2-(TERT-butyl)-4-methylphenyl appended 1,4-diazepane based novel TFEB activating compounds as autophagy modulators for neurodegenerative disorders including alzheimer's disease (AD)

Abstract

Provided are compounds for activating TFEB, the compounds being according to Formula (I) or Formula (II) as well as pharmaceutical compositions including such compounds and methods for using the compounds for the treatment of diseases and disorders.

Description

[0001] Atty. Dkt. No.: 115872-3160 (SK2023-044-02) 2-(TERT-BUTYL)-4-METHYLPHENYL APPENDED 1,4-DIAZEPANE BASED NOVEL TFEB ACTIVATING COMPOUNDS AS AUTOPHAGY MODULATORS FOR NEURODEGENERATIVE DISORDERS INCLUDING ALZHEIMER'S DISEASE (AD) CROSS-REFERENCE TO RELATED PATENT APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application 63 / 633,431, filed April 12, 2024, the entirety of which is incorporated herein by reference. BACKGROUND Alzheimer’s disease (AD) is one of the most common forms of progressive neurodegenerative disorders marked by memory loss, changes in thinking and behavior. There are currently 6.9 million Americans live with AD costing $360 billion in 2024 [1]. Histopathologically, AD is mainly characterized by the presence of extracellular amyloid plaques consisting of β-amyloid peptides (Aβ) and intracellular neurofibrillary tangles (NFTs) composed of hyperphosphorylated Tau (pTau) protein in the brain [2]. Genetic and animal studies strongly indicate that Aβ, Tau and neuroinflammation play an important role in the pathogenesis of AD [2]. Therefore, drugs that target such processes and pathways are actively being pursued [3, 4]. Currently, there is no treatment that significantly slows the AD progression. However, recently two monoclonal antibody-based treatments, Aducanumab (Aduhelm®) and Lecanemab (Leqembi®) have been approved by the FDA in 2021 and 2023 respectively, for the treatment of early AD. Aducanumab targets Aβ aggregates and plaques in the brains of people with early AD and reduce its build up, while Lecanemab targets protofibrils to prevent the Aβ depositions[5] [6]. However, in January 2024, Aducanumab (Aduhelm®) was abandoned by its manufacturer (Biogen), for financial reasons [7]. There is no Tau targeting therapies available in the market. Therefore, there is an urgent need for disease modifying therapies that could either delay the AD progression or cure the disease. Tau, encoded by the microtubule-associated protein tau gene (MAPT), is typically localized to the axons where it binds and stabilizes microtubules. Aberrant Tau phosphorylation leads its disassembly from microtubules to form aggregates and -1- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) redistribution to cell bodies and dendrites. Clinically, NFT pathology better correlates with dementia than amyloid plaques in AD [8, 9]. Development of Tau-based therapies have mainly been focused on small molecules that include Tau kinase inhibitors, microtubule stabilizers, HSP90 inhibitors, O-GlcNAcase inhibitors, fibrillization blockers, and immunotherapeutic ranging from active vaccines to antibodies [3, 4, 10-13]. PROTACs (PROteolysis TArgeting Chimeras) have also been developed for tau degradation [14, 15]. However, crossing of the blood brain barrier (BBB) of such molecules is a formidable challenge for AD treatment. There remains a need developing small molecule-based pharmaceuticals that cross the BBB and promote the clearance of Tau aggregates and NFTs, and α-synuclein in AD, tauopathies, and PD SUMMARY In an aspect, this disclosure provides a compound according to Formula (I): or a pharmaceutical salt thereof, wherein: R1, R2, R3, R4, R5, R6, R7, and R8are each independently selected from H or D; and x is an integer from 1 to 7. In another aspect, this disclosure provides a compound according to Formula (II): -2- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) or a pharmaceutical salt thereof, wherein: R9, R10, R11, R12, R13, R14, R15, R16, R17and R18are each independently selected from H or D; and y is an integer from 1 to 7. In yet another aspect, this disclosure provides a compound selected from: -3- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) thereof. In another aspect, this disclosure provides a composition comprising a compound of any embodiment disclosed herein, and a pharmaceutically acceptable carrier. In yet another aspect, provided is a pharmaceutical composition for treating a disease or disorder, wherein the pharmaceutical composition comprises a therapeutically -4- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) effective amount of a compound described herein, and a pharmaceutically acceptable carrier. In another aspect, this disclosure is directed to a pharmaceutical composition for activating TFEB, wherein the pharmaceutical composition comprises a therapeutically effective amount of a compound described herein, and a pharmaceutically acceptable carrier In another aspect, provided is a method of treating a disease or disorder in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a compound described herein or a pharmaceutical composition described herein In yet another aspect, provided is a method of activating TFEB in a patient, the method comprising administering to the patient a therapeutically effective amount of a compound described herein or a pharmaceutical composition described herein. In another aspect, this disclosure is directed to the use of a therapeutically effective amount of a compound described herein for treating a disease or disorder in a patient in need thereof. In any embodiment herein, the disease or disorder is a neurological disease or disorder (e.g., Alzheimer’s disease, Parkinson’s disease, or other protein aggregation diseases), a lysosomal storage disease (e.g., Gaucher disease, Fabry disease, Niemann-Pick disease, or the mucopolysaccharidoses), or a Transcription Factor EB (TFEB)-mediated disease (e.g., kidney diseases, cancer, metabolic disorders, inflammation, or liver diseases). In yet another aspect, this disclosure is directed to the use of a therapeutically effective amount of a compound described herein for activating TFEB. BRIEF DESCRIPTION OF THE DRAWINGS FIG.1 is a schematic of mTORC1 dependent and independent autophagic pathways. Numbers 1-5 indicate multiple steps of autophagy starting from 1) TFEB translocation; 2) Autophagosome formation and maturation; 3) Autophagosome fusion with lysosome; 4) -5- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Degradation of contents within the autolysosome; 5) example showing the possible clearance of toxic Tau aggregates in the neurons. FIGs.2A-C show the development and characterization of the mCherry-TFEB reporter assay. FIG.2A shows an image of U2OS mCherry-TFEB cells after 5h treatment, DAPI (top), mCherry (bottom). Both Torin1 and chloroquine induce the translocation of TFEB. Time and concentration dependence of TFEB translocation with Torin1 is shown in FIG.2B and chloroquine in FIG.2C. FIG.3 shows the primary mCherry-TFEB translocation assay to find hits from HTS. FIGs.4A-4C show a schematic of the tandem-tagged LC3 reporter assay for monitoring autophagic flux using mTagRFP-mWasabi-LC3’ expression vector. A confocal image of merged cells before compound treatment is shown in FIG.4A; a confocal image of cells upon induction of autophagy is shown in FIG.4B; and a confocal image of the formation of active lysosome quenching the green (mWasabi) fluorescence proteins is shown in FIG.4C (assay results are not shown here). FIG.5 is a western blot (bottom, left) showing LC3-I to LC3-II conversion assay upon compound treatment of M1 for 5 hrs in U2OS cells. Quantification (bottom, middle) of LC3-II / Total LC3 indicates that M1 activates autophagy at a 10 μM concentration. LC3 reporter assay of M1 at 5 μM concentration showing the formation of both yellow and red puncta indicative of the initiation of autophagy. FIGs.6A-6D show that M65 activates TFEB. FIG.6A shows immunoblot analysis of TFEB in U2OS treated for 5 hours with DMSO, Torin (1 μM), or M65; FIG.6B is a representative image of U2OS mCherry-TFEB treated with DMSO or M65 (2.5 and 1.25 μM) for 5 hours; FIG.6C shows the quantification of Nuclear to Cytoplasmic ratio of mCherry-TFEB at 5 hours of treatment with M65. n=3; FIG.6D shows the relative gene expression of TFEB target genes in U2OS cells at 24 hours treatment of M65 (2.5 μM). n= 3. Data was analyzed by two-way ANOVA with multiple comparisons (vs. DMSO) *p < 0.05, **p < 0.01, and ***p < 0.001. -6- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) FIGs.7A-7C show that M65 induces autophagosome and autolysosome formation. FIG.7A is an immunoblot analysis of autophagosome marker LC3 in U2OS treated for 5 hours with DMSO or M65, and the quantification of LC3-II ratio. Data was analyzed by one-way ANOVA with multiple comparisons (vs. DMSO) *p < 0.05, **p < 0.01, and ***p < 0.001. FIG.7B shows representative images of U2OS mTagFRP-Wasabi-LC3 after 24 hour treatment of DMSO or M65. FIG.7C shows the quantification of Autolysosomes (red only LC3 pucnta) per cell in U2OS mTagFRP-Wasabi-LC3 after 24 hour treatment of DMSO or M65. Data was analyzed by one-way ANOVA with multiple comparisons (vs. DMSO) *p < 0.05, **p < 0.01, and ***p < 0.001. FIGs.8A-8B show that M65 increases lysosomal protease activity, where FIG.8A provides representative images of DQ-BSA (50 ug / mL) in U2OS cells 6 hours after treatment of DMSO or M65, and FIG.8B illustrates quantification of DQ-BSA fluorescence intensity for DMSO, Torin (1 μM), Bafilomycin A1 (100 nM). Data analyzed by one-way ANOVA with multiple comparisons (vs. DMSO) *p < 0.05, **p < 0.01, and ***p < 0.001. FIG.9 shows that m65 increases degradation of autophagic cargo. Immunoblot analysis of GFP in U2OS GFP-LC3 after 4 hours of treatment with either no treatment or cotreatment or final 1 hour of bafilomycin (100 nM) treatment. FIG.10 shows that M65 Degrades Tau Aggregates. Tau aggregate level in Ruby- Clover-TauP301S reporter cell line with 24 hours M65 (4 and 2 μM) treatment at the time of aggregate seeding. Data analyzed by one way ANOVA with multiple comparisons**** p<0.0001 FIGs.11A-11C demonstrate that m65 activates autophagy in neurons. FIG.11A shows immunoblot analysis of autophagosome marker LC3 in DIV14 neurons with 5 hours of M65 treatment (top) and the quantification of LC3-II (bottom). FIG.11B shows immunoblot analysis of phosphorylated tau (AT8) and total tau (HT7) in DIV14 neurons with 5 hours of M65 treatment. FIG.11 C shows the relative expression of TFEB target genes in DIV14 neurons following 24 hours of M65 treatment. Data was analyzed by two- way ANOVA with multiple comparisons (vs. DMSO) *p < 0.05, **p < 0.01, and ***p < 0.001. -7- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) FIG.12 shows the assessment of Metabolic Stability in Mouse Hepatocytes for M6, M65 and M100, along with reference compounds Testosterone (very low metabolic stability), Imipramine and 7-Hydroxycoumarine (low metabolic stability). FIGs.13A-13E demonstrate that M65 crosses the BBB in vivo (B6 female mice, at 10 mg / kg dose) (p.o; plasma, FIG.13A, and brain, FIG.13B); (i.v plasma, FIG.13C, and brain, FIG.13D) with saline as vehicle. PK parameters for i.v injection shown in FIG.13E. FIGs.14A-14B shows the M65 pharmacokinetic profile. FIG.14A shows M65 detection in plasma of black 6 mice following M65 (30 mg / kg) i.v or i.p. administration. FIG.14B shows M65 detection in brain of black 6 mice following M65 (30 mg / kg) i.v or i.p. administration. FIG.15 shows histamine H3 antagonists do not activate autophagy. Immunoblot analysis of LC3 after 5 hours of DMSO, M65 (2.5 μM), and H3 antagonists Thioperamide and Clobenpropit treatment. Quantification of LC3-II to total LC3 ratio. FIG.16 shows that M65 does not inhibit mTOR Immunoblot analysis of mTOR substrates S6-Kinase and 4E-BP1 from U2OS cells treated with DMSO, Torin (125 nM), M65 (1.25 μM) for 30 mins. Quantification of the ratio of phosphorylated proteins to total proteins. Data was analyzed by two-way ANOVA with multiple comparisons (vs. DMSO) *p < 0.05, **p < 0.01, and ***p < 0.001. FIGs.17A-17D show the photoaffinity probe development. FIG.17A shows the structure of photoaffinity probe M92; FIG.17B shows representative images of U2OS mCherry-TFEB cells after 5 hours of treatment with M92 (5 uM) and DMSO; FIG.17C shows immunoblot analysis of LC3 after 5 hours of M92 treatment(right). Quantification of LC3-II to total LC3 ratio (left). Data analyzed by one-way ANOVA with multiple comparisons (vs. DMSO) *p < 0.05, **p < 0.01, and ***p < 0.001; FIG.17D Representative images of and quantification of DQ-BSA (50 ug / mL) fluorescence intensity over time. Each data point is representative of the mean of 3 wells per experiment. FIGs.18A-18D show M92 Labeling in intact cells. FIG.18A shows M92 (0.5 μM) labeling with AF647 in U2OS cells with or without M65 (5 μM) pretreatment; -8- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Immunofluorescent labeling of M92 (0.5μM) labeled U2OS looking at ER (FIG.18B), Mitochondria (FIG.18C), and Lysosomal (FIG.18D) localization. FIGs.19A-19B show the proteomic analysis of M92. FIG.19A shows TAMRA in- gel fluorescence of M92 (1 μM) labeled proteins from intact U2OS cells with or without parental M65 (10 μM) blocking. FIG.19B shows significantly enriched proteins from M92 labeled U2OS membranes. FIG.20 shows the proposed pathway of SCARB2, GBA, and PSAP’S in glucosylceramide metabolism. FIGs.21A-21B demonstrate that M92 labels SCARB2, GBA, and PSAP. FIG.21A is an immunoblot analysis of SCARB2, GBA AND psapin M92 (0.5 μM) labeled U2OS membranes with or without M65 (5 μM). FIG.21B shows immunofluorescent labeling of SCARB2 and M92 in intact U2OS cells. FIGs.22A-22C show the effect of M65 on targets. FIG.22A shows relative gene expression of SCARB2, GBA, and PSAP in U2OS Cells following 24 hour M65 treatment as assessed by qPCR. Data analyzed by two-way ANOVA with multiple comparisons (vs. DMSO) *p < 0.05, **p < 0.01, and ***p < 0.001 B / C) GBA activity assay for U2OS Cells treated with DMSO or M65 for 5 hours (FIG.22B) or mouse brains (FIG.22C) following 24 hour treatment of M65 at 30mg / kg via IP or IV administration Data analyzed by one-way ANOVA with multiple comparisons (vs. DMSO) *p < 0.05, **p < 0.01, and ***p < 0.001. FIGs.23A-23B shows that GBA inhibition does not activate autophagy. FIG.23A is an immunoblot analysis of autophagosome marker LC3 and p62 in U2OS cells treated for 5 hours with DMSO or GBA inhibitor CBE (50 μM). FIG.23B shows the quantification of DQ-BSA fluorescence intensity over time after 12 hours of DMSO or CBE (50 μM) treatment. FIGs.24A-24C show glucosylceramide synthase inhibition activates autophagy. FIG.24A shows the results of U2OS mCherry-TFEB after 5 hours of DMSO or glucosylceramide synthase inhibitor Eliglustat (100 or 50 μM). Quantification of TFEB nuclear to cytoplasmic ratio. FIG.24B is an immunoblot analysis of autophagosome marker -9- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) LC3 and p62 in U2OS cells treated for 5 hours with DMSO or Eliglustat (100 or 50 μM). FIG.24C shows representative images of DQ-BSA (50ug / mL) after 12 hours of DMSO or Eliglustat (50 μM) treatment. Quantification of DQ-BSA florescence intensity over time. Data analyzed by one-way ANOVA with multiple comparisons (vs. DMSO) *p < 0.05, **p < 0.01, and ***p < 0.001. FIGs.25A-25B demonstrate that in U2OS cells, compounds M65 and M146 were able to significantly upregulate the expression of TFEB, the master regulator of autophagy and lysosomal biogenesis, target genes, including lysosomal membrane proteins (LAMP1 MCOLN1, ATP6V1H) and proteases (CTSD, GLA), and genes involved in autophagosome formation (ATG7 / 14, UVRAG, VPS18, WRD45, LC3) and the recognition of autophagic cargo (NBR1, SQSTM1) (FIG.25A). M65 was able to significantly upregulate the expression of TFEB target genes in the brain tissue of Black-6 Mice following IP treatment at 30 mg / kg (FIG.25B). FIGs.26A-26B demonstrate that M146 and M151 cross the BBB in vivo with Tmax at 4 hr. The calculated brain concentrations at Tmax are 3.94 µM (M146, FIG.26A, left) and 4.12 µM (M151, FIG.26A, right). Plasma analyses indicate the half-life (t1 / 2) of 17.17 h (M146, FIG.26B, left) and 18.68 h (M151, FIG.26B, right). DETAILED DESCRIPTION Before the present disclosure is further described, it is to be understood that the disclosure is not limited to the particular embodiments set forth herein, and it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Definitions The following terms are used throughout as defined below. As used herein and in the appended claims, singular articles such as “a” and “an” and “the” and similar referents in the context of describing the elements (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of -10- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the embodiments and does not pose a limitation on the scope of the claims unless otherwise stated. No language in the specification should be construed as indicating any non-claimed element as essential. As used herein, “about” will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art, given the context in which it is used, “about” will mean up to plus or minus 10% of the particular term – for example, “about 10 wt.%” would be understood to mean “9 wt.% to 11 wt.%.” It is to be understood that when “about” precedes a term, the term is to be construed as disclosing “about” the term as well as the term without modification by “about” – for example, “about 10 wt.%” discloses “9 wt.% to 11 wt.%” as well as disclosing “10 wt.%.” The phrase “and / or” as used in the present disclosure will be understood to mean any one of the recited members individually or a combination of any two or more thereof – for example, “A, B, and / or C” would mean “A, B, C, A and B, A and C, B and C, or the combination of A, B, and C.” Generally, reference to a certain element such as hydrogen or H is meant to include all isotopes of that element. For example, if an R group is defined to include hydrogen or H, it also includes deuterium and tritium. Compounds comprising radioisotopes such as tritium, C14, P32and S35are thus within the scope of the present technology. Procedures for inserting such labels into the compounds of the present technology will be readily apparent to those skilled in the art based on the disclosure herein. In general, “substituted” refers to an organic group as defined below (e.g., an alkyl group) in which one or more bonds to a hydrogen atom contained therein are replaced by a bond to non-hydrogen or non-carbon atoms. Substituted groups also include groups in -11- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) which one or more bonds to a carbon(s) or hydrogen(s) atom are replaced by one or more bonds, including double or triple bonds, to a heteroatom. Thus, a substituted group is substituted with one or more substituents, unless otherwise specified. In some embodiments, a substituted group is substituted with 1, 2, 3, 4, 5, or 6 substituents. Examples of substituent groups include: halogens (i.e., F, Cl, Br, and I); hydroxyls; alkoxy, alkenoxy, aryloxy, aralkyloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, and heterocyclylalkoxy groups; carbonyls (oxo); carboxylates; esters; urethanes; oximes; hydroxylamines; alkoxyamines; aralkoxyamines; thiols; sulfides; sulfoxides; sulfones; sulfonyls; pentafluorosulfanyl (i.e., SF5), sulfonamides; amines; N-oxides; hydrazines; hydrazides; hydrazones; azides; amides; ureas; amidines; guanidines; enamines; imides; isocyanates; isothiocyanates; cyanates; thiocyanates; imines; nitro groups; and nitriles (i.e., CN). Substituted ring groups such as substituted cycloalkyl, aryl, heterocyclyl and heteroaryl groups also include rings and ring systems in which a bond to a hydrogen atom is replaced with a bond to a carbon atom. Therefore, substituted cycloalkyl, aryl, heterocyclyl and heteroaryl groups may also be substituted with substituted or unsubstituted alkyl, alkenyl, and alkynyl groups as defined below. Alkyl groups include straight chain and branched chain alkyl groups having from 1 to 12 carbon atoms, and typically from 1 to 10 carbons or, in some embodiments, from 1 to 8, 1 to 6, or 1 to 4 carbon atoms. Alkyl groups may be substituted or unsubstituted. Examples of straight chain alkyl groups include groups such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, tert-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups. Representative substituted alkyl groups may be substituted one or more times with substituents such as those listed above, and include without limitation haloalkyl (e.g., trifluoromethyl), hydroxyalkyl, thioalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, alkoxyalkyl, carboxyalkyl, and the like. Cycloalkyl groups include mono-, bi- or tricyclic alkyl groups having from 3 to 12 carbon atoms in the ring(s), or, in some embodiments, 3 to 10, 3 to 8, or 3 to 4, 5, or 6 carbon atoms. Cycloalkyl groups may be substituted or unsubstituted. Exemplary -12- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) monocyclic cycloalkyl groups include, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, the cycloalkyl group has 3 to 8 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 5, 3 to 6, or 3 to 7. Bi- and tricyclic ring systems include both bridged cycloalkyl groups and fused rings, such as, but not limited to, bicyclo[2.1.1]hexane, adamantyl, decalinyl, and the like. Substituted cycloalkyl groups may be substituted one or more times with non-hydrogen and non-carbon groups as defined above. However, substituted cycloalkyl groups also include rings that are substituted with straight or branched chain alkyl groups as defined above. Representative substituted cycloalkyl groups may be mono-substituted or substituted more than once, such as, but not limited to, 2,2-, 2,3-, 2,4- 2,5- or 2,6-disubstituted cyclohexyl groups, which may be substituted with substituents such as those listed above. Cycloalkylalkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a cycloalkyl group as defined above. Cycloalkylalkyl groups may be substituted or unsubstituted. In some embodiments, cycloalkylalkyl groups have from 4 to 16 carbon atoms, 4 to 12 carbon atoms, and typically 4 to 10 carbon atoms. Substituted cycloalkylalkyl groups may be substituted at the alkyl, the cycloalkyl or both the alkyl and cycloalkyl portions of the group. Representative substituted cycloalkylalkyl groups may be mono-substituted or substituted more than once, such as, but not limited to, mono-, di- or tri-substituted with substituents such as those listed above. Alkenyl groups include straight and branched chain alkyl groups as defined above, except that at least one double bond exists between two carbon atoms. Alkenyl groups may be substituted or unsubstituted. Alkenyl groups have from 2 to 12 carbon atoms, and typically from 2 to 10 carbons or, in some embodiments, from 2 to 8, 2 to 6, or 2 to 4 carbon atoms. In some embodiments, the alkenyl group has one, two, or three carbon- carbon double bonds. Examples include, but are not limited to vinyl, allyl, -CH=CH(CH3), -CH=C(CH3)2, -C(CH3)=CH2, -C(CH3)=CH(CH3), -C(CH2CH3)=CH2, among others. Representative substituted alkenyl groups may be mono-substituted or substituted more than once, such as, but not limited to, mono-, di- or tri-substituted with substituents such as those listed above. -13- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Cycloalkenyl groups include cycloalkyl groups as defined above, having at least one double bond between two carbon atoms. Cycloalkenyl groups may be substituted or unsubstituted. In some embodiments the cycloalkenyl group may have one, two or three double bonds but does not include aromatic compounds. Cycloalkenyl groups have from 4 to 14 carbon atoms, or, in some embodiments, 5 to 14 carbon atoms, 5 to 10 carbon atoms, or even 5, 6, 7, or 8 carbon atoms. Examples of cycloalkenyl groups include cyclohexenyl, cyclopentenyl, cyclohexadienyl, cyclobutadienyl, and cyclopentadienyl. Cycloalkenylalkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of the alkyl group is replaced with a bond to a cycloalkenyl group as defined above. Cycloalkenylalkyl groups may be substituted or unsubstituted. Substituted cycloalkenylalkyl groups may be substituted at the alkyl, the cycloalkenyl or both the alkyl and cycloalkenyl portions of the group. Representative substituted cycloalkenylalkyl groups may be substituted one or more times with substituents such as those listed above. Alkynyl groups include straight and branched chain alkyl groups as defined above, except that at least one triple bond exists between two carbon atoms. Alkynyl groups may be substituted or unsubstituted. Alkynyl groups have from 2 to 12 carbon atoms, and typically from 2 to 10 carbons or, in some embodiments, from 2 to 8, 2 to 6, or 2 to 4 carbon atoms. In some embodiments, the alkynyl group has one, two, or three carbon- carbon triple bonds. Examples include, but are not limited to – C≡CH, -C≡CCH3, -CH2C≡CCH3, and -C≡CCH2CH(CH2CH3)2, among others. Representative substituted alkynyl groups may be mono-substituted or substituted more than once, such as, but not limited to, mono-, di- or tri-substituted with substituents such as those listed above. Aryl groups are cyclic aromatic hydrocarbons that do not contain heteroatoms. Aryl groups herein include monocyclic, bicyclic and tricyclic ring systems. Aryl groups may be substituted or unsubstituted. Thus, aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, fluorenyl, phenanthrenyl, anthracenyl, indenyl, indanyl, pentalenyl, and naphthyl groups. In some embodiments, aryl groups contain 6-14 carbons, and in others from 6 to 12 or even 6-10 carbon atoms in the ring portions of the groups. In some embodiments, the aryl groups are phenyl or naphthyl. The phrase “aryl groups” -14- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) includes groups containing fused rings, such as fused aromatic-aliphatic ring systems (e.g., indanyl, tetrahydronaphthyl, and the like). Representative substituted aryl groups may be mono-substituted (e.g., tolyl) or substituted more than once. For example, monosubstituted aryl groups include, but are not limited to, 2-, 3-, 4-, 5-, or 6-substituted phenyl or naphthyl groups, which may be substituted with substituents such as those listed above. Aralkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined above. Aralkyl groups may be substituted or unsubstituted. In some embodiments, aralkyl groups contain 7 to 16 carbon atoms, 7 to 14 carbon atoms, or 7 to 10 carbon atoms. Substituted aralkyl groups may be substituted at the alkyl, the aryl or both the alkyl and aryl portions of the group. Representative aralkyl groups include but are not limited to benzyl and phenethyl groups and fused (cycloalkylaryl)alkyl groups such as 4-indanylethyl. Representative substituted aralkyl groups may be substituted one or more times with substituents such as those listed above. Heterocyclyl groups include aromatic (also referred to as heteroaryl) and non- aromatic ring compounds containing 3 or more ring members, of which one or more is a heteroatom such as, but not limited to, N, O, and S. Heterocyclyl groups may be substituted or unsubstituted. In some embodiments, the heterocyclyl group contains 1, 2, 3 or 4 heteroatoms. In some embodiments, heterocyclyl groups include mono-, bi- and tricyclic rings having 3 to 16 ring members, whereas other such groups have 3 to 6, 3 to 10, 3 to 12, or 3 to 14 ring members. Heterocyclyl groups encompass aromatic, partially unsaturated and saturated ring systems, such as, for example, imidazolyl, imidazolinyl and imidazolidinyl groups. The phrase “heterocyclyl group” includes fused ring species including those comprising fused aromatic and non-aromatic groups, such as, for example, benzotriazolyl, 2,3-dihydrobenzo[1,4]dioxinyl, and benzo[1,3]dioxolyl. The phrase also includes bridged polycyclic ring systems containing a heteroatom such as, but not limited to, quinuclidyl. The phrase includes heterocyclyl groups that have other groups, such as alkyl, oxo or halo groups, bonded to one of the ring members, referred to as “substituted heterocyclyl groups”. Heterocyclyl groups include, but are not limited to, aziridinyl, azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, thiazolidinyl, tetrahydrothiophenyl, tetrahydrofuranyl, dioxolyl, furanyl, thiophenyl, pyrrolyl, pyrrolinyl, imidazolyl, -15- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) imidazolinyl, pyrazolyl, pyrazolinyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, thiazolinyl, isothiazolyl, thiadiazolyl, oxadiazolyl, piperidyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydropyranyl, tetrahydrothiopyranyl, oxathiane, dioxyl, dithianyl, pyranyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, dihydropyridyl, dihydrodithiinyl, dihydrodithionyl, homopiperazinyl, quinuclidyl, indolyl, indolinyl, isoindolyl,azaindolyl (pyrrolopyridyl), indazolyl, indolizinyl, benzotriazolyl, benzimidazolyl, benzofuranyl, benzothiophenyl, benzthiazolyl, benzoxadiazolyl, benzoxazinyl, benzodithiinyl, benzoxathiinyl, benzothiazinyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[1,3]dioxolyl, pyrazolopyridyl, imidazopyridyl (azabenzimidazolyl), triazolopyridyl, isoxazolopyridyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, quinolizinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, naphthyridinyl, pteridinyl, thianaphthyl, dihydrobenzothiazinyl, dihydrobenzofuranyl, dihydroindolyl, dihydrobenzodioxinyl, tetrahydroindolyl, tetrahydroindazolyl, tetrahydrobenzimidazolyl, tetrahydrobenzotriazolyl, tetrahydropyrrolopyridyl, tetrahydropyrazolopyridyl, tetrahydroimidazopyridyl, tetrahydrotriazolopyridyl, and tetrahydroquinolinyl groups. Representative substituted heterocyclyl groups may be mono-substituted or substituted more than once, such as, but not limited to, pyridyl or morpholinyl groups, which are 2-, 3-, 4-, 5-, or 6-substituted, or disubstituted with various substituents such as those listed above. Heteroaryl groups are aromatic ring compounds containing 5 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N, O, and S. Heteroaryl groups may be substituted or unsubstituted. Heteroaryl groups include, but are not limited to, groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiophenyl, benzothiophenyl, furanyl, benzofuranyl, indolyl, azaindolyl (pyrrolopyridinyl), indazolyl, benzimidazolyl, imidazopyridinyl (azabenzimidazolyl), pyrazolopyridinyl, triazolopyridinyl, benzotriazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Heteroaryl groups include fused ring compounds in which all rings are aromatic such as indolyl groups and include fused ring compounds in which only one of the rings is aromatic, such as 2,3-dihydro -16- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) indolyl groups. Representative substituted heteroaryl groups may be substituted one or more times with various substituents such as those listed above. Heterocyclylalkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a heterocyclyl group as defined above. Heterocyclylalkyl groups may be substituted or unsubstituted. Substituted heterocyclylalkyl groups may be substituted at the alkyl, the heterocyclyl or both the alkyl and heterocyclyl portions of the group. Representative heterocyclyl alkyl groups include, but are not limited to, morpholin-4-yl-ethyl, furan-2-yl-methyl, imidazol-4-yl-methyl, pyridin-3-yl-methyl, tetrahydrofuran-2-yl-ethyl, and indol-2-yl-propyl. Representative substituted heterocyclylalkyl groups may be substituted one or more times with substituents such as those listed above. Heteroaralkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a heteroaryl group as defined above. Heteroaralkyl groups may be substituted or unsubstituted. Substituted heteroaralkyl groups may be substituted at the alkyl, the heteroaryl or both the alkyl and heteroaryl portions of the group. Representative substituted heteroaralkyl groups may be substituted one or more times with substituents such as those listed above. Groups described herein having two or more points of attachment (i.e., divalent, trivalent, or polyvalent) within the compound of the present technology are designated by use of the suffix, “ene.” For example, divalent alkyl groups are alkylene groups, divalent aryl groups are arylene groups, divalent heteroaryl groups are divalent heteroarylene groups, and so forth. Substituted groups having a single point of attachment to the compound of the present technology are not referred to using the “ene” designation. Thus, e.g., chloroethyl is not referred to herein as chloroethylene. Alkoxy groups are hydroxyl groups (-OH) in which the bond to the hydrogen atom is replaced by a bond to a carbon atom of a substituted or unsubstituted alkyl group as defined above. Alkoxy groups may be substituted or unsubstituted. Examples of linear alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy, and the like. Examples of branched alkoxy groups include but are not limited to isopropoxy, sec-butoxy, tert-butoxy, isopentoxy, isohexoxy, and the like. Examples of -17- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) cycloalkoxy groups include but are not limited to cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. Representative substituted alkoxy groups may be substituted one or more times with substituents such as those listed above. The terms “alkanoyl” and “alkanoyloxy” as used herein can refer, respectively, to – C(O)–alkyl groups and –O–C(O)–alkyl groups, each containing 2–5 carbon atoms. Similarly, “aryloyl” and “aryloyloxy” refer to –C(O)–aryl groups and –O–C(O)–aryl groups. The terms "aryloxy" and “arylalkoxy” refer to, respectively, a substituted or unsubstituted aryl group bonded to an oxygen atom and a substituted or unsubstituted aralkyl group bonded to the oxygen atom at the alkyl. Examples include but are not limited to phenoxy, naphthyloxy, and benzyloxy. Representative substituted aryloxy and arylalkoxy groups may be substituted one or more times with substituents such as those listed above. The term “carboxylate” as used herein refers to a -COOH group. The term “ester” as used herein refers to –COOR70and –C(O)O-G groups. R70is a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heterocyclylalkyl or heterocyclyl group as defined herein. G is a carboxylate protecting group. Carboxylate protecting groups are well known to one of ordinary skill in the art. An extensive list of protecting groups for the carboxylate group functionality may be found in Protective Groups in Organic Synthesis, Greene, T.W.; Wuts, P. G. M., John Wiley & Sons, New York, NY, (3rd Edition, 1999) which can be added or removed using the procedures set forth therein and which is hereby incorporated by reference in its entirety and for any and all purposes as if fully set forth herein. The term “amide” (or “amido”) includes C- and N-amide groups, i.e., -C(O)NR71R72, and –NR71C(O)R72groups, respectively. R71and R72are independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl or heterocyclyl group as defined herein. Amido groups therefore include but are not limited to carbamoyl groups (-C(O)NH2) and formamide groups (-NHC(O)H). In some embodiments, the amide is –NR71C(O)-(C1-5alkyl) and the group is termed -18- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) "carbonylamino," and in others the amide is –NHC(O)-alkyl and the group is termed "alkanoylamino." The term “nitrile” or “cyano” as used herein refers to the –CN group. Urethane groups include N- and O-urethane groups, i.e., -NR73C(O)OR74and -OC(O)NR73R74groups, respectively. R73and R74are independently a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl, or heterocyclyl group as defined herein. R73may also be H. The term “amine” (or “amino”) as used herein refers to –NR75R76groups, wherein R75and R76are independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl or heterocyclyl group as defined herein. In some embodiments, the amine is alkylamino, dialkylamino, arylamino, or alkylarylamino. In other embodiments, the amine is NH2, methylamino, dimethylamino, ethylamino, diethylamino, propylamino, isopropylamino, phenylamino, or benzylamino. The term “sulfonamido” includes S- and N-sulfonamide groups, i.e., -SO2NR78R79and –NR78SO2R79groups, respectively. R78and R79are independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl, or heterocyclyl group as defined herein. Sulfonamido groups therefore include but are not limited to sulfamoyl groups (-SO2NH2). In some embodiments herein, the sulfonamido is –NHSO2-alkyl and is referred to as the "alkylsulfonylamino" group. The term “thiol” refers to –SH groups, while “sulfides” include –SR80groups, “sulfoxides” include –S(O)R81groups, “sulfones” include -SO2R82groups, and “sulfonyls” include –SO2OR83. R80, R81, R82, and R83are each independently a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein. In some embodiments the sulfide is an alkylthio group, -S-alkyl. The term “urea” refers to –NR84-C(O)-NR85R86groups. R84, R85, and R86groups are independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclyl, or heterocyclylalkyl group as defined herein. -19- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) The term “amidine” refers to –C(NR87)NR88R89and –NR87C(NR88)R89, wherein R87, R88, and R89are each independently hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein. The term “guanidine” refers to –NR90C(NR91)NR92R93, wherein R90, R91, R92and R93are each independently hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein. The term “enamine” refers to –C(R94)=C(R95)NR96R97and –NR94C(R95)=C(R96)R97, wherein R94, R95, R96and R97are each independently hydrogen, a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein. The term “halogen” or “halo” as used herein refers to bromine, chlorine, fluorine, or iodine. In some embodiments, the halogen is fluorine. In other embodiments, the halogen is chlorine or bromine. The term “hydroxyl” as used herein can refer to –OH or its ionized form, –O–. A “hydroxyalkyl” group is a hydroxyl-substituted alkyl group, such as HO-CH2-. The term “imide” refers to –C(O)NR98C(O)R99, wherein R98and R99are each independently hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein. The term “imine” refers to –CR100(NR101) and –N(CR100R101) groups, wherein R100and R101are each independently hydrogen or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein, with the proviso that R100and R101are not both simultaneously hydrogen. The term “nitro” as used herein refers to an –NO2 group. The term “trifluoromethyl” as used herein refers to –CF3. The term “trifluoromethoxy” as used herein refers to –OCF3. -20- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) The term “azido” refers to –N3. The term “trialkyl ammonium” refers to a –N(alkyl)3 group. A trialkylammonium group is positively charged and thus typically has an associated anion, such as halogen anion. The term “isocyano” refers to –NC. The term “isothiocyano” refers to –NCS. The term “pentafluorosulfanyl” refers to –SF5. As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 atoms refers to groups having 1, 2, or 3 atoms. Similarly, a group having 1-5 atoms refers to groups having 1, 2, 3, 4, or 5 atoms, and so forth. As understood by one of ordinary skill in the art, “molecular weight” (also known as “relative molar mass”) is a dimensionless quantity but is converted to molar mass by multiplying by 1 gram / mole or by multiplying by 1 Da – for example, a compound with a weight-average molecular weight of 5,000 has a weight-average molar mass of 5,000 g / mol and a weight-average molar mass of 5,000 Da. Pharmaceutically acceptable salts of compounds described herein are within the scope of the present technology and include acid or base addition salts which retain the desired pharmacological activity and is not biologically undesirable (e.g., the salt is not -21- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) unduly toxic, allergenic, or irritating, and is bioavailable). When the compound of the present technology has a basic group, such as, for example, an amino group, pharmaceutically acceptable salts can be formed with inorganic acids (such as hydrochloric acid, hydroboric acid, nitric acid, sulfuric acid, and phosphoric acid), organic acids (e.g., alginate, formic acid, acetic acid, benzoic acid, gluconic acid, fumaric acid, oxalic acid, tartaric acid, lactic acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, naphthalene sulfonic acid, and p-toluenesulfonic acid) or acidic amino acids (such as aspartic acid and glutamic acid). When the compound of the present technology has an acidic group, such as for example, a carboxylic acid group, it can form salts with metals, such as alkali and earth alkali metals (e.g., Na+, Li+, K+, Ca2+, Mg2+, Zn2+), ammonia or organic amines (e.g., dicyclohexylamine, trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine) or basic amino acids (e.g., arginine, lysine and ornithine). Such salts can be prepared in situ during isolation and purification of the compounds or by separately reacting the purified compound in its free base or free acid form with a suitable acid or base, respectively, and isolating the salt thus formed. Those of skill in the art will appreciate that compounds of the present technology may exhibit the phenomena of tautomerism, conformational isomerism, geometric isomerism, and / or stereoisomerism. As the formula drawings within the specification and claims can represent only one of the possible tautomeric, conformational isomeric, stereochemical or geometric isomeric forms, it should be understood that the present technology encompasses any tautomeric, conformational isomeric, stereochemical and / or geometric isomeric forms of the compounds having one or more of the utilities described herein, as well as mixtures of these various different forms. “Tautomers” refers to isomeric forms of a compound that are in equilibrium with each other. The presence and concentrations of the isomeric forms will depend on the environment the compound is found in and may be different depending upon, for example, whether the compound is a solid or is in an organic or aqueous solution. For example, in aqueous solution, quinazolinones may exhibit the following isomeric forms, which are referred to as tautomers of each other: -22- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) . As another example, guanidines may exhibit the following isomeric forms in protic organic solution, also referred to as tautomers of each other: . Because of the limits of representing compounds by structural formulas, it is to be understood that all chemical formulas of the compounds described herein represent all tautomeric forms of compounds and are within the scope of the present technology. Stereoisomers of compounds (also known as optical isomers) include all chiral, diastereomeric, and racemic forms of a structure, unless the specific stereochemistry is expressly indicated. Thus, compounds used in the present technology include enriched or resolved optical isomers at any or all asymmetric atoms as are apparent from the depictions. Both racemic and diastereomeric mixtures, as well as the individual optical isomers can be isolated or synthesized so as to be substantially free of their enantiomeric or diastereomeric partners, and these stereoisomers are all within the scope of the present technology. The compounds of the present technology may exist as solvates, especially hydrates. Hydrates may form during manufacture of the compounds or compositions comprising the compounds, or hydrates may form over time due to the hygroscopic nature of the compounds. Compounds of the present technology may exist as organic solvates as well, including DMF, ether, and alcohol solvates among others. The identification and preparation of any particular solvate is within the skill of the ordinary artisan of synthetic organic or medicinal chemistry. The terms “patient” or “subject” are used interchangeably to refer to a human or a non-human animal (e.g., a mammal). -23- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) The terms “treat”, “treating”, treatment” and the like refer to a course of action that eliminates, reduces, suppresses, mitigates, ameliorates, or prevents the worsening of, either temporarily or permanently, a disease, disorder or condition to which the term applies, or at least one of the symptoms associated therewith. Treatment includes alleviation of symptoms, diminishment of extent of disease, inhibiting (e.g., arresting the development or further development of the disease, disorder or condition or clinical symptoms association therewith) an active disease, delaying or slowing of disease progression, improving the quality of life, and / or prolonging survival of a subject as compared to expected survival if not receiving treatment or as compared to a published standard of care therapy for a particular disease. Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. Also within this disclosure are Arabic numerals referring to referenced citations, the full bibliographic details of which are provided immediately preceding the claims. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure to more fully describe the present technology. The Present Technology Macroautophagy (herein referred to as autophagy) is a conserved process by which long-lived intracellular organelles and protein aggregates are engulfed and degraded through a series of events, including the initiation and elongation of precursor double- membraned autophagosomes, maturation of autophagosomes, fusion of autophagosomes with lysosomes, and degradation of engulfed materials by the lysosomes

[0016] (FIG.1). Autophagy disfunction has been implicated in multiple neurodegenerative diseases with abnormal protein aggregates [2, 16-21]. Several studies have showed that brains of AD patients have lower expression of Beclin-1, an upstream component of autophagy [22, 23], and that neurites from AD patients have an excess of autophagic vacuoles, presumably consequential of failed autophagosome clearance by lysosomes [24, 25]. Increasing evidence also suggests that clearance of aggregated Tau and NFTs is mediated by the Autophagy-Lysosome Pathway (ALP)

[0026] . -24- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Transcription Factor EB (TFEB) is considered as the master transcriptional regulator of lysosomal biogenesis and autophagy [27, 28]. Under normal physiological conditions, TFEB normally resides in the cytoplasm. However, under certain conditions, TFEB translocates into the nucleus and activates target genes that are required for lysosome biogenesis and autophagy (FIG.1). Phosphorylation / dephosphorylation has been found to play an important role in controlling the TFEB pathway [29-33]. For example: 1) Phosphorylation of TFEB at the serine 211 by the mammalian target of rapamycin complex 1 (mTORC1) retains TFEB in the cytoplasm [29, 30].2) RIP1, the serine-threonine kinase, activates ERK, that then phosphorylates TFEB at the serine 142, preventing TFEB translocation

[0033] .3) Furthermore, calcineurin dephosphorylates TFEB, activating TFEB signaling

[0032] .4) phosphorylation of TFEB on three serine residues located in its last 15 amino acids by PKCβ promotes the translocation of TFEB into the nucleus

[0031] . 5) PPARγ coactivator 1α (PGC-1α) can initiate TFEB activity

[0034] . Thus, various pathways can modulate TFEB transcriptional activity. In addition, mTORC1 inactivation has been demonstrated to be a general upstream event for TFEB activation

[0030] . However, mTORC1 is a crucial regulator of various cellular processes and a converging point for multiple signaling pathways. As such, inhibition of mTORC1 will impact a plethora of normal biological events such as protein synthesis, cellular metabolism and growth, and cell viability

[0035] . Notably, a mild upregulation of TFEB in the brain of mouse model using AAV-mediated expression effectively clears toxic Tau species without overt adverse effects

[0036] . In addition, the activation of TFEB has been shown to help in clearance of aggregated proteins, including α-synuclein, polyglutamine- expanded androgen receptor, huntingtin, Aβ and APP[34, 37-42]. Therefore, TFEB activation and subsequent promotion of the endolysosomal and autophagic network represents an appealing therapeutic strategy for AD treatment; and if this can be achieved without inhibiting mTORC1 general activity, various potential adverse side effects associated with mTORC1 inhibition can be avoided. Moreover, enhancing the clearance of toxic protein aggregates represents a promising strategy for treatment of AD and other neurodegenerative disorders. Upregulation of TFEB can specifically clear toxic protein aggregates, such as Tau. Therefore, in this -25- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) regard, described herein is a series of novel small molecules that promote ALP via TFEB activation in an mTORC1 independent pathway to promote autophagy flux thereby clearing the toxic Tau protein, one of the hallmarks of AD. Herein, provided are: a phenotypic screening method to find hits; a chemical library synthesis and structure-activity relationship (SAR) studies to generate the compounds as described herein; the validation of autophagic activity of the described compounds using several cell-based assays; in vitro ADME, in vivo pharmacokinetic (PK) and maximum tolerated dose (MTD) of the described compounds in B6 female mice; the design and synthesis of trifunctional photoaffinity probe for novel protein target identification via photoaffinity labelling (PAL), and mechanism of action of the described compounds on their target. Compounds The present disclosure generally relates to compounds that activate Transcription Factor EB (TFEB). In one aspect, the disclosure is directed to a compound according to Formula (I): or a pharmaceutical salt thereof, wherein: R1, R2, R3, R4, R5, R6, R7, and R8are each independently selected from H or D; and x is 1, 2, 3, 4, 5, 6, or 7. In any embodiment herein, it may be that R1, R2, R3, R4, R5, R6, R7, and R8are each H. In any embodiment herein, it may be that x is 1. In any embodiment herein, it may be that x is 3. In any embodiment herein, it may be that x is 5. In any embodiment herein, it may be that x is 7. In any embodiment herein, it may be that the compound is: -26- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) In an aspect, the disclosure is directed to a compound according to Formula (II): or a pharmaceutical salt thereof, wherein: R9, R10, R11, R12, R13, R14, R15, R16, R17and R18are each independently selected from H or D, and y is 1, 2, 3, 4, 5, 6, or 7. In any embodiment herein, it may be that R9, R10, R11, R12, R13, R14, R15, R16, R17and R18are each H. In any embodiment herein, it may be that R9, R10, R11, R12, R13, R14, R17and R18are each H, and R15and R16are each D. In any embodiment herein, it may be that R9, R10, R11, R12, R13, R14, R15, and R16are each H, and R17and R18are each D. In any embodiment herein, it may be that R9, R10, R11, R12, R13, R14, R15, and R16are each H, and R17and R18are each D. In any embodiment herein, it may be that y is 1. In any -27- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) embodiment herein, it may be that y is 3. In any embodiment herein, it may be that y is 5. In any embodiment herein, it may be that y is 7. In any embodiment herein, it may be that the compound is: thereof. In an aspect, the disclosure is directed to a compound selected from: -28- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) , -29- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) pharmaceutically acceptable salt thereof. Therapeutic Uses The present disclosure relates to methods of treating a disease or disorder in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a compound described herein or a pharmaceutical composition described herein. The present disclosure relates to the use of a therapeutically effective amount of a compound described herein or a pharmaceutical composition described herein for treating a disease or disorder in a patient in need thereof. In any embodiment herein, it may be that the disease or disorder is a neurological disease or disorder. Non-limiting examples of the neurological disease or disorder include Alzheimer’s disease, Parkinson’s disease, or other protein aggregation diseases (e.g., Huntington's disease and amyotrophic lateral sclerosis (ALS)). In any embodiment herein, it may be that the disease or disorder is a lysosomal storage disease. Non-limiting examples of the lysosomal storage disease include Gaucher disease, Fabry disease, Niemann-Pick disease, or the mucopolysaccharidoses (e.g., MPS I (Hurler syndrome), MPS II (Hunter syndrome), MPS III (Sanfilippo syndrome), MPS IV -30- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) (Morquio syndrome), MPS V (Scheie syndrome), MPS VI (Maroteaux-Lamy syndrome), or MPS VII (Sly syndrome)). In any embodiment herein, it may be that the disease or disorder is a Transcription Factor EB (TFEB)-mediated disease. Non-limiting examples of the TFEB-mediated disease include kidney diseases, cancer, metabolic disorders, inflammation, or liver diseases. The present disclosure relates to methods of activating TFEB in a patient, the method comprising administering to the patient a therapeutically effective amount of a compound described herein or a pharmaceutical composition described herein. The present disclosure relates to the use of a therapeutically effective amount of a compound described herein for activating TFEB. Routes of Administration In any embodiment herein, it may be that pharmaceutical compositions containing a compound according to this disclosure may be in a form suitable for oral administration. Oral administration may involve swallowing the formulation thereby allowing the compound to be absorbed into the bloodstream in the gastrointestinal tract. Alternatively, oral administration may involve buccal, lingual or sublingual administration, thereby allowing the compound to be absorbed into the blood stream through oral mucosa. In another embodiment, the pharmaceutical compositions containing a compound according to this disclosure may be in a form suitable for parenteral administration. Forms of parenteral administration include, but are not limited to, intravenous, intraarterial, intramuscular, intradermal, intraperitoneal, intrathecal, intracisternal, intracerebral, intracerebroventricular, intraventricular, and subcutaneous. Pharmaceutical compositions suitable for parenteral administration may be formulated using suitable aqueous or non- aqueous carriers. Depot injections, which are generally administered subcutaneously or intramuscularly, may also be utilized to release the compounds disclosed herein over a defined period of time. -31- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Other routes of administration are also contemplated by this disclosure, including, but not limited to, nasal, vaginal, intraocular, rectal, topical (e.g., transdermal), and inhalation. Pharmaceutical Compositions The compounds of the present disclosure may be in the form of compositions suitable for administration to a subject. In general, such compositions are pharmaceutical compositions comprising a compound according to this disclosure or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable excipients. In certain embodiments, the compound may be present in an effective amount. The pharmaceutical compositions may be used in the methods of the present disclosure; thus, for example, the pharmaceutical compositions comprising a compound according to this disclosure can be administered to a subject in order to practice the therapeutic methods and uses described herein. The pharmaceutical compositions of the present disclosure can be formulated to be compatible with the intended method or route of administration. Routes of administration may include those known in the art. Exemplary routes of administration are oral and parenteral. Furthermore, the pharmaceutical compositions may be used in combination with one or more other therapies described herein in order to treat or prevent the diseases, disorders and conditions as contemplated by the present disclosure. In one embodiment, one or more other therapeutic agents contemplated by this disclosure are included in the same pharmaceutical composition that comprises the compound according to this disclosure. In another embodiment, the one or more other therapeutical agents are in a composition that is separate from the pharmaceutical composition comprising the compound according to this disclosure. In one aspect, the compounds described herein may be administered orally. Oral administration may be via, for example, capsule or tablets. In making the pharmaceutical compositions that include the compounds of the present disclosure (e.g., a compound of Formula I), or a pharmaceutically acceptable salt thereof, the tablet or capsule includes at least one pharmaceutically acceptable excipient. Non-limiting examples of pharmaceutically acceptable excipients include lactose, dextrose, sucrose, sorbitol, -32- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, polyethylene glycol, cellulose, sterile water, syrup, and methyl cellulose. Additional pharmaceutically acceptable excipients include lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl and propylhydroxy-benzoates. In another aspect, the compounds of the present disclosure, or a pharmaceutically acceptable salt thereof, may be administered parenterally, for example by intravenous injection. A pharmaceutical composition appropriate for parenteral administration may be formulated in solution for injection or may be reconstituted for injection in an appropriate system such as a physiological solution. Such solutions may include sterile water for injection, salts, buffers, and tonicity excipients in amounts appropriate to achieve isotonicity with the appropriate physiology. The pharmaceutical compositions described herein may be stored in an appropriate sterile container or containers. In any embodiment herein, it may be that the container is designed to maintain stability for the pharmaceutical composition over a given period of time. Administering In general, the disclosed methods comprise administering a compound described herein, or a composition thereof, in an effective amount to a subject in need thereof. An “effective amount” with reference to a TFEB activator of the present disclosure means an amount of the compound that is sufficient to engage the target (e.g., by activating the target) at a level that is indicative of the potency of the compound. For TFEB, target engagement can be determined by one or more biochemical or cellular assays resulting in an EC50, ED50, EC90, IC50, or similar value which can be used as one assessment of the potency of the compound. Assays for determining target engagement include, but are not limited to, those described in the Examples. The effective amount may be administered as a single quantity or as multiple, smaller quantities (e.g., as one tablet with “x” amount, as two tablets each with “x / 2” amount, etc.). -33- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) In any embodiment herein, it may be that the disclosed methods include administering a therapeutically effective amount of a compound described herein to a subject in need thereof. As used herein, the phrase “therapeutically effective amount” with reference to compound disclosed herein means a dose regimen (i.e., amount and interval) of the compound that provides the specific pharmacological effect for which the compound is administered to a subject in need of such treatment. For prophylactic use, a therapeutically effective amount may be effective to eliminate or reduce the risk, lessen the severity, or delay the onset of the disease, including biochemical, histological and / or behavioral signs or symptoms of the disease. For treatment, a therapeutically effective amount may be effective to reduce, ameliorate, or eliminate one or more signs or symptoms associated with a disease, delay disease progression, prolong survival, decrease the dose of other medication(s) required to treat the disease, or a combination thereof. With respect to cancer specifically, a therapeutically effective amount may, for example, result in the killing of cancer cells, reduce cancer cell counts, reduce tumor burden, eliminate tumors or metastasis, or reduce metastatic spread. A therapeutically effective amount may vary based on, for example, one or more of the following: the age and weight of the subject, the subject’s overall health, the stage of the subject’s disease, the route of administration, and prior or concomitant treatments. Administration may include one or more (e.g., one, two, or three or more) dosing cycles. EXAMPLES High content assay (HCA) for TEFB translocation TFEB was chosen as the target for the discovery and development of novel small molecule autophagy modulators. TFEB is a master regulator of lysosomal biogenesis and autophagy, coordinating the expression of lysosomal genes involved in multiple steps of autophagy

[0019] (FIG.1). Before initiating the high throughput screen (HTS), a TFEB- translocation high content assay (HCA) was developed by constructing TFEB and mCherry fusion proteins with mCherry on the N-terminal or C-terminal end of TFEB, named mCherry-TFEB or TFEB-mCherry, respectively. The TFEB-fused reporter assay allows for monitoring of TFEB translocation under different conditions. Both cell lines were -34- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) characterized under treatments of starvation, Torin1 or chloroquine, all of which have been reported to activate TFEB signaling. In these cell lines, TFEB resides in the cytoplasm of the cells under normal conditions and translocates to the nucleus upon starvation or treatment with the mTORC1 inhibitor Torin1 or the lysosomotropic agent chloroquine, validating that the TFEB reporters can detect the activation of TFEB. Therefore, this mCherry-TFEB high content assay can be used to identify agents for potentiating TFEB signaling. Treatment with Torin1 (500 nM) or chloroquine (100 μM) 5h induced strong TFEB translocation to the nucleus (FIG.2A). This assay system also allowed for measuring compound toxicity by counting nuclei with DAPI staining. It was found that chloroquine at 100 μM was toxic to cells when it is incubated for 24 hrs. Further, time dependence of TFEB translocation was determined (FIG.2B and FIG.2C). Torin1 had a dose response curve starting from 30 min (FIG.2B), whereas chloroquine needed at least 120 min. EC50 values for Torin1 and chloroquine were approximately 10 nM and 20 μM, respectively. The same EC50 data were observed at 5h incubation. Moreover, these findings suggest that mTORC1 inhibition is faster than impairment of lysosomal activity for the activation of TFEB activity, and that this assay can detect both early and late events for TFEB activation. The same results were also observed with the TFEB-mCherry cells. Therefore, the mCherry-TFEB cells were chosen for the chemical library screen. This assay has been miniaturized to a 384-well format with Z-prime (Z’) = 0.8 (DMSO and Torin1 used as high and low controls, respectively). The value above 0.5 indicates that this is a robust assay for HTS. To assess the hit rates and feasibility of the mCherry-TFEB assay, a pilot screening was performed of a chemical library consisting of 9794 biologically active and structurally diverse compounds including chemicals from Microsource, Prestwick, Seqchem, Toris, Sigma and Selleck. Cells were seeded on the 384 well plates and grown for 2 days. Then, compounds from the library at a final concentration of 5 μM were added to the wells using Bravo Automated Liquid Handling Platform. After 5h of incubation, cells were fixed and stained with Hoechst, then washed with BioTek ELx406 Washer Dispenser. Next, the cells were imaged at 10x magnification on the INCell6000. Images were analyzed using the GE Workstation algorithm. The primary readout was % cells with TFEB Nuclear / Cytoplasm -35- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) ratio >1. Toxic compounds and fluorescent compounds were filtered out. Data tables were merged concatenated using the KNIME Analytica Platform. Z’ for all plates was above 0.8, indicating the robustness of this assay.27 compounds were identified as hits, with the hit rate of approximately 0.28%. Among these hits, many compounds targeted the TKR-PI3K- mTOR axis, such as Torin1, Torin2, AZD8055, PKI-587, INK128, BEZ235, Wortmannin and Foretninb, in which the mechanism of action has well been established. In addition, Bortezomib, a proteosome inhibitor, potently promotes TFEB translocation. Hits that enhance translocation of TFEB without a clearly defined mechanism were also identified. Thus, this pilot screen further validated that this high content assay can be used to screen larger chemical libraries for the identification of TFEB modulators. High Throughput Screening (HTS) to find hits After developing and validating the high content TFEB translocation assay using mCherry-TFEB stable cell lines, followed by the pilot screen of chemical library, the high throughput screen of a large chemical library was used to find the novel TFEB modulator compounds for therapeutic use in neurodegenerative disorders, including AD. a) Primary mCherry-TFEB translocation assay to find hits The HTS was conducted on the same automated system that was used in the pilot plot to identify compounds that activate the TFEB to translocate to the nucleus. In the primary mCherry-TFEB translocation assay, MSK’s in house library was used, which consisted of approximately 280,000 unique compounds that span a broad range of chemical space represented in relatively small cluster sizes, potentially yielding preliminary structure activity relationship from the primary HTS. The collection of compound library was assembled from several established vendors with compounds filtered according to Lipinski’s rule [43-45] for drug-likeliness and additional criteria to eliminate reactive and otherwise undesirable functional groups / moieties. In this assay, mCherry tagged TFEB was stably expressed in U2OS cells. In normal conditions, mCherry-TFEB was localized in the cytoplasm and the ratio of mCherry-TFEB in nucleus to cytoplasm was <1. However, upon compound treatment, if a compound activates, mCherry-TFEB translocates into the nucleus giving the ratio of mCherry-TFEB in nucleus to cytoplasm of >1 (FIG.3).346 unique compounds were found as primary hits. -36- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) b) Tandem-tagged LC3 reporter assay as secondary or confirmatory assay To validate the autophagy induction and the complete autophagic flux formation of 346 hits from primary mCherry-TFEB screen the tandem-tagged LC3 reporter assay was used as a secondary or confirmative assay. LC3, a microtubule-associated protein 1 light chain 3 (or MAP1-LC3) is a soluble protein present in mammalian tissues and cultured cells[46-48]. During the autophagy initiation (FIG.1.), the cytosolic form LC3-I undergoes a post-translational modification and conjugated to phosphatidylethanolamine to form LC3- II, which is then recruited on to the autophagosome. Autophagosome fuses with lysosome along with the LC3-II to form an autolysosome, in which engulfed components along with LC3-II are degraded by lysosomal hydrolases and released to the cytoplasm for recycling and cell survival. Therefore, LC3-II has been widely used as autophagy marker to monitor the autophagic activity using western blotting, immunoprecipitation, and immunofluorescence. In this assay, a tandem-tagged ‘mTagRFP-mWasabi-LC3’ expression vector was stably transfected into U2OS cells (FIGs.4A-4C). The principle of this assay was based on different pH stability of red (mTagRFP) and green (mWasabi) fluorescence proteins. Upon the compound treatment (FIG.4A), if the compound initiates the autophagy, the acidic environment within the lysosome will quench the signal of green (mWasabi) fluorescent protein, while the signal of red (mTagRFP) fluorescent protein was barely or unaffected. In the green and red merged channel, autophagosome was shown yellow (FIG.4B), and autolysosome was red (FIG. 4C). The anticipated result with autophagy inducer treatment would be the increase of total puncta number as well as the percent of red puncta (FIG.4C). With this assay, the 346 primary hit compounds were screened, and 39 compounds were found to initiate the complete flux of autophagy. Compound M1 was chosen as initial point for library synthesis, SAR studies and lead generation. The M1 hit possessed decent drug-like and physicochemical properties (e.g., M.Wt.304.48, tPSA: 24.5, CLogP: 4.512, pKa: 8.958 and 3.942) with room for further modification to generate library of compounds for SAR. Initial autophagic activity of M1 and other analogues was evaluated using a) LC3-I to LC3-II conversion assay (see experimental session for assay details) visualizing it on the western blot and quantified, b) LC3 reporter assay followed by c) TFEB translocation assay. Following the initial LC3-I to LC3-II conversion assay, most active analogues were fully -37- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) evaluated their autophagic activity using several cell-based assays (as shown in FIG.1) including TFEB translocation assay using mCherry-TFEB reporter cell line; Tandem-tagged LC3 reporter assay using mTagRFP-Mwasabi-LC3 expression vector; DQ-BSA (Dye quenched bovine-serum-albumin) assay; and finally Tau degradation or inhibition assay to evaluate the ability of the compound to modulate TFEB for the clearance of toxic Tau proteins through the autophagy pathway. Molecular design and medicinal chemistry for hit to lead generation (see experimental section for the synthesis and characterization data) The hit molecule 1-(4-(2-(tert-butyl)-4-methylphenoxy)butyl)piperazine, M1 (FIG. 5) was resynthesized and fully characterized using1H,13C NMR and HRMS (see experimental section for synthesis of all compounds). Initial autophagic activity of M1 was evaluated using the LC3-I to LC3-II conversion assay and LC3 reporter assay using U2OS cells, visualized on the western blot and quantified (FIG.5). Western blot of M1 showed the initiation of autophagy at 10 μM, and activity at 5 μM in LC3 reporter assay and was the starting point for the development of a library of compounds for SAR. Phenyl ring exploration After evaluating the LC3 activity (LC3-I to LC3-II conversion and LC3 reporter assays) of the M1 hit, a library of compounds for SAR was developed to evaluate the complete flux of autophagy and to generate the lead compound. In this regard, all the new compounds were screened with the LC3 reporter assay to quickly evaluate the autophagic activity by visualizing the puncta formation upon the compound treatment as shown in FIG.5. Initially, optimization of the phenyl ring in M1 was explored. Replacement of 2- (tert-butyl)-4-methylphenyl ring in M1 to a simple neutral phenyl (M2, which was previously reported as histamine H3-antagonist

[0049] ), electron withdrawing 4- fluorophenyl (M3) and 4-trifluoromethyl phenyl (M4) groups resulted in the loss of activity (Table 1), and it established that 2 -(tert-butyl)-4-methylphenyl ring in M1 is mandatory for LC3 activity. Table 1. Effects of substitutions at the phenyl ring. -38- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Note: -- indicates not tested. Exploration on the substitution on the piperazine ring NH The effect of free base in the piperazine ring and N-alkylation of M1 were then explored. Surprisingly, hydrogen replacement on the NH with a simple methyl group (M1D) resulted in the loss of LC3 reporter activity (Table 2). Further derivatization on the NH group of M1 resulted in additional analogues (see experimental section for synthesis) M1C, M2A, M3A and M4A, with slight activity for M1C and M4A at elevated concentrations (10 μM) than M1. Table 2. Effects of substitutions at the piperazine ring. -39- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Note: -- indicates not tested. Alkyl chain length exploration After establishing the fact that free base in the piperazine ring and 2 -(tert-butyl)-4- methylphenyl rings were necessary for the LC3 activity, the alkyl-chain length optimization was explored. First, shortening the alkyl chain length of M1 from 4 carbons to 3 carbons resulted in M5, and M5A as its dimer, and both showed loss of activity indicative of the minimal alkyl chain length to be 4 carbons (Table 3). Therefore, further optimization in the alkyl chain length to 6 carbons (M25), 8 carbons (M6), and 10 carbons (M27) resulted in the improved LC3 reporter activity for M6 (1 μM for LC3-western, 3 μM for LC3 reporter and 5 μM for TFEB reporter) with 8 carbon chain length and established as optimal alkyl chain length for LC3 reporter activity. M27 also showed similar LC3 reporter activity as M6 but with less activity on LC3-western (3 μM). Further modification of M6 replacing the 2 -(tert-butyl)-4-methylphenyl ring with 4-trifluoromethylphenyl ring resulted the new compound M7, but diminished LC3 activity showing the importance of the bulky 2 -(tert- butyl)-4-methylphenyl group on M6 for activity. Additional compounds M6A (dimer of M6), M7A (dimer of M7), M23 (hetero dimer of M6 and M4), M24 (hetero dimer of M1 and M4), M26 (dimer of M25) and M28 (dimer of M27) with various chain lengths showed no activity due the absence of free NH (Table 4). Table 3. Effects of alkyl chain length. -40- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Note: -- indicates not tested. Table 4. Effects of alkyl chain length at piperazine ring. -41- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Note: -- indicates not tested. Alkyl chain modulation After alkyl chain length optimization resulted M6 as the new potent compound with improved LC3 reporter (3 μM) and LC3-western (1 μM) activity, the modulation of the alkyl chain was explored with the introduction of functional groups such as double bonds, hydroxyl groups and triazole moiety into the alkyl chain while leaving the piperazine as free base. First, the cis double bond (z-isomer) was introduced on to the alkyl chain to generate M49 (N-Boc protected) and M50 as free base, and the M51 with trans double bond. However, all these compounds with strained alkyl chains showed no superior activity than M1 (except M50 with >LC3 activity) in both LC3 reporter and LC3-western assays, indicating the importance of non-strained alkyl chain in the molecule for activity (Table 5). Then, additional analogues were developed with the introduction of triazole moieties in the alkyl chain to further validate the effect of functional group or strain on the alkyl chain. First, M56 was designed and synthesized keeping the triazole moiety in the center of alkyl chain leaving the mandatory 2 -(tert-butyl)-4-methylphenyl ring and piperazine (free base) moieties intact. However, this compound showed some activity (10 μM) in LC3 reporter assay. Additional analogues of M56 with varying alkyl chain lengths (1 carbon or 3 -42- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) carbons) between triazole and 2 -(tert-butyl)-4-methylphenoxy group, and replacing the parental 2 -(tert-butyl)-4- methylphenyl ring with 1,3-diisopropylphenyl or 2,6- dimethylphenyl or 2-(tert-butyl)-4-methoxyphenyl group resulted additional seven analogues M58-M64. All these triazole compounds did not show any improved activity either in LC3 reporter or LC3-western assay, indicating the necessity of free alkyl chain for autophagic activity. Table 5. Effects of alkyl chain modulation. -43- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Note: -- indicates not tested. Exploration of piperazine ring After optimizing the phenyl ring moiety and alkyl chain length in M1 resulted M6 as the potent compound, the piperazine ring optimization was explored while keeping the free NH intact as it is mandatory for the autophagy activation. Therefore, a new piperazine analogue was designed with the ring expansion to seven membered ring by replacing the piperazine with 1,4-diazepane resulted new analogue M65. Surprisingly, M65 showed better LC3-western (0.625 μM) LC3-reporter (1.25 μM) and TFEB (1.25 μM) activity than M6 (Table 6). -44- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) 1,4-diazipane ring exploration Additional analogues based off 1,4-diazepane from M65 resulted M66, M69, M91 and M93 with varying alkyl chain or phenyl group, with lower autophagic activity than M65 but higher than the previous lead M6. It indicates that the improved autophagic activity of these analogues is due to 1,4-diazepane. Further modifications on the 1,4- diazepane resulted additional analogues with chiral methyl group M94 and M95 ((S)- isomer); chiral primary amines M100 and M102 ((S)-isomer and (R)-isomer); and with ring expanded analogues aza-^-Caprolactam M110, 1,5-diazacone M134 (8- membered ring). Steric hindrance from chiral methyl group in M94 and M95 resulted in decreased activity while chiral amine group in M101 and M102 preserved similar LC3 activity but less TFEB activity. Surprisingly, 8-membered ring analogues aza-^-Caprolactam M110 and 1,5- diazacone M134 showed complete diminishing of autophagic activity. Thus, based on the initial LC3 and TFEB activity data from M1, 44 analogues were designed and synthesized and M65 was identified as the lead molecule for further evaluation of autophagic activity using DQBSA assay, Tau degradation assay. Table 6. Effects of piperazine ring and 1,4-diazipane ring exploration. -45- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Results and Discussion Autophagy validation of the lead M65 -46- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) a) M65 activates TFEB translocation As shown in FIG.1, under abnormal conditions such as starvation or oxidative stress, autophagy begins with the activation of TFEB to translocate to the nucleus and initiates autophagy and lysosomal related gene expression. This initiation occurs following dephosphorylation of TFEB resulting apparent downshift in molecular weight of TFEB which can be observed by visualizing western blotting. When U2OS cells were treated with M65, clear TFEB translocation was observed at 5 and 2.5 μM, while partial or incomplete shift was observed at 1.25 and 0.625 μM (FIG.6A). To track the localization of TFEB more directly in cells, the U2OS mCherry-TFEB reporter cell line was used. In agreement with western blot data, near complete TFEB nuclear translocation was observed at both 5 and 2.5 μM, with partial translocation seen at 1.25 μM. M65’s EC50 for TFEB translocation was determined to be 1.3 μM, and nuclear translocation was onset between 1 and 2 hours of treatment (FIGs.6B-6C). Using quantitative polymerase chain reaction (qPCR), it was found that the M65 induced TFEB translocation was functional and resulted in the transcription of its target genes. In this assay, both lysosomal and autophagy genes were included as probed genes. M65 was found to significantly upregulate lysosomal genes (LAMP1, MCOLIN, CTST and GLA) as well as key components of autophagosome formation machinery (ATG7, ATG14, UVRAG and VPS18). In addition, an adapter protein for specific cargo degradation (NBR1) was also found to be significantly upregulated upon M65 treatment (FIG.6D). b) M65 activates autophagosome and autolysosome formation After evaluating that M65 activated TFEB, downstream autophagosome and autolysosome formation was explored upon M65 treatment. The autophagosome marker LC3 was used to identify autophagosome formation in two different methods. First, immunoblotting was used for monitoring the conversion of LC3-I to LC3-II in M65 treated cells. During autophagosome formation, LC3-I is lipidated with phosphatidylethanolamine to form LC3-II which then be recruited on to the autophagosome membranes. This lipidation event allows for the differentiation of LC3-I and LC3-II on western blots, with an increased LC3-II to total LC3 ratio being indicative of autophagosome formation. In U2OS, this increased ratio is observed in a dose dependent manner between 2.5 and 0.625 μM -47- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) (FIG.7A). Additionally, autolysosome formation was observed in the mtagRFP-Wasabi- LC3 reporter cell line. An increase in red-only LC3 puncta formation, indicative of autolysosome formation was found at concentrations as low as 1.25 μM (FIGs.7B and 7C). c) M65 treatment activates lysosomal degradation As a survival response in cells, autophagy is often time initiated during times of lysosomal dysfunction; therefore, it was sought to validate that M65’s apparent autophagic activity resulted in complete autophagic flux and was not the result of purely lysosomal distress. To look at general lysosomal activity, the dye quenched bovine serum albumin (DQ-BSA) assay was utilized, a derivative of bovine serum albumin (BSA) that is very heavily labeled with BODIPY TR-X dye. This high level of labeling results in a self- quenching effect such that fluorescence is not observable. However, when DQ-BSA is trafficked to lysosomes, proteases will begin to cleave BSA, giving rise to BSA fragments with strong fluorescent signals. The change in DQ-BSA intensity over time was utilized as a means of measuring lysosomal degradative capacity. Lysosomal proteolytic cleavage of DQ-BSA was not impaired by M65. When compared to DMSO treated cells, M65 resulted in an almost four-fold change of increased cleavage of substrates at concentrations as low as 0.3125 μM (FIG.8). Further, it was determined that 0.078 μM was the lowest active concentration of M65. To look at autophagosome derived cargo degradation more specifically, specific autophagy adapter protein p62 was measured utilizing GFP-LC3 cleavage assays. As an adapter protein for specific autophagic cargo, p62 is localized in the inner membrane of autophagosomes and degraded along with cargo during functional autophagy. p62 is however, also a target gene of TFEB meaning it may also be upregulated when autophagy is induced. Bafilomycin A1, a lysosomal v-type ATPase inhibitor, can be used to deconvolute p62’s genetic upregulation from potential impaired degradation. In U2OS cells, p62 levels were not significantly changed by M65 treatment at baseline. However, with Bafilomycin A1 cotreatment an increase in both LC3 and p62 levels were observed. If complete autophagic flux was inhibited by M65, such an increase in protein levels would not be observed with Bafilomycin cotreatment. Additionally, in GFP-LC3 cleavage assays M65 -48- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) treatment resulted in the release of free GFP at 2.5 and 1.25 μM. This cleavage was inhibited by Bafilomycin A1 cotreatment (FIG.9). d) M65 treatment degrades Tau aggregates in cellular models as treatment for AD As M65’s functional autophagy was fully evaluated using various cell-based assays mentioned in the results and discussion, determining its utility as a therapeutic intervention for neurodegenerative disorders, especially AD, was explored. M65’s ability to degrade aggregated Tau, a hallmark of AD, was investigated using a modified Tau biosensor assay (FIG.10). In the original assay

[0050] , monoclonal HEK293T cell line was stably expressed the mutant P301S repeat domain of tau fused to either cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP), to allow for the detection of tau aggregates by FRET signal detection. This system was modified by replacing the CFP-YFP FRET pair with fluorophores Ruby and Clover and expressing it in U2OS cells. In this system, rapid tau aggregation can be initiated with the introduction of pre-formed recombinant P301S tau fibrils to cells with lipofectamine treatment. Using this Tau biosensor cell lines, two different paradigms of treatment were explored. First, M65’s ability to decrease the formation of tau aggregates was explored. When cells were co-treated with M65 at the time of aggregate seeding, an approximately 50% decrease in the amount of aggregation formed was observed. In the second treatment paradigm, the ability of M65 to clear already formed aggregates was explored. M65 treatment began 24 hours after seeding was initiated. In these experiments a decrease of approximately 25% of the aggregates was observed. e) M65 treatment activates autophagy in Neurons Initial autophagy studies of M65 were primarily carried out in U2OS cells. As the goal is to utilize M65 in the treatment of neurodegenerative disorders, more disease relevant cells neurons were used to test M65’s ability to activate autophagy. Primary neurons were cultured from embryonic mouse pups and allowed to mature for 14 days in culture before beginning M65 treatment. Western blot analysis revealed an increase in LC3-II levels indicating autophagosome formation while total Tau levels were unchanged; however, phosphorylated tau was specifically decreased upon M65 treatment (FIGs.11A and 11B). qPCR analysis revealed the upregulation of autophagy (SQSTM1, ATG14, NBR1) and lysosomal (ATP6V1H, MCOLN1) genes (FIG.11C). -49- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) In vitro ADME of M65, M6 and M100 The assessment of in vitro Absorption, Distribution, Metabolism, and Excretion (ADME) properties of a lead or drug like molecule is a critical component in support of lead selection, optimization, and development of compounds within drug discovery and development research space. Therefore, after establishing the autophagic activity of M65 in both U2OS and primary neuronal cells, in vitro ADME of M65 was evaluated along with medium autophagic active compounds M6 and M100 as reference compounds. Note: All ADME properties were evaluated outsourcing to an external contract research organization (CRO). a) Assessment of MDR1-MDCKII permeability for M6, M65 and M100 The purpose of this study was to evaluate the A-B / B-A permeability of compounds M6, M65, and M100 in the MDR1-MDCKII assay including the identification of P- glycoprotein substrate (Pgp-mediated transport). In addition, these results can be correlated to the in vivo blood brain barrier (BBB) of a test compound for CNS targeting drug. Therefore, assessment of in vitro permeability of M65 was necessary before evaluating its in vivo PK / PD. All compounds under standard experimental conditions exhibited moderate permeability in A-B direction, did not undergo active efflux (Table 7), and were not P-gp substrates in presence of Cyclosporine A (Table 8). Table 7. A-B and B-A permeability data for test and reference compounds. *Efflux ratio is expressed as the quotient of Papp(BA) to Papp(AB). -50- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Table 8. Data of A-B and B-A permeability in the presence of Cyclosporine A. * Efflux ratio is expressed as the quotient of Papp(BA) to Papp(AB). b) Assessment of Metabolic Stability in Mouse Hepatocytes for 3 Compounds: M6, M65 and M100 The objective of this study was to determine metabolic stability of M6, M65 and M100, and reference compounds (Testosterone, 7-Hydroxycoumarin and Imipramine) in primary mouse hepatocytes. Under standard experimental conditions, all three compounds exhibited moderate metabolic stability (FIG.12). c) Analysis of Mouse Plasma Protein Binding for M6, M65 and M100 The objective of this study was to determine binding capabilities of compounds M6, M65 and M100, and reference compound (Verapamil) to mouse plasma proteins. Under standard experimental conditions, all 3 compounds showed low or instability in mice plasma, which may be due to low solubility of test compounds in mice plasma (Table 9). Table 9. Assessment of Mouse Plasma Protein Binding for M6, M65 and M100, and reference compound (Verapamil, a highly bound and highly stable compound). -51- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) d) Blood Plasma Partitioning Assay to determine red blood cells to plasma partitioning (KRBC / Pl) of compounds M6, M65 and M100 Knowledge of blood cells partitioning is important to predicting the test compound concentration in blood during in vivo tests. A significant partitioning ratio may indicate compound accumulation in red blood cells. Thus, in vitro Blood Plasma Partitioning assessment is important before measuring in vivo PK / PD. The data in Table 10, for Blood Plasma Partitioning (Mouse blood to plasma ratio) of M6, M65 and M100, along with reference compounds Verapamil (low partitioning) and Chlorthalidone (high partitioning), indicated moderate partitioning for M6 and M65. Table 10. Blood Plasma Partitioning data for M6, M65 and M100 along with reference compounds Verapamil and Chlorthalidone. -52- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) 6) In vivo PK of M65 in B6 female mice: After evaluating the in vitro permeability, metabolic stability, and blood plasma partitioning properties of M65, in vivo PK was explored to assess the compounds’ ability to cross BBB in B6 female mice model. B6 female mice (6-8 weeks old) were dosed with M65 in saline (vehicle) at 10 mg / kg (p.o, FIGs.13A and 13B; i. v. FIGs.13 C and 13D), following single administration, 2 mice from each group were sacrificed (sac’d) at different time points in 24 hr period. Blood and brain samples were analyzed by LC-MS. Quantitative results indicated that M65 successfully crossed BBB as drug detected at 4 hr and 24 hr timepoints. PK parameters for i.v. experiment calculated, and t1 / 2 found to be 10.54 hr (FIG.13E). However, when administered at higher dose, 30 mg / kg (single dose) in a modified vehicle, 10% HPBCD in water (water for injection) via i.v or i.p administration (single administration), an improved PK profile was found compared to previous experiment. The t1 / 2 values of both plasma and brain in i.v., and plasma in i.p experiment are shown in FIGs.14A-14B. The brain t1 / 2 in i.p experiment was not calculated due to insufficient clearance. No toxic effects were detected in 24 hr time point post M65 administration. The pharmacodynamic (PD) studies of this experiment will be reported in due course. -53- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) 7) M65 autophagy is independent of Histamine H3 antagonism. Initial hit M1 shared a scaffold with previously reported Histamine H3 receptor antagonist M2

[0049] and other scaffolds

[0051] . To determine if H3 receptor antagonism could be responsible for autophagy induction, two structurally distinct H3 receptor antagonists were tested, thioperamide and clobenpropit, in the autophagy assays. Treatment with these antagonists did not result in increased cellular autophagy and did not increase the LC3-II to total LC3 ratios (FIG.15), indicative of autophagy activation of M65 through a novel mechanism of action. 8) M65 autophagy initiation is independent of canonical mTORC1 inhibition. mTOR inhibition is one of the most well-known and studied methods of TFEB activation. Treating U2OS cells with Torin, an mTOR1 / 2 inhibitor, there is a loss of phosphorylation of mTOR substrates, including S6-Kinase and 4E-BP1; this loss of phosphorylation was not observed with M65 treatment. M65 treated cells (FIG.16) retained phosphorylation of these substrates to levels like DMSO treated cells, thus suggesting M65 autophagic activity was not due to canonicalmTORC1 inhibition but via a different mechanism. 9) Novel protein target identification using photoaffinity labelling (PAL). It was demonstrated that M65 activates autophagy neither through mTROC1 dependent nor Histamine H3-anatagonism pathways, but through a novel mechanism. Therefore, to identify the novel drug target and mechanism of action of M65, the Photoaffinity Labelling (PAL) approach was utilized. PAL is a widely used chemoproteomics technique to covalently attach labels to biological macromolecules such as proteins

[0052] . PAL uses chemical probe that binds covalently to its target upon activation by light (usually UV radiation). PAL has been widely used in medicinal chemistry and drug discovery to study protein-ligand interactions and became a powerful tool to identify novel drug targets for new therapeutics development. In general, PAL consists of a pharmacophore which selectively binds to its target; photo reactive group

[0053] (diazirine or benzophenone or aryl azide) which covalently crosslinks to the protein upon light -54- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) activation; and a reporter group to identify and isolate the labeled proteins (Biotin tag) or to visualize the localization of the target (a fluorophore such as TAMRA). a) Synthesis of trifunctional photoaffinity probe M92 and its autophagy validation A trifunctional photoaffinity probe M92 (FIG.17 A) was designed and synthesized based off M65 (see experimental section for synthesis) in such a way that the autophagic activity would not be affected upon functionalization with benzophenone derivative. M92 contains a pharmacophore (modified M65) to bind to the protein target, a benzophenone photoreactive group to covalently crosslink to the protein, and an alkyl handle which can be conjugated to the reporter tag (Biotin) via Copper(I) click chemistry [54, 55] to isolate the protein of interest. Initial autophagic activity of M92 was evaluated to confirm that the modification on M65 would not hider its activity. M92 treatment resulted in TFEB translocation at 10 and 5 μM (data not shown), increased LC3-II levels were observed on western blot in M92 treated cells (FIGs.17B-17C). Finally, increased lysosomal activity was observed by increased DQ-BSA cleavage (FIG.17 D). b) Photoaffinity Labeling and Target Identification With autophagy activity preserved, probe M92 was used for in-cell target identification. The alkyne handle on M92 allowed for investigating labeled proteins in two different methodologies. The first method involves utilizing M92 in a manner similar to immunofluorescence with a CY3-like fluorophore (AF647) being added to the probe via click chemistry to look at the probe’s cellular localization in intact cells. Specific M92 labeling was achieved as parental M65 was able to block the signal (FIG.18 A). Live cells were labeled with M92, fixed, and then stained with either LAMP1, TIMM23 or PDI, for markers of lysosomal, mitochondrial, and endoplasmic reticulum subcellular localizations, respectively (FIGs.18B-18D). Fluorescence microscopy revealed colocalization only between lysosomes and M92, suggesting that M65’s target could be a lysosomal protein. The second method of labeling with M92, involves using Copper (I) ‘click’ chemistry to attach a DDE-TAMRA-Biotin, allowing for the isolation of labeled proteins by cleavage of streptavidin beads. Photoaffinity labeling in live U2OS cells revealed an approximately 75 kDa membrane protein, when visualized by in-gel fluorescence (FIG. -55- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) 19A). This labeling was blocked by parental M65 pretreatment suggesting specific labeling. The photoaffinity labeling experiment was scaled up and eluted proteins were submitted for mass spectrum analysis in triplicate.305 total proteins were labeled by M92 as identified by proteomic analysis.17 of these proteins were found to have significant enrichment in M92 labeled samples as compared to those samples pre-blocked by parental M65 (FIGs.19B). In agreement with the immunofluorescence experiments, a significant enrichment of lysosomal proteins was observed (SCARB2, PSAP, PPT1, ACP2, GBA, SLC1A5, SLC3A2, CD63, SLC16A1, SLC2A1). Lysosomal integral membrane protein 2 (LIMP2 / SCARB2) was the most enriched protein found in M92 labeled membranes. SCARB2 is a type-iii glycoprotein found in the limiting membranes on lysosomes and endosomes. SCARB2 has a role in lysosomal maintenance, homeostasis, and cholesterol export

[0056]

[0057] . Additionally, SCARB2 chaperones the lysosomal hydrolase β-Glucocerebrosidase (GBA), through the ER to the Golgi and finally importing GBA into the lysosomal membrane where it serves a role in glucosylceramide metabolism

[0058] . Of note, GBA and its activating cofactor PSAP, were also enriched in M92 samples (FIG.20). As three proteins all known to co-interact and serve a role in glucosylceramide metabolism were pulled down in the photolabeling experiments, their validation was explored. Photolabeling experiments with streptavidin enrichment were repeated and the resulting labeled proteins were cleaved from streptavidin beads, run on SDS-PAGE and transferred to PVDF membranes for western blotting. Specific labeling of SCARB2, GBA, and PSAP were validated (FIG.21A). Intact cell labeling studies with subsequent immunofluorescence also corroborated M92 labeling of targets. Colocalization of SCARB2 and M92 were observed (FIG.21 B). c) M65’s effect on Targets With labeling validated, the potential role of M65 on these targets was investigated. Increased PSAP and GBA mRNA expression was observed while SCARB2 was not significantly affected (FIG.22A). Next, it was sought to identify if M65 had an effect on GBA Activity. M65 treatment resulted in increased GBA activity as observed by cleavage of 4MU-glucosylcerbrosdase substrate (FIG.22B). Additionally, increased GBA activity -56- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) was also observed in brain tissue from Black6 mice treated with 30 mg / kg and 15 mg / kg M65 via IP administration (FIG.22 C). d) Role of Glucosylceramide and Autophagy: it was next sought out to investigate the broader role of glucosylceramide metabolism on autophagy. Conduritol B epoxide (CBE) is an irreversible inhibitor of GBA. CBE treatment did not result in increased LC3-II ratio or lysosomal activity as assayed by DQ-BSA assays (FIGs.23A-23B), further validating the hypothesis that M65 activity could be the result of GBA activation rather than inhibition. Eliglustat is an FDA approved inhibitor of glucosylceramide synthase, the enzyme responsible for making GBA’s substrate glucosylceramide. Eliglustat treatment resulted in nuclear TFEB translocation in mCherry-TFEB reporter cells at 100 and 50 μM (FIG.24 A). Potential autophagy initiation by eliglustat was further confirmed through western blotting for LC3-II, where the ratio of LC3-II to total LC3 was increased compared to that of DMSO treated cells (FIG.24 B). Additionally, 50 μM eliglustat treatment was found to increase lysosomal activity by DQ-BSA (FIG.24 C). New analogues with improved drug like properties and in vivo PK parameters. -57- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) New analogues were pursued to improve drug-like properties and in vivo PK parameters. In this series, several analogues were designed and synthesized (see experimental section for synthesis and characterization) and structures of all newly synthesized compounds (M137, M138, M146, M147, M148, M149, M150, M151 and M152) are shown above. Autophagic activity of these new analogues were assed (Table 11) and found that M146 and M151 were shown to be more potent than initial lead M65. Initial in vivo PK studies of M146 and M151 in B6-female mice showed improved PK parameters (FIG.26A and FIG.26B) compared to M65 (FIGs.12-14). Table 11. Results of autophagy assays for selected compounds: -58- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) M1 Compound Summary Data: Lowest active concentration for compounds in various autophagy assays probing auto-phagosome / -lysosome formation (LC3 Flux and LC3 Reporter), Lysosomal Activity (DQ-BSA), and TFEB nuclear localization (TFEB Reporter). - denotes compound was not tested in a particular assay. TFEB Gene Expression of the lead compounds M65 and M146: U2OS Cells: After 24-hour treatment in U2OS cells, lead compounds M65 (FIG. 11C) and M146 (FIG.25A) were able to significantly upregulate the expression of TFEB, the master regulator of autophagy and lysosomal biogeneis, target genes. These genes include lysosomal membrane proteins (LAMP1 MCOLN1, ATP6V1H) and proteases (CTSD, GLA). Additionally, genes involved in autophagosome formation (ATG7 / 14, UVRAG, VPS18, WRD45, LC3) and the recognition of autophagic cargo (NBR1, SQSTM1). Primary Mouse Neurons: After 24-hour treatment in primary mouse neurons, lead compounds M65 and M146 were able to significantly upregulate the expression of TFEB, the master regulator of autophagy and lysosomal biogenesis, target genes. These genes include lysosomal membrane proteins (MCOLN1, ATP6V1H. Additionally, genes involved in autophagosome formation (ATG14, FLCN1) and the recognition of autophagic cargo (NBR1, SQSTM1). -59- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) IP Treated Mice: M65 was able to significantly upregulate the expression of TFEB, the master regulator of autophagy and lysosomal biogenesis, target genes in the brain tissue of Black-6 Mice following IP treatment at 30 mg / kg (FIG.25B). Cellular GBA Activity Lead compounds M65, M146, and M151 increased GBA activity as observed by the increased cleavage of 4MU-glucosylceramidase substrate in comparison to DMSO in pretreated U2OS cell lysates. Table 12. GBA activity of selected compounds. Results of Tau Aggregate Formation Assay for M65, M146 and M151: These results indicate that the lead compounds M65, M146 and M151 inhibited the tau aggregate formation at 2 and 4 µM concentrations compared to DMSO (control). Table 13. Results of Tau Aggregate Formation Assay. In vivo PK data for M146 and M151 to check whether these compounds cross BBB: -60- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) B6 female mice, 6-8 weeks (n = 24 mice) were administered with M146.HCl or M151.HCl in 10% HPBCD in Water for Injection (15 mg / kg i. p. once) and sacrificed three animals at 15 min, 30 min, 1 hr, 4 hr, 8 hr and 24 hr time points (vehicle mice at 24 h). Both brain and plasma were collected and analyzed by LCMS. The results show that both compounds (M146 and M151) cross the BBB in vivo with Tmax at 4 hr (FIG.26A). The calculated brain concentrations at Tmax are 3.94 µM (M146, FIG.26A (left)) and 4.12 µM (M151, FIG.26A (right)). Plasma analyses indicate the half-life (t1 / 2) of 17.17 h (M146, FIG.26B (left)) and 18.68 h (M151, FIG.26B (right)). Table 14. M146 PK Parameters in brain Table 15. M151 PK Parameters in brain. Table 16. M146 PK Parameters in plasma -61- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Table 17. M151 PK Parameters in plasma 10) Summary and Conclusions Impairment in the protein degradation pathway appears to be a consistent phenomenon in neurodegenerative diseases such as AD, Parkinson’s, and Huntington’s diseases. Autophagic dysfunction contributes to the pathogenesis of AD. Therefore, the autophagy-lysosome pathway (ALP) plays a critical role in the clearance of cellular macromolecules and has emerged as therapeutic targets for AD and other neurodegenerative disorders. Transcription Factor EB (TFEB) is considered as the master transcriptional regulator of lysosomal biogenesis and autophagy, and a small upregulation in the TFEB expression proved to clearing of toxic Tau aggregates in mouse brain. Therefore, discovery and development of TFEB activating compounds (independent of mTORC1 activation) to modulate ALP pathway is a promising strategy for AD and other neurodegenerative disorders. In this regard, HTS campaign was initialized utilizing Memorial Sloan-Kettering Cancer Center’s (MSK’s) in house library consisting of ~ 280,000 unique compounds. -62- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Screening of these compounds on primary TFEB translocation assay followed by secondary or confirmative tandem tagged LC3-reporter assay found 39 hits. Considering drug like properties and the ease of functionalization to generate library of analogues, M1 was chosen as a hit molecule as starting point for further development to generate a lead molecule. A library of novel analogues (~44 compounds) was systematically designed and synthesized, and SAR studies found M65 as a lead autophagy modulator. M65 has a novel chemical identity with decent drug like properties (M.Wt 374.61, tPSA 24.5, cLogP 6.78) . In U2OS cells, it was demonstrated that M651) promoted TFEB translocation, a master regulator of ALP; 2) enhanced the autophagic flux (LC3 reporter); and the degradation activity (DQ- BSA); 3) led to tau aggregate (hallmark of AD) clearance in cellular models. M65 also activated autophagy in neurons, and it was proved that M65’s autophagy activation is independent of canonical mTORC1 inhibition. M65 possesses good in vitro ADME properties including permeability, metabolic stability, and blood-plasma partitioning. Initial in vivo PK (in B6 female mice) of M65 when dosed at 10 mg / kg or 30 mg / kg in saline or 10% HPBCD as vehicle, revealed that M65 crosses blood brain barrier (BBB), as it is the most important parameter in CNS drug development. Following the discovery of a M65, a trifunctional photo affinity probe M92 was designed and synthesized to identify its target protein by Photoaffinity labeling (PAL) approach and evaluated its autophagic activity. In the PAL experiments, M92 significantly labeled to 17 lysosomal proteins and chemoproteomics analysis revealed the SCARB2 and β-Glucocerebrosidase (GBA) as highly enriched proteins. Preliminary studies utilizing recombinant GBA showed evidence of M65 increasing GBA’s activity in vitro. Experiments including the use of glucosylceramidase synthase inhibitors support the hypothesis that this increase in GBA activity is causative for its autophagy activity rather than an effect of TFEB activation. Inhibition of glucosylceramide synthase with eliglustat resulted in autophagy activation as observed by TFEB translocation, autophagosome formation, and increased lysosomal proteolytic cleavage. In terms of glucosylceramide levels in cells, inhibition of glucosylceramide synthase would have a similar effect as increased GBA activity, increasing ceramide and glucose levels, while decreasing glucosylceramide. This suggests that the autophagy regulated by M65 could be related to the levels of glucosylceramide precursors. Such a hypothesis is further supported by -63- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) ceremide being implicated in autophagy activation

[0059] . Ceramide has been demonstrated to activate JNK leading to increased Beclin1 expression and activity which contributes to autophagosome formation

[0060] . Additionally, C18-ceramide has been shown to direct mitochondrial degradation and mitophagy, through binding with LC3-II during autophagosome formation [61, 62]. Ceramide is a reported activator of protein phosphatase 2A (PP2A) [63-66], which is a known regulator of TFEB phosphorylation

[0067] . Traditionally, glucose negatively regulates autophagy, and autophagy is initiated during times of glucose starvation [68, 69]. However, increased glucose levels have been demonstrated to initiate autophagy

[0070] . Glucose has also been linked to increase lysosomal pH through interactions with vacuolar H+ ATPase

[0071] . Additionally, trehalose, a natural product disaccharide molecule made up of two glucose molecules connected by a linker, is a known activator of autophagy[72, 73]. However, further work is necessary to fully understand this mechanism connecting increased GBA activity to TFEB nuclear translocation. Moreover, GBA plays an important role in Parkinson’s disease development

[0074] , and found to be a risk factor in AD

[0075] . Therefore, further studies on the mechanism of action of M65 on GBA, and pharmacodynamic models in PS19 (Tau) mice would help to further develop of M65 as the novel autophagy modulator for neurodegenerative disorders including AD and Parkinson’s disease. Lead optimization of M65 resulted in several new analogues of which M146 and M151 showed superior autophagic activity with improved drug-like properties. Initial in vivo PK assessment in B6-female mice showed these new analogues also crossed BBB when administered at 15 mg / kg (i.p, single dose). In vitro tau aggregation assay results indicated that these new compounds inhibit tau aggregate formation in vitro when cells were treated at different concentrations. Therefore, additional work on pharmacodynamic (PD) studies is necessary to establish the therapeutic utility of these compounds to treat neurodegenerative disorders including AD and PD etc. General materials and methods for chemical synthesis: All reagents were purchased from Aldrich chemicals (www.sigma-aldrich.com) or from Fisher scientific (www.fishersci.org) or from CROs Ambeed, Chemscene, or WuXi -64- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) LabNetwork etc., and used without further purification. All solvents including HPLC or Optima grade solvents were purchased from Fisher Scientific (www.fishersci.com) and used without further purification. All reactions were performed in oven dried (overnight or 24 h) or flame-dried glassware under positive Ar atmosphere. Liquid reagents and solutions were transferred through rubber septa via syringes flushed with Ar prior to use. Solvent evaporation was performed on Buchi Interface I-300 Pro rotary evaporator. TLC analysis was performed on 1) EMD silica gel 60 F254 plates (aluminum sheets) and visualized under UV light (254 nm) or by staining with potassium permanganate (KMnO4), or cerium ammonium molybdenate (CAM) solution; 2) RediSep TLC plates (Basic alumina glass plates, cat# 69-2203-403) with 254nm fluorescent indicator (5 x 10 cm plates) visualizing under UV light (254 nm). Silica gel, reverse-phase and basic-alumina flash chromatography columns were purchased from Teledyne ISCO CombiFlash instruments and used as received. Flash chromatography was performed manually on ISCO CombiFlash Rf+Lumen instrument with RediSep silica gel normal phase columns or RediSep Gold silica gel normal phase columns with UV detection at 254 nm. NMR spectra were recorded on (in MSK’s NMR core facility) on a Bruker UltraShield Plus 600 MHz Avance III NMR with DCH CryoProbe or UltraShield Plus 500 MHz Avance III NMR or at 24 °C in CDCl3 or DMSO-d6 or MeOD or unless otherwise indicated. Chemical shifts (δ) are reported in parts per million (ppm) units relative to TMS (1 H, 0 ppm) or solvent signals. Coupling constants (J) are expressed in hertz (Hz). The first-order peak patterns are indicated as (singlet), or d (doublet), and t (triplet). Non-first order, complex peak patterns are expressed as m (multiplet). NMR spectra were processed using Bruker TopSpin, Mnova (www.mestrelab.com / software / mnova-nmr). Low-resolution mass spectra were obtained at the MSKCC Analytical Core Facility on a Waters Acuity SQD LC-MS. High resolution mass spectra were obtained on a Waters Acuity Premiere XE TOF LC-MS by electrospray ionization (ESI). Biological assay details: -65- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Western Blotting: Following treatment cells were washed with PBS and lysed in RIPA + PI +PMSF shaking at 4C. Samples were normalized prior to the addition of 4X ameli’s buffer then boiled for 10 min at 95C. Samples were run on 4-20% Criterion TGX gels. Transferred on PVDF Membrane and blocked for 1 Hour in 1:1 Intercept TBS Blocking Buffer (LICOR) and TBS at room temp. Membranes are incubated overnight at 4C in primary antibodies. TFEB Translocation: mCherry-TFEB U2OS cells were pre-stained with Hoechst to label nuclei for 30 minutes. Following nuclear staining cells were treated with compounds at specified concentrations. Live cell imagining was performed once an hour for 5 hours at 10x magnification on BioTek Cytation 5 Cell Imaging Multimode Reader with DAPI, and Texas Red filters. Cellular analysis was performed using Biotek Gen5. Cells were identified by their nucleus using the DAPI channel as the primary mask; a secondary mask was generated by expanding the primary mask by 30 um. Nuclear and Cytosolic TFEB were determined by the mean intensity of the Texas Red channel, in the primary and secondary masks, respectively. The half maximal effective concentration (EC50) were determined based on the 5 hour time point. LC3- Reporter: mTagRFP-mWasabi-LC3 U2OS cells were treated with compounds at specified concentrations. Live cell imagining was performed every 6 hours for 24 hours at 20x magnification on BioTek Cytation 5 Cell Imaging Multimode Reader with DAPI, GFP, and RFP filters. Cellular analysis was performed using Biotek Gen5. Cells were identified by their nucleus using the DAPI channel as the primary mask; a secondary mask was -66- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) generated by expanding the primary mask by 30 um. Puncta per cell were counted in the RFP and GFP channels in the secondary masks. Autolysosome number per cell was determined by subtracting the number of GFP puncta per cell from the number of RFP puncta per cell. Dye Quenched- BSA (DQ-BSA): U2OS were preincubated with Hoechst to label nuclei FBS and DQ-BSA red (50 ug / mL; Thermo Fisher D12051) for 1 hour. Excess DQ- BSA was washed away with PBS and cells were treated with compounds at the specified concentrations. Live cell imagining was performed every 3 hours for 12 hours at 10x magnification on BioTek Cytation 5 Cell Imaging Multimode Reader with DAPI, and Texas Red filters. Cellular analysis was performed using Biotek Gen5. Cells were identified by their nucleus using the DAPI channel as the primary mask; a secondary mask was generated by expanding the primary mask by 30 um. DQ-BSA intensity was quantified as the mean intensity of the Texas Red channel in the secondary masks. GFP-LC3 Cleavage: U2OS were treated with compounds for 4 hours. Following treatment cells were washed with PBS and lysed in RIPA + PI +PMSF shaking at 4C. Samples were normalized prior to the addition of 4X Lameli’s buffer then boiled for 10 min at 95C. Samples were run on 4-20% Criterion TGX gels. Transferred on PVDF Membrane and blocked for 1 Hour in 1:1 Intercept TBS Blocking Buffer (LICOR) and TBS at room temp. Membranes are incubated overnight at 4C in GFP (Thermofisher A-11122), and Tubulin (Abcam ab56676) primary antibodies. Membranes were washed three times in TBST, prior to 1 hour incubation with IRDye® (LICOR) secondary antibodies. Western blots were imaged on Odyssey CLx Imager, and levels of Free GFP was quantified on Image Studio. LC3 Flux Western: U2OS were treated with compounds for 5 hours. Following treatment cells were washed with PBS and lysed in RIPA + PI +PMSF shaking at 4C. Samples were normalized prior to the addition of 4X Lameli’s buffer then boiled for 10 min at 95C. Samples were run on 4-20% Criterion TGX gels. Transferred on PVDF Membrane and blocked for 1 Hour in 1:1 Intercept TBS Blocking Buffer (LICOR) and TBS at room temp. Membranes are incubated overnight at 4C in LC3B (Fisher Scientific NB100-2220), p62 (MBL PM045), and Tubulin (Abcam ab56676) primary antibodies. Membranes were -67- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) washed three times in TBST, prior to 1 hour incubation with IRDye® (LICOR) secondary antibodies. Western blots were imaged on Odyssey CLx Imager, and was levels of LC3-I, LC3-II and LC3-II / LC3-I ratio were quantified on Image Studio. Cellular GBA Activity: U2OS cells were treated with compounds for 5 hours prior to lysis. The Glucosylceramidase Activity Assay Kit (Fluorometric) (abcam ab273339) was followed per manufacturer’s protocol. Briefly, cells were lysed in assay buffer through passage of a 25 gauge needle and incubated of ice for 10 minutes. Debris was cleared from lysate via centrifugation at 13000 rpm. Total protein per sample was quantified via DC Assay (Biorad). Cell lysates were incubated with GBA substrate for 30 minutes at 37C. Stop solution was added, and the resulting fluorescence was measured. The fluorescence intensity of experimental standards was compared to that of the standard curve to determine the amount of substrate processed during the experiment. GBA Activity was then calculated as the (pmol of substrate processed) / ((total protein concentration x ul of sample x time)). qPCR: RNA was extracted from cells with RNeasy Plus Mini Kits (QIAGEN, 74134) according to manufacturer's protocol. cDNA with SuperScript™ III First-Strand Synthesis SuperMix for qRT-PCR (Thermofisher, 11752050) according to manufacturer's protocol. Quantification of gene expression was performed on QuantStudio™ 5 instrument (Applied Biosystems). Samples were heated at 95°C for 20 sec and amplified in 40 cycles for 1 sec at 95°C and 20 sec at 60°C. The comparative Ctmethod was used to measure the target mRNA expression level and normalized to GAPDH expression level. -68- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Primary Neuronal Culture: Primary mouse cortical neurons using pregnant C57 / bl6 mice at embryonic day 16.5 (E16.5) experimental protocol was approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center. Cortices from embryos were collected and meninges were removed. Cortices were then washed with ice-cold HBSS before dissociation with 0.25% trypsin and 0.15 μg / mL DNase at 37°C for 5 minutes. Dissociated tissue was triturated with 30 passages through a P1000 tip to fully dissociate cells and then resuspended with Neurobasal Media plus 10% FBS to inactivate trypsin. Cells were spun down at 500 x g for 5 minutes, supernatant was aspirated, and the cell pellet was resuspended in Neurobasal Media plus 2% B27 supplement, L-glutamax, and 5mM HEPES. Cells were filtered using a 40 μm strainer and then seeded on plates coated with PDL / PLL. Complete media was changed at 1 day in vitro (DIV) and half media was replaced every 2-3 days thereafter. Photoaffinity Labeling: U2OS cells were pre-treated with either DMSO or M65 (5 uM) for 30 minutes, followed by additional treatment of M92 (0.5 uM) for 30 minutes. Cell media was changed to PBS and UV irradiated at 360 nM for 15 mins on ice blocks. Cells were collected in resuspension buffer (50 mM HEPES, 150 Mm NaCl, pH 7.2) and lysed by probe sonication. Cytoplasmic and membrane cellular fractions were separated by ultracentrifugation at 100,000 x g for 45 minutes at 4C. Click chemistry was performed with 10 mM CuSO4, 10 -69- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) mM TCEP, 1 mM TBTA, and 100 μM TAMRA-DDE-Biotin Azide for 1 hour rotating at room temperature. Proteins were precipitated with ultracentrifugation at 100,000 x g for 45 minutes at 4C and resuspended by sonication. Samples were incubated with streptavidin beads rotating for 2 hours at room temperature. SA beads were washed over night with RIPA at 4C. The beads were further washed with RIPA, TBS-T (3 times), and PBS. Proteins were cleaved off beads with cleavage buffer (2% hydrazine, 0.05% SDS in water) shaking for 30 mins at 14000 rpm at room temperature. Photoaffinity Labeling for Immunofluorescence: U2OS cells were pre-treated with either DMSO or M65 (5 uM) for 30 minutes, followed by additional treatment of M92 (0.5 uM) for 30 minutes. Cell media was changed to PBS and UV irradiated at 360 nM for 10 mins on ice blocks. Cells were fixed with ice cold methanol for 10s. Click chemistry was performed in 2 mM BTTES, 1 mM CuSO4, 20 mM Na-L-ascorbate in 1x PBS with 50 uM Cy3-N3for 1 hour at room temperature. Cells were washed 3x with PBS and immunofluorescence was performed. Tau biosensor assay experimental details: FM5-4RD Tau(P301S)-Clover / FM5-4RD Tau(P301S)-mRuby2 These lentiviral packaging vectors were generated based on the FM5-4RD Tau(P301S)-CFP / YFP vectors that were generous gifts from Dr. David Holtzman’s laboratory (Washington University, St. Louis). Basically, Clover and mRuby2 genes were cloned out from the vectors purchased from Addgene (#54537 and #54768). Then the CFP and YFP genes were replaced with Clover and mRuby2 using restriction free cloning method. Lentivirus production HEK Lenti-X 293T cells were plated on T75 culture flask at a density of 1x105cells per mL. The following day, cells were co-transfected with FM5-4RD Tau-Clover / mRuby2, packaging plasmid psPAX2 and envelope plasmid pMD2.G (4:3:1 in amount) using Lipofectamine 3000 transfection reagent based on manufacture’s protocol. Culture medium was changed to RPMI with 10%FBS and Pen-Strep 8 hours after transfection. Lentivirus -70- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) containing medium was collected 48 hours afterwards. Medium was passed through 0.45um filter before aliquoting and freezing in -80°C. Tau biosensor stable cell line generation U-2 OS cells were grown in T25 flask with Tau-Clover and Tau-mRuby2 virus present for 72h before dissociation and subjected to fluorescence-activated single cell sorting (FACS). Single positive cells were sorted and maintained as polyclonal cell line. Population with Clover and mRuby2 dual-positive cells were seeded into 96-well plate for further expansion and generation of monoclonal cell lines. The derived monoclonal biosensor cell lines were tested for best FRET signal to noise, and the picked monoclonal cell line was used for all experiments. Recombinant Tau expression, purification and fibrillization pRK172-Tau RD construct for expressing tau monomer with P301S mutation was a generous gift from Dr. David Holtzman’s laboratory (Washington University, St. Louis). Recombinant tau monomer was prepared as previously described [76-78]. Lyophilized tau monomer was aliquoted with 24 ug in each tube and stored in -80°C. To fibrilize the tau, one aliquot of tau monomer was re-suspended in 40uL of 10mM HEPES (pH=7.4), 40uL of 100mM NaCl and 4uL of 25mM DTT and incubated at room temperature for 45 minutes. Then 50uL of 40mM HEPES, 50uL of 400mM NaCl, 32uL of 50uM Heparin and 24uL of H2O was added to the mixture and incubated at 37°C with agitation for 48 hours. Fibrils were then pelleted at 100,000g for 1 hour, resuspended in 100uL of PBS without Ca2+or Mg2+, and bath sonicated for 5 minutes (amplitude 65, 10 seconds on, 10 seconds off). Fibrils were then aliquoted in -80C for further use. Tau seeds transduction with lipofectamine Tau fibrils were bath sonicated for 10 minutes (amplitude 65, 10 seconds on, 10 seconds off) before incubating with lipofectamine 3000 (from Thermo Fisher) by following the manufacturer’s protocol. After incubation for 15 min at room temperature, the transduction mixture was added into the medium. For Tau formation assay, treatment was added together with tau seeds for 18 hours, after which extracellular tau was washed off -71- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) with PBS and fresh medium with treatment was added. At 24 hours, cells were subjected to FRET flow cytometry analysis. Tau FRET flow cytometry analysis Cells were dissociated with 0.05% trypsin and resuspended in FACS buffer (1x PBS, 0.5mM EDTA), then analyzed on the CytoFLEX LX flow cytometer (Beckman Coulter). To measure Clover and FRET signal, cells were excited with a 488 nm laser and fluorescent signal was captured with 525 / 40 nm and 610 / 20 nm filter, respectively. The mRuby2 signal was excited with a 561 nm laser and captured with 610 / 20 nm filter. Cells only with lipofectamine were used as FRET-negative controls to set up gating. Two- dimensional scatterplot of Clover vs. FRET was utilized to assess FRET-positive cell population. Experimental procedure for the synthesis of all compounds (1H and 13C NMR, HRMS data was reported) General scheme 1 for M1 Conditions: a) K2CO3, Acetone, reflux, 24 h, 33%; b) tert-butyl piperazine-1-carboxylate (N-Boc-piperazine), K2CO3, DMF, r.t, overnight, 85%; c) TFA:DCM, r.t, 2 h, 96%. Step 1: 1-(4-bromobutoxy)-2-(tert-butyl)-4-methylbenzene (1). To a mixture of 2- (tert-butyl)-4-methylphenol (2.0 g, 12.17 mmol) and K2CO3(3.45 g, 25 mmol, 2.0 eq) in acetone (35 mL) was added 1,4-dibromobutane (2.84 g, 13.17 mmol, 1.1 eq). The reaction mixture was refluxed under N2atm for 24 h. The solids were removed by filtration and the filtrate was concentrated under vacuum. The product was purified by column chromatography on silica gel (5-10% CH2Cl2 in Hexanes) to afford 1 (1.2 g, 33%) as a colorless oil.1H NMR1H NMR (600 MHz, CDCl3) δ 7.08 (d, J = 2.3 Hz, 1H), 6.96 (dd, J = -72- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) 7.9, 1.7 Hz, 1H), 6.75 (d, J = 8.2 Hz, 1H), 3.99 (t, J = 6.1 Hz, 2H), 3.51 (t, J = 6.6 Hz, 2H), 2.28 (s, 3H), 2.15 – 2.07 (m, 2H), 2.04 – 1.95 (m, 2H), 1.37 (s, 9H).13C NMR (151 MHz, CDCl3) δ 155.47, 137.66, 129.20, 127.54, 127.08, 111.72, 66.63, 34.71, 33.53, 29.86, 29.72, 28.15, 20.78. Step 2: To a solution of 1-Boc-piperazine (372.5 mg, 2.0 mmol, 1.2 eq.) in dry DMF (4 mL) were added anhydrous K2CO3(1.15 g, 8.35 mmol, 5 eq.) and Bromo alkyl phenol derivative 1 (500 mg, 1.67 mmol, 1.0 eq.). The reaction mixture was stirred at RT for 12 h (ESI MS: [M+H]+405.51). The K2CO3 was removed by suction filtration and the solvents were removed under vacuum. The residue was added water and extracted with dichloromethane. The organics were washed with water and brine. The combined organic portions were dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by flash column chromatography using MeOH:DCM (5-10%) to give 574 mg (85%) of Boc protected intermediate 2 (structure not shown) as thick oily product and was directly used in the next step for Boc deprotection reaction. Step 3: Boc protected intermediate 2 (642 mg, 1.59 mmol) was dissolved in dry dichloromethane (2 mL) and added 0.5 mL of TFA at RT. The reaction mixture was stirred at RT for 2 h. Upon completion (by LC / MS), the solvents were evaporated under vacuum and residue was redissolved in DCM, washed with 1M aq. NaOH solution, then with water and brine. Combined organic portions were dried over anhydrous Na2SO4, filtered, and concentrated under vacuum to give a thick oily product M1 (463 mg, 96%).1H NMR (600 MHz, CDCl3) δ 7.08 (d, J = 2.2 Hz, 1H), 6.96 (dd, J = 8.1, 1.4 Hz, 1H), 6.74 (d, J = 8.2 Hz, 1H), 3.96 (t, J = 6.2 Hz, 2H), 3.17 (t, J = 5.0 Hz, 4H), 2.72 (s, 4H), 2.50 (t, J = 7.3 Hz, 2H), 2.28 (s, 3H), 1.90 – 1.81 (m, 2H), 1.72 (qd, J = 8.8, 6.1 Hz, 2H), 1.37 (s, 9H).13C NMR (151 MHz, CDCl3) δ 155.69, 137.73, 129.24, 127.65, 127.23, 111.87, 67.49, 58.03, 51.03, 44.38, 34.85, 30.00, 27.44, 23.55, 20.92. HRMS Calculated for C19H33N2O [M+H]+305.2593, Found 305.2594. General scheme 2 for M1C (dimer) -73- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Conditions: a) K2CO3, DMF, r.t, 12 h, > 85%. To a solution of M1 (200 mg, 0.66 mmol, 1 eq.) in dry DMF (4 mL) were added anhydrous K2CO3 (454 mg, 3.284 mmol, 5 eq.) and Bromo alkyl phenol derivative 1 (197.5 mg, 1.0 eq.). The reaction mixture was stirred at RT for 12 h. The K2CO3was removed by suction filtration and the solvents were removed under vacuum. The residue was added water and extracted with dichloromethane. The organics were washed with water and brine. The combined organic portions were dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by flash column chromatography using MeOH:DCM (gradient) to give 303 mg (85%) of M1C.1H NMR (600 MHz, CDCl3) δ 7.07 (d, J = 2.2 Hz, 2H), 6.95 (dd, J = 8.1, 1.5 Hz, 2H), 6.74 (d, J = 8.2 Hz, 2H), 3.97 (t, J = 6.1 Hz, 4H), 2.50 (br s, 12H), 2.28 (s, 6H), 1.87 (s, 4H), 1.74 (s, 4H), 1.37 (s, 18H).13C NMR (151 MHz, CDCl3) δ 155.54, 137.61, 129.10, 127.50, 127.09, 111.79, 67.37, 57.91, 52.30, 34.71, 29.86, 27.43, 23.21, 20.78. HRMS Calculated for C34H55N2O2[M+H]+523.4264, Found 523.4254. General scheme 3 for the synthesis of M4 -bromobutoxy)-4-(trifluoromethyl)benzene Conditions: a) K2CO3, Acetone, reflux, 24 h (40%); b) N-Boc-Piperazine, K2CO3, DMF, r.t, overnight, 80%; c) TFA / DCM, r.t, 2 h, 91%. -74- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Step 1: 1-(4-bromobutoxy)-4-(trifluoromethyl)benzene was synthesized following the procedure from General Scheme 1, Step 1, using 4-(trifluoromethyl)phenol and 1,4- dibromobutane (40%).1H NMR (600 MHz, CDCl3) δ 7.54 (d, J = 8.0 Hz, 2H), 6.94 (d, J = 8.3 Hz, 2H), 4.04 (t, J = 6.1 Hz, 2H), 3.50 (t, J = 6.6 Hz, 2H), 2.11 – 2.04 (m, 2H), 2.01 – 1.94 (m, 2H).13C NMR (151 MHz, CDCl3) δ 161.28 (d, J = 1.51 Hz), 126.91 (q, J =3.8 Hz), 124.43 (q, J =270.29 Hz), 122.87 (q, J = 32.7 Hz), 114.37, 67.06, 33.31, 29.32, 27.72. Step 2: M4 was synthesized following conditions from general Scheme 1, steps 2 and 3, reacting 1-(4-bromobutoxy)-4-(trifluoromethyl)benzene intermediate with N-Boc- piperazine (80%, directly used without further characterization), followed by Boc deprotection (91%).1H NMR (600 MHz, CDCl3) δ 7.59 – 7.49 (m, 2H), 7.00 – 6.88 (m, 2H), 4.01 (t, J = 6.1 Hz, 2H), 3.27 (t, J = 5.0 Hz, 4H), 2.84 (s, 4H), 2.54 (s, 2H), 1.82 (dt, J = 8.4, 6.2 Hz, 2H), 1.70 (d, J = 10.7 Hz, 2H).13C NMR (151 MHz, CDCl3) δ 161.29, 126.91 (q, J = 3.7 Hz), 124.42 (q, J =-270.29 Hz), 122.18 (q, J = 33.22 Hz), 114.35, 67.59, 57.45, 49.56, 43.58, 26.72, 22.98. HRMS: Calculated for C15H22F3N2O [M+H]+303.1684, Found 303.1696. Scheme 3a for the synthesis of hetero dimer M24 1-(4-bromobutoxy)-4-(trifluoromethyl)benzene Conditions: a) K2CO3, DMF, r.t, 12 h, 80%. M24 was synthesized following the procedure from Scheme 2, using M1 and 1-(4- bromobutoxy)-4-(trifluoromethyl)benzene (80%).1H NMR (600 MHz, CDCl3) δ 7.46 (d, J = 8.6 Hz, 2H), 7.00 (d, J = 2.3 Hz, 1H), 6.90 – 6.84 (m, 3H), 6.68 (d, J = 8.2 Hz, 1H), 3.95 (t, J = 6.3 Hz, 2H), 3.89 (t, J = 6.3 Hz, 2H), 2.80 – 2.27 (m, 12H), 2.21 (s, 3H), 1.83 – 1.72 (m, 4H), 1.72 – 1.60 (m, 4H), 1.30 (s, 9H).13C NMR (151 MHz, CDCl3) δ 160.39 (d, J = 1.51 Hz), 154.58, 136.61, 128.00, 126.46,126.04, 125.83 (q, J =3.7 Hz), 123.43 (q, J = 271.8 Hz), 121.66 (q, J =32.6 Hz), 113.35, 110.75, 66.80, 66.49, 57.11, 56.96, 51.73, 51.71, -75- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) 33.69, 28.83, 26.50, 26.03, 22.46, 22.13, 19.76. HRMS: Calculated for C30H44F3N2O2 [M+H]+521.3355, Found 521.3358. General Scheme 4 for synthesis of M1D: To a solution of M1 (100 mg, 0.334 mmol, 1 eq.) in MeOH (5 mL) was added 1- methylpiperazine (66.8 mg, 73 µL, 0.668 mmol, 2 eq.). The reaction mixture was refluxed at 70oC for 20 h while monitoring the reaction progress by TLC. Upon disappearance of starting material M1, solvent was evaporated under vacuum. Residue was dissolved in methylene chloride, washed with 10% aq. NaOH, followed by water and brine. Organics were dried over anhydrous Na2SO4, filtered and concentrated to give the crude, which was directly loaded onto the silica gel column and purified by flash column chromatography eluting with MeOH:DCM (5-10% MeOH) to give 41 mg of M1D (39%) as white solid.1H NMR (600 MHz, CDCl3) δ 7.08 (d, J = 2.3 Hz, 1H), 6.96 (dd, J = 8.4, 2.2 Hz, 1H), 6.74 (d, J = 8.2 Hz, 1H), 3.98 (t, J = 5.8 Hz, 2H), 2.95 (s, 8H), 2.67 (s, 2H), 2.61 (s, 3H), 2.28 (s, 3H), 1.93 – 1.77 (m, 4H), 1.37 (s, 9H).13C NMR (151 MHz, CDCl3) δ 155.43, 137.55, 129.25, 127.55, 127.13, 111.80, 67.11, 57.22, 53.33, 50.56, 44.39, 34.71, 29.88, 27.18, 22.87, 20.78. HRMS: Calculated for C20H35N2O [M+H]+319.2749, found 319.2747. General Scheme 5 for one pot synthesis of M2 and M2A: (M2 is a known compound from literature) [1] -76- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Step 1: (4-bromobutoxy)benzene was synthesized following general Scheme 1, Step 1 using phenol and 1,4-dibromobutane.1H NMR (600 MHz, CDCl3) δ 7.30 – 7.25 (m, 2H), 6.96 – 6.91 (m, 1H), 6.88 (dd, J = 8.8, 1.0 Hz, 2H), 3.98 (t, J = 6.1 Hz, 2H), 3.48 (t, J = 6.7 Hz, 2H), 2.06 (m, 2H), 1.98 – 1.89 (m, 2H).13C NMR (151 MHz, CDCl3) δ 158.79, 129.45, 120.69, 114.39, 66.62, 33.53, 29.46, 27.88. Step 2: To a solution of (4-bromobutoxy)benzene (500 mg, 2.18 mmol, 1 eq.) in MeOH (10 mL) was added piperazine (939 mg, 10.91 mmol, 5 eq.). The reaction mixture was refluxed at 70oC for 20 h while monitoring the reaction progress by TLC. Upon disappearance of starting material, solvent was evaporated under vacuum. Residue was dissolved in methylene chloride, washed with 10% aq. NaOH, followed by water and brine. Organics were dried over anhydrous Na2SO4, filtered and concentrated to give the crude, which was directly loaded onto the silica gel column and purified by flash column chromatography eluting with MeOH:DCM (5-10% MeOH) to give 73 mg of monomer M2 (31%) as and 160 mg of dimer M2A (19%) as white powders. M2:1H NMR (600 MHz, CDCl3) δ 7.31 – 7.25 (m, 2H), 6.94 (t, J = 7.3 Hz, 1H), 6.88 (d, J = 8.1 Hz, 2H), 3.98 (t, J = 6.1 Hz, 2H), 3.25 (s, 4H), 2.81 (s, 4H), 2.53 (s, 2H), 1.81 (p, J = 6.2 Hz, 2H), 1.68 (p, J = 7.4 Hz, 2H).13C NMR (151 MHz, CDCl3) δ 158.82, 129.47, 120.69, 114.40, 67.21, 57.53, 49.57, 43.62, 26.87, 23.05. HRMS: Calculated for C14H23N2O [M+H]+235.1810, found 235.1805. M2A:1H NMR (600 MHz, CDCl3) δ 7.30 – 7.24 (m, 4H), 6.93 (t, J = 7.3 Hz, 2H), 6.89 (d, J = 7.7 Hz, 4H), 3.97 (t, J = 6.4 Hz, 4H), 2.42 (t, J = 7.7 Hz, 12H), 1.80 (p, J = 6.6 Hz, 4H), 1.73 – 1.64 (m, 4H).13C NMR (151 MHz, CDCl3) δ 158.97, 129.41, 120.52, 114.44, 67.52, 58.26, 53.11, 27.32, 23.44. HRMS: Calculated for C24H35N2O2[M+H]+383.2699, found 383.2690. General Scheme 6 for the synthesis of M3 and M3A: -77- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Step 1: 1-(4-bromobutoxy)-4-fluorobenzene intermediate was synthesized following general Scheme 1, Step 1 using 4-fluorophenol and 1,4-dibromobutane (32%).1H NMR (600 MHz, CDCl3) δ 6.99 – 6.93 (m, 2H), 6.85 – 6.79 (m, 2H), 3.95 (t, J = 6.1 Hz, 2H), 3.49 (t, J = 6.6 Hz, 2H), 2.11 – 2.02 (m, 2H), 1.97 – 1.89 (m, 2H).13C NMR (151 MHz, CDCl3) δ 157.2 (d, J = 238.58 Hz), 154.95 (d, J = 1.51 Hz), 115.78 (d, J = 22.65 Jz), 115.34 (d, J = 7.55 Hz), 67.41, 33.46, 29.42, 27.89. Step 2: M3 and M3A (dimer) were synthesized in one pot reaction following General Scheme 5, Step 2 using 1-(4-bromobutoxy)-4-fluorobenzene and piperazine. M3: (32% yield)1H NMR (600 MHz, CDCl3) δ 7.00 – 6.94 (m, 2H), 6.84 – 6.79 (m, 2H), 3.93 (t, J = 6.1 Hz, 2H), 3.27 (t, J = 5.0 Hz, 4H), 2.83 (t, J = 4.9 Hz, 4H), 2.52 (t, J = 7.2 Hz, 2H), 1.84 – 1.75 (m, 2H), 1.67 (p, J = 7.5 Hz, 2H).13C NMR (151 MHz, CDCl3) δ 157.18 (d, J =238.58 Hz), 154.97 (d, J = 1.51 Hz), 115.81 (d, J = 22.65 Hz), 115.32 (d, J = 7.55 Hz), 67.97, 57.52, 49.55, 43.61, 26.89, 23.03. HRMS: Calculated for C14H22FN2O [M+H]+253.1716, found 253.1715. M3A: (20% yield)1H NMR (600 MHz, CDCl3) δ 7.00 – 6.92 (m, 4H), 6.85 – 6.79 (m, 4H), 3.93 (t, J = 6.1 Hz, 4H), 2.94 – 2.37 (m, 12H), 1.83 – 1.77 (m, 4H), 1.73 (s, 4H).13C NMR (151 MHz, CDCl3) δ 157.16 (d, J =238.58 Hz), 155.02 (d, J =3.02 Hz), 115.76 (d, J = 22.65 Hz), 115.34 (d, J = 7.55 Hz), 68.12, 57.94, 52.50, 27.16, 23.06. HRMS: Calculated for C24H33F2N2O2[M+H]+419.2510, found 419.2500. General Scheme for the synthesis of M5: -78- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Conditions: a) K2CO3, Acetone, reflux, 24 h, 39%; b) tert-butyl piperazine-1-carboxylate, K2CO3, DMF, r.t, overnight; c) TFA / DCM, r.t, 2 h, (82% over 2 steps). Step 1: 1-(3-bromopropoxy)-2-(tert-butyl)-4-methylbenzene intermediate was synthesized following general Scheme 1, Step 1, using 2-(tert-butyl)-4-methylphenol and 1,3-dibromopropane (39%).1H NMR (600 MHz, CDCl3) δ 7.09 (d, J = 2.3 Hz, 1H), 6.97 (ddd, J = 8.1, 2.3, 0.8 Hz, 1H), 6.78 (d, J = 8.2 Hz, 1H), 4.10 (t, J = 5.8 Hz, 2H), 3.65 (t, J = 6.5 Hz, 2H), 2.40 – 2.33 (m, 2H), 2.28 (s, 3H), 1.37 (s, 9H).13C NMR (151 MHz, CDCl3) δ 155.22, 137.63, 129.42, 127.58, 127.14, 111.88, 65.26, 34.68, 32.60, 30.48, 29.90, 20.78. Step 2: Compound M5 was synthesized following general Scheme 1, Steps 2 and 3, using 1-(3-bromopropoxy)-2-(tert-butyl)-4-methylbenzene and N-Boc-piperazine (82% over two steps).1H NMR (600 MHz, CDCl3) δ 7.07 (d, J = 2.3 Hz, 1H), 6.98 – 6.94 (m, 1H), 6.76 (d, J = 8.2 Hz, 1H), 4.00 (t, J = 6.2 Hz, 2H), 2.91 (t, J = 4.9 Hz, 4H), 2.60 – 2.54 (m, 2H), 2.46 (br s, 4H), 2.28 (s, 3H), 2.07 – 2.00 (m, 2H), 1.37 (s, 9H).13C NMR (151 MHz, CDCl3) δ 155.59, 137.64, 129.01, 127.47, 127.06, 111.76, 66.07, 56.31, 54.62, 46.08, 34.71, 29.86, 26.74, 20.78. HRMS: Calculated for C18H31N2O [M+H]+291.2436, found 291.2444. General Scheme for the synthesis of M6: Conditions: a) K2CO3, Acetone, reflux, 24 h (34%); b) N-Boc-Piperazine, K2CO3, DMF, r.t, overnight; c) TFA / DCM, r.t, 2 h, 82% (over two steps). -79- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Step 1: The bromo intermediate 1-((8-bromooctyl)oxy)-2-(tert-butyl)-4- methylbenzene was synthesized following the experimental procedure from general Scheme 1, Step 1, using 2-(tert-butyl)-4-methylphenol and 1,8-dibromooctane as starting materials (34%).1H NMR (600 MHz, CDCl3) δ 7.07 (d, J = 2.3 Hz, 1H), 6.95 (dd, J = 8.1, 2.3 Hz, 1H), 6.75 (d, J = 8.2 Hz, 1H), 3.94 (t, J = 6.4 Hz, 2H), 3.41 (t, J = 6.9 Hz, 2H), 2.28 (s, 3H), 1.91 – 1.78 (m, 4H), 1.56 – 1.42 (m, 4H), 1.38 (br s, 13H).13C NMR (151 MHz, CDCl3) δ 155.73, 137.68, 128.86, 127.44, 127.04, 111.73, 67.69, 34.72, 34.00, 32.78, 29.83, 29.47, 29.14, 28.67, 28.09, 26.28, 20.78. Step 2: 1-(8-(2-(tert-butyl)-4-methylphenoxy)octyl)piperazine (M6), was synthesized following the general Scheme 1, Steps 2 and 3, using 1-((8-bromooctyl)oxy)-2- (tert-butyl)-4-methylbenzene and N-Boc-piperazine (82% over 2 steps).1H NMR (600 MHz, CDCl3) δ 7.07 (d, J = 1.5 Hz, 1H), 6.98 – 6.93 (m, 1H), 6.75 (d, J = 8.2 Hz, 1H), 3.94 (t, J = 6.5 Hz, 2H), 2.90 (t, J = 4.9 Hz, 4H), 2.41 (br s, 3H), 2.33 – 2.29 (m, 2H), 2.28 (s, 3H), 1.90 – 1.76 (m, 4H), 1.55 – 1.45 (m, 4H), 1.38 (m, 15H).13C NMR (151 MHz, CDCl3) δ 155.73, 137.65, 128.81, 127.42, 127.02, 111.71, 67.73, 59.47, 54.61, 46.06, 34.71, 29.82, 29.48, 29.26, 27.55, 26.63, 26.31, 20.78. HRMS: Calculated for C23H41N2O [M+H]+361.3219, found 361.3203. General Scheme for the synthesis of M7: Conditions: a) K2CO3, Acetone, reflux, 24 h (49%); b) N-Boc-Piperazine, K2CO3, DMF, r.t, overnight; c) TFA / DCM, r.t, 2 h, 78% (over two steps). Step 1: The bromo intermediate 1-((8-bromooctyl)oxy)-4-(trifluoromethyl)benzene was synthesized following the general Scheme 1, Step 1, using 4-(trifluoromethyl)phenol and 1,8-dibromooctane as starting materials (49%).1H NMR (600 MHz, CDCl3) δ 7.53 (d, J = 8.5 Hz, 2H), 6.94 (d, J = 8.4 Hz, 2H), 3.99 (t, J = 6.5 Hz, 2H), 3.42 (t, J = 6.8 Hz, 2H), 1.86 (p, J = 7.0 Hz, 2H), 1.80 (dt, J = 14.8, 6.6 Hz, 2H), 1.51 – 1.42 (m, 4H), 1.42 – 1.31 -80- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) (m, 4H).13C NMR (151 MHz, CDCl3) δ 161.54, 126.84 (q, J =3.7 Hz), 124.48 (q, J = 377.5 Hz), 122.59 (q, J =32.7 Hz), 114.38, 68.12, 34.01, 32.75, 29.15, 29.04, 28.67, 28.07, 25.89. Step 2: 1-(8-(4-(trifluoromethyl)phenoxy)octyl)piperazine (M7) was synthesized following the general Scheme 1, Steps 2 and 3, using 1-((8-bromooctyl)oxy)-4- (trifluoromethyl)benzene and N-Boc-piperazine (78% over two steps).1H NMR (600 MHz, CDCl3) δ 7.53 (d, J = 8.7 Hz, 2H), 6.94 (d, J = 8.6 Hz, 2H), 3.98 (t, J = 6.5 Hz, 2H), 2.90 (t, J = 4.9 Hz, 4H), 2.41 (s, 3H), 2.34 – 2.28 (m, 2H), 2.05 (s, 2H), 1.83 – 1.75 (m, 2H), 1.54 – 1.41 (m, 4H), 1.41 – 1.26 (m, 6H).13C NMR (151 MHz, CDCl3) δ 161.56 (q, J = 1.2 Hz), 126.83 (q, J =3.8 Hz), 124.5 (q, J =271.8 Hz), 122.55 (q, J =32.7 Hz), 114.39, 68.18, 59.44, 54.57, 46.02, 29.48, 29.26, 29.06, 27.54, 26.63, 25.92. HRMS: Calculated for C19H30F3N2O [M+H]+359.2310, found 359.2304. Synthesis of M23, a hetero dimer: Conditions: a) MeOH, 70oC, 20 h, 65%. Step 1: 1-(8-(2-(tert-butyl)-4-methylphenoxy)octyl)-4-(4-(4- (trifluoromethyl)phenoxy)butyl)piperazine (M23) was synthesized following the general Scheme 4, using M6 and 1-(4-bromobutoxy)-4-(trifluoromethyl)benzene as starting materials (65%).1H NMR (600 MHz, CDCl3) δ 7.46 (d, J = 8.3 Hz, 2H), 7.00 (d, J = 1.9 Hz, 1H), 6.91 – 6.84 (m, 3H), 6.68 (d, J = 8.2 Hz, 1H), 3.95 (t, J = 6.0 Hz, 2H), 3.86 (t, J = 6.4 Hz, 2H), 3.21 – 2.39 (m, 12H), 2.20 (s, 3H), 1.82 – 1.58 (m, 8H), 1.48 – 1.38 (m, 2H), 1.30 (m, 15H).13C NMR (151 MHz, CDCl3) δ 160.24, 154.68, 136.61, 127.85, 126.42, 126.03, 125.88 (q, J = 3.8 Hz), 123.40 (q, J =271.40 Hz), 121.81 (q, J = 32.6 Hz), 113.35, 110.70, 66.60, 66.52, 56.74, 56.25, 50.64, 49.70, 33.69, 28.81, 28.40, 28.09, 25.96, 25.76, -81- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) 25.21, 23.87, 21.66, 19.76. HRMS: Calculated for C34H52F3N2O2 [M+H]+577.3981, found 577.4003. Synthesis of M25: Conditions: a) K2CO3, DMF, 80oC, 4 h (29%); b) N-Boc-Piperazine, MeOH, 70oC, 20 h, reflux; c) TFA / DCM, r.t, 2h (73% from two steps). Step 1: Synthesis of 1-((6-bromohexyl)oxy)-2-(tert-butyl)-4-methylbenzene: To a mixture of 2-(tert-butyl)-4-methylphenol (1.0 g, 6.08 mmol) and K2CO3 (1.008 g, 7.3 mmol, 1.2 eq) in dry DMF (12 mL) was added 1,6-dibromohexane (2.966 g, 12.16 mmol, 2.0 eq). The reaction mixture was heated at 80oC under N2atm for 4-5 h. Solvents were evaporated under reduced pressure (55oC bath temperature, 10 mbar pressure), residue was added water, extracted with methylene chloride (3x). Combined organics were washed with water, brine and dried over anhydrous Na2SO4, filtered, and evaporated the solvents under vacuum. The product was purified by flash column chromatography on silica gel (5-10% CH2Cl2 in Hexanes) to afford the bromo intermediate as a colorless oil (577 mg, 29%).1H NMR (600 MHz, CDCl3) δ 7.11 (d, J = 2.3 Hz, 1H), 7.01 – 6.96 (m, 1H), 6.78 (d, J = 8.2 Hz, 1H), 3.98 (t, J = 6.4 Hz, 2H), 3.46 (t, J = 6.8 Hz, 2H), 2.31 (s, 3H), 1.94 (p, J = 6.9 Hz, 2H), 1.91 – 1.84 (m, 2H), 1.57 (m, 4H), 1.41 (s, 9H).13C NMR (151 MHz, CDCl3) δ 155.68, 137.68, 128.97, 127.50, 127.08, 111.74, 67.51, 34.75, 33.86, 32.71, 29.85, 29.38, 27.94, 25.64, 20.81. Step 2: 1-(6-(2-(tert-butyl)-4-methylphenoxy)hexyl)piperazine M25, was synthesized following the experimental procedure from general Scheme 4 using 1-((6- bromohexyl)oxy)-2-(tert-butyl)-4-methylbenzene and N-Boc-piperazine followed by the Boc deprotection using TFA / DCM to give colorless thick oily product (73% in two steps).1H NMR (600 MHz, CDCl3) δ 7.01 (d, J = 2.2 Hz, 1H), 6.91 – 6.86 (m, 1H), 6.68 (d, J = 8.2 -82- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Hz, 1H), 3.87 (t, J = 6.4 Hz, 2H), 3.18 (s, 4H), 2.74 (s, 4H), 2.41 (s, 2H), 2.21 (s, 3H), 1.81 – 1.71 (m, 2H), 1.46 (p, J = 7.6 Hz, 4H), 1.30 (s, 11H).13C NMR (151 MHz, CDCl3) δ 155.67, 137.64, 128.98, 127.50, 127.09, 111.73, 67.51, 57.93, 49.97, 43.62, 34.74, 29.86, 29.43, 26.91, 26.21(2C), 20.81. HRMS: Calculated for C21H37N2O [M+H]+333.2906, found 333.2905. Synthesis of M26 (homo dimer from M25): 1,4-bis(6-(2-(tert-butyl)-4-methylphenoxy)hexyl)piperazine M26, was synthesized in one step using M25 and the bromo intermediate 1-((6-bromohexyl)oxy)-2-(tert-butyl)-4- methylbenzene following the general Scheme 2 (63%).1H NMR (600 MHz, CDCl3) δ 7.00 (d, J = 2.2 Hz, 2H), 6.90 – 6.86 (m, 2H), 6.68 (d, J = 8.2 Hz, 2H), 3.87 (t, J = 6.4 Hz, 4H), 2.74 – 2.24 (m, 12H), 2.21 (s, 6H), 1.80 – 1.72 (m, 4H), 1.53 – 1.41 (m, 8H), 1.30 (s, 22H).13C NMR (151 MHz, CDCl3) δ 155.72, 137.67, 128.88, 127.47, 127.06, 111.71, 67.63, 58.66, 53.15, 34.74, 29.85, 29.46, 27.33, 26.76, 26.35, 20.81. HRMS: Calculated for C38H63N2O2[M+H]+579.4890, found 579.4900. Synthesis of M27: Conditions: a) K2CO3, Acetone, reflux, 24 h (30%); b) N-Boc-Piperazine, K2CO3, DMF, r.t, overnight; c) TFA / DCM, r.t, 2 h, 79% (over two steps). Step 1: The bromo intermediate 1-((10-bromodecyl)oxy)-2-(tert-butyl)-4- methylbenzene was synthesized following the general Scheme 1, Step 1, using 2-(tert- butyl)-4-methylphenol and 1,10-dibromodecane (30%).1H NMR (600 MHz, CDCl3) δ 7.11 (d, J = 1.8 Hz, 1H), 7.02 – 6.96 (m, 1H), 6.79 (d, J = 8.2 Hz, 1H), 3.97 (t, J = 6.5 Hz, 2H), -83- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) 3.44 (t, J = 6.9 Hz, 2H), 2.31 (s, 3H), 1.93 – 1.81 (m, 4H), 1.57 – 1.50 (m, 2H), 1.50 – 1.44 (m, 2H), 1.42 (br s, 17H).13C NMR (151 MHz, CDCl3) δ 155.78, 137.69, 128.85, 127.46, 127.06, 111.74, 67.76, 34.75, 34.11, 32.85, 29.85, 29.53, 29.45, 29.38, 29.31, 28.77, 28.20, 26.37, 20.82. Step 2: 1-(10-(2-(tert-butyl)-4-methylphenoxy)decyl)piperazine, M27 was synthesized following the general procedure from Scheme 1, steps 2 and 3, using bromo intermediate 1-((10-bromodecyl)oxy)-2-(tert-butyl)-4-methylbenzene and N-Boc-Piperazine (79% over two steps).1H NMR (600 MHz, CDCl3) δ 7.10 (d, J = 2.2 Hz, 1H), 6.98 (ddd, J = 8.2, 2.3, 0.9 Hz, 1H), 6.78 (d, J = 8.2 Hz, 1H), 3.96 (t, J = 6.5 Hz, 2H), 2.93 (t, J = 4.9 Hz, 4H), 2.43 (s, 3H), 2.35 – 2.31 (m, 2H), 2.31 (s, 3H), 1.90 – 1.80 (m, 2H), 1.52 (tdd, J = 11.5, 9.0, 3.5 Hz, 4H), 1.41 (br s, 19H).13C NMR (151 MHz, CDCl3) δ 155.78, 137.68, 128.82, 127.44, 127.05, 111.73, 67.78, 59.56, 54.73, 46.18, 34.74, 29.85, 29.60, 29.53(2C), 29.52, 29.36, 27.67, 26.72, 26.39, 20.81. HRMS: Calculated for C25H45N2O [M+H]+389.3532 , found 389.3520. General scheme for the synthesis of M49 and M57 Conditions: a) HIO4 / H2O, 1,4-Dioxane, 0oC - rt, 2.5 h; b) NaBH4, EtOH, Et2O, 0oC to r.t, (75% over two steps); c) CBr4, PPh3, DCM, 0oC to r.t, (73%); d) N-Boc Piperazine, TEA, -84- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) DCM, r.t, 18 h (~ 35%); e) K2CO3, acetone, reflux, 12 h (> 76%); f) OsO4, NMO,tBuOH:acetone (1:1), 0oC, 8 h, argon atm (78%); g) TFA:DCM, r.t, 2 h (> 88%). References: For steps 1, 2, 3:[2, 3]; For step 4:[4]; For step 6:[5] Step 1, Step 2: Synthesis of (Z)-oct-4-ene-1,8-diol 2 (in two steps from known lit procedure)1. To a solution of (Z)-9-oxabicyclo[6.1.0]non-4-ene (Millipore Sigma)(2.0 g, 16.105 mmol, 1 equiv.) in 1,4-dioxane (20 mL) at 0oC was added periodic acid (4.221 g, 1.15 equiv) dropwise in 20 mL of water. The reaction mixture was allowed to warm to room temperature over 2.5 h. Then reaction mixture was extracted with Et2O (3 X 50 mL), combined organic layers were dried over anhydrous Na2SO4, filtered, and evaporated the solvents. The crude 1 was directly used in the next step for NaBH4reduction reaction by adding fresh Et2O (50 mL) to the crude, cooled the reaction mixture to 0oC. Then added the NaBH4 (1 g, in 15 mL of absolute EtOH) dropwise. Stir the contents for 3 h at 0oC, then at room temperature for 1 h. The reaction was quenched by the careful addition of water (20- 30 mL), extracted with EtOAc (3 x 50 mL), dried over anhydrous Na2SO4, filtered, and evaporated the solvents under reduced pressure. The resulting crude was purified by flash column chromatography on silica gel column eluting with 5-10% MeOH in DCM to give 1.75 g of diol 2 as clear oil (75% over two steps).1H NMR (600 MHz, CDCl3) δ 5.40 (ddd, J = 5.7, 4.5, 1.1 Hz, 2H), 3.64 (t, J = 6.2 Hz, 4H), 2.23 (s, 2H), 2.21 – 2.16 (m, 4H), 1.67 – 1.58 (m, 4H).13C NMR (151 MHz, CDCl3) δ 129.87, 61.85, 32.25, 23.24. ESI-MS detected 145.2 [M+H]+. Step 3: Synthesis of (Z)-1,8-dibromooct-4-ene 3 (synthesized following literature procedure2: A round bottom flask was charged with diol 2 (3.0 g, 20 mmol, 1 equiv) in CH2Cl2(100 mL). CBr4(13.93 g, 42 mmol, 2.1 equiv) was added, and the reaction mixture was cooled to 0oC. Then PPh3 (11.54 g, 44 mmol, 2.2 equiv) was added, and the reaction mixture was allowed to warm to room temperature over 16 h. Then solvent was evaporated under reduced pressure to give a pale-yellow oily product. Then hexane (60 mL) was added to crash out the solids, and the suspension was sonicated and filtered off. The filtrate was concentrated under vacuum to give a pale-yellow oil, which was purified by flash column chromatography on silica gel column (5% EtOAc in hexanes) to give dibromide 3 as a clear oil (4.04 g, 73%).1H NMR (600 MHz, CDCl3) δ 5.44 – 5.36 (m, 2H), 3.42 (t, J = 6.7 Hz, -85- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) 4H), 2.28 – 2.19 (m, 4H), 1.93 (p, J = 6.9 Hz, 4H).13C NMR (151 MHz, CDCl3) δ 129.28, 33.34, 32.50, 25.70. Step 4: Synthesis of tert-butyl (Z)-4-(8-bromooct-4-en-1-yl)piperazine-1- carboxylate 4: To a solution of N-Boc-piperazine (1.724 g, 9.25 mmol, 1.0 equiv) in DCM (30 mL) was added triethylamine (1.124 g, 11.11 mmol, 1.2 equiv) and contents were cooled to 0oC. Then added dibromide 3 (3.0 g, 1.2 equiv) in 5 mL of DCM, and stirred at room temperature for 24 h. The reaction mixture was diluted with DCM, washed with saturated aq. NaHCO3 solution (~ 15 mL) followed by washing with water and brine. The organic portion was dried over anhydrous Na2SO4, filtered, and concentrated to give a colorless oil, which was purified by flash column chromatography on silica gel column eluting with 10-20% EtOAc in hexanes to give 1.2 g of colorless oily product 4 (35%).1H NMR (600 MHz, CDCl3) δ 5.47 – 5.40 (m, 1H), 5.36 – 5.29 (m, 1H), 3.42 (m, 6H), 2.36 (m, 6H), 2.20 (q, J = 6.4 Hz, 2H), 2.09 (q, J = 7.4 Hz, 2H), 1.91 (p, J = 6.8 Hz, 2H), 1.56 (p, J = 7.5 Hz, 2H), 1.46 (s, 9H).13C NMR (151 MHz, CDCl3) δ 154.76, 130.95, 128.05, 79.57, 58.09, 53.03, 44.11*, 43.07*, 33.45, 32.51, 28.43, 26.74, 25.58, 25.12. (* peak split due to change in piperazine ring configuration). HRMS: Calculated for C17H32BrN2O2[M+H]+375.1647, found 375.1655, 377.1639. Step 5: Synthesis of tert-butyl (Z)-4-(8-(2-(tert-butyl)-4-methylphenoxy)oct-4-en-1- yl)piperazine-1-carboxylate M49. A round-bottom flask was charged with Boc protected intermediate 4 (1.0 g, 2.66 mmol, 1.0 equiv), 2-(tert-butyl)-4-methylphenol (1.75 g, 4.0 equiv), K2CO3 (2.942 g, 21.28 mmol, 8.0 equiv) and acetone (30 mL). The contents were refluxed under argon atm for overnight. Reaction mixture was cooled to room temperature, filtered off the solid, filtrate was concentrated to give a thick oily compound. The crude was purified by flash column chromatography on silica gel column eluting with 20-30% EtOAc in hexanes to afford the 0.93 g oily product M49 (76%).1H NMR (600 MHz, CDCl3) δ 7.07 (d, J = 2.4 Hz, 1H), 6.95 (ddd, J = 8.2, 2.3, 0.9 Hz, 1H), 6.74 (d, J = 8.2 Hz, 1H), 5.48 – 5.37 (m, 2H), 3.94 (t, J = 6.2 Hz, 2H), 3.47 – 3.33 (m, 4H), 2.42 – 2.23 (m, 11H), 2.06 (q, J = 6.7 Hz, 2H), 1.92 – 1.83 (m, 2H), 1.59 – 1.49 (m, 2H), 1.46 (s, 9H), 1.39 (s, 9H).13C NMR (151 MHz, CDCl3) δ 155.67, 154.76, 137.59, 130.22, 129.20, 128.92, 127.46, 127.05, 111.65, 79.55, 66.95, 58.17, 52.99, 44.10*, 43.06*, 34.72, 29.88, 29.54, 28.43, 26.76, 25.07, -86- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) 24.14, 20.79. (* peak split). HRMS: Calculated for C28H47N2O3 [M+H]+459.3587, found 459.3606. Step 6: A round bottom flask was charged with alkene M49 (50 mg, 0.109 mmol, 1 equiv) and t-Butanol / Acetone (1:1 mL), and cooled to 0oC with an ice water bath. Then added NMO (14.046 mg, 0.1199 mmol, 1.1 equiv), catalytic amount of OsO4 (2 mol%, 0.554 mg (13.85 µL)) and the reaction mixture stirred at 0oC for 12 h under argon atm. After completion of the reaction, added saturated aq. Na2SO3, extract with Et2O, wash with brine, combined organics dry over anhydrous MgSO4, filtered, concentrated. The crude was purified by flash column chromatography on silica gel column eluting with 5-10% MeOH / DCM to give 42 mg of dehydroxylated intermediate as thick oil (78%), which was directly used in the next step for Boc deprotection reaction.1H NMR (600 MHz, CDCl3) δ 7.07 (d, J = 2.3 Hz, 1H), 6.95 (ddd, J = 8.2, 2.3, 0.8 Hz, 1H), 6.76 (d, J = 8.2 Hz, 1H), 4.05 – 3.93 (m, 2H), 3.67 (dt, J = 9.5, 3.6 Hz, 1H), 3.47 (dt, J = 9.5, 3.3 Hz, 5H, ovelap), 2.57 (br s, 2H), 2.47 – 2.41 (m, 2H), 2.41 – 2.31 (m, 2H), 2.28 (s, 3H), 2.15 – 2.03 (m, 1H), 1.90 (ddq, J = 12.8, 10.0, 6.3 Hz, 1H), 1.86 – 1.52 (m, 6H), 1.45 (s, 9H), 1.37 (s, 9H).13C NMR (151 MHz, CDCl3) δ 155.65, 154.65, 137.64, 128.96, 127.43, 127.07, 111.86, 79.89, 74.33, 74.32, 67.80, 58.80, 52.76, 43.59*, 42.57*, 34.71, 31.15, 29.87, 28.83, 28.38, 26.30, 24.11, 20.78. (* peak split). HRMS: Calculated for C28H49N2O5 [M+H]+493.3641, found 493.3653. Step 7: A round bottom flask was charged with dehydroxylated intermediate (40 mg, 0.0812 mmol) and dissolved in 2 mL of dry DCM. Then added trifluoro acetic acid (TFA) (0.5 mL) and the contents were stirred at room temperature for 2 h. The solvents were evaporated under vacuum, co-evaporate with DCM (x3). Then the residue was dissolved in fresh DCM, washed with 1M aq. NaOH, then with water and brine, dry over anhydrous Na2SO4, filtered and concentrated. The crude was purified by flash column chromatography on basic alumina eluting with 5-10% MeOH in DCM to afford the 28 mg of pure M57 (88%).1H NMR (600 MHz, CDCl3) δ 7.07 (d, J = 2.3 Hz, 1H), 6.95 (dd, J = 8.2, 1.5 Hz, 1H), 6.76 (d, J = 8.2 Hz, 1H), 4.06 – 3.93 (m, 2H), 3.68 (dt, J = 9.5, 3.6 Hz, 1H), 3.48 (ddd, J = 10.0, 3.8, 1.6 Hz, 1H), 2.93 (dt, J = 6.2, 3.1 Hz, 4H), 2.62 (s, 2H), 2.51 – 2.31 (m, 4H), 2.28 (s, 3H), 2.17 – 2.03 (m, 2H), 1.90 (ddq, J = 12.8, 9.9, 6.3 Hz, 2H), 1.83 (ddt, J = 14.2, 7.2, 2.1 Hz, 1H), 1.80 – 1.55 (m, 6H), 1.38 (s, 9H).13C NMR (151 MHz, -87- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) CDCl3) δ 155.67, 137.65, 128.93, 127.42, 127.07, 111.85, 74.34, 74.27, 67.81, 59.33, 54.10, 45.56, 34.71, 31.23, 29.86, 28.82, 26.34, 24.00, 20.78. HRMS: Calculated for C23H41N2O3 [M+H]+393.3117, found 393.3128. General scheme 5 for M50 Conditions: a) TFA:DCM, r.t, 2 h, 93%. Step 1: A round bottom flask was charged with Boc protected alkene M49 (235 mg, 0.512 mmol), dissolved in 8 mL of DCM, then add 2 mL of TFA at room temperature. The reaction mixture was stirred at room temperature for 2 h, evaporated solvents, co- evaporated (x3) with DCM. Then add fresh DCM to dissolve the crude, washed with 1M aq. NaOH, then with water and brine, dry over anhydrous Na2SO4, filter and concentrate. The residue was purified by flash column chromatography on basic alumina eluting with 5-10% MeOH in DCM to afford 170 mg of colorless oily product M50 (93%).1H NMR (600 MHz, CDCl3) δ 7.07 (d, J = 2.2 Hz, 1H), 6.95 (dd, J = 8.3, 2.2 Hz, 1H), 6.74 (d, J = 8.2 Hz, 1H), 5.47 – 5.39 (m, 2H), 3.95 (t, J = 6.2 Hz, 2H), 2.88 (t, J = 4.9 Hz, 4H), 2.37 (br s, 4H), 2.31 – 2.23 (m, 7H), 2.10 – 2.02 (m, 2H), 1.92 – 1.85 (m, 2H), 1.54 (p, J = 7.6 Hz, 2H), 1.39 (s, 9H).13C NMR (151 MHz, CDCl3) δ 155.68, 137.60, 130.26, 129.16, 128.89, 127.45, 127.05, 111.67, 67.00, 58.75, 54.25, 45.88, 34.73, 29.88, 29.56, 26.58, 25.13, 24.15, 20.78. HRMS: Calculated for C23H39N2O [M+H]+359.3062, found 359.3075. General scheme 6 for E isomer M51 (ratio not calculated the E / Z ratio) -88- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Conditions: a) Grubb's 2ndgen catalyst, DCM, r.t, 24 h, argon atm, 64% (ref.); b) 1, K2CO3, acetone, reflux, 24 h, 31%; c) N-Boc piperazine, TEA, DCM, r.t, 18 h, 48%; d) TFA:DCM, r.t, 2 h, 80%. Step 1: Synthesis of 1,8-dibromooct-4-ene was achieved by following the literature procedure3and used directly in the next step of the reaction (E:Z ratio not calculated). Step 2: Synthesis of 1-((8-bromooct-4-en-1-yl)oxy)-2-(tert-butyl)-4-methylbenzene: A round bottom flask was charged with 1,8-dibromooct-4-ene (740 mg, 2.74 mmol, 1.0 equiv), 2-(tert-butyl)-4-methylphenol (449.87 mg, 1 equiv), K2CO3 (753.33 mg,5.48 mmol, 2.0 equiv) and acetone (10 mL). The reaction mixture was heated to reflux under inert atmosphere for 24 h. Then cooled to room temperature, filtered off solids, filtrate was concentrated and purified by flash column chromatography on silica gel eluting with 1-2% DCM in hexanes to give 300 mg of the title compound as colorless oil (31%) (E:Z ratio not calculated).1H NMR (600 MHz, CDCl3) δ 7.08 (d, J = 2.2 Hz, 1H), 6.96 (dd, J = 8.3, 2.2 Hz, 1H), 6.75 (d, J = 8.2 Hz, 1H), 5.58 – 5.47 (m, 1H), 5.47 – 5.34 (m, 1H), 3.99 – 3.91 (m, 2H), 3.39 (m, 2H), 2.28 (s, 3H), 2.26 – 2.19 (m, 2H), 2.19 – 2.12 (m, 2H), 1.95 – 1.86 (m, 4H), 1.39 (s, 2H), 1.38 (s, 7H).13C NMR (151 MHz, CDCl3) δ 155.65, 155.64*, 137.66, 137.61*, 130.98, 130.46, 128.95*, 128.92, 128.50, 127.47*, 127.46, 127.06*, 127.05, -89- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) 111.70, 111.68*, 67.03*, 66.97, 34.72, 33.35, 33.29*, 32.56*, 32.31, 30.84, 29.87*, 29.85, 29.57*, 29.36, 29.32, 25.63, 24.27, 20.78. (*minor isomer peaks). Step 3: Synthesis of tert-butyl 4-(8-(2-(tert-butyl)-4-methylphenoxy)oct-4-en-1- yl)piperazine-1-carboxylate. A round bottom flask was charged with N-Boc-piperazine (237.213 mg, 1.5 equiv), DCM (4 mL) and triethylamine (103.09 mg (141.41 µL), 1.5 equiv) and cooled to 0oC. Then add tert-butyl 4-(8-(2-(tert-butyl)-4-methylphenoxy)oct-4- en-1-yl)piperazine-1-carboxylate (300 mg, 0.849 mmol, 1 equiv) in 4 mL of dry DCM, and stir the reaction mixture at room temperature for 18 h under inert atmosphere while monitoring the reaction by TLC (5% MeOH in DCM). The reaction mixture was diluted with DCM, washed with saturated aq. NaHCO3, followed by water and brine. The organic portion was dried over anhydrous MgSO4, filtered, concentrated under vacuum to give crude, which was further purified by flash column chromatography on silica gel column eluting with 5-10% MeOH in DCM to afford 185 mg of product as colorless oil (48%).1H NMR (600 MHz, CDCl3) δ 7.07 (d, J = 2.2 Hz, 1H), 6.98 – 6.92 (m, 1H), 6.74 (d, J = 8.2 Hz, 1H), 5.52 – 5.38 (m, 2H), 3.94 (t, J = 6.4 Hz, 2H), 3.42 (br s, 4H), 2.42 – 2.29 (m, 6H), 2.28 (s, 3H), 2.24 – 2.17 (m, 2H), 2.10 – 1.98 (m, 2H), 1.89 (dq, J = 8.5, 6.5 Hz, 2H), 1.60 – 1.51 (m, 2H), 1.46 (s, 9H), 1.39 (s, 2H), 1.38 (s, 7H). (E:Z ratio not calculated).13C NMR (151 MHz, CDCl3) δ 155.67, 154.75, 137.65*, 137.59*, 130.56, 130.19*, 129.74, 129.22*, 128.91*, 128.89, 127.46*, 127.44, 127.05*, 127.03, 111.69, 111.65,79.58, 79.57*, 66.97, 66.94*, 58.15*, 58.13, 52.98, 44.05, 43.02, 34.72, 30.41, 29.88, 29.85, 29.82*, 29.53*, 29.35, 29.31, 28.42, 28.39*, 26.72*, 26.55, 25.06*, 24.14*, 20.78. (* minor isomer peaks). ESI-MS [M+H]+detected 459.59. Step 4: Synthesis of 1-(8-(2-(tert-butyl)-4-methylphenoxy)oct-4-en-1-yl)piperazine M51: In a 4 dram screw vial with a stir bar was charged tert-butyl 4-(8-(2-(tert-butyl)-4- methylphenoxy)oct-4-en-1-yl)piperazine-1-carboxylate (80 mg, 0.174 mmol), DCM (2 mL) and contents were cooled to 0oC. Then added TFA (0.5 mL), reaction mixture was stirred at room temperature for 2 h. Then, solvents were evaporated at room temperature under reduced pressure, co-evaporated with DCM (x3), washed with 1M aq. NaOH, water and brine. Organic portion was dried over anhydrous Na2SO4, filtered, concentrated, purified by flash column chromatography on basic alumina eluting with 5-10% MeOH:DCM to afford 50 mg of M51 as colorless thick oil (80%).1H NMR (600 MHz, CDCl3) δ 7.07 (d, J = 2.3 -90- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Hz, 1H), 6.95 (dd, J = 8.2, 2.2 Hz, 1H), 6.74 (d, J = 8.2 Hz, 1H), 5.50 – 5.39 (m, 2H), 3.94 (t, J = 6.4 Hz, 2H), 2.95 – 2.84 (m, 4H), 2.53 – 2.33 (m, 4H), 2.33 – 2.28 (m, 2H), 2.28 (s, 3H), 2.24 – 2.17 (m, 2H), 2.09 – 1.98 (m, 2H), 1.89 (dt, J = 8.3, 6.6 Hz, 2H), 1.55 (m, 2H), 1.39 (s, 1.66 H), 1.38 (s, 6.93H). (~ 80:20, E:Z isomeric ratio by1H NMR).13C NMR (151 MHz, CDCl3) δ 155.68, 137.66, 137.60*, 130.69, 130.30*, 129.62, 129.12*, 128.88*, 128.86, 127.45*, 127.43, 127.04*, 127.02, 111.70, 111.67*, 67.01*, 67.00, 58.83*, 58.81, 54.54, 54.51*, 46.03, 34.72, 30.51, 29.88*, 29.84, 29.81*, 29.57*, 29.37, 29.31, 26.61*, 26.44, 25.16*, 24.15*, 20.78. (* minor z isomer peaks). HRMS: Calculated for C23H39N2O [M+H]+359.3062, found 359.3071. General scheme for the synthesis of M56 (a triazole analogue) 1) Synthesis of azide intermediate A 2) Synthesis of alkyne intermediate B 3) Triazole analogue synthesis from A and B using Cu(I) 'click' chemistry Conditions: a) TEA, DCM, 24 h, r.t, > 70%; b) NaN3, DMF, 60oC, 2 days, N2 atm, 96%; c) Cs2CO3, KI, MeCN, 85oC, overnight, >88%; d) CuSO4·5H2O, sodium ascorbate, THF:H2O, r.t, 2 h, Ar atm, 84%; e) TFA:DCM,. r.t, 2 h, > 98%. 1) Synthesis of azide intermediate A: [7] -91- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Step 1: tert-butyl 4-(3-bromopropyl)piperazine-1-carboxylate synthesis: To a cold solution (ice bath) of N-Boc-piperazine (2.0 g, 10.7 mmol, 1 equiv) and triethylamine (1.5 mL, 1.1 equiv) in dichloromethane (30 mL) was added 1,3-dibromopropane (1.12 mL, 1.1 equiv), and the solution was stirred for 24 h at room temperature under argon atmosphere. Then the reaction mixture was diluted with dichloromethane (10 mL), washed with a saturated aq. NaHCO3(10 mL), water (10 mL), and brine (10 mL), dried over anhydrous Na2SO4, filtered, and then concentrated. The residue was purified by flash chromatography on silica gel column eluting with 20-30% EtOAc in hexanes afforded 2.3 g of bromo intermediate as an oil (70%).1H NMR (600 MHz, CDCl3) δ 3.47 (t, J = 6.6 Hz, 2H), 3.42 (t, J = 5.2 Hz, 4H), 2.49 (t, J = 7.0 Hz, 2H), 2.38 (t, J = 5.0 Hz, 4H), 2.03 (p, J = 6.8 Hz, 2H), 1.46 (s, 9H).13C NMR (151 MHz, CDCl3) δ 154.73, 79.63, 56.43, 53.02, 44.10, 43.10, 31.71, 29.86, 28.42. Step 2: Synthesis of tert-butyl 4-(3-azidopropyl)piperazine-1-carboxylate A: A round bottom flask was charged with tert-butyl 4-(3-bromopropyl)piperazine-1-carboxylate (200 g, 0.65 mmol, 1.0 equiv), anhydrous DMF (4 mL), and Sodium azide (93.09 mg, 1.43 mmol, 2.2 equiv). The resultant mixture was heated in an oil bath at 60 °C under nitrogen atmosphere for 2 days. The reaction mixture was cooled to room temperature, ethyl acetate (15 mL) and saturated aqueous sodium chloride (NaCl) (15 mL) were added. The organic layer was separated, washed with saturated aqueous NaCl (15 mL), dried over magnesium sulfate, filtered, and concentrated to give 170 mg of the title compound as a dark oil (96% yield).1H NMR (600 MHz, CDCl3) δ 3.43 (t, J = 5.1 Hz, 4H), 3.35 (t, J = 6.7 Hz, 2H), 2.43 (t, J = 7.1 Hz, 2H), 2.38 (t, J = 5.1 Hz, 4H), 1.77 (p, J = 6.9 Hz, 2H), 1.46 (s, 9H).13C NMR (151 MHz, CDCl3) δ 154.74, 79.64, 55.24, 52.95, 49.47, 44.08, 43.08, 28.42, 26.17. ESI- MS: [M+H]+270.4. 2) Synthesis of alkyne intermediate B: [8] Synthesis of 2-(tert-butyl)-4-methyl-1-(pent-4-yn-1-yloxy)benzene B: A mixture of phenol (2.463 g, 15.0 mmol, 1.0 equiv), 5-chloro-pent-1-yne (2.0 g, 19.50 mmol, 1.3 equiv), Cs2CO3 (7.33 g, 22.5 mmol, 1.5 equiv) and KI (249 mg, 1.5 mmol, 0.1 equiv) in 50 mL CH3CN was stirred at 85 °C overnight. Then cooled to room temperature, quenched by adding water and extracted with EtOAc three times. The combined organic layers were -92- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) dried over anhydrous Na2SO4, filtered, and removal of the solvent under reduced pressure afforded the crude product as a liquid. The crude was purified by flash column chromatography on silica gel column eluting with 5-10% DCM in hexanes to afford 3.05 g of alkyne B as a liquid (88%).1H NMR (600 MHz, CDCl3) δ 7.08 (d, J = 2.2 Hz, 1H), 6.99 – 6.94 (m, 1H), 6.78 (d, J = 8.2 Hz, 1H), 4.06 (t, J = 6.0 Hz, 2H), 2.46 (td, J = 7.1, 2.7 Hz, 2H), 2.28 (s, 3H), 2.11 – 2.02 (m, 2H), 1.97 (t, J = 2.6 Hz, 1H), 1.37 (s, 9H).13C NMR (151 MHz, CDCl3) δ 155.45, 137.65, 129.17, 127.52, 127.09, 111.78, 83.52, 68.88, 66.04, 34.70, 29.87, 28.45, 20.78, 15.69. General procedure for the synthesis of M56 with Cu(I) ‘click’ chemistry: Step 1: General procedure for Cu(I) ‘click’ reaction4: for the synthesis of tert-butyl 4-(3-(4-(3-(2-(tert-butyl)-4-methylphenoxy)propyl)-1H-1,2,3-triazol-1-yl)propyl)piperazine- 1-carboxylate: In a round-bottom flask equipped with a magnetic stir bar, alkyne derivative B (34.21 mg, 0.185 mmol, 1.25 equiv), azide derivative A (40 mg, 0.1485 mmol, 1 equiv), CuSO4·5H2O (1.82 mg, 0.0074 mmol, 5 mol%), sodium ascorbate (2.94 mg, 0.01485 mmol, 10 mol%), THF (1 mL), and H2O (1 mL) were added. The resulting mixture was sonicated and stirred at room temperature under argon atmosphere for about 2 h. The reaction progress was monitored by TLC (silica gel, EtOAc in hexanes, 3:1). After completion of the reaction, water (1 mL) was added, and the reaction mixture was extracted with EtOAc (3 × 5 mL). The combined organic layer was washed with brine (5 mL), dried over anhydrous Na2SO4, filtered, and concentrated under vacuum to afford the crude product. The crude was purified by flash column chromatography on silica gel column eluting with 5-10% MeOH in DCM to afford 68 mg of the triazole product as thick oil (84%).1H NMR (600 MHz, CDCl3) δ 7.30 (s, 1H), 7.08 (d, J = 2.2 Hz, 1H), 6.97 – 6.92 (m, 1H), 6.73 (d, J = 8.2 Hz, 1H), 4.39 (t, J = 6.9 Hz, 2H), 3.98 (t, J = 6.1 Hz, 2H), 3.40 (t, J = 5.0 Hz, 4H), 2.97 (t, J = 7.6 Hz, 2H), 2.32 (m, 6H), 2.28 (s, 3H), 2.24 (tt, J = 7.6, 6.2 Hz, 2H), 2.06 (p, J = 6.9 Hz, 2H), 1.46 (s, 9H), 1.40 (s, 9H).13C NMR (151 MHz, CDCl3) δ 155.50, 154.70, 147.03, 137.58, 129.12, 127.51, 127.10, 121.23, 111.69, 79.70, 66.58, 54.64, 52.86, 47.90, 44.04, 43.04, 34.74, 29.90, 29.12, 28.41, 27.35, 22.52, 20.78. HRMS: Calculated for C28H46N5O3 [M+H]+500.3601, found 500.3621. -93- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Step 2: Boc deprotection procedure for the synthesis of 1-(3-(4-(3-(2-(tert-butyl)-4- methylphenoxy)propyl)-1H-1,2,3-triazol-1-yl)propyl)piperazine, M56: In a 4 dram vail containing a magnetic stir bar was added tert-butyl 4-(3-(4-(3-(2-(tert-butyl)-4- methylphenoxy)propyl)-1H-1,2,3-triazol-1-yl)propyl)piperazine-1-carboxylate (50 mg, 0.1 mmol), DCM (2 mL), and TFA (0.5 mL). The reaction mixture was stirred at room temperature for 2 h. Solvents were evaporated under reduced pressure, co-evaporated with more DCM (3x5 mL). Then added fresh DCM and washed with 10% aq. NaOH solution, followed by water and brine. Combined organics were dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure to give the 39 mg of the title compound M56 (98%).1H NMR (600 MHz, CDCl3) δ 7.31 (s, 1H), 7.08 (d, J = 2.3 Hz, 1H), 6.97 – 6.93 (m, 1H), 6.73 (d, J = 8.2 Hz, 1H), 4.39 (t, J = 7.0 Hz, 2H), 3.99 (t, J = 6.2 Hz, 2H), 2.97 (t, J = 7.5 Hz, 2H), 2.87 (t, J = 4.9 Hz, 4H), 2.36 (s, 4H), 2.30 (t, J = 6.9 Hz, 2H), 2.28 (s, 3H), 2.24 (tt, J = 7.5, 6.2 Hz, 2H), 2.05 (p, J = 6.9 Hz, 2H), 1.40 (s, 9H).13C NMR (151 MHz, CDCl3) δ 155.51, 146.98, 137.59, 129.10, 127.50, 127.09, 121.29, 111.70, 66.59, 55.20, 54.39, 47.98, 46.05, 34.74, 29.89, 29.15, 27.22, 22.52, 20.78. HRMS: Calculated for C23H38N5O [M+H]+400.3076, found 400.3087. General scheme for the synthesis of M58: Conditions: a) K2CO3, Acetone, reflux, overnight, 98%; b) CuSO4·5H2O, sodium ascorbate, THF:H2O, r.t, 2 h, Ar atm, 79%; c) TFA:DCM,. r.t, 2 h, > 98%. Step 1: 2-(tert-butyl)-4-methyl-1-(prop-2-yn-1-yloxy)benzene was synthesized following the literature procedure5. To a solution of 2-(tert-butyl)-4-methylphenol (1.0 g, 6.088 mmol, 1.0 equiv) in acetone (45 mL) was added K2CO3 (3.36 g, 24.353 mmol, 4.0 equiv) and 3-bromoprop-1-yne (0.941 g, 7.914 mmol, 1.3 equiv). The resulting mixture was stirred at reflux for overnight, then cooled to room temperature, filtered off the solids and -94- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) the filtrate was concentrated under reduced pressure. The crude product was purified by flash column chromatography on silica gel eluting with 5-10% DCM in hexanes to give 1.2 g of the title compound (98%).1H NMR (600 MHz, CDCl3) δ 7.10 (d, J = 2.2 Hz, 1H), 7.00 – 6.96 (m, 1H), 6.86 (d, J = 8.2 Hz, 1H), 4.70 (d, J = 2.4 Hz, 2H), 2.47 (t, J = 2.4 Hz, 1H), 2.29 (s, 3H), 1.38 (s, 9H).13C NMR (151 MHz, CDCl3) δ 154.39, 138.49, 130.23, 127.69, 127.04, 112.74, 79.12, 74.86, 55.69, 34.68, 29.89, 20.84. Step 2: Boc protected, triazole intermediate tert-butyl 4-(3-(4-((2-(tert-butyl)-4- methylphenoxy)methyl)-1H-1,2,3-triazol-1-yl)propyl)piperazine-1-carboxylate was synthesized following the general ‘click’ reaction procedure from M56 synthesis, using 2- (tert-butyl)-4-methyl-1-(prop-2-yn-1-yloxy)benzene and tert-butyl 4-(3- azidopropyl)piperazine-1-carboxylate A. (69% yield).1H NMR (600 MHz, CDCl3) δ 7.57 (s, 1H), 7.10 (d, J = 2.2 Hz, 1H), 6.97 (ddd, J = 8.2, 2.3, 0.9 Hz, 1H), 6.87 (d, J = 8.2 Hz, 1H), 5.25 (s, 2H), 4.46 (t, J = 6.8 Hz, 2H), 3.40 (s, 4H), 2.38 – 2.22 (m, 9H), 2.09 (p, J = 6.8 Hz, 2H), 1.46 (s, 9H), 1.37 (s, 9H).13C NMR (151 MHz, CDCl3) δ 154.72, 154.68, 144.79, 137.92, 129.90, 127.66, 127.22, 122.71, 112.46, 79.71, 62.32, 54.31, 52.82, 48.00, 44.06*, 43.04 *, 34.70, 29.87, 28.41, 27.13, 20.81. (* peak split). HRMS: Calculated for C26H42N5O3 [M+H]+472.3288, found 472.3308. Step 3: M58 was prepared by the Boc-deprotection of tert-butyl 4-(3-(4-((2-(tert- butyl)-4-methylphenoxy)methyl)-1H-1,2,3-triazol-1-yl)propyl)piperazine-1-carboxylate following the general procedure from M56 synthesis, (98% yield).1H NMR (600 MHz, CDCl3) δ 7.58 (s, 1H), 7.09 (d, J = 2.3 Hz, 1H), 6.97 (dd, J = 8.4, 2.2 Hz, 1H), 6.88 (d, J = 8.2 Hz, 1H), 5.25 (s, 2H), 4.45 (t, J = 6.8 Hz, 2H), 2.88 (s, 4H), 2.51 – 2.30 (m, 4H), 2.28 (s, t, 5H, overlap), 2.08 (p, J = 6.8 Hz, 2H), 1.37 (s, 9H).13C NMR (151 MHz, CDCl3) δ 154.77, 144.71, 137.92, 129.87, 127.64, 127.22, 122.72, 112.47, 62.38, 54.86, 54.31, 48.04, 46.05, 34.70, 29.86, 27.03, 20.81. HRMS: Calculated for C21H34N5O [M+H]+372.2763, found 372.2763. General scheme for the synthesis of M59: -95- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Conditions: a) Cs2CO3, KI, MeCN, 85oC, overnight, 96%; b) CuSO4·5H2O, sodium ascorbate, THF:H2O, r.t, 2 h, Ar atm, 85%; c) TFA:DCM, r.t, 2 h, > 94%. Step 1: Alkyne intermediate 1,3-diisopropyl-2-(pent-4-yn-1-yloxy)benzene, was prepared following the literature procedure6. A round bottom flask equipped with a stir bar was charged with 2,6-diisopropylphenol (0.5 g, 2.805 mmol, 1.0 equiv), Cs2CO3(1.37 g, 4.2075 mmol, 1.5 equiv), KI (46.56 mg, 0.2805 mmol, 0.1 equiv), acetonitrile (10 mL) and the contents were stirred at room temperature for 10 min. Then, 5-chloropent-1-yne (373.98 mg, 3.64 mmol, 1.3 equiv (0.386 mL)) was added via syringe, and the resulting mixture was heated at 85oC for overnight under argon atmosphere. Then the reaction mixture was cooled to room temperature, filtered, concentrated under reduced pressure. The crude product was purified by flash column chromatography on silica gel column eluting with 5- 10% DCM in hexanes to afford 660 mg of the title compound as thick oil (96%).1H NMR (600 MHz, CDCl3) δ 7.10 (d, J = 1.3 Hz, 3H), 3.84 (t, J = 6.1 Hz, 2H), 3.32 (hept, J = 6.9 Hz, 2H), 2.50 (td, J = 7.0, 2.7 Hz, 2H), 2.06 – 2.00 (m, 2H), 1.99 (t, J = 2.7 Hz, 1H), 1.23 (d, J = 6.9 Hz, 12H).13C NMR (151 MHz, CDCl3) δ 153.03, 141.83, 124.50, 123.97, 83.63, 72.56, 68.73, 29.20, 26.33, 24.10, 15.21. Step 2: The boc-protected triazole derivative tert-butyl 4-(3-(4-(3-(2,6- diisopropylphenoxy)propyl)-1H-1,2,3-triazol-1-yl)propyl)piperazine-1-carboxylate was prepared following the general ‘click’ reaction procedure from the synthesis of M56, using 1,3-diisopropyl-2-(pent-4-yn-1-yloxy)benzene and tert-butyl 4-(3-azidopropyl)piperazine-1- carboxylate, (85%).1H NMR (600 MHz, CDCl3) δ 7.34 (s, 1H), 7.12 – 7.06 (m, 3H), 4.41 (t, J = 7.0 Hz, 2H), 3.80 (t, J = 6.3 Hz, 2H), 3.43 (s, 4H), 3.29 (hept, J = 6.9 Hz, 2H), 3.04 – 2.95 (m, 2H), 2.35 (m, 6H), 2.28 – 2.17 (m, 2H), 2.12 – 2.02 (m, 2H), 1.46 (s, 9H), 1.21 (d, J = 6.9 Hz, 12H).13C NMR (151 MHz, CDCl3) δ 154.71, 153.17, 147.50, 141.72, 124.49, -96- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) 123.97, 120.93, 79.72, 73.86, 54.70, 52.90, 47.96, 44.06*, 43.05*, 30.13, 28.41, 27.41, 26.41, 24.10, 22.48. (* peak split). HRMS: Calculated for C29H48N5O3 [M+H]+514.3757, found 514.3740. Step 3: 1-(3-(4-(3-(2,6-diisopropylphenoxy)propyl)-1H-1,2,3-triazol-1- yl)propyl)piperazine M59 ,was synthesized by the Boc-deprotection of tert-butyl 4-(3-(4-(3- (2,6-diisopropylphenoxy)propyl)-1H-1,2,3-triazol-1-yl)propyl)piperazine-1-carboxylate, following the general procedure from M56 synthesis. (Yield 94%).1H NMR (600 MHz, CDCl3) δ 7.35 (s, 1H), 7.09 (m, 3H), 4.41 (t, J = 7.0 Hz, 2H), 3.80 (t, J = 6.3 Hz, 2H), 3.29 (hept, J = 6.9 Hz, 2H), 3.04 – 2.95 (m, 2H), 2.89 (t, J = 4.9 Hz, 4H), 2.39 (s, 4H), 2.33 (t, J = 6.9 Hz, 2H), 2.26 – 2.18 (m, 2H), 2.07 (p, J = 6.9 Hz, 2H), 1.21 (d, J = 6.9 Hz, 12H).13C NMR (151 MHz, CDCl3) δ 153.19, 147.44, 141.73, 124.47, 123.97, 120.97, 73.88, 55.27, 54.46, 48.04, 46.10, 30.15, 27.30, 26.42, 24.10, 22.48. HRMS: Calculated for C24H40N5O [M+H]+414.3233, found 414.3223. Synthesis of M60: Conditions: a) K2CO3, Acetone, reflux, overnight, 99%; b) CuSO4•5H2O, sodium ascorbate, THF:H2O, r.t, 2 h, Ar atm, 99%; c) TFA:DCM, r.t, 2 h, > 87%. Step 1: The alkyne intermediate 1,3-diisopropyl-2-(prop-2-yn-1-yloxy)benzene was prepared by following the Step 1 of the general procedure in M58 synthesis, using 2,6- diisopropylphenol and 3-bromoprop-1-yne (99% yield).1H NMR (600 MHz, CDCl3) δ 7.11 (d, J = 1.1 Hz, 3H), 4.46 (d, J = 2.4 Hz, 2H), 3.39 (hept, J = 6.9 Hz, 2H), 2.56 (t, J = 2.4 Hz, 1H), 1.24 (d, J = 6.9 Hz, 12H).13C NMR (151 MHz, CDCl3) δ 152.64, 141.96, 125.20, 124.09, 79.17, 75.03, 61.93, 26.65, 24.07. -97- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Step 2: The boc-protected triazole derivative tert-butyl 4-(3-(4-((2,6- diisopropylphenoxy)methyl)-1H-1,2,3-triazol-1-yl)propyl)piperazine-1-carboxylate was prepared following the general ‘click’ reaction procedure from M56 synthesis, using 1,3- diisopropyl-2-(prop-2-yn-1-yloxy)benzene and tert-butyl 4-(3-azidopropyl)piperazine-1- carboxylate (99% yield).1H NMR (600 MHz, CDCl3) δ 7.68 (s, 1H), 7.14 (s, 3H), 4.95 (s, 2H), 4.49 (t, J = 6.9 Hz, 2H), 3.44 (t, J = 4.9 Hz, 4H), 3.38 (p, J = 6.9 Hz, 2H), 2.38 (m, 6H), 2.12 (p, J = 6.8 Hz, 2H), 1.46 (s, 9H), 1.24 (d, J = 6.9 Hz, 12H).13C NMR (151 MHz, CDCl3) δ 154.70, 152.65, 144.70, 141.85, 125.04, 124.15, 122.71, 79.73, 68.25, 54.63, 52.90, 48.18, 44.12*, 43.12*, 28.41, 27.30, 26.59, 24.10. (* peak split). HRMS: Calculated for C27H44N5O3[M+H]+486.3444, found 486.3467. Step 3: 1-(3-(4-((2,6-diisopropylphenoxy)methyl)-1H-1,2,3-triazol-1- yl)propyl)piperazine M60, was prepared by the Boc-deprotection of tert-butyl 4-(3-(4-((2,6- diisopropylphenoxy)methyl)-1H-1,2,3-triazol-1-yl)propyl)piperazine-1-carboxylate, following the general procedure from M56 synthesis (>87% yield).1H NMR (600 MHz, CDCl3) δ 7.69 (s, 1H), 7.14 (s, 3H), 4.95 (s, 2H), 4.48 (t, J = 7.0 Hz, 2H), 3.39 (hept, J = 6.9 Hz, 2H), 2.91 (t, J = 4.9 Hz, 4H), 2.41 (br s, 4H), 2.35 (t, J = 6.8 Hz, 2H), 2.12 (p, J = 6.9 Hz, 2H), 1.24 (d, J = 6.9 Hz, 12H).13C NMR (151 MHz, CDCl3) δ 152.68, 144.63, 141.86, 125.02, 124.14, 122.77, 68.27, 55.12, 54.43, 48.20, 46.11, 27.18, 26.59, 24.10. HRMS: Calculated for C22H36N5O [M+H]+386.2920, found 386.2906. Synthesis of M61: Conditions: a) K2CO3, Acetone, reflux, overnight, 85%; b) CuSO4•5H2O, sodium ascorbate, THF:H2O, r.t, 2 h, Ar atm, 92%; c) TFA:DCM, r.t, 2 h, 96%. -98- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Step 1: The alkyne intermediate 1,3-dimethyl-2-(prop-2-yn-1-yloxy)benzene, was prepared following the general procedure of Step-1 from M58 synthesis, using 2,6- dimethylphenol and 3-bromoprop-1-yne (85% yield).1H NMR (600 MHz, CDCl3) δ 7.01 (d, J = 6.8 Hz, 2H), 6.97 – 6.93 (m, 1H), 4.50 (d, J = 2.4 Hz, 2H), 2.51 (t, J = 2.5 Hz, 1H), 2.32 (s, 6H).13C NMR (151 MHz, CDCl3) δ 155.22, 131.17, 128.84, 124.42, 79.35, 74.90, 59.72, 16.50. Step 2: The Boc-protected triazole intermediate tert-butyl 4-(3-(4-((2,6- dimethylphenoxy)methyl)-1H-1,2,3-triazol-1-yl)propyl)piperazine-1-carboxylate was prepared following the general ‘click’ reaction procedure from M56 synthesis, using 1,3- dimethyl-2-(prop-2-yn-1-yloxy)benzene and tert-butyl 4-(3-azidopropyl)piperazine-1- carboxylate (92% yield).1H NMR (600 MHz, CDCl3) δ 7.65 (s, 1H), 7.03 (d, J = 7.3 Hz, 2H), 6.96 (dd, J = 8.1, 6.8 Hz, 1H), 4.97 (s, 2H), 4.47 (t, J = 6.9 Hz, 2H), 3.43 (t, J = 5.2 Hz, 4H), 2.36 (t, J = 6.5 Hz, 6H, overlap), 2.30 (s, 6H), 2.11 (p, J = 6.9 Hz, 2H), 1.46 (s, 9H).13C NMR (151 MHz, CDCl3) δ 155.34, 154.71, 144.65, 131.03, 128.91, 124.26, 122.84, 79.73, 65.69, 54.58, 52.88, 48.15, 44.04*, 43.05*, 28.41, 27.31, 16.38. (* peak split). ESI- MS: m / z 430.48 [M+H]+. Step 3: 1-(3-(4-((2,6-dimethylphenoxy)methyl)-1H-1,2,3-triazol-1- yl)propyl)piperazine M61, was prepared by the Boc-deprotection of tert-butyl 4-(3-(4-((2,6- dimethylphenoxy)methyl)-1H-1,2,3-triazol-1-yl)propyl)piperazine-1-carboxylate, following the general procedure from M56 synthesis (96% yield).1H NMR (600 MHz, CDCl3) δ 7.66 (s, 1H), 7.03 (d, J = 7.4 Hz, 2H), 6.96 (dd, J = 8.1, 6.8 Hz, 1H), 4.98 (s, 2H), 4.47 (t, J = 6.9 Hz, 2H), 2.90 (t, J = 4.9 Hz, 4H), 2.40 (br s, 4H), 2.34 (t, J = 6.9 Hz, 2H), 2.30 (s, 6H), 2.10 (p, J = 6.9 Hz, 2H).13C NMR (151 MHz, CDCl3) δ 155.37, 144.59, 131.04, 128.90, 124.24, 122.90, 65.71, 55.11, 54.40, 48.20, 46.08, 27.19, 16.38. HRMS: Calculated for C18H28N5O [M+H]+330.2294, found 330.2282. Synthesis of M62: -99- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Conditions: a) Cs2CO3, KI, MeCN, 85oC, overnight, 82%; b) CuSO4·5H2O, sodium ascorbate, THF:H2O, r.t, 2 h, Ar atm, 90%; c) TFA:DCM, r.t, 2 h, 95%. Step 1: The alkyne intermediate 1,3-dimethyl-2-(pent-4-yn-1-yloxy)benzene was prepared using 2,6-dimethylphenol and 5-chloropent-1-yne following the general procedure from Step-1 of M59 synthesis (82% yield).1H NMR (600 MHz, CDCl3) δ 7.01 (d, J = 7.4 Hz, 2H), 6.92 (dd, J = 8.0, 6.9 Hz, 1H), 3.87 (t, J = 6.1 Hz, 2H), 2.49 (td, J = 7.0, 2.7 Hz, 2H), 2.28 (s, 6H), 2.05 – 1.99 (m, 2H), 1.98 (t, J = 2.7 Hz, 1H).13C NMR (151 MHz, CDCl3) δ 155.69, 130.96, 128.80, 123.77, 83.76, 70.00, 68.76, 29.22, 16.24, 15.25. Step 2: The Boc-protected triazole intermediate tert-butyl 4-(3-(4-(3-(2,6- dimethylphenoxy)propyl)-1H-1,2,3-triazol-1-yl)propyl)piperazine-1-carboxylate was prepared following the general ‘click’ reaction procedure from M56 synthesis, using 1,3- dimethyl-2-(pent-4-yn-1-yloxy)benzene and tert-butyl 4-(3-azidopropyl)piperazine-1- carboxylate (90% yield).1H NMR (600 MHz, CDCl3) δ 7.34 (s, 1H), 7.00 (d, J = 8.0 Hz, 2H), 6.94 – 6.89 (m, 1H), 4.41 (t, J = 6.9 Hz, 2H), 3.83 (t, J = 6.3 Hz, 2H), 3.49 – 3.36 (m, 4H), 3.02 – 2.95 (m, 2H), 2.35 (q, J = 6.4 Hz, 6H, overlap), 2.27 (s, 6H), 2.25 – 2.15 (m, 2H), 2.08 (p, J = 6.9 Hz, 2H), 1.46 (s, 9H).13C NMR (151 MHz, CDCl3) δ 155.82, 154.71, 147.50, 130.85, 128.81, 123.75, 121.00, 79.72, 71.23, 54.68, 52.90, 47.94, 44.07*, 43.05*, 30.14, 28.41, 27.39, 22.48, 16.31. (* peak split, from piperazine ring C). ESI-MS, C25H40N5O3 m / z 458.51 [M+H]+. Step 3: 1-(3-(4-(3-(2,6-dimethylphenoxy)propyl)-1H-1,2,3-triazol-1- yl)propyl)piperazine M62, was prepared by the Boc-deprotection of tert-butyl 4-(3-(4-(3- (2,6-dimethylphenoxy)propyl)-1H-1,2,3-triazol-1-yl)propyl)piperazine-1-carboxylate, following the general procedure from M56 synthesis (95% yield).1H NMR (600 MHz, -100- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) CDCl3) δ 7.35 (s, 1H), 7.00 (d, J = 7.3 Hz, 2H), 6.94 – 6.88 (m, 1H), 4.41 (t, J = 7.0 Hz, 2H), 3.83 (t, J = 6.3 Hz, 2H), 3.02 – 2.95 (m, 2H), 2.89 (t, J = 4.9 Hz, 4H), 2.38 (br s, 4H), 2.32 (t, J = 6.9 Hz, 2H), 2.27 (s, 6H), 2.25 – 2.15 (m, 2H), 2.07 (p, J = 6.9 Hz, 2H).13C NMR (151 MHz, CDCl3) δ 155.83, 147.43, 130.86, 128.80, 123.74, 121.04, 71.23, 55.25, 54.44, 48.02, 46.08, 30.15, 27.27, 22.48, 16.31. HRMS: Calculated for C20H32N5O [M+H]+358.2607, found 358.2604. Synthesis of M63: Conditions: a) K2CO3, Acetone, reflux, overnight, 66%; b) CuSO4•5H2O, sodium ascorbate, THF:H2O, r.t, 2 h, Ar atm, 92%; c) TFA:DCM, r.t, 2 h, 76%. Step 1: The alkyne intermediate 2-(tert-butyl)-4-methoxy-1-(prop-2-yn-1- yloxy)benzene was prepared following the procedure from Step-1 of M58 synthesis, using 2-(tert-butyl)-4-methoxyphenol and 3-bromoprop-1-yne (66% yield).1H NMR (600 MHz, CDCl3) δ 6.92 – 6.88 (m, 2H), 6.69 (dd, J = 8.8, 3.1 Hz, 1H), 4.68 (d, J = 2.4 Hz, 2H), 3.77 (s, 3H), 2.48 (t, J = 2.4 Hz, 1H), 1.38 (s, 9H).13C NMR (151 MHz, CDCl3) δ 153.80, 150.81, 140.47, 114.36, 113.63, 109.67, 79.21, 74.86, 56.24, 55.55, 34.91, 29.78. Step 2: The Boc-Protected triazole intermediate tert-butyl 4-(3-(4-((2-(tert-butyl)-4- methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-yl)propyl)piperazine-1-carboxylate was prepared following the general ‘click’ reaction procedure from M56 synthesis, using the 2- (tert-butyl)-4-methoxy-1-(prop-2-yn-1-yloxy)benzene and tert-butyl 4-(3- azidopropyl)piperazine-1-carboxylate (92% yield).1H NMR (600 MHz, CDCl3) δ 7.57 (s, 1H), 6.93 – 6.88 (m, 2H), 6.69 (dd, J = 8.8, 3.1 Hz, 1H), 5.22 (s, 2H), 4.46 (t, J = 6.8 Hz, 2H), 3.77 (s, 3H), 3.41 (br t, J = 4.8 Hz, 4H), 2.32 (dt, J = 13.4, 5.7 Hz, 6H, overlap), 2.10 (p, J = 6.8 Hz, 2H), 1.46 (s, 9H), 1.37 (s, 9H).13C NMR (151 MHz, CDCl3) δ 154.69, -101- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) 153.58, 151.14, 144.77, 139.85, 122.66, 114.46, 113.25, 109.76, 79.73, 62.81, 55.57, 54.37, 52.82, 48.02, 44.03, 43.04, 34.92, 29.75, 28.41, 27.15. ). ESI-MS, C26H42N5O4 m / z 488.49 [M+H]+. Step 3: 1-(3-(4-((2-(tert-butyl)-4-methoxyphenoxy)methyl)-1H-1,2,3-triazol-1- yl)propyl)piperazine M63, was prepared by the Boc-deprotection of tert-butyl 4-(3-(4-((2- (tert-butyl)-4-methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-yl)propyl)piperazine-1- carboxylate, following the general procedure from M56 synthesis (76% yield).1H NMR (600 MHz, CDCl3) δ 7.58 (s, 1H), 6.94 – 6.88 (m, 2H), 6.68 (dd, J = 8.8, 3.1 Hz, 1H), 5.23 (s, 2H), 4.46 (t, J = 6.8 Hz, 2H), 3.77 (s, 3H), 2.87 (t, J = 4.9 Hz, 4H), 2.35 (br s, 4H), 2.28 (t, J = 6.8 Hz, 2H), 2.08 (p, J = 6.8 Hz, 2H), 1.37 (s, 9H).13C NMR (151 MHz, CDCl3) δ 153.55, 151.16, 144.71, 139.86, 122.72, 114.47, 113.23, 109.74, 62.84, 55.57, 54.88, 54.36, 48.06, 46.10, 34.93, 29.75, 27.04. HRMS: Calculated for C21H34N5O2 [M+H]+388.2713, found 388.2712. Synthesis of M64: Conditions: a) Cs2CO3, KI, MeCN, 85oC, overnight, 50%; b) CuSO4•5H2O, sodium ascorbate, THF:H2O, r.t, 2 h, Ar atm, 90%; c) TFA:DCM, r.t, 2 h, 75%. Step 1: The alkyne intermediate 2-(tert-butyl)-4-methoxy-1-(pent-4-yn-1- yloxy)benzene was prepared following the procedure from Step-1 of M59 synthesis, using 2-(tert-butyl)-4-methoxyphenol and 5-chloropent-1-yne (50% yield).1H NMR (600 MHz, CDCl3) δ 6.89 (d, J = 3.1 Hz, 1H), 6.80 (d, J = 8.8 Hz, 1H), 6.68 (dd, J = 8.8, 3.1 Hz, 1H), 4.04 (t, J = 6.1 Hz, 2H), 3.77 (s, 3H), 2.46 (td, J = 7.1, 2.7 Hz, 2H), 2.10 – 2.02 (m, 2H), 1.98 (t, J = 2.7 Hz, 1H), 1.37 (s, 9H).13C NMR (151 MHz, CDCl3) δ 153.13, 151.88, 139.54, 114.40, 112.35, 109.67, 83.53, 68.89, 66.47, 55.62, 34.93, 29.75, 28.50, 15.68. -102- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Step 2: The Boc-Protected triazole intermediate tert-butyl 4-(3-(4-(3-(2-(tert-butyl)- 4-methoxyphenoxy)propyl)-1H-1,2,3-triazol-1-yl)propyl)piperazine-1-carboxylate was prepared following the general ‘click’ reaction procedure from M56 synthesis, using 2-(tert- butyl)-4-methoxy-1-(pent-4-yn-1-yloxy)benzene and tert-butyl 4-(3-azidopropyl)piperazine- 1-carboxylate (90% yield).1H NMR (600 MHz, CDCl3) δ 7.31 (s, 1H), 6.89 (d, J = 3.1 Hz, 1H), 6.76 (d, J = 8.8 Hz, 1H), 6.66 (dd, J = 8.8, 3.1 Hz, 1H), 4.40 (t, J = 6.9 Hz, 2H), 3.97 (t, J = 6.2 Hz, 2H), 3.76 (s, 3H), 3.41 (br t, J = 5.1 Hz, 4H), 2.96 (t, J = 7.6 Hz, 2H), 2.33 (t, J = 6.7 Hz, 6H), 2.23 (tt, J = 7.6, 6.2 Hz, 2H), 2.07 (p, J = 6.9 Hz, 2H), 1.46 (s, 9H), 1.39 (s, 9H).13C NMR (151 MHz, CDCl3) δ 154.69, 153.09, 151.93, 147.08, 139.45, 121.21, 114.40, 112.23, 109.64, 79.71, 67.05, 55.61, 54.65, 52.85, 47.92, 44.03*, 43.01*, 34.96, 29.77, 29.20, 28.41, 27.33, 22.53. (* peak split, from piperazine ring). ESI-MS, C28H46N5O4 m / z 516.58 [M+H]+. Step 3: 1-(3-(4-(3-(2-(tert-butyl)-4-methoxyphenoxy)propyl)-1H-1,2,3-triazol-1- yl)propyl)piperazine, M64 was prepared by the Boc-deprotection of tert-butyl 4-(3-(4-(3-(2- (tert-butyl)-4-methoxyphenoxy)propyl)-1H-1,2,3-triazol-1-yl)propyl)piperazine-1- carboxylate, following the general procedure from M56 synthesis (75% yield).1H NMR (600 MHz, CDCl3) δ 7.32 (s, 1H), 6.89 (d, J = 3.1 Hz, 1H), 6.75 (d, J = 8.8 Hz, 1H), 6.69 – 6.63 (m, 1H), 4.39 (t, J = 6.9 Hz, 2H), 3.97 (t, J = 6.2 Hz, 2H), 3.76 (s, 3H), 2.96 (t, J = 7.6 Hz, 2H), 2.87 (t, J = 4.9 Hz, 4H), 2.36 (br s, 4H), 2.30 (t, J = 6.9 Hz, 2H), 2.23 (dq, J = 7.6, 6.2 Hz, 2H), 2.06 (p, J = 6.9 Hz, 2H), 1.39 (s, 9H).13C NMR (151 MHz, CDCl3) δ 153.07, 151.94, 147.01, 139.45, 121.27, 114.40, 112.22, 109.64, 67.04, 55.61, 55.21, 54.40, 47.98, 46.06, 34.96, 29.77, 29.21, 27.22, 22.53. HRMS: Calculated for C23H38N5O2[M+H]+416.3026, found 416.3016. Synthesis of M66: Conditions: a) TEA, DCM, r.t, 24 h (~33%); b) NaN3, DMF, 60oC, 3 days (85%); c) 1 = 1,3-diisopropyl-2-(prop-2-yn-1-yloxy)benzene)*, CuSO4•5H2O, sodium ascorbate, -103- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) THF:H2O, r.t, 2 h, Ar atm; d) TFA:DCM, r.t, 2 h, 70% over two steps. (* alkyne intermediate 1 was prepared in Step-1 of M60 synthesis) Step 1: The bromo derivative tert-butyl 4-(3-bromopropyl)-1,4-diazepane-1- carboxylate was prepared following the general procedure used to prepare ‘azido intermediate’ in the Step-1 of M56 synthesis, using 1,3-dibromopropane and 1-Boc- hexahydro-1,4-diazepine (33% yield).1H NMR (600 MHz, CDCl3) δ 3.61 (d, J = 2.2 Hz, 0.6H), 3.47 (qd, J = 6.3, 4.2 Hz, 3.5 H), 3.45 – 3.38 (m, 2H), 2.69 – 2.56 (m, 6H), 1.99 (pd, J = 6.6, 2.7 Hz, 1.6 H), 1.91 (td, J = 6.8, 2.7 Hz, 0.6H), 1.85 – 1.75 (m, 2H), 1.46 (s, 9H). (* peak split due to ring isomerization).13C NMR (151 MHz, CDCl3) δ 155.61, 155.50*, 79.30, 79.27*, 56.02, 55.96*, 55.45, 55.41*, 54.96, 54.53*, 54.40, 54.37*, 46.88, 46.30*, 46.03, 45.14*, 43.17, 31.94, 31.91*, 30.64, 30.54*, 28.50, 27.81*. (* peak split, rotamer peaks). Step 2: The azido intermediate tert-butyl 4-(3-azidopropyl)-1,4-diazepane-1- carboxylate was prepared following the general procedure in Step-2 of M56 synthesis, reacting the bromo intermediate tert-butyl 4-(3-bromopropyl)-1,4-diazepane-1-carboxylate with NaN3, in DMF, at 60oC for ~ 3 days (85% yield). LCMS: m / z 284.4 [M+H]+. (directly used in next step for ‘click’ reaction without further characterization). Step 3: Cu(I) ‘click’ reaction was performed following the general procedure from M56 synthesis using tert-butyl 4-(3-azidopropyl)-1,4-diazepane-1-carboxylate, and alkyne intermediate 1 to afford a Boc-protected triazole intermediate, which was directly used in the Boc-deprotection step to give M66 as colorless, thick oil (70% over two steps).1H NMR (600 MHz, CDCl3) δ 7.69 (s, 1H), 7.14 (s, 3H), 4.95 (s, 2H), 4.50 (t, J = 6.9 Hz, 2H), 3.39 (hept, J = 6.9 Hz, 2H), 2.96 (t, J = 6.1 Hz, 2H), 2.95 – 2.90 (m, 2H), 2.71 – 2.68 (m, 2H), 2.68 – 2.64 (m, 2H), 2.52 (t, J = 6.7 Hz, 2H), 2.09 (p, J = 6.8 Hz, 2H), 1.79 (p, J = 6.0 Hz, 2H), 1.24 (d, J = 6.9 Hz, 12H). (NH integration was ignored for clarity).13C NMR (151 MHz, CDCl3) δ 152.68, 144.59, 141.87, 125.02, 124.14, 122.79, 68.27, 58.12, 54.34, 54.29, 49.02, 48.12, 47.39, 30.32, 28.35, 26.59, 24.10. HRMS: Calculated for C23H38N5O [M+H]+400.3076, found 400.3081. Synthesis of M65: -104- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Conditions: a) 1-Boc-hexahydro-1,4-diazepine, K2CO3, DMF, r.t, overnight, 80%; b) TFA / DCM, r.t, 2 h, 91%. M65 was synthesized following the general procedure from M6 synthesis using C8- Br (2.17 g, 1.06mmol, 1.0 equiv) intermediate and 1-Boc-hexahydro-1,4-diazepine (1.223g, 1.0 equiv) in two steps. Step 1: tert-butyl 4-(8-(2-(tert-butyl)-4-methylphenoxy)octyl)-1,4-diazepane-1- carboxylate (2.4 g, 83%).1H NMR (600 MHz, CDCl3) δ 7.07 (d, J = 2.2 Hz, 1H), 6.97 – 6.93 (m, 1H), 6.75 (d, J = 8.2 Hz, 1H), 3.94 (t, J = 6.5 Hz, 2H), 3.54 – 3.39 (m, 4H), 2.68 – 2.56 (m, 4H), 2.48 – 2.41 (m, 2H), 2.28 (s, 3H), 1.89 – 1.76 (m, 4H), 1.46 (m, s, 13H, overlap), 1.38 (s, m, 15H, overlap).13C NMR (151 MHz, CDCl3) δ 155.74, 155.65, 155.55*, 137.67, 128.84, 128.82*, 127.43, 127.03, 111.72, 79.22, 79.19*, 67.74, 57.94, 57.91*, 56.13, 55.95*, 54.92, 54.67*, 46.73, 46.14*, 45.99, 45.03*, 34.72, 29.83, 29.49, 29.47, 29.30, 28.51, 27.84, 27.72*, 27.45(2C), 27.40*, 26.32, 20.78. (* rotamer peak). Step 2: tert-butyl 4-(8-(2-(tert-butyl)-4-methylphenoxy)octyl)-1,4-diazepane-1- carboxylate, M65. Using 1.41 g, 2.97 mmol of Boc-protected intermediate from Step-1, afforded M65 as thick oil (91% yield).1H NMR (600 MHz, CDCl3) δ 7.07 (d, J = 2.2 Hz, 1H), 6.95 (dd, J = 8.2, 2.2 Hz, 1H), 6.75 (d, J = 8.2 Hz, 1H), 3.94 (t, J = 6.5 Hz, 2H), 2.94 (t, J = 6.1 Hz, 2H), 2.92 – 2.88 (t, J = 6 Hz, 2H), 2.71 – 2.67 (t, J = 6 Hz, 2H), 2.67 – 2.63 (t, J = 6 Hz, 2H), 2.50 – 2.45 (t, J = 6 Hz, 2H), 2.28 (s, 3H), 1.86 – 1.79 (m, 2H), 1.77 (p, J = 6.0 Hz, 3H), 1.49 (m, 4H), 1.38 (s, m, 15H, overlap).13C NMR (151 MHz, CDCl3) δ -105- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) 155.74, 137.66, 128.82, 127.42, 127.03, 111.71, 67.74, 58.61, 58.49, 54.57, 48.79, 47.42, 34.72, 30.41, 29.82, 29.50, 29.49, 29.30, 27.50, 27.49, 26.32, 20.78. HRMS: Calculated for C24H43N2O [M+H]+375.3375, found 375.3391. Synthesis of M69: Conditions: a) K2CO3, Acetone, reflux, 24 h (54%); b) 1-Boc-hexahydro-1,4-diazepine, K2CO3, DMF, r.t, overnight (74%); c) TFA / DCM, r.t, 2 h (96%). M69 was synthesized in two steps following the general procedure from M6 synthesis. Step 1: The bromo-intermediate 2-(4-bromobutoxy)-1,3-diisopropylbenzene was prepared (0.958 g, 54% yield) 2,6-diisopropylphenol (1.0 g, 5.6 mmol, 1.0 equiv) and 1,4- dibromobutane (2.42 g, 11.22 mmol, 2.0 equiv).1H NMR (600 MHz, CDCl3) δ 7.13 – 7.06 (m, 3H), 3.77 (t, J = 6.2 Hz, 2H), 3.53 (t, J = 6.7 Hz, 2H), 3.27 (hept, J = 6.9 Hz, 2H), 2.19 – 2.10 (m, 2H), 2.02 – 1.93 (m, 2H), 1.23 (s, 12H).13C NMR (151 MHz, CDCl3) δ 153.18, 141.72, 124.50, 123.98, 73.66, 33.71, 29.61, 29.14, 26.47, 24.08. Step 2: 1-(4-(2,6-diisopropylphenoxy)butyl)-1,4-diazepane, M69 was prepared in two consecutive steps using the bromo-intermediate 2-(4-bromobutoxy)-1,3- diisopropylbenzene (93.8 mg, 1.2 equiv), and 1-Boc-hexahydro-1,4-diazepine (50 mg, 0.25 mmol, 1.0 equiv) to afford a Boc-protected intermediate (80 mg, 74% yield), which was directly used in the next step for Boc-deprotection to afford M69 (96%).1H NMR (600 MHz, CDCl3) δ 7.13 – 7.04 (m, 3H), 3.74 (t, J = 6.5 Hz, 2H), 3.31 (hept, J = 6.9 Hz, 2H), 2.98 – 2.91 (m, 4H), 2.75 – 2.71 (m, 2H), 2.71 – 2.68 (m, 2H), 2.61 – 2.57 (m, 2H), 1.87 – 1.81 (m, 2H), 1.79 (p, J = 5.9 Hz, 2H), 1.71 (tt, J = 9.8, 6.2 Hz, 2H), 1.22 (d, J = 6.9 Hz, 12H).13C NMR (151 MHz, CDCl3) δ 153.36, 141.79, 124.36, 123.93, 74.66, 58.41, 58.29, 54.54, 48.91, 47.43, 30.43, 28.37, 26.41, 24.13, 24.11. HRMS: Calculated for C21H37N2O [M+H]+333.2906, found 333.2919. -106- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Synthesis of M70: Conditions: a) K2CO3, Acetone, reflux, 24 h (41%); b) 1-Boc-1,4-diazepane, K2CO3, DMF, r.t, overnight (83%); c) TFA / DCM, r.t, 2 h (91%). M70 was synthesized in two steps following the general procedure from M6 synthesis. Step 1: The bromo-intermediate 2-((8-bromooctyl)oxy)-1,3-diisopropylbenzene was prepared using 2,6-diisopropylphenol (1.0 g, 5.6 mmol, 1.0 equiv) and 1,8-dibromooctane (3.052 g, 11.22 mmol, 2.0 equiv) (848 mg, 41% yield).1H NMR (600 MHz, CDCl3) δ 7.12 – 7.05 (m, 3H), 3.72 (t, J = 6.6 Hz, 2H), 3.43 (t, J = 6.8 Hz, 2H), 3.31 (hept, J = 6.9 Hz, 2H), 1.91 – 1.85 (m, 2H), 1.85 – 1.78 (m, 2H), 1.54 – 1.44 (m, 4H), 1.44 – 1.34 (m, 4H), 1.23 (d, J = 6.9 Hz, 12H).13C NMR (151 MHz, CDCl3) δ 153.39, 141.81, 124.33, 123.93, 74.81, 34.03, 32.78, 30.37, 29.33, 28.73, 28.13, 26.37, 25.99, 24.12. Step 2: 1-(8-(2,6-diisopropylphenoxy)octyl)-1,4-diazepane, M70 was prepared in two consecutive steps using bromo-intermediate 2-((8-bromooctyl)oxy)-1,3- diisopropylbenzene (110.82 mg, 0.3 mmol, 1.2 equiv) and 1-Boc-hexahydro-1,4-diazepine (50 mg, 0.249 mmol, 1.0 equiv) to afford a Boc-protected intermediate (101.3 mg, 83%), which was directly used in the Boc-deprotection step to afford M70 (~ 60 mg, 91% yield).1H NMR (600 MHz, CDCl3) δ 7.12 – 7.05 (m, 3H), 3.72 (t, J = 6.6 Hz, 2H), 3.31 (hept, J = 6.9 Hz, 2H), 2.97 – 2.88 (m, 4H), 2.72 – 2.68 (m, 2H), 2.68 – 2.63 (m, 2H), 2.51 – 2.45 (m, 2H), 1.86 – 1.79 (m, 2H), 1.77 (p, J = 6.0 Hz, 3H, overlap)), 1.49 (tdd, J = 12.2, 6.2, 3.6 Hz, 4H), 1.42 – 1.27 (m, 6H), 1.22 (d, J = 6.9 Hz, 12H).13C NMR (151 MHz, CDCl3) δ 153.42, 141.82, 124.31, 123.92, 74.89, 58.61, 58.50, 54.57, 48.80, 47.41, 30.40, 29.57, 29.50, 27.56, 27.55, 26.36 (2C), 26.03, 24.12. HRMS: Calculated for C25H45N2O [M+H]+389.3532, found 389.3523. Synthesis of M84: -107- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Conditions: a) K2CO3, DMF, r.t, 12 h, 71%. Step 1: Synthesis of (4-((4-(4-(2-(tert-butyl)-4-methylphenoxy)butyl)piperazin-1- yl)methyl)phenyl)(4-(prop-2-yn-1-yloxy)phenyl)methanone, M84: This compound was synthesized in one step following the procedure from Step-2 of M1 synthesis, using M1 (15.52 mg, 0.051 mmol, 1.0 equiv) and (4-(bromomethyl)phenyl)(4-(prop-2-yn-1- yloxy)phenyl)methanone (20 mg, 0.061 mmol, 1.2 equiv) as starting materials to afford 20 mg (71% yield) of M84 as light brown solid.1H NMR (600 MHz, CDCl3) δ 7.84 (d, J = 8.8 Hz, 2H), 7.72 (d, J = 8.2 Hz, 2H), 7.44 (d, J = 8.1 Hz, 2H), 7.07 (d, J = 2.3 Hz, 1H), 7.05 (d, J = 8.8 Hz, 2H), 6.95 (dd, J = 8.2, 1.5 Hz, 1H), 6.74 (d, J = 8.2 Hz, 1H), 4.78 (d, J = 2.4 Hz, 2H), 3.96 (t, J = 6.3 Hz, 2H), 3.59 (s, 2H), 2.79 – 2.36 (m, 11H), 2.27 (s, 3H), 1.88-1.83 (m, 2H), 1.75-1.70 (m, 2H), 1.37 (s, 9H).13C NMR (151 MHz, CDCl3) δ 195.39, 161.08, 155.79, 142.95, 137.82, 137.06, 132.54, 131.24, 130.02, 129.12, 129.00, 127.61, 127.19, 114.54, 111.92, 77.96, 76.30, 67.73, 62.79, 58.37, 56.02, 53.19, 34.85, 30.00, 29.84, 27.75, 23.77, 20.92. HRMS: HRMS: Calculated for C36H45N2O3[M+H]+553.3430, found 553.3431. Synthesis of M91 and the probe M92: -108- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Conditions: a) K2CO3, acetone, reflux, overnight, 31%; b) 1-Boc-hexahydro-1,4-diazepine, K2CO3, DMF, r.t, overnight, 64%; c) MeOH, THF, 4N aq.NaOH, 80oC, 2 h, reflux; 1N aq.HCl (pH~3), > 90%; d) 1, HOBt, EDC,TEA, DMF, r.t, 4 h, 80%; e) TFA:DCM, r.t, 2 h, 93%. Step 1: Synthesis of methyl 4-((8-bromooctyl)oxy)benzoate: A round bottom flask containing a stir bar was added methyl 4-hydroxybenzoate (3.0 g, 19.71 mmol, 1.0 equiv) and 1,8-dibromooctane (5.36 g (3.62 mL), 1.0 equiv), K2CO3(5.44 g, 39.42 mmol, 2.0 equiv) and acetone (20 mL). The reaction mixture was stirred at reflux for ~ 12 h under argon atmosphere. Then the reaction mixture was cooled to room temperature, filtered off the solid, concentrate the filtrate to obtain the crude. The crude was purified by flash column chromatography on silica gel column eluting with ExOAc:Hexanes (0-10%, gradient) to afford the title compound as colorless oil (2.1 g, 31%).1H NMR (600 MHz, CDCl3) δ 7.98 (d, J = 8.9 Hz, 2H), 6.90 (d, J = 8.9 Hz, 2H), 4.00 (t, J = 6.5 Hz, 2H), 3.88 (s, 3H), 3.41 (t, J = 6.8 Hz, 2H), 1.86 (dt, J = 14.7, 6.9 Hz, 2H), 1.83 – 1.76 (m, 2H), 1.51 – 1.41 (m, 4H), 1.41 – 1.31 (m, 4H).13C NMR (151 MHz, CDCl3) δ 166.92, 162.90, 131.57, 122.34, 114.04, 68.09, 51.85, 34.00, 32.75, 29.15, 29.06, 28.67, 28.07, 25.90. Step 2: Synthesis of tert-butyl 4-(8-(4-(methoxycarbonyl)phenoxy)octyl)-1,4- diazepane-1-carboxylate: In a 4 dram screw vial, added methyl 4-((8- bromooctyl)oxy)benzoate (0.5 g, 1.46 mmol, 1.0 equiv), 1-Boc-hexahydro-1,4-diazepine (291.3 mg, 1.0 equiv), K2CO3(1.008 g, 7.3 mmol, 5.0 equiv) and dry DMF (5 mL). The reaction mixture was purged and sealed under argon. The reaction mixture was stirred at room temperature for 8 h. Then the solvent was evaporated under reduced pressure, residue was added water (2 mL), extracted with DCM (3x5mL). The combined organic portion was -109- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) washed with water (10 mL) and brine, and organic portion was dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure to obtain a crude product. The crude was further purified by flash column chromatography on silica gel column eluting with EtOAc:Hexanes (20-60%, gradient) to afford the title compound as thick oil (430 mg, 64%).1H NMR (600 MHz, CDCl3) δ 7.98 (d, J = 8.9 Hz, 2H), 6.90 (d, J = 8.9 Hz, 2H), 4.00 (t, J = 6.5 Hz, 2H), 3.88 (s, 3H), 3.52 – 3.49 (m, 1H), 3.47 (t, J = 6.3 Hz, 1H), 3.45 – 3.39 (m, 2H), 2.67 – 2.63 (m, 1H), 2.63 – 2.61 (m, 1H), 2.61 – 2.56 (m, 2H), 2.47 – 2.41 (m, 2H), 1.81 (tt, J = 14.8, 6.2 Hz, 4H), 1.46 (s, 12H), 1.41 – 1.24 (m, 6H).13C NMR (151 MHz, CDCl3) δ 166.92, 162.93, 155.64, 155.54*, 131.56, 122.32, 122.30*, 114.04, 79.21, 79.19*, 68.15, 57.90, 57.84*, 56.09, 55.96*, 54.95, 54.66*, 51.84, 46.74, 46.14*, 45.99, 45.04*, 29.48, 29.47*, 29.31, 29.09*, 28.51, 27.87, 27.74*, 27.48, 27.45*, 27.43, 25.95. (* rotamer peak). Step 3: Synthesis of 4-((8-(4-(tert-butoxycarbonyl)-1,4-diazepan-1- yl)octyl)oxy)benzoic acid7: A round bottom flask was charged with Boc-protected intermediate tert-butyl 4-(8-(4-(methoxycarbonyl)phenoxy)octyl)-1,4-diazepane-1- carboxylate (200 mg, 0.432 mmol), dissolved in MeOH:THF solvent mixture (2:1 mL). Then added 4N aq. NaOH (0.75 mL) and the contents were refluxed at 80oC while monitoring the reaction progress by LCMS. After 2 h, solvents were evaporated under vacuum, residue was taken up in water (4 mL), acidified with 1N Aq. HCl (~ 2-3 mL) to reach the pH ~ 3. The resulting precipitate was filtered off, washed with cold water (2 mL), dried under vacuum overnight to afford the carboxylic acid derivate as white solid (175 mg, ~ 90% yield, ESI-MS, m / z 449.4 [M+H]+found), which was directly used in the next step of the reaction for amide coupling. Step 4: Synthesis of tert-butyl 4-(8-(4-((4-(4-(prop-2-yn-1- yloxy)benzoyl)benzyl)carbamoyl)phenoxy)octyl)-1,4-diazepane-1-carboxylate: A 4 dram screw vail containing a magnetic stir bar was charged with 4-((8-(4-(tert-butoxycarbonyl)- 1,4-diazepan-1-yl)octyl)oxy)benzoic acid (22.4 mg, 0.05 mmol, 1.0 equiv), HOBt (10.134 mg, 1.5 equiv), EDC (14.377 mg, 1.5 equiv), dry DMF (2 mL), TEA (5.1 mg, 1.0 equiv) and the contents were stirred at room temperature for 10 mins. Then added (4- (aminomethyl)phenyl)(4-(prop-2-yn-1-yloxy)phenyl)methanone (20 mg, 0.075 mmol, 1.5 equiv) and the rection mixture was stirred at room temperature for 3 h. The solvent was removed under reduced pressure, the residue was added 4% aq. NaOH, the aqueous phase -110- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) was extracted three times with EtOAc. The combined organic portions were dried over anhydrous NaSO4, filtered and concentrated. The crude was purified by flash column chromatography on silica gel column eluting with 0-10% MeOH:DCM to afford 28 mg of the pure Boc-protected, benzophenone-alkyne functionalized title compound as colorless thick oil (80% yield).1H NMR (600 MHz, CDCl3) δ 7.82 (d, J = 8.8 Hz, 2H), 7.78 (dd, J = 8.7, 3.7 Hz, 2H), 7.74 (d, J = 8.1 Hz, 2H), 7.45 (d, J = 8.0 Hz, 2H), 7.04 (d, J = 8.8 Hz, 2H), 6.92 (d, J = 8.8 Hz, 2H), 6.59 – 6.49 (m, 1H), 4.78 (d, J = 2.4 Hz, 2H), 4.72 (d, J = 5.9 Hz, 2H), 3.99 (t, J = 6.5 Hz, 2H), 3.55 – 3.36 (m, 4H), 2.70 – 2.58 (m, 4H), 2.57 (t, J = 2.4 Hz, 1H), 2.46 (t, J = 7.7 Hz, 2H), 1.88 – 1.75 (m, 4H), 1.45 (s, 13H), 1.41 – 1.27 (m, 6H).13C NMR (151 MHz, CDCl3) δ 195.05, 167.01, 161.96, 161.02, 155.64, 155.53*, 142.85, 142.81*, 137.27, 137.25*, 132.42, 130.90, 130.27, 128.80, 127.48, 127.47*, 126.08, 114.43, 114.31, 79.28, 77.76, 76.19, 68.15, 57.86, 55.99, 55.95*, 55.88, 54.96, 54.56*, 46.58, 45.92* (overlap), 45.00*, 43.65, 29.42, 29.37*, 29.25, 29.17*, 29.07, 29.04*, 28.51, 27.72, 27.59*, 27.39, 27.33*(2C, overlap), 25.93, 25.89*. (* rotamer peak split). ESI-MS: Calculated for C42H54N3O6 [M+H]+696.9, Found 696.6. Step 5: Synthesis of 4-((8-(1,4-diazepan-1-yl)octyl)oxy)-N-(4-(4-(prop-2-yn-1- yloxy)benzoyl)benzyl)benzamide, M92: An oven dried round bottom flask was added the Boc-protected tert-butyl 4-(8-(4-((4-(4-(prop-2-yn-1- yloxy)benzoyl)benzyl)carbamoyl)phenoxy)octyl)-1,4-diazepane-1-carboxylate (20 mg, 0.29 mmol), dry DCM (2.0 mL) and cooled the reaction mixture to 0-4oC in a ice-water bath, then TFA (0.5 mL) via a syringe. The reaction mixture was stirred at room temperature for 2 h. Solvents were evaporated under reduced pressure, co-evaporated with more DCM (x3). Then the residue was added 1M aq. NaOH, extracted with DCM (x3), organic portion was washed with water and brine, dried over anhydrous Na2SO4. filtered off the solids, filtrate was concentrated under reduced pressure to afford 16 mg of the title compound M92 as light brown solid (93%).1H NMR (600 MHz, CDCl3) δ 7.82 (d, J = 8.8 Hz, 2H), 7.78 (d, J = 8.8 Hz, 2H), 7.74 (d, J = 8.2 Hz, 2H), 7.45 (d, J = 8.4 Hz, 2H), 7.04 (d, J = 8.8 Hz, 2H), 6.92 (d, J = 8.8 Hz, 2H), 6.50 (t, J = 5.9 Hz, 1H), 4.78 (d, J = 2.4 Hz, 2H), 4.72 (d, J = 5.8 Hz, 2H), 3.99 (t, J = 6.5 Hz, 2H), 2.93 (t, J = 6.1 Hz, 2H), 2.92 – 2.89 (m, 2H), 2.71 – 2.67 (m, 2H), 2.67 – 2.64 (m, 2H), 2.57 (t, J = 2.4 Hz, 1H), 2.50 – 2.45 (m, 2H), 1.84 – 1.73 (m, 4H), 1.52 – 1.41 (m, 4H), 1.40 – 1.27 (m, 6H).13C NMR (151 MHz, CDCl3) δ 195.05, -111- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) 167.02, 161.98, 161.02, 142.81, 137.28, 132.42, 130.89, 130.28, 128.78, 127.48, 126.07, 114.44, 114.32, 77.76, 76.19, 68.18, 58.54, 58.33, 55.88, 54.56, 48.69, 47.35, 43.66, 30.29, 29.47, 29.29, 29.09, 27.47, 27.45, 25.93. HRMS: Calculated for C37H46N3O4[M+H]+596.3488, found 596.3495. Synthesis of M91: Conditions: a) TFA:DCM, r.t, 2 h, 90%. Methyl 4-((8-(1,4-diazepan-1-yl)octyl)oxy)benzoate, M91 was prepared in one step from the Boc-protected methyl ester intermediate tert-butyl 4-(8-(4- (methoxycarbonyl)phenoxy)octyl)-1,4-diazepane-1-carboxylate (using 20 mg, 0.043 mmol) following the experimental procedure from Step-5 of M92 synthesis (14.4 mg, 90% yield)1H NMR (600 MHz, CDCl3) δ 7.98 (d, J = 8.9 Hz, 2H), 6.90 (d, J = 8.9 Hz, 2H), 4.00 (t, J = 6.5 Hz, 2H), 3.88 (s, 3H), 2.96 (t, J = 6.1 Hz, 2H), 2.94 – 2.91 (m, 2H), 2.72 – 2.65 (m, 4H), 2.52 – 2.41 (m, 3H), 1.84 – 1.75 (m, 4H), 1.52 – 1.42 (m, 4H), 1.40 – 1.27 (m, 6H).13C NMR (151 MHz, CDCl3) δ 166.93, 162.93, 131.56, 122.30, 114.04, 68.15, 58.53, 58.03, 54.52, 51.84, 48.61, 47.22, 30.07, 29.48, 29.31, 29.09, 27.48, 27.46, 25.94. HRMS: Calculated for C21H35N2O3[M+H]+363.2648, found 363.2645. Synthesis of M96: -112- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Conditions: a) K2CO3, acetone, reflux, overnight, 37%; b) 1-Boc-hexahydro-1,4-diazepine, K2CO3, DMF, r.t, overnight, 64%; c) MeOH, THF, 4N aq.NaOH, 80oC, 2 h, reflux; 1N aq.HCl (pH~3) >90%; d) 1, HOBt, EDC,TEA, DMF, r.t, 4 h, 57%; e) TFA:DCM, r.t, 2 h, 85%. M96 was synthesized following the procedure from M92 synthesis. Step 1: Methyl 4-(4-bromobutoxy)benzoate was synthesized using methyl 4- hydroxybenzoate and 1,4-dibromobutane (37% yield).1H NMR (600 MHz, CDCl3) δ 7.99 (d, J = 8.9 Hz, 2H), 6.90 (d, J = 8.9 Hz, 2H), 4.05 (t, J = 6.1 Hz, 2H), 3.89 (s, 3H), 3.50 (t, J = 6.6 Hz, 2H), 2.12 – 2.03 (m, 2H), 2.02 – 1.93 (m, 2H).13C NMR (151 MHz, CDCl3) δ 166.85, 162.60, 131.61, 122.62, 114.01, 67.01, 51.88, 33.32, 29.35, 27.74. Step 2: tert-butyl 4-(4-(4-(methoxycarbonyl)phenoxy)butyl)-1,4-diazepane-1- carboxylate was synthesized following the procedure from Step-2 of M92 synthesis using Methyl 4-(4-bromobutoxy)benzoate and 1-Boc-hexahydro-1,4-diazepine (64%).1H NMR (600 MHz, CDCl3) δ 7.98 (d, J = 8.8 Hz, 2H), 6.90 (d, J = 8.8 Hz, 2H), 4.03 (td, J = 6.4, 2.3 Hz, 2H), 3.88 (s, 3H), 3.54 – 3.38 (m, 4H), 2.71 – 2.57 (m, 4H), 2.53 (t, J = 7.4 Hz, 2H), 1.88 – 1.76 (m, 4H), 1.65 (q, J = 6.7 Hz, 2H), 1.46 (s, 9H).13C NMR (151 MHz, CDCl3) δ 166.90, 166.89*, 162.82, 162.81*, 155.63, 155.51*, 131.57, 122.41, 122.38*, 114.03, 79.25, 79.23*, 67.91, 57.37, 57.28*, 56.07, 55.97*, 54.87, 54.60*, 51.85, 46.83, 46.22*, 46.00, 45.07*, 28.51, 27.93, 27.78*, 27.01, 26.99*, 24.04, 23.99*. (* rotamer peak). ESI-MS: calculated for C22H35N2O5406.5, found m / z 407.3 [M+H]+. -113- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Step 3: 4-(4-(4-(tert-butoxycarbonyl)-1,4-diazepan-1-yl)butoxy)benzoic acid was synthesized in one step following the procedure from Step-3 of M92 synthesis by base hydrolysis of tert-butyl 4-(4-(4-(methoxycarbonyl)phenoxy)butyl)-1,4-diazepane-1- carboxylate (>90% yield) (not characterized, used directly in the next step). Step 4: tert-butyl 4-(4-(4-((4-(4-(prop-2-yn-1- yloxy)benzoyl)benzyl)carbamoyl)phenoxy)butyl)-1,4-diazepane-1-carboxylate was synthesized following the procedure from Step-4 of M92 synthesis using 4-(4-(4-(tert- butoxycarbonyl)-1,4-diazepan-1-yl)butoxy)benzoic acid and (4-(aminomethyl)phenyl)(4- (prop-2-yn-1-yloxy)phenyl)methanone (57%).1H NMR (600 MHz, CDCl3) δ 7.82 (d, J = 8.1 Hz, 2H), 7.78 (d, J = 8.6 Hz, 2H), 7.74 (d, J = 7.9 Hz, 2H), 7.45 (d, J = 7.9 Hz, 2H), 7.07 – 7.02 (m, 2H), 6.92 (d, J = 8.5 Hz, 2H), 6.51 (t, J = 5.9 Hz, 1H), 4.78 (dd, J = 2.4, 0.8 Hz, 2H), 4.72 (d, J = 5.8 Hz, 2H), 4.02 (td, J = 6.4, 1.6 Hz, 2H), 3.55 – 3.38 (m, 4H), 2.65 (br s, 4H), 2.59 – 2.56 (m, 1H), 2.54 (t, J = 6 Hz, 2H), 1.90 – 1.74 (m, 4H), 1.66 (br s, 2H), 1.46 (s, 9H*). (* peak split, rotamer).13C NMR (151 MHz, CDCl3) δ 195.04, 167.01, 166.99*, 161.84, 161.02*, 155.62, 155.50*, 142.80, 142.79*, 137.28, 132.42, 130.89, 130.27, 128.80, 127.48, 126.18, 126.17*, 114.44, 114.32, 114.31*, 79.29, 77.76, 76.20, 67.90, 57.36, 57.28*, 55.96, 55.88*, 54.91, 54.53*, 46.75, 46.11*, 45.94, 45.06*, 43.66, 28.51, 27.77 (br), 26.98, 26.96*, 23.96 (br). (* rotamer peak, br = broad). ESI-MS: C38H46N3O6calculated 639.7, found m / z 640.4 [M+H]+. Step 5: 4-(4-(1,4-diazepan-1-yl)butoxy)-N-(4-(4-(prop-2-yn-1- yloxy)benzoyl)benzyl)benzamide, M96 was prepared by Boc deprotection of tert-butyl 4-(4- (4-((4-(4-(prop-2-yn-1-yloxy)benzoyl)benzyl)carbamoyl)phenoxy)butyl)-1,4-diazepane-1- carboxylate, following the boc-deprotection procedure from Step-5 of M92 synthesis (85%).1H NMR (600 MHz, CDCl3) δ 7.82 (d, J = 8.8 Hz, 2H), 7.78 (d, J = 8.8 Hz, 2H), 7.74 (d, J = 8.2 Hz, 2H), 7.45 (d, J = 8.2 Hz, 2H), 7.04 (d, J = 8.9 Hz, 2H), 6.92 (d, J = 8.9 Hz, 2H), 6.53 (t, J = 5.9 Hz, 1H), 4.78 (d, J = 2.4 Hz, 2H), 4.72 (d, J = 5.9 Hz, 2H), 4.03 (t, J = 6.4 Hz, 2H), 2.96 (t, J = 6.0 Hz, 2H), 2.95 – 2.91 (m, 2H), 2.73 – 2.66 (m, 4H), 2.59 – 2.54 (m, 3H, overlap), 1.80 (m, 4H, overlap), 1.70 – 1.61 (m, 2H).13C NMR (151 MHz, CDCl3) δ 195.05, 167.01, 161.86, 161.02, 142.82, 137.27, 132.42, 130.89, 130.27, 128.81, 127.48, 126.15, 114.44, 114.32, 77.76, 76.20, 67.95, 57.96, 57.58, 55.88, 54.47, 48.59, -114- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) 47.07, 43.66, 29.89, 29.71, 27.01, 24.04. HRMS: Calculated for C33H38N3O4 [M+H]+540.2862, found 540.2861. Synthesis of M93: Follow M1 synthesis for general procedure. Conditions: a) tert-butyl 1,4-diazepane-1-carboxylate, K2CO3, DMF, r.t, overnight; b) TFA:DCM, r.t, 2 h, 64% over two steps. 1-(4-(2-(tert-butyl)-4-methylphenoxy)butyl)-1,4-diazepane, M93 was synthesized in two steps from the bromo intermediate 1, adopting the general procedure from Step-2 and Step-3 of M1 synthesis, using 50 mg (0.249 mmol, 1 equiv) of 1-(4-(2-(tert-butyl)-4- methylphenoxy)butyl)-1,4-diazepane (Boc intermediate was used in next step without further characterization), to afford M93 as thick oil (50 mg, 63% over two steps).1H NMR (600 MHz, CDCl3) δ 7.08 (d, J = 2.3 Hz, 1H), 6.98 – 6.92 (m, 1H), 6.75 (d, J = 8.2 Hz, 1H), 3.97 (t, J = 6.4 Hz, 2H), 2.94 (t, J = 6.1 Hz, 2H), 2.93 – 2.90 (m, 2H), 2.73 – 2.69 (m, 2H), 2.69 – 2.66 (m, 2H), 2.60 – 2.55 (m, 2H), 2.28 (s, 3H), 1.90 – 1.81 (m, 3H), 1.77 (p, J = 6.0 Hz, 2H), 1.70 (p, J = 7.6 Hz, 2H), 1.38 (s, 9H).13C NMR (151 MHz, CDCl3) δ 155.69, 137.67, 128.93, 127.46, 127.05, 111.76, 67.66, 58.30, 58.06, 54.44, 48.90, 47.42, 34.72, 30.43, 29.85, 27.54, 24.42, 20.78. HRMS: Calculated for C20H35N2O [M+H]+319.2749, found 319.2756. Synthesis of M95: Follow M1 synthesis for general procedure. -115- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Conditions: a) tert-butyl (S)-3-methyl-1,4-diazepane-1-carboxylate, K2CO3, DMF, r.t, overnight; b) TFA:DCM, r.t, 2 h, 45% over two steps. (S)-1-(4-(2-(tert-butyl)-4-methylphenoxy)butyl)-3-methyl-1,4-diazepane, M95 was synthesized was synthesized in two steps from the bromo intermediate 1, adopting the general procedure from Step-2 and Step-3 of M1 synthesis, using tert-butyl (S)-3-methyl- 1,4-diazepane-1-carboxylate (Boc intermediate was used in next step without further characterization), to afford M95 as thick oil ( 10 mg, 45% over two steps). 1H NMR (600 MHz, CDCl3) δ 7.08 (d, J = 2.2 Hz, 1H), 6.95 (dd, J = 8.3, 2.2 Hz, 1H), 6.75 (d, J = 8.2 Hz, 1H), 3.97 (t, J = 6.3 Hz, 2H), 3.06 – 2.93 (m, 2H), 2.88 (ddd, J = 13.4, 9.0, 4.2 Hz, 1H), 2.83 – 2.72 (m, 2H), 2.64 (ddd, J = 12.9, 8.1, 4.8 Hz, 1H), 2.57 (t, J = 6 Hz, 2H), 2.32 (dd, J = 13.2, 8.7 Hz, 1H), 2.28 (s, 3H), 1.85 (dt, J = 15.1, 6.6 Hz, 2H), 1.79 (dq, J = 9.9, 5.1 Hz, 1H), 1.74 (dt, J = 8.7, 5.2 Hz, 1H), 1.69 (ddd, J = 14.9, 8.8, 6.1 Hz, 2H), 1.38 (s, 9H), 1.05 (d, J = 6.5 Hz, 3H).13C NMR (151 MHz, CDCl3) δ 155.68, 137.66, 128.93, 127.46, 127.05, 111.74, 67.64, 64.44, 58.38, 54.60, 54.16, 45.71, 34.72, 30.73, 29.85, 27.49, 24.48, 20.91, 20.78. HRMS: Calculated for C21H37N2O [M+H]+333.2906, found 333.2909. Synthesis of M94. Follow M65 synthesis from C8-Br intermediate. Conditions: a) tert-butyl (S)-3-methyl-1,4-diazepane-1-carboxylate, K2CO3, DMF, r.t, overnight; b) TFA:DCM, r.t, 2 h, 50% over two steps. (S)-1-(8-(2-(tert-butyl)-4-methylphenoxy)octyl)-3-methyl-1,4-diazepane, M94 was synthesized using C8-Br intermediate and tert-butyl (S)-3-methyl-1,4-diazepane-1- carboxylate in two steps following the synthesis of M65 (50% yield, over two steps) (Boc- intermediate was not characterized, and used directly in the Boc-deprotection step).1H -116- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) NMR (600 MHz, CDCl3) δ 7.07 (d, J = 2.3 Hz, 1H), 6.97 – 6.93 (m, 1H), 6.75 (d, J = 8.2 Hz, 1H), 3.94 (t, J = 6.5 Hz, 2H), 3.03 – 2.93 (m, 2H), 2.87 (ddd, J = 13.4, 8.9, 4.4 Hz, 1H), 2.81 – 2.72 (m, 2H), 2.60 (ddd, J = 12.9, 8.3, 4.7 Hz, 1H), 2.50 – 2.45 (m, 2H), 2.28 (s, 4H, overlap), 1.87 – 1.66 (m, 6H), 1.53 – 1.42 (m, 4H), 1.38 (s, 15H, overlap), 1.04 (d, J = 6.5 Hz, 3H).13C NMR (151 MHz, CDCl3) δ 155.76, 137.68, 128.83, 127.43, 127.03, 111.73, 67.76, 64.77, 58.91, 54.68, 54.06, 45.83, 34.72, 30.83, 29.83, 29.50, 29.50, 29.33, 27.48, 27.46, 26.33, 21.11, 20.78. HRMS: Calculated for C25H45N2O [M+H]+389.3532, found 389.3543. Synthesis of M100: Follow M65 synthesis from C8-Br intermediate Conditions: a) tert-butyl (S)-azepan-4-ylcarbamate, K2CO3, DMF, r.t, overnight; b) TFA:DCM, r.t, 2 h, 53% over two steps. (S)-1-(8-(2-(tert-butyl)-4-methylphenoxy)octyl)azepan-4-amine, M100 was synthesized using C8-Br intermediate and tert-butyl (S)-azepan-4-ylcarbamate, following the general procedure from M65 synthesis (Boc-intermediate was not characterized, directly used in Boc-deprotection step). (53% over two steps).1H NMR (600 MHz, CDCl3) δ 7.07 (d, J = 2.2 Hz, 1H), 6.97 – 6.93 (m, 1H), 6.75 (d, J = 8.2 Hz, 1H), 3.94 (t, J = 6.5 Hz, 2H), 3.03 (tt, J = 8.6, 4.2 Hz, 1H), 2.71 (ddd, J = 13.3, 7.2, 3.5 Hz, 1H), 2.66 – 2.56 (m, 2H), 2.52 (ddd, J = 12.8, 9.0, 3.1 Hz, 1H), 2.46 – 2.39 (m, 2H), 2.28 (s, 3H), 1.89 – 1.77 (m, 4H), 1.71 (dtt, J = 14.6, 7.2, 3.6 Hz, 1H), 1.57 (dtd, J = 14.4, 9.0, 3.5 Hz, 2H), 1.53 – 1.41 (m, 6H), 1.38 (s, 9H), 1.37 – 1.27 (m, 6H).13C NMR (151 MHz, CDCl3) δ 155.76, 137.68, 128.82, 127.43, 127.03, 111.74, 67.77, 58.73, 55.56, 51.49, 51.46, 37.69, 37.46, 34.72, 29.83, 29.51, 29.50, 29.32, 27.57, 27.51, 26.33, 24.60, 20.78. HRMS: Calculated for C25H45N2O [M+H]+389.3532, found 389.3536. Synthesis of M102: Follow M65 synthesis from C8-Br intermediate -117- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Conditions: a) tert-butyl (R)-azepan-4-ylcarbamate, K2CO3, DMF, r.t, overnight; b) TFA:DCM, r.t, 2 h, 55% over two steps. (R)-1-(8-(2-(tert-butyl)-4-methylphenoxy)octyl)azepan-4-amine, M102 was synthesized using C8-Br intermediate and tert-butyl (R)-azepan-4-ylcarbamate, following the general procedure from M65 synthesis (Boc-intermediate was not characterized, directly used in Boc-deprotection step). (55% yield, over two steps).1H NMR (600 MHz, CDCl3) δ 7.07 (d, J = 2.2 Hz, 1H), 6.95 (dd, J = 8.2, 2.2 Hz, 1H), 6.75 (d, J = 8.2 Hz, 1H), 3.94 (t, J = 6.5 Hz, 2H), 3.04 (tt, J = 8.6, 4.2 Hz, 1H), 2.73 (ddd, J = 13.3, 7.3, 3.4 Hz, 1H), 2.69 – 2.58 (m, 2H), 2.54 (ddd, J = 12.8, 8.9, 3.1 Hz, 1H), 2.48 – 2.41 (m, 2H), 2.28 (s, 3H), 1.90 – 1.78 (m, 4H, overlap), 1.72 (dtt, J = 14.5, 7.3, 3.6 Hz, 2H), 1.62-1.56 (m, 3H), 1.53 – 1.42 (m, 6H), 1.38 (s, m, 15H, overlap).13C NMR (151 MHz, CDCl3) δ 155.75, 137.67, 128.82, 127.42, 127.03, 111.73, 67.75, 58.68, 55.48, 51.39, 51.34, 37.36 (2C), 34.72, 29.83, 29.49, 29.30, 27.48, 27.42, 26.32, 24.41(2C), 20.78. HRMS: Calculated for C25H45N2O [M+H]+389.3532, found 389.3542. Synthesis of M110: Conditions: a) 1,4-diazepan-5-one acetic acid, K2CO3, DMF, r.t, overnight, 56%. Step 1: Synthesis of 1-(8-(2-(tert-butyl)-4-methylphenoxy)octyl)-1,4-diazepan-5-one, M110: In a 4 dram screw vial with a magnetic stir bar, added 1,4-diazepan-5-one acetic acid (40.6 mg, 0.233 mmol, 1.0 equiv), 1-((8-bromooctyl)oxy)-2-(tert-butyl)-4- -118- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) methylbenzene (99.5 mg, 0.28 mmol, 1.2 equiv), dry DMF (2.0 mL), K2CO3 (161.7 mg, 1.17 mmol, 5.0 equiv), and purged with argon gas for 5 mins. The reaction mixture was stirred at room temperature for overnight. Then the solvent was evaporated under reduced pressure, residue was added 1M aq. NaOH (~ 2 mL) and extracted with DCM (3X 5 mL). Combined organic portions were washed with water and brine, dried over anhydrous Na2SO4, filtered off the solid. The filtrate was concentrated to give the crude product, which was purified by flash column chromatography on basic alumina column eluting with 0-5% MeOH:DCM (gradient) afforded ~ 50 mg of the title compound M110 as thick oil (56% yield).1H NMR (600 MHz, CDCl3) δ 7.07 (d, J = 2.3 Hz, 1H), 6.95 (ddd, J = 8.2, 2.3, 0.8 Hz, 1H), 6.75 (d, J = 8.2 Hz, 1H), 5.87 (br s, 1H), 3.94 (t, J = 6.4 Hz, 2H), 3.30 (td, J = 5.8, 3.3 Hz, 2H), 2.68 – 2.57 (m, 6H), 2.47 – 2.40 (m, 2H), 2.28 (s, 3H), 1.86 – 1.78 (m, 2H), 1.52-1.45 (m, 4H, overlap), 1.40-1.31 (s, m, 15H, overlap).13C NMR (151 MHz, CDCl3) δ 177.74, 155.74, 137.68, 128.88, 127.45, 127.04, 111.73, 67.71, 58.85, 57.18, 50.63, 42.86, 37.90, 34.72, 29.84, 29.48, 29.43, 29.26, 27.38, 26.96, 26.32, 20.78. HRMS: Calculated for C24H41N2O2 [M+H]+389.3168, found 389.3163. Synthesis of M134: Conditions: a) tert-butyl 1,5-diazocane-1-carboxylate, K2CO3, DMF, r.t, overnight; b) TFA:DCM, r.t, 2 h, 40% over two steps. 1-(8-(2-(tert-butyl)-4-methylphenoxy)octyl)-1,5-diazocane, M134 was synthesized using the C8-Br intermediate and tert-butyl 1,5-diazocane-1-carboxylate, following the general procedure from Step-1 and Step-2 of M65 synthesis. (Boc-intermediate was not characterized, directly used in Boc-deprotection step). (40% yield, over two steps).1H NMR (600 MHz, CDCl3) δ 7.07 (d, J = 2.3 Hz, 1H), 6.95 (dd, J = 8.2, 2.3 Hz, 1H), 6.75 (d, J = 8.2 Hz, 1H), 3.94 (t, J = 6.4 Hz, 2H), 3.11 – 2.86 (m, 4H), 2.66 (t, J = 5.8 Hz, 4H), 2.42 – 2.34 (m, 2H), 2.28 (s, 3H), 1.86 – 1.79 (m, 2H), 1.66 (p, J = 5.8 Hz, 4H), 1.50 (p, J = 7.2 -119- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Hz, 2H), 1.44 (p, J = 7.4 Hz, 2H), 1.38 (s, m (overlap), 15H).13C NMR (151 MHz, CDCl3) δ 155.76, 137.68, 128.82, 127.42, 127.03, 111.74, 111.73, 67.76, 59.21, 53.07, 47.71, 34.72, 29.84, 29.50, 29.33, 28.25, 27.98 (2C), 27.42, 26.35, 20.78. HRMS: Calculated for C25H45N2O [M+H]+389.3532, found 389.3536. Synthesis of M137: Conditions: a) K2CO3, DMF, r.t, overnight, 62%; b) 1-(4-bromobutoxy)-2-(tert-butyl)-4- methylbenzene, NaH, DMF, 0° C, 30 mins, r.t, 5 h, 43%; c) TFA / DCM, r.t, 2 h, 64%. Step 1: Synthesis of tert-butyl 4-(3-hydroxypropyl)-1,4-diazepane-1-carboxylate: In a 4 dram vail with a magnetic stir bar, added tert-butyl 1,4-diazepane-1-carboxylate (0.5 g, 2.49 mmol), 3-bromopropan-1-ol (346.99 mg, 1 equiv), K2CO3(1.72 g, 12.45 mmol, 5 equiv) and 4 mL of dry DMF. The contents were stirred at room temperature overnight under argon atmosphere. Then DMF was evaporated under reduced pressure. The residue was added water and extracted with DCM, washed organic phases with water and brine, dried over anhydrous Na2SO4, filtered off and evaporated solvents. The crude was purified by flash column chromatography using silica gel column eluting with MeOH in DCM (gradient) to obtain 400 mg of the Boc intermediate (62% yield). LCMS: Calculated for C13H26N2O3 [M+H]+258.3; found 259.3. Step 2: Synthesis of 1-(3-(4-(2-(tert-butyl)-4-methylphenoxy)butoxy)propyl)-1,4- diazepane M137: In an oven dried 10 mL RB flask equipped with a magnetic stir bar was added tert-butyl 4-(3-hydroxypropyl)-1,4-diazepane-1-carboxylate (100 mg, 0.387mmol) and 2 mL of dry DMF under argon atmosphere. The contents were stirred at 0֯ C under argon atmosphere for 10 mins. Then added NaH (0.58 mmol, 1.5 equiv, 23.2 mg (60% in oil)) once and stirred for 30 mins at 0֯ C. Then added 1-(4-bromobutoxy)-2-(tert-butyl)-4- methylbenzene (173.74 mg, 1.5 equiv; compound 1 from general scheme 1) in 1 mL of dry DMF. The contents were stirred at room temperature for 5 h and monitored by LCMS. The -120- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) reaction mixture was cooled down to 40֯C and quenched by dropwise addition of water. Contents were added diethyl ether (10 mL), stirred for 10 mins, then aqueous phase was washed with more ether. Combined ether portions were washed with water and brine, dried over anhydrous Na2SO4. The crude was purified by silica gel flash column chromatography eluting with MeOH in DCM (gradient) to obtain 80 mg of the boc-protected intermediate as light-yellow oily product (43 % yield) and was characterized by only LCMS (Calculated for: C28H48N2O4[M+H]+476.7; found 477.7). Boc deprotection was performed using standard conditions (from the synthesis of M65) using TFA / DCM to obtain 40 mg (64% yield) of the title compound M137 as colorless oily product.1H NMR (600 MHz, CDCl3):1H NMR (600 MHz, CDCl3) δ 7.07 (d, J = 2.3 Hz, 1H), 6.95 (ddd, J = 8.2, 2.3, 0.9 Hz, 1H), 6.75 (d, J = 8.2 Hz, 1H), 3.97 (t, J = 6.4 Hz, 2H), 3.48 (dt, J = 12.8, 6.4 Hz, 4H), 2.98 – 2.88 (m, 4H), 2.74 – 2.63 (m, 4H), 2.62 – 2.54 (m, 2H), 2.28 (s, 3H), 1.95 – 1.88 (m, 2H), 1.88 – 1.83 (m, 1H), 1.83 – 1.71 (m, 6H), 1.37 (s, 9H).13C NMR (151 MHz, CDCl3) δ 155.68, 137.69, 128.94, 127.46, 127.05, 111.76, 70.48, 69.16, 67.57, 58.39, 55.30, 54.52, 48.89, 47.42, 34.72, 30.48, 29.87, 27.86, 26.67, 26.44, 20.78. HRMS: Calculated for C23H41N2O2 [M+H]+377.3168, found 377.3170. Synthesis of M138: Conditions: a) K2CO3, Acetone, reflux, overnight, 33%; b) tert-butyl 1,4-diazepane-1- carboxylate, K2CO3, DMF, r.t, 24 h; c) TFA / DCM, r.t, 2 h, 64% over two steps. Step 1: Synthesis of 1-((8-bromooctyl)oxy)-4-(tert-butyl)benzene was achieved by following the step 1 of general scheme 1, using 4-(tert-butyl)phenol and 1,8-dibromooctane (33% yield).1H NMR (600 MHz, CDCl3) δ 7.29 (d, J = 9.0 Hz, 2H), 6.83 (d, J = 8.8 Hz, 2H), 3.93 (t, J = 6.5 Hz, 2H), 3.41 (t, J = 6.8 Hz, 2H), 1.86 (p, J = 7.0 Hz, 2H), 1.77 (p, J = 6.7 Hz, 2H), 1.50 – 1.32 (m, 10H), 1.32 – 1.26 (m, 9H).13C NMR (151 MHz, CDCl3) δ -121- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) 156.83, 143.13, 126.17, 113.90, 67.82, 34.04, 34.00, 32.79, 31.53, 29.30, 29.19, 28.70, 28.10, 25.99. Step 2: Synthesis of 1-(8-(4-(tert-butyl)phenoxy)octyl)-1,4-diazepane, M138 was achieved by following step 2 and 3 in general scheme 1, using 1-((8-bromooctyl)oxy)-4- (tert-butyl)benzene followed by deprotection of Boc with TFA / DCM (48% in two steps).1H NMR (600 MHz, CDCl3) δ 7.29 (d, J = 8.8 Hz, 2H), 6.83 (d, J = 8.8 Hz, 2H), 3.93 (t, J = 6.5 Hz, 2H), 2.94 (t, J = 6.1 Hz, 2H), 2.93 – 2.90 (m, 2H), 2.70 – 2.64 (m, 4H), 2.50 – 2.45 (m, 2H), 1.81 – 1.72 (m, 4H), 1.51 – 1.40 (m, 4H), 1.30 (s, 15H (overlap)).13C NMR (151 MHz, CDCl3) δ 156.85, 143.10, 126.16, 113.90, 67.89, 58.58, 58.30, 54.56, 48.72, 47.35, 34.04, 31.54, 30.29, 29.52, 29.36, 29.34, 27.50 (2), 26.04. HRMS: Calculated for C23H41N2O [M+H]+361.3219, found 361.3206. Synthesis of M146: Conditions: a) K2CO3, DMF, r.t, 8 h (55% yield); b) TFA / DCM, r.t, 2 h (60%). Step 1: Synthesis of 1-(6-(2-(tert-butyl)-4-methylphenoxy)hexyl)-1,4-diazepane, M146: In a 4 dram vial, added 1-((6-bromohexyl)oxy)-2-(tert-butyl)-4-methylbenzene (an intermediate compound from M25 synthesis) (200 mg, 0.611 mmol, 1 equiv.;), tert-butyl 1,4-diazepane-1-carboxylate (134.62 mg, 0.672 mmol, 1.1 equiv.), dry DMF (4 mL) and K2CO3(337.76 mg, 2.44 mmol, 4 equiv.). The vial was flushed with argon gas, sealed and stirred at room temperature for 8 h. Upon completion of the reaction monitored by LCMS, DMF was evaporated on rotary evaporator (~ 45 ֯C). The crude was added water and extracted with DCM (3 x 5 mL), combined DCM portions were washed with water and brine, dried over anhydrous Na2SO4, filtered, filtrate was concentrated under reduced pressure. The residue was purified by flash column chromatography using silica gel column eluting with MeOH:DCM (gradient elution) to obtain 150 mg (55% yield) of Boc protected intermediate, which was directly used in the next step of Boc deprotection reaction. -122- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Boc deprotection: 100 mg (134.5 mmol) of the Boc intermediate was taken into a 4 dram vail, dissolved in 4 mL of DCM and added 1 mL of TFA. The contents were stirred at room temperature for 2 h, solvents were evaporated under reduced pressure, co-evaporated with more DCM (x3). The residue was dissolved in fresh DCM (4 mL), added ~1 mL of saturated aq. NaHCO3 and stirred for 30 mins at room temperature, then extracted the aqueous phase with DCM (3 x 5 mL), combined DCM portions washed with water and brine, dried over anhydrous Na2SO4, filtered, filtrate was concentrated under reduced pressure to obtain the crude. The crude was purified on the flash column chromatography using basic Al2O3column and eluted with MeOH:DCM (gradient) to obtain the title compound M146 as light yellow oily product (77.6 mg, 60% yield).1H NMR (600 MHz, CDCl3) δ 7.07 (d, J = 2.3 Hz, 1H), 6.95 (dd, J = 8.2, 2.3 Hz, 1H), 6.75 (d, J = 8.2 Hz, 1H), 3.94 (t, J = 6.4 Hz, 2H), 2.99 – 2.90 (m, 4H), 2.74 – 2.65 (m, 4H), 2.54 – 2.45 (m, 2H), 2.28 (s, 3H), 1.82 (m, 4H), 1.57 – 1.46 (m, 4H), 1.38 (s, 11H (overlap)).13C NMR (151 MHz, CDCl3) δ 155.73, 137.68, 128.86, 127.44, 127.04, 111.73, 67.66, 58.44, 57.99, 54.53, 48.57, 47.22, 34.72, 30.03, 29.84, 29.49, 27.41, 27.25, 26.34, 20.78. HRMS: Calculated for C22H39N2O [M+H]+347.3062, found 347.3073. Synthesis of M147: Conditions: a) K2CO3, DMF, r.t, overnight, 64%; b) Aq. NaOH (1M), MeOH / THF, 80 ֯C, 4 h; c) tert-butyl 1,4-diazepane-1-carboxylate, HATU, DIPEA, DMF, r.t, 20 h, (86% over two steps); d) TFA / DCM, r.t, 2 h (81% isolated yield). Step 1: Synthesis of methyl 6-(2-(tert-butyl)-4-methylphenoxy)hexanoate: In a reaction vial (4 dram), added 2-(tert-butyl)-4-methylphenol (0.5 g, 3.04 mmol, 1.0 equiv.), methyl 6-bromohexanoate (954 mg, 4.5 mmol, 1.5 equiv.), K2CO3 (840 mg, 6.08 mmol, 2 equiv.) and dry DMF (5 mL). The vial was flushed with argon, sealed and stirred at room -123- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) temperature for overnight. DMF was evaporated, residue was added water, extracted with DCM. Combined DCM portions were washed with water and brine, dried over anhydrous Na2SO4, filtered off and the filtrate was concentrated under reduce pressure. Tyeh crude was purified by silica gel flash column chromatography eluting with EtOAc:Hex (gradient) to obtain 570 mg of colorless oily product (64% yield).1H NMR (600 MHz, CDCl3) δ 7.07 (d, J = 2.3 Hz, 1H), 6.95 (dd, J = 8.2, 2.2 Hz, 1H), 6.74 (d, J = 8.2 Hz, 1H), 3.95 (t, J = 6.4 Hz, 2H), 3.67 (s, 3H), 2.36 (t, J = 7.5 Hz, 2H), 2.27 (s, 3H), 1.88 – 1.81 (m, 2H), 1.72 (p, J = 7.6 Hz, 2H), 1.57 – 1.51 (m, 2H), 1.37 (s, 9H).13C NMR (151 MHz, CDCl3) δ 174.10, 155.65, 137.68, 128.96, 127.46, 127.05, 111.74, 67.44, 51.52, 34.71, 34.03, 29.84, 29.22, 26.01, 24.72, 20.78. Step 2 and 3: Synthesis of tert-butyl 4-(6-(2-(tert-butyl)-4- methylphenoxy)hexanoyl)-1,4-diazepane-1-carboxylate: In a 100 mL round bottomed flask equipped with a magnetic stir bar was charged with methyl 6-(2-(tert-butyl)-4- methylphenoxy)hexanoate (570 mg, 1.94 mmol, 1 equiv.), aq. NaOH (~0.5 mL, 19.5 mmol, ~ 10 equiv.) and MeOH:THF (3:1.5 mL). The reaction mixture was refluxed at 80֯C while monitoring the reaction progress by LCMS. Upon completion in 4 h, MeOH and THF were evaporated under reduced pressure and the residue was acidified with 1M HCl (until pH = ~3). The crude was extracted with EtOAc, washed with brine, dried over anhydrous Na2SO4, filtered and evaporated the solvent to obtain 350 mg of 6-(2-(tert-butyl)-4- methylphenoxy)hexanoic acid intermediate as white thick semi solid (65% yield) and was directly used in the next step of the amide coupling reaction. 6-(2-(tert-butyl)-4-methylphenoxy)hexanoic acid (350 mg, 1.26 mmol, 1 equiv.), HATU (718.81 mg, 1.89 mmol, 1.5 equiv.), DIPEA (329 uL (244.3 mg), 1.5 equiv.) and DMF (10 mL) were added into an oven dried round bottomed flask and stirred the contents at room temperature under argon for 20 mins. Then, added tert-butyl 1,4-diazepane-1- carboxylate (302.26 mg, 1.51 mmol, 1.2 equiv.) in 2 mL of DMF via syringe. The contents were stirred at the same temperature for 20 h. Upon completion of the reaction, the solvents were evaporated, residue was added water and extracted with DCM, combined organic portions were washed with water and brine, dried over anhydrous Na2SO4, filtered, filtrate was concentrated under reduced pressure to obtain the crude which was purified by silica gel flash column chromatography eluting with MeOH:DCM (gradient) to obtain 550 mg of -124- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) the Boc intermediate tert-butyl 4-(6-(2-(tert-butyl)-4-methylphenoxy)hexanoyl)-1,4- diazepane-1-carboxylate (86% yield). LCMS: Calculated for C27H45N2O4 [M+H]+461.6, found 461.58. In a 4 dram vial, the Boc intermediate (500 mg, 1.085 mmol) was dissolved in 10 mL of DCM and added 2 mL of TFA. The contents were stirred at room temperature for 2 h. Then solvents were evaporated, co-evaporated with more DCM (x3) followed by the addition of fresh DCM and saturated aq. NaHCO3. The contents were stirred at room temperature for 30 mins, extracted with DCM, washed with water and brine, dried over anhydrous Na2SO4. Filtered off, filtrate were concentrated under reduced pressure to obtain the crude which was purified by flash column chromatography using basic Al2O3column eluting with MeOH:DCM (gradient) to obtain 316 mg of the title product 6-(2-(tert-butyl)- 4-methylphenoxy)-1-(1,4-diazepan-1-yl)hexan-1-one, M147 as light yellow oily product (81%).1H NMR (600 MHz, CDCl3) δ 7.07 (d, J = 2.3 Hz, 1H), 6.95 (dd, J = 8.3, 2.3 Hz, 1H), 6.75 (d, J = 8.2 Hz, 1H), 3.96 (t, J = 6.4 Hz, 2H), 3.63 (dt, J = 14.3, 5.9 Hz, 2H), 3.55 (t, J = 6.3 Hz, 1H), 3.52 – 3.46 (m, 1H), 3.00 – 2.91 (m, 2H), 2.87 (dt, J = 16.7, 5.8 Hz, 2H), 2.36 (dt, J = 14.5, 7.6 Hz, 2H), 2.27 (s, 3H), 1.91 – 1.69 (m, 8H), 1.61 – 1.52 (m, 2H), 1.37 (s, 9H).13C NMR (151 MHz, CDCl3) δ 172.55, 172.50, 155.69, 137.65, 128.92, 128.91, 127.44, 127.07, 111.80, 67.56, 50.89, 50.25, 49.11, 48.75, 48.59, 47.80, 47.01, 44.67, 34.72, 33.30, 33.09, 30.93, 29.86, 29.59, 29.43, 26.37, 26.36, 25.11, 25.03, 20.78. HRMS: Calculated for C22H37N2O2 [M+H]+361.2855, found 361.2870. Synthesis of M148: Conditions: a) K2CO3, Acetone, reflux, overnight (32%); b) tert-butyl 1,4-diazepane-1- carboxylate, K2CO3, DMF, r.t, overnight (90%); c) TFA / DCM, r.t, 2 h (34% isolated yield). -125- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Step 1: 1-((5-bromopentyl)oxy)-2-(tert-butyl)-4-methylbenzene was synthesized following the general procedure from step 1 of the M5 synthesis, using 2-(tert-butyl)-4- methylphenol and 1,5-dibromopentane.1H NMR (600 MHz, CDCl3) δ 7.08 (d, J = 2.3 Hz, 1H), 6.95 (dd, J = 8.2, 2.3 Hz, 1H), 6.75 (d, J = 8.2 Hz, 1H), 3.96 (t, J = 6.3 Hz, 2H), 3.45 (t, J = 6.8 Hz, 2H), 2.28 (s, 3H), 1.95 (p, J = 7.0 Hz, 2H), 1.91 – 1.81 (m, 2H), 1.72 – 1.64 (m, 2H), 1.38 (s, 9H).13C NMR (151 MHz, CDCl3) δ 155.60, 137.71, 129.05, 127.50, 127.06, 111.74, 67.36, 34.72, 33.59, 32.49, 29.86, 28.74, 25.13, 20.78. Step 2: 1-(5-(2-(tert-butyl)-4-methylphenoxy)pentyl)-1,4-diazepane, M148 was synthesized following the general procedure from step 2 of the M5 synthesis using tert- butyl 1,4-diazepane-1-carboxylate, followed by Boc deprotection.1H NMR (600 MHz, MeOD) δ 7.03 (d, J = 2.3 Hz, 1H), 6.95 – 6.88 (m, 1H), 6.78 (d, J = 8.2 Hz, 1H), 3.96 (t, J = 6.2 Hz, 2H), 3.31 (p, J = 1.6 Hz, 2H), 2.96 – 2.87 (m, 4H), 2.79 – 2.68 (m, 4H), 2.59 – 2.50 (m, 2H), 2.23 (s, 3H), 1.84 (m, 4H), 1.66 – 1.50 (m, 4H), 1.36 (s, 9H).13C NMR (151 MHz, MeOD) δ 155.64, 137.12, 128.60, 126.80, 126.71, 111.53, 67.22, 57.95, 56.49, 54.24, 46.61, 46.02, 34.14, 29.21, 29.04, 28.10, 26.43, 24.21, 19.45. HRMS: Calculated for C21H37N2O [M+H]+333.2906, found 333.2895. Synthesis of M149: Condition: a) K2CO3, DMF, r.t, overnight, 45%. Step 1: 1-(6-(2-(tert-butyl)-4-methylphenoxy)hexyl)-1,4-diazepan-5-one, M149 was synthesized in one step using 1-((6-bromohexyl)oxy)-2-(tert-butyl)-4-methylbenzene and 1,4-diazepan-5-one (following procedure from step 1 of M146 synthesis).1H NMR (600 MHz, CDCl3) δ 7.07 (d, J = 2.3 Hz, 1H), 6.95 (dd, J = 8.1, 2.2 Hz, 1H), 6.75 (d, J = 8.2 Hz, 1H), 6.04 (t, J = 5.7 Hz, 1H), 3.94 (t, J = 6.4 Hz, 2H), 3.30 (q, J = 5.1 Hz, 2H), 2.62 (m, 6H (overlap)), 2.50 – 2.40 (m, 2H), 2.28 (s, 3H), 1.88 – 1.79 (m, 2H), 1.53 (m, 4H), 1.37 (s, 11H (overlap)).13C NMR (151 MHz, CDCl3) δ 177.81, 155.69, 137.65, 128.93, 127.47, -126- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) 127.05, 111.71, 67.59, 58.70, 57.19, 50.64, 42.83, 37.91, 34.72, 29.85, 29.47, 27.16, 26.92, 26.30, 20.78. HRMS: Calculated for C22H37N2O2 [M+H]+361.2855, found 361.2845. Synthesis of M150: Conditions: a) LiAlD4, THF, 0 °C - r.t, 3 h, 44% (isolated yield) Step 1: Synthesis of 1-(6-(2-(tert-butyl)-4-methylphenoxy)hexyl)-1,4-diazepane-5,5- d2, M150: An oven dried round bottomed flask was added 1-(6-(2-(tert-butyl)-4- methylphenoxy)hexyl)-1,4-diazepan-5-one, M149 (83 mg, 0.2302 mmol, 1 equiv.), dissolved in anhydrous THF (2 mL) under argon atmosphere, cooled the reaction flask to 0 ֯C and stirred for 15 mins. Then LiAlD4 (19.32 mg, 0.460 mmol, 2 equiv.) was added portion wise at 0֯C, stirred the reaction mixture at room temperature for 2 h under argon atmosphere while monitoring by LCMS. Then added additional 1 equivalent of LiAlD4and stirred for 2 hrs at room temperature. The reaction flask was cooled down to 40 C֯, quenched by careful addition of water, extracted with DCM (3 x 10 mL), washed DCM portions with water and brine, dried over anhydrous Na2SO4, filtered, filtrate was concentrated under reduce pressure. The crude was purified by basic Al2O3flash column chromatography eluting with MeOH:DCM (gradient) to afford 35 mg of M150 as colorless thick oily product (44% isolated yield).1H NMR (600 MHz, CDCl3) δ 7.07 (d, J = 2.3 Hz, 1H), 6.95 (dd, J = 8.2, 2.2 Hz, 1H), 6.75 (d, J = 8.2 Hz, 1H), 3.94 (t, J = 6.4 Hz, 2H), 2.96 – 2.85 (m, 2H), 2.75 – 2.59 (m, 4H), 2.54 – 2.41 (m, 2H), 2.28 (s, 3H), 2.06 (s, 1H), 1.83 (dt, J = 12.7, 6.6 Hz, 2H), 1.75 (t, J = 5.9 Hz, 2H), 1.52 (m, 4H), 1.38 (s, 11H (overlap)).13C NMR (151 MHz, CDCl3) δ 155.73, 137.67, 128.85, 127.44, 127.03, 111.72, 67.67, 58.46, 58.42, 54.54, 48.70, 46.87, 46.73, 46.60, 46.46, 46.33, 34.72, 30.15, 29.84, 29.50, 27.47, 27.27, 26.35, 20.78. HRMS: Calculated for C22H37D2N2O [M+H]+349.3188, found 349.3203. Synthesis of M151: -127- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Conditions: a) LiAlD4, THF, 0 °C - r.t, 2 h (98%); b) CBr4, PPh3, DCM, 0 °C - r.t, 24 h (57%); c) tert-butyl 1,4-diazepane-1-carboxylate, K2CO3, DMF, r.t, overnight (77%); d) TFA / DCM, r.t, 2 h (49% isolated yield). Step 1: Synthesis of 6-(2-(tert-butyl)-4-methylphenoxy)hexan-1,1-d2-1-ol: An oven dried round bottomed flask was charged with methyl 6-(2-(tert-butyl)-4- methylphenoxy)hexanoate (1.0 g, 3.2 mmol, 1.0 equiv., an intermediate compound from the synthesis of M147), anhydrous THF (15 mL) and cooled to 0֯C under argon atmosphere while stirring for 10 mins. Then LiAlD4(287.12 mg, 6.84 mmol, 2 equiv.) was added portion wise and the reaction mixture was stirred at room temperature for 2 h while monitoring by LCMS. After completion, the reaction flask was cooled down to 4 ֯C (ice bath) and quenched by slow addition of EtOAc (5 mL) followed by saturated aq. Na2SO4 solution (5 mL) while thoroughly stirring (ppt formed). Then added excess of EtOAc (20 mL) and stirred for additional 20 mins. Filtered off the solids through pad of celite, washed solids with more EtOAc. The filtrate was washed with water and brine, dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure to obtain 898 mg of the desired product as thick colorless oil (98%) which was directly used in the next step of the reaction.1H NMR (600 MHz, CDCl3) δ 7.07 (d, J = 2.2 Hz, 1H), 6.95 (dd, J = 8.2, 2.2 Hz, 1H), 6.75 (d, J = 8.2 Hz, 1H), 3.95 (t, J = 6.4 Hz, 2H), 2.28 (s, 3H), 1.89 – 1.81 (m, 2H), 1.63 – 1.51 (m, 5H), 1.50 – 1.42 (m, 2H), 1.38 (s, 9H).13C NMR (151 MHz, CDCl3) δ 155.71, 137.69, 128.90, 127.45, 127.04, 111.74, 67.62, 62.50, 62.35, 62.21, 62.07, 61.93, 34.72, 32.48, 29.83, 29.49, 26.23, 25.45, 20.78. Step 2: Synthesis of 1-((6-bromohexyl-6,6-d2)oxy)-2-(tert-butyl)-4-methylbenzene: Into a 100 mL round bottomed flask added 6-(2-(tert-butyl)-4-methylphenoxy)hexan-1,1-d2- 1-ol (898 mg, 3.37 mmol, 1 equiv.), dissolved in dry DCM (20 mL), then added CBr4 (1.23 -128- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) g, 3.71 mmol, 1.1 equiv.), cooled the flask to 0֯C and stirred for 20 mins. Then added PPh3 (1.06 g, 4.0444 mmol, 1.2 equiv.) and allowed the reaction mixture to warm up to room temperature and stirred for 2 days at room temperature while monitoring the reaction progress by TLC. The reaction mixture was concentrated under reduced pressure and directly loaded onto the silica gel column and purified by flash column chromatography eluting with EtOAc in Hexanes (gradient elution, 0-5% EtOAc) afforded the bromo intermediate (630 mg, 57% isolated yield) as colorless thick oily product.1H NMR (600 MHz, CDCl3) δ 7.08 (d, J = 2.3 Hz, 1H), 6.95 (dd, J = 8.3, 2.2 Hz, 1H), 6.75 (d, J = 8.2 Hz, 1H), 3.95 (t, J = 6.4 Hz, 2H), 2.28 (s, 3H), 1.89 (t, J = 6.9 Hz, 2H), 1.87 – 1.81 (m, 2H), 1.58 – 1.50 (m, 4 H (overlap with H2O)), 1.38 (s, 9H).13C NMR (151 MHz, CDCl3) δ 155.67, 137.69, 128.96, 127.47, 127.05, 111.74, 67.51, 34.72, 33.62, 33.46, 33.31, 33.16, 33.01, 29.84, 29.36, 27.86, 25.63, 20.78. Step 3: Synthesis of tert-butyl 4-(6-(2-(tert-butyl)-4-methylphenoxy)hexyl-1,1-d2)- 1,4-diazepane-1-carboxylate: This compound was synthesize following the procedure from the synthesis of M149 using 1-((6-bromohexyl-6,6-d2)oxy)-2-(tert-butyl)-4-methylbenzene (630 mg, 1.913 mmol, 1.0 equiv.) and tert-butyl 1,4-diazepane-1-carboxylate (421.46 mg, 2.104 mmol, 1.1 equiv.) afforded the Boc- intermediate (664 mg, 77% isolated yield). This compound was directly used in the Boc- deprotection step without further characterization. HRMS: Calculated for C27H45N2O3D2[M+H]+449.3712, found 449.3713. Step 4: Synthesis of 1-(6-(2-(tert-butyl)-4-methylphenoxy)hexyl-1,1-d2)-1,4- diazepane, M151: Boc- deprotection of 1-(6-(2-(tert-butyl)-4-methylphenoxy)hexyl-1,1-d2)- 1,4-diazepane (663 mg, 1.47 mmol) was performed following the procedure from the synthesis of M146 with the isolated yield of 49% (254 mg).1H NMR (600 MHz, CDCl3) δ 7.07 (d, J = 2.3 Hz, 1H), 6.95 (dd, J = 8.2, 2.3 Hz, 1H), 6.75 (d, J = 8.2 Hz, 1H), 3.94 (t, J = 6.4 Hz, 2H), 2.93 (t, J = 6.1 Hz, 2H), 2.92 – 2.88 (m, 2H), 2.69 (t, J = 5.8 Hz, 2H), 2.67 – 2.63 (m, 2H), 2.28 (s, 3H), 1.86 – 1.81 (m, 3H), 1.76 (p, J = 6.0 Hz, 2H), 1.57 – 1.46 (m, 4H), 1.39-1.34 (m, 11H (overlap)).13C NMR (151 MHz, CDCl3) δ 155.73, 137.68, 128.85, 127.44, 127.03, 111.72, 67.67, 58.44, 57.91, 57.78, 57.65, 57.52, 57.38, 54.52, 48.86, 47.43, 34.72, 30.45, 29.84, 29.50, 27.24, 27.23, 26.36, 20.78. HRMS: Calculated for C22H37N2OD2[M+H]+349.3188, found 349.3191. -129- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Synthesis of M152: Conditions: a) K2CO3, Acetone, reflux, overnight (48%); b) tert-butyl 1,4-diazepane-1- carboxylate, K2CO3, DMF, r.t, overnight (82%); c) TFA / DCM, r.t, 2 h (39% isolated yield). Step 1: Synthesis of 1-((6-bromohexyl)oxy)-2-(tert-butyl)-4-fluorobenzene: The bromo- intermediate was synthesized following the procedure from step 1 of M146 synthesis using 2-(tert-butyl)-4-fluorophenol (250 mg, 1.486 mmol, 1 equiv.) afforded 236 mg of the product (48%). This intermediate was directly used in the next step of the reaction without further characterization. Step 2: Synthesis of tert-butyl 4-(6-(2-(tert-butyl)-4-fluorophenoxy)hexyl)-1,4- diazepane-1-carboxylate: Boc- intermediate was synthesized following the procedure from step 2 of the M146 synthesis, using 1-((6-bromohexyl)oxy)-2-(tert-butyl)-4-fluorobenzene (236 mg, 0.712 mmol, 1.0 equiv.) and tert-butyl 1,4-diazepane-1-carboxylate (156.82 mg, 0.783 mmol, 1.1 equiv.) afforded 270 mg of the colorless oily product (82% yield).1H NMR (600 MHz, CDCl3) δ 6.99 (dd, J = 10.9, 3.1 Hz, 1H), 6.82 (ddd, J = 8.8, 7.5, 3.1 Hz, 1H), 6.75 (dd, J = 8.9, 4.9 Hz, 1H), 3.93 (t, J = 6.4 Hz, 2H), 3.54 – 3.37 (m, 4H), 2.70 – 2.55 (m, 4H), 2.46 (t, J = 7.6 Hz, 2H), 1.85 – 1.79 (m, 4H), 1.51 (s, 4H (br)), 1.46 (s, 9H), 1.36 (s, 11H (overlap)).13C NMR (151 MHz, CDCl3) δ 156.72 (d, J = 237.07 Hz), 155.64, 155.54*, 153.90 (d, J = 3.02 Hz) , 139.99 (d, J = 6.04 Hz), 113.88 (d, J = 22.65 Hz), 112.24 (d, J = 1.51 Hz), 112.13 (d, J = 15.1 Hz), 79.21, 68.20, 57.80, 57.73*, 56.12, 55.99*, 54.97, 54.69*, 46.79, 46.19*, 46.01, 45.06*, 34.96, 29.56, 29.45, 28.52, 27.91, 27.77*, 27.46, -130- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) 27.41*, 27.22, 26.31, 26.29*. (* rotamer peaks due to confirmational change of Boc- diazepane ring). HRMS: Calculated for C26H44N2O3 [M+H]+451.3336, found 451.3351. Step 3: Boc deprotection of tert-butyl 4-(6-(2-(tert-butyl)-4-fluorophenoxy)hexyl)- 1,4-diazepane-1-carboxylate was carried out using standard conditions from step 3 of the M146 synthesis to afford 82 mg (39% isolated yield) of the title compound 1-(6-(2-(tert- butyl)-4-fluorophenoxy)hexyl)-1,4-diazepane, M152 as colorless thick oil.1H NMR (600 MHz, CDCl3) δ 6.99 (dd, J = 10.8, 3.1 Hz, 1H), 6.84 – 6.78 (m, 1H), 6.75 (dd, J = 8.9, 4.8 Hz, 1H), 3.93 (t, J = 6.4 Hz, 2H), 2.94 – 2.90 (m, 4H), 2.69 (t, J = 5.8 Hz, 2H), 2.67 – 2.63 (m, 2H), 2.50 (dd, J = 8.9, 6.3 Hz, 2H), 1.83 (dt, J = 12.9, 6.5 Hz, 2H), 1.76 (p, J = 6.1 Hz, 2H), 1.66 (s, 1H), 1.55 -1.49 (m, 4H), 1.37 (s, 11H (overlap)).13C NMR (151 MHz, CDCl3) δ 156.70 (d, J = 237.07 Hz), 153.92 (d, J = 1.51 Hz), 140.0 (d, J = 6.04 Hz), 113.88 (d, J = 24.16 Hz), 112.23 (d, J = 3.02 Hz), 112.13 (d, J = 16.61 Hz), 68.22, 58.58, 58.44, 54.59, 48.88, 47.47, 34.97, 34.96, 30.49, 29.56, 29.46, 27.50, 27.27, 26.33.19F NMR (471 MHz, CDCl3) δ -124.45 (ddd, J = 11.2, 7.4, 4.9 Hz). HRMS: Calculated for C21H36N2OF [M+H]+351.2812, found 351.2813. While certain embodiments have been illustrated and described, a person with ordinary skill in the art, after reading the foregoing specification, can effect changes, substitutions of equivalents and other types of alterations to the compounds of the present technology or salts, pharmaceutical compositions, derivatives, prodrugs, metabolites, tautomers or racemic mixtures thereof as set forth herein. Each aspect and embodiment described above can also have included or incorporated therewith such variations or aspects as disclosed in regard to any or all of the other aspects and embodiments. The present technology is also not to be limited in terms of the particular aspects described herein, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. It is to be understood that this present technology is not limited to -131- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) particular methods, reagents, compounds, compositions, labeled compounds or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting. Thus, it is intended that the specification be considered as exemplary only with the breadth, scope and spirit of the present technology indicated only by the appended claims, definitions therein and any equivalents thereof. The embodiments, illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” “containing,” etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the claimed technology. Additionally, the phrase “consisting essentially of” will be understood to include those elements specifically recited and those additional elements that do not materially affect the basic and novel characteristics of the claimed technology. The phrase “consisting of” excludes any element not specified. In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and -132- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. All publications, patent applications, issued patents, and other documents (for example, journals, articles and / or textbooks) referred to in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure. The present technology may include, but is not limited to, the features and combinations of features recited in the following lettered paragraphs, it being understood that the following paragraphs should not be interpreted as limiting the scope of the claims as appended hereto or mandating that all such features must necessarily be included in such claims: A. A compound according to Formula (I): or a pharmaceutical salt thereof, wherein R1, R2, R3, R4, R5, R6, R7, and R8are each independently selected from H or D: and x is an integer from 1, 2, 3, 4, 5, 6, or 7. B. The compound of Paragraph A, wherein R1, R2, R3, R4, R5, R6, R7, and R8are each H. -133- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) C. The compound of Paragraph A or B, wherein x is 1. D. The compound of Paragraph A or B, wherein x is 3. E. The compound of Paragraph A or B, wherein x is 5. F. The compound of Paragraph A or B, wherein x is 7. G. The compound of any one of Paragraphs A–F, wherein the compound is: H. A compound according to Formula (II): -134- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) or a pharmaceutical salt thereof, wherein: R9, R10, R11, R12, R13, R14, R15, R16, R17and R18are each independently selected from H or D, and y is 1, 2, 3, 4, 5, 6, or 7. I. The compound of Paragraph H, wherein R9, R10, R11, R12, R13, R14, R15, R16, R17and R18are each H. J. The compound of Paragraph H, wherein R9, R10, R11, R12, R13, R14, R17and R18are each H, and R15and R16are each D. K. The compound of Paragraph H, wherein R9, R10, R11, R12, R13, R14, R15, and R16are each H, and R17and R18are each D. L. The compound of Paragraph H, wherein R9, R10, R11, R12, R13, R14, R15, and R16are each H, and R17and R18are each D. M. The compound of any one of Paragraphs H–L, wherein y is 1. N. The compound of any one of Paragraphs H–L, wherein y is 3. O. The compound of any one of Paragraphs H–L, wherein y is 5. P. The compound of any one of Paragraphs H–L, wherein y is 7. Q. The compound of any one of Paragraphs H–P, wherein the compound is: , -135- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) pharmaceutically acceptable salt thereof. R. A compound selected from: , -136- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) NH , -137- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) , or a pharmaceutically acceptable salt thereof. S. A composition comprising a compound of any one of Paragraphs A – R; and a pharmaceutically acceptable carrier. T. A pharmaceutical composition for treating a disease or disorder, wherein the pharmaceutical composition comprises a therapeutically effective amount of the compound of any one of Paragraphs A – R for treating the disease or disorder; and a pharmaceutically acceptable carrier. U. The pharmaceutical composition of Paragraph T, wherein the disease or disorder comprises a neurological disease or disorder. V. The pharmaceutical composition of Paragraph U, wherein the neurological disease or disorder comprises Alzheimer’s disease, Parkinson’s disease, or other protein aggregation diseases. W. The pharmaceutical composition of Paragraph T, wherein the disease or disorder comprises a lysosomal storage disease. X. The pharmaceutical composition of Paragraph W, wherein the lysosomal storage disease comprises Gaucher disease, Fabry disease, Niemann-Pick disease, or the mucopolysaccharidoses. -138- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) Y. The pharmaceutical composition of Paragraph T, wherein the disease or disorder comprises a Transcription Factor EB (TFEB)-mediated disease. Z. The pharmaceutical composition of Paragraph Y, wherein the TFEB-mediated disease comprises kidney diseases, cancer, metabolic disorders, inflammation, or liver diseases. AA. A pharmaceutical composition for activating Transcription Factor EB (TFEB), wherein the pharmaceutical composition comprises a therapeutically effective amount of the compound of any one of Paragraphs A–R for activating TFEB; and a pharmaceutically acceptable carrier. AB. A method of treating a disease or disorder in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of the compound of any one of Paragraphs A–R for treating the disease or disorder or administering to the patient the pharmaceutical composition of any one of Paragraphs S- AA. AC. The method of Paragraph AB, wherein the disease or disorder comprises a neurological disease or disorder. AD. The method of Paragraph AC, wherein the neurological disease or disorder comprises Alzheimer’s disease, Parkinson’s disease, or other protein aggregation diseases. AE. The method of Paragraph AB, wherein the disease or disorder comprises a lysosomal storage disease. AF. The method of Paragraph AE, wherein the lysosomal storage disease comprises Gaucher disease, Fabry disease, Niemann-Pick disease, or the mucopolysaccharidoses. AG. The method of Paragraph AB, wherein the disease or disorder comprises a Transcription Factor EB (TFEB)-mediated disease. AH. The method of Paragraph AG, wherein the TFEB-mediated disease comprises kidney diseases, cancer, metabolic disorders, inflammation, or liver diseases. -139- Atty. Dkt. No.: 115872-3160 (SK2023-044-02) AI. A method of activating Transcription Factor EB (TFEB) in a patient, the method comprising administering to the patient a therapeutically effective amount of the compound of any one of Paragraphs A–R activating TFEB or administering to the patient the pharmaceutical composition of any one of Paragraphs S-AA. AJ. Use of a therapeutically effective amount of the compound of any one of Paragraphs A– R for treating a disease or disorder in a patient in need thereof. AK. The use of Paragraph AJ, wherein the disease or disorder comprises a neurological disease or disorder. AL. The use of Paragraph AK, wherein the neurological disease or disorder comprises Alzheimer’s disease, Parkinson’s disease, or other protein aggregation diseases. AM. The use of Paragraph AJ, wherein the disease or disorder comprises a lysosomal storage disease. AN. The use of Paragraph AM, wherein the lysosomal storage disease comprises Gaucher disease, Fabry disease, Niemann-Pick disease, or the mucopolysaccharidoses. AO. The use of Paragraph AJ, wherein the disease or disorder comprises a Transcription Factor EB (TFEB)-mediated disease. AP. The use of Paragraph AO, wherein the TFEB-mediated disease comprises kidney diseases, cancer, metabolic disorders, inflammation, or liver diseases. AQ. Use of a therapeutically effective amount of the compound of any one of Paragraphs A–R for activating Transcription Factor EB (TFEB). Other embodiments are set forth in the following claims, along with the full scope of equivalents to which such claims are entitled. REFERENCES 1. 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Claims

Atty. Dkt. No.: 115872-3160 (SK2023-044-02) WHAT IS CLAIMED IS:

1. A compound according to Formula (I):or a pharmaceutical salt thereof, wherein: R1, R2, R3, R4, R5, R6, R7, and R8are each independently selected from H or D, and x is 1, 2, 3, 4, 5, 6, or 7.

2. The compound of claim 1, wherein R1, R2, R3, R4, R5, R6, R7, and R8are each H.

3. The compound of claim 1, wherein x is 1.

4. The compound of claim 1, wherein x is 3.

5. The compound of claim 1, wherein x is 5.

6. The compound of claim 1, wherein x is 7. -147-Atty. Dkt. No.: 115872-3160 (SK2023-044-02) 7. The compound of claim 1, wherein the compound is:.

8. A compound according to Formula (II):or a pharmaceutical salt thereof, wherein: R9, R10, R11, R12, R13, R14, R15, R16, R17and R18are each independently selected from H or D, and y is is 1, 2, 3, 4, 5, 6, or 7.

9. The compound of claim 8, wherein R9, R10, R11, R12, R13, R14, R15, R16, R17and R18are each H.

10. The compound of claim 8, wherein R9, R10, R11, R12, R13, R14, R17and R18are each H, and R15and R16are each D.

11. The compound of claim 8, wherein R9, R10, R11, R12, R13, R14, R15, and R16are each H, and R17and R18are each D. -148-Atty. Dkt. No.: 115872-3160 (SK2023-044-02) 12. The compound of claim 8, wherein R9, R10, R11, R12, R13, R14, R15, and R16are each H, and R17and R18are each D.

13. The compound of claim 8, wherein y is 1.

14. The compound of claim 8, wherein y is 3.

15. The compound of claim 8, wherein y is 5.

16. The compound of claim 8, wherein y is 7.

17. The compound of claim 8, wherein the compound is:thereof. -149-Atty. Dkt. No.: 115872-3160 (SK2023-044-02) 18. A compound selected from:-150-Atty. Dkt. No.: 115872-3160 (SK2023-044-02)thereof.

19. A composition comprising a compound of any one of claims 1-18; and a pharmaceutically acceptable carrier.

20. A pharmaceutical composition for treating a disease or disorder, wherein the pharmaceutical composition comprises a therapeutically effective amount of the compound of any one of claims 1- 18 for treating the disease or disorder; and a pharmaceutically acceptable carrier.

21. The pharmaceutical composition of claim 20, wherein the disease or disorder comprises a neurological disease or disorder.

22. The pharmaceutical composition of claim 21, wherein the neurological disease or disorder comprises Alzheimer’s disease, Parkinson’s disease, or other protein aggregation diseases.

23. The pharmaceutical composition of claim 20, wherein the disease or disorder comprises a lysosomal storage disease. -151-Atty. Dkt. No.: 115872-3160 (SK2023-044-02) 24. The pharmaceutical composition of claim 23, wherein the lysosomal storage disease comprises Gaucher disease, Fabry disease, Niemann-Pick disease, or the mucopolysaccharidoses.

25. The pharmaceutical composition of claim 20, wherein the disease or disorder comprises a Transcription Factor EB (TFEB)-mediated disease.

26. The pharmaceutical composition of claim 25, wherein the TFEB-mediated disease comprises kidney diseases, cancer, metabolic disorders, inflammation, or liver diseases.

27. A pharmaceutical composition for activating Transcription Factor EB (TFEB), wherein the pharmaceutical composition comprises a therapeutically effective amount of the compound of any one of claims 1- 18 for activating TFEB; and a pharmaceutically acceptable carrier.

28. A method of treating a disease or disorder in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of the compound of any one of claims 1-18 for treating the disease or disorder.

29. The method of claim 28, wherein the disease or disorder comprises a neurological disease or disorder.

30. The method of claim 29, wherein the neurological disease or disorder comprises Alzheimer’s disease, Parkinson’s disease, or other protein aggregation diseases.

31. The method of claim 28, wherein the disease or disorder comprises a lysosomal storage disease.

32. The method of claim 31, wherein the lysosomal storage disease comprises Gaucher disease, Fabry disease, Niemann-Pick disease, or the mucopolysaccharidoses.

33. The method of claim 28, wherein the disease or disorder comprises a Transcription Factor EB (TFEB)-mediated disease.

34. The method of claim 33, wherein the TFEB-mediated disease comprises kidney diseases, cancer, metabolic disorders, inflammation, or liver diseases. -152-Atty. Dkt. No.: 115872-3160 (SK2023-044-02) 35. A method of activating Transcription Factor EB (TFEB) in a patient, the method comprising administering to the patient a therapeutically effective amount of the compound of any one of claims 1-18 for activating TFEB.

36. Use of a therapeutically effective amount of the compound of any one of claims 1-18 for treating a disease or disorder in a patient in need thereof.

37. The use of claim 36, wherein the disease or disorder comprises a neurological disease or disorder.

38. The use of claim 37, wherein the neurological disease or disorder comprises Alzheimer’s disease, Parkinson’s disease, or other protein aggregation diseases.

39. The use of claim 36, wherein the disease or disorder comprises a lysosomal storage disease.

40. The use of claim 39, wherein the lysosomal storage disease comprises Gaucher disease, Fabry disease, Niemann-Pick disease, or the mucopolysaccharidoses.

41. The use of claim 36, wherein the disease or disorder comprises a Transcription Factor EB (TFEB)-mediated disease.

42. The use of claim 41, wherein the TFEB-mediated disease comprises kidney diseases, cancer, metabolic disorders, inflammation, or liver diseases.

43. Use of a therapeutically effective amount of the compound of any one of claims 1-18 for activating Transcription Factor EB (TFEB). -153-

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