Methods of lowering LRRK2

Abstract

The present disclosure describes that the 15-lipoxygenase (15-LO) product 4-hydroxynonenal (4HNE or 4-HNE) post-translationally modifies LRRK2 and is believed to induce a pathologically elevated, chronic LRRK2 kinase activity associated with a LRRK2 mediated disease or condition, e.g., Parkinson's disease or frontotemporal dementia. Production of LRRK2-4HNE adducts, e.g., LRRK2 Cys2024-4HNE and / or LRRK2 Cys2025-4HNE adduct described herein, may be an indication of a LRRK2 mediated disease or condition. Inhibiting the production of excessive 4HNE in cells by 15-LO inhibitors converted an elevated LRRK2 activity to a normal basal LRRK2 activity level as demonstrated in cellular experiments in the Examples. The 15-LO inhibition of 4HNE production is believed to only remove elevated LRRK2 activity associated with LRRK2 mediated diseases or conditions and not normal LRRK2 activity that may be required for other cellular functions. Accordingly, 15-LO inhibition of elevated LRRK2 activity and / or expression may afford a safe and effective treatment for LRRK2 mediated diseases or conditions.

Description

Attorney Docket No.: 058367-511001WO METHODS OF LOWERING LRRK2 CROSS-REFERENCE TO RELATED APPLICATIONS

[0000] This application claims the benefit of U.S. provisional application 63 / 658,683 filed June 11, 2024, which is herein incorporated by reference in its entirety for all purposes. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0001] This invention was made with government support under grant R56 NS131137-01 awarded by the United States National Institutes of Health. The government has certain rights in the invention. BACKGROUND

[0002] Leucine-rich repeat kinase 2 (LRRK2) is a large (2527 amino acids) protein consisting of several domains with different functions. The central portion of LRRK2 contains a Ras of Complex (Roc) GTPase and a C-terminus of Roc (COR) domain, followed by serine-threonine kinase domains.

[0003] Missense mutations in LRRK2 are a common cause of familial Parkinson’s disease (PD). Both coding and non-coding variants in LRRK2 have been shown to increase the risk of developing PD. Most of these mutations are located in the Roc-Cor GTPase or kinase domain of LRRK2 and often result in elevated kinase activity, leading to a toxic gain-of-function. Evidence also suggests wild-type (WT) LRRK2 kinase activity is enhanced in idiopathic PD (iPD). Thus, LRRK2 might be a link between genetic and idiopathic forms of PD. However, the upstream signaling mechanisms that promote WT LRRK2 kinase activation are not well understood.

[0004] LRRK2 uses a classical kinase switch activation mechanism, where the dynamics of the kinase domain drive its activation. Upon activation, LRRK2 phosphorylates its substrates, including a subset of Rab GTPases (e.g., Rab10 and Rab12), which regulate endolysosomal function. Excessive LRRK2 kinase activity can also cause endolysosomal dysfunction, creating a complex feedback loop.

[0005] The kinase domain of LRRK2 is similar to the redox-sensitive MAP kinase, ASK1, which undergoes redox-induced functional changes. Redox stress, long implicated in PD pathogenesis, can induce WT LRRK2 kinase activity, potentially to the same extent as the kinase activating G2019S mutation. Interestingly, LRRK2 can be activated by various stimuli that induce oxidative damage, lysosomal damage, membrane rupture or remodeling, vesicular trafficking deficits, and signaling through Stimulator of Interferon Genes (STING).

[0006] A potential common factor among these stimuli is their association with membrane damage and lipid peroxidation, leading to the accumulation of 4-hydroxynonenal (4HNE), a reactive aldehyde that plays a role in activating other kinases. The formation of 4HNE occurs either nonenzymatically when lipids react with reactive oxygen species (ROS), or enzymatically through the hydroperoxidation of membrane phospholipids by lipoxygenase enzymes, primarily 15-lipoxygenase (15-LO).4HNE can form irreversible Michael-adducts with cysteine residues, and the kinase activation loop of LRRK2 contains highly conserved vicinal cysteine residues, Cys2024 and Cys2025, which may be important for redox sensing.

[0007] There remains a need to detect and to treat LRRK2-mediated diseases and conditions, such as Parkinson’s disease, in human subjects. The subject may be suffering from or suspected from suffering from a disease or condition. While prospective LRRK2 kinase inhibitors are currently being developed in the clinic, many of the inhibitors suffer from adverse side effects, such as lung toxicity, that is believed to result from excessive inhibition of LRRK2 activity. LRRK2 kinase inhibitors may also suffer from non-target toxicity associated with inhibition of other kinases. This disclosure addresses these needs and others. BRIEF OVERVIEW OF THE INVENTION

[0008] The present disclosure describes that the 15-lipoxygenase (15-LO) product 4- hydroxynonenal (4HNE or 4-HNE) post-translationally modifies LRRK2 and is believed to induce a pathologically elevated, chronic LRRK2 kinase activity associated with a LRRK2 mediated disease or condition, e.g., Parkinson’s disease or frontotemporal dementia. Production of LRRK2-4HNE adducts, e.g., LRRK2 Cys2024-4HNE and / or LRRK2 Cys2025-4HNE adduct described herein, may be an indication of a LRRK2 mediated disease or condition. Inhibiting the production of excessive 4HNE in cells by 15-LO inhibitors converted an elevated LRRK2 activity to a normal basal LRRK2 activity level as demonstrated in cellular experiments in the Examples. The 15-LO inhibition of 4HNEproduction is believed to only remove elevated LRRK2 activity associated with LRRK2 mediated diseases or conditions and not normal LRRK2 activity that may be required for other cellular functions. Accordingly, 15-LO inhibition of elevated LRRK2 activity and / or expression may afford a safe and effective treatment for LRRK2 mediated diseases or conditions.

[0009] In some embodiments, the present disclosure provides evidence that oxidative stimulation of endogenous LRRK2 kinase activity by H2O2, rotenone, chloroquine, or monensin depends on cysteine residues 2024 and 2025 and involves the formation of adducts between 4HNE and LRRK2. The experiments illustrated in the data provided by the experiments in Example 3 show that blockade of 4HNE production with a 15-LO inhibitor or by genetic ablation prevents LRRK2 kinase activation in certain cell types.

[0010] In one aspect, the present invention provides a method of lowering LRRK2 activity and / or expression in a cell, comprising contacting the cell with an effective amount of a 15- lipoxygenase (15-LO) inhibitor, wherein the LRRK2 activity and / or expression is higher than a control LRRK2 activity and / or expression in a control cell.

[0011] In some embodiments, a method of the present disclosure is a method of reducing 4- hydroxynonenal (4HNE) in a cell, comprising administering an effective amount of a 15- lipooxygenase (15-LO) inhibitor.

[0012] In some embodiments, a method of the present disclosure is a method of treating a disease or condition characterized by increased LRRK2 activity and / or expression in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a 15-lipooxygenase (15-LO) inhibitor. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG.1 shows that H2O2 elicits rapid LRRK2 autophosphorylation. (A) Endogenous LRRK2 kinase activity was assessed by proximity ligation (PL) assay between pS1292 and total LRRK2 (PL pS1292–LRRK2) in LRRK2WT / WTHEK293 cells treated with 5μM H2O2 for 5, 10, 15, or 30 minutes. There was a robust increase in PL pS1292-LRRK2 signal (red) within 10 minutes of treatment that was sustained. (B) Quantification of LRRK2 activity time course in response to H2O2. Each symbol represents the average PL pS1292-LRRK2 fluorescence intensity from 4 independent experiments obtained from 100-150 cells per treatment group for each independent experiment. (C) H2O2(5μM) induced LRRK2 kinaseactivity was assessed by PL pS1292-LRRK2 in LRRK2WT / WTand LRRK2- / -HEK293 cells. H2O2 elicited a strong increase in PL pS1292-LRRK2 signal compared to vehicle in LRRK2WT / WT. Pretreatment with NAC prevented the H2O2induced increase in PL pS1292- LRRK2 signal, and no signal was detected in LRRK2- / -cells. (D) Quantification of PL pS1292–LRRK2 fluorescence intensity. Each symbol represents the average PL pS1292– LRRK2 fluorescence intensity obtained from 100-150 cells per treatment group for each independent experiment; N=3 independent experiments. Statistical testing by two-way ANOVA with Tukey correction; ****denotes p<0.0001; ns denotes not significant.

[0014] FIG.2 shows that stimulated LRRK2 activity is ROS dependent. (A) Rotenone (50nM) induced LRRK2 kinase activity was assessed by PL pS1292-LRRK2 in LRRK2WT / WTand LRRK2- / -HEK293 cells and RAW264.7 macrophages. Rotenone treatment led to an increase in PL pS1292-LRRK2 (red dots) in both LRRK2WT / WTHEK293 and RAW264.7 macrophages compared to vehicle. NAC cotreatment prevented rotenone induced PL pS1292-LRRK2 signal and no signal was detected in LRRK2- / -cells. (B) Quantification of PL pS1292–LRRK2 Objects / DAPI in HEK293 cells. (C) Quantification of PL pS1292– LRRK2 Objects / DAPI in RAW264.7 macrophages. (D) Chloroquine (100μM) and (F) Monensin (10μM) elicited an increase in PL pS1292-LRRK2 signal in LRRK2WT / WTHEK293 cells. NAC cotreatment prevented both (D) chloroquine and (F) monensin induced PL pS1292-LRRK2 signal. No signal was observed in LRRK2- / -cells. (E) Quantification of PL pS1292–LRRK2 Objects / DAPI in presence of chloroquine. (G) Quantification of PL pS1292–LRRK2 Objects / DAPI in presence of monensin. For each graph (B, C, E, G), each symbol represents the average number of PL pS1292-LRRK2 objects / DAPI obtained from 100-150 cells per treatment group for each independent experiment; N=3 independent experiments. Statistical testing by two-way ANOVA with post-hoc Tukey correction; ****denotes p<0.0001***denotes p<0.001; **denotes p<0.005; ns denotes not significant.

[0015] FIG.3 shows Cys2024 and Cys2025 are important for stimulated LRRK2 kinase activity. Endogenous LRRK2 kinase activity was assessed by PL pS1292-LRRK2 in LRRK2WT / WT, LRRK2C2024A, LRRK2C2025A, and LRRK2C2024A+C2025A(denoted as C2024A,C2025A, C2024A / C2025A respectively) HEK293 cells. (A) H2O2 (5μM) (C) rotenone(50nM) (E) chloroquine (100μM) and (G) monensin (10μM) elicited an increase in PL pS1292-LRRK2 signal in LRRK2WT / WT, which was prevented in LRRK2C2024A, LRRK2C2025A, and LRRK2C2024A+C2025AHEK293 cells. Note that the PL signal is green for H2O2 and rotenone and red for chloroquine and monensin. No signal was observed inLRRK2- / -cells (A, C, E, G). Quantification of LRRK2 activity by PL pS1292–LRRK2fluorescence signal in presence of (B) H2O2, (D) rotenone, (F) chloroquine, and (H)monensin. For all graphs (B, D, F, H), each symbol represents the average PL pS1292– LRRK2 fluorescence intensity obtained from 100-150 cells per treatment group for each independent experiment; N=3 independent experiments. Statistical testing by two-way ANOVA with Tukey correction; ****denotes p<0.0001; ns denotes not significant.

[0016] FIG.4 shows the lipid peroxidation product, 4HNE, interacts with LRRK2 and induces kinase activation. (A) PL between LRRK2 and 4HNE (PL LRRK2–4HNE) was used as an index of LRRK2-4HNE adduct formation in LRRK2WT / WT, C2024A / C2025A, and LRRK2- / -HEK293 cells. Exogenous 4HNE treatment (10μM, 30μM, 100μM) led to a dose- dependent increase in PL LRRK2–4HNE signal (red dots) in LRRK2WT / WTcells.4HNE treatment did not increase PL LRRK2–4HNE signal in C2024A / C2025A and LRRK2- / -HEK293 cells. (B) Quantification of PL LRRK2-4HNE. (C) 4HNE-induced LRRK2 kinase activity was assessed by PL pS1292-LRRK2. Baseline kinase activity in LRRK2WT / WTand C2024A / C2025A cells was inhibited by the LRRK2 kinase inhibitor PF360 (1μM), and 4HNE treatment dose-dependently increased PL pS1292-LRRK2 signal (red dots) in LRRK2WT / WTcells, but not in C2024A / C2025A cells. (D) Quantification of LRRK2 activity by PL pS1292–LRRK2 Objects / DAPI in HEK293 cells. For graphs (B and D), each symbol represents the average signal from 100-150 cells per treatment group for each independent experiment; N=3 independent experiments. Statistical testing by two-way ANOVA with post- hoc Tukey correction. (E) LRRK2–4HNE interaction was assessed via PL LRRK2–4HNE (red dots) in TH+dopaminergic neurons (blue) in in the substantia nigra of rats treated with vehicle or rotenone. Rotenone treatment caused an increase in PL LRRK2–4HNE in TH+dopaminergic neurons compared to vehicle treated rats. (F) Quantification of the PL LRRK2–4HNE signal in dopamine neurons. Each symbol represents the average number of PL LRRK2–4HNE objects / TH+neuron from an individual animal with two sections of brain tissue stained per animal; N=4 animals per treatment group. Statistical testing by two-tailed unpaired t-test with Welch’s correction. **denotes p<0.01; ***denotes p<0.0005; ****denotes p<0.0001; ns denotes not significant.

[0017] FIG.5: 15-LO-derived 4HNE is a key mediator of LRRK2 hyperactivation. (A) Rotenone-induced endogenous LRRK2–4HNE adduct formation was assessed by PL LRRK2–4HNE in 15-LOWT / WTand 15-LO- / -HAP1 cells. Rotenone treatment promoted an increase in PL LRRK2–4HNE signal that was prevented by α-tocopherol in 15-LOWT / WT, andnot observed in 15-LO- / -cells. (B) Quantification of PL LRRK2–4HNE. (C) Rotenone- induced LRRK2 kinase activity was assessed by PL pS1292 – LRRK2. Rotenone increased PL pS1292 – LRRK2, an effect that was that was mitigated by cotreatment with the 15-LO inhibitor, CU12991, and completely prevented by PF360.15-LO- / -cells had a normal basal PL pS1292 – LRRK2 signal, which was not further increased by rotenone or affected by CU12991. (D) Quantification of LRRK2 activity by PL pS1292–LRRK2. (E) 4HNE (100^M) treatment led to an increase in PL pS1292–LRRK2 signal in both 15-LOWT / WTand 15-LO- / -cells. This was prevented by pretreatment with 250^M NAC. (F) Quantification of LRRK2 activity by PL pS1292–LRRK2. For graphs (B, D, and F), each symbol represents the average signal from 100-150 cells per treatment group for each independent experiment; N=3 independent experiments. Statistical testing by two-way ANOVA with post-hoc Tukey correction. (G) Cellular 4HNE signal in LCLs derived from healthy control subjects, G2019S mutation carriers, and iPD patients. The increased 4HNE signal in G2019S and iPD LCLs was reduced to healthy control levels CU12991. (H) Quantification of 4HNE. Each symbol represents an individual donor line.4HNE signal was taken from 90-120 cells / line; 4 donor lines per group. Statistical testing by two-way ANOVA with post-hoc Tukey correction. **denotes p<0.005; *** denotes p<0.0005; ****denotes p<0.0001; ns denotes not significant. For panel D, red symbols indicate additional statistical testing by one-wayANOVA, examining each genotype (15-LOWT / WT and 15-LO- / -) separately. ns# denotes notsignificant compared to vehicle-treated cells; # denotes p< 0.05 compared to vehicle-treated or CU12991-treated cells.

[0018] FIG. 6: 15-LO activity regulates stimulated LRRK2 kinase activity via 4HNE. (A)Chloroquine, monensin, and rotenone-induced kinase activity was assessed by PL pS1292- LRRK2 in LRRK2WT / WTRAW264.7 macrophages. Chloroquine, monensin and rotenone caused increased PL pS1292-LRRK2 signal (red dots) which was prevented by cotreatment with the 15-LO inhibitor, CU12991. (B) Quantification of LRRK2 activity by PL pS1292– LRRK2. (C) LRRK2WT / WTRAW264.7 macrophages were treated with either 12(S)-HpETE (top panel, black outline) or 15(S)-HpETE (bottom panel, red outline) and kinase activity was measured by PL pS1292-LRRK2.12(S)-HpETE did not increase PL pS1292-LRRK2 signal above baseline (red dots).2.5μM and 25μM 15(S)-HpETE led to an increase in PL pS1292- LRRK2 signal relative to vehicle. Cotreatment with NAC prevented the increase in PL pS1292-LRRK2 signal elicited by 25μM 15(S)-HpETE. (D) Quantification of LRRK2 activity by PL pS1292–LRRK2. (E) LRRK2-4HNE adduct formation was assessed by PLLRRK2-4HNE in LRRK2WT / WTRAW264.7 macrophages. Chloroquine, monensin, and rotenone led to an increase PL LRRK2-4HNE signal (red dots). The increase in PL LRRK2- 4HNE signal was prevented by cotreatment with CU12991. (F) Quantification of LRRK2 activity by PL pS1292–LRRK2. For each graph (B, D, and F), each symbol represents the average number of PL pS1292-LRRK2 Objects / DAPI obtained from 100-150 cells per treatment group for each independent experiment; N=3-4 independent experiments. Statistical testing by two-way ANOVA with post-hoc Tukey correction. (G) LRRK2 activity measured by PL pS1292 – LRRK2 in LCLs derived from healthy control subjects, G2019S mutation carriers, and iPD patients. The increased PL pS1292 – LRRK2 signal in G2019S and iPD LCLs was reduced to healthy control basal levels by CU12991 and completely blocked by PF360. (H) Quantification of LRRK2 activity by PL pS1292–LRRK2 Objects / DAPI. by PL pS1292–LRRK2 Objects / DAPI was taken from 90-120 cells / line; 4 donor lines per group. Statistical testing by two-way ANOVA with post-hoc Tukey correction. *denotes p<0.05; **denotes p<0.005; ***denotes p<0.0005; ****denotes p<0.0001; ns denotes not significant.

[0019] FIG.7: PEBP1 and 15-LO interaction is increased in the rotenone model andpatient derived samples. (A) Immunofluorescent staining for 15-LO (green), PEBP1 (red),and their colocalization (yellow) in LRRK2WT / WTRAW264.7 macrophages. Rotenone led to an increase in overlap between 15-LO and PEPB1 relative to vehicle. Inset is 200X zoom. (B) Quantification of the % overlap of 15-LO and PEBP1 objects / Total Number of 15-LO Objects. Each symbol represents the average % overlap of 15-LO and PEBP1 objects / Total Number of 15-LO Objects obtained from 100-150 cells per treatment group for each independent experiment; N=3 independent experiments. Statistical testing by two tailed unpaired t-test with Welch’s correction. *** denotes p<0.001 (C) PL between PEBP1 and 15- LO was used as an index for PEPB1–15-LO interaction in LRRK2WT / WTRAW264.7 macrophages. Rotenone treatment led to an increase in PL PEBP1–5-LO signal (red dots) compared to vehicle. (D) Quantification of PL PEBP1–15-LO . Each symbol represents the average number of PL PEPB1–5-LO Objects / DAPI obtained from 100-150 cells per treatment group for each independent experiment; N=3 independent experiments. Statistical testing by two tailed unpaired t-test with Welch’s correction. **denotes p<0.01 (E) PEBP1 – 15-LO interaction was assessed via PL PEBP1–15-LO (red dots) in TH+dopaminergic neurons (blue) in in the substantia nigra of rats treated with vehicle or rotenone. Rotenone treatment caused an increase in PL PEBP1–15-LO in TH+dopaminergic neurons compared to vehicle-treated rats. (F) Quantification of the PL PEBP1–15-LO signal in dopamine neurons.Each symbol represents the average number of PL PEBP1–15-LO HNE objects / TH+neuron from an individual animal with two sections of brain tissue stained per animal; N=4 animals per treatment group. Statistical testing by two-tailed unpaired t-test with Welch’s correction.***denotes p<0.0005. (G) PL PEBP1–15-LO was assessed in LCLs derived from healthy control subjects, G2019S mutation carriers, and iPD patients. There was an increase in PL PEBP1–15-LO signal in G2019S and iPD derived LCLs relative to healthy control. (H) Quantification of PL PEBP1–15-LO Objects / DAPI. Each symbol represents an individual donor line.4HNE signal was taken from 90-120 cells / line; 4 donor lines per group. Statistical testing by one-way ANOVA with post-hoc Tukey correction *denotes p<0.05; **denotes p<0.005.

[0020] FIG. 8: N-acetylcysteine prevents rotenone induced Rab10 phosphorylation. (A)Representative western blots for total Rab10 (top) and pThr73-Rab10 (bottom) in LRRK2WT / WTHEK293 cells (left) and RAW264.7 macrophages (right). Rotenone elicited an increase in pT73-Rab10 / Total Rab10 compared to vehicle in both cell types. Cotreatment with NAC prevented the rotenone induced increase pT73-Rab10 / Total Rab10. Total Rab10 levels were not changed. (B) Quantification of pT73-Rab10 / Total Rab10 normalized to LRRK2WT / WTvehicle HEK293 cells. Each symbol represents the average of pRab10 / Total Rab10 obtained from two technical replicates per treatment group for each independent experiment: N=5 independent experiments. Statistical testing by two-way ANOVA with Tukey correction. *denotes p<0.05; ns denotes not significant. (C) Quantification of pRab10 / Total Rab10 normalized to LRRK2WT / WTvehicle RAW264.7 macrophages. Each symbol represents the average of pT73-Rab10 / Total Rab10 obtained from two technical replicates per treatment group for each experiment; N=4 independent experiments. Statistical testing by two-way ANOVA with Tukey correction. (D) Proximity ligation (PL) assay between pT73-Rab10 and total Rab10 (PL pT73(Rab10)–Rab10) was used to assess levels of Rab10 phosphorylation. In LRRK2WT / WTHEK293 and RAW264.7 macrophages, rotenone elicited an increase in PL pT73(Rab10)–Rab10 signal relative to the vehicle. This was not observed in cells cotreated with NAC or in LRRK2- / -cells. (E) Quantification of Rab10 phosphorylation by PL pT73(Rab10)–Rab10 Objects / DAPI in HEK293 cells. Each symbol represents the average number of PL pT73(Rab10)–Rab10 Objects / DAPI obtained from 100- 150 cells per treatment group for each independent experiment; N=3 independent experiments. Statistical testing by two-way ANOVA with post-hoc Tukey correction. (F) Quantification of Rab10 phosphorylation by PL pT73(Rab10) – Rab10 Objects / DAPI inRAW264.7 macrophages. Each symbol represents the average number of PL pT73(Rab10)– Rab10 Objects / DAPI obtained from 100-150 cells per treatment group for each independent experiment; N=3 independent experiments. Statistical testing by two-way ANOVA with post- hoc Tukey correction. *denotes p<0.05; ***denotes p<0.0005; ****denotes p<0.0001; ns denotes not significant.

[0021] FIG.9: PL pRab10-Rab10 Validation. (A) Validation of PL between pT73-Rab10 and total Rab10 (PL pT73(Rab10)–Rab10) to detect endogenous Rab10 phosphorylation in LRRK2WT / WTand LRRK2- / -HEK293 cells. At baseline, there was PL signal in LRRK2WT / WTwhich was significantly decreased in presence of the LRRK2 kinase inhibitor PF360. In contrast, there was no signal in LRRK2- / -HEK293 cells. (B) Quantification of Rab10 phosphorylation by PL pT73(Rab10)–Rab10 Objects / DAPI in HEK293 cells. Each symbol represents the average number of PL pT73(Rab10)–Rab10 Objects / DAPI obtained from 100 - 150 cells per treatment group for each independent experiment; N=3 independent experiments. Statistical testing by two-way ANOVA with post-hoc Tukey correction; ****denotes p<0.0001; ns denotes not significant.

[0022] FIG.10: Chloroquine and monensin cause ROS production and lipid peroxidation. (A) ROS production was assessed by dihydroethidium (DHE) fluorescence (red). Chloroquine (100μM, 3 hours) and monensin (10μM, 4 hours) resulted in an increase in DHE signal compared to vehicle in LRRK2WT / WTHEK293 cells. (B) Quantification of normalized DHE signal. Each symbol represents the normalized average DHE signal measured from 100- 150 cells per treatment group per experiment. N=3 independent experiments. Statistical testing by one-way ANOVA with post-hoc Tukey correction. (C) Immunofluorescence staining for 4HNE (green) in LRRK2WT / WTHEK293 cells. LRRK2WT / WTHEK293 cells exposed to chloroquine had an increase in 4HNE signal at 1 hour and 3 hours after treatment, which was prevented by co-treatment with NAC. (D) Quantification of cellular 4HNE signal. Each symbol represents the average number of 4HNE objects / DAPI values obtained from 100-150 cells per treatment group per experiment. N=3 independent experiments. Statistical testing by one-way ANOVA with post-hoc Tukey correction. (E) LRRK2WT / WTHEK293 cells treated with monensin displayed an increase in 4HNE signal at 4 hours, which was prevented by NAC cotreatment. (F) Quantification of cellular 4HNE signal. Each symbol represents the average number of 4HNE objects / DAPI obtained from 100-150 cells per treatment group per experiment. N=3 independent experiments. *denotes p<0.05; **denotes p<0.005; ***denotes p<0.001; ns denotes not significant.

[0023] FIG.11: C2024A or C2025A mutation do not affect baseline kinase activity. (A) LRRK2 kinase activity was assessed by PL pS1292–LRRK2. Under vehicle conditions, C2024A, C2025A, and C2024A / C2025A had PL pS1292–LRRK2 signal (red dots) that was not different than LRRK2WT / WT. Treatment with PF360 reduced PL pS1292-LRRK2 signal in LRRK2WT / WT, C2024A, C2025A, and C2024A / C2025A. No signal was observed in LRRK2- / -. (B) Quantification of LRRK2 activity by PL pS1292–LRRK2 Objects / DAPI. Each symbol represents the average number of PL pS1292-LRRK2 objects / DAPI obtained from 100-150 cells per treatment group for each independent experiment; N=3 independent experiments. Statistical testing by two-way ANOVA with post-hoc Tukey correction. (C) PL pT73(Rab10)–Rab10 was used to assess baseline Rab10 phosphorylation. Under vehicle conditions, C2024A, C2025A, and C2024A / C2025A PL pRab10–Rab10 signal (red dots) that was not different than LRRK2WT / WT. Treatment with PF360 reduced PL pT73(Rab10)–Rab10 signal in LRRK2WT / WT, C2024A, C2025A, and C2024A / C2025A. No signal was observed in LRRK2- / -. (D) Quantification of Rab10 phosphorylation by PL pT73(Rab10)–Rab10 Objects / DAPI. Each symbol represents the average number PL pT73(Rab10)–Rab10 objects / DAPI obtained from 100-150 cells per treatment group for each independent experiment; N=3 independent biological experiments. Statistical testing by two-way ANOVA with post-hoc Tukey correction. ****denotes p<0.0001; ns denotes not significant.

[0024] FIG.12: C2024A or C2025A or double mutants prevent rotenone induced ROS production. (A) ROS production was assessed by DHE fluorescence (red). Rotenone increased ROS in LRRK2WT / WTHEK293 cells but not in LRRK2- / -, C2024A, C2025A, C2024A / C2025A cells. (B) Quantification of normalized DHE signal. Each symbol represents the normalized average DHE signal measured from 100-150 cells per treatment group per experiment. N=3 independent experiments. Statistical testing by one-way ANOVA with post-hoc Tukey correction. ****denotes p<0.0001; ns denotes not significant.

[0025] FIG.13: Exogenous 4-hydroxynonenal (4HNE) forms adducts with LRRK2. (A) PL between LRRK2 and 4HNE (PL LRRK2–4HNE) was used as an index of LRRK2-4HNE adduct formation in LRRK2WT / WTand LRRK2- / -HEK293 cells. LRRK2WT / WTcells treated with 100 μM 4HNE for 1 hour had strong PL LRRK2-4HNE signal compared to vehicle. This was prevented by NAC treatment and not observed in LRRK2- / -as an assay control. (B) Quantification of PL LRRK2-4HNE Objects / DAPI. Each symbol represents the average number of PL LRRK2–4HNE Objects / DAPI obtained from 100-150 cells per treatment group for each independent experiment; N=3 independent experiments. Statistical testing bytwo-way ANOVA with post-hoc Tukey correction. ****denotes p<0.0001; ns denotes not significant.

[0026] FIG.14: Exogenous 4HNE induces LRRK2 kinase activation. (A) LRRK2 kinase activity was assessed by PL pS1292–LRRK2. LRRK2WT / WTHEK293 cells treated with 100μM 4HNE had an increase in PL pS1292–LRRK2 signal (red dots) relative to vehicle, which was prevented by pretreatment with NAC. (B) Quantification of LRRK2 activity by PL pS1292–LRRK2 Objects / DAPI. Each symbol represents the average number of PL pS1292-LRRK2 objects / DAPI 100-150 cells per treatment group for each independent experiment; N=4 independent experiments. Statistical testing by two-way ANOVA with post- hoc Tukey correction. (C) PL pT73(Rab10)–Rab10 was used to assess 4HNE induced Rab10 phosphorylation as a surrogate marker for LRRK2 kinase activity. LRRK2WT / WTHEK293 cells treated with 100μM 4HNE had an increase in PL pT73(Rab10)–Rab10 signal (red dots) relative to vehicle, which was prevented by pretreatment with NAC. (B) Quantification of Rab10 phosphorylation by PL pT73(Rab10)–Rab10 Objects / DAPI. Each symbol represents the average number PL pT73(Rab10)–Rab10 Objects / DAPI obtained from 100-150 cells per treatment group for each independent experiment; N=4 independent experiments. Statistical testing by two-way ANOVA with post-hoc Tukey correction. **denotes p<0.005; ***denotes p<0.0005; ****denotes p<0.0001.

[0027] FIG.15: Correlation between LRRK2-4HNE adduct formation and LRRK2 kinase activity. LRRK2WT / WTHEK293 cells were treated with 4HNE (10μM, 30μM, 100μM) and, in separate experiments, PL LRRK2-4HNE and PL pS1292-LRRK2 were measured as seen in Figures 4C and 4D. For each concentration of 4HNE, mean values for PL LRRK2-4HNE and PL pS1292-LRRK2 were plotted and correlation analysis was performed. R2= 0.996; p<0.005.

[0028] FIG.16: Rotenone induces endogenous LRRK2-4HNE adduct formation. Endogenous LRRK2 – 4HNE adduct formation was assessed by PL LRRK2–4HNE in LRRK2WT / WTand LRRK2- / -(A) HEK293 and (C) RAW264.7 macrophages. Rotenone treatment increased PL LRRK2–4HNE signal (red dots) in LRRK2WT / WT(A) HEK293 and (C) RAW264.7 macrophages. Rotenone-induced PL LRRK2–4HNE signal was prevented by cotreatment with the lipid soluble antioxidant, ^^-tocopherol. (B) Quantification of PL LRRK2–4HNE Objects / DAPI in HEK293 cells. (D) Quantification of PL LRRK2–4HNE Objects / DAPI in RAW264.7 macrophages. For each graph, each symbol represents theaverage number of PL LRRK2–4HNE Objects / DAPI obtained from 100-150 cells per treatment group for each independent experiment; N=3 independent biological experiments. Statistical testing by two-way ANOVA with post-hoc Tukey correction. ****denotes p<0.0001; ns denotes not significant.

[0029] FIG.17: LRRK2 activating stimuli promote the endogenous LRRK2 – 4HNEadduct formation. (A) PL between LRRK2 and 4HNE (PL LRRK2–4HNE) was used as anindex of endogenous LRRK2 – 4HNE adduct formation in LRRK2WT / WTand LRRK2- / -HEK293 cells. LRRK2WT / WTtreated with chloroquine had an increase in PL LRRK2-4HNE (red dots), that was prevented by co-treatment with ^^-tocopherol. (B) Quantification of PL LRRK2-4HNE Objects / DAPI in HEK293 cells. Each symbol represents the average number of PL LRRK2-4HNE Objects / DAPI obtained from 100-150 cells per treatment group for each independent experiment; N=3 independent experiments. Statistical testing by two-way ANOVA with post-hoc Tukey correction. (C) LRRK2WT / WTHEK293 cells exposed to monensin displayed an increase in PL LRRK2-4HNE signal compared to vehicle. This was prevented by co-treatment with ^^-tocopherol. (D) Quantification of PL LRRK2-4HNE Objects / DAPI in HEK293 cells. Each symbol represents the average number of PL LRRK2- 4HNE Objects / DAPI obtained from 100-150 cells per treatment group for each independent experiment; N=3 independent experiments. Statistical testing by two-way ANOVA with post- hoc Tukey correction. ****denotes p<0.0001.

[0030] FIG.18: Structure and potency of CU12991. (A) Structure of the specific 15-LO inhibitor, CU12991. (B) Dose response curve of cellular 4HNE signal vs. CU12991 concentration in LRRK2G2019Sfibroblasts treated with the glutathione peroxidase 4 inactivator and ferroptosis inducer, RSL3. Dashed line represents the baseline 4HNE signal. (C) Dose response curve of cellular 4HNE signal vs. CU12991 concentration in LRRK2WT / WTRAW264.7 macrophages exposed to 50nM rotenone. Dashed line represents the baseline 4HNE signal.

[0031] FIG.19: 15-LO inhibition prevents rotenone induced Rab10 phosphorylation. (A) Immunofluorescence staining for 4HNE (green) in 15-LOWT / WTand 15-LO- / -HAP1 cells. Rotenone treatment elicited an increase in 4HNE signal in 15-LOWT / WT, which was prevented by the 15-LO inhibitor, CU12991. In 15-LO- / -HAP1 cells, rotenone induced 4HNE accumulation was prevented. (B) Quantification of cellular 4HNE signal. Each symbol represents the average number of 4HNE objects / DAPI values obtained from 100-150 cellsper treatment group per experiment; N=3 independent experiments. Statistical testing by two- way ANOVA with post-hoc Tukey correction. (C) PL pT73(Rab10)–Rab10 (red dots) was used to assess rotenone induced Rab10 phosphorylation as a surrogate marker for LRRK2 kinase activity.15-LOWT / WTcells treated with rotenone had an increase in PL pT73(Rab10)– Rab10 signal (red dots) relative to vehicle, which was prevented by cotreatment with CU12991 or completely abolished by PF360. Baseline pT73(Rab10)–Rab10 signal was similar between 15-LO- / -and 15-LOWT / WT, however rotenone did not result in an increase in PL pT73(Rab10)–Rab10 signal in 15-LO- / -. (D) Quantification of Rab10 phosphorylation by PL pT73(Rab10)–Rab10 Objects / DAPI. Each symbol represents the average number PL pT73(Rab10)–Rab10 Objects / DAPI obtained from 100-150 cells per treatment group for each independent experiment; N=3 independent experiments. Statistical testing by two-way ANOVA with post-hoc Tukey correction. ****denotes p<0.001.

[0032] FIG.20: CU12991 does not interfere with 4HNE. (A) 4HNE induced LRRK2 kinase activity measured by PL pS1292 – LRRK2 was assessed in 15-LOWT / WTand 15-LO- / -HAP1 cells in presence or absence of CU12991.4HNE treatment elicited an increase in PL pS1292 – LRRK2 in both in 15-LOWT / WTand 15-LO- / -. CU12991 cotreatment had no effect on 4HNE induced PL pS1292 – LRRK2 in 15-LOWT / WTand 15-LO- / -. (B) Quantification of LRRK2 activity by PL pS1292–LRRK2 Objects / DAPI. Each symbol represents the average number of PL pS1292-LRRK2 objects / DAPI obtained from 100-150 cells per treatment group for each independent experiment; N=3 independent experiments. Statistical testing by two-way ANOVA with post-hoc Tukey correction. *denotes p<0.05.

[0033] FIG.21: CU12991 prevents stimulated Rab10 phosphorylation. Representative western blots for total Rab10 and pThr73-Rab10 in LRRK2WT / WTRAW264.7 macrophages treated with (A) rotenone, (C) chloroquine, or (E) monensin. (A) Rotenone, (C) chloroquine, and (E) monensin elicited an increase in pT73-Rab10 / Total Rab10 compared to vehicle, which was prevented by cotreatment with CU12991. (B) Quantification of rotenone induced pRab10 / Total Rab10 normalized to vehicle. Each symbol represents the average of pT73- Rab10 / Total Rab10 obtained from two technical replicates per treatment group for each experiment; N=4 independent experiments. Statistical testing by two-way ANOVA with post- hoc Tukey correction. (D) Quantification of chloroquine induced pRab10 / Total Rab10 normalized to vehicle. Each symbol represents the average of pT73-Rab10 / Total Rab10 obtained from two technical replicates per treatment group for each experiment; N=6 independent experiments. Statistical testing by two-way ANOVA with post-hoc Tukeycorrection. (F) Quantification of monensin induced pRab10 / Total Rab10 normalized to vehicle. Each symbol represents the average of pT73-Rab10 / Total Rab10 obtained from two technical replicates per treatment group for each experiment; N=5 independent experiments. Statistical testing by two-way ANOVA with Tukey correction. *denotes p<0.05; ** denotes p<0.005; ns denotes not significant.

[0034] FIG. 22: 4HNE mediated LRRK2 activation. (1) Under physiological conditionsLRRK2 has basal kinase activity and is primarily cytosolic. Similarly, 15-LO resides in the cytosol with low catalytic activity. Under PD-related pathogenic conditions (i.e. mitochondrial dysfunction, lysosomal dyshomeostasis, or vesicular trafficking impairment), the scaffolding protein, PEBP1, associates with 15-LO. (2) The newly formed PEBP1 – 15- LO complex, then translocates to membrane compartments. This in turn, allows 15-LO to preferentially oxygenate membrane phospholipids, the key step in the enzymatic generation of 4HNE. Sustained 15-LO activation as a result of these cellular perturbations, leads to the excessive generation and accumulation of 4HNE at membranes and in the cytoplasm. Since 4HNE is a reactive aldehyde, it can form adducts with proteins, such as LRRK2 and influence their function. (3) LRRK2 – 4HNE adduct formation results in LRRK2 hyperactivation. Inhibition of 15-LO, 15-LO depletion, or scavenging the endogenous generation of 4HNE mitigates pathological LRRK2 activation. Thus, 15-LO regulates pathologic LRRK2 kinase activity through the lipid peroxidation product, 4HNE. DETAILED DESCRIPTIONI. GENERAL

[0035] The present disclosure describes that the 15-lipoxygenase (15-LO) product 4- hydroxynonenal (4HNE or 4-HNE) post-translationally modifies LRRK2 and is believed to induce a pathologically elevated, chronic LRRK2 kinase activity associated with a LRRK2 mediated disease or condition, e.g., Parkinson’s disease or frontotemporal dementia. Production of LRRK2-4HNE adducts, e.g., LRRK2 Cys2024-4HNE and / or LRRK2 Cys2025-4HNE adduct described herein, may be an indication of a LRRK2 mediated disease or condition. Inhibiting the production of excessive 4HNE in cells by 15-LO inhibitors converted an elevated LRRK2 activity to a normal basal LRRK2 activity level as demonstrated in cellular experiments in the Examples. The 15-LO inhibition of 4HNE production is believed to only remove elevated LRRK2 activity associated with LRRK2 mediated diseases or conditions and not normal LRRK2 activity that may be required forother cellular functions. Accordingly, 15-LO inhibition of elevated LRRK2 activity and / or expression may afford a safe and effective treatment for LRRK2 mediated diseases or conditions.

[0036] The Examples in the application provide illustrative embodiments of the invention. For instance, Example 3 of the application showed that in preventing 4HNE production in cells by genetic knock-out of 15-LO or by administering a 15-LO inhibitor, for instance, CU12991, pathologically elevated chronic LRRK2 kinase activity was prevented. Without wishing to be bound by theory, 15-LO inhibition can prevent pathologically elevated LRRK2 kinase activity while basal (physiological) LRRK2 kinase activity remained unaffected. Existing LRRK2 kinase inhibitors in development in the clinic have been shown to inhibit both basal (physiological) and pathologically elevated LRRK2 kinase activity. Because LRRK2 kinase inhibitors have exhibited a toxicology signature in human patients, the data in the present disclosure suggests that a 15-LO inhibitor may be a safer method for inhibiting pathologically elevated, chronic LRRK2 kinase activity driving Parkinson’s disease pathology.II. DEFINITIONS

[0037] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0038] The prefix “Cu-Cv” or “Cu-v”indicates that the following group has from u to v carbon atoms. For example, “C1-8alkyl” indicates that the alkyl group has from 1 to 8 carbon atoms.

[0039] “Alkyl” is a linear or branched saturated monovalent or divalent hydrocarbon. For example, an alkyl group can have 1 to 10 carbon atoms (i.e., C1-10alkyl) or 1 to 8 carbon atoms (i.e., C1-8 alkyl) or 1 to 6 carbon atoms (i.e., C1-6 alkyl) or 1 to 4 carbon atoms (i.e., (C1-4alkyl). Examples of alkyl groups include, but are not limited to, methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, i-butyl, -CH2CH(CH3)2), 2-butyl (s-Bu, s-butyl, -CH(CH3)CH2CH3), 2-methyl-2-propyl (t- Bu, t-butyl, -C(CH3)3), 1-pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-1-butyl (-CH2CH2CH(CH3)2), 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (- CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (- CH(CH3)C(CH3)3, and octyl (-(CH2)7CH3).

[0040] “Alkenyl” refers to a straight chain or branched hydrocarbon having at least 2 carbon atoms and at least one double bond. Alkenyl can include any number of carbons, such as C2, C2-3, C2-4, C2-5, C2-6, C2-7, C2-8, C2-9, C2-10, C3, C3-4, C3-5, C3-6, C4, C4-5, C4-6, C5, C5-6, and C6. Alkenyl groups can have any suitable number of double bonds, including, but not limited to, 1, 2, 3, 4, 5 or more. Examples of alkenyl groups include, but are not limited to, vinyl (ethenyl), propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, or 1,3,5-hexatrienyl. Alkenyl groups can be substituted or unsubstituted.

[0041] “Alkynyl” refers to either a straight chain or branched hydrocarbon having at least 2 carbon atoms and at least one triple bond. Alkynyl can include any number of carbons, such as C2, C2-3, C2-4, C2-5, C2-6, C2-7, C2-8, C2-9, C2-10, C3, C3-4, C3-5, C3-6, C4, C4-5, C4-6, C5, C5-6, and C6. Examples of alkynyl groups include, but are not limited to, acetylenyl, propynyl, 1-butynyl, 2-butynyl, butadiynyl, 1-pentynyl, 2-pentynyl, isopentynyl, 1,3-pentadiynyl, 1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadiynyl, 1,4-hexadiynyl, 1,5-hexadiynyl, 2,4-hexadiynyl, or 1,3,5-hexatriynyl. Alkynyl groups can be substituted or unsubstituted.

[0042] “Alkoxy” means a group having the formula –O-alkyl, in which an alkyl group, as defined above, is attached to the parent molecule via an oxygen atom. The alkyl portion of an alkoxy group can have 1 to 20 carbon atoms (i.e., C1-C20 alkoxy), 1 to 12 carbon atoms (i.e., C1-C12alkoxy), 1 to 8 carbon atoms (i.e., C1-C8alkoxy), 1 to 6 carbon atoms (i.e., C1-C6alkoxy) or 1 to 3 carbon atoms (i.e., C1-C3 alkoxy). Examples of suitable alkoxy groups include, but are not limited to, methoxy (-O-CH3or –OMe), ethoxy (-OCH2CH3or -OEt), isopropoxy (-O-CH(CH3)2), t-butoxy (-O-C(CH3)3 or –OtBu) and the like. Other examples of suitable alkoxy groups include, but are not limited to, sec-butoxy, tert-butoxy, pentoxy, hexoxy, and the like.

[0043] “Alkoxyalkyl” refers an alkoxy group linked to an alkyl group which is linked to the remainder of the compound. Alkoxyalkyl can have any suitable number of carbons, such as from 2 to 6 (C2-6alkoxyalkyl), 2 to 5 (C2-5alkoxyalkyl), 2 to 4 (C2-4alkoxyalkyl), or 2 to 3 (C2-3 alkoxyalkyl). Alkoxy and alkyl are as defined above. Examples of “alkoxyalkyl” include, but are not limited to, methoxymethyl (CH3OCH2-), and methoxyethyl (CH3OCH2CH2).

[0044] “Hydroxyalkyl” refers to a hydroxy group, -OH, linked to an alkyl group which is linked to the remainder of the compound such that the alkyl group is divalent. Hydroxyalkyl can have any suitable number of carbons, such as from 1 to 8 (C1-8hydroxyalkyl), 1 to 6 (C1-6 hydroxyalkyl), 2 to 6 (C2-6 hydroxyalkyl), 2 to 4 (C2-4 hydroxyalkyl), or 2 to 3 (C2-3hydroxyalkyl). Alkyl is as defined above where the alkyl is divalent.

[0045] “Aminoalkyl” is an alkyl group, as defined above, in which one or more hydrogen atoms of the alkyl group is replaced with an amine group (-NR2, where R = H or alkyl). The alkyl portion of an aminoalkyl group can have 1 to 20 carbon atoms (i.e., C1-C20aminoalkyl), 1 to 12 carbon atoms (i.e., C1-C12 aminoalkyl), 1 to 8 carbon atoms (i.e., C1-C8 aminoalkyl), 1 to 6 carbon atoms (i.e., C1-C6aminoalkyl) or 1 to 3 carbon atoms (i.e., C1-C3aminoalkyl). The alkyl groups can be substituted with 1, 2, 3, 4, 5, 6, 7, 8, 9 or more amines. Examples of suitable aminoalkyl groups include, but are not limited to, -CH2NH2, -CH2NHCH3, -CH2N(CH3)2, -CH2CH2NHCH3, -CH2CH2N(CH3)2, and -CH2CH2CH2N(CH3)2.

[0046] “Halo” or “halogen” as used herein refers to fluoro (-F), chloro (-Cl), bromo (-Br) and iodo (-I).

[0047] “Haloalkyl” is an alkyl group, as defined above, in which one or more hydrogen atoms of the alkyl group is replaced with a halogen atom. The alkyl portion of a haloalkyl group can have 1 to 20 carbon atoms (i.e., C1-C20haloalkyl), 1 to 12 carbon atoms (i.e., C1-C12haloalkyl), 1 to 8 carbon atoms (i.e., C1-C8 haloalkyl), 1 to 6 carbon atoms (i.e., C1-C6 alkyl) or 1 to 3 carbon atoms (i.e., C1-C3alkyl). The alkyl groups can be substituted with 1, 2, 3, 4, 5, 6, 7, 8, 9 or more halogens. Examples of suitable haloalkyl groups include, but are not limited to, -CF3, -CHF2, -CFH2, -CH2CF3, fluorochloromethyl, difluorochloromethyl, 1,1,1- trifluoroethyl and pentafluoroethyl.

[0048] “Cycloalkyl” refers to a single saturated or partially unsaturated all carbon ring having 3 to 20 annular carbon atoms (i.e., C3-20 cycloalkyl), for example from 3 to 12 annular atoms, for example from 3 to 10 annular atoms, or 3 to 8 annular atoms, or 3 to 6 annular atoms, or 3 to 5 annular atoms, or 3 to 4 annular atoms. The term “cycloalkyl” also includes multiple condensed, saturated and partially unsaturated all carbon ring systems (e.g., ring systems comprising 2, 3 or 4 carbocyclic rings). Accordingly, cycloalkyl includes multicyclic carbocycles such as a bicyclic carbocycles (e.g., bicyclic carbocycles having about 6 to 12 annular carbon atoms such as bicyclo[3.1.0]hexane and bicyclo[2.1.1]hexane), and polycyclic carbocycles (e.g. tricyclic and tetracyclic carbocycles with up to about 20 annular carbon atoms). The rings of a multiple condensed ring system can be connected to each other via fused, spiro and bridged bonds when allowed by valency requirements. Non- limiting examples of monocyclic cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, 1- cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1- cyclohex-2-enyl and 1-cyclohex-3-enyl.

[0049] “Alkyl-cycloalkyl” refers to a radical having an alkyl component and a cycloalkyl component, where the alkyl component links the cycloalkyl component to the point of attachment. The alkyl component is as defined above, except that the alkyl component is at least divalent, an alkylene, to link to the cycloalkyl component and to the point of attachment. In some instances, the alkyl component can be absent. The alkyl component can include any number of carbons, such as C1-6, C1-2, C1-3, C1-4, C1-5, C2-3, C2-4, C2-5, C2-6, C3-4, C3-5, C3-6, C4-5, C4-6 and C5-6. The cycloalkyl component is as defined within. Exemplary alkyl-cycloalkyl groups include, but are not limited to, methyl-cyclopropyl, methyl-cyclobutyl, methyl- cyclopentyl and methyl-cyclohexyl.

[0050] “Heterocyclyl” or “heterocycle” or “heterocycloalkyl” as used herein refers to a single saturated or partially unsaturated non-aromatic ring or a non-aromatic multiple ring system that has at least one heteroatom in the ring (i.e., at least one annular heteroatom selected from oxygen, nitrogen, and sulfur). Unless otherwise specified, a heterocyclyl group has from 3 to about 20 annular atoms, for example from 3 to 12 annular atoms, for example from 3 to 10 annular atoms, or 3 to 8 annular atoms, or 3 to 6 annular atoms, or 3 to 5 annular atoms, or 4 to 6 annular atoms, or 4 to 5 annular atoms. Thus, the term includes single saturated or partially unsaturated rings (e.g., 3, 4, 5, 6 or 7-membered rings) having from about 1 to 6 annular carbon atoms and from about 1 to 3 annular heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur in the ring. The rings of the multiplecondensed ring (e.g. bicyclic heterocyclyl) system can be connected to each other via fused, spiro and bridged bonds when allowed by valency requirements. Heterocycles include, but are not limited to, azetidine, aziridine, imidazolidine, morpholine, oxirane (epoxide), oxetane, thietane, piperazine, piperidine, pyrazolidine, piperidine, pyrrolidine, pyrrolidinone, tetrahydrofuran, tetrahydrothiophene, dihydropyridine, tetrahydropyridine, quinuclidine,, 2- oxa-6-azaspiro[3.3]heptan-6-yl, 6-oxa-1-azaspiro[3.3]heptan-1-yl, 2-thia-6- azaspiro[3.3]heptan-6-yl, 2,6-diazaspiro[3.3]heptan-2-yl, 2-azabicyclo[3.1.0]hexan-2-yl, 3- azabicyclo[3.1.0]hexanyl, 2-azabicyclo[2.1.1]hexanyl, 2-azabicyclo[2.2.1]heptan-2-yl, 4- azaspiro[2.4]heptanyl, 5-azaspiro[2.4]heptanyl, and the like. The heterocycle can be unsubstituted or substituted.

[0051] “Alkyl-heterocycloalkyl” refers to a radical having an alkyl component and a heterocycloalkyl component, where the alkyl component links the heterocycloalkyl component to the point of attachment. The alkyl component is as defined above, except that the alkyl component is at least divalent, an alkylene, to link to the heterocycloalkyl component and to the point of attachment. The alkyl component can include any number of carbons, such as C0-6, C1-2, C1-3, C1-4, C1-5, C1-6, C2-3, C2-4, C2-5, C2-6, C3-4, C3-5, C3-6, C4-5, C4-6 and C5-6. In some instances, the alkyl component can be absent. The heterocycloalkyl component is as defined above.

[0052] A “bicyclic nitrogen-containing heterocyclic ring” as used herein is a bicyclic heterocycle that has at least one nitrogen within the ring. The bicyclic nitrogen-containing heterocyclic ring can be bridged, spiro, or fused. Exemplary bicyclic nitrogen-containing heterocycles include 3,8-diazabicyclo[3.2.1]octane, 2,5-dimethyl-2,5- diazabicyclo[2.2.2]octane, 3,9-diazabicyclo[3.3.1]nonane, 2,6-diazaspiro[3.3]heptane, and 2,5-dimethyloctahydro-1H-pyrrolo[3,4-c]pyridine. The bicyclic nitrogen-containing heterocyclic ring can be unsubstituted or substituted.

[0053] “Aryl” means an aromatic hydrocarbon radical derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system. For example, an aryl group can have 6 to 20 carbon atoms, 6 to 14 carbon atoms, or 6 to 10 carbon atoms. Exemplary aryl groups include, but are not limited to, radicals derived from benzene (e.g., phenyl), naphthalene, anthracene, biphenyl, and the like.

[0054] “Alkyl-aryl” refers to a radical having an alkyl component and an aryl component, where the alkyl component links the aryl component to the point of attachment. The alkylcomponent is as defined above, except that the alkyl component is at least divalent, an alkylene, to link to the aryl component and to the point of attachment. The alkyl component can include any number of carbons, such as C0-6, C1-2, C1-3, C1-4, C1-5, C1-6, C2-3, C2-4, C2-5, C2-6, C3-4, C3-5, C3-6, C4-5, C4-6 and C5-6. In some instances, the alkyl component can be absent. The aryl component is as defined above. Examples of alkyl-aryl groups include, but are not limited to, benzyl and ethyl-benzene.

[0055] “Heteroaryl” as used herein refers to a single aromatic ring that has at least one atom other than carbon in the ring, wherein the atom is selected from the group consisting of oxygen, nitrogen and sulfur; “heteroaryl” also includes multiple condensed ring systems that have at least one such aromatic ring, which multiple condensed ring systems are further described below. Thus, “heteroaryl” includes single aromatic rings of from about 1 to 6 carbon atoms and about 1-4 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur. The sulfur and nitrogen atoms may also be present in an oxidized form provided the ring is aromatic. Exemplary heteroaryl ring systems include but are not limited to pyridyl, pyrimidinyl, oxazolyl or furyl. “Heteroaryl” also includes multiple condensed ring systems (e.g., ring systems comprising 2, 3 or 4 rings) wherein a heteroaryl group, as defined above, is condensed with one or more rings selected from heteroaryls (to form for example 1,8-naphthyridinyl), heterocycles, (to form for example 1,2,3,4-tetrahydro-1,8- naphthyridinyl), carbocycles (to form for example 5,6,7,8-tetrahydroquinolyl) and aryls (to form for example indazolyl) to form the multiple condensed ring system. Thus, a heteroaryl (a single aromatic ring or multiple condensed ring system) has about 1-20 carbon atoms and about 1-6 heteroatoms within the heteroaryl ring. Such multiple condensed ring systems may be optionally substituted with one or more (e.g., 1, 2, 3 or 4) oxo groups on the carbocycle or heterocycle portions of the condensed ring. The rings of the multiple condensed ring system can be connected to each other via fused, spiro and bridged bonds when allowed by valency requirements. It is to be understood that the individual rings of the multiple condensed ring system may be connected in any order relative to one another. It is to be understood that the point of attachment for a heteroaryl or heteroaryl multiple condensed ring system can be at any suitable atom of the heteroaryl or heteroaryl multiple condensed ring system including a carbon atom and a heteroatom (e.g., a nitrogen). It also to be understood that when a reference is made to a certain atom-range membered heteroaryl (e.g., a 5 to 10 membered heteroaryl), the atom range is for the total ring atoms of the heteroaryl and includes carbon atoms and heteroatoms. For example, a 5-membered heteroaryl would include a thiazolyland a 10-membered heteroaryl would include a quinolinyl. Exemplary heteroaryls include but are not limited to pyridyl, pyrrolyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrazolyl, thienyl, indolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, furyl, oxadiazolyl, thiadiazolyl, quinolyl, isoquinolyl, benzothiazolyl, benzoxazolyl, indazolyl, quinoxalyl, quinazolyl, 5,6,7,8- tetrahydroisoquinolinyl benzofuranyl, benzimidazolyl, thianaphthenyl, pyrrolo[2,3- b]pyridinyl, quinazolinyl-4(3H)-one, phenothiazinyl, and triazolyl. The heteroaryl can be substituted or unsubstituted.

[0056] “Phenothiazine” as used herein refers to 10H-phenothiazine, having the structure: , and tautomers thereof.

[0057] “Alkyl-heteroaryl” refers to a radical having an alkyl component and a heteroaryl component, where the alkyl component links the heteroaryl component to the point of attachment. The alkyl component is as defined above, except that the alkyl component is at least divalent, an alkylene, to link to the heteroaryl component and to the point of attachment. The alkyl component can include any number of carbons, such as C0-6, C1-2, C1-3, C1-4, C1-5, C1-6, C2-3, C2-4, C2-5, C2-6, C3-4, C3-5, C3-6, C4-5, C4-6and C5-6. In some instances, the alkyl component can be absent. The heteroaryl component is as defined within.

[0058] “Oxo” refers to the group (=O) or (O).

[0059] “Tautomer” refers to alternate forms of a compound that differ in the position of a proton, such as enol-keto and imine-enamine tautomers, or the tautomeric forms of heteroaryl groups containing a ring atom attached to both a ring -NH- and a ring =N- such as pyrazoles, imidazoles, benzimidazoles, triazoles, and tetrazoles.

[0060] A “compound of the present disclosure” includes compounds disclosed herein, for example a compound of the present disclosure includes compounds of Formula I, Ia, II, III, and / or IV, including the compounds of the Examples.III. COMPOUNDS

[0061] The present disclosure relates to methods and / or uses of 15-lipoxygenase (15-LO) inhibitors. Any 15-LO inhibitor can be used with the methods and / or uses of the disclosure asdescribed herein. In some embodiments, the 15-LO inhibitor is any one of the compounds described in Sadeghian, H. and Jabbari, A. “15-Lipoxygenase inhibitors: a patent review,” Expert Opinion on Therapeutic Patents 2016; 26(1): pages 65-88; Kahn-Kirby AH, et al. “Targeting ferroptosis: A novel therapeutic strategy for the treatment of mitochondrial disease-related epilepsy,” PLoS One.2019 Mar 28;14(3):e0214250, and in references cited therein. In some embodiments, the 15-LO inhibitor is any one of the compounds described in US Patent Nos.3,388,133, 3,682,952, 3,853,908, 4,132,714, 4,797,495, 6,630,507, 6,858,739, 8,048,900, 8,791,155, 10,287,279, and 11,174,212; US application publication nos.20050065198, 20060106014, 20090186918, and 20120053220; and PCT Publications WO 96 / 38144, 97 / 12613, 99 / 32433, 01 / 96298, 01 / 96336, 2004 / 080999, 2011 / 088322, 2011 / 028651, 2021 / 077034, which are each incorporated by reference herein.

[0062] In some embodiments, the 15-LO inhibitor is a compound of Formula I: , or a pharmaceuticallywherein each R1is independently -F, -Cl, -Br, -I, -ORa, -SRa, -NRaRb, -NO2, -CN, C1-6 alkyl, C2- 6 alkenyl, or C2-6alkynyl, wherein the alkyl, alkenyl, or alkynyl is substituted with 0, 1, 2, or 3 groups independently selected from -F, -Cl, -Br, -I, -ORa, -SRa, -NRaRb, oxo, -NO2, and -CN; each R2is independently -F, -Cl, -Br, -I, -OH, -ORa, -SRa, -NRaRb, -CN, C1-6 alkyl, C2- 6 alkenyl, or C2-6alkynyl, wherein the alkyl, alkenyl, or alkynyl is substituted with 0, 1, 2, or 3 groups independently selected from -F, -Cl, -Br, -I, -ORa, -SRa, -NRaRb, oxo, -NO2, and -CN; each Rais independently H or C1-6 alkyl; each Rbis independently H or C1-6alkyl; the subscript m is 0, 1, 2 or 3; the subscript n is 0, 1, 2, or 3; and is a bicyclic nitrogen-containing heterocyclic ring.

[0063] In some embodiments of the compound or pharmaceutically acceptable salt thereof, each R1is independently -F, -Cl, -Br, -I, -ORa, -SRa, -NRaRb, -CN, or C1-6 alkyl.

[0064] In some embodiments of the compound or pharmaceutically acceptable salt thereof, each R2is independently -F, -Cl, -Br, -I, -ORa, -SRa, -NRaRb, -CN, or C1-6alkyl.

[0065] In some embodiments of the compound or pharmaceutically acceptable salt thereof, each Rais independently H or C1-3 alkyl.

[0066] In some embodiments of the compound or pharmaceutically acceptable salt thereof, each Rbis independently H or C1-3alkyl.

[0067] In some embodiments of the compound or pharmaceutically acceptable salt thereof, the subscript m is 0 or 1.

[0068] In some embodiments of the compound or pharmaceutically acceptable salt thereof, the subscript n is 0 or 1.

[0069] In some embodiments of the compound or pharmaceutically acceptable salt thereof, the compound has the structure of Formula Ia: .of the compound or pharmaceutically acceptable salt thereof, is a bridged bicyclic nitrogen-containing heterocyclic ring.In some embodiments of the compound or pharmaceutically acceptable salt thereof, is attached to the phenothiazine through a nitrogen.In some embodiments of the compound or pharmaceutically acceptable salt thereof, has the structure:, , , R3is H, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 cycloalkyl, 4- to 8-membered heterocyclyl, phenyl, or 5- to 10-membered heteroaryl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, phenyl, or heteroaryl is substituted with 0, 1, 2, or 3 groups independently selected from -F, -Cl, -Br, -I, -OR3a, -SR3a, -NR3aR3b, oxo, -NO2, and –CN; each R3ais independently H or C1-6 alkyl; and each R3bis independently H or C1-6 alkyl.

[0073] In some embodiments of the compound or pharmaceutically acceptable salt thereof,has the structure: ,66C2-6alkynyl, C3-8cycloalkyl, 4- to 8-membered heterocyclyl, phenyl, or 5- to 10-membered heteroaryl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, phenyl, or heteroaryl is substituted with 0, 1, 2, or 3 groups independently selected from -F, -Cl, -Br, -I, -OR3a, -SR3a, -NR3aR3b, oxo, -NO2, and –CN; each R3ais independently H or C1-6 alkyl; and each R3bis independently H or C1-6alkyl.

[0074] In some embodiments of the compound or pharmaceutically acceptable salt thereof, .of the compound or pharmaceutically acceptable salt thereof, the heterocyclyl has 1, 2, or 3 atoms selected from N, O, and S.

[0076] In some embodiments of the compound or pharmaceutically acceptable salt thereof, the heteroaryl has 1, 2, or 3 atoms selected from N, O, and S.

[0077] In some embodiments of the compound or pharmaceutically acceptable salt thereof, R3is H, C1-6alkyl, or C3-8cycloalkyl, wherein the alkyl or cycloalkyl is substituted with 0, 1, 2, or 3 groups independently selected from -F, -Cl, -Br, -I, -OR3a, -SR3a, -NR3aR3b, oxo, -NO2, and –CN. In some embodiments of the compound or pharmaceutically acceptable salt thereof, R3is H, C1-6 alkyl, or C3-8 cycloalkyl. In some embodiments, R3is H, C1-3 alkyl, or C3-6cycloalkyl. In some embodiments, R3is H, CH3, CH2CH3, CH(CH3)2, or cyclopropyl. In some embodiments, R3is H, CH2CH3, CH(CH3)2, or cyclopropyl.

[0078] In some embodiments of a compound of the present disclosure or pharmaceutically acceptable salt thereof, the compound has a structure as shown in Table 1. Table 1. Compounds Compound Structure / Name 1 -610131721242832[ ] n some em o men s, e compoun o e presen sc osure s a compound of Formula II: R13bR13a, or a pharmaceuticallywhereineach R11 and R12 is independently H, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 haloalkyl, C1-6hydroxyalkyl, C1-6aminoalkyl, (C0-6alkyl)(C3-8cycloalkyl), (C0-6 alkyl)(heterocyclyl), (C0-6 alkyl)(C6-10 aryl), (C0-6 alkyl)(heteroaryl), halogen, ORa, SRa, NRaRb, NO2, or CN, wherein the alkyl, alkenyl, alkynyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is substituted with 0, 1, 2, or 3 R11a; orR11and R12taken together with the carbon atoms to which they are attached form a C5-10cycloalkyl, heterocyclyl, C6-10 aryl, or heteroaryl ring each substituted with 0, 1, 2, or 3 R11b; each R11aand R11bis independently ORc, SRc, NRcRd, oxo, NO2, or CN;each Rc and Rd is independently H or C1-6 alkyl;each R13a, R13b, R14a, R14b, R15a, R15b, R16a, and R16bis independently H, C1-6 alkyl, C1-6 alkyl, C2-6alkenyl, C2-6alkynyl, C1-6haloalkyl, C1-6hydroxyalkyl, C1-6aminoalkyl; or two of R13a, R13b, R14a, R14b, R15a, R15b, R16a, and R16btaken together with the atoms to which they are attached form a C3-8cycloalkyl or a heterocyclyl; X is –C(R17a)(R17b)-, -N(R18)-, -O-, or –S-; R17ais (C0-6alkyl)-N(R17a1)(R17a2), (C0-6alkyl)-OR17a1, or (C0-6alkyl)-SR17a1; R17bis H or C1-6 alkyl; or R17aand R17btaken together form an oxo; each R17a1and R17a2is independently H, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C2-6 alkoxyalkyl, C1-6haloalkyl, C1-6hydroxyalkyl, C1-6aminoalkyl, (C2-6alkyl)-N(R17c)C(O)-(R17d), (C2-6 alkyl)-N(R17c)C(O)(OR17d), (C2-6 alkyl)-N(R17c)S(O)2R17d, (C0-6 alkyl)(C3-8 cycloalkyl), (C0-6alkyl)(heterocyclyl), (C0-6alkyl)(C6-10aryl), (C0-6alkyl)(heteroaryl), (C0-6 alkyl)-C(O)-R17c, (C0-6 alkyl)-C(O)O-R17c, (C0-6 alkyl)-C(O)-N(R17c)(R17d), (C0- 6 alkyl)-S(O)R17c, (C0-6alkyl)-S(O)(NH)R17c, (C0-6alkyl)-S(O)2R17c, (C0-6alkyl)-S(O)2N(R17c)(R17d), or (C0-6alkyl)-S(O)(NR17c)R17d, wherein the cycloalkyl, heterocyclyl, aryl, or heteroaryl is substituted with 0, 1, 2, or 3 R17e; or R17a1and R17a2taken together with the atoms to which they are attached form a heterocyclyl, which is substituted with 0, 1, 2, or 3 R17e; R18is H, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C2-6alkoxyalkyl, C1-6haloalkyl, C1-6hydroxyalkyl, C1-6aminoalkyl, (C2-6alkyl)-N(R18a)C(O)-(R18b), (C2-6alkyl)-N(R18a)C(O)(OR18b), (C2-6alkyl)-N(R18a)S(O)2R18b, (C0-6alkyl)(C3-8cycloalkyl), (C0-6alkyl)(heterocyclyl), (C0-6alkyl)(C6-10aryl), (C0-6alkyl)(heteroaryl), (C0-6alkyl)-C(O)-R18a, (C0-6alkyl)-C(O)O-R18a, (C0-6alkyl)-C(O)-N(R18a)(R18b), (C0-6 alkyl)-S(O)R18a, (C0-6 alkyl)-S(O)(NH)R18a, (C0-6 alkyl)-S(O)2R18a, (C0- 6 alkyl)-S(O)2N(R18a)(R18b), or (C0-6alkyl)-S(O)(NR18a)R18b, wherein the cycloalkyl, heterocyclyl, aryl, or heteroaryl is substituted with 0, 1, 2, or 3 R18c; each R17eand R18cis independently C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C2-6alkoxyalkyl, C1-6 haloalkyl, C1-6 hydroxyalkyl, C1-6 aminoalkyl, C3-8 cycloalkyl, heterocyclyl, C6-10 aryl, heteroaryl, (C0-6alkyl)-C(O)-R19a, (C0-6alkyl)-C(O)O-R19a, (C0-6alkyl)-C(O)-N(R19a)(R19b), (C0-6alkyl)-S(O)R19a, -S(O)(NH)R19a, -S(O)2R19a, -S(O)2N(R19a)(R19b), or -S(O)(NR19a)R19b; each R17c, R17d, R18a, R18b, R19a, and R19bis independently H, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C2-6 alkoxyalkyl, C1-6 haloalkyl, C1-6 hydroxyalkyl, C1-6 aminoalkyl, (C0- 6 alkyl)(C3-8cycloalkyl), (C0-6alkyl)(heterocyclyl), (C0-6alkyl)(C6-10aryl), or (C0-6 alkyl)(heteroaryl), wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is substituted with 0 to 4 Z1; each Z1is independently C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C2-6 alkoxyalkyl, C1-6 haloalkyl, C3-8cycloalkyl, heterocyclyl, C6-10aryl, heteroaryl, halogen, oxo, -OH, -CN, - NO2, -NH2, -N3, -SH, -O(C1-6 alkyl), -O(C1-6 haloalkyl), -NH(C1-6 alkyl), -NH(C1-6 haloalkyl), -N(C1-6alkyl)2, -N(C1-6haloalkyl)2, -N(C1-6alkyl)(C1-6haloalkyl), -C(O)(C1-6 alkyl), -C(O)(C1-6 haloalkyl), -C(O)O(C1-6 alkyl), -C(O)O(C1-6 haloalkyl), -C(O)NH2, -C(O)NH(C1-6alkyl), -C(O)NH(C1-6haloalkyl), -C(O)N(C1-6alkyl)2, -C(O)N(C1-6 haloalkyl)2, -NHC(O)(C1-6 alkyl), -NHC(O)(C1-6 haloalkyl), - NHC(O)O(C1-6alkyl), -NHC(O)O(C1-6haloalkyl), -NHC(O)NH(C1-6alkyl), -NHC(O)NH(C1-6 haloalkyl), -NHS(O)(C1-6 alkyl), -N(C1-6 alkyl)(S(O)(C1-6 alkyl), -S(C1-6alkyl), -S(C1-6haloalkyl), -S(O)N(C1-6alkyl)2, -S(O)(C1-6alkyl), - S(O)(C1-6 haloalkyl), -S(O)2(C1-6 alkyl), -S(O)2(C1-6 haloalkyl), -S(O)(NH)(C1-6 alkyl), -S(O)2NH(C1-6alkyl), or -S(O)2N(C1-6alkyl)2; each heterocyclyl is a 4- to 10-membered ring having 1, 2, 3, or 4 heteroatoms selected from N, O, and S; and each heteroaryl is a 5- to 10-membered ring having 1, 2, 3, or 4 heteroatoms selected from N, O, and S.

[0080] In some embodiments of the compound of the present disclosure or a pharmaceutically acceptable salt thereof, each R11is independently C1-6alkyl, F, Cl, Br, I, ORc, SRc, NRcRd, or CN.

[0081] In some embodiments of the compound of the present disclosure or a pharmaceutically acceptable salt thereof, each R12is independently C1-6alkyl, F, Cl, Br, I, ORc, SRc, NRcRd, or CN.

[0082] In some embodiments of the compound of the present disclosure or apharmaceutically acceptable salt thereof, each R11 and R12 is independently H, C1-6 alkyl, C2-6alkenyl, C2-6alkynyl, C1-6haloalkyl, wherein the alkyl, alkenyl, alkynyl, or haloalkyl issubstituted with 0, 1, 2, or 3 R11a. In some embodiments, each R11 and R12 is independentlyC1-6 alkyl. In some embodiments, each R11 and R12 is methyl.

[0083] In some embodiments of the compound of the present disclosure or a pharmaceutically acceptable salt thereof, each R11ais independently ORc, NRcRd, oxo, or CN.

[0084] In some embodiments of the compound of the present disclosure or a pharmaceutically acceptable salt thereof, each R11bis independently C1-6 alkyl, C2-6 alkenyl, C2-6alkynyl, C1-6haloalkyl, halogen, or CN.

[0085] In some embodiments of the compound of the present disclosure or a pharmaceutically acceptable salt thereof, each Rcis independently H or C1-3 alkyl.

[0086] In some embodiments of the compound of the present disclosure or a pharmaceutically acceptable salt thereof, each Rdis independently H or C1-3alkyl.

[0087] In some embodiments of the compound of the present disclosure or a pharmaceutically acceptable salt thereof, each R13a, R13b, R16a, and R16bis H.

[0088] In some embodiments of the compound of the present disclosure or a pharmaceutically acceptable salt thereof, each R13a,R13b,R14a,R14b,R15a,R15b,R16a,and R16bis H.

[0089] In some embodiments, the compound of Formula (II) or a pharmaceutically acceptable salt thereof has the structure of Formula III: .

[0090] In some embodimentsdisclosure or a pharmaceutically acceptable salt thereof, R11and R12taken together with the carbon atoms to which they are attached form C6-10 aryl or heteroaryl ring substituted with 0, 1, 2, or 3 R11b.

[0091] In some embodiments, the compound of Formula (II) or a pharmaceutically acceptable salt thereof has the structure of Formula IV:V), wherein p is 0, 1,

[0092] In some embodiments of the compound of the present disclosure or a pharmaceutically acceptable salt thereof, p is 1, 2, or 3. In some embodiments, p is 3. In some embodiments, p is 2. In some embodiments, p is 0 or 1. In some embodiments, p is 1. In some embodiments, p is 0.

[0093] In some embodiments of the compound of the present disclosure or a pharmaceutically acceptable salt thereof, X is –C(R17a)(R17b)- or -N(R18)-. In some embodiments, X is –C(R17a)(R17b)- . In some embodiments, X is -N(R18)-.

[0094] In some embodiments of the compound of the present disclosure or a pharmaceutically acceptable salt thereof, R17ais -N(R17a1)(R17a2); and R17bis H.

[0095] In some embodiments of the compound of the present disclosure or a pharmaceutically acceptable salt thereof, each R17a1and R17a2is independently H, C1-6 alkyl, C2-6 alkoxyalkyl, C1-6 haloalkyl, C1-6 hydroxyalkyl, C1-6 aminoalkyl, (C2-6 alkyl)-N(R17c)C(O)- (R17d), (C2-6 alkyl)-N(R17c)C(O)(OR17d), (C2-6 alkyl)-N(R17c)S(O)2R17d, (C0-6 alkyl)(C3-8 cycloalkyl), (C0-6 alkyl)(heterocyclyl), (C0-6 alkyl)(C6-10 aryl), (C0-6 alkyl)(heteroaryl), (C0- 6 alkyl)-C(O)-R17c, (C0-6 alkyl)-C(O)O-R17c, (C0-6 alkyl)-C(O)-N(R17c)(R17d), (C0- 6 alkyl)-S(O)R17c, (C0-6 alkyl)-S(O)(NH)R17c, (C0-6 alkyl)-S(O)2R17c, (C0-6alkyl)-S(O)2N(R17c)(R17d), or (C0-6alkyl)-S(O)(NR17c)R17d, wherein the cycloalkyl, heterocyclyl, aryl, or heteroaryl is substituted with 0, 1, or 2 R17e.

[0096] In some embodiments of the compound of the present disclosure or a pharmaceutically acceptable salt thereof, each R17a1and R17a2is independently H, C1-6alkyl, C2-6 alkoxyalkyl, C1-6 haloalkyl, C1-6 hydroxyalkyl, C1-6 aminoalkyl, (C2-6 alkyl)-N(R17c)C(O)- (R17d), (C2-6alkyl)-N(R17c)C(O)(OR17d), (C2-6alkyl)-N(R17c)S(O)2R17d, (C0-6alkyl)(C3-8cycloalkyl), (C0-6 alkyl)(heterocyclyl), (C0-6 alkyl)(C6-10 aryl), (C0-6 alkyl)(heteroaryl), (C0-6alkyl)-C(O)-R17c, (C0-6alkyl)-C(O)O-R17c, (C0-6alkyl)-C(O)-N(R17c)(R17d), (C0-6 alkyl)-S(O)R17c, (C0-6 alkyl)-S(O)(NH)R17c, (C0-6 alkyl)-S(O)2R17c, (C0-6alkyl)-S(O)2N(R17c)(R17d), or (C0-6alkyl)-S(O)(NR17c)R17d. In some embodiments, each R17a1and R17a2is independently H, C1-3 alkyl, C2-3 alkoxyalkyl, C1-3 haloalkyl, C1-3 hydroxyalkyl, C1-3aminoalkyl, (C2-3alkyl)-N(R17c)C(O)-(R17d), (C2-3alkyl)-N(R17c)C(O)(OR17d), (C2-3 alkyl)-N(R17c)S(O)2R17d, (C0-3 alkyl)(C3-8 cycloalkyl), (C0-3 alkyl)(heterocyclyl), (C0-3alkyl)(C6aryl), (C0-3alkyl)(heteroaryl), (C0-3alkyl)-C(O)-R17c, (C0-3alkyl)-C(O)O-R17c, (C0-3 alkyl)-C(O)-N(R17c)(R17d), (C0-3 alkyl)-S(O)R17c, (C0-3 alkyl)-S(O)(NH)R17c, (C0-3 alkyl)- S(O)2R17c, (C0-3alkyl)-S(O)2N(R17c)(R17d), or (C0-3alkyl)-S(O)(NR17c)R17d. In some embodiments, each R17a1and R17a2is independently H, C1-3 alkyl, C2-3 alkoxyalkyl, C1-3haloalkyl, C1-3hydroxyalkyl, C1-3aminoalkyl, (C2-3alkyl)-N(R17c)C(O)-(R17d), (C2-3 alkyl)-N(R17c)C(O)(OR17d), (C2-3 alkyl)-N(R17c)S(O)2R17d, C3-8 cycloalkyl, heterocyclyl, phenyl, heteroaryl, -C(O)-R17c, -C(O)O-R17c, -C(O)-N(R17c)(R17d), -S(O)R17c, -S(O)(NH)R17c, -S(O)2R17c, -S(O)2N(R17c)(R17d), or -S(O)(NR17c)R17d.

[0097] In some embodiments of the compound of the present disclosure or a pharmaceutically acceptable salt thereof, R17a1and R17a2taken together with the atoms to which they are attached form a heterocyclyl, which is substituted with 0, 1, 2, or 3 R17e.

[0098] In some embodiments of the compound of the present disclosure or pharmaceutically acceptable salt thereof, each R17eis independently C1-6alkyl, C2-6 alkoxyalkyl, C1-6 haloalkyl, C1-6 hydroxyalkyl, C1-6 aminoalkyl, C3-8 cycloalkyl, heterocyclyl, C6-10aryl, heteroaryl, (C0-3alkyl)-C(O)-R19a, (C0-3alkyl)-C(O)O-R19a, (C0-3alkyl)-C(O)- N(R19a)(R19b), (C0-3 alkyl)-S(O)R19a, -S(O)(NH)R19a, -S(O)2R19a, -S(O)2N(R19a)(R19b), or - S(O)(NR19a)R19b.

[0099] In some embodiments of the compound of the present disclosure or a pharmaceutically acceptable salt thereof, R18is H, C1-6 alkyl, C2-6 alkoxyalkyl, C1-6 haloalkyl, C1-6hydroxyalkyl, C1-6aminoalkyl, (C2-6alkyl)-N(R18a)C(O)-(R18b), (C2-6 alkyl)-N(R18a)C(O)(OR18b), (C2-6 alkyl)-N(R18a)S(O)2R18b, (C0-6 alkyl)(C3-8 cycloalkyl), (C0-6alkyl)(heterocyclyl), (C0-6alkyl)(C6-10aryl), (C0-6alkyl)(heteroaryl), (C0-6alkyl)-C(O)-R18a, (C0-6 alkyl)-C(O)O-R18a, (C0-6 alkyl)-C(O)-N(R18a)(R18b), (C0-6 alkyl)-S(O)R18a, (C0-6 alkyl)- S(O)(NH)R18a, (C0-6alkyl)-S(O)2R18a, (C0-6alkyl)-S(O)2N(R18a)(R18b), or (C0-6alkyl)- S(O)(NR18a)R18b, wherein the cycloalkyl, heterocyclyl, aryl, or heteroaryl is substituted with 0, 1, or 2 R18c.

[0100] In some embodiments of the compound of the present disclosure or a pharmaceutically acceptable salt thereof, each R18cis independently C1-6 alkyl, C2-6alkoxyalkyl, C1-6haloalkyl, C1-6hydroxyalkyl, C1-6aminoalkyl, C3-8cycloalkyl, heterocyclyl, C6-10 aryl, heteroaryl, (C0-6 alkyl)-C(O)-R19a, (C0-6 alkyl)-C(O)O-R19a, (C0-6 alkyl)-C(O)- N(R19a)(R19b), (C0-6alkyl)-S(O)R19a, -S(O)(NH)R19a, -S(O)2R19a, -S(O)2N(R19a)(R19b), or - S(O)(NR19a)R19b.

[0101] In some embodiments of the compound of the present disclosure or a pharmaceutically acceptable salt thereof, R17ais NH2, N(CH2CH3)2, NHCH(CH3)2, NHCH2CH2OCH3, NHCH2CH2F, NHCH2CH2OH, NHCH2CH2CF3, NHCH2CH2Ph, ,

[0102] In some embodiments of the compound of the present disclosure or a pharmaceutically acceptable salt thereof, R18is H, CH3, CH2CH3, CH(CH3)2, CH(CH2CH3)2, CH2CH2OCH3, CH2CH2F, CH2CH2OH, CH2CH2CF3, CH2CH2N(CH3)2, CH2CH2CH2NHCH3, CH2CH2CH2N(CH3)2, CH2CH2CH2N(CH3)C(O)(C(CH3)3), cyclopentyl, CH2CH2Ph, ,

[0103] In some embodiments of the compound of the present disclosure or a pharmaceutically acceptable salt thereof, the heterocyclyl has 1, 2, or 3 atoms selected from N, O, and S.

[0104] In some embodiments of the compound of the present disclosure or a pharmaceutically acceptable salt thereof, the heteroaryl has 1, 2, or 3 atoms selected from N, O, and S.

[0105] In some embodiments of a compound of the present disclosure or a pharmaceutically acceptable salt thereof, e.g., a compound of Formula II, III, and / or IV, the compound has a structure as shown in Table 2 or Table 3. Table 2. Compounds Compound Structure / Name101Compound Structure / Name106 -Compound Structure / Name111Compound Structure / Name116Compound Structure / Name121Compound Structure / Name126 O -Compound Structure / Name3-(2-hydroxyethyl)-2,3,4,5-tetrahydro-1H-naphtho[2,3-Table 3. Compounds Compound Structure / Name201Compound Structure / Name206 eCompound Structure / Name211 - -Compound Structure / Name219

[0106] As known in the art, stereoisomers refer to compounds that differ in the chirality of one or more stereocenters. Stereoisomers include enantiomers and diastereomers. The compounds may exist in stereoisomeric form if they possess one or more asymmetric centers or a double bond with asymmetric substitution and, therefore, can be produced as individual stereoisomers or as mixtures. Unless otherwise indicated, the description is intended to include individual stereoisomers as well as mixtures. The methods for the determination of stereochemistry and the separation of stereoisomers are well-known in the art (see, e.g., Chapter 4 of Advanced Organic Chemistry, 4th ed., J. March, John Wiley and Sons, New York, 1992).

[0107] The compounds described herein may be prepared and / or formulated as pharmaceutically acceptable salts or when appropriate as a free base. Pharmaceutically acceptable salts are non-toxic salts of a free base form of a compound that possess the desired pharmacological activity of the free base. These salts may be derived from inorganic or organic acids or bases. For example, a compound that contains a basic nitrogen may be prepared as a pharmaceutically acceptable salt by contacting the compound with an inorganic or organic acid. Non-limiting examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogen-phosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, methylsulfonates, propylsulfonates, besylates, xylenesulfonates, naphthalene-1- sulfonates, naphthalene-2-sulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, γ-hydroxybutyrates, glycolates, tartrates, and mandelates. Lists of othersuitable pharmaceutically acceptable salts are found in Remington: The Science and Practice of Pharmacy, 21stEdition, Lippincott Wiliams and Wilkins, Philadelphia, Pa., 2006.

[0108] Examples of pharmaceutically acceptable salts of the compounds disclosed herein also include salts derived from an appropriate base, such as an alkali metal (for example, sodium, potassium), an alkaline earth metal (for example, magnesium), ammonium and NX4+(wherein X is C1^C4 alkyl). Also included are base addition salts, such as sodium or potassium salts.

[0109] The compounds of the present disclosure generally possess sufficient solubility to permit dissolution in biological media, such as blood or plasma. Kinetic solubility assays can be assessed by diluting a solution of the compound of the present disclosure prepared in DMSO into aqueous buffer, simulated intestinal fluid (SIF), or simulated gastric fluid (SGF). See, United States Pharmacopeia, USP40-NF35 (2017). In some embodiments, a compound of Formula I, Ia, II, III, or IV, or a pharmaceutically acceptable salt thereof, has a solubility of greater than about 5 µM, such as greater than about 10 µM, greater than about 20 µM, greater than about 30 µM, greater than about 40 µM, greater than about 50 µM, greater than about 100 µM, greater than about 200 µM, or greater than about 300 µM. In some embodiments, a compound of Formula I, Ia, II, III, or IV, or a pharmaceutically acceptable salt thereof, has a solubility of from about 5 µM to about 300 µM, such as from about 10 µM to about 200 µM or from about 20 µM to about 200 µM.IV. COMPOSITIONS

[0110] In some embodiments, the pharmaceutical composition of the present invention is a pharmaceutical composition comprising a 15-LO inhibitor or pharmaceutically acceptable salt as described herein, and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a compound of Formula I, Ia, II, III, or IV, or pharmaceutically acceptable salt as described herein, and a pharmaceutically acceptable excipient.

[0111] As understood in the art, a pharmaceutically effective amount refers to an amount of the compound of the present disclosure in a formulation or combination thereof, that provides the desired therapeutic or pharmaceutical result.

[0112] Generally, a therapeutically effective amount refers to an amount that is effective to elicit the desired biological or medical response, including the amount of the compound that,when administered to a subject for treating a disease, is sufficient to effect such treatment for the disease. The effective amount will vary depending on the compound, the disease, and its severity and the age, weight, etc., of the subject to be treated. The effective amount can include a range of amounts. As is understood in the art, an effective amount may be in one or more doses, i.e., a single dose or multiple doses may be required to achieve the desired treatment endpoint. An effective amount may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable or beneficial result may be or is achieved. Suitable doses of any co-administered compounds may optionally be lowered due to the combined action (e.g., additive or synergistic effects) of the compounds.

[0113] The compound can be administered by any useful route and means, such as by oral or parenteral (e.g., intravenous) administration. Therapeutically effective amounts of the compound may include from about 0.00001 mg / kg body weight per day to about 10 mg / kg body weight per day, such as from about 0.0001 mg / kg body weight per day to about 10 mg / kg body weight per day, or such as from about 0.001 mg / kg body weight per day to about 1 mg / kg body weight per day, or such as from about 0.01 mg / kg body weight per day to about 1 mg / kg body weight per day, or such as from about 0.05 mg / kg body weight per day to about 0.5 mg / kg body weight per day, or such as from about 0.3 mg to about 30 mg per day, or such as from about 30 mg to about 300 mg per day. A. Formulation

[0114] For preparing pharmaceutical compositions from the compound or pharmaceutically acceptable salt of the present invention, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, cachets, and dispersible granules. A solid carrier can be one or more substances, which may also act as diluents, binders, preservatives, disintegrating agents, or an encapsulating material. Details on techniques for formulation and administration are well described in the scientific and patent literature, see, e.g., the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton PA ("Remington's").

[0115] As known in the art, pharmaceutically acceptable or physiologically acceptable refer to compounds, salts, compositions, dosage forms and other materials which are useful inpreparing a pharmaceutical composition that is suitable for veterinary or human pharmaceutical use.

[0116] A pharmaceutically acceptable excipient can include without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.

[0117] In powders, the carrier is a finely divided solid, which is in a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the binding properties in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain from 5% or 10% to 70% of the conjugates of the present invention.

[0118] Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water / propylene glycol solutions. For parenteral injection, liquid preparations can be formulated in solution in aqueous polyethylene glycol solution.

[0119] Aqueous solutions suitable for oral use can be prepared by dissolving the compound or pharmaceutically acceptable salt of the present invention in water and adding suitable colorants, flavors, stabilizers, and thickening agents as desired. Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethylene oxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensation product of ethylene oxide with a partial ester derived from fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). The aqueous suspension can also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose, aspartame or saccharin. Formulations can be adjusted for osmolality.

[0120] Also included are solid form preparations, which are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.

[0121] Oil suspensions can be formulated by suspending the compound or pharmaceutically acceptable salt of the present invention in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin; or a mixture of these. The oil suspensions can contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents can be added to provide a palatable oral preparation, such as glycerol, sorbitol or sucrose. These formulations can be preserved by the addition of an antioxidant such as ascorbic acid. As an example of an injectable oil vehicle, see Minto, J. Pharmacol. Exp. Ther.281 :93-102, 1997. The pharmaceutical formulations of the invention can also be in the form of oil-in- water emulsions. The oily phase can be a vegetable oil or a mineral oil, described above, or a mixture of these. Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. The emulsion can also contain sweetening agents and flavoring agents, as in the formulation of syrups and elixirs. Such formulations can also contain a demulcent, a preservative, or a coloring agent.

[0122] The compositions of the present invention can also be delivered as microspheres for slow release in the body. For example, microspheres can be formulated for administration via intradermal injection of drug-containing microspheres, which slowly release subcutaneously (see Rao, J. Biomater Sci. Polym. Ed.7:623-645, 1995; as biodegradable and injectable gel formulations (see, e.g., Gao Pharm. Res.12:857-863, 1995); or, as microspheres for oral administration (see, e.g., Eyles, J. Pharm. Pharmacol.49:669-674, 1997). Both transdermal and intradermal routes afford constant delivery for weeks or months.

[0123] In another embodiment, the compositions of the present invention can be formulated for parenteral administration into a body cavity. The formulations for administration will commonly comprise a solution of the compositions of the present invention dissolved in a pharmaceutically acceptable carrier. Among the acceptable vehicles and solvents that can beemployed are water and Ringer's solution, an isotonic sodium chloride. In addition, sterile fixed oils can conventionally be employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can likewise be used in the preparation of injectables. These solutions are sterile and generally free of undesirable matter. These formulations may be sterilized by conventional, well known sterilization techniques. The formulations may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of the compositions of the present invention in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs. For IV, intratumoral, or intravitreal administration, the formulation can be a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic parenterally-acceptable diluent or solvent, such as a solution of 1,3-butanediol.

[0124] In another embodiment, the formulations of the compositions of the present invention can be delivered by the use of liposomes which fuse with the cellular membrane or are endocytosed, i.e., by employing ligands attached to the liposome, or attached directly to the oligonucleotide, that bind to surface membrane protein receptors of the cell resulting in endocytosis. By using liposomes, particularly where the liposome surface carries ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the compositions of the present invention into the target cells in vivo. (See, e.g., Al-Muhammed, J. Microencapsul.13:293-306, 1996; Chonn, Curr. Opin. Biotechnol.6:698-708, 1995; Ostro, Am. J. Hosp. Pharm.46: 1576-1587, 1989).

[0125] Lipid-based drug delivery systems include lipid solutions, lipid emulsions, lipid dispersions, self-emulsifying drug delivery systems (SEDDS) and self-microemulsifying drug delivery systems (SMEDDS). In particular, SEDDS and SMEDDS are isotropic mixtures of lipids, surfactants and co-surfactants that can disperse spontaneously in aqueous media and form fine emulsions (SEDDS) or microemulsions (SMEDDS). Lipids useful in the formulations of the present invention include any natural or synthetic lipids including, but notlimited to, sesame seed oil, olive oil, castor oil, peanut oil, fatty acid esters, glycerol esters, Labrafil®, Labrasol®, Cremophor®, Solutol®, Tween®, Capryol®, Capmul®, Captex®, and Peceol®. B. Administration

[0126] The compound or pharmaceutically acceptable salt and compositions of the present invention can be delivered by any suitable means, including oral, parenteral and topical methods.

[0127] A compound or composition of the present disclosure may be administered to an individual in accordance with an effective dosing regimen for a desired period of time or duration, such as at least about one month, at least about 2 months, at least about 3 months, at least about 6 months, or at least about 12 months or longer. In one variation, the compound is administered on a daily or intermittent schedule for the duration of the individual’s life.

[0128] The dosage or dosing frequency of a compound or composition of the present disclosure may be adjusted over the course of the treatment, based on the judgment of the administering physician.

[0129] The compound or composition may be administered to an individual (e.g., a human) in an effective amount. In some embodiments, the compound is administered once daily.

[0130] The pharmaceutical preparation is preferably in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the compounds and compositions of the present invention. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules.

[0131] The compounds and compositions of the present invention can be co-administered with other agents. Co-administration includes administering the compound or composition of the present invention within 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, or 24 hours of the other agent. Co-administration also includes administering simultaneously, approximately simultaneously (e.g., within about 1, 5, 10, 15, 20, or 30 minutes of each other), or sequentially in any order. Moreover, the compounds and compositions of the present invention can each be administered once a day, or two, three, or more times per day so as to provide the preferred dosage level per day.

[0132] In some embodiments, co-administration can be accomplished by co-formulation, i.e., preparing a single pharmaceutical composition including the compounds and compositions of the present invention and any other agent. Alternatively, the various components can be formulated separately.

[0133] The compounds and compositions of the present invention, and any other agents, can be present in any suitable amount, and can depend on various factors including, but not limited to, weight and age of the subject, state of the disease, etc. Suitable dosage ranges include from about 0.1 mg to about 10,000 mg, or about 1 mg to about 1000 mg, or about 10 mg to about 750 mg, or about 25 mg to about 500 mg, or about 50 mg to about 250 mg. Suitable dosages also include about 1 mg, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 mg. The composition can also contain other compatible therapeutic agents. The compounds described herein can be used in combination with one another, with other active agents known to be useful in modulating ferroptosis, or with adjunctive agents that may not be effective alone, but may contribute to the efficacy of the active agent.V. METHODSA. Methods of Reducing LRRK2 Activity and / or Expression

[0134] In some embodiments, the method of the present invention is a method for reducing LRRK2 activity and / or expression, e.g., in a cell. As illustrated in Example 3 herein, compounds or compositions of the present disclosure are useful for lowering LRRK2 activity and / or expression in a cell. Accordingly, in some embodiments, a method of lowering LRRK2 activity and / or expression in a cell comprises administering to the cell an effective amount of a 15-LO inhibitor. In some embodiments, the method described herein is a method of lowering LRRK2 activity and / or expression in a cell, comprising contacting the cell with an effective amount of a 15-lipoxygenase (15-LO) inhibitor, wherein the LRRK2 activity and / or expression is higher than a control LRRK2 activity and / or expression in a control cell. In some embodiments, the 15-LO inhibitor is a compound of the present disclosure or pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure. In some embodiments, the method comprises administering the compound of the present disclosure or pharmaceutically acceptable salt thereof in vitro, ex vivo, or in vivo.

[0135] In some embodiments, a therapeutically effective amount or effective amount refers to an amount that is effective to elicit the desired biological or medical response, including the amount of the compound that, when administered to a subject for treating a disease, is sufficient to effect such treatment for the disease. The effective amount will vary depending on the compound, the disease, and its severity and the age, weight, etc., of the subject to be treated. The effective amount can include a range of amounts. As is understood in the art, an effective amount may be in one or more doses, i.e., a single dose or multiple doses may be required to achieve the desired treatment endpoint. An effective amount may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable or beneficial result may be or is achieved. Suitable doses of any co- administered compounds may optionally be lowered due to the combined action (e.g., additive or synergistic effects) of the compounds.

[0136] Mutations in leucine-rich repeat kinase 2 (LRRK2) that increase its kinase activity are strongly linked to genetic forms of Parkinson’s disease (PD). However, the regulation of endogenous wild-type (WT) LRRK2 kinase activity remains poorly understood, despite its frequent elevation in idiopathic PD (iPD) patients. Various stressors such as mitochondrial dysfunction, lysosomal dyshomeostasis, or vesicle trafficking deficits can activate WT LRRK2 kinase, but the specific molecular mechanisms are not fully understood. The production of 4-hydroxynonenal (4HNE), a lipid hydroperoxidation end-product, is believed to be a common biochemical response to these diverse stimuli. As illustrated in Example 3, 4- HNE forms post-translational adducts with Cys2024 and Cys2025 in the kinase activation loop of WT LRRK2 in certain cell types, significantly increasing its kinase activity. Additionally, the 4-HNE responsible for regulating LRRK2 is believed to be generated by the action of 15-lipoxygenase (15-LO), making 15-LO an upstream regulator of the pathogenic activation or hyperactivation of LRRK2 kinase activity. Pharmacological inhibition or genetic ablation of 15-LO with a pharmacological inhibitor such as CU12991 prevented 4- HNE post-translational modification of LRRK2 kinase and its subsequent pathogenic hyperactivation in these cells. Therefore, 15-LO inhibitors, or methods to lower 4-HNE levels, or the targeting of Cys2024 / 2025 may provide new therapeutic strategies to modulate LRRK2 kinase activity and treat diseases or conditions such as Parkinson’s disease.

[0137] In some embodiments, a method of the present disclosure is a method of reducing a LRRK2 activity and / or expression in a cell, comprising contacting the cell with an effectiveamount of a compound of Formula I, Ia, II, III, or IV, or pharmaceutically acceptable salt thereof.

[0138] In some embodiments, reduction of a LRRK2 activity and / or expression in a method as described herein is a reduction in the amount of a LRRK2 nucleic acid, e.g., RNA and / or DNA, and / or a reduction in the amount of a LRRK2 protein. In some embodiments, the reduction in the amount of a LRRK2 DNA is determined by any assay method, including assays known in the art and the assays described in the present disclosure. In some embodiments, the reduction in the amount of a LRRK2 RNA is determined by any assay method, including assays known in the art and the assays described in the present disclosure.

[0139] In some embodiments, reduction of a LRRK2 activity and / or expression in a method as described herein is a reduction in the amount of LRRK2 protein and / or a reduction in the activity of LRRK2 protein. In some embodiments, the reduction of a LRRK2 activity and / or expression is a reduction in the amount of a LRRK2 protein as determined by any assay method, including assays known in the art and the assays described in the present disclosure, that results in a reduction in the LRRK2 activity.

[0140] In some embodiments, LRRK2 activity can comprise kinase activity and / or GTPase activity. In some embodiments, phosphorylation status of LRRK2 on Serine1292 and / or Serine935 can be detected by western blot, ELISA (including (DNA) aptamer- and antibody- based methods), proximity ligation assay (including (DNA) aptamer- and antibody-based methods), FRET (including (DNA) aptamer- and antibody-based methods), flow cytometry (including (DNA) aptamer- and antibody-based methods), or mass spectroscopy. In some embodiments, phosphorylation status of the LRRK2 substrate Rab10 on Threonine73 can be detected by western blot, ELISA (including (DNA) aptamer- and antibody-based methods), proximity ligation assay (including (DNA) aptamer- and antibody-based methods), FRET (including (DNA) aptamer- and antibody-based methods), flow cytometry (including (DNA) aptamer- and antibody-based methods), and mass spectroscopy. Assays to measure LRRK2 kinase activity include those described in Example 3 herein. LRRK2 activity assays are also known in the art, including TR-FRET and cellular kinase assays as described in US Patent No.10,087,186; LanthaScreen assay described in US Patent No.9,493,440; and those described in US Patent Nos.9,156,845; 9,440,952; 9,718,818; and 10,294,235; which are each incorporated by reference herein. Further cellular assays for measuring LRRK2 kinase activity are described in US Patent Nos.9,675,594; 9,695,171; 10,039,753; 10,913,744; and10,975,081; which are each incorporated by reference herein, and references cited therein, as well as the biological assay described in Example 3 herein. LRRK2 GTPase assays are also known in the art, such as those described in Xiong, Y. et al. (2010) GTPase Activity Plays a Key Role in the Pathobiology of LRRK2. PLoS Genet 6(4): e1000902.

[0141] Methods of measuring LRRK2 expression are known in the art. For instance, expression can be measured at the mRNA level by northern blot, qfRT-PCR, RNA-Seq, mRNA microarray, Nanopore sequencing, and at the protein level by western blot, ELISA (including aptamer- and antibody-based methods), flow cytometry (including aptamer- and antibody-based methods), immunocytochemistry and immunohistochemistry (including aptamer- and antibody-based methods), and mass spectroscopy. See, e.g., Mabrouk, O. S. et al. “Quantitative measurements of LRRK2 in human cerebrospinal fluid demonstrates increased levels in G2019S patients.” Frontiers in Neuroscience 2020, 14: Article 526, and references cited therein.

[0142] Any suitable cell can be used in a method of the present disclosure, e.g., a method of reducing, or downregulating, LRRK2 activity and / or expression described herein. Cultured cells may be derived from a subject (e.g., a patient) or control samples; and may be modified to generate genetically-modified cells, in vitro differentiated cells, cells exposed to a candidate therapeutic agent; and the like. In some embodiments, the cell is a peripheral blood mononuclear cell (PBMC), peripheral blood lymphocyte, red blood cell (RBC), fibroblast (such as skin fibroblast), iPSC, or iPSC-derived cell of the central nervous system (e.g., neuron, microglia, astrocyte, and / or oligodendrocyte). In some embodiments, the cell is a skin cell. In some embodiments, the cell is a muscle cell. For example, the muscle cell can be a cardiac cell, that is, a cardiomyocyte. In some embodiments, the cell is a renal cell. In some embodiments, the cell is a liver cell. In some embodiments, the cell is a neuronal cell. The method can be performed in a cell in vitro, ex vivo, or in vivo. In some embodiments, the reducing LRRK2 activity and / or expression is in vitro or ex vivo. In some embodiments, the reducing LRRK2 activity and / or expression is in vivo.

[0143] Any suitable biological sample can be used in the methods of the present disclosure. The methods can be performed with a biological sample obtained from a subject, including without limitation biological samples such as tissue samples (e.g., from human brain), cerebrospinal fluid (CSF), whole blood, plasma, serum, fibroblasts (such as skin fibroblasts), and urine. In some embodiments, the biological sample comprises CSF. A biological samplecan comprise any type of cell described herein. As understood in the art, a biological sample from a subject is compared with the same type and amount of a control sample, for example, a PBMC sample from a subject is compared with an equivalent amount of a control PBMC sample.

[0144] Control samples used in a method of the present invention can be obtained by standard approaches known in the art. In some embodiments, a control sample can be obtained from a healthy volunteer who is not suffering from and not believed to be suffering from a disease or condition characterized by increased LRRK2 activity and / or expression. In some embodiments, a control sample is derived from multiple pooled donors who are not suffering from and not believed to be suffering from a disease or condition characterized by increased LRRK2 activity and / or expression. A control sample can be obtained commercially, e.g., from BioIVT (Westbury, NY USA).

[0145] In some embodiments, a 15-LO inhibitor, e.g., a compound of the present disclosure, e.g., a compound of Formula I, Ia, II, III, or IV, or pharmaceutically acceptable salt thereof, reduces the activity and / or expression of LRRK2 to a normal range. For example, in some embodiments, a LRRK2 normal range can be the range observed between untreated or naïve healthy fibroblast or iPSC DA neuron cells (top of the range) and mitochondrial stressor-challenged healthy fibroblast or iPSC DA neuron cells (bottom of the range).

[0146] In some embodiments, the cell has a higher LRRK2 activity and / or expression compared to a control cell. In some embodiments, the control cell is a normal cell, that is, not one that has or is suspected of having an elevated LRRK2 level. In some embodiments, the control LRRK2 activity and / or expression is a normal LRRK2 activity and / or expression activity and / or expression in a healthy cell.

[0147] In some embodiments, a 15-LO inhibitor, e.g., a compound of Formula I, Ia, II, III, or IV, or pharmaceutically acceptable salt thereof reduces, or downregulates, the activity and / or expression of LRRK2 to within about 50%, about 40%, about 30%, or about 20% relative to a control activity and / or expression of LRRK2. In some embodiments, the control activity and / or expression of LRRK2 is measured in a control cell from a control subject that does not have or is not suspected of having a disease or disorder mediated by an increased LRRK2 level. In some embodiments, a 15-LO inhibitor reduces the LRRK2 activity and / or expression in a cell to within about 50%, e.g., within about 40%, about 30%, about 20%, orwithin about 10%, relative to a control activity and / or expression LRRK2 in a control cell. For example, a compound of Formula I, Ia, II, III, or IV, or pharmaceutically acceptable salt thereof can downregulate the LRRK2 activity and / or expression in a neuronal cell from a Parkinson’s disease patient to within about 50% relative to a control activity and / or expression LRRK2 in a control neuronal cell from an age-matched patient that does not have or is not suspected of having Parkinson’s disease.

[0148] The activity and / or expression of LRRK2 in a cell after contacting with a 15-LO inhibitor, e.g., a compound of Formula I, Ia, II, III, or IV, or pharmaceutically acceptable salt thereof, can be higher than the control activity and / or expression of LRRK2 in the control cell. In some embodiments, the activity and / or expression of LRRK2 in a cell after contacting with the 15-LO inhibitor is about 20%, about 30%, about 40%, or about 50% higher than the control activity and / or expression of LRRK2 in the control cell. In some embodiments, the activity and / or expression of LRRK2 in a cell after contacting with 15-LO inhibitor is from about 20% to about 50% higher than the control activity and / or expression of LRRK2 in the control cell. In some embodiments, the activity and / or expression of LRRK2 in a cell after contacting with 15-LO inhibitor is within about three times standard deviation of the control activity and / or expression of LRRK2 in the control cell.

[0149] Any suitable concentration of a 15-LO inhibitor, e.g., a compound of Formula I, Ia, II, III, or IV, or pharmaceutically acceptable salt thereof in a cell can be used to effect reducing the LRRK2 activity and / or expression in the cell to a desired level. In some embodiments, the concentration of the 15-LO inhibitor in the cell can be from about 1 nM to about 100 µM, such as from about 1 nM to about 10 µM, from about 1 nM to about 1 µM, from about 10 nM to about 100 µM, from about 10 nM to about 10 µM, from about 10 nM to about 1 µM, from about 100 nM to about 100 µM, from about 100 nM to about 10 µM, from about 100 nM to about 1 µM, from about 1 µM to about 100 µM, or from about 1 µM to about 10 µM.

[0150] In some embodiments, the control cell is an untreated cell, that is, a cell that has not been administered a 15-LO inhibitor as described herein.

[0151] In some embodiments, the 15-LO inhibitor, e.g., a compound of Formula I, Ia, II, III, or IV, or pharmaceutically acceptable salt thereof reduces the activity and / or expression of LRRK2 by about 20% or more, for example, about 30% or more, about 40% or more, or about 50% or more, about 60% or more, about 70% or more, or about 80% or more, e.g.about 90%, or about 95% relative to an untreated control not contacted with the 15-LO inhibitor. In some embodiments, the 15-LO inhibitor reduces the activity and / or expression of LRRK2 by about 20% relative to an untreated control not contacted with the 15-LO inhibitor. In some embodiments, the 15-LO inhibitor reduces the activity and / or expression of LRRK2 by about 30% relative to an untreated control not contacted with the 15-LO inhibitor. In some embodiments, the 15-LO inhibitor reduces the activity and / or expression of LRRK2 by about 40% relative to an untreated control not contacted with the 15-LO inhibitor. In some embodiments, the 15-LO inhibitor reduces the activity and / or expression of LRRK2 by about 50% relative to an untreated control not contacted with the 15-LO inhibitor. In some embodiments, the 15-LO inhibitor reduces the activity and / or expression of LRRK2 by about 60% relative to an untreated control not contacted with the 15-LO inhibitor. In some embodiments, the 15-LO inhibitor reduces the activity and / or expression of LRRK2 by about 70% relative to an untreated control not contacted with the 15-LO inhibitor. In some embodiments, the 15-LO inhibitor reduces the activity and / or expression of LRRK2 by about 80% relative to an untreated control not contacted with the 15-LO inhibitor. In some embodiments, the 15-LO inhibitor reduces the activity and / or expression of LRRK2 by about 90% relative to an untreated control not contacted with the 15-LO inhibitor.

[0152] Further provided herein is a method of reducing 4-hydroxynonenal (4HNE) in a cell, comprising administering an effective amount of a 15-lipooxygenase (15-LO) inhibitor, e.g., a compound of the present disclosure, e.g., a compound of Formula I, Ia, II, III, or IV, or a pharmaceutically acceptable salt thereof. B. Methods of Selection for 15-LO Treatment

[0153] The present invention, for instance, the experimental data provided in Example 3 and the Figures, is believed to be the first report of LRRK2-4HNE adduct formation that afforded increased LRRK2 activity. The increased LRRK2 activity phenotype in turn was shown to be able to be reversed to a basal level of LRRK2 activity by treatment with 15-LO inhibitor CU12991. Measurement of LRRK2-4HNE adduct formation may be used as a diagnostic biomarker for Parkinson’s disease (PD) or as a biomarker of 15-LO target engagement in the central nervous system (CNS).

[0154] Any suitable biological sample can be used in the methods of the present disclosure. The methods can be performed with a biological sample obtained from a subject, including without limitation biological samples such as tissue samples (e.g., from human brain),cerebrospinal fluid (CSF), whole blood, plasma, serum, fibroblasts (such as skin fibroblasts), and urine. In some embodiments, the biological sample comprises CSF. A biological sample can comprise any type of cell described herein. As understood in the art, a biological sample from a subject is compared with the same type and amount of a control sample, for example, a PBMC sample from a subject is compared with an equivalent amount of a control PBMC sample.

[0155] In some embodiments, an increased LRRK2-4HNE adduct level in a biological sample from a subject compared to control LRRK2-4HNE adduct level in a control sample is used in a method for diagnosis of a disease or condition characterized by increased LRRK2 activity and / or expression, such as Parkinson’s disease. A biological sample from a subject may be any biological sample as described elsewhere herein. For instance, a CSF sample from a subject may be used to measure LRRK2-4HNE adduct level and compared with a control LRRK2-4HNE adduct level in a control sample. In some embodiments, if the LRRK2-4HNE adduct level is higher than the control LRRK2-4HNE adduct level, then the subject can be diagnosed with Parkinson’s disease. In some embodiments, the subject can be treated with a 15-LO inhibitor described herein. In some embodiments, the method comprises a kit comprising an assay measuring LRRK2-4HNE adduct formation described herein.

[0156] In some embodiments, an increased LRRK2-4HNE adduct level in a biological sample from a subject compared to control LRRK2-4HNE adduct level in a control sample could be used in a method to select the subject for treatment of a disease or condition characterized by increased LRRK2 activity and / or expression, such as Parkinson’s disease, with a 15-LO inhibitor of the present disclosure, such as a compound of Formula I, Ia, II, III, or IV, or pharmaceutically acceptable salt thereof. For instance, Parkinson’s disease patients who exhibit an increased LRRK2-4HNE adduct level may be more responsive to treatment with a 15-LO inhibitor described herein.

[0157] 4-Hydroxynonenal, also known as 4-hydroxy-2E-nonenal, 4-hydroxy-2-nonenal, 4- HNE, 4HNE, or HNE, is an α, β-unsaturated hydroxyalkenal that is produced by lipid hydroperoxidation in cells. 4HNE has the chemical structure: .

[0158] Without wishing to be bound by theory, 4HNE adducts with proteins are believed to form via chemical reaction with a protein side chain, e.g., Cys, His, Lys, or Arg. For instance, 4HNE adducts with proteins through Cys side chain thiols are believed to occur via sulfur 1,4-addition at the 4HNE α, β-unsaturation.

[0159] Accordingly, in some embodiments, a method of the present invention is a method of selecting a subject for treatment of a disease or condition characterized by increased LRRK2 activity and / or expression, comprising measuring an increased LRRK2-4HNE adduct level in a biological sample from the subject compared to control LRRK2-4HNE adduct level in a control sample.

[0160] The LRRK2-4HNE adduct can be any covalent adduct formed between LRRK2 enzyme and 4HNE and in any ratio of LRRK2 and 4HNE, e.g., 1:1 or 1:2. In some embodiments, the LRRK2-4HNE adduct is in the LRRK2 kinase domain. In some embodiments, the LRRK2-4HNE adduct is a LRRK2 Cys-4HNE adduct, e.g., formed via Cys thiol side chain and 4HNE. In some embodiments, the LRRK2-4HNE adduct comprises a LRRK2 Cys2024-4HNE adduct. In some embodiments, the LRRK2-4HNE adduct comprises a LRRK2 Cys2025-4HNE adduct. In some embodiments, the LRRK2-4HNE adduct comprises a LRRK2 Cys2024-4HNE adduct and a LRRK2 Cys2025-4HNE adduct.

[0161] A LRRK2-4HNE adduct level can be measured by any method in the art, including but not limited to affinity assays, immuno-PCR, mass spectrometry, and PET tracers.

[0162] Affinity Assays, which are often immunoassays, are an assay or analytic procedure that relies on the binding of the target molecule, e.g., a LRRK2-4HNE adduct, to antibodies or binding fragments thereof. A detection method is used to determine the presence and extent of the binding complexes that are formed. Many formats for such assays are known and used in the art, and are suitable for detection of a LRRK2-4HNE adduct. In some embodiments, the assay format is suitable for high-throughput analysis.

[0163] Included in suitable assay formats are immunoassays that utilize antibodies or binding fragments thereof specific for a LRRK2-4HNE adduct, such as a LRRK2 Cys-4HNE adduct, e.g., a LRRK2 Cys2024-4HNE and / or a LRRK2 Cys2025-4HNE adduct.

[0164] Assays of interest include, for example, Western blots; immunohistochemistry; immunoprecipitation; mass spectrometry; etc., and include immunoassays such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and enzyme immunoassay (EIA).

[0165] Enzyme-linked immunosorbent assays (ELISAs) are used to qualitatively and quantitatively analyze the presence or concentration of a particular soluble antigen such as a LRRK2-4HNE adduct, in liquid samples, such as cell lysates. These assays generally make use of the ability of multiwell plates or others to bind antibodies which trap the cognate antigen. Usually a colorimetric endpoint that can be detected via absorbance wavelength and quantitated from a known standard curve of antigen or antibody dilutions is used. The detection antibody is often labelled with an enzyme such as horseradish peroxidase or alkaline phosphatase, or a fluorescent tag, or an electrochemiluminescent label or through an intermediary label such as biotin.

[0166] Common ELISA formats include the sandwich ELISA, so named because the analyte is between two different antibodies. The capture substrate in this format is a capture antibody, often a monoclonal antibody, to increase the specificity of the assay and reduce background noise. The analyte is bound to the capture antibody, then detected by binding to a detection antibody. A variation of sandwich ELISA assay, called Single-Molecule Assay (Simoa), uses beads are coated with a capture antibody; each bead is bound to either one or zero target molecule, and individual beads are detected with another antibody (detection antibody) and a labeling enzyme.

[0167] Other ELISA formats include indirect ELISA, where the capture substrate is the specific antigen that is being tested and the detection step is mediated by a primary antibody and an enzyme-conjugated secondary antibody which is reactive against the primary antibody. Thus, the primary antibody that recognizes the antigen is not labeled. In a direct ELISA the capture substrate is the specific antigen that is being tested, and the enzyme that catalyzes the color-change reaction is conjugated to the antigen detector antibody.

[0168] Immuno-PCR (I-PCR) is a technique that combines the sensitivity of the nucleic acid amplification by PCR with the specificity of the antibody-based assays resulting in an increase of the detection sensitivity.

[0169] Mass spectrometry (MS) is an instrumental technique for separation of electricallycharged species in the gas phase. The charged species (ions) are produced in the ion source.In some embodiments, the ion source assists the transfer of solid-phase or liquid-phase analytes into the gas phase. The gas-phase ions subsequently are transferred into the massanalyzer. The mass analyzer sorts the ions—in space or time—according to the mass-to- charge ratios (m / z). The separated ions are detected by an ion detector in the space or time domain. Electric signals, produced by the ion detector, are subsequently processed to produce mass spectra. In fact, mass spectra can be viewed as histograms, which provide information on the number of ions at different m / z values. The detected ions may correspond to the original molecules, their fragments or other species formed during the ionization process. MS enables direct identification of molecules based on the mass-to-charge ratio as well as fragmentation patterns. Commercial MS instruments, such as Thermo Scientific Orbitrap Astral Mass Spectrometer, permit quantitative MS measurements.

[0170] In some embodiments, quantitative MS measures fragments from a protease digest of a biological sample. In some embodiments, the protease digest is an elastase digest, thermolysin digest, pepsin digest, trypsin digest, or chymotrypsin digest. In some embodiments, the protease digest is a trypsin digest. In some embodiments, the trypsin digest comprises a LRRK2 fragment comprising IADYGIAQYCCR (SEQ ID NO: 7). In some embodiments, the LRRK2 fragment comprises a Cys-4HNE adduct.

[0171] Positron-emitting tomography (PET) tracers can also be prepared using an antibody or binding fragment thereof that specifically binds to a LRRK2-4HNE adduct, such as a LRRK2 Cys-4HNE adduct, e.g., a LRRK2 Cys2024-4HNE and / or a LRRK2 Cys2025-4HNE adduct. Antibody-based PET tracers have been reported for imaging in solid tumors (Manafi- Farid, R. et al. Frontiers in Medicine 2022, vol.9, Article 916693) and for detection of inflammation in chronic inflammatory diseases (Lee, H. J. et al. Chembiochem 2019, 20(4): 422-436). Antibody-based PET tracers (for Aβ, Tau, and α-Synuclein) have also been reported for imaging in brain for neurodegenerative diseases. In some embodiments, the PET tracer comprises124I,18F,68Ga,61 / 64Cu,89Zr,11C, or44Sc radiolabel.

[0172] In some embodiments, LRRK2 Cys-4HNE adduct specific PET tracers can be used to quantify levels of 4-HNE / LRRK2 adduct species (at amino acid residues Cys2024 and / or Cys2025) in various brain subregions in human subjects. Such a tool may be used as a diagnostic biomarker for PD, as a biomarker of 15-LO target engagement in the CNS, and as a predictive biomarker (e.g., a measure of how likely a subject is to benefit from a particular treatment, e.g., a 15-LO inhibitor).

[0173] Suitable anti-LRRK2-4HNE adduct antibodies or binding fragments thereof for use in a method of the present disclosure include polyclonal, monoclonal, genetically engineered,and / or other antibodies otherwise modified in nature, including but not limited to chimeric antibodies, humanized antibodies, human antibodies, primatized antibodies, single chain antibodies, etc. In some embodiments, the anti-LRRK2-4HNE adduct antibodies comprise all or a portion of a constant region of an antibody. In some embodiments, the constant region is an isotype selected from: IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3 or IgG4), and IgM. Examples of antibody binding fragments include by way of example and not limitation, Fab, Fab′, F(ab′)2, Fv fragments, single chain Fv fragments and single domain fragments. Methods for production of such antibodies or binding fragments thereof are known in the art.

[0174] An increased LRRK2-4HNE adduct level in a biological sample from the subject compared to control LRRK2-4HNE adduct level in a control sample can be any increased level, e.g., from about 1.1 to about 30 times higher LRRK2-4HNE adduct level in the biological sample compared to the control sample. In some embodiments, a LRRK2-4HNE adduct level in a biological sample from the subject is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or about 30 times higher, or any range bounded by any of the aforementioned numbers, than control LRRK2-4HNE adduct level in a control sample. In some embodiments, a LRRK2-4HNE adduct level in a biological sample from the subject is at least about three standard deviations higher than control LRRK2-4HNE adduct level in a control sample.

[0175] In some embodiments, the disease or condition characterized by increased LRRK2 activity and / or expression is a neurodegenerative disease. In some embodiments, the neurodegenerative disease is Parkinson's disease, Alzheimer's disease, cerebral amyloid angiopathy (CM), prion-mediated diseases, amyotrophic lateral sclerosis (ALS), Huntington's disease, Friedreich's ataxia (FRDA), or Leber's Hereditary Optic Neuropathy (LHON). In some embodiments, the neurodegenerative disease is Parkinson's disease.

[0176] In some embodiments, the disease or condition characterized by increased LRRK2 activity and / or expression is an immunological disease. LRRK2 has been associated with immune-related disorders. See, e.g., Russo, I. et al. “LRRK2 as a target for modulating immune system responses.” Neurobiology of Disease 2022, vol.169, 105724. In some embodiments, the disease or condition characterized by increased LRRK2 activity and / or expression is an inflammatory bowel disease (IBD), such as Crohn’s disease or ulcerative colitis, or leprosy.

[0177] In some embodiments, a method of selecting a subject described herein can be used in a method of treatment or use of the present invention described further herein.

[0178] In some embodiments of the methods described herein, the subject is any mammal, such as a mouse, a rat, a dog, a cat, including veterinary animals, such as a goat, a pig, a horse, a cow, or a donkey, and primates, such as non-human primates, e.g., a cynomolgous monkey, rhesus monkey, or chimpanzee, as well as humans. In some embodiments, the subject is a human. In some embodiments, a subject is a patient. C. Methods and / or Uses of Treatment with 15-LO Inhibitors

[0179] Without wishing to be bound by theory, the present method as illustrated in the experimental information in Example 3 is believed to be capable of reducing the increased LRRK2 activity and / or expression as a result of a disease or condition characterized by elevated LRRK2, but without eliminating LRRK2 basal activity present in normal, non- diseased cells. By doing so, the present method may be superior to treatment with direct LRRK2 kinase inhibitors, which can inhibit all LRRK2 kinase activity and have exhibited toxicity that may result from inhibiting critical LRRK2 basal activity. See, e.g., report of lung toxicity on chronic administration of GNE-7915: Miller, G. K. et al. “Effects of LRRK2 inhibitors in nonhuman primates,” Toxicologic Pathology 2023; 51(5): pages 232-245.

[0180] The compounds or compositions of the present disclosure are believed to be useful for inhibiting 15-LO in the treatment of a disease or condition characterized by increased LRRK2 activity and / or expression. Accordingly, in some embodiments, a method of treating a disease or condition characterized by increased LRRK2 activity and / or expression in a subject in need thereof comprises administering to the subject a therapeutically effective amount of a 15-LO inhibitor, e.g., a compound of the present disclosure, e.g., a compound of Formula I, Ia, II, III, or IV, or pharmaceutically acceptable salt thereof. In some embodiments, the 15-LO inhibitor is a compound of the present disclosure or pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure.

[0181] In some embodiments, treatment refers to an approach for obtaining beneficial or desired results. For purposes of the present disclosure, beneficial or desired results include, but are not limited to, alleviation of a symptom and / or diminishment of the extent of a symptom and / or preventing a worsening of a symptom associated with a disease or condition.In one embodiment, treatment or treating includes one or more of the following: a) inhibiting the disease or condition (e.g., decreasing one or more symptoms resulting from the disease or condition, and / or diminishing the extent of the disease or condition); b) slowing or arresting the development of one or more symptoms associated with the disease or condition (e.g., stabilizing the disease or condition, delaying the worsening or progression of the disease or condition); and c) relieving the disease or condition, e.g., causing the regression of clinical symptoms, ameliorating the disease state, delaying the progression of the disease, increasing the quality of life, and / or prolonging survival.

[0182] In some embodiments, the disease or condition characterized by increased LRRK2 activity and / or expression is a neurodegenerative disease. In some embodiments, the neurodegenerative disease is Parkinson's disease, Alzheimer's disease, cerebral amyloid angiopathy (CM), prion-mediated diseases, amyotrophic lateral sclerosis (ALS), Huntington's disease, Friedreich's ataxia (FRDA), or Leber's Hereditary Optic Neuropathy (LHON). In some embodiments, the neurodegenerative disease is Parkinson's disease.

[0183] Parkinson's disease (PD) is a neurodegenerative disease resulting from the progressive loss of dopamine-producing neurons and which affects up to 4% of the population over age 80. PD is characterized by both motor symptoms, such as tremor at rest, rigidity, akinesia and postural instability as well as non-motor symptoms such as impairment of cognition, sleep and sense of smell. Studies have linked LRRK2 to PD and many patients with point mutations in LRRK2 present symptoms that are indistinguishable from those with idiopathic PD. Over 20 LRRK2 mutations have been associated with autosomal-dominant Parkinsonism. R1441C, R1441G, R1441H, Y1699C, G2019S, I2020T and N1437H missense mutations are considered to be pathogenic. The LRRK2 R1441G mutation has been shown to increase the release of proinflammatory cytokines (higher levels of TNF-a, IL- 1 β, IL-12 and lower levels of IL-10) in microglial cells from transgenic mice and thus may result in direct toxicity to neurons (Gillardon, F. et al. Neuroscience 2012, 208, 41 - 48). In a murine model of neuroinflammation, induction of LRRK2 in microglia was observed and inhibition of LRRK2 kinase activity with small molecule LRRK2 inhibitors (LRRK2-IN-1 or sunitinib) or LRRK2 knockout resulted in attenuation of TNF-a secretion and nitric oxide synthase (iNOS) induction (Moehle, M. et al. J. Neurosci.2012, 32(5), 1602-1611 ). The most common of the LRRK2 mutations, G2019S, is present in more than 85% of PD patients carrying LRRK2 mutations. This mutation, which is present in the LRRK2 kinase domain, leads to an enhancement of LRRK2 kinase activity. In the human brain LRRK2 expression ishighest in the same regions of the brain that are impacted by PD, and LRRK2 is found in Lewy Bodies, a hallmark of PD. Recent studies indicate that a potent, selective, brain- penetrant kinase inhibitor for LRRK2 could be a therapeutic treatment for PD.

[0184] Dementia results from a wide variety of distinctive pathological processes. The most common pathological processes causing dementia are Alzheimer’s disease (AD), cerebral amyloid angiopathy (CM) and prion-mediated diseases (see, e.g., Haan et al., Clin. Neurol. Neurosurg.1990, 92(4):305-310; Glenner et al., J. Neurol. Sci.1989, 94: 1 -28). AD is a progressive, neurodegenerative disorder characterized by memory impairment and cognitive dysfunction. AD affects nearly half of all people past the age of 85, the most rapidly growing portion of the United States population. As such, the number of AD patients in the United States is expected to increase from about 4 million to about 14 million by 2050. LRRK2 mutations have been associated with AD-like pathology, which suggests that there may be a partial overlap between the neurodegenerative pathways in both AD and PD (Zimprach, A. et al. Neuron 2004, 44, 601-607). In addition, the LRRK2 R1628P variant (COR domain) has been associated with an increased incidence of AD in a certain population, perhaps resulting from increased apoptosis and cell death (Zhao, Y. et al. ; Neurobiology of Aging 2011, 32, 1990-1993).

[0185] Friedreich's ataxia is an autosomal recessive neurodegenerative and cardiodegenerative disorder caused by decreased levels of the protein Frataxin. The disease causes the progressive loss of voluntary motor coordination (ataxia) and cardiac complications. Symptoms typically begin in childhood, and the disease progressively worsens as the patient grows older; patients eventually become wheelchair-bound due to motor disabilities.

[0186] Leber's Hereditary Optic Neuropathy (LHON) is a disease characterized by blindness which occurs on average between 27 and 34 years of age. Other symptoms may also occur, such as cardiac abnormalities and neurological complications.

[0187] Amyotrophic lateral sclerosis (ALS) is caused by selective degeneration of motor neurons in the brain and spinal cord, which may be mediated by mitochondrial dysfunction. Studies suggest that ferroptosis mediates selective motor neuron death in amyotrophic lateral sclerosis. See, Wang, T. et al. Cell Death Differ 29, 1187–1198 (2022).

[0188] In some embodiments, the disease or condition characterized by increased LRRK2 activity and / or expression is an immunological disease. LRRK2 has been associated withimmune-related disorders. See, e.g., Russo, I. et al. “LRRK2 as a target for modulating immune system responses.” Neurobiology of Disease 2022, vol.169, 105724. In some embodiments, the disease or condition characterized by increased LRRK2 activity and / or expression is an inflammatory bowel disease (IBD), such as Crohn’s disease or ulcerative colitis, or leprosy.

[0189] As understood in the art, treatment or treating refers to an approach for obtaining beneficial or desired results. For purposes of the present disclosure, beneficial or desired results include, but are not limited to, alleviation of a symptom and / or diminishment of the extent of a symptom and / or preventing a worsening of a symptom associated with a disease or condition. In one embodiment, treatment or treating includes one or more of the following: a) inhibiting the disease or condition (e.g., decreasing one or more symptoms resulting from the disease or condition, and / or diminishing the extent of the disease or condition); b) slowing or arresting the development of one or more symptoms associated with the disease or condition (e.g., stabilizing the disease or condition, delaying the worsening or progression of the disease or condition); and c) relieving the disease or condition, e.g., causing the regression of clinical symptoms, ameliorating the disease state, delaying the progression of the disease, increasing the quality of life, and / or prolonging survival.

[0190] In some embodiments, a subject is a mammal, such as a mouse, a rat, a dog, a cat, including veterinary animals, such as a goat, a pig, a horse, a cow, or a donkey, and primates, such as non-human primates, e.g., a cynomolgous monkey, rhesus monkey, or chimpanzee, as well as humans. In some embodiments, the subject is a human. In some embodiments, a subject is a patient.

[0191] In some embodiments, a method of the present invention is a method of treating a subject suffering from or suspected of suffering from a disease or condition characterized by increased LRRK2 activity and / or expression, comprising administering a therapeutically effective amount of a 15-LO inhibitor if an increased LRRK2-4HNE adduct level was measured in a biological sample from the subject compared to control LRRK2-4HNE adduct level in a control sample.

[0192] Compounds of the disclosure or pharmaceutically acceptable salts thereof are also useful for methods of aiding the treatment of a disease or condition characterized by increased LRRK2 level, such as Parkinson’s disease. As used herein, a “method of aiding” generally refers to methods of assisting in performing or practicing a method disclosedherein, for example, methods of assisting in (i) performing, (ii) practicing, and / or (iii) making a determination concerning the detection, classification, treatment regiment, or nature, of a mitochondrial disease (e.g., Parkinson’s disease), a disease, a disorder, and / or a health condition.

[0193] Accordingly, in some embodiments, a method of aiding in the treatment of disease or condition characterized by increased LRRK2 activity and / or expression in a subject in need thereof comprises administering to the subject a therapeutically effective amount of a compound of the present disclosure or pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure. In some embodiments, the method of aiding in the treatment comprises administering a pharmaceutical composition of the compound of the present disclosure as described in Section IV above.

[0194] In some embodiments, a use of the present disclosure comprises a compound of the present disclosure or pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure for the manufacture of a medicament for aiding in the treatment of a disease or condition characterized by increased LRRK2 activity and / or expression in a subject in need thereof. In some embodiments, the use comprises a pharmaceutical composition of the compound of the present disclosure as described in Section IV above.

[0195] In some embodiments, a compound of the present disclosure or pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure is for use in aiding in the treatment of a disease or condition characterized by increased LRRK2 activity and / or expression in a subject in need thereof. In some embodiments, the compound for use comprises a pharmaceutical composition of the compound of the present disclosure as described in Section IV above.

[0196] In some embodiments, a use of the present disclosure comprises a compound of the present disclosure or pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure for the manufacture of a medicament for treating a disease or condition characterized by increased LRRK2 activity and / or expression in a subject in need thereof. In some embodiments, the use comprises a pharmaceutical composition of the compound of the present disclosure as described in Section IV above.

[0197] In some embodiments, a compound of the present disclosure or pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure is for use intreating a disease or condition characterized by increased LRRK2 activity and / or expression in a subject in need thereof. In some embodiments, the compound for use comprises a pharmaceutical composition of the compound of the present disclosure as described in Section IV above.

[0198] Kits that comprise a compound of the present disclosure, or pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing any of the above, are also included in the present disclosure. In some embodiments, a kit further includes instructions for use. In some embodiments, a kit includes a compound of the disclosure, or a pharmaceutically acceptable salt thereof, and a label and / or instructions for use of the compounds in the treatment of the indications, such as the diseases or conditions, described herein. In some embodiments, kits comprising a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, in combination with one or more (e.g., one, two, three, four, one or two, or one to three, or one to four) additional therapeutic agents are provided.

[0199] Provided herein are also articles of manufacture that include a compound of the present disclosure or a pharmaceutically acceptable salt thereof in a suitable container. The container may be a vial, jar, ampoule, preloaded syringe, and intravenous bag.VI. EXAMPLES

[0200] The following examples are provided to further aid in understanding the embodiments disclosed in the application, and presuppose an understanding of conventional methods well known to those persons having ordinary skill in the art to which the examples pertain. The particular materials and conditions described hereunder are intended to exemplify particular aspects of embodiments disclosed herein and should not be construed to limit the reasonable scope thereof.

[0201] Many general references providing commonly known chemical synthetic schemes and conditions useful for synthesizing the disclosed compounds are available (see, e.g., Smith, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 7thedition, Wiley-Interscience, 2013.)

[0202] Compounds as described herein can be purified by any of the means known in the art, including chromatographic means, such as high performance liquid chromatography (HPLC), preparative thin layer chromatography, flash column chromatography and ionexchange chromatography. Any suitable stationary phase can be used, including normal and reversed phases as well as ionic resins. For example, disclosed compounds can be purified via silica gel chromatography. See, e.g., Introduction to Modern Liquid Chromatography, 2nd ed., ed. L. R. Snyder and J. J. Kirkland, John Wiley and Sons, 1979; and Thin Layer Chromatography, E. Stahl (ed.), Springer-Verlag, New York, 1969.

[0203] Compounds were characterized using standard instrumentation methods. Identification of the compound was carried out by hydrogen nuclear magnetic resonance spectrum (1H-NMR) and mass spectrum (MS).1H-NMR was measured at 400 MHz, unless otherwise specified. In some cases, exchangeable hydrogen could not be clearly observed depending on the compound and measurement conditions. The designation br. or broad, used herein, refers to a broad signal. HPLC preparative chromatography was carried out by a commercially available ODS column in a gradient mode using water / methanol (containing formic acid) as eluents, unless otherwise specified.

[0204] The Examples provided herein describe the synthesis of compounds disclosed herein as well as intermediates used to prepare the compounds. It is to be understood that individual steps described herein may be combined. It is also to be understood that separate batches of a compound may be combined and then carried forth in the next synthetic step.

[0205] Representative syntheses of compounds of the present disclosure are described in schemes below, and the particular examples that follow.

[0206] Abbreviations. Certain abbreviations and acronyms are used in describing the experimental details. Although most of these would be understood by one skilled in the art, contains a list of many of these abbreviations and acronyms. Table 4. List of abbreviations and acronyms. Abbreviation MeaningDIPEA N,N-diisopropylethylamine DMSO dimethylsulfoxide kExample 1. Compound Synthesis

[0207] HPLCMS Method A: The column was an Xbridge Shield RP18 2.1*50mm, (5 um particles). UV detection was by diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was 10 mM ammonium bicarbonate in water. Mobile phase B was HPLC grade acetonitrile. The gradient was 5-95% B in 2.05 min. The flow rate was 1.0 mL / min.

[0208] HPLCMS Method B: The column was a ZORBAX Eclipse XDB-C182.1*30 mm, (3.5 um particles). UV detection was by diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was 0.037% trifluoroacetic acid in water. Mobile phase B was 0.018% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 2.20 min. The flow rate was 1.0 mL / min.

[0209] Preparative HPLC Method: The column was a GromSil 80 Si NP-15µm, 250mm x4.6mm column. The mobile phase was heptane / ethanol with a gradient of 5%-95% ethanol over 15 min. Synthesis of Intermediate 8-ethyl-3,8-diazabicyclo[3.2.1]octane (INT-2)

[0210] To a mixture of INT-2A (20 g, 94.21 mmol) and K2CO3(26.04 g, 188.42 mmol) in acetonitrile (200 mL) was added ethyl iodide (14.69 g, 94.21 mmol) at 15°C. The mixture was stirred at 40°C for 12 hours under N2. LCMS indicated starting material was consumed, and desired product was detected. The reaction mixture was cooled to 15°C. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (ethyl acetate) to give INT-2B (14 g, 58.25 mmol, 62% yield) as a white solid. HPLCMS Method A (ESI+): m / z 241.2 (MH+), RT 0.765 min.

[0211] To a mixture of INT-2B (14 g, 58.25 mmol) in methanol (100 mL) was added HCl / MeOH (4 M, 87.50 mL) at 20°C. The mixture was stirred at 20°C for 12 hrs. The reaction mixture was concentrated under reduced pressure to give crude 8-ethyl-3,8-diazabicyclo[3.2.1]octane (INT-2) dihydrochloride (10 g, 46.92 mmol, 80% yield) as a yellowsolid. Synthesis of Intermediate 3-ethyl-3,8-diazabicyclo[3.2.1]octane (INT-3)

[0212] To a mixture of INT-3A (20 g, 94.21 mmol) and K2CO3(26.04 g, 188.42 mmol) in acetonitrile (200 mL) was added ethyl iodide (14.69 g, 94.21 mmol) at 15°C. The mixturewas stirred at 40°C for 12 hours under N2. LCMS indicated starting material was consumed, and desired product was detected. The reaction mixture was cooled to 15°C. The mixture was filtered, and the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography on silica gel (ethyl acetate) to give INT-3B (14 g, 58.25 mmol, 62% yield) as a white solid. HPLCMS Method A (ESI+): m / z 241.2 (MH+), RT 0.853 min.

[0213] To a mixture of INT-3B (14 g, 58.25 mmol) in methanol (100 mL) was added HCl / MeOH (4 M, 87.50 mL) at 20°C. The mixture was stirred at 20°C for 12 hrs. The reaction mixture was concentrated under reduced pressure to give crude 3-ethyl-3,8- diazabicyclo[3.2.1]octane (INT-3) dihydrochloride (10 g, 46.92 mmol, 80% yield) as a yellow solid. Synthesis of 7-((1R,5S)-3-ethyl-3,8-diazabicyclo[3.2.1]octan-8-yl)-3H-phenothiazin-3-one (Compound 1)

[0214] To aadded dropwise a mixture of iodine (38.21 g, 150 mmol) in CHCl3 (750 mL) at 5°C for 1 hr. The mixture was stirred at 5°C for 3 hours. The mixture was filtered, and the filter cake was washed with CHCl3 (200 mL). The filter cake was dried by high vacuum to give crude A2 (10 g) as a darkblue solid. 1H NMR (400 MHz, (CD3)2SO) δ 7.30-7.76 (m, 3H), 7.85-8.19 (m, 4H), 8.36-8.63 (m, 1H).

[0215] To a mixture of A2 (7.6 g, 23.37 mmol) and 3-ethyl-3,8-diazabicyclo[3.2.1]octane dihydrochloride (9.96 g, 46.75 mmol, 2 eq) in CHCl3 (450 mL) was added N-ethyl-N- isopropylpropan-2-amine (15.10 g, 116.86 mmol, 5 eq) at 20oC. The mixture was stirred at 20°C for 48 hrs. The mixture was concentrated under reduced pressure, and the residue was washed with methyl tert-butyl ether (100 mL) and dried by high vacuum to give crude A4 (9 g) as a blue solid. HPLCMS Method B (ESI+): m / z 474.3 (M+), RT 0.176 min.potassium hydroxide (8 N, 150 mL) at 20°C. The mixture was stirred at 70°C for 2 hrs. The mixture was cooled to 15°C and concentrated under reduced pressure, and the residue was extracted with ethyl acetate (300 mL). The organic layer was concentrated under reduced pressure, and the crude product was purified by column chromatography on silica gel (ethyl acetate: methanol = 1: 1) three times, and then purified twice by normal-phase preparative HPLC to give after lyophilization Compound 1 (55 mg) as a brown solid.1H NMR (400MHz, CDCl3) δ 1.02 (t, J=7.28 Hz, 3H), 1.98-2.03 (m, 2H), 2.07-2.15 (m, 2H), 2.30-2.41 (m,4H), 2.72 (dd, J=10.79, 2.26 Hz, 2H), 4.34 (br d, J=2.01 Hz, 2H), 6.67 (dd, J=12.80, 2.76 Hz, 2H), 6.85 (ddd, J=11.54, 9.29, 2.26 Hz, 2H), 7.56 (d, J=10.04 Hz, 1H), 7.69 (d, J=9.03 Hz,1H). HPLCMS Method A (ESI+): m / z 352.1 (MH+), RT 2.621 min.Synthesis of 7-((1R,5S)-8-ethyl-3,8-diazabicyclo[3.2.1]octan-3-yl)-3H-phenothiazin-3-one (Compound 2)

[0217] octane dihydrochloride (9.96 g, 46.75 mmol, 2 eq) in CHCl3 (450 mL) was added N-ethyl-N- isopropylpropan-2-amine (15.10 g, 116.86 mmol, 5 eq) at 20oC. The mixture was stirred at 20 °C for 48 hours. The mixture was concentrated under reduced pressure, and the residue was washed with methyl tert-butyl ether (100 mL) and dried by high vacuum to give crude A3 (9 g) as a blue solid. HPLCMS Method B (ESI+): m / z 474.3 (M+), RT 0.461 min., . . The mixture was cooled to 15°C and concentrated under reduced pressure to remove most of the organic solvents. The residue was extracted with ethyl acetate (300 mL). The organic layer was concentrated under reduced pressure, and the crude product was purified by column chromatography on silica gel (from ethyl acetate to ethyl acetate: methanol = 1: 1) three times, and then purified twice by normal-phase preparative HPLC to give after lyophilization Compound 2 (60 mg) as a brown solid. 1H NMR (400MHz, CDCl3) δ 1.15 (t, J=7.28 Hz,3H), 1.70 (d, J=7.53 Hz, 2H), 2.00- , 2.45-2.55 (m, 2H), 3.26 (dd, J=11.04, 2.01Hz, 2H), 3.46 (br d, J=1.51 Hz, 2H), 3.49-3.55 (m, 2H), 6.68 (t, J=2.51 Hz, 2H), 6.84 (dd, J=9.79, 2.26 Hz, 1H), 6.92 (dd, J=9.03, 3.01 Hz, 1H), 7.55 (d, J=9.54 Hz, 1H), 7.70 (d, J=9.03 Hz, 1H). HPLCMS Method A (ESI+): m / z 352.1 (MH+), RT 2.352 min.

[0219] The following compounds were prepared by similar methods. ,, 1.70 (d, J=7.53 Hz, 2H), 2.00-2.07 (m, 2H), 2.60-2.70 (m, 1H), 3.26 (dd, J=11.04, 2.01 Hz, 2H), 3.46 (br d, J=1.51 Hz, 2H), 3.49-3.55 (m, 2H), 6.68 (t, J=2.51 Hz, 2H), 6.84(dd, J=9.79, 2.26 Hz, 1H), 6.92 (dd, J=9.03, 3.01 Hz, 1H), 7.55 (d, J=9.54 Hz, 1H), 7.70 (d, J=9.03 Hz, 1H). HPLCMS Method A (ESI+): m / z 366.1 (MH+), RT 2.492 min.(600 MHz, CD2Cl2) δ 7.66 (d, J = 9.2 Hz, 1H), 7.51 (d, J = 9.7 Hz, 1H), 6.93 (dd, J = 2.8, 9.2 Hz, 1H), 6.74 (dd, J =2.1, 9.8 Hz, 1H), 6.72 (d, J = 2.6 Hz, 1H), 6.61 (d, J = 2.2 Hz, 1H), 3.55 - 3.47 (m, 4H), 3.16 (br d, J = 10.6 Hz, 2H), 2.12 - 2.05 (m, 2H), 1.90 (br s, 1H), 1.71 (br d, J = 7.6 Hz, 2H), 0.52 - 0.43 (m, 4H) HPLCMS Method A (ESI+): m / z 364.1 (MH+), RT 2.411 min. Compound 101: tert-butyl 6,11-dioxo-1,2,4,5,6,11-hexahydro-3H-naphtho[2,3-d]azepine-3- carboxylate(4.13 g, 9.48 mmol, 1.5 eq) and naphthalene-1,4-dione (1 g, 6.32 mmol, 1 eq) in H2O (25 mL) and MeCN (50 mL) was added AgNO3(1.29 g, 7.59 mmol, 1.2 eq) at 20°C. The mixture was stirred at 100°C for 10 min. Then to the mixture a reaction mixture was added the solution of ammonium persulfate (5.77 g, 25.29 mmol, 5.50 mL, 4 eq) in H2O (10 mL) and MeCN (20 mL) dropwise over 30 min at 20°C. The mixture was stirred at 100°C for 3 hrs. LCMS showed the starting material was consumed, and desired mass was detected. Then the mixture was cooled to 25°C and adjust to pH=8 with NaHCO3. The mixture was diluted with THF (50 mL). Then Boc2O (2.76 g, 12.65 mmol, 2.91 mL, 2 eq) was added to the mixture at 25°C. Then the mixture was stirred for 3 hrs at 25°C. LCMS showed the reaction worked well. The mixture was diluted with water (100 mL), the mixture was extracted with EtOAc (80 mL*3). The combined organic phase was washed with brine (50mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. Theresidue was purified by flash column on silica gel (petroleum ether: ethyl acetate=93:7) and the eluent was concentrated under reduced pressure to give Compound 1 (335 mg, 992.60 μmol, 15.70% yield, 97% purity) obtained as light-yellow solid:1H NMR (400 MHz, CHLOROFORM-d) δ 8.15 - 8.05 (m, 2H), 7.77 - 7.68 (m, 2H), 3.60 (br d, J = 4.8 Hz, 4H), 3.09 - 2.98 (m, 4H), 1.48 (s, 9H); Method A LCMS (ESI+): 2.881 min, m / z 350 (M+Na). Compound 102: 2,3,4,5-tetrahydro-1H-naphtho[2,3-d]azepine-6,11-dione

[0224] To(5 mL) was added HCl / EtOAc (4 M, 12.88 mL, 53.67 eq) at 25°C. The mixture was stirred at 25 °C for 8 hrs. LCMS showed the starting material was consumed, and one major peak with desired Mass was detected. The reaction mixture was concentrated under reduced pressure. The residue was triturated with ethyl acetate (5 mL) and filtered. The cake was washed with ethyl acetate (5 mL). The cake was dried by high vacuum to give Compound 102 (205 mg, 777.34 μmol, 80.97% yield, HCl salt) was obtained as light-yellow solid:1H NMR (400 MHz, DMSO-d6) δ 9.26 (br s, 2H), 8.12 - 7.94 (m, 2H), 7.92 - 7.77 (m, 2H), 3.22 (br s, 4H), 3.17 - 3.08 (m, 4H). Compound 103: 3-isopropyl-2,3,4,5-tetrahydro-1H-naphtho[2,3-d]azepine-6,11-dione

[0225] To a solution of Compound 102 (200 mg, 697.71 μmol, 1 eq, HCl) in IPA (10 mL) was added acetone (202.61 mg, 3.49 mmol, 256.47 μL, 5 eq), AcOH (41.90 mg, 697.71 μmol, 39.94 μL, 1 eq) and NaOAc (171.70 mg, 2.09 mmol, 3 eq) at 25°C. Then the mixture was stirred for 1 hr at 40°C. Then NaBH3CN (131.53 mg, 2.09 mmol, 3 eq) was added to the mixture at 25°C. The mixture was stirred for 12 hrs at 40°C. LCMS showed the reactionworked well. The mixture was added saturated Na2CO3(aqueous) to pH = 8, then the mixture was extracted with ethyl acetate (3 x 20 mL). The combined organic phases were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (neutral condition, column: Waters Xbridge BEH C18100*30 mm*10 um; mobile phase: [H2O (10 mM NH4HCO3)-MeCN]; gradient: 25%-55% B over 8.0 min) and lyophilized to give Compound 103 (64 mg, 225.50 μmol, 32.32% yield, 94.9% purity) as light purple solid:1H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.01 - 8.23 (m, 2 H), 7.58 - 7.82 (m, 2 H), 2.86 - 3.16 (m, 5 H), 2.66 (br s, 4 H), 1.05 (br d, J=6.50 Hz, 6 H); Method A LCMS (ESI+): 1.687 min, m / z 268 (M+H). Compound 104: 3-cyclopentyl-2,3,4,5-tetrahydro-1H-naphtho[2,3-d]azepine-6,11-dione(20 mL) was added cyclopentanone (293.44 mg, 3.49 mmol, 308.89 μL, 5 eq), AcOH (41.90 mg, 697.71 μmol, 39.94 μL, 1 eq) and NaOAc (171.70 mg, 2.09 mmol, 3 eq) at 25°C. The mixture was stirred for 1 hr at 40°C. Then NaBH3CN (131.53 mg, 2.09 mmol, 3 eq) was added to the mixture at 25°C. The mixture was stirred for 2 hrs at 40°C. LCMS showed the reaction worked well. The mixture was added saturated Na2CO3 (aqueous) to pH = 8. Then the mixture was extracted with ethyl acetate (3 x 10 mL). The combined organic phases were washed with brine (10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. One additional vial in 200 mg scale was set up as described above, the residue was combined. The crude product was triturated with MeOH 5 mL, filtered. The filter cake was dried in high vacuo to give Compound 104 (86 mg, 287.66 μmol, 20.61% yield, 98.8% purity) as light brown solid:1H NMR (400 MHz, DMSO-d6) δ ppm 7.97 - 8.05 (m, 2 H), 7.84 (dd, J=5.69, 3.31 Hz, 2 H), 2.86 - 2.92 (m, 4 H), 2.82 (br d, J=8.38 Hz, 1 H), 2.57 - 2.64 (m, 4 H), 1.71 - 1.83 (m, 2 H), 1.55 - 1.66 (m, 2 H), 1.48 (br dd, J=7.44, 4.82 Hz, 2 H), 1.29 - 1.41 (m, 2 H); Method A LCMS (ESI+): 1.832 min, m / z 296 (M+H).Compound 105: 3-(pentan-3-yl)-2,3,4,5-tetrahydro-1H-naphtho[2,3-d]azepine-6,11-dione105 was prepared according to the general procedures described in other Examples in the application. Compound 106: 3-phenethyl-2,3,4,5-tetrahydro-1H-naphtho[2,3-d]azepine-6,11-dionea mg, eq, phenylacetaldehyde (419.14 mg, 3.49 mmol, 272.17 μL, 5 eq) in MeOH (20 mL) was added NaOAc (171.70 mg, 2.09 mmol, 3 eq) and AcOH (41.90 mg, 697.71 μmol, 39.94 μL, 1 eq) at 25°C. Then the mixture was stirred for 1 hr at 25°C. Then NaBH3CN (131.53 mg, 2.09 mmol, 3 eq) was added in portions to the mixture at 25°C. The mixture was stirred for 2 hrs at 25°C. One additional vial in 20 mg scale was set up as described above and these two reactions were combined. The combined mixture was added saturated Na2CO3(aqueous) to pH = 8. Then the mixture was extracted with ethyl acetate (3 x 30 mL). The combined organic phases were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 4 g SepaFlash® Silica Flash Column, Eluent of 0~20% Ethyl acetate in Petroleum ether gradient @ 60 mL / min) to give crude product. Then the crude product was triturated with MeOH (2 mL) and then filtered. The filter cake was dried in high vacuum to give Compound 106 (48 mg, 141.94 μmol, 18.49% yield, 98.0% purity) as a light brown solid:1H NMR (400 MHz, DMSO-d6) δ ppm 7.98 - 8.06 (m, 2 H), 7.84 (dd, J=5.69, 3.31 Hz, 2 H), 7.20 - 7.29 (m, 4 H), 7.14 - 7.20 (m, 1 H), 2.87 - 2.94 (m, 4H), 2.72 - 2.78 (m, 2 H), 2.62 - 2.71 (m, 6 H); Method A LCMS (ESI+): 2.042 min, m / z 332 (M+H). Compound 107: 3-(3,3,3-trifluoropropyl)-2,3,4,5-tetrahydro-1H-naphtho[2,3-d]azepine-6,11- dionein DMF (10 mL) was added K2CO3(534.54 mg, 3.87 mmol, 4 eq) at 25°C. The mixture was stirred for 0.5 hr at 25°C. Then 1,1,1-trifluoro-3-iodo-propane (649.68 mg, 2.90 mmol, 339.97 μL, 3 eq) was added to the mixture at 25°C and stirred for 8 hrs at 50°C. LCMS showed the reaction worked and desired mass was detected. One additional vial in 50 mg scale was set up as described above. The mixture was combined and diluted with water (20 mL) and extracted with ethyl acetate (3 x 8 mL). The combined organic phases were washed with brine (3 x 10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (neutral condition, column: Waters Xbridge Prep OBD C18150*40 mm*10 um; mobile phase: [H2O (10 mM NH4HCO3)- MeCN]; gradient: 45%-75% B over 8.0 min) and lyophilization to give crude product. The crude product was triturated with methanol (1 mL), filtered and the filter cake was dried in high vacuum to give Compound 107 (23 mg, 68.15 μmol, 6.59% yield, 95.8% purity) as a deep-purple solid:1H NMR (400 MHz, METHANOL-d4) δ ppm 8.08 (dd, J=5.69, 3.31 Hz, 2 H), 7.77 (dd, J=5.69, 3.31 Hz, 2 H), 2.95 - 3.10 (m, 4 H), 2.74 - 2.85 (m, 2 H), 2.65 - 2.74 (m, 4 H), 2.33 - 2.53 (m, 2 H); Method A LCMS (ESI+): 1.815 min, m / z 324 (M+H). Compound 108: 3-(4-(trifluoromethyl)benzoyl)-2,3,4,5-tetrahydro-1H-naphtho[2,3- d]azepine-6,11-dione[0230 . , . , , 10 mL) was added 4-(trifluoromethyl)benzoic acid (216.27 mg, 1.14 mmol, 1.5 eq), NMI (186.80 mg, 2.28 mmol, 181.36 μL, 3 eq) and TCFH (425.57 mg, 1.52 mmol, 2 eq) at 25°C in order. The mixture was stirred at 25°C for 12 hours. The mixture was diluted with ethyl acetate (10 mL) and water (10 mL). The mixture was extracted with ethyl acetate (3 x 10 mL). The organic layer was dried with Na2SO4, filtered and concentrated under reduced pressure to give the crude product. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=92%) and then triturated with ethyl acetate (2 mL). The solid was dried in high vacuum to give Compound 108 (101 mg, 247.59 μmol, 32.65% yield, 97.9% purity) as yellow solid:1H NMR (DMSO-d6, 400 MHz) δ (ppm) 8.02 (br dd, J = 10.8, 4.6 Hz, 1H), 7.78-7.90 (m, 2H), 7.65 (d, J = 8.0 Hz, 1H), 3.82 (br s, 1H), 3.46 (br s, 1H), 3.10 (br s, 1H), 2.94 (br s, 1H); Method A LCMS (ESI+): 2.832 min, m / z 400 (M+H). Compound 109: 1,2,4,5-tetrahydronaphtho[2,3-d]oxepine-6,11-dione

[0231] To a solution of naphthalene-1,4-dione (400 mg, 2.53 mmol, 1 eq) and 3,3'-oxydipropionic acid (410.08 mg, 2.53 mmol, 1 eq) in acetonitrile (40 mL) and H2O (20 mL) was added AgNO3 (515.57 mg, 3.04 mmol, 1.2 eq) in portions at 25°C under N2. The mixture was stirred at 100°C under N2for 30 minutes. Then the mixture was cooled to 25°C and to a reaction mixture was added the solution of ammonium persulfate (1.85 g, 8.09 mmol, 1.76 mL, 3.2 eq) in acetonitrile (40 mL) and H2O (20 mL) dropwise at 25°C under N2. The mixture was stirred at 100°C under N2 for 3 hours. The mixture was cooled to 25°C.The mixture was quenched with aqueous of saturated sodium sulfite (300 mL). The aqueous phase was extracted with ethyl acetate (3 x 300 mL). The combined organic phase was dried with anhydrous Na2SO4, filtered and concentrated in vacuum to give the residue. The residue was purified by prep-HPLC (NH4HCO3condition; Column: Waters Xbridge BEH C18 100*30mm*10um;mobile phase: [H2O(10mM NH4HCO3)-ACN];gradient:24%-56% B over 8.0 min) and the desired eluent was lyophilized to give Compound 109 (45.6 mg, 177.61 μmol, 7.02% yield, 88.9% purity) as light brown solid:1H NMR (400 MHz, DMSO-d6) δ 2.96-3.03 (m, 4H), 3.65-3.71 (m, 4H), 7.81-7.88 (m, 2H), 7.98-8.06 (m, 2H); Method B LCMS (ESI-): 2.798 min. Compound 110: 3-(pyrimidin-4-yl)-2,3,4,5-tetrahydro-1H-naphtho[2,3-d]azepine-6,11-dione

[0232] To a solution of Compound 102 (250 mg, 853.18 μmol, 1 eq, HCl salt) and 4- chloropyrimidine (195.43 mg, 1.71 mmol, 2 eq) in THF (6 mL) was added TEA (259.00 mg, 2.56 mmol, 356.26 μL, 3 eq) in one portion at 25°C under N2. The mixture was stirred at 60°C for 12 hours under N2. One additional vial in 100 mg scale was set up as described above and these two reactions were combined. The mixture was diluted with H2O (50 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic layers were washed with brine (60 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (neutral condition, column: Waters Xbridge Prep OBD C18150*40mm*10um; mobile phase: [H2O (10mM NH4HCO3)-ACN]; gradient:30%-60% B over 8.0 min) and the desired eluent was lyophilized to give Compound 110 (34 mg, 110.24 μmol, 9.23% yield, 99% purity) as a light yellow solid:1H NMR (400 MHz, DMSO-d6) δ ppm 2.07 (s, 1 H) 2.96 - 3.10 (m, 4 H) 3.89 (br s, 4 H) 6.85 (dd, J=6.32, 1.19 Hz, 1 H) 7.78 - 7.89 (m, 2 H) 7.97 - 8.05 (m, 2 H) 8.18 (d, J=6.13 Hz, 1 H) 8.51 (s, 1 H); Method A LCMS (ESI+): 1.896 min, m / z 306 (M+H). Compound 111: 3-(pyrimidin-2-yl)-2,3,4,5-tetrahydro-1H-naphtho[2,3-d]azepine-6,11-dione

[0233] To a solution of Compound 102 (250 mg, 853.18 μmol, 1 eq, HCl salt) and 4- chloropyrimidine (195.43 mg, 1.71 mmol, 2 eq) in THF (6 mL) was added TEA (259.00 mg, 2.56 mmol, 356.26 μL, 3 eq) in one portion at 25°C under N2. The mixture was stirred at 60°C for 12 hours under N2. One additional vial in 100 mg scale was set up as described above and these two reactions were combined. The mixture was diluted with H2O (50 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic layers were washed with brine (60 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (neutral condition, column: Waters Xbridge Prep OBD C18150*40mm*10um; mobile phase: [H2O (10mM NH4HCO3)-ACN]; gradient:30%-60% B over 8.0 min) and the desired eluent was lyophilized to give Compound 111 (16 mg, 50.78 μmol, 4.25% yield, 96.9% purity) as a purple solid:1H NMR (400 MHz, DMSO-d6) δ 2.95 - 3.07 (m, 4 H) 3.91 - 4.05 (m, 4 H) 6.61 (t, J=4.75 Hz, 1 H) 7.84 (dd, J=5.57, 3.31 Hz, 2 H) 8.01 (dd, J=5.75, 3.25 Hz, 2 H) 8.39 (d, J=4.75 Hz, 2 H); Method A LCMS (ESI+): 2.404 min, m / z 306 (M+H). Compound 112: 6,7,9,10-tetrahydro-5H-cyclohepta[b]naphthalene-5,8,11-trione

[0234] To a solution of naphthalene-1,4-dione (2 g, 12.65 mmol, 1 eq) and 4- oxoheptanedioic acid (2.20 g, 12.65 mmol, 1 eq) in acetonitrile (200 mL) and H2O (100 mL) was added AgNO3 (2.58 g, 15.18 mmol, 1.2 eq) in portions at 25°C under N2. The reaction was stirred at 100°C for 30 minutes. Then the mixture was cooled to 25°C and to a reaction mixture was added the solution of ammonium persulfate (9.23 g, 40.47 mmol, 8.79 mL, 3.2eq) in acetonitrile (200 mL) and H2O (100 mL) dropwise at 25°C under N2. The mixture was stirred at 100°C under N2 for 3 hours. The mixture was cooled to 25°C. The mixture was quenched with aqueous of saturated sodium sulfite (500 mL). The aqueous phase was extracted with ethyl acetate (3 x 200 mL). The combined organic phase was dried with anhydrous Na2SO4, filtered and concentrated in vacuum to give the residue. The residue was purified by prep-HPLC (NH4HCO3 condition; column: Welch Xtimate C18 250*70mm#10um;mobile phase: [H2O(10mM NH4HCO3)-ACN];gradient:30%-60% B over 17.0 min) and the desired eluent was lyophilized to give the Compound 112 (211.6 mg, 830.53 μmol, 6.57% yield, 94.3% purity) as light yellow solid:1H NMR (400 MHz, DMSO- d6) δ 2.55-2.60 (m, 4H), 2.94 (br d, J=5.00 Hz, 4H), 7.86 (br dd, J=5.25, 3.25 Hz, 2H), 8.04 (dd, J=5.57, 3.31 Hz, 2H); Method B LCMS (ESI-): 2.599 min, m / z 239 (M-1). Compound 113: 8-(isopropylamino)-7,8,9,10-tetrahydro-5H-cyclohepta[b]naphthalene- 5,11(6H)-dione

[0235] To a mixture of Compound 112 (60 mg, 224.76 μmol, 1 eq, purity 90%) and propan-2-amine (39.86 mg, 674.29 μmol, 57.93 μL, 3 eq) in dichloromethane (1 mL) was added AcOH (13.50 mg, 224.76 μmol, 12.87 μL, 1 eq) dropwise at 25°C. Then the reaction was stirred at 25°C for 30 minutes. The sodium triacetoxyboranuide (119.09 mg, 561.91 μmol, 2.5 eq) was added to the mixture at 25°C. The reaction mixture was stirred at 25°C for 1 hour. The mixture was concentrated under reduced pressure to give the residue. The residue was purified by prep-HPLC (HCl condition; column: Phenomenex luna C18 100*40mm*5 um;mobile phase: [H2O(0.04% HCl)-ACN];gradient:1%-35% B over 8.0 min) and the desired eluent was lyophilized to give Compound 113 (63.8 mg, 197.49 μmol, 67.59% yield, 99% purity, HCl salt) as yellow solid:1H NMR (400 MHz, DMSO-d6) δ 1.48 (q, J=11.42 Hz, 2H), 2.23-2.34 (m, 2H), 2.40 (br dd, J=14.76, 11.63 Hz, 2H), 2.98 (br dd, J=10.32, 6.32 Hz, 2H), 3.12-3.24 (m, 2H), 3.29 (br d, J=7.63 Hz, 2H), 3.47 (br d, J=1.00 Hz,1H), 7.21-7.40 (m, 5H), 7.80-7.90 (m, 2H), 7.97-8.07 (m, 2H), 9.02 (br s, 2H); Method A LCMS (ESI+): 1.896 min, m / z 284 (M+1). Compound 114: 8-hydroxy-7,8,9,10-tetrahydro-5H-cyclohepta[b]naphthalene-5,11(6H)- dione

[0236] To a mixture of Compound 112 (500 mg, 1.98 mmol, 1 eq, purity 95%) in methanol (10 mL) was added NaBH4 (149.60 mg, 3.95 mmol, 2 eq) in portions at 0°C. The mixture was stirred for 10 minutes at 25°C. The mixture was quenched with ice saturated aqueous of ammonium chloride (100 mL) and extracted with ethyl acetate (3 x 30 mL). The combined organic phases were washed with brine (30mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give Compound 114 (572 mg, 2.31 mmol, 97.53% yield, 98% purity) as yellow solid:1H NMR (400 MHz, DMSO-d6) δ 1.27-1.40 (m, 2H), 1.77-1.89 (m, 2H), 2.42 (br dd, J=14.26, 10.38 Hz, 2H), 3.07 (br dd, J=14.38, 9.26 Hz, 2H), 3.79 (td, J=8.13, 3.88 Hz, 1H), 4.76 (d, J=4.13 Hz, 1H), 7.75-7.89 (m, 2H), 7.93-8.07 (m, 2H); Method A LCMS (ESI+): 2.222 min, m / z 243 (M+1). Compound 115: 3-(2-(pyrrolidin-1-yl)pyrimidin-4-yl)-2,3,4,5-tetrahydro-1H-naphtho[2,3- d]azepine-6,11-dione

[0237] To a solution of Compound 102 (150 mg, 568.79 μmol, 1 eq, HCl) in HFIP (4 mL) was added 4-chloro-2-(pyrrolidin-1-yl)pyrimidine (164.92 mg, 853.18 μmol, 1.5 eq)dropwise at 25°C under N2. The mixture was stirred at 50°C for 12 hours under N2. Then the mixture was concentrated under reduced pressure to remove the solvent. The crude was purified by prep-HPLC (neutral condition, column: Waters Xbridge Prep OBD C18 150*40mm*10um;mobile phase: [H2O(10mM NH4HCO3)-ACN];gradient:40%-80% B over 8.0 min) and the desired eluent was lyophilized to give Compound 115 (17 mg, 39.30 μmol, 6.91% yield, 95% purity, HCl) as a yellow solid:1H NMR (400 MHz, DMSO-d6) δ 1.82 - 2.11 (m, 4 H) 2.99 - 3.15 (m, 4 H) 3.43 - 3.67 (m, 4 H) 3.81 - 4.21 (m, 4 H) 6.57 (d, J=7.50 Hz, 1 H) 7.78 - 7.91 (m, 3 H) 8.02 (dd, J=5.69, 3.31 Hz, 2 H) 12.14 (br d, J=1.75 Hz, 1 H); Method A LCMS (ESI+): 2.238 min, m / z 375 (M+H). Compound 116: 3-(4-(pyrrolidin-1-yl)pyrimidin-2-yl)-2,3,4,5-tetrahydro-1H-naphtho[2,3- d]azepine-6,11-dione

[0238] To a solution of Compound 102 (150 mg, 568.79 μmol, 1 eq, HCl) in HFIP (5 mL)was added 2-chloro-4-(pyrrolidin-1-yl)pyrimidine (164.92 mg, 853.18 μmol, 1.5 eq) in oneportion at 25°C under N2. The mixture was stirred at 50°C for 72 hours under N2. Then the mixture was concentrated under reduced pressure to remove the solvent. The crude was purified by prep-HPLC (neutral condition, column: Waters Xbridge Prep OBD C18 150*40mm*10um; mobile phase: [H2O (10mM NH4HCO3)-ACN]; gradient:15%-75% B over 8.0 min) and the desired eluent was lyophilized to give Compound 116 (25 mg, 62.89 μmol, 11.06% yield, 94.29% purity) as an orange solid:1H NMR (400 MHz, DMSO-d6) δ 1.91 (br s, 4 H) 2.91 - 3.04 (m, 4 H) 3.32 - 3.62 (m, 4 H) 3.84 - 3.99 (m, 4 H) 5.72 (d, J=5.75 Hz, 1 H) 7.78 - 7.88 (m, 3 H) 7.96 - 8.04 (m, 2 H); Method A LCMS (ESI+): 2.244 min, m / z 375 (M+H). Compound 117: 8-(4-isopropylpiperazin-1-yl)-7,8,9,10-tetrahydro-5H- cyclohepta[b]naphthalene-5,11(6H)-dione[02 o a u e o o pou g, . μ o , eq, pu y a 1- isopropylpiperazine (115.27 mg, 899.05 μmol, 128.65 μL, 3 eq) in dichloromethane (1 mL) was added AcOH (18.00 mg, 299.68 μmol, 17.16 μL, 1 eq) dropwise at 25°C. Then the reaction was stirred at 25°C for 30 minutes. Then sodium triacetoxyboranuide (158.79 mg, 749.21 μmol, 2.5 eq) was added to the mixture at 25°C. The reaction mixture was stirred at 25°C for 1 hour. The mixture was concentrated under reduced pressure to give the residue. The residue was purified by prep-HPLC (HCl condition; column: Phenomenex luna C18 100*40mm*5 um;mobile phase: [H2O(0.04% HCl)-ACN];gradient:1%-30% B over 8.0 min) and the desired eluent was lyophilized to give Compound 117 (78.4 mg, 201.58 μmol, 61.15% yield, 100% purity, HCl) as off-white solid:1H NMR (400 MHz, METHANOL-d4) δ 1.43 (d, J=6.63 Hz, 6H), 1.64 (q, J=11.92 Hz, 2H), 2.33-2.49 (m, 4H), 3.56-3.87 (m, 12H), 7.76-7.83 (m, 2H), 8.06-8.13 (m, 2H); Method A LCMS (ESI+): 1.749 min, m / z 353 (M+1). Compound 118: 3-(methylsulfonyl)-2,3,4,5-tetrahydro-1H-naphtho[2,3-d]azepine-6,11-dione

[0240] To a solution of Compound 102 (30 mg, 113.76 μmol, 1 eq, HCl) in DCM (3 mL) was added methoxysulfinyl methyl sulfite (29.72 mg, 170.64 μmol, 1.5 eq) and TEA (23.02 mg, 227.51 μmol, 31.67 μL, 2 eq) at 0°C. The mixture was stirred at 25°C for 2 hours. The mixture was diluted with water (10 mL). The aqueous phase was extracted with dichloromethane (3 x 10 mL). The organic layer was washed with HCl solution (1 M, 10 mL), dried with Na2SO4, filtered and concentrated under reduced pressure to give the crude product. The crude product was triturated with methyl alcohol (5 mL) at 25oC for 10 min and then filtered. The solid was dried in high vacuum to give Compound 118 (6.5 mg, 20.84 μmol, 18.32% yield, 97.9% purity) as yellow solid: 1H NMR (DMSO-d6, 400 MHz) δ (ppm)8.03 (dd, J = 5.7, 3.3 Hz, 2H), 7.82-7.91 (m, 2H), 3.37-3.44 (m, 4H), 2.98-3.05 (m, 4H), 2.90 (s, 3H); Method A LCMS (ESI+): 2.359 min, m / z 306(M+H). Compound 119: 8-(pyrrolidin-1-yl)-7,8,9,10-tetrahydro-5H-cyclohepta[b]naphthalene- 5,11(6H)-dionemg, 899.05 μmol, 75.05 μL, 3 eq, purity 90%) in dichloromethane (2 mL) was added AcOH (18.00 mg, 299.68 μmol, 17.16 μL, 1 eq) dropwise at 25°C. Then the reaction was stirred at 25°C for 30 minutes. Then sodium triacetoxyboranuide (158.79 mg, 749.21 μmol, 2.5 eq) was added to the mixture at 25°C. The reaction mixture was stirred at 25°C for 1 hour. The mixture was concentrated under reduced pressure to give the residue. The residue was purified by prep-HPLC (HCl condition; column: Phenomenex luna C18100*40mm*5 um;mobile phase: [H2O(0.04% HCl)-ACN];gradient:1%-30% B over 8.0 min) and the desired eluent was lyophilized to give Compound 119 (45.7 mg, 137.31 μmol, 45.82% yield, 99.7% purity, HCl) as brown solid:1H NMR (400 MHz, DMSO-d6) δ 1.56 (q, J=11.51 Hz, 2H), 1.80-2.00 (m, 4H), 2.29 (br t, J=8.94 Hz, 2H), 2.40 (br dd, J=14.95, 11.44 Hz, 2H), 3.08 (br dd, J=10.69, 7.19 Hz, 2H), 3.29 (br s, 1H), 3.33-3.37 (m, 1H), 3.43 (br dd, J=10.07, 4.69 Hz, 2H), 3.48-3.58 (m, 1H), 7.80-7.90 (m, 2H), 7.97-8.07 (m, 2H), 10.80 (br d, J=1.75 Hz, 1H); Method A LCMS (ESI+): 1.878 min, m / z 296 (M+1). Compound 120: 8-(phenethylamino)-7,8,9,10-tetrahydro-5H-cyclohepta[b]naphthalene- 5,11(6H)-dione

[0242] To a mixture of Compound 112 (60 mg, 224.76 μmol, 1 eq, purity 90%) and 2- phenylethanamine (81.71 mg, 674.29 μmol, 84.67 μL, 3 eq) in dichloromethane (2 mL) was added AcOH (13.50 mg, 224.76 μmol, 12.87 μL, 1 eq) dropwise at 25°C. Then the reaction was stirred at 25°C for 30 minutes. Then sodium triacetoxyboranuide (119.09 mg, 561.91 μmol, 2.5 eq) was added to the mixture at 25°C. The reaction mixture was stirred at 25°C for 1 hour. The mixture was concentrated under reduced pressure to give the residue. The residue was purified by prep-HPLC (HCl condition; column: Phenomenex luna C18 100*40mm*5 um;mobile phase: [H2O(0.04% HCl)-ACN];gradient:1%-40% B over 8.0 min) and the desired eluent was lyophilized to give Compound 120 (58.9 mg, 152.69 μmol, 58.56% yield, 99% purity, HCl) as light yellow solid:1H NMR (400 MHz, DMSO-d6) δ 1.48 (q, J=11.42 Hz, 2H), 2.23-2.34 (m, 2H), 2.40 (br dd, J=14.76, 11.63 Hz, 2H), 2.98 (br dd, J=10.32, 6.32 Hz, 2H), 3.12-3.24 (m, 2H), 3.29 (br d, J=7.63 Hz, 2H), 3.47 (br d, J=1.00 Hz, 1H), 7.21-7.40 (m, 5H), 7.80-7.90 (m, 2H), 7.97-8.07 (m, 2H), 9.02 (br s, 2H); Method A LCMS (ESI+): 2.186 min, m / z 346 (M+1). Compound 121: 8-((3,3,3-trifluoropropyl)amino)-7,8,9,10-tetrahydro-5H- cyclohepta[b]naphthalene-5,11(6H)-dione

[0243] To a solution of Compound 112 (80 mg, 299.68 μmol, 1 eq, purity 90%) and 3,3,3- trifluoropropan-1-amine (134.45 mg, 899.05 μmol, 3 eq, HCl) in dichloromethane (1 mL) was added AcOH (18.00 mg, 299.68 μmol, 17.16 μL, 1 eq) dropwise at 25°C. Then the reaction was stirred at 25°C for 30 minutes. The sodium triacetoxyboranuide (158.79 mg, 749.21 μmol, 2.5 eq) was added to the mixture at 25°C. The reaction mixture was stirred at 25°C for 1 hour. The mixture was concentrated under reduced pressure to give the residue. The residue was triturated with methanol. Then the filter cake was collected and the cake was concentrated under reduced pressure to give Compound 121 (9 mg, 24.03 μmol, 8.02% yield, 99.8% purity, HCl) as light yellow solid. The rest of the residue was purified by prep- HPLC (HCl condition; column: Phenomenex luna C18100*40mm*5 um;mobile phase:[H2O(0.04% HCl)-ACN];gradient:1%-40% B over 8.0 min) and the desired eluent was lyophilized to give Compound 121 (29.1 mg, 71.23 μmol, 23.77% yield, 91.5% purity, HCl) as light yellow solid:1H NMR (400 MHz, DMSO-d6) δ 1.48 (q, J=11.46 Hz, 2H), 2.27 (br t, J=8.63 Hz, 2H), 2.39 (br dd, J=14.63, 11.63 Hz, 2H), 2.71-2.89 (m, 2H), 3.15-3.25 (m, 2H), 3.28 (br d, J=7.75 Hz, 2H), 3.49 (br s, 1H), 7.77-7.93 (m, 2H), 8.02 (dd, J=5.69, 3.31 Hz, 2H), 9.28 (br d, J=1.63 Hz, 2H);19F NMR (377 MHz, DMSO-d6) δ -64.07 (s, 3F); Method A LCMS (ESI+): 1.995 min, m / z 338 (M+1). Compound 122: 8-((4-(trifluoromethyl)phenyl)amino)-7,8,9,10-tetrahydro-5H- cyclohepta[b]naphthalene-5,11(6H)-dione

[0244] To a mixture of Compound 112 (35 mg, 138.40 μmol, 1 eq, purity 95%) and 4- (trifluoromethyl)aniline (66.90 mg, 415.19 μmol, 51.62 μL, 3 eq) in dichloromethane (1 mL) was added AcOH (8.31 mg, 138.40 μmol, 7.92 μL, 1 eq) dropwise at 25°C and stirred at 25°C for 30 minutes. Then sodium triacetoxyboranuide (73.33 mg, 345.99 μmol, 2.5 eq) was added to the mixture at 25°C. The reaction mixture was stirred at 25°C for 3 hours. The mixture was concentrated under reduced pressure to give the residue. The residue was purified by prep-HPLC (HCl condition; column: Phenomenex luna C18100*40mm*5 um;mobile phase: [H2O(0.04% HCl)-ACN];gradient:45%-75% B over 8.0 min) and the desired eluent was lyophilized to give Compound 122 (8.4 mg, 19.91 μmol, 6.31% yield, 100% purity, HCl) as yellow solid:1H NMR (400 MHz, CHLOROFORM-d) δ 1.28-1.41 (m, 2H), 2.22-2.35 (m, 2H), 2.42 (br dd, J=14.38, 12.01 Hz, 2H), 3.44 (dd, J=15.13, 7.88 Hz, 2H), 3.63-3.77 (m, 1H), 6.65 (br d, J=2.63 Hz, 2H), 7.44 (br d, J=8.25 Hz, 2H), 7.68-7.78 (m, 2H), 8.07-8.18 (m, 2H);19F NMR (377 MHz, CHLOROFORM-d) δ -61.10 (br s, 3F); Method A LCMS (ESI+): 3.220 min, m / z 386 (M+1). Compound 123: tert-butyl (3-(6,11-dioxo-1,2,4,5,6,11-hexahydro-3H-naphtho[2,3-d]azepin- 3-yl)propyl)(methyl)carbamate

[0245] To a solution of Compound 102 (200 mg, 758.38 μmol, 1 eq, HCl) and tert-butyl N-methyl-N-(3-oxopropyl)carbamate (425.99 mg, 2.28 mmol, 3 eq) in MeOH (8 mL) was added NaOAc (186.63 mg, 2.28 mmol, 3 eq) and AcOH (45.54 mg, 758.38 μmol, 43.41 μL, 1 eq) in order at 25°C. The mixture was stirred for 1 hr at 40°C. Then NaBH3CN (142.97 mg, 2.28 mmol, 3 eq) was added to the mixture in portions and stirred for 12 hrs at 25°C. The mixture was added saturated NaHCO3 (aqueous) to pH = 8. Then the mixture was extracted with ethyl acetate (3 x 10 mL). The combined organic phases were washed with brine (10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Ethyl acetate in Petroleum ether = 0-45%) to give a crude product. The crude product was purified byprep-HPLC (HCl condition, column: Phenomenex luna C18100*40 mm*5 um; mobilephase: [H2O (0.04% HCl)-ACN]; gradient:1%-40% B over 8.0 min) to give Compound 123 (30 mg, 66.01 μmol, 8.70% yield, 95.7% purity, HCl) as an off-white solid:1H NMR (400 MHz, METHANOL-d4) δ ppm 8.12 (dd, J=5.75, 3.38 Hz, 2 H), 7.82 (dd, J=5.75, 3.38 Hz, 2 H), 3.70 - 3.84 (m, 2 H), 3.53 - 3.68 (m, 2 H), 3.38 (t, J=6.69 Hz, 2 H), 3.13 - 3.29 (m, 4 H), 2.78 - 3.09 (m, 5 H), 1.99 - 2.10 (m, 2 H), 1.48 (s, 9 H); Method A LCMS (ESI+): 2.130 min, m / z 399.2 (M+H). Compound 124: 3-(3-(methylamino)propyl)-2,3,4,5-tetrahydro-1H-naphtho[2,3-d]azepine- 6,11-dione

[0246] A solution of Compound 123 (30 mg, 75.28 μmol, 1 eq) in EtOAc (1 mL) and HCl / EtOAc (1 mL, 4 mol / L) was stirred for 2 hrs at 25°C. The residue was purified by prep-HPLC (HCl condition, column: Phenomenex luna C18100*40 mm*5 um;mobile phase: [H2O (0.04% HCl)-ACN]; gradient: 1%-20% B over 8.0 min) and lyophilized to give Compound 124 (11 mg, 28.91 μmol, 38.41% yield, 97.6% purity, 2HCl) as a light yellow solid:1H NMR (400 MHz, DMSO-d6) δ ppm 11.34 - 11.53 (m, 1 H), 8.96 (br s, 2 H), 7.99 - 8.12 (m, 2 H), 7.88 (dd, J=5.69, 3.31 Hz, 2 H), 3.63 (br d, J=1.00 Hz, 2 H), 3.34 - 3.35 (m, 2 H), 3.24 (br s, 2 H), 3.05 - 3.19 (m, 4 H), 3.00 (br s, 2 H), 2.54 (br s, 3 H), 2.10 (br s, 2 H); Method A LCMS (ESI+): 1.40 min, m / z 299.1 (M+H). Compound 125: 3-acetyl-2,3,4,5-tetrahydro-1H-naphtho[2,3-d]azepine-6,11-dione

[0247] a mg, eq, (3 mL) was added TEA (115.11 mg, 1.14 mmol, 158.34 μL, 3 eq) and acetyl chloride (59.53 mg, 758.38 μmol, 53.92 μL, 2 eq) in portions at 25°C under N2. The mixture was stirred at 25°C under N2 for 2 hours. One additional vial in 20 mg scale was set up as described above and these two reactions were combined. Then the mixture was diluted with H2O (20 mL) and extracted with ethyl acetate (3 x 40 mL). The combined organic phases were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude. The crude product was triturated with tert-butyl methyl ether (10 mL) at 25°C for 30 minutes. The mixture was filtered and the filter cake was concentrated under reduced pressure to give Compound 125 (57 mg, 211.03 μmol, 46.38% yield, 99.7% purity) as a yellow solid:1H NMR (400 MHz, DMSO-d6) δ ppm 2.06 (s, 3 H) 2.82 - 3.07 (m, 4 H) 3.62 (td, J=5.16, 2.56 Hz, 4 H) 7.78 - 7.91 (m, 2 H) 7.96 - 8.07 (m, 2 H); Method A LCMS (ESI+): 2.140 min, m / z 270 (M+H). Compound 126: 3-(2-(dimethylamino)ethyl)-2,3,4,5-tetrahydro-1H-naphtho[2,3-d]azepine- 6,11-dioneO N was prepared according to the general procedures described in otherCompound 127: 3-(3-(dimethylamino)propyl)-2,3,4,5-tetrahydro-1H-naphtho[2,3-d]azepine- 6,11-dionea mg, eq, (1 mL) was added NaOAc (13.78 mg, 167.94 μmol, 3 eq), (HCHO)n (10.09 mg, 335.88 μmol, 6 eq) and AcOH (3.36 mg, 55.98 μmol, 3.20 μL, 1 eq) in order at 25°C. The mixture was stirred for 1 hr at 25°C. Then NaBH3CN (10.55 mg, 167.94 μmol, 3 eq) was added to the mixture at 25°C. The mixture was stirred for 12 hrs at 25°C. One additional vial in 5 mg scale was set up as described above. The mixtures were combined and filtered. The filtrate was purified by prep-HPLC (HCl condition, column: Phenomenex Luna C1875*30 mm*3 um; mobile phase: [H2O (0.04% HCl)-ACN]; gradient: 1%-25% B over 8.0 min) and lyophilized to give Compound 127 (8.9 mg, 22.66 μmol, 32.38% yield, 98.1% purity, 2HCl) as a yellow solid:1H NMR (400 MHz, DMSO-d6) δ ppm 8.04 (dd, J=5.69, 3.31 Hz, 2 H), 7.87 (dd, J=5.69, 3.31 Hz, 2 H), 3.61 (br d, J=1.00 Hz, 2 H), 3.29 - 3.40 (m, 2 H), 3.05 - 3.25 (m, 6 H), 2.90 - 3.02 (m, 2 H), 2.78 (s, 6 H), 2.00 - 2.13 (m, 2 H); Method A LCMS (ESI+): 1.48 min, m / z 313.1 (M+H). Compound 128: 8-amino-7,8,9,10-tetrahydro-5H-cyclohepta[b]naphthalene-5,11(6H)-dione

[0250] To a solution of Compound 112 (300 mg, 1.25 mmol, 1 eq) and NH4Cl (80.15 mg, 1.50 mmol, 1.2 eq) in DCM (6 mL) was added HOAc (74.98 mg, 1.25 mmol, 71.48 μL, 1 eq) at 25°C. The mixture was stirred at 25°C for 1 hour. Then NaBH(OAc)3 (661.61 mg, 3.12 mmol, 2.5 eq) was added to the mixture. The mixture was stirred at 25°C for 12 hours. The mixture was concentrated under reduced pressure to give the crude product. The crude product was purified by prep-HPLC (column: Phenomenex luna C18100*40mm*5 um; mobile phase: [H2O (0.04% HCl)-ACN]; gradient:5%-30% B over 8.0 min) and lyophilized to give Compound 128 (24.9 mg, 103.20 μmol, 8.26% yield) as a yellow solid:1H NMR (DMSO-d6, 400 MHz) δ (ppm) 8.07 (br s, 2H), 7.97-8.04 (m, 2H), 7.80-7.91 (m, 2H), 3.37- 3.48 (m, 1H), 3.23-3.30 (m, 2H), 2.41 (br dd, J = 14.4, 12.1 Hz, 2H), 2.05-2.19 (m, 2H), 1.28- 1.43 (m, 2H); Method A LCMS (ESI+): 1.709 min, m / z 242 (M+1). Compound 129: 8-(diethylamino)-7,8,9,10-tetrahydro-5H-cyclohepta[b]naphthalene- 5,11(6H)-dione

[0251] To a mixture of Compound 112 (200 mg, 790.83 μmol, 1 eq, purity 95%) and N- ethylethanamine (578.39 mg, 7.91 mmol, 814.63 μL, 10 eq) in dichloromethane (4 mL) was added AcOH (47.49 mg, 790.83 μmol, 45.27 μL, 1 eq) dropwise at 25°C. Then the reaction was stirred at 40°C for 12 hours in a 40 mL of sealed tube. Then sodium triacetoxyboranuide (419.02 mg, 1.98 mmol, 2.5 eq) was added to the mixture at 25°C. The reaction mixture was stirred at 25°C for 16 hours in a 40 mL of sealed tube. The mixture was concentrated under reduced pressure to give the residue. The residue was purified by prep-HPLC (HCl condition; column: Phenomenex luna C18100*40mm*5 um;mobile phase: [H2O(0.04%HCl)-ACN]; gradient:1%-45% B over 8.0 min) and the desired eluent was lyophilized to give the product. The product was further purified again by prep-HPLC (HCl condition; column: Phenomenex luna C18100*40mm*5 um;mobile phase: [H2O(0.04% HCl)- ACN];gradient:1%-35% B over 8.0 min) and the desired eluent was lyophilized to give Compound 129 (26.3 mg, 77.20 μmol, 9.76% yield, 98% purity, HCl) as yellow solid:1H NMR (400 MHz, DMSO-d6) δ 1.27 (br t, J=7.19 Hz, 6H), 1.52 (q, J=11.97 Hz, 2H), 2.22- 2.30 (m, 2H), 2.36-2.43 (m, 2H), 3.04 (dt, J=13.26, 6.63 Hz, 2H), 3.11-3.18 (m, 2H), 3.38 (br s, 2H), 3.67-3.77 (m, 1H), 7.82-7.90 (m, 2H), 8.03 (td, J=3.60, 1.56 Hz, 2H), 9.55 (br s, 1H); Method A LCMS (ESI+): 1.919 min, m / z 298 (M+1). Compound 130: 3-methyl-2,3,4,5-tetrahydro-1H-naphtho[2,3-d]azepine-6,11-dione

[0252] To a solution of Compound 102 (150 mg, 568.79 μmol, 1 eq, HCl) and (HCHO)n (51.24 mg, 1.71 mmol, 47.00 μL, 3 eq) in MeOH (4 mL) was added AcOH (34.16 mg, 568.79 μmol, 32.56 μL, 1 eq) and NaOAc (139.97 mg, 1.71 mmol, 3 eq) in portions at 25°C under N2. The mixture was stirred at 40°C under N2for 1 hour. Then to the mixture was added NaBH3CN (107.23 mg, 1.71 mmol, 3 eq) in one portion at 25°C under N2. The mixture was stirred at 40°C under N2for 1 hour. The combined mixture was added saturated aqueous Na2CO3 to adjusted to pH=8. Then the mixture was extracted with ethyl acetate (3 x 40 mL). The combined organic phases were washed with brine (40 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a crude. The crude was purified by prep-HPLC (neutral condition, column: Waters Xbridge Prep OBD C18150*40mm*10um; mobile phase: [H2O(10mM NH4HCO3)-ACN]; gradient:30%-70% B over 8.0 min) and the desired eluent was lyophilized to give Compound 130 (69 mg, 277.10 μmol, 48.72% yield, 96.9% purity) as a dark purple solid:1H NMR (400 MHz, DMSO-d6) δ ppm 2.25 (s, 3 H) 2.41 - 2.47 (m, 4 H) 2.85 - 2.93 (m, 4 H) 7.84 (br dd, J=5.50, 3.38 Hz, 2 H) 7.96 - 8.07 (m, 2 H); Method A LCMS (ESI+): 1.622 min, m / z 242 (M+H). Compound 131: 3-ethyl-2,3,4,5-tetrahydro-1H-naphtho[2,3-d]azepine-6,11-dione

[0253] o a so u o o o pou g, . μ o , eq, MF (3 mL) was added TEA (115.11 mg, 1.14 mmol, 158.34 μL, 3 eq) and iodoethane (295.70 mg, 758.38 μmol, 151.64 μL, 2 eq) in portions at 25°C under N2. The mixture was stirred at 25°C under N2 for 2 hours. One additional vial in 10 mg scale was set up as described above and these two reactions were combined. The combined mixture was diluted with H2O (30 mL) and extracted with ethyl acetate (3 x 40 mL). Then the combined organic phases were washed with brine (3 x 30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a crude. The crude was purified by prep-HPLC (HCl condition, column: Phenomenex luna C18100*40mm*5 um;mobile phase: [H2O(0.04% HCl)- ACN];gradient:1%-30% B over 8.0 min) and the desired eluent was lyophilized to give Compound 131 (22 mg, 74.35 μmol, 17.82% yield, 98.6% purity, HCl) as a light-yellow solid:1H NMR (400 MHz, DMSO-d6) δ ppm 1.25 (t, J=7.25 Hz, 3 H) 2.85 - 3.03 (m, 2 H) 3.04 - 3.23 (m, 4 H) 3.37 - 3.38 (m, 2 H) 3.39 - 3.49 (m, 2 H) 3.54 - 3.72 (m, 2 H) 7.83 - 7.93 (m, 2 H) 8.00 - 8.10 (m, 2 H) 10.08 - 10.72 (m, 1 H); Method A LCMS (ESI+): 1.672 min, m / z 256 (M+H). Compound 132: 3-(2-methoxyethyl)-2,3,4,5-tetrahydro-1H-naphtho[2,3-d]azepine-6,11- dione O O O

[0254] To a solution of Compound 102 (150 mg, 568.79 μmol, 1 eq, HCl) in DMF (3 mL) was added TEA (172.67 mg, 1.71 mmol, 237.50 μL, 3 eq) and 1-bromo-2-methoxy-ethane (158.11 mg, 1.14 mmol, 106.90 μL, 2 eq) in portions at 25°C under N2. The mixture wasstirred at 25°C under N2for 12 hours. The mixture was diluted with H2O (40 mL) and extracted with ethyl acetate (3 x 40 mL). The combined organic layers were washed with brine (3 x 40 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (HCl condition, column: Phenomenex luna C18100*40mm*5 um;mobile phase: [H2O(0.04% HCl)- ACN];gradient:1%-40% B over 8.0 min) and the desired eluent was lyophilized to give Compound 132 (13 mg, 39.99 μmol, 7.03% yield, 99% purity, HCl) as a light-yellow solid:1H NMR (400 MHz, DMSO-d6) δ ppm 2.95 - 3.10 (m, 2 H) 3.14 - 3.26 (m, 2 H) 3.37 (br d, J=5.00 Hz, 7 H) 3.58 - 3.68 (m, 2 H) 3.71 (br t, J=4.82 Hz, 2 H) 7.82 - 7.94 (m, 2 H) 7.99 - 8.11 (m, 2 H) 10.55 (br s, 1 H); Method A LCMS (ESI+): 1.720 min, m / z 286 (M+H). Compound 133: 3-(2-hydroxyethyl)-2,3,4,5-tetrahydro-1H-naphtho[2,3-d]azepine-6,11-dione

[0255] To a solution of Compound 102 (150 mg, 568.79 μmol, 1 eq, HCl) in DMF (3 mL) was added TEA (172.67 mg, 1.71 mmol, 237.50 μL, 3 eq) and 2-iodoethanol (195.62 mg, 1.14 mmol, 88.92 μL, 2 eq) in portions at 25°C under N2. The mixture was stirred at 25°C under N2for 12 hours. One additional vial in 10 mg scale was set up as described above and these two reactions were combined. The mixture was diluted with H2O (50 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic layers were washed with brine (3 x 60 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (HCl condition, column: Phenomenex luna C18100*40mm*5 um;mobile phase: [H2O(0.04% HCl)- ACN];gradient:1%-30% B over 8.0 min) and the desired eluent was lyophilized to give Compound 133 (23 mg, 73.98 μmol, 12.27% yield, 99% purity, HCl) as a light-yellow solid:1H NMR (400 MHz, DMSO-d6) δ ppm 2.99 - 3.12 (m, 2 H) 3.14 - 3.32 (m, 6 H) 3.67 (br dd, J=12.01, 7.75 Hz, 2 H) 3.79 (br s, 2 H) 5.28 - 5.47 (m, 1 H) 7.82 - 7.94 (m, 2 H) 7.99 - 8.10 (m, 2 H) 10.47 (br d, J=2.13 Hz, 1 H); Method A LCMS (ESI+): 1.598 min, m / z 272 (M+H). Compound 134: 3-(2-fluoroethyl)-2,3,4,5-tetrahydro-1H-naphtho[2,3-d]azepine-6,11-dione

[0256] To a solution of Compound 102 (150 mg, 568.79 μmol, 1 eq, HCl) in DMF (3 mL) was added TEA (172.67 mg, 1.71 mmol, 237.50 μL, 3 eq) and 1-fluoro-2-iodo-ethane (197.89 mg, 1.14 mmol, 2 eq) in portions at 25°C under N2. The mixture was stirred at 25°C under N2 for 12 hours. The mixture was diluted with H2O (50 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic layers were washed with brine (3 x 60 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (HCl condition, column: Phenomenex luna C18 100*40mm*5 um;mobile phase: [H2O(0.04% HCl)-ACN];gradient:1%-30% B over 8.0 min) and the desired eluent was lyophilized to give Compound 134 (45 mg, 145.27 μmol, 25.54% yield, 100% purity, HCl) as a light yellow solid:1H NMR (400 MHz, DMSO-d6) δ ppm 2.94 - 3.10 (m, 2 H) 3.19 - 3.29 (m, 2 H) 3.35 - 3.46 (m, 2 H) 3.50 - 3.77 (m, 4 H) 4.75 - 5.02 (m, 2 H) 7.82 - 7.94 (m, 2 H) 8.00 - 8.10 (m, 2 H) 10.93 (br d, J=1.13 Hz, 1 H);19F NMR (376 MHz, DMSO-d6) δ ppm -218.76 (s, 1 F); Method A LCMS (ESI+): 1.673 min, m / z 274 (M+H). Compound 135: 8-morpholino-7,8,9,10-tetrahydro-5H-cyclohepta[b]naphthalene-5,11(6H)- dione

[0257] To a mixture of Compound 112 (100 mg, 395.42 μmol, 1 eq, purity 95%) and morpholine (103.35 mg, 1.19 mmol, 104.39 μL, 3 eq) in dichloromethane (2 mL) was added AcOH (23.75 mg, 395.42 μmol, 22.64 μL, 1 eq) dropwise at 25°C. The reaction mixture was stirred at 25°C for 30 minutes. Then NaBH(OAc)3 (209.51 mg, 988.54 μmol, 2.5 eq) wasadded to the mixture at 25°C. The reaction mixture was stirred at 25°C for 3 hours. The mixture was added saturated NaHCO3 (aqueous) to pH = 8. Then the mixture was extracted with ethyl acetate (3 x 40 mL). The combined organic phases were washed with brine (40 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (NH4HCO3condition; column: Waters Xbridge Prep OBD C18150*40mm*10um;mobile phase: [H2O(10mM NH4HCO3)-ACN];gradient:30%- 60% B over 8.0 min) and the desired eluent was lyophilized to give Compound 135 (100.4 mg, 315.99 μmol, 66.60% yield, 98% purity) as yellow solid:1H NMR (400 MHz, DMSO- d6) δ 1.28-1.41 (m, 2H), 1.85-1.96 (m, 2H), 2.07 (s, 1H), 2.22-2.34 (m, 2H), 2.43-2.48 (m, 4H), 2.61 (ddd, J=10.41, 7.54, 3.06 Hz, 1H), 3.23 (dd, J=14.45, 7.69 Hz, 2H), 3.49-3.59 (m, 4H), 7.83 (dd, J=5.69, 3.31 Hz, 2H), 7.97-8.04 (m, 2H); Method A LCMS (ESI+): 1.817 min, m / z 312 (M+1). Compound 136: 8-((2-methoxyethyl)amino)-7,8,9,10-tetrahydro-5H- cyclohepta[b]naphthalene-5,11(6H)-dione

[0258] To a solution of Compound 112 (100 mg, 395.42 μmol, 1 eq) and 2- methoxyethanamine (89.10 mg, 1.19 mmol, 103.12 μL, 3 eq) in DCM (2 mL) was added AcOH (23.74 mg, 395.42 μmol, 22.64 μL, 1 eq) in one portion at 25°C under N2. The reaction mixture was stirred at 25°C for 30 minutes. Then NaBH(OAc)3 (209.51 mg, 988.54 μmol, 2.5 eq) was added to the mixture in one portion at 25°C under N2. The mixture was stirred at 25°C under N2 for 1 hour. One additional vial in 20 mg scale was set up as described above and these two reactions were combined. The combined mixture was added saturated aqueous Na2CO3 to adjusted to pH=8. Then the mixture was extracted with ethyl acetate (3 x 40 mL). The combined organic phases were washed with brine (40 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The crude product was triturated with tert-butyl methyl ether (10 mL) at 25°C for 30 minutes. The mixture was filtered and the filter cake was concentrated under reduced pressure to giveCompound 136 (49 mg, 157.13 μmol, 33.12% yield, 96.4% purity) as a yellow solid:1H NMR (400 MHz, DMSO-d6) δ ppm 1.31 - 1.53 (m, 2 H) 2.14 - 2.31 (m, 2 H) 2.38 (br dd, J=14.57, 11.57 Hz, 2 H) 3.04 - 3.14 (m, 2 H) 3.23 - 3.32 (m, 6 H) 3.60 (t, J=5.13 Hz, 2 H) 7.79 - 7.92 (m, 2 H) 7.97 - 8.08 (m, 2 H) 8.53 - 9.07 (m, 1 H); Method A LCMS (ESI+): 1.864 min, m / z 300 (M+H). Compound 137: 8-((2-hydroxyethyl)amino)-7,8,9,10-tetrahydro-5H- cyclohepta[b]naphthalene-5,11(6H)-dione2- aminoethan-1-ol (72.46 mg, 1.19 mmol, 71.60 μL, 3 eq) in dichloromethane (1 mL) was added AcOH (23.75 mg, 395.42 μmol, 22.64 μL, 1 eq) dropwise at 25°C. The reaction mixture was stirred at 25°C for 30 minutes. Then sodium triacetoxyboranuide (209.51 mg, 988.54 μmol, 2.5 eq) was added to the mixture at 25°C. The reaction mixture was stirred at 25°C for 3 hours. The combined mixture was added saturated Na2CO3(aqueous) to pH = 8. Then the mixture was extracted with ethyl acetate (3 x 40 mL). The combined organic phases were washed with brine (40 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was triturated with tert-butyl methyl ether: ethyl acetate = 5:1. The mixture was filtered and the filter cake was collected. The filter cake was dried by high vacuum to give the Compound 137 (65.4 mg, 221.41 μmol, 46.66% yield, 96.6% purity) as orange solid:1H NMR (400 MHz, DMSO-d6) δ 1.15-1.28 (m, 2H), 1.85-1.96 (m, 2H), 2.41 (br dd, J=14.38, 10.76 Hz, 2H), 2.63 (t, J=5.75 Hz, 2H), 2.73-2.84 (m, 1H), 3.07-3.19 (m, 2H), 3.45 (br d, J=2.25 Hz, 2H), 4.51 (br s, 1H), 7.80-7.86 (m, 2H), 7.97-8.04 (m, 2H); Method A LCMS (ESI+): 1.776 min, m / z 286 (M+1). Compound 138: 8-((2-fluoroethyl)amino)-7,8,9,10-tetrahydro-5H-cyclohepta[b]naphthalene- 5,11(6H)-dioneo a so u o o o pou g, . μ o , eq, pu y a - fluoroethanamine (118.07 mg, 1.19 mmol, 3 eq, HCl) in dichloromethane (2 mL) was added AcOH (23.75 mg, 395.42 μmol, 22.64 μL, 1 eq) dropwise at 25°C. The reaction mixture was stirred at 25°C for 1 hour. Then sodium triacetoxyboranuide (209.51 mg, 988.54 μmol, 2.5 eq) was added to the mixture at 25°C. The reaction mixture was stirred at 25°C for 3 hours. The combined mixture was added saturated NaHCO3 (aqueous) to pH = 8. Then the mixture was extracted with ethyl acetate (3 x 40 mL). The combined organic phases were washed with brine (40 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (NH4HCO3condition; column: Waters Xbridge Prep OBD C18150*40mm*10um;mobile phase: [H2O(10mM NH4HCO3)- ACN];gradient:15%-45% B over 8.0 min) and the desired eluent was lyophilized to give the product, which was purified again by prep-HPLC (HCl condition; column: Phenomenex luna C18100*40mm*5 um;mobile phase: [H2O(0.04% HCl)-ACN];gradient:5%-50% B over 8.0 min) and the desired eluent was lyophilized to give the Compound 138 (47.6 mg, 145.54 μmol, 30.67% yield, 99% purity, HCl) was as light yellow solid:1H NMR (400 MHz, DMSO-d6) δ 1.39 (q, J=11.72 Hz, 2H), 2.20-2.30 (m, 2H), 2.37 (br dd, J=14.70, 11.94 Hz, 2H), 3.25-3.34 (m, 3H), 3.35-3.40 (m, 1H), 3.47 (ddd, J=11.07, 7.63, 3.44 Hz, 1H), 4.63-4.80 (m, 2H), 7.80-7.88 (m, 2H), 8.01 (dd, J=5.75, 3.38 Hz, 2H);19F NMR (376 MHz, DMSO-d6) δ -222.39 (s, 1F); Method A LCMS (ESI+): 1.812 min, m / z 288 (M+1). Compound 201: 3-isopropyl-7,8-dimethyl-2,3,4,5-tetrahydro-1H-benzo[d]azepine-6,9-dione, mg,4.10 mmol,), NaOAc (134.40 mg, 1.64 mmol), 4A molecular sieve (100 mg) in MeOH (5mL) was stirred at 20°C for 0.2 hr. Then was added AcOH (24.60 mg, 409.58 μmol) at 20°C. The mixture was stirred at 40°C for 1 hr before NaBH3CN (51.48 mg, 819.15 μmol) was added into the reaction at 20°C. The resulting mixture was stirred at 40°C for 3 hrs. Additional two reactions were set up as described above. The reaction mixtures were combined and purified follows. The solution was poured into ice NaHCO3(20 mL). The aqueous phase was extracted with EtOAc (10 mL × 3). The combined organic phase was washed with brine (10 mL × 3), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by prep-HPLC to give Compound 201 (66.2 mg, 14.91% yield, 100% purity, TFA) as a yellow solid:1H NMR: 400 MHz, DEUTERIUM OXIDE δ = 3.70 - 3.61 (m, 1H), 3.57 (br dd, J = 6.8, Hz, 2H), 3.37 (br dd, J = 6.5, 17.8 Hz, 2H),3.12 (br t, J = 12.1 Hz, 2H), 2.79 - 2.64 (m, 2H), 1.97 (s, 6H), 1.30 (d, J = 6.6 Hz, 6H); Method A LCMS (ESI+):1.611 min, m / z 448.1. Compound 202: 3-cyclopentyl-7,8-dimethyl-2,3,4,5-tetrahydro-1H-benzo[d]azepine-6,9- dioneHCl) and cyclopentanone (226.20 mg, 2.69 mmol, 238.10 μL, 5 eq) in MeOH (15 mL) wasadded NaOAc (132.36 mg, 1.61 mmol, 3 eq) and AcOH (32.30 mg, 537.83 μmol, 30.79 μL, 1 eq) at 25°C. The mixture was stirred for 1 hr at 40°C. Then NaBH3CN (101.39 mg, 1.61 mmol, 3 eq) was added to the mixture in portions at 25°C. The mixture was stirred for 2 hrs at 40°C. To the mixture was added saturated Na2CO3(aqueous) to pH = 8, extracted with ethyl acetate (3 x 15 mL). The combined organic phases were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Ethyl acetate in Petroleum ether = 0 to 100%) to give Compound 202 (56 mg, 201.57 μmol, 37.48% yield, 98.4% purity) as a light brown solid:1H NMR (400 MHz, METHANOL-d4) δ ppm 2.87 - 2.95 (m, 1 H), 2.80 - 2.87 (m, 4 H), 2.65 - 2.74 (m, 4 H), 2.00 (s, 6 H), 1.86 - 1.96 (m, 2 H), 1.66 - 1.76 (m, 2 H),1.54 - 1.63 (m, 2 H), 1.39 - 1.51 (m, 2 H); Method A LCMS (ESI+): 1.750 min, m / z 274 (M+H). Compound 203: 7,8-dimethyl-3-(pentan-3-yl)-2,3,4,5-tetrahydro-1H-benzo[d]azepine-6,9- dione(1 mL) was added pentan-3-one (48.11 mg, 558.51 μmol, 59.18 μL, 3 eq), ZnCl2(25.38 mg, 186.17 μmol, 8.73 μL, 1 eq) and NaBH3CN (35.10 mg, 558.51 μmol, 3 eq) in portions at 25°C under N2. The mixture was stirred at 70°C under N2for 6 hours with microwave. Additional vials at 10 mg and 40 mg scale were set up as described above and these three reactions were combined. The residue was diluted with H2O (20 mL) and extracted with ethyl acetate (3 x 30 mL). The combined organic layers were washed with brine (30 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (HCl condition, column: Phenomenex luna C18100*40mm*5 um; mobile phase: [H2O(0.04% HCl)- ACN];gradient:1%-35% B over 8.0 min) and the desired eluent was lyophilized to give Compound 203 in two batches (17 mg, 51.79 μmol, 11.13% yield, 95% purity, HCl salt; 26 mg, 80.87 μmol, 17.38% yield, 97% purity, HCl salt) as light yellow solids:1H NMR (400 MHz, DMSO-d6) δ ppm 0.96 (t, J=7.44 Hz, 6 H) 1.50 - 1.67 (m, 2 H) 1.84 (dqd, J=14.39, 7.40, 7.40, 7.40, 4.19 Hz, 2 H) 1.98 (s, 6 H) 2.89 - 3.03 (m, 2 H) 3.06 - 3.24 (m, 5 H) 3.50 (br dd, J=12.63, 6.38 Hz, 2 H) 10.07 (br dd, J=3.94, 1.94 Hz, 1 H); Method A LCMS (ESI+): 1.882 min, m / z 276 (M+H). Compound 204: tert-butyl 7,8-dimethyl-6,9-dioxo-1,2,4,5,6,9-hexahydro-3H- benzo[d]azepine-3-carboxylate Compound 205: 7,8-dimethyl-2,3,4,5-tetrahydro-1H-benzo[d]azepine-6,9-dioneOH O MnO2

[0264] To a in acetone (60mL) was added g, mixture was stirred at 20°C for 4 hrs. The mixture was filtered, and the filtrate was concentrated under reduced pressure to give Intermediate 204-1 (2,3-dimethylcyclohexa-2,5-diene-1,4-dione, 7 g, 79.92% yield, 90% purity) as a yellow solid:1H NMR (400 MHz, CHLOROFORM-d) δ 6.73 (s, 2H), 2.04 (s, 6H).29.75mmol) in H2O (300 mL) and ACN (400 mL) was added AgNO3 (4.04 g, 23.80 mmol) and Intermediate 204-1 (3 g, 19.83 mmol) at 20°C. The mixture was stirred at 100°C for 0.2hr. Then the mixture was cooled to 20°C, and to a reaction mixture was added the solution of (NH4)2S2O8(14.48 g, 63.46 mmol) in H2O (300 mL) and ACN (400 mL) dropwise at 20°C. The mixture was stirred at 100°C for 3hrs. The mixture was cooled to 20°C. The solution was poured into water (400 mL). The aqueous phase was extracted with EtOAc (200 mL × 3). The combined organic phase was washed with brine (200mL × 3), dried with anhydrous Na2SO, filtered and concentrated in vacuum. The residue was purified by prep-HPLC to give Compound 204 (200 mg, 3.01% yield, 91% purity) and Compound 205 in two batches (1.2 g, 18.78% yield, 75% purity, HCl; 0.3 g, 1.23 mmol, 6.20% yield, 99% purity, HCl) as brown solids.

[0266] The crude product Compound 204 (200 mg, 3.01% yield, 91% purity) was re-purified by prep-HPLC to give the compound (130.6 mg, 77.4% yield, 96.4% purity) asbrown solid:1H NMR (400 MHz, CHLOROFORM-d) δ = 3.54 (br s, 4H), 2.93 - 2.82 (m, 4H), 2.04 (s, 6H), 1.48 (s, 9H).

[0267] The crude product Compound 205 was purified by prep-HPLC [Column: Phenomenex luna C18100*40mm*5 um;mobile phase: [H2O(0.04% HCl)- ACN];gradient:5%-30% B over 8.0 min] to give purified Compound 205 (35.2 mg, 7.15% yield, 97.37% purity, HCl) as brown solid:1H NMR: (400 MHz, DEUTERIUM OXIDE) δ = 3.37 - 3.27 (m, 4H), 3.08 - 3.00 (m, 4H), 1.99 (s, 6H); Method A LCMS (ESI+): rt = 1.474 min, m / z 206.1 (M+H)+. Compound 206: 7,8-dimethyl-3-phenethyl-2,3,4,5-tetrahydro-1H-benzo[d]azepine-6,9-dionephenylacetaldehyde (268.42 mg, 2.23 mmol, 174.30 μL, 3 eq) in MeOH (3 mL) was added AcOH (44.72 mg, 744.68 μmol, 42.63 μL, 1 eq) and NaOAc (183.26 mg, 2.23 mmol, 3 eq) in portions at 25°C under N2. The mixture was stirred at 40°C under N2 for 1 hour. Then to the mixture was added NaBH3CN (140.39 mg, 2.23 mmol, 3 eq) in one portion at 25°C under N2. The mixture was stirred at 40°C under N2 for 12 hours. One additional vial in 15 mg scale was set up as described above and these two reactions were combined. The mixture was diluted with H2O (30 mL) and extracted with ethyl acetate (3 x 30 mL). The combined organic layers were washed with brine (30 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep- HPLC (HCl condition; column: Phenomenex luna C18100*40mm*5 um;mobile phase: [H2O(0.04% HCl)-ACN];gradient:1%-40% B over 8.0 min) and the desired eluent was lyophilized to give Compound 206 (48 mg, 138.78 μmol, 17.34% yield, 100% purity, HCl salt) as a light yellow solid:1H NMR (400 MHz, DMSO-d6) δ ppm 1.99 (s, 6 H) 2.77 - 2.93 (m, 2 H) 2.98 - 3.07 (m, 2 H) 3.14 - 3.24 (m, 4 H) 3.36 - 3.43 (m, 2 H) 3.68 (ddd, J=9.57, 7.75, 2.06 Hz, 2 H) 7.21 - 7.42 (m, 5 H) 10.04 - 10.28 (m, 1 H); Method A LCMS (ESI+): 2.053 min, m / z 310 (M+H).Compound 207: 7,8-dimethyl-3-(3,3,3-trifluoropropyl)-2,3,4,5-tetrahydro-1H- benzo[d]azepine-6,9-dione

[0269] To a mixture of Compound 205 (250 mg, 930.85 μmol, 1 eq, HCl salt) in DMF (8 mL) was added K2CO3(514.61 mg, 3.72 mmol, 4 eq) at 25°C. The mixture was stirred for 0.5 hr at 25°C. Then 1,1,1-trifluoro-3-iodo-propane (625.43 mg, 2.79 mmol, 327.28 μL, 3 eq) was added to the mixture at 25°C and stirred for 8 hrs at 50°C. The mixture was diluted with water (20 mL) and extracted with ethyl acetate (3 x 8 mL). The combined organic phases were washed with brine (3 x 10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. Two additional vial in 50 mg scale was set up as described above. The residue was combined and purified by prep-HPLC (neutral condition, column: Waters Xbridge Prep OBD C18150*40 mm*10 um; mobile phase: [H2O (10 mM NH4HCO3)-MeCN]; gradient: 45%-75% B over 8.0 min) and lyophilization to give Compound 207 (29 mg, 94.03 μmol, 7.22% yield, 97.7% purity) as a gray solid:1H NMR (400 MHz, METHANOL-d4) δ ppm 2.80 - 2.88 (m, 4 H), 2.73 - 2.79 (m, 2 H), 2.58 - 2.63 (m, 4 H), 2.34 - 2.48 (m, 2 H), 2.01 (s, 6 H); Method A LCMS (ESI+): 1.699 min, m / z 302 (M+H). Compound 208: 7,8-dimethyl-3-(4-(trifluoromethyl)benzoyl)-2,3,4,5-tetrahydro-1H- benzo[d]azepine-6,9-dione

[0270] To a solution of Compound 205 (120 mg, 496.46 μmol, 1 eq, HCl) in DMF (10 mL) was added 4-(trifluoromethyl)benzoic acid (141.58 mg, 744.68 μmol, 1.5 eq), NMI (122.28 mg, 1.49 mmol, 118.72 μL, 3 eq) and 1-chloro-N,N,N′,N′-tetramethylformamidinium hexafluorophosphate (TCFH, 278.59 mg, 992.91 μmol, 2 eq) at 25°C in order. The mixture was stirred at 25°C for 12 hours. The mixture was diluted with ethyl acetate (10 mL) and water (10 mL). The mixture was extracted with ethyl acetate (3 x 10 mL). The organic layer was dried with Na2SO4, filtered and concentrated under reduced pressure to give the crude product. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=92%) and then triturated with ethyl acetate (2 mL). The solid was dried in high vacuum to give Compound 208 (56 mg, 144.39 μmol, 29.08% yield, 97.3% purity) as yellow solid: 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 7.82 (d, J = 8.0 Hz, 2H), 7.63 (d, J = 8.0 Hz, 2H), 3.75 (br s, 2H), 3.40 (br s, 2H), 2.94 (br s, 2H), 2.77 (br s, 2H), 1.97 (br s, 6H); Method A LCMS (ESI+): 2.806 min, m / z 378 (M+H). Compound 209: 7,8-dimethyl-1,2,4,5-tetrahydrobenzo[d]oxepine-6,9-dione

[0271] To a mixture of Intermediate 204-1 (400 mg, 2.94 mmol, 1 eq) and 3,3'- oxydipropionic acid (476.37 mg, 2.94 mmol, 1 eq) in MeCN (40 mL) and H2O (25 mL) was added AgNO3 (598.90 mg, 3.53 mmol, 1.2 eq) in portions at 25°C. Then the mixture was stirred for 30 min at 100°C. Then a solution of (NH4)2S2O8 (2.15 g, 9.40 mmol, 2.04 mL, 3.2 eq) in MeCN (40 mL) and H2O (25 mL) was added to the mixture dropwise at 25°C. The mixture was stirred for 3 hrs at 100°C. The mixture was extracted with EtOAc (3 x 50 mL). The combined organic phase was washed with saturated Na2S2O3(aqueous, 50 mL), dried with anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (neutral condition, column: Waters Xbridge BEH C18100*30 mm*10 um; mobile phase: [H2O (10 mM NH4HCO3)-MeCN]; gradient: 24%-56%, B over 8.0 min) and lyophilization of appropriate fractions to give Compound 209 (8.6 mg, 41.70 μmol, 1.42% yield) as a yellow solid:1H NMR (400 MHz,DMSO-d6) δ ppm 3.58 - 3.67 (m, 4 H), 2.78 - 2.86 (m, 4 H), 1.96 (s, 6 H); Method B LCMS (ESI-): 2.566 min; GCMS: m / z: 206. Compound 210: 7,8-dimethyl-3-(pyrimidin-4-yl)-2,3,4,5-tetrahydro-1H-benzo[d]azepine-6,9- dione

[0272] To a solution of Compound 205 (200 mg, 744.68 μmol, 1 eq, HCl) and 4- chloropyrimidine (170.58 mg, 1.49 mmol, 2 eq) in THF (4 mL) was added TEA (226.06 mg, 2.23 mmol, 310.95 μL, 3 eq) in one portion at 25°C under N2. The mixture was stirred at 60°C for 12 hours under N2. The mixture was diluted with H2O (50 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic layers were washed with brine (60 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (neutral condition, column: Waters Xbridge Prep OBD C18150*40mm*10um; mobile phase: [H2O (10mM NH4HCO3)-ACN]; gradient: 25%-55% B over 8.0 min) and the desired eluent was lyophilized to give Compound 210 (42 mg, 148.24 μmol, 19.91% yield, 100% purity) as a light yellow solid:1H NMR (400 MHz, DMSO-d6) δ ppm 1.85 - 2.04 (m, 6 H) 2.07 (s, 1 H) 2.78 - 2.96 (m, 4 H) 3.81 (br s, 4 H) 6.80 (dd, J=6.25, 0.88 Hz, 1 H) 8.17 (d, J=6.25 Hz, 1 H) 8.49 (s, 1 H); Method A LCMS (ESI+): 1.828 min, m / z 284 (M+H). Compound 211: 7,8-dimethyl-3-(pyrimidin-2-yl)-2,3,4,5-tetrahydro-1H-benzo[d]azepine-6,9- dione

[0273] To a solution of Compound 205 (200 mg, 744.68 μmol, 1 eq, HCl salt) and 2- chloropyrimidine (170.58 mg, 1.49 mmol, 2 eq) in THF (4 mL) was added TEA (226.06 mg, 2.23 mmol, 310.95 μL, 3 eq) in one portion at 25°C under N2. The mixture was stirred at 60°C for 12 hours under N2. One additional vial in 100 mg scale was set up as described above and these two reactions were combined. The mixture was diluted with H2O (50 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic layers were washed with brine (60 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (neutral condition,column: Waters Xbridge BEH C18100*30mm*10um;mobile phase: [H2O (10mM NH4HCO3)-ACN];gradient:30%-60% B over 8.0 min) and the desired eluent was lyophilized to give Compound 211 (26 mg, 84.43 μmol, 7.56% yield, 92% purity) as a light green solid:1H NMR (400 MHz, DMSO-d6) δ ppm 1.95 (s, 6 H) 2.80 - 2.89 (m, 4 H) 3.84 - 3.95 (m, 4 H) 6.61 (t, J=4.75 Hz, 1 H) 8.37 (d, J=4.75 Hz, 2 H); Method A LCMS (ESI+): 2.311 min, m / z 284 (M+H). Compound 212: 2,3-dimethyl-5,6,8,9-tetrahydro-1H-benzo[7]annulene-1,4,7-trione

[0274] To a solution of Intermediate 204-1 (1 g, 7.34 mmol, 1 eq) in ACN (100mL) and H2O (50 mL) was added 4-oxoheptanedioic acid (1.92 g, 11.02 mmol, 1.5 eq) andAgNO3 (1.50 g, 8.81 mmol, 1.2 eq) at 25°C. The mixture was stirred at 100°C for 45 mins.Then a mixture of ammonium persulfate (5.36 g, 23.50 mmol, 5.11 mL, 3.2 eq) in ACN (100 mL) and H2O (50 mL) was added to the solution dropwise at 25°C. The mixture was stirred at 100°C for 3 hours. TLC showed the reaction worked. The mixture was diluted with ethyl acetate (100 mL) and water (50 mL). The mixture was extracted with ethyl acetate (3 x 80 mL). The organic layer was washed with saturated sodium sulfite solution (100 mL), dried with Na2SO4, filtered and concentrated under reduced pressure to give the crude product. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=8%), and then purified by prep-HPLC (HCl system; column: Phenomenex luna C18100*40mm*5 um; mobile phase: [H2O (0.04% HCl)-ACN]; gradient: 25%-45% B over 8.0 min) and lyophilized to give Compound 212 (39.4 mg, 180.53 μmol, 2.46% yield) as a yellow solid:1H NMR (METHANOL-d4, 400 MHz) δ (ppm) 2.88-2.94 (m, 4H), 2.59-2.65 (m, 4H), 2.05 (s, 6H); Method A LCMS (ESI+): 2.245 min, m / z 219 (M+1). Compound 213: 7-(isopropylamino)-2,3-dimethyl-6,7,8,9-tetrahydro-1H-benzo[7]annulene- 1,4(5H)-dionea mg, eq, according to the general procedure for Compound 12) and propan-2-amine (21.94 mg, 371.14 μmol, 31.89 μL, 3 eq) in dichloromethane (1 mL) was added AcOH (7.43 mg, 123.71 μmol, 7.08 μL, 1 eq) dropwise at 25°C. The reaction mixture was stirred at 25°C for 30 minutes. Then sodium triacetoxyboranuide (65.55 mg, 309.28 μmol, 2.5 eq) was added to the mixture at 25°C. The reaction mixture was stirred at 25°C for 1 hour. The mixture was concentrated under reduced pressure to give the residue. The residue was purified by prep-HPLC (NH4HCO3 condition; column: Waters Xbridge Prep OBD C18150*40mm*10um;mobile phase: [H2O(10mM NH4HCO3)-ACN];gradient:25%-65% B over 8.0 min) and the desired eluent was lyophilized to give the product. The product was purified again by prep-HPLC (NH4HCO3condition; column: Waters Xbridge BEH C18100*25mm*10um;mobile phase: [H2O(10mM NH4HCO3)-ACN];gradient:15%-45% B over 8.0 min) and the desired eluent was lyophilized to give Compound 213 (9.2 mg, 34.22 μmol, 21.27% yield, 97.2% purity) as yellow solid:1H NMR (400 MHz, DMSO-d6) δ 0.95 (d, J=6.25 Hz, 6H), 1.00-1.11 (m, 2H), 1.13-1.27 (m, 1H), 1.74-1.87 (m, 2H), 1.95 (br s, 6H), 2.24 (br t, J=12.07 Hz, 2H), 2.74-2.82 (m, 1H), 2.82-2.88 (m, 1H), 2.88-3.00 (m, 2H); Method A LCMS (ESI+): 2.238 min, m / z 262 (M+1).Compound 214: 7,8-dimethyl-3-(2-(pyrrolidin-1-yl)pyrimidin-4-yl)-2,3,4,5-tetrahydro-1H- benzo[d]azepine-6,9-dione

[0276] To a solution of 2,4-dichloropyrimidine (5.7 g, 38.26 mmol, 1 eq) in THF (60 mL) was added pyrrolidine (5.44 g, 76.52 mmol, 6.39 mL, 2 eq) dropwise at 25°C. The mixture was stirred at 25°C for 2 hours under N2. One additional vial in 0.3 g scale was set up as described above and these two reactions were combined. Then the mixture was diluted with H2O (80 mL) and extracted with ethyl acetate (3 x 100 mL). The combined organic phases were washed with brine (100 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude. The crude was purified by reversed-phase HPLC (0.1% HCl condition). The desired eluent was concentrated under reduced pressure to remove the solvent and extracted with ethyl acetate (2 x 100 mL). The combined organic phases were dried over Na2SO4, filtered and concentrated under reduced pressure. Intermediate 214-1 (3 g, 15.52 mmol, 38.63% yield, 95% purity) and Intermediate 214-2 (1 g, 5.17 mmol, 12.88% yield, 95% purity) were obtained as white solid: 214-1:1H NMR (400 MHz, DMSO-d6) δ ppm 1.80 - 2.03 (m, 4 H) 3.33 - 3.50 (m, 4 H) 6.48 (d, J=6.13 Hz, 1 H) 8.01 (d, J=6.00 Hz, 1 H); 214-2:1H NMR (400 MHz, DMSO-d6) δ ppm 1.85 - 1.99 (m, 4 H) 3.45 (br s, 4 H) 6.67 (d, J=5.13 Hz, 1 H) 8.27 (d, J=5.13 Hz, 1 H).

[0277] To a solution of Compound 205 (150 mg, 620.57 μmol, 1 eq, HCl) in HFIP (5 mL) was added Intermediate 214-2 (179.94 mg, 930.85 μmol, 1.5 eq) dropwise at 25°C under N2.The mixture was stirred at 50°C for 12 hours under N2. LCMS showed 30% of starting material remained and 40% of desired mass was detected. Then the mixture was concentrated under reduced pressure to remove the solvent. The crude was purified by prep- HPLC (neutral condition, column: Waters Xbridge Prep OBD C18150*40mm*10um; mobile phase: [H2O (10mM NH4HCO3)-ACN];gradient:40%-80% B over 8.0 min) and the desired eluent was lyophilized to give Compound 214 (22 mg, 56.57 μmol, 9.12% yield, HCl) as a yellow solid:1H NMR (400 MHz, DMSO-d6) δ ppm 1.84 - 1.91 (m, 4 H) 1.94 (s, 6 H) 2.81 - 2.90 (m, 4 H) 3.42 (br t, J=6.44 Hz, 4 H) 3.77 (br s, 4 H) 6.05 (d, J=6.13 Hz, 1 H) 7.82 (d, J=6.13 Hz, 1 H); Method A LCMS (ESI+): 2.188 min, m / z 353 (M+H). Compound 215: 7,8-dimethyl-3-(4-(pyrrolidin-1-yl)pyrimidin-2-yl)-2,3,4,5-tetrahydro-1H- benzo[d]azepine-6,9-dione

[0278] To a solution of Compound 205 (150 mg, 620.57 μmol, 1 eq, HCl) in HFIP (5 mL) was added Intermediate 214-1 (179.94 mg, 930.85 μmol, 1.5 eq) in one portion at 25°C under N2. The mixture was stirred at 50°C for 72 hours under N2. Then the mixture was concentrated under reduced pressure to remove the solvent. The crude was purified by prep- HPLC (neutral condition, column: Waters Xbridge Prep OBD C18150*40mm*10um; mobile phase: [H2O (10mM NH4HCO3)-ACN]; gradient:15%-75% B over 8.0 min) and the desired eluent was lyophilized to give Compound 215 (30 mg, 83.42 μmol, 13.44% yield, 98% purity) as an orange solid:1H NMR (400 MHz, DMSO-d6) δ ppm 1.80 - 2.03 (m, 10 H) 2.81 (br s, 4 H) 3.32 - 3.55 (m, 4 H) 3.74 - 3.91 (m, 4 H) 5.73 (d, J=5.88 Hz, 1 H) 7.82 (d, J=5.88 Hz, 1 H); LCMS (ESI+): 2.205 min, m / z 353 (M+H). Compound 216: tert-butyl (3-(7,8-dimethyl-6,9-dioxo-1,2,4,5,6,9-hexahydro-3H- benzo[d]azepin-3-yl)propyl)(methyl)carbamate

[0279] To a solution of Compound 205 (300 mg, 1.24 mmol, 1 eq, HCl) and tert-butyl N- methyl-N-(3-oxopropyl)carbamate (697.16 mg, 3.72 mmol, 3 eq) in MeOH (4 mL) was added NaOAc (305.43 mg, 3.72 mmol, 3 eq) and AcOH (74.53 mg, 1.24 mmol, 71.05 μL, 1 eq) in order at 25°C. The mixture was stirred for 1 hr at 25°C. Then NaBH3CN (233.98 mg, 3.72 mmol, 3 eq) was added to the mixture and stirred for 12 hrs at 25°C. The mixturewas filtered. The filtrate was purified by prep-HPLC (FA condition, column: Phenomenexluna C18100*40 mm*3 um; mobile phase: [H2O (0.2% FA)-ACN]; gradient: 5%-40% B over 8.0 min) and lyophilized to give Compound 216 (255 mg, crude) as a purple solid:1H NMR (400 MHz, DMSO-d6) δ ppm 3.14 - 3.19 (m, 2 H), 2.76 (br s, 3 H), 2.70 - 2.74 (m, 4 H), 2.51 - 2.53 (m, 2 H), 2.49 - 2.49 (m, 2 H), 2.39 (br t, J=7.00 Hz, 2 H), 1.95 (s, 6 H), 1.58 - 1.64 (m, 2 H), 1.39 (s, 9 H). Compound 217: 7,8-dimethyl-3-(3-(methylamino)propyl)-2,3,4,5-tetrahydro-1H- benzo[d]azepine-6,9-dione

[0280] A mixture of Compound 216 (35 mg, 84.76 μmol, 1 eq, HCl) in EtOAc (2 mL) and HCl / EtOAc (2 mL) was stirred for 2 hrs at 25°C. The mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (HCl condition, column: Phenomenex luna C18100*40 mm*5 um; mobile phase: [H2O (0.04% HCl)-ACN]; gradient: 1%-20% B over 8.0 min) and lyophilized to give Compound 217 (5.6 mg, 15.63 μmol, 18.44% yield, 97.5% purity, 2HCl) as a yellow solid:1H NMR (400 MHz,METHANOL-d4) δ ppm 3.74 (br dd, J=13.01, 7.13 Hz, 2 H), 3.32 - 3.44 (m, 4 H), 3.21 (br t, J=12.01 Hz, 2 H), 3.10 - 3.15 (m, 2 H), 2.95 (br dd, J=17.70, 10.69 Hz, 2 H), 2.75 (s, 3 H), 2.17 - 2.26 (m, 2 H), 2.04 (s, 6 H); Method A LCMS (ESI+): m / z 277.1 (M+H). Compound 218: 3-(3-(dimethylamino)propyl)-7,8-dimethyl-2,3,4,5-tetrahydro-1H- benzo[d]azepine-6,9-dione 1.(HCHO)nO AcOH (1 eq) O HClNaOAc (3 eq)HCl25o1 hrwas added NaOAc (40.07 mg, 488.47 μmol, 3 eq), (HCHO)n (15.65 mg, 488.47 μmol, 3 eq) and AcOH (9.78 mg, 162.82 μmol, 9.32 μL, 1 eq) in order at 25°C. The mixture was stirred for 1 hr at 25°C. Then NaBH3CN (30.70 mg, 488.47 μmol, 3 eq) was added to the mixture in portions at 25°C. The mixture was stirred for 12 hrs at 25°C. One additional vial in 30 mg scale was set up as described above. The mixture was combined and filtered, and the filter cake was washed with 2 mL MeOH. The filtrate was purified by prep-HPLC (HCl condition, column: Phenomenex Luna C1875*30 mm*3 um; mobile phase: [H2O (0.04% HCl)-ACN]; gradient:1%-25% B over 8.0 min) and lyophilized to give Compound 218 (14.1 mg, 46.85 μmol, 21.78% yield, 96.5% purity) as a brick-red solid:1H NMR (400 MHz, DMSO-d6) δ ppm 3.56 - 3.66 (m, 2 H), 3.08 - 3.22 (m, 8 H), 2.84 - 2.92 (m, 2 H), 2.78 (s, 6 H), 2.07 - 2.18 (m, 2 H), 1.97 (s, 6 H); Method A LCMS (ESI+): m / z 293.1 (M+H). Compound 219: 3-(2-(dimethylamino)ethyl)-7,8-dimethyl-2,3,4,5-tetrahydro-1H- benzo[d]azepine-6,9-dionewas prepared according to the general procedures described in other Examples in the application.Example 2.15-LO Biological Data

[0283] Human fibroblast survival assay. All fibroblasts were obtained from the National Institute of Neurological Disorders and Stroke (NINDS) cell line repository housed at Rutgers University. Compounds were tested in at least one of the following Parkinson’s disease subject-derived fibroblast lines: Line ND29802 (LRRK2 G2019S mutation carrier), Line ND40996 (SNCA A53T mutation carrier), or Line ND34263 (GBA N370S mutation carrier).

[0284] Human fibroblasts were seeded into 96-well plates on Day 0. On Day 1, fibroblasts were challenged with RSL3 (a ferroptosis activator, CAS No.1219810-16-8) alone or in co- treatment with test compound administered in dose response. On Day 2, fibroblasts were fixed and stained by the nuclear stain 4’, 6-diamidino-2-phenylindole (DAPI), and the number of DAPI-positive nuclei were counted for each condition using the Yokogawa CQ1 imaging platform to quantify the EC50 for cell survival. Exemplary results are shown in Table 5 below. Table 5. Compound Activity Compound EC50 (nM)

[0285] Compounds were tested in the following survival assay. Human fibroblasts were maintained and passaged with standard protocols used by the field. Fibroblasts are split and plated at 3K / well in 96-well culture plates. After 24 hours, cells were treated with either DMSO (vehicle), 75nM RSL3 (challenge), or 75nM RSL3 with 100nM, 33nM, or 5nM doses of each compound. Each condition was performed in quadruplicate wells. After 24 hours, images of the cells were captured using the Sartorius IncuCyte SX5 imaging system and cell confluency was calculated using the built-in confluency calculation software. To determine % Cell Viability Rescue, the DMSO (vehicle) cell confluency was first set to 100% and the 75nM RSL3 (challenge) cell confluency was set to 0%. The data shown in Table 6 representsthe cell confluency of each condition (the average of quadruplicate wells) normalized to that scale. Table 6. Cell Viability of Human Fibroblasts with Compound after RSL3 Challenge % Cell Viability Rescue from RSL3 Challenge% Cell Viability Rescue from RSL3 ChallengeExample 3.15-Lipoxygenase-Mediated Lipid Peroxidation Regulates LRRK2 Kinase Activity A. Materials and Methods

[0286] Study Design: This study was designed to explore the mechanism(s) that driveendogenous WT LRRK2 kinase activation. To this end we used CRISPR / Cas9 gene-edited HEK293 cells, CRISPR / Cas9 gene-edited HAP1 cells, as well as RAW264.7 macrophages, patient-derived LCLs, and an in vivo rotenone rat model of PD to explore what physiological players regulate LRRK2 activation. To enhance scientific rigor, we generally used orthogonal approaches to support each conclusion. Furthermore, as target validation of 15-LO, specifically, 15-LO derived 4HNE, being upstream LRRK2 activation, we employed both a specific and highly potent pharmacological inhibitor and genetic knockout approaches to support key findings. To ensure clinical relevance and translational potential, key results were confirmed in patient-derived LCLs and in an in vivo model of PD. All in vitro experimentswere replicated independently at least three times and performed by multiple experimenters. In vivo experiments were conducted using a single cohort of rats that were assigned randomly to treatment group.

[0287] Cell Culture: HEK293 cells and RAW264.7 macrophages were cultured in DMEM / F12 (cat:11320082, Thermo Fisher) supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin (pen-strep).15-LOWT / WTand CRISPR / Cas9 gene-edited 15-LO- / -HAP1 cells were purchased from Horizon ( HZGHC007334c00) and cultured in IMDM (cat: 30-2005, ATCC) supplemented with 10% FBS and pen-strep. Cells were maintained at 37°C and 5% CO2. CRISPR / Cas9 gene-edited HEK293 cells were generated as previously described (9). LRRK2- / -(sc-6004) and LRRK2WT / WT(parental) (sc-6003) RAW264.7 macrophages were obtained from ATCC. Cell passage number did not exceed 20 for HEK293 or RAW264.7 macrophages. Cells were plated on poly-d-lysine coated coverslips in a 24-well plate or 16-well Nunc Lab Tek chamber slides (cat: 178599, Thermo Fisher). Details for reagents used for cell treatments can be found in Table 7. For H2O2, 4HNE, 12(S)HpETE, and 15(S)HpETE treatments, DMEM / F12 supplemented with 5% fetal bovine serum (FBS) and penicillin-streptomycin (pen-strep) was used. Table 7. Reagents used for cell culture treatments Reagent Catalog Vendor / Source Concentration Time Rotenone R8875-1G Sigma Aldrich 50nM 24-hourse ur ) orReagent Catalog Vendor / Source Concentration Time CU12991 Acurex Biosciences 20nM 24-hoursBiorepository through an MTA and sample ID numbers may be found in Table 8. Cells were maintained in RPMI medium GlutaMAX supplement (cat: 61870127, Thermo Fisher Scientific) with 15% FBS and pen-strep. Cells were plated on poly-d-lysine coated coverslips in a 24-well plate or 16-well Nunc Lab Tek chamber slides (cat: 178599, Thermo Fisher) and attached by light centrifugation at 690 x g at room temperature. Cells were passaged every 3 days and did not exceed 20 passages. Table 8. Lymphoblastoid cell line information Cell line ID LRRK2 Age at Mutation Clinical Status Sex sampling

[0289] CRISPR / Cas9 gene edited HEK293 cells (C2024A, C2025A, C2024A / C2025A): LRRK2C2024A / C2024A, LRRK2C2025A / C2025Aand double cysteine mutants were created in-house. A guide RNA targeting exon 41 of the LRRK2 gene (5’- CTCAGTACTGCTGTAGAATG- 3’ (SEQ ID NO: 3)) was commercially synthesized (ThermoFisher) and used to form ribonucleoprotein (RNP) complexes when combined with recombinant Cas9 protein (New England Biolabs). Ribonucleoprotein complexes were formed using the method of Kouranova49. RNP complexes were formed by incubating the recombinant Cas9 protein and guide RNA at 1:1 mass ratio (1: 4.6 molar ratio of the Cas9 protein to sgRNA) at 37°C for 5 min. RNPs were delivered to HEK293 cells using Transit-X2 (Mirius) transfection reagent as described by the manufacturer. Single stranded repair templates specific to the C2024 or C2025 residues were included in the transfection reactions to facilitate the cysteine to alanineconversion by homologous recombination. The double mutant C2024A / C2025A was generated in a similar fashion using the repair template specific to the double mutation. Transfected cells were collected and enriched by FACS sorting. Sorted cells were grown and expanded for PCR and DNA sequencing analyses. All clones generated by these methods were sequenced on both strands to confirm their identity. Protein expression – or lack thereof – was confirmed by western blotting. Using the same technology, LRRK2G2019S / G2019S, LRRK2R1441G / R1441G, LRRK2- / -cells were created. Details for CRISPR / Cas9 reagents (SEQ ID NO: 1 through 6, respectively) can be found in Table 9. Table 9. CRISPR / Cas9 reagentsy g g p previously (44). A 3 mM stock solution of DHE (cat: D11347, Thermo Fisher Scientific) was reconstituted in DMSO and stored at -20°C in single use aliquots. Prior to use, the DHE stock solution was thawed, protected from light, and diluted in cell culture media to reach a final concentration of 3μM DHE. Old media was aspirated and replaced with the fresh media containing 3μM DHE. Cells were incubated for 20 minutes at 37°C and 5% CO2, protected from light. Cells were then gently washed with phosphate buffer saline (PBS) at room temperature for 5 minutes and then fixed with 4% paraformaldehyde (PFA) (cat:15710-S, Electron Microscopy Science) for 20 minutes. Following fixation, cells underwent three 10- minute washes in PBS. Coverslips were mounted on glass slides (cat: 22-037-246, Fisher Scientific) using gelvatol mounting medium (see (34) for recipe).

[0291] Immunofluorescence: Cells were fixed in 4% paraformaldehyde in phosphate buffer saline (PBS) for 20 minutes at room temperature, followed by three 10-minute washes in PBSon a shaker. Cells were blocked and permeabilized in 10% normal donkey serum (NDS) + 0.02% triton-x in PBS for 1 hour at room temperature on a shaker. After blocking / permeabilization, cells underwent three washes in PBS and then were incubated in primary antibody (Table 10, for antibody details) in PBS containing 1% NDS overnight at 4ºC on a shaker. The following day, cells underwent three 10-minute washes in PBS and were incubated in secondary antibody in PBS containing 1% NDS for one hour at room temperature on a shaker protected from light. Cells underwent three 10-minute washes in PBS and stained with DAPI for two minutes, followed by two quick washes with PBS. Coverslips were mounted on glass slides (cat: 22-037-246, Fisher Scientific) using gelvatol mounting medium. Table 10. List of antibodies using for immunofluorescence, proximity ligation assay and western blot Antibody Catalog Vendor Dilution Rit 12 2 LRRK2 2 1 1 A1:1,000 (PL pS1292- ) ) – 1 )

[0292] Proximity Ligation Assay

[0293] Cells (Figures 1 and 3): Proximity ligation assay (PLA) was performed on an individual coverslip in 24 well plates using Duolink In Situ PLA reagents (DUO92002, DUO920024, DUO92007 (orange) or DUO92014 (green), Sigma Aldrich). Cells were fixed, blocked / permeabilized, and incubated in primary antibody solution as above and as previously described (see reference 47). Following overnight primary antibody incubation (Table 10, for antibody details), cells underwent three 10-minute washes in PBS. After washes, PLA was performed as described in (see reference 47). PLA incubation steps took place in a humidified incubator at 37ºC with gentle agitation taking care that samples did not dry out. For details of other products used, see reference 47.

[0294] Cells: Proximity ligation assay (PLA) was performed in 16-well Nunc Lab Tekchamber slides (cat: 178599, Thermo Fisher) using a NaveniFlex MR Kit (cat: NF.MR.100,Navinci). Cells were fixed, blocked / permeabilized, and incubated in primary antibody solution as above and as previously described (see reference 47). Following overnight primary antibody incubation (Table 10, for antibody details), cells underwent three 10-minute washes in PBS. After washes, PLA was performed according to manufacturer’s protocol. PLA incubation steps took place in a humidified incubator at 37ºC with gentle agitation taking care that samples did not dry out. For details of other products used, see reference 47.

[0295] Rat brain tissue: PLA was performed in blocked and permeabilized brain tissue as previously described (see reference 47) using NaveniFlex Tissue MR Red (NT.MR.100.RED). Free floating tissue sections were blocked and permeabilized in 1% triton-x (in PBS) containing 10% NDS for one hour at room temperature on a shaker. Sections were then washed three times (10 minutes per wash) and incubated in primary antibody solution (1% NDS in PBS) to label tyrosine hydroxylase (Table 10, for antibody details) overnight at 4ºC on a shaker. The following day the tissue sections underwent three 10-minute washes in PBS and were incubated in secondary antibody in PBS containing 1% NDS for one hour at room temperature on a shaker protected from light. After secondary antibody labeling, tissue sections were incubated overnight 4ºC on a shaker in primary antibody solution containing the antibodies for PLA (Table 10, for antibody details). Next day, tissue sections underwent three 10-minute washes in PBS and then were mounted on glass slides (cat: 22-037-246, Fisher Scientific). See reference 47 for detailed description and video for mounting of tissue prior to PLA. PLA was performed following the manufacturer’s protocol. PLA incubation steps took place at 37ºC ensuring that samples did not dry out.After PLA was completed, sections were covered with a glass coverslip with gelvatol mounting medium.

[0296] Animals: All experiments utilizing animals were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh. Male Lewis rats (8-9 months old) received a single daily interperitoneal (i.p.) injection of 2.8 mg / kg of rotenone resuspended in 2% DMSO, 98% miglyol 812 N as previously described (23, 48) until they reached behavioral endpoint (7-10 injections). Animals were euthanized using 0.3 mg / kg pentobarbital, followed by transcardial perfusion. Brains were removed and postfixed in 4% paraformaldehyde for 24-h before placing them 30% sucrose. Free-floating sections (35μm) were collected using a microtome and stored in cyroprotectant at −20 °C until use.

[0297] Cell lysis and western blot: Cells were collected on ice in 1X Cell Signaling lysis buffer (cat:9803, Cell Signaling) containing 1:33 Halt Phosphatase Inhibitors (cat:78427, Thermo Fisher Scientific) and 1:100 Halt Protease and Phosphatase Inhibitors (cat:78446, Thermo Fisher Scientific). Cells were lysed using a freeze-thaw lysis method where cells were incubated in pulverized dry ice with ethanol for three minutes followed by a warm water bath for three minutes and was repeated three times. Lysates were clarified by centrifugation at 12,000 rpm for 10 minutes 4°C. Supernatant of the cell lysates were collected, and protein quantified by Pierce BCA kit.25-45μg of samples (dependent on target) was mixed with 4X NuPAGE LDS Sample Buffer (supplemented with 5% β- mercaptoethanol and 10% glycerol). Samples were run in duplicates on 4-12% NuPAGE or 10% NuPAGE Bis-Tris midi gels with MOPS Running Buffer at 120 Volts for eight minutes followed by 200 Volts for 45 minutes. At the end of electrophoresis, samples were transferred onto nitrocellulose membranes (0.2μm) at 100 Volts for 60 minutes on ice in prechilled transfer buffer (25mM Tris-Base, 198mM Glycine, 20% methanol v / v). Transferred membranes were blocked in Fish Serum Blocking Buffer (cat:37527, Thermo Scientific) diluted in PBS (1:1 ratio) for one hour at room temperature on a shaker. Membranes were incubated in primary antibodies (Table 10, for antibody details) diluted in Fish Serum Blocking Buffer / PBS overnight at 4ºC on a shaker. The following day, membranes were washed in PBS three times 10 minutes each and incubated in LiCor secondary antibodies(Table 10, for antibody details). diluted in Fish Serum Blocking Buffer / PBS for 2 hours at room temperature on a shaker. Membranes were washed three times in PBS for a duration of 10 minutes each and imaged on an Odyssey LiCor CLx imaging system. Analysis was performed using Image Studio software.

[0298] Confocal microscopy and analysis: Images were acquired on a Nikon Eclipse Ti2 Resonance Scan Spectral Confocal Microscope at 60X magnification using Nyquist criterion and resonant scanning. Laser parameters were set up on positive and negative controls (e.g. primary antibody delete) to ensure there was no pixel saturation. For quantitative comparisons all imaging parameters (e.g. laser power, gain, pinhole) were held constant across an experiment. Confocal images were analyzed using Nikon NIS-Elements Advanced Research software. For DHE fluorescence intensity analysis, regions of interest were drawn around the signal on a per cell basis to obtain the fluorescence intensity. For all other analyses, a binary layer was created using threshold parameters for each experiment and used to identify each puncta or ‘object’. For colocalization analysis the intersection function was used to determine the number of PEBP1 objects that overlapped with the 15-LO objects. These threshold parameters were held constant across images for each experiment.

[0299] For Figures 1 and 3 images were acquired on Olympus BX61 confocal microscope at 60X magnification (UPlansApo 60X) and Fluoview 1000 software (Melville, NY). Imaging parameters were monitored to ensure that images were above background level and below pixel saturation. For quantitative comparisons between groups, all imaging parameters (e.g. laser power, exposure, and pinhole) were held constant across. For PL pS1292-LRRK2 (Figures 1 and 3, Sigma) intensity analysis, regions of interest were drawn around the signal on a per cell basis to obtain the fluorescence intensity.

[0300] Statistical Analysis: Results are presented as mean ± SEM and are derived from 3 - 5 independent experiments. For simple comparisons of two experimental conditions, two- tailed unpaired t-tests were used. Where variances were unequal Welch’s correction was used. For comparisons of multiple experimental conditions, one-way or two-way ANOVA was used, and if significant overall, post hoc corrections (with Tukey tests) for multiple comparisons were made. P-values less than 0.05 were considered significant. B. ResultsStimulated endogenous LRRK2 activity is redox dependent

[0301] The regulation of WT LRRK2 kinase activity has yet to be fully elucidated, but there is accumulating evidence that it is a sensitive to ROS (8). Several reports indicate that LRRK2 kinase activity is increased by H2O2 and decreased by the antioxidant curcumin (9, 18, 31, 32). The kinase activity of WT endogenous LRRK2 can be elevated dose dependentlyby physiological concentrations of H2O2, and this can be blocked by the ROS scavenger α- tocopherol (9), which is both a ROS scavenger and 15-LO inhibitor (33). To further investigate its regulation, we examined how quickly H2O2could increase endogenous WT LRRK2 activation in a cellular context. Human embryonic kidney 293 cells (HEK293) were treated with 5μM H2O2for 5, 10, and 15 minutes and endogenous WT LRRK2 kinase activity was assessed by a proximity ligation (PL) assay that detects phosphorylation status of the autophosphorylation site, Ser1292, (PL pSer1292–LRRK2) (9, 34). We found there was robust PL pSer1292–LRRK2 signal within 5 minutes after H2O2 exposure, that was maximal by about 10 minutes and sustained for at least 30 minutes (Fig.1A-B). Pretreatment of cells with the thiol antioxidant, N-acetylcysteine (NAC), prevented the H2O2 induced increase in PL pSer1292-LRRK2 signal in WT HEK293 cells compared to vehicle (p<0.0001; two-way analysis of variance (ANOVA) with Tukey correction) (Fig.1C-D), thereby confirming the redox sensitivity of LRRK2 kinase activation.

[0302] Activation of WT LRRK2 can occur in response to different cellular stressors such as mitochondrial dysfunction, lysosomal dyshomeostasis, and impaired trafficking deficits (9, 20-22, 34). Therefore, we next explored whether known LRRK2 activators, such as rotenone, chloroquine, and monensin might also function in LRRK2 activation via an oxidative mechanism. Rotenone is a mitochondrial complex I inhibitor that causes ROS production and mitochondrial dysfunction in cells (35). In WT HEK293 cells, sublethal rotenone treatment elicited an increase in PL pSer1292-LRRK2 signal compared to vehicle (p<0.0001; two-way ANOVA with Tukey correction) (Fig.2A, B). The rotenone induced increase in PL pSer1292-LRRK2 signal was prevented by co-treatment with NAC (p<0.0001; two-way ANOVA with Tukey correction) (Fig.2A, B), suggesting that rotenone induced LRRK2 activation is ROS dependent. Congruent results were obtained by western blotting for the phosphorylation state of the LRRK2 substrate, Rab10 (14) (FIG.8). Rotenone treatment led to an increase in pThr73-Rab10 compared to vehicle (p<0.05; two-way ANOVA with Tukey correction), and this increase was prevented by NAC co-treatment (p=0.05; one-way ANOVA with Tukey correction) (FIG.8A, B). We confirmed these findings using a third method, a PL assay to amplify the specific signal of pThr73-Rab10: PL (pThr73-Rab10) – Rab10. Using this assay, there was a baseline pThr73-Rab10– Rab10 signal in WT HEK293 cells that was reduced in the presence of the LRRK2 kinase inhibitor, PF360 (p<0.0001; two- way ANOVA with Tukey correction) (FIG.9A, B). Importantly, the baseline pThr73-Rab10– Rab10 signal in WT HEK293 cells was higher than that in LRRK2- / -HEK293 cells(p<0.0001; two-way ANOVA with Tukey correction) (FIG.9A, B). Similar to the results obtained with the PL pS1292-LRRK2 and pThr73-Rab10 western blotting, rotenone elicited an increase in PL pThr73-Rab10 – Rab10 signal (p<0.05; two-way ANOVA with Tukey correction), that was prevented by co-treatment with NAC (p<0.01; two-way ANOVA with Tukey correction) (FIG.8E, F).

[0303] Because LRRK2 is highly expressed in immune cells, and there is accumulating evidence suggesting that LRRK2 plays a key role in inflammatory responses, we assessed whether rotenone induced kinase activation was also redox dependent in RAW264.7 macrophages. Consistent with our results obtained in HEK293 cells; rotenone elicited an increase in endogenous LRRK2 activity as assessed by PL pSer1292–LRRK2 in WT RAW264.7 macrophages (Fig.2A, C). Co-treatment with NAC prevented the rotenone induced increase in PL pSer1292–LRRK2 signal (Fig.2A, C). Congruent results were obtained using the orthogonal assays of pThr73-Rab10 detection by western blot (p<0.05; two-way ANOVA with Tukey correction) (FIG.8A, C) and PL pThr73-Rab10–Rab10 (p<0.0001; two-way ANOVA with Tukey correction) (FIG.8D, F). Treatment with NAC effectively blocked rotenone induced increase in pThr73-Rab10 western blot signal (p<0.05; two-way ANOVA with Tukey correction) (FIG.8A, C) and PL pThr73-Rab10–Rab10 signal (p<0.0001; two-way ANOVA with Tukey correction) (FIG.8D, F). Thus, using 3 three separate assays (PL pSer1292-LRRK2, pThr73-Rab10 immunoblot, PL pThr73-Rab10– Rab10) and two different cell lines (HEK293 and RAW264.7 macrophages), we found that that rotenone stimulates endogenous LRRK2 kinase activity in ROS-dependent manner.

[0304] As noted, disruption of endolysosomal function or trafficking using chloroquine or monensin activates WT LRRK2 kinase (21, 22, 34). How these induced deficits in the endolysosomal system regulate LRRK2 kinase activity is unclear however. Given our results with rotenone, we examined whether chloroquine or monensin activates LRRK2 kinase in a ROS dependent fashion. First, we assessed if chloroquine or monensin causes ROS production or oxidative damage. Using parameters that have been shown to elicit endogenous LRRK2 activity (chloroquine: 100 μM for 3 hours (34); monensin: 10 μM for 4 hours (22)) we assessed cytoplasmic superoxide production using dihydroethidium (DHE). Under these conditions, both chloroquine and monensin elicited a significant increase in DHE signal compared to vehicle (chloroquine: p<0.005, monensin: p<0.001; one-way ANOVA with Tukey correction) (FIG.10A, B) in WT HEK293 cells. Thus, these compounds cause ROS production at concentrations and time points that are known to induce LRRK2 activity. Wealso examined ROS production at an earlier time point of 1hour. WT HEK293 cells were preloaded with DHE and 100 μM chloroquine (1 hour), 10 μM monensin (1 hour) and ROS production was compared to treatment with 5 μM H2O2(for 1 hour) or 100 μM H2O2(for 15 minutes). Both chloroquine and monensin resulted in an elevated DHE signal one hour after treatment compared to vehicle (chloroquine: p<0.05, monensin: p<0.005; one-way ANOVA with Tukey correction) (FIG.10C, D). The DHE signal in response to chloroquine or monensin was comparable to that measured in presence of 5 μM H2O2or 100 μM H2O2(FIG. 10C, D).

[0305] Excessive ROS production or inadequate antioxidant activity may lead to lipid peroxidation and accumulation of the lipid peroxidation end-product, 4-hydroxynoneal (4HNE). Consistent with the ROS production it induced, chloroquine caused an increase in 4HNE at 1 hour (p<0.05; two-way ANOVA with Tukey correction) and 3 hours (p<0.001; two-way ANOVA with Tukey correction), which was prevented by cotreatment with NAC (FIG.10E, F). Similarly, monensin caused an increase in 4HNE at 4 hours (p<0.001; two- way ANOVA with Tukey correction), which was prevented by cotreatment with NAC (FIG. 10G, H). Given that these LRRK2 kinase activators cause ROS production and lipid peroxidation that is prevented by NAC, we explored whether NAC could prevent chloroquine or monensin induced LRRK2 kinase activity. In fact, both chloroquine (Fig.2D, E) and monensin (Fig.2F, G) treatment of WT HEK293 cells led to an increase in PL pSer1292– LRRK2 signal (p<0.0001; two-way ANOVA with Tukey correction), that was prevented by NAC cotreatment. Together, these results indicate that known LRRK2 kinase activators that result in either mitochondrial dysfunction (rotenone), lysosomal dyshomeostasis (chloroquine), or pleiotropic trafficking deficits (monensin) may activate LRRK2 through a common oxidative mechanism. Kinase activation loop cysteines 2024 and 2025 are important for oxidative activation of LRRK2

[0306] The dynamics of the kinase activation loop plays a critical role in driving LRRK2 activation (12, 13). Cysteine residues on proteins can readily undergo oxidative modifications which, in turn, can influence function. Interestingly, the kinase activation loop of LRRK2 contains two solvent-exposed vicinal cysteine residues, Cys2024 and 2025. These residues are conserved across many species (36) and are not present in LRRK1 (37), suggesting that they may have a specific role in LRRK2 function. As such, we hypothesized that Cys2024and / or 2025 are important for sensing the state of the local subcellular environment (12) and may play a role in LRRK2 activation. Therefore, we generated CRISPR / Cas9 gene edited mutant HEK293 cells with each these cysteines mutated individually to an alanine (LRRK2C2024Aand LRRK2C2025A) and a double mutant (LRRK2C2024A+C2025A). As a prerequisite to using these cells, we first needed to be certain that the mutations did not alter (“kill”) baseline LRRK2 kinase activity. Consequently, we assessed how the mutations influenced basal kinase activity using 2 assays: PL pSer1292–LRRK2 and PL pThr73- Rab10–Rab10 (FIG.11A-D). Under vehicle conditions, LRRK2C2024A, LRRK2C2025A, and LRRK2C2024A+C2025Acells had PL pSer1292–LRRK2 and PL pThr73-Rab10–Rab10 signals equivalent to LRRK2WT / WT(FIG.11A-D). Importantly, PF360 treatment decreased basal PL pSer1292–LRRK2 and PL pThr73-Rab10–Rab10 signals (p<0.0001; two-way ANOVA with Tukey correction) (FIG.11A-D) in all cell lines.

[0307] Having demonstrated that that baseline kinase activity of endogenous LRRK2 was not reduced in any of the cysteine mutant cell lines, we examined their responses to oxidative challenges. We found that while H2O2 exposure elevated PL pS1292–LRRK2 signal in WT HEK293 cells (p<0.0001; two-way ANOVA with Tukey correction), there was no increase in PL pS1292–LRRK2 signal in any of the cysteine mutant cell lines (Fig.3A, B). We next explored if rotenone, chloroquine, or monensin were able to enhance kinase activity in LRRK2C2024A, LRRK2C2025A, and LRRK2C2024A+C2025AHEK293 cells. In WT HEK293 cells, rotenone, chloroquine, and monensin treatment each led to an increase in PL pSer1292– LRRK2 signal compared to vehicle (p<0.0001; two-way ANOVA with Tukey correction) (Fig.3C-H). Strikingly, in LRRK2C2024A, LRRK2C2025A, and LRRK2C2024A+C2025Acells there was no increase in PL pSer1292–LRRK2 signal in response to rotenone, chloroquine or monensin (Fig.3C-H).

[0308] Although mutation of Cys2024 and / or Cys2025 does not affect basal LRRK2 activity, it has been previously shown that endogenous LRRK2 activity regulates rotenone induced ROS production (38). To assess whether there was a downstream consequence of mutating these cysteine residues (thereby blocking the ability of rotenone to stimulate kinase activity) rotenone induced ROS production was assessed in each of the cell lines (FIG.12). Rotenone treatment led to an increase in DHE signal in WT HEK293 cells (p<0.0001; two- way ANOVA with Tukey correction), but not in LRRK2C2024A, LRRK2C2025Aor LRRK2C2024A+C2025Acells (FIG.12). Together, these results indicate that Cys2024 and Cys2025 are important residues that regulate kinase activation in a redox responsive fashion.4HNE is a critical signaling mediator of LRRK2 kinase activity

[0309] LRRK2 kinase activity has been shown to be provoked by membrane damaging agents (19, 20) which also cause lipid peroxidation and consequent accumulation of the peroxidation end-product, 4HNE, a reactive aldehyde.4HNE has been reported to interact with and activate several kinases, including ERK, JNK, Src and p38 MAPK (25-28). As such, we examined whether exogenous 4HNE was able to interact with and activate endogenous WT LRRK2. For this purpose, we devised a new PL assay, PL LRRK2–4HNE, using antibodies that recognize 4HNE and total LRRK2, as an index of the formation of 4HNE- LRRK2 adducts. When WT HEK293 cells were treated with a pathophysiological concentration of 4HNE (100 μM for 1 hour), there was an increase in PL LRRK2–4HNE signal, which was prevented by pretreatment with NAC (FIG.13). Corresponding with the increase in PL LRRK2–4HNE signal, there was a concomitant increase in endogenous WT LRRK2 kinase activity as measured by PL pSer1292–LRRK2 (p<0.005; two-way ANOVA with Tukey correction) (FIG.14A, B), and PL pThr73-Rab10–Rab10 (p<0.005; two-way ANOVA with Tukey correction (FIG.14C, D). NAC pretreatment prevented the 4HNE- induced increase in both PL pSer1292-LRRK2 (FIG.14A, B), and PL pThr73-Rab10–Rab10 (FIG.14C, D). These results demonstrate that exogenous 4HNE can form adducts with and activate endogenous WT LRRK2.

[0310] To further examine the relationship between LRRK2-4HNE adducts and LRRK2 kinase activity, WT HEK293 cells were treated with varying concentrations of 4HNE (10 – 100 µM for 1 hour) and LRRK2-4HNE adducts and LRRK2 activity were measured by PL. In WT HEK293 cells, increasing concentrations of 4HNE caused parallel, dose-dependent increases in PL LRRK2-4HNE and PL pSer1292-LRRK2 signals (Fig.4A-D). In fact, within this concentration range, there was a highly significant linear relationship between the degree of LRRK2-4HNE adduct formation and LRRK2 kinase activity (p<0.005; R2=0.99) (FIG. 15). Given that 4HNE has a high affinity for cysteine residues, we also investigated whether the cysteine mutants influence LRRK2-4HNE adduct formation and 4HNE induced activity. Surprisingly, in LRRK2C2024A+C2025AHEK293 cells 4HNE treatment did not increase PL LRRK2-4HNE or PL pSer1292-LRRK2 signals (Fig.4A-D). This suggests that, within this concentration range, 4HNE binds preferentially to the Cys2024 and Cys2025 residues of LRRK2 in a cellular context.

[0311] To explore whether the interaction between LRRK2 and 4HNE occurred with endogenous generation of 4HNE in response to stimuli that activate LRRK2, we examined the effects of rotenone, chloroquine and monensin. Compared to vehicle, rotenone elicited an increase in PL LRRK2–4HNE signal in LRRK2WT / WTHEK293 (p<0.0001; two-way ANOVA with Tukey correction) and RAW267.4 macrophages (p<0.0001; two-way ANOVA with Tukey correction) (FIG.16). Cotreatment with ^^-tocopherol, a potent ROS scavenger that can protect against lipid peroxidation (42, 43), prevented the rotenone-induced increase in PL LRRK2-4HNE signal (FIG.16). Similar to rotenone, both chloroquine and monensin treatment led to an increase in PL LRRK2-4HNE signal compared to vehicle (p<0.0001; two- way ANOVA with Tukey correction) (FIG.17). Cotreatment with ^^-tocopherol prevented the chloroquine- or monensin-induced increase in PL LRRK2-4HNE (FIG.17).

[0312] We previously showed there is 4HNE accumulation and elevated LRRK2 kinase activity in dopaminergic neurons of the substantia nigra (SN) in the rotenone rat model of PD (9, 44, 45). Therefore, we investigated whether LRRK2-4HNE adduct formation also occurred in this model. As described (45), middle-aged rats were treated with rotenone for 7- 10 days until they reached behavioral endpoint, at which time they were euthanized for analysis. Rotenone treatment led to a marked increase in PL LRRK2–4HNE signal in SN dopamine neurons compared to vehicle (Fig.4E, G)) (p<0.01; two tailed unpaired t-test with Welch’s correction), thereby providing in vivo relevance of LRRK2–4HNE adduct formation. Together, these data demonstrate that the lipid peroxidation product, 4HNE, may be a key signaling mediator in LRRK2 activation that links membrane damage and LRRK2 kinase activation. 15-Lipoxygenase regulates LRRK2 kinase activity

[0313] Because 4HNE forms adducts with LRRK2 and regulates its kinase activation, we explored the possibility of manipulating upstream sources of this reactive aldehyde.4HNE can be produced nonenzymatically when membrane lipids react directly with ROS; alternatively, it can be produced enzymatically as the end-product of 15-lipoxygenase (15- LO) activity (29, 30). Our data demonstrate that ^^-tocopherol can prevent stimulus-induced PL LRRK2-4HNE in an endogenous context, but whether this is due to its ROS scavenging properties or its ability to inhibit 15-LO is uncertain (33). To specifically assess the role of 15-LO in producing the pool of 4HNE that activates LRRK2, we took both genetic and pharmacological approaches. We utilized CRISPR / Cas9 gene-edited 15-LO knockout (15-LO- / -) HAP1 cells and a novel, potent and selective small molecule 15-LO inhibitor, CU12991, that has an IC50 of 2.5 nM for inhibition of rotenone-induced 4HNE production (FIG.18). As a prerequisite, we assessed rotenone-induced 4HNE production in WT and 15- LO- / -cells (FIG.19A, B). In WT cells, rotenone elicited an increase in cellular 4HNE, which was prevented by cotreatment with 20 nM CU12991 (p < 0.0001; two-way ANOVA with Tukey correction) (FIG.19A, B). There was no rotenone-induced 4HNE accumulation in 15- LO- / -cells and CU12991 had no effect in these cells (FIG.19A, B). Thus, in absence of 15- LO, the enzymatic source of 4HNE, there was no rotenone-induced 4HNE production.

[0314] We next explored whether 15-LO-derived 4HNE is a mediator of rotenone-induced LRRK2 activity. First, we assessed LRRK2–4HNE adduct formation via PL LRRK2–4HNE. Congruent with our previous observations (FIG.4 and FIG.16), in WT cells, rotenone elicited an increase in PL LRRK2–4HNE signal compared vehicle (p<0.0005; two-way ANOVA with Tukey correction) that was prevented by cotreatment with ^^-tocopherol (Fig. 5A, B). Strikingly, there was no rotenone-induced PL LRRK2–4HNE signal in 15-LO- / -cells (Fig.5A, B). Next, we assessed kinase activation by both autophosphorylation (Fig.5C, D), and substrate phosphorylation (FIG.19C, D). In WT cells, rotenone elicited an increase in PL pS1292 – LRRK2 (p<0.0001; two-way ANOVA with Tukey correction) (Fig.5C, D) and PL pT73(Rab10) – Rab10 (p<0.0001; two-way ANOVA with Tukey correction) (FIG.19C, D). Interestingly, 15-LO inhibition by CU12991 mitigated rotenone-induced PL pS1292 – LRRK2 (Fig.5C, D) and PL pT73(Rab10) – Rab10 (FIG.19C, D), but did not reduce LRRK2 kinase activity below basal levels (Fig.5C, D). In contrast, PF360 treatment completely abolished both basal and rotenone-induced kinase activity (PL pS1292 – LRRK2, Fig.5C, D) (PL pT73(Rab10) – Rab10, FIG.19C, D).

[0315] Congruent with our results using CU12991, in 15-LO- / -cells there was no rotenone- induced PL pS1292–LRRK2 (Fig.5C, D) or PL pT73(Rab10)–Rab10 (FIG.19C, D), but basal kinase activity was preserved. Importantly CU12991 did not impact LRRK2 activity in 15-LO- / -cells (Fig.5C, D and FIG.19C, D). Furthermore, to ensure CU12991 was not directly interfering with 4HNE, we treated cells with exogenous 4HNE and found that induced kinase activity was unaffected by CU12991 (FIG.20).

[0316] To ensure that the stimulatory effect of 15-LO on LRRK2 kinase activity was due to its production of 4HNE, we treated cells with exogenous 4HNE (Fig.5E, F). In both WT and 15-LO- / -cells, 4HNE elicited an increase in PL pS1292 – LRRK2 signal (p<0.005; two-wayANOVA with Tukey correction), which was prevented by treatment with a saturating concentration of NAC (Fig.5E, F). Thus, the negative impact on LRRK2 stimulation of pharmacological inhibition or genetic deletion of 15-LO can be overcome by exogenous 4HNE. Collectively, these results demonstrate that 4HNE generated by 15-LO forms adducts with LRRK2, which in turn leads to LRRK2 hyperactivation. Thus, LRRK2 kinase activation is regulated specifically by the 4HNE that is produced by 15-LO as an end-product of lipid peroxidation.

[0317] Accumulation of 4HNE in the SN of PD post-mortem human brain tissue has been recognized for 30 years (46); however, a functional role of 15-LO-derived 4HNE in PD has yet to be explored. Therefore, we assessed 4HNE levels in patient-derived lymphoblastoid cell lines (LCLs) derived from healthy control subjects, G2019S-LRRK2 mutation carriers, and iPD patients. Relative to healthy controls, 4HNE was elevated in LCLs derived from subjects with iPD as well as subjects harboring a G2019S mutation (p < 0.0001 for both iPD and G2019S compared to healthy controls; two-way ANOVA with Tukey correction) (Fig. 5H). The elevated 4HNE signal in the iPD subjects and G2019S mutation carriers was reduced to healthy control levels by treatment with CU12991 (p < 0.0001 for both iPD and G2019S; two-way ANOVA with Tukey correction two-way ANOVA with Tukey correction) (Fig.5H). This suggests that 15-LO is responsible for the pathologic accumulation of 4HNE observed in G2019S and iPD patient samples.

[0318] We next investigated whether 15-LO-derived 4HNE was responsible for LRRK2– 4HNE adduct formation elicited by other known LRRK2 activators: chloroquine, monensin, or rotenone. Consistent with our results in HEK cells (FIG.16, FIG.17), in WT RAW264.7 macrophages, chloroquine, monensin, and rotenone each elicited an increase in PL LRRK2– 4HNE signal (Fig.6A, B). As observed in 15-LO- / -HAP1 cells (Fig.5A, B), 15-LO inhibition with CU12991 prevented formation rotenone-induced LRRK2–4HNE adducts (Fig.6A, B) in WT RAW264.7 macrophages. Similarly, CU12991 cotreatment prevented chloroquine and monensin induced PL LRRK2–4HNE signal in WT RAW264.7 macrophages. Under the same conditions, chloroquine, monensin, and rotenone each increased PL pS1292–LRRK2 signal significantly in WT RAW264.7 macrophages (Fig.6C, D). Strikingly, CU12991 cotreatment completely prevented rotenone, chloroquine and monensin induced PL pS1292–LRRK2 signal (Fig.6A, B). As an orthogonal approach for LRRK2 kinase activity, we measured substrate phosphorylation via pT73-Rab10 western blot. Chloroquine, monensin, and rotenone each led to an increase in pT73-Rab10, which wasprevented by cotreatment with CU12991 (FIG.21). Thus, using genetic and pharmacological approaches, and 3 independent LRRK2 activity assays (PL pS1292–LRRK2, pThr-73-Rab10 western blot, and PL pT73-(Rab10)–Rab10 our results indicate that endogenous stimulated LRRK2 activity is regulated by 15-LO.

[0319] Although both 15-LO and 12-lipoxygenase (12-LO) can participate in lipid peroxidation, only 15-LO activity results in formation of 4HNE. The initial step in this enzymatic pathway (for both 15-LO and 12-LO) produces their respective hydroperoxy lipid metabolites: 15(S)-HpETE for 15-LO and 12(S)-HpETE for 12-LO (29, 30). Further peroxidation of 15(S)-HpETE, but not 12(S)-HpETE, produces 4HNE (47). To further validate the 15-LO pathway and subsequent 4HNE formation as a regulator of LRRK2 kinase activity, we treated WT RAW264.7 macrophages with either 15(S)-HpETE or 12(S)-HpETE and assessed endogenous LRRK2 kinase activation by PL pS1292–LRRK2.12(S)-HpETE treatment (0.25 – 25 µM) did not increase PL pS1292–LRRK2 signal (Fig.6E, F); however, 15(S)-HpETE dose-dependently increased PL pS1292–LRRK2 signal (p<0.0001; two-way ANOVA with Tukey correction) (Fig.6E, F). NAC pretreatment prevented 15(S)-HpETE- induced PL pS1292–LRRK2 signal (Fig.6E, F). Together, these results indicate that LRRK2 kinase activation was driven by the peroxidation of 15(S)HpETE to 4HNE.

[0320] To determine the PD relevance of 15-LO regulated LRRK2 activity, we next investigated if LRRK2 activity was elevated in G2019S mutation carriers and iPD patient- derived LCLs. Relative to healthy controls, there was elevated PL pS1292 -LRRK2 in both G2019S (p < 0.0001; two-way ANOVA with Tukey correction) and iPD LCLs (p < 0.005; two-way ANOVA with Tukey correction) that was completely abolished by PF360 (Fig.6G, H). Strikingly, CU12991 reduced pathologic hyperactive LRRK2 activity in both G2019S and iPD LCLs back to basal healthy control levels but did not completely inhibit LRRK2 activity (Fig.6G, H). Collectively, these data provide strong evidence that the 15-LO activity regulates stimulated endogenous LRRK2 kinase activity through the enzymatic production of 4HNE and its subsequent adduct formation.

[0321] The ability of 15-LO to peroxidize membrane phospholipids depends on its association with the small scaffolding protein, phosphatidylethanolamine-binding protein (PEBP1) (48). Typically, PEBP1 is in complex with RAF1 kinase; however, upon phosphorylation, it is liberated and interacts with new proteins (49), including 15-LO. In the unbound state, 15-LO can only oxygenate free fatty acids, not those incorporated intomembranes. However, when complexed with PEPB1 it preferentially oxygenates membrane phosphatidylethanolamine (48, 50), the first step in production of 4HNE. Therefore, we explored the interaction between PEPB1 and 15-LO in a system (rotenone treated RAW264.7 macrophages) in which endogenous 4HNE production is stimulated with resultant LRRK2 kinase activation. Quantitative confocal immunofluorescence staining revealed that rotenone treatment markedly increased the colocalization of the PEBP1 and 15-LO objects (p < 0.005; two tailed unpaired t-test with Welch’s correction) (Fig.7A, B). Using a complimentary approach, we developed a PL assay that detects the interaction between PEBP1 and 15-LO (PL PEBP1–15-LO). Similar to our colocalization results, rotenone induced a strong PL PEBP1–15-LO signal compared to vehicle (Fig.7C, D).

[0322] We also examined if this occurred in vivo in the rotenone rat model of PD (Fig.7E, F). Analogous to our findings in macrophages, there was an increase in PL PEBP1–15-LO signal in dopaminergic neurons in rotenone treated rats compared to vehicle (p < 0.0005; two tailed unpaired t-test with Welch’s correction) (Fig.7E, F). To investigate if this was relevant in the human disease, we sought to determine if PL PEBP1–15-LO was increased LCLs derived from G2019S and iPD patients compared to healthy controls. Indeed, there was elevated PL PEBP1–15-LO signal in G2019S- (p < 0.05; one-way ANOVA with Tukey correction) and iPD- (p=0.005; one-way ANOVA with Tukey correction) derived LCLs compared to healthy controls (Fig.7G, H). Thus, using complimentary approaches in multiple model systems and PD patient samples, the data demonstrate that under conditions that generate endogenous 4HNE, and which stimulate LRRK2 kinase activity, there is an enhanced association between 15-LO and PEBP1. C. Discussion

[0323] Pathogenic mutations in LRRK2 that cause elevated kinase activity are predominantly driven by altered kinetics, altered GTP hydrolysis or enhanced substrate specificity (8, 12, 13, 51). Although pathogenic mutations can each increase LRRK2 kinase activity, different mutations do so in distinct ways, thus pointing to the complexity LRRK2 activation. When assessing kinase activity, there are advantages to cellular in situ assays as opposed to in vitro kinase assays, as cellular context is important for LRRK2 activation (6), and upstream signaling events may be critical with regard to how and where endogenous WT LRRK2 activation occurs and what substrates are involved. In the work presented here, we endeavored to elucidate what regulates endogenous LRRK2 activation. To bolster ourconclusions, kinase activity was assessed using 3 complementary approaches: (i) PL pS1292– LRRK2, (ii) pT73-Rab10 western blot and (iii) PL pT73-Rab10–Rab10, each of which yielded similar results.

[0324] In our studies, treatment of cells with a physiological concentration of H2O2led quickly (within 5-10 minutes) to an increase in LRRK2 activation which was prevented by quenching ROS with NAC, thus suggesting that LRRK2 itself may be redox sensitive. In similar fashion, different cellular perturbations that promote LRRK2 activation, such as mitochondrial dysfunction (rotenone), lysosomal dyshomeostasis (chloroquine), and trafficking deficits (monensin) have been shown to also result in oxidative damage (21, 22, 35, 36, 52-54) – and here we have shown that they can stimulate 4HNE accumulation and subsequent adduct formation with LRRK2 (FIG.22).

[0325] The dynamics of the kinase domain have strong influence on the activation state of LRRK2 (12, 13). We found that Cys2024 / 2025 in the kinase activation loop are important residues for stimulated LRRK2 activation. Recent work by others has also suggested that these cysteine residues may be important for kinase activation (37); however, they used in vitro kinase assays and experiments (37), whereas we maintained an endogenous cellular context and used multiple complementary assays to support our conclusions. We found that while the cysteine-to-alanine mutations preserved basal kinase activity, stimulated LRRK2 activity was completely prevented. The fact that LRRK2 bearing these cysteine-to-alanine mutations had little, if any, propensity to form adducts with 4HNE suggests that 4HNE preferentially targets these solvent-exposed Cys2024 / 2025 residues. It is important to note, however, that the Cys2024 / 2025 mutants are not “kinase-dead”; they retain normal basal activity but simply cannot be further activated. A previous study postulated that the development of LRRK2 kinase inhibitors directed towards Cys2024 and Cys2025 may hold therapeutic benefit (55). Our study supports this hypothesis and, indicates these residues are critical for LRRK2 hyperactivation. It is unlikely that oxidative stress causes Cys2024 / 2025 to form an intramolecular disulfide bridge in monomeric LRRK2 as disulfide bridge formation between vicinal cysteine residues is an extremely rare biological phenomenon (56). Whether intermolecular disulfide bonds involving these cysteine residues might occur in higher order oligomeric species of LRRK2 (57) is unknown.

[0326] LRRK2 activation has been shown to be associated with damaged membranes, especially in the endolysosomal system (20-23, 35). As noted, endolysosomal impairmentand mitochondrial dysfunction lead to the formation the lipid peroxidation product 4HNE. This raises the possibility that this reactive aldehyde may be important in LRRK2 activation and might provide a link between membrane damage and LRRK2 activation. Given that 4HNE is known to activate other kinases (25-28), there is precedent for this idea. Indeed, we found that treatment of cells with exogenous 4HNE led to dose-dependent increases in LRRK2-4HNE adduct formation and LRRK2 activation – with a strong linear correlation between the two. Similarly, other LRRK2 activating stimuli (rotenone, chloroquine, monensin, and H2O2) which lead to the endogenous accumulation of 4HNE also resulted in LRRK2-4HNE adduct formation. Interestingly, Rab GTPases, which have been shown to activate LRRK2 (58, 59), may also be modified by 4HNE (60). Furthermore, STING pathway activation, which leads to lipid peroxidation (61), plays a role in activation of LRRK2 at damaged lysosomes (23). As such, 4HNE formation appears to be a convergent event shared by these seemingly disparate LRRK2 activating stimuli. Thus, 4HNE may be a final common regulator of LRRK2 kinase activation.

[0327] Levels of 4HNE and 4HNE protein adducts are elevated in PD patients compared to healthy controls (46, 62).4HNE can be formed nonenzymatically or enzymatically though the 15-LO pathway (29, 30). Therefore, we explored whether 15-LO activity regulates 4HNE production, LRRK2–4HNE adduct formation and LRRK2 kinase activation. Remarkably, 15- LO knockout or 15-LO inhibition with CU12991 potently blocked rotenone-stimulated 4HNE production, LRRK2-4HNE adducts and LRRK2 kinase hyperactivation, but did not inhibit basal kinase activity. Moreover, the upstream 15-LO specific metabolite, 15(S)-HpETE, which undergoes peroxidation to form 4HNE, dose-dependently increased LRRK2 activity. Importantly, we demonstrated in patient-derived iPD and G2019S LCLs that there were elevated 4HNE levels and LRRK2 activity that could be reduced to healthy control levels by inhibiting 15-LO with CU12991. Thus, we conclude that the specific end-product of 15-LO activity, 4HNE, is a physiological modulator of endogenous pathologic LRRK2 activity (FIG.22).

[0328] Normally, 15-LO can only oxygenate free fatty acids, not those incorporated into membranes. However, its substrate specificity is regulated by interaction with PEBP1 (48). When 15-LO is in complex with PEPB1, it acquires the ability to oxygenate membrane phospholipids (48), which is the first step in the formation of 4HNE. In this regard, we found that rotenone led to an increase in association of 15-LO with PEBP1 in vitro and in vivo. We found that stimuli that increase endogenous WT LRRK2 kinase activity also (i) enhance thePEBP1–15-LO interaction, (ii) increase 4HNE production, (iii) cause LRRK2-4HNE adducts to form, and (iv) activate LRRK2 kinase (FIG.22). Strikingly, this association was observed in G2019S and iPD patient-derived samples, confirming the relevance of this mechanism to the human disease. These data suggest that the increase in 15-LO–PEBP1 interaction leads to elevated 4HNE levels, which in turn promotes the hyperactivation of LRRK2. The fact that this sequence of events is demonstratable in G2019S and iPD patient derived samples provides strong evidence for the 15-LO pathway as a key contributor to PD pathogenesis through the regulation of LRRK2 activity.

[0329] Our study has some limitations. We acknowledge that, while our data strongly implicates cysteine residues 2024 and 2025 in the oxidative activation of LRRK2, we did not show directly that they undergo a specific 4HNE post-translational modification. Additionally, we have not determined the subcellular localization of LRRK2 activation in response to these stimuli. It will be important to determine if LRRK2 activation and LRRK2– 4HNE interaction occur in one specific cellular compartment, or if it is dependent on the location of the inciting insult (e.g., lysosomes, mitochondria).

[0330] In summary, we have defined a new mechanism of cellular perturbation-induced LRRK2 kinase activation. Our data demonstrate that LRRK2 is a redox-sensitive kinase and Cys2024 / 2025 are critical for the oxidative stress-induced stimulation of activity. Moreover, we found that seemingly disparate LRRK2 activating stimuli (chloroquine, H2O2, monensin, and rotenone), each with a different proximal mechanism of action, actually activate LRRK2 through a convergent redox-dependent mechanism that is mediated by the lipid peroxidation product, 4HNE, which in turn, forms adducts with LRRK2. Further, 15-LO, the enzymatic source of 4HNE, was identified as the upstream mediator of stimulated LRRK2 kinase activation (FIG.22). These results suggest that 15-LO inhibitors or drugs that otherwise reduce lipid peroxidation may provide a new therapeutic avenue to target the LRRK2 pathway in PD. Moreover, that fact that inhibition of 15-LO prevented stimulus-induced LRRK2 kinase hyperactivation in cell lines, and disease-associated hyperactivity in LCLs, without impacting basal kinase activity, may allow therapeutic modulation of LRRK2 while avoiding on-target liabilities.

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Ray et al., The Parkinson disease-linked LRRK2 protein mutation I2020T stabilizes an active state conformation leading to increased kinase activity. J Biol Chem 289, 13042-13053 (2014). 56. O. Carugo et al., Vicinal disulfide turns. Protein Eng 16, 637-639 (2003). 57. H. Zhu, F. Tonelli, D. R. Alessi, J. Sun, Structural basis of human LRRK2 membrane recruitment and activation. bioRxiv, 2022.2004.2026.489605 (2022). 58. H. S. Dhekne et al., Genome-wide screen reveals Rab12 GTPase as a critical activator of Parkinson's disease-linked LRRK2 kinase. Elife 12, (2023). 59. X. Wang et al., Rab12 is a regulator of LRRK2 and its activation by damaged lysosomes. Elife 12, (2023). 60. B. K. Chacko et al., Pleiotropic effects of 4-hydroxynonenal on oxidative burst and phagocytosis in neutrophils. Redox Biol 9, 57-66 (2016). 61. J. Wu et al., STING-dependent induction of lipid peroxidation mediates intestinal ischemia-reperfusion injury. Free Radic Biol Med 163, 135-140 (2021). 62. M. L. Selley, (E)-4-hydroxy-2-nonenal may be involved in the pathogenesis of Parkinson's disease. Free Radic Biol Med 25, 169-174 (1998).63. J. R. Cannon et al., A highly reproducible rotenone model of Parkinson's disease. Neurobiol Dis 34, 279-290 (2009). 64. N. Kumar et al., Global and transcription-coupled repair of 8-oxoG is initiated by nucleotide excision repair proteins. Nat Commun 13, 974 (2022).

[0332] Although the foregoing invention has been described in some detail by way of illustration and Example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference. Where a conflict exists between the instant application and a reference provided herein, the instant application shall dominate.

Claims

Attorney Docket No.: 058367-511001WO WHAT IS CLAIMED IS:

1. A method of lowering LRRK2 activity and / or expression in a cell, comprising contacting the cell with an effective amount of a 15-lipoxygenase (15-LO) inhibitor, wherein the LRRK2 activity and / or expression is higher than a control LRRK2 activity and / or expression in a control cell.

2. The method of claim 1, wherein the control LRRK2 activity and / or expression is a normal LRRK2 activity and / or expression in a healthy control cell.

3. The method of claim 1, wherein the 15-LO inhibitor has the structure of a compound of Formula I: , or a pharmaceuticallywherein each R1is independently -F, -Cl, -Br, -I, -ORa, -SRa, -NRaRb, -NO2, -CN, C1-6alkyl, C2-6 alkenyl, or C2-6 alkynyl, wherein the alkyl, alkenyl, or alkynyl is substituted with 0, 1, 2, or 3 groups independently selected from -F, -Cl, -Br, -I, -ORa, -SRa, -NRaRb, oxo, -NO2, and -CN; each R2is independently -F, -Cl, -Br, -I, -OH, -ORa, -SRa, -NRaRb, -CN, C1-6alkyl, C2-6 alkenyl, or C2-6 alkynyl, wherein the alkyl, alkenyl, or alkynyl is substituted with 0, 1, 2, or 3 groups independently selected from -F, -Cl, -Br, -I, -ORa, -SRa, -NRaRb, oxo, -NO2, and -CN; each Rais independently H or C1-6 alkyl; each Rbis independently H or C1-6 alkyl; the subscript m is 0, 1, 2 or 3; the subscript n is 0, 1, 2, or 3; and is a bicyclic nitrogen-containing heterocyclic ring.

4. The method of claim 1 or 3, wherein the compound or pharmaceutically acceptable salt thereof has the structure of Formula Ia:Attorney Docket No.: 058367-511001WO .one of claims 1 to 4, wherein ,R3is H, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 cycloalkyl, 4- to 8-membered heterocyclyl, phenyl, or 5- to 10-membered heteroaryl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, phenyl, or heteroaryl is substituted with 0, 1, 2, or 3 groups independently selected from -F, -Cl, -Br, -I, -OR3a, -SR3a, -NR3aR3b, oxo, -NO2, and –CN; each R3ais independently H or C1-6alkyl; and each R3bis independently H or C1-6 alkyl.

6. The method of any one of claims 1 to 5, wherein the compound has the structure as shown in Table 1.

7. The method of claim 1, wherein the 15-LO inhibitor has the structure of a compound of Formula II: ,or a pharmaceutically whereinAttorney Docket No.: 058367-511001WOeach R11 and R12 is independently H, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 haloalkyl, C1-6 hydroxyalkyl, C1-6 aminoalkyl, (C0-6 alkyl)(C3-8 cycloalkyl), (C0- 6 alkyl)(heterocyclyl), (C0-6alkyl)(C6-10aryl), (C0-6alkyl)(heteroaryl), halogen, ORa, SRa, NRaRb, NO2, or CN, wherein the alkyl, alkenyl, alkynyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is substituted with 0, 1, 2, or 3 R11a; or R11and R12taken together with the carbon atoms to which they are attached form a C5-10 cycloalkyl, heterocyclyl, C6-10aryl, or heteroaryl ring each substituted with 0, 1, 2, or 3 R11b; each R11aand R11bis independently ORc, SRc, NRcRd, oxo, NO2, or CN;each Rc and Rd is independently H or C1-6 alkyl;each R13a,R13b,R14a,R14b,R15a,R15b,R16a,and R16bis independently H, C1-6alkyl, C1-6alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 haloalkyl, C1-6 hydroxyalkyl, C1-6 aminoalkyl; or two of R13a,R13b,R14a,R14b,R15a,R15b,R16a,and R16btaken together with the atoms to which they are attached form a C3-8 cycloalkyl or a heterocyclyl; X is –C(R17a)(R17b)-, -N(R18)-, -O-, or –S-; R17ais (C0-6 alkyl)-N(R17a1)(R17a2), (C0-6 alkyl)-OR17a1, or (C0-6 alkyl)-SR17a1; R17bis H or C1-6alkyl; or R17aand R17btaken together form an oxo; each R17a1and R17a2is independently H, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C2-6alkoxyalkyl, C1-6haloalkyl, C1-6hydroxyalkyl, C1-6aminoalkyl, (C2-6alkyl)-N(R17c)C(O)-(R17d), (C2-6alkyl)-N(R17c)C(O)(OR17d), (C2-6alkyl)-N(R17c)S(O)2R17d, (C0-6alkyl)(C3-8cycloalkyl), (C0-6alkyl)(heterocyclyl), (C0-6alkyl)(C6-10aryl), (C0-6alkyl)(heteroaryl), (C0-6alkyl)-C(O)-R17c, (C0-6alkyl)-C(O)O-R17c, (C0-6alkyl)-C(O)-N(R17c)(R17d), (C0-6alkyl)-S(O)R17c, (C0-6alkyl)-S(O)(NH)R17c, (C0-6alkyl)-S(O)2R17c, (C0-6alkyl)-S(O)2N(R17c)(R17d), or (C0-6alkyl)-S(O)(NR17c)R17d, wherein the cycloalkyl, heterocyclyl, aryl, or heteroaryl is substituted with 0, 1, 2, or 3 R17e; or R17a1and R17a2taken together with the atoms to which they are attached form a heterocyclyl, which is substituted with 0, 1, 2, or 3 R17e; R18is H, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C2-6 alkoxyalkyl, C1-6 haloalkyl, C1- 6 hydroxyalkyl, C1-6aminoalkyl, (C2-6alkyl)-N(R18a)C(O)-(R18b), (C2-6 alkyl)-N(R18a)C(O)(OR18b), (C2-6 alkyl)-N(R18a)S(O)2R18b, (C0-6 alkyl)(C3-8 cycloalkyl), (C0-6alkyl)(heterocyclyl), (C0-6alkyl)(C6-10aryl), (C0-6alkyl)(heteroaryl), (C0-6 alkyl)-C(O)-R18a, (C0-6 alkyl)-C(O)O-R18a, (C0-6 alkyl)-C(O)-N(R18a)(R18b), (C0- 6 alkyl)-S(O)R18a, (C0-6alkyl)-S(O)(NH)R18a, (C0-6alkyl)-S(O)2R18a, (C0-Attorney Docket No.: 058367-511001WO 6 alkyl)-S(O)2N(R18a)(R18b), or (C0-6alkyl)-S(O)(NR18a)R18b, wherein the cycloalkyl, heterocyclyl, aryl, or heteroaryl is substituted with 0, 1, 2, or 3 R18c; each R17eand R18cis independently C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C2-6alkoxyalkyl, C1-6 haloalkyl, C1-6 hydroxyalkyl, C1-6 aminoalkyl, C3-8 cycloalkyl, heterocyclyl, C6-10 aryl, heteroaryl, (C0-6alkyl)-C(O)-R19a, (C0-6alkyl)-C(O)O-R19a, (C0-6alkyl)-C(O)- N(R19a)(R19b), (C0-6 alkyl)-S(O)R19a, -S(O)(NH)R19a, -S(O)2R19a, -S(O)2N(R19a)(R19b), or -S(O)(NR19a)R19b; each R17c, R17d, R18a, R18b, R19a, and R19bis independently H, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C2-6alkoxyalkyl, C1-6haloalkyl, C1-6hydroxyalkyl, C1-6aminoalkyl, (C0-6 alkyl)(C3-8 cycloalkyl), (C0-6 alkyl)(heterocyclyl), (C0-6 alkyl)(C6-10 aryl), or (C0- 6 alkyl)(heteroaryl), wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is substituted with 0 to 4 Z1; each Z1is independently C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C2-6alkoxyalkyl, C1-6haloalkyl, C3-8 cycloalkyl, heterocyclyl, C6-10 aryl, heteroaryl, halogen, oxo, -OH, -CN, - NO2, -NH2, -N3, -SH, -O(C1-6alkyl), -O(C1-6haloalkyl), -NH(C1-6alkyl), -NH(C1-6haloalkyl), -N(C1-6 alkyl)2, -N(C1-6 haloalkyl)2, -N(C1-6 alkyl)(C1-6 haloalkyl), -C(O)(C1-6alkyl), -C(O)(C1-6haloalkyl), -C(O)O(C1-6alkyl), -C(O)O(C1-6haloalkyl), -C(O)NH2, -C(O)NH(C1-6 alkyl), -C(O)NH(C1-6 haloalkyl), -C(O)N(C1-6 alkyl)2, -C(O)N(C1-6haloalkyl)2, -NHC(O)(C1-6alkyl), -NHC(O)(C1-6haloalkyl), - NHC(O)O(C1-6alkyl), -NHC(O)O(C1-6haloalkyl), -NHC(O)NH(C1-6alkyl), -NHC(O)NH(C1-6haloalkyl), -NHS(O)(C1-6alkyl), -N(C1-6alkyl)(S(O)(C1-6alkyl), -S(C1-6alkyl), -S(C1-6haloalkyl), -S(O)N(C1-6alkyl)2, -S(O)(C1-6alkyl), - S(O)(C1-6haloalkyl), -S(O)2(C1-6alkyl), -S(O)2(C1-6haloalkyl), -S(O)(NH)(C1-6alkyl), -S(O)2NH(C1-6alkyl), or -S(O)2N(C1-6alkyl)2; each heterocyclyl is a 4- to 10-membered ring having 1, 2, 3, or 4 heteroatoms selected from N, O, and S; and each heteroaryl is a 5- to 10-membered ring having 1, 2, 3, or 4 heteroatoms selected from N, O, and S.

8. A method of reducing 4-hydroxynonenal (4HNE) in a cell, comprising administering an effective amount of a 15-lipooxygenase (15-LO) inhibitor.

9. The method of claim 8, wherein the 15-LO inhibitor has the structure of a compound of Formula I:Attorney Docket No.: 058367-511001WO , or a pharmaceuticallywherein each R1is independently -F, -Cl, -Br, -I, -ORa, -SRa, -NRaRb, -NO2, -CN, C1-6 alkyl, C2-6alkenyl, or C2-6alkynyl, wherein the alkyl, alkenyl, or alkynyl is substituted with 0, 1, 2, or 3 groups independently selected from -F, -Cl, -Br, -I, -ORa, -SRa, -NRaRb, oxo, -NO2, and -CN; each R2is independently -F, -Cl, -Br, -I, -OH, -ORa, -SRa, -NRaRb, -CN, C1-6 alkyl, C2- 6 alkenyl, or C2-6alkynyl, wherein the alkyl, alkenyl, or alkynyl is substituted with 0, 1, 2, or 3 groups independently selected from -F, -Cl, -Br, -I, -ORa, -SRa, -NRaRb, oxo, -NO2, and -CN; each Rais independently H or C1-6 alkyl; each Rbis independently H or C1-6alkyl; the subscript m is 0, 1, 2 or 3; the subscript n is 0, 1, 2, or 3; and is a bicyclic nitrogen-containing heterocyclic ring.

10. The method of claim 8 or 9, wherein the compound or pharmaceutically acceptable salt thereof has the structure of Formula Ia: .any one of claims 8 to 10, wherein ,Attorney Docket No.: 058367-511001WO R3is H, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C3-8cycloalkyl, 4- to 8-membered heterocyclyl, phenyl, or 5- to 10-membered heteroaryl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, phenyl, or heteroaryl is substituted with 0, 1, 2, or 3 groups independently selected from -F, -Cl, -Br, -I, -OR3a, -SR3a, -NR3aR3b, oxo, -NO2, and –CN; each R3ais independently H or C1-6 alkyl; and each R3bis independently H or C1-6alkyl.

12. The method of any one of claims 8 to 11, wherein the compound has the structure as shown in Table 1.

13. The method of claim 8, wherein the 15-LO inhibitor has the structure of a compound of Formula II: , or a pharmaceuticallywhereineach R11 and R12 is independently H, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 haloalkyl, C1-6hydroxyalkyl, C1-6aminoalkyl, (C0-6alkyl)(C3-8cycloalkyl), (C0-6 alkyl)(heterocyclyl), (C0-6 alkyl)(C6-10 aryl), (C0-6 alkyl)(heteroaryl), halogen, ORa, SRa, NRaRb, NO2, or CN, wherein the alkyl, alkenyl, alkynyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is substituted with 0, 1, 2, or 3 R11a; or R11and R12taken together with the carbon atoms to which they are attached form a C5-10cycloalkyl, heterocyclyl, C6-10 aryl, or heteroaryl ring each substituted with 0, 1, 2, or 3 R11b; each R11aand R11bis independently ORc, SRc, NRcRd, oxo, NO2, or CN;each Rc and Rd is independently H or C1-6 alkyl;each R13a, R13b, R14a, R14b, R15a, R15b, R16a, and R16bis independently H, C1-6 alkyl, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 haloalkyl, C1-6 hydroxyalkyl, C1-6 aminoalkyl; orAttorney Docket No.: 058367-511001WO two of R13a,R13b,R14a,R14b,R15a,R15b,R16a,and R16btaken together with the atoms to which they are attached form a C3-8 cycloalkyl or a heterocyclyl; X is –C(R17a)(R17b)-, -N(R18)-, -O-, or –S-; R17ais (C0-6 alkyl)-N(R17a1)(R17a2), (C0-6 alkyl)-OR17a1, or (C0-6 alkyl)-SR17a1; R17bis H or C1-6alkyl; or R17aand R17btaken together form an oxo; each R17a1and R17a2is independently H, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C2-6alkoxyalkyl, C1-6 haloalkyl, C1-6 hydroxyalkyl, C1-6 aminoalkyl, (C2-6 alkyl)-N(R17c)C(O)-(R17d), (C2-6alkyl)-N(R17c)C(O)(OR17d), (C2-6alkyl)-N(R17c)S(O)2R17d, (C0-6alkyl)(C3-8cycloalkyl), (C0-6 alkyl)(heterocyclyl), (C0-6 alkyl)(C6-10 aryl), (C0-6 alkyl)(heteroaryl), (C0-6alkyl)-C(O)-R17c, (C0-6alkyl)-C(O)O-R17c, (C0-6alkyl)-C(O)-N(R17c)(R17d), (C0-6 alkyl)-S(O)R17c, (C0-6 alkyl)-S(O)(NH)R17c, (C0-6 alkyl)-S(O)2R17c, (C0- 6 alkyl)-S(O)2N(R17c)(R17d), or (C0-6alkyl)-S(O)(NR17c)R17d, wherein the cycloalkyl, heterocyclyl, aryl, or heteroaryl is substituted with 0, 1, 2, or 3 R17e; or R17a1and R17a2taken together with the atoms to which they are attached form a heterocyclyl, which is substituted with 0, 1, 2, or 3 R17e; R18is H, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C2-6alkoxyalkyl, C1-6haloalkyl, C1-6 hydroxyalkyl, C1-6 aminoalkyl, (C2-6 alkyl)-N(R18a)C(O)-(R18b), (C2- 6 alkyl)-N(R18a)C(O)(OR18b), (C2-6alkyl)-N(R18a)S(O)2R18b, (C0-6alkyl)(C3-8cycloalkyl), (C0-6alkyl)(heterocyclyl), (C0-6alkyl)(C6-10aryl), (C0-6alkyl)(heteroaryl), (C0-6alkyl)-C(O)-R18a, (C0-6alkyl)-C(O)O-R18a, (C0-6alkyl)-C(O)-N(R18a)(R18b), (C0-6alkyl)-S(O)R18a, (C0-6alkyl)-S(O)(NH)R18a, (C0-6alkyl)-S(O)2R18a, (C0-6alkyl)-S(O)2N(R18a)(R18b), or (C0-6alkyl)-S(O)(NR18a)R18b, wherein the cycloalkyl, heterocyclyl, aryl, or heteroaryl is substituted with 0, 1, 2, or 3 R18c; each R17eand R18cis independently C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C2-6alkoxyalkyl, C1-6haloalkyl, C1-6hydroxyalkyl, C1-6aminoalkyl, C3-8cycloalkyl, heterocyclyl, C6-10aryl, heteroaryl, (C0-6alkyl)-C(O)-R19a, (C0-6alkyl)-C(O)O-R19a, (C0-6alkyl)-C(O)- N(R19a)(R19b), (C0-6alkyl)-S(O)R19a, -S(O)(NH)R19a, -S(O)2R19a, -S(O)2N(R19a)(R19b), or -S(O)(NR19a)R19b; each R17c, R17d, R18a, R18b, R19a, and R19bis independently H, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C2-6 alkoxyalkyl, C1-6 haloalkyl, C1-6 hydroxyalkyl, C1-6 aminoalkyl, (C0- 6 alkyl)(C3-8cycloalkyl), (C0-6alkyl)(heterocyclyl), (C0-6alkyl)(C6-10aryl), or (C0-6 alkyl)(heteroaryl), wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is substituted with 0 to 4 Z1;Attorney Docket No.: 058367-511001WO each Z1is independently C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C2-6alkoxyalkyl, C1-6haloalkyl, C3-8 cycloalkyl, heterocyclyl, C6-10 aryl, heteroaryl, halogen, oxo, -OH, -CN, - NO2, -NH2, -N3, -SH, -O(C1-6alkyl), -O(C1-6haloalkyl), -NH(C1-6alkyl), -NH(C1-6haloalkyl), -N(C1-6 alkyl)2, -N(C1-6 haloalkyl)2, -N(C1-6 alkyl)(C1-6 haloalkyl), -C(O)(C1-6alkyl), -C(O)(C1-6haloalkyl), -C(O)O(C1-6alkyl), -C(O)O(C1-6haloalkyl), -C(O)NH2, -C(O)NH(C1-6 alkyl), -C(O)NH(C1-6 haloalkyl), -C(O)N(C1-6 alkyl)2, -C(O)N(C1-6haloalkyl)2, -NHC(O)(C1-6alkyl), -NHC(O)(C1-6haloalkyl), - NHC(O)O(C1-6 alkyl), -NHC(O)O(C1-6 haloalkyl), -NHC(O)NH(C1-6 alkyl), -NHC(O)NH(C1-6haloalkyl), -NHS(O)(C1-6alkyl), -N(C1-6alkyl)(S(O)(C1-6alkyl), -S(C1-6 alkyl), -S(C1-6 haloalkyl), -S(O)N(C1-6 alkyl)2, -S(O)(C1-6 alkyl), - S(O)(C1-6haloalkyl), -S(O)2(C1-6alkyl), -S(O)2(C1-6haloalkyl), -S(O)(NH)(C1-6alkyl), -S(O)2NH(C1-6 alkyl), or -S(O)2N(C1-6 alkyl)2; each heterocyclyl is a 4- to 10-membered ring having 1, 2, 3, or 4 heteroatoms selected from N, O, and S; and each heteroaryl is a 5- to 10-membered ring having 1, 2, 3, or 4 heteroatoms selected from N, O, and S.

14. A method of treating a disease or condition characterized by increased LRRK2 activity and / or expression in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a 15-lipooxygenase (15-LO) inhibitor.

15. The method of claim 14, wherein the subject is a human.

16. The method of claim 14, wherein the disease or condition characterized by increased LRRK2 activity and / or expression is a neurodegenerative disease.

17. The method of any one of claims 14 to 16, wherein the disease or condition characterized by increased LRRK2 activity and / or expression is Parkinson’s disease.

18. The method of any one of claims 14 to 17, wherein the 15-LO inhibitor has the structure of a compound of Formula I: ,Attorney Docket No.: 058367-511001WO or a pharmaceutically acceptable salt thereof, wherein each R1is independently -F, -Cl, -Br, -I, -ORa, -SRa, -NRaRb, -NO2, -CN, C1-6alkyl, C2-6 alkenyl, or C2-6 alkynyl, wherein the alkyl, alkenyl, or alkynyl is substituted with 0, 1, 2, or 3 groups independently selected from -F, -Cl, -Br, -I, -ORa, -SRa, -NRaRb, oxo, -NO2, and -CN; each R2is independently -F, -Cl, -Br, -I, -OH, -ORa, -SRa, -NRaRb, -CN, C1-6alkyl, C2-6 alkenyl, or C2-6 alkynyl, wherein the alkyl, alkenyl, or alkynyl is substituted with 0, 1, 2, or 3 groups independently selected from -F, -Cl, -Br, -I, -ORa, -SRa, -NRaRb, oxo, -NO2, and -CN; each Rais independently H or C1-6alkyl; each Rbis independently H or C1-6 alkyl; the subscript m is 0, 1, 2 or 3; the subscript n is 0, 1, 2, or 3; and is a bicyclic nitrogen-containing heterocyclic ring.

19. The method of any one of claims 14 to 18, wherein the compound or pharmaceutically acceptable salt thereof has the structure of Formula Ia: .one of claims 14 to 19, wherein ,R3is H, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 cycloalkyl, 4- to 8-membered heterocyclyl, phenyl, or 5- to 10-membered heteroaryl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, phenyl, or heteroaryl is substitutedAttorney Docket No.: 058367-511001WO with 0, 1, 2, or 3 groups independently selected from -F, -Cl, -Br, -I, -OR3a, -SR3a, -NR3aR3b, oxo, -NO2, and –CN; each R3ais independently H or C1-6alkyl; and each R3bis independently H or C1-6 alkyl.

21. The method of any one of claims 14 to 20, wherein the compound has the structure as shown in Table 1.

22. The method of claim 14, wherein the 15-LO inhibitor has the structure of a compound of Formula II: , or a pharmaceuticallywhereineach R11 and R12 is independently H, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 haloalkyl, C1-6 hydroxyalkyl, C1-6 aminoalkyl, (C0-6 alkyl)(C3-8 cycloalkyl), (C0- 6 alkyl)(heterocyclyl), (C0-6alkyl)(C6-10aryl), (C0-6alkyl)(heteroaryl), halogen, ORa, SRa, NRaRb, NO2, or CN, wherein the alkyl, alkenyl, alkynyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is substituted with 0, 1, 2, or 3 R11a; or R11and R12taken together with the carbon atoms to which they are attached form a C5-10 cycloalkyl, heterocyclyl, C6-10aryl, or heteroaryl ring each substituted with 0, 1, 2, or 3 R11b; each R11aand R11bis independently ORc, SRc, NRcRd, oxo, NO2, or CN;each Rc and Rd is independently H or C1-6 alkyl;each R13a,R13b,R14a,R14b,R15a,R15b,R16a,and R16bis independently H, C1-6alkyl, C1-6alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 haloalkyl, C1-6 hydroxyalkyl, C1-6 aminoalkyl; or two of R13a,R13b,R14a,R14b,R15a,R15b,R16a,and R16btaken together with the atoms to which they are attached form a C3-8 cycloalkyl or a heterocyclyl; X is –C(R17a)(R17b)-, -N(R18)-, -O-, or –S-;Attorney Docket No.: 058367-511001WO R17ais (C0-6alkyl)-N(R17a1)(R17a2), (C0-6alkyl)-OR17a1, or (C0-6alkyl)-SR17a1; R17bis H or C1-6 alkyl; or R17aand R17btaken together form an oxo; each R17a1and R17a2is independently H, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C2-6 alkoxyalkyl, C1-6haloalkyl, C1-6hydroxyalkyl, C1-6aminoalkyl, (C2-6alkyl)-N(R17c)C(O)-(R17d), (C2-6 alkyl)-N(R17c)C(O)(OR17d), (C2-6 alkyl)-N(R17c)S(O)2R17d, (C0-6 alkyl)(C3-8 cycloalkyl), (C0-6alkyl)(heterocyclyl), (C0-6alkyl)(C6-10aryl), (C0-6alkyl)(heteroaryl), (C0-6 alkyl)-C(O)-R17c, (C0-6 alkyl)-C(O)O-R17c, (C0-6 alkyl)-C(O)-N(R17c)(R17d), (C0- 6 alkyl)-S(O)R17c, (C0-6alkyl)-S(O)(NH)R17c, (C0-6alkyl)-S(O)2R17c, (C0-6 alkyl)-S(O)2N(R17c)(R17d), or (C0-6 alkyl)-S(O)(NR17c)R17d, wherein the cycloalkyl, heterocyclyl, aryl, or heteroaryl is substituted with 0, 1, 2, or 3 R17e; or R17a1and R17a2taken together with the atoms to which they are attached form a heterocyclyl, which is substituted with 0, 1, 2, or 3 R17e; R18is H, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C2-6 alkoxyalkyl, C1-6 haloalkyl, C1- 6 hydroxyalkyl, C1-6aminoalkyl, (C2-6alkyl)-N(R18a)C(O)-(R18b), (C2-6 alkyl)-N(R18a)C(O)(OR18b), (C2-6 alkyl)-N(R18a)S(O)2R18b, (C0-6 alkyl)(C3-8 cycloalkyl), (C0-6alkyl)(heterocyclyl), (C0-6alkyl)(C6-10aryl), (C0-6alkyl)(heteroaryl), (C0-6 alkyl)-C(O)-R18a, (C0-6 alkyl)-C(O)O-R18a, (C0-6 alkyl)-C(O)-N(R18a)(R18b), (C0- 6 alkyl)-S(O)R18a, (C0-6alkyl)-S(O)(NH)R18a, (C0-6alkyl)-S(O)2R18a, (C0-6alkyl)-S(O)2N(R18a)(R18b), or (C0-6alkyl)-S(O)(NR18a)R18b, wherein the cycloalkyl, heterocyclyl, aryl, or heteroaryl is substituted with 0, 1, 2, or 3 R18c; each R17eand R18cis independently C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C2-6alkoxyalkyl, C1-6haloalkyl, C1-6hydroxyalkyl, C1-6aminoalkyl, C3-8cycloalkyl, heterocyclyl, C6-10aryl, heteroaryl, (C0-6alkyl)-C(O)-R19a, (C0-6alkyl)-C(O)O-R19a, (C0-6alkyl)-C(O)- N(R19a)(R19b), (C0-6alkyl)-S(O)R19a, -S(O)(NH)R19a, -S(O)2R19a, -S(O)2N(R19a)(R19b), or -S(O)(NR19a)R19b; each R17c, R17d, R18a, R18b, R19a, and R19bis independently H, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C2-6alkoxyalkyl, C1-6haloalkyl, C1-6hydroxyalkyl, C1-6aminoalkyl, (C0-6 alkyl)(C3-8 cycloalkyl), (C0-6 alkyl)(heterocyclyl), (C0-6 alkyl)(C6-10 aryl), or (C0- 6 alkyl)(heteroaryl), wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is substituted with 0 to 4 Z1; each Z1is independently C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C2-6alkoxyalkyl, C1-6haloalkyl, C3-8 cycloalkyl, heterocyclyl, C6-10 aryl, heteroaryl, halogen, oxo, -OH, -CN, - NO2, -NH2, -N3, -SH, -O(C1-6alkyl), -O(C1-6haloalkyl), -NH(C1-6alkyl), -NH(C1-6Attorney Docket No.: 058367-511001WO haloalkyl), -N(C1-6alkyl)2, -N(C1-6haloalkyl)2, -N(C1-6alkyl)(C1-6haloalkyl), -C(O)(C1-6 alkyl), -C(O)(C1-6 haloalkyl), -C(O)O(C1-6 alkyl), -C(O)O(C1-6 haloalkyl), -C(O)NH2, -C(O)NH(C1-6alkyl), -C(O)NH(C1-6haloalkyl), -C(O)N(C1-6alkyl)2, -C(O)N(C1-6 haloalkyl)2, -NHC(O)(C1-6 alkyl), -NHC(O)(C1-6 haloalkyl), - NHC(O)O(C1-6alkyl), -NHC(O)O(C1-6haloalkyl), -NHC(O)NH(C1-6alkyl), -NHC(O)NH(C1-6 haloalkyl), -NHS(O)(C1-6 alkyl), -N(C1-6 alkyl)(S(O)(C1-6 alkyl), -S(C1-6alkyl), -S(C1-6haloalkyl), -S(O)N(C1-6alkyl)2, -S(O)(C1-6alkyl), - S(O)(C1-6 haloalkyl), -S(O)2(C1-6 alkyl), -S(O)2(C1-6 haloalkyl), -S(O)(NH)(C1-6 alkyl), -S(O)2NH(C1-6alkyl), or -S(O)2N(C1-6alkyl)2; each heterocyclyl is a 4- to 10-membered ring having 1, 2, 3, or 4 heteroatoms selected from N, O, and S; and each heteroaryl is a 5- to 10-membered ring having 1, 2, 3, or 4 heteroatoms selected from N, O, and S.

23. A method of selecting a subject for treatment of a disease or condition characterized by increased LRRK2 activity and / or expression, comprising measuring an increased LRRK2-4HNE adduct level in a biological sample from the subject compared to control LRRK2-4HNE adduct level in a control sample.

24. A method of treating a subject suffering from or suspected of suffering from a disease or condition characterized by increased LRRK2 activity and / or expression, comprising administering a therapeutically effective amount of a 15-LO inhibitor if an increased LRRK2-4HNE adduct level was measured in a biological sample from the subject compared to control LRRK2-4HNE adduct level in a control sample.

25. The method of claim 24, wherein the 15-LO inhibitor has the structure of a compound of Formula I: ,or a pharmaceutically whereinAttorney Docket No.: 058367-511001WO each R1is independently -F, -Cl, -Br, -I, -ORa, -SRa, -NRaRb, -NO2, -CN, C1-6alkyl, C2-6 alkenyl, or C2-6 alkynyl, wherein the alkyl, alkenyl, or alkynyl is substituted with 0, 1, 2, or 3 groups independently selected from -F, -Cl, -Br, -I, -ORa, -SRa, -NRaRb, oxo, -NO2, and -CN; each R2is independently -F, -Cl, -Br, -I, -OH, -ORa, -SRa, -NRaRb, -CN, C1-6alkyl, C2-6 alkenyl, or C2-6 alkynyl, wherein the alkyl, alkenyl, or alkynyl is substituted with 0, 1, 2, or 3 groups independently selected from -F, -Cl, -Br, -I, -ORa, -SRa, -NRaRb, oxo, -NO2, and -CN; each Rais independently H or C1-6alkyl; each Rbis independently H or C1-6 alkyl; the subscript m is 0, 1, 2 or 3; the subscript n is 0, 1, 2, or 3; and is a bicyclic nitrogen-containing heterocyclic ring.

26. The method of claim 24, wherein the 15-LO inhibitor has the structure of a compound of Formula II: , or a pharmaceuticallywhereineach R11 and R12 is independently H, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 haloalkyl, C1-6hydroxyalkyl, C1-6aminoalkyl, (C0-6alkyl)(C3-8cycloalkyl), (C0-6 alkyl)(heterocyclyl), (C0-6 alkyl)(C6-10 aryl), (C0-6 alkyl)(heteroaryl), halogen, ORa, SRa, NRaRb, NO2, or CN, wherein the alkyl, alkenyl, alkynyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is substituted with 0, 1, 2, or 3 R11a; or R11and R12taken together with the carbon atoms to which they are attached form a C5-10cycloalkyl, heterocyclyl, C6-10 aryl, or heteroaryl ring each substituted with 0, 1, 2, or 3 R11b;Attorney Docket No.: 058367-511001WO each R11aand R11bis independently ORc, SRc, NRcRd, oxo, NO2, or CN;each Rc and Rd is independently H or C1-6 alkyl;each R13a,R13b,R14a,R14b,R15a,R15b,R16a,and R16bis independently H, C1-6alkyl, C1-6alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 haloalkyl, C1-6 hydroxyalkyl, C1-6 aminoalkyl; or two of R13a,R13b,R14a,R14b,R15a,R15b,R16a,and R16btaken together with the atoms to which they are attached form a C3-8 cycloalkyl or a heterocyclyl; X is –C(R17a)(R17b)-, -N(R18)-, -O-, or –S-; R17ais (C0-6 alkyl)-N(R17a1)(R17a2), (C0-6 alkyl)-OR17a1, or (C0-6 alkyl)-SR17a1; R17bis H or C1-6alkyl; or R17aand R17btaken together form an oxo; each R17a1and R17a2is independently H, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C2-6alkoxyalkyl, C1-6 haloalkyl, C1-6 hydroxyalkyl, C1-6 aminoalkyl, (C2-6 alkyl)-N(R17c)C(O)-(R17d), (C2-6alkyl)-N(R17c)C(O)(OR17d), (C2-6alkyl)-N(R17c)S(O)2R17d, (C0-6alkyl)(C3-8cycloalkyl), (C0-6 alkyl)(heterocyclyl), (C0-6 alkyl)(C6-10 aryl), (C0-6 alkyl)(heteroaryl), (C0-6alkyl)-C(O)-R17c, (C0-6alkyl)-C(O)O-R17c, (C0-6alkyl)-C(O)-N(R17c)(R17d), (C0-6 alkyl)-S(O)R17c, (C0-6 alkyl)-S(O)(NH)R17c, (C0-6 alkyl)-S(O)2R17c, (C0- 6 alkyl)-S(O)2N(R17c)(R17d), or (C0-6alkyl)-S(O)(NR17c)R17d, wherein the cycloalkyl, heterocyclyl, aryl, or heteroaryl is substituted with 0, 1, 2, or 3 R17e; or R17a1and R17a2taken together with the atoms to which they are attached form a heterocyclyl, which is substituted with 0, 1, 2, or 3 R17e; R18is H, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C2-6alkoxyalkyl, C1-6haloalkyl, C1-6hydroxyalkyl, C1-6aminoalkyl, (C2-6alkyl)-N(R18a)C(O)-(R18b), (C2-6alkyl)-N(R18a)C(O)(OR18b), (C2-6alkyl)-N(R18a)S(O)2R18b, (C0-6alkyl)(C3-8cycloalkyl), (C0-6alkyl)(heterocyclyl), (C0-6alkyl)(C6-10aryl), (C0-6alkyl)(heteroaryl), (C0-6alkyl)-C(O)-R18a, (C0-6alkyl)-C(O)O-R18a, (C0-6alkyl)-C(O)-N(R18a)(R18b), (C0-6alkyl)-S(O)R18a, (C0-6alkyl)-S(O)(NH)R18a, (C0-6alkyl)-S(O)2R18a, (C0-6alkyl)-S(O)2N(R18a)(R18b), or (C0-6alkyl)-S(O)(NR18a)R18b, wherein the cycloalkyl, heterocyclyl, aryl, or heteroaryl is substituted with 0, 1, 2, or 3 R18c; each R17eand R18cis independently C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C2-6 alkoxyalkyl, C1- 6 haloalkyl, C1-6hydroxyalkyl, C1-6aminoalkyl, C3-8cycloalkyl, heterocyclyl, C6-10aryl, heteroaryl, (C0-6 alkyl)-C(O)-R19a, (C0-6 alkyl)-C(O)O-R19a, (C0-6 alkyl)-C(O)- N(R19a)(R19b), (C0-6alkyl)-S(O)R19a, -S(O)(NH)R19a, -S(O)2R19a, -S(O)2N(R19a)(R19b), or -S(O)(NR19a)R19b;Attorney Docket No.: 058367-511001WO each R17c, R17d, R18a, R18b, R19a, and R19bis independently H, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C2-6 alkoxyalkyl, C1-6 haloalkyl, C1-6 hydroxyalkyl, C1-6 aminoalkyl, (C0- 6 alkyl)(C3-8cycloalkyl), (C0-6alkyl)(heterocyclyl), (C0-6alkyl)(C6-10aryl), or (C0-6 alkyl)(heteroaryl), wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is substituted with 0 to 4 Z1; each Z1is independently C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C2-6 alkoxyalkyl, C1-6 haloalkyl, C3-8cycloalkyl, heterocyclyl, C6-10aryl, heteroaryl, halogen, oxo, -OH, -CN, - NO2, -NH2, -N3, -SH, -O(C1-6 alkyl), -O(C1-6 haloalkyl), -NH(C1-6 alkyl), -NH(C1-6 haloalkyl), -N(C1-6alkyl)2, -N(C1-6haloalkyl)2, -N(C1-6alkyl)(C1-6haloalkyl), -C(O)(C1-6 alkyl), -C(O)(C1-6 haloalkyl), -C(O)O(C1-6 alkyl), -C(O)O(C1-6 haloalkyl), -C(O)NH2, -C(O)NH(C1-6alkyl), -C(O)NH(C1-6haloalkyl), -C(O)N(C1-6alkyl)2, -C(O)N(C1-6 haloalkyl)2, -NHC(O)(C1-6 alkyl), -NHC(O)(C1-6 haloalkyl), - NHC(O)O(C1-6alkyl), -NHC(O)O(C1-6haloalkyl), -NHC(O)NH(C1-6alkyl), -NHC(O)NH(C1-6 haloalkyl), -NHS(O)(C1-6 alkyl), -N(C1-6 alkyl)(S(O)(C1-6 alkyl), -S(C1-6alkyl), -S(C1-6haloalkyl), -S(O)N(C1-6alkyl)2, -S(O)(C1-6alkyl), - S(O)(C1-6 haloalkyl), -S(O)2(C1-6 alkyl), -S(O)2(C1-6 haloalkyl), -S(O)(NH)(C1-6 alkyl), -S(O)2NH(C1-6alkyl), or -S(O)2N(C1-6alkyl)2; each heterocyclyl is a 4- to 10-membered ring having 1, 2, 3, or 4 heteroatoms selected from N, O, and S; and each heteroaryl is a 5- to 10-membered ring having 1, 2, 3, or 4 heteroatoms selected from N, O, and S.

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