Conjugates for delivery across the blood-brain barrier

Abstract

The present invention provides compounds that can be conjugated to biological macromolecules and conjugated biological macromolecules to enable delivery of the biological macromolecule across the blood brain barrier. The present invention also provides methods of delivering a biological macromolecule to a target within the blood brain barrier and methods of using a biological macromolecule to treat a patient having a nervous system disease.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application 63 / 594,143, filed October 30, 2023, the entire contents of which are incorporated herein by reference.

[0003] sequence list

[0004] This application contains an electronic sequence list. The contents of the electronic sequence list are as follows: Name: NOVEL CONJUGATES ANDUSES THEREOF 4888-9300-7090.1.xml; Size: 4,140 bytes; Creation date: October 22, 2024, incorporated herein by reference in its entirety. Invention Field

[0005] This disclosure relates to compounds that can be conjugated to biological macromolecules and conjugated biological macromolecules for delivery of said biological macromolecules across the blood-brain barrier. Background of the Invention

[0007] Biomolecules such as proteins and nucleic acids have been applied in various therapeutic areas. Oligonucleotides have become an important class of therapeutic agents, used to treat liver diseases, metabolic diseases, and rare diseases, and more than 20 RNA-based therapies have been approved by the FDA. However, the presence of the blood-brain barrier (BBB) ​​makes the efficient and safe delivery of these biomolecules to brain tissue a significant challenge. The difficulty in reaching the brain severely hinders their application in treating central nervous system diseases.

[0008] Researchers have explored various methods to enable therapeutic drugs (such as oligonucleotides) to cross the blood-brain barrier, including lipid conjugation, peptide / protein modification, adeno-associated viruses, and synthetic biomaterials. For example, cholesterol or α-tocopherol conjugation can facilitate the intravenous transport of DNA / RNA heteroduplex oligonucleotides to brain tissue. In mouse and non-human primate models, antisense oligonucleotides (ASOs) bound to transferrin can cross the blood-brain barrier (BBB) ​​via human transferrin receptor 1 (TfR1). A bivalent siRNA structure that effectively silences the huntingtin gene in animals via cerebrospinal fluid injection has been developed. Engineered AAV capsids can achieve transgenic expression in the central nervous system of rhesus monkeys after systemic administration. Fucoidan-based nanoparticles can bind to caveolin-1 and enter the brain via transcytosis. Nb62 is an anti-mouse transferrin receptor VHH that can deliver neuropeptides to the mouse brain. Previous studies have shown that various active receptor-mediated transcytosis can promote crossing the blood-brain barrier (BBB) ​​through systemic administration. Examples include transferrin receptors, low-density lipoprotein receptors, and insulin receptors located on the luminal membrane of the blood-brain barrier. These receptors can initiate endocytosis and form transcytotic vesicles that are transported into the brain. Conjugation of palmitoyl-modified siRNAs via intrathecal or intraventricular injection has been found to effectively silence genes in the central nervous system (CNS). An Fc fragment that binds to human transferrin receptor 1 (TfR1) has been developed, capable of crossing the blood-brain barrier and delivering antisense oligonucleotides (ASOs) in animal models. Recently, Tofersen, targeting the superoxide dismutase 1 (SOD1) gene, was approved by the FDA for intrathecal injection to treat amyotrophic lateral sclerosis (ALS). However, these administration methods are generally associated with increased risks of adverse events (e.g., infection) and invasive procedures.

[0009] Furthermore, previous studies have reported that many other small molecule drugs can cross the blood-brain barrier (BBB) ​​via multiple pathways. For example, tryptophan derivatives can cross the BBB via serotonin reuptake transporters. Cinnamic acid derivatives can penetrate the brain and interact with the β-sheet structure of β-amyloid protein. MK-0752 reduces Aβ production after crossing the BBB. SR-57227 can bind to 5-HT3 receptors, thereby transporting the molecule to the brain. However, new methods for delivering therapeutic drugs across the BBB are still needed. Therefore, there is an urgent need to develop new technologies that enable systemic administration to overcome the BBB and enhance the delivery of biomolecular therapeutics to the central nervous system (CNS). Invention Overview

[0011] On the one hand, conjugates of formula (I) are provided:

[0012]

[0013] (I)

[0014] in:

[0015] M represents the transshipment section, which is selected independently in each case;

[0016] W represents a biological macromolecule or small molecule drug;

[0017] R 1 For hydrogen or ;

[0018] R 2 In each case, it is independently selected from (C1-C) 20 )alkyl or (C2-C 20 ) alkyl residues, wherein one to six methylene groups are replaced by -O-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-;

[0019] m is independently selected from integers 1 to 9 in each case; and

[0020] L p A linker base that includes one or more of the following:

[0021] (a)-C(=O)(CH2) q NH-, where q is an integer from 1 to 6;

[0022] (b) Polyamides containing 1 to 5 ω-amino acids;

[0023] (c) Triazole

[0024] (d) 3-Thiopyrrolidine-2,5-dione

[0025] (e) disulfide bonds or

[0026] (f) C2 to C 20 oxa-alkylene;

[0027] [CONH] indicates that the carboxyl terminus and amino terminus can be located at any connection point.

[0028] On the one hand, conjugates of formula (Ia) are provided:

[0029]

[0030] (Ia)

[0031] in:

[0032] M represents the transshipment section, which is selected independently in each case;

[0033] W represents a biological macromolecule or small molecule drug;

[0034] R 1 For hydrogen or ;

[0035] R 2 In each case, it is independently selected from (C1-C) 20 )alkyl or (C2-C 20 ) alkyl residues, wherein one to six methylene groups are replaced by -O-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-;

[0036] m is independently selected from integers 1 to 9 in each case; and

[0037] n is an integer selected from 1 to 15; and

[0038] [CONH] indicates that the carboxyl terminus and amino terminus can be located at any connection point.

[0039] On the one hand, compounds of formula (II) are provided:

[0040]

[0041] (II)

[0042] in:

[0043] M represents the transshipment section, which is selected independently in each case;

[0044] R 1 For hydrogen or ;

[0045] R 2 In each case, it is independently selected from (C1-C) 20 )alkyl or (C2-C 20 ) alkyl residues, wherein one to six methylene groups are replaced by -O-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-;

[0046] m is independently selected from integers 1 to 9 in each case; and

[0047] n is an integer selected from 1 to 15; and

[0048] In formula (II) Represents a bond with hydrogen, or with a biological macromolecule or small molecule pharmaceutical agent; and

[0049] [CONH] indicates that the carboxyl terminus and amino terminus can be located at any connection point.

[0050] On the one hand, compounds of formula (IIa) or formula (IIb) are provided:

[0051]

[0052] (IIa)

[0053]

[0054] (IIb)

[0055] in:

[0056] M represents the transshipment section, which is selected independently in each case;

[0057] R 1 For hydrogen or ;

[0058] R 2 In each case, it is independently selected from (C1-C) 20 )alkyl or (C2-C 20 ) alkyl residues, wherein one to six methylene groups are replaced by -O-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-;

[0059] m is independently selected from integers 1 to 9 in each case; and

[0060] n is an integer selected from 1 to 15;

[0061] In formula (IIa) Represents a bond with a hydroxyl group, or with a biomolecule or small molecule pharmaceutical agent; and

[0062] In equation (IIb) Represents a bond with hydrogen, or with biological macromolecules or small molecule pharmaceuticals.

[0063] On the one hand, compounds of formula (IIIa) are provided:

[0064]

[0065] (IIIa)

[0066] in:

[0067] M represents the transshipment section, which is selected independently in each case;

[0068] R 1 For hydrogen or ;

[0069] R 2 In each case, it is independently selected from (C1-C) 20 )alkyl or (C2-C 20) alkyl residues, wherein one to six methylene groups are replaced by -O-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-;

[0070] m is independently selected from integers 1 to 9 in each case; and

[0071] Q is a linker containing one or more of the following groups:

[0072] (a) An acylalkyl group with 1 to 10 carbon atoms;

[0073] (b) Polyamides containing 1 to 5 ω-amino acids;

[0074] (c) alkylene groups with 1 to 10 carbon atoms; or

[0075] (d) Alkyl groups with 1 to 10 carbon atoms;

[0076] R 10 Selected from:

[0077] (a)N3;

[0078] (b)-C≡CH;

[0079] (c) ;

[0080] (d)-SH;

[0081] (e) ;

[0082] (f)-NH2; and

[0083] (g)-COOH.

[0084] On the one hand, pharmaceutical formulations are provided that comprise a conjugate of formula (I) or formula (Ia) or a compound of formula (IIa), formula (IIb) or formula (IIIa), and a pharmaceutically acceptable carrier.

[0085] On the one hand, a method for delivering a biomacromolecule to a target within the blood-brain barrier is provided, the method comprising providing the target with a conjugate of formula (I) or formula (Ia), wherein the target may be in vivo or in vitro.

[0086] On the one hand, a method for delivering a biomacromolecule to a target within the blood-brain barrier is provided, the method comprising conjugating the biomacromolecule to a compound of formula (II), (IIa), (IIb) or (IIIa) to form a conjugated biomacromolecule, and providing the conjugated biomacromolecule to the target, wherein the target may be in vivo or in vitro.

[0087] On the one hand, a method for treating a subject with a neurological disorder is provided, the method comprising administering to the subject a conjugate of formula (I) or formula (Ia), the conjugate comprising a biomolecule, wherein the biomolecule is a medicament for treating the neurological disorder, and wherein the conjugate is capable of crossing the blood-brain barrier or an in vitro model thereof.

[0088] On the one hand, a method for treating a subject with a neurological disorder is provided, the method comprising conjugating a biomacromolecule, which is a medicament for treating the neurological disorder, with a compound of formula (II), (IIa), (IIb) or (IIIa) to form a conjugated biomacromolecule, and administering the conjugated biomacromolecule to the subject, wherein the conjugated biomacromolecule is capable of crossing the blood-brain barrier or an in vitro model thereof.

[0089] These and other objects, features and advantages of the present invention will become apparent from the following detailed description of various aspects of the invention taken in conjunction with the accompanying drawings. Attached Figure Description

[0090] Figure 1A , Figure 1B and Figure 1C An assessment of the brain-penetrating ability of the blood-brain barrier-penetrating conjugate (BCC) carrying Cy5 was demonstrated. Figure 1A Representative images of mouse brains treated with intravenous BCC-Cy5 are shown. Figure 1B The fluorescence intensity of mice treated with intravenous BCC-Cy5 is shown. Fluorescence intensity was normalized with reference to the free Cy5 group (n=3). Data are expressed as mean ± standard deviation (SD). Statistical significance was determined by one-way ANOVA followed by Dunnett's multiple comparison test. Figure 1C The fluorescence intensity of mice treated with intravenous BCC-Cy5 is shown. Fluorescence intensity is a percentage of the injected dose per gram of brain tissue (%ID / g) (n = 3 mice).

[0091] Figure 2A and Figure 2B The synthetic routes for BCC10-Oligo and BCC10-mCherry are shown respectively. Figure 2C The structure of BCC10-ASO is shown.

[0092] Figure 3A The fluorescence intensity of mice treated with intravenous DNA-Cy5, Chol-DNA-Cy5, and BCC-DNA-Cy5 is shown. Fluorescence intensity was normalized to a reference level of the free DNA-Cy5 group (n=3). Figure 3BThe results of FACS analysis of Cy5 signaling in different brain cells are shown. Data are expressed as mean ± standard deviation (SD). Statistical significance was determined by one-way ANOVA followed by Dunnett's multiple comparison test. Figure 3C and Figure 3D The distribution of BCC10-Oligo at two different concentrations in different cell types of brain tissue was shown by flow cytometry analysis.

[0093] Figure 4A , Figure 4B and Figure 4C This indicates that BCC10-ASO Malat1 It can reduce the level of Malat1 mRNA in multiple brain regions and cell types in mice.

[0094] Figure 5A and Figure 5B SOD1 was displayed G93A ALS mice were treated with BCC10-ASO Sod1 After treatment, the level of Sod1 mRNA in their brain ( Figure 5A ) or SOD1 protein concentration ( Figure 5B Quantitative analysis of ).

[0095] Figure 6 shows the results of treatment with BCC10-Cy5 in an in vitro transwell model to mimic the blood-brain barrier (BBB).

[0096] Figure 7 The results show the results of FACS analysis comparing the expression levels of mCherry in different brain cells after treatment with PBS, mCherry, and BCC10-mCherry.

[0097] Figure 8 The results show the results of flow cytometry analysis comparing the expression levels of mCherry in different brain cells after treatment with PBS, mCherry, and BCC10-mCherry.

[0098] Figure 9 The results show the results of flow cytometry analysis comparing the expression levels of TdTomato in different brain cells after treatment with PBS, mCherry, and BCC10-mCherry. Invention Details

[0100] We have designed a modular platform that enhances the delivery of intravenously administered biopharmaceutical therapeutics across the blood-brain barrier (BBB) ​​into the central nervous system (CNS) via multiple transcytosis pathways. To achieve this, a novel platform has been conceived, constructed by covalently conjugating specific BBB-crossing small-molecule agents to biopharmaceuticals. Inspired by their BBB-penetration potential, we designed and synthesized four classes of BBB-penetrating conjugates derived from small molecules (cinnamic acid derivatives, tryptophan derivatives, MK-0752, and SR57227) to promote BBB penetration and deliver the carrier to the brain via systemic administration. Without adhering to any single theory, it is assumed that these BBB-penetrating conjugates (BCCs) can facilitate the crossing of biopharmaceuticals across the BBB and modulate multiple types of brain cells.

[0101] In some embodiments, the conjugate is a compound of formula (I):

[0102]

[0103] (I)

[0104] in:

[0105] M represents the transshipment section, which is selected independently in each case;

[0106] W represents a biological macromolecule or small molecule drug;

[0107] R 1 For hydrogen or ;

[0108] R 2 In each case, it is independently selected from (C1-C) 20 )alkyl or (C2-C 20 ) alkyl residues, wherein one to six methylene groups are replaced by -O-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-;

[0109] m is independently selected from integers 1 to 9 in each case; and

[0110] L p A linker base that includes one or more of the following:

[0111] (a)-C(=O)(CH2) q NH-, where q is an integer from 1 to 6;

[0112] (b) Polyamides containing 1 to 5 ω-amino acids;

[0113] (c) Triazole

[0114] (d) 3-Thiopyrrolidine-2,5-dione

[0115] (e) disulfide bond or

[0116] (f) C2 to C 20 oxa-alkylene;

[0117] [CONH] indicates that the carboxyl terminus and amino terminus can be located at any connection point.

[0118] In some embodiments, the conjugate is a compound of formula (Ia):

[0119]

[0120] (Ia)

[0121] in:

[0122] M represents the transshipment section, which is selected independently in each case;

[0123] W represents a biological macromolecule or small molecule drug;

[0124] R 1 For hydrogen or ;

[0125] R 2 In each case, it is independently selected from (C1-C) 20 )alkyl or (C2-C 20 ) alkyl residues, wherein one to six methylene groups are replaced by -O-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-;

[0126] m is independently selected from integers 1 to 9 in each case; and

[0127] n is an integer selected from 1 to 15; and

[0128] [CONH] indicates that the carboxyl terminus and amino terminus can be located at any connection point.

[0129] In some embodiments, the compound is a compound of formula (II):

[0130]

[0131] (II)

[0132] in:

[0133] M represents the transshipment section, which is selected independently in each case;

[0134] R 1 For hydrogen or ;

[0135] R 2 In each case, it is independently selected from (C1-C) 20 )alkyl or (C2-C 20 ) alkyl residues, wherein one to six methylene groups are replaced by -O-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-;

[0136] m is independently selected from integers 1 to 9 in each case; and

[0137] n is an integer selected from 1 to 15; and

[0138] In formula (II) Represents a bond with hydrogen, or with a biological macromolecule or small molecule pharmaceutical agent; and

[0139] [CONH] indicates that the carboxyl terminus and amino terminus can be located at any connection point.

[0140] In some embodiments, the compound is a compound of formula (IIa) or formula (IIb):

[0141]

[0142] (IIa)

[0143]

[0144] (IIb)

[0145] in:

[0146] M represents the transshipment section, which is selected independently in each case;

[0147] R 1 For hydrogen or ;

[0148] R 2 In each case, it is independently selected from (C1-C) 20 )alkyl or (C2-C 20 ) alkyl residues, wherein one to six methylene groups are replaced by -O-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-;

[0149] m is independently selected from integers 1 to 9 in each case; and

[0150] n is an integer selected from 1 to 15;

[0151] In formula (IIa) Represents a bond with a hydroxyl group, or with a biomolecule or small molecule pharmaceutical agent; and

[0152] In equation (IIb) Represents a bond with hydrogen, or with biological macromolecules or small molecule pharmaceuticals.

[0153] In some embodiments, the compound is a compound of formula (IIIa):

[0154]

[0155] (IIIa)

[0156] in:

[0157] M represents the transshipment section, which is selected independently in each case;

[0158] R 1 For hydrogen or ;

[0159] R 2 In each case, it is independently selected from (C1-C) 20 )alkyl or (C2-C 20 ) alkyl residues, wherein one to six methylene groups are replaced by -O-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-;

[0160] m is independently selected from integers 1 to 9 in each case; and

[0161] Q is a linker containing one or more of the following groups:

[0162] (a) An acylalkyl group with 1 to 10 carbon atoms;

[0163] (b) Polyamides containing 1 to 5 ω-amino acids;

[0164] (c) alkylene groups with 1 to 10 carbon atoms; or

[0165] (d) Alkyl groups with 1 to 10 carbon atoms;

[0166] R 10 Selected from:

[0167] (a)N3;

[0168] (b)-C≡CH;

[0169] (c) ;

[0170] (d)-SH;

[0171] (e) ;

[0172] (f)-NH2; and

[0173] (a)-COOH.

[0174] Throughout this specification, terms and substituents retain their definitions. Substituents (e.g., R...) n It is typically defined at the time of introduction and is maintained throughout the specification and all independent claims.

[0175] In some embodiments, M is the transport moiety, chosen independently in each case. In some embodiments, M is cinnamic acid. In other embodiments, M is a cinnamic acid derivative. In some embodiments, M is tryptamine. In other embodiments, M is a tryptamine derivative. In some embodiments, M is MK-0752. In other embodiments, M is SR-57227. Each M is chosen independently, so that in the same conjugate, all instances of M can be the same, or in a conjugate, each instance of M can be different, or in a conjugate, two instances of M can be the same while a third instance of M can be different.

[0176] In some embodiments, W is a biological macromolecule. In other embodiments, W is a small molecule pharmaceutical agent. In some embodiments, W is a protein. In other embodiments, W is a peptide. In other embodiments, W is a nucleic acid. In some embodiments, W is an oligonucleotide. In some embodiments, W is a dye. In other embodiments, W is a fluorescent dye. In some embodiments, W is a cyan dye. In other embodiments, W is cyanin 5 (also known as "Cy5"). In some embodiments, W is a fluorescent protein. In some embodiments, W is mCherry.

[0177] In some implementation schemes, R 2 In each case, it is independently selected from (C1-C) 20 )alkyl or (C2-C 20 ) alkyl residues, wherein one to six methylene groups are replaced by -O-, -C(=O)O-, -OC(=O)-, -C(=O)NH-, or -NHC(=O)-. In other embodiments, R 2 Selected from: -CH2CH2OCH2CH2OCH2CH2-, -(CH2)6-, -(CH2)8-, -(CH2CH2O)3CH2CH2-, -(CH2)4-, -CH2CH2OCH2CH2-, -(CH2)3C(=O)NH(CH2)3-, -(CH2)3NHC(=O)(CH2)3-, -(CH2)3C(=O)O(CH2)3- and -(CH2)3OC(=O)(CH2)3-. In other embodiments, R 2 For (C2-C) 20)Oxyalkyl. In some embodiments, R 2 It is -CH2CH2OCH2CH2OCH2CH2-. In some implementations, R 2 For (C1-C 20 )alkyl. In other embodiments, R 2 It is a (C6) alkyl group. In other embodiments, R 2 It is a (C5) alkyl group. In other embodiments, R 2 It is a (C7) alkyl group. In other embodiments, R 2 It is a (C4) alkyl group. In other embodiments, R 2 It is a (C8) alkyl group. To illustrate this more clearly, R... 2 All instances can be the same part (e.g., as a non-restricted instance, R). 2 All instances can be -CH2CH2OCH2CH2OCH2CH2-, or all can be (C6) alkyl, or all can be (C8) alkyl, etc., or R 2 An instance can be different from R 2 One or more other instances (e.g., as a non-restricted instance, R) 2 One example could be -CH2CH2OCH2CH2OCH2CH2-, one example could be (C6) alkyl, and a third example (if present) could be (C4) alkyl.

[0178] In some implementations, m is independently selected from an integer from 1 to 9 in each case. In some implementations, m is 1. In some implementations, m is 2. In some implementations, m is 3. In some implementations, m is 4. In some implementations, m is 5. In some implementations, m is 6. In some implementations, m is 7. In some implementations, m is 8. In some implementations, m is 9. In some implementations, m is independently selected from 1, 2, and 3 in each case. In some implementations, m is independently selected from 1 and 2 in each case. In some implementations, m is independently selected from 1, 2, 3, or 4 in each case. To illustrate more clearly, all instances of m can have the same value (e.g., as a non-limiting instance, all instances of m can be 1, or can be 2, or can be 3, etc.), or one instance of m can be different from one or more other instances of m (e.g., as a non-limiting instance, one instance of m can be 1, one instance can be 2, and a third instance (if present) can be 1).

[0179] In some implementations, n is selected from integers 1 to 15. In some implementations, n is 1. In some implementations, n is 2. In some implementations, n is 3. In some implementations, n is 4. In some implementations, n is 5. In some implementations, n is 6. In some implementations, n is 7. In some implementations, n is 8. In some implementations, n is 9. In some implementations, n is 10. In some implementations, n is 11. In some implementations, n is 12. In some implementations, n is 13. In some implementations, n is 14. In some implementations, n is 15. In some implementations, n is selected from 3, 4, 5, 6, and 7. In some implementations, n is selected from 1, 2, 3, 4, 5, and 6. In some implementations, n is selected from 2, 3, 4, and 5. In some implementations, n is selected from 2, 3, and 4. In some implementations, n is selected from 2 and 3. In some implementations, n is selected from 3 and 4.

[0180] In some implementations of formula (IIa), Represents the bond with a hydroxyl group. In some embodiments of formula (IIa), Represents a bond with a biomolecule or small molecule pharmaceutical agent. In some embodiments of formula (IIb), Represents a bond with hydrogen. In some embodiments of formula (IIb), It represents the bond with biological macromolecules or small molecule drugs.

[0181] In some implementation schemes, R 1 It is hydrogen. In other embodiments, R 1 for M, m and R 2 As defined above.

[0182] In some implementation schemes, R 1 for And R 2 It is -CH2CH2OCH2CH2OCH2CH2-. In other embodiments, R 1 for And m is 1. In other implementations, R 1 for R 2 It is -CH2CH2OCH2CH2OCH2CH2-, and m is 1.

[0183] As shown in formula (I), the biomacromolecule or small molecule drug W is linked by the linker L p Connected to R 1 On the nitrogen atom attached to the carbon atom. In some implementations, L pA linker containing one or more of the following groups: -C(=O)(CH2) q NH-, where q is an integer from 1 to 6; polyamide containing 1 to 5 ω-amino acids; triazole; 3-thiopyrrolidine-2,5-dione; disulfide bond; or C2 to C 20 Oxyalkane. In other embodiments, L p It is a linker comprising 1 to 4 ω-amino acids and 8,9-dihydro-3H-dibenzo[b,f][1,2,3]triazolo[4,5-d]azacyclooctatetraene. In some embodiments, L p It is a linker comprising an acylalkylamine and 8,9-dihydro-3H-dibenzo[b,f][1,2,3]triazolo[4,5-d]azacyclooctatetraene. In other embodiments, L p It is a linker comprising 1 to 4 ω-amino acids and a 3-thiopyrrolidine-2,5-dione. In some embodiments, L p It is a linker containing an acylalkylamine and a 3-thiopyrrolidine-2,5-dione.

[0184] In some implementations, q is 1, or q is 2, or q is 3, or q is 4, or q is 5, or q is 6.

[0185] Common biological macromolecules or small molecule drugs include proteins, peptides, nucleic acids, and oligonucleotides.

[0186] Cyanide 5 is a typical small molecule reagent. Cyanide 5 dyes are a class of commercially available fluorescent molecules recognized in the art, which are bisindole compounds linked by conjugated olefin chains. The indoles may be substituted, for example, with sulfonic acids. One indole nitrogen is quaternized, and the other indole nitrogen (which may or may not be quaternized) is typically alkylated with a hexanoic acid side chain, thereby providing a linking site. All of these molecules are referred to as cyanide 5 dyes.

[0187] mCherry is a fluorescent protein derived from the mushroom coral (Discosoma sp.) and consists of 236 amino acids. Its structure and function were initially published by Shaner et al. in 2004 (Nature Biotechnology, 22(12), 1567-1572.(2004). doi: 10.1038 / nbt1037), and subsequently further reported by Shu et al. (Shu et al., Biochemistry.2006 Aug 15; 45(32): 9639-47. doi: 10.1021 / bi060773l.). mCherry is widely known and used in this field. In addition to its terminal residues, the protein contains a large number of lysine and glutamic acid residues, which can be linked by amide bonds or via sulfur atoms using the Traut reagent. (The amino group in a protein reacts with 2-iminothione to form mercaptobutamine, and then the mercapto group reacts with maleimide to form 3-thiopyrrolidine-2,5-dione.) Other proteins and peptides may also contain cysteine ​​residues, which can be directly linked by the sulfur atom of their cysteine ​​residues.

[0188] Besides these two linking methods (formation of amide bonds and formation of thiopyrrolidone diones), W can also be linked via click chemistry. Click chemistry has been well-known in the field for over two decades and is the basis of the 2022 Nobel Prize in Chemistry. Its basic principle is the reaction between an azide group (N3) and an alkynyl group (-C≡CH-) to generate a triazole. An alkynyl bond can be introduced into a cyclic structure, such as dibenzocyclooctyne (DBCO), which introduces sufficient ring strain to allow the click reaction to proceed without a copper catalyst. The product is still a triazole, in this case 8,9-dihydro-3H-dibenzo[b,f][1,2,3]triazolo[4,5-d]azacyclooctatetraene:

[0189] .

[0190] As those skilled in the art will understand, the connection base L p Therefore, it can be assembled in two ways. It can act as a fully assembled linker attached to both ends; in this case, the penultimate step is the reaction of compound (IIIa) with W.

[0191]

[0192] (IIIa)

[0193] Among them, R 10 Reaction with W or derivatized W to form amide bonds (when R 10When -NH2 or -COOH), thiopyrrolidone (when R) 10 (when it is -SH or maleimide) or triazine (when R 10 When W is an azide or alkynyl group, and W has been derivatized to a corresponding (converse) alkynyl or azide group, the resulting amide, thiopyrrolidone, or amide becomes L. p Part of it. The derivation methods for W are as described above and are well known in the art. Alternatively, L can be... p Part of it is connected to R 1 On adjacent nitrogen atoms, and with L p Another part is connected to W, and then L is connected to W. p These two parts are coupled to form a complete L p Regardless of the method used, the intermediate is always a compound of formula IIIa, wherein R 10 Selected from: -N3; ​​-C≡CH; ;-SH; -NH2; and -COOH.

[0194] From the BCC synthesis described below, those skilled in the art will understand that group M can be attached to the 2-amino-2-(hydroxymethyl)propane-1,3-diol (Tris) core in a manner similar to that described above. In the synthesis described below, standard peptide coupling reagents known in the art were used to attach M to Tris via an amide bond formed between a commercially available amino-terminal structural unit and a carboxyl-terminal structural unit. Similarly, various R... 2 Units can be assembled from commercially available diamines, dicarboxylic acids, and ω-amino acids using standard peptide coupling operations [see...]. Principles of Peptide Synthesis , M. Bodanszky, Springer Science & Business Media, 2012 ISBN3642967639, 9783642967634].

[0195] It should be understood that the examples of click chemistry disclosed herein are not exhaustive, and those skilled in the art can use other variations of click chemistry.

[0196] The conjugates disclosed herein can be used to treat subjects with neurological disorders. These conjugates are capable of crossing the blood-brain barrier to deliver biomolecules for the treatment of neurological disorders. Therefore, these conjugates can be used to treat a range of central nervous system disorders, including, but not limited to, the following: amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease, Huntington's disease, ischemic stroke, multiple sclerosis, schizophrenia, epilepsy, and addictions (including drug addiction) and brain or spinal cancer.

[0197] definition

[0198] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. A complete list of abbreviations used by organic chemists (i.e., those of ordinary skill in the art) is published in Journal of Organic Chemistry The list is included in the first issue of each volume. This list is typically presented in a table titled "List of Standard Abbreviations" and is incorporated herein by reference. If multiple definitions exist for a term cited herein, the definition in this section shall prevail unless otherwise stated.

[0199] As used herein, the terms “comprising” and “including” or their grammatical variations shall be regarded as specifying the stated feature, integer, step, or component, but do not preclude the addition of one or more other features, integers, steps, components, or combinations thereof. The term encompasses both “consisting of” and “substantially consisting of”.

[0200] The phrase “consistently of…” or its grammatical variations as used herein shall be regarded as specifying the stated feature, integer, step or component, but do not preclude the addition of one or more other features, integers, steps, components or combinations thereof, provided that such additional features, integers, steps, components or combinations thereof do not materially alter the fundamental and novel characteristics of the claimed composition or method.

[0201] The symbol "[CONH]" indicates that the carboxyl terminus and amino terminus can be located at either connection point, i.e., M-[CONH]-R 2 It can be represented as M-[CONH]-R 2 Or M-[HNOC]-R 2 In some cases, -C=O- or -NH in -[CONH]- can be part of the M part or part of the W (biological macromolecule or small molecule drug).

[0202] Alkyl groups are a subset of hydrocarbons. Unless otherwise stated, alkyl (or alkylene) is intended to include straight-chain or branched saturated hydrocarbon structures and combinations thereof. In some embodiments, alkyl refers to an alkyl group having 1 to 20 carbon atoms, or 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, etc. The name “(C…” in this document…” x Alkyl group (where x is an integer) is equivalent to -(CH2). x For example, "(C6)alkyl" has the same meaning as -(CH2)6-.

[0203] “Oxaalkyl” or “oxaalkane” or “oxaalkylene” refers to an alkyl residue in which one or more carbon atoms (and the hydrogen atoms to which they are attached) have been replaced by an oxygen atom. Examples include methoxypropoxy, 3,6,9-trioxadecyl, etc. The term oxaalkyl is understood in the same way as it is understood in the art [see Naming and Indexing of Chemical Substances for Chemical Abstracts, 196, but not limited to the limitations of clause 127(a)], that is, it refers to a compound in which oxygen is bonded to its adjacent atoms by a single bond (forming an ether bond); it does not refer to double oxygen as is present in a carbonyl group.

[0204] MK-0752 has been designated as CAS number 471905-41-6, and its structure is as follows: .

[0205] SR-57227 has been designated as CAS number 77145-61-0, and its structure is as follows: .

[0206] Cinnamic acid has the following structure Cinnamic acid derivatives are well known to those skilled in the art, and non-limiting examples of such derivatives include hydroxycinnamic acid, caffeic acid, and ferulic acid. Other non-limiting examples can be found in Ruwizhi N, Aderibigbe BA. Int J Mol Sci. 2020 Aug 9; 21(16):5712. doi: 10.3390 / ijms21165712. PMID: 32784935; PMCID: PMC7460980.

[0207] Tryptamine has the following structure Tryptophan derivatives are well known to those skilled in the art, and non-limiting examples of such derivatives include serotonin, halogenated or alkoxylated tryptophan, etc. Other non-limiting examples can be found in Kousara et al., J Pharmacovigil 2017, 5:5 DOI: 10.4172 / 2329-6887.1000239.

[0208] "Derivatives" also include cinnamic acid or tryptamine that are substituted or modified by covalent linkage, such as by alkylation, glycosylation, acetylation, phosphorylation, and other modifications known in the art.

[0209] As used herein, the terms "subject" and "patient" are used interchangeably and include humans and other animals, particularly mammals. Therefore, the methods described are applicable to both human treatment and veterinary applications. Examples of "subjects" include, but are not limited to, humans, rats, mice, guinea pigs, monkeys, pigs, goats, cattle, horses, dogs, cats, birds, and poultry. In some embodiments, the subject is a mammal, such as a primate. In some embodiments, the subject is a human. In one embodiment, the subject is an infant, adolescent, or adult.

[0210] Unless the context otherwise requires, the term “treatment” refers to therapeutic procedures and preventative measures taken to prevent recurrence, with the aim of inhibiting or slowing (alleviating) adverse physiological changes or disorders, such as the progression or spread of cancer or viral infection.

[0211] For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to: symptom relief, reduction in disease severity, stable disease status (i.e., no worsening), delayed or slowed disease progression, improvement or reduction of disease status, and (partial or complete) remission (whether detectable or undetectable). "Treatment" may also refer to extended survival compared to expected survival without treatment. Individuals in need of treatment include those who already have the condition or disorder, and those who are susceptible to the condition or disorder.

[0212] Treatment may include administering the conjugates or compounds described herein to patients diagnosed with a disease, or to patients without active symptoms. Conversely, treatment may include administering the compositions or formulations to patients at risk of developing a specific disease, or to patients who report physiological symptoms of one or more diseases (even if the disease has not yet been diagnosed).

[0213] Therapeuticly effective amounts of the conjugates and compounds described herein are administered to subjects. Therapeuticly effective amounts of the conjugates and compounds described herein generally refer to amounts sufficient to achieve the desired effect and may vary depending on the nature and severity of the disease condition and the potency of the compound. It should be understood that concentrations used for prophylaxis may differ from concentrations used for treating active disease. A therapeutic benefit is achieved when one or more physiological symptoms associated with the underlying disorder are improved, resulting in an observed improvement in the patient's condition, although the patient may still have the underlying disorder. It should also be understood that the therapeutically effective amounts may involve biomolecules that act as agents for treating neurological disorders.

[0214] Therefore, therapeutic effects can be a reduction in the severity of symptoms associated with the disorder, and / or a (partial or complete) suppression of the disorder's progression, or an improvement, cure, prevention, or elimination of the disorder or its side effects. The amount required to elicit a therapeutic response can be determined based on the subject's age, health status, body size, and sex. The optimal amount can also be determined based on monitoring the subject's therapeutic response. The term "treatment" can include effectively suppressing, inhibiting, or terminating symptoms, thereby preventing or delaying the onset of the condition, slowing its progression, or improving the symptoms of the condition.

[0215] As used herein, and as those skilled in the art will understand, unless further expressly limited, the expression “compound” or “conjugate” is intended to include a pharmaceutically acceptable salt of that compound or conjugate, provided that a pharmaceutically acceptable salt is appropriate.

[0216] The term "pharmaceutically acceptable salt" refers to a salt prepared from a pharmaceutically acceptable non-toxic acid or base (including inorganic acids and bases, as well as organic acids and bases). When the compounds of the present invention are basic, their salts can be prepared from pharmaceutically acceptable non-toxic acids (including inorganic acids and organic acids). Pharmaceutically acceptable acid addition salts applicable to the compounds of this invention include salts of the following acids: acetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid (benzenesulfonate), benzoic acid, boric acid, butyric acid, camphoric acid, camphorsulfonic acid, carbonic acid, citric acid, ethanedisulfonic acid, ethanesulfonic acid, ethylenediaminetetraacetic acid, formic acid, fumaric acid, glucoheponic acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, hydroxynaphthoic acid, hydroxyethanesulfonic acid, lactic acid, lactobionic acid, lauryl sulfonic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, viscous acid, naphthalenesulfonic acid, nitric acid, oleic acid, dihydroxynaphthoic acid, pantothenic acid, phosphoric acid, neopentanoic acid, polygalacturonic acid, salicylic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, tartaric acid, theoclatic acid, p-toluenesulfonic acid, etc. When the compounds of the present invention contain acidic side chains, pharmaceutically acceptable base addition salts suitable for the compounds of the present invention include, but are not limited to: metal salts made of aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc, or organic salts made of lysine, arginine, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucosamine), and procaine. Other pharmaceutically acceptable salts include (if applicable) non-toxic ammonium cations and carboxylate, sulfonate, and phosphonate anions linked to alkyl groups having 1 to 20 carbon atoms.

[0217] Unless otherwise stated or described, the structures described herein are also intended to include all stereoisomers of the structure (e.g., enantiomers, diastereomers, and cis-trans isomers); for example, R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, single stereochemical isomers of the compounds of the present invention, as well as mixtures of enantiomers, diastereomers, and cis-trans isomers (or conformational isomers), are all within the scope of the present invention. Unless otherwise stated, all tautomers of the compounds of the present invention are within the scope of the present invention. Furthermore, unless otherwise stated, the structures described herein are also intended to include compounds that differ only in one or more isotopically enriched atoms. For example, compounds having the structure of the present invention, but with hydrogen replaced by deuterium or tritium, or carbon replaced by... 13 C- or 14 Compounds enriched with carbon (C-) are all within the scope of this invention. For example, such compounds can be used as analytical tools or probes in bioassays.

[0218] The illustrations of racemic, ambiscalemic, scalemic, or enantiomerically pure compounds used in this paper are taken from a modified version indicated in Maehr J. Chem. Ed. 62, 114-120 (1985): simple lines provide no information about stereochemistry, only indicating connectivity; solid and dashed wedges are used to represent the absolute configuration of chiral atoms; thick solid and dashed lines are geometric descriptors, indicating the relative configuration shown, but not necessarily the racemic nature; and wedge outlines and dotted or dashed lines represent enantiomerically pure compounds with indeterminate absolute configurations. For example, see the illustration below.

[0219]

[0220] It indicates two trans: one or both of the trans enantiomers, in any ratio, from pure enantiomers to racemic mixtures.

[0221]

[0222] As shown in the image below:

[0223]

[0224] This represents a single enantiomer whose absolute stereochemical configuration is unknown; that is, it can be either of the two aforementioned structures, as a substantially pure single enantiomer. Finally, as shown in the diagram below:

[0225]

[0226] The term "pure" or "substantially pure" enantiomers are intended to mean that the enantiomer has at least 95% of the indicated configuration and that other enantiomers comprise 5% or less. Similarly, "pure" or "substantially pure" diastereomers are intended to mean that the diastereomer has at least 95% of the indicated relative configuration and that other diastereomers comprise 5% or less. In some embodiments, the purity of the compound is at least 99%.

[0227] In either of these possibilities, the compound can be a single stereoisomer or a mixture. If it is a mixture, the mixture is most commonly a racemic mixture, but not necessarily. A substantially pure single stereoisomer of a bioactive compound (such as those described herein) is generally advantageous over its racemic mixture.

[0228] Enantiomer purity means an enantiomer excess of more than 80%, preferably more than 90%. For the purposes of this disclosure, a stereoisomer that is "pure" or "substantially pure" is intended to mean that the stereoisomer has at least 95% of the indicated configuration and other stereoisomers account for 5% or less; or has at least 97% of the indicated configuration and other stereoisomers account for 3% or less; or has at least 99% of the indicated configuration and other stereoisomers account for 1% or less.

[0229] During the examination process, it may be discovered that certain species and genera in this application are not patentable for the inventor. In such cases, the species and genera excluded in the applicant's claims should be considered as a product of the patent examination process and do not reflect the inventor's conception or description of their invention, since the inventor's conception or description of their invention covers all members of that genus that are not yet publicly known.

[0230] While the conjugates and compounds disclosed herein may be administered as raw material chemicals, in some embodiments they are provided as pharmaceutical formulations. According to another aspect, the present invention provides pharmaceutical formulations comprising the conjugates or compounds disclosed herein, and one or more pharmaceutically acceptable carriers. The carrier must be "acceptable," meaning it is compatible with other components in the formulation and harmless to the recipient. In one embodiment, the pharmaceutically acceptable carrier is selected from liquid fillers, solid fillers, diluents, excipients, solvents, and encapsulating materials.

[0231] Implementation Plan

[0232] Various preferred embodiments of the present invention [EMB. 1] to [EMB. 7] are described below:

[0233] [EMB. 1] Conjugate of formula (I):

[0234]

[0235] (I)

[0236] Where: M represents the transport component, which is selected independently in each case; W represents the biomolecule or small molecule agent; R 1 For hydrogen or ;R 2 In each case, it is independently selected from (C1-C) 20 )alkyl or (C2-C 20 ) oxazane; m is independently selected from an integer from 1 to 9 in each case; and n is selected from an integer from 1 to 15; and [CONH] indicates that the carboxyl terminus and amino terminus can be located at either connection point.

[0237] [EMB. 2] [EMB. 1] conjugates, wherein: M is selected from cinnamic acid or a cinnamic acid derivative, tryptamine or a tryptamine derivative, MK-0752 or SR-57227; and / or W is selected from proteins, nucleic acids, blue pigment 5 or mCherry; and / or R 2 In each case, it is independently selected from -CH2CH2OCH2CH2OCH2CH2- or (C6) alkyl; and / or m is 1; and / or n is 5.

[0238] [EMB. 3] [EMB. 1] conjugate, wherein: M is MK-0752; and / or R 2 It is (C6) alkyl; and / or m is 1; and / or n is 5.

[0239] [EMB. 4] Compound of formula (II):

[0240]

[0241] (II)

[0242] Where: M represents the transshipment portion, which is selected independently in each case; R 1 For hydrogen or ;R 2 In each case, it is independently selected from (C1-C) 20 )alkyl or (C2-C 20 ) oxazane; m is independently selected from integers 1 to 9 in each case; and n is selected from integers 1 to 15; and in formula (II) Represents a bond with hydrogen, or with a biomolecule or small molecule drug; and [CONH] indicates that the carboxyl terminus and amino terminus can be located at either connection point.

[0243] [EMB. 5] [EMB. 4] compounds, wherein: M is selected from cinnamic acid or a cinnamic acid derivative, tryptamine or a tryptamine derivative, MK-0752 or SR-57227; and / or R 2 In each case, it is independently selected from -CH2CH2OCH2CH2OCH2CH2- or (C6) alkyl; and / or m is 1; and / or n is 5.

[0244] [EMB. 6] [EMB. 4] compounds, wherein: M is MK-0752; and / or R 2 It is (C6) alkyl; and / or m is 1; and / or n is 5.

[0245] [EMB. 7] A pharmaceutical preparation comprising a conjugate of any one of [EMB. 1], [EMB. 2] or [EMB. 3] or a compound of any one of [EMB. 4], [EMB. 5] or [EMB. 6] and a pharmaceutically acceptable carrier.

[0246] Various preferred embodiments of the present invention [A] to [AU] are described below:

[0247] [Implementation Scheme A] Conjugate of Formula (I):

[0248]

[0249] (I)

[0250] in:

[0251] M represents the transshipment section, which is selected independently in each case;

[0252] W represents a biological macromolecule or small molecule drug;

[0253] R 1 For hydrogen or ;

[0254] R 2 In each case, it is independently selected from (C1-C) 20 )alkyl or divalent (C2-C 20 ) alkyl residues, wherein one to six methylene groups are replaced by -O-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-;

[0255] m is independently selected from integers 1 to 9 in each case; and

[0256] L p A linker base that includes one or more of the following:

[0257] (a)-C(=O)(CH2)q NH-, where q is an integer from 1 to 6;

[0258] (b) Polyamides containing 1 to 5 ω-amino acids;

[0259] (c) Triazole

[0260] (d) 3-Thiopyrrolidine-2,5-dione

[0261] (e) disulfide bond or

[0262] (f) C2 to C 20 Oxyalkane;

[0263] [CONH] indicates that the carboxyl terminus and amino terminus can be located at any connection point.

[0264] [Scheme B] The conjugate of Scheme [A] above, or a conjugate according to other embodiments of the present invention, wherein L p It is a linker containing 1 to 4 ω-amino acids and 8,9-dihydro-3H-dibenzo[b,f][1,2,3]triazolo[4,5-d]azacyclooctatetraene.

[0265] [Scheme C] The conjugate of Scheme [A] above, or a conjugate according to other embodiments of the present invention, wherein L p It is a linker group comprising an acylalkylamine and 8,9-dihydro-3H-dibenzo[b,f][1,2,3]triazolo[4,5-d]azacyclooctatetraene.

[0266] [Scheme D] The conjugate of Scheme [A] above, or a conjugate according to other embodiments of the present invention, wherein L p It is a linker consisting of 1 to 4 ω-amino acids and 3-thiopyrrolidine-2,5-dione.

[0267] [Scheme E] The conjugate of Scheme [A] above, or a conjugate according to other embodiments of the present invention, wherein L p It is a linker containing an acylalkylamine and a 3-thiopyrrolidine-2,5-dione.

[0268] [Scheme F] The conjugate of Scheme [A] above, or a conjugate according to other embodiments of the present invention, wherein the conjugate is a compound of formula (Ia):

[0269]

[0270] (Ia)

[0271] in:

[0272] M represents the transshipment section, which is selected independently in each case;

[0273] W represents a biological macromolecule or small molecule drug;

[0274] R 1 For hydrogen or ;

[0275] R 2 In each case, it is independently selected from (C1-C) 20 )alkyl or (C2-C 20 ) alkyl residues, wherein one to six methylene groups are replaced by -O-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-;

[0276] m is independently selected from integers 1 to 9 in each case; and

[0277] n is an integer selected from 1 to 15; and

[0278] [CONH] indicates that the carboxyl terminus and amino terminus can be located at any connection point.

[0279] [Scheme G] A conjugate of any one of the above embodiments [A] to [F], or a conjugate according to other embodiments of the present invention, wherein W is selected from proteins, peptides, nucleic acids, oligonucleotides or fluorescent dyes.

[0280] [Scheme H] A conjugate of any one of the above embodiments [A] to [F], or a conjugate according to other embodiments of the present invention, wherein W is selected from proteins, peptides, nucleic acids, oligonucleotides, blue pigment 5 or mCherry.

[0281] [Scheme I] A conjugate of any one of the above embodiments [A] to [F], or a conjugate according to other embodiments of the present invention, wherein W is a protein.

[0282] [Scheme J] A conjugate of any one of the above embodiments [A] to [F], or a conjugate according to other embodiments of the present invention, wherein W is a peptide.

[0283] [Scheme K] A conjugate of any one of the above schemes [A] to [F], or a conjugate according to other schemes of the present invention, wherein W is a nucleic acid.

[0284] [Scheme L] A conjugate of any one of the above embodiments [A] to [F], or a conjugate according to other embodiments of the present invention, wherein W is an oligonucleotide.

[0285] [Scheme M] A conjugate of any one of the above embodiments [A] to [F], or a conjugate according to other embodiments of the present invention, wherein W is a fluorescent dye.

[0286] [Scheme N] The conjugate of any one of the above embodiments [A] to [F], or the conjugate according to other embodiments of the present invention, wherein W is cyanin 5.

[0287] [Scheme O] A conjugate of any one of the above schemes [A] to [F], or a conjugate according to other schemes of the present invention, wherein W is mCherry.

[0288] [Implementation Scheme P] Compound of Formula (II):

[0289]

[0290] (II)

[0291] in:

[0292] M represents the transshipment section, which is selected independently in each case;

[0293] R 1 For hydrogen or ;

[0294] R 2 In each case, it is independently selected from (C1-C) 20 )alkyl or (C2-C 20 ) alkyl residues, wherein one to six methylene groups are replaced by -O-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-;

[0295] m is independently selected from integers 1 to 9 in each case; and

[0296] n is an integer selected from 1 to 15; and

[0297] In formula (II) Represents a bond with hydrogen, or with a biological macromolecule or small molecule pharmaceutical agent; and

[0298] [CONH] indicates that the carboxyl terminus and amino terminus can be located at any connection point.

[0299] [Implementation Scheme Q] Compound of Formula (IIa):

[0300]

[0301] (IIa)

[0302] in:

[0303] M represents the transshipment section, which is selected independently in each case;

[0304] R 1 For hydrogen or ;

[0305] R 2 In each case, it is independently selected from (C1-C) 20 )alkyl or (C2-C 20 ) alkyl residues, wherein one to six methylene groups are replaced by -O-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-;

[0306] m is independently selected from integers 1 to 9 in each case; and

[0307] n is an integer selected from 1 to 15;

[0308] In formula (IIa) Represents a bond with a hydroxyl group, or with a biological macromolecule or small molecule drug.

[0309] [Scheme R] The compound of the above-described embodiment [Q], or the compound according to other embodiments of the present invention, wherein the compound of formula (IIa) Represents the bond with the hydroxyl group.

[0310] [Implementation S] Compound of formula (IIb):

[0311]

[0312] (IIb)

[0313] in:

[0314] M represents the transshipment section, which is selected independently in each case;

[0315] R 1 For hydrogen or ;

[0316] R 2 In each case, it is independently selected from (C1-C) 20 )alkyl or (C2-C 20 ) alkyl residues, wherein one to six methylene groups are replaced by -O-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-;

[0317] m is independently selected from integers 1 to 9 in each case; and

[0318] n is an integer selected from 1 to 15; and

[0319] In equation (IIb) Represents a bond with hydrogen, or with biological macromolecules or small molecule pharmaceuticals.

[0320] [Scheme T] The compound of the above-described embodiment [S], or the compound according to other embodiments of the present invention, wherein the compound of formula (IIb) It represents the bond with hydrogen.

[0321] [Scheme U] The compound of the above-described scheme [Q] or scheme [S], or the compound according to other embodiments of the present invention, wherein Represents the bond with biological macromolecules.

[0322] [Scheme V] The compound of the above-described scheme [Q] or scheme [S], or the compound according to other embodiments of the present invention, wherein Represents the bond with small molecule drugs.

[0323] [Scheme W] The conjugate or compound of any one of the above embodiments [F] to [V], or the conjugate or compound according to other embodiments of the present invention, wherein n is selected from 1, 2, 3, 4, 5 and 6.

[0324] [Scheme X] A conjugate or compound of any one of the above embodiments [F] to [V], or a conjugate or compound according to other embodiments of the present invention, wherein n is selected from 3, 4 and 5.

[0325] [Scheme Y] A conjugate or compound of any one of the above embodiments [F] to [V], or a conjugate or compound according to other embodiments of the present invention, wherein n is 3.

[0326] [Implementation Scheme Z] Compound of Formula (IIIa):

[0327]

[0328] (IIIa)

[0329] in:

[0330] M represents the transshipment section, which is selected independently in each case;

[0331] R 1 For hydrogen or ;

[0332] R 2 In each case, it is independently selected from (C1-C) 20 )alkyl or (C2-C 20 ) alkyl residues, wherein one to six methylene groups are replaced by -O-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-;

[0333] m is independently selected from integers 1 to 9 in each case; and

[0334] Q is a linker containing one or more of the following groups: an acylalkyl group with 1 to 10 carbons; a polyamide containing 1 to 5 ω-amino acids; an oxaalkylene group with 1 to 10 carbons; or an alkyl group with 1 to 10 carbons;

[0335] R 10 Selected from N3; -C≡CH; ;-SH; -NH2; and -COOH.

[0336] [Scheme AA] The compound of the above-described embodiment [Z], or the compound of other embodiments according to the present invention, wherein Q is a linker comprising one or more of the following groups: an acylalkyl group of 1 to 10 carbons; a polyamide comprising 1 to 5 ω-amino acids; an oxaalkylene group of 1 to 10 carbons; or an alkyl group of 1 to 10 carbons;

[0337] [Implementation Scheme AB] The conjugate or compound of any one of the above embodiments [A] to [AA], or the conjugate or compound according to other embodiments of the present invention, wherein M is individually selected from cinnamic acid or a cinnamic acid derivative, tryptamine or a tryptamine derivative, MK-0752 or SR-57227 in each case.

[0338] [Scheme AC] The conjugate or compound of any one of the above embodiments [A] to [AA], or the conjugate or compound according to other embodiments of the present invention, wherein M is cinnamic acid or a cinnamic acid derivative in each case.

[0339] [Scheme AD] The conjugate or compound of any one of the above embodiments [A] to [AA], or the conjugate or compound of other embodiments according to the present invention, wherein M is tryptamine or a tryptamine derivative in each case.

[0340] [Implementation Scheme AE] The conjugate or compound of any one of the above embodiments [A] to [AA], or the conjugate or compound according to other embodiments of the present invention, wherein M is MK-0752 in each case.

[0341] [Implementation Scheme AF] The conjugate or compound of any one of the above embodiments [A] to [AA], or the conjugate or compound according to other embodiments of the present invention, wherein M is SR-57227 in each case.

[0342] [Implementation Scheme AG] The conjugate or compound of any one of the above embodiments [A] to [AF], or the conjugate or compound according to other embodiments of the present invention, wherein m is independently selected from 1, 2, 3 or 4 in each case.

[0343] [Scheme AH] The conjugate or compound of any one of the above embodiments [A] to [AF], or the conjugate or compound according to other embodiments of the present invention, wherein in at least one case m is 1.

[0344] [Implementation Scheme AI] The conjugate or compound of any one of the above embodiments [A] to [AF], or the conjugate or compound according to other embodiments of the present invention, wherein m is 1 in all cases.

[0345] [Implementation Scheme AJ] A conjugate or compound of any one of the above embodiments [A] to [AI], or a conjugate or compound according to other embodiments of the present invention, wherein R 1 for .

[0346] [Scheme AK] A conjugate or compound of any one of the above embodiments [A] to [AI], or a conjugate or compound according to other embodiments of the present invention, wherein R 1 It is hydrogen.

[0347] [Scheme AL] A conjugate or compound of any one of the above embodiments [A] to [AK], or a conjugate or compound according to other embodiments of the present invention, wherein R 2 Selected from -CH2CH2OCH2CH2OCH2CH2-, -(CH2)6-, -(CH2)8-, -(CH2CH2O)3CH2CH2-, -(CH2)4-, -CH2CH2OCH2CH2-, -(CH2)3C(=O)NH(CH2)3-, -(CH2)3NHC(=O)(CH2)3-, -(CH2)3C(=O)O(CH2)3- and -(CH2)3OC(=O)(CH2)3-.

[0348] [Scheme AM] A conjugate or compound of any one of the above embodiments [A] to [AK], or a conjugate or compound according to other embodiments of the present invention, wherein R 2 For (C2-C) 20 )O-alkyl.

[0349] [Implementation Scheme AN] A conjugate or compound of any one of the above embodiments [A] to [AK], or a conjugate or compound according to other embodiments of the present invention, wherein R 2It is -CH2CH2OCH2CH2OCH2CH2-.

[0350] [Scheme AO] A conjugate or compound of any one of the above embodiments [A] to [AK], or a conjugate or compound according to other embodiments of the present invention, wherein R 2 For (C1-C 20 )alkyl.

[0351] [Implementation Scheme AP] A conjugate or compound of any one of the above embodiments [A] to [AK], or a conjugate or compound according to other embodiments of the present invention, wherein R 2 It is a (C6) alkyl group.

[0352] [Implementation Scheme AQ] A pharmaceutical preparation comprising a conjugate or compound of any one of the above embodiments [A] to [AP] or a conjugate or compound according to other embodiments of the present invention, and a pharmaceutically acceptable carrier.

[0353] [Implementation Scheme AR] A method for delivering a biomacromolecule to a target within the blood-brain barrier, the method comprising providing the target with a conjugate, compound, or pharmaceutical composition of any one of the above embodiments [A] to [AQ], or a conjugate, compound, or pharmaceutical composition according to other embodiments of the invention, wherein the target may be in vivo or in vitro.

[0354] [Implementation Scheme AS] A method for delivering a biomacromolecule to a target within the blood-brain barrier, the method comprising conjugating the biomacromolecule to a compound of any one of the above embodiments [P] to [AQ], or a compound according to other embodiments of the invention, to form a conjugated biomacromolecule, and providing the conjugated biomacromolecule to the target, wherein the target may be in vivo or in vitro.

[0355] [Implementation Scheme AT] A method for treating a subject with a neurological disorder, the method comprising administering to the subject a conjugate of any one of the above-described embodiments [A] to [O], [W] to [Y], or [AA] to [AS], or a conjugate according to other embodiments of the invention, the conjugate comprising a biomacromolecule, wherein the biomacromolecule is an agent for treating the neurological disorder, and wherein the conjugate is capable of crossing the blood-brain barrier or an in vitro model thereof.

[0356] [Implementation Scheme AU] A method for treating a subject suffering from a neurological disease, the method comprising conjugating a biomacromolecule, which is a medicament for treating the neurological disease, with a compound of any one of the above embodiments [P] to [AQ], or a compound according to other embodiments of the invention, to form a conjugated biomacromolecule, and administering the conjugated biomacromolecule to the subject, wherein the conjugated biomacromolecule is capable of crossing the blood-brain barrier or an in vitro model thereof. Example

[0357] Based on the structure and function of small molecule ligands capable of crossing the blood-brain barrier, four classes of blood-brain barrier crossing conjugates (BCCs) were designed and synthesized, including cinnamic acid derivatives, tryptamine derivatives, MK-0752 derivatives, and SR-57227 derivatives. Specifically, the cinnamic acid derivative can cross the blood-brain barrier and interact with the β-sheet structure of β-amyloid protein (Aβ). The tryptamine derivative, on the other hand, depends on Mg²⁺. + It and adenosine triphosphate (ATP) are transported into the brain via active transport. MK-0752 is a γ-secretase inhibitor that effectively crosses the blood-brain barrier and reduces Aβ production. The drug has been evaluated for safety in clinical trials, showing an overall acceptable safety profile, and most patients tolerated the treatment well. SR-57227 interacts with 5-HT3 receptors, thereby promoting its transport into the brain.

[0358] Nirogacestat and MK-0752 were purchased from MedChemExpress (NJ, USA). All oligonucleotides used in the experiments were purchased from Integrated DNA Technologies (IA, USA). mCherry was obtained from Creative Biomart (NY, USA). TAT-Cre recombinase was obtained from Excellgen (MD, USA). Unless otherwise specified, all other chemicals and solvents were purchased from Thermo Fisher Scientific. Blood-brain barrier-penetrating conjugates were purified by column chromatography (gradient elution using a CombiFlash Rf system with RediSep Gold Resolution silica columns (Teledyne Isco). 1¹H NMR spectroscopy was performed on a Bruker Avance 400 MHz instrument. Mass spectrometry measurements were performed using a 6210 TOF LC / MS (Agilent) and a microFlex LRF MALDI-TOF-MS (Bruker Daltonics). Mouse brain endothelial cell line bEnd.3 (CRL-2299, BCEC) was purchased from ATCC and cultured according to the manufacturer's protocol.

[0359] All animal experiments conducted in this study followed the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of the Icahn School of Medicine at Mount Sinai (IPROTO 202200000134) and complied with local, state, and federal regulations. C57BL / 6 mice, Ai14 mice, and mice expressing the G93A mutant form of human SOD1 were used. G93A Transgenic mice (6-8 weeks old) were purchased from Jackson Laboratories. The number of mice analyzed is provided in the text and / or legend.

[0360] A series of blood-brain barrier-penetrating conjugates coupled with cyanin 5 (Cy5), a fluorescent dye, were synthesized. Each newly synthesized BCC consists of bitennary and triantennary small molecule ligands and is linked to a cyanin 5 (Cy5) fluorescent imaging probe. Two types of spacers were installed: alkane spacers and ethylene glycol spacers. These structures are shown below (BCC1-Cy5 to BCC12-Cy5).

[0361]

[0362]

[0363] BCC Synthesis

[0364] An exemplary synthetic route for the BCC10-Cy5 molecule is shown in Scheme 1 above. In short, the synthesis of compound 2 involves the reaction of tris(hydroxymethyl)aminomethane with tert-butyl acrylate, followed by condensation with Fmoc-GABA, and then deprotection to give compound 4. Compound 4 is then condensed in the presence of the amino group of compound 5 to give compound 6, which is then deprotected with Fmoc to give BCC10. BCC10 undergoes coupling reactions with Cy5-NHS, maleimide-SH, and click reactions with, for example, DBCO-Azide to give BCC10-Cy5, BCC10-Mal, and BCC10-DBCO (DBCO = dibenzocyclooctylene, NHS = N-hydroxysuccinimide), respectively. All compounds were purified by rapid chromatography and... 1 The results were verified by 1H NMR and mass spectrometry. A detailed description of the synthesis of BCC10-Cy5 shown in Scheme 1 is as follows.

[0365] Compound 2 was synthesized according to a previously reported method (D. Spitzer, LL Rodrigues, D. Straßburger, M. Mezger, P. Besenius, Angewandte Chemie International Edition 2017, 56, 15461-15465).

[0366] To a solution of 4-(Fmoc-amino)butyric acid (1.02 g, 3.1 mmol), DMF (5 mL), and N,N-diisopropylethylamine (1 mL, 5.7 mmol), (N,N,N',N'-tetramethyl-O-(3,4-dihydro-4-oxo-1,2,3-benzotriazine-3-yl)urea tetrafluoroborate) (1.1 g, 3.2 mmol) was added. The resulting solution was stirred at room temperature for 20 minutes. Then, compound 2 (1.2 g, 2.4 mmol) was added, and the solution was stirred at room temperature overnight. The mixture was added to 50 mL of NaHCO3 solution, extracted with ethyl acetate, washed three times with deionized water, and further purified by Combiflash column chromatography on a RediSep Gold Resolution silica gel column (eluting with a 100% hexane to hexane / ethyl acetate (40 / 60, v / v) gradient) to give compound 3 (1.35 g, 71% yield). 1H NMR (400 MHz, CDCl3) δ 7.74 (d, J = 7.4 Hz,2H), 7.59 (d, J = 7.3 Hz, 2H), 7.37 (t, J = 7.3 Hz, 2H), 7.28 (t, J = 7.4 Hz,2H), 4.38 – 4.29 (m, 2H), 4.19 (t, J = 6.9 Hz, 1H), 3.74 (m, 2H), 3.66 (m,12H), 3.24 (m, 2H), 2.43 (t, J = 6.2 Hz, 6H), 2.23 (m, 2H), 1.94 – 1.70 (m,2H), 1.43 (s, 27H). C 44 H 64 N2O 12 ESI-MS ([M+H]) + Calculated value: 813.5, Measured value: 813.5.

[0367] Then, compound 3 (0.51 g, 0.6 mmol) was dissolved in 10 mL of dichloromethane and 2 mL of CF3COOH, and the mixture was stirred overnight. After the reaction was completed by TLC, the solvent was removed under reduced pressure. The resulting mixture was further purified by Combiflash column chromatography on a RediSep Gold Resolution silica gel column (eluting with a gradient of 100% CH2Cl2 to CH2Cl2 / MeCN (30 / 70, v / v)) to give compound 4 (0.30 g, 76%). 1 H NMR (400 MHz, CDCl3)δ 7.76 (d, J = 6.7 Hz, 2H), 7.57 (d, J = 6.6 Hz, 2H), 7.44 – 7.35 (t, 2H),7.30 (t, J = 7.4 Hz, 2H), 4.51 – 4.33 (m, C 32 H 40 N2O 12 ESI-MS ([M+H]) + Calculated value: 645.2, Measured value: 645.2.

[0368] The synthesis of compound 5 (N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-3-((1r,4s)-4-((4-chlorophenyl)sulfonyl)-4-(2,5-difluorophenyl)cyclohexyl)acrylamide) is shown below:

[0369] .

[0370] A solution was prepared consisting of compound 4 (80 mg, 0.12 mmol), DMF (5 mL), and N,N-diisopropylethylamine (0.1 mL, 0.57 mmol). Subsequently, (N,N,N',N'-tetramethyl-O-(3,4-dihydro-4-oxo-1,2,3-benzotriazine-3-yl)urea tetrafluoroborate) (198 mg, 0.57 mmol) was added to this solution. The resulting mixture was stirred at room temperature for 20 minutes. Then, compound 5 (295 mg, 0.52 mmol) was added, and the solution was stirred overnight at room temperature. The resulting mixture was added to a 5% NaHCO3 solution (200 mL) and then extracted with ethyl acetate. The extracted solution was then concentrated and further purified by Combiflash column chromatography on a RediSep Gold Resolution silica column (eluting with a gradient from 100% CH2Cl2 to CH2Cl2 / MeOH (85 / 15, v / v)) to give compound 6 (94 mg, 34% yield). 1 H NMR(400 MHz, CDCl3) δ 7.76 (d, J = 7.5 Hz, 2H), 7.57 (t, J = 15.8 Hz, 2H), 7.42– 7.27 (m, 12H), 7.08 – 6.93 (m, 4H), 6.91 – 6.74 (m, 4H), 6.57 – 6.33 (m, 3H), 4.45 – 4.33 (m, 2H), 4.19 (t, J = 12.8 Hz, 1H), 3.80 – 3.57 (m, 18H), 3.53 (m, 12H), 3.47 – 3.39 (m, 8H), 3.25 (t, J = 12.1 Hz, 2H), 2.42 (m, 12H), 2.30 – 2.13 (m, 6H), 1.88 – 1.62 (m, 20H), 1.45 (m, 6H). C 113 H 139 Cl3F6N8O 24 S3's ESI-MS ([M+H) +Calculated value: 2307.8, Measured value: 2307.8.

[0371] Compound 6 (50 mg, 0.21 mmol) was dissolved in 5 mL of dichloromethane and 0.2 mL of piperidine, and the mixture was stirred for 30 minutes. After the reaction was completed by TLC, the solvent was removed under reduced pressure. The resulting solid was further purified by Combiflash column chromatography on a RediSep Gold Resolution silica gel column (eluting with a gradient of 100% CH2Cl2 to CH2Cl2 / MeOH / NH4OH (70 / 30 / 1, v / v / v)) to give compound BCC10 (41 mg, 91%). 1 H NMR (400MHz, MeOD) δ 7.49 (d, J = 8.5 Hz, 6H), 7.34 (d, J = 8.5 Hz, 6H), 7.19 – 7.05(m, 6H), 7.02 – 6.90 (m, 3H), 3.67 (s, 18H), 3.60 C 98 H 129 Cl3F6N8O 22 S3's ESI-MS ([M+2H) 2+ Calculated value: 1043.4, Measured value: 1043.4.

[0372] NHS-Cy5 was added to a solution of BCC10, DMF, and N,N-diisopropylethylamine. The resulting mixture was stirred overnight at room temperature. After removing the solvent under reduced pressure, the resulting solid BCC10-Cy5 was obtained. 130 H 166 Cl3F6N 10 O 23 S3 + ESI-MS ([M+H]) 2+ Calculated value: 1275.5, Measured value: 1275.5.

[0373] The synthesis steps of BCC10-Mal and BCC-DBCO are similar to those of BCC10-Cy5. ESI-MS of BCC-Mal:C 110 H 142 Cl3F6N9O 25 S3 ([M+H)+ Calculated value: 2304.8; Measured value: 2304.8. ESI-MS of BCC10-DBCO: C 119 H 146 Cl3F6N9O 24 S3 ([M+H) + Calculated value: 2400.9; Measured value: 2400.9.

[0374] The synthesis steps for BCC1-Cy5---BCC12-Cy5 are similar to those for BCC10-Cy5.

[0375] BCC1: 1 H NMR (400 MHz, MeOD) δ 7.44 (dd, J = 17.6, 12.3 Hz, 6H), 6.74 (d, J = 8.9 Hz, 4H), 6.40 (d, J = 15.6 Hz, 2H), 4.17 – 4.08 (m, 1H), 3.70 –3.33 (m, 24H), 3.23 – 3.15 (m, 2H), 3.01 (s, 12H), 2.91 – 2.82 (m, 2H), 2.49– 2.38 (m, 6H), 2.39 – 2.30 (m, 2H), 1.87 (m, 2H), and C 47 H 74 N8O 11 ESI-MS ([M+H]) + Calculated value: 1392.8, Measured value: 1392.8. ESI-MS of BCC2-Cy5: C 79 H 111 N 10 O 12 + ([M+H)) 2+ Calculated value: 696.4, Measured value: 696.4.

[0376] BCC2: 1H NMR (400 MHz, MeOD) δ 7.42 (dd, J = 19.7, 12.1 Hz, 9H), 6.67 (d, J = 8.6 Hz, 6H), 6.37 (d, J = 15.6 Hz, 3H), 3.72 – 3.22 (m, 36H), 2.96 (s, 18H), 2.92 – 2.83 (m, 2H), 2.41 (t, J = 5.6 Hz, 8H), 1.85 – 1.68 (m, 2H), and C 68 H 105 N 11 O 16 ESI-MS ([M+2H]) + Calculated value: 1332.8, Measured value: 1332.8. ESI-MS of BCC2-Cy5: C 100 H 142 N 13 O 17 + ([M+2H)) 2+ Calculated value: 899.5, Measured value: 899.5.

[0377] BCC3: 1 H NMR (400 MHz, MeOD) δ 7.56 (d, J = 7.8 Hz, 2H), 7.33 (d, J =8.0 Hz, 2H), 7.13 – 6.95 (m, 6H), 4.12 (m, 1H), 3.76 – 3.61 (m, 4H), 3.57 –3.42 (m, 4H), 3.33 (m, 4H), 3.23 – 2.98 (m, 12H), 2.70 – 2.48 (m, 6H), 2.48 –2.35 (m, 4H), 2.24 (t, J = 7.4 Hz, 2H), 1.85 – 1.12 (m, 20H), and C 47 H 70 ESI-MS of N8O7 ([M+H)) + Calculated value: 859.5, Measured value: 859.5. ESI-MS of BCC3-Cy5: C 79 H 107 N 10 O8 + ([M+H)) 2+ Calculated value: 662.4, Measured value: 662.4.

[0378] BCC4: 1 H NMR (400 MHz, MeOD) δ 7.56 (d, J = 7.8 Hz, 3H), 7.33 (d, J =8.1 Hz, 3H), 7.16 – 6.89 (m, 9H), 3.66 (m, 12H), 3.33– 2.90 (m, 24H), 2.56(t, J = 7.4 Hz, 6H), 2.41 (t, J = 6.0 Hz, 6H), 2.26 (t, J = 7.2 Hz, 2H), 1.54– 1.08 (m, 24H), and C 68 H 99 N 11 O 10 ESI-MS ([M+H]) + Calculated value: 1230.8, Measured value: 1230.8. ESI-MS of BCC4-Cy5: C 100 H 136 N 13 O 11 + ([M+2H)) 2+ Calculated value: 848.5, Measured value: 848.5.

[0379] BCC5: 1 H NMR (400 MHz, MeOD) δ 7.57 (d, J = 7.9 Hz, 2H), 7.34 (d, J =8.1 Hz, 2H), 7.14 – 6.96 (m, 6H), 4.18 – 4.03 (m, 2H), 3.71 – 2.98 (m, 24H), 2.63 (m, 6H), 2.49 – 2.36 (m, 6H), 2.37 – 2.26 (m, 2H), and C 47 H 70 N8O 11 ESI-MS ([M+H]) + Calculated value: 923.5, Measured value: 923.5. ESI-MS of BCC5-Cy5: C 79 H 107 N 10 O 12 + ([M+H)) 2+ Calculated value: 694.4, Measured value: 694.4.

[0380] BCC6:1 H NMR (400 MHz, MeOD) δ 7.54 (d, J = 7.9 Hz, 3H), 7.30 (d, J = 8.1 Hz, 3H), 7.10 – 6.90 (m, 9H), 3.68 – 3.56 (m, 12H), 3.46– 3.25 (m, 44H), 3.10 – 2.93 (m, 8H), 2.55 (t, J = 7.4 Hz, 6H), 2.37 (t, J = 5.9 Hz, 6H), 2.28– 2.14 (m, 2H), and C 68 H 99 N 11 O 16 ESI-MS ([M+H]) + Calculated value: 1326.7, Measured value: 1326.7. ESI-MS of BCC6-Cy5: C 100 H 136 N 13 O 17 + ([M+2H)) 2+ Calculated value: 896.5, Measured value: 896.5.

[0381] BCC7: 1 H NMR (400 MHz, MeOD) δ 7.50 (d, J = 8.1 Hz, 4H), 7.36 (d, J =8.3 Hz, 4H), 7.14 (m, 4H), 6.97 (m, 2H), 4.11 (m, 1H), 3.76 – 3.61 (m, 6H),3.57 – 3.42 (m, 6H), 3.23 – 2.98 (m, 12H), 2.70 – 2.48 (m, 12H), 2.48 – 2.35(m, 6H), 2.24 (t, J = 7.4 Hz, 2H), 1.85 – 1.12 (m, 42H), and C 67 H 90 Cl2F4N6O 11 S2's ESI-MS ([M+H) + Calculated value: 1365.5, Measured value: 1365.5. ESI-MS BCC7-Cy5: C 99 H 127 Cl2F4N8O 12 S2 +([M+2H)) 2+ Calculated value: 915.9, Measured value: 915.9.

[0382] BCC8: 1 H NMR (400 MHz, MeOD) δ 7.50 (d, J = 8.1 Hz, 6H), 7.36 (d, J =8.3 Hz, 6H), 7.14 (m, 6H), 6.97 (m, 3H), 3.76 – 3.61 (m, 4H), 3.57 – 3.42 (m,4H), 3.23 – 2.98 (m, 12H), 2.70 – 2.48 (m, 18H), 2.48 – 2.35 (m, 6H), 2.24(t, J = 7.4 Hz, 2H), 1.85 – 1.12 (m, 30H), and C 98 H 129 Cl3F6N8O 16 S3's ESI-MS ([M+H) + Calculated value: 1989.8, Measured value: 1989.8. ESI-MS of BCC8-Cy5: C 130 H 166 Cl3F6N 10 O 17 S3 + ([M+2H)) 2+ Calculated value: 1228.0, Measured value: 1228.0.

[0383] BCC9: 1 H NMR (400 MHz, MeOD) 1 H NMR (400 MHz, MeOD) δ 7.50 (d, J = 8.1 Hz, 4H), 7.36 (d, J = 8.3 Hz, 4H), 7.14 (m, 4H), 6.97 (m, 2H), 4.11 (m, 1H), 3.75 – 3.43 (m, 34H), 2.94 (s, 20H), 2.55 – 2.29 (m, 12H), 2.24 (m, 6H), 1.89– 1.22 (m, 18H), and C 82 H 100 Cl2F4N6O 17 S2's ESI-MS ([M+H) +Calculated value: 1651.6, Measured value: 1651.6. ESI-MS for BCC9-Cy5 C 99 H 127 Cl2F4N8O 16 S2 + ([M+2H)) 2+ Calculated value: 947.4, Measured value: 947.4.

[0384] BCC10: 1 H NMR (400 MHz, MeOD) δ 7.49 (d, J = 8.5 Hz, 6H), 7.34 (d, J= 8.5 Hz, 6H), 7.19 – 7.05 (m, 6H), 7.02 – 6.90 (m, 3H), 3.67 (s, 18H), C 98 H 129 Cl3F6N8O 22 S3's ESI-MS ([M+2H) 2+ Calculated value: 1043.4, Measured value: 1043.4. ESI-MS of BCC10-Cy5: C 130 H 166 Cl3F6N 10 O 23 S3 + ([M+H)) 2+ Calculated value: 1275.5, Measured value: 1275.5.

[0385] BCC11: 1 H NMR (400 MHz, MeOD) δ 7.45 (t, J = 7.9 Hz, 2H), 6.69 (d, J= 8.4 Hz, 2H), 6.57 (d, J = 7.5 Hz, 2H), 4.16 – 4.03 (m, 1H), 3.78 – 3.40 (m,28H), 3.14 – 3.05 (m, 6H), 2.99 (t, J = 11.8 Hz, 4H), 2.89 (t, J = 7.4 Hz,2H), 2.51 – 1.24 (m, 24H), and C 47 H74 Cl2N 10 O 11 ESI-MS ([M+H]) + Calculated value: 1025.5, Measured value: 1025.5. ESI-MS of BCC11-Cy5: C 79 H 111 Cl2N 12 O 12 + ([M+H)) 2+ Calculated value: 745.4, Measured value: 745.4.

[0386] BCC12: 1 H NMR (400 MHz, MeOD) δ 7.45 (t, J = 7.9 Hz, 3H), 6.68 (d, J = 8.4 Hz, 3H), 6.56 (d, J = 7.4 Hz, 3H), 3.84 – 3.00 (m, 44H), 2.85 – 2.71 (m, 2H), 2.44 (t, J = 6.0 Hz, 12H), 2.33 – 2.22 (m, 2H), 2.00 – 1.33 (m, 28H), and C 68 H 105 Cl3N 14 O 16 ESI-MS ([M+H]) + Calculated value: 1479.7, Measured value: 1479.7. ESI-MS of BCC12-Cy5: C 100 H 142 Cl3N 16 O 17 + ([M+H)) 2+ Calculated value: 972.5, Measured value: 972.5.

[0387] Fluorescence intensity was measured by intravenous injection.

[0388] BCC-Cy5 was administered intravenously to 8-10 week old C57BL / 6 mice (n=3 per group) at a dose of 1 mg / kg Cy5. One hour later, the mice were euthanized, and brain tissue was collected for observation using a biophotonic IVIS (Spectrum) system. To analyze the percentage of Cy5 accumulation in brain tissue, brain tissue was collected after perfusion following intravenous injection of different BCC-Cy5 compounds. Each brain tissue was then weighed and homogenized. The Cy5 content was quantified using a fluorescence spectrophotometer according to the calibration curve of each BCC and expressed as the percentage of injected dose per gram of brain tissue (%ID / g). Compared with free Cy5, most BCC-Cy5 compounds resulted in an increase in the percentage of injected dose per gram of brain tissue (%ID / g). Figure 1A and Figure 1C In particular, the levels of trianthraquinone derivatives, tryptophan derivatives, and SR derivatives were 62.5 times, 97.0 times, and 141.8 times higher than those of free Cy5, respectively. Figure 1C Overall, the tri-antennae BCC exhibited superior BBB penetration compared to the bi-antennae BCC. Interestingly, BCC10 derived from the tri-antennae MK-0752 accumulated in the mouse brain at a rate 220.5 times higher than that of free Cy5.

[0389] Eight- to ten-week-old C57BL / 6J mice (n=3 per group) were intravenously injected with BCC at a dose of 1 mg / kg. One hour after injection, major organs, including the brain, were dissected and imaged using an IVIS imaging system (Xenogen). The BCC-Cy5 described in this study was injected intravenously (iv) into mice, and the accumulation of BCC-Cy5 in the brain after administration was quantitatively analyzed. Subsequently, the fluorescence intensity of these BCCs after intravenous injection into mice was quantitatively analyzed. Interestingly, the study found that BCC10-Cy5 could effectively cross the blood-brain barrier and exhibited a strong fluorescent signal in the mouse brain. Figure 1A and Figure 1B Compared to free Cy5, BCC10-Cy5 exhibited a 12-fold higher fluorescence intensity. The brain penetration of these biantennary and triantennary BCCs with Cy5 was assessed after intravenous injection in mice. Most BCCs induced an increase in intrabrain fluorescence intensity compared to free Cy5. Specifically, the fluorescence intensities of the triantennary cinnamic acid derivative, tryptamine derivative, and SR derivative were 3.2, 5.6, and 5.6 times higher than Cy5, respectively. Importantly, BCC10 derived from triantennary MK-0752 showed a 12-fold higher fluorescence intensity in the brain than Cy5.

[0390] As described below, four classes of blood-brain barrier-penetrating conjugates were designed and synthesized by conjugating these molecules with various reagents such as proteins and nucleic acids. Representative compounds and conjugates prepared and described in this article are as follows: Figure 2A , Figure 2B and Figure 2C As shown.

[0391] BCC10 conjugation with DNA

[0392] BCC10-DNA-Cy5 was synthesized following previously reported procedures (SM Sarett et al., Proceedings of the National Academy of Sciences 2017, 114, E6490-E6497). Azide-DNA-Cy5 was purchased from Integrated DNA Technologies (IA, USA) and reacted with a five-fold molar excess of BCC10-DBCO at a DNA concentration of 1 mg / mL in a mixture of 30% methanol and 70% water at 25°C for 12 h. The reaction mixture was lyophilized and then quantitatively precipitated by sequentially adding 50 μL of 1 M acetic acid, 25 μL of 3 M sodium acetate buffer (pH 5.0), and 500 μL of ethanol, followed by incubation at -20°C for 2 h. The DNA was separated by centrifugation, and the resulting DNA precipitate was washed with ice-cooled 90% (v / v) ethanol to obtain BCC10-DNA-Cy5.

[0393] The potential of BCC10 to deliver oligonucleotides to the brain was tested. First, BCC10 was conjugated with Cy5-tagged DNA (similar to...). Figure 2A However, the term "Oligo" was replaced with "DNA-Cy5". Agarose gel electrophoresis showed that BCC10-DNA-Cy5 had a decreased migration rate compared to DNA-Cy5. The molecular weight of BCC10-DNA-Cy5 was confirmed by MALDI-MS. Mice were then intravenously injected with free DNA-Cy5, Chol-DNA-Cy5 (a previously reported DNA conjugate), and BCC10-DNA-Cy5. BCC10-DNA-Cy5 showed significantly higher fluorescence intensity than both DNA-Cy5 and Chol-DNA-Cy5. Figure 3A (p<0.0001). FACS analysis showed that BCC10-DNA-Cy5 could effectively deliver DNA to microglia, neurons, astrocytes, and BCECs (p<0.0001). Figure 3B ).

[0394] BCC conjugation with oligonucleotides

[0395] To test the potential of BCC10 to deliver oligonucleotides (Oligo) to the brain, BCC10 was first conjugated to Oligo using a previously reported method (Sarett, SM et al., Proceedings of the National Academy of Sciences 114, E6490-E6497 (2017)). Figure 2A Azide-Oligo was reacted with a 20-fold molar excess of BCC10-DBCO at a concentration of 1 mg / mL Oligo in 30% methanol and 70% water at 25°C for 12 hours. The resulting reaction mixture was lyophilized and then precipitated by sequentially adding 25 μL of 3M sodium acetate (pH 5.0) and 500 μL of ethanol. The mixture was then incubated at -20°C for three hours. Oligo was separated by centrifugation, and the resulting DNA precipitate was washed with ice-cooled 90% ethanol to obtain BCC10-Oligo, with a MALDI-MS calculated value of 8142.14 and a measured value of 8142.28.

[0396] BCC10 enhances the blood-brain barrier penetration and gene silencing effects of oligonucleotides.

[0397] BCC-Oligo, free Oligo, and Chol-Oligo were administered intravenously to C57BL / 6 mice (n=3) at doses of 1 mg / kg and 25 mg / kg Oligo, respectively. One hour post-administration, major organs, including brain, heart, liver, spleen, lung, and kidney, were collected via perfusion. Oligo concentrations in each organ were determined using established methods. Oligo concentrations in these tissues were quantified based on established calibration curves for BCC-Oligo, Oligo, and Chol-Oligo, and expressed as a percentage of the injected dose per gram of tissue (%ID / g). Pharmacokinetics of BCC-Oligo, Oligo, and Chol-Oligo were evaluated using established methods. Blood samples (50 μL) were collected at specific time points post-injection for analysis. Oligo concentrations in the supernatant were determined using a fluorescence spectrophotometer, based on the calibration curves for BCC-Oligo, Oligo, and Chol-Oligo, respectively. Free Oligo, cholesterol-conjugated oligonucleotides (Chol-Oligo, a previously reported oligonucleotide conjugate; Kariolis, MS et al., Science Translational Medicine 12, eaay1359 (2020)), and BCC10-Oligo were intravenously injected into mouse models at doses of 1 mg / kg and 25 mg / kg Oligo. Quantitative results showed that in the BCC10-Oligo treatment group, 0.49%ID / g Oligo accumulated in brain tissue at an Oligo dose of 1 mg / kg. When the dose was increased to 25 mg / kg, the Oligo accumulation in brain tissue of the BCC10-Oligo treatment group was 1.48%ID / g, which was 66.0-fold and 7.3-fold higher than that of free Oligo and Chol-Oligo, respectively (p < 0.0001). The accumulation of BCC10 in the rat brain was also quantified. Following intravenous injection, the accumulation (ID / g) of BCC10-Oligo in rat brain tissue was 20.8 times higher than that of free Oligo. BCC10-Oligo promoted greater accumulation of Oligo in brain tissue, which was mainly attributed to γ-secretase-mediated transcytosis.

[0398] Pharmacokinetic studies were also conducted on Oligo, Chol-Oligo, and BCC10-Oligo. Plasma half-life (t...) 1 / 2 The time values ​​were 7.6 hours (Oligo), 11.2 hours (Chol-Oligo), and 10.9 hours (BCC10-Oligo) (n = 3 rats, administered via a single intravenous injection of 1 mg / kg Oligo). Biodistribution data indicated that BCC10-Oligo primarily accumulated in the kidneys, liver, brain, and lungs.

[0399] Flow cytometry analysis

[0400] The distribution of BCC10-Oligo in different cell types in brain tissue was then investigated by flow cytometry. Single-cell brain cell suspensions were prepared according to the previously described method¹³. Briefly, brain tissue was dissected and removed after perfusion with ice-cooled PBS. The brain tissue was then cut into small pieces and placed in test tubes containing TrypLE Express (Gibco, 15 mL) and DNase I (Sigma), and digested at 37°C for 15 minutes.

[0401] Brain tissue was mechanically dissociated using a pipette, followed by the addition of culture medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin to terminate digestion. Single cells were collected using a 70 μm nylon mesh cell filter (Thermo Fisher). The obtained single-cell suspension was used for immunofluorescence staining to identify different cell types using specific markers: NeuN (1:100, Abcam) for neurons, GFAP (1:100, Abcam) for astrocytes, CD11b (1:100, Abcam) for microglia, and CD31 (1:100, Abcam) for brain capillary endothelial cells (BCEC). Finally, the cells were stained with 4',6-diamino-2-phenylindole dihydrochloride (DAPI) and analyzed by flow cytometry, recording 20,000 DAPI-positive events. Mouse brain tissue was dissociated using the above method, and then cells were separated using specific kits: microglia-specific anti-CD11b microbead kit, anti-astrocytocyte surface antigen-2 microbead kit, and neuron-specific neuron separation kit (all from Miltenyi Biotec), and the process was performed using MACS technology according to the manufacturer's instructions.

[0402] BCC10-Oligo showed strong positive signals in multiple cell types, including microglia (2.5%), neurons (4.6%), astrocytes (9.6%), and brain capillary endothelial cells (BCEC, 2.3%). Figure 3C When the Oligo dose was increased to 25 mg / kg, higher delivery efficiency was observed in these cell types: microglia (26.1%), neurons (23.9%), and brain capillary endothelial cells (19.8%). Particularly in BCC10-Oligo treatment, a positive signal was observed in 43.9% of astrocytes. Figure 3D ).

[0403] To observe the distribution of BCC10-Oligo in the brain, a neuron-specific (NeuN) assay was used. +) and astrocyte-specific (GFAP) + Immunofluorescence imaging of mouse brain tissue was performed using the antibody. Significant co-localization of BCC10-Oligo with neurons and astrocytes in the hippocampus, cerebral cortex, and striatum was observed, confirming its presence; in contrast, no signal of free Oligo was detected in any brain region at the same dose (data not shown). These results indicate that BCC10 can promote the crossing of the blood-brain barrier by Oligo, enabling its distribution in a wide range of brain regions.

[0404] BCC-ASO induces gene silencing in wild-type mice

[0405] To further investigate whether the BCC10-Oligo conjugate could induce functional activity in the brain, we conjugated BCC10 with an antisense oligonucleotide (ASO) that targets the non-coding RNA lung adenocarcinoma metastasis-associated transcript 1 (Malat1), which is expressed in all types of brain cells. Free ASO was then... Malat1 Or BCC10 conjugated ASO Malat1 ASO at doses ranging from 1 mg / kg to 50 mg / kg Malat1 The dose was administered intravenously, and the Malat1 mRNA level was detected by quantitative PCR after reverse transcription (qPCR) (n = 3 mice). All data are expressed as mean ± standard deviation (SD). Statistical significance was calculated by unpaired two-tailed Student's t-test.

[0406] Free ASO Malat1 Treatment showed negligible knockdown of Malat1, consistent with previous findings that ASO alone, administered systemically, is ineffective in inducing gene knockdown in the brain. (BCC10-ASO) Malat1 The conjugate effectively reduced Malat1 mRNA levels by 26.0% to 62.6% in a dose-dependent manner. Figure 4A A single intravenous injection of 25 mg / kg ASO into different brain regions (including the brainstem, hippocampus, striatum, cerebellum, and cerebral cortex). Malat1 After dose, BCC10-ASO Malat1 This reduced Malat1 levels by approximately 40% to approximately 70%. Figure 4C Simultaneously, from a single intravenous injection of 25 mg / kg ASO Malat1 BCC10-ASO Malat1 After isolating neurons, astrocytes, and microglia from the brains of treated mice, the levels of Malat1 in these cells were measured. The levels in neurons, astrocytes, and microglia decreased by 45.3%, 62.1%, and 56.5%, respectively. Figure 4B Importantly, when mice were pretreated with nilogasstat, BCC10-ASO Malat1 The silencing activity was significantly reduced. These results indicate that γ-secretase plays an important role in the process of BCC10 crossing the blood-brain barrier.

[0407] PBS, ASO Malat1 and dosages ranging from 1 mg / kg to 50 mg / kg ASO Malat1 BCC10-ASO Malat1 C57BL / 6 mice were administered the drug intravenously. 72 hours after administration, the mice were anesthetized, and brain tissue was collected. Target genes were analyzed by quantitative real-time PCR. Briefly, partial brain tissue was collected and stored at -20°C in RNAlater solution (Thermo Fisher Scientific) until RNA extraction. RNA was isolated from the brain tissue using the RNeasy Mini Kit (QIAGEN) according to the manufacturer's protocol and quantified using a Nanodrop One micro-UV-Vis spectrophotometer (Thermo Fisher Scientific). cDNA was then synthesized using the TaqMan Reverse Transcription Kit (Thermo Fisher) according to the manufacturer's protocol. Finally, q-PCR was performed using TaqMan Gene Expression Detection (Thermo Fisher) with the cDNA template (10 ng). Gapdh was selected as the internal control gene for data analysis. RNA expression levels are expressed as a percentage of PBS. The following TaqMan probes were used: MmMalat1 (Thermo Fisher) and MmGapdh (Thermo Fisher). Additionally, PBS and free ASO were used. Mapt Or a dose of 25 mg / kg ASO Mapt BCC10-ASO Mapt Intravenous injection was administered to C57BL / 6 mice, and a similar procedure to Malat1 knockdown was performed using the Taqman probe MmMapt (Thermo Fisher). Furthermore, we examined an antisense oligonucleotide (ASO) targeting the microtubule-associated protein tau (Mapt) mRNA, a promising target for the treatment of Alzheimer's disease (AD). BCC10-ASO Mapt A single intravenous injection significantly reduced Mapt mRNA levels by 63.5% (p < 0.0001).

[0408] Due to significant differences in genetic, cellular, and physiological characteristics between mice and humans, results from mouse brain studies typically correlate poorly with those from human brain cell studies. To investigate its role in primary human cells, we evaluated BCC10-ASO in fresh human brain tissue ex vivo samples. Malat1 The gene silencing activity was observed. Human cortical tissue was obtained from two adult Parkinson's disease patients who underwent standard deep brain stimulation (DBS) surgery. Specifically, during DBS, a technique called cauterization was used to assist in the safe implantation of DBS electrodes. Catheterization results in a small loss of volume of "non-functional" cortical tissue, meaning that this loss has no noticeable functional impact on the patients. As part of the Living Brain Project (LBP) protocol (TE00001203), this technique was used to obtain a small biopsy tissue from the prefrontal cortex (PFC) region, which would otherwise be discarded after cauterization. Prior to cauterization, a biopsy sample of the prefrontal cortex (PFC) was obtained using a 4 mm biopsy perforator and microdissector. The obtained cortical tissue biopsy sample was immediately placed on ice and sliced ​​into 300 μm thick sections within 30 minutes. Subsequently, these sections were placed in DMDM / F12 medium supplemented with growth factors and cultured for one hour to stabilize them, after which they were subjected to PBS and BCC10-ASO, respectively. Malat1 The brain tissue was treated with 400 nM. After 48 hours, brain tissue was collected, and the levels of target genes were detected by q-PCR using the method described above. The following TaqMan probes were used in the experiment: HsMalat1 (Thermo Fisher) and HsGapdh (Thermo Fisher). Human brain tissue was cut into small pieces and treated with BCC10-ASO... Malat1 The cells were co-incubated, and the mRNA level of Malat1 was detected by qPCR. Quantitative results showed that, compared with the PBS group, BCC10-ASO... Malat1 Treatment reduced Malat1 mRNA levels by 81.3% (p < 0.0001). In summary, these results collectively demonstrate that BCC10-ASO can effectively deliver antisense oligonucleotides to the brain and achieve potent gene silencing in mouse brains and isolated human brain tissue.

[0409] BCC10 silences SOD1 in an ALS mouse model

[0410] Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease. Mutations in the superoxide dismutase 1 (SOD1) gene (which encodes superoxide dismutase 1) are one of the genetic causes of ALS, leading to the accumulation of toxic proteins and damage to motor neurons. To evaluate the translational potential of the BCC platform in ALS disease models, we expressed a G93A mutant form of human SOD1.G93A BCC10 was tested in a transgenic mouse model. SOD1 was used. G93A Transgenic mice (ALS mice) were used to evaluate BCC-ASO. Sod1 Induced knockdown efficiency. First, using a method similar to the one described above, ASO targeting SOD1 was conjugated with BCC10, followed by BCC10-ASO... Sod1 Or control treatment (PBS, free ASO) Sod1 and Chol-ASO Sod1 ) at 25 mg / kg ASO Sod1 Intravenous administration of SOD1 at a dose of G93A Transgenic mice. Seventy-two hours after administration, the mice were anesthetized, and their brains were collected. The level of the target gene (SOD1) was detected using q-PCR according to the method described above. The following Taqman probes were used in the experiment: HsSod1 (Thermo Fisher) and HsGapdh (Thermo Fisher). The concentration of Sod1 protein in brain tissue was detected by ELISA according to the manufacturer's protocol.

[0411] Following treatment, we observed that, compared to the PBS group, BCC10-ASO [the following was observed after a single intravenous injection]. Sod1 It reduced Sod1 mRNA levels to the lowest level, decreasing by 44.2%. Figure 5A Meanwhile, compared to the PBS group, those receiving free ASO... Sod1 and Chol-ASO Sod1 The treated mice showed a slight decrease in Sod1 mRNA levels. More importantly, based on ELISA results, BCC10-ASO Sod1 Reduced Sod1 protein concentration ( Figure 5B Given the therapeutic benefits of reducing Sod1 levels observed in clinical data, BCC-ASO, which can effectively silence Sod1 mRNA and corresponding protein expression, is a promising candidate. Sod1 It is a practical method for treating ALS.

[0412] BCC10-ASO was studied. Sod1 In SOD1 G93A Safety in ALS mice. To further evaluate the systemic toxicity of BCC, major organs were collected, stained with hematoxylin and eosin (H&E), and observed under a light microscope. BCC10-ASO Sod1 In SOD1 G93A Safety in ALS mice. To further evaluate the systemic toxicity of BCC, major organs of mice were collected, stained with hematoxylin and eosin (H&E), and observed under a light microscope. To evaluate the safety of BCC10-ASO... Sod1Effects on liver and kidney function, after intravenous administration of PBS and free ASO Sod1 Chol-ASO Sod1 Or BCC10-ASO Sod1 Subsequently, key metabolic and excretory biomarkers in plasma samples, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN), were measured using an ELISA kit (Abcam). These biomarkers were measured in different groups (PBS group, free ASO group, etc.). Sod1 Group, Chol-ASO Sod1 Group or BCC10-ASO Sod1 The similar levels among the groups indicate that BCC10-ASO Sod1 The administration of BCC10-ASO did not affect liver or kidney function. Further examination of major organs by H&E staining showed that, compared with the PBS group, intravenous administration of BCC10-ASO... Sod1 No obvious organ damage or inflammatory lesions were subsequently observed.

[0413] In order to study BCC10-ASO Sod1 To investigate whether it modulates γ-secretase-related pathways in different organs, we performed RNA sequencing (RNA-seq) analysis to study the expression levels of Notch-related mRNA transcripts in the liver, kidney, and brain. For batch RNA sequencing studies, 25 mg / kg ASO was used. SOD1 Mice were intravenously injected with BCC10-ASO at the specified dose. Sod1 MK-0752 was administered orally. 72 hours later, brain, liver, and kidney tissues from the treated mice were harvested and stored at -20°C in RNAlater solution (Thermo Fisher Scientific) until RNA extraction. mRNA was extracted from brain, liver, and kidney tissues using the method described above. Total RNA was quantified using a Qubit 4 fluorometer (Thermo Fisher Scientific), and RNA integrity was assessed using an Agilent 2100 bioanalyzer (Agilent Technologies). RNA-seq library construction began with mRNA enrichment using Oligo(dT) magnetic beads, followed by random fragmentation. Complementary DNA (cDNA) was synthesized using random hexameric primers. Subsequent steps included end repair, adapter ligation, fragment size selection, and PCR enrichment to construct a double-stranded cDNA library (Illumina). The constructed cDNA library was then sequenced on an Illumina NovaSeq 6000 system for deep sequencing.

[0414] Mice administered MK-0752 showed significant alterations in certain key γ-secretase-related transcripts, consistent with results reported in the literature. Simultaneously, at 25 mg / kg ASO... Sod1 The dose received by BCC10-ASO Sod1 In treated mice, the expression levels of most γ-secretase-related genes in the liver, kidneys, and brain were comparable to those in the PBS group. Furthermore, the expression levels of BCC10-ASO were also investigated. Sod1 Effects on the expression of multiple integrated membrane proteins (type I transmembrane proteins) in the brain. Results also showed that BCC10-ASO... Sod1 The gene expression levels of the treated mice were similar to those of the mice treated with PBS.

[0415] According to the manufacturer's protocol, 32 mouse cytokines, chemokines, and growth factors in plasma were simultaneously detected using the Mouse Cytokine 32-Plex Discovery Assay Kit (Eve Technologies). This was to analyze BCC10-ASO. Sod1 The spectrum of cytokines, chemokines, and growth factors in BCC10-ASO after intravenous injection. Sod1 Plasma samples were then collected from the mice for comprehensive blood biochemical analysis. Results showed that, compared to the PBS group, BCC10-ASO... Sod1 The cytokine, chemokine, and growth factor profiles in the treatment group showed only minor changes. Notably, in free ASO... Sod1 and Chol-ASO Sod1 In the group, the levels of certain cytokines (e.g., IL-1α, IL-1β, IL-4, IL-6, IL-12P70) were elevated. These results indicate that BCC10-ASO at the tested dose... Sod1 In SOD1 G93A It is well tolerated in ALS mice.

[0416] Molecular docking

[0417] To further understand the interaction between BCCs and γ-secretase, molecular docking studies were conducted on several representative BCCs (BCC8, BCC9, and BCC10) and γ-secretase. γ-secretase is a transmembrane protein complex composed of presenilin-1 (PS1), nicastrin, APH-1, and PEN-2. Previous cryo-electron microscopy (Cryo-EM) studies have provided structural understanding of γ-secretase and its inhibitors, and determined that γ-secretase inhibitors occupy the binding pocket in PS1.

[0418] First, target ligands (BCC8, BCC9, and BCC10) were constructed using ChemDraw (RRID: SCR_016768), and then relaxed using the Glide Maestro macromolecular minimization module. For receptor preparation, the PS1 domain of the γ-secretase was obtained from a protein database (PDB ID: 7d8x). Two models of the secretases were constructed by replicating and positioning the PS1 domains so that their allosteric sites were aligned with each other. The distance between the domains was adjusted to maintain a 10 Å buffer region to prevent steric hindrance conflicts. The constructed ligands were docked to the receptor using AutoDock Vina, generating 40 conformations of the ethylene glycol spacer-ligand and 20 conformations of the alkyl spacer-ligand. All generated conformations were visually examined, and the first 14 conformations of the ethylene glycol spacer binding at the interface of the two secretases and 4 conformations of the alkyl spacer were selected for further analysis.

[0419] Based on these findings, molecular docking results show that BCCs tend to bind at the interface between two secretases rather than a single secretase. Unbound by any single theory, docking score analysis indicates that trivalent BCCs are more favorable for binding than divalent ones due to enhanced ligand-protein interactions and a larger contact area, reflected in superior docking scores (-7.9 to -8.2 kcal / mol vs. -6.4 to -7.6 kcal / mol). The ethylene glycol linker in BCC10 promotes broader contact with γ-secretase, thereby improving binding efficiency. In contrast, the alkane linker in BCC9 tends to fold and coil, resulting in reduced contact with γ-secretase and weaker binding. This observation is consistent with the overall docking scores of BCC10 (-8.2 kcal / mol) and BCC8 (-7.9 kcal / mol). These calculated data are consistent with experimental results.

[0420] Blood-brain barrier penetration mechanism

[0421] BCC10 is derived from Tripterygium MK-0752 (a γ-secretase inhibitor). While many studies have reported the functions of γ-secretase, such as cleavage of amyloid precursor protein (APP), it appears that γ-secretase has not previously been considered a mediator of transcytosis associated with biomolecules. Unconstrained by any single hypothesis, γ-secretase may mediate transcytosis and enhance the permeability of BCC10 bound to γ-secretase across the blood-brain barrier. To test this hypothesis, an in vitro transwell model was constructed to mimic the blood-brain barrier. Briefly, a brain endothelial cell line (bEnd.3 cells) was seeded into the upper chamber of a 24-well transwell (Corning, USA, 1×10⁻⁶ cells) with a pore size of 0.4 μm.4 Incubation was performed in wells (10 nM each) for 72 hours to establish an in vitro blood-brain barrier model. A permeability test using dextran-FITC was performed to verify the integrity and tightness of the transwell model. Nilogasstat (10 nM) and MK-0752 (10 nM) were added to the upper chamber. After 1 hour of incubation, BCC10-Cy5 was added to the upper chamber. The percentage of BCC10-Cy5 in the lower chamber was then quantified using the method described above. Compared to the untreated group, the percentage of BCC10-Cy5 was significantly reduced after pretreatment with MK-0752. Figure 6A Subsequently, the transcytosis mechanism of BCC10 was investigated by blocking γ-secretase in transwell assays. To inhibit γ-secretase, bEnd.3 cells were pretreated with nilogasstat, an FDA-approved γ-secretase inhibitor. Results showed that nilogasstat significantly reduced the percentage of BCC10-Cy5 in the brain compared to the untreated group. Figure 6A ).

[0422] To further investigate γ-secretase-mediated transcytosis in vivo, C57BL / 6 mice were first treated orally with nilogasstat (30 mg / kg), followed by intravenous injection of BCC10-Cy5 one hour later. One hour after that, brain tissue was collected, and BCC10-Cy5 levels in the brain tissue were quantitatively analyzed using the methods described above. Consistent with in vitro results, compared to mice not treated with nilogasstat, the percentage of injected BCC10-Cy5 in brain tissue was reduced by 78.1% in the presence of nilogasstat (p = 0.0003). Figure 6B These findings demonstrate that γ-secretase plays a crucial role in the transport of BCC10-Cy5 across the blood-brain barrier. These data consistently indicate that inhibiting γ-secretase significantly blocks BCC10 from crossing the blood-brain barrier and confirms its important role in molecular transport to the brain. Given that this pathway differs from other known transcytosis receptor pathways (such as transferrin and insulin), this represents a new paradigm for delivering various therapeutic payloads to the brain.

[0423] The combination of BCC10 and mCherry

[0424] To further test the BBB penetration capability of the BCC platform, BCC10 was conjugated with the fluorescent protein mCherry. The conjugation of BCC10 with mCherry was performed according to a previously established protocol (J. Xu et al., Advanced Materials 2020, 32, 1905145). Briefly, 0.1 mg / mL of mCherry in PBS was reacted with 5 equivalents of Traut reagent (20 mg / mL in PBS), and the reaction was incubated overnight at room temperature. Then, 10 equivalents of BCC10-mCherry (20 mg / mL in DMSO) were added. The reaction mixture was incubated at room temperature in the dark for 1 day and filtered using a 7K Zeba size size exclusion column.

[0425] BCC10-mCherry significantly enhanced mCherry transport in the brain (2.2-fold increase compared to free mCherry). Flow cytometry was used to analyze various brain cell types and quantitatively determine their cell distribution. Figure 7 As shown, BCC10-mCherry effectively delivers mCherry to microglia, neurons, astrocytes, and brain capillary endothelial cells (BCEC). Compared to free mCherry, BCC10-mCherry significantly increased mCherry signaling in various brain cell types, including neurons (2.2-fold), astrocytes (16.3-fold), BCEC (21.1-fold), and microglia (1.9-fold). Furthermore, the BCC10-DNA conjugate exhibited a 2.5-fold higher fluorescence signal compared to free DNA and Chol-DNA. In conclusion, these BCCs provide an innovative platform for overcoming the blood-brain barrier and delivering therapeutic agents to brain cells.

[0426] Assessing the blood-brain barrier penetration of BCC-mCherry and BCC-Cre recombinases in mice.

[0427] We conducted a study to demonstrate the suitability of the BCC platform for protein delivery. In the experiments, BCC10 was conjugated to mCherry and Cre recombinases, respectively. The conjugation of BCC to mCherry and Cre was performed according to a previously described protocol (Li, B. et al. Bioorganic & Medicinal Chemistry 24, 5855-5860 (2016)). Briefly, a PBS solution containing 0.1 mg / mL mCherry or Cre was mixed with 5 equivalents of Traut reagent (20 mg / mL PBS solution), and the reaction was incubated overnight. Subsequently, 10 equivalents of BCC (20 mg / mL DMSO solution) were added. The reaction mixture was then incubated at 4°C in the dark for 1 day and filtered using a 7K Zeba size size exclusion column.

[0428] To assess mCherry delivery, wild-type C57BL / 6 mice (n=3) were intravenously injected with mCherry and BCC10-mCherry at a dose of 1 mg / kg mCherry. Brain tissue was collected one hour later and quantitatively analyzed by ELISA (Abcam). To assess the transfection / editing efficiency of TAT-Cre recombinase, Ai14 mice were intravenously injected with TAT-Cre recombinase and BCC10-Cre at a dose of 10 mg / kg TAT-Cre recombinase. Brain tissue was collected five days later and analyzed by flow cytometry.

[0429] BCC10-mCherry significantly enhanced mCherry transport into the brain (3.9-fold increase compared to mCherry alone, p = 0.0014), and BCC10-mCherry delivered mCherry to microglia, neurons, astrocytes, and brain capillary endothelial cells (BCEC) significantly more efficiently than mCherry alone. Figure 8 In the Ai14 mouse model, based on flow cytometry analysis, BCC-Cre showed more efficient editing effects than free Cre in multiple brain cell types. Figure 9 These findings further reinforce the effectiveness of the BCC platform and highlight its potential as a reliable method for transporting therapeutic drugs across the blood-brain barrier.

[0430] antisense oligonucleotide sequences

[0431] The sequence is explained here, where "#" represents 2-methoxyethoxy (2'-MOE), and "+" indicates that locked nucleic acid (LNA) is linked to the subsequent nucleotide base. "" indicates the thiophosphate backbone linkage. The exact Malat1 ASO sequence. Mapt sequence and SOD1 sequence See Table S1.

[0432] Table S1. Sequences of oligonucleotides

[0433]

[0434] Statistical analysis

[0435] Statistical analyses were performed using Graph Pad Prism software. One-way ANOVA was performed for statistical analyses comparing multiple data groups, followed by Tukey's multiple comparison test. Unpaired Student's t-test was used for statistical analyses comparing two groups. Data are expressed as mean ± SD.

[0436] In summary, we have developed a novel BBC platform capable of crossing the blood-brain barrier and delivering biomolecules to a variety of brain cells. This platform holds great promise for applications in a wide range of biomedical fields.

[0437] While various aspects of the invention have been described and illustrated herein, those skilled in the art may implement alternatives to achieve the same objectives. Therefore, the appended claims are intended to cover all such alternative aspects that fall within the true spirit and scope of the invention.

Claims

1. Conjugate of formula (I): (I) in: M represents the transshipment section, which is selected independently in each case; W represents a biological macromolecule or small molecule drug; R 1 For hydrogen or ; R 2 In each case, it is independently selected from (C1-C) 20 )alkyl or (C2-C 20 ) alkyl residues, wherein one to six methylene groups are replaced by -O-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-; m is independently selected from integers 1 to 9 in each case; and L p A linker base that includes one or more of the following: (g)-C(=O)(CH2) q NH-, where q is an integer from 1 to 6; (h) Polyamides containing 1 to 5 ω-amino acids; (i) Triazole (j) 3-Thiopyrrolidine-2,5-dione (k) disulfide bond or (l)C2 to C 20 Oxyalkane; [CONH] indicates that the carboxyl terminus and amino terminus can be located at any connection point.

2. The conjugate of claim 1, wherein L p As a linker base, it includes: (a) 1 to 4 ω-amino acids and 8,9-dihydro-3H-dibenzo[b,f][1,2,3]triazolo[4,5-d]azacyclooctatetraene; (b) Acylalkylamines and 8,9-dihydro-3H-dibenzo[b,f][1,2,3]triazolo[4,5-d]azacyclooctatetraene; (c) 1 to 4 ω-amino acids and 3-thiopyrrolidine-2,5-dione; or (d) Acylalkylamines and 3-thiopyrrolidine-2,5-dione.

3. The conjugate of claim 1, wherein the conjugate is a conjugate of formula (Ia): (him) in: M represents the transshipment section, which is selected independently in each case; W represents a biological macromolecule or small molecule drug; R 1 For hydrogen or ; R 2 In each case, it is independently selected from (C1-C) 20 )alkyl or (C2-C 20 )oxane; m is independently selected from integers 1 to 9 in each case; and n is an integer selected from 1 to 15; and [CONH] indicates that the carboxyl terminus and amino terminus can be located at any connection point.

4. The conjugate of any one of claims 1, 2 or 3, wherein: M is individually selected from cinnamic acid or a cinnamic acid derivative, tryptamine or a tryptamine derivative, MK-0752 or SR-57227 in each case; and / or W is selected from proteins, peptides, nucleic acids, oligonucleotides, or fluorescent dyes.

5. The conjugate of claim 4, wherein: M is individually selected from cinnamic acid or a cinnamic acid derivative, tryptamine or a tryptamine derivative, MK-0752 or SR-57227 in each case; and / or W is selected from proteins, peptides, nucleic acids, oligonucleotides, blue pigment 5 or mCherry; and / or R 2 In each case, it is independently selected from -CH2CH2OCH2CH2OCH2CH2- or (C6) alkyl; and / or m is 1; and / or n is 3.

6. The conjugate of claim 1, wherein: R 1 for ; and / or R 2 In each case, it is independently selected from (C2-C) 20 )O-alkyl.

7. The conjugate of claim 1, wherein: M is MK-0752; and / or R 2 -CH2CH2OCH2CH2OCH2CH2-; and / or m is 1.

8. Compound of formula (II): (II) in: M represents the transshipment section, which is selected independently in each case; R 1 For hydrogen or ; R 2 In each case, it is independently selected from (C1-C) 20 )alkyl or (C2-C 20 ) alkyl residues, wherein one to six methylene groups are replaced by -O-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-; m is independently selected from integers 1 to 9 in each case; and n is an integer selected from 1 to 15; and In formula (II) Represents a bond with hydrogen, or with a biological macromolecule or small molecule pharmaceutical agent; and [CONH] indicates that the carboxyl terminus and amino terminus can be located at any connection point.

9. The compound of claim 8, wherein: M is individually selected in each case from cinnamic acid or a cinnamic acid derivative, tryptamine or a tryptamine derivative, MK-0752 or SR-57227; and R 2 In each case, it is independently selected from -CH2CH2OCH2CH2OCH2CH2- or (C6) alkyl; and / or R 1 for ; and / or m is 1; and / or n is 3.

10. The compound of claim 9, wherein: M is MK-0752; and / or R 2 -CH2CH2OCH2CH2OCH2CH2-; and / or m is 1; and / or n is 3.

11. Compounds of formula (IIa) or (IIb): (IIa) (IIb) in: M represents the transshipment section, which is selected independently in each case; R 1 For hydrogen or ; R 2 In each case, it is independently selected from (C1-C) 20 )alkyl or (C2-C 20 ) alkyl residues, wherein one to six methylene groups are replaced by -O-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-; m is independently selected from integers 1 to 9 in each case; and n is an integer selected from 1 to 15; In formula (IIa) Represents a bond with a hydroxyl group, or with a biological macromolecule or small molecule drug; and In equation (IIb) Represents a bond with hydrogen, or with biological macromolecules or small molecule pharmaceuticals.

12. The compound of claim 11, wherein: M is individually selected in each case from cinnamic acid or a cinnamic acid derivative, tryptamine or a tryptamine derivative, MK-0752 or SR-57227; and R 2 In each case, it is independently selected from -CH2CH2OCH2CH2OCH2CH2- or (C6) alkyl; and / or R 1 for ; and / or m is 1; and / or n is 3.

13. The compound of claim 12, wherein: M is MK-0752; and / or R 2 -CH2CH2OCH2CH2OCH2CH2-; and / or m is 1; and / or n is 3.

14. Compound of formula (IIIa): (IIIa) in: M represents the transshipment section, which is selected independently in each case; R 1 For hydrogen or ; R 2 In each case, it is independently selected from (C1-C) 20 )alkyl or (C2-C 20 ) alkyl residues, wherein one to six methylene groups are replaced by -O-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-; m is independently selected from integers 1 to 9 in each case; and Q is a linker base that includes one or more of the following: (a) An acylalkyl group with 1 to 10 carbon atoms; (b) Polyamides containing 1 to 5 ω-amino acids; (c) alkylene groups with 1 to 10 carbon atoms; or (d) Alkyl groups with 1 to 10 carbon atoms; R 10 Selected from: (a)N3; (b)-C≡CH; (c) ； (d)-SH; (e) ； (f)-NH2; and (a)-COOH.

15. The compound of claim 14, wherein M is individually selected from cinnamic acid or a cinnamic acid derivative, tryptamine or a tryptamine derivative, MK-0752 or SR-57227 in each case.

16. A pharmaceutical preparation comprising a conjugate of any one of claims 1 to 7, or a compound of any one of claims 8 to 15, and a pharmaceutically acceptable carrier.

17. A method for delivering a biomacromolecule to a target within the blood-brain barrier, the method comprising providing the target with a conjugate of any one of claims 1, 2, 3, 6 or 7, wherein the target may be in vivo or in vitro.

18. A method for delivering a biomacromolecule to a target within the blood-brain barrier, the method comprising conjugating the biomacromolecule to a compound of any one of claims 8 to 15 to form a conjugated biomacromolecule, and providing the conjugated biomacromolecule to the target, wherein the target may be in vivo or in vitro.

19. A method of treating a subject with a neurological disorder, the method comprising administering to the subject a conjugate of any one of claims 1, 2, 3, 6 or 7, the conjugate comprising a biomolecule, wherein the biomolecule is an agent for treating the neurological disorder, and wherein the conjugate is capable of crossing the blood-brain barrier or an in vitro model thereof.

20. A method of treating a subject suffering from a neurological disease, the method comprising conjugating a biomacromolecule, which is a medicament for treating the neurological disease, with a compound of any one of claims 8 to 15 to form a conjugated biomacromolecule, and administering the conjugated biomacromolecule to the subject, wherein the conjugated biomacromolecule is capable of crossing the blood-brain barrier or an in vitro model thereof.

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