PSD-95 inhibitors and their uses

Abstract

The present invention relates to compounds capable of binding to the PDZ domain of PSD-95 and their medical use as inhibitors of protein-protein interactions mediated by PSD-95.

Description

[Technical field]

[0001] The present invention relates to compounds capable of binding to the PDZ domain of PSD-95 and their medical use as inhibitors of protein-protein interactions mediated by PSD-95. [Background technology]

[0002] PSD-95 is a scaffolding protein in neuronal synapses that interacts with N-methyl-D-aspartate (NMDA) receptors (Kornau et al. 1995) and neuronal nitric oxide synthase (nNOS) via its PSD-95 / Discs-large / ZO-1 (PDZ) domain. During cerebral ischemia, excessive release of glutamate leads to excessive activation of NMDA receptors and intracellular Ca 2+ and NO levels, which ultimately induces excitotoxicity leading to neuronal death and brain damage (Aarts et al., 2002; Dawson et al., 1991; Huang et al., 1994; Sattler et al., 1999). PSD-95 is being explored as a drug target in acute ischemic stroke (AIS) and related ischemic pathologies of the brain.

[0003] The 20-mer peptide nerinetide (also known as Tat-NR2B9c or NA-1) was recently investigated in the Phase 3 clinical trial ESCAPE-NA1 (Hill et al., 2020), which evaluated whether treatment with nerinetide in addition to standard of care such as alteplase (tissue plasminogen activator, tPA) would improve clinical outcomes for patients with ischemic stroke and undergoing endovascular thrombectomy (Hill et al., 2020). A total of 1105 patients were enrolled in the study, of which 659 patients received tPA treatment and 446 patients did not. Nerinetide was not effective in patients receiving alteplase, but in the group not receiving alteplase, nerinetide was found to result in improved functional outcomes, reduced mortality, and reduced infarct volume (Hill et al., 2020). It has recently been demonstrated that the lack of efficacy in the tPA-treated group can be explained by a drug-drug interaction between nerinetide and tPA. Specifically, tPA generates plasmin, a serine protease that cleaves nerinetide (Mayor-Nunez et al. 2021). It is therefore highly advantageous that the drug is compatible with the administration of thrombolytic agents, the standard of care for AIS.

[0004] Nerinetide suffers from a relatively low affinity for its target PSD-95, which prompted the design of dimeric compounds such as Tat-N-dimer and O-dimer, also known as UCCB01-144 (AB144) and UCCB01-125, respectively (Bach et al., 2009, 2012; WO2010 / 004003; WO2012 / 156308; Kucharz et al., 2017). In an in vitro fluorescence polarization (FP) assay, the K iIn contrast, AB144 and UCCB01-125, due to their dimeric structure and bivalent nature, bind simultaneously to PDZ1 and PDZ2 of PSD-95 with 500-1000-fold higher affinity for PDZ1-2 compared to that of monomeric nerinetide (K of 4.6 and 9.5 nM, respectively). i In mice subjected to permanent middle cerebral artery occlusion (pMCAO), a single intravenous (iv) bolus injection of AB144 (3 nmol / g) given 30 min after ischemia reduced infarct volume by 40% and 37% at 6 and 48 h postischemic survival, respectively, compared with the effect of saline (Bach et al., 2012). Furthermore, functional outcomes such as grip strength and rotarod performance improved, thus correlating with the reduction in infarct size. Under the same experimental conditions and dose (3 nmol / g), nerinetide did not show significant neuroprotective properties (Bach et al., 2012). Similarly, UCCB01-125, which does not possess the Tat moiety, did not reach the brain and did not show any neuroprotective properties (Bach et al., 2012).

[0005] Therefore, PSD-95 is considered a particularly promising drug target for the treatment of acute pathologies such as subarachnoid hemorrhage (SAH) and AIS. Importantly, since PSD-95 is located intracellularly, any drug targeting PSD-95 needs to efficiently cross the cell membrane and bind to PSD-95. Summary of the Invention

[0006] The present inventors have developed a series of novel compounds with high affinity to PSD-95. Compared with known PSD-95 inhibitors such as nerinetide, the compounds of the present invention have improved cellular uptake and much higher plasmin stability. Therefore, the compounds of the present invention are useful in the treatment of excitotoxicity-related diseases such as AIS and SAH, and are compatible with standard treatments such as thrombolytic agents.

[0007] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: a. a first peptide (P1) comprising or consisting of the amino acid sequence X2TX3V (SEQ ID NO:53), X2 is selected from the group consisting of E and S; X3 is selected from the group consisting of L, T, V, R, and D; b. A second peptide (P2) comprising or consisting of the amino acid sequence X5TX6V (SEQ ID NO:55), X5 is selected from the group consisting of E and S; X6 is selected from the group consisting of L, T, V, R, and D; and c. A cell membrane penetrating peptide (CPP) selected from the group consisting of: i. poly-L-arginine peptides consisting of 3 to 20 L-arginine residues (poly-Arg); ii.yGrkkrrqrrr (SEQ ID NO:9, D-TAT), iii. RQIKIWFQNRRMKWKK (SEQ ID NO: 14, L-Pen); iv.rqikiwfqnrrmkwkk (SEQ ID NO: 15, D-Pen), v.RKKRRRESRKKRRRES (SEQ ID NO: 17, L-DPV3), vi. LLIILRRRIRKQAHAHSK (SEQ ID NO: 18, L-pVEC), vii. KLALKLALKALKAALKLA (SEQ ID NO: 16, L-MAP), viii. PLIYLRLLRGQF (SEQ ID NO: 19, L-TP2), ix. |(Dap)KAPETALD| (SEQ ID NO: 21, MiniAp4), and x.|Ff(Nal2)RrRrQ|GABA-K (SEQ ID NO: 22, CPP12), wherein the CPP is linked to a linker through its C-terminus, and P1 and P2 are conjugated to the linker through their N-terminus, and the compound has the formula (I): [ka] or a pharma- ceutically acceptable salt thereof.

[0008] In another aspect, the invention relates to a compound as defined herein for use as a medicament.

[0009] In another aspect, the invention relates to a compound as defined herein for use in treating, preventing, reducing and / or delaying the onset of an excitotoxicity-related disorder, such as stroke selected from acute ischemic stroke and subarachnoid hemorrhage.

[0010] In a further aspect, the invention relates to a compound as defined herein for use in the treatment or prophylaxis of pain.

[0011] The present invention further relates to processes for preparing the compounds defined herein. [Brief description of the drawings]

[0012] [Figure 1] Affinity of O-PEG4 dimers (compounds 1-7) for PSD-95 PDZ1-2 as determined by FP. Data are presented as mean + SEM, n=3. [Diagram 2] Affinity of dimeric ligands fused to Ac-TAT and Ac-polyArg for PSD-95 PDZ1-2 as determined by FP. Data are presented as mean + SEM, n=3. [Diagram 3] Affinity of N-PEG4(IETDV)2 fused to various CPP tags (SEQ ID NOs: 8-22) for PSD-95 PDZ1-2 as determined by FP. Data are presented as mean + SEM, n=3. [Figure 4] Affinity of NPEG4-(KETLV)2 dimeric ligand fused to a poly-Arg CPP tag for PSD-95 PDZ1-2 as determined by FP. Data are presented as mean + SEM, n = 3. [Diagram 5]Half-lives of dimeric ligands fused to Ac-TAT and Ac-polyArg as determined in an in vitro plasmin stability assay. Data are presented as mean + SEM, n=3. [Figure 6] Half-life of N-PEG4(IETDV)2 fused to various CPP tags (SEQ ID NOs: 8-22) as determined in an in vitro plasmin stability assay. Data are presented as mean + SEM, n=3. [Figure 7] Intracellular uptake of dimeric ligands fused to Ac-TAT and Ac-polyArg as determined by CAPA. Data are presented as mean + SEM, n=3. [Figure 8] Intracellular uptake of N-PEG4(IETDV)2 fused to various CPP tags (SEQ ID NOs: 8-22) as determined by CAPA. Data are presented as mean + SEM, n=3. [Figure 9] Intracellular uptake of N-PEG4(IETDV)2 and N-PEG4(KETLV)2 fused to various poly-Arg CPP tags as determined by CAPA. Data are presented as mean + SEM, n=3. [Figure 10] Half-lives of N-PEG4(IETDV)2 and N-PEG4(KETLV)2 fused to a poly-Arg CPP tag as determined in a human plasma stability assay. Data are presented as mean ± SEM, n=3. [Figure 11] General structure of compounds that bind to PDZ1-2 of PSD-95. R1 through R5 are amino acid side chains as described herein, and R6 represents a CPP tag as described herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] definition Amide bond: As used herein, the term "amide bond" is a chemical bond formed by the reaction between a carboxylic acid and an amine (with the concomitant release of water). When the reaction is between two amino acid residues, the bond formed as a result of the reaction is known as a peptide linkage (peptide bond).

[0014] Comprises: The term "comprises" as used herein should be understood in an inclusive manner. Thus, by way of example, a composition comprising compound X may comprise compound X and optionally additional compounds.

[0015] Dimer: The term dimer as used herein refers to two identical or non-identical chemical moieties associated by chemical or physical interactions. As an example, a dimer can be a homodimer, such as two identical chemical moieties linked by a linker. A dimer can also be a heterodimer, such as two different chemical moieties linked by a linker. An example of a dimer is the PSD-95 inhibitor of the present invention, which is a compound comprising two peptides covalently linked by means of a linker, and these peptides can simultaneously bind to or interact with the PDZ1 and PDZ2 domains of PSD-95.

[0016] Dipeptide: As used herein, the term "dipeptide" refers to two natural or unnatural amino acids linked by a peptide bond.

[0017] Fatty acid: The term fatty acid (abbreviated as FA) as used herein typically refers to a carboxylic acid with a long aliphatic carbon chain, which may be either saturated or unsaturated. The fatty acid may be selected from short chain fatty acids (SCFA), medium chain fatty acids (MCFA), long chain fatty acids (LCFA), and very long chain fatty acids (VLCFA). Short chain fatty acids (SCFA) are fatty acids with an aliphatic tail of less than 6 carbons (i.e. butyric acid). Medium chain fatty acids (MCFA) are fatty acids with an aliphatic tail of 6 to 12 carbons that can form medium chain triglycerides. Long chain fatty acids (LCFA) are fatty acids with an aliphatic tail of 13 to 21 carbons. Very long chain fatty acids (VLCFA) are fatty acids with an aliphatic tail longer than 22 carbons. The fatty acid of the present invention may be any suitable fatty acid or fatty acid derivative known to those skilled in the art.

[0018] Nonproteinogenic Amino Acids: Nonproteinogenic amino acids (also called non-coded, nonstandard, or unnatural amino acids) are amino acids that are not coded for by the genetic code. A non-exhaustive list of non-proteinogenic amino acids includes: gamma-aminobutyric acid, L-3-(2-naphthyl)alanine, L-2,3-diaminopropionic acid, alpha-amino-n-butyric acid, norvaline, norleucine, isoleucine, alloisoleucine, tert-leucine, alpha-amino-n-heptanoic acid, pipecolic acid, alpha,beta-diaminopropionic acid, alpha,gamma-diaminobutyric acid, ornithine, allothreonine, homocysteine, homoserine, beta-alanine, beta-amino-n-butyric acid, beta-aminoisobutyric acid, alpha-aminoisobutyric acid, isovaline, sarcosine, N-ethylglycine, N-propylglycine, N-isopropylglycine, N-methylalanine, N-ethylalanine, N-methylbeta-alanine, N-ethylbeta-alanine, isoserine, and alpha-hydroxy-gamma-aminobutyric acid.

[0019] Proteinogenic Amino Acids: Proteinogenic amino acids (also known as natural amino acids) include alanine, cysteine, selenocysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, pyrrolysine, glutamine, arginine, serine, threonine, valine, tryptophan, and tyrosine. Capitalized abbreviations indicate L-amino acids, while lowercase abbreviations indicate D-amino acids.

[0020] PDZ: As used herein, the term "PDZ" refers to Postsynaptic density protein-95 (PSD-95), Drosophila homologue discs large tumor suppressor (DlgA), and Zonula occludens-1 protein (zo-1).

[0021] PSD-95: As used herein, the term "PSD-95" refers to postsynaptic density protein-95.

[0022] PSD-95 inhibitor: As used herein, the term "PSD-95 inhibitor" refers to a compound that binds to PDZ1, PDZ2, or both PDZ1 and PDZ2 of PSD-95 and inhibits protein-protein interactions promoted by these PDZ domains in cells. An example of an interaction that is inhibited by a PSD-95 inhibitor is the ternary complex formation between nNOS, PSD-95, and the NMDA receptor.

[0023] compound In one aspect, the present invention relates to a compound comprising a first peptide (P1) linked to a cell membrane penetrating peptide (CPP). In one embodiment, P1 is linked to the CPP via a linker. In one embodiment, the compound further comprises a second peptide (P2). In one embodiment, the CPP is linked to P1 and P2 via a linker. Thus, in one embodiment, the CPP is linked to the linker via its C-terminus, and P1 and P2 are conjugated to the linker via their N-terminus, and the compound has the general structure of formula (I): [ka]

[0024] Thus, in one embodiment, the compound of the present invention comprises a first peptide (P1) and a second peptide (P2), both of which are capable of binding simultaneously to the PDZ1 and PDZ2 domains of PSD-95, i.e., the compound is a dimeric PSD-95 inhibitor.

[0025] In one aspect, the present invention relates to a compound comprising a peptide (P1) comprising or consisting of the amino acid sequence X2TX3V (SEQ ID NO: 53), in which a. X2 is selected from the group consisting of E and S; b. X3 is selected from the group consisting of L, T, V, R, and D; P1 is linked to a cell membrane penetrating peptide (CPP).

[0026] In a further aspect, the present invention provides a compound comprising a first peptide (P1) linked to a second peptide (P2) via a linker, e.g., a compound represented by formula (XXIV): [ka] wherein the linker, P1, and P2 are as defined herein. Preferably, P1 and P2 comprise or consist of KETLV (SEQ ID NO:2), KETTV (SEQ ID NO:3), KETVV (SEQ ID NO:4), KETRV (SEQ ID NO:5), ISTDV (SEQ ID NO:6), or VETVV (SEQ ID NO:7). Optionally, the linker is further conjugated to a cell membrane penetrating peptide (CPP) or an albumin binding moiety, thereby increasing the passage of the peptide through the membrane or blood-brain barrier. The albumin binding moiety can be any suitable chemical group that binds to albumin. Preferably, the albumin binding moiety is a fatty acid. In one embodiment, the linker is further conjugated to a CPP, i.e., the compound has the general structure of formula (I).

[0027] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: a. a first peptide (P1) comprising or consisting of the amino acid sequence X2TX3V (SEQ ID NO:53), X2 is selected from the group consisting of E and S; X3 is selected from the group consisting of L, T, V, R, and D; b. A second peptide (P2) comprising or consisting of the amino acid sequence X5TX6V (SEQ ID NO:55), X5 is selected from the group consisting of E and S; X6 is selected from the group consisting of L, T, V, R, and D; and c. A cell membrane penetrating peptide (CPP) selected from the group consisting of: i. poly-L-arginine peptides consisting of 3 to 20 L-arginine residues (poly-Arg); ii.yGrkkrrqrrr (SEQ ID NO:9, D-TAT), iii. RQIKIWFQNRRMKWKK (SEQ ID NO: 14, L-Pen); iv.rqikiwfqnrrmkwkk (SEQ ID NO: 15, D-Pen), v.RKKRRRESRKKRRRES (SEQ ID NO: 17, L-DPV3), vi. LLIILRRRIRKQAHAHSK (SEQ ID NO: 18, L-pVEC), vii. KLALKLALKALKAALKLA (SEQ ID NO: 16, L-MAP), viii. PLIYLRLLRGQF (SEQ ID NO: 19, L-TP2), ix. |(Dap)KAPETALD| (SEQ ID NO: 21, MiniAp4), and x.|Ff(Nal2)RrRrQ|GABA-K (SEQ ID NO: 22, CPP12), wherein the CPP is linked to a linker through its C-terminus, and P1 and P2 are conjugated to the linker through their N-terminus, and the compound has the formula (I): [ka] or a pharma- ceutically acceptable salt thereof.

[0028] Binding peptides P1 and P2 Peptides P1 and P2 have high affinity for the PDZ1 and PDZ2 domains of PSD-95. In one embodiment, the compound is a PSD-95 inhibitor, i.e., the compound is capable of binding to one or more of the PDZ domains of PSD-95.

[0029] In one embodiment, the compound comprises a first peptide (P1) comprising or consisting of the amino acid sequence X2TX3V (SEQ ID NO:53), wherein: a. X2 is selected from the group consisting of E and S; b. X3 is selected from the group consisting of L, T, V, R, and D.

[0030] In one embodiment, P1 comprises or consists of the sequence X1X2TX3V (SEQ ID NO:54), wherein: a. X1 is selected from the group consisting of K, V, and I; b. X2 is selected from the group consisting of E and S; X3 is selected from the group consisting of L, T, V, R, and D.

[0031] In one embodiment, P1 comprises or consists of IETDV (SEQ ID NO:1). In one embodiment, P1 comprises or consists of KETLV (SEQ ID NO:2). In one embodiment, P1 comprises or consists of KETTV (SEQ ID NO:3). In one embodiment, P1 comprises or consists of KETVV (SEQ ID NO:4). In one embodiment, P1 comprises or consists of KETRV (SEQ ID NO:5). In one embodiment, P1 comprises or consists of ISTDV (SEQ ID NO:6). In one embodiment, P1 comprises or consists of VETVV (SEQ ID NO:7).

[0032] In one embodiment, P1 consists of 4 to 10 amino acid residues, such as 5 amino acid residues, such as 6 amino acid residues. The amino acid residues beyond those given in SEQ ID NO: 53 or SEQ ID NO: 54 may be proteinogenic or non-proteinogenic amino acids.

[0033] In one embodiment, the compound further comprises a second peptide (P2) comprising or consisting of the amino acid sequence X5TX6V (SEQ ID NO:55), wherein: a. X5 is selected from the group consisting of E and S; b. X6 is selected from the group consisting of L, T, V, R, and D.

[0034] In one embodiment, P2 comprises or consists of the sequence X4X5TX6V (SEQ ID NO:56), wherein: a. X4 is selected from the group consisting of K, V, and I; b. X5 is selected from the group consisting of E and S; c. X6 is selected from the group consisting of L, T, V, R, and D.

[0035] In one embodiment, P2 comprises or consists of IETDV (SEQ ID NO:1). In one embodiment, P2 comprises or consists of KETLV (SEQ ID NO:2). In one embodiment, P2 comprises or consists of KETTV (SEQ ID NO:3). In one embodiment, P2 comprises or consists of KETVV (SEQ ID NO:4). In one embodiment, P2 comprises or consists of KETRV (SEQ ID NO:5). In one embodiment, P2 comprises or consists of ISTDV (SEQ ID NO:6). In one embodiment, P2 comprises or consists of VETVV (SEQ ID NO:7).

[0036] In one embodiment, P2 consists of 4 to 10 amino acid residues, such as 5 amino acid residues, such as 6 amino acid residues. The amino acid residues beyond those given in SEQ ID NO: 55 or SEQ ID NO: 56 may be proteinogenic or non-proteinogenic amino acids.

[0037] In one embodiment, P1 and P2 are the same.

[0038] In one embodiment, when X2 is E, X3 is not D. In one embodiment, when X5 is E, X6 is not D. In one embodiment, P1 and / or P2 are not IETDV (SEQ ID NO: 1).

[0039] In a preferred embodiment, peptides P1 and P2 are conjugated to a linker or CPP via their N-terminus. In one embodiment, the amino acid sequence X2TX3V (SEQ ID NO: 53) is the C-terminus of P1. In one embodiment, the amino acid sequence X5TX6V (SEQ ID NO: 55) is the C-terminus of P2.

[0040] Cell-penetrating peptides (CPPs) The compounds of the invention comprise peptide P1 linked to a cell membrane penetrating peptide (CPP), which is characterized by its ability to cross the blood-brain barrier (BBB) ​​and / or plasma membrane of mammalian cells, thereby allowing the intracellular delivery of a cargo molecule, such as a peptide, protein, oligonucleotide, etc., to which it is linked.

[0041] In one embodiment, the CPP is a poly-L-arginine peptide (poly-Arg). In one embodiment, the poly-Arg consists of 3 to 20 L-arginine residues. In one embodiment, the poly-Arg consists of 5 to 10 L-arginine residues, such as 7 to 10 L-arginine residues, such as 8 or 9 L-arginine residues. In one embodiment, the poly-Arg consists of 3 to 10 L-arginine residues. In one embodiment, the CPP comprises or consists of the amino acid sequence RRRRRRRRR (L-Arg9, SEQ ID NO: 12). In one embodiment, the CPP comprises or consists of the amino acid sequence RRRRRRRR (L-Arg8, SEQ ID NO: 57). In one embodiment, the CPP comprises or consists of the amino acid sequence RRRRRRR (L-Arg7, SEQ ID NO: 58). In one embodiment, the CPP comprises or consists of the amino acid sequence RRRRRR (L-Arg6, SEQ ID NO: 59). In one embodiment, the CPP comprises or consists of the amino acid sequence RRRRR (L-Arg5, SEQ ID NO: 60). In one embodiment, the CPP comprises or consists of the amino acid sequence RRRR (L-Arg4, SEQ ID NO: 61). In one embodiment, the CPP comprises or consists of the amino acid sequence RRR (L-Arg3).

[0042] In one embodiment, the CPP is selected from the CPPs provided in Table 1. [Table 1]

[0043] In one embodiment, the CPP comprises a peptide selected from the group consisting of poly-L-arginine peptides (poly-Arg), such as L-Arg9, D-TAT, L-Pen, D-Pen, L-DPV3, L-pVEC, L-MAP, L-TP2, MiniAp4, and CPP12. In one embodiment, the CPP is selected from the group consisting of poly-L-arginine peptides (poly-Arg), such as L-Arg9, D-TAT, L-Pen, D-Pen, L-DPV3, L-pVEC, L-MAP, L-TP2, MiniAp4, and CPP12. In one embodiment, the CPP is D-TAT. In one embodiment, the CPP is L-Pen. In one embodiment, the CPP is D-Pen. In one embodiment, the CPP is L-DPV3. In one embodiment, the CPP is L-pVEC. In one embodiment, the CPP is L-MAP. In one embodiment, the CPP is L-TP2. In one embodiment, the CPP is MiniAp4. In one embodiment, the CPP is CPP12.

[0044] In one embodiment the CPP comprises 20 or less amino acid residues, such as 19 or less, such as 18 or less, for example 17 or less, such as 16 or less, for example 15 or less, such as 14 or less, for example 13 or less, such as 12 or less, for example 11 or less, such as 10 or less, for example 9 or less, such as 8 or less, for example 7 or less amino acid residues.

[0045] In some embodiments, the CPP is conjugated to a non-peptide moiety. For example, the CPP may be methylated or acetylated. In some embodiments, when a peptide is defined herein as consisting of a sequence of specific amino acid residues, the peptide is not conjugated to any other amino acid residue, but the peptide may be conjugated to a non-peptide moiety, as long as the conjugation to the non-peptide moiety does not result in a different amino acid sequence. In some embodiments, the N-terminus of the CPP is conjugated to a non-peptide moiety. In some embodiments, the C-terminus of the CPP is conjugated to a non-peptide moiety. In some embodiments, the N-terminus of the CPP is acetylated, i.e., linked to the chemical structure CH3C(O)-. For example, the CPP may be a poly-Arg peptide consisting of nine L-arginine residues, with the N-terminus being acetylated. In some embodiments, the N-terminus of the CPP is conjugated to a chloroalkane tag (CA) having the following structure: [ka]

[0046] In one embodiment, the N-terminus of the CPP is methylated. In another embodiment, the N-terminus of the CPP is formylated.

[0047] Linker The term "linker" as used herein refers to one or more atoms that form a connection from one chemical entity to another. As an example, the "linker" referred to herein may join two PDZ domain-binding peptides P1 and P2 by forming a link to each of their N-termini. Various linkers are known in the art. The linker may be, for example, a chemical linker or a peptide linker, or a combination thereof. In one embodiment, the linker comprises an active functional group, such as an electrophilic or nucleophilic functional group, which can be used to attach the linker to each peptide.

[0048] In one embodiment, the linker comprises one or more polyethylene glycol (PEG) units.

[0049] PEG has the chemical formula C 2n H 4n+2 O n+1 and the following repeating structure: [ka] where n is an integer.

[0050] Thus, a PEG unit has the following structure: [ka]

[0051] For example, a polymer of four PEG moieties, or PEG4, corresponds to a polymer of four ethylene glycol moieties (n=4).

[0052] The terms "PEG unit" and "PEG moiety" are used interchangeably herein.

[0053] In one embodiment, at least one oxygen atom of one of the PEG units is replaced with a nitrogen atom to provide NPEG. As used herein, the term "ethylene glycol moiety" refers to a structural unit that constitutes a PEG or NPEG linker.

[0054] In one embodiment, the N-termini of two PDZ domain-binding peptides, P1 and P2, are linked to each other via a linker comprising one or more PEG units, at least one oxygen atom of the PEG units being optionally replaced by a nitrogen atom. In one embodiment, P1 and / or P2 are individually linked to the PEG / NPEG units via a spacer group, e.g., via a short alkane chain.

[0055] In one embodiment, the linker comprises one or more PEG units, wherein at least one oxygen atom of one of the PEG units is replaced with a nitrogen atom resulting in NPEG.

[0056] In one embodiment, the linker comprises an NPEG unit, and the CPP is linked to the linker via a chemical bond, either direct or indirect, to a nitrogen atom in the backbone of the NPEG linker. The attachment of the CPP to the nitrogen of the NPEG linker may be mediated via an amide bond, a 1,3-dipolar cycloaddition such as copper-catalyzed azide-alkyne cycloaddition, maleimide coupling, a disulfide bond, or an amino-reactive electrophilic group selected from among N-hydroxysuccinimide (NHS) esters, p-nitrophenyl esters, succinimidyl carbonate, p-nitrophenyl carbonate, succinimidyl urethane, isocyanates, isothiocyanates, acyl azides, sulfonyl chlorides, aldehydes, carbonates, imidioesters, or anhydrides; and a thio-reactive group selected from among haloacetyls, alkyl halide derivatives, aziridines, and acryloyl derivatives arylating agents. In one embodiment, the linker comprises one or more PEG units, where at least one oxygen atom of one of the PEG units is replaced with a nitrogen atom to provide an NPEG, and the CPP is linked to the nitrogen atom of the linker by an amide bond. Alternatively, the attachment of the CPP to the nitrogen of the linker comprising an NPEG unit may be mediated through a spacer group, where a suitable spacer group may be, for example, any amino acid(s), a short alkane chain, or a short PEG / NPEG chain.

[0057] In one embodiment, the linker comprises an NPEG unit and the CPP is linked to the nitrogen atom of the linker by an amide bond. For example, in one embodiment, the compound has formula (III): [ka] wherein: CPP, P1 and P2 are as defined herein; p is an integer from 0 to 10, q is an integer from 0 to 10.

[0058] In one embodiment, p = q. In one embodiment, p > q. In one embodiment, p < q. In one embodiment, the sum of p and q is an integer from 1 to 20. In one embodiment, p is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In one embodiment, p is an integer from 0 to 4. In one embodiment, q is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In one embodiment, q is an integer from 0 to 4. In one embodiment, the total number of ethylene glycol moieties p + q is 2 to 12, such as 2, such as 4, such as 6, such as 8, such as 10, such as 12. In one embodiment, the total number of ethylene glycol moieties p + q is 4. In one embodiment, p is 2 and q is 2.

[0059] Overall dimer structure In one embodiment, the compound has the formula (XXV):

Chemical formula

Chemical formula

[0060] In one embodiment, p and q are 2, and the compound has the general structure of formula (XXVI).

Chemical formula

[0061] In one embodiment, the compound is of formula (XXVI), wherein X a is -O- and P1 and P2 are KETLV (SEQ ID NO:2), thus forming compound 2, i.e., OPEG4-(KETLV)2.

[0062] In one embodiment, the compound is of formula (XXVI), wherein X a is -O- and P1 and P2 are KETTV (SEQ ID NO:3), thus forming compound 3, OPEG4-(KETTV)2.

[0063] In one embodiment, the compound is of formula (XXVI), wherein X a is -O- and P1 and P2 are KETVV (SEQ ID NO: 4), thus forming compound 4, i.e., OPEG4-(KETVV)2.

[0064] In one embodiment, the compound is of formula (XXVI), wherein X a is -O- and P1 and P2 are KETRV (SEQ ID NO:5), thus forming compound 5, i.e., OPEG4-(KETRV)2.

[0065] In one embodiment, the compound is of formula (XXVI), wherein X a is -O- and P1 and P2 are ISTDV (SEQ ID NO: 6), thus forming compound 6, i.e., OPEG4-(ISTDV)2.

[0066] In one embodiment, the compound is of formula (XXVI), wherein X a is -O- and P1 and P2 are VETVV (SEQ ID NO: 7), thus forming compound 7, i.e., OPEG4-(VETVV)2.

[0067] In one embodiment, the compound has the formula (V): [ka] wherein: CPP is as defined herein, X1 is selected from the group consisting of K, V, and I; X2 is selected from the group consisting of E and S; X3 is selected from the group consisting of L, T, V, R, and D; X4 is selected from the group consisting of K, V, and I; X5 is selected from the group consisting of E and S; X6 is selected from the group consisting of L, T, V, R, and D; p is an integer from 0 to 10; q is an integer from 0 to 10.

[0068] Compounds of formula (V), where p and q are 2, are further illustrated in Figure 11. R1 through R5 are the amino acid side chains of peptides X1X2TX3V (SEQ ID NO:54) and X4X5TX6V (SEQ ID NO:56), and R6 represents the CPP tag described herein.

[0069] Poly-Arg as CPP In one embodiment, the CPP is a poly-L-arginine peptide (poly-Arg). Thus, in one embodiment, the compound has the following formula (II): [ka] where poly-Arg, the linker, P1 and P2 are as defined herein.

[0070] In one embodiment, the CPP is a poly-L-arginine peptide of 3-20 L-arginine residues (poly-Arg). In one embodiment, the CPP is a poly-L-arginine peptide of 3-15 L-arginine residues (poly-Arg). In one embodiment, the CPP is a poly-L-arginine peptide of 3-12 L-arginine residues (poly-Arg). In one embodiment, the CPP is a poly-L-arginine peptide of 3-10 L-arginine residues (poly-Arg). In one embodiment, the N-terminus of the poly-Arg is optionally modified, e.g., optionally acetylated.

[0071] Compounds of the invention with a poly-Arg CPP tag, such as L-Arg9 (SEQ ID NO: 12), have low CP 50 values ​​(Example 4, Figures 7-9), i.e., intracellular uptake was highly efficient, suggesting better BBB penetration and intracellular delivery. Surprisingly, compounds containing CPP L-Arg9 (SEQ ID NO: 12) showed in vitro plasmin half-life times comparable to compounds containing D-amino acids or macrocyclic CPPs (Figures 5-6). Interestingly, the half-life of L-Arg9-containing compounds was significantly better than L-TAT (SEQ ID NO: 8) (Figure 5).

[0072] In one embodiment, the CPP is poly-Arg, the linker is a PEG linker comprising NPEG units, and the CPP is linked to the nitrogen atom of the linker by an amide bond. Thus, in one embodiment, the compound has formula (IV): [ka] wherein: Poly-Arg, P1 and P2 are as defined herein; p is an integer from 0 to 10; q is an integer from 0 to 10.

[0073] In one embodiment, p is 2 and q is 2. Thus, in one embodiment, the compound has formula (VII): [ka] where poly-Arg, P1 and P2 are as defined herein.

[0074] In one embodiment, the compound has formula (VI): [ka] wherein: X1 is selected from the group consisting of K, V, and I; X2 is selected from the group consisting of E and S; X3 is selected from the group consisting of L, T, V, R, and D; X4 is selected from the group consisting of K, V, and I; X5 is selected from the group consisting of E and S; X6 is selected from the group consisting of L, T, V, R, and D; p is an integer from 0 to 10; q is an integer from 0 to 10.

[0075] In one embodiment, p is 2 and q is 2. Thus, in one embodiment, the compound has formula (VIII): [ka] where poly-Arg, X1, X2, X3, X4, X5, and X6 are as defined herein.

[0076] In one embodiment, the CPP is L-Arg9, p is 2, and q is 2. Thus, in one embodiment, the compound has formula (IX): [ka] where X1, X2, X3, X4, X5, and X6 are as defined herein.

[0077] In one embodiment, the compound is selected from the group consisting of formulas (X)-(XVI): [ka] TIFF2024520234000021.tif60159

[0078] In one embodiment, any of peptides P1, P2, and / or CPP is further modified. For example, in one embodiment, the N-terminus of CPP is optionally acetylated. Thus, in one embodiment, the compound is selected from the group consisting of formulas (XVII) to (XXIII): [ka] TIFF2024520234000023.tif119159

[0079] Specific Compounds In one embodiment, the compound has the general structure CPP-NPEG4-(P1)(P2), i.e., the compound has the formula (XXVIII): [ka] where CPP, P1 and P2 are as defined in Table 2. In one embodiment, the compound is selected from the group consisting of compounds 9-63. [Table 2] TIFF2024520234000026.tif239159TIFF2024520234000027.tif238159TIFF2024520234000028.tif238159TIFF2024520234000029.tif36159

[0080] In one embodiment, the compound has formula (XXVII): [ka] where P1 and P2 are as defined in Table 3. In one embodiment, the compound is selected from the group consisting of compounds 2-7. [Table 3]

[0081] In one embodiment, the compound is selected from the group consisting of formulas (XXIX) to (XXXIV): [ka]

[0082] In one embodiment, P1 and P2 consist of the amino acid sequence IETDV (SEQ ID NO: 1), and the CPP is selected from the group consisting of D-Pen (rqikiwfqnrrmkwkk, SEQ ID NO: 15), L-pVEC (LLIILRRRIRKQAHAHSK, SEQ ID NO: 18), L-TP2 (PLIYLRLLRGQF, SEQ ID NO: 19), MiniAp4 (|(Dap)KAPETALD|, SEQ ID NO: 21), and CPP12 (|Ff(Nal2)RrRrQ|GABA-K, SEQ ID NO: 22).

[0083] Salts and Prodrugs The compounds defined herein may be in the form of a pharma- ceutically acceptable salt or prodrug of the compound. In one embodiment of the present invention, the compounds defined herein may be in the form of a pharma- ceutically acceptable addition salt or hydrate of the compound, such as, but not limited to, K + , Na + , as well as non-salts, e.g., H + It can be formulated as:

[0084] Pharmaceutical Compositions In one aspect, the present invention relates to a pharmaceutical composition comprising the compound defined herein.The formulation of the compound of the present invention into a pharmaceutical composition is well known in the art and is further described in Gennaro (ed.), 2000, Remington: The Science and Practice of Pharmacy, 20th ed., Lippincott, Williams & Wilkins (2000), and Ansel et al., 1999, Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed., Lippincott Williams & Wilkins Publishers.

[0085] membrane permeability Since PSD-95 is located intracellularly, it is essential that any drug targeting PSD-95 efficiently crosses the cell membrane. To assess the cell permeability and delivery to the cytosol of the compounds defined herein, the Cellular Chloroalkane Permeation Assay (CAPA) may be used (Peraro et al. 2018). This assay utilizes modified haloalkane dehalogenases designed to covalently bind to chloroalkane (CA) molecules. Cytosolic delivery is reported using a HeLa cell line expressing a fusion protein containing HaloTag, green fluorescent protein (GFP), and a mitochondrial targeting peptide. The general format of CAPA is a pulse-chase assay (Deprey & Kritzer, 2020). Cells expressing the HaloTag enzyme are incubated with CA-tag fusion peptides. As these CA-peptides permeate the cell membrane and reach the cytosol, they will bind to and react with HaloTag (pulse step). Following a washing step, cells are incubated with a CA-tagged fusion dye (chase step) that quantitatively permeabilizes the cell membrane and reacts with remaining unreacted HaloTag sites. The fluorescence intensity of the cells is measured using flow cytometry and can be used to assess cytosolic delivery, as the measured fluorescence is inversely proportional to the amount of CA-peptide that reaches the permeabilized cells. The data obtained are typically expressed as CP, the concentration at which 50% cell permeation is observed. 50 The CP of the compound is expressed as a value. 50 Values ​​may be measured as described in Example 4. Example 4 shows that the cellular uptake of the described compounds was measured with CP values ​​ranging from 86.1 μM (compound 22) to 0.68 μM (compound 30). 50 The intracellular uptake efficiency of NPEG4(IETDV)2 conjugated to various CPP tags (SEQ ID NOs: 8 to 22) was measured and shown to be highly dependent on the CPP used (Figure 7). Furthermore, the intracellular uptake efficiency of NPEG4(IETDV)2 conjugated to various CPP tags (SEQ ID NOs: 8 to 22) varied in an unpredictable range of CPPs. 50 values ​​were obtained (Figure 8).

[0086] In one embodiment the compound has a CP of 250 μM or less, such as 200 μM or less, for example 150 μM or less, such as 100 μM or less, for example 80 μM or less, such as 70 μM or less, for example 60 μM or less, such as 50 μM or less, for example 40 μM or less, such as 30 μM or less, for example 20 μM or less, such as 15 μM or less, for example 10 μM or less, such as 5 μM or less. 50 Preferably, the compound has a CP value of 60 μM or less. 50 It has a value.

[0087] Plasmin Stability Ischemic stroke (also known as "brain ischemia" or "cerebral ischemia") is usually caused by blockage of an artery that supplies blood to the brain. The blockage reduces blood flow and oxygen to the brain, leading to damage or death of brain cells. The blockage of the blood vessel can be removed using various mechanical devices or using "clot busting agents" delivered intravenously or intraarterially. Among such thrombolytic agents is tissue plasminogen factor (tPA), which generates plasmin from plasminogen. Examples of recombinant tPA are alteplase, reteplase, and tenecteplase, while other thrombolytic drugs that break down blood clots include streptokinase, urokinase, and desmotaplase.

[0088] In one embodiment, the compounds of the invention are administered to subjects receiving tPA or recombinant tPA, the standard of care for AIS, and therefore it is essential that the compounds are compatible with the administration of tPA, including the generation of the serine protease plasmin.

[0089] The in vitro plasmin stability of the compounds of the invention was determined in Example 3. In one embodiment, the compound has a half-life in the presence of plasmin in the plasmin stability assay described in Example 3 of at least 10 minutes, such as at least 30 minutes, for example at least 1 hour, such as at least 2 hours, for example at least 3 hours, such as at least 4 hours, for example at least 5 hours, such as at least 6 hours, for example at least 7 hours, such as at least 8 hours, for example at least 9 hours, such as at least 10 hours, for example at least 15 hours, such as at least 20 hours, for example at least 30 hours.

[0090] Affinity of PSD-95 for PDZ1-2 Preferably, the compounds of the present invention have nanomolar range affinity for PDZ1-2 of PSD-95, making them highly potent inhibitors. The affinity of PSD-95 for PDZ1-2 is essential in reducing the threshold concentration of a drug required to achieve a therapeutic effect, which is particularly important when a drug must cross the blood-brain barrier (BBB) ​​to reach its target, as the BBB tends to limit accumulation of drug concentration at the target.

[0091] In some embodiments, the compounds of the invention are dimeric PSD-95 inhibitors that bind simultaneously to PDZ1 and PDZ2, which may explain their high affinity for these domains.

[0092] As described in Example 2, the resulting affinities for PDZ1-2 of PSD-95 for compounds of the invention are in the low nanomolar range and are similar to L-TAT-NPEG4(IETDV)2.

[0093] In one embodiment, the K of the compound for PDZ1-2 of PSD-95 i Values ​​may be 100nM or less, such as 80nM or less, for example 70nM or less, such as 60nM or less, for example 50nM or less, such as 40nM or less, for example 30nM or less, such as 20nM or less, for example 10nM or less.

[0094] Medical Use In one aspect of the invention, the compounds defined herein are for use as a pharmaceutical.

[0095] The PDZ1 and PDZ2 domains of PSD-95 interact with several proteins, including the simultaneous binding of NMDA-type ionotropic glutamate receptors and the nitric oxide (NO)-producing enzyme nNOS. NMDA receptors are the major mediators of excitotoxicity, i.e., glutamate-mediated neurotoxicity, which has been implicated in neurodegenerative diseases and acute brain injury. PSD-95 simultaneously binds to NMDA receptors, mainly GluN2A and GluN2B subunits, and nNOS via PDZ1 and PDZ2, respectively. Activation of NMDA receptors causes an influx of calcium ions, which activates nNOS, resulting in NO production. Thus, PSD-95 mediates a specific partnership between NMDA receptor activation and NO production, which can be harmful to cells if sustained for a long period of time, and is a major promoter of glutamate-mediated neurotoxicity. Inhibition of the nNOS / PSD-95 / NMDA receptor ternary complex interaction by targeting PSD-95 is known to prevent ischemic brain injury in mice by impairing the functional link between calcium ion influx and NO production, while leaving intact physiological functions such as ion influx and the pro-survival signaling pathway of the NMDA receptor. Specific inhibition of excitotoxicity can be obtained by disrupting the intracellular nNOS / PSD-95 / NMDA receptor complex using PSD-95 inhibitors.

[0096] The compounds of the present invention are PSD-95 inhibitors, and therefore can inhibit excitotoxicity.Therefore, the compounds of the present invention are useful for treating various diseases, particularly neurological diseases, especially diseases that are partially mediated by excitotoxicity.Such diseases and pathologies include stroke, epilepsy, hypoxia, traumatic injury to the CNS not related to stroke, such as traumatic brain injury and spinal cord injury, other cerebral ischemia, Alzheimer's disease, and Parkinson's disease.

[0097] In one aspect, the invention relates to a compound as defined herein for use in preventing, treating, reducing, and / or delaying the onset of an excitotoxicity-related disorder. In one embodiment, the excitotoxicity-related disorder is stroke. In one embodiment, the excitotoxicity-related disorder is ischemic stroke. In one embodiment, the excitotoxicity-related disorder is cerebral ischemia. In one embodiment, the excitotoxicity-related disorder is acute ischemic stroke. In one embodiment, the excitotoxicity-related disorder is subarachnoid hemorrhage.

[0098] In one aspect, the invention relates to the use of a compound defined herein for the manufacture of a medicament for preventing, treating, reducing and / or delaying the onset of an excitotoxicity-related disorder.

[0099] In one aspect, the invention relates to a method for preventing, treating, reducing, and / or delaying the onset of excitotoxicity-related disorders, the method comprising administering a therapeutically effective amount of a compound defined herein.

[0100] In one aspect, the present invention relates to a compound for use in reducing and / or protecting against the damaging effects of excitotoxicity.In one embodiment, the compound is for use in reducing the damaging effects of stroke.In one embodiment, the compound is for use in treating the damaging effects of acute ischemic stroke.In one embodiment, the compound is for use in treating the damaging effects of subarachnoid hemorrhage.

[0101] In one aspect, the invention relates to a method for protecting against and / or reducing the damaging effects of excitotoxicity to the brain or spinal cord in a subject, the method comprising the step of administering to the subject an effective amount of a compound defined herein to protect against and / or reduce the damaging effects.

[0102] In one aspect, the invention relates to a method of treating, reducing or delaying the onset of a condition mediated by excitotoxicity, the method comprising administering to a human subject having or at risk of the condition a compound defined herein.

[0103] In one aspect, the present invention relates to a method for treating or inhibiting or delaying at least one sign or symptom of a pathology mediated by excitotoxicity in a subject, comprising administering a compound as defined herein to a subject having the pathology or a risk factor associated with the pathology.In one embodiment, the pathology is stroke or traumatic injury to CNS.In one embodiment, the excitotoxicity-related disease is ischemic injury or traumatic injury to / in / the CNS.

[0104] In one aspect, the invention relates to a method of reducing the damaging effects of a stroke in a subject having a stroke, the method comprising administering to the subject an effective amount of a compound defined herein to reduce the damaging effects of the stroke.

[0105] As used herein, "stroke" is a general term referring to a condition caused by occlusion or hemorrhage of one or more blood vessels supplying the brain, leading to cell death. As used herein, "ischemic stroke" refers to a stroke caused by occlusion of one or more blood vessels supplying the brain. Types of ischemic stroke include, for example, embolic stroke, cardiogenic stroke, thrombotic stroke, large vessel thrombosis, lacunar infarction, artery-artery stroke, and cryptogenic stroke. "Cerebral ischemia" is a condition in which a blockage in an artery limits the delivery of oxygen-rich blood to the brain, resulting in damage to brain tissue. Cerebral ischemia may be referred to as cerebral ischemia or cerebrovascular ischemia.

[0106] As used herein, "hemorrhagic stroke" refers to a stroke caused by bleeding in one or more blood vessels supplying the brain. Types of hemorrhagic stroke include, for example, subdural stroke, intraparenchymal stroke, epidural stroke, and subarachnoid stroke.

[0107] In one embodiment, the disease treatable by the compound of the present invention is ischemic or traumatic injury of CNS.In one aspect, the present invention relates to a method for reducing the traumatic or ischemic injury effect on the brain or spinal cord in a subject, comprising treating the subject with the compound defined herein to achieve said reduction.

[0108] In one aspect, the invention relates to a method for inhibiting cerebral ischemia resulting from endovascular surgery, the method comprising administering to a subject undergoing endovascular surgery a compound defined herein in a regimen effective to inhibit cerebral ischemia.

[0109] In one aspect, the invention relates to a method of inhibiting ischemic damage due to endovascular surgery to treat an aneurysm, diagnostic angiography, or carotid artery stenting, comprising administering a compound as defined herein in an effective regimen to a subject undergoing endovascular surgery to treat an aneurysm or diagnostic angiography.

[0110] In one aspect, the invention relates to a compound as defined herein for use in inhibiting ischemic damage due to neurosurgery, which in one embodiment is a diagnostic angiogram of the brain or an endovascular procedure to treat an aneurysm.

[0111] In some embodiments, the compound is administered in combination with reperfusion therapy, hi one embodiment, the compound and the reperfusion are administered to a subject simultaneously, sequentially, or separately.

[0112] The term "reperfusion therapy" as used herein refers to a medical procedure to restore blood flow either through or around a blocked artery. Reperfusion therapy includes medical agents and mechanical reperfusion. The medical agent may be a thrombolytic or fibrinolytic agent used in a process called thrombolysis. In some embodiments, reperfusion therapy is performed by administering a thrombolytic agent such as a plasminogen activator, e.g., tPA.

[0113] In one embodiment, the compound is compound 8 (AB144) and the compound is administered in combination with a plasminogen activator, such as tPA.

[0114] In some embodiments, the reperfusion therapy is mechanical reperfusion, including surgery. The surgery performed may be a minimally invasive endovascular procedure.

[0115] Among the mechanical reperfusion devices are intra-arterial caters, balloons, stents, and various thrombectomy devices.

[0116] In one embodiment, the compound is administered in combination with a thrombolytic agent, and the compound and the thrombolytic agent are administered to a subject simultaneously, sequentially, or separately.

[0117] In one aspect, the invention relates to a method of treating the damaging effects of ischemia on the central nervous system, the method comprising: a) administering to a subject having or at risk of ischemia a compound as defined herein; b) administering reperfusion therapy to the subject; The compounds and the reperfusion therapy treat the damaging effects of ischemia on the subject's central nervous system.

[0118] In one aspect, the invention relates to a compound as defined herein for use in treating the damaging effects of ischemia on the central nervous system in a subject having or at risk of ischemia, wherein a reperfusion therapy is administered to the subject, and the compound and the reperfusion therapy treat the damaging effects of ischemia on the subject's central nervous system.

[0119] In one embodiment, the method further comprises simultaneously, sequentially or separately administering a thrombolytic agent to the subject.

[0120] In one aspect, the present invention relates to a kit of parts comprising at least two separate unit dosage forms (A) and (B), (A) comprises a compound as defined herein; (B) contains a thrombolytic agent.

[0121] In one aspect, the kit of parts defined herein is for use in treating the damaging effects of ischemia on the central nervous system, wherein (A) and (B) are administered simultaneously, sequentially or separately to a subject.

[0122] In one aspect, the present invention relates to a compound as defined herein for use in treating the damaging effects of subarachnoid hemorrhage. As used herein, the term "subarachnoid hemorrhage" refers to a bleeding condition in the subarachnoid space.

[0123] In one aspect, the invention relates to a method of treating subarachnoid hemorrhage in a subject, the method comprising administering a compound defined herein to a subject having a subarachnoid hemorrhage, wherein the development of a neurocognitive deficit in the subject is inhibited.

[0124] In one aspect, the invention relates to a method of inhibiting the development of neurological or neurocognitive deficits of a subarachnoid hemorrhage in a subject, the method comprising administering a compound defined herein to a subject having a subarachnoid hemorrhage, wherein the development of the neurological or neurocognitive deficits in the subject is inhibited.

[0125] Other neurological disorders that are not known to be associated with excitotoxicity and can be treated by the compounds of the present invention include anxiety and pain.Dimeric ligands that target PSD-95 are undergoing preclinical / clinical evaluation as treatments for chronic pain and ischemic stroke (Andreasen et al., Neuropharmacol, 2013, 67, 193-200; Bach et al, PNAS USA, 2012, 109, 3317-3322).In one embodiment, the compounds defined herein are for use in treating or preventing pain.

[0126] In one embodiment, the subject referred to herein is a mammal, such as a human.

[0127] synthesis The compounds of the invention as defined herein are a) the general steps of synthesizing peptides P1 / P2 as defined herein; b) Peptide (a) is PEGylated n or NPEG n the general step of dimerizing via a linker, where "n" is the number of PEG moieties; c) the general step of attaching a CPP tag at the N of the NPEG linker, for example by using an automated peptide synthesizer; It may be produced by a method comprising:

[0128] In one embodiment, the compound is a) general steps of providing a Ns-NPEG diacid linker; b) general steps for preparing peptides P1 / P2 using Fmoc-based solid phase peptide synthesis; c) general step of dimerizing Fmoc-deprotected peptides P1 / P2 with a Ns-NPEG diacid linker to form a linker-dimer conjugate; d) the general step of attaching a CPP to the N of the NPEG linker of the linker-dimer conjugate, for example by using an automated peptide synthesizer; The present invention is produced by a method comprising the steps of:

[0129] In one embodiment, the compounds of the invention are synthesized as described below.

[0130] Ns-NPEG diacid linker: "Ns-NPEG diacid linker" is a structure in which the NPEG linker is protected on the nitrogen by an ortho-nitrobenzenesulfonyl (Ns) protecting group on the linker nitrogen and the terminus of the NPEG linker contains a carboxylic acid. This chemical reagent or building block is used to dimerize two peptide moieties, P1 and P2.

[0131] Ortho-nitrobenzenesulfonyl (Ns) protected NPEG linkers are produced either on solid phase or in solution.

[0132] The solid phase procedure typically begins by loading Fmoc-NH-PEG-CHCHCOOH onto a solid support useful for solid phase peptide synthesis, such as 2-chlorotrityl chloride resin, using an organic solvent appropriate for the particular resin (e.g., DCM, DMF, ACN, THF) and base (e.g., DIPEA, DBU, collidine, NMM).

[0133] The Fmoc group can be removed with a base (eg, piperidine, dimethylamine, morpholine, piperazine, dicyclohexylamine, DMAP) in a suitable solvent (eg, DMF, DCM, ACN, THF).

[0134] Ortho-nitrobenzenesulfonyl chloride can be coupled to the free amine using a base (e.g., DIPEA, DBU, collidine, NMM) and an appropriate solvent (e.g., THF, DCM) to give Ns-NH-PEG-CH2CH2COO-resin.

[0135] The second portion of the linker product can be attached to a resin-bound linker moiety by use of Mitsunobu chemistry: the resin is treated with triphenylphosphine, HO-PEG-CH2CH2COOtBu, a solvent, and an ester or amide reagent of azodicarboxylic acid (e.g., diisopropyl azodicarboxylate, DIAD; diethyl azodicarboxylate, DEAD; 1,1'-(azodicarbonyl)-dipiperidine, ADDP).

[0136] Treatment of the resin with an acid such as trifluoroacetic acid (TFA) affords the final Ns-NPEG diacid linker.

[0137] A solution phase procedure can be performed by Ns protection of the amine group of NH2-PEG-CH2CH2COOtBu, followed by Mitsunobu chemistry in solution using triphenylphosphine and DIAD, DEAD, or ADDP, or similar reagents, HO-PEG-CH2CH2COOtBu, and a suitable solvent (THF, DCM). Treatment with an acid such as TFA then gives the final Ns protected NPEG-linker.

[0138] Peptide synthesis: Peptide sequences are synthesized by Fmoc-based solid-phase peptide synthesis using a solid support such as 2-chlorotrityl chloride resin or Wang resin, Fmoc-protected amino acids, bases, coupling reagents (e.g., HBTU [N,N,N',N'-tetramethyl-O(1H-benzotriazol-1-yl)uronium hexafluorophosphate], O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate [HATU], PyBOB, DIC / HOBt), and solvents. As an alternative to coupling reagents, activated esters of Fmoc-protected amino acids (e.g., pentafluorophenyl, succinimide) can be used.

[0139] Dimerization: The Fmoc-deprotected resin-bound peptide is dimerized with the Ns-NPEG diacid linker by an on-resin dimerization process by repeatedly treating the resin with substoichiometric amounts (e.g., 1 / 6) of Ns-NPEG diacid linker, base, coupling reagent, and a suitable solvent (e.g., DMF, DCM, THF). As an alternative to coupling reagents, activated esters of the Ns-NPEG linker can be used.

[0140] The dimerization process can also be formed in solution using an activated ester of the Ns-NPEG linker (e.g., pentafluorophenyl, succinimide) together with either 1-hydroxy-7-azabenzotriazole (HOAt) or hydroxybenzotriazole (HOBt) in a solvent (e.g., ACN, DMF, DCM, THF) and a suitable side chain protection peptide (e.g., tert-butyl). Dimerization in solution can also be carried out using the Ns-NPEG diacid linker, a coupling reagent (e.g., HBTU, HATU, etc.), a base, and a solvent.

[0141] The Ns group is removed by mercaptoethanol and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) or by sodium thiophenolate.

[0142] Attachment of the CPP to the NH moiety of the NPEG linker of the linker-dimer conjugate: First, an amino acid is coupled to the NH moiety of the NPEG linker, and then the CPP is synthesized according to standard (manual / automated) Fmoc SPPS on resin.

[0143] The ester protecting group can be removed by stirring the cleaved product in aqueous base (eg, NaOH, LiOH) and acetonitrile, followed by acidification with TFA or HCl.

[0144] Lyophilization and purification by HPLC or similar chromatographic method affords the final compound of the invention.

[0145] In a further embodiment, the synthesis of the compounds of the invention is carried out as outlined in Example 1.

[0146] Terms 1. A first peptide (P1) comprising or consisting of the amino acid sequence X2TX3V (SEQ ID NO:53), wherein: a. X2 is selected from the group consisting of E and S; b. X3 is selected from the group consisting of L, T, V, R, and D; A compound, wherein P1 is linked to a cell membrane penetrating peptide (CPP).

[0147] 2. A compound comprising a first peptide (P1) linked to a second peptide (P2) by a linker, wherein P1 and P2 comprise or consist of an amino acid sequence selected from the group consisting of KETLV (SEQ ID NO:2), KETTV (SEQ ID NO:3), KETVV (SEQ ID NO:4), KETRV (SEQ ID NO:5), ISTDV (SEQ ID NO:6), and VETVV (SEQ ID NO:7).

[0148] 3. The compound according to clause 2, wherein P1 and P2 are linked to a cell membrane penetrating peptide (CPP) or an albumin binding moiety via said linker, thereby increasing the passage of said peptide through a membrane or the blood-brain barrier.

[0149] 4. The compound has a CP of 250 μM or less, such as 200 μM or less, for example 150 μM or less, such as 100 μM or less, for example 80 μM or less, such as 70 μM or less, for example 60 μM or less, such as 50 μM or less, for example 40 μM or less, such as 30 μM or less, for example 20 μM or less, such as 15 μM or less, for example 10 μM or less, for example 5 μM or less. 50 4. The compound according to any one of the preceding clauses, having a value:

[0150] 5. The compound according to any one of the preceding clauses, wherein said compound has a half-life in a plasmin stability assay of at least 10 minutes, such as at least 30 minutes, for example at least 1 hour, such as at least 2 hours, for example at least 3 hours, such as at least 4 hours, for example at least 5 hours, such as at least 6 hours, for example at least 7 hours, such as at least 8 hours, for example at least 9 hours, such as at least 10 hours, for example at least 15 hours, such as at least 20 hours, for example at least 30 hours.

[0151] 6. K of the compound for PDZ1 to PDZ2 of the PSD-95 i A compound according to any one of the preceding clauses, wherein the value is 100nM or less, such as 80nM or less, for example 70nM or less, such as 60nM or less, for example 50nM or less, such as 40nM or less, for example 30nM or less, such as 20nM or less, for example 10nM or less.

[0152] 7. The compound according to any one of the preceding clauses, wherein the CPP is a poly-L-arginine peptide (poly-Arg).

[0153] 8. The compound according to clause 7, wherein the poly-Arg consists of 5 to 10 L-arginine residues, such as 7 to 10 L-arginine residues, such as 8 or 9 L-arginine residues.

[0154] 9. The compound according to clause 7, wherein said poly-Arg comprises or consists of the amino acid sequence RRRRRRRRR (SEQ ID NO:12).

[0155] 10. The compound according to any one of clauses 1-6, wherein said CPP is L-TAT (YGRKKRRQRRR, SEQ ID NO: 8).

[0156] 11. The compound according to any one of clauses 1-6, wherein said CPP is D-TAT (yGrkkrrqrrr, SEQ ID NO: 9).

[0157] 12. The compound according to any one of clauses 1-6, wherein said CPP is mTAT (rRrGrKkRr, SEQ ID NO: 10).

[0158] 13. The compound according to any one of clauses 1-6, wherein said CPP is riTAT (rrrqrrkkr, SEQ ID NO: 11).

[0159] 14. The compound according to any one of clauses 1-6, wherein said CPP is D-Arg9 (rrrrrrrrr, SEQ ID NO: 13).

[0160] 15. The compound according to any one of clauses 1 to 6, wherein said CPP is L-Pen (RQIKIWFQNRRMKWKK, SEQ ID NO: 14).

[0161] 16. The compound according to any one of clauses 1 to 6, wherein said CPP is D-Pen (rqikiwfqnrrmkwkk, SEQ ID NO: 15).

[0162] 17. The compound according to any one of clauses 1-6, wherein said CPP is L-DPV3 (RKKRRRESRKKRRRES, SEQ ID NO: 17).

[0163] 18. The compound according to any one of clauses 1 to 6, wherein said CPP is L-pVEC (LLIILRRRIRKQAHAHSK, SEQ ID NO: 18).

[0164] 19. The compound according to any one of clauses 1-6, wherein said CPP is L-MAP (KLALKLALKALKAALKLA, SEQ ID NO: 16).

[0165] 20. The compound according to any one of clauses 1-6, wherein said CPP is L-TP2 (PLIYLRLLRGQF, SEQ ID NO: 19).

[0166] 21. The compound according to any one of clauses 1-6, wherein said CPP is D-TP2 (pliylrllrGqf, SEQ ID NO: 20).

[0167] 22. The compound according to any one of clauses 1-6, wherein said CPP is MiniAp4 (|(Dap)KAPETALD|, SEQ ID NO: 21).

[0168] 23. The compound according to any one of clauses 1-6, wherein said CPP is CPP12 (|Ff(Nal2)RrRrQ|GABA-K, SEQ ID NO: 22).

[0169] 24. The compound according to any one of the preceding clauses, wherein the amino acid sequence X2TX3V (SEQ ID NO:53) is the C-terminus of P1.

[0170] 25. A compound according to any one of the preceding clauses, wherein P1 consists of 4 to 10 amino acid residues, such as 5 amino acid residues, such as 6 amino acid residues.

[0171] 26. P1, comprising or consisting of the sequence X1X2TX3V (SEQ ID NO:54), a. X1 is selected from the group consisting of K, V, and I; b. X2 is selected from the group consisting of E and S; c. X3 is selected from the group consisting of L, T, V, R, and D; 4. A compound according to any one of the preceding clauses.

[0172] 27. The compound according to any one of the preceding clauses, wherein P1 comprises or consists of KETLV (SEQ ID NO:2), KETTV (SEQ ID NO:3), KETVV (SEQ ID NO:4), KETRV (SEQ ID NO:5), ISTDV (SEQ ID NO:6), VETVV (SEQ ID NO:7), and IETDV (SEQ ID NO:1).

[0173] 28. The compound comprises a second peptide (P2) comprising or consisting of the amino acid sequence X5TX6V (SEQ ID NO:55), wherein: a. X5 is selected from the group consisting of E and S; b. X6 is selected from the group consisting of L, T, V, R, and D; 4. A compound according to any one of the preceding clauses.

[0174] 29. The compound according to any one of the preceding clauses, wherein the amino acid sequence X5TX6V (SEQ ID NO:55) is the C-terminus of P2.

[0175] 30. A compound according to any one of the preceding clauses, wherein P2 consists of 4 to 10 amino acid residues, such as 5 amino acid residues, such as 6 amino acid residues.

[0176] 31. P2 comprises or consists of the sequence X4X5TX6V (SEQ ID NO:56), wherein: a. X4 is selected from the group consisting of K, V, and I; b. X5 is selected from the group consisting of E and S; c. X6 is selected from the group consisting of L, T, V, R, and D; 4. A compound according to any one of the preceding clauses.

[0177] 32. The compound according to any one of the preceding clauses, wherein P2 comprises or consists of an amino acid sequence selected from the group consisting of KETLV (SEQ ID NO:2), KETTV (SEQ ID NO:3), KETVV (SEQ ID NO:4), KETRV (SEQ ID NO:5), ISTDV (SEQ ID NO:6), VETVV (SEQ ID NO:7), and IETDV (SEQ ID NO:1).

[0178] 33. The compound according to any one of the preceding clauses, wherein P1 and P2 are identical.

[0179] 34. The compound according to any one of the preceding clauses, wherein P1 and P2 are conjugated to the linker via their N-terminus.

[0180] 35. The compound according to any one of the preceding clauses, wherein the CPP, such as poly-Arg, is linked to P1 via a linker.

[0181] 36. The compound according to any one of the preceding clauses, wherein the CPP, such as poly-Arg, is linked to P1 and P2 via a linker.

[0182] 37. The compound according to any one of the preceding clauses, wherein the CPP, such as poly-Arg, is linked to the linker via its C-terminus, and P1 and P2 are conjugated to the linker via their N-terminus, and the compound has the general structure of formula (I):

[0183] [ka]

[0184] 38. The compound according to any one of the preceding clauses, wherein the CPP is poly-Arg and the compound has the general structure of formula (II): [ka]

[0185] 39. The compound according to any one of the preceding clauses, wherein said linker comprises one or more PEG units.

[0186] 40. The compound according to clause 39, wherein at least one oxygen atom of one of said PEG units is replaced with a nitrogen atom resulting in NPEG.

[0187] 41. The compound according to any one of the preceding clauses, wherein the linker comprises an NPEG unit and the CPP, such as poly-Arg, is linked to the nitrogen atom of the linker by an amide bond.

[0188] 42. The compound has the formula (III): [ka] wherein: p is an integer from 0 to 10; q is an integer from 0 to 10; 4. A compound according to any one of the preceding clauses.

[0189] 43. The compound has the formula (IV): [ka] wherein: p is an integer from 0 to 10; q is an integer from 0 to 10; 4. A compound according to any one of the preceding clauses.

[0190] 44. The compound has the formula (V): [ka] wherein: X1 is selected from the group consisting of K, V, and I; X2 is selected from the group consisting of E and S; X3 is selected from the group consisting of L, T, V, R, and D; X4 is selected from the group consisting of K, V, and I; X5 is selected from the group consisting of E and S; X6 is selected from the group consisting of L, T, V, R, and D; p is an integer from 0 to 10; q is an integer from 0 to 10; 4. A compound according to any one of the preceding clauses.

[0191] 45. The compound has the formula (VI): [ka] wherein: X1 is selected from the group consisting of K, V, and I; X2 is selected from the group consisting of E and S; X3 is selected from the group consisting of L, T, V, R, and D; X4 is selected from the group consisting of K, V, and I; X5 is selected from the group consisting of E and S; X6 is selected from the group consisting of L, T, V, R, and D, p is an integer from 0 to 10, q is an integer from 0 to 10, A compound according to any one of the preceding clauses.

[0192] 46. A compound according to any one of the preceding clauses, wherein p = q.

[0193] 47. A compound according to any one of the preceding clauses, wherein p > q.

[0194] 48. A compound according to any one of the preceding clauses, wherein p < q.

[0195] 49. A compound according to any one of the preceding clauses, wherein the sum of p and q is an integer from 1 to 20.

[0196] 50. A compound according to any one of the preceding clauses, wherein the number p of ethylene glycol moieties is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 ethylene glycol moieties.

[0197] 51. A compound according to any one of the preceding clauses, wherein the number p of ethylene glycol moieties is from 0 to 4.

[0198] 52. A compound according to any one of the preceding clauses, wherein the number q of ethylene glycol moieties is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 ethylene glycol moieties.

[0199] 53. A compound according to any one of the preceding clauses, wherein the number q of ethylene glycol moieties is from 0 to 4.

[0200] 54. A compound according to any one of the preceding clauses, wherein the total number p + q of the ethylene glycol moieties is 2 to 12, for example 2, for example 4, for example 6, for example 8, for example 10, for example 12.

[0201] 55. The compound according to any one of the preceding clauses, wherein the total number of ethylene glycol moieties, p+q, is 4.

[0202] 56. A compound according to any one of the preceding clauses, wherein p is 2 and q is 2.

[0203] 57. The compound according to any one of the preceding clauses, wherein the compound has the general structure of formula (VII): [ka]

[0204] 58. The compound according to any one of the preceding clauses, wherein the compound has the general structure of formula (VIII): [ka]

[0205] 59. The compound according to any one of the preceding clauses, wherein the compound has the general structure of formula (IX): [ka]

[0206] 60. The compound according to any one of the preceding clauses, wherein the compound is selected from the group consisting of formulas (X) to (XVI): [ka] TIFF2024520234000043.tif182159

[0207] 61. The compound according to any one of the preceding clauses, wherein the N-terminus of the CPP is acetylated.

[0208] 62. The compound according to any one of clauses 1-60, wherein the N-terminus of the CPP is conjugated to a chloroalkane tag (CA) having the structure: [ka]

[0209] 63. The compound according to any one of clauses 1-60, wherein the N-terminus of the CPP is methylated.

[0210] 64. The compound according to any one of clauses 1-60, wherein the N-terminus of the CPP is formylated.

[0211] 65. A compound according to any one of the preceding clauses, in the form of a pharma- ceutically acceptable salt or prodrug of said compound.

[0212] 66. a. a first peptide (P1) comprising or consisting of the amino acid sequence X2TX3V (SEQ ID NO:53), X2 is selected from the group consisting of E and S; X3 is selected from the group consisting of L, T, V, R, and D; b. A second peptide (P2) comprising or consisting of the amino acid sequence X5TX6V (SEQ ID NO:55), X5 is selected from the group consisting of E and S; X6 is selected from the group consisting of L, T, V, R, and D; and c. cell-penetrating peptides (CPPs), wherein the CPP is linked to a linker through its C-terminus, and P1 and P2 are conjugated to the linker through their N-terminus, and the compound comprises the formula (I): [ka] and wherein the compound has a CP of 20 μM or less. 50 The compound having a value.

[0213] 67. P1 comprises or consists of the sequence X1X2TX3V (SEQ ID NO: 54), wherein: X1 is selected from the group consisting of K, V, and I; X2 is selected from the group consisting of E and S; X3 is selected from the group consisting of L, T, V, R, and D; P2 comprises or consists of the sequence X4X5TX6V (SEQ ID NO:56), in which X4 is selected from the group consisting of K, V, and I; X5 is selected from the group consisting of E and S; X6 is selected from the group consisting of L, T, V, R, and D; 67. A compound as defined in clause 66.

[0214] 68. The compound according to any one of clauses 66 or 67, wherein P1 and / or P2 consists of 4 to 10 amino acid residues, such as 5 amino acid residues.

[0215] 69. The compound according to any one of clauses 66-68, wherein X3 and X6 are selected from the group consisting of L, T, V, and R.

[0216] 70. The compound according to any one of clauses 66 to 68, wherein P1 and P2 consist of an amino acid sequence selected from the group consisting of KETLV (SEQ ID NO:2), KETTV (SEQ ID NO:3), KETVV (SEQ ID NO:4), KETRV (SEQ ID NO:5), ISTDV (SEQ ID NO:6), and VETVV (SEQ ID NO:7).

[0217] 71. The linker comprises one or more PEG units, at least one oxygen atom of one of the PEG units is replaced with a nitrogen atom to provide NPEG, the CPP is linked to the nitrogen atom of the linker by an amide bond, and the compound has the formula (III): [ka] wherein: p is an integer from 0 to 10; q is an integer from 0 to 10; 71. The compound according to any one of clauses 66 to 70.

[0218] 72. The compound according to any one of clauses 66 to 71, wherein said CPP is selected from the group consisting of poly-L-arginine peptide (poly-Arg), D-TAT (yGrkkrrqrrr, SEQ ID NO: 9), L-Pen (RQIKIWFQNRRMKWKK, SEQ ID NO: 14), D-Pen (rqikiwfqnrrmkwkk, SEQ ID NO: 15), L-DPV3 (RKKRRRESRKKRRRES, SEQ ID NO: 17), L-pVEC (LLIILRRRIRKQAHAHSK, SEQ ID NO: 18), L-MAP (KLALKLALKALKAALKLA, SEQ ID NO: 16), L-TP2 (PLIYLRLLRGQF, SEQ ID NO: 19), MiniAp4 (|(Dap)KAPETALD|, SEQ ID NO: 21), and CPP12 (|Ff(Nal2)RrRrQ|GABA-K, SEQ ID NO: 22).

[0219] 73. The compound according to any one of clauses 66 to 72, wherein the CPP comprises or consists of a poly-L-arginine peptide (poly-Arg), such as a poly-Arg consisting of 5 to 10 L-arginine residues, such as 8 or 9 L-arginine residues, e.g. said poly-Arg comprises or consists of the amino acid sequence RRRRRRRRR (SEQ ID NO: 12).

[0220] 74. The compound has the formula (VI): [ka] wherein: X1 is selected from the group consisting of K, V, and I; X2 is selected from the group consisting of E and S; X3 is selected from the group consisting of L, T, V, R, and D; X4 is selected from the group consisting of K, V, and I; X5 is selected from the group consisting of E and S; X6 is selected from the group consisting of L, T, V, R, and D; p is an integer from 0 to 10; q is an integer from 0 to 10; 74. The compound according to any one of clauses 66 to 73.

[0221] 75. The compound according to any one of clauses 66 to 74, wherein p is 2 and q is 2.

[0222] 76. The compound according to any one of clauses 66 to 75, wherein the N-terminus of the CPP is acetylated or the N-terminus of the CPP is conjugated to a chloroalkane tag (CA) having the structure: [ka]

[0223] 77. The compound according to any one of the preceding clauses, wherein the compound is a PSD-95 inhibitor.

[0224] 78. A pharmaceutical composition comprising a compound according to any one of the preceding clauses.

[0225] 79. A compound according to any one of the preceding clauses for use as a medicament.

[0226] 80. A compound according to any one of clauses 1 to 77 for use in treating, preventing, reducing and / or delaying the onset of an excitotoxicity-related disorder.

[0227] 81. The compound for use according to clause 80, wherein said excitotoxicity-related disease is stroke.

[0228] 82. The compound for use according to clause 80, wherein said excitotoxicity-related disease is ischemic stroke.

[0229] 83. The compound for use according to clause 80, wherein said excitotoxicity-related disease is cerebral ischemia.

[0230] 84. The compound for use according to clause 80, wherein said excitotoxicity-related disease is acute ischemic stroke.

[0231] 85. The compound for use according to clause 80, wherein said excitotoxicity-related disorder is subarachnoid hemorrhage.

[0232] 86. A compound according to any one of clauses 1 to 77 for use in the treatment or prevention of pain.

[0233] 87. A kit of parts comprising at least two separate unit dosage forms (A) and (B), (A) comprises a compound according to any one of clauses 1 to 77; (B) comprises a thrombolytic agent; A kit of the above parts.

[0234] 88. The kit of parts according to clause 87 for use in treating, preventing, reducing and / or delaying the onset of an excitotoxicity-related disease and / or pain, wherein (A) and (B) are administered to said subject simultaneously, sequentially or separately. EXAMPLES

[0235] Example 1: Synthesis Resin-bound compounds 2–7 (OPEG4-KETLV, OPEG4-KETTV, OPEG4-KETVV, OPEG4-KETRV, OPEG4-ISTDV, and OPEG4-VETVV) were synthesized as previously described in Bach et al., Angew. Chem. Int. Ed., 2009, 48, 9685.

[0236] Resin-bound NPEG4-IETDV, NPEG4-KETLV, NPEG4-KETTV, NPEG4-KETVV, NPEG4-KETRV, NPEG4-ISTDV, and NPEG4-VETVV were synthesized as previously described by Bach et al., Proc. Natl. Acad. Sci. USA, 2012, 109, 3317. Linear CPPs (L-TAT, DTAT, mTAT, riTAT, PolyR, D-PolyR, L-Pen, D-Pen, L-pVEC, L-MAP, L-DPV3, L-TP2, D-TP2, MiniAp4, CPP12) The first C-terminal amino acid (Fmoc-Ala-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-Asp(OAll)-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-L-Lys(Ns)-OH, Fmoc-L-Phe-OH, Fmoc-D-Phe-OH, Fmoc-L-Ser(tBu)-OH) were manually coupled three times to the nitrogen of the NPEG4 linker using O-(1H-6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) as the coupling reagent. The synthesis of the CPP was then accomplished using an automated peptide synthesizer (Prelude X, Gyros Protein Technologies, US). For the synthesis using Prelude X, all reagents were prepared as solutions in DMF: Fmoc-protected amino acids (0.2 M), HCTU (0.4 M), and DIPEA (1.0 M). Sequence elongation was achieved using the following protocol: deprotection (2x2 min, room temperature, shaking at 350 rpm) and coupling (3x10 min, 50°C, shaking at 350 rpm). Amino acids were coupled in triplicate using a mixture of Fmoc-amino acids / HCTU / DIPEA (1:1:2.5) in 5-fold excess over resin loading. Cyclic CPP (MiniAp4 and CPP12)-containing peptides were treated with 20 equivalents of PhSiH3 and 0.2 equivalents of Pd(PPh3)4 in dichloromethane (DCM) for 2x15 min under nitrogen to remove allyl and alloc protecting groups. The resin was washed with DCM followed by dimethylformamide (DMF).The cyclization reaction between the side chain of Asp / Dap and / or the C-terminus of Glu and the N-terminus of Phe was carried out by treating the peptide with 2 equivalents of (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP) and 2 equivalents of DIPEA in DMF for 16 h at room temperature, followed by washing with DMF. After successful synthesis of the CPP-tag fused dimeric peptide, the resin was split into two. The N-terminus of one part of the resin-bound peptide was capped twice for 10 min using a mixture of DMF:acetic anhydride:DIPEA (8:1.5:0.5). The N-terminus of the other part of the resin-bound peptide was functionalized with a chloroalkane tag (CA). The CA tag was coupled to the N-terminal nitrogen group of the CPP-tag fused peptide using a mixture of CA:PyBOP:DIPEA (3:3:10) in DMF for 16 h. For CPP12-containing peptides, the ortho-nitrobenzenesulfonyl (Ns) group was removed prior to coupling of the CA tag by adding 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 0.5 mmol) in DMF (2 mL) and mercaptoethanol (0.5 mmol) in DMF (2 mL) and shaking for 30 min. After flow washing in DMF, the mercaptoethanol / DBU treatment was repeated four times. After a final flow wash in DMF, the CA tag was coupled to the side chain nitrogen of the Lys residue.

[0237] Peptide compounds 2-75 were cleaved from the dried resin using TFA / triisopropylsilane / 2,2'-(ethylenedioxy)diethanethiol (DODT) / HO (90 / 2.5 / 2.5) for 2 h, followed by filtration, evaporation, precipitation with ice-cold ether, lyophilization, and purification by preparative RP-HPLC. The final peptide ligands were characterized by LC-MS for molecular weight determination and UPLC (214 nm) for purity (>95%).

[0238] The syntheses as described above can also be carried out on a 40 mmol or up to 100 mmol scale to provide multigram preparations of the peptide compounds reported herein.

[0239] The peptide compounds described herein exhibited excellent solubility properties reaching concentrations of 25 mg / mL and above.

[0240] This example demonstrates that the ligand can be synthesized, purified and obtained in pure form.

[0241] Example 2: Determination of affinity of PSD-95 for PDZ1-2 The binding affinities of compounds 1-75 to PDZ1-2 of PSD-95 were measured using an in vitro fluorescence polarization (FP) assay as described by Bach et al., Proc. Natl. Acad. Sci. USA, 2012, 109, 3317. First, the K D Saturation binding curves were obtained to determine IC values. Increasing concentrations of PDZ1-2 (0.015-30 nM) were added to a fixed concentration of probe (0.5 nM). Fluorescence polarization (FP) of the samples was measured at excitation / emission wavelengths of 635 / 670 nm, and the generated FP values ​​were fitted to a one-site binding model using the software GraphPad Prism. The affinity of unlabeled peptides for PSD-95 PDZ12 was then assessed in a heterologous competitive binding assay by adding increasing concentrations of the test peptide (0.4-960 nM, final concentrations) to a fixed amount of PSD-95 PDZ12 (4 nM) and probe (0.5 nM) using the same conditions as for the saturation binding experiments. The FP values ​​were fitted to a one-site competition (variable slope) model in GraphPad prism. The resulting IC 50 Values ​​were calculated as K as previously described by Nikolovska-Coleska et al., Anal. Biochem., 2004, 332, 261. i converted to a value.

[0242] This example describes a method to determine the affinity of ligands binding to PDZ1-2 of PSD-95. The results are presented in Tables 4-7 and Figures 1-4. In conclusion, the compounds tested have affinities (K ivalue), which is equivalent to that of compound 1. [Table 4] [Table 5] TIFF2024520234000051.tif60159 [Table 6] [Table 7]

[0243] Example 3: In vitro plasmin stability of ligands In vitro plasmin stability of compounds 8-62 was determined by incubating 100 μM of ligand in phosphate buffered saline (PBS) supplemented with plasmin (5 ug / mL) for 0-1440 min at 37°C. At selected time points during incubation, ligand was extracted from 80 μL of assay matrix by treatment with 80 μL of 50% acetonitrile (ACN). Samples were filtered and analyzed by UPLC to determine the amount of ligand remaining. LC-MS analysis was performed to confirm ligand integrity and identify cleavage sites.

[0244] This example describes a method to determine the in vitro plasmin stability of ligands that bind to PDZ1-2 of PSD-95. The results are presented in Tables 8 and 9, and in Figures 5 and 6. In conclusion, the tested compounds containing the CPP L-Arg9 (SEQ ID NO: 12) show in vitro plasmin half-life times comparable to compounds containing D-amino acids or macrocyclic CPPs (Figure 6). Unexpectedly, the half-life of the L-Arg9-containing compounds was significantly better than L-TAT (SEQ ID NO: 8) (Figures 5 and 6). [Table 8] TIFF2024520234000055.tif109159 [Table 9] TIFF2024520234000057.tif58159

[0245] Example 4: Determination of membrane permeability and intracellular uptake of ligands The membrane permeability of compounds 10-75 was determined in HeLa cells stably expressing halo-GFP, which is exclusively located in the cytosol. Cells were seeded at a density of 40.000 cells / well 1 day before the experiment. After aspirating the growth medium and replacing it with 100 μL of Opti-MEM, 25 μL of the prepared serial dilutions of the ligands in Opti-MEM were added to the cells (constant DMSO concentration) and the plate was incubated for 4 h at 37 °C and 5% CO2. The contents of the wells were aspirated and the cells were washed for 15 min with fresh Opti-MEM. After aspiration of the wash, the cells were incubated with TAMRA-CA (5 μM) for 15 min. After aspiration of the chase solution, the cells were washed for 30 min with Opti-MEM. Following removal of the wash, the cells were trypsinized, resuspended in PBS (2% FBS) and analyzed using a benchtop flow cytometer. The resulting fluorescence intensity data were normalized using no ligand and no TAMRA-CA control wells and plotted as dose-response curves. 50 Values ​​represent the half-maximal amount of red fluorescence that behaves inversely proportional to the cell penetration of the ligand.

[0246] This example describes a method to determine the membrane permeability and intracellular uptake of CA tagged fusion compounds that bind to PDZ1-2 of PSD-95. The results are presented in Tables 10-12 and Figures 7-9. In conclusion, the tested compounds show significantly different intracellular uptake efficiencies, which are highly dependent on the CPP used. Surprisingly, CPP tags L-Arg9 (SEQ ID NO: 12) and L-pVEC (SEQ ID NO: 18) show the lowest CP 50 Depending on the dimeric ligand, L-Arg9 also induces the CP 50values ​​below 1 μM (Figure 7), promoting highly efficient cellular uptake, suggesting better BBB penetration and intracellular delivery. Furthermore, the L-Arg9CPP tag can be truncated to 7, 6, 5, or 4 arginines without losing its cell-penetrating properties (see Table 12) (Figure 9). [Table 10] TIFF2024520234000059.tif136159 [Table 11] TIFF2024520234000061.tif74159 [Table 12]

[0247] Example 5: In vitro plasma stability determination The in vitro plasma stability of compounds 8, 23, 35, 67, 69, 71, and 73 was determined by incubating 100 μM of the ligand in undiluted pooled human plasma for 1440 min at 37° C. At selected time points during the incubation, the ligand was extracted from the assay matrix by adding it to 6 M urea, followed by precipitation using 20% ​​TCA in acetone. After overnight incubation, samples were filtered and analyzed by UPLC to determine the amount of ligand remaining. LC-MS analysis was performed to confirm ligand integrity and identify the cleavage site.

[0248] This example describes a method to determine the in vitro plasma stability of ligands that bind to PDZ1-2 of PSD-95. The results are presented in Table 13 and Figure 10. In conclusion, the L-Arg9CPP tag can be truncated to 7, 6, 5, or 4 arginines without a significant decrease in stability in human plasma (see Table 13). [Table 13]

[0249] Example 6: Determination of in vivo brain levels after intravenous injection Female Balb / c mice (four groups of three animals) are administered the compound of interest at 18 nmol / g body weight using isotonic saline as vehicle. Group 1 is used to obtain blood concentration profiles at selected time points after intravenous injection (up to 4 h post-injection). Groups 2-4 are used to determine blood and brain concentrations at three different time points. Thus, animals are sacrificed at selected time points after intravenous injection and relevant tissues are collected and analyzed using appropriate methods (LC-MS / MS).

[0250] This example describes a method to determine in vivo brain levels of a ligand that binds to PDZ1-2 of PSD-95. Additionally, blood-to-brain ratios and associated tissue targeting efficiencies are determined, providing information about the ability of a compound to cross the blood-brain barrier and reach targets in the brain.

[0251] Example 7: Determination of in vivo neuroprotective effects against stroke-induced damage using the pMCAO mouse model For experimental and surgical details, see Bach et al, Proc. Natl. Acad. Sci. USA, 2012, 109, 3317. Briefly, the extent of ischemic infarction was measured in a randomized, double-blind, placebo-controlled study using a permanent middle cerebral artery occlusion (pMCAO) model in C57BL / 6J mice. Following anesthesia and arterial occlusion, animals were dosed 30 minutes after surgery. Animals surviving 6 hours after surgery were sacrificed and the brains were carefully removed. Following fixation and sectioning of the brains, infarct volume analysis was performed and total infarct volume was calculated.

[0252] This example describes a method to determine the in vivo neuroprotective effect of ligands that bind to PDZ1-2 of PSD-95. The readout parameter is infarct volume in compound-treated groups versus placebo control groups. TIFF2024520234000064.tif232159TIFF2024520234000065.tif234159TIFF2024520234000066.tif233159TIFF2024520234000067.tif79159References Aarts, M., Liu, Y., Liu, L., Besshoh, S., Arundine, M., Gurd, J.W., Wang, Y.T., Salter, M.W., Tymianski, M., 2002. Treatment of ischemic brain damage by perturbing NMDA receptor-PSD-95 protein interactions. Science 298, 846-850. J.T. Andreasen, A. Bach, M. Gynther, A. Nasser, J. Mogensen, K. Stromgaard, D.S. Pickering, UCCB01-125, a dimeric inhibitor of PSD-95, reduces inflammatory pain without disrupting cognitive or motor performance: comparison with the NMDA receptor antagonist MK-801, Neuropharmacology, 67, 193-200(2013). Bach, A., Chi, C.N., Pang, G.F., Olsen, L., Kristensen, A.S., Jemth, P., Stromgaard, K., Design and synthesis of highly potent and plasma-stable dimeric inhibitors of the PSD-95-NMDA receptor interaction. Angew. Chem. Int. Ed. 48, 9685-9689(2009). Bach,A.,Clausen,B.H.,Moller,M.,Vestergaard,B.,Chi,C.N.,Round,A.,Sorensen,P.L.,Nissen,K.B.,Kastrup,J.S.,Gajhede,M.,Jemth,P.,Kristensen,A.S.,Lundstrom,P.,Lambertsen,K.L.,Stromgaard,K.,A high-affinity,dimeric inhibitor of PSD-95 bivalently interacts with PDZ1-2 and protects against ischemic brain damage.Proc.Natl.Acad.Sci.U.S.A.109,3317-3322(2012). Dawson,V.L.,Dawson,T.M.,London,E.D.,Bredt,D.S.,Snyder,S.H.,1991.Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures.Proc.Natl.Acad.Sci.U.S.A 88,6368-6371. Deprey,K.;Kritzer,J.A.,2020.Quantitative measurement of cytosolic penetration using the chloroalkane penetration assay.Method.Enzymol.641,277-309. Hill et al.2020.Efficacy and safety of nerinetide for the treatment of acute ischaemic stroke(ESCAPE-NA1):A multicentre,double-blind,randomised controlled trial.Lancet 395,878-887. Huang,Z.,Huang,P.L.,Panahian,N.,Dalkara,T.,Fishman,M.C.,Moskowitz,M.A.,1994.Effects of cerebral ischemia in mice deficient in neuronal nitric oxide synthase.Science 265,1883-1885. Kornau,H.C.,Schenker,L.T., Kennedy,M.B.,Seeburg,P.H.,1995.Domain interaction between NMDA receptor subunits and the postsynaptic density protein PSD-95.Science 269,1737-1740. Kucharz,K.,Rasmussen,I.S.,Bach,A.,Stromgaard,K.,Lauritzen,M.,PSD-95 uncoupling from NMDA receptors by Tat-N-dimer ameliorates neuronal depolarization in cortical spreading depression.J.Cereb.Blood Flow Metab.37,1820-1828(2017). Mayor-Nunez,D.,Ji,Z.,Sun,X.,Teves,L.,Garman,J.D.,Tymianski,M.,2021.Plasmin-resistant PSD-95 inhibitors resolve effect-modifying drug-drug interactions between alteplase and nerinetide in acute stroke,Sci.Transl.Med.13,eabb1498. Peraro,L.;Deprey,K.L.;Moser,M.K.;Zou,Z.;Ball,H.L.;Levine,B.;Kritzer,J.A.,2018.Cell Penetration Profiling Using the Chloroalkane Penetration Assay.J.Am.Chem.Soc.140,11360-11369. Sattler,R.,Xiong,Z.,Lu,W.Y.,Hafner,M.,MacDonald,J.F.,Tymianski,M.,1999.Specific coupling of NMDA receptor activation to nitric oxide neurotoxicity by PSD-95 protein.Science 284,1845-1848.

Claims

1. a. Amino acid sequence X ２ T.X. ３ A first peptide (P) comprising or consisting of V (SEQ ID NO:53). １ ) (in the sequence, X ２ is selected from the group consisting of E and S; X ３ is selected from the group consisting of L, T, V, R, and D; b. Amino acid sequence X ５ T.X. ６ A second peptide (P) comprising or consisting of V (SEQ ID NO:55). ２ ) (in the sequence, X ５ is selected from the group consisting of E and S; X ６ is selected from the group consisting of L, T, V, R, and D; and c. A cell membrane penetrating peptide (CPP) selected from the group consisting of: i. poly-L-arginine peptides consisting of 3 to 9 L-arginine residues (poly-Arg); ii. yGrkkrrqrrr (SEQ ID NO: 9, D-TAT); iii. RQIKIWFQNRRMKWKK (SEQ ID NO: 14, L-Pen); iv. rqikiwfqnrrmkwkk (SEQ ID NO: 15, D-Pen); v. RKKRRRESRKKRRRES (SEQ ID NO: 17, L-DPV3); vi. LLIILRRRRIRKQAHAHSK (SEQ ID NO: 18, L-pVEC); vii. KLALKLALKALKAALKAALKLA (SEQ ID NO: 16, L-MAP); viii. PLIYLRLLRGQF (SEQ ID NO: 19, L-TP2); ix. |(Dap)KAPETALD| (SEQ ID NO: 21, MiniAp4), and x. |Ff(Nal2)RrRrQ|GABA-K (SEQ ID NO: 22, CPP12), A PSD-95 inhibitor comprising: １ and P ２ are conjugated to the linker via their N-terminus, and the PSD-95 inhibitor has the formula (I): 【Chemistry 1】 The PSD-95 inhibitor has the general structure:

2. P １ But array X １ X ２ T.X. ３ V (SEQ ID NO:54), in which: X １ is selected from the group consisting of K, V, and I; X ２ is selected from the group consisting of E and S; X ３ is selected from the group consisting of L, T, V, R, and D; P ２ But array X ４ X ５ T.X. ６ V (SEQ ID NO:56), in which: X ４ is selected from the group consisting of K, V, and I; X ５ is selected from the group consisting of E and S; X ６ is selected from the group consisting of L, T, V, R, and D; The PSD-95 inhibitor according to claim 1.

3. P １ and / or P ２ The PSD-95 inhibitor according to claim 1 or 2, wherein consists of 4 to 10 amino acid residues, for example 5 amino acid residues.

4. X ３ and X ６ The PSD-95 inhibitor of claim 1, wherein is selected from the group consisting of L, T, V, and R.

5. The linker comprises one or more PEG units, at least one oxygen atom of one of the PEG units is replaced with a nitrogen atom resulting in NPEG, the CPP is linked to the nitrogen atom of the linker by an amide bond, and the PSD-95 inhibitor has the formula (III): 【Chemistry 2】 wherein: p is an integer from 0 to 10; The PSD-95 inhibitor according to claim 1, wherein q is an integer of 0 to 10.

6. P １ and P ２ The PSD-95 inhibitor according to claim 1, which is composed of the amino acid sequences: KETLV (SEQ ID NO: 2), KETTV (SEQ ID NO: 3), KETVV (SEQ ID NO: 4), KETRV (SEQ ID NO: 5), ISTDV (SEQ ID NO: 6), VETVV (SEQ ID NO: 7).

7. The PSD-95 inhibitor has the formula (VI): 【Chemistry 3】 wherein: X １ is selected from the group consisting of K, V, and I; X ２ is selected from the group consisting of E and S; X ３ is selected from the group consisting of L, T, V, R, and D; X ４ is selected from the group consisting of K, V, and I; X ５ is selected from the group consisting of E and S; X ６ is selected from the group consisting of L, T, V, R, and D; p is an integer from 0 to 10; q is an integer from 0 to 10; Poly-Arg is a poly-L-arginine peptide consisting of 3 to 9 L-arginine residues; The PSD-95 inhibitor according to claim 1.

8. 2. The PSD-95 inhibitor of claim 1, wherein the CPP is RRRRRRRRRR (SEQ ID NO: 12), RRRRRRRRRR (SEQ ID NO: 57), RRRRRRRRR (SEQ ID NO: 58), RRRRRR (SEQ ID NO: 59), RRRRR (SEQ ID NO: 60), RRRR (SEQ ID NO: 61), or RRR.

9. The PSD-95 inhibitor according to any one of claims 5 and 7, wherein p is 2 and q is 2.

10. 2. The PSD-95 inhibitor of claim 1, wherein the N-terminus of the CPP is acetylated or the N-terminus of the CPP is conjugated to a chloroalkane tag (CA) having the structure: 【Chemistry 4】

11. The PSD-95 inhibitor has the formula (XXVIII): 【Chemistry 5】 wherein P １ , P ２ and CPP is as follows: 【change】 the N-terminus of the CPP is unsubstituted or acetylated; or 2. The PSD-95 inhibitor of claim 1, wherein the PSD-95 inhibitor has the general structure of formula (XXVIII), P 1 and P 2 consist of the amino acid sequence IETDV (SEQ ID NO:1), and the CPP is selected from the group consisting of rqikiwfqnrrmkwkk (SEQ ID NO:15, D-Pen), LLIILRRRRIRKQAHAHSK (SEQ ID NO:18, L-pVEC), PLIYLRLLRGQF (SEQ ID NO:19, L-TP2), |(Dap)KAPETALD| (SEQ ID NO:21, MiniAp4), and |Ff(Nal2)RrRrQ|GABA-K (SEQ ID NO:22, CPP12).

12. The PSD-95 inhibitor according to claim 1, wherein the PSD-95 inhibitor is selected from the group consisting of the following formulas (X) to (XXIII): 【Chemistry 6】 【change】

13. A pharmaceutical composition comprising the PSD-95 inhibitor described in claim 1.

14. 2. A medicament comprising a compound of a PSD-95 inhibitor according to claim 1 for use in treating, preventing, reducing and / or delaying the onset of an excitotoxicity-related disorder, such as stroke selected from acute ischemic stroke and subarachnoid hemorrhage.

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