Selective AMPA receptor modulator

Abstract

Provided are compounds of formula (I), related compositions, and related methods, including therapeutic methods for treating neurological diseases and disorders, including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS), and methods for in vivo imaging of AMPA receptors or their surrogates in the brain.

Description

[Technical Field]

[0001]

[0001] This disclosure relates to small molecule inhibitors of AMPAR-mediated neurotoxicity. [Background technology]

[0002]

[0002] Excess glutamate, which acts mainly via NMDAR and AMPAR, Ca 2+ It can promote influx and produce excitotoxicity under pathological conditions such as ischemia, trauma, hypoglycemia, and epileptic seizures. Glutamate receptor-mediated excitotoxicity is involved in demyelination, neuronal / oligodendrocyte loss, and axonal damage, which are causes of neurological damage in multiple sclerosis (MS). There is evidence that this neurotransmission system can also modulate immune system function and may be involved in the mechanisms of inflammatory neuronal damage. However, because glutamate receptors are essential for normal brain function, prevention of glutamate-mediated excitotoxicity is difficult.

[0003]

[0003] A protein complex composed of the GluR2 subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptor (AMPAR) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been shown to play a role in glutamate-mediated neurotoxicity, and peptides that inhibited the GluR2-GAPDH complex dramatically improved neurological outcomes in mouse models of MS and experimental autoimmune encephalomyelitis (EAE) models (Zhai et al., Annals Clin. Transl. Neurology 2015 2:388~400). Inhibition of the GluR2-GAPDH complex prevented AMPAR-mediated excitotoxicity, improved neurological outcomes in the EAE model without the side effects of immunosuppressants, and without impairing AMPAR-mediated neurotransmission. It was also found that the GluR2-GAPDH complex was significantly increased in postmortem tissue from chronic MS patients.

[0004]

[0004] US 20130336951 (Liu, Centre for Addiction and Mental Health) provides a method for modulating AMPA receptor-mediated excitotoxicity using inhibitory peptides that modulate GAPDH binding with GluR2 or p53. [Overview of the project] [Problems that the invention aims to solve]

[0005]

[0005] Further compositions and methods are needed to modulate this interaction and AMPA receptor-mediated excitotoxicity. [Means for solving the problem]

[0006]

[0006] Compounds of formula (I) and pharmaceutically acceptable salts thereof:

[0007] [ka]

[0008] (In the formula, R1, R2, R3, and R4 are each independently H or halo.) At least two of R1, R2, R3, and R4 are H, and (At least two of R1, R2, R3, and R4 are halos.) It will be provided.

[0009]

[0007] The compound may also include cases where R1 and R3 are independently selected from fluoro and chloro, respectively, and R2 and R4 are H, respectively.

[0008] The compound may also include cases where R2 and R3 are independently selected from fluoro and chloro, respectively, and R1 and R4 are H, respectively.

[0010]

[0009] The compound may also include cases where R2 and R4 are independently selected from fluoro and chloro, respectively, and R1 and R3 are H, respectively.

[0010] In some embodiments, the compound is

[0011]

[0011] [ka]

[0012]

[0012]

[0013] [ka]

[0014] and

[0013]

[0015] [ka]

[0016] It is selected from the group consisting of the following.

[0014] A pharmaceutical composition is also provided comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier or excipient.

[0017]

[0015] The pharmaceutical composition may also include cases in which the composition is formulated as an oral dosage form.

[0016] The pharmaceutical composition may also include cases where one oral dosage form contains about 0.1 to 250 mg or about 1 to 25 mg of the compound.

[0018]

[0017] Also provided is a method for treating a subject in need of treatment for a neurological disorder or impairment, wherein at least one symptom of the neurological disorder or impairment is reduced by inhibiting AMPA-mediated neurotoxicity, and the method comprises the step of administering to the subject a pharmaceutical composition comprising an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0019]

[0018] The method may also include cases where the neurological disorder or impairment is characterized by GluR2 / AMPAR neurotoxicity.

[0019] The method may also include cases where the neurological disorder or disability is selected from neuroinflammation, stroke, and epilepsy.

[0020]

[0020] The method may also include cases where the neurological disorder or impairment is selected from Parkinson's disease, Alzheimer's disease, dementia, and Huntington's disease.

[0021] A method is also provided for treating a subject in need of treatment for a neurological disorder or disability selected from the group consisting of multiple sclerosis (MS), spasticity associated with multiple sclerosis, and amyotrophic lateral sclerosis (ALS), the method comprising the step of administering to the subject a pharmaceutical composition comprising an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0021]

[0022] The method may also include cases where the pharmaceutical composition is in oral dosage form and the route of administration is oral.

[0023] The methods may include cases where the subject is a human being.

[0022]

[0024] Also provided are in vitro or ex vivo methods for inhibiting glutamate receptor-mediated excitotoxicity, inhibiting GluR2 / AMPAR neurotoxicity, and / or inhibiting GAPDH-GluR2 complex formation in cells, tissues, or organoids, the method comprising the step of contacting cells, tissues, or organoids with a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0023]

[0025] Other technical features may be readily apparent to those skilled in the art from the following drawings, description, and claims. [Brief explanation of the drawing]

[0024] [Figure 1]

[0026] The clinical scores of EAE mice treated with compound 262, 1 mg / kg intraperitoneal (ip) (diamond), 2.5 mg / kg intraperitoneal (closed square), 5 mg / kg (triangle), or untreated (inverted triangle) are shown. [Figure 2A]

[0027] This section describes the testing in the Cuprison model system. [Figure 2B]

[0028] The graph shows cuprizon-induced behavioral defects in mice, measured in two open-field tests, total distance traveled (left bar set) and speed (right bar set), for control mice fed a normal diet (Ctr), untreated mice fed a 0.2% cuprizon diet and saline for 6 weeks (Cup), and mice fed a 0.2% cuprizon diet and compound 262, 5mpk, orally (po) for 4 weeks (Cup+262). [Figure 2C]

[0029] This shows cuprizon-induced behavioral defects in mice, measured by two sets of rotorod tests, time to fall (left bar set) and speed at fall (right bar set), in control mice fed a normal diet (Ctr), untreated mice fed a 0.2% cuprizon diet and saline for 6 weeks (Cup), and mice fed a 0.2% cuprizon diet and compound 262, 5mpk orally for 4 weeks (Cup+262). [Figure 3A]

[0030] This shows the quantification of myelin levels, measured as the mean fluoromyelin green intensity in representative electron micrographs of the medial corpus callosum (left bar) and lateral corpus callosum (right bar) for control mice fed a normal diet (Ctr), untreated mice fed a 0.2% cuprizon diet and saline for 6 weeks (Cup), and mice fed a 0.2% cuprizon diet and compound 262, 5mpk orally for 4 weeks (Cup+262). [Figure 3B]

[0031] This shows the quantification of myelinated axons per 100 square microns in representative corpus callosum electron micrographs of control mice fed a normal diet (Ctr), untreated mice fed a 0.2% cuprizon diet and saline for 6 weeks (Cup), and mice fed a 0.2% cuprizon diet and compound 262, 5mpk orally for 4 weeks (Cup+262). [Figure 3C]

[0032] This paper quantifies the myelin thickness (microns) in corpus callosum electron micrographs of control mice fed a normal diet (Ctr), untreated mice fed a 0.2% cuprizon diet and saline for 6 weeks (Cup), and mice fed a 0.2% cuprizon diet and compound 262, 5mpk orally for 4 weeks (Cup+262). [Figure 3D]

[0033] The graph shows the ratio of inner diameter to outer diameter (g ratio) of axons in electron micrographs of the corpus callosum for control mice fed a normal diet (Ctr), untreated mice fed a 0.2% cuprizon diet and saline for 6 weeks (Cup), and mice fed a 0.2% cuprizon diet and compound 262, 5mpk orally for 4 weeks (Cup+262). This ratio reflects the function and integrity of the axons. Since the thickness of the myelin sheath increases or decreases linearly with respect to the axon diameter, the g ratio is plotted against the axon diameter in the chart on the left, indicating that the differences in treatment were not due to biased axon sampling. [Figure 4]

[0034] This shows the quantification of oligodendrocyte survival in the corpus callosum as the number of olig2 cells per square millimeter (olig2 cells / mm2) for three mice per group in three groups: control mice fed a normal diet (Ctr), untreated mice fed a 0.2% cuprizon diet and saline for 6 weeks (Cup), and mice fed a 0.2% cuprizon diet and compound 262, 5mpk orally for 4 weeks (Cup+262). *p-values ​​compared to the control group, p-values ​​compared to the saline (untreated) group, one-way ANOVA. [Figure 5]

[0035] This shows the quantification of microglia density in the corpus callosum as the number of lba1+ cells per square millimeter (lba1+ cells / mm2) in untreated mice (Cup+veh) fed a 0.2% cuprizon diet and saline for 6 weeks, control mice (Ctr) fed a normal diet, and mice (Cup+262) fed a 0.2% cuprizon diet and compound 262, 5mpk orally for 4 weeks, with 3 mice per group. *p-values ​​compared to the control group, p-values ​​compared to the saline (untreated) group, one-way ANOVA. [Figure 6A]

[0036] Figure 6A shows the relationship between fEPSP peak amplitude (mV) and stimulus intensity (mA) for C57Bl6 / J mice treated with compound 262, 5mpk, orally (gray triangles) or via vehicle (black circles) one hour prior to measurement. Data were calculated using 16 slices from 4 mice in each group; p=0.92 (treatment), p=0.50 (treatment x stimulus), two-way ANOVA comparison. This is a measure of baseline synaptic transmission. [Figure 6B]

[0037] Figure 6B shows the input-output relationship between fEPSP (mV) and prefibre salvo (mV) amplitude for C57Bl6 / J mice treated with compound 262, 5mpk, orally (gray triangle), or via vehicle (black circle) one hour prior to measurement. Data were calculated using 16 slices from 4 mice in each group; treatment: t(47)=-0.14, p=340 0.89, β=-0.028, standardized coefficient=0.12; treatment x fibre salvo: t(132)=1.43, p=0.15, β=0.44, standardized coefficient=0.12; general linear model. This data indicates that baseline synaptic transmission results were not biased by electrode placement, which may affect the fEPSP-stimulus relationship. [Figure 7A]

[0038] Figure 7A, PET images of rats 0-5 minutes after intravenous injection of representative radiolabeled compounds of formula I, [11C]-262, in several embodiments. [Figure 7B]

[0039] Figure 7B is a line graph of standardized uptake (SUV) against time in rats (circles, n=3) and mice (squares, n=2), comparing the uptake of [11C]-262. [Modes for carrying out the invention]

[0025]

[0040] This disclosure relates to compounds of formula (I) and salts thereof, as well as related compositions and related methods, including therapeutic methods. Compounds of formula (I) exhibit low nanomolar efficacy for inhibiting AMPA-mediated neurotoxicity, along with very high CNS permeability and low efflux, resulting in excellent in vivo CNS exposure. Representative compounds also exhibit excellent oral bioavailability, low clearance, and dose-dependent efficacy in animal model systems of neurological diseases and disorders.

[0026] Compounds of the Disclosure

[0041] Compounds of formula (I) and their pharmaceutically acceptable salts

[0027] [ka]

[0028] (In the formula, R1, R2, R3, and R4 are each independently H or halo.) At least two of R1, R2, R3, and R4 are H, and (At least two of R1, R2, R3, and R4 are halos.) It will be provided.

[0029]

[0042] In this embodiment, R1 and R3 are independently selected from fluoro and chloro, respectively, and R2 and R4 are H, respectively.

[0043] In this embodiment, R2 and R3 are independently selected from fluoro and chloro, respectively, and R1 and R4 are H, respectively.

[0030]

[0044] In this embodiment, R2 and R4 are independently selected from fluoro and chloro, respectively, and R1 and R3 are H, respectively.

[0045] In this embodiment, the compound is

[0046]

[0031] [ka]

[0032]

[0047]

[0033] [ka]

[0034] or

[0048]

[0035] [ka]

[0036] Alternatively, a selection is made from its pharmaceutically acceptable salts. Labeled compound of formula I

[0049] Labeled compounds of formula I, such as radiolabeled compounds and fluorescently labeled compounds, are also provided. In one embodiment, the radiolabeled compound of formula I contains a fluorine isotope. In one embodiment, the fluorine isotope is fluorine-18( 18 F) and fluorine-19 ( 19 Selected from F). In one embodiment, the radiolabeled compound of formula I contains a carbon isotope. In one embodiment, the carbon isotope is carbon-11 ( 11 C) In one embodiment, the radiolabeled compound of formula I contains an iodine isotope. In one embodiment, the iodine isotope is iodine-123( 123 I) or Iodine-131 131 I) In one embodiment, the radiolabeled compound of formula I is tritium ( 3 Includes H).

[0037]

[0050] In this manner, 18 F-261,18 F-262, and 18 A compound of Formula I selected from F-263 18 F-labeled compound is provided. In an embodiment, 11 C-261, 11 C-262, and 11 A compound of Formula I selected from C-263 11 C-labeled compound is provided.

[0038] Synthesis Method

[0051] Methods for the chemical synthesis of the compound of Formula (I) are described herein. These methods and / or other well-known methods can be modified and / or adapted in known ways to facilitate the synthesis of further compounds (as described herein).

[0039]

[0052] In an embodiment, 2-chloropyrimidine-4-carbonyl chloride is prepared by the reaction of 2-chloropyrimidine-4-carboxylic acid with a suitable chlorinating agent, such as oxalyl chloride, in a suitable solvent, for example DCM, in the presence of a suitable catalyst, for example DMF. An example of such a method is shown in Scheme 1.

[0040]

Chemical Formula

[0041]

[0053] In one embodiment, 2-chloro-N,N-dimethylpyrimidine-4-carboxamide is prepared by amination of 2-chloropyrimidine-4-carbonyl chloride using dimethylamine hydrochloride in a suitable solvent, for example DCM, in the presence of a suitable base, for example N,N-diisopropylethylamine. The pure product can then be isolated by a suitable chromatographic method, for example, medium pressure liquid chromatography (MPLC). An example of such a method is shown in Scheme 2.

[0042]

Chemical Formula

[0043]

[0054] In one embodiment, (R)-2-(3-hydroxypyrrolidine-1-yl)-N,N-dimethylpyrimidine-4-carboxamide is prepared by the reaction of (R)-pyrrolidine-3-ol with 2-chloro-N,N-dimethylpyrimidine-4-carboxamide in a suitable solvent, such as acetonitrile, in the presence of a suitable base, such as N,N-diisopropylethylamine. An example of such a method is shown in Scheme 3.

[0044] [ka]

[0045]

[0055] In one embodiment, N,N-dimethyl-2-(3-oxopyrrolidine-1-yl)pyrimidine-4-carboxamide can be prepared from (R)-2-(3-hydroxypyrrolidine-1-yl)-N,N-dimethylpyrimidine-4-carboxamide by a reaction using a suitable oxidizing agent, such as Des-Martin periodinane (DMP), in a suitable solvent, such as DCM. The pure product can then be isolated by a suitable chromatographic method, such as MPLC. An example of such a method is shown in Scheme 4.

[0046] [ka]

[0047]

[0056] In one embodiment, 2-(3-((dimethylamino)methylene)-4-oxopyrrolidine-1-yl)-N,N-dimethylpyrimidine-4-carboxamide can be prepared by the reaction of N,N-dimethyl-2-(3-oxopyrrolidine-1-yl)pyrimidine-4-carboxamide with N,N-dimethylformamide dimethylacetyl (DMF-DMA) in a suitable solvent, such as tetrahydrofuran (THF). The pure product can then be isolated by a suitable chromatographic method, such as MPLC. An example of such a method is shown in Scheme 5.

[0048] [ka]

[0049]

[0057] In one embodiment, 2-(2,6-dihydropyrrolo[3,4-c]pyrazole-5(4H)-yl)-N,N-dimethylpyrimidine-4-carboxamide can be prepared by the reaction of 2-(3-((dimethylamino)methylene)-4-oxopyrrolidine-1-yl)-N,N-dimethylpyrimidine-4-carboxamide with hydrazine in a suitable solvent, such as ethanol. An example of such a method is shown in Scheme 6.

[0050] [ka]

[0051]

[0058] In one embodiment, 2-(2-benzyl-2,6-dihydropyrrolo[3,4-c]pyrazole-5(4H)-yl)-N,N-dimethylpyrimidine-4-carboxamide can be prepared by reaction of 2-(2,6-dihydropyrrolo[3,4-c]pyrazole-5(4H)-yl)-N,N-dimethylpyrimidine-4-carboxamide with a suitably substituted benzyl bromide or benzyl chloride, for example, by deprotonation of dihydropyrrolopyrazole with sodium hydride, followed by reaction with benzyl bromide in a suitable solvent, such as THF, in the presence of a suitable catalyst, such as tetra-n-butylammonium iodide (TBAI). The reaction yields a mixture of two positional isomers, which can be separated by a suitable chromatographic method, such as preparative HPLC. An example of such a method is shown in Scheme 7.

[0052] [ka]

[0053]

[0059] In one embodiment, 2-substituted-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole can be prepared by reacting a well-substituted hydrazine, prepared for example by the reaction of a substituted benzyl chloride and hydrazine (see, e.g., US2008 / 0255156), or by the reductive amination of a suitable aldehyde, with tert-butyl(3Z)-3-[(dimethylamino)methylene]-4-oxopyrrolidine-1-carboxylate, prepared from the reaction of a commercially available tert-butyl 3-oxopyrrolidine-1-carboxylate and DMF-DMA, in ethanol in a sealed tube (see, e.g., WO2009 / 025784). The protecting group can then be removed under acidic conditions, for example, using 4M hydrochloric acid. An example of such a method is shown in Scheme 8.

[0054] [ka]

[0055]

[0060] In another embodiment, the 2-substituted pyrrolopyrazole can then be reacted with a desired heteroaryl group having a suitable leaving group such as a halogen, in the presence of a suitable catalyst, such as Pd(OAc)2, XPhos, and Cs2CO3, to confer a desired substitution to the pyrrole. An example of such a method is shown in Scheme 9.

[0056] [ka]

[0057]

[0061] In another embodiment, the unsubstituted 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole ("pyrrolopyrazole") skeleton can be first prepared by the reaction of tert-butyl(3Z)-3-[(dimethylamino)methylene]-4-oxopyrrolidine-1-carboxylate with hydrazine (see, e.g., WO2011 / 146358), and then deprotected as described above. The pyrrolopyrazole can then be reacted with a suitable aryl or heteroaryl group on pyrrole nitrogen as described above, and the pyrazole can then be deprotonated with a suitable base, e.g., NaH in THF, and reacted with a suitable benzyl bromide or benzyl chloride to obtain the desired product (as a mixture of positional isomers, which can be separated by high-speed flash chromatography, see, e.g., WO2009 / 025784). An example of such a method is shown in Scheme 10.

[0058] [ka]

[0059]

[0062] In another embodiment, the pyrrole-substituted 5-substituted-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole skeleton can be first prepared by the reaction of tert-butyl(3Z)-3-[(dimethylamino)methylene]-4-oxopyrrolidine-1-carboxylate with hydrazine (see, e.g., WO2011 / 146358) and then substituted on the pyrazole as described above. The resulting product can then be deprotected and further reacted as shown in Scheme 10. An example of such a method is shown in Scheme 11.

[0060] [ka]

[0061]

[0063] A method for synthesizing the radiolabeled compound of formula I is also provided. 11 C- and 18 The following methods exemplify the preparation of F-labeled compounds. 11 Synthesis of 1C-labeled compound 262

[0064] 11 The synthesis of the 1C-labeled compound 262 involved the synthesis of its desmethyl variant according to Scheme 12.

[0062] [ka]

[0063]

[0065] Compound 3: Compound 2 (4.75 g, 22.9 mmol, 1.20 equivalent) was added to a solution of Compound 1 (4.00 g, 19.1 mmol, 1.00 equivalent) and Cs2CO3 (18.7 g, 57.4 mmol, 3.00 equivalent) in MeCN (50.0 mL). The mixture was stirred at 80°C for 2 hours under N2. TLC (petroleum ether:ethyl acetate = 1:1) was performed on the spot (R f This indicates that (=0.48) was the product. The reaction mixture was quenched with 50.0 mL of H2O at 25°C, then diluted with 20.0 mL of H2O, and extracted with 100 mL of ethyl acetate (50.0 mL x 2). The combined organic layer was washed with 90.0 mL of brine (30.0 mL x 3), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by flash silica gel chromatography (ISCO®; 120 g SepaFlash® silica flash column, eluent 0-50% ethyl acetate / petroleum ether gradient at 100 mL / min). 1 This was confirmed by 1H NMR (EW54448-4-P1A) and 1F NMR (EW54448-4-P1A). Compound 3 (2.30 g, 6.86 mmol, yield 35.9%) was obtained as a yellow oil. 1 H NMR: EW54448-4-P1A (400 MHz, CDCl3). δ (ppm) 7.13-7.25 (m, 2H), 6.74-6.91 (m, 2H), 5.27-5.29 (m, 1H), 5.28 (d, J = 4.4 Hz, 1H), 4.33-4.55 (m, 4H), 1.49 (s, 9H).

[0066] Compound 4 was dissolved in HCl / dioxane (20.0 mL) in a solution of Compound 3 (2.30 g, 6.86 mmol, 1.00 equivalent). The mixture was stirred at 25°C for 12 hours. LC-MS (EW54692-1-R1A) showed that 0% of reactant 1 remained. The reaction mixture was concentrated under reduced pressure to obtain the residue. The reaction mixture was partitioned between 30.0 mL of saturated Na2CO3 and 50.0 mL of siRNA. The organic phase was separated, washed with 30.0 mL of brine (15.0 mL x 2), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. TLC (dichloromethane:methanol = 10:1) was performed on the spot (R f This indicates that (=0.12) was the product. 1 1H NMR (EW54692-1-P1A), 19 This was confirmed by 1F NMR (EW54692-1-P1A). Compound 4 (1.31 g, crude) was obtained as yellow oil. 1 H NMR: (400 MHz, CDCl3). δ (ppm) 7.14-7.20 (m, 1H), 7.10 (s, 1H), 6.77-6.85 (m, 2H), 5.25 (s, 2H), 3.97 (d, J = 1.6 Hz, 4H), 2.60 (br s, 1H).

[0067] Compound 6: A mixture of Compound 4 (800 mg, 3.40 mmol, 1.00 equivalent), Compound 5 (704 mg, 4.08 mmol, 1.20 equivalent), Cs2CO3 (3.32 g, 10.2 mmol, 3.00 equivalent), XPhos (324 mg, 680 μmol, 0.20 equivalent), and Pd(OAc)2 (76.4 mg, 340 μmol, 0.10 equivalent) in dioxane (8.00 mL) was degassed, purged three times with N2, and then stirred at 80°C for 12 hours under an N2 atmosphere. LC-MS (EW54692-2-R1A) showed that 0% of reactant 1 remained. Several new peaks were shown in LC-MS, and 26% of the desired compound (Rt=0.831) was detected. TLC (petroleum ether:ethyl acetate = 1:1) is spot (R fThis indicates that (=0.45) was the product. The reaction mixture was filtered and concentrated under reduced pressure to obtain the residue. The crude product was triturated with 10.0 mL of CH2Cl2 at 25°C for 10 minutes. 1 1H NMR (EW54692-2-P1A), 19 The compound was confirmed by 1F NMR (EW54692-2-P1A) and LC-MS (EW54692-2-P1A). Compound 6 (797 mg, 2.15 mmol, yield 63.1%) was obtained as a white solid. 1 H NMR: EW54692-2-P1A (400 MHz, DMSO-d6). δ (ppm) 8.66 (d, J = 4.8 Hz, 1H), 7.72 (s, 1H), 7.33-7.43 (m, 1H), 7.28 (td, J = 9.6, 2.4 Hz, 1H), 7.18 (d, J = 4.8 Hz, 1H), 7.09 (td, J = 8.4, 2.4 Hz, 1H), 5.37 (s, 2H), 4.58 (br d, J = 7.6 Hz, 4H), 3.88 (s, 3H).

[0068] Desmethyl variant of compound 262: To a solution of compound 6 (797 mg, 2.15 mmol, 1.00 equivalent) in THF (7 mL), methanamine (2 M, 10.73 mL, 10.0 equivalent) was added. The mixture was stirred at 25°C for 12 hours. Methanamine (2 M, 80.5 mL, 75.0 equivalent) was added. The mixture was stirred at 25°C for 12 hours, and LC-MS (EW54692-3-R1A) showed that 0% of reactant 1 remained. Several new peaks were shown on LC-MS, indicating 75% of the desired compound (R t A concentration of 0.817 (=0.817) was detected. The reaction mixture was concentrated at <40°C. The residue was purified by flash silica gel chromatography (ISCO®; 20g SepaFlash® silica flash column, eluent 0-10% ethyl acetate / petroleum ether gradient at 30 mL / min). TLC (dichloromethane:methanol = 10:1) was performed using spot (R f This indicates that (=0.42) was the product. 1 1H NMR (EW54692-3-P1A1),19 F NMR (EW54692-3-P1A1), 13 Confirmation was made by 13C NMR (EW54692-3-P1B) and LC-MS (EW54692-3-P1B1). Desmethyl-262 (160 mg, 432 μmol, 20.1% yield, 100% purity) was obtained as a white solid. 1 H NMR: EW54923-3-P1A1 (400 MHz, DMSO-d6). δ (ppm) 8.71 (br s, 1H), 8.61 (d, J = 4.8 Hz, 1H), 7.64-7.84 (m, 1H), 7.23-7.40 (m, 2H), 7.16 (d, J = 4.8 Hz, 1H), 7.09 (td, J = 8.8, 2.4 Hz, 1H), 5.38 (s, 2H), 4.55-4.72 (m, 4H), 2.83 (d, J = 4.8 Hz, 3H). 13 C NMR: EW54692-3-P1B (400 MHz, CDCl3). Radiolabeling method

[0069] Pressurized gas target containing nitrogen:oxygen mixture (99.5:0.5) in Scanditronix 17 MeV cyclotron (Scanditronix; Uppsala, Sweden) 14 N(p,α) 11 Due to the C nuclear reaction, without the addition of a carrier [ 11 C] Carbon dioxide ([ 11 [C]CO2) was produced. Automated radiochemistry was performed using TRACERlab software on the GE FX2 C automated radiochemistry module. 11 [C]CO2 was supplied from the cyclotron target to the GE FX 2C via a 3.175 mm (1 / 8") stainless steel delivery line under nitrogen pressure. A column packed with 0.3 g of molecular sieve and 0.2 g of nickel (Shimalite-Ni (for reduction), Shimadzu Inc.) was used. 11 [C]CO2 was captured at room temperature. The column was sealed under hydrogen and heated to 350°C for 60 seconds. 11C]CO2 11 It was reduced to C]CH4, and then, 11 [C]CH4 was passed through a phosphorus pentoxide (P2O5) column and captured on a carbosphere column cooled to -75°C with liquid nitrogen. When the carbosphere column was heated to 80°C, [ 11 [C]CH4 is released into a circulating loop, which includes a membrane gas pump, an iodine column heated to 100°C, a quartz glass iodine reaction tube at 740°C, two Askalite columns, and a Porapak Q column at room temperature. 11 [C]CH3I (approximately 10-20 GBq) was released from the Porapak column by heating to 190°C and supplied directly to the reactor by a controlled helium flow (10 mL / min). The reaction vessel was pre-loaded with a mixture of desmethyl-262 (0.5 mg) and 12N KOH (7 μL) in 300 μL anhydrous dimethylformamide (DMF). The reaction vessel was heated to 80°C for 5 minutes, cooled to 30°C, and quenched using a semi-preparative HPLC mobile phase (1.5 mL). The reaction mixture was injected into a semi-preparative HPLC column (LUNA C18(2), 10 μm, 10 × 250 mm, Phenomenex; Torrance, California, US) and eluted with 0.1 M ammonium formate:acetonitrile (60:40) at 5.0 mL / min. Purification was monitored by UV (λ=254 nm) and a radioactivity detector was connected in series. 11 [C]-262 was collected around 13 minutes and loaded into a solid-phase extraction cartridge (SepPak tC18 Plus, Waters; Milford, Massachusetts, US), and washed with 10 mL of sterile water. The radioactive tracer was then eluted with 1 mL of ethanol and prepared for injection with 9 mL of 0.9% sodium chloride.

[0064]

[0070] 11 The 1C-labeled compound 262 was successfully synthesized according to Scheme 13.

[0065] [ka]

[0066]

[0071] A reaction mixture containing 0.5 mg of desmethyl-262 in 300 μL of anhydrous DMF and 7 μL of 12N KOH [ 11 [C]CH3I was ventilated. 11 After aeration of [C]CH3I into the reactor, the reaction mixture was heated to 80°C for 5 minutes, cooled to 30°C, and then quenched using 1.5 mL of semi-preparative HPLC mobile phase. The crude mixture was loaded into a semi-preparative HPLC for purification and re-prepared for injection in 90% sterile saline:10% ethanol. Radiochemical yield (RCY) ranged from 0.5% to 6% RCY (n=5), and radiochemical purity was >99%.

[0067] 18 Synthesis of 262 fluorine-labeled compound

[0068] [ka]

[0069]

[0072] To a solution of compound 1 (10.0 g, 47.8 mmol, 1.00 equivalent) in ACN (120 mL), Cs2CO3 (46.7 g, 143 mmol, 3.00 equivalent) was added, followed by compound 2 (15.4 g, 57.4 mmol, 1.20 equivalent) being added to the mixture. The mixture was stirred at 80°C for 12 hours. LC-MS showed that compound 1 was completely consumed. The reaction mixture was concentrated to obtain the residue, which was then diluted with ethyl acetate (100 mL) and H2O (100 mL), separated, and the organic matter was concentrated to obtain the residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0~0 / 1) and TLC (petroleum ether / ethyl acetate = 5:1, Rf(P1) = 0.3). Compound 3 (11.0 g, 26.5 mmol, yield 55.4%, purity 95.3%) was obtained as yellow oil. 1 H NMR: (400 MHz, CDCl3). δ 7.06 (m, 4H), 5.29 (s, 2H), 4.42 - 5.50 (m, 4H), 1.52 (s, 9H).

[0073]

[0070] [ka]

[0071]

[0074] To a solution of compound 4 (1.50 g, 5.07 mmol, 1.00 equivalent) and compound 5 (1.05 g, 6.08 mmol, 1.20 equivalent) in THF (20 mL), DIEA (1.96 g, 15.2 mmol, 2.65 mL, 3.00 equivalent) was added, and the mixture was stirred at 20-30°C for 12 hours. The reaction mixture was filtered, and the filtered cake was evaporated to obtain the residue. Compound 6 (2.00 g, 4.63 mmol, yield 91.4%) was obtained as a white solid. 1 H NMR: (400 MHz, DMSO). δ 8.64 (d, J = 4.8 Hz, 1H), 7.72 (s, 1H), 7.57(d, J = 8.8 Hz, 1H), 7.42(d, J = 8.0 Hz, 1H), 7.16 - 7.23 (m, 2H), 5.36 (s, 2H), 4.56(d, J = 8.4 Hz, 4H), 3.87 (s, 3H).

[0075]

[0072] [ka]

[0073]

[0076] Compound 6 (1.50 g, 3.47 mmol, 1.00 equivalent) was dissolved in N-methylmethaneamine (2 M, 10.4 mL, 6.00 equivalent) and stirred at 60°C for 8 hours. The reaction mixture was concentrated to obtain the residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0~0 / 1) and TLC (petroleum ether / ethyl acetate = 1:1, Rf(P1) = 0.3). Compound 7 (600 mg, 1.35 mmol, yield 38.8%) was obtained as a white solid.

[0074]

[0077]

[0075] [ka]

[0076]

[0078] To a solution of compound 7 (200 mg, 449 μmol, 1.00 equivalent) in dioxane (3 mL), Pd(PPh3)4 (51.9 mg, 44.9 μmol, 0.10 equivalent) and trimethyl(trimethylstannyl) stannane (325 mg, 992 μmol, 205 μL, 2.21 equivalents) were added, and the mixture was stirred at 100°C for 30 minutes. The reaction mixture was quenched with saturated KF aqueous solution (10 mL), then extracted with EA (10 mL x 3), dried over Na2SO4, filtered, and concentrated to obtain the residue. The residue was purified by column chromatography to obtain compound 262-P (59.0 mg, 104 μmol, yield 23.3%, purity 93.8%) as a white solid. 1 H NMR: (400 MHz, DMSO) δ 8.53 (d, J = 4.8 Hz, 1H), 7.70 (s, 1H), 7.23 - 7.31(m, 3H), 6.74 (d, J = 5.2 Hz, 1H), 5.37 (s, 2H), 4.53 - 4.59 (m, 4H), 2.97 (d, J = 11.2 Hz, 6H), 0.27(s, 9H).

[0077] Radiolabeling method

[0079] H2 generated in a cyclotron 18 O middle [ 18 Transfer the [F] fluoride aqueous solution to a V vial in a lead-shielded hot cell, and here [ 18 The [F] fluoride anion was captured on a pre-activated anion exchange resin cartridge (Chromafix-PS-HCO3) by continuous elution using EtOH (5 mL), potassium triflate aqueous solution (KOTf, 90 mg / mL, 5 mL), and deionized (DI) water (5 mL). Then, [ 18Potassium fluoride [F] was eluted from the cartridge into a 2 mL V vial containing an aqueous solution of KOTf (10 mg / mL, 0.45 mL) and K2CO3 (1 mg / mL, 50 μL), and MeCN (0.5 mL). The eluent was dried by azeotropic heating at 110 °C, with two additions of anhydrous MeCN (1.0 mL × 2) during the process. Next, a solution of precursor 262-P (1.7~3.0 mg) in anhydrous N,N-dimethylacetamide (DMA, 0.4 mL) was added to the reaction vial, followed by the addition of a pyridine solution (1 M in DMA, 0.1 mL) and copper(II) triflate (0.2 M in DMA, 67 μL). The reaction mixture was then heated at 110 °C for 20 minutes, diluted with HPLC mobile phase (1.5 mL), and purified by HPLC. The eluent was monitored using a UV detector (254 nm) and a radioactivity detector. 18 The fraction containing F]-262 was collected, diluted with DI water (50 mL), passed through a C18 SepPak (WTWAT051910, Waters), washed with 0.001 N HCl (10 mL), and dried with 10 mL of air. The product was eluted with EtOH (1 mL), diluted with USP-grade saline (3 mL), passed through a sterile membrane filter (0.22 μm), and collected in a sterile vial pre-filled with 7 mL of USP saline and 320 μL of 8.4% NaHCO3 to obtain a solution ready for administration. Analytical HPLC conditions were used in the quality control process.

[0078] Preparation of example compounds Synthesis 1: 2-Chloropyrimidine-4-carbonyl chloride

[0079] [ka]

[0080]

[0080] 12 g of 2-chloropyrimidine-4-carboxylic acid was treated at 0°C with 16.2 mL (2.5 equivalents) of oxalyl chloride in DCM containing a catalytic amount of DMF. The temperature was raised to room temperature and the mixture was stirred for 2 hours. The progress of the reaction was monitored by TLC. Once the reaction was complete, the reaction mixture was evaporated to dryness under N2 to obtain 24 g of crude 2-chloropyrimidine-4-carbonyl chloride, which was used without further purification.

[0081] Synthesis 2: 2-Chloro-N,N-dimethylpyrimidine-4-carboxamide

[0082] [ka]

[0083]

[0081] Crude 2-chloropyrimidine-4-carbonyl chloride was dissolved in DCM and treated with DIPEA (46 mL, 3.5 equivalents) and dimethylamine hydrochloride (11.1 g, 1.8 equivalents) at 0°C. The solution was heated to room temperature and stirred for 3 hours. At this point, the reaction mixture was diluted with DCM and washed with water. The DCM was evaporated, and the resulting mixture was purified with MPLC (35-45% toluene:hexane) to obtain 10 g of the title compound. 1 H NMR (400 MHz, DMSO-d6) δ 8.92 (d, J = 4.8 Hz, 1H), 7.70 (d, J = 4.8 Hz, 1H), 3.00 (s,3H), 2.91 (s,3H).m / z(ESI)(Relative intensity)186.4[M+H] + (100).

[0084] Synthesis 3: (R)-2-(3-hydroxypyrrolidine-1-yl)-N,N-dimethylpyrimidine-4-carboxamide

[0085] [ka]

[0086]

[0082] 10 g of 2-chloro-N,N-dimethylpyrimidine-4-carboxamide was dissolved in acetonitrile and treated with (R)-pyrrolidine-3-ol (7.0 g, 1.5 equivalents) DIPEA (26.1 mL, 3 equivalents). The reaction mixture was stirred at 80°C for 16 hours. The solvent was evaporated, and the resulting crude mixture was purified with MPLC (0.1% MeOH / DCM) to obtain 8 g of the title compound.

[0087] Synthesis 4: N,N-dimethyl-2-(3-oxopyrrolidine-1-yl)pyrimidine-4-carboxamide

[0088] [ka]

[0089]

[0083] (R)-2-(3-hydroxypyrrolidine-1-yl)-N,N-dimethylpyrimidine-4-carboxamide (8 g) was dissolved in DCM and treated with DMP (14.37 g, 1.0 equivalent) at room temperature for 5 hours. The mixture was diluted with DCM, washed with NaHCO3 (aq), evaporated, and the crude product was purified with MPLC (0.1% MeOH / DCM) to obtain 4.0 g of the title compound.

[0090] Synthesis 5: (E,Z)-2-(3-((dimethylamino)methylene)-4-oxopyrrolidine-1-yl)-N,N-dimethylpyrimidine-4-carboxamide

[0091] [ka]

[0092]

[0084] N,N-dimethyl-2-(3-oxopyrrolidine-1-yl)pyrimidine-4-carboxamide (4 g) was dissolved in THF and treated with DMF-DMA (6.1 g, 3.0 equivalents), and stirred at room temperature for 20 hours. The solvent was evaporated, and the crude product was washed with n-hexane to obtain 3.7 g of the title compound.

[0093] Synthesis 6: 2-(2,6-dihydropyrrolo[3,4-c]pyrazole-5(4H)-yl)-N,N-dimethylpyrimidine-4-carboxamide

[0094] [ka]

[0095]

[0085] (E,Z)-2-(3-((dimethylamino)methylene)-4-oxopyrrolidine-1-yl)-N,N-dimethylpyrimidine-4-carboxamide (3.2 g) was dissolved in ethanol (150 mL) and treated with hydrazine hydrate (0.709 g, 2.0 equivalents) at room temperature. The mixture was stirred under reflux for 3 days. The reaction mixture was cooled to room temperature and 2 M HCl (2.4 mL, 6 equivalents) was added. The mixture was stirred for a further 20 hours, then the solvent was evaporated, the residue was dissolved in DCM, washed with NaHCO3 (aq), and purified by MPLC (0.1% MeOH / DCM) to obtain 1.4 g of the title compound.

[0096] Synthesis 7: 2-(2-(3,4-difluorobenzyl)-2,6-dihydropyrrolo[3,4-c]pyrazole-5(4H)-yl)-N,N-dimethylpyrimidine-4-carboxamide (compound 261)

[0097] [ka]

[0098]

[0086] 2-(2,6-dihydropyrrolo[3,4-c]pyrazole-5(4H)-yl)-N,N-dimethylpyrimidine-4-carboxamide (1.5 g) was dissolved in THF at 0°C and treated with NaH (279 mg, 2.0 equivalents). 1-(bromomethyl)-3,4-difluorobenzene (0.74 mL, 1.1 equivalents) and a catalytic amount of tetra-n-butylammonium iodide (TBAI) (214 mg, 0.1 equivalents) were added, and the mixture was stirred at room temperature for 16 hours. The solvent was evaporated, the residue was dissolved in ELISA, and washed with NH4Cl(aq). The crude product was obtained by evaporation as a mixture of positional isomers (2.5 g), which was purified by preparative HPLC to obtain the title compound (280 mg) as a white solid. 1 ¹H NMR (400 MHz, DMSO-d6) δ 8.53 (d, J = 4.4 Hz, 1H), 7.75 (s, 1H), 7.43 - 7.34 (m, 2H), 7.13 (m, 1H), 6.74 (d, J = 4.8 Hz, 1H), 5.34 (s, 2H), 4.58 (s, 2H), 4.55 (s, 2H), 2.99 (s, 3H), 2.96 (s, 3H); NOE between 5.34 and 7.75 (400 MHz, DMSO-d6), confirming the presence of the correct positional isomers; m / z (ESI) (relative intensity) 385.1 [M+H] + (100).

[0099] Synthesis 8: 2-(2-(2,4-difluorobenzyl)-2,6-dihydropyrrolo[3,4-c]pyrazole-5(4H)-yl)-N,N-dimethylpyrimidine-4-carboxamide (compound 262)

[0100] [ka]

[0101]

[0087] 2-(2,6-dihydropyrrolo[3,4-c]pyrazole-5(4H)-yl)-N,N-dimethylpyrimidine-4-carboxamide (1 g) was dissolved in THF at 0°C and treated with NaH (181 mg, 2.0 equivalents). 1-(bromomethyl)-2,4-difluorobenzene (0.6 mL, 1.1 equivalents) and a catalytic amount of tetra-n-butylammonium iodide (TBAI) (140 mg, 0.1 equivalents) were added, and the mixture was stirred at room temperature for 16 hours. The solvent was evaporated, the residue was dissolved in ELISA, and washed with NH4Cl(aq). The crude product was obtained by evaporation as a mixture of positional isomers (2.3 g), which was purified by preparative HPLC to obtain the title compound (275 mg) as a white solid. 1 ¹H NMR (400 MHz, CDCl3) δ 8.49 (d, J = 4.9 Hz, 1H), 7.31 - 7.17 (m, 2H), 6.92 - 6.83 (m, 2H), 6.77 (s, 1H), 5.34 (s, 2H), 4.71 (s, 2H), 4.68 (s, 2H), 3.14 (s, 3H), 3.11 (s, 3H); NOE between 5.34 and 7.75 (400 MHz, DMSO-d6), confirming the presence of the correct positional isomers; 13 C NMR (101 MHz, CDCl3) δ 167.7, 163.0 (dd. J C-F = 237.5, 12.1 Hz), 162.3, 160.5 (dd, J C-F = 237.5, 12.1 Hz), 159.6, 159.2, 155.2, 131.2 (dd, J C-F = 9.8, 5.3 Hz), 123.1, 120.0 (dd, J C-F = 14.9, 3.9 Hz), 119.7, 111.8 (dd, J C-F = 21.4, 3.7 Hz), 107.9, 104.1 (t, J C-F = 25.4 Hz), 49.4, 46.5 , 46.2, 38.7, 35.4; 19F NMR (376 MHz, CDCl3) δ -109.4 (d, J = 7.8 Hz, 1F), -114.3 (m, 1F);HRMS(ESI) C 19 H 19 F2N6O[M+H] + The calculated value for this was 385.1583, and the measured value was 385.1582 (Δ-0.35 ppm).

[0102] Synthesis 9: 2-(2-(3,5-difluorobenzyl)-2,6-dihydropyrrolo[3,4-c]pyrazole-5(4H)-yl)-N,N-dimethylpyrimidine-4-carboxamide (compound 263)

[0103] [ka]

[0104]

[0088] 2-(2,6-dihydropyrrolo[3,4-c]pyrazole-5(4H)-yl)-N,N-dimethylpyrimidine-4-carboxamide was reacted with 1-(bromomethyl)-3,5-difluorobenzene according to the procedure described in Synthesis 8. 1 ¹H NMR (400 MHz, DMSO-d6) δ 8.53 (d, J = 4.4 Hz, 1H), 7.75 (s, 1H), 7.17 (t, J = 4.8 Hz, 1H), 6.97 (s, 1H), 6.95 (s, 1H), 6.74 (d, J = 4.8 Hz, 1H), 5.38 (s, 2H), 4.59 (s, 2H), 4.57 (s, 2H), 2.99 (s, 3H), 2.96 (s, 3H); NOE (400 MHz, DMSO-d6) between 5.38 and 7.78, confirming the presence of the correct positional isomers; m / z (ESI) (relative intensity) 385.1 [M+H] + (100).

[0105]

[0089] Synthesis 10: tert-butyl(Z)-3-((dimethylamino)methylene)-4-oxopyrrolidine-1-carboxylate

[0106] [ka]

[0107]

[0090] Using the method described by Biftu et al. (J. Med. Chem., 57, 3205-3212, 2014), a solution of N-Boc-3-pyrrolidinone (11 g, 59.4 mmol) and N,N-dimethylformamide dimethylacetal (21.2 g, 178.2 mmol, 23.7 mL) in THF (20 mL) was stirred at 70°C for 16 hours. The reaction mixture was cooled to 23°C and the solvent was evaporated under reduced pressure. The residue was triturated with hexane, filtered, washed with hexane, and dried under reduced pressure. The title compound was obtained as a yellow-orange powder (11.7 g, 82%), which was used in the next synthetic step without further purification. 1 H NMR (400 MHz, CDCl3) δ 7.32 (s, 1H), 4.59 (s, 2H), 3.82 (s, 2H), 3.11 (s, 6H), 1.49 (s, 9H).m / z(ESI)(Relative intensity)241.1[M+H] + (100).

[0108]

[0091] Synthesis 11: tert-butyl 4,6-dihydropyrrolo[3,4-c]pyrazole-5(1H)-carboxylate

[0109] [ka]

[0110]

[0092] Using the improved method described by Chung et al. (Org. Process Res. Dev. 2015, 19(11), 1760~1768), 1.33 g of hydrazine hydrochloride (1.33 g, 19.4 mmol) was added at 23°C to a solution of tert-butyl(Z)-3-((dimethylamino)methylene)-4-oxopyrrolidine-1-carboxylate (3.1 g, 12.9 mmol) in EtOH (10 mL). The resulting suspension was stirred at 23°C for 2 hours and then heated at 50°C for 40 hours. The solvent was then removed under reduced pressure, and the residue was diluted with H2O (20 mL). The aqueous phase was extracted with SiO (3 × 10 mL) and CH2Cl2 (3 × 10 mL). The combined organic layers were dried over Na2SO4 and concentrated under vacuum to obtain the title compound (2.42 g, 90%) as an orange oil, which was used in the next step without further purification. m / z(ESI)(relative intensity) 210.1[M+H] + (100).

[0111]

[0093] Synthesis 12: tert-butyl 2-(4-fluorobenzyl)-2,6-dihydropyrrolo[3,4-c]pyrazole-5(4H)-carboxylate and tert-butyl 1-(2,4-difluorobenzyl)-4,6-dihydropyrrolo[3,4-c]pyrazole-5(1H)-carboxylate

[0112] [ka]

[0113]

[0094] To a solution of tert-butyl 4,6-dihydropyrrolo[3,4-c]pyrazole-5(1H)-carboxylate (9 g, 43 mmol) in THF, NaH (1.55 g, 64.5 mmol, 60% in mineral oil) was added at 0°C. After stirring at 0°C for 30 minutes, 1-(bromomethyl)-2,4-fluorobenzene (10.3 g, 50 mmol) was added. The resulting reaction mixture was stirred at 23°C for 30 minutes, then heated to 60°C for 24 hours, and quenched with a saturated solution of NH4Cl (10 mL) at 0°C, and diluted with siRNA (50 mL). The layers were separated, and the organic layers were extracted with H2O (2 × 10 mL) and CH2Cl2 (2 × 10 mL). The combined organic layers were dried over Na2SO4 and concentrated under vacuum to obtain the title compound as a mixture of positional isomers. 1 H NMR (400 MHz, CDCl3) δ 7.24 - 7.29 (m, 2H), 6.85 (m, 2H), 5.22 and 5.29 (s, 2H), 4.29 - 4.74 (m, 4H), 1.49 (s, 9H).m / z(ESI)(Relative intensity)336.6[M+H] + (100).

[0114]

[0095] Synthesis 13: 2-(4-fluorobenzyl)-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole and 1-(2,4-difluorobenzyl)-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole

[0115] [ka]

[0116]

[0096] The mixture obtained from synthesis 12 (12 g, 35 mmol) was dissolved in 1,4-dioxane, and 4N HCl (3 equivalents) in the 1,4-dioxane was added at 0°C. After stirring at room temperature for 3 hours, the mixture was neutralized with NaHCO3(aq), and the title compound was precipitated as a solid and collected under suction. The mixture was purified by flash chromatography to obtain a mixture of the title compound (6 g). 1H NMR (400 MHz, DMSO-d6) δ 7.00 - 7.49 (m, 4H), 5.28 (m, 2H), 3.73 - 3.82 (m, 4H).m / z(ESI)(Relative intensity)336.6[M+H] + (100). m / z(ESI)(relative intensity)236.2[M+H] + (100).

[0117]

[0097] Synthesis 14: 2-(2-(2,4-difluorobenzyl)-2,6-dihydropyrrolo[3,4-c]pyrazole-5(4H)-yl)-N,N-dimethylpyrimidine-4-carboxamide (compound 262)

[0118] [ka]

[0119]

[0098] A mixture (5.4 g) of 2-(2,6-dihydropyrrolo[3,4-c]pyrazole-5(4H)-yl)-N,N-dimethylpyrimidine-4-carboxamide and 1-(2,4-difluorobenzyl)-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole was dissolved in DMF at 0°C and treated with 2-chloro-N,N-dimethylpyrimidine-4-carboxamide (4.0 g, 1 equivalent) and DIPEA (5.9 g, 2.0 equivalent). The mixture was stirred at 100°C for 4 hours. After cooling, the mixture was diluted with DCM, washed with water, evaporated, and the resulting crude material was purified by flash column chromatography to obtain a mixture of positional isomers (6.3 g). 1 H NMR (400 MHz, DMSO-d6) δ 8.53 (d, J = 5.2 Hz, 1H), 7.71 (s, 1H), 7.26 - 7.36 (m, 3H), 7.10 (m, 1H), 6.75 (m, 1H), 5.37 (s, 2H), 4.50 - 4.57 (m, 4H), 2.94 - 2.99 (m, 6H);m / z(ESI)(relative intensity)385.8[M+H] +(100). The compound was purified by supercritical fluid chromatography to obtain the title compound as described in Synthesis 8.

[0120] composition

[0099] The disclosure also includes pharmaceutical compositions comprising a compound of formula (I) as defined above, or a pharmaceutically acceptable salt, solvate, and prodrug thereof, and a pharmaceutically acceptable carrier or diluent.

[0121]

[0100] Pharmaceutical compositions containing the compound of formula (I) described herein may be formulated using one or more pharmaceutically acceptable components, such as pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, colorants, flavoring agents, and sweeteners. When the composition is formulated as individual units, such as capsules, pills, tablets, or ampoules, each unit contains a predetermined amount (dosage) of the compound appropriate for the intended delivery route and indication.

[0122]

[0101] Compositions containing the compound of formula (I) can be prepared by known methods for preparing pharmaceutically acceptable compositions that can be administered to a subject, such that an effective amount of the active substance is mixed with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (2003-20th edition) and the United States Pharmacopeia: National Collection of Medicines (USP24 NF19), published in 1999. Based on this, the composition comprises a solution of the substance contained in a buffered solution having a suitable pH and osmotic pressure equal to that of a physiological solution, in combination with one or more pharmaceutically acceptable vehicles or diluents, though not exclusively.

[0123]

[0102] Suitable carriers, diluents, excipients, etc., can be found in standard pharmaceutical textbooks, e.g., Remington's textbook and the Handbook of Pharmaceutical Excipients, 5th edition, 2005. Formulations can be prepared by any method known in the field of pharmaceutical technology. Such methods involve associating a compound with a carrier that constitutes one or more minor components. Generally, formulations are prepared by homogeneously and closely associating a compound with a carrier (e.g., a liquid carrier, a micronized solid carrier, etc.) and then, if necessary, forming the product. Formulations can be prepared to provide rapid or slow release; immediate, delayed, timed, or sustained release; or a combination thereof.

[0124]

[0103] In some embodiments, the pharmaceutical composition may be in the form of a liquid, a liquid (e.g., aqueous, non-aqueous), a suspension (e.g., aqueous, non-aqueous), an emulsion (e.g., oil in water, water in oil), an elixir, a syrup, a lick, an oral rinse, a drop, a tablet (e.g., including coated tablets), a granule, a powder, a lozenge, a pastil, a capsule (e.g., including hard and soft gelatin capsules), a cachet, a pill, an ampoule, a bolus, a suppository, a pessary, a tincture, a gel, a paste, an ointment, a cream, a lotion, an oil, a foam, a spray, a mist, or an aerosol.

[0125]

[0104] In some embodiments, the pharmaceutical composition may be provided as a patch, bandage, dressing, or similar, impregnated with one or more compounds and optionally one or more other pharmaceutically acceptable components, such as penetration, permeation, and absorption enhancers. In some embodiments, the pharmaceutical composition may be provided in the form of a depot or reservoir.

[0126]

[0105] In some embodiments, the pharmaceutical composition may include a compound of formula (I) dissolved in, suspended in, or mixed with one or more pharmaceutically acceptable components.

[0127]

[0106] In some embodiments, the pharmaceutical composition may include liposomes or other microparticles containing the compound of formula (I), which are designed to target, for example, a specific biological compartment or tissue.

[0128]

[0107] In some embodiments, the pharmaceutical composition is formulated for oral administration (e.g., by ingestion) and is in the form of a liquid, a liquid (e.g., aqueous, non-aqueous), a suspension (e.g., aqueous, non-aqueous), an emulsion (e.g., oil in water, water in oil), an elixir, a syrup, a lick, a tablet, a granule, a powder, a capsule, a cachet, a pill, an ampoule, or a bolus.

[0129]

[0108] In some embodiments, a pharmaceutical composition formulated for oral administration may contain about 0.1 to 250 mg or about 1 to 25 mg of a compound of formula (I) or a subset thereof.

[0109] In some embodiments, the pharmaceutical composition is formulated for sublingual administration and is in the form of tablets, lozenges, pastilles, capsules, or pills.

[0130]

[0110] In some embodiments, the pharmaceutical composition is formulated for oral transmucosal administration and is in the form of a liquid, a solution (e.g., aqueous, non-aqueous), a suspension (e.g., aqueous, non-aqueous), an emulsion (e.g., oil in water, water in oil), an oral rinse, a lozenge, a pastille, a patch, a bandage, a depot, or a reservoir.

[0131]

[0111] Tablets can be prepared by conventional means, for example, by compression or molding using one or more optional components. Compressed tablets can be prepared by compressing a compound in a fluid form such as powder or granules, mixed with one or more optional binders (e.g., povidone, gelatin, gum arabic, sorbitol, tragacanth, hydroxypropyl methylcellulose); excipients or diluents (e.g., lactose, crystalline cellulose, calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, silica); disintegrants (e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethylcellulose); surfactants or dispersants or wetting agents (e.g., sodium lauryl sulfate); preservatives (e.g., methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, sorbic acid); flavorings, flavor enhancers, and sweeteners, in a suitable machine. Wet tablets can be prepared by molding a mixture of powdered compounds moistened with an inert liquid diluent in a suitable machine. Tablets may optionally be coated or scored, and may be formulated to provide sustained or controlled release of the compound in the tablet, for example, by using hydroxypropyl methylcellulose in various ratios to produce a desired release profile. Tablets may optionally have coatings to affect release, for example, enteric coatings to provide release in the part of the intestine other than the stomach.

[0132]

[0112] In some embodiments, the pharmaceutical composition is formulated for intranasal administration, in which case the carrier is a liquid, and the composition may be in the form of a spray nasal solution or nasal solution, or in the form of a liquid suitable for aerosolization and administration by a nebulizer, and comprises an aqueous solution or oily solution of the compound.

[0133]

[0113] In some embodiments, the pharmaceutical composition is formulated for intranasal administration, in which case the carrier is solid, and the composition may be in the form of a dry powder suitable for inhalation.

[0114] In some embodiments, the pharmaceutical composition is formulated for pulmonary administration (e.g., by inhalation or gas infusion therapy) either as a dry powder for administration by a suitable device such as a dry powder inhaler or sprayer, or as a propellant-based formulation administered as an aerosol spray from a pressurized pack using, for example, a pressurized metered-dose inhaler (MDI) device.

[0134]

[0115] In some embodiments, the pharmaceutical composition is formulated for ophthalmic use, for example, as a liquid or gel ophthalmic formulation suitable for administration to the eye.

[0116] In some embodiments, the pharmaceutical composition is formulated for parenteral administration (e.g., by injection) and may contain an aqueous or nonaqueous, isotonic, pyrogen-free sterile liquid (e.g., a solution, a suspension) in which the compound is dissolved, suspended, or otherwise provided (e.g., in liposomes or other microparticles). Such a liquid may further contain other pharmaceutically acceptable components, such as antioxidants, buffers, preservatives, stabilizers, bacteriostatic agents, suspending agents, thickeners, and solutes to make the formulation isotonic with the blood (or other relevant body fluids) of the recipient to whom the formulation is intended. Examples of excipients include, for example, water, alcohol, polyol, glycerol, and vegetable oil. Examples of isotonic carriers suitable for use in such formulations include sodium chloride injection, Ringer's solution, or Ringer's lactate injection. Typically, the concentration of the compound in the liquid is about 1 ng / mL to about 10 μg / mL, for example, about 10 ng / mL to about 1 μg / mL. The formulations may be supplied in sealed containers of single or multi-dose quantities, such as ampoules and vials, and may be stored in a freeze-dried state requiring only the addition of a sterile liquid carrier, such as sterile water for injection, immediately before use. Immediate injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[0135]

[0117] Administration can be achieved as a single dose, continuously, or intermittently throughout the treatment process (for example, in divided doses at appropriate intervals). Single doses or multiple doses may be administered.

[0136]

[0118] In some embodiments, a composition comprising the compound of formula (I) may be administered in a combination therapeutic regimen with another active agent that can be administered simultaneously or sequentially, or via different routes in individually varying dosing schedules. For example, when administered sequentially, the composition may be administered at short intervals (e.g., every 5-10 minutes) or at longer intervals (e.g., every 1, 2, 3, 4 hours or more, or longer if necessary), and the exact dosing regimen will depend on the characteristics of the therapeutic agent. The composition (i.e., the compound of formula (I) and one or more other active agents) may be formulated together into a single dosage form, or the individual agents may be formulated separately and given together in the form of a kit, optionally with instructions for their use.

[0137]

[0119] This disclosure includes compounds of formula (I) as described herein, or compositions comprising compounds as described herein, for example, preferably provided in a suitable container and / or in appropriate packaging; and kits including instructions for use, for example, written instructions on how to administer the compound or composition. The written instructions may include a list of indications for which the active ingredient is an appropriate treatment.

[0138]

[0120] In some embodiments, the kit further comprises one or more (e.g., 1, 2, 3, 4) additional agents or therapeutic agents in a separate container from the compound of formula (I), or in the same container.

[0139] Treatment method

[0121] AMPA-mediated excitotoxicity is involved in neurological diseases and disorders, including neurodegenerative disorders such as ALS (amyotrophic lateral sclerosis), in which motor neurons are primarily affected. Post-translational processing of GluR2 mRNA is altered in spinal motor neurons derived from ALS patients, resulting in a higher proportion of calcium-permeable AMPA receptors containing GluR with unedited Q / R sites.

[0140]

[0122] Excessive levels of glutamate that act primarily via NMDAR and AMPAR promote Ca 2+ influx, which can result in excitotoxicity under pathological conditions including ischemia, trauma, hypoglycemia and epileptic seizures. Glutamate receptor-mediated excitotoxicity is also involved in demyelination, neuron / oligodendrocyte loss and axonal damage that cause neurological disorders in multiple sclerosis (MS). Mice with Gria3 mutations that do not express functional GluR3 AMPA receptor subunits are resistant to demyelination and neurological sequelae in the EAE mouse model of autoimmune encephalomyelitis (EAE), the most common mouse model of MS. In contrast, mGluR4 knockout mice are more susceptible to the EAE model.

[0141]

[0123] In an embodiment, the present disclosure provides a method of modulating AMPA receptor signaling using a compound of Formula (I). In an embodiment, the compound of Formula (I) modulates AMPA receptor signaling in vitro, ex vivo, or in vivo. In an embodiment, the compound of Formula (I) modulates AMPA receptor signaling by interfering with or reducing the GluR2-GAPDH interaction. In an embodiment, the compound of Formula (I) inhibits AMPAR-mediated neurotoxicity, preferably without impairing AMPAR-mediated neurotransmission.

[0142]

[0124] In an embodiment, the present disclosure provides a method of inhibiting the interaction between GluR2 and GAPDH, comprising contacting a cell, preferably a neuron, with a compound of Formula (I). In some embodiments, the contacting is in vitro or ex vivo.

[0143]

[0125] In an embodiment, the present disclosure provides a method of inhibiting glutamate receptor-mediated excitotoxicity without impairing AMPAR-mediated neurotransmission, comprising contacting a cell, preferably a neuron, with a compound of Formula (I). In some embodiments, the contacting is in vitro or ex vivo.

[0144]

[0126] In some embodiments, the present disclosure provides a method for inhibiting calcium influx of glutamate receptor-mediated excitotoxicity without impairing AMPAR-mediated neurotransmission, comprising contacting cells, preferably neurons, with a compound of formula (I). In some embodiments, the contact is in vitro or ex vivo.

[0145]

[0127] In an embodiment, the Disclosure also provides a method for treating a disease or condition in which modulation of AMPA receptor signaling is beneficial, preferably by inhibiting or reducing glutamate receptor-mediated excitotoxicity without impairing AMPA-mediated neurotransmission, comprising administering an effective amount of a compound of formula (I) as defined herein to a subject. In another embodiment, a method is provided for treating a disease or condition in which improvement is achieved by inhibiting glutamate receptor-mediated excitotoxicity without impairing AMPA-mediated neurotransmission, comprising administering an effective amount of a compound of formula (I) as defined herein to a subject.

[0146]

[0128] Also provided is the use of a compound of formula (I) for the preparation of a drug, wherein the compound or drug is for the treatment of a disease or condition in which modulation of AMPA receptor signaling that inhibits or reduces glutamate receptor-mediated excitotoxicity is beneficial. Also provided is the use of a compound of formula (I) for the preparation of a drug, wherein the compound or drug is for the treatment of a disease or condition in which the compound or drug is improved by inhibiting glutamate receptor-mediated excitotoxicity without impairing AMPA-mediated neurotransmission.

[0147]

[0129] In some embodiments, the disease or condition involves inappropriate AMPA receptor signaling. In some embodiments, the disease or condition is a neurological disorder or condition in which modulation of the interaction between GluR2 and glyceraldehyde-3-phosphate dehydrogenase is beneficial, or a disorder or condition in which modulation of AMPA receptor signaling that inhibits or reduces glutamate receptor-mediated excitotoxicity is beneficial. In some embodiments, the neurological disorder or condition is characterized by excessive glutamate receptor-mediated excitotoxicity and includes, but is not limited to, GluR2 / AMPAR neurotoxicity, including neuroinflammation, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), stroke, and epilepsy.

[0148]

[0130] Accordingly, the Disclosure provides the use of a compound of formula (I) in a method for treating neurological diseases or conditions characterized by GluR2 / AMPAR neurotoxicity, including multiple sclerosis (MS), spasticity associated with multiple sclerosis, or amyotrophic lateral sclerosis (ALS), wherein the method comprises administering an effective amount of the compound of formula (I) to a subject. In some embodiments, the Disclosure provides the use of a compound of formula (I) in the preparation of agents for treating neurological diseases or conditions such as multiple sclerosis (MS), spasticity associated with multiple sclerosis, or amyotrophic lateral sclerosis (ALS).

[0149]

[0131] The Disclosure provides the use of a compound of formula (I) in a method for treating neurodegenerative disorders selected from Parkinson's disease, Alzheimer's disease, dementia, Huntington's disease, and amyotrophic lateral sclerosis (ALS), wherein the method comprises administering an effective amount of the compound of formula (I) to a subject. In some embodiments, the Disclosure provides the use of a compound of formula (I) in the preparation of agents for treating neurodegenerative disorders selected from Parkinson's disease, Alzheimer's disease, dementia, Huntington's disease, and amyotrophic lateral sclerosis (ALS).

[0150]

[0132] In some embodiments, the use of a compound of formula (I) in a method for treating neuroinflammation is also provided, wherein the method comprises administering an effective amount of the compound of formula (I) to a subject. In some embodiments, the present disclosure provides the use of a compound of formula (I) in the preparation of a pharmacopoeia for treating neuroinflammation.

[0151]

[0133] In this embodiment, the use of the compound of formula (I) in a method for treating neuropathic pain, inflammatory pain, pain associated with multiple sclerosis, pain of an unknown origin, pain associated with cancer or cancer chemotherapy, pain associated with diabetic neuropathy, or pain associated with surgery is also provided.

[0152]

[0134] In a part, the use of the compound of formula (I) in a method for treating eating disorders, such as cachexia or anorexia associated with cancer chemotherapy or cachexia or anorexia associated with HIV / AIDS is also provided.

[0153]

[0135] In some embodiments, the use of compounds of formula (I) in methods for treating rheumatoid arthritis, inflammatory bowel disease, psoriatic arthritis, chronic obstructive pulmonary disease, ankylosing spondylitis, immune responses resulting in organ rejection or graft rejection after transplantation, or psoriasis is also provided.

[0154]

[0136] In an embodiment, the use of compounds of formula (I) in a method for treating anxiety, including panic disorder, social anxiety disorder, and post-traumatic stress disorder is also provided. In an embodiment, the mental disorder is mania or schizophrenia.

[0155]

[0137] In any embodiment of the treatment method described above, the route of administration may be either an oral route or a parenteral route.

[0138] In accordance with the embodiments of the treatment method described above, the subjects to whom the compound of formula (I) is administered are human subjects. In some embodiments, a human is an adult, child, or elderly human, as is understood by physicians, for example, as defined by the U.S. Food and Drug Administration. In some embodiments, a human subject is a person who requires treatment for the disease, disorder, or condition being treated, for example, a person who has received a clinical diagnosis of the disease, disorder, or condition.

[0156]

[0139] According to the treatment method described above, the effective dose of the compound of formula (I) is in the range of approximately 50 μg to approximately 250 mg per kg of body weight per day (more typically approximately 100 micrograms (μg) to approximately 10 mg or approximately 500 micrograms (μg) to approximately 5 mg). If the compound is a salt, ester, amide, prodrug, or similar, the amount administered is calculated based on the parent compound, so the actual weight used increases proportionally.

[0157]

[0140] A method of radioisotope therapy (RIT, or "radiotherapy") using a radiolabeled compound of formula I is also provided.

[0158] Imaging methods

[0141] Despite a considerable amount of basic research demonstrating the role of AMPA in neurological disorders, clinical interpretations have been partially limited by unsuitable imaging tools for detecting and quantifying AMPA receptors or their surrogates in the living human brain. The present invention addresses the need for such imaging tools by providing a labeled compound of formula I.

[0159]

[0142] The labeled compound of formula I may be useful in optical imaging, such as positron emission tomography (PET) imaging, gadolinium contrast-enhanced imaging (cMRI), or fluorescence imaging. The labeled compound of formula I may also be useful in preclinical imaging studies.

[0160]

[0143] PET is a molecular imaging technology useful for visualizing biochemical and physiological processes, and is widely used in clinical imaging and diagnosis. PET is also a useful tool in drug development, as it can be used in studies of pharmacokinetics, receptor occupancy and target engagement, target validation, and drug metabolism.

[0161]

[0144] In an embodiment, there is provided a compound of Formula I for use in preclinical imaging of AMPA receptors or surrogates thereof in the brain 11 C-labeled compound.

[0145] In an embodiment, there is provided a compound of Formula I for use in PET imaging 18 F-labeled compound. In an embodiment, there is provided a compound of Formula I for use in non-PET imaging 19 F-labeled compound.

[0162]

[0146] There is also provided a method for imaging an AMPA receptor or a surrogate thereof in the brain of a subject in vivo, the method comprising administering a labeled compound of Formula I to the subject, wherein the compound comprises 11 C, 18 F, 19 a carbon or fluorine isotope selected from F, 123 I, 131 I or 3 H. In an embodiment, the labeled compound of Formula I is 11 C-261, 11 C-262, and 11 C-263; or 18 F-261, 18 F-262, and 18 F-263. In an embodiment, the labeled compound of Formula I is administered orally, intraperitoneally, or intravenously. In an embodiment, the subject is a rodent, dog, rabbit, or non-human primate. In an embodiment, the subject is a human.

[0163] Imaging Examples

[0147] Dynamic PET imaging was performed in healthy adult Sprague Dolly rats and Dorr mice. 11 Representative PET images of C-262 at 0-5 minutes are shown in Figure 7A, which show the localization of the labeled compound to the brain. [ ] 11 Following bolus intravenous (iv) injection of [C]-262, baseline cerebral uptake reached a maximum of 3.6 standardized uptake units (SUV) in the whole brain of rats and a maximum of 2.05 SUV in the whole brain of mice. After 5 minutes, the labeled compound [ 11 [C]-262 rapidly decreased to 1.7 SUV and 1.0 SUV in rats and mice, respectively (Figure 7B). After 60 minutes, the labeled compound [ 11 [C]-262 was further reduced to 0.74 SUV and 0.5 SUV in rats and mice, respectively. 11 The rapid uptake and rapid elimination of [C]-262 make this compound a suitable candidate as a brain PET contrast agent for measuring AMPA and its changes in the brain.

[0164] Biological evaluation

[0148] As will be discussed in detail in the following sections, compounds within the range of Formula I exhibited specific binding to the AMPA GluR2 subunit and also showed therapeutically relevant biological activities, including the prevention of AMPA-mediated excitotoxicity in a Cuprison-fed mouse model of multiple sclerosis (MS) and neurological function rescue in experimental autoimmune encephalomyelitis (EAE) mice.

[0165]

[0149] Representative compounds described herein were first tested for in vitro metabolic stability and exposure to the central nervous system, particularly the brain. In vitro studies were also performed to evaluate the ability of compounds to inhibit AMPA-mediated neurotoxicity. Several compounds, 261, 262, and 263, were identified that exhibited significantly improved predicted CNS penetration, determined from in vitro models [low efflux / high permeability], and high metabolic stability [rat / human microsomes] while maintaining nanomolar potency for inhibiting AMPA-mediated neurotoxicity. Representative compounds 261 and 262 also showed improved unbound brain concentrations and in vivo exposure in rat models compared to reference compounds. Furthermore, compounds 261 and 262 had considerably better oral bioavailability compared to the reference. Finally, the disease-specific efficacy of compound 262 was evaluated using two in vivo models of neurological disorders: an experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis and an experimental cuprizon-induced behavioral deficit model. As will be discussed in more detail below, compound 262 demonstrated dose-dependent efficacy in animal models.

[0166]

[0150] In summary, the results presented herein demonstrate that the compounds in this disclosure exhibit not only very high permeability with low nanomolar potency for inhibiting AMPA-mediated neurotoxicity and low efflux resulting in excellent in vivo CNS exposure, but also excellent oral bioavailability, low clearance, and dose-dependent efficacy in animal model systems of neurological diseases and disorders.

[0167]

[0151] The small molecule AMPAR modulator is described in PCT / IB20 / 51920 (filed March 5, 2020). However, the lead compound ZCN155, which inhibited AMPAR-mediated neurotoxicity with a potency of 35 nM, was found by the inventors to have insufficient exposure to the central nervous system (CNS). The inventors found that ZCN155 is actively removed from the brain by the P-gp efflux transporter, and this was considered to be the main cause of its insufficient CNS exposure. To test this, the permeability and efflux of ZCN155 were evaluated in a blood-brain barrier model cell line (details of the assay can be seen in the Methods section below). Two other reference compounds, ZCN210 and ZCN217, were selected for comparative testing. The results are shown in Table 1. The data showed that the reference compound ZCN217 had significantly better permeability and improved emission ratio compared to ZCN155 or ZCN210 (hereinafter referred to as "REF155" and "REF210" in this specification).

[0168] [Table 1]

[0169]

[0152] In addition to increased permeability and decreased efflux, another means of improving CNS exposure is to increase metabolic stability. Metabolic stability was measured by determining the compound elimination rate when incubated in the presence of human and rat liver microsomes, as described in the Methods section below. The ability of the compounds to inhibit AMPA-mediated neurotoxicity was simultaneously evaluated, as increased metabolic stability had to be achieved without loss of biological potency, and it was impossible to accurately predict the effect of predetermined substituent changes. Biological potency was determined based on the ability of the compounds to inhibit AMPA-mediated neurotoxicity, as determined using an in vitro cytotoxicity assay (also described below). The results of the metabolic stability and biological potency assays are shown in Table 2. In the table, "RLM" and "HLM" refer to rat and human liver microsomes, respectively.

[0170]

[0153] The data show unpredictable effects from simply changing the position of the halogen substituent on the benzyl moiety. As exemplified by compounds 236 and 264, changing the position of the fluoro moiety on REF155 or REF210 resulted in an unacceptable loss of biological potency. Thus, REF155 had a potency of 35 nM, which decreased to less than one-hundredth, to 5000 nM, by changing 4-fluoro to 3-fluoro (compound 236). Similarly, REF210 had a potency of 82 nM, which decreased to almost one-tenth, to 733 nM, by changing 4-fluoro to 2-fluoro (compound 264).

[0171]

[0154] REF217 exhibited significantly better permeability and improved efflux ratio compared to both REF155 and REF210, making it a better candidate for further development. However, changing the halogen position on the benzyl moiety from 4-fluoro to 2-fluoro (compound 254) or 3-fluoro (compound 237), as shown below, also reduced potency, though not as dramatically as seen in compounds 236 and 264. Here, potency was reduced to approximately one-sixth in each case. This change also failed to produce increased metabolic stability compared to the parent compound REF217, instead showing an unexpected decrease in rat liver microsomes (compound 237), as well as in both human and rat microsomes (compound 254). The inadequate performance of the remaining compounds in Table 2 with respect to metabolic stability indicates that changing the position of the halogen substituent on the benzyl moiety, adding a second halogen, or changing from fluoro to chloro was insufficient, respectively, to obtain molecules that exhibited the desired increase in metabolic stability while also retaining biological potency.

[0172] [Table 2-1]

[0173] [Table 2-2]

[0174]

[0155] In exploring the development of compounds that possess the CNS exposure required to be useful as CNS-targeted therapeutics, particularly compounds with high brain exposure, the present inventors have unexpectedly discovered that certain compounds of Formula I exhibit desirable in vivo biological potency and high CNS exposure following both intravenous and oral administration in a rat system. The superior properties of these compounds of Formula I are driven by their low efflux, high permeability, high metabolic stability, and high bioavailability, all of which are significantly and unexpectedly superior to reference compounds.

[0175]

[0156] The biological properties of these compounds are shown in Table 3. Surprisingly, the addition of two fluorine groups to REF210 resulted in considerable improvements in both permeability and efflux, which was a clear and unprecedented result. While fluorine can be added as a substituent to improve metabolic stability, it is not known to be expected to affect other pharmacokinetic properties. However, this modification did not produce uniform or predictable results. Instead, Compound 262 showed significant improvements in both permeability and efflux, while Compound 261 showed improvements only in efflux, and Compound 263 showed no improvement in permeability at all and only a slight improvement in efflux.

[0176]

[0157] The dramatic improvement in metabolic stability observed for each of Compounds 261, 262, and 263 compared to the reference compounds, combined with the improved potency, was also unexpected. As discussed above, the addition of fluorine groups to other reference compounds almost without exception resulted in a considerable decrease in metabolic stability. Additionally, since the addition of a 2-fluoro moiety significantly reduced the potency of REF210, the fact that adding an additional fluoro moiety not only is tolerated but also results in increased potency was unexpected and unpredictable.

[0177] [[ID=17】Table 3

[0178]

[0158] These in vitro data predicted that Compound 262 has significantly improved unbound brain concentration and exposure compared to the reference compound. This was tested in a rat model and found to be true. Furthermore, it was even more unexpectedly found that both Compound 261 and Compound 262 have considerably better oral bioavailability than both REF155 and REF217. These data are summarized in Table 4.

[0179] [Table 4]

[0180]

[0159] Based on these in vitro and rat pharmacokinetic data, Compound 262 was selected for evaluation in two animal models of neurological diseases, the experimental autoimmune encephalomyelitis (EAE) model for multiple sclerosis and the experimental cuprizone-induced behavioral deficit model.

[0181]

[0160] An experimental autoimmune encephalomyelitis (EAE) model for multiple sclerosis was one model system used to evaluate the in vivo efficacy of 262 representative compounds. Chronic relapsing EAE was induced as follows: Female C57BL / 6 mice (8-12 weeks old) were purchased from Charles River Laboratories. 200 μL of recombinant MOG35-55 (200 μg, Biomatik Corporation, Cambridge, ON, Canada), emulsified in Freund's complete adjuvant (Sigma, Oakville, ON, Canada) and supplemented with 4 mg / mL Mycobacterium tuberculosis (H37Ra strain, BD Biosciences, Mississauga, ON, Canada), was subcutaneously injected (50 μL per site) at four different sites on the back of the mice. 200 nanograms of pertussis toxin (List Biological Laboratories, Inc., Campbell, CA, USA) were administered intraperitoneally (ip) at 0 and 2 days after immunization. Mice were evaluated daily for clinical signs of disease, starting on the day of immunization. Clinical scoring was performed according to the following criteria: 0, asymptomatic; 0.5, distal caudal paresis; 1, complete caudal paralysis; 1.5, caudal paresis and mild hindlimb paresis; 2, unilateral severe hindlimb paresis; 2.5, bilateral severe hindlimb paresis; 3, complete bilateral hindlimb paralysis; 3.5, complete bilateral hindlimb paralysis; 4, complete paralysis (quadriplegia). All animal procedures used complied with the approved Centre for Addiction and Mental Health Animal Research Ethics Committee protocols.

[0182]

[0161] As shown in Figure 1, treatment with compound 262 improved the clinical score of EAE mice. These results indicate that administration of compound 262 starting 11 days post-immunization (1-2 days after the onset of symptoms) significantly improved neurological function in a dose-dependent manner.

[0183]

[0162] The experimental cuprizon diet-induced demyelination model was a second model system used to evaluate the in vivo efficacy of compound 262, a representative compound. This model is induced by providing free access to a high-protein diet containing 0.2% cuprizon, purchased from Envigo (Indianapolis, IN, USA). Control animals are given a high-protein diet without cuprizon. Female 7-week-old C57BL / 6 mice were switched from their standard diet to either a cuprizon or control diet. Oral administration of 262 was started after 2 weeks on the cuprizon diet. Animals were tested for behavioral measures of motor function and then sacrificed for myelin evaluation after 6 weeks on the cuprizon diet. Mice given cuprizon exhibited motor dysfunction and myelin loss.

[0184]

[0163] Figure 2A illustrates a protocol for a cuprizon model in which myelin loss is induced using a high-protein diet supplemented with 0.2% cuprizon, which typically peaks after 6 weeks of the diet.

[0185]

[0164] Figure 2B shows that cuprizon-induced behavioral deficits were reduced by treatment with compound 262, as measured by an open-field test, and displays the distance traveled and movement speed measured over one hour in an empty arena.

[0186]

[0165] Figure 2C shows that cuprizon-induced behavioral deficits were remissioned by treatment with compound 262, as measured by the time to fall and the rotations per minute (rpm) at the time of fall in the rotarod test, which includes mice that maintain balance on a rotating axel that gradually accelerates until they fall.

[0187]

[0166] As shown in Figure 3A, compound 262 promotes the recovery of cuprizone-induced demyelination of the medial and lateral corpus callosum, as measured in vivo using Fluoromyelin® green staining.

[0188]

[0167] As shown in Figure 3B, compound 262 reverses the cuprizone-induced decrease in the number of myelinated axons in the corpus callosum, as measured in vivo using a transmission electron microscope.

[0168] As shown in Figure 3C, compound 262 reverses the cuprizone-induced decrease in mean myelin thickness of myelinated axons in the corpus callosum, as measured in vivo using a transmission electron microscope.

[0189]

[0169] As shown in Figure 3D, compound 262 reverses the cuprizone-induced increase in the ratio of inner diameter to outer diameter (g ratio) of axons in the corpus callosum, as measured in vivo using a transmission electron microscope.

[0190]

[0170] As shown in Figure 4, compound 262 promoted oligodendrocyte survival in the cuprizone model.

[0171] As shown in Figure 5, compound 262 reduces cuprizone-induced inflammation in vivo.

[0191]

[0172] As shown in Figure 6A, the input-output curves of the stimulation intensity fEPSP amplitude did not show a significant difference between the compound 262 treatment group and the control group.

[0173] As shown in Figure 6B, the input-output relationship of fEPSP with respect to the prefiber salvo amplitude did not show a significant difference between the compound 262 treatment group and the control group.

[0192]

[0174] In summary, the data shown in Figures 6A and 6B indicate that treatment with compound 262 does not alter basic neurotransmission.

[0193] In vitro method In vitro metabolic stability

[0175] Metabolic stability was measured by determining the rate of compound disappearance when incubated in the presence of human or rat liver microsomes. Liver microsomes are prepared from the endoplasmic reticulum of liver cells and are the primary source of the most important enzymes involved in drug metabolism (cytochrome P450). Testing drug stability in the presence of liver microsomes is accepted as a valuable model that enables rapid prediction of in vivo drug stability.

[0194] Protocol Overview:

[0176] Human or rat liver microsomes were obtained from commercial sources. Test compounds (3 μM) were incubated with pooled liver microsomes (male and female). Samples were incubated for 45 minutes, removed at 5 time points, and the test compounds were analyzed by LC-MS / MS.

[0195]

[0177] Microsomes (final protein concentration 0.5 mg / mL), 0.1 M phosphate buffer pH 7.4, and test compound (final concentration 3 μM; diluted from 10 mM stock solution to a final DMSO concentration of 0.25%) were incubated at 37°C, then NADPH (final concentration 1 mM) was added to initiate the reaction. The final incubation volume was 25 μL. Control incubations were included for each compound tested, in which 0.1 M phosphate buffer pH 7.4 was added instead of NADPH. Control compounds testosterone and 7-hydroxycoumarin were included in each experiment, and all incubations were performed singly for each compound.

[0196]

[0178] Each compound was incubated for 0, 5, 15, 30, and 45 minutes. Controls (with NADPH omitted) were incubated for 45 minutes only. The reaction was stopped by adding 50 μL of methanol containing internal standard at the appropriate time points. The incubation plate was centrifuged at 2500 rpm for 20 minutes at 4°C to precipitate the protein.

[0197] Quantitative Analysis:

[0179] After protein precipitation, the sample supernatant was mixed in cassettes of up to four compounds and analyzed using standard LC-MS / MS conditions.

[0198] Data analysis:

[0180] The slope of the line was determined from the plot of the natural logarithm of the peak area ratio (i.e., the ratio of compound peak area to internal standard peak area) against time. Subsequently, the half-life and intrinsic clearance were calculated using the following equation: The rate of disappearance constant (k) = (-gradient). Half-life (t 1 / 2 )(min)=0.063 / k. Intrinsic clearance (CL) int )(μL / min / 1 million cells)=(Vx0.693) / t 1 / 2 . In the formula, V = incubation volume (μL / mg of microsomal protein).

[0199] In vitro permeability and discharge

[0181] Maidin Darby canine kidney (MDCK) cells are an epithelial cell line of canine kidney origin. These cells can be stably transfected to express active P-glycoprotein (MDCK-MDR1) and are ideal for testing drug efflux. Permeability is measured by adding the test compound to either the apical or basolateral side of a confluent monolayer of MDCK-MDR1 cells and monitoring the appearance of the test compound to the opposite side of the monolayer using LC-MS / MS. When performing a bidirectional assay, the efflux ratio (ER) is calculated from the ratio of BA and AB permeability.

[0200] Protocol Overview:

[0182] Use MDCK-MDR1 cells obtained from NIH (Rockville, MD, USA) at passage 6-30. Arrange the cells on a Millipore Multiscreen Transwell plate in a 3.4x10⁶ format. 5 cells / cm 2Seeds are seeded. Cells are cultured in DMEM, and the medium is changed on day 3. A permeability test is performed on day 4. Cell culture and assay incubation are carried out at 37°C in a 5% CO2 atmosphere with a relative humidity of 95%. On the day of assay, a monolayer is prepared by rinsing both the apical and basal outer surfaces twice with Hanks equilibrium salt solution (HBSS) at the desired pH and warmed to 37°C. Then, the cells are incubated with HBSS at the desired pH in both the apical and basal outer compartments for 40 minutes to stabilize physiological parameters.

[0201]

[0183] Prepare the drug solution by diluting the test compound with assay buffer to a final test compound concentration of 10 μM (final DMSO concentration 1% volume / volume). The fluorescent integrity marker Lucifer Yellow is also included in the drug solution. Prepare the analytical standard from the DMSO dilution of the test compound, transfer it to buffer, and maintain a 1% volume / volume DMSO concentration.

[0202]

[0184] While a standard assay buffer consists of additional HBSS pH 7.4, various other buffers and pH values ​​can be used to evaluate AB permeability. Remove the HBSS from the apical compartment and replace it with the test compound dosing solution. Then, place the apical compartment insert in a companion plate containing fresh buffer (containing 1% volume / volt DMSO). For BA permeability evaluation, remove the HBSS from the companion plate and replace it with the test compound dosing solution. Add fresh buffer (containing 1% volume / volt DMSO) to the apical compartment insert and then place it in a companion plate.

[0203]

[0185] After 60 minutes, separate the apical compartment insert and companion plate, and dilute the apical and basal outer samples for analysis.

[0186] Evaluate the permeability of the test compound in a dual assay. A compound with known permeability characteristics is used as a control in each assay plate. The test compound and control compound are quantified by LC-MS / MS cassette analysis using a 7-point calibration with appropriate sample dilutions. Standard analytical conditions are used. Determine the starting concentration (C0) from the drug solution, and calculate the experimental recovery from C0 and the concentrations in both the apical and basal outer compartments.

[0204]

[0187] The integrity of the monolayer during the experiment is confirmed by monitoring Lucifer Yellow transmission using fluorescence analysis. If the monolayer is damaged, Lucifer Yellow transmission is high. app The value in one well is higher than a predetermined threshold, but the P value derived for the test compound or marker compound in that well is higher. app If the results are qualitatively similar to those determined in the remaining replication wells (within the Lucifer Yellow threshold), the cell monolayer may be considered acceptable based on the scientific judgment of the responsible scientist. If this is not the case, the results from the affected monolayer are excluded, the n=1 result is reported, or the compound may be retested. Lucifer Yellow P app If the value exceeds the threshold in both replication wells for the test compound, the compound is retested. If this recurs repeatedly in both wells, toxicity or intrinsic fluorescence of the test compound is inferred. In this case, no further experiments are performed.

[0205] Data analysis:

[0188] The permeability coefficient (Papp) for each compound is calculated using the following equation.

[0206]

number

[0207]

[0189] In the formula, dQ / dt is the rate of drug permeation through the cell, C0 is the donor compartment concentration at time zero, and A is the area of ​​the cell monolayer. C0 is obtained from the analysis of the drug solution.

[0208]

[0190] For bidirectional experiments, the emission ratio (ER) is calculated from the average AB and BA data. This is because

[0209]

number

[0210] It is derived from this.

[0191] Two control compounds, propranolol (highly permeable) and prazosin (P-glycoprotein substrate), are screened in parallel with the test compound.

[0211] Inhibition of AMPA-mediated neurotoxicity

[0192] The ability of compounds to inhibit AMPA-mediated neurotoxicity was determined using the CellTox® Green Cytotoxicity Assay (G8743) kit from Promega. In this assay, GAPDH and GluR2 are expressed in HEK-293 cells. AMPA (500 μM) was added to induce the AMPA-mediated cell death signaling cascade. This is initiated by GAPDH-GluR2 complex formation and subsequent nuclear translocation of the complex, which activates the p53-mediated cell death pathway. The presence of compounds that can block the dimerization of GAPDH and GluR2 prevents nuclear translocation and cell death; therefore, this assay is an effective measure of the ability of compounds to prevent AMPA-mediated neurotoxicity and thus demonstrates therapeutic efficacy in the treatment of MS.

[0212]

[0193] The assay was performed according to the manufacturer's instructions. Briefly, 293 cells stably expressing GluR1 and GluR2VENUS were transfected with pcDNA-RluC-GAPDH. AMPA (500 μM) was added to induce cell death. CTZ (100 μM) was added to prevent AMPAR desensitization. The protective effect of the candidate molecule was tested by adding it before AMPA treatment. Untreated cells and the AMPA-treated group were used as negative and positive controls, respectively. CellTox® Green reagent (approximately 5x) was then added to achieve a final concentration of 1x, and the cells were incubated at room temperature for 15 minutes (shielded from ambient light). Fluorescence intensity at 485-500 nmEx / 520-530 nmEm was then measured and calculated as a percentage value for the positive control. The concentration at which 50% of cell death was prevented (IC) was calculated. 50 The following was determined for each compound:

[0213] In vivo method CNS exposure test

[0194] The plasma pharmacokinetics and brain distribution of the compound were investigated in male Sprague Dolly rats after a single intravenous and oral administration.

[0214] Protocol Overview:

[0195] 54 male rats were divided into two groups, Group 1 (n=27) and Group 2 (n=27), using a sparse sampling design of 3 rats per time point. A liquid formulation of the compound at a dose of 5 or 10 mg / kg was administered intravenously to the animals in Group 1 as a slow bolus injection via the tail vein. The animals in Group 2 were administered a liquid formulation of the compound at a dose of 5 or 10 mg / kg via the oral route.

[0215]

[0196] For both groups, the formulation vehicles were 5% volume / volt NMP, 5% volume / volt Solutol HS-5, and 90% volume / volt physiological saline.

[0197] Blood samples (approximately 120 μL) were collected from groups of three rats under mild isoflurane anesthesia (Surgivet®) from the retro orbital plexus at pre-drug administration (PO only), 0.08 (IV only), 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after administration. In addition, brain samples were collected from three rats per time point, along with terminal blood samples, at pre-drug administration (PO only), 0.08 (IV only), 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after administration. Immediately after blood collection, plasma was collected by centrifugation at 4000 rpm for 10 minutes at 4°C, and the samples were stored at -70±10°C until bioanalysis. After blood collection, the animals were immediately slaughtered, the abdominal vena cava was incised, and the entire body was perfused from the heart using 10 mL of physiological saline.

[0216]

[0198] Brain samples were collected from each individual rat at specific time points in a set of three rats. After separation, tissue samples were rinsed three times in ice-cold saline (5–10 seconds per rinse, using approximately 5–10 mL of saline in a disposable Petri dish for each rinse) and dried on blotting paper. Tissue samples were homogenized using ice-cold phosphate-buffered saline (pH 7.4). The total homogenate volume was three times the tissue weight. All homogenates were stored below -70±10°C until bioanalysis.

[0217]

[0199] All samples were processed for analysis by protein precipitation and analyzed using appropriate LC-MS / MS methods (LLOQ = 1.02 ng / mL for plasma and brain). Pharmacokinetic parameters of plasma and brain were estimated using the non-compartmental analysis tool of Phoenix® WinNonlin software (version 8.0).

[0218]

[0200] As used herein, the term "halo" refers to a halogen atom, and includes fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).

[0201] The abbreviation "Me" in the chemical structure refers to a methyl substituent.

[0219]

[0202] The term “pharmaceutically acceptable salt” means, for example, a salt that retains the desired biological activity of the compounds of this disclosure and does not impart any undesirable toxicological effects to them; it may also mean an acid addition salt or a base addition salt.

[0220]

[0203] As used herein, the term “solvate” means a compound or a pharmaceutically acceptable salt thereof in which molecules of a suitable solvent are incorporated into the crystal lattice. A suitable solvent is one that is physiologically acceptable at the dosage administered. Examples of suitable solvents include ethanol and water. When water is the solvent, the molecule is called a “hydrate.” The formation of solvates varies depending on the compound and the solvate. Generally, solvates are formed by dissolving a compound in a suitable solvent and isolating the solvate by cooling or using a poor solvent. Solvates are usually dried or azeotropically formed under ambient conditions.

[0221]

[0204] In embodiments of the present disclosure, compounds may have chiral centers. These compounds exist as enantiomers. If a compound has more than one chiral center, they may exist as diastereomers. Naturally, all such isomers and mixtures thereof in any ratio are included within the scope of the present disclosure and are generally known as stereoisomers. Naturally, the stereochemistry of a compound may be as shown in any given compound listed herein, but such a compound may contain a certain amount (e.g., less than 20%, preferably less than 10%, more preferably less than 5%) of a compound of the present disclosure having other possible stereochemistrys. For example, a compound of the present disclosure shown without specifying any stereochemistry is understood to be a racemic mixture (i.e., containing equal amounts of each possible enantiomer or diastereomer). Naturally, however, all enantiomers and diastereomers, including mixtures thereof in any ratio, are included within the scope of the present disclosure.

[0222]

[0205] As used herein, the terms “effective dose,” “therapeutic effective dose,” or “pharmaceutical effective dose” mean the amount that is effective in the dosage and duration required to achieve the desired result. For example, in a situation where a subject has a disease or condition in which modulation of AMPA receptor signaling, such as pain, is beneficial, the effective dose is, for example, the amount that results in some reduction, remission, relief, and / or decrease of the amount of pain experienced by the subject.

[0223]

[0206] The effective dose may vary depending on factors such as the patient's condition, age, sex, and / or weight. The amount of a given compound equivalent to such a dose will vary depending on various factors such as the drug or compound given, formulation, route of administration, condition, type of disease or disorder, identity of the person being treated, and concomitant treatments, but it can still be routinely determined by those skilled in the art.

[0224]

[0207] In the foregoing and in the following claims, it should be understood that: The phrases “at least one,” “one or more,” and “and / or” as used herein are open-ended expressions that are both conjunctive and disjunctive in their use. The terms “a,” “an,” “the,” “first,” “second,” etc., do not exclude plurals. For example, the term “a” or “an” entity as used herein refers to one or more such entities. Thus, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

[0225]

[0208] Abbreviations used herein have their conventional meanings within the technical fields of chemistry and biology. Chemical structures and formulas shown herein are constructed in accordance with the standard rules of chemical valence known in chemical science.

[0226]

[0209] As used herein, “subject” refers to all members of the animal kingdom, including mammals, and appropriately refers to humans. Members of the animal kingdom include, but are not limited to, mammals (e.g., humans, primates, pigs, sheep, cows, equids, horses, camels, canids, dogs, felids, cats, tigers, leopards, civets, minks, martens, polecats, domestic pets, livestock, rabbits, mice, rats, guinea pigs or other rodents, marine mammals of the sea lion and seal families, whales, etc.), fish, amphibians, reptiles, and birds (e.g., waterfowl, migratory birds, quail, ducks, geese, poultry, or chickens). In one embodiment of the present disclosure, the subject requires a compound or composition of the present disclosure. In a preferred embodiment, the subject is a human subject.

[0227]

[0210] As used herein, the terms “treatment” or “to treat” typically describe the management and care of subjects, including human and animal subjects (e.g., veterinary applications), achieving therapeutic effects with respect to a disease or disorder in a subject, such as reducing, relieving, or alleviating one or more symptoms or complications of a disease or disorder, slowing the progression of a disease or disorder, including reducing the rate of progression or stopping its progression, and curing the condition.

[0228]

[0211] The term "prodrug" refers to a substance prepared in an inactive form that is converted into an active form (i.e., a drug) in the body or within its cells, for example, by the action of endogenous enzymes or other chemical substances and / or states. Prodrug derivatives of the compound of formula (I) or its pharmaceutically acceptable salts or solvates can be prepared by methods known to those skilled in the art.

[0229]

[0212] The term "small organic molecule" refers to small organic compounds, including heteroorganic compounds and organometallic compounds, having a molecular weight of less than approximately 1,000 grams per mole or less than approximately 500 grams per mole, as well as salts, esters, and other pharmaceutically acceptable forms of the above compounds.

[0230]

[0213] The inventions disclosed herein have been described using specific embodiments and applications thereof, but numerous modifications and variations can be made by those skilled in the art without departing from the scope of the invention as set forth in the claims.

[0231]

[0214] Naturally, the present invention is described in varying degrees of detail in this application. In some cases, details that are not necessary for a person skilled in the art to understand the invention, or that would make it difficult to understand other details, may have been omitted. The terms used herein are for the purpose of describing specific embodiments and are not intended to be limited beyond the scope of the appended claims. Unless otherwise defined, the technical terms used herein should be understood as they are generally understood by a person skilled in the art to which this disclosure belongs.

[0232]

[0215] In the claims, the term “contains / contains” does not exclude the existence of other elements, components, features, areas, integers, processes, operations, etc. Furthermore, individual features may be included in different claims, but they may be advantageously combined, and their inclusion in different claims does not imply that the combination of features is unfeasible and / or unfavorable. The transitional phrase “essentially from” (and grammatical variations) should be interpreted as encompassing the enumerated materials or processes and “materials or processes that do not substantially affect the basic and novel features” of the enumerated embodiments. Accordingly, the term “essentially from” as used herein should not be interpreted as equivalent to “contains.”

Claims

1. Compounds of formula (I), or pharmaceutically acceptable salts thereof: 【Chemistry 1】 (In the formula, R １ , R ２ , R ３ and R ４ Each of these is independently H or halo, R １ , R ２ , R ３ , and R ４ At least two of them are H, and R １ , R ２ , R ３ , and R ４ at least two of which are halo. Optionally, the methyl group is at least one radioactive isotope of carbon, optionally carbon-11 ( １１ (Including C)).

2. R １ and R ３ However, R is independently selected from fluoro and chloro. ２ and R ４ Each of them is H, and optionally at least one fluoro is a radioactive isotope of fluorine, and optionally the isotope is fluorine-18 ( １８ The compound according to claim 1, which is F).

3. R ２ and R ３ However, R is independently selected from fluoro and chloro. １ and R ４ Each of them is H, and optionally at least one fluoro is a radioactive isotope of fluorine, and optionally the isotope is fluorine-18 ( １８ The compound according to claim 1, which is F).

4. R ２ and R ４ However, R is independently selected from fluoro and chloro. １ and R ３ Each of them is H, and optionally at least one fluoro is a radioactive isotope of fluorine, and optionally the isotope is fluorine-18 ( １８ The compound according to claim 1, which is F). 【Request Item 5】 【Chemistry 2】 Selected from the group consisting of, optionally, the methyl group is at least one radioactive isotope of carbon, optionally carbon-11 ( １１ C) comprises; or at least one fluoro is a radioactive isotope of fluorine, and optionally the isotope is fluorine-18 ( １８ F) The compound according to any one of claims 1 to 4.

6. A pharmaceutical composition comprising a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier or excipient.

7. The pharmaceutical composition according to claim 6, formulated as an oral dosage form.

8. The pharmaceutical composition according to claim 7, wherein one oral dosage form contains about 0.1 to 250 mg or about 1 to 25 mg of the compound.

9. The compound comprises the compound according to any one of claims 1 to 5, wherein the methyl group comprises at least one radioactive isotope of carbon, optionally carbon-11 ( １１ C) comprises; or at least one fluoro is a radioactive isotope of fluorine, and optionally the isotope is fluorine-18 ( １８ F) An imaging composition.

10. A method for treating a subject in need of treatment for a neurological disorder or impairment, wherein at least one symptom of the neurological disorder or impairment is reduced by inhibiting AMPA-mediated neurotoxicity, the method comprising administering to the subject a pharmaceutical composition comprising an effective amount of any one of claims 1 to 5.

11. The method according to claim 10, wherein the neurological disease or disorder is characterized by GluR2 / AMPAR neurotoxicity.

12. The method according to claim 10, wherein the neurological disease or disorder is selected from neuroinflammation, stroke, and epilepsy.

13. The method according to claim 10, wherein the neurological disorder or disability is selected from Parkinson's disease, Alzheimer's disease, dementia, and Huntington's disease.

14. A method for treating a subject in need of treatment for a neurological disorder or disability selected from the group consisting of multiple sclerosis (MS), spasticity associated with multiple sclerosis, and amyotrophic lateral sclerosis (ALS), comprising the step of administering to the subject a pharmaceutical composition containing an effective amount of any one of claims 1 to 5.

15. The method according to any one of claims 10 to 14, wherein the pharmaceutical composition is in oral dosage form and the route of administration is oral.

16. The method according to any one of claims 10 to 15, wherein the subject is a human.

17. The method according to any one of claims 10 to 16, wherein the effective amount of the compound is about 0.1 to 250 mg or about 1 to 25 mg per day.

18. An in vitro or ex vivo method for inhibiting glutamate receptor-mediated excitotoxicity, inhibiting GluR2 / AMPAR neurotoxicity, and / or inhibiting GAPDH-GluR2 complex formation in cells, tissues, or organoids, comprising the step of contacting the cells, tissues, or organoids with a compound according to any one of claims 1 to 5.

19. A method for in vivo imaging of AMPA receptors or their surrogates in the brain of a target, comprising the step of administering a radiolabeled compound of formula I to the target, wherein the compound is １１ C, １８ F, １９ A carbon or fluorine isotope selected from F, １２３ I, １３１ I or ３ A method that includes H.

20. The labeled compound of formula I is １１ C-261, １１ C-262, １１ C-263, １８ F-261, １８ F-262, and １８ The method according to claim 19, selected from F-263.

21. The method according to claim 19 or 20, wherein the labeled compound of formula I is administered orally, intraperitoneally, or intravenously.

22. The method according to any one of claims 19 to 21, wherein the subject is a rodent, a dog, a rabbit, a non-human primate, or a human.

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