Novel linkers for sustained delivery of therapeutic agents

Abstract

A linker for linking a therapeutic agent to another moiety, such as the Fc region of an antibody. A linker having two or more maleimide functional groups capable of undergoing a nucleophilic conjugate addition reaction. A method for increasing the duration of action of a therapeutic agent by conjugating it to a novel linker and the Fc region of an antibody. An antibody-drug conjugate having an increased duration of action over the drug alone.

Description

[Technical Field]

[0001] (Sequence Listing) This application has been submitted with a Sequence Listing in ST.26 XML format. The Sequence Listing is provided in a file titled "30359 Sequence Listing.xml," created on December 9, 2022, and is 14.6 kilobytes in size. The Sequence Listing information in ST.26 XML format is incorporated herein by reference in its entirety.

[0002] FIELD OF THE INVENTION The present invention relates to novel compounds comprising novel linkers for improving the duration of action of therapeutic agents, such as peptides. The present invention also relates to novel conjugate moieties comprising a linker, an Fc region, and optionally a therapeutic agent. The present invention also relates to novel compounds comprising two or more maleimide functional groups, which may be useful for coupling to regions bearing two or more nucleophilic groups, such as the Fc region of an antibody. [Background technology]

[0003] Peptide therapeutics, such as insulin, have previously been used to treat diseases such as diabetes. However, the duration of action of many peptides can be very short. Therefore, patients are required to administer them every few hours, typically by injection, which can be cumbersome and time-consuming for the patient.

[0004] One strategy for increasing duration of action is to use antibody-drug conjugates (ADCs) in which the drug component is a peptide therapeutic. However, such ADCs are often difficult to produce, and production methods can require the peptide therapeutic to be exposed to harsh degradation conditions. One way to shield the peptide therapeutic during ADC formation is to modify and / or protect the peptide therapeutic, but such modifications can affect the efficacy of the peptide therapeutic. Known ADC production methods also cannot be used with peptide therapeutics that incorporate synthetic amino acids.

[0005] Thus, there is a need for ADCs that can incorporate unprotected / unmodified peptide therapeutics, with or without unnatural amino acids.

[0006] The identity of the linker can affect the reaction conditions that the therapeutic must experience during production of the ADC. Thus, there is also a need for improved linkers that can incorporate unprotected / unmodified peptide therapeutics, with or without unnatural amino acids. DETAILED DESCRIPTION OF THE INVENTION

[0007] The present invention relates to novel linkers, such as novel conjugates comprising novel linkers, for the delivery of therapeutic agents to animals. The novel conjugates may also include an Fc region and a therapeutic agent, such as a peptide therapeutic.

[0008] It has surprisingly been found that by conjugating a therapeutic agent to one of the disclosed novel linkers and another moiety, such as the Fc region of an antibody, the duration of action of the therapeutic agent can be improved and / or extended. Without wishing to be bound by theory, it is believed that conjugation of the therapeutic agent to the linker and Fc region may increase the duration of action of the therapeutic agent by expanding the overall moiety and preventing degradation of the therapeutic agent. The reaction conditions for the conjugation reaction between the novel linker and the Fc region are relatively mild, and therefore the payload, i.e., the therapeutic agent, does not need to be modified to protect it during the reaction.

[0009] It has also been surprisingly found that the disclosed novel linkers are capable of generating ADCs that can incorporate unprotected / unmodified peptide therapeutics, with or without unnatural amino acids.

[0010] [ka]

[0011] Disclosed herein are novel conjugate compounds of formula IA, where Fc comprises an Fc region, e.g., the Fc region of an antibody, L comprises a linker, and Z comprises a therapeutic agent, a functional group suitable for subsequent attachment of a therapeutic agent, or a functional group suitable for the conjugate compound to exist without an attached therapeutic agent.

[0012] [ka]

[0013] Also disclosed herein are novel conjugate compounds of formula IB, where Fc comprises one or more Fc regions, L comprises a linker, and Z comprises a therapeutic agent, a functional group suitable for subsequent attachment of a therapeutic agent, or a functional group suitable for the conjugate compound to exist without an attached therapeutic agent.

[0014] [ka]

[0015] Also disclosed herein are novel conjugate compounds of formula IC, where Fc comprises a single Fc region, e.g., the Fc region of an antibody, L comprises a linker, and Z comprises a therapeutic agent, a functional group suitable for subsequent attachment of a therapeutic agent, or a functional group suitable for the conjugate compound to exist without an attached therapeutic agent.

[0016] [ka]

[0017] Also provided herein are novel compounds of Formula II-A, wherein R1 and R2 are independently C1-C5 alkyl, optionally substituted with one or more of -NH2, -CH2NH2, and -CH2CH2NH2; R3 and R4 are independently C1-C5 alkyl or absent; U is a tertiary amine or a 1,3,5-substituted phenyl; Z is H, OH, NH2, NH, a therapeutic agent, a C1-C5 alkyl group, or a 1,3,5-substituted phenyl group. 30Alkyl, (OCH2CH2) m or combinations thereof, wherein m is an integer from 1 to 30, as well as pharmaceutically acceptable salts thereof.

[0018] [ka]

[0019] Also described herein are novel compounds of Formula II-B, wherein R1 and R2 are independently C1-C5 alkyl, optionally substituted with one or more of -NH2, -CH2NH2, or -CH2CH2NH2; R3 and R4 are independently C1-C5 alkyl; and Z is H, OH, NH2, NH, a therapeutic agent, a C1-C5 alkyl group, or a C1-C5 alkyl group. 30 Alkyl, (OCH2CH2) m or combinations thereof, wherein m is an integer from 1 to 30, as well as pharmaceutically acceptable salts thereof.

[0020] [ka]

[0021] Also described herein are novel compounds of Formula II-C, wherein R and R are independently C-C alkyl, optionally substituted with one or more of -NH, -CHNH, or -CHCHNH; and Z is H, OH, NH, NH, a therapeutic agent, a C-C alkyl group, or a C-C alkyl group. 30 Alkyl, (OCH2CH2) m or combinations thereof, wherein m is an integer from 1 to 30, as well as pharmaceutically acceptable salts thereof.

[0022] [ka]

[0023] Also provided herein are novel compounds of Formula III-A, wherein R1 and R2 are independently C1-C5 alkyl, optionally substituted with one or more of -NH2, -CH2NH2, and -CH2CH2NH2; R3 and R4 are independently C1-C5 alkyl or absent; U is a tertiary amine or a 1,3,5-substituted phenyl; Y1 and Y2 independently comprise an amide, an amine, an imine, a sulfonamide, a thiourea, a urea, a thioether, or a combination thereof; Z is H, OH, NH2, NH, a therapeutic agent, a C1-C5 alkyl, or a 1,3,5-substituted phenyl; 30 Alkyl, (OCH2CH2) m or combinations thereof, wherein m is an integer from 1 to 30, as well as pharmaceutically acceptable salts thereof.

[0024] [ka]

[0025] Also provided herein are novel compounds of Formula III-B, wherein R1 and R2 are independently C1-C5 alkyl, optionally substituted with one or more of -NH2, -CH2NH2, and -CH2CH2NH2; R3 and R4 are independently C1-C5 alkyl or absent; U is a tertiary amine or a 1,3,5-substituted phenyl; Z is H, OH, NH2, NH, a therapeutic agent, a C1-C5 alkyl group, or a 1,3,5-substituted phenyl group. 30 Alkyl, (OCH2CH2) m or a combination thereof, wherein m is an integer of 1 to 30, Fc comprises an Fc region, and S is a sulfur atom derived from the Fc region, as well as pharmaceutically acceptable salts thereof.

[0026] [ka]

[0027] Also provided herein are novel compounds of Formula III-C, wherein R1 and R2 are independently C1-C5 alkyl, optionally substituted with one or more of -NH2, -CH2NH2, and -CH2CH2NH2; R3 and R4 are independently C1-C5 alkyl or absent; and Z is H, OH, NH2, NH, a therapeutic agent, a C1-C5 alkyl group, or a C1-C5 alkyl group. 30 Alkyl, (OCH2CH2) m or a combination thereof, wherein m is an integer of 1 to 30, Fc comprises an Fc region, and S is a sulfur atom derived from the Fc region, as well as pharmaceutically acceptable salts thereof.

[0028] [ka]

[0029] Also described herein are novel compounds of Formula III-D, wherein R and R are independently C-C alkyl, optionally substituted with one or more of -NH, -CHNH, and -CHCHNH; and Z is H, OH, NH, NH, a therapeutic agent, a C-C alkyl group, or a C-C alkyl group. 30 Alkyl, (OCH2CH2) m or a combination thereof, wherein m is an integer of 1 to 30, Fc comprises an Fc region, and S is a sulfur atom derived from the Fc region, as well as pharmaceutically acceptable salts thereof.

[0030] [ka]

[0031] Also provided herein are novel compounds of Formula IV-A, wherein R1 and R2 are independently C1-C5 alkyl, optionally substituted with one or more of -NH2, -CH2NH2, and -CH2CH2NH2; R3 and R4 are independently C1-C5 alkyl or absent; U is a tertiary amine or a 1,3,5-substituted phenyl; Z is H, OH, NH2, NH, a therapeutic agent, a C1-C5 alkyl group, or a 1,3,5-substituted phenyl group.30 Alkyl, (OCH2CH2) m or combinations thereof, wherein m is an integer from 1 to 30, as well as pharmaceutically acceptable salts thereof.

[0032] [ka]

[0033] Also described herein are novel compounds of Formula IV-B, wherein R1 and R2 are independently C1-C5 alkyl, optionally substituted with one or more of -NH2, -CH2NH2, or -CH2CH2NH2; R3 and R4 are C1-C5 alkyl; and Z is H, OH, NH2, NH, a therapeutic agent, a C1-C5 alkyl group, or a C1-C5 alkyl group. 30 Alkyl, (OCH2CH2) m or combinations thereof, wherein m is an integer from 1 to 30, as well as pharmaceutically acceptable salts thereof.

[0034] [ka]

[0035] Also described herein are novel compounds of Formula IV-C, wherein R and R are independently C-C alkyl, optionally substituted with one or more of -NH, -CHNH, or -CHCHNH; and Z is H, OH, NH, NH, a therapeutic agent, a C-C alkyl group, or a C-C alkyl group. 30 Alkyl, (OCH2CH2) m or combinations thereof, wherein m is an integer from 1 to 30, as well as pharmaceutically acceptable salts thereof.

[0036] Linker The present invention includes linkers L for connecting Z to another moiety, such as one or more cysteine ​​amino acids in the Fc region of an antibody, as shown in Formulas IA, IB, and / or IC, or to another suitable nucleophilic group, such as an amide, amine, imine, sulfonamide, thiourea, urea, thioether, thiol, cysteine, or combinations thereof, as shown in Formulas III-A, III-B, and / or III-C, via a nucleophilic conjugate addition reaction.

[0037] The linker can include a central trivalent linking unit U, as shown in Formulae II-A, III-A, and IV-A. One of the linker moieties can be attached to Z. The other two moieties of the linker contain maleimide functional groups, electrophilic groups, which can be used to selectively connect Z to two nucleophilic groups in a nucleophilic conjugate addition reaction.

[0038] Thus, as disclosed herein, the linkers of the present invention comprise two or more maleimide functional groups, preferably two maleimide functional groups, prior to the nucleophilic conjugate addition reaction.

[0039] Suitable trivalent linking units can include any atom or molecule that can be trisubstituted, such as a trisubstituted C5-C8 aryl, a trisubstituted C5-C8 heteroaryl having 1-3 heteroatoms in the ring system, a trisubstituted C5-C8 cycloalkyl, a trisubstituted C5-C8 heterocycle having 1-3 heteroatoms in the ring system, a tertiary amine, a tertiary phosphine, or combinations thereof. Preferably, U is a 1,3,5-trisubstituted phenyl or a tertiary amine.

[0040] Each arm attached to U and the maleimide functional group can be represented by formula V:

[0041] [ka]

[0042] In the first of the two arms represented by Formula V, R1 can be a C1-C5 alkyl optionally substituted with one or more of -NH2, -CH2NH2, and -CH2CH2NH2, and R3 can be a C1-C5 alkyl or absent. In the second of the two arms represented by Formula V, R2 can be a C1-C5 alkyl optionally substituted with one or more of -NH2, -CH2NH2, and -CH2CH2NH2, and R4 can be a C1-C5 alkyl or absent.

[0043] R1 and / or R2 may preferably be C2 alkyl, optionally substituted with one or more of -NH2, -CH2NH2, or -CH2CH2NH2. R3 and / or R4 may preferably be C2 alkyl when U is a tertiary amine, or may be absent when U is 1,3,5-trisubstituted phenyl.

[0044] When both arms are attached to U, R1 and R2 can be the same or independently C1-C5 alkyl, optionally substituted with one or more of -NH2, -CH2NH2, and -CH2CH2NH2. When both arms are attached to U, R3 and R4 can be the same or independently C1-C5 alkyl, or absent. When R3 and / or R4 are absent, the amide nitrogen in Formula V is directly attached to U, as when U is 1,3,5-trisubstituted phenyl as in Formula IV-C.

[0045] The third of the three arms attached to U contains Z. Z can be H, OH, NH, NH, a therapeutic agent, C-C 30 Alkyl, (OCH2CH2) mor a combination thereof, where m is an integer from 1 to 30. Z can be bonded directly to U, or Z can be bonded to a carbonyl functional group. The carbon atom of the carbonyl functional group can be bonded directly to U and Z, as shown in Formulas II-IV.

[0046] Thus, disclosed herein are novel linkers as shown in Formula II-A, Formula II-B, and Formula II-C, which have two maleimide functional groups prior to any nucleophilic conjugate addition reaction.

[0047] Nucleophilic conjugate addition reaction with maleimide functional groups As disclosed herein, a linker, L, can be attached to Z to yield a linked payload, or, when Z includes a therapeutic agent, to yield a linked therapeutic agent. The linkers disclosed herein contain two or more, or preferably two, maleimide functional groups, as shown in Formula V. The maleimide functional groups can be used to attach Z to a different moiety, such as an Fc region, via a nucleophilic conjugate addition reaction.

[0048] In nucleophilic conjugate addition reactions, maleimides act as electrophiles and can react with nucleophilic groups to form new covalent bonds between the nucleophilic atom and the electrophilic carbon atom, resulting in ring-opening of the maleimide functionality as shown in Formula VI.

[0049] [ka]

[0050] A variety of nucleophilic groups can be used to react with the electrophilic maleimide functional group. Suitable nucleophilic groups include moieties containing one or more nitrogen and / or sulfur atoms in the functional group, which can function as electron-rich nucleophilic groups. Suitable examples of nucleophilic groups include moieties containing one or more of the following functional groups: amide, amine, imine, sulfonamide, thiol, thiourea, urea, or combinations thereof, also shown in Formula VII.

[0051] [ka]

[0052] In particular, the amino acid cysteine, found in many amino acid sequences of peptides, antibodies, proteins, etc., contains a thiol functional group and can be used as a nucleophile to form a thioether between the amino acid sequence and the linker, linked payload, and / or linked therapeutic agent, as shown in Formula VIII within the peptide chain.

[0053] [ka]

[0054] Specific conditions for the conjugate nucleophilic conjugate addition reaction are well known to those skilled in the art. Specific conditions may vary depending on the nucleophilic and electrophilic groups used. Thus, disclosed herein are novel compounds and / or conjugate compounds comprising linked payloads bound to another moiety via a nucleophilic conjugate addition reaction, as shown in Formula III-A, Formula III-B, Formula III-C, Formula III-D, Formula IV-A, Formula IV-B, and / or Formula IV-C.

[0055] Furthermore, the use of the novel linkers disclosed herein can be advantageous because the reaction conditions are milder compared to other nucleophilic conjugate addition reactions disclosed using other linkers. For example, the disclosed linkers can be combined with the Fc region of an antibody with very little heating. Specifically, the disclosed linkers can be conjugated to the Fc region of an antibody by mixing the linked therapeutic agent and the reduced Fc region at 30° C. or below or 25° C. or below for 2 hours or less to obtain a conjugate.

[0056] Because the reaction conditions for conjugation are relatively mild, the payload, i.e., the therapeutic agent, does not need to be modified to be protected during the reaction. Other linkers require peptide therapeutics, such as insulin, to be modified by recombinant extension. The disclosed conjugated compounds remain effective with a longer duration of action without the need to modify the therapeutic agent.

[0057] Fc area In some embodiments, the nucleophilic group capable of reacting with two or more maleimide functional groups on the linker is the Fc region. The Fc region may also be known as the fragment crystallizable region. The Fc region is the tail region of an antibody that interacts with several cell surface receptors called Fc receptors. The Fc region may comprise a peptide, protein, or portion of an antibody capable of reacting with an Fc receptor, or an entire monoclonal antibody. In particular, the Fc region of certain antibodies, such as IgA, IgD, IgE, IgG, IgM, or a combination thereof, may have two or more cysteine ​​amino acids that form disulfide bridges.

[0058] In some embodiments, the Fc region of the antibody may be modified prior to attachment to the linker via a nucleophilic conjugate addition reaction. Suitable modifications may include modifications to the antibody, as further described herein, to minimize reactivity between the antibody and the linker other than the desired nucleophilic conjugate addition reaction.

[0059] In some embodiments, the Fc region can be a modified Fc region of an antibody. Suitable modifications include blunting and / or removal of certain portions of the Fc region, such as the hinge region.

[0060] The disulfide bridges present in the appropriate Fc region can be reduced, and the two reduced thiols can be suitable nucleophilic groups that can undergo a nucleophilic conjugate addition reaction with two maleimide functional groups of a linker of Formula II-A, Formula II-B, and / or Formula II-C to provide a compound of Formula IV-A, Formula IV-B, and / or Formula IV-C.

[0061] Treatment drugs In some embodiments, Z can comprise a therapeutic agent, which is a drug or active agent that, when administered to a patient with a target disease, can treat the target disease.

[0062] Suitable therapeutic agents may include one or more amino acids, or peptides having from about 2 to 500 amino acids, from about 2 to 250 amino acids, or from about 2 to about 100 amino acids.

[0063] Suitable therapeutic agents can include peptide hormones, such as adrenocorticotropic hormone (ACTH), adropin, amylin, angiotensin, atrial natriuretic peptide (ANP), calcitonin, cholecystokinin (CCK), gastrin, ghrelin, glucagon, growth hormone, follicle-stimulating hormone (FSH), insulin, leptin, luteinizing hormone (LH), melanocyte-stimulating hormone (MSH), oxyntomodulin, oxytocin, parathyroid hormone (PTH), prolactin, renin, somatostatin, thyroid-stimulating hormone (TSH), thyrotropin-releasing hormone (TRH), vasopressin, vasoactive intestinal peptide (VIP), also known as arginine vasopressin (AVP) or antidiuretic hormone (ADH), combinations thereof, and / or analogs thereof. In some embodiments, the therapeutic agent can be insulin, oxyntomodulin, and / or analogs thereof.

[0064] Other suitable therapeutic agents include monoclonal antibodies, interleukins, interferons, protein kinase inhibitors, hematopoietic factors, proteins, fusion proteins, oligonucleotides, or combinations thereof.

[0065] Conjugates In some embodiments, the novel compounds may be conjugates and / or antibody-drug conjugates (ADCs) when Z comprises a therapeutic agent and the linker is conjugated to at least a portion of an Fc region of an antibody. As described herein, it has surprisingly been found that when a therapeutic agent, such as oxyntomodulin, is attached to the Fc region of an antibody via the novel linkers disclosed herein, the therapeutic agent has an improved duration of action compared to the therapeutic agent alone.

[0066] Treatment method Also disclosed herein are methods for increasing the duration of action of a therapeutic agent, which may include attaching a therapeutic agent to a trivalent linker to provide a linker therapeutic agent, such as a compound of Formula II-A, Formula II-B, and / or Formula II-C, where Z comprises a therapeutic agent.

[0067] The method may also include attaching the linked therapeutic agent to at least a portion of an Fc region of an antibody, e.g., an IgG, via a nucleophilic conjugate addition reaction between the linked therapeutic agent and two nucleophilic groups in the Fc region, e.g., two sulfur atoms and / or anions, to obtain a conjugate compound.

[0068] The conjugated compound can include a therapeutic agent attached to a novel linker disclosed herein, which is then conjugated to at least a portion of an Fc region of an antibody. The conjugated compound can have a longer duration of action than the unattached therapeutic agent. The duration of action of the conjugated compound can be at least 12 hours, 1 day, 2 days, or 3 days longer than the duration of action of the therapeutic agent.

[0069] Furthermore, the use of the novel linkers disclosed herein can be advantageous because the reaction conditions are milder compared to other nucleophilic conjugate addition reactions disclosed using other linkers. For example, the disclosed linkers can be combined with the Fc region of an antibody with very little heating. Specifically, the disclosed linkers can be conjugated to the Fc region of an antibody by mixing the linked therapeutic agent and the reduced Fc region at 30° C. or below or 25° C. or below for 2 hours or less to obtain a conjugate.

[0070] formulation The compounds disclosed herein can be included in pharmaceutical preparations. Pharmaceutical preparations can include one or more carriers, diluents, and excipients that are compatible with the compounds and other components of the composition or formulation and are not harmful to the patient. Examples of pharmaceutical compositions and processes for their preparation can be found in "Remington: The Science and Practice of Pharmacy", Loyd, V., et al. Eds., 2002. nd Ed., Mack Publishing Co., 2012.

[0071] definition As used herein, the terms "a," "an," "the," and similar terms as used in the context of this disclosure (particularly in the context of the claims) should be construed to cover both the singular and the plural, unless otherwise specified herein or clearly contradicted by context.

[0072] As used herein, the term "alkyl" means a saturated straight or branched chain monovalent or divalent hydrocarbon group containing the indicated number of carbon atoms. For example, "C1-C 20 "Alkyl" means a radical having from 1 to 20 carbon atoms in a linear or branched arrangement.

[0073] Certain abbreviations used herein are defined as follows: "BEA" refers to 5-methyl-1,2,4-triazolo[3,4-b]benzothiazole, "Boc" refers to tert-butoxycarbonyl, "DABA" refers to 3,5-diaminobenzoic acid, "DCM" refers to dichloromethane, "DIC" refers to diisopropylcarbodiimide, "DIEA" refers to diisopropylethylamine, "DMAP" refers to 4-dimethylaminopyridine, and "EtOAc" refers to ethyl acetate. acetate), "Fmoc" refers to fluorenylmethyloxycarbonyl, "hr / hrs" refers to time, "FPLC" refers to fast protein liquid chromatography, "MalDab" refers to (S)-2-((tert-butoxycarbonyl)amino)-4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoic acid, and "Maldap" refers to (S)-3-((tert-butoxycarbonyl)amino)-2 -(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoic acid, β-MalDap refers to (S)-2-((tert-butoxycarbonyl)amino)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoic acid, "MeOH" refers to methanol, "min" refers to minutes / minutes, and "MTT" refers to methylthialazole tetrazolium. tetrazolium), "OXM" refers to oxyntomodulin, "Oxyma" refers to ethyl cyanohydroxyiminoacetate, "polyethylene glycol, PEG" refers to polyethylene glycol, "PyBOP" refers to benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate, "tBu" refers to tertiary butyl, "TCEP" refers to tris(2-carboxyethyl)phosphine hydrochloride, and "THF" refers to tetrahydrofuran.

[0074] preparation Preparation 1 (S)-2-((tert-butoxycarbonyl)amino)-4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoic acid

[0075] [ka]

[0076] Triethylamine (1 mL, 7.17 mmol) was added to a mixture of (2S)-4-amino-2-(tert-butoxycarbonylamino)butanoic acid (500 mg, 2.29 mmol) in 1,4-dioxane (10 mL), THF (5 mL), and water (5 mL). Stirring was continued until a homogeneous mixture was obtained, and then methyl 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate (370 mg, 2.31 mmol) was added. The mixture was mixed at ambient temperature for 1 hour. The mixture was diluted with 50 mL of water, and the pH was adjusted to ∼6 by adding 5N HCl. The aqueous layer was extracted with EtOAc (30 mL) and chloroform / isopropanol (3 × 30 mL). The organic layers were combined, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography eluting with 0-30% MeOH in DCM to give the title compound (580 mg, 81%). ES / MS m / z: 199 (M-tBu).

[0077] Preparation 2 2,5-Dioxopyrrolidin-1-yl (S)-2-((tert-butoxycarbonyl)amino)-4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoate

[0078] [ka]

[0079] Dicyclohexylcarbodiimide (320 mg, 1.53 mmol) was added to a solution of (S)-2-((tert-butoxycarbonyl)amino)-4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoic acid (400 mg, 1.27 mmol) and N-hydroxysuccinimide (180 mg, 1.53 mmol) in THF (6 mL). Stirring was allowed to continue at ambient temperature for 3 hours. The solids were removed by filtration, and the filtrate was then concentrated under reduced pressure to give the title compound (605 mg, 84%), which was used without further purification. ES / MS m / z: 296 (M-tBu).

[0080] Preparation 3 (S)-2-((tert-butoxycarbonyl)amino)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoic acid

[0081] [ka]

[0082] (2S)-3-Amino-2-(tert-butoxycarbonylamino)propanoic acid (5.00 g, 24.5 mmol) was mixed with 1 M aqueous sodium bicarbonate (130 mL, 130 mmol). Stirring was continued at ambient temperature for 10 minutes until a clear solution was formed. The solution was cooled in an ice-water bath, and methyl 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate (4.00 g, 25.0 mmol) was added in three portions over 15 minutes. Stirring was continued at 0°C for 3 hours. 100 mL of EtOAc was added and stirred in the cold, followed by the addition of concentrated HCl to adjust the pH to 1. The layers were separated, and the aqueous layer was extracted with DCM (4 × 50 mL). The organic layers were combined, washed with saturated aqueous NaCl, and dried over sodium sulfate. The solvent was removed under reduced pressure. Purification by silica gel chromatography eluting with 0-40% MeOH in DCM gave the title compound (5.70 g, 66%). ES / MS m / z: 185 (M-tBu).

[0083] Preparation 4 2,5-Dioxopyrrolidin-1-yl (S)-2-((tert-butoxycarbonyl)amino)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate

[0084] [ka]

[0085] Dicyclohexylcarbodiimide (484 mg, 2.32 mmol) was added to a mixture of (S)-2-((tert-butoxycarbonyl)amino)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoic acid (550 mg, 1.54 mmol) and N-hydroxysuccinimide (272 mg, 2.31 mmol) in THF (5 mL). Stirring was continued at ambient temperature until a precipitate appeared. The solid was removed by filtration and washed with DCM. The filtrate was concentrated under reduced pressure to give the title compound (1.18 g, 85%). ES / MS m / z: 282 (M+H-Boc).

[0086] Preparation 5 4-(bis(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)amino)-4-oxobutanoic acid

[0087] [ka]

[0088] A solution of N-(9-fluorenylmethoxycarbonyloxy)succinimide (19.4 g, 56.4 mmol) in DCM (80 mL) was added over 45 min to a solution of diethylenetriamine (3.1 mL, 28 mmol) in DCM (30 mL) cooled at -78 °C. After 2 h, the mixture was warmed to ambient temperature, and succinic anhydride (9.91 g, 98 mmol) and DMAP (691 mg, 5.60 mmol) were added. The mixture was stirred at ambient temperature for 15 h. The pH was adjusted to 5 by slow addition of 1 N HCl (20 mL). The phases were separated, and the aqueous phase was extracted with DCM (2 × 100 mL). The organic layers were combined, washed with saturated aqueous NaCl, dried over magnesium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography eluting with 0–10% MeOH in DCM to give the title compound (911.84 g, 65%) as a white powder. ES / MS m / z: 648(M+1). [Example]

[0089] Scheme 1

[0090] [ka]

[0091] General synthesis of conjugates containing an Fc region, a linker, and a peptide

[0092] A general synthesis of linker-peptides is outlined in Scheme 1. In each case, an Fmoc-protected (in two of the three available arms) trivalent core, either a 1,3,5-trisubstituted phenyl (commercially available) or a tertiary amine (Preparation 5), was first reacted with the peptide on the resin in reaction (1). The Fmoc group was removed in reaction (2). In reaction (3), the primary amine functionality resulting from removal of the Fmoc protecting group was reacted with an NHS ester, such as MalDap (commercially available), MapDab (Preparation 2), or β-MalDap (Preparation 4), to liberate the peptide from the resin. In reaction (4), the bismaleimide functionality reacted with two reduced thiol functionalities in the Fc region in a nucleophilic conjugate addition reaction to yield a conjugate comprising the Fc region, linker, and therapeutic agent.

[0093] The linker peptide is generated by solid-phase peptide synthesis using the Fmoc / t-Bu strategy on a SymphonyX automated peptide synthesizer (PTI Protein Technologies Inc.) starting with RAPP AM-Rink Amide resin (H40023 Polystyrene AM RAM, Rapp polymere GmbH). Amino acid coupling is performed using 5 equivalents of amino acid, 1.2 M DIC, and 0.9 M Oxyma in DMF for 2 h at 25 °C. Deprotection is performed using a 25% piperidine solution in DMF.

[0094] The N-terminal amino acid is a boc-protected proline, allowing Fmoc coupling reactions to be performed at the C-terminal K ε-amine. The MTT protecting group present at the C-terminal K is removed using 30% hexafluoroisopropanol (HFIP) in DCM. Further coupling / deprotection cycles using the Fmoc / t-Bu strategy for side chain extension include Fmoc-Peg24-OH (Broadpharm, catalog number BP-22036), an Fmoc-protected BEA (bisethylamine) aliphatic linker, and Boc-Maldap (CAS number 1491152-23-8). For PEG coupling, 2 equivalents of Fmoc-Peg24-OH, 2 equivalents of PyBOP, and 4 equivalents of DIEA were coupled at 37°C for 2 hours. For BEA, 3 equivalents of BEA were used with 3 equivalents of PyBop and 6 equivalents of DIEA, and the coupling was performed at 37°C for 3 hours. Boc-Maldap was coupled to each of the free amines of BEA using 5 equivalents of Boc-Maldap, 0.9 M Oxyma, and 1.2 M DIC in DMF for 6 hours at 25 °C. Deprotection of PEG and BEA was carried out using a 25% piperidine solution in DMF. Simultaneous cleavage from the resin and removal of side chain protecting groups were carried out in a solution containing trifluoroacetic acid (TFA):triisopropylsilane:MilliQ HO:thioanisole 85:5:5:5 (v / v) for 2 hours at 25 °C, followed by precipitation with cold ether. The crude peptide was purified by reverse-phase HPLC on a Waters Symmetry Prep C18 column (19 × 250 mm, 100 A, 5 μm), and the appropriate fractions were pooled and lyophilized.

[0095] Once the linker-therapeutic agent is cleaved from the resin, it can react with the reduced Fc region to form a conjugate comprising the Fc region, linker, and therapeutic agent. A summary of possible conjugates is provided in Table 1.

[0096] The following linkers were synthesized by the process described above:

[0097] [Table 1-1]

[0098] [Table 1-2]

[0099] SEQ ID NO: 7: H-Aib-QGTFTSDYSKYLDEKKAQEFVEWLLEGGPSSGK(Peg24-BEA-Maldap2)-NH2

[0100] [ka]

[0101] In this sequence, the N-terminus is free and the C-terminus is amidated as a primary amide. The C-terminal K is chemically modified by conjugation to the ε-amino group of the K side chain using Peg24-BEA-Maldap2. The amine is MalDap, the tertiary core is a tertiary amine, and the bridge between the peptide and the linker is PEG24. This was synthesized using the general synthesis described above.

[0102] SEQ ID NO: 8: H-Aib-QGTFTSDYSKYLDEKKAK(Peg24-BEA-Maldap2)EFVEWLLEGGPSSG-NH2

[0103] [ka]

[0104] In this sequence, the N-terminus is free and the C-terminus is amidated as a primary amide. The K at position 20 is chemically modified by conjugation of the K side chain to the ε-amino group using Peg24-BEA-Maldap2. The amine is MalDap, the tertiary core is a tertiary amine, and the bridge between the peptide and linker is PEG24, which was synthesized using the general synthesis described above.

[0105] SEQ ID NO: 9: NH-SPPPAGSSPGG-H-RFSPCGLGSYHGIRDIKGGFCSSRGKE-(PEG-G-PAPAPAPAPAPA)-DABA-(Maldab)

[0106] [ka]

[0107] SEQ ID NO: 9, in which the amine is MalDab, the tertiary core is 1,3,5-phenyl, and the bridge between the peptide and the linker is PEG12, was prepared using the same general synthesis as sequences 7, 8, and 9.

[0108] SEQ ID NO: 10: NH-SPPPAGSSPGG-H-RFSPCGLGSYHGIRDIKGGFCSSRGKE-(PEG-DABA-(Maldab)

[0109] [ka]

[0110] SEQ ID NO: 10, in which the amine is MalDab, the tertiary core is 1,3,5-phenyl, and the bridge between the peptide and linker is PEG24, was prepared using the same general synthesis as sequences 7, 8, and 9.

[0111] Peptides SEQ ID NOs: 7-12 were produced by solid-phase peptide synthesis using the Fmoc / t-Bu strategy on a SymphonyX automated peptide synthesizer (PTI Protein Technologies Inc.) starting with RAPP AM-Rink Amide resin (H40023 Polystyrene AM RAM, Rapp polymere GmbH). Amino acid coupling was carried out using 5 equivalents of amino acid, 1.2 M DIC, and 0.9 M Oxyma in DMF at 25°C for 2 hours. Deprotection was carried out using a 25% piperidine solution in DMF.

[0112] The N-terminal amino acid is a Boc-protected proline, allowing Fmoc coupling to be performed at the ε-amine of K. The MTT protecting group present at K at position 20 is removed using 30% hexafluoroisopropanol (HFIP) in DCM. Further coupling / deprotection cycles using the Fmoc / t-Bu strategy for side chain extension include Fmoc-Peg24-OH (Broadpharm, catalog number BP-22036), an Fmoc-protected BEA (bisethylamine) aliphatic linker, and Boc-Maldap (CAS number 1491152-23-8). For Peg coupling, 2 equivalents of Fmoc-Peg24-OH, 2 equivalents of PyBOP, and 4 equivalents of DIEA were coupled at 37°C for 2 hours. For BEA, 3 equivalents of BEA were used with 3 equivalents of PyBop and 6 equivalents of DIEA, and the coupling was performed at 37°C for 3 hours. Boc-Maldap was coupled to each of the free amines of BEA using 5 equivalents of Boc-Maldap, 0.9 M Oxyma, and 1.2 M DIC in DMF for 6 hours at 25° C. Deprotection of PEG and BEA was achieved using a 25% piperidine solution in DMF.

[0113] IgG4 Fc region The Fc region of human IgG4 was prepared and purified by well-known methods. Chinese hamster ovary cells (CHO) were transiently or stably transfected with an expression system for secreting Fc using a single vector encoding a blunt-ended Fc heavy chain nucleotide sequence. The clarified medium into which the blunt-ended Fc was secreted was purified using commonly used techniques known to those skilled in the art.

[0114] The IgG4 Fc region contained a human IgG4 Fc region with a blunt-ended hinge. To improve expression and minimize the possibility of unstructured peptides that could interfere with the conjugation process, a very short dipeptide consisting of alanine and glycine was incorporated upstream of the cysteine ​​at position 229 (EU index numbering). The traditional human IgG4 hinge (SEQ ID NO: 1) was blunted to remove the cysteine ​​at position 226 (EU index numbering), which is involved in the upper hinge disulfide bond, and replaced with a shortened hinge (SEQ ID NO: 2) consisting of an alanine-glycine N-terminal dipeptide followed by a single disulfide derived from the native cysteine ​​at position 229. Removing the upper hinge disulfide eliminates potential mixed conjugation species by limiting the available cysteine ​​thiols that can react with maleimides, simplifying the conjugation process. The complete human IgG4 Fc containing the blunted hinge with a single disulfide is shown in SEQ ID NO: 3.

[0115] Two alternative human IgG4 blunt-ended Fc mutants and their combinations have been explored: 1) Mutations focused on increasing the isoelectric point (pI) of the Fc to potentially improve the biophysical properties of the final conjugate include the Q274K, Q355R, and E419Q (EU index numbering) mutations (SEQ ID NO: 4). Mutations potentially improving the half-life of the conjugate include the M252Y, S254T, and T256E mutations, which are known to enhance neonatal Fc receptor (FcRn) binding at low pH, which in turn leads to higher levels of antibody recycling (SEQ ID NO: 5). SEQ ID NO: 6 is a combination of both the Q274K, Q355R, E419Q and M252Y, S254T, T256E mutations.

[0116] Attachment of the linker peptide to the Fc region The disulfide bond in the hinge of Fc was reduced by adding 2 equivalents of TCEP prepared in PBS to Fc and incubating at 37°C for 1 hour. The reduction was followed by LCMS-TOF using a Zorbax 300 SB-C3 analytical column, RRHD, 2.1 x 100 mm, and analyzed using MassHunter software. Once the ~52 kD peak was completely reduced to a ~26 kD peak, the Fc was ready for re-crosslinking.

[0117] The re-crosslinking reaction was carried out by diluting the reduced Fc five-fold into 50 mM sodium acetate pH 5.5 buffer containing 20% ​​acetonitrile. Two equivalents of peptide were dissolved in cold MilliQ HO containing 20% ​​acetonitrile and 0.1% TFA and then immediately added to the buffered Fc solution. The reaction was monitored by LCMS-TOF. The ∼26 kD peak was converted to a ∼58 kD peak as each peptide maleimide re-crosslinked the reduced cysteine.

[0118] The Fc-peptide recrosslinked conjugate was purified in two steps using an AKTA FPLC. First, the reaction mixture was loaded onto a HiTrap™ MabSelect Xtra™ Protein A column (Cytiva) in a two-step purification. The reaction mixture was loaded onto a system equilibrated in PBS pH 7.2 to bind the Fc-containing product and remove the peptide. After peptide removal, the Fc-containing material was collected by changing the mobile phase to 20 mM citrate pH 3 to disrupt the interaction of Fc with the column. The pH of the eluted fractions was increased by adding 1 M T8, and buffer exchange to 20 mM Tris pH 8 was performed by centrifugation using amicon-ultra vials with a 10,000 mw cutoff.

[0119] If necessary, a second purification step was performed to remove unbound Fc components by strong anion exchange using Mono-Q 5 / 50 GL (Cytiva). Samples were loaded with 20 mM T8 and eluted with an increasing gradient of 20 mM T8 + 1 M NaCl. LCMS-TOF was used to determine the fractions containing the desired material, which were then buffer-exchanged again to remove NaCl and obtain a final solution of 20 mM T8.

[0120] Example 1: OXMP-2Fc

[0121] [ka]

[0122] Example 1 was synthesized using the procedure described above for attaching a peptide to a linker and then conjugating the peptide-linker to an Fc region. Example 1 is a conjugate comprising the peptide of SEQ ID NO: 11 and the Fc region of SEQ ID NO: 6. The linker used to link SEQ ID NO: 11 to SEQ ID NO: 6 was Example D in Table 1.

[0123] Example 2: Natriuretic peptides

[0124] [ka]

[0125] Example 2 was synthesized using the procedure described above for attaching the peptide to a linker and then conjugating the peptide-linker to an Fc region. Example 2 is a conjugate comprising the peptide-linker of SEQ ID NO: 10 to the Fc region of SEQ ID NO: 6.

[0126] Example 3: Natriuretic peptides

[0127] [ka]

[0128] Example 3 was synthesized using the procedure described above for attaching the peptide to a linker and then conjugating the peptide-linker to an Fc region. Example 3 is a conjugate comprising the peptide-linker of SEQ ID NO:9 attached to the Fc region of SEQ ID NO:6.

[0129] Example 4 Example 4 was synthesized using the procedure described above for attaching a peptide to a linker and then conjugating the peptide-linker to an Fc region. Example 4 is a conjugate comprising the peptide of SEQ ID NO: 12 and the Fc region of SEQ ID NO: 6. The linker used to link SEQ ID NO: 12 to SEQ ID NO: 6 was Example E in Table 1.

[0130] Example 5 Example 5 was synthesized using the procedure described above for attaching the peptide to a linker and then conjugating the peptide-linker to an Fc region. Example 3 is a conjugate comprising the peptide of SEQ ID NO: 12 and the Fc region of SEQ ID NO: 6. The linker used to link SEQ ID NO: 12 to SEQ ID NO: 6 was Example F in Table 1.

[0131] Hydrolysis of the maleimide functional group Maleimide hydrolysis occurs on these constructs at pH ≥ 8, opening the maleimide ring and causing a mass increase of 18 Daltons. Re-crosslinked constructs each have two maleimides, which accounts for a total mass shift of 36 Daltons. Ring opening (hydrolysis) is followed by mass using LCMS-TOF. Autohydrolysis of the linker

[0132] Linker autohydrolysis was assessed by incubating the linkers in triple phosphate buffers with corresponding pHs of 5.0, 6.0, 7.0, and 8.0. Lyophilized linker powder was solubilized in 100% DMSO to create an 80 mM stock solution. Each linker was incubated in the buffer at a final linker concentration of 1.5 mM, and succinimide hydrolysis was measured at room temperature for 1 hour. The effect of pH on linker autohydrolysis was assessed at specific intervals by reverse-phase HPLC mass spectrometry. Tables 2A and 2B show the effect of pH on specific conjugates.

[0133] Surprisingly, Tables 2A and 2B show that linkers containing βMaldap were more stable to higher pH conditions. Example 4 included a conjugate containing a natriuretic peptide (SEQ ID NO: 12) linked to an Fc region (SEQ ID NO: 6) via the Maldap linker of Example E in Table 1. Example 5 included a conjugate containing a natriuretic peptide (SEQ ID NO: 12) linked to an Fc region (SEQ ID NO: 6) via the Maldap linker of Example F in Table 1.

[0134] [Table 2]

[0135] [Table 3]

[0136] Duration of action of the therapeutic drug DIO mice were weighed, fed fresh TD95217 diet, and acclimated to handling, daily body weight, and food intake before the start of the study. Animals were randomized in a block order based on BW. For acute studies, mice received a single injection of 10 nmol / kg of each peptide / conjugate shown in Table 3. Daily food intake and body weight were monitored for 15 days after injection. For chronic studies, each dose group (0.3 nmol / kg, 1 nmol / kg, 3 nmol / kg, 10 nmol / kg, and 30 nmol / kg) received weekly injections for 4 weeks, with food intake and body weight monitored until 3 weeks after the last injection. QNMR was measured every 2 weeks.

[0137] Table 3 shows the effect of conjugating a therapeutic agent to the Fc region via the disclosed linkers. Two different injections were administered to two cohorts of randomized DIO mice. The first group received a control injection containing no therapeutic agent. The second group was injected with an oxyntomodulin analog (Example 1) conjugated to the blunt-ended Fc region of IgG4.

[0138] Daily food intake in DIO mice fed the control dose did not change over time (2.7–3.1 g of food per day).

[0139] Surprisingly, the daily food intake of DIO mice injected with Example 1 (oxyntomodulin derivative conjugated via a linker to the Fc region of IgG4) decreased to 0.94 g by day 2 and remained below 1 g until day 5. By day 7, the food intake of DIO mice injected with Example 1 returned to levels comparable to mice given control injections.

[0140] In particular, it is well known to those skilled in the art that the equivalent oxyntomodulin analog of Example 1 that is not conjugated to an Fc region and / or the disclosed linker rarely has an extended duration of action beyond the initial effect immediately after administration. See, e.g., Camacho et al. Conjugation of a peptide to an antibody engineered with free cysteines dramatically improves half-life and activity. Mabs. 2020 Jan-Dec;12(1):1794687. doi:10.1080 / 19420862.2020.1794687, which states that "endodenous peptide hormones have extremely short half-lives, making them impractical as therapeutic agents." Thus, without wishing to be bound by theory, it is believed that conjugation of the oxyntomodulin analog to the Fc region via the disclosed linker extended the duration of action of the oxyntomodulin analog by approximately 7 days.

[0141] Thus, conjugation to the Fc region via one of the disclosed linkers resulted in an increase in the duration of action of the therapeutic agent by one week.

[0142] [Table 4]

[0143] array SEQ ID NO:1; ESKYGPPCPSCP SEQ ID NO:2; AGCP SEQ ID NO: 3: Blunt-ended human IgG4 Fc with a single hinge disulfide AGCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG SEQ ID NO: 4: Blunt-ended human IgG4 Fc with a single hinge disulfide (Q274K, Q355R and E419Q mutants) AGCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVKFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSLG SEQ ID NO: 5: Blunt-ended human IgG4 FcM252Y, S254T and T256E mutants with a single hinge disulfide AGCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG SEQ ID NO: 6: Blunt-ended human IgG4 Fc with a single hinge disulfide (Q274K, Q355R, E419Q and M252Y, S254T, T256E combination mutant) AGCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSQEDPEVKFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSLG SEQ ID NO: 7: H-Aib-QGTFTSDYSKYLDEKKAQEFVEWLLEGGPSSGK(Peg24-BEA-Maldap2)-NH2 SEQ ID NO: 8: H-Aib-QGTFTSDYSKYLDEKKAK(Peg24-BEA Maldap2)EFVEWLLEGGPSSG-NH2 SEQ ID NO: 9: NH-SPPPAGSSPGG-H-RFSPCGLGSYHGIRDIKGGFCSSRGKE-(PEG-G-PAPAPAPAPAPA)-DABA-(Maldab) SEQ ID NO: 10 NH2-SPPPAGSSPGG-H-RFSPCGLGSYHGIRDIKGGFCSSRGKE-(PEG24-DABA-(Maldab)2 SEQ ID NO: 11 OXMP-2 H-Aib-QGTFTSDYSKYLDEKKAQEFVEWLLEGGPSSGK SEQ ID NO: 12 natriuretic peptide EKGRSS C FGGKIDRIGHYSGLG C PSFR-H-GGPSSGAPPPS |-------------------------------| Forms an intramolecular bond with cysteine ​​at position 33

Claims

1. formula: 【Chemistry 1】 A compound of the formula During the ceremony, R １ and R ２ are independently １ ~C ５ alkyl, which are -NH ２ , -CH ２ NH ２ , and -CH ２ CH ２ NH ２ and optionally substituted with one or more of R ３ and R ４ are independently １ ~C ５ alkyl or absent, U is a tertiary amine or a 1,3,5-substituted phenyl; Z is H, OH, NH ２ ,NH,Therapeutic drug,C １ ~C ３０ Alkyl, (OCH ２ CH ２ ) ｍ or a combination thereof, wherein m is an integer from 1 to 30; and pharmaceutically acceptable salts thereof.

2. formula: 【Chemistry 2】 A compound of the formula During the ceremony, R １ and R ２ are independently １ ~C ５ alkyl, which are -NH ２ , -CH ２ NH ２ , or -CH ２ CH ２ NH ２ and optionally substituted with one or more of R ３ and R ４ is C １ ~C ５ is alkyl, Z is H, OH, NH ２ ,NH,Therapeutic drug,C １ ~C ３０ Alkyl, (OCH ２ CH ２ ) ｍ or a combination thereof, wherein m is an integer from 1 to 30; and and pharmaceutically acceptable salts thereof.

3. formula: 【Transformation 3】 A compound of the formula During the ceremony, R １ and R ２ are independently １ ~C ５ straight chain alkyls, which are -NH ２ , -CH ２ NH ２ , or -CH ２ CH ２ NH ２ and optionally substituted with one or more of Z is H, OH, NH ２ ,NH,Therapeutic drug,C １ ~C ３０ Alkyl, (OCH ２ CH ２ ) ｍ or a combination thereof, wherein m is an integer from 1 to 30; and and pharmaceutically acceptable salts thereof.

4. formula: 【Chemistry 4】 A compound of the formula During the ceremony, R １ and R ２ are independently １ ~C ５ alkyl, which are NH ２ , C.H. ２ NH ２ , and C.H. ２ CH ２ NH ２ and optionally substituted with one or more of R ３ and R ４ are independently １ ~C ５ alkyl or absent, U is a tertiary amine or a 1,3,5-substituted phenyl; Y １ and Y ２ independently comprises an amide, an amine, an imine, a sulfonamide, a thiourea, a urea, or a thioether; Z is H, OH, NH ２ ,NH,Therapeutic drug,C １ ~C ３０ Alkyl, (OCH ２ CH ２ ) ｍ or a combination thereof, wherein m is an integer from 1 to 30; and pharmaceutically acceptable salts thereof.

5. formula: 【Transformation 5】 A compound of the formula During the ceremony, R １ and R ２ are independently １ ~C ５ These are straight chain alkyls, and ２ , C.H. ２ NH ２ , and C.H. ２ CH ２ NH ２ and optionally substituted with one or more of R ３ and R ４ are independently １ ~C ５ linear alkyl or absent, U is a tertiary amine or a 1,3,5-substituted phenyl; Z is H, OH, NH ２ ,NH,Therapeutic drug,C １ ~C ３０ Alkyl, (OCH ２ CH ２ ) ｍ or a combination thereof, wherein m is an integer from 1 to 30; Fc comprises an Fc region; 5. The compound of claim 4, wherein S is a sulfur atom from the Fc region.

6. formula: 【Transformation 6】 A compound of the formula During the ceremony, R １ and R ２ are independently １ ~C ５ straight chain alkyls, which are -NH ２ , -CH ２ NH ２ , and -CH ２ CH ２ NH ２ and optionally substituted with one or more of Z is H, OH, NH ２ ,NH,Therapeutic drug,C １ ~C ３０ Alkyl, (OCH ２ CH ２ ) ｍ or a combination thereof, wherein m is an integer from 1 to 30; Fc comprises an Fc region; 5. The compound of claim 4, wherein S is a sulfur atom from the Fc region; and and pharmaceutically acceptable salts thereof.

7. R １ and R ２ But independently, C ２ alkyl, which are -NH ２ , -CH ２ NH ２ , or -CH ２ CH ２ NH ２ 7. The compound of claim 1, or a pharmaceutically acceptable salt thereof, optionally substituted with one or more of:

8. R １ and R ２ The compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, wherein

9. R ３ and R ４ 9. The compound of claim 1, 2, 4, 5, 7 or 8, or a pharmaceutically acceptable salt thereof, wherein:

10. R ３ and R ４ is C ２ 10. The compound of any one of claims 1, 2, 4, 5, or 7 to 9, or a pharmaceutically acceptable salt thereof, wherein:

11. Z is NH, C １ ~C ３０ Alkyl, (OCH ２ CH ２ ) ｍ 11. The compound of any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, comprising:

12. Z is H, OH, NH ２ , therapeutic drug, or C １ ~C ３０ 12. The compound of any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, which is alkyl.

13. The compound of any one of claims 1 to 12, wherein Z comprises a therapeutic agent.

14. 14. The compound of claim 13, or a pharmaceutically acceptable salt thereof, wherein the therapeutic agent comprises an amino acid or a peptide.

15. 15. The compound of claim 14, or a pharmaceutically acceptable salt thereof, wherein the peptide comprises 2 to 100 amino acids.

16. 16. The compound of any one of claims 13 to 15, or a pharmaceutically acceptable salt thereof, wherein the therapeutic agent comprises adrenocorticotropic hormone (ACTH), adropin, amylin, angiotensin, atrial natriuretic peptide (ANP), calcitonin, cholecystokinin (CCK), gastrin, ghrelin, glucagon, growth hormone, follicle-stimulating hormone (FSH), insulin, leptin, luteinizing hormone (LH), melanocyte-stimulating hormone (MSH), natriuretic peptides, oxyntomodulin, oxytocin, parathyroid hormone (PTH), prolactin, renin, somatostatin, thyroid-stimulating hormone (TSH), thyrotropin-releasing hormone (TRH), vasopressin, vasoactive intestinal peptide (VIP), also known as arginine vasopressin (AVP) or antidiuretic hormone (ADH), an analog thereof, or a combination thereof.

17. 17. The compound of any one of claims 13 to 16, or a pharmaceutically acceptable salt thereof, wherein the therapeutic agent comprises oxyntomodulin, a natriuretic peptide, an analog thereof, or a combination thereof.

18. 17. The compound of any one of claims 13 to 16, or a pharmaceutically acceptable salt thereof, wherein the therapeutic agent comprises SEQ ID NO: 11 or SEQ ID NO:

12.

19. 14. The compound of claim 13, or a pharmaceutically acceptable salt thereof, wherein the therapeutic agent comprises a monoclonal antibody, an interleukin, an interferon, a protein kinase inhibitor, a hematopoietic factor, a protein, a fusion protein, an oligonucleotide, or a combination thereof.

20. Y １ and Y ２ The compound of claim 4, wherein said compound comprises an Fc region.

21. 21. The compound of any one of claims 5 to 20, or a pharmaceutically acceptable salt thereof, wherein the compound is linked to a single Fc region.

22. The compound according to any one of claims 5 to 21, or a pharmaceutically acceptable salt thereof, wherein the Fc region comprises the Fc region of a monoclonal antibody.

23. The compound according to any one of claims 5 to 22, or a pharmaceutically acceptable salt thereof, wherein the Fc region comprises at least a portion of IgA, IgD, IgE, IgG, IgM, or a combination thereof.

24. 24. The compound of any one of claims 5 to 23, or a pharmaceutically acceptable salt thereof, wherein the Fc region comprises SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:

6.

25. 25. The compound of claim 24, wherein the Fc region comprises SEQ ID NO:

6.

26. 26. The compound of claim 25, wherein Z comprises SEQ ID NO:11 or SEQ ID NO:12.

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