoyl chloride (10.4 mg, 0.0797 mmol). The mixture stirred for 3 hours with temperature increasing from 0 °C to 25 °C before the reaction was quenched with H2O. The aqueous layer was extracted in triplicate with DCM. The combined organic extracts were washed with brine, dried with NazSCU, and concentrated under vacuum. The crude product was purified by column chromatography (0- 100% DCM:MeOH over 10 min, holding at 15% MeOH for 5 min) to afford the desired product as a clear oil: 12.2 mg (68%). H NMR (500 MHz, MeOD) 5 7.36 - 7.29 (m, 4H), 7.27 (t, J = 6.76 Hz, 1 H), 4.20 (dd, 7 = 1 1.21 , 6.45 Hz, 1H), 4.07 (dd, J = 1 1 .19, 7.38 Hz, 1 H), 3.69 (d, 7 = 3.12 Hz, 2H), 2.99 (t, J = 8.71 Hz, 1H), 2.85 (d, J = 7.98 Hz, 1H), 2.76 (s, 3H), 2.53 (d, J = 9.75 Hz, 1H), 2.46 (t, J = 7.34 Hz, 2H), 2.41 (d, J = 8.90 Hz, 1H), 2.28 - 2.15 (m, 5H), 1.95 (t, 7 = 13.08 Hz, 1H), 1.85 - 1.67 (m, 13H),

1.33 (d, 7 = 12.83 Hz, 2H), 1.22 (d, 7 = 12.68 Hz, 1H), 1.01 (d, 7 = 11.76 Hz, 2H). ¹³C NMR (125 MHz, MeOD) 8 173.18, 137.86, 128.67, 128.03, 127.01, 68.95, 63.13, 62.58, 59.83, 55.96, 51.21, 50.64, 46.25, 40.62, 34.97, 33.64, 32.27, 31.02, 29.46, 25.86, 25.41, 23.50, 17.03. IR (neat)

= 2922, 2850, 2797, 1583, 1495, 1450, 1377, 1265, 1072, 1028, 984. HRMS (ESI) C29H42N2O2. Calculated: [M+H]⁺, 451.3325 Found: [M+H]⁺, 451.3325.

(2-benzyl-8-(cyclohexylmethyl)-2,8-diazaspiro[4.5]decan-4-yl)methyl 4-(l-(13-oxo-17-(2- oxohexahydro-lH-thieno[3,4-d]imidazol-4-yl)-3,6,9-trioxa-12-azaheptadecyl)-lH-l,2,3- triazol-5- yl)butanoate (Biotin-RHl 115)- To a flame dried flask that was purged with argon was added equipped a magnetic stir bar. To the flask was added 2-benzyl-8-(cyclohexyhnethyl)-2,8- diazaspiro[4.5]decan-4- yl)methyl hex-5-ynoate (12.2 mg, 0.0271 mmol), Azide-PEG3-biotin conjugate (14.4 mg, 0.0325 mmol), and CUSO4-5H2O (20 moi%) as a solution in 4:1 H2O:THF. The mixture was healed to 90 °C and stirred for 48 hours. The mixture was concentrated and purified by column chromatography (0-100% DCM:MeOH over 35 min, holding at 18% MeOH for 10 min) to afford the desired product as a slightly yellow solid: 8.7 mg (36%). ¹H NMR (500 MHz, MeOD) 6 7.80 (s, 1H), 7.31 (d, J = 6.59 Hz, 4H), 7.25 (d, J = 6.59 Hz, 1H), 5.23 (t, 1H), 4.54 (t, J = 5.04 Hz, 2H), 4.48 (t, J = 6.44 Hz, 1H), 4.36 - 4.27 (m, 3H), 4.23 - 4.13 (m, 3H), 4.03 (dd, J = 11.07, 7.88 Hz, 1H), 3.88 (t, J = 5.08 Hz, 2H), 3.63 (s, 1H), 3.60 (s, 3H), 3.58 (s, 3H), 3.52 (t, J= 5.41 Hz, 2H), 3.21 - 3.15 (m, 1H), 2.91 (dd, J = 11.69, 5.89 Hz, 2H), 2.80 (d, J = 9.67 Hz, 1H), 2.76 - 2.66 (m, 3H), 2.44 - 2.30 (m, 7H), 2.20 (t, J = 7.39 Hz, 2H), 2.04 (d, J = 2.65 Hz, 5H), 2.02 (s, 2H), 1.95 (t, J = 7.48 Hz, 2H), 1.78 - 1.70 (m, 6H), 1.66 - 1.58 (m, 8H), 1.53 (d, J = 6.40 Hz, 4H), 1.43 (q, J = 7.73, 7.00 Hz, 3H). ¹³C NMR (125 MHz, MeOD) 88 173.31, 166.78, 166.22, 128.84, 128.68, 128.03, 127.99, 126.94, 122.82, 76.84, 70.14, 70.08, 70.02, 69.86, 69.22, 69.02, 63.32, 62.82, 61.96, 60.23, 59.94, 56.06, 55.59, 54.85, 51.51, 50.77, 39.66, 38.94, 35.34, 32.81, 31.67, 31.36, 29.34, 29.06, 28.36, 28.11, 26.71, 26.05, 25.58, 25.45, 24.41, 24.21, 22.33, 18.66, 15.89, 13.03. IR (neat) Umax = 3137, 3079, 2922, 2854, 1698, 1607, 1538, 1501, 1435, 1402, 1347, 1331, 1309, 1276, 1254, 1219, 1114, 1086, 1048, 900, 879, 847, 814, 744, 733, 687, 607, 592, 585, 564. HRMS (ESI) C47H74N8O7S. Calculated: [M+H]⁺, 895.5479 Found: [M+H]⁺, 895.5.

Tert-butyl 4-(2-methoxy-2-oxoethylidene)piperidine-l-carboxylate: To a flame dried flask equipped with a stir bar was added NaH (402.5 mg, 8.39 mmol) as a 60% dispersion in mineral oil and was subsequently suspended in DMF (0.1M). The solution was cooled to 0 °C and then trimethyl phosphonoacetate (2.29 g, 12.58 mmol) was added dropwise and allowed to react for 30 minutes. Tert-butyl 4-oxopiperidine- 1 -carboxylate (1 g, 8.39 mmol) was then added and the reaction was allowed to stir at room temperature for 6 hours. Reaction was stirred for 6 hours at room temperature and was subsequently quenched with aqueous NH4CL The reaction was extracted with ethyl acetate 3x and the organic layer was subsequently dried with sodium sulfate and concentrated under vacuum. The product was purified using column chromatography (0-20% EtOAc:hexane over 7 minutes) to afford the desired product as a white solid: 1.198 g (94%).

'H NMR (500 MHz, CDCI3) 6 5.44 (s, 1H), 3.40 (s, 3H), 3.23 (t, J = 5.9 Hz, 2H), 3.19 (t, J = 5.9 Hz, 2H), 2.66 (t, J = 6.1 Hz, 2H), 2.01 (t, J = 6.0 Hz, 2H), 1.19 (s, 9H).

¹³C NMR (125 MHz, CDCI3) 5 166.15, 157.85, 154.13, 114.56, 79.33, 50.52, 44.28, 36.12, 29.24, 28.08. HRMS (ESI): C13H22NO4+, Calculated: [M+H-Boc]+, 156.1025; Found: [M+H-Boc]+, 156.1019.

IR (neat): 2976, 2950, 1696, 1654, 1421 cm ¹.

8' (tert-butyl) 4-methyl 2-benzyl-2,8-diazaspiro[4.5]decane-4,8-dicarboxylate: To a flame dried flask equipped with a stir bar was added solid Tert-butyl 4-(2-methoxy-2-oxoethylidene)piperidine-l -carboxylate (504.3 mg, 1.98 mmol) and LiF (153.7 mg, 5.93 mmol). N-(Methoxymethyl)-N-

( trimethylsilylmethyl) benzylamine was then added (937.9 mg, 3.95 mmol) and the reaction was heated to 140 °C for 24 hours. The reaction was removed from heat and filtered to remove excess LiF. The product was purified using column chromatography (0-100% DCM:ACN over 10 minutes) to afford the desired product as a yellow oil 653.7 mg (84%).

'H NMR (500 MHz, CDCI3) 8 7.27 (d, J = 6.7 Hz, 4H), 7.23 - 7.18 (m, 1H), 3.82 (s, 2H), 3.63 (s, 3H), 3.62 (d, J = 4.2 Hz, 2H), 2.94 (t, J = 8.6 Hz, 1H), 2.83 (d, J = 10.3 Hz, 1H), 2.78 (t, J = 8.8 Hz, 2H), 2.69 (t, J = 8.0 Hz, 1H), 2.31 (d, J = 9.2 Hz, 1H), 1.81 - 1.72 (m, 1H), 1.62 (d, J = 13.2 Hz, 1H), 1.51 (d, J = 12.5 Hz, 1H), 1.41 (s, 9H), 1.35 (d, J = 10.6 Hz, 1H).

¹³C NMR (125 MHz, CDCh) 8 173.23, 154.77, 138.83, 128.42, 128.27, 126.98, 79.37, 61.93, 59.84, 55.27, 53.42, 51.46, 44.03, 41.32, 36.88, 32.48, 28.40.

HRMS (ESI): Calculated: [M+H]+, 389.2440; Found: [M+H]+, 389.2439.

IR (neat): 2923, 2851, 2801, 1735, 1692, 1421 cm'¹.

Methyl 2-benzyl-2,8-diazaspiro[4.5]decane-4-carboxylate: Following General Procedure Q, 8-(tert-butyl)

4-mcthyl 2-bcnzyl-2,8-diazaspiro[4.5]dccanc-4,8-dicarboxylatc (653.7 mg, 1.68 mmol) was used. The product was taken crude to the next step.

HRMS (ESI): C17H24N2O2+, Calculated: [M+H]+, 289.1916; Found: [M+H]+, 289.1912.

IR (neat): 3405, 2921, 2804, 1733, 1677, 1453, 1436 cm'¹.

Methyl 2-benzyl-8-(cyclohexylmethyl)-2,8-diazaspiro[4.5]decane-4-carboxylate: Following General Procedure L, methyl 2-benzyl-2,8-diazaspiro[4.5]decane-4-carboxylate (416.3 mg, 1.444 mmol) and cyclohexane carboxaldehyde (809.87 mg, 7.22 mmol) were used. The product was purified using column chromatography (0-10% DCM:MeOH over 10 minutes) to afford the desired product as a yellow oil 453.1 mg (82%).

'H NMR (500 MHz, Methanol-^) δ 7.35 - 7.28 (m, 4H), 7.27 - 7.22 (m, 1H), 3.69 (s, 3H), 3.66 (d, J = 3.8 Hz, 2H), 3.03 (d, J = 14.5 Hz, 2H), 2.95 - 2.90 (m, 1H), 2.85 - 2.76 (m, 3H), 2.48 - 2.42 (m, 3H), 2.38 (s, 1H), 2.06 (td, J = 13.1, 4.1 Hz, 1H), 1.81 - 1.58 (m, 10H), 1.28 (qt, J = 12.5, 3.4 Hz, 2H), 1.18 (qt, J = 12.0, 2.6 Hz, 1H), 0.93 (qd, J = 13.3, 4.0 Hz, 2H).

¹³C NMR (125 MHz, Methanol-^) δ 172.86, 138.07, 128.59, 128.03, 126.97, 64.33, 62.08, 59.65, 54.63, 52.78, 51.25, 50.76, 50.66, 42.73, 34.98, 33.85, 31.16, 30.87, 25.97, 25.49.

HRMS (ESI): C24H37N2O2+, Calculated: [M+H]+, 385.2855; Found: [M+H]+, 385.2859.

IR (neat): 2921, 2849, 2802, 1735, 1695, 1450 cm'¹.

Methyl 8-(cyclohexylmethyl)-2-(phenylsulfonyl)-2,8-diazaspiro[4.5]decane-4-carboxylate: Following

General Procedure M, Methyl 2-benzyl-8-(cyclohexylmethyl)-2,8-diazaspiro[4.5]decane-4-carboxylate (45.8 mg, 0.119 mmol) and benzenesulfonyl chloride (20.98 mg, 0.1188 mmol) were used. The product was purified using column chromatography (0-10% DCM:MeOH over 15 minutes) to afford the desired product as an orange solid: 19.8 mg (38%).

'H NMR (500 MHz, CDC1₃) 6 7.86 (d, J = 7.3 Hz, 2H), 7.61 (t, J = 7.5 Hz, 1H), 7.55 (t, J = 7.6 Hz, 2H), 3.60 - 3.57 (m, 1H), 3.55 (s, 4H), 3.47 (dd, J = 10.4, 6.4 Hz, 1H), 3.26 (s, 2H), 2.72 (t, J = 7.1 Hz, 1H), 2.06 (d, J = 4.1 Hz, 2H), 1.98 (s, 1H), 1.88 (s, 2H), 1.68 (q, J = 15.7 Hz, 6H), 1.42 (d, J = 13.7 Hz, 3H), 1.25 (s, 1H), 1.15 (dq, J = 24.3, 13.0 Hz, 5H), 0.83 (t, J = 11.6 Hz, 3H).

¹³C NMR (125 MHz, CDCI3) 5 171.50, 136.70, 132.79, 129.05, 127.45, 65.58, 55.03, 52.19, 51.83, 51.14, 50.76, 48.28, 44.00, 35.05, 31.89, 30.09, 26.69, 26.08.

HRMS (ESI): C23H35N2O4S+, Calculated: [M+H]+, 435.2312; Found: [M+H]+, 435.2315.

AD-1059: Following General Procedure O, methyl 8-(cyclohexylmethyl)-2-(phenylsulfonyl)-2,8- diazaspiro[4.5]decane-4-carboxylate (35.95 mg, 0.083 mmol) was used. The product was purified using column chromatography (0-10% DCM:MeOH over 13 minutes) to afford the desired product as an orange solid: 12.9 mg (17%).

'H NMR (500 MHz, CDCI3) 6 7.84 (d, J = 7.1 Hz, 2H), 7.61 (t, J = 7.4 Hz, 1H), 7.54 (t, J = 7.5 Hz, 2H), 3.68 (dd, J = 11.1, 6.1 Hz, 1H), 3.64 (s, 1H), 3.48 - 3.37 (m, 4H), 3.17 (dd, J = 10.3, 7.1 Hz, 1H), 3.06 (d, J = 10.1 Hz, 1H), 2.96 - 2.85 (m, 2H), 2.36 (s, 2H), 2.23 (s, 2H), 2.12 - 1.98 (m, 2H), 1.93 (s, 1H), 1.79 (d, J = 12.1 Hz, 2H), 1.71 (d, J = 13.0 Hz, 2H), 1.65 (d, J = 11.9 Hz, 1H), 1.58 (s, 1H), 1.31 (d, J = 15.2 Hz, 2H), 1.27 - 1.22 (m, 3H), 1.22 - 1.08 (m, 3H), 0.91 (q, J = 13.4 Hz, 3H).

¹³C NMR (125 MHz, CDCk) 5 136.51, 132.86, 129.10, 127.32, 70.53, 64.95, 60.57, 55.92, 51.22, 50.94, 49.13, 41.65, 34.22, 33.25, 31.70, 27.15, 26.22, 25.79.

HRMS (ESI): C22H35N2O3S+, Calculated: [M+H]+, 407.2363; Found: [M+H]+, 407.2374.

Methyl 8-(cyclohexylmethyl)-2-tosyl-2,8-diazaspiro[4.5]decane-4-carboxylate: Following General

Procedure M, methyl 2-benzyl-8-(cyclohexylmethyl)-2,8-diazaspiro[4.5]decane-4-carboxylate (107.98 mg, 0.281 mmol) and tosylsulfonyl chloride (58.91 mg, 0.309 mmol) were used. The product was purified using column chromatography (0-10% DCM:MeOH over 15 minutes) to afford the desired product as an orange solid: 70.48 mg (56%).

'H NMR (500 MHz, CDC1₃) 6 7.72 (d, J = 7.8 Hz, 2H), 7.33 (d, J = 7.9 Hz, 2H), 3.56 (s, 3H), 3.55 - 3.51 (m, 1H), 3.45 (dd, J = 10.4, 6.8 Hz, 1H), 3.23 (t, J = 8.3 Hz, 2H), 2.70 (t, J = 7.3 Hz, 1H), 2.56 (d, J = 20.1 Hz, 2H), 2.43 (s, 3H), 2.05 (d, J = 19.9 Hz, 2H), 1.93 (d, J = 31.3 Hz, 2H), 1.80 (s, 1H), 1.68 (q, J = 16.2 Hz, 6H), 1.48 - 1.32 (m, 3H), 1.24 (d, J = 6.3 Hz, 1H), 1.14 (dq, J = 24.2, 12.5 Hz, 4H), 0.82 (q, J = 11.2 Hz, 2H).

¹³C NMR (125 MHz, CDCh) 5 171.52, 143.54, 133.74, 129.66, 127.55, 65.61, 55.08, 52.27, 51.81, 51.20, 50.77, 48.25, 44.01, 35.09, 31.90, 30.20, 29.71, 26.72, 26.10, 21.57.

HRMS (ESI): C24H37N2O4S+, Calculated: [M+H]+, 449.2469; Found: [M+H]+, 449.2477.

AD-1069: Following General Procedure O, methyl 8-(cyclohexylmethyl)-2-tosyl-2,8-diazaspiro[4.5]decane- 4-carboxylate (70.48 mg, 0.083 mmol) was used. The product was purified using column chromatography (0-10% DCM:MeOH over 13 minutes) to afford the desired product as an orange solid: 33.7 mg (51%).

'H NMR (500 MHz, CDCI3) 6 7.70 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 7.9 Hz, 2H), 3.65 (dd, J = 11.8, 6.4 Hz, 1H), 3.42 (dd, J = 10.1, 7.7 Hz, 1H), 3.36 (t, J = 9.4 Hz, 2H), 3.15 (dd, J = 10.1, 6.9 Hz, 1H), 3.02 (d, J = 10.1 Hz, 1H), 2.76 (d, J = 11.5 Hz, 2H), 2.42 (s, 3H), 2.25 (d, J = 6.9 Hz, 2H), 2.11 (s, 2H), 1.97 (p, J = 7.1 Hz, 1H), 1.74 (d, J = 11.5 Hz, 3H), 1.66 (dd, J = 22.7, 12.3 Hz, 4H), 1.25 (d, J = 14.9 Hz, 3H), 1.23 - 1.05 (m, 4H), 0.86 (q, J = 12.1 Hz, 2H).

¹³C NMR (125 MHz, CDCh) 5 143.64, 133.51, 129.71, 127.43, 70.53, 65.24, 60.61, 55.93, 51.22, 50.91, 49.37, 48.70, 41.84, 34.50, 33.91, 31.78, 29.70, 27.79, 26.41, 25.92, 21.58.

HRMS (ESI): C23H37N2O3S+, Calculated: [M+H]+, 421.2519; Found: [M+H]+, 421.2526.

Methyl 8-(cyclohexylmethyl)-2-phenethyl-2,8-diazaspiro[4.5]decane-4-carboxylate: Following General

Procedure N, methyl 2-henzyl-8-(cyclohexylmethyl)-2,8-diazaspiro[4.5]decane-4-carboxylate (141.5 mg, 0.368 mmol) and phenethyl iodide (93.9 mg, 0.4058 mmol) were used. The product was purified using column chromatography (0-10% DCM:MeOH over 10 minutes) to afford the desired product as a yellow oil: 87.8 mg (60%).

'H NMR (500 MHz, Methanol-^) 5 7.29 - 7.23 (m, 2H), 7.23 - 7.19 (m, 2H), 7.19 - 7.14 (m, 1H), 3.69 (s, 3H), 3.02 (dd, J = 9.5, 7.3 Hz, 1H), 2.89 (d, J = 9.6 Hz, 1H), 2.84 - 2.77 (m, 3H), 2.77 - 2.71 (m, 3H), 2.71 - 2.61 (m, 2H), 2.37 (d, J = 9.6 Hz, 1H), 2.11 (d, J = 7.0 Hz, 2H), 2.03 (t, J = 11.2 Hz, 1H), 1.94 (td, J = 12.2, 3.9 Hz, 2H), 1.80 - 1.69 (m, 4H), 1.64 (d, J = 13.9 Hz, 2H), 1.55 - 1.45 (m, 3H), 1.33 - 1.13 (m, 3H), 0.89 (qd, J = 12.3, 3.2 Hz, 2H).

¹³C NMR (125 MHz, Methanol-^) δ 172.81, 139.79, 128.32, 128.09, 125.84, 65.05, 62.55, 57.94, 54.94, 52.84, 51.44, 50.77, 50.75, 42.92, 35.70, 34.48, 34.36, 31.51, 31.48, 26.17, 25.68.

HRMS (ESI): C25H39N2O2+, Calculated: [M+H]+, 399.3006; Found: [M+H]+, 399.016.

IR (neat): 2925, 2851, 1733, 1670, 1603, 1585, 1450 cm-¹.

AD-1190: Following General Procedure P, methyl 8-(cyclohexylmethyl)-2-phenethyl-2,8- diazaspiro[4.5]decane-4-carboxylate (85.3 mg, 0.214 mmol) was used. The product was purified using column chromatography (0-10% DCM:MeOH on neutral alumina over 8 minutes) to afford the desired product as a yellow oil: 66.7 mg (84%).

'H NMR (500 MHz, Methanol-^) 8 7.26 (t, J = 7.5 Hz, 2H), 7.20 (d, J = 6.8 Hz, 2H), 7.19 - 7.14 (m, 1H), 3.72 (dd, J = 10.6, 5.5 Hz, 1H), 3.50 (dd, J = 10.6, 8.3 Hz, 1H), 3.09 - 3.02 (m, 1H), 2.85 - 2.77 (m, 3H), 2.75 - 2.63 (m, 4H), 2.45 - 2.32 (m, 2H), 2.12 (d, J = 6.9 Hz, 2H), 2.02 (t, J = 10.6 Hz, 1H), 1.99 - 1.89 (m, 2H), 1.84 - 1.74 (m, 3H), 1.70 (td, J = 12.7, 3.9 Hz, 4H), 1.58 - 1.45 (m, 3H), 1.32 - 1.13 (m, 3H), 0.90 (qd, J = 12.1, 3.2 Hz, 2H).

¹³C NMR (125 MHz, Methanol-^) 8 139.86, 128.28, 128.09, 125.81, 65.87, 63.79, 61.01, 58.59, 57.14, 51.86, 51.08, 49.74, 40.83, 36.91, 34.86, 34.53, 31.83, 30.90, 26.36, 25.86.

HRMS (ESI): C24H39N2O+, Calculated: [M+H]+, 371.3062; Found: [M+H]+, 371.3056.

IR (neat): 3345, 2919, 2848, 2801, 1495, 1449 cm ¹.

Methyl 2,8-bis(cyclohexylmethyl)-2,8-diazaspiro[4.5]decane-4-carboxylate: To a flame dried flask equipped with a stir bar was added palladium on carbon (10%) (244.9 mg, 0.23 mmol) and a slurry was formed with a small volume of methanol. Methyl 2-benzyl-8-(cyclohexylmethyl)-2,8-diazaspiro[4.5]decane- 4-carboxylate (442.5 mg, 1.15 mmol) was then added as a solution in methanol (0.1M) and the reaction was heated to 35 °C for 6 hours. The reaction was filtered through celite and then concentrated under vacuum. The crude product was then dissolved in methanol (0.5M) and cyclohexyl carboxaldehyde (645.4 mg, 0.4058 mmol), and TFA (1573.5 mg, 13.8 mmol) were added. The reaction was heated to 50 °C for 6 hours before the addition of sodium borohydride (217.52 mg, 5.75 mmol). The reaction was stirred for 12 hours before being removed from heat. The reaction was quenched with H2O and was extracted 3x with EtOAc and NaHC’Ch. The organic layer was dried with sodium sulfate and subsequently concentrated under vacuum. The product was purified using column chromatography (0-10% DCM:MeOH over 10 minutes) to afford the desired product as a yellow oil: 156.4 mg (35%).

'H NMR (500 MHz, Methanol-d4) δ 3.70 (s, 3H), 3.09 (dd, J = 25.0, 11.6 Hz, 2H), 3.00 (d, J = 8.7 Hz, 1H), 2.90 - 2.85 (m, 1H), 2.85 - 2.79 (m, 1H), 2.55 (d, J = 7.0 Hz, 3H), 2.50 (d, J = 9.7 Hz, 1H), 2.40 (qd, J = 11.9, 7.1 Hz, 2H), 2.14 - 2.04 (m, 1H), 1.86 - 1.75 (m, 6H), 1.75 - 1.61 (m, 9H), 1.51 (ttt, J = 10.7, 6.9, 3.4 Hz, 1H), 1.35 - 1.25 (m, 4H), 1.24 - 1.14 (m, 2H), 1.01 - 0.86 (m, 4H).

¹³C NMR (125 MHz, Methanol-^) 8 172.64, 64.10, 62.98, 62.47, 55.15, 52.41, 51.12, 50.85, 50.56, 42.53, 36.36, 34.56, 33.70, 31.46, 31.36, 31.07, 30.46, 26.38, 25.94, 25.73, 25.46.

HRMS (ESI): C24H43N2O2+, Calculated: [M+H]+, 391.3319; Found: [M+H]+, 391.3325. IR (neat): 2924, 2851, 1735, 1674, 1585, 1449 cm'¹.

AD-2052: Following General Procedure P, methyl 2,8-bis(cyclohexylmethyl)-2,8-diazaspiro[4.5]decane-4- carboxylate (77.3 mg, 0.198 mmol) was used. The product was purified using column chromatography (0- 10% DCM:MeOH on neutral alumina over 8 minutes) to afford the desired product as a yellow oil: 39.8 mg (54%).

NMR (500 MHz, Methanol-d4) 8 3.70 (dd, J = 10.7, 5.4 Hz, 1H), 3.56 (dd, J = 10.7, 7.0 Hz, 1H), 3.08 (dd, J = 10.0, 7.1 Hz, 1H), 2.87 (d, J = 7.3 Hz, 2H), 2.82 (d, J = 10.3 Hz, 1H), 2.62 (d, J = 10.1 Hz, 1H), 2.60

- 2.54 (m, 1H), 2.50 - 2.41 (m, 2H), 2.32 (s, 3H), 2.28 - 2.19 (m, 1H), 2.02 (p, J = 7.2 Hz, 1H), 1.88 - 1.50 (m, 17H), 1.29 (q, J = 12.5 Hz, 5H), 1.24 - 1.15 (m, 2H), 0.94 (qd, J = 11.8, 3.4 Hz, 4H).

¹³C NMR (125 MHz, Methanol-d4) 3 65.10, 63.72, 63.36, 60.85, 57.25, 51.43, 51.02, 40.74, 36.09, 34.39, 31.50, 31.31, 31.27, 30.10, 26.22, 26.16, 25.66. HRMS (ESI): C23H43N2O+, Calculated: [M+H]+, 363.3370; Found: [M+H]+, 363.3375.

IR (neat): 3355, 2920, 2850, 2800, 2771, 1448 cm'¹.

Methyl 8-((lH-indol-3-yl)methyl)-2-benzyl-2,8-diazaspiro[4.5]decane-4-carboxylate: Following General Procedure L, methyl 2-benzyl-2,8-diazaspiro[4.5]decane-4-carboxylate (198.1 mg, 0.687mmol) and 1 H- Indole-3-carbaldehyde (498.62 mg, 3.435 mmol) were used. The product was purified using column chromatography (0-10% DCM:MeOH on neutral alumina over 10 minutes) to afford the desired product as a yellow oil: 44 mg (15%).

'H NMR (600 MHz, Methanol-*) 57.60 (dt, J = 7.9, 1.0 Hz, 1H), 7.36 (dt, J = 8.1, 0.9 Hz, 1H), 7.33 - 7.29 (m, 4H), 7.25 (ddt, J = 8.5, 5.6, 2.9 Hz, 1H), 7.19 (s, 1H), 7.10 (ddd, J = 8.2, 6.9, 1.2 Hz, 1H), 7.03 (ddd, J = 7.9, 7.0, 1.1 Hz, 1H), 3.68 (s, 2H), 3.61 (d, J = 3.1 Hz, 5H), 2.88 (dd, J = 9.4, 7.2 Hz, 1H), 2.82 (d, J = 10.6 Hz, 1H), 2.79 - 2.73 (m, 3H), 2.72 - 2.67 (m, 1H), 2.33 (d, J = 9.5 Hz, 1H), 2.13 (d, J = 36.5 Hz, 2H), 1.98 - 1.90 (m, 1H), 1.65 (dq, J = 13.4, 3.0 Hz, 1H), 1.56 - 1.45 (m, 2H).

¹³C NMR (150 MHz, Methanol-*) 5 173.18, 138.17, 136.44, 128.63, 128.12, 127.96, 126.88, 124.87, 120.97, 118.64, 118.22, 110.88, 109.39, 62.75, 59.80, 54.80, 53.09, 52.35, 50.58, 50.48, 49.80, 43.20, 36.58, 32.23.

HRMS (ESI): C₂₆H₃₂N₃O2+, Calculated: [M+H]+, 418.2489; Found: [M+H]+, 418.2499.

IR (neat): 3416, 3398, 2938, 2921, 2806, 1732, 1454, 1436 cm*

AD-2081: Following General Procedure P, methyl 8-((lH-indol-3-yl)methyl)-2-benzyl-2,8- diazaspiro[4.5]decane-4-carboxylate (22.0 mg, 0.054 mmol) was used. The product was purified using column chromatography (0-10% DCM:MeOH on neutral alumina over 8 minutes) to afford the desired product as a white solid: 5 mg (23%).

'H NMR (600 MHz, Methanol-*) 57.61 (d, J = 7.9 Hz, 1H), 7.38 (d, J = 8.1 Hz, 1H), 7.29 (d, J = 4.8 Hz, 4H), 7.26 - 7.20 (m, 2H), 7.14 - 7.09 (m, 1H), 7.05 (td, J = 7.5, 1.1 Hz, 1H), 3.76 (s, 2H), 3.66 (dd, J = 10.7,

5.6 Hz, 1H), 3.55 (s, 2H), 3.44 (dd, J = 10.7, 8.1 Hz, 1H), 2.93 - 2.83 (m, 3H), 2.66 (d, J = 9.8 Hz, 1H), 2.34 - 2.28 (m, 2H), 2.23 (s, 1H), 2.16 (s, 1H), 1.96 - 1.89 (m, 1H), 1.78 (td, J = 12.9, 4.1 Hz, 1H), 1.70 (td, J = 12.9, 4.0 Hz, 1H), 1.56 (d, J = 13.6 Hz, 1H), 1.47 (d, J = 12.9 Hz, 1H).

¹³C NMR (150 MHz, Methanol-^) 8 138.08, 136.45, 128.81, 128.08, 128.02, 126.96, 125.57, 121.21, 118.95, 118.23, 111.15, 108.33, 63.36, 61.08, 60.20, 56.78, 52.27, 50.53, 49.88, 49.60, 40.66, 36.42, 30.47.

HRMS (ESI): C2₅H₃₂N₃O+, Calculated: [M+H]+, 390.2540; Found: [M+H]+, 390.2554.

IR (neat): 3403, 3253, 3058, 3030, 2920, 2808, 1493, 1470, 1454 cm’¹.

8- (tert-butyl) 4-methyl 2-phenethyl-2,8-diazaspiro[4.5]decane-4,8-dicarboxylate: Following General Procedure N, 8-(tert-butyl) 4-methyl 2-benzyl-2,8-diazaspiro[4.5]decane-4,8-dicarboxylate (409.4 mg, 1.05 mmol) and phenethyl iodide (268.03 mg, 1.155 mmol) were used. The product was purified using column chromatography (0-10% ACN:MeOH over 13 minutes) to afford the desired product as a clear oil: 238.4 mg (55%).

'H NMR (500 MHz, CDC1₃) 5 7.13 (t, J = 7.4 Hz, 2H), 7.05 (dd, J = 14.0, 7.0 Hz, 3H), 3.74 (s, 2H), 3.52 (s, 3H), 3.36 (s, 1H), 2.91 (t, J = 8.6 Hz, 1H), 2.76 (d, J = 9.3 Hz, 2H), 2.72 - 2.63 (m, 4H), 2.59 (td, J = 8.6, 5.6 Hz, 3H), 2.24 (d, J = 9.3 Hz, 1H), 1.66 (td, J = 12.4, 4.6 Hz, 1H), 1.50 (d, J = 15.5 Hz, 1H), 1.38 (d, J = 13.0 Hz, 1H), 1.33 (s, 9H), 1.27 - 1.17 (m, 1H).

¹³C NMR (125 MHz, CDC1₃) 5 172.92, 154.66, 139.95, 128.52, 128.24, 125.97, 79.31, 62.20, 57.72, 55.25, 53.16, 51.36, 49.96, 43.68, 41.15, 36.71, 35.00, 32.42, 28.30.

HRMS (ESI): C2₃H₃₅N₂O₄+, Calculated: [M+H]+, 403.2597; Found: [M+H]+, 403.2596. IR (neat): 2933, 2860, 2802, 1734, 1692, 1424 cm'¹.

Methyl 2-phenethyl-2,8-diazaspiro[4.5]decane-4-carboxylate: Following General Procedure Q, 8-(tert- butyl) 4-methyl 2-phenethyl-2,8-diazaspiro[4.5]decane-4,8-dicarboxylate (249.8 mg, 0.62 mmol) was used. The product was taken crude to the next step.

HRMS (ESI): C18H27N2O2+, Calculated: [M+H]+, 303.2073; Found: [M+H]+, 303.2074. IR (neat): 3397, 2932, 2804, 1732, 1687, 1453, 1436 cm'¹.

Methyl 8-((lH-indol-3-yl)methyl)-2-phenethyl-2,8-diazaspiro[4.5]deeane-4-carboxylate: Following

General Procedure R, methyl 2-phenethyl-2,8-diazaspiro[4.5]decane-4-carboxylate (133.02 mg, 0.44 mmol) and lH-Indole-3-carbaldehyde (96.5 mg, 0.66 mmol) were used. The product was purified using column chromatography (0-100% ACN:MeOH (1% NH4OH) on silica over 11 minutes) to afford the desired product as a white solid: 59.2 mg (40%).

'H NMR (500 MHz, Methanol-d4) 6 7.61 (d, J = 7.9 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H), 7.24 (t, J = 7.3 Hz, 2H), 7.21 - 7.17 (m, 3H), 7.17 - 7.12 (m, 1H), 7.12 - 7.06 (m, 1H), 7.06 - 6.99 (m, 1H), 3.71 (s, 2H), 3.62 (s, 3H), 3.04 - 2.95 (m, 1H), 2.88 - 2.60 (m, 10H), 2.33 (d, J = 9.6 Hz, 1H), 2.27 - 2.18 (m, 1H), 2.17 - 2.08 (m, 1H), 1.94 (td, J = 12.8, 4.3 Hz, 1H), 1.64 (d, J = 13.3 Hz, 1H), 1.56 - 1.43 (m, 2H).

¹³C NMR (125 MHz, Methanol-^) 6 173.03, 139.88, 136.43, 128.25, 128.13, 128.00, 125.74, 124.87, 120.93, 118.59, 118.18, 110.86, 109.29, 62.95, 57.98, 55.07, 52.93, 52.29, 50.49, 49.67, 48.43, 43.06, 36.54, 34.50, 32.18.

HRMS (ESI): C27H34N3O2+, Calculated: [M+H]+, 432.2646; Found: [M+H]+, 432.2653.

IR (neat): 3399, 3058, 3026, 2923, 2849, 2849, 2805, 1731, 1633, 1604 cm'¹.

AD-2143: Following General Procedure P, methyl 8-((lH-indol-3-yl)methyl)-2-phenethyl-2,8- diazaspiro[4.5]decane-4-carboxylate (59.2 mg, 0.045 mmol) was used. The product was purified using column chromatography (0-100% DCM:MeOH (1% NH4OH) on silica over 10 minutes) to afford the desired product as a white solid: 41.3 mg (75%).

'H NMR (500 MHz, Methanol-^) 5 7.63 (d, J = 7.8 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.31 (s, 1H), 7.27 - 7.14 (m, 5H), 7.14 - 7.09 (m, 1H), 7.05 (td, J = 7.5, 1.2 Hz, 1H), 3.90 (s, 2H), 3.69 (dd, J = 10.7, 5.6 Hz, 1H), 3.49 (dd, J = 10.7, 7.9 Hz, 1H), 3.09 (dd, J = 9.8, 7.2 Hz, 1H), 2.98 (d, J = 10.7 Hz, 2H), 2.85 - 2.64 (m, 5H), 2.49 - 2.28 (m, 4H), 1.99 (p, J = 7.7 Hz, 1H), 1.91 - 1.82 (m, 1H), 1.82 - 1.71 (m, 1H), 1.60 (t, J = 14.3 Hz, 2H).

¹³C NMR (125 MHz, Methanol-^) 5 139.62, 136.43, 128.25, 128.04, 127.99, 125.81, 125.75, 121.16, 118.91, 118.05, 113.01, 111.02, 107.62, 63.38, 60.71, 58.27, 56.85, 52.07, 50.25, 49.62, 49.34, 40.59, 36.05, 34.23, 30.10.

HRMS (ESI): C₂6H₃4N₃O+, Calculated: [M+H]+, 404.2696; Found: [M+H]+, 404.2707.

IR (neat): 3231, 3058, 3027, 2921, 2850, 2808, 1651, 1621, 1603, 1545, 1494, 1455 cm’¹.

Methyl 2-phenethyl-8-(pyridin-2-ylmethyl)-2,8-diazaspiro[4.5]decane-4-carboxylate: Following

General Procedure L, methyl 2-phenethyl-2,8-diazaspiro[4.5]decane-4-carboxylate (432.5 mg, 1.43 mmol) and pyridine-2-carbaldehyde (765.8 mg, 7.15 mmol) were used. The product was purified using column chromatography (0-100% ACN:MeOH (1% NH4OH) on silica over 11 minutes) to afford the desired product as a yellow oil: 44 mg (13%).

'H NMR (500 MHz, Methanol-d4) 8 8.45 (d, J = 6.4 Hz, 1H), 7.76 (t, J = 7.7 Hz, 1H), 7.49 (d, J = 7.9 Hz, 1H), 7.24 (t, J = 7.4 Hz, 3H), 7.21 - 7.11 (m, 3H), 3.65 (s, 3H), 3.58 (s, 2H), 2.97 (t, J = 8.2 Hz, 1H), 2.86 (d, J = 9.6 Hz, 1H), 2.80 - 2.72 (m, 4H), 2.72 - 2.62 (m, 4H), 2.33 (d, J = 9.6 Hz, 1H), 2.23 - 2.13 (m, 1H), 2.13 - 2.04 (m, 1H), 1.93 (td, J = 12.4, 4.1 Hz, 1H), 1.62 (d, J = 13.4 Hz, 1H), 1.50 (s, 2H).

¹³C NMR (125 MHz, Methanol-^) 5 172.91, 157.92, 148.26, 139.99, 137.22, 128.42, 128.17, 125.89, 123.76, 122.52, 63.74, 62.85, 58.03, 55.12, 53.11, 51.30, 50.73, 50.48, 43.07, 36.80, 34.69, 32.55.

HRMS (ESI): C₂4H₃₂N₃O2+, Calculated: [M+H]+, 394.2489; Found: [M+H]+, 394.2501.

IR (neat): 3025, 292, 2805, 2769, 1732, 1677, 1589, 1570, 1474, 1434 cm ¹.

AD-2128: Following General Procedure P, AD-2135 (373.8 mg, 0.95 mmol) was used. The product was purified using column chromatography (0-100% DCM:MeOH (1% NH4OH) on silica over 14 minutes) to afford the desired product as a yellow oil: 318.0 mg (92%). 'H NMR (500 MHz, Methanol-^) 5 8.46 (d, J = 3.3 Hz, 1H), 7.77 (td, J = 7.7, 1.8 Hz, 1H), 7.50 (d, J = 7.9 Hz, 1H), 7.31 - 7.22 (m, 3H), 7.22 - 7.11 (m, 3H), 3.72 (dd, J = 10.7, 5.5 Hz, 1H), 3.59 (s, 2H), 3.51 (dd, J = 10.7, 8.1 Hz, 1H), 3.09 - 3.02 (m, 1H), 2.86 - 2.75 (m, 3H), 2.75 - 2.60 (m, 4H), 2.41 (t, J = 10.7 Hz, 2H), 2.23 - 2.13 (m, 1H), 2.10 (t, J = 10.8 Hz, 1H), 1.99 (p, J = 8.0 Hz, 1H), 1.80 (td, J = 12.7, 4.1 Hz, 1H), 1.71 (td, J = 12.5, 3.9 Hz, 1H), 1.54 (d, J = 13.5 Hz, 1H), 1.49 (d, J = 11.8 Hz, 1H).

¹³C NMR (125 MHz, Methanol-A) 3 157.73, 148.22, 139.77, 137.30, 128.38, 128.21, 125.95, 123.91, 122.60, 63.78, 63.69, 60.97, 58.52, 57.07, 51.39, 50.63, 49.65, 40.73, 37.09, 34.46, 31.10.

HRMS (ESI): C₂3H3₂N₃O+, Calculated: [M+H]+, 366.2540; Found: [M+H]+, 366.2545. IR (neat): 3329, 3061, 3025, 2917, 2830, 1591, 1570, 1496, 1475, 1455, 1434 cm’ .

Methyl 2-benzyl-8-(pyridin-2-ylmethyl)-2,8-diazaspiro[4.5]decane-4-carboxylate: Following General Procedure L, 2-benzyl-2,8-diazaspiro[4.5]decane-4-carboxylate (161.8 mg, 0.561 mmol) and pyridine-3- carbaldehyde (300.4 mg, 2.81 mmol) were used. The product was purified using column chromatography (0- 100% ACN:MeOH (1% NH4OH) on silica over 10 minutes) to afford the desired product as a yellow oil: 83.8 mg (39%).

'H NMR (500 MHz, Methanol-^) 5 8.46 (s, 1H), 8.42 (d, J = 5.0 Hz, 1H), 7.78 (d, J = 7.9 Hz, 1H), 7.38 (dd, J = 7.8, 4.9 Hz, 1H), 7.34 - 7.27 (m, 4H), 7.25 - 7.21 (m, 1H), 3.65 (s, 3H), 3.62 (d, J = 5.1 Hz, 2H), 3.47 (s, 2H), 2.89 (dd, J = 9.2, 7.2 Hz, 1H), 2.79 - 2.62 (m, 5H), 2.34 (d, J = 9.4 Hz, 1H), 2.11 - 1.97 (m, 2H), 1.90 (td, J = 12.6, 4.1 Hz, 1 H), 1 .65 (dd, J = 13.2, 3.2 Hz, 1 H), 1.54 (dd, J = 12.9, 3.9 Hz, 1H), 1.50 - 1.40 (m, 1H).

¹³C NMR (125 MHZ, Methanol-^) 5 173.07, 149.65, 147.61, 138.30, 137.99, 133.94, 128.59, 128.01, 126.90, 123.70, 62.46, 59.81, 59.54, 54.85, 53.20, 50.98, 50.60, 50.21, 43.20, 36.65, 32.44.

HRMS (ESI): C23H30N3O2+, Calculated: [M+H]+, 380.2333; Found: [M+H]+, 380.2340.

IR (neat): 3364, 2920, 2849, 2803, 1732, 1692, 1528, 1468, 1453, 1434 cm'¹.

AD-2144: Following General Procedure P, methyl 2-benzyl-8-(pyridin-2-ylmethyl)-2,8- diazaspiro[4.5]decane-4-carboxylate (79.0 mg, 0.208 mmol) was used. The product was purified using column chromatography (0-100% DCM:MeOH (1% NH4OH) on silica over 10 minutes) to afford the desired product as a yellow oil: 31.8 mg (44%).

'H NMR (500 MHz, Methanol-d4) 5 8.47 (d, J = 1.5 Hz, 1H), 8.43 (dd, J = 4.9, 1.7 Hz, 1H), 7.80 (dt, J =

7.8, 1.9 Hz, 1H), 7.39 (dd, J = 7.8, 4.9 Hz, 1H), 7.34 - 7.27 (m, 4H), 7.27 - 7.21 (m, 1H), 3.69 (dd, J = 10.6, 5.7 Hz, 1H), 3.61 (s, 2H), 3.52 - 3.44 (m, 3H), 2.94 (dd, J = 9.5, 7.1 Hz, 1H), 2.73 (d, J = 9.8 Hz, 1H), 2.69 (d, J = 12.1 Hz, 2H), 2.35 (dd, J = 20.1, 9.6 Hz, 2H), 2.12 - 1.98 (m, 2H), 1.97 - 1.91 (m, 1H), 1.72 (dtd, J =

24.8, 12.5, 4.1 Hz, 2H), 1.54 (td, J = 16.2, 3.1 Hz, 2H).

¹³C NMR (125 MHz, Methanol-d4) 3 149.63, 147.54, 138.20, 138.03, 134.02, 128.76, 127.96, 126.88, 123.67, 63.42, 61.09, 60.32, 59.65, 56.94, 51.16, 50.39, 49.81, 40.79, 37.11, 31.18.

HRMS (ESI): C22H30N3O+, Calculated: [M+H]+, 352.2383; Found: [M+H]+, 352.2393.

IR (neat): 3285, 3028, 2914, 2800, 1594, 1578, 1493, 1475, 1451, 1427 cm’¹.

Methyl 2-phenethyl-8-(pyridin-3-ylmethyl)-2,8-diazaspiro[4.5]decane-4-carboxylate: Following

General Procedure L, methyl 2-phenethyl-2,8-diazaspiro[4.5]decane-4-carboxylate (93.6 mg, 0.31 mmol) and pyridine-3-carbaldehyde (166.02 mg, 1.55 mmol) were used. The product was purified using column chromatography (0-100% ACN:MeOH (1% NH4OH) on silica over 10 minutes) to afford the desired product as a yellow oil: 62.2 mg (51%).

'H NMR (500 MHz, Methanol-d4) δ 8.48 (d, J = 2.8 Hz, 1H), 8.44 (dd, J = 5.0, 1.7 Hz, 1H), 7.81 (dt, J = 7.9, 1.9 Hz, 1H), 7.40 (dd, J = 8.3, 4.5 Hz, 1H), 7.25 (t, J = 7.3 Hz, 2H), 7.20 (d, J = 6.6 Hz, 2H), 7.16 (t, J = 7.2 Hz, 1H), 3.67 (s, 3H), 3.52 (s, 2H), 3.04 - 2.98 (m, 1H), 2.88 (d, J = 9.6 Hz, 1H), 2.79 (td, J = 8.7, 4.8 Hz, 3H), 2.75 - 2.63 (m, 5H), 2.37 (d, J = 9.5 Hz, 1H), 2.16 (t, J = 10.5 Hz, 1H), 2.08 (t, J = 11.6 Hz, 1H), 1.92 (td, J = 12.6, 4.1 Hz, 1H), 1.65 (dd, J = 13.3, 3.1 Hz, 1H), 1.53 (d, J = 11.6 Hz, 1H), 1.50 - 1.43 (m, 1H).

¹³C NMR (125 MHz, Methanol-d4) δ 172.95, 149.64, 147.62, 139.91, 138.01, 133.92, 128.30, 128.06, 125.80, 123.71, 62.77, 59.55, 58.01, 55.03, 53.05, 50.92, 50.62, 50.12, 43.04, 36.62, 34.56, 32.39.

HRMS (ESI): C24H₃₂N₃O2-I-, Calculated: [M+H]+, 394.2489; Found: [M+H]+, 394.2499.

IR (neat): 3027, 2941, 2924, 2803, 2770, 1732, 1476, 1453, 1431 cm ¹.

AD-2167: Following General Procedure P, methyl 2-phenethyl-8-(pyridin-3-ylmethyl)-2,8- diazaspiro[4.5]decane-4-carboxylate (62.2 mg, 0.158 mmol) was used. The product was purified using column chromatography (0-100% DCM:MeOH (1% NH4OH) on silica over 11 minutes) to afford the desired product as a yellow oil: 38.3 mg (66%).

'H NMR (500 MHz, Methanol-d4) δ 8.49 (d, J = 2.4 Hz, 1H), 8.44 (dd, J = 4.9, 1.7 Hz, 1H), 7.82 (dt, J = 7.9, 1.9 Hz, 1H), 7.41 (dd, J = 7.8, 4.9 Hz, 1H), 7.29 - 7.23 (m, 2H), 7.22 - 7.12 (m, 3H), 3.70 (dd, J = 10.7, 5.6 Hz, 1H), 3.53 (s, 2H), 3.51 - 3.43 (m, 1H), 3.06 (dd, J = 9.6, 7.1 Hz, 1H), 2.82 (dd, J = 20.0, 8.7 Hz, 3H), 2.76 - 2.62 (m, 4H), 2.40 (dd, J = 18.5, 9.8 Hz, 2H), 2.15 (t, J = 10.5 Hz, 1H), 2.08 (t, J = 10.5 Hz, 1H), 2.03 - 1.92 (m, 1H), 1.79 (td, J = 12.6, 4.1 Hz, 1H), 1.74 - 1.64 (m, 1H), 1.54 (t, J = 14.3 Hz, 2H).

¹³C NMR (125 MHz, Methanol-^) δ 149.63, 147.56, 139.84, 138.03, 134.01, 128.25, 128.07, 125.79, 123.69, 63.70, 60.93, 59.66, 58.52, 57.04, 51.10, 50.30, 49.71, 40.72, 37.07, 34.49, 31.07.

HRMS (ESI): C23H32N3O+, Calculated: [M+H]+, 366.2540; Found: [M+H]+, 366.2547.

IR (neat): 3346, 3027, 2941, 2922, 2846, 2803, 2770, 1732, 1674, 1591, 1578, 1476, 1453, 1430 cm'¹.

Methyl 8-((lH-imidazol-2-yl)methyl)-2-benzyl-2,8-diazaspiro[4.5]decane-4-carboxylate: Following

General Procedure L, 2-benzyl-2,8-diazaspiro[4.5]decane-4-carboxylate (158.8 mg, 0.551 mmol) and 2- imidazolecarboxaldehyde (264.7 mg, 2.755 mmol) were used. The product was purified using column chromatography (0-100% ACN:MeOH (1% NH4OH) on silica over 11 minutes) to afford the desired product as a yellow oil: 43.7 mg (21%).

'H NMR (500 MHz, Methanol-d4) 8 7.34 - 7.27 (m, 4H), 7.25 - 7.20 (m, 1H), 6.96 (s, 2H), 4.91 (s, 1H), 3.65 (s, 3H), 3.62 (d, J = 3.2 Hz, 2H), 3.54 (s, 2H), 2.94 - 2.85 (m, 1H), 2.82 - 2.59 (m, 5H), 2.32 (d, J = 9.5 Hz, 1H), 2.12 (t, J = 12.2 Hz, 1H), 2.06 (t, J = 10.4 Hz, 1H), 1.92 (td, J = 12.4, 4.0 Hz, 1H), 1.65 (d, J = 9.9 Hz, 1H), 1.57 - 1.43 (m, 2H).

¹³C NMR (125 MHz, Methanol-^) 8 173.11, 144.47, 138.20, 128.59, 127.96, 126.87, 121.87, 62.48, 59.77, 54.80, 54.50, 53.15, 50.88, 50.55, 50.04, 43.04, 36.73, 32.46.

HRMS (ESI): C21H29N4O2+, Calculated: [M+H]+, 369.2285; Found: [M+H]+, 369.2293.

IR (neat): 3150, 3059, 3029, 2920, 2806, 1730, 1563, 1451, 1438 cm'¹.

AD-2145: Following General Procedure P, methyl 8-((lH-imidazol-2-yl)mcthyl)-2-bcnzyl-2,8- diazaspiro[4.5]decane-4-carboxylate (43.7 mg, 0.119 mmol) was used. The product was purified using column chromatography (0-100% ACN:MeOH (1% NH4OH) on silica over 10 minutes) to afford the desired product as a yellow oil: 37.3 mg (92%).

'H NMR (500 MHz, Methanol-d4) 37.35 - 7.28 (m, 4H), 7.27 - 7.21 (m, 1H), 6.97 (s, 2H), 4.93 (s, 2H), 3.68 (dd, J = 10.6, 5.8 Hz, 1H), 3.62 (s, 2H), 3.54 (s, 2H), 3.48 (dd, J = 10.6, 8.1 Hz, 1H), 2.94 (dd, J = 9.6, 7.1 Hz, 1H), 2.71 (t, J = 9.4 Hz, 3H), 2.36 (dd, J = 15.4, 9.6 Hz, 2H), 2.08 (dt, J = 22.4, 11.5 Hz, 2H), 2.00 - 1.90 (m, 1H), 1.74 (dtd, J = 24.8, 12.5, 4.1 Hz, 2H), 1.55 (t, J = 13.8 Hz, 2H).

¹³C NMR (125 MHz, Methanol-A) 3 144.50, 137.99, 128.79, 127.95, 126.92, 121.38, 63.33, 61.04, 60.25, 56.81, 54.58, 51.01, 50.28, 49.74, 40.66, 37.15, 31.19.

HRMS (ESI): C20H29N4O+, Calculated: [M+H]+, 341.2336; Found: [M+H]+, 341.2338.

IR (neat): 3158, 3060, 3029, 2916, 2802, 1558, 1494, 1471, 1454 cm-¹.

Methyl 2-benzyl-8-(cyclohexylmethyl)-2,8-diazaspiro[4.5]decane-4-carboxylate: Following General Procedure R, 2-benzyl-2,8-diazaspiro[4.5]decane-4-carboxylate (396.7 mg, 1.376 mmol) and tetrahydropyran-4-carbaldehyde (235.5 mg, 2.064 mmol) were used. The product was purified using column chromatography (0-100% ACN:MeOH on silica over 11 minutes) to afford the desired product as a yellow oil: 91.4 mg (17%).

'H NMR (500 MHz, Methanol-d4) 57.36 - 7.28 (m, 4H), 7.26 - 7.22 (m, 1H), 3.90 (dd, J = 10.8, 3.4 Hz, 2H), 3.67 (s, 3H), 3.63 (d, J = 5.1 Hz, 2H), 3.38 (td, J = 11.7, 2.2 Hz, 2H), 2.91 (dd, J = 9.1, 7.2 Hz, 1H), 2.80 - 2.65 (m, 5H), 2.34 (d, J = 9.5 Hz, 1H), 2.13 (d, J = 5.7 Hz, 2H), 1.98 (t, J = 11.6 Hz, 1H), 1.92 (td, J = 11.5, 2.9 Hz, 2H), 1.76 (ttt, J = 10.9, 7.1, 3.7 Hz, 1H), 1.64 (dd, J = 13.2, 2.1 Hz, 3H), 1.53 (dd, J = 13.4, 2.3 Hz, 1H), 1.51 - 1.44 (m, 1H), 1.26 - 1.16 (m, 2H).

¹³C NMR (125 MHz, Methanol-d4) δ 173.18, 138.24, 128.60, 127.97, 126.88, 67.44, 64.93, 62.48, 59.84, 54.85, 53.26, 51.69, 50.95, 50.56, 43.34, 36.63, 32.42, 32.16, 31.56.

HRMS (ESI): Calculated: [M+H]+, 387.2642; Found: [M+H]+, 387.2657.

IR (neat): 2945, 2919, 2840, 2804, 1734, 1451, 1437 cm'¹.

AD-2178: Following General Procedure P, methyl 2-bcnzyl-8-(cyclohcxylmethyl)-2,8- diazaspiro[4.5]decane-4-carboxylate (35.4 mg, 0.95 mmol) was used. The product was purified using column chromatography (0-100% DCM:MeOH (1% NH4OH) on silica over 11 minutes) to afford the desired product as a yellow oil: 15 mg (46%).

1H NMR (500 MHz, Methanol-^) 5 7.36 - 7.29 (m, 4H), 7.28 - 7.23 (m, 1H), 3.91 (dd, J = 11.4, 2.8 Hz, 2H), 3.70 (dd, J = 10.6, 5.6 Hz, 1H), 3.66 (s, 2H), 3.50 (dd, J = 10.6, 8.1 Hz, 1H), 3.40 (td, J = 11.8, 2.2 Hz, 2H), 2.98 (dd, J = 9.6, 7.0 Hz, 1H), 2.76 (d, J = 10.4 Hz, 3H), 2.43 (d, J = 10.0 Hz, 1H), 2.39 (t, J = 8.9 Hz, 1H), 2.20 (d, J = 6.9 Hz, 2H), 2.08 - 2.00 (m, 1H), 2.00 - 1.90 (m, 2H), 1.83 - 1.75 (m, 2H), 1.75 - 1.69 (m, 1H), 1.66 (d, J = 13.0 Hz, 2H), 1.56 (t, J = 15.6 Hz, 2H), 1.31 - 1.17 (m, 2H).

¹³C NMR (125 MHz, Methanol-^) 6 137.83, 128.83, 127.96, 126.96, 67.39, 64.83, 63.34, 61.02, 60.23, 56.83, 51.73, 51.05, 49.69, 40.85, 36.77, 32.01, 31.49, 30.84.

HRMS (ESI): C22H35N2O2+, Calculated: [M+H]+, 359.2693; Found: [M+H]+, 359.2703.

IR (neat): 3367, 2919, 1847, 2802, 2775, 1495, 1468, 1453 cm ¹.

Methyl 8-(cyclohexylmethyl)-2-phenethyl-2,8-diazaspiro[4.5]decane-4-carboxylate: Following General Procedure L, methyl 2-phenethyl-2,8-diazaspiro[4.5]decane-4-carboxylate (93.6 mg, 0.31 mmol) and tetrahydropyran-4-carbaldehyde (176.9 mg, 1.55 mmol) were used. The product was purified using column chromatography (0-100% ACN:MeOH (1% NH4OH) on silica over 9 minutes) to afford the desired product as a yellow solid: 43.9 mg (35%).

'H NMR (500 MHz, Methanol-d4) 5 7.26 (t, J = 7.3 Hz, 2H), 7.21 (d, J = 6.7 Hz, 2H), 7.17 (t, J = 7.2 Hz, 1H), 3.91 (dd, J = 11.4, 2.4 Hz, 2H), 3.69 (s, 3H), 3.40 (td, J = 11.7, 2.2 Hz, 2H), 3.03 (dd, J = 9.4, 7.3 Hz, 1H), 2.89 (d, J = 9.6 Hz, 1H), 2.84 - 2.78 (m, 3H), 2.78 - 2.72 (m, 3H), 2.71 - 2.64 (m, 2H), 2.38 (d, J = 9.5 Hz, 1H), 2.17 (d, J = 7.2 Hz, 2H), 2.06 (t, J = 11.0 Hz, 1H), 1.99 (d, J = 11.1 Hz, 1H), 1.93 (td, J = 12.5, 4.0 Hz, 1H), 1.79 (dddt, J = 14.6, 11.0, 7.1, 3.5 Hz, 1H), 1.65 (ddt, J = 10.2, 4.3, 2.2 Hz, 3H), 1.56 - 1.44 (m, 2H), 1.27 - 1.16 (m, 2H). ¹³C NMR (125 MHz, Methanol-d4) 6 173.04, 139.90, 128.28, 128.05, 125.78, 67.45, 64.95, 62.81, 58.08, 55.05, 53.12, 51.64, 50.88, 50.59, 43.19, 36.60, 34.54, 32.38, 32.17, 31.57.

HRMS (ESI): C24H37N2O3+, Calculated: [M+H]+, 401.2799; Found: [M+H]+, 401.2807.

IR (neat): 2942, 2919, 2838, 2803, 2771, 1733, 1631, 1604, 1451, 1436 cm'¹.

AD-2166: Following General Procedure P, methyl 8-(cyclohexylmethyl)-2-phenethyl-2,8- diazaspiro[4.5]decane-4-carboxylate (43.9 mg, 0.11 mmol) was used. The product was purified using column chromatography (0-100% DCM:MeOH (1% NH4OH) on silica over 11 minutes) to afford the desired product as a yellow oil: 32.9 mg (80%).

'H NMR (500 MHz, Methanol-^) 5 7.33 - 7.23 (m, 2H), 7.23 - 7.11 (m, 3H), 3.91 (dd, J = 11.7, 2.2 Hz, 2H), 3.72 (dd, J = 10.6, 5.5 Hz, 1H), 3.50 (dd, J = 10.7, 8.3 Hz, 1H), 3.40 (td, J = 11.9, 2.2 Hz, 2H), 3.07 (dd, J = 9.6, 7.1 Hz, 1H), 2.89 - 2.78 (m, 3H), 2.78 - 2.62 (m, 4H), 2.41 (dd, J = 15.1, 9.2 Hz, 2H), 2.18 (d, J = 6.9 Hz, 2H), 2.05 (t, J = 12.3 Hz, 1H), 2.02 - 1.90 (m, 2H), 1.86 - 1.72 (m, 2H), 1.71 - 1.62 (m, 3H), 1.54 (t, J = 14.3 Hz, 2H), 1.23 (qd, J = 11.9, 4.5 Hz, 2H).

¹³C NMR (125 MHz, Methanol-^) 5 139.84, 128.25, 128.06, 125.78, 67.45, 65.06, 63.76, 60.98, 58.57, 57.10, 51.80, 51.03, 49.73, 40.83, 36.99, 34.48, 32.15, 31.59, 30.99.

HRMS (ESI): C23H37N2O2+, Calculated: [M+H]+, 373.2850; Found: [M+H]+, 373.2857.

IR (neat): 3392, 2917, 2840, 2803, 2771, 1683, 1496, 1467, 1455 cm'¹.

l-propyl-lH-indole-3-carbaldehyde: Following General Procedure S, lH-indole-3-carbaldehyde (1.00 g, 6.88 mmol) and 1-propyl bromide (4.231 g, 34.4 mmol) were used. The product was purified using column chromatography (0-50% hexanes:EtOAc on silica over 10 minutes) to afford the desired product as a yellow solid: 1.129 g (87%).

'H NMR (500 MHz, CDC1₃) 5 9.99 (s, 1H), 8.34 - 8.28 (m, 1H), 7.71 (s, 1H), 7.40 - 7.27 (m, 3H), 4.13 (t, J = 7.1 Hz, 2H), 1 .93 (h, J = 7.3 Hz, 2H), 0.97 (t, .1 = 7.4 Hz, 3H). ¹³C NMR (125 MHZ, CDC1₃) 5 184.46, 138.39, 137.24, 125.46, 123.88, 122.84, 122.11, 117.96, 110.11, 48.89, 23.05, 11.36.

HRMS (ESI): C12H14NO+, Calculated: [M+H]+, 188.1070; Found: [M+H]+, 118.1073.

IR (neat): 3103, 3047, 2966, 2935, 2877, 2808, 1657, 1532, 1468 cm'¹.

Methyl 2-phenethyl-8-((l-propyl-lH-indol-3-yl)methyl)-2,8-diazaspiro[4.5]decane-4-carboxylate: Following General Procedure R, methyl 2-phcncthyl-2,8-diazaspiro[4.5]dccanc-4-carboxylatc (219.2 mg, 0.725 mmol) and 1 -propyl- lH-indole-3-carbaldehy de (203.62 mg, 1.0875 mmol) were used. The product was purified using column chromatography (0-100% ACN:MeOH (1% NH4OH) on silica over 9 minutes) to afford the desired product as a white solid: 153.8 mg (45%).

'H NMR (500 MHz, Methanol-^) 5 7.65 (d, J = 8.0 Hz, 1H), 7.37 (d, J = 8.3 Hz, 1H), 7.29 (s, 1H), 7.22 (t, J = 7.3 Hz, 2H), 7.19 - 7.11 (m, 4H), 7.09 (ddd, J = 8.0, 7.0, 1.0 Hz, 1H), 4.09 (t, J = 7.0 Hz, 2H), 3.96 (s, 2H), 3.60 (s, 3H), 3.02 (d, J = 11.2 Hz, 1H), 2.99 - 2.90 (m, 2H), 2.85 - 2.59 (m, 8H), 2.54 (d, J = 10.6 Hz, 1H), 2.50 - 2.41 (m, 1H), 2.39 (d, J = 9.7 Hz, 1H), 2.04 - 1.95 (m, 1H), 1.82 (h, J = 7.3 Hz, 2H), 1.70 (dd, J = 14.1, 2.3 Hz, 1H), 1.63 - 1.57 (m, 1H), 1.57 - 1.49 (m, 1H), 0.87 (t, J = 7.4 Hz, 3H).

¹³C NMR (125 MHz, Methanol-^) 5 172.66, 139.76, 136.29, 129.58, 128.50, 128.30, 128.08, 125.83, 121.42, 119.26, 118.51, 109.52, 105.85, 62.47, 57.75, 54.89, 52.53, 51.74, 50.70, 49.94, 49.28, 47.38, 42.59, 35.32, 34.45, 31.08, 23.20, 10.36.

HRMS (ESI): C30H40N3O2+, Calculated: rM+H]+, 474.3115; Found: [M+H]+, 474.3125.

IR (neat): 3027, 2925, 2877, 2849, 2803, 1732, 1689, 1675, 1469, 1455, 1437 cm'¹.

AD-3020: Following General Procedure P, methyl 2-phenethyl-8-((l-propyl-lH-indol-3-yl)methyl)-2,8- diazaspiro[4.5]decane-4-carboxylate (106.7 mg, 0.225 mmol) was used. The product was purified using column chromatography (0-100% DCM:MeOH (1% NH4OH) on silica over 9 minutes) to afford the desired product as a white solid: 69.0 mg (69%).

'H NMR (500 MHz, Methanol-^) 5 7.61 (d, J = 7.9 Hz, 1H), 7.34 (d, J = 8.3 Hz, 1H), 7.26 - 7.21 (m, 2H), 7.17 (d, J = 4.0 Hz, 2H), 7.16 - 7.08 (m, 3H), 7.04 (ddd, J = 8.0, 7.0, 1.0 Hz, 1H), 4.08 (t, J = 6.9 Hz, 2H), 3.69 (s, 2H), 3.67 (d, J = 5.4 Hz, 1H), 3.46 (dd, J = 10.6, 8.3 Hz, 1H), 3.05 - 2.99 (m, 1H), 2.81 (s, 2H), 2.75 (t, J = 8.2 Hz, 3H), 2.69 - 2.56 (m, 2H), 2.38 - 2.29 (m, 2H), 2.18 (t, J = 11.1 Hz, 1H), 2.08 (d, J = 10.9 Hz, 1H), 1.98 - 1.90 (m, 1H), 1.82 (dt, J = 14.3, 7.2 Hz, 2H), 1.79 - 1.72 (m, 1H), 1.66 (td, J = 12.7, 3.9 Hz, 1H), 1.55 (d, J = 2.8 Hz, 1H), 1.50 - 1.44 (m, 1H), 0.86 (t, J = 7.4 Hz, 3H).

¹³C NMR (125 MHz, Methanol-d4) δ 139.85, 136.22, 128.79, 128.39, 128.24, 128.05, 125.77, 120.99, 118.68, 118.64, 109.18, 108.58, 63.66, 60.97, 58.50, 57.13, 52.25, 50.65, 49.84, 49.70, 47.22, 40.67, 36.90, 34.50, 30.89, 23.22, 10.33.

HRMS (ESI): C29H40N3O+, Calculated: [M+H]+, 446.3166; Found: [M+H]+, 446.3178.

IR (neat): 3345, 3026, 2925, 2805, 1629, 1554, 1546, 1468 cm ¹.

Methyl 2-benzyl-8-((l-propyl-lH-indol-3-yl)methyl)-2,8-diazaspiro[4.5]decane-4-carboxylate:

Following General Procedure R, methyl 2-benzyl-2,8-diazaspiro[4.5]decane-4-carboxylate (340.2 mg, 1.18 mmol) and 1 -propyl- lH-indole-3-carbaldehyde (331.4 mg, 1.77 mmol) were used. The product was purified using column chromatography (0-100% ACN:MeOH (1% NH4OH) on silica over 10 minutes) to afford the desired product as a white solid: 100.5 mg (19%).

'H NMR (500 MHz, Methanol-d4) 8 7.58 (d, J = 8.0 Hz, 1H), 7.34 (d, J = 8.3 Hz, 1H), 7.31 - 7.26 (m, 4H), 7.22 (ddt, J = 8.5, 5.5, 3.0 Hz, 1H), 7.16 - 7.09 (m, 2H), 7.03 (ddd, J = 8.0, 7.0, 1.0 Hz, 1H), 4.08 (t, J = 7.0 Hz, 2H), 3.68 (s, 2H), 3.61 - 3.54 (m, 5H), 2.92 - 2.83 (m, 1H), 2.80 (d, J = 11.1 Hz, 1H), 2.77 - 2.64 (m, 4H), 2.29 (d, J = 9.5 Hz, 1H), 2.20 - 2.04 (m, 2H), 1.92 (td, J = 12.6, 4.1 Hz, 1H), 1.81 (h, J = 7.3 Hz, 2H), 1.64 (d, J = 14.1 Hz, 1H), 1.55 - 1.43 (m, 2H), 0.86 (t, J = 7.4 Hz, 3H).

¹³C NMR (125 MHz, Methanol-^) 6 173.12, 138.15, 136.23, 128.72, 128.58, 128.44, 127.94, 126.85, 120.98, 118.69, 118.60, 109.15, 108.35, 62.58, 59.75, 54.80, 53.03, 52.07, 50.47, 50.41, 49.66, 47.22, 43.14, 36.46, 32.14, 23.21, 10.28.

HRMS (ESI): C29H38N3O2+, Calculated: [M+H]+, 460.2959; Found: [M+H]+, 460.2972.

IR (neat): 3057, 3028, 2932, 2874, 2802, 1732, 1672, 1467, 1452, 1437 cm'¹.

AD-3021: Following General Procedure P, methyl 2-benzyl-8-((l-propyl-lH-indol-3-yl)methyl)-2,8- diazaspiro[4.5]decane-4-carboxylate (57.2 mg, 0.124 mmol) was used. The product was purified using column chromatography (0-100% DCM:MeOH (1% NH4OH) on silica over 9 minutes) to afford the desired product as a white solid: 36.8 mg (69%).

'H NMR (500 MHz, Methanol-^) 57.59 (d, J = 8.0 Hz, 1 H), 7.33 (d, J = 8.3 Hz, 1 H), 7.29 (d, J = 4.6 Hz, 4H), 7.26 - 7.21 (m, 1H), 7.15 (s, 1H), 7.14 - 7.10 (m, 1H), 7.03 (ddd, J = 8.0, 7.0, 1.1 Hz, 1H), 4.08 (t, J = 7.0 Hz, 2H), 3.68 (d, J = 5.5 Hz, 1H), 3.66 (s, 2H), 3.57 (s, 2H), 3.44 (dd, J = 10.6, 8.3 Hz, 1H), 2.92 (dd, J = 9.5, 7.0 Hz, 1H), 2.80 (d, J = 11.6 Hz, 2H), 2.67 (d, J = 9.8 Hz, 1H), 2.35 - 2.26 (m, 2H), 2.17 - 2.08 (m, 1H), 2.05 (t, J = 11.3 Hz, 1H), 1.95 - 1.88 (m, 1H), 1.82 (p, J = 7.2 Hz, 2H), 1.74 (td, J = 12.9, 4.2 Hz, 1H), 1.66 (td, J = 12.7, 4.1 Hz, 1H), 1.55 (dd, J = 13.5, 2.7 Hz, 1H), 1.48 (d, J = 12.7 Hz, 1H), 0.85 (t, J = 7.4 Hz, 3H).

¹³C NMR (125 MHz, Methanol-

5 138.11, 136.20, 128.76, 128.36, 127.92, 126.85, 120.96, 118.65, 118.61, 109.13, 108.58, 63.39, 61.10, 60.27, 56.93, 52.23, 50.68, 49.92, 49.80, 47.20, 40.74, 36.89, 30.97, 23.22, 10.30.

HRMS (ESI): C28H38N3O+, Calculated: [M+H]+, 432.3009; Found: [M+H]+, 432.3020.

IR (neat): 3348, 3057, 3028, 2920, 2874, 2801, 2778, 1633, 1614, 1546, 1467, 1454 cm'¹.

Methyl 8-((l-methyl-lH-indol-3-yl)methyl)-2-phenethyl-2,8-diazaspiro[4.5]decane-4-carboxylate:

Following General Procedure R, methyl 2-phenethyl-2,8-diazaspiro[4.5]decane-4-carboxylate (109.7 mg, 0.363 mmol) and l-methyl-lH-indole-3-carbaldehyde (86.68 mg, 0.5445 mmol) were used. The product was purified using column chromatography (0-100% ACN:MeOH (1% NH4OH) on silica over 11 minutes) to afford the desired product as a white solid: 76.2 mg (47%).

'H NMR (500 MHz, Methanol-d4) δ 7.61 (d, J = 8.0 Hz, 1H), 7.33 (d, J = 8.2 Hz, 1H), 7.23 (t, J = 7.3 Hz, 2H), 7.20 - 7.12 (m, 4H), 7.12 (s, 1H), 7.06 (t, J = 7.5 Hz, 1H), 3.77 (s, 3H), 3.69 (s, 2H), 3.62 (s, 3H), 2.98 (dd, J = 9.5, 7.3 Hz, 1H), 2.86 - 2.73 (m, 6H), 2.73 - 2.60 (m, 3H), 2.32 (d, J = 9.6 Hz, 1H), 2.26 - 2.17 (m, 1H), 2.17 - 2.06 (m, 1H), 1.94 (td, J = 13.2, 4.3 Hz, 1H), 1.64 (d, J = 12.3 Hz, 1H), 1.55 - 1.42 (m, 2H).

¹³C NMR (125 MHz, Methanol-d4) 6 172.98, 139.87, 136.95, 129.28, 128.66, 128.26, 128.01, 125.74, 121.10, 118.73, 118.49, 108.85, 108.58, 62.92, 57.97, 55.03, 52.94, 52.09, 50.51, 50.46, 49.65, 43.02, 36.50, 34.49, 32.17, 31.37.

HRMS (ESI): C₂8H₃6N₃O2+, Calculated: [M+H]+, 446.2802; Found: [M+H]+, 446.2816.

IR (neat): 3056, 3026, 2934, 2803, 2776, 1731, 1472, 1454, 1435 ent¹.

AD-3036: Following General Procedure P, methyl 8-((l-methyl-lH-indol-3-yl)methyl)-2-phenethyl-2,8- diazaspiro[4.5]decane-4-carboxylate (57.7 mg, 0.129 mmol) was used. The product was purified using column chromatography (0-100% DCM:MeOH (1% NH4OH) on silica over 10 minutes) to afford the desired product as a white solid: 47.5 mg (88%).

'H NMR (500 MHz, Methanol-d4) δ 7.62 (d, J = 7.9 Hz, 1H), 7.32 (d, J = 8.3 Hz, 1H), 7.23 (t, J = 7.3 Hz, 2H), 7.20 - 7.10 (m, 5H), 7.06 (t, J = 7.0 Hz, 1H), 3.75 (s, 2H), 3.74 (s, 3H), 3.68 (dd, J = 10.7, 5.5 Hz, 1H), 3.46 (dd, J = 10.7, 8.2 Hz, 1H), 3.04 (dd, J = 9.7, 7.1 Hz, 1H), 2.87 (d, J = 10.0 Hz, 2H), 2.76 (t, J = 8.0 Hz, 3H), 2.71 - 2.58 (m, 2H), 2.37 (t, J = 9.7 Hz, 2H), 2.27 (d, J = 10.7 Hz, 1H), 2.17 (d, J = 9.8 Hz, 1H), 1.95 (p, J = 8.2 Hz, 1 H), 1.79 (td, J = 12.9, 4.1 Hz, 1 H), 1.75 - 1 .64 (m, 1 H), 1.56 (d, J = 13.4 Hz, 1 H), 1 .50 (d, J = 13.0 Hz, 1H).

¹³C NMR (125 MHZ, Methanol-^) 8 139.75, 136.94, 129.58, 128.63, 128.26, 128.05, 125.80, 121.20, 118.87, 118.47, 108.96, 108.00, 63.59, 60.86, 58.39, 56.98, 52.09, 50.52, 49.79, 49.51, 40.64, 36.55, 34.37, 31.43, 30.56.

HRMS (ESI): C27H₃₆N₃O+, Calculated: [M+H]+, 418.2853; Found: [M+H]+, 418.2866.

IR (neat): 3332, 3056, 3026, 2918, 2804, 2780, 1545, 1472 cm'¹.

Methyl 8-((lH-pyrrolo[2,3-b]pyridin-3-yl)methyl)-2-phenethyl-2,8-diazaspiro[4.5]decane-4- carboxylate: Following General Procedure R, methyl 2-phenethyl-2,8-diazaspiro[4.5]decane-4-carboxylate (133.0 mg, 0.44 mmol) and lH-pyrrolo[2,3-b]pyridine-3-carbaldehyde (96.46 mg, 0.66 mmol) were used. The product was purified using column chromatography (0-100% ACN:MeOH (1% NH4OH) on silica over 10 minutes) to afford the desired product as a yellow oil: 153.8 mg (45%).

'H NMR (500 MHz, Methanol-d4) δ 8.18 (d, J = 3.3 Hz, 1H), 8.09 (dd, J = 7.9, 1.5 Hz, 1H), 7.35 (s, 1H), 7.22 (d, J = 7.2 Hz, 2H), 7.20 - 7.15 (m, 2H), 7.14 - 7.06 (m, 2H), 3.70 (s, 2H), 3.62 (s, 3H), 3.04 - 2.96 (m, 1H), 2.89 - 2.60 (m, 10H), 2.33 (d, J = 9.6 Hz, 1H), 2.22 (d, J = 13.1 Hz, 1H), 2.13 (d, J = 7.4 Hz, 1H), 1.93 (td, J = 13.4, 4.2 Hz, 1H), 1.65 (d, J = 11.1 Hz, 1H), 1.53 (d, J = 12.3 Hz, 1H), 1.47 (td, J = 12.2, 3.9 Hz, 1H).

¹³C NMR (125 MHz, Methanol-d4) δ 172.94, 147.84, 141.87, 139.84, 128.27, 128.02, 127.82, 125.90, 125.77, 121.07, 115.21, 108.83, 62.84, 57.97, 55.04, 52.90, 52.27, 50.56, 49.73, 44.72, 43.01, 36.47, 34.50, 32.18.

HRMS (ESI): C26H33N4O2+, Calculated: [M+H]+, 433.2598; Found: [M+H]+, 433.2609. IR (neat): 3148, 3085, 3026, 2928, 2804, 1731, 1604, 1580, 1540, 1452, 1436 cm'¹.

AD-3056: Following General Procedure P, methyl 8-((lH-pyrrolo[2,3-b]pyridin-3-yl)methyl)-2-phenethyl- 2,8-diazaspiro[4.5]decane-4-carboxylate (54.0 mg, 0.125 mmol) was used. The product was purified using column chromatography (0-100% DCM:MeOH (1% NH4OH) on silica over 9 minutes) to afford the desired product as a yellow oil: 41.1 mg (81%).

'H NMR (500 MHz, Methanol-d4) 5 8.18 (d, J = 4.9 Hz, 1H), 8.11 (d, J = 7.8 Hz, 1H), 7.38 (s, 1H), 7.23 (t, J = 7.4, 7.4 Hz, 2H), 7.20 - 7.13 (m, 3H), 7.11 (dd, J = 7.9, 4.8 Hz, 1H), 3.74 (s, 2H), 3.69 (dd, J = 10.7, 5.5 Hz, 1H), 3.49 (dd, J = 10.7, 8.1 Hz, 1H), 3.12 - 3.04 (m, 1H), 2.89 - 2.76 (m, 5H), 2.71 (q, J = 9.0, 8.0, 8.0 Hz, 2H), 2.43 (d, J = 9.9 Hz, 2H), 2.30 - 2.19 (m, 1H), 2.15 (t, J = 11.4, 11.4 Hz, 1H), 1.98 (p, J = 8.2, 8.2, 8.0, 8.0 Hz, 1H), 1.80 (td, J = 12.7, 12.7, 4.0 Hz, 1H), 1.71 (td, J = 12.4, 12.1, 3.8 Hz, 1H), 1.56 (dd, J = 21.6, 13.3 Hz, 2H).

¹³C NMR (125 MHz, Methanol-d4) δ 147.86, 141.91, 139.65, 128.25, 128.05, 127.82, 126.01, 125.82, 121.05, 115.24, 108.67, 63.54, 60.80, 58.36, 56.95, 52.33, 50.63, 49.91, 49.47, 40.75, 36.66, 34.27, 30.67. HRMS (ESI): C25H33N4O+, Calculated: [M+H]+, 405.2649; Found: [M+H]+, 405.2654. IR (neat): 3172, 3027, 2922, 2806, 1604, 1580, 1539, 1494, 1452 cm'¹.

Methyl 8-((lH-imidazol-4-yl)methyl)-2-phenethyl-2,8-diazaspiro[4.5Jdecane-4-carboxylate: Following

General Procedure R, methyl 2-phenethyl-2,8-diazaspiro[4.5]decane-4-carboxylate (109.7 mg, 0.363 mmol) and lH-imidazole-4-carbaldehyde (52.32 mg, 0.5445 mmol) were used. The product was purified using column chromatography (0-100% ACN:MeOH (1% NH4OH) on silica over 10 minutes) to afford the desired product as a yellow oil: 80.9 mg (58%).

'H NMR (500 MHz, Methanol-d4) 5 7.63 (s, 1 H), 7.28 - 7.23 (m, 2H), 7.22 - 7.18 (m, 2H), 7.16 (tt, J = 6.5, 1.5 Hz, 1H), 7.01 (s, 1H), 3.66 (s, 3H), 3.56 (s, 2H), 3.04 (dd, J = 9.7, 7.4 Hz, 1H), 2.92 - 2.61 (m, 10H), 2.39 (d, J = 9.7 Hz, 1H), 2.24 (t, J = 11.0 Hz, 1H), 2.20 - 2.09 (m, 1H), 1.95 (td, J = 13.3, 4.3 Hz, 1H), 1.68 (d, J = 11.6 Hz, 1H), 1.56 (d, J = 12.7 Hz, 1H), 1.53 - 1.45 (m, 1H).

¹³C NMR (125 MHz, Methanol-d4) 5 172.85, 139.77, 134.99, 131.94, 128.30, 128.07, 125.83, 119.50, 62.69, 57.94, 54.96, 53.43, 52.83, 50.65, 50.36, 49.56, 42.90, 36.24, 34.41, 32.00.

HRMS (ESI): C22H31N4O2+, Calculated: [M+H]+, 383.2442; Found: [M+H]+, 383.2453.

IR (neat): 3084, 3026, 2932, 2805, 1729, 1472, 1452, 1436 cm'¹.

AD-3037: Following General Procedure P, methyl 8-((lH-imidazol-4-yl)methyl)-2-phenethyl-2,8- diazaspiro[4.5]decane-4-carboxylate (78.4 mg, 0.205 mmol) was used. The product was purified using column chromatography (0-100% DCM:MeOH (1% NH4OH) on silica over 9 minutes) to afford the desired product as a yellow oil: 64.6 mg (89%).

'H NMR (500 MHz, Methanol-d4) δ 7.63 (s, 1H), 7.26 (t, J = 7.4 Hz, 2H), 7.23 - 7.12 (m, 3H), 7.00 (s, 1H), 4.95 (s, 6H), 3.70 (dd, J = 10.7, 5.5 Hz, 1H), 3.54 (s, 2H), 3.50 (dd, J = 10.7, 8.1 Hz, 1H), 3.13 - 3.05 (m, 1H), 2.82 (dd, J = 17.7, 8.7 Hz, 5H), 2.77 - 2.63 (m, 2H), 2.48 - 2.38 (m, 2H), 2.20 (t, J = 11.6 Hz, 1H), 2.13 (t, J = 11.7 Hz, 1H), 1.99 (p, J = 8.2 Hz, 1H), 1.79 (td, J = 12.7, 4.2 Hz, 1H), 1.70 (td, J = 12.5, 4.0 Hz, 1H), 1.57 (dd, J = 17.8, 13.4 Hz, 2H). ¹³C NMR (125 MHz, Methanol-^) 6 139.67, 134.89, 132.23, 128.27, 128.08, 125.84, 119.38, 63.55, 60.82,

58.41, 56.96, 53.58, 50.54, 49.81, 49.52, 40.68, 36.75, 34.30, 30.75.

HRMS (ESI): C21H31N4O+, Calculated: [M+H]+, 355.2492; Found: [M+H]+, 355.2498.

IR (neat): 3111, 3027, 2919, 2807, 1649, 1603, 1494, 1471, 1454 cm'¹.

Methyl 8-((lH-imidazol-4-yl)methyl)-2-benzyl-2,8-diazaspiro[4.5]decane-4-carboxylate: Following

General Procedure R methyl 2-benzyl-2,8-diazaspiro[4.5]decane-4-carboxylate (224.94 mg, 0.78 mmol) and lH-imidazole-4-carbaldehyde (58.7 mg, 0.6105 mmol) were used. The product was purified using column chromatography (0-100% ACN:MeOH (1% NH4OH) on silica over 10 minutes) to afford the semi-crude product as a yellow oil: 113.2 mg (39%). Product was taken forward without further purification.

'H NMR (600 MHz, Methanol-^) 67.62 (d, J = 1.2 Hz, 1H), 7.36 - 7.29 (m, 4H), 7.25 (tt, J = 6.0, 1.8 Hz, 1H), 6.97 (s, 1H), 5.00 (s, 1H), 3.66 (s, 3H), 3.63 (d, J = 2.1 Hz, 2H), 3.49 (s, 2H), 2.94 - 2.87 (m, 1H), 2.81 - 2.67 (m, 5H), 2.34 (d, J = 9.5 Hz, 1H), 2.16 - 2.08 (m, 1H), 2.05 (t, J = 10.1 Hz, 1H), 1.94 (td, J = 12.5, 4.1 Hz, 1H), 1.67 (dd, J = 13.4, 3.1 Hz, 1H), 1.56 (d, J = 12.2 Hz, 1H), 1.53 - 1.45 (m, 1H).

¹³C NMR (150 MHz, Methanol-d4) 5 173.12, 138.33, 134.86, 129.22, 128.61, 128.03, 127.48, 126.91, 62.58, 59.82, 54.86, 53.61, 53.18, 52.21, 50.61, 49.79, 44.89, 43.19, 36.65, 32.35.

HRMS (ESI): C21H29N4O2+, Calculated: [M+H]+, 369.2285; Found: [M+H]+, 369.2292.

IR (neat): 3085, 2943, 2921, 2807, 1730, 1494, 1471, 1451, 1437 cm'¹.

AD-3064: Following General Procedure P, methyl 8-((lH-imidazol-4-yl)methyl)-2-benzyl-2,8- diazaspiro[4.5]decane-4-carboxylate (56.6 mg, 0.154 mmol) was used. The product was purified using column chromatography (0-100% DCM:McOH (1% NH4OH) on silica over 9 minutes) to afford the desired product as a yellow oil: 45.7 mg (20%).

'H NMR (600 MHz, Methanol-d4) 57.67 (s, 1H), 7.38 (d, J = 7.2 Hz, 2H), 7.34 (t, J = 7.5 Hz, 2H), 7.28 (t, J = 7.2 Hz, 1H), 7.12 (s, 1H), 3.74 (s, 2H), 3.70 (d, J = 7.9 Hz, 3H), 3.52 (dd, J = 10.7, 7.6 Hz, 1H), 3.10 - 3.01 (m, 1H), 2.93 (t, J = 13.7 Hz, 2H), 2.81 (d, J = 10.1 Hz, 1H), 2.58 - 2.44 (m, 2H), 2.35 (dd, J = 28.9, 11.6 Hz, 2H), 2.03 (p, J = 7.6 Hz, 1H), 1.84 (dtd, J = 32.9, 12.9, 4.3 Hz, 2H), 1.66 (t, J = 14.2 Hz, 2H). ¹³C NMR (150 MHz, Methanol-d4) 5 137.20, 135.37, 130.86, 129.00, 128.92, 128.12, 127.25, 127.19, 120.14, 62.95, 60.74, 60.69, 60.00, 56.52, 56.46, 53.15, 52.43, 51.96, 50.26, 49.72, 49.26, 43.69, 40.69, 40.06, 35.96, 35.46, 30.06, 29.58.

HRMS (ESI): C20H29N4O+, Calculated: [M+H]+, 341.2336; Found: [M+H]+, 341.2342.

IR (neat): 3186, 2918, 1806, 1493, 1472, 1452 cm ¹.

Methyl 8-((l-methyl-lH-pyrrolo[2,3-b]pyridin-3-yl)methyl)-2-phenethyl-2,8-diazaspiro[4.5]decane-4- carboxylate: Following General Procedure R, methyl 2-phenethyl-2,8-diazaspiro[4.5]decane-4-carboxylate (89.2 mg, 0.295 mmol) and l-methyl-lH-pyrrolo[2,3-b]pyridine-3-carbaldehyde (70.88 mg, 0.4425 mmol) were used. The product was purified using column chromatography (0-100% ACN:MeOH (1% NH4OH) on silica over 10 minutes) to afford the desired product as a yellow oil: 74.6 mg (57%).

'H NMR (500 MHz, Methanol-d4) 5 8.23 (dd, J = 4.8, 1.6 Hz, 1H), 8.08 (dd, J = 7.8, 1.5 Hz, 1H), 7.31 (s, 1H), 7.23 (t, J = 7.3 Hz, 2H), 7.18 (d, J = 6.7 Hz, 2H), 7.16 - 7.08 (m, 2H), 3.84 (s, 3H), 3.67 (s, 2H), 3.63 (s, 3H), 2.99 (dd, J = 9.5, 7.3 Hz, 1H), 2.83 - 2.60 (m, 9H), 2.32 (d, J = 9.6 Hz, 1H), 2.18 (t, J = 11.2 Hz, 1H), 2.14 - 2.04 (m, 1 H), 1.93 (td, .1 = 12.5, 4.0 Hz, 1 H), 1.64 (d, J = 13.8 Hz, 1H), 1.52 (d, J = 15.3 Hz, 1H), 1.50 - 1.41 (m, 1H).

¹³C NMR (125 MHz, Methanol-^) 5 172.97, 147.26, 141.87, 139.89, 129.82, 128.25, 128.00, 127.94, 125.74, 121.47, 115.04, 108.19, 62.87, 57.96, 55.04, 52.97, 52.20, 50.57, 50.52, 49.77, 43.05, 36.59, 34.52, 32.28, 30.07.

HRMS (ESI): C27H₃₅N₄O2+, Calculated: [M+H]+, 447.2755; Found: [M+H]+, 447.2763.

IR (neat): 3025, 2936, 2801, 1732, 1601, 1541, 1490, 1460, 1435 cm'¹-

AD-3112: Following General Procedure P, methyl 8-((l-methyl-lH-pyrrolo[2,3-b]pyridin-3-yl)methyl)-2- phenethyl-2,8-diazaspiro[4.5]decane-4-carboxylate (49.0 mg, 0.110 mmol) was used. The product was purified using column chromatography (0-100% DCM:MeOH (1% NH4OH) on silica over 9 minutes) to afford the desired product as a yellow oil: 32.4 mg (70%).

'H NMR (500 MHz, Methanol-d4) 5 8.23 (dd, J = 4.8, 1.5 Hz, 1H), 8.10 (dd, J = 7.8, 1.5 Hz, 1H), 7.37 (s, 1H), 7.23 (t, J = 7.3 Hz, 2H), 7.18 (d, J = 6.7 Hz, 2H), 7.17 - 7.07 (m, 2H), 3.84 (s, 3H), 3.76 (s, 2H), 3.69 (dd, J = 10.7, 5.6 Hz, 1H), 3.50 (dd, J = 10.8, 7.8 Hz, 1H), 3.16 - 3.08 (m, 1H), 2.94 - 2.83 (m, 3H), 2.81 (t, J = 7.5 Hz, 2H), 2.78 - 2.67 (m, 2H), 2.54 - 2.44 (m, 2H), 2.27 (t, J = 10.6 Hz, 1H), 2.19 (d, J = 10.7 Hz, 1H), 2.01 (q, J = 7.2 Hz, 1H), 1.82 (td, J = 12.4, 4.0 Hz, 1H), 1.73 (td, J = 13.1, 4.0 Hz, 1H), 1.58 (dd, J = 20.7, 13.9 Hz, 2H).

¹³C NMR (125 MHz, Methanol-d4) δ 147.26, 141.98, 139.48, 130.22, 128.28, 128.07, 127.93, 125.86, 121.41, 115.18, 107.42, 63.42, 60.68, 58.27, 56.83, 52.11, 50.50, 49.84, 49.33, 40.77, 36.44, 34.10, 30.47, 30.12.

HRMS (ESI): C26H35N4O+, Calculated: [M+H]+, 419.2805; Found: [M+H]+, 419.2826.

IR (neat): 3343, 2920, 2849, 2805, 1650, 1602, 1541, 1492, 1460 cm-¹.

5-fluoro-l-propyl-lH-indole-3-carbaldehyde: Following General Procedure S, 5-fluoro-lH-indole-3- carbaldehyde (200.0 mg, 1.14 mmol) and 1-propyl bromide (701.04 mg, 5.7 mmol) were used. The product was purified using column chromatography (0-100% hexanes:EtOAc on silica over 10 minutes) to afford the desired product as a yellow solid: 1.129 g (87%).

'H NMR (500 MHz, CDCI3) 8 9.85 (s, 1H), 7.90 (dd, J = 9.3, 2.6 Hz, 1H), 7.66 (s, 1H), 7.22 (dd, J = 9.0, 4.2 Hz, 1H), 7.01 - 6.92 (m, 1H), 4.03 (t, J = 7.1 Hz, 2H), 1.83 (h, J = 7.3 Hz, 2H), 0.88 (t, J = 7.4 Hz, 3H). ¹³C NMR (125 MHz, CDCI3 δ 184.24, 160.59, 158.69, 139.57, 133.68, 125.92, 125.83, 117.70, 117.66, 112.17, 111.96, 111.15, 111.07, 107.35, 107.15, 49.03, 22.96, 11.21.

HRMS (ESI): CI₂HI₃FNO+, Calculated: [M+H]+, 206.0976; Found: [M+H]+, 206.0988.

IR (neat): 3129, 3099, 3048, 2966, 2937, 2921, 2878, 1646, 1624, 1533, 1481, 1451 cm ¹.

5-methoxy- 1-propyl- lH-indole-3-carbaldehy de: Following General Procedure S, 5-methoxy-lH-indole-3- carbaldehyde (200.0 mg, 1.14 mmol) and 1-propyl bromide (701.04 mg, 5.7 mmol) were used. The product was purified using column chromatography (0-100% hexanes:EtOAc on silica over 10 minutes) to afford the desired product as a yellow solid: 1.129 g (87%).

'H NMR (500 MHz, CDC1₃) 6 9.82 (s, 1H), 7.74 (s, 1H), 7.52 (s, 1H), 7.14 (d, J = 8.9 Hz, 1H), 6.86 (dd, J = 8.9, 2.6 Hz, 1H), 3.92 (t, J = 7.1 Hz, 2H), 3.78 (s, 3H), 1.75 (h, J = 7.4 Hz, 2H), 0.81 (t, J = 7.4 Hz, 3H).

¹³C NMR (125 MHz, CDCh) 5 184.37, 156.50, 138.81, 132.10, 126.06, 117.55, 114.01, 111.03, 103.40, 55.66, 48.83, 22.97, 11.19.

HRMS (ESI): C13H16NO2+, Calculated: [M+H]+, 218.1176; Found: [M+H]+, 218.1182.

IR (neat): 3102, 2965, 2935, 2923, 2878, 2847, 1654, 1621, 1530, 1485, 1470 cm-¹.

l-propyl-lH-pyrrolo[2,3-b]pyridine-3-carbaldehyde: Following General Procedure S, lH-pyrrolo[2,3- b]pyridine-3-carbaldehyde (500.0 mg, 3.42 mmol) and 1-propyl bromide (2103.13 mg, 17.1 mmol) were used along with the addition of potassium iodide (57.0 mg, 0.342 mmol). The product was purified using column chromatography (0-100% hexanes:EtOAc on silica over 10 minutes) to afford the desired product as a yellow solid: 323.3 mg (50%).

'H NMR (600 MHz, Methanol-^) 5 9.84 (s, 1H), 8.44 (dd, J = 7.8, 1.6 Hz, 1H), 8.33 (dd, J = 4.8, 1.6 Hz, 1H), 8.24 (s, 1H), 7.24 (dd, J = 7.8, 4.8 Hz, 1H), 4.29 - 4.24 (m, 2H), 1.89 (h, J = 7.3 Hz, 2H), 0.91 (t, J = 7.4 Hz, 3H).

¹³C NMR (150 MHz, Methanol-A) 3 185.45, 148.21, 144.28, 140.44, 130.09, 118.48, 117.64, 115.83, 46.66, 22.89, 10.04.

HRMS (ESI): C11H13N2O+, Calculated: [M+H]+, 189.1028; Found: [M+H]+, 189.1020. IR (neat): 3101, 2966, 2922, 1661, 1599, 1533, 1432 cm ¹.

Methyl 8-((5-fluoro-l-propyl-lH-indol-3-yl)methyl)-2-phenethyl-2,8-diazaspiro[4.5]decane-4- carboxylate: Following General Procedure R, methyl 2-phenethyl-2,8-diazaspiro[4.5]decane-4-carboxylate (69.2 mg, 0.229 mmol) and 5-fluoro-l-propyl-lH-indole-3-carbaldehyde (70.5 mg, 0.3435 mmol) were used. The product was purified using column chromatography (0-100% ACN:MeOH (1% NH4OH) on silica over 10 minutes) to afford the desired product as a yellow oil: 42.7 mg (38%).

'H NMR (500 MHz, Methanol-d4) δ 7.35 - 7.27 (m, 2H), 7.26 - 7.21 (m, 3H), 7.20 - 7.12 (m, 3H), 6.90 (td, J = 9.1, 2.5 Hz, 1H), 4.08 (t, J = 7.0 Hz, 2H), 3.64 (s, 2H), 3.63 (s, 3H), 2.98 (dd, J = 9.5, 7.4 Hz, 1H), 2.82 - 2.60 (m, 9H), 2.32 (d, J = 9.7 Hz, 1H), 2.18 (t, J = 11.0 Hz, 1H), 2.10 (t, J = 10.4 Hz, 1H), 1.93 (td, J = 12.7, 4.2 Hz, 1H), 1.82 (h, J = 7.3 Hz, 2H), 1.63 (d, J = 12.0 Hz, 1H), 1.52 (d, J = 15.4 Hz, 1H), 1.50 - 1.42 (m, 1H), 0.87 (t, J = 7.4 Hz, 3H).

¹³C NMR (125 MHz, Methanol-d4) δ 172.98, 158.65, 156.80, 139.87, 132.91, 130.18, 129.04, 128.97, 128.26, 128.02, 125.76, 110.13, 110.06, 109.19, 108.98, 108.81, 103.46, 103.27, 62.81, 57.96, 55.02, 52.95, 52.16, 50.52, 50.46, 49.66, 47.49, 43.05, 36.50, 34.51, 32.20, 23.20, 10.25.

HRMS (ESI): C30H39FN3O2+, Calculated: [M+H]+, 492.3021; Found: [M+H]+, 492.3038.

IR (neat): 3026, 2935, 2803, 1732, 1622, 1578, 1485, 1453 cm'¹.

AD-3127: Following General Procedure P, methyl 8-((5-fluoro-l-propyl-lH-indol-3-yl)methyl)-2- phenethyl-2,8-diazaspiro[4.5]decane-4-carboxylate (42.7 mg, 0.087 mmol) was used. The product was purified using column chromatography (0-100% DCM:MeOH (1% NH4OH) on silica over 9 minutes) to afford the desired product as a yellow oil: 31.0 mg (77%).

'H NMR (500 MHz, Methanol-d4) 57.36 - 7.28 (m, 3H), 7.24 (t, J = 7.3 Hz, 2H), 7.21 - 7.12 (m, 3H), 6.92 (td, J = 9.1, 2.5 Hz, 1H), 4.10 (t, J = 7.0 Hz, 2H), 3.73 (s, 2H), 3.69 (dd, J = 10.7, 5.6 Hz, 1H), 3.48 (dd, J = 10.7, 8.0 Hz, 1H), 3.08 (dd, J = 9.8, 7.2 Hz, 1H), 2.87 (d, J = 12.1 Hz, 2H), 2.84 - 2.76 (m, 3H), 2.75 - 2.63 (m, 2H), 2.47 - 2.37 (m, 2H), 2.27 (t, J = 10.8 Hz, 1H), 2.18 (d, J = 12.5 Hz, 1H), 1.98 (p, J = 8.1 Hz, 1H), 1.82 (dt, J = 14.5, 7.3 Hz, 3H), 1.77 - 1.65 (m, 1H), 1.57 (dd, J = 21.1, 13.2 Hz, 2H), 0.87 (t, J = 7.4 Hz, 3H).

¹³C NMR (125 MHz, Methanol-d4) δ 158.73, 156.87, 139.67, 132.89, 130.53, 129.02, 128.94, 128.25, 128.05, 125.81, 110.23, 110.15, 109.28, 109.06, 108.07, 103.41, 103.22, 63.46, 60.80, 58.36, 56.95, 52.09, 50.43, 49.73, 49.48, 47.52, 40.69, 36.50, 34.30, 30.52, 23.19, 10.24. HRMS (ESI): C₂₉H₃₉FN₃O+, Calculated: [M+H]+, 464.3072; 464.3072: [M+H]+, 464.3079.

IR (neat): 3279, 2962, 2921, 2879, 2849, 2807, 1623, 1578, 1545, 1486, 1455 cm'¹.

Methyl 8-((5-methoxy-l-propyl-lH-indol-3-yl)methyl)-2-phenethyl-2,8-diazaspiro[4.5]decane-4- carboxylate: Following General Procedure R, methyl 2-phenethyl-2,8-diazaspiro[4.5]decane-4-carboxylate (69.2 mg, 0.229 mmol) and 5-methoxy-l-propyl-lH-indole-3-carbaldehyde (74.6 mg, 0.3435 mmol) were used. The product was purified using column chromatography (0-100% ACN:MeOH (1% NFUOH) on silica over 10 minutes) to afford the desired product as a yellow oil: 45.5 mg (39%).

'H NMR (500 MHz, Methanol-d4) δ 7.28 - 7.19 (m, 4H), 7.19 - 7.10 (m, 4H), 6.81 (dd, J = 8.9, 2.5 Hz, 1H), 4.05 (t, J = 7.0 Hz, 2H), 3.82 (s, 3H), 3.79 (s, 2H), 3.61 (s, 3H), 3.05 - 2.96 (m, 1H), 2.91 (d, J = 12.0 Hz, 1H), 2.87 - 2.61 (m, 9H), 2.35 (d, J = 9.7 Hz, 2H), 2.26 (s, 1H), 2.02 - 1.92 (m, 1H), 1.81 (h, J = 7.3 Hz, 2H), 1.69 (d, J = 13.5 Hz, 1H), 1.60 - 1.48 (m, 2H), 0.86 (t, J = 7.4 Hz, 3H).

¹³C NMR (125 MHz, Methanol-^) 5 172.90, 154.10, 139.81, 131.62, 129.56, 129.02, 128.27, 128.03, 125.78, 111.27, 110.05, 106.85, 100.68, 62.72, 57.85, 55.02, 54.92, 52.69, 52.01, 50.60, 50.05, 49.35, 44.59, 42.87, 35.95, 34.44, 31.60, 23.26, 10.30.

HRMS (ESI): C₃IH₃₂N₃O₃+, Calculated: [M+H]+, 504.3221; Found: [M+H]+, 504.3236.

IR (neat): 3395, 3026, 2935, 2801, 1729, 1621, 1488, 1453, 1439 cm'¹.

AD-3128: Following General Procedure P, methyl 8-((5-methoxy-l-propyl-lH-indol-3-yl)methyl)-2- phenethyl-2,8-diazaspiro[4.5]decane-4-carboxylate (45.5 mg, 0.090 mmol) was used. The product was purified using column chromatography (0-100% DCM:MeOH (1% NFUOH) on silica over 9 minutes) to afford the desired product as a yellow oil: 29.3 mg (68%).

'H NMR (500 MHz, Methanol-d4) 57.29 - 7.21 (m, 3H), 7.21 - 7.16 (m, 3H), 7.16 - 7.11 (m, 2H), 6.81 (dd, J = 8.8, 2.4 Hz, 1H), 4.06 (t, J = 6.9 Hz, 2H), 3.82 (s, 3H), 3.77 (s, 2H), 3.69 (dd, J = 10.7, 5.6 Hz, 1H), 3.48 (dd, J = 10.7, 8.0 Hz, 1H), 3.06 (dd, J = 9.7, 7.1 Hz, 1H), 2.91 (d, J = 11.7 Hz, 2H), 2.83 - 2.75 (m, 3H), 2.74 - 2.62 (m, 2H), 2.45 - 2.36 (m, 2H), 2.36 - 2.27 (m, 1H), 2.27 - 2.15 (m, 1H), 1.97 (p, J = 7.8 Hz, 1H), 1.81 (h, J = 7.3 Hz, 3H), 1.74 (td, J = 12.1, 3.6 Hz, 1H), 1.57 (dd, J = 23.3, 13.7 Hz, 2H), 0.86 (t, J = 7.4 Hz, 3H).

¹³C NMR (125 MHz, Methanol-^) 5 154.05, 139.72, 131.60, 129.41, 129.02, 128.24, 128.05, 125.79, 111.22, 109.99, 107.21, 100.65, 63.49, 60.83, 58.37, 56.95, 54.90, 52.15, 50.36, 49.66, 49.51, 40.65, 36.42, 34.35, 30.42, 23.25, 10.28.

HRMS (ESI): C₃oH₄2N₃02+, Calculated: [M+H]+, 476.3272; Found: [M+H]+, 476.3295.

IR (neat): 3333, 2922, 2804, 1731, 1620, 1577, 1487, 1454 crrr¹.

Methyl 2-phenethyl-8-((l-propyl-lH-pyrrolo[2,3-b]pyridin-3-yl)methyl)-2,8-diazaspiro[4.5]decane-4- carboxylate: Following General Procedure R, methyl 2-phenethyl-2,8-diazaspiro[4.5]decane-4-carboxylate (99.5 mg, 0.329 mmol) and l-propyl-lH-pyrrolo[2,3-b]pyridine-3-carbaldehyde (92.9 mg, 0.4935 mmol) were used. The product was purified using column chromatography (0-100% ACN:MeOH (1% NH₄OH) on silica over 10 minutes) to afford the desired product as a yellow oil: 81.4 mg (82%).

'H NMR (500 MHz, Methanol-^) 6 8.23 - 8.19 (m, 1H), 8.06 (dd, J = 7.9, 1.6 Hz, 1H), 7.35 (s, 1H), 7.21 (t, J = 7.3 Hz, 2H), 7.18 - 7.11 (m, 3H), 7.09 (dd, J = 7.9, 4.8 Hz, 1H), 4.21 (t, J = 7.1 Hz, 2H), 3.67 (s, 2H), 3.62 (s, 3H), 2.96 (dd, J = 9.5, 7.4 Hz, 1H), 2.82 - 2.59 (m, 9H), 2.31 (d, J = 9.5 Hz, 1H), 2.24 - 2.14 (m, 1H), 2.09 (d, J = 13.3 Hz, 1H), 1.92 (td, J = 13.1, 4.1 Hz, 1H), 1.84 (h, J = 7.4 Hz, 2H), 1.63 (d, J = 13.7 Hz, 1H), 1.52 (d, J = 13.7 Hz, 1H), 1.50 - 1.41 (m, 1H), 0.88 (t, J = 7.4 Hz, 3H).

¹³C NMR (125 MHz, Methanol-^) 6 172.90, 146.93, 141.82, 139.86, 128.90, 128.29, 128.04, 127.94, 125.78, 121.47, 115.13, 108.00, 62.81, 57.95, 55.04, 52.94, 52.25, 50.57, 49.77, 48.49, 45.74, 43.04, 36.54, 34.54, 32.24, 23.31, 10.20.

HRMS (ESI): C29H₃₉N₄O2+, Calculated: [M+H]+, 475.3068; Found: [M+H]+, 475.3083.

IR (neat): 3026, 2935, 2801 , 1732, 1600, 1541 , 1455, 1434 cm'¹.

501-A-001: Following General Procedure P, methyl 2-phenethyl-8-((l -propyl- lH-pyrrolo[2, 3-b]pyridin-3- yl)methyl)-2,8-diazaspiro[4.5]decane-4-carboxylate (40.7 mg, 0.0857 mmol) was used. The product was purified using column chromatography (0-100% DCM:MeOH (1% NH4OH) on silica over 9 minutes) to afford the desired product as a clear oil: 12.4 mg (38%).

'H NMR (600 MHz, Methanol-^) 5 8.12 (dd, J = 4.8, 1.6 Hz, 1H), 8.01 (dd, J = 7.9, 1.6 Hz, 1H), 7.29 (s, 1H), 7.16 - 7.12 (m, 2H), 7.09 (dd, J = 8.3, 1.4 Hz, 2H), 7.07 - 7.04 (m, 1H), 7.02 (dd, J = 7.9, 4.8 Hz, 1H), 4.15 (t, J = 7.1 Hz, 2H), 3.61 (s, 2H), 3.60 - 3.57 (m, 1H), 3.38 (dd, J = 10.6, 8.3 Hz, 1H), 2.95 (dd, J = 9.6, 7.1 Hz, 1H), 2.72 (s, 2H), 2.68 (t, J = 8.6 Hz, 3H), 2.63 - 2.52 (m, 2H), 2.28 (dd, J = 9.3, 6.7 Hz, 2H), 2.14 - 2.05 (m, 1H), 2.01 (t, J = 11.6 Hz, 1H), 1.89 - 1.83 (m, 1H), 1.77 (h, J = 7.4 Hz, 2H), 1.69 (td, J = 12.9, 4.2 Hz, 1H), 1.60 (td, J = 12.5, 4.0 Hz, 1H), 1.50 - 1.41 (m, 2H), 0.80 (t, J = 7.4 Hz, 3H).

¹³C NMR (150 MHz, Methanol-d4) 6 147.00, 141.89, 140.02, 128.93, 128.38, 128.16, 128.03, 125.87, 121.48, 115.18, 108.25, 63.70, 60.96, 58.53, 57.19, 52.43, 50.81, 50.01, 49.77, 45.73, 40.77, 37.08, 34.56, 31.04, 23.37, 10.29.

HRMS (ESI): C28H39N4O+, Calculated: [M+H]+, 447.3118; Found: [M+H]+, 447.3122.

IR (neat): 3327, 2926, 2801, 1663, 1600, 1538, 1455 cm ¹.

Methyl 2-benzyl-8-((5-fluoro-l-propyl-lH-indol-3-yl)methyl)-2,8-diazaspiro[4.5]decane-4-carboxylate:

Following General Procedure R methyl 2-benzyl-2,8-diazaspiro[4.5]decane-4-carboxylate (124.9 mg, 0.433 mmol) and 5-fluoro-l-propyl-lH-indole-3-carbaldehyde (133.3 mg, 0.65 mmol) were used. The product was purified using column chromatography (0-100% ACN:MeOH (1% NH4OH) on silica over 10 minutes) to afford the desired product as a yellow oil: 90.2 mg (44%).

'H NMR (600 MHz, Methanol-d4) δ 7.39 - 7.28 (m, 7H), 7.28 - 7.22 (m, 1H), 6.95 (td, J = 9.1, 2.6 Hz, 1H), 4.11 (t, J = 7.0 Hz, 2H), 3.84 (s, 2H), 3.68 - 3.58 (m, 5H), 2.96 (d, J = 12.5 Hz, 1H), 2.94 - 2.86 (m, 2H), 2.79 (t, J = 9.0 Hz, 1H), 2.77 - 2.71 (m, 2H), 2.38 (d, J = 9.5 Hz, 2H), 2.34 (s, 1H), 2.00 (td, J = 12.8, 4.3 Hz, 1H), 1.83 (h, J = 7.3 Hz, 2H), 1.73 (dq, J = 13.9, 3.4 Hz, 1H), 1.62 (dq, J = 13.5, 2.9 Hz, 1H), 1.58 - 1.51 (m, 1H), 0.88 (t, J = 7.4 Hz, 3H).

¹³C NMR (150 MHz, Methanol-d4) δ 172.94, 158.70, 157.16, 138.15, 132.95, 131.00, 128.86, 128.80, 128.58, 128.00, 126.92, 110.44, 110.37, 109.53, 109.36, 106.79, 103.42, 103.26, 62.30, 59.67, 54.72, 52.83, 51.84, 50.62, 50.16, 49.47, 47.62, 42.88, 35.69, 31.45, 23.20, 10.27.

HRMS (ESI): C29H37FN3O2+, Calculated: [M+H]+, 478.2870; Found: [M+H]+, 478.2871.

IR (neat): 2936, 2805, 1732, 1624, 1579, 1486, 1452 cm’¹.

501-A-031: Following General Procedure P, methyl 2-benzyl-8-((5-fluoro-l-propyl-lH-indol-3-yl)methyl)- 2,8-diazaspiro[4.5]decane-4-carboxylate (45.1 mg, 0.0944 mmol) was used. The product was purified using column chromatography (0-100% DCM:MeOH (1% NH4OH) on silica over 9 minutes) to afford the desired product as a yellow oil: 38.2 mg (90%).

'H NMR (600 MHz, Methanol-d4) δ 7.26 - 7.17 (m, 6H), 7.17 - 7.12 (m, 2H), 6.81 (td, J = 9.1, 2.5 Hz, 1H), 4.00 (t, J = 7.0 Hz, 2H), 3.58 (q, J = 5.5 Hz, 3H), 3.50 (s, 2H), 3.37 (dd, J = 10.6, 8.3 Hz, 1H), 2.84 (dd, J = 9.6, 7.1 Hz, 1H), 2.73 (d, J = 8.0 Hz, 2H), 2.60 (d, J = 9.9 Hz, 1H), 2.25 (dd, J = 16.0, 9.4 Hz, 2H), 2.07 (s, 1H), 2.01 (s, 1H), 1.87 - 1.81 (m, 1H), 1.76 - 1.65 (m, 3H), 1.60 (td, J = 12.9, 3.9 Hz, 1H), 1.49 (d, J = 13.8 Hz, 1H), 1.44 (d, J = 12.3 Hz, 1H), 0.76 (t, J = 7.4 Hz, 3H).

¹³C NMR (150 MHz, Methanol-d4) 5 158.55, 157.00, 138.01, 132.87, 130.42, 129.01, 128.95, 128.81, 127.95, 126.91, 110.17, 110.10, 109.21, 109.03, 103.39, 103.24, 61.04, 60.26, 56.88, 52.13, 50.58, 49.84, 48.46, 47.50, 40.72, 36.71, 30.82, 27.26, 23.22, 10.24.

HRMS (ESI): C28H37FN3O+, Calculated: [M+H]+, 450.2915: Found: [M+H]+, 450.2942.

IR (neat): 3339, 2919, 2804, 1485, 1451 cm ¹.

Methyl 2-benzyl-8-((5-methoxy-l-propyl-lH-indol-3-yl)methyl)-2,8-diazaspiro[4.5]decane-4- carboxylate: Following General Procedure R methyl 2-benzyl-2,8-diazaspiro[4.5]decane-4-carboxylate (124.9 mg, 0.433 mmol) and 5-methoxy-l-propyl-lH-indole-3-carbaldehyde (141.11 mg, 0.65 mmol) were used. The product was purified using column chromatography (0-100% ACN:MeOH (1% NH4OH) on silica over 10 minutes) to afford the desired product as a yellow oil: 69.9 mg (35%).

'H NMR (600 MHz, Methanol-d4) 57.36 - 7.29 (m, 6H), 7.28 - 7.24 (m, 1H), 7.19 (d, J = 2.5 Hz, 1H), 6.89 - 6.86 (m, 1H), 4.15 - 4.10 (m, 4H), 3.86 (s, 3H), 3.66 (dd, J = 19.4, 11.3 Hz, 5H), 3.19 (d, J = 15.1 Hz, 1H), 3.14 (s, 1H), 2.93 (t, J = 8.5 Hz, 1H), 2.84 (t, J = 8.9 Hz, 1H), 2.81 - 2.76 (m, 2H), 2.72 (s, 2H), 2.47 (d, J = 9.5 Hz, 1H), 2.14 - 2.04 (m, 1H), 1.85 (h, J = 7.3 Hz, 3H), 1.74 (d, J = 12.9 Hz, 1H), 1.68 - 1.60 (m, 1H), 0.90 (t, J = 7.4 Hz, 3H).

¹³C NMR (150 MHz, Methanol^) 6 172.76, 154.58, 138.14, 131.63, 130.68, 128.69, 128.54, 128.01, 126.94, 111.82, 110.44, 100.39, 61.98, 59.52, 54.97, 54.66, 52.50, 51.59, 50.72, 49.64, 49.07, 47.65, 42.50, 34.68, 30.46, 23.22, 10.25.

HRMS (ESI): C30H42N3O3+, Calculated: [M+H]+, 490.3070; Found: [M+H]+, 490.3073.

IR (neat): 2924, 2804, 1732, 1622, 1489, 1453 cm'¹.

501-A-032: Following General Procedure P, methyl 2-benzyl-8-((5-methoxy-l-propyl-lH-indol-3- yl)methyl)-2,8-diazaspiro[4.5]decane-4-carboxylate (34.95 mg, 0.0714 mmol) was used. The product was purified using column chromatography (0-100% DCM:MeOH (1% NH4OH) on silica over 9 minutes) to afford the desired product as a yellow oil: 21.6 mg (58%).

'H NMR (500 MHz, Methanol-d4) 57.35 - 7.29 (m, 4H), 7.26 (dd, J = 8.8, 1.8 Hz, 2H), 7.15 (s, 1H), 7.12 (d, J = 2.3 Hz, 1H), 6.82 (dd, J = 8.9, 2.4 Hz, 1H), 4.08 (t, J = 6.9 Hz, 2H), 3.84 (s, 3H), 3.70 (dd, J = 10.5, 5.5 Hz, 1H), 3.67 (s, 2H), 3.61 (s, 2H), 3.48 (dd, J = 10.7, 8.4 Hz, 1H), 2.95 (dd, J = 9.5, 7.1 Hz, 1H), 2.84 (d, J = 11.3 Hz, 2H), 2.71 (d, J = 9.8 Hz, 1H), 2.40 - 2.32 (m, 2H), 2.13 (dt, J = 33.9, 10.8 Hz, 2H), 1.99 - 1.91 (m, 1H), 1.87 - 1.66 (m, 4H), 1.59 (dd, J = 13.4, 2.5 Hz, 1H), 1.53 (dd, J = 12.7, 3.2 Hz, 1H), 0.88 (t, J = 7.4 Hz, 3H).

¹³C NMR (125 MHZ, Methanol-A) 3 153.90, 138.13, 131.61, 129.02, 128.78, 127.93, 126.86, 111.13, 109.88, 108.12, 100.66, 63.40, 61.12, 60.28, 56.94, 54.88, 52.36, 50.67, 49.92, 49.75, 47.39, 40.80, 36.88, 30.94, 23.28, 10.28.

HRMS (ESI): C29H40N3O2+, Calculated: [M+H]+, 462.3115; Found: [M+H]+, 462.3184.

IR (neat): 3343, 2921, 2805, 1487, 1452 cm'¹.

Methyl 8-((5-fluoro-lH-indol-3-yl)methyl)-2-phenethyl-2,8-diazaspiro[4.5]decane-4-carboxylate:

Following General Procedure R, methyl 2-phenethyl-2,8-diazaspiro[4.5]decane-4-carboxylate (66.83 mg, 0.221 mmol) and 5-fluoro-lH-indole-3-carbaldehyde (54.08 mg, 0.331 mmol) were used. The product was purified using column chromatography (0-100% ACN:MeOH (1% NH4OH) on silica over 10 minutes) to afford the desired product as a yellow oil: 80.2 mg (81%).

'H NMR (500 MHz, Methanol-d4) 57.22 - 7.16 (m, 2H), 7.15 - 7.09 (m, 3H), 7.04 (dd, J = 15.9, 7.2 Hz, 3H), 6.76 (td, J = 9.2, 2.5 Hz, 1H), 3.62 - 3.57 (m, 1H), 3.54 (s, 2H), 3.50 (s, 3H), 2.85 (t, J = 8.5 Hz, 1H), 2.69 (d, J = 9.3 Hz, 2H), 2.64 (t, J = 8.3 Hz, 3H), 2.60 - 2.48 (m, 3H), 2.20 (d, J = 9.6 Hz, 1H), 2.13 - 2.04 (m, 1H), 2.03 - 1.95 (m, 1H), 1.82 (td, J = 12.6, 4.1 Hz, 1H), 1.76 - 1.70 (m, 1H), 1.52 (d, J = 11.6 Hz, 1H), 1.43 - 1.32 (m, 2H).

¹³C NMR (125 MHz, Methanol-*) 6 172.97, 158.64, 156.79, 139.88, 133.03, 128.50, 128.42, 128.31, 128.06, 127.00, 125.80, 111.80, 111.73, 109.59, 109.55, 109.29, 109.08, 103.08, 102.90, 67.46, 62.91, 57.97, 55.03, 52.93, 52.34, 50.58, 50.50, 49.69, 43.04, 36.45, 34.53, 32.14, 29.56, 25.11.

HRMS (ESI): C₂7H₃₃FN₃O2+, Calculated: [M+H]+, 450.2557; Found: [M+H]+, 450.2585.

IR (neat): 3367, 2921, 2805, 1731, 1583, 1486, 1438 cm*

501-A-053: Following General Procedure P, methyl 8-((5-fluoro-lH-indol-3-yl)methyl)-2-phenethyl-2,8- diazaspiro[4.5]decane-4-carboxylate (40.1 mg, 0.0892 mmol) was used. The product was purified using column chromatography (0-100% DCM:MeOH (1% NF4OH) on silica over 9 minutes) to afford the desired product as a clear oil: 29.6 mg (79%).

'H NMR (500 MHz, Methanol-d4) 57.22 - 7.16 (m, 3H), 7.13 (t, J = 7.3 Hz, 2H), 7.05 (dd, J = 15.9, 7.4 Hz, 3H), 6.76 (td, J = 9.2, 2.5 Hz, 1H), 3.57 (q, J = 5.5 Hz, 3H), 3.36 (dd, J = 10.8, 8.2 Hz, 1H), 2.94 (dd, J = 9.8, 7.0 Hz, 1H), 2.73 (d, J = 9.3 Hz, 2H), 2.66 (t, J = 6.7 Hz, 3H), 2.55 (q, J = 7.2 Hz, 2H), 2.27 (t, J = 9.2 Hz, 2H), 2.15 - 2.07 (m, 1H), 2.02 (d, J = 11.2 Hz, 1H), 1.89 - 1.81 (m, 1H), 1.68 (td, J = 12.7, 4.0 Hz, 1H), 1.58 (td, J = 12.7, 4.1 Hz, 1H), 1.45 (d, J = 13.4 Hz, 1H), 1.39 (d, J = 13.3 Hz, 1H). ¹³C NMR (125 MHZ, Methanol-

5 158.66, 156.81, 139.78, 133.03, 128.49, 128.41, 128.29, 128.08, 127.14, 125.82, 111.84, 111.76, 109.32, 109.10, 103.05, 102.86, 63.62, 60.91, 58.42, 57.04, 52.40, 50.62, 49.88, 49.58, 40.71, 36.67, 34.42, 30.68.

HRMS (ESI): C26H33FN3O+, Calculated: [M+H]+, 422.2602; Found: [M+H]+, 422.2612.

IR (neat): 3216, 2919, 2807, 1583, 1485, 1453 cm ¹.

Methyl 8-((5-methoxy-lH-indol-3-yl)methyl)-2-phenethyl-2,8-diazaspiro[4.5]decane-4-carboxylate:

Following General Procedure R, methyl 2-phenethyl-2,8-diazaspiro[4.5]decane-4-carboxylate (66.83 mg, 0.221 mmol) and 5-methoxy-lH-indole-3-carbaldehyde (58.07 mg, 0.331 mmol) were used. The product was purified using column chromatography (0-100% ACN:MeOH (1% NH4OH) on silica over 10 minutes) to afford the desired product as a yellow oil: 60.9 mg (81%).

'H NMR (500 MHz, Methanol-^) 6 7.15 (dd, J = 7.9, 6.9 Hz, 3H), 7.11 - 7.04 (m, 4H), 7.02 (d, J = 2.4 Hz, 1H), 6.67 (dd, J = 8.8, 2.4 Hz, 1H), 3.73 (s, 3H), 3.60 (s, 2H), 3.53 (s, 3H), 2.91 (dd, J = 9.5, 7.5 Hz, 1H), 2.84 - 2.52 (m, 10H), 2.26 (d, J = 9.6 Hz, 1H), 2.15 (s, 1H), 2.07 (s, 1H), 1.86 (td, J = 12.7, 4.3 Hz, 1H), 1.57 (dd, J = 13.6, 1.7 Hz, 1H), 1.47 - 1.36 (m, 2H).

¹³C NMR (125 MHz, Methanol-^) 5 173.00, 153.86, 139.89, 131.70, 128.45, 128.30, 128.05, 125.78, 125.71 , 1 1 1.58, 1 1 1.23, 109.10, 100.38, 67.46, 62.91 , 57.99, 55.04, 54.91 , 52.96, 52.48, 50.55, 50.51 , 49.69, 43.08, 36.50, 34.53, 32.17, 29.54, 25.10.

HRMS (ESI): C28H36N3O3+, Calculated: [M+H]+, 462.2757; Found: [M+H]+, 462.2763.

IR (neat): 3390, 2937, 2802, 1731, 1584, 1484, 1439 cm'¹.

501-A-054: Following General Procedure P, methyl 8-((5-methoxy-lH-indol-3-yl)methyl)-2-phenethyl-2,8- diazaspiro[4.5]decane-4-carboxylate (30.45 mg, 0.066 mmol) was used. The product was purified using column chromatography (0-100% DCM:MeOH (1% NH4OH) on silica over 9 minutes) to afford the desired product as a clear oil: 25.2 mg (88%). 'H NMR (500 MHz, Methanol-d4) δ 7.16 - 7.11 (m, 3H), 7.10 - 7.02 (m, 4H), 7.02 (d, J = 2.3 Hz, 1H), 6.67 (dd, J = 8.9, 2.4 Hz, 1H), 3.71 (s, 3H), 3.61 - 3.56 (m, 3H), 3.36 (dd, J = 10.7, 8.2 Hz, 1H), 2.94 (dd, J = 9.6, 7.2 Hz, 1H), 2.75 (s, 2H), 2.70 - 2.65 (m, 3H), 2.62 - 2.49 (m, 2H), 2.27 (dt, J = 8.7, 4.0 Hz, 2H), 2.16 - 2.08 (m, 1H), 2.08 - 1.98 (m, 1H), 1.89 - 1.81 (m, 1H), 1.68 (td, J = 12.7, 4.0 Hz, 1H), 1.58 (td, J = 12.5, 4.0 Hz, 1H), 1.46 (dd, J = 13.6, 2.7 Hz, 1H), 1.40 (dd, J = 12.9, 3.0 Hz, 1H).

¹³C NMR (125 MHz, Methanol-A) 3 153.87, 139.87, 131.70, 128.44, 128.27, 128.05, 125.78, 125.76, 111.57, 111.23, 108.99, 100.34, 63.71, 60.98, 58.48, 57.11, 54.91, 52.56, 50.68, 49.90, 48.47, 40.73, 36.79, 34.49, 30.78.

HRMS (ESI): C27H₃₆N₃O2+, Calculated: [M+H]+, 434.2808; Found: [M+H]+, 434.2811.

IR (neat): 3233, 2920, 2808, 1583, 1486 cm'¹.

Methyl 8-(benzofuran-3-ylmethyl)-2-phenethyl-2,8-diazaspiro[4.5]decane-4-carboxylate: Following

General Procedure R, methyl 2-phenethyl-2,8-diazaspiro[4.5]decane-4-carboxylate (119.45 mg, 0.395 mmol) and benzofuran-3-carbaldehyde (86.59 mg, 0.331 mmol) were used. The product was purified using column chromatography (0-100% ACN:MeOH (1% NH4OH) on silica over 10 minutes) to afford the desired product as a yellow oil: 107.4 mg (63%).

'H NMR (500 MHz, Methanol-d4) δ 7.56 (dd, J = 7.1, 1.4 Hz, 1H), 7.54 (s, 1H), 7.33 (d, J = 8.1 Hz, 1 H), 7.16 (td, J = 7.7, 1.4 Hz, 1H), 7.14 - 7.09 (m, 3H), 7.07 - 7.00 (m, 3H), 3.51 (s, 3H), 3.51 (s, 2H), 2.86 (dd, J = 9.5, 7.3 Hz, 1H), 2.72 - 2.48 (m, 10H), 2.19 (d, J = 9.6 Hz, 1H), 2.11 - 2.02 (m, 1H), 2.02 - 1.93 (m, 1H), 1.81 (td, J = 12.6, 4.2 Hz, 1H), 1.52 (d, J = 14.0 Hz, 1H), 1.43 - 1.32 (m, 2H).

¹³C NMR (125 MHZ, Methanol-d4) δ 172.99, 155.40, 144.03, 139.90, 128.31, 128.11, 128.07, 125.81, 124.08, 122.34, 119.89, 115.89, 110.89, 62.89, 57.99, 55.09, 52.99, 50.80, 50.76, 50.60, 50.01, 43.05, 36.70, 34.57, 32.40.

HRMS (ESI): C27H₃₃N₂O₃+, Calculated: [M+H]+, 433.2491; Found: [M+H]+, 433.2486.

IR (neat): 2940, 2805, 1732, 1451 cm'¹.

501-A-066: Following General Procedure P, methyl 8-(benzofuran-3-ylmethyl)-2-phenethyl-2,8- diazaspiro[4.5]decane-4-carboxylate (53.7 mg, 0.124 mmol) was used. The product was purified using column chromatography (0-100% DCM:MeOH (1% NH4OH) on silica over 9 minutes) to afford the desired product as a clear oil: 29.5 mg (59%).

'H NMR (500 MHz, Methanol-d4) 5 7.56 (dd, J = 7.0, 1.4 Hz, 1H), 7.53 (s, 1H), 7.32 (d, J = 7.5 Hz, 1H), 7.17 - 7.08 (m, 4H), 7.07 - 7.00 (m, 3H), 3.57 (dd, J = 10.6, 5.6 Hz, 1H), 3.47 (s, 2H), 3.36 (dd, J = 10.7, 8.2 Hz, 1H), 2.92 (dd, J = 9.6, 7.0 Hz, 1H), 2.70 - 2.61 (m, 5H), 2.58 - 2.47 (m, 2H), 2.29 - 2.21 (m, 2H), 2.07 - 1.98 (m, 1H), 1.98 - 1.89 (m, 1H), 1.87 - 1.80 (m, 1H), 1.65 (td, J = 12.7, 4.1 Hz, 1H), 1.55 (td, J = 12.4, 4.0 Hz, 1H), 1.42 (d, J = 13.4 Hz, 1H), 1.35 (d, J = 12.4 Hz, 1H).

¹³C NMR (125 MHz, Methanol-d4) δ 155.39, 143.97, 139.80, 128.33, 128.13, 125.87, 124.10, 122.36, 119.95, 116.06, 110.92, 63.69, 60.96, 58.48, 57.07, 51.05, 50.95, 50.27, 49.64, 40.74, 37.09, 34.47, 31.08. HRMS (ESI): C26H33N2O2+, Calculated: 1M+H]+, 405.2537; Found: [M+H]+, 405.2543.

IR (neat): 3339, 2919, 2805, 1451 cm ¹.

8- (tert-butyl) 4-methyl 2-(2-(pyridin-2-yl)ethyl)-2,8-diazaspiro[4.5]decane-4,8-dicarboxylate:

Following General Procedure N, 8-(tert-butyl) 4-methyl 2-benzyl-2,8-diazaspiro[4.5]decane-4,8- dicarboxylate (445.95 mg, 1.145 mmol) and 2-(2-chloroethyl)pyridine hydrochloride (246.04 mg, 1.37 mmol) were used along with the addition of TBAI (422.93 mg, 1.145 mmol). The product was purified using column chromatography (0-100% EtOAc:hexanes on neutral alumina over 10 minutes) to afford the desired product as a yellow oil: 51.2 mg (11%).

'H NMR (600 MHz, Methanol-^) 5 8.46 (ddd, J = 5.0, 1.9, 1.0 Hz, 1H), 7.78 (td, J = 7.6, 1.8 Hz, 1H), 7.39 (dt, J = 7.8, 1.0 Hz, 1H), 7.28 (ddd, J = 7.6, 5.0, 1.2 Hz, 1H), 3.91 - 3.82 (m, 2H), 3.70 (s, 3H), 3.07 - 2.95 (m, 4H), 2.95 - 2.82 (m, 5H), 2.77 (t, J = 8.1 Hz, 1H), 2.52 (d, J = 9.5 Hz, 1H), 1.78 (td, J = 12.6, 4.5 Hz, 1H), 1.66 - 1.60 (m, 1H), 1.53 (dq, J = 13.2, 3.0 Hz, 1H), 1.47 (s, 9H), 1.36 - 1.29 (m, 1H). ¹³C NMR (150 MHz, Methanol-

5 172.98, 159.77, 155.11, 148.21, 137.29, 123.65, 121.64, 79.70, 61.85, 55.68, 54.85, 53.62, 53.11, 50.66, 43.58, 41.06, 36.48, 36.27, 32.26, 27.28.

HRMS (ESI): C22H₃4N₃O₄+, Calculated: [M+H]+, 404.2544; Found: [M+H]+, 404.2584.

IR (neat): 3364, 2972, 2934, 2858, 2808, 1733, 1690, 1476, 1432 cm'¹.

Methyl 2-(2-(pyridin-2-yl)ethyl)-2,8-diazaspiro[4.5]decane-4-carboxylate: Following General Procedure

Q, 8-(tert-butyl) 4-methyl 2-(2-(pyridin-2-yl)ethyl)-2,8-diazaspiro[4.5]decane-4,8-dicarboxylate (67.4 mg, 0.167 mmol) was used. The product was taken crude to the next step.

HRMS (ESI): C17H26N3O2+, Calculated: [M+H]+, 304.2020; Found: [M+H]+, 304.2027.

IR (neat): 3422, 3014, 2965, 2851, 2755, 1733, 1672, 1477, 1439 cm'¹.

Methyl 8-(cyclohexylmethyl)-2-(2-(pyridin-2-yl)ethyl)-2,8-diazaspiro[4.5]decane-4-carboxylate:

Following General Procedure R, methyl 2-(2-(pyridin-2-yl)cthyl)-2,8-diazaspiro[4.5]dccanc-4-carboxylate (57.34 mg, 0.189 mmol) and cyclohexyl carboxaldehyde (31.8 mg, 0.284 mmol) were used. The product was purified using column chromatography (0-100% ACN:MeOH (1% NH₄OH) on silica over 10 minutes) to afford the desired product as a yellow oil: 25.0 mg (33%).

'H NMR (600 MHz, Methanol-^) 5 8.48 (ddd, J = 4.8, 1.8, 1.0 Hz, 1H), 7.79 (td, J = 7.6, 1.8 Hz, 1H), 7.45 (d, J = 7.9 Hz, 1H), 7.30 (ddd, J = 7.5, 5.0, 1.2 Hz, 1H), 3.77 (s, 3H), 3.38 (s, 1H), 3.32 - 3.12 (m, 8H), 3.05 (d, J = 7.3 Hz, 1H), 2.97 - 2.67 (m, 4H), 2.24 - 2.14 (m, 1H), 1.98 (d, J = 13.8 Hz, 1H), 1.89 - 1.74 (m, 8H), 1.71 (dt, J = 12.9, 3.4 Hz, 1H), 1.35 (qt, J = 12.8, 3.3 Hz, 2H), 1.23 (qt, J = 12.6, 3.4 Hz, 1H), 1.06 (q, J = 12.6 Hz, 2H).

¹³C NMR (150 MHz, Methanol-d4) δ 172.16, 158.69, 148.35, 137.43, 123.87, 121.90, 63.25, 61.04, 55.49, 54.37, 51.67, 51.15, 50.45, 50.11, 42.57, 34.86, 33.26, 33.12, 30.87, 29.24, 25.77, 25.35.

HRMS (ESI): C24H38N3O2+, Calculated: [M+H]+, 400.2964; Found: [M+H]+, 400.2968.

IR (neat): 2921, 2849, 1732, 1592, 1474, 1436 cm'¹.

501-A-106: Following General Procedure P, methyl 8-(cyclohexylmethyl)-2-(2-(pyridin-2-yl)ethyl)-2,8- diazaspiro[4.5]decane-4-carboxylate (12.5 mg, 0.0313 mmol) was used. The product was purified using column chromatography (0-100% DCM:MeOH (1% NH4OH) on silica over 9 minutes) to afford the desired product as a clear oil: 10.9 mg (94%).

'H NMR (600 MHz, Methanol-d4) δ 8.51 - 8.46 (m, 1H), 7.80 (td, J = 7.8, 1.9 Hz, 1H), 7.46 (d, J = 7.8 Hz, 1H), 7.30 (ddd, J = 7.7, 5.0, 1.1 Hz, 1H), 3.75 (dd, J = 11.1, 5.5 Hz, 1H), 3.68 (dd, J = 11.0, 5.9 Hz, 1H), 3.47 (s, 1 H), 3.32 - 3.16 (m, 7H), 3.08 (d, J = 56.9 Hz, 2H), 2.73 (d, J = 39.9 Hz, 4H), 2.29 - 2.21 (m, 1 H), 2.10 (q, J = 13.9 Hz, 2H), 1.87 (dd, J = 23.3, 13.8 Hz, 4H), 1.77 (dd, J = 10.2, 3.4 Hz, 3H), 1.70 (d, J = 12.8 Hz, 1H), 1.34 (qt, J = 12.9, 3.3 Hz, 2H), 1.23 (qt, J = 12.6, 3.4 Hz, 1H), 1.09 - 0.99 (m, 2H).

¹³C NMR (150 MHz, Methanol-d4) δ 13C NMR 158.26, 148.44, 137.48, 123.87, 122.03, 63.67, 62.03, 59.94, 55.90, 55.57, 52.27, 50.58, 50.45, 40.88, 33.89, 33.87, 33.41, 31.01, 28.07, 25.85, 25.42.

HRMS (ESI): C23H38N3O+, Calculated: [M+H]+, 372.3009; Found: [M+H]+, 372.3016.

IR (neat): 3346, 2922, 2850, 2804, 1651, 1594, 1475, 1440 cm'¹.

8-(tert-butyl) 4-methyl 2-(2-morpholinoethyl)-2,8-diazaspiro[4.5]decane-4,8-dicarboxylate: Following General Procedure N, 8-(tert-butyl) 4-methyl 2-benzyl-2,8-diazaspiro[4.5]decane-4,8-dicarboxylate (210.6 mg, 0.542 mmol) and 4-(2-chloroethyl)morpholine (445.95 mg, 1.145 mmol) were used along with the addition of TBAI (422.93 mg, 1.145 mmol). The product was purified using column chromatography (0- 100% EtOAc:hexanes on neutral alumina over 10 minutes) to afford the desired product as a yellow oil: 122.5 mg (26%).

'H NMR (500 MHz, Methanol-d4) δ 3.80 - 3.68 (m, 2H), 3.65 - 3.52 (m, 7H), 2.96 - 2.67 (m, 5H), 2.67 - 2.52 (m, 3H), 2.48 - 2.39 (m, 6H), 2.36 (d, J = 9.5 Hz, 1H), 1.67 (td, J = 12.6, 4.6 Hz, 1H), 1.52 (d, J = 13.4 Hz, 1H), 1.45 - 1.39 (m, 1H), 1.35 (s, 9H), 1.21 (td, J = 12.7, 4.3 Hz, 1H). ¹³C NMR (125 MHz, Methanol-d4) δ 172.91, 155.09, 79.70, 66.24, 64.04, 62.37, 56.88, 56.63, 55.15, 53.70,

53.64, 53.03, 52.79, 50.69, 43.50, 41.25, 36.44, 32.28, 27.29.

HRMS (ESI): C2iH₃₈N₃O₅+, Calculated: [M+H]+, 412.2806; Found: [M+H]+, 412.2835.

IR (neat): 2948, 2934, 2853, 2808, 1733, 1691, 1476, 1450, 1424 cm'¹.

Methyl 2-(2-morpholinoethyl)-2,8-diazaspiro[4.5]decane-4-carboxylate: Following General Procedure

Q, 8-(tert-butyl) 4-methyl 2-(2-morpholinoethyl)-2,8-diazaspiro[4.5]decane-4,8-dicarboxylate (67.8 mg, 0.165 mmol) was used. The product was taken crude to the next step.

HRMS (ESI): CI₆H₃₀N₃O₃+, Calculated: [M+H]+, 312.2282; Found: [M+H]+, 312.2288.

IR (neat): 3445, 3013, 2971, 2861, 2757, 1732, 1670, 1457, 1438 cm'¹.

Methyl 8-(cyclohexylmethyl)-2-(2-morpholinoethyl)-2,8-diazaspiro[4.5]decane-4-carboxylate:

Following General Procedure R, methyl 2-(2-morpholinoethyl)-2,8-diazaspiro[4.5]decane-4-carboxylate (51.38 mg, 0.165 mmol) and cyclohexyl carboxaldehyde (27.76 mg, 0.248 mmol) were used. The product was purified using column chromatography (0-100% ACN:MeOH (1% NH4OH) on silica over 10 minutes) to afford the desired product as a yellow oil: 23.1 mg (34%).

'H NMR (500 MHz, Methanol-d4) δ 3.60 (d, J = 3.5 Hz, 7H), 2.99 - 2.93 (m, 1H), 2.86 - 2.57 (m, 7H), 2.49 - 2.41 (m, 5H), 2.37 - 2.34 (m, 1H), 2.19 - 1.97 (m, 4H), 1.88 (td, J = 12.7, 4.1 Hz, 1H), 1.72 - 1.54 (m, 7H), 1.52 - 1.39 (m, 3H), 1.25 - 1.04 (m, 3H), 0.83 (qd, J = 12.3, 3.3 Hz, 2H).

¹³C NMR (125 MHz, Methanol-d4) δ 172.94, 66.23, 65.24, 62.87, 56.75, 56.54, 55.18, 53.68, 52.78, 51.44, 50.79, 50.70, 43.84, 43.03, 41.27, 35.84, 34.56, 31.64, 31.61, 26.24, 25.74.

HRMS (ESI): C2₃H₄2N₃O₃+, Calculated: [M+H]+, 408.3221; Found: [M+H]+, 408.3228.

IR (neat): 3400, 2923, 2850, 2805, 2771, 1733, 1669, 1651, 1450 cm'¹.

501-A-092: Following General Procedure P, methyl 8-(cyclohexylmethyl)-2-(2-morpholinoethyl)-2,8- diazaspiro[4.5]decane-4-carboxylate (11.55 mg, 0.0352 mmol) was used. The product was purified using column chromatography (0-100% DCM:MeOH (1% NH4OH) on silica over 9 minutes) to afford the desired product as a clear oil: 6 mg (56%).

'H NMR (500 MHz, Methanol-^) 6 3.74 (q, J = 4.3 Hz, 6H), 3.63 (t, J = 8.5 Hz, 1H), 3.37 (s, 3H), 3.32 - 3.17 (m, 4H), 2.95 (d, J = 29.0 Hz, 2H), 2.91 - 2.77 (m, 4H), 2.64 (s, 3H), 2.32 (s, 1H), 2.15 (dd, J = 20.2, 10.4 Hz, 2H), 2.00 (d, J = 13.6 Hz, 2H), 1.79 (dt, J = 13.4, 3.4 Hz, 5H), 1.76 - 1.68 (m, 1H), 1.42 - 1.31 (m, 3H), 1.24 (qt, J = 12.5, 3.3 Hz, 1H), 1.07 (q, J = 11.4 Hz, 2H).

¹³C NMR (125 MHz, Methanol-A) 5 66.24, 62.95, 61.99, 59.60, 55.88, 53.86, 53.17, 52.07, 50.33, 41.64, 40.64, 32.98, 32.90, 30.74, 29.37, 27.37, 25.71, 25.30.

HRMS (ESI): C22H42N3O2+, Calculated: [M+H]+, 380.3272; Found: [M+H]+, 380.3278.

IR (neat): 3365, 2923, 2851, 2815, 1651, 1451 cm ¹.

8- (tert-butyl) 4-methyl 2-(3-(dimethylamino)propyl)-2,8-diazaspiro[4.5]decane-4,8-dicarboxylate: To a flame dried flask equipped with a stir bar was added palladium on carbon (10%) (115.46 mg, 0.1086 mmol) and a slurry was formed with a small volume of methanol. Methyl 2-benzyl-8-(cyclohexylmethyl)-2,8- diazaspiro[4.5]decane-4-carboxylate (210.57 mg, 0.542 mmol) was then added as a solution in methanol (0.1M) and the reaction was heated to 35 °C for 6 hours. The reaction was filtered through celite and then concentrated under vacuum. The crude product was then dissolved in DCM (0.5M) and 3,3-dimethylbutanal (81.42 mg, 0.813 mmol), and acetic acid (32.5 mg, 0.542 mmol) were added. The reaction was heated to 35 °C for 1 hour before the addition of sodium triacetoxyborohydride (172.29 mg, 0.813 mmol). The reaction was stirred for 12 hours before being removed from heat. The reaction was quenched with H2O and was extracted 3x with EtOAc and NaHCO3. The organic layer was dried with sodium sulfate and subsequently concentrated under vacuum. The product was purified using column chromatography (0-10% DCM:MeOH over 10 minutes) to afford the desired product as a yellow oil: 76.3 mg (37%). 'H NMR (500 MHz, Methanol-d4) δ 3.80 - 3.71 (m, 2H), 3.59 (s, 3H), 2.92 (dd, J = 9.0, 6.6 Hz, 1H), 2.85 (d, J = 9.9 Hz, 2H), 2.71 - 2.63 (m, 2H), 2.50 - 2.39 (m, 2H), 2.32 (d, J = 10.0 Hz, 1H), 1.68 (td, J = 12.7, 4.7 Hz, 1H), 1.53 (d, J = 13.5 Hz, 1H), 1.45 - 1.40 (m, 1H), 1.35 (s, 12H), 1.25 - 1.18 (m, 1H), 0.84 (s, 9H). ¹³C NMR (125 MHz, Methanol-d4) δ 172.74, 155.07, 79.70, 62.30, 55.01, 53.03, 52.48, 50.70, 43.31, 41.37, 36.46, 32.47, 29.19, 28.48, 27.28.

HRMS (ESI): C21H39N2O4+, Calculated: [M+H]+, 383.2904; Found: [M+H]+, 383.2925.

IR (neat): 2952, 2865, 2807, 1736, 1695, 1475, 1423 cm’¹.

Methyl 2-(3,3-dimethylbutyl)-2,8-diazaspiro[4.5]decane-4-carboxylate: Following General Procedure Q, 8-(tert-butyl) 4-methyl 2-(3-(dimethylamino)propyl)-2,8-diazaspiro[4.5]decane-4,8-dicarboxylate (76.3 mg, 0.199 mmol) was used. The product was taken crude to the next step.

HRMS (ESI): C16H31N2O2+, Calculated: [M+H]+, 283.2380; Found: [M+H]+, 283.2393.

IR (neat): 3422, 2953, 2864, 2810, 1733, 1678, 1472, 1436 cm'¹.

Methyl 8-(cyclohexylmethyl)-2-(3-(dimethylamino)propyl)-2,8-diazaspiro[4.5]decane-4-carboxylate:

Following General Procedure R, methyl 2-(3,3-dimethylbutyl)-2,8-diazaspiro[4.5]decane-4-carboxylate (71.17 mg, 0.252 mmol) and cyclohexyl carboxaldehyde (42.40 mg, 0.378 mmol) were used. The product was purified using column chromatography (0-100% ACN:MeOH (1% NH4OH) on silica over 10 minutes) to afford the desired product as a yellow oil: 70.6 mg (74%).

'H NMR (500 MHz, Methanol-d4) δ 3.60 (s, 3H), 2.93 - 2.87 (m, 1 H), 2.79 (d, J = 9.8 Hz, 1H), 2.69 - 2.63 (m, 3H), 2.60 (d, J = 11.5 Hz, 1H), 2.49 - 2.35 (m, 2H), 2.21 (d, J = 9.7 Hz, 1H), 2.03 (d, J = 7.2 Hz, 2H), 1.96 (t, J = 11.2 Hz, 1H), 1.85 (td, J = 12.7, 4.0 Hz, 2H), 1.70 - 1.60 (m, 4H), 1.60 - 1.52 (m, 2H), 1.46 - 1.30 (m, 5H), 1.21 - 1.04 (m, 3H), 0.84 (s, 11H).

¹³C NMR (125 MHz, Methanol-A) 8 172.77, 65.64, 62.91, 55.13, 52.96, 52.54, 51.67, 50.91, 50.75, 43.06, 41.33, 36.35, 34.86, 32.30, 31.81, 29.28, 28.69, 26.44, 25.90.

HRMS (ESI): C23H43N2O2+, Calculated: [M+H]+, 379.3319; Found: [M+H]+, 379.3333. IR (neat): 2921, 2850, 2803, 2768, 1735, 1682, 1471, 1449, 1436 cm'¹.

501-A-087: Following General Procedure P, Methyl 8-(cyclohexylmethyl)-2-(3-(dimethylamino)propyl)- 2,8-diazaspiro[4.5]decane-4-carboxylate (35.3 mg, 0.0932 mmol) was used. The product was purified using column chromatography (0- 100% DCM:MeOH (1 % NH4OH) on silica over 9 minutes) to afford the desired product as a clear oil: 30.6 mg (94%).

'H NMR (500 MHz, Methanol-d4) δ 3.62 (dd, J = 10.8, 5.5 Hz, 1H), 3.45 (dd, J = 10.8, 7.8 Hz, 1H), 3.12 (dd, J = 10.3, 7.2 Hz, 1H), 2.89 (d, J = 10.4 Hz, 1H), 2.71 (d, J = 11.9 Hz, 2H), 2.61 (q, J = 7.1 Hz, 2H), 2.57

- 2.47 (m, 2H), 2.13 (d, J = 7.0 Hz, 2H), 2.11 - 1.94 (m, 3H), 1.76 (td, J = 12.7, 4.0 Hz, 1H), 1.72 - 1.40 (m, 11H), 1.24 - 1.04 (m, 3H), 0.85 (s, 11H).

¹³C NMR (125 MHz, Methanol-d4) 8 65.38, 63.26, 60.36, 56.65, 53.02, 51.51, 50.86, 49.05, 40.91, 40.43, 35.98, 34.63, 31.67, 30.15, 29.20, 28.44, 26.29, 25.79.

HRMS (ESI): C22H43N2O+, Calculated: [M+H]+, 351.3370; Found: [M+H]+, 351.3378.

IR (neat): 3340, 2921, 2850, 2801, 2766, 1470, 1449 cm'¹.

501-A-064: Following General Procedure P, methyl 2-benzyl-8-(cyclohexylmethyl)-2,8- diazaspiro[4.5]decane-4-carboxylate (500.0 mg, 1.3 mmol) was used. The product was purified using column chromatography (0-100% DCM:MeOH (1% NH4OH) on silica over 9 minutes) to afford the desired product as a clear oil: 168.9 mg (36%).

'H NMR (500 MHz, Methanol-d4) δ 7.25 - 7.18 (m, 4H), 7.17 - 7.12 (m, 1H), 3.60 (dd, J = 10.5, 5.5 Hz, 1H), 3.53 (s, 2H), 3.38 (dd, J = 10.6, 8.3 Hz, 1H), 2.86 (dd, J = 9.5, 7.1 Hz, 1H), 2.63 (d, J = 9.8 Hz, 3H), 2.28 (dd, J = 19.8, 9.7 Hz, 2H), 2.01 (d, J = 6.9 Hz, 2H), 1.92 - 1.79 (m, 3H), 1.72 - 1.54 (m, 7H), 1.49 - 1.34 (m, 3H), 1.21 - 1.02 (m, 3H), 0.79 (qd, J = 12.1, 2.9 Hz, 2H).

¹³C NMR (125 MHz, Methanol-d4) δ 138.10, 128.80, 127.97, 126.91, 65.76, 63.47, 61.14, 60.33, 56.96, 51.85, 51.12, 49.80, 40.88, 36.82, 34.79, 31.79, 30.89, 26.32, 25.83.

HRMS (ESI): C23H37N2O+, Calculated: [M+H]+, 357.2906; Found: [M+H]+, 357.2900.

IR (neat): 3342, 2919, 2849, 1494, 1469, 1449 cm'¹.

501-A-069: To a flame -dried flask equipped with a stir bar was added (2-benzyl-8-(cyclohexylmethyl)-2,8- diazaspiro[4.5]decan-4-yl)methanol (40.05 mg, 0.112 mmol) as a solution in ACN (0.5 M). NaH (60%) (5.39 mg, 0.135 mmol) was then added and allowed to react for 30 minutes. Propyl bromide (13.82 mg, 0.112 mmol) and TBAI (41.4 mg, 0.112 mmol) were then added, and the reaction was stirred for 12 hours at room temperature. The reaction was quenched with FLO and was extracted 3x with EtOAc and NaHCO3. The organic layer was dried with sodium sulfate and subsequently concentrated under vacuum. The product was purified using column chromatography (0-20% EtOAc :hexanes on neutral alumina over 9 minutes) to afford the desired product as a clear oil: 3.9 mg (9%).

'H NMR (500 MHz, Methanol-A) 8 7.25 - 7.18 (m, 4H), 7.17 - 7.12 (m, 1H), 3.51 (d, J = 2.7 Hz, 2H), 3.42 (dd, J = 9.3, 6.1 Hz, 1H), 3.30 - 3.23 (m, 3H), 2.83 (dd, J = 9.5, 7.2 Hz, 1H), 2.68 (d, J = 9.8 Hz, 1H), 2.65 - 2.58 (m, 2H), 2.24 (d, J = 9.8 Hz, 1H), 2.19 (t, J = 9.2 Hz, 1H), 2.00 (d, J = 6.9 Hz, 2H), 1.93 (p, J = 7.6 Hz, 1H), 1.83 (dt, J = 24.9, 11.4 Hz, 2H), 1.73 - 1.53 (m, 7H), 1.50 - 1.35 (m, 5H), 1.22 - 1.02 (m, 4H), 0.85 - 0.74 (m, 5H).

¹³C NMR (125 MHz, Methanol-^) 8 138.13, 128.81, 127.93, 126.87, 72.50, 69.87, 65.81, 63.35, 60.36, 56.83, 51.88, 51.08, 40.97, 36.75, 34.86, 31.83, 30.95, 26.32, 25.83, 22.50, 9.61.

HRMS (ESI): C26H43N2O+, Calculated: [M+H]+, 399.3375; Found: [M+H]+, 399.3376. IR (neat): 2921, 2851, 2799, 1676, 1450 cm'¹.

2-benzyl-8-(cyclohexylmethyl)-2,8-diazaspiro[4.5]decane-4-carboxylic acid: To a flame-dried flask equipped with a stir bar was added methyl 2-benzyl-8-(cyclohexylmethyl)-2,8-diazaspiro[4.5]decane-4- carboxylate (276.0 mg, 0.718 mmol) as a solution in THF (0.2 M). Lithium hydroxide (68.83 mg, 2.87 mmol) was then added, and the solution was diluted with water to a final concentration of 0.1 M. The reaction was heated to 50 °C and stirred for 36 hours. The reaction was quenched with IM HC1, and extracted 3x with DCM. The organic layer was dried with sodium sulfate and subsequently concentrated under vacuum. The product was taken crude to the next step.

HRMS (ESI): C23H35N2O2+, Calculated: [M+H]+, 371.2699; Found: [M+H]+, 371.2702.

IR (neat): 3376, 2922, 2851, 1590, 1450 cm'¹.

501-A-112: To a flame -dried flask equipped with a stir bar was 2-benzyl-8-(cyclohexylmethyl)-2,8- diazaspiro[4.5]decane-4-carboxylic acid (62.90 mg, 0.17 mmol) as a solution in DCM (0.5 M). The solution was cooled to 0 °C and EDC (39.5 mg, 0.255 mmol) was added. The solution was stirred for 30 minutes after which HOBt hydrate (43.0 mg, 0.255 mmol) and DIPEA (43.9 mg, 0.340 mmol) were added. The solution was stirred for an additional 30 minutes, after which isopropylamine (15.05 mg, 0.255 mmol) was added. The reaction was stirred at room temperature for 24 hours. The reaction was quenched with H2O and extracted 3x with DCM. The organic layer was dried with sodium sulfate and subsequently concentrated under vacuum. The product was purified using column chromatography (0-100% DCM:MeOH on silica over 9 minutes) to afford the desired product as a yellow oil: 27.8 mg (40%).

'H NMR (600 MHz, Methanol-A) 5 7.41 - 7.32 (m, 4H), 7.31 - 7.26 (m, 1H), 3.97 (hept, J = 6.6 Hz, 1H), 3.81 (d, J = 12.9 Hz, 1H), 3.74 (d, J = 12.8 Hz, 1H), 3.24 - 3.15 (m, 1H), 3.13 (s, 1H), 3.04 - 2.97 (m, 1H), 2.91 - 2.83 (m, 2H), 2.76 (s, 1H), 2.70 - 2.55 (m, 5H), 2.02 - 1.95 (m, 1H), 1.95 - 1.88 (m, 1H), 1.88 - 1.67 (m, 9H), 1.37 - 1.29 (m, 2H), 1.23 (tt, J = 12.6, 3.4 Hz, 1H), 1.17 (dd, J = 6.6, 1.9 Hz, 6H), 1.05 - 0.93 (m, 2H).

¹³C NMR (150 MHz, Methanol-^) 6 171.91, 137.68, 128.73, 128.15, 127.21, 63.75, 62.14, 59.44, 55.40, 53.40, 50.93, 50.71, 42.46, 41.17, 38.78, 34.89, 33.46, 31.15, 30.96, 30.40, 25.83, 25.36, 21.46, 21.23, 19.86.

HRMS (ESI): C26H42N3O+, Calculated: [M+H]+, 412.3328; Found: [M+H]+, 412.3333. IR (neat): 3269, 2923, 2851, 2804, 1645, 1543, 1451 cm'¹.

501-A-113: To a flame -dried flask equipped with a stir bar was 2-benzyl-8-(cyclohexyhnethyl)-2,8- diazaspiro[4.5]decane-4-carboxylic acid (62.90 mg, 0.17 mmol) as a solution in DCM (0.5 M). The solution was cooled to 0 °C and EDC (39.5 mg, 0.255 mmol) was added. The solution was stirred for 30 minutes after which HOBt hydrate (43.0 mg, 0.255 mmol) and DIPEA (43.9 mg, 0.340 mmol) were added. The solution was stirred for an additional 30 minutes, after which diethylamine (18.60 mg, 0.255 mmol) was added. The reaction was stirred at room temperature for 24 hours. The reaction was quenched with H2O and extracted 3x with DCM. The organic layer was dried with sodium sulfate and subsequently concentrated under vacuum. The product was purified using column chromatography (0-100% DCM:MeOH on silica over 9 minutes) to afford the desired product as a yellow oil: 19.6 mg (27%).

'H NMR (600 MHz, Methanol-d4) δ 7.39 (d, J = 6.7 Hz, 2H), 7.34 (t, J = 7.6 Hz, 2H), 7.28 (t, J = 7.3 Hz, 1H), 3.82 - 3.73 (m, 2H), 3.65 (dp, J = 13.6, 7.4 Hz, 2H), 3.37 - 3.30 (m, 2H), 3.20 - 2.93 (m, 6H), 2.86 - 2.79 (m, 1H), 2.46 (s, 3H), 2.39 (d, J = 9.4 Hz, 1H), 2.32 (s, 1H), 2.02 (d, J = 14.2 Hz, 1H), 1.93 (td, J = 13.1, 4.5 Hz, 1H), 1.82 - 1.61 (m, 8H), 1.31 (tdd, J = 12.6, 9.2, 3.2 Hz, 2H), 1.21 (t, J = 7.1 Hz, 4H), 1.15 (t, J = 7.1 Hz, 3H), 0.96 (qd, J = 12.3, 3.3 Hz, 2H).

¹³C NMR (150 MHz, Methanol-d4) 171.96, 137.78, 128.69, 128.10, 127.12, 64.49, 61.50, 59.54, 56.83, 51.31, 51.14, 48.73, 43.52, 42.76, 40.70, 35.26, 33.92, 31.26, 31.22, 26.00, 25.52, 13.67, 12.08.

HRMS (ESI): C27H44N3O+, Calculated: [M+H]+, 426.3484; Found: [M+H]+, 426.3483. IR (neat): 2923, 2850, 2801, 1633, 1449 cm'¹.

5-fluoro-l-methyl-lH-indole-3-carbaldehyde: Following General Procedure S, 5-fluoro-lH-indole-3- carbaldehyde (300.0 mg, 1.84 mmol) and methyl iodide (287.1 mg, 2.02 mmol, 1.2 eq) were used. The product was purified using column chromatography (0-100% hexanes:EtOAc on silica over 10 minutes) to afford the desired product as an orange solid: 231.4 mg (71%).

'H NMR (600 MHz, CDCh) δ 9.88 (s, 1H), 7.90 (dd, J = 9.2, 2.5 Hz, 1H), 7.62 (s, 1H), 7.21 (dd, J = 8.9, 4.2 Hz, 1H), 7.02 (td, J = 9.0, 2.6 Hz, 1H), 3.80 (s, 3H).

¹³C NMR (150 MHz, CDCh) δ 184.14, 160.68, 159.09, 140.07, 134.38, 125.93, 125.85, 117.92, 112.49, 112.31, 110.79, 110.72, 107.56, 107.40, 33.97.

HRMS (ESI): C10H9FNO+, Calculated: [M+H]+, 178.0663; Found: [M+H]+, 178.0668. IR (neat): 2970, 2922, 2813, 1656, 1627, 1534, 1477 cm ¹.

5-methoxy-l-methyl-lH-indole-3-carbaldehyde: Following General Procedure S, 5-methoxy-lH-indole- 3-carbaldehyde (300.0 mg, 1.71 mmol) and methyl iodide (267.38 mg, 1.88 mmol, 1.2 eq) were used. The product was purified using column chromatography (0-100% hexanes:EtOAc on silica over 10 minutes) to afford the desired product as a yellow solid: 198.7 mg (61%).

'H NMR (600 MHz, CDC1₃) δ 9.88 (s, 1 H), 7.72 (d, J = 2.5 Hz, 1 H), 7.55 (s, 1 H), 7.18 (d, J = 8.9 Hz, 1 H), 6.91 (dd, J = 8.8, 2.5 Hz, 1H), 3.83 (s, 3H), 3.78 (s, 3H).

¹³C NMR (150 MHz, CDCh) 8 184.37, 156.72, 139.35, 132.84, 126.02, 117.82, 114.48, 110.72, 103.35, 55.84, 33.84.

HRMS (ESI): C11H12NO2+, Calculated: [M+H]+, 190.0863; Found: [M+H]+, 190.0863. IR (neat): 3107, 2939, 1832, 1652, 1624, 1534, 1485, 1460 cm'¹.

Methyl 8-((5-fluoro-l-methyl-lH-indol-3-yl)methyl)-2-phenethyl-2,8-diazaspiro[4.5]decane-4- carboxylate: Following General Procedure R, methyl 2-phenethyl-2,8-diazaspiro[4.5]decane-4-carboxylate (113.79 mg, 0.376 mmol) and 5-fluoro-l-methyl-lH-indole-3-carbaldehyde (100.0 mg, 0.564 mmol) were used. The product was purified using column chromatography (0-100% ACN:MeOH (1% NH4OH) on silica over 10 minutes) to afford the desired product as a white solid: 94.6 mg (54%).

¹H NMR (600 MHz, Methanol-d4) δ 7.31 (ddd, J = 9.4, 6.7, 3.4 Hz, 2H), 7.28 - 7.23 (m, 2H), 7.21 - 7.14 (m, 4H), 6.94 (td, J = 9.1, 2.5 Hz, 1H), 3.77 (s, 3H), 3.65 (s, 3H), 3.63 (s, 2H), 3.03 - 2.97 (m, 1H), 2.86 - 2.61 (m, 9H), 2.34 (d, J = 9.7 Hz, 1H), 2.25 - 2.15 (m, 1H), 2.11 (d, J = 9.6 Hz, 1H), 1.99 - 1.91 (m, 1H), 1.65 (dq, J = 13.5, 3.1 Hz, 1H), 1.53 (dd, J = 12.9, 3.2 Hz, 1H), 1.51 - 1.45 (m, 1H).

¹³C NMR (150 MHz, Methanol-d4) δ 173.01, 158.62, 157.08, 139.91, 133.68, 131.07, 128.97, 128.90, 128.29, 128.04, 125.77, 109.89, 109.82, 109.27, 109.10, 109.02, 108.99, 103.41, 103.25, 62.92, 57.98, 55.04, 53.01, 52.19, 50.55, 49.72, 43.09, 36.57, 34.53, 32.26, 31.68.

HRMS (ESI): C28H35FN3O2+, Calculated: [M+H]+, 464.2708; Found: [M+H]+, 464.2715. IR (neat): 2938, 2803, 1731, 1622, 1579, 1490, 1453 cm'¹.

501-A-128: Following General Procedure P, methyl 8-((5-fluoro-l-methyl-lH-indol-3-yl)methyl)-2- phenethyl-2,8-diazaspiro[4.5]decane-4-carboxylate (43.0 mg, 0.093 mmol) was used. The product was purified using column chromatography (0-100% DCM:MeOH (1% NH4OH) on silica over 9 minutes) to afford the desired product as a clear oil: 35.8 mg (89%).

1H NMR (600 MHz, Methanol-d4) 57.35 (dd, J = 9.8, 2.5 Hz, 1H), 7.30 (q, J = 4.4 Hz, 2H), 7.27 - 7.23 (m, 2H), 7.22 - 7.18 (m, 2H), 7.18 - 7.15 (m, 1H), 6.95 (td, J = 9.2, 2.5 Hz, 1H), 3.80 (s, 2H), 3.76 (s, 3H), 3.70 (dd, J = 10.8, 5.6 Hz, 1H), 3.51 (dd, J = 10.8, 7.8 Hz, 1H), 3.15 - 3.09 (m, 1H), 2.93 (d, J = 8.8 Hz, 2H), 2.87 - 2.69 (m, 5H), 2.49 (d, J = 10.1 Hz, 2H), 2.36 (s, 1H), 2.28 (s, 1H), 2.02 (p, J = 7.8 Hz, 1H), 1.86 (td, J = 13.6, 4.1 Hz, 1H), 1.78 (td, J = 13.6, 4.0 Hz, 1H), 1.62 (d, J = 13.6 Hz, 1H), 1.57 (d, J = 13.2 Hz, 1H).

¹³C NMR (150 MHz, Methanol-d4) 6 158.76, 157.22, 139.54, 133.66, 131.82, 128.93, 128.87, 128.32, 128.11, 125.89, 110.11, 110.04, 109.48, 109.30, 107.52, 103.37, 103.21, 63.36, 60.69, 58.24, 56.80, 51.96, 50.29, 49.69, 49.28, 40.72, 36.13, 34.13, 31.78, 30.17.

HRMS (ESI): C27H35FN3O+, Calculated: [M+H]+, 436.2759: Found: [M+H]+, 436.2775.

IR (neat): 3361, 3027, 2920, 2849, 2806, 1625, 1578, 1546, 1490, 1455, 1426 cm'¹.

Methyl 8-((5-methoxy-l-methyl-lH-indol-3-yl)methyl)-2-phenethyl-2,8-diazaspiro[4.5]decane-4- carboxylate: Following General Procedure R, methyl 2-phenethyl-2,8-diazaspiro[4.5]decane-4-carboxylate (106.55 mg, 0.352 mmol) and 5-methoxy-l-methyl-lH-indole-3-carbaldehyde (100.0 mg, 0.529 mmol) were used. The product was purified using column chromatography (0-100% ACN:MeOH (1% NH4OH) on silica over 10 minutes) to afford the desired product as an orange oil: 42.7 mg (25%).

1H NMR (600 MHz, Methanol-d4) 57.31 - 7.22 (m, 5H), 7.22 - 7.18 (m, 2H), 7.18 - 7.14 (m, 1H), 6.88 (dd, J = 8.8, 2.3 Hz, 1H), 4.12 (s, 2H), 3.86 (s, 3H), 3.77 (s, 3H), 3.65 (s, 3H), 3.18 (s, 1H), 3.12 (s, 1H), 3.03 - 2.98 (m, 1H), 2.90 - 2.85 (m, 2H), 2.82 - 2.67 (m, 7H), 2.51 (d, J = 9.8 Hz, 1H), 2.08 (ddd, J = 14.1, 11.9, 4.3 Hz, 1H), 1.82 - 1.77 (m, 1H), 1.72 - 1.67 (m, 1H), 1.66 - 1.59 (m, 1H). ¹³C NMR (150 MHz, Methanol-d4) 5 172.58, 154.66, 139.78, 132.38, 131.58, 128.67, 128.32, 128.08, 125.84, 111.87, 110.19, 103.97, 100.46, 62.32, 57.62, 55.04, 54.80, 52.35, 51.63, 50.81, 49.67, 49.05, 42.43, 34.65, 34.40, 31.83, 30.43.

HRMS (ESI): C29H38N3O3+, Calculated: [M+H]+, 476.2908; Found: [M+H]+, 476.2924.

IR (neat): 2942, 2804, 1730, 1622, 1578, 1544, 1491, 1454, 1428 cm ¹.

501-A-129: Following General Procedure P, methyl 8-((5-methoxy-l-methyl-lH-indol-3-yl)methyl)-2- phenethyl-2,8-diazaspiro[4.5]decane-4-carboxylate (45.2 mg, 0.095 mmol) was used. The product was purified using column chromatography (0-100% DCM:MeOH (1% NH4OH) on silica over 9 minutes) to afford the desired product as a yellow oil: 36.6 mg (86%).

'H NMR (600 MHz, Methanol-d4) δ 7.26 - 7.22 (m, 2H), 7.21 - 7.13 (m, 6H), 6.85 (dd, J = 8.8, 2.3 Hz, 1H), 3.85 (s, 2H), 3.83 (s, 3H), 3.69 (s, 4H), 3.49 (dd, J = 10.7, 7.8 Hz, 1H), 3.08 (dd, J = 9.8, 7.2 Hz, 1H), 2.97 (d, J = 6.9 Hz, 2H), 2.83 - 2.76 (m, 3H), 2.75 - 2.65 (m, 2H), 2.45 (d, J = 10.1 Hz, 2H), 2.41 (s, 1H), 2.33 (s, 1H), 2.00 (p, J = 7.8 Hz, 1H), 1.92 - 1.84 (m, 1H), 1.83 - 1.76 (m, 1H), 1.62 (d, J = 12.2 Hz, 1H), 1.55 (d, J = 12.5 Hz, 1H).

¹³C NMR (150 MHz, Methanol-d4) 5 154.30, 139.61, 132.38, 130.80, 128.96, 128.34, 128.12, 125.90,

1 1 1.43, 109.92, 106.44, 100.66, 63.37, 60.72, 58.21 , 56.76, 55.00, 51.97, 50.16, 49.57, 49.29, 40.65, 35.94, 34.19, 31.70, 29.97.

HRMS (ESI): C28H38N3O2+, Calculated: [M+H]+, 448.2959; Found: [M+H]+, 448.2970.

IR (neat): 3365, 3060, 3026, 2920, 2805, 1622, 1578, 1545 ,1491, 1454, 1425 cm'¹.

Methyl 8-((lH-imidazol-2-yl)methyl)-2-phenethyl-2,8-diazaspiro[4.5]decane-4-carboxylate: Following

General Procedure R, methyl 2-phenethyl-2,8-diazaspiro[4.5]decane-4-carboxylate (53.0 mg, 0.18 mmol) and 2-imidazolecarboxaldehyde (22.5 mg, 0.234 mmol) were used. The product was extracted with a 9: 1 mixture of CHCl3:i-PrOH and then purified using column chromatography (0-20% ACN:MeOH (1% NH4OH) on silica over 10 minutes) to afford the desired product as a brown solid: 38.2 mg (56%).

'H NMR (500 MHz, CDCI3) 6 9.82 (s, 1H), 7.39 - 7.30 (m, 2H), 7.28 - 7.21 (m, 3H), 7.05 (s, OH), 3.74 (s, 3H), 3.67 (s, 2H), 3.09 (dd, J = 8.9, 7.1 Hz, 1H), 2.94 (d, J = 9.2 Hz, 1H), 2.88 - 2.65 (m, 6H), 2.36 (d, J = 9.1 Hz, 1H), 2.27 (td, J = 11.7, 2.8 Hz, 1H), 2.18 (td, J = 11.5, 2.8 Hz, 1H), 2.00 - 1.91 (m, 1H), 1.77 - 1.69 (m, 1H), 1.65 - 1.49 (m, 2H).

¹³C NMR (125 MHz, CDCi3) 5 173.48, 146.01, 140.42, 128.78, 128.48, 128.40, 126.18, 115.74, 63.25, 58.08, 56.35, 55.68, 53.64, 51.97, 51.64, 51.19, 43.50, 37.61, 35.43, 33.17.

HRMS (ESI): C22H31N4O2+, Calculated: [M+H]+, 383.2442; Found: [M+H]+, 383.2444.

TW1162: Following General Procedure P, methyl 8-((lH-imidazol-2-yr)methyl)-2-phenethyl-2,8- diazaspirof4.5]decane-4-carboxylate (32.6 mg, 0.085 mmol) was used. The product was purified using column chromatography (0-30% DCM:MeOH (1% NH4OH) on silica over 9 minutes) to afford the desired product as a white solid: 13.6 mg (48%).

'H NMR (500 MHz, Methanol-^) 57.30 - 7.25 (m, 2H), 7.25 - 7.16 (m, 3H), 6.99 (s, 2H), 3.70 (dd, J = 10.7, 5.5 Hz, 1H), 3.59 (s, 2H), 3.54 (dd, J = 10.7, 7.6 Hz, 1H), 3.16 (dd, J = 9.9, 7.2 Hz, 1H), 2.90 (d, J =

10.2 Hz, 1H), 2.87 - 2.77 (m, 4H), 2.73 (dd, J = 1 1 .7, 4.3 Hz, 2H), 2.56 (t, J = 9.7 Hz, 2H), 2.24 - 2.10 (m, 2H), 2.06 - 1.98 (m, 1H), 1.77 (dtd, J = 36.4, 12.7, 3.9 Hz, 2H), 1.61 - 1.53 (tn, 2H).

¹³C NMR (125 MHz, Methanol-d4) δ 144.55, 139.24, 128.24, 128.12, 125.95, 121.25, 63.44, 60.67, 58.25, 56.84, 54.55, 50.83, 50.17, 49.28, 40.77, 36.93, 33.99, 30.90.

HRMS (ESI): C21H31N4O+, Calculated: [M+H]+, 355.2492; Found: [M+H]+, 355.2499.

Methyl 8-((lH-pyrrolo[2,3-b]pyridin-3-yl)methyl)-2-benzyl-2,8-diazaspiro[4.5]decane-4-carboxylate:

Following General Procedure R, methyl 2-benzyl-2,8-diazaspiro[4.5]decane-4-carboxylate (105.0 mg, 0.364 mmol) and lH-pyrrolo[2,3-b]pyridine-3-carbaldehyde (69.2 mg, 0.473 mmol) were used. The product was extracted with a 9:1 mixture of CHC13:i-PrOH and then purified using column chromatography (0-20% DCM:MeOH (1% NH4OH) on silica over 10 minutes) to afford the desired product as a white solid: 73.8 mg (48%).

'H NMR (500 MHz, Methanol-d4) δ 8.15 (dd, J = 4.8, 1.5 Hz, 1H), 8.06 (dd, J = 7.9, 1.5 Hz, 1H), 7.37 (s, 1H), 7.30 - 7.15 (m, 7H), 7.08 (dd, J = 7.9, 4.8 Hz, 1H), 3.80 (s, 2H), 3.62 - 3.54 (m, 6H), 2.92 - 2.65 (m, 8H), 2.32 (dq, J = 21.5, 9.4 Hz, 4H), 1.92 (s, 1H), 1.71 - 1.63 (m, 1H), 1.59 - 1.45 (m, 3H).

¹³C NMR (125 MHz, Methanol-d4) δ 174.3, 149.3, 143.5, 139.5, 130.0, 129.4, 129.1, 128.3, 128.0, 122.2, 116.8, 108.7, 63.7, 61.1, 56.1, 54.2, 53.4, 52.0, 51.7, 51.0, 45.4, 44.3, 37.2, 37.0, 33.0.

HRMS (ESI): C25H31N4O2+, Calculated: [M+H]+, 419.2442; Found: [M+H]+, 419.2447.

TW2052: Following General Procedure P, methyl 8-((lH-pyrrolo[2,3-b]pyridin-3-yl)methyl)-2-benzyl-2,8- diazaspiro[4.5]decane-4-carboxylate (59.0 mg, 0.1415 mmol) was used. The product was purified using column chromatography (0-30% DCM:MeOH (1% NH4OH) on silica over 9 minutes) to afford the desired product as a white solid: 33.4 mg (61%).

'H NMR (500 MHz, Methanol-d4) 3 8.14 (d, J = 5.0 Hz, 1H), 8.06 (d, J = 7.9 Hz, 1H), 7.32 (s, 1H), 7.27 (t, J = 5.5 Hz, 5H), 7.21 (d, J = 6.6 Hz, 1H), 7.10 - 7.04 (m, 1H), 3.68 (s, 2H), 3.63 (dd, J = 10.4, 5.6 Hz, 1H), 3.58 (d, J = 8.4 Hz, 2H), 3.47 - 3.40 (m, 1H), 2.91 (dd, J = 9.5, 6.9 Hz, 1H), 2.80 (d, J = 11.7 Hz, 2H), 2.68 (d, J = 9.6 Hz, 2H), 2.33 (dt, J = 22.3, 9.1 Hz, 2H), 2.21 (s, 1H), 2.13 (dd, J = 28.1, 16.0 Hz, 2H), 1.94 - 1.87 (m, 1H), 1.77 - 1.62 (m, 2H), 1.59 - 1.46 (m, 2H).

¹³C NMR (125 MHz, Methanol-d4) δ 147.83, 141.87, 137.99, 128.76, 127.92, 127.81, 126.89, 125.90, 120.99, 115.19, 108.78, 63.36, 61.04, 60.23, 56.82, 52.97, 52.38, 50.77, 50.06, 49.67, 40.77, 36.81, 30.86. HRMS (ESI): C24H31N4O+, Calculated: [M+H]+, 391.2492; Found: [M+H]+, 391.2495.

Methyl 2-benzyl-8-((l-methyl-lH-indol-3-yl)methyl)-2,8-diazaspiro[4.5]decane-4-carboxylate:

Following General Procedure R, methyl 2-benzyl-2,8-diazaspiro[4.5]decane-4-carboxylate (105.0 mg, 0.364 δmol) and 1 -methyl- lH-indole-3-carbaldehy de (75.3 mg, 0.473 mmol) were used. The product was extracted with a 9:1 mixture of CHCl₃:i-PrOH and then purified using column chromatography (0-20% DCM:MeOH (1% NH4OH) on silica over 10 minutes) to afford the desired product as a clear oil: 79.9 mg (51%).

'H NMR (500 MHz, Methanol-d4) 57.57 (d, J = 8.0 Hz, 1H), 7.31 (dt, J = 8.3, 0.9 Hz, 1H), 7.29 - 7.22 (m, 5H), 7.19 (ddd, J = 5.9, 4.5, 2.6 Hz, 1H), 7.17 - 7.12 (m, 2H), 7.04 (ddd, J = 8.0, 7.1, 1.0 Hz, 1H), 3.84 (s, 2H), 3.73 (s, 3H), 3.60 - 3.51 (m, 5H), 2.96 - 2.81 (m, 3H), 2.75 - 2.65 (m, 3H), 2.33 (d, J = 9.5 Hz, 3H), 1.98 - 1.89 (m, 1H), 1.67 (d, J = 13.8 Hz, 1H), 1.59 - 1.45 (m, 2H).

¹³C NMR (125 MHz, Methanol-d4 δ 172.91, 138.14, 136.99, 130.12, 129.31, 128.56, 128.38, 127.97, 126.88, 121.41, 119.15, 118.35, 109.08, 106.48, 62.41, 59.64, 54.70, 52.77, 51.78, 50.56, 50.13, 49.44, 42.80, 35.62, 31.49, 31.33.

HRMS (ESI): C27H34N3O2+, Calculated: [M+H]+, 432.2646; Found: [M+H]+, 432.2656.

TW2053: Following General Procedure P, methyl 2-benzyl-8-((l -methyl- lH-indol-3-yl)methyl)-2, 8- diazaspiro[4.5]decane-4-carboxylate (63.9 mg, 0.148 mmol) was used. The product was purified using column chromatography (0-20% DCM:MeOH (1% NH4OH) on silica over 9 minutes) to afford the desired product as a white solid: 37.3 mg (62%).

'H NMR (500 MHz, Methanol-d4) δ 7.63 (d, J = 8.0 Hz, 1H), 7.36 (d, J = 8.2 Hz, 1H), 7.31 (d, J = 4.7 Hz, 4H), 7.25 (s, 1H), 7.22 - 7.16 (m, 2H), 7.09 (t, J = 7.5 Hz, 1H), 3.91 (s, 2H), 3.78 (s, 3H), 3.63 (d, J = 13.8 Hz, 3H), 3.48 (dd, J = 10.6, 7.7 Hz, 1H), 3.05 - 2.91 (m, 3H), 2.73 (d, J = 9.8 Hz, 1H), 2.49 - 2.29 (m, 4H), 1.97 (q, J = 7.3 Hz, 1H), 1.86 - 1.72 (m, 2H), 1.63 (t, J = 16.5 Hz, 2H).

¹³C NMR (125 MHz, Methanol-d4) 8 137.90, 136.99, 130.15, 128.74, 128.35, 127.97, 126.95, 121.44, 119.17, 118.33, 109.09, 106.39, 63.18, 60.90, 60.08, 56.59, 51.85, 50.29, 49.72, 49.40, 40.56, 35.98, 31.49, 30.01.

HRMS (ESI): C₂6H₃4N₃O-I-, Calculated: [M+H]+, 404.2696; Found: [M+H]+, 404.2708.

Methyl 2-benzyl-8-((l-methyl-lH-pyrrolo[2,3-b]pyridin-3-yl)methyl)-2,8-diazaspiro[4.5]decane-4- carboxylate: Following General Procedure R, methyl 2-benzyl-2,8-diazaspiro[4.5]decane-4-carboxylate (158.0 mg, 0.548 mmol) and l-methyl-lH-pyrrolo[2,3-b]pyridine-3-carbaldehyde (176.0 mg, 1.10 mmol) were used. The product was extracted with a 9:1 mixture of CHCh:/-PrOH and then purified using column chromatography (0-20% DCM:MeOH (1% NH4OH) on silica over 10 minutes) to afford the desired product as a yellow oil: 99.1 mg (42%).

'H NMR (500 MHz, CDCls) 6 8.28 (dd, J = 4.7, 1.5 Hz, 1H), 7.91 (dd, J = 7.8, 1.6 Hz, 1H), 7.27 - 7.22 (m, 4H), 7.21 - 7.16 (m, 1H), 7.10 (s, 1H), 7.00 (dd, J = 7.8, 4.7 Hz, 1 H), 3.80 (s, 3H), 3.63 (s, 2H), 3.59 (s, 3H), 3.58 (s, 2H), 2.90 (t, J = 7.8 Hz, 1H), 2.80 - 2.62 (m, 5H), 2.22 (d, J = 9.2 Hz, 1H), 2.11 (s, 1H), 2.00 (d, J = 23.5 Hz, 1H), 1.94 - 1.84 (m, 1H), 1.66 (d, J = 13.2 Hz, 1H), 1.51 (q, J = 4.3 Hz, 2H).

¹³C NMR (125 MHz, CDC1₃) 5 173.63, 148.05, 143.07, 139.12, 128.79, 128.56, 128.36, 127.65, 127.03, 120.83, 115.43, 62.92, 60.02, 55.56, 53.49, 51.54, 51.23, 50.59, 43.92, 37.27, 32.74, 31.21.

HRMS (ESI): C26H33N4O2+, Calculated: [M+H]+, 433.2598; Found: [M+H]+, 433.2608.

TW2086: Following General Procedure P, methyl 2-benzyl-8-((l-methyl-lH-pyrrolo[2,3-b]pyridin-3- yl)methyl)-2,8-diazaspiro[4.5]decane-4-carboxylate (77.7 mg, 0.180 mmol) was used. The product was purified using column chromatography (0-20% DCM:MeOH (1% NH4OH) on silica over 9 minutes) to afford the desired product as a yellow solid: 55.6 mg (76%).

'H NMR (500 MHz, Methanol-d4) δ 8.19 (dd, J = 4.8, 1.5 Hz, 1H), 8.04 (dd, J = 7.9, 1.5 Hz, 1H), 7.32 (s, 1H), 7.28 - 7.22 (m, 4H), 7.19 (ddt, J= 8.5, 5.3, 2.3 Hz, 1H), 7.08 (dd, J = 7.9, 4.8 Hz, 1H), 3.79 (s, 3H), 3.77 (s, 2H), 3.62 - 3.56 (m, 3H), 3.43 (dd, J = 10.7, 7.7 Hz, 1H), 2.94 - 2.82 (m, 3H), 2.70 (d, J= 9.9 Hz, 1H), 2.41 - 2.15 (m, 4H), 1.91 (p, 7 = 7.4 Hz, 1H), 1.72 (dtd, J = 23.4, 12.6, 4.0 Hz, 2H), 1.59 - 1.50 (m, 2H).

¹³C NMR (125 MHz, Methanol-d4) 6 147.25, 142.11, 137.56, 130.46, 128.83, 128.01, 127.90, 127.08, 121.25, 115.28, 106.67, 63.16, 60.85, 60.08, 56.62, 51.97, 50.47, 49.88, 49.42, 40.70, 36.31, 30.36, 30.12. HRMS (ESI): C₂5H₃₃N₄O+, Calculated: [M+H]+, 405.2649; Found: [M+H]+, 405.2655.

Methyl 2-benzyl-8-((l-propyl-lH-pyrrolo|2,3-bJpyridin-3-yl)methyl)-2,8-diazaspiro|4.5Jdecane-4- carboxylate: Following General Procedure R, methyl 2-benzyl-2,8-diazaspiro[4.5]decane-4-carboxylate (151.0 mg, 0.52 mmol) and 1 -propyl- lH-pyrrolo[2,3-b]pyridine-3-carbaldehyde (198.0 mg, 1.05 mmol) were used. The product was extracted with a 9:1 mixture of CHCl₃:z-PrOH and then purified using column chromatography (0-20% DCM:MeOH (1% NH4OH) on silica over 10 minutes) to afford the desired product as a brown oil: 1 10.0 mg (45%).

'H NMR (500 MHz, CDC1₃) 5 8.26 (dd, J = 4.7, 1.5 Hz, 1H), 7.91 (dd, J = 7.8, 1.6 Hz, 1H), 7.28 - 7.22 (m, 4H), 7.19 (ddt, J = 8.5, 5.3, 2.6 Hz, 1H), 7.13 (s, 1H), 6.99 (dd, J = 7.9, 4.7 Hz, 1H), 4.16 (t, J = 7.2 Hz, 2H), 3.63 (s, 2H), 3.59 (d, J = 6.0 Hz, 5H), 2.90 (t, J = 7.6 Hz, 1H), 2.79 - 2.63 (m, 5H), 2.21 (d, J = 9.2 Hz, 1H), 2.12 - 1.94 (m, 2H), 1.94 - 1.78 (m, 3H), 1.66 (d, J = 13.3 Hz, 1H), 1.50 (d, J = 11.6 Hz, 2H), 0.87 (t, J = 7.4 Hz, 3H).

¹³C NMR (125 MHz, CDCh) 5 173.66, 147.75, 142.88, 139.17, 128.55, 128.36, 127.65, 127.01, 120.88, 115.40, 62.92, 60.04, 55.59, 53.64, 53.53, 51.53, 51.26, 50.62, 46.20, 43.96, 37.40, 32.86, 23.75, 11.49. HRMS (ESI): C₂₈H₃₇N₄O₂+, Calculated: [M+H]+, 461.2911; Found: [M+H]+, 461.2919.

TW2087: Following General Procedure P, methyl 2-benzyl-8-((l-propyl-lH-pyrrolo[2,3-b]pyridin-3- yl)methyl)-2,8-diazaspiro[4.5]decane-4-carboxylate (88.0 mg, 0.191 mmol) was used. The product was purified using column chromatography (0-20% DCM:MeOH (1% NH₄OH) on silica over 9 minutes) to afford the desired product as a white solid: 49.7 mg (60%).

'H NMR (500 MHz, Methanol-^) 5 8.18 (dd, J = 4.8, 1.5 Hz, 1H), 8.04 (dd, J = 7.9, 1.6 Hz, 1H), 7.35 (s, 1H), 7.29 - 7.22 (m, 4H), 7.20 (ddt, J = 8.4, 5.0, 2.5 Hz, 1H), 7.07 (dd, J = 7.9, 4.8 Hz, 1H), 4.18 (t, J = 7.1 Hz, 2H), 3.73 (s, 2H), 3.65 - 3.55 (m, 3H), 3.43 (dd, J = 10.6, 7.9 Hz, 1H), 2.92 (dd, J = 9.6, 7.2 Hz, 1H), 2.83 (s, 2H), 2.68 (d, J = 9.8 Hz, 1H), 2.37 - 2.30 (m, 2H), 2.18 (dd, J = 27.6, 13.7 Hz, 2H), 1.95 - 1.86 (m, 1H), 1.84 - 1.64 (m, 4H), 1.59 - 1.49 (m, 2H), 0.83 (t, J = 7.4 Hz, 3H).

¹³C NMR (125 MHz, Methanol-d4) δ 146.90, 141.90, 137.77, 129.27, 128.78, 127.97, 127.93, 126.98, 121.37, 115.23, 107.13, 63.19, 60.94, 60.15, 56.75, 52.08, 50.55, 49.90, 49.54, 45.75, 40.73, 36.56, 30.61, 23.26, 10.09.

HRMS (ESI): C27H37N4O+, Calculated: [M+H]+, 433.2962; Found: [M+H]+, 433.2970.

Methyl 2-benzyl-8-((5-fluoro-lH-indol-3-yl)methyl)-2,8-diazaspiro[4.5]decane-4-carboxylate:

Following General Procedure R, methyl 2-benzyl-2,8-diazaspiro[4.5]decane-4-carboxylate (100.0 mg, 0.347 mmol) and 5-fluoro-lH-indole-3-carbaldehyde (113.2 mg, 0.694 mmol) were used. The product was extracted with a 9:1 mixture of CHCf:/-PrOH and then purified using column chromatography (0-20% DCM:MeOH (1% NH4OH) on silica over 10 minutes) to afford the crude product as a white solid: 71.0 mg (47%). The product was taken forward without further purification.

HRMS (ESI): C26H31FN3O2+, Calculated: [M+H]+, 436.2395; Found: [M+H]+, 436.2404.

TW2103: Following General Procedure P methyl 2-benzyl-8-((5-fluoro-lH-indol-3-yl)methyl)-2,8- diazaspiro[4.5]decane-4-carboxylate (71.0 mg, 0.163 mmol) was used. The product was purified using column chromatography (0-20% DCM:MeOH (1% NH4OH) on silica over 9 minutes) to afford the desired product as a white solid: 51.2 mg (77%).

'H NMR (500 MHz, Methanol-d4) δ 7.37 - 7.24 (m, 7H), 6.90 (td, J = 9.1, 2.5 Hz, 1H), 3.70 (d, J = 6.2 Hz, 3H), 3.67 - 3.57 (m, 2H), 3.49 (dd, J = 10.6, 8.2 Hz, 1H), 2.96 (dd, J = 9.5, 7.1 Hz, 1H), 2.88 (d, J = 11.7 Hz, 2H), 2.76 (d, J = 9.8 Hz, 1H), 2.43 - 2.33 (m, 2H), 2.29 - 2.09 (m, 2H), 2.00 - 1.91 (m, 1H), 1.84 - 1.67 (m, 2H), 1.64 - 1.52 (m, 2H). ¹³C NMR (125 MHZ, Methanol-d4) 5 158.61, 156.77, 138.04, 132.98, 128.78, 128.70, 128.38, 128.30, 127.92, 127.00, 126.87, 111.70, 111.63, 109.24, 109.03, 102.96, 102.77, 63.38, 61.05, 60.23, 56.82, 52.40, 50.70, 49.98, 40.73, 36.71, 30.78.

HRMS (ESI): C25H31FN3O+, Calculated: [M+H]+, 408.2446; Found: [M+H]+, 408.2458.

Methyl 2-benzyl-8-((5-methoxy-lH-indol-3-yl)methyl)-2,8-diazaspiro[4.5]decane-4-carboxylate:

Following General Procedure R, methyl 2-benzyl-2,8-diazaspiro[4.5]decane-4-carboxylate (100.0 mg, 0.347 mmol) and 5-fluoro-1 H-indole-3-carbaldehyde (121 .6 mg, 0.694 mmol) were used. The product was extracted with a 9: 1 mixture of CHCU:z-PrOH and then purified using column chromatography (0-20% DCM:MeOH (1% NH4OH) on silica over 10 minutes) to afford the crude product as a white solid: 72.0 mg (47%). The product was taken forward without further purification.

HRMS (ESI): Calculated: [M+H]+, 448.2595; Found: [M+H]+, 448.2598.

TW2104: Following General Procedure P methyl 2-benzyl-8-((5-methoxy-lH-indol-3-yl)methyl)-2,8- diazaspiro[4.5]decane-4-carboxylate (72.0 mg, 0.161 mmol) was used. The product was purified using column chromatography (0-20% DCM:MeOH (1% NH4OH) on silica over 9 minutes) to afford the desired product as a white solid: 52.5 mg (78%).

'H NMR (500 MHz, Methanol-d4) δ 7.31 - 7.19 (m, 6H), 7.16 (d, J = 2.3 Hz, 1H), 7.08 (d, J = 2.4 Hz, 1H), 6.74 (dt, J = 8.9, 2.4 Hz, 1H), 3.79 (d, J = 2.3 Hz, 2H), 3.72 (s, 2H), 3.66 - 3.61 (m, 1H), 3.60 - 3.56 (m, 2H), 3.44 (ddd, J = 10.6, 8.2, 2.3 Hz, 1H), 2.96 - 2.81 (m, 3H), 2.70 (d, J = 9.9 Hz, 1H), 2.38 - 2.12 (m, 4H), 1.96 - 1.86 (m, 1H), 1.80 - 1.64 (m, 2H), 1.55 (dd, J = 25.0, 13.2 Hz, 2H).

¹³C NMR (125 MHz, Methanol-d4) δ 153.93, 138.06, 131.64, 128.76, 128.27, 127.92, 126.87, 125.96, 111.58, 111.33, 108.26, 100.15, 63.36, 61.03, 60.19, 56.78, 54.85, 52.42, 50.59, 49.90, 49.59, 40.70, 36.50, 30.56.

HRMS (ESI): C26H34N3O2+, Calculated: [M+H]+, 420.2646; Found: [M+H]+, 420.2650.

Methyl 2-benzyl-8-((5-methoxy-lH-indol-3-yl)methyl)-2,8-diazaspiro[4.5]decane-4-carboxylate:

Following General Procedure R, methyl 2-benzyl-2,8-diazaspiro[4.5]decane-4-carboxylate (100.0 mg, 0.347 mmol) and benzofuran-3-carbaldehyde (101.0 mg, 0.694 mmol) were used. The product was extracted with a 9:1 mixture of CHCh:/-PrOH and then purified using column chromatography (0-5% DCM:MeOH (1% NH4OH) on silica over 10 minutes) to afford the crude product as a yellow oil: 82.3 mg (57%). The product was taken forward without further purification.

HRMS (ESI): C26H31N2O3+, Calculated: [M+H]+, 419.2329; Found: [M+H]+, 419.2340.

TW2102: Following General Procedure P methyl 2-benzyl-8-((5-methoxy-lH-indol-3-yl)methyl)-2,8- diazaspiro[4.5]decane-4-carboxylate (72.0 mg, 0.161 mmol) was used. The product was purified using column chromatography (0-40% DCM:MeOH (1% NH4OH) on silica over 9 minutes) to afford the desired product as a clear oil: 23.9 mg (31%).

'H NMR (500 MHz, Methanol-^) 57.73 (d, J = 7.0 Hz, 2H), 7.49 (d, J = 8.2 Hz, 1H), 7.40 - 7.30 (m, 6H), 7.27 (td, J = 7.5, 1.1 Hz, 1H), 3.79 (s, 2H), 3.75 - 3.68 (m, 3H), 3.55 (dd, J = 10.7, 7.6 Hz, 1H), 3.10 (dd, J = 10.0, 7.2 Hz, 1H), 2.89 (t, J = 9.2 Hz, 3H), 2.64 - 2.50 (m, 2H), 2.24 (dt, J = 25.7, 11.7 Hz, 2H), 2.04 (p, J = 7.4 Hz, 1H), 1.80 (dtd, J = 28.5, 12.6, 3.9 Hz, 2H), 1.67 - 1.57 (m, 2H).

¹³C NMR (125 MHz, Methanol-d4) δ 155.39, 144.29, 136.59, 129.05, 128.14, 127.88, 127.43, 124.11, 122.34, 119.80, 115.44, 110.83, 63.08, 60.67, 59.98, 56.51, 50.80, 50.65, 50.20, 49.19, 40.88, 36.53, 30.65. HRMS (ESI): C25H31N2O2+, Calculated: [M+H]+, 391.2380; Found: [M+H]+, 391.2390.

Example 3

Small molecule modulator of neuronal lysosome positioning and function resolves Alzheimer’s Disease-linked pathologies in cultured human neurons: In this example, compound RH1115 was shown to rescue both the axonal lysosome accumulation pathology as well as the aberrant Aβ42 production found in JIP3 KO human iPSC-derived neurons (i³Neurons). RH1115 and other compounds of the present disclosure can also reduce the buildup of autophagic vacuoles within neuronal processes of JIP3 KO i³Neurons suggesting that it acts to enhance retrograde transport of these axonal organelles, and hence their maturation. This rescue of axonal lysosome buildup in JIP3 KO i³Neurons by RH1115 requires JIP4, an adaptor highly related to JIP3, that has been implicated in regulating dynein-based retrograde lysosome movement in cells. RH1115 also leads to increased levels of the lysosomal transmembrane protein TMEM55B, a JIP4-interacting protein shown to recruit it to lysosomes and mediate starvation- induced lysosome movement in cultured cells. This example provides a proof of principle that restoring normal axonal lysosome transport and clearing organelles that build up there, can modulate amyloid production and is therefore a viable therapeutic avenue to pursue using compounds of the present disclosure. In further support of enhancement of axonal lysosome transport efficiency as a strategy to restore optimal neuronal health and function, it is shown that the compounds of the present disclosure can rescue locomotor defects in JIP3 KO zebrafish larvae.

In addition to its effect on axonal lysosomes, RH1115 mobilized lysosomes within the neuronal cell body to a more perinuclear location and enhanced their degradative capacity. Lysosome positioning and motility within cells is regulated by several factors including nutrient status. In turn, lysosome positioning can affect different cellular functions including signaling, autophagy, cell migration and adhesion. Lysosomes differ in their intraluminal pH and degradative capacity depending on their positioning within the cells, with peripheral lysosomes being less degradative and perinuclear lysosomes being more degradative and conducive to receiving autophagic cargo for turnover. Given the ability of the small molecule to mobilize lysosomes to the perinuclear region and its ability to enhance lysosome function, it may be relevant to the treatment of other diseases characterized by altered lysosome function and distribution including lysosomal storage diseases. iPSC Culture and i³Neuron Differentiation: The JIP3 KO and JIP3/4 DKO iPSC lines (generated from WTC-11 iPSC parental line) were described previously (Gowrishankar et aL, 2021). iPSC cell lines were maintained in E8 media (Life Technologies) supplemented with .05% Penicillin/Streptomycin (Gibco) and were passaged when 70% confluent using Accutase (Corning). The iPSCs were differentiated into i³Neurons by standard methods. i³Neurons were plated at 30,000 cells per 35 mm glass-bottom dishes (MatTek Life Sciences) for live imaging experiments or on 35mm glass coverslips (Carolina Biologicals) for immunofluorescence studies. Glass was pre-coated with 0.1 mg/ml Poly-L- Ornithine (Sigma Aldrich) and 10 |tg/ml mouse Laminin (Gibco). I³Neurons were plated and maintained in Cortical Neuron Culture Medium containing KO DMEM F12 (Gibco) B27 supplement (Thermo Fisher), 10 ng/ml BDNF (PeproTech) and 10 ng/ml NT3 (PeproTech), 1 |ig/ml mouse Laminin (Gibco), and 2 |ig/ml Doxycycline (Fisher Bioreagents). Immunoblotting: Lysis of i³Neurons and western blotting was carried out as described previously (Gowrishankar et al., 2021). Briefly, DIV21 i³Neurons were washed three times with cold PBS and lysed in lysis buffer [1 % Triton-X in PBS, Benzonase (Millipore Sigma, E1014), protease inhibitor (Thermo Fisher Scientific) and phosphatase inhibitor (PhosStop, Roche)]. Prior to immunoblotting, samples were run using SDS-PAGE for 1 hour and 20 minutes at 90V followed by transfer onto a nitrocellulose membrane.

Immunofluorescence analysis of i ³Neurons: i³Neurons differentiated for 1-2 weeks on 24 mm glass coverslips or 35 mm Mattek glass-bottom dishes were processed for immunostaining as described previously.

I³Neuron Viability Assay: DIV 10 i³Neurons on 35mm glass coverslips were treated with 0.1% DMSO or I 5LIM RH1115 for 72 hours before fixation and immunostaining for Tau and LAMP1 as described previously. Images were acquired (5 to 6 areas at random) using a high magnification objective on the Keyence BZ-X810 microscope (Osaka, Japan), and the number of neurons per unit area was computed. Tau staining was used to confirm neuronal viability (normal morphology and neurite integrity). Mean ± SEM of three independent experiments was computed.

Analysis of Autophagosome and Autolysosome Distribution and Motility: DIV 15-16 i³Neurons stably expressing LC3-RFP-GFP were imaged live at 37°C for approximately 20 minutes. Neuronal processes were selected at random after ensuring that they were sufficiently dispersed to allow for easy identification of individual processes. Time-lapse images in both green and red channels were acquired at 2.5 FPS using Fast Airyscan mode on LSM880 microscope (Zeiss) with a 63x oil objective (1.4 NA) and 2.5x optical zoom. Motile fraction was analyzed. Briefly, RFP and GFP intensities were scaled based on control condition and then autophagosomes (Green + Red) and autolysosomes (Red) were identified and tracked. Every neurite (5-17 per time series) of at least 10pm was analyzed. Neurites were traced using the ‘freehand line’ tool, followed by straightening using ‘straighten’ tool, and then ‘resliced’ to create a kymograph (ImagcJ). Vesicles were identified as autolysosomes if they did not contain any green channel puncta. Vesicles were identified as motile if they moved 2pm or more during the period of the movie (2 minutes). Vesicle density was quantified from a single timeframe (t= 10) for all neuronal processes examined.

% Autolysosomes (RFP only) per neuronal process was also computed from timeframe t= 10.

Quantification of Axonal Lysosome Accumulation Index: Axonal lysosome buildup was quantified using endogenous LAMP1 staining or from LAMP1-GFP fluorescence. For experiments involving analysis of endogenous LAMP1 staining, DIV10-12 Control and JIP3 KO i³Neurons were treated with 0.15% DMSO or 15pM RH1115 for 72 hours and fixed and stained for LAMP1. For exogenous LAMP1-GFP experiments, DIV10-14 JIP3 KO or DIV 8 JIP3/JIP4 DKO LAMP1-GFP FNeurons were fixed and imaged. 3x3 stitched z-stack images were acquired using Airyscan SR (super resolution) imaging mode on an LSM88O Confocal microscope (Zeiss) with a 63x oil immersion objection (1.4 NA) to capture 200pm² area per image, at high resolution. Stitched images were analyzed by setting the ‘threshold’ for visualizing LAMP1+ swellings based on the DMSO condition in JIP3 KO i³Neurons. All LAMP1+ swellings above that threshold were counted using the ‘line’ tool followed by the ‘measure’ tool. The lysosome accumulation index was determined by counting the number of swellings greater than 5pm, 10pm, and 20pm (for JIP3/4 DKO only) per 200pm² image area and then normalizing to the number of neurites in that area, this number was then multiplied by 10. The lysosome accumulation index is normalized to the mean of the control condition.

Analysis of Lysosomal vesicle Properties in i³Neurons

ImageJ ‘analyze particles’ tool was used to measure lysosomal vesicle size, integrated density, and number within i³Neuron somas immunostained for LAMP1. Each soma was outlined using the ‘freehand selections’ tool, and then using ‘threshold’ function, a threshold was set based on optimal coverage of most vesicles possible without compromising vesicle size and shape (avoiding false negatives or collapse/fusion of different vesicles). The ‘analyze particles’ tool was then used to define the parameters for the measured vesicles, with a minimum size of 0.002 pm² and no maximum size. Mean size, intensity of vesicles, and count per neuron were computed.

Evaluation of Lysosome Positioning in JIP3 KO i³Neurons: DIV 10 i³Neurons on 35 mm glass coverslips treated with 0.1% DMSO, or RH1115 (15 pM) for 72 hours were fixed and stained for Tau, LAMP1 and DAPI. Images were acquired using a 60X objective on the Keyence BZ-X810 microscope. Lysosome distribution was classified as “clustered”, “normal”, or “intermediate” based on proximity to nucleus (DAPI signal). Imaging and analysis were carried out in a double -blind fashion. Mean ± SEM of percent neurons exhibiting perinuclear clustering of lysosomes from three independent experiments were computed.

Evaluation of Lysosome Positioning in Control Hcla cells expressing LAMP1-KBS-GFP: Control HcLa cells were transfected with LAMP1-KBS-GFP plasmid (Pu ct al., 2015) using Fugcnc reagent (Promega) for 48 hours and then treated with 0.15% DMSO or 15pM RH1115 for 6 hours, fixed, and stained with anti-GFP (Invitrogen) antibody and DAPI (SouthernBiotech). Images were acquired on an LSM880 in confocal mode using 63x oil immersion objective at 1.5x optical zoom. Cells of comparable GFP intensities, and in smaller clusters of 2-5 cells were chosen to ensure uniformity in morphology. Cells were categorized into three groups, blinded to condition, based on where the majority of LAMP1-GFP signal was seen within the cell: majority seen in periphery, majority seen in perinuclear region, or mixed distribution (equally dispersed or equal intensity in periphery and perinuclear).

Evaluation of Lysosome Acidification in i³Neurons: DIV 10-13 Control and JIP3 KO i³Neurons were incubated with lOOnM of LysoTracker-Red for 10 minutes in culture medium at 37 °C. After incubation, i³Neurons were washed in warm imaging media twice before imaging live for approximately 15 minutes per condition at 37°C using an LSM88O microscope in Airyscan super resolution mode using a 63x oil immersion objective (Zeiss). Images were acquired after focusing on central plane of the soma. Images were processed using Airyscan processing function of Zen software. Lysotracker intensity was measured in ImageJ using the ‘freehand’ selection tool to outline the soma followed by the ‘measure’ tool. Mean intensity per cell was computed and population mean across multiple independent experiments across treatments were compared.

Lysosomal Degradation Assay: Lysosomal degradation was measured using DQ-Red BSA. I³Neurons were preloaded with DQ-BSA prior to treatment with 0.15% DMSO or 15pM RH1115. Conditioned media from DIV12-13 i³Neurons was saved and DQ-Red BSA (25pg/mL) that was equilibrated in 10% culture media at 37°C for 5 minutes was added to the i³Neurons and incubated for 4 hours at 37°C, 5% CO2. DQ-Red BSA solution was removed and i³Neurons were washed with warm PBS twice before a 2- hour chase in the conditioned media. Conditioned media was replaced with 10% culture media containing 0.15% DMSO or 15pM RH1115 for 4.5 hours. i³Neurons were washed twice with warm imaging media (20 mM HEPES, 5 mM KCL, 1 mM CaC12, 150 mM NaCl, 1 mM MgC12, 1.9 mg/mL glucose and BSA, pH 7.4) prior to imaging live at 37°C on an LSM88O in Airyscan super resolution mode, on a 63x oil immersion objective, focusing on the central plane of the somas. Images were processed using ‘Airyscan processing’ function of Zen software and the intensity was analyzed in ImageJ using the ‘freehand’ selection tool to outline the soma followed by the ‘measure’ tool.

Measurement of Extracellular A [542: Control and JIP3 KO i³Neurons were differentiated for 42 days. At DIV42 the media was removed and replaced with KO DMEMF12 (equilibrated overnight at 37°C and 5% CO₂) with DMSO (0.15%), BACE1-I (5pM) or small molecule (RH1115 15pM). i³Neurons were maintained for 7 days in KO DMEMF12 + treatment, with a media change and addition of new DMSO/BACE1-I/RH1115 4 days after the first treatment (Fig 2 C, D). On DIV49/50 i³Neurons were lysed and conditioned media was spun down at 5000g for 5 min and the supernatant saved at -80°C. CM was measured using ELISA A [542 high sensitivity kit (Wako) according to the manufacturer’s instructions.

Generation and Maintenance of Zebrafish lines: A previously described JIP3 knockout (KO) zebrafish line (JIP3¹¹¹⁷) was obtained. Briefly, JIP3 KO fish were generated using ENU mutagenesis and found to have a single nucleotide substitution that resulted in an early stop codon, Spel restriction enzyme site, and truncated JIP3 protein. Genotyping was conducted using PCR to generate a 385- base pair (bp) amplicon followed by an overnight restriction enzyme digest with Spel (New England Biosystems, Ipswich MA) to generate double band homozygous amplicons (243 and 142 bp) detected using gel electrophoresis.

Animals were raised in quarantine and housed at the University of Alabama at Birmingham (UAB) Zebrafish Research Facility and all procedures were approved by the UAB Institutional Animal Care and Use Committee. Zebrafish embryos were raised in a 28.5 °C incubator on a 14-hour light, 10-hour dark cycle to 5-days post-fertilization in 1 x E3B made in recirculating, filtered system water. After 5 days, animals were transferred to tanks on the recirculating water system (Aquaneering, Inc., San Diego, CA). Heterozygous JIP3 adult fish were in crossed (1 male:l female) to generate wild type, heterozygous, and homozygous embryos.

Zebrafish Locomotion Assay: Embryos were placed in petri dishes containing either 0.05% DMSO dissolved in E3 blue (no drug controls) or 0.05% DMSO with 0.15 pM RH1115 and kept in the incubator with steady temperature and light cycles. Water changes were completed daily in the morning to refresh drug and maintain a steady concentration during screening. At 6-days post fertilization larvae were transferred to 4 x 48-well plates, allowed to acclimate, and then run on the Zantiks (Zantiks Ltd, Cambridge UK) for 1 hour to observe locomotor activity. Using a camera and infrared light tracing, total distance travelled (mm) and average velocity (mm/s) were calculated for individual larvae. Larvae were then transferred to a PCR genotyping plate and DNA was extracted using NaOH, neutralized with TRIS-HCL, and genotyped as described above. One-way ANOVA with Tukey’s test for multiple comparisons was used to calculate significance across genotypes for locomotor phenotypes and all analyses were completed using GraphPad Prism Software (GraphPad Software, LLC, San Diego, CA).

Statistical Analysis: Data are represented as superplots consisting of mean + SEM across experiments as well as individual data points across all the experiments, unless otherwise specified. Statistical analysis was performed using GraphPad Prism 10 software. Groups (means from the different experimental repeats) were compared using students t-test or one-way ANOVA multiple comparison test as appropriate. Detailed statistical information [number of independent experiments(N), number of individual measurements(n), statistical test performed and p values] are described in the respective figure legends.

Loss of JIP3 in human PNeurons reduces motility of both autophagosomes and autolysosomes, but not fusion of autophagosomes with endo-lysosomes: Mammalian JIP3/ MAPK8IP3 as well as its orthologs in the nematode worm and zebrafish have been shown to play a role in axonal lysosome transport with their loss resulting in an abnormal accumulation of endo-lysosomes within axonal swellings. Autophagosome biogenesis and trafficking in axons is spatially organized, and dependent on fusion with endo-lysosomes for acquiring adaptors that aid in their retrograde transport to the soma for degradation. Given the role of the lysosome adaptor JIP3 in regulating retrograde lysosome transport, it was predicted that its loss would affect the movement and distribution of autophagic vacuoles in neurons lacking JIP3. Towards this end, the dynamics of LC3-containing vesicles in the neurites of Control and JIP3 KO i³Neurons generated from iPSCs stably expressing LC3-RFP-GFP were examined. It was found that there were far more autophagic vacuoles in the neurites of JIP3 KO PNeurons, both within axonal swellings (FIG. 11 ; white arrowheads) and outside of the swellings, compared with the neurites of Control PNeurons (FIG. 11). Furthermore, the timelapse imaging studies revealed a significant decrease in the motile fraction of both autophagosomes (both RFP- and GFP-positive) and the more acidic autolysosomes (only RFP- positive) (FIGS. 12 and 13) in JIP3 KO i³Neurons compared to Control i³Neurons. Whether the loss of JIP3 had any effect on fusion of autophagosomes with the endolysosomes in the neurites was also examined. Interestingly, it was found that the percentage of autolysosomes in the neurites of JIP3 KO i³Neurons was comparable to that in Control i³Neurons (FIG. 14), suggesting that JIP3 is not necessary for the fusion of autophagosomes with endo-lysosomes in these neuronal processes.

RH1115 clears autophagic vacuole buildup from neuronal processes of JIP3 KO FNeurons: RH1115 enhances autophagic flux as well as alters lysosome positioning in both HeLa cells and the cell bodies of FNeurons. Since RH1115 strongly increased perinuclear localization of lysosomes in Control FNeurons, suggestive of increased net retrograde lysosome movement, it was tested whether the small molecule could mobilize and effect retrograde movement of the autophagic vacuoles that build up in the neuronal processes of JIP3 KO FNeurons. It was found that RH1115 treatment led to a dramatic decrease in the autophagic vacuole accumulation observed in JIP3 KO FNeurons (FIGS. 11, 15, 16). There is an almost four-fold increase in autophagic vacuole density in neuronal processes of JIP3 KO FNeurons, even outside of axonal swellings, which is rescued to levels comparable to that of Control FNeurons (FIG. 16). The small molecule can reduce even the massive autophagic vacuole build up observed within the axonal swellings of JIP3 KO FNeurons (FIGS. 11, 15, 17). FIGS. 11-17 show that loss of JIP3 impairs motility of autophagosomes and autolysosomes, causing their buildup in neuronal processes, which is rescued by small molecule activator of ALP.

RH1115 reduces both axonal lysosome build up and A [542 pathology of JIP3 KO FNeurons and increases net retrograde movement of the lysosomes in the soma: Given the strong reduction of autophagic vacuole buildup effected by RH1115 in JIP3 KO FNeurons, and their previously established dependence on axonal lysosomes for their transport to the soma for clearance, how RH1115 affected axonal lysosome pathology in these FNeurons was examined. As previously shown, JIP3 KO FNeurons exhibit a strong axonal lysosome accumulation phenotype which is both robust and penetrant (FIG. 18). RH1115 treatment strongly reduced the axonal lysosome buildup (FIGS. 18 and 19). In fact, the small molecule almost eliminated (97% reduction) the larger lysosome accumulations that would often extend well over 10 microns in length in these JIP3 KO FNeurons (FIGS. 18 and 19). In addition, RH1115 strongly suppressed the overall axonal lysosome buildup (FIGS. 18 and 19), leading to a lysosome distribution like that observed in Control FNeurons (FIG. 18). It was also observed that a similar rescue of axonal lysosome buildup in JIP3 KO FNeurons stably expressing LAMP1-GFP upon treatment with RH1115. FIGS. 18-27 show that RH1115 reduces the axonal lysosome buildup observed in JIP3 KO FNeurons and increases perinuclear lysosome clustering in neuronal cell bodies of these FNeurons. In addition to the axonal lysosome phenotype, JIP3 KO i³Neurons have previously been shown to have higher levels of intraneuronal Amyloid [342 (A042). It was found that JIP3 KO i³Neurons in fact also have higher levels (50% increase) of extracellular or secreted Af42, as measured from the culture media (FIGS. 20 and 21). Given links between altered axonal lysosome transport, maturation and increased amyloidogenesis, and the dramatic rescue of axonal lysosome pathology by RH1115, it was examined whether the small molecule had any effect on A042 production in these JIP3 KO i³Neurons. Indeed, it was found that RH1115 treatment significantly decreased extracellular Af42 in JIP3 KO i³Neurons, to levels comparable to Control i³Neurons (FIGS. 20 and 21). The small molecule had no negative impact on neuronal viability at the dosage at which it rescues axonal lysosome buildup in JIP3 KO PNeurons.

Given the clearance of axonal lysosome buildup in J1P3 KO i³Neurons brought about by RH1115, it was next examined how it modulated movement as well as properties of lysosomes in the neuronal cell body. It was found that RH1115 causes a strong perinuclear localization of lysosomes within the soma (FIGS. 22 and 23), as previously observed in Control i³Neurons. It was also observed that an increase in the size and intensity of LAMP1 -positive vesicles, in the soma of JIP3 KO i³Neurons (FIGS. 22, 24 and 25). Based on the mobilization and clearance of axonal lysosomes and the perinuclear clustering of soma lysosomes induced by RH1115, it was hypothesized that the small molecule causes an increase in the net retrograde movement of lysosomes. This was tested by examining the effect of RH1115 on distribution of LAMP1-KBS-GFP expressed in HeLa cells. This construct includes LAMP1 with three copies of the Kinesin light chain binding sequence of SKIP, tagged with GFP. This bypasses the requirement for ARL8B, SKIP and BORC, allowing for direct binding between LAMP1 and kinesin- 1, which results in a strong peripheral accumulation of lysosomes. As expected, a little over 60% of DMSO-treated LAMP1-KBS-GFP expressing cells show a strong peripheral LAMP1-KBS-GFP accumulation, with only about 20% cells showing a perinuclear LAMP1 distribution (FIGS. 26 and 27). However, in the case of RH1115 treatment, there is a dramatic shift in lysosome positioning in most cells, with greater than 60% of cells showing LAMP1-KBS-GFP strongly enriched in the perinuclear region, and almost none having lysosomes mainly in the periphery (FIGS. 26 and 27).

RH1115 increases neuronal lysosomal degradative capacity: Lysosome positioning in nonneuronal cells has been linked to functional differences, with lysosomes that are more perinuclear being more degradative than peripheral lysosomes. Given the effect of RH1115 in mobilizing neuronal lysosomes towards the perinuclear region, its impact on lysosome acidification was examined. Control and JIP3 KO i³Neurons treated with RH1115 for 72 hours were labelled with Lysotracker Red, a fluorescent dye that preferentially identifies acidic organelles. RH1115 treatment led to a ten-fold increase in intensity of Lysotracker Red in both Control and JIP3 KO i³Neurons (FIGS. 28 and 29), suggestive of an increase in acidic organelles in the neuronal cell body. The effect of RH1115 on the proteolytic activity of lysosomes in both Control and JIP3 KO i³Neurons using DQ-Red BSA (dye-quenched Bovine Serum Albumin), a cargo that fluoresces when proleoiytically cleaved, in i -’Neurons was also examined. To isolate the effect of RH1115 on lysosomal degradation alone and remove effects on DQ-Red BSA intensity arising from differences in endocytic uptake, lysosomes in both Control and JIP3 KO i³Neurons were preloaded with DQ-Red BSA prior to treatment with RH1115 or DMSO. The DQ-Red BSA intensity was decreased in both Control and JIP3 KO i³Neurons upon treatment with Bafilomycin Al post lysosomal loading, confirming that it reports on lysosomal proteolytic activity. RH1115 treatment resulted in a striking increase in the DQ-Red BSA mean intensity in both Control and JIP3 KO i³Neurons, (FIGS. 30-33), suggestive of increased proteolytic activity in the lysosomes.

The rescue of axonal lysosome pathology in J1P3 KO PNeurons by RH1115 requires JIP4: Application of the small molecule RH1115, results in clearance of axonal lysosome accumulations in JIP3 KO i³Neurons and mobilizes lysosomes to the perinuclear region within the soma, suggesting an increase in retrograde lysosome transport. While JIP3 has an established role in regulating retrograde axonal lysosome movement, the highly related adaptor JIP4 has some overlapping function in this pathway as well, and is known to play a role in dynein-dependent lysosome movement from the periphery to the perinuclear region in non-neuronal cells. The lysosomal transmembrane protein TMEM55B, has been shown to recruit JIP4 to lysosomes to induce this dynein-dependent movement in these cells. Given this prior evidence for regulation of TMEM55B expression as a means of regulating lysosome movement, it was examined whether RH1115 treatment altered TMEM55B expression in these i³Neurons. Indeed, RH1115 treatment led to increased levels of TMEM55B protein in both Control and JIP3 KO i³Neurons (FIGS. 34 and 35). Considering the observed increase in TMEM55B levels and its known role in recruiting JIP4 to lysosomes to bring about retrograde movement, it was interrogated if this rescue of axonal lysosome pathology in JIP3 KO i³Neurons depends on JIP4. For this, the effect of RH1115 on the lysosomal pathology in JIP3/4 double KO i³Neurons stably expressing LAMP1-GFP was examined. The LAMP1 accumulations, which arc more severe in the JIP3/4 double KO i³Neurons were not rescued by RH1115 treatment (FIGS. 35-38), suggesting that RH1115 requires JIP4 to mobilize the lysosomes and clear the axonal lysosome pathology, and thus likely enhances retrograde lysosome movement in neurons via a JIP4/TMEM55B- mediated pathway. Furthermore, the small molecule failed to mobilize lysosomes to the perinuclear region in the neuronal cell bodies of JIP3/4 double KO i³Neurons unlike what was observed in JIP3 KO i³Neurons, suggesting a role for the adaptor JIP4 in retrograde lysosome movement in the neuronal cell body.

RH1115 rescues locomotor defects in JIP3KO zebrafish larvae: Loss of JIP3 in zebrafish has been shown to alter retrograde axonal lysosome transport. This example, which examined locomotion behavior in zebrafish larvae, revealed that JIP3 KO zebrafish move less distance and travel at slower velocity on average, compared to wild type clutch mates (FIGS. 39 and 40). Treatment of JIP3 KO zebrafish with RH1115 for 6 days, immediately following fertilization, resulted in a striking rescue of the locomotor defects in fish that completely lack JIP3 protein, with significant increases in total distance travelled and average velocity (FIGS. 39 and 40). In fact, the small molecule restored locomotor properties in the JIP3 KO zebrafish to levels observed in the wildtype zebrafish (FIGS. 39 and 40), with no negative impacts on zebrafish larvae of either genotype.

Perturbations to axonal lysosome transport that result in an imbalance in lysosomal distribution in axons has been linked to defects in neurodevelopment and to the pathophysiology of neurological diseases. The abnormal accumulation of protease-deficient axonal lysosomes near plaques, which were linked to APP processing, suggested that defects in retrograde transport and maturation of these organelles could contribute to plaque development. In support of such a model, loss of the brain- enriched lysosomal adaptor JIP3, recently shown to be a dynein activator, resulted in axonal lysosome buildup as well as increased A042 production in both primary mouse neurons and human iPSC-derived neurons. Furthermore, reducing dosage of JIP3 in a mouse model of AD resulted in increased soluble A [142 levels and increased neuritic plaque abundance and size. Together, these prior studies suggest that efficient retrograde axonal lysosome transport is anti-amyloidogenic and an attractive pathway for therapeutic intervention. In this study, it was shown that RH1115 clears axonal lysosome accumulation and reduces the extracellular A [142 levels in human i³Neurons lacking JIP3. RH1115 treatment clears not only the endolysosomal buildup, but also the autophagic vacuole accumulation from JIP3 KO axons, suggesting that it can restore protein and organelle homeostasis in neurons where axonal ALP is impaired. Thus, elucidating the mechanism of action for RH1115 in human neurons could be impactful in different neurodegenerative diseases where impaired ALP is a contributing factor.

Previous studies based on JIP3 depletion in PC 12 cells and exogenous JIP3 expression in neurons postulated that JIP3 interacts with AVs marked by both LAMP1 and LC3, and that JIP3 modulates movement of more mature AVs in middle and proximal axons. Consistent with these reports, examples herein suggest that JIP3 affects the motility of autolysosomes (FIGS. 11-13). Here, it was found that loss of JIP3 also impacts the motility of autophagosomes in addition to the movement of autolysosomes (FIGS. 11-13). Interestingly, several accessory proteins in addition to JIP3 have been identified to regulate the retrograde movement of AVs, including JIP1, HAP1- Huntingtin and RILP. These adaptors have been suggested to act sequentially on AVs depending on the AV maturation state and spatial distribution within the axon. The results from imaging stably expressed LC3-RFP-GFP in (’Neurons suggest that loss of JIP3 leads to a decrease in the motile fraction of both autophagosomes and autolysosomes but does not affect fusion of the axonal autophagosomes with endo-lysosomes. Given evidence from previous studies that the less mature autophagosomes are more frequent in the distal axon, JIP3 likely has a role in regulating organelle transport in the distal axon as well. This is supported by endogenous JIP3 localization in distal axons, including in the tips of growth cones in neurons.

It was also shown that this rescue of axonal lysosome buildup in JIP3 KO i³Neurons requires the related lysosome adaptor JIP4 and that the small molecule RH1115 increases levels of the JIP4-interacting, transmembrane lysosomal protein TMEM55B in PNeurons (FIGS. 34-38). TMEM55B has been shown to recruit JIP4 to lysosomes, and its upregulation linked to dynein- dependent lysosome movement in response to nutrient starvation in cultured cell lines. Thus, it is possible that RH1115-mediated TMEM55B upregulation recruits more JIP4 to axonal lysosomes and aids in their retrograde movement. A recent study demonstrated that TMEM55B interacts with an E3 Ubiquitin Ligase, NEDD4, in response to oxidative stress, which competes with the TMEM55B-JIP4 interaction. It will be interesting to determine if RH1115 can modulate this NEDD4-TMEM55B interaction in response to oxidative stress. Of relevance to Parkinson’s disease, expression of a hyperactive LRRK2 in neurons led to increased JIP4 recruitment to A Vs and also reduced their processive retrograde movement. The studies in the JIP3/4 DKO i³Neurons suggest that RH1115- mediated retrograde axonal lysosome movement requires JIP4. Given that prior studies implicated a JIP4-mediated bias towards kinesin activation on these AVs under pathological conditions, it may be possible that RH1115 could rescue the AV transport defect in the LRRK2 mutant neurons.

Prior target identification studies revealed a specific interaction of RH1115 with LAMPE LAMP1 is an abundant glycoprotein on the surface of late endosomes, degradative lysosomes, and biosynthetic precursor organelles. While LAMP1 has been widely used as a ‘marker’ for this spectrum of organelles, its precise function in the context of lysosome transport is not fully understood. Based on the interaction with RH1115 and its effect on increasing net retrograde lysosome movement, it is tempting to speculate that LAMP1 may be involved in lysosome transport. Consistent with this possibility, LAMP1-APEX studies in neurons suggest that LAMP1 interacts with two GTPases involved in lysosome biogenesis and transport, namely ARL8B and Rab7. It currently is believed that RH1115 is the first compound targeting LAMP1 that alters lysosome movement.

RH1115 appears to enhance neuronal lysosome acidification (FIGS. 28-33). This change in lysosomal physiology may be mediated by its interaction with LAMP1 or its effect on TMEM55B. LAMP1 has been shown to interact with multiple subunits of the vacuolar ATPase via LAMP 1 -APEX proteomic analysis. Previous research has shown that changes in nutrient status can modulate the association between the transmembrane Vo subunit and the membrane associated Vi subunit on the lysosomal membrane, determining lysosomal acidification, thus it is possible that RH1115 may positively modulate vATPase subunit Vo or Vi interaction with LAMP1, increasing vATPase assembly. In addition, LAMP1 has also been shown to bind to and inhibit the proton leak channel in lysosomes, TMEM175, thus increasing acidification of lysosomes. Alternatively, TMEM55B may also have a role in vATPase assembly on lysosomes that could contribute to observed effects of RH1115. TMEM55B deficiency in Hela cells causes a decrease in the levels of the ATP6V1 A subunit of the vATPase in the detergent resistant membrane fraction (representing lipid rafts present in lysosomal membranes) suggesting that TMEM55B may have a role in anchoring or increasing the association of the VI subunit with the lysosomal membrane (Hashimoto et aL, 2018). Therefore, RH1115 increasing TMEM55B may increase lysosomal acidification through modulating vATPase association/assembly on lysosomal membranes. Future studies using the small molecule could also shed light on whether LAMP1 plays a role in modulating neuronal lysosomal acidification.

Additionally, the small molecule RH1115 increases perinuclear localization of lysosomes within the neuronal cell body/soma of JIP3 KO PNeurons and increases lysosomal size, and lysosomal degradative capacity (FIGS. 22-25 and 28-33). Defects in lysosomal degradative capacity are a major contributing factor to the pathology of lysosomal storage disorders, therefore, effect of RH1115 on neuronal lysosomes could have a broad clinical relevance. In addition to rescuing autophagic and lysosomal defects in human neurons, RH1115 treatment rescues locomotor defects in JIP3 KO zebrafish larvae (FIGS. 39 and 40), suggesting that restoration of axonal lysosome transport can positively impact neuronal health and functioning.

The movement and positioning of lysosomes within the neuronal cell body could be influenced by a variety of cellular factors. For instance, in non-neuronal cells, lysosome positioning is altered by nutrient status. In turn, the positioning of lysosomes affects cellular functioning including autophagy, cell migration, cancer cell invasion and antigen-presentation. Future studies will determine how the small molecule could modulate these different processes in the distinct cell types.

Examples of the present disclosure establish that compounds disclosed herein can enhance net retrograde lysosome movement and that increasing efficient axonal lysosome transport can be anti- amyloidogenic. As such, compounds of the present disclosure provide a new molecular tool to both modulate lysosome position-dependent cellular functions and to elucidate the role of LAMP1 in regulating lysosome transport, acidification, and degradation.

In 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

We claim:

1. A compound of formula I or II, or a pharmaceutically acceptable salt thereof,

wherein

Ai, Aj, A3 and A4 are independently selected from C1-8 alkyl, C6-10aryl, 5- to 10-membered heteroaryl, Cs-iocycloalkyl and 4- to 10-membered heterocyclyl, and a group of formula -NR3R4, wherein the alkyl, aryl, heteroaryl, cycloalkyl and heterocyclyl are optionally substituted with one or more groups independently selected from - C(O)Rs, -C(O)NR5R6, -SO2R5, C6-10aryl(Ci-6alkyl), 5- to 10-membered heteroaryl(C1-6alkyl), C 1-10cycloalkyl(C1-6alkyl), 4- to 10-membered heterocyclyl(Ci-6alkyl), urea (- NHC(O)NH2), sulfonamide (-SC2amino), C1-6alkyl, halo, amino, hydroxy, or amide groups, or a combination thereof;

Li, and L4 are independently absent or C1-C4 alkylene;

L3 is absent or is selected from O, NR5, S, C(O), and C1-C4 alkylene;

L2 and L5 are independently absent or selected from C1-C4 alkylene and -SO2-;

Z is selected from -C(O)R₅, -C(O)NR₈R9, -SO2R5, hydroxy, C1-C4 alkoxy, -C(O)OR₅, -OC(O)R₅, and -C(O)R5;

Ri, R2, R3, R4, and R5 are independently selected from H, -C(O)R6, -C(O)NR8N9, -SC2R6, C1-6alkyl, - O(C1-6alkyl), halo, C6-10aryl. C6-10arylalkyl, 5- to 10-membered heteroaryl, 5- to 10-membered heteroaryl(Ci. 6alkyl), C3-10cycloalkyl, C3-10cycloalkyl(C1-6alkyl), 4- to 10-membered heterocyclyl, and 4- to 10-membered heterocyclylalkyl;

R6 and R7 are independently selected from H, C1-10alkyl, C2-10alknyl, C6-10aryl, C6-10aryl(C1-6alkyl), 5- to 10-membered heteroaryl, 5- to 10-membered heteroaryl(Ci-ealkyl), C3-10cycloalkyl, C3-10cycloalkyl(C1- 6alkyl), 4- to 10-membered heterocyclyl, and 4- to 10-membered heterocyclylalkyl; and

R8 and R9 are independently selected from H, C1-10alkyl, C2-10alknyl, C3-10aryl, C3-10aryl(Ci-ealkyl), 5- to 10-membered heteroaryl, 5- to 10-membered heteroaryl(C1-6alkyl), C3-10cycloalkyl, C3-10cycloalkyl(C1- 6alkyl), 4- to 10-membered heterocyclyl, and 4- to 10-membered heterocyclylalkyl, or R8 and R9 together with the nitrogen to which they are attached form a 5- to 7-membered heterocyclyl, optionally including 1, 2 or 3 additional heteroatoms selected from N, O or S, and optionally substituted with 1 , 2, or 3 substituents selected from C1-6alkyl, -CHiphenyl, or -C(O)OC1-6alkyl.

2. The compound according to claim 1 wherein Li, L3 and L4 arc -CH2-

3. The compound according to any one of claims 1-2 wherein Ai, A2, A3 and A4 are independently selected from

4. The compound of any one of claims 1-3, wherein A1, A2, A3 and A4 are independently selected from

5. The compound according to any one of claims 1-4, having a structure according to Formula

6. The compound according to any one of claims 1 -5 wherein the compound has a structure according to formula I-A or I-B

7. The compound according to any one of claims 1-5 wherein the compound has a structure according to formula I-C, I-D or I-E

8. The compound of any one of claims 1-7, wherein R4 and R5 are each independently selected from H or C1-8alkyl.

9. The compound of any one of claims 1-8, wherein Z is OH.

10. The compound of any one of claims 1 -8, wherein Z is C1-8alkyl.

11. The compound of any one of claims 1-10, wherein the -L2A2 moiety is -(C ialkyl)phenyL optionally substituted with from 1 to 5 substituents selected from Ci-4alkyl, halo, hydroxy, or a combination thereof.

12. The compound of claim 11, wherein the -L2A2 moiety is -CH2phenyl or -CH2CH2phenyl, optionally substituted with from 1 to 5 substituents selected from C1-4alkyl, halo, hydroxy, or a combination thereof.

13. The compound of any one of claims 1-10, wherein the -L2A2 moiety is — (C1- 4alkyl)cyclohexane.

14. The compound of claim 13, wherein the -L2A2 moiety is -CHicyclohcxanc, or - CH2CH2cyclohexane.

15. The compound of any one of claims 1-14, wherein the -L1A1 moiety is -(Ci- 4alkyl)cyclohexane

16. The compound of claim 15, wherein the -L1A1 moiety is -CfTcyclohexane. or - CH2CH2cyclohexane.

17. The compound of any one of claims 1-14, wherein the -L1A1 moiety is -(Ci- 4alkyl)heteroaryl, optionally substituted with from 1 to 5 substituents selected from Chalky!, CMalkoxy, halo, hydroxy, or a combination thereof, where Ri is as previously defined.

18. The compound of claim 17, wherein the -LjAi moiety

, optionally substituted with from 1 to 5 substituents selected from Cwalkyl, Ci.4alkoxy, halo, hydroxy, or a combination thereof.

19. The compound of claim 18, wherein the -L1A1 moiety i

substituted with a halo atom or a Cualkoxy group.

20. The compound according to claim 1 selected from

or a pharmaceutically acceptable salt thereof.

21. The compound according to any one of claims 1-4, having a structure according to Formula

22. The compound of claim 21, wherein the compound has a structure according to Formula

23. The compound according to claim 21 or claim 22, selected from

or a pharmaceutically acceptable salt thereof.

24. A composition comprising a compound according to any one of claims 1-23 and a pharmaceutically acceptable diluent or excipient.

25. A method of treating a neurodegenerative disease or condition comprising administering to a subject in need of treatment an effective amount of a compound according to any one of claims 1-23, or the composition of claim 24.

26. The method of claim 25, wherein the neurodegenerative disease or condition is selected from Huntington’s disease (HD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and Alzheimer’s disease.

27. The method of claim 26, wherein the neurodegenerative disease or condition is Alzheimer’s disease.

28. A use of a compound according to any one of claims 1-23 in the manufacture of a medicament for the treatment of a neurodegenerative disease.