Small molecule enhancers of secreted clusterin (SCLU)

Abstract

Described herein are compounds that increase levels of sCLU (clusterin secreted isoform) and associated methods of treating Alzheimer's disease, diabetes and cardiovascular disease.

Description

[0001] SMALL MOLECULE ENHANCERS OF SECRETED CLUSTERIN (SCLU)

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 537,391, filed September 8, 2023; the entire contents of which are incorporated herein by reference.

[0004] GOVERNMENT SUPPORT

[0005] This invention was made with government support under AG068116 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] BACKGROUND

[0007] Alzheimer's Disease (AD) is the most common form of dementia and is characterized by the gradual accumulation of amyloid-P (AP) plaques and tau neurofibrillary tangles in the brain, leading to significant inflammation, synaptic injury, and progressive neuronal degeneration. Currently, more than 50 million people worldwide are living with AD, making it the seventh leading cause of death in the United States. In 2021 alone, AD cost the nation $355 billion, and it is projected that this number of cases will triple by 2050, imposing an unprecedented burden on society and the healthcare system. Despite numerous therapeutic approaches, including 143 agents in clinical trials as of 2022, treatment options for AD patients remain severely limited. Of the three classes of FDA- approved drugs, namely acetylcholinesterase inhibitors and N-methyl D-aspartyl (NMD A) receptor antagonists, only provide temporary symptomatic relief without halting disease progression. Recently, two Ap- directed antibodies, aducanumab and lecanemab, demonstrated the ability to reduce Ap plaques and slow cognitive decline in clinical trials. These therapies received authorization under the FDA's accelerated approval pathway, highlighting the urgent need for novel disease-modifying therapeutic targets. Furthermore, it may be necessary to develop multifaceted therapies that target both the primary disease pathogenesis and the additional cellular stress responses associated with AD pathophysiology.

[0008] SUMMARY OF THE INVENTION

[0009] In certain aspects, provided herein is a compound of Formula (I), or a pharmaceutically acceptable salt thereof:

[0010]

[0011] Formula (I) wherein X is selected from OR1and NR2R3;

[0012] R1is selected from H, optionally substituted aryl, optionally substituted alkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted cycloalkyl, and alkynyl;

[0013] R2and R3are independently selected from H, optionally substituted aryl, hydroxyl, optionally substituted alkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted cycloalkyl, and alkynyl; provided that R2and R3are not both hydroxyl; or

[0014] R2and R3taken together with the nitrogen atom to which they are attached form an optionally substituted 4-7 membered ring; n is 0-5; and

[0015] Y is independently selected from F, Cl, Br, and I; wherein the compound is not

[0016] BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 shows molecular mechanisms of secreted clusterin for preventing Alzheimer’s disease. (A) sCLU binds and prevent the aggregation of Ap. (B) sCLU interacts with LRP2 to transports Ap species across the blood-brain barrier into the periphery. (C) sCLU interacts with TREM2 to transport Ap into glial cells for lysosomal degradation. (D) sCLU interacts with APOER2 and VLDR to induce reelin signaling, which decreases tau phosphorylation by inhibiting GSK3p. (E) sCLU prevents oxidative stress and associated CASP3-mediated apoptosis. (F) sCLU prevents assembly of the membrane attack complex and associated apoptosis. (G) sCLU interacts with VGLUT1 to stimulate excitatory neurotransmission. Figure 2 shows faulty transvascular clearance of brain Ap across the blood-brain barrier (BBB) plays an important role in Ap accumulation in AD. The two major risk factors for AD, apolipoprotein E (APOE) and clusterin (CLU), interact with Ap and regulate its clearance from the brain. Transvascular brain-to-blood transport across the BBB clears most of the Ap from the brain (-85%) (Adapted from Nelson et al. 2017).

[0018] Figure 3 shows phagocytic uptake of Ap inlo microglial cells is mediated by CLU and TREM2. (a) Ap is taken up more efficiently by microglia in the presence of CLU (adapted from Retana et al. 2019). (b) Uptake of Ab-CLU complex is TREM2 dependent (adapted from Yeh et al, 2016).

[0019] Figure 4 shows peripheral administration of human recombinant sCLU reduces brain Ap levels in an AD mouse model. Cerebral Ap levels in 15-month-old APP23 mice treated subchronically with saline, rHDL-rApoJ nanodiscs, or free rApOJ (a) Cerebral soluble AP40 levels, (b) Cerebral insoluble AP40. (c) Cerebral insoluble AP42, detected by ELISA, (d) Representative images showing ThS-positive vessels and the corresponding in brain sections, (e) Representative images showing ThS-positive deposits, quantification of the number of ThS-positive deposits per area (px2), and quantification of the mean size of those deposits, (f) Representative images of anti-Ap (4G8)-positive deposits in the brain parenchyma, quantification of the number or Ap-positive deposits per area (px2), and quantification of the mean size of those deposits (adapted from Retana et al., 2019).

[0020] Figure 5 shows testing of various reagent concentrations. U-87 MG cells were treated with Vorinostat (0, 2.5 pM) for 24 hours. sCLU AlphaLISA was performed on the EMEM media and Cell Supernatant (0, 2.5 pM Vorinostat) using increasing concentrations of J84- Biotinylated Ab and P-Acc Beads. Highly significant differences in between conditions was observed at 2x and 4x concentrations of J84-Bio / p-Acc Mix.

[0021] Figure 6 shows testing various treatments time points. U-87 MG cells were treated with Vorinostat (0, 2.5 pM) and the supernatant was collected at 12, 18, 24, 30, and 36 hours. sCLU levels were measured using AlphaLISA. Similar increases in sCLU levels (-1.5 fold) were observed at 24, 30, and 36 hours. Z’ statistic shows the good separation between the distributions of the positive and negative controls at 24, 30, and 36 hours.

[0022] Figure 7 shows the validation of HTS conditions. New reagents to be used for the HTS were tested: P -chain Acceptor Beads (2 pL; 50 pg / mL), J84-Biotinylated Ab (2 pL; 10 nM), and Donor Beads- (2 pL; 60x dilution). Differences between the two conditions (0 pM, 2.5 pM Vorinostat) was highly significant (-1.5-fold, p<0.00001).

[0023] FIG. 8 shows high-throughput screening identifies HD AC and BET inhibitors to increase sCLU levels. (A) sCLU levels from a high-throughput screening of LOPAC and Prestwick chemical libraries at a concentration of 5 pM in U-87 MG glioblastoma cells. (B) sCLU levels from the retesting of 32 compounds selected from the high-throughput screen in triplicate. (C) Schematic showing the molecular relationship between HD AC inhibitor, vorinostat, and BET inhibitor, I-BET151. (D, E) sCLU and nCLU levels following treatment with HD AC inhibitors (vorinostat and belinostat), and BET inhibitors (I-BET151 and (+)- JQ1) at a concentration of 5 pM. (F) Dose-response curves showing sCLU levels and calculated EC50 values for vorinostat (2042 nM), belinostat (1122 nM), I-BET151 (617 nM), and (+)-JQl (313 nM), following treatment at concentrations of 2, 7, 206, 62, 185, 556, 1667 and 5000 nM. (G) Live U-87 MG cell measures following treatment with vorinostat, belinostat, I-BET151, and (+)-JQl, at 23, 69, 206, 617, 1852, 5556, 16667 and 50000 nM. All results graphed as mean ± SEM. All statistics were performed with a two-way ANOVA (*p < 0.05; **p < 0.001; ***p < 0.0001; ****p < 0.00001). (H) sCLU following testing of bromodomain selective inhibitors, MS436 and ABBV744, independently and jointly at concentrations of 0.2, 1 and 5 pM. (I) Live and dead U-87 MG cell measures following treatment with vorinostat or LBET151 at 23, 69, 206, 617, 1852, 5556, 16667 and 50000 nM. All results graphed as mean ± SEM. All statistics were performed with a two-way ANOVA (*p < 0.05; **p < 0.001; ***p < 0.0001; ****p < 0.00001).

[0024] Fig. 9 shows testing of selective inhibitors provides insights into the molecular mechanisms underlying sCLU enhancement. (A) sCLU levels following testing with isoform selective HDAC inhibitors (CM-675, santacruzamate A, RGFP966, tasquinimod, BRD4354, MPT0G211, HDAC8-IN-1, SIS 17) and pan HDAC inhibitor vorinostat at a concentration of 5 pM. (B) Dose-response curves showing sCLU levels and calculated EC50 values for HDAC1 inhibitor, CM-675 (6.0 pM), HDAC3 inhibitor, RGFP966 (10.1 pM), and HDAC6 inhibitor, MPT0G211 (10.0 pM), following treatment at concentrations of 2, 7, 206, 62, 185, 556, 1667 and 5000 nM. (C) Dose-response curves showing sCLU levels and calculated EC50 values for selective bromodomain 1 inhibitor, MS436, and bromodomain 2 inhibitor, ABBV- 774 (1.1 pM), following treatment at concentrations of 2, 7, 206, 62, 185, 556, 1667 and 5000 nM. (D) sCLU following testing of vorinostat and I-BET151 independently and jointly at concentrations of 0.2, 1 and 5 pM. All statistics were performed with a two-way ANOVA (*p < 0.05; **p < 0.001; ***p < 0.0001; ****p < 0.00001). Fig. 10 (A, B) shows an LC-MS, Total ion chromatogram (TIC) and extracted ion chromatogram (EIC) for DDL-356. (C) HPLC UV-Vis spectra at 280 nm. (D) A 64-scan proton NMR (*H-NMR) utilizing default parameters.!H NMR (400 MHz, CDC13) 57.44 - 7.36 (m, 2H), 7.36 - 7.30 (m, 2H), 5.52 (tt, J = 6.4, 5.3 Hz, 1H), 4.91 (tdd, J = 7.5, 6.3, 1.0 Hz, 2H), 4.72 (dddd, J = 7.5, 5.0, 3.8, 0.9 Hz, 2H), 4.60 (t, J = 6.3 Hz, 1H), 3.77 - 3.60 (m, 2H), 2.72-2.67 (s, 3H), 2.46-2.39 (s, 3H), 1.71-1.65 (s, 3H).

[0025] Fig. 11 (A, B) shows LC-MS, Total ion chromatogram (TIC) and extracted ion chromatogram (EIC) for DDL-357 (adduct is m / z = 527.25). (C) HPLC UV-Vis spectra at 250 nm. (D) A 64-scan proton NMR (*H-NMR) utilizing default parameters. 51H NMR (400 MHz, CDC13) 5^ NMR (300 MHz, CDC13) 57.43 - 7.31 (m, 4H), 4.61 (t, J = 7.0 Hz, 1H), 4.19 (tt, J = 6.2, 2.9 Hz, 1H), 3.59 (d, J = 7.1 Hz, 2H), 2.70 (s, 3H), 2.42 (s, 3H), 1.25 (s, 3H), 0.79 - 0.73 (m, 4H).

[0026] Fig. 12 shows in-silico molecular docking guides design of (+)-JQ-l analogs with favorable protein-ligand binding energies. (A) Molecular docking of (+)-JQ-l in BD1 domain of BRD4 (pdb: 3MXF) and (B) predicted protein-ligand physiochemical interactions. (C) Molecular docking of DDL-357 in BD1 domain of BRD4 (pdb: 3MXE), (D) predicted protein-ligand physiochemical interactions, and (E) results of a 100 ns simulation (RMSD, nm; root mean square deviations).

[0027] Fig. 13 shows dose-response curves identify ester containing (+)-JQ-l analogs to be significantly more potent. (A) Dose-response curves showing sCLU levels and calculated EC50 values for (+)-JQ-l and ten novel analogs (DDL-351 - DDL-360), following treatment at concentrations of 2, 7, 206, 62, 185, 556, 1667 and 5000 nM. (B) Dose-response curves showing BRD2 inhibition (%) and calculated IC50 values for (+)-JQ-l and ten novel analogs (DDL-351 - DDL-360), following treatment at concentrations of 2, 7, 206, 62, 185, 556, 1667 and 5000 nM. (C) Dose-response curves showing BRD4 inhibition (%) and calculated IC50 values for (+)-JQ-l and ten novel analogs (DDL-351 - DDL-360), following treatment at concentrations of 2, 7, 206, 62, 185, 556, 1667 and 5000 nM. (D) Dose-response curves showing BRD4 bromodomain 1 (BD1) and bromodomain 2 (BD2) inhibition (%) and calculated IC50 values for lead candidate, DDL-357, following treatment at concentrations of 2, 7, 206, 62, 185, 556, 1667 and 5000 nM.

[0028] FIG. 14 shows chronic administration of DDL-357 increases sCLU levels and improves memory in AD mouse models. (A) DDL-357 brain concentrations (total and unbound), 1, 2, 4, 6 and 8 hours following oral administration at a dose of 30 mg / kg. (B) Live cell measures in U-87 MG, SH-SY5Y, HEK293T, and HEPG2 cells following treatment with lead candidate, DDL-357, at 23, 69, 206, 617, 1852, 5556, 16667 and 50000 nM. (C) sCLU levels in the hippocampus of E4 / FAD and 3xTG-AD mice following chronic administration of vehicle DDL-357 at a dose of 15 mg / kg. (D) Latency to escape hole for DDL-357-treated and vehicle-treated 3xTG-AD mice in both training a probe trials in the Barnes Maze assessment of spatial learning and memory. (E) Amyloid P 1-42 (Ap42) levels in the hippocampus of E4 / FAD and 3xTG-AD mice following chronic administration of vehicle or DDL-357 at a dose of 15 mg / kg. (F) Phosphorylated tau (pTau) / total tau levels at S396. (G) phosphorylated tau (pTau) / total tau levels at S202 / T205 in the hippocampus of 3xTG-AD mice following chronic administration of vehicle or DDL-357 at a dose of 15 mg / kg. (H) A scatter plot showing the inverse correlation between phosphorylated tau (pTau) / total tau levels at S396 and S202 / T205 vs sCLU levels in the hippocampus of 3xTG-AD mice following chronic administration of vehicle or DDL-357 at a dose of 15 mg / kg. All results graphed as mean ± SEM. All statistics were performed with a two-way ANOVA (*p < 0.05; **p < 0.001; ***p < 0.0001; ****p < 0.00001).

[0029] FIG. 15 shows candidate sCLU enhancers reach therapeutically relevant brain concentrations in-vivo. (A) DDL-357 brain concentrations (total and unbound), 1 hour following oral administration at a dose of 3, 10, or 30 mg / kg. (B) Human ether-a-go-go-related gene (hERG) potassium channel activity following treatment with lead candidate, DDL-357, at 0.39, 1.56, 6.25, 25, and 100 pM. (C) Phosphorylated tau (pTau) / total tau levels at S396 in the hippocampus of 3xTG-AD, separated by gender, mice following chronic administration of vehicle or DDL-357 at a dose of 15 mg / kg (D) Phosphorylated tau (pTau) / total tau levels at S202 / T205 in the hippocampus of 3xTG-AD mice, separated by gender, following chronic administration of vehicle or DDL-357 at a dose of 15 mg / kg. (E) DDL-356 brain concentrations (total and unbound), 1 hour following oral administration at a dose of 3, 10, or 30 mg / kg. (F) DDL-356 brain concentrations (total and unbound), 1, 2, 4, 6 and 8 hours following oral administration at a dose of 30 mg / kg. All results graphed as mean ± SEM. All statistics were performed with a two-way ANOVA (*p < 0.05; **p < 0.001; ***p < 0.0001; ****p < 0.00001).

[0030] FIG. 16 shows Proteomics identifies DDL-357 to increase mitochondrial function, synaptic plasticity, and aggregate clearance. (A) Differences in the abundances of proteins, illustrated via volcano plot, in the hippocampus of 3xTG-AD and E4 / FAD mice following chronic administration of DDL-357 at a dose of 15 mg / kg. The-logio (p-value) is plotted against logio (abundance ratio: DDL-357 / Vehicle). (B) Results of gene set pathway enrichment analysis performed on genes significantly upregulated by DDL-357 in-vivo (C) A Venn diagram illustrating the association of DDL-357-upregulated genes with mitochondrial function, protein homeostasis, or synaptic plasticity. (D) Basal, ATP-linked, and Maximal oxygen consumption rate (OCR) in U-87 MG glioblastoma cells and (E) SH-SY5Y neuroblastoma cells, following treatment with DDL-357 at concentrations of 0, 8, 40 and 200 nM. (F) Humanin levels in SH-SY5Y cells, following treatment with DDL-357 at concentrations of 200 nM. (G) Sirtuin 1 levels in SH-SY5Y cells, following treatment with DDL-357 at concentrations of 200 nM. (H) Confocal image of Calcein-AM stained iPSC- derived neurons 48 hours after treatment with 0.5% DMSO and (I) DDL-357 at a concentration of 6.9 nM. (J) Measurements of neurite outgrowth in iPSC-derived neurons following treatment with 0.5% DMSO or DDL-357 at concentrations. (K) Gene ontology enrichment analysis using proteins significantly upregulated in the hippocampus of DDL-357 treated E4 / FAD mice, relative to vehicle treated controls. (L) A schematic illustrating the DDL- 357 -upregulated genes important for maintaining AD-relevant biological functions, including mitochondrial function, protein homeostasis, and synaptic plasticity. All results graphed as mean ± SEM. All statistics were performed with a two-way ANOVA (*p < 0.05; **p < 0.001; ***p < 0.0001; ****p < 0.00001).

[0031] Fig. 17 shows DDL-357 dose-response curves for neuroprotective targets in SH-SY5Y neuroblastoma cells. (A) Dose-response curve showing sCLU levels in SH-SY5Y cells and the calculated EC50 values for DDL-357 (152 nM), following treatment at concentrations of 2, 7, 206, 62, 185, 556, 1667 and 5000 nM. (B) Dose-response curve showing SIRT1 levels in SH-SY5Y cells and the calculated EC50 values for DDL-357 (105 nM), following treatment at concentrations of 2, 7, 206, 62, 185, 556, 1667 and 5000 nM. (C) Humanin levels in SH-SY5Y cells following testing of DDL-357 at concentrations of 0.2, 1 and 5 pM. All statistics were performed with a two-way ANOVA (*p < 0.05; **p < 0.001; ***p < 0.0001; ****p < 0.00001).

[0032] Fig. 18 shows the neuroprotective effects of DDL-357.

[0033] DETAILED DESCRIPTION OF THE INVENTION

[0034] Genome-wide association studies (GWAS) show that the third strongest genetic risk factor for late-onset Alzheimer's disease (LOAD) is a single nucleotide polymorphism (SNP) in a gene that encodes for clusterin (CLU), a multifunctional chaperone protein directly involved in a wide range of AD-associated biological processes, including Ap and Tau metabolism, lipid transport, immune modulation, oxidative stress, and cell apoptosis.4Interestingly, seven CLU gene variants have been associated with AD, and individuals carrying the C allele of the rsl 1136000 SNP show increased Ap and Tau deposition, faster cognitive decline, and decreased expression of the secreted clusterin isoform (sCLU) compared to other isoforms.5,6Consequently, extensive research on CLU in different disease states, including neurodegeneration, cardiovascular disease, and cancer, has led to the prevailing hypothesis that sCLU promotes cytoprotection, while intracellular isoforms (nCLU) have pro-apoptotic properties.4

[0035] The most significant protective function of sCLU is its role in regulating protein homeostasis within the central nervous system (CNS). Research shows that sCLU binds and prevents the protein aggregation of disease relevant toxic, misfolded proteins, including a- synuclein, TDP-43, Ap, and Tau7'10(Fig. 1A). sCLU is a crucial mediator of Ap clearance from the brain due to its strong affinity for Ap and endocytic receptors like LRP2 and TREM2, facilitating the transport of toxic Ap species across the blood-brain barrier (BBB) or into glial cells for lysosomal degradation11 12(Fig. IB, 1C). Additionally, sCLU binds to ApoER2 and VLDR, activating signaling pathways such as Reelin that can reduce tau phosphorylation (p- tau)13’14(Fig. ID). sCLU also protects against oxidative stress, mitochondrial dysfunction, harmful immunological changes, and synaptic deficits, all contributing to AD progression15'19(Fig. IE, IF, 1G). Not surprisingly, peripheral administration of recombinant sCLU in an AD mouse model reduces Ap accumulation, neuronal loss and levels of pro-inflammatory markers.20As research on this target protein continues to accumulate, the therapeutic potential of sCLU in preventing the molecular processes underlying AD becomes increasingly apparent.

[0036] In certain aspects, provided herein is a compound of Formula (I), or a pharmaceutically acceptable salt thereof:

[0037] Formula (I) wherein X is selected from OR1and NR2R3; R1is selected from H, optionally substituted aryl, optionally substituted alkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted cycloalkyl, and alkynyl;

[0038] R2and R3are independently selected from H, optionally substituted aryl, hydroxyl, optionally substituted alkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted cycloalkyl, and alkynyl; provided that R2and R3are not both hydroxyl; or

[0039] R2and R3taken together with the nitrogen atom to which they are attached form an optionally substituted 4-7 membered ring; n is 0-5; and

[0040] Y is independently selected from F, Cl, Br, and I; wherein the compound is not

[0041] In certain embodiments, n is 1. In certain embodiments, the compound of Formula (I) has the structure . In certain such embodiments, Y is Cl. In certain embodiments, X is OR1.

[0042] In certain embodiments, R1is selected from alkyl, cycloalkyl, and heterocyclyl. In certain embodiments, R1is selected from C1-C4 alkyl, cyclohexyl, cyclobutyl, , and . In certain embodiments, R1is selected from . In certain embodiments, In certain embodiments, X is NR2R3. In certain embodiments, R2is H. In certain embodiments, R3is selected from -OH, cycloalkyl, optionally substituted alkyl, and heterocyclyl. In certain embodiments, R is selected from -OH, cyclopentyl s , and \ In certain embodiments, R2and R3taken together with the nitrogen atom to which they are attached form a 4-7 membered ring. In certain embodiments, R2and R3taken together with the nitrogen atom to which they are attached form a 6-membered ring.

[0043] In certain embodiments, the structure is selected from: embodiments, the structure is selected from:

[0044]

[0045] In certain embodiments, the structure is

[0046] In certain embodiments, the invention provides a pharmaceutical composition comprising the compound of Formula (I) and a pharmaceutically acceptable excipient.

[0047] In certain embodiments, the invention provides methods of treating or preventing a disease selected from Alzheimer’s disease, diabetes, and cardiovascular disease, comprising administering to a subject in need thereof a compound of Formula I, or a pharmaceutically acceptable salt thereof:

[0048] Formula (I) wherein X is selected from OR1and NR2R3; R1is selected from H, optionally substituted aryl, optionally substituted alkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted cycloalkyl, and alkynyl;

[0049] R2and R3are independently selected from H, optionally substituted aryl, hydroxyl, optionally substituted alkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted cycloalkyl, and alkynyl; provided that R2and R3are not both hydroxyl; or

[0050] R2and R3taken together with the nitrogen atom to which they are attached form an optionally substituted 4-7 membered ring; n is 0-5; and

[0051] Y is selected from F, Cl, Br, and I.

[0052] In certain embodiments, a therapeutically effective amount of the compound of

[0053] Formula (I) is administered to a subject in need thereof. In certain embodiments, n is 1. In certain embodiments, the compound of Formula (I) has the structure certain embodiments, X is OR1.

[0054] In certain embodiments, R1is selected from alkyl, cycloalkyl, and heterocyclyl. In certain embodiments, R1is selected from C1-C4 alkyl, cyclohexyl, cyclobutyl, , and . In certain embodiments, R1is selected from . In certain embodiments,

[0055] In certain embodiments, X is NR2R3. In certain embodiments, R2is H. In certain embodiments, R3is selected from -OH, cycloalkyl, optionally substituted alkyl, and heterocyclyl. In certain embodiments, R is selected from -OH, cyclopentyl, = , and in certain embodiments, R2and R3taken together with the nitrogen atom to which they are attached form a 4-7 membered ring. In certain embodiments, R2and R3taken together with the nitrogen atom to which they are attached form a 6-membered ring.

[0056] In certain embodiments, the structure is selected from: embodiments, the structure is selected from: In certain embodiments, the structure

[0057] In certain embodiments, the disease is Alzheimer’s disease. In certain embodiments, the Alzheimer’s disease is late-onset Alzheimer’s disease.

[0058] The study described herein represents the first description of a drug discovery effort aimed at identifying potent, brain-permeable small molecules that increase levels of sCLU, and evaluating their effects on AD. A high-throughput screening (HTS) approach successfully identified three compounds (vorinostat, I-BET151, piperlongumine) that significantly increased sCLU levels secreted from glioblastoma cells. Interestingly, all three compounds shared a common theme of inhibiting proteins that recognize acetyl functional groups on lysine residues. Two of these compounds, vorinostat and piperlongumine, had been classified as HD AC inhibitors. HDACs are enzymes that regulate the transcription process by removing acetyl groups from lysine residues of histones and transcription factors and have been associated with various neurodegenerative diseases, including AD. Interestingly, HD AC inhibition has been described as a promising therapeutic strategy offering neuroprotection by reversing the hypo acetylation commonly observed in AD, preventing Ap-induced hyperphosphorylation of tau, and promoting the expression of crucial genes associated with synaptic plasticity, learning, and memory. Not surprisingly, vorinostat is already being tested in phase 1 clinical trials for treating AD. The other compound identified in the HTS was the well-known BET inhibitor, I-BET151. Similar to HDACs, BET proteins are widely acknowledged as master transcriptional regulators in response to physiological and pathophysiological cues. This function is facilitated by two acetyl lysine-recognizing bromodomains (BDs) that recruit transcription factors and coactivators to target gene sites, activating the transcriptional machinery. Therefore, pharmacological inhibition of HDACs may exert downstream effects on BET proteins. Accordingly, the inhibition of BET proteins has also been recognized as a potential therapeutic strategy for treating AD. BET inhibitors have been shown to reduce neuroinflammation and tau phosphorylation, while also promoting brain plasticity and cognitive function. Many of the molecular mechanisms underlying these neurotrophic changes are not well characterized and may be mediated by sCLU. These findings reveal a shared molecular pathway connecting impaired protein acetylation with sCLU and AD pathogenesis.

[0059] Through testing a structurally diverse array of HD AC and BET inhibitors, a comprehensive investigation was conducted into the molecular mechanisms underlying sCLU expression. While the availability of entirely selective inhibitors somewhat constrained the experiments, the results suggest that three HDACs (HDAC1, HDAC3, HDAC6) and BD2 of BRD4 regulate sCLU expression. The results described herein could be utilized in the pursuit of developing more selective sCLU-enhancing therapeutics that avoid disrupting the normal homeostatic functions of all HD AC or BET proteins simultaneously. Limiting drug promiscuity is often critical in preventing adverse reactions / effects that contribute to drug attrition. The findings also revealed BET inhibitors to be considerably more potent and less toxic than HD AC inhibitors, effectively elevating sCLU levels at low nanomolar concentrations without causing significant cellular toxicity. Consequently, that the results described herein suggest that BET inhibition represents a more valuable therapeutic approach, as pharmacologically relevant drug levels were much more attainable in vivo, and less likely to cause unwanted side effects.

[0060] In pursuit of identifying BET inhibitors likely to be successful therapeutics for treating CNS disorders in-vivo, various in-vitro assays were used to evaluate physicochemical properties that influence drug ADME. While a compound may exhibit good in-vitro drug activity, it does not automatically guarantee favorable in-vivo activity. Bioavailability and half-life should be considered. Moreover, the escalating costs associated with the development of novel therapeutics and the high rate of candidate attrition have prompted a shift in drug discovery strategies towards the simultaneous evaluation of comprehensive drug physicochemical and ADME properties alongside efficacy. This comprehensive assessment holds particular significance for CNS disorders due to the selective exclusion of nearly 100% of large-molecule neuro therapeutics and over 98% of all small-molecule drugs by the blood-brain barrier (BBB). Additional optimization efforts also resulted in the design and synthesis of ten novel JQ1 analogs. Of the nineteen compounds assessed, only one known BET inhibitor and three NCEs met the stringent go / no-go ADME criteria required for further evaluation in-vivo. Moreover, DDL-357 exhibited enhanced potency, BBB permeability, and metabolic stability, along with decreased brain tissue and plasma binding. As a result, it was selected as the lead sCLU enhancing drug candidate.

[0061] Proof-of-concept pharmacokinetic and pharmacodynamic analyses were subsequently conducted to determine if DDL-357 could reach therapeutically relevant concentrations in the brain and effectively increase sCLU levels. As predicted by the in-vitro ADME assessment, DDL-357 exhibited excellent brain bioavailability, reaching micromolar concentrations one- hour post-administration at a dose of 30 mg / kg. Following this, DDL-357 demonstrated a significant increase in hippocampal sCLU levels in the E4 / FAD AD mouse model after two weeks of treatment. Interestingly, a corresponding decrease in Ap42 levels was also observed, but due to large variability, it was not deemed statistically significant. It is possible that a longer study in a larger cohort of mice would yield different results. Unfortunately, the effects of DDL-357 on p-tau levels were unable to be assessed, as the mice do not express the human form of tau.

[0062] To further characterize the pharmacological effects of DDL-357 in vivo, an unbiased proteomics analysis was utilized to identify additional differentially expressed proteins. This approach revealed increased expression of various proteins critical to mitochondrial function, synaptic plasticity, and protein homeostasis processes known to be disrupted during AD pathogenesis. This included several proteins from complex I, NADHmbiquinone oxidoreductase (NDUFA13, NDUFC2, NDUFS5, NDUFB4), and complex III, ubiquinol- cytochrome c oxidoreductase (UQCRFS1), of the mitochondrial respiratory chain. These proteins are notably decreased in AD patient brains, and in some cases, this decrease has been suggested to be causative of the disease. Increasing these proteins has important implications for maintaining mitochondrial bioenergetics vital to supporting neuronal function and preventing oxidative damage in AD. Interestingly, there was also an upregulation of several autophagy -regulating proteins (SMCR8, TRPV2, RAB8A, RAB27B, GRPEL1) known to enhance autophagic-lysosomal clearance of protein aggregates, including Ap and alpha- synuclein (asyn). This was accompanied by increases in important molecular chaperones (CCT2, CCT3, VBP1), as well as additional components of the endo-lysosome autophagy pathway (VSP39) and the ubiquitin-proteasome system degradation pathway (UBE2L3, PSMD3). Several proteins critical for maintaining synaptic function, dendritic spine morphology, long-term potentiation, and associated learning and memory processes were also upregulated (SNAP29, ALCAM, ITSN1, DLGAP2, NPTXR). Interestingly, one of these proteins, ITSN1, is a prominent component of the reelin signaling pathway and may, therefore, work in conjunction with sCLU to reduce levels of p-tau. Encouraged by these findings, DDL-357 was tested on human iPSC-derived neurons and shown to significantly enhance neurite outgrowth. While the effects of DDL-357 on mitochondrial respiration have yet to be tested in these cells, it is hypothesized that a Seahorse-based bioenergetics analysis will show pronounced increases in ATP production. This discovery of a novel class of sCLU enhancers presents an opportunity for further evaluation and development of these compounds as novel therapeutics for treating Alzheimer’s disease. The results described herein provide evidence of numerous diseaserelevant biological processes mediated by BET proteins. Moreover, it provides evidence that BET inhibition may represent a mechanistically-related, yet superior therapeutic approach when compared to HD AC inhibition, which is currently being evaluated in clinical trials. The lead drug candidate, DDL-357, specifically shows promise as a potent, multifaceted therapeutic that may inhibit primary disease pathogenesis while also promoting various neuroprotective functions such as mitochondrial function, synaptic plasticity, and protein homeostasis. There is confidence that a long-term in-vivo study in the 3xTg-AD mouse model will provide further evidence of drug efficacy and spawn the emergence of a promising new clinical candidate that may, one day, be used to treat MCI and AD patients. While additional research is needed to evaluate the full extent of sCLU's involvement, this drug discovery effort has undoubtedly shed light on a valuable therapeutic approach that warrants further evaluation as a treatment for AD.

[0063] The invention described herein relates to potent, brain-permeable small molecules that enhance brain sCLU levels and evaluating their impact on Alzheimer's disease (AD) pathology, memory function, and neuroprotective proteins. High-throughput screening (HTS) was employed to identify small molecule compounds enhancing sCLU. Following confirmation and prioritization of hits, we conducted exploratory medicinal chemistry based on a known, potent BET inhibitor to design novel sCLU enhancers. In vitro absorption, distribution, metabolism, excretion, and toxicity (ADMET) assays, as well as in vivo pharmacokinetics (PK) analyses, we assessed drug-like properties and oral brain permeability were assessed to guide lead selection for further preclinical efficacy testing in two AD mouse models. The lead drug candidate, DDL-357, described herein, represents a promising new therapeutic approach with potential to mitigate AD progression via multiple mechanisms. These include reducing disease-associated Ap plaques and Tau neurofibrillary tangles, while also enhancing various neuroprotective functions, including mitochondrial function, synaptic plasticity, and protein homeostasis.

[0064] The mechanisms described herein suggest that increasing sCLU levels could be represent a broader therapeutic approach for treating additional proteinopathies, such as Parkinson's disease and amyotrophic lateral sclerosis.

[0065] Described herein is research to identify potent, brain permeable, small molecules that increase levels of sCLU and to evaluate their effect on AD pathology. This was accomplished using drug discovery and development strategy methods involving a high-throughput screening (HTS) to identify compounds or ‘hits’ with potent sCLU enhancing ability, in-vitro absorption, distribution, metabolism, excretion and toxicity (ADMET) assays and in-vivo pharmacokinetics (PK) analyses to assess drug-likeness and guide hit selection, exploratory medicinal chemistry to design novel sCLU enhancers, and further efficacy testing in AD patient-derived neural cells and 5XFAD-ApoE4 (E4FAD) mice. This demonstrates the value using this approach to identify novel therapeutics and to determine which are likely to be effective in-vivo. The lead drug candidates described here represent a promising new therapeutic paradigm with the potential to prevent AD progression through numerous mechanisms, including, but not limited to the reducing disease-causing Ap plaques and Tau neurofibrillary tangles, as well as promoting mitochondrial function, synaptic plasticity, and protein homeostasis.

[0066] More than 5 million Americans are currently living with Alzheimer’s disease (AD), which is now the sixth leading cause of death in the United States. AD will cost the nation $305 billion in 2020 alone, and the number of cases is expected to triple by 2050 putting an unprecedented burden on our society and healthcare system (https: / / www.alz.org / alzheimers- dementia / facts-figures). There are currently no available disease-modifying therapies that target specific pathological mechanisms responsible for AD progression. While familial forms of the disease typically result from mutations in genes for amyloid precursor protein (APP), presenilin (PSEN1) and presenilin 2 (PSEN2), the etiology of sporadic or late-onset Alzheimer’s disease (LOAD) is still largely unknown. Representing -95% of all AD cases, LOAD is considered multifactorial disease dependent on the complex interaction of both genetic and environmental risk factors. Among currently known genetic risk factors for LOAD, two members of the apolipoprotein family, apolipoprotein E (APOE) and apolipoprotein J (APOJ), also known as clusterin (CLU), are among the most significant, and are both known to regulate Ap clearance from brain (Fig. 2). It is important to highlight that only the mature, secreted CLU isoform (sCLU), commonly recognized as an extracellular chaperone, facilitates Ap metabolism.

[0067] Interestingly, several CLU gene variants have been associated with AD, and the majority rsl 1136000C SNP result in reduced CLU expression and a decreased ratio of sCLU relative to the intracellular isoform (iCLU). Recent studies investigated the relationship between CLU SNPs and pathological markers of AD, reporting that individuals carrying the C allele of rsl 1136000 have increased Ap deposition and tau levels than noncarriers. Additionally, the C allele is linked with faster cognitive decline, altered memory processing, and structural deficits in the brain of both AD patients and healthy individuals. Altogether, this evidence supports the hypothesis that increased sCLU in brain may be protective in AD by preventing aggregation and increasing clearance of Ap. Also, considering that the rsl 1136000C risk allele is prominent amongst certain demographics, including -88% of Caucasians, developing novel therapeutics to influence this major genetic risk factor is of great significance to public health. As the literature surrounding CLU accumulates, its importance as a potential therapeutic target for AD becomes more evident. Specifically, the secreted isoform of CLU (sCLU) is believed to be a neuroprotective protein with the ability to reverse several cellular processes underlying AD pathophysiology. Once novel drug candidates that enhance circulating levels of sCLU have been identified, they were utilized to assess the potential of sCLU as disease-modifying therapy for treating AD.

[0068] World- wide, AD is the most common form of dementia and is characterized by the gradual accumulation of amyloid-P (AP) plaques and tau neurofibrillary tangles in the brain that results in synaptic injury and progressive neuronal degeneration. These pathological Ap plaques originate from the transmembrane APP that undergoes sequential proteolytic cleavage by P-secretase [‘P-site-APP cleaving enzyme 1’ (BACE1)] and y-secretase to generate aggregation-prone Ap40 or Ap42 peptides. Despite a myriad of therapeutic strategies targeting Ap-related mechanisms, no drug has been approved for treating AD since 2003, suggesting the paramount need for novel therapeutic targets for this multifactorial disease. Moreover, multifaceted therapies that inhibit both primary disease pathogenesis as well as additional, unwanted cellular stress responses associated with AD pathophysiology, may be necessary. Interestingly, CLU, the third strongest risk factor for LOAD, is a multifunctional protein directly involved in Ap clearance as well as a wide range of additional biological processes relevant in AD including lipid transport, immune modulation, oxidative stress and cell apoptosis.

[0069] CLU is conventionally described as a secreted, heterodimeric glycoprotein that is upregulated following cellular stress or in response to neurotoxicity. Though in recent years, examination of brain CLU in various rodent and human models has revealed the existence of alternative protein isoforms with distinct functions and cellular and subcellular localizations. While it is well documented that alternative splicing can impart distinct functions through isoform- specific protein-protein interactions, it is rare that isoforms of the same protein have completely opposing effects on biochemical processes. Interestingly, numerous studies on CLU across many different disease states including neurodegeneration, cardiovascular disease and cancer, has led to the predominant hypotheses that the mature, secreted isoform (sCLU) supports cytoprotection, while the intracellular isoform (iCLU) is pro-apoptotic in nature. Additionally, there is a recently discovered, mitochondrial isoform (mitoCLU) with lesser known functions that may also play important roles in cellular homeostasis and disease.

[0070] The most pronounced protective role of sCLU is regulating protein homeostasis in the brain. sCLU has long been demonstrated to bind and prevent the aggregation of toxic, misfolded protein species such as Ap. It is also known as an essential mediator of Ap clearance from the brain. The affinity of sCLU for both Ap and endocytotic receptors such as LRP2 and TREM2 allows it to readily transport toxic Ap species across the blood-brain barrier (BBB) or into glial cells for lysosomal degradation, respectively (Fig. 2, 3). Not surprisingly, peripheral administration of human recombinant sCLU reduces brain Ap levels in AD mouse models (Fig. 4). It is also hypothesized that sCLU may disrupt tau-mediated cytotoxicity by inhibiting matrix-dependent endocytosis of tau aggregates. However, despite the obvious therapeutic potential of CLU, inconsistent results have fueled debate over its neuroprotective role. Interestingly, Ap and tau induce the expression of pro-apototic iCLU in neurons and astrocytes. This can explain results of CLU-mediated toxicity in AD and further demonstrates the importance of distinguishing between the different CLU isoforms. Additionally, we believe that the predominant use of blunt force protein knock-out (KO) experiments that abolish all CLU isoforms have led to reports that may obscure the functionally distinct biological processes performed by the individual isoforms. Overall, majority of research highlights the potentially beneficial effects of CLU in mediating Ap metabolism, but there is still much insight to be gained from understanding controlled expression of the functionally distinct isoforms.

[0071] Numerous lines of evidence provide additional support for the functional dichotomy between CLU isoforms. Clear antagonistic functions can be demonstrated through their individual interactions with components of the survival and apoptosis pathways. Additionally, there is strong evidence supporting the protective nature of sCLU against oxidative stress, which is a prominent pathological feature contributing to AD progression. Furthermore, by regulating membrane attack complex (MAC) formation, facilitating the clearance of late apoptotic cells by macrophages and preventing apoptotic cell-induced autoimmune responses, sCLU plays an important role in managing detrimental immunological changes prevalent in AD. While countless links have been established between CLU and AD related pathologies, there is often a lack of clarity as to which CLU isoforms are involved. Nevertheless, these studies highlight how different CLU isoforms can impart either cytoprotective or apoptotic functions. Furthermore, as scientific literature accumulates, the therapeutic potential of the mature, secreted isoform as a multifaceted mediator of inflammation and other pathological features of AD is becoming more evident. In this study, we will identify compounds that increase extracellular sCLU levels with the goal of providing a novel therapeutic option for treating AD.

[0072] To identify compounds that can enhance the expression of sCLU in different cellular systems, the UCLA small molecule libraries were screened. The primary HTS was performed in U-87 MG cells (a human glioblastoma cell line) (Figs. 5-7). For the primary screen, U-87 MG cells were seeded at 10K cells / well and treated at 5 pM for 24 hours. At the end of this time, extracellular levels of sCLU in the supernatant were assessed using an AlphaLISA immunoassay optimized to detect sCLU. The “hits”, compounds that increased intracellular sCLU over control (DMSO) were validated / retested to confirm an increase in sCLU over control. After secondary screening in SH- SY5Y neuroblastoma cells, tertiary screening were performed in human iPSCs.

[0073] Abbreviations

[0074] Definitions

[0075] Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics and protein and nucleic acid chemistry, described herein, are those well-known and commonly used in the art.

[0076] The methods and techniques of the present disclosure are generally performed, unless otherwise indicated, according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this specification. See, e.g., “Principles of Neural Science”, McGraw-Hill Medical, New York, N.Y. (2000); Motulsky, “Intuitive Biostatistics”, Oxford University Press, Inc. (1995); Lodish et al., “Molecular Cell Biology, 4th ed.”, W. H. Freeman & Co., New York (2000); Griffiths et al., “Introduction to Genetic Analysis, 7th ed.”, W. H. Freeman & Co., N.Y. (1999); and Gilbert et al., “Developmental Biology, 6th ed.”, Sinauer Associates, Inc., Sunderland, MA (2000).

[0077] Chemistry terms used herein, unless otherwise defined herein, are used according to conventional usage in the art, as exemplified by “The McGraw-Hill Dictionary of Chemical Terms”, Parker S., Ed., McGraw-Hill, San Francisco, C.A. (1985).

[0078] All of the above, and any other publications, patents and published patent applications referred to in this application are specifically incorporated by reference herein. In case of conflict, the present specification, including its specific definitions, will control.

[0079] The term “agent” is used herein to denote a chemical compound (such as an organic or inorganic compound, a mixture of chemical compounds), a biological macromolecule (such as a nucleic acid, an antibody, including parts thereof as well as humanized, chimeric and human antibodies and monoclonal antibodies, a protein or portion thereof, e.g., a peptide, a lipid, a carbohydrate), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. Agents include, for example, agents whose structure is known, and those whose structure is not known. The ability of such agents to inhibit AR or promote AR degradation may render them suitable as “therapeutic agents” in the methods and compositions of this disclosure.

[0080] A “patient,” “subject,” or “individual” are used interchangeably and refer to either a human or a non-human animal. These terms include mammals, such as humans, primates, livestock animals (including bovines, porcines, etc.), companion animals (e.g., canines, felines, etc.) and rodents (e.g., mice and rats).

[0081] “Treating” a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. As used herein, and as well understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0082] The term “preventing” is art-recognized, and when used in relation to a condition, such as a local recurrence (e.g., pain), a disease such as cancer, a syndrome complex such as heart failure or any other medical condition, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition. Thus, prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving a prophylactic treatment relative to an untreated control population, and / or delaying the appearance of detectable cancerous growths in a treated population versus an untreated control population, e.g., by a statistically and / or clinically significant amount.

[0083] “Administering” or “administration of’ a substance, a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or an agent can be administered, intravenously, arterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct). A compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the compound or agent. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0084] Appropriate methods of administering a substance, a compound or an agent to a subject will also depend, for example, on the age and / or the physical condition of the subject and the chemical and biological properties of the compound or agent (e.g., solubility, digestibility, bioavailability, stability and toxicity). In some embodiments, a compound or an agent is administered orally, e.g., to a subject by ingestion. In some embodiments, the orally administered compound or agent is in an extended release or slow-release formulation, or administered using a device for such slow or extended release.

[0085] As used herein, the phrase “conjoint administration” refers to any form of administration of two or more different therapeutic agents such that the second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., the two agents are simultaneously effective in the patient, which may include synergistic effects of the two agents). For example, the different therapeutic compounds can be administered either in the same formulation or in separate formulations, either concomitantly or sequentially. Thus, an individual who receives such treatment can benefit from a combined effect of different therapeutic agents.

[0086] A “therapeutically effective amount” or a “therapeutically effective dose” of a drug or agent is an amount of a drug or an agent that, when administered to a subject will have the intended therapeutic effect. The full therapeutic effect does not necessarily occur by administration of one dose and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. The precise effective amount needed for a subject will depend upon, for example, the subject’s size, health and age, and the nature and extent of the condition being treated, such as a neurological, metabolic, or cardiovascular disease. The skilled worker can readily determine the effective amount for a given situation by routine experimentation.

[0087] As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance may occur or may not occur, and that the description includes instances where the event or circumstance occurs as well as instances in which it does not. For example, “optionally substituted alkyl” refers to the alkyl may be substituted as well as where the alkyl is not substituted.

[0088] It is understood that substituents and substitution patterns on the compounds of the present invention can be selected by one of ordinary skilled person in the art to result chemically stable compounds which can be readily synthesized by techniques known in the art, as well as those methods set forth below, from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure results.

[0089] As used herein, the term “optionally substituted” refers to the replacement of one to six hydrogen radicals in a given structure with the radical of a specified substituent including, but not limited to: hydroxyl, hydroxyalkyl, alkoxy, halogen, alkyl, nitro, silyl, acyl, acyloxy, aryl, cycloalkyl, heterocyclyl, amino, aminoalkyl, cyano, haloalkyl, haloalkoxy, -OCO-CH2- O-alkyl, -OP(O)(O-alkyl)2 or -CH2-OP(O)(O-alkyl)2. Preferably, “optionally substituted” refers to the replacement of one to four hydrogen radicals in a given structure with the substituents mentioned above. More preferably, one to three hydrogen radicals are replaced by the substituents as mentioned above. It is understood that the substituent can be further substituted.

[0090] As used herein, the term “alkyl” refers to saturated aliphatic groups, including but not limited to C1-C10 straight-chain alkyl groups or C1-C10 branched-chain alkyl groups. Preferably, the “alkyl” group refers to Ci-Ce straight-chain alkyl groups or Ci-Ce branched- chain alkyl groups. Most preferably, the “alkyl” group refers to C1-C4 straight-chain alkyl groups or C1-C4 branched-chain alkyl groups. Examples of “alkyl” include, but are not limited to, methyl, ethyl, 1 -propyl, 2-propyl, n-butyl, sec-butyl, tert-butyl, 1 -pentyl, 2-pentyl, 3-pentyl, neo-pentyl, 1 -hexyl, 2-hexyl, 3-hexyl, 1 -heptyl, 2-heptyl, 3-heptyl, 4-heptyl, 1- octyl, 2-octyl, 3-octyl or 4-octyl and the like. The “alkyl” group may be optionally substituted.

[0091] The term “acyl” is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.

[0092] The term “acylamino” is art-recognized and refers to an amino group substituted with an acyl group and may be represented, for example, by the formula hydrocarbylC(O)NH-.

[0093] The term “acyloxy” is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)O-, preferably alkylC(O)O-.

[0094] The term “alkoxy” refers to an alkyl group having an oxygen attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy and the like.

[0095] The term “alkoxyalkyl” refers to an alkyl group substituted with an alkoxy group and may be represented by the general formula alkyl-O-alkyl.

[0096] The term “alkyl” refers to saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., Ci- 30 for straight chains, C3-30 for branched chains), and more preferably 20 or fewer.

[0097] Moreover, the term “alkyl” as used throughout the specification, examples, and claims is intended to include both unsubstituted and substituted alkyl groups, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone, including haloalkyl groups such as trifluoromethyl and 2,2,2- trifluoroethyl, etc.

[0098] The term “Cx.y” or “Cx-Cy”, when used in conjunction with a chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups that contain from x to y carbons in the chain. Coalkyl indicates a hydrogen where the group is in a terminal position, a bond if internal. A Ci-ealkyl group, for example, contains from one to six carbon atoms in the chain.

[0099] The term “alkylamino”, as used herein, refers to an amino group substituted with at least one alkyl group.

[0100] The term “alkylthio”, as used herein, refers to a thiol group substituted with an alkyl group and may be represented by the general formula alkylS-.

[0101] The term “amide”, as used herein, refers to a group

[0102] R10wherein R9and R10each independently represent a hydrogen or hydrocarbyl group, or R9and R10taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure.

[0103] The terms “amine” and “amino” are art-recognized and refer to both unsubstituted and substituted amines and salts thereof, e.g., a moiety that can be represented by wherein R9, R10, and R10’ each independently represent a hydrogen or a hydrocarbyl group, or R9and R10taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure.

[0104] The term “aminoalkyl”, as used herein, refers to an alkyl group substituted with an amino group.

[0105] The term “aralkyl”, as used herein, refers to an alkyl group substituted with an aryl group.

[0106] The term “aryl” as used herein include substituted or unsubstituted single-ring aromatic groups in which each atom of the ring is carbon. Preferably the ring is a 5- to 7- membered ring, more preferably a 6-membered ring. The term “aryl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like. The term “carbamate” is art-recognized and refers to a group io wherein R9and R10independently represent hydrogen or a hydrocarbyl group.

[0107] The term “carbocyclylalkyl”, as used herein, refers to an alkyl group substituted with a carbocycle group.

[0108] The term “carbocycle” includes 5-7 membered monocyclic and 8-12 membered bicyclic rings. Each ring of a bicyclic carbocycle may be selected from saturated, unsaturated and aromatic rings. Carbocycle includes bicyclic molecules in which one, two or three or more atoms are shared between the two rings. The term “fused carbocycle” refers to a bicyclic carbocycle in which each of the rings shares two adjacent atoms with the other ring. Each ring of a fused carbocycle may be selected from saturated, unsaturated and aromatic rings. In an exemplary embodiment, an aromatic ring, e.g., phenyl, may be fused to a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Any combination of saturated, unsaturated and aromatic bicyclic rings, as valence permits, is included in the definition of carbocyclic. Exemplary “carbocycles” include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5 -cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct-3-ene, naphthalene and adamantane. Exemplary fused carbocycles include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro- IH-indene and bicyclo[4.1.0]hept-3-ene. “Carbocycles” may be substituted at any one or more positions capable of bearing a hydrogen atom.

[0109] The term “carbocyclylalkyl”, as used herein, refers to an alkyl group substituted with a carbocycle group.

[0110] The term “carbonate” is art-recognized and refers to a group -OCO2-.

[0111] The term “carboxy”, as used herein, refers to a group represented by the formula -CO2H.

[0112] The term “ester”, as used herein, refers to a group -C(O)OR9wherein R9represents a hydrocarbyl group.

[0113] The term “ether”, as used herein, refers to a hydrocarbyl group linked through an oxygen to another hydrocarbyl group. Accordingly, an ether substituent of a hydrocarbyl group may be hydrocarbyl-O-. Ethers may be either symmetrical or unsymmetrical. Examples of ethers include, but are not limited to, heterocycle-O-heterocycle and aryl-O- heterocycle. Ethers include “alkoxyalkyl” groups, which may be represented by the general formula alkyl-O-alkyl.

[0114] The terms “halo” and “halogen” as used herein means halogen and includes chloro, fluoro, bromo, and iodo.

[0115] The terms “hetaralkyl” and “heteroaralkyl”, as used herein, refers to an alkyl group substituted with a hetaryl group.

[0116] The terms “heteroaryl” and “hetaryl” include substituted or unsubstituted aromatic single ring structures, preferably 5- to 7-membered rings, more preferably 5- to 6-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms “heteroaryl” and “hetaryl” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like.

[0117] The term “heteroatom” as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

[0118] The term “heterocyclylalkyl”, as used herein, refers to an alkyl group substituted with a heterocycle group.

[0119] The terms “heterocyclyl”, “heterocycle”, and “heterocyclic” refer to substituted or unsubstituted non-aromatic ring structures, preferably 3- to 10-membered rings, more preferably 3- to 7-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms “heterocyclyl” and “heterocyclic” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heterocyclic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls. Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.

[0120] The term “hydrocarbyl”, as used herein, refers to a group that is bonded through a carbon atom that does not have a =0 or =S substituent, and typically has at least one carbonhydrogen bond and a primarily carbon backbone, but may optionally include heteroatoms. Thus, groups like methyl, ethoxyethyl, 2-pyridyl, and even trifluoromethyl are considered to be hydrocarbyl for the purposes of this application, but substituents such as acetyl (which has a =0 substituent on the linking carbon) and ethoxy (which is linked through oxygen, not carbon) are not. Hydrocarbyl groups include, but are not limited to aryl, heteroaryl, carbocycle, heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof.

[0121] The term “hydroxy alkyl”, as used herein, refers to an alkyl group substituted with a hydroxy group.

[0122] The term “lower” when used in conjunction with a chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups where there are ten or fewer atoms in the substituent, preferably six or fewer. A “lower alkyl”, for example, refers to an alkyl group that contains ten or fewer carbon atoms, preferably six or fewer. In certain embodiments, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents defined herein are respectively lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, whether they appear alone or in combination with other substituents, such as in the recitations hydroxyalkyl and aralkyl (in which case, for example, the atoms within the aryl group are not counted when counting the carbon atoms in the alkyl substituent).

[0123] The terms “polycyclyl”, “polycycle”, and “polycyclic” refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls) in which two or more atoms are common to two adjoining rings, e.g., the rings are “fused rings”. Each of the rings of the polycycle can be substituted or unsubstituted. In certain embodiments, each ring of the polycycle contains from 3 to 10 atoms in the ring, preferably from 5 to 7.

[0124] The term “sulfate” is art-recognized and refers to the group -OSO3H, or a pharmaceutically acceptable salt thereof.

[0125] The term “sulfonamide” is art-recognized and refers to the group represented by the general formulae wherein R9and R10independently represents hydrogen or hydrocarbyl.

[0126] The term “sulfoxide” is art-recognized and refers to the group-S(O)-.

[0127] The term “sulfonate” is art-recognized and refers to the group SO3H, or a pharmaceutically acceptable salt thereof.

[0128] The term “sulfone” is art-recognized and refers to the group -S(O)2-. The term “substituted” refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. Substituents can include any substituents described herein, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate.

[0129] The term “thioalkyl”, as used herein, refers to an alkyl group substituted with a thiol group.

[0130] The term “thioester”, as used herein, refers to a group -C(O)SR9or -SC(O)R9wherein R9represents a hydrocarbyl.

[0131] The term “thioether”, as used herein, is equivalent to an ether, wherein the oxygen is replaced with a sulfur.

[0132] The term “urea” is art-recognized and may be represented by the general formula wherein R9and R10independently represent hydrogen or a hydrocarbyl. The term “modulate” as used herein includes the inhibition or suppression of a function or activity (such as cell proliferation) as well as the enhancement of a function or activity.

[0133] The phrase “pharmaceutically acceptable” is art-recognized. In certain embodiments, the term includes compositions, excipients, adjuvants, polymers and other materials and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0134] “Pharmaceutically acceptable salt” or “salt” is used herein to refer to an acid addition salt or a basic addition salt which is suitable for or compatible with the treatment of patients.

[0135] The term “pharmaceutically acceptable acid addition salt” as used herein means any non-toxic organic or inorganic salt of any base compounds represented by Formula I. Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids that form suitable salts include mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylic acids, as well as sulfonic acids such as p-toluene sulfonic and methanesulfonic acids. Either the mono or di-acid salts can be formed, and such salts may exist in either a hydrated, solvated or substantially anhydrous form. In general, the acid addition salts of compounds of Formula I are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms. The selection of the appropriate salt will be known to one skilled in the art. Other non-pharmaceutically acceptable salts, e.g., oxalates, may be used, for example, in the isolation of compounds of Formula I for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt.

[0136] The term “pharmaceutically acceptable basic addition salt” as used herein means any non-toxic organic or inorganic base addition salt of any acid compounds represented by Formula I or any of their intermediates. Illustrative inorganic bases which form suitable salts include lithium, sodium, potassium, calcium, magnesium, or barium hydroxide. Illustrative organic bases which form suitable salts include aliphatic, alicyclic, or aromatic organic amines such as methylamine, trimethylamine and picoline or ammonia. The selection of the appropriate salt will be known to a person skilled in the art. Many of the compounds useful in the methods and compositions of this disclosure have at least one stereogenic center in their structure. This stereogenic center may be present in a R or a S configuration, said R and S notation is used in correspondence with the rules described in Pure Appl. Chem. (1976), 45, 11-30. The disclosure contemplates all stereoisomeric forms such as enantiomeric and diastereoisomeric forms of the compounds, salts, prodrugs or mixtures thereof (including all possible mixtures of stereoisomers). See, e.g., WO 01 / 062726.

[0137] Furthermore, certain compounds which contain alkenyl groups may exist as Z (zusammen) or E (entgegen) isomers. In each instance, the disclosure includes both mixture and separate individual isomers.

[0138] Some of the compounds may also exist in tautomeric forms. Such forms, although not explicitly indicated in the formulae described herein, are intended to be included within the scope of the present disclosure.

[0139] “Prodrug” or “pharmaceutically acceptable prodrug” refers to a compound that is metabolized, for example hydrolyzed or oxidized, in the host after administration to form the compound of the present disclosure (e.g., compounds of formula I). Typical examples of prodrugs include compounds that have biologically labile or cleavable (protecting) groups on a functional moiety of the active compound. Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, or dephosphorylated to produce the active compound. Examples of prodrugs using ester or phosphoramidate as biologically labile or cleavable (protecting) groups are disclosed in U.S. Patents 6,875,751, 7,585,851, and 7,964,580, the disclosures of which are incorporated herein by reference. The prodrugs of this disclosure are metabolized to produce a compound of Formula I. The present disclosure includes within its scope, prodrugs of the compounds described herein. Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in “Design of Prodrugs” Ed. H. Bundgaard, Elsevier, 1985.

[0140] The phrase “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filter, diluent, excipient, solvent or encapsulating material useful for formulating a drug for medicinal or therapeutic use.

[0141] The term “Log of solubility”, “LogS” or “logS” as used herein is used in the art to quantify the aqueous solubility of a compound. The aqueous solubility of a compound significantly affects its absorption and distribution characteristics. A low solubility often goes along with a poor absorption. LogS value is a unit stripped logarithm (base 10) of the solubility measured in mol / liter.

[0142] EXAMPLES

[0143] Example 1 - HTS identifies HD AC and BET inhibitors that increase sCLU levels

[0144] The studies described herein were designed to identify potent, brain-permeable small molecules that increase sCLU levels and evaluate their effects in Alzheimer's disease (AD) models. For the HTS campaign, a customized AlphaLISA immunoassay was developed to quantify sCLU secreted from human U-87 MG glioblastoma cells and used to screen a subset of the UCLA compound libraries at a concentration of 5 pM (Fig. 8A). After the initial screening round, three of the thirty-two hit compounds were retested in triplicate and confirmed to significantly increase sCLU levels relative to DMSO controls (Fig. 8B). These validated hits included two HD AC inhibitors, vorinostat and piperlongumine, as well as one BET inhibitor, I-BET151. Notably, all three compounds inhibit epigenetic protein families that recognize acetyl groups on lysine residues (Fig. 8C). HDACs regulate transcription by removing acetyl groups from histones and transcription factors and are linked to neurodegenerative diseases, including AD21. Interestingly, HD AC inhibition has been described as a promising therapeutic strategy offering neuroprotection by reversing hypoacetylation in AD, preventing Ap-induced tau hyperphosphorylation, and promoting genes related to synaptic plasticity, learning, and memory21. Not surprisingly, vorinostat has already been evaluated in phase 1 clinical trials. Similarly, BET proteins are recognized as master transcriptional regulators, using two acetyl lysine-recognizing bromodomains to recruit transcription factors and coactivators to target gene sites22. Accordingly, BET inhibition has also demonstrated beneficial effects in AD mouse models, reducing neuroinflammation and tau phosphorylation, and promoting brain plasticity and cognitive function22'25. Furthermore, the BET inhibitor, Apabetalone, has been associated with improved cognition in clinical testing26. Many of the molecular mechanisms underlying these neurotrophic changes are not well characterized and may be mediated by sCLU.

[0145] Next, testing of additional structurally diverse HD AC and BET inhibitors obtained commercially was conducted to determine increase of sCLU in U-87 MG cells without affecting nCLU concentrations (Fig. 8D, E). Dose-response analyses revealed BET inhibitors to be significantly more potent than HD AC inhibitors (Fig. 8F). I-BET151 and (+)-JQl both demonstrated nanomolar potency, increasing sCLU with EC50 values of 617 nM and 313 nM, respectively. In contrast, vorinostat and belinostat had micromolar EC50 values of 2042 nM and 1122 nM, respectively. Additionally, cytotoxicity assays showed HD AC inhibitors to be significantly cytotoxic to U-87 MG cells at concentrations above 5 pM, while BET inhibitors were well tolerated with Ctox > 50 M (Fig. 8G). Consequently, our drug discovery efforts focused on advancing exploratory medicinal chemistry to identify potent, brain-permeable BET inhibitors that enhance sCLU levels with a favorable therapeutic index, to be evaluated for efficacy in reducing pathology and improving memory in AD.

[0146] To further investigate the molecular mechanisms underlying sCLU expression from our HTS hits, we also tested other commercially available, selective HD AC and BET inhibitors. Inhibition of HDAC1, HDAC3, and HDAC6 increased sCLU levels but was less effective and less potent than pan-HDAC inhibition with vorinostat (Fig. 9A, 9B). Similarly, bromodomain selective inhibitors were significantly less potent than pan-BET inhibitors (Fig. 9C). This finding is important because more selective compounds, though less potent, significantly reduce adverse effects, the primary cause of drug attrition27. Testing of bromodomain (BD) selective inhibitors identified BD2 of BET proteins to be essential the mechanism of enhancing sCLU (Fig. 8H). BD2 selective inhibitor, ABBV744, increased sCLU levels significantly at all tested concentrations. BD1 selective inhibitor, MS436, only increased sCLU at 5 pM, a concentration at which it is no longer selective for just BD1. Interestingly, dual inhibition of both HD AC and BET proteins by combined treatment with both HD AC and BET inhibitors resulted in a significant and synergistic increase in sCLU levels compared to individual treatments, suggesting potential for more effective therapies as well (Fig. 9D). However, this effect was only observed at micromolar concentrations, prompting our focused in medicinal chemistry efforts to develop brain-permeable sCLU enhancers based on BET inhibition. Evaluation of both compounds in a cytotoxicity assay identified vorinostat to be significantly toxic to U-87 MG cells at concentrations greater than 5 pM, while I-BET151 was well tolerated up to 50 pM (Fig. 81).

[0147] Methods

[0148] High Throughput Screening

[0149] Human glioblastoma U-87 MG cells were proliferated in EMEM with 10% FBS and 1% P / S (complete medium) in 10 cm dishes until approximately 90% confluent. Following trypsinization, a suspension of 2 x 105 cells / mL (25 pL) were added to 384-well plates containing complete medium (25 pL) and library compounds from 9 plates (5 pM). After incubation for 30 hours at 37 °C and 5% CO2, 2 pL of cell culture supernatant were analyzed via human sCLU AlphaLISA as described above. Cell culture medium and cells were added to the 384-well plates using the MultiDrop. The BioMek FX was used to pin the drugs and transfer the cell culture supernatant. The MANTIS liquid handler was used to added human sCEU AlphaEISA reagents. Primary screen

[0150] Getting started

[0151] Prepare complete medium by complementing EMEM with 10% FBS and 1% P / S. Proliferate U-87 MG cells in 10cm dishes until 70-90% confluent. Warm reagent to 37°C in water bath. Spray bottles down with 70% ethanol. Aspirate spent culture media, wash the cells with 5mL of sterile PBS. Aspirate PBS, add ImL of trypsin-EDTA solution and return the plate to the incubator for 3-5 minutes. Once the cells have detached add 5mL of complete medium and harvest the cells into a 15mL falcon tube. Centrifuge cells for approximately 4 minutes at 200g (about 1000 rpm, depending on the centrifuge). Aspirate the media above the cell pellet and resuspend the cells in lOmL of complete medium. Count the cells with a cell counter. Adjust the cell density to: for 384 well plate 4 x 105cells / mL (to obtain 10K cells / 25 uL to be added to each well).

[0152] Prepare vorinostat (positive control), by diluting primary stock (10 mM) 1000-fold to make secondary stock of 10 uM. Treat cells with final concentration of 5 uM vorinostat for 24 h.

[0153] During screen

[0154] Warm complete medium to 37°C in water bath. Spray bottle down with 70% ethanol. Prepare the 384 well plates by adding 25 pL of complemented EMEM using the MultiDrop to columns 2-23 of the 384 well plate. Add 25 uL of positive control to columns 1 & 24. Then pin 50 nL of the compounds from the drug library. Next, add 25ul media containing cells (4x105 cells / mL; 10k cell / well) using the MultiDrop. Allow the treatment to take place for 24 hours in the incubator at 37°C and 5% CO2.

[0155] Primary screen

[0156] After the 24-hour treatment, add 2 uL of lx HiBlock buffer to a 384-well proxiplate using the Multidrop. Then, transfer 2 uL of cell culture supernatant to the proxiplate using the BioMek FX.

[0157] Prepare the sCLU AlphaLISA reagents as follows: Dilute 10X HiBlock Buffer 10- fold in Ultrapure- H2O. Dilute mAb J84, biotinylated (500 nM) and mAb CLU aa 22-227, Acceptor bead conjugated (5 mg / mL) 200-fold in lx HiBlock buffer. Dilute AlphaScreen Streptavidin Donor beads 60-fold in lx HiBlock buffer. Use the MANTIS liquid handler to dispense 2 uL of the antibody mixture to each well and incubate for 1-hour at room temperature. Use the MANTIS to dispense 2 uL of Donor bead solution to each well and incubate for 45 minutes at room temperature, in the dark. Read AlphaLISA signal using the Envision Plate reader.

[0158] Secondary screen

[0159] Validation of selected hits were done in the MSSR. Briefly, U-87 MG cells (lOk / well in 384 well format) were treated at 5 uM in triplicate (n = 3) with selected hits for 24 h followed by detection of sCLU in the supernatant. The same protocol described above was used.

[0160] Reagent / Stock Preparation

[0161] • Complete medium:

[0162] EMEM with 10% FBS and 1% P / S.

[0163] • lx HiBlock Buffer

[0164] Dilute 10X HiBlock Buffer 10-fold in Ultrapure-H2O

[0165] • Acceptor Bead / Biotinylated Antibody Mixture

[0166] Dilute mAb J84, biotinylated (500 nM) and mAb CLU aa 22-227, Acceptor bead conjugated (500 nM) 200-fold in lx HiBlock buffer.

[0167] • Donor Bead Solution

[0168] Dilute AlphaScreen Streptavidin Donor beads 60-fold in IxHiBlock buffer.

[0169] Human sCLU AlphaLISA

[0170] Cell culture supernatant (2 pL) was added to a 384- well proxy plate preloaded with AlphaLISA 388 HiBlock buffer (2 pL, IX, CAT#AL004C, Perkin Elmer). Then an anti- sCLU antibody mixture (2 389 pL, mAb J84, biotinylated (10 nM, Cat# 3717-6-1000, Mabtech) and mAb CLU aa 22-227 390 (Cat#AF7084, R&D Systems), Acceptor bead conjugated (50 pg / mL, Cat#6772001, Perkin 391 Elmer)) was added and incubated at room temperature for 1 hour. AlphaScreen Streptavidin Donor beads (2 pL, Cat#6760002S, Perkin Elmer) were added, incubated for an additional 30 minutes in the dark, and the plate was read in an Envision plate reader. Mouse sCLU ELISA

[0171] Brain tissue was homogenized and diluted to a protein concentration of 50 ng / mL. The Mouse Clusterin ELISA Kit (Cat#abl99079, Abeam) was performed according to the manufacturer’s protocol.

[0172] Dose Response Analysis

[0173] Human glioblastoma U-87 MG cells were proliferated in EMEM with 10% FBS and 1% P / S (complete medium) in 10 cm dishes until approximately 90% confluent. Following trypsinization, a suspension of 2 x 105 cells / mL (25 pL) were added to 384-well plates containing complete medium (25 L) with various concentrations of compounds (5, 14, 41, 123, 370, 1111, 3333, 10000 nM). After incubation for 30 hours at 37 °C and 5% CO2, 2 pL of cell culture supernatant were analyzed via human sCLU AlphaLISA as described above.

[0174] Example 2 - Design, synthesis and evaluation of novel sCLU enhancers.

[0175] To develop more novel, potent and brain permeable BET inhibitors, ten novel (+)-JQl analogs were designed and synthesized as shown below.

[0176] Scheme 1. Synthetic scheme for the synthesis of novel sCLU enhancers (A) General synthetic scheme used for the synthesis of ester-containing (+)-JQ 1 analogs, and the biological activity (sCLU ECso, BRD2 / 4 ICso’s) for ester-containing analogs, DDL-351, -355, -356, -357, and -358 (B) General synthetic scheme used for the synthesis of amide-containing (+)-JQ 1 analogs, and the biological activity (sCLU ECso, BRD2 / 4 ICso’s) for amide- containing analogs DDL DDL-352, -353, -354, -359, and -360.

[0177]

[0178] Computer-aided molecular docking of these small molecules in bromodomain 1 of the bromodomain-containing protein 4 (BRD4) crystal structure (PDB: 3MXF) were used to model potential protein-ligand physiochemical interactions and facilitate design of new chemical entities with favorable binding energies, prior to their synthesis and testing, such as DDL-357 (Fig. 12A-2E). Two synthetic approaches were used to create these analogs of the BET inhibitor (+)-JQl. Analogs DDL-351, -355, -356, -357, and -358 were synthesized via an ester coupling reaction between (+)-JQl carboxylic acid and an alcohol-containing substituent (2 equiv), in the presence of 4-dimethylaminopyridine (DMAP; 2 equiv) and diisopropylcarbodiimide (DIC; 2 equiv) (Scheme 1). Analogs DDL-352, -353, -354, and -359 were synthesized via an amide coupling reaction between the (+)-JQl carboxylic acid (1 equiv) and an amide-containing substituent (2 equiv), in the presence of l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC; 2 equiv.), hydroxybenzotriazole (HOBt; 2 equiv.), and N-N, diisopropylethylamine (DIPEA; 2.5 equiv.) (Scheme 1). Dose-response curves clearly demonstrated that ester-containing analogs, such as DDL-351, exhibit significantly greater potency compared to their amide-containing counterparts, such as DDL-353 (Scheme 1; Fig. 13A). Bromodomain-containing protein 2 and 4 (BRD2 / 4) inhibition assays confirmed a similar trend in potency against direct molecular drug targets (Fig. 13B, 13C). Of the NCEs, synthesized DDL-355 exhibited the greatest potency in-vitro with BRD2 / 4 IC50’s of 163 and 19 nM and a sCLU EC50 of 99 nM.

[0179] [DDL-351]

[0180] (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][l,2,4]triazolo[4,3- a][l,4]diazepin-6-yl)acetic acid (JQ1 -carboxylic acid, 51.24 mg, 1 equiv), pentanol (32.23 mg, 3 equiv), and 4-Dimethylaminopyridine (30.42 mg, 2 equiv) in 700 pL of methylene chloride was brought to 0 °C. Diisopropylcarbodiimide (31.42 mg, 2 equiv) was added dropwise over the course of 30 min. The reaction was slowly brought to room temperature. After 24 h, the reaction mixture was filtered through a filter funnel and concentrated. The crude product was purified via flash column chromatography. A mobile phase of hexanes:ethyl acetate (time / % ethyl acetate: 0 / 0, 2 / 0, 7 / 50, 15 / 100, 20 / 0) followed by DCM:MeOH (time / %MeOH: 0 / 0, 4 / 0, 35 / 15, 42 / 20, 45 / 50, 50 / 0) afforded pure DDL-351. Peaks corresponding to DDL-351 were identified via LC / MS. Purity of DDL-351 was determined via NMR and HPLC UV-Vis. Pure DDL-351 was obtained as a white solid (47.76 mg, 80% yield). 5!H NMR (400 MHz, CDC13) 5 7.41 (m, 2H), 7.32 (m, 2H), 5.25 (m, 1H), 4.12 (q, J = 6.47 Hz, 1H), 3.57 (m, 1H), 2.68 (s, 3H), 2.41 (s, 3H), 1.90 (m, 2H), 1.77 (m, 2H), 1.69 (s, 3H), 1.60 (m, 2H), 1.25 (t, J = 7.06 Hz, 2H). LC-MS m / z [M+H] + 483.17.

[0181] [DDL-352]

[0182] JQ1 -carboxylic acid (80.00 mg, 0.199 mmol, 1 equiv.) in 3 mL of anhydrous DMF was added into a 250mL round bottom flask. Pyrrolidine (32.68 uL, 0.398 mmol, 2 equiv.) was added into the tube followed by the addition of ED AC (76.5 mg, 0.398 mmol, 2 equiv.), HOBt (29.67 mg, 0.219 mmol, 1.1 equiv.), and N-N, Diisopropylethylamine (0.104 mL, 0.597 mmol, 3 equiv.), and the resulting reaction mixture were stirred at room temperature overnight. After stirring overnight, the solution changed colors to light brownish. EIA was performed to check the reaction. The reaction mixture was dried and reconstituted using 20 mL of DCM, transferred to a separatory funnel, and the organic phase was washed with brine (2 x 20 mL), dried with anhydrous Na2SO4, filtered, concentrated, and evaporated. The resultant crude compound was purified by using a 4 g silica flash column, eluted with DCM: MeOH (time / % MeOH: 5 / 0, 20 / 10, 35 / 20). Purification by flash column chromatography afforded DDL 352. The fractions that were corresponding to the interest peaks were dried in a speed vacuum to yield (49.66 mg, Viscous white solid, 54.9%, DDL 352, LC-MS m / z [M+H]+454.5 ’ H NMR (400 MHz, CDC13) 5 7.39 (d, J = 8 Hz, 2H), 7.31 (d, J = 8 Hz, 2H), 4.80 (t, J = 4 Hz 1H), 3.69 - 3.49 (m, 4H), 2.95 (s, 3H), 2.88 (d, J = 0.6 Hz, 2H), 2.66 (s, 3H), 2.39 (s, 3H), 2.09 - 1.85 (m, 4H). LC- MS m / z [M+H] + 468.17

[0183] [DDL-353]

[0184] JQ1 -carboxylic acid (25.0 mg, 0.062 mmol, 1 equiv.) in 3 mL of DMF, Cyclopentylamine (0.124 mmol, 2 equiv.), ED AC (0.124 mmol, 2 equiv.), HOBt (0.124 mmol, 2 equiv.), and N- N, Diisopropylethylamine (0.186 mmol, 3 equiv.) and 10 mL of anhydrous DCM was added into a 250mL round bottom flask. The resulting reaction mixture was stirred at room temperature overnight, then filtered through a filter funnel and concentrated. The crude product was purified via flash column chromatography using a mobile phase of hexanes:ethyl acetate (time / % ethyl acetate: 0 / 0, 2 / 0, 7 / 50, 15 / 100, 20 / 0) followed by DCM:MeOH (time / %MeOH: 0 / 0, 4 / 0, 35 / 15, 42 / 20, 45 / 50, 50 / 0) afforded pure DDL-353. Eractions corresponding to DDL-353 were identified via LC / MS. Purity of DDL-353 was determined via NMR and HPLC UV-Vis. Pure DDL-353 was obtained as a white solid (15.6 mg, 54% yield).JH NMR (400 MHz, CDCh) 6 7.46 - 7.32 (m, 4H), 4.68 (t, J = 7.1 Hz, 1H), 4.22 (h, J = 6.9 Hz, 1H), 3.54 (dd, J= 14.3, 7.3 Hz, 1H), 3.38 (dd, J= 14.3, 6.8 Hz, 1H), 2.72 (s, 3H), 2.41 (s, 3H), 2.23 (s, 3H), 2.06 - 1.88 (m, 2H), 1.65 - 1.46 (m, 6H). LC-MS m / z [M+H]+468.17.

[0185]

[0186] [DDL-354]

[0187] JQ1 -carboxylic acid (25.0 mg, 0.062 mmol, 1 equiv.) in 3 mL of DMF, propan-2-yl 2- aminoacetate (0.124 mmol, 2 equiv.), ED AC (0.124 mmol, 2 equiv.), HOBt (0.124 mmol, 2 equiv.), and N-N, Diisopropylethylamine (0.186 mmol, 3 equiv.) and 10 mL of anhydrous DCM was added into a 250mL round bottom flask. The resulting reaction mixture was stirred at room temperature overnight, then filtered through a filter funnel and concentrated. The crude product was purified via flash column chromatography using a mobile phase of hexanes:ethyl acetate (time / % ethyl acetate: 0 / 0, 2 / 0, 7 / 50, 15 / 100, 20 / 0) followed by DCM:MeOH (time / %MeOH: 0 / 0, 4 / 0, 35 / 15, 42 / 20, 45 / 50, 50 / 0) afforded pure DDL-354. Eractions corresponding to DDL-354 were identified via LC / MS. Purity of DDL-354 was determined via NMR and HPLC UV-Vis. Pure DDL-354 was obtained as a white solid (11.1 mg, 36% yield).JH NMR (300 MHz, CDC13) 5 7.50 - 7.32 (m, 4H), 5.08 (t, J = 7.1 Hz, 1H), 4.74 (m, 1H), 3.62 (s, 2H), 3.12 (d, J= 6.7 Hz, 2H), 2.73 (s, 3H), 2.45 (s, 3H), 1.73 (s, 3H), 1.39-1.51 (d, J = 6.9 Hz, 6H). LC-MS m / z [M+H]+ 500.16. LC-MS m / z

[0188] [DDL-355]

[0189] JQ1 -carboxylic acid (51.13 mg, 1 equiv), cyclobutanol (26.98 mg, 3 equiv), and 4- Dimethylaminopyridine (31.48 mg, 2 equiv) in 700 pL of methylene chloride was brought to 0 °C. Diisopropylcarbodiimide (31.42 mg, 2 equiv) was added dropwise over the course of 30 min. The reaction was slowly brought to room temperature. After 24 h, the reaction mixture was filtered through a filter funnel and concentrated. The crude product was purified via flash column chromatography. A mobile phase of hexanes :ethyl acetate (time / % ethyl acetate: 0 / 0, 2 / 0, 7 / 50, 15 / 100, 20 / 0) followed by DCM:MeOH (time / %MeOH: 0 / 0, 4 / 0, 35 / 15, 42 / 20, 45 / 50, 50 / 0) afforded pure DDL-355. Peaks corresponding to DDL-355 were identified via LC / MS. Purity of DDL-355 was determined via NMR and HPLC UV-Vis. Pure DDL-355 was obtained as a white solid (48.82 mg, 84% yield).!H NMR (300 MHz, CDC13) 5 7.50 - 7.32 (m, 4H), 5.16 - 5.04 (m, 1H), 4.64 (dd, J = 7.5, 6.6 Hz, 1H), 3.70 - 3.59 (m, 2H), 2.73 (s, 3H), 2.45 (s, 3H), 2.26 - 2.11 (m, 2H), 1.93 - 1.79 (m, 1H), 1.73 (s, 3H), 1.69 - 1.59 (m, 1H), 1.30 (dt, J = 7.1, 4.1 Hz, 1H), 0.90 (dd, J = 14.6, 6.8 Hz, 1H). LC-MS m / z [M+H] + 455.14

[0190] DDL-356: Oxetan-3-yl (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f] [ 1 ,2,4] triazolo[4,3-a] [ 1 ,4]diazepin-6-yl)acetate

[0191] Scheme 2: Reaction scheme for DDL-356.

[0192] Method

[0193] 50 mg Scale A 25 mL round bottom flask equipped with a stir bar was charged with (S)-2-(4-(4- chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][l,2,4]triazolo[4,3-a][l,4]diazepin-6-yl)acetic acid (59.34 mg, 1 equiv), oxetan-3-ol (29.20 mg, 3 equiv), and 4-Dimethylaminopyridine (44.93 mg, 2 equiv). The starting materials and reagents were solubilized in methylene chloride (800 pL) and was brought to 0 °C in an ice bath. A solution of diisopropylcarbodiimide (31.48 mg, 2 equiv) in methylene chloride (200 p L) was added dropwise over the course of 30 min. The reaction was slowly brought to room temperature. After 24 h, insoluble urea product was clearly visible, and the reaction mixture was filtered through a filter funnel and concentrated in vacuo. The crude product was purified via flash column chromatography. A mobile phase of hexanes:ethyl acetate (time / % ethyl acetate: 0 / 0, 2 / 0, 7 / 50, 15 / 100, 20 / 0) was first utilized to elute any existing impurities, and the product was eluted in the DCM:MeOH mobile phase (time / %MeOH: 0 / 0, 4 / 0, 35 / 15, 42 / 20, 45 / 50, 50 / 0). Peaks corresponding to DDL-356 were identified via LC / MS. Purity of DDL-356 was determined via NMR and HPLC UV-Vis. Magnetic resonance spectroscopy was done at the UCLA Molecular Instrumentation Center (MIC; Ignacio Martini, Ph.D., Director). DDL-356 (2.23 mg) was solubilized in chloroform-d (750 pL) and analyzed using an AV400 NMR spectrometer (Bruker) containing a 5 mm broadband Z-gradient probe with Automatic Tune and Match (ATM). The analysis method consisted of a 64-scan proton NMR (*HNMR) utilizing default parameters. The experimental data was visualized and interpreted using Mnova (Mestrelab Research).!H NMR (400 MHz, CDC13) 5 7.44 - 7.36 (m, 2H), 7.36 - 7.30 (m, 2H), 5.52 (tt, J = 6.4, 5.3 Hz, 1H), 4.91 (tdd, J = 7.5, 6.3, 1.0 Hz, 2H), 4.72 (dddd, J = 7.5, 5.0, 3.8, 0.9 Hz, 2H), 4.60 (t, J = 6.3 Hz, 1H), 3.77 - 3.60 (m, 2H), 2.72-2.67 (s, 3H), 2.46-2.39 (s, 3H), 1.71-1.65 (s, 3H). LC-MS m / z [M+H]+457.6. (Fig. 11, Table 1).

[0194] Table 1. DDL-356 final mass and purity percent

[0195] DDL-357: Cyclopropyl (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f] [ 1 ,2,4] triazolo[4,3-a] [ 1 ,4]diazepin-6-yl)acetate .

[0196]

[0197] [DDL-357]

[0198] Scheme 3: Reaction scheme for DDL-357.

[0199] Method

[0200] 50 mg Scale

[0201] A 25 mL round bottom flask equipped with a stir bar was charged with (S)-2-(4-(4- chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][l,2,4]triazolo[4,3-a][l,4]diazepin-6-yl)acetic acid (162.6 mg, 1 equiv), propanol (69.7 mg, 3 equiv), and 4-Dimethylaminopyridine (100.7 mg, 2 equiv). The starting materials and reagents were dissolved in of methylene chloride (3 mL) and was brought to 0 °C in an ice bath. A solution of diisopropylcarbodiimide (100.7 mg, 2 equiv) in methylene chloride (0.2 mL) was added dropwise over the course of 30 min. The reaction was slowly brought to room temperature. After 24 h, insoluble urea product was clearly visible, and the reaction mixture was filtered through a filter funnel and concentrated in vacuo. The crude product was purified via flash column chromatography. A mobile phase of hexanes:ethyl acetate (time / % ethyl acetate: 0 / 0, 2 / 0, 7 / 50, 15 / 100, 20 / 0) was first utilized to elute any existing impurities, and the product was eluted in the DCM:MeOH mobile phase (time / %MeOH: 0 / 0, 4 / 0, 35 / 15, 42 / 20, 45 / 50, 50 / 0). Peaks corresponding to DDL-357 were identified via LC / MS. Purity of DDL-357 was determined via NMR and HPLC UV-Vis. Magnetic resonance spectroscopy was done at the UCLA Molecular Instrumentation Center (MIC; Ignacio Martini, Ph.D., Director). DDL-357 (2.23 mg) was solubilized in chloroform-d (750 pL) and analyzed using an AV400 NMR spectrometer (Bruker) containing a 5 mm broadband Z-gradient probe with Automatic Tune and Match (ATM). The analysis method consisted of a 64-scan proton NMR (*H NMR) utilizing default parameters. The experimental data was visualized and interpreted using Mnova (Mestrelab Research). 5!H NMR (400 MHz, CDC13) 5^ NMR (300 MHz, CDC13) 57.43 - 7.31 (m, 4H), 4.61 (t, J = 7.0 Hz, 1H), 4.19 (tt, J = 6.2, 2.9 Hz, 1H), 3.59 (d, J = 7.1 Hz, 2H), 2.70 (s, 3H), 2.42 (s, 3H), 1.25 (s, 3H), 0.79 - 0.73 (m, 4H). LC-MS m / z [M+H]+441.67 (Fig. 11, Table 2).

[0202] Table 2, DDL-357 final mass and purity percent

[0203] [DDL-358]

[0204] JQ1 -carboxylic acid (67 mg, 1 equiv), 1-butanol (28 mg, 3 equiv), and 4- Dimethylaminopyridine (33 mg, 2 equiv) in 1 mL of methylene chloride was brought to 0 °C. Diisopropylcarbodiimide (31.55 mg, 2 equiv) was added dropwise over the course of 30 min. The reaction was slowly brought to room temperature. After 24 h, the reaction mixture was filtered through a filter funnel and concentrated. The crude product was purified via flash column chromatography. A mobile phase of hexanes:ethyl acetate (time / % ethyl acetate: 0 / 0, 2 / 0, 7 / 50, 15 / 100, 20 / 0) followed by DCM:MeOH (time / %MeOH: 0 / 0, 4 / 0, 35 / 15, 42 / 20, 45 / 50, 50 / 0) afforded pure DDL-358. Peaks corresponding to DDL-358 were identified via LC / MS. Purity of DDL-358 was determined via NMR and HPLC UV-Vis. Pure DDL-358 was obtained as a white solid (13 mg, 23% yield). 51H NMR (400 MHz, CDC13) 57.44 - 7.30 (m, 4H), 4.63 (t, J = 6.7 Hz, 1H), 4.18 (t, J = 6.6 Hz, 2H), 3.62 (d, J = 1.4 Hz, 2H), 2.73 (s, 3H), 2.42 (s, 3H), 1.69 (s, 3H), 1.68 - 1.62 (m, 2H), 1.47 - 1.34 (m, 2H), 0.94 (t, J = 7.4 Hz, 3H). LC-MS m / z [M+H] + 457.15.

[0205] [DDL-359]

[0206] JQ1 -carboxylic acid (25.0 mg, 0.062 mmol, 1 equiv.) in 3 mL of DMF, (S)-Isopropyl 2- aminopropanoate (0.124 mmol, 2 equiv.), ED AC (0.124 mmol, 2 equiv.), HOBt (0.124 mmol, 2 equiv.), and N-N, Diisopropylethylamine (0.186 mmol, 3 equiv.) and 10 mL of anhydrous DCM was added into a 250mL round bottom flask. The resulting reaction mixture was stirred at room temperature overnight, then filtered through a filter funnel and concentrated. The crude product was purified via flash column chromatography using a mobile phase of hexanes: ethyl acetate (time / % ethyl acetate: 0 / 0, 2 / 0, 7 / 50, 15 / 100, 20 / 0) followed by DCM:MeOH (time / %MeOH: 0 / 0, 4 / 0, 35 / 15, 42 / 20, 45 / 50, 50 / 0) afforded pure DDL-359. Eractions corresponding to DDL-359 were identified via LC / MS. Purity of DDL- 359 was determined via NMR and HPLC UV-Vis. Pure DDL-359 was obtained as a white solid (14.0 mg, 44% yield).JH NMR (400 MHz, CDC13) 5 7.39 (d, J = 4 Hz, 2H), 7.32 (d, J = 4 Hz, 2H), 5.03 (t, J = 8 Hz, 1H), 4.54 (sept, J = 4 Hz, 1H), 3.54 (q, 4Hz, 1H), 2.66 (s, 2H), 2.61 (s, 6H), 2.39 (s, 3H) 1.45 (d, J = 4 Hz, 3H), 1.23 (d, J = 4 Hz, 3H), 1.205 (d, J = 2 Hz, 3H). LC-MS m / z [M+H] + 514.17

[0207] [DDL-360]

[0208] To a mixture of JQ1 -carboxylic acid (0.0998mmol, 1 Equiv.), EDC (0.1497mmol, 1.5 Equiv.), and HOBt (0.1497mmol, 1.5 Equiv.) in anhydrous DMF were added DIPEA (0.299mmol, 3 Equiv.). The mixture was stirred at RT for 10 min followed by the addition of hydroxyl amine HC1 (0.1497mmol, 1.5 Equiv.). The mixture was stirred at rt for 24hr. The resultant crude compound was purified by using a 4 g silica flash column, eluted with DCM: MeOH (time / % MeOH: 0 / 0, 3 / 0, 20 / 10, and 30 / 100). The fractions that were corresponding to the interest peaks were dried in a speed vacuum to yield (13.41 mg, white solid, 32.4%, DDL 360, LC-MS m / z [M+H]+416.10.JH NMR (400 MHz, CDC13) 57.46 (d, J = 8 Hz, 2H), 7.41 (d, J = 8 Hz, 2H), 4.46 (t, J = 8 Hz, 1H), 3.09 (d, J = 8 Hz), 2.56 (s, 3H), 2.38 (s, 3H), 1.59 (s, 3H), 1.20 (s, 1H). LC-MS m / z [M+H] + 416.10.An ion-extracted chromatogram (IEC) using the m / z corresponding to the [M+H]+ion (416.10) was utilized to identify the chromatographic peak for each compound. Furthermore, the purity percent was calculated by dividing the chromatographic peak area for each compound by the sum of all the non-background peak areas in the UV-Vis absorption spectrum (Chem 3138).

[0209] Dose-response curves clearly demonstrated that ester-containing analogs, such as DDL-351, exhibit significantly greater potency compared to their amide-containing counterparts, such as DDL-353 (Fig. 10A). Bromodomain-containing protein 2 and 4 (BRD2 / 4) inhibition assays confirmed a similar trend in potency against direct molecular drug targets (Fig.lOB-D). Of the NCEs, synthesized DDL-355 exhibited the greatest potency in-vitro with BRD2 / 4 IC50’s of 163 and 19 nM and a sCLU EC50 of 99 nM. Table 3. Physicochemical Properties of Candidate sCLU Enhancers.

[0210] To identify compounds suitable for treating CNS disorders in vivo, various in vitro assays were used to evaluate physicochemical properties influencing drug ADME, including solubility, liver microsome stability, plasma binding, brain tissue binding, and blood-brain barrier permeability (Table 3). Good in vitro activity alone does not guarantee favorable in vivo activity without adequate bioavailability and half-life28. Due to high development costs and candidate attrition rates, drug discovery strategies now emphasize simultaneous evaluation of drug physicochemical and ADME properties alongside efficacy29. This is especially important for CNS disorders, as the blood-brain barrier (BBB) selectively excludes nearly 100% of large-molecule neuro therapeutics and over 98% of small-molecule drugs30. Desired in vitro ADME property values and criteria for continued in vivo evaluation are: Kinetic Solubility > 50 pM; Liver Microsomal Stability tl / 2 > 1 hour; Plasma Binding < 90%; Brain Tissue Binding < 80%; BBB Permeability Pm > 1.0 = CNS+; sCLU EC50 < 313 nM. Of the ten compounds assessed, only DDL-357 met these stringent criteria and was therefore selected as the lead candidate for further in vivo efficacy testing. Methods

[0211] Molecular Docking and Scoring of Protein-Ligand Binding Energy

[0212] Molecular docking and scoring were performed in Flare (Cresset, v.7) using the Lead Finder (O. V. 424 Stroganov et al., Lead Finder: an approach to improve accuracy of proteinligand docking, binding energy estimation, and virtual screening, J. Chem. Inf. Model. 2008, 48, 2371-2385) docking algorithm. Lead Finder combines a generic algorithm search with local optimization procedures. Three different scoring functions are employed and optimized for the accurate prediction of 3D docked ligand poses, protein-ligand binding energy and rank-ordering of active and inactive compounds in virtual screening experiments. Pocket Detection in Flare is based on the fpocket (V. Le Guilloux et al., Fpocket: An open source platform for ligand pocket detection, BMC Bioinformatics 2009, 10, 168) and MDpocket (P. Schmidtke et al., MDpocket: open-source cavity detection and characterization on molecular dynamics trajectories, Bioinformatics 2011, 27, 3276- 85) methods, and can be used to identify and characterize pockets and cavities within a protein structure, enabling the identification of possible drug binding active sites, water binding pockets, channels and small cavities, large solvent-exposed sites.

[0213] Compound Synthesis via Amide Coupling

[0214] (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][l,2,4]triazolo[4,3- a][l,4]diazepin-6- yl)acetic acid (1 equiv), amine-containing reagent (2 equiv), l-Ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC; 2 equiv.), Hydroxybenzotriazole (HOBt; 2 equiv.), and N-N, Diisopropylethylamine (DIPEA; 2.5 equiv.) were added to a round bottom flask. The 14 reagents were solubilized using methylene chloride and the reaction mixture was stirred at room temperature for 16 hours. The crude product was concentrated in vacuo and then purified via flash column chromatography. A mobile phase of hexanes:ethyl acetate (time / % ethyl acetate: 0 / 0, 2 / 0, 7 / 50, 15 / 100, 20 / 0) was first utilized to elute any existing impurities, and the product was eluted in the DCM:MeOH mobile phase (time / %MeOH: 0 / 0, 4 / 0, 35 / 15, 42 / 20, 45 / 50, 50 / 0). Identity and purity of each compound was confirmed via LC- MS, LC-UV / Vis, and1H NMR.

[0215] Compound Synthesis via Ester Coupling

[0216] (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][l,2,4]triazolo[4,3- a][l,4]diazepin-6- yl)acetic acid (1 equiv), alcohol-containing reagent (3 equiv), and 4- Dimethylaminopyridine (DMAP; 2 equiv) were added to a round bottom flask. The reagents were solubilized using methylene chloride (800 pL) and was brought to 0 °C in an ice bath. A solution of diisopropylcarbodiimide (DIC; 2 equiv) in methylene chloride (200 p L) was added dropwise over the course of 30 minutes and then slowly brought to room temperature. After stirring for 24 hours, the reaction mixture was filtered through a filter funnel and concentrated in vacuo. The crude product was purified via flash column chromatography. A mobile phase of hexanes: ethyl acetate (time / % ethyl acetate: 0 / 0, 2 / 0, 7 / 50, 15 / 100, 20 / 0) was first utilized to elute any existing impurities, and the product was eluted in the DCM:MeOH mobile phase (time / %MeOH: 0 / 0, 4 / 0, 460 35 / 15, 42 / 20, 45 / 50, 50 / 0). Identity and purity of each compound was confirmed via LC-MS, LC-UV / Vis, andXH NMR .

[0217] Purity Analysis via Proton Nuclear Magnetic Resonance Spectroscopy (XH NMR)

[0218] Analysis of purity by magnetic resonance spectroscopy was done at the UCLA Molecular Instrumentation Center. -4 mg of compound were diluted in 750 pL of MeOH-D4 and analyzed using a AV400 nmr spectrometer (Bruker) containing a 5 mm broadband Z- gradient probe with Automatic Tune and Match (ATM). The analysis method consisted of a 64-scan proton nmr (*H-NMR) utilizing default parameters. Predicted *H-NMR spectra were obtained using the ChemNMR *H estimation function in ChemDraw (PerkinElmer). The experimental data was visualized and interpreted using Mnova (Mestrelab Research).

[0219] Liquid Chromatography-Tandem Mass Spectrometry

[0220] Analysis of compound levels was done at the UCLA Pasarow Mass Spectrometry Lab. A targeted LC-MS / MS assay was developed for each compound using the multiple reaction monitoring (MRM) acquisition method on a 6460 triple quadrupole mass spectrometer (Agilent Technologies) coupled to a 1290 Infinity HPLC system (Agilent Technologies) with a Phenomenex analytical column (Kinetex 1.7 pm C18 100 A 100 x 2.1 mm). The HPLC method utilized a mixture of solvent A (99.9 / 1 Water / Formic Acid) and solvent B (99.9 / 1 Acetonitrile / Formic Acid) and a gradient was use for the elution of the 17 compounds (min / %B: 0 / 1, 3 / 1, 19 / 99, 20 / 1, 30 / 1). Two fragment ions originating from each compound were monitored at specific LC retention times to ensure specificity and accurate quantification in the complex biological samples. The normalized chromatographic peak areas were determined by taking the ratio of measured chromatographic peak areas corresponding to each compound over that of the internal standard (Analyte / IS). Kinetic Solubility

[0221] Test compound (10 mM, 100% DMSO) was diluted separately into aqueous buffer (100 pM; PBS pH 7.4) and DMSO at various concentrations (1000, 100, 10, 1, 0.1 pM). The solutions were then incubated at 37 o C for 90 min and centrifuged (16000xg, 5 min). An aliquot of each supernatant was analyzed by UV / Vis (if possible) or LC-MS / MS. A standard curve was made by plotting the known amount of analyte per standard in DMSO vs. absorbance or chromatographic peak area. Kinetic solubility (mM) was calculated using the trendline equation with maximum absorbance or chromatographic peak area observed in the aqueous sample.

[0222] Blood-Brain Barrier (BBB) Permeability Assay

[0223] A liquid chromatography-ultraviolet / visible spectroscopy (LC-UV / Vis) assay was performed on a 1290 Infinity HPLC system (Agilent Technologies) with an HPLC column containing immobilized phosphatidylcholine (IAM.PC.DD, Regis Technologies, Cat#774011, 5 pm 300 A 100 x 4.6 mm). The HPLC method was a mixture of 6.7 mM phosphate buffer saline (pH 7.4; solvent A) and acetonitrile (solvent B), and a gradient was used for the elution of the compounds (min / %B: 0 / 20, 20 / 60, 21 / 20, 30 / 20). The retention time of the compound (tr) and void volume time of the column (tO) were recorded. Bloodbrain barrier (BBB) permeability (Pm) was calculated using the following equations as described by Yoon et al (DOI: 10.1177 / 1087057105281656): KIAM = (tr - tO) / tO ; Pm = (KIAM / MW4 ) x 1010 Compounds with a Pm >0.85 were determined to be BBB permeable (CNS+) at pH 7.

[0224] Plasma and Brain Tissue Binding Assays

[0225] Brain tissue was homogenized in PBS (pH 7.4) (1: 3 weight(mg) / volume(pL)) and the protein concentration was determined using the Micro BCA™ Protein Assay Kit (Thermo Fisher Scientific, Cat#23235). Brain homogenate was clarified and diluted to 20 mg / mL in PBS (pH 7.4). Either brain homogenate or plasma was and added to Slide- A-Lyzer™ MINI Dialysis Devices, 10K MWCO dialysis cups (Thermo Fisher Scientific, Cat#PI88401) in a 48-well plate containing PBS (500 pL; pH 7.4). 1 pL of 1 mM compound was added to the brain homogenate (Final Concentration: 2 pM compound, 0.5% DMSO) and incubated on a rocker for 4.5 hours at 37 °C. 50 pL of brain homogenate or plasma (within the dialysis cup) and PBS (within the 48-well plate) were transferred to new microcentrifuge tubes containing 400 pL of quenching reagent (100% Acetonitrile) containing internal standard. Solutions were clarified by centrifugation (16,000 x g, 5 min), and the supernatants were transferred to new tubes and lyophilized. Samples were reconstituted in 100 pL of 50 / 50 / 0.1

[0226] ( Water / Acetonitrile / Formic Acid) prior to analysis via liquid chromatography-tandem mass spectrometry (LC-MS / MS). The % of the unbound drug (fu, bound) was calculated using the following equation: % Bound = [1- (PBS chromatographic peak area / brain homogenate or plasma chromatographic peak area)] x 100

[0227] Liver Microsome Stability Assay

[0228] An aliquot (1 pL) of test compound (1 mM, 100% DMSO) was added to an aqueous liver microsome solution (1000 pL, PBS pH 7.4, 0.5mg / mL human liver microsomes (Thermo Fisher Scientific, Cat#HMMPL), 2 mM NADPH, 2 mM MgC12) and incubated at 37 °C for 120 min. Aliquots (50 pL) of the microsome solution were taken at various time points (0, 5, 10, 15, 30, 60, 90, 120 min) and added to a reaction quenching solution (200 pL 100% Acetonitrile) containing an internal standard. Solutions were clarified by centrifugation (16,000 x g, 5 min), and the supernatants were transferred to new tubes and lyophilized. Samples were reconstituted in 100 pL of 50 / 50 / 0.1 (Water / Acetonitrile / Formic Acid) prior to analysis via liquid chromatography-tandem mass spectrometry (LC-MS / MS). Chromatographic peak areas normalized to the internal standard were plotted at each time point and the half-life (t 1 / 2) of compound in liver microsomes was determined by using the trendline equation to calculate the time at which compound abundance was 50% of that at time point 0 (tO).

[0229] Bromodomain-Containing Protein Inhibition Assays

[0230] An AlphaScreen binding assay format was utilized by Reaction Biology Corporation, Malvern PA, to measure compound binding to the Human BRD4 Full length or BRD2-Tndm- His at various concentrations (0.25, 0.76, 2.3, 6.9, 20.6, 61.7, 185.2, 555.6, 16666.7, 5000 nM) using the Histone H4 peptide (1-21) K5 / 8 / 12 / 16Ac-Biotin substrate.

[0231] Example 3 - DDL-357 increases sCLU levels and improves memory in AD mouse models.

[0232] Proof-of-concept pharmacokinetic analyses were conducted to determine if DDL-357 could reach therapeutically relevant brain concentrations. As predicted by in-vitro ADME assessments, DDL-357 showed excellent brain bioavailability, reaching a maximum concentration of 1850 nM one hour after oral administration at 30 mg / kg (Fig. 14A). Though, looking solely at the total brain drug concentration can be misleading, as only unbound drug exerts pharmacological effects according to the free drug hypothesis31. Therefore, integrating in vitro brain tissue binding studies with in vivo pharmacokinetics is essential. When DDL- 357’s brain tissue binding (75.3%) is considered, the unbound brain concentration is still 457 nM, nearly twice its sCLU EC50 or minimum target concentration (EC50 = 223 nM) (Fig. 14A). Further analyses confirmed that unbound brain concentrations of DDL-357 one hour post administration at doses of either 3 or 10 mg / kg were below the sCLU EC50 (Fig. 15A). Based on these results, bidaily treatments of 15 mg / kg were selected for preclinical efficacy testing in two AD mouse models (ApoE4-TR:5xFAD, 9 3xTG-AD). Given the key role of drug toxicity in attrition during clinical development and a concern at micromolar concentrations, DDL-357 cytotoxicity was evaluated in CNS-derived gliotoxicity (U-87 MG) and neurotoxicity (SH-SY5Y), as well as two cell types commonly used to assess nephrotoxicity (HEK 293T) and hepatotoxicity (HEPG2)32,33(Fig. 14B). Encouragingly, DDL-357 was shown to be well-tolerated in these cells at concentrations as high as 50 pM (Ctox > 50uM). DDL-357 was also evaluated in a human ether- a- go-go-related gene (hERG) potassium channel inhibition assay and showed minimal effect with an EC50 > 100 M (Fig. 15B). Overall, these analyses support DDL-357 as a safe and effective sCLU-enhancing candidate for in-vivo development and highlights the importance of pharmacokinetics and toxicology studies for establishing a therapeutic index for appropriate dosing at which medications will be effective without adverse events.

[0233] Subchronic two-week oral administration of DDL-357 in the ApoE4-TR:5xFAD (E4 / FAD) resulted in a significant increase in sCLU levels in the hippocampus (Fig. 14C). Next, we conducted chronic oral administration of DDL-357 over six weeks and observed that it significantly improved learning and memory in 3xTG-AD mice compared to the vehicle-treated group, as assessed via the Barnes Maze (Fig. 14D). An analysis of the hippocampus tissue revealed DDL-357 to decrease in pathological markers of AD, including Amyloid P 1-42 (Ap42), the major component of amyloid plaques, and tau phosphorylation at residues Ser202 / Thr205 and Ser396, the primary components of intraneuronal neurofibrillary tangles (Fig. 14E-14G). However, only the reduction in pTau at Ser396 was determined to be statistically significant, likely due to high analyte variability (Fig. 14H). Importantly, levels of pTau / Tau were noticeably different between genders, with male mice exhibiting more tau phosphorylation than females (Fig. 15C, 15D). This underscores a significant issue with reproducibility that hampers the effective use of AD mouse models in preclinical research34,35. We strongly believe that longer, higher-powered studies with gender- specific analyses would yield more significant results. Interestingly, an inverse correlation between sCLU levels and the p-tau / tau ratio was observed within each mouse, suggesting that increased sCLU may be activating signaling pathway such as Reelin that could lead to inhibition of tau phosphorylation (Fig. 14H). This finding provides compelling evidence for further investigation of DDL-357 and highlights the potential impact of future studies where markers of AD pathology can be non-invasively sampled, providing longitudinal measurements within each mouse. Recent pharmacokinetics experiments also indicate that an additional sCLU enhancing candidate, DDL-356, may be even more effective than DDL-357 in-vivo, as it reaches higher concentrations of unbound drug in the brain and has lower sCLU EC50 value (Fig. 15E, 15F).

[0234] Methods

[0235] In-Vivo Pharmacokinetics

[0236] Following oral administration of compound, mice brain tissue and plasma were collected after euthanasia and perfusion at 1, 2, 4, 6 and 8 hours. Brain tissue were homogenized in a bead beater using 5 volumes of ice-cold 80% acetonitrile (1 / 5; mg of brain / pL of 80% ACN). Plasma analytes were extracted using 4 volumes of ice-cold acetonitrile (1 / 4; pL of plasma / pL of ACN). Solutions were clarified by centrifugation (16,000 x g, 5 min) and the supernatants were transferred to new tubes and lyophilized. Samples were reconstituted in 100 pL of 50 / 50 / 0.1 (Water / Acetonitrile / Formic Acid) prior to analysis via liquid chromatography-tandem mass spectrometry (LC-MS / MS). An internal standard (IS) was added to every sample to account for compound loss during sample processing. Standards were made in drug naive plasma and brain lysates with increasing amounts of analyte (S1,S2: 0 pmol / S3,S4: 1 pmol / S5,S6: 10 pmol / S7,S8: 100 pmol, S9,S10: 1000 pmol). The standard curve was made by plotting the known amount of analyte per standard vs. the ratio of measured chromatographic peak areas corresponding to the analyte over that of the IS (analyte / IS). The trendline equation was then used to calculate the absolute concentrations of each compound in plasma and brain tissue.

[0237] Cell Viability and Toxicity Analysis

[0238] Cell viability and toxicity were simultaneously assessed using the MultiTox-Fluor Multiplex 420 Cytotoxicity Assay (Cat# PRG9200, Fisher Scientific) according to the manufacturer’s protocol. In-Vivo Testing of DDL-357

[0239] Male and female 3-month-old mice were administered DDL-357 via pipette feeding at a dose of 15 mg / kg BID (30 mg / kg / day). Mice were weighed before the first dose for the calculation of volume to be administered. The formulation used comprised DDL-357 as a 15 mg / mL solution dissolved in a strawberry flavored solution (propylene glycol / water / strawberry syrup, 2 / 1 / 1, v / v / v). E4FAD mice were dosed for two weeks. 3xTg- AD mice were dosed for six weeks before entering Barnes Maze testing.

[0240] Barnes Maze Assessment of Spatial Learning and Memory

[0241] Barnes Maze testing was performed as describe by Attar et al. Briefly, in a room with visual cues on the walls, mice are introduced in a circular maze containing 20 holes where 19 holes are closed during training and all 20 holes are closed during the probe trials. On day 1 of the habituation stage, mice are placed at the center of the maze inside the start chamber. After 10 s the start chamber is removed, and mice are allowed to explore the maze for 3 min. After 3 min of exploration, the mice are guided to the target (escape) hole and allowed to stay there for 2 min. After habituation, mice undergo 3 consecutives training days, and each training day is comprised of 3 trials. Each trial starts with the mice being placed at the center of the maze inside the start chamber for 10 s. After this the start chamber is removed and mice are allowed to explore the maze for 3 min. If during this time they do not find the target hole, they are guided to it and allowed to remain inside for 1 min. At the end of each training day mice were dosed at 10 mg / kg. Next are the 24- and 48-hours probe days. During the probe days, the target hole is closed, and spatial memory of the mouse is determined during a 90 s probe trial. Mice are placed at the center of the maze inside the start chamber, after 10 s the start chamber is removed, and mice are allowed to explore the maze for 90 s. At the end of the trial, the mice are returned to the home cage and dose at 10 mg / kg. The ANY-maze software (Stoelting) was used to analyze the behavior of the mice in the Barnes Maze. Over the course of the training and probe trials, the software-calculated parameters included, but were not limited to, total distance traveled, speed, number of holes visited, latency to visiting the target / escape hole, percent time spent in the four quadrants of the maze

[0242] Statistical Analysis

[0243] All statistical analyses for in vitro and in vivo studies was performed using GraphPad Prism® software; for one-way ANOVA, with level of significance set at p < 0.05. A two- tailed unpaired Student’s t-test was utilized where appropriate, with a p < 0.05 considered significant. Note than in efficacy studies, the NTg mice are not littermates of E4FAD mice (which have no NTg littermates), therefore, for statistical analysis of treatment effects, only the E4FAD groups are compared.

[0244] Example 4 - DDL-357 promotes mitochondrial function, synaptic plasticity and aggregate clearance.

[0245] To further characterize the pharmacological effects of DDL-357 in-vivo, an unbiased proteomics analysis was utilized to identify differentially expressed proteins in the hippocampus of the vehicle versus DDL-357 treated E4 / FAD and 3xTg-AD mice (Fig. 16A). A subsequent gene-set enrichment analysis determined many of the proteins significantly upregulated by DDL-357 treatment to be associated with biologically relevant processes, including pathways of neurodegeneration (Fig. 16B). This also revealed increased expression of various proteins important for mitochondrial function, synaptic plasticity, and protein aggregate clearance - processes disrupted during AD pathogenesis36'38(Fig. 16C, Table 4). Notably, this included various proteins (TRPV2, RAB8A, TMED10, FMNL2, RPSA, SNX6, PRKCE, HNRNPA3) known to be involved in the clearance of various pathological protein aggregates, including Ap, p-tau, alpha-synuclein 10 (asyn), and dipeptide repeat (DPR) proteins (Table 4). This was accompanied by increases in other autophagic, endolysosomal, and ubiquitin-proteasome system pathway components (TRAP1, AP3B1, SNX1, VAPB, VSP39, LAMTOR3, PSMD6, UBE2L3) (Table 4). Given that these critical regulatory mechanisms are known to become progressively impaired with aging, identifying interventions that regulate proteostasis network machinery may be instrumental in effectively controlling the accumulation and clearance of disease relevant aggregation-prone proteins39.

[0246] The proteomics analysis also identified upregulation of several proteins from mitochondrial respiratory chain complex I (NDUFS6, NDUFV3) as well as notable regulators of mitochondrial dynamics (MFF, OSCP1, VPS39), autophagy (VAPB, TRAP1, VPS39), antioxidant defense (TMED10, PRDX3, GCSH, TRAP1), and bioenergetics (PTMA, CKB, TRAP1, OSCP1) (Table 4). Many of these proteins are notably decreased in AD patient brains and this has been suggested to be causative of the disease (Table 4). Their upregulation suggests a potential role in mitigating mitochondrial dysfunction, increasingly recognized as a driver of cognitive decline in AD40. Not surprisingly, DDL-357 was subsequently shown to increase the oxygen consumption rate — an established measure of mitochondrial respiration — in both glial and neuronal cell lines in-vitro41(Fig. 16D, 16E). Additionally, treatment of SH-SY5Y cells with DDL-357 at a concentration of 200 nM significantly increased expression of master metabolic regulator sirtuin 1 (SIRT1) and mitochondrial- derived peptide humanin, both of which have numerous neuroprotective functions shown to lessen AD pathology and improve cognition42'44(Fig. 16F, 16G). Importantly, significant increases in these cytoprotective analytes and mitochondrial function were observed at low nanomolar concentrations achieved by the free drug in the brain during these in-vivo studies (Fig. 16D, 16E; Fig. 17A, 17B, 17C). This discovery is highly encouraging, as numerous mitochondria-targeting interventions are currently being investigated in clinical trials as valuable therapeutic strategies for preventing AD progression45.

[0247] Lastly, DDL-357 also increased numerous proteins critical for maintaining synaptic function, dendritic spine morphology, long-term potentiation, and associated learning and memory processes, many of which proteins are notably decreased in AD (NPTN, KCNJ6, ITM2B, DUSP3, PLCB3, LRRC4B, MBNL1, DLGAP2, NCS1, RPSA, ALCAM, BRK1, GUCY1B1, CKB, OSCP1) (Table 4). Encouraged by these findings, DDL-357 was evaluated for its ability to promote neurite outgrowth in human iPSC-derived neurons, which was visibly enhanced after 48 hours compared to DMSO-treated controls (Fig. 16H, 161). Again, this effect occurred at concentrations achieved by the free unbound drug in the brain, supporting the observed improvements in learning and memory in DDL-357-treated mice (Fig. 16J). Interestingly, a positive allosteric modulator of GUCY1B1, which is upregulated by DDL-357, boosts cGMP production, enhancing neuronal function, cerebral blood flow, reducing neuroinflammation, and improving cellular bioenergetics in preclinical models, and is currently in clinical trials46. Based on these results, it is evident that DDL-357 not only enhances sCLU expression but also induces a neuroprotective gene expression profile, bolstering mitochondrial function, synaptic plasticity, and disease relevant protein aggregate clearance.

[0248] This discovery of a novel class of BET inhibitors as sCLU enhancers presents an exciting opportunity for developing new therapies for AD. The results described provide evidence of numerous disease-relevant biological processes mediated by BET proteins. Moreover, it provides evidence that BET inhibition may represent a mechanistically related, yet superior therapeutic approach when compared to HD AC inhibition, which has already been evaluated in clinical trials. This is further supported by cognitive improvement using a BET inhibitor in the clinic26. Despite the limitations of mouse models for AD, preclinical testing of lead candidate, DDL-357, demonstrate that sCLU enhancers suppress p-tau, improve memory and key neurotrophic proteins associated with mitochondrial function, synaptic plasticity, and disease relevant protein aggregate clearance, as well as lack adverse effects after six weeks of oral treatment in mice (Fig. 18). Given the limited success of current treatments focused on altering a single protein target associated with AD pathology, this finding may represent a new class of next-generation therapeutics that increase the expression of neuroprotective protein networks. While further analysis of AD-related biomarkers, off-target effects in the brain, and IND-enabling studies are needed to fully understand the impact of sCLU enhancers on AD pathology and to develop this class of compounds, this drug discovery effort underscores their potential as valuable therapeutic avenue. Importantly, it has not escaped our notice that the results described here suggest these therapeutics may also represent a broader approach for treating other neurodegenerative proteinopathies, such as Parkinson's disease and amyotrophic lateral sclerosis.

[0249] Table 4. Alzheimer’s Disease-relevant proteins unregulated in E4 / FAD and 3xTG-AD mice following DDL-357 treatment. References in Table 4 begin at SI. Methods

[0250] Proteomics

[0251] Hippocampus were homogenized in lysis buffer (12 mM sodium lauroyl sarcosine, 0.5% sodium deoxycholate, 50 mM triethylammonium bicarbonate (TEAB), Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific)), the samples were reduced and alkylated with tris(2- carboxyethyl)phosphine (10 mM) and chloro acetamide (40 mM) for 30 minutes at 95 o C, and then digested with trypsin (20 ug) for 16 hours at 37 °C. The samples were then desalted using a modified version of Rappsilber's protocol [Ref. 1] and were isotopically labeled (TMT18plex Isobaric Label Reagent Set, Thermo Fisher Scientific) according to the manufacturer’s protocol to provide relative quantitation between samples. The samples were then fractionated separately via high pH reversed-phase chromatography (Pierce™ High pH Reversed-Phase Peptide Fractionation Kit) as per manufacturer’s protocol for increased proteome coverage. Aliquots of each fraction were injected onto a reverse phase nanobore HPLC column (AcuTech Scientific, Cl 8, 1.8um particle size, 360 um x 20 cm, 150 um ID), equilibrated in solvent A (water / acetonitrile / FA, 98 / 2 / 0.1, v / v / v) and eluted (300 nL / min) with an increasing concentration of solvent B (acetonitrile / water / FA, 98 / 2 / 0.1, v / v / v: min / % F; 0 / 0, 5 / 3, 18 / 7, 74 / 12, 144 / 24, 153 / 27, 162 / 40, 164 / 80, 174 / 80, 176 / 0, 180 / 0) using an EASY-nLC II (Thermo Fisher Scientific). The effluent from the column was directed to a nano spray ionization source connected to a hybrid quadrupoleOrbitrap mass spectrometer (Q Exactive Plus, Thermo Fisher Scientific) acquiring mass spectra in a data-dependent mode alternating between a full scan (m / z 350-1700, automated gain control (AGC) target 3 x 106, 50 ms maximum injection time, FWHM resolution 70,000 at m / z 200) and up to 15 MS / MS scans (quadrupole isolation of charge states 2-7, isolation window 0.7 m / z) with previously optimized fragmentation conditions (normalized collision energy of 32, dynamic exclusion of 30 s, AGC target 1 x 105, 100 ms maximum injection time, FWHM resolution 35,000 at m / z 200). Raw proteomic data were searched against a Uniprot database containing the complete human proteome using SEQUEST-HT (including dynamic modifications: oxidation (+15.995) on M, deamidation (+0.984) on N / Q, and carbamidomethyl (+57.021), phosphorylation (+79.966) on S / T / Y) in Proteome Discoverer (Version 2,4, Thermo Scientific), which provided measurements of relative abundance of the identified peptides. Decoy database searching was used to generate high confidence tryptic peptides (FDR < 1%). Tryptic peptides containing amino acid sequences unique to individual proteins were used to identify and provide relative quantification between different proteins in each sample. Post- translationally modified peptides from each protein were normalized to protein abundance and peptides exhibiting a p-value < 0.05 with a log2-fold change > 0.5 were analyzed using a series of bioinformatics tools including functional protein association network analysis, comprehensive gene set enrichment gene ontology (GO) classification and pathway analysis, as well as kinase substrate enrichment analyses [13, 14, 17].

[0252] Neurite Outgrowth Assay in iPSC-Derived Human Neurons.

[0253] The iPSC derived human neurons were provided by the Komblum lab to be cultured in normal 623 growth conditions (37°C, 5% CO2) for 2-3 days. Cells were cultured in CELLSTAR uClear 384 well plates, PS, F bottom at seeding density of 5000 cells per well coated overnight with 30ug / mL 18 poly-D-lysine and 2 ug / mL laminin. Media was replaced every 3 days with Lonza Primary Neuron Basal Medium (PNBM) supplemented with 2mM L-glutamine, GA- 1000, and 2% NSF-1. On day 3, cells were treated with DDL357 in doses ranging from 15,000nM to 0.76nM for 48 hours. All compounds were diluted in 0.5% DMSO and pinned to 384 well plates with 25pL of cell media per well with pin size of 250nL using BioMek FX (Beckman Coulter, CA). After 48 hours, media was replaced twice to ensure removal of any remaining compound residues before imaging for neurite outgrowth with Calcein-AM and Hoescht. Imaging was done at UCEA Molecular Screening Shared Resource core facility (MSSR; Robert Damoiseaux, Ph.D., Director) with ImageXPress Confocal (Molecular Devices, CA) using a lOx objective. Total neurite outgrowth was measured via a MetaXpress (Molecular Devices, CA) neurite outgrowth analysis algorithm. Neurite outgrowth was set to detect cell bodies with approximate width of > 20pm and outgrowth with maximal width of 5pm and length of > 100pm with intensity of 1000 grey scales over background. Mean neurite outgrowth per drug exposure was determined by normalizing to vehicle control (n = 60). Neurite outgrowth assay data were compared using a two-way ANOVA for drug effect and concentration. Internal standards were designed in each plate with controls of untreated neurons (n = 24) and vehicle controls of neurons treated with 0.5% DMSO (n = 60) since all compounds were dissolved in 0.5% DMSO - the concentration that showed no signs of cell toxicity from previous studies. REFERENCES

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[0374] INCORPORATION BY REFERENCE

[0375] All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0376] EQUIVALENTS

[0377] While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

CLAIMSWe claim:

1. A compound of Formula (I), or a pharmaceutically acceptable salt thereof:Formula (I) wherein X is selected from OR1and NR2R3;R1is selected from H, optionally substituted aryl, optionally substituted alkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted cycloalkyl, and alkynyl;R2and R3are independently selected from H, optionally substituted aryl, hydroxyl, optionally substituted alkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted cycloalkyl, and alkynyl; provided that R2and R3are not both hydroxyl; orR2and R3taken together with the nitrogen atom to which they are attached form an optionally substituted 4-7 membered ring; n is 0-5; andY is independently selected from F, Cl, Br, and I; wherein the compound is not2. The compound of claim 1, wherein n is 1.

3. The compound of claim 1 or 2, wherein the compound of Formula (I) has the structure4. The compound of any one of claims 1-3, wherein Y is Cl.

5. The compound of any one of claims 1-4, wherein X is OR1.

6. The compound of any one of claims 1-5, wherein R1is selected from alkyl, cycloalkyl, and heterocyclyl.

7. The compound of any one of claims 1-6, wherein R1is selected from C1-C4 alkyl, cyclohexyl, cyclobutyl,18. The compound of any one of claims 1-7, wherein R is selected fromand9. The compound of any one of claims 1-8, wherein R is ?10. The compound of any one of claims 1-4, wherein X is NR2R3.

11. The compound of claim 10, wherein R2is H.

12. The compound of claim 10 or 11, wherein R3is selected from -OH, cycloalkyl, optionally substituted alkyl, and heterocyclyl.

13. The compound of claim 23, wherein R3is selected from -OH, cyclopentyl 114. The compound of claim 10, wherein R2and R3taken together with the nitrogen atomo which they are attached form a 4-7 membered ring.

15. The compound of claim 14, wherein R2and R3taken together with the nitrogen atomo which they are attached form a 6-membered ring.

16. The compounds of any one of claims 1-15, wherein the structure is selected from:

17. The compound of any one of claims 1-16, wherein the structure is selected from:d of any one of claims 1-17, wherein the structure is19. A pharmaceutical composition comprising the compound of any one of claims 1-18 and a pharmaceutically acceptable excipient.

20. A method of treating or preventing a disease selected from Alzheimer’s disease, diabetes, and cardiovascular disease, comprising administering to a subject in need thereof a compound of Formula I, or a pharmaceutically acceptable salt thereof:Formula (I) wherein X is selected from OR1and NR2R3;R1is selected from H, optionally substituted aryl, optionally substituted alkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted cycloalkyl, and alkynyl;R2and R3are independently selected from H, optionally substituted aryl, hydroxyl, optionally substituted alkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted cycloalkyl, and alkynyl; provided that R2and R3are not both hydroxyl; orR2and R3taken together with the nitrogen atom to which they are attached form an optionally substituted 4-7 membered ring; n is 0-5; andY is selected from F, Cl, Br, and I.

21. The method of claims 20, wherein a therapeutically effective amount of the compound of Formula (I) is administered to a subject in need thereof.

22. The method of claim 20 or 21, wherein n is 1.23 The method of any one of claims 20-22, wherein the compound of Formula (I) has the structure24. The method of any one of claims 20-24, wherein Y is Cl.

25. The method of any one of claims 20-24, wherein X is OR1.

26. The method of any one of claims 20-25, wherein R1is selected from alkyl, cycloalkyl, and heterocyclyl.

27. The method of any one of claims 20-26, wherein R1is selected from C1-C4 alkyl, cyclohexyl, cyclobutyl,28. The method of any one of claims 20-27, wherein R1is selected from29. The method of any one of claims 20-28, wherein R1is30. The method of any one of claims 20-29, wherein X is NR2R3.

31. The method of claim 30, wherein R2is H.

32. The method of claim 30 or 31, wherein R3is selected from -OH, cycloalkyl, optionally substituted alkyl, and heterocyclyl.

33. The method of claim 32, wherein R is selected from -OH, cyclopentyl, = , and34. The method of claim 30, wherein R2and R3taken together with the nitrogen atom to which they are attached form a 4-7 membered ring.

35. The method of claim 34, wherein R2and R3taken together with the nitrogen atom to which they are attached form a 6-membered ring.

36. The method of any one of claims 21-35, wherein the structure is selected from:

37. The method of any one of claims 20-36, wherein the structure is selected from:f any one of claims 20-37, wherein the structure is39. The method of any one of claims 20-38, wherein the disease is Alzheimer’s disease.

40. The method of claim 39, wherein the Alzheimer’s disease is late-onset Alzheimer’s disease.

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