Ester derivatives of binders that target CA-IV

Abstract

The present invention provides compositions and methods comprising brinzolamide and brinzolamide derivatives as shuttles for crossing the blood-brain barrier via receptor carbonic anhydrase IV (CA-IV), for example. The present invention provides rationally designed reactive small molecule binders based on brinzolamide that can function as shuttles to facilitate CA-IV-mediated brain delivery. The reactive small molecule shuttles can be conjugated to different therapeutic cargo modalities, including nanobodies, therapeutic IgG antibodies, small interfering RNA (siRNA), or antisense oligonucleotides (ASOs), for example, via a single-step N-hydroxysuccinimide (NHS) esterification reaction.

Description

[Technical Field]

[0001] Government Licensing Authority Statement This invention was made with government support under grant numbers NS111369 and GM118191, awarded by the National Institutes of Health. The government has certain rights in this invention.

[0002] Field of Invention This invention relates to a method and shuttle for crossing the blood-brain barrier. [Background technology]

[0003] background The blood-brain barrier (BBB) ​​presents a fundamental bottleneck for the development of effective research tools and therapeutics for the central nervous system (CNS). This structure, primarily composed of brain endothelial cells, requires macromolecules to be delivered via invasive intracranial injection, technically challenging intensive ultrasound, or receptor-mediated transcytosis. The rational design of BBB-crossing macromolecules has long been hampered by an incomplete understanding of the mechanisms involved in transcytosis, and only a few targets, such as transferrin receptors, have been validated for research and therapeutic purposes.

[0004] Therefore, the identification of cross-brain barrier targets, mechanisms, molecules, and methods is necessary to improve the efficiency of research tools and treatments for the central nervous system (CNS). [Overview of the project] [Means for solving the problem]

[0005] overview The present invention provides compositions and methods comprising, for example, brinzolamide and brinzolamide derivatives as shuttles for crossing the blood-brain barrier via receptor carbonic anhydrase IV (CA-IV).

[0006] The present invention provides a rationally designed reactive small molecule binder based on brinzolamide that can function as a shuttle to facilitate CA-IV-mediated brain delivery. The reactive small molecule shuttle can be conjugated to different therapeutic cargo modalities, including nanobodies, therapeutic IgG antibodies, small interfering RNA (siRNA), or antisense oligonucleotides (ASOs), for example, via a single-step N-hydroxysuccinimide (NHS) esterification reaction.

[0007] The present invention provides a robust and adaptable approach for transporting therapeutic substances across the blood-brain barrier using CA-IV-mediated delivery, which is introduced by linking a therapeutic cargo to a brinzolamide derivative CA-IV binder via bioconjugation.

[0008] Aspects of the present invention provide a conjugate comprising a blood-brain barrier (BBB) ​​shuttle selected from brinzolamide or its derivatives, and a therapeutic cargo conjugated to the shuttle.

[0009] Brinzolamide derivatives may, for example, be brinzolamide esters. Brinzolamide derivatives may advantageously have higher specificity for CA-IV compared to brinzolamide. Brinzolamide derivatives may have reduced specificity for CA II compared to brinzolamide.

[0010] Brinzolamide derivatives may contain N-hydroxysuccinimide (NHS) esters. Brinzolamide derivatives containing NHS esters can be formed from a single-step esterification reaction. Brinzolamide derivatives can also be formed from alkylation, tosylation, amination, oxidation, deprotection, Jones oxidation, and esterification of brinzolamide.

[0011] Brinzolamide derivatives can be covalently bonded to NHS esters via 1-8 carbon alkyl linkers. For example, brinzolamide esters are given by formula: [ka] The formula has the following characteristics, where n is 1 to 8, preferably n is 1, 3, 4, 6, or 8.

[0012] Brinzolamide derivatives include the following compounds: [ka] [ka] [ka] You can choose from among them.

[0013] In aspects of the present invention, the brinzolamide derivative may include a pentafluorophenyl (PFP) ester. In this derivative, brinzolamide may be covalently bonded to the PFP ester via polyethylene glycol.

[0014] Brinzolamide derivatives include the following compounds: [ka] You can choose from among them.

[0015] The conjugate may contain multiple small molecule shuttles conjugated to a therapeutic cargo. For example, the conjugate may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more than 14 brinzolamide or brinzolamide derivatives conjugated to a single therapeutic cargo.

[0016] Therapeutic cargo can be biological molecules, such as nucleic acids (e.g., RNA, siRNA, DNA, or ASO), proteins (e.g., enzymes), peptides, antibodies, nanobodies, lipids, polysaccharides, or combinations thereof. Therapeutic cargo can also be non-biological molecules, such as small molecules or dyes.

[0017] The therapeutic cargo can be conjugated to brinzolamide or a brinzolamide derivative via N-hydroxysuccinimide (NHS) ester coupling or copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC).

[0018] Advantageously, the shuttle could be a carbonic anhydrase IV (CA-IV) shuttle. Thus, when provided to cells expressing CA-IV as a surface protein, the binding of the shuttle to the CA-IV protein mediates the transcytosis of therapeutic cargo across the BBB.

[0019] A therapeutic cargo may be a therapeutic cargo for treating disorders affecting the central nervous system. A CA-IV shuttle may be a shuttle for human CA-IV.

[0020] Aspects of the present invention further provide a method for delivering a therapeutic cargo across the target blood-brain barrier (BBB). The method of the present invention includes the step of providing a conjugate to a target comprising brinzolamide or a brinzolamide derivative and a therapeutic cargo conjugated to a shuttle. [Brief explanation of the drawing]

[0021] [Figure 1] Figure 1 is a schematic diagram of transcytosis and antibody conjugation mediated by CA-IV according to the present invention. [Figure 2] Figure 2A shows a structural analysis of the mouse CA-IV binding pocket and its interactions with designed AAV, BZA, and activated BZA (BZA-2C-NHS). Figure 2B shows the structural alignment of BZA across CA-IV homologs. [Figure 3] Figure 3 shows the 1H-NMR verification of the chemical synthesis of NHS-ester-brinzolamide. [Figure 4A] Figure 4A is a structural schematic showing the Ate conjugation to BZA using linker 2C. [Figure 4B]Figures 4B-4G show LC-MS graphs of the BZA-2C-Ate conjugation. [Figure 4C] Same as above. [Figure 4D] Same as above. [Figure 4E] Same as above. [Figure 4F] Same as above. [Figure 4G] Same as above. [Figure 5A-D] Figures 5A-D are graphs showing the results from surface plasmon resonance (SPR) assays of CA-IV and BZA-2C-Ate. [Figure 5E] Figure 5E shows immunofluorescence images of living CA-IV-expressing cells incubated with unconjugated or BZA-conjugated Ate. [Figure 5F] Figures 5F–G show the mean and counted spot intensities of CA-IV-expressing HeLa cells incubated either unconjugated or with a BZA-2C-Ate conjugate, respectively. [Figure 5G] Same as above. [Figure 6A] Figure 6A shows the structure of the selected BZA-Ate linker variant. [Figure 6B] Figures 6B-6M show LC-MS graphs of the unmodified Ate and BZA-Ate linker variant conjugates. [Figure 6C] Same as above. [Figure 6D] Same as above. [Figure 6E] Same as above. [Figure 6F] Same as above. [Figure 6G] Same as above. [Figure 6H] Same as above. [Figure 6I] Same as above. [Figure 6J] Same as above. [Figure 6K] Same as above. [Figure 6L] Same as above. [Figure 6M] Same as above. [Figure 7A-B]Figures 7A-7C are graphs showing the results of the SPR assay for the binding of CA-IV to the BZA-Ate linker variant. [Figure 7C-D] Figures 7A–C are graphs of the results from SPR assays of binding between CA-IV and BZA-Ate linker variants. Figure 7D shows immunofluorescence images of living CA-IV expressing cells incubated with either unconjugated or BZA-conjugated Ate linker variants. [Figure 7E-F] Figures 7E-7F show representative live cell images of CA-IV-expressing HeLa cells incubated with either unconjugated or BZA-conjugated antibodies, after 3 hours and 6 hours of incubation with the antibody, respectively. [Figure 7G-H] Figures 7G–4H show the mean and counted spot intensities of CA-IV-expressing HeLa cells incubated with either unconjugated or BZA-conjugated linker variants, respectively, after 3 hours. [Figure 7I-J] Figures 7I–4J show the mean and counted spot intensities of CA-IV-expressing HeLa cells incubated with either unconjugated or BZA-conjugated linker variants, respectively, after 6 hours. [Figure 8A] Figure 8A shows the structures of two alternative CA-IV binders, acetazolamide (AZA) and dorzolamide (DZA), as NHS ester variants. [Figure 8B] Figures 8B–E show LC-MS graphs of the NHS ester variants of two alternative CA-IV binders, AZA and DZA, respectively. [Figure 8C] Same as above. [Figure 8D] Same as above. [Figure 8E] Same as above. [Figure 8F-G]Figures 8F-G are graphs showing the results from SPR assays with CA-IV and two alternative CA-IV binders, NHS ester variants of AZA and DZA, respectively. [Figure 8H-I] Figures 8H-8I show live cell images of CA-IV-expressing HeLa cells incubated with either AZA-conjugated or DZA-conjugated Ate, respectively. [Figure 8J] Figures 8J-K show the mean and counted spot intensities of CA-IV-expressing HeLa cells incubated with either unconjugated, AZA-conjugated, or DZA-conjugated Ate, respectively. [Figure 8K] Same as above. [Figure 9A] Figure 9A shows the structure of the IgG antibody BZA variant conjugate. [Figure 9B] Figures 9B-9K show LC-MS graphs of BZA-IgG antibody conjugates containing Don. [Figure 9C] Same as above. [Figure 9D] Same as above. [Figure 9E] Same as above. [Figure 9F] Same as above. [Figure 9G] Same as above. [Figure 9H] Same as above. [Figure 9I] Same as above. [Figure 9J] Same as above. [Figure 9K] Same as above. [Figure 10A-D] Figures 10A-E show graphs of the results from SPR assays with CA-IV and BZA-conjugated Don, unconjugated Don, and BZA-conjugated higG1 isotypes, respectively. [Figure 10E-F]Figures 10A–E show graphs of results from SPR assays with CA-IV and BZA-conjugated Don, unconjugated Don, and BZA-conjugated higG1 isotypes, respectively. Figure 10F shows representative immunofluorescence and live-cell images of CA-IV-expressing HeLa cells incubated with either unconjugated-IgG or BZA-conjugated variant antibodies containing Don. [Figure 10G] Figures 10G-H show the mean and counted spot intensities of CA-IV-expressing HeLa cells incubated with either an unconjugated or BZA-conjugated antibody variant containing Don, respectively. [Figure 10H] Same as above. [Figure 11A-B] Figures 11A–D show the SPRs of modified and unmodified therapeutic IgG antibodies, including atezolizumab and Don, tested against purified human and mouse CA-IV. [Figure 11C-D] Same as above. [Figure 11E] Figures 11E-F show the internalization assays in cultured HeLa cells using BZA-modified IgG antibodies containing atezolizumab and Don, as well as unmodified IgG antibodies. [Figure 11F] Same as above. [Figure 12A-B] Figures 12A and 12B show the structures of the BZA-nanobody variants. [Figure 12C] Figures 12C-D show LC-MS analysis of unmodified nanobodies and nanobodies conjugated with BZA. [Figure 12D] Same as above. [Figure 12E-H] Figures 12E-H show graphs of results from SPR assays of binding interactions between unconjugated or BZA-conjugated nanobodies and Fc-tagged CA-IV proteins. [Figure 12I]Figure 12I shows a representative immunofluorescence image of CA-IV-expressing HeLa cells incubated with either unconjugated or BZA-conjugated anti-GFP VHH nanobodies. [Figure 13A-B] Figures 13A and 13B show graphs of results from SPR assays of modified and unmodified nanobodies tested against purified human CA-IV and mouse CA-IV. [Figure 13C] Figures 13C-E show representative immunofluorescence images of CA-IV-expressing HeLa cells incubated with anti-GFP VHH-conjugated nanobodies, BZA-2C-conjugated nanobodies, or BZA-3C-conjugated nanobodies. [Figure 13D] Same as above. [Figure 13E] Same as above. [Figure 14A] Figure 14A shows the structure of the BZA shuttle-siRNA variant. [Figure 14B-E] Figures 14B–D show graphs of results from SPR assays of binding interactions between unconjugated or BZA-conjugated siRNA double helix and Fc-tagged CA-IV protein. [Figure 14F] Figure 14E shows a representative immunofluorescence image of CA-IV-expressing HeLa cells incubated with either unconjugated fluorescently labeled siRNA or BZA-2C conjugated siRNA. [Figure 15A] Figure 15A shows the structure of the BZA shuttle-siRNA variant. [Figure 15B-C] Figures 15B-C show graphs of results from SPR assays of binding interactions between unconjugated or BZA-conjugated siRNA double helix and Fc-tagged CA-IV protein. [Figure 16A-B]Figure 16A shows the structure of the BZA shuttle-small molecule cargo variant. Figure 16B shows representative immunofluorescence images of CA-IV-expressing HeLa cells incubated with either an unconjugated small molecule dye or a BZA-conjugated small molecule dye. [Figure 16C-D] Figure 16C shows the structure of the BZA shuttle-small molecule cargo variant. This schematic diagram illustrates the process of conjugating a fluorescent molecule (Alexa Fluor 647) to brinzolamide using the CuAAC reaction. Figure 16D shows images from an internalization assay using HEK cells overexpressing the CA-IV receptor. The bottom row shows a zoomed-in image of a representative field of view in the image in the top row. [Figure 16E] Figure 16E shows representative images of brain and liver slices from mice injected with BZA-conjugated or unconjugated fluorophores. [Figure 16F] Figure 16F shows fluorescence readings of lysed tissue from animals injected with BZA-conjugated or unconjugated fluorophores, using a plate reader. [Figure 17A-B] This is a diagram. [Figure 17C-D] This is a diagram. [Figure 17E-F] This is a diagram. [Figure 17G-H] This is a diagram. [Figure 18A] Figures 18A-B show immunofluorescence images of whole liver sections from mice systemically administered with unconjugated and BZA-conjugated Ate antibodies. [Figure 18B] Same as above. [Figure 18C] Figure 18C shows the quantification of BZA-conjugated and unconjugated Ate antibodies in peripheral organs at day 7 in mice systemically administered unconjugated and BZA-conjugated Ate antibodies. [Modes for carrying out the invention]

[0022] Detailed explanation The present invention provides compositions and methods comprising, for example, brinzolamide and brinzolamide derivatives as shuttles for crossing the blood-brain barrier via receptor carbonic anhydrase IV (CA-IV).

[0023] Receptors for enhanced blood-brain barrier crossing The blood-brain barrier (BBB) ​​has emerged as a complex, dynamic, and adaptable interface that controls the exchange of substances between the central nervous system (CNS) and the blood, preventing the uncontrolled leakage of substances from the blood into the brain. The cells that make up the structure of the BBB include, for the most part, brain endothelial cells, which are in constant contact with other cells of the CNS (e.g., astrocytes, microglia, neurons, mast cells and pericytes, as well as circulating immune cells) to adapt their behavior to meet the demands of the CNS, respond to pathological conditions, and in some cases are involved in the onset, maintenance, or progression of disease. The complexity of BBB function explains much of the difficulty in developing drugs that can cross the BBB. Utilizing receptors on the BBB interface may provide a way to cross the BBB.

[0024] The present invention provides receptors on the BBB interface, specifically shuttles for carbonic anhydrase IV, and methods of using them to enhance BBB transconjugation and CNS efficacy, for example, by increasing BBB permeability and delivering therapeutic agents across the BBB to the nervous system. While not bound by any particular theory, the novel target receptors disclosed herein may promote enhanced BBB receptor-mediated transcytosis across various mammalian species, including humans.

[0025] In some embodiments, a method for increasing BBB permeability includes the step of providing a shuttle capable of binding to a BBB transverse receptor (e.g., carbonic anhydrase IV), thereby increasing BBB permeability (e.g., via transcytosis). In some embodiments, the activity of at least one of the BBB transverse receptors (e.g., carbonic anhydrase IV) may be reduced via binding to a small molecule. Therefore, in some embodiments, a method for increasing BBB permeability includes the step of reducing the activity of carbonic anhydrase IV, thereby increasing BBB permeability. In some embodiments, the shuttle binds to one or more of the zinc-binding sites (e.g., catalytic pockets) and substrate-binding sites of carbonic anhydrase IV. Carbonic anhydrase IV can be vertebrate carbonic anhydrase IV, including non-human primates and humans. In some embodiments, carbonic anhydrase IV is mouse carbonic anhydrase IV (Car4), human carbonic anhydrase IV (CA4), or a variant or homolog thereof. CA-IV, as used herein, is understood to refer to any variant.

[0026] Carbonic anhydrase IV Carbonic anhydrase (or dehydratase) (CA) is a family of enzymes that catalyze the interconversion of carbon dioxide and water with the dissociated ion of carbonic acid. CA is involved in a variety of biological processes, including respiration, calcification, acid-base balance, bone resorption, and the formation of aqueous humor, cerebrospinal fluid, saliva, and gastric acid.

[0027] This invention provides a BBB crossing shuttle (CA-IV shuttle) that utilizes receptor carbonic anhydrase IV, capable of facilitating drug delivery across the blood-brain barrier. Carbonic anhydrase IV is responsible for the reversible hydration reaction of CO2 (H2O + CO2 ⇔ HCO3). - +H + This enzyme catalyzes CO2 and H + and HCO3 -Carbonic anhydrase is an isozyme belonging to the family of zinc metalloenzymes, which are capable of regulating intracellular and extracellular concentrations of carbon dioxide. Carbonic anhydrase is involved in various biological processes, including respiration, calcification, acid-base balance, bone resorption, and the formation of aqueous humor, cerebrospinal fluid, saliva, and gastric acid. Carbonic anhydrase exhibits a wide range of diversity in tissue distribution and their intracellular localization. Mammalian carbonic anhydrase has at least seven genetically distinct isozymes, called I-VII, each of which catalyzes the reversible hydration of carbon dioxide via the zinc-hydroxylase mechanism. Physiological functions regulated by carbonic anhydrase include, for example, the saturation of HCO3 in the lungs during respiration. - Removal of HCO3 in the kidneys - This includes the reuse of carbon dioxide, the production of aqueous humor in the eye, cerebrospinal fluid in the brain, gastric juice production in the stomach, pancreatic juice production, and bone resorption by osteoclasts. Carbonic anhydrase family members also play important roles in metabolic processes including urea production, gluconeogenesis, and lipid biosynthesis.

[0028] Unlike other carbonic anhydrases, which are either soluble or bound to the plasma membrane by a transmembrane domain, carbonic anhydrase IV is a membrane isozyme anchored with glycosylphosphatidyl-inositol. Carbonic anhydrase IV is widely conserved across vertebrates and has a similar CNS expression profile in humans, and recent single-cell analyses of human cerebral vascular structures have confirmed CA-IV expression in the human BBB. Carbonic anhydrase IV has been shown to regulate pH, which may be related to nerve discharge and affect neuronal function via ion gate channels.

[0029] In some embodiments, the carbonic anhydrase IV disclosed herein is human carbonic anhydrase IV. CA-IV is known to localize on the luminal surface of endothelial cells throughout the cortex and cerebellum, where it enzymatically modulates the carbon dioxide-bicarbonate balance. Human CA-IV has "high activity" in CO2 hydration and HCO3 -It has been previously characterized as a 35 kDa protein with higher activity than other isozymes in catalyzing dehydration. Generally, human CA-IV contains a 260-amino acid "CA domain" with an 18-amino acid signal sequence at the N-terminus of the protein for endoplasmic reticulum (ER) translocation and active site amino acid residues showing 30-36% homology with cytoplasmic CA. At the C-terminus, a further 27 amino acid residues containing a 21-amino acid hydrophobic sequence sufficient for transmembrane transport are preceded by a 6-amino acid signal sequence for GPI anchoring. Amino acid residue Ser266 was identified as the site for GPI anchor binding. Removal of the C-terminal hydrophobic domain found in CA-IV precursors has a significant impact on GPI anchoring, cell surface expression, and the realization of enzyme activity. Based on the amino acid sequence inferred from the nucleotide sequence, human CA-IV does not contain the classical consensus site (Asn-Xxx-Ser / Thr) for N-glycosylation. Human CA-IV does not contain oligosaccharide chains, whereas other mammalian carbonic anhydrase IV (e.g., mouse carbonic anhydrase IV) is a glycoprotein that has one to several oligosaccharide side chains.

[0030] In some embodiments, the carbonic anhydrase IV disclosed herein is mouse carbonic anhydrase IV. CA-IV has recently been found to be the mouse protein most strongly positively correlated with plasma-protein uptake in the brain (slightly stronger than the often targeted transferrin receptor). This property is useful for identifying receptors for enhanced BBB cross-section. CA-IV is expressed in the GI tubules, kidneys and lungs, as well as in taste receptor cells, which enable it to sense carbonation. Mouse and human CA-IV are highly homologous and contain the same amino acid (e.g., histidine residue 64 (His64)) at a position crucial to enzymatic activity, with some differences including, for example, that mouse CA-IV is an N-linked glycoprotein and that the CO2 hydration rate catalyzed by mouse CA-IV is considerably lower than that of human CA-IV. While not bound by any theory, the lower enzymatic activity of mouse CA-IV may be related to the substitution of Gly63 with Gln63 in human CA-IV, among several other amino acid substitutions. Another difference between mouse CA-IV and human CA-IV is the Val-131 to Asp-136 segment (segments in the 130s), which forms an α-helix in mice and an extended loop in human CA-IV.

[0031] In some embodiments, the carbonic anhydrase IV disclosed herein as a receptor for enhancing BBB cross-section may be any carbonic anhydrase IV, e.g., mouse CA-IV, human CA-IV, or its homolog or variant. Carbonic anhydrase IV homologs and / or variants may originate from vertebrate species, including but not limited to mice, rats, humans, cattle, rabbits, monkeys, pigs, horses, rainbow trout, chimpanzees, squirrels, chickens, goats, and sheep. Carbonic anhydrase IV homologs from various species can be found in public databases identifiable to those skilled in the art, including, for example, UniProt, NCBI, and Swiss-Prot.

[0032] In some embodiments, small molecules can interact with carbonic anhydrase IV (e.g., mouse CA-IV, human CA-IV or its homolog or variant) disclosed herein, thereby increasing blood-brain barrier (BBB) ​​permeability (e.g., via transcytosis). In some embodiments, the increase in BBB permeability is achieved by altering (e.g., increasing or decreasing) the carbonic anhydrase IV activity, for example, by reducing its activity.

[0033] In some embodiments, modification of carbonic anhydrase IV activity is achieved by a shuttle that interacts with one or more active sites of carbonic anhydrase IV, including a zinc-binding site and a hydrophobic substrate-binding pocket. For example, the shuttle can interact with the zinc-binding site, the hydrophobic substrate-binding pocket, or both.

[0034] The zinc-binding site in carbonic anhydrase IV has a conserved structure governed by a β-sheet higher-order structure, with a metal-binding site formed by at least three His residues. While not bound by any particular theory, the zinc-binding site is thought to lie on one face of the β-sheet at the bottom of a 15 Å deep conical active site cleft where three His residues with tetrahedral geometry and hydroxide ions coordinate to zinc. The hydrophobic substrate-binding pocket is adjacent to the zinc-bound hydroxide and is largely formed by bulky residues, such as Val at its base and Val, Trp, and Leu at its neck. This pocket is highly conserved across all active isozymes based on phylogenetic comparisons. While not bound by any particular theory, the hydrophobic pocket has the minimum width and depth necessary for efficient catalysis, and linear free energy relationships suggest that the volume of amino acid residues at the base of the pocket and the hydrophobicity of residues at the neck are important for activity. Both the zinc binding site and the hydrophobic substrate binding pocket are highly conserved among carbonic anhydrase isozymes.

[0035] Brinzolamide and brinzolamide derivatives Brinzolamide (BZA) is a highly specific, non-competitive, reversible carbonic anhydrase II (CA II) inhibitor that has been shown to reduce intraocular pressure in patients with ocular hypertension or open-angle glaucoma. Brinzolamide was approved by the FDA as an external preparation in 1998 under the trade name AZOPT, and was subsequently approved as a combination product with timolol under the trade name AZARGA, and as brimonidine tartrate under the trade name SIMBRINZA.

[0036] N-desethyl brinzolamide is an active metabolite of brinzolamide, which exhibits CA1 inhibitory activity when brinzolamide is present, and also accumulates in red blood cells. However, none of the other known metabolites of brinzolamide (N-desmethoxypropyl brinzolamide and O-desmethyl brinzolamide) have activity, or their activities are currently unknown.

[0037] The structure of brinzolamide (Brizolamide) is

Chemical Formula

[0038] It has been discovered according to the present invention that brinzolamide, in particular brinzolamide derivatives, can act as a shuttle for receptors on the BBB interface, specifically carbonic anhydrase IV, and methods of using the same to enhance BBB crossing and CNS efficacy, for example, as a method for increasing permeability of the BBB and delivering therapeutic agents across the BBB to the nervous system.

[0039] This invention proposes an innovative method to overcome this challenge using receptor-mediated transcytosis (RMT) that utilizes carbonic anhydrase IV (CA-IV) as a BBB receptor. While not limited by mechanism of action, this invention is the first to demonstrate that the carbonic anhydrase inhibitor brinzolamide binds to mouse CA-IV at the same catalytic site as some BBB-crossing capsids (including 9P31 and 9P36), suggesting a potential mechanism for transporting therapeutic agents across the BBB.

[0040] Brinzolamide exhibits binding affinity to CA-IV at a half-maximal inhibitory concentration (IC50) of 45 nM. Its safety profile is well-established, with an oral LD50 in rats between 1000 mg / kg and 2000 mg / kg, and a very long half-life (111 days) in whole blood due to its strong adhesion to carbonic anhydrase-containing erythrocytes and minimal metabolism. Importantly, the brinzolamide binding pocket on CA-IV is conserved across species, and this invention has demonstrated the potential for therapeutic molecules conjugated with brinzolamide to be used across animals and animal models.

[0041] As further described below, chemical ligation reactions, such as lysine-N-hydroxysuccinimide (NHS) ester coupling or copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), may be used in the present invention to conjugate brinzolamide (BZA) derivatives with a therapeutic cargo comprising nanobodies, therapeutic IgG antibodies, and small molecules. These derivatives are specifically designed to function as effective binders to CA-IV.

[0042] As described herein, cell-based assays, surface plasmon resonance (SPR), and in vivo intravenous delivery have been used to verify the successful binding of these conjugated molecules to CA-IV and their subsequent internalization.

[0043] Accordingly, aspects of the present invention provide selected brinzolamide derivatives. For example, aspects of the present invention provide brinzolamide derivatives synthesized using linkers and esters as described below.

[0044] BZA-2C-NHS(1~6) [ka] [ka] AcOH = Acetic acid, THF = Tetrahydrofuran, DCM = Dichloromethane, DMAP = 4-Dimethylaminopyridine, EDCI = N-(3-Dimethylaminopropyl)-N'-Ethylcarbodiimide hydrochloride, TSTU = N,N,N',N'-Tetramethyl-O-(N-Succinimidyl)uronium tetrafluoroborate, DIPEA = N,N-Diisopropylethylamine

[0045] Under nitrogen, NaBH4 (4.93 mg, 0.13 mmol, 1.0 equivalent) was added to an oven-dried drum vial equipped with a Teflon®-coated stirring bar. The solid was suspended in anhydrous THF (0.44 mL), and the suspension was cooled to 0°C in an ice bath. Acetic acid (33.6 μL, 35.2 mg, 0.59 mmol, 4.5 equivalents) was slowly added (gas generation). The resulting solution was stirred at 0°C for 30 minutes, then warmed to room temperature (23°C), and stirred for another 30 minutes. Brinzolamide (1) (50.0 mg, 0.13 mmol, 1.0 equivalent) and acetic acid (11.2 μL, 11.7 mg, 0.20 mmol, 1.5 equivalents) were subsequently added. After completely dissolving brinzolamide (for approximately 5 minutes), acetaldehyde (5M in THF, 26.1 μL, 0.13 mmol, 1.0 equivalent) was added dropwise. The reaction mixture was stirred at room temperature for 18 hours. Then, saturated aqueous NaHCO3 (1.0 mL) was added. The aqueous phase was extracted with phenylethylamine (3 × 3 mL). The combined organic phase was dried over Na2SO4, filtered, and the volatile materials were evaporated under reduced pressure. The crude material was purified by column chromatography on silica (hexane:phenylethylamine = 2:3) to obtain tertiary amine 2 as a colorless oil (53.7 mg, 0.13 mmol, quantitative). 1 H NMR (500 MHz, CDCl3): δ 7.63 (s, 1H), 5.50 (s, 2H), 4.27 (dd, J = 10.7, 5.3 Hz, 1H), 4.14-4.03 (m, 1H), 3.61-3.48 (m, 3H), 3.44 (ddd, J = 9.6, 6.2, 5.3 Hz, 1H), 3.34 (s, 3H), 3.10 (dt, J = 13.9, 7.0 Hz, 1H), 2.57 (qq, J = 13.5, 6.9, 6.3 Hz, 4H), 1.92 (dqd, J = 12.1, 6.6, 6.2, 3.4Hz, 2H), 1.06 (t, J = 7.1 Hz, 6H). 13 C{ 1H} NMR (126 MHz, CDCl3): δ 146.7, 145.7, 139.3, 130.8, 69.3, 58.8, 52.8, 47.6, 46.0, 44.7, 29.6, 15.0. FTIR (NaCl, thin film): 3271, 2968, 2932, 1459, 1344, 1172, 1158, 1104, 1040, 1016, 653cm -1 . HRMS:(ESI-TOF)C 14 H 26 N3O5S3[M+H] + The calculated value is 412.1029, and the measured value is 412.1065. TLC (1:4 hexane:SiO), R f :0.64(UV).

[0046] [ka] Under nitrogen, a suspension of 2 (104 mg, 253 μmol, 1.0 equivalent) of boron tribromide in 5.1 mL of CH2Cl2 in two drum vials was cooled to -78°C in a dry ice-acetone bath. Boron tribromide (1.01 mL, 1.01 mmol, 4.0 equivalents) was added dropwise to the suspension. The reaction mixture was stirred at -78°C for 10 minutes, and then gradually warmed to room temperature (23°C). After stirring at room temperature for 20 hours, it was cooled to 0°C in an ice bath, and MeOH (2.5 mL) was carefully added. The resulting homogeneous mixture was stirred at room temperature for 30 minutes. Subsequently, the volatile material was evaporated, and the crude material was purified by preparative HPLC (C18 column, 9.4 × 250 mm) eluting with 20% MeCN over 2.5 minutes at a flow rate of 5 mL / min, followed by a gradient of 20 to 70% MeCN over 10 minutes at a flow rate of 5 mL / min, followed by 100% MeCN over 2.5 minutes at a flow rate of 5 mL / min. Bromide 4 (14.8 mg, 32.1 μmol, 13%) was obtained as a colorless oil, and alcohol 3 (42.4 mg, 107 μmol, 42%) was obtained as a colorless oil.

[0047] Analysis data for alcohol 3: 1 H NMR (400 MHz, CD3CN): δ 7.67 (s, 1H), 6.24 (s, 2H), 4.45 (s, 1H), 4.03 (dd, J = 15.0, 10.5 Hz, 1H), 3.77-3.69 (m, 1H), 3.57 (t, J = 6.1 Hz, 2H), 3.49 (dt, J = 14.3, 7.4 Hz, 1H), 3.09 (dt, J = 13.6, 6.8 Hz, 1H), 2.85-2.59 (m, 4H), 1.96 (s, 1H), 1.87-1.75 (m, 2H), 1.08 (t, J = 7.2 Hz, 6H). 13 C{ 1 H} NMR (101 MHz, CD3CN): δ 148.7, 139.2, 131.3, 59.3, 53.3, 47.6, 46.3, 45.6, 35.4, 32.6, 14.9. FTIR (NaCl, thin film): 3347, 2963, 2653, 2263, 1558, 1453, 1346, 1156, 918, 741cm -1 . HRMS:(ESI-TOF)C 13 H 24 N3O5S3[M+H] + The calculated value is 398.0873, and the measured value is 398.0881. TLC(95:5 CH2Cl2:MeOH), R f :0.28(UV).

[0048] Analysis data for bromide 4: 1H NMR (400 MHz, CD3CN): δ 7.58 (s, 1H), 6.14 (s, 2H), 4.39 (dd, J = 10.7, 5.5 Hz, 1H), 4.02 (ddd, J = 15.1, 10.8, 1.1 Hz, 1H), 3.67 (dd, J = 15.0, 5.5 Hz, 1H), 3.62 - 3.48 (m, 3H), 3.16 (dt, J = 13.9, 6.4 Hz, 1H), 2.57 (q, J = 7.1 Hz, 4H), 2.22-2.12 (m, 2H), 1.05 (t, J = 7.1 Hz, 6H). 13 C{ 1 H} NMR (101 MHz, CD3CN): δ 148.7, 147.4, 138.7, 131.2, 53.3, 48.2, 47.7, 45.3, 32.8, 31.6, 15.2. FTIR (NaCl, thin film): 3356, 3274, 3094, 2969, 1152, 1452, 1342, 1157, 911, 648 cm⁻¹ -1 . HRMS:(ESI-TOF)C 13 H 23 BrN3O4S3[M+H] + The calculated value is 460.0029 and the measured value is 460.0034. TLC(3:7 ヘキサン:EtOAc)、R f 0.77 (UV).

[0049]

change

[0050] [ka] In a 20 mL scintillation vial equipped with a stirring bar, carboxylic acid 5 (25.0 mg, 60.8 μmol, 1.0 equivalent) was dissolved in a mixture of MeCN (2.28 mL) and H2O (759 μL) and cooled to 0°C. To this solution, N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU) (36.6 mg, 122 μmol, 2.0 equivalent) and N,N-diisopropylethylamine (21.2 μL, 15.7 mg, 122 μmol, 2.0 equivalent) were added. After 20 minutes at 0°C, the volatile materials were evaporated under reduced pressure. The crude reaction mixture was taken into MeCN (approximately 1.5 mL) and purified by preparative HPLC (C18 column, 9.4 × 250 mm) eluting with a 40 to 50% MeCN gradient over 11 minutes at a flow rate of 5 mL / min. NHS-ester 6 (9.5 mg, 18.7 μmol, 31%) was obtained as a colorless oily substance. 1 H NMR (400 MHz, CD3CN) δ 7.58 (s, 1H), 6.14 (s, 2H), 4.39 (dd, J = 10.8, 5.5 Hz, 1H), 4.02 (dddd, J = 15.0, 10.3, 9.3, 0.9 Hz, 1H), 3.81-3.55 (m, 2H), 3.39 (ddt, J = 53.6, 14.0, 6.9 Hz, 1H), 3.10-2.95 (m, 1H), 2.78 (s, 2H), 2.67 (td, J = 6.7, 1.8 Hz, 1H), 2.62-2.48 (m, 4H), 1.96 (s, 2H), 1.04 (td, J = 7.1, 1.4 Hz, 6H). 13 C{ 1 H} NMR (101 MHz, CD3CN) δ 173.1, 172.8, 171.0, 168.2, 148.9, 147.6, 138.3, 131.2, 53.6, 49.2, 45.3, 34.9, 32.8, 26.4, 15.2. HRMS:(ESI-TOF)C 17 H 25 N4O8S3[M+H]+ The calculated value is 509.0829, and the measured value is 509.0839.

[0051] BZA-N3(7) [ka] In a drum vial equipped with a stirring bar, bromide 4 (14.8 mg, 32.6 μmol, 1.0 equivalent) and sodium azide (6.35 mg, 97.7 μmol, 3.0 equivalent) were dissolved in dimethylformamide (163 μL). The solution was heated overnight at 80°C. The mixture was then diluted with HCl (1.5 mL) and washed with brine (1.5 mL). The aqueous layer was extracted three times with HCl (3 mL), and the combined organic layers were dried over MgSO4. The dried organic layers were concentrated under vacuum, and the resulting residue was then treated with silica and purified by column chromatography (HCl:hexane = 6:4). Azide 7 (6.20 mg, 14.7 μmol, 45%) was isolated as a colorless oil. 1 H NMR (500 MHz, CD3Cl): δ 7.69 (s, 1H), 5.24 (s, 2H), 4.30 (s, 1H), 4.14 (dd, J = 14.7, 10.6 Hz, 1H), 3.67-3.36 (m, 4H), 3.07 (dt, J = 13.7, 6.7 Hz, 1H), 2.61 (tq, J = 13.1, 6.4 Hz, 4H), 2.00-1.84 (m, 2H), 1.07 (t, J = 7.1 Hz, 6H). 13 C{ 1 H} NMR (101 MHz, CD3Cl): δ 146.9, 139.6, 130.9, 52.9, 48.5, 47.9, 46.3, 44.9, 32.1, 29.9, 28.9, 14.9. FTIR (NaCl, thin film): 3334, 2923, 2358, 2097, 1618, 1458, 1342, 1259, 1154, 845, 608cm -1 . HRMS:(ESI-TOF)C 13H 23 N6O4S3[M+H] + The calculated value is 423.0937, and the measured value is 423.1033. TLC (6:4 hexane:alkyl), R f :0.67(UV).

[0052] BZA-nC-NHS (n=1, 3, 4, 6, and 8) General procedure 1: Alkylation of the BZA core (8) [ka] In a 50 mL round-bottom flask equipped with a stirring bar, (S)-6-chloro-4-hydroxy-3,4-dihydro-2H-thieno[3,2-e][1,2]thiazine 1,1-dioxide(8) (2.40 g, 10.0 mmol, 1.0 equivalent) was dissolved in DMSO (12.0 mL), and potassium carbonate (4.15 g, 30.0 mmol, 3.0 equivalents) was added. The suspension was stirred at room temperature, and a solution of bromoalkylmethyl ether (12.0 mmol, 1.2 equivalents) in DMSO (2.0 mL) was added dropwise over a period of 1 hour. After the addition was complete, the reaction mixture was stirred for a further 1.5 hours. The mixture was then poured into brine (20 mL) and extracted with Et2O (3×). The combined extracts were washed with 1M NaOH (30 mL) and 1:1 bleach / water (40 mL), dried over Na2SO4, filtered, and the volatile materials were evaporated under reduced pressure. The crude material was purified by column chromatography on silica to obtain alkylated BZA cores.

[0053] General procedure 2: Tosylation of alcohol [ka] In a 100 mL round-bottom flask equipped with a stir bar, the corresponding secondary alcohol (1.0 eq.) and triethylamine (2.5 eq.) were dissolved in THF (0.5 M with respect to the alcohol), and the solution was cooled to 0°C in an ice bath. To this solution, a solution of p-toluenesulfonyl chloride (2.0 eq.) in THF (2 M with respect to p-TsCl) was added dropwise. After the addition, the reaction mixture was warmed to room temperature and stirred for 3 hours. Water was then added, and the aqueous phase was extracted with ethyl acetate (3×). The combined organic layers were dried over Na₂SO₄, filtered, and volatile materials were evaporated under reduced pressure. The crude material was purified by column chromatography on silica to give the tosyl-protected alcohol.

[0054] General Procedure 3: Amination of Tosylate

Chemical Structure

[0055] General Procedure 4: Formation of Benzyl Sulfide

Chemical Structure

[0056] General Procedure 5: Oxidation of Benzyl Sulfide

Chemical Structure

[0057] General Procedure 6: Deprotection of Methoxy Group

Chemical Structure

[0058] General procedure 7: Jones oxidation of carboxylic acid [ka] The corresponding alcohol (1.0 equivalent) was dissolved in acetone (0.075 M), and the solution was cooled to 0°C in an ice bath. Jones' reagent (2.5 M solution in 3:1 water / concentrated H2SO4, 4.7 equivalents) was added dropwise. The reaction mixture was stirred at 0°C for 2 hours, after which neopentyl alcohol (1 mg per 1 μmol of starting material) was added all at once. The mixture was stirred at room temperature for 1 hour. The mixture was then filtered through a cotton plug and washed with acetone. Unless otherwise indicated, silica was added to the solution, the suspension was concentrated, and the corresponding carboxylic acid was obtained by purification by column chromatography on silica.

[0059] General Procedure 8: NHS Ester Formation [ka] In a 20 mL scintillation vial equipped with a stirring bar, the corresponding carboxylic acid (1.0 equivalent) was dissolved in a mixture of MeCN and H2O (3:1, 0.02 M) and cooled to 0°C. To this solution, N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU) (2.0 equivalents) and N,N-diisopropylethylamine (2.0 equivalents) were added. After 20 minutes at 0°C, the volatile materials were evaporated under reduced pressure. The crude reaction mixture was placed in MeCN (approximately 1.5 mL) and purified by preparative HPLC (C18 column, 9.4 × 250 mm) elution with a water / acetonitrile mixture to obtain the corresponding NHS-ester.

[0060] BZA-1C-NHS(9) Compound S1 [ka] Compound S1 was synthesized on a 10.0 mmol scale according to general procedure 1 using 1-bromo-2-methoxyethane (1.13 mL, 1.67 g, 12.0 mmol, 1.2 equivalents). After column chromatography on silica (hex:HCl = 4:1 to 7:3), the target compound was isolated as a colorless oil (3.00 g, 10.0 mmol, quantitative). 1 H NMR (400 MHz, CDCl3): δ 6.98 (s, 1H), 4.56 (ddd, J = 8.6, 4.2, 2.8 Hz, 1H), 4.36 (dd, J = 15.8, 4.2 Hz, 1H), 4.11 (d, J = 8.8 Hz, 1H), 3.90-3.80 (m, 2H), 3.75 (ddd, J = 10.5, 7.6, 2.7 Hz, 1H), 3.60 (ddd, J = 10.8, 5.9, 2.9 Hz, 1H), 3.42 (ddd, J = 14.8, 7.5, 2.8 Hz, 1H), 3.30 (s, 3H), 1.60 (s, 1H). 13 C{ 1H} NMR (101 MHz, CDCl3): δ 143.8, 135.9, 133.2, 126.4, 71.8, 62.0, 58.9, 55.2, 50.0. FTIR (NaCl, thin film): 3425, 2924, 2359, 1420, 1331, 1156, 1026, 833, 734, 668cm -1 . HRMS:(ESI-TOF)C 19 H 12 ClNaNO4S2[M+Na] + The calculated value is 319.9788, and the measured value is 319.9802. TLC (3:7 hexane:HCl), R f :0.58(UV).

[0061] Compound S2 [ka] Compound S2 was synthesized on a 10.0 mmol scale according to general procedure 2. After column chromatography on silica (hex:pharmaceutically pharmaceutically = 3:1), the target compound was isolated as a colorless oil (3.70 g, 8.17 mmol, 82%). 1 H NMR (500 MHz, CDCl3): δ 7.90-7.76 (m, 2H), 7.46-7.38 (m, 2H), 6.64 (s, 1H), 5.37 (dd, J = 4.1, 3.1 Hz, 1H), 4.24 (dd, J = 16.2, 4.1 Hz, 1H), 4.11 (dd, J = 16.2, 3.1 Hz, 1H), 3.69-3.62 (m, 2H), 3.62-3.53 (m, 2H), 3.41-3.36 (m, 1H), 3.35 (s, 3H), 2.51 (s, 3H). 13 C{ 1{13C} NMR (125 MHz, CDCl3): δ 146.1, 136.9, 136.4, 135.7, 133.0, 130.4, 128.1, 126.2, 72.2, 68.3, 59.1, 52.1, 49.7, 21.9. FTIR (NaCl, thin film): 2922, 2357, 1597, 1427, 1356, 1168, 1119 cm -1 -1. HRMS (ESI-TOF) for C 16 H 18 NNaO6S3 [M+Na] + : calculated 473.9877, found 473.9896. TLC (7:3 hexane:EtOAc), R f f = 0.64 (UV).

[0062] Compound S3

Chemical Structure

[0063] Compound S4 [ka] Compound S4 was synthesized on a 2.05 mmol scale according to general procedure 4. After column chromatography on silica (hex:HCl=85:15), the target compound was isolated as a yellow oil (634 mg, 1.44 mmol, 70%). 1 H NMR (500 MHz, CDCl3): δ 7.35-7.15 (m, 5H), 6.89 (s, 1H), 4.23 (dd, J = 10.7, 5.3 Hz, 1H), 4.14-3.94 (m, 3H), 3.70 (dd, J = 14.6, 5.3 Hz, 1H), 3.66-3.49 (m, 3H), 3.36 (s, 3H), 3.25-3.15 (m, 1H), 2.48 (qd, J = 7.2, 5.2 Hz, 4H), 1.01 (t, J = 7.1 Hz, 6H). 13 C{ 1 H} NMR (125 MHz, CDCl3): δ 145.9, 140.0, 136.6, 136.0, 132.1, 128.9, 128.6, 127.7, 72.3, 58.9, 52.1, 48.0, 47.9, 44.4, 43.0, 14.8. FTIR (NaCl, thin film): 2923, 2550, 2331, 1724, 1333, 1142, 678cm-1 . HRMS:(ESI-TOF)C 20 H 28 N2O3S3[M+H] + The calculated value is 440.1262, and the measured value is 441.1323. TLC(85:15 Hexane:ethyl), R f :0.13(UV).

[0064] Compound S5 [ka] Compound S5 was synthesized on a 1.37 mmol scale according to general procedure 5. Following column chromatography on silica (DCM:MeOH = 99:1), the target compound was isolated as a brown oily substance (444 mg, 1.12 mmol, 81%). The compound was isolated along with aromatic impurities that could be separated in subsequent steps. 1 H NMR (400 MHz, CDCl3): δ 7.64 (s, 1H), 5.33 (bs, 2H), 4.43 (d, J = 5.7 Hz, 1H), 4.34 (dd, J = 10.7, 5.3 Hz, 1H), 4.13 (dd, J = 14.7, 10.7 Hz, 1H), 3.79 (dd, J = 14.7, 5.3 Hz, 1H), 3.66-3.56 (m, 3H), 3.36 (s, 3H), 3.32-3.19 (m, 1H), 2.56 (dtt, J = 19.9, 12.9, 7.0 Hz, 4H), 1.05 (t, J = 7.1 Hz, 6H). 13 C{ 1 H} NMR (101 MHz, CDCl3): δ 146.6, 146.1, 139.6, 130.9, 72.3, 59.1, 52.7, 48.3, 47.7, 44.6, 44.0, 23.5, 14.8. FTIR (NaCl, thin film): 3330, 2925, 2850, 2356, 2336, 1714, 1458, 1270, 718, 615cm -1 . HRMS: (ESI-TOF)C 13 H 23 N3O5S3[M] + calculated 397.0800, found 397.0582. TLC (99:1 DCM:MeOH), R f f: 0.16 (UV).

[0065] Compound S6

Chemical Structure

[0066] Compound S7

Chemical Structure

[0067] Compound 9

Chemical Structure

[0068] BZA-3C-NHS (10) Compound S8

Chemical Structure

[0069] Compound S9 [ka] Compound S9 was synthesized on a 10.0 mmol scale according to general procedure 2. After column chromatography on silica (hex:HCl = 4:1 to 3:2), the target compound was isolated as a colorless oil (4.82 g, 10.0 mmol, quantitative). 1 H NMR (500 MHz, CDCl3): δ 7.92-7.71 (m, 2H), 7.50-7.35 (m, 2H), 6.59 (s, 1H), 5.32 (dd, J = 3.9, 2.4 Hz, 1H), 4.21 (ddd, J = 16.3, 3.9, 0.8 Hz, 1H), 3.90 (dd, J = 16.3, 2.5 Hz, 1H), 3.39 (t, J = 6.0 Hz, 2H), 3.34 (s, 3H), 3.16 (ddd, J = 13.3, 7.3, 5.6 Hz, 1H), 2.51 (s, 3H), 1.80-1.47 (m, 4H). 13 C{ 1 H} NMR (125 MHz, CDCl3): δ 146.3, 136.4, 136.4, 135.6, 132.9, 130.5, 128.0, 126.1, 72.0, 67.9, 58.8, 50.5, 49.6, 26.6, 25.0, 21.9. FTIR (NaCl, thin film): 2927, 2357, 1339, 1169, 1114, 921, 739cm -1 . HRMS:(ESI-TOF)C 18 H 26 ClN2O6S3[M+NH4] + The calculated value is 497.0636, and the measured value is 497.0645. TLC (7:3 hexane:HCl), R f :0.58(UV).

[0070] Compound S10 [ka] Compound S10 was synthesized on a 10.0 mmol scale according to general procedure 3. After column chromatography on silica (hex:alkyl=85:15), the target compound was isolated as a colorless oil (2.10 g, 5.51 mmol, 55%). 1 H NMR (500 MHz, CDCl3): δ 6.95 (s, 1H), 4.18 (dd, J = 10.9, 5.1 Hz, 1H), 4.07 (dd, J = 14.7, 10.8 Hz, 1H), 3.52 (ddt, J = 20.5, 14.2, 4.3 Hz, 2H), 3.41 (t, J = 6.0 Hz, 2H), 3.33 (s, 3H), 2.98 (ddd, J = 13.2, 7.2, 5.5 Hz, 1H), 2.67-2.46 (m, 4H), 1.85-1.59 (m, 4H), 1.06 (t, J = 7.1 Hz, 6H). 13 C{ 1 H} NMR (125 MHz, CDCl3): δ 145.4, 135.5, 133.0, 126.2, 72.1, 58.8, 52.1, 48.0, 46.3, 44.7, 26.7, 25.6, 15.0. FTIR (NaCl, thin film): 2933, 2355, 1417, 1339, 1165, 1115, 733cm -1 . HRMS:(ESI-TOF)C 15 H 26 ClN2O3S2[M+H] + The calculated value is 381.1068, and the measured value is 381.1289. TLC (8:2 hexane:ethyl), R f :0.46(UV).

[0071] Compound S11 [ka] Compound S11 was synthesized on a 5.49 mmol scale according to general procedure 4. After column chromatography on silica (hex:toluene=85:15), the target compound was isolated as a yellow oil (1.39 g, 2.96 mmol, 54%). 1 H NMR (500 MHz, CDCl3): δ 7.32-7.19 (m, 5H), 6.86 (s, 1H), 4.13 (dd, J = 10.8, 5.2 Hz, 1H), 4.05 (s, 2H), 4.03-3.94 (m, 1H), 3.47 (ddd, J = 20.6, 14.6, 6.5 Hz, 2H), 3.41 (t, J = 5.9 Hz, 2H), 3.33 (s, 3H), 2.95 (ddd, J = 13.2, 7.3, 5.5 Hz, 1H), 2.49 (qd, J = 7.1, 3.8 Hz, 4H), 1.88-1.50 (m, 4H), 1.01 (t, J = 7.1 Hzi, 6H). 13 C{ 1 H} NMR (125 MHz, CDCl3): δ 145.4, 140.1, 136.7, 136.3, 132.1, 129.1, 128.8, 127.8, 72.1, 58.8, 52.2, 47.9, 46.7, 44.7, 43.2, 26.7, 25.6, 15.0. FTIR (NaCl, thin film): 2924, 2358, 2356, 1338, 1166, 1114, 737, 668cm -1 . HRMS:(ESI-TOF)C 22 H 33 N2O3S3[M+H] + The calculated value is 469.1648, and the measured value is 469.1647. TLC (8:2 hexane:ethyl), R f :0.56(UV).

[0072] Compound S12 [ka] Compound S12 was synthesized on a 2.94 mmol scale according to general procedure 5. After column chromatography on silica (DCM:MeOH=99:1), the target compound was isolated as a brown oily substance (855 mg, 2.01 mmol, 68%). 1 H NMR (400 MHz, CDCl3): δ 7.63 (s, 1H), 5.31 (bs, 2H), 4.24 (dd, J = 10.8, 5.2 Hz, 1H), 4.14-3.99 (m, 1H), 3.58 (dd, J = 14.8, 5.3 Hz, 1H), 3.53-3.44 (m, 1H), 3.42 (t, J = 5.9 Hz, 2H), 3.33 (s, 3H), 3.00 (ddd, J = 13.3, 7.2, 5.5 Hz, 1H), 2.58 (dddd, J = 20.0, 13.1, 7.1, 5.9 Hz, 4H), 1.86-1.52 (m, 4H), 1.06 (t, J = 7.1 Hz, 6H). 13 C{ 1 H} NMR (101 MHz, CDCl3): δ 146.6, 139.8, 130.9, 128.9, 72.1, 58.8, 52.7, 48.2, 46.5, 44.8, 44.0, 26.6, 25.7, 23.5, 15.0. FTIR (NaCl, thin film): 3733, 3256, 2356, 2354, 1521, 1167, 831, 733, 673cm -1 . HRMS:(ESI-TOF)C 15 H 28 N3O5S3[M+H] + The calculated value is 426.1186, and the measured value is 426.1209. TLC(99:1 DCM:MeOH), R f :0.1(UV).

[0073] Compound S13 [ka] Compound S13 was synthesized on a 2.01 mmol scale according to general procedure 6. After column chromatography on silica (DCM:MeOH=97:3), the target compound was isolated as a yellow oily substance (759 mg, 1.84 mmol, 92%). 1 H NMR (400 MHz, CD3CN): δ 7.57 (s, 1H), 6.14 (bs, 2H), 4.37 (dd, J = 10.7, 5.5 Hz, 1H), 3.99 (dd, J = 15.0, 10.7 Hz, 1H), 3.68 (dd, J = 15.0, 2.69-2.47 (m, 5H), 1.76-1.61 (m, 2H), 1.52 (ddt, J = 13.0, 8.6, 6.5 Hz, 2H), 1.05 (t, J = 7.1 Hz, 6H). 13 C{ 1 H} NMR (101 MHz, CD3CN): δ 148.5, 147.4, 131.2, 129.3, 128.3, 61.9, 53.3, 49.0, 47.4, 45.3, 30.8, 30.3, 26.0, 15.2. FTIR (NaCl, thin film): 3734, 3220, 2356, 1354, 1522, 1110, 739cm -1 . HRMS:(ESI-TOF)C 14 H 26 N3O5S3[M+H] + The calculated value is 412.1029, and the measured value is 412.0804. TLC (3:7 hexane:HCl), R f :0.15(UV).

[0074] Compound S14 [ka] Compound S14 was synthesized on a 1.83 mmol scale according to general procedure 7. Since the compound is not stable on silica, the crude reaction mixture was used directly in the next step after filtration through a cotton plug and evaporation.

[0075] compound 10 [ka] Compound 10 was synthesized on a 117 μmol scale according to general procedure 8. After purification by preparative HPLC (C18 column, 9.4 × 250 mm), the target compound was isolated as a colorless oil (22.2 mg, 42.5 μmol, 36%). 1 H NMR (400 MHz, CD3CN): δ 7.58 (s, 1H), 6.13 (bs, 2H), 4.38 (dd, J = 10.7, 5.6 Hz, 1H), 4.02 (ddd, J = 15.0, 10.7, 0.9 Hz, 1H), 3.69 (dd, J = 15.0, 5.5 Hz, 1H), 3.55-3.42 (m, 1H), 3.13 (ddd, J = 13.7, 7.2, 6.1 Hz, 1H), 2.83-2.66 (m, 6H), 2.57 (qd, J = 7.1, 1.5 Hz, 4H), 2.08-1.98 (m, 2H), 1.04 (t, J = 7.1 Hz, 6H). 13 C{ 1 H} NMR (101 MHz, CD3CN): δ 171.0, 169.7, 148.6, 147.4, 138.7, 131.2, 53.3, 48.12, 48.08, 45.3, 28.4, 26.3, 25.0, 15.2. HRMS:(ESI-TOF)C 18 H 27 N4O8S3[M+H] + The calculated value is 523.0986, and the measured value is 523.1107.

[0076] BZA-4C-NHS(11) Compound S15 [ka] Compound S15 was synthesized on a 9.50 mmol scale according to general procedure 1 using 1-bromo-5-methoxypentane (1.40 mL, 2.06 g, 11.4 mmol, 1.2 equivalents). After column chromatography on silica (hex:siRNA=4:1), the target compound was isolated as a colorless oil (2.47 g, 7.26 mmol, 76%). 1 H NMR (500 MHz, CDCl3): δ 6.96 (s, 1H), 4.72 (d, J = 5.1 Hz, 1H), 4.02 (dd, J = 15.3, 4.4 Hz, 1H), 3.82 (dd, J = 15.3, 4.9 Hz, 1H), 3.46-3.35 (m, 3H), 3.35-3.25 (m, 4H), 2.50 (bs, 1H), 1.70 (p, J = 7.2 Hz, 2H), 1.65-1.56 (m, 2H), 1.50-1.39 (m, 2H). 13 C{ 1 H} NMR (125 MHz, CDCl3): δ 142.9, 136.4, 133.0, 125.8, 72.6, 61.8, 58.7, 53.0, 49.4, 29.1, 28.4, 23.3. FTIR (NaCl, thin film): 3375, 2929, 2358, 1424, 1335, 1120cm -1 . HRMS:(ESI-TOF)C 12 H 19 ClNO4S2[M+H] + The calculated value is 340.0439, and the measured value is 340.0459. TLC (7:3 hexane:HCl), R f :0.66(UV).

[0077] Compound S16 [ka] Compound S16 was synthesized on a 7.24 mmol scale according to general procedure 2. After column chromatography on silica (hex:siRNA = 4:1 to 7:3), the target compound was isolated as a colorless oil (2.84 g, 5.76 mmol, 80%). 1 H NMR (500 MHz, CDCl3): δ 7.89-7.72 (m, 2H), 7.52-7.33 (m, 2H), 6.57 (s, 1H), 5.32 (dd, J = 3.9, 2.4 Hz, 1H), 4.22 (ddd, J = 16.4, 3.9, 0.8 Hz, 1H), 3.90 (dd, J = 16.4, 2.5 Hz, 1H), 3.46 (dt, J = 13.4, 7.6 Hz, 1H), 3.37 (t, J = 6.4 Hz, 2H), 3.34 (s, 3H), 3.12 (ddd, J = 13.4, 7.3, 6.1Hz, 1H), 2.51 (s, 3H), 1.68-1.49 (m, 4H), 1.46-1.31 (m, 2H). 13 C{ 1 H} NMR (125 MHz, CDCl3): δ 146.3, 136.40, 136.39, 135.6, 132.9, 130.5, 128.0, 126.1, 72.6, 67.9, 58.7, 50.5, 49.8, 29.2, 27.9, 23.2, 21.9. FTIR (NaCl, thin film): 2924, 2359, 1345, 1168, 823, 737, 668cm -1 . HRMS:(ESI-TOF)C 19 H 28 ClN2O6S3[M+NH4] + The calculated value is 511.0793, and the measured value is 511.0804. TLC (7:3 hexane:HCl), R f :0.4(UV).

[0078] Compound S17 [ka] Compound S17 was synthesized on a 5.75 mmol scale according to general procedure 3. After column chromatography on silica (hex:toluene=85:15), the target compound was isolated as a colorless oil (1.05 g, 2.67 mmol, 46%). 1 H NMR (500 MHz, CDCl3): δ 6.95 (s, 1H), 4.17 (dd, J = 10.8, 5.1 Hz, 1H), 4.11-4.00 (m, 1H), 3.56-3.43 (m, 2H), 3.38 (t, J = 6.4 Hz, 2H), 3.33 (s, 3H), 2.94 (dt, J = 13.5, 6.7 Hz, 1H), 2.6 -2.42 (m, 4H), 1.77-1.54 (m, 4H), 1.52-1.37 (m, 2H), 1.06 (t, J = 7.1 Hz, 6H). 13 C{ 1 H} NMR (125 MHz, CDCl3): δ 145.3, 135.5, 133.0, 126.2, 72.6, 58.6, 52.1, 48.2, 46.5, 44.7, 29.3, 28.7, 23.4, 15.0. FTIR (NaCl, thin film): 2927, 2358, 1338, 1163, 1115, 739, 668cm -1 . HRMS:(ESI-TOF)C 16 H 28 ClN2O3S2[M+H] + The calculated value is 395.1224, and the measured value is 395.1268. TLC(81:15 Hexane:SiO), R f :0.5(UV).

[0079] Compound S18 [ka] Compound S18 was synthesized on a 2.66 mmol scale according to general procedure 4. After column chromatography on silica (hex:toluene = 85:15), the target compound was isolated as a yellow oil (813 mg, 1.69 mmol, 63%). 1 H NMR (500 MHz, CDCl3): δ 7.34-7.16 (m, 5H), 6.86 (s, 1H), 4.12 (dd, J = 10.8, 5.3 Hz, 1H), 4.05 (s, 2H), 4.03-3.95 (m, 1H), 3.53-3.41 (m, 2H), 3.38 (t, J = 6.4 Hz, 2H), 3.33 (s, 3H), 2.90 (dt, J = 13.5, 6.8 Hz, 1H), 2.49 (qd, J = 7.0, 3.6 Hz, 4H), 1.73-1.54 (m, 4H), 1.51-1.38 (m, 2H), 1.01 (t, J = 7.1 Hz, 6H). 13 C{ 1 H} NMR (125 MHz, CDCl3): δ 145.3, 140.1, 136.7, 136.4, 132.1, 129.1, 128.8, 127.8, 72.6, 58.7, 52.2, 48.1, 46.9, 44.8, 43.2, 29.3, 28.7, 23.4, 15.1. FTIR (NaCl, thin film): 2928, 2359, 1339, 1166, 1117, 742cm -1 . HRMS:(ESI-TOF)C 23 H 35 N2O3S3[M+H] + The calculated value is 483.1804, and the measured value is 483.2046. TLC (3:7 hexane:HCl), R f :0.65(UV).

[0080] Compound S19 [ka] Compound S19 was synthesized on a 1.20 mmol scale according to general procedure 5. After column chromatography on silica (DCM:MeOH = 99:1), the target compound was isolated as a brown oily substance (347 mg, 0.79 mmol, 66%). 1 H NMR (400 MHz, CDCl3): δ 7.63 (s, 1H), 5.32 (bs, 2H), 4.24 (dd, J = 10.8, 5.2 Hz, 1H), 4.11-4.04 (m, 1H), 3.55 (dd, J = 14.8, 5.3 Hz, 1H), 3.47 (dt, J = 13.3, 7.6 Hz, 1H), 3.38 (t, J = 6.3 Hz, 2H), 3.32 (s, 3H), 2.96 (dt, J = 13.5, 6.7 Hz, 1H), 2.67-2.48 (m, 4H), 1.73-1.55 (m, 4H), 1.50-1.37 (m, 2H), 1.06 (t, J = 7.1 Hz, 6H). 13 C{ 1 H} NMR (101 MHz, CDCl3): δ 146.6, 145.5, 139.7, 130.8, 72.6, 58.7, 52.7, 48.4, 46.7, 44.8, 29.2, 28.7, 23.4, 15.0. FTIR (NaCl, thin film): 3733, 3220, 2927, 2359, 1339, 1157, 1101cm -1 . HRMS:(ESI-TOF)C 16 H 30 N3O5S3[M+H] + The calculated value is 440.1342, and the measured value is 440.1344. TLC(99:1 DCM:MeOH), R f :0.1(UV).

[0081] Compound S20 [ka] Compound S20 was synthesized on a 735 μmol scale according to general procedure 6. After column chromatography on silica (DCM:MeOH = 97:3), the target compound was isolated as a yellow oil (240 mg, 563 μmol, 77%). 1 H NMR (400 MHz, CD3CN): δ 7.57 (s, 1H), 6.12 (bs, 2H), 4.37 (dd, J = 10.7, 5.5 Hz, 1H), 3.99 (dd, J = 15.0, 10.7 Hz, 1H), 3.67 (dd, J = 15.0, 5.5 Hz, 1H), 3.53-3.46 (m, 2H), 3.39 (dt, J = 13.1, 7.7 Hz, 1H), 3.08-2.97 (m, 1H), 2.66-2.44 (m, 5H), 1.71-1.59 (m, 2H), 1.57-1.46 (m, 2H), 1.44-1.32 (m, 2H), 1.05 (t, J = 7.1 Hz, 6H). 13 C{ 1 ¹H} NMR (101 MHz, CD3CN): δ 148.1, 147.0, 130.8, 61.9, 52.9, 48.7, 47.0, 44.9, 32.6, 30.5, 28.8, 23.2, 14.9 (one signal is obscured directly below the solvent signal). FTIR (NaCl, thin film): 3734, 2947, 2356, 2347, 1507, 1165, 826, 739, 668cm -1 . HRMS:(ESI-TOF)C 15 H 28 N3O5S3[M+H] + The calculated value is 426.1186, and the measured value is 425.1194. TLC(97:3 DCM:MeOH), R f :0.1(UV).

[0082] Compound S21 [ka] Compound S21 was synthesized on a 557 μmol scale according to general procedure 7. Since the compound is not stable on silica, the crude reaction mixture was used directly in the next step after filtration through a cotton plug and evaporation.

[0083] compound 11 [ka] Compound 11 was synthesized on a 114 μmol scale according to general procedure 8. After purification by preparative HPLC (C18 column, 9.4 × 250 mm), the target compound was isolated as a colorless oil (6.20 mg, 11.6 μmol, 10%). 1 H NMR (400 MHz, CD3CN): δ 7.58 (s, 1H), 6.13 (bs, 2H), 4.38 (dd, J = 10.7, 5.6 Hz, 1H), 3.99 (ddd, J = 15.0, 10.8, 1.0 Hz, 1H), 3.68 (dd, J = 15.0, 5.6 Hz, 1H), 3.47-3.35 (m, 1H), 3.08 (dt, J = 13.4, 5.9 Hz, 1H), 2.77 (s, 3H), 2.71-2.52 (m, 6H), 2.35-2.06 (m, 2H), 1.83-1.59 (m, 4H), 1.05 (t, J = 7.1 Hz, 6H). 13 C{ 1 H} NMR (101 MHz, CD3CN): δ 171.1, 169.9, 148.5, 147.3, 138.9, 131.2, 53.2, 48.5, 47.4, 45.3, 30.9, 28.2, 26.3, 22.4, 15.2. HRMS:(ESI-TOF)C 19 H 29 N4O8S3[M+H] + The calculated value is 537.1142, and the measured value is 537.1162.

[0084] BZA-6C-NHS(12) Compound S22 [ka] Compound S22 was synthesized on an 8.50 mmol scale according to general procedure 1 using 1-bromo-7-methoxypentane (1.44 mL, 2.13 g, 10.2 mmol, 1.2 equivalents). After column chromatography on silica (hex:siRNA=4:1), the target compound was isolated as a colorless oil (2.44 g, 6.64 mmol, 78%). 1 H NMR (500 MHz, CDCl3): δ 6.96 (s, 1H), 4.72 (bs, 1H), 4.01 (dd, J = 15.3, 4.4 Hz, 1H), 3.82 (dd, J = 15.3, 4.4 Hz, 1H), 3.37 (dd, J = 7.4, 5.7 Hz, 3H), 3.32 (s, 3H), 3.28 (dd, J = 13.6, 7.1 Hz, 1H), 2.52 (s, 1H), 1.67 (p, J = 7.2 Hz, 2H), 1.55 (q, J = 6.8, Hz, 2H), 1.36 (dd, J = 6.0, 2.6 Hz, 6H). 13 C{ 1 H} NMR (125 MHz, CDCl3): δ 142.9, 136.4, 133.0, 125.8, 72.9, 61.7, 58.7, 53.0, 49.5, 29.6, 29.0, 28.5, 26.5, 26.1. FTIR (NaCl, thin film): 3362, 2928, 2361, 1422, 1334, 1169cm -1 . HRMS:(ESI-TOF)C 14 H 22 ClNO4S2[M] + The calculated value is 367.0679, and the measured value is 367.0770. TLC (8:2 hexane:ethyl), R f :0.5(UV).

[0085] Compound S23 [ka] Compound S23 was synthesized on a 6.63 mmol scale according to general procedure 2. Following column chromatography on silica (hex:HCl = 4:1 to 7:3), the target compound was isolated as a colorless oil (3.17 g, 6.63 mmol, 91%). 1 H NMR (500 MHz, CDCl3): δ 7.88-7.78 (m, 2H), 7.47-7.39 (m, 2H), 6.57 (s, 1H), 5.32 (dd, J = 3.9, 2.4 Hz, 1H), 4.29-4.17 (m, 1H), 3.90 (dd, J = 16.4, 2.5 Hz, 1H), 3.44 (dt, J = 13.3, 7.6 Hz, 1H), 3.37 (t, J = 6.6 Hz, 2H), 3.34 (s, 3H), 3.10 (ddd, J = 13.4, 7.5, 6.1 Hz, 1H), 2.51 (s, 3H), 1.67-1.51 (m, 4H), 1.39-1.28 (m, 6H). 13 C{ 1 H} NMR (125 MHz, CDCl3): δ 146.3, 136.39, 136.36, 135.6, 133.0, 130.5, 128.0, 126.1, 72.9, 67.9, 58.7, 50.4, 49.7, 29.7, 29.1, 28.0, 26.5, 26.2, 21.9. FTIR (NaCl, thin film): 2920, 2358, 1356, 1169cm -1 . HRMS:(ESI-TOF)C 21 H 32 ClN2O6S3[M+NH4] + The calculated value is 539.1106, and the measured value is 539.1121. TLC (7:3 hexane:HCl), R f :0.83(UV).

[0086] Compound S24 [ka] Compound S24 was synthesized on a 6.05 mmol scale according to general procedure 3. After column chromatography on silica (hex:HCl=85:15), the target compound was isolated as a colorless oil (1.27 g, 3.00 mmol, 50%). 1 H NMR (500 MHz, CDCl3): δ 6.95 (s, 1H), 4.17 (dd, J = 10.8, 5.1 Hz, 1H), 4.11-4.02 (m, 1H), 3.57-3.41 (m, 2H), 3.36 (t, J = 6.5 Hz, 2H), 3.33 (s, 3H), 2.92 (dt, J = 13.4, 6.8 Hz, 1H), 2.67-2.46 (m, 4H), 1.69-1.49 (m, 5H), 1.46-1.30 (m, 6H), 1.06 (t, J = 7.1 Hz, 6H). 13 C{ 1 H} NMR (125 MHz, CDCl3): δ 145.3, 135.5, 133.1, 126.2, 72.9, 58.7, 52.1, 48.2, 46.3, 44.7, 29.7, 29.2, 28.7, 26.6, 26.2, 15.0. FTIR (NaCl, thin film): 2930, 2857, 1416, 1343, 1168, 1117cm -1 . HRMS:(ESI-TOF)C 18 H 31 ClN2O3S2[M+H] + The calculated value is 423.1537, and the measured value is 423.1776. TLC(85:15 Hexane:ethyl), R f :0.6(UV).

[0087] Compound S25 [ka] Compound S25 was synthesized on a 2.98 mmol scale according to general procedure 4. After column chromatography on silica (hex:toluene = 85:15), the target compound was isolated as a yellow oil (1.12 g, 2.19 mmol, 74%). 1 H NMR (500 MHz, CDCl3): 7.31-7.20 (m, 5H), 6.86 (s, 1H), 4.12 (dd, J = 10.8, 5.2 Hz, 1H), 4.04 (s, 2H), 4.02-3.94 (m, 1H), 3.45 (ddd, J = 22.8, 14.9, 6.5 Hz, 2H), 3.36 (t, J = 6.6 Hz, 2H), 3.33 (s, 3H), 2.89 (dt, J = 13.4, 6.7 Hz, 1H), 2.49 (qd, J = 6.7, 6.2, 3.1 Hz, 4H), 1.71-1.50 (m, 4H), 1.45-1.31 (m, 6H), 1.01 (t, J = 7.1 Hz, 5H). 13 C{ 1 H} NMR (125 MHz, CDCl3): δ 145.3, 140.1, 136.7, 136.4, 132.1, 129.1, 128.8, 127.8, 72.9, 58.7, 52.2, 48.1, 46.8, 44.8, 43.2, 29.7, 29.2, 28.7, 26.6, 26.2, 15.1. FTIR (NaCl, thin film): 1929, 1857, 1357, 1456, 1339, 1160, 1111, 732cm -1 . HRMS:(ESI-TOF)C 25 H 39 N2O3S3[M+H] + The calculated value is 511.2117, and the measured value is 511.2327. TLC(85:15 Hexane:ethyl), R f :0.74(UV).

[0088] Compound S26 [ka] Compound S26 was synthesized on a 2.17 mmol scale according to general procedure 5. After column chromatography on silica (DCM:MeOH = 99:1), the target compound was isolated as a brown oily substance (139 mg, 0.30 mmol, 14%). 1 H NMR (500 MHz, CDCl3): δ 7.63 (s, 1H), 5.32 (bs, 2H), 4.24 (dd, J = 10.8, 5.2 Hz, 1H), 4.11-4.04 (m, 1H), 3.55 (dd, J = 14.8, 5.3 Hz, 1H), 3.47 (dt, J = 13.3, 7.6 Hz, 1H), 3.38 (t, J = 6.3 Hz, 2H), 3.32 (s, 3H), 3.02 (dt, J = 13.5, 6.7 Hz, 1H), 2.71-2.47 (m, 4H), 1.71-1.61 (m, 2H), 1.59-1.50 (m, 2H), 1.44-1.31 (m, 6H), 1.06 (t, J = 7.1 Hz, 6H). 13 C{ 1 H} NMR (101 MHz, CDCl3): δ 146.6, 145.5, 139.8, 130.8, 72.9, 58.7, 52.8, 48.4, 46.5, 44.8, 29.6, 29.1, 28.8, 26.6, 26.1, 15.0. FTIR (NaCl, thin film): 3296, 2854, 2360, 1454, 1346, 1170, 1161, 1101, 738cm -1 . HRMS:(ESI-TOF)C 18 H 34 N3O5S3[M+H] + The calculated value is 468.1655, and the measured value is 468.1665. TLC(99:1 DCM:MeOH), R f :0.13(UV).

[0089] Compound S27 [ka] Compound S27 was synthesized on a 14.5 mmol scale according to general procedure 6. After column chromatography on silica (DCM:MeOH=97:3), the target compound was isolated as a yellow oily substance (147 mg, 2.42 mmol, 13%). 1 H NMR (400 MHz, acetone-d6): δ 7.60 (s, 1H), 7.22 (bs, 1H), 4.52 (dd, J = 10.8, 5.6 Hz, 1H), 4.05 (dd, J = 14.9, 10.8 Hz, 1H), 3.79 (dd, J = 14.9, 5.6 Hz, 1H), 3.52 (t, J = 6.5 Hz, 3H), 3.49-3.31 (m, 2H), 3.15 (ddd, J = 13.4, 7.4, 5.9 Hz, 1H), 2.62 (qd, J = 6.9, 3.1 Hz, 4H), 1.79-1.60 (m, 3H), 1.57-1.45 (m, 2H), 1.43-1.33 (m, 7H), 1.07 (t, J = 7.1 Hz, 6H). 13 C{ 1 ¹H} NMR (10¹ MHz, acetone-d6): δ 149.4, 146.9, 139.2, 130.7, 62.5, 53.4, 49.0, 47.4, 45.4, 33.8, 27.4, 26.7, 15.4. FTIR (NaCl, thin film): 3367, 2934, 2361, 29359, 1269, 1110, 778, 740, 668cm -1 . HRMS:(ESI-TOF)C 17 H 32 N3O5S3[M+H] + The calculated value is 454.1499, and the measured value is 454.1537. TLC (3:7 hexane:HCl), R f :0.77(UV).

[0090] Compound S28 [ka] Compound S28 was synthesized on a 310 μmol scale according to general procedure 7. After purification by preparative HPLC (C18 column, 9.4 × 250 mm), the target compound was isolated as a colorless oil (107 mg, 229 μmol, 74%). 1 H NMR (400 MHz, acetone-d6): δ 7.60 (s, 1H), 7.21 (bs, 2H), 4.52 (dd, J = 10.7, 5.5 Hz, 1H), 4.05 (dd, J = 14.9, 10.7 Hz, 1H), 3.80 (dd, J = 14.9, 5.5 Hz, 1H), 3.43 (dt, J = 13.4, 7.6 Hz, 1H), 3.15 (ddd, J = 13.5, 7.7, 5.8 Hz, 1H), 2.62 (qd, J = 6.8, 3.4 Hz, 4H), 2.29 (t, J = 7.4 Hz, 2H), 1.79-1.54 (m, 4H), 1.47-1.33 (m, 4H), 1.06 (t, J = 7.1 Hz, 6H). 13 C{ 1 ¹H} NMR (10¹ MHz, acetone-d6): δ 174.7, 149.3, 146.9, 139.1, 130.7, 53.4, 49.0, 47.4, 45.3, 34.1, 29.41, 29.39, 27.0, 25.6, 15.4. HRMS:(ESI-TOF)C 17 H 30 N3O6S3[M+H] + The calculated value is 468.1291, and the measured value is 468.1299.

[0091] compound 12 [ka] Compound 12 was synthesized on a 45.3 μmol scale according to general procedure 8. After purification by preparative HPLC (C18 column, 9.4 × 250 mm), the target compound was isolated as a colorless oil (7.60 mg, 13.5 μmol, 30%). 1 H NMR (400 MHz, CD3CN): δ 7.57 (s, 1H), 6.12 (bs, 2H), 4.37 (dd, J = 10.7, 5.6 Hz, 1H), 3.98 (ddd, J = 14.5, 10.8, 3.7 Hz, 1H), 3.72-3.62 (m, 1H), 3.43-3.28 (m, 1H), 3.03 (ddd, J = 13.6, 7.7, 5.9 Hz, 1H), 2.76 (s, 4H), 2.59 (dt, J = 20.2, 7.1 Hz, 6H), 1.78-1.55 (m, 4H), 1.49-1.32 (m, 4H), 1.05 (t, J = 7.0 Hz, 6H). 13 C{ 1 H} NMR (101 MHz, CD3CN): δ 171.1, 170.1, 148.5, 147.3, 139.0, 131.2, 53.3, 49.0, 47.5, 45.3, 31.3, 29.1, 28.8, 26.6, 26.3, 25.2, 15.2. HRMS:(ESI-TOF)C 21 H 33 N4O8S3[M+H] + The calculated value is 565.1455, and the measured value is 565.1451.

[0092] BZA-8C-NHS(13) Compound S29 [ka] Compound S29 was synthesized on a 10.0 mmol scale according to general procedure 1 using 1-bromo-9-methoxynonane (1.92 mL, 2.85 g, 12.0 mmol, 1.2 equivalents). After column chromatography on silica (hex:siRNA=4:1), the target compound was isolated as a colorless oil (4.20 g, 10.6 mmol, quantitative). 1 H NMR (400 MHz, CDCl3): δ 6.95 (s, 1H), 3.99 (dd, J = 15.4, 4.4 Hz, 1H), 3.82 (dd, J = 15.3, 5.2 Hz, 1H), 3.53-3.22 (m, 7H), 2.72-2.58 (m, 1H), 1.72-1.60 (m, 2H), 1.60-1.49 (m, 2H), 1.37-1.27 (m, 10H). 13 C{ 1 H} NMR (101 MHz, CDCl3): δ 143.1, 136.3, 132.9, 125.8, 73.1, 61.7, 58.7, 52.9, 49.5, 29.7, 29.5, 29.4, 29.1, 28.6, 26.5, 26.2. FTIR (NaCl, thin film): 3422, 2927, 2855, 2357, 1339, 1154cm -1 . HRMS:(ESI-TOF)C 16 H 27 ClNO4S2[M+H] + The calculated value is 396.1065, and the measured value is 396.1139. TLC (8:2 hexane:ethyl), R f :0.53(UV).

[0093] Compound S30 [ka] Compound S30 was synthesized on a 10.0 mmol scale according to general procedure 2. After column chromatography on silica (hex: Depositphotos = 3:1), the target compound was isolated as a colorless oil (5.44 g, 9.90 mmol, 99%). 1 H NMR (500 MHz, CDCl3): δ 7.89-7.78 (m, 2H), 7.46-7.40 (m, 2H), 6.57 (s, 1H), 4.25-4.16 (m, 1H), 3.90 (dd, J = 16.3, 2.5 Hz, 1H), 3.47-3.35 (m, 3H), 3.34 (s, 3H), 3.10 (ddd, J = 13.4, 7.5, 6.1 Hz, 1H), 2.51 (s, 3H), 1.66-1.49 (m, 4H), 1.41-1.24 (m, 10H). 13 C{ 1 H} NMR (125 MHz, CDCl3): δ 146.2, 136.4, 136.4, 135.6, 133.0, 130.5, 128.0, 126.1, 73.0, 67.9, 58.7, 50.3, 49.8, 29.8, 29.6, 29.5, 29.2, 28.0, 26.5, 26.3, 21.9. FTIR (NaCl, thin film): 2926, 2854, 2359, 1716, 1454, 1360, 1166, 940, 732cm -1 . HRMS:(ESI-TOF)C 23 H 36 ClN2O6S3[M+NH4] + The calculated value is 567.1419, and the measured value is 567.1434. TLC (7:3 hexane:HCl), R f :0.92(UV).

[0094] Compound S31 [ka] Compound S31 was synthesized on a 9.89 mmol scale according to general procedure 3. After column chromatography on silica (hex:toluene=85:15), the target compound was isolated as a colorless oil (2.15 g, 4.77 mmol, 48%). 1 H NMR (500 MHz, CDCl3): δ 6.95 (s, 1H), 4.17 (dd, J = 10.8, 5.1 Hz, 1H), 4.06 (ddd, J = 14.7, 10.8, 0.9 Hz, 1H), 3.53-3.42 (m, 2H), 3.36 (t, J = 6.6 Hz, 2H), 3.33 (s, 3H), 2.92 (dt, J = 13.5, 6.8 Hz, 1H), 2.67-2.48 (m, 4H), 1.70-1.51 (m, 4H), 1.42-1.26 (m, 10H), 1.06 (t, J = 7.1 Hz, 6H). 13 C{ 1 H} NMR (125 MHz, CDCl3): δ 145.2, 135.3, 133.0, 126.1, 72.9, 58.6, 51.9, 48.1, 46.2, 44.6, 29.6, 29.4, 29.4, 29.1, 28.6, 26.5, 26.1, 14.9. FTIR (NaCl, thin film): 2924, 2853, 2358, 1539, 1455, 1348, 1166, 749cm -1 . HRMS:(ESI-TOF)C 20 H 36 ClN2O3S2[M+H] + The calculated value is 451.1850, and the measured value is 451.1867. TLC(85:15 Hexane:ethyl), R f :0.66(UV).

[0095] Compound S32 [ka] Compound S32 was synthesized on a 4.77 mmol scale according to general procedure 4. After column chromatography on silica (hex:HCl=85:15), the target compound was isolated as a yellow oil (1.34 g, 2.49 mmol, 52%). 1 H NMR (400 MHz, CDCl3): δ 7.34-7.17 (m, 5H), 6.86 (s, 1H), 4.12 (dd, J = 10.8, 5.1 Hz, 1H), 4.04 (s, 2H), 3.98 (dd, J = 14.7, 10.8 Hz, 1H), 3.52-3.40 (m, 2H), 3.36 (t, J = 6.6 Hz, 2H), 3.33 (s, 3H), 2.88 (dt, J = 13.5, 6.8 Hz, 1H), 2.49 (qd, J = 7.0, 2.0 Hz, 4H), 1.58 (dq, J = 20.8, 6.9 Hz, 4H), 1.39-1.25 (m, 10H), 1.00 (t, J = 7.1 Hz, 6H). 13 C{ 1 H} NMR (101 MHz, CDCl3): δ 145.3, 140.0, 136.7, 136.5, 132.1, 129.1, 128.8, 127.8, 73.1, 58.7, 52.3, 48.1, 46.8, 44.8, 43.2, 29.8, 29.6, 29.5, 29.3, 28.8, 26.7, 26.2. FTIR (NaCl, thin film): 2925, 2853, 2359, 1540, 1450, 1362, 1169, 770cm -1 . HRMS:(ESI-TOF)C 27 H 43 N2O3S3[M+H] + The calculated value is 539.2430, and the measured value is 539.2675. TLC(85:15 Hexane:ethyl), R f :0.83(UV).

[0096] Compound S33 [ka] Compound S33 was synthesized on a 2.49 mmol scale according to general procedure 5. After column chromatography on silica (hex:RINKAN=3:2), the target compound was isolated as a brown oily substance (833 mg, 1.68 mmol, 68%). 1 H NMR (500 MHz, CDCl3): δ 7.63 (s, 1H), 5.40 (bs, 2H), 4.24 (dd, J = 10.8, 5.2 Hz, 1H), 4.12-4.03 (m, 1H), 3.55 (dd, J = 14.9, 5.3 Hz, 1H), 3.45 (dt, J = 13.5, 7.6 Hz, 1H), 3.37 (t, J = 6.6 Hz, 2H), 3.33 (s, 3H), 2.96 (dt, J = 13.5, 6.8 Hz, 1H), 2.69-2.48 (m, 4H), 1.70-1.60 (m, 2H), 1.59-1.51 (m, 2H), 1.41-1.27 (m, 10H), 1.07 (t, J = 7.1 Hz, 6H). 13 C{ 1 H} NMR (125 MHz, CDCl3): δ 146.6, 145.4, 139.8, 130.8, 73.1, 58.7, 52.7, 48.4, 46.5, 44.8, 29.7, 29.5, 29.5, 29.1, 28.7, 26.6, 26.2, 15.0. FTIR (NaCl, thin film): 3334, 2928, 2357, 1540, 1352, 1161, 780cm -1 . HRMS:(ESI-TOF)C 20 H 38 N3O5S3[M+H] + The calculated value is 496.1968, and the measured value is 495.2363. TLC (6:4 hexane:alkyl), R f :0.16(UV).

[0097] Compound S34 [ka] Compound S34 was synthesized on a 1.67 mmol scale according to general procedure 6. After column chromatography on silica (DCM:MeOH=97:3), the target compound was isolated as a yellow oil (344 mg, 0.71 mmol, 43%). 1 H NMR (400 MHz, acetone-d6): δ 7.60 (s, 1H), 7.22 (bs, 2H), 4.51 (dd, J = 10.7, 5.6 Hz, 1H), 4.05 (ddd, J = 14.9, 10.7, 1.0 Hz, 1H), 3.79 (dd, J = 15.0, 5.6 Hz, 1H), 3.52 (dt, J = 9.2, 4.4 Hz, 2H), 3.48-3.35 (m, 2H), 3.14 (ddd, J = 13.5, 7.5, 5.9 Hz, 1H), 2.62 (qd, J = 7.1, 3.1Hz, 4H), 1.72-1.64 (m, 2H), 1.54-1.45 (m, 2H), 1.41-1.26 (m, 10H), 1.06 (t, J = 7.1 Hz, 6H). 13 C{ 1 ¹H} NMR (10¹ MHz, acetone-d6): δ 149.3, 146.8, 139.1, 130.6, 62.5, 53.3, 48.9, 47.3, 45.3, 33.8, 30.3, 30.2, 27.2, 26.7, 15.3. FTIR (NaCl, thin film): 3337, 2930, 2854, 1453, 1344, 1170, 1157, 681cm -1 . HRMS:(ESI-TOF)C 19 H 36 N3O5S3[M+H] + The calculated value is 482.1812, and the measured value is 482.1766. TLC(97:3 DCM:MeOH), R f :0.18(UV).

[0098] Compound S35 [ka] Compound S35 was synthesized on a 688 μmol scale according to general procedure 7. Since the compound is not stable on silica, the crude reaction mixture was used directly in the next step after filtration through a cotton plug and evaporation.

[0099] compound 13 [ka] Compound 13 was synthesized on a 10¹ μmol scale according to general procedure 8. After purification by preparative HPLC (C18 column, 9.4 × 250 mm), the target compound was isolated as a colorless oil (25.2 mg, 42.5 μmol, 42%). 1 H NMR (400 MHz, CD3CN): δ 7.57 (s, 1H), 6.12 (bs, 2H), 4.36 (dd, J = 10.7, 5.5 Hz, 1H), 3.98 (dd, J = 15.0, 10.7 Hz, 1H), 3.66 (dd, J = 15.0, 5.5 Hz, 1H), 3.37 (dt, J = 13.5, 7.7 Hz, 1H), 3.02 (ddd, J = 13.4, 7.4, 5.9 Hz, 1H), 2.76 (s, 4H), 2.68-2.51 (m, 6H), 1.75-1.55 (m, 4H), 1.46-1.28 (m, 8H), 1.04 (t, J = 7.1 Hz, 6H). 13 C{ 1 H} NMR (101 MHz, CD3CN): δ 171.1, 170.1, 148.4, 147.3, 139.0, 131.2, 53.2, 49.0, 47.4, 45.3, 31.4, 29.5, 29.5, 29.2, 29.2, 27.0, 26.3, 25.3, 15.2. HRMS:(ESI-TOF)C 23 H 37 N4O8S3[M+H] +The calculated value is 593.1768, and the measured value is 593.1794.

[0100] BZA-PEG-PFP(14) Compound S36 and Compound S37 [ka] In a 250 mL round-bottom flask equipped with a stirring bar, tert-butyl 3-(2-(2-hydroxyethoxy)ethoxy)propanoate (S36) (5.00 g, 21.3 mmol, 1.0 equivalent) was dissolved in DCM (61.0 mL). Carbon tetrabromide (8.85 g, 26.7 mmol, 1.25 equivalents) and triphenylphosphine (7.28 g, 27.7 mmol, 1.3 equivalents) were slowly added (exothermic reaction). The reaction mixture was stirred at room temperature for 2 hours, after which the volatile materials were evaporated. The residue was purified by column chromatography on silica (hex:siRNA=9:1) to obtain alkyl bromide S37 (3.69 g, 12.4 mmol, 58%) as a colorless oil. 1 H NMR (400 MHz, CDCl3): δ 3.80 (t, J = 6.3 Hz, 2H), 3.72 (t, J = 6.5 Hz, 2H), 3.68-3.58 (m, 4H), 3.46 (t, J = 6.4 Hz, 2H), 2.50 (t, J = 6.5 Hz, 2H), 1.44 (s, 9H). 13 C{ 1 H} NMR (101 MHz, CDCl3): δ 171.0, 80.7, 71.4, 70.6, 70.5, 67.1, 36.4, 30.4, 28.2. FTIR (NaCl, thin film): 2977, 2928, 2872, 1731, 1457, 1392, 1366, 1255, 1156, 848cm -1 . HRMS:(ESI-TOF)C 11 H 21 BrNaO4[M+Na] + The calculated value is 319.0515, and the measured value is 319.0513. TLC (4:1 hexane:SiO), R f :0.40(KMnO4).

[0101] Compound S38 [ka] Compound S38 was synthesized on a 10.0 mmol scale according to general procedure 1 using S37 (3.57 g, 12.0 mmol, 1.2 equivalents). After column chromatography on silica (hex: HCl = 3:2 to 2:3), the target compound was isolated as a colorless oil (4.78 g, 10.5 mmol, quantitative). 1 H NMR (400 MHz, CDCl3): δ 6.99 (s, 1H), 4.72 (q, J = 4.4 Hz, 1H), 4.34 (d, J = 5.9 Hz, 1H), 4.22 (dd, J = 15.5, 4.3 Hz, 1H), 4.07 (dd, J = 15.5, 3.9 Hz, 1H), 3.81-3.73 (m, 3H), 3.70 (t, J = 6.2 Hz, 2H), 3.64-3.40 (m, 5H), 2.50 (t, J = 6.2 Hz, 2H), 1.45 (s, 9H). 13 C{ 1 H} NMR (101 MHz, CDCl3): δ 171.5, 144.1, 135.7, 132.7, 126.5, 81.2, 71.0, 70.3, 70.0, 61.7, 54.2, 48.8, 36.4, 31.1, 28.2. FTIR (NaCl, thin film): 3445, 2977, 2875, 1725, 1423, 1367, 1341, 1167, 1027, 845cm -1 . HRMS:(ESI-TOF)C 17 H 26 ClNNaO7S2[M+Na] + The calculated value is 478.0731, and the measured value is 478.0758. TLC (2:3 hexane:ethyl), R f:0.46(UV).

[0102] Compound S39 [ka] Compound S39 was synthesized on a 10.0 mmol scale according to general procedure 2. After column chromatography on silica (hex: Depositphotos = 3:1), the target compound was isolated as a colorless oil (6.05 g, 9.92 mmol, 99%). 1 H NMR (400 MHz, CDCl3): δ 7.91-7.77 (m, 2H), 7.45-7.37 (m, 2H), 6.60 (s, 1H), 5.40 (t, J = 3.7 Hz, 1H), 4.26 (dd, J = 16.2, 4.1 Hz, 1H), 4.18-4.11 (m, 1H), 3.76-3.56 (m, 9H), 3.44-3.31 (m, 1H), 2.56-2.43 (m, 5H), 1.44 (s, 9H). 13 C{ 1 H} NMR (101 MHz, CDCl3): δ 171.0, 146.1, 137.0, 136.3, 135.8, 133.1, 130.4, 128.1, 126.2, 80.7, 70.8, 70.7, 70.4, 68.1, 67.1, 52.3, 49.8, 36.4, 28.2, 21.9. FTIR (NaCl, thin film): 3349, 3095, 2850, 2315, 1728, 1605, 1520, 1415, 1335, 1169cm -1 . HRMS:(ESI-TOF)C 24 H 33 ClNO9S3[M+H] + The calculated value is 610.1000, and the measured value is 610.1057. TLC (3:1 hexane:SiO), R f :0.11(UV).

[0103] Compound S40 [ka] Compound S40 was synthesized on a 9.92 mmol scale according to general procedure 3. After column chromatography on silica (hex:HCl=75:25), the target compound was isolated as a colorless oil (2.51 g, 4.92 mmol, 50%). 1 H NMR (400 MHz, CDCl3): δ 6.96 (s, 1H), 4.26 (dd, J = 10.7, 5.1 Hz, 1H), 4.11 (dd, J = 14.6, 10.8 Hz, 1H), 3.78 (dd, J = 14.7, 5.2 Hz, 1H), 3.73-3.66 (m, 4H), 3.66-3.54 (m, 5H), 3.22 (ddd, J = 14.3, 6.7, 4.8 Hz, 1H), 2.65-2.42 (m, 6H), 1.44 (s, 9H), 1.05 (t, J = 7.0 Hz, 6H). 13 C{ 1 H} NMR (101 MHz, CDCl3): δ 170.9, 146.0, 135.5, 132.9, 126.4, 80.7, 71.1, 70.7, 67.0, 52.2, 48.2, 47.9, 44.6, 36.4, 28.2, 15.0. FTIR (NaCl, thin film): 3096, 2969, 2873, 1737, 1731, 1454, 1416, 1338, 1160, 1005cm -1 . HRMS:(ESI-TOF)C 21 H 36 ClN2O6S3[M+H] + The calculated value is 511.1698, and the measured value is 511.1815. TLC (3:1 hexane:SiO), R f :0.28(UV).

[0104] Compound S41 [ka] Compound S41 was synthesized on a 4.91 mmol scale according to general procedure 4. After column chromatography on silica (hex:toluene = 85:15), the target compound was isolated as a yellow oil (1.78 g, 2.97 mmol, 60%). 1 H NMR (400 MHz, CDCl3): 7.32-7.18 (m, 5H), 6.88 (s, 1H), 4.21 (dd, J = 10.7, 5.3 Hz, 1H), 4.10-3.96 (m, 3H), 3.79-3.66 (m, 5H), 3.64-3.53 (m, 5H), 3.19 (dt, J = 14.3, 5.5 Hz, 1H), 2.48 (dt, J = 12.2, 6.8 Hz, 6H), 1.44 (s, 9H), 0.99 (t, J = 7.1 Hz, 6H). 13 C{ 1 H} NMR (101 MHz, CDCl3): δ 170.9, 140.1, 136.7, 136.2, 132.3, 129.0, 128.8, 127.8, 80.7, 71.2, 70.7, 70.4, 67.0, 52.4, 48.3, 48.2, 44.6, 43.2, 36.4, 28.2, 15.0. FTIR (NaCl, thin film): 3061, 2970, 2925, 2869, 1730, 1453, 1364, 1136, 948, 848cm -1 . HRMS:(ESI-TOF)C 28 H 43 N2O6S3[M+H] + The calculated value is 599.2278, and the measured value is 599.2428. TLC (3:1 hexane:SiO), R f :0.21(UV).

[0105] Compound S42 [ka] In a 50 mL round-bottom flask equipped with a stirring bar, tert-butyl ester S41 (1.16 g, 1.94 mmol, 1.0 equivalent) was dissolved in THF (9.69 mL). The flask was cooled to 0°C, and lithium aluminum hydride (110 mg, 2.91 mmol, 1.5 equivalents) was added in installments. The ice bath was removed, and the reaction was stirred at room temperature for 4 hours. After Fieser workup, the crude material was purified by column chromatography on silica (hex:SiO = 2:3 to 100% SiO) to obtain alcohol S42 (814 mg, 1.54 mmol, 79%) as a brown oily substance. 1 H NMR (400 MHz, CDCl3): δ 7.33-7.18 (m, 5H), 6.88 (s, 1H), 4.20 (dd, J = 10.7, 5.3 Hz, 1H), 4.04 (s, 3H), 3.82-3.52 (m, 12H), 3.18 (dt, J = 14.4, 5.8 Hz, 1H), 2.48 (q, J = 7.1 Hz, 4H), 1.83 (p, J = 5.7 Hz, 2H), 1.00 (t, J = 7.0 Hz, 6H). 13 C{ 1 H} NMR (101 MHz, CDCl3): δ 145.9, 140.1, 136.7, 136.1, 132.3, 129.0, 128.8, 127.8, 71.2, 70.6, 70.5, 70.2, 61.9, 52.4, 48.5, 48.3, 44.6, 43.2, 32.0, 15.0. FTIR (NaCl, thin film): 3432, 3027, 2873, 2474, 1952, 1670, 1453, 1335, 1163, 1110cm -1 . HRMS:(ESI-TOF)C 24 H 37 N2O5S3[M+H] + The calculated value is 529.1859, and the measured value is 529.1980. TLC (1:4 hexane:SiO), R f :0.36(UV).

[0106] Compound S43 [ka] In a two-drum vial equipped with a stirring bar, alcohol S42 (264 mg, 0.50 mmol, 1.0 equivalent) was dissolved in DCM (1.0 mL). The solution was cooled to 0°C, and triethylamine (76.7 μL, 55.7 mg, 0.55 mmol, 1.1 equivalent) and acetyl chloride (35.6 μL, 39.2 mg, 0.50 mmol, 1.0 equivalent) were added. The ice bath was removed, and the reaction was stirred overnight at room temperature. Then, water (2.0 mL) was added, and the aqueous phase was extracted with DCM (3×). The combined organic phase was dried over Na₂SO₄, filtered, and the volatile materials were evaporated under reduced pressure. The crude material was purified by column chromatography on silica (hex:siRNA=1:1) to obtain acetate S43 (209 mg, 367 μmol, 73%) as a colorless oil. 1 H NMR (400 MHz, CDCl3): δ 7.33-7.18 (m, 5H), 6.88 (s, 1H), 4.21 (dd, J = 10.7, 5.2 Hz, 1H), 4.15 (t, J = 6.5 Hz, 2H), 4.08-3.97 (m, 3H), 3.77-3.67 (m, 3H), 3.65-3.47 (m, 7H), 3.19 (dt, J = 14.3, 5.6 Hz, 1H), 2.54-2.39 (m, 4H), 2.04 (s, 3H), 1.90 (p, J = 6.4 Hz, 2H), 0.99 (t, J = 7.0 Hz, 6H). 13 C{ 1 H} NMR (101 MHz, CDCl3): δ 171.2, 145.9, 140.1, 136.7, 136.1, 132.2, 129.0, 128.8, 127.8, 71.1, 70.7, 70.3, 67.9, 61.8, 52.4, 48.3, 48.2, 44.6, 43.2, 29.1, 21.1, 15.0. FTIR (NaCl, thin film): 3449, 3029, 2967, 2468, 1737, 1437, 1341, 1245, 1140, 953, 829, 718cm -1 . HRMS:(ESI-TOF)C 26 H 39 N2O6S3[M+H] + The calculated value is 571.1965, and the measured value is 571.2076. TLC (1:1 hexane:SiO), R f :0.63(UV).

[0107] Compound S44 [ka] Compound S44 was synthesized on a 345 μmol scale according to general procedure 6. Hydrolysis of the acetate protecting group was observed during the reaction. After column chromatography on silica (DCM:MeOH = 95:5), the free alcohol S44 was isolated as a colorless oil (44.1 mg, 90.8 μmol, 26%). 1 H NMR (400 MHz, CD3CN): δ 7.58 (s, 1H), 6.28 (bs, 2H), 4.44 (dd, J = 10.7, 5.6 Hz, 1H), 4.03 (ddd, J = 14.9, 10.8, 1.0 Hz, 1H), 3.85 (dd, J = 14.9, 5.6 Hz, 1H), 3.69-3.62 (m, 2H), 3.60-3.46 (m, 9H), 3.28 (ddd, J = 14.4, 6.4, 4.4 Hz, 1H), 2.57 (qd, J = 7.0, 1.1 Hz, 4H), 1.71 (p, J = 6.2 Hz, 2H), 1.05 (t, J = 7.1 Hz, 6H). 13 C{ 1H} NMR (101 MHz, CD3CN): δ 148.5, 147.6, 139.0, 131.2, 71.1, 70.7, 70.6, 69.3, 60.2, 53.4, 48.9, 48.2, 45.2, 33.4, 22.5, 15.2. TLC(19:1 DCM:MeOH), R f :0.25(UV).

[0108] Compound S45 [ka] Compound S45 was synthesized on a 90.6 μmol scale according to general procedure 7. After purification by preparative HPLC (C18 column, 9.4 × 250 mm), the target compound was isolated as a colorless oil (26.7 mg, 53.4 μmol, 59%). 1 H NMR (400 MHz, CD3CN): δ 7.58 (s, 1H), 6.15 (bs, 2H), 4.45 (dd, J = 10.6, 5.6 Hz, 1H), 4.04 (dd, J = 15.0, 10.6 Hz, 1H), 3.87 (dd, J = 15.0, 2.50 (t, J = 6.2 Hz, 2H), 1.05 (t, J = 7.1 Hz, 6H). 13 C{ 1 H} NMR (101 MHz, CD3CN): δ 173.3, 148.5, 147.2, 139.1, 131.2, 71.0, 70.8, 70.6, 67.3, 53.4, 48.9, 48.3, 45.3, 35.4, 15.0. HRMS:(ESI-TOF)C 17 H 30 N3O8S3[M+H] + The calculated value is 500.1190, and the measured value is 500.1209.

[0109] compound 14 [ka] Acid S45 (10.0 mg, 20.0 μmol, 1.0 equivalent) was dissolved in DCM:MeCN (9:1, 200 μL) in a drum vial equipped with a stirring bar. N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (4.20 mg, 22.0 μmol, 1.1 equivalent), pentafluorophenol (4.10 mg, 22.0 μmol, 1.1 equivalent), and DMAP (0.3 mg, 2.00 μmol, 0.1 equivalent) were added to the solution. The reaction was stirred overnight at room temperature. Subsequently, the volatile materials were evaporated under reduced pressure. The crude material was purified by preparative HPLC (C18 column, 9.4 × 250 mm) to obtain target compound 14 (7.9 mg, 11.9 μmol, 59%) as a colorless oil. 1 H NMR (400 MHz, CD3CN): δ 7.57 (s, 1H), 6.16 (bs, 2H), 4.42 (dd, J = 10.7, 5.6 Hz, 1H), 4.02 (ddd, J = 15.0, 10.7, 1.0 Hz, 1H), 3.90-3.78 (m, 3H), 3.69-3.64 (m, 2H), 3.62-3.50 (m, 5H), 3.25 (ddd, J = 14.4, 6.4, 4.4 Hz, 1H), 2.94 (t, J = 6.0 Hz, 2H), 2.56 (q, J = 7.1 Hz, 4H), 1.04 (t, J = 7.1 Hz, 6H). 13 C{ 1 ¹H} NMR (10¹ MHz, CD3CN): δ 168.9, 148.5, 147.5, 131.2, 71.0, 70.6, 66.7, 53.3, 48.80, 48.1, 45.2, 35.1, 15.1. Note: Phenol carbon is not observed due to CF coupling. 19 F{ 1H} NMR (376 MHz, CD3CN): δ -154.67 (d, J = 16.6 Hz), -160.41 (t, J = 20.9 Hz), -164.58 (dd, J = 20.7, 16.8 Hz). HRMS:(ESI-TOF)C 23 H 29 F5N3O8S3[M+H] + The calculated value is 666.1031, and the measured value is 666.1042.

[0110] BZA-2C-PFP(15) Compound S46 [ka] Compound S46 was synthesized on a 10.0 mmol scale according to general procedure 1 using 1-bromo-3-methoxypropane (1.36 mL, 1.84 g, 12.0 mmol, 1.2 equivalents). After column chromatography on silica (hex:HCl = 4:1 to 7:3), the target compound was isolated as a colorless oil (3.25 g, 10.4 mmol, quantitative). 1 H NMR (400 MHz, CDCl3): δ 6.95 (s, 1H), 4.63 (dt, J = 8.3, 4.1 Hz, 1H), 4.13-3.98 (m, 1H), 3.76 (dd, J = 15.3, 4.2 Hz, 1H), 3.65 (ddd, J = 12.6, 7.8, 6.4 Hz, 1H), 3.51 (qdd, J = 9.6, 6.7, 4.5 Hz, 2H), 3.42-3.27 (m, 2H), 3.24 (s, 3H), 2.03-1.80 (m, 2H). 13 C{ 1 H} NMR (101 MHz, CDCl3): δ 143.1, 136.1, 133.2, 126.0, 70.2, 61.8, 58.7, 53.9, 47.1, 29.0. FTIR (NaCl, thin film): 3434, 2931, 2881, 2104, 1644, 1422, 1333, 1163, 1112, 1027, 684cm -1 . HRMS:(ESI-TOF)C 10 H 14 ClNaNO4S2[M+Na] + The calculated value is 333.9945, and the measured value is 333.9983. TLC (3:1 hexane:SiO), R f :0.11(UV).

[0111] Compound S47 [ka] Compound S47 was synthesized on a 10.0 mmol scale according to general procedure 2. After column chromatography on silica (hex:HCl=3:1), the target compound was isolated as a colorless oil (4.66 g, 10.0 mmol, quantitative). 1 H NMR (500 MHz, CDCl3): δ 7.90-7.76 (m, 2H), 7.47-7.40 (m, 2H), 6.61 (s, 1H), 5.34 (dd, J = 3.9, 2.6 Hz, 1H), 4.23 (ddd, J = 16.2, 3.9, 0.8 Hz, 1H), 3.92 (dd, J = 16.3, 2.6 Hz, 1H), 3.55 (dt, J = 13.8, 6.9 Hz, 1H), 3.47-3.38 (m, 2H), 3.32 (s, 3H), 3.26 (dt, J = 13.7, 6.8 Hz, 1H), 2.51 (s, 3H), 1.91-1.81 (m, 2H). 13 C{ 1 H} NMR (125 MHz, CDCl3): δ 146.2, 136.5, 136.4, 135.6, 133.0, 130.5, 128.1, 126.1, 69.2, 67.8, 58.8, 51.4, 47.5, 29.0, 21.9. FTIR (NaCl, thin film): 3439, 2111, 1659, 1645, 1423, 1346, 1190, 1175, 1116, 939cm -1 . HRMS:(ESI-TOF)C 17 H 21 NClO6S3[M+H] + The calculated value is 466.0214, and the measured value is 466.0268. TLC (3:1 hexane:SiO), R f :0.38(UV).

[0112] Compound S48 [ka] Compound S48 was synthesized on an 8.28 mmol scale according to general procedure 3. After column chromatography on silica (hex:toluene=85:15), the target compound was isolated as a colorless oil (1.70 g, 4.63 mmol, 56%). 1 H NMR (500 MHz, CDCl3): δ 6.96 (s, 1H), 4.20 (dd, J = 10.8, 5.1 Hz, 1H), 4.10 (ddd, J = 14.6, 10.8, 0.9 Hz, 1H), 3.62-3.48 (m, 3H), 3.43 (ddd, J = 9.5, 6.4, 5.3 Hz, 1H), 3.34 (s, 3H), 3.08 (dt, J = 13.9, 7.0 Hz, 1H), 2.68-2.45 (m, 4H), 1.99-1.83 (m, 2H), 1.06 (t, J = 7.1Hz, 6H). 13 C{ 1 H} NMR (125 MHz, CDCl3): δ 145.5, 135.6, 132.9, 126.3, 69.4, 58.9, 52.2, 47.5, 45.9, 44.7, 29.7, 15.0. FTIR (NaCl, thin film): 3433, 2968, 2932, 2872, 2831, 2100, 1644, 1342, 1169, 1115cm-1 . HRMS:(ESI-TOF)C 14 H 24 ClN2O3S2[M+H] + The calculated value is 367.0911, and the measured value is 367.0940. TLC (3:1 hexane:SiO), R f :0.43(UV).

[0113] Compound S49 [ka] Compound S49 was synthesized on a 4.61 mmol scale according to general procedure 4. After column chromatography on silica (hex:siRNA=85:15), the target compound was isolated as a yellow oil (1.02 g, 2.25 mmol, 49%). 1 H NMR (500 MHz, CDCl3): δ 7.33-7.17 (m, 5H), 6.86 (s, 1H), 4.14 (dd, J = 10.8, 5.2 Hz, 1H), 4.07-3.92 (m, 3H), 3.59-3.37 (m, 4H), 3.34 (s, 3H), 3.05 (dt, J = 13.8, 7.0 Hz, 1H), 2.48 (qd, J = 7.1, 1.5 Hz, 4H), 1.98-1.79 (m, 2H), 1.00 (t, J = 7.1 Hz, 6H). 13 C{ 1 H} NMR (125 MHz, CDCl3): δ 145.5, 140.2, 136.7, 136.2, 132.2, 129.1, 128.8, 127.8, 69.5, 58.9, 52.4, 47.9, 45.8, 44.7, 43.2, 29.7, 15.1. FTIR (NaCl, thin film): 3422, 2967, 2114, 1657, 1643, 1463, 1377, 1339, 1165, 1115cm -1 . HRMS:(ESI-TOF)C 21 H31 N2O3S3[M+H] + The calculated value is 455.1491, and the measured value is 455.1520. TLC (3:1 hexane:SiO), R f :0.36(UV).

[0114] compound 2 [ka] Compound 2 was synthesized on a 2.24 mmol scale according to general procedure 5. After column chromatography on silica (DCM:MeOH = 99:1), the target compound was isolated as a brown oily substance (733 mg, 1.78 mmol, 79%).

[0115] The spectroscopic data were consistent with the data reported above.

[0116] compound 3 [ka] Compound 3 was synthesized on a 1.77 mmol scale according to general procedure 6. After column chromatography on silica (DCM:MeOH=97:3), the target compound was isolated as a yellow oil (64.0 mg, 161 μmol, 9.1%).

[0117] The spectroscopic data were consistent with the data reported above.

[0118] compound 5 [ka] Compound 5 was synthesized on a 153 μmol scale according to general procedure 7. After purification by column chromatography (CH2Cl2:MeOH = 93:7 to 9:1), the carboxylic acid (22.5 mg, 54.7 μmol, 36%) was isolated as a colorless oil.

[0119] The spectroscopic data were consistent with the data reported above.

[0120] compound 15 [ka] Acid 5 (10.0 mg, 24.3 μmol, 1.0 equivalent) was dissolved in DCM:MeCN (9:1, 243 μL) in a drum vial equipped with a stirring bar. N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (5.12 mg, 26.7 μmol, 1.1 equivalent), pentafluorophenol (4.92 mg, 26.7 μmol, 1.1 equivalent), and DMAP (0.3 mg, 2.43 μmol, 0.1 equivalent) were added to the solution. The reaction was stirred overnight at room temperature. The volatile materials were then evaporated under reduced pressure. The crude material was purified by preparative HPLC (C18 column, 9.4 × 250 mm) to obtain target compound 15 (8.2 mg, 14.2 μmol, 58%) as a colorless oil. 1 H NMR (400 MHz, CD3CN): δ 7.60 (s, 1H), 6.19 (bs, 2H), 4.42 (dd, J = 10.8, 5.5 Hz, 1H), 4.05 (ddd, J = 15.0, 10.8, 0.8 Hz, 1H), 3.86-3.65 (m, 2H), 3.46 (dt, J = 14.3, 6.7 Hz, 1H), 3.12 (td, J = 6.5, 1.6 Hz, 2H), 2.58 (qd, J = 7.1, 1.9 Hz, 4H), 1.05 (t, J = 7.1 Hz, 6H). 13 C{ 1 ¹H} NMR (10¹ MHz, CD3CN): δ 168.6, 148.9, 147.4, 138.3, 131.2, 53.5, 49.0, 45.3, 45.3, 34.8, 15.2. Note: Phenol carbon is not observed due to CF coupling. 19 F{ 1H} NMR (376 MHz, CD3CN): δ -154.33 - -154.53 (m), -160.16 (t, J = 21.0 Hz), -164.10 - -164.94 (m). HRMS:(ESI-TOF)C 19 H 21 F5N3O6S3[M+H] + The calculated value is 578.0507, and the measured value is 578.0510.

[0121] Chemical synthesis of NHS ester brinzolamide using click chemistry [ka] [ka]

[0122] General overview A reactive handle can be attached to brinzolamide (BZA). Reductive amination of the secondary amine of brinzolamide (1) and subsequent treatment of the intermediate methoxy ether (2) with boron tribromide provides access to either a primary alcohol (3) or a primary alkyl bromide (4). Both are useful intermediates for further derivatization. Oxidation of the primary alcohol to a carboxylic acid (5) and EDC coupling with N-hydroxysuccinimide yields an NHS ester (6). Other carboxylic acid derivatives for chemical conjugation can also be prepared by this method. Alternatively, the alkyl bromide (4) can be converted to an alkyl azide (7) which can be used in click cycloaddition reactions to produce further NHS derivatives. Alkyl azides can also be used to conjugate low molecular weight cargoes, such as fluorophores, to brinzolamide.

[0123] Payload delivery across the BBB Methods and delivery systems for delivering a payload (e.g., a therapeutic agent) to the nervous system are included in the disclosures herein. The method includes the step of providing a small molecule or a derivative thereof capable of interacting with carbonic anhydrase IV. The small molecule may be part of a delivery system, which may include a payload to be delivered to the nervous system. The method may further include the step of administering the delivery system to a target.

[0124] In some embodiments, the delivery system includes nanoparticles, nanotubes, nanowires, dendrimers, liposomes, ethosomes and aquasomes, polymerosomes and niosomes, foams, hydrogels, cubosomes, quantum dots, exosomes, macrophages, and combinations thereof. In some embodiments, the delivery system includes nanoparticles selected from lipid-based nanoparticles, polymeric nanoparticles, inorganic nanoparticles, surfactant-based emulsions, nanowires, silica nanoparticles, virus-like particles, peptide or protein-based particles, lipid-polymer particles, nanolipoprotein particles, and combinations thereof.

[0125] For example, the payload may include antibacterial agents, therapeutic agents, prodrugs, peptides, proteins, enzymes, lipids, biological response modifiers, pharmaceuticals, lymphokines, heterologous antibodies or fragments thereof, detectable labels, polyethylene glycol (PEG) molecules, or two or more combinations of these agents.

[0126] The payload may include neurotropic polypeptides, such as neurotrophic factors, endocrine factors, growth factors, paracrine factors, hypothalamic release factors, neurotransmitter polypeptides, polypeptide agonists for receptors expressed by CNS cells, polypeptides involved in lysosomal storage disorders, or any combination thereof. Another example of a payload is IL-1 receptor antagonist (IL-1Ra), dalargin, interferon-β, glial neurotrophic factor (GDNF), tumor necrosis factor receptor (TNFR), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-4 / 5, neurotrophin (NT)-3, neuruturin, neuregulin, netrin, ciliary neurotrophic factor (CNTF), stem cell factor (SCF), semaphorin, hepatocyte growth factor (HGF), epidermal growth factor (EGF), and transfer This may include forming growth factor (TGF)-cx, TGF-B, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), heregulin, artemin, parcephin, interleukin, granulocyte colony-stimulating factor (CSF), granulocyte-macrophage-CSF, cardiotrophin-1, hedgehog, leukemia suppressor (LIF), midkine, pleiotrophin, erythropoietin (EPO), bone morphogenetic protein (BMP), netrin, saposin, any fragment thereof, or any combination thereof.

[0127] Aspects of the present invention also provide delivery of a conjugate to a target for transporting a therapeutic agent across the blood-brain barrier (BBB). In aspects of the present invention, the delivery of the therapeutic payload may be for the treatment of a disease, disorder, or injury of the central nervous system (CNS). In aspects of the present invention, the therapeutic agent may be released from the conjugate after entering the CNS. In certain aspects, diseases, disorders, or injuries of the CNS include, without limitation, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease, spinal cord injury, traumatic brain injury, stroke, neuropathic pain, neurodegeneration, neuroinflammation, progressive multifocal leukoencephalopathy (PML), encephalomyelitis (EPL), central pontine myelolysis (CPM), adrenoleukodystrophy, Alexander disease, Pelizaeus-Merzbach disease (PMZ), and globoid cell leukodystrophy. This may include leucodystrophy (Krabbe disease), Wallerian degeneration, optic neuritis, transverse myelitis, post-irradiation injury, neurological complications of chemotherapy, acute ischemic optic neuropathy, vitamin E deficiency, isolated vitamin E deficiency syndrome, Bassen-Kohnzweig syndrome, Marchia-Fava-Bignami syndrome, metachromatic leukodystrophy, trigeminal neuralgia, Bell's palsy, primary tumor, secondary metastasis, or any combination thereof. [Examples]

[0128] Experimental example research design The blood-brain barrier (BBB) ​​functions as a highly selective, semipermeable membrane that separates circulating blood from the brain and extracellular fluid in the central nervous system (CNS). Due to its selective nature, this barrier restricts the entry of numerous therapeutic agents, particularly large molecules, into the brain. Mechanism-agnostic manipulative strategies, such as the directional evolution of adeno-associated virus capsids, have successfully enabled BBB-crossing delivery in animals. However, the brain shuttle molecules generated through this strategy possess unpredictable translatability across species, due to an unknown mechanism. Receptor-mediated transport mechanisms have emerged as a promising avenue for rationally manipulating transferable molecular shuttles that facilitate the transport of therapeutic agents across the BBB. However, all previously identified receptors are ubiquitously expressed in both the CNS and peripheral tissues, leading to off-target tissue delivery and potential safety disadvantages, such as reduced reticulocyte counts. Furthermore, most of these binder-cargo conjugates require individual optimization. There is still a need for more specific and versatile methods that can address the delivery of biologics to the brain.

[0129] The present invention provides a rationally designed reactive small molecule binder based on the carbonic anhydrase inhibitor brinzolamide, which can function as a shuttle to facilitate CA-IV-mediated brain delivery.

[0130] Design of activated CA-IV binders for bioconjugation One important factor to consider when designing the shuttle of the present invention when targeting an epitope is that the efficiency of receptor-mediated transcytosis can vary depending on the specific epitope on the BBB receptor targeted by the binder. AAV capsids, which have been proposed to rely on CA-IV to cross the blood-brain barrier, bind to deep pockets on their surface that function as catalytic centers for their enzymatic processes, implying that binding to these pockets may be sufficient to induce efficient CA-IV-mediated transcytosis.

[0131] Figure 1 is a schematic diagram of transcytosis and antibody conjugation mediated by CA-IV according to the present invention.

[0132] Carbonic anhydrase binders have been modified with reactive groups designed to facilitate one-step bioconjugation to therapeutic cargo while minimizing any impact on CA-IV binding. The conjugate can bind to CA-IV receptors present on brain endothelial cells, thereby inducing transcytosis across the blood-brain barrier (BBB).

[0133] To effectively target the CA-IV catalytic pocket, we did not utilize existing binder molecules developed for other intracellular carbonic anhydrase family members. These carbonic anhydrase inhibitors may exhibit cross-reactivity with CA-IV and can function as parent compounds for our shuttle molecule. An example of this is brinzolamide (BZA), an FDA-approved generic inhibitor of intracellular CA-II with desirable physicochemical properties and safety profile. This compound was found to bind to CA-IV using a binding site that may overlap with that of brain-transducing AAV.

[0134] To enable bioconjugation of brinzolamide with therapeutic cargo, brinzolamide was modified by introducing an N-hydroxysuccinimide (NHS) ester group. The NHS ester was chosen for its ability to react with a wide range of protein molecules having primary amines exposed on their surfaces under mild buffer conditions and at room temperature. Structural analysis of brinzolamide in complex with the mouse CA-IV receptor revealed that the methyl ether moiety of brinzolamide was exposed on the receptor surface.

[0135] Figure 2A shows a structural analysis of the mouse CA-IV binding pocket and its interactions with the designed AAV, BZA, and activated BZA (BZA-2C-NHS).

[0136] Figure 2B shows the structural alignment of BZA across CA-IV homologs.

[0137] The alignment shows that all residues immediately adjacent to the BZA binding pocket have the same identity across all homologs. BZA-mediated receptor binding and targeted delivery may potentially be transferred across species.

[0138] While not limited by the mechanism of action, this suggests that the therapeutic cargo can be conjugated to the receptor without facing any steric hindrance. Testing of CA-IV structures from diverse mammalian species verified the high conservation of this binding pocket, as detailed below by amino acid sequence alignment and the bolded residues that form the binding pocket: [Table 1]

[0139] Although not limited by the mechanism of action, it has been proposed that brinzolamide binds to various CA-IV homologs in a similar manner. Following structural analysis, brinzolamide derivatives characterized by reactive NHS esters substituting methyl ether groups were designed. To simplify the synthesis of NHS ester brinzolamide, an extra ethyl group was introduced to convert the secondary amine to a tertiary amine, thereby minimizing self-reactions.

[0140] Production of BZA-conjugated cargo via NHS-esterification ligation NHS-ester ligation was performed in a neutral pH buffer to maintain the stability of the cargo molecules. For protein cargoes, the reaction buffer was DPBS containing 0.001% Pluronic® F-68 (2.7 mM KCl, 1.5 mM KH2PO4, 136.9 mM NaCl, 8.1 mM Na2HPO4, pH 7.4; 0.001% Pluronic F-68). For siRNA cargoes (described below), the reaction buffer was nuclease-free double-strand buffer (30 mM HEPES, pH 7.5; 100 mM potassium acetate).

[0141] NHS-ester-brinzolamide powder was dissolved in DMSO at 50 mM, divided into aliquots, and stored at -80°C. Immediately before the reaction, the stock NHS-ester-brinzolamide solution was dissolved in 100 μL of reaction buffer at 10× the target final concentration. The 10× solution was mixed in a 2 mL centrifuge tube with purified cargo molecules diluted in 900 μL of the same reaction buffer. The tube was rotated and stirred during the reaction at 25°C. The reaction was then stopped by adding 1 μL of Tris-based buffer (e.g., 1 M Tris-HCl pH 7.5) containing at least 500 mM amine groups. The resulting product was purified using a PD-10 column according to the manufacturer's protocol, and the buffer was replaced with fresh DPBS containing 0.001% Pluronic F-68. The product was concentrated to a maximum of 10 mg / mL using a 30 kD MWCO protein concentrator. The final product was sterilized using a 0.22 μM filter. The final concentration was measured using nanodrop. [Table 2-1] [Table 2-2]

[0142] Production of BZA-conjugated cargo via CuAAC ligation Azido-brinzolamide and alkyne-Alexa647 were dissolved together in a 1:1 mixture of water and tert-butyl alcohol at 5 mM. Sodium ascorbate was dissolved in water at 12.5 mM (10 × working concentration). Copper(II) sulfate pentahydrate was dissolved in water at 12.5 mM (100 × working concentration). The reaction was carried out in a 2 mL centrifuge tube by mixing 50 μL of azido-brinzolamide, 50 μL of alkyne-Alexa647, 10 μL of sodium ascorbate, and 1 μL of copper(II) sulfate pentahydrate. The tube was rotated and stirred overnight at 25°C during the reaction. The product was stored at -20°C before testing.

[0143] Synthesis of BZA-2C-NHS from commercially available brinzolamide molecules [ka] [ka] AcOH = Acetic acid, THF = Tetrahydrofuran, DCM = Dichloromethane, DMAP = 4-Dimethylaminopyridine, EDCI = N-(3-Dimethylaminopropyl)-N'-Ethylcarbodiimide hydrochloride, TSTU = N,N,N',N'-Tetramethyl-O-(N-Succinimidyl)uronium tetrafluoroborate, DIPEA = N,N-Diisopropylethylamine

[0144] Figure 3 shows the chemical synthesis of NHS-ester-brinzolamide. 1 H-NMR verification is shown.

[0145] In vitro interaction between CA-IV and biologics conjugated with carbonic anhydrase binders. To test whether NHS-ester brinzolamide can be conjugated to a biological cargo, BZA-2C-NHS was used to react it with the commercially available anti-human PD-L1 antibody atezolizumab biosimilar (Ate), a model for the human antibody cargo. After purification, the conjugation product was analyzed using liquid chromatography-mass spectrometry (LC-MS) and compared to unconjugated Ate.

[0146] Figure 4A is a structural schematic diagram showing the Ate conjugation to a BZA using linker 2C to generate BZA-2C-Ate.

[0147] Figures 4B-4G show LC-MS graphs of the BZA-Ate conjugation.

[0148] Approximately half of the light chain and half of the heavy chain were found to contain a conjugation of at least one copy of BZA (approximately 400 Da) after reacting with BZA-2C-NHS. This indicates that more than 90% of the IgG complex, each composed of two light chains and two heavy chains, binds to at least one copy of the BZA compound.

[0149] LC-MS using electrospray shows that more than half of both the heavy and light chains of the IgG antibody are conjugated with 1–3 brinzolamide moieties (approximately 390 Da) compared to the unmodified atezolizumab control.

[0150] Cell-based binding assays using HEK293T cells and HeLa cells, cell-based internalization assays using pHrodo dyes, SPR assays, in vivo distribution and pharmacokinetic characterization, and ELIZA assays were performed as described below:

[0151] Selected experimental methods Cell-based binding assay - HEK293T HEK293T cells were seeded at 80% confluence in 6-well plates and maintained at 37°C in 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 5% fetal bovine serum (FBS), 1% non-essential amino acids (NEAA), and penicillin-streptomycin (100 U / ml). Membrane-bound human and mouse CA-IV were transfected with polyethyleneimine (PolySciences, no. 23966). Cells were seeded in 24-well plates 24 hours after transfection on sterile German glass coverslips coated with Neuvitro poly-d-lysine (Fisher Scientific, no. NC0343705). Brinzolamide-conjugated nanobodies and antibodies were added to the medium at 0.4 μM and 0.2 μM, respectively, incubated at 37°C in 5% CO2 for 1 hour, and then fixed in 4% PFA. At ambient temperature, the coverslips were blocked for 30 minutes in 1× Tris-buffered saline (TBS) containing 3% bovine serum albumin (BSA) and 0.3% Triton®-X100 for permeabilization conditions, and incubated for 60 minutes in 1× TBS with 0.05% Triton X-100 for permeabilization conditions and in secondary antibody (nanobody, 1:1000 dilution; GenScript, A01994; antibody, 1:1000 dilution; Invitrogen, A-21445) in 3% BSA. The coverslips were washed three times in 1× TBS. The coverslips were mounted on slides using diamond antifade mounting media (Invitrogen, P36931) containing 4',6-diamidino-2-phenylindole. Fluorescence microscope images were captured using a confocal laser scanning microscope (LSM 880, Carl Zeiss, USA).

[0152] Cell-based binding assay - HeLa HeLa cells were seeded at 80% confluence in 6-well plates and maintained at 37°C in 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% non-essential amino acids (NEAA), and penicillin-streptomycin (100 U / ml). Membrane-bound human and mouse CA-IV were transfected with polyethyleneimine (PolySciences, no. 23966). Cells were seeded 24 hours after transfection in black 96-well glass-bottom plates coated with poly-L-ornithine (Cellvis, no. P96-1.5HN; Sigma Aldrich, no. P4957). Brinzolamide-conjugated nanobodies and antibodies were added to the medium at 0.4 μM and 0.2 μM, respectively, incubated at 37°C in 5% CO2 for 1 hour, and then fixed in 4% PFA. At ambient temperature, cells were blocked for 30 minutes in 1× Tris-buffered saline (TBS) containing 3% bovine serum albumin (BSA) and 0.3% Triton-X100 for permeabilization conditions, and then incubated for 60 minutes in 1× TBS with 0.05% Triton X-100 for permeabilization conditions and in secondary antibody (nanobody, 1:1000 dilution; GenScript, A01994; antibody, 1:1000 dilution; Invitrogen, A-21445) in 3% BSA. Cells were washed three times in 1× TBS. Fluorescence microscopy images were captured on a confocal laser scanning microscope (LSM 980, Carl Zeiss, USA).

[0153] Cell-based internalization assay using pHrodo dyes HeLa cells were seeded at 80% confluence in 6-well plates and maintained at 37°C in 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% non-essential amino acids (NEAA), and penicillin-streptomycin (100 U / ml). Membrane-bound CA-IV of human, mouse, marmoset, or rhesus monkey origin was co-transfected in a 1:1 ratio with CAG-mNeonGreen using polyethyleneimine (PolySciences, no. 23966). The following day, cells were seeded in black 96-well plates with optically clear polystyrene bottoms (Greiner Bio-One, No. 655096) and maintained at 37°C in 5% CO2 in FluoroBrite® Dulbecco's Modified Eagle Medium (DMEM) supplemented with 5% fetal bovine serum (FBS), 1% non-essential amino acids (NEAA), penicillin-streptomycin (100 U / ml), 1×GlutaMAX, and 15 mM HEPES. Once cells had adhered to the plates, antibodies conjugated into small molecules by various linkers were labeled using pH-sensing dyes (Invitrogen, Z25612) according to the manufacturer's protocol, with adjustments made as needed for drug administration. Briefly, antibodies were labeled by mixing an 800 nM solution of the treated antibody with a 960 nM solution of the labeling reagent in a 1:1 v / v ratio. After 10 minutes at room temperature, labeled antibodies were added to the cells in a 1:1 v / v ratio. After 3 or 6 hours of incubation, the cells were stained with NucBlue LiveReady Probes (Invitrogen, R37065) and imaged on a high-content confocal microscope (Micro Confocal ImageXPress, Molecular Devices) using a 40× objective.

[0154] Surface plasmon resonance (SPR) A Sierra SPR-32 (Bruker) equipped with a Protein A sensor chip was used. For nanobodies and siRNA cargoes, Fc-fused CA-IV receptor protein was immobilized at 200 nM in HBS-EP+ buffer (GE Healthcare). Conjugated and unconjugated cargo molecules at the stated concentrations in the same buffer were injected. For IgG antibody cargoes, the setup was reversed, and cargo proteins were immobilized at 200 nM in HBS-EP+ buffer (GE Healthcare), and untagged CA-IV receptor protein at the stated concentrations was injected. The analytes were injected at a flow rate of 10 μL / min for 240 seconds, followed by dissociation for 600 seconds. A regeneration step using 10 mM glycine pH 1.5 was performed between each cycle. All kinetic data were double-referenced subtracted.

[0155] In vivo distribution and pharmacokinetic characterization Eight-week-old C57BL / 6J mice were intravenously injected with 0.05 mg of IgG antibody. At the indicated time points, to collect the target tissues, the mice were anesthetized with Euthasol (sodium pentobarbital and sodium phenytoin solution, Virbac AH) and perfused cardiacally with 30-50 mL of 0.1 M phosphate-buffered saline (PBS) (pH 7.4), followed by 30-50 mL of 4% paraformaldehyde (PFA) in 0.1 M PBS. Organs were collected and post-fixed in 4% PFA at 4°C for 24-48 hours. After this, the tissues were washed twice with 0.1 M PBS and stored at 4°C in fresh PBS-azide (0.1 M PBS containing 0.05% sodium azide). For immunochemistry, the brain and liver were sectioned into 100 μm thick slices using a vibratome. Slices were initially incubated with 1:200 anti-human Igg-Alexa647 antibody in blocking buffer (0.1 M PBS, pH 7.4, 10% normal donkey serum (NDS), 0.1% Triton X-100, and 0.01% sodium azide). The slices were then washed three times in 0.1 M PBS for a total duration of 5–6 hours. For DNA staining, tissues were incubated with 4',6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, 10236276001, 1:1000) in 0.1 M PBS for 15 minutes, followed by a single wash in 0.1 M PBS for 10 minutes. DAPI and / or antibody-stained tissue sections were mounted using a ProLong Diamond Antifade Mountant (Thermo Fisher Scientific, P36970) before imaging them under a microscope.

[0156] ELISA Ate and BZA-Ate concentrations were quantified using the Anti-Human IgG ELISA Kit (abcam, no. ab195215). PBS-perfused organs from mice were collected in tubes pre-filled with beads (Benchmark no. D1032-30, no. D1032-15, no. D1033-28). These tubes were then filled with 1 mL of cell extraction buffer from the ELISA kit according to the protocol. The tubes were then placed in a Benchmark BeadBlaster 24 homogenization machine at a speed of 7 m / s for two total cycles with 15-second cycles and a 30-second pause between each cycle. After two cycles, a manual 1-minute pause was given. This homogenization and pause process was repeated two more times. The samples were centrifuged at 18,000 g at 4°C for 30 minutes. 700 μL of supernatant was collected into two wells (350 μL each) without disturbing the debris near the beads. The supernatant was then diluted in DI water, and 50 μL of the diluted sample was pipetteed into each well of a pre-coated ELISA plate from the kit. Diluted antibody solutions and standards from the kit were prepared according to their protocols. Standard concentrations ranged from 0.23 ng / mL to 15 ng / mL. Next, 50 μL of diluted antibody solution was added to all wells, and the plate was incubated on a shaker for 50 minutes. The plate was then washed 3× with wash buffer from the kit, 100 μL of TMB solution was added to each well, and the plate was incubated on a shaker for 35 minutes. Next, 100 μL of stop solution was added to the wells, the plate was sealed, and the absorbance at 450 nm was read.

[0157] Figures 5A-D are graphs showing the results of SPR assays between CA-IV and BZA-2C-Ate.

[0158] The binding interaction between conjugated or unconjugated IgG antibodies and purified CA-IV protein was evaluated using SPR assays. 200 nM IgG antibodies were immobilized on pre-coated capture sensors with protein A, followed by introduction of different concentrations of CA-IV protein. Receptor concentrations are shown in the inset.

[0159] A concentration-dependent binding signal was observed between CA-IV and BZA-2C-Ate, indicating an estimated dissociation constant in the range of 100 nM to 200 nM. Subsequently, tests were conducted to determine whether the conjugated antibody could bind to cultured cells overexpressing CA-IV.

[0160] Figure 5E shows immunofluorescence images of living CA-IV-expressing cells incubated with either unconjugated or BZA-conjugated Ate.

[0161] In immunofluorescence imaging, cells were fixed after 1 hour incubation with 0.2 μM cargo antibody. After fixation, the cells were washed with detergent-containing buffer and permeabilized. Next, the fixed cells were stained with an anti-human antibody conjugated with Alexa Fluor 647 and subsequently imaged. Panels labeled IF(MIP) show the maximum intensity projection of the cells, while panels labeled IF(z-slice) show sections near the bottom of the well. In live-cell imaging, the cargo antibody was pre-labeled with the fluorescent pH indicator pHrodo, which shows increased fluorescence intensity in acidic environments such as endosomes, and then applied to cells at 0.4 μM. The treated cells were imaged 6 hours after incubation with the antibody. Spotted structures were clearly visible inside the permeabilized cells before staining, suggesting internalization of the antibody via endocytosis. Binding and internalization can be observed using CA-IV from various species, e.g., mouse, human, and rhesus monkey.

[0162] Consistent with findings from SPR experiments, immunofluorescence analysis revealed the presence of BZA-2C-Ate signaling on CA-IV-expressing cells. These signals were observed at or within cell boundaries in the form of mottled patterns.

[0163] To provide further confirmation that the spots originated from endocytosis of BZA-conjugated antibodies, antibody samples were labeled with the pH-sensitive dye pHrodo. Subsequently, CA-IV-expressing cells were incubated with the labeled antibody samples. The pHrodo dye showed a significant increase in fluorescence as the pH decreased, thus serving as an indicator of internalization. After a 6-hour incubation period, the consistent presence of spotted structures was observed, which closely resembled the pattern observed in immunofluorescence imaging.

[0164] Quantified and intensified intracellular spots revealed that BZA-2C-Ate produced significantly higher numbers of spots (p<0.0001) with significantly higher average intensity (p<0.0001) compared to unconjugated Ate.

[0165] Figures 5F–G show the mean and counted spot intensities of CA-IV-expressing HeLa cells incubated either unconjugated or with a BZA-2C-Ate conjugate, respectively.

[0166] In vitro findings demonstrated consistent binding of BZA-2C-Ate to CA-IV and its subsequent internalization in CA-IV-expressing cells across various species, including humans, mice, and macaques. This observation revealed the conservation of a critical residue for brinzolamide interactions, enabling in vivo evaluation in rodent and non-human primate models.

[0167] BZA-NHS Linker Variant We developed a synthetic method for producing NHS-ester brinzolamide molecules with different linker variations. The nature of the synthesized products was confirmed using LC-MS and NMR.

[0168] BZA derivatives were synthesized and analyzed as follows:

[0169] De novo synthesis of NHS-ester-BZA variants using hydrocarbon linkers. [ka]

[0170] We analyzed carbonic anhydrase inhibitor-based shuttles containing linker variants of NHS-ester-BZA.

[0171] Figure 6A shows the structure of the selected BZA-Ate linker variant.

[0172] Figures 6B-6M show LC-MS graphs of the unmodified Ate and BZA-Ate linker variant conjugates.

[0173] As shown, the efficiency of conjugation depends on both the linker length and the binder type.

[0174] Figures 7A-7C are graphs showing the results of the SPR assay for the binding of CA-IV to the BZA-Ate linker variant.

[0175] Figure 7D shows immunofluorescence images of living CA-IV expressing cells incubated with either an unconjugated or BZA-conjugated Ate linker variant.

[0176] Z-slice images of HeLa cells stained for human IgG, incubated with 0.2 μM BZA-conjugated Ate using BZA-2C-Ate, BZA-3C-Ate, and BZA-4C-Ate, show clear spots indicating antibody internalization inside the cells that were permeabilized before staining.

[0177] Figures 7E-7F show representative live cell images of CA-IV-expressing HeLa cells incubated with either unconjugated or BZA-conjugated antibodies, after 3 hours and 6 hours of incubation with the antibody, respectively.

[0178] In living cells, for identified spots, the image is segmented, the intensity contained within each spot is quantified, and the result is averaged for each cell.

[0179] Figures 7G–4H show the mean and counted spot intensities of CA-IV-expressing HeLa cells incubated with either unconjugated or BZA-conjugated linker variants, respectively, after 3 hours.

[0180] Figures 7I–4J show the mean and counted spot intensities of CA-IV-expressing HeLa cells incubated with either unconjugated or BZA-conjugated linker variants, respectively, after 6 hours.

[0181] The average spot intensity for each cell was calculated, outliers were highlighted, and the results were shown as a box plot. Statistical significance was determined using Student's t-test. The number of spots for each cell was counted, outliers were highlighted, and the results were shown as a box plot. Statistical significance was determined using Poisson's means test (statistical significance, * :0.01 <p<=0.05、 ** :0.001 <p<=0.01、 *** :0.0001 <p<=0.01、 **** :p<=0.0001).

[0182] When using BZA-2C-Ate, BZA-3C-Ate, and BZA-4C-Ate, clear spots indicating antibody internalization are present inside cells that have been permeabilized before staining.

[0183] Of the NHS-ester-BZA linker variants tested, BZA-3C-NHS showed the highest drug-to-antibody ratio.

[0184] BZA-2C-NHS facilitates highly efficient internalization and could be easily synthesized using commercially available starting materials (4 steps compared to 8 steps for other linker variants).

[0185] NHS-ester derivatives of other CA-IV binders In addition to testing BZA, we also tested a range of other carbonic anhydrase inhibitor-based shuttles, including other CA-IV binders, namely NHS-ester derivatives of acetazolamide (AZA) and dorzolamide (DZA).

[0186] AZA and DZA In vitro characterization of IgG cargo conjugated with alternative CA-IV binders

[0187] Figure 8A shows the structures of two alternative CA-IV binders, acetazolamide (AZA) and dorzolamide (DZA), as NHS ester variants.

[0188] Figures 8B–E show LC-MS graphs of the NHS ester variants of two alternative CA-IV binders, AZA and DZA, respectively.

[0189] Figures 8F-G are graphs showing the results from SPR assays with CA-IV and two alternative CA-IV binders, NHS ester variants of AZA and DZA, respectively.

[0190] Figures 8H-8I show live cell images of CA-IV-expressing HeLa cells incubated with either AZA-conjugated or DZA-conjugated Ate, respectively.

[0191] In living cells, for identified spots, the image is segmented, the intensity contained within each spot is quantified, and the result is averaged for each cell.

[0192] Figures 8J-K show the mean and counted spot intensities of CA-IV-expressing HeLa cells incubated with either unconjugated, AZA-conjugated, or DZA-conjugated Ate, respectively.

[0193] The average spot intensity for each cell was calculated, outliers were highlighted, and the results were shown as a box plot. Statistical significance was determined using Student's t-test. The number of spots for each cell was counted, outliers were highlighted, and the results were shown as a box plot. Statistical significance was determined using Poisson's mean test (statistical significance, * :0.01 <p<=0.05、 ** :0.001 <p<=0.01、 *** :0.0001 <p<=0.01、 **** :p<=0.0001).

[0194] Compared to BZA-based variants, compounds utilizing AZA and DZA as binding cores demonstrated inferior conjugation efficiency and binding signal intensity. Functional binding between Ate antibodies and their target antigens is not affected by the conjugation of these compounds to lysine residues, regardless of the type of linker and binding core used.

[0195] BZA and BZA-2C-IgG Cargo Variants In addition to conjugating with Ate antibodies, we explored the potential to extend this bioconjugation approach to other biological molecules and modalities to enhance receptor binding. We tested further in vitro characterization of BZA-NHS conjugated to different therapeutic modalities, including nanobodies, siRNA, and small molecules.

[0196] IgG antibody (Don) Immunoglobulin G (IgG) antibodies are crucial components of the immune response, capable of neutralizing pathogens and signaling to immune cells. Focusing on their interaction with CA-IV, we explored the potential therapeutic applications of BZA-modified human IgG1.

[0197] In addition to Ate, it has been shown that multiple IgG antibodies, including donanemab (Don), can be conjugated to BZA and promote binding to CA-IV in vitro.

[0198] BZA-modified IgG antibodies were generated, including donanemab, which targets beta-amyloid aggregates, and atezolizumab, which targets PD-L1. For donanemab, two reaction conditions were used: a high-label-density condition (reaction condition 1, 40 μM NHS-ester-BZA: 0.2 μM antibody) and a low-label-density condition (reaction condition 2, 4 μM NHS-ester-BZA: 0.2 μM antibody). For atezolizumab, only one reaction condition was used (200 μM NHS-ester-BZA: 10 μM antibody).

[0199] Figure 9A shows the structure of the IgG antibody BZA variant conjugate.

[0200] Figures 9B-9K show LC-MS graphs of BZA-IgG antibody conjugates containing Don.

[0201] Figures 10A-E show graphs of the results from SPR assays with CA-IV and BZA-conjugated Don, unconjugated Don, and BZA-conjugated higG1 isotypes, respectively.

[0202] Figure 10F shows representative immunofluorescence and live-cell images of CA-IV expressing HeLa cells incubated with either an unconjugated-IgG or BZA-conjugated variant antibody containing Don.

[0203] In living cells, for identified spots, the image is segmented, the intensity contained within each spot is quantified, and the result is averaged for each cell.

[0204] Figures 10G-H show the mean and counted spot intensities of CA-IV-expressing HeLa cells incubated with either an unconjugated or BZA-conjugated antibody variant containing Don, respectively.

[0205] Figures 11A–D show the SPRs of modified and unmodified therapeutic IgG antibodies, including atezolizumab and Don, tested against purified human and mouse CA-IV.

[0206] IgG antibodies were immobilized on a protein A chip, and CA-IV protein at different concentrations was injected. Receptor concentrations are shown in the insert.

[0207] Figures 11E-F show the internalization assays in cultured HeLa cells using BZA-modified IgG antibodies containing atezolizumab and Don, as well as unmodified IgG antibodies.

[0208] Hela cells were transfected with the indicated receptor and incubated with the indicated antibody. After fixation, the cells were washed with either a detergent-containing (permeabilized) or detergent-free (not permeabilized) buffer. The fixed cells were then stained with an anti-human antibody conjugated with Alexa Fluor 647 and imaged. Maximum intensity projection images of cells incubated with 1C, BZA-modified and unmodified antibodies.

[0209] Only the BZA-modified antibody shows clear binding to cells overexpressing the CA-IV protein. Z-slice images of HeLa cells incubated with BZA-modified and unmodified atezolizumab show distinct spots indicating antibody endocytosis inside the cells, which were permeabilized before staining.

[0210] Binding and endocytosis can be observed using CA-IV from different species, including mice, humans, and rhesus monkeys.

[0211] In immunofluorescence imaging, cells were fixed after 1 hour incubation with 0.2 μM cargo antibody. After fixation, the cells were washed with detergent-containing buffer and permeabilized. Next, the fixed cells were stained with anti-human antibody conjugated with Alexa Fluor 647 and subsequently imaged. Panels labeled IF(z slice) show sections near the bottom of the well. In live cell imaging, cargo antibodies were pre-labeled with the fluorescent pH indicator pHrodo, which shows increased fluorescence intensity in acidic environments such as endosomes, and then used to treat the cells. Treated cells were imaged 6 hours after incubation with the antibody. Spotted structures were clearly visible inside the permeabilized cells before staining, suggesting internalization of the antibody via endocytosis. Binding and internalization could be observed using CA-IV from various species.

[0212] BZA-modified therapeutic human IgG1 showed binding affinity to purified CA-IV with a Kd of 100–300 nM. This is similar to the Kd of Denali's TfR-binding ATV, suggesting comparable efficacy. As with the modified nanobodies, products from high-label-density conditions showed stronger binding to the CA-IV protein, indicating that controlling label density may be a potential way to modulate the binding affinity of the modified protein. The BZA-modified IgG molecule also showed binding to cultured cells overexpressing CA-IV. Importantly, BZA-modified IgG can bind not only to human CA-IV protein but also to CA-IV proteins from mouse and rhesus monkeys. This allows the modified IgG molecule to be tested in both rodent and non-human primate (NHP) models.

[0213] When the ability of BZA-modified IgG antibodies to penetrate and localize into the brain in mice was tested, BZA-modified atezolizumab was found to exhibit a sustained brain-enriched signal compared to its unmodified counterpart. Sagittal sections of mouse brains injected intravenously with the antibody showed a clearly stronger brain enrichment for the BZA-modified atezolizumab biosimilar, starting 48 hours after injection, compared to the unmodified version. In particular, certain sections of the cerebral cortex co-stained with neuronal markers demonstrated that the BZA-modified atezolizumab biosimilar molecule was able to cross the blood-brain barrier and bind to neurons. To confirm that this enrichment was not ubiquitous across all organs, liver sections from mice injected intravenously with both types of atezolizumab were tested, but no significant differences were observed.

[0214] Nanobody Nanobody or camelid single-domain antibodies have been highlighted for their high stability and high yield, and have demonstrated therapeutic value alongside several FDA-approved drugs currently in use.

[0215] In particular, these nanobodies derived from Camelus dromedarius possess three surface-exposed lysine residues within a conserved region. This structure allows for chemical modification without interfering with target binding, thereby making them ideal for further manipulation in therapeutic applications.

[0216] Commercially available nanobody anti-GFP VHH was conjugated to brinzolamide using NHS ester ligation. The protein concentration did not show significant changes after the reaction, indicating the stability of the reaction product and the potential for high yield. Two reaction conditions were used: a high-label-density condition (reaction condition 1, 40 μM NHS-ester-BZA: 2 μM nanobody) and a low-label-density condition (reaction condition 2, 4 μM NHS-ester-BZA: 2 μM nanobody).

[0217] Figures 12A and 12B show the structures of the BZA-nanobody variants.

[0218] Figures 12C-D show LC-MS analysis of unmodified nanobodies and nanobodies conjugated with BZA.

[0219] The results demonstrate that approximately half of the nanobody was conjugated with one to two BZA regions (approximately 390 Da).

[0220] Figures 12E-H show graphs of results from SPR assays of binding interactions between unconjugated or BZA-conjugated nanobodies and Fc-tagged CA-IV proteins.

[0221] 200 nM Fc-tagged CA-IV protein was immobilized on a capture sensor pre-coated with protein A, followed by the introduction of nanobodies at different concentrations. Nanobody concentrations are shown in the inset. Background binding signals observed in unconjugated nanobodies were likely due to weak interactions between the nanobodies and protein A.

[0222] Figure 12I shows representative immunofluorescence images of CA-IV-expressing HeLa cells incubated with unconjugated anti-GFP VHH nanobodies, BZA-2C-conjugated nanobodies, or BZA-3C-conjugated nanobodies.

[0223] For immunofluorescence imaging, cells were fixed after 1 hour incubation with 0.4 μM cargo nanobodies. After fixation, the cells were washed with detergent-containing buffer and permeabilized. The fixed cells were then stained with an anti-camellid antibody conjugated to Alexa Fluor 647 and subsequently imaged. Maximum intensity projection images are shown. Scale bar, 100 μm.

[0224] Figures 13A and 13B show graphs of results from SPR assays of modified and unmodified nanobodies tested against purified human CA-IV and mouse CA-IV.

[0225] The CA-IV protein was fused to a human Fc tag and immobilized on a protein A chip. Nanobodies were injected at different concentrations as shown in the insert. The background signal observed in the unmodified nanobodies is likely due to sporadic interactions between the nanobodies and protein A.

[0226] Figures 13C-D show the internalization assays in cultured HEK293 cells using BZA-modified and unmodified nanobodies.

[0227] After fixation, the cells incubated with nanobodies were washed with either a detergent-containing (permeabilized) or detergent-free (not permeabilized) buffer. The fixed cells were then stained with an anti-camel antibody conjugated to Alexa Fluor 647.

[0228] For immunofluorescence imaging, cells were fixed after 1 hour incubation with 0.4 μM cargo nanobodies. After fixation, the cells were washed with detergent-containing buffer and permeabilized. The fixed cells were then stained with an anti-camellid antibody conjugated to Alexa Fluor 647 and subsequently imaged. Maximum intensity projection images are shown. Scale bar, 100 μm.

[0229] Evidence from surface plasmon resonance (SPR) and cell-based binding assays suggests a direct binding interaction between BZA-modified nanobodies and purified human sulfonic carbonic anhydrase IV (hCA-IV) protein. Products from high-label-density conditions showed stronger binding to the CA-IV protein, suggesting that controlling label density may be a potential way to modulate the binding affinity of the modified protein. Furthermore, BZA-modified nanobodies were also able to bind to purified mouse C-IV (mCA-IV).

[0230] The results demonstrate that conjugation to nanobodies also enables binding to CA-IV.

[0231] siRNA Small interfering RNAs (siRNAs) are emerging as a central therapeutic modality in the world of molecular medicine. Fundamentally, siRNAs function by specifically targeting and degrading messenger RNA (mRNA) sequences, thereby halting the production of targeted proteins. This provides a highly selective mechanism for downregulating disease-causing genes. The importance of siRNAs is highlighted by their potential to treat many diseases, particularly those that conventional therapies fail to address. Unlike conventional therapeutic modalities that can inadvertently affect multiple targets and result in off-target effects, siRNAs provide precise gene silencing, ensuring a higher degree of specificity. Furthermore, siRNAs' ability to target genes that were previously "undruggable" offers new hope for conditions that were previously elusive to treat.

[0232] Despite its potential, systemic delivery of siRNA to the brain has been a challenging problem.

[0233] Figure 14A shows the structure of the BZA shuttle-siRNA variant.

[0234] Figures 14B–D show graphs of results from SPR assays of binding interactions between unconjugated or BZA-conjugated siRNA double helix and Fc-tagged CA-IV protein.

[0235] 200 nM Fc-tagged CA-IV protein was immobilized on a pre-coated capture sensor with protein A, followed by the introduction of siRNA at different concentrations. siRNA concentrations are shown in the inset.

[0236] Figure 14E shows a representative immunofluorescence image of CA-IV-expressing HeLa cells incubated with either unconjugated fluorescently labeled siRNA or BZA-2C conjugated siRNA.

[0237] Representative immunofluorescence image showing CA-IV expressing HeLa cells incubated with either unconjugated or BZA-conjugated fluorescently labeled siRNA molecules containing GFP-targeting sequences at 1.0 μM. Scale bar, 100 μm.

[0238] Figure 15A shows the structure of the BZA shuttle-siRNA variant.

[0239] Sequence and design of GFP-targeted siRNA double helix. The sense and antisense strands of the double helix were modified using cholesterol-TEG and a primary amine, respectively.

[0240] Figures 15B-C show graphs of results from SPR assays of binding interactions between unconjugated or BZA-conjugated siRNA double helix and Fc-tagged CA-IV protein.

[0241] 200 nM Fc-tagged CA-IV protein was immobilized on a pre-coated capture sensor with protein A, followed by the introduction of siRNA at different concentrations. siRNA concentrations are shown in the inset.

[0242] The results demonstrate that conjugation of oligonucleotide molecules, such as primary amine-modified siRNA molecules, with brinzolamide promotes CA-IV binding and internalization in CA-IV expressing cells.

[0243] small molecules Small molecules possess unique properties that make them desirable for use in drug development, including the ability to penetrate cell membranes and bind to specific targets. BZA derivatives were conjugated to fluorophores, a type of small molecule, to evaluate their ability to bind to cells and internalize them.

[0244] To determine whether BZA can mediate the binding and internalization of low-molecular-weight cargoes, BZA-conjugated fluorophores were synthesized using CuAAC.

[0245] Figure 16A shows the structure of the BZA shuttle-small molecule cargo variant.

[0246] Figure 16B shows a representative immunofluorescence image of CA-IV-expressing HeLa cells incubated with either an unconjugated small molecular weight dye or a BZA-conjugated small molecular weight dye.

[0247] Figure 16C shows the structure of the BZA shuttle-small molecule cargo variant.

[0248] This schematic diagram illustrates the process of conjugating a fluorescent molecule (Alexa Fluor 647) to brinzolamide using the CuAAC reaction.

[0249] Figure 16D shows images from an internalization assay using HEK cells overexpressing the CA-IV receptor. The bottom row shows a zoomed-in image of a representative field of view in the image in the top row.

[0250] The results indicate that the BZA fluorophore binds to and is internalized in cells that overexpress either hCA-IV or mCA-IV.

[0251] Figure 16E shows representative images of brain and liver slices from mice injected with BZA-conjugated or unconjugated fluorophores. Animals injected with BZA-conjugated fluorophores show stained vascular structures in both the brain and liver.

[0252] Figure 16F shows fluorescence readings of lysed tissue from animals injected with BZA-conjugated or unconjugated fluorophores, using a plate reader.

[0253] Preliminary data suggested that this BZA-conjugated small molecule fluorophore bound to and internalized the cell membrane. This was confirmed by observations made to the presence of cell contours in the absence of detergent and spots in the presence of detergent. In vivo studies showed that the BZA-modified fluorophore localized on brain endothelial cells. Interestingly, this was not the case for the fluorophore without BZA modification. Quantification of fluorescence distribution in different organs showed that BZA modification increased the retention of the small molecule in multiple organs, including the brain.

[0254] The results demonstrate that conjugation of small molecules, such as fluorophores, with brinzolamide can lead to the localization of fluorophores within cells.

[0255] In particular, BZA-conjugated fluorophore molecules signal throughout the cytosol, suggesting their ability to transmembrane and potentially interact with intracellular carbonic anhydrases. This difference in membrane permeability and access to carbonic anhydrase family members may lead to different intracellular distributions of BZA-conjugated small molecules versus BZA-conjugated large molecules.

[0256] The results suggest that BZA-modified nanobodies, therapeutic IgG antibodies, and small molecules have strong potential for broad application in targeted therapeutics. Their binding affinity to hCA-IV, as well as their ability to mediate binding and internalization, indicate promising transfer potential.

[0257] Biologics conjugated to a carbonic anhydrase binder that cross the blood-brain barrier in mice. The BZA-conjugation technique was analyzed in vivo in mice. Both unconjugated and BZA-conjugated ate were administered intravenously to mice. Animal tissues were collected at various time intervals for analysis via immunofluorescence imaging. Brain tissue was collected at different time points after intravenous injection, then fixed and stained for human IgG. N=3 animals at each time point.

[0258] Figure 17A shows immunofluorescence images of mice systemically administered with unconjugated and BZA-conjugated Ate antibodies in sagittal brain sections. Scale bar, 1 mm.

[0259] Figure 17B shows immunofluorescence images of mice systemically administered with unconjugated and BZA-conjugated Ate antibodies in the cerebral cortex. Scale bar, 100 μm.

[0260] Figure 17C shows a zoomed-in image of the cerebral cortex of mice systemically administered with unconjugated and BZA-conjugated Ate antibodies, co-stained with neuronal markers, 120 hours after injection. Scale bar, 100 μm.

[0261] Quantification of BZA-conjugated and unconjugated Ate antibodies was determined by IgG concentration via anti-human IgG ELISA, and then normalized based on total protein concentration measured by BCA. N=3 animals at each time point.

[0262] Figure 17D shows a graph of the in vivo distribution of BZA-conjugated ate compared to unconjugated control on day 7.

[0263] Left: Fold change of BZA-conjugated IgG concentration compared to unconjugated IgG. Right: Normalized IgG concentrations in the whole brain, vascular structure fraction, and brain parenchyma fraction.

[0264] Figures 17E-F show graphs of the pharmacokinetics of BZA-conjugated and unconjugated ate in the whole brain and liver, respectively, over a 14-day period.

[0265] Figures 17G-H show graphs of the pharmacokinetics of BZA-conjugated and unconjugated ate in cerebral vascular structures and brain parenchyma, respectively, over a 14-day period.

[0266] BZA-conjugated ate showed extended enrichment in the brain, including the cortex, compared to unconjugated ate.

[0267] Immunofluorescence imaging and ELISA quantification both showed peaks in brain IgG signaling between days 5 and 7, followed by a decline over the 14-day period. ELISA-based quantification on day 7 showed that the concentration of BZA-conjugated Ate in the brain was an order of magnitude higher than that of the unconjugated control. The duration of brain exposure for the CA-IV-conjugated brain shuttle was greater than that of the TfR-based shuttle.

[0268] Further analysis demonstrated that BZA-conjugated ate can efficiently cross the blood-brain barrier and reach the brain parenchyma. This was confirmed by co-localization of IgG signals with neurons in immunofluorescence imaging after capillary depletion and by ELISA results, which supports a significant increase in brain distribution within the brain parenchymal fraction.

[0269] Furthermore, enrichment induced by BZA showed a strong preference for the brain.

[0270] Figures 18A and 18B show immunofluorescence images of whole liver sections from mice systemically administered with unconjugated and BZA-conjugated Ate antibodies, with scale bars of 1 mm and 100 μm, respectively.

[0271] Liver tissue was collected at different time points after intravenous injection, then fixed and stained for human IgG. N=3 animals at each time point.

[0272] Figure 18C shows the quantification of BZA-conjugated and unconjugated Ate antibodies in peripheral organs at day 7 in mice systemically administered unconjugated and BZA-conjugated Ate antibodies.

[0273] IgG concentrations were determined via anti-human IgG ELISA and then normalized based on total protein concentrations measured by BCA. N=3 animals at each time point.

[0274] The data demonstrated that on day 7, there was no significant increase in distribution in the major peripheral organs evaluated. The distribution pattern was consistent with the dominant expression of receptors in the brain and lungs. Both ELISA-based quantification and immunofluorescence imaging confirmed that the liver distribution of BZA-conjugated ate was not significantly different from that of unconjugated antibody over a 14-day period.

[0275] Consideration The transport of IgG cargo to the brain via the CA-IV receptor has been demonstrated for the first time by this invention. Our results not only validate CA-IV as a BBB receptor for nonviral cargoes but also highlight its significant advantages over widely distributed BBB receptors, such as TfR and CD98hc. The increased brain selectivity of CA-IV-mediated delivery offers the potential to mitigate the toxicity associated with off-target delivery.

[0276] One common challenge in the development of brain delivery technologies is that binders designed for the BBB receptor in one species may not be able to bind to the corresponding receptor in another species. Examples of this problem have been reported with TfR binders and CD98hc binders. However, the receptor binder of the present invention (called "Brain CAB" - Brain Access via Carbonic Anhydrase Binder Bioconjugation) can successfully bind to CA-IV homologs across various species, such as rodents and non-human primates, due to a conserved binding pocket for CA-IV. This ability enables broad application and evaluation of this technology in diverse model organisms and disease models.

[0277] The single-step bioconjugation method used in this invention demonstrates versatility in accepting various therapeutic agents, including nanobodies and oligonucleotides, highlighting its broad applicability. Furthermore, the uncomplicated nature of this bioconjugation process enables seamless scaling of production. Collectively, these characteristics position the invention as a versatile technology with practical transferability for developing therapeutic agents for neurological indications.

[0278] The single-step bioconjugation process-driven brain CAB technology has demonstrated considerable potential in both in vitro and in vivo studies. Along with its broad applicability and simple production process, its high BBB cross-efficiency, enhanced brain specificity, and extended pharmacokinetics suggest this approach holds great potential for delivering therapeutic agents to states previously hindered by the complexity of the blood-brain barrier.

[0279] Overall, the conjugated molecules of the present invention were shown to exhibit successful binding to CA-IV proteins from different mammalian species, and subsequent internalization was validated via surface plasmon resonance (SPR) and cell-based assays. In vivo mouse experiments demonstrated that intravenously administered conjugated therapeutic IgG antibodies efficiently penetrated the brain barrier (BBB). Antibody levels in the brain parenchyma increased tenfold over 7 days compared to unconjugated controls, and this parenchymal boost was localized in the brain and consistent with CA-IV receptor expression. The broad potential of this technology lays the foundation for pioneering therapeutic strategies across diverse CNS indications.

[0280] Import by reference References and citations to other materials, such as patents, patent applications, patent publications, journals, books, articles, and web content, have been made throughout this disclosure. All such materials are incorporated herein by reference in their entirety for all purposes.

[0281] Equal portions Various modifications of the present invention and its many further embodiments will be apparent to those skilled in the art from the entirety of this material, including references to scientific and patent documents cited herein, in addition to those shown and described herein. The subject matter of this specification includes important information, examples, and guidance that may be adapted for the implementation of the invention in its various embodiments and equivalents.

Claims

1. A blood-brain barrier (BBB) ​​shuttle selected from brinzolamide or its derivatives; and The therapeutic cargo conjugated to the aforementioned shuttle A conjugate that includes this.

2. The conjugate according to claim 1, wherein the shuttle is a brinzolamide ester.

3. The conjugate according to claim 2, wherein the brinzolamide derivative comprises an N-hydroxysuccinimide (NHS) ester.

4. The conjugate according to claim 3, wherein in the brinzolamide derivative, brinzolamide is covalently bonded to the NHS ester via an alkyl linker of 1 to 8 carbon atoms.

5. The aforementioned brinzolamide ester has the formula: 【Chemical 84】 The conjugate according to claim 4, wherein n is 1 to 8 in the formula.

6. The brinzolamide derivative is the following compound: 【Chemical 85】 [Chem. 86] 【Chemistry 87】 A conjugate according to claim 5, selected from among the following.

7. The conjugate according to claim 2, wherein the brinzolamide derivative comprises a pentafluorophenyl (PFP) ester.

8. The conjugate according to claim 7, wherein in the brinzolamide derivative, brinzolamide is covalently bonded to the PFP ester via polyethylene glycol.

9. The brinzolamide derivative is the following compound: [[Chemical 88]] 【Chemistry 89】 A conjugate according to claim 8, selected from among the following.

10. The conjugate according to claim 1, comprising a plurality of low-molecular-weight shuttles conjugated to the therapeutic cargo.

11. The conjugate according to claim 1, wherein the therapeutic cargo is a biological molecule.

12. The conjugate according to claim 11, wherein the biological molecule is selected from the group consisting of nucleic acids, proteins, peptides, antibodies, nanobodies, lipids, polysaccharides, and combinations thereof.

13. The conjugate according to claim 1, wherein the therapeutic cargo is a small molecule.

14. The conjugate according to claim 1, wherein the shuttle is a carbonic anhydrase IV (CA-IV) shuttle.

15. The conjugate according to claim 14, wherein, when provided to cells expressing CA-IV as a surface protein, the binding of the shuttle to the CA-IV protein mediates the transcytosis of the therapeutic cargo across the BBB.

16. A method for delivering therapeutic cargo across a target BBB, A blood-brain barrier (BBB) ​​shuttle selected from brinzolamide or its derivatives; and The therapeutic cargo conjugated to the aforementioned shuttle A method that includes steps to provide for a conjugate that includes

17. The method according to claim 16, wherein the shuttle is a brinzolamide ester.

18. The method according to claim 17, wherein the brinzolamide derivative comprises an N-hydroxysuccinimide (NHS) ester.

19. The method according to claim 18, wherein in the brinzolamide derivative, brinzolamide is covalently bonded to the NHS ester via a 1-8 carbon alkyl linker.

20. The brinzolamide derivative is given by formula: [Chemical 90] The method according to claim 19, wherein n is 1 to 8 in the formula.

21. The brinzolamide derivative is the following compound: 【Chemistry 91】 【Chemistry 92】 【Chemistry 93】 The method according to claim 20, selected from among the following.

22. The method according to claim 17, wherein the brinzolamide derivative comprises a pentafluorophenyl (PFP) ester.

23. The method according to claim 22, wherein in the brinzolamide derivative, brinzolamide is covalently bonded to the PFP ester via polyethylene glycol.

24. The brinzolamide derivative is the following compound: 【Chemical 94】 【Chemical 95】 The method according to claim 23, selected from among the following.

25. The method according to claim 16, wherein the conjugate comprises a plurality of low-molecular-weight shuttles conjugated to the therapeutic cargo.

26. The method according to claim 16, wherein the therapeutic cargo is a biological molecule.

27. The method according to claim 26, wherein the biological molecule is selected from the group consisting of nucleic acids, proteins, peptides, antibodies, nanobodies, lipids, polysaccharides, and combinations thereof.

28. The method according to claim 16, wherein the therapeutic cargo is a small molecule.

29. The method according to claim 16, wherein the shuttle is a carbonic anhydrase IV (CA-IV) shuttle.

30. The method according to claim 29, wherein, when provided to cells expressing CA-IV as a surface protein, the binding of the shuttle to the CA-IV protein mediates the transcytosis of the therapeutic cargo across the BBB.

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