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WO2025076226A1 - Hétérocycles fusionnés pour le traitement de maladies neurodégénératives - Google Patents

Hétérocycles fusionnés pour le traitement de maladies neurodégénératives Download PDF

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WO2025076226A1
WO2025076226A1 PCT/US2024/049800 US2024049800W WO2025076226A1 WO 2025076226 A1 WO2025076226 A1 WO 2025076226A1 US 2024049800 W US2024049800 W US 2024049800W WO 2025076226 A1 WO2025076226 A1 WO 2025076226A1
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compound
nmr
mmol
mhz
6alkyl
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Leslie ALDRICH
Andrew DOBRIA
Ryan S. HIPPMAN
Thomas WHITMARSH-EVERISS
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University of Illinois at Urbana Champaign
University of Illinois System
US Department of Health and Human Services
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University of Illinois at Urbana Champaign
University of Illinois System
US Department of Health and Human Services
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/10—Spiro-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Definitions

  • 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.
  • 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.
  • 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.
  • PSMs peptide spectrum matches
  • 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. 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. 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 i 3 Neurons treated with RH1115 (15 pM) or DMSO (0.1%) for 72 hours.
  • FIG. 5B shows that quantification of LC3II/LC3I ratio shows that i 3 Neurons treated with RH1115 exhibit significantly increased LC3 lipidation in comparison to DMSO treated i 3 Neurons. Mean ⁇ SEM from four independent experiments, **P ⁇ 0.01.
  • FIG. 5C Confocal images of DIVIO i 3 Neurons 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 i 3 Neurons treated with RH1115 (15 pM) or BafAl (100 nM) compared to DMSO.
  • FIG. 5E shows quantification of percent of autolysosomes in i 3 Neurons treated with RH1115 (15 pM) or BafAl (100 nM) compared to DMSO.
  • FIG. 5F shows quantification of mean size of autolysosomes in i 3 Neurons treated with RH1115 (15 pM) compared to DMSO.
  • FIG. 5G shows quantification of intensity of autolysosomes in i 3 Neurons treated with RH1115 (15 pM) compared to DMSO.
  • RH1115 15 pM
  • 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.
  • 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 i 3 Neurons. 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 i 3 Neurons.
  • Superplots show mean ⁇ SEM as well as individual data (represented as large and small symbols respectively. Circles, triangles, and squares represent each independent experiment).
  • FIG. 14 shows quantification showing percentage of total autophagic vacuoles that arc autolysosomes in DIV15-16 Control and JIP3 KO i 3 Neurons.
  • Superplots show mean + SEM as well as individual data (represented as large and small circles, triangles and squares).
  • 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).
  • 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.
  • 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).
  • 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).
  • 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).
  • 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.
  • 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 i 3 Neurons treated with 0.15% DMSO or 15pM RH1115.
  • DMSO treated n 61 swellings
  • RH1115 treated n 68 swellings.
  • FIG. 39 shows that RH1115 treatment rescues locomotor defects in JIP3 KO larval zebrafish.
  • FIG. 40 shows that RH1115 treatment rescues locomotor defects in JIP3 KO larval zebrafish.
  • 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.
  • 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.
  • these compound embodiments can additionally be mixtures of diastereomers.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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 -CH 2 -, -CH 2 CH 2 -, - CH 2 CH 2 CHC(CH 3 )-, and -CH 2 CH(CH 2 CH 3 )CH 2 -.
  • 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.
  • alkylene group is -(CH 2 )n-
  • An alkyl, e.g., methyl, or alkylene, e.g., — CH 2 CH 2 — , 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 a R b wherein R a and R b are independently selected from hydrogen and C
  • -C alkyl. Acetylamino means a -NHC( O)CH 3 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.
  • 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
  • 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.
  • the aryl may be substituted by one or more halo, alkyl, haloalkyl, or alkoxy groups.
  • 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.
  • 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, pyr
  • heterocyclyl may be substituted by one or more halo, alkyl, haloalkyl, or alkoxy groups.
  • 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.
  • 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.
  • 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, pival
  • 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.
  • 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.
  • compounds of the disclosure are effective modulators of neurodegenerative diseases or conditions.
  • the neurodegenerative disease or condition is selected from Huntington’s disease (HD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and Alzheimer’s disease.
  • 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.
  • 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).
  • AD Alzheimer’ s disease
  • AD patients live for 8 to 10 years after they are diagnosed, though the disease can last up to 20 years.
  • 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 is an idiopathic, slowly progressive, degenerative CNS disorder characterized by slow and decreased movement, muscular rigidity, resting tremor, and postural instability.
  • ALS Amyotrophic lateral sclerosis
  • 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.
  • 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.
  • CAG trinucleotide repeat
  • 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.
  • 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.
  • 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.
  • 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)R 5 .
  • 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 R 8 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.
  • L1, L3 and L4 are -CH2-.
  • L2 is -CH2-. In another aspect, L2 is -CH2CH2-. In a further aspect, L2 is -SO2-.
  • n is 1.
  • Ai, A2, A3 and A4 are independently selected from
  • Ri is H, C1-6alkyl, or halo, and in certain aspects, R1 is methyl, H, or propyl.
  • R2 is H. In other aspects, R2 is alkyl, such as methyl; -Oalkyl, such as methoxy; or halo, such as F or Cl.
  • 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.
  • Rs and R6 are independently selected from H or C 1-6alkyl, such as methyl, ethyl, propyl, or isopropyl.
  • R5 is H and R6 is C1-6alkyl. In other aspects, both R5 and R6 independently are C1-6alkyl.
  • 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.
  • this disclosure provides a compound of formula I-A:
  • this disclosure provides a compound of formula I-B:
  • the compound has a formula I-C
  • the compound has a formula I-D
  • 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.
  • 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.
  • the -L1A1 moiety may be -(Ci-4alkyl)heteroaryl, such as , which is substituted with halogen (preferably F) or C1-4alkoxy (preferably methoxy).
  • 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:
  • this disclosure provides compounds according to formula II.
  • such compounds can be of a formula:
  • the compound can be of a formula:
  • Exemplary compounds according to Formula II include, but are not limited to
  • 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.
  • this disclosure provides a pharmaceutical composition
  • 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.
  • this disclosure provides a pharmaceutical composition
  • 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.
  • excipients include liquids such as water, saline, glycerol, polyethylene glycol, hyaluronic acid, ethanol, and the like.
  • pharmaceutically acceptable vehicle refers to a diluent, adjuvant, excipient or carrier with which a compound of the disclosure is administered.
  • 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).
  • 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.
  • sodium benzoate, sorbic acid and esters of p- hydroxybenzoic acid can be added as preservatives. Id. at 1449.
  • 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).
  • 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.
  • buffer solutions include saline, phosphate buffered saline, Tris buffered saline, Hank’s buffered saline, and the like.
  • 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.
  • 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.
  • 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.
  • oral including buccal and sub-lingual
  • rectal including 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • polycations chitosan and its quaternary ammonium derivatives, poly-L-arginine, aminated gelatin
  • polyanions N-carboxymethyl chitosan, poly-acrylic acid
  • thiolated polymers carboxymethyl cellulose-cysteine, polycarbophil-cysteine, chitosan-thiobutyl amidine, chitosan- thioglycoli
  • 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.
  • 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.
  • 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.
  • 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.
  • suitable inert carrier such as ethanol, glycerol, water, and the like
  • 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.
  • 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.
  • the acceptable vehicles and solvents that can be employed are water, Ringer’s solution and isotonic sodium chloride solution.
  • sterile, fixed oils, fatty esters or polyols are conventionally employed as solvents or suspending media.
  • parenteral administration can involve the use of a slow release or sustained release system such that a constant level of dosage is maintained.
  • 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.
  • 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.
  • Suitable vehicles and solvents that can be employed are water, Ringer’s solution and isotonic sodium chloride solution.
  • sterile, fixed oils, fatty esters or polyols are conventionally employed as solvents or suspending media.
  • parenteral administration can involve the use of a slow release or sustained release system such that a constant level of dosage is maintained.
  • 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.
  • 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.
  • 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.
  • 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.
  • the pharmaceutical compositions of the disclosure can be administered in the form of suppositories for rectal administration.
  • suppositories 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.
  • suitable nonirritating excipient include cocoa butter, beeswax and polyethylene glycols.
  • 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.
  • 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 is one that will provide for optimum drug delivery.
  • 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.
  • 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.
  • 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.
  • suitable skin contact adhesive materials include, but are not limited to, polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates, polyurethanes, and the like.
  • 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.
  • 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.
  • CFC chlorofluorocarbon
  • the aerosol can conveniently also contain a surfactant such as lecithin.
  • the dose of drug can be controlled by a metered valve.
  • 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).
  • 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.
  • 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.
  • the effective amount for a given situation can be determined by routine experimentation.
  • 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.
  • 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.
  • formulations can be prepared with enteric coatings adapted for sustained or controlled release administration of the active ingredient.
  • 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.
  • a neurodegenerative disease or condition is selected from Huntington’s disease (HD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and Alzheimer’s disease.
  • compounds of the present disclosure can be used to treat diseases characterized by altered lysosome function and distribution, such as lysosomal storage diseases.
  • 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.
  • 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.
  • 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.
  • the compounds modulate the activity of Lamin A/C and LAMP1 in cells.
  • 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)R 5 , -C(O)NR 8 R9, -SO2R5, hydroxy, C1-C4 alkoxy, -C(O)OR 5 , -OC(O)R 5 , 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.
  • 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.
  • Li, L3 and L4 are -CH2-.
  • Ai, A 2 , A 3 and A 4 are independently selected from
  • Z is C1-8alkyl.
  • 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.
  • 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.
  • the -L2A2 moiety is -(Ci-4alkyl)cyclohexane.
  • the -L2A2 moiety is -CH2cyclohexane, or -CH2CH2cyclohexane.
  • the -L1A1 moiety is-(Ci-4alkyl)cyclohexane
  • the -L1A1 moiety is -CH2cyclohexane, or -CH2CH2cyclohexane.
  • 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.
  • 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.
  • 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.
  • the compound has a structure according to Formula
  • the compound is selected from or a pharmaceutically acceptable salt thereof.
  • 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.
  • the neurodegenerative disease or condition is selected from Huntington’s disease (HD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and Alzheimer’s disease.
  • HD Huntington’s disease
  • PD Parkinson’s disease
  • ALS amyotrophic lateral sclerosis
  • Alzheimer’s disease is selected from Huntington’s disease (HD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and Alzheimer’s disease.
  • the neurodegenerative disease or condition is Alzheimer’s disease.
  • 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.
  • 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% CO 2 .
  • 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.
  • 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.
  • Z Z factor
  • 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.
  • 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 10 6 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.
  • 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.
  • LC-MS Liquid Chromatography-Mass Spectrometry
  • 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.
  • 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.
  • HeLa cells were plated at a density of 3.75 x 10 5 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.
  • Pierce IP Lysis Buffer Thermo, #87788
  • Pierce protease and phosphatase inhibitor tablet Thermo, #A32959
  • Lysate was centrifuged at 18,000 g for 30 minutes at 4 °C to remove debris and the supernatant was 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.
  • A549 cells were plated at a density of 2.5 x 10 6 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 10 7 cells/mL in PBS supplemented with Pierce protease inhibitor tablet.
  • 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.
  • 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
  • FIGS. 8-10 Exemplary results are provided in FIGS. 8-10.
  • Mean puncta/cell is used to determine activation based on the following formula:
  • 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.
  • PE phosphatidylethanolamine
  • 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.
  • 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.
  • the GFP fluorescence is quenched due to the low pH, leaving the red color from the mCherry.
  • 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).
  • SAR structure-activity relationships
  • 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.
  • the tertiary amine in the R3 position was necessary for activity.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • RH1115 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.
  • CETSA cellular thermal shift assay
  • LAMP1 staining in these neurons revealed that the compound treatment resulted in a profound effect on lysosome positioning in these i 3 Neurons (FIG. 4B and 4C).
  • 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).
  • 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.
  • phenotypic screening provides an unbiased approach to drug discovery.
  • 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.
  • 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).
  • 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.
  • Lamin A/C in most cells types, healthy neurons notably have little to no Lamin A expression, which allows for improved flexibility and plasticity.
  • 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.
  • 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.
  • 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.
  • NPC Niemann-Pick type Cl
  • 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.
  • 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.
  • this compound alters positioning of lysosomes and increases autophagic flux in human iPSC-derived neurons.
  • 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.
  • 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%).
  • 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.
  • 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%).
  • 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%).
  • 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%).
  • 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%).
  • AD-2052 Following General Procedure F, AD-2040 (77.3 mg, 0.198 mmol) and LiAIFU (15.02 mg, 0.396 mmol) were used.
  • 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%).
  • 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%).
  • 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%).
  • 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%).
  • 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%).
  • 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%).
  • 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%).
  • 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%).
  • 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%).
  • 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%).
  • 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%).
  • 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.
  • 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%).
  • 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%).
  • 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%).
  • 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%).
  • 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%).
  • 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%).
  • 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%).
  • 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%).
  • 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%).
  • 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%).
  • 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%).
  • 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%).
  • 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%).
  • 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%).
  • 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%).
  • 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%).
  • 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%).
  • 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%).
  • 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%).
  • 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%).
  • 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%).
  • 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.
  • 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%).
  • 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%).
  • 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%).
  • 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%).
  • 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%).
  • 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% NH 4 OH) on silica over 9 minutes) to afford the desired product as a white solid: 49.7 mg (60%).
  • 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%).
  • 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%).
  • 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.
  • the compounds of the present disclosure can rescue locomotor defects in JIP3 KO zebrafish larvae.
  • 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.
  • iPSC Culture and i 3 Neuron 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).
  • iPSCs were differentiated into i 3 Neurons by standard methods. i 3 Neurons 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
  • I 3 Neurons 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
  • Immunoblotting Lysis of i 3 Neurons and western blotting was carried out as described previously (Gowrishankar et al., 2021).
  • DIV21 i 3 Neurons 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 3 Neurons i 3 Neurons differentiated for 1-2 weeks on 24 mm glass coverslips or 35 mm Mattek glass-bottom dishes were processed for immunostaining as described previously.
  • I 3 Neuron Viability Assay DIV 10 i 3 Neurons 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.
  • DIV 15-16 i 3 Neurons 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.

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Abstract

L'invention concerne des aspects de composés selon la formule I et la formule II. L'invention concerne également des compositions pharmaceutiques comprenant les composés et un procédé de fabrication et d'utilisation des composés. Les composés sont utiles en tant que modulateurs d'autophagie. En outre, les composés divulgués peuvent être utiles en tant qu'agents thérapeutiques pour traiter et/ou prévenir des maladies neurodégénératives notamment la maladie d'Alzheimer.
PCT/US2024/049800 2023-10-04 2024-10-03 Hétérocycles fusionnés pour le traitement de maladies neurodégénératives Pending WO2025076226A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023064343A1 (fr) * 2021-10-13 2023-04-20 Vanqua Bio, Inc. Modulateurs à petites molécules de l'activité glucocérébrosidase et leurs utilisations
WO2023147513A2 (fr) * 2022-01-28 2023-08-03 Washington University Compositions d'agents de modulation d'autophagie et leurs utilisations

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023064343A1 (fr) * 2021-10-13 2023-04-20 Vanqua Bio, Inc. Modulateurs à petites molécules de l'activité glucocérébrosidase et leurs utilisations
WO2023147513A2 (fr) * 2022-01-28 2023-08-03 Washington University Compositions d'agents de modulation d'autophagie et leurs utilisations

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
"Remington's Pharmaceutical Sciences", 1990, MACK PUBLISHING COMPANY
BENCE ET AL., SCIENCE, vol. 292, 2001, pages 1552 - 1555
ELIEL, EWILEN, S.: "Stereochemistry of Organic Compounds", 1994, JOHN WILEY & SONS, INC.
S. P. PARKER: "McGraw-Hill Dictionary of Chemical Terms", 1984, MCGRAW-HILL BOOK COMPANY
WALTER ET AL., MOL. BIOL. CELL, vol. 12, 2001, pages 1393 - 1407

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