Novel linkers for sustained delivery of therapeutic agents

Abstract

Linkers for linking a therapeutic agent to other moieties, such as an Fc region of an antibody. A linker having two or more maleimide functional groups, which may be subjected to a nucleophilic conjugate addition reaction. A method for increasing the duration of action of a therapeutic agent by linking a novel linker to the Fc region of an antibody. Antibody drug conjugates having a longer duration of action than the drug alone.

Description

Sequence Listing

[0001] This application is submitted with a sequence listing in ST.26 XML format. The sequence listing is provided as a file named "30359sequencelisting.xml," which was 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. Technical Field

[0002] The present invention relates to novel compounds comprising novel linkers for improving the duration of action of therapeutic agents (e.g., peptides). The present invention also relates to novel conjugate moieties comprising linkers, Fc regions, and optional therapeutic agents. The present invention also relates to novel compounds comprising two or more maleimide functional groups, which can be used to link regions having two or more nucleophiles (e.g., Fc regions of antibodies). Background of the Invention

[0003] Peptide therapeutics, such as insulin, have previously been used to treat diseases like diabetes. However, many peptides can have a very short duration of action. Consequently, patients need to take them every few hours, typically via injection, which can be burdensome and time-consuming for them.

[0004] One strategy to increase the 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 the production process may require exposing the peptide therapeutic to harsh degradation conditions. One way to protect 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 are also unable to use peptide therapeutics containing synthetic amino acids.

[0005] Therefore, there is a need for ADCs that are capable of incorporating unprotected / unmodified peptide therapeutics with or without unnatural amino acids.

[0006] The properties of the linker can affect the reaction conditions that the therapeutic must undergo during ADC production. Thus, there is a need for improved linkers that can be incorporated into unprotected / unmodified peptide therapeutics with or without unnatural amino acids. Detailed Description of the Invention

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

[0008] Surprisingly, it has been discovered that by linking a therapeutic agent to one of the disclosed novel linkers and another moiety (e.g., the Fc region of an antibody), the duration of action of the therapeutic agent can be improved and / or prolonged. While not wishing to be bound by theory, it is believed that linking the therapeutic agent to the linker and the Fc region can increase the duration of action of the therapeutic agent by enlarging the entire moiety and preventing the therapeutic agent from decomposing. The reaction conditions for the linking reaction of the novel linker to the Fc region are relatively mild, so there is no need to modify the payload or therapeutic agent to protect it during the reaction.

[0009] It has also been unexpectedly discovered that the disclosed novel linkers can produce ADCs that can incorporate unprotected / unmodified peptide therapeutics with or without unnatural amino acids. Formula IA.

[0010] Disclosed herein are novel conjugated compounds of Formula IA, wherein Fc comprises an Fc region, such as an antibody Fc region, L comprises a linker, and Z comprises a therapeutic agent, a functional group suitable for subsequent attachment to a therapeutic agent, or a functional group suitable for allowing the conjugated compound to exist without an attached therapeutic agent. Formula IB.

[0011] Also disclosed herein are novel conjugated compounds of Formula IB, wherein Fc comprises one or more Fc regions, L comprises a linker, and Z comprises a therapeutic agent, a functional group suitable for subsequent attachment to a therapeutic agent, or a functional group suitable for allowing the conjugated compound to exist without an attached therapeutic agent. Type IC.

[0012] Also disclosed herein are novel conjugated compounds of Formula IC, wherein Fc comprises a single Fc region, such as an Fc region of an antibody, L comprises a linker, and Z comprises a therapeutic agent, a functional group suitable for subsequent attachment to a therapeutic agent, or a functional group suitable for allowing the conjugated compound to exist without an attached therapeutic agent. Formula II-A.

[0013] Also disclosed herein are novel compounds of formula II-A, wherein R1 and R2 are independently C1 to C5 alkyl, which are optionally substituted with one or more -NH2, -CH2NH2 and -CH2CH2NH2; R3 and R4 are independently C1 to C5 alkyl or absent; U is a tertiary amine or a 1,3,5-substituted phenyl; Z comprises H, OH, NH2, NH, a therapeutic agent, a C1 to C 30 Alkyl, (OCH2CH2) mor a combination thereof, and wherein m is an integer from 1 to 30; and pharmaceutically acceptable salts thereof. Formula II-B.

[0014] Also disclosed herein are novel compounds of formula II-B, wherein R1 and R2 are independently C1 to C5 alkyl, which are optionally substituted with one or more -NH2, -CH2NH2 or -CH2CH2NH2; R3 and R4 are independently C1 to C5 alkyl; Z comprises H, OH, NH2, NH, a therapeutic agent, C1 to C 30 Alkyl, (OCH2CH2) m or a combination thereof, and wherein m is an integer from 1 to 30; and pharmaceutically acceptable salts thereof. Formula II-C.

[0015] Also disclosed herein are novel compounds of formula II-C, wherein R1 and R2 are independently C1 to C5 alkyl, which are optionally substituted with one or more -NH2, -CH2NH2 or -CH2CH2NH2; Z comprises H, OH, NH2, NH, a therapeutic agent, C1 to C 30 Alkyl, (OCH2CH2) m or a combination thereof, and wherein m is an integer from 1 to 30; and pharmaceutically acceptable salts thereof. Formula III-A.

[0016] Also disclosed herein are novel compounds of formula III-A, wherein R1 and R2 are independently C1 to C5 alkyl, which are optionally substituted with one or more NH2, CH2NH2 and CH2CH2NH2; R3 and R4 are independently C1 to 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; and Z comprises H, OH, NH2, NH, a therapeutic agent, a C1 to C 30 Alkyl, (OCH2CH2) m or a combination thereof, and wherein m is an integer from 1 to 30; and pharmaceutically acceptable salts thereof. Formula III-B.

[0017] Also disclosed herein are novel compounds of formula III-B, wherein R1 and R2 are independently C1 to C5 alkyl, which are optionally substituted with one or more NH2, CH2NH2 and CH2CH2NH2; R3 and R4 are independently C1 to C5 alkyl or absent; U is a tertiary amine or a 1,3,5-substituted phenyl; Z comprises H, OH, NH2, NH, a therapeutic agent, a C1 to C 30 Alkyl, (OCH2CH2) m or a combination thereof, and wherein m is an integer from 1 to 30; Fc comprises an Fc region; S is a sulfur atom from the Fc region; and pharmaceutically acceptable salts thereof. Formula III-C.

[0018] Also disclosed herein are novel compounds of formula III-C, wherein R1 and R2 are independently C1 to C5 alkyl, which are optionally substituted with one or more -NH2, -CH2NH2 and -CH2CH2NH2; R3 and R4 are independently C1 to C5 alkyl or absent; Z comprises H, OH, NH2, NH, a therapeutic agent, C1 to C 30 Alkyl, (OCH2CH2) m or a combination thereof, and wherein m is an integer from 1 to 30; Fc comprises an Fc region; S is a sulfur atom from the Fc region; and pharmaceutically acceptable salts thereof. Formula III-D.

[0019] Also disclosed herein are novel compounds of formula III-D, wherein R1 and R2 are independently C1 to C5 alkyl, which are optionally substituted with one or more -NH2, -CH2NH2, and -CH2CH2NH2; Z comprises H, OH, NH2, NH, a therapeutic agent, C1 to C 30 Alkyl, (OCH2CH2) m or a combination thereof, and wherein m is an integer from 1 to 30; Fc comprises an Fc region; S is a sulfur atom from the Fc region; and pharmaceutically acceptable salts thereof. Formula IV-A.

[0020] Also disclosed herein are novel compounds of formula IV-A, wherein: R1 and R2 are independently C1 to C5 alkyl, which are optionally substituted with one or more -NH2, -CH2NH2 and -CH2CH2NH2; R3 and R4 are independently C1 to C5 alkyl or absent; U is a tertiary amine or a 1,3,5-substituted phenyl; Z comprises H, OH, NH2, NH, a therapeutic agent, a C1 to C 30 Alkyl, (OCH2CH2) mor a combination thereof, and wherein m is an integer from 1 to 30; and pharmaceutically acceptable salts thereof. Formula IV-B.

[0021] Also disclosed herein are novel compounds of Formula IV-B, wherein: R1 and R2 are independently C1 to C5 alkyl, which are optionally substituted with one or more -NH2, -CH2NH2, or -CH2CH2NH2; R3 and R4 are C1 to C5 alkyl; and Z comprises H, OH, NH2, NH, a therapeutic agent, a C1 to C 30 Alkyl, (OCH2CH2) m or a combination thereof, and wherein m is an integer from 1 to 30; and pharmaceutically acceptable salts thereof. Formula IV-C.

[0022] Also disclosed herein are novel compounds of formula IV-C, wherein R1 and R2 are independently C1 to C5 alkyl, which are optionally substituted with one or more -NH2, -CH2NH2 or -CH2CH2NH2; Z comprises H, OH, NH2, NH, a therapeutic agent, C1 to C 30 Alkyl, (OCH2CH2) m or a combination thereof, and wherein m is an integer from 1 to 30; and pharmaceutically acceptable salts thereof. Linker

[0023] The present invention includes linkers L that link 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 a combination thereof (as shown in Formulas III-A, III-B and / or III-C) via a nucleophilic conjugate addition reaction.

[0024] The linker can include a central trivalent linking unit U, as shown in Formula II-A, III-A, and IV-A. One portion of the linker can be connected to Z. The other two portions of the linker contain maleimide functional groups, i.e., electrophilic groups, which can be used to selectively connect Z to two nucleophilic groups in a nucleophilic conjugate addition reaction.

[0025] Thus, as disclosed herein, prior to the nucleophilic conjugate addition reaction, the linker of the present invention comprises two or more maleimide functional groups, preferably two maleimide functional groups.

[0026] Suitable trivalent linking units may include any atom or molecule that can be trisubstituted, such as a trisubstituted C5 to C8 aryl group, a trisubstituted C5 to C8 heteroaryl group containing 1 to 3 heteroatoms in the ring system, a trisubstituted C5 to C8 cycloalkyl group, a trisubstituted C5 to C8 heterocycle containing 1 to 3 heteroatoms in the ring system, a tertiary amine, a tertiary phosphine, or a combination thereof. Preferably, U is a 1,3,5-trisubstituted phenyl group or a tertiary amine.

[0027] Each arm connected to U and the maleimide functional group can be represented by formula V. Formula V.

[0028] In the first of the two arms represented by Formula V, R1 can be a C1 to C5 alkyl group, which is optionally substituted with one or more of -NH2, -CH2NH2, and -CH2CH2NH2, and R3 can be a C1 to C5 alkyl group or not present. In the second of the two arms represented by Formula V, R2 can be a C1 to C5 alkyl group, which is optionally substituted with one or more of -NH2, -CH2NH2, and -CH2CH2NH2; and R4 can be a C1 to C5 alkyl group or not present.

[0029] Preferably, R1 and / or R2 can be C2 alkyl, which is optionally substituted by one or more -NH2, -CH2NH2 or -CH2CH2NH2. Preferably, when U is a tertiary amine and is absent, when U is a 1,3,5-trisubstituted phenyl, R3 and / or R4 can be C2 alkyl.

[0030] When both arms are connected to U, R1 and R2 can be the same, or they can independently be C1 to C5 alkyl, which is optionally substituted with one or more -NH2, -CH2NH2 and -CH2CH2NH2. When both arms are connected to U, R3 and R4 can be the same, or they can independently be C1 to C5 alkyl or absent. When R3 and / or R4 are absent, the amide nitrogen in Formula V is directly connected to U, for example, when U is a 1,3,5-trisubstituted phenyl, as shown in Formula IV-C.

[0031] The third of the three arms connected to U includes Z. Z may include H, OH, NH2, NH, therapeutic agents, C1 to C 30 Alkyl, (OCH2CH2) m or a combination thereof, and wherein m is an integer from 1 to 30. Z can be directly connected to U, or Z can be connected to the carbonyl functional group. The carbon atom in the carbonyl functional group can be directly connected to U and Z, as shown in Formula II-IV.

[0032] Thus, disclosed herein are novel linkers, as shown in Formula II-A, Formula II-B, and Formula II-C, which contain two maleimide functional groups prior to the nucleophilic conjugate addition reaction. Nucleophilic conjugate addition reaction with maleimide functional groups

[0033] As disclosed herein, a linker L can be attached to Z to obtain a linked payload, or if Z comprises a therapeutic agent, a linked therapeutic agent. The linkers disclosed herein comprise two or more, or preferably two, maleimide functional groups, as shown in Formula V. The maleimide functional group can be used to attach Z to a different moiety, such as an Fc region, via a nucleophilic conjugate addition reaction.

[0034] In the nucleophilic conjugate addition reaction, maleimide acts as an electrophile and reacts with the nucleophile to form a new covalent bond between the nucleophilic atom and the electrophilic carbon atom, which results in the opening of the maleimide functional group, as shown in Formula VI. Formula VI.

[0035] Various nucleophiles can be used for reacting with the electrophilic maleimide functional group.Suitable nucleophiles are included in the part that comprises one or more nitrogen and / or sulphur atoms in the functional group, wherein nitrogen and / or sulphur atoms can be used as electron-rich nucleophiles.Suitable examples of nucleophiles include the part that comprises following one or more functional groups: amides, amine, imines, sulfonamide, thiol, thiourea, urea or its combination, it is as shown in Formula VII. Formula VII.

[0036] In particular, the amino acid cysteine ​​(as shown in Formula VIII in the peptide chain), which is present in many amino acid sequences of peptides, antibodies, proteins, etc., includes a thiol functionality that can serve as a nucleophile to form a thioether between the amino acid sequence and the linker, attached payload, and / or attached therapeutic agent. Formula VIII.

[0037] The specific conditions for coupling nucleophilic conjugate addition reactions are well known to those of ordinary skill in the art. The specific conditions can vary depending on the nucleophilic and electrophilic groups used. Therefore, disclosed herein are novel compounds and / or coupled compounds comprising a connected payload, which is connected 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.

[0038] Furthermore, the use of the novel linkers disclosed herein may be advantageous because the reaction conditions are mild relative to other nucleophilic conjugate addition reactions disclosed using other linkers. For example, the disclosed linkers can be conjugated to the Fc region of an antibody using minimal 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 a temperature not exceeding 30°C or not exceeding 25°C for not more than 2 hours to produce a conjugate.

[0039] Because the conjugation reaction conditions are relatively mild, there's no need to modify the payload or therapeutic agent during the reaction to protect it. Other linkers require the use of recombinant extensions to modify peptide therapeutics, such as insulin. The disclosed conjugated compounds remain effective and have a longer-lasting effect, without the need for modifying the therapeutic agent. Fc region

[0040] In some embodiments, the nucleophilic group capable of reacting with two or more maleimide functional groups on the linker is an Fc region. The Fc region may also be referred to as a fragment crystallizable region. The Fc region is the tail region of an antibody that interacts with certain cell surface receptors called Fc receptors. The Fc region may include a portion of a peptide, protein, or antibody, or a complete monoclonal antibody capable of reacting with Fc receptors. 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.

[0041] In some embodiments, the Fc region of the antibody can be modified prior to being linked to the linker via a nucleophilic conjugate addition reaction. Suitable modifications may include modifications to the antibody to minimize reactivity between the antibody and the linker other than the desired nucleophilic conjugate addition reaction, as described elsewhere herein.

[0042] In some embodiments, the Fc region can be that of a modified antibody. Suitable modifications include blunting and / or removing certain portions of the Fc region, such as the hinge region.

[0043] The disulfide bonds present in the appropriate Fc region can be reduced, and the two reduced sulfhydryl groups can be suitable nucleophilic groups, which can undergo nucleophilic conjugate addition reactions with the two maleimide functional groups of the linker of Formula II-A, Formula II-B and / or Formula II-C to produce compounds of Formula IV-A, Formula IV-B and / or Formula IV-C. therapeutic agents

[0044] In some embodiments, Z may comprise a therapeutic agent. A therapeutic agent is a drug or active agent that, if administered to a patient suffering from the disease of interest, can treat the disease of interest.

[0045] Suitable therapeutic agents may comprise one or more amino acids or peptides having from 2 to about 500 amino acids, from 2 to about 250 amino acids, or from 2 to about 100 amino acids.

[0046] 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), vasopressor hormones, also known as arginine vasopressor hormone (AVP) or antidiuretic hormone (ADH), vasoactive intestinal peptide (VIP), combinations thereof, and / or analogs thereof. In some embodiments, the therapeutic agent can be insulin, oxyntomodulin, and / or analogs thereof.

[0047] Other suitable therapeutic agents include monoclonal antibodies, interleukins, interferons, protein kinase inhibitors, hematopoietic growth factors, proteins, fusion proteins, oligonucleotides, or combinations thereof. Conjugate

[0048] In some embodiments, when Z comprises a therapeutic agent and the linker is coupled to the Fc region of at least a portion of an antibody, the novel compound can be a conjugate and / or antibody drug conjugate (ADC). As described herein, it was surprisingly found that when a therapeutic agent (e.g., oxyntomodulin) is linked to the Fc region of an antibody via the novel linkers disclosed herein, the therapeutic agent has an improved duration of action relative to the therapeutic agent alone. Treatment

[0049] Also disclosed herein are methods for increasing the duration of action of a therapeutic agent. The method can include the step of linking the therapeutic agent to a trivalent linker to produce a linker therapeutic agent, such as a compound of Formula II-A, Formula II-B, and / or Formula II-C, wherein Z comprises a therapeutic agent.

[0050] The method may further comprise the step of linking the linked therapeutic agent to the Fc region of at least a portion of an antibody (e.g., IgG) by a nucleophilic conjugate addition reaction between the linked therapeutic agent and two nucleophilic groups (e.g., two sulfur atoms and / or anions) of the Fc region to produce a conjugated compound.

[0051] The conjugated compound may comprise a therapeutic agent linked to a novel linker disclosed herein, which is in turn linked to the Fc region of at least a portion of an antibody. The duration of action of the conjugated compound may be longer than that of the unbound therapeutic agent. The duration of action of the conjugated compound may be at least 12 hours, 1 day, 2 days, or 3 days longer than that of the therapeutic agent.

[0052] Furthermore, the use of the novel linkers disclosed herein may be advantageous because the reaction conditions are mild relative to other nucleophilic conjugate addition reactions disclosed using other linkers. For example, the disclosed linkers can be conjugated to the Fc region of an antibody using minimal 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 a temperature not exceeding 30°C or not exceeding 25°C for not more than 2 hours to produce a conjugate. preparation

[0053] 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 compound and other components of the composition or preparation and are harmless to the patient. Examples of pharmaceutical compositions and their preparation processes can be found in: "Remington: The Science and Practice of Pharmacy", edited by Loyd, V et al., 22nd edition, Mack Publishing Company, 2012. definition

[0054] As used herein, the terms “a,” “an,” and “the,” and similar referents used in the context of the disclosure (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0055] The term "alkyl" as used herein refers to a saturated straight or branched monovalent or divalent hydrocarbon radical containing the specified number of carbon atoms. For example, "C1-C 20 "Alkyl" refers to a group having 1 to 20 carbon atoms arranged in a straight or branched chain.

[0056] 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-butyloxycarbonyl; “DABA” refers to 3,5-diaminobenzoic acid; “DCM” refers to dichloromethane; “DIC” refers to diisopropylcarbodiimide; “DIEA” refers to diisopropylethylamine; “DMAP” refers to 4-dimethylaminopyridine; “EtOAc” refers to ethyl acetate; “Fmoc” refers to fluorenylmethoxycarbonyl; “hr / hrs” refers to hours; “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; "Maldap" refers to (S)-3-((tert-Butoxycarbonyl)amino)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoic acid; Beta-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; "MTT" refers to methylthialazole tetrazolium “OXM” refers to oxyntomodulin; “Oxyma” refers to ethyl cyanohydroxyimidoacetate; “PEG” refers to polyethylene glycol; “PyBOP” refers to benzotriazol-1-yl-oxy-tripyrrolidinium hexafluorophosphate; “t-Bu” refers to tert-butyl; “TCEP” refers to tris(2-carboxyethyl)phosphine hydrochloride; and “THF” refers to tetrahydrofuran. Preparation Example Preparation Example 1 (S)-2-((tert-Butoxycarbonyl)amino)-4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoic acid

[0057] 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 performed until a homogeneous mixture was obtained, followed by the addition of methyl 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate (370 mg, 2.31 mmol). Mixing was carried out at ambient temperature for 1 hour. Diluted with 50 mL of water, the pH was adjusted to ~6 by adding 5N HCl. The aqueous solution was extracted with EtOAc (30 mL) and chloroform / isopropanol (3 x 30 mL). The organic phases were combined, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography, eluting with 0-30% MeOH in DCM to give the title compound (580 mg, 81%). ES / MS m / z: 199 (M- tBu). Preparation Example 2 (S)-2-((tert-Butoxycarbonyl)amino)-4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoate, 2,5-dioxopyrrolidin-1-yl

[0058] 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). The mixture was stirred at ambient temperature for 3 hrs. The solids were removed by filtration, and the filtrate was concentrated under reduced pressure to give the title compound (605 mg, 84%), which was used without purification. ES / MS m / z: 296 (M-tBu). Preparation Example 3 (S)-2-((tert-Butoxycarbonyl)amino)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoic acid

[0059] Combine (2S)-3-amino-2-(tert-butoxycarbonylamino)propanoic acid (5.00 g, 24.5 mmol) with a 1M aqueous solution of sodium bicarbonate (130 mL, 130 mmol). Stir at ambient temperature for 10 minutes until a clear solution forms. Cool the solution in an ice-water bath, and add methyl 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate (4.00 g, 25.0 mmol) in three portions over 15 minutes. Stirring is continued at 0°C for 3 hours. Add 100 mL of EtOAc and stir while cooling, then add concentrated HCl to adjust the pH to 1. Separate the layers and extract the aqueous solution with DCM (4 x 50 mL). Combine the organic layers, wash with a saturated aqueous NaCl solution, and dry over sodium sulfate. Remove the solvent under reduced pressure. Purify via silica gel column chromatography eluting with 0-40% MeOH in DCM to give the title compound (5.70 g, 66%). ES / MS m / z: 185 (M-tBu). Preparation Example 4 (S)-2,5-dioxopyrrolidin-1-yl 2-((tert-Butoxycarbonyl)amino)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate

[0060] 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 carried out 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). Preparation Example 5 4-(Bis(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)amino)-4-oxobutanoic acid

[0061] A solution of N-(9-fluorenylmethoxycarbonyloxy)succinimide (19.4 g, 56.4 mmol) in DCM (80 mL) was added over 45 minutes to a solution of diethylenetriamine (3.1 mL, 28 mmol) in DCM (30 mL) at -78°C. After 2 hours, 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 hours. The pH was adjusted to 5 by the slow addition of 1N HCl (20 mL). The phases were separated, and the aqueous solution was extracted with DCM (2 x 100 mL). The organic layers were combined and 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 afford the title compound (911.84 g, 65%) as a white solid. ES / MS m / z: 648 (M+1). Example Solution 1 Universal synthesis of conjugates including Fc region, linker and peptide

[0062] A summary of the general synthesis of linker-peptides is provided in Scheme 1. In each case, in reaction (1), a trivalent core of an Fmoc-protected (protected on two of the three available arms) 1,3,5-trisubstituted phenyl (commercially available) or tertiary amine (Preparation 5) is initially reacted with the peptide on the resin. In reaction (2), the Fmoc group is removed. In reaction (3), the primary amine function resulting from removal of the Fmoc protecting group is reacted with an NHS ester, such as MalDap (commercially available), MapDab (Preparation 2), or β-MalDap (Preparation 4), to release the peptide from the resin. In reaction (4), the bismaleimide function reacts with two reduced thiol functions in the Fc region in a nucleophilic conjugate addition reaction to provide a conjugate comprising the Fc region, the linker, and the therapeutic agent.

[0063] Linker-peptides were generated by solid-phase peptide synthesis using an Fmoc / t-Bu strategy on a SymphonyX automated peptide synthesizer (PTI Protein Technologies Inc.) starting from RAPP AM-Rink amide resin (H40023 polystyrene AM RAM, Rapp polymere GmbH). Amino acid coupling was performed using 5 equivalents of amino acid, 1.2 M DIC, and 0.9 M Oxyma in DMF for 2 hours at 25°C. Deprotection was performed using 25% piperidine in DMF.

[0064] The N-terminal amino acid was a Boc-protected proline to allow Fmoc chemistry to proceed at the C-terminal Kε-amine. The MTT protecting group present in the C-terminal K was removed using 30% hexafluoroisopropanol (HFIP) in DCM. Additional coupling / deprotection cycles to extend the side chain using an Fmoc / t-Bu strategy involved Fmoc-Peg24-OH (Broadpharm, catalog number BP-22036), an Fmoc-protected BEA (bisethylamine) aliphatic linker, and Boc-Maldap (CAS number 1491152-23-8). For the 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. BEA was coupled using 3 equivalents of BEA, 3 equivalents of PyBop, and 6 equivalents of DIEA at 37°C for 3 hours. Boc-Maldap was coupled to each free amine of BEA using 5 equivalents of Boc-Maldap, 0.9 M Oxyma, and 1.2 M DIC in DMF at 25°C for 6 hours. PEG and BEA were deprotected using 25% piperidine in DMF. Cleavage from the resin and removal of side-chain protecting groups were performed simultaneously in a solution containing trifluoroacetic acid (TFA): triisopropylsilane: MilliQ H2O: thioanisole 85:5:5:5 (v / v) at 25°C for 2 hours, followed by precipitation with cold ether. The crude peptide was purified by reverse-phase HPLC on a Waters Symmetry preparative C18 column (19 x 250 mm 100A, 5 μm), and appropriate fractions were pooled and lyophilized.

[0065] After the linker-therapeutic agent is cleaved from the resin, it can be reacted with the reduced Fc region to form a conjugate comprising the Fc region, the linker, and the therapeutic agent. A summary of possible conjugates is provided in Table 1.

[0066] The following linkers were synthesized by the method described above: Table 1. Conjugates

[0067] SEQ ID NO. 7: H-Aib-QGTFTSDYSKYLDEKKAQEFVEWLLEGGPSSGK(Peg24-BEA-Maldap2)-NH2

[0068] In this sequence, the N-terminus is free, while the C-terminus is amidated as a primary amide. The C-terminal K was chemically modified by coupling Peg24-BEA-Maldap2 to the ε-amino group of the K side chain. It was synthesized using the general synthesis described above, where the amine is MalDap, the trivalent core is a tertiary amine, and the bridge between the peptide and the linker is PEG24.

[0069] SEQ ID NO. 8 : H-Aib-QGTFTSDYSKYLDEKKAK(Peg24-BEA-Maldap2)EFVEWLLEGPSSG-NH2

[0070] In this sequence, the N-terminus is free, while the C-terminus is amidated as a primary amide. K at position 20 was chemically modified by coupling Peg24-BEA-Maldap2 to the ε-amino group of the K side chain. It was synthesized using the general synthesis described above, where the amine is MalDap, the trivalent core is a tertiary amine, and the bridge between the peptide and the linker is PEG24.

[0071] SEQ ID NO 9: NH2-SPPPAGSPGG-H-RFSPCGLGSYHGIRDIKGGFCSSRGKE-(PEG12-G-PAPAPAPAPAPA)-DABA-(Maldab)2

[0072] SEQ ID NO 9 was prepared using a general synthesis similar to sequences 7, 8 and 9, wherein the amine is MalDab, the trivalent core is 1,3,5-phenyl, and the bridge between the peptide and the linker is PEG12.

[0073] SEQ ID NO 10: NH2-SPPPAGSPGG-H-RFSPCGLGSYHGIRDIKGGFCSSRGKE-(PEG24-DABA-(Maldab)2

[0074] SEQ ID NO 10 was prepared using a general synthesis similar to sequences 7, 8 and 9, wherein the amine is MalDab, the trivalent core is 1,3,5-phenyl, and the bridge between the peptide and the linker is PEG24.

[0075] The peptides of SEQ ID NOs 7-12 were generated by solid-phase peptide synthesis using an Fmoc / t-Bu strategy on a SymphonyX automated peptide synthesizer (PTI Protein Technologies Inc.) starting from RAPP AM-Rink amide resin (H40023 polystyrene AM RAM, Rapp polymere GmbH). Amino acid coupling was performed using 5 equivalents of amino acid, 1.2 M DIC, and 0.9 M Oxyma in DMF for 2 hours at 25°C. Deprotection was performed using 25% piperidine in DMF.

[0076] The N-terminal amino acid is a proline protected by Boc, to allow Fmoc chemical reaction to proceed at Kε-amine. The MTT protecting group present in the K at position 20 is removed using a DCM solution of 30% hexafluoroisopropanol (HFIP). Fmoc-Peg24-OH (Broadpharm, catalog number (Cat. No.) BP-22036) is involved in the other coupling / deprotection cycle of the extended side chain, Fmoc-Peg24-OH, BEA (bisethylamine) aliphatic linker and Boc-Maldap (CAS No. 1491152-23-8) protected by Fmoc are involved in the Peg coupling. For the Peg coupling, 2 equivalents of Fmoc-Peg24-OH, 2 equivalents of PyBOP and 4 equivalents of DIEA are coupled at 37°C for 2 hours. Using 3 equivalents of BEA and 3 equivalents of PyBop and 6 equivalents of DIEA, BEA is coupled at 37°C for 3 hours. Boc-Maldap was coupled to each free amine 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. PEG and BEA were deprotected using 25% piperidine in DMF. IgG4 Fc region

[0077] Human IgG4 Fc regions were prepared and purified using known methods. Chinese hamster ovary (CHO) cells were transiently or stably transfected with a single vector encoding the inactivated Fc heavy chain nucleotide sequence using an Fc secretory expression system. Clarified culture medium from which the inactivated Fc was secreted was purified using conventional techniques known to those of ordinary skill in the art.

[0078] The IgG4 Fc region includes a human IgG4 Fc region with an inactivated hinge. To improve expression and minimize the potential for unstructured peptides that could interfere with the conjugation process, a very short dipeptide consisting of alanine and glycine was incorporated upstream of cysteine ​​229 (EU index numbering). The traditional human IgG4 hinge (SEQ ID NO 1) was inactivated by removing the cysteine ​​at position 226 (EU index numbering) that participates in the upper hinge disulfide bond and replacing it with a shortened hinge consisting of an alanine-glycine N-terminal dipeptide (SEQ ID NO 2) just before the single disulfide bond from the native cysteine ​​at position 229. Removal of the upper hinge disulfide bond simplifies the conjugation process by limiting the available cysteine ​​thiols that could react with maleimide, eliminating potential mixed conjugation species. The complete human IgG4 Fc containing the inactivated hinge with a single disulfide bond is shown in SEQ ID NO 3.

[0079] Two alternative human IgG4 inactivated Fc variants 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, including Q274K, Q355R, and E419Q (EU index numbering) (SEQ ID NO 4) mutations to potentially improve the half-life of the conjugate, including M252Y, S254T, and T256E, which are known to enhance neonatal Fc receptor (FcRn) binding at low pH, leading to higher levels of antibody recycling (SEQ ID NO 5). SEQ ID NO 6 is a combination of the two mutations Q274K, Q355R, E419Q and M252Y, S254T, T256E. Linker - connection of peptide to Fc region

[0080] The disulfide bonds in the Fc hinge were reduced by adding 2 equivalents of TCEP prepared in PBS to the 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 × 100 mm) and analyzed using MassHunter software. The complete reduction of the ~52 kD peak to a ~26 kD peak indicates Fc rebridging.

[0081] The re-bridging reaction was completed by diluting the reduced Fc fivefold 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 immediately added to the buffered Fc solution. The reaction was monitored by LCMS-TOF. As each peptide maleimide was re-bridged with the reduced cysteine, the ~26 kD peak shifted to a ~58 kD peak.

[0082] The Fc peptide rebridged conjugate was purified in two steps using AKTA FPLC. First, in the two-step purification, the reaction was loaded onto a HiTrap TM MabSelect Xtra TM Protein A column (Cytiva). The reaction mixture was loaded into a system balanced in PBS pH 7.2 to bind the Fc-containing product and remove the peptide. After the peptide was removed, the Fc-containing material was collected by changing the mobile phase to 20mM citrate pH 3, thereby disrupting the interaction between the Fc and the column. 1M T8 was added to increase the pH value of the fraction during elution, and the buffer was exchanged to 20mM Tris pH 8 by centrifugation using an amicon-ultra vial with a 10,000mW cutoff.

[0083] If necessary, a second purification step is performed to remove unconjugated Fc components by strong anion exchange using Mono-Q 5 / 50 GL (Cytiva). The sample is loaded with 20 mM T8 and eluted with an increasing gradient of 20 mM T8 + 1 M NaCl. Fractions containing the desired material are analyzed by LCMS-TOF and then buffer exchanged again to remove NaCl, resulting in a final solution of 20 mM T8. Example 1: OXMP-2Fc

[0084] Example 1 was synthesized using the above-described method of linking a peptide to a linker and then coupling the peptide-linker to an Fc region. Example 1 is a conjugate comprising a peptide of SEQ ID NO 11 and an Fc region of SEQ ID NO 6. The linker used to link SEQ ID NO 11 to SEQ ID NO 6 is Example D in Table 1. Example 2: Natriuretic Peptides

[0085] Using the above method of linking the peptide to the linker and then coupling the peptide-linker to the Fc region, Example 2 was synthesized. Example 2 is a conjugate comprising the peptide-linker of SEQ ID NO 10 and the Fc region of SEQ ID NO 6. Example 3: Natriuretic Peptides

[0086] Using the above method of linking the peptide to the linker and then coupling the peptide-linker to the Fc region, Example 3 was synthesized. Example 3 is a conjugate comprising the peptide-linker of SEQ ID NO 9 linked to the Fc region of SEQ ID NO 6. Example 4

[0087] Example 4 was synthesized using the above-described method of linking a peptide to a linker and then coupling the peptide-linker to an Fc region. Example 4 is a conjugate comprising a peptide of SEQ ID NO 12 and an Fc region of SEQ ID NO 6. The linker used to link SEQ ID NO 12 to SEQ ID NO 6 is Example E in Table 1. Example 5

[0088] Example 5 was synthesized using the above-described method of linking a peptide to a linker and then coupling the peptide-linker to an Fc region. Example 3 is a conjugate comprising a peptide of SEQ ID NO 12 and an Fc region of SEQ ID NO 6. The linker used to link SEQ ID NO 12 to SEQ ID NO 6 is Example F in Table 1. Hydrolysis of maleimide functional groups

[0089] When pH ≥ 8, maleimide hydrolysis occurs on these constructs, resulting in maleimide ring opening and an 18 Dalton mass increase. The rebridged constructs each have 2 maleimides with a total mass shift of 36 Daltons. Ring opening (hydrolysis) was tracked by mass using LCMS-TOF. Autohydrolysis of the linker

[0090] The autohydrolysis of the linker was assessed by incubating the linker in triphosphate buffer with the corresponding pH 5.0, 6.0, 7.0, and 8.0. Lyophilized linker powder was dissolved in 100% DMSO to make an 80 mM stock solution. Each linker was incubated in the buffer at a final linker concentration of 1.5 mM at room temperature for 1 hour to determine the hydrolysis of succinimide. The effect of pH on linker autohydrolysis was assessed at specific time intervals by reverse phase HPLC mass spectrometry. Tables 2A and 2B show the effect of pH on specific conjugates.

[0091] Surprisingly, Tables 2A and 2B show that the linker comprising β Maldap is more stable under higher pH conditions. Example 4 includes a conjugate comprising 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 includes a conjugate comprising 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. Table 2A. pH Stability of Example 4 Table 2B. pH Stability of Example 5 Duration of action of the therapeutic agent

[0092] Prior to the start of the study, DIO mice were weighed and given a fresh TD95217 diet and acclimated to handling and daily weighing and food intake. Animals were randomized by body weight in block order. For acute studies, each peptide / conjugate shown in Table 3 was injected once at a dose of 10 nmol / kg. Daily food intake and body weight were tracked 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) was injected once weekly for 4 weeks, and food intake and body weight were tracked until 3 weeks after the last injection. QNMR was measured every two weeks.

[0093] Table 3 shows the effects of coupling a therapeutic agent to an Fc region via a disclosed linker. Two groups of randomized DIO mice were injected with two different methods. The first group received a control injection containing no therapeutic agent. The second group received an injection of an oxyntomodulin analog (Example 1) linked to a deactivated Fc region of IgG4.

[0094] Daily food intake of control-dosed DIO mice did not change over time (2.7-3.1 g food per day).

[0095] Surprisingly, the daily food intake of DIO mice injected with Example 1 (oxyntomodulin analog linked to the Fc region of IgG4 via a linker) dropped to 0.94 g on day 2 and remained at 1 g or less until day 5. By day 7, the food intake of DIO mice injected with Example 1 had returned to a level comparable to that of mice injected with the control group.

[0096] It is noteworthy that it is well known to those skilled in the art that the equivalent oxyntomodulin analogs of Example 1 that are not connected to the Fc region and / or the disclosed linker have almost no extended duration of action except for the initial effect that occurs immediately after administration. For example, see 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 "endogenous peptide hormones have very short half-lives and are therefore not suitable for use as therapeutic agents." Therefore, although not wishing to be bound by theory, it is believed that connecting the oxyntomodulin analog to the Fc region via the disclosed linker can extend the duration of action of the oxyntomodulin analog by nearly 7 days.

[0097] Thus, conjugation to an Fc region via one of the disclosed linkers can increase the duration of action of a therapeutic agent by one week. Table 3. Effect of coupling to the Fc region via a linker sequence SEQ ID NO 1: ESKYGPPCPSCP SEQ ID NO 2: AGCP SEQ ID NO 3: Inactivated human IgG4 Fc with single hinge disulfide bond AGCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG SEQ ID NO 4: Inactivated human IgG4 Fc with single hinge disulfide bonds (Q274K, Q355R and E419Q mutations) AGCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVKFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSLG SEQ ID NO 5: Inactivated human IgG4 Fc with single hinge disulfide bonds (M252Y, S254T and T256E mutations) AGCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG SEQ ID NO 6: Human IgG4 Fc with inactivated single hinge disulfide bonds (Q274K, Q355R, E419Q and M252Y, S254T, T256E combined mutations) AGCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSQEDPEVKFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSLG 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: NH2-SPPPAGSPGG-H-RFSPCGLGSYHGIRDIKGGFCSSRGKE-(PEG12-G-PAPAPAPAPAPA)-DABA-(Maldab)2 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. A compound of the formula: , in: R1 and R2 are independently C1 to C5 alkyl, optionally substituted with one or more of -NH2, -CH2NH2 and -CH2CH2NH2; R3 and R4 are independently C1 to C5 alkyl or absent; U is a tertiary amine or a 1,3,5-substituted phenyl group; Z contains H, OH, NH2, NH, therapeutic agent, C1 to C 30 Alkyl, (OCH2CH2) m or a combination thereof, and wherein m is an integer from 1 to 30; and or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, wherein the compound is of the formula: ， in: R1 and R2 are independently C1 to C5 alkyl, optionally substituted with one or more -NH2, -CH2NH2 or -CH2CH2NH2; R3 and R4 are C1 to C5 alkyl; Z contains H, OH, NH2, NH, therapeutic agent, C1 to C 30 Alkyl, (OCH2CH2) m or a combination thereof, and wherein m is an integer from 1 to 30; and or a pharmaceutically acceptable salt thereof.

3. The compound of claim 1, wherein the compound is of the formula: ， in: R1 and R2 are independently C1 to C5 straight chain alkyl, which is optionally substituted with one or more -NH2, -CH2NH2 or -CH2CH2NH2; Z contains H, OH, NH2, NH, therapeutic agent, C1 to C 30 Alkyl, (OCH2CH2) m or a combination thereof, and wherein m is an integer from 1 to 30; and or a pharmaceutically acceptable salt thereof.

4. Compounds of the formula: ， in: R1 and R2 are independently C1 to C5 alkyl, optionally substituted with one or more NH2, CH2NH2 and CH2CH2NH2; R3 and R4 are independently C1 to C5 alkyl or absent; U is a tertiary amine or a 1,3,5-substituted phenyl group; Y1 and Y2 independently include amide, amine, imine, sulfonamide, thiourea, urea or thioether; Z contains H, OH, NH2, NH, therapeutic agent, C1 to C 30 Alkyl, (OCH2CH2) m or a combination thereof, and wherein m is an integer from 1 to 30; and or a pharmaceutically acceptable salt thereof.

5. The compound of claim 4, wherein the compound is of the formula: ， in: R1 and R2 are independently C1 to C5 straight chain alkyl, which is optionally substituted with one or more NH2, CH2NH2 and CH2CH2NH2; R3 and R4 are independently C1 to C5 linear alkyl or absent; U is a tertiary amine or a 1,3,5-substituted phenyl group; Z contains H, OH, NH2, NH, therapeutic agent, C1 to C 30 Alkyl, (OCH2CH2) m or a combination thereof, and wherein m is an integer from 1 to 30; Fc contains the Fc region; S is a sulfur atom from the Fc region; and 6. The compound of claim 4, wherein the compound is of the formula: ， in: R1 and R2 are independently C1 to C5 straight chain alkyl, which is optionally substituted with one or more of -NH2, -CH2NH2 and -CH2CH2NH2; Z contains H, OH, NH2, NH, therapeutic agent, C1 to C 30 Alkyl, (OCH2CH2) m or a combination thereof, and wherein m is an integer from 1 to 30; Fc contains the Fc region; S is a sulfur atom from the Fc region; and or a pharmaceutically acceptable salt thereof.

7. The compound of claims 1 to 6, wherein R1 and R2 are independently C2 alkyl, optionally substituted with one or more -NH2, -CH2NH2 or -CH2CH2NH2, or a pharmaceutically acceptable salt thereof.

8. The compound of any one of claims 1 to 7, wherein R1 and R2 are the same, or a pharmaceutically acceptable salt thereof.

9. The compound of any one of claims 1, 2, 4, 5, 7 or 8, wherein R3 and R4 are the same, or a pharmaceutically acceptable salt thereof.

10. The compound of any one of claims 1, 2, 4, 5, or 7 to 9, wherein R3 and R4 are C2 alkyl, or a pharmaceutically acceptable salt thereof.

11. The compound of any one of claims 1 to 10, wherein Z comprises NH, C1 to C 30 Alkyl, (OCH2CH2) m or a combination thereof and a therapeutic agent, or a pharmaceutically acceptable salt thereof.

12. The compound of any one of claims 1 to 11, wherein Z is H, OH, NH2, a therapeutic agent, or C1 to C 30 alkyl, or a pharmaceutically acceptable salt thereof.

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

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

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

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

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

18. The compound of any one of claims 13 to 16, wherein the therapeutic agent comprises SEQ ID NO 11 or SEQ ID NO 12, or a pharmaceutically acceptable salt thereof.

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

20. The compound of claim 4, wherein Y1 and Y2 comprise an Fc region.

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

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

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

24. The compound of any one of claims 5 to 23, wherein the Fc region comprises SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, or SEQ ID NO 6, or a pharmaceutically acceptable salt thereof.

25. The compound of claim 24, wherein the Fc region comprises SEQ ID NO 6.

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

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