N-methyl-D-aspartate receptor allosteric modulators and their usage

Abstract

This invention relates to therapeutic compounds and compositions, and methods of using them in the prevention or treatment of neurological disorders. In particular, this invention relates to N-methyl-D-aspartate receptor (NMDAR) allosteric modulators and methods of using them in the prevention or treatment of conditions or illnesses caused by or related to NMDAR dysfunction. This invention also relates to methods for identifying NMDAR allosteric modulators.

Description

Technical Field

[0001] This invention relates to therapeutic compounds and compositions, and methods of using them in the prevention or treatment of neurological disorders. In particular, this invention relates to N-methyl-D-aspartate receptor (NMDAR) allosteric modulators and methods of using them in the prevention or treatment of disorders or conditions caused by or related to NMDAR dysfunction. This invention also relates to methods for identifying NMDAR allosteric modulators. Background of the Invention

[0003] N-methyl-D-aspartate receptors (NMDARs) are a subfamily of ionotropic glutamate receptors in the brain, mediating brain functions such as learning and memory (Tang, YP et al., Nature (1999) 401(6748):63-69), and chronic neurodegenerative diseases such as Alzheimer's disease (Paoletti, P. et al., Nat Rev Neurosci (2013) 14(6):383-400), Huntington's disease (Fan, M. & Raymond, L. Prog Neurobiol (2007) 81(5-):272-293), and Parkinson's disease (Schmidt, BJAnn NYAcad Sci (1998) 860:189-202), as well as traumatic brain injury (Shohami, E. & Biegon, A. CNS Neurol Disord Drug). It plays a key role in the pathogenesis of conditions such as acute brain injury (Liu, Y. et al. J Neurosci (2007) 27(11): 2846-2857) and Targets (2014) 13(4): 567-573) and acute brain injury such as stroke (Liu, Y. et al. J Neurosci (2007) 27(11): 2846-2857).

[0004] Stroke is a leading cause of severe, long-term disability (Mozaffarian, D. et al., Circulation (2015) 131(4):e29-322). Furthermore, 87% of all strokes are ischemic and are caused by blood clots forming within blood vessels that block blood flow to the brain (Haast et al., J Cereb Blood Flow Metab (2012) 32(12):2100-2107; Mozaffarian, D. et al., Circulation (2015) 131(4):e29-322; Zhang, Y. et al., Circulation (2008) 118(15):1577-1584). This blockage ultimately leads to rapid loss of brain function, as well as slow cell death caused by cellular hypoxia, programmed cell death, free radical formation, and uncontrolled cell death (necrosis), all of which have detrimental effects on the brain (Northington, FJ et al., Ann Neurol (2011) 69(5):743-758).

[0005] The NMDAR subunits form heterotetrameric transmembrane channels, which are composed of a combination of the essential GluN1 subunit (formerly known as NR1) with GluN2A-D (formerly known as NR2A-D) and / or GluN3 (A&B) subunits (Collingridge, GL et al. Neuropharmacology (2009) 56(1):2-5). Different GluN2 subunits (GluN2A-D) endow the receptor complex with different electrophysiological and pharmacological properties and couple them to different signal transduction mechanisms (Bliss, T. & Schoepfer, R. Science (2004) 304(5673):973-974; Seeburg, PHTrends Neurosci (1993) 16(9):359-365; Seeburg, PHTrends Pharmacol Sci (1993) 14(8):297-303). Recent evidence suggests that NMDARs play distinct roles in mediating synaptic plasticity and cell survival, depending on the presence of the GluN2 subunit. Typically, GluN2B-containing NMDARs activate cell death signaling, thereby mediating excitotoxic neuronal damage, while GluN2A-containing NMDARs promote the induction of long-term enhancements important for learning, memory, and neuronal survival, thus protecting neurons from excitotoxic damage.

[0006] The dual function of NMDARs in neuronal survival and death may be at least partly attributable to the lack of success of NMDAR modulators in recent clinical trials. Conventional NMDAR antagonists target surface receptors, essentially blocking both neuronal survival-signaling pathways and neuronal death-signaling pathways, as well as the normal functioning of the receptors, causing undesirable side effects. In contrast, enhancing NMDARs containing GluN2A can protect neurons from ischemic damage by specifically promoting neuronal survival mechanisms, resulting in fewer side effects and a wider therapeutic window.

[0007] Current treatment options for ischemic stroke have been limited to restoring blood flow through reperfusion after stroke via vascular-based therapies, or blocking signaling pathways leading to ischemic cell death through neuroprotective strategies (Woodruff TM et al., Mol Neurodegener (2011) 6(1):11). To date, there has been no progress in the pharmacological enhancement of NMDAR function using positive modulation strategies of receptor channels. Invention Overview

[0009] This invention relates to compounds that modulate NMDAR activity. Specifically, the compounds identified herein exhibit positive regulation of NMDAR GluN1 / GluN2A and / or GluN1 / GluN2B isoforms.

[0010] In one aspect of the invention, a compound of formula 1 or a pharmaceutically acceptable salt thereof is provided for use in individuals in need of prevention or treatment of conditions or illnesses caused by or related to NMDAR dysfunction.

[0011]

[0012] in

[0013] R 1 R 2 R 3 R 4 and R 5 Each of them independently is H, OH, halogen, CN, NO2, NRR', COOR, CONRR', C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkoxy, C2-C6 alkenyloxy, or C2-C6 alkoxy.

[0014] R 15 It is an H or C1-C6 alkyl group;

[0015] R 11 It is an H or C1-C6 alkyl group;

[0016] G is a direct bond, O, NR, S, OCR'R”, SCR'R”, NRCR'R”, NRC(O), NRC(O)NR', ​​NRC(O)CR'R” or NRC(O)CR'R”O, and G is connected to carbonyl and A in any direction;

[0017] A is A-1

[0018]

[0019] X is CR 7 Or N;

[0020] Y is CR 8 Or N;

[0021] R 6 R 7 R 8 R 9 and R 10 Each of these elements independently is H, OH, halogen, CN, NO2, NRR', NRC(O)R 14 COOR, CONRR', C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, C2-C6 alkenyloxy, or C2-C6 alkynyloxy; or R 7 and R 8 Together or R 8 and R 9 Together they can form 5- or 6-membered saturated, partially unsaturated or aromatic monocyclic rings, which optionally contain 1 to 3 heteroatoms selected from O, N and S;

[0022] R 14 It is H, C1-C6 alkyl, or C substituted with one or more C1-C6 alkyl groups. 5- C 10 Aryl;

[0023] R, R', and R” each time they appear independently is H or a C1-C6 alkyl group; and

[0024] The alkyl, alkenyl, alkynyl, alkoxy, alkenyloxy, and alkynyloxy groups are each optionally substituted with one or more groups selected from OH and halogens.

[0025] In another aspect of the invention, a method is provided for the prevention or treatment in an individual of a condition or illness caused by or related to NMDAR dysfunction, comprising administering to the individual a preventive or therapeutically effective amount of the compound of the invention or a pharmaceutically acceptable salt thereof.

[0026] In another aspect of the invention, the use of the compounds of the invention or pharmaceutically acceptable salts thereof in the preparation of medicaments for the prevention or treatment in individuals in need of preventing or treating conditions or illnesses caused by or related to NMDAR dysfunction.

[0027] In another aspect of the invention, a pharmaceutical composition is provided comprising a compound of the invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition may be used to modulate NMDAR activity for the prevention or treatment in individuals in need of such treatment of conditions or illnesses caused by or related to NMDAR dysfunction.

[0028] In another aspect of the invention, a compound is provided that specifically binds to a target site at the interface between the GluN1 and GluN2A subunits in the N-terminal domain (NTD), wherein said target site is defined by at least one or more amino acid residues 135 of GluN1 and amino acid residues 79, 111, 115, 177, and 178 of GluN2A. In some embodiments, said compound specifically enhances NMDAR containing GluN1 / GluN2A.

[0029] In another aspect of the invention, a computer-aided method is provided for identifying compounds that specifically enhance NMDAR containing GluN1 / GluN2A, the method comprising the following steps:

[0030] i) The structure of the candidate compound is docked with the binding pocket between the GluN1 and GluN2A interfaces of the NMDAR receptor in the NTD, wherein the binding pocket is defined by at least one or more amino acid residues 135 of the GluN1 subunit and amino acid residues 79, 111, 115, 177, and 178 of the GluN2A subunit, and

[0031] ii) Identify candidate compounds that can specifically enhance NMDAR containing GluN1 / GluN2A.

[0032] The method may also include synthesizing or obtaining identified candidate compounds and determining whether the compounds specifically enhance NMDAR containing GluN1 / GluN2A. Brief description of the attached diagram

[0034] Figure 1 The enhancement and inhibition effects of compounds that were "hit" in the initial screening were recorded using whole-cell voltage clamp.

[0035] Figure 2 The regulatory effects of different Npam02 analogues in HEK293 cells expressing GluN1 / GluN2A and GluN1 / GluN2B NMDAR.

[0036] Figure 3 In HEK293 cells transfected with GluN1 / GluN2A subunits, Npam02 enhances GluN1 / GluN2A-mediated NMDAR currents by direct binding, without altering GluN1 / GluN2B-mediated NMDAR currents.

[0037] Figure 4 In cultured hippocampal neurons, Npam02 enhanced neuronal NMDAR function, and this enhancement was blocked by GluN2A antagonists.

[0038] Figure 5 Npam02 was used to enhance GluN2A NMDAR-mediated currents in cortical cultured neurons lacking the GluN2B subunit gene.

[0039] Figure 6 In neuronal cultures, Npam02 does not affect the current response induced by AMPA and GABA.

[0040] Figure 7 The 2D chemical structure of Npam02.

[0041] Figure 8 GluN2A F177 and Q111 form an Npam02 binding pocket between the GluN1 and GluN2A interfaces of the NMDAR receptor in the NTD.

[0042] Figure 9 The regulatory effect of Npam43 in HEK293 cells expressing GluN1 / GluN2A and GluN1 / GluN2B NMDAR and its dose-dependent curves in the two expression systems.

[0043] Figure 10 The key amino acid residues at the GluN1 / GluN2A interface in the N-terminal domain that reduce the enhancing effect of Npam43.

[0044] Figure 11 Glutamate dose-response in HEK293 cells expressing wild-type GluN1 / GluN2A, mutant GluN1(L135Q), and mutant GluN1(L135Q) / GluN2A(F115S).

[0045] Figure 12 In cultured hippocampal neurons, Npam43 specifically targets NMDAR containing GluN2A.

[0046] Figure 13In cultured hippocampal neurons, Npam43 dose-dependently enhances NMDAR-mediated currents and modulates NMDA antagonist binding.

[0047] Figure 14 Npam43 increases intracellular calcium levels via NMDAR containing GluN1 / GluN2A. 2+ .

[0048] Figure 15 In cortical neurons, Npam43 enhances CREB phosphorylation (pCREB), which is a well-characterized indicator of activation of cell survival signaling.

[0049] Figure 16 Npam43 protects cortical neurons from NMDA-induced excitotoxicity.

[0050] Figure 17 In cortical neurons, Npam43 also protects against non-NMDA-dependent, H2O2-induced oxidative cytotoxicity by increasing the activation of NMDAR containing GluN2A.

[0051] Figure 18 Npam43 enhances the GluN2A component of synaptic transmission in hippocampal slices.

[0052] Figure 19 Npam43 promotes long-term potentiation (LTP) in hippocampal slices.

[0053] Figure 20 Npam43 enhances NMDA-induced currents in CA1 neurons of hippocampal slices from wild-type but non-GluN2A knockout mice.

[0054] Figure 21 Npam43 increased pCREB levels in hippocampal slices prepared acutely from mature rats.

[0055] Figure 22 In mature rats, Npam43 crosses the blood-brain barrier after intravenous injection.

[0056] Figure 23 In mature rats, Npam43 increased pCREB levels in the hippocampus and cortical tissues after IV injection.

[0057] Figure 24 Npam43 reduces the cerebral necrosis volume of ischemic brain in mice in vivo.

[0058] Figure 25 Npam43 reduces the volume of cerebral necrosis after stroke when using long-term assessment points.

[0059] Figure 26In vivo administration of Npam43 28 days after stroke to improve behavioral performance in post-stroke treatment. Invention Details

[0061] Novel regulatory binding sites on GluN1 / GluN2A NMDAR

[0062] The structural system of NMDAR can be characterized as having an extracellular N-terminal domain (NTD), three transmembrane domains (M1, M3, M4) with a folded loop (M2) forming a pore channel, a bilobed ligand-binding domain (called the S1 domain) formed by the distal portion after the NTD, and a large extracellular loop connecting M3 and M4 (called the S2 domain), plus an intracellular C-terminal domain (CTD) (Paoletti et al., 2013).

[0063] Through 3D structural analysis and site-directed mutagenesis experiments, the inventors identified a novel allosteric regulatory binding site at the interface between the GluN1 and GluN2A subunits of the NTD. Specifically, the binding pocket is formed by amino acid residues at the interface between the GluN1 and GluN2A NTDs of the NMDAR receptor, including (but not limited to) GluN1 (Leu135) and GluN2A (Phe177, Pro79, Phe115, Gln111, and Pro178). Regulators suited to this pocket and interacting with Leu135 of GluN1 and Phe177, Pro79, Phe115, Gln111, or Pro178 of GluN2A may be able to enhance NMDAR, for example, specifically enhancing GluN2A containing GluN2A.

[0064] Therefore, in one aspect of the invention, a compound is provided that specifically binds to a target site at the interface between the GluN1 and GluN2A subunits in the N-terminal domain (NTD).

[0065] In some embodiments, the amino acid sequence of the GluN1 subunit is shown in SEQ ID NO:1 (UniProtKB / Swiss-Prot:P35439). In some embodiments, the amino acid sequence of the GluN2A subunit is shown in SEQ ID NO:2 (UniProtKB / Swiss-Prot:Q00959).

[0066] In some embodiments, the target site is defined by at least one or more of amino acid residue 135 of GluN1 and amino acid residues 79, 111, 115, 177, and 178 of GluN2A. In some preferred embodiments, the target site is defined by at least one or more of L135 of GluN1 and P79, Q111, F115, F177, and P178 of GluN2A. In some preferred embodiments, the target site is defined by at least L135 of GluN1 and P79, Q111, F115, F177, and P178 of GluN2A. In some embodiments, the compound interacts with one or more of amino acid residues 135 of GluN1 and amino acid residues 79, 111, 115, 177, and 178 of GluN2A. In some embodiments, the compound interacts with amino acid residues Q111 and F177 of GluN2A. In some embodiments, the compound interacts with amino acid residues L135 of GluN1 and amino acid residues P79, Q111, F115, F177, and P178 of GluN2A.

[0067] In some embodiments, the compound is an allosteric modulator of the N-methyl-D-aspartate receptor (NMDAR) containing GluN1 / GluN2A. In some embodiments, the modulator specifically enhances the NMDAR containing GluN1 / GluN2A.

[0068] In another aspect of the invention, a computer-aided method is provided for identifying compounds that specifically enhance NMDAR containing GluN1 / GluN2A, the method comprising the following steps:

[0069] i) The structure of the candidate compound is docked with the binding pocket between the GluN1 and GluN2A interfaces of the NMDAR receptor in the NTD, wherein the binding pocket is defined by at least one or more amino acid residues 135 of the GluN1 subunit and amino acid residues 79, 111, 115, 177, and 178 of the GluN2A subunit, and

[0070] ii) Identify candidate compounds that can specifically enhance NMDAR containing GluN1 / GluN2A.

[0071] In some preferred embodiments, the binding pocket is defined by at least one or more of L135 of GluN1 and P79, Q111, F115, F177, and P178 of GluN2A.

[0072] It is known in the art that substrates, cofactors, antagonists, agonists, or allosteric modulators can be identified using computer modeling with docking procedures such as GRAM, DOCK, or AUTODOCK (Dunbrack et al., 1997). Computer programs can also be used to evaluate the attraction, repulsion, and steric hindrance of candidate compounds to receptors.

[0073] In some embodiments, the method further includes synthesizing or obtaining identified candidate compounds and determining whether the compounds specifically enhance NMDAR containing GluN1 / GluN2A.

[0074] To determine whether a candidate specifically enhances NMDAR containing GluN1 / GluN2A, the methods described in the working embodiments of this application can be used.

[0075] compound

[0076] The present invention also provides compounds of Formula 1 or pharmaceutically acceptable salts thereof for use in individuals in need of prevention or treatment of conditions or illnesses caused by or related to NMDAR dysfunction.

[0077]

[0078] in

[0079] R 1 R 2 R 3 R 4 and R 5 Each of them independently is H, OH, halogen, CN, NO2, NRR', COOR, CONRR', C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkoxy, C2-C6 alkenyloxy, or C2-C6 alkoxy.

[0080] R 15 It is an H or C1-C6 alkyl group;

[0081] R 11 It is an H or C1-C6 alkyl group;

[0082] G is a direct bond, O, NR, S, OCR'R”, SCR'R”, NRCR'R”, NRC(O), NRC(O)NR', ​​NRC(O)CR'R” or NRC(O)CR'R”O, and G is connected to carbonyl and A in any direction;

[0083] A is A-1

[0084]

[0085] X is CR7 Or N;

[0086] Y is CR 8 Or N;

[0087] R 6 R 7 R 8 R 9 and R 10 Each of these elements independently is H, OH, halogen, CN, NO2, NRR', NRC(O)R 14 COOR, CONRR', C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, C2-C6 alkenyloxy, or C2-C6 alkynyloxy; or R 7 and R 8 Together or R 8 and R 9 Together they can form 5- or 6-membered saturated, partially unsaturated or aromatic monocyclic rings, which optionally contain 1 to 3 heteroatoms selected from O, N and S;

[0088] R 14 It is H, C1-C6 alkyl, or C substituted with one or more C1-C6 alkyl groups. 5- C 10 Aryl;

[0089] R, R', and R” each time they appear independently is H or a C1-C6 alkyl group; and

[0090] The alkyl, alkenyl, alkynyl, alkoxy, alkenyloxy, and alkynyloxy groups are each optionally substituted with one or more groups selected from OH and halogens.

[0091] In a preferred embodiment, G is a direct bond, NMeC(O), NHC(O)CH2, NHCH2, NHC(O)CH2O, or CH2C(O)NH.

[0092] In a preferred embodiment, X and Y are not both N.

[0093] In the preferred embodiment, R 11 and R 15 Both are H; R 4 It is H, halogen, NO2, or C2-C4 alkenyl; R 5 It is H or halogen; R 6 It is H or C1-C4 alkyl; and R 10 It is H, OH, halogen, NH2, C1-C4 alkyl or C1-C4 alkoxy.

[0094] In the preferred embodiment, R 1It is H, OH, C1-C4 alkoxy or C2-C4 alkynoxy; R 2 It is H, OH, C1-C4 alkoxy or halogen; and R 3 It is H or OH; and the halogen represents F, Cl, Br or I, preferably Cl or Br.

[0095] In the preferred embodiment, R 7 It is H, OH, halogen, or C1-C4 alkyl; R 8 It is H, OH, halogen, C1-C4 alkyl, C1-C4 alkoxy or NHC(O)R 14 , where R 14 It is a phenyl group substituted with a C1-C4 alkyl group; R 9 It is H, OH, halogen, NO2 or C 1- C4 alkyl; or R 7 and R 8 Together they form a phenyl group; or R 8 and R 9 Together they form 1,4-dioxanecyclohexyl; and the halogen represents F, Cl, Br or I.

[0096] In some embodiments, the compounds of the present invention have the structure of Formula I.

[0097]

[0098] in

[0099] R1=H, OH, OMe, OC2H2, OEt, NH2, NEt2, NHEt, NHMe, COOH, CH2OH, NMe2, NO2, CONH2;

[0100] R2=H, OH, OMe, OEt, OtBu, OPr, Pr, Me, Et, NHMe, NHEt, NEt2, NMe2, COOH, NO2, I, tBu, CF3, OIsoPr, IsoPr, CH2OH, CN, CBr3, CCl3;

[0101] R3=H, OH, NH2, Me, OMe, OEt, F, Cl, Br, NHMe, NHEt, Et, NEt2, NMe2, COOH, NO2, I, tBu, CF3, CH2OH, CN, CBr3, CCl3;

[0102] R4=H, OH, NH2, OMe, OEt, F, Cl, Br, Me, CH2C2H4, NHMe, NHEt, Et, NEt2, NMe2, COOH, NO2, I, tBu, CF3, OtBu, OisoPr, IsoPr, CN, CH2OH, CBr3, CCl3;

[0103] R5=H, OH, NH2, OMe, F, Cl, Br, Me, Et, NHMe, NHEt, NEt2, NMe2, COOH, NO2, I, tBu, CF3, CN, CH2OH, CBr3, CCl3;

[0104] R6=H, OH, NH2, F, Cl, Br, Me, OMe, Et, OEt, NHMe, NHEt, NEt2, NMe2, COOH, NO2, I, tBu, CF3, CN, CH2OH, CBr3, CCl3;

[0105] R7=H, OH, NH2, F, Cl, Br, Me, OMe, Et, OEt, NHMe, NHEt, NEt2, NMe2, COOH, NO2, I, tBu, CF3, CN, CH2OH, CBr3, CCl3;

[0106] R8=H, OH, NH2, OMe, F, Cl, Br, Me, Et, OEt, NHMe, NHEt, NEt2, NMe2, COOH, NO2, I, tBu, CF3, CN, CH2OH, CBr3, CCl3;

[0107] R9=H, OH, NH2, F, Cl, Br, Me, OMe, Et, OEt, NHMe, NHEt, NEt2, NMe2, COOH, NO2, I, tBu, CF3, CN, CH2OH, CBr3, CCl3;

[0108] R10=H, OH, NH2, F, Cl, Br, Me, OMe, Et, OEt, NHMe, NHEt, NEt2, NMe2, COOH, NO2, I, tBu, CF3, CN, CH2OH, CCl3, CBr3;

[0109] R11 = H, Me;

[0110] R12 = (=O);

[0111] R15 = H, Me, Et, tertiary-Bu.

[0112] In some embodiments, the compounds of the present invention have the structure of Formula II.

[0113]

[0114] wherein

[0115] R1 = H, OH, OMe, OC2H2, OEt, NH2, NEt2, NHEt, NHMe, COOH, CH2OH, NMe2, NO2, CONH2;

[0116] R2 = H, OH, OMe, OEt, OtBu, OPr, Pr, Me, Et, NHMe, NHEt, NEt2, NMe2, COOH, NO2, I, tBu, CF3, OIsoPr, IsoPr, CH2OH, CN, CBr3, CCl3;

[0117] R3 = H, OH, NH2, Me, OMe, OEt, F, Cl, Br, NHMe, NHEt, Et, NEt2, NMe2, COOH, NO2, I, tBu, CF3, CH2OH, CN, CBr3, CCl3;

[0118] R4 = H, OH, NH2, OMe, OEt, F, Cl, Br, Me, CH2C2H4, NHMe, NHEt, Et, NEt2, NMe2, COOH, NO2, I, tBu, CF3, OtBu, OisoPr, IsoPr, CN, CH2OH, CBr3, CCl3;

[0119] R5 = H, OH, NH2, OMe, F, Cl, Br, Me, Et, NHMe, NHEt, NEt2, NMe2, COOH, NO2, I, tBu, CF3, CN, CH2OH, CBr3, CCl3;

[0120] R11 = H, Me;

[0121] R12 = (=O);

[0122] R15 = H, Me, Et, tert - Bu;

[0123]

[0124] R6 = H, OH, NH2, F, Cl, Br, Me, OMe, Et, OEt, NHMe, NHEt, NEt2, NMe2, COOH, NO2, I, tBu, CF3, CN, CH2OH, CBr3, CCl3;

[0125] R7=H, OH, NH2, F, Cl, Br, Me, OMe, Et, OEt, NHMe, NHEt, NEt2, NMe2, COOH, NO2, I, tBu, CF3, CN, CH2OH, CBr3, CCl3;

[0126] R8=H, OH, NH2, OMe, F, Cl, Br, Me, Et, OEt, NHMe, NHEt, NEt2, NMe2, COOH, NO2, I, tBu, CF3, CN, CH2OH, CBr3, CCl3;

[0127] R9=H, OH, NH2, F, Cl, Br, Me, OMe, Et, OEt, NHMe, NHEt, NEt2, NMe2, COOH, NO2, I, tBu, CF3, CN, CH2OH, CBr3, CCl3;

[0128] R10=H, OH, NH2, F, Cl, Br, Me, OMe, Et, OEt, NHMe, NHEt, NEt2, NMe2, COOH, NO2, I, tBu, CF3, CN, CH2OH, CCl3, CBr3;

[0129] R15 = H, Me, Et, tertiary-Bu;

[0130] G9 = O, N, S.

[0131] In some embodiments, the compounds of the present invention are one or more of the compounds shown in Table A or Table B:

[0132] Table A.

[0133]

[0134]

[0135]

[0136]

[0137]

[0138] Table B.

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145] The compounds of the present invention are allosteric modulators of NMDAR, and preferably are positive NMDAR modulators selectively containing GluN2A and / or positive NMDAR modulators selectively containing GluN2B.

[0146] As used in this article, the following definitions apply.

[0147] The term "alkyl" refers to a branched or straight-chain saturated aliphatic hydrocarbon group having a specified number of carbon atoms. Unless otherwise specified, "alkyl" refers to C1-C6 alkyl. For example, "C1-C6 alkyl" is defined as a group comprising 1, 2, 3, 4, 5, or 6 carbon atoms arranged in a straight or branched chain. For example, "C1-C6 alkyl" includes, but is not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, and hexyl.

[0148] The term "alkenyl" refers to a branched or straight-chain hydrocarbon group having a specified number of carbon atoms and at least one carbon-carbon double bond. In some embodiments, one carbon-carbon double bond is present, and up to three carbon-carbon double bonds may be present. Thus, "C2-C6 alkenyl" refers to an alkenyl group having 2, 3, 4, 5, or 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds. For example, "C2-C6 alkenyl" includes, but is not limited to, vinyl, propenyl, butenyl, and 2-methylbutenyl.

[0149] The term "alkynyl" refers to a branched or straight-chain hydrocarbon group having a specified number of carbon atoms and at least one carbon-carbon triple bond. In some embodiments, one carbon-carbon triple bond is present, and up to three carbon-carbon triple bonds may be present. Thus, "C2-C6 alkynyl" refers to an alkynyl group having 2, 3, 4, 5, or 6 carbon atoms and 1, 2, or 3 carbon-carbon triple bonds. For example, "C2-C6 alkynyl" includes, but is not limited to, ethynyl, propynyl, butynyl, and 3-methylbutynyl.

[0150] The terms “alkoxy,” “alkenoxy,” and “alkynoxy” refer to the alkyl, alkenyl, and alkynyl groups defined above, respectively, but connected by an oxygen bridge on any available carbon atom. Thus, for example, “C1-C6 alkoxy” refers to an alkyl group having 1, 2, 3, 4, 5, or 6 carbon atoms connected by an oxygen bridge, including but not limited to methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, isobutoxy, pentoxy, and hexoxy.

[0151] Those skilled in the art will understand that COOH and NRR' in the compounds of the present invention can exist in the form of corresponding ions, such as carboxylate ions and ammonium ions. Alternatively, in the case of ions, those skilled in the art will understand that anti-charge ions may also be present.

[0152] Those skilled in the art will understand that the points where the group is covalently linked to the compound described herein can, for example, but not limited to, cleave under specific conditions. Specific conditions may include, for example, enzymatic or non-enzymatic pathways in vivo. The cleavage of the group can, for example, but not limited to, occur spontaneously, or be catalyzed or induced by another agent or changes in physical or environmental parameters, such as enzymes, light, acids, temperature, or pH. The group can, for example, but not limited to, a protecting group for masking functional groups, a group that serves as a substrate for one or more active or passive transport mechanisms, or a group that imparts or enhances properties of the compound (e.g., solubility, bioavailability, or localization).

[0153] The compounds described herein may be in their free form or in salt form. In some embodiments, the compounds described herein may be in the form of pharmaceutically acceptable salts, which are known in the art (Berge SM et al., JPharm Sci (1977) 66(1): 1-19).

[0154] Pharmaceutically acceptable salts as used herein include, for example, salts having the desired pharmacological activity of a parent compound (retaining the biological efficacy and / or properties of the parent compound and not being a salt that is biologically and / or otherwise undesirable). For example, compounds described herein having one or more functional groups capable of forming salts can form pharmaceutically acceptable salts.

[0155] Compounds containing one or more basic functional groups may form pharmaceutically acceptable salts with, for example, pharmaceutically acceptable organic or inorganic acids. Pharmaceutically acceptable salts can be derived from, for example, but not limited to, acetic acid, adipic acid, alginic acid, aspartic acid, ascorbic acid, benzoic acid, benzenesulfonic acid, butyric acid, cinnamic acid, citric acid, camphoric acid, camphorsulfonic acid, cyclopentylpropionic acid, diethylacetic acid, digluconic acid, dodecyl sulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, glucoheponic acid, gluconic acid, glycerophosphate, glycolic acid, and hemisulfonic acid. (acid), heptanoic acid, hexanoic acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, 2-hydroxyethanesulfonic acid, isonicotinic acid, lactic acid, malic acid, maleic acid, malonic acid, mandelic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, p-toluenesulfonic acid, nicotinic acid, nitric acid, oxalic acid, dihydroxynaphthalic acid, pectinic acid, 3-phenylpropionic acid, phosphoric acid, picric acid, pimelic acid, pimelic acid, pentanoic acid, propionic acid, pyruvic acid, salicylic acid, succinic acid, sulfuric acid, aminosulfonic acid, tartaric acid, thiocyanate, or undecanoic acid.

[0156] Compounds containing one or more acidic functional groups may form pharmaceutically acceptable salts with pharmaceutically acceptable bases, such as, but not limited to, inorganic bases based on alkali metals or alkaline earth metals, or organic bases such as primary amines, secondary amines, tertiary amines, quaternary amines, substituted amines, naturally occurring substituted amines, cyclic amines, or basic ion exchange resins. Pharmaceutically acceptable salts may be derived from, for example, but not limited to, hydroxides, carbonates, or bicarbonates of pharmaceutically acceptable metal cations (such as ammonium, sodium, potassium, lithium, calcium, magnesium, iron, zinc, copper, manganese, or aluminum), ammonia, benzathine penicillin, meglumine, methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, isopropylamine, tripropylamine, tributylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, and histidine. Caffeine, heparin, choline, betaine, ethylenediamine, glucosamine, succinylglucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, procaine, N-ethylpiperidine, theobromine, tetramethylammonium compounds, tetraethylamine compounds, pyridine, N,N-dimethylaniline, N-methylpiperidine, morpholine, N-methylmorpholine, N-ethylmorpholine, dicyclohexylamine, dibenzylamine, N,N-dibenzylphenethylamine, 1-diphenylhydroxymethylamine, N,N'-dibenzylethylenediamine, or polyamine resin.

[0157] In some embodiments, the compounds described herein may contain acidic and basic groups and may be in the form of an internal salt or a zwitterion, such as, but not limited to, betaine.

[0158] Salts as described herein can be prepared by conventional methods known to those skilled in the art, such as, but not limited to, by reacting the free form with an organic or inorganic acid or base, or by anion or cation exchange from other salts. Those skilled in the art will understand that salt preparation can occur in situ during the isolation and purification of compounds, or that salt preparation can occur from compounds that have been isolated and purified by separate reactions.

[0159] In some embodiments, the compound and all its different forms (e.g., free form, salt, polymorph, isomer) can be solvation forms, such as solvates. Solvates contain a stoichiometric or non-stoichiometric solvent that is physically bound to the compound or its salt. The solvent can be, for example, but not limited to, pharmaceutically acceptable solvents. For example, a hydrate is formed when the solvent is water, or an alcohol is formed when the solvent is an alcohol.

[0160] In some embodiments, the compounds described herein and all their different forms (e.g., free form, salt, solvate, isomer) may include crystalline and amorphous forms, such as polymorphs, pseudopolymorphs, conformational polymorphs, amorphous forms, or combinations thereof. Polymorphs comprise different crystalline arrangements of the same elemental composition of the compound. Polymorphs typically have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardness, crystal shapes, optical and electrical properties, stability, and / or solubility. Those skilled in the art will understand that various factors, including recrystallization solvents, crystallization rates, and storage temperatures, can cause the single-crystal form to predominate.

[0161] In some embodiments, the compounds described herein and all their different forms (e.g., free forms, salts, solvates, polymorphs) include isomers such as geometric isomers, optical isomers based on asymmetric carbon, stereoisomers, tautomers, enantiomers alone, diastereomers alone, racemates, mixtures of diastereomers, and combinations thereof, and are not limited to the description of the formulas shown for convenience.

[0162] Pharmaceutical compositions and formulations

[0163] This invention provides pharmaceutical compositions comprising a compound described herein or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient. The pharmaceutical compositions are used to modulate NMDAR activity, for example, to specifically enhance NMDAR, such as NMDAR containing GluN2A.

[0164] The pharmaceutical compositions of the present invention can be provided in commercial packaging containing instructions for using the composition to modulate NMDAR activity.

[0165] Pharmaceutical formulations typically contain one or more carriers, excipients, or diluents that are acceptable for the formulation's route of administration, such as by injection, inhalation, local administration, irrigation, or other methods suitable for the chosen treatment. Suitable carriers, excipients, or diluents (used interchangeably herein) are those known in the art for such routes of administration.

[0166] Suitable pharmaceutical compositions can be formulated by methods known in the art, and their routes of administration and dosages can be determined by those skilled in the art. For parenteral administration, the compound can be dissolved in sterile water or saline or in pharmaceutically acceptable carriers for the administration of non-water-soluble compounds, such as those for vitamin K. For enteral administration, the compound can be administered in tablet, capsule, or liquid form. Tablets or capsules can be enteric-coated or formulations for sustained release. Many suitable formulations are known, including polymer or protein microparticles encapsulating the compound to be released, ointments, pastes, gels, hydrogels, or solutions for topically or locally administered compounds. Sustained-release patches or implants can be used to provide extended-time release. Many techniques known to those skilled in the art are described in Remington: The Science & Practice of Pharmacy by Alfonso Gennaro, 20th edition, Lippencott Williams & Wilkins, (2000). Formulations for parenteral administration may contain, for example, excipients, polyalkylene glycols (such as polyethylene glycol), vegetable oils, or hydrogenated naphthalene. Biocompatible, biodegradable lactide polymers, lactide / glycolic acid copolymers, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compound. Other potentially useful parenteral delivery systems for regulating the compound include ethylene vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients such as lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate, and deoxycholate, or may be oil-containing solutions administered as nasal drops, or gels.

[0167] Compounds or pharmaceutical compositions described or used herein can be administered via medical devices or instruments, such as implants, grafts, prostheses, stents, etc. Furthermore, implants can be designed to contain and release such compounds or compositions. An example is an implant made of polymeric materials suitable for releasing compounds over a period of time.

[0168] The term "effective amount" for a pharmaceutical composition as described herein includes a therapeutically effective amount. A "therapeuticly effective amount" refers to the amount that effectively achieves the desired therapeutic outcome (such as reducing cerebral lesion size, reducing neuronal damage, improving behavioral performance, increasing lifespan, or increasing life expectancy) at the necessary dose and for the required duration. Therapeuticly effective amounts of a compound can vary depending on factors such as brain injury or an individual's disease state, age, sex, and weight, as well as the compound's ability to elicit the desired response in the individual.

[0169] Dosing regimens can be adjusted to provide the best therapeutic response. Therapeutic effective doses are those in which the beneficial therapeutic effect outweighs any toxic or harmful effects of the compound. "Prophylactic effective doses" refer to the amount at which the desired preventative outcome (reduction of infarct size, reduction of neuronal damage, improvement of behavioral performance, increase in lifespan, or increase in life expectancy) is effectively achieved at the necessary dose and for the required time period. Typically, prophylactic doses are administered to individuals before or in the early stages of disease, so that the prophylactic effective dose may be less than the therapeutic effective dose.

[0170] It should be noted that dosage values ​​may vary depending on the severity of the condition to be alleviated. For any given individual, a specific dosing regimen may be adjusted over time based on individual needs and the professional judgment of the person administering or managing the dosing composition. The dosage ranges described herein are merely exemplary and do not limit the dosage ranges that may be chosen by a physician. The amount of the active compound in the composition may vary depending on factors such as an individual's disease state, age, sex, and weight. Dosing regimens may be adjusted to provide the best therapeutic response. For example, a single bolus injection may be administered, several separate doses may be administered over time, or the dose may be proportionally reduced or increased depending on the urgency of the treatment situation. Formulating parenteral compositions in dosage units may be advantageous to facilitate administration and uniformity of dosage.

[0171] In some embodiments, the compounds described herein and all their different forms may, for example but not limited to, be used in combination with other treatments for at least one indication selected from Alzheimer's disease, Huntington's disease, Parkinson's disease, traumatic brain injury, and acute brain injury such as stroke. For example, the compounds described herein and all their different forms may be used as neoadjuvant (before), adjuvant (during), and / or adjuvant (after) treatment with tissue plasminogen activator (TPA) or other therapies.

[0172] Medical use

[0173] The present invention provides a method for modulating NMDAR activity, for example, specifically enhancing NMDAR, such as NMDAR containing GluN2A, said method comprising administering to mammalian cells a compound described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein.

[0174] Alternatively, the present invention provides a method for modulating NMDAR activity, for example, specifically enhancing NMDAR, such as NMDAR containing GluN2A, said method comprising administering to an individual in need a compound described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein.

[0175] By modulating NMDAR activity, the compounds and compositions of the present invention can be used to prevent or treat conditions or illnesses caused by or related to NMDAR dysfunction. In particular, the condition or illness can be at least one selected from: learning and memory disorders, migraines, epilepsy, Alzheimer's disease, Huntington's disease, Parkinson's disease, traumatic brain injury, acute brain injury such as stroke, schizophrenia, neuropathic pain, depression, and drug addiction. For example, the condition or illness is stroke, especially ischemic stroke. The compounds and compositions of the present invention can also be used to improve learning, cognition, or memory.

[0176] Mammalian cells can be human cells. These cells can be neurons.

[0177] Individuals may be suspected of having, or at risk of having, a neuropathological condition or neurodegenerative disease. Neuropathological conditions may include acute brain injury, including stroke or traumatic brain injury. Neurodegenerative diseases may include Alzheimer's disease, Parkinson's disease, or Huntington's disease, or mental illnesses such as schizophrenia, anxiety, and depression.

[0178] Generally, the compounds described herein should be used without causing substantial toxicity. The toxicity of the compounds described herein can be determined using standard techniques, for example, by testing in cell cultures or laboratory animals and determining the therapeutic index, i.e., the ratio of the LD50 (the dose lethal to 50% of the population) to the LD100 (the dose lethal to 100% of the population). However, in certain situations, such as in severe disease conditions, administering a substantial overdose of the composition may be appropriate. Some of the compounds described herein may be toxic at certain concentrations. Titration studies can be used to determine toxic and non-toxic concentrations. Toxicity can be assessed by detecting the specificity of a particular compound or composition across cell lines. Animal studies can be used to provide indications of whether the compound has any effect on other tissues.

[0179] The compounds described herein can be administered to an individual. As used herein, "individual" can be a human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc. The individual may be suspected of having or at risk of having a neurodegenerative disease (such as Alzheimer's disease, Parkinson's disease, Huntington's disease, or mental illness) or a neuropathological condition (such as traumatic brain injury or acute brain injury such as stroke). Diagnostic methods for various neuropathological and neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease, Huntington's disease, mental illness, traumatic brain injury, or acute brain injury such as stroke are known to those skilled in the art.

[0180] This document describes various alternative embodiments and examples of the invention. These embodiments and examples are illustrative and should not be construed as limiting the scope of the invention. Example

[0181] General methods and materials

[0182] 1. Computer simulation approach (In silico pipeline)

[0183] 1.1 Selecting compounds from the ZINC library

[0184] A subset of lead sample compounds was screened from the ZINC library based on various molecular descriptors. This lead sample set was then further screened using Lupinski's rule of three for developing CNS compounds (Pajouhesh & Lenz, 2005), along with other chemical properties including the absence of carboxyl groups, the absence of peptide-like structures, a polar surface area of ​​60-80 Ų, and the presence of at least one nitrogen, one oxygen, and one aromatic ring. All candidate compounds were screened based on whether they contained chemical motifs or known toxic groups. The resulting database was finalized by adding hydrogen and removing minor components (salts), deprotonating strong acids, and protonating strong bases. Finally, the energy of the screened database was minimized to obtain the three-dimensional structures of the compounds in their lowest energy states. The final optimized structures were then prepared for interstitial docking with the N-terminal domain (NTD) of the GluN1 / GluN2A heterodimer.

[0185] 1.2 Homology Model

[0186] Homology models were established based on the crystal structures of GluN1 / GluN2B. A homology model of the rat NMDAR N-terminal extracellular domain of the NR1 / NR2A receptor was constructed using the X-ray structure of the similar GluN1 / GluN2B N-terminal domain (PDB code: 3QEK). Although the GluN1 / GluN2A receptor is a tetrameric receptor, the homology model was built on a dimer consisting of one GluN1 subunit and one GluN2A subunit. The NTDs of GluN2A and GluN2B showed 72% sequence identity and 82% homology in sequence alignment.

[0187] 1.3 Virtual screening (docking) for selectivity modifiers of NMDAR containing GluN1 / GluN2A

[0188] Using a previously described consensus-based computer simulation method (Axerio-Cilies et al., 2011; Lack et al., 2011), the inventors virtually screened 200,000 commercially available chemicals pre-screened from the lead sample ZINC chemical library (Irwin & Shoichet, 2005) (Axerio-Cilies et al., 2009; Pajouhesh & Lenz, 2005) to identify specific binders that might be capable of positive allosteric modulation (PAM) of NMDAR containing GluN1 / GluN2A. The results of each stage of this multi-parameter method were edited, and the compounds were ranked using a consensus scoring procedure. Up to 10,000 of the top-ranked compounds were conceptualized, and 200 initial candidates predicted to have high binding potential at the GluN1 / GluN2A interface were selected for empirical testing.

[0189] 1.4 Analog Search

[0190] A similarity search is performed to obtain a large number of chemical substances from which structure-activity relationships (SARs) are generated. The chemical substance database is searched using a molecular fingerprint-based similarity search, with the active compound (Npam02) as a template / query. If the feature is present in the molecule, the bit is set to "1"; if the feature is not present, the bit is set to "0," thus forming a unique fingerprint for each chemical structure. Similarity between two molecules is identified by comparing the bit strings of the molecules and quantifying them into Tanimoto coefficients (Tc) (Bajusz, Racz, & Heberger, 2015). The compounds generated in the search are combined with compounds designed for chemical synthesis in computer simulations to produce a list of compounds, which can be tested using whole-cell voltage-clamp recordings in cortical neurons to generate SARs.

[0191] 1.5 Chemical Synthesis of Npam Compounds

[0192] The chemical synthesis of Npam compounds is carried out by reacting a suitably substituted benzaldehyde with a suitably substituted hydrazine, or by reacting a suitably substituted benzaldehyde with hydrazine and then reacting the product with a suitably acyl halide. Proton nuclear magnetic resonance (NMR) is then performed. 1 H-NMR and electrospray ionization mass spectrometry (ESI-MS) were used to verify the structure and purity of each compound.

[0193] 5.1 Synthesis of Npam 43

[0194]

[0195] Step I: 2,3-Dichloro-5-ethoxy-6-hydroxybenzaldehyde (Int-1)

[0196] 2-Hydroxy-3-ethoxybenzaldehyde (1.0 g, 5.2 mmol) was dissolved in acetic acid (20 mL), and N-chlorosuccinimide (NCS) (1.4 g, 11 mmol) was added all at once. The reaction mixture was stirred overnight at 80 °C and then cooled to room temperature. Water and CH2Cl2 were then added to separate the phases. The aqueous phase was further extracted with CH2Cl2, dried over MgSO4, and evaporated under vacuum. The crude product was purified by rapid chromatography (CH2Cl2 / hexane) to give a pure product (1.2 g, 89%) as a yellow solid.

[0197] Step II: (E)-2-bromo-N'-(2,3-dichloro-5-ethoxy-6-hydroxybenzyl)benzoylhydrazide (Npam43)

[0198] Equimolar amounts of 2-bromobenzoyl hydrazine (0.30 mmol) and Int-1 (0.30 mmol) were dissolved in THF (0.5 M solution). Two equivalents of MgSO4 were added and the mixture was heated under reflux for 1 hour. The product may precipitate. If no precipitation occurs, the reaction mixture was examined by TLC or NMR to determine the consumption of Int-1. If the product precipitates, it was recovered by filtration and washed with water (2 × 2 ml) to remove residual MgSO4. If no precipitation occurs, the reaction mixture was diluted with water until precipitation occurred. The mixture was acidified to pH 3 with diluted HCl to remove residual hydrazine and filtered, recrystallized if necessary. The compound is a high-melting-point solid (melting point 210–212 °C). Extraction was avoided. If no precipitation was observed, the solvent was evaporated under vacuum and the compound was purified by rapid column chromatography.

[0199] 1 H NMR (400MHz, DMSO-d6): δ = 12.59 (s, 1H), 10.41 (s, 1H), 8.89 (s, 1H), 7.61-7.34 (m, 4H), 7.08 (s, 1H), 4.11 (q, J = 6.9Hz, 2H), 1.41 (t, J = 7.0Hz, 3H).

[0200] MS(EI):(C 16 H 13 BrCl2N2O3+H) + The calculated value is 433.0, and the measured value is 433.0; (C 16 H 13 The calculated value of BrCl2N2O3+Na is 455.0, and the measured value is also 455.0.

[0201] 5.2 Synthesis of other Npam compounds

[0202] The following compounds were synthesized in a similar manner.

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210] 2. In vitro identification of compounds

[0211] 2.1 HEK293 cell culture and plasmid transfection

[0212] Cells were transfected using a combination of pcDNA3-CMV expression vectors, each expressing one rat recombinant subunit (GluN2AWT, GluN1WT, GluN2BWT). The sequences of all plasmids were confirmed by automated DNA sequencing. Human embryonic kidney 293 (HEK293 cells) were cultured in Dulbecco modified Eagle medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS). When HEK293 cells reached 90% confluence, one of the GluN1 & GluN2A or GluN1 & GluN2B plasmids was co-transfected into the cells using Lipofectamine 2000 (Invitrogen, 11668019) according to the manufacturer's instructions. HEK293 cells were then maintained in a 37°C incubator with 95% O2 and 5% CO2 for 48 hours before use in experiments. The transfection ratio of the NMDAR subunit combinations was 1:1 (GluN1 / GluN2A or GluN1 / GluN2B).

[0213] 2.2 Primary Culture of Cortical Neurons

[0214] As previously described, dissociated cultures of rat cortical neurons were prepared from 18-day-old Sprague Dawley rat embryos. To obtain neuron-rich mixed cortical cultures, uridine (10 μM) and 5-fluoro-2'-deoxyuridine (10 μM) were added to the culture medium on day 3 in vitro (DIV) and maintained for 48 hours before returning to normal culture medium to inhibit non-neuronal cell proliferation. Mature neurons (11–14 DIV) were used for experiments. Mouse cortical cultures were prepared from 18-day-post-mating embryos of heterozygous GluN2A+ / - or GluN2B+ / - litters. To obtain homozygous and wild-type (WT) litter control neuronal cultures, cortical cells from single embryos were plated separately. Genotyping was performed using tail samples collected from each embryo as previously described. To induce neuronal apoptosis, cortical cultures were stimulated for 20 minutes with NMDA (50 μM) and glycine (10 μM), or in a Mg-free medium containing the following (in mM). 2+ Stimulation with STS (100 nM) for 1 hour in extracellular solution (ECS): 25 HEPES acid, 140 NaCl, 33 glucose, 5.4 KCl, and 1.3 CaCl2, pH 7.35, osmolality 320-330 mOsm. This was achieved by Mg-free... 2+ In ECS, treatment with (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycloheptene-5,10-imine maleate (MK-801) (10 μM) in the presence of paeonol for 10–15 min, followed by thorough washing with ECS containing 1 mmol MgCl2 (normal ECS) to remove any trace amounts of MK-801, achieves specific blockade of synaptic NMDA receptors. The GluN2A-specific antagonist NVP-AAM077 (0.4 μM; a gift from YP Auberson, Novartis Pharma AG, Basel, Switzerland) or the GluN2B-specific antagonist Ro 25-6981 (0.5 μM) was added to the bath medium 10 min before and throughout the treatment.

[0215] 2.3 In vitro electrophysiology

[0216] Whole-cell patch-clamp recording was performed in voltage-clamp mode using an Axopatch 200B or 1D patch-clamp amplifier (Molecular Devices). Unless otherwise specified, whole-cell currents were recorded at a hold voltage of -60 mV, with the signal filtered at 2 kHz and digitized at 10 kHz (Digidata 1322A). Recording pipettes (3–5 MΩ) were filled with an intracellular solution containing the following (mM): CsCl 140, HEPES 10, Mg-ATP 4, QX-314 5, pH 7.20; osmolality, 290–295 mOsm. BAPTA (10 mM) was added to the intracellular solution (unless otherwise specified). Coverslips were continuously perfused with an extracellular solution containing the following (mM): NaCl 140, KCl 5.4, HEPES 10, CaCl2 1.3, glucose 20, pH 7.4; osmolality, 305–315 mOsm. NMDA-induced currents were implemented via a perfusion rapid step (Warner Instruments). NMDA application was achieved using a bicornuate barrel system via a perfusion rapid step system, with CNQX (10 μM), TTX (0.5 μM), or BIC (10 μM) added to the extracellular solution, depending on the age of the cultured neurons, to minimize activation of ionotropic glutamate receptors and voltage-gated sodium channels, respectively. All experiments were performed at room temperature. All active compounds were recorded from at least six HEK293 cells / neurons. Data from HEK293 cells or primary neurons were pooled and composite dose-response data were fitted using the following equation: Percentage of response = 100 × Relative potency / [1 + (EC50 / concentration)nH], where EC50 is the agonist concentration producing the half-maximal response, relative potency is the response at the maximum effective concentration relative to glutamate, and nH is the Hill slope.

[0217] 2.4 Ex vivo electrophysiology

[0218] Whole-cell patch-clamp recording was performed in voltage-clamp mode using an Axopatch 200B or 1D patch-clamp amplifier (Molecular Devices). Unless otherwise specified, whole-cell currents were recorded at a hold voltage of -60 mV, with the signal filtered at 2 kHz and digitized at 10 kHz (Digidata 1322A). Recording pipettes (3-5 MΩ) were filled with an intracellular solution containing the following (mM): CsCl 140, HEPES 10, Mg-ATP 4, QX-314 5, pH 7.20; osmolality, 290-295 mOsm. BAPTA (10 mM) was added to the intracellular solution (unless otherwise specified). Coverslips were continuously perfused with an extracellular solution containing the following (mM): NaCl 140, KCl 5.4, HEPES 10, CaCl2 1.3, glucose 20, pH 7.4; osmolality, 305-315 mOsm. NMDA, GABA, or AMPA-induced currents were applied via a rapid perfusion step (Warner Instruments). The rapid perfusion step system was used to apply NMDA, GABA, or AMPA through double-barreled glass tubes, with CNQX (10 μM) and TTX (0.5 μM) added to the extracellular solution according to the age of the cultured neurons to minimize activation of ionotropic glutamate receptors and voltage-gated sodium channels, respectively. All experiments were performed at room temperature. All active compounds were recorded from at least six HEK293 cells / neurons. Data from HEK293 cells or primary neurons were pooled and composite dose-response data were fitted using the following equation: Percentage of response = 100 × Relative potency / [1 + (EC...] 50 / concentration) nH ], of which EC 50 It is the agonist concentration that produces the half-maximal response; relative potency is the response at the maximum effective concentration relative to the maximum response of glutamate, n. H It is the slope of Hill.

[0219] 2.5 Slice Recording

[0220] Six- to eight-week-old C57 / B16 mice or rats underwent cervical dislocation followed by decapitation. The brain was immediately transferred to an ice-cold NMDG-based cutting fluid consisting of the following (in mM): 120 NMDG, 2.5 KCl, 1.2 NaH2PO4, 25 NaHCO3, 1.0 CaCl2, 7.0 MgCl2, 2.4 Sodium pyruvate, 1.3 Sodium ascorbate, and 20 D-glucose, and the pH was adjusted to 7.35 with HCl acid (unless otherwise specified, all chemicals and drugs were purchased from Sigma or BioShop, Canada). The hippocampus was dissected, and transverse hippocampal sections (400 μm) were obtained using a manual histiocentesis machine (Stoelting, Wood Dale, IL, USA). Sections were incubated for 1 hour in a heated (30°C) culture chamber containing ACSF of the following composition (in mM): 124 NaCl, 3 KCl, 1.25 NaH2PO4, 1 MgSO4·7H2O, 2 CaCl2, 26 NaHCO3, and 15 D-glucose, continuously bubbled with carbokinase (95% O2 / 5% CO2) (pH to 7.3). After another 30 minutes at room temperature, sections were transferred to an immersed recording chamber and continuously perfused with carbokinized ACSF (2–3 ml / min). Whole-cell recordings of CA1 pyramidal neurons were performed using a “blind” method with a MultiClamp 700B amplifier. EPSC (excitatory postsynaptic current) was elicited by stimulation of the SC pathway. To isolate NMDAR currents, cells were voltage-clamped at +40 mV. Recording pipettes were filled with a solution containing the following (in mM): 122.5 μM cesium mesylate, 17.5 μM CsCl, 2 μM MgCl2, 10 μM EGTA, 10 μM HEPES, 4 μM ATP(K), and 5 μM OX-314, and the pH was adjusted to 7.2 with CsOH. GABA receptor-mediated inhibitory synaptic currents were blocked with methyl iodine (10 μM; Abcam), and AMPAR-mediated currents were blocked with CNQX (10 μM; Abcam) to further separate NMDAR currents. To specifically separate the NR2A and NR2B components of the NMDAR current, NVP or efendil was added to inhibit these receptors, respectively. Residual synaptic currents mediated by NMDAR were confirmed by APV at the end of the experiment. EPSCs were recorded and analyzed using WinLTP. Statistical analysis was performed using GraphPad InStat. ANOVA and Tugi post-hoc tests were used to compare NMDAR currents in response to various drug cocktails to determine differences between treatments. Statistical significance was defined as p < 0.05, where n = cell number. Data are expressed as mean ± SEM. The conditions for extracellular recording (fEPSP) sectioning were similar to those for whole-cell preparation. The stimulating electrode was placed in the SC pathway, and the recording electrode was placed in the radiative layer of CA1.Records were acquired and analyzed using WinLTP. The initial slope of the fEPSP was measured to quantify synaptic strength (Johnston and Wu, 1995). Statistical comparisons of mean fEPSP slopes between groups were performed using the Student's t-test. All values ​​are shown as mean ± SEM, n = number of slices.

[0221] 2.6 In vitro electrophysiology (GluN2A knockout mice)

[0222] Male and female wild-type or GluN2A KO mice aged 20–25 days were anesthetized with isoflurane and rapidly decapitated. The brain was removed and immersed for 30 seconds in a cold (2–4°C) cutting fluid containing the following: 92 mM NMDG, 2.5 mM KCl, 1.25 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 4.5 mM D-glucose, 5 mM sodium ascorbate, 3 mM sodium pyruvate, 0.5 mM CaCl2, and 10 mM MgCl2. The brain was sealed in melted 3% agar-A (CAS#9002-18-0, Bio Basic Canada Inc.), then glued to a sectioning platform and sectioned coronally at a thickness of 320 μm using a vibratory microtome (Leica VT 1000S) containing a cold (2–4°C) bubble (95% O2 / 5% CO2) cutting fluid. Sections including those from the frontal cortex were transferred to a preheated (32-34°C) cutting fluid for 12 minutes for initial recovery. Then, prior to recording, the sections were transferred to a room-temperature carbomer-treated retention solution containing the following for at least 30 minutes of recovery: 119 mM NaCl, 2.5 mM KCl, 1.2 mM NaH₂PO₄, 24 mM NaHCO₃, 12.5 mM D-glucose, 5 mM sodium ascorbate, 3 mM sodium pyruvate, 2 mM CaCl₂, and 2 mM MgCl₂.

[0223] 2.7 GluN2A-knockout mice

[0224] Wild-type (WT) and GluN2A knockout (GluN2A- / -) mice with a C57BL / 6J background (Sakimura et al., 1995; Townsend et al., 2003) were housed in groups in standard cages (2-3 mice per cage, minimum enrichment) at 21°C. The animals were kept in a 12-hour light / dark cycle with free access to food and water. Each sample (2 μL DNA) was incubated in a PCR master mixture consisting of: 14.85 μL nuclease-free H₂O, 2.5 μL 10% PE reaction buffer, 1.4 μL (50 mM) MgCl₂, 2.0 μL (2.5 mM) dNTPs, 0.5 μL NR₂A₁ primer, 1.0 μL NR₂A₃ primer, 0.5 μL Neo₂A primer, and 0.25 μL (5 U / μL) Taq DNA polymerase (Invitrogen Canada; Burlington, Ontario, Canada). The cycling parameters used were as follows: the first cycle was at 94 °C for 4 min, followed by 29 cycles at 94 °C for 30 s, 60 °C for 40 s, and 72 °C for 60 s. Samples were then placed at 72 °C for 7 min and stored at 4 °C until use. The primers used were NR2A1 (5'-TCTGGG GCC TGG TCT TCA ACA ATT CTG TGC-3'), NR2A3 (5'-CCC GTT AGC CCG TTG AGT CACCCC T-3'), and Neo2A (5'-GCC TGC TTG CCG AAT ATC ATG GTG GAA AAT-3') (Invitrogen Canada; Burlington, Ontario, Canada). PCR products were run on a SYBR-safe 1.5% agarose gel and visualized using a transmission illuminator.

[0225] 2.8 Brain slice preparation (GluN2A-knockout mice)

[0226] Male and female wild-type or GluN2A KO mice aged 20–25 days were anesthetized with isoflurane and rapidly decapitated. The brain was removed and immersed for 30 seconds in a cold (2–4°C) cutting fluid containing the following: 92 mM NMDG, 2.5 mM KCl, 1.25 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 4.5 mM D-glucose, 5 mM sodium ascorbate, 3 mM sodium pyruvate, 0.5 mM CaCl2, and 10 mM MgCl2. The brain was sealed in melted 3% agar-A (CAS#9002-18-0, Bio Basic Canada Inc.), then glued to a sectioning platform and sectioned coronally at a thickness of 320 μm using a vibratory microtome (Leica VT 1000S) containing a cold (2–4°C) bubble (95% O2 / 5% CO2) cutting fluid. Sections including those from the frontal cortex were transferred to a preheated (32-34°C) cutting fluid for 12 minutes for initial recovery. Then, prior to recording, the sections were transferred to a room-temperature carbomer-treated retention solution containing the following for at least 30 minutes of recovery: 119 mM NaCl, 2.5 mM KCl, 1.2 mM NaH₂PO₄, 24 mM NaHCO₃, 12.5 mM D-glucose, 5 mM sodium ascorbate, 3 mM sodium pyruvate, 2 mM CaCl₂, and 2 mM MgCl₂.

[0227] 2.9 Preparation of hippocampal sections for immunoblotting using pCREB

[0228] Male Sprague Dawley rats aged 4-8 weeks (125-200 gm) were decapitated, and their brains were rapidly dissected and placed in ice-cold cutting saline [containing (in mM): 110 sucrose, 60 NaCl, 3 KCl, 1.25 NaH2PO4, 28 NaHCO3, 5 D-glucose, 0.5 CaCl2, 7 MgCl2, and 0.6 ascorbate, saturated with 95% O2 / 5% CO2]. 400 μm transverse sections were then prepared using a Vibratome Series 1000 (Pelco, Ted Pella, Redding, CA). Immediately transfer the sections to a 1:1 mixture of cutting saline and normal ACSF [containing (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 10 D-glucose, 2 CaCl2, and 1 MgCl2, saturated with 95% O2 / 5% CO2] and maintain at room temperature for at least 90 minutes. Then, before pharmacological stimulation, transfer the sections to ACSF in an immersion chamber at 32°C for 45–60 minutes.

[0229] 2.10 Electrophysiological methods (GluN2A knockout mice)

[0230] Brain slices were transferred to the recording chamber of an upright Nikon FN1 microscope and continuously perfused with a 32°C Carbomeride aCSF recording solution containing the following: 121.85 mM NaCl, 2.5 mM KCl, 1.2 mM NaH2PO4, 24 mM NaHCO3, 12.5 mM D-glucose, 5 mM sodium ascorbate, 3 mM sodium pyruvate, 2 mM CaCl2, and 0.1 mM MgCl2. Cone neurons in layer V of the prefrontal cortex were identified using video-monitored infrared differential interference contrast illumination microscopy with a CFI APO 40X W NIR objective (0.80 numerical aperture, 3.5 mm working distance). Whole-cell patch-clamp recording was performed using patch pipettes with a resistance of 5–8 MΩ. Recording pipettes were prepared from borosilicate glass capillaries (1B150F-4, WPI, USA) and filled with a pipette solution containing the following (280-290 mol / M, pH 7.4): 135 mM CsMeSO4, 0.6 mM EGTA, 10 mM HEPES, 2.5 mM MgCl2, 5 mM Tris phosphate, 3 mM Mg-ATP, 0.2 mM GTP Tris, and 5 mM QX314 chloride. Voltage-clamp recordings of the current signals were amplified using a MultiClamp 700B amplifier (Molecular Devices), low-pass filtered at 4 kHz, sampled at 10 kHz using a Digidata 1440A data acquisition system (Molecular Devices), and recorded using pCLAMP 10.2 acquisition software (Molecular Devices). Cells were maintained at -65 mV with liquid junction potential correction and 40% series resistance correction.

[0231] 2.11 Site-directed mutagenesis

[0232] Site-directed mutagenesis of the GluN1 or GluN2A subunits was performed using the QuikChange method (Stratagene). All mutant clones were confirmed by DNA sequencing. Wild-type or mutant subunits were transfected into HEK293 cells, and electrophysiological assays were performed. Cells were transfected with a combination of pcDNA3-CMV expression vectors, each expressing the rat recombinant GluN2A. WT GluN1 WT GluN2B WT One of the subunits. Co-transfection with enhanced green fluorescent protein (GFP) pcDNA3-GFP to facilitate microscopic observation. PFU DNA polymerase was used via GluN1. WT and GluN2A WT Site-directed mutagenesis to construct GluN2AA108G GluN2A P79A GluN2A P178G GluN2A Q111A GluN2A F115Y GluN2A F115S GluN2A F177S GluN2A I176Y GluN2A M112I GluN1 R115E GluN1 L135Q Plasmids. The sequences of all plasmids were confirmed by automated DNA sequencing.

[0233] 2.12 Using Ca 2+ Sensitive dye measurement of Ca in rat cortical cultures 2+

[0234] Rat neurons isolated from the entire cortex were seeded into poly-d-lysine-coated 96-well plates. After 12–14 days of culture, intracellular calcium levels were determined using the Fluo-4No Wash calcium assay kit according to the manufacturer’s guidelines (Thermo Fisher Scientific). Briefly, the neuronal culture medium was removed and replaced with calcium assay buffer (CAB) containing a mixture of 1×HBSS, 20 mM HEPES, 2.5 mM probenecid, and Fluo4-NW dye (pH 7.4; Thermo Fisher Scientific). Cells were then incubated at 37°C for 45 min to load the dye, followed by incubation at 25°C for 15 min. To isolate NMDAR-mediated calcium signaling, NMDA (10 μM) was used as an agonist with the addition of 2 μM glycine. GluN1 / GluN2A NMDAR was blocked with the antagonist NVP-AAM007. After recording for 60 seconds using a FLEXStation II benchtop scanning fluorometer (Molecular Devices), calcium fluorescence was measured at 25°C; then, the NMDAR agonist NMDA (10 μM) / glycine (2 μM) was added at 180 seconds. A total of 30 minutes of continuous fluorescence plate readings were performed using excitation at 485 nM, emission at 538 nM, and cutoff at 530 nM. Data were recorded using SoftMax Pro software (Molecular Devices).

[0235] 2.13 Lactate dehydrogenase assay

[0236] Lactate dehydrogenase (LDH) is a cytoplasmic enzyme that converts nicotinamide adenine dinucleotide (NAD) to NADH (reduced form). LDH is released from cells into the culture medium when plasma membrane integrity is compromised. Therefore, the amount of LDH released represents the degree of cell death. In this study, extracellular LDH levels were measured using an in vitro toxicology assay kit obtained from Sigma-Aldrich (no. TOX-7). The LDH assay was based on the following steps: (1) LDH reduced NAD to NADH, (2) the resulting NADH was then used for the stoichiometric conversion of a tetrazolium dye, and (3) the resulting colored compound was measured at a wavelength of 490 nm using a spectrophotometric microplate reader. Cell death rate was expressed as the ratio (%) between the absorbance of the treatment group and the absorbance of the control group.

[0237] 2.14 Immunoblotting

[0238] Brain tissue or cultured cells were lysed on ice in lysis buffer, and the solution was then centrifuged at 14,000 rpm for 10 minutes at 4°C. The supernatant was then collected and protein concentration was determined using a BCA protein assay kit (Thermo Scientific, 23227). An equal volume of protein sample was mixed with 4 times sample buffer, boiled at 100°C for 5 minutes, and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred to an Immobilon-PTM polyvinylidene fluoride (PVDF) membrane (Bio-Rad, 162-0177). The membrane was blocked with Tris-buffered saline containing 0.1% Tween-20 (TBST) in 5% skim milk for 1 hour at room temperature, followed by overnight incubation with primary antibody at 4°C. After washing in TBST for 3 × 5 minutes, the proteins were visualized in a Bio-Rad imaging system using ECL Western blot substrate (Pierce, 32016). To detect phosphorylated-CREB, samples prepared on the same day were used. A polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA) was incubated with a primary antibody against phosphorylated-CREB (Ser133) (Cell Signaling Technology, Beverly, MA). To detect total CREB, the same polyvinylidene fluoride membrane was peeled off and re-detected with a primary antibody against total CREB (Cell Signaling Technology). The band density of each protein was quantified using Bio-Rad Quantity One software, and the relative optical density compared to total CREB loaded onto the same membrane was analyzed.

[0239] 2.15 Excitotoxicity assay

[0240] Cortical cultures were treated with Npam43, and NMDA-induced excitotoxicity was assessed 20–24 hours after treatment by measuring lactate dehydrogenase (LDH) release. Briefly, cells were treated with 75 μM NMDA for 1.5 h, followed by washing neurons once with fresh neural basal medium and replacing the medium with conditioned medium. LDH release was measured using an in vitro toxicology assay kit from Sigma-Aldrich (no. TOX-7). Cell death rate was expressed as the ratio (%) between the absorbance of the treated group and the absorbance of the control group.

[0241] 2.16 H2O2 Cytotoxicity Assay

[0242] Cultured cortical neurons were exposed to 600 μM H2O2 for 1 hour to induce neuronal cell death. To demonstrate the selectivity of Npam43 for GluN1 / GluN2A NMDAR, neurons were treated with either the GluN1 / GluN2B selective antagonist efendil (3 μM) or the GluN1 / GluN2B selective antagonists NVP-AAM077 (0.2 μM) and TCN-201 (10 μM).

[0243] 2.17 Reagents. Phosphate buffer solutions were prepared using NaH₂PO₄·2H₂O and Na₂HPO₄·12H₂O, with the pH adjusted by varying the molar ratio of NaH₂PO₄·2H₂O to Na₂HPO₄·12H₂O. Other chemicals used were of analytical grade or better quality, and Milli-Q ultrapure water (>18 MU cm) was used throughout the experiments.

[0244] 2.18 Instrumentation and Chromatographic Conditions. Npam43 was separated from CSF and serum matrices using high-performance liquid chromatography (HPLC) and quantified by electrochemical detection. The system consisted of an ESA 582 pump (Bedford, MA), a pulse damper (Scientific Systems Inc., State College, PA), a Rheodyne Inert manual injector (model 9125i, 20 μL injection loop; Rohnert Park, CA), a Tosoh Bioscience Super ODS TSK column (2 μm particles, 2 mm × 10 mm; Montgomeryville, PA), and an Antec Leyden IntroElectrochemical detector (V) with a VT-03 flow cell and an Ag / AgCl reference electrode. 施加= +800mV; Leyden, The Netherlands). The mobile phase was a 20mM phosphate buffer-acetonitrile (80:20, v / v) mixture, pH 7.0, flowing through the system at 0.1 mL / min. The column was maintained at 40°C throughout the analysis, with an injection volume of 8 μL. Before use, the mobile phase was filtered through a 0.22 mm membrane and degassed using a vacuum pump, and kept under helium purging during the experimental tests. Chromatographic data were acquired and analyzed using EZChrome Elite software (ScientificSoftware, Pleasanton, CA).

[0245] 2.19 Sample Preparation

[0246] Samples were prepared by mixing the original sample with an aliquot of acetonitrile (50:50). The samples were mixed, allowed to stand at ambient temperature for 10 minutes, and then centrifuged at 5000g for 5 minutes. 8 μL of the supernatant was injected.

[0247] 2.20 CSF and serum extraction and HPLC-ECD analysis

[0248] Thaw the CSF and serum samples generated in vivo and transfer 8 μl to separate Eppendorf tubes. Add 2 μl of 0.5 μg / mL Npam50 internal standard (IS), followed by 22 μl of acetonitrile. Vortex the sample for 5–10 seconds and centrifuge at 20,000 × g for 5 min to precipitate any precipitated proteins. Transfer the clear supernatant to an HPLC vial for analysis. Prepare standards using blank rat CSF and serum in a similar manner. Sample preparation and subsequent HPLC-ECD analysis were performed using optimal grade (Fisher Scientific) solvent and 18 MΩ water (Millipore). Calibration standards were 0.1–50 μM (6 spots, CSF equivalent level), R 2 >0.99. The detection limit is >0.8 μM Npam43. Pre- and post-spiking serum compared to pure standards showed approximately 10% inhibition and 95% extraction efficiency. Any samples outside the calibration range should be diluted 10-fold for reanalysis.

[0249] 3. In vivo analysis of compounds

[0250] 3.1 Cerebral ischemia

[0251] Adult male Sprague Dawley rats weighing approximately 200g were anesthetized, and the middle cerebral artery (MCA) was exposed by making a craniotomy window (2mm in diameter) 1mm apical at the anterior junction of the zygomatic and squamous bones. Tri-vessel occlusion was achieved using a suture ligation method. The exposed MCA was ligated with a square knot using 10-0 nylon sutures. Next, the bilateral common carotid arteries (CCAs) were clamped with non-traumatic arterial clips. A significant decrease in local cerebral blood flow monitored by laser Doppler flowmeter (PF-5010, Periflux system; Perimed AB) confirmed the success of the procedure. After 90 minutes of ischemia, the sutures and clips were removed to allow immediate reperfusion. The rats were subdivided to receive different doses of Npam43 or saline / carrier (as specified) via femoral vein injection. The size of the necrotic lesion in stained brain sections was assessed using 2,3,5-triphenyltetrazolium chloride (TTC). Npam43 and saline / carrier were administered via bolus injection 3.5 hours after stroke onset. To achieve optimal results, two additional doses of Npam43 were administered on the second and third days, respectively. The rats were then allowed to recover for varying periods until further experiments.

[0252] 3.2 Magnetic Resonance Imaging

[0253] Rats were anesthetized, and their body temperature was maintained at 37.0 ± 0.5 °C using a heating pad during imaging. T2-weighted spin-echo imaging (T2WI) sequences were performed using a 3.0T General Electric imaging system (R4, GE) with the following parameters: repetition time, 4000 ms; echo time, 105 ms; 6–8 consecutive coronal sections, each 2 mm thick. At this stage of stroke development (7 days post-ischemia), cerebral infarction appears as a high signal (bright white) on magnetic resonance imaging (MRI). Non-infarcted areas were manually plotted from section to section, and their volume was measured using Voxtool analysis software (General Electric). Scleretic size was quantified by subtracting the non-scleretic volume of the ischemic hemisphere from the total volume of the contralateral hemisphere.

[0254] 3.3 Neurobehavioral Testing

[0255] To assess the functional recovery of neural circuits damaged by ischemic injury, three modes of motor activity (sensomotor) deficit were tested: 1) vertical movement (the total number of beam interruptions occurring in the vertical sensor); 2) the number of vertical movements (the number of times the animal's hind legs were upright); and 3) the duration of vertical movement using a VersaMax Animal Activity Monitor (Accuscan Instruments) (the amount of time the animal's hind legs were upright, in seconds). Ischemic rats were placed in a recording chamber during the "dark" phase of the day / night cycle, and the duration of vertical movement (in seconds) was automatically recorded by computer over 2 hours. The total length of vertical movement represents the recovery of motor circuits damaged by ischemic stroke.

[0256] Example 1: Identification of positive allosteric modifiers

[0257] The GluN1 / GluN2A NMDAR isoform is an attractive target for positive allosteric modulators. The inventors employed a computer-simulated drug discovery approach to virtually screen commercially available lead-sample compounds from the ZINC database (Irwin, J. et al. Abstr Pap Am Chem Soc (2005) 230: U1009) and a number of synthetic compounds to identify potential GluN1 / GluN2A-NMDAR modulators. The computer simulation method included large-scale docking at the gap interface of the GluN1 / GluN2A heterodimer and consistency scoring of compounds selected for empirical testing. Candidate compounds were further screened using GluN1 / GluN2A transiently transfected in human embryonic kidney (HEK293) cells, and the tests were recorded using whole-cell patch-clamp.

[0258] Table 1 shows the synthetic compounds and compounds from the ZINC database that were identified as having a positive regulatory effect on GluN1 / GluN2A NMDAR.

[0259] Table 1. Enhancement effects of synthesized (syn) compounds and compounds from the ZINC database

[0260]

[0261]

[0262]

[0263] Example 2: Identification of regulators that selectively enhance NMDAR containing GluN1 / GluN2A

[0264] The inventors used transiently transfected HEK293 cells expressing NMDAR of GluN1 / GluN2A or GluN1 / GluN2B and used whole-cell voltage patch-clamp recording assays to observe whether the selected compounds controlled the subtype selectivity of the drugs.

[0265] Table 2 shows some of the identified compounds that exhibit positive regulation of GluN1 / GluN2A NMDAR and / or inhibitory effects on GluN1 / GluN2B NMDAR. Figure 1 Data from whole-cell patch electrophysiological recordings showed various regulatory effects of several Npam compounds on NMDAR-mediated currents. Figure 1 The Npam compound was shown to have a positive enhancing effect on recombinant GluN1 / GluN2ANMDAR and / or an inhibitory effect on GluN1 / GluN2B NMDAR, and / or little to no effect on GluN1 / GluN2B NMDAR.

[0266] Table 2. The hit compounds identified in the first screening showed positive regulation of GluN1 / GluN2A NMDAR and / or inhibition of GluN1 / GluN2B NMDAR.

[0267]

[0268] *:50μM

[0269] **:25μM

[0270] Figure 2 This demonstrates the enhanced selectivity of NMDAR containing GluN1 / GluN2A in the presence of the selected Npam compound. Figure 2 It was demonstrated that Npam02, Npam58, Npam72, and Npam43 are selective for GluN1 / GluN2ANMDAR compared to Npam01, Npam04, and Npam59.

[0271] Two compounds, Npam02 and Npam43, which selectively enhance NMDAR containing GluN1 / GluN2A, were further investigated. Npam43 was obtained through a structure-activity relationship (SAR) study based on Npam02.

[0272] Example 3: Characteristics of Npam02

[0273] 3.1 Evaluation of the enhancing effect of Npam02 in HEK293 cells transfected with GluN1 / GluN2A or GluN1 / GluN2B

[0274] Whole-cell patch-clamp recordings were performed to measure the glutamate-induced current using a chloride-based pipette solution at a holding voltage of -60 mV.

[0275] To rule out the possibility that Npam02 itself might induce any current in HEK293 cells expressing GluN1 / GluN2A or GluN1 / GluN2B, administration of Npam02 alone (100 μM) showed no change in inward or outward current. Figure 3 a,d). Compared with glutamate alone ( Figure 3 a, d), co-administration of Npam02 (100 μM) and a co-agonist moderately enhanced NMDA-mediated currents in HEK293 cells expressing GluN1 / GluN2A receptors (100 μM; n = 6; 38.85 ± 3.70%; P < 0.001; and 200 μM; n = 6; 71.69 ± 5.03%; P < 0.001). Figure 3 (b, d). In the presence of both co-agonists, co-administration of the selective GluN1 / GluN2A antagonist NVP-AAM007 (0.2 μM) completely blocked the increased NMDAR current, confirming that there were no secondary effects attributable to endogenous proteins in HEK293 cells. Figure 3 a,d). Conversely, HEK293 cells expressing the GluN1 / GluN2B combination in the presence of Npam02 (100 μM) did not exhibit enhanced NMDAR current ( Figure 3 c, e). Similarly, Npam02 itself does not induce currents, which is attributed to the successful blocking of the NMDAR current of the GluN1 / GluN2B receptor by the GluN2B-specific antagonist efendil (IF; 3 μM). Figure 3 c,e).

[0276] 3.2 Evaluation of the enhancing effect of Npam02 in mature cortical wild-type and GluN2B-deficient neurons using whole-cell voltage-clamp recording.

[0277] To test whether the compound could enhance NMDAR in mature 14-18 day old cortical neurons with high GluN2A receptor expression, the inventors used NVP-AAM007 (a selective GluN1 / GluN2A antagonist at low concentrations) to block receptors containing GluN1 / GluN2A. Figure 4 As shown in a, in the presence of the co-agonists NMDA (5 μM) and glycine (2 μM) in hippocampal neurons, perfusion application of Npam02 (100 μM) moderately modulated the NMDAR current (n = 6, 43.04 ± 6.55%; P < 0.001) compared with the NMDA control group.

[0278] Consistent with HEK293 cell data, Npam02 itself cannot induce inward or outward currents. Figure 4 a). Furthermore, the application of APV (50 μM) blocked all currents, indicating that the enhancement was mediated by NMDAR and not by secondary effects attributed to other endogenous proteins. On the other hand, the enhancement effect of Npam02 could be blocked by the presence of NVP (0.2 μM; n = 6, 0.06 ± 1.36%; P > 0.05), indicating that the enhancement effect originated from NMDAR containing GluN2A ( Figure 4 b).

[0279] Npam01 and Npam04 were tested in a similar manner, but unlike Npam02, no observable enhancement or suppression effect was observed in the NMDAR-current. Figure 4 c). This suggests that the previously observed enhancing effects of Npam01 and Npam04 in HEK293 cells expressing GluN1 and 2A NMDAR may be masked by the inhibitory effects of GluN1 / GluN2B-containing NMDAR in neurons, resulting in zero net change in NMDAR current.

[0280] To further evaluate whether the enhancement effect of Npam02 is selective for NMDAR containing GluN1 / GluN2A, Npam02 was tested in neurons lacking the GluN2B subunit. Co-administration of NVP-AAM007 (0.2 μM) and NMDA (20 μM) in conditional GluN2B knockout neurons almost completely blocked NMDAR currents (n = 4; 90.73 ± 1.11%; P < 0.001). Figure 5 a, b) indicate that a small portion of the residual current still originates from GluN2B. Compared to the NMDA control group, perfusion application of Npam02 (100 μM) positively modulated NMDAR current in neurons from GluN2B-deficient mice in the presence of the co-agonists NMDA (20 μM) and glycine (2 μM) (n = 4; 44.72 ± 4.29%; P < 0.001). Figure 5 (a, b). This is consistent with observations in HEK293 cells expressing GluN1 / GluN2A-NMDAR and the enhancement observed in wild-type neurons.

[0281] Furthermore, to further isolate the pure GluN2A-fraction, co-administration of the GluN2B antagonist efendil (IF) and Npam02, compared with the NMDA control group, resulted in an enhancement similar to previously observed (n = 4; 42.22 ± 4.03%; P < 0.001). Figure 5 a,b).

[0282] Finally, GluN2B-knockout cortical neurons were used to determine whether Npam02 was selective for NMDAR containing GluN1 / GluN2A. GluN2A-mediated currents were blocked using a selective GluN2A antagonist, and it was confirmed whether the compound differentiated between the two subtypes. Perfusion administration of both co-agonists and the GluN2A-NMDAR antagonist NVP-AAM007 (0.2 μM) and Npam02, compared to the NMDA control, prevented the enhancement of NMDAR currents by Npam02 (100 μM; n = 4; 88.87 ± 3.45% P > 0.001) (Figure 5a, b). This indicates that the residual GluN2B-NMDAR currents were not enhanced.

[0283] 3.3 In mature cortical neurons, Npam02 had no visible regulatory effect on AMPAR and GABAR-mediated currents.

[0284] Next, the inventors evaluated the selectivity of Npam02 for two other key ionotropic receptors in the central nervous system (CNS): the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor AMPAR, known as a non-NMDA ionotropic transmembrane receptor for glutamate, which mediates rapid synaptic transmission in the CNS, and the GABA receptor, which is a major inhibitory ligand-gated ion channel receptor. Whole-cell patch-clamp recordings were performed in cultured hippocampal neurons. The tests showed that Npam02 selectively targets AMPAR (… Figure 6 a,c) or GABAR ( Figure 6 The currents mediated by (b, c) have no observable effect.

[0285] 3.4 Npam02 binding sites at the GluN1 / GluN2A interface

[0286] Structural analysis revealed that the GluN1 / GluN2A dimer interface is primarily surrounded by hydrophobic residues, such as (GluN2A... F115 GluN2A M112 GluN2A P79 GluN1 F113 GluN1 Y109 GluN1 L135 GluN2A F177 GluN2A P178 It is distributed throughout the pocket and around the Npam02 ligand. In fact, the Npam02 ligand is located in the area formed by GluN2A. F115 GluN2A P79 GluN1 F113 and GluN1 Y109The described hydrophobic cage is characterized by residues consisting of a ring structure in its side chain. This feature is particularly interesting because ideally, the ring from the ligand within the cage could anchor itself within the region via strong hydrophobic interactions mediated by two Π systems (aryl-aryl). Npam02 appears to fit this possibility because its aromatic ring ( Figure 7 Positions 1-6) are located within this region and are associated with the aromatic rings surrounding Npam02 ( Figure 7 All the above residues at positions 1-6) are hydrophobically linked. More specifically, for GluN2A... F115 and Npam02 aromatic ring ( Figure 7 The interaction between the two Π systems (aryl-aryl) at positions 1-6 is a strong interaction between the T-side-to-face conformations and appears to be energy-attractive and favorable. A similar effect is observed on the opposite side of the ligand, where another aromatic ring ( Figure 7 (Middle position 11-16) and GluN1 L135 GluN2A F177 and GluN2A P178 Strong interaction. GluN1 L135 It appears that it can directly interact with the second aromatic ring of Npam02 through edge-to-face interactions, while other residues of GluN2A can also interact hydrophobically, but most likely to a lesser extent, because they are farther from the ligand. Conversely, GluN2A... Q111 It exhibits polar hydrogen bonds, accepting the hydroxyl proton of NpamO2 ( Figure 7 The interaction at position 18 in the middle.

[0287] 3.5 Site-directed mutations of predicted binding sites in the N-terminal domain (NTD)

[0288] To verify whether the drug binds to the dimer interface between GluN1 and GluN2A, site-directed mutagenesis was performed on the N-terminal domain (NTD) pocket. Two residues considered crucial for direct drug interaction were selected. Extensive induced fit docking indicated that Npam02 can occupy and utilize the space between the upper lobe (R1) of the NTD. Figure 8 a). Therefore, the inventors tested whether the enhancement of Npam02 to NMDAR containing GluN1 / GluN2A was altered by mutations in residues around the pocket interface, which replaced side chains by selectively blocking compound interactions without interfering with the overall protein structure. The docking model of Npam02 at the predicted regulatory sites showed that the two residues GluN2A (GluN1 / GluN2A) were mutated. 111 ) and GluN2A(F 177 ()( Figure 8b) Directly interacts with Npam02, where GluN2A(Gln) 111 ) and hydroxyl ( Figure 7 Hydrogen bonds are formed at position 18) of GluN2A(F 177 This may cause interference with the aromatic ring ( Figure 7 Hydrophobic interactions at positions 11-16. In fact, a significant reduction in the degree of regulation was achieved through two mutations compared to the regulatory effect observed in wild-type GluN1 / GluN2A receptors. In wild-type neurons (n ​​= 7, 41.75 ± 2.62%) Figure 8 c) When GluN2A is from Gln 111 When Ala is mutated, the regulatory effect of Npam02 (100 μM) is significantly reduced (n = 7, 30.69 ± 1.60%, P > 0.05). Figure 8 c), and Phe 177 Ser mutations further reduced the effect (n = 7, 22.86 ± 1.12%, P > 0.01) (Figure 8c). The incorporation of these two mutations and the subsequent reduction in Npam02 regulation suggest that Npam02 can bind to this interfacial site.

[0289] Example 4: Characteristics of Npam43

[0290] 4.1 In transfected HEK293 cells, Npam43 selectively enhances NMDAR-mediated currents containing GluN1 / GluN2A.

[0291] like Figure 9 As shown, compared with glutamate alone, co-administration of Npam43 (10 μM) with a co-agonist significantly enhanced NMDA-mediated currents in HEK cells expressing GluN1 / GluN2A receptors, indicating that Npam43 can act as an Npam agent. Conversely, HEK cells expressing the GluN1 / GluN2B isoform did not show an enhancement of NDMAR currents in the presence of Npam43 (10 μM). Figure 9 Similarly, Npam43 itself does not generate current. (e.g.) Figure 9 As shown, the enhanced GluN1 / GluN2ANMDAR-mediated current in HEK cells exhibits a dose-dependent relationship in the presence of Npam43, saturating at approximately 350% relative to baseline with the co-agonist alone, EC50. 50 The pEC50 was -0.614 ± 0.05 μM (0.24 ± 0.05 μM). No dose-dependent enhancement of Npam43 current was observed in NMDAR recordings containing GluN2B.

[0292] 4.2 Key amino acid residues at the GluN1 / GluN2A interface in the N-terminal domain that reduce the enhancing effect

[0293] The inventors tested whether the enhancement of Npam43 to NMDAR containing GluN1 / GluN2A was altered by mutations in residues around the pocket interface, which selectively substituted side chains that block compound interactions without interfering with the overall protein structure. The primary sequences of the GluN1 and GluN2A subunits are shown... Figure 10 In a, the bolded residues are defined as those required for the site pocket based on the GluN1 / GluN2A 3-D model. Figure 10 b). The docking model of Npam43 at the predicted regulatory sites shows GluN1-(Leu 135 ()( Figure 10 b) (which forms a bend before the β chain 5) may play a role in its interaction with ligands.

[0294] In fact, GluN1-(Leu 135 Gln significantly reduced the degree of regulation, decreasing positive regulation by more than 70% (n = 6; -77.5 ± 2.12%; P < 0.001; normalized to Npam43 reaction in wild-type GluN1 / GluN2A), indicating that this amino acid is crucial for hydrophobic contact with the compound. Figure 10 c). The model also shows GluN2A-(Gln) in α-helix 80. 111 ) forms a key H-bond interaction with Npam43 ( Figure 10 b), therefore when Gln 111 Mutation to GluN2A-(Gln) 111 When Ala), an enhanced reduction was also observed ( Figure 10 d), consistent with the role of this region in mediating ligand binding (n = 6; -67.4 ± 11.8%; P < 0.001; normalized to Npam43 reaction in wild-type GluN1 / GluN2A). Furthermore, GluN2A-(Phe) located at α-helix 80... 115 The Ser mutation reduced positive regulation (n = 6; -67.4 ± 11.8%; P < 0.001; normalized to the Npam43 response in wild-type GluN1 / GluN2A), indicating that the hydrophobic feature at this position is essential. Figure 10 d). Figure 11 a shows GluN1-(Leu 135 Gln) and GluN2A-(Phe 115Neither the independent nor the co-mutation of Ser directly reflects a change in the overall protein structure but rather affects ligand binding. This is also confirmed by the fact that the dose-response of L-glutamate in these two mutations is unchanged compared to wild-type GluN1 / GluN2A.

[0295] The summary in Table 3 Figure 10 The other eight mutations shown in d, within the defined pocket, also exhibited a significant reduction in enhancement, ranging from 10% to 62%, strongly suggesting that ligand binding occurs at this junction in the upper lobe (R1) of the NTD. Four negative control mutations, GluN1-(Arg), were also mutated. 115 Glu), GluN2A-(Met) 112 Ile), GluN2A-(Ile) 176 Tyr) and GluN2A-(Ala 108 Gly), defined as residues within the binding pocket that do not contribute to ligand binding, is used to further demonstrate the accuracy of the model's binding site. Figure 10 d). Table 3 does not show any single-point mutations that can eliminate Npam43 binding, therefore it is a double mutation; construct a mutation from GluN1 (Leu 135 Gln) and a substance from GluN2A (Phe 115 Mutations in Ser were investigated, and it was tested whether the residual positive regulation could be further reduced. Double mutations of the interacting NTDs individually weakened the response, as did the additive reduction in the enhanced response, which further reduced Npam43 binding (n = 6; -93.9 ± 10.3%; P < 0.001; normalized to Npam43 response in wild-type GluN1 / GluN2A). Figure 10 e Figure 10 f highlights the two point mutations, their relative positions, and how strongly they affect the interaction at the binding site. To determine whether this reduction is due to Npam43 binding or receptor desensitization, the dose-response relationship of glutamate activation was investigated in the wild-type double mutation. Figure 11 The results showed that the double-mutated glutamate dose-response was not significantly altered compared to the wild-type GluN1 / GluN2A receptor, indicating that the mutation does not affect protein function. To assess the accuracy of the structural model for the GluN1 / GluN2A NTD, correlation plotting was performed between the relative enhancement of Npam43 observed in the mutant forms of GluN1 or GluN2A in HEK293 cells, and binding energies were predicted by docking analysis. Figure 11 b). A strong correlation exists between the observed enhancement and the predicted binding energy (R² = 0.93), indicating that the model is accurate enough to simulate the compound within this binding pocket. Figure 11 b).

[0296] Table 3. Enhancement of Npam43-induced GluN1 or GluN2A mutants

[0297] <![CDATA[GluN1 WT / GluN2A WT ]]> 209±6.7％ 6 <![CDATA[GluN1 / GluN2A Q111A ]]> 129±6.7％ It is the / H-key -38.4±0.78％ 4 <![CDATA[GluN1 / GluN2A F115Y ]]> 100±4.2％ It is / Hyd-C -52.2±0.52％ 6 <![CDATA[GluN1 / GluN2A F177S ]]> 79.9±2.6％ It is / Hyd-C -61.7±0.03％ 5 <![CDATA[GluN1 / GluN2A I176Y ]]> 189±3.8％ no / - -10.1±0.12％ 3 <![CDATA[GluN1 / GluN2A M112I ]]> 255±19％ It is / Hyd-C +22.2±0.94％ 3 <![CDATA[GluN1 / GluN2A P178G ]]> 95.5±3.8％ It is / Hyd-C -54.3±0.42％ 4 <![CDATA[GluN1 / GluN2A P79A ]]> 99.3±2.8％ It is / Hyd-C -52.5±0.20％ 5 <![CDATA[GluN1 / GluN2A F115S ]]> 68.1±14.1％ It is / Hyd-C -67.4±11.8％ 6 <![CDATA[GluN1 / GluN2A A108G ]]> 210±9.0％ no / - +0.48±0.01％ 3 <![CDATA[GluN1 R115E / GluN2A]]> 255±3.5％ no / - +22.0±0.40％ 4 <![CDATA[GluN1 L135Q / GluN2A]]> 47.1±2.8％ It is / Hyd-C -77.5±2.12％ 7 <![CDATA[GluN1 L135Q / GluN2A F115S ]]> 12.7±1.8％ Yes, yes / Hyd-C,Hyd-C -93.9±10.3％ 5

[0298] 4.3 In cultured rat hippocampal neurons, Npam43 modulates NMDAR containing GluN1 / GluN2A, enhancing NMDAR-mediated currents.

[0299] Figure 12 We demonstrated that Npam43 selectively enhances GluN1 / GluN2ANMDAR in hippocampal neurons. Figure 12 Increased enhancement was observed in the presence of Npam43 or by co-administration of Npam43 and the GluN1 / GluN2B specific antagonist ivermil (IF; 3 μM). Figure 12 Further analysis showed that treatment with the GluN1 / GluN2A specific antagonist NVP-AAM077 and the NMDA blocker AP5 reduced the enhancement and elimination currents, respectively. Dose-response analysis showed that Npam43 dose-dependently enhanced the NMDAR current, EC 50 The value was 0.25 ± 0.12 μM. Figure 13 It reached a saturation point at 10 μM (n = 6; 322 ± 27% above baseline control). Additionally, the NMDA dose-response curve was found to be pEC. 50 = 1.484 ± 0.082 μM or 30.5 ± 11.7 μM, wherein the left shift occurs in the presence of Npam43 (5 μM), thereby enhancing... Figure 13 The affinity of the NMDA agonist is shown.

[0300] 4.4 Npam43 increases intracellular calcium levels via NMDAR containing GluN1 / GluN2A. 2+

[0301] Cell-based Ca2+ using primary cultured rat neurons 2+ Influx assays were used to determine whether Npam43 regulation contributes to intracellular Ca2+. 2+ The increase and whether this effect is mediated by NMDAR containing GluN1 / GluN2A were investigated. NMDA (10 μM) and glycine (2 μM) were added to cell cultures to activate NMDAR. Npam43 administration to cultured neurons increased [the level of NMDAR]. Figure 14 The Ca shown 2+ Inflow fluorescence signal. To determine Ca 2+Whether the influx is through NMDAR containing GluN1 / GluN2A, the inventors co-administered the GluN2A antagonist NVP-AAM0077 and observed the Ca response to Npam43. 2+ The significant reduction in influx indicates enhanced calcium influx mediated by GluN2A receptors, such as... Figure 14 As shown in the image.

[0302] Example 5: Neuroprotective effect of Npam43

[0303] 5.1 Phosphorylation of CREB (pCREB)

[0304] Phosphorylation of CREB is a reliable indicator of activation of cell survival pathways. Figure 15 A study demonstrated that treatment with Npam43 increased CREB phosphorylation (pCREB) in the presence or absence of the GluN1 / GluN2B antagonist imipendiol. Figure 15 Further analysis showed that, compared with the positive control pachymenine (BIC; 10 μM), the GluN1 / GluN2A specific antagonist NVP-AAM007 reduced Npam43-induced pCREB. Figure 15 b demonstrates the dose-dependent effect of Npam43 on increased pCREB.

[0305] 5.2 Decrease in NMDA-induced excitotoxicity and H2O2-induced cytotoxicity

[0306] Figure 16-17 The results showed that Npam43 treatment reduced NMDA-induced excitotoxicity and H2O2-induced non-NMDAR-dependent cytotoxicity in cortical neurons in the absence of the GluN1 / GluN2A antagonist NVP-AAM007. Figure 16 Further, the dose-dependent effect of Npam43 on NMDA-induced cytotoxicity was demonstrated. Figure 17 Exposure to H2O2 showed that it increased neuronal cell death. Figure 17 Further studies showed that H2O2-induced cytotoxicity was reduced in the presence of Npam43, except with co-treatment with GluN1 / GluN2A antagonists (NVP-AAM077, 0.2 μM and TCN-201, 10 μM).

[0307] Characteristics of Npam43 in slices from Example 6 (ex vivo)

[0308] 6.1 The GluN2A component enhanced both synaptic transmission and long-term potentiation (LTP).

[0309] Electrophysiological recordings were performed to directly determine the effect of Npam43 on NMDAR current in hippocampal slices. The inventors pharmacologically separated the GluN2A and GluN2B components of synaptic transmission in the presence of GluN2A or GluN2B antagonists (NVP-AAM007(NVP), 0.2 μM or afenidil, (IF) 3 μM). Figure 18 We demonstrated that Npam43 selectively enhances the GluN2A component of NMDAR currents in mouse hippocampal slices, but has no effect on GluN2B NMDAR. Since GluN2A activation has been shown to promote synaptic plasticity, electrophysiological recordings were also performed to demonstrate that GluN2A enhancement promotes long-term potentiation (LTP), thereby contributing to increased synaptic strength. Figure 19 Npam43 was shown to promote the induction of LTP in mouse hippocampal slices.

[0310] 6.2 Cortical sections from knockout (KO) GluN2A mice demonstrated the enhancing effect of Npam43 on GluN2A NMDAR.

[0311] To directly test the selectivity of Npam43 for synaptically activated NMDAR, data were recorded in slices from GluN2A knockout (KO) mice and wild-type (WT) mice. Figure 20 The study showed that in WT mice, a significant enhancement of NMDAR EPSC was observed when Npam43 was administered to brain slices. In GluN2A KO mice, Npam43 administration had no effect on NMDAR EPSC, suggesting that the enhancement was attributable to GluN2A NMDAR.

[0312] 6.3 Npam43 increases pCREB levels in hippocampal slices

[0313] like Figure 21 The image shows an immunocytochemical analysis of baseline levels of CREB phosphorylation on serine 133 (pCREB) in hippocampal slices treated with bipinnatine (BiC; 10 μM; exposure for 30 min) or Npam43 (10 μM; exposure for 30 min) in the presence and absence of NVP-AAM077 (0.2 μM). Stimulation of neurons with BiC increased pCREB levels, and this effect was reduced by NVP-AAM007 (0.2 μM). Figure 21 As shown, treatment with Npam43 (10 μM) significantly enhanced pCREB levels, which were completely blocked in the presence of NVP-AAM007 (0.2 μM).

[0314] Example 7 Pharmacological characteristics of Npam43

[0315] Npam43 crosses the blood-brain barrier after intravenous injection in mature rats.

[0316] The in vivo pharmacological characteristics of Npam43 were assessed using mature Sprague Dawley rats (~300g). Npam43 was administered intravenously at doses ranging from 0.5–5 mg / kg (iv). Cerebrospinal fluid (CSF) and serum samples were collected and analyzed by high-performance liquid chromatography-electrochemical detection (HPLC-ECD). Figure 22 As shown, pharmacokinetic analysis of Npam43 after intravenous administration demonstrated its effective crossing of the blood-brain barrier (BBB) ​​and moderate metabolic stability in CSF and serum. A linear relationship between the injected dose and the final concentration in CSF was observed 1 hour after injection. Figure 22 As shown, the half-life of Npam43 in CSF is estimated to be 2.95 ± 0.6 h, which is similar to the decay in serum.

[0317] Example 8: In vivo efficacy of Npam43

[0318] 8.1 Npam43 enhances pCREB levels in hippocampal and cortical tissues

[0319] like Figure 23 As shown, hippocampal and cortical brain slices were harvested 1 hour after intravenous Npam43 (1 mg / kg) treatment, and pCTEB and tCREB levels in the samples were detected by immunoblotting. Consistent with in vitro assays, pCTEB levels were elevated.

[0320] 8.2 Reduction of neuronal damage after ischemic brain injury

[0321] Infarct size can be used as a measure of brain injury after an attack. Figure 24-25 The results showed that Npam43 treatment reduced infarct volume in a rat model of middle cerebral artery occlusion (MCAo) compared with the carrier / saline control. Figure 24 This study demonstrated the dose-dependent effect of Npam43 on cytoplasmic lesion volume 24 hours after ischemia. Figure 25 MRI scans showed that Npam43 reduced rim necrosis size using long-term assessment points (7 days).

[0322] 8.3 Improved behavioral manifestations after focal ischemic brain injury

[0323] Neurobehavioral assays were performed to assess the functional recovery of neurons after stroke. Figure 26 The study demonstrated that, compared with untreated control rats 28 days after stroke, rats treated with Npam43 exhibited enhanced post-stroke motor behavior. sequence list <110> Qingdao Pumeisheng Pharmaceutical Technology Co., Ltd. <120> N-methyl-D-aspartate receptor allosteric modulators and their usage <130> P2017TC382 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 938 <212> PRT <213> Rattus norvegicus <400> 1 Met Ser Thr Met His Leu Leu Thr Phe Ala Leu Leu Phe Ser Cys Ser 1 5 10 15 Phe Ala Arg Ala Ala Cys Asp Pro Lys Ile Val Asn Ile Gly Ala Val 20 25 30 Leu Ser Thr Arg Lys His Glu Gln Met Phe Arg Glu Ala Val Asn Gln 35 40 45 Ala Asn Lys Arg His Gly Ser Trp Lys Ile Gln Leu Asn Ala Thr Ser 50 55 60 Val Thr His Lys Pro Asn Ala Ile Gln Met Ala Leu Ser Val Cys Glu 65 70 75 80 Asp Leu Ile Ser Ser Gln Val Tyr Ala Ile Leu Val Ser His Pro Pro 85 90 95 Thr Pro Asn Asp His Phe Thr Pro Thr Pro Val Ser Tyr Thr Ala Gly 100 105 110 Phe Tyr Arg Ile Pro Val Leu Gly Leu Thr Thr Arg Met Ser Ile Tyr 115 120 125 Ser Asp Lys Ser Ile His Leu Ser Phe Leu Arg Thr Val Pro Pro Tyr 130 135 140 Ser His Gln Ser Ser Val Trp Phe Glu Met Met Arg Val Tyr Asn Trp 145 150 155 160 Asn His Ile Ile Leu Leu Val Ser Asp Asp His Glu Gly Arg Ala Ala 165 170 175 Gln Lys Arg Leu Glu Thr Leu Leu Glu Glu Arg Glu Ser Lys Ala Glu 180 185 190 Lys Val Leu Gln Phe Asp Pro Gly Thr Lys Asn Val Thr Ala Leu Leu 195 200 205 Met Glu Ala Arg Glu Leu Glu Ala Arg Val Ile Ile Leu Ser Ala Ser 210 215 220 Glu Asp Asp Ala Ala Thr Val Tyr Arg Ala Ala Ala Met Leu Asn Met 225 230 235 240 Thr Gly Ser Gly Tyr Val Trp Leu Val Gly Glu Arg Glu Ile Ser Gly 245 250 255 Asn Ala Leu Arg Tyr Ala Pro Asp Gly Ile Ile Gly Leu Gln Leu Ile 260 265 270 Asn Gly Lys Asn Glu Ser Ala His Ile Ser Asp Ala Val Gly Val Val 275 280 285 Ala Gln Ala Val His Glu Leu Leu Glu Lys Glu Asn Ile Thr Asp Pro 290 295 300 Pro Arg Gly Cys Val Gly Asn Thr Asn Ile Trp Lys Thr Gly Pro Leu 305 310 315 320 Phe Lys Arg Val Leu Met Ser Ser Lys Tyr Ala Asp Gly Val Thr Gly 325 330 335 Arg Val Glu Phe Asn Glu Asp Gly Asp Arg Lys Phe Ala Asn Tyr Ser 340 345 350 Ile Met Asn Leu Gln Asn Arg Lys Leu Val Gln Val Gly Ile Tyr Asn 355 360 365 Gly Thr His Val Ile Pro Asn Asp Arg Lys Ile Ile Trp Pro Gly Gly 370 375 380 Glu Thr Glu Lys Pro Arg Gly Tyr Gln Met Ser Thr Arg Leu Lys Ile 385 390 395 400 Val Thr Ile His Gln Glu Pro Phe Val Tyr Val Lys Pro Thr Met Ser 405 410 415 Asp Gly Thr Cys Lys Glu Glu Phe Thr Val Asn Gly Asp Pro Val Lys 420 425 430 Lys Val Ile Cys Thr Gly Pro Asn Asp Thr Ser Pro Gly Ser Pro Arg 435 440 445 His Thr Val Pro Gln Cys Cys Tyr Gly Phe Cys Ile Asp Leu Leu Ile 450 455 460 Lys Leu Ala Arg Thr Met Asn Phe Thr Tyr Glu Val His Leu Val Ala 465 470 475 480 Asp Gly Lys Phe Gly Thr Gln Glu Arg Val Asn Asn Ser Asn Lys Lys 485 490 495 Glu Trp Asn Gly Met Met Gly Glu Leu Leu Ser Gly Gln Ala Asp Met 500 505 510 Ile Val Ala Pro Leu Thr Ile Asn Asn Glu Arg Ala Gln Tyr Ile Glu 515 520 525 Phe Ser Lys Pro Phe Lys Tyr Gln Gly Leu Thr Ile Leu Val Lys Lys 530 535 540 Glu Ile Pro Arg Ser Thr Leu Asp Ser Phe Met Gln Pro Phe Gln Ser 545 550 555 560 Thr Leu Trp Leu Leu Val Gly Leu Ser Val His Val Val Ala Val Met 565 570 575 Leu Tyr Leu Leu Asp Arg Phe Ser Pro Phe Gly Arg Phe Lys Val Asn 580 585 590 Ser Glu Glu Glu Glu Glu Asp Ala Leu Thr Leu Ser Ser Ala Met Trp 595 600 605 Phe Ser Trp Gly Val Leu Leu Asn Ser Gly Ile Gly Glu Gly Ala Pro 610 615 620 Arg Ser Phe Ser Ala Arg Ile Leu Gly Met Val Trp Ala Gly Phe Ala 625 630 635 640 Met Ile Ile Val Ala Ser Tyr Thr Ala Asn Leu Ala Ala Phe Leu Val 645 650 655 Leu Asp Arg Pro Glu Glu Arg Ile Thr Gly Ile Asn Asp Pro Arg Leu 660 665 670 Arg Asn Pro Ser Asp Lys Phe Ile Tyr Ala Thr Val Lys Gln Ser Ser 675 680 685 Val Asp Ile Tyr Phe Arg Arg Gln Val Glu Leu Ser Thr Met Tyr Arg 690 695 700 His Met Glu Lys His Asn Tyr Glu Ser Ala Ala Glu Ala Ile Gln Ala 705 710 715 720 Val Arg Asp Asn Lys Leu His Ala Phe Ile Trp Asp Ser Ala Val Leu 725 730 735 Glu Phe Glu Ala Ser Gln Lys Cys Asp Leu Val Thr Thr Gly Glu Leu 740 745 750 Phe Phe Arg Ser Gly Phe Gly Ile Gly Met Arg Lys Asp Ser Pro Trp 755 760 765 Lys Gln Asn Val Ser Leu Ser Ile Leu Lys Ser His Glu Asn Gly Phe 770 775 780 Met Glu Asp Leu Asp Lys Thr Trp Val Arg Tyr Gln Glu Cys Asp Ser 785 790 795 800 Arg Ser Asn Ala Pro Ala Thr Leu Thr Phe Glu Asn Met Ala Gly Val 805 810 815 Phe Met Leu Val Ala Gly Gly Ile Val Ala Gly Ile Phe Leu Ile Phe 820 825 830 Ile Glu Ile Ala Tyr Lys Arg His Lys Asp Ala Arg Arg Lys Gln Met 835 840 845 Gln Leu Ala Phe Ala Ala Val Asn Val Trp Arg Lys Asn Leu Gln Asp 850 855 860 Arg Lys Ser Gly Arg Ala Glu Pro Asp Pro Lys Lys Lys Ala Thr Phe 865 870 875 880 Arg Ala Ile Thr Ser Thr Leu Ala Ser Ser Phe Lys Arg Arg Arg Ser 885 890 895 Ser Lys Asp Thr Ser Thr Gly Gly Gly Arg Gly Ala Leu Gln Asn Gln 900 905 910 Lys Asp Thr Val Leu Pro Arg Arg Ala Ile Glu Arg Glu Glu Gly Gln 915 920 925 Leu Gln Leu Cys Ser Arg His Arg Glu Ser 930 935 <210> 2 <211> 1464 <212> PRT <213> Rattus norvegicus <400> 2 Met Gly Arg Leu Gly Tyr Trp Thr Leu Leu Val Leu Pro Ala Leu Leu 1 5 10 15 Val Trp Arg Asp Pro Ala Gln Asn Ala Ala Ala Glu Lys Gly Pro Pro 20 25 30 Ala Leu Asn Ile Ala Val Leu Leu Gly His Ser His Asp Val Thr Glu 35 40 45 Arg Glu Leu Arg Asn Leu Trp Gly Pro Glu Gln Ala Thr Gly Leu Pro 50 55 60 Leu Asp Val Asn Val Val Ala Leu Leu Met Asn Arg Thr Asp Pro Lys 65 70 75 80 Ser Leu Ile Thr His Val Cys Asp Leu Met Ser Gly Ala Arg Ile His 85 90 95 Gly Leu Val Phe Gly Asp Asp Thr Asp Gln Glu Ala Val Ala Gln Met 100 105 110 Leu Asp Phe Ile Ser Ser Gln Thr Phe Ile Pro Ile Leu Gly Ile His 115 120 125 Gly Gly Ala Ser Met Ile Met Ala Asp Lys Asp Pro Thr Ser Thr Phe 130 135 140 Phe Gln Phe Gly Ala Ser Ile Gln Gln Gln Ala Thr Val Met Leu Lys 145 150 155 160 Ile Met Gln Asp Tyr Asp Trp His Val Phe Ser Leu Val Thr Thr Ile 165 170 175 Phe Pro Gly Tyr Arg Asp Phe Ile Ser Phe Ile Lys Thr Thr Val Asp 180 185 190 Asn Ser Phe Val Gly Trp Asp Met Gln Asn Val Ile Thr Leu Asp Thr 195 200 205 Ser Phe Glu Asp Ala Lys Thr Gln Val Gln Leu Lys Lys Ile His Ser 210 215 220 Ser Val Ile Leu Leu Tyr Cys Ser Lys Asp Glu Ala Val Leu Ile Leu 225 230 235 240 Ser Glu Ala Arg Ser Leu Gly Leu Thr Gly Tyr Asp Phe Phe Trp Ile 245 250 255 Val Pro Ser Leu Val Ser Gly Asn Thr Glu Leu Ile Pro Lys Glu Phe 260 265 270 Pro Ser Gly Leu Ile Ser Val Ser Tyr Asp Asp Trp Asp Tyr Ser Leu 275 280 285 Glu Ala Arg Val Arg Asp Gly Leu Gly Ile Leu Thr Thr Ala Ala Ser 290 295 300 Ser Met Leu Glu Lys Phe Ser Tyr Ile Pro Glu Ala Lys Ala Ser Cys 305 310 315 320 Tyr Gly Gln Ala Glu Lys Pro Glu Thr Pro Leu His Thr Leu His Gln 325 330 335 Phe Met Val Asn Val Thr Trp Asp Gly Lys Asp Leu Ser Phe Thr Glu 340 345 350 Glu Gly Tyr Gln Val His Pro Arg Leu Val Val Ile Val Leu Asn Lys 355 360 365 Asp Arg Glu Trp Glu Lys Val Gly Lys Trp Glu Asn Gln Thr Leu Ser 370 375 380 Leu Arg His Ala Val Trp Pro Arg Tyr Lys Ser Phe Ser Asp Cys Glu 385 390 395 400 Pro Asp Asp Asn His Leu Ser Ile Val Thr Leu Glu Glu Ala Pro Phe 405 410 415 Val Ile Val Glu Asp Ile Asp Pro Leu Thr Glu Thr Cys Val Arg Asn 420 425 430 Thr Val Pro Cys Arg Lys Phe Val Lys Ile Asn Asn Ser Thr Asn Glu 435 440 445 Gly Met Asn Val Lys Lys Cys Cys Lys Gly Phe Cys Ile Asp Ile Leu 450 455 460 Lys Lys Leu Ser Arg Thr Val Lys Phe Thr Tyr Asp Leu Tyr Leu Val 465 470 475 480 Thr Asn Gly Lys His Gly Lys Lys Val Asn Asn Val Trp Asn Gly Met 485 490 495 Ile Gly Glu Val Val Tyr Gln Arg Ala Val Met Ala Val Gly Ser Leu 500 505 510 Thr Ile Asn Glu Glu Arg Ser Glu Val Val Asp Phe Ser Val Pro Phe 515 520 525 Val Glu Thr Gly Ile Ser Val Met Val Ser Arg Ser Asn Gly Thr Val 530 535 540 Ser Pro Ser Ala Phe Leu Glu Pro Phe Ser Ala Ser Val Trp Val Met 545 550 555 560 Met Phe Val Met Leu Leu Ile Val Ser Ala Ile Ala Val Phe Val Phe 565 570 575 Glu Tyr Phe Ser Pro Val Gly Tyr Asn Arg Asn Leu Ala Lys Gly Lys 580 585 590 Ala Pro His Gly Pro Ser Phe Thr Ile Gly Lys Ala Ile Trp Leu Leu 595 600 605 Trp Gly Leu Val Phe Asn Asn Ser Val Pro Val Gln Asn Pro Lys Gly 610 615 620 Thr Thr Ser Lys Ile Met Val Ser Val Trp Ala Phe Phe Ala Val Ile 625 630 635 640 Phe Leu Ala Ser Tyr Thr Ala Asn Leu Ala Ala Phe Met Ile Gln Glu 645 650 655 Glu Phe Val Asp Gln Val Thr Gly Leu Ser Asp Lys Lys Phe Gln Arg 660 665 670 Pro His Asp Tyr Ser Pro Pro Phe Arg Phe Gly Thr Val Pro Asn Gly 675 680 685 Ser Thr Glu Arg Asn Ile Arg Asn Asn Tyr Pro Tyr Met His Gln Tyr 690 695 700 Met Thr Arg Phe Asn Gln Arg Gly Val Glu Asp Ala Leu Val Ser Leu 705 710 715 720 Lys Thr Gly Lys Leu Asp Ala Phe Ile Tyr Asp Ala Ala Val Leu Asn 725 730 735 Tyr Lys Ala Gly Arg Asp Glu Gly Cys Lys Leu Val Thr Ile Gly Ser 740 745 750 Gly Tyr Ile Phe Ala Ser Thr Gly Tyr Gly Ile Ala Leu Gln Lys Gly 755 760 765 Ser Pro Trp Lys Arg Gln Ile Asp Leu Ala Leu Leu Gln Phe Val Gly 770 775 780 Asp Gly Glu Met Glu Glu Leu Glu Thr Leu Trp Leu Thr Gly Ile Cys 785 790 795 800 His Asn Glu Lys Asn Glu Val Met Ser Ser Gln Leu Asp Ile Asp Asn 805 810 815 Met Ala Gly Val Phe Tyr Met Leu Ala Ala Ala Met Ala Leu Ser Leu 820 825 830 Ile Thr Phe Ile Trp Glu His Leu Phe Tyr Trp Lys Leu Arg Phe Cys 835 840 845 Phe Thr Gly Val Cys Ser Asp Arg Pro Gly Leu Leu Phe Ser Ile Ser 850 855 860 Arg Gly Ile Tyr Ser Cys Ile His Gly Val His Ile Glu Glu Lys Lys 865 870 875 880 Lys Ser Pro Asp Phe Asn Leu Thr Gly Ser Gln Ser Asn Met Leu Lys 885 890 895 Leu Leu Arg Ser Ala Lys Asn Ile Ser Asn Met Ser Asn Met Asn Ser 900 905 910 Ser Arg Met Asp Ser Pro Lys Arg Ala Thr Asp Phe Ile Gln Arg Gly 915 920 925 Ser Leu Ile Val Asp Met Val Ser Asp Lys Gly Asn Leu Ile Tyr Ser 930 935 940 Asp Asn Arg Ser Phe Gln Gly Lys Asp Ser Ile Phe Gly Asp Asn Met 945 950 955 960 Asn Glu Leu Gln Thr Phe Val Ala Asn Arg His Lys Asp Asn Leu Ser 965 970 975 Asn Tyr Val Phe Gln Gly Gln His Pro Leu Thr Leu Asn Glu Ser Asn 980 985 990 Pro Asn Thr Val Glu Val Ala Val Ser Thr Glu Ser Lys Gly Asn Ser 995 1000 1005 Arg Pro Arg Gln Leu Trp Lys Lys Ser Met Glu Ser Leu Arg Gln 1010 1015 1020 Asp Ser Leu Asn Gln Asn Pro Val Ser Gln Arg Asp Glu Lys Thr 1025 1030 1035 Ala Glu Asn Arg Thr His Ser Leu Lys Ser Pro Arg Tyr Leu Pro 1040 1045 1050 Glu Glu Val Ala His Ser Asp Ile Ser Glu Thr Ser Ser Arg Ala 1055 1060 1065 Thr Cys His Arg Glu Pro Asp Asn Asn Lys Asn His Lys Thr Lys 1070 1075 1080 Asp Asn Phe Lys Arg Ser Met Ala Ser Lys Tyr Pro Lys Asp Cys 1085 1090 1095 Ser Asp Val Asp Arg Thr Tyr Met Lys Thr Lys Ala Ser Ser Pro 1100 1105 1110 Arg Asp Lys Ile Tyr Thr Ile Asp Gly Glu Lys Glu Pro Ser Phe 1115 1120 1125 His Leu Asp Pro Pro Gln Phe Val Glu Asn Ile Thr Leu Pro Glu 1130 1135 1140 Asn Val Gly Phe Pro Asp Thr Tyr Gln Asp His Asn Glu Asn Phe 1145 1150 1155 Arg Lys Gly Asp Ser Thr Leu Pro Met Asn Arg Asn Pro Leu His 1160 1165 1170 Asn Glu Asp Gly Leu Pro Asn Asn Asp Gln Tyr Lys Leu Tyr Ala 1175 1180 1185 Lys His Phe Thr Leu Lys Asp Lys Gly Ser Pro His Ser Glu Gly 1190 1195 1200 Ser Asp Arg Tyr Arg Gln Asn Ser Thr His Cys Arg Ser Cys Leu 1205 1210 1215 Ser Asn Leu Pro Thr Tyr Ser Gly His Phe Thr Met Arg Ser Pro 1220 1225 1230 Phe Lys Cys Asp Ala Cys Leu Arg Met Gly Asn Leu Tyr Asp Ile 1235 1240 1245 Asp Glu Asp Gln Met Leu Gln Glu Thr Gly Asn Pro Ala Thr Arg 1250 1255 1260 Glu Glu Val Tyr Gln Gln Asp Trp Ser Gln Asn Asn Ala Leu Gln 1265 1270 1275 Phe Gln Lys Asn Lys Leu Arg Ile Asn Arg Gln His Ser Tyr Asp 1280 1285 1290 Asn Ile Leu Asp Lys Pro Arg Glu Ile Asp Leu Ser Arg Pro Ser 1295 1300 1305 Arg Ser Ile Ser Leu Lys Asp Arg Glu Arg Leu Leu Glu Gly Asn 1310 1315 1320 Leu Tyr Gly Ser Leu Phe Ser Val Pro Ser Ser Lys Leu Leu Gly 1325 1330 1335 Asn Lys Ser Ser Leu Phe Pro Gln Gly Leu Glu Asp Ser Lys Arg 1340 1345 1350 Ser Lys Ser Leu Leu Pro Asp His Ala Ser Asp Asn Pro Phe Leu 1355 1360 1365 His Thr Tyr Gly Asp Asp Gln Arg Leu Val Ile Gly Arg Cys Pro 1370 1375 1380 Ser Asp Pro Tyr Lys His Ser Leu Pro Ser Gln Ala Val Asn Asp 1385 1390 1395 Ser Tyr Leu Arg Ser Ser Leu Arg Ser Thr Ala Ser Tyr Cys Ser 1400 1405 1410 Arg Asp Ser Arg Gly His Ser Asp Val Tyr Ile Ser Glu His Val 1415 1420 1425 Met Pro Tyr Ala Ala Asn Lys Asn Thr Met Tyr Ser Thr Pro Arg 1430 1435 1440 Val Leu Asn Ser Cys Ser Asn Arg Arg Val Tyr Lys Lys Met Pro 1445 1450 1455 Ser Ile Glu Ser Asp Val 1460

Claims

1. Use of a compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention or treatment in an individual in need of a condition or illness caused by or related to N-methyl-D-aspartate receptor (NMDAR) dysfunction, wherein said condition or illness is stroke, and said compound is: 。

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