Chemically Linkable Nuclear Targeting Tags

Abstract

A nuclear targeting tag having a phenylboronate targeting moiety, a linker, a chemically linkable end group suitable for linking the targeting moiety to a molecule of interest, and optionally a spacer between the linker and the chemically linkable end group. Also provided is a compound comprising a nuclear targeting tag covalently linked to a molecule of interest, such as a drug, a probe, a dye, a peptide, a protein, a drug candidate, or a natural product. Also provided is a method of using the nuclear targeting tag to introduce a molecule of interest into the nucleus of a cell. Also provided is a method for producing the nuclear targeting tag.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 183,894, filed May 4, 2021, the entire contents of which are incorporated herein in their entirety.

[0002] The delivery of molecules of interest, such as drugs, probes, dyes, peptides, proteins, etc., to the nucleus of a cell has attracted significant interest from researchers across a wide variety of research fields. For example, targeting research probes to the nucleus allows for further elucidation of molecular mechanisms specific to cell organelles. Nuclear targeting of therapeutic agents is desirable to increase therapeutic efficacy while reducing off-target side effects. [Background technology]

[0003] Conventional methods include the use of short peptides or proteins to transport molecules of interest to the nucleus, but there are several problems associated with the use of such peptides or proteins, including instability, short shelf life, incompatibility with some molecules of interest, difficulty in linking to some molecules of interest, and difficulty in manufacturing. Summary of the Invention [Problem to be solved by the invention]

[0004] There is a need for novel nuclear targeting tags that have greater stability and shelf life, can be easily linked to a myriad of molecules of interest, and can be manufactured in a cost-effective manner. [Means for solving the problem]

[0005] Provided is a nuclear targeting tag that has a phenylboronate targeting moiety, a linker that is bonded to the phenyl group of the phenylboronate targeting moiety and a chemically linkable end group.The linker is selected from carbamate, ether, alkylenyl group and PEG group.In certain embodiments, the phenylboronate targeting moiety is a phenylboronic acid group, and in other embodiments, the phenylboronate targeting moiety is a phenylboronic acid pinacol ester group.

[0006] In some embodiments, the nuclear targeting tag also comprises a spacer between the linker and the chemically linkable end group. Preferred spacers include alkylenyl spacers and PEG spacers.

[0007] Chemically linkable end groups include halo, amino, carboxyl, C 2 ~C 10 Alkynyl, Hydroxyl, C 1 ~C 10 It may be selected from alkoxy, azide, sulfinate, thiol, fluorosulfate and boronic acid.

[0008] In some embodiments, the nuclear targeting tag may also include a molecule covalently attached to the chemically linkable end group. Suitable molecules include, but are not limited to, drugs, probes, dyes, peptides, proteins, drug candidates, and natural products.

[0009] In one embodiment, the compound of formula I: [ka] wherein X is a boronic acid or boronic ester, L is a linker, S is a spacer, s is 0-10, and Y is a chemically linkable end group.

[0010] In a preferred embodiment, X is selected from a boronic acid group and a boronic acid pinacol ester group, and L is a carbamate, an ether, a C1 ~C 20 Alkylenyl groups and -[-O-CH 2 CH 2 -] n - (wherein n is 1 to 12), and S is selected from C 1 ~C 20 Alkylenyl groups and -[-O-CH 2 CH 2 -] n - (wherein n is 1 to 12), s is 0 to 10, and Y is selected from halo, amino, carboxyl, C 2 ~C 10 Alkynyl, Hydroxyl, C 1 ~C 10 It is selected from alkoxy, azide, sulfinate, thiol, fluorosulfate and boronic acid.

[0011] In some embodiments, the compound of formula I further comprises a molecule conjugated to the chemically linkable terminal group. Such molecules may include drugs, probes, dyes, peptides, proteins, drug candidates and natural products.

[0012] In a second embodiment, the compound of formula II [ka] wherein X is a boronic acid or a boronic ester, S is a spacer, s is 0-10, and Y is a chemically linkable end group. In a preferred embodiment, X is selected from a boronic acid group and a boronic acid pinacol ester group, and S is C 1 ~C 20 Alkylenyl and -[-O-CH 2 CH 2 -] n - (wherein n is 1 to 12), s is 0 to 10, and Y is selected from halo, amino, carboxyl, C 2 ~C 10 Alkynyl, Hydroxyl, C 1 ~C 10 It is selected from alkoxy, azide, sulfinate, thiol, fluorosulfate and boronic acid.

[0013] In some preferred embodiments, the compound of formula II also includes a molecule conjugated to the chemically linkable end group, in such embodiments, the molecule is selected from the group consisting of a drug, a probe, a dye, a peptide, a protein, a drug candidate, and a natural product.

[0014] Formula III [ka] Also provided is a compound of the formula: where X is a boronic acid or boronic ester, S is a spacer, s is 0-10, and Y is a chemically linkable end group. In a preferred embodiment, X is selected from a boronic acid group and a boronic acid pinacol ester group, and S is C 1 ~C 20 Alkylenyl and -[-O-CH 2 CH 2 -] n - (wherein n is 1 to 12), s is 0 to 10, and Y is selected from halo, amino, carboxyl, C 2 ~C 10 Alkynyl, Hydroxyl, C 1 ~C 10 It is selected from alkoxy, azide, sulfinate, thiol, fluorosulfate and boronic acid.

[0015] In some preferred embodiments, the compound of formula II also includes a molecule conjugated to the chemically linkable end group, in such embodiments, the molecule is selected from the group consisting of a drug, a probe, a dye, a peptide, a protein, a drug candidate, and a natural product.

[0016] Further provided is a method of delivering a molecule to the nucleus within a cell by contacting the cell with a nuclear targeting tag of Formula I or II that comprises a molecule of interest bound to the nuclear targeting tag.

[0017] Further provided are methods of producing the nuclear targeting tags described herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Compounds are provided that comprise a phenylboronate nuclear targeting moiety linked to a conjugation moiety that allows for the conjugation of many different molecules of interest, indeed many different classes of molecules of interest, to the targeting moiety and allows for the uptake of the molecule of interest into the cell nucleus, thereby being able to provide many different functions.

[0019] In some embodiments, the phenylboronate targeting moiety can be conjugated to a small molecule, i.e., a drug, probe, or dye, via a linker and optionally a spacer, while in other embodiments it can also be used with peptides or proteins.

[0020] The resulting tag-containing compounds preferentially target the nucleus over other cellular compartments, allowing enrichment in the nucleus. Nuclear targeting research probes can allow further elucidation of organelle-specific molecular mechanisms, while nuclear targeting of therapeutic agents can increase therapeutic efficacy while reducing off-target side effects. The compounds provided herein have the advantage of higher stability and longer shelf life than current nuclear targeting tags, which are mainly short amino acid peptides or proteins. In addition, these nuclear targeting tags are smaller, more easily linked to each target molecule, and less expensive to produce.

[0021] The nuclear targeting tag comprises a phenylboronate targeting moiety at one end and a chemically linkable end group at the other end, attached by a linker and optionally a spacer.

[0022] In certain embodiments, the phenylboronate targeting moiety is a boronic acid group, and in other embodiments, the phenylboronate targeting moiety is a phenylboronic acid pinacol ester, i.e., a 4,4,5,5-tetramethyl-1,3,2-dioxaborolan2-yl group. Other arylboronate targeting moieties can be envisioned by one skilled in the art.

[0023] The linker is selected from carbamates, ethers, alkylenyl groups, and polyethylene glycols (PEGs). As used herein, the term alkylenyl refers to a divalent analog of a straight-chain or branched alkyl group. In embodiments where the linker is an alkylenyl group, the linker is C 1 ~C 20 In some embodiments, the linker is C 1 ~C 10 It may be alkylenyl. In some embodiments, the alkylenyl linker is a straight chain alkyl, and in other embodiments, the alkylenyl linker may be a branched alkylenyl. In embodiments where the linker is PEG, the linker may comprise 1-12 PEG units. In preferred embodiments, the linker is in the para position relative to the boronate group.

[0024] In some embodiments, the nucleus targeting tag also comprises a spacer between the linker and the chemically linkable end group. The spacer can be used, for example, to provide separation between the phenylboronate targeting moiety and the molecule of interest, thereby allowing the molecule of interest to interact with its target within the nucleus. In some embodiments, the spacer can incorporate a disulfide group, a photolabile group, or other group that allows the release of the molecule of interest once inside the nucleus. A preferred spacer is C 1 ~C 20 Alkylenyl and -[-O-CH 2 CH 2 -] n- (where n is 1 to 12), although other spacers can be envisaged for introducing equivalent or additional functionality.

[0025] The chemically linkable end group can be selected from many different end groups that allow for conjugation or chemical bonding of a molecule of interest. Some preferred chemically linkable end groups are halo, amino, carboxyl, C 2 ~C 10 Alkynyl, Hydroxyl, C 1 ~C 10 Including, but not limited to, alkoxy, azide, sulfinate, thiol, fluorosulfate and boronic acid.

[0026] In some embodiments, the nuclear targeting tag may also include a molecule covalently attached to the chemically linkable end group. Suitable molecules include, but are not limited to, drugs, probes, dyes, peptides, proteins, drug candidates, and natural products.

[0027] Examples of drugs and drug candidates include small molecule drugs, peptide drugs, natural products, protein therapeutics, antibodies, antibody-like binding agents and antibody fragments, such as single-chain antigen-binding fragments. Specific drugs may include, but are not limited to, levimastat, fluconazole, tanomastat and similar drugs. Specific peptide drugs may include, but are not limited to, ziconotide, enfuvirtide, octreotide, lanreotide and pasireotide. Drug candidates may include any small molecule or peptide that has been selected for or is undergoing clinical trials.

[0028] Peptides suitable for use with the nuclear targeting tags described herein can include any peptide having two or more amino acids, substituted amino acids, amino acid derivatives, or substituted amino acid derivatives, including standard, non-standard, and chemically synthesized amino acids, including L- and D-isomers, linked together by amide bonds. Substituted amino acids are those that typically contain one or more substituents in the side chain. Amino acid derivatives are those that have been chemically modified at the α-amino or acyl groups. An exemplary amino acid for use with the nuclear targeting tags described herein is azatyrosine.

[0029] Proteins suitable for use with the nuclear targeting tags described herein include, but are not limited to, natural proteins, including antibodies, synthetic proteins and protein fragments.Some exemplary proteins include, but are not limited to, gemtuzumab and brentuximab.

[0030] Examples of probes suitable for use with the nuclear targeting tags described herein include, but are not limited to, single-stranded DNA or RNA that bind to complementary DNA sequences, histones, nuclear transport proteins, and nuclear location proteins.

[0031] Examples of dyes suitable for use with the nuclear targeting tags described herein include, but are not limited to, heptamethines, cyanines, and fluoresceins.

[0032] Examples of natural products suitable for use with the nuclear targeting tags described herein include, but are not limited to, anthramycin and maytansinoids.

[0033] In one embodiment, the compound of formula I: [ka] wherein X is a boronic acid or boronic ester, L is a linker, and S Sis a spacer, and Y is a chemically linkable end group.

[0034] In a preferred embodiment, X is selected from boronic acid groups and boronic acid pinacol esters, ie, 4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl groups.

[0035] The linker L may be a carbamate group, an ether, or a C 1 ~C 20 Alkylenyl group, or -[-O-CH 2 CH 2 -] n -, where n is 1 to 12. In one preferred embodiment, the linker L is a carbamate group. In another preferred embodiment, the linker L is an ether.

[0036] Spacer S s is an optional spacer unit. Spacer S s The use of and the selection of a particular spacer will depend on the molecule of interest, its function and the maintenance of the interaction of the linked molecule to its target. The spacer unit provides additional tunability to the nuclear localization tags provided herein and can be varied to provide optimal structures for localization depending on both the molecule and the nuclear target.

[0037] In some embodiments, the spacer S s When present, the spacer S s is C 1 ~C 20 Alkylenyl groups, preferably C 4 ~C 18 alkylenyl groups, and s is 1 to 10. s When is alkylenyl, it may be linear or branched. s When is alkylenyl, it can contain one or more substituents. sWhen is alkylenyl, it can contain one or more unsaturated bonds. In another embodiment, the spacer S s When present, the spacer S s is the formula -[-O-CH 2 CH 2 -] n -PEG, where n is 1 to 12, preferably 4 to 12, and s may be 1 to 10. In yet another embodiment, the spacer S s may be made up of multiple alkylenyl units, PEG units, or a combination of alkylenyl and PEG units. In such embodiments, s may be 2-10.

[0038] The chemically linkable end group Y is selected to provide a convenient manner of conjugating to or forming a covalent bond with a molecule of interest. In various embodiments, the chemically linkable end group is halo, e.g., fluoro, chloro, bromo, iodo; amino, including primary amines, secondary amines, and amino acids; carboxyl, including organic acids and amino acids; 2 ~C 10 Alkynyl, for example, methynyl, ethynyl, propynyl, butynyl, etc.; hydroxyl; C 1 ~C 10 Alkoxy, including for example methoxy, ethoxy, propoxy, butoxy, etc.; azide, sulfinate, thio, fluorosulfate, and boronic acid.

[0039] In some embodiments, the compound of formula I further comprises a molecule conjugated to the chemically linkable terminal group. Such molecules may include drugs, probes, dyes, peptides, proteins, drug candidates and natural products, each as outlined above.

[0040] In a second embodiment, the compound of formula II [ka] wherein X is a boronic acid or boronic ester, S is an optional spacer, s is 0-10, and Y is a chemically linkable end group. In a preferred embodiment, X is selected from a boronic acid group and a boronic acid pinacol ester, i.e., 4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl group.

[0041] When the spacer S is present, the spacer is preferably selected from an alkylenyl spacer and a PEG spacer. When S is an alkylenyl spacer, it is preferably C 1 ~C 20 An alkylenyl group. S is of the formula -[-O-CH 2 CH 2 -] n When the spacer is -PEG, n is preferably 1 to 12. In some embodiments, the spacer S may be made up of multiple alkylenyl units, PEG units, or a combination of alkylenyl and PEG units. In such embodiments, s may be 2 to 10.

[0042] The chemically linkable end group Y is selected to provide a convenient manner of conjugating to or forming a covalent bond with a molecule of interest as described above. In various embodiments, the chemically linkable end group is selected from halo, e.g., fluoro, chloro, bromo, iodo; amino, e.g., including primary amines, secondary amines, and amino acids; carboxyl, e.g., including organic acids and amino acids; 2 ~C 10 Alkynyl, for example, methynyl, ethynyl, propynyl, butynyl, etc.; hydroxyl; C 1 ~C 10 Alkoxy, including for example methoxy, ethoxy, propoxy, butoxy, etc.; azide, sulfinate, thio, fluorosulfate, and boronic acid.

[0043] In some preferred embodiments, the compound of formula II also includes a molecule conjugated to the chemically linkable end group, in such embodiments, the molecule is selected from the group consisting of a drug, a probe, a dye, a peptide, a protein, a drug candidate, and a natural product.

[0044] In some preferred embodiments, the nuclear targeting tags provided herein may be (4-((((2-aminoethyl)carbamoyl)oxy)methyl)phenyl)boronic acid hydrochloride, (4-((((2-bromoethyl)carbamoyl)oxy)methyl)phenyl)boronic acid, 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl(bromoethyl)carbamate, 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl(2-aminoethyl)carbamate, and 2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)ethan-1-amine.

[0045] Further provided is a method of delivering a molecule to the nucleus within a cell by contacting the cell with a nuclear targeting tag of formula I or II, which comprises a molecule of interest bound to the nuclear targeting tag.

[0046] Also included is a method of delivering a molecule of interest to a cell nucleus in a subject, comprising administering to a subject in need of such treatment a nuclear targeting tag as described herein that comprises a molecule of interest attached to a chemically linkable end group. In various embodiments, the subject may be a human subject or a veterinary subject.

[0047] The present disclosure further provides the following embodiments, as described in the following clauses.

[0048] {1} A first method for producing a nuclear targeting tag, comprising: a. reacting a pinacol boronate phenyl alcohol with carbonyldiimidazole to form a boronate carbonyldiimidazole intermediate; b. reacting the boronate carbonyldiimidazole intermediate of step (a) with ethylenediamine to form the nuclear targeting tag. The method includes:

[0049] {2} The method according to clause {1}, wherein step (a) is carried out in acetonitrile.

[0050] {3} The method according to any of clauses {1} or {2}, wherein step (a) is carried out at an elevated temperature, preferably 50°C.

[0051] {4} The process according to any of clauses {1} to {3}, wherein step (b) is carried out in tetrahydrofuran (THF).

[0052] {5} The method according to any one of clauses {1} to {4}, further comprising the steps of: c. Purifying the nuclear targeting tag.

[0053] {6} A method for producing a nuclear targeting tag, comprising: a. reacting a pinacol boronate phenyl alcohol (1) with carbonyldiimidazole (2) to form a boronate carbonyldiimidazole intermediate (3); [ka] and b. reacting the boronate carbonyldiimidazole intermediate (3) with ethylenediamine to form the nuclear targeting tag (4). [ka] A method comprising:

[0054] {7} A second method for producing a nuclear targeting tag, comprising: a. reacting a pinacol boronate phenyl alcohol with carbonyldiimidazole to form a boronate carbonyldiimidazole intermediate; and b. reacting the boronate carbonyldiimidazole intermediate of step (a) with a bromoalkylamine hydrobromide to form the nuclear targeting tag. A method comprising:

[0055] {8} The method according to clause {7}, wherein step (a) is carried out in acetonitrile.

[0056] {9} The method according to any of clauses {7} or {8}, wherein step (a) is carried out at an elevated temperature, preferably 50°C.

[0057] {10} The method according to any one of clauses {7} to {9}, wherein step (b) is carried out in dimethylformamide (DMF).

[0058] {11} The method according to any one of clauses {7} to {10}, further comprising the steps of: c. Purifying the nuclear targeting tag.

[0059] {12} A method for producing a nuclear targeting tag, comprising: a. reacting a pinacol boronate phenyl alcohol (1) with carbonyldiimidazole (2) to form a boronate carbonyldiimidazole intermediate (3); [ka] and b. reacting the boronate carbonyldiimidazole intermediate (3) with ethylenediamine to form the nuclear targeting tag (5). [ka] A method comprising:

[0060] {13} A third method for producing a nuclear tag, comprising: a. reacting a pinacol boronate phenyl alcohol with carbonyldiimidazole to form a boronate carbonyldiimidazole intermediate; b. reacting the boronate carbonyldiimidazole intermediate of step (a) with an amine-protected ethylenediamine to form an amine-protected pinacolylboronic ester intermediate; c. reacting the amine-protected pinacolyl boronate intermediate of step (b) with diethanolamine to form an amine-protected pinacolyl borate DEA complex; d. reacting the amine protected pinacolylborate DEA complex of step (c) with diethyl ether to form a nuclear targeting tag; and e. Optionally, deprotecting the amine. A method comprising:

[0061] {14} The method according to clause {13}, wherein step (a) is carried out in acetonitrile.

[0062] {15} The method according to any of clauses {13} or {14}, wherein step (a) is carried out at an elevated temperature, preferably 50°C.

[0063] {16} The method according to any of clauses {13} to {15}, wherein step (b) is carried out in THF.

[0064] {17} The method of any of clauses {13} to {16}, wherein step (c) is carried out in the presence of isopropyl alcohol.

[0065] {18} The method according to any of clauses {13} to {17}, wherein step (c) is carried out in diethyl ether.

[0066] {19} The method of any of clauses {13} to {18}, wherein step (d) is carried out in the presence of isopropyl alcohol.

[0067] {20} The method according to any one of clauses {13} to {19}, wherein step (d) is carried out in acetonitrile.

[0068] {21} The method according to any one of clauses {13} to {20}, further comprising a step of purifying the nuclear targeting tag.

[0069] {22} A method for producing a nuclear tag, comprising the steps of: a. reacting a pinacol boronate phenyl alcohol (1) with carbonyldiimidazole (2) to form a boronate carbonyldiimidazole intermediate (3); [ka] b. reacting the boronate carbonyldiimidazole intermediate (3) with a protected ethylenediamine to form the amine protected pinacolyldiethanolamine intermediate (6); [ka] c. reacting the amine-protected pinacolyl boronate intermediate (6) with diethanolamine to form the amine-protected pinacolyl borate DEA complex (7); [ka] and d. Reacting the amine-protected pinacolylborate DEA complex (7) with diethyl ether and deprotecting the amine to form the nuclear targeting tag (8). [ka] A method comprising:

[0070] {23} A fourth method for producing a nuclear targeting tag, comprising: a. reacting a pinacol boronate phenyl alcohol with carbonyldiimidazole to form a boronate carbonyldiimidazole intermediate; b. reacting the boronate carbonyldiimidazole intermediate of step (a) with a bromoalkylamine hydrobromide to form a bromo-pinacolylboronic ester intermediate; c. reacting the bromo-pinacolyl boronate intermediate of step (b) with diethanolamine to form a bromo-pinacolyl borate DEA complex; d. reacting the bromo-pinacolylborate DEA complex of step (c) with diethyl ether to form the nuclear targeting tag. A method comprising:

[0071] {24} The method according to clause {23}, wherein step (a) is carried out in acetonitrile.

[0072] {25} The method according to any of clauses {23} or {24}, wherein step (a) is carried out at an elevated temperature, preferably 50°C.

[0073] {26} The method according to any of clauses {23} to {25}, wherein step (b) is carried out in DMF.

[0074] {27} The method according to any of clauses {23} to {26}, wherein step (b) is carried out in the presence of diethyl ether.

[0075] {28} The method of any of clauses {23} to {27}, wherein step (c) is carried out in the presence of isopropyl alcohol.

[0076] {29} The method according to any of clauses {23} to {28}, wherein step (c) is carried out in diethyl ether.

[0077] {30} The method according to any one of clauses {23} to {29}, wherein step (d) is carried out in diethyl ether.

[0078] {31} The process according to any of clauses {23} to {30}, wherein step (d) is carried out in the presence of an acid, preferably in the presence of hydrochloric acid.

[0079] {32} The method according to any one of clauses {23} to {31}, further comprising the steps of: e. Purifying the nuclear targeting tag.

[0080] {33} A method for producing a nuclear targeting tag, comprising: a. reacting a pinacol boronate phenyl alcohol (1) with carbonyldiimidazole (2) to form a boronate carbonyldiimidazole intermediate (3); [ka] b. reacting the boronate carbonyldiimidazole intermediate (3) with a bromoalkylamine hydrobromide to form a bromo-pinacolylboronic ester intermediate (9); [ka] c. reacting the bromo-pinacolyl boronate intermediate (9) with diethanolamine to form the bromo-pinacolyl borate DEA complex (10); [ka] d. Reacting the bromo-pinacolylborate DEA complex (10) with diethyl ether to form the nuclear targeting tag (11). [ka] A method comprising:

[0081] {34} A fifth method for producing a nuclear targeting tag, comprising: a. reacting N-(2-hydroxyethyl)phthalimide with 4-bromobenzyl bromide to form a 2-(2(4-bromobenzyl)oxyethylisoindoline-1,3-dione intermediate; b. reacting the 2-(2(4-bromobenzyl)oxyethylisoindoline-1,3-dione intermediate of step (a) with pinacol bis-boronic acid to form the 2-(2-(4,4,5,5-tetramethyl-1,3,2-dioxoboronate-2-yl)benzyl)oxy)ethyl)isoindoline-1,3-dione intermediate; c. reacting the 2-(2-(4,4,5,5-tetramethyl-1,3,2-dioxoboronate-2-yl)benzyl)oxy)ethyl)isoindoline-1,3-dione intermediate of step (c) with hydrazine hydrate to form the nuclear targeting tag. A method comprising:

[0082] {35} The method according to clause {34}, wherein step (a) is carried out in DMF.

[0083] {36} The process of either clause {34} or {35}, wherein step (a) is carried out in the presence of sodium hydride.

[0084] {37} The method according to clause {36}, wherein DMF and sodium hydride are combined and cooled, preferably to 0°C to 5°C, before adding N-(2-hydroxyethyl)phthalimide.

[0085] {38} The method according to clause {37}, wherein N-(2-hydroxyethyl)phthalimide in DMF and NaH is heated, preferably to 40° C., before the addition of 4-bromobenzyl bromide.

[0086] {39} The method according to clause {38}, wherein the reaction of step (a) is carried out at elevated temperature, preferably at 70°C.

[0087] {40} The method according to any of clauses {34} to {39}, wherein step b is carried out in the presence of a catalyst.

[0088] {41} The method according to clause {40}, wherein the catalyst is a palladium catalyst.

[0089] {42} The catalyst is PdCl 2 The method according to clause {41}, wherein (dppf).

[0090] {43} The method according to any of clauses {34} to {42}, wherein step (b) is carried out in dichloromethane.

[0091] {44} The method according to any of clauses {34} to {43}, wherein step (b) is carried out in the presence of potassium acetate.

[0092] {45} The method according to any of clauses {34} to {44}, wherein step (b) is carried out at an elevated temperature, preferably 85°C.

[0093] {46} The method according to any of clauses {34} to {45}, wherein step (c) is carried out in ethanol.

[0094] {47} The method according to any one of clauses {34} to {46}, further comprising the steps of: d. Purifying the nuclear targeting tag.

[0095] {48} A method for producing a nuclear targeting tag, comprising: a. reacting 4-bromobenzyl bromide (12) with N-(2-hydroxyethyl)phthalimide (13) to form 2-(2(4-bromobenzyl)oxyethylisoindoline-1,3-dione intermediate (14); [ka] b. reacting 2-(2(4-bromobenzyl)oxyethylisoindoline-1,3-dione intermediate (14) with pinacol bis-boronic acid in the presence of a catalyst to form 2-(2-(4,4,5,5-tetramethyl-1,3,2-dioxoboronate-2-yl)benzyl)oxy)ethyl)isoindoline-1,3-dione intermediate (15); [ka] c. reacting the 2-(2-(4,4,5,5-tetramethyl-1,3,2-dioxoboronate-2-yl)benzyl)oxy)ethyl)isoindoline-1,3-dione intermediate (15) with hydrazine hydrate to form the nuclear targeting tag (16). A method comprising: EXAMPLES

[0096] Example 1. Synthesis of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl (2-aminoethyl)carbamate [ka] A 500 mL four neck round bottom flask was flame dried and fitted with a condenser, thermopocket, septum and magnetic stir bar. The entire system was evacuated with nitrogen gas. Boronate alcohol (20 g, 85 mmol), carbonyldiimidazole (18 g, 111 mmol) and 200 ml of acetonitrile were charged to the flask and the reactant mass was stirred at 50° C. for approximately 2 hours. After the reaction was complete as indicated by TLC, the reaction mass was cooled and acetonitrile was distilled off under reduced pressure at 40° C. 50 mL of n-hexane was charged to the reaction vessel and stirred for 30 minutes. The reaction mass was filtered to obtain a white solid. The material was dried under vacuum at 40° C. for 2 hours to obtain 26 g of white product.

[0097] A 500 mL four-neck round bottom flask was flame dried and equipped with a nitrogen bubbler, septum and thermometer. 20 ml of boronate CDI (10 g, 30 mmol) in 250 ml of THF was charged to the flask. Ethylenediamine (20 ml, 300 mmol) was added to the flask and the reaction mixture was stirred at room temperature for 24 hours. After the reaction was complete as shown by TLC, the solvent was evaporated and 25 mL of dichloromethane and 10 ml of water were added to the residue. The reaction was stirred for 5 minutes. The mixture was separated into two layers. The organic layer was washed with water and dried over sodium sulfate. The organic layer was concentrated under reduced pressure to yield 3 g of the title compound.

[0098] Example 2. Synthesis of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl (2-bromoethyl)carbamate [ka] A 1 L three-neck round bottom flask was fitted with a reflux condenser, nitrogen bubbler and thermometer. Carbonyldiimidazole (18 g, 111 mmol) in dry acetonitrile (200 mL) was added to the flask. Pinacol boronate phenyl alcohol (20 g, 85 mmol) was added to the reaction flask under nitrogen atmosphere. The resulting mixture was heated to 50° C. and reacted for approximately 2 hours until TLC analysis showed no presence of any starting material. After completion of the reaction, the reaction mixture was cooled to room temperature and concentrated under reduced pressure to produce a yellowish residue. The crude residue was triturated using pentane or hexane to produce 25 g of carbamate as a white solid.

[0099] A 500 mL four-neck round-bottom flask was flame dried and equipped with a condenser, thermopocket, septum, and magnetic stir bar. The system was flushed with N 2 Evacuate with gas for 15 minutes. Charge the reaction flask with boronate CDI (12 g, 36.5 mmol), 2-bromoethylamine hydrobromide (19 g, 92 mmol) and 120 mL (10.0 vol) dry DMF. Stir the reaction mass for 10 minutes to produce a homogeneous clear solution, then charge triethylamine (15 g, 146 mmol) to the reaction flask via syringe. Stir the resulting heterogeneous mixture until the reaction is complete as indicated by TLC. After completion, charge 360 ​​mL diethyl ether to the reaction mass and stir for 10 minutes. Filter the reaction mass and extract the filtrate with ice water, brine and dry over anhydrous sodium sulfate. Concentrate the organic layer under reduced pressure to produce the crude product, which was purified by silica gel chromatography to produce 5.3 g of solid title product.

[0100] Example 3. Synthesis of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl (2-bromoethyl)carbamate hydrochloride [ka] A 1 L three-necked round bottom flask was equipped with a reflux condenser, nitrogen bubbler and thermometer and charged with carbonyldiimidazole (18 g, 111 mmol) in dry acetonitrile (200 mL). Pinacol boronate phenyl alcohol (20 g, 85 mmol) was added to the reaction mixture under nitrogen atmosphere. The resulting mixture was heated to 50° C. and reacted for approximately 2 hours until TLC analysis showed no starting material. After completion of the reaction, the reaction mixture was cooled to room temperature and concentrated under reduced pressure to yield a yellowish residue. The crude residue was triturated with pentane or hexane to yield 25 g of carbamate as a white solid.

[0101] To a solution of imidazole carbamate (20 g, 61 mmol) in 300 mL dry THF in a 1 L three-neck round bottom flask equipped with a nitrogen bubbler, thermometer and stopper, N-Boc-ethylenediamine (18 g, 74 mmol) was added. The reaction mixture was stirred at room temperature under nitrogen for approximately 20 hours. The solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography to give 18 g of product as a colorless, low melting solid.

[0102] A 1 L three-neck round bottom flask equipped with a nitrogen bubbler, thermometer and septum was flame dried and evacuated with nitrogen gas for 15 minutes. N-boc pinacolyl boronic ester (16 g, 38 mmol), diethanolamine (5 g, 45 mmol), 32 ml of IPA and 480 ml of diethyl ether were charged to the flask under nitrogen atmosphere. The resulting mixture was stirred at room temperature for approximately 72 hours. After completion of the reaction, the reaction mixture was filtered and the product was washed with diethyl ether. Finally, the product was dried under vacuum at 45 degrees to give 14 g of a white solid.

[0103] A 250 mL three-neck round bottom flask was flame dried and equipped with a nitrogen bubbler, thermometer and septum and evacuated with nitrogen gas for 15 minutes. The flask was charged with N-boc-pinacolyl boronate DEA complex (6.0 g, 15 mmol), 180 mL of 0.1 M diethyl ether, 12 mL of IPA and 300 mL of acetonitrile. The reaction was stirred at room temperature for 2 hours. It was kept at room temperature overnight. After completion of the reaction, the organics were distilled and the residue was dissolved in 300 mL of diethyl ether. The ether layer was washed with water and Na 2 SO 4 It was dried at 40° C. and concentrated to give 3.5 g of the title compound as a white crystalline material.

[0104] Example 4. Synthesis of (4-((((2-bromoethyl)carbamoyl)oxy)methyl)phenyl)boronic acid [ka] A 1 L three-neck round bottom flask was equipped with a reflux condenser, nitrogen bubbler and thermometer. Carbonyldiimidazole (18 g, 111 mmol) in dry acetonitrile (200 mL) was added. Pinacol boronate phenyl alcohol (20 g, 85 mmol) was added to the reaction mixture under nitrogen atmosphere. The resulting mixture was heated to 50° C. for approximately 2 hours until TLC analysis showed no starting material. After completion of the reaction, the reaction mixture was cooled to room temperature and concentrated under reduced pressure to produce a yellowish residue. The crude residue was triturated with pentane or hexane to produce 25 g of carbamate as a white solid.

[0105] A 500 mL four-neck round-bottom flask was flame dried and equipped with a condenser, thermopocket, septum, and magnetic stir bar. The system was flushed with N 2Evacuate with gas for 15 minutes. Charge boronate CDI (12 g, 36.5 mmol), 2-bromoethylamine hydrobromide (19 g, 92 mmol) and 120 mL (10.0 vol) of dry DMF to the reaction flask. The reaction mass was stirred for 10 minutes to produce a homogeneous clear solution. Then, triethylamine (15 g, 146 mmol) was syringed into the reaction flask. The heterogeneous mixture was stirred until completion of the reaction. After the reaction was complete as indicated by TLC, 360 mL of diethyl ether was charged to the reaction mass and stirred for 10 minutes. The reaction mass was filtered and the filtrate was extracted with ice water, brine and dried over anhydrous sodium sulfate. The organic layer was concentrated under reduced pressure to produce the crude product. This was purified by silica gel chromatography to produce 5.3 g of solid product.

[0106] A 1 L three-neck round bottom flask was flame dried and equipped with a nitrogen bubbler, thermometer and septum and evacuated with nitrogen gas for 15 minutes. The flask was charged with bromo-pinacolylboronic acid ester (5.3 g, 14 mmol), diethanolamine (1.6 g, 15 mmol), 10.6 ml of IPA and 106 ml of diethyl ether under nitrogen atmosphere. The resulting mixture was stirred at room temperature for approximately 72 hours. After the reaction was complete, the reaction mixture was filtered and the product was washed with diethyl ether. Finally, the product was dried under vacuum at 45° C. to yield 4 g of a white solid.

[0107] A 250 mL three-neck round bottom flask was flame dried and equipped with a nitrogen bubbler, thermometer and septum and evacuated with nitrogen gas for 15 minutes. The flask was charged with bromo-pinacolyl boronate DEA complex (4.0 g, 11 mmol), 123 mL of diethyl ether and 123 mL of 0.1 M aqueous HCl. The reaction was stirred at room temperature for 2 hours and kept at room temperature overnight. After the reaction was complete, 125 mL of ether was added and the layers were allowed to separate. The organic layer was separated and the aqueous layer was further extracted with ether. The combined organic layers were washed with brine and Na 2 SO 4 Finally, after concentration of the organic layer, 2.7 g of the title compound was obtained as a white crystalline material.

[0108] Example 5. Synthesis of 2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)ethan-1-amine [ka] A 1 L three-neck round bottom flask was flame dried and equipped with a reflux condenser, nitrogen bubbler, thermometer and septum and evacuated with nitrogen gas for 15 min. 125 mL of dry DMF and NaH (6.24 g, 156 mmol) were added to the reaction flask and the reaction flask was cooled to 0-5 °C. N-(2-hydroxyethyl)phthalimide (23 g, 120 mmol) dissolved in 68 ml of DMF was slowly added to the reaction mixture and heated at 40 °C for 30 min. 4-Bromobenzyl bromide (30 g, 120 mmol) dissolved in 60 ml of DMF was slowly added to the reaction mixture and heated to 70 °C for 2 h. After the reaction was complete, the reaction mixture was cooled to room temperature and 500 mL of water was added. The product was extracted into ethyl acetate and the ethyl acetate layer was concentrated under reduced pressure to give a light yellow crude product. The crude residue was purified by silica gel column chromatography to give 13 g of product.

[0109] A 1 L three-neck round-bottom flask was flame dried and equipped with a reflux condenser, nitrogen bubbler, thermometer and septum, and evacuated with nitrogen gas for 15 min. 2-(2(4-bromobenzyl)oxyethylisoindoline-1,3-dione (13 g, 36 mmol), bis-pinacol borate (10 g, 40 mmol), 10.6 g potassium acetate and PdCl 2 (dppf) was charged to a reaction flask and DCM (1.47 g, 1.8 mmol) was added under nitrogen atmosphere. The resulting mixture was heated to 85° C. for 20 h under a continuous flow of nitrogen. After the reaction was complete, the flask was cooled to room temperature and 130 mL of water was added. The product was extracted into ethyl acetate. The ethyl acetate layer was washed with water and brine and dried over sodium sulfate. The organic layer was concentrated under reduced pressure and the residue was purified using silica gel column chromatography to give 12 g of a white solid product.

[0110] A 1 L four-neck round bottom flask was fitted with a reflux condenser, nitrogen bubbler, thermometer and septum, and 2-(2-(4,4,5,5-tetramethyl-1,3,2-dioxoboronate-2-yl)benzyl)oxy)ethyl)isoindoline-1,3-dione (6.5 g, 16 mmol) in 325 ml ethanol was added, and then the reaction vessel was evacuated with nitrogen gas for 15 minutes. To this reaction, 10 mL of hydrazine hydrate was added at room temperature, and the reaction mixture was refluxed for 12 hours. The reaction mixture was cooled to room temperature and stirred at room temperature for 24 hours. After the reaction was complete, the reaction mixture was filtered through a celite pad and the mother liquor was concentrated under vacuum to give a residue, which was dissolved in diethyl ether. The ether layer was washed with brine and shrunk under vacuum to give 2.6 g of the title product as a white solid.

[0111] The examples herein are for illustrative purposes and are not intended to limit the scope of the invention, which is defined in the claims.

Claims

1. Formula I 【Chemistry 1】 (In the formula, X is selected from a boronic acid group and a boronic acid pinacol ester group; L is a linker, S is a spacer and s is 0 to 10; Y is a chemically linkable end group. Compound.

2. L is a carbamate, ether, C １ ~C ２０ Alkylenyl and -[-O-CH ２ CH ２ -] ｎ - selected from the group consisting of: (wherein n is 1 to 12); S is selected from the group consisting of C 1 -C 20 alkylenyl and -[-O-CH 2 CH 2 -] n -, where n is 1 to 12; s is 1 to 10; Y is selected from the group consisting of halo, amino, carboxyl, C 2 -C 10 alkynyl, hydroxyl, C 1 -C 10 alkoxy, azide, sulfinate, thiol, fluorosulfate, and boronic acid; The compound of claim 1.

3. The compound of claim 2 further comprising a molecule conjugated to the chemically linkable end group.

4. The compound of claim 3 , wherein the molecule is selected from the group consisting of a drug, a probe, a dye, a peptide, a protein, a drug candidate and a natural product.

5. Formula II 【Chemistry 2】 (In the formula, X is selected from a boronic acid group and a boronic acid pinacol ester group; S is a spacer and s is 0 to 10; Y is a chemically linkable end group. Compound.

6. S is C １ ~C ２０ Alkylenyl and -[-O-CH ２ CH ２ -] ｎ - selected from the group consisting of: (wherein n is 1 to 12); s is 1 to 10; Y is selected from the group consisting of halo, amino, carboxyl, C 2 -C 10 alkynyl, hydroxyl, C 1 -C 10 alkoxy, azide, sulfinate, thiol, fluorosulfate, and boronic acid; The compound according to claim 5.

7. The compound of claim 6 further comprising a molecule conjugated to the chemically linkable end group.

8. The compound of claim 7, wherein the molecule is selected from the group consisting of a drug, a probe, a dye, a peptide, a protein, a drug candidate and a natural product.

9. Formula III 【Chemistry 3】 (In the formula, X is selected from a boronic acid group and a boronic acid pinacol ester group; S is a spacer and s is 0 to 10; Y is a chemically linkable end group. Compound.

10. S is C １ ~C ２０ Alkylenyl and -[-O-CH ２ CH ２ -] ｎ - selected from the group consisting of: (wherein n is 1 to 12); s is 1 to 10; Y is selected from the group consisting of halo, amino, carboxyl, C 2 -C 10 alkynyl, hydroxyl, C 1 -C 10 alkoxy, azide, sulfinate, thiol, fluorosulfate, and boronic acid; 10. The compound according to claim 9.

11. The compound of claim 10 further comprising a molecule conjugated to the chemically linkable end group.

12. The compound of claim 11 , wherein the molecule is selected from the group consisting of a drug, a probe, a dye, a peptide, a protein, a drug candidate and a natural product.

13. 2. A compound selected from the group consisting of (4-((((2-aminoethyl)carbamoyl)oxy)methyl)phenyl)boronic acid hydrochloride, (4-((((2-bromoethyl)carbamoyl)oxy)methyl)phenyl)boronic acid, 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl(bromoethyl)carbamate, 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl(2-aminoethyl)carbamate and 2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)ethan-1-amine.

14. 19. A method of delivering a molecule to the nucleus in a cell, comprising contacting a cell with a compound of any of claims 4, 8 or 12.

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