Polymorphs of nitrogen-containing heterocyclic compounds and uses thereof

Abstract

The present invention relates to polymorphs of a nitrogen-containing heterocyclic compound. The polymorphs of the present invention can effectively enhance or reactivate synaptic plasticity, and inhibit or eliminate the accumulation of beta-amyloid, without causing side effects, thereby exerting a therapeutic effect on various nervous system diseases or neurodegenerative diseases. In addition, the polymorphs of the present invention exhibit excellent physicochemical properties, such as stability, solubility, hygroscopicity, and mechanical strength, and show excellent permeability to the blood-brain barrier (BBB).

Description

Technical Field

[0001] This application claims priority to Korean Patent Application No. 10-2023-0131187, filed on September 27, 2023, the disclosure of which is incorporated herein by reference in its entirety.

[0002] This invention relates to polymorphs of nitrogen-containing heterocyclic compounds. Specifically, this invention relates to polymorphs of compounds used for the prevention or treatment of nervous system diseases or neurodegenerative diseases, methods for their preparation, pharmaceutical compositions, and uses. Background Technology

[0003] Synaptic plasticity and critical period A neural network is a network composed of neurons, and synapses are the nodes in a neural network where neurons exchange information with each other. In a neural network, synapses exhibit "plasticity" because they are added to, removed from, or refined by continuous neural activity in response to synaptic input. The number and strength of synapses change throughout an individual's life. Synaptic plasticity refers to the modification of synapses by neural activity, manifested as changes in the strength and / or efficiency of synaptic transmission.

[0004] Higher functions such as learning, cognition, synthesis, judgment, art, and creativity depend on the ability to form neural networks (called cortical neural networks) in the cerebral cortex. Synaptic plasticity of cortical neural networks peaks early in life; this period is known as the critical period of synaptic plasticity. After the critical period, cortical synaptic plasticity gradually declines with age, eventually leading to a state where experience no longer elicits active changes in the cerebral cortex. Interestingly, recent studies have shown that under certain conditions, even after the critical period has ended, synaptic plasticity can still recover to a high level: in animal models, it has been reported that peripheral nerve injury can reactivate synaptic plasticity in the somatosensory thalamic cortex circuit that transmits peripheral sensory signals to the cerebral cortex (see Yu et al., 2012, Neuron. 74:731-42; Petrus et al., 2014, Neuron. 81:664-73; Gao et al., 2014, Cell 159.4: 775-788; and Gao et al., 2014, J Neurosci. 34(32): 10770-10779).

[0005] Decreased synaptic plasticity is also associated with the decline of brain function in adults. In particular, synaptic plasticity in the thalamic cortex circuits is significantly limited with age. As mentioned above, if the critical period can be reopened, reactivating or enhancing cortical synaptic plasticity through therapeutic means may help treat diseases caused by decreased synaptic plasticity.

[0006] β -Amyloid protein Alzheimer's disease (AD) accounts for the largest proportion of dementia cases and is a degenerative brain disease for which no effective treatment has yet been found. Alzheimer's disease is characterized by the accumulation of β-amyloid-beta (Aβ) and tau proteins in the brain, chronic neuroinflammation, and mitochondrial dysfunction. Previous research on Alzheimer's disease has primarily focused on the accumulation of β-amyloid and tau proteins, but the development of new drugs targeting these two pathogenic substances has largely failed. Aducanumab, which targeted β-amyloid and received clinical approval in 2021, has had its approval withdrawn in Europe due to lower-than-expected efficacy and side effects. Therefore, in addition to the two pathogenic substances mentioned above, therapeutic agents targeting the pathological phenotypes of Alzheimer's disease are still needed.

[0007] Crystal form of the compound Crystal form influences the physical properties of compounds to a certain extent. Due to differences in crystal lattice structure, pharmacological compounds with multiple crystal forms not only have different appearances (e.g., color; morphology such as needle-like crystals, crystal flakes, and crystal particles), but also different physical properties (e.g., melting point, solubility, density, stability, and hygroscopicity), and exhibit different solubility and absorption characteristics in vivo. This may affect the clinical efficacy and stability of pharmacological compounds.

[0008] Specific crystal forms exhibit different thermodynamic behaviors compared to amorphous or other crystal forms. To characterize the thermal properties that distinguish specific crystal forms, amorphous states, and other crystal forms, methods such as melting point apparatus, thermogravimetric analysis (TGA), or differential scanning calorimetry (DSC) can be used. Some crystal forms may possess unique spectral properties. For example, X-ray powder diffraction patterns (XRPD) can characterize specific crystal forms.

[0009] [Existing technical documents] [Non-patent literature] (Non-patent literature 1) Yu, Xin, et al. "Thalamocortical inputs show post-critical-period plasticity." Neuron 74.4 (2012): 731-742. (Non-patent document 2) Petrus, Emily, et al. "Crossmodal induction ofthalamocortical potentiation leads to enhanced information processing in theauditory cortex." Neuron 81.3 (2014): 664-673. (Non-patent literature 3) Gao, Peng, et al. "Deterministic progenitor behavior and unitary production of neurons in the neocortex." Cell 159.4 (2014): 775-788. (Non-patent document 4) Gao, Ming, et al. "Rebound potentiation of inhibition in juvenile visual cortex requires vision-induced BDNF expression." Journal of Neuroscience 34.32 (2014): 10770-10779. Summary of the Invention Technical issues The purpose of this invention is to solve all the above-mentioned problems.

[0010] One object of the present invention is to provide a novel crystal form of a nitrogen-containing heterocyclic compound for enhancing synaptic plasticity and / or inhibiting or eliminating the accumulation of β-amyloid protein (Aβ).

[0011] Another object of the present invention is to provide a pharmaceutical composition comprising, as an active ingredient, a crystalline form of a nitrogen-containing heterocyclic compound for enhancing synaptic plasticity and / or inhibiting or eliminating β-amyloid protein accumulation.

[0012] Another object of the present invention is to provide a method for treating or preventing neurological diseases or neurodegenerative diseases, comprising administering to a subject a crystal form of a nitrogen-containing heterocyclic compound for enhancing synaptic plasticity and / or inhibiting or eliminating β-amyloid protein accumulation.

[0013] Another object of the present invention is to provide a pharmaceutical composition, kit, or method for co-administering a crystal form of a nitrogen-containing heterocyclic compound for enhancing synaptic plasticity and / or inhibiting or eliminating β-amyloid accumulation with a second therapeutic agent.

[0014] The objectives of this invention are not limited to those described above. The objectives of this invention will become more apparent from the following description and will be achieved by the means and combinations thereof set forth in the claims.

[0015] Technical solution The present invention provides a new polymorph of compound of formula (1), namely (3S,6R)-1-(2-(2-chloro-4-fluorophenyl)acetyl)-6-methylpiperidine-3-carboxylic acid, pharmaceutical compositions thereof, methods of preparation thereof, and use for the prevention and / or treatment of nervous system diseases or neurodegenerative diseases.

[0016] In one aspect of the invention, a crystal form of the compound represented by formula (1) is provided.

[0017] Equation (1)

[0018] In one aspect of the invention, a crystal form A of the compound represented by formula (1) is provided, characterized in that its X-ray powder diffraction pattern has diffraction peaks at diffraction angles 2θ (°) of 9.9±0.10, 14.9±0.10, 18.4±0.10, 24.5±0.10, 24.8±0.10, 25.2±0.10 and 28.7±0.10.

[0019] In one embodiment, the X-ray powder diffraction pattern of crystal form A may further exhibit diffraction peaks at diffraction angles 2θ (°) of 5.0±0.10, 13.4±0.10, 19.7±0.10, 19.8±0.10, 20.5±0.10, 22.5±0.10, 24.0±0.10, and 35.0±0.10.

[0020] In one embodiment, in addition to the diffraction peaks described above, the X-ray powder diffraction pattern of crystal form A may further exhibit diffraction peaks at diffraction angles 2θ (°) of 12.1±0.10, 16.2±0.10, 20.1±0.10, 21.5±0.10, 27.4±0.10, 29.6±0.10, 30.0±0.10, 31.2±0.10, 31.5±0.10, and 36.8±0.10.

[0021] In another aspect of the invention, crystal form B of the compound shown in formula (1) is provided, characterized in that its X-ray powder diffraction pattern has diffraction peaks at diffraction angles 2θ (°) of 8.6±0.10, 9.1±0.10, 9.2±0.10, 15.8±0.10, 20.0±0.10, 20.3±0.10, 20.7±0.10, 23.9±0.10 and 32.6±0.10.

[0022] In one embodiment, the X-ray powder diffraction pattern of crystal form B may further exhibit diffraction peaks at diffraction angles 2θ (°) of 8.2±0.10, 14.2±0.10, 14.7±0.10, 18.0±0.10, 18.3±0.10, 22.9±0.10, 26.4±0.10, 26.7±0.10, 27.6±0.10, 28.1±0.10, and 29.1±0.10.

[0023] In one embodiment, in addition to the diffraction peaks described above, the X-ray powder diffraction pattern of crystal form B may further exhibit diffraction peaks at diffraction angles 2θ (°) of 11.9±0.10, 15.1±0.10, 16.5±0.10, 19.3±0.10, 22.1±0.10, 22.6±0.10, 24.5±0.10, 24.8±0.10, 25.9±0.10, 27.0±0.10, 27.8±0.10, 28.9±0.10, and 36.5±0.10.

[0024] In another aspect of the invention, a crystal form C of the compound represented by formula (1) is provided, characterized in that its X-ray powder diffraction pattern has diffraction peaks at diffraction angles 2θ (°) of 8.6±0.10, 10.0±0.10, 18.0±0.10 and 26.4±0.10.

[0025] In one embodiment, the X-ray powder diffraction pattern of crystal form C may further have diffraction peaks at diffraction angles 2θ (°) of 17.2 ± 0.10, 20.2 ± 0.10, and 25.5 ± 0.10.

[0026] In one embodiment, in addition to the diffraction peaks mentioned above, the X-ray powder diffraction pattern of crystal form C may further have diffraction peaks at diffraction angles 2θ (°) of 15.1±0.10, 16.0±0.10, 21.1±0.10, 23.4±0.10, 26.1±0.10 and 34.2±0.10.

[0027] In another aspect of the invention, a crystal form D of the compound shown in formula (1) is provided, characterized in that its X-ray powder diffraction pattern has diffraction peaks at diffraction angles 2θ (°) of 8.8±0.10, 10.7±0.10, 17.1±0.10, 17.6±0.10, 19.1±0.10, 21.5±0.10, 22.2±0.10 and 23.4±0.10.

[0028] In one embodiment, the X-ray powder diffraction pattern of crystal form D may further exhibit diffraction peaks at diffraction angles 2θ (°) of 12.5±0.10, 15.4±0.10, 16.1±0.10, 16.7±0.10, 18.3±0.10, 22.5±0.10, 22.9±0.10, 27.4±0.10, 27.8±0.10, and 29.8±0.10.

[0029] In one embodiment, in addition to the diffraction peaks described above, the X-ray powder diffraction pattern of crystal form D may further exhibit diffraction peaks at diffraction angles 2θ (°) of 9.5±0.10, 15.7±0.10, 21.0±0.10, 25.1±0.10, 25.3±0.10, 27.0±0.10, 28.6±0.10, 30.9±0.10, 32.1±0.10, 32.6±0.10, and 34.1±0.10.

[0030] In another aspect of the invention, a crystal form E of the compound shown in formula (1) is provided, characterized in that its X-ray powder diffraction pattern has diffraction peaks at diffraction angles 2θ (°) of 10.2±0.10, 11.3±0.10, 18.3±0.10, 20.5±0.10, 22.8±0.10, 23.5±0.10, 25.2±0.10, 26.1±0.10, 28.6±0.10, 28.9±0.10 and 31.2±0.10.

[0031] In one embodiment, the X-ray powder diffraction pattern of crystal form E may further exhibit diffraction peaks at diffraction angles 2θ (°) of 9.9±0.10, 13.4±0.10, 17.9±0.10, 18.6±0.10, 19.6±0.10, 20.8±0.10, 21.0±0.10, 24.4±0.10, 24.8±0.10, 29.5±0.10, 29.8±0.10, and 32.5±0.10.

[0032] In one embodiment, in addition to the diffraction peaks described above, the X-ray powder diffraction pattern of crystal form E may further exhibit diffraction peaks at diffraction angles 2θ (°) of 4.9±0.10, 7.2±0.10, 14.8±0.10, 16.1±0.10, 16.4±0.10, 19.8±0.10, 20.0±0.10, 21.5±0.10, 22.5±0.10, 24.0±0.10, 25.4±0.10, 27.4±0.10, 32.8±0.10, and 39.5±0.10.

[0033] In one embodiment, the crystal form of the present invention may have high BBB permeability.

[0034] In another aspect of the invention, a pharmaceutical composition is provided comprising any of the crystal forms of the invention as an active ingredient.

[0035] In one embodiment, the composition can be used to prevent or treat nervous system diseases or neurodegenerative diseases.

[0036] In another aspect of the invention, a method for preventing or treating nervous system diseases or neurodegenerative diseases is provided, comprising administering any one of the crystal forms of the invention to a subject who requires prevention or treatment of a nervous system disease or neurodegenerative disease.

[0037] In one implementation, the disease may be Alzheimer's disease.

[0038] In one implementation, the disease may be post-traumatic stress disorder (PTSD).

[0039] In one embodiment, any of the crystal forms of the present invention may be used in combination with a second therapeutic agent.

[0040] In one embodiment, the second therapeutic agent may be a drug used to prevent or treat nervous system diseases or neurodegenerative diseases.

[0041] In one embodiment, the second therapeutic agent may be selected from the group consisting of donepezil, rivastigmine, galantamine, memantine, verubecestat, solanumab, bapinzumab, aducanumab, lencanemab, tideglusib, epothilone D, and ABBV-8E12.

[0042] In one embodiment, the second therapeutic agent may be an antibody that specifically binds to β-amyloid protein (e.g., aducanumab).

[0043] Beneficial effects The novel crystalline form of this invention can effectively enhance or reactivate synaptic plasticity and inhibit or eliminate the accumulation of β-amyloid protein without producing side effects, thereby exerting therapeutic effects on various neurological diseases or neurodegenerative diseases. Furthermore, the crystalline form of this invention exhibits excellent physicochemical properties, such as stability, solubility, hygroscopicity, and mechanical strength. In addition, the crystalline form of this invention shows excellent permeability to the blood-brain barrier (BBB). Therefore, the novel crystalline form of this invention can be developed into an excellent therapeutic agent for neurological diseases or neurodegenerative diseases such as Alzheimer's disease or PTSD. Attached Figure Description

[0044] Figure 1a This is a graph showing the X-ray powder diffraction (XRPD) analysis results of crystal form A of compound (1).

[0045] Figure 1b It is the crystal form A of the compound represented by formula (1) 1 A chart of H-nuclear magnetic resonance (NMR) analysis results.

[0046] Figure 1c This is a graph showing the results of differential scanning calorimetry (DSC) analysis of crystal form A of compound (1).

[0047] Figure 1d This is a graph showing the thermogravimetric analysis (TGA) results of crystal form A of compound (1).

[0048] Figure 2a This is a graph showing the X-ray powder diffraction (XRPD) analysis results of crystal form B of compound (1).

[0049] Figure 2b This is a graph showing the results of differential scanning calorimetry (DSC) analysis of crystal form B of compound (1).

[0050] Figure 2c This is a graph showing the thermogravimetric analysis (TGA) results of crystal form B of compound (1).

[0051] Figure 3a The graph shows the X-ray powder diffraction (XRPD) analysis results of crystal form C of compound (1).

[0052] Figure 3b It is the crystal form C of the compound represented by formula (1) 1 A chart of H-nuclear magnetic resonance (NMR) analysis results.

[0053] Figure 3c It is a graph representing the differential scanning calorimetry (DSC) analysis results of crystal form C of compound (1).

[0054] Figure 3d This is a graph showing the thermogravimetric analysis (TGA) results of crystal form C of compound (1).

[0055] Figure 4a This is a graph showing the X-ray powder diffraction (XRPD) analysis results of crystal form D of compound (1).

[0056] Figure 4b It represents the crystal form D of the compound of formula (1). 1 A chart of H-nuclear magnetic resonance (NMR) analysis results.

[0057] Figure 4c It is a graph representing the differential scanning calorimetry (DSC) analysis results of crystal form D of compound (1).

[0058] Figure 4d This is a graph showing the thermogravimetric analysis (TGA) results of crystal form D of the compound of formula (1).

[0059] Figure 5a This is a graph showing the X-ray powder diffraction (XRPD) analysis results of crystal form E of compound (1).

[0060] Figure 5b It is the crystal form E of the compound represented by formula (1) 1 A chart of H-nuclear magnetic resonance (NMR) analysis results.

[0061] Figure 5c It is a graph showing the results of differential scanning calorimetry (DSC) analysis of the crystal form E of the compound of formula (1).

[0062] Figure 5d This is a graph showing the thermogravimetric analysis (TGA) results of crystal form E of compound (1).

[0063] Figure 6 It is a diagram showing the interconversion relationships of crystal forms A, B, C, D and E of the compound of formula (1).

[0064] Figure 7a This is a graph representing the first-order X-ray powder diffraction (XRPD) superimposed pattern of a sample obtained in a competitive equilibrium experiment at 5°C.

[0065] Figure 7b This is a graph representing the superimposed second-order X-ray powder diffraction (XRPD) patterns of a sample obtained in a competitive equilibrium experiment at 5°C.

[0066] Figure 7c This is a graph representing the first-order X-ray powder diffraction (XRPD) superimposed pattern of a sample obtained in a competitive equilibrium experiment at 25°C.

[0067] Figure 7d This is a graph representing the superimposed second-order X-ray powder diffraction (XRPD) patterns of a sample obtained in a competitive equilibrium experiment at 25°C.

[0068] Figure 7e This is a graph representing the first-order X-ray powder diffraction (XRPD) superimposed pattern of a sample obtained in a competitive equilibrium experiment at 50°C.

[0069] Figure 7f This is a graph representing the superimposed second-order X-ray powder diffraction (XRPD) patterns of a sample obtained in a competitive equilibrium experiment at 50°C.

[0070] Figure 8 It is a graph showing the X-ray powder diffraction (XRPD) superimposed pattern of the sample obtained after applying stress to the crystal form C of the compound of formula (1).

[0071] Figure 9a and Figure 9b The figure shows the dynamic vapor adsorption (DVS) analysis results of crystal form C of compound (1).

[0072] Figure 9c It is a graph showing the X-ray powder diffraction (XRPD) superimposed images of crystal form C of compound (1) before and after DVS testing.

[0073] Figure 10 This is a graph showing the X-ray powder diffraction (XRPD) overlay pattern of the sample obtained by compressing the crystal form C of compound (1).

[0074] Figure 11 This is a graph showing the X-ray powder diffraction (XRPD) overlay pattern of a sample obtained by grinding the crystal form C of compound (1).

[0075] Figure 12 It is a graph showing the X-ray powder diffraction (XRPD) overlay pattern of the sample obtained by granulating the crystal form C of the compound of granulation (1).

[0076] Figure 13a Dosing regimens are shown for analyzing the efficacy of monotherapy of compound C of formula (1) (denoted as BnH-015B) or aducanumab, as well as their combination, in enhancing ex vivo synaptic plasticity in animal models of Alzheimer's disease.

[0077] Figure 13b The results show the LTP measurements of excitatory postsynaptic potentials in the hippocampus of an Alzheimer's disease mouse model after administration of crystal form C (denoted as BnH-015B) of compound (1) alone or in combination with aducanumab.

[0078] Figure 13c The results show the excitation / inhibition balance in the sensory cortex of an Alzheimer's disease mouse model after administration of crystal form C (denoted as BnH-015B) of compound (1) alone or in combination (right) and the changes in TC EPSC efficacy (left).

[0079] Figure 14a The changes in TC EPSC efficacy in the barrel cortex (layer 4) of mice after oral administration of (1 mg / kg) of the compound of formula (1) in crystal form C (denoted as BnH-015B enantiomer (3S, 6R, 98% <)) were shown.

[0080] Figure 14b The results show the excitation / inhibition balance in the barrel cortex (layer 4) of mice after oral administration of (1 mg / kg) of the compound of formula (1) in crystal form C (denoted as BnH-015B enantiomer (3S, 6R, 98% <)).

[0081] Figure 15 The results of a one-tailed independent samples t-test are shown for IL-33 and OPN expression levels after administration of crystal form C (denoted as 015B) in mouse proteomic array analysis.

[0082] Figure 16a The results of Bielschowsky silver staining in the cerebral cortex of APP / PS1 mice after administration of the medium control and crystal form C (denoted as 015B) are shown.

[0083] Figure 16b The results of a one-tailed independent samples t-test show the percentage of Aβ plaque area after application of the medium control and crystal form C (denoted as BnH-015B). Detailed Implementation

[0084] The following detailed description of the invention will be made with reference to specific accompanying drawings (if any) to illustrate particular embodiments in which the invention may be practiced, but the invention is not limited thereto, but is defined only by the appended claims within the same or equivalent scope as those claims. It should be understood that the various embodiments / exemplaries of the invention differ from one another, but are not necessarily mutually exclusive. For example, specific shapes, structures, and characteristics described herein may be varied from one embodiment / exemplary to another, or implemented by combining multiple embodiments / exemplary, without departing from the technical spirit and scope of the invention. Technical and academic terms used in this specification, unless otherwise defined, have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the definitions set forth below will apply, and terms expressed in the singular form shall be construed as also referring to the plural form (i.e., at least one), unless the context is inappropriate, and vice versa.

[0085] definition The term "and / or" is used herein to mean "and" or "or" unless otherwise stated.

[0086] The term "at least one" includes each of the objects mentioned after this statement, as well as various combinations of two or more of the objects, unless otherwise understood in context and usage.

[0087] The terms “comprising,” “containing,” “including,” “having,” “owning,” “containing,” or “covering” (including their grammatical equivalents) should generally be understood as open-ended and non-restrictive, and should not be construed as excluding elements or steps not otherwise mentioned unless the context clearly specifies or understands otherwise.

[0088] The term "about" means approximately, roughly, approximately, or close to. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the upper and lower boundaries of the value. Typically, the term "about" is used herein to modify the upper and lower values ​​of the value by a variation of 10%.

[0089] The terms "individual," "patient," and "subject" are used interchangeably and can refer to mammals, such as primates (e.g., humans), companion animals (e.g., dogs, cats, etc.), livestock (e.g., cattle, pigs, horses, sheep, goats, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, etc.). In one embodiment, the subject is a human.

[0090] The term "treatment" generally refers to achieving a desired pharmacological and / or physiological effect. Such effects have a therapeutic effect in partially or completely curing a disease and / or the adverse reactions caused by such a disease. Desired therapeutic effects include, but are not limited to: preventing the onset or recurrence of disease, improving symptoms, alleviating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and achieving remission or improved prognosis. Preferably, "treatment" may refer to a medical intervention for an existing disease or disorder.

[0091] The term "pharmaceutically acceptable" includes molecular entities and preparations that, when properly administered to animals or humans, will not produce harmful reactions, allergic reactions, or other adverse reactions.

[0092] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effects and properties of the corresponding free base or free acid. For example, pharmaceutically acceptable salts can be prepared by adding an inorganic or organic base to a free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethylamine, lysine, arginine, N-ethylpiperidine, piperidine, and piperazine.

[0093] All solvents used herein are commercially available. The following abbreviations are used in this disclosure: EtOH refers to ethanol; MeOH refers to methanol; MEK refers to methyl ethyl ketone; ACN refers to acetonitrile; EA refers to ethyl acetate; IPAc refers to isopropyl acetate; MTBE refers to methyl tert-butyl ether; THF refers to tetrahydrofuran; 2-MeTHF refers to 2-methyltetrahydrofuran; DCM refers to dichloromethane; DMSO refers to dimethyl sulfoxide; DMF refers to N,N-dimethylformamide; FeSSIF-v1 refers to simulated intestinal fluid version 1 during the feeding state (the period of digestion and absorption of nutrients); FaSSIF-v1 refers to simulated intestinal fluid version 1 during the fasting state; and PBS refers to phosphate-buffered saline.

[0094] New polymorphs of nitrogen-containing heterocyclic compounds This invention provides a novel polymorph of the compound shown in formula (1), namely (3S,6R)-1-(2-(2-chloro-4-fluorophenyl)acetyl)-6-methylpiperidine-3-carboxylic acid, pharmaceutical compositions thereof, methods of preparation thereof, use for the prevention or treatment of nervous system diseases or neurodegenerative diseases, and methods for the prevention or treatment of nervous system diseases or neurodegenerative diseases: Equation (1)

[0095] The compound represented by formula (1) is described in detail in Korean Patent Application No. 10-2023-0040835 and International Patent Application No. PCT / KR2023 / 004138, the entire contents of which are incorporated herein by reference.

[0096] The term "compound" means a chemical substance having the structure shown in formula (1) described herein, its stereoisomers (including mixtures of stereoisomers), its polymorphs, or its pharmaceutically acceptable salts, hydrates, solvates or prodrugs.

[0097] The term "active agent" is used to refer to a biologically active substance, its stereoisomers (including mixtures of stereoisomers), its polymorphs, or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof. In one embodiment, the "active agent" is a compound of the present invention that has pharmaceutical applicability, particularly a polymorph of the present invention. For example, the active agent may be a therapeutic agent for nervous system diseases or neurodegenerative diseases.

[0098] It has been confirmed that the new crystal form of the compound shown in formula (1) ((3S,6R)-1-(2-(2-chloro-4-fluorophenyl)acetyl)-6-methylpiperidine-3-carboxylic acid) has excellent chemical and physical properties that are beneficial to the manufacture and formulation of the compound, as well as excellent pharmacological effects, such as enhancing synaptic plasticity and inhibiting or eliminating β-amyloid (Aβ) accumulation, and excellent BBB permeability.

[0099] In one aspect of the invention, crystal forms of the compound of formula (1) are provided, and five polymorphs of the compound of formula (1) are also provided, namely polymorphs named crystal form A, crystal form B, crystal form C, crystal form D and crystal form E.

[0100] In one aspect of the invention, a crystal form is provided having X-ray powder diffraction patterns with diffraction peaks at diffraction angles 2θ (°) of 9.9±0.10, 14.9±0.10, 18.4±0.10, 24.5±0.10, 24.8±0.10, 25.2±0.10, and 28.7±0.10, which is defined herein as crystal form A.

[0101] Preferably, the X-ray powder diffraction pattern of crystal form A further exhibits diffraction peaks at diffraction angles 2θ (°) of 5.0±0.10, 13.4±0.10, 19.7±0.10, 19.8±0.10, 20.5±0.10, 22.5±0.10, 24.0±0.10, and 35.0±0.10.

[0102] More preferably, the X-ray powder diffraction pattern of crystal form A further exhibits diffraction peaks at diffraction angles 2θ (°) of 12.1±0.10, 16.2±0.10, 20.1±0.10, 21.5±0.10, 27.4±0.10, 29.6±0.10, 30.0±0.10, 31.2±0.10, 31.5±0.10, and 36.8±0.10.

[0103] The XRPD pattern of crystal form A is shown in Figure 1a The analytical data of the XRPD spectra of the intermediate and crystal form A are shown in Table 1.

[0104] [Table 1] Analysis data of XRPD pattern of crystal form A

[0105] Crystal form A shows essentially as Figure 1b shown 1 H-NMR spectrum. Crystal form A 1 The H-NMR spectrum indicates the absence of detectable residual solvent. Crystal form A shows essentially the same... Figure 1cThe DSC spectrum shown is shown below. The DSC spectrum of crystal form A shows a melting peak at 128.3 °C with an enthalpy of approximately 61 J / g. Crystal form A exhibits essentially the following... Figure 1d The TGA spectrum shown is shown. The TGA spectrum of crystal form A shows a weight loss of approximately 0.7% when heated from room temperature to 120°C.

[0106] In another aspect of the invention, a crystal form is provided having X-ray powder diffraction patterns with diffraction peaks at diffraction angles 2θ (°) of 8.6±0.10, 9.1±0.10 and / or 9.2±0.10, 15.8±0.10, 20.0±0.10, 20.3±0.10, 20.7±0.10, 23.9±0.10 and 32.6±0.10, which is defined herein as crystal form B.

[0107] Preferably, the X-ray powder diffraction pattern of crystal form B further exhibits diffraction peaks at diffraction angles 2θ (°) of 8.2±0.10, 14.2±0.10, 14.7±0.10, 18.0±0.10, 18.3±0.10, 22.9±0.10, 26.4±0.10, 26.7±0.10, 27.6±0.10, 28.1±0.10, and 29.1±0.10.

[0108] More preferably, the X-ray powder diffraction pattern of crystal form B further exhibits diffraction peaks at diffraction angles 2θ (°) of 11.9±0.10, 15.1±0.10, 16.5±0.10, 19.3±0.10, 22.1±0.10, 22.6±0.10, 24.5±0.10, 24.8±0.10, 25.9±0.10, 27.0±0.10, 27.8±0.10, 28.9±0.10, and 36.5±0.10.

[0109] The XRPD pattern of crystal form B is shown in Figure 2a The analytical data of the XRPD spectra of the intermediate and crystal form B are shown in Table 2.

[0110] [Table 2] Analysis data of XRPD pattern of crystal form B

[0111] Crystal form B shows essentially as Figure 2b The DSC spectrum shown is for crystal form B. The DSC spectrum of crystal form B shows a melting peak with an enthalpy of approximately 64 J / g at 111.4 °C and an endothermic peak with an enthalpy of approximately 2 J / g at 126.8 °C. Crystal form B exhibits essentially the following characteristics: Figure 2c The TGA spectrum shown is shown. The TGA spectrum of crystal form B shows a weight loss of approximately 0.4% when heated from room temperature to 110°C.

[0112] In another aspect of the invention, a crystal form is provided having X-ray powder diffraction patterns with diffraction peaks at diffraction angles 2θ (°) of 8.6 ± 0.10, 10.0 ± 0.10, 18.0 ± 0.10, and 26.4 ± 0.10, which is defined herein as crystal form C.

[0113] Preferably, the X-ray powder diffraction pattern of crystal form C further exhibits diffraction peaks at diffraction angles 2θ (°) of 17.2±0.10, 20.2±0.10, and 25.5±0.10.

[0114] More preferably, the X-ray powder diffraction pattern of crystal form C further exhibits diffraction peaks at diffraction angles 2θ (°) of 15.1±0.10, 16.0±0.10, 21.1±0.10, 23.4±0.10, 26.1±0.10 and 34.2±0.10.

[0115] The XRPD pattern of crystal form C is shown in Figure 3a The analytical data of the XRPD spectra of medium and crystalline C are shown in Table 3.

[0116] [Table 3] Analysis data of XRPD spectra of crystalline form C

[0117] Crystal form C shows essentially as Figure 3b shown 1 H-NMR spectrum. Crystal form C shows essentially the same... Figure 3c The DSC spectrum shown is shown below. The DSC spectrum of crystalline form C shows a melting peak at 116.1 °C with an enthalpy of approximately 76 J / g. Crystalline form C exhibits essentially the following... Figure 3d The TGA spectrum shown is shown. The TGA spectrum of crystal form C shows a weight loss of approximately 0.3% when heated from room temperature to approximately 110°C.

[0118] In another aspect of the invention, a crystal form is provided having X-ray powder diffraction patterns with diffraction peaks at diffraction angles 2θ (°) of 8.8±0.10, 10.7±0.10, 17.1±0.10, 17.6±0.10, 19.1±0.10, 21.5±0.10, 22.2±0.10, and 23.4±0.10, which is defined herein as crystal form D.

[0119] Preferably, the X-ray powder diffraction pattern of crystal form D further exhibits diffraction peaks at diffraction angles 2θ (°) of 12.5±0.10, 15.4±0.10, 16.1±0.10, 16.7±0.10, 18.3±0.10, 22.5±0.10, 22.9±0.10, 27.4±0.10, 27.8±0.10, and 29.8±0.10.

[0120] More preferably, the X-ray powder diffraction pattern of crystal form D further exhibits diffraction peaks at diffraction angles 2θ (°) of 9.5±0.10, 15.7±0.10, 21.0±0.10, 25.1±0.10, 25.3±0.10, 27.0±0.10, 28.6±0.10, 30.9±0.10, 32.1±0.10, 32.6±0.10, and 34.1±0.10.

[0121] The XRPD pattern of crystal form D is shown in Figure 4a The analytical data of the XRPD spectra of the intermediate and crystal forms D are shown in Table 4.

[0122] [Table 4] Analysis data of XRPD spectra of crystal form D

[0123] Crystal form D shows essentially as Figure 4b shown 1 H-NMR spectrum. Crystal form D 1 H-NMR spectra showed 5.5% toluene residue by weight (0.2 molar equivalents of toluene). Crystal form D showed essentially the same... Figure 4c The DSC spectrum shown is as follows. The DSC spectrum of crystal form D shows a desolvation peak at 62 °C, an endothermic peak with an enthalpy of approximately 39 J / g at 114.4 °C, and an endothermic peak with an enthalpy of approximately 22 J / g at 128.1 °C. Crystal form D shows essentially the following... Figure 4d The TGA spectrum shown is as follows. The TGA spectrum of crystal form D shows a weight loss of approximately 4.8% when heated from room temperature to approximately 80°C, and a weight loss of approximately 2.5% when heated from approximately 80°C to 170°C.

[0124] In another aspect of the invention, a crystal form is provided having X-ray powder diffraction patterns with diffraction peaks at diffraction angles 2θ (°) of 10.2±0.10, 11.3±0.10, 18.3±0.10, 20.5±0.10, 22.8±0.10, 23.5±0.10, 25.2±0.10, 26.1±0.10, 28.6±0.10, 28.9±0.10, and 31.2±0.10, which is defined herein as crystal form E.

[0125] Preferably, the X-ray powder diffraction pattern of crystal form E further exhibits diffraction peaks at diffraction angles 2θ (°) of 9.9±0.10, 13.4±0.10, 17.9±0.10, 18.6±0.10, 19.6±0.10, 20.8±0.10, 21.0±0.10, 24.4±0.10, 24.8±0.10, 29.5±0.10, 29.8±0.10, and 32.5±0.10.

[0126] More preferably, the X-ray powder diffraction pattern of crystal form E further exhibits diffraction peaks at diffraction angles 2θ (°) of 4.9±0.10, 7.2±0.10, 14.8±0.10, 16.1±0.10, 16.4±0.10, 19.8±0.10, 20.0±0.10, 21.5±0.10, 22.5±0.10, 24.0±0.10, 25.4±0.10, 27.4±0.10, 32.8±0.10, and 39.5±0.10.

[0127] The XRPD pattern of crystal form E is shown in Figure 5a The analytical data of the XRPD spectra of the intermediate and crystal forms E are shown in Table 5.

[0128] [Table 5] Analysis data of XRPD spectra of crystal form E

[0129] Crystal form E shows essentially as Figure 5b shown 1 H-NMR spectrum. Crystal form E 1 H-NMR spectra showed 9.3% ethanol residue by weight (0.7 molar equivalents of ethanol). Crystal form E showed essentially the same... Figure 5c The DSC spectrum shown is shown. The DSC spectrum of crystal form E shows a desolvation peak at approximately 40 °C. Crystal form E shows essentially the following... Figure 5d The TGA spectrum shown is shown. The TGA spectrum of crystal form E shows a weight loss of approximately 10.2% when heated from room temperature to approximately 130°C.

[0130] The crystal form of formula (1) disclosed herein, especially crystal form C, exhibits excellent physicochemical properties, such as stability, solubility, hygroscopicity and mechanical strength, and excellent permeability to the blood-brain barrier (BBB) ​​(see Examples 7 to 12).

[0131] Furthermore, the crystal form of formula (1) disclosed herein, especially crystal form C, can effectively enhance or reactivate synaptic plasticity without producing side effects, and effectively inhibit or eliminate the accumulation of β-amyloid protein, thereby exerting excellent preventive or therapeutic effects on various nervous system diseases or neurodegenerative diseases (see Examples 13 to 15).

[0132] XRPD map, 1 H-NMR spectra, DSC spectra, and TGA spectra can be determined by methods known in the art, including those described in the examples below. Due to instrumental errors or differences between operators, those skilled in the art will understand that the parameters determining the physical properties of crystal forms may vary. Therefore, while the above parameters are helpful in characterizing the polymorphs provided by this invention, they should not be construed as limiting the polymorphs disclosed herein.

[0133] Preparation method The compounds of formula (1) of the present invention can be prepared by methods known in the art. Furthermore, the compounds of the present invention can be prepared, for example, by the methods described in Example 1 and Reaction Scheme 1.

[0134] Crystal form A of the present invention can be prepared, for example, from the compound of formula (1) by the method of Example 2. A schematic diagram showing the interconversion relationships of crystal forms B, C, D, and E of the compound of formula (1) can be obtained from crystal form A. Figure 6 For representative preparation examples of the crystal forms of compounds of formula (1), please refer to the Examples.

[0135] Pharmaceutical Composition According to another aspect of the present invention, a pharmaceutical composition is provided comprising a compound of formula (1) or a novel polymorph thereof as an active ingredient. The novel polymorphs of the present invention may include polymorphs A to E, characterized by comprising analytical data of XRPD spectra listed in Tables 1 to 5.

[0136] In one embodiment, the composition comprises one or more crystal forms selected from A, B, C, D and E of the present invention.

[0137] In one embodiment, the composition comprises an effective amount of crystal form A of the present invention.

[0138] In one embodiment, the composition comprises an effective amount of crystal form B of the present invention.

[0139] In one embodiment, the composition comprises an effective amount of the crystal form C of the present invention.

[0140] In one embodiment, the composition comprises an effective amount of the crystal form D of the present invention.

[0141] In one embodiment, the composition comprises an effective amount of the crystal form E of the present invention.

[0142] In one embodiment, the composition can be used to prevent or treat nervous system diseases or neurodegenerative diseases.

[0143] In one implementation, the disease may be Alzheimer's disease or PTSD.

[0144] In one embodiment, the pharmaceutical composition of the present invention may further comprise one or more pharmaceutically acceptable excipients and / or carriers. Specifically, the one or more pharmaceutically acceptable excipients are selected from the group consisting of diluents, buffers, preservatives, stabilizers, solubilizers, or any combination thereof.

[0145] The terms "pharmaceuticalally acceptable excipient" and "pharmaceuticalally acceptable carrier" refer to substances that facilitate administration of the active agent to a subject and absorption of the active agent by the subject, and mean substances that can be included in the compositions of the present invention without causing significant adverse toxicological effects on the patient.

[0146] Non-limiting examples of pharmaceutically acceptable excipients or carriers include, but are not limited to: buffers (e.g., phosphates, citrates, or other organic acids); antioxidants (e.g., ascorbic acid or methionine); preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride, benzyl ammonium chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methyl or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (about 10 residues or less) peptides; proteins (e.g., serum albumin, gelatin, or leucovorin). Immunoglobulins); hydrophilic polymers (e.g., polyvinylpyrrolidone); amino acids (e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine); monosaccharides, disaccharides, and other carbohydrates, such as glucose, mannose, or dextrin; chelating agents (e.g., EDTA); sugars (e.g., sucrose, mannitol, trehalose, or sorbitol); salt-forming counterions (e.g., sodium); metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants (e.g., TWEEN®, PLURONICS®, or polyethylene glycol (PEG)).

[0147] The pharmaceutically acceptable excipients or carriers may be sterilized and, if desired, mixed with adjuvants that will not adversely react with the compounds of the present invention, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, colorants, and / or aromatic substances.

[0148] In one embodiment, the pharmaceutical composition may be formulated as a liquid dosage form suitable for oral, intracavitary, intradermal, intramuscular, intrathecal, intravenous, subcutaneous, or intraventricular administration. For example, see the literature [Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984)].

[0149] In one embodiment, the liquid dosage form of the pharmaceutical composition disclosed herein may further include a diluent. In some embodiments, the inert diluent is saline solution.

[0150] In one embodiment, the liquid dosage form of the pharmaceutical composition as described herein may further comprise a buffer. For example, buffers suitable for use in this invention include organic acids and inorganic acids and their salts, such as citrate buffers (e.g., a mixture of monosodium citrate and disodium citrate, a mixture of citrate and trisodium citrate, a mixture of citrate and monosodium citrate), succinate buffers (e.g., a mixture of succinate and monosodium succinate, a mixture of succinate and sodium hydroxide, a mixture of succinate and disodium succinate), tartrate buffers (e.g., a mixture of tartaric acid and sodium tartrate, a mixture of tartaric acid and potassium tartrate, a mixture of tartaric acid and sodium hydroxide), and fumarate buffers (e.g., monosodium fumarate). Mixtures, fumarate-disodium fumarate mixtures, monosodium fumarate-disodium fumarate mixtures, gluconate buffers (e.g., gluconic acid-sodium gluconate mixtures, gluconic acid-sodium hydroxide mixtures, gluconic acid-potassium gluconate mixtures), oxalate buffers (e.g., oxalate-sodium oxalate mixtures, oxalate-sodium hydroxide mixtures, oxalate-potassium oxalate mixtures, etc.), lactate buffers (e.g., lactate-sodium lactate mixtures, lactate-sodium hydroxide mixtures, lactate-potassium lactate mixtures, etc.), and acetate buffers (e.g., acetate-sodium acetate mixtures, acetate-sodium hydroxide mixtures, etc.). Additionally, phosphate buffers, histidine buffers, and trimethylamine salts, such as Tris, can be used.

[0151] In one embodiment, the liquid dosage form of the pharmaceutical composition disclosed herein may further comprise a preservative. For example, preservatives suitable for use in this invention include, but are not limited to, phenol, benzyl alcohol, m-cresol, methylparaben, propylparaben, octadecyl dimethyl benzyl ammonium chloride, benzalkonium chloride (e.g., chloride, bromide, and iodide), hexamethyl chloride, and alkylparabens (e.g., methylparaben or propylparaben, catechol, resorcinol, cyclohexanol, and 3-pentanol).

[0152] In one embodiment, the liquid dosage form of the pharmaceutical composition disclosed herein may further include a stabilizer. Suitable stabilizers include, for example, polyols, triols and more, amino acids, organic sugars or sugar alcohols, polyvinylpyrrolidone, monosaccharides, trisaccharides, polysaccharides, proteins, sulfur-containing reducing agents, amino acid polymers, and polyethylene glycol.

[0153] In one embodiment, the liquid dosage form of the pharmaceutical composition disclosed herein may further include a solubilizer. Specifically, the solubilizer is an ionic surfactant. Examples of nonionic surfactants include, but are not limited to, polysorbates, poloxamer, poloxamer polyols, and polyoxyethylene sorbitan monoether.

[0154] In one embodiment, the pharmaceutical compositions disclosed herein can be prepared into lyophilized formulations or aqueous solutions for storage by mixing the composition with any pharmaceutically acceptable carrier, excipient, or stabilizer (e.g., buffers, stabilizers, preservatives, isotonic agents, nonionic detergents, antioxidants, and various other additives) commonly used in the art.

[0155] The term "application" means the provision of a substance (e.g., a pharmaceutical composition comprising the compound of formula (1) of the present invention or a new crystal form thereof as an active ingredient) to a subject for the purpose of prevention or treatment (e.g., a neurological disease or a neurodegenerative disease).

[0156] Administration can be performed via any route, including oral, parenteral, and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or percutaneous). Parenteral administration refers to administration by injection (e.g., bolus) or infusion. Parenteral administration includes subcutaneous injection, intravenous injection, intramuscular injection, intra-arterial injection, intraperitoneal injection, intracerebral injection, intrathecal injection, or intraventricular injection. Other delivery methods include, but are not limited to, the use of liposomal formulations and transdermal patches.

[0157] When the compound is administered orally, it can be formulated into pills, capsules, pouches, tablets, etc., together with pharmaceutically acceptable excipients.

[0158] Compressed tablets can be prepared by compressing an active ingredient in a free-flowing form (e.g., powder or granules) in a suitable machine, the active ingredient optionally mixed with a binder, lubricant, inert diluent, preservative, surfactant, or dispersant. Molded tablets can be prepared by molding a mixture of powdered active ingredients wetted with an inert liquid diluent in a suitable machine. Tablets may optionally be coated or scored and may optionally be formulated to provide a slow or controlled release of the active ingredient. Tablets, lozenges, sugar lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules (e.g., gelatin capsules), syrups, or elixirs can all be prepared for oral administration. Formulations of the compounds of the present invention intended for oral administration can be prepared according to any method known in the art for preparing pharmaceutical compositions, and such compositions may contain one or more agents, including sweeteners, flavoring agents, coloring agents, and preservatives, to provide a palatable formulation. Tablets containing the active ingredient are suitable for mixing with non-toxic, pharmaceutically acceptable excipients suitable for tablet preparation. Such excipients may include, for example, inert diluents such as calcium carbonate or sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrants such as corn starch or alginate; binders such as starch, gelatin or gum arabic; and lubricants such as magnesium stearate, stearic acid or talc. Tablets may be uncoated or coated using known techniques, including microencapsulation, to delay disintegration and absorption in the gastrointestinal tract and provide sustained action over a longer period. For example, delaying agents such as glyceryl monostearate or glyceryl distearate may be used alone or in combination with waxes.

[0159] In one embodiment, the pharmaceutical composition of the present invention may be administered to a subject, for example, by oral, intracavitary, intradermal, intramuscular, intrathecal, intravenous, subcutaneous, or intraventricular administration.

[0160] The pharmaceutical formulation of the present invention can be applied to mammals, such as humans, but can also be applied to other mammals, such as animals requiring veterinary treatment, such as domestic animals (e.g., dogs, cats, etc.), farm animals (e.g., cattle, sheep, pigs, horses, etc.) and laboratory animals (e.g., rats, mice, guinea pigs, etc.).

[0161] The active ingredient and its effective amount in the pharmaceutical composition may vary depending on the rate of absorption, inactivation, and excretion of the drug, as well as other factors known to those skilled in the art. It should also be noted that the therapeutically effective amount will vary depending on the severity of the disease to be alleviated. It should be further understood that, for any particular subject, the specific dosing regimen should be adjusted over time based on the subject's needs and the professional judgment of the person administering or supervising the administration of the composition, and the concentration ranges set forth herein are merely exemplary and not intended to limit the scope or implementation of the claimed compositions. The active ingredient may be administered once or divided into multiple smaller doses administered at different time intervals.

[0162] The term "effective amount" or "therapeutic effective amount" refers to an amount of compound, composition, or medicine sufficient to treat a specified disease, condition, or disorder, for example by enhancing a specified function, such as enhancing or increasing desired and / or beneficial function, or reducing or weakening undesired function; or improving, temporarily relieving, alleviating, and / or delaying one or more symptoms.

[0163] In some embodiments, the pharmaceutical composition may comprise about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1250 mg, about 1500 mg, about 1750 mg, about 2000 mg, about 2250 mg, about 2500 mg, about 2750 mg, or about 3000 mg of the compound.

[0164] In some embodiments, the pharmaceutical composition may contain about 0.01 mg to about 125 mg, preferably about 1 mg to about 50 mg per kg of subject body weight, administered once or more daily to achieve the desired therapeutic effect.

[0165] In one embodiment, based on the total weight of the composition, the composition may contain, for example, 0.0001 to 99.9% by weight, preferably 0.001 to 50% by weight of the compound. The content ratio is based on a dry weight after removing the solvent.

[0166] In another embodiment, the composition may comprise a 5% (w / v), 10% (w / v), 15% (w / v), 20% (w / v), 25% (w / v), 30% (w / v), 35% (w / v), or 40% (w / v) aqueous solution of one or more compounds. In some embodiments, an effective amount of the composition may comprise a 25% (w / v) solution of one or more compounds.

[0167] Biological assay This article describes a method for selecting a crystal form that can be used in this invention.

[0168] Blood-brain barrier permeability is a crucial property in decision-making for evaluating compounds as potential drug candidates for brain diseases. Drugs typically need to cross the blood-brain barrier (BBB) ​​to reach their targets, making this an important property for assessing a compound's ability to passively cross this membrane. The BBB is composed of tightly connected brain endothelial cells. In drug discovery, rapid early BBB penetration screening of compounds is highly desirable.

[0169] In one embodiment, the crystal form of the present invention is selected based on its BBB film permeability.

[0170] Detailed examples of methods that can be used to select the crystal form of the present invention are described herein.

[0171] Therapeutic uses of the compounds of this invention In another aspect of the invention, a method for preventing or treating a neurological disease or neurodegenerative disease is provided, comprising administering to a subject who requires prevention or treatment of a neurological disease or neurodegenerative disease a crystalline form of the compound of formula (1) disclosed herein.

[0172] The term "neurological disorders" includes neurodegenerative diseases, neuropsychiatric disorders, amnesia, cognitive impairment / disorders, and extinction learning disabilities.

[0173] The term "neurodegenerative disease" refers to any disorder that can be reversed, inhibited, managed, treated, improved, or eliminated by agents that stimulate the generation of new neurons.

[0174] The compounds of this invention can be used to enhance synaptic plasticity. In one embodiment, the compounds described herein may have therapeutic uses because reduced synaptic plasticity is also associated with the decline of brain function in adults. In particular, synaptic plasticity in thalamic cortical circuits is significantly limited with age, and therefore, reactivating or enhancing cortical synaptic plasticity through therapeutic means may help treat diseases caused by reduced synaptic plasticity. Therefore, the crystal forms described herein may be useful in cases where brain pathology is associated with or mediated by reduced synaptic plasticity.

[0175] The compounds of the present invention can be used to inhibit or eliminate β-amyloid protein accumulation. In one embodiment, the compounds described herein can be used to treat Alzheimer's disease caused by inhibiting or eliminating β-amyloid protein accumulation. That is, the crystal forms described herein may be useful in cases where brain pathology is associated with or mediated by β-amyloid protein accumulation.

[0176] Neurogenesis, or the birth of new neurons, was once thought to occur only in developing organisms. However, recent research has demonstrated that neurogenesis continues into adulthood and persists throughout adulthood. This ongoing neurogenesis is considered a crucial mechanism underlying neural plasticity, enabling organisms to adapt to environmental changes and influencing learning and memory throughout life.

[0177] In one embodiment, the present invention includes a method for increasing synaptic density in a subject, comprising administering the compound of the present invention to a subject who requires an increase in synaptic density.

[0178] In another embodiment, the present invention includes a method for increasing synaptic plasticity in a subject, comprising administering the compound of the present invention to a subject who requires an increase in synaptic plasticity.

[0179] In yet another embodiment, the present invention includes a method for increasing the density of neuronal dendrites in a subject, comprising administering the compound of the present invention to a subject who requires an increase in the density of neuronal dendrites.

[0180] This invention provides a method for improving memory in subjects with memory impairments. Examples of memory impairment types include, but are not limited to: Alzheimer's disease, amnesia, bromine poisoning, fugue state, Korsakov's syndrome, prosopagnosia, Ribot's Law, repressed memory, false memory syndrome, memory distrust syndrome, memory overload syndrome, amnesia (e.g., childhood amnesia, partial amnesia, post-traumatic amnesia, psychogenic amnesia, anterograde amnesia, retrograde amnesia, selective amnesia, source amnesia, transient epileptic amnesia, transient generalized amnesia, blackout (alcohol-related amnesia)), source monitoring error, the seven sins of memory, the tip of the tongue phenomenon, and twilight sleep.

[0181] In one implementation, the memory impairment may be Alzheimer's disease. Such methods include optionally administering an inhibitor to a subject with Alzheimer's disease and monitoring the subject to determine whether previously lost memories have been recovered. The subject can be monitored using standard tests known in the art.

[0182] In one embodiment, the Alzheimer's disease subject may be a subject suffering from advanced Alzheimer's disease. Many compounds proposed for the treatment of Alzheimer's disease aim to treat the early stages of the disease by preventing plaque buildup. The compounds of the present invention improve memory and cognition while preventing plaque buildup, and are therefore suitable for treating both early and late-stage dementia.

[0183] In one embodiment, a disease or disorder mediated by decreased synaptic plasticity and / or β-amyloid accumulation can be cognitive impairment / damage. Therefore, a method for enhancing the cognitive function of a subject suffering from cognitive impairment / damage is also provided, comprising administering an effective amount of at least one of the compounds described herein to the subject in need. The present invention also provides a method for enhancing the cognitive function of a normal subject.

[0184] The subject's "cognitive function" can be defined as intellectual activities or processes. Examples of intellectual activities or processes include, but are not limited to, attention, processing speed, learning and memory, executive function, verbal fluency, and working memory. For example, when the composition of the present invention improves one or more intellectual activities or processes in a subject with impaired cognitive function, it can improve cognitive function.

[0185] The term "memory" refers to the ability to retrieve information about past events or knowledge.

[0186] The term "age-related memory loss" refers to any continuous state characterized by deterioration of neurological function but not reaching the level of dementia, as further defined in this paper and with reference to the definition in the literature [Diagnostic and Statistical Manual of Mental Disorders: 4th Edition of the American Psychiatric Association (DSM-IV, 1994)].

[0187] The term "injury-related memory loss" refers to memory loss that is present in the presence of brain injury and may also be caused by neurological damage.

[0188] The term "impaired cognitive function" refers to cognitive function that is not as robust as that observed in age-matched normal subjects, and includes a state of cognitive decline.

[0189] In some cases, cognitive function may be reduced by approximately 5%, 10%, 30%, or more compared to that measured in age-matched normal subjects. Cognitive function can improve to any detectable degree, but in humans, improvement is preferably sufficient to enable subjects with impaired cognitive function to perform normal daily activities.

[0190] Cognitive function can be assessed by one or more tests or measurements of cognitive function, and can optionally be defined accordingly. Non-limiting examples of cognitive function tests or measurements may include, but are not limited to: CANTAB (e.g., see [Fray et al. "CANTAB battery: proposed utility in neurotoxicology." Neurotoxicol Teratol 1996; 18(4):499-504]), Stroop test, TrailMaking test, Wechsler Digit Span, or CogState computerized cognitive test (see [Dehaene et al. "Reward-dependent learning in neuronal networks for planning and decision making." Brain Res. 2000; 126:21729]; [Iverson et al. "Interpreting change on the WAIS-III / WMS-I11 in clinical samples." Arch Clin Neuropsychol. 2001;16(2):183-91]; and [Weaver et al. "Mild memory impairment in healthy older adults is distinct from normal aging." CogState. [2006;60(2):146-55]). The methods of the present invention can be used to enhance the cognitive function of normal subjects, treat and / or alleviate cognitive impairment in subjects with cognitive impairment, and / or prevent cognitive impairment in subjects. As used herein, normal subjects are subjects who have not been diagnosed with a cognitive impairment-related disorder.

[0191] In one implementation scheme, cognitive impairment or impairment may be associated with, but is not limited to, the following: Alzheimer's disease, Huntington's disease, memory loss due to seizures, schizophrenia, Rubinstein-Taybi syndrome, Rett syndrome, Fragile X syndrome, Lewy body dementia, vascular dementia, social, cognitive, and learning impairments associated with bipolar disorder and autism spectrum disorder (ASD), attention deficit hyperactivity disorder (ADHD), dyslexia, learning disabilities, traumatic head injury, cognitive and motor impairments due to stroke, traumatic brain injury, cognitive impairment mediated by neurodegeneration and neuronal loss, and attention deficit disorder.

[0192] In one implementation, the cognitive impairment or impairment may be associated with, but is not limited to, anxiety disorders, conditioned fear responses, panic disorder, obsessive-compulsive disorder, post-traumatic stress disorder, phobias, social anxiety disorder, substance dependence recovery or age-related memory disorder (AAMI), and age-related cognitive decline (ARCD).

[0193] In one embodiment, a disease or disorder mediated by reduced synaptic plasticity and / or β-amyloid accumulation may be an extinction learning disorder, such as a fear extinction deficit. Therefore, a method for treating or preventing an extinction learning disorder in a subject is also provided, comprising administering an effective amount of at least one of the compounds described herein to the subject suffering from the extinction learning disorder.

[0194] The compounds of this invention can be used to promote the psychological process of extinction learning, and therefore can be used to treat, alleviate, and / or prevent neuropsychiatric disorders and other related disorders. Unlike traditional anti-anxiety medications that are administered long-term and treat the physiological symptoms of anxiety, the compounds of this invention can be used in combination with secondary therapies (e.g., psychotherapy) for long-term or short-term use.

[0195] In some embodiments, diseases or disorders mediated by decreased synaptic plasticity and / or β-amyloid accumulation may be immune disorders, which may be co-treated with TNFα or other immunomodulators when administered to a subject in a therapeutically effective amount of at least one of the compounds described herein. Therefore, a method for treating immune disorders mediated by decreased synaptic plasticity and / or β-amyloid accumulation in a subject is also provided, comprising administering to a subject requiring treatment of the immune disorder a therapeutically effective amount of at least one of the compounds described herein before, during, or after treatment with TNFα or other immunomodulators.

[0196] In one embodiment, diseases or disorders mediated by decreased synaptic plasticity and / or β-amyloid accumulation include disorders that can also be treated with precursor / stem cell therapy, such as: diabetes-related disorders (e.g., organ failure, cirrhosis, and hepatitis); central nervous system (CNS) disorders associated with precursor cell dysregulation in the brain (e.g., PTSD); tumors (e.g., retinoblastoma); disorders affecting oligodendrocyte precursor cells (e.g., astrocytoma and ependymoma); multiple sclerosis; demyelinating disorders, such as leukodystrophy; neuropathy associated with white matter loss; cerebellar disorders, such as ataxia; and olfactory precursor disorders (e.g., anosmia). Therefore, a method for treating a disorder mediated by decreased synaptic plasticity and / or β-amyloid accumulation in a subject is also provided, comprising administering a therapeutically effective amount of at least one of the compounds described herein to a subject requiring treatment for a disorder mediated by decreased synaptic plasticity and / or β-amyloid accumulation before, during, or after treatment with precursor / stem cell therapy.

[0197] In one embodiment, diseases or disorders mediated by decreased synaptic plasticity and / or β-amyloid accumulation may include blood pressure disorders related to nitric oxide (NO) regulation (e.g., hypertension, erectile dysfunction, asthma; and eye disorders, such as glaucoma). Therefore, a method for treating a subject with blood pressure disorders mediated by decreased synaptic plasticity and / or β-amyloid accumulation related to nitric oxide (NO) regulation is also provided, comprising administering a therapeutically effective amount of at least one of the compounds described herein to a subject requiring treatment for blood pressure disorders mediated by decreased synaptic plasticity and / or β-amyloid accumulation related to nitric oxide (NO) regulation.

[0198] In one embodiment, the disease or disorder may be cardiac hypertrophy disorder. Therefore, a method for treating cardiac hypertrophy disorder mediated by decreased synaptic plasticity and / or β-amyloid accumulation in a subject is also provided, comprising administering a therapeutically effective amount of at least one of the compounds described herein to a subject requiring treatment for cardiac hypertrophy disorder mediated by decreased synaptic plasticity and / or β-amyloid accumulation.

[0199] In one embodiment, diseases or disorders mediated by decreased synaptic plasticity and / or β-amyloid accumulation may include hematological disorders such as thalassemia, anemia, and sickle cell anemia. Therefore, a method for treating hematological disorders mediated by decreased synaptic plasticity and / or β-amyloid accumulation in a subject is also provided, comprising administering a therapeutically effective amount of at least one of the compounds described herein to a subject requiring treatment for a hematological disorder mediated by decreased synaptic plasticity and / or β-amyloid accumulation.

[0200] In one embodiment, a disease or disorder mediated by decreased synaptic plasticity and / or β-amyloid accumulation may include a metabolic disease, such as prediabetes or diabetes (type I or type II). Therefore, a method for treating a subject with a metabolic disease mediated by decreased synaptic plasticity and / or β-amyloid accumulation (e.g., prediabetes or diabetes (type I or type II)) is also provided, comprising administering a therapeutically effective amount of at least one of the compounds described herein to a subject requiring treatment for a metabolic disease.

[0201] In one aspect of the invention, a pharmaceutical composition or kit is provided, further comprising a second therapeutic agent, for treating a subject suffering from a disease or disorder mediated by decreased synaptic plasticity and / or β-amyloid accumulation. In one embodiment, the second therapeutic agent may be administered in combination to treat a disease or disorder mediated by decreased synaptic plasticity and / or β-amyloid accumulation.

[0202] The term "combined administration" may mean that the compositions described herein are intended to be administered simultaneously, immediately before, or immediately after the application of one or more other therapies (e.g., anticancer agents, chemotherapeutic agents, or neurodegenerative disease treatments). In one embodiment, the compounds of the present invention may be administered to a subject alone or in combination. Combined administration is intended to include the simultaneous or sequential administration of the compounds, alone or in combination (one or more compounds or formulations). Thus, if desired (e.g., to reduce metabolic degradation), the formulations may also be combined with other active substances.

[0203] In addition, a method is provided for treating a subject with a disease or disorder mediated by reduced synaptic plasticity and / or β-amyloid accumulation, wherein at least one compound described herein or a pharmaceutically acceptable salt thereof may be administered to the subject in combination with one or more additional active agents (second therapeutic agents).

[0204] In one embodiment, the second therapeutic agent may be a drug for the prevention or treatment of neurological diseases or neurodegenerative diseases. Preferably, it may be a drug that exhibits a synergistic effect when used with the compounds of the present invention, or a drug that can improve the preventive or therapeutic effect by complementing each other.

[0205] In one embodiment, the second therapeutic agent may be selected from, but is not limited to, the group consisting of: cholinesterase inhibitors, NMDA receptor antagonists, humanized antibodies targeting tau protein, humanized antibodies targeting β-amyloid protein, and BACE inhibitors. The humanized antibodies targeting tau protein or β-amyloid protein may be antibodies that inhibit or reduce the accumulation of tau protein or β-amyloid protein, respectively.

[0206] In one embodiment, the second therapeutic agent may be selected from, but is not limited to, the group consisting of donepezil, rivastigmine, galantamine, memantine, verubecestat, solanumab, bapinzumab, aducanumab, lencanemab, tideglusib, epothilone D, and ABBV-8E12.

[0207] Typical therapies included in this invention include combination therapies. For example, the combination therapy may be a combination of pharmacological therapy (i.e., the compounds of this invention) and behavioral therapy. Behavioral therapy includes, but is not limited to, electroconvulsive therapy, exercise, group therapy, talk therapy, or conditioning. In another embodiment, the behavioral therapy may be cognitive behavioral therapy. Examples of behavioral therapies that can be used in this method can be found, for example, in the literature [Cognitive-Behavioral Therapies by K. Dobson, ed., Guilford Publications, Inc., 2002] and [The new Handbook of Cognitive Therapy: Basics and Beyond by Judith SS Beck, Guilford Publications, Inc. 1995], the entire contents of which are incorporated herein by reference.

[0208] Furthermore, any pharmaceutically active ingredient can be used as a pharmacological agent to enhance learning or conditioned reflexes in the treatment methods of the present invention, and the pharmacological agent to enhance learning or conditioned reflexes can be appropriately selected by those skilled in the art. For example, one class of pharmaceutically active ingredients considered herein includes compounds that increase norepinephrine levels in the brain. Such compounds may include compounds that are norepinephrine reuptake inhibitors (e.g., atomoxetine, reboxetine, duloxetine, venlafaxine, and mirtazapine) and compounds that induce norepinephrine release (e.g., amphetamine, dextroamphetamine, pimoxetine, and methylphenidate). Another class of such pharmaceutically active ingredients may be compounds that increase acetylcholine levels in the brain, and may include, for example, compounds that block the degradation of acetylcholine. Examples of such compounds may include, but are not limited to, the cholinesterase activity inhibitor Aricept (generic name: donepezil hydrochloride) and tacrine capsules.

[0209] Methods for use in combination therapy may include administering one or more courses of a combination therapy regimen to a subject, wherein the combination therapy regimen may include a short-term, therapeutically effective dose of a compound of the present invention for enhancing learning or conditioning, administered in conjunction with a psychotherapy course. In one embodiment, exposure to the compound may occur within approximately 24 hours prior to the start of the psychotherapy course, preferably within approximately 12 hours, and more preferably within approximately 6 hours. The entire course of treatment for neuropsychiatric disorders may involve at least one course of such a combination therapy regimen.

[0210] The invention will be described in more detail below through the following embodiments. These embodiments are provided to aid in understanding the invention and are not intended to limit its scope in any way, nor should they be construed as limiting its scope.

[0211] The methods for obtaining the compounds described herein or their pharmaceutically acceptable salts will be obvious to those skilled in the art, and suitable procedures are described, for example, in the following preparation examples and in the references cited herein.

[0212] Example Examples of representative polymorphs included within the scope of this invention are described in the following embodiments. The polymorphs described in the following preparation examples are provided to enable those skilled in the art to more clearly understand and practice this invention.

[0213] Generally, the nomenclature used in this invention is based on AUTONOM (trademark) v.4.0, a computerized system of the Beilstein Institute used to generate IUPAC system names. If the structure shown differs from the name given to that structure, the structure shown shall prevail. Furthermore, if the stereochemistry of a structure or part thereof is not indicated, for example, by bold or dashed lines, then that structure or part thereof shall be interpreted to encompass all its stereoisomers.

[0214] Experimental methods Experimental Method 1. Nuclear Magnetic Resonance Spectroscopy (nmR) Instrument model: Bruker Avance-AV 400M (for ¹H-NMR) Detailed 1 The H-NMR parameters are as follows.

[0215] Frequency: 400MHz Probe: 5mMPABBO BB / 19F- 1 H / D Z-GRD Z108618 / 0406 Number of scans: 8 Temperature: Approximately 24℃ Relaxation time: 1 second Experimental Method 2. X-ray Powder Diffraction (XRPD) Instrument Model: Bruker D8 Advance X-ray Diffractometer The detailed XRPD parameters are as follows.

[0216] X-ray tube: Cu, kα1 (λ=1.54056Å) X-ray tube voltage: 40kV, X-ray tube current: 40mA Scanning range: 2 to 40 degrees or 3 to 40 degrees Step size: 0.02 degrees Step duration: 0.3 seconds or 0.12 seconds Sample stage rotation speed: 15 rpm Experimental Method 3. Differential Scanning Calorimetry (DSC) Instrument Model: TA Discovery 2500 Differential Scanning Calorimeter The sample (approximately 1 to 2 mg) was placed in a DSC aluminum dish and tested. The sample was heated from 0°C to 250°C at a heating rate of 10°C / min under N2 conditions of 50 mL / min.

[0217] Experimental Method 4. Thermogravimetric Analysis (TGA) Instrument Model: Discovery 5500 Thermogravimetric Analyzer The sample (approximately 2 to 10 mg) was placed in a TGA platinum dish and tested. The sample was heated from room temperature to 300°C or until the weight was reduced by 20% under N2 conditions at a heating rate of 10°C / min.

[0218] Experimental Method 5. Dynamic Vapor Adsorption (DVS) Instrument Model: SMS DVS Advantage Dynamic Moisture Adsorption Analyzer Test method: Take a sample (10 to 15 mg) and place it on the DVS sample tray for testing.

[0219] The detailed DVS parameters are as follows.

[0220] Temperature: 25℃ Balance dm / dt = 0.002% / minute: (Minimum: 60 minutes, Maximum: 240 minutes) Relative humidity (RH (%)) measurement gradient: 10% Relative humidity (RH (%)) measurement gradient cycle: 40-0-95-0-40% The following description is a criterion for judging hygroscopicity: [Table 6] Hygroscopicity classification standards

[0221] * Indicates the weight increase due to moisture absorption at 25℃ / 80%RH.

[0222] Experimental Method 6. High-performance liquid chromatography (HPLC) The HPLC analysis methods are shown in Table 7.

[0223] [Table 7]

[0224] Experimental Method 7. Parallel Artificial Membrane Permeability Measurement (PAMPA) - PAMPA: PMBBB-096 Reagent Kit - Measurement method: 96-well plate - Reference substance: Imipramine (H) (10 μM) - Test substance concentration: 10 μM - Number of repetitions: 3 - Reaction time: 6 hours at 25°C on a shaker (75-80 rpm). - Analysis method: LC-MS / MS - Data processing: P e (cm / s) Example 1. Preparation of compound of formula (1) By means of non-limiting examples, the compound of formula (1) ((3S,6R)-1-(2-(2-chloro-4-fluorophenyl)acetyl)-6-methylpiperidine-3-carboxylic acid) can be prepared as shown in reaction scheme 1 below and in the examples disclosed herein.

[0225] Equation (1)

[0226] [Reaction Scheme 1]

[0227] Example 1.1. Preparation of methyl (3S,6R)-1-(2-(2-chloro-4-fluorophenyl)acetyl)-6-methylpiperidine-3-carboxylic acid (Step 1) 1.7 kg of 2-(2-chloro-4-fluorophenyl)acetic acid, 1.05 eq. of methyl (3S,6R)-6-methylpiperidin-3-carboxylic acid, 1.5 eq. of DIPEA, 0.3 eq. of HOBt, and 1.2 eq. of EDC HCl were added to a 2-MeTHF solution (10 V). The mixture was heated at 25-35 °C for 1.3 hours. The mixture was washed twice with H₂O (5 V) to obtain methyl (3S,6R)-1-(2-(2-chloro-4-fluorophenyl)acetyl)-6-methylpiperidin-3-carboxylic acid with a purity of 99.6%.

[0228] Example 1.2. Preparation of ((3S,6R)-1-(2-(2-chloro-4-fluorophenyl)acetyl)-6-methylpiperidine-3-carboxylic acid) (Step 2) Methyl (3S,6R)-1-(2-(2-chloro-4-fluorophenyl)acetyl)-6-methylpiperidine-3-carboxylic acid was scaled up to 2.953 kg. Methyl (3S,6R)-1-(2-(2-chloro-4-fluorophenyl)acetyl)-6-methylpiperidine-3-carboxylic acid was added to 2-MeTHF (10 V) with 1N NaOH (1.3 eq.). The mixture was heated at 25 °C for 3 hours. Crystallization in 2-MeTHF / n-heptane yielded 2.754 kg of (3S,6R)-1-(2-(2-chloro-4-fluorophenyl)acetyl)-6-methylpiperidine-3-carboxylic acid with impurities of 0.1% or less.

[0229] Example 1.3. Purification of ((3S,6R)-1-(2-(2-chloro-4-fluorophenyl)acetyl)-6-methylpiperidine-3-carboxylic acid (Step 3) (3S,6R)-1-(2-(2-chloro-4-fluorophenyl)acetyl)-6-methylpiperidine-3-carboxylic acid (2.57 kg) was added to EA (5 V) and n-heptane (15 V). The mixture was heated from 0 °C to 45 °C to obtain 2.404 kg of white solid dried filter cake.

[0230] Example 2. Preparation of crystal form A (anhydrous form) of compound (1) Crystal form A of compound (1) is prepared by the following method.

[0231] (1) Equilibrate at 25°C for 2 weeks or at 50°C for 1 week in MEK, MTBE, IPAc, THF / heptane (v:v=1:1), 2-MeTHF / heptane (v:v=1:1), DMSO / water (v:v=24:76; aw=0.9), and DMF / water (v:v=31:69; aw=0.9). (2) Equilibrate at 25°C for 2 weeks in acetone or DCM / heptane (v:v=1:1). (3) Equilibrate in toluene at 50°C for 1 week. (4) Equilibrate for 10 cycles in acetone, ACN, or 1,4-dioxane / heptane (v:v=1:1) at a temperature cycling range of 5°C to 50°C, with a heating / cooling rate of 0.1°C / min. (5) Slow evaporation (approximately 20-25°C, 40-70%RH) and rapid evaporation (using dry nitrogen at approximately 20-25°C) can be carried out in most solvent systems (methanol, ethanol, acetone, ACN, etc.). (6) Slowly cool in acetone (dissolve in a minimum amount of solvent at 50°C, then filter through a 0.45 μm syringe membrane filter, cool the resulting clear solution to 5°C at a rate of 0.1°C / min, and further cool to -20°C). (7) Rapidly cool in acetone or ACN (dissolve in a minimum amount of solvent at 50°C, then filter through a 0.45 μm syringe membrane filter, and stir the resulting clear solution in an ice bath at 0°C). (8) Crystallization in ethanol by adding an antisolvent (water) (dissolving in a minimal amount of solvent at room temperature, then filtering through a 0.45 μm syringe membrane filter, adding 4 to 8 times the volume of the antisolvent to the resulting clear solution to form a precipitate), and (9) Crystallize by vapor diffusion using 1,4-dioxane, MEK or acetone (dissolve in a minimum amount of solvent at room temperature of about 20 to 25°C, then filter through a 0.45 μm syringe membrane filter, transfer the resulting clear solution to an open 4 mL glass vial, then transfer the 4 mL vial to a 40 mL glass vial, and add the antisolvent (heptane, heptane or MTBE); then tighten the cap and let stand at room temperature for up to 14 days).

[0232] In the above method, equilibration is carried out using a stir bar on a magnetic stirring plate at 300 to 400 rpm, and the final precipitate is centrifuged at 14,000 rpm through a 0.45 μm syringe membrane filter to obtain a solid of crystal form A.

[0233] Meanwhile, the crystal forms B, C, D and E of compound (1) are prepared from crystal form A, and a schematic diagram of the interconversion relationships between crystal forms A, B, C, D and E is shown in [the diagram]. Figure 6 The specific preparation methods for crystal forms B, C, D and E of the compound of formula (1) are described in detail in the following examples.

[0234] Solubility analysis was performed on the obtained crystal form A, and the analysis was conducted using X-ray powder diffraction (XRPD), nuclear magnetic resonance (¹H-NMR), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA) according to the methods described in Experimental Methods 1 to 4. The results are shown in [Table 1]. Figures 1a to 1d middle.

[0235] XRPD results show that it has peaks at diffraction angles 2θ (°) of 4.96, 9.88, 12.09, 13.4, 14.85, 16.2, 18.4, 19.65, 19.83, 20.06, 20.49, 21.47, 22.49, 22.51, 24.04, 24.45, 24.84, 25.23, 27.39, 28.68, 29.64, 30.01, 31.15, 31.5, 35.03, and 36.77. XRPD results indicate that crystal form A has high crystallinity. Figure 1a ).

[0236] A melting peak (128.3℃) with an enthalpy of approximately 61 J / g was confirmed by DSC results. Figure 1c TGA results confirmed that crystal form A exhibits a 0.7% weight loss when heated from room temperature to 120°C. Figure 1d ).pass 1 H-NMR results confirmed the absence of detectable residual solvent. Figure 1b ).

[0237] Solubility assay For solubility analysis, approximately 5 mg or 10 mg of crystal form A was weighed into 2 mL glass vials. 20 μL aliquots of each of the 17 solvents listed in Table 8 were added to the vials to dissolve the drug substance. For approximately 5 mg of crystal form A, the drug substance was dissolved at 25 °C; for approximately 10 mg of crystal form A, the drug substance was dissolved at 50 °C. To aid dissolution, vortexing and sonication were applied, and the maximum volume of each solvent added was 1 mL. The solubility results are shown in Table 8 below.

[0238] [Table 8] Solubility of crystalline form A at 25℃ and 50℃

[0239] Example 3. Preparation of crystal form B (anhydrous form) of compound (1) In ACN and EA, slurry equilibrium via salt screening yielded crystal form B of compound (1) from crystal form A. The obtained solids were also analyzed by XRPD, DSC, and TGA, and the results are shown in [Figures to be inserted]. Figures 2a to 2c middle.

[0240] XRPD results show that it has peaks at diffraction angles of 2θ (°) of 8.22, 8.62, 8.64, 9.09, 9.12, 11.88, 14.21, 14.7, 15.09, 15.78, 16.48, 18.01, 18.28, 19.26, 19.99, 20.34, 20.67, 22.14, 22.61, 22.9, 23.88, 24.49, 24.78, 25.86, 26.4, 26.7, 26.95, 27.58, 27.77, 28.11, 28.89, 29.05, 32.59, and 36.46. Figure 2a XRPD results showed that crystal form B has high crystallinity. DSC results confirmed a melting peak of approximately 64 J / g enthalpy (111.4 °C) and an endothermic peak of approximately 2 J / g enthalpy (126.8 °C). Figure 2b TGA results confirmed that crystal form B exhibits a 0.4% weight loss when heated from room temperature to 110°C. Figure 2c Crystal form B exhibits lower reproducibility.

[0241] Example 4. Preparation of crystal form C (anhydrous form) of compound (1) Crystal form C was prepared by the following process: (1) equilibration at 25°C for 2 weeks in ACN or EA; (2) equilibration at 5°C to 50°C for 10 cycles at a heating / cooling rate of 0.1°C / min in EA; (3) rapid cooling in MEK, EA, MTBE, IPAc, THF / heptane (v:v=1:1), 2-MeTHF / heptane (v:v=1:1), and acetone / water (v:v=35:65, aw=0.9); (4) slow cooling in EA; (5) addition of an antisolvent (water or heptane, respectively) in acetone or EA; and (6) competitive equilibration of crystal forms A and C at 50°C or lower in acetone, ACN, EA, IPAc, and acetone / water (v:v=35:65, aw=0.9).

[0242] In the above method, equilibration, rapid cooling, slow cooling, addition of antisolvent, and recovery of the final precipitate are carried out in the same manner as described in the preparation of crystal form A.

[0243] The specific method for achieving competitive equilibrium is as follows: 500 mg of crystal form A was weighed into an 8 mL glass vial. 2 mL of EA (suspension) was added to the vial at room temperature (approximately 20-25°C). Approximately 10 mg of crystal form C seed crystals (crystal form A) was added to the suspension. The suspension was continuously stirred at 25°C for approximately 2 days. The suspension was cooled to 5°C and continuously stirred at 5°C for approximately 2 days. To conduct the competitive equilibrium experiment, approximately 156 mg of crystal form C was separated by centrifugation. 300 mg of crystal form A (suspension) was added to the suspension. The suspension was heated to 40°C and continuously stirred at 40°C for approximately 3 days. The solid was collected by filtration. Approximately 397 mg of crystal form C was obtained as a white solid, with a yield of 49.6%.

[0244] The obtained solid was also processed by XRPD, 1 Analysis was performed using H-NMR, DSC, and TGA, and the results are shown in [Figures 1-4]. Figures 3a to 3d middle.

[0245] XRPD results show that it has peaks at diffraction angles of 2θ (°) of 8.55, 8.57, 10.03, 15.06, 15.96, 17.18, 17.96, 17.98, 20.17, 21.05, 23.38, 25.51, 26.06, 26.43, and 34.24. Figure 3a XRPD results indicate that crystal form C has high crystallinity.

[0246] A melting peak (116.1℃) with an enthalpy of approximately 76 J / g was confirmed by DSC results. Figure 3c TGA results confirmed that crystal form C exhibits a 0.3% weight loss when heated from room temperature to 110°C. Figure 3d ).

[0247] Example 5. Preparation of crystal form D (toluene solvate) of compound (1) Crystal form D of compound of formula (1) was obtained from crystal form A by equilibration at 25 °C and temperature cycling in toluene. Temperature cycling was performed in 0.1 to 0.2 mL of solvent at a heating / cooling rate of 0.1 °C / min, at a temperature cycling rate of 5 °C to 50 °C. Equilibration was carried out using a stirring bar on a magnetic stirring plate at a speed of 300 to 400 rpm. The obtained solid was also obtained by XRPD, 1 Analysis was performed using H-NMR, DSC, and TGA, and the results are shown in [Figures 1-4]. Figures 4a to 4d middle.

[0248] XRPD results show that it has peaks at diffraction angles of 2θ (°) of 8.76, 9.52, 10.72, 12.5, 15.36, 15.65, 16.1, 16.69, 17.14, 17.57, 18.32, 19.1, 21, 21.53, 22.16, 22.51, 22.88, 23.41, 25.07, 25.25, 26.99, 27.37, 27.78, 28.64, 29.81, 30.87, 32.09, 32.56, and 34.07. Figure 4a XRPD results indicate that crystal form D has high crystallinity.

[0249] The DSC results confirmed the presence of a desolvation peak (approximately 62℃), an endothermic peak with an enthalpy of approximately 39 J / g (114.4℃), and an endothermic peak with an enthalpy of approximately 22 J / g (128.1℃). Figure 4c The TGA results confirmed that crystal form D exhibits approximately 4.8% weight loss when heated from room temperature to approximately 80°C, and approximately 2.5% weight loss when heated from approximately 80°C to 170°C. Figure 4d ). 1 H-NMR results showed the presence of 5.5% toluene residue by weight (0.2 molar equivalents of toluene). Figure 4b ).

[0250] Example 6. Preparation of crystal form E (ethanol solvate) of compound (1) Crystal form E of compound (1) was obtained by cooling crystal form A in ethanol to -20°C. The obtained solid was also subjected to XRPD, 1 Analysis was performed using H-NMR, DSC, and TGA, and the results are shown in [Figures 1-4]. Figures 5a to 5d middle.

[0251] XRPD results show that it has peaks at diffraction angles 2θ (°) of 4.93, 7.16, 9.86, 10.17, 11.29, 13.38, 14.81, 16.1, 16.36, 17.88, 18.33, 18.59, 19.62, 19.82, 20.03, 20.45, 20.82, 21.03, 21.46, 22.46, 22.81, 23.54, 24.01, 24.42, 24.81, 25.2, 25.42, 26.1, 27.35, 28.63, 28.86, 29.53, 29.84, 31.23, 32.52, 32.81, and 39.47. Figure 5a XRPD results showed that crystal form E has high crystallinity. The desolvation peak (approximately 40℃) was confirmed by DSC results. Figure 5cTGA results confirmed that crystal form E exhibits a 10.2% weight loss when heated from room temperature to 130°C. 1 H-NMR results showed the presence of 9.3% ethanol residue by weight (equivalent to 0.7 molar equivalents of ethanol). Figure 5b ).

[0252] Example 7. Competition equilibrium experiment between crystal forms A and C To determine the relative stability of crystal forms A and C, competitive equilibrium experiments were conducted in various solvent systems.

[0253] Approximately 15 mg of crystal form A and 15 mg of crystal form C were added to 0.2 mL of saturated solutions of the selected solvents (acetone, acetonitrile, ethyl acetate, isopropyl acetate, and acetone / water (v:v=35:65)) (Table 9). The resulting suspensions were stirred for one week at 5°C, 25°C, and 50°C, respectively. The solid fractions (wet filter cake) were separated by centrifugation and filtration, and analyzed by XRPD and DSC. The results are shown in Table 9. Figures 7a to 7f middle.

[0254] [Table 9] Competition equilibrium experimental conditions for crystal forms A and C

[0255] XRPD results confirmed that crystal form C was obtained in all solvents listed in Table 9, indicating that crystal form C is the most thermodynamically stable crystal form below 50°C.

[0256] Example 8. Batch stability of crystal form C Crystal form C was placed in open containers at 25℃ / 92.5%RH, open containers at 40℃ / 75%RH, and closed containers at 60℃ for one week, respectively. After stress treatment, the samples were characterized by XRPD and HPLC, and color changes were examined.

[0257] HPLC conditions are described in Experimental Method 6. HPLC analysis results are shown in Table 10 below, and XRPD analysis results are shown in... Figure 8 middle.

[0258] [Table 10] Batch stability results of crystal form C

[0259] HPLC and XRPD results show that crystal form C exhibits high batch stability in both physical and chemical aspects.

[0260] Example 9. Solubility of Crystal Form C Weigh approximately 4 mg of crystalline form C into a 4 mL or 8 mL glass vial, add 4 to 9 mL of dissolving medium, and dissolve the drug substance at 25°C. Approximate solubility is determined by visual observation.

[0261] The solvent and solubility results are shown in Table 11 below (solubility of crystal form C at 37°C).

[0262] [Table 11]

[0263] Example 10. Hygroscopicity of Crystal Form C The water adsorption and desorption behavior of crystal form C was studied using the DVS experiment (method 5). After the DVS test, the change in crystal form was confirmed by measuring XRPD.

[0264] The DVS results are shown in Table 12 below (Water adsorption and desorption experiments of crystal form C (DVS)). Figure 9a and Figure 9b The XRPD results after the DVS test are shown in the figure. Figure 9c middle.

[0265] [Table 12]

[0266] The DVS results show that crystal form C is non-hygroscopic, with a moisture absorption rate of less than 0.2% at RH levels up to 95%. Furthermore, the XRPD results indicate that the sample obtained after the DVS test still exhibits crystallographic pattern C.

[0267] Example 11. Mechanical strength of crystal form C To test the mechanical strength of crystal form C, compression simulation experiments, dry grinding simulation experiments, and wet granulation simulation experiments were conducted.

[0268] Compression simulation experiment Approximately 5 mg of crystalline C was compressed for 5 minutes at 2 MPa, 5 MPa, and 10 MPa using a hydraulic press. Potential changes in crystalline form and crystallinity were evaluated by XRPD.

[0269] The XRPD results of the compression simulation experiment are shown in Figure 10 In China Figure 10 CSE1, CSE2, and CSE3 represent the experimental numbers at pressures of 2MPa, 5MPa, and 10MPa, respectively.

[0270] Dry grinding simulation experiment Approximately 5 mg of crystalline form C was manually ground for 1 minute, 3 minutes, and 5 minutes using a mortar and pestle. Potential changes in crystalline form and crystallinity were assessed by XRPD.

[0271] The XRPD results of the dry grinding simulation experiment are shown in Figure 11 In China Figure 11 GSE1, GSE2, and GSE3 represent the experiment numbers for grinding for 1 minute, 3 minutes, and 5 minutes, respectively.

[0272] Wet granulation simulation experiment Water or ethanol was added dropwise to approximately 10 mg of crystal form C until it was fully wetted. The wet sample was then gently ground using a mortar and pestle. After granulation, the sample was dried under ambient conditions for 10 minutes. Potential changes in crystal form and crystallinity were assessed by XRPD.

[0273] The XRPD results of the wet granulation simulation experiment are shown in Figure 12 In China Figure 12 GNSE1 and GNSE2 represent the experiment numbers for water and ethanol, respectively.

[0274] The above experimental results show that crystal form C exhibits excellent tolerance to compression and granulation with water, without any change in crystal form or a significant decrease in crystallinity. On the other hand, when ground or granulated with ethanol, crystal form C does not show any change in crystal form, but its crystallinity decreases significantly.

[0275] Example 12. BBB Transmittance of Crystal Forms A and C To confirm the BBB permeability of crystal forms A and C of the present invention, PAMPA (parallel artificial membrane permeability measurement) was performed. For specific experimental conditions, please refer to Experimental Method 7.

[0276] PAMPA assay Test compounds: The test compounds (crystal forms A and C) were dissolved in DMSO to prepare a 10 mM (1 mg / mL) standard solution. Next, the dried brain lipids were resuspended in 600 μL of dodecane to prepare a BBB lipid solution in dodecane. The solution was repeatedly aspirated and pipetted (approximately 100 times) until all the dried brain lipids were dissolved (vortexing or sonication may be helpful). A 10 μM reaction solution containing 5% DMSO in PBS was prepared.

[0277] Using the prepared reaction solutions of each test compound and the BBB lipid solution, experiments were conducted in 96-well plates according to the following assay procedure.

[0278] 1. Prepare 500 μL of 500 μM test compound solution in a separate centrifuge tube (mix 25 μL of 10 mM test compound with 475 μL of PBS (pH 7.2) in DMSO). When using the permeability control, dilute it to 500 μM with PBS (mix 25 μL of permeability control with 475 μL of PBS (pH 7.2)).

[0279] 2. Prepare a 200 μM equilibration standard for each test compound and control in separate tubes (mix 80 μL of a 500 μM test compound or control with 120 μL of PBS (pH 7.2)). If the compound can permeate the membrane and reach complete equilibration, 200 μM will be the final concentration of the solution in the donor and acceptor wells. Next, prepare a blank control by mixing 5 μL of DMSO and 245 μL of PBS (pH 7.2) in a separate tube. Set aside the equilibration standards and blank control separately for analysis the following day.

[0280] 3. Add 300 μL of PBS (pH 7.2) to the wells of the receptor plate.

[0281] 4. Leave the donor plate in the tray and add 5 μL of BBB lipid solution dissolved in dodecane directly onto the well membrane of the donor plate. When adding, be careful not to puncture the membrane with the pipette tip.

[0282] 5. To replicate the wells of the donor plate, 200 μL of 500 μM test compound and 500 μM permeability control were added to each well.

[0283] 6. Carefully place the donor plate onto the recipient plate well and incubate at room temperature or 37°C for 18 hours or the desired incubation time (e.g., 16 to 24 hours).

[0284] 7. Carefully remove the donor plate, add solvent, and collect the liquid (recipient solution) from the wells of the acceptor plate for analysis.

[0285] 8. For each test compound and permeability control, add 100 μL of acceptor solution and 100 μL of equilibration standard, and add 100 μL of blank control to the wells of the UV plate (Cat # P96UV).

[0286] 9. To determine the peak absorbance of the test compound, absorbance spectra were measured at 10 nm intervals from 200 nm to 500 nm. Blank controls were used to confirm that the peak was caused by the test compound and not by DMSO in solution. The peak absorbances of the high-transmittance and low-transmittance controls were 250 nm and 270 nm, respectively.

[0287] Data Analysis For the compound and the permeability control, the permeability (P) was determined using the following formula. e ):

[0288] Among them, OD A It is the absorbance of the acceptor solution minus the blank, OD E This is the absorbance of the balance standard minus the blank; each absorbance value uses the peak absorbance determined for each test. In the transmittance formula, C, when using 18 hours of incubation, is C = 7.72 × 10⁻⁶. -6 .

[0289] The PAMPA analysis results obtained using the above method are shown in Table 13 below.

[0290] [Table 13]

[0291] As shown in Table 13, it was confirmed that crystal forms A and C of the present invention have BBB permeability, and it was confirmed that the BBB permeability of crystal form C is better than that of crystal form A.

[0292] Example 13. Efficacy analysis of enhancing isolated synaptic plasticity in an animal model of Alzheimer's disease. The APP / PS1 mouse model of Alzheimer's disease (Taconic Bioscience, 15 months old) was used to confirm the efficacy of crystalline C or aducanumab (Creative Biolabs) administered alone or in combination. The control group and four experimental groups were prepared as follows (see Table 14).

[0293] [Table 14]

[0294] As a control antibody, an allotype antibody was administered intravenously; and as a medium, 200 μL of a mixed solution in the ratio of DMSO (10):PEG400 (44):physiological saline (55) was administered orally. The dosing regimen is as follows: Figure 13a As shown in the figure. Five mice were used in each treatment group.

[0295] For intravenous infusion, such as Figure 13a As shown, aducanumab or control antibody was prepared as a 1 mg / mL stock solution for the experiment. Aducanumab was administered at a dose of 5 mg / kg, and the control antibody was administered at a dose of 5 mg / kg in an inactivated form of aducanumab. For intravenous infusion, it was injected into the tail vein a total of four times, starting on the experimental start date and every week thereafter.

[0296] For oral administration, approximately 2 weeks after the start of the experiment, 1 mg / kg of the compound of the present invention (crystal form C, BnH-015B) or the medium was administered orally once daily for approximately 11 to 13 days.

[0297] Four weeks after the start of the experiment, mice were sacrificed, and changes in the slope of fEPSP (field excitatory postsynaptic potential) in the CA1 region of the Schaffer collateral circuit in the hippocampus were observed. Results are as follows: Figure 13b The results confirmed that the use of crystalline C alone, aducanumab alone, and the combination of crystalline C and aducanumab all increased synaptic efficacy. In particular, the increase in synaptic efficacy was significant in the cases of crystalline C alone or in combination with aducanumab. Furthermore, measurements of the long-term potentiation (LTP) of fEPSP in the CA1 region of the Schaffer collateral circuit in the hippocampus also confirmed a significant increase in the long-term potentiation (LTP) of the excitatory SC pathway. The increase in fEPSP slope and LTP indicates enhanced synaptic plasticity; therefore, these results suggest that the use of crystalline C alone and in combination with aducanumab increases the synaptic efficacy of the SC pathway in the hippocampus, indicating that the use of crystalline C alone and in combination with aducanumab is effective in treating Alzheimer's disease.

[0298] On the other hand, the aim was to confirm the efficacy of the compound against Alzheimer's disease by measuring efficacy and the GABA / AMPA ratio in the sensory cortex. Specifically, the aim was to compare the effects of monotherapy and combination therapy with aducanumab. When crystalline C was administered alone or in combination with aducanumab to APP / PS1 mice, as confirmed by measurements of the GABA / AMPA ratio, it maintained excitation / inhibition balance (…). Figure 13c The right-hand diagram shows an increase in excitatory TC synaptic efficacy (TC efficacy). Figure 13c (Left figure). Increased TC efficacy indicates enhanced synaptic plasticity; therefore, these results suggest that administration of crystalline C alone or in combination with aducanumab can increase TC synaptic efficacy in the sensory cortex while maintaining excitation / inhibition balance. This indicates that administration of crystalline C alone or in combination with aducanumab is effective in treating Alzheimer's disease. In this case, the combination of crystalline C and aducanumab showed significantly better efficacy compared to administration of crystalline C and aducanumab alone.

[0299] Example 14. Efficacy analysis of enhancing isolated synaptic plasticity The efficacy of the compound was confirmed by measuring its potency in the barrel cortex and the GABA / AMPA ratio after oral administration of crystal form C.

[0300] Eight-week-old C57BL / 6J mice were orally administered crystalline form C at a dose of 1 mg / kg. For the control group, 200 μL of a mixture of DMSO (10):PEG400 (44): physiological saline (55) was administered orally as a mediator. Five mice were used in each of the control (mediator) and compound administration groups. Results confirmed that crystalline form C can increase TC synaptic efficacy in the sensory cortex while maintaining excitation / inhibition balance. Figure 14a and Figure 14b ).

[0301] Example 15. Analysis of the inhibitory efficacy of β-amyloid protein accumulation To confirm the inhibitory efficacy of crystalline form C of the present invention on β-amyloid protein accumulation, mouse proteomic profiling array analysis and Bielschowsky silver staining were performed. Fifteen-week-old APP / PS1 mice were used in the experiment, and the crystalline form C of the present invention (1 mg / kg of BnH-015B) or a carrier was administered orally.

[0302] Osteopontin (OPN) has attracted attention as a next-generation AD treatment candidate because it may help improve IL-33-mediated AD-related phenotypes. OPN is a cytokine expressed by activated peripheral dendritic cells and some macrophages, and it is known to regulate innate and adaptive immune responses through interaction with integrin receptors (see [Patarca et al., 1989]; [Ashkar et al., 2000; Inoue & Shinohara, 2015]; [Danzaki et al., 2016; Aggarwal et al., 2021]). Furthermore, it is known that the level of CD11c+ microglia that produce OPN is strongly correlated with cognitive deficits and the severity of AD neuropathy (see [Qiu et al., 2023]), and it has been reported that OPN gene knockout or administration of monoclonal anti-OPN antibodies improves cognitive function in 5XFAD mice by reducing the number of pro-inflammatory microglia, plaque formation, and atrophic neurites (see [Qiu et al., 2023]).

[0303] β-amyloid-beta (Aβ) is one of the risk-associated molecular patterns targeting microglia. Microglia are known to be activated by Aβ and promote inflammatory responses by secreting a variety of cytokines, such as IL-1β, IL-6, and TNF-α (Clark & ​​Vissel, 2015).

[0304] Therefore, experiments confirmed that crystal form C regulates neuroinflammation by modulating microglia expressing OPN in AD mice.

[0305] Mouse proteomic array analysis The relative levels of mouse cytokines, chemokines, and growth factors were determined using a membrane-based antibody array kit (Proteome Profiler Mouse XL Cytokine Array Kit, ARY028 R&D Systems, Inc., Minneapolis, USA) containing 111 capture antibodies coated on nitrocellulose membranes using a double-point sampling method. Somatosensory cortex homogenates of APP / PS1 mice were prepared and tested according to the manufacturer's instructions. The array was detected using Super RX X-ray film (Fujifilm, Tokyo, Japan), and the images were analyzed using an Epson scanner (L655 series, Epson, Suwa, Japan) with Protein Array Analyzer Macro and ImageJ (National Institutes of Health, Bethesda, Maryland, USA) after reading the film.

[0306] The study confirmed whether crystalline C could alter the expression of IL-33 and OPN in the cerebral cortex of 15-month-old APP / PS1 mice, and identified 14 positively stained spots of different cytokines. After standardizing the staining results, the relative expression levels of the 14 cytokines in the crystalline C-treated group (14 days) of 15-month-old APP / PS1 mice were compared with those in the medium-treated control group.

[0307] IL-33 expression levels were significantly increased in mice administered crystalline C compared to the mediator-treated control group; conversely, OPN expression was suppressed in mice administered crystalline C compared to the mediator-treated control group. Figure 15 ).

[0308] Bielschowsky silver staining Bielschowsky silver staining was performed according to the manufacturer's protocol (Bielschowsky Silver Staining Kit, ab245877, abcam, Cambridge, UK). 40 μm thick brain sections from APP / PS1 mice were incubated in warm 20% silver nitrate solution at 40°C for 15 minutes, then washed four times with distilled water and rinsed three times with water for 3 minutes each time. Slides were incubated in ammoniacal silver solution at 40°C for 10 minutes, then immersed in developing solution (50 mL H₂O containing 8 drops of 20% formalin, 8 drops of citric acid, and 4 drops of nitric acid) for a few seconds until the tissue sections turned yellow / brown, and then immersed in ammonia water for 30 seconds. The sections were washed three times with water for 2 minutes each time and incubated in 5% thiosulfate solution for 2 minutes. Then, the slide was dehydrated, covered with a coverslip using Permount mounting medium (Thermo Fisher Scientific), and images were acquired using an Olympus BX 53 optical microscope at 10x magnification via bright-field mode cellSens software.

[0309] Aβ plaques in the brain were visualized using Bielschowsky silver staining targeting Aβ monomers. Figure 16a Crystalline C reduced Aβ plaques by enhancing the phagocytic activity of microglia in the cerebral cortex of 15-month-old APP / PS1 mice. Figure 16b ).

[0310] Data Statistical Analysis Statistical analysis was performed using GraphPad Prism 9.5.0 software (San Diego, California, USA). To determine statistical significance between the two experimental groups, a one-tailed unpaired t-test with Welch correction was used. Differences in mean values ​​between groups were considered significant at p < 0.05 (*) and p < 0.01 (**). All data are expressed as mean ± SEM.

Claims

1. The crystal form of the compound shown in formula (1): Equation (1) 。 2. The crystal form A of the compound as described in claim 1, characterized in that... Its X-ray powder diffraction pattern shows diffraction peaks at diffraction angles 2θ (°) of 9.9±0.10, 14.9±0.10, 18.4±0.10, 24.5±0.10, 24.8±0.10, 25.2±0.10 and 28.7±0.

10.

3. The crystal form A as described in claim 2, characterized in that... The X-ray powder diffraction pattern of the crystal form further exhibits diffraction peaks at diffraction angles 2θ (°) of 5.0±0.10, 13.4±0.10, 19.7±0.10, 19.8±0.10, 20.5±0.10, 22.5±0.10, 24.0±0.10, and 35.0±0.

10.

4. The crystal form A as described in claim 3, characterized in that... The X-ray powder diffraction pattern of the crystal form further exhibits diffraction peaks at diffraction angles 2θ (°) of 12.1±0.10, 16.2±0.10, 20.1±0.10, 21.5±0.10, 27.4±0.10, 29.6±0.10, 30.0±0.10, 31.2±0.10, 31.5±0.10, and 36.8±0.

10.

5. The crystal form B of the compound as described in claim 1, characterized in that its X-ray powder diffraction pattern has diffraction peaks at diffraction angles 2θ (°) of 8.6±0.10, 9.1±0.10 and / or 9.2±0.10, 15.8±0.10, 20.0±0.10, 20.3±0.10, 20.7±0.10, 23.9±0.10 and 32.6±0.

10.

6. The crystal form B as described in claim 5, characterized in that the X-ray powder diffraction pattern of the crystal form further exhibits diffraction peaks at diffraction angles 2θ (°) of 8.2±0.10, 14.2±0.10, 14.7±0.10, 18.0±0.10, 18.3±0.10, 22.9±0.10, 26.4±0.10, 26.7±0.10, 27.6±0.10, 28.1±0.10, and 29.1±0.

10.

7. Crystal form B as described in claim 6, characterized in that the X-ray powder diffraction pattern of the crystal form further exhibits diffraction peaks at diffraction angles 2θ (°) of 11.9±0.10, 15.1±0.10, 16.5±0.10, 19.3±0.10, 22.1±0.10, 22.6±0.10, 24.5±0.10, 24.8±0.10, 25.9±0.10, 27.0±0.10, 27.8±0.10, 28.9±0.10, and 36.5±0.

10.

8. The crystal form C of the compound as described in claim 1, characterized in that... Its X-ray powder diffraction pattern shows diffraction peaks at diffraction angles 2θ (°) of 8.6±0.10, 10.0±0.10, 18.0±0.10 and 26.4±0.

10.

9. The crystal form C as described in claim 8, characterized in that... The X-ray powder diffraction pattern of the crystal form further exhibits diffraction peaks at diffraction angles 2θ (°) of 17.2±0.10, 20.2±0.10, and 25.5±0.

10.

10. The crystal form C as described in claim 9, characterized in that... The X-ray powder diffraction pattern of the crystal form further exhibits diffraction peaks at diffraction angles 2θ (°) of 15.1±0.10, 16.0±0.10, 21.1±0.10, 23.4±0.10, 26.1±0.10 and 34.2±0.

10.

11. The crystal form D of the compound as described in claim 1, characterized in that... Its X-ray powder diffraction pattern shows diffraction peaks at diffraction angles 2θ (°) of 8.8±0.10, 10.7±0.10, 17.1±0.10, 17.6±0.10, 19.1±0.10, 21.5±0.10, 22.2±0.10 and 23.4±0.

10.

12. The crystal form D as described in claim 11, characterized in that... The X-ray powder diffraction pattern of the crystal form further exhibits diffraction peaks at diffraction angles 2θ (°) of 12.5±0.10, 15.4±0.10, 16.1±0.10, 16.7±0.10, 18.3±0.10, 22.5±0.10, 22.9±0.10, 27.4±0.10, 27.8±0.10, and 29.8±0.

10.

13. The crystal form D as described in claim 12, characterized in that... The X-ray powder diffraction pattern of the crystal form further exhibits diffraction peaks at diffraction angles 2θ (°) of 9.5±0.10, 15.7±0.10, 21.0±0.10, 25.1±0.10, 25.3±0.10, 27.0±0.10, 28.6±0.10, 30.9±0.10, 32.1±0.10, 32.6±0.10, and 34.1±0.

10.

14. The crystal form E of the compound as claimed in claim 1, characterized in that its X-ray powder diffraction pattern has diffraction peaks at diffraction angles 2θ (°) of 10.2±0.10, 11.3±0.10, 18.3±0.10, 20.5±0.10, 22.8±0.10, 23.5±0.10, 25.2±0.10, 26.1±0.10, 28.6±0.10, 28.9±0.10 and 31.2±0.

10.

15. The crystal form E as described in claim 14, characterized in that the X-ray powder diffraction pattern of the crystal form further exhibits diffraction peaks at diffraction angles 2θ (°) of 9.9±0.10, 13.4±0.10, 17.9±0.10, 18.6±0.10, 19.6±0.10, 20.8±0.10, 21.0±0.10, 24.4±0.10, 24.8±0.10, 29.5±0.10, 29.8±0.10, and 32.5±0.

10.

16. The crystal form E as described in claim 15, characterized in that the X-ray powder diffraction pattern of the crystal form further has diffraction peaks at diffraction angles 2θ (°) of 4.9±0.10, 7.2±0.10, 14.8±0.10, 16.1±0.10, 16.4±0.10, 19.8±0.10, 20.0±0.10, 21.5±0.10, 22.5±0.10, 24.0±0.10, 25.4±0.10, 27.4±0.10, 32.8±0.10, and 39.5±0.

10.

17. The crystal form of the compound according to any one of claims 1-16, wherein the crystal form exhibits improved BBB permeability compared to the amorphous form.

18. A pharmaceutical composition comprising, as an active ingredient, the crystalline form of the compound as described in any one of claims 1-16.

19. The pharmaceutical composition of claim 18, wherein the composition is used for the prevention or treatment of nervous system diseases or neurodegenerative diseases.

20. The pharmaceutical composition of claim 19, wherein the disease is Alzheimer's disease.

21. The pharmaceutical composition of claim 19, wherein the disease is post-traumatic stress disorder (PTSD).

22. The pharmaceutical composition of claim 18, further comprising a second therapeutic agent.

23. The pharmaceutical composition of claim 22, wherein the second therapeutic agent is selected from the group consisting of donepezil, rivastigmine, galantamine, memantine, verubecestat, solanumab, bapinzumab, aducanumab, lencanemab, tideglusib, epothilone D, and ABBV-8E12.

24. The pharmaceutical composition of claim 22, wherein the second therapeutic agent is an antibody that specifically binds to β-amyloid protein and inhibits or degrades its accumulation.

25. The pharmaceutical composition of claim 22, wherein the second therapeutic agent is aducanumab.

26. A method for preventing or treating a neurological disease or neurodegenerative disease, comprising administering to a subject requiring prevention or treatment of a neurological disease or neurodegenerative disease a crystalline form of the compound as described in any one of claims 1-16.

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