Derivatives of substituted morpholines and their use

Abstract

The compounds of formula I include their stereoisomers and / or salts thereof, where R 1 X is a substituted alkane group, a heterocyclic group, or a pyridine group, and X is hydrogen, a halogen, an amino acid residue, a substituted amino acid residue, an alkyl group, or an ester. Such compounds (I) may be used in pharmaceutical compositions and for the treatment of central nervous system (CNS) disorders. [Formula 1] JPEG2026513169000244.jpg51159

Description

[Technical Field]

[0001] Priority This application claims priority to U.S. Provisional Patent Application No. 63 / 454,930, filed on 27 March 2023, which is incorporated herein by reference in whole.

[0002] This technology broadly relates to derivatives of substituted morpholines, their use in pharmaceutical compositions, and their use for the treatment of central nervous system (CNS) disorders. [Background technology]

[0003] (R,S)-2-[(2-ethoxyphenoxy)methyl]morpholine: [ka] It is a bicyclic morpholine derivative and has been assigned CAS number 46817-91-8 (the HCl salt has CAS number 35604-67-2). Formula C 13 H 19 It is characterized by NO3 and has a molecular weight of 237.295 g / mol.

[0004] 2-((2-ethoxyphenoxy)methyl)morpholine is known to have several desirable pharmacological uses, including, among others, the treatment of depression, nocturnal enuresis, narcolepsy, sleep disorders, and alcoholism. 2-((2-ethoxyphenoxy)methyl)morpholine was previously marketed in several European countries for the treatment of major depressive disorder (MDD). 2-((2-ethoxyphenoxy)methyl)morpholine is a norepinephrine reuptake inhibitor ("NRI"), but may also enhance the release of serotonin from storage sites in nerve cells.

[0005] However, treatment with 2-((2-ethoxyphenoxy)methyl)morpholine has been associated with numerous side effects, including nausea, vomiting, loss of appetite, increased erythrocyte sedimentation rate, EKG and EEG abnormalities, epigastric pain, diarrhea, constipation, dizziness, orthostatic hypotension, lower extremity edema, dysarthria, tremors, psychomotor agitation, mental confusion, inappropriate secretion of antidiuretic hormone, increased transaminases, and seizures.

[0006] To minimize the side effects associated with 2-((2-ethoxyphenoxy)methyl)morpholine, chemists have synthesized derivatives and analogs that retain the pharmacological properties of 2-((2-ethoxyphenoxy)methyl)morpholine. Derivatives of substituted morpholines have been previously disclosed in the art, for example, in UK Patent No. 1243391 and UK Patent No. 1260886. The present inventors synthesize novel derivatives of substituted morpholines using a different approach. Prodrugs are often a class of derivatives that have little or no pharmacological activity and are converted into therapeutically active compounds in vivo. In some cases, the prodrug itself may have biological activity. Activation of a prodrug can occur by enzymatic or non-enzymatic cleavage of a transient bond between the carrier and the drug molecule, or a sequential or simultaneous combination of both.

[0007] Novelly synthesized derivatives of substituted morpholines, obtained by derivatization of the amine group of morpholine in the structure of 2-((2-ethoxyphenoxy)methyl)morpholine, produce chemically stable compounds that function as novel compounds. These derivatives of 2-((2-ethoxyphenoxy)methyl)morpholine can be used in pharmaceutical compositions and for the treatment of central nervous system (CNS) disorders. [Overview of the Initiative]

[0008] In some embodiments, treatment of a central nervous system (CNS) disorder is provided, the treatment comprising administering to a subject that needs it a pharmaceutical composition comprising a derivative of a substituted morpholine (including a compound of Formula I, II, III, or IV). In one embodiment, a compound of Formula I, its stereoisomers, or its salts: [Chemical formula] is utilized, which is a derivative of a substituted morpholine. In Formula I, R 1 can be alkyl, heterocyclyl, or pyridyl, and R 2 can be alkyl, aryl, heteroaryl, or heterocyclyl, and R 3 ~R 14 can each independently be H, F, Cl, Br, I, CN, NO2, alkyl, aryl, heteroaryl, or heterocyclyl, and X can be H, F, Cl, Br, I, an amino acid residue, a substituted amino acid residue, alkyl, or an ester.

[0009] In some embodiments, the technology relates to derivatives of substituted morpholines corresponding to a compound of Formula II, its stereoisomers, and / or its salts, and these are utilized. [Chemical formula] In Formula II, L can be alkyl, a substituted pyridinecarboxylic acid, or a substituted azanediylacetate, and R 2 can be alkyl, aryl, heteroaryl, or heterocyclyl, and R 3 ~R 14 14 can each independently be H, F, Cl, Br, I, CN, NO2, alkyl, aryl, heteroaryl, or heterocyclyl.

[0010] In some embodiments, the technology utilizes derivatives of substituted morpholines corresponding to a compound of Formula III, its stereoisomers, and / or its salts. [Chemical formula] In formula III, Y can be F, Cl, Br, I, an amino acid residue, a substituted amino acid residue, an alkyl, or an ester, and R 2 R can be alkyl, aryl, heteroaryl, or heterocyclyl, 3 ~R 14 Each of these elements can independently be H, F, Cl, Br, I, CN, NO2, alkyl, aryl, heteroaryl, or heterocyclyl.

[0011] In some embodiments, derivatives of substituted morpholines are formula IV, its stereoisomers, and / or salts thereof: [ka] Something equivalent will be used. In formula IV, Z can be H, F, Cl, Br, I, an amino acid residue, a substituted amino acid residue, or a nitrogen-containing group, and R 2 R can be alkyl, aryl, heteroaryl, or heterocyclyl, 3 ~R 14 Each of these elements can independently be H, F, Cl, Br, I, CN, NO2, alkyl, aryl, heteroaryl, or heterocyclyl.

[0012] In some embodiments, CNS disorders include, but are not limited to, depression, attention deficit hyperactivity disorder (ADHD), sleep disorders (e.g., cataplexy, narcolepsy, REM sleep behavior disorder), emotional blunting, cognition, anxiety, orthostatic hypotension, and pain, as well as neurological disorders (e.g., Parkinson's disease, Alzheimer's disease, Lewy body dementia, multiple system atrophy). Preferably, the subject suffering from the CNS disorder is a human. [Brief explanation of the drawing]

[0013] [Figure 1A] The in vitro stability of compound A is demonstrated by showing the retention rate of compound A in human plasma. [Figure 1B]The in vitro stability of compound B is demonstrated by showing the retention rate of compound B in human plasma. [Figure 1C] The in vitro stability of compound C is demonstrated by showing the retention rate of compound C in human plasma. [Figure 2] This study demonstrates the stability of compound A in three different fluid matrices, each modeling a different body fluid: SGF (stomach, pH 2.0), SIF (upper intestinal tract, pH 6.0), and PBS (whole body, pH 7.4). [Figure 3] This shows the stability of compound A in human blood components (red blood cells, plasma, and whole blood, respectively). [Figure 4] The stability of compound A in human blood is shown for use with a protease inhibitor, without a protease inhibitor, and incubated at pH 6.0, respectively. [Figure 5] A represents the hemolytic activity of compound A in human blood. B represents the hemolytic activity of amphotericin B (positive assay control) in human blood. [Figure 6] This shows the stability of compound A in rat blood components (red blood cells, plasma, and whole blood, respectively). [Figure 7] A shows the hemolytic activity of compound A in rat blood. B shows the hemolytic activity of amphotericin B (positive assay control) in rat blood. [Figure 8] This shows the stability of compound A in human and rat feces at pH 6.0 or 7.4. [Figure 9A] This shows the stability of compound A in phosphate-buffered saline (PBS) in the presence of amidase. The stability is represented by peak area. [Figure 9B] This shows the stability of compound A in phosphate-buffered saline (PBS) in the presence of amidase. The change is expressed as a relative percentage change to the control. [Figure 9C] This shows the stability of compound A in phosphate-buffered saline in the presence of amidase and an amidase inhibitor (chloroacetone or MAFP). [Figure 10]A and B show the stability of compound A in human plasma in the presence of an amidase inhibitor (chloroacetone or MAFP). A is expressed as peak area, and B is expressed as the percentage change relative to the control. [Figure 11] A and B show the stability of compound A in human plasma in the presence of an amidase inhibitor and / or esterase inhibitor. The amidase inhibitors tested were chloroacetone and MAFP. The esterase inhibitor tested was everactone. A is expressed as peak area, and B is expressed as the percentage change relative to the control. [Figure 12] Figures A and B show the stability of compound A in the presence of a protease inhibitor cocktail in human plasma. This cocktail contained 104 mM AEBSF, 80 μM aprotinin, 4 mM bestatin, 1.4 mM E-64, 2 mM leupeptin, and 1.5 mM pepstatin A. Dilutions of the protease inhibitor cocktail were prepared in plasma at final dilutions of 1:50 and 1:10. Figure A is expressed as peak area, and Figure B is expressed as the percentage change relative to the control. [Figure 13] A and B show the stability of compound A in rat plasma in the presence of an amidase inhibitor (chloroacetone or MAFP). A is expressed as peak area, and B is expressed as the percentage change relative to the control. [Figure 14] A and B show the stability of compound A in rat plasma in the presence of an amidase inhibitor and / or an esterase inhibitor. The amidase inhibitors tested were chloroacetone and MAFP. The esterase inhibitor tested was everactone. A is expressed as peak area, and B is expressed as the percentage change relative to the control. [Figure 15] A and B show the stability of compound A in rat plasma in the presence of a protease inhibitor cocktail. Dilutions of the protease inhibitor cocktail were prepared in plasma at final dilutions of 1:50 and 1:10. A is expressed as peak area, and B is expressed as the percentage change relative to the control. [Figure 16]This report compares enzyme inhibition after a 60-minute incubation. Tests were conducted under three conditions: without inhibitor, in the presence of MAFP, and in the presence of everactone A. [Figure 17] A and B show the individual and mean concentration-time profiles of biloxazine in plasma (A) and brain (B) after IV administration of compound A to male CD-1 mice at 9.911 mg / kg. [Figure 18] A and B show the individual and mean concentration-time profiles of biloxazine in plasma (A) and brain (B) after IV administration of compound B at 10.439 mg / kg to male CD-1 mice. [Figure 19] A and B show the individual and mean concentration-time profiles of biloxazine in plasma (A) and brain (B) after IV administration of compound C at 10.557 mg / kg to male CD-1 mice. [Figure 20A] The individual and mean plasma concentration-time profiles of S(-)-piroxazine after a single PO administration to male CD-1 mice are shown. The plasma concentration-time profiles of S(-)-piroxazine after administration of compound A to mice at 19.82 mg / kg are also shown. [Figure 20B] The individual and mean plasma concentration-time profiles of R(+)-biloxazine after a single PO administration to male CD-1 mice are shown. The plasma concentration-time profiles of R(+)-biloxazine after administration of compound A to mice at 19.82 mg / kg are also shown. [Figure 20C] The individual and mean plasma concentration-time profiles of S(-)-biloxazine after a single PO administration to male CD-1 mice are shown. The plasma concentration-time profiles of S(-)- after administration of compound B to mice at 21.03 mg / kg are also shown. [Figure 20D] The individual and mean plasma concentration-time profiles of R(+)- biloxazine after a single PO administration to male CD-1 mice are shown. The plasma concentration-time profiles of R(+)- after administration of compound B to mice at 21.03 mg / kg are also shown. [Figure 20E]The individual and mean plasma concentration-time profiles of S(-)-biloxazine after a single PO administration to male CD-1 mice are shown. The plasma concentration-time profiles of S(-)- after administration of compound C to mice at 21.11 mg / kg are also shown. [Figure 20F] The individual and mean plasma concentration-time profiles of R(+)- biloxazine after a single PO administration to male CD-1 mice are shown. The plasma concentration-time profiles of R(+)- after administration of compound C to mice at 21.11 mg / kg are also shown. [Figure 20G] The individual and mean plasma concentration-time profiles of S(-)- piroxazine after a single PO administration to male CD-1 mice are shown. The plasma concentration-time profiles of S(-)- after administration of racemic piroxazine at 11.64 mg / kg to mice are also shown. [Figure 20H] The individual and mean plasma concentration-time profiles of R(+)- biloxazine after a single PO administration to male CD-1 mice are shown. The plasma concentration-time profiles of R(+)- after administration of racemic biloxazine at 11.64 mg / kg to mice are also shown. [Figure 21] Individual and mean plasma concentration-time profiles of S(-)-biloxazine after a single oral administration of 40 mg / kg to fasted male Sprague-Dawley rats are shown. [Figure 22] A and B show the individual and mean plasma concentration-time profiles of S(-)-biloxazine after a single oral administration of compound A at 60 mg / kg (A) or 120 mg / kg (B) to fasted male Sprague-Dawley rats. [Figure 23] The plasma concentration-time profiles of S(-)-biloxazine after a single PO administration of compound A at 80 mg / kg to male (top) and female (center) beagle dogs, as well as the corresponding mean (bottom) plasma concentration-time profiles, are shown. [Figure 24]The plasma concentration-time profiles of R(+)-biloxazine after a single PO administration of compound A at 80 mg / kg to male (top) and female (center) beagle dogs, as well as the corresponding mean (bottom) plasma concentration-time profiles, are shown. [Figure 25] The plasma concentration-time profiles of S(-)-biloxazine after a single PO administration of compound B at 80 mg / kg to male (top) and female (center) beagle dogs, as well as the corresponding mean (bottom) plasma concentration-time profiles, are shown. [Figure 26] The plasma concentration-time profiles of R(+)-biloxazine after a single PO administration of compound B at 80 mg / kg to male (top) and female (center) beagle dogs, as well as the corresponding mean (bottom) plasma concentration-time profiles, are shown. [Figure 27] The plasma concentration-time profiles of compound C after a single PO administration of 80 mg / kg to male (top) and female (center) beagle dogs, as well as the corresponding mean (bottom) plasma concentration-time profiles, are shown. [Figure 28] The plasma concentration-time profiles of S(-)-biloxazine after a single PO administration of compound C at 80 mg / kg to male (top) and female (center) beagle dogs, as well as the corresponding mean (bottom) plasma concentration-time profiles, are shown. [Figure 29] The plasma concentration-time profiles of R(+)-biloxazine after a single PO administration of compound C at 80 mg / kg to male (top) and female (center) beagle dogs, as well as the corresponding mean (bottom) plasma concentration-time profiles, are shown. [Figure 30] The effect of compound C on the percentage of immobility time during 240 minutes of tail suspension is shown. The rods are mean ± SEM. The group included male C57Bl / 6 mice (N=10-11 / group) treated with compound C(po) 30 minutes prior to the test. [Figure 31]The effect of compound C on the percentage of immobility time during tail suspension for 240 seconds is shown. Bars are mean ± SEM. The groups included male C57Bl / 6 mice (N=10-groups) treated with compound C(PO), vehicle(PO), or imipramine(IP) 30 minutes prior to the test. * indicates p<0.05 compared to vehicle. [Figure 32] A schematic diagram of the experiment is shown. The activity levels of mice were evaluated over 24 hours before and 24 hours after oral treatment with compound C. [Figure 33A] This graph shows the effect of compound C on activity time using data recorded by SmartCage®. The gray bars represent the dark period from 6 PM to 6 AM. [Figure 33B] This shows the effect of compound C on walking distance, using data recorded by SmartCage®. The gray bars represent the dark period from 6 PM to 6 AM. [Figure 33C] This shows the effect of compound C on velocity using data recorded by SmartCage®. The gray bars represent the dark period from 6 PM to 6 AM. [Figure 33D] This shows the effect of compound C on standing behavior using data recorded by SmartCage®. The gray bars represent the dark period from 6 PM to 6 AM. [Figure 34A] This shows the effect of compound C on the nocturnal activity ratio (ratio of nocturnal activity time) before and after drug administration. Data are shown as mean ± SEM. [Figure 34B] This shows the effect of compound C on the nocturnal activity ratio (ratio of nighttime standing up) before and after drug administration. Data are shown as mean ± SEM. [Figure 34C] This shows the effect of compound C on the nocturnal activity ratio (ratio of nocturnal travel distance) before and after drug administration. Data are shown as mean ± SEM. [Figure 34D] This shows the effect of compound C on the nocturnal activity ratio (nocturnal movement speed ratio) before and after drug administration. Data are shown as mean ± SEM. [Figure 35A]This shows the effect of compound C on the daytime activity ratio (daytime activity time ratio) before and after drug administration. Data are shown as mean ± SEM. [Figure 35B] This shows the effect of compound C on the daytime activity ratio (ratio of daytime activity onsets) before and after drug administration. Data are shown as mean ± SEM. [Figure 35C] This shows the effect of compound C on the daytime activity ratio (daytime travel distance ratio) before and after drug administration. Data are shown as mean ± SEM. [Figure 35D] This shows the effect of compound C on the daytime activity ratio (daytime mobility ratio) before and after drug administration. Data are shown as mean ± SEM. [Figure 36] This study demonstrates the effect of compound C on prepulse suppression (PPI) of the acoustic startle response in a rat apomorphine-induced injury model. The effects of apomorphine (0.5 mg / kg), a reference dose of haloperidol, or three doses of compound C on reducing the apomorphine-induced PPI at three prepulse intensities (75, 80, and 85 dB) are shown. Bars represent the mean ± SEM for each treatment (N=10). VEH = vehicle, APO = apomorphine. HAL = haloperidol. * = significant difference compared to VEH-related response (p<0.05). + = significant difference compared to VEH-APO-related response (p<10.05). [Figure 37] The following shows the number of trials to achieve a learning threshold for cognitive flexibility indicators, including visual cue discrimination, shift to response discrimination, and reversal, corresponding to the administration of vehicle + saline, vehicle + PCP, compound C (40 mg / kg) + PCP, compound C (80 mg / kg) + PCP, compound C (120 mg / kg) + PCP, and compound C (160 mg / kg) + PCP, respectively. PCP impaired learning, and compound C could not reverse these effects. *p<0.05, **p<0.01 compared to vehicle + saline. [Figure 38]The total number of errors on indicators of cognitive flexibility, including visual cue discrimination, response discrimination shift, and reversal, is shown for administration of vehicle + saline, vehicle + PCP, compound C (40 mg / kg) + PCP, compound C (80 mg / kg) + PCP, compound C (120 mg / kg) + PCP, and compound C (160 mg / kg) + PCP, respectively. PCP increased errors, and compound C was unable to reverse these effects. *p<0.05, **p<0.01, ***p<0.001 compared to vehicle + saline. [Figure 39] Each regimen exhibited persistence errors, indicating a reversal of behavior to previous task contingencies in visual cue discrimination, response discrimination shift, and reversal, corresponding to the administration of vehicle + saline, vehicle + PCP, compound C (40 mg / kg) + PCP, compound C (80 mg / kg) + PCP, compound C (120 mg / kg) + PCP, and compound C (160 mg / kg) + PCP, respectively. *p<0.05, ***p<0.001 compared to vehicle + saline. [Figure 40] The rats showed no response to visual cue discrimination, response discrimination shift, and reversal in response to administration of vehicle + saline, vehicle + PCP, compound C (40 mg / kg) + PCP, compound C (80 mg / kg) + PCP, compound C (120 mg / kg) + PCP, and compound C (160 mg / kg) + PCP, respectively. Compound C increased no response compared to PCP-treated rats. *p<0.05 compared to vehicle + PCP. [Figure 41] This shows a visual representation of binary discrimination in a ranked, uncorrelated feature space. The metric derived from the overlap of the cloud is discrimination probability = 1 - overlap, which measures how reliably the classifier can be trained to discriminate the two groups beyond the chance level, where 0 corresponds to 100% overlap and the inability to distinguish the two groups beyond the chance level, while 100% means error-free discrimination. [Figure 42A] The class and subclass analyses of the test compound are shown. [Figure 42B] The legend corresponding to Figure 42A is shown. [Figure 43] The DRFA analysis of the active dose of the test compound compared to vehicle cloud and atomoxetine, amphetamine, and modafinil cloud is shown. [Figure 44] The DRFA analysis of the active dose of the test compound compared to vehicle cloud and atomoxetine cloud is shown. [Figure 45] The DRFA analysis of the active dose of the test compound compared to vehicle cloud and amphetamine cloud is shown. [Figure 46] The DRFA analysis of the active dose of the test compound compared to vehicle cloud and modafinil cloud is shown. [Figure 47] The DRFA analysis of the active dose of the test compound compared to vehicle cloud and donepezil cloud is shown. [Figure 48] The DRFA analysis of the active dose of the test compound compared to vehicle cloud and morphine cloud is shown. [Figure 49] The DRFA analysis of the active dose of the test compound compared to vehicle cloud and amitriptyline cloud is shown. [Figure 50] The DRFA analysis of the active dose of the test compound compared to vehicle cloud and desipramine cloud is shown. [Figure 51] The DRFA analysis of the active dose of the test compound compared to vehicle cloud and lorcaserin cloud is shown. [Figure 52] The DRFA analysis of the active dose of the test compound compared to vehicle cloud and thioperamide cloud is shown. [Figure 53] The DRFA analysis of the active dose of the test compound compared to vehicle cloud and memantine cloud is shown. [Figure 54] The DRFA analysis of the active dose of the test compound compared to vehicle cloud and bupropion cloud is shown. [Figure 55] The DRFA analysis of the active dose of compound A compared to vehicle cloud and thioperamide cloud is shown. [Figure 56]The DRFA analysis of the active dose of compound A compared to vehicle cloud and memantine cloud is shown. [Figure 57] The DRFA analysis of the active dose of compound A compared to vehicle cloud and donepezil cloud is shown. [Figure 58] The DRFA analysis of the active dose of compound C compared to vehicle cloud and thiperamide cloud is shown. [Figure 59] The DRFA analysis of the active dose of compound C compared to vehicle cloud and memantine cloud is shown. [Figure 60] The DRFA analysis of the active dose of compound C compared to vehicle cloud and donepezil cloud is shown. [Figure 61] This shows the DRFA analysis of the active dose of biloxazine compared to vehicle cloud and memantine cloud. [Figure 62] This shows the DRFA analysis of the active dose of biloxazine compared to vehicle cloud and thioperamide cloud. [Figure 63] This shows a DRFA analysis of the active dose of biloxazine compared to vehicle cloud and donepezil cloud. [Figure 64] A schematic diagram of the experiment in Example 20 is shown. The activity levels of mice were evaluated over 24 hours before and 24 hours after oral treatment with compound A. [Figure 65A] This shows the effect of compound A on activity time using data recorded by SmartCage®. The gray bars represent the dark period from 6 PM to 6 AM. [Figure 65B] This shows the effect of compound A on walking distance, using data recorded by SmartCage®. The gray bars represent the dark period from 6 PM to 6 AM. [Figure 65C] This shows the effect of compound A on speed using data recorded by SmartCage®. The gray bars represent the dark period from 6 PM to 6 AM. [Figure 65D] This shows the effect of compound A on standing behavior using data recorded by SmartCage®. The gray bars represent the dark period from 6 PM to 6 AM. [Figure 66] This shows the effect of compound A on the nocturnal activity ratio before and after drug administration. Data are shown as mean ± SEM. [Figure 67] This shows the effect of compound A on the daytime activity ratio before and after drug administration. Data are shown as mean ± SEM. [Figure 68] This shows the effect of compound A on the immobility rate during tail suspension tests. Data are shown as mean ± SEM. **p ≤ 0.01 compared to vehicle. [Figure 69] Figures A-C show the effects of compound A (abbreviated as "SPN" in the legend) in rats using an elevated cusp maze (EPM). The effects of three doses of compound A (30, 60, and 120 mg / kg po) on the number of open arm entries (A), percentage of time spent on open arms (B), and total distance traveled (C) are shown. The inset shows the effect of a single dose of the positive control compound, midazolam (0.5 mg / kg ip). The bars represent the mean ± SEM for each treatment (N=8). VEH = vehicle, SPN = compound A, Mid = midazolam. * = significant difference from VEH-related response (p<0.05), *** = p<0.001. [Figure 70] Figures A-C show the effects of S-biloxazine (S-VLX) in an elevated cusp maze (EPM) in rats. The effects of three doses of S-VLX (15, 30, and 60 mg / kg po) on the number of open arm entries (A), percentage of time spent in open arms (B), and total distance traveled (C) are shown. The inset shows the effect of a single dose of the positive control compound, midazolam (0.5 mg / kg ip). The bars represent the mean ± SEM for each treatment (N=8-9). VEH = vehicle, S-VLX = S-biloxazine, Mid = midazolam. * = significant difference from VEH-related response (p<0.05), ** = p<0.01. [Figure 71]This study demonstrates the effect of donepezil (2.0 mg / kg) on ​​performance in a spontaneous novel object recognition task in young adult Wistar rats. The mean (±SEM) search times (A / B holding sessions) for known and novel objects after a 48-hour delay are shown in the main figure (A). Inset (B) shows the mean (±SEM) discrimination (d²) ratio. d² ratio = (novel - known) / (novel + known). +p<0.02, novel vs known object; *p<0.05 vs VEH. N=10-13 in each group. VEH = vehicle, DON = donepezil. [Figure 72] This figure shows the dose-related effect of compound A (abbreviated as "SPN" in the figure) on performance on a spontaneous novel object recognition task in young adult Wistar rats. The mean (±SEM) search time (A / B holding session) for known and novel objects after a 48-hour delay is shown in the main figure (A). The inset (B) shows the mean (±SEM) discrimination (d²) ratio. d² ratio = (novel - known) / (novel + known). +p<0.05, ++p<0.01, +++p<0.001, novel vs known object; *p<0.05 vs VEH. N=11-12 in each group. VEH = vehicle. [Figure 73] This study demonstrates the effect of vortioxetine (10.0 mg / kg) on ​​the performance of a scopolamine-impaired model in a spontaneous novel object recognition task in young adult Wistar rats. The mean (±SEM) search times (A / B holding sessions) for known and novel objects after a 3-hour delay are shown in the main figure (A). Inset (B) shows the mean (±SEM) discrimination (d²) ratio. d² ratio = (novel - known) / (novel + known). +++p<0.001, novel vs known object; *p<0.05 vs VEH-VEH. N=9~11 in each group. VEH = vehicle, SCOP = scopolamine, VORT = vortioxetine. [Figure 74]This study demonstrates the dose-related effects of compound A (abbreviated as "SPN" in the figure) on the performance of a scopolamine-impaired model in a spontaneous novel object recognition task in young adult Wistar rats. The mean (±SEM) search times (A / B holding sessions) for known and novel objects after a 3-hour delay are shown in the main figure (A). Inset (B) shows the mean (±SEM) discrimination (d²) ratio. d² ratio = (novel - known) / (novel + known). ++p<0.01, +++p<0.001, novel vs known object; *p<0.05 vs VEH-VEH; #p<0.05 vs VEH-SCOP. N=9-11 in each group. VEH = vehicle, SCOP = scopolamine. [Figure 75] The latency (mean ± SEM) from the first six consecutive epochs of NR (upper panel) and the first three consecutive epochs of REM (lower panel) during time ZT19-ZT24 (second half of the dark period) is shown. * = Significant difference compared to Veh (p<0.05). [Figure 76] The percentage of time spent in W, NR, REM, and C at one-hour intervals during time ZT19-ZT24 (second half of the dark period) in the vs. Veh group after administration of compound A (30, 90, and 120 mg / kg). Medication was administered immediately before ZT12 (beginning of time ZT13). Asterisks in the legend indicate overall conditional effects that are significantly different from Veh (p<0.05). Top left: Percentage of time in W. ANOVA is significant only for the treatment. Top right: Percentage of time in NR. ANOVA is significant only for the treatment. Bottom left: Percentage of time in REM. ANOVA is not significant (NS). Bottom right: Percentage of time in C. ANOVA is significant only for the treatment. [Figure 77]The graphs show the cumulative time spent in W, NR, REM, and C during time ZT19-ZT24 (second half of the dark period) in the vs. Veh group after administration of compound A (30, 90, and 120 mg / kg). Medication was administered immediately before ZT12 (beginning of time ZT13). The asterisks at the top of the graph (color-coded according to the condition) indicate points in time where there is a significant difference compared to Veh (p<0.05). The asterisks in the legend indicate the overall condition effect where there is a significant difference compared to Veh (p<0.05). Top left: Cumulative time in W. ANOVA is significant for treatment and time-specific treatment. Top right: Cumulative time in NR. ANOVA is significant for treatment and time-specific treatment. Bottom left: Cumulative time in REM. ANOVA is significant only for treatment. Bottom right: Cumulative time in C. ANOVA is significant for treatment and time-specific treatment. [Figure 78] The upper panel shows the total time spent in wakefulness, NREM, REM, and c (the latter half of the dark period) over the entire 6-hour period from ZT19 to ZT24, as well as the REM:NR ratio (the lower panel). Upper panel: Total time spent in wakefulness, NREM, REM, and c. Lower panel: REM:NR ratio. * = Significantly different from Veh (p<0.05). [Figure 79] This graph shows the mean bout duration at time ZT19-ZT24 (second half of the dark period) for Vs. Veh after administration of compound A (30, 90, and 120 mg / kg). Medication was administered immediately before ZT12 (beginning of time ZT13). The asterisks at the top of the graph (color-coded according to the condition) indicate points in time where there is a significant difference compared to Vs. Veh (p<0.05). The asterisks in the legend indicate the overall condition effect where there is a significant difference compared to Vs. Veh (p<0.05). Top left panel: Time-averaged W bout duration. ANOVA is NS. Top right panel: Time-averaged NR bout duration. ANOVA is NS. Bottom left panel: Time-averaged REM bout duration. ANOVA is significant for treatment and time-specific treatment. Bottom right panel: Time-averaged C bout duration. ANOVA could not be performed for some conditions because there were no C bouts for several hours. [Figure 80]The mean number of bouts at time ZT19-ZT24 (second half of the dark period) after administration of compound A (30, 90, and 120 mg / kg) versus Veh is shown. Medication was administered immediately before ZT12 (beginning of time ZT13). Asterisks in the legend indicate overall conditional effects that are significantly different from Veh (p<0.05). Top left panel: Time-mean number of W bouts. ANOVA is significant only for treatment. Top right panel: Time-mean number of NR bouts. ANOVA is significant only for treatment. Bottom left panel: Time-mean number of REM bouts. ANOVA is NS. Bottom right panel: Time-mean number of C bouts. ANOVA is significant only for treatment. [Figure 81] The time-mean LMA and body temperature in Vs. Veh after administration of compound A (30, 90, and 120 mg / kg) are shown from time ZT19 to ZT24 (second half of the dark period). Medication was administered immediately before ZT12 (beginning of time ZT13). Upper panel: Time-mean activity. ANOVA is NS. Lower panel: Time-mean body temperature. ANOVA is NS. [Figure 82] The latency (mean ± SEM in this figure and all subsequent figures) is shown for the first six consecutive epochs of NR (upper panel) and the first three consecutive epochs of REM (lower panel). * = Significant difference in the condition compared to Veh (p<0.05). [Figure 83] The mean times for wakefulness, NREM, REM, and c, as well as the REM:NR ratio, are shown for the entire 6-hour recording period. Upper panel: mean times for wakefulness, NREM, REM, and c. Lower panel: REM:NR ratio. * = statistically significant difference compared to Veh (p<0.05). [Figure 84]The graphs show the percentage of time spent in W, NR, REM, and C at one-hour intervals in the W, NR, REM, and C phases after administration of amphetamine (Amph) (2 mg / kg) and compound A (10, 30, 90, and 120 mg / kg) in the Veh group. Medication was administered immediately before the start of ZT12. The asterisks at the top of the graph (color-coded according to the condition) indicate time points with a significant difference compared to Veh (p<0.05). The asterisks in the legend indicate overall condition effects with a significant difference compared to Veh (p<0.05). Top left: Percentage of time in W. ANOVA is significant for treatment and time-specific treatments. Top right: Percentage of time in NR. ANOVA is significant for treatment and time-specific treatments. Bottom left: Percentage of time in REM. ANOVA is significant only for treatment. Bottom right: Percentage of time in C. ANOVA is significant for treatment and time-specific treatments. [Figure 85] The graphs show the cumulative time spent at W, NR, REM, and C every hour after administration of Amph (2 mg / kg) and compound A (10, 30, 90, and 120 mg / kg) versus Veh. Medication was administered immediately before the start of ZT12. The asterisks at the top of the graph (color-coded according to the condition) indicate time points with a significant difference from Veh (p<0.05). The asterisks in the legend indicate overall condition effects with a significant difference from Veh (p<0.05). Top left: Cumulative time at W. ANOVA is significant for treatment and time-specific treatments. Top right: Cumulative time at NR. ANOVA is significant for treatment and time-specific treatments. Bottom left: Cumulative time at REM. ANOVA is significant for treatment and time-specific treatments. Bottom right: Cumulative time at C. ANOVA is significant for treatment and time-specific treatments. [Figure 86]The mean bout durations after administration of Amph (2 mg / kg) and compound A (10, 30, 90, and 120 mg / kg) versus Veh are shown. Medication was administered immediately before the start of ZT12. The asterisks at the top of the graph (color-coded according to the condition) indicate points in time where there is a significant difference compared to Veh (p<0.05). The asterisks in the legend indicate overall condition effects where there is a significant difference compared to Veh (p<0.05). Top left panel: Time-mean W bout duration. ANOVA is significant for treatment and time-specific treatment. Top right panel: Time-mean NR bout duration. ANOVA is significant only for time-specific treatment. Bottom left panel: Time-mean REM bout duration. ANOVA could not be performed in some conditions because REM was absent for several hours. Bottom right panel: Time-mean C bout duration. ANOVA could not be performed in some conditions because C was absent for several hours. [Figure 87] The graph shows the average number of bouts in vs. Veh after administration of Amph (2 mg / kg) and compound A (10, 30, 90, and 120 mg / kg). Medication was administered immediately before the start of ZT12. The asterisks at the top of the graph (color-coded according to the condition) indicate time points where there is a significant difference compared to Veh (p<0.05). The asterisks in the legend indicate overall condition effects where there is a significant difference compared to Veh (p<0.05). Top left panel: Time-averaged number of W bouts. ANOVA is significant for treatment and time-specific treatment. Top right panel: Time-averaged number of NR bouts. ANOVA is significant for treatment and time-specific treatment. Bottom left panel: Time-averaged number of REM bouts. ANOVA is significant for treatment and time-specific treatment. Bottom right panel: Time-averaged number of C bouts. ANOVA is significant for treatment and time-specific treatment. [Figure 88]The following panels show the entire normalized EEG spectrum (0.3–100 Hz) of W in vs. Veh after administration of Amph (2 mg / kg) and compound A (10, 30, 90, and 120 mg / kg). Top left panel: Normalized EEG spectrum of W 1 hour after administration. Top right panel: Normalized EEG spectrum of W 2 hours after administration. Center left panel: Normalized EEG spectrum of W 3 hours after administration. Center right panel: Normalized EEG spectrum of W 4 hours after administration. Bottom left panel: Normalized EEG spectrum of W 5 hours after administration. Bottom right panel: Normalized EEG spectrum of W 6 hours after administration. [Figure 89] This graph shows time-averaged EEG power in W for six standard frequency bands (delta, theta, alpha, beta, low gamma, and high gamma) in comparison to Veh after administration of Amph (2 mg / kg) and compound A (10, 30, 90, and 120 mg / kg). Data were normalized to a 6-hour average after the Veh control. Asterisks at the top of the graph (color-coded according to the condition) indicate time points where there is a significant difference compared to Veh. Asterisks in the legend indicate overall condition effects where there is a significant difference compared to Veh. Top left panel: Time-averaged power in W in the delta frequency range. ANOVA is significant for treatment and time-specific treatments. Top right panel: Time-averaged power in W in the theta frequency range. ANOVA is significant only for time-specific treatments. Center left panel: Time-averaged power in W in the alpha frequency range. ANOVA is significant for treatment and time-specific treatments. Center right panel: Time-averaged power in W in the beta frequency range. ANOVA is significant only for treatment. Lower left panel: Time-averaged power at W in the low gamma frequency range. ANOVA is not significant. Lower right panel: Time-averaged power at W in the high gamma frequency range. ANOVA is significant for treatment and for time-specific treatments. [Figure 90]The following panels show the entire normalized EEG spectrum (0.3–100 Hz) of NR in vs. Veh after administration of Amph (2 mg / kg) and compound A (10, 30, 90, and 120 mg / kg). Top left panel: Normalized EEG spectrum of NR 1 hour after administration. Top right panel: Normalized EEG spectrum of NR 2 hours after administration. Center left panel: Normalized EEG spectrum of NR 3 hours after administration. Center right panel: Normalized EEG spectrum of NR 4 hours after administration. Bottom left panel: Normalized EEG spectrum of NR 5 hours after administration. Bottom right panel: Normalized EEG spectrum of NR 6 hours after administration. [Figure 91] This graph shows time-averaged EEG power in NR for six standard frequency bands (delta, theta, alpha, beta, low gamma, and high gamma) in comparison to Veh after administration of Amph (2 mg / kg) and compound A (10, 30, 90, and 120 mg / kg). Data were normalized to a 6-hour average after the Veh control. Asterisks at the top of the graph (color-coded according to the condition) indicate time points where there is a significant difference compared to Veh. Asterisks in the legend indicate overall condition effects where there is a significant difference compared to Veh. Top left panel: Time-averaged power in NR in the delta frequency range. ANOVA is significant for treatment and time-specific treatments. Top right panel: Time-averaged power in NR in the theta frequency range. ANOVA is significant for treatment and time-specific treatments. Center left panel: Time-averaged power in NR in the alpha frequency range. ANOVA is significant for treatment and time-specific treatments. Center right panel: Time-averaged power in NR in the beta frequency range. ANOVA is significant for both treatment and time-dependent treatment. Lower left panel: Time-averaged power in NR in the low gamma frequency range. ANOVA is significant for both treatment and time-dependent treatment. Lower right panel: Time-averaged power in NR in the high gamma frequency range. ANOVA is significant for both treatment and time-dependent treatment. [Figure 92]The following panels show the entire normalized REM EEG spectrum (0.3–100 Hz) in vs. Veh after administration of Amph (2 mg / kg) and compound A (10, 30, 90, and 120 mg / kg). Top left panel: Normalized REM EEG spectrum 1 hour after administration. Top right panel: Normalized REM EEG spectrum 2 hours after administration. Center left panel: Normalized REM EEG spectrum 3 hours after administration. Center right panel: Normalized REM EEG spectrum 4 hours after administration. Bottom left panel: Normalized REM EEG spectrum 5 hours after administration. Bottom right panel: Normalized REM EEG spectrum 6 hours after administration. [Figure 93] This panel shows time-averaged EEG power in REM for six standard frequency bands (delta, theta, alpha, beta, low gamma, and high gamma) in vs. Veh after administration of Amph (2 mg / kg) and compound A (10, 30, 90, and 120 mg / kg). Data were normalized to a 6-hour average after the Veh control. In some conditions, sufficient REM did not occur to perform an ANOVA. Top left panel: Time-averaged power in REM in the delta frequency range. Top right panel: Time-averaged power in REM in the theta frequency range. Center left panel: Time-averaged power in REM in the alpha frequency range. Center right panel: Time-averaged power in REM in the beta frequency range. Bottom left panel: Time-averaged power in REM in the low gamma frequency range. Bottom right panel: Time-averaged power in REM in the high gamma frequency range. [Figure 94] The following panels show the entire normalized EEG spectrum (0.3–100 Hz) of C in vs. Veh after administration of Amph (2 mg / kg) and compound A (10, 30, 90, and 120 mg / kg). Top left panel: Normalized EEG spectrum of C 1 hour after administration. Top right panel: Normalized EEG spectrum of C 2 hours after administration. Center left panel: Normalized EEG spectrum of C 3 hours after administration. Center right panel: Normalized EEG spectrum of C 4 hours after administration. Bottom left panel: Normalized EEG spectrum of C 5 hours after administration. Bottom right panel: Normalized EEG spectrum of C 6 hours after administration. [Figure 95] This panel shows time-averaged EEG power in C for six standard frequency bands (delta, theta, alpha, beta, low gamma, and high gamma) in vs. Veh after administration of Amph (2 mg / kg) and compound A (10, 30, 90, and 120 mg / kg). Data were normalized to a 6-hour average after the Veh control. In some conditions, sufficient C was not generated to perform an ANOVA. Top left panel: Time-averaged power in C in the delta frequency range. Top right panel: Time-averaged power in C in the theta frequency range. Center left panel: Time-averaged power in C in the alpha frequency range. Center right panel: Time-averaged power in C in the beta frequency range. Bottom left panel: Time-averaged power in C in the low gamma frequency range. Bottom right panel: Time-averaged power in C at high gamma. [Figure 96] The time-mean LMA and body temperature after administration of Amph (2 mg / kg) and compound A (10, 30, 90, and 120 mg / kg) versus Veh are shown. Medication was administered immediately before the start of ZT12. A "+" in the legend indicates an overall condition effect with a significant difference compared to Veh. Upper panel: Time-mean activity. ANOVA is significant only for the treatment. Lower panel: Time-mean body temperature. ANOVA is significant only for the treatment. [Figure 97] This shows the plasma concentration of biloxazine after polyoxygenated administration of compound D at a dose of 19.74 mg / kg. [Figure 98] This shows the plasma concentration of biloxazine after PO administration of compound E at 20.99 mg / kg. [Figure 99A] This shows the plasma concentration of compound D after IP administration at 10.978 mg / kg. [Figure 99B] This shows the plasma concentration of biloxazine after intravenous administration of compound D at 10.978 mg / kg. [Figure 99C] This shows the plasma concentration of compound B after IP administration at 10.948 mg / kg. [Figure 99D] This shows the plasma concentration of biloxazine after intravenous administration of compound B at 10.948 mg / kg. [Figure 99E]This shows the plasma concentration of biloxazine after intravenous administration at a dose of 11.82 mg / kg. [Figure 99F] This shows a comparison of plasma concentrations of piroxazine between the prodrug and piroxazine itself after IP administration. [Figure 100A] This shows the stability of compound D in the rat liver S9 fraction. [Figure 100B] This shows the stability of compound E in the S9 fraction of rat liver. [Figure 100C] This shows the stability of 7-EC (7-ethoxycoumarin) in the S9 fraction of rat liver. [Figure 100D] This shows the stability of 7-HC (7-hydroxycoumarin) in the S9 fraction of rat liver. [Figure 101A] This shows the stability of compound D in the S9 fraction of canine liver. [Figure 101B] This shows the stability of compound E in the S9 fraction of canine liver. [Figure 101C] This shows the stability of 7-EC (7-ethoxycoumarin) in the S9 fraction of canine liver. [Figure 101D] This shows the stability of 7-HC (7-hydroxycoumarin) in the S9 fraction of canine liver. [Figure 102A] This demonstrates the stability of compound D in the S9 fraction of human liver. [Figure 102B] This demonstrates the stability of compound E in the human liver S9 fraction. [Figure 102C] This shows the stability of 7-EC (7-ethoxycoumarin) in the S9 fraction of human liver. [Figure 102D] This shows the stability of 7-HC (7-hydroxycoumarin) in the S9 fraction of human liver. [Figure 103A] This shows the plasma stability of the test compound. It also shows the stability of compound D in rat plasma. [Figure 103B] This shows the plasma stability of the test compound. It also shows the stability of compound E in rat plasma. [Figure 103C] This shows the plasma stability of the test compound. It also shows the stability of the positive control enalapril in rat plasma. [Figure 103D] This shows the plasma stability of the test compound. It also shows the stability of compound D in human plasma. [Figure 103E] This shows the plasma stability of the test compound. It shows the stability of compound E in human plasma. [Figure 103F] This shows the plasma stability of the test compound. It also shows the stability of the positive control propantheline in human plasma. [Figure 104A] This shows the stability of compounds in human intestinal homogenate. It also shows the stability of compound D in human intestinal homogenate. [Figure 104B] This shows the stability of the compound in human intestinal homogenate. It also shows the stability of compound E in human intestinal homogenate. [Figure 104C] This shows the stability of the compound in human intestinal homogenate. It also shows the stability of the positive control testosterone in human intestinal homogenate. [Figure 104D] This shows the stability of the compound in human intestinal homogenate. It also shows the stability of the positive control 7-HC(7-hydroxycoumarin) in human intestinal homogenate. [Modes for carrying out the invention]

[0014] definition The following terms will be used throughout the text according to their definitions below.

[0015] As used herein, the terms “biloxazine” or “2-(2-ethoxyphenoxy)methyl)morpholine” mean (R,S)-2-[(2-ethoxyphenoxy)methyl]morpholine (including its pharmaceutically acceptable salts or esters), comprising either a single (-) enantiomer or a single (+) enantiomer, or in the form of a racemic or non-racemic mixture of enantiomers containing varying amounts of (-) and (+) enantiomers.

[0016] Where used herein and in the appended claims, singular terms such as "a," "an," and "the" and similar references in the context of describing elements (particularly in the context of the following claims) should be interpreted as encompassing both singular and plural, unless otherwise stated herein or unless the context clearly contradicts this interpretation. Descriptions of ranges of values ​​herein are intended, unless otherwise stated herein, merely as an abbreviation to indicate each distinct value falling within that range individually, and each distinct value is incorporated herein as if it were described separately. All methods described herein may be performed in any preferred order, unless otherwise stated herein or unless the context clearly contradicts this interpretation. Any examples or illustrative language presented herein (e.g., "such as") are intended, unless otherwise specified, merely to make embodiments easier to understand and do not limit the scope of the claims. Nothing in this specification should be interpreted as indicating that any unclaimed element is essential.

[0017] Where used herein, “approximately” is as understood by those skilled in the art and may vary to some extent depending on the context in which it is used. Where there is use of an item that is not obvious to those skilled in the art, “approximately” shall mean plus or minus 10% of the specific item, given the context in which it is used.

[0018] Generally, references to specific elements such as hydrogen or H are intended to include all isotopes of that element. For example, if the R group is defined as containing hydrogen or H, then deuterium and tritium are also included. Thus, tritium, C 14 , P 32 , and S 35 Compounds containing radioactive isotopes such as those mentioned herein are included within the scope of this technology. Procedures for inserting such labels into compounds of this technology will be readily apparent to those skilled in the art based on the disclosures herein.

[0019] Generally, "substituted" refers to an organic group (e.g., an alkyl group) in which one or more bonds to hydrogen atoms are replaced by bonds to non-hydrogen or non-carbon atoms, as defined below. Substituted groups also include those in which one or more bonds to carbon atoms or hydrogen atoms are replaced by one or more bonds (including double or triple bonds) to heteroatoms. Therefore, unless otherwise specified, substituted groups are substituted with one or more substituents. In some embodiments, substituted groups are substituted with one, two, three, four, five, or six substituents. Examples of substituents include halogens (i.e., F, Cl, Br, and I); hydroxyl; alkoxy, alkeneoxy, aryloxy, aralkyloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, and heterocyclylalkoxy groups; carbonyl (oxo); carboxylate; ester; urethane; oxime; hydroxylamine; alkoxyamine; aralkoxyamine; thiol; sulfide; sulfoxide; sulfone; sulfonyl; pentafluorosulfanil (i.e., SF5), sulfonamide; amine; N-oxide; hydrazine; hydrazide; hydrazone; azide; amide; urea; amidine; guanidine; enamine; imide; isocyanate; isothiocyanate; cyanate; thiocyanate; imine; nitro group; nitrile (i.e., CN), etc.

[0020] As used herein, the term "carboxylate" refers to the conjugate base of a carboxylic acid having the chemical formula -COO.

[0021] The term "ester" as used herein refers to -COOR 2 - Refers to the - group and the -C(O)OG group. 2is a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heterocyclylalkyl, or heterocyclyl group as defined herein. G is a carboxylic acid protecting group. Carboxylic acid protecting groups are well known to those skilled in the art. A detailed list of carboxylic acid functional protecting groups is found in Protective Groups in Organic Synthesis, Greene, TW; Wuts, PGM, John Wiley & Sons, New York, NY, (3rd Edition, 1999), which can be added or removed using the procedures described therein and are incorporated herein by reference for all purposes as described herein in whole.

[0022] The term "amide" (or "amido") refers to the C-amide group and the N-amide group, i.e., C(O)NR, respectively. 3 R 4 And it contains the -NRC(O)-R group. 3 and R 4 This is independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl, or heterocyclyl group as defined herein. Thus, the amide group includes, but is not limited to, a carbamoyl group (-C(O)NH2) and a formamide group (NHC(O)H). In some embodiments, the amide is -NRC(O)-(C 1~5 In other embodiments, the amide is -NHC(O)-alkyl, and its group is called "carbonylamino".

[0023] As used herein, the term "amine" (or "amino") is defined as -NR 5 R 6 It refers to the base, and in the formula, R 5 and R 6This is independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl, or heterocyclyl group as defined herein. In some embodiments, the amine is an alkylamino, dialkylamino, arylamino, or alkylarylamino. In other embodiments, the amine is NH2, methylamino, dimethylamino, ethylamino, diethylamino, propylamino, isopropylamino, phenylamino, or benzylamino.

[0024] As used herein, the terms "halogen" or "halo" refer to bromine (Br), chlorine (Cl), fluorine (F), or iodine (I). In some embodiments, the halogen is chlorine (Cl).

[0025] As used herein, the terms “polypeptide” or “peptide” refer to two or more amino acids linked by a peptide (i.e., amide) bond between the carboxyl terminus of one amino acid and the amino terminus of another. The term “peptide” may be combined with a prefix indicating the number of amino acids in the peptide; for example, “pentapeptide” is a peptide of five amino acids.

[0026] The term “amino acid” is recognized in the art and broadly refers to natural or unnatural alpha-amino acids or beta-amino acids. The term “amino acid” includes, but is not limited to, any of the 21 standard L-amino acids commonly found in natural peptides.

[0027] As used herein, the term “amino acid residue containing a hydrophobic side chain” refers to the following amino acids: alanine (Ala), valine (Val), isoleucine (Ile), leucine (Leu), methionine (Met), phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp). In some embodiments, the amino acid residue containing a hydrophobic side chain is valine (Val). In other embodiments, the amino acid residue containing a hydrophobic side chain is phenylalanine (Phe).

[0028] As used herein, the term "acetyl" refers to a methyl group bonded to a carbonyl group (CH3CO-).

[0029] As used herein, the term "pyridine" group refers to a group in a heterocyclic organic compound having the chemical formula C5H5N.

[0030] As used herein, the term "pyridinecarboxylic acid" refers to a compound having a pyridine ring and a carboxyl group.

[0031] As used herein, the term "azandiyl" refers to a functional group having the formula -NH, which is attached to the remainder of the compound by two single bonds.

[0032] pharmaceutically acceptable salts of the compounds described herein are within the scope of this art and include acid-addition or base-addition salts that retain the desired pharmacological activity and are not biologically undesirable (e.g., the salts are not excessively toxic, allergenic, or irritating and are bioavailable). If the compounds of this art have a basic group, such as an amino group, pharmaceutically acceptable salts can be formed using inorganic acids (e.g., hydrochloric acid, hydroboric acid, nitric acid, sulfuric acid, and phosphoric acid), organic acids (e.g., alginic acid, formic acid, acetic acid, benzoic acid, gluconic acid, fumaric acid, oxalic acid, tartaric acid, lactic acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid, and p-toluenesulfonic acid) or acidic amino acids (e.g., aspartic acid and glutamic acid). If the compounds of this art have an acidic group, such as a carboxylic acid group, they can be formed using metals such as alkali metals and alkaline earth metals (e.g., Na + Li + , K + Ca 2+ Mg 2+ , or Zn 2+ ), ammonia or organic amines (e.g., dicyclohexylamine, trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, or triethanolamine), or basic amino acids (e.g., arginine, lysine, or ornithine) can form salts. Such salts can be prepared in situ during the isolation and purification of the compound, or they can be prepared by separately reacting the purified compound in the form of a free base or free acid with a suitable acid or base, and then isolating the resulting salts.

[0033] The stereoisomers (also known as optical isomers) of a compound include all chiral, diastereomer, and racemic forms of the structure unless otherwise specified. That is, the compounds used in this technique, as is evident from the description, include concentrated or divided optical isomers at any or all chiral atoms. Both racemic and diastereomer mixtures, as well as individual optical isomers, can be isolated or synthesized so as to substantially omit one of their enantiomers or diastereomers, and all such stereoisomers are within the scope of this technique.

[0034] The term "pharmaceutically acceptable excipient" refers to substances that are widely recognized by industry and regulatory authorities, such as those listed in monographs in abstracts such as the USP-NF, Food Chemicals Codex, Code of Federal Regulations (CFR), and FDA Inactive Ingredients Guide, as well as in 21 CFR Parts 182 and 184, which list substances that are generally considered safe (GRAS) food ingredients.

[0035] In one embodiment, a compound represented by formula I, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof: [ka] It will be provided. In the compound of formula I, R 1 R can be alkyl, heterocyclyl, or pyridyl, 2 R can be alkyl, aryl, heteroaryl, or heterocyclyl, 3 ~R 14 Each of these can independently be H, F, Cl, Br, I, CN, NO2, alkyl, aryl, heteroaryl, or heterocyclyl, and X can be H, halogen, amino acid residue, substituted amino acid residue, alkyl, or ester. In some preferred embodiments, R 2 R is ethyl. In any of the above embodiments, R 1X may be CH2, CH2CH2, CH2CH2CH2, CH2CH2CH2CH2, (CH3)2C, (CH3)2CHCH2, or (CH3)3CCH2. In any of the above embodiments, X may be an amino acid residue. In such embodiments, the amino acid residue may further include a hydrophobic side chain. In any of the above embodiments, the amino acid residue may be valine or phenylalanine. In any of the above embodiments, R 3 ~R 14 Each of these can independently be H, F, Cl, Br, I, or alkyl. In some such embodiments, R 3 ~R 14 Each of these can independently be H or a C1-C6 alkyl group. In some embodiments, R 3 ~R 14 All of the above embodiments are H. 1 R can be CH2CH2 or CH2CH2CH2CH2. In the various embodiments described above, R1 can be CH2 or C2H5, and / or X can be an ester. In the various embodiments described above, R 1 can be a pyridyl group, and X can be F, Cl, Br, or I.

[0036] In various embodiments, the compound represented by formula I is one or more of the following compounds, but it should be understood that each representation also includes any R, S, or racemic structure if a chiral center is present: [ka] , [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] or [ka] .

[0037] In some embodiments, the compound represented by formula I is [ka] That is the case. In the above formula, R 15 is H, alkyl, or -C(O)OR 17 It could be, R 16 is H, alkyl, or -C(O)OR 17 It could be, R 17 This can be H or alkyl. In some embodiments, R 15 R can be alkyl, 16 R can be H or alkyl. In such embodiments, R 15 It can be methyl, R 16 R can be H or methyl. In some embodiments, R15 and R 16 is methyl. In some embodiments, R 15 is -C(O)OR 17 And R 16 H is R 17 It is methyl.

[0038] In another embodiment, the compound represented by formula II, or its stereoisomer, and / or its salt: [ka] It will be provided. In formula II, L is an alkyl, substituted pyridinecarboxylic acid, or substituted azanediyl acetate, and R 2 is an alkyl, aryl, heteroaryl, or heterocyclyl, and R 3 ~R 14 Each of these is independently H, F, Cl, Br, I, CN, NO2, alkyl, aryl, heteroaryl, or heterocyclyl. In some embodiments, R 2 It is ethyl.

[0039] In some embodiments, the compound represented by formula II is [ka] That is the case.

[0040] In various embodiments, the compound of formula II is [ka] [ka] or [ka] It is one or more of the following.

[0041] In another embodiment, a compound represented by formula III, or its stereoisomer, and / or its salt: [ka] It will be provided. In formula III, Y can be F, Cl, Br, I, an amino acid residue, a substituted amino acid residue, an alkyl, or an ester, and R 2 R can be alkyl, aryl, heteroaryl, or heterocyclyl, 3 ~R 14 Each of these can independently be H, F, Cl, Br, I, CN, NO2, alkyl, aryl, heteroaryl, or heterocyclyl. In some embodiments, R 2 It is ethyl.

[0042] In some embodiments, the compound represented by formula III is [ka] That is the case.

[0043] In some embodiments, the compound represented by formula III is [ka] That is the case.

[0044] In another embodiment, a compound represented by formula IV, or its stereoisomer, and / or its salt: [ka] It will be provided. In formula III, Z can be H, F, Cl, Br, I, an amino acid residue, a substituted amino acid residue, or a nitrogen-containing group, and R 2 R can be alkyl, aryl, heteroaryl, or heterocyclyl, 3 ~R 14 Each of these can independently be H, F, Cl, Br, I, CN, NO2, alkyl, aryl, heteroaryl, or heterocyclyl. In some embodiments, R 2 It is ethyl.

[0045] In some embodiments, the compound represented by formula IV is [ka] That is the case.

[0046] In some embodiments, the compound represented by formula IV is [ka] That is the case.

[0047] In some embodiments, the composition includes derivatives of substituted morpholines of formula I, II, III, or IV, their stereoisomers, and / or salts thereof, as well as at least one pharmaceutically acceptable excipient or carrier.

[0048] In some embodiments, the pharmaceutical composition includes a derivative of a substituted morpholine of formula I, II, III, or IV, its stereoisomer, and / or a salt thereof, together with a pharmaceutically acceptable carrier or excipient. The pharmaceutical formulation may be in a suitable dosage form. Exemplary dosage forms include, but are not limited to, injections, oral preparations, suppositories, cachets, pouches, and dermatological preparations.

[0049] In another embodiment, treatment of CNS disorders is provided by administering a composition comprising a derivative of a substituted morpholine of formula I, II, III, or IV described herein, or a salt thereof, to a subject requiring such treatment.

[0050] In another embodiment, a method is provided for administering a composition comprising a compound of formula I, II, III, or IV or a salt thereof to a subject. In one embodiment, the subject is a mammal. In a further embodiment, the mammalian subject is a human. In a particular embodiment, the mammalian subject is a human adult or a human child.

[0051] In some embodiments, the methods described herein involve administering a derivative of a substituted morpholine of formula I, II, III, or IV, its stereoisomer, and / or a salt thereof in combination with at least one additional pharmaceutical agent. In some embodiments, the at least one additional pharmaceutical agent is another agent for CNS disorders. In further embodiments, the at least one additional pharmaceutical agent is 2-((2-ethoxyphenoxy)methyl)morpholine or a salt thereof.

[0052] In one embodiment, derivatives of substituted morpholines can be prepared from 2-((2-ethoxyphenoxy)methyl)morpholine or a salt thereof.

[0053] In one embodiment, a derivative of substituted morpholine can be prepared by reacting 2-((2-ethoxyphenoxy)methyl)morpholine or a salt thereof with sodium bicarbonate to form intermediate 1 having the following structure: [ka]

[0054] In one embodiment, a derivative of substituted morpholine can be prepared by reacting 2-((2-ethoxyphenoxy)methyl)morpholine or a salt thereof with 1-chloromethyl chloroformate to form intermediate 2 having the following structure: [ka]

[0055] In one embodiment, a derivative of substituted morpholine can be prepared by reacting 2-((2-ethoxyphenoxy)methyl)morpholine or a salt thereof with 1-chloroethyl chloroformate to form intermediate 3 having the following structure: [ka]

[0056] In one embodiment, derivatives of substituted morpholines of formulas I, II, III, or IV are prepared by reacting 2-((2-ethoxyphenoxy)methyl)morpholine or a salt thereof with intermediate 1, intermediate 2, or intermediate 3.

[0057] In another embodiment, a method is provided for preparing derivatives of substituted morpholines of formulas I, II, III, or IV.

[0058] Derivatives of substituted morpholines can be analyzed by liquid chromatography-mass spectrometry (LCMS) and nuclear magnetic resonance (NMR) spectroscopy.

[0059] Although the present invention has been broadly described, it will be more readily understood by referring to the following examples. The examples are presented as illustrations and are not intended to limit the present invention. [Examples]

[0060] Example 1. Preparation of the compound of the present invention Procedure for preparing intermediates. In some structures, the chiral center is shown in either an R or S configuration, but it should be understood that other configurations are also disclosed herein.

[0061] Intermediate 1: Synthesis of 2-((2-ethoxyphenoxy)methyl)morpholine-4-carbonyl chloride. [ka]

[0062] A 50 ml solution of 2-((2-ethoxyphenoxy)methyl)-morpholine hydrochloride (500 mg, 1.83 mmol) in dichloromethane was added dropwise to a slurry of sodium bicarbonate (460 mg, 5.48 mmol). The reaction mixture was stirred for 30 minutes. A 25 ml solution of triphosgene (358 mg, 1.21 mmol) in dichloromethane was added over 15 minutes at 10-15°C. The reaction mixture was stirred at room temperature for 3 hours. The reaction mass was filtered to remove sodium chloride, and the filtrate was concentrated under vacuum to obtain 438 mg of ethylmethylcarbamoyl chloride as a pale yellow oil (yield: 80%).

[0063] 1 H NMR (CDCl3, 400MHz): δ ppm 6.88-6.91 (m, 4H), 4.39-4.47 (br t, 1H), 3.96-4.25 (m, 6H), 3.83-3.87 (br t, 1H), 3.61-3.71 (br t, 1H), 3.03-3.38 (m, 2H), 1.44 (t, 3H).

[0064] Intermediate 2: Chloromethyl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate. [ka]

[0065] 1-chloromethyl chloroformate was added dropwise to a stirred, ice-cold mixture of 2-((2-ethoxyphenoxy)methyl)morpholine hydrochloride (1.3 gm, 4.52 mmol) and trimethylamine (1.01 gm, 9.95 mmol) dichloromethane. The reaction mixture was stirred at 10-15°C, returned to room temperature, and stirred for 5 hours. The precipitated solid was filtered, and the filtrate was concentrated. The crude product was purified by column chromatography (hexane: siRNA 7:3) to obtain a 1.2 gm (80%) white solid.

[0066] 1H NMR (CDCl3, 400MHz): δ ppm 1.46 (t, 3 H), 1.59 (s, 4 H), 3.05 (d, 2 H), 3.63 (d, 1H), 3.92 - 4.02 (m, 2 H), 4.04 - 4.15 (m, 4 H), 4.23 (br. s., 1 H), 5.76 - 5.86 (m, 2 H), 6.84-7.00 (m, 4 H).

[0067] Intermediate 3: 1-Chloroethyl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate. [ka]

[0068] 2-((2-ethoxyphenoxy)-methyl)morpholine hydrochloride (2 gm, 6.96 mmol) and trimethylamine (1.01 g, 9.95 mmol) dichloromethane were stirred and cooled on ice, to which 1-chloroethyl chloroformate (1.19 g, 83.5 mmol) was added dropwise. The reaction mixture was stirred at 10-15°C and then allowed to return to room temperature, where it was stirred for 5 hours. The precipitated solid was filtered, and the filtrate was concentrated. The crude product was purified by column chromatography (hexane: Depositphotos 7:3) to obtain a 1.42 gm (59.3%) white solid.

[0069] 1 H NMR (400 MHz, CDCl3): δ ppm 1.39-1.51 (m, 3 H), 1.83 (d, 3 H), 2.92-3.12 (m, 2 H), 3.54-3.72 (m, 1 H), 3.85 (br. s., 1 H), 3.89-4.13 (m, 7 H), 4.20 (d, 1 H), 6.61 (m, 1 H), 6.84-7.01 (m, 4 H).

[0070] Procedure for synthesizing compounds of formulas I, II, III, or IV:

[0071] SP-16: ((D-valyl)oxy)methyl 2-((2-ethoxyphenoxy)methyl)morpholine-4 carboxylate.

[0072] Step 1. [ka] N-Boc-D-valine (175 mg, 0.80 mmol) and cesium carbonate (130 mg, 0.4 mmol) were mixed with methanol (3.3 ml). The reaction mixture was stirred at room temperature for 3 hours, then the methanol was evaporated, and the residue was redissolved in DMF (1 ml). Chloromethyl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate (intermediate 2) (177 mg, 0.52 mmol) was added to the reaction mixture. The resulting mixture was stirred at 80°C for 20 hours. The DMF was evaporated under vacuum, the residue was dissolved in chloroform, and purified by column chromatography (hexane:SiO 1:1) to obtain 112 mg (39.4%) of a semi-solid oil.

[0073] Step 2: [ka] A dioxane solution of SP-16A (65 mg, 0.12 mmol) and 2 M HCl was stirred overnight at room temperature. The solvent was evaporated, and the mixture was dried under vacuum to obtain 50 mg (95.6%) of the pure desired product (SP-16) as a brown semi-solid. Purity measured by LC-MS with an ELS detector: 96.27%. MS: M+H=411.14. 1 H NMR (CDCl3, 400MHz): δ ppm 1.12 (t, 6 H), 1.44 (t, 3 H), 2.46 (br. s, 1 H), 2.90 - 3.10 (m, 2H), 3.52-3.66 (m, 1 H), 3.85-420 (m, 10 H), 5.83 (br. s, 1 H), 5.95 (d, 1 H), 6.85-6.96 (m, 4 H), 8.24 (br. s, 2 H).

[0074] SP-17: 1-((L-Valyl)oxy)ethyl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate.

[0075] Step 1.

Chem.

[0076] Step 2:

Chem.

[0077] SP-18: (2R)-2-Amino-N-((2-((2-ethoxyphenoxy)methyl)morpholino)methyl)-3-methylbutanamide bis hydrochloride.

[0078] Step 1:

Chem.

[0079] Step 2:

Chem.

[0080] SP-19: Pyridin-2-yl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate [ka]

[0081] A 1 ml solution of triphosgene (163 mg, 0.55 mmol) in dichloromethane (DCM) was stirred in an ice bath at 0-5°C for 15 minutes, and a 1 ml solution of 2-hydroxypyridine (150 mg, 1.58 mmol) and N,N-diisopropylethylamine (DIPEA; 208 mg, 1.61 mmol) in DCM was added dropwise. The reaction mixture was allowed to return to room temperature. The completion of the reaction was monitored by TLC. After the reaction was complete, the reaction mixture was evaporated, redissolved in DCM, and evaporated (3 times) to remove excess triphosgene. The residue was redissolved in DCM, and a 363 mg solution of 2-((2-ethoxyphenoxy)methyl)morpholine hydrochloride (363 mg, 1.26 mmol) and a 13.6 mg solution of TEA (1.34 mmol) in DCM were added, and the mixture was stirred overnight at room temperature. The reaction mixture was absorbed into silica and purified by column chromatography using hexane-ethyl acetate (2:1) to obtain 56 mg (12.3%) of the target compound (SP-19) as a semi-solid. LCMS: M+H = 359.08. Purity 100% as measured by ELS detector. 1 H NMR (400 MHz, CDCL3): δ 1.25-1.46 (m, 3 H), 3.02-3.34 (m, 2 H), 3.69-3.76 (m, 1 H), 3.95-4.16 (m, 7 H), 4.28-4.42 (d, 1 H), 6.85-6.99 (m, 4 H), 7.11 (dd, 1 H), 7.21 (dd, 1 H), 7.75 - 7.83 (m, 1 H), 8.39 (dd, 1 H).

[0082] SP-20: 2-Chloropyridine-4-yl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate. [ka]

[0083] 2-chloro-4-hydroxypyridine (95 mg, 0.73 mmol) was dissolved in anhydrous THF (10 mL), stirred, and cooled on ice. 2-((2-ethoxyphenoxy)methyl)morpholine-4-carbonyl chloride (intermediate 1) (273 mg, 0.33 mmol) was added, followed by dropwise addition of NaH (60% in oil, 35 mg, 0.146 mmol). The reaction mixture was stirred under argon at room temperature for 14 hours. After evaporating the solvent under vacuum, water (5 mL) was added, and the mixture was extracted with ether (3 × 10 mL). The organic phase was washed with dilute NaOH (pH 10-11), dried, and evaporated to dryness under vacuum. Purification by column chromatography (hexane:siRNA 2:1) yielded 83 mg (29%) of semi-solid (SP-20). LCMS: 100% purity as measured by ELS detector. MS: M+H=393.08. 1 H NMR (CDCl3, 400MHz): δ ppm 1.38-1.47 (m, 3 H), 3.04-3.32 (m, 2 H), 3.70 (t, 1 H) 3.9-3.93 (m, 1 H), 4.03-4.08 (m, 5 H), 4.15-4.18 (m, 1 H), 4.30-4.35 (m, 1 H), 6.87-6.99 (m, 4 H), 7.11-7.12 (m, 1 H), 7.23 (d, 1 H), 8.37 (d, 1 H).

[0084] SP-21: Methylenebis(2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate). [ka]

[0085] A DMF solution (2 ml) of 2-((2-ethoxyphenoxy)methyl)morpholine hydrochloride (108 mg, 0.4 mmol) and methylene dibromide (50 mg) was added with a slurry of cesium carbonate (100 mg, 1.2 mmol). Carbon dioxide gas was passed through the reactants for 30 minutes, and the mixture was stirred at room temperature for 48 hours. The reaction mass was filtered, and the filtrate was concentrated in vacuo. The residue was dissolved in chloroform and purified by column chromatography (hexane:EtOAc 4:1) to obtain 52 mg (22.6%) of a solid. Purity 100% by ELS detector. MS: M+H = 575.15. 1 H NMR (CDCl3, 400 MHz): δ ppm 6.7 - 7.00 (m, 8H), 5.83 (s, 2H), 3.8 - 4.2 (m, 16H), 3.5 - 3.6 (m, 2H), 2.8 - 3.0 (m, 4H), 1.43 - 1.47 (t, 6H).

[0086] SP-22: 1-((L-phenylalanyl)oxy)ethyl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate.

[0087] Step 1. [Chemical Structure] A reaction mixture of N-Boc-phenylalanine (175 mg, 0.66 mmol) and cesium carbonate (107 mg, 0.33 mmol) mixed in methanol (1.3 ml) was stirred at room temperature for 3 hours, then methanol was evaporated, and the residue was redissolved in DMF (1 ml). To the reaction mixture was added 1-chloroethyl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate (Intermediate 3) (150 mg, 0.42 mmol). The resulting mixture was stirred at 80 °C for 20 hours. Then, DMF was evaporated under vacuum, the residue was dissolved in chloroform, and then purified by column chromatography (hexane:EtOAc 8:2) to obtain 232 mg (61%) of a semi-solid oil.

[0088] Step 2: [ka] A dioxane solution containing SP-22A (140 mg, 0.238 mmol) and 2 M HCl was stirred overnight at room temperature. The solvent was evaporated, and the mixture was dried under vacuum to obtain 58 mg (52%) of the pure desired product as a light brown solid. Purity measured by LC-MS with an ELS detector: 100%. MS: M+H = 495.24. 1 H NMR (CDCl3, 400MHz): δ ppm 1.25 - 1.50 (m, 6 H), 2.95 - 3.06 (m, 2 H), 3.35-3.71 (m, 4 H) 3.76- 4.13 (m, 8 H) 4.34 - 4.40 (m, 2 H) 6.85 - 6.92 (m, 5 H), 7.25-7.36 (m, 5 H), 8.70 (br. s., 1 H), 8.79 (br. s., 1 H).

[0089] SP-23 1-((dimethyl-L-valyl)oxy)ethyl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate, hydrochloride. [ka]

[0090] L-Val-N,N-dimethyl (100 mg, 0.68 mmol) and cesium carbonate (110 mg, 0.34 mmol) were mixed with methanol (0.75 ml). The reaction mixture was stirred at room temperature for 3 hours, then the methanol was evaporated, and the residue was redissolved in DMF (1 ml). 1-chloroethyl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate (intermediate 3) (160 mg, 0.44 mmol) was added to the reaction mixture. The resulting mixture was stirred at 80°C for 20 hours. The DMF was evaporated under vacuum, the residue was dissolved in chloroform, and purified by column chromatography (hexane:SiO 3:2) to obtain 91 mg (45.7%) of semi-solid.

[0091] 77 mg of the parent compound was dissolved in 2 ml of chloroform, and 0.17 ml of 2 M HCl / dioxane was added. The reaction mixture was stirred at room temperature for 2 hours. The solvent was then evaporated under argon, and then under vacuum, to obtain 81 mg of an oily substance. Purity measured by LC-MS with an ELS detector: 99.61%. MS: M+H = 453.30, M+Na = 475.28. 1 H NMR (CDCl3, 400MHz): δ ppm 0.89 (dd, 3 H), 0.97 (d, 3 H), 1.45 (t, 3 H), 1.53 (d, 3 H), 1.63 (s, 1 H), 2.01 (dt, 6.54 Hz, 1 H), 2.31 (s, 6 H), 2.72 (m, 1 H), 3.04 (br. s., 2 H), 3.59 (d,1 H), 3.81-4.18 (m, 8 H), 6.88-6.91 (m, 5 H).

[0092] SP-24: 1-((acetyl-L-valyl)oxy)ethyl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate. [ka]

[0093] A reaction mixture of N-acetylvaline (120 mg, 0.69 mmol) and cesium carbonate (110 mg, 0.34 mmol) in methanol (0.9 ml) was stirred at room temperature for 3 hours. The methanol was then evaporated, and the residue was redissolved in DMF (1 ml). 1-chloroethyl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate (intermediate 3) (160 mg, 0.44 mmol) was added to the reaction mixture. The resulting mixture was stirred at 80°C for 20 hours. The DMF was evaporated under vacuum, and the residue was dissolved in chloroform and purified by column chromatography (hexane:siRNA 3:2) to obtain 75 mg (37%) of an oily substance. LCMS: Purity by ELS detector: 100%. MS: M+H = 473.26, M+Na = 495.24. 1H NMR (CDCl3, 400MHz): δ ppm 0.82-1.03 (m, 3 H), 0.93 (d, 3 H), 1.45 (br. s., 3 H), 1.52-1.53 ​​(m, 3 H), 2.04 (d, 3 H), 2.17 (m, 1 H), 2.94 - 3.10 (m, 2 H), 3.57-3.60 (m, 1H), 3.84 - 4.17 (m, 8 H), 4.55-4.62 (m, 1 H), 5.97 (br. s., 1 H), 6.89-6.95 (m, 5 H).

[0094] SP-25: 1-((methyl-D-valyl)oxy)ethyl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate, trifluoroacetate.

[0095] Step 1. [ka] N-Boc-D-valine (160 mg, 0.69 mmol) and cesium carbonate (110 mg, 0.35 mmol) were mixed in methanol (1.2 ml). The reaction mixture was stirred at room temperature for 3 hours, then the methanol was evaporated, and the residue was redissolved in DMF (1 ml). 1-chloroethyl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate (intermediate 3) (160 mg, 0.44 mmol) was added to the reaction mixture. The resulting mixture was stirred at 80°C for 20 hours. The DMF was evaporated under vacuum, the residue was dissolved in chloroform, and purified by column chromatography (hexane:siRNA 2:1) to obtain 90 mg (36.1%) of a semi-solid oil.

[0096] Step 2: [ka] A solution of SP-25A (50 mg, 0.09 mmol) in DCM (1 ml) and TFA (0.1 ml) was stirred at room temperature overnight (18 h). Then the solvent was evaporated and dried under vacuum to give 35.8 mg (85.3%) of the pure desired product as a yellow oil. LCMS: Purity by ELS detector: 100%. MS: M+H = 439.24 . 1 H NMR (CDCl3, 400 MHz): δ 0.98 - 1.15 (m, 6 H), 1.36 - 1.49 (m, 3 H), 1.57 (d, 3 H), 2.37 (br.s., 1 H), 2.78 (s, 3 H), 2.88 - 3.10 (m, 2 H), 3.14 (d, 1 H), 3.49 - 3.64 (m, 1 H), 3.67 (br.s., 1 H), 3.84 (br.s., 1 H), 3.89 (br.s., 1 H), 3.97 (br.s., 2 H), 4.00 - 4.11 (m, 3H), 4.16 (d, 2 H), 6.84 - 7.00 (m, 5 H).

[0097] SP-26: 1-((D-Valyl)oxy)-2-methylpropyl 2-((2-ethoxyphenoxy)methyl)-morpholine-4-carboxylate HCl salt.

[0098] Step 1: [Chemical formula] To a stirred ice-cooled mixture of 2-((2-ethoxyphenoxy)-methyl)morpholine hydrochloride (350 mg, 1.22 mmol), trimethylamine (271 mg, 2.68 mmol) in dichloromethane, 1-chloro-2-methylpropyl chloroformate (210 mg, 1.46 mmol) was added dropwise. The reaction mixture was stirred at 10 - 15 °C and returned to room temperature and stirred for 2 h. The precipitated solid was filtered and the filtrate was concentrated. The crude product was purified by column chromatography (hexane:EtOAc 4:1) to give 0.55 gm (59.3%) of an oil. 1H NMR (CDCl3, 400MHz): δ ppm 1.06-1.09 (m, 6 H), 1.43-1.46 (t, 3 H), 2.18-2.22 (m, 1 H), 2.95-3.20 (m, 2 H), 3.55-3.69 (m, 1 H), 3.86-4.27 (m, 8 H), 6.36-6.37 (d, 1H), 6.86-6.97 (m, 4 H).

[0099] Step 2. [ka] N-Boc-D-valine (200 mg, 0.92 mmol) and cesium carbonate (150 mg, 0.46 mmol) were mixed in methanol (1.5 ml). The reaction mixture was stirred at room temperature for 2 hours, then the methanol was evaporated, and the residue was redissolved in DMF (1 ml). 1-chloro-2-methylpropyl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate (SP-26A) (230 mg, 0.59 mmol) was added to the reaction mixture. The resulting mixture was stirred at 80°C for 20 hours. The DMF was evaporated under vacuum, the residue was dissolved in chloroform, and purified by column chromatography (hexane:siRNA 4:1) to obtain 170 mg (52.1%) of a semi-solid oil.

[0100] Step 3: [ka] A solution of SP-26B (110 mg, 0.2 mmol) dissolved in dioxane (1 ml) and 2 M HCl / dioxane (0.4 ml) was stirred overnight (18 hours) at room temperature. The solvent was then evaporated, and the mixture was dried under vacuum to obtain 80 mg (88%) of the pure desired product as an oily substance. Purity measured by LC-MS: ELS detector: 100%. MS: M+H=453.22. 1H NMR (CDCl3, 400MHz): δ ppm 0.92-1.04 (m, 6 H), 1.04-1.23 (m, 6 H), 1.42 (t, 3 H), 2.09 (br, 1 H), 2.48 (br, 1 H), 2.89-3.17 (m, 1 H), 3.49-4.25 (m, 13 H), 6.81-6.73 (m, 1H), 6.88-6.92 (m, 4 H), 8.70-8.76 (d, 2 H).

[0101] SP-27: 1-(((R)-2-(aminomethyl)-3-methylbutanoyl)oxy)ethyl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate trifluoroacetate.

[0102] Step 1. [ka] N-Boc-3-amino-2-isopropionic acid (100 mg, 0.43 mmol) and cesium carbonate (70 mg, 0.22 mmol) were mixed with methanol (0.75 ml). The reaction mixture was stirred at room temperature for 2 hours ("h"), then the methanol was evaporated, and the residue was redissolved in DMF (0.75 ml). Chloromethyl 2-1-chloroethyl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate (intermediate 3) (99 mg, 0.28 mmol) was added to the reaction mixture. The resulting mixture was stirred at 80°C for 20 hours. The DMF was evaporated under vacuum, and the residue was dissolved in chloroform and purified by column chromatography (hexane:siRNA 4:1) to obtain 117 mg (77.6%) of semi-solid.

[0103] Step 2: [ka] A solution of SP-27A (58 mg, 0.012 mmol) dissolved in chloroform (1 ml) and TFA (0.2 ml) was stirred at room temperature for 24 hours. The solvent was then evaporated, and the mixture was dried under vacuum to obtain 52 mg (90%) of the pure desired product as an oily substance. Purity measured by LC-MS with an ELS detector: 100%. MS: M+H=439.21. 1 H NMR (CDCl3, 400MHz): δ ppm 0.86-1.04 (m, 6 H), 1.35-1.49 (m, 3 H), 1.53 (br. s., 3 H), 2.96-3.09 (m, 1 H), 3.10-3.31 (m, 2 H), 3.79-3.91 (m, 2 H), 3.92-4.16 (m, 6 H), 6.85-7.01 (m, 4 H), 7.65 (br. s., 3 H).

[0104] SP-28: 1-(((R)-2-(aminomethyl)-3-methylbutanoyl)oxy)ethyl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate trifluoroacetate.

[0105] Step 1. [ka] Boc-Val-Val (150 mg, 0.47 mmol) and cesium carbonate (80 mg, 0.24 mmol) were mixed with methanol (1.13 ml). The reaction mixture was stirred at room temperature for 2 hours, then the methanol was evaporated, and the residue was redissolved in DMF (1 ml). 1-Chloroethyl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate (intermediate 3) (110 mg, 0.3 mmol) was added to the reaction mixture. The resulting mixture was stirred at 80°C for 18 hours. The DMF was evaporated under vacuum, the residue was dissolved in DCM, and purified by column chromatography (hexane SiO 1:1) to obtain 35 mg (11.9%) of semi-solid.

[0106] Step 2: [ka] A solution of SP-28A (32 mg, 0.005 mmol) dissolved in chloroform (1 ml) and TFA (0.085 ml) was stirred at room temperature for 6 hours. The solvent was then evaporated, and the mixture was dried under vacuum to obtain 33 mg (98%) of the pure desired product as a yellow semi-solid. Purity measured by LC-MS with an ELS detector: 100%. MS: M+H = 524.27. 1 H NMR (CDCl3, 400MHz): δ ppm 0.87-1.16 (m, 11 H), 1.36-1.56 (m, 6 H), 2.18 (br. s., 2 H), 2.99-3.05 (m, 2 H), 3.59-4.24 (m, 11 H), 6.18 (br. s., 2 H), 6.84 - 7.05 (m, 5H), 7.34-7.53 (m, 1 H), 8.10 (br. s., 2 H).

[0107] SP-29: (((R)-3-amino-4-methylpentanoyl)oxy)methyl 2-((2-ethoxyphenoxy)-methyl)morpholine-4-carboxylate, trifluoroacetate

[0108] Step 1. [ka] Boc-L-β-leucine (150 mg, 0.65 mmol) and cesium carbonate (110 mg, 0.146 mmol) were mixed in methanol (1.13 ml). The reaction mixture was stirred at room temperature for 2 hours, then the methanol was evaporated, and the residue was redissolved in DMF (1 ml). Chloromethyl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate (intermediate 2) (140 mg, 0.42 mmol) was added to the reaction mixture. The resulting mixture was stirred at 80°C for 18 hours. The DMF was evaporated under vacuum, the residue was dissolved in DCM, and purified by column chromatography (hexane:SiO 4:1) to obtain 120 mg (54.5%) of semi-solid.

[0109] Step 2: [ka] A solution of SP-29A (58 mg, 0.11 mmol) dissolved in chloroform (1 ml) and TFA (0.55 ml) was stirred at room temperature for 24 hours. The solvent was then evaporated, and the mixture was dried under vacuum to obtain 50 mg (90%) of the pure desired product as an oily substance. Purity measured by LC-MS: ELS detector: 100%. MS: M+H = 425.19. 1 H NMR (CDCl3, 400MHz): δ ppm 1.03 (dd, 6 H), 1.36-1.48 (m, 3 H), 2.04 (m, 1 H), 2.79 (d, 2 H), 2.93-3.22 (m, 2 H), 3.46 (br. s. 1 H), 3.57-3.65 (m, 1 H), 3.90- 4.18 (m, 6 H), 5.72-5.91 (m, 2 H), 6.86-7.02 (m, 3 H), 7.43-7.73 (m, 3 H), 8.35 (br. s., 3 H).

[0110] SP-30: Bis(((2-((2-ethoxyphenoxy)methyl)morpholine-4-carbonyl)oxy)methyl)pyridine-3,5-dicarboxylate [ka]

[0111] 3,5-Pyridinecarboxylic acid (75 mg, 0.4 mmol) and cesium carbonate (190 mg, 0.6 mmol) were mixed in methanol (0.6 ml). The reaction mixture was stirred at room temperature for 2 hours, then the methanol was evaporated, and the residue was redissolved in DMF (1 ml). Chloromethyl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate (intermediate 2) (370 mg, 1.1 mmol) was added to the reaction mixture. The resulting mixture was stirred at 80°C for 18 hours. The DMF was evaporated under vacuum, the residue was dissolved in DCM, and purified by column chromatography (hexane:SiO 1:1) to obtain 56 mg (18.5%) of a semi-solid oil. Purity by LCMS:ELS detector: 100%. MS: M+H = 754.21. 1 H NMR (CDCl3, 400MHz): δ ppm 1.44 (t, 6 H), 2.92-3.21 (m, 4 H), 3.57-3.67 (m, 2 H), 3.84 (br. s, 2 H) 3.93-4.12 (m, 12 H), 4.18-4.27 (m, 2 H), 6.07-6.11 (m, 4 H), 6.82-7.04 (m, 8 H), 8.93 (s, 1 H), 9.43 (s, 2 H).

[0112] SP-31: ((2,2'-(methylazandiyl)bis(acetyl))bis(oxy))bis(methylene)bis(2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate). [ka]

[0113] A reaction mixture of methyliminodiacetic acid (50 mg, 0.3 mmol) and cesium carbonate (144 mg, 0.4 mmol) was mixed with methanol (0.4 ml). The reaction mixture was stirred at room temperature for 2 hours, then the methanol was evaporated, and the residue was redissolved in DMF (1 ml). Chloromethyl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate (intermediate 2) (280 mg, 0.8 mmol) was added to the reaction mixture. The resulting mixture was stirred at 80°C for 18 hours. The DMF was evaporated under vacuum, the residue was dissolved in DCM, and purified by column chromatography (hexane:siRNA 1:1). The resulting product was re-purified by reverse-phase C18 column using a gradient mixture of acetonitrile and water to obtain 30.5 mg (13.8%) of a pure semi-solid oil product. Purity by LCMS:ELS detector: 100%. MS: M+H = 734.23. 1 H NMR (CDCl3, 400MHz): δ ppm 1.43-1.48 (t, 6 H), 2.55 (s, 3 H), 2.89 - 3.18 (m, 4 H), 3.51-3.69 (m, 6 H), 3.82-4.24 (m, 16 H), 5.82 (s, 4 H), 6.83-7.00 (m, 8 H).

[0114] SP-32: (((R)-2-(aminomethyl)-3-methylbutanoyl)oxy)methyl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate, trifluoroacetate.

[0115] Step 1. [ka] N-Boc-3-amino-2-isopropylpropionic acid (10 mg, 0.4 mmol), cesium carbonate (78 mg, 0.2 mmol) were mixed in methanol (0.85 ml), and the reaction mixture was stirred at room temperature for 2 hours. Then, methanol was evaporated and the residue was redissolved in DMF (1 ml). To the reaction mixture, chloromethyl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate (Intermediate 2) (95 mg, 0.3 mmol) was added. The resulting mixture was stirred at 80 °C for 18 hours. DMF was evaporated under vacuum, the residue was dissolved in DCM, and purified by column chromatography (hexane:EtOAc 4:1) to obtain 80 mg (50.8%) of a semi-solid.

[0116] Step 2:

Chem.

[0117] In vitro stability study Example 2. In vitro stability of Compound A, Compound B and Compound C in human plasma In vitro studies were conducted to evaluate the metabolic stability of compounds A, B, and C in human plasma by monitoring the elimination of prodrugs and the formation of metabolites.

[0118] The structures of compound A, compound B, and compound C are shown below. [ka] [ka] [ka]

[0119] Solution preparation Test substance solutions: 10 mM stock solutions of compound A, compound B, and compound C were prepared with dimethyl sulfoxide (DMSO). Further dilution with DMSO was performed to prepare 2.0 mM solutions. A 50 μM working solution was prepared using 20% ​​MeOH / water. Positive control stock solution and working solution: A positive control stock solution of propantheline was prepared with DMSO and stored at approximately -20°C. Before use, the stock solution was brought to room temperature, thoroughly mixed, and prepared in the same manner as above. Internal standard solution and stop solution: Stock solutions of the internal standards tolbutamide (1 mg / mL) and labetalol (1 mg / mL) were prepared with DMSO and stored at approximately -20°C. The stop solution was prepared by spike-adding the stock solution to acetonitrile to achieve a final concentration of 200 ng / mL.

[0120] Assay procedure All types of frozen plasma were thawed in a water bath at approximately 37°C and centrifuged at 3220×g for 5 minutes to remove debris. Incubation was performed in a 96-well plate format. The time points defined for this study were 0 minutes, 5 minutes, 15 minutes, 30 minutes, and 60 minutes.

[0121] Appropriate amounts of plasma derived from the test species were added to 96 deep-well plates, and working solutions of compounds A, B, and C were spiked into the plasma in double increments to achieve a final concentration of 2.0 μM. As positive controls, rat plasma was spiked with enalapril, canine plasma with bisacodyl, and human plasma with propantheline. All spiked plasma sample plates were incubated in a 37°C water bath with shaking.

[0122] At the end of each time point, the samples were immediately quenched with 3 times the volume of cold quench solution. The entire sample plate was thoroughly mixed by shaking for approximately 10 minutes and centrifuged at 3220 × g for 15 minutes. Then, 100 μL of supernatant was taken from each well and mixed with 100 μL of water in a new 96-well plate for LC-MS / MS analysis.

[0123] result The in vitro stability and piroxazine formation results for compounds A, B, and C are summarized in Table 1 and illustrated in Figures 1A-1C. The survival rates at 60 minutes were 68%, 91.5%, and 84.4% for compounds A, B, and C, respectively. The formation of the metabolite piroxazine was found to increase over time for all three compounds. [Table 1]

[0124] Therefore, the results indicated that the metabolite piroxazine was formed, and that compounds A, B, and C were metabolized in human plasma.

[0125] Example 3. In vitro stability of compound A in the intestines, whole blood, and blood components. In vitro studies were conducted to evaluate the stability of compound A in the intestines, whole blood, and blood components.

[0126] Solution preparation Artificial gastric fluid (SGF) was prepared using 34.2 mM NaCl, 80 μM sodium taurocholate, 20 μM L-alphaphosphatidylcholine / lecithin, and 0.1 mg / mL pepsin. The pH was adjusted to 1.96 by adding 1 M HCl dropwise. Artificial intestinal fluid (SIF) was prepared using monobasic sodium phosphate, 105.9 mM NaCl, 3 μM sodium taurocholate, and 750 μM L-alphaphosphatidylcholine / lecithin. The pH was adjusted to 5.98 by adding 1 N NaOH dropwise. PBS with a pH of 6.0 was prepared by titrating pH 7.4 phosphate-buffered saline (PBS) with 1 M HCl.

[0127] Stability of the test substance at different pH values. A 10 mM stock solution of compound A was prepared with DMSO. From this stock solution, 10 μL aliquots of a single concentration were added to three fluid matrices: SGF (stomach) at pH 2.0, SIF (upper intestinal tract) at pH 6.0, and PBS (whole body) at pH 7.4. The final volume was 1 mL (0.1 mM). The preparations were then incubated at 37°C, and samples were collected at 0, 15, 30, 60, and 120 minutes for analysis of the parent substance by LC / MS / MS. The collected samples were quenched in an acetonitrile / 0.1% formic acid mixture. Red blood cells (RBCs): Human and rat blood were centrifuged at 1174 × g for 15 minutes to separate red blood cells. The upper plasma surface was aspirated with a micropipette and placed in bleach for disposal. The RBC pellet was resuspended in sterile isotonic saline. The pellet was gently resuspended by shaking the tube. The sample was then centrifuged again to collect the RBC pellet, which was then resuspended and washed one more time using the same procedure. This was done a total of three times. Following the final wash and centrifugation, the RBCs were resuspended in PBS and mixed by inversion. Aliquots of the resuspended and washed RBCs were then placed into four reaction tubes. These tubes were centrifuged to collect the RBCs. The fluid level was marked in each tube, and the supernatant was then aspirated and removed. The cells were resuspended to the original mark in PBS pH 7.4 at room temperature. Aliquots of the RBC suspension were added to four sterile tubes containing PBS pH 7.4 at room temperature.

[0128] Assay procedure Enteric permeability and metabolic stability. Compound A (100 μL of 100 μM solution, or 4.25 μg) was applied to the apical surface of an epilntestinal model. Samples were collected from the basal outer compartment of the epilntestinal model at 0, 15, 30, 60, and 120 minutes. Metabolic stability was determined by the loss of the parent compound using LC / MS / MS. The collected samples were quenched in an acetonitrile / 0.1% formic acid mixture.

[0129] Stability and hemolysis in human and rat erythrocytes. Stability experiment: The stability of compound A in whole blood, erythrocytes only, and plasma was determined to assess its stability within each blood compartment. 50 μL of the test substance was placed in each blood matrix. The final volume was 1 mL (0.5 mM). Samples were collected at 0, 15, 30, 60, and 120 minutes. Stability was determined using LC / MS / MS of the parent compound. The collected samples were quenched in an acetonitrile / 0.1% formic acid mixture. Hemolysis test: Compound A (10 μL) and diluted RBCs (190 μL) derived from human or rat blood were mixed in clear 96-well V-bottom polystyrene plates at final concentrations of 25, 50, and 100 μM. The reaction mixture was incubated at 37°C for 60 minutes. Following the exposure period, the plates were centrifuged. After centrifugation, 70 μL of supernatant (without disturbing the pellet) was transferred to a clean, clear polystyrene flat-bottom 96-well plate, and the absorbance of the sample at a single wavelength was measured using a Bio Tech Synergy HCl plate reader at 410 nm. The positive control for this assay was the amphotericin B test at concentrations of 1, 10, 30, and 100 μM. Protease inhibitor study: Human whole blood (9 mL) was centrifuged at 1174 × g for 30 minutes. Aliquots (950 μL) were transferred to 1.5 mL centrifuge tubes. Then, the inhibitor cocktail was added to the tubes in 10 μL in 1:100 tubes and 5 μL in 1:200 tubes. The remaining plasma (approximately 3 mL) was transferred to a new 15 mL canonical-bottom tube. The blood was then titrated with 1N HCl to a final pH of 5.99. Aliquots (950 μL) of this matrix were transferred to 1.5 mL centrifuge tubes. All tubes were preheated to 37°C. Aliquots (50 μL) of 10 mM compound A stock solution were added to the tubes. The tubes were then pulse-vortexed for 15 seconds, a 50 μL aliquot of the sample was taken, quenched with 0.1% formic acid / acetonitrile, and pulse-vortexed for 3 seconds. The tubes were then incubated for 15 and 30 minutes, and samples were taken using the same procedure. All samples were frozen at -80°C until analysis. Stability was measured by determining the loss of the parent compound using LC / MS / MS.

[0130] Stability in the gut microbiota of rats and humans. Rat feces were obtained and weighed. One capsule of live probiotic was crushed before use and weighed. The weighed samples were then solubilized in PBS at pH 6.0 and 7.4 to extract the microorganisms. The samples were centrifuged at 18,000 × g for 10 minutes to remove granular material, and the supernatant was used to evaluate the stability of the test substance. Aliquots (50 μL) of compound A stock solution were added to the fecal extract (final concentration 0.5 mM) and incubated at 37°C. Aliquots were collected at 0, 15, 30, 60, and 120 minutes and analyzed by LC / MS / MS to determine the stability of the parent molecule. The collected samples were quenched in an acetonitrile / 0.1% formic acid mixture.

[0131] result Figure 2 shows the stability of the test substance in different matrices corresponding to different body fluids. After incubation of compound A in SGF (pl-I 2.0), the LC / MS / MS peak area decreased in a time-dependent manner up to 20 minutes. After 20 minutes, compound A remained stable in SGF. After incubation of compound A in SIP (pH 6.0), the amount of the parent compound remained at 100% at all time points. After incubation of compound A in PBS (pH 7.4), the amount of the parent compound decreased in a time-dependent manner.

[0132] Figure 3 shows the stability of the test substance in human blood. After incubation of compound A in red blood cells and whole blood, the amount of the parent compound present decreased in a time-dependent manner. Figure 4 shows the stability of the test substance in human blood with added protease inhibitors. After incubation of compound A in plasma containing a protease inhibitor cocktail, the amount of the parent compound present decreased in a time-dependent manner. When compound A was incubated with pH 6.0 plasma, there was a time-dependent increase, and its degradation was slower compared to other matrices.

[0133] Figures 5A and 5B show the hemolytic activity of the test substances in human blood. After 1 hour of exposure to compound A, there was no dose-dependent response in RBC solubility percentage in human blood. No detectable hemolysis was observed under the conditions used in this study. The EC50 of this sample was outside the test concentration range. After 60 minutes of exposure to amphotericin B (positive assay control), RBC solubility percentage increased in a dose-dependent manner. After administration of 10 μg / mL of amphotericin B, a response of over 50% was observed, and the EC50 was calculated to be 7.06 μM.

[0134] Figure 6 shows the stability of the test substance in rat blood. After incubation of compound A in all three matrices (red blood cells, plasma, and whole blood, respectively), the amount of the parent compound present decreased in a time-dependent manner.

[0135] Figures 7A and 7B show the hemolytic activity of the test substances in rat blood. After 1 hour of exposure to compound A in rat blood, there was no dose-dependent response in RBC solubility percentage. No detectable hemolysis occurred under the conditions used in this study. The EC50 of this sample was outside the test concentration range. After 60 minutes of exposure to amphotericin B (positive assay control), RBC solubility percentage increased in a dose-dependent manner. After administration of 10 μg / mL of amphotericin B, a response of over 50% was observed, and the EC50 was calculated to be 4.73 μM.

[0136] Figure 8 shows the stability of the test substance in the gut microbiota of humans and rats. After exposure to compound A in rat feces at pH 6 and pH 7.4, the amount of the parent compound decreased in a time-dependent manner. After exposure to compound A in human feces at pH 6 and 7.4, the amount of the parent compound remained close to 100% at all time points.

[0137] Therefore, compound A was stable in all bodily fluid matrices. This drug was unstable in human and rat blood, as well as in the rat gut microbiota. Furthermore, it did not permeate the intestines. Finally, under the conditions tested, no hemolytic activity was detected in human or rat blood. In comparison, the positive control (amphotericin B) resulted in a clear dose-related increase in hemolysis in human and rat blood.

[0138] Example 4. In vitro stability of compound A in the intestines, whole blood, and blood components. In vitro studies were conducted to evaluate the stability of compound A in blood and the intestines. This study also aimed to identify the enzyme family responsible for the degradation of compound A in human and rat plasma and intestinal (bacterial) enzymes.

[0139] Plasma preparation. Whole blood was centrifuged at 1174 × g for 30 minutes. Then, aliquots of the supernatant were transferred to 5 mL centrifuge tubes.

[0140] Stability of compound A in the presence of amidase in a pure system. To demonstrate amidase-dependent degradation of compound A, its stability was measured in PBS in the presence of amidase. The stock solution of the test substance (compound A) was prepared at a concentration of 10 mM in 100% DMSO. Aliquots (50 μL) of the stock solution of the test substance were added to 950 μL of PBS with and without amidase (50 units) in a 1.5 mL tube, the mixture was vortexed, and incubated at 37°C and 5% CO2 for 120 minutes. After vortexing, samples (50 μL) were taken at 0, 15, 30, 60, and 120 minutes. The samples were quenched in 150 μL of acetonitrile / 0.1% formic acid solution, vortexed briefly, and immediately frozen at -80°C. Stability was determined by measuring the loss of the parent compound using LC / MS / MS. Each exposure condition and time point was performed in triplicate.

[0141] Stability of compound A in phosphate-buffered saline (PBS) in the presence of amidase and amidase inhibitors. To confirm amidase inhibition, individual inhibitors were added to PBS containing amidase (50 units). Solutions without amidase and without inhibitors were also tested as controls. The individual inhibitors tested were 200 μM chloroacetone and 40 nM MAFP. The amidase solution was prepared as described above, and the inhibitor was added to the solution to the intended concentration. The solution was then aliquoted into 1.5 mL tubes and pre-incubated for 15 minutes before adding the test substance stock solution. An aliquot (50 μL) of the test substance stock solution was added to the PBS solution, the tube was vortexed, and then incubated with CO2 at 37°C. Decomposition began upon addition of the test substance. After vortexing, samples (50 μL) were taken at 0, 15, 30, 60, and 120 minutes. The samples were quenched in 150 μL of acetonitrile / 0.1% formic acid solution, vortexed briefly, and then immediately frozen at -80°C. Stability was determined by measuring the loss of the parent compound using LC / MS / MS. Each exposure condition and time point was performed in triplicate.

[0142] Stability of compound A in human and rat plasma (circulating enzymes) in the presence of a protease inhibitor cocktail. Compound A was added to plasma with and without the protease inhibitor cocktail and incubated to study the inhibition of the degradation of the test substance (compound A). Plasma was prepared as described above, and dilutions of the protease inhibitor cocktail (Sigma-Aldrich, catalog no. P8340, containing 104 mM AEBSF, 80 μM aprotinin, 4 mM bestatin, 1.4 mM E-64, 2 mM leupeptin, and 1.5 mM pepstatin A) were prepared in plasma at final dilutions of 1:50 and 1:10. Plasma with and without the inhibitor cocktail was aliquoted into 1.5 mL tubes to a volume of 950 μL and then pre-incubated for 15 minutes. Aliquots (50 μL) of the test substance stock solution were added to the plasma with and without the inhibitor, the tubes were vortexed, and then incubated at 37°C. Decomposition began upon addition of the test substance. After vortexing, aliquots (50 μL) were taken at 0, 15, 30, 60, and 120 minutes, quenched in 150 μL of acetonitrile / 0.1% formic acid solution, vortexed briefly, and immediately frozen at -80°C. Stability was determined by measuring the loss of the parent compound using LC / MS / MS. Each exposure condition and time point was performed in triplicate.

[0143] The stability of compound A in human and rat plasma (circulating enzymes) in the presence of individual protease inhibitors. In addition to using a cocktail of protease inhibitors, individual inhibitors were used in plasma. Chloroacetone (amidase inhibitor) was tested at 200 μM and 500 μM. MAFP (amidase inhibitor) was tested at 40 nM and 100 nM. Everactone A (esterase inhibitor) was tested at 0.15 μg / rnL. A mixture of all three individual inhibitors was also tested up to the final concentrations of 500 μM chloroacetone, 100 nM MAFP, and 0.15 μg / mL everactone A. Plasma with and without inhibitors was prepared as described above. Plasma with and without inhibitors was aliquoted into 1.5 mL tubes and pre-incubated at 37°C for 15 minutes before adding the test substance (compound A). Degradation was initiated by adding 50 μL of the test substance stock solution to 950 μL aliquots of plasma with and without inhibitors. After vortexing, aliquots (50 μL) were collected at 0, 15, 30, 60, and 120 minutes. The samples were quenched in 150 μL of acetonitrile / 0.1% formic acid solution, vortexed briefly, and immediately frozen at -80°C. Stability was determined by measuring the loss of the parent compound using LC / MS / MS. Each exposure condition and time point was performed in triplicate.

[0144] result By using PBS (phosphate-buffered saline) containing amidase enzymes together with the test substance (compound A), it was found that amidase activity plays a crucial role (Figures 9A-9B). By using fresh human and rat plasma in combination with amidase and esterase inhibitors, it was confirmed that nearly 100% of the degradation of the test substance (compound A) could be explained by esterase and amidase enzymes (Figures 10-15). Human proteolytic activity was approximately three times higher than rat proteolytic activity (Figure 16). Both amidase and esterase contribute almost equally to the degradation of the test substance under these in vitro conditions (Figure 16).

[0145] The test substance, compound A, was evaluated in both human and rat plasma. Both amidase and esterase proteases were capable of degrading the test substance. This was demonstrated by the fact that when amidase-specific inhibitors (chloroacetone and MAFP) and an esterase-specific inhibitor (everactone A) were included in the plasma incubation, complete inhibition of proteolysis (and esterase degradation) occurred when the incubation with inhibitors was compared to the incubation without inhibitors. Both amidase and esterase enzymes showed similar degradation of the test substance (compound A), and complete inhibition occurred after 1 hour in the presence of sufficient amounts of the inhibitor. The protease inhibitors showed similar results under the in vitro conditions evaluated.

[0146] In vitro metabolic stability study Example 5. In vitro metabolic stability of compounds A, B, and C in human intestinal homogenate. In vitro studies were conducted to evaluate the metabolic stability of compounds A, B, and C in human intestinal homogenates by monitoring both the elimination of prodrugs and the formation of metabolites.

[0147] Preparation of solutions. Incubation buffer (PBK): 50 mM potassium phosphate buffer (PBK) was prepared from 1 M potassium phosphate buffer pH 7.2. Test substance solutions: 10 mM stock solutions of compound A, compound B, and compound C were prepared in DMSO. A 1 mM dilution of the intermediate was prepared from the 10 mM stock solution and 90% methanol / water, and then a 10 μM working solution was prepared by diluting the 1 mM intermediate with 50 mM PBK. All working solutions were prepared on the day of the experiment and disposed of after use. Positive control stock solution and working solution: Positive control stock solutions of testosterone and 7-hydroxycoumarin were prepared in 10 mM in DMSO and stored at approximately -20°C. Before use, the stock solutions were brought to room temperature and mixed thoroughly. Working solutions were prepared on the day of use in the same manner as described for the working solutions of the test substances. Internal standard solutions and stop solutions: Stock solutions of the internal standards tolbutamide and labetalol were prepared in DMSO and stored at approximately -20°C. The stop solution was prepared by spike-adding the stock solution (1 mg / mL) to acetonitrile to achieve a final concentration of 200 ng / mL. Test system: Human intestinal homogenate was diluted with 50 mM PBK buffer to prepare a 0.625 mg / mL protein mixture solution. Cofactor solution: The cofactor solution was prepared with 10 mM NADPH (nicotinamide-adenine dinucleotide phosphate, reduced form) in 50 mM PBK buffer.

[0148] Assay procedure Incubation was performed in a 96-well plate. The intestinal homogenate mixture solution was added in two batches to a 96-well plate (80 μL / well). The plate was pre-incubated in a 37°C water bath for 10 minutes, and then 10 μM of compound A, compound B, and compound C, or the positive control working solution (10 μL each), was spiked into the corresponding wells. The reaction was initiated by adding the cofactor solution at a rate of 10 μL / well. The plate was incubated in a 37°C water bath with shaking. The final incubation mixture contained 1 μM of compound A, compound B, and compound C, or the positive control, as well as 0.5 mg / mL of human intestinal homogenate and 1 mM NADPH. The final organic solvent content during incubation was less than 1%.

[0149] The NCF60 reaction (without cofactor at 60 minutes, and without NADPH exchanged with 50 mM PBK buffer) was stopped by adding 3 times the volume of stop solution at specific time points, namely 5, 10, 20, 30, and 60 minutes. The 0 hour (T0) sample was prepared by adding 3 times the volume of stop solution to the intestinal homogenate sample, followed by the test substance or control and 10 μL / well of cofactor solution.

[0150] All sample plates were thoroughly mixed by shaking for approximately 10 minutes, and then centrifuged at 3220 × g for 20 minutes. Subsequently, 100 μL of supernatant was taken from each well, diluted with 100 μL of pure water, and analyzed by LC / MS / MS.

[0151] result Survival rate, t 1 / 2 (minutes), and inherent clearance value (CL) int(HIH) Table 2a summarizes the metabolic stability results of compounds A, B, and C in human intestinal homogenates, including ), and Table 2b summarizes the formation of the metabolite biloxazine. [Table 2] [Table 3]

[0152] Compounds A, B, and C were rapidly metabolized in human intestinal homogenate, and the formation of the metabolite biloxazine at 5 minutes was significantly higher than at 0 hours, but this did not increase further with incubation time.

[0153] Example 6. In vitro metabolic stability of compounds A, B, and C in liver S9 of Sprague Dawley rats, beagle dogs, and humans. In vitro studies were conducted to evaluate the metabolic stability of compounds A, B, and C in rat, dog, and human liver S9 by monitoring both prodrug elimination and metabolite formation.

[0154] Preparation of solutions. Incubation buffer (PBK): 50 mM potassium phosphate buffer was prepared from 1 M potassium phosphate buffer pH 7.2. Test substance solutions: 10 mM stock solutions of compound A, compound B, and compound C were prepared in DMSO. A 1 mM dilution of the intermediate was prepared in 90% methanol / 10% DMSO, and then a 10 μM working solution was prepared by diluting 0.1 mM of the intermediate with 50 mM PBK. All working solutions were prepared on the day of the experiment and disposed of after use. Positive control stock solution and working solution: Positive control stock solutions of 7-ethoxycoumarin and 7-hydroxycoumarin were prepared in 10 mM in DMSO and stored at approximately -20°C. Before use, the stock solutions were brought to room temperature and thoroughly mixed. Working solutions were prepared on the day of use in the same manner as described for the working solutions of the test substances. Internal standard solution and stop solution: Stock solutions of the internal standards tolbutamide and labetalol were prepared in DMSO and stored at approximately -20°C. The stop solution was prepared by spike-adding the stock solution (1 mg / mL) to acetonitrile to achieve a final concentration of 200 ng / mL. S9 working solution: Liver S9 derived from the test species was diluted with PBK buffer to prepare a working solution of 0.625 mg / mL. Cofactor solution: A solution of the cofactor mixture was prepared in PBK buffer with 10 mM NADPH and 10 mM UDPGA.

[0155] Assay procedure Incubation was performed in a 96-well plate. Liver S9 working solution was added in double batches to a 96-well plate (80 μL / well). The plate was pre-incubated in a 37°C water bath for 10 minutes, and then 10 μM of compound A, compound B, and compound C, or the working solution of the positive control (10 μL each), was spiked into the corresponding wells separately. The reaction was initiated by adding the cofactor mixture at 10 μL / well. The plate was incubated in a 37°C water bath with shaking. The final incubation mixture contained 1 μM of compound A, compound B, and compound C, or the positive control, as well as 0.5 mg / mL of liver S9, 1 mM NADPH, and 1 mM UDPGA. The final organic solvent content during incubation was less than 1%.

[0156] The NCF60 reaction (without NADPH (nicotinamide-adenine dinucleotide phosphate, reduced form) and UDPGA (uridine 5'-diphosphoglucuronide trisodium salt) exchanged for PBK buffer) was stopped at specific time points, namely 5, 10, 20, 30, and 60 minutes, by adding 3 times the volume of stop solution. The 0-hour (T0) sample was prepared by adding 3 times the volume of stop solution to the liver S9 sample, followed by the addition of 10 μL / well of the test substance or control and cofactor mixture solution.

[0157] All sample plates were thoroughly mixed by shaking for approximately 10 minutes, and then centrifuged at 3220 × g for 20 minutes. Subsequently, 100 μL of the supernatant was collected.

[0158] result Survival rate, t 1 / 2 (minutes), and inherent clearance (CL int(LS9) Tables 3a-3d summarize the metabolic stability results of compounds A, B, and C in liver segment S9 of the test species, including the values ​​for ). Table 3e summarizes the formation of the metabolite biloxazine. [Table 4] [Table 5] [Table 6] [Table 7] [Table 8]

[0159] Compounds A, B, and C were rapidly metabolized with half-lives of 4–12 minutes in rat, dog, and human liver S9 incubation. At 60 minutes, all samples, both in the presence and absence of cofactors, showed a residual rate of less than 5% of the test compound, suggesting that degradation was cofactor-independent. The amount of biloxazine in samples from 5-minute incubation was significantly higher than in 0-hour samples, but remained at similar levels even with longer incubation times.

[0160] In vitro coupling studies Example 7. In vitro tissue binding of compounds A, B, and C in CD-1 mouse brain homogenate. In vitro studies were conducted to determine the tissue binding properties of compounds A, B, and C in CD-1 mouse brain homogenate and to monitor the formation of their metabolite, biloxazine.

[0161] Preparation of solutions. Test Substance Solutions: 10 mM stock solutions of test substances A, B, and C, and a 3.6 mM stock solution of biloxazine were prepared in DMSO. For each compound, a 0.4 mM working solution was prepared from the stock solution in DMSO. All working solutions were prepared on the day of the experiment and disposed of after use. Positive Control Stock Solution and Working Solution: Propranolol stock solution (10 mM) was prepared in DMSO and stored at approximately -20°C. A 0.4 mM working solution was prepared on the day of the experiment in the same manner as described for the test substance working solution. Internal Standard Solution and Stop Solution: Stock solutions of tolbutamide (1 mg / mL) and labetalol (1 mg / mL), which are the internal standards for the positive control, were prepared in DMSO and stored at approximately -20°C. The stop solution was prepared by spike-adding the stock solution (2 mg / mL) to acetonitrile to achieve a final concentration of 200 ng / mL. Dialysis buffer: In this study, phosphate-buffered saline (PBS), pH 7.4, was used. Test system: Frozen mouse and human brain homogenates were thawed in a 37°C water bath and centrifuged at 3220 × g for 5 minutes to remove debris.

[0162] Assay procedure 5 μL each of compound A, compound B, and compound C, along with either a 0.4 mM working solution of viroxazine or a 0.4 mM working solution of propranolol, were separately added to a blank mouse brain homogenate (995 μL) using spike injection. The final concentrations of compound A, compound B, compound C, viroxazine, and propranolol in the assay mixture were 2 μM. The concentration of organic solvent (DMSO) in the assay mixture was 0.5%.

[0163] After thorough mixing, 50 μL aliquots of spiked brain homogenate were transferred in triplicate to a sample collection plate, immediately combined with 50 μL of dialysis buffer, and then 300 μL of stop solution to prepare the time-0 sample (C0). These samples were thoroughly mixed and stored at 2–8°C until further processing with other post-dialysis samples.

[0164] In the dialysis device, the remaining 150 μL of spiked brain homogenate was loaded into the donor chamber, and 150 μL of dialysis buffer was added in triplicate to the receiver chamber. The dialysis device was then sealed with a breathable sealer and incubated for 4 hours at 37°C in a humidified incubator containing 5% CO2, using a constant rotation on a 3D analog weaving platform shaker. (Compound A was incubated for 1 hour). At the end of dialysis, 50 μL aliquots were taken separately from both chambers and transferred to a new 96-well plate. Each sample was combined with an equal volume of blank buffer or blank brain homogenate as needed to a final volume of 100 μL, followed by the addition of 300 μL of stop solution.

[0165] All samples (including the 0 hour sample) were thoroughly mixed and centrifuged at 3220 × g for 20 minutes. 100 μL aliquots were taken from the supernatant of each well, mixed with 100 μL of ultrapure water in a new 96-well plate, and subjected to LC-MS / MS analysis.

[0166] result Table 4a shows the binding results of 2 μM compounds A, B, and C to mouse brain homogenates, and Table 4b shows the formation of biloxazine. Compounds A, B, and C were found to be unstable in mouse brain homogenates, and therefore the binding percentage values ​​under experimental conditions may be biased. The binding results of biloxazine to mouse and human brain homogenates are summarized in Table 4c. In mouse and human brain homogenates, the tissue binding affinity of biloxazine was 80.5% and 74.0%, and the recovery rates were 91.4% and 94.8%, respectively. [Table 9] [Table 10] [Table 11]

[0167] Example 8. In vitro protein binding of compounds A, B, and C in CD-1 mouse and human plasma. In vitro studies were conducted to determine the protein binding properties of compounds A, B, and C in CD-1 mouse and human plasma.

[0168] Preparation of solutions. Test Substance Solutions: 10 mM stock solutions of test substances A, B, and C, and a 3.6 mM stock solution of piroxazine (metabolite) were prepared in DMSO. For each compound, a 0.4 mM working solution was prepared from the stock solution in DMSO. All working solutions were prepared on the day of the experiment and disposed of after use. Positive Control Stock Solution and Working Solution: Warfarin stock solution (10 mM) was prepared in DMSO and stored at approximately -20°C. A 0.4 mM working solution was prepared on the day of the experiment in the same manner as described for the test substance working solution. Internal Standard Solution and Stop Solution: Stock solutions of tolbutamide and labetalol, the internal standards for the positive control, were prepared in DMSO and stored at approximately -20°C. The stop solution was prepared by spike-adding the stock solution (2 mg / mL) to CAN (acetonitrile) to achieve a final concentration of 200 ng / mL. Dialysis Buffer: Phosphate-buffered saline (PBS), pH 7.4, was used in this study. Test setup: Frozen plasma was thawed in a 37°C water bath and centrifuged at 3220×g for 5 minutes to remove debris.

[0169] Assay procedure Mouse and human plasma samples (995 μL / well) were either spiked with 5 μL of 0.4 mM working solutions of compounds A, B, and C, and biloxazine, or with warfarin working solution (0.4 mM). The final concentrations of compounds A, B, and C, as well as biloxazine or warfarin, in the samples were 2 μM. The concentration of organic solvent (DMSO) during the final incubation was 0.5%.

[0170] After thorough mixing, 50 μL aliquots of spiked plasma were transferred in triplicate to a sample collection plate, combined with 50 μL of dialysis buffer, and immediately afterward, 300 μL of stop solution was added to prepare the time-0 sample (C0). These samples were thoroughly mixed and stored at 2-8°C until further processing was carried out with the other post-dialysis samples.

[0171] 150 μL of spiked plasma was loaded into the donor chamber in a triple configuration, and 150 μL of dialysis buffer was loaded into the receiver chamber of the dialysis device. The dialysis device was then sealed with a breathable sealer and incubated for 4 hours at 37°C in a humidified incubator containing 5% CO2, using a constant rotation on a 3D analog waving platform shaker. (Compound A was incubated for 1 hour). At the end of dialysis, 50 μL aliquots were taken separately from both chambers and transferred to a new 96-well plate. Each sample was combined with an equal volume of blank buffer or plasma as needed to bring the final volume to 100 μL, and 300 μL of stop solution was added immediately afterward.

[0172] All samples (including the 0 hour) were thoroughly mixed for approximately 30 minutes and centrifuged at 3220 × g for 15 minutes. A 100 μL aliquot of the supernatant was taken from each well and mixed with 100 μL of ultrapure water in a new 96-well plate for analysis by LC-MS / MS.

[0173] result Plasma protein binding of compounds A, B, and C. Because these three compounds are unstable in mouse plasma, plasma binding and recovery rates were not reported.

[0174] Plasma protein binding of biloxazine (metabolite). The percentage of biloxazine protein binding was 80.5% and 74.0% in mouse and human plasma, respectively. The recovery rate of biloxazine from all dialysis wells ranged from 101% to 105%, indicating that this compound was stable during the dialysis process in this study. The results are summarized in Table 5 below. [Table 12]

[0175] In vivo research Example 9. Evaluation of cerebral permeability of biloxazine after single intravenous administration of an analog (S) to male CD-1 mice. Studies were conducted to determine the pharmacokinetic (PK) profiles of biloxazine in plasma and brain after a single intravenous (IV) administration of three analogues, namely compound A, compound B, and compound C, to male CD-1 mice.

[0176] Study Design: Forty-five male CD-1 mice were divided into three treatment groups (n=15 in each group). The animals were not fasted before administration and were given free access to food and water throughout the study period. Compounds A, B, and C, all analogs of biloxazine, were administered to one mouse group in each of the three groups at doses of 9.911, 10.439, and 10.557 mg / kg, respectively. These doses of the three biloxazine analogs were equivalent to 5 mg / kg of free base biloxazine. After treatment, three mice were euthanized at 0.083 hours, 0.25 hours, 0.5 hours, 1 hour, and 4 hours, respectively, and plasma and brain samples were collected. Biroxazine concentrations in plasma and brain homogenates were measured by LC-MS / MS. Mean concentration-time data of biloxazine in plasma and brain were used to evaluate PK properties and brain-plasma ratios.

[0177] Composition of the test substances. The drug solution was prepared before administration on the day of the study. The vehicle used for the drug preparation was the same for all three analogs, consisting of DMSO, PEG400, and 30% HP-β-CD / H2O in ratios of 4, 30, and 64 (v / v / v). The target concentrations of the drug solutions for compounds A, B, and C were 1.982, 2.088, and 2.111 mg / mL, respectively. All final formulations appeared as clear solutions, and each was administered at a dose of 5 mL / kg, achieving target doses of 9.991, 10.439, and 10.557 mg / kg, respectively. The dose of each analog was equivalent to 5 mg / kg of free base biloxazine.

[0178] result Clinical findings: Immediately after administration, all animals appeared lethargic and exhibited a slow, shallow breathing pattern. The adverse effect lasted for approximately 2 minutes, after which all animals recovered. Slowing down the administration of the compound over approximately 30 seconds reduced the adverse effect.

[0179] Table 6 summarizes the PK parameters of biloxazine in plasma and brain. Figures 17, 18, and 19 show the concentration-time profiles of biloxazine in plasma and brain after IV administration of compounds A, B, and C, respectively. [Table 13]

[0180] The data indicates that when these three piroxazine analogs, compound A, compound B, and compound C, were administered intravenously at doses equivalent to 5 mg / kg of free base piroxazine, similar piroxazine pharmacokinetic profiles were produced in both plasma and the brain. The brain-to-plasma ratio of piroxazine, measured by either concentration or AUC, was also similar among the three analogs after IV administration (the brain / plasma ratio was approximately 4–5). The high brain concentrations or AUC of piroxazine suggest that this compound can efficiently cross the blood-brain barrier (BBB), likely via active uptake transport. These data indicate that, as measured by the PK profile or brain permeability characteristics of piroxazine, the three piroxazine analogs were indistinguishable in terms of conversion to piroxazine.

[0181] Example 10. Evaluation of the pharmacokinetics of S(-)-piroxazine and R(+)-piroxazine after single oral administration of a prodrug and racemic piroxazine to male CD-1 mice. To evaluate the pharmacokinetic (PK) characteristics of S(-)-biloxazine and R(+)-biloxazine after single oral (PO) administration of three prodrugs, namely compound A, compound B, compound C, and racemic biloxazine, to male CD-1 mice, we conducted a study.

[0182] Study Design: Twelve male CD-1 mice were divided into four treatment groups (n=3 in each group). The animals were fasted overnight before administration. Food was returned two hours after administration. Water was always freely available to the animals throughout the study. Three prodrugs, compound A, compound B, and compound C, and racemic biloxazine were administered orally to one group of mice at doses of 19.82, 21.03, 21.11, and 11.64 mg / kg, respectively. The doses of the prodrugs and racemic biloxazine were equivalent to 10 mg / kg of free base biloxazine. To determine the concentrations of S(-)-biloxazine and R(+)-biloxazine, blood samples were collected from each animal at 0.25, 0.5, 1, 2, 4, 8, and 24 hours after administration, and the plasma was extracted by centrifugation. The concentrations of the two isomers in plasma were quantified by liquid chromatography-tandem mass spectrometry (LC-MS / MS). In this bioanalysis assay, the limit of quantification (LLOQ) for both isomers was 1.5 ng / mL, and the linear range was up to 1500 ng / mL. Plasma concentration vs. time data were analyzed using Phoenix WinNonlin 6.3 to determine the PK characteristics of S(-)-biloxazine and R(+)-biloxazine. A non-compartmental analysis model and a linear log-trapezoid method were applied to the PK calculations.

[0183] Composition of the test substances. The drug solution was prepared immediately before administration on the day of the study. The formulations of the three prodrugs and racemic biloxazine were prepared in the same manner using 4% DMSO, 30% PEG 400, and 66% HPβCD (30% in H2O, w / v) as the vehicle. The target concentrations of compound A, compound B, compound C, and racemic biloxazine were 19.82, 21.03, 21.11, and 11.64 mg / kg, respectively, all corresponding to 10 mg / mL of free base biloxazine. The nominal dose volume was 10 mL / kg for all four compounds administered. All formulations appeared as clear solutions at the time of administration.

[0184] result Clinical findings: Compounds A, B, C, and racemic biloxazine were well tolerated in all animals at the administered doses. No adverse effects were observed during the study period.

[0185] The corresponding PK parameters are summarized in Tables 7a to 7d. The plasma concentration-time profiles of S(-)-piroxazine and R(+)-piroxazine after PO administration are shown in Figures 20A to 20H, respectively. [Table 14] [Table 15] [Table 16] [Table 17]

[0186] These data show that after PO administration of three prodrugs, S(-)-biloxazine C max and AUC 0-last However, it consistently showed higher yields (approximately 10 times) than those of R(+)-biloxazine. For both isomers, the yields were numerically higher for compounds A and B compared to compound C. C of the two isomers after administration of racemic biloxazine max and AUC 0-last They were equivalent. 1 / 2 and MRT 0-last This was similar between the two isomers, regardless of whether they were prodrugs.

[0187] Example 11. Evaluation of the pharmacokinetics of S-biloxazine in male Sprague-Dawley rats. This study was conducted to evaluate the pharmacokinetic (PK) characteristics of S-biloxazine after a single oral (PO) administration to male Sprague-Dawley rats.

[0188] Study Design: Three male Sprague-Dawley rats were administered a single dose of 40 mg / kg of S-biloxazine, equivalent to 40 mg / kg of free base VLX (biloxazine), via forced oral administration. The animals were fasted overnight prior to administration. Food was returned 2 hours after administration. Water was continuously available to the animals throughout the study. Blood samples were collected from each animal at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after administration to determine the plasma concentrations of R(+)-VLX and S(-)-VLX. The plasma concentrations of these two analytes were quantified by liquid chromatography-tandem mass spectrometry (LC-MS / MS). In this bioanalysis assay, the lower limit of quantification (LLOQ) was 1.5 ng / mL and the upper limit of quantification (ULOQ) was 1500 ng / mL for both R(+)-VLX and S(-)-VLX. To elucidate the pharmacokinetic (PK) properties of the analytes, plasma concentration-time data were analyzed using Phoenix WinNonlin (version 8.3). A non-compartmental analysis model and a linear / log-trapezoidal method were applied to calculate the PK parameters.

[0189] Composition of the test substance. The drug solution was prepared immediately before administration on the day of the study. The mixture appeared as a clear solution, and the final concentration was 40 mg / mL in 0.9% saline.

[0190] result Clinical findings: S-biloxazine at the administered dose was well tolerated in all animals. No adverse effects were observed during the study period.

[0191] The PK parameters of S(-)-VLX are summarized in Table 8. The plasma concentration-time profile of S(-)-VLX is shown in Figure 21. [Table 18]

[0192] After a single oral administration of S-biloxazine at 40 mg / kg, the enantiomer R(+)VLX was undetectable at any time point, and therefore all PK parameters could not be determined. Peak plasma concentration of S(-)VLX (C max The respective values ​​were 2973 ± 1201 ng / mL. max This was achieved 0.417 ± 0.144 hours after administration (T max ). Area under the plasma concentration-time curve (AUC) of S(-)-VLX from 0 hours to the time of final quantification. 0-last The terminal elimination half-life (T) of S(-)-VLX was 3594±1641 ng·h / mL. 1 / 2 The average residence time (MRT) from 0 hours to the final quantifiable time is 0.97 ± 0.271 hours. 0-last The duration was 1.10 ± 0.106 hours.

[0193] S-biloxazine was detected in plasma with typical PK levels, and no detectable conversion to R-biloxazine occurred; the level was below the lower limit of quantification.

[0194] Example 12. Evaluation of the pharmacokinetics of compound A in male Sprague-Dawley rats. This study evaluated the pharmacokinetic (PK) properties of compound A (also known as compound A), a biloxazine (VLX) derivative, after a single oral (PO) administration to male Sprague-Dawley rats.

[0195] Study Design: Animals were fasted overnight before drug administration. Feed was returned 2 hours after drug administration. Water was always freely available to the animals throughout the study. In Group 1, three male Sprague-Dawley rats were administered a single dose of compound A at 60 mg / kg, equivalent to 36 mg / kg of free base VLX, via forced oral administration. In Group 2, three male Sprague-Dawley rats were administered a single dose of compound A at 120 mg / kg, equivalent to 72 mg / kg of free base VLX, via forced oral administration. Blood samples were collected from each animal at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after drug administration to determine the plasma concentrations of compound A, as well as R(+)-VLX and S(-)-VLX. The plasma concentrations of these three analytes were quantified by liquid chromatography-tandem mass spectrometry (LC-MS / MS). In this bioanalysis assay, the lower limit of quantification (LLOQ) was 1.5 ng / mL and the upper limit of quantification (ULOQ) was 1500 ng / mL for both R(+)-VLX and S(-)-VLX. The LLOQ and ULOQ for the analysis of compound A were 3 ng / mL and 3000 ng / mL, respectively. To elucidate the PK characteristics of the analytes, plasma concentration-time data were analyzed using Phoenix WinNonlin (version 8.3). A non-compartmental analysis model and a linear / logarithmic trapezoidal method were applied to calculate the PK parameters.

[0196] Composition of the test substance. The drug solution was prepared immediately before administration on the day of the study. The mixture appeared as a clear solution, and the final concentrations were 60 mg / mL (Group 1) and 120 mg / mL (Group 2) in 0.9% saline.

[0197] result Clinical findings: Compound A was well tolerated in all animals at the administered dose. No adverse effects were observed during the study period.

[0198] Following a single oral administration of compound A, the derivative itself was undetectable, and therefore no data to report. The enantiomer R(+)-VLX was undetectable for most time points and only had low concentration values ​​insufficient for PK calculations. The PK parameters of S(-)-VLX are summarized in Table 9. The plasma concentration-time profiles of R(+)-VLX and S(-)-VLX are shown in Figures 22A and 22B, respectively. [Table 19] [Table 20]

[0199] Peak plasma concentrations of S(-)-VLX in Group 1 and Group 2 (C max The values ​​were 815±325 and 3806.7±1103.5 ng / mL, respectively. max In group 1, this occurred 0.333 ± 0.144 hours after drug administration (T max ), in group 2, 0.667±0.289 hours after drug administration (T max This was achieved in the area under the plasma concentration-time curve (AUC) of S(-)-VLX from 0 hours to the final quantification time. 0-last The glycemic acid levels were 981±442 ng·h / mL in group 1 and 5753±402 ng·h / mL in group 2. In group 1, the terminal elimination half-life (T) of S(-)-VLX was 1 / 2 The average residence time (MRT) from 0 hours to the final quantifiable time is 0.92 ± 0.22 hours. 0-last The half-life (T) was 1.14 ± 0.133 hours, and in group 2, the half-life (T) was 1.14 ± 0.133 hours. 1 / 2 ) is 0.89 ± 0.0327 hours, MRT 0-last The duration was 1.36 ± 0.18 hours.

[0200] These data indicate that compound A is rapidly metabolized upon absorption, as it was less than LLOQ in all collected samples. The S-enantiomer C max and AUC 0-lastAs evidenced by the significantly higher value, the degradation of compound A resulted in more S(-)-VLX than R(+)-VLX. Between the two dose groups, T max They are the same, MRT 0-last They were equivalent.

[0201] Example 13. Evaluation of the pharmacokinetics of biloxazine analogs, as well as S(-)-biloxazine and R(+)-biloxazine, after a single oral administration to male and female beagle dogs. A study was conducted to evaluate the pharmacokinetic (PK) characteristics of three biloxazine analogs, namely compound A, compound B, and compound C, after single oral (PO) administration to male and female beagle dogs, as well as S(-)-biloxazine and R(+)-biloxazine.

[0202] Study Design: Three biloxazine analogs, compound A, compound B, and compound C, were administered orally at a dose of 80 mg / kg to two male and two female beagle dogs. The animals were fasted overnight before administration. Food was returned 4 hours after administration. Water was continuously available to the animals throughout the study. To determine the concentrations of the analogs and the two biloxazine isomers, S(-)-biloxazine and R(+)-biloxazine, blood samples were collected from each animal at 0.5, 1, 2, 4, 6, 8, and 24 hours after administration, and the plasma was extracted by centrifugation. The concentrations of the analytes in the plasma were quantified by liquid chromatography-tandem mass spectrometry (LC-MS / MS). In this bioanalysis assay, the lower limit of quantification (LLOQ) was 1.5 ng / mL and the upper limit of quantification (ULOQ) was 1500 ng / mL for the two biloxazine isomers, while the LLOQ was 3.0 ng / mL and the ULOQ was 3000 ng / mL for the three analogs. Plasma concentration vs. time data were analyzed using Phoenix WinNonlin 8.3 to determine the PK characteristics of the analytes. A non-compartmental analysis model and a linear log-trapezoidal method were applied to the PK calculations.

[0203] Composition of the test substance. The drug solution was prepared immediately before administration on the day of the study. Compositions of the three analogues were similarly prepared using HPLC water as the vehicle. The target concentrations of compound A, compound B, and compound C were 16 mg / mL. When administered at a nominal dose volume of 5 mL / kg, the target dose of each prodrug was 80 mg / kg.

[0204] result Clinical findings: Compounds A, B, and C were well tolerated in all animals at the administered doses. No adverse effects were observed during the study period.

[0205] For compound A and compound B, their plasma concentrations were mostly below LLOQ in both male and female beagle dogs after oral administration at 80 mg / kg. Therefore, the PK properties of compound A and compound B were not determined. The corresponding PK parameters are summarized in Tables 10a to 10c. The plasma concentration-time profiles of PV-0448, as well as S(-)-biloxazine and R(+)-biloxazine, are shown in Figures 23 to 28. [Table 21] [Table 22] [Table 23]

[0206] These data show that after PO administration of three analogues, S(-)-piroxazine C max and AUC 0-lastHowever, the S(-) isomer yield was consistently higher (approximately 10 times) than that of R(+)-biloxazine, and the yield of the S(-) isomer was better than that of the R(+) isomer in all three analogs. The PK properties of each isomer were comparable among the three analogs. The only difference among the three analogs was that the plasma concentration of compound C was significantly above LLOQ, while the plasma concentrations of the other two analogs were mostly below LLOQ. In this study, no significant differences were observed in the measured PK parameters between male and female dogs.

[0207] Example 14. Dose-response evaluation of S-piroxazine prodrug compound C in mice using the tail suspension test (TST). The tail suspension test is an experimental method used in scientific studies to measure helplessness in rodents (especially mice). It is based on the observation that mice cease to struggle when exposed to short-term, unavoidable stress. Immobility is quantified by measuring the amount of time spent without overall activity. A decrease in immobility time (seconds) after treatment indicates that the drug may have an antidepressant effect.

[0208] A study was conducted to evaluate the behavioral effects of acute administration of S-piroxazine prodrug compound C in the tail suspension test. Adult C57Bl / 6 mice were administered one of four doses (PO) of compound C, and the tail suspension test (TST) was performed 30 minutes later. Immobility time (suggesting depressive-like behavior) during the last 4 minutes of the TST was quantified using blinded offline video manual scoring.

[0209] Methods and materials Animals. Adult (7-8 weeks old) male mice (C57Bl / 6, Charles River Laboratories) were used in the study. Upon arrival, the animals were housed in groups (5 mice / cage) and given free access to food and water. The animals were kept in a controlled room with controlled temperature (22±2℃) and humidity (approximately 50%) under a 12 / 12-hour light / dark cycle. The animals were numbered sequentially by tail marks. Each cage was identified by a card showing the study number, sex, animal number, and date of birth.

[0210] Substances and composition. The test substances were formulated according to the table below. [Table 24]

[0211] The test substance formulation was prepared on each administration day based on weight versus volume in the vehicle. Vehicle formulation composition: 0.8% DMSO, 6% PEG400, 93.2% HPβCD (6% in H2O), diluted from a 5x concentrated (5X) stock solution of 4% DMSO + 30% PEG400 + 66% HβCD (30%) in water (1x volume of stock solution + 4x volume of H2O). The formulation was clear in appearance and no precipitate was observed. The solution of compound C was slightly pink. The compound was protected from light and kept on ice until administration. 30 minutes before TST, animals (10-11 animals / group) were force-administered orally (10 mL / kg, po). Doses of compound C included 10, 30, 60, and 90 mg / kg. A vehicle group was included for comparison.

[0212] Monitoring. Video recordings were made for scoring using piezoelectric sensors operated by the SmartCage system. After the animals were acclimatized to the laboratory, each mouse's tail was placed on a piezoelectric sensor board, and the mouse was suspended upside down. This immediately triggered recording of struggling behavior, which continued for 6 hours. The mouse was then released and returned to its home cage. Manual scoring of video recordings during periods of 120 to 360 seconds in the TST was used to quantify immobility time suggestive of depression-like behavior. The percentage of immobility time was calculated as (immobility time / 240 seconds) * 100.

[0213] result At all doses, compound C did not result in a significant reduction in immobility as determined by ANOVA (F4,47=0.8366, p=0.5089). Mice treated with compound C (30 mg / kg, PO) tended to show reduced immobility compared to the vehicle-treated group (Figure 30).

[0214] Example 15. Follow-up evaluation of compound C in mice using the tail suspension test (TST). A study was conducted to evaluate the behavioral effects of acute treatment with S-biloxazine prodrug compound C in the tail suspension test. This was a follow-up study of Example 14, a dose-response selection study of compound C in the tail suspension test (TST). Previous studies showed low immobility levels in the negative control group and lacked a positive control group to support task reliability. Therefore, this follow-up study was conducted to clarify the data from previous studies and to evaluate a promising dose of compound C (30 mg / kg) in conjunction with positive and negative control groups. To this end, adult C57Bl / 6 mice were administered one of three treatments: compound C at a dose of 30 mg / kg (PO), vehicle (negative control), or imipramine (positive control), and the TST was performed 30 minutes later. Immobility time (suggesting depressive-like behavior) during the last 4 minutes of the TST was quantified using blinded offline video manual scoring.

[0215] Methods and materials Animals. Adult male mice (C57Bl / 6, supplied by Charles River Laboratories), approximately 8 weeks old and weighing 20-25g, were used in the study. Upon arrival, the animals were housed in groups (5 mice / cage) and given free access to food and water. The animals were kept in a controlled room with controlled temperature (22±2℃) and humidity (35-50%) under a 12 / 12 light / dark cycle. The animals were numbered sequentially by tail ID. Each cage was identified by a colored card indicating the study number, sex, animal number, and date of birth.

[0216] Substances and composition. The test substances were formulated according to the table below. [Table 25]

[0217] On the day of administration, the test substance formulation was prepared immediately based on the weight-to-volume ratio in the vehicle. Vehicle formulation composition: 0.8% DMSO, 6% PEG400, 93.2% HPβCD (6% in H2O), diluted in water (1x volume of stock solution + 4x volume of H2O) from a 5x concentrated (5X) stock solution of 4% DMSO + 30% PEG400 + 66% HPβCD (30%). The formulation was clear in appearance and no precipitate was present. The solution of compound C was slightly pink. The compound was protected from light and kept on ice until administration. 30 minutes before TST, animals (10 animals / group) were force-administered orally (10 mL / kg, po). Compound C at a dose of 30 mg / kg was tested together with a vehicle-negative control group and an imipramine (15 mg / kg)-positive control group, which were included for comparison.

[0218] Monitoring. Video recordings were made for scoring using piezoelectric sensors operated by the SmartCage system. After the animals were acclimatized to the laboratory, each mouse's tail was placed on a piezoelectric sensor board, and the mouse was suspended upside down. This immediately triggered recording of struggling behavior, which continued for 6 hours. The mouse was then released and returned to its home cage. Manual scoring of video recordings during periods of 120 to 360 seconds in the TST was used to quantify immobility time suggestive of depression-like behavior. The percentage of immobility time was calculated as (immobility time / 240 seconds) * 100.

[0219] result In all included treatment groups, a significant effect of the treatment on immobility time was demonstrated by one-way ANOVA. Post-hoc testing showed that this effect was primarily facilitated by the positive control imipramine, which significantly reduced immobility time compared to vehicle (p=0.01, Figure 31). However, compound C at a dose of 30 mg / kg did not result in a significant reduction in immobility compared to vehicle (p=0.43).

[0220] Example 16. Dose-response evaluation of S-piroxazine prodrug compound C in mice using SmartCage for walking activity. Using SmartCage®, we conducted a study to evaluate the behavioral effects of acute administration of S-piroxazine prodrug compound C on 24-hour walking activity.

[0221] Methods and materials Animals. A cohort of 39 adult male mice (C57BL / 6, 7-8 weeks old, 20-25g body weight, supplied by Charles River Laboratories) was used in this study. Upon arrival at the facility, the animals were housed in groups (5 animals / cage) and given free access to food and water. The animals were kept in a controlled room with controlled temperature (22±2℃) and humidity (approximately 35-50%) on a 12 / 12 light / dark cycle. Each animal in its home cage was sequentially numbered using a tail ID marker. Each home cage was identified by a colored ID card showing the study number, sex, animal number, and date of birth. The animals used in this study were the same ones previously used in the TST evaluation (Example 14).

[0222] Substances and composition. The test substances were formulated according to the table below. [Table 26]

[0223] The test substance formulation was prepared on each administration day based on the weight-to-volume ratio in the vehicle. The vehicle formulation included 0.8% DMSO, 6% PEG400, and 93.2% HPβCD (6% in H2O), which were diluted with water (1x volume of the stock solution + 4x volume of H2O) from a 5x concentrated (5X) stock solution of 4% DMSO + 30% PEG400 + 66% HPβCD (30%).

[0224] Preparation of the stock solution. The compound was progressively prepared in 4% DMSO + 30% PEG400 + 66% HPβCD (30%) at 5 times the final concentration (mg / ml). Subsequently, HPβCD (30%) was added after PEG400. The formulation was clear in appearance and no precipitate was observed. The compound was protected from light and kept on ice until administration.

[0225] After recording baseline activity over a 24-hour period, the animals (7-8 animals / group) were force-administered orally (10 mL / kg, po; around 12-1 PM). For recording walking activity, the dosage of compound C included 10, 30, 60, and 90 mg / kg.

[0226] Behavioral testing methods Monitoring of walking activity. Mice were monitored for 48 hours using Smart Cages (experimental schematic diagram, Figure 32). Baseline activity was measured during the first 24 hours. From day 1 onward, animals were administered one of four doses (PO) of the test substance and monitored for the next 24 hours. Animal activity during the monitoring period was quantified using parameters such as distance traveled, activity time, and standing behavior.

[0227] SmartCage. After a drug-free period of at least 24 hours following TST evaluation in which animals were housed in groups (5 mice / cage), individual mice were placed in newly prepared home cages, and their activity was simultaneously recorded using the SmartCage® system and a video camera positioned above the SmartCage® system. Activity in a predetermined time block (1-hour block) was automatically calculated using CageScore® (a program associated with the SmartCage® system; AfaSci, Inc.).

[0228] Active wakefulness was defined as active movement, standing, and exploratory behavior. Variables of home cage activity included activity count (i.e., photocell beam interruptions), walking (distance traveled and speed), and standing count. Activity count was obtained from a downward horizontal infrared (IR) sensor (along the X and Y axes). Similarly, distance traveled in centimeters was obtained from the downward horizontal IR and calculated considering the animal's path. Walking was defined as traveling a distance longer than the body length of the subject. The calculated distance traveled (during any predetermined period called a “block” or “total measurement period”) and speed were the two main parameters of walking activity. The number of photocell beam interruptions on the Z axis reflected the number of beam interruptions in the IR sensor in the upper column and indicated standing or climbing behavior, which is considered part of the exploratory behavior parameter. All IR data (beam interruption activity count, walking, standing, and turning) were continuously recorded at a sampling rate of 4 Hz. The absolute and percentage values ​​of the time spent in this wakeful state in selected blocks (or “time bins”) were also collected. Mice were most active within the first hour after being transferred to an unused or new cage, after which their activity levels gradually decreased. Treatment with the test substance was initiated once the mice's activity level stabilized.

[0229] Endpoints / Parameters: Behavioral assessment using SmartCage over a 48-hour period, measured in 1-hour blocks: ● Activity time ● Distance traveled (and speed of travel) ● Startup

[0230] Scoring Method: The SmartCage system calculates distance traveled and speed by integrating X and Y coordinates and elapsed time using an automated scoring algorithm. The upper row of IR sensors detects Z photobeam interruption accounts indicating standing behavior. Data analysis included calculating the ratio of light (daytime) to dark (nighttime) periods. The nighttime ratio was calculated using the entire 12-hour dark period (T31-42) after drug administration (T26) and compared to the same time before drug treatment (i.e., T7-T18) using IR data. The daytime ratio was calculated using the 5-hour period after drug administration (T26-30) and compared to the same time before drug treatment (T2-6). At the end of the experiment, mice were euthanized using CO2 and subsequent secondary methods. No tissues were collected.

[0231] result Walking activity. Following administration of compound C, there was no significant increase in activity time, nor any improvement in walking parameters such as distance traveled, speed, or number of stands (Figures 33A-33D). When comparing average activity data for nighttime (Figures 34A-34D) and daytime (Figures 35A-35D), there were no differences in any of the measured activity parameters before and after treatment with compound C. Therefore, the activity of the home cages exhibited a typical circadian rhythm regardless of compound C administration (10, 30, 60, and 90 mg / kg, PO). Compound C did not significantly affect daytime or nighttime activity measurements, including activity, stands, distance, or speed.

[0232] Example 17. Evaluation of compound C for prepulse suppression in rats. A study was conducted to evaluate the effect of compound C on prepulse suppression of the acoustic startle response in an experimental animal model. Prepulse suppression is the preattentional part of information processing (i.e., sensorimotor gating) characterized by the suppression of the startle reflex when preceded by a weak prepulse stimulus. This process is impaired in schizophrenia and other mental disorders (e.g., obsessive-compulsive disorder). Apomorphine, a dopamine agonist, reduces prepulse suppression and serves as a model for screening antipsychotic drugs in a prepulse suppression task. Compound C was evaluated for its ability to attenuate the impaired effect of apomorphine on prepulse suppression, which supports the possibility of improving cognitive abilities in schizophrenia. A single dose of haloperidol, a typical antipsychotic, was included as a positive control during the evaluation process for each dose of compound C. The main measurement parameters in this study were prepulse suppression % and mean startle intensity (arbitrary units - AU).

[0233] Experimental Design Study subjects: Sixty adult (approximately 3 months old) male Wistar rats (Envigo, Inc., Indianapolis, IN) were used in the study. Two rats were housed in each polycarbonate cage (45 x 30 x 18 cm) filled with corn cob bedding in a room with a constant temperature (21-23°C) and humidity (40-50%). Lighting was maintained on a 12-hour light-dark cycle (7:00 AM to 7:00 PM), and the rats were given free access to water and food throughout the study period. All behavioral tests were conducted during the light phase of the light / dark cycle (9:00 AM to 5:00 PM) (Monday to Friday).

[0234] Prepulse suppression (PPI) of acoustic startle response - Procedure. Four standard startle chambers (San Diego Instruments, San Diego, CA) were used. These startle chambers consisted of plexiglass tubes (8.2 cm in diameter, 25 cm in length) placed inside soundproof chambers, with rats individually placed inside. The tubes were mounted on plastic frames, with piezoelectric accelerometers mounted below to record and convert the tube's movement. Three days before drug testing, each subject was placed in one of the startle test chambers for 10 minutes as an initial acclimatization period to the apparatus (no startle stimulus was applied). Two days before drug testing, the animals were placed back into the test chambers and then exposed to 12 startle stimuli and each prepulse level three times (see below). This procedure was performed to reduce the high variability in response to initial exposure to startle stimuli and to ensure that prepulse stimuli (alone) did not have a significant effect on the startle response. One day prior to the drug trials, all subjects underwent a total of 60 PPI trial sessions (details of the drug trial experiments are described below). Subsequently, drug trial groups (N=8-10) were created and balanced to ensure similar average startle intensities.

[0235] On the day of the drug test, the rats were moved to a startle chamber and kept calm for at least 30 minutes. Afterward, the rats were placed in the chamber and remained there. Following this period, the rats underwent 12 startle trials, 12 no-stimulation trials, and 12 pre-pulse / startle trials (see below), for a total of 60 trials. Intervals between trials ranged from 10 to 30 seconds, and each session lasted approximately 25 to 30 minutes. The startle trial consisted of a single 120 dB white noise burst lasting 20 milliseconds (ms).

[0236] The prepulse suppression trials consisted of a prepulse (a 20 ms burst of white noise at an intensity of 75, 80, or 85 dB) followed 100 ms later by a startle stimulus (120 dB, 20 ms white noise). During the no-stimulus trials, no startle sound was presented, but rat movements were recorded. This served as a control test to detect differences in overall activity. Sixty different trials were presented quasi-randomly, with each trial being presented 12 times, ensuring that no two consecutive trials were identical. The resulting rat movements in the startle chamber were measured within 100 ms after the start of the startle stimulus (sampling frequency 1 kHz), rectified, amplified, and input into a computer to determine the maximum response during the 100 ms period. The baseline startle intensity was determined as the average intensity of the 12 startle trials. Prepulse suppression was calculated according to the formula 100 - 100% × (PPx / P120). In the formula, PPx is the average of 12 prepulse suppression trials (i.e., at each individual prepulse level), and p120 is the baseline shock intensity. The mean level of the PPI was also calculated (mean response to pp75, pp80, or pp85) and analyzed separately.

[0237] Drug preparation and administration. The doses of all compounds were calculated based on the free base of each compound. The doses of apomorphine (0.5 mg / kg, sc) and the positive control compound haloperidol (0.3 mg / kg) are based on previously reported studies. Phenotypic screening in mice showed that compound C exhibited a cognitive enhancement signature at 120 and 150 mg / kg (po) and an antipsychotic signature at 150 mg / kg. To include similar plasma concentrations of compound C in rats, this study utilized compound C at doses of 60, 90, and 120 mg / kg, administered at 2.0 mL / kg (po).

[0238] The test substance formulations were prepared immediately before use based on weight versus volume in the vehicle. The vehicle was added after weighing the compounds. The physical properties of the formulations, including appearance (solution vs. suspension), color, and precipitate, were recorded. During preparation, all compounds in this study were completely dissolved (i.e., particle-free solution) and clear.

[0239] After preparation, all compound solutions were protected from light and kept on ice until administration. Administration was carried out within 8 hours of preparation. The final concentration composition of the vehicle for compound C was 0.8% DMSO, 6% PEG400, and 93.2% HPβCD (6% in H2O), which was obtained by diluting a 5x concentrated stock solution of 4% DMSO + 30% PEG400 + 66% HPβCD (30%), which was prepared stepwise, with water (1x volume of stock solution + 4x volume of H2O). The drug was dissolved using low heat (37°C water bath). Subsequently, PEG400 was added, followed by HPβCD (30%). [Table 27]

[0240] result Figure 36 shows the effects of compound C and the positive control antipsychotic haloperidol on PPIs at different prepulse levels, and Table 11 shows the mean PPI response (mean of all prepulse levels). Statistical significance was found in the response to various drug treatments [F(5,54)=4.2, p=0.003] and the response to different prepulse levels [F(2,108)=343.1, p<0.001], and the interaction between treatment and prepulse intensity was significant [F(10,108)=2.0, p=0.044]. Post-hoc analysis showed that apomorphine significantly reduced PPIs compared to the vehicle at all three prepulse levels (p<0.05). Haloperidol (0.3 mg / kg) significantly antagonistized the effect of apomorphine on PPIs at all three prepulse levels (75, 80, and 85 dB; p<0.05). Averaging the data across all prepulse intensities, the effect of haloperidol on apomorphine-related responses was also significant (p<0.05) (see Table 1). None of the compound C doses were associated with a statistically significant attenuation of the effect of apomorphine on PPIs. Furthermore, this study did not find any statistically significant treatment-related effects on mean startle intensity (Table 11). [Table 28]

[0241] In summary, under vehicle (control) conditions, the prepulse stimuli used (75, 80, and 85 dB) clearly suppressed the startle response to a 120 dB acoustic stimulus in a decibel level-dependent manner (i.e., higher prepulse decibel levels resulted in greater suppression of the startle response). Apomorphine clearly reduced the effect of prepulse stimuli on the acoustic startle response. A single dose of the positive control antipsychotic haloperidol was effective in attenuating the damaging effects of apomorphine at all three prepulse levels. Compound C did not significantly attenuate the effects of apomorphine at any prepulse level. Therefore, based on this study, which is based on the dopamine hypothesis of schizophrenia, compound C (at least at the three doses evaluated) did not appear to exhibit a significant cognitive enhancement ability to normalize attentional preprocessing function after dopaminergic impairment (i.e., apomorphine-induced reduction).

[0242] Example 18. Evaluation of compound C against PCP in a rat operant set shift test. The use of the operant-set shift test in rats has shown that PCP induces a decline in cognitive flexibility (i.e., the ability to modify / adapt behavior in accordance with changes in rules). This assay serves as a model for executive dysfunction associated with neuropsychiatric disorders such as schizophrenia. The aim of this study was to evaluate whether compound C reverses the decline in cognitive flexibility observed in PCP using the operant-set shift test in rats.

[0243] Methods and materials Substance and composition. Compound C (white powder) was first dissolved in 4% dimethyl sulfoxide (DMSO) at 5 times its final concentration (mg / mL), then in 30% polyethylene glycol (PEG) 400, then in 66% 30% 2-hydroxypropyl-β-cyclodextrin (HPβCD), and finally diluted with water (1x volume of stock solution + 4x volume of H2O).

[0244] The dosage was prepared separately by weighing (W / V). Compound C formulations were prepared immediately before use on each administration day, and administration was carried out within 8 hours of formulation. White powder phencyclidine (PCP) hydrochloride was dissolved in physiological saline by magnetic agitation on each administration day. A vehicle (20% distilled water containing 4% DMSO, 30% PEG 400, 66% 30% HPβCD, and 80% distilled water) was used as a control substance.

[0245] Test system. 78 male Wistar rats were supplied for this study. ● Seventy-two rats aged 8-9 weeks, weighing 218-299g at the start of the visual discrimination test, were subjected to the whole-behavioral test and randomized into a match group based on their performance during the second visual discrimination session. ● Six reserve rats were subjected to lever-pushing, side-bias, and visual discrimination tests, but were excluded during the test period (set shift and reversal).

[0246] The animals were sent to the laboratory at least 5 days before the start of the experiment (lever pressing) to allow them to acclimate to laboratory conditions. The animals were housed in pairs in macrolon cages (SAFE, 89290 Augy, France) with wood shavings as bedding. Water was freely available, but access to food (code A04-SAFE, 89290 Augy, France) was restricted. Environmental enrichment (chewing material and nesting material) was provided. The animal enclosure was maintained under artificial lighting (12 hours) from 7:00 to 19:00, with an ambient temperature controlled at 22±2℃ and a relative humidity of 30-70%.

[0247] During this experiment, no deaths or signs of abnormal behavior were observed. The animals were sacrificed by exposure to CO2. No autopsies were performed on the animals.

[0248] Operantset shift test in rats (reference CNS 6.3R system) The experimental protocol for detecting the effect on behavioral flexibility follows that described by Floresco et al. (Behav. Brain Res., 190, 85-96 (2008)).

[0249] The apparatus consists of a standard Med Associates ENV-008 Skinner box (30 x 25 x 30 cm) equipped with a house light, two retractable levers, and a feed pellet dispenser. The levers are positioned on either side of the feed tray, which is connected to the pellet dispenser. An LED stimulation light is positioned above and centered on each lever and served as a stimulus for visual cue discrimination learning.

[0250] The Skinner box is housed in a standard Med Associates soundproof enclosure, the ENV-022MD, and connected to the MED-PC programming system for automated experiment control and data acquisition.

[0251] Feeding schedule. From arrival, the animals were subjected to restricted feeding (15g / day) to acclimate them to the fasting schedule to be used during the experiment. This fasting schedule was maintained throughout the experiment and was necessary to motivate the animals to perform the task. On the test day, after the last animal was tested, the animals were given 15g of food in their home cages.

[0252] The day before the lever-pushing experiment began, the animals were given several 45mg food pellets in their home cages to help them adapt to the new food.

[0253] Lever pressing training. The goal of this stage was to train the animal to press the lever in order to receive a reward of food pellets.

[0254] In an experimental chamber, animals were subjected to several learning sessions according to a fixed ratio (FR1) reinforcement schedule. Reinforcement consisted of a feed pellet (45 mg) supplied after each lever press.

[0255] Rats were first subjected to lever-pushing learning sessions 2-6 times a day, in which food pellets were supplied based on responses using either the left or right lever. The lever was inserted into the chamber at the start of the session and withdrawn at the end of the session. The house light was turned on at the start of the session and turned off at the end of the session. The session ended after 30 minutes or after the animal had performed 50 lever responses, whichever came first.

[0256] Animals that quickly learned to press the lever were allowed to rest until the remaining animals learned to press the lever. Then, before moving on to the next stage, all the animals were tested together in a final lever-pressing session.

[0257] Subsequently, the rats were subjected to sessions in which either the left or right lever was presented in a pseudo-random manner every 20 seconds. Each session consisted of 90 lever presentations (i.e., 1 trial = 1 lever presentation), starting with the levers retracted and the chamber darkened. Every 20 seconds, a trial was initiated by turning on the house light and inserting one of the two levers into the chamber. If the rat did not respond to the lever within 10 seconds, the lever was retracted, the chamber was darkened, and the trial was scored as unresponsive. If the rat responded within 10 seconds, the lever was retracted, one pellet was dispensed, and the house light was left on for an additional 4 seconds. Importantly, the stimulus light above each lever was never turned on during these training sessions.

[0258] Rats were given daily sessions until they achieved a criterion of fewer than 5 no responses to 90 lever presentations in a single final session, after which they proceeded to the next stage (3 or 4 sessions in this study).

[0259] Side bias. On the final day of training, the rats' side bias was assessed. This session was similar to the lever-pushing session, except that both levers were inserted into the chamber together (i.e., 90 lever presentations at 20-second intervals). Again, the stimulus lights above each lever did not illuminate during this training session. In the initial lever presentation, a food pellet was dispensed after a response to either lever. In subsequent lever insertions, food was dispensed only if the rat responded to the lever opposite to the one initially selected. If the rat selected the same lever as initially selected, no food was dispensed. This continued until the rat selected the lever opposite to the one initially selected. After both levers had been selected, a new trial was started. Thus, one trial of the side bias process consisted of responding to both levers. The session ended after 90 lever presentations or after 21 trials were completed.

[0260] The lever (right or left) to which the rat responded in the initial choice of each trial was recorded and added to the side bias. The lever that the rat initially selected more often over 21 trials was considered the side bias. However, if the rat responded excessively (i.e., more than a 2:1 ratio) to one lever throughout the entire session, that lever was considered the side bias.

[0261] Visual cue discrimination learning. After determining side bias, animals were subjected to visual cue discrimination learning.

[0262] In this discrimination study, rats were asked to respond to a lever (either left or right, presented randomly) with an illuminated visual cue light above it. The session began with both levers retracted and the chamber dark (inter-trial state). Every 20 seconds, one of the cue lights above one lever was illuminated to initiate a trial. After 3 seconds, the house light was turned on and both levers were inserted into the chamber. A response to the lever with the illuminated cue light (positive response) resulted in the lever being retracted, the cue light being turned off, and a food pellet being supplied. After feeding, the house light was left on for an additional 4 seconds, and then the chamber was returned to the inter-trial state. If the rat responded to the other lever (negative response), both levers were immediately retracted without feeding, and the chamber was returned to the inter-trial state. If the rat did not respond to either lever within 10 seconds, both levers were retracted, the house light was turned off, and the trial was recorded as unresponsive. In each pair of trials, the left or right stimulus light was activated once, and the order of the pairs of trials was randomized. For each trial, the position of the lever and stimulus light selected by the animal was recorded. Trials continued until the rat achieved a baseline performance of 10 consecutive positive responses after a minimum of 30 trials, or 150 trials, whichever came first. Trials with no response were not included in the count of trials to achieve the baseline.

[0263] The following day, a second visual cue discrimination session was conducted using the same baseline performance to confirm that the rats had learned visual cue discrimination.

[0264] Response discrimination shift. Acquiring this discrimination required the animal to cease using a visual cue discrimination strategy to obtain food rewards and instead use an egocentric, spatial response strategy. A correct response here required responding with the lever opposite the side bias (left or right) as defined above, regardless of the position of the stimulus light illuminated above one of the levers. As with the initial visual cue discrimination, the left or right stimulus light was illuminated once in each pair of trials, and the order of the pairs of trials was randomized. The trials were conducted in the same manner as the visual cue discrimination, and again, the level and position of the stimulus light selected by the animal were recorded for each trial. Trials continued until the rat achieved a criterion performance of 10 consecutive correct responses after a minimum of 30 trials, or 150 trials, whichever came first. Trials with no response were not included in the count of trials to achieve the criterion.

[0265] In this case as well, a second response discrimination shift session was performed the following day (without drugs) to confirm that the rats had shifted to response discrimination.

[0266] Reverse learning. Finally, the rats were moved to the reverse test. This was as described for response discrimination, however, the correct response here required responding to the same lever as the side bias (left or right), regardless of the position of the stimulus light illuminating above one of the levers.

[0267] In this case as well, the reversed session was performed the following day (without drugs) to confirm that the rats had learned the rule.

[0268] Recorded behavioral indicators: ● Number of attempts, number of no responses, and number of errors to achieve the criterion of 10 consecutive correct responses in each session (visual cue learning, set-shift to response discrimination, and reverse learning). ● Furthermore, in response discrimination set shift, the number of errors is reported as the total number of errors and the number of persistent errors. Therefore, the number of errors was divided into the following two types: ○ Persistence error: In a trial where the rat was required to press the opposite lever, the rat responded (as in visual cue learning) to the lever where the upper stimulus light was lit. ○ Never-Reinforced Error: When a rat presses the wrong lever in a trial where a visual cue light is illuminated above the correct lever (i.e., the selection was not reinforced during the initial visual cue discrimination or response discrimination shift).

[0269] Drug testing procedure. Twelve rats were tested per group. The study was conducted in a blinded manner, divided into two sub-experiments, with the same number of animals per group in each sub-experiment. PCP (2 mg / kg sc), administered again 30 minutes before the first response discrimination shift and before the first reversal session, was used as the reagent substance. Compound C was administered at doses of 40, 80, 120, and 160 mg / kg 10 minutes after PCP (i.e., 20 minutes before the test session) and evaluated, and compared with the vehicle control group.

[0270] result An operant set shift test was conducted in rats. Almost all rats achieved the criterion of 10 consecutive correct responses in fewer than 150 trials (78 trials as an average score during one visual cue discrimination session). Acquisition of visual cue discrimination was confirmed during the second session. These animals were randomized into treatment groups of n=12 based on their performance during the second visual cue discrimination session, ensuring no significant differences between groups in this metric before the drug test. [Table 29]

[0271] Animals treated with saline solution achieved the criterion of 10 consecutive correct responses with an average score of 70.8 trials or less during the first response discrimination set-shift session. Error analysis showed that these were primarily persistent errors during the set-shift, supporting the influence of visual cue discrimination learning (24.0 ± 2.3 persistent errors out of a total of 25.7 ± 2.3 errors). Subsequently, the criterion of 10 consecutive correct responses was achieved with an average score of 95.9 trials or less during the first reversal session. This suggests adaptation to the new rules.

[0272] PCP (2 mg / kg) administered via sc 30 minutes prior to the first response discrimination set shift session and the first reversal session significantly increased the number of trials required to reach the baseline compared to the saline control (+45% and +35%, t(22)=3.09, p<0.01 for the first response discrimination set shift session, and t(22)=3.26, p<0.01 for the first reversal session). As a result, the number of errors significantly increased in the first response discrimination set shift session and the first reversal session, respectively (+47% and +66%, t(22)=2.42, p<0.05 and t(22)=3.65, p<0.01), with persistence errors being the primary cause of the significant increase in the first response discrimination set shift session (mean total errors 37.8 vs. mean persistence errors 35.8). However, in the second session (treatment without drugs), rule acquisition was confirmed, as indicated by a clear decrease in the number of trials compared to the first session. However, there was a slight but significant increase compared to the saline control (+31%, t(22)=2.55, p<0.05).

[0273] Compound C (40, 80, 120, and 160 mg / kg) administered post-administered 20 minutes prior to the first response discrimination set-shift session and the first reversal session (i.e., 10 minutes after PCP) did not significantly affect the number of trials to reach the baseline (one-way Anova: F(4,55) = 0.729, NS, and 2.009, NS, respectively) or the number of persistence errors (one-way Anova: F(4,55) = 2.198, NS, and 1.197, NS, respectively) compared to PCP controls within the tested dose range.

[0274] However, the number of unresponsive episodes increased across all doses, particularly during the first response discrimination set-shift session (one-way Anova: F(4,55) = 2.593, p<0.05 and 1.438, respectively, for the first set-shift session and the first reversal session, NS). A post-hoc Dunnett t-test showed a significant effect at the 120 mg / kg dose (p<0.05).

[0275] These results, using the operant-set shift test in rats, demonstrate that PCP induces a decline in cognitive flexibility (i.e., the ability to modify / adapt behavior in accordance with rule changes). These findings support the suitability of this assay as a model for executive dysfunction associated with neuropsychiatric disorders such as schizophrenia.

[0276] Example 19. SMARTCUBE® Phenotypic Screening To evaluate the CNS-like efficacy of the novel test compound, we conducted a study using the SmartCube® system.

[0277] Methods and materials Animals. Male C57 / Bl6 mice from Taconic Laboratories (Germantown, NY) were used. Upon receipt, the mice were housed in groups of four per cage in OPTIMice® ventilated cages. The mice were acclimatized to the colony room for at least one week prior to the experiment, and then tested at approximately 8-9 weeks of age. Before the start of the study, all animals were examined, handled, and weighed to ensure sufficient health and fitness, and to minimize nonspecific stress associated with the procedure. A 12 / 12 light / dark cycle was maintained throughout the study. Room temperature was maintained at 20-23°C, and relative humidity at 30-70%. Feed and water were freely available throughout the study.

[0278] Test compounds. All compounds were administered orally at a dose volume of 10 mL / kg 20 minutes prior to the test. The formulations were prepared on the day of the test, and administration was carried out within 1 hour of preparation. The compounds were prepared according to the table below. Twelve mice were used in each treatment group. [Table 30]

[0279] The structures of compounds D and E are shown below. [ka] [ka]

[0280] SmartCube®. The SmartCube® system is designed to measure a wide range of spontaneous behaviors and responses to loads in the same test environment, and it is able to do so successfully. The hardware includes force sensors and several aversive stimuli to elicit behaviors. Three high-resolution video cameras provide a continuous 3D view of the mouse inside the SmartCube® apparatus (SC) throughout the entire test period. The mouse is subjected to a series of loads during 45-minute test sessions. The cube is cleaned between each run.

[0281] To construct a reference dataset, drugs were injected 15 minutes prior to the trial, and multiple loads were applied throughout the trial session. The digital video of the subjects was processed using a computer segmentation algorithm to fit a geometric model to each mouse frame image. The resulting fitted parameters were then analyzed using a behavioral classifier algorithm to extract behavioral states such as standing, walking, and immobility. Using the data thus obtained, drug signatures of known reference compounds in PGI were defined, and therapeutic class signatures enabling comparison of test samples were established.

[0282] The data mining process utilizes several analytical methods, including Bayesian probability density models and decision trees. The algorithm considers approximately 2,600 indicators, including the frequency and duration of behavioral states such as grooming and standing, as well as many other features obtained during the test sessions.

[0283] We routinely perform analyses of two main types: classes and subclasses.

[0284] For class and subclass analysis, we constructed a reference dataset from hundreds of drug doses in multiple drug classes and control groups. Each reference drug was tested in mice at multiple appropriate doses. We selected the best-performing classifier from our evaluation tests and constructed two different types of classifiers that make independent predictions at the class and subclass levels of drugs. A class consists of drugs currently on the market or drugs clinically validated for their specific indications. A subclass is a larger set than a class, consisting of both marketed drugs and other compounds that are mechanistically validated.

[0285] Data from screening was processed using proprietary computer vision and data mining algorithms, and the results were compared to the signatures of reference compounds in our database. A combined analysis of the data was performed to quantitatively make independent predictions of drug classes and drug subclasses. These classifiers were used to evaluate the behavioral signatures of the investigational drugs and predict their potential therapeutic utility.

[0286] The results of class analysis and subclass analysis are presented as standardized bar graphs using percentages that sum to 100 for each dose. The results of classification at the drug level are presented as individual similarities.

[0287] Similarity analysis using a "cloud framework." The outcomes from SmartCube® are a large set of features (behavioral parameters) that can be used for various analyses. Many of these features are correlated. Therefore, statistically independent combinations of the original features (hereinafter referred to as uncorrelated features) were formed. Since each uncorrelated feature extracted information from the entire cluster of original features, the dimensionality of the new feature space is reduced. Next, a proprietary feature ranking algorithm was applied to score the discriminative power (the ability to distinguish between two groups, such as vehicles and treatments) of each feature. Ranking is an important part of the analysis because it weights the changes in each feature according to their importance. Each feature was ranked by applying a feature ranking algorithm derived from support vectors of support vector machine learning. If there is a significant change in some unimportant features measured for a particular phenotype, the low rank of these features automatically reduces the impact of that change in our analysis, thus eliminating the need to rely on conventional "feature selection" methods or to discard information buried in features with less information.

[0288] We then examined the uncorrelated and ranked features as "clouds" (Gaussian distributions approximating groups of mice (e.g., reference compound and vehicle) in the ranked uncorrelated feature space) and calculated a quantitative measure of separability between the two groups ("distinguishability") (Figure 41). The top two uncorrelated features were selected to form a 2D coordinate plane for visualization purposes. For visualization purposes, each cloud was plotted on a semi-axis equal to one standard deviation along the corresponding dimension. Next, we calculated the proximity of the test compound to the reference compound(s). Proximity of over 80% suggests strong proximity.

[0289] result The class and subclass analyses of all tested compounds can be seen in Figures 42A-42B.

[0290] Biloxazine (15 mg / kg) showed low behavioral activity and a prominent vehicle-like signature, as indicated by the white bars. This compound showed moderate activity at 30 mg / kg and high activity at 60 and 90 mg / kg. All doses showed a mixed signature with a prominent "unknown" signature at the class level and a mixed signature at the subclass level. The unknown portion of the signature indicates that the compound was active, but the classification system, while detecting a difference from the vehicle, could not reliably assign any specific features or patterns (which may be novel, or simply insufficient changes in strength shown at low doses) to any CNS class.

[0291] Compound E (40 and 80 mg / kg) showed moderate activity and a mixed signature with a prominent "unknown" signature in both its class and subclass.

[0292] Compound D (40 and 80 mg / kg) showed dose-dependent increases in activity and mixed signatures in both its class and subclasses. The prominent class signature was "unknown," while the subclass showed a mixed signature.

[0293] Compound A (30-120 mg / kg) and Compound B (90 and 120 mg / kg) exhibited mixed signatures with high behavioral activity and a prominent "unknown" signature within their respective classes. Subclass analysis also showed that Compounds A and B primarily exhibited nootropic-like mixed signatures.

[0294] Compound C (60–150 mg / kg) showed high behavioral activity and mixed signatures in its class and subclasses. While 60, 90, and 120 mg / kg showed a prominent "unknown" signature in its class, the highest dose tested exhibited a prominent antipsychotic-like signature.

[0295] Interestingly, the subclass signatures for all active compounds were mixed. The prominent signatures observed were nootropic and analgesic.

[0296] The subsequent data analysis, which involved performing a similarity analysis (DRFA) using a cloud framework to evaluate active doses for various reference compounds, is summarized in Figures 43–54. Discrimination values ​​were related to the vehicle cloud and the reference compound cloud, while proximity values ​​indicated the relative distance between the test compound cloud and the reference compound cloud. Unique features were those present in the test cloud that were not similar to either the reference cloud or the vehicle cloud, and could be nonspecific effects.

[0297] None of the compounds showed proximity to the attention-deficit hyperactivity disorder (ADHD) compound complex cloud used for the analysis results in Figure 43. However, when the reference compounds were compared individually in Figures 44-46, the level of proximity varied between active doses, but the similarity to amphetamine and modafinil remained relatively moderate to low. Notably, compound C showed proximity of over 75% to atomoxetine and amphetamine.

[0298] Interestingly, when compared to donepezil, biloxazine, compound B, and compound C showed proximity of over 70%, and biloxazine and compound B showed similar proximity to thioperamide. Compounds B and C also showed proximity of over 70% to morphine. Compound C showed proximity to the antidepressants desipramine and amitriptyline. Biroxazine and compound B also showed proximity to these two antidepressants, but to a relatively lower degree of over 60%. In a comparison between biloxazine and bupropion, proximity of over 70% was shown, while compounds B and C were 69%.

[0299] Subsequently, biloxazine, compound A, and compound C were analyzed at different doses and compared with a subset of the same reference compounds (see Figures 55-63). Compound A showed increased proximity to both thioperamide and donepezil with increasing dose. At the two highest doses (90 and 120 mg / kg), proximity peaked at over 75% and over 65%, respectively. Overall, compound C and biloxazine showed similar patterns, peaking at approximately 100% proximity when compared to each other. Furthermore, biloxazine showed a dose-dependent increase in proximity to memantine, although it only reached 57%.

[0300] In summary, several compounds were orally administered with a 20-minute pretreatment time before being tested with SmartCube®. Most doses showed high activity, with mixed signatures exhibiting at the class and subclass levels, and in most cases, subclass analysis. Biroxazine, compound A, compound B, and compound C also tended to exhibit additional nootropic signatures at higher test doses, similar to H3 antagonists. Notably, the class analysis of 90 mg / kg of biloxazine showed a significant analgesic class signature not seen in the other test compounds. Compound C at 120 mg / kg and 150 mg / kg also exhibited additional analgesic subclass and antipsychotic class features, respectively. Subsequently, proximity analysis to selective reference compounds(s) revealed that biloxazine, compound B, and compound C exhibited proximity to donepezil, desipramine, thioperamide, and amitriptyline. Compounds B and C showed proximity to morphine of over 70%. Biroxazine, compound B, and compound C showed proximity to bupropion of approximately 70%. Compound C also showed proximity to atomoxetine and amphetamine of over 70% when analyzed separately from modafinil, another ADHD compound. Dose-based analyses of biloxazine, compound A, and compound C generally showed a dose-dependent increase in proximity to thioperamide and donepezil.

[0301] Example 20. Dose-response evaluation of S-piroxazine prodrug compound A in mice using SmartCage and tail suspension tests for walking activity. Evaluating walking activity is a simple way to demonstrate spontaneous walking activity, arousal, and exploratory drive in rodents. It is one of the most common rodent tests and can be used to test the effects of various drugs on animal behavior in both wild-type and genetically modified animals. This study used the SmartCage® system, an automated, non-invasive rodent behavior monitoring system that allows biomedical researchers to perform various neurobehavioral assays by consistently and accurately monitoring the activity and behavior of rodents in their home cages.

[0302] The tail suspension test is a mouse behavioral test useful for screening potential antidepressants and evaluating other operations that are expected to affect depression-related behaviors. The tail suspension test is an experimental method used in scientific studies to measure helplessness in rodents (especially mice). This is based on the observation that when mice are exposed to short-term, unavoidable stress, they give up struggling. "Immobility" is quantified by measuring the amount of time an animal is inactive. A reduction in "immobility" time (seconds) after treatment indicates that the drug may have an antidepressant effect.

[0303] Using SmartCage, we conducted studies to evaluate the behavioral effects of acute administration of S-piroxazine prodrug compound A on 24-hour walking activity, and to assess potential antidepressant effects using the tail suspension test (TST). The results may be used for dose range selection in follow-up EEG sleep / wake studies.

[0304] Methods and materials Animals. A cohort of 32 adult male mice (C57BL / 6, 7-8 weeks old, weighing 18-25g, supplied by Charles River Laboratories) was used for walking activity, and another cohort of 40 male mice was used for the TST study. Upon arrival at the facility, the animals were housed in groups (5 animals / cage) and given free access to food and water. The animals were kept in a controlled room with controlled temperature (22±2℃) and humidity (approximately 50%) on a 12 / 12 light / dark cycle. Each animal in its home cage was sequentially numbered using a tail ID marker. Each home cage was identified by a colored ID card showing the study number, sex, animal number, and date of birth.

[0305] Substances and composition. The test substances were formulated according to the instructions in the table below. [Table 31]

[0306] On each day of administration, the test substance formulation was prepared immediately based on weight versus volume in the vehicle. (Vehicle composition: 4% DMSO, 30% PEG400, 66% HPβCD (30% in H2O)). The compound was weighed before adding the vehicle. The formulation was clear and no precipitate was observed. The compound was protected from light and kept on ice until administration.

[0307] Thirty minutes before the TST, animals (8 mice / group) were force-administered orally (10 mL / kg, po). To monitor walking activity, baseline activity was recorded over a 24-hour period, after which mice were force-administered orally (10 mL / kg, po, around 12-1 PM). For walking activity recording, the dosages of compound A included 10, 30, and 90 mg / kg. For the TST, the dosages of compound A included 4, 8, 16, and 32 mg / kg.

[0308] Dose selection: The dose range for monitoring with SmartCage was based on previous behavioral studies. SmartCube phenotypic screening at 90 mg / kg (po) showed that compound A produced biological activity and there was no evidence of side effects.

[0309] Behavioral testing methods Monitoring of walking activity. Mice were monitored for 48 hours using Smart Cages (experimental schematic diagram, Figure 64). Baseline activity was measured during the first 24 hours. From day 1 onward, animals were administered one of three doses (PO) of the test substance and monitored for the next 24 hours. Animal activity during the monitoring period was quantified using parameters such as distance traveled, activity time, and standing behavior.

[0310] SmartCage. Individual mice were placed in newly prepared home cages and simultaneously recorded using the SmartCage® system and a video camera positioned above the SmartCage® system. The CageScore® program, associated with the SmartCage® system (AfaSci, Inc.), automatically calculated activity within a defined time block.

[0311] The active arousal state is defined as vigorous movement, standing, and exploratory behavior. Variables of home cage activity include activity count (i.e., photocell beam interruptions), walking (distance traveled and speed), and standing count. Activity count is obtained from a downward horizontal infrared (IR) sensor (along the X and Y axes). Similarly, distance traveled in centimeters is obtained from the downward horizontal IR and calculated considering the animal's path. Walking is defined as a distance traveled longer than the body length of the subject. The calculated distance traveled (during any predetermined period called a “block” or “total measurement period”) and speed are the two main parameters of walking activity. The photocell beam interruption count on the z axis reflects the number of beam interruptions in the IR sensor in the upper column and indicates standing or climbing behavior, which is considered part of the exploratory behavior parameter. All IR data (beam interruption activity count, walking, standing, and turning) were continuously recorded at a sampling rate of 4 Hz. The absolute and percentage values ​​of the time spent in this arousal state in selected blocks (or “time bins”) were also collected. Mice are most active within the first hour after being moved to an unused or new cage, after which their activity level gradually decreases. Treatment with the test substance was initiated once the mice's activity level stabilized.

[0312] Endpoints / Parameters: Behavioral assessment using SmartCage over a 48-hour period, measured in 2-hour blocks: ○ Activity time ○ Distance traveled (and speed of travel) ○ Starting up

[0313] Scoring method. The SmartCage system uses an automatic scoring algorithm to calculate distance traveled and speed by integrating X and Y coordinates and elapsed time. The upper row of IR sensors detects Z photobeam interruption accounts indicating rise behavior.

[0314] Data analysis included calculating the ratio of light (daytime) to dark (nighttime) periods. The nighttime ratio was calculated using the entire 12-hour dark period after drug administration (T34-46) on T28, and the IR data was compared to the same time period before drug treatment (i.e., T10-T22). The daytime ratio was calculated using the 2-hour period after drug administration (T30-32) and comparing it to the same time period before drug treatment (T6-8).

[0315] At the end of the experiment, the mice were euthanized using CO2 and subsequent secondary methods. No tissue was collected.

[0316] Tail suspension test (TST). Video recording was performed using a piezoelectric sensor operated by the SmartCage system for scoring. After acclimatizing the animals to the laboratory, the TST was initiated by placing the tail of the test mouse on a piezoelectric sensor board and suspending the mouse upside down. The recording period began when the mouse was inverted and continued for 6 minutes. After the recording period, the mouse was released from the recording device and returned to its home cage. Manual scoring of 120-360 second video recordings during the TST was used to quantify immobility time suggestive of depressive-like behavior. The percentage of immobility time was calculated as (immobility time (seconds) / 240 seconds) * 100.

[0317] result Basic statistical analyses were performed using GraphPad Prism and expressed as mean ± standard error of the mean (SEM). The data were evaluated for normal distribution and uniform variance. Non-normally distributed data were analyzed using alternative nonparametric tests. Group differences found to be normally distributed were evaluated using one-way ANOVA followed by Dunnett's multiple comparison test, comparing all treatment groups to the vehicle control. Statistical significance was set at p ≤ 0.05.

[0318] Walking activity. After administration of compound A, there was no significant increase in activity time, nor were there any improvements in walking parameters such as distance traveled, speed, and number of times standing up (Figures 65A-65D). When comparing the average activity data for nighttime (Figure 66) and daytime (Figure 67), there was no difference in any of the measured activity parameters between before and after treatment with compound A.

[0319] Tail suspension test. Compound A resulted in a significant reduction in immobility (F4,35=4.398, p=0.006). Post-hoc testing revealed that at a dose of 32 mg / kg (p≦0.01), mouse mobility was significantly higher compared to the vehicle-treated group (Figure 68).

[0320] In summary, home cage activity exhibited a typical circadian rhythm regardless of compound A administration (10, 30, and 90 mg / kg, PO). Compound A did not significantly affect diurnal or nocturnal activity measurements, including activity, rising, distance, or speed. In contrast, the highest dose of compound A (32 mg / kg, PO) significantly reduced mouse immobility in the TST by 30% compared to the vehicle, suggesting an antidepressant effect.

[0321] Example 21. Effects of compound A and S-biloxazine on the elevated cusp maze in rats. A study was conducted to evaluate the anxiolytic behavioral effects of two compounds, compound A and S-biloxazine, in the elevated cross maze (EPM) task in rats. The EPM is a widely used behavioral assay in rodents that has been validated to evaluate the anxiolytic effects of pharmacological agents. The EPM typically consists of two open arms and two closed (walled) arms elevated above the laboratory floor and arranged in a cross shape. The EPM relies on the innate and unconditional fear that rodents have of open spaces and heights. Anxious animals spend more time in the closed arms, while increased activity (duration and / or number of entries) in the open arms reflects anxiolytic (calming) behavior.

[0322] In this study, a single dose of midazolam, a benzodiazepine tranquilizer, was evaluated as a positive control / reference compound during the evaluation process for each compound. The main measurement parameters in this study were the number of open arm entries, the percentage of time spent in the open arm, and the total distance traveled, but various additional anxiety-related behaviors were also analyzed.

[0323] Methods and materials Study subjects: 97 adult (approximately 3 months old) male Wistar rats (Envigo, Inc., Indianapolis, IN) were used in the study. Two rats were housed in each polycarbonate cage (45 x 30 x 18 cm) filled with corn cob bedding in a room with a constant temperature (21-23°C) and humidity (40-50%). Lighting was maintained on a 12-hour light-dark cycle (7:00 AM to 7:00 PM), and the rats were given free access to water and food throughout the study period. All behavioral tests were conducted during the light phase of the light / dark cycle (9:00 AM to 5:00 PM) (Monday to Friday).

[0324] Methods. After the test subjects were delivered to the testing facility, they were allowed to acclimate to the new containment environment for at least one week. At the beginning of the week of drug testing, each subject was handled for approximately 5 minutes per day for two days. Then, on the third day, the test subjects were placed in an open-field walking activity chamber (43.2 × 43.2 cm, Med Associates, St. Albans, VT) for 30 minutes. This handling and open-field process was performed to reduce initial anxiety and variability associated with handling, movement, and exposure to a new environment. The EPM (Habitest® Modular Systems, Coulbourn Instruments, Allentown PA) used in these studies was made of black acrylic and consisted of two open arms (44.5 cm × 10 cm) and two closed arms of the same size, each with a wall 29.5 cm high. The maze was configured with arms of the same type facing each other, and the apparatus was elevated to a height of 53 cm from the floor. These arms were connected by a 10 cm × 10 cm square in the center. All arms and the central square were equally illuminated at approximately 200 lux. For the trial, each animal was placed in the center of the maze facing an open arm and observed for 5 minutes. During the 5-minute trial period, the number of entries into each open and closed arm, the duration of stay, and the total distance traveled were recorded. The percentage of time spent in the open arm [(time spent in the open arm / (time spent in the open arm + time spent in the closed arm)) × 100] and the total number of entries (number of entries into the open arm + number of entries into the closed arm) were also calculated. Entry into an arm was recorded when all four limbs were inside the arm. Additional anxiety-related behaviors were also evaluated: head dipping (exploratory head / shoulder movements beyond the lateral side of the open arm), stretch-attend posture (an exploratory posture in which rodents stretch forward and return to their original position without walking forward), and retreat to the closed arm (moving out of the closed arm using only the forelimbs and returning to the same arm; see Rogers and Johnson, 1995). All trials were video-tracked, and the above outcome measures were determined using EthoVision XT 14 software. Eight to nine animals were used per treatment group.

[0325] Drug preparation and administration. Compound A was prepared using 0.8% DMSO, 6% PEG400, and 93.2% HPβCD (6% in H2O). The vehicle composition of compound A was 0.8% DMSO, 6% PEG400, and 93.2% HPβCD (6% in H2O), which was diluted from a 5x concentrated (5X) stock solution of 4% DMSO + 30% PEG400 + 66% HβCD (30%) in water (1x volume of stock solution + 4x volume of H2O). This stock solution was dissolved in 0.8% DMSO and 6% PEG400, and then diluted with water containing 6% HPβCD to the desired final concentration. Administration was carried out within 1 hour of preparation / remixing on each administration day. The appearance of both formulations, including appearance (solution vs. suspension), color, and precipitate, was recorded. The final preparation of compound A for administration was a pale yellow solution with no precipitate. The compounds were protected from light and kept on ice until administration. The vehicle for S-VLX was purified water, while the vehicle for compound A was 0.8% DMSO, 6% PEG400, and 93.2% HPβCD (6% in H2O) (see the composition table below). [Table 32]

[0326] The required amount of S-VLX was weighed into a brown beaker (or a beaker wrapped in aluminum foil), mixed with a small amount of vehicle to moisten the test substance, stirred with a magnetic stirrer, and transferred to a measuring cylinder / volume flask. The remaining vehicle was added to achieve the required concentration. S-VLX was prepared to the highest concentration based on weight-to-volume in water (Milli-Q). The final preparation of S-VLX for administration was a colorless solution without precipitate. The required amount of midazolam was dissolved in 0.9% saline and further diluted with 0.9% saline to achieve the concentration required for injection.

[0327] Dosage Basis: Compounds were administered by forced oral administration. Doses were calculated based on the free base of each compound. Based on previous in vivo pharmacological studies, compound A was found to exhibit an antidepressant-like effect in the tail suspension test in mice at a dose of 32 mg / kg (po). Furthermore, 100 mg of immediate-release piroxazine (racemate) administered (for its antidepressant effect) resulted in plasma levels similar to 400 mg of sustained-release piroxazine, which, using allometric scaling, was equivalent to 34 mg / kg VLX or approximately 21 mg / kg S-VLX in rats.

[0328] For confirmation, when administered at 40 mg / kg, racemic VLX showed approximately half the C of S-VLX when S-VLX was administered alone to rats at 40 mg / kg (po). max This resulted in a higher level of S-VLX. Conversely, compound A required a higher dose to obtain a similar plasma concentration of S-VLX compared to when S-VLX was administered alone. Therefore, based on these data, the doses of compound A orally delivered to rats in this study were 30, 60, and 120 mg / kg, and the doses of S-VLX in rats were the molar equivalents of compound A at 15, 30, and 60 mg / kg.

[0329] According to PK data, the t max The values ​​were 0.33 and 0.42, respectively. However, the higher dose of compound A (120 mg / kg) was 0.67. max This was demonstrated. Therefore, since the EPM task can be completed in a short time (5 minutes), the two lower doses of compound A (30 and 60 mg / kg) and all doses of S-VLX were administered orally 20 minutes before the EPM task. The higher dose of compound A (120 mg / kg) was administered 40 minutes before the task.

[0330] For the positive control / reference tranquilizer midazolam, the dosage and route of administration (0.5 mg / kg ip 30 minutes prior to the study) were based on previously published literature (Gazarini, et al., Neuroscience. 2011;179:179-187).

[0331] result Data were imported into SigmaPlot® 11.0 for statistical analysis. Independent t-tests and one-factor analysis of variance (ANOVA) were used (where appropriate), followed by Dunnett's multiple comparison post-hoc test. All results were expressed as mean (±SEM). Differences in the mean from the experimental group were considered significant at the p<0.05 level.

[0332] Study of compound A. The effects of compound A in EPM are shown in Figure 69 and Tables 12 and 13. Of all the outcome measures analyzed, the only statistically significant difference between compound A and the vehicle was in total distance traveled [F(3,28)=6.28, p=0.002]. Post-hoc analysis showed that rats administered with 60 and 120 mg / kg doses of compound A traveled significantly shorter distances than animals treated with the vehicle (p<0.001 and p<0.05 for 60 and 120 mg / kg doses, respectively).

[0333] The effects of midazolam in Study 1 are shown in the inset of Figure 69 and in Tables 12 and 13. Compared to the vehicle, midazolam showed significant (p<0.05) tranquilizing activity, as indicated by the following outcomes: increased number of open arm entries, t=2.9, df=14, p=0.01; open arm stay time, t=2.6, df=14, p=0.02; open arm stay rate (%), t=2.6, df=14, p=0.02; peering into the open arm, t=2.3, df=14, p=0.04; and decreased closed arm stay time, t=3.5, df=14, p=0.003.

[0334] Studies on S-biloxazine. The effects of S-biloxazine (S-VLX) in EPM are shown in Figure 70 and Tables 12 and 13. As with compound A, the only statistically significant difference between S-VLX and the vehicle among all the outcome measures analyzed was total distance traveled [F(3,28)=4.22, p=0.01]. Post-hoc analysis showed that rats administered S-VLX at doses of 15, 30, and 60 mg / kg traveled significantly shorter distances than animals treated with the vehicle (p<0.05 for all three doses compared to the vehicle).

[0335] The effects of midazolam in Study 2 are shown in the inset of Figure 70 and in Tables 12 and 13. Compared to the vehicle, midazolam showed significant (p<0.05) tranquilizing activity, as indicated by the following outcomes: increased number of open arm entries, t=2.6, df=15, p=0.02; open arm stay time, t=2.2, df=15, p=0.047; open arm stay rate (%), t=2.2, df=15, p=0.047; peering into the open arm, t=2.3, df=15, p=0.04; and decreased closed arm stay time, t=2.7, df=15, p=0.02. Interestingly, in Study 2, midazolam was also associated with an increase in the number of closed arm entries (t=2.9, df=15, p=0.01); a decrease in closed arm reversals (t=3.7, df=15, p=0.002); an increase in the total number of arm entries (t=3.7, df=15, p=0.002); and an increase in the total distance traveled (t=3.7, df=15, p=0.002). [Table 33] [Table 34]

[0336] In summary, none of the doses of compound A were associated with tranquilizing activity. The two higher doses of compound A were associated with a decrease in total travel distance in the EPM. None of the doses of S-piroxazine were associated with tranquilizing activity. All three doses of S-piroxazine were associated with a moderate decrease in total travel distance in the EPM. In both studies, the positive control / reference compound midazolam demonstrated tranquilizing activity, as indicated by a significantly increased number of open arm entries, open arm dwell time (and percentage of dwell time), peering into the open arm, and decreased closed arm dwell time. The validity of the EPM task was demonstrated by the tranquilizing effect of midazolam, a benzodiazepine.

[0337] Example 22. Effect of compound A on novel object recognition in rats. A study was conducted to evaluate compound A in a spontaneous novel object recognition (NOR) task in rats. The study included a 48-hour retention interval version of the task and a 3-hour retention interval scopolamine impairment model. NOR is a rodent model of (non-spatial) cognitive memory commonly used for the preclinical evaluation of novel compounds that may have cognitive-enhancing effects. This test is based on the innate tendency of rodents to explore novel objects rather than known ones. During the dose-effect evaluation of compound A, a single dose of donepezil, an acetylcholinesterase inhibitor and Alzheimer's disease treatment, was evaluated as a positive control / reference compound in the 48-hour retention interval version of the task. In the scopolamine impairment version of the task, a single dose of vortioxetine, an antidepressant, was evaluated as a positive control / reference compound. The primary measurement parameters in this study were time spent on novel and known objects, and the discrimination (d2) ratio in A / B sessions.

[0338] Methods and materials Study subjects: 143 adult (approximately 3 months old) male Wistar rats (Envigo, Inc., Indianapolis, IN) were used in the study. Two rats were housed in each polycarbonate cage (45 x 30 x 18 cm) filled with corn cob bedding in a room with a constant temperature (21-23°C) and humidity (40-50%). Lighting was maintained on a 12-hour light-dark cycle (7:00 AM to 7:00 PM), and the rats were given free access to water and food throughout the study period. All behavioral tests were conducted during the light phase of the light / dark cycle (9:00 AM to 5:00 PM) (Monday to Friday).

[0339] Methods. The NOR task was adapted from Ennaceur and Delacour (1988), as previously reported by us (Callahan et al, Neuropharmacology 67:201-212, Callahan et al, Psychopharmacology 231,3695-3706, Callahan et al, Neuropharmacology 117:422-433). Briefly, the test subjects were acclimatized to laboratory conditions for at least three days prior to the experiment (i.e., tail marking, daily handling, and weight measurement). During the experiment, the animals were moved to the laboratory and acclimatized for 30 minutes before starting the experimental phase. After the completion of the study, the animals were kept in the laboratory for 15 minutes.

[0340] Habituation – Animals were acclimatized, weighed, and individually placed in a dimly lit (10 lux) training / test environment (opaque plastic chamber with bedding on the floor, 78.7 cm × 39.4 cm × 31.7 cm) and allowed to explore the chamber for 10 minutes. The NOR chamber was placed on a table positioned along the short-side wall of the laboratory. External background noise was reduced by masking noise from HVAC ventilation, and there were no room orientation cues or wall-mounted visual cues (except for a small camera positioned above the NOR chamber). At the start of each series of NOR experiments, fresh bedding material was placed in the chamber before acclimatization to saturate it with animal scent. Animal feces were removed between experimental sessions. However, the same bedding material was used for the remainder of each study (i.e., during training and testing) to ensure there were no specific olfactory cues during the experimental period.

[0341] Training Trials – 24 hours after the acclimatization session, animals were acclimatized, weighed, injected with the test compound (drug or vehicle), and after an appropriate pre-treatment period, placed in a chamber with their noses facing the center of the long side wall and allowed to search for two identical objects for 10 minutes. The animals' behavior was observed and digitally recorded with a CCTV camera positioned 69 cm above the chamber. The principal investigator sat quietly 10–15 feet away from the NOR chamber. To further the study of the experimental subjects and conduct test trials, it was required that each individual object was searched for for at least 4 seconds during the training session, with a total object search time of at least 12 seconds.

[0342] In the trial-standard version of the NOR task, a delay interval (e.g., 48 hours after the training session) was used that reliably resulted in forgetting the known object. In the scopolamine impairment study, a delay interval (3 hours) was used that reliably allowed for the recall of the known object under vehicle conditions. In the NOR task, two objects (the same object as in training (known) and a new object) were placed in a chamber, and animals were asked to search for these objects for 5 minutes. The experimental objects to be discriminated were a ceramic, green, conical Christmas tree salt / pepper shaker (12 cm high, 5 cm in diameter) paired with a tower (12 cm high, 6 cm wide) made of multicolored plastic Duplo-Lego blocks. Duplicates of all objects were prepared. These objects were placed 19.3 cm from the two short walls of the chamber and 19.3 cm from the long wall, with a distance of approximately 40 cm between the two objects. The roles of known and new objects, as well as their positions within the chamber, were randomly assigned between subjects and between treatments. Objects were cleaned with a 50% diluted (volume / volume) ethanol solution between sessions to eliminate olfactory cues. The criteria for observers to classify interaction with an object as exploratory behavior were direct interaction through the nostrils or the head being at a maximum distance of 2 cm from the object. Physically climbing, standing, and digging around an object were not scored as object exploration. The primary behavioral index was the time (in seconds) spent exploring each object. A discrimination index (d²) was calculated for each test trial. This was defined as the difference in time spent exploring new and known objects divided by the total time spent exploring both objects: d² index = (new - known) / (new + known). This index is considered an index of cognitive memory and takes into account individual differences in the total amount of exploration time. To include data, rats were required to search each object for at least 4 seconds, with a total object search time of at least 12 seconds. Each experimental group consisted of at least 8 rats per treatment (or test) condition. This constituted a sufficient sample size to observe statistical significance. Animals were tested only once, and object search time was scored both in real time and via video recording under a blinded test method (i.e., the principal investigator was unaware of the treatment assignment).

[0343] Drug administration. Compound A was administered by forced oral administration at a volume of 2.0 mL / kg (see below for consistency with vortioxetine). The dose was calculated based on the free base of the compound. Based on previous in vivo pharmacological studies, compound A was found to exhibit cognitive enhancement phenotypic signatures in mice at 90 and 120 mg / kg(po), and antidepressant-like effects in the tail suspension test at 32 mg / kg(po). Furthermore, in humans, a daily dose of 400 mg of racemic biloxazine was observed to improve mental function. Using allometric scaling of this therapeutic dose to obtain the human equivalent, this could result in 34 mg / kg of racemic biloxazine. In rats, 40 mg / kg(po) of racemic biloxazine was equivalent to 1600 ng / mL of S-VLX C max This results in higher doses of compound A to obtain similar plasma concentrations of S-VLX in rats. Furthermore, compared to rats orally treated with compound A at 60 mg / kg, mice showed higher C at 20 mg / kg. max This was demonstrated. Therefore, based on these data, the doses of compound A orally administered to rats in the 48-hour delayed version of the NOR in this study were 15, 30, 60, and 90 mg / kg. The effects of these doses were evaluated, and then three doses were selected for additional testing in a scopolamine injury model (see below).

[0344] According to PK data, the tmax of compound A when administered at 60 mg / kg is 0.33 h. However, for higher doses of compound A (120 mg / kg), the tmax is 0.67 h. max This was demonstrated. Therefore, since the NOR training session was short (10 minutes to complete), the lower dose of compound A (15, 30, and 60 mg / kg) was administered orally 20 minutes before the NOR task training session. The higher dose of compound A (90 mg / kg) was administered 40 minutes before the task training session.

[0345] In the 48-hour delayed version of the NOR, as previously reported by us, the positive control compound donepezil was dissolved in physiological saline (0.9% NaCl) and administered by intraperitoneal (ip) injection at a rate of 1.0 ml / kg 30 minutes before the A / A session. (Terry et al., The Journal of Pharmacology and Experimental Therapeutics, 352(2), 405-418)

[0346] Preparation of Compound A. Compound A was weighed and prepared in an appropriate volume of vehicle (0.8% DMSO, 6% PEG400, 93.2% HPβCD (6% in H2O)). The vehicle composition of Compound A, 0.8% DMSO, 6% PEG400, 93.2% HPβCD (6% in H2O), was prepared by diluting a 5x concentrated (5X) stock solution of 4% DMSO + 30% PEG400 + 66% HβCD (30%) with water (1x volume of stock solution + 4x volume of H2O). This stock solution was dissolved in 0.8% DMSO and 6% PEG400, and then diluted with water containing 6% HPβCD to the desired final concentration. Dosage was carried out within 1 hour of preparation / remixing on each dosing day. The appearance of the formulation, including appearance (solution vs. suspension), color, precipitate, etc., was recorded. Compound A was protected from light and kept on ice until administration. The vehicle for compound A was 0.8% DMSO, 6% PEG400, and 93.2% HPβCD (6% in H2O).

[0347] Scopolamine: Dosage and formulation. For the scopolamine reversal study, (-)-scopolamine hydrobromide (CAS number 6533-68-2) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Scopolamine was dissolved in physiological saline and administered by intraperitoneal (ip) injection at a dose of 0.2 mg / kg 30 minutes before the training session. This scopolamine administration method is based on our recently published study (Callahan et al., Scientific Reports 11(1):9843).

[0348] Vortioxetine has shown cognitive enhancement capabilities in healthy rodents, rodents subjected to pharmacological stress, and animal models of depression, and these were used as positive controls in scopolamine reversal studies. The dose of vortioxetine (10 mg / kg, sc) was based on previously reported studies showing sustained memory after a 24-hour delay and previously reported studies in scopolamine stress models (Mork et al., Pharmacology, Biochemistry and Behavior, 105, 41-50; Pehrson et al., J Pharmacol Exp Ther, 358, 472-482). Vortioxetine was dissolved in a 20% hydroxypropyl-β-cyclodextrin aqueous solution (w / v) and administered at a dose of 2.0 mL / kg one hour before the training session of NOR.

[0349] result Data were imported into SigmaPlot® 11.0 or GraphPad Prism 9 for statistical analysis. Independent t-tests and one-factor or two-factor analysis of variance (ANOVA) were used (where appropriate), followed by Dunnett or Student-Newman-Coils post-hoc tests, respectively. All results were expressed as mean ± mean standard error (SEM). Mean differences from the experimental group were considered significant at a p<0.05 level.

[0350] 48-hour delayed study. Figure 71 shows the effect of the AD treatment drug donepezil (2.0 mg / kg) on ​​the NOR task (A / B retention session) after a 48-hour retention interval. As shown, donepezil was associated with increased preference for novel objects: main effect of treatment [F(1,21)=8.60, p=0.008]; object type [F(1,21)=1.68, p=0.209]; interaction between treatment and object type [F(1,21)=4.61, p=0.044]. Post-hoc analysis showed that donepezil was associated with a significant preference for novel objects (p=0.017 compared to known). This effect of donepezil was also observed when the d2 ratio was analyzed [t(21)=2.466 p=0.02].

[0351] The effects of compound A in the NOR task (A / B retention session) after a 48-hour retention interval are shown in Figure 72. As shown, compound A was associated with a dose-dependent increase in preference for the novel object: main effect of dose [F(4,54)=1.18, p=0.329]; object type [F(1,54)=25.06, p<0.001]; and interaction between dose and object type [F(4,54)=2.96, p=0.028]. Post-hoc analysis showed that compound A 15 mg / kg was associated with a non-significant tendency to prefer the novel object over the known object compared to the vehicle control (p=0.054). Doses of 30, 60, and 90 mg / kg were associated with a significant (p<0.05) preference for the novel object compared to the known object. Analysis of the d2 ratio yielded the following statistical result: dose effect [F(4,54)=2.91, p=0.030]. Post-hoc analysis showed that doses of 60 and 90 mg / kg were associated with a significant difference (p<0.05) compared to the vehicle control response.

[0352] Scopolamine reversal study. Figure 73 shows the effect of the antidepressant vortioxetine (10.0 mg / kg) on ​​the scopolamine reversal task (A / B retention session) after a 3-hour holding interval. In the analysis of exploration time, neither the effect of the treatment nor the interaction of treatment × object type was significant (p>0.05). However, the effect of object type was significant [F(1,26)=12.43, p=0.002]. Post-hoc analysis showed that preference for novel objects was significant in the vehicle-treated group (p=0.002), but not in the group administered the vehicle-scopolamine or vortioxetine-scopolamine combination. Analyzing the d2 ratio yielded the following statistical result: effect of the treatment [F(2,26)=3.26, p=0.055]. Post-hoc analysis revealed a significant difference (p=0.04) in the response of vehicle-scopolamine treated animals compared to vehicle controls, but not with the vortioxetine-scopolamine combination. It is also important to note that 3 out of 12 rats administered the vortioxetine-scopolamine combination exhibited adverse reactions (tactile lability, vocalization, labored breathing) during the A / A session, and therefore were not further evaluated.

[0353] Figure 74 shows the effect of compound A in the scopolamine reversal task (A / B retention session) after a 3-hour retention interval. Analysis of exploration time yielded the following statistical results: main effect of treatment [F(4,47)=0.60, p=0.67]; object type [F(1,47)=55.48, p<0.001]; interaction between treatment and object type [F(4,47)=2.66, p=0.044]. Post-hoc analysis showed that subjects administered with vehicle and compound A + scopolamine at doses of 30, 60, and 90 mg / kg were associated with a significant (p<0.05) preference for the novel object compared to known objects. In contrast, subjects administered with the vehicle-scopolamine combination did not show a significant preference for the novel object. Analysis of the d2 ratio yielded the following statistical results: dose effect [F(4,47)=2.75, p=0.039]. Post-hoc analysis showed that subjects administered the vehicle-scopolamine combination were significantly impaired compared to vehicle controls (p<0.05), and the highest dose of compound A (90 mg / kg) significantly attenuated the effect of this scopolamine.

[0354] In summary, the positive control / reference compound donepezil, evaluated using the 48-hour delayed version of the NOR, was associated with significant memory improvement in both object search analysis and d2 ratio analysis in the A / B sessions. These results demonstrate the validity of the NOR task for detecting cognitive enhancement effects in rats. Compound A was associated with a statistically significant and dose-dependent improvement in NOR performance in the 48-hour delayed version of the NOR. In the object search analysis in the A / B sessions, the three higher doses of compound A were statistically different from the vehicle control, while in the d2 ratio analysis, the two highest doses (60 and 90 mg / kg) were statistically different from the vehicle control. In the scopolamine inverted version of the NOR, the reference compound vortioxetine was not associated with significant memory improvement (i.e., attenuation of scopolamine impairment). Three of the twelve rats administered the vortioxetine-scopolamine combination showed adverse reactions during the A / A session and were not further evaluated. Compound A was associated with a statistically significant improvement in NOR performance in a scopolamine-reversed version of the NOR task. In object exploration analysis in A / B sessions, all three doses of compound A evaluated (30, 60, and 90 mg / kg) were associated with statistically different responses (i.e., preference for novel objects) compared to the vehicle-scopolamine treatment response. However, in d2 ratio analysis, the highest dose of compound A (90 mg / kg) was associated with a significant attenuation of the scopolamine-impairing effect. These studies, conducted in two versions of the NOR task, support the potential for further development of compound A as a memory-enhancing compound.

[0355] Example 23. Evaluation of the effect of compound A on EEG parameters during sleep / wake and activity periods in an orexin / Tta;Tet-O / diphtheria toxin A mouse model of narcolepsy. A study was conducted to investigate the dose-related effects of test compound A in a novel inducible mouse model of narcolepsy. Telemetry electroencephalography (EEG) was used to determine whether compound A had a therapeutic effect on symptoms after induction of the narcolepsy phenotype. EEG patterns, electromyography (EMG), core body temperature (Tb), and total walking activity (LMA) were collected and analyzed.

[0356] Methods and materials The animals were housed in a temperature-controlled recording chamber under a 12 / 12 light / dark cycle, with free access to food and water. Room temperature (24±2℃), humidity (relative humidity 50±20%), and lighting conditions were monitored and recorded daily.

[0357] Breeding of Orexin / tTA;Tet-O Diphtheria Toxin A ("DTA") Mice. This study used a conditional model of hypocretin neuron ablation (orexin / tTA;Tet-O diphtheria toxin A, i.e., "DTA mice"). In this narcolepsy model, degeneration of hypocretin / orexin neurons occurs when the neurotoxic diphtheria toxin subunit A (DTA) protein is synthesized in these neurons. DTA transgene expression is regulated via the tetracycline trans-activator (Tet-off) system. When doxycycline (Dox) is present in the diet, it binds to tetracycline trans-activator (tTA), thereby preventing tTA from binding to the Tet-O regulatory site upstream of the preprohypocretin / DTA transgene. Removal of Dox from the diet allows tTA to bind to Tet-O, ​​thereby initiating transgene transcription. Since the Tet-O binding site is present only in hypocretin / orexin (Hcrt) neurons, dietary elimination of Dox (Dox(-)) leads to the accumulation of neurotoxic DTA proteins in these cells, causing degeneration of Hcrt neurons. Six weeks after Dox(-) status, over 97% of Hcrt cells are degenerated, and the major features of narcolepsy, including arousal fragmentation and cataplexy, are readily apparent.

[0358] The male DTA mice used in this study were bred using SRI and their genotype was confirmed by identification. The mice were fed a Dox+ diet until approximately 14 weeks of age, after which a 6-week degeneration period was initiated by removing Dox from the diet. Therefore, the mice were approximately 20 weeks old at the start of the experimental period.

[0359] Surgical procedure. In this study, eight male DTA mice were implanted with long-term recording devices for continuous recording of EEG, EMG, Tb, and LMA via telemetry. Under isoflurane anesthesia (1-4%), hair was shaved from the vertebral and mid-vertebral regions. After disinfecting the skin with chlorhexidine and sterile water, a dorsal midline incision of approximately 2.5 cm was made on the vertebral region. A subcutaneous pocket was bluntly incised along the left dorsal flank and then irrigated with 1.5-3.0 ml of sterile saline. A sterile miniature transmitter (HD-X02, Data Sciences Inc., St. Paul, MN) was then inserted through the incision made in the subcutaneous pocket. The temporalis muscle was then contracted to cauterize the skull, and it was thoroughly cleaned with 3% hydrogen peroxide solution. Holes were made in the skull along the midline at coordinates of -2.0 mm AP and 2.0 mm ML from the bregma, and -1 mm AP from the lambda. Two biopotential leads used as EEG electrodes were inserted into holes and attached to the skull with dental acrylic. Two biopotential leads used as EMG electrodes were sutured to the muscle tissue of the neck. The incisions were closed with absorbable sutures.

[0360] Animals were administered anti-inflammatory drugs (NSAIDs, e.g., meloxicam), analgesics (opioids, e.g., buprenorphine), and saline during postoperative recovery from anesthesia. Animals were closely monitored during recovery until they were able to walk. Thereafter, they were carefully observed daily (approximately 5 minutes / day) until the incision healed and sutures were removed (1-2 weeks post-surgery). NSAIDs were then administered once daily for 72 hours, and opioids once daily for 24 hours post-surgery, or as needed for signs of pain. Signs of pain included decreased activity, decreased food / water intake, weight loss, kyphosis, abnormal respiratory rate or characteristics, chattering / teeth grinding, ruffled hair, changes in facial features (e.g., position / condition of ears, eyes, whiskers), absence of grooming, or excessive grooming.

[0361] Experimental Design: Using a repeated measures counterbalanced design, compound A (10, 30, 90, and 120 mg / kg, po) and amphetamine (Amph; 2 mg / kg, ip) were administered at 10 ml / kg to DTA mice. Their effects on cataplexy, sleep / wake parameters, Tb, and LMA were tested compared to a vehicle control (Veh; 4% DMSO, 30% PEG400, 66% HPβCD [30% in H2O]). Injections were administered immediately before the start of the dark period (before the start of Zeitgeber time [ZT] 12). Amph was dissolved in saline, not the vehicle control. EEG, EMG, Tb, and LMA were recorded telemetry along with video recording using Ponemah 6.41 software (Data Sciences Inc., St. Paul, MN). A minimum of 3 days was allowed between treatments, and 6 doses were administered per animal over a 3-week period. The animals were acclimatized to the handling process and given 0.2 ml of water multiple times (po) during the week prior to the first experimental day.

[0362] result Compound A: Following administration of Compound A, strong dose-related effects were observed for most of the parameters tested here. REM latency increased at all concentrations of Compound A (Figure 82). Total W time decreased after administration of the two highest doses of Compound A (90 and 120 mg / kg), and total NREM time increased after administration of the three highest doses of Compound A (30, 90, and 120 mg / kg) (Figure 83). Total REM time decreased at all concentrations of Compound A, and total C time decreased after administration of Compound A at 30, 90, and 120 mg / kg. The REM:NR ratio decreased under all test conditions, reflecting the suppression of REM sleep (Figure 83).

[0363] W decreased in ZT12-14 and overall after administration of compound A at 120 mg / kg (treatment effect), and also decreased in ZT12-13 and overall after administration of compound A at 90 mg / kg (Figure 84). W also decreased in ZT12 after administration of compound A at 30 mg / kg. NREM increased overall after administration of compound A at 30, 60, and 120 mg / kg. Between ZT12 and ZT13, compound A at 30 and 90 mg / kg increased NREM, and at 10 mg / kg, NREM increased in ZT13. The highest dose of 120 mg / kg increased NREM between ZT12 and ZT16. REM decreased overall after administration of compound A at all concentrations, and C decreased after administration of compound A at all concentrations except 10 mg / kg. C also decreased between ZT13 and ZT15-ZT17 after administration of compound A at 90 and 120 mg / kg, between ZT13 and ZT15-16 after administration of compound A at 30 mg / kg, and during ZT13 after administration of compound A at 10 mg / kg.

[0364] After administration of compound A at 30, 90, and 120 mg / kg, cumulative NREM increased overall, while cumulative W, REM, and C decreased overall (Figure 85). Cumulative REM also decreased after administration of compound A at 10 mg / kg. After administration of compound A at 90 and 120 mg / kg, cumulative W and REM decreased and cumulative NREM increased at all times during the recording period. After administration of compound A at 30 mg / kg, cumulative W decreased between ZT12 and ZT15, cumulative NREM increased between ZT12 and ZT17, and cumulative REM decreased between ZT13 and ZT17. After administration of the lowest concentration of compound A, cumulative NREM increased between ZT13 and ZT14, and cumulative REM decreased between ZT12 and ZT17. Cumulative C decreased between ZT13 and ZT17 after administration of compound A at 30, 90, and 120 mg / kg, and between ZT13 and ZT14 after administration of compound A at 10 mg / kg.

[0365] Changes in sleep-wake cycles after compound A administration were primarily due to changes in the number of sleep-wake bouts, rather than changes in bout duration (Figures 86-87). W bout duration decreased between ZT13-ZT14 after 120 mg / kg compound A administration and between ZT12 after 90 mg / kg compound A administration, but increased between ZT14 after 30 mg / kg compound A administration (Figure 86). The number of W bouts decreased overall after 90 mg / kg compound A administration, while the number of NREM bouts increased overall after 30, 90, and 120 mg / kg compound A administration (Figure 87). The number of REM bouts decreased overall after all concentrations of compound A administration, and the number of C bouts decreased overall after 30, 90, and 120 mg / kg compound A administration. The number of W bouts decreased between ZT15 and ZT17 after administration of compound A at 90 mg / kg, and between ZT14 and ZT15 after administration of compound A at 30 mg / kg. The number of NR bouts increased between ZT12, ZT14 and ZT16 after administration of compound A at 90 and 120 mg / kg, and between ZT12, ZT13 and ZT17 after administration of compound A at 30 mg / kg. The number of REM bouts decreased between ZT12 and ZT16 after administration of 90 and 120 mg / kg at 3466 doses, between ZT12 and ZT14 after administration of 30 mg / kg at 3466 doses, and between ZT12 and ZT13 after administration of compound A at 10 mg / kg. The number of C-bouts decreased between ZT12 and ZT16 after administration of compound A at 30, 90, and 120 mg / kg, and also between ZT12 after administration of compound A at 10 mg / kg.

[0366] The EEG spectrum also changed significantly after administration of compound A (Figures 88-95). During W, a significant overall decrease in EEG power was observed in the delta, alpha, beta, and high-gamma frequency bands after administration of 120 mg / kg of compound A (Figure 89). After administration of 120 mg / kg of compound A, W's delta decreased between ZT14-ZT17, W's alpha decreased between ZT12-ZT14 and ZT16, and W's high-gamma decreased between ZT12-ZT13 and ZT16. During NREM sleep, a substantial decrease in EEG power was observed across the entire spectrum (Figures 90-91). The delta power of NREM decreased overall after administration of compound A at 120 mg / kg, the theta and low-gamma power of NREM decreased overall after administration of compound A at 30, 90, and 120 mg / kg, and the alpha, beta, and high-gamma power of NREM decreased overall after administration of compound A at all concentrations. Furthermore, power decreased significantly in all power bands after compound A administration at many hourly time points, including all time in all power bands after administration of compound A at 120 mg / kg (Figure 91). For REM and C, statistical analysis of their power spectra could not be performed because the time spent in these states after compound A administration was too short (Figures 92-95).

[0367] LMA decreased overall after administration of compound A at 90 and 120 mg / kg, and Tb decreased after administration of 3466 at 120 mg / kg (Figure 96).

[0368] Amphetamine: The expected results were obtained with amphetamine, namely, increased latency to the onset of both NREM and REM sleep (Figure 82), increased W, decreased NREM (Figure 83), and an overall increase in LMA (Figure 96). C levels were generally unaffected by Amph (Figures 83-85). Although not designed for quantitative comparison with compound A, these observations collectively demonstrate the validity of the bioassay.

[0369] In summary, after administration of compound A, both REM and cataplexy were very strongly suppressed, while NREM significantly increased and W decreased in a dose-dependent manner. After administration of compound A at the two highest concentrations, 90 and 120 mg / kg, REM and C virtually disappeared during the 6-hour recording, thus reducing the REM:NR ratio to virtually zero. Changes in the time spent in each state were mainly due to changes in the number of bouts in each state. A very significant decrease in EEG power during NREM sleep was observed across the power spectrum, indicating brain penetration by compound A and the involvement of underlying molecular targets in the brain networks contributing to EEG activity. Even at the lowest concentration of compound A tested, a significant decrease in NREM EEG power was observed. Since other indicators were only slightly affected by the 10 mg / kg concentration, both REM sleep duration and NREM EEG power may be particularly sensitive to the effects of compound A on the brain networks contributing to ongoing EEG activity. It should be noted that the 30 mg / kg dose did not have a significant effect on the total amount of W over the entire 6-hour period analyzed, and the effect on NREM sleep was transient and significant only during the first hour after administration.

[0370] At its highest dose, compound A reduced high-gamma range spectral activity in W during the first 1–2 hours after administration. This EEG band is often associated with cognition in joint electrophysiological and behavioral tasks. While it is unclear whether it affects cognition assessed in other environments, the overwhelming effect of compound A, at least when measured in known home cage environments, is to increase NR sleep and suppress EEG spectral power, particularly at a concentration of 120 mg / kg.

[0371] The strong suppression of C after administration of compound A supports the treatment of narcolepsy. The strong promotion of NREM suggests that this compound is preferably administered during the inactive or sleep phase.

[0372] Example 24. Additional analysis of the in vivo effects of compound A, a biloxazine derivative, in orexin-DTA transgenic mice. A study was conducted to evaluate the in vivo effects of compound A, a biloxazine derivative, in orexin-DTA transgenic mice. Six administration conditions were used: one dose of positive control d-amphetamine sulfate (2 mg / kg), four doses of the test compound, compound A (10, 30, 90, and 120 mg / kg), and one dose of vehicle control. Telemetry using a DSI data acquisition system collected EEG, electromyography (EMG), body temperature (Tb, sc), and walking activity (LMA) (N=8 mice). Dose administration was performed immediately before light offset, the primary activity period for nocturnal rodents such as mice. Only the first six hours immediately following dose administration were scored and analyzed first for reporting. The results indicated that further analysis was needed to elucidate the time course of cataplexy suppression. At the end of the first 6 hours after administration of d-amphetamine and compound A at a concentration of 10 mg / kg, cataplexy was at baseline levels, and therefore no further analysis of these conditions was performed. For the second 6-hour period, further analysis was performed under four conditions: compound A at 30, 90, and 120 mg / kg, as well as a vehicle control.

[0373] Sleep latency, hourly and cumulative sleep / wake cycles, and a combined sleep / wake / cataplexy index (duration of bouts and number of bouts per hour) were evaluated over the last 6 hours of the dark period (Zeitgeber time [ZT]19-ZT24). EEG and EMG recordings were scored in 10-second epochs for wakefulness (W), rapid eye movement sleep (REM), non-rapid eye movement sleep (NREM), and cataplexy (C).

[0374] The latency to REM increased after administration of compound A at 90 and 120 mg / kg (Figure 75). After administration of compound A at 30 mg / kg, W increased overall (treatment effect) and NREM decreased overall with respect to hourly, cumulative, and total time effects (Figures 76-78). After administration of compound A at 120 mg / kg, NREM increased overall with respect to hourly, cumulative, and total time effects. C remained significantly decreased after administration of compound A at the two highest concentrations, and decreased overall with respect to both hourly and cumulative data as well as total time. Cumulative REM decreased overall after administration of compound A at 90 and 120 mg / kg. The REM:NR ratio decreased after administration of compound A at 90 and 120 mg / kg (Figure 78).

[0375] The duration of REM bouts decreased overall after administration of compound A at 120 mg / kg (Figure 79). The number of W bouts decreased overall after administration of compound A at 90 mg / kg (Figure 80). After administration of compound A at 90 and 120 mg / kg, the number of C bouts decreased, but the number of NREM bouts increased.

[0376] No significant effect on subcutaneous body temperature or LMA was observed (Figure 81).

[0377] In summary, while many of the effects on sleep / wake parameters described in the initial report decreased or were absent in the latter half of the dark period, C remained reduced at the two highest concentrations of compound A during ZT19–24. C was at vehicle levels after administration of 30 mg / kg of compound A. However, the increase in W and decrease in NREM after administration of 30 mg / kg of compound A suggested a rebound in wakefulness during this period. Interestingly, in the last hour of the recording period analyzed here (ZT24), most parameters for all compound A conditions were equivalent to vehicle values. The exception was C after administration of the highest concentration of compound A. C occurred during ZT24 after administration of 120 mg / kg of compound A, but its level was approximately half of the vehicle level (Figure 76).

[0378] In vitro assay Example 25. In vitro pharmacology of each compound: Compound D, Compound E, Compound A, Compound B, and Compound C. Studies were conducted to test five compounds using functional assays of cells and nuclear receptors, as well as enzyme and uptake assays.

[0379] Methods and materials In vitro pharmacology: Functional assays of cells and nuclear receptors. [Table 35]

[0380] The results are expressed as a percentage of the control agonist response or inverse agonist response obtained in the presence of the test compound, i.e.

number

number

[0381] The EC50 value (concentration that elicits a half-dose response) and IC50 value (concentration that elicits half-dose inhibition of the control agonist response) were determined by nonlinear regression analysis of concentration-response curves generated using mean replicate values ​​with curve fitting according to Hill's equation.

number

[0382] For antagonists, the apparent dissociation constant (KB) is given by a modified Cheng-Prusoff formula.

number

[0383] In vitro pharmacology: Enzyme and uptake assays [Table 36]

[0384] The results are obtained in the presence of the test compound, as a percentage of the specific activity of the control, i.e.

number

number

[0385] The IC50 value (concentration that causes half-level inhibition of the control's specific activity), the EC50 value (concentration that causes half-level increase in the control's basal activity), and the Hill coefficient (nH) were determined by nonlinear regression analysis of inhibition / concentration-response curves generated from repeated trial mean values ​​using curve fitting according to Hill's equation.

number

[0386] result In vitro pharmacology. Results showing inhibition (or stimulation in assays performed under basal conditions) higher than 50% were considered to represent a significant effect of the test compound. Results showing inhibition (or stimulation) between 25% and 50% suggested a weak to moderate effect. Results showing inhibition (or stimulation) lower than 25% were not considered significant and were attributed mainly to signal variability centered around the control level.

[0387] Results showing inhibition or stimulation higher than 50% were considered to represent a significant effect of the test compound. Such effects were observed and are listed in Table 14. [Table 37]

[0388] Example 26. hNav1.5 in vitro functional assay of each compound: Compound D, Compound E, Compound A, Compound B, and Compound C. Using the QPatch HT electrophysiological platform, electrophysiological assays were performed to profile the activity of five compounds against ion channel targets.

[0389] Methods and materials CYL6004QP2DR Nav1.5 Human Sodium Ion Channel Cell-Based Automated Patch-Clamp Assay. Cells were held at -120mV for 100ms, stepped to -130mV for 100ms, and then stepped back to -120mV for 100ms, and leakage current was measured. Na channels exist in a) a quiescent or closed state at -120mV, and b) a transient open state, from which they become inactivated to c) an inactivated state at -10mV. Inhibition of Na current within 1-2ms of channel opening at -10mV is open channel inhibition (pulse 1). To completely inactivate the Na channels and promote inactivation-dependent drug binding, the channels were kept open (-10mV) for a longer period (pulsed for 500ms), then stepped back to -120mV for 20ms to allow them to recover from inactivation to a resting or closed state (however, drug-bound channels did not recover from inactivation and remained closed), and then stepped back to -10mV for 50ms (pulse 2) to measure the number of Na channels that could be opened. The higher inhibition observed in pulse 2 is due to inactivation-dependent inhibition. Pulses 1 and 2 were used to investigate drug binding to open and inactivated Na channels, respectively. Compounds of each concentration were applied for 5 minutes.

[0390] Data analysis and results Current amplitudes exceeding 200 pA were analyzed during the control phase. Current amplitude was calculated by measuring the difference between the peak inward current (i.e., the current peak) at the stepping point to -10 mV and the remaining current at the end of the step. Current was evaluated under vehicle control conditions and then at the end of each 5-minute compound application. Reference standards were performed as an essential part of each assay to ensure the validity of the results. The results are summarized in Tables 15-16. [Table 38] [Table 39]

[0391] Example 27. Study of Compound D and Compound E Studies were conducted to test compounds D and E in binding, enzyme, and uptake assays.

[0392] Compounds D and E were tested with 1.0E-05M.

[0393] The results are the percentage of specific binding of the control obtained in the presence of the test compound, i.e.

number

number

[0394] The IC50 value (the concentration that causes half-mass inhibition of specific binding in the control) and the Hill coefficient (nH) were determined by nonlinear regression analysis of the competition curve generated from the repeated trial mean values ​​using curve fitting according to Hill's equation.

number

[0395] The inhibition coefficient (Ki) is given by the Cheng-Prusoff formula.

number

[0396] In vitro pharmacology: Enzyme and uptake assays

[0397] The results are obtained in the presence of the test compound, as a percentage of the specific activity of the control, i.e.

number

number

[0398] The IC50 value (concentration that causes half-level inhibition of the control's specific activity), the EC50 value (concentration that causes half-level increase in the control's basal activity), and the Hill coefficient (nH) were determined by nonlinear regression analysis of inhibition / concentration-response curves generated from repeated trial mean values ​​using curve fitting according to Hill's equation.

number

[0399] result In vitro pharmacology. Results showing inhibition (or stimulation in assays performed under basal conditions) higher than 50% were considered to represent a significant effect of the test compound. Results showing inhibition (or stimulation) between 25% and 50% suggested a weak to moderate effect. Results showing inhibition (or stimulation) lower than 25% were not considered significant and were attributed mainly to signal variability centered around the control level.

[0400] Results showing inhibition or stimulation higher than 50% were considered to represent a significant effect of the test compound. Such effects were observed and are listed in Table 17. [Table 40]

[0401] Example 28a. Preclinical PK of Compound D and Compound E We conducted a study on the preclinical pharmacokinetic (PK) performance of compounds D and E. For details of this study, in which compounds D or E were orally administered to fasted mice, please refer to the following.

[0402] Research details. [Table 41]

[0403] result The results are shown in Figures 97-98. The relevant data are summarized in the table below (ND = Not determined (parameter could not be determined because the terminal elimination phase was not sufficiently defined). BQL = Less than the lower limit of quantification (LLOQ). If the adjusted rsq (linear regression coefficient of the concentration value in the terminal phase) is less than 0.9, T1 / 2 may not be accurately estimated. %AUC) Extra If >20%, AUC 0-inf , Cl, MRT 0-inf , and Vdss may not be accurately estimated. %AUMC Extra If >20%, MRT 0-inf and Vd ssThis may not be accurately estimated. a: Bioavailability (%) is AUC0-inf(%AUC0) at the nominal dose. Extra <20% or AUC0-last(%AUC Extra (Calculated using >20%) [Table 42] [Table 43]

[0404] Example 28b. Preclinical PK of compound D, compound B, and viroxazine. We conducted studies on the preclinical pharmacokinetic (PK) performance of compounds D and B. For details on the study of intraperitoneal injection of compounds B and D in fasted mice, please refer to the following.

[0405] Research details. [Table 44]

[0406] result The results are shown in Figures 99a-99f. Relevant data are summarized in the table below (ND = Terminal elimination phase was not well defined or could not be determined due to insufficient values). BQL = Below the lower limit of quantification (LLOQ). If the adjusted rsq (linear regression coefficient of the concentration value in the terminal phase) is less than 0.9, T1 / 2 may not be accurately estimated. %AUC Extra If >20%, AUC 0-inf , Cl, MRT 0-inf , and Vdss may not be accurately estimated. %AUMC Extra If >20%, MRT 0-inf and Vd ss This may not be accurately estimated. a: Bioavailability (%) is AUC0-inf(%AUC0) at the nominal dose. Extra <20% or AUC0-last(%AUC Extra (Calculated using >20%) [Table 45] [Table 46] [Table 47] [Table 48] [Table 49]

[0407] Example 29. Metabolic stability of the 1 μM test compound in the liver S9 fraction. Test compounds: Compound D, Compound E, and biloxazine. Positive controls: 7-ethoxycoumarin and 7-hydroxycoumarin.

[0408] [Table 50]

[0409] Data analysis:

number

[0410] result The results are shown in Figures 100A-100D, 101A-101D, and 102A-102D. Related data is summarized in the table below. [Table 51]

[0411] Example 30. Plasma stability. A study was conducted to investigate plasma stability. Test compounds: Compound D, Compound E, and biloxazine. Positive controls: Enalapril and propantheline.

[0412] [Table 52]

[0413] Data analysis:

number

[0414] The stability results for compound D in rat plasma are shown in Figure 103A. The stability results for compound E in rat plasma are shown in Figure 103B. The results for the positive control in rat plasma are shown in Figure 103C. The stability results for compound D in human plasma are shown in Figure 103D. The stability results for compound E in human plasma are shown in Figure 103E. The results for the positive control in human plasma are shown in Figure 103F. The relevant data are also summarized in the table below. [Table 53]

[0415] Example 31. Metabolic stability of the test compound in human intestinal homogenate. A study was conducted to investigate metabolic stability. Test compounds: Compound D, Compound E, and biloxazine. Positive controls: 7-hydroxycoumarin and testosterone.

[0416] [Table 54]

[0417] Data analysis:

number

[0418] The stability results for compound D, compound E, and the positive control are shown in Figures 104A-104D. Relevant data are also summarized in the table below. [Table 55]

[0419] Example 32. Bioanalysis data on compounds in rat plasma. A study was conducted to examine bioanalysis data on compounds in rat plasma. The results are summarized in the table below. Frozen Sprague Dawley rat (male mixed) and human (sex mixed) plasma were used in the study. The test compound was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution. A working solution was prepared using MeOH / water. Compound E (2 μM) was incubated in a double-batch water bath at 37°C with plasma. Sampling time points included 0, 5, 15, 30, and 60 minutes. Enalapril and propantheline were used as positive controls in rat and human plasma at 2 μM. [Table 56] [Table 57] [Table 58] [Table 59] [Table 60] [Table 61]

[0420] Example 33. Bioanalysis data on compounds in rat plasma. A study was conducted to examine bioanalysis data on compounds in rat plasma. The results are summarized in the table below. Frozen Sprague Dawley rat (male mixed) and human (sex mixed) plasma were used in the study. The test compound was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution. A working solution was prepared using MeOH / water. Compound E (2 μM) was incubated in a double-batch water bath at 37°C with plasma. Sampling time points included 0, 5, 15, 30, and 60 minutes. Enalapril and propantheline were used as positive controls in rat and human plasma at 2 μM. [Table 62] [Table 63] [Table 64] [Table 65] [Table 66] [Table 67]

[0421] While specific embodiments have been illustrated and described, it should be understood that modifications and alterations may be made by those skilled in the art without departing from the broader embodiments of the Art as defined in the claims.

[0422] The embodiments described herein as examples are suitably implementable without any elements(s) or limitations(s) not specifically disclosed herein. Therefore, terms such as “comprising,” “including,” and “containing” should be interpreted in an open and non-restrictive manner. Furthermore, the terms and expressions used herein are for illustrative purposes only, not limitation, and the use of such terms and expressions is not intended to exclude equivalents of the presented and described features or any part thereof, and it should be recognized that various modifications are possible within the scope of the claimed technology. Furthermore, the phrase “consisting essentially of” should be understood to include the elements specifically described, as well as additional elements that do not substantially affect the fundamental and novel features of the claimed technology. The phrase “consisting of” excludes any elements not specified.

[0423] This disclosure is not limited to the specific embodiments described herein. Many modifications and changes can be made without departing from the spirit and scope, as will be apparent to those skilled in the art. In addition to those enumerated herein, functionally equivalent methods and compositions within the scope of this disclosure will be apparent to those skilled in the art from the foregoing description. Such modifications and changes shall be included within the scope of the appended claims. This disclosure is limited only to the terminology of the appended claims and the entire scope of the equivalents of those claims to which rights are granted. It should be understood that this disclosure is not limited to specific methods, reagents, compounds, compositions, or biological systems, which can naturally vary. Furthermore, it should be understood that the terminology used herein is intended solely to describe specific embodiments and is not intended to limit them.

[0424] Furthermore, where any feature or aspect of this disclosure is described as a Markush group, a person skilled in the art will understand that this disclosure is also described as an individual component or a subgroup of a component of the Markush group.

[0425] As will be understood by those skilled in the art, for all purposes, and especially in terms of providing written explanations, all scopes disclosed herein include all possible sub-scopes and combinations of sub-scopes. Each described scope can be divided into at least two, three, four, five, ten, etc., and this is readily apparent. As a non-limiting example, each scope described herein can be readily divided into a lower third, a middle third, an upper third, etc. Again, as will be understood by those skilled in the art, all phrases such as “up to,” “at least,” “greater than,” and “less than” include the stated numbers and indicate scopes that can be divided later into the sub-scopes described above. Finally, as will be understood by those skilled in the art, a scope includes each individual component.

[0426] All publications, patent applications, granted patents, and other documents referenced herein are incorporated by reference in the same manner as each individual publication, patent application, granted patent, or other document is specifically and individually indicated to be incorporated by reference as a whole. Definitions contained in any text incorporated by reference are excluded to the extent that they conflict with the definitions in this disclosure.

[0427] In one embodiment, a process for preparing 2-((2-ethoxyphenoxy)methyl)morpholine-4-carbonyl chloride (intermediate 1) is provided, and this process is The reaction involves reacting 2-((2-ethoxyphenoxy)methyl)-morpholine with a reaction mixture containing triphosgene. [ka]

[0428] In further embodiments, the reaction mixture contains dichloromethane. In other embodiments, the reaction mixture contains sodium bicarbonate.

[0429] In some embodiments, intermediate 1 is used to prepare 2-chloropyridine-4-yl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate (SP-20), The reaction comprises reacting 2-((2-ethoxyphenoxy)methyl)morpholine-4-carbonyl chloride (intermediate 1) with a reaction mixture containing 2-chloro-4-hydroxypyridine. [ka]

[0430] In further embodiments, the reaction mixture further comprises anhydrous tetrahydrofuran.

[0431] In some embodiments, a process is provided for preparing chloromethyl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate (intermediate 2), which is a process for preparing chloromethyl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate (intermediate 2). The reaction involves reacting 2-((2-ethoxyphenoxy)methyl)-morpholine with a reaction mixture containing 1-chloromethyl chloroformate. [ka]

[0432] In further embodiments, the reaction mixture contains trimethylamine. In other embodiments, the reaction mixture contains dichloromethane.

[0433] In some embodiments, intermediate 2 is used to prepare ((D-valyl)oxy)methyl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate (SP-16), (a) Reacting intermediate 2 with a reaction mixture containing N-Boc-D-valine to form (SP-16A), [ka] (b) Reacting SP-16A with dioxane in an organic acid, etc. [ka]

[0434] In further embodiments, the reaction mixture contains cesium carbonate. In other embodiments, the reaction mixture contains methanol. In some embodiments, the organic acid is hydrochloric acid.

[0435] In some embodiments, intermediate 2 is used to prepare (((R)-3-amino-4-methylpentanoyl)oxy)methyl 2-((2-ethoxyphenoxy)-methyl)morpholine-4-carboxylate (SP-29), (a) Forming SP-29A by reacting intermediate 2 with a reaction mixture containing Boc-L-β-leucine, [ka] (b) Stirring SP-29A in chloroform and trifluoroacetic acid. [ka]

[0436] In further embodiments, the reaction mixture contains cesium carbonate. In other embodiments, the reaction mixture contains methanol.

[0437] In other embodiments, intermediate 2 is used to prepare bis(((2-((2-ethoxyphenoxy)methyl)morpholine-4-carbonyl)oxy)methyl)pyridine-3,5-dicarboxylate (SP-30), The process involves reacting intermediate 2 with a reaction mixture containing 3,5-pyridinedicarboxylic acid. [ka]

[0438] In further embodiments, the reaction mixture contains cesium carbonate. In other embodiments, the reaction mixture contains methanol.

[0439] In some embodiments, intermediate 2 is used to prepare ((2,2'-(methylazandiyl)bis(acetyl))bis(oxy))bis(methylene)bis(2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate)(SP-31), The reaction involves reacting chloromethyl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate (intermediate 2) with a reaction mixture containing methyliminodiacetic acid. [ka]

[0440] In further embodiments, the reaction mixture contains cesium carbonate. In other embodiments, the reaction mixture contains methanol.

[0441] In other embodiments, intermediate 2 is used to prepare (((R)-2-(aminomethyl)-3-methylbutanoyl)oxy)methyl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate, trifluoroacetate (SP-32), (a) Forming SP-32A by reacting intermediate 2 with a reaction mixture containing N-Boc-3-amino-2-isopropylpropionic acid, [ka] (b) Stirring SP-32A in chloroform and trifluoroacetic acid. [ka]

[0442] In further embodiments, the reaction mixture contains cesium carbonate. In other embodiments, the reaction mixture contains methanol.

[0443] In another embodiment, a process for preparing 1-chloroethyl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate (intermediate 3) is provided, which process is The reaction involves reacting 2-((2-ethoxyphenoxy)methyl)-morpholine with a reaction mixture containing 1-chloroethyl chloroformate. [ka]

[0444] In further embodiments, the reaction mixture contains trimethylamine. In other embodiments, the reaction mixture contains dichloromethane.

[0445] In some embodiments, intermediate 3 is used to prepare 1-((L-valyl)oxy)ethyl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate (SP-17), (a) Forming SP-17A by reacting intermediate 3 with a reaction mixture containing N-Boc-L-valine, [ka] (b) Reacting SP-17A with dioxane in an organic acid, etc. [ka]

[0446] In further embodiments, the reaction mixture contains methanol. In other embodiments, the reaction mixture contains cesium carbonate. In some further embodiments, the organic acid is hydrochloric acid.

[0447] In some embodiments, intermediate 3 is used to prepare 1-((l-phenylalanyl)oxy)ethyl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate (SP-22), (a) Forming SP-22A by reacting a reaction mixture containing N-Boc-phenylalanine with intermediate 3, [ka] (b) Reacting SP-22A with dioxane in an organic acid, etc. [ka]

[0448] In further embodiments, the reaction mixture further comprises cesium carbonate. In other embodiments, the reaction mixture further comprises methanol. In alternative embodiments, the organic acid is hydrochloric acid.

[0449] In some embodiments, intermediate 3 is used to prepare 1-((dimethyl-L-valyl)oxy)ethyl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate (SP-23), The reaction involves reacting intermediate 3 with a reaction mixture containing L-Val-N,N-dimethyl. [ka]

[0450] In further embodiments, the reaction mixture contains cesium carbonate. In some embodiments, the reaction mixture contains methanol.

[0451] In some embodiments, intermediate 3 is used to prepare 1-((acetyl-L-valyl)oxy)ethyl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate (SP-24), The reaction comprises reacting an intermediate 3 with a reaction mixture containing N-acetylvaline. [ka]

[0452] In further embodiments, the reaction mixture contains cesium carbonate. In some embodiments, the reaction mixture contains methanol.

[0453] In some embodiments, intermediate 3 is used to prepare 1-((methyl-D-valyl)oxy)ethyl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate (SP-25), (a) Forming SP-25A by reacting a reaction mixture containing N-Boc-D-valine with intermediate 3, [ka] (b) Reacting SP-25A with dichloromethane and trifluoroacetic acid, etc. [ka]

[0454] In further embodiments, the reaction mixture contains methanol. In some embodiments, the reaction mixture contains cesium carbonate.

[0455] In some embodiments, intermediate 3 is used to prepare 1-(((R)-2-(aminomethyl)-3-methylbutanoyl)oxy)ethyl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate (SP-27), (a) Forming SP-27A by reacting intermediate 3 with a reaction mixture containing N-Boc-3-amino-2-isopropionic acid, [ka] (b) Stirring a solution of SP-27A in chloroform and trifluoroacetic acid. [ka]

[0456] In further embodiments, the reaction mixture further comprises cesium carbonate. In other embodiments, the reaction mixture further comprises methanol.

[0457] In some embodiments, intermediate 3 is used to prepare 1-(((R)-2-(aminomethyl)-3-methylbutanoyl)oxy)ethyl 2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate (SP-28), (a) Forming SP-28A by reacting a reaction mixture containing Boc-Val-Val with intermediate 3, [ka] (b) Stirring a solution of SP-28A in chloroform and trifluoroacetic acid. [ka]

[0458] In further embodiments, the reaction mixture further comprises cesium carbonate. In some embodiments, the reaction mixture further comprises methanol.

[0459] In one embodiment, a process for preparing (2R)-2-amino-N-((2-((2-ethoxyphenoxy)methyl)morpholino)methyl)-3-methylbutanamide (SP-18) is provided, which process is (a) Forming SP-18A by reacting 2-((2-ethoxyphenoxy)methyl)morpholine with a reaction mixture containing polyformaldehyde, [ka]

[0460] In further embodiments, the reaction mixture further comprises tetrahydrofuran. In some embodiments, the organic acid is hydrochloric acid.

[0461] In another embodiment, a process for preparing pyridine-2-yl 2((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate (SP-19) is provided, which process is (a) Forming pyridine-2-yl carbonochloride by reacting a reaction mixture containing N,N-diisopropylethylamine with 2-hydroxypyridine, [ka] (b) Reacting pyridine-2-ylcarbonochloride with a second reaction mixture containing 2-((2-ethoxyphenoxy)methyl)morpholine. [ka]

[0462] In further embodiments, the reaction mixture contains triphosgene. In some embodiments, the reaction mixture contains dichloromethane. In other embodiments, the second reaction mixture contains dichloromethane. In yet another embodiment, the second reaction mixture contains triethylamine.

[0463] In one embodiment, a process for preparing methylenebis(2-((2-ethoxyphenoxy)methyl)morpholine-4-carboxylate (SP-21) is provided, which process is The reaction involves reacting a reaction mixture containing methylene dibromide with (2-((2-ethoxyphenoxy)methyl)morpholine. [ka]

[0464] In further embodiments, the reaction mixture contains dimethylformamide. In some embodiments, carbon dioxide gas is passed through the reaction mixture.

[0465] In another embodiment, a process for preparing 1-((D-valyl)oxy)-2-methylpropyl 2-((2-ethoxyphenoxy)methyl)-morphylin-4-carboxylate (SP-26) is provided, which process is (a) Forming SP-26A by reacting (2-((2-ethoxyphenoxy)-methyl)morpholine with a first reaction mixture containing 1-chloro-2-methylpropyl chloroformate, [ka] (b) Forming SP-26B by reacting SP-26A with a second reaction mixture containing N-Boc-D-valine, [ka] (c) Reacting SP-26B with dioxane in an organic acid, etc. [ka]

[0466] In further embodiments, the first reaction mixture contains trimethylamine. In some embodiments, the first reaction mixture contains dichloromethane. In other embodiments, the second reaction mixture contains cesium carbonate. In some embodiments, the second reaction mixture contains methanol. In other embodiments, the organic acid is hydrochloric acid.

[0467] Other embodiments are described in the following claims.

Claims

1. A method for treating central nervous system disorders, wherein the method involves a compound of formula I, its stereoisomer, or a salt thereof as the subject: 【Chemistry 1】 The formula includes administering, R １ These are alkyl, heterocyclyl, or pyridyl compounds. R ２ These are alkyl, aryl, heteroaryl, or heterocyclyl compounds. R ３ ~R １４ These are H, F, Cl, Br, I, CN, NO, each independently. ２ , alkyl, aryl, heteroaryl, or heterocyclyl, The method wherein X is H, halogen, amino acid residue, substituted amino acid residue, alkyl, or ester.

2. R １ is CH ２ 、CH ２ CH ２ 、CH ３ CH、CH ２ CH ２ CH ２ 、CH ２ CH ２ CH ２ CH ２ 、(CH ３ ) ２ C、(CH ３ ) ２ CHCH、or (CH ３ ) ３ CCHand the method according to claim 1

3. The method according to claim 2, wherein X is an amino acid residue.

4. The method according to claim 3, wherein the amino acid residue includes a hydrophobic side chain.

5. The method according to claim 4, wherein the amino acid residue containing the hydrophobic side chain is valine.

6. R １ However, CH ２ ,CH ３ CH, or (CH ３ ) ２ The method according to claim 5, wherein CHCH.

7. The method according to claim 6, wherein the compound is 【Chemistry 2】 【Transformation 3】 or 【Chemistry 4】 The method having the structure described above.

8. The method according to claim 4, wherein the amino acid residue containing the hydrophobic side chain is phenylalanine.

9. R １ However, CH ３ The method according to claim 8, wherein CH.

10. The method according to claim 9, wherein the compound is 【Transformation 5】 The method having the structure described above.

11. R ３ ~R １４ The method according to claim 3, wherein each of them is independently H, F, Cl, Br, I, or alkyl.

12. R ３ ~R １４ Each of them independently is H or C １ ~C ６ The method according to claim 11, wherein the alkyl group is alkyl.

13. R ３ ~R １４ The method according to claim 12, wherein all are H.

14. R １ However, CH ２ ,CH ２ CH ２ ,CH ３ CHienCH ２ CH ２ CH ２ CH ２ , or CH ３ CH ２ CH ２ CH, or (CH ３ ) ３ The method according to claim 13, wherein CCH.

15. The method according to claim 1, wherein the compound is 【Transformation 6】 It has the structure, and in the formula, R １５ is H, alkyl, -C(O)OR １７ , or -C(O)R １７ And, R １６ is H, alkyl, -C(O)OR １７ , or -C(O)R １７ And, R １７ The method wherein is H or alkyl.

16. R １５ is alkyl, R １６ The method according to claim 15, wherein is H or alkyl.

17. R １５ is methyl, R １６ The method according to claim 16, wherein is H or methyl.

18. The method according to claim 17, wherein the compound is 【Transformation 7】 The method having the structure described above.

19. R １５ and R １６ The method according to claim 17, wherein is methyl.

20. The method according to claim 19, wherein the compound is 【Transformation 8】 The method having the structure described above.

21. R １５ ga-C(O)R １７ And R １６ H is R １７ The method according to claim 15, wherein is methyl.

22. The method according to claim 21, wherein the compound is 【Chemistry 9】 The method having the structure described above.

23. R １ However, CH ２ or CH ３ The method according to claim 1, wherein CH.

24. The method according to claim 23, wherein X is an ester.

25. The method according to claim 24, wherein the compound is 【Chemistry 10】 【Chemistry 11】 【Chemistry 12】 or 【Chemistry 13】 The method having the structure described above.

26. The method according to claim 1, wherein the compound is 【Chemistry 14】 or 【Chemistry 15】 The method having the structure described above.

27. R １ The method according to claim 1, wherein is a pyridyl group and X is H.

28. The method according to claim 27, wherein the compound is 【Chemistry 16】 The method having the structure described above.

29. R １ The method according to claim 1, wherein is a pyridyl group and X is F, Cl, Br, or I.

30. The method according to claim 29, wherein the compound is 【Chemistry 17】 The method having the structure described above.

31. Compounds of formula II, their stereoisomers, and / or salts thereof: [Chemistry 18] (In the formula, L is an alkyl, substituted pyridinecarboxylic acid, or substituted azanediyl acetate. R ２ These are alkyl, aryl, heteroaryl, or heterocyclyl compounds. R ３ ~R １４ These are H, F, Cl, Br, I, CN, NO, each independently. ２ (These are alkyl, aryl, heteroaryl, or heterocyclyl compounds.)

32. L is CH ２ The compound according to claim 31. 【Request Item 33】 【Chemistry 19】 The compound according to claim 31 or 32.

34. The compound according to claim 31, wherein L is a substituted pyridinecarboxylic acid group.

35. The compound according to claim 34, wherein the substituted pyridinecarboxylic acid group is dimethylpyridine-dicarboxylate. 【Request Item 36】 【Chemistry 20】 The compound according to claim 31 or 35.

37. The compound according to claim 31, wherein the substituted azandiylacetate group is methylazandiylacetate. 【Request Item 38】 【Chemistry 21】 The compound according to claim 31 or 37.

39. A method for treating central nervous system disorders, wherein the method involves a compound of formula III, its stereoisomer, and / or salt thereof as the subject: 【Chemistry 22】 The formula includes administering, Y is F, Cl, Br, I, an amino acid residue, a substituted amino acid residue, an alkyl group, or an ester. R ２ These are alkyl, aryl, heteroaryl, or heterocyclyl compounds. R ３ ~R １４ These are H, F, Cl, Br, I, CN, NO, each independently. ２ The method, wherein the alkyl, aryl, heteroaryl, or heterocyclyl is used.

40. The method according to claim 39, wherein Y is Cl.

41. The method according to claim 39 or 40, wherein the compound is 【Chemistry 23】 The method having the structure described above.

42. A method for treating central nervous system disorders, wherein the method involves a compound of formula IV, its stereoisomer, and / or salt thereof: 【Chemistry 24】 The formula includes administering, Z is H, F, Cl, Br, I, an amino acid residue, a substituted amino acid residue, or a nitrogen-containing group. R ２ These are alkyl, aryl, heteroaryl, or heterocyclyl compounds. R ３ ~R １４ These are H, F, Cl, Br, I, CN, NO, each independently. ２ The method, wherein the alkyl, aryl, heteroaryl, or heterocyclyl is used.

43. The method according to claim 42, wherein Z is a nitrogen-containing group.

44. The method according to claim 43, wherein the nitrogen-containing group is an amide.

45. The method according to claim 42 or 44, wherein the compound is 【Chemistry 25】 The method having the structure described above.

46. The method according to any one of claims 1 to 30 and 38 to 45, wherein the disorder is a sleep disorder.

47. The method according to any one of claims 1 to 30 and 38 to 45, wherein the disorder is narcolepsy.

48. The method according to claim 47, wherein the administration is performed during an inactive or sleep phase.

49. A method for improving the recognition of an object, wherein the method involves applying to the object a compound of formula I, a stereoisomer thereof, or a salt thereof: 【Chemistry 26】 The formula includes administering, R １ These are alkyl, heterocyclyl, or pyridyl compounds. R ２ These are alkyl, aryl, heteroaryl, or heterocyclyl compounds. R ３ to R １４ are each independently H, F, Cl, Br, I, CN, NO ２ , alkyl, aryl, heteroaryl, or heterocyclyl, and The method wherein X is H, halogen, amino acid residue, substituted amino acid residue, alkyl, or ester.

50. The method according to claim 49, wherein the compound is 【Chemistry 27】 or 【Chemistry 28】 The method having the structure described above.

51. A method according to claim 49 or 50, wherein the compound is 【Chemistry 29】 【Transformation 30】 or 【Chemistry 31】 The method having the structure described above.

52. A method for improving the memory of a subject, wherein the method involves applying a compound of formula I, a stereoisomer thereof, or a salt thereof to the subject: 【Chemistry 32】 The formula includes administering, R １ These are alkyl, heterocyclyl, or pyridyl compounds. R ２ These are alkyl, aryl, heteroaryl, or heterocyclyl compounds. R ３ ~R １４ each independently represents H, F, Cl, Br, I, CN, NO ２ , alkyl, aryl, heteroaryl, or heterocyclyl, The method wherein X is H, halogen, amino acid residue, substituted amino acid residue, alkyl, or ester.

53. A method according to claim 52, wherein the compound is 【Transformation 33】 The method having the structure described above.

54. A method according to claim 51 or 52, wherein the compound is 【Transformation 34】 The method having the structure described above.

55. A method for treating a mental illness, wherein the method involves applying to the subject a compound of formula I, a stereoisomer thereof, or a salt thereof: 【Chemistry 35】 The formula includes administering, R １ These are alkyl, heterocyclyl, or pyridyl compounds. R ２ These are alkyl, aryl, heteroaryl, or heterocyclyl compounds. R ３ ~R １４ These are H, F, Cl, Br, I, CN, NO, each independently. ２ , alkyl, aryl, heteroaryl, or heterocyclyl, The method wherein X is H, halogen, amino acid residue, substituted amino acid residue, alkyl, or ester.

56. The method according to claim 55, wherein the compound is 【Transformation 36】 The method having the structure described above.

57. A method according to claim 55 or 56, wherein the compound is 【Chemistry 37】 The method having the structure described above.

58. A method for treating pain in a subject or inducing an analgesic effect, wherein the method involves applying to the subject a compound of formula I, a stereoisomer thereof, or a salt thereof: 【Transformation 38】 The formula includes administering, R １ These are alkyl, heterocyclyl, or pyridyl compounds. R ２ These are alkyl, aryl, heteroaryl, or heterocyclyl compounds. R ３ ~R １４ These are H, F, Cl, Br, I, CN, NO, each independently. ２ , alkyl, aryl, heteroaryl, or heterocyclyl, The method wherein X is H, halogen, amino acid residue, substituted amino acid residue, alkyl, or ester.

59. The method according to claim 58, wherein the compound is 【Chemistry 39】 The method having the structure described above.

60. The method according to claim 58 or 59, wherein the compound is 【Chemistry 40】 The method having the structure described above.

61. A method for treating schizophrenia, wherein the method involves applying to the subject a compound of formula I, a stereoisomer thereof, or a salt thereof: 【Chemistry 41】 The formula includes administering, R １ These are alkyl, heterocyclyl, or pyridyl compounds. R ２ These are alkyl, aryl, heteroaryl, or heterocyclyl compounds. R ３ ~R １４ These are H, F, Cl, Br, I, CN, NO, each independently. ２ , alkyl, aryl, heteroaryl, or heterocyclyl, The method wherein X is H, halogen, amino acid residue, substituted amino acid residue, alkyl, or ester.

62. The method according to claim 61, wherein the compound is 【Chemistry 42】 The method having the structure described above.

63. The method according to claim 61 or 62, wherein the compound is 【Chemistry 43】 The method having the structure described above.

64. A method for treating neuropsychiatric disorders in patients, wherein the method involves a compound of formula I, its stereoisomer, or a salt thereof as the subject: 【Chemistry 44】 The formula includes administering, R １ These are alkyl, heterocyclyl, or pyridyl compounds. R ２ These are alkyl, aryl, heteroaryl, or heterocyclyl compounds. R ３ ~R １４ These are H, F, Cl, Br, I, CN, NO, each independently. ２ , alkyl, aryl, heteroaryl, or heterocyclyl, The method wherein X is H, halogen, amino acid residue, substituted amino acid residue, alkyl, or ester.

65. The method according to claim 64, wherein the compound is 【Chemistry 45】 The method having the structure described above.

66. The method according to claim 64 or 65, wherein the compound is 【Chemistry 46】 The method having the structure described above.

67. A method for treating anxiety in a subject, wherein the method involves providing the subject with a compound of formula I, a stereoisomer thereof, or a salt thereof: 【Chemistry 47】 The formula includes administering, R １ These are alkyl, heterocyclyl, or pyridyl compounds. R ２ These are alkyl, aryl, heteroaryl, or heterocyclyl compounds. R ３ ~R １４ These are H, F, Cl, Br, I, CN, NO, each independently. ２ , alkyl, aryl, heteroaryl, or heterocyclyl, The method wherein X is H, halogen, amino acid residue, substituted amino acid residue, alkyl, or ester.

68. The method according to claim 67, wherein the compound is 【Chemistry 48】 The method having the structure described above.

69. The method according to claim 67 or 68, wherein the compound is 【Chemistry 49】 The method having the structure described above.

70. A method for treating depression in a subject, wherein the method involves applying to the subject a compound of formula I, a stereoisomer thereof, or a salt thereof: [Transformation 50] The formula includes administering, R １ These are alkyl, heterocyclyl, or pyridyl compounds. R ２ These are alkyl, aryl, heteroaryl, or heterocyclyl compounds. R ３ ~R １４ These are H, F, Cl, Br, I, CN, NO, each independently. ２ , alkyl, aryl, heteroaryl, or heterocyclyl, The method wherein X is H, halogen, amino acid residue, substituted amino acid residue, alkyl, or ester.

71. A method according to claim 70, wherein the compound is 【Chemistry 51】 or 【Chemistry 52】 The method having the structure described above.

72. A method according to claim 70, wherein the compound is 【Chemistry 53】 【Chemistry 54】 or 【Transformation 55】 The method having the structure described above.

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