Blood-brain-barrier penetrant HCN channel inhibitors and method of use

Abstract

The present disclosure is directed to blood-brain barrier penetrant channel inhibitors and their use in the treatment of major depressive disorders and other diseases. Patients with major depressive disorder exhibit alterations in their dopamine signaling pathways through the midbrain ventral tegmental area and a similar observation is also detected in preclinical models of stress - mice exhibit behavioral and physiological impairments following chronic social defeat stress. Prior studies have shown chronic social defeat stress-susceptible mice have increased ventral tegmental area dopamine neuronal excitability in part driven by an upregulation in hyperpolarization activated, cyclic nucleotide gated, Na+ / K+ (HCN) channels. Inhibiting these channels with known inhibitors such as Cilobradine has been shown to alleviate the negative behavioral effects of chronic social defeat stress.

Description

Attorney Docket: 27527-0224WO1 BLOOD-BRAIN-BARRIER PENETRANT HCN CHANNEL INHIBITORS AND METHOD OF USE FIELD OF THE INVENTION The present disclosure is directed to HCN channel inhibitors. More particularly, the present disclosure is directed to blood-brain barrier penetrant HCN channel inhibitors and their use in the treatment of major depressive disorder and other diseases. BACKROUND OF THE INVENTION

[0001] Major Depressive Disorder (MDD) is a devastating disease that affects over 300million people globally, representing one of the leading causes of disability worldwide (1). It is characterized by behavioral symptoms such as feelings of sadness, anhedonia, and physical symptoms such as fatigue, amongst others, due to a variety of causes including environmental stressors, genetics, and gender (2). Depression has been viewed as a reduction in the concentration of monoamines including dopamine (DA) (3). Studies have shown that human patients with depression show DA neuron dysregulation in the ventral tegmental area (VTA) of the brain, which is a critical regulator in the mesolimbic pathway of reward learning and memory (4,5). This VTA DA dysregulation is shared with rodent models of social stress-induced anhedonia and social avoidance (6). Antidepressant drugs targeting monoamine reuptake inhibitors are the main pharmacological treatment for depression, yet the majority of people who take them do not respond and are considered treatment-resistant (7,8). The high percentage of treatment-resistant MDD patients underscores the need for novel, quicker-acting and long-lasting treatment strategies for MDD. Recent breakthrough drugs Esketamine and Brexanolone are rapid acting, long-lasting, and act on the glutamatergic and GABAergic systems, respectively, as opposed to monoamine transporters (9,10). These drugs align with recent research depicting depression as a disruption of neuronal excitability, which is itself due to aberrant activity of a variety of ion channels that delicately control neuronal firing rate (FR) in the brain. While many channels are dysregulated during depression, the hyperpolarization-activated, cyclic nucleotide-gated (HCN) Na+ / K+ion channel is known to be dysregulated in mouse models of depression, and thus represents an interesting pharmacological target (11–14).Attorney Docket: 27527-0224WO1

[0002] HCN channels are critical regulators of membrane excitability. HCN channels pass amixed sodium and potassium current (called the Ih current) in a ratio of 1:4, respectively, resultingin a reversal potential of approximately -40 mV, near the resting membrane potential of the cell (15). They open upon hyperpolarization following an action potential or when cells deviate from their resting membrane potential, returning the neurons to resting membrane potential and facilitating their ability to fire again (15). In doing so, they ensure rhythmic FRs of many neurons. They are widely expressed throughout the brain, but are mainly found in the cortex, thalamus, hippocampus, and midbrain, where they regulate higher cognitive functions, thalamocortical rhythms, and somatic sensation or pain (11,15,16). HCN channel activity in the brain is particularly important in monitoring and maintaining the rhythmic FR of tonically-firing neurons, such as the DA neurons in the VTA (17,18). Researchers have determined that the dysregulation of HCN channels in the nervous system is associated with multiple brain dysfunctions and mood disorders such as anxiety and depression (in the VTA) (11,15,17–19).

[0003] In particular, the expression of HCN in the DA neurons of the VTA - a critical hub ofreward and motivation – has been associated with a variety of neuropsychiatric disorders, chiefly depression (19–23). This association has been determined both in human depression and in mouse models mirroring human depression (4,6). The chronic social defeat stress (CSDS) protocol induces social avoidance, anhedonia, altered reward processing and cognitive inflexibility in both male and female mice susceptible to the paradigm as measured by the Social Interaction (SI) test(6,24–26). These susceptible mice also exhibit a significant upregulation of the Ih current in theirVTA DA neurons, which in turn increases the spontaneous FR of these neurons (17,18,27).Notably, because Ih currents allow neurons to return to resting membrane potential, CSDS-inducedaltered Ih function results in VTA DA neurons’ increased tonic and bursting activity. Tonic andbursting patterns of activity mediate dopamine functions throughout the brain, and VTA DA neurons’ bursting activity is critical in encoding rewards, reward prediction error, salient and aversive stimuli (28,29). Previous research has causally linked increased VTA DA bursting activity with the observed social avoidance and reduced preference for sucrose solution inducedby CSDS (18). Therefore, we hypothesize that inhibiting the Ih current will decrease the FR, andthus bursting activity, and return the resultant phenotype back to baseline. There is a series of compounds – Ivabradine, Cilobradine, Zatebradine – that block HCN channels, with Ivabradine asAttorney Docket: 27527-0224WO1 an FDA approved drug to treat many cardiac conditions (17,30). Previous studies suggest that Ivabradine has limited abilities to cross the blood-brain barrier (BBB) (31,32). However, it is unclear if Cilobradine and Zatebradine can cross the BBB (17,33). Our lab has shown that an IP injection of 20 mg / kg of Cilobradine exhibits a ketamine-like rapid-acting and long-lasting ameliorating effect on stress-induced social deficits and anhedonia in CSDS-susceptible mice (34).

[0004] SUMMARY OF THE INVENTIONIn an embodiment, the present disclosure includes (S)-7,8-dimethoxy-3-((1-(2-(3-methoxyphenoxy)ethyl)piperidin-3-yl)methyl)-1,3,4,5- tetrahydro-2H-benzo[d]azepin-2-one (1,MS7710); (S)-3-((1-(2-(3,4-dimethoxyphenoxy)ethyl)piperidin-3-yl)methyl)-7,8-dimethoxy-1,3,4,5- tetrahydro-2H-benzo[d]azepin-2-one (1); (S)-7,8-dimethoxy-3-((1-(2-phenoxyethyl)piperidin-3-yl)methyl)-1,3,4,5-tetrahydro-2H- benzo[d]azepin-2-one (3); (S)-3-((1-(2-(2-fluorophenoxy)ethyl)piperidin-3-yl)methyl)-7,8-dimethoxy-1,3,4,5- tetrahydro-2H-benzo[d]azepin-2-one (4); (S)-3-((1-(2-(4-fluorophenoxy)ethyl)piperidin-3-yl)methyl)-7,8-dimethoxy-1,3,4,5- tetrahydro-2H-benzo[d]azepin-2-one (5, MS7712); (S)-3-((4-(3,4-dimethoxyphenethyl)morpholin-2-yl)methyl)-7,8-dimethoxy-1,3,4,5- tetrahydro-2H-benzo[d]azepin-2-one (6); (S)-3-((4-(2-(4-fluorophenoxy)ethyl)morpholin-2-yl)methyl)-7,8-dimethoxy-1,3,4,5- tetrahydro-2H-benzo[d]azepin-2-one (7); and 3-(3-((2-(3,4-dimethoxyphenyl)-2,2-difluoroethyl)(methyl)amino)propyl)-7,8-dimethoxy- 1,3,4,5-tetrahydro-2H-benzo[d]azepin-2-one (8); and pharmaceutically acceptable salts thereof. In an embodiment, the disclosure includes compositions including a compound as disclosed above and a pharmaceutically acceptable carrier.Attorney Docket: 27527-0224WO1 In an embodiment, the disclosure includes a method of treating major depressive disorder, including administering a therapeutically effective amount of a composition including a compound disclosed above to a subject in need thereof. In an embodiment, the composition is administered at a dosage of from 1 to 5 mg / kg / day. In an embodiment, the dosage is from 1 to 10 mg / kg / day. BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Figure 1: Chemical structures of Cilobradine, Ivabradine and Zatebradine andtheir analogs that are designed and synthesized to improve BBB permeability. a Structures ofCilobradine, Ivabradine and Zatebradine. b Novel analogs that are designed and synthesized andused in this study. Modifications to the substituents of the left-hand side phenyl ring are highlighted in blue. The alteration of the central piperidine ring of Cilobradine to a morpholine ring is represented by the introduced oxygen highlighted in orange. The replacement of the benzylic methylene group of Zatebradine with a difluoromethylene group is highlighted in pink.

[0006] Figure 2: Novel HCN inhibitors exhibit a range of efficacy on VTA dopamineneurons compared to parent compounds. a Experimental schematic of brain sliceelectrophysiology with local application. b Example image of VTA DA neuron (denoted with >and a circle), the patch pipette (denoted with >>), and the local application pipette (denoted with>>>). c Sample trace of Ih inhibition evoked at -120 mV of VTA DA neurons before (gray) andafter (black) compound application. Sample traces and percent inhibition of Ih currents in VTADA neurons following local compound application of: d Ivabradine, Zatebradine, and 8 (n = 4-9,, H4 / 26 = 16.82, p = 0.0004; Zatebradine vs. vehicle, ** p = 0.0064; Ivabradine vs. vehicle, * p =0.0144), and e Cilobradine and Cilobradine novel analogs (n = 4-11, H9 / 60= 42.94, p < 0.0001; 1vs. vehicle, ** p = 0.0049; MS7712 vs. vehicle, ** p = 0.0012; MS7710 vs. vehicle, *** p =0.0002; Cilobradine vs. vehicle, *** p < 0.0001). Sample traces and percent inhibition of FR inVTA DA neurons following local compound application of: f Ivabradine and Zatebradine(n=6-10,H3 / 25= 16.66, p = 0.0002; Zatebradine vs. vehicle, ** p = 0.0040; Ivabradine vs. vehicle, *** p =0.0003), and g Cilobradine and top analogs (n=7-13, H3 / 29 = 10.03, p = 0.0067; MS7712 vs.Cilobradine, ** p = 0.0066; MS7710 vs. Cilobradine, * p = 0.0244). All sample trace scale barsare 200 pA vs.1 second for Ih currents and 1 mV vs. 2 seconds, all baseline traces are in gray, andAttorney Docket: 27527-0224WO1 all colored traces are post-compound application. All data are presented as mean ± s.e.m, analyzed by the Kruskal-Wallis one-way ANOVA and corrected for multiple comparisons with the Dunn's test.* p < 0.05, ** p <0.01, *** p < 0.001.

[0007] Figure 3: Novel HCN inhibitors MS7710 and MS7712 exhibit improved BBBpermeability compared to their parent compound Cilobradine. a Experimental timeline forBBB permeability testing in stress-naïve, male, C57BL / 6 mice – brain and blood samples were taken at time points 15 minutes, 1 hour, and 4 hours post-IP injection of 20 mg / kg of eachcompound. b Schematic of sample collection. c Cilobradine brain and plasma concentrations andratio of 0.076. d MS7710 brain and plasma concentrations and ratio of 0.28. e MS7712 brain andplasma concentrations and ratio of 0.57. As a general yet flexible rule, a ratio of approximately 0.3-0.5 is sufficient for a drug to cross a healthy BBB, and a ratio < 0.1 is insufficient (55). Data represented by mean ± s.e.m., n =3 per timepoint.

[0008] Figure 4: Novel HCN inhibitors MS7710 and MS7712 reverse CSDS-inducedhyperdopaminergia in male, CSDS-susceptible mice. a Experimental timeline for CSDS and invivo electrophysiology. b Example in vivo electrophysiology trace of FR in VTA DA neurons inCSDS-susceptible mice. c Examples of tonic activity (yellow box labeled i) and phasic (bursting)activity (red box labeled ii). d Classical waveform of a VTA DA neuron, averaged over 50 spikes(scale bar 20 mV vs. 1 ms). e Sample traces of baseline FR and FR following a 20 mg / kg IPinjection of Cilobradine (scale bars 0.05 mV vs.1 s). Summary and inset cumulative distribution(dotted horizontal line represents 100%) of: f FR in VTA DA neurons during baseline conditions(0 mg / kg Cilobradine; n = 57 / 17) or post-IP injection of 20 mg / kg Cilobradine (n=44 / 11; * p =0.0184), and g bursting activity in VTA DA neurons during baseline conditions (0 mg / kgCilobradine; n = 57 / 17) or post-IP injection of 20 mg / kg Cilobradine (n=44 / 11; *** p = 0.0001 ). h Sample traces of baseline FR and FR following an IP injection of 1 mg / kg and 10 mg / kgMS7712. Summary and inset cumulative distribution of: i FR in VTA DA neurons during baselineconditions (0 mg / kg MS7712; n=17 / 6) or post-IP injection of 1 mg / kg (n=21 / 3) or 10 mg / kg(n=22 / 3) MS7712 (H3 / 60 = 23.42, p < 0.0001; 10 mg / kg MS7712 vs. baseline, *** p < 0.0001),and j bursting activity in VTA DA neurons during baseline conditions (0 mg / kg MS7712; n=17 / 6)or post-IP injection of 1 mg / kg (n=21 / 3) or 10 mg / kg (n =22 / 3) MS7712 (H3 / 60 = 11.38, p = 0.0034;Attorney Docket: 27527-0224WO110 mg / kg MS7712 vs. baseline, ** p = 0.0023). k Sample traces of FR following an IP injectionof 1 mg / kg and 10 mg / kg MS7710. Summary and inset cumulative distribution of l FR in VTADA neurons during baseline conditions (0 mg / kg MS7710; n=27 / 8) or post-IP injection of 1 mg / kg(n=15 / 3) or 10 mg / kg (n=18 / 3) MS7710 (H3 / 59 = 14.97, p = 0.0006; 1 mg / kg MS7710 vs. baseline,* p = 0.0150; 10 mg / kg MS7710 vs. baseline, *** p = 0.0007) and m bursting activity in VTA DAneurons during baseline conditions (0 mg / kg MS7710; n=27 / 8) or post-IP injection of 1 mg / kg(n=15 / 3) or 10 mg / kg (n=18 / 3) MS7710 (H3 / 59 = 7.752, p = 0.0207; 1 mg / kg MS7710 vs. baseline,* p = 0.0494; 10 mg / kg MS7710 vs. baseline, * p = 0.0364). All sample trace scale bars 0.1 mVvs. 1 s unless otherwise noted. Notation of n=ntotal cells / ntotal mice. Data are presented as median ± quartiles ± min and max, analyzed by the Mann-Whitney test for two groups or the Kruskal-Wallis one-way ANOVA for three groups followed by the Dunn's test for multiple comparisons. * p < 0.05, ** p <0.01, *** p < 0.001.

[0009] Figure 5: Novel HCN inhibitor MS7710 reverses CSDS-inducedhyperdopaminergia in female, CSDS-susceptible mice. a Experimental timeline for femaleCSDS and in vivo electrophysiology. b Example in vivo electrophysiology traces of FR in VTADA neurons in female, CSDS-susceptible mice without MS7710 (0 mg / kg, baseline) and followinga 1 mg / kg IP injection of MS7710 (scale bars 0.1 mV vs. 1 s). Summary and inset cumulativedistribution (dotted horizontal line represents 100%) of: c FR in VTA DA neurons during baselineconditions (0 mg / kg MS7710; n = 11 / 3) or post-IP injection of 1 mg / kg MS7710 (n=16 / 3; ** p =0.0010), and d bursting activity in VTA DA neurons during baseline conditions (0 mg / kg MS7710;n = 11 / 3) or post-IP injection of 1 mg / kg MS7710 (n=16 / 3; * p = 0.0246). Notation of n=ntotalcells / ntotal mice. Data are presented as median ± quartiles ± min and max, analyzed by the Mann- Whitney test for two groups. * p < 0.05, ** p <0.01.

[0010] Figure 6: Novel HCN inhibitor MS7710 improves CSDS-induced social, reward,and cognitive deficits in male mice. a Timeline of CSDS and PRLT experimental paradigm. bScheme of SI test. c Schema of PRLT including placement of SI tests. d Social interaction testbefore PRLT and near the end of PRLT, 14 days after IP injection of either saline (n=11) or 5mg / kg MS7710 (n=13). 5 mg / kg MS7710 improved SI ratios (paired t test: * p = 0.0238). e PRLTcorrect choice percentage each trial during learning for control mice (n=18) and susceptible miceAttorney Docket: 27527-0224WO1(n=25). f PRLT correct choice percentage each trial during the reversal post-injection for controlmice (n=18), susceptible + saline mice (n=12) and susceptible + 5 mg / kg mice MS7710 (n=13;two-way mixed effects ANOVA: effect of time: F3.932, 156.3 = 64.62, *** p < 0.0001; effect of condition: F2,42= 3.295, p = 0.0505 for sessions 1-13). The inset depicts the number of trials until the criteria is reached for each condition (one way ANOVA: F2,40= 5.526, p = 0.008; Tukey’s testfor multiple comparisons: control vs. susceptible + saline mice: p = 0.007; susceptible + MS7710vs. susceptible + saline mice: p = 0.04). Data are presented as mean ± s.e.m. * p < 0.05, ** p <0.01, *** p < 0.001.

[0011] Figure 7: 1H NMR spectrum of MS7710.

[0012] Figure 8: 13C NMR spectrum of MS7710.

[0013] Figure 9: LC-MS spectrum of MS7710.

[0014] Figure 10: 1H NMR spectrum of MS7712.

[0015] Figure 11: 13C NMR spectrum of MS7712.

[0016] Figure 12: LC-MS spectrum of MS7712.

[0017] Figure 13: Effect of 1 mg / kg of MS7710 on the Si teset of CSDS-susceptible mice.Pilot data of the effect of an IP injection of 1 mg / kg MS7710 demonstrates that while the dose seems like it might be efficacious at 24 hours, there is a loss of efficacy by 4 days. This data led us to up the dosage to 5 mg / kg for our main behavioral study.

[0018] Figure 14(a) and 4(b): Locomotion and velocity of CSDS-susceptible mice beforeand after injection of saline or MS7710.

[0019] DETAILED DESCRIPTION

[0020] Modulation of dopamine activity through HCN channel functions has revealed itspotency as a novel treatment for stress-induced behavioral and physiological alterations (11,19,34– 36). Yet, current HCN inhibitors lack ideal efficacy due to their limited or unknown BBB permeability (31,32). In this study we created a series of analogs of Cilobradine (also known as DH-AK-269) and one analog of Zatebradine, all specifically designed to improve BBB permeability and inhibitory efficacy as a basis for a novel class of antidepressants. We thenAttorney Docket: 27527-0224WO1characterized their effects on VTA DA neuron Ih currents and FR utilizing ex vivoelectrophysiology. We determined that two novel compounds, MS7710 and MS7712, hadimproved inhibitory efficacy compared to Cilobradine. These compounds differ by the presenceof either a methoxy (MS7710) or a p-fluoro (MS7712) substituent on the left-hand side phenylring of the molecule. Pharmacokinetic analysis confirmed that MS7710 and MS7712demonstrated significant improvements in BBB permeability compared to their parent compound Cilobradine. To further validate the potent stress-ameliorating effects of the newly designed compounds we employed CSDS mouse model. This model allows researchers to examine how a stressful experience in mice leads to symptoms such as social and reward-seeking impairments as well as decreased motivation, which mirrors human responses to stress and associated depression symptoms (24,26,27,37). Following CSDS, we performed in vivo electrophysiological recordings of VTA DA neurons in CSDS-susceptible mice and tested the pharmacological efficacy ofMS7710 and MS7712. We determined that MS7710 restored VTA DA neuron FR and burstingactivity in CSDS male mice at lower doses than MS7712 or Cilobradine. Notably, we recapitulatedour main effect in female mice. Finally, we observed that MS7710 restored social interactionbehaviors as well as reward and cognitive deficits measured during operant behaviors in male, CSDS-susceptible mice, two weeks post-IP injection. Together, our results show that HCN inhibitors with improved BBB permeability may yield promising antidepressant effects. METHODS Compound synthesis and chemical characterization

[0021] The synthesis and characterization of compounds 1-8, including top compoundsMS7710 (2) and MS7712 (5), are described below. The 1H NMR & 13C NMR as well as LCMSspectra of compounds MS7710 (2) and MS7712 (5), are provided in the Figures.BBB permeability and pharmacokinetic testing

[0022] Pharmacokinetic (PK) testing for Cilobradine, MS7710, and MS7712 was conductedby Sai Life Sciences. Each compound was formulated in 5% v / v NMP, 45% v / v PG and 50% v / v PEG-400 and injected intraperitoneally (IP) at a dose of 20 mg / kg, volume of 10 mL / kg. A total of 9 male, stress-naïve C57BL / 6 – 3 mice / timepoint – were assessed for each compound, withAttorney Docket: 27527-0224WO1 timepoints at 15 minutes, 1 hour, and 4 hours post-injection. At each timepoint, the mice were sacrificed, their blood was collected for plasma samples and their brains were homogenized. Analytes from each were extracted and run on an LC-MS / MS to quantify the compound’s concentration. Mice

[0023] The studies utilized C57BL / 6J male and female mice (8 weeks old), CD1 male retiredbreeders and F1 ER -Cre males, progeny of ER -Cre mice and CD1 female breeders (all fromJackson Labs). C57BL / 6J mice were group-housed before the CSDS and singly housed after theCSDS. CD1 mice and F1 ER -Cre mice were singly-housed before and after the CSDS. All micewere maintained on a 12 hour light / dark cycle with ad libitum access to food and water. Control C57BL / 6J mice were also singly housed after the CSDS period. All experiments and protocols were approved by the Mount Sinai Institutional Animal Care and Use Committee and the National Institute of Health for Care and Use of Laboratory Animals. CSDS paradigm

[0024] The CSDS paradigm was performed as previously established (38,39). Briefly, male(CD1 mice) and female (F1 ER -Cre mice) aggressors were screened for aggression prior toCSDS. The F1 ER -Cre mice are male mice with upregulated ER activity, resulting inuncharacteristic aggression upon female mice. During the male and female CSDS paradigms, the CD57BL / 6J test mouse (intruder) was placed into the home cage of the aggressor mouse (resident) for 10 minutes, during which the resident will attack and defeat the intruder, followed by 24 hours of sensory stress. This process was repeated consecutively for a total of 10 defeats in 10 days with different resident mice each day. The stress-naïve non-defeated control mice were pair-housed separated by a clear, perforated barrier, handled every day, and rotated to another cage. SI test

[0025] The SI test was performed as previously described, 24 hours post-CSDS (38,39). Thesocially defeated mouse or stress-naïve control mouse first experienced 2.5 minutes of exploration in an open field test (OFT) box with no target present (“No Target” phase). Then, the experimentalAttorney Docket: 27527-0224WO1 mouse was removed, an unfamiliar aggressor was placed within the wire mesh cage on one side of the box, and the experimental mouse was then placed back in the box for 2.5 minutes of exploration with the target present (“Target” phase). The experimental mouse was then placed back in its home cage until future use. The time the experimental mouse spent within the social interaction (SI) zone (a predefined area surrounding the wire mesh cage) was recorded using the Ethovision tracking system. The SI ratio for each mouse was calculated as: time spent in SI zone during the “target” phase divided by time spent in the SI zone during the “no target” phase. The mice were then segregated into the “susceptible” group (SI ratio < 1) or the “resilient” group (SI ratio 1). This segregation is predictive of the presence of a stress-associated alterations in neuronal excitability and accompanying behavioral deficits (susceptible mice) or the lack thereof (resilient mice) (6,17). Only the susceptible mice were used for future studies. PRL task

[0026] The PRLT task was performed similarly to previous protocols in male mice post-CSDS(40–42). During the course of the experiment, mice were water restricted to 40 minutes perday following the operant session (1 session / day). During the task, reward delivery consisted ofthe delivery of 10 L of 0.2% saccharin solution. First, mice underwent three sessions of 30-minutehabituation to the chamber; during this time, both levers, a sound, and a reward were presented. Second, mice were trained (10-sec trial, 60 trials / session) to press any levers to induce reward delivery at deterministic fix-ratio 1. Mice remained in this training phase until the completion of two sessions with greater than 35 rewarded trials, and then exposed to the same training at FR2 until the completion of two sessions with greater than 35 rewarded trials. Probabilistic learning and reversal: In these sessions, both levers were extended simultaneously, with two consecutive presses on the right / left levers providing an 80% / 20% chance of reward, respectively. Upon achieving the criterion, reward contingencies were reversed; the previous high, 80% reward lever rewarded on only 20% of trials and vice versa. The total number of 80_lever selections and 20_lever selections were monitored throughout the task, and the percentage of 80_lever choices was calculated as [%80_lever choices = 80_lever choices / (80_lever choices+20_lever choices)*100. DrugsAttorney Docket: 27527-0224WO1

[0027] For all in vivo experiments, compounds were formulated in 5% v / v NMP, 45% v / v PGand 50% v / v PEG-400 at a dose of 20 mg / kg, and freshly dissolved in saline on the day of experiment for lower doses. Compound doses ranged from 1 mg / kg to 20 mg / kg. Ex vivo electrophysiological recordings

[0028] Acute, coronal VTA brain slices were prepared and recorded from as previouslypublished (Fig. 1a) (18,19). Male, 8-12 week old mice underwent perfusion with cold, artificial cerebrospinal fluid (ACSF) containing (in mM): 128 NaCl, 3 KCl, 1.25 NaH2PO4, 10 D-glucose,25 NaHCO3, 2 CaCl2, and 2 MgSO4 (oxygenated with 95% O2, 5% CO2). Then, the brain wasremoved, placed into an oxygenated, sucrose-based ACSF (replacing NaCl with 254 mM sucrose), and sectioned into 250 µM coronal VTA slices with a vibratome (DTK-1000, Ted Pella). Following 1 hour of recovery in 37ºC ACSF, a slice was then placed in the recording chamber of the electrophysiology rig (perfused with 35ºC, 95% O2 / 5% CO2 ACSF at 2.5 mL / min). A glassrecording pipette (3-7 M ) was filled with internal solution containing (in mM): 115 K-gluconate,20 KCl, 1.5 MgCl2, 10 phosphocreatine-tris, 10 HEPES, 2 Mg-ATP and 0.5 Na-GTP (pH 7.2,285 mOsm), and a glass application pipette was filled with 750 µM solutions of compoundsdissolved directly in ACSF, fresh for each experiment. Compounds were applied by local application via the glass application pipette (Fig.1b) using the PicoSpritzer III (Parker Hannifin). DA neurons were identified by location, size, shape, and FR (or lack thereof) and the presence ofan Ih current if the cell was held in a whole cell configuration. FR was recorded using the cell-attached configuration; continuously recording, the cell was held in I=0 for a 3 minute baseline, the compound was applied for 3 minute, and the compound was removed and the cell observed for the next 3 minutes to observe partial washout. If there was no washout present, the cell’s data wasnot included for analysis. Ih current was recorded using the whole-cell configuration; the cellexperienced a 3 second -120 mV pulse (from a holding potential of -60 mV) to yield a baseline Ih,the compound was then applied for 3 minutes, at the end of which another -120 mV pulse was applied. Series resistance was noted for every recording. All data were collected utilizing theDigidata 1440A digitizer, Multiclamp 700B amplifier, and pClamp 10.2 (Axon Instruments), andanalyzed using pClamp 10.2 and R Statistical Software (43). In vivo electrophysiological recordingsAttorney Docket: 27527-0224WO1

[0029] Single-unit, juxtacellular in vivo recordings of VTA DA neuron activity in anesthetizedmice (IP injection, chloral hydrate 8%, 400 mg / kg) were conducted as previously described (44– 47). CSDS-susceptible male and female mice aged 10-12 weeks old were anesthetized with an IP injection of 8% chloral hydrate (400 mg / kg), their head was fixed flat on a stereotaxic apparatus (Kopf), their skull was exposed to identify bregma, a small hole anterior to bregma was drilled for the reference wire, and a window to their brain was opened to allow access to the VTA. From bregma, the VTA is located at coordinates anterior / posterior -3.0 to -3.7, medial / lateral ± 0.2 to 0.7, dorsal / ventral -4.0 to -5.0(44). The pipette - 1-2 mm tip, filled with an internal of 2M NaCl – was lowered slowly through the brain within the DV -4.0 to -5.0 track using a micromanipulator (Sutter Instruments DP-311). DA neurons were distinguished by the presence of a triphasic action potential (AP) with a long duration (>2.0 ms), an AP width from start to negative deflection 1.1 ms and a baseline FR between 0.5 – 10 Hz with irregular single spiking pattern (tonic) and discreteshort bursting activity as previously established (0.3–5 kHz band-pass) (45,48–51). For each cell,APs were collected, followed by 3 minutes of recording FR activity. For each mouse, at least three cells were recorded pre-drug injection, and multiple cells were recorded post-injection (> 30 minutes post-injection). Data was acquired with the Axon Digidata 1440A Data AcquisitionSystem and, following signal amplification and filtering (0.3–1 kHz band-pass), analyzed in realtime and stored for additional analyses in the pCLAMP system. Extracted data was run through R Statistical Software to analyze FR and bursting activity (45–47). FR was measured via a 15 s sliding window of spikes per 60 s, and averaged over the course of the recording. Bursts were analyzed as percent spikes within burst (% SWB, number of spikes within a burst divided by the total number of spikes). Bursts were identified by an onset of two consecutive spikes within an interval <80 ms and terminated when the interval to the next spike was > 160 ms. Statistics

[0030] All statistical analyses were performed using GraphPad Prism 10.0.2 (52). The datawas tested for normality using the D’Agostino and Pearson test. For an analysis between two groups, the unpaired t test for normal data, or Mann-Whitney U test for nonparametric data was used for unpaired data, and the paired t test was used for normal, paired data. For an analysis between three or more groups differing in one factor, an ordinary one-way ANOVA followed by Dunnett's test for multiple comparisons for normal data, or the Kruskal Wallis one-way ANOVAAttorney Docket: 27527-0224WO1 followed by Dunn’s test for multiple comparisons was used. For an analysis between three of more groups differing in two factors, the two-way mixed effects ANOVA followed by Tukey’s test for multiple comparisons was used. Data are presented as mean ± s.e.m. RESULTS HCN inhibitor analogs were designed to increase neural tropism

[0031] Cilobradine is a known HCN channel inhibitor that rapidly reduces stress-inducedbehavioral outcomes in mice, viewed as a model of symptoms mirroring those seen in human depression (34). However, Cilobradine, Zatebradine, and Ivabradine were not designed to target brain function and cross the BBB. Therefore, in this study, we created a series of analogs of Cilobradine and Zatebradine aiming to improve BBB permeability as well as HCN modulation of the dopaminergic system and associated stress-induced behaviors. We employed a central nervous system multiparameter optimization (CNS MPO) approach to improve BBB permeability of Cilobradine and Zatebradine while maintaining or improving their HCN channel inhibitory activity (53,54). This MPO approach focuses on six fundamental physicochemical properties: (1) lipophilicity, calculated partition coefficient (ClogP); (2) calculated distribution coefficient at pH 7.4 (ClogD); (3) molecular weight (MW); (4) topological polar surface area (TPSA); (5) number of hydrogen-bond donors (HBDs); and (6) most basic center (pKa) that were determined as important factors for CNS drugs (53,54). We specifically focused on two of these parameters, MW and pKa and tried to reduce both while keeping the other four parameters still favorable to avoid disrupting binding of these compounds to the HCN channel. To this end, we kept the right-hand side dimethoxy-tetrahydro-benzoazepin-2-one moiety, which is a common structural feature of Cilobradine, Ivabradine and Zatebradine (Fig. 1a). On the other hand, to reduce the pKa of the piperidine moiety of Cilobradine, we introduced an oxygen to the benzylic position of the left- hand side dimethoxy phenyl group while simultaneously modifying the substituents on this phenyl ring to reduce the MW as illustrated by compounds 1-5 (Fig. 1b, modified groups highlighted inblue). To reduce the MW, we started by removing one of the methoxy groups first (compound 1to compound 2, named MS7710), and then both methoxy groups (compound 3) and furthermodified the phenyl group to obtain o-fluoro (compound 4) or p-fluoro (compound 5, namedMS7712) derivatives. Compound 6 was designed to replace the central piperidine ring ofCilobradine with a morpholine ring (the added ring oxygen is highlighted in orange, Fig.1b), whileAttorney Docket: 27527-0224WO1compound 7 featured both a left-hand side modification replacing dimethoxy phenyl group withpara-fluoro phenyl group and the morpholine ring replacement (Fig. 1b, modified groups arehighlighted in blue and orange). Furthermore, a Zatebradine analog (compound 8) where the left-hand side benzylic methylene group was replaced with the difluoromethylene group was also designed (Fig. 1b, modified groups highlighted in pink). All these structural modifications were aimed at reducing the pKa of the most basic center as well as the molecular weight of the compounds to improve BBB permeability.Analogs exhibited a variety of Ih inhibition in VTA DA neurons compared to parentcompounds

[0032] Following analog design and synthesis, we first tested these compounds for inhibitionof Ih currents in VTA DA neurons of stress naive, male, C57 / BL6 mice utilizing local applicationof compounds and whole-cell electrophysiology (Fig. 2a, b). Ih current sizes were compared pre-and post- compound application (Fig. 2c). As expected, parent compounds Ivabradine andZatebradine demonstrated significant inhibition of the Ih current when compared to the applicationof external solution with no compound present (vehicle; Fig. 2d). The Zatebradine analog, 8,demonstrated no efficacy against the HCN channel (Fig.2d). In agreement with previous reports,parent compound Cilobradine demonstrated significant inhibition of the VTA DA neuron Ihcurrent when compared to the vehicle (Fig.2e) (34). Cilobradine’s analogs exhibited a variety ofeffects; 11 demonstrated less than half the efficacy of Cilobradine, while 6, 4, and 3 improvedupon 11’s efficacy but were still less efficacious than Cilobradine. Compounds 1, 5 (MS7712) and2 (MS7710) had a similar efficacy to Cilobradine (Fig. 2e). Based on these results, MS7712 andMS7710 were chosen as the top two compounds of interest (sample traces in left panel of Fig 2e), as they have particularly interesting structural changes and lower MW for improvement of BBB permeability, and additionally, display high HCN inhibition. MS7710 and MS7712 improved FR inhibition in VTA DA neurons compared to parent compound Cilobradine

[0033] To test the modulatory effects of the newly designed HCN inhibitors on VTA DAneuronal activity, we examined the effect of the parent and top two compounds on the FR of VTAAttorney Docket: 27527-0224WO1 DA neurons utilizing cell-attached electrophysiology. We observed an expected decrease in FR following application of the parent compounds Zatebradine (59.7%) and Ivabradine (61.1%) ascompared to the vehicle (Fig. 1f). MS7712 and MS7710 also significantly decreased the FR of theVTA DA neurons as compared to Cilobradine, with average inhibition percentages of 87.8% and 90.6%, respectively, as compared to 61.8% (Fig. 1g). Together our results demonstrate that thenewly designed analogs of Cilobradine, MS7712, and MS7710, display inhibitory effects on Ihcurrents and FR of VTA DA neurons. MS7710 and MS7712 display improved BBB permeability

[0034] Following determination that Cilobradine analogs MS7710 and MS7712 inhibitedVTA DA neuron Ih currents and FRs, we conducted a mouse pharmacokinetic (PK) study forMS7710 and MS7712 with Cilobradine as a control in healthy, stress-naïve C57BL / 6 male mice.Each compound was assessed after a single 20 mg / kg IP injection in triplicate at 15 minutes, 1 hour, and 4 hours post-injection for their brain and plasma concentrations (Fig. 3a, 3b). As a general rule within a healthy brain, compounds with a brain plasma ratio less than 0.1 are considered impermeant of the BBB, compounds with a ratio of 0.3 to 0.5 are considered sufficiently permeant of the BBB, and compounds with a ratio greater than 1 can freely cross theBBB (55). Cilobradine had a low brain plasma ratio (0.076) (Fig. 3c). Both MS7710 and MS7712demonstrated improved brain plasma ratios of 0.28 and 0.57, respectively (Fig.3d, 3e). MS7710 decreased FR and bursting activity in VTA DA neurons of male and female mice

[0035] Our in vitro electrophysiology approaches give us access to the electrophysiologicalfiring properties of VTA DA neurons and Ih current amplitudes in response to local compoundapplications. However, in vivo, VTA DA neuron activity ranges from slow single-spike tonicfiring to high frequency, phasic burst firing, which is lost in acute ex vivo slice preparations but maintained in anesthetized animals (56). The distinct dopamine release induced by these transitions is crucial in integrating and processing salient, reward, and stressful events. Previous reports, including our own, established that VTA DA neurons of CSDS-susceptible mice display hypertonic and hyper bursting spontaneous FR compared to stress naïve mice, which in turnAttorney Docket: 27527-0224WO1 contributes to the induction of social and reward-related behavioral deficits (18). Thus, it is crucialto also assess in vivo the VTA DA neuron response to the newly designed HCN inhibitors.

[0036] CSDS-susceptible mice were anesthetized and VTA DA neuron activity was recordedusing in vivo, juxta-cellular, electrophysiological approaches (Fig. 4a). VTA DA neurons were identified using well-established in vivo electrophysiological criteria (Fig. 4b-d) (57). First, baseline measurements of VTA DA neuron spontaneous FR activity was recorded (Fig.4e-m). A 20 mg / kg IP injection of Cilobradine significantly reduced both the FR (Fig.4e, f) and the burstingactivity (Fig. 4g) of VTA DA neurons. We further tested the modulatory effects of MS7712 andMS7710 on VTA DA neurons in CSDS-susceptible mice. To determine dosage, we chose to startat a lower concentration of 10 mg / kg for MS7710 and MS7712 because of their increased brain-plasma ratio, as well as their increased inhibition of ex vivo VTA DA neuron FR, compared toCilobradine. At 10 mg / kg, MS7712 significantly reduced VTA DA neuron FR (Fig. 4h, i) andbursting activity (Fig. 4j), whereas an IP injection of 1 mg / kg did not (Fig. 4h-j). For MS7710,both 10 mg / kg and 1 mg / kg IP injections yielded significant reductions in VTA DA neuron FRs(Fig. 4k,l) and bursting activity (Fig. 4m). Importantly, the effect of MS7710 at 1 mg / kg, ourlowest tested dosage with significant inhibitory efficacy, was validated in female, CSDS- susceptible mice (Fig.5a), resulting in significant reductions in VTA DA neuron FR (Fig. 5b, c)and bursting activity (Fig.5d). Based on our in vitro and in vivo electrophysiological data, MS7710successfully modulates VTA DA neurons activity and rescues CSDS-induced VTA DAhyperactivity in vivo at lower doses than Cilobradine and MS7712.MS7710 improved SI ratios and cognitive flexibility in PRLT

[0037] We next tested if MS7710 ameliorates the behavioral phenotypes induced by CSDSexposure. Our previous studies and others have established that CSDS induces social and reward- related behavioral deficits, behavioral features that are similar to depressive symptoms in human patients(6). First, we piloted the effect of 1 mg / kg (IP injection) on CSDS-susceptible mice SI behaviors. We observed that treatment of 1 mg / kg was insufficient to restore long-lasting SIbehaviors in CSDS-susceptible mice (Fig. 13). Therefore, we tested a dose of 5 mg / kg MS7710on social behaviors. For this purpose, the mice underwent CSDS, followed by an SI test, and thenAttorney Docket: 27527-0224WO1 the PRLT, with a final SI test 2 weeks after compound injection (Fig.6a-c). The SI test measures the social interaction of the CSDS-susceptible mice with an unfamiliar mouse, and determines that mice are susceptible to CSDS if they exhibit an SI ratio below 1 (Fig.6b). The 5 mg / kg dose of MS7710 led to a significant improvement in the SI ratios of CSDS-susceptible mice two weeks after a single IP injection, from an average SI ratio of 0.69 to an average SI ratio of 1.05 (Fig.6d). Following the IP injection, there was no significant difference between the saline-treated or the MS7710-treated groups locomotion or velocity in the open field portion of the SI test (Fig.14).

[0038] Lastly, we tested the effect of MS7710 on reward-related behavioral deficits using thePRLT. Previous reports have established that chronic stress exposure alters reversal reward learning (24). During the PRLT, the mice first underwent a training phase to associate lever pressing with the delivery of a saccharin reward (Fig. 6b). They next underwent a probabilistic phase where they associated one lever with 80% chance of reward, and the other lever with 20% chance of reward delivery (Fig. 6b). Similar to previous reports, we observed that CSDS- susceptible mice successfully associated lever presses with reward deliveries at a similar progression to control mice (Fig 6e) (24). Following reward learning, the susceptible mice (n= 25)were injected with either saline (n=12) or 5 mg / kg MS7710 (n=13). The following day, theyunderwent the reversal phase, during which the contingencies were reversed between the two levers. We determined via a mixed-model ANOVA that there was a significant effect of time and a trending effect of condition (p = 0.0505) for sessions 1-13 of the reversal phase (Fig. 6f). Additionally, the number of trials until criteria is reached is significantly different across condition, driven by the difference between the susceptible + saline mice vs. the control mice or thesusceptible + MS7710 mice (Fig. 6f inset). Therefore, it took longer for the susceptible mice +saline to alter their behavior after the paradigm was reversed, demonstrating cognitive inflexibility.However, the administration of MS7710 to susceptible mice improved this deficit; their cognitiveflexibility was no different than that of control mice. Overall, MS7710 demonstrated good BBBpermeability and inhibition of Ih currents, FR, and bursting activity, which in turn translated intoamelioration of behavioral alterations in the SI test and PRLT, and represents an interesting potential therapeutic for future research for stress-related disorders. DISCUSSIONAttorney Docket: 27527-0224WO1

[0039] MDD is a multifactorial disease, for which chronic stress is a major trigger (2,58,59).While available treatments may reduce symptoms in MDD patients, their positive effects may take multiple months to work and be lacking in many patients, underscoring the need for novel alternative therapeutic strategies for MDD (60). In our study we utilized preclinical models for stress-induced physiological and behavioral impairments in mice. Similar to humans, mice exposed to chronic stress experience decreased motivation and impairments in social and reward processes – key features of human MDD. DA is a key factor in these behavioral alterations, but as shown in current antidepressants, targeting monoamines has limited efficacy. This is likely in part due to non-circuit-specific modulation and thus off-target side effects. Here, we leveraged HCN channels to regulate DA neuron activity and reduce the detrimental behavioral outcomes of chronic stress. We first designed analogs of existing HCN inhibitors to improve BBB permeability and increase the neural tropism of the compounds. Using ex vivo electrophysiological approaches, wevalidated their inhibitory effects on the Ih current in VTA DA neurons, and tested VTA DA FRinhibition of top compounds MS7710 and MS7712 and parent compounds. Seeing a stronginhibitory effect, we then affirmed the expected BBB permeability improvements of MS7710 andMS7712 compared to Cilobradine. As tonic and bursting VTA DA neuron activity are critical in the expression of CSDS-induced behavioral deficits, we confirmed, in vivo, the inhibitory effectof compounds MS7710 and MS7712 in CSDS-susceptible male mice on tonic and burstingactivity. Additionally, multiple studies have shown the high propensity of women to develop MDD and mood disorders compared to men (26,61,62). Critically, we recapitulated our in vivo physiological validation in CSDS-susceptible female mice. To finally establish the therapeuticeffect of MS7710, we tested and observed its rescue of social behaviors, reward-related behavioralresponses, and cognitive flexibility. Together, our results established the long-lasting effect of MS7710 on physiological and behavioral deficits induced by chronic stress exposure.

[0040] We tested analogs of a series of bradycardic agents: Cilobradine, Zatebradine, andIvabradine (63). All of these compounds are known to have some aspect of use-dependency, meaning that they favor a certain state of the channel such as open or closed, or a transition of those states, to cause their effect (63–66). These compounds are known to block most favorably as channels transition between states, therefore the rapid opening and closing of the channels such as the rhythmic or burst firing of VTA DA neurons enhances the blocking effect. Interestingly, weAttorney Docket: 27527-0224WO1saw that compounds MS7710 and MS7712 had a much stronger inhibitory effect than the parentcompounds on VTA DA FR activity. While their effect on a single Ih current was the same, wehypothesize that these compounds have an improved use-dependency, allowing them to exhibit an enhanced block upon repeated activation as seen in spontaneously active VTA DA neurons. This is an important characteristic of the compounds, because it means that the higher the FR (as seen in VTA DA neurons of CSDS-susceptible mice), the stronger the effect of the compounds. This also means that other HCN channels throughout the brain that are associated with slower firing neurons will not experience the same effect as those that are aberrantly hyperactive.

[0041] Mice susceptible to CSDS exhibit increased bursting and tonic FR activity in VTA DAneurons (18). Our previous research has causally linked VTA DA hyper-bursting activity to the expression of social avoidance behaviors induced by CSDS (17,18,67). More specifically, circuit- specific hyperactivity of VTA DA projections to the striatum, VTA-NAc, results in the social avoidance behaviors induced by CSDS (18). Notably, VTA-NAc projecting DA neurons,compared to other projections, have large Ih currents in stress-naïve mice, which are furtheraugmented following CSDS (19,44,68). Therefore, inhibiting the Ih current allows us to selectivelytarget the VTA-NAc pathway with limited effect on other VTA projections. Together with ourresults, the data indicate that compounds MS7710 and MS7712 may preferentially modulate thehyperactive HCN channels expressed by VTA-NAc DA neurons in CSDS-susceptible mice, therefore minimizing side effects from multiple sources. However, our focused studies of HCN inhibitors on VTA DA neuronal functions may constitute a limiting aspect of our investigations.Future studies investigating the effect of MS7710 across brain areas to examine potentiallysynergistic effects would help the greater understanding of how MS7710 ameliorates stress-induced behaviors. Our results established that MS7710 alleviated the impact of stress on VTADA neuronal activity in both male and female mice, due to a decrease in HCN channel activity in these neurons.

[0042] The goal of our compound design, in addition to improved efficacy, was to improvethe neural tropism of the analogs. While it was unknown if Cilobradine crossed the BBB, it is important for compounds targeting neuronal function to cross the BBB. Our experiments haveshown that Cilobradine has very limited BBB permeability, whereas both MS7710 and MS7712Attorney Docket: 27527-0224WO1 have improved BBB permeability. This is further evidenced by the efficacy of the compounds in in vivo electrophysiology and in vivo behavior following an IP injection. Assessment of BBB permeability was performed in stress-naïve mice. Recent studies have established that chronic stress exposure increases the BBB permeability in discrete brain areas, including within the mesolimbic system, contributing to the emergence of the observed pathological behaviors (69– 71). Therefore, we expect that a further augmented permeability of our compounds and Cilobradine in socially stressed subjects compared to stress-naïve individuals. Together, thisinformation suggests that MS7710 will yield improved BBB permeability and alleviate behavioralimpairments in stressed subjects.

[0043] A current challenge in the treatment of MDD is achieving rapid and long-lastingtherapeutic effects. Classical antidepressants such as fluoxetine must be taken chronically, and are known to take weeks to months before they begin to reduce MDD symptoms (60,72). Similarly, multiple weeks of daily treatment of these drugs is required to observe the amelioration of stress- induced behaviors in CSDS-susceptible mice (17,73). In this study, we observed a significanteffect of MS7710 within two weeks, following just a single, 5 mg / kg (but not 1 mg / kg) IP injection,a dose lower than that used for acute cilobradine or chronic fluoxetine administration in ourprevious research (17,34). MS7710 shows a moderate onset time for efficacy, with a long-lastingimpact following a single injection. Future studies investigating the molecular mechanisms of thelong-lasting and dose-dependent stress-ameliorating activity of MS7710 will provide valuableinformation to define the molecular underpinnings of long-lasting antidepressant effects.

[0044] In conclusion, we designed novel Cilobradine analogs MS7710 and MS7712 thatdemonstrated improved neural tropism via improved BBB permeability while maintaining theirinhibitory efficacy on HCN channel Ih currents and enhancing their inhibitory efficacy on FR inVTA DA neurons. MS7710 and MS7712 reduced the FR and bursting activity of VTA DAneurons from CSDS-susceptible mice at concentrations lower than Cilobradine, a finding that wasrecapitulated for MS7710 in female mice. Critically, we observed sustained positive effects ofMS7710 on CSDS-induced social deficits and cognitive inflexibility. These findings yield a compound with novel stress-alleviating effects that could provide a basis for future antidepressantAttorney Docket: 27527-0224WO1 drug discovery, with the potential to improve the symptoms of people with treatment-resistant depression. General Chemistry Methods

[0045] All chemical reagents were purchased from commercial vendors and used withoutfurther purification. The flash column chromatography was conducted using a Teledyne ISCO CombiFlash Rf+ instrument. This instrument was also equipped with a variable-wavelength UV detector and a fraction collector. RediSep Rf Gold C18 columns were used for purification. High- performance liquid chromatography (HPLC) spectra for compounds were acquired using an Agilent 1200 Series system with a DAD detector. Chromatography was performed on a 2.1 × 150 mm Zorbax 300SB-C185 m column with water containing 0.1% formic acid as solvent A and acetonitrile containing 0.1% formic acid as solvent B at a flow rate of 0.4 mL / min. The gradientprogram was as follows: 1% B (0 1 min), 1 99% B (1 4 min), and 99% B (4 8 min). Ultra-performance liquid chromatography (UPLC) spectra for compounds were acquired using a Waters Acquity I-Class UPLC system with a PDA detector. Chromatography was performed on a 2.1 × 30 mm ACQUITY UPLC BEH C181.7 m column with water containing 3% acetonitrile, 0.1% formic acid as solvent A and acetonitrile containing 0.1% formic acid as solvent B at a flow rateof 0.8 mL / min. The gradient program was as follows: 1 99% B (1 1.5 min), and 99 1% B(1.5 2.5 min). High-resolution mass spectra (HRMS) data were acquired in the positive ion modeusing Agilent G1969A API-TOF with an electrospray ionization (ESI) source. Nuclear magnetic resonance (NMR) spectra were acquired on a Bruker DXI 800 MHz spectrometer with 800 MHz for proton (1H NMR) or a Bruker DRX-600 spectrometer with 600 MHz for proton (1H NMR) or a Bruker DRX-500 spectrometer with 500 MHz for proton (1H NMR) or 400 MHz for proton (1HNMR) and 101 MHz for carbon (13C NMR). Chemical shifts are reported in ppm ( ). PreparativeHPLC was performed using an Agilent Prep 1200 series with UV detector set to 220 nm. Samples were injected into a Phenomenex Luna 75 × 30 mm, 5 m, C18 column at room temperature. The flow rate was 40 mL / min. A linear gradient was used with 10% of Acetonitrile (A) in H2O (with 0.1% TFA) (B) to 100% of Acetonitrile (A). All final compounds had > 95% purity using the UPLC and HPLC methods described above.Attorney Docket: 27527-0224WO1 Synthesis and Characterization of the Compounds 1-7Scheme 1. The syntheses of Compounds 1-7.benzo[d]azepin-2-one, NaH, DMF, 0°C-rt, 2h; (c)1. TFA, DCM, 2. K2CO3, DMF, rt, overnight.

[0046] tert-butyl(R)-3-((7,8-dimethoxy-2-oxo-1,2,4,5-tetrahydro-3H-benzo[d]azepin-3-yl)methyl)piperidine-1-carboxylate (11)

[0047] A solution of commercially available tert-butyl (S)-3-(hydroxymethyl)piperidine-1-carboxylate (9) (215 mg, 1 mmol) and CBr4(497 mg, 1.5 mmol) in 5 mL of DCM was treated PPh3 (288 mg, 1.1 mmol). The resulting mixture was stirred at room temperature (rt) for 1h. After the completion of the reaction, the mixture was poured into ice water, aqueous phase was extracted with ethyl acetate (EtOAc). The combined organic phase was washed with brine twice, dried and concentrated. The residue was purified by flash column chromatography (EtOAc / Petroleum ether= 1:10) to afford the desired tert-butyl (S)-3-(bromomethyl)piperidine-1-carboxylate as a whitesolid (195 mg, 70%). MS (ESI) m / z = 278.3 [M + H]+. This intermediate was immediately usedfor the next step without further characterization.Attorney Docket: 27527-0224WO1

[0048] A solution of 7,8-dimethoxy-1,3,4,5-tetrahydro-2H-benzo[d]azepin-2-one (221 mg, 1mmol) and NaH (48 mg, 1.2 mmol) in 5 mL of DMF was stirred at 0 °C for 0.5h. Then tert-butyl(S)-3-(bromomethyl)piperidine-1-carboxylate (278 mg, 1 mmol) described above was added to the solution and the resulting mixture was stirred at rt for 2h. After the reaction was completed, the reaction mixture then was poured into ice water, aqueous phase was extracted with ethyl acetate. The combined organic phase was washed with brine twice, dried and concentrated. The residue was purified by flash column chromatography with reverse phase C18 column (10% - 100%methanol / 0.1% TFA in water) to afford the compound 11 as a white solid (272 mg, 65%). MS(ESI) m / z = 419.5 [M + H]+, calculated for 419.3.

[0049] (S)-3-((1-(2-(3,4-dimethoxyphenoxy)ethyl)piperidin-3-yl)methyl)-7,8-dimethoxy-1,3,4,5-tetrahydro-2H-benzo[d]azepin-2-one (1)

[0050] Intermediate 11 (418 mg, 1 mmol) was dissolved in 4 ml 4 N HCl in Dioxane, and themixture was stirred at rt for 1h. After the reaction was completed, the reaction mixture was evaporated and the residue was purified by reverse phase C18 column (10% - 100% methanol / 0.1% TFA in water) to afford the intermediate, which was directly used for the next step without further purification.

[0051] A solution of the above deprotected intermediate (100 mg, 0.3 mmol) and NaH (17 mg,0.5 mmol) in 3 mL of DMF was stirred at 0 °C for 0.5h. Then after the addition of 4-(2- bromoethoxy)-1,2-dimethoxybenzene (94 mg, 0.36 mmol) the resulting reaction mixture was stirred at rt overnight. After the reaction was completed, the resulting mixture was purified bypreparative HPLC to give the compound 1 as a white solid (74.8 mg, 50%). 1H NMR (800 MHz,Methanol-d4) 6.86 (d, J = 8.8 Hz, 1H), 6.70 – 6.58 (m, 3H), 6.44 (dt, J3.3 Hz, 1H), 4.24(d, J = 5.1 Hz, 2H), 3.82 (d, J = 13.0 Hz, 4H), 3.80 – 3.74 (m, 12H), 3.63 (dt, J = 12.1, 3.0 Hz,1H), 3.51 (dddd, J = 45.3, 19.4, 13.8, 6.9 Hz, 4H), 3.28 (dd, J = 13.8, 5.7 Hz, 1H), 3.07 (t, J = 6.2Hz, 2H), 3.00 – 2.95 (m, 1H), 2.80 (t, J = 12.2 Hz, 1H), 2.29 (s, 1H), 2.00 (d, J = 13.7 Hz, 1H),1.86 (t, J = 14.2 Hz, 2H), 1.32 – 1.22 (m, 1H). MS (ESI) m / z = 499.3 [M + H]+, calculated for499.3.Attorney Docket: 27527-0224WO1

[0052] The same synthetic route (Scheme 1) and procedure for the synthesis of compound 1described above was also used to obtain compounds 2-5.

[0053] (S)-7,8-dimethoxy-3-((1-(2-(3-methoxyphenoxy)ethyl)piperidin-3-yl)methyl)-1,3,4,5-tetrahydro-2H-benzo[d]azepin-2-one (2, MS7710) 1H NMR (400 MHz, CD3OD) 7.16(t, J = 8.3 Hz, 1H), 6.64 (s, 1H), 6.60 (s, 1H), 6.54 – 6.49 (m, 3H), 4.36 – 4.26 (m, 2H), 3.79 –3.68 (m, 13H), 3.59 – 3.40 (m, 5H), 3.27 – 3.19 (m, 1H), 3.10 – 2.91 (m, 3H), 2.78 (t, J = 12.4 Hz,1H), 2.41 – 2.24 (m, 1H), 2.04 – 1.85 (m, 2H), 1.82 – 1.75 (m, 1H), 1.34 – 1.12 (m, 1H).13C NMR (101 MHz, CD3OD) 175.68, 162.39, 160.17, 149.44, 148.54, 131.21, 129.19, 124.20, 115.26, 114.98, 108.21, 107.71, 102.24, 63.15, 57.59, 57.34, 56.43, 56.38, 55.81, 54.59, 50.32, 47.96,42.71, 35.59, 32.60, 27.03, 23.71. HRMS (ESI-TOF) m / z: [M + H]+ calcd for C27H37N2O5,469.2697; found, 469.2692.

[0054] (S)-7,8-dimethoxy-3-((1-(2-phenoxyethyl)piperidin-3-yl)methyl)-1,3,4,5-tetrahydro-2H-benzo[d]azepin-2-one (3)

[0055] 1H NMR (800 MHz, Methanol-d4) 7.31 (t, J = 7.7 Hz, 2H), 7.00 (t, J = 7.3 Hz, 1H),6.95 (d, J = 8.0 Hz, 2H), 6.67 (d, J = 14.6 Hz, 2H), 4.31 (s, 2H), 3.82 (dd, J = 11.5, 5.3 Hz, 3H),3.79 – 3.75 (m, 7H), 3.64 (d, J = 12.7 Hz, 1H), 3.57 – 3.47 (m, 4H), 3.29 (dd, J = 13.8, 5.7 Hz,1H), 3.07 (t, J = 6.2 Hz, 2H), 3.00 (t, J = 12.7 Hz, 1H), 2.82 (t, J = 12.3 Hz, 1H), 2.27 (s, 1H), 2.01(d, J = 15.0 Hz, 1H), 1.85 (d, J = 13.1 Hz, 2H), 1.30 (dd, J = 15.5, 11.7 Hz, 1H). MS (ESI) m / z =439.4 [M + H]+, calculated for 439.3.

[0056] (S)-3-((1-(2-(2-fluorophenoxy)ethyl)piperidin-3-yl)methyl)-7,8-dimethoxy-1,3,4,5-tetrahydro-2H-benzo[d]azepin-2-one (4)

[0057] 1H NMR (800 MHz, Methanol-d4) 7.13 (p, J = 8.0, 7.0 Hz, 3H), 7.04 – 6.99 (m, 1H),6.67 (d, J = 14.8 Hz, 2H), 4.40 (q, J = 5.5 Hz, 2H), 3.91 – 3.81 (m, 4H), 3.78 (s, 3H), 3.77 – 3.75(m, 4H), 3.70 (dq, J = 12.1, 2.9 Hz, 1H), 3.62 – 3.55 (m, 3H), 3.52 (dd, J = 13.7, 8.5 Hz, 1H), 3.08(t, J = 6.1 Hz, 2H), 3.06 – 2.98 (m, 1H), 2.86 (t, J = 12.2 Hz, 1H), 2.33 – 2.26 (m, 1H), 2.04 – 1.98(m, 1H), 1.86 (d, J = 12.8 Hz, 2H), 1.33 – 1.25 (m, 1H). MS (ESI) m / z = 457.4 [M + H]+, calculatedfor 457.2.Attorney Docket: 27527-0224WO1

[0058] (S)-3-((1-(2-(4-fluorophenoxy)ethyl)piperidin-3-yl)methyl)-7,8-dimethoxy-1,3,4,5-tetrahydro-2H-benzo[d]azepin-2-one (5, MS7712). 1H NMR (400 MHz, CD3OD) 7.00(t, J = 8.4 Hz, 2H), 6.92 – 6.86 (m, 2H), 6.65 (s, 1H), 6.60 (s, 1H), 4.30 – 4.21 (m, 2H), 3.83 –3.71 (m, 10H), 3.61 – 3.40 (m, 5H), 3.26 – 3.16 (m, 1H), 3.09 – 2.92 (m, 3H), 2.86 – 2.73 (m, 1H),2.36 – 2.23 (m, 1H), 2.02 – 1.90 (m, 1H), 1.89 – 1.76 (m, 2H), 1.33 – 1.16 (m, 1H). 13C NMR (101MHz, CD3OD) 175.87, 159.17 (d, 1JC-F = 236.8 Hz), 155.26, 155.24, 149.51, 148.62, 129.16,124.22, 117.02 (3JC-F= 7.9 Hz), 116.82, 115.12 (2JC-F= 30.5 Hz), 63.81, 57.68, 57.44, 56.41, 56.37, 54.61, 50.50, 48.08, 42.67, 35.73, 32.73, 27.06, 23.75. HRMS (ESI-TOF) m / z: [M + H]+calcd for C26H34FN2O4, 457.2497; found, 457.2531.

[0059] The same procedure for the synthesis of compound 1, which was described above(Scheme 1) was employed using tert-butyl (S)-2-(hydroxymethyl)morpholine-4-carboxylate (10)as the starting material instead of 9 to obtain the compounds 6 and 7.

[0060] (S)-3-((4-(3,4-dimethoxyphenethyl)morpholin-2-yl)methyl)-7,8-dimethoxy-1,3,4,5-tetrahydro-2H-benzo[d]azepin-2-one (6)

[0061] 1H NMR (800 MHz, Methanol-d4) 6.91 (d, J = 8.1 Hz, 1H), 6.86 (s, 1H), 6.79 (d, J =8.1 Hz, 1H), 6.72 (s, 1H), 6.70 (s, 1H), 4.16 (d, J = 13.4 Hz, 1H), 3.97 (d, J = 9.4 Hz, 1H), 3.90(ddd, J = 15.3, 11.0, 4.4 Hz, 4H), 3.84 (s, 3H), 3.81 (s, 3H), 3.78 (s, 6H), 3.70 (dd, J = 14.7, 3.9Hz, 1H), 3.59 – 3.48 (m, 4H), 3.33 (s, 1H), 3.11 (dd, J = 16.0, 10.4 Hz, 4H), 2.92 (t, J = 8.5 Hz,2H), 2.81 (t, J = 11.9 Hz, 1H). MS (ESI) m / z = 485.4 [M + H]+. calculated for 485.3.

[0062] (S)-3-((4-(2-(4-fluorophenoxy)ethyl)morpholin-2-yl)methyl)-7,8-dimethoxy-1,3,4,5-tetrahydro-2H-benzo[d]azepin-2-one (7)

[0063] 1H NMR (500 MHz, Methanol-d4) 7.07 – 7.01 (m, 2H), 7.00 – 6.95 (m, 2H), 6.70 (s,1H), 6.67 (s, 1H), 4.32 (td, J = 4.9, 1.5 Hz, 2H), 4.15 (dd, J = 13.1, 3.7 Hz, 1H), 4.02 (t, J = 6.5Hz, 1H), 3.93 – 3.85 (m, 4H), 3.82 (s, 1H), 3.79 (s, 3H), 3.78 (s, 3H), 3.69 (dd, J = 14.4, 4.2 Hz,1H), 3.57 (ddt, J = 14.4, 9.8, 5.5 Hz, 5H), 3.21 (t, J = 11.1 Hz, 1H), 3.13 – 3.07 (m, 2H), 2.95 (t, J= 11.8 Hz, 1H). MS (ESI) m / z = 459.3 [M + H]+, calculated for 459.2.Attorney Docket: 27527-0224WO1

[0064] Scheme 2. Synthesis of compound 8.F FMeF FMeMeOIa, bMeO NHcMeO NHdrt,2h; (c) BH3.THF, THF, 0 °C- 65 °C, 40h; (d) 1,3-dibromopropane, K2CO3, Acetone, rt, overnight; (e) 7,8-dimethoxy-1,3,4,5-tetrahydro-2H-benzo[d]azepin-2-one, NaH, DMF, 0 °C-rt, 2h.

[0066] 2-(3,4-dimethoxyphenyl)-2,2-difluoro-N-methylacetamide (14)

[0067] To a solution of 4-iodo-1,2-dimethoxybenzene (13) (290 mg, 1.1 mmol) and ethyl 2-bromo-2,2-difluoroacetate (203 mg, 1 mmol) in 2 mL of DMSO was added Cu power (147 mg, 2.3 mmol) and the resulting mixture was stirred at 50°C for 15h. After the reaction was completed, the reaction mixture was poured into ice water, aqueous phase was extracted with ethyl acetate. The combined organic phase was washed with brine twice, dried and concentrated. The residue was purified by flash column (EtOAc / Petroleum ether= 1:5) to afford the desired ethyl 2-(3,4- dimethoxyphenyl)-2,2-difluoroacetate as a white solid (182 mg, 70%), which was used for the next step without further characterization.

[0068] A solution of ethyl 2-(3,4-dimethoxyphenyl)-2,2-difluoroacetate (260 mg, 1 mmol) and2N CH3NH2in THF (5 mL) was stirred at rt for 2h. After the reaction was completed, the reaction mixture was poured into ice water, aqueous phase was extracted with ethyl acetate. The combined organic phase was washed with brine twice, dried and concentrated. The residue was purified by flash column chromatography by using reverse phase C18 column (10% - 100% methanol / 0.1%Attorney Docket: 27527-0224WO1TFA in water) to afford the compound 18 as a colorless liquid (147 mg, 60%). MS (ESI) m / z =246.5 [M + H]+, calculated for 246.1.

[0069] This intermediate was used for the next step without further characterization.

[0070] 2-(3,4-dimethoxyphenyl)-2,2-difluoro-N-methylethan-1-amine (15)

[0071] A solution of compound 14 (245 mg, 1 mmol) in 3 mL of THF was stirred at 0 °C, thenaddition of 3mL of 2N BH3.THF. The resulting mixture was stirred at 65 °C for 40h. After the reaction was completed, the reaction mixture was poured into ice water, aqueous phase was extracted with ethyl acetate. The combined organic phase was washed with brine twice, dried and concentrated. The residue was purified by reverse phase C18 column (10% - 100% methanol / 0.1% TFA in water) to afford the compound 15 as a white solid (139 mg, 60%). MS (ESI) m / z =232.5 [M + H]+, calculated for 232.1. This intermediate was used for the next step without furthercharacterization.

[0072] 3-bromo-N-(2-(3,4-dimethoxyphenyl)-2,2-difluoroethyl)-N-methylpropan-1-amine (16)

[0073] A solution of compound 15 (231 mg, 1 mmol) and 1,3-dibromopropane (1.01 g, 5mmol) in 5 mL of Acetone was stirred at rt overnight. After the reaction was completed, the reaction mixture was poured into ice water, aqueous phase was extracted with ethyl acetate. The combined organic phase was washed with brine twice, dried and concentrated. The residue was purified by reverse phase C18 column (10% - 100% methanol / 0.1% TFA in water) to afford thecompound 16 as a white solid in TFA salt form (176 mg, 50%). MS (ESI) m / z = 352.5 [M + H]+,calculated for 352.1. This intermediate was used for the next step without further characterization.

[0074] 3-(3-((2-(3,4-dimethoxyphenyl)-2,2-difluoroethyl)(methyl)amino)propyl)-7,8-dimethoxy-1,3,4,5-tetrahydro-2H-benzo[d]azepin-2-one (8)

[0075] A solution of 7,8-dimethoxy-1,3,4,5-tetrahydro-2H-benzo[d]azepin-2-one (110 mg,0.5 mmol) and NaH (32 mg, 0.8 mmol) in 5 mL of DMF was stirred at 0 °C for 0.5h. Then additionof 16 (176 mg, 0.5 mmol) and the mixture was stirred at rt for 2h. After the reaction was completed,the reaction mixture was poured into ice water, aqueous phase was extracted with ethyl acetate. The combined organic phase was washed with brine twice, dried and concentrated. The resultingmixture was purified by preparative HPLC to give the compound 8 as a white solid (74 mg, 30%).Attorney Docket: 27527-0224WO11H NMR (600 MHz, Methanol-d4) 7.18 (dd, J = 8.4, 2.2 Hz, 1H), 7.14 (d, J = 2.2 Hz, 1H), 7.08(d, J = 8.4 Hz, 1H), 6.71 (s, 1H), 6.69 (s, 1H), 4.07 – 3.97 (m, 2H), 3.88 (s, 3H), 3.87 (s, 3H), 3.86– 3.84 (m, 4H), 3.79 (s, 3H), 3.78 (s, 3H), 3.54 (t, J = 6.6 Hz, 2H), 3.27 (t, J = 7.8 Hz, 2H), 3.11(dd, J = 8.1, 4.2 Hz, 2H), 3.05 (s, 3H), 2.10 (p, J = 6.9 Hz, 2H).Attorney Docket: 27527-0224WO1 REFERENCES 1. 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Claims

Attorney Docket: 27527-0224WO1 WE CLAIM:

1. (S)-7,8-dimethoxy-3-((1-(2-(3-methoxyphenoxy)ethyl)piperidin-3-yl)methyl)-1,3,4,5-tetrahydro-2H-benzo[d]azepin-2-one (1,MS7710) and pharmaceutically acceptable salts thereof.

2. (S)-3-((1-(2-(3,4-dimethoxyphenoxy)ethyl)piperidin-3-yl)methyl)-7,8-dimethoxy-1,3,4,5-tetrahydro-2H-benzo[d]azepin-2-one (1) and pharmaceutically acceptable salts thereof.

3. (S)-7,8-dimethoxy-3-((1-(2-phenoxyethyl)piperidin-3-yl)methyl)-1,3,4,5-tetrahydro-2H-benzo[d]azepin-2-one (3) and pharmaceutically acceptable salts thereof.

4. (S)-3-((1-(2-(2-fluorophenoxy)ethyl)piperidin-3-yl)methyl)-7,8-dimethoxy-1,3,4,5-tetrahydro-2H-benzo[d]azepin-2-one (4) and pharmaceutically acceptable salts thereof.

5. (S)-3-((1-(2-(4-fluorophenoxy)ethyl)piperidin-3-yl)methyl)-7,8-dimethoxy-1,3,4,5-tetrahydro-2H-benzo[d]azepin-2-one (5, MS7712) and pharmaceutically acceptable salts thereof.

6. (S)-3-((4-(3,4-dimethoxyphenethyl)morpholin-2-yl)methyl)-7,8-dimethoxy-1,3,4,5-tetrahydro-2H-benzo[d]azepin-2-one (6) and pharmaceutically acceptable salts thereof.

7. (S)-3-((4-(2-(4-fluorophenoxy)ethyl)morpholin-2-yl)methyl)-7,8-dimethoxy-1,3,4,5-tetrahydro-2H-benzo[d]azepin-2-one (7) and pharmaceutically acceptable salts thereof.

8. 3-(3-((2-(3,4-dimethoxyphenyl)-2,2-difluoroethyl)(methyl)amino)propyl)-7,8-dimethoxy-1,3,4,5-tetrahydro-2H-benzo[d]azepin-2-one (8) and pharmaceutically acceptable salts thereof.

9. A composition comprising a compound according to any one of claims 1 – 8, and apharmaceutically acceptable carrier.

10. A method of treating major depressive disorder, comprising administering atherapeutically effective amount of a compound according to any of claims 1 – 8 to a subject in need thereof.

11. A method of treating major depressive disorder, comprising administering atherapeutically effective amount of a composition according to claim 9 to a subject in need thereof.

12. A method according to any one of claims 10 or 11, wherein the composition isadministered at a dosage of from 1 to 50 mg / kg / day.Attorney Docket: 27527-0224WO113. The method of claim 12, wherein the dosage is from 1 to 20 mg / kg / day.

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