Novel 5-HT4 receptor inverse agonists for the prevention and treatment of stress-induced psychiatric disorders

Abstract

Certain serotonin type 4 receptor (5-HT4Rs) inverse agonists or antagonists are protective against stress-induced behavior and can treat stress-induced behavior. These 5-HT4R inverse agonists or antagonists can be administered to subjects in need, to help prevent and treat stress-induced behavior psychiatric disorders.

Description

NOVEL 5-HT4 RECEPTOR INVERSE AGONISTS FOR THE PREVENTION AND TREATMENT OF STRESS-INDUCED PSYCHIATRIC DISORDERSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 691,565, filed September 6, 2024, to The Trustees of Columbia University and The Research Foundation for Mental Hygiene, Inc., titled “NOVEL 5-HT4 RECEPTOR INVERSE AGONISTS PROTECT AGAINST STRESS-INDUCED MALADAPTIVE BEHAVIOR,’" the entirety of the disclosure of which is hereby incorporated by this reference. The entire contents of the above-identified applications are hereby fully incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under TR001873 awarded by the National Institutes of Health. The government has certain rights in the invention.TECHNICAL FIELD

[0003] The subj ect matter disclosed herein is generally directed to 5-HT4R inverse agonists or antagonists that are protective against stress-induced behavior and can treat stress-induced behaviors.BACKGROUND

[0004] Depressive disorders impact over 65 million worldwide making them a leading cause of disability (1,2). Those diagnosed with major depressive disorder (MDD) exhibit high comorbidity with other psychiatric disorders, such as generalized anxiety disorder (GAD) (3), which is associated with reduced symptom remission following pharmacological treatment (4). Additionally, there is a health disparity in gender, as women are twice as likely as men to be diagnosed with MDD or GAD during their lifetime (5-7). Although the etiology of these disorders is not fully understood, stressful or traumatic life events are a major risk factor for symptom onset and severity (8-11).

[0005] In adults with MDD, only half receive any treatment (12). Many individuals report worsening of symptoms upon treatment onset (13-16) and about 31 % of patients do not achieve remission after full courses of two or more antidepressants (17) indicating a need for new pharmacotherapies. Recent work from Applicants and others has identified the serotonin type4 receptor (5-HT4R) as a promising target to treat depression and anxiety (18-22). The 5-HT4R is a G-coupled receptor that primarily activates the cyclic adenosine monophosphate (cAMP) - protein kinase A (PKA) pathway, resulting in excitation of neurons in response to serotonin. 5-HT4RS are found throughout regions implicated in anxiety and depression, including the hippocampus (HPC), medial prefrontal cortex, amygdala, and striatum (23-26).

[0006] In previous preclinical work, subchronic administration of the 5-HT4R partial agonist RS-67,333 reduced anxiety and depressive-like behaviors following corticosterone exposure (27) and acute administration of RS-67,333 or the 5-HT4R agonist prucalopride decreased immobility in the FST test (28) indicating 5-HT4R activation produces rapid-acting antidepressant-like effects. Additionally, Applicants recently identified a role for 5-HT4R agonists in enhancing stress resilience. A prophylactic injection of prucalopride or PF- 04995274 attenuated learned fear and decreased depressive-like behavior in male mice. Applicants established that prophylactic administration of prucalopride reduced large AMP A receptor-mediated bursts in CA3 of the HPC, highlighting a common mechanism with the fastacting antidepressant and prophylactic (R,S)-ketamine (18,29).

[0007] Current treatment options selectively targeting the 5-HT4R are limited. Expression of 5-HT4RS occurs throughout the gastrointestinal (30-32) and cardiovascular system (33,34), making peripheral side effects common. However, there is an effort to identify 5-HT4R compounds that will improve psychiatric symptoms. In the United States, the high-affinity 5- HT4R agonist prucalopride is approved for chronic idiopathic constipation (35,36) and recent work in healthy volunteers found that subchronic administration improves memory and alters neural activity, particularly in the HPC (37,38). Recently, a clinical trial was conducted to determine if the purported partial agonist PF-04995274 improves emotional processing and neural activity in individuals with treatment-resistant depression (NCT03515733) (39).

[0008] As there are currently no FDA-approved 5-HT4R agonists to treat psychiatric symptoms, there is a need to develop new compounds.

[0009] Citation or identification of any document in this application is not an admission that such a document is available as prior art to the present invention.SUMMARY

[0010] In one aspect, the present invention provides for a 5-HT4R inverse agonist or antagonist having the structure:wherein X is O, S, SO, SO2or Se, wherein Ar is fused heteroaromatic and wherein R is heterocyclic, bridged cyclic, spiro cyclic or fused cyclic.

[0011] In certain embodiments, Ar has the structure:

[0012] In certain embodiments, R has the structure:

[0013] In another aspect, the present invention provides for a 5-HT4R inverse agonist or antagonist selected from the group consisting of:

[0014] In another aspect, the present invention provides for a method of protecting against stress-induced maladaptive behavior in a subject in need thereof comprising administering a prophylactically effective amount of the 5-HT4R inverse agonist or antagonist of any embodiment herein to the subject. In certain embodiments, the 5-HT4R inverse agonist or antagonist is administered after stress but before stress-induced maladaptive behavior onset. In certain embodiments, the 5-HT4R inverse agonist or antagonist is administered before stress. In certain embodiments, the subject is an adult. In certain embodiments, the subject is aged. In certain embodiments, the subject is a female or male. In certain embodiments, the subject is diagnosed with PTSD, an anxiety disorder, a phobia, major depressive disorder, bipolar depression, OCD, ASD, schizophrenia, Huntington’s disease, Parkinson’s disease, an eating disorder, or treatment-resistant depression. In certain embodiments, the maladaptive behavior is selected from the group consisting of depression, anxiety, anorexia, compulsiveness, freezing, intrusive thoughts and flashbacks, avoidance, negative changes in mood and cognition, hyperarousal, dissociation, difficulty concentrating and making decisions, and memory loss. In certain embodiments, a female subject is treated with D5 or D8 after a stress to prevent an anxiety or eating disorder symptom. In certain embodiments, a male subject is treated with D7 or D8 after a stress to prevent depression. In certain embodiments, a male subject is treated with D7 to prevent a PTSD, anxiety, or phobia symptom. In certain embodiments, a male subject is treated with D5, D7, or D8 to prevent major depressive disorder or bipolar depression. In certain embodiments, a male subject is treated with D5 or D7 to prevent a compulsive symptom associated with OCD, ASD, schizophrenia, Huntington’s disease, or Parkinson’s disease. In certain embodiments, a male subject is treated with D5 or D8 to prevent anxiety or an eating disorder symptom. In certain embodiments, an aged male subject is treated with D7 to prevent major depressive disorder or bipolar depression. In certain embodiments, a female subject is treated with D7 or D8 to prevent a compulsive symptom associated with OCD, ASD, schizophrenia, Huntington’s disease, or Parkinson’s disease. In certain embodiments, a female subject is treated with D5 to prevent anxiety or an eating disorder symptom.

[0015] These and other aspects, objects, features, and advantages of the example embodiments will become apparent to those having ordinary skill in the art upon consideration of the following detailed description of example embodiments.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0017] An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention may be utilized, and the accompanying drawings of which:

[0018] FIG. 1 - Chemical composition of eight novel 5- HT4R- targeting compounds. Compounds were designed based on PF-04995274. Images of compounds generated in Chem Draw.

[0019] FIG. 2A-FIG. 2H - 5-HT4R activity in a direct miniG and a cAMP accumulation competition assay identifies novel inverse agonists. (FIG. 2A) Schematic of the miniGs direct recruitment assay, showing the 5-HT4R fused to nanoluciferase at its C- terminus and miniGs fused to the acceptor mVenus. Receptor activation due to ligand binding results in recruitment of miniG proteins from the cytoplasm to the plasma membrane and leads to an increase in BRET between the donor and acceptor molecules. (FIG. 2B) Dose response curves for the miniGs assays with standards serotonin, prucalopride, partial agonist RS-67,333, and parental compound PF-04995274, which acts as an inverse agonist. (FIG. 2C) Compound D4 acts as a weak partial agonist compared to serotonin, while compounds D1-D3 act as weak inverse agonists. (FIG. 2D) Compounds D5, D7, and D8 seem to elicit no effects in this system, while compound D6 acts as an inverse agonist. (FIG. 2E) Schematic of the cAMP assay, showing activation of the Gα subunit of the Gsheterotrimeric complex in response to receptor activation, resulting in an increase in intracellular cAMP. Changes in intracellular cAMP are monitored using the cAMP sensor CAMYEL, an EP AC protein that is fused to both a donor luciferase and acceptor fluorescent protein. As cAMP is produced, it binds to EP AC and causes conformational changes that lead to a decrease in the BRET signal. The experiments in (FIG. 2F, G, H) are competition assays against an EC80 concentration of serotonin (35nM). (FIG. 2F) RS-67,333 decreases residual cAMP levels in a concentration dependent manner, replacing serotonin but acting as a partial agonist. PF-04995274 also leads to concentration dependent decrease in cAMP levels but to a value below baseline, consistent with its actions as an inverse agonist. (FIG. 2G-H) Compounds DI, D2, D4, D5, and D7 all lead to concentration dependentdecreases in residual cAMP to near baseline values. Compounds D6 and D8 lead to decreases in residual cAMP below the vehicle baseline similar to PF-04995274. In contrast, compound D3 is inactive with no effect on residual cAMP levels compared to EC80 serotonin alone. Data represented as a percentage normalized to Emaxof serotonin (FIG. 2B-D) or EC80 serotonin (FIG. 2F-H). Dose response curves were fit using a 3-parameter model and values represent the mean of 3 - 7 individual replicates. Each replicate was performed with triplicate determinations. Error bars represent ± SEM; cAMP, cyclic adenosine monophosphate; EC80, effective concentration 80% nluc, nanoluciferase.

[0020] FIG. 3A-FIG. 3O - A single prophylactic injection of a novel 5-HT4R compound protects against stress-induced maladaptive behaviors in male mice. (FIG. 3A) Experimental protocol. (FIG. 3B-C) Freezing was comparable across all groups during CFC training. (FIG. 3D-E) During context re-exposure, PF-04995274 (10 mg / kg) and D7 (3 mg / kg) decreased fear expression when compared with saline mice. (FIG. 3F-G) On day 1 of the FST, immobility time was comparable across groups. (FIG. 3H-I) On day 2 of the FST, PF- 04995274, D5 (3 and 10 mg / kg), D7 (3 mg / kg), and D8 (10 mg / kg) decreased immobility time when compared with saline. (FIG. 3J) In the MB task, D5 (10 mg / kg) and D7 (3 and 10 mg / kg) decreased the number of marbles buried compared to saline. (FIG. 3K) Prior to administration of NSF, animals lost a comparable amount of weight during food restriction across groups. (FIG. 3L-M) In the NSF, PF-04995274 (10 mg / kg), D5 (3 mg / kg), and D8 (3 mg / kg) reduced the latency to feed in the OF. In the home cage, groups exhibited comparable (FIG. 3N) latency to feed and (FIG. 3O) amount of food consumed, n = 5-13 male mice per group; error bars represent ± SEM; *p < 0.05, ** p < 0.01, *** p < 0.001. CFC, contextual fear conditioning; FST, forced swim test; HC, home cage; MB, marble burying; NSF, novelty-suppressed feeding; OF, open field; PF, PF-04995274; Sal, saline. Behavioral timeline created with BioRender.com.

[0021] FIG. 4A-FIG. 4M - A single injection of a novel 5-HT4R compound decreases behavioral despair when administered following stress in male mice. (FIG. 4A) Experimental design. (FIG. 4B-C) Freezing was comparable across all groups during CFC exposure. (FIG. 4D-E) On day 1 of FST, compounds D5, D7, and D8 (all 3 mg / kg) increased immobility time. (FIG. 4F-G) On day 2 of the FST, PF-04995274 (10 mg / kg), D7 (10 mg / kg), and D8 (10 mg / kg) reduced immobility time compared with saline. (FIG. 4H) In the MB task, all groups buried a comparable number of marbles. (FIG. 4I) In the NSF, all groups lost asimilar amount of weight when compared directly to saline although there was an overall effect of Drug. (FIG. 4J-K) During the NSF, there was an overall effect of Drug but no drug decreased the latency to feed in the OF when compared to saline. In the home cage, (FIG. 4L) latency to feed and (FIG. 4M) food consumed was comparable across all groups, n = 5 - 14 male mice per group; error bars represent ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001. CFC, contextual fear conditioning; FST, forced swim test; MB, marble burying; NSF, novelty suppressed feeding; OF, open field; HC, home cage; Sal, saline; PF, PF-04995274; min, minutes; sec, seconds; mg, milligram; kg, kilogram. Behavioral timeline created with BioRender.com.

[0022] FIG. 5A-FIG. 5H - Prophylactic administration of novel 5-HT4R compounds reduce large AMPA-driven synaptic bursts in CA3. (FIG. 5A) Experimental design. Mice were administered saline, D5 (3 mg / kg), D7 (3 mg / kg), or D8 (3 mg / kg) 1 week before wholecell voltage clamp electrophysiology. Representative EPSCs following administration of (FIG. SB) saline, (FIG. 5C) D5, (FIG. 5D) D7, and (FIG. 5E) D8. (FIG. 5F) The mean amplitude was attenuated in D5- and D7-administered mice when compared to saline. D8-administered mice exhibited increased mean amplitude when compared with saline. (FIG. 5G) The mean burst amplitude was significantly increased by D8, but not D5 and D7 when compared with saline. (FIG. 5H) The mean frequency of all AMPAR-mediated EPSCs within a 20-second recording period was significantly decreased by D5 and D7, but not by D8, when compared with saline, n = 3-6 cells per group; error bars represent ± SEM; *p < 0.05, ** p < 0.01, *** p < 0.001. AMP AR, AMP A receptor; CA3, cornu ammonis area 3; EPSC, excitatory postsynaptic cunent; Sal, saline; ms, millisecond; pA, picoampere. Behavioral timeline created with BioRender.com.

[0023] FIG. 6A-FIG. 6M - D7 reduces behavioral despair when administered after learned helplessness in male mice. (FIG. 6 A) Experimental design. (FIG. 6B) Total activity during habituation period was comparable across groups. (FIG. 6C) Total session latencies were comparable across groups. (FIG. 6D) Mean latency to escape the shock was comparable across all groups when compared to saline. (FIG. 6E) Mean latency to escape in trials 21 - 30 was comparable across groups. (FIG. 6F-G) On day 1 of the FST, all groups exhibited comparable immobility time compared to saline. (FIG. 6H-I) On day 2 of the FST, PF- 04995274 (10 mg / kg) and D7 (10 mg / kg) reduced immobility time compared with saline. (FIG. 6J) Representative images of c-Fos expression in the HPC. (FIG. 6K) PF-04995274, D5, D7,and D8 (10 mg / kg) increased c-Fos expression in the DG when compared with saline. (FIG. 6L) PF-04995274, D5, and D7 (10 mg / kg) increased c-Fos expression in CA3 when compared with saline. (FIG. 6M) c-Fos expression was comparable across groups in CA1. n = 4 - 11 male mice per group; error bars represent SEM; * p < 0.05, ** p < 0.01, *** p < 0.001. CFC, contextual fear conditioning; ; CA3, comu ammonis area 3; CA1, comu ammonis area 1; DG, dentate gyrus; FST, forced swim test; HC, home cage; HPC, hippocampus; MB, marble burying; NSF, novelty suppressed feeding; OF, open field; PF, PF-04995274; Sal, saline; min, minutes; sec, seconds; mg, milligram; kg, kilogram. Behavioral timeline created with BioRender.com.

[0024] FIG. 7A-FIG. 70 - Prophylactic D7 exerts therapeutic potential in aged male mice. (FIG. 7 A) Experimental timeline for aged (~17 month) male mice. (FIG. 7B-C) Freezing during CFC training was comparable across groups. (FIG. 7D-E) During CFC exposure, freezing behavior was comparable across groups. (FIG. 7F) On day 1 of the FST, RS-67,333 (10 mg / kg) reduced immobility across the entire experimental session compared to saline, but (FIG. 7G) average immobility time for 3 - 6 min was comparable across groups. (FIG. 7H-I) On day 2 of the FST, RS-67,333 (10 mg / kg), PF-04995274 (10 mg / kg), and D7 (3 mg / kg) reduced immobility compared to saline. (FIG. 7J) In the MB task, all groups buried a similar number of marbles compared to saline. (FIG. 7K) During food restriction phase of the NSF assay, drug groups lost a comparable amount of weight when compared directly to saline although there was a main effect of Drug. (FIG. 7L-M) Latency to feed in the OF in the NSF was comparable across groups. In the home cage, (FIG. TN) latency to feed and (FIG. 70) the amount of food consumed was comparable between the groups. 7 - 11 male mice per group; error bars represent ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001. CFC, contextual fear conditioning; FST, forced swim test; MB, marble burying; NSF, novelty suppressed feeding; OF, open field; HC, home cage; Sal, saline; PF, PF-04995274; P, prucalopride; RS, RS-67,333; min, minutes; sec, seconds; mg, milligram; kg, kilogram.

[0025] FIG. 8A-FIG. 8J - 5-HT4RS exhibit high levels of constitutive activity. (FIG. 8A) Schematic of the cAMP Response Element (CRE) Gene Expression assay. The reporter contains the firefly luciferase gene under the control of multimerized CRE located upstream of a minimal promoter. Elevation of the intracellular cAMP level activates cAMP response element binding protein (CREB) to bind CRE and induces the expression of luciferase, which is quantitated by luminescence. As a control, a constitutively active Renilla-luciferase geneunder the CMV promoter is included to determine transfection efficiency. (FIG. 8B) Fold changes in CRE-luciferase in the presence of varying amounts of transiently transfected 5-HT4receptor. Slight differences are seen between the receptor alone condition and the receptor + 10 μM serotonin, suggesting a high degree of constitutive activity. (FIG. 8C) Pooled dose response curves for cAMP CAMYEL, average of reps 1 - 7 showing results for standard ligands 5-HT, prucalopride, RS- 67,333, and PF-04995274. (FIG. 8D-J) Dose response curves for the cAMP CAMYEL. Each panel shows one replicate and shows the variation seen in the cAMP assay from experiment to experiment with the standard ligands 5-HT, prucalopride, RS- 67,333, and PF-04995274. While 5-HT consistently increase cAMP, there is great variation in the extent of partial agonism of the other ligands across replicates. Dose response curves were fit using a 3 -parameter model and values represent 1 individual replicate ± SEM. Each replicate was performed with triplicate determinations. Emaxis expressed as a percentage relative to serotonin. cAMP, cyclic adenosine monophosphate; Emax, maximum effect.

[0026] FIG. 9A-FIG. 90 - Prophylactic D5 attenuates hyponeophagia while D7 and D8 attenuate perseverative behavior in adult female 129S6 / SvEv mice. (FIG. 9A) Experimental protocol. (FIG. 9B) Freezing was comparable between all groups during CFC training. (FIG. 9C) Average freezing time was comparable between all groups during CFC training. (FIG. 9D) During context re-exposure, freezing was comparable between all groups. (FIG. 9E) Average freezing time was comparable between all groups during context reexposure. (FIG. 9F) During day 1 of the FST, compounds D5, D7, and D8 (3 mg / kg) increased immobility compared to saline control across the entire test session, (FIG. 9G) but only D8 (3 mg / kg) increased immobility compared to saline during minutes 3 - 6. (FIG. 9H, I) On day 2 of the FST, all groups exhibited comparable immobility time when compared with saline- administered mice. (FIG. 9J) In the MB task, D7 and D8 (3 mg / kg) buried fewer marbles compared to saline control. (FIG. 9K) Weight loss was similar across groups during food restriction phase ofNSF. (FIG. 9L, M) In theNSF, PF-04995274 and D5 (3 mg / kg) exhibited reduced latency to feed in the OF. In the home cage, all groups exhibited comparable (FIG. 9N) latency to feed and (FIG. 90) amount of food consumed, n = 5-9 female mice per group; error bars represent ± SEM; *p < 0.05, ** p < 0.01, *** p < 0.001. CFC, contextual fear conditioning; FST, forced swim test; HC, home cage; MB, marble burying; NSF, novelty- suppressed feeding; OF, open field; PF, PF-04995274; Sal, saline. Behavioral timeline created with BioRender.com.

[0027] FIG. 10A-FIG. 10M - Post-stress administration of D7 and D8 decrease fear expression, while D5 and D8 decrease hyponeophagia in adult female 129S6 / SvEv mice. (FIG. 10A) Experimental design. (FIG. 10B, C) Freezing was comparable across all groups during CFC exposure. (FIG. 10D, E) On day 1 of FST, all groups exhibited comparable immobility time. (FIG. 10F, G) On day 2 of FST all groups exhibited comparable immobility time. (FIG. 10H) In the MB task, all groups buried a comparable number of marbles. (FIG. 10I) In the NSF, all groups lost a comparable amount of weight during food restriction period. (FIG. 10J, K) PF-04995274, D5, and D8 (3 mg / kg) decreased the latency to feed in the open arena. (FIG. 10L) Latency to feed in the home cage was comparable across groups as was (FIG. 10M) the amount of food consumed in the home cage, n = 5 - 9 female mice per group; error bars represent ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001. CFC, contextual fear conditioning; FST, forced swim test; MB, marble burying; NSF, novelty suppressed feeding; OF, open field; HC, home cage; Sal, saline; PF, PF-04995274; min, minutes; sec, seconds; mg, milligram; kg, kilogram. Behavioral timeline created with BioRender.com.

[0028] FIG. 11A-FIG. 11M - 5-HT4R agonists do not improve stress induced maladaptive behavior when administered after stress in adult male C57BL / 6J mice. (FIG. 11A) Experimental timeline. (FIG. 11B, C) Freezing across groups is comparable during CFC exposure. (FIG. 11D, E) On day 1 of FST and (FIG. 11F, G) day 2 of FST, immobility time was comparable across all groups. (FIG. 11H) In the MB task, PF-04995274 (3 mg / kg) increased the number of marbles buried compared to saline. (FIG. 11l) Weight loss during food restriction in the NSF is comparable across groups. (FIG. 11J, K) During the NSF latency to eat in the open arena was comparable across groups. (FIG. 11L) Latency to feed in the home cage and (FIG. 11M) food consumed in the home cage was comparable across groups, n = 5 male mice per group; error bars represent ± SEM; ** p < 0.01. CFC, contextual fear conditioning; FST, forced swim test; MB, marble burying; NSF, novelty suppressed feeding; OF, open field; HC, home cage; Sal, saline; PF, PF-04995274; Prue, prucalopride; min, minutes; sec, seconds; mg, milligram; kg, kilogram. Behavioral timeline created with BioRender.com.

[0029] FIG. 12A-FIG. 12M - PF-04995274 and prucalopride improve perseverative behavior when administered after stress in adult female C57BL / 6J mice. (FIG. 12A) Experimental timeline. (FIG. 12B-C) Freezing across groups is comparable during CFC exposure. (FIG. 12D-E) On day 1 of FST and (FIG. 12F, G) day 2 of FST immobility timewas comparable across all groups. (FIG. 12H) In the MB task, PF-04995274 (3 mg / kg) and prucalopride (10 mg / kg) reduces the number of marbles buried compared to saline. (FIG. 121) Weight loss during food restriction in the NSF across drug groups is comparable to saline controls. (FIG. 12J, K) During the NSF latency to eat in the open arena across drug groups was comparable to saline. (FIG. 12L) PF-04995274 (10 mg / kg) increased latency to feed in the home cage but (FIG. 12M) food consumed in the home cage was comparable across groups, n = 4 - 9 female mice per group; error bars represent ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001. CFC, contextual fear conditioning; FST, forced swim test; MB, marble burying; NSF, novelty suppressed feeding; OF, open field; HC, home cage; Sal, saline; PF, PF- 04995274; Prue, prucalopride; min, minutes; sec, seconds; mg, milligram; kg, kilogram. Behavioral timeline created with BioRender.com.

[0030] FIG. 13A-FIG. 130 - Prophylactic RS-67,333 attenuates stress-induced depressive-like behavior and prophylactic RS-67,333, prucalopride, and PF-04995274 attenuate perseverative behavior in aged female 129S6 / SvEv mice. (FIG. 13A) Experimental timeline for aged (~17 month) female mice. (FIG. 13B, C) Freezing across CFC training is comparable across groups. (FIG. 13D, E) During CFC exposure freezing is comparable across groups. (FIG. 13F, G) On day 1 of FST immobility was comparable across groups. (FIG. 13H, I) On day 2 of FST, RS-67,333 (3 mg / kg) reduced immobility compared to saline. (FIG. 13J) In the MB task, RS-67,333, prucalopride, and PF-04995274 (all 3 mg / kg) reduced the number of marbles buried compared to saline. (FIG. 13K) During the food restriction phase of NSF, groups lost a comparable amount of. (FIG. 13L, M) Latency to feed in the open arena in the NSF was comparable across groups. (FIG. 13N) RS-67,333 (3 mg / kg) increased latency to feed in the home cage compared to saline but (FIG. 130) amount of food consumed was comparable across all groups. 5 - 9 female mice per group; error bars represent ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001. CFC, contextual fear conditioning; FST, forced swim test; MB, marble burying; NSF, novelty suppressed feeding; OF, open field; HC, home cage; Sal, saline; PF, PF-04995274; P, prucalopride; RS, RS-67,333; min, minutes; sec, seconds; mg, milligram; kg, kilogram. Behavioral timeline created with BioRender.com.

[0031] The figures herein are for illustrative purposes only and are not necessarily drawn to scale.DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTSGeneral Definitions

[0032] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of common terms and techniques in molecular biology may be found in Molecular Cloning: A Laboratory Manual, 2ndedition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4thedition (2012) (Green and Sambrook); Cunent Protocols in Molecular Biology (1987) (F.M. Ausubel et al. eds.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (1995) (M.J. MacPherson, B.D. Hames, and G.R. Taylor eds.): Antibodies, A Laboratory Manual (1988) (Harlow and Lane, eds.): Antibodies A Laboratory Manual, 2ndedition 2013 (E.A. Greenfield ed.); Animal Cell Culture (1987) (R.I. Freshney, ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); Singleton etal., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2ndedition (2011).

[0033] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.

[0034] The term “optional” or “optionally” means that the subsequent described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0035] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0036] The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of + / -10% or less, + / -5% or less, + / -1% or less, and + / -0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that thevalue to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.

[0037] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.

[0038] As used herein an “inverse agonist” refers to a compound that binds to a receptor, producing an effect opposite to that of an agonist and reducing the receptor's basal (constitutive) activity, stabilizing it in an inactive form. Many receptors are “constitutively active,” meaning they have a certain level of activity (receptor basal activity) even without an agonist present. An inverse agonist actively suppresses the receptor basal activity below the baseline, requiring the receptor to have some spontaneous activity to be effective. An inverse agonist stabilizes the receptor in a conformation that reduces its basal activity, leading to an effect opposite to what an agonist would produce.

[0039] As used herein an “antagonist” refers to a compound that blocks receptor activity without affecting the basal level. An antagonist blocks the binding of agonists, but does not affect the basal receptor activity.

[0040] As used herein a “partial agonist” refers to a compound that binds to a receptor but does not fully activate it, producing a response less than the maximum possible.

[0041] As used herein a “neutral antagonist” has zero efficacy, meaning it has no effect on its own but blocks both agonists and inverse agonists.

[0042] As used herein, “anxiety disorder” refers to a group of mental health conditions characterized by excessive fear, worry, or apprehension, often accompanied by behavioral and physiological symptoms such as restlessness, muscle tension, sleep disturbance, or hypervigilance. Examples include generalized anxiety disorder, panic disorder, and social anxiety disorder, as defined in standard psychiatric classification systems.

[0043] As used herein, “post-traumatic stress disorder (PTSD)” is a mental health condition that develops after experiencing or witnessing atraumatic event, such as a natural disaster, war, or violent crime. The symptoms of PTSD can vary widely, but typically include intrusive thoughts and flashbacks (e.g., reliving the traumatic event in vivid detail), avoidance (e.g., avoiding reminders of the trauma, such as people, places, or activities), negative changes inmood and cognition (e.g., feeling guilty, ashamed, or numb), hyperarousal (e.g., increased anxiety, irritability, and difficulty sleeping), dissociation (e.g., feeling detached from oneself or the world), and difficulty concentrating and making decisions. Treatment for PTSD typically involves a combination of psychotherapy (e.g., talk therapy, such as cognitive behavioral therapy (CBT)), medication (e.g., antidepressants and anti-anxiety medications), and support groups. PTSD may be diagnosed according to criteria set forth in the Diagnostic and Statistical Manual of Mental Disorders (DSM) or the International Classification of Diseases (ICD).

[0044] As used herein, “obsessive-compulsive disorder (OCD)” is a psychiatric disorder characterized by the presence of obsessions (intrusive, recurrent thoughts, urges, or images) and / or compulsions (repetitive behaviors or mental acts performed to reduce distress or prevent a feared event), which are time-consuming or cause clinically significant impairment.

[0045] As used herein, “autism spectrum disorder (ASD)” is a developmental disability caused by differences in the brain. People with ASD often have problems with social communication and interaction, and restricted or repetitive behaviors or interests. People with ASD may also have different ways of learning, moving, or paying attention.

[0046] As used herein, “phobia” refers to a type of anxiety disorder involving marked and persistent fear of a specific object, situation, or activity, leading to avoidance behavior and distress. Examples include specific phobias (e.g., fear of animals, heights, or blood) and social phobia (social anxiety disorder).

[0047] As used herein, “major depressive disorder” or “MDD” refers to a mood disorder characterized by one or more major depressive episodes, including symptoms such as depressed mood, diminished interest or pleasure, changes in appetite or weight, sleep disturbances, psychomotor changes, fatigue, feelings of worthlessness or guilt, diminished concentration, and recunent thoughts of death or suicide. Diagnosis may be made according to DSM or ICD criteria.

[0048] As used herein, “bipolar depression” refers to the depressive phase of bipolar disorder, a mood disorder characterized by episodes of depression alternating with episodes of mania or hypomania. The depressive episodes may resemble those of MDD but occur within the context of a bipolar illness.

[0049] As used herein, “schizophrenia” refers to a chronic and severe psychiatric disorder involving disturbances in thought processes, perceptions, emotional responsiveness, and behavior. Symptoms can include delusions, hallucinations, disorganized speech, grosslydisorganized or catatonic behavior, and negative symptoms such as diminished emotional expression or avolition.

[0050] As used herein, “Huntington’s disease” refers to a progressive neurodegenerative disorder caused by a genetic mutation in the huntingtin gene, characterized clinically by motor dysfunction (e.g., chorea, dystonia), cognitive decline, and psychiatric disturbances.

[0051] As used herein, “Parkinson’s disease” refers to a progressive neurodegenerative disorder of the central nervous system, primarily affecting movement. Hallmark motor symptoms include resting tremor, rigidity, bradykinesia, and postural instability, and may be accompanied by non-motor symptoms such as mood changes, sleep disturbances, and autonomic dysfunction.

[0052] As used herein, “eating disorder” refers to a psychiatric condition characterized by persistent disturbances in eating behavior and related thoughts or emotions that significantly impair physical health or psychosocial functioning. Examples include anorexia nervosa, bulimia nervosa, and binge-eating disorder.

[0053] As used herein, “treatment-resistant depression” or “TRD” refers to a major depressive disorder that has failed to respond adequately to at least one, and typically two or more, prior antidepressant treatments of adequate dose and duration.

[0054] Various embodiments are described hereinafter. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment(s). Reference throughout this specification to “one embodiment”, “an embodiment,” “an example embodiment,” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” or “an example embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within thescope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0055] All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference.OVERVIEW

[0056] Embodiments disclosed herein provide novel 5-HT4 receptor inverse agonists or antagonists useful for protecting against stress-induced maladaptive behavior. Stress is a significant risk factor for developing psychiatric disorders, including major depressive disorder (MDD). Studies have shown that serotonin type 4 receptor (5-HT4Rs) agonists hold promise as novel rapid-acting treatments of mood disorders. However, a lack of selectivity and numerous side effects have been limiting factors for their clinical use. Here, Applicants developed and characterized novel-composition 5-HT4R compounds in mouse models of stress.

[0057] Eight 5-HT4R-targeting compounds were designed and synthesized based on PF- 04995274, a high-affinity 5-HT4R ligand reported to be a partial agonist. G-protein and cAMP assays were utilized to characterize molecular activity. Saline, PF-04995274, or novel compounds were administered before or after a stressor in both male and female mice. Drug effects were assayed using behavioral tests. Patch clamp electrophysiology was used to determine the effect of drug administration on glutamatergic activity in hippocampal comu ammonis 3 (CA3).

[0058] Prophylactic administration of D5, D7, or D8 was effective at reducing stress- induced maladaptive behaviors in adult mice. When administered following learned helplessness, D7 reduced behavioral despair and increased c-Fos in the dentate gyrus and CA3. Only D5 and D7 attenuated large-amplitude AMP A receptor-mediated bursts in ventral CA3 (vCA3). In aged males, prophylactic administration of D7 was effective in reducing depressive- like behavior.

[0059] These results characterize three new 5-HT4R-targeting compounds for stress- induced psychiatric disease. These drugs have the potential to address unmet needs in adult andaged patients with stress-induced psychiatric illness. Future work will characterize their mechanism of action with the goal of clinical development.5-HT4R-targeting compounds

[0060] In some embodiments, the novel 5-HT4 receptor inverse agonist of the present invention has the following structure:

[0061] Wherein X is O, S, SO, SO2or Se, wherein Ar is fused heteroaromatic and wherein R is heterocyclic, bridged cyclic, spiro cyclic or fused cyclic.

[0062] In some embodiments, Ar is

[0063] In some embodiments, R is

[0064] In some embodiments, the novel 5-HT4 receptor inverse agonist of the present invention is

[0065]

[0066] In some embodiments, the novel 5-HT4 receptor inverse agonist or antagonist is a compound in Table 1.Table 1. Compound Chemical InformationMethods of Treatment

[0067] The present invention relates to methods of treating psychiatric and neurological disorders in human subjects by administering compounds that demonstrate efficacy in specific preclinical behavioral mouse models (see, e.g., Porsolt RD. Animal models of depression: utility for transgenic research. Rev Neurosci. 2000;l l(l):53-58). In example embodiments, human subjects in need thereof are treated with one or more of the disclosed compositions. In example embodiments, subjects are treated with a dose of a compound in the range of 0.15-50 mg / day. In example embodiments, about 10-15 mg / day are administered. In example embodiments, 3-10 mg / kg / day are administered. In example embodiments, subjects are treated for 1-7 days or longer.Indications Treated

[0068] In example embodiments, rodent assays have predictive validity for human psychiatric conditions, and improvements in these assays correlate with established clinical efficacy of marketed drugs. Accordingly, the invention provides therapeutic methods for treating symptoms associated with anxiety disorders, mood disorders, obsessive-compulsive spectrum disorders, autism spectrum disorder, schizophrenia, and related conditions, by administering a pharmaceutically effective amount of a compound described herein that improves behavioral performance in one or more of the following mouse models: learned fear, behavioral despair, perseverative behavior, hyponeophagia, and learned helplessness.

[0069] In example embodiments, the compounds disclosed herein are used to treat subj ects susceptible to stress-induced maladaptive behavior, such as, but not limited to, subjects with PTSD, an anxiety disorder, a phobia, major depressive disorder, bipolar depression, obsessive- compulsive disorder (OCD), (ASD), schizophrenia, Huntington’s disease, Parkinson’s disease, an eating disorder, or treatment-resistant depression. In example embodiments, the compounds disclosed herein are used to prevent the onset of symptoms including, but not limited to depression, anxiety, anorexia, compulsiveness, freezing, intrusive thoughts and flashbacks, avoidance, negative changes in mood and cognition, hyperarousal, dissociation, difficulty concentrating and making decisions, and memory loss.

[0070] Learned fear in rodents is typically evaluated using classical fear conditioning models, in which a neutral stimulus (e.g., auditory tone) is paired with an aversive stimulus (e.g., mild foot shock). Compounds that reduce conditioned fear responses or facilitate extinction in these models have predictive validity for medications used to treat anxietydisorders in humans. Selective serotonin reuptake inhibitors (SSRIs) such as citalopram reduce conditioned fear expression in mice (Burghardt NS, Sullivan GM, McEwen BS, Gorman JM, LeDoux JE. The selective serotonin reuptake inhibitor citalopram increases fear after acute treatment but reduces fear with chronic treatment: a comparison with tianeptine. Biol Psychiatry. 2004;55(12): 1171-1178). Citalopram (marketed as Celexa) is approved by the U.S. Food and Drug Administration (FDA) for the treatment of Major Depressive Disorder (MDD) in adults. Additionally, SSRI administration (citalopram or fluoxetine) to rats increases conditioned fear expression (Burghardt NS, Bush DE, McEwen BS, LeDoux JE. Acute selective serotonin reuptake inhibitors increase conditioned fear expression: blockade with a 5-HT(2C) receptor antagonist. Biol Psychiatry. 2007;62(10):ll 11-1118). Fluoxetine (brand names include Prozac®, Sarafem®, and others) is a selective serotonin reuptake inhibitor (SSRI) antidepressant. Thus, compounds active in learned fear assays in mice are suitable for treating anxiety -related disorders in humans.

[0071] Behavioral despair is assessed in rodents using the forced swim test (FST) or tail suspension test (TST), where increased immobility reflects a depressive-like phenotype (see, e.g., Yankelevitch-Yahav R, Franko M, Huly A, Doron R. The forced swim test as a model of depressive-like behavior. J Vis Exp. 2015;(97):52587). Compounds that decrease immobility are predictive of antidepressant effects. For example, ketamine produces a rapid and robust reduction of immobility in mice and has been shown to exert rapid antidepressant effects in patients with treatment-resistant depression (Autry AE, Adachi M, Nosyreva E, et al. NMD A receptor blockade at rest triggers rapid behavioural antidepressant responses. Nature. 2011;475(7354):91-95). Thus, compounds improving performance in behavioral despair assays are useful for treating MDD, bipolar depression, and stress-related depression.

[0072] In addition to the FST and TST, behavioral deficits associated with the learned helplessness paradigm are also sensitive to a broad spectrum of antidepressant drugs (Leshner Al, Remler H, Biegon A, Samuel D. Desmethylimipramine (DMI) counteracts learned helplessness in rats. Psychopharmacology (Berl). 1979;66(2):207-208; Martin P, Soubrie P, Puech AJ. Reversal of helpless behavior by serotonin uptake blockers in rats. Psychopharmacology (Berl). 1990:101(3):403-407; Sherman AD, Petty F. Additivity of neurochemical changes in learned helplessness and imipramine. Behav Neural Biol 1982;35(4):344-353). Learned helplessness is induced by exposing rodents to uncontrollable stressors, leading to deficits in escape or avoidance behaviors. The learned helplessnessparadigm incorporates the motivational and emotional aspects of stress-induced depression. Specifically, in this paradigm animals subjected to repeated inescapable uncontrollable shock demonstrate escape deficits when tested at a later time (Overmier and Seligman 1967). These deficits can be reversed by a variety of antidepressant drugs (see, Blendy J A. The role of CREB in depression and antidepressant treatment. Biol Psychiatry. 2006;59(12):1144-1150). Thus, compounds that alleviate learned helplessness are useful for treating major depression, treatment-resistant depression, PTSD, and stress-related disorders.

[0073] Perseverative or stereotyped behavior in rodents may be measured through excessive grooming, marble burying, or deficits in set-shifting tasks. Compounds that reduce perseveration or restore cognitive flexibility are relevant to disorders characterized by compulsivity. For example, fluoxetine reduces marble-burying behaviors in rodents and is effective in obsessive-compulsive disorder (Greene-Schloesser DM, Van der Zee EA, Sheppard DK, et al. Predictive validity of a non-induced mouse model of compulsive-like behavior. Behav Brain Res. 2011;221(l):55-62). Thus, compounds that reduce perseverative behavior are useful in treating OCD, which is common in ASD, schizophrenia, and related disorders.

[0074] The use of hyponeophagia, such as the novelty-suppressed feeding test, have become effective paradigms for testing treatment with drugs such as anxiolytics and antidepressants. Compounds that reduce feeding latency in novelty-suppressed feeding models are predictive of medications used to treat anxiety disorders in humans (see, e.g., Samuels, B.A., Hen, R. (2011). Novelty-Suppressed Feeding in the Mouse. Tn: Gould, T. (eds) Mood and Anxiety Related Phenotypes in Mice. Neuromethods, vol 63. Humana Press). Therefore, compounds that improve hyponeophagia are suitable for treating anxiety disorders and eating- related disorders with anxiety components.

[0075] Collectively, these models represent validated translational assays. Improvements in rodent learned fear, behavioral despair, perseverative behavior, hyponeophagia, or learned helplessness are predictive of therapeutic benefit in human psychiatric disorders, including but not limited to anxiety disorders, PTSD, generalized anxiety disorder (GAD), panic disorder, phobias, mood disorders (e.g., major depression, bipolar depression, treatment-resistant depression), obsessive-compulsive disorder, autism spectrum disorder, schizophrenia and related executive dysfunction, and eating disorders with anxiety features.

[0076] Accordingly, the invention provides methods of treating these disorders by administering a compound as described herein that has demonstrated improvement in one or more of the above-described rodent models.

[0077] In example embodiments, aged or adult humans are treated with a compound described herein. Comparing ages between mice and humans requires considering different aging stages, as there is not a simple linear conversion for their entire lifespans. For instance, a 3-6-month-old mature mouse is roughly equivalent to a 20-30-year-old human, a 10-14- month-old mouse correlates to a 38-47-year-old human, and an 18-24-month-old mouse corresponds to a 56-69-y ear-old human. Some researchers consider a mouse to be an adult at 8-12 weeks. The end of reproductive function (a mouse equivalent of menopause) occurs around 15 months of age in mice, which is comparable to 51 years old in humans. As used herein ‘"adult” refers to a human 18 or older. As used herein “aged” refers to a human older than 60 years old. As used herein, experiments conducted with 9-week-old mice are considered adult and experiments conducted with 17-month-old are considered aged.

[0078] In example embodiments, subjects are treated with a compound before a stress. In example embodiments, subjects are treated before experiencing symptoms induced by a stress. In example embodiments, subjects are treated immediately after a stress.

[0079] In example embodiments, males or females are treated. In examples, males and females are treated differently according to embodiments herein. In example embodiments, specific compounds are more effective in males than females. In example embodiments, specific compounds are more effective in females than males. In example embodiments, specific compounds are equally effective in males and females.

[0080] In example embodiments, a female subject is treated with D5 or D8 after a stress to prevent an anxiety or eating disorder symptom. In example embodiments, a male subject is treated with D7 or D8 after a stress to prevent depression. In example embodiments, a male subject is treated with D7 to prevent a PTSD, anxiety, or phobia symptom. In example embodiments, a male subject is treated with D5, D7, or D8 to prevent major depressive disorder or bipolar depression. In example embodiments, a male subject is treated with D5 or D7 to prevent a compulsive symptom associated with OCD, ASD, schizophrenia, Huntington’s disease, or Parkinson’s disease. In example embodiments, a male subject is treated with D5 or D8 to prevent anxiety or an eating disorder symptom. In example embodiments, an aged male subject is treated with D7 to prevent major depressive disorder or bipolar depression. Inexample embodiments, a female subject is treated with D7 or D8 to prevent a compulsive symptom associated with OCD, ASD, schizophrenia, Huntington’s disease, or Parkinson’s disease. In example embodiments, a female subject is treated with D5 to prevent anxiety or an eating disorder symptom.Combination Treatments

[0081] In example embodiments, one or more of the compounds disclosed herein are administered in combination with other drugs or treatments associated with anxiety or stress- induced maladaptive behaviors. Non-limiting examples include benzodiazepines, antipsychotics, selective serotonin reuptake inhibitors (SSRIs), ketamine, mood stabilizers (e.g., lithium and certain anticonvulsant medications, such as valproic acid and lamotrigine), or electroconvulsive therapy (ECT).

[0082] Further embodiments are illustrated in the following Examples which are given for illustrative purposes only and are not intended to limit the scope of the invention.EXAMPLESExample 1 - 5-HT4R Agonist results

[0083] Applicants developed 8 novel compounds at Columbia University’s Organic Chemistry Collaborative Center to target 5-HT4R. Three compounds were selected for in vivo testing based on cAMP inhibition and G protein recruitment assays. To determine if novel drugs were prophylactic against stress, Applicants administered three of these compounds - D5, D7, or D8 - to male and female mice prior to acute stress. As stressful events can be unpredictable, Applicants also administered compounds following stress. Applicants then utilized slice electrophysiology and quantification of the immediate early gene c-Fos (40) to explore how novel compounds influenced HPC activity. Overall, by utilizing a diverse set of behavioral tasks, Applicants identify novel compounds that improve stress-induced maladaptive behaviors. Altogether, the data suggest these novel drugs show promise as future treatments for psychiatric disorders.Synthesis of novel 5-HT4R-tareeting compounds

[0084] Based on the structure of the purported 5-HT4R partial agonist, PF-04995274, Applicants designed and synthesized novel chemical entities likely to modulate 5-HT4R. PF- 04995274 has an aromatic and an aliphatic domain. The aromatic domain is notable for a chiral substituent resolved to the R-configuration. The substituent and the chiral configuration areimportant for activity and thus this domain was left unaltered in the library except DI. The aliphatic domain is notable for a central piperidine ring and distal oxane ring, each regiochemically positioned to render this domain achiral.

[0085] Applicants varied the aliphatic domain replacing the piperidine ring with no ring (D3), a nitrogen-containing bicycloalkane ring system (D2, D4, and D6), or with a nitrogencontaining spiro ring system (D5). The bicycloalkane ring system represents three classes: D2 based on a 3-aza-bicyclo[3,1,1]hept-6-yl moiety, D4 based on a 3-aza-bicyclo[3,2,1]oct-8-yl moiety , and D6 from a 3-aza-bicyclo[3,l,0]hex-6-yl moiety with the nitrogen constituent and site of attachment to the aromatic domain positioned to render the aliphatic domain achiral. The spiro analog D5 entailed diastereomers which were tested without resolution in preliminary assessment of this synthetically difficult class of modification.

[0086] The oxane ring was replaced by a thiane ring to yield two complementary pairs of analogs. Thus, thiane D8 complements the oxane-based PF-04995274 and thiane D7 complements oxane D6. Of note, the spiro analog D5 was prepared as the thiane, enabling comparison to D7. DI, with a modified aromatic domain, provided a control for evaluation of D2.Novel compounds exhibit variable affinity for 5-HT4RS and function as weak or inverse agonists

[0087] The 5-HT4R is reported to have constitutive activation of Gsand stimulation of adenylate cyclase (41 ,42). To assess the ability of the receptor to increase cAMP in the presence and absence of agonist, Applicants employed a CRE gene reporter assay sensitive to increases in cAMP (Figure 8A). Transfecting HEK293 cells with increasing amounts of cDNA encoding 5-HT4R increased luminescence signal, consistent with constitutive activity; addition of 10 μM 5-HT led to a small increase (Figure 8B). The impact of 5-HT was smaller than that of increasing receptor expression, and there was substantial overlap in points with and without 5- HT. This variability extended to the measurements of acute changes in cAMP levels using a BRET-based CAMYEL cAMP sensor (Figure 8C). While Applicants observed a consistent increase in cAMP with 5-HT, the dynamic range of the raw BRET change varied across replicates. Normalizing to 5-HT, Applicants consistently saw partial agonism by prucalopride, but RS-67,333, which has been reported to be a partial agonist (43), gave variable responses (Figure 8D-J). PF-04995274, which is purportedly a partial agonist, behaved as a neutral antagonist or a weak inverse agonist. These results are in sharp contrast to previous use of this assay for the study of other GPCRs, (44,45) where Applicants observe more consistent results.

[0088] To more consistently measure potency and efficacy of the novel ligands Applicants created a 5-HT4R fused at its C terminus with Nanoluc® luciferase and set up a direct recruitment assay with miniGs fused to mVenus. MiniGs is a Gsprotein engineered to reside in the cytoplasm until recruited to an activated receptor at the membrane and measures a 1:1 interaction between receptor and activated G protein (Figure 2A) (46). This led to highly reproducible results, with prucalopride behaving as a partial agonist with Emax~80% of 5-HT, and RS-67,333 a more potent partial agonist with Emax~25% of 5-HT. PF-04995274 still behaved as an inverse agonist, lowering the Emaxto ~20% below vehicle baseline (Figure 2B). While D4 acted as a low potency weak partial agonist, DI, D2 and D3, and D6 all had low inverse agonist activity like PF-04995274. In contrast, D5, D7, and D8 were without apparent effect (Figure 2C-D) making it difficult to determine whether compounds were acting at the receptor or were inert.

[0089] To better elucidate the activity of these compounds, Applicants conducted an inhibition mode assay, competing with an EC80 concentration of the full agonist 5-HT (Figure 2E). Applicants obtained reliable inhibition curves for RS-67,333 that plateaued at about 25%, consistent with its action as a partial agonist (Figure 2F). PF-04995274 again behaved as an inverse agonist. D3 proved to be inert, having lost affinity for 5-HT4, and D4 acted as a low potency weak partial agonist. D1, D2, D5 and D7 all inhibited completely, consistent with neutral antagonism, whereas D6 and D8 behaved as inverse agonists like the parental compound (Figure 2G-H). As representative compounds for subsequent studies, Applicants chose to test D5 and D7, which acted as neutral antagonists, as well as D8, which exhibited inverse agonist activity. All three compounds had lower affinity than PF-04995274 (Table 2).Table 2. cAMP EC80A prophylactic injection of a novel 5-HT4R compound protects against stress

[0090] Previously, Applicants have shown that a single prophylactic administration of prucalopride or PF -04995274 attenuates learned fear and behavioral despair in male mice (18). Here, Applicants sought to determine if a novel 5-HT4R compound was also effective at preventing maladaptive behaviors. Male mice were administered saline, PF-04995274 (10 mg / kg), or D5, D7, or D8 (3 or 10 mg / kg) (Figure 3A). One week later, mice were administered 3-shock CFC. Drug administration did not alter freezing during CFC training (Figure 3B-C). Five days later, mice were returned to the context for a retrieval session. Prophylactic PF- 04995274, as previously demonstrated (18), and D7 (3 mg / kg) attenuated learned fear (Figure 3D-E).

[0091] On FST day 2, but not day 1, PF-04995274 reduced immobility time, consistent with previous findings (18). D5 (3 and 10 mg / kg), D7 (3 mg / kg), and D8 (10 mg / kg) also reduced immobility time (Figure 3F-I). In the marble burying (MB) task, D5 and D7 reduced marble burying, indicating a reduction in perseverative behavior (Figure 3J). In the novelty suppressed feeding (NSF) task, PF-04995274, D5, and D8 reduced the latency to feed in the open field (OF) arena (Figure 3K-O). These data highlight the prophylactic efficacy of D5, D7, or D8 to impact stress-induced maladaptive behaviors.

[0092] Female mice were administered saline, PF-04995274 (3 mg / kg), or D5, D7, or D8 (3 mg / kg) (Figure 9A). Drug administration did not alter freezing during CFC training orcontext re-exposure (Figure 9B-E). On FST day 1, D8 increased immobility time in female mice (Figure 9F-G). On FST day 2, all mice exhibited comparable immobility time (Figure 9H-I). In the MB task, D7 and D8 significantly reduced the number of marbles buried (Figure 9J). In the NSF task, PF-04995274 and D5 reduced the latency to feed in the OF (Figure 3K- O). These data highlight the prophylactic efficacy of D7 and D8 for perseverative behavior and D5 for hyponeophagia in female mice.Novel 5-HT4R compounds are effective when administered, following stress

[0093] Applicants then sought to determine efficacy of these compounds when administered following acute stress (Figure 4A). Five minutes following 3-shock CFC, mice were administered saline, PF-04995274 (10 mg / kg), or D5, D7, or D8 (3 or 10 mg / kg). Drug administration did not alter freezing during CFC training (data not shown) or context reexposure (Figure 4B-C). During FST day 1, D5, D7, and D8 increased immobility time (Figure 4D-E). During FST day 2, PF -04995274, D7 (10 mg / kg), and D8 (10 mg / kg) decreased immobility time (Figure 4F-G). Drug administration did not alter behavior in the MB (Figure 4H) or NSF tasks (Figure 4I-4M).

[0094] In female mice, five minutes following 3-shock CFC, mice were administered saline, PF-04995274 (10 mg / kg), or D5, D7, or D8 (3 or 10 mg / kg) (Figure 10A). Drug administration did not alter freezing during CFC training (data not shown) or context reexposure (Figure 10B-C). During the FST and MB, all mice exhibited comparable behavior (Figure 10D-H). In the NSF, PF-04995274, D5, and D8 decreased the latency to feed in the OF arena (Figure 10I-M).5-HT4R compounds decrease perseverative behavior in C57BL / 6J female, but not male mice

[0095] To extend these findings to other mouse strains, Applicants administered PF- 04995274 or prucalopride (3 or 10 mg / kg) following stress in C57BL / 6J mice, a more resilient strain (47-49). Neither drug improved stress-induced maladaptive behavior in male mice; unexpectedly PF-04995274 increased perseverative behavior in the MB task (Figure 11). In C57BL / 6J female mice, PF-04995274 (3 mg / kg) and prucalopride (10 mg / kg) decreased the number of marbles buried (Figure 12). Together, the findings indicate that acute administration of a 5-HT4R agonist does not significantly impact behavior in a mouse line resilient to stress.Novel 5-HT4R compounds reduce AMPA receptor-driven synaptic bursts in CA3

[0096] Applicants have previously shown that prophylactic administration of (R,S)- ketamine, (2R,6R)-hydroxynorketamine, prucalopride, and fluoroethylnormemantine reducelarge AMP A receptor (AMPAR)-mediated excitatory postsynaptic currents (EPSCs) in CA3 (18,50,51) To determine if the new compounds share this effect, Applicants administered saline, D5, D7, or D8. One week later, mice were euthanized, and Applicants conducted wholecell voltage clamp recordings of spontaneous EPSCs in CA3 (Figure 5A). D5 and D7 reduced mean EPSC amplitude (-46.00 ± 2.03 pA) (Figure 5B-D, F) and frequency (Figure 5H) compared to saline. Conversely, D8 increased the mean amplitude (Figure 5E, F) and mean burst amplitude compared to saline (-352.24 ± 16.69 pA) (Figure 5G). These data indicate that although all three drugs target 5-HT4Rs and reduce behavioral despair, their effects on HPC signaling diverge.Novel compound D7 is antidepressant when administered, foliowins learned helplessness

[0097] To extend the capacity of these compounds for other stressors in male mice, Applicants next utilized learned helplessness (LH) (52-55). Five minutes following LH training (Figure 6A-B), mice were administered saline, PF-04995274, or D5, D7, or D8 (10 mg / kg). Five days later, mice underwent a shock escape protocol. There were no group differences in total session latency or latency to escape (Figure 6C-E).

[0098] On FST day 1, all groups exhibited comparable immobility time (Figure 6F-G). On FST day 2, PF-04995274 and D7 reduced immobility time (Figure 6H-I). Sixty minutes following the FST, animals were euthanized, and brains were processed for neural activity using an antibody against c-Fos (Figure 6J). Administration of all drugs increased c-Fos expression in the DG (Figure 6K), while PF-04995274, D5, and D7 increased c-Fos expression in CA3 (Figure 6L). Drug administration did not alter c-Fos expression in CA1 (Figure 6M). Together, this data suggests these drugs produce long-lasting neural changes in the DG and CA3, regions Applicants have previously shown are modulated by prophylactic (R,S)-ketamine (51,56).Prophylactic D7 attenuates stress-induced behavioral despair in aged male mice

[0099] Applicants previously found that the (R,S)-ketamine is not effective at improving stress-induced behavioral despair in aged mice (57,58). To determine if these compounds are effective in aged mice, Applicants administered RS-67,333, prucalopride, PF-04995274, or D7 (3 or 10 mg / kg) one week prior to CFC (Figure 7 A). In male mice, drug administration did not influence freezing behavior during CFC training or retrieval (Figure 7B-E). On FST day 1, all groups exhibited comparable immobility (Figure 7F-G). On FST day 2, RS-67,333, PF-04995274, and D7 reduced immobility time (Figure 7H-I). There was no effect of drug administration in the MB or NSF assays (Figure 7J-O).

[0100] In female mice, there was no effect of drug administration on freezing during CFC training or retrieval or during FST day 1 (Figure 13A-G). However, on FST day 2, RS-67,333 decreased immobility time (Figure 13H-I). In the MB assay, RS-67,333, prucalopride, and PF- 04995274 decreased the number of marbles buried (Figure 13J). In the NSF task, drug administration did not alter behavior in the OF, but RS-67,333 increased the latency to feed in the home cage (Figure 13K-O). Together these experiments illustrate efficacy of RS-67,333, PF-04995274, and D7 to improve stress-induced behavioral despair behavior in aged mice.Example 2 - Discussion

[0101] Applicants developed 8 novel 5-HT4R compounds based on the compound PF- 04995274 for application to stress-induced psychiatric disease. Applicants report that PF- 04995274 behaved as an inverse agonist despite previously being reported to be a partial agonist (59,60). Results of a competing EC80 serotonin inhibition assay established that novel compounds D5 and D7 function as neutral antagonists, whereas D8 functions as an inverse agonist. Behavioral testing found that in male mice, prophylactic D5, D7, and D8 improved stress-induced behavioral despair; when administered following stress, D7 and D8 were also effective. In female mice, prophylactic D5 improved hyponeophagia while D7 and D8 reduced perseverative behavior; after stress, D5 and D8 were again effective at improving hyponeophagia. A single prophylactic administration of D5 or D7, but not D8, reduced spontaneous EPSCs in CA3 illustrating a potential mechanism by which these drugs may attenuate stress-induced maladaptive behavior. Notably, prophylactic administration of D7 was also effective at reducing behavioral despair in aged male mice. Applicants primarily utilized 129S6 / SvEv mice in these experiments as they are more susceptible to stress (47-49,61), but tested prucalopride and PF-04995274 in C57BL / 6J mice. PF-04995274 or prucalopride improved perseverative behavior in female C57BL / 6J mice but were not effective in males.

[0102] It is widely accepted that 5-HT4RS stimulate cAMP production (62,63), indicating an interaction with the Gsprotein pathway (64,65). Here, Applicants identified high levels of constitutive activity of 5HT4R associated with Gsactivation. Somewhat unexpectedly, in the signaling assays in heterologous cells, PF-04995274 and other effective novel compounds behaved as inverse agonists or neutral antagonists - decreasing 5HT-induced activity below orto baseline levels. Within the HPC, cAMP signaling pathways are crucial for long term potentiation and, ultimately, memory consolidation (66,67). In a mouse model of chronic unpredictable stress (CUS), increased behavioral despair was accompanied by a downregulation of cAMP signaling (68) while increasing cAMP signaling improved behavioral responses to stress (69). Although Applicants could not characterize in vivo activity of 5- HT4Rs, inverse agonism or neutral antagonism, leading to a reduction of receptor activity in neural circuits, perhaps by competition with endogenous 5HT, may counterintuitively underlie drug efficacy.

[0103] It is also possible that signaling pathways other than Gsand cAMP may play a critical role in the behavioral effects Applicants have observed. Recent work has documented 5-HT4R interactions with other G-proteins, including Gi, (70,71). In addition, 5-HT4RS also recruit arrestin (72) and activate the extracellular signal-regulated kinase (ERK) pathway through interaction with the non-receptor tyrosine kinase Src, independent of G-protein signaling (73-75). In addition to influencing cellular differentiation and growth (76,77), activation of the ERK pathway is implicated in synaptic plasticity. Pharmacologically inhibiting ERK signaling in CA1 of the HPC impairs LTP (78) while inhibition in the lateral amygdala impairs LTP and long-term memory of Pavlovian fear conditioning (79,80). Future work characterizing how PF-04995274 and novel compounds alter Src or Girelated pathways may be informative.

[0104] Applicants previously described an electrophysiological signature following prophylactic administration of several compounds (e.g., (R,S)-ketamine) characterized by a reduction of large AMPAR-driven synaptic bursts in CA3 characteristic of sharp wave ripples (SPW-Rs). These SPW-Rs are notable for their role in memory and can contribute emotional salience to events (81-83). Elevated 5-HT can quell SPW-Rs (84). Here, prophylactic D5 and D7, which acted at Gsas neutral antagonists, reduced mean amplitude of EPSCs and mean frequency of large AMP AR bursts in CA3, suggesting these may reduce SPW-Rs. Although considered a NMD A antagonist, recent work suggests (R,S)-ketamine and its metabolites also influence AMPAR-mediated signaling, which may contribute to antidepressant efficacy (18,85-88). It is possible that inverse agonists may impact AMP AR signaling by influencing cAMP-PKA or other ERK signaling pathways. Some work suggests that modulators of AMP AR signaling increase BDNF, which underlies antidepressant efficacy (89,90), but recentwork failed to show a robust relationship between 5-HT4R activity and BDNF (91). Future work will elucidate if these novel compounds similarly alter activity in other brain regions.

[0105] Following a test of behavioral despair, PF-04995274, D5, D7, and D8 increased neural activity in the DG, while PF-04995274, D5 and D7 increased activity in CA3 as measured by increased c-Fos+cells. In the HPC, 5-HT4Rs are highly expressed on GABAergic neurons (92,93). Dampening activation of 5-HT4Rs via inverse agonism on these neurons may ultimately disinhibit neural circuits underlying depressive-like behavior. Previous work has shown that 5-HT4R activation can modulate GABAergic activity within the HPC and other regions in a complex, bidirectional manner, with receptor activation either increasing or decreasing GABA activity (94-96) Although the current experiments were not designed to monitor GABAergic release, future work will explore how these compounds influence neurotransmission within this region.

[0106] The potential for 5-HT4R compounds to improve behavior in aged mice is promising. In older populations, treatment options for late-life depression (LLD) are lacking (97-99) as treatments such as SSRIs and esketamine show reduced efficacy (100-103). As LLD is a modifiable risk factor for cognitive impairment, (104-106) new therapeutics are needed. Unlike other serotonin receptors that exhibit reduced binding with age, 5-HT4R expression is stable (107-109) making them a promising therapeutic target for LLD. Our group previously found that prophylactic (R,S)-ketamine is not effective in aged mice (57,58), but here Applicants report that RS-67,333, PF-04995274, and D7 are effective at reducing behavioral despair. 5-HT4R agonists may also improve cognitive function directly. Previous work found RS-67,333 improved object recognition in aged rats (110) and clinically, a subchronic, low-dose of prucalopride improved cognitive function in an image recall task (37) and enhanced connectivity between the central executive network and cingulate cortex (38). Future work will be needed to determine if administration of the neutral antagonist D7 may also improve cognitive functioning.

[0107] Applicants identified sex differences in how 5-HT4targeting compounds influence behavior. In male mice, prophylactic administration of novel compounds improved behavioral despair, hyponeophagia and perseverative behavior, but in females, efficacy is observed in reductions in hyponeophagia and perseverative behavior (Table 3). In males, compounds administered after stress reduce behavioral despair; in females compounds reduce hyponeophagia (Table 4). Although sex differences in 5-HT signaling have been documented(111-113) differences in 5-HT4RS are underexplored. Post-mortem studies did not identify sex differences in 5-HT4R density (25,114) however, PET imaging identified lower 5-HT4R binding in the HPC (115) and limbic regions (107) of women compared to men. Hormone status may also influence 5-HT4R activity; in healthy men, testosterone level was negatively correlated with 5-HT4R binding, particularly in the HPC (116). In women, oral contraception use resulted in a 12% reduction in global 5-HT4R binding compared to controls (117). If hormone status decreases binding due to a decline in expression of the 5-HT4R or altered serotoninergic tone remains to be elucidated.

[0108] 5-HT neurotransmission, and 5-HT4R activity, has been implicated in feeding behaviors. Increased 5-HT tone is anorexiogenic (118,119). Stimulating 5-HT4RS in the nucleus accumbens induces hypophagia, conversely, inactivating 5-HT4RS in this region with an antagonist, increases feeding (120). 5-HT4R knock-out (KO) mice do not exhibit stress- induced hypophagia (121). Given their role in feeding behaviors, 5-HT4RS have been implicated in anorexia nervosa restricting-type (AN) (122,123), a disease marked by restricted food intake (124-127). Although Applicants did not test these compounds in a model of AN, Applicants find that following a brief period of food restriction (12 hours) administration of inverse agonists PF-04995274 or D8, as well as the neutral antagonist D5, improved feeding behavior in the NSF in both sexes. Applicants hypothesize that by blunting 5-HT4R activity, these compounds may protect against stress-induced hyponeophagia Future testing using an activity-based anorexia model (128) will provide valuable insight into efficacy of inverse agonists to improve behavior in AN.

[0109] In untreated patients with MDD, there is a 7% reduction in global 5-HT4R binding compared to healthy controls (129). There is also anegative correlation between global 5-HT4R binding and anxiety symptoms in patients with MDD, with more severe anxiety symptoms associated with lower 5-HT4R binding (130). In the United States, only two 5-HT4R agonists are FDA-approved, tegaserod and prucalopride, for gastrointestinal disorders; (35,36,131) no 5-HT4R therapeutics are approved for psychiatric use. As Applicants have demonstrated that a single administration of a 5-HT4R inverse agonist is effective, future clinical work might utilize acute or subchronic administration, reducing the likelihood of peripheral effects while maintaining efficacy. Additional positive findings from future metabolic and safety tests will allow these compounds to move into clinical trials and may ultimately expand treatment options for those living with psychiatric illness.Table 3. Prophylactic SummaryTable 4. Post stress summaryExample 3 - Methods And MaterialsMice

[0110] Male and female 129S6 / SvEvTac mice were purchased from Taconic (Hudson, NY) at 7 - 8 weeks of age or bred in house. Male and female C57BL / 6J were bred in house. Experiments conducted with adult (9- week-old) or aged (17-month-old) mice. Mice were housed 4-5 / cage in a 12-h (06:00 - 18:00) light-dark colony room at 22°C with food and water available ad libitum. Behavioral tests were conducted during the light phase. All procedures were conducted in accordance with the National Institutes of Health (NIH) regulations and approved by the Institutional Animal Care and Use Committees (IACUCS) of New York State Psychiatric Institute (NYSPI) and Columbia University Irving Medical Center (CUIMC).Drugs

[0111] All drug injections were administered intraperitoneally (i.p.) in volumes of 0.1 cc per 10 mg body weight. PF-04995274 (Sigma, Catalog No 1331782-27-4, St. Lois, MO), prucalopride (Sigma, Catalog No 179474-8108, St. Lois MO), and RS-67,333 (Sigma, Catalog No 168986-60-5, St. Lois, MO) were dissolved in 0.5 mL dimethyl sulfoxide (DMSO) and saline (BioLogics, Model 3000, Manassas, VA). Novel 5-HT4R compounds were dissolved in a 10% 2-hydroxypropyl-beta-cyclodextrin (HBCD) solution. All drugs were administered in a single injection either 1 week before stress or 5 min after stress at 3 or 10 mg / kg of body weight based on our previous work (20).Molecular AssaysMaterials

[0112] HEK-293T cells were obtained from the American Type Culture Collection (Rockville, MD) and were cultured in a 5% CO2atmosphere at 37 °C in Dulbecco's Modified Eagle Medium (DMEM) (Life Technologies; Grand Island, NY) supplemented with 10% Fetal Bovine Serum (FBS) and lOO IU ml"1penicillin and 100 μg ml-1streptomycin (Coming; Tewksbury, MA). The following chemicals were used without further modification: Serotonin (Tocris; Bristol, United Kingdom), Coelenterazine H (Dalton Pharma Services, Toronto, ON, Canada), Polyethylenimine (PEI; Polysciences; Warrington, PA), Firefly D-luciferin (Nanolight Technologies; Norman, OK), Prucalopride (Sigma, Catalog No. 179474-8108, St. Louis, MO), RS-67,333 (Sigma, Catalog No. 168986-60-5, St. Louis, MO), and PF-04995274 (Sigma, Catalog No. 1331782-27-4, St. Louis, MO).DNA Constructs

[0113] The vector coding for 5-HT4bwas purchased from cDNA.org. 5-HT4cwas created by modifying the 5-HT4bcDNA using standard procedures and was sequence verified (Plasmidsaurus, Eugene, OR). The CRE luc-glo vector was constructed by Dr. Nicole Perry- Hauser using standard techniques in molecular biology and confirmed by DNA sequencing. It contains a cAMP response element (CRE) that drives transcription of the luciferase reporter gene luc2P and a co-transfected control Renilla luciferase reporter that is used to normalize the results for transfection efficiency. The plasmids coding for CAMYEL, Venus-miniGs and 5- HT4b-nanoluc (5-HT4b-nluc) were gifts from Dr. Nevin Lambert at the Medical College of Georgia.Transfection

[0114] For the CRE gene reporter assay, the miniGs or the CAMYEL BRET assays, a total of 1, 5, or 20 μg of cDNA, respectively, was transiently transfected into HEK-293T cells (350,000 cells for CRE in 12-well plate format; 2 x 106cells per plate for miniGs and 4 x 106cells per plate for CAMYEL and arrestin in 10 cm dishes). For the gene expression CRE assay, 600 ng of CRE-luc / glo and a range of receptor amounts were transiently transfected using PEI in 2:1 ratio (up to 400 ng, Q.S. with empty vector). For the miniGs assay, 1 μg receptor-nluc, 1 μg Venus-miniGs, and 3 μg empty vector were transfected using PEI in a 6:1 ratio (diluted in DMEM). For the CAMYEL assay, 1.25 μg of receptor, 10 μg of CAMYEL sensor, and 8.75 μg of empty vector were transiently transfected using PEI in a 2: 1 ratio (diluted in DMEM). For the arrestin assay, 0.2 μg receptor-nluc, 15 μg of arrestin-3-venus, and 4.8 μg of GRK3 were transiently transfected using PEI in a 2: 1 ratio (diluted in DMEM). Cells were maintained and transfected in the HEK-293T media described above, then switched to a serum free version of the media (lacking Fetal Bovine Serum (FBS)). For the CRE gene expression assays, media was switched to serum free 24 hours post transfection and ligand was added at the same time and left to incubate overnight. For the CAMYEL, arrestin, and miniGs assay, media was switched to serum free 2-6 hours post transfection. The CRE gene expression experiments were performed 24 hours post transfection, while the CAMYEL, arrestin, and miniGs assays were performed 48 hours after transfection.Bioluminescence Resonance Energy Transfer (BRET)

[0115] Experiments were performed as described previously (Donthamsetti P, Quejada JR, Javitch JA, Gurevich V V., Lambert NA (2015): Using Bioluminescence Resonance EnergyTransfer (BRET) to Characterize Agonist-Induced Arrestin Recruitment to Modified and Unmodified G Protein-Coupled Receptors. Curr Protoc Pharmacol 70: 2.14.1-2.14.14). Briefly, transfected cells were dissociated and resuspended in Dulbecco’s Phosphate Buffered Saline (dPBS). Cells were added to a black-framed, white well 96-well plate (Perkin Elmer; Waltham, MA). The luciferase substrate coelenterazine H (5 μM) was added to each well at time zero. Five min later, ligands were added. After a 10 min incubation, the BRET signal was measured. BRET measurements were performed using a PHERAstar FS plate reader or a LUMIstar plate reader (BMG Labtech, Cary, NC). The BRET signal was calculated as the ratio of the light emitted by the acceptor (510-540 nm) over the light emitted by the donor (475 nm). Dose-response curves were fit using a three-parameter logistic equation in GraphPad Prism 8 (La Jolla, CA). All experiments were repeated in at least three independent trials each with triplicate determinations.Gene expression cAMP Response Element (CRE) Assay

[0116] Experimental setups were adapted from methods described previously (Baker JM, Boyce FM (2014): High-throughput functional screening using a homemade dual-glow luciferase assay. Journal of Visualized Experiments 88: e50282; Perry -Hauser NA, VanDyck MW, Lee KH, Shi L, Javitch JA (2022): Disentangling autoproteolytic cleavage from tethered agonist-dependent activation of the adhesion receptor ADGRL3. Journal of Biological Chemistry 298(12): 1 - 9) using a dual-glo luciferase assay. Briefly, transfected cells were dissociated and resuspended in 275 μL of dPBS and 80 μL of this mixture was added to a black-framed, white well 96-well plate (Perkin Elmer; Waltham, MA, USA) in triplicate. 40 μL of Firefly luciferase buffer containing firefly D-luciferin was added to each well and allowed to incubate for 10 min. Following this incubation, a LUMIstar plate reader (BMG Labtech; Cary, NC, USA) was used to measure the firefly luciferase counts at 535nm. Next, a renilla luciferase buffer containing coelenterazine H was added to each well and allowed to incubate for an additional 10 min prior to reading the renilla luciferase counts at 475nm. Luciferase counts were normalized by calculating the ratio of firefly emission over renilla emission. Fold changes were calculated by dividing the normalized ratio by the average of the triplicate ‘control’ points, (control was [no drug; no receptor] or [X ng receptor; no drug] conditions).Behavioral AssaysContextual Fear Conditioning (CFC)

[0117] A 3-shock CFC was administered as previously described (Chen BK, Mendez- David I, Luna VM, Faye C, Gardier AM, David DJ, Denny CA (2020): Prophylactic efficacy of 5-HT4R agonists against stress. Neuropsychopharmacology 45: 542-552; Denny CA, Burghardt NS, Schachter DM, Hen R, Drew MR (2012): 4- to 6-week-old adult-bom hippocampal neurons influence novelty-evoked exploration and contextual fear conditioning. Hippocampus 22: 1188-1201). CFC was conducted in chambers obtained from Actimetrics (Lafayette, IN), with internal dimensions of 7.4" L x 8.1” D x 7.9’ H. The chambers had metal walls on each side, clear plastic front and back walls and ceilings, and stainless-steel bars on the floor. A house light (CM1820 bulb, 28v, 100mA) mounted directly above the chamber provided illumination. Each chamber was located inside a larger, insulated, plastic cabinet that provided protection from outside light and noise. Each cabinet contained a ventilation fan that was operated during the sessions. A paper towel dabbed with lemon solution (Pure Lemon Extract; McCormick®, Hunt Valley, MD) was placed underneath the chamber floor. To regulate the amount of lemon solution, Q-Tips were used to place 4 equal dots of lemon scent on 4 comers of the paper towel lining the chamber. Mice were held outside the experimental room in their home cages prior to testing and transported to the conditioning apparatus individually in standard mouse cages. Chambers were cleaned with 70% EtOH after each run. Mice were placed into the conditioning chamber and received shocks at 180 s, 240 s, and 300 s (2 s duration, 0.75 mA). Fifteen seconds after the last shock, mice were removed from the chamber. Overall, the training session lasted 317 s. During re-exposure, mice were placed in the conditioning chamber for 3 minutes and did not receive any shocks. All sessions were scored for freezing using FreezeFrame4.Learned Helplessness (LH)[0118J LH was used to induce depressive-like behavior through exposure to repeated unpredictable foot shocks and as previously described (Brachman RA, McGowan JC, Perusini JN, Lim SC, Pham TH, Faye C, et al. (2016): Ketamine as a Prophylactic Against Stress- Induced Depressive-like Behavior. Biol Psychiatry 79: 776-786). In this paradigm, mice are exposed to unpredictable and uncontrollable stress (shocks) and then develop coping deficits to deal with the inescapable shocks. Applicants utilized a two-chamber shuttle box (ENV 010MD; Med Associates, St. Albans VT) within a sound-attenuated cubicle. The grid floor wasmade of stainless steel and connected to a shock generator. The scrambled shock generator (ENV 414S, Med Associates, St. Albans VT) created varying electrical potential differences between bars preventing an animal from avoiding shock.

[0119] Inescapable shock (training): For each shuttle box, 2 animals were administered the protocol at the same time; the central door was closed, with one animal in the chamber on each side. After a 3 min habituation period, the shock deliveries began. The training protocol consisted of 70 shocks, each with a 3 s average duration, at 0.5 mA, and with an intertrial interval (ITI) of approximately 15 s.

[0120] Shock escape (testing): Mice were tested in the same shuttle box used in the inescapable shock training. The box consisted of two identical chambers (17 1 x 20 w x 17 h), separated by an automated door that opened vertically. The shuttle box was equipped with 8 infrared beams (4 on each side) for detecting position and activity of the animal (Med Associates, St. Albans, VT). Five days after training, each mouse was placed into the right chamber with the door raised and was allowed to freely explore both chambers for 3 min. Then the door then closed automatically. At the beginning of each trial, the door was raised and 5 s later a foot shock (0.5 mA) was delivered. The subject's exit from the shocked side ended the trial. If the mouse did not exit after 15 s, the shock was turned off and the trial ended. The door was lowered at the end of the trial. A session consisted of 30 trials, each separated by a 30 s ITI. Escape latencies were computed as the time from shock onset to the end of trial. If the subject failed to make a transition the maximum 15 s was used for the escape latency score.Forced. Swim Test (FST)

[0121] FST was administered as previously described (Chen et al., 2020; Brachman et al., 2016) to gauge behavioral despair. Briefly, mice were placed into clear plastic buckets 20 cm in diameter and 23 cm deep filled 2 / 3rdof the way with 22°C water. Mice were recorded from the side for 6 min and were exposed to the test on 2 consecutive days. Immobility was scored by an experimenter blind to experiment group and was used as a measure of depressive behavior.Marble Burying (MB)

[0122] The MB assay was conducted in a clean cage (10.5 in x 5.5 in) containing soft pliable Beta Chip bedding (Northeastern Products Corp, Warrensburg, NY). The cage contained 16 marbles set up in 4 rows of 4 across. Mice were given 30 minutes to explore andbury. Following task completion, the percentage of marbles buried was calculated, a marble was counted as buried if at least if 2 / 3rdwas covered.Novelty-Suppressed. Feeding (NSF)

[0123] Testing was performed as previously described (Brachman et al., 2016; Chen BK, Luna VM, Shannon ME, Hunsberger HC, Mastrodonato A, Stackmann M, et al. (2021): Fluoroethylnormemantine, a Novel NMDA Receptor Antagonist, for the Prevention and Treatment of Stress-Induced Maladaptive Behavior. Biol Psychiatry 90: 458-472; Chen BK, Luna VM, LaGamma CT, Xu X, Deng SX, Suckow RF, et al. (2020): Sex-specific neurobiological actions of prophylactic (R,S)-ketamine, (2R,6R)-hydroxynorketamine, and (2S,6S)-hydroxynorketamine. Neuropsychopharmacology 45: 1545-1556). Briefly, the NSF testing apparatus consisted of a plastic box (50 x 50 x 20 cm). The floor of which was covered with approximately 2 cm of wooden bedding and the arena was brightly lit. Mice were food restricted for 12 h prior to testing. At the time of testing, a single pellet of food (regular chow) was placed on a white paper platform positioned in the center of the box. Each animal was placed in a comer of the box, and a stopwatch was immediately started. The latency of the mice to begin eating in the arena was recorded. Immediately after the latency was recorded, the food pellet was removed from the arena. The mice were then placed back into their home cage. The latency to eat and the amount of food consumed in 5 min were measured (home cage consumption), followed by an assessment of post-restriction weight. A Kaplan-Meier survival analysis was used due to the lack of normal distribution of data. The Mantel-Cox log-rank test was used to evaluate differences between the experimental groups.Electrophysiology

[0124] Slice electrophysiology was conducted as previously described (Chen et al., 2020; Chen et al., 2021; Chen et al., 2020b; Luna VM, Anacker C, Burghardt NS, Khandaker H, Andreu V, Millette A, et al. (2019): Adult-bom hippocampal neurons bidirectionally modulate entorhinal inputs into the dentate gyrus. Science 364(6440): 578-583). One week after saline, PF-0499527, or novel 5-HT4R agonists administration (3 mg / kg) mice were anesthetized by isoflurane inhalation, decapitated, and brains rapidly removed. CA3 slices (350 μm) were cut on a vibratome (Leica VT1000S) in ice cold partial sucrose artificial cerebrospinal fluid (aCSF) solution (in mM): 80 NaCl, 3.5 KCl, 4.5 MgSO4, 0.5 CaCl2, 1.25 H2PO4, 25 NaHCO3, 10 glucose, and 90 sucrose equilibrated with 95% 02 / 5% CO2and stored in the same solution at 37°C for 30 min, then at RT until use. Recordings were made at 30 - 32°C (TC234-B, WarnerInstrument Corp.) in aCSF (in mM: 124 NaCl, 8.5 KCl, 1 NaH2PO4, 25 NaHCO3, 20 glucose, 1 MgCl2, 2 CaCl2). Whole-cell voltage clamp recordings (-70 mV) were obtained using a patch pipette (4-6 M MΩ) containing in mM: 135 K-Gluconate, 5 KCl, 0.1 EGTA-Na, 10 HEPES, 2 NaCl, 5 ATP, 0.4 GTP, 10 phosphocreatine (pH 7.2; 280 - 290 mOsm). Bicuculline (5 μM) was also included in the bath solution to inhibit GABAA receptors. Patch pipettes were made from borosilicate glass (A-M Systems, Sequium, WA) using a micropipette puller (Model P- 1000, Sutter Instruments). Recordings were made without correction for junction potentials. Pyramidal cells were visualized and targeted via infrared-differential interference contrast (IR- DIC, 40x objective) optics on an Axioskop-2 FS (Zeiss, Oberkochen, Germany).Imnumohistochemistrv

[0125] Immunohistochemistry was performed as previously described (Baker and Boyce, 2014; Denny et al., 2012). Mice were deeply anesthetized with a ketamine / xylazine mixture 1 hour following FST and were transcardially perfused with 50 mL ice-cold lx PBS followed by 50 mL 4% paraformaldehyde (PF A). Brains were removed and left in 4% PF A overnight and then cut on a vibratome (Leica VT1000S, Deer Park, IL, USA) and stored in lx PBS with 0.1% sodium azide prior to processing. Sections (50 μm) were washed 3x in lx PBS and blocked in 1 x PBS with 0.5% Triton X-100 (PBST) and 10% normal donkey serum (NDS) for 2 hours at room temperature (RT). Sections were then incubated in primary rat anti-c-Fos for 24 hr at 4°C (Synaptic Systems, 226 017, 1:5,000). The next day tissue was washed 3x in lx PBS and then incubated for 2 hours in secondary donkey anti-rat Alexa Fluor 488 at RT (ThermoFisher, A21208, 1:500). Tissue was incubated with Hoechst in lx PBS at RT (Hoechst, 33342, ThermoFisher, 1:10k) and washed again in lx PBS. All slices were mounted on glass slides and cover slipped with Fluoromount G (Electron Microscopy Sciences, Hatfield, PA, 17984- 25).Confocal Microscopy

[0126] Fluorescent confocal micrographs were taken at 20x magnification with a Leica SP8 microscope and with LAS X software as previously described (Chen et al., 2021). Hippocampal sections were imaged throughout the rostro-caudal axis of the HPC. Identification of hippocampal regions (CA1, CA3, DG,) involved acquiring 5 - 8 sections per mouse due to variability in sectioning. Images were scanned and captured at a z-increment of 3 μm. Z-stack analysis was performed using LAS X image browser to determine expression of c-Fos which was compared across all sections using identical exposure conditions.Cell Quantification

[0127] A researcher blinded to treatment group used FIJI software (NIH) to manually count c-Fos immunoreactive cells in the DG, CA3 and CA1 regions of the HPC of one hemisphere across the rostrocadual axis (-1.34 to -2.18 AP). The number of c-Fos+ cells across groups is presented in Figure 6.Statistical Analysis

[0128] Results from all data analyses are expressed as means ± SEM. Alpha was set to0.05. Data were analyzed using Prism v8.0 or 10.0 (GraphPad Software, La Jolla, CA). One or two-way ANOVAs with repeated measures were applied to data as appropriate. Significant main effects and / or interactions were followed by Fisher's LSD. All statistical tests and p values are included in Table 5.Table 5. Statistical Analysis4352Table 6. Key ResourcesReferences1. Collins PY, Patel V, Joestl SS, March D, Insel TR, Daar AS, et al. (2011): Grand challenges in global mental health. Nature 475: 27-30.2. Smith K, De Torres I (2014): A world of depression. Nature 515: 10-38.3. Kalin NH (2020): The critical relationship between anxiety and depression. American Journal of Psychiatry 177: 365-367.4. Fava M, Rush AJ, Alpert JE, Balasubramani GK, Wisniewski SR, Carmin CN, et al. (2008): Difference in treatment outcome in outpatients with anxious versus nonanxious depression: A STAR*D report. 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[0129] Various modifications and variations of the described methods, pharmaceutical compositions, and kits of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it will be understood that it is capable of furthermodifications and that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known customary practice within the art to which the invention pertains and may be applied to the essential features herein before set forth.

Claims

CLAIMSWhat is claimed is:

1. A 5-HT4R inverse agonist or antagonist having the structure:wherein X is O, S, SO, SO2or Se, wherein Ar is fused heteroaromatic and wherein R is heterocyclic, bridged cyclic, spiro cyclic or fused cyclic.

2. The 5-HT4R inverse agonist or antagonist of claim 1, wherein Ar has the structure:

3. The 5-HT4R inverse agonist or antagonist of claim 1, wherein R has the structure:

4. A 5-HT4R inverse agonist or antagonist selected from the group consisting of:

5. A method of protecting against stress-induced maladaptive behavior in a subject in need thereof comprising administering a prophylactically effective amount of the 5-HT4R inverse agonist or antagonist of any one of claims 1-4 to the subject.

6. The method of claim 5, wherein the 5-HT4R inverse agonist or antagonist is administered after stress but before stress-induced maladaptive behavior onset.

7. The method of claim 5, wherein the 5-HT4R inverse agonist or antagonist is administered before stress.

8. The method of any of claims 5-7, wherein the subject is an adult.

9. The method of claims 8, wherein the subject is aged.

10. The method of any of claims 5-9, wherein the subject is a female or male.

11. The method of any of claims 5-11, wherein the subject is diagnosed with PTSD, an anxiety disorder, a phobia, major depressive disorder, bipolar depression, OCD, ASD, schizophrenia, Huntington’s disease, Parkinson’s disease, an eating disorder, or treatmentresistant depression.

12. The method of any of claims 5-12, wherein the maladaptive behavior is selected from the group consisting of depression, anxiety, anorexia, compulsiveness, freezing, intrusivethoughts and flashbacks, avoidance, negative changes in mood and cognition, hyperarousal, dissociation, difficulty concentrating and making decisions, and memory loss.

13. The method of any of claims 5-13, wherein a female subject is treated with D5 or D8 after a stress to prevent an anxiety or eating disorder symptom.

14. The method of any of claims 5-13, wherein a male subject is treated with D7 or D8 after a stress to prevent depression.

15. The method of any of claims 5-13, wherein a male subject is treated with D7 to prevent a PTSD, anxiety, or phobia symptom.

16. The method of any of claims 5-13, wherein a male subject is treated with D5, D7, or D8 to prevent major depressive disorder or bipolar depression.

17. The method of any of claims 5-13, wherein a male subject is treated with D5 or D7 to prevent a compulsive symptom associated with OCD, ASD, schizophrenia, Huntington’s disease, or Parkinson’s disease.

18. The method of any of claims 5-13, wherein a male subject is treated with D5 or D8 to prevent anxiety or an eating disorder symptom.

19. The method of any of claims 5-13, wherein an aged male subject is treated with D7 to prevent major depressive disorder or bipolar depression.

20. The method of any of claims 5-13, wherein a female subject is treated with D7 or D8 to prevent a compulsive symptom associated with OCD, ASD, schizophrenia, Huntington’s disease, or Parkinson’s disease.

21. The method of any of claims 5-13, wherein a female subject is treated with D5 to prevent anxiety or an eating disorder symptom.

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