Agents for use in the treatment of endoplasmic stress

Abstract

Inhibitors of C / EBP homologous protein (CHOP) are provided. The inhibitors find use in the modulation of endoplasmic reticulum (ER) stress, and the treatment of conditions involving ER stress, including neuronal conditions.

Description

AGENTS FOR USE IN THE TREATMENT OF ENDOPLASMIC STRESS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] Pursuant to 35 U.S.C. § 119 (e), this application claims priority to the filing date of United States Provisional Patent Application Serial No.63 / 574,763, filed April 4, 2024 thedisclosure of which application is herein incorporated by reference. GOVERNMENT SUPPORT RESEARCH

[0002] This invention was made with Government support under contract EY023295 awarded by the National Institutes of Health. The Government has certain rights in the invention. INTRODUCTION

[0003] Glaucoma is the most common cause of irreversible blindness and will affect more than 100 million people between 40 to 80 years old by 2040. $2.9 billion in direct medical costs is required to care for about 2 million American patients with glaucoma. Glaucoma is characterized by ON neuropathy with thinning of the nerve fiber layer (NFL) followed by progressive RGC degeneration. Elevated intraocular pressure (IOP) is the most recognized risk factor. The only available treatments act by reducing IOP. However, irreversible RGC death continues even after significant IOP reduction, suggesting that a degeneration mechanism continues after the initial insult. Preventing degeneration of RGCs and their axons would therefore have a great impact on the treatment of glaucoma. Similar to other chronic neurodegenerative diseases, neuroprotectants have long been sought for glaucoma but have not been found. Deciphering the key upstream signals that trigger the degeneration cascade is useful in identification of therapeutic targets and lead to innovative and efficient neuroprotective treatments to fulfill this significant unmet clinical need.

[0004] ER stress occurs in many acute and chronic neurodegenerative diseases. Modulation of ER stress protects injured neurons and improves functional recovery in experimental spinal cord injury, stroke, Alzheimer’s disease, Parkinson’s disease, ALS, prion disease and, most relevantly, retinal cell degenerations, including RGC and photoreceptor cells. In addition to the traditional ER stress stimuli, such as hypoxia, nutrient deprivation, disruption of calcium homeostasis and high-fat diet; axon injury induces neuronal ER stress in RGCs. CHOP inhibition can preserve the structure and function of both RGC somata and axons, indicating the critical importance of ER stress in the pathophysiology of glaucoma and of a broad range of neurodegenerative diseases. Identification of small modulators of ER stress to block CHOP is an important step toward developing effective neuroprotectants for glaucoma.

[0005] When the ER is overwhelmed by unfolded and misfolded proteins, cells experience ER stress and activate a complex cascade of reactions, in general called the unfolded protein 1response (UPR). Three evolutionarily conserved UPR pathways are initiated by three ER- resident stress-sensing proteins: inositol-requiring protein-1 (IRE1α), activating transcription factor-6 (ATF6) and protein kinase RNA-like ER kinase (PERK).

[0006] IRE1α, a bi- functional enzyme that contains both a Ser / Thr kinase domain and an endoribonuclease (RNase) domain, initiates the protective UPR pathway by mediating the splicing of X-box binding protein 1 (XBP-1) mRNA to generate an active (spliced) form of the transcription factor, XBP-1s. The IRE1α-XBP-1s pathway targets genes that increase ER protein-folding capacity and facilitate degradation of misfolded proteins. In addition to its transcriptional function, XBP-1 directly interacts with Forkhead box O1 (FOXO1) to assist its degradation and block FOXO- dependent apoptosis. On the other hand, IRE1α kinase activity also activates pro-apoptotic c-Jun kinase (JNK)55, which may contribute to Bax-dependent IRE1α-induced apoptosis. Interestingly, both JNK and Bax have been suggested as major regulators of axon injury-induced RGC death.

[0007] ATF6 is cleaved in Golgi to generate an active transcription factor, ATF6 N-terminal domain (ATF6N). ATF6N acts alone or forms heterodimers with XBP-1s to induce expression of ER chaperones, foldases and lipid synthesis genes to promote protein folding and protein degradation, which is generally considered cytoprotective. Activation of the ATF6 transcriptional program prevents the aberrant secretion of proteins associated with degenerative diseases and attenuates extracellular protein aggregation. Mutation of ATF6 has recently been linked with achromatopsia, a cone degeneration disease. ATF6 deletion mice develop rod and cone dysfunction with ageing, and selective activation of ATF6 prevents mutant rhodopsin accumulation.

[0008] PERK phosphorylates and inactivates eukaryotic translation initiation factor 2α (eIF2α) to attenuate global cap-dependent mRNA translation and therefore reduce protein load on the ER. However, phosphorylated eIF2α (eIF2α-P) induces the expression of a pro-apoptotic molecule, C / EBP homologous protein (CHOP), by selectively activating translation of transcription factor ATF4 through upstream open reading frames. As a negative feedback mechanism, ATF4 and CHOP induce expression of growth arrest and DNA-damage-inducible protein 34 (GADD34) to facilitate dephosphorylation of eIF2α-P and resume global mRNA translation. CHOP mediates ER stress-induced apoptosis through down-regulating anti- apoptotic Bcl2, up-regulating pro-apoptotic BH-3 only molecules Bim and PUMA, increasing expression of death receptor 5 (DR5) and caspase 8 cleavage. CHOP also can form heterodimers with ATF4 to cause cell death by upregulating protein synthesis and inducing oxidative stress. Inhibition of CHOP is beneficial in many disease models.

[0009] Provided herein are methods and agents for the modulation of ER stress. 2SUMMARY

[0010] Inhibitors of C / EBP homologous protein (CHOP) are identified herein. The inhibitors find use in the modulation of endoplasmic reticulum (ER) stress, and the treatment of conditions involving ER stress. ER stress occurs in many acute and chronic neurodegenerative diseases. In some embodiments modulation of ER stress protects injured neurons and improves functional recovery in a neuronal condition. In some embodiments a neuronal condition for treatment with a CHOP inhibitor includes, without limitation, spinal cord injury, stroke, Alzheimer’s disease, Parkinson’s disease, ALS, prion disease and retinal cell degenerations, including RGC and photoreceptor neurons.

[0011] In some embodiments, compositions and methods are provided for treating a mammalian subject for neuronal conditions associated with ER stress. In some embodiments the compositions are used for reducing or ameliorating degeneration of axons and / or soma of retinal ganglion cells (RGCs) by administering an effective dose of a CHOP inhibitor. Aspects of the disclosure include formulations of a CHOP inhibitor and a pharmaceutically acceptable excipient, including an ophthalmologic excipient. The formulations are optionally suitable for delivery to the eye, and may provide for sustained release delivery to the eye. A variety of neuropathies, including optical neuron (ON) conditions, may be treated by practicing the methods, including without limitation retinal ganglion cell degeneration, glaucoma, optic neuritis, ON traumatic injury and other ON-related diseases. The subject may be diagnosed with an ON neuropathy prior to treatment.

[0012] In some embodiments the CHOP inhibitor is a compound of formula I I where R1, R2, Rm H; halo, e.g. Br, Cl, F, I; - OR8, where R8may be an alkyl, including a lower alkyl, branched alkyl, cycloalkyl; -CN; - CO2R9, where R9 may be H, halo, an alkyl, including a lower alkyl, branched alkyl, cycloalkyl, amine, including a substituted amine; -NO2; amine, including a substituted amine; -CF3; alkyl; including a lower alkyl, cycloalkyl; etc.; and R5, R6, and R7are independently selected from H; halo; -CO2R9, -CN; -CF3; tetrazole, -CH2OH, -CF2H, -SO3H; NHCO2R’, -SO2NR’R”, -CONR’R”, where R’, R” are independently H, 3lower alkyl, branched alkyl, substituted lower alkyl. The compound of formula II may be provided as a derivative or prodrug.

[0013] In some embodiments R1, R2, R3 and R4 are independently selected from H; Br; C; F; I; CF3; and -OCH3; and R5 is CO2R9, where R9 is H or lower alkyl; or CONR’R”, where R’, R” are independently H, lower alkyl, or substituted lower alkyl.

[0014] In some embodiments, at least two of R5, R6, and R7are H. In some embodiments R6and R7are H. In some embodiments R5and R7are halo. In some embodiments R6is halo, R5and R7are H.

[0015] In some embodiments the CHOP inhibitor is a compound set forth in Table 1. In some embodiment the CHOP inhibitor is selected from:4T-5a28

[0016] In some embodiments the CHOP inhibitor has an IC50 on human CHOP of less than about 5 µM, less than about 3 µM, less tha ut 1 µM, less than about 0.5 µM, less than about 0.1 µM, and may be from about 0.01 µM to 5 µM.

[0017] The CHOP inhibitors can modulate ER stress, e.g. ER stress in a neuron. The effects of CHOP inhibition can include a range of outcomes. For instance, neuroprotective effects may include, without limitation, preservation of the structure and function of neurons, a reduction in neuronal cell body death, a reduction in neuronal axon death, an improvement in visual acuity when compared to the absence of treatment, an increase in ganglion cell complex size relative to the absence of treatment, and the like.

[0018] In an embodiment, a method is provided for treating a mammalian subject for a disorder relating to ER stress, the method comprising administering a therapeutically effective dose of a CHOP inhibitor of the disclosure. In some embodiments the disorder is a neuronal disorder, for example spinal cord injury, stroke, Alzheimer’s disease, Parkinson’s disease, ALS, prion disease and retinal cell degeneration. In some embodiments the disorder is an optical neuron disorder. In some embodiments a method is provided for reducing or ameliorating degeneration of axons and / or soma of retinal ganglion cells (RGCs) by administering a therapeutically effective dose of a CHOP inhibitor as set forth herein. In some embodiments, administration is ocular.

[0019] In some embodiments a formulation is provided, comprising a a compound of formula I. In some embodiments, the compound is set forth in Table 1. The formulation may be provided in a unit dose for treatment of a disorder relating to ER stress, comprising a therapeutically effective dose of the compound. The formulation may be provided for oral administration, parenteral administration, in an eye drop, for intra-vitreal injection, etc. The formulation may be provided for sustained release to the eye. In some embodiments the formulation is suitable for ocular administration, e.g. eye drops, sustained release to the eye, intra-vitreal injection, and the like.

[0020] When the formulation is administered, it may be administered at a time that is dependent on the type of neuropathy being treated. For example, if the neuropathy is the result of traumatic injury, the composition may be administered within hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, one week, two weeks or more than two weeks after traumatic injury.

[0021] In some embodiments, the composition of the present disclosure may be administered with a secondary treatment modality that is used to further treat the neuropathy, e.g. using agents known in the art for therapy of the condition. In some embodiments, an agent that activates XBP-1 is provided in the combination. If the optic neuropathy is glaucoma, a number of different secondary treatment modalities may be administered. For instance, secondary 5treatment modalities for glaucoma may include, without limitation, prostaglandins (e.g. latanoprost, travoprost, tafluprost, or bimatoprost), rho kinase inhibitors (e.g. netarsudil), nitric oxides (e.g. latanoprostene bunod), miotic or cholinergic agents (e.g. pilocarpine), alpha- adrenergic agonists (e.g. apraclonidine or brimonidine), beta blockers (e.g. betaxolol or timolol), carbonic anhydrase inhibitors (e.g. dorzolamide or brinzolamide), etc. BRIEFDESCRIPTION OF THEFIGURES

[0022] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

[0023] FIG.1A-1B. SAR Analog Design of CHOP inhibitor C5a. A, Inhibition of the CHOP-LUC reporter activity in HEK293T cells treated with 1 µM Tunicamycin (Tm) + 1 µM Thapsigarginin (Tg) in the presence of C5a or 5a28, 24 h after exposure. IC50 is calculated with nonlinear regression through dose-dependent fits of CHOP-Luc activities (relative to DMSO) of individual compound. Data are presented as means ± s.e.m., n = 4 independent replicates. B, Analog design of CHOP inhibitor targeting Ring A (Red), Ring B (green), and the linker region based on the core scaffold of C5a.

[0024] FIG.2A-2C. The top 10 analog compounds of CHOP inhibition effect. A, The top 10 CHOP inhibitors are shown with the chemical structure, IC50 (μM) of CHOP-LUC activity of the cells treated with 1 µM Tm + 1 µM Tg for 24 h, relative to the DMSO treated cells. Benchmark compounds are C5a and C5a28. B, Inhibition of the CHOP-LUC reporter in HEK293T cells treated with 1 µM Tm + 1 µM Tg for 24 h in the presence of top 10 small molecule CHOP inhibitors at 8-point dose. Data are presented as means ± s.e.m., n = 4 independent replicates. C, Quantification of hATF4 and hCHOP mRNA levels by RT-qPCR in HEK293T cells treated with 1 µM Tm + 1 µM Tg for 24 h in the presence of top 10 small molecule CHOP inhibitors (10 µM). Data are presented as means ± s.e.m., n = 4-6 independent replicates.

[0025] FIG. 3A. Inhibitory effects of the top 10 CHOP inhibitors on CHOP protein levels. A, Representative immunoblots of ATF4 and CHOP in HEK293T cells treated with 1 µM Tm + 1 µM Tg for 24 h, in the presence of top 10 small molecule CHOP inhibitors (10 µM). Quantification of ATF4 or CHOP protein levels are attached. Data are presented as means ± s.e.m., n = 4 independent replicates. One-way ANOVA with Dunnett’s post hoc test (compared to DMSO), *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Compounds with strong CHOP-LUC inhibitory effects (***p < 0.001 and ****p < 0.0001) are highlighted in red. 6

[0026] FIGS.4A-4B. CHOP-Luc cell-based assay and WB identify 5a, T-22 and 5a28 as top inhibitors. A, IC50 calculated with nonlinear regression through dose-dependent fits of CHOP promoter-driven luciferase (CHOP-Luc) activities (relative to DMSO) of top CHOP inhibitors 5a, 5a28 and T-22 at 8 indicated concentration in the presence of Tm / Tg (1 µM), 24 h after exposure. Data are presented as means ± s.e.m., n = 4 independent replicates. The structure and and IC50of dose-dependent responses of 12 “hits” (group 1) compounds in CHOP-Luc inhibition is as shown in Table 1 of the specification. B, Immunoblots of HEK293T cell lysates showing the effects of the compounds T-22, T-2029, T-2054, T-2031, 5a and 5a28 on the ATF4-CHOP pathway. Quantification of ATF4 and CHOP 24 h after DMSO or compound (10 µM) treatment in the presence of Tm / Tg (1 µM), relative to control (without Tm / Tg). n = 3-5 independent replicates. Data in this figure are presented as means ± s.e.m,. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA with Dunnett’s multiple comparisons test.

[0027] FIGS.5A-5B. Histological results of 5a / T-22 / 5a28 compound treatment in mouse SOHU glaucoma model. A, Quantification of surviving RBPMS+RGC somata in peripheral, middle and central regions of wholemount retinas at 3 weeks post silicon oil (SO) injection (3wpi), represented as percentage in SOHU eyes compared to the sham contralateral control eyes. n = 18 mice in control group, n = 9 for both 5a and T-22 groups, n=10 for 5a28 group. B, representative confocal images of peripheral, middle and central regions of wholemount retinas, showing surviving RBPMS+ (red) RGCs at 3wpi. Scale bar, 20 µm. For compound 5a treatment, each eye received an intravitreal injection twice per week with 1 µl of 5 mM compound for 3 weeks after SO injection. For compound T-22 / 5a28 treatment, each eye received an intravitreal injection twice per week with 1 µl of 2 mM compound for 3 weeks after SO injection. Data in this figure are presented as means ± s.e.m. *p < 0.05, ns, no significance, independent t-test for the comparisons between control and treatment groups.

[0028] FIGS. 6A-6D. In vivo imaging and visual function results of 5a / T-22 / 5a28 compound treatment in mouse SOHU glaucoma model. A, Quantification of GCC thickness measured by OCT at 3wpi, represented as percentage of GCC thickness in the SOHU eyes with treatment compared to the sham contralateral control eyes. n = 17 mice in PBS group, n = 7 for 5a group, n = 9 for T-22 group, n=10 for 5a28 group. B, quantification of P1-N2 amplitude of PERG at 3wpi, represented as percentage in SOHU eyes with treatment compared to the sham contralateral control eyes. n = 17 mice in PBS group, n = 9 for both 5a and T-22 groups, n=10 for 5a28 group. C, visual acuity measured by OKR at 3wpi, represented as percentage of cycles / degree value in the treatment eyes compared to the sham contralateral control eyes. n = 17 mice in PBS group, n = 9 for T-22 group, n=10 for both 5a and 5a28 groups. D, IOP measurements at 3wpi. n = 17 mice in PBS group, n = 9 for T-22 group, n=10 for both 5a and 5a28 groups. SOHU: intracameral injection of SO to induce ocular hypertension. All data in this 7figure are presented as means ± s.e.m., *p < 0.05, ns, no significance, independent t-test for the comparisons between control and treatment groups. DEFINITIONS

[0029] The terms “specific binding,” “specifically binds,” and the like, refer to non-covalent or covalent preferential binding to a molecule relative to other molecules or moieties in a solution or reaction mixture (e.g., an antibody specifically binds to a particular polypeptide or epitope relative to other available polypeptides). In some embodiments, the affinity of one molecule for another molecule to which it specifically binds is characterized by a KD (dissociation constant) of 10-5M or less (e.g., 10-6M or less, 10-7M or less, 10-8M or less, 10-9M or less, 10-10M or less, 10-11M or less, 10-12M or less, 10-13M or less, 10-14M or less, 10-15M or less, or 10-16M or less). "Affinity" refers to the strength of binding, increased binding affinity being correlated with a lower Kd.

[0030] The term “Alkyl” refers to a C1-C20alkyl that may be linear, branched, or cyclic. “Lower alkyl”, as in “lower alkyl”, or “substituted lower alkyl”, means a C1-C10 alkyl. The term “alkyl”, “lower alkyl” or “cycloalkyl” includes methyl, ethyl, isopropyl, propyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl, hexyl, cyclopropyl, cyclopropylmethyl, cyclobutyl, cyclobutylmethyl, cyclopentyl, cyclopentylmethyl, cyclohexyl, cyclohexylmethyl, C6to C12spirocycles, cyclopropylethyl, cyclobutylethyl, decalinyl, Bicyclo-[1.1.1]-pentyl, norboranyl, bicylo-[2.2.2]- octyl, cubyl, adamantanyl and related cage hydrocarbon moieties. In certain embodiments, the alkyl is a C1-C20 alkyl. In certain embodiments the alkyl group is poly deuterated.

[0031] A “substituted alkyl” is an alkyl which is typically mono-, di-, or tri-substituted with heterocycloalkyl, aryl, substituted aryl, heteroaryl, nitro, cyano (also referred to herein as nitrile), azido, halo, −OR, -SR, -SF5, -CHO, −COR, −C(O)OR, -C(O)-NR2, −OC(O)R, -OC(O)NR2, - OC(O)OR,--P(O)(OR)2, -OP(O)(OR)2, −NR2, -N+R3(wherein a counterion may be present), −CONR2, −NRCOR, -NHC(O)OR, -NHC(O)NR2, -NHC(NH)NR2,SO3- , -SO2OR, -OSO2R, -SO2NR2, or -NRSO2R, where each R is, independently, hydrogen, lower alkyl, R′-substituted lower alkyl, aryl, R′-substituted aryl, heteroaryl, heteroaryl(alkyl), R′-substituted aryl(alkyl), or aryl(alkyl) and each R′ is, independently, hydroxy, halo, alkyloxy, cyano, thio, SF5, nitro, alkyl, halo- alkyl, or amino. Substituted alkyls which are substituted with one to three of the substituents selected from the group consisting of alkynyl, cyano, halo, alkyloxy, thio, nitro, amino, or hydroxy are particularly of interest.

[0032] The term “Aryl” refers to an aromatic ring having (4n+2) pi electrons that may contain 6 to 20 ring carbon atoms, and be composed of a single ring (e.g., phenyl), or two or more condensed rings, such as 2 to 3 condensed rings (e.g., naphthyl), or two or more aromatic rings, such as 2 to 3 aromatic rings, which are linked by a single bond (e.g., biphenylyl). In certain 8cases, the aryl is C6-C16or C6to C14. In certain embodiments the alkyl group has one or more hydrogen atoms replaced with deuterium.

[0033] Heteroaryl means an aromatic ring system containing (4n+2)pi electrons and comprised of 1 to 10 ring carbon atoms and 1 to 5 heteroatoms selected from O, N, S, Se, having a single ring (e.g., thiophene, pyridine, pyrazine, imidazole, oxazole, tetrazole, etc.), or two or more condensed rings, for example 2 to 3 condensed rings (e.g., indole, benzimidazole, quinolone, quinoxaline, phenothiazine, etc.), or two or more aromatic rings, such as 2 to 3 aromatic rings, which are linked by a single bond (e.g., bipyridyl). In some cases, the heteroaryl is C1-C16, and a selection of 1 to 5 heteroatoms consisting of S, Se, N, and O.

[0034] The term “heterocycloalkyl”, “heterocycle”, “heterocyclic group” or “heterocyclyl” refers to a saturated or unsaturated nonaromatic ring system containing 1 to 10 ring carbon atoms and 1 to 5 heteroatoms selected from O, N, S, Se, having a single ring (e.g., tetrahydrofuran, aziridine, azetidine, pyrrolidine, piperidine, tetrathiopyran, hexamethylene oxide, oxazepane, etc.), or two or more condensed rings, such as 2 to 3 condensed rings (e.g., indoline, tetrahydrobenzodiazapines, etc., including fused, bridged and spiro ring systems, having 3-15 ring atoms, included 1 to 4 heteroatoms. In certain cases, the heterocycloalky is C1-C16, and a selection of 1 to 5 heteroatoms consisting of S, Se, N, and O. In fused ring systems, one or more of the rings can be cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, provided that the point of attachment is through the non-aromatic ring. In certain embodiments, the nitrogen and / or sulfur atom(s) of the heterocyclic group are optionally oxidized to provide for the N-oxide, -S(O)-, or – SO2- moieties.

[0035] Examples of heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, benzimidazole, pyrazole, benzopyrazole, tetrazole, 1,2,3-triazole, benzotriazole, 1,2,4-triazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, benzoisothiazole, phenazine, isoxazole, benzoisooxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7- tetrahydrobenzo[b]thiophene, thiazole, benzothiazole, thiazolidine, furan, benzofuran, thiophene, benzothiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to as thiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, benzotetrahydrofuranyl, and the like.

[0036] Substituted heterocycloalkyl, aryl, heteroaryl are optionally substituted with, hydrogen, 1 to 3 alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkyl(alkyl), aryl, substituted aryl, aryl(alkyl), -SO2NR5R5, -PO3H2, -NR5SO2R6or –NR5C(=O)R6, wherein R5and R6are independently, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkyl(alkyl), aryl, optionally 9substituted heterocycloalkyl, aryloxy, heteroaryl, heteroaryl(alkyl), or R5and R6together are -(CH2)3-6- or -(CH2)0-3X(CH2)0-3- where X= NR, O, S, SO2, substituted aryl(alkyl), halo(alkyl), SF5, NR53+, azido, cyano (also referred to herein as nitrile), -OR5, -SR5, -NR5R6, halogen, nitro, SCH3, OCF3, SO2CH3, SCF3, SO2CF3, CF3, -SO2OR5, -OSO2R5, CCl3, -C(=O)R5, -C(=O)OR5; -C(=O)NR5R6, -OC(=O)R5.

[0037] By "substituted" as in "substituted alkyl," "substituted aryl," and the like, as alluded to in some of the aforementioned definitions, is meant that in the hydrocarbyl, alkyl, aryl, or other moiety, at least one hydrogen atom bound to a carbon (or other) atom is replaced with one or more non-hydrogen substituents. Examples of such substituents include, without limitation, functional groups, and the hydrocarbyl moieties C1-C24 alkyl (including C1-C18 alkyl, further including C1-C12 alkyl, and further including C1-C6 alkyl), C2-C24 alkenyl (including C2-C18 alkenyl, further including C2-C12 alkenyl, and further including C2-C6 alkenyl), C2-C24 alkynyl (including C2-C18 alkynyl, further including C2-C12 alkynyl, and further including C2-C6 alkynyl), C5-C30 aryl (including C5-C20 aryl, and further including C5-C12 aryl), and C6-C30 aralkyl (including C6-C20 aralkyl, and further including C6-C12 aralkyl). The above-mentioned hydrocarbyl moieties may be further substituted with one or more functional groups or additional hydrocarbyl moieties such as those specifically enumerated. Unless otherwise indicated, any of the groups described herein are to be interpreted as including substituted and / or heteroatom- containing moieties, in addition to unsubstituted groups.

[0038] “Sulfonyl” refers to the group SO2-alkyl, SO2-substituted alkyl, SO2-alkenyl, SO2- substituted alkenyl, SO2-alkynyl, SO2-substituted alkynyl, SO2-cycloalkyl, SO2-substituted cylcoalkyl, SO2-cycloalkenyl, SO2-substituted cylcoalkenyl, SO2-aryl, SO2-substituted aryl, SO2- heteroaryl, SO2-substituted heteroaryl, SO2-heterocyclic, and SO2-substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. Sulfonyl includes, by way of example, methyl-SO2-, phenyl-SO2-, and 4-methylphenyl- SO2-. Sulfonimidoyl refers to S(O)(NH)-bonded as for sulfonyl defined above.

[0039] The term "water-soluble group" refers to a functional group that is well solvated in aqueous environments and that imparts improved water solubility to the compound to which it is attached. Water-soluble groups of interest include, but are not limited to, polyalcohols, straight chain or cyclic saccharides, primary, secondary, tertiary, or quaternary amines and polyamines, sulfate groups, sulfonate groups, sulfinate groups, carboxylate groups, phosphate groups, phosphonate groups, phosphinate groups, ascorbate groups, glycols, including polyethylene glycols (PEG) and modified PEGs, and polyethers. In some instances, water-soluble groups are primary, secondary, tertiary, and quaternary amines, carboxylates, phosphonates, phosphates, sulfonates, sulfates, -N(H)0-1(CH2CH2OH)1-2, -NHCH2CH2N(CH3)2-3, -NHCH2CH2SO3H, - 10NHCH2CH2PO3H2and -NHCH2CH2CO2H, --(CH2CH2O)yyCH2CH2XRyy, -- (CH2CH2O)yyCH2CH2X--, --X(CH2CH2O)yyCH2CH2--, glycol, oligoethylene glycol, and polyethylene glycol, wherein yy is selected from 1 to 1000, X is selected from O, S, and NRZZ, and RZZand RYYare independently selected from H and C1-3 alkyl.

[0040] The term “carboxy isostere” refers to standard medicinal bioisosteric replacement groups for carboxylic acids, amides and ester. These include, but are not limited to: acyl cyanamide, tetrazoles, hydroxychromes, 3-hydroxy-1,2,4-triazoles, 1-hydroxy pyrazoles, 2,4- dihydroxy imidazoles, 1-hydroxy imidazole, 1-hydroxy 1,2,3-triazole, alkylsulfonyl carboxamides, hydroxy isoxazoles, 5-hydroxy 1,2,4-oxadiazoles, thiazoles, 1,2,4-oxadiazoles, 1,2,4-oxadiazolones, oxazoles, triazoles, thiazoles, others hydroxamic acids, sulfonimide, acylsulfonamide, sulfonylureas, oxadiazolone, thiazolidinediones, oxadiazole, thiadiazole, isothiazoles, difluorophenols, tetramic acids, tetronic acids, squaric acids, hydroxyquinoline- ones, hydroxyquinoline-2-ones, boronic acids and phosphoric acids.

[0041] As used herein the term “PEG” refers to a polyethylene glycol or a modified polyethylene glycol. Modified polyethylene glycol polymers include a methoxypolyethylene glycol, and polymers that are unsubstituted or substituted at one end with an alkyl, a substituted alkyl or a substituent (e.g., as described herein).

[0042] By the term “functional groups” is meant chemical groups such as halo, hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C20 aryloxy, acyl (including C2-C24 alkylcarbonyl (-CO-alkyl) and C6-C20 arylcarbonyl (-CO-aryl)), acyloxy (-O- acyl), C2-C24 alkoxycarbonyl (-(CO)-O-alkyl), C6-C20 aryloxycarbonyl (-(CO)-O-aryl), halocarbonyl (-CO)-X where X is halo), C2-C24 alkylcarbonato (-O-(CO)-O-alkyl), C6-C20 arylcarbonato (-O-(CO)-O-aryl), carboxy (-COOH), carboxylato (-COO- ), carbamoyl (-(CO)- NH2), mono-substituted C1-C24 alkylcarbamoyl (-(CO)-NH(C1-C24 alkyl)), di-substituted alkylcarbamoyl (-(CO)-N(C1-C24 alkyl)2), mono-substituted arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH2), carbamido (-NH-(CO)-NH2), cyano (-C≡N), isocyano (-N+≡C-), cyanato (-O-C≡N), isocyanato (-O-N+≡C-), isothiocyanato (-S-C≡N), azido (-N=N+=N-), formyl (-(CO)-H), thioformyl (-(CS)-H), amino (-NH2), mono- and di-(C1-C24 alkyl)-substituted amino, mono- and di-(C5-C20 aryl)-substituted amino, C2-C24 alkylamido (-NH-(CO)-alkyl), C5-C20 arylamido (-NH-(CO)-aryl), imino (-CR=NH where R = hydrogen, C1-C24 alkyl, C5-C20 aryl, C6-C20 alkaryl, C6-C20 aralkyl, etc.), alkylimino (-CR=N(alkyl), where R = hydrogen, alkyl, aryl, alkaryl, etc.), arylimino (-CR=N(aryl), where R = hydrogen, alkyl, aryl, alkaryl, etc.), nitro (-NO2), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-SO2-O-), C1-C24 alkylsulfanyl (-S-alkyl; also termed "alkylthio"), arylsulfanyl (-S-aryl; also termed "arylthio"), C1-C24 alkylsulfinyl (-(SO)-alkyl), C5- C20 arylsulfinyl (-(SO)-aryl), C1-C24 alkylsulfonyl (-SO2-alkyl), C5-C20 arylsulfonyl (-SO2-aryl), phosphono (-P(O)(OH)2), phosphonato (-P(O)(O-)2), phosphinato (-P(O)(O-)), phospho (-PO2), and phosphino (-PH2), mono- and di-(C1-C24 alkyl)-substituted phosphino, mono- and di-(C5- 11C20 aryl)-substituted phosphine. In addition, the aforementioned functional groups may, if a particular group permits, be further substituted with one or more additional functional groups or with one or more hydrocarbyl moieties such as those specifically enumerated above.

[0043] When the term "substituted" appears prior to a list of possible substituted groups, it is intended that the term apply to every member of that group. For example, the phrase "substituted alkyl and aryl" is to be interpreted as "substituted alkyl and substituted aryl."

[0044] In addition to the disclosure herein, the term “substituted,” when used to modify a specified group or radical, can also mean that one or more hydrogen atoms of the specified group or radical are each, independently of one another, replaced with the same or different substituent groups as defined below.

[0045] In addition to the groups disclosed with respect to the individual terms herein, substituent groups for substituting for one or more hydrogens (any two hydrogens on a single carbon can be replaced with =O, =NR70, =N-OR70, =N2or =S) on saturated carbon atoms in the specified group or radical are, unless otherwise specified, -R60, halo, =O, -OR70, -SR70, -NR80R80, trihalomethyl, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -SO2R70, -SO2O–M+, -SO2OR70, -OSO2R70, -OSO2O–M+, -OSO2OR70, -P(O)(O–)2(M+)2, -P(O)(OR70)O–M+, -P(O)(OR70) 2, -C(O)R70, -C(S)R70, -C(NR70)R70, -C(O)O–M+, -C(O)OR70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(O)R70, -OC(S)R70, -OC(O)O- M+, -OC(O)OR70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70CO2–M+, -NR70CO2R70, -NR70C(S)OR70, -NR70C(O)NR80R80, -NR70C(NR70)R70and -NR70C(NR70)NR80R80, where R60is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl, each R70is independently hydrogen or R60; each R80is independently R70or alternatively, two R80’s, taken together with the nitrogen atom to which they are bonded, form a 5-, 6- or 7-membered heterocycloalkyl which may optionally include from 1 to 4 of the same or different additional heteroatoms selected from the group consisting of O, N and S, of which N may have -H or C1-C3 alkyl substitution; and each M+is a counter ion with a net single positive charge. Each M+may independently be, for example, an alkali ion, such as K+, Na+, Li+; an ammonium ion, such as+N(R60)4; or an alkaline earth ion, such as [Ca2+]0.5, [Mg2+]0.5, or [Ba2+]0.5(“subscript 0.5 means that one of the counter ions for such divalent alkali earth ions can be an ionized form of a compound of the invention and the other a typical counter ion such as chloride, or two ionized compounds disclosed herein can serve as counter ions for such divalent alkali earth ions, or a doubly ionized compound of the invention can serve as the counter ion for such divalent alkali earth ions). As specific examples, -NR80R80is meant to include -NH2, -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N-methyl-piperazin-1-yl, N-morpholinyl, - N(H)0-1(CH2CH2OH)1-2, -NHCH2CH2N(CH3)2-3, -NHCH2CH2SO3H, -NHCH2CH2PO3H2and -12

[0046] In addition to the disclosure herein, substituent groups for hydrogens on unsaturated carbon atoms in “substituted” alkene, alkyne, aryl and heteroaryl groups are, unless otherwise specified, -R60, halo, -O-M+, -OR70, -SR70, -S–M+, -NR80R80, trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -SO2R70, -SO3–)O–O2–O2–O2–R70hatn case o substtuted a ene or a yne, t e substtuents are not -O , -O , -S , or -SM+.

[0047] In addition to the groups disclosed with respect to the individual terms herein, substituent groups for hydrogens on nitrogen atoms in “substituted” heteroalkyl and cycloheteroalkyl groups are, unless otherwise specified, -R60, -O-M+, -OR70, -SR70, -S-M+, -NR80R80, trihalomethyl, -CF3, -CN, -NO, -NO2, -S(O)2R70, -S(O)2O-M+, -S(O)2OR70, -OS(O)2R70, -OS(O)2 70, 0, - R7as

[0048] Salts include but are not limited to: Na, K, Ca, Mg, ammonium, tetraalkyl ammonium, aryl and alkyl sulfonates, phosphates, carboxylates, sulfates, Cl, Br, and guanidinium.

[0049] Unless otherwise specified, reference to an atom is meant to include isotopes of that atom. For example, reference to H is meant to include1H,2H (i.e., D) and3H (i.e., T), and reference to C is meant to include12C and all isotopes of carbon (such as13C).

[0050] In addition to the disclosure herein, in a certain embodiment, a group that is substituted has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.

[0051] Unless indicated otherwise, the nomenclature of substituents that are not explicitly defined herein are arrived at by naming the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment. For example, the substituent “heterocycloalkyl(alkyl)” refers to the group (heterocycloalkyl)-(alkyl)-.

[0052] As to any of the groups disclosed herein which contain one or more substituents, it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and / or synthetically non-feasible. In addition, the subject compounds include all stereochemical isomers arising from the substitution of these compounds. 13

[0053] In certain embodiments, a substituent may contribute to optical isomerism and / or stereo isomerism of a compound. Salts, solvates, hydrates, and prodrug forms of a compound are also of interest. Polymorphic, pseudo-polymorphic, amorphous and co-crystal forms of a compound are also of interest. All such forms are embraced by the present disclosure. Thus, the compounds described herein include salts, solvates, hydrates, prodrug and isomer forms thereof, including the pharmaceutically acceptable salts, solvates, hydrates, prodrugs and isomers thereof. In certain embodiments, a compound may be a metabolized into a pharmaceutically active derivative.

[0054] Turning to the administration of therapeutics, the compounds of the invention may be administered as described herein, or in a form from which the active agent can be derived, such as a prodrug. A "prodrug" is a derivative of a compound described herein, the pharmacologic action of which results from the conversion by chemical or metabolic processes in vivo to the active compound. Prodrugs include compounds wherein an amino acid residue, or a polypeptide chain of two or more (e.g., two, three or four) amino acid residues is covalently joined through an amide or ester bond to a free amino, hydroxyl or carboxylic acid group of the compound. Additional types of prodrugs are also encompassed. For instance, free carboxyl groups can be derivatized as amides or alkyl esters. Prodrug esters as employed herein includes esters and carbonates formed by reacting one or more hydroxyls of compounds of the method of the invention with alkyl, alkoxy, or aryl substituted acylating agents employing procedures known to those skilled in the art to generate acetates, pivalates, methylcarbonates, benzoates and the like. As further examples, free hydroxyl groups may be derivatized using groups including but not limited to hemisuccinates, phosphate esters, dimethylaminoacetates, and phosphoryloxymethyloxycarbonyls, as outlined in Advanced Drug Delivery Reviews, 1996, 19, 115. Carbamate prodrugs of hydroxyl and amino groups are also included, as are carbonate prodrugs, sulfonate prodrugs, sulfonate esters and sulfate esters of hydroxyl groups. Free amines can also be derivatized to amides, sulfonamides or phosphonamides. All of the stated prodrug moieties may incorporate groups including but not limited to ether, amine and carboxylic acid functionalities. Moreover, any compound that can be converted in vivo to provide the bioactive agent (e.g., a compound of formula I) is a prodrug within the scope of the invention. Various forms of prodrugs are well known in the art. A comprehensive description of prodrugs and prodrug derivatives are described in: (a) The Practice of Medicinal Chemistry, Camille G. Wermuth et al., (Academic Press, 1996); (b) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985); (c) A Textbook of Drug Design and Development, P. Krogsgaard-Larson and H. Bundgaard, eds., (Harwood Academic Publishers, 1991).

[0055] Half maximal inhibitory concentration (IC50) is a measure of the potency of a substance in inhibiting a specific biological or biochemical function. IC50is a quantitative measure that indicates how much of a particular inhibitory substance (e.g. drug) is needed to inhibit, in vitro, 14a given biological process or biological component by 50%. The biological component may be an enzyme, e.g. CHOP. IC50 values are typically expressed as molar concentration.

[0056] The terms "treatment," "treating," "treat" and the like are used herein to generally refer to obtaining a desired pharmacologic and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom(s) thereof and / or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and / or adverse effect attributable to the disease. The term “treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease and / or symptom(s) from occurring in a subject who may be predisposed to the disease or symptom(s) but has not yet been diagnosed as having it; (b) inhibiting the disease and / or symptom(s), i.e., arresting development of a disease and / or the associated symptoms; or (c) relieving the disease and the associated symptom(s), i.e., causing regression of the disease and / or symptom(s). Those in need of treatment can include those already inflicted (e.g., those with optic neuropathies) as well as those in which prevention is desired (e.g., those with increased susceptibility to neuropathies; those with neuropathies; those suspected of having neuropathies; etc.).

[0057] The terms “recipient,” “individual,” “subject,” “host,” and “patient” are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, camels, etc. In some embodiments, the mammal is human.

[0058] The terms "co-administration" and "in combination with" include the administration of two or more therapeutic agents either simultaneously, concurrently or sequentially within no specific time limits. In one embodiment, the agents are present in the cell or in the subject's body at the same time or exert their biological or therapeutic effect at the same time. In one embodiment, the therapeutic agents are in the same composition or unit dosage form. In other embodiments, the therapeutic agents are in separate compositions or unit dosage forms. In certain embodiments, a first agent can be administered prior to (e.g., minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks) after the administration of a second therapeutic agent.

[0059] The term “sample” as used herein relates to a material or mixture of materials, typically, although not necessarily, in fluid, i.e., aqueous, form, containing one or more components of 15interest. Samples may be derived from a variety of sources such as from food stuffs, environmental materials, a biological sample or solid, such as tissue or fluid isolated from an individual, including but not limited to, for example, plasma, serum, spinal fluid, semen, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs, and also samples of in vitro cell culture constituents (including but not limited to conditioned medium resulting from the growth of cells in cell culture medium, putatively virally infected cells, recombinant cells, and cell components). In certain embodiments of the method, the sample includes a cell. In some instances of the method, the cell is in vitro. In some instances of the method, the cell is in vivo.

[0060] The term 'neuroprotective' as used herein refers to the ability to protect neurons or their axons or synapses in the central or peripheral nervous system from damage or death. Many different types of insult can lead to neuronal damage or death, for example: metabolic stress caused by hypoxia, hypoglycemia, diabetes, loss of ionic homeostasis or other deleterious process, physical injury of neurons, exposure to toxic agents and numerous diseases affecting the nervous system including inherited disorders. The presence of an agent that is neuroprotective enables a neuron to remain viable upon exposure to insults that would otherwise cause a loss of functional integrity in an unprotected neuron.

[0061] The term 'injury' as used herein refers to damage inflicted on the neuron, whether in the cell body or in axonal or dendritic processes. This can be a physical injury in the conventional sense i.e. traumatic injury to the brain, spinal cord or peripheral nerves caused by an external force applied to a subject. Other damaging external factors are for example environmental toxins such as mercury and other heavy metals, pesticides and solvents. Alternatively, injury can result from an insult to the neuron originating from within the subject, for example: reduced oxygen and energy supply as in ischemic stroke and diabetic neuropathy, autoimmune attack as in multiple sclerosis or oxidative stress and free-radical generation as is believed to be important in amyotrophic lateral sclerosis. Injury is also used here to refer to any defect in the mechanism of axonal transport.

[0062] In some embodiments, the CHOP inhibitor is intended for use as a neuroprotective medicament wherein the medicament is used to treat a neurodegenerative disorder. In one embodiment the neurodegenerative disorder is associated with ER stress. Examples of disorders where such degeneration may be of importance include Alexander's disease, Alper's disease, Alzheimer's disease, Amyotrophic lateral sclerosis, Ataxia telangiectasia, Batten disease, Canavan disease, Cerebral palsy, Cockayne syndrome, Corticobasal degeneration, Creutzfeldt- Jakob disease, Diabetic neuropathy, Frontotemporal lobar degeneration, Glaucoma, Guillain- Barre syndrome, Hereditary spastic paraplegia, Huntington's disease, HIV associated dementia, Kennedy's disease, Krabbe's disease, Lewy body dementia, Motor neuron disease, Multiple System Atrophy, Multiple sclerosis, Narcolepsy, Neuroborreliosis, 16Niemann Pick disease, Parkinson's disease, Pelizaeus- Merzbacher Disease, Peripheral neuropathy, Pick's disease, Primary lateral sclerosis, Prion diseases, Progressive Supranuclear Palsy, Refsum's disease, Sandhoff's disease, Schilder's disease, Spinocerebellar ataxia, Spinal cord injury, Spinal muscular atrophy, Steele- Richardson-Olszewski disease, Stroke and other ischaemic disorders, Tabes dorsalis or Traumatic brain injury. In some embodiments the disorder is glaucoma. DETAILEDDESCRIPTION

[0063] Before the present invention is described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0064] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0065] Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0066] 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 to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

[0067] All publications and patents cited in this specification are herein incorporated by reference in their entirety, as if each individual publication or patent were specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should 17not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0068] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0069] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0070] While the compositions and methods have been or will be described for the sake of grammatical fluidity with functional explanations, it is to be expressly understood that the claims, unless expressly formulated under 35 U.S.C. §112, are not to be construed as necessarily limited in any way by the construction of "means" or "steps" limitations, but are to be accorded the full scope of the meaning and equivalents of the definition provided by the claims under the judicial doctrine of equivalents, and in the case where the claims are expressly formulated under 35 U.S.C. §112 are to be accorded full statutory equivalents under 35 U.S.C. §112.

[0071] Inhibitors of CHOP are identified herein. High-throughput screening using the methods disclosed herein identified novel neuroprotective agents based on their ability to modulate C / EBP homologous protein (CHOP) based endoplasmic reticulum (ER) stress. Neuroprotective agents can block ER stress-induced CHOP expression, suppress the unfolded protein response (UPR) pathway, and protect neurons, including retinal ganglion cells (RGCs), optic nerve cells (ON), and visual functions in glaucoma and traumatic injury. 18

[0072] In some embodiments the CHOP inhibitor is a compound of formula I I where R1, R2, R3 m H; halo, e.g. Br, Cl, F, I; -OR8, where R8 may be an alkyl, including a lower alkyl, branched alkyl, cycloalkyl; -CN; - CO2R9, where R9 may be H, halo, an alkyl, including a lower alkyl, branched alkyl, cycloalkyl, amine, including a substituted amine; -NO2; amine, including a substituted amine; -CF3; alkyl; including a lower alkyl, cycloalkyl; etc.; and R5, R6, and R7 are independently selected from H; halo; -CO2R9, -CN; -CF3; tetrazole, -CH2OH, -CF2H, -SO3H; NHCO2R’, -SO2NR’R”, -CONR’R”, where R’, R” are independently H, lower alkyl, branched alkyl, substituted lower alkyl. The compound of formula II may be provided as a derivative or prodrug.

[0073] In some embodiments R1, R2, R3and R4are independently selected from H; Br; C; F; I; CF3; and -OCH3;and R5is CO2R9, where R9is H or lower alkyl; or CONR’R”, where R’, R” are independently H, lower alkyl, or substituted lower alkyl.

[0074] In some embodiments, at least two of R5, R6, and R7 are H. In some embodiments R6 and R7 are H. In some embodiments R5 and R7 are halo. In some embodiments R6 is halo, R5 and R7 are H.

[0075] In some embodiments R1, R2, R3and R4are independently selected from H; Br; C; F; I; CF3; and -OCH3;R5is CO2R9, where R9is H or lower alkyl; or CONR’R”, where R’, R” are independently H, lower alkyl, or substituted lower alkyl; R6and R7are H. The compound of formula 1 may be provided as a derivative or prodrug.

[0076] In some embodiments an isolated compound of Formula I is provided. In some embodiments the isolated compound is other than T-5a and T-5a28.

[0077] In some embodiments the CHOP inhibitor has an IC50on human CHOP of less than about 5 µM, less than about 3 µM, less than about 1 µM, less than about 0.5 µM, less than about 0.1 µM, and may be from about 0.01 µM to 5 µM.

[0078] In some embodiments the CHOP inhibitor is a compound set forth in Table 1. In some embodiment the CHOP inhibitor is selected from: 19Table 1 Tar et-No Structure IC0202122233.40 μM2425262728293031[ ] p p p pathways play a critical role in both neuronal cell body and axon degeneration. For example, has been shown that ON injury induces ER stress in RGCs. ER stress activates a complex cascade of reactions, in general called the unfolded protein response (UPR). Striking neuroprotection has been accomplished by manipulating downstream signaling molecules individually or combined of ER stress / UPR, including C / EBP homologous protein (CHOP), X- Box-Binding Protein 1 (XBP-1), eukaryotic translation initiation factor 2 alpha (eIF2α), Activating Transcription Factor 6 (ATF6) and Activating Transcription Factor 4 (ATF4). Of special interest, it was found that modulating these molecules could achieve neuroprotection in three optic neuropathy models, indicating that ER stress is a common mechanism for neurodegeneration. Thus, targeting ER stress / UPR molecules, e.g. CHOP, is therapeutic in neural injury / diseases associated neurodegeneration. Pharmacologic Formulations

[0080] Formulations of CHOP inhibitors of the disclosure can be prepared for any suitable method of delivery, including ocular, intra-vitreal, oral, parenteral, etc. Parenteral infusions include intramuscular, intravenous (bolus or slow drip), intraarterial, intraperitoneal, intrathecal 32or subcutaneous administration. An agent can be administered in any manner which is medically acceptable. Sustained release administration is also specifically included in the disclosure, by such means as depot injections or erodible implants.

[0081] As noted above, an agent can be formulated with an a pharmaceutically acceptable carrier (one or more organic or inorganic ingredients, natural or synthetic, with which a subject agent is combined to facilitate its application). A suitable carrier includes sterile saline although other aqueous and non-aqueous isotonic sterile solutions and sterile suspensions known to be pharmaceutically acceptable are known to those of ordinary skill in the art. An "effective amount" refers to that amount which is capable of ameliorating or delaying progression of the diseased, degenerative or damaged condition. An effective amount can be determined on an individual basis and will be based, in part, on consideration of the symptoms to be treated and results sought. An effective amount can be determined by one of ordinary skill in the art employing such factors and using no more than routine experimentation.

[0082] For example a therapeutic dose may be at least about 0.01 µg / kg body weight, at least about 0.05 µg / kg body weight; at least about 0.1 µg / kg body weight, at least about 0.5 µg / kg body weight, at least about 1 µg / kg body weight, at least about 2.5 µg / kg body weight, at least about 5 µg / kg body weight, at least about 10 µg / kg body weight; at least about 50 µg / kg body weight; at least about 100 µg / kg body weight; at least about 500 µg / kg body weight; at least about 1 mg / kg body weight; at least about 10 mg / kg body weight; up to about 100 mg / kg body weight, and may be not more than about 1000 mg / kg body weight., not more than about 500 mg / kg body weight, not more than about 100 mg / kg body weight. It will be understood by one of skill in the art that such guidelines will be adjusted for the molecular weight of the active agent. The dosage may also be varied for localized administration, e.g. intraocular, oral, inhalation, etc., or for systemic administration, e.g. i.m., i.p., i.v., and the like.

[0083] In some embodiments an effective dose of a CHOP inhibitor is from 0.1 µg / retina to about 1 mg / retina or more, e.g. from about 0.5 µg, about 1 µg, about 5 µg, about 10 µg, about 25 µg, about 50 µg, about 75 µg, about 100 µg, about 250 µg, about 500 µg, about 750 µg, about 1 mg. In some embodiments the agent is provided in a sustained release formulation that delivers the agent of a period of over about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or more, e.g. over 2 weeks, 3 weeks 4 weeks, or more. In some embodiments the CHOP inhibitor is delivered topically to the eye, e.g. in the form of eye drops. In some embodiments the CHOP inhibitor is delivered by intravitreal injection.

[0084] The volume in intravitreal injection, per injection, may be not more than about 500 µl, not more than about 200 µl, not more than about 100 µl, and may be from about 1 µl to about 200 µl, from about 5 µl to about 100 µl, from about 25 µl to about 100 µl, and may be around 50 µl. 33

[0085] The compounds may be dissolved in an aqueous diluent for topical administration to the eye. Any suitable aqueous diluent may be used, for example normal saline (0.9% NaCl), phosphate-buffered saline, a balanced salt solution, etc. For example a balance salt solution may be a sterile balanced salt solution, each mL containing sodium chloride (NaCl) 0.64%, potassium chloride (KCl) 0.075%, calcium chloride dihydrate (CaCl2·2H2O) 0.048%, magnesium chloride hexahydrate (MgCl26·H2O) 0.03%, sodium acetate trihydrate (C2H3NaO2·3H2O) 0.39%, sodium citrate dihydrate (C6H5Na3O7·2H2O) 0.17%, sodium hydroxide and / or hydrochloric acid (to adjust pH), and water for injection. The pH is approximately 7.5. The osmolality is approximately 300 mOsm / Kg.

[0086] A typical dose is from 1 to 2 drops per eye, where a drop may be from about 5 ml; from about 10 ml; from about 15 ml; from about 20 ml; from about 25 ml; from about 30 ml; up to about 50 ml; up to about 40 ml.

[0087] The effective dose for topical delivery may be at least about 0.01 mg / eye; at least about 0.1 mg / eye; at least about 0.5 mg / eye; at least about 0.75 mg / eye; at least about 1 mg / eye; at least about 1.5 mg / eye; at least about 2 mg / eye; at least about 2.5 mg / eye; at least about 3 mg / eye; at least about 3.5 mg / eye; at least about 4 mg / eye; at least about 4.5 mg / eye; at least about 5 mg / eye; and up to about 50 mg / eye; and up to about 30 mg / eye; and up to about 25 mg / eye; and up to about 20 mg / eye; and up to about 25 mg / eye; and up to about 15 mg / eye; and up to about 10 mg / eye.

[0088] In certain embodiments, multiple therapeutically effective doses are administered according to a daily dosing regimen, or intermittently. For example, a therapeutically effective dose can be administered, one day a week, two days a week, three days a week, four days a week, or five days a week, and so forth. By "intermittent" administration is intended the therapeutically effective dose can be administered, for example, every other day, every two days, every three days, once a week, once every two weeks, once every three weeks, once a month, and so forth. For example, in some embodiments, an antibody is administered once every two to four weeks for an extended period of time, such as for 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 24 months, and so forth. By "twice-weekly" or "two times per week" is intended that two therapeutically effective doses of the agent in question is administered to the subject within a 7 day period, beginning on day 1 of the first week of administration, with a minimum of 72 hours, between doses and a maximum of 96 hours between doses. By "thrice weekly" or "three times per week" is intended that three therapeutically effective doses are administered to the subject within a 7 day period, allowing for a minimum of 48 hours between doses and a maximum of 72 hours between doses. For purposes of the present invention, this type of dosing is referred to as "intermittent" therapy. In accordance with the methods of the present invention, a subject can receive intermittent therapy for one or more weekly or monthly cycles until the desired 34therapeutic response is achieved. The agents can be administered by any acceptable route of administration as noted herein below.

[0089] Formulations suitable for injection can be administered by an intravitreal, intraocular, or other route of administration, e.g., injection into the retina.

[0090] An agent can be administered as a pharmaceutical composition comprising a pharmaceutically acceptable excipient. The preferred form depends on the intended mode of administration and therapeutic application. The compositions can also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.

[0091] As used herein, compounds which are "commercially available" may be obtained from commercial sources including but not limited to Acros Organics (Pittsburgh PA), Aldrich Chemical (Milwaukee WI, including Sigma Chemical and Fluka), Apin Chemicals Ltd. (Milton Park UK), Avocado Research (Lancashire U.K.), BDH Inc. (Toronto, Canada), Bionet (Cornwall, U.K.), Chemservice Inc. (West Chester PA), Crescent Chemical Co. (Hauppauge NY), Eastman Organic Chemicals, Eastman Kodak Company (Rochester NY), Fisher Scientific Co. (Pittsburgh PA), Fisons Chemicals (Leicestershire UK), Frontier Scientific (Logan UT), ICN Biomedicals, Inc. (Costa Mesa CA), Key Organics (Cornwall U.K.), Lancaster Synthesis (Windham NH), Maybridge Chemical Co. Ltd. (Cornwall U.K.), Parish Chemical Co. (Orem UT), Pfaltz & Bauer, Inc. (Waterbury CN), Polyorganix (Houston TX), Pierce Chemical Co. (Rockford IL), Riedel de Haen AG (Hannover, Germany), Spectrum Quality Product, Inc. (New Brunswick, NJ), TCI America (Portland OR), Trans World Chemicals, Inc. (Rockville MD), Wako Chemicals USA, Inc. (Richmond VA), Novabiochem and Argonaut Technology.

[0092] Compounds can also be made by methods known to one of ordinary skill in the art. As used herein, "methods known to one of ordinary skill in the art" may be identified though various reference books and databases. Suitable reference books and treatises that detail the synthesis of reactants useful in the preparation of compounds of the present invention, or provide references to articles that describe the preparation, include for example, "Synthetic Organic Chemistry", John Wiley & Sons, Inc., New York; S. R. Sandler et al., "Organic Functional Group Preparations," 2nd Ed., Academic Press, New York, 1983; H. O. House, "Modern Synthetic Reactions", 2nd Ed., W. A. Benjamin, Inc. Menlo Park, Calif.1972; T. L. Gilchrist, “Heterocyclic Chemistry”, 2nd Ed., John Wiley & Sons, New York, 1992; J. March, “Advanced Organic Chemistry: Reactions, Mechanisms and Structure”, 4th Ed., Wiley-Interscience, New York, 351992. Specific and analogous reactants may also be identified through the indices of known chemicals prepared by the Chemical Abstract Service of the American Chemical Society, which are available in most public and university libraries, as well as through on-line databases (the American Chemical Society, Washington, D.C. may be contacted for more details). Chemicals that are known but not commercially available in catalogs may be prepared by custom chemical synthesis houses, where many of the standard chemical supply houses (e.g., those listed above) provide custom synthesis services.

[0093] In some embodiments, pharmaceutical compositions can also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized SepharoseTM, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).

[0094] A carrier may bear the agents in a variety of ways, including covalent bonding either directly or via a linker group, and non-covalent associations. Suitable covalent-bond carriers include proteins such as albumins, peptides, and polysaccharides such as aminodextran, each of which have multiple sites for the attachment of moieties. The nature of the carrier can be either soluble or insoluble for purposes of the invention.

[0095] Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyidimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEENTM, PLURONICSTMor polyethylene glycol (PEG). Formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0096] The active ingredients may also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, 36microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0097] Compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997. The agents of this invention can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient. The pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0098] Toxicity of the active agents can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used in further optimizing and / or defining a therapeutic dosage range and / or a sub-therapeutic dosage range (e.g., for use in humans). The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. Conditions for Treatment

[0099] The CHOP inhibitors of the disclosure are useful as a neuroprotective medicament wherein the neuropathy is associated with ER stress. Neuropathies associated with endoplasmic reticulum (ER) stress involve disruptions in the normal functioning of the endoplasmic reticulum, a cellular organelle responsible for protein synthesis, folding, and processing. When cells experience stress, such as an accumulation of misfolded proteins, the ER initiates a signaling pathway called the unfolded protein response (UPR) to restore cellular homeostasis. However, chronic or severe ER stress can lead to the development of neuropathies.

[0100] Several mechanisms contribute to ER stress-induced neuropathies. Misfolded Protein Accumulation: ER stress can result from the accumulation of misfolded proteins within the endoplasmic reticulum. This can occur due to genetic mutations, environmental factors, or disease conditions. The UPR is activated to address the issue, but prolonged stress can lead to nerve damage. Oxidative Stress: ER stress often triggers oxidative stress, causing an imbalance between the production of reactive oxygen species (ROS) and the ability of the cell to detoxify them. Oxidative stress can damage cellular components, including proteins and lipids, 37contributing to neuropathies. Inflammation: ER stress can activate inflammatory responses, involving the release of pro-inflammatory cytokines. Chronic inflammation can contribute to nerve damage and is implicated in various neuropathic conditions. Apoptosis (Cell Death): Prolonged or severe ER stress may activate apoptotic pathways, leading to cell death. In the context of neuropathies, this can result in the loss of nerve cells and disruption of normal neural function. Impaired Autophagy: Autophagy is a cellular process responsible for removing damaged or dysfunctional cellular components. ER stress can interfere with autophagy, leading to the accumulation of cellular debris and contributing to nerve dysfunction.

[0101] Neuropathies associated with ER stress are often observed in the context of certain neurodegenerative diseases, metabolic disorders, and conditions like diabetes. For instance, diabetic neuropathy is linked to ER stress in nerve cells due to the metabolic challenges associated with diabetes.

[0102] Conditions for treatment include central and peripheral nervous systems axonopathies. The axonopathy may be the result of disease or trauma, including for example: CNS axonapathies such as multiple sclerosis, amyotrophic lateral sclerosis (ALS) and hereditary spastic paraplegia (HSP); PNS nerve injury; traumatic brain injury; spinal cord injury or neuronal injury induced by a toxic agent such as a chemotherapeutic agent; and the like. In an embodiment, the neuropathy is a neuronal injury induced by a chemotherapeutic agent, e.g. a taxane, vincristine, etc. In some embodiments the neuropathy is an optic nerve neuropathy. In some embodiments the optic nerve neuropathy is glaucoma, e.g. open-angle glaucoma, angle- closure glaucoma, etc. In other embodiments an optic neuropathy is non-arteritic ischemic optic neuropathy (NAION), optic neuritis, ischemic optic neuropathy, inflammatory (non- demyelinating) and traumatic optic neuropathy, etc.

[0103] In one embodiment, the neurodegenerative disorder is an ophthalmic disorder such as glaucoma. Glaucomas are a group of eye disorders characterized by progressive optic nerve damage in which an important part is a relative increase in intraocular pressure (IOP) that can lead to irreversible loss of vision. Glaucomas are categorized as open-angle glaucoma or angle- closure glaucoma. The “angle” refers to the angle formed by the junction of the iris and cornea at the periphery of the anterior chamber. The angle is where > 98% of the aqueous humor exits the eye via either the trabecular meshwork and the Schlemm canal or the ciliary body face and choroidal vasculature. Glaucomas are further subdivided into primary (cause of outflow resistance or angle closure is unknown) and secondary (outflow resistance results from a known disorder), accounting for > 20 adult types. Another group of glaucoma patients does not have IOP elevation, which in general is called normal tension glaucoma (NTG). NTG is also associated with progressive optic nerve degeneration and RGC death. Thus they are also subject to this gene therapy treatment. 38

[0104] Axons of retinal ganglion cells travel through the optic nerve carrying visual information from the eye to the brain. Damage to these axons causes ganglion cell death with resultant optic nerve atrophy and patchy vision loss. Elevated intraocular pressure (IOP; in unaffected eyes, the average range is 11 to 21 mm Hg) plays a role in axonal damage, either by direct nerve compression or diminution of blood flow. However, the relationship between externally measured pressure and nerve damage is complicated. Of people with IOP > 21 mm Hg (ie, ocular hypertension), only about 1 to 2% / year (about 10% over 5 years) develop glaucoma. Additionally, about one third of patients with glaucoma do not have IOP > 21 mm Hg (known as low-tension glaucoma or normal-tension glaucoma).

[0105] IOP is determined by the balance of aqueous secretion and drainage. Elevated IOP is caused by inhibited or obstructed outflow, not oversecretion; a combination of factors in the trabecular meshwork (eg, dysregulation of extracellular matrix, cytoskeletal abnormalities) appear to be involved. In open-angle glaucoma, IOP is elevated because outflow is inadequate despite an angle that appears unobstructed. In angle-closure glaucoma, IOP is elevated when a physical distortion of the peripheral iris mechanically blocks outflow.

[0106] Symptoms and signs of glaucoma vary with the type of glaucoma, but the defining characteristic is optic nerve damage as evidenced by an abnormal optic disk and certain types of visual field deficits. Glaucoma is diagnosed when characteristic findings of optic nerve damage are present and other causes have been excluded. Elevated IOP makes the diagnosis more likely, but elevated IOP can occur in the absence of glaucoma and is not essential for making the diagnosis. METHODS

[0107] As summarized above, aspects of the instant disclosure include methods of treating a subject for a neuropathy associated with ER stress. In some embodiments, provided herein is a method of treating a neuropathy in a mammalian subject in need thereof, comprising administering a compound of the disclosure to the subject, thereby treating the neuropathy. In some embodiments, provided herein is a method of reducing or ameliorating degeneration of axons and / or soma of neurons, comprising intravitreally, orally, systemically, etc. administering the composition into a mammalian subject experiencing or at imminent risk of a neuropathy.

[0108] In some aspects provided herein is a method of inducing neuroprotection / increasing survival / promoting functional recovery of RGC somata and axons, comprising administering a compound of the disclosure into a mammalian subject experiencing or at risk of an ON neuropathy. In some embodiments of the method, the ON neuropathy is retinal ganglion cell degeneration, including glaucoma, optic neuritis, ON traumatic injury and other ON-related diseases. 39

[0109] Various subjects may be treated in the methods of the present disclosure. In some instances, treated subjects may be mammals, including but not limited to e.g., rodents (e.g., rats, mice, etc.), non-human primates (e.g., macaques, marmosets, tamarins, spider monkeys, owl monkeys, vervet monkeys, squirrel monkeys, baboons, chimpanzees, etc.), humans, and the like. In some instances, a treated subject may be an animal model (e.g., a rodent model, a non-human primate model, etc.) of an optic neuropathy and / or neurodegenerative disorder. In some instances, a treated subject may be a human subject, including but not limited to e.g., a human subject having a neuropathy and / or neurodegenerative disorder, a human subject at increased risk of developing a neuropathy and / or neurodegenerative disorder, a human subject of advanced age (e.g., at least 60 years of age, at least 65 years of age, at least 70 years of age, at least 75 years of age, at least 80 years of age, at least 85 years of age, at least 90 years of age, etc.), or a combination thereof. Treated subjects may or may not be symptomatic, e.g., subject may or may not display or have previously displayed one or more symptoms of an optic neuropathy and / or neurodegenerative disorder, including but not limited to e.g., those optic neuropathies and / or neurodegenerative disorders described herein.

[0110] The compositions of this disclosure can be supplied in the form of a pharmaceutical composition. Any suitable pharmaceutical composition may be employed, described in more detail below. As such, in some instances, methods of the present disclosure may include administering one or more agents in a composition comprising an excipient (e.g., an isotonic excipient) prepared under sufficiently sterile conditions for administration to a mammal, e.g., a human.

[0111] Administration of an agent to a subject, as described herein, may be performed employing various routes of administration. The route of administration may be selected according to a variety of factors including, but not necessarily limited to, the condition to be treated, the formulation and / or device used, the patient to be treated, and the like. Routes of administration useful in the disclosed methods include but are not limited to oral and parenteral routes, such as topical eye drops, intravenous (iv), intravitreal, intraperitoneal (ip), rectal, topical, ophthalmic, nasal, and transdermal. Formulations for these dosage forms are described herein.

[0112] Those of skill in the art will readily appreciate that dose levels can vary as a function of the specific compound, the nature of the delivery vehicle, and the like. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.

[0113] In those embodiments where an effective amount of an active agent is administered to the subject, the amount or dosage is effective when administered for a suitable period of time, such as one week or longer, including two weeks or longer, such as 3 weeks or longer, 4 weeks or longer, 8 weeks or longer, etc., so as to evidence a reduction in the disorder, e.g., a reduction in a symptom of the disorder or in a marker of disease pathology. For example, an effective dose is the dose that, when administered for a suitable period of time, such as at least about 40one week, and maybe about two weeks, or more, up to a period of about 3 weeks, 4 weeks, 8 weeks, or longer, will reduce a symptom of the disorder, for example, by about 10% or more, by about 20% or more, e.g., by 30% or more, by 40% or more, or by 50% or more, in some instances by 60% or more, by 70% or more, by 80% or more, or by 90% or more, for example, and will halt progression of the disorder in the subject. In some instances, an effective amount or dose of active agent will not only slow or halt the progression of the disease condition but will also induce the reversal of the condition, i.e., will cause an improvement in the neurological health of the subject. For example, in some instances, an effective amount is the amount that when administered for a suitable period of time, for example, at least about one week, and / or about two weeks, or more, up to a period of about 3 weeks, 4 weeks, 8 weeks, or longer will improve, stabilize, or at least reduce the progression of a disorder in subject, for example 1.5- fold, 2-fold, 3-fold, 4-fold, 5-fold, in some instances 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more relative to the subject’s condition prior to administration.

[0114] In some instances, in those embodiments where an effective amount of an active agent is administered to the subject, the amount or dosage is effective when administered for a suitable period of time to result in a reduction in neuronal degeneration in the subject. Such a reduction may manifest in various ways, including but not limited to e.g., an increase in the number, size or length of neurons, or a reduction in the amount of degeneration of neurons, or their axons or soma, or the like. In some instances, methods of the present disclosure may result in at least a 5%, e.g., at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 30%, at least a 35%, at least a 40%, at least a 45%, at least a 50%, at least a 55%, at least a 60%, at least a 65%, at least a 70% at least a 75%, at least a 80%, e.g., reduction in neuronal degeneration. In some instances, methods of the present disclosure may result in at least a 5%, e.g., at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 30%, at least a 35%, at least a 40%, at least a 45%, at least a 50%, at least a 55%, at least a 60%, at least a 65%, at least a 70% at least a 75%, at least a 80%, e.g., increase in neuronal number, size or length of neuronal axons or somata. Various methods of assessing the amount of degeneration or increase in number, size or length of axons or somata may be employed, including invasive and non- invasive techniques, such as electrophysiology measurement for neuronal function, visual acuity, OCT imaging, fundus imaging, histology studies of somata and axons morphology.

[0115] A “therapeutically effective amount”, a "therapeutically effective dose" or “therapeutic dose” is an amount sufficient to effect desired clinical results (i.e., achieve therapeutic efficacy, achieve a desired therapeutic response, etc.). A therapeutically effective dose can be administered in one or more administrations. For purposes of this disclosure, a therapeutically effective dose of an agent that inhibits activity of CHOP and / or compositions is an amount that is sufficient, when administered to the individual, to palliate, ameliorate, stabilize, reverse, prevent, slow or delay the progression of the disease state (e.g., an optic neuropathy). 41

[0116] An effective amount of a composition comprising a compound of the disclosure will depend, at least, on the particular method of use, the subject being treated, the severity of the affliction, and the manner of administration of the therapeutic composition. A "therapeutically effective amount" of a composition is a quantity of a specified compound sufficient to achieve a desired effect in a subject (host) being treated.

[0117] Therapeutically effective doses of a composition comprising a compound of the disclosure or pharmaceutical composition can be determined by one of skill in the art, with a goal of achieving local (e.g., tissue) concentrations that are at least as high as the IC50 of an applicable compound disclosed herein.

[0118] The specific dose level and frequency of dosage for any particular subject may be varied and will depend upon a variety of factors, including the activity of the composition comprising a neuroprotective agent, the metabolic stability and length of action of that composition, the age, body weight, general health, sex and diet of the subject, mode and time of administration, rate of excretion, drug combination, and severity of the condition of the host undergoing therapy.

[0119] Conversion of an animal dose to human equivalent doses (HED) may, in some instances, be performed using the conversion table and / or algorithm provided by the U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER) in, e.g., Guidance for Industry: Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers (2005) Food and Drug Administration, 5600 Fishers Lane, Rockville, MD 20857; (the disclosure of which is incorporated herein by reference). 42Conversion of Animal Doses to Human Equivalent Doses Based on Body Surface Area To Convert Animal Dose in mg / kg to HEDain To Convert Animal Dose in mg / kg, Either: Species mg / kg to Dose in mg / m², Divide Multi l bels.REAGENTS, DEVICES AND KITS

[0120] Also provided are reagents, devices and kits thereof for practicing one or more of the above-described methods. The subject reagents, devices and kits thereof may vary greatly. Reagents and devices of interest include those mentioned above with respect to the methods of treating a neurodegenerative condition in a subject, including by administering to the subject an effective amount of an agent that modulates ER stress. The subject kits may include any combination of components (e.g., reagents, cell lines, etc.) for performing the subject methods, such as e.g., methods of treating a neurodegenerative condition.

[0121] In some embodiments, a subject kit may be employed in a method of identifying a target gene associated with degeneration of retinal ganglion cells. Such kits may vary and may, but need not necessarily, include one or more RGC populations. In some embodiments, a subject kit may include one or more, including a plurality of or a library of, CRISPR-based gene silencing agents. In some embodiments, the subject kits may include a nucleic acid for expressing a Cas9 polypeptide within a particular cell type, such as a retinal ganglion cell. In some instances, a cell line contained within a subject kit may be configured (e.g., genetically modified) to express a Cas9 polypeptide. 43

[0122] In addition to the above components, the subject kits will further include instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, etc. Yet another means would be a computer readable medium, e.g., diskette, CD, portable flash drive, etc., on which the information has been recorded. Yet another means that may be present is a website address which may be used via the internet to access the information at a removed site. Any convenient means may be present in the kits. EXAMPLES

[0123] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

[0124] General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference. Example 1

[0125] ER stress modulators are identified by screening chemical libraries with cell-based high throughput screen (HTS) assays; and then validated for promoting RGC and ON survival and preserve visual function in glaucoma models. The ER stress modulators identified by these assays provide valuable tools and have important implications for treatment of other illnesses associated with ER stress, including CNS neurodegenerations, cancer and metabolic diseases. These compounds may also be tested with axon degeneration molecules in our RGC / ON system, which will greatly enhance our understanding of the role of NAD+ metabolism in glaucomatous ON degeneration. Combining modulation of neuronal ER stress and NAD+ 44metabolism generates combinatory strategies that can lead to more efficient neuroprotection in patients.

[0126] Treatments for glaucoma that promote RGC soma and axon protection and preserve visual function are urgently needed. We pioneered in elucidating the role of axon-injury induced neuronal ER stress in optic neuropathies, which generated the new concept that neuronal ER stress is a general mechanism of both neuronal soma and axon neurodegeneration. Our proposed work in clinically relevant scenarios has the potential to translate our findings into therapies to prevent glaucomatous degeneration. We will test conceptually and practically new approaches to develop complementary screening methods to identify ER stress modulators that achieve robust neuroprotection by blocking CHOP; using several visual functional assays and advanced live imaging techniques to determine the effectiveness of our interventions.

[0127] We have used mouse models of acute ON traumatic injury and chronic ocular hypertension / glaucoma to demonstrate that ON injury-induced neuronal ER stress plays a critical role in RGC / ON degeneration: 1) Inhibition of CHOP and activation of XBP-1 synergistically promote RGC and axon survival and preserve visual function; 2) Previously we used two strategies to reduce CHOP levels specifically in RGCs after IOP elevation, to simulate the clinical setting: We injected AAV-CHOP shRNA + AAV-XBP-1s or AAV-scramble shRNA + AAV-GFP (control) in WT mice at 1WPI, when IOP reaches its peak. Since AAV- mediated gene expression in RGCs normally peaks at least 2 weeks after infection, CHOP is likely to be inhibited and XBP-1s expressed in RGCs 2 weeks after IOP elevation. Importantly, this delayed ER stress modulation significantly protected RGC somata and axons. The treated eyes showed significantly less prolonged N1 latency and P1 latency than the control eyes. In eIF2α A / A transgenic mouse, we injected AAV-Cre+ AAV-XBP-1s at 1WPI, which deletes the WT eIF2α and is accompanied by expression of unphosphorylated eIF2α S51A mutant and GFP specifically in RGCs. As with direct knock-down of CHOP, delayed inhibition of eIF2α-P and XBP-1 activation significantly protected RGC somata and axons and spared visual function. These results suggest that these downstream molecules of the two UPR pathways, XBP-1 and CHOP, play opposite roles in controlling neuronal survival after axon injury. The results therefore also reveal the neuroprotection potential of targeting ER stress and provide a unique opportunity for us to identify ER stress modulators that can be used as neuroprotectants for glaucoma.

[0128] To use complementary cell-based high throughput screening (HTS) to identify chemical modulators of CHOP, and test their efficacy in mouse glaucoma models. The next step in bringing our findings to the clinic is to identify potent ER stress-modulating small molecules that can be administered to patients with glaucoma to protect RGCs and preserve vision. Cell-based HTS assays have been employed successfully to identify ER stress modulators targeting PERK / ATF4, ATF6, BiP and IRE1α. Inhibitors of CHOP upstream molecules have been found: 45GSK2606414 is a potent PERK kinase inhibitor and ISRIB is a blocker of eIF2α-P identified by HTS with a luciferase reporter driven by 5’UTR of ATF4 mRNA. However, both PERK and eIF2α-P have multiple effectors in addition to CHOP and inhibiting these CHOP upstream molecules will generate complicated outputs that are not necessarily neuroprotective. We identify selective CHOP inhibitors that are more directly relevant to neuroprotection.

[0129] To identify small molecules that inhibitCHOP expression: CHOP expression isregulated primarily at the transcription level; the promoter region (-954 to +96) of human CHOP is responsible for CHOP expression. We therefore acquired the construct of human CHOP promoter-driven luciferase and developed stable lines of HEK293T cells. The same construct stably expressed in CHO cells has been used successfully to identify CHOP activators and inhibitors through small scale HTS assays. HEK293T cell line has been used extensively in neuroscience research and HTS assays. No human RGC cell line is available; RGC-5 is of mouse photoreceptor origin. Tunicamycin (Tm) and thapsigargin (Tg) are widely used to induce ER stress and cause accumulation of unfolded proteins in the ER: Tm, by inhibiting N-linked glycosylation; Tg, by specifically inhibiting SERCA, which decreases ER calcium levels and inhibits calcium-dependent ER chaperones. The stable CHOP-Luc line shows good dose response to Tm and Tg in 96-well plate format. To confirm our cell lines are able to identify positive hits, we tested PERK inhibitor GSK2606414 and IRE1 inhibitor KIRA6, which shows significant inhibition of Tm or Tg induced CHOP expression and XBP-1 splicing. We have further miniaturized the assay with 1536-well plates and determined that 500 cells / well gives the best response curve. We used this format in the HTS assays.

[0130] The traditional HTS screen of chemical libraries uses a single concentration of each compound and has many inherent limitations. For example, the single-concentration screening is often associated with high rates of both false positives and false negatives due to ineffective concentrations and requires follow-up screenings that are expensive and time consuming. The titration-based qHTS significantly improves the traditional single-dose HTS by generating a concentration-response curve (CRC) directly from a single experiment. Based on the CRC data, we can identify hit compounds with a much wider range of activities and much lower false negative and false positive rates; we will be able to classify hit compounds with knowledge of their potencies, efficacies and structure-activity relationships (SAR) from the primary screen. We will first perform titration- based qHTS in 1536-well plates with several sets of representative chemical libraries, including a comprehensive collection of approved drugs (The NCGC Pharmaceutical Collection), a collection of 2,000 compounds that act by diverse mechanisms, and a large collection of over 8,000 pharmacologically annotated investigational drugs and tool compounds that have been deposited in NIH. Following a published protocol, an interpolated asymmetric dilution series of compounds was constructed in the 1536-well source plates (1:2 dilution for top 4 concentrations and half-log / 3.16 fold dilution for bottom 4 concentrations) to 46achieve a concentration covering a range > 600-fold for each compound. For the CHOP inhibitor assay, test compounds are added together with Tm or Tg to evaluate CHOP inhibition after ER stress stimulation. Inhibition of the luciferase activity will be calculated as the percent reduction in relative luminescence normalized to Tg / Tm treatment (0% inhibition) and the no-ER stress control (100% inhibition). A tentative threshold for “hit” CHOP inhibitors is defined as a compound that decreases luciferase activity to ≤ 50% of Tg / Tm or that lies beyond three standard deviations from the Tg / Tm treatment mean. We perform HTS with a larger chemical library, MSLMR (Molecular Libraries Small Molecule Repository), which comprises 350,478 compounds and continues to expand. The qHTS is done at National Center for Advancing Translational Sciences (NCATS) in NIH.

[0131] To exclude hit compounds that activate CHOP expression by counter- screening. The CHOP-Luc and XBP-1-Luc HEK293T cell lines are two complementary cell lines that can be used for counter-screening with the hit compounds. Counter-screening allow isolation of compounds that selectively inhibit CHOP but not XBP-1 or activate XBP-1 but not CHOP, and so to exclude compounds that activate or inhibit both CHOP and XBP-1. Most importantly, this strategy will allow us to identify the ideal agents that activate XBP-1 as well as inhibit CHOP to achieve the more potent synergistic neuroprotection.

[0132] To exclude toxic hit compounds by cytotoxicity assay. The toxicity of a compound may decrease luciferase activity due to cell death, which would preclude future application as a therapeutic reagent. A cell viability assay, CellTiter-Glo™ from Promega, is employed to exclude toxic hit compounds that induce cell death.

[0133] To validate the neuroprotective effects of the lead compounds in vivo. Hit compounds are tested in the in vivo ON crush and glaucoma mouse models to isolate the most promising lead compounds for RGC neuroprotection. Pilot experiments with a serial dilution of hit compounds injected in naïve mouse vitreous chamber determine the safe concentration range of compounds that can effectively inhibit CHOP in RGCs without significant toxic effects. We first test the neuroprotective effects of these compounds after ON crush because it is an acute and severe degeneration model in which protection effects will be easily noticed. We include a mix of 10 male and female mice in each experimental group; the group sample size was determined by a power analysis based on our pilot experiments and previous studies. This sample size achieves 80% power to detect a standardized mean difference in RGC numbers between groups of ~ 1.4 (Cohen’s d) using estimated group coefficient of variation of ±15%, two-sided hypothesis testing and statistical significance set to alpha equal to 0.05. Numbers of surviving RGCs and axons are determined 2 weeks after ON crush. One way ANOVA and Bonferroni's Multiple Comparison Test are used to compare the means of all groups (including CHOP KO mice as positive control) and p<0.05 is considered as significant. CHOP expression 47and XBP-1 splicing in RGCs are monitored by in situ hybridization to confirm a compound’s effect.

[0134] The most effective compounds are further tested in two glaucoma models by RGC and ON morphology studies, as well as visual function analysis. We induce high IOP in left eyes by anterior chamber microbead injection; sham-injected right eyes will serve as controls. The IOP of both eyes will be monitored once a week using the TonoLab tonometer. If the average IOP of the left eye is less than 7mmHg above baseline or any other ocular pathology develops, the mouse is excluded from further analysis. All experimental groups are assessed by OKR, VEP and PERG before microbead injection for the baseline visual acuity, contrast threshold, VEP and PERG signals. They are tested similarly at multiple time points after microbead injection and IOP elevation to assess preservation of visual functions and correlation with protection of RGC somata and axons. We validate the lead compounds in the DBA / 2J mouse line, which more closely mimics human hereditary glaucoma.

[0135] Shown in FIG.1 is the activity in HEK293T cells treated with 1 µM Tunicamycin (Tm) + 1 µM Thapsigarginin (Tg) in the presence of C5a or 5a28, 24 h after exposure. IC50 is calculated with nonlinear regression through dose-dependent fits of CHOP-Luc activities (relative to DMSO) of individual compound. Data are presented as means ± s.e.m., n = 4 independent replicates. B, Analog design of CHOP inhibitor targeting Ring A (Red), Ring B (green), and the linker region based on the core scaffold of C5a.

[0136] The top 10 analog compounds are shown in FIG.2 for the CHOP inhibition effect. A, The top 10 CHOP inhibitors are shown with the chemical structure, IC50 (μM) of CHOP-LUC activity of the cells treated with 1 µM Tm + 1 µM Tg for 24 h, relative to the DMSO treated cells. Benchmark compounds C5a and C5a28 are highlighted in yellow. B, Inhibition of the CHOP- LUC reporter in HEK293T cells treated with 1 µM Tm + 1 µM Tg for 24 h in the presence of top 10 small molecule CHOP inhibitors at 8-point dose. Data are presented as means ± s.e.m., n = 4 independent replicates. C, Quantification of hATF4 and hCHOP mRNA levels by RT-qPCR in HEK293T cells treated with 1 µM Tm + 1 µM Tg for 24 h in the presence of top 10 small molecule CHOP inhibitors (10 µM). Data are presented as means ± s.e.m., n = 4-6 independent replicates.

[0137] Inhibitory effects of the top 10 CHOP inhibitors on CHOP protein levels is shown in FIG. 3. A, Representative immunoblots of ATF4 and CHOP in HEK293T cells treated with 1 µM Tm + 1 µM Tg for 24 h, in the presence of top 10 small molecule CHOP inhibitors (10 µM). Quantification of ATF4 or CHOP protein levels are attached. Data are presented as means ± s.e.m., n = 4 independent replicates. One-way ANOVA with Dunnett’s post hoc test (compared to DMSO), *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Compounds with strong CHOP- LUC inhibitory effects (***p < 0.001 and ****p < 0.0001) are highlighted in red. 48

[0138] The novel ER stress modulators identified through this study provide an important opportunity to explore the utility of UPR-based therapies, but shed light on the fundamental mechanism that maintains the healthy balance of UPR branches in neurons. Example 2 Synthesis of methyl 2-((4-fluorobenzo[d]thiazol-2-yl)amino)benzo[d]thiazole-6-carboxylate (T- 2028) [0013944 mmol) in DMF (2 mL) were added K2CO3 (0.152 g, 1.1 mmol) and 4-fluorobenzo[d]thiazol-2- amine (0.044 g, 0.26 mmol) and stirred the reaction mixture at 70 oC for 30h. Reaction mixture was poured in ice-water (100 mL) during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (10 mL x 3) and dried under high vacuum to afford the crude mass, which was triturated with a mixture of methanol-ethyl acetate (1:1; 20 mL x 3) followed by ACN (2 mL x 2) and dried under high vacuum to obtain methyl 2-((4-fluorobenzo[d]thiazol-2-yl)amino)benzo[d]thiazole-6-carboxylate (T-2028) (0.030 g, 19.02%) as a light-yellow solid.1H NMR (400 MHz, DMSO-d6-D2O) δ 8.22 (s, 1H), 7.81- 7.79(d, J=8.4 Hz, 1H), 7.49-7.47 (d, J=7.6 Hz, 1H), 7.42-7.40 (d, J=8.4 Hz, 1H), 7.07-6.98 (m, 2H), 3.81 (s, 3H). MS: [MH]+ 360.2. Note: NH proton signal appeared at δ 13.25 (br, 1H) in 1H NMR (400 MHz, DMSO-d6). Example 3 Synthesis of methyl 2-((4,6-difluorobenzo[d]thiazol-2-yl)amino)benzo[d]thiazole-6-carboxylate (T-2029)

[0140] To a stirred solution of methyl 2-chlorobenzo[d]thiazole-6-carboxylate (0.100 g, 0.44 mmol) in DMF (2 mL) were added K2CO3(0.152 g, 1.1 mmol) and 4,6-difluorobenzo[d]thiazol- 492-amine (0.049 g, 0.26 mmol) and stirred the reaction mixture at 70oC for 24h. Reaction mixture was poured in ice-water (100 mL) during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (10 mL x 4) and dried under high vacuum to afford the crude mass, which was triturated with a mixture of methanol-ethyl acetate (2:8; 20 mL x 3) followed by pentane (5 mL x 2) and dried under high vacuum to obtain methyl 2-((4,6-difluorobenzo[d]thiazol-2-yl)amino)benzo[d]thiazole-6-carboxylate (T-2029) (0.020 g, 12.08%) as a cream solid.1H NMR (400 MHz, DMSO-d6-D2O-a drop of TFA) δ 8.52 (s, 1H), 7.99-7.97 (d, J=8.4 Hz,1H), 7.96-7.64 (m, 2H), 7.30-7.25 (t, J=9.6 Hz, 1H), 3.84 (s, 3H). MS: [MH]- 376.1. Note: NH proton signal appeared at δ 13.28 (br, 1H) in1H NMR (400 MHz, DMSO-d6). Example 4 Synthesis of methyl 2-((5,6-dichlorobenzo[d]thiazol-2-yl)amino)benzo[d]thiazole-6-carboxylate (T-2030)[001, 4 mmol) in DMF (2 mL) were added K2CO3 (0.152 g, 1.1 mmol) and 5,6-dichlorobenzo[d]thiazol- 2-amine (0.057 g, 0.26 mmol) and stirred the reaction mixture at 70oC for 24h. Reaction mixture was poured in ice-water (100 mL) during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (10 mL x 3) and dried under high vacuum to afford the crude mass, which was triturated with a mixture of methanol-ethyl acetate (1:9; 20 mL x 3) followed by pentane (5 mL x 3) and dried under high vacuum to obtain methyl 2-((5,6-dichlorobenzo[d]thiazol-2-yl)amino)benzo[d]thiazole-6- carboxylate (T-2030) (0.030 g, 16.70%) as a yellow solid.1H NMR (400 MHz, DMSO-d6) δ 13.30 (br, 1H), 8.48 (s,1H), 8.21 (s, 1H), 7.96-7.94 (d, J=8.4 Hz, 1H), 7.81 (s, 1H), 7.63-7.61 (d, J=8.4 Hz, 1H), 3.86 (s, 3H). MS: [MH]+410.0 / [MH+2]+412.0 / [MH+4]+414.0 50Example 5 Synthesis of methyl 2-((4,5-dichlorobenzo[d]thiazol-2-yl)amino)benzo[d]thiazole-6- carboxylate (T-2031) [00142 4mmol) in DMF (2 mL) were added K2CO3 (0.152 g, 1.1 mmol) and 4,5-dichlorobenzo[d]thiazol- 2-amine (0.057 g, 0.26 mmol) and stirred the reaction mixture at 70oC for 24h. Reaction mixture was poured in ice-water (100 mL) during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (10 mL x 3) and dried under high vacuum to afford the crude mass, which was triturated with a mixture of methanol-ethyl acetate (5:5; 20 mL x 3) followed by diethyl ether (5 mL x 2) and dried under high vacuum to obtain methyl 2-((4,5-dichlorobenzo[d]thiazol-2-yl)amino)benzo[d]thiazole-6- carboxylate (T-2031) (0.021 g, 11.7%) as a pale yellow solid.1H NMR (400 MHz, DMSO-d6 - D2O) δ 8.25 (s, 1H), 7.84-7.81 (d, J=8.4 Hz,1H), 7.64-7.61 (d, J=8.4 Hz, 1H), 7.47-7.45 (d, J=8.4 Hz, 1H), 7.20-7.17 (d, J=8.4 Hz, 1H), 3.85 (s, 3H; merged in moisture from DMSO-d6). MS: [MH]+410.1 / [MH+2]+412.0 / [MH+4]+414.0 Note: NH proton signal appeared at δ 13.48 (br, 1H) in1H NMR (400 MHz, DMSO-d6). Example 6 Synthesis of methyl 2-((6-bromobenzo[d]thiazol-2-yl)amino)benzo[d]thiazole-6-carboxylate (T-2032)

[0143] To a stirred solution of methyl 2-chlorobenzo[d]thiazole-6-carboxylte (0.129 g, 0.56 mmol) and 6- bromobenzo[d]thiazol-2-amine (0.100g, 0.43 mmol) in DMF (2 ml) were added Cs2CO3(0.420 g, 1.29 mmol) and reaction mixture was stirred at room temperature for 16h. Reaction mixture was poured in water (100 mL) during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (10 mL x 4) and 51dried under high vacuum to afford the crude mass, which was triturated with a mixture of dichloromethane-methanol (9:1; 20 mL x 5) and dried under high vacuum to obtain methyl 2- ((6-bromobenzo[d]thiazol-2-yl)amino)benzo[d]thiazole-6-carboxylate (T-2032) (0.012 g, 6.56%) as a cream solid.1H NMR (400 MHz, DMSO-d6-D2O-a drop of TFA) δ 8.57-8.57 (d, J=1.2 Hz,1H), 8.21-8.21 (d, J=1.6 Hz,1H), 8.02-8.00 (dd, J=8.4 Hz, 1.6 Hz, 1H), 7.71-7.69 (d, J=8.4 Hz, 1H), 7.62-7.56 (m, 2H), 3.87 (s, 3H). MS: [MH]+420 / [MH+2]+422. Note: NH proton signal [001. , . F (1 mL) were added Cs2CO3 (0.480 g, 1.47 mmol) and 2-chloro-6-nitrobenzo[d]thiazole (0.164 g, 0.76 mmol) at room temperature under nitrogen and stirred for 16h at the same temperature. Reaction mixture was poured in ice-water (100 mL) during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (10 mL x 3) and dried under high vacuum to afford the crude mass, which was triturated with a mixture of dichloromethane-methanol (9:1; 20 mL x 5) followed by acetonitrile (10 mL x 2) and dried under high vacuum to obtain 6-fluoro-N-(6-nitrobenzo[d]thiazol-2-yl)benzo[d]thiazol-2-amine (T-2033) (0.025 g, 12.15%) as a saffron solid.1H NMR (400 MHz, DMSO-d6) δ 13.46 (br, 1H), 8.69 (s, 1H), 8.13-8.11 (dd, J =9.2 Hz, 2.4 Hz, 1H), 7.72-7.69 (dd, J =8.8 Hz, 2.4 Hz, 1H), 7.56-7.52 (m, 1H), 7.50-7.47 (d, J = 8.8 Hz,1H), 7.17-7.12 (dt, J =9.2 Hz, 2.4 Hz, 1H). MS: [MH]+347.1.52Example 8 Synthesis of bis(6-bromobenzo[d]thiazol-2-yl)amine (T-2034)

[0014] o a s e sou o o - o o- -c oo e o[ ] a oe ( . g, . o) DMF (2 mL) were added K2CO3(0.139 g, 1.01 mmol) and 6-bromobenzo[d]thiazol-2-amine (0.063 g, 0.28 mmol) sequentially at room temperature and resulting mixture was at 70oC for 36h. Reaction mixture was poured in ice-water (100 mL) during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (15 mL x 3) and dried under high vacuum to afford the crude mass, which was triturated with a mixture of dichloromethane-methanol (9:1; 20 mL x 5) followed by pentane (5 mL x 3) and dried under high vacuum to obtain bis(6-bromobenzo[d]thiazol-2-yl)amine (T-2034) (0.020 g, 11.25%) as a Cream solid.1H NMR (400 MHz, DMSO-d6) δ 13.00 (s, 1H), 8.21 (s, 2H), 7.57-7.55 (d, J=7.2 Hz, 4H). MS: [MH]+439.9 / [MH+2]+441.9 / / [MH+4]+443.8.Example 9 Synthesis of bis(6-fluorobenzo[d]thiazol-2-yl)amine (T-2036)

[0146] To a stirred solution of 2-chloro-6-fluorobenzo[d]thiazole (0.100 g, 0.53 mmol) in DMF (2 mL) were added K2CO3(0.183 g, 1.32 mmol) and 6-fluorobenzo[d]thiazol-2-amine (0.054 g, 0.32 mmol) sequentially at room temperature and the resulting mixture was stirred at 70oC for 24h. Reaction mixture was poured in ice-water (100 mL) during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (20 mL x 3) and dried under high vacuum to afford the crude mass, which was triturated with a mixture of hot methanol-ethyl acetate (5:5; 30 mL x 5) followed by methanol (5 mL x 3) and dried under high vacuum to obtain bis(6-fluorobenzo[d]thiazol-2-yl)amine (T-2036) (0.011 g, 536.43%) as a light brown solid.1H NMR (400 MHz, DMSO-d6) δ 12.74 (s, 1H), 7.88 (brs, 2H), 7.71 (brs, 2H), 7.279 (brs, 2H). MS: [MH]+320.09. Example 10 Synthesis of 2-((6-fluorobenzo[d]thiazol-2-yl)amino)benzo[d]thiazole-6-carboxylic acid (T- 2037)

[001] y ess o e y -(( - uoo e o[ ] a o- -y)a o) e o[ ] a oe-6- carboxylate (T-0009). To a stirred solution of methyl 2-chlorobenzo[d]thiazole-6-carboxylate (0.500 g, 2.19 mmol) in DMF (10 mL) were added K2CO3(0.750 g, 5.49 mmol) and 6- fluorobenzo[d]thiazol-2-amine (0.220 g, 1.31 mmol) sequentially at room temperature and the resulting mixture was stirred at 50oC for 24h. Reaction mixture was poured in ice-water (500 mL) during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (100 mL x 3) and dried under high vacuum to afford the crude mass, which was triturated with a mixture of dichloromethane-methanol (9:1; 50 mL x 5) followed by acetonitrile (50 mL x 3) and dried under high vacuum to obtain methyl 2-((6- fluorobenzo[d]thiazol-2- yl)amino)benzo[d]thiazole-6-carboxylate (T-0009) (0.300 g, 38.01%). MS: [MH]+260.13.

[0148] Synthesis of 2-((6-fluorobenzo[d]thiazol-2-yl)amino)benzo[d]thiazole-6-carboxylic acid (T-2037). To a stirred solution of methyl 2-((6-fluorobenzo[d]thiazol-2- yl)amino)benzo[d]thiazole-6-carboxylate (T-0009) (0.200 g, 0.55 mmol) in methanol-water (5:1; 12 mL) was added LiOH.H2O (0.053 g, 2.22 mmol) at room temperature and stirred the reaction mixture at the same temperature for 24h. Reaction mixture was acidified with 1N HCl (100 mL) and was extracted with ethyl acetate (50 mL x 3). The combined organic layer was washed with water, dried over Na2SO4and concentrated in vacuum. Obtained crude was triturated with a mixture of dichloromethane-methanol (9:1; 100 mL x 3) followed by acetonitrile (20 mL x 3) and dried under high vacuum to obtain 2-((6-fluorobenzo[d]thiazol-2- yl)amino)benzo[d]thiazole-6-carboxylic acid (T-2037) (0.150 g, 78.04%) as a green solid.1H NMR (400 MHz, DMSO-d6 -D2O) δ 8.52 (s, 1H), 8.00-7.97 (dd, J=8.4 Hz, 1.6 Hz, 1H), 7.88- 7.85 (dd, J=8.4 Hz, 2.4 Hz, 1H), 7.69 (br, 2H), 7.31-7.26 ((dd, J=8.8 Hz, 2.4 Hz, 1H). MS: 54[MH]+346.16. Note: NH proton signal appeared at δ 13.09 (br, 1H) in1H NMR (400 MHz, DMSO-d6). Example 11 Synthesis of isopropyl 2-((6-fluorobenzo[d]thiazol-2-yl)amino)benzo[d]thiazole-6-carboxylate (T-2038) [00149thiazole-6- carboxylic acid (T-2037) (0.100 g, 0.29 mmol) in isopropanol (2 ml) was added concentrated H2SO4 (catalytic amount) at room temperature and stirred the reaction mixture at 80oC for 24h. Reaction mixture was quenched with 10% NaHCO3 (50 mL) and was extracted with ethyl acetate (50 mL x 3). Combined organic extracts were washed with water (20 mL), dried over Na2SO4and concentrated under in vacuo. Obtained crude was triturated with methanol (20 mL x 3) followed by pentane (5 mL x 3) and dried under high vacuum to obtain isopropyl 2- ((6- fluorobenzo[d]thiazol-2-yl)amino)benzo[d]thiazole-6-carboxylate (T-2038) (0.020 g, 17.83%) as an off white solid.1H NMR (400 MHz, DMSO-d6) δ 12.93 (br, 1H), 8.50 (s, 1H), 7.97-7.94 (d, J=8.4 Hz,1H), 7.85-7.83 (d, J=7.6 Hz 1H), 7.64 (brs, 2H), 7.28-7.23 (t, J=8.0 Hz,1H), 5.16-5.13 (m, 1H), 1.35-1.33 (d, J=6.4 Hz, 6H). MS: [MH]+388.2. Example 12 Synthesis of 2-((6-fluorobenzo[d]thiazol-2-yl)amino)-N-methylbenzo[d]thiazole-6- carboxamide (STL16-T-2039)55

[0150] To a stirred solution of methyl 2-((6-fluorobenzo[d]thiazol-2- yl)amino)benzo[d]thiazole-6-carboxylate (T-0009) (0.100g, 0.27 mmol) in THF (2 mL) were added MeNH2 (0.012 g, 0.41 mmol) and TMA (2M in toluene; 0.83 ml, 1.67 mmol) followed by DIPEA (0.143g, 0.19 mL, 1.11 mmol) at room temperature under nitrogen and stirred the resulting mixture at 80oC for 6h. Reaction mixture was poured in ice-water (100 mL) during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (50 mL x 3) and dried under high vacuum to afford the crude mass, which was purified by flash column chromatography (3-5 % MeOH in DCM) to afford 2-((6- fluorobenzo[d]thiazol-2-yl)amino)-N- methylbenzo[d]thiazole-6-carboxamide (T-2039) (0.020 g, 20.05%) as a pale-yellow solid.1H NMR (400 MHz, DMSO-d6) δ 12.97 (s, 1H), 8.47-8.46 (d, J=4.4 Hz, 1H), 8.37 (s,1H), 7.89-7.87 (d, J=8.4 Hz 2H), 7.67 (br, 2H), 7.30-7.25 (dt, J=8.8 Hz, 2.4 Hz, 1H), 2.81-2.80 (d, J=4.4Hz, 3H). MS: [MH]+359.2. Example 13 Synthesis of 2,2'-azanediylbis(benzo[d]thiazole-5-carbonitrile) (T-2041)

[0151] To a stirred solution of 2-chlorobenzo[d]thiazole-5-carbonitrile (0.100 g, 0.51 mmol) in DMF (2 ml) were added K2CO3 (0.178 g, 1.28 mmol) and 2-aminobenzo[d]thiazole-5- carbonitrile (0.053 g, 0.30 mmol) sequentially at room temperature under nitrogen and resulting reaction mixture was stirred at 70oC for 24h. Reaction mixture was poured in ice- water (100 mL) during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (20 mL x 3) and dried under high vacuum to afford the crude mass, which was triturated with hot methanol (20 mL x 4) followed by pentane (5 mL x 3) and dried under high vacuum to obtain 2,2'-azanediylbis(benzo[d]thiazole- 5-carbonitrile) (T-2041) (0.016 g, 9.32%) as a pale-yellow solid.1H NMR (400 MHz, DMSO-d6) δ 13.27 (s, 1H), 8.21-8.19 (d, J=7.6 Hz, 2H), 8.11 (br, 2H), 7.69-7.67 (d, J=8.0 Hz 2H). MS: [MH]+334.1. Example 14 Synthesis of 2-((6-fluorobenzo[d]thiazol-2-yl)amino)benzo[d]thiazole-6-carbonitrile (T-2042) 56[0015in DMF (2 mL) were added K2CO3 (0.178 g, 1.28 mmol) and 6-fluorobenzo[d]thiazol-2-amine (0.051 g, 0.30 mmol) sequentially atroom temperature under nitrogen and resulting reactionmixture was stirred at 70oC for 24h. Reaction mixture was poured in ice-water (100 mL) during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (20 mL x 3) and dried under high vacuum to afford the crude mass, which was triturated with a methanol (10 mL x 4) followed by pentane (5 mL x 3) and dried under high vacuum to obtain 2-((6-fluorobenzo[d]thiazol-2-yl)amino)benzo[d]thiazole-6- carbonitrile (T-2036) (0.021 g, 12.49%) as a yellow solid.1H NMR (400 MHz, DMSO-d6) δ 13.07 (br, 1H), 8.20 (s, 1H), 7.69-7.67 (dd, J=8.8 Hz, 2.0 Hz, 1H), 7.62-7.60 (d, J=8.0 Hz, 1H), 7.52- 7.49 (m, 2H), 7.15-7.10 (dt, J=9.2 Hz, 2.4 Hz, 1H). MS: [MH]+327.1. Example 15 Synthesis of 5-fluoro-N-(6-(methylsulfonyl)benzo[d]thiazol-2-yl)benzo[d]thiazol-2-amine (STL16-T-2043)

[00153] To a stirred solution of 2-chloro-5-fluorobenzo[d]thiazole (0.200 g, 1.06 mmol) in DMF (5 mL) were added K2CO3 (0.369 g, 2.67 mmol) and 6-(methylsulfonyl)benzo[d]thiazol-2-amine (0.146 g, 0.64 mmol) sequentially at room temperature under nitrogen and resulting reaction mixture was stirred at 70oC for 24h. Reaction mixture was poured in ice-water (150 mL) during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (60 mL x 3) and dried under high vacuum to afford the crude mass, which was triturated with a mixture of methanol-dichloromethane (1:9; 100 mL x 3) followed by diethyl ether (5 mL x 3) and dried under high vacuum to obtain 5-fluoro-N-(6- 57(methylsulfonyl)benzo[d]thiazol-2-yl)benzo[d]thiazol-2-amine (T-2043) (0.020 g, 4.93%) as an off white solid.1H NMR (400 MHz, DMSO-d6 -D2O) δ 8.49 (s, 1H), 7.94-7.90 (m, 2H), 7.82- 7.80 (d, J=8.4 Hz, 1H), 7.48-7.46 (d, J=8.8 Hz, 1H), 7.17-7.12 (dt, J=9.2 Hz, 2.4 Hz, 1H), 3.20 (s, 3H). MS: [MH]+380.2. Note: NH proton signal appeared at δ 13.22 (br, 1H) in1H NMR (400 MHz, DMSO-d6). Example 16 Synthesis of bis(6-(trifluoromethyl)benzo[d]thiazol-2-yl)amine (T-2044)

[0154] To a stirred solution of 2-chloro-6-(trifluoromethyl)benzo[d]thiazole (0.100 g, 0.42 mmol) in DMF (2 mL) were added K2CO3 (0.145 g, 1.05 mmol) and 6- (trifluoromethyl)benzo[d]thiazol-2-amine (0.054 g, 0.25 mmol) sequentially at room temperature under nitrogen and resulting reaction mixture was stirred at 70oC for 24h. Reaction mixture was poured in ice-water (100 mL) during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (60 mL x 3) and dried under high vacuum to afford the crude mass, which was triturated with a mixture of methanol-dichloromethane (1:9; 50 mL x 3) followed by diethyl ether (10 mL x 3) and dried under high vacuum to obtain bis(6- (trifluoromethyl)benzo[d]thiazol-2-yl)amine (T-2044) (0.012 g, 6.80%) as an off white solid.1H NMR (400 MHz, DMSO-d6) δ 13.33 (s, 1H), 8.45 (s, 2H), 7.82 (brs, 2H), 7.76-7.74 (d, J=7.6 Hz, 2H). MS: [MH]+420.1. Example 17 Synthesis of methyl 2-((6-(methylsulfonyl)benzo[d]thiazol-2-yl)amino)benzo[d]thiazole-6- carboxylate(T-2045)58

[0155] To a stirred solution of methyl 2-chlorobenzo[d]thiazole-6-carboxylate (0.100g, 0.44 mmol) in DMF (1 mL) were added K2CO3 (0.152 g, 1.10 mmol) and 6- (methylsulfonyl)benzo[d]thiazol-2-amine (0.060 g, 0.26 mmol) sequentially at room temperature under nitrogen and resulting reaction mixture was stirred at 70oC for 24h. Reaction mixture was poured in ice-water (100 mL) during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (40 mL x 3) and dried under high vacuum to afford the crude mass, which was triturated with a mixture of methan ol- ethyl acetate (2:8; 50 mL x 3) followed by dichloromethane (10 mL x 3) and dried under high vacuum to obtain methyl 2-((6- (methylsulfonyl)benzo[d]thiazol-2- yl)amino)benzo[d]thiazole-6-carboxylate (T-2045) (0.020 g, 10.83%) as a light yellow solid.1H NMR (400 MHz, DMSO-d6 -D2O + a drop of TFA) δ 8.44-8.43 (d, J=1.6 Hz, 2H), 7.96-7.93 (dd, J=8.4 Hz, 1.2 Hz, 1H), 7.89-7.87 (dd, J=8.4 Hz, 1.6 Hz, 1H), 7.79-7.77 (d, J=8.4 Hz, 1H), 7.67-7.65 (d, J=8.8 Hz, 1H), 3.81 (s, 3H), 3.18 (s, 3H). MS: [MH]+420.12. Note: NH proton signal appeared at δ 13.45 (br, 1H) in1H NMR (400 MHz, DMSO-d6). Example 18 Synthesis of bis(4-fluorobenzo[d]thiazol-2-yl)amine (T-2046)

[0156] To a stirred solution of 2-chloro-4-fluorobenzo[d]thiazole (0.100g, 0.53 mmol) in DMF (2 mL) were added K2CO3 (0.184 g, 1.33 mmol) and 4-fluorobenzo[d]thiazol-2-amine (0.053 g, 0.32 mmol) sequentially at room temperature under nitrogen and resulting reaction mixture was stirred at 70oC for 24h. Reaction mixture was poured in ice-water (100 mL) during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (40 mL x 3) and dried under high vacuum to afford the crude mass, which was triturated with a mixture of methanol-ethyl acetate (5:5; 50 mL x 3) followed by CAN (5 mL x 3) and dried under high vacuum to obtain bis(4-fluorobenzo[d]thiazol-2-yl)amine (T-2046) (0.025 g, 14.65%) as an off white solid.1H NMR (400 MHz, DMSO-d6) δ 13.18 (s, 1H), 7.82- 7.81 (d, J=6.0 Hz, 2H), 7.32-7.28 (m, 4H). MS: [MH]+320.09 59Example 19 Synthesis of 5-fluoro-N-(6-nitrobenzo[d]thiazol-2-yl)benzo[d]thiazol-2-amine (T-2047) [001DMF (5 mL) were added K2CO3 (0.322 g, 2.33 mmol) and 5-fluorobenzo[d]thiazol-2-amine (0.094 g, 0.56 mmol) sequentially at room temperature under nitrogen and resulting reaction mixture was stirred at 70oC for 24h. Reaction mixture was poured in ice-water (100 mL) during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (60 mL x 3) and dried under high vacuum to afford the crude mass, which was triturated with a mixture of methanol-ethyl acetate (5:5; 100 mL x 3) followed by ACN (10 mL x 3) and dried under high vacuum to obtain 5-fluoro-N-(6-nitrobenzo[d]thiazol-2- yl)benzo[d]thiazol-2- amine (T-2047) (0.012g, 3.71%) as a light-yellow solid.1H NMR (400 MHz, DMSO-d6) δ 13.36 (s, 1H), 8.99 (s, 1H), 8.29-8.26 (dd, J=8.8 Hz, 2.0 Hz, 1H), 8.02-7.99 (m, 1H), 7.79 (br, 1H), 7.50 (br, 1H), 7.22-7.18 (dd, J=9.2 Hz, 2.4 Hz, 1H). MS: [MH]+347.11. Example 20 Synthesis of N-(6-(1H-tetrazol-5-yl)benzo[d]thiazol-2-yl)-6-fluorobenzo[d]thiazol-2-amin (T- 2048)

[0158] To a stirred solution of 2-((6-fluorobenzo[d]thiazol-2-yl)amino)benzo[d]thiazole-6- carbonitrile (T-2042) (0.120 g, 0.36 mmol) in DMF (5 mL) were added NaN3 (0.071 g, 1.10 mmol) and NH4Cl (0.058 g, 1.10 mmol) at room temperature and the resulting reaction mixture was stirred at 120oC for 72h. After completion of reaction as indicated by TLC, the reaction mixture was quenched with water (50 mL) and was extracted with EtOAc (3 x 30 mL). Combined organic layers were washed with water (10 mL x 3), dried over Na2SO4 and 60concentrated under reduced pressure to afford the crude mass, which was triturated with a mixture of methanol- ethyl acetate (5:5; 100 mL x 3) followed by acetonitrile (10 mL x 3) and dried under high vacuum to obtain N-(6-(1H-tetrazol-5-yl)benzo[d]thiazol-2-yl)-6- fluorobenzo[d]thiazol-2-amine (T-2048) (0.030g, 22.09%) as a pale yellow solid.1H NMR (400 MHz, DMSO-d6) δ 13.00 (br, 1H), 8.48 (s, 1H), 8.06- 8.04 (dd, J=8.4 Hz, 1.6 Hz, 1H), 7.89- 7.86 (dd, J=8.8 Hz, 2.8 Hz, 1H), 7.69-7.66 (m, 2H), 7.30-7.25 (dt, J=9.2 Hz, 2.4 Hz, 1H). MS: [MH]- 368.09. Example 21 Synthesis of 2,2’-azanediylbis(benzo[d]thiazole-5-carboxamide) (T-2049) [00159e) (T-2041) (0.150 g, 0.45 mmol) in THF: H2O (8:2) was added KOH (0.252 g, 4.50 mmol). Reaction mixture was stirred at 90oC for 40h. After completion of reaction as indicated by LCMS, the reaction mixture was quenched with 5N HCl (50 mL) during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (50 mL x 3) and dried under high vacuum to afford the crude mass, which was purify by Prep HPLC (CP-2021-009, CAN / 0.1% formic acid in milliQ water) to obtain 2,2’- azanediylbis(benzo[d]thiazole-5-carboxamide) (T-2049) (0.010 g, 6.02%) as an off white solid. 1H NMR (400 MHz, DMSO-d6) δ 13.06 (s, 1H), 8.22 (br, 1H), 8.13 (s, 3H), 8.02-8.00 (d, J=6.4 Hz, 2H), 7.80-7.78 (d, J=8.0 Hz, 2H), 7.45 (s, 2H). MS: [MH]+370.21. 61Example 22 Synthesis of bis(5-bromobenzo[d]thiazol-2-yl)amine (T-2051) [0016 DMF(2 mL) were added K2CO3 (0.138 g, 1.0 mmol) and 5-bromobenzo[d]thiazol-2-amine (0.055 g, 0.24 mmol) sequentially at room temperature under nitrogen and resulting reaction mixture was stirred at 700C for 24h. Reaction mixture was poured in ice-water (100 mL) during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (40 mL x 3) and dried under high vacuum to afford the crude mass, which was triturated with a mixture of dichloromethane-methanol (5:5; 100 mL x 3) followed by acetonitrile (10 mL x 3) and dried under high vacuum to obtain bis(5-bromobenzo[d]thiazol-2- yl)amine (T-2051) (0.020 g, 11.27%) as an off white solid.1H NMR (400 MHz, DMSO-d6) δ 13.09 (s, 1H), 7.91-7.89 (d, J=8.4 Hz, 2H), 7.80 (s, 2H), 7.42-7.40 (d, J=8.4 Hz, 2H). MS: [MH]+439.9 / [MH+2]+441.9 / / [MH+4]+443.9 Example 23 Synthesis of bis(5-bromobenzo[d]thiazol-2-yl)amine (T-2052)

[0161] Synthesis of N-((perfluorophenyl)carbamothioyl)benzamide (T-2052A1). A solution of benzoyl chloride (4.03 g, 28.68mmol) and potassium Thiocyanate (2.91g, 29.99mmol) in dry 62acetone (25 mL) was refluxed for 1.5h. 2,3,4,5,6-pentafluoroaniline (5.0g, 27.32mmol) was, then, added to the refluxing solution and stirring was continued for an additional 2h at the same temperature. After cooling, the solvent was evaporated in vacuo, and diluted with water (30 mL). The mixture was stirred for 10 min during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (50 mL x 3) and dried under high vacuum to obtain N-((perfluorophenyl)carbamothioyl)benzamide (T-2052A1) (7.0 g, 74.05%) as a white solid. MS: [MH]- 345.

[0162] Synthesis of 1-(perfluorophenyl)thiourea (T-2052A2). A mixture of N- ((perfluorophenyl)carbamothioyl)benzamide (T-2052A1) (7.0 g, 20.23mmol) in an aqueous solution of 10% NaOH (7V) was stirred at 80 °C for 2h. The reaction mixture was cooled, acidified (pH~3) with addition of an aqueous solution of HCl (1M). Then, an aqueous solution of NH3 (25%) was added to reach pH ~ 9. The mixture was stirred at 5 °C for 30 min. Obtained precipitate was filtered, washed with water and dried under high vacuum to obtain 1- (perfluorophenyl)thiourea.(T-2052A2) (3.0 g, 61.28%) as an off- white solid. MS: [MH]+242.97.

[0163] Synthesis of 4,5,6,7-tetrafluorobenzo[d]thiazol-2-amine.(T-2052A3). To a stirred solution of 1-(perfluorophenyl)thiourea (T-2052A2) (3.0 g, 12.39 mmol) in DMF (30 mL) was added NaH (60% dispersion in mineral oil; 0.515 g, 12.89 mmol) at room temperature and the resulting mixture was heated to 80 °C under stirring for 3h under nitrogen. After cooling to room temperature, water (400 mL) was added slowly during which a solid was precipitated out. The precipitate was filtered, washed with water and recrystallized from EtOH-H2O (1:1) to obtain 4,5,6,7-tetrafluorobenzo[d]thiazol-2-amine (T-2052A3) (1.5- g, 54.50%) as a white solid. MS: [MH]+222.98.

[0164] Synthesis of 4,5,6,7-tetrafluorobenzo[d]thiazol-2-amine.( T-2052). To a stirred solution of methyl 2-chlorobenzo[d]thiazole-6-carboxylate (0.150 g, 0.65 mmol) in DMF (2 ml) were added K2CO3(0.224 g, 1.62 mmol) and 4,5,6,7-tetrafluorobenzo[d]thiazol-2-amine (T-2052A3) (0.087 g, 0.39 mmol) sequentially at room temperature under nitrogen and resulting reaction mixture was stirred at 700C for 24h. Reaction mixture was poured in ice-water (100 mL) during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (40 mL x 3) and dried under high vacuum to afford the crude mass, which was triturated with a methanol (50 mL x 3) followed by pentane (10 mL x 3) and dried under high vacuum to obtain 4,5,6,7-tetrafluorobenzo[d]thiazol-2-amine.(T-2052) (0.015 g, 5.51%) as an off white solid.1H NMR (400 MHz, DMSO-d6) δ 13.53 (s, 1H), 8.48 (s, 1H), 7.96- 7.93 (d, J=8.4 Hz, 1H), 7.63 (s, 1H), 3.86 (s, 3H). MS: [MH]+414.13. Example 24 Synthesis of methyl 2-((5,7-dichlorobenzo[d]thiazol-2-yl)amino)benzo[d]thiazole-6- carboxylate (T-2053) 63[0016 88mmol) in DMF (5 ml) were added K2CO3 (0.304 g, 2.20 mmol) and 5,7-dichlorobenzo[d]thiazol- 2-amine (0.115 g, 0.52 mmol) sequentially at room temperature under nitrogen and resulting reaction mixture was stirred at 700C for 24h. Reaction mixture was poured in ice-water (100 mL) during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (40 mL x 3) and dried under high vacuum to afford the crude mass, which was purify by Prep HPLC (CP-2021-009, CAN / 0.05% TRIFUROACETIC ACID IN MILIQUE WATER) to obtain methyl 2-((5,7- dichlorobenzo[d]thiazol-2- yl)amino)benzo[d]thiazole-6-carboxylate (T-2053) (0.006 g, 1.67%) as an off white solid.1H NMR (400 MHz, DMSO-d6 -D2O) δ 8.53 (s, 1H), 8.01-7.99 (d, J=8.8 Hz, 1H), 7.71 (brs, 2H), 7.50 (s, 1H), 3.86 (s, 3H). MS: [MH]+410.1 / [MH+2]+412.0 / [MH+4]+414.0 Note: NH proton signal appeared at δ 13.42 (br, 1H) in1H NMR (400 MHz, DMSO-d6). Example 25 Synthesis of methyl 2-((6,7-dichlorobenzo[d]thiazol-2-yl)amino)benzo[d]thiazole-6- carboxylate (T-2054)[001600 g, 0.44 mmol) in DMF (1 mL) were added K2CO3(0.152 g, 1.1 mmol) and 6,7-dichlorobenzo[d]thiazol- 2-amine (0.057 g, 0.26 mmol) sequentially at room temperature under nitrogen and resulting reaction mixture was stirred at 700C for 24h. Reaction mixture was poured in ice-water (100 mL) during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (50 mL x 3) and dried under high vacuum to afford the crude mass, which was triturated with a mixture of methanol-dichloromethane (1:9; 100 mL x 3) followed by pentane (5 mL x 3) and dried under high vacuum to obtain methyl 2-((6,7- dichlorobenzo[d]thiazol-2-yl)amino)benzo[d]thiazole-6-carboxylate (T-2054) (0.025 g, 13.88%) 64as a pale- yellow solid.1H NMR (400 MHz, DMSO-d6) δ 13.37 (s, 1H), 8.5 (s, 1H), 8.00-7.98 (d, J=8.4 Hz, 1H), 7.68- 7.64 (m, 3H), 3.87 (s, 3H). MS: [MH]+410.1 / [MH+2]+412.0 / [MH+4]+414.0 Example 26 Synthesis of 2,2’-azanediylbis(benzo[d]thiazole-6-carbonitrile) (T-2055) [0016mmol) in DMF (2 ml) were added K2CO3(0.178 g, 1.28 mmol) and 2-aminobenzo[d]thiazole-6- carbonitrile (0.054 g, 0.30 mmol) sequentially at room temperature under nitrogen and resulting reaction mixture was stirred at 700C for 24h. Reaction mixture was poured in ice-water (100 mL) during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (50 mL x 3) and dried under high vacuum to afford the crude mass, which was triturated with ethyl acetate (30 mL x 3) followed by pentane (20 mL x 3) and dried under high vacuum to obtain 2,2’-azanediylbis(benzo[d]thiazole-6- carbonitrile) (T-2055) (0.030 g, 17.47%) as a yellow solid.1H NMR (400 MHz, DMSO-d6– D2O) δ 8.17 (s, 2H), 7.61-7.59 (d, J= 8.4 Hz, 2H), 7.53-7.51 (d, J= 84 H -z, 2H). MS: [MH] 332. Example 27 Synthesis of 2-((6-cyanobenzo[d]thiazol-2-yl)amino)benzo[d]thiazole-5-carbonitrile (T-2056)

[0168] To a stirred solution of 2-chlorobenzo[d]thiazole-5-carbonitrile (0.150 g, 0.77 mmol) in DMF (2 ml) were added K2CO3(0.267 g, 1.93 mmol) and 2-aminobenzo[d]thiazole-6- carbonitrile (0.081 g, 0.46 mmol) sequentially at room temperature under nitrogen and resulting 65reaction mixture was stirred at 700C for 24h. Reaction mixture was poured in ice-water (100 mL) during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (50 mL x 3) and dried under high vacuum to afford the crude mass, which was triturated with methanol-dichloromethane (1:9; 50 mL x 3) followed by pentane (10 mL x 3) and dried under high vacuum to obtain 2-((6- cyanobenzo[d]thiazol-2- yl)amino)benzo[d]thiazole-5-carbonitrile (T-2056) (0.020 g, 7.77%) as a pale-yellow solid.1H NMR (400 MHz, DMSO-d6-D2O) δ 8.10 (s, 1H), 7.86-7.84 (d, J=7.6 Hz, 1H), 7.75 (s, 1H), 7.55-7.50 (d, J=8.4 Hz, 1H), 7.47-7.45 (d, J=8.4 Hz, 1H), 7.37-7.35 (d, J=8.4 Hz, 1H). MS: [MH]+334.11. Example 28 Synthesis of 6-bromo-N-(5-fluorobenzo[d]thiazol-2-yl)benzo[d]thiazol-2-amine (T-2057)

[0169] To a stirred solution of 6-bromo-2-chlorobenzo[d]thiazole (0.100g, 0.40 mmol) in DMF (2 ml) were added K2CO3 (0.139 g, 1.01 mmol) and 5-fluorobenzo[d]thiazol-2-amine (0.040 g, 0.24 mmol) sequentially at room temperature under nitrogen and resulting reaction mixture was stirred at 700C for 24h. Reaction mixture was poured in ice-water (100 mL) during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (50 mL x 3) and dried under high vacuum to afford the crude mass, which was triturated with ethyl acetate-diethyl ether (5:5; 50 mL x 3) followed by pentane (10 mL x 3) and dried under high vacuum to obtain 6-bromo-N-(5-fluorobenzo[d]thiazol-2-yl)benzo[d]thiazol- 2-amine (T- 2057) (0.015 g, 9.77%) as an off white solid.1H NMR (400 MHz, DMSO-d6) δ 12.93 (s, 1H), 8.18 (s, 1H), 7.96-7.92 (t, J=8.0 Hz, 1H), 7.55 (s, 2H), 7.45-7.43 (d, J=6.8 Hz, 1H), 7.15-7.11 (t, J=7.6 Hz, 1H). MS: [MH]+380.1 / [MH+2]+382.1. 66Example 29 Synthesis of methyl 2-((4,6-dichlorobenzo[d]thiazol-2-yl)amino)benzo[d]thiazole-6- carboxylate (T-2058) [0017 44mmol) in DMF (2 ml) were added K2CO3 (0.152 g, 1.1 mmol) and 4,6- dichlorobenzo[d]thiazol-2-amine (0.056 g, 0.26 mmol) sequentially at room temperature under nitrogen and resulting reaction mixture was stirred at 700C for 24h. Reaction mixture was poured in ice-water (100 mL) during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (50 mL x 3) and dried under high vacuum to afford the crude mass, which was triturated with ethyl acetate-diethyl ether (5:5; 50 mL x 3) followed by pentane (5mL x 3) and dried under high vacuum to obtain methyl 2-((4,6-dichlorobenzo[d]thiazol-2-yl)amino)benzo[d]thiazole-6-carboxylate (T-2058) (0.024 g, 13.32%) as an off white solid.1H NMR (400 MHz, DMSO-d6) δ 13.43 (brs, 1H), 8.57 (brs, 1H), 8.09 (brs, 1H), 8.01-7.99 (d, J=8.0 Hz, 1H), 7.64 (br, 2H), 3.87 (s, 3H). MS: [MH]+410.1 / [MH+2]+412.0 / [MH+4]+414.0 Example 30 Synthesis of 6-bromo-N-(6-fluorobenzo[d]thiazol-2-yl)benzo[d]thiazol-2-amine (T-2059)

[0017] o a s rre sou on o - romo- -c oro enzo[ ] azoe ( . g, . mmol) in DMF (2 ml) were added K2CO3(0.141 g, 1.02 mmol) and 6-fluorobenzo[d]thiazol-2-amine (0.040 g, 0.24 mmol) sequentially at room temperature under nitrogen and resulting reaction mixture was stirred at 700C for 24h. Reaction mixture was poured in ice-water (100 mL) during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (50 mL x 3) and dried under high vacuum to afford the crude mass, which 67was triturated with methanol-diethyl ether (2:8; 50 mL x 3) followed by pentane (5 mL x 3) and dried under high vacuum to obtain 6-bromo-N-(6-fluorobenzo[d]thiazol-2-yl)benzo[d]thiazol- 2-amine (T-2059) (0.015 g, 9.77%) as an off white solid.1H NMR (400 MHz, DMSO-d6) δ 12.89 (s, 1H), 8.20 (s, 1H), 7.88-7.87(d, J=6.4 Hz, 1H), 7.71 (br, 1H), 7.57-7.55 (d, J=6.0 Hz, 2H), 7.30- 7.26 (dt, J=8.8 Hz, 2.4 Hz, 1H). MS: [MH]+380.1 / [MH+2]+382.1. Example 31 Synthesis of bis(4,6-difluorobenzo[d]thiazol-2-yl)amine (T-2060) [0017mmol) in DMF (2 ml) were added K2CO3(0.252 g, 1.82 mmol) and 4,6-difluorobenzo[d]thiazol-2-amine (0.081 g, 0.43 mmol) sequentially at room temperature under nitrogen and resulting reaction mixture was stirred at 700C for 24h. Reaction mixture was poured in ice-water (100 mL) during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (50 mL x 3) and dried under high vacuum to afford the crude mass, which was triturated with ethyl acetate-diethyl ether (2:8; 50 mL x 3) followed by pentane (5 mL x 3) and dried under high vacuum to obtain bis(4,6-difluorobenzo[d]thiazol-2-yl)amine (T- 2060) (0.011 g, 4.23%) as a light brown solid.1H NMR (400 MHz, DMSO-d6– D2O) δ 7.77- 7.75 (d, J= 7.6 Hz, 2H), 7.36-7.31 (t, J=9.6 Hz, 2H). MS: [MH]+356.13. Note: NH proton signal appeared at δ 13.15 (br, 1H) in1H NMR (400 MHz, DMSO-d6). Example 32 Synthesis of methyl 2-((5-fluorobenzo[d]thiazol-2-yl)amino)benzo[d]thiazole-6-carboxylate (T- 2061)68

[0173] To a stirred solution of methyl 2-chlorobenzo[d]thiazole-6-carboxylate (0.100 g, 0.44 mmol) in DMF (2 ml) were added K2CO3 (0.152 g, 1.1 mmol) and 5-fluorobenzo[d]thiazol-2- amine (0.043 g, 0.26 mmol) sequentially at room temperature under nitrogen and resulting reaction mixture was stirred at 700C for 24h. Reaction mixture was poured in ice-water (100 mL) during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (50 mL x 3) and dried under high vacuum to afford the crude mass, which was purify by Prep HPLC (CP-2022-001, CAN / 0.05% trifluoroacetic acid in milliQ water) to obtain methyl 2-((5-fluorobenzo[d]thiazol-2- yl)amino)benzo[d]thiazole- 6-carboxylate (T-2061) (0.018 g, 11.40%) as an off white solid.1H NMR (400 MHz, DMSO- d6 – D2O) δ 8.53 (s, 1H), 8.01-7.98 (dd, J=8.4 Hz,1.6 Hz, 1H), 7.95-7.91 (dd, J=8.4 Hz, 5.6 Hz, 1H), 7.70 (br, 1H), 7.48-7.47 (d, J= 7.2Hz, 1H), 7.17-7.12 (dd, J=9.2 Hz,2.4 Hz, 1H), 3.85 (s, 3H). MS: [MH]+360.13. Note: NH proton signal appeared at δ 13.18 (br, 1H) in1H NMR (400 MHz, DMSO-d6). Example 33 Synthesis of 5-bromo-N-(5-fluorobenzo[d]thiazol-2-yl)benzo[d]thiazol-2-amine (T-2062) [00174. , . ol) in DMF (1.5 ml) were added K2CO3 (0.184 g, 1.33 mmol) and 5-bromobenzo[d]thiazol-2-amine (0.072 g, 0.32 mmol) sequentially at room temperature under nitrogen and resulting reaction mixture was stirred at 700C for 24h. Reaction mixture was poured in ice-water (100 mL) during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (50 mL x 3) and dried under high vacuum to afford the crude mass, which was triturated with ethyl acetate-diethyl ether (2:8; 50 mL x 3) followed by pentane (10 mL x 3) and dried under high vacuum to obtain 5-bromo-N-(5-fluorobenzo[d]thiazol-2-yl)benzo[d]thiazol- 2-amine (T- 2062) (0.011g, 5.43%) as an off white solid.1H NMR (400 MHz, DMSO-d6 - D2O) δ 7.94-7.91 (t, J= 8.4 Hz, 1H), 7.89-7.87 (d, J= 8.0 Hz, 1H), 7.81 (s, 1H), 7.47-7.41 (m, 2H), 7.16- 7.12 (t, J= 7.2 Hz, 1H). MS: [MH]+380.1 / [MH+2]+382.1. Note: NH proton signal appeared at δ 13.02 (br, 1H) in1H NMR (400 MHz, DMSO-d6). 69Example 34 Synthesis of methyl 2-((5,7-difluorobenzo[d]thiazol-2-yl)amino)benzo[d]thiazole-6-carboxylate (T-2063) [00 .44mmol) in DMF (2 mL) were added K2CO3(0.152 g, 1.1 mmol) and 5,7-difluorobenzo[d]thiazol- 2-amine (0.049 g, 0.26 mmol) sequentially at room temperature under nitrogen and resulting reaction mixture was stirred at 700C for 24h. Reaction mixture was poured in ice-water (100 mL) during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (50 mL x 3) and dried under high vacuum to afford the crude mass, which was triturated with a mixture of methanol-dichloromethane (1:9; 50 mL x 3) followed by pentane (5 mL x 3) and dried under high vacuum to obtain methyl 2-((5,7- difluorobenzo[d]thiazol-2-yl)amino)benzo[d]thiazole-6-carboxylate (T-2063) (0.025 g, 15.10%) as a pale- yellow solid.1H NMR (400 MHz, DMSO-d6 – D2O) δ 8.53 (s, 1H), 8.01-7.98 (dd, J=8.4 Hz, 1.6 Hz, 1H), 7.71-7.70 (d, J= 6.8 Hz, 1H), 7.41-7.39 (d, J= 9.2 Hz, 1H), 7.24-7.19 (dt, J= 9.2 Hz, 2,4 Hz, 1H), 3.86 (s, 3H). MS: [MH]+378.2. Note: NH proton signal appeared at δ 13.35 (br, 1H) in1H NMR (400 MHz, DMSO-d6). Example 35 Synthesis of 6-fluoro-N-(6-(methylsulfonyl)benzo[d]thiazol-2-yl)benzo[d]thiazol-2-amine (T- 2064) (FR-1)

[00176] o a s rre sou on o -c oro- - uoro- - enzo[ ][ , ]oxa oe ( . g, 0.53 mmol) in DMF(1 ml) were added K2CO3 (0.185 g, 1.32 mmol) and 6- (methylsulfonyl)benzo[d]thiazol-2-amine (0.072 g, 0.31 mmol) sequentially at room temperature under nitrogen and resulting reaction mixture was stirred at 700C for 24h. Reaction 70mixture was poured in ice-water (100 mL) during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (50 mL x 3) and dried under high vacuum to afford the crude mass, which was purify by Prep HPLC (CP- 2021-008, CAN / 0.1% formic acid in milliQ water) to obtain 6-fluoro-N-(6- (methylsulfonyl)benzo[d]thiazol-2-yl)benzo[d]thiazol-2-amine (T-2064) (FR-1) (0.020 g, 6.91%) as an off white solid.1H NMR (400 MHz, DMSO-d6– D2O) δ 8.48 (s, 1H), 7.91-7.89 (dd, J=8.8 Hz, 1.6 Hz, 1H), 7.82- 7.78 (m, 2H), 7.69- 7.65 (dd, J=8.4 Hz, 4.8 Hz, 1H), 7.30- 7.25 (dt, J= 9.2 Hz, 2,4 Hz, 1H), 3.20 (s, 3H). MS: [MH]+380.2. Note: NH proton signal appeared at δ 13.14 (br, 1H) in1H NMR (400 MHz, DMSO-d6).Example 36 Synthesis of 2-((4-fluorobenzo[d]thiazol-2-yl)amino)benzo[d]thiazole-6-carbonitrile (T-2065)

[00177] To a stirred solution of 2-chloro-4-fluorobenzo[d]thiazole (0.100g, 0.53 mmol) in DMF (2 ml) were added K2CO3(0.182 g, 1.32 mmol) and 2-aminobenzo[d]thiazole-6-carbonitrile (0.056 g, 0.32 mmol) sequentially at room temperature under nitrogen and resulting reaction mixture was stirred at 700C for 24h. Reaction mixture was poured in ice-water (100 mL) during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (50 mL x 3) and dried under high vacuum to afford the crude mass, which was triturated with a mixture of methanol-ethyl acetate (1:9; 50 mL x 3) followed by diethyl ether (10 mL x 3) and dried under high vacuum to obtain 2-((4-fluorobenzo[d]thiazol-2- yl)amino)benzo[d]thiazole-6-carbonitrile (T-2065) (0.025 g, 14.30%) as a yellow solid.1H NMR (400 MHz, DMSO-d6) δ 13.32 (s, 1H), 8.53 (s, 1H), 7.84-7.75 (m, 3H), 7.34-7.29 (m, 2H). MS: [MH]+327.10. Example 37 Synthesis of 2-((5-fluorobenzo[d]thiazol-2-yl)amino)benzo[d]thiazole-6-carbonitrile (T-2066) 71[0017 77 mmol) in DMF (2 ml) were added K2CO3 (0.267 g, 1.93 mmol) and 5-fluorobenzo[d]thiazol-2-amine (0.077 g, 0.46 mmol) sequentially at room temperature under nitrogen and resulting reaction mixture was stirred at 700C for 24h. Reaction mixture was poured in ice-water (100 mL) during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (50 mL x 3) and dried under high vacuum to afford the crude mass, which was triturated with a mixture of methanol-diethyl ether (5:5; 50 mL x 3) followed by pentane (10 mL x 3) and dried under high vacuum to obtain 2-((5-fluorobenzo[d]thiazol-2- yl)amino)benzo[d]thiazole-6- carbonitrile(T-2066) (0.020 g, 7.93%) as a light-yellow solid.1HNMR (400 MHz, DMSO-d6) δ 13.16 (s, 1H), 8.48 (s, 1H), 7.97 (br, 1H), 7.82-7.75 (m, 2H), 7.47 (br, 1H),7.18- 7.14 (t, J= 8.8 Hz, 1H).MS: [MH]+327.10.Example 38 Synthesis of bis(5-fluorobenzo[d]thiazol-2-yl)amine (T-2067) [00179. , . ) in DMF (2 ml) were added K2CO3 (0.27 g, 2.0 mmol) and 2-chloro-5-fluorobenzo[d]thiazole (0.080 g, 0.48 mmol) sequentially at room temperature under nitrogen and resulting reaction mixture was stirred at 700C for 24h. Reaction mixture was poured in ice-water (100 mL) during which a solid was precipitated out. Solid residue was filtered through a Buchner funnel, residue was washed with water (50 mL x 3) and dried under high vacuum to afford the crude mass, which was triturated with ethyl acetate-diethyl ether (5:5; 50 mL x 3) followed by pentane (10 mL x 3) and dried under high vacuum to obtain bis(5-fluorobenzo[d]thiazol-2-yl)amine (T-2067) (0.010 g, 3.91%) as an off white solid.1H NMR (400 MHz, DMSO-d6) δ 12.99 (brs, 1H), 7.97(brs, 2H), 7.49 (br, 2H), 7.18-7.14 (t, J=8.0 Hz, 2H). MS: [MH]+320.08. 72Example 39 Synthesis of 2-((5,7-difluorobenzo[d]thiazol-2-yl)amino)benzo[d]thiazole-6-carbonitrile (T- 2068) [00180F (5 ml) were added K2CO3 (0.44 g, 3.22 mmol) and 2-chlorobenzo[d]thiazole-6-carbonitrile (0.250 g, 1.28 mmol) sequentially at room temperature under nitrogen and resulting reaction mixture was stirred at 700C for 24h. the reaction mixture was quenched with water (100 mL) and extracted with Ethyl Acetate (3x50 mL). The combined organic layer was washed with water (10 mL x 3), dried over Na2SO4 and concentrated under vacuum to afford the crude mass, which was purify by Prep HPLC (CP-2022-001, CAN / 0.05% ammonia in milliQ water) to obtain 2-((5,7-difluorobenzo[d]thiazol-2-yl)amino)benzo[d]thiazole-6-carbonitrile (T-2068) (0.020g, 4.51%) as a pale-yellow solid.1H NMR (400 MHz, DMSO-d6 – D2O) δ 8.42 (s, 1H), 7.80-7.78 (d, J=8.4 Hz, 1H), 7.75-7.73 (d, J=8.4 Hz, 1H), 7.39-7.37 (d, J=9.2 Hz, 1H), 7.23- 7.18 (t, J= 9.6 Hz, 1H). MS: [MH]+345.11. Note: NH proton signal appeared at δ 13.47 (br, 1H) in1H NMR (400 MHz, DMSO-d6). Example 40 Synthesis of 2,2’-azanediylbis(benzo[d]thiazole-5-carboxylic acid) (T-2069)

[0181] To a stirred solution of 2,2-azanediylbis(benzo[d]thiazole-5-carbonitrile) (T-2041) (0.150g, 0.45 mmol) in THF: H2O (8:2) was added KOH (0.252 g, 4.50 mmol). Reaction mixture was stirred at 90oC for 5day. After completion of reaction as indicated by LCMS, the reaction mixture was quenched with 6N HCl (50 mL) during which a solid was precipitated out. Solid 73residue was filtered through a Buchner funnel, residue was washed with water (50 mL x 3) and dried under high vacuum to afford the crude mass, which was triturated with MeOH (20 mL x 3) followed by pentane (10 mL x 3) and dried under high vacuum to obtain 2,2’ – azanediylbis(benzo[d]thiazole-5-carboxylic acid) (T-2069) (46.66 g, 27.92%) as a pale-yellow solid.1H NMR (400 MHz, DMSO-d6) δ 13.10 (brs, 3H), 8.14 (brs, 2H), 8.06- 8.04 (d, J=8.4 Hz, 2H), 7.84- 7.82(dd, J=8.4 Hz, 1.2 Hz, 2H). MS: [MH]+372.14. Example 41

[0182] As shown in FIGS.4A-4B, a CHOP-Luc cell-based assay and WB identified compounds 5a, T-22 and 5a28 (see Table 1) as top inhibitors.

[0183] The histological results of treatment with 5a / T-22 / 5a28 compounds in a mouse SOHU glaucoma model are shown in FIGS.5A-5B. The data show an increase in RGC survival after treatment with the compounds. FIGS. 6A-6B show the effect of 5a / T-22 / 5a28 compound treatment with in vivo imaging and visual function results in mouse SOHU glaucoma model. Methods

[0184] Immunoblotting. After treatment, cell lysates were prepared in RIPA buffer (Themo Fisher Scientific, 89901) supplemented with Halt Protease inhibitor cocktail (Themo Fisher Scientific, PI78437). The total protein concentration of lysates was measured by the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, 23227). 20 µg protein of each sample were denatured at 95 °C for 5 min in 1× Blue Protein Loading Dye with DTT (New England Biolabs) before being separated by SDS-PAGE. Protein samples were then transferred to 0.2 µm nitrocellulose membranes (Bio-Rad, 1610097) and blocked with 3% non-fat milk for 2 h. Subsequently, the membranes were incubated with primary antibodies (ATF4, Cell Signaling, 11815S 1:1000, or CHOP, Cell Signaling, 2895S 1:1000 in 4% BSA) overnight at 4 °C. After washing in TBST, these membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling, 7074S and 7076S, 1:8000) and visualized using a GE- AI600 imaging system.

[0185] SOHU glaucoma model, compound treatment and IOP measurement. The detailed procedure has been published before. Briefly, mice were anesthetized by an intraperitoneal injection of Avertin (0.3 mg / g) and received the silicone oil (1000 mPa.s, Silikon, Alcon Laboratories, Fort Worth, Texas) injection at 9 weeks of age. Prior to injection, 0.5% proparacaine hydrochloride (Akorn, Somerset, New Jersey) was applied to the cornea to reduce its sensitivity during the procedure. A 32 G needle was tunneled through the layers of the cornea at the superotemporal side close to the limbus to reach the anterior chamber without injuring lens or iris. Following this entry, ~2 µl silicone oil (SO) was injected slowly into the anterior chamber using a sterile glass micropipette, until the oil droplet expanded to cover most areas of 74the iris (diameter ~1.8–2.2 mm). After the injection, veterinary antibiotic ointment (BNP ophthalmic ointment, Vetropolycin, Dechra, Overland Park, Kansas) was applied to the surface of the injected eye. The contralateral control eyes received mock injection with 2 µl normal PBS to the anterior chamber.

[0186] For compound treatment, each eye received an intravitreal injection twice per week with 1 µl of 5mM 5a, 1 µl of 2mM 5a28 / T-22 for 3 weeks after SO injection. Control groups received the same volume of vehicle as control (10% DMSO, 10% Kolliphor®HS 15, and 80% PBS).

[0187] The IOP of both eyes was measured by the TonoLab tonometer (Colonial Medical Supply, Espoo, Finland) according to product instructions. Briefly, mice were anesthetized and 1% Tropicamide sterile ophthalmic solution (Akorn, Somerset, New Jersey) was applied three times at 3-minute intervals to fully dilate the pupils before taking measurements. The average of six measurements by the TonoLab was considered as one machine-generated reading and three machine-generated readings were obtained from each eye; the mean was calculated to determine the IOP.

[0188] Spectral-domain optical coherence tomography (SD-OCT) imaging. Fundus OCT imaging was performed under OCT mode by switching to a 30olicensed lens (Heidelberg Engineering), as previously described. Briefly, At 3wpi, the mouse retina was scanned with the ring scan mode centered by the ON head at 100 frames average under high-resolution mode (each B-scan consisted of 1536 A scans). The average thickness of GCC (includes retinal nerve fiber layer, ganglion cell layer, and inner plexiform layer) around the ON head was measured with the Heidelberg software (Heidelberg Engineering, Franklin, MA). The mean of the GCC thickness in the treated retina was compared to that in the contralateral control (CL) retina to yield a percentage of GCC thickness value.

[0189] Pattern electroretinogram (PERG) recording. PERG recording of both eyes was performed with the Miami PERG system (Intelligent Hearing Systems, Miami, Florida) according to product instructions. A heating pad (TCAT-2LV, Physitemp Instruments Inc., Clifton, New Jersey) maintained animal core temperature at 37 °C. A small lubricant eye drop (Systane) was applied before recording to prevent corneal opacities. Two 14 cm×14 cm LED-based stimulators were placed in front so that the center of each screen was 10 cm from each eye. The pattern remained at a contrast of 85% with a luminance of 800 cd / m2, and consisted of four cycles of black-gray elements, with a spatial frequency of 0.052 c / d. Upon stimulation, the independent PERG signals were recorded from the snout and simultaneously by asynchronous binocular acquisition. With each trace recording up to 1020 ms, two consecutive recordings of 100 and 300 traces were averaged to achieve one readout. The first positive peak in the waveform was designated as P1 and the second negative peak as N2. The mean amplitude of the P1-N2 amplitude in the treated eye was compared to that in the contralateral control eye to yield a percentage of amplitude change. 75

[0190] Optokinetic tracking response (OKR). The spatial vision of both eyes was measured using the OptoMotry system (CerebralMechanics Inc., Lethbridge, Alberta, Canada) dependent on opto-kinetic response (OKR). In brief, mice were placed unrestrained on a platform in the center of four 17-inch LCD computer monitors (Dell, Phoenix, Arizona); their movement was captured by a video camera above the platform. A rotating cylinder with vertical sine wave grating was computed and projected to the four monitors by OptoMotry software (CerebralMechanics Inc., Lethbridge, Alberta, Canada). The sine wave grating provides a virtual-reality environment to measure the spatial acuity of left eye when rotated clockwise and right eye when rotated counterclockwise. When the mouse calmed down and stopped moving, the gray of the monitor immediately switched to a low spatial frequency (0.1 cycle / degree) for five seconds, in which the mouse was assessed by judging whether the head turned to track the grating. The mice were judged to be capable of tracking the grating. The spatial frequency increased repeatedly until a maximum frequency was identified and recorded. The % of vision acuity was yielded by comparing the maximum frequency of the treated eye to that of the contralateral eye.

[0191] Immunohistochemistry of whole mount retina. After perfusion fixation with 4% PFA in PBS, mice eyeballs were dissected and post-fixed with 4% PFA for 2 h at room temperature. Retinas were dissected for whole-mount retina immunostaining. Retinas were blocked with 10% goat serum (Sigma, G9023) for 2 h before incubating with primary antibody anti-RBPMS 1:4000 overnight at 4 °C. After washing 3 times with PBS, samples were incubated with secondary antibody (1:400; Jackson ImmunoResearch, West Grove, Pennsylvania) at room temperature for 2 h. Tissues were washed with PBS 3 times before mounting with Fluoromount-G (SouthernBiotech, Alabama). Images of immunostained wholemounts were acquired with a Keyence epifluorescence microscope (BZ-X800) or Zeiss confocal microscope (LSM 880). For RGC counting, 8 circles drawn by Concentric Circle plugin of NIH Fiji / ImageJ were used to define the peripheral, middle, and central areas of the retina. Multiple 250 × 250 μm counting frames were applied by Fiji / ImageJ and the number of surviving RGCs was counted by RGCode software (https: / / gitlab.com / NCDRlab / rgcode). The percentage of RGC survival was calculated as the ratio of surviving RGC numbers in treated eyes compared to contralateral eyes. References

[0192] Zhang, J. et al. Silicone oil-induced ocular hypertension and glaucomatous neurodegeneration in mouse. eLife 8, e45881 (2019).

[0193] Li, L. et al. Longitudinal morphological and functional assessment of RGC neurodegeneration after optic nerve crush in mouse. Front Cell Neurosci.14, 109 (2020). 76

[0194] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0195] Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.

[0196] The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 112 (f) or 35 U.S.C. §112(6) is not invoked. 77

Claims

WHAT IS CLAIMED IS:

1. A pharmaceutical formulation comprising a compound of Formula I: I wherein R1, R2, m H; halo; -OR8, where R8 isan alkyl; -CN; -CO2R9, where R9is H, halo, alkyl, amine; -NO2; amine; substituted amine; - CF3; and alkyl; and R5, R6, and R7are independently selected from H; halo; -CO2R9; -CN; -CF3; tetrazole, -CH2OH, -CF2H, -SO3H; NHCO2R’, -SO2NR’R”, -CONR’R”, where R’, R” are independently H, lower alkyl, branched alkyl, substituted lower alkyl; or a prodrug or derivative thereof; and a pharmaceutically acceptable excipient.

2. The pharmaceutical formulation of claim 1, wherein R1, R2, R3and R4are independently selected from H; Br; C; F; I; CF3; OCH3;and R5 is CO2R9, where R9 is H or lower alkyl; or CONR’R”, where R’, R” are independently H, lower alkyl, or substituted lower alkyl.

3. The pharmaceutical formulation of any of claims 1, wherein at least two of R5, R6, and R7are H.

4. The pharmaceutical composition of claim 1, wherein R6 and R7 are H.

5. The pharmaceutical composition of claim 1, wherein R6 is halo, R5 and R7 are H.

6. The pharmaceutical composition of any of claims 1-5, wherein the compound is set forth in Table 1.

7. The pharmaceutical composition of any of claims 1-6, wherein the compound is selected from:

788. A pharmaceutical formulation according to any of claims 1-7, in a unit dosage for therapeutic inhibition of CHOP.

9. The pharmaceutical formulation of any of claims 1-8 in a formulation suitable for ocular administration. 7910. An isolated compound of Formula I: Iwherein R1, R2, R3 and R4 are independently selected from H; halo; -OR8, where R8 is an alkyl; -CN; -CO2R9, where R9 is H, halo, alkyl, amine; -NO2; amine; substituted amine; - CF3; and alkyl; and R5, R6, and R7are independently selected from H; halo; -CO2R9; -CN; -CF3; tetrazole, -CH2OH, -CF2H, -SO3H; NHCO2R’, -SO2NR’R”, -CONR’R”, where R’, R” are independently H, lower alkyl, branched alkyl, substituted lower alkyl; or a prodrug or derivative thereof; and a pharmaceutically acceptable excipient.

11. A method of modulating endoplasmic reticulum (ER) stress in an individual in need thereof, the method comprising: administering an effective dose of a pharmaceutical formulation of any of claims 1-9 in dose effective to inhibit CHOP and modulate ER stress.

12. The method of claim 11, wherein the ER stress is associated with a neuropathy.

13. The method of claim 11 or claim 12, wherein the neuropathy is an optic nerve (ON) neuropathy.

14. The method of claim 13, wherein the ON neuropathy is retinal ganglion cell degeneration.

15. The method of claim 14, wherein the retinal ganglion cell degeneration is glaucoma, optic neuritis, or ON traumatic injury.

16. The method of any of claims 11-15, wherein the administration reduces or ameliorates degeneration of axons and / or soma of retinal ganglion cells (RGCs). 8017. The method of any one of claims 11-16, wherein the individual is human.

18. The method of any of claims 11-17, wherein the composition is administered to the eye.

19. The method of any of claims 11-17, wherein the composition administered systemically. 81

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