Fc-acylated polypeptide conjugates

Abstract

To provide a treatment option that provides an extended duration of therapeutic action while reducing dosing frequency in the treatment of diabetes.SOLUTION: Provided are compounds comprising a therapeutic polypeptide Fc conjugate having activity at one or more of the GIP, GLP-1, and glucagon receptors. These compounds provide activity at one or more of these receptors and have structural features that extend the duration of action. Also provided are methods for treating diseases and / or conditions such as obesity, chronic weight management, and type 2 diabetes.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to novel compounds, specifically therapeutic polypeptide-Fc conjugates, designed to exert long-lasting therapeutic effects on, for example, glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1), and / or glucagon (GCG) receptors. These conjugates incorporate structural modifications that optimize receptor activity and enhance duration of action, providing improved pharmacological profiles for therapeutic use. [Background technology]

[0002] The prevalence of diabetes continues to increase and represents a chronic condition characterized by persistent hyperglycemia due to disruption of insulin secretion, insulin function, or both. Type 2 diabetes mellitus (T2DM) is the most common form, accounting for approximately 90% of cases. In T2DM, elevated blood glucose levels result from a combination of impaired insulin secretion and insulin resistance.

[0003] Uncontrolled diabetes in individuals can contribute to a variety of health complications that affect both quality of life and overall survival. Excess weight is a significant risk factor for T2DM, and the majority of individuals with T2DM, approximately 90%, are classified as living with overweight or obesity. Research suggests that reducing body adiposity can contribute to improving obesity-related health conditions.

[0004] Standard approaches to managing T2DM involve lifestyle changes, including diet and exercise, along with pharmacological interventions such as oral and injectable incretin-based therapies and insulin therapy. Currently available injectable incretin-based therapies include GLP-1 receptor agonists such as dulaglutide and semaglutide, and the dual GIP and GLP-1 receptor agonist tirzepatide. Therapeutics containing semaglutide and tirzepatide have also been approved to support weight loss and long-term weight management in individuals who are overweight or living with obesity. Research is being conducted on additional compounds described as having dual agonist activity at GIP and GLP-1 receptors, such as those described in U.S. Patent Application Publication No. 20200024322, and compounds described as having triple agonist activity at GIP, GLP-1, and glucagon receptors, such as those described in U.S. Patent Application Publication No. 20240270821. Furthermore, insulin therapy remains the cornerstone of diabetes management, especially for individuals with advanced disease or severe insulin deficiency.

[0005] However, currently available injectable incretin-based and insulin therapies generally require at least weekly administration. Many individuals may prefer options requiring fewer injections, and expanding the availability of treatments to include less frequent administration may increase patient adherence, acceptance, and overall treatment success.

[0006] Several approaches have been proposed to enhance the duration of action of protein or peptide therapeutics. Such approaches include conjugation of the therapeutic to the Fc region of an antibody (see, e.g., WO 2016 / 131893) and lipidation with a fatty acid moiety (see, e.g., U.S. Patent Application Publication No. 2020 / 0283492). Furthermore, A. Zaykov, et al. (2024), RSC Chem. Biol., describe the effect of combining lipidation and Fc conjugation on the pharmacokinetic and pharmacodynamic profiles of insulin molecules.

[0007] However, there remains a need for innovative treatment options that provide an extended duration of therapeutic action while requiring less frequent administration compared to currently available therapies. Addressing this need could increase patient adherence, improve treatment outcomes, and provide a more convenient approach to disease management. Summary of the Invention [Means for solving the problem]

[0008] In a first aspect, the present disclosure provides a compound of formula:

[0009] [ka] or a pharmaceutically acceptable salt thereof, wherein X TPP is a therapeutic polypeptide conjugated to a fatty acid moiety, and Z is O, C(O), NH, C1-C 30 Alkyl, (OCH2CH2) m , [C(O)NH-CH2CH2OCH2] m , amino acid, peptide, or a combination thereof, m is an integer of 1 to 30, U is C1 to C5 alkyl, N, phenyl, or phenylcarbonyl, or a combination thereof, R 1 and R 2 are independently absent, a covalent bond, or a C1-C5 alkyl; R 3 and R 4 is independently absent or amide, and R 5 and R 6 is independently absent or selected from the group consisting of C1-C5 alkyl or phenyl, which includes NH2, CH2NH2, and CH2CH2NH2, CH2O(CH2CH2O) n wherein n is an integer from 1 to 5; and R 7 and R 8is independently absent, or

[0010] [ka] wherein: * ) is R 9 and R 10 is the connection point to ( ** ) is R 5 or R 6 is the connection point to R 9 and R 10 are each an antibody or fragment thereof. In one embodiment, the therapeutic polypeptide comprises an agonist at one or more of the GIP, GLP-1 and glucagon receptors.

[0011] In a second aspect, the present disclosure provides a compound of formula:

[0012] [ka] or a pharmaceutically acceptable salt thereof, wherein X TPP is a therapeutic polypeptide, YAibEGTX6TSDX 10 X 11 X 12 X 13 LX 15 X 16 KAQX 20 X 21 FIX 24 X 25 LX 27 X 28 X 29 X 30 X 31 X 32 X 33 X 34 X 35 X 36 X 37 X 38 X 39 X 40 X 41 (SEQ ID NO: 1219), X6 is αMeF(2F), F, or αMeF; 10 is 4-Pal, Y, or V, and X 11 is Aib or S, and X 12 is I or S, and X 13 is L or αMeL, and X 15 is D or E, and X 16 is E or Orn, and X 20 is Aib, αMeL, or Iva, and X 21 is E, Q, D, or Orn, and X 24 is K, Q, E, or D-Glu, and X 25 is αMeY or Y, and X 27 is I, L or V, and X 28 is K, E or A, and X 29 is Aib, G, or Q, and X 30 is G or S, and X 31 is P, G, E, or Orn, and X 32 is absent or is K, S, or P, and X 32 If is S or P, then X 33 is S and X 33 If S, then X 34 is G or Aib, and X 34 If is G or Aib, then X 35 is absent, A or Orn, and X 35 If is A or Orn, then X 36 is absent or P, and X 36 If P, then X 37 is absent or P, and X 37 If P, then X 38 is absent or P, and X 38 If P, then X 39 is absent, S, Orn, or G, and X 39 If is S, Orn, or G, then X 40 is absent, K, or G, and X 40 If is K or G, then X 41 is absent or is S or G, and X 32 If does not exist or if K, then X 33~X 41 There is no X 35 If does not exist, X 36 ~X 41 There is no X 36 If does not exist, X 37 ~X 41 There is no X 37 If does not exist, X 38 ~X 41 There is no X 38 If does not exist, X 39 ~X 41 There is no X 39 If does not exist, X 40 and X 41 There is no X 40 If does not exist, X 41 does not exist, and L is R 9 , R 10 , and X TPP and R 9 and R 10 are each an antibody or a fragment thereof.

[0013] In one embodiment, the therapeutic polypeptide has dual agonist activity at the GIP receptor and the GLP-1 receptor.

[0014] In a third aspect, the present disclosure provides a compound of formula:

[0015] [ka] or a pharmaceutically acceptable salt thereof, wherein X TPP is a therapeutic polypeptide, X1X2X3X4TX6TSDX 10 X 11 X 12 X 13 LX 15 X 16 KAQX 20 X 21 FIX 24 X 25 LX 27 X 28 X29 X 30 X 31 X 32 X 33 X 34 X 35 X 36 X 37 X 38 X 39 X 40 X 41 (SEQ ID NO: 1220), X1 is H, NMeY, or Y; X2 is Ac4c, Aib, αMeS, Iva, or D-Ala; X3 is Q, E, or H; X4 is G or D-Ala; X6 is αMeF(2F), F, or αMeF; 10 is 4-Pal, Y, or V, and X 11 is Aib, S, or αMeS, and X 12 is I or S, and X 13 is L or αMeL, and X 15 is D or E, and X 16 is E or Orn, and X 20 is Aib, αMeL, Iva, or αMe4Pal, and X 21 is E, Q, D, or Orn, and X 24 is K, Q, E, D-Glu, or D-Gln, and X 25 is αMeY or Y, and X 27 is I, L or V, and X 28 is K, E or A, and X 29 is Aib, G, or Q, and X 30 is G or S, and X 31 is P, G, E, or Orn, X32 is absent, S, or P, and X 32 If is S or P, then X 33 is S and X 33 If S, then X 34 is G or Aib, and X 34 If is G or Aib, then X 35 is absent or is A or Orn, and X 35 If is A or Orn, then X 36 is absent or P, and X 36If P, then X 37 is absent or P, and X 37 If P, then X 38 is absent or P, and X 38 If P, then X 39 is absent or is S, Orn, or G, and X 39 If is S, Orn, or G, then X 40 is absent, K, or G, and X 40 If is K or G, then X 41 is absent or is S or G, and X 32 If does not exist or is K, then X 33 ~X 41 There is no X 35 If does not exist, X 36 ~X 41 There is no X 36 If does not exist, X 37 ~X 41 There is no X 37 If does not exist, X 38 ~X 41 There is no X 38 If does not exist, X 39 ~X 41 There is no X 39 If does not exist, X 40 and X 41 There is no X 40 If there is no X41, there is no L, and R 9 , R 10 , and X TPP and R 9 and R 10 is an antibody or antibody fragment thereof.

[0016] In one embodiment, the therapeutic polypeptide has triple agonist activity at the GIP, GLP-1 and glucagon receptors.

[0017] In a fourth aspect, the present disclosure provides a pharmaceutical composition comprising a compound described herein, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, diluent, or excipient.

[0018] In a fifth aspect, the present disclosure provides a method of treating a disease or condition, the method comprising administering to an individual in need thereof an effective amount of a compound described herein or a pharmaceutically acceptable salt thereof. [Brief explanation of the drawings]

[0019] [Figure 1] 1 shows the molecular structure of compound 164 (SEQ ID NO: 622). [Figure 2] 1 shows the molecular structure of Compound 1 (SEQ ID NO: 459). [Figure 3] 1 shows the molecular structure of compound 32 (SEQ ID NO: 490). [Figure 4] 1 shows the molecular structure of compound 47 (SEQ ID NO: 505). [Figure 5] 1 shows the molecular structure of compound 127 (SEQ ID NO: 585). [Figure 6] 1 shows the molecular structure of compound 129 (SEQ ID NO: 587). [Figure 7] 1 shows the molecular structure of compound 136 (SEQ ID NO: 594). [Figure 8] 1 shows the molecular structure of compound 158 (SEQ ID NO: 616). [Figure 9] 1 shows the molecular structure of compound 168 (SEQ ID NO: 626). [Figure 10] 1 shows the molecular structure of compound 169 (SEQ ID NO: 627). [Figure 11] 1 shows the molecular structure of compound 170 (SEQ ID NO: 628). [Figure 12] 1 shows the molecular structure of compound 172 (SEQ ID NO: 630). [Figure 13] 1 shows the molecular structure of compound 173 (SEQ ID NO: 631). [Figure 14] 1 shows the molecular structure of compound 181 (SEQ ID NO: 639). [Figure 15] 1 shows the molecular structure of compound 183 (SEQ ID NO: 641). [Figure 16] 1 shows the molecular structure of compound 184 (SEQ ID NO: 642). [Figure 17] 1 shows the molecular structure of compound 185 (SEQ ID NO: 643). [Figure 18] 1 shows the molecular structure of compound 187 (SEQ ID NO: 645). [Figure 19] 1 shows the molecular structure of compound 188 (SEQ ID NO: 646). [Figure 20] 1 shows the molecular structure of compound 204 (SEQ ID NO: 662). [Figure 21] 1 shows the molecular structure of compound 479 (SEQ ID NO: 1099). [Figure 22] 1 shows the molecular structure of compound 480 (SEQ ID NO: 698). [Figure 23] 1 shows the molecular structure of compound 481 (SEQ ID NO: 699). [Figure 24] 1 shows the molecular structure of compound 482 (SEQ ID NO: 700). [Figure 25] 1 shows the molecular structure of compound 336 (SEQ ID NO: 792). [Figure 26] 1 shows the molecular structure of compound 387 (SEQ ID NO: 843). [Figure 27] 1 shows the molecular structure of compound 403 (SEQ ID NO: 859). [Figure 28] 1 shows the molecular structure of compound 413 (SEQ ID NO: 869). [Figure 29] 1 shows the molecular structure of compound 433 (SEQ ID NO: 889). [Figure 30] 1 shows the molecular structure of compound 435 (SEQ ID NO: 891). [Figure 31] 1 shows the molecular structure of compound 444 (SEQ ID NO: 900). [Figure 32] 1 shows the molecular structure of compound 483 (SEQ ID NO: 1100). [Figure 33] 1 shows the molecular structure of compound 484 (SEQ ID NO: 1101). [Figure 34] 1 shows the molecular structure of compound 485 (SEQ ID NO: 1102). [Figure 35] 1 shows the molecular structure of compound 456 (SEQ ID NO: 912). [Figure 36] 1 shows the molecular structure of compound 486 (SEQ ID NO: 1103). [Figure 37] 1 shows the molecular structure of compound 487 (SEQ ID NO: 1104). [Figure 38] 1 shows the molecular structure of compound 459 (SEQ ID NO: 915). [Figure 39] 1 shows the molecular structure of compound 493 (SEQ ID NO: 1110). [Figure 40] 1 shows the molecular structure of compound 555 (SEQ ID NO: 1172). [Figure 41] 1 shows the molecular structure of compound 556 (SEQ ID NO: 1173). [Figure 42] 1 shows the molecular structure of compound 557 (SEQ ID NO: 1174). [Figure 43] 1 shows the molecular structure of compound 558 (SEQ ID NO: 1175). [Figure 44] Weekly administration of Compound 170 (SEQ ID NO: 628) demonstrates a dose-dependent reduction in body weight in diet-induced obese mice. [Figure 45] Weekly administration of Compound 172 (SEQ ID NO: 630) demonstrates a dose-dependent reduction in body weight in diet-induced obese mice. [Figure 46] Weekly administration of Compound 387 (SEQ ID NO: 843) shows a dose-dependent reduction in body weight in diet-induced obese mice. [Figure 47] Weekly administration of compound 456 (SEQ ID NO: 912) shows a dose-dependent reduction in body weight in diet-induced obese mice. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present disclosure relates to methods and compositions comprising therapeutic polypeptide-Fc conjugates for facilitating delivery of therapeutic polypeptides to patients. These conjugates comprise a therapeutic polypeptide covalently linked via a linker to an antibody or fragment thereof, e.g., the Fc region. In particular, the conjugation reaction between the therapeutic polypeptide, the disclosed linkers, and the antibody or fragment thereof occurs under relatively mild conditions, eliminating the need for safeguards during the reaction process.

[0021] Surprisingly, by conjugating an acylated therapeutic polypeptide to an antibody or fragment thereof, e.g., the Fc region, using a linker disclosed herein, the duration of action of the therapeutic polypeptide is significantly improved and / or extended beyond that expected by using either acylation or conjugation to the Fc region alone. Conjugation of the therapeutic polypeptide to a linker and an Fc region may increase the duration of action of the therapeutic polypeptide by one or more of increasing the overall hydrodynamic size of the moiety, FcRn binding and / or recycling, and preventing degradation of the therapeutic polypeptide. Because the reaction conditions for the conjugation reaction between the linkers disclosed herein and an antibody or antibody fragment, e.g., the Fc region, are relatively mild, the therapeutic polypeptide does not need to be modified to protect it during the reaction. Acylation of the therapeutic polypeptide may further increase the duration of action of the therapeutic polypeptide by one or more of promoting binding to serum albumin. In certain embodiments, the duration of action of the therapeutic polypeptide may be further increased by including structural features that improve the proteolytic stability of the therapeutic polypeptide.

[0022] Therapeutic Polypeptide Fc Conjugates The present disclosure provides several different embodiments of compounds that include features that result in increased duration of action.

[0023] In certain embodiments, the present disclosure provides compounds of formula I:

[0024] [ka] or a pharmaceutically acceptable salt thereof, wherein: Therapeutic Polypeptide X TPP is conjugated to a fatty acid moiety, Z is a linker, U is C1-C5 alkyl, N, phenyl, or phenylcarbonyl, or a combination thereof, and R 1 and R 2are independently absent, a covalent bond, or a C1-C5 alkyl; R 3 and R 4 is independently absent or amide, and R 5 and R 6 is independently absent or selected from the group consisting of C1-C5 alkyl or phenyl, which includes NH2, CH2NH2, and CH2CH2NH2, CH2O(CH2CH2O) n wherein n is an integer from 1 to 5; and R 7 and R 8 is independently absent, or

[0025] [ka] is selected from the group consisting of In the formula, ( * ) is R 9 and R 10 is the connection point to ( ** ) is R 5 or R 6 is the connection point to R 9 and R 10 are each an antibody or a fragment thereof.

[0026] In certain embodiments, Z is O, C(O), NH, C-C 30 Alkyl, (OCH2CH2) m , [C(O)NH-CH2CH2OCH2] m , (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) m , amino acids, peptides, or combinations thereof, and m is an integer of 1 to 30.

[0027] In certain embodiments, R 1 and R 2 are the same. In certain embodiments, R 3 and R 4 are the same. In certain embodiments, R 5 and R 6are the same. In certain embodiments, R 7 and R 8 are the same. In certain embodiments, R 9 and R 10 is an Fc region. In certain embodiments, R 9 and R 10 is the same.

[0028] In certain embodiments, the therapeutic polypeptide has agonist activity at one or more of the GIP, GLP-1 and / or glucagon receptors.

[0029] In some embodiments, the disclosure provides compounds of formula I, wherein R 7 and R 8 teeth,

[0030] [ka] is.

[0031] In some embodiments, R 5 and R 6 Each is a C alkyl substituted with NH. In some embodiments, R 3 and R 4 Each is an amide. In some embodiments, R 5 and R 6 are each C alkyl. In some embodiments, U is N or phenyl.

[0032] In some embodiments, the present disclosure provides compounds of formula IA:

[0033] [ka] or a pharmaceutically acceptable salt thereof.

[0034] In some embodiments, the disclosure provides compounds of formula I, wherein R 7 and R8 teeth,

[0035] [ka] is.

[0036] In some embodiments, R 5 and R 6 are (OCH2CH2) m and m is 3. In some embodiments, R 3 and R 4 and R are each amides. 1 and R 2 are each C alkyl. In some embodiments, U is N or phenyl.

[0037] In some embodiments, the present disclosure provides compounds of formula IB:

[0038] [ka] or a pharmaceutically acceptable salt thereof.

[0039] In some embodiments, the disclosure provides compounds of formula I, wherein R 7 and R 8 teeth,

[0040] [ka] is.

[0041] In some embodiments, R 5 and R 6 are (OCH2CH2) m and m is 3. In some embodiments, R 3 and R 4 and R are each amides.1 and R 2 are each C alkyl. In some embodiments, U is N or phenyl.

[0042] In some embodiments, the present disclosure provides compounds of formula IC:

[0043] [ka] or a pharmaceutically acceptable salt thereof.

[0044] In some embodiments of the compound of Formula I, R 7 and R 8 are each absent. In some embodiments, R 5 and R 6 are each C alkyl. In some embodiments, R 3 and R 4 and R are each amides. 1 and R 2 are each C alkyl. In some embodiments, U is N or phenyl.

[0045] In some embodiments, the present disclosure provides a compound of formula ID:

[0046] [ka] or a pharmaceutically acceptable salt thereof.

[0047] In some embodiments, the present disclosure provides compounds of formula IE:

[0048] [ka] or a pharmaceutically acceptable salt thereof.

[0049] In certain embodiments, the present disclosure provides compounds of formula II:

[0050] [ka] or a pharmaceutically acceptable salt thereof, wherein: X TPP is a therapeutic polypeptide, L is a linker conjugated to the therapeutic polypeptide, and R 9 and R 10 , and R 9 and R 10 and R are each an antibody or antibody fragment thereof. 9 and R 10 are the Fc regions.

[0051] In some embodiments, a therapeutic polypeptide has dual agonist activity at the GIP receptor and the GLP-1 receptor, hi some embodiments, a therapeutic polypeptide has triagonist activity at the GIP, GLP-1, and glucagon receptors.

[0052] In certain embodiments, the present disclosure provides compounds of formula III:

[0053] [ka] or a pharmaceutically acceptable salt thereof, wherein: Fc1 and Fc2 are absent or each is an Fc region, L is a linker, Z is a functional group of a small molecule, an amino acid polymer, or a combination thereof, and X is a therapeutic polypeptide, optionally conjugated to a fatty acid Y.

[0054] In certain embodiments, the present disclosure provides compounds of formula IV:

[0055] [ka] or a pharmaceutically acceptable salt thereof, wherein: R 1 and R 2 is not present or, independently,

[0056] [ka] wherein: ( * ) is the attachment point to the sulfur atom in the cysteine ​​residue of Fc1 or Fc2, and ( ** ) is R 3 or R 4 is the connection point to R 3 and R 4 is independently a C1-C5 alkyl, which is optionally substituted with one or more of NH2, CH2NH2, and CH2CH2NH2; R 5 and R 6 are independently a covalent bond or a C1-C5 alkyl; U is a tertiary amine or a 1,3,5-substituted phenyl; Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof; m is an integer from 1 to 30; X is a therapeutic polypeptide; Y is absent or a fatty acid; and Fc1 and Fc2 are independently an Fc region.

[0057] In some embodiments, the present disclosure provides compounds of formula IV-A:

[0058] [ka] or a pharmaceutically acceptable salt thereof, wherein: R 3 and R 4 is independently a C1-C5 alkyl, which is optionally substituted with one or more of NH2, CH2NH2, and CH2CH2NH2; R 5 and R 6are independently a covalent bond or a C1-C5 alkyl; U is a tertiary amine or a 1,3,5-substituted phenyl; Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof; m is an integer from 1 to 30; X is a therapeutic polypeptide; Y is absent or a fatty acid; and Fc1 and Fc2 are independently an Fc region.

[0059] In another embodiment, the present disclosure provides:

[0060] [ka] or a pharmaceutically acceptable salt thereof, wherein: R3 and R4 are independently C1-C5 alkyl, which is optionally substituted with one or more of NH2, CH2NH2, and CH2CH2NH2; R 5 and R 6 are independently a covalent bond or a C1-C5 alkyl, and Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof; m is an integer from 1 to 30; X is a therapeutic polypeptide; Y is absent or a fatty acid; and Fc1 and Fc2 are independently an Fc region.

[0061] In yet another embodiment, the present disclosure provides:

[0062] [ka] or a pharmaceutically acceptable salt thereof, wherein: R3 and R4 are independently C1-C5 alkyl, which is optionally substituted with one or more of NH2, CH2NH2, and CH2CH2NH2; Z is O, NH, C1-C30 alkyl, (OCH2CH2)m , an amino acid polymer, or a combination thereof; m is an integer from 1 to 30; X is a therapeutic polypeptide; Y is absent or a fatty acid; and Fc1 and Fc2 are independently an Fc region.

[0063] In some embodiments, the present disclosure provides compounds of formula IV-B:

[0064] [ka] or a pharmaceutically acceptable salt thereof, wherein: R 3 and R 4 is independently a C1-C5 alkyl, which is optionally substituted with one or more of NH2, CH2NH2, and CH2CH2NH2; R 5 and R 6 are independently a covalent bond or a C1-C5 alkyl; U is a tertiary amine or a 1,3,5-substituted phenyl; Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof; m is an integer from 1 to 30; X is a therapeutic polypeptide; Y is absent or a fatty acid; and Fc1 and Fc2 are independently an Fc region.

[0065] In another embodiment, the present disclosure provides:

[0066] [ka] or a pharmaceutically acceptable salt thereof, wherein: R 3 and R 4 is independently a C1-C5 alkyl, which is optionally substituted with one or more of NH2, CH2NH2, and CH2CH2NH2; R 5 and R 6are independently a covalent bond or a C1-C5 alkyl, and Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof; m is an integer from 1 to 30; X is a therapeutic polypeptide; Y is absent or a fatty acid; and Fc1 and Fc2 are independently an Fc region.

[0067] In yet another embodiment, the present disclosure provides:

[0068] [ka] or a pharmaceutically acceptable salt thereof, wherein: R 3 and R 4 is independently a C1-C5 alkyl, which is optionally substituted with one or more of NH2, CH2NH2, and CH2CH2NH2; Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof; m is an integer from 1 to 30; X is a therapeutic polypeptide; Y is absent or a fatty acid; and Fc1 and Fc2 are independently an Fc region.

[0069] In some embodiments, the present disclosure provides compounds of formula IV-C:

[0070] [ka] or a pharmaceutically acceptable salt thereof, wherein: R 3 and R 4 is independently a C1-C5 alkyl, which is optionally substituted with one or more of NH2, CH2NH2, and CH2CH2NH2; R 5 and R 6are independently a covalent bond or a C1-C5 alkyl; U is a tertiary amine or a 1,3,5-substituted phenyl; Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof; m is an integer from 1 to 30; X is a therapeutic polypeptide; Y is absent or a fatty acid; and Fc1 and Fc2 are independently an Fc region.

[0071] In another embodiment, the present disclosure provides:

[0072] [ka] or a pharmaceutically acceptable salt thereof, wherein: R 3 and R 4 is independently a C1-C5 alkyl, which is optionally substituted with one or more of NH2, CH2NH2, and CH2CH2NH2; R 5 and R 6 are independently a covalent bond or a C1-C5 alkyl, and Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof; m is an integer from 1 to 30; X is a therapeutic polypeptide; Y is absent or a fatty acid; and Fc1 and Fc2 are independently an Fc region.

[0073] In yet another embodiment, the present disclosure provides:

[0074] [ka] or a pharmaceutically acceptable salt thereof, wherein: R 3 and R 4is independently a C1-C5 alkyl, which is optionally substituted with one or more of NH2, CH2NH2, and CH2CH2NH2; Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof; m is an integer from 1 to 30; X is a therapeutic polypeptide; Y is absent or a fatty acid; and Fc1 and Fc2 are independently an Fc region.

[0075] In some embodiments, the present disclosure provides compounds of formula IV-D:

[0076] [ka] or a pharmaceutically acceptable salt thereof, wherein: R 3 and R 4 is independently a C1-C5 alkyl, which is optionally substituted with one or more of NH2, CH2NH2, and CH2CH2NH2; R 5 and R 6 are independently a covalent bond or a C1-C5 alkyl; U is a tertiary amine or a 1,3,5-substituted phenyl; Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof; m is an integer from 1 to 30; X is a therapeutic polypeptide; Y is absent or a fatty acid; and Fc1 and Fc2 are independently an Fc region.

[0077] In another embodiment, the present disclosure provides:

[0078] [ka] or a pharmaceutically acceptable salt thereof, wherein: R 3 and R 4is independently a C1-C5 alkyl, which is optionally substituted with one or more of NH2, CH2NH2, and CH2CH2NH2; R 5 and R 6 are independently a covalent bond or a C1-C5 alkyl, and Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof; m is an integer from 1 to 30; X is a therapeutic polypeptide; Y is absent or a fatty acid; and Fc1 and Fc2 are independently an Fc region.

[0079] In yet another embodiment, the present disclosure provides:

[0080] [ka] or a pharmaceutically acceptable salt thereof, wherein: R 3 and R 4 is independently a C1-C5 alkyl, which is optionally substituted with one or more of NH2, CH2NH2, and CH2CH2NH2; Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof; m is an integer from 1 to 30; X is a therapeutic polypeptide; Y is absent or a fatty acid; and Fc1 and Fc2 are independently an Fc region.

[0081] Intermediate Therapeutic Polypeptide Conjugates The present disclosure provides intermediate compounds comprising a therapeutic polypeptide, which is conjugated to a linker and subsequently conjugated to an antibody or antibody fragment thereof, e.g., an Fc region. Non-limiting examples of such intermediate compounds are provided below.

[0082] Maleimide intermediate compound In some embodiments, the present disclosure provides:

[0083] [ka] or a pharmaceutically acceptable salt thereof, wherein R 3 and R 4 is independently a C1-C5 alkyl, which is optionally substituted with one or more of NH2, CH2NH2, and CH2CH2NH2; R 5 and R 6 are independently a covalent bond or a C1-C5 alkyl; U is a tertiary amine or a 1,3,5-substituted phenyl; Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof, m is an integer from 1 to 30, X is a therapeutic polypeptide, and Y is absent or a fatty acid.

[0084] In another embodiment, the present disclosure provides:

[0085] [ka] or a pharmaceutically acceptable salt thereof, wherein R 3 and R 4 is independently a C1-C5 alkyl, which is optionally substituted with one or more of NH2, CH2NH2, and CH2CH2NH2; R 5 and R 6 are independently a covalent bond or a C1-C5 alkyl, and Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof, m is an integer from 1 to 30, X is a therapeutic polypeptide, and Y is absent or a fatty acid.

[0086] In yet another embodiment, the present disclosure provides:

[0087] [ka] or a pharmaceutically acceptable salt thereof, wherein R 3 and R 4 is independently a C1-C5 alkyl, which is optionally substituted with one or more of NH2, CH2NH2, and CH2CH2NH2; Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof, m is an integer from 1 to 30, X is a therapeutic polypeptide, and Y is absent or a fatty acid.

[0088] [ka]

[0089] The present specification also discloses a compound of the above structure or a pharmaceutically acceptable salt thereof, wherein R 3 and R 4 is independently a C1-C5 alkyl, which is optionally substituted with one or more of NH2, CH2NH2, and CH2CH2NH2; R 5 and R 6 are independently a covalent bond or a C1-C5 alkyl; U is a tertiary amine or a 1,3,5-substituted phenyl; Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof, m is an integer from 1 to 30, X is a therapeutic polypeptide, Y is absent or a fatty acid, and D1 and D2 independently comprise an amide, an amine, an imine, a sulfonamide, a thiourea, a urea, a thioether, or a combination thereof.

[0090] [ka]

[0091] The present specification also discloses a compound of the above structure or a pharmaceutically acceptable salt thereof, wherein R 3 and R 4 is independently a C1-C5 alkyl, which is optionally substituted with one or more of NH2, CH2NH2, and CH2CH2NH2; R 5 and R 6 are independently a covalent bond or a C1-C5 alkyl; U is a tertiary amine or a 1,3,5-substituted phenyl; Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof, m is an integer from 1 to 30, X is a therapeutic polypeptide, and Y is absent or a fatty acid.

[0092] [ka]

[0093] The present specification also discloses a compound of the above structure or a pharmaceutically acceptable salt thereof, wherein R 3 and R 4 is independently a C1-C5 alkyl, which is optionally substituted with one or more of NH2, CH2NH2, and CH2CH2NH2; R 5 and R 6 are independently a covalent bond or a C1-C5 alkyl, and Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof, m is an integer from 1 to 30, X is a therapeutic polypeptide, and Y is absent or a fatty acid.

[0094] [ka]

[0095] The present specification also discloses a compound of the above structure or a pharmaceutically acceptable salt thereof, wherein R 3 and R 4is independently a C1-C5 alkyl, which is optionally substituted with one or more of NH2, CH2NH2, and CH2CH2NH2; Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof, m is an integer from 1 to 30, X is a therapeutic polypeptide, and Y is absent or a fatty acid.

[0096] Tetrazole intermediate compound In some embodiments, the present disclosure provides:

[0097] [ka] or a pharmaceutically acceptable salt thereof, wherein R 3 and R 4 is independently a C1-C5 alkyl, which is optionally substituted with one or more of NH2, CH2NH2, and CH2CH2NH2; R 5 and R 6 are independently a covalent bond or a C1-C5 alkyl; U is a tertiary amine or a 1,3,5-substituted phenyl; Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof, m is an integer from 1 to 30, X is a therapeutic polypeptide, and Y is absent or a fatty acid.

[0098] In another embodiment, the present disclosure provides:

[0099] [ka] or a pharmaceutically acceptable salt thereof, wherein R 3 and R 4is independently a C1-C5 alkyl, which is optionally substituted with one or more of NH2, CH2NH2, and CH2CH2NH2; R 5 and R 6 are independently a covalent bond or a C1-C5 alkyl, and Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof, m is an integer from 1 to 30, X is a therapeutic polypeptide, and Y is absent or a fatty acid.

[0100] In yet another embodiment, the present disclosure provides:

[0101] [ka] or a pharmaceutically acceptable salt thereof, wherein R 3 and R 4 is independently a C1-C5 alkyl, which is optionally substituted with one or more of NH2, CH2NH2, and CH2CH2NH2; Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof, m is an integer from 1 to 30, X is a therapeutic polypeptide, and Y is absent or a fatty acid.

[0102] [ka]

[0103] The present specification also discloses a compound of the above structure or a pharmaceutically acceptable salt thereof, wherein R 3 and R 4 is independently a C1-C5 alkyl, which is optionally substituted with one or more of NH2, CH2NH2, and CH2CH2NH2; R 5 and R 6are independently a covalent bond or a C1-C5 alkyl; U is a tertiary amine or a 1,3,5-substituted phenyl; Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof, m is an integer from 1 to 30, X is a therapeutic polypeptide, Y is absent or a fatty acid, and D1 and D2 independently comprise an amide, an amine, an imine, a sulfonamide, a thiourea, a urea, a thioether, or a combination thereof.

[0104] [ka]

[0105] The present specification also discloses a compound of the above structure or a pharmaceutically acceptable salt thereof, wherein R 3 and R 4 is independently a C1-C5 alkyl, which is optionally substituted with one or more of NH2, CH2NH2, and CH2CH2NH2; R 5 and R 6 are independently a covalent bond or a C1-C5 alkyl; U is a tertiary amine or a 1,3,5-substituted phenyl; Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof, m is an integer from 1 to 30, X is a therapeutic polypeptide, and Y is absent or a fatty acid.

[0106] [ka]

[0107] The present specification also discloses a compound of the above structure or a pharmaceutically acceptable salt thereof, wherein R 3 and R 4 is independently a C1-C5 alkyl, which is optionally substituted with one or more of NH2, CH2NH2, and CH2CH2NH2; R 5 and R6 are independently a covalent bond or a C1-C5 alkyl, and Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof, m is an integer from 1 to 30, X is a therapeutic polypeptide, and Y is absent or a fatty acid.

[0108] [ka]

[0109] The present specification also discloses a compound of the above structure or a pharmaceutically acceptable salt thereof, wherein R 3 and R 4 is independently a C1-C5 alkyl, which is optionally substituted with one or more of NH2, CH2NH2, and CH2CH2NH2; Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof, m is an integer from 1 to 30, X is a therapeutic polypeptide, and Y is absent or a fatty acid.

[0110] Oxidizole intermediate compound In some embodiments, the present disclosure provides:

[0111] [ka] or a pharmaceutically acceptable salt thereof, wherein R 3 and R 4 is independently a C1-C5 alkyl, which is optionally substituted with one or more of NH2, CH2NH2, and CH2CH2NH2; R 5 and R 6 are independently a covalent bond or a C1-C5 alkyl; U is a tertiary amine or a 1,3,5-substituted phenyl; Z is O, NH, C1-C 30 Alkyl, (OCH2CH2)m , an amino acid polymer, or a combination thereof, m is an integer from 1 to 30, X is a therapeutic polypeptide, and Y is absent or a fatty acid.

[0112] In another embodiment, the present disclosure provides:

[0113] [ka] or a pharmaceutically acceptable salt thereof, wherein R 3 and R 4 is independently a C1-C5 alkyl, which is optionally substituted with one or more of NH2, CH2NH2, and CH2CH2NH2; R 5 and R 6 are independently a covalent bond or a C1-C5 alkyl, and Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof, m is an integer from 1 to 30, X is a therapeutic polypeptide, and Y is absent or a fatty acid.

[0114] In yet another embodiment, the present disclosure provides:

[0115] [ka] or a pharmaceutically acceptable salt thereof, wherein R 3 and R 4 is independently a C1-C5 alkyl, which is optionally substituted with one or more of NH2, CH2NH2, and CH2CH2NH2; Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof, m is an integer from 1 to 30, X is a therapeutic polypeptide, and Y is absent or a fatty acid.

[0116] [ka]

[0117] The present specification also discloses a compound of the above structure or a pharmaceutically acceptable salt thereof, wherein R 3 and R 4 is independently a C1-C5 alkyl, which is optionally substituted with one or more of NH2, CH2NH2, and CH2CH2NH2; R 5 and R 6 are independently a covalent bond or a C1-C5 alkyl; U is a tertiary amine or a 1,3,5-substituted phenyl; Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof, m is an integer from 1 to 30, X is a therapeutic polypeptide, Y is absent or a fatty acid, and D1 and D2 independently comprise an amide, an amine, an imine, a sulfonamide, a thiourea, a urea, a thioether, or a combination thereof.

[0118] [ka]

[0119] The present specification also discloses a compound of the above structure or a pharmaceutically acceptable salt thereof, wherein R 3 and R 4 is independently a C1-C5 alkyl, which is optionally substituted with one or more of NH2, CH2NH2, and CH2CH2NH2; R 5 and R 6 are independently a covalent bond or a C1-C5 alkyl; U is a tertiary amine or a 1,3,5-substituted phenyl; Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof, m is an integer from 1 to 30, X is a therapeutic polypeptide, and Y is absent or a fatty acid.

[0120] [ka]

[0121] The present specification also discloses a compound of the above structure or a pharmaceutically acceptable salt thereof, wherein R 3 and R 4 is independently a C1-C5 alkyl, which is optionally substituted with one or more of NH2, CH2NH2, and CH2CH2NH2; R 5 and R 6 are independently a covalent bond or a C1-C5 alkyl, and Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof, m is an integer from 1 to 30, X is a therapeutic polypeptide, and Y is absent or a fatty acid.

[0122] [ka]

[0123] The present specification also discloses a compound of the above structure or a pharmaceutically acceptable salt thereof, wherein R 3 and R 4 is independently a C1-C5 alkyl, which is optionally substituted with one or more of NH2, CH2NH2, and CH2CH2NH2; Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof, m is an integer from 1 to 30, X is a therapeutic polypeptide, and Y is absent or a fatty acid.

[0124] In some embodiments described herein, the therapeutic polypeptide is conjugated to Z, wherein Z is O, C(O), NH, C1-C30 alkyl, (OCH2CH2) m , [C(O)NH-CH2CH2OCH2] m, (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) m , an amino acid, a peptide, or a combination thereof, and m is an integer from 1 to 30. In some embodiments, Z comprises a peptide comprising one or more of the following sequences:

[0125] [Table 1]

[0126] Linker In some embodiments, Formula II includes a linker, L, for connecting the therapeutic polypeptide to an antibody or fragment thereof, e.g., an Fc region. In certain embodiments, L in Formula II is

[0127] [ka] wherein *** ) includes a connection point to a therapeutic polypeptide, and ( * ) is R 9 or R 10 Includes connection points to

[0128] In some other embodiments, L in formula II is

[0129] [ka] wherein *** ) includes a connection point to a therapeutic polypeptide, and ( * ) is R 9 or R 10 Includes connection points to

[0130] In some embodiments, the present disclosure includes a linker, L, that connects Z to another moiety via a nucleophilic conjugate addition reaction. In some embodiments, the moiety is one or more cysteine ​​amino acids in the Fc region of an antibody or fragment thereof. In some embodiments, the moiety is a sulfur atom from a cysteine ​​residue in the Fc region. In some embodiments, Z is connected via a nucleophilic conjugate addition reaction to another nucleophile, such as an amide, amine, imine, sulfonamide, thiourea, urea, thioether, thiol, cysteine, or a combination thereof, as shown in some of the reactions depicted and described below.

[0131] In some embodiments, the linker can include a central trivalent linking unit U, as shown in Formulas IV-A, IV-B, and IV-C above. In some embodiments, one portion of U can be attached to Z. In some embodiments, the other two portions of U include maleimide functional groups, e.g., electrophiles, which can be used to selectively connect Z to two nucleophiles in a nucleophilic conjugate addition reaction.

[0132] Thus, the linkers of the present invention contain two or more maleimide functional groups, preferably two maleimide functional groups, prior to the nucleophilic conjugate addition reaction. Alternatively, the linkers of the present invention contain two or more methylsulfonyltetrazole functional groups, or preferably two methylsulfonyltetrazole functional groups, prior to the aromatic nucleophilic substitution reaction. Alternatively, the linkers of the present invention contain two or more methylsulfonyloxadiazole functional groups, or preferably two methylsulfonyloxadiazole functional groups, prior to the aromatic nucleophilic substitution reaction. Alternatively, the linkers of the present invention contain two or more bromoacetyl functional groups, or preferably two bromoacetyl functional groups, prior to the nucleophilic substitution reaction.

[0133] 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.

[0134] In some embodiments, the structure attached to U is represented as follows:

[0135] [ka]

[0136] In these structures above, R3 can be a C1-C5 alkyl, which is optionally substituted with one or more of -NH2, -CH2NH2, and -CH2CH2NH2, and R5 can be a C1-C5 alkyl or is absent. R4 can be a C1-C5 alkyl, which is optionally substituted with one or more of -NH2, -CH2NH2, and -CH2CH2NH2. 2、 -CHNH 2、 and —CH 2 CH 2 NH 2 , and R 6 can be a C 1 -C 5 alkyl or is absent.

[0137] R3 and / or R4 may preferably be C2 alkyl, optionally substituted with one or more of -NH2, -CH2NH2, or -CH2CH2NH2. R5 and / or R6 may preferably be C2 alkyl when U is a tertiary amine, or absent when U is 1,3,5-trisubstituted phenyl.

[0138] When both of the above structures are attached to U, R3 and R4 can be the same or independently C1-C5 alkyl, optionally substituted with one or more of -NH2, -CH2NH2, and -CH2CH2NH2. When both of the above structures are attached to U, R5 and R6 can be the same or independently C1-C5 alkyl, or absent. When R5 and / or R6 are absent, the amide nitrogen in the structure is directly connected to U, such as when U is 1,3,5-trisubstituted phenyl.

[0139] The third arm of the trivalent linking unit attached to U can comprise Z. Z can be O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof, wherein m is an integer from 1 to 30, X is a therapeutic polypeptide, Y is absent or a fatty acid, and Fc1 and Fc2 are independently Fc regions. Z can be directly attached to U or a carbonyl functional group. For example, the carbon atom of the carbonyl functional group can be directly attached to U and Z.

[0140] Alternatively, each arm attached to U and the tetrazole functionality can be represented by the following structure:

[0141] [ka]

[0142] Alternatively, each arm attached to U and the oxadiazole functionality can be represented by the formula:

[0143] [ka]

[0144] In one embodiment, R3, the first of the two arms represented by the above structure, is phenyl-(OCH2CH2) mIn another embodiment, R4, the second of the two arms represented by the above structure, can be phenyl-(OCH2CH2) m OCH2, and R6 can be a C1-C5 alkyl or is absent.

[0145] R3 and / or R4 may preferably be phenyl-(OCH2CH2)2OCH2. R5 and / or R6 may preferably be C2 alkyl when U is a tertiary amine, or absent when U is 1,3,5-trisubstituted phenyl.

[0146] When both arms are attached to U, R3 and R4 may be the same or may independently be phenyl-(OCH2CH2)2OCH2. When both arms are attached to U, R5 and R6 may be the same or may independently be C1-C5 alkyl or may be absent. When R5 and / or R6 are absent, the amide nitrogen in the above structure is directly connected to U, such as when U is 1,3,5-trisubstituted phenyl.

[0147] The third of the three arms bonded to U includes Z. Z is H, OH, NH, NH, a therapeutic agent, C1-C 30 Alkyl, (OCH2CH2) m or a combination thereof, where m is an integer from 1 to 30. Z can be directly connected to U, or Z can be connected to a carbonyl functional group. The carbon atom of the carbonyl functional group can be directly bonded to U and Z.

[0148] Alternatively, each arm attached to a U and an acetyl functionality can be represented by the following structure:

[0149] [ka]

[0150] In one embodiment, R3, the first of the two arms represented by the structure above, can be a C1-C5 alkyl and R5 can be a C1-C5 alkyl or is absent. In another embodiment, R4, the second of the two arms represented by the structure above, can be a C1-C5 alkyl and R6 can be a C1-C5 alkyl or is absent.

[0151] R3 and / or R4 may preferably be C2 alkyl. R5 and / or R6 may preferably be C2 alkyl when U is a tertiary amine, and absent when U is 1,3,5-trisubstituted phenyl.

[0152] When both arms are attached to U, R3 and R4 can be the same or independently C1-C5 alkyl. When both arms are attached to U, R5 and R6 can be the same or independently C1-C5 alkyl, or absent. When R5 and / or R6 are absent, the amide nitrogen of Formula VB or Formula VC is directly connected to U, such as when U is 1,3,5-trisubstituted phenyl.

[0153] The third of the three arms bonded to U includes Z. Z is H, OH, NH, NH, a therapeutic agent, C1-C 30 Alkyl, (OCH2CH2) m or a combination thereof, where m is an integer from 1 to 30. Z can be directly attached to U, or Z can be attached to a carbonyl functional group. The carbon atom of the carbonyl functional group can be directly attached to U and Z, as shown in Formulas II-IV.

[0154] 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 disclosed in the art require the therapeutic polypeptide, e.g., insulin, to be modified with a recombinant extension. The conjugate compounds disclosed herein have a longer duration of action and are effective without the need to modify the therapeutic polypeptide.

[0155] [ka]

[0156] In some embodiments disclosed herein, the compounds are of the structure above and pharmaceutically acceptable salts thereof, wherein R3 and R4 are independently selected from C1-C5 alkyl; R5 and R6 are independently C1-C5 alkyl or absent; U is a tertiary amine or 1,3,5-substituted phenyl; Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof, wherein X comprises a therapeutic polypeptide, Y comprises a fatty acid, m is an integer from 1 to 30, and Fc1 and Fc2 each comprise an Fc region.

[0157] [ka]

[0158] In some embodiments disclosed herein, the compound has the structure shown above, or a pharmaceutically acceptable salt thereof, wherein R3 and R4 are independently selected from C1-C5 alkyl; R5 and R6 are independently C1-C5 alkyl; and Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof, wherein X comprises a therapeutic polypeptide, Y comprises a fatty acid, m is an integer from 1 to 30, and Fc1 and Fc2 each comprise an Fc region.

[0159] [ka]

[0160] In some embodiments disclosed herein, the compound has the structure shown above, or a pharmaceutically acceptable salt thereof, wherein R and R are independently selected from C-C alkyl; and Z is O, NH, C-C alkyl; 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof, wherein X comprises a therapeutic polypeptide, Y comprises a fatty acid, m is an integer from 1 to 30, and Fc1 and Fc2 each comprise an Fc region.

[0161] [ka]

[0162] In some embodiments disclosed herein, the compound has the structure shown above, or a pharmaceutically acceptable salt thereof, wherein R3 and R4 are independently selected C1-C5 alkyl; R5 and R6 are independently C1-C5 alkyl or absent; U is a tertiary amine or a 1,3,5-substituted phenyl; and Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof, wherein X comprises a therapeutic polypeptide, Y comprises a fatty acid, m is an integer from 1 to 30, and D1 and D2 independently comprise an amide, an amine, an imine, a sulfonamide, a thiourea, a urea, a thioether, or a combination thereof.

[0163] [ka]

[0164] In some embodiments disclosed herein, the compound has the structure shown above, or a pharmaceutically acceptable salt thereof, wherein R3 and R4 are independently selected from C1-C5 alkyl; R5 and R6 are independently C1-C5 alkyl or absent; U is a tertiary amine or a 1,3,5-substituted phenyl; and Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof, wherein X comprises a therapeutic polypeptide, Y comprises a fatty acid, and m is an integer from 1 to 30.

[0165] [ka]

[0166] In some embodiments disclosed herein, the compound has the structure shown above, or a pharmaceutically acceptable salt thereof, wherein R3 and R4 are independently selected C1-C5 alkyl, R5 and R6 are C1-C5 alkyl, and Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof, wherein X comprises a therapeutic polypeptide, Y comprises a fatty acid, and m is an integer from 1 to 30.

[0167] [ka]

[0168] In some embodiments disclosed herein, the compound has the structure shown above, or a pharmaceutically acceptable salt thereof, wherein R and R are independently selected from C-C alkyl; and Z is O, NH, C-C alkyl; 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof, wherein X comprises a therapeutic polypeptide, Y comprises a fatty acid, and m is an integer from 1 to 30.

[0169] In some embodiments, compounds of the present disclosure comprise a linker, L, that connects Z to another moiety, such as one or more cysteine ​​amino acids in the Fc region of an antibody or fragment thereof, as shown in Formula II, or to another suitable nucleophile, such as an amide, amine, imine, sulfonamide, thiourea, urea, thioether, thiol, cysteine, or combinations thereof, as shown in the structures disclosed herein, via a nucleophilic conjugate addition reaction.

[0170] In some embodiments, the linker can include a central trivalent linking unit U, as shown in the structures disclosed herein. One of the linker moieties can bind to Z. The other two moieties of the linker contain maleimide functional groups, electrophiles, that can be used to selectively connect Z to two nucleophiles in a nucleophilic conjugate addition reaction.

[0171] Nucleophilic conjugate addition reaction with maleimide functional groups As disclosed herein, linker L can be attached to Z to provide a linked payload, where Z further comprises a therapeutic agent X, e.g., a linked therapeutic agent. In some embodiments, linker L comprises two maleimide functional groups. The maleimide functional groups can be used to attach Z to a different moiety, such as the Fc region of an antibody or fragment thereof, via a nucleophilic conjugate addition reaction.

[0172] In nucleophilic conjugate addition reactions, maleimides are electrophiles and react with nucleophiles to form new covalent bonds between electrophilic carbon atoms and nucleophilic atoms, which can lead to ring-opening of the maleimide functionality as shown below.

[0173] [ka]

[0174] A variety of nucleophiles can be used to react with the electrophilic maleimide functional group. Suitable nucleophiles include moieties containing one or more nitrogen and / or sulfur atoms in the functional group, which can function as electron-rich nucleophiles. Suitable examples of nucleophiles include moieties containing one or more of the following functional groups: amide, amine, imine, sulfonamide, thiol, thiourea, urea, or combinations thereof, as shown in the following structures:

[0175] [ka]

[0176] In particular, the amino acid cysteine, as shown below, is found in many peptides, antibodies, proteins, etc. and contains a thiol functional group that can be used as a nucleophile to form a thioether between a peptide, antibody, protein, etc. and a linker, linked payload, and / or linked therapeutic agent.

[0177] [ka]

[0178] The conditions for nucleophilic conjugate addition reactions are well known to those skilled in the art. The conditions may vary depending on the selected nucleophile and electrophile used in a particular reaction. As disclosed herein, there are compounds and / or conjugate compounds that include a linked payload that is attached to another moiety via a nucleophilic conjugate addition reaction.

[0179] Nucleophilic aromatic substitution (SNAr) reactions with methylsulfonyl-tetrazole or methylsulfonyl-oxadiazole functional groups As disclosed herein, a linker L can be attached to Z to provide a linked payload, where Z further comprises a therapeutic agent X, e.g., a linked therapeutic agent. In some embodiments, the linker L comprises two methylsulfonyl-tetrazole functional groups or two methylsulfonyl-oxadiazole functional groups. The methylsulfonyl-tetrazole or methylsulfonyl-oxadiazole functional groups can be used to attach Z to a different moiety, such as the Fc region of an antibody or fragment thereof, via a nucleophilic aromatic substitution reaction.

[0180] In nucleophilic aromatic substitution reactions, the methylsulfonyl of tetrazole or oxadiazole acts as a leaving group that is displaced by the nucleophile to form a new covalent bond between the nucleophilic atom and the electrophilic carbon atom, as shown below.

[0181] [ka]

[0182] A variety of nucleophiles can be used to react with the electrophilic methylsulfonyltetrazole or methylsulfonyloxadiazole functional group. Suitable nucleophiles include moieties containing one or more nitrogen and / or sulfur atoms in the functional group, which can function as electron-rich nucleophiles. Suitable examples of nucleophiles include thiol-containing moieties, as shown below.

[0183] [ka]

[0184] In particular, the amino acid cysteine, as shown below, is found in many peptides, antibodies, proteins, etc. and contains a thiol functional group that can be used as a nucleophile to form a thioether between a peptide, antibody, protein, etc. and a linker, linked payload, and / or linked therapeutic agent.

[0185] [ka]

[0186] The conditions for aromatic nucleophilic substitution reactions are well known to those skilled in the art. The conditions may vary depending on the selected nucleophile and electrophile used in a particular reaction. As disclosed herein, there are compounds and / or conjugated compounds that include linked payloads that are attached to another moiety via an aromatic nucleophilic substitution reaction.

[0187] Nucleophilic substitution with the bromoacetyl functional group (S N 2) Reaction As disclosed herein, a linker L can be attached to Z to provide a linked payload, or, when Z includes a therapeutic agent, to provide a linked therapeutic agent. The linkers disclosed herein contain two or more, or preferably two, bromoacetyl functional groups, as shown in formula VD. The bromoacetyl functional groups can be used to link Z to a different moiety, such as an Fc region, via a nucleophilic substitution reaction.

[0188] In a nucleophilic substitution reaction, the bromide in bromoacetyl acts as a leaving group that is displaced by the nucleophile to form a new covalent bond between the nucleophilic atom and the electrophilic carbon atom as shown below.

[0189] [ka]

[0190] A variety of nucleophiles can be used to react with the electrophilic bromoacetyl functional group. Suitable nucleophiles include moieties containing one or more nitrogen and / or sulfur atoms in the functional group, which can function as electron-rich nucleophiles. Suitable examples of nucleophiles include moieties containing one or more of the following functional groups: amide, amine, imine, sulfonamide, thiol, thiourea, urea, or combinations thereof, as shown in the following structures:

[0191] [ka]

[0192] In particular, the amino acid cysteine, as shown below, is found in many peptides, antibodies, proteins, etc. and contains a thiol functional group that can be used as a nucleophile to form a thioether between a peptide, antibody, protein, etc. and a linker, linked payload, and / or linked therapeutic agent.

[0193] [ka]

[0194] The conditions for nucleophilic substitution reactions are well known to those skilled in the art. The conditions may vary depending on the selected nucleophile and electrophile used in a particular reaction. Disclosed herein are compounds and / or conjugate compounds that include linked payloads that are attached to another moiety via a nucleophilic substitution reaction.

[0195] Duration of action The compounds described herein include features that extend the duration of action of a therapeutic polypeptide. These features include conjugation to an antibody or fragment thereof, e.g., the Fc region; albumin-binding moieties; acylation of the therapeutic polypeptide with, e.g., a fatty acid; and / or incorporation of structural features into the therapeutic polypeptide itself that result in enhanced proteolytic stability. In certain embodiments, compounds incorporating each or all of these features have an extended duration of action, allowing for less frequent administration compared to currently available therapies. For example, in the context of therapeutic polypeptides that have binding and activity at one or more of the GIP, GLP-1, and / or glucagon receptors, certain compounds described herein have a duration of action that allows for their use in treatment methods with infrequent dosing, such as once a month.

[0196] Also disclosed herein are methods for increasing the duration of action of a therapeutic agent, which can include attaching a therapeutic agent to a trivalent linker to provide a linker therapeutic agent.

[0197] The method can further include conjugating the linked therapeutic agent to at least a portion of the antibody or fragment thereof, e.g., the Fc region, via a nucleophilic addition or substitution reaction, which occurs between the linked therapeutic agent and two nucleophiles (such as a sulfur atom and / or anion) present in the Fc region, resulting in the formation of a conjugate compound.

[0198] The resulting conjugate compound contains a therapeutic molecule covalently attached to the disclosed linker, which is then conjugated to an Fc fragment. The conjugation extends the duration of action beyond that of the unconjugated therapeutic agent.

[0199] In certain embodiments, the linkers disclosed herein offer notable advantages because the reaction conditions are mild compared to other nucleophilic addition or substitution reactions disclosed using other linkers. For example, the disclosed linkers can be combined with the Fc region of an antibody or fragment thereof with minimal heating. Specifically, the disclosed linkers can be conjugated to the Fc region of an antibody or fragment thereof by mixing the linked therapeutic agent with the reduced Fc region for no more than 2 hours at a temperature not exceeding 30°C, or in some cases at 25°C.

[0200] Linker Preparation Preparation 1 4-(bis(2-((tert-butoxycarbonyl)amino)ethyl)amino)-4-oxobutanoic acid

[0201] [ka] A solution of succinic anhydride (0.990 g, 9.88 mmol) in DCM (40 mL) was added to a solution of 10-oxa-2,5,8-triazadodecanoic acid, 11,11-dimethyl-9-oxo-,1,1-dimethylethyl ester (3.0 g, 9.88 mmol) in DCM (40 mL) over 30 minutes at ambient temperature under N. The reaction was stirred at ambient temperature for 48 hours, diluted with HO (50 mL), and extracted with DCM (3 × 40 mL). The organic layers were combined and washed with saturated aqueous NHCl, aqueous NaHCO, and brine. The organic layer was dried over MgSO, filtered, and concentrated in vacuo. The material was adsorbed onto silica gel with DCM and then concentrated under reduced pressure to give a free-flowing residue. The residue was purified by normal phase silica gel chromatography, eluting with DCM (5 min) and then DCM / MeOH (9:1) to give the title compound (2.84 g, 70%) as a white powder. ES / MS (m / z): 402 (M-).

[0202] Preparation 2 1-(4-(5-(methylsulfonyl)-1H-tetrazol-1-yl)phenoxy)-12-(2-(2-(2-(2-(4-(5-(methylsulfonyl)-1H-tetrazol-1-yl)phenoxy)ethoxy)ethoxy)acetamido)ethyl)-8,13-dioxo-3,6-dioxa-9,12-diazahexadecan-16-oic acid

[0203] [ka] 4-(bis(2-((tert-butoxycarbonyl)amino)ethyl)amino)-4-oxobutanoic acid (0.47 g, 1.05 mmol) was dissolved in DCM (3 mL) and then TFA (2 mL) was added. The reaction was stirred at ambient temperature for 3 hours and then concentrated in vacuo. Toluene (10 mL) was added to the residue and the mixture was concentrated in vacuo. This process was repeated once more. The residue was dried under vacuum for 2 hours and then dissolved in DMF (5 mL). DIEA (0.41 g, 3.15 mmol) was added, followed by a solution of 2,5-dioxopyrrolidin-1-yl 2-(2-(2-(4-(5-(methylsulfonyl)-1H-tetrazol-1-yl)phenoxy)ethoxy)ethoxy)acetate (1.07 g, 2.10 mmol) in DMF (4 mL). The reaction was stirred at ambient temperature for 1 hour, then diluted with DCM (100 mL) and washed with brine followed by HO. The organic layer was dried over NaSO, filtered, and concentrated in vacuo. The residue was purified by normal phase silica gel chromatography. The column was eluted with DCM for 5 minutes, then 0% to 20% methanol / DCM over 20 minutes to afford the title compound (0.86 g, 82%) as a white solid. ES / MS (m / z): 940 (M+H).

[0204] Preparation 3 (S)-2-((tert-butoxycarbonyl)amino)-4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoic acid

[0205] [ka]

[0206] 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 approximately 6 by adding 5N HCl. The aqueous solution was extracted with EtOAc (30 mL) and chloroform / isopropanol (3 × 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 chromatography eluting with 0-30% MeOH in DCM to give the title compound (580 mg, 81%). ES / MS m / z: 199 (M-tBu).

[0207] Preparation 4 2,5-Dioxopyrrolidin-1-yl (S)-2-((tert-butoxycarbonyl)amino)-4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoate

[0208] [ka] 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).

[0209] Preparation 5 (S)-2-((tert-butoxycarbonyl)amino)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoic acid

[0210] [ka] (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 solution 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).

[0211] Preparation 6 2,5-Dioxopyrrolidin-1-yl (S)-2-((tert-butoxycarbonyl)amino)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate

[0212] [ka] 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).

[0213] Fc area In certain embodiments, the nucleophile capable of reacting with two or more functional groups on the linker is an Fc region, also known as a fragment crystallizable region. The Fc region comprises the lower constant domain consisting of a complete or partial hinge along with the CH2 and CH3 domains or portions thereof. This Fc fragment may have effector function by fully binding to Fc receptors, or may contain mutations that limit or abolish Fc receptor binding. In particular, the Fc region of certain antibody classes, such as IgA, IgD, IgE, IgG, IgM, or a combination thereof, may contain two or more cysteine ​​residues that form disulfide bridges within the N-terminal portion of the molecule or hinge region.

[0214] In some embodiments, the Fc region of an antibody may be modified prior to attachment to a linker via a nucleophilic conjugate addition reaction. Such modifications may be designed to minimize unintended reactivity between the antibody and the linker, ensuring that the desired nucleophilic conjugation reaction proceeds efficiently.

[0215] In certain embodiments, the Fc region may be a modified version of an antibody Fc domain, with alterations including blunting or removal of particular segments such as the hinge region.

[0216] Disulfide bridges within the appropriate Fc region can be reduced to provide two thiol groups that function as nucleophiles capable of undergoing nucleophilic conjugate addition reactions with the maleimide functional groups of the linkers described herein.

[0217] Alternatively, reduced disulfide bridges in suitable Fc regions may provide thiol nucleophiles that can participate in aromatic nucleophilic addition reactions with the methylsulfonyltetrazole or methylsulfonyloxadiazole functional groups of the linkers described herein.

[0218] Exemplary Fc Regions The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions.

[0219] As used herein, the term "variant Fc region," "Fc region variant," or "Fc region mutation" refers to the amino acid sequence of an Fc region that differs from the sequence of a parent Fc region (or fragment thereof) by at least one amino acid substitution.

[0220] As used herein, an "amino acid substitution" refers to the replacement of at least one existing amino acid residue in a given amino acid sequence with another, different, "replacement" amino acid residue.

[0221] Furthermore, substitutions are named herein by the amino acid in the parent Fc, followed by the position number where the substitution occurs, followed by the amino acid that replaces the amino acid in the parent Fc region at the same position (e.g., human IgG1 Fc region variant P247I indicates that the proline residue at position 247 of the parent human IgG1 Fc region has been replaced by an isoleucine residue).

[0222] In one embodiment, a human IgG heavy chain Fc region extends from Cys229 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0223] In certain embodiments, the human IgG4 Fc amino acid sequence (hIgG4) is utilized for re-crosslinking conjugation.

[0224] [Table 2]

[0225] The Fc region of SEQ ID NO: 935 is a native IgG4 with a blunt hinge beginning at position 229 (EU index numbering) with an additional N-terminal dipeptide composed of alanine and glycine (AG). To minimize effector function, the Fc contains two mutations in the CH2 domain, F234A and L235A (EU index numbering), as shown in bold and underlined. Unless otherwise specified, references to "Fc" in the nomenclature of the compounds set forth in Table 3 below and described herein refer to SEQ ID NO: 935.

[0226] An IgG4 Fc region containing the mutations Q274K, Q355R, and E419Q (SEQ ID NO: 936) can be used to increase the overall pI of the Fc to aid in biophysical properties when more neutral peptides are used for conjugation.

[0227] [Table 3]

[0228] Unless otherwise specified, references to "Fc KRQ" in the nomenclature of the compounds set out in Table 3 below and described herein refer to SEQ ID NO:936.

[0229] To mitigate N-terminal misprocessing in the CHO expression system, several specific signal peptides were identified that provide high titer and minimal N-terminal clipping of the first alanine residue.

[0230] [Table 4]

[0231] Instead of changing the signal peptide, six alternative residues were explored for the N-terminal alanine, including cysteine ​​(C), arginine (R), isoleucine (I), leucine (L), tyrosine (Y), and threonine (T), all of which prevented clipping. Alternatively, any of 20 amino acids, excluding alanine, serine, and glycine, can be used to replace the N-terminal residue. Examples include an isoleucine N-terminal Fc (Fc IG KRQ) and a cysteine ​​N-terminal KRQ Fc (Fc N-Cys KRQ), which contain the F234A, L235A, Q274K, Q355R, and E419Q mutations, respectively, as shown in the following sequences:

[0232] [Table 5]

[0233] Another example is a cysteine ​​N-terminal Fc (Fc N-Cys), which contains the F234A and L235A mutations as shown in the following sequence:

[0234] [Table 6]

[0235] Unless otherwise specified, references to "Fc IG KRQ" in the nomenclature of the compounds shown in Table 3 below refer to SEQ ID NO: 941, references to "Fc N-Cys KRQ" in the nomenclature of the compounds shown in Table 3 below refer to SEQ ID NO: 942, and references to "Fc N-Cys" in the nomenclature of the compounds shown in Table 3 below refer to SEQ ID NO: 1178.

[0236] Besides the IgG4 subclass, the IgG1 Fc was also probed using an IgG1 with effector knockout mutations F234A and L235A and P329A (EU index numbering).

[0237] [Table 7]

[0238] In some embodiments, a wild-type IgG1 Fc with full effector function can be utilized in place of the effector knockout version.

[0239] [Table 8]

[0240] As an alternative to the re-crosslinking approach described above, engineered cysteines were introduced at positions 358, 398, and 415 of an IgG4 Fc with an intact hinge containing the S228P mutation, starting at position 216, to enhance hinge stability. The Fc fragment contains two engineered cysteines, resulting in a symmetric molecule with each half or two peptides per Fc conjugate.

[0241] [Table 9]

[0242] To generate an Fc with a single peptide conjugated via an engineered cysteine, an Fc lacking an eCys site and with an intact hinge starting at position 216 was generated using the mutations F405L and R409K in the CH3 domain. Using an Fc swap approach, the half-Fc lacking the eCys site was paired with a half-Fc containing the desired eCys site, followed by conjugation.

[0243] [Table 10]

[0244] In certain embodiments, compounds of the present disclosure comprise an Fc region having at least 90% sequence identity, or at least 95% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to any of SEQ ID NOs: 935-936, 941-948, 1178-1181.

[0245] General methodology and conditions for addition of FC Scheme 1

[0246] [ka] Scheme 1, Step A, shows H2N-PEG produced by solid-phase peptide synthesis using the Fmoc / t-Bu strategy on a Classic Symphony automated peptide synthesizer, starting from RAPP AM-Rink Amide resin. 24Step B shows the synthesis of the PEGylated peptide 2 to give compound 2. Step B shows the reaction of PEGylated peptide 2 with a bis-Fmoc-protected trivalent core (U is either a 1,3,5-trisubstituted phenyl or a tertiary amine (Preparation 1)) to give compound 3. In Step C, the Fmoc group was removed, and the resulting primary amine was reacted with NHS ester MalDap (commercially available), MalDab (Preparation 15), or β-MalDap (Preparation 17) to give compound 4. In Step D, the peptide was cleaved from the resin. The bismaleimide functionality reacted with two reduced thiol functional groups in the Fc region in a nucleophilic conjugate addition reaction to give conjugate 5.

[0247] Scheme 2

[0248] [ka] Scheme 2, Step A shows H2N-PEG produced by solid-phase peptide synthesis. 24 Step B shows the synthesis of the PEGylated peptide 21 to give compound 1. Step B shows the coupling of the PEGylated peptide 21 with a bis-(methylsulfonyl)tetrazole analog (Preparation 12) to give compound 2. In Step C, the peptide was cleaved from the resin. The bis-(methylsulfonyl)tetrazole functionality reacted with two reduced thiol functional groups on the Fc region in a nucleophilic conjugate addition reaction to give conjugate 3.

[0249] Scheme 3

[0250] [ka] Scheme 3, Step A shows H2N-PEG produced by solid-phase peptide synthesis. 24Step B shows the synthesis of a PEGylated peptide (2) to give compound (1). Step B shows the coupling of PEGylated peptide (2) with a bis-Fmoc-protected trivalent core (U is either a 1,3,5-trisubstituted phenyl or a tertiary amine (Preparation 1)) to give compound (2). Step C shows the removal of the Fmoc group and the reaction of the resulting primary amine with 2,5-dioxopyrrolidin-1-yl 2-bromoacetate to give compound (3). The functional groups of compound (3) react with two reduced thiol functional groups in the Fc region in a nucleophilic conjugate addition reaction to give conjugate (4).

[0251] Therapeutic Polypeptides The term "therapeutic polypeptide" refers to any amino acid polymer having activity suitable for use in the treatment and / or prevention of a disease or condition. The conjugate structures herein can be used to provide an extended duration of action for various therapeutic polypeptides. Non-limiting examples of such therapeutic agents include the following, or variants thereof: GIP, GLP-1, GLP-2, GIP, oxyntomodulin (OXM), amylin, apelin, urocortin (UCN), ANP, BNP, CNP, parathyroid hormone (PTH), MC4 agonist, somatostatin, GDF15, peptide YY (PYY), pancreatic polypeptide, ACTH, VIP, oxytocin, ghrelin, gastrin, insulin, human growth hormone (hGH), fibroblast growth factor 21 (FGF21), interleukin 2 (IL-2), and IL-1. 2, IL2), IL10, IL15, IL22, IL7, pepstatin, calcitonin, erythropoietin (EPO), thrombopoietin and receptor agonists, angiotensin, vasopressin, thymosin beta-4, guanylate cyclase-C agonists, epidermal growth factor (EGF), alpha-1-antitrypsin, corticotropin releasing hormone (CRH), GnRH, thymosin alpha-1, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), thyroid releasing hormone (TRH), melanacortin stimulating hormone (MSH), hormone (MSH), apo-CII mimetic peptide, factor IX, factor VIII, factor X, granulocyte colony-stimulating factor, interferon-α, β-amyloid inhibitor, CD40 ligand, CD137 agonist CD24, CTLA4, TNFR, ENPP1, SIRP-α, and urinstatin.

[0252] In certain embodiments, the therapeutic polypeptide comprises an agonist at one or more of the GIP, GLP-1, and glucagon receptors. GIP is a 42 amino acid peptide and an incretin that plays a physiological role in glucose homeostasis by stimulating insulin secretion from pancreatic beta cells in the presence of glucose. GLP-1 is a 36 amino acid peptide and an incretin that stimulates glucose-dependent insulin secretion and has been shown to prevent hyperglycemia in diabetic patients. The primary biologically active fragment of GLP-1 is a 30 amino acid C-terminal amidated peptide (GLP-1 7-36 Glucagon is a 29-amino acid peptide that helps maintain blood glucose by binding to and activating glucagon receptors on liver cells, causing the liver to release glucose stored in the form of glycogen through a process called glycogenolysis.

[0253] In certain embodiments, exemplary compounds of the present disclosure include dual agonists at the GIP receptor and the GLP-1 receptor, hi some other embodiments, exemplary compounds of the present disclosure include triple agonists at the GIP, GLP-1, and glucagon receptors.

[0254] Compounds of the present disclosure having activity at one or more of the GIP, GLP-1 and / or glucagon receptors may be useful in treating T2DM, obesity, dyslipidemia, cardiovascular disease (CVD), major adverse cardiovascular events (MACE), chronic kidney disease (CKD), heart failure (HF), hypertension (HT), peripheral arterial disease (PAD), obstructive sleep apnea (OSA), metabolic syndrome, type 2 diabetes mellitus (T2DM), glycemic control, osteoarthritis (OA), chronic lower back pain (CLBP), neurodegenerative and / or cognitive disorders, such as Alzheimer's disease (AD), Parkinson's disease, metabolic steatohepatitis (MASH), cirrhosis, fatty liver disease, and the like. It has potential therapeutic value in many conditions, diseases or disorders, including, but not limited to, non-alcoholic fatty liver disease (FLD), polycystic ovary syndrome and / or alcohol abuse disorder, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), metabolic syndrome, and bone-related disorders.For example, Jall et al. (2017) Mol.Metab.6:440-446, Carbone et al. (2016) J.Gastroenterol.Hepatol.31:23-31, Finan et al. (2016) Trends Mol.Med.22:359-376, Choi et al. (2017) Potent body weight loss and efficacy in a NASH animal model by a novel long-acting GLP-1 / Glucagon / GIP triple-agonist(HM15211),ADA Poster 1139-P, Ding(2008)J.Bone Miner.Res.23:536-543, Tai et al.(2018)Brain Res.1678:64-74, Muller et al. al.(2017)Physiol.Rev.97:721-766, Finan et al. See al. (2013) Sci. Transl. Med. 5:209, Holscher (2014) Biochem. Soc. Trans. 42:593-600.

[0255] In certain embodiments, the therapeutic polypeptide is X1X2X3X4TX6TSDX 10 X 11 X 12 X 13 LX 15 X 16 KAX 19 X 20 X 21 FIX 24 X 25 LX 27 X 28 X 29 X 30 X 31 X 32 X 33 X 34 X 35 X 36 X 37 X 38 X 39 X 40 X 41 (SEQ ID NO: 1218), X1 is H, NMeY, or Y; X2 is Ac4c, Aib, αMeS, Iva, or D-Ala; X3 is Q, E, or H; X4 is G or D-Ala, X6 is αMeF(2F), F, or αMeF; X 10 is 4-Pal, Y, or V, X 11 is Aib, S, or αMeS, X 12 is I or S, X 13 is L or αMeL, X 15 is D or E, X 16 is K, E, or Orn, X 19 is A or Q, X 20 is Aib, αMeL, Iva, or αMe4Pal, X 21 is E, Q, D, or Orn, X 24 is K, Q, E, D-Glu, or D-Gln, X 25 is αMeY, W, or Y, X 27 is I, L, or V, X 28 is K, E, or A, X 29 is Aib, G, or Q, X 30 is G or S, X 31 is P, G, E, or Orn, X 32 is absent, S or P, X 32 If is S or P, then X 33 is S, X 33 If S, then X 34 is G or Aib, X 34 If is G or Aib, then X 35 is absent or is A or Orn, X 35 If is A or Orn, then X 36 is absent or is P, X 36 If P, then X 37 is absent or is P, X 37 If P, then X 38 is absent or is P, X 38 If P, then X 39 is absent or is S, Orn, or G, X 39 If is S, Orn, or G, then X 40 is absent, K, or G, X 40 If is K or G, then X 41 is absent or is S or G, X 32 If does not exist, X 33 ~X 41 There is no X 35 If does not exist, X 36 ~X 41 There is no X 36 If does not exist, X 37 ~X 41 There is no X 37 If does not exist, X 38 ~X 41 There is no X 38 If does not exist, X 39 ~X 41 There is no X 39 If does not exist, X 40 and X 41 There is no X 40 If does not exist, X 41 does not exist.

[0256] In certain embodiments, the therapeutic polypeptide comprises 90% sequence identity to any of SEQ ID NO:1 through SEQ ID NO:186 or SEQ ID NO:996 through SEQ ID NO:1038. In certain embodiments, the therapeutic polypeptide comprises any of SEQ ID NO:1 through SEQ ID NO:186 or SEQ ID NO:996 through SEQ ID NO:1038.

[0257] In certain embodiments, the therapeutic polypeptide is YAibEGTX6TSDX 10 X 11 X 12 X 13 LX 15 X 16 KAQX 20 X 21 FIX 24 X 25 LX 27 X 28 X 29 X 30 X 31 X 32 X 33 X 34 X 35 X 36 X 37 X 38 X 39 X 40 X 41 (SEQ ID NO: 1219), X6 is αMeF(2F), F, or αMeF; X 10 is 4-Pal, Y, or V, X 11 is Aib or S, X 12 is I or S, X 13 is L or αMeL, X 15 is D or E, X 16 is E or Orn, X 20 is Aib, αMeL, or Iva, X 21 is E, Q, D, or Orn, X 24is K, Q, E, or D-Glu; X 25 is αMeY or Y, X 27 is I, L, or V, X 28 is K, E, or A, X 29 is Aib, G, or Q, X 30 is G or S, X 31 is P, G, E, or Orn, X 32 is absent, S or P, X 32 If is S or P, then X 33 is S, X 33 If S, then X 34 is G or Aib, X 34 If is G or Aib, then X 35 is absent, A, or Orn, X 35 If is A or Orn, then X 36 is absent or is P, X 36 If P, then X 37 is absent or is P, X 37 If P, then X 38 is absent or is P, X 38 If P, then X 39 is absent, S, Orn, or G, X 39 If is S, Orn, or G, then X 40 is absent, K, or G, X 40 If is K or G, then X 41 is absent, S, or G, X 32 If does not exist, X 33 ~X 41 There is no X 35If does not exist, X 36 ~X 41 There is no X 36 If does not exist, X 37 ~X 41 There is no X 37 If does not exist, X 38 ~X 41 There is no X 38 If does not exist, X 39 ~X 41 There is no X 39 If does not exist, X 40 and X 41 There is no X 40 If does not exist, X 41 does not exist.

[0258] In some embodiments, the polypeptide of SEQ ID NO: 1219 has dual agonist activity at the GIP receptor and the GLP-1 receptor. In some embodiments, the polypeptide comprises at least one of the following: X 10 is Y and X 11 is Aib and X 24 is E and X 30 is S. In some embodiments, the polypeptide comprises at least two of the following: X 10 is Y and X 11 is Aib and X 24 is E and X 30 is S. In some embodiments, the polypeptide comprises each of the following: X 10 is Y and X 11 is Aib and X 24 is E and X 30 is S.

[0259] In some embodiments, the polypeptide further comprises at least one of the following: X6 is αMeF(2F), and X 12 is I and X 13 is αMeL and X 15is D and X 16 is Orn and X 20 is Aib and X 21 is E or Q, and X 25 is αMeY and X 27 is I and X 28 is K or E, and X 29 is Aib or G, and X 31 is P or G, and X 32 is absent or is S and X 40 In some embodiments, the polypeptide comprises each of the following: X6 is αMeF(2F), and X 12 is I and X 13 is αMeL and X 15 is D and X 16 is Orn and X 20 is Aib and X 21 is E or Q, and X 25 is αMeY and X 27 is I and X 28 is K or E, and X 29 is Aib or G, and X 31 is P or G, and X 32 is absent or is S and X 40 is K. In some embodiments, X 21 is E and X 28 is E and X 29 is G and X 31 is P and X 32 is S.

[0260] In certain embodiments, the therapeutic polypeptide comprises each of the following: X1 is Y, X2 is Aib, X3 is E, X4 is G, X6 is αMeF(2F), and X 10 is 4-Pal or Y, and X 11 is Aib or S, and X 12 is I and X 13 is αMeL and X 15 is D and X 16 is E or Orn, X1 is Q, and X20 is Aib and X 21 is E and X 24 is Q, E, or D-Glu, and X 25 is αMeY and X 27 is I and X 28 is E and X 29 is Aib or G, and X 30 is G or S, and X 31 is P and X 32 is G or S, and X 32 If S, then X 33 is S and X 34 is G and X 35 is A and X 36 is P and X 37 is P and X 38 is P and X 39 is S and X 40 is K and X 41 does not exist, and X 32 If does not exist, X 33 ~X 41 In certain embodiments, the therapeutic polypeptide comprises at least one of the following: X 10 is Y or 4-Pal, and X 11 is Aib or S, and X 24 is Q, E, or d-Glu, and X 30 is G or S. In certain embodiments, the therapeutic polypeptide comprises each of the following: X 10 is Y or 4-Pal, and X 11 is Aib and X 24 is E and X 30 is S and X 40 is K. In some embodiments, one or more of the above amino acid modifications result in a therapeutic polypeptide with improved proteolytic stability. In some embodiments, one or more of the above amino acid modifications result in a therapeutic polypeptide with increased activity at the GIP and / or GLP-1 receptor.

[0261] In some embodiments, a therapeutic polypeptide comprises 90% sequence identity to any of SEQ ID NO:1-92 or SEQ ID NO:996-999. In some embodiments, a therapeutic polypeptide comprises one or more conservative amino acid substitutions relative to any of SEQ ID NO:1-92 or SEQ ID NO:996-999. In some embodiments, a therapeutic polypeptide comprises no more than five, no more than four, no more than three, no more than two, or no more than one conservative amino acid substitution relative to any of SEQ ID NO:1-92 or SEQ ID NO:996-999. In some embodiments, a therapeutic polypeptide comprises any of SEQ ID NO:1-92 or SEQ ID NO:996-999. In some embodiments, a therapeutic polypeptide comprises any of SEQ ID NO:1, 2, 22, 43, 45, 48, 55, 56, 58, 59, 61, 66, 68, and 47.

[0262] In other embodiments, the therapeutic polypeptide is X1X2X3X4TX6TSDX 10 X 11 X 12 X 13 LX 15 X 16 KAQX 20 X 21 FIX 24 X 25 LX 27 X 28 X 29 X 30 X 31 X 32 X 33 X 34 X 35 X 36 X 37 X 38 X 39 X 40 X 41 (SEQ ID NO: 1220), X1 is H, NMeY, or Y; X2 is Ac4c, Aib, αMeS, Iva, or D-Ala; X3 is Q, E, or H; X4 is G or D-Ala, X6 is αMeF(2F), F, or αMeF; X 10 is 4-Pal, Y, or V, X 11 is Aib, S, or αMeS, X 12 is I or S, X 13 is L or αMeL, X 15 is D or E, X 16 is E or Orn, X 20 is Aib, αMeL, Iva, or αMe4Pal, X 21 is E, Q, D, or Orn, X 24 is K, Q, E, D-Glu, or D-Gln, X 25 is αMeY or Y, X 27 is I, L, or V, X 28 is K, E, or A, X 29 is Aib, G, or Q, X 30 is G or S, X 31 is P, G, E, or Orn, X 32 is absent, S or P, X 32 If is S or P, then X 33 is S, X 33 If S, then X 34 is G or Aib, X 34 If is G or Aib, then X 35 is absent or is A or Orn, X 35 If is A or Orn, then X 36 is absent or is P, X36 If P, then X 37 is absent or is P, X 37 If P, then X 38 is absent or is P, X 38 If P, then X 39 is absent or is S, Orn, or G, X 39 If is S, Orn, or G, then X 40 is absent, K, or G, X 40 If is K or G, then X 41 is absent or is S or G, X 32 If does not exist, X 33 ~X 41 There is no X 35 If does not exist, X 36 ~X 41 There is no X 36 If does not exist, X 37 ~X 41 There is no X 37 If does not exist, X 38 ~X 41 There is no X 38 If does not exist, X 39 ~X 41 There is no X 39 If does not exist, X 40 and X 41 There is no X 40 If does not exist, X 41 does not exist.

[0263] In certain embodiments, the therapeutic polypeptide of SEQ ID NO: 1220 has triagonist activity at the GIP, GLP-1, and glucagon receptors. 10 is Y and X 24 is E and X 30is S and X 40 is K. In some embodiments, the therapeutic polypeptide comprises at least one of 10 is Y and X 24 is E and X 30 is S and X 40 K.

[0264] In some embodiments, the therapeutic polypeptide comprises at least one of the following: X1 is H or Y, X2 is Ac4c or Aib, X3 is Q, X4 is G, X6 is αMeF(2F), and X 11 is αMeS, and X 12 is I and X 13 is αMeL and X 15 is D and X 16 is Orn and X 20 is αMe4Pal, and X 21 is E or Orn, and X 25 is αMeY and X 27 is I and X 28 is E and X 29 is Aib or G, and X 31 is P and X 32 is S and X 33 is S and X 34 is G and X 35 is A and X 36 is P and X 37 is P and X 38 is P and X 39 is S. In some embodiments, a therapeutic polypeptide comprises each of the following: X1 is H or Y, X2 is Ac4c or Aib, X3 is Q, X4 is G, X6 is αMeF(2F), and X 11 is αMeS, and X 12 is I and X 13 is αMeL and X 15 is D and X 16 is Orn and X 20 is αMe4Pal, and X 21is E or Orn, and X 25 is αMeY and X 27 is I and X 28 is E and X 29 is Aib or G, and X 31 is P and X 32 is S and X 33 is S and X 34 is G and X 35 is A and X 36 is P and X 37 is P and X 38 is P and X 39 is S. In some embodiments, X1 is Y, X2 is Aib, X3 is Q, and X 21 is E and X 29 is G.

[0265] In some embodiments, the therapeutic polypeptide comprises each of the following: X1 is H or Y, X2 is Aib, X3 is Q, X4 is G, X6 is αMeF(2F), and X 10 is Y or 4-Pal, and X 11 is S or αMeS, and X 12 is I and X 13 is αMeL and X 15 is D and X 20 is Aib or αMe4Pal, and X 21 is E or Orn, and X 24 is E or d-Glu, and X 25 is αMeY and X 27 is L or I, and X 28 is E and X 29 ~X 39 is GSPSSGAPPPS, X 40 is K. In some embodiments, the therapeutic polypeptide comprises at least one of the following: X 10 is Y or 4-Pal, and X 24 is E or d-Glu, and X 30 is S. In some embodiments, the therapeutic polypeptide comprises each of the following: X10 is Y or 4-Pal, and X 24 is E or d-Glu, and X 30 is S and X 40 is K and is conjugated to a linker.

[0266] In some embodiments, one or more of the above amino acid modifications result in a therapeutic polypeptide with improved proteolytic stability, hi some embodiments, one or more of the above amino acid modifications result in a therapeutic polypeptide with increased activity at the GIP and / or GLP-1 receptors.

[0267] In some embodiments, a therapeutic polypeptide comprises 90% sequence identity to any of SEQ ID NOs: 93-186 or 1000-1038. In some embodiments, a therapeutic polypeptide comprises one or more conservative amino acid substitutions relative to any of SEQ ID NOs: 93-186 or 1000-1038. In some embodiments, a therapeutic polypeptide comprises no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 conservative amino acid substitution relative to any of SEQ ID NOs: 93-186 or 1000-1038. In some embodiments, a therapeutic polypeptide comprises any of SEQ ID NOs: 93-186 or 1000-1038. In some embodiments, a therapeutic polypeptide comprises any of SEQ ID NOs: 97, 113, 128, 130, 144, 171, 183.

[0268] The structural features of the compounds described in certain embodiments herein allow them to have adequate activity at each of the GIP, GLP-1, and glucagon receptors (i.e., triple agonist activity) to obtain the desired effects of activity at each receptor, but not so much activity at any one receptor that it would either overwhelm the activity at the other two receptors or result in undesirable side effects when administered at doses sufficient to provide activity at all three receptors.

[0269] The affinity of the polypeptides described herein for each of the GIP, GLP-1, and glucagon receptors can be measured using techniques known in the art for measuring receptor binding levels, and is generally expressed as an inhibition constant (Ki) value. The activity of the polypeptides described herein at each of the receptors can also be measured using techniques known in the art, including, for example, the in vitro activity assays described below, and is generally expressed as an effective concentration 50 (EC 50 ) value, which is the concentration of compound that causes half-maximal simulation of the dose-response curve.

[0270] In some embodiments, the polypeptides described herein are partial agonists at the GLP-1 receptor, as demonstrated by the HEK293 cell GLP-1 receptor internalization assay described herein, which is comparable to the activity of native GLP-1. 7~36 (SEQ ID NO: 1221). In other embodiments, the polypeptides described herein are full agonists at the GLP-1 receptor, as demonstrated by the HEK293 cell GLP-1 receptor internalization assay described herein, which shows 80% or less agonism compared to native GLP-1. 7~36 In some embodiments, the polypeptides described herein exhibit 80% or greater agonism compared to native glucagon (SEQ ID NO: 1222), GIP (SEQ ID NO: 1223), and GLP-1 7-36 (SEQ ID NO: 1221) has greater potency at one or more of the glucagon, GIP and GLP-1 receptors.

[0271] In addition to the sequences described herein, the polypeptides described herein may include one or more conservative amino acid substitutions, provided that the polypeptide is still capable of binding to and activating GIP, GLP-1, and glucagon receptors.

[0272] The structural features of certain embodiments of the compounds described herein also result in the compounds possessing many other beneficial attributes relevant to their potential development as therapeutic treatments, including improved solubility of the analogs in aqueous solutions near neutral pH, improved chemical and physical formulation stability, improved peptide membrane permeability in the presence of penetration enhancers, an extended pharmacokinetic profile, and minimized potential for injection site reactions or immunogenicity.

[0273] It should be noted that the combination of beneficial features of the exemplary analogs described herein is not the result of any single modification in isolation, but instead is achieved through novel combinations of the structural features described herein.

[0274] In some embodiments, the polypeptides described herein are amidated. In some embodiments, the polypeptides described herein have a C-terminal group modification, where the modification is NH2 or absent. In some embodiments, the polypeptides described herein have an OH group at the C-terminus. A non-limiting list of exemplary therapeutic polypeptides is provided in Table 1 below.

[0275] [Table 11-1]

[0276] [Table 11-2]

[0277] [Table 11-3]

[0278] [Table 11-4]

[0279] [Table 11-5]

[0280] [Table 11-6]

[0281] [Table 11-7]

[0282] [Table 11-8]

[0283] [Table 11-9]

[0284] Acylation of therapeutic polypeptides As previously mentioned, in some embodiments, certain polypeptides described herein include an albumin-binding moiety, such as a fatty acid moiety, conjugated, for example, by a direct bond or a linker, to a natural or unnatural amino acid having a functional group available for conjugation. Such conjugation is sometimes referred to as acylation. In some cases, the amino acid having a functional group available for conjugation can be K, C, E, and D. In a specific example, the amino acid having a functional group available for conjugation is K, and the conjugation is to the epsilon-amino group of the K side chain.

[0285] In some embodiments, the polypeptides described herein include C 1H 2 O 4 O 5 O 6 O 7 O 8 O 9 O 10 O 11 O 12 O 13 O 14 O 15 O 16 O 17 O 18 O 19 O 20 O 21 O 22 O 23 O 24 O 25 O 26 O 27 O 28 O 29 O 30 O 31 O 32 O 33 O 44 O 45 O 56 O 57 O 60 O 61 O 72 O 73 O 74 O 75 O 86 O 87 O 88 O 89 O 90 O 91 O 92 O 93 O 94 O 95 O 96 O 97 O 98 O 99 O 100 O 101 O 102 O 103 O 104 O 10 16 ~C 22The length and composition of fatty acids influence the half-life of polypeptides, their efficacy in vivo in animal models, and their solubility and stability. 16 ~C 22 Conjugation to saturated fatty mono- or diacids results in polypeptides that exhibit desirable half-lives, desirable in vivo efficacy in animal models, and desirable solubility and stability characteristics.

[0286] Saturated C for use herein 16 ~C 22 Examples of fatty acids include palmitic acid (hexadecanoic acid) (C 16 monoacid), hexadecanedioic acid (C 16 diacid), margaric acid (heptadecanoic acid) (C 17 monoacid), heptadecanedioic acid (C 17 diacid), stearic acid (C 18 monoacid), octadecanedioic acid (C 18 diacid), nonadecylic acid (nonadecanoic acid) (C 19 monoacid), nonadecanedioic acid (C 19 diacid), alachadic acid (eicosanoic acid) (C 20 monoacid), eicosanedioic acid (C 20 diacid), heneicosylic acid (heneicosanoic acid) (C 21 monoacid), heneicosanedioic acid (C 21 diacid), behenic acid (docosanoic acid) (C 22 monoacid), docosanedioic acid (C 22 Diacids), including, but not limited to, branched and substituted derivatives thereof.

[0287] In some cases, C 16 ~C 22 Fatty acids are saturated C 18 monoacid, saturated C 18 Diacid, saturated C 19 monoacid, saturated C 19 Diacid, saturated C 20 monoacid, saturated C 20 Diacids and branched and substituted derivatives thereof. 16 ~C 22 The fatty acid is octadecanedioic acid (C18 diacid) or eicosanedioic acid (C 20 diacid).

[0288] In some embodiments, the lysine at position 17 is conjugated to a fatty acid. 16 ~C 22 It is a fatty acid.

[0289] In some embodiments, the therapeutic polypeptide is conjugated to the fatty acid by a linker. In some embodiments, the linker comprises 1 to 5 amino acids. In examples where the linker comprises at least one amino acid, the amino acid can be 1 to 5 Glu or γGlu amino acid residues. In some examples, the linker can comprise 1, 2, 3, 4, or 5 Glu or γGlu amino acid residues, where the Glu or γGlu amino acid residues include their D-forms. For example, the linker can comprise any of 1, 2, 3, or 4 γGlu amino acid residues. Alternatively, the linker can comprise 1 to 5 (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) ("AEEA") and / or 1 to 5 amino acid residues (e.g., Glu or γGlu amino acids) used in combination with 1 to 5 εK moieties. Specifically, the linker can be a combination of 1-5 Glu or γGlu amino acids with 1-5 (2-[2-(2-aminoethoxy)-ethoxy]-acetyl) moieties, or a combination of 1-5 Glu or γGlu amino acids with 1-5 εK moieties. In some examples, the linker can be a combination of 1, 2, or 3 γGlu amino acids with 1 or 2 (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) or εK moieties.

[0290] For example, in some embodiments, the polypeptides described herein have the following formula: (γGlu) a -(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) b -(γGlu) c -CO-(CH2)p It has a linker having a —CO 2 H structure and a fatty acid moiety, wherein a is 0, 1, or 2, b is 0, 1, or 2, c is 0, 1, 2, or 3, and p is an integer of 14 to 20.

[0291] In some preferred embodiments, a is 0 or 1; b is 0, 1, or 2; c is 1, 2, or 3; and p is an integer from 14 to 20.

[0292] In some embodiments, a is 0, b is 1, c is 1 or 2, and p is 16 or 18.

[0293] For example, in some embodiments, a is 0, b is 1, c is 1, and p is 16, the structure of which is shown below.

[0294] [ka]

[0295] For example, in some embodiments, a is 0, b is 1, c is 1, and p is 18, the structure of which is shown below.

[0296] [ka]

[0297] In some embodiments, a is 0, b is 1, c is 2, and p is 16, the structure of which is shown below.

[0298] [ka]

[0299] In some embodiments, a is 0, b is 1, c is 2, and p is 18, the structure of which is shown below.

[0300] [ka]

[0301] In some embodiments, a is 0, b is 2, c is 1, and p is 16 or 18.

[0302] For example, in some embodiments, a is 0, b is 2, c is 1, and p is 16, the structure of which is shown below.

[0303] [ka]

[0304] In some embodiments, a is 0, b is 2, c is 1, and p is 18, the structure of which is shown below.

[0305] [ka]

[0306] In some embodiments, a is 0, b is 0, c is 2, and p is 16 or 18.

[0307] For example, in some embodiments, a is 0, b is 0, c is 2, and p is 16, the structure of which is shown below.

[0308] [ka]

[0309] In some embodiments, a is 0, b is 0, c is 2, and p is 18, the structure of which is shown below.

[0310] [ka]

[0311] In some embodiments, a is 0, b is 0, c is 3, and p is 16 or 18.

[0312] For example, in some embodiments, a is 0, b is 0, c is 3, and p is 16, the structure of which is shown below.

[0313] [ka]

[0314] In some embodiments, a is 0, b is 0, c is 3, and p is 18, the structure of which is shown below.

[0315] [ka]

[0316] In some embodiments, a is 1, b is 1, c is 1, and p is 16 or 18.

[0317] For example, in some embodiments, a is 1, b is 1, c is 1, and p is 16, the structure of which is shown below.

[0318] [ka]

[0319] For example, in some embodiments, a is 1, b is 1, c is 1, and p is 18, the structure of which is shown below.

[0320] [ka]

[0321] In some embodiments, the polypeptides described herein have the following formula: (γGlu) d -(εK) e -(γGlu) f -CO-(CH2) q It has a linker having a —CO 2 H structure and a fatty acid moiety, wherein d is 0, 1 or 2, e is 0, 1 or 2, f is 0, 1, 2 or 3, and q is an integer of 14 to 20.

[0322] For example, in one embodiment, d is 0, e is 2, f is 1, and q is an integer from 14 to 20. In some embodiments, d is 0, e is 2, f is 1, and q is 16 or 18.

[0323] For example, in some embodiments, d is 0, e is 2, f is 1, and q is 16, the structure of which is shown below.

[0324] [ka]

[0325] For example, in some embodiments, d is 0, e is 2, f is 1, and q is 18, the structure of which is shown below.

[0326] [ka]

[0327] A non-limiting list of exemplary acylated therapeutic polypeptides is provided in Table 2 below.

[0328] [Table 12-1]

[0329] Table 12-2

[0330] Table 12-3

[0331] Table 12-4

[0332] Table 12-5

[0333] Table 12-6

[0334] Table 12-7

[0335] Table 12-8

[0336] Table 12-9

[0337] Table 12-10

[0338] Table 12-11

[0339] Table 12-12

[0340] Table 12-13

[0341] Table 12-14

[0342] Table 12-15

[0343] Table 12-16

[0344] Table 12-17

[0345] Table 12-18

[0346] Table 12-19

[0347] Table 12-20

[0348] Table 12-21

[0349] Other definitions and abbreviations The amino acid sequences of the therapeutic polypeptides described herein typically incorporate naturally occurring amino acids, which are shown herein using the standard single-letter code (e.g., L=leucine), as well as α-methyl substituted residues of natural amino acids (e.g., α-methyl-leucine (αMeL)), and certain other unnatural amino acids, such as α-aminoisobutyric acid (Aib), the structures of which are shown below.

[0350] [ka]

[0351] [ka]

[0352] As used herein, "Orn" refers to L-ornithine. As used herein, "4-Pal" or "4Pal" refers to 3-(4-pyridyl)-L-alanine or (S)-2-amino-3-(pyridin-4-yl)propanoic acid. As used herein, "3-Pal" or "3Pal" refers to 3-(3-pyridyl)-L-alanine or (S)-2-amino-3-(pyridin-3-yl)propanoic acid. As used herein, "αMe-4-Pal" or "αMe4Pal" refers to alpha-methyl-3-(4-pyridyl)-L-alanine. As used herein, "αMeY" refers to alpha-methyl-L-tyrosine. As used herein, "αMeL" refers to alpha-methyl-leucine. As used herein, "Ac3c" refers to 1-aminocyclopropanecarboxylic acid. As used herein, "Ac4c" means 1-aminocyclobutane-1-carboxylic acid. As used herein, "D-Ala" and "a" each mean D-alanine. As used herein, "D-Glu" and "e" each mean D-glutamic acid. As used herein, "Aib" means 2-aminoisobutyric acid. As used herein, "NMeY" means N-methyl-tyrosine. As used herein, "Dap" means (S)-2,3-diaminopropanoic acid. As used herein, "Dab" means (S)-2,4-diaminobutanoic acid. As used herein, "Hyp" means hydroxy-L-proline. As used herein, "K(Ac)" means N 6As used herein, "γGlu" means gamma-L-glutamic acid. As used herein, "Aad" means (S)-2-aminohexanedioic acid. As used herein, "F(4CN)" means 4-cyano-L-phenylalanine or (S)-2-amino-3-(4-cyanophenyl)propanoic acid. As used herein, "F(4NO2)" means 4-nitro-L-phenylalanine or (S)-2-amino-3-(4-nitrophenyl)propanoic acid. As used herein, "αMeS" means alpha-methyl-L-serine. As used herein, "αMeF" means alpha-methyl-L-phenylalanine. As used herein, "αMeF(2F)" means alpha-methyl-2-fluoro-L-phenylalanine or (S)-2-amino-3-(2-fluorophenyl)-2-methylpropanoic acid. As used herein, "L-Iva" and "Iva" refer to L-isovaline. As used herein, "D-Gln" and "q" refer to D-glutamine, respectively.

[0353] Certain abbreviations used herein are defined as follows: "AcOH" refers to acetic acid, "ACN" refers to acetonitrile, "BEA" refers to 4-(bis(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)amino)-4-oxobutanoic acid, "Boc" refers to tert-butoxycarbonyl, "t-Bu" refers to tert-butyl, "DABA" refers to 3,5-diaminobenzoic acid, and "DCM" refers to dichloromethane. "DMAP" refers to 4-dimethylaminopyridine, "DIC" refers to diisopropylcarbodiimide, "EtO" refers to diethyl ether, "DIEA" refers to diisopropylethylamine, "DMF" refers to dimethylformamide, "DMAP" refers to 4-dimethylaminopyridine, "ivDde" refers to 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl, "EtOH" refers to ethanol, "EtOAc" refers to ethyl acetate, "eq" refers to equivalents, "Fmoc" refers to fluorenylmethyloxycarbonyl, "FPLC" refers to fast protein liquid chromatography, "HFIP" refers to hexafluoroisopropanol, "IPA" refers to isopropyl alcohol, and "MalDab" refers to (S)-2-((tert-butyl)methylpropanol. "MalDap" refers to (S)-3-((tert-butoxycarbonyl)amino)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoic acid; and 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 minute / minutes, "MTT" refers to methylthiazole tetrazolium, "Mtt" refers to 4-methyltrityl, "Oxyma" refers to ethyl cyanohydroxyiminoacetate, "PEG" refers to polyethylene glycol, "PyBOP" refers to benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate, "RT (room temperature)" refers to room temperature, "t-Bu" refers to tertiary butyl, "SPPS (solid-phase peptide synthesis)" refers to solid-phase peptide synthesis, "soln" refers to solution, "TFA" refers to trifluoroacetic acid, "TIPS" refers to triisopropylsilane, "TCEP" refers to tris(2-carboxyethyl)phosphine hydrochloride, and "THF" refers to tetrahydrofuran. ,

[0354] As used herein, "BEA-MalDap2" refers to the following structure:

[0355] [ka]

[0356] As used herein, "dCAP" refers to the following structure:

[0357] [ka]

[0358] As used herein, "BEA-MSTP2" refers to the following structure:

[0359] [ka]

[0360] As used herein, "BEA-acetyl 2" refers to the following structure:

[0361] [ka]

[0362] As used herein, "BEA-OD2" refers to the following structure:

[0363] [ka]

[0364] As used herein, "BEA-β-MalDap2" refers to the following structure:

[0365] [ka]

[0366] As used herein, "about" means within a statistically significant range of one or more values, such as, for example, a stated concentration, length, molecular weight, pH, sequence identity, time frame, temperature, or volume. Such values ​​or ranges may be within an order of magnitude, typically within 20%, more typically within 10%, and even more typically within 5% of a given value or range. The allowable variation encompassed by "about" will depend on the particular system under study and can be readily appreciated by one of ordinary skill in the art.

[0367] As used herein, "activity," "activate," "activating," and the like, in reference to one or more of the GIP, GLP-1, or glucagon receptors, refer to the ability of a compound, e.g., the ability of a polypeptide described herein, to bind to the receptor and elicit a response at the receptor, as measured using assays known in the art, e.g., the in vitro assays described below.

[0368] As used herein, "amino acid having a functional group available for conjugation" means any natural (encoded) or unnatural (non-encoded) amino acid having a functional group that can be conjugated to a fatty acid directly or, for example, via a linker. Examples of such functional groups include, but are not limited to, alkynyl, alkenyl, amino, azido, bromo, carboxyl, chloro, iodo, and thiol groups. Examples of natural amino acids containing such functional groups include K (amino), C (thiol), E (carboxyl), and D (carboxyl).

[0369] As used herein, "conservative amino acid substitution" refers to the substitution of an amino acid with an amino acid that has similar properties (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone structure, rigidity, etc.) and that has minimal effect on the biological activity of the resulting substituted peptide or polypeptide. Conservative substitution of functionally similar amino acids is well known in the art and need not be exhaustively described herein.

[0370] As used herein, "C 16 ~C 22 "Fatty acid" means a carboxylic acid having 16 to 22 carbon atoms. 16 ~C 22 Fatty acids can be saturated monoacids or saturated diacids. As used herein, "saturated" means that the fatty acid does not contain carbon-carbon double or triple bonds. Unless otherwise specified, fatty acids as referred to herein may also contain other acidic groups, such as phosphonic or sulfonic acid groups.

[0371] As used herein, "effective amount" refers to an amount, concentration, or dosage of one or more polypeptides described herein or pharmaceutically acceptable salts thereof that, after administration of a single or multiple doses to an individual in need thereof, provides a desired effect to such an individual under diagnosis or treatment. An effective amount can be readily determined by one skilled in the art by using known techniques and observing results obtained under similar circumstances. In determining an effective amount for an individual, several factors are taken into consideration, including, but not limited to, the mammalian species; its size, age, and general health; the specific disease or disorder involved; the extent or involvement or severity of the disease or disorder; the individual patient's response; the specific polypeptide administered; the mode of administration; the bioavailability characteristics of the administered preparation; the selected dosing regimen; the use of concomitant medications; and other relevant circumstances.

[0372] As used herein, "extended duration of action" means that the binding affinity and activity of the polypeptide continues for a longer period than native human GIP, GLP-1, and glucagon, allowing for administration less frequently, such as at least once daily, three times weekly, twice weekly, once weekly, every other week (every two weeks), or once monthly. The time-action profile of the polypeptide can be measured using known pharmacokinetic testing methods, such as those utilized in the Examples below.

[0373] As used herein, "polypeptide" or "peptide" refers to a polymer composed of multiple amino acid residues covalently linked in a specific sequence. The amino acids are linked through amide (peptide) bonds, which are formed when the carboxyl group of one amino acid reacts with the amino group of another amino acid, releasing a water molecule. The term applies to polymers containing naturally occurring amino acids as well as polymers containing one or more non-naturally occurring amino acids.

[0374] As used herein, "individual in need thereof" means a mammal, such as a human, having a condition, disease, disorder, or symptom that requires treatment or therapy, including, for example, those listed herein.

[0375] As used herein, "treat," "treating," "to treat," and the like refer to inhibiting, slowing, halting, or reversing the progression or severity of an existing condition, disease, disorder, or symptom.

[0376] As used herein, with respect to a compound, "dual agonist activity" refers to a compound that has activity at each of the GIP and GLP-1 receptors, and in particular to a polypeptide that has sufficient activity at each receptor to provide the benefits of agonism at that receptor while avoiding the undesirable side effects associated with excessive activity. Furthermore, polypeptides with dual agonist activity have an extended duration of action at each of the GIP and GLP-1 receptors, which advantageously allows for infrequent dosing, such as once weekly, twice monthly, monthly, or quarterly.

[0377] As used herein, with respect to a compound, "triple agonist activity" refers to a compound having activity at each of the GIP, GLP-1, and glucagon receptors, and particularly to a polypeptide having sufficient activity at each receptor to provide the benefits of agonism at that receptor while avoiding undesirable side effects associated with excessive activity. Furthermore, polypeptides with triple agonist activity have an extended duration of action at the GIP, GLP-1, and glucagon receptors, which advantageously allows for infrequent dosing, such as once weekly, twice monthly, monthly, or quarterly.

[0378] As used herein, the term "sequence identity" refers to the degree of similarity between two sequences. The degree of sequence identity between two polypeptides is expressed as a percentage and can be calculated as follows: % sequence identity = 100% * (number of identical amino acids) / (length of shortest consensus sequence).

[0379] In certain embodiments of a polypeptide of any of the formulas described herein, the polypeptide is an isotopic derivative of any one of the polypeptides described herein or a pharmaceutically acceptable salt thereof. It is understood that isotopic derivatives can be prepared using any of a variety of art-recognized techniques. For example, isotopic derivatives can generally be prepared by performing the procedures disclosed in the examples described herein by substituting an isotopically labeled reagent for a non-isotopically labeled reagent. In certain embodiments of a polypeptide of any of the formulas described herein or a pharmaceutically acceptable salt thereof, the polypeptide is a deuterated derivative of any one of the polypeptides described herein.

[0380] In the polypeptides of the present invention, any atom not specifically designated as a particular isotope is intended to represent any stable isotope of that atom. Unless otherwise specified, when an atom is specifically designated as "H" or "hydrogen," the atom is understood to have hydrogen at its natural abundance isotopic composition. Also, unless otherwise specified, when an atom is specifically designated as "D" or "deuterium," the atom is understood to have deuterium at an abundance substantially greater than the natural abundance of deuterium, which is 0.015%.

[0381] The polypeptides described herein can react with a number of inorganic and organic acids / bases to form pharmaceutically acceptable acid / base addition salts. Pharmaceutically acceptable salts and general techniques for preparing them are well known in the art (see, e.g., Stahl et al., "Handbook of Pharmaceutical Salts: Properties, Selection and Use," 2004). ndRevised Edition (Wiley-VCH, 2011). Pharmaceutically acceptable salts for use herein include sodium salts, potassium salts, trifluoroacetate salts, hydrochloride salts, and / or acetate salts. Thus, in some embodiments, pharmaceutically acceptable salt forms of the polypeptides are provided herein. In some embodiments, the pharmaceutically acceptable form is selected from the sodium salt or the potassium salt. In some embodiments, the pharmaceutically acceptable form is selected from the group consisting of sodium salts, potassium salts. In some embodiments, the pharmaceutically acceptable salt is the sodium salt.

[0382] In another embodiment, provided herein is a pharmaceutical composition comprising a polypeptide described herein or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, or excipient. Several pharmaceutical compositions and techniques for their preparation are well known in the art. See, for example, Remington: The Science and Practice of Pharmacy (Troy, Ed., 2001). st Edition, Lippincott, Williams & Wilkins, 2006).

[0383] In some embodiments, the pharmaceutical composition is suitable for administration by a parenteral route (e.g., subcutaneous, intravenous, intraperitoneal, intramuscular, or transdermal). In some embodiments, the pharmaceutical composition is suitable for oral administration (e.g., tablet, capsule). In some embodiments, the pharmaceutical composition is administered parenterally. In some embodiments, the pharmaceutical composition is administered orally.

[0384] The present disclosure also provides and therefore encompasses novel intermediates and methods for synthesizing the polypeptides described herein or pharmaceutically acceptable salts thereof. The intermediates and polypeptides described herein can be prepared by various techniques known in the art. For example, methods using chemical synthesis are illustrated in the examples below, or using biological expression. The specific synthetic steps for each of the described pathways can be combined into different methods for preparing the polypeptides described herein. The reagents and starting materials are readily available to those skilled in the art.

[0385] For chemical synthesis, standard manual or automated solid-phase synthesis procedures can be used. For example, automated peptide synthesizers are commercially available from, for example, CEM (Charlotte, North Carolina), CSBio (Menlo Park, California), and Gyros Protein Technologies Inc. (Tucson, Arizona). Reagents for solid-phase synthesis are readily available from commercial suppliers. Solid-phase synthesizers can be used according to the manufacturer's instructions for blocking interfering groups, protecting amino acids during the reaction, coupling, deprotecting, and capping unreacted amino acids.

[0386] The compounds disclosed herein include all possible stereoisomers, geometric (e.g., cis / trans), and structural isomers, unless otherwise specified. Such isomers may exist due to chiral centers, double bonds, or other steric elements in the molecular structure. Unless otherwise specified, the nomenclature used and depicted structures are intended to encompass racemic mixtures, individual enantiomers, diastereomers, positional and regioisomers, and mixtures thereof. The present invention extends to any pharmaceutically acceptable forms of these compounds, including salts, solvates, and polymorphs, regardless of their isomeric composition.

[0387] For biological expression, standard recombinant techniques can be used to construct a polynucleotide having a nucleic acid sequence encoding all or part of the amino acid sequence of a polypeptide, incorporate the polynucleotide into a recombinant expression vector, and introduce the vector into host cells, such as bacteria, yeast, and mammalian cells, to produce the polypeptide. See, for example, Green & Sambrook, "Molecular Cloning: A Laboratory Manual" (Cold Spring Harbor Laboratory Press, 4th ed., 2012). Polypeptides can be easily produced in mammalian cells, such as CHO, NSO, 20 HEK293, BHK, or COS cells; bacterial cells, such as E. coli, Bacillus subtilis, or Pseudomonas fluorescens; insect cells; or fungal or yeast cells, which are cultured using techniques known in the art. Vectors containing the polynucleotide sequence of interest can be transferred into host cells by well-known methods, which vary depending on the type of cellular host. Various methods of protein purification may be used and are known in the art.

[0388] The compounds described herein can be used to treat a variety of conditions, disorders, diseases, or symptoms. Specifically, methods are provided for treating obesity in an individual, comprising at least administering to an individual in need of such treatment an effective amount of a conjugate described herein, or a pharmaceutically acceptable salt thereof.

[0389] Additionally, methods for chronic weight management in an individual are provided, such methods comprising at least the step of administering to an individual in need of such treatment an effective amount of a conjugate described herein, or a pharmaceutically acceptable salt thereof.

[0390] Additionally, methods are provided for treating type 2 diabetes mellitus (T2DM) in an individual, such methods comprising at least the step of administering to an individual in need of such treatment an effective amount of a conjugate described herein, or a pharmaceutically acceptable salt thereof.

[0391] Additionally, methods are provided for treating non-alcoholic fatty liver disease (NAFLD) in an individual, such methods comprising at least the step of administering to an individual in need of such treatment an effective amount of a conjugate described herein, or a pharmaceutically acceptable salt thereof.

[0392] Additionally, methods are provided for treating non-alcoholic steatohepatitis (NASH) in an individual, such methods comprising at least the step of administering to an individual in need of such treatment an effective amount of a conjugate described herein, or a pharmaceutically acceptable salt thereof.

[0393] Additionally, methods for treating dyslipidemia in an individual are provided, such methods comprising at least the step of administering to an individual in need of such treatment an effective amount of a polypeptide described herein or a pharmaceutically acceptable salt thereof.

[0394] Additionally, methods for treating metabolic syndrome in an individual are provided, such methods comprising at least the step of administering to an individual in need of such treatment an effective amount of a conjugate described herein, or a pharmaceutically acceptable salt thereof.

[0395] Additionally, methods for treating osteoarthritis (OA) in an individual are provided, such methods comprising at least the step of administering to an individual in need of such treatment an effective amount of a conjugate described herein, or a pharmaceutically acceptable salt thereof.

[0396] Additionally, methods are provided for treating obesity-related sleep apnea (OSA) in an individual, such methods comprising at least the step of administering to an individual in need of such treatment an effective amount of a conjugate described herein, or a pharmaceutically acceptable salt thereof.

[0397] Additionally, methods are provided for treating polycystic ovary syndrome (PCOS) in an individual, such methods comprising at least the step of administering to an individual in need of such treatment an effective amount of a conjugate described herein, or a pharmaceutically acceptable salt thereof.

[0398] Additionally, methods are provided for inducing non-therapeutic weight loss in an individual, such methods comprising at least the step of administering to an individual in need of such treatment an effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof.

[0399] In these methods, the effectiveness of the compound can be assessed, for example, by observing a significant decrease in blood glucose, observing a significant increase in insulin, observing a significant decrease in HbA1c, and / or observing a significant decrease in body weight.

[0400] Alternatively, the compounds described herein or pharmaceutically acceptable salts thereof can be used to improve bone strength in individuals in need thereof. In some instances, the individuals in need thereof have hypoostosis or osteoid hypoplasia, or are recovering from a fracture, joint muscle restoration surgery, prosthetic implants, dental implants, and / or spinal fusion surgery. The polypeptides described herein can also be used to treat other disorders, such as Parkinson's disease or Alzheimer's disease.

[0401] Additionally, provided herein are compounds described herein, or pharmaceutically acceptable salts thereof, for use in treatment. In some embodiments, provided herein are polypeptides described herein, or pharmaceutically acceptable salts thereof, for use in the treatment of obesity, chronic weight management, type 2 diabetes, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), metabolic dysfunction-associated steatohepatitis (MASH), dyslipidemia, metabolic syndrome, osteoarthritis (OA), obesity-related sleep apnea (OSA), and polycystic ovary syndrome (PCOS). Also provided are polypeptides described herein, or pharmaceutically acceptable salts thereof, for use in the treatment of non-therapeutic weight loss.

[0402] Additionally provided is the use of a compound described herein, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating obesity, chronic weight management, type 2 diabetes, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), dyslipidemia, metabolic syndrome, osteoarthritis (OA), obesity-related sleep apnea (OSA) and polycystic ovary syndrome (PCOS). Also provided is the use of a polypeptide described herein, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for inducing non-therapeutic weight loss.

[0403] The polypeptides, compounds, or pharmaceutical compositions described herein may be provided as part of a kit. In some examples, the kit includes a device, such as a syringe, automatic injector, or pump, for administering at least one polypeptide (and optionally at least one additional therapeutic agent) to an individual. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the polypeptides, pharmaceutical compositions, and methods, the preferred methods and materials are described herein.

[0404] The reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires that there is only one element. Thus, the indefinite article "a" or "an" normally means "at least one."

[0405] An additional non-limiting list of embodiments is provided below. Embodiment 1. Formula:

[0406] [ka] or a pharmaceutically acceptable salt thereof, wherein R1 and R2 are independently

[0407] [ka] is selected from the group consisting of R3 and R4 independently comprise a C1-C5 alkyl, optionally substituted with one or more of NH2, CH2NH2, and CH2CH2NH2; * is the attachment point to the sulfur atom in a cysteine ​​residue in Fc1 or Fc2, ** is the connection point to R5 or R6, R5 and R6 are independently selected from a covalent bond or a C1-C5 alkyl; U comprises a tertiary amine or a 1,3,5-substituted phenyl; Z is O, NH, C1-C 30 Alkyl, (OCH2CH2) m , an amino acid polymer, or a combination thereof; X comprises a therapeutic polypeptide; Y comprises a fatty acid; m is an integer from 1 to 30; Fc1 and Fc2 each represent a compound containing an Fc region, or a pharmaceutically acceptable salt thereof.

[0408] Embodiment 2. The compound is:

[0409] [ka] or a pharmaceutically acceptable salt thereof.

[0410] Embodiment 3. The compound is

[0411] [ka] or a pharmaceutically acceptable salt thereof.

[0412] Embodiment 4. The compound is

[0413] [ka] or a pharmaceutically acceptable salt thereof.

[0414] Embodiment 5. The compound is

[0415] [ka] or a pharmaceutically acceptable salt thereof.

[0416] Embodiment 6. The polypeptide comprises: X1X2X3X4TX6TSDX 10 X 11 X 12 X 13 LX 15 X 16 KAQX 20 X 21 FIX 24 X 25 LX 27 X 28 X 29 X30 X 31 X 32 X 33 X 34 X 35 X 36 X 37 X 38 X 39 X 40 X 41 Including, X1 is H, NMeY, or Y; X2 is Ac4c, Aib, αMeS, Iva, or D-Ala; X3 is Q, E, or H; X4 is G or D-Ala, X6 is αMeF(2F), F, or αMeF; X 10 is 4-Pal, Y, or V, X 11 is Aib, S, or αMeS, X 12 is I or S, X 13 is L or αMeL, X 15 is D or E, X 16 is E or Orn, X 20 is Aib, Iva, or αMe4Pal, X 21 is E, Q, D, or Orn X 24 is K, Q, E, D-Glu, or D-Gln, X 25 is αMeY or Y, X 27 is L or V, X 28 is K, E, or A, X 29 is Aib or G, X 30 is G or S, X 31 is P, G, E, or Orn X 32is absent, S or P, X 32 If is S or P, then X 33 is S, X 33 If S, then X 34 is G or Aib, X 34 If is G or Aib, then X 35 is absent or is A or Orn, X 35 If is A or Orn, then X 36 is absent or is P, X 36 If P, then X 37 is absent or is P, X 37 If P, then X 38 is absent or is P, X 38 If P, then X 39 is absent or is S, Orn, or G, X 39 If is S, Orn, or G, then X 40 is absent, K, or G, X 40 If is K or G, then X 41 is absent or is S or G, X 32 If does not exist, X 33 ~X 41 There is no X 35 If does not exist, X 36 ~X 41 There is no X 36 If does not exist, X 37 ~X 41 There is no X 37 If does not exist, X 38 ~X 41 There is no X 38 If does not exist, X 39 ~X 41 There is no X39 If does not exist, X 40 and X 41 There is no X 40 If does not exist, X 41 does not exist, The compound of any one of embodiments 1-5, wherein the C-terminal amino acid is optionally amidated, or a pharmaceutically acceptable salt thereof.

[0417] Embodiment 7. The compound of embodiment 6, wherein Z is conjugated to an amino acid at any one of positions 24, 28, 31 or 40 of the polypeptide.

[0418] Embodiment 8. The compound of any one of embodiments 1 to 7, or a pharmaceutically acceptable salt thereof, wherein Z comprises an amino acid polymer.

[0419] Embodiment 9. The compound of embodiment 8, or a pharmaceutically acceptable salt thereof, wherein the amino acid polymer comprises any of (GGGGS)n, (SGGGG)n, (GGGGQ)n, (EAAAK)n, (KAAAE)n, (AEEA)n, G(PA)n, G(PA)n, G(PE)n, G(PK)n, (AP)n, (AP)n, or G(EP)n, where n is an integer from 1 to 10.

[0420] Embodiment 10. The lysine at position 17 is a C 16 ~C 22 via a linker between the fatty acid and C 16 ~C 22 10. The compound of any one of embodiments 1-9, or a pharmaceutically acceptable salt thereof, conjugated to a fatty acid.

[0421] Embodiment 11.C 16 ~C 22 11. The compound of embodiment 10, or a pharmaceutically acceptable salt thereof, wherein the fatty acid is conjugated to the lysine at position 17 via a linker.

[0422] Embodiment 12. The compound of embodiment 11, or a pharmaceutically acceptable salt thereof, wherein the linker comprises 1 to 4 amino acids.

[0423] Embodiment 13. The compound of embodiment 12, or a pharmaceutically acceptable salt thereof, wherein the amino acid included in the linker is Glu, γGlu, or a combination thereof.

[0424] Embodiment 14. The compound of any one of embodiments 11 to 13, or a pharmaceutically acceptable salt thereof, wherein the linker comprises one to four (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) moieties.

[0425] Embodiment 15. The linker is (γGlu) a -(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) b -(γGlu) c -CO-(CH2) p -COH structure, wherein a is 0 or 1, b is 0, 1 or 2, c is 1, 2 or 3, p is an integer from 14 to 20, or a pharmaceutically acceptable salt thereof.

[0426] Embodiment 16. A compound according to embodiment 15, wherein a is 0, b is 1, and c is 1, or a pharmaceutically acceptable salt thereof.

[0427] Embodiment 17. The compound of any one of embodiments 1 to 16, or a pharmaceutically acceptable salt thereof, wherein the polypeptide comprises greater than 90% sequence identity to any of SEQ ID NOs: 1 to 482.

[0428] Embodiment 18. The compound of embodiment 17, or a pharmaceutically acceptable salt thereof, wherein the polypeptide comprises any of the sequences set forth in SEQ ID NOs: 1-482.

[0429] Embodiment 19. Fc1 and Fc2 are a.AGCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 935), b.AGCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVKFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 936), c.IGCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVKFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 941), d.IGCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVKFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 941), e.AGCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 943), f.AGCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 944), g.ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVKFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREECTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 1179), h.ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVKFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVCDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 1180), i.ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVKFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKCRWQQGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 1181) j. The compound of any one of embodiments 1-18, selected from the group consisting of ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVKFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 948), or a pharmaceutically acceptable salt thereof.

[0430] Embodiment 20. The compound of any one of embodiments 1 to 19, or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt is selected from sodium, potassium, trifluoroacetate, hydrochloride, and acetate.

[0431] Embodiment 21 A compound comprising any of the following Fc-acylated polypeptides:

[0432] [Table 13-1]

[0433] [Table 13-2]

[0434] Table 13-3

[0435] Table 13-4

[0436] Table 13-5

[0437] Table 13-6

[0438] Table 13-7

[0439] Table 13-8

[0440] Table 13-9

[0441] Table 13-10

[0442] Table 13-11

[0443] Table 13-12

[0444] Table 13-13

[0445] Table 13-14

[0446] Table 13-15

[0447] Table 13-16

[0448] Table 13-17

[0449] Table 13-18

[0450] Table 13-19

[0451] Table 13-20

[0452] Table 13-21

[0453] Table 13-22

[0454] Table 13-23

[0455] [Table 13-24]

[0456] [Table 13-25]

[0457] [Table 13-26]

[0458] [Table 13-27]

[0459] [Table 13-28]

[0460] [Table 13-29]

[0461] [Table 13-30]

[0462] [Table 13-31]

[0463] Embodiment 22 A compound comprising any of the following Fc-acylated polypeptides:

[0464] [ka]

[0465]

change

[0466]

change

[0467]

change

[0468]

change

[0469]

change

[0470]

change

[0471]

change

[0472]

change

[0473]

change

[0474]

change

[0475]

change

[0476]

change

[0477]

change

[0478]

change

[0479]

change

[0480]

change

[0481]

change

[0482]

change

[0483]

change

[0484]

change

[0485]

change

[0486] [ka]

[0487] [ka]

[0488] [ka]

[0489] [ka]

[0490] Embodiment 22. A compound according to any one of embodiments 1 to 22, or a pharmaceutically acceptable salt thereof, wherein the compound has greater potency at the GIP and GLP-1 receptors compared to native GIP (SEQ ID NO: 1223) and GLP-17-36 (SEQ ID NO: 1221), respectively.

[0491] Embodiment 23. The compound binds to native glucagon (SEQ ID NO: 1222), GIP (SEQ ID NO: 1223) and GLP-1 7-36 23. The compound of any one of embodiments 1 to 22, or a pharmaceutically acceptable salt thereof, having greater potency at each of the glucagon, GIP and GLP-1 receptors compared to (SEQ ID NO: 1221).

[0492] Embodiment 24. The compound of any one of embodiments 1 to 23, or a pharmaceutically acceptable salt thereof, wherein the compound has a sufficiently extended duration of action to allow for dosing as infrequently as once a month.

[0493] Embodiment 25. A pharmaceutical composition comprising a compound according to any one of embodiments 1 to 24, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, diluent, or excipient.

[0494] Embodiment 26. The pharmaceutical composition of embodiment 25, wherein the composition is formulated for subcutaneous administration.

[0495] Embodiment 27. A method of treating a disease or condition selected from the group consisting of diabetes, obesity, chronic weight management, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), dyslipidemia, metabolic syndrome, chronic kidney disease (CKD), osteoarthritis (OA), obesity-related sleep apnea (OSA), polycystic ovary syndrome (PCOS), Parkinson's disease, and Alzheimer's disease, comprising administering to an individual in need thereof an effective amount of a compound of any one of embodiments 1 to 26, or a pharmaceutically acceptable salt thereof.

[0496] This invention is further illustrated by the following examples which should not be construed as limiting. [Example]

[0497] Preparation of therapeutic polypeptides Processes for making exemplary compounds of the present disclosure are described below, it being understood that these processes are not exhaustive and that compounds of the present disclosure can also be prepared using other processes known to those skilled in the art.

[0498] Peptide intermediates intended for Fc conjugation are fully synthesized following a conventional fluorenylmethyloxycarbonyl (Fmoc) / tert-butyl (t-Bu) solid-phase peptide synthesis (SPPS) protocol, performed using a Symphony 12-Channel Multiplex Peptide Synthesizer (Protein Technologies, Inc., Tucson, AZ).

[0499] A linear peptide backbone is first synthesized with orthogonally protected lysine residues (4-methyltrityl (Mtt) and ivDde) ​​at designated sequence positions. This is followed by successive deprotection and coupling steps to introduce a "linker-fatty acid" moiety, followed by a "linker-Fc conjugation" moiety. These modifications complete the synthesis of peptide intermediates, which are cleaved from the solid support and subsequently purified, as described below.

[0500] Procedure for peptide synthesis Solid-phase peptide synthesis (SPPS) is performed using 1% DVB cross-linked polystyrene resin (Fmoc-Rink-MBHA, 100-200 mesh, Chem-Impex International) with a substitution range of 0.3-0.6 mmol / g. The peptide backbone is assembled with N-Fmoc-protected amino acids containing standard side chain protecting groups, except for the residue at position 1 (e.g., Boc-L-Tyr(OtBu)-OH or Boc-L-His(Boc)-OH), the lysine residue at position 17 (Fmoc-L-Lys(Mtt)-OH), and the lysine residues at positions 24, 28, 32, or 40 (Fmoc-L-Lys(ivDde)-OH). Before each coupling step, the Fmoc group is removed using 20% ​​piperidine in DMF (two treatments of 8 min each). To initiate coupling, equimolar amounts of Fmoc amino acid (0.3 M in DMF), diisopropylcarbodiimide (0.9 M in DCM), and Oxyma (0.9 M in DMF) are combined in a 9-fold molar excess relative to theoretical peptide loading, while the reaction is maintained at 60 °C. Coupling times are varied; standard Fmoc-amino acids are coupled for 40 min, while Fmoc-α-methylated amino acids require 3 h, as Fmoc-amino acids are coupled onto α-methylated residues. An extended coupling time of 6 h applies to Fmoc-amino acids at positions 5 and 6, while residues at positions 1–4 undergo coupling for 3 h. After peptide backbone synthesis is complete, the resin is thoroughly washed with DCM to remove residual DMF.

[0501] Procedure for assembly of the linker-fatty acid moiety After peptide backbone synthesis, the Mtt protecting group on lysine 17 is selectively removed by treatment with 30% hexafluoroisopropanol in DCM (four treatments of 30 min each), followed by extensive washing with DCM. The linker-fatty acid moiety is assembled using a similar coupling approach to the previous step, optionally employing selected N-Fmoc-protected amino acid derivatives. These may include 2-[2-(2-Fmoc-amino-ethoxy)-ethoxy]-acetic acid (Fmoc-AEEA-OH), Fmoc-glutamic acid α-t-butyl ester (Fmoc-Glu(OH)-OtBu), Nα-tert-butoxycarbonyl-Nε-Fmoc-lysine (Boc-L-Lys(Fmoc)-OH), and 18-(tert-butoxy)-18-oxo-octadecanoic acid. Each derivative is coupled in the presence of diisopropylcarbodiimide (9 equivalents, 0.9 M in DCM) and Oxyma (9 equivalents, 0.9 M in DMF) in amounts ranging from 4 to 9 equivalents relative to the resin charge (0.15-0.3 M in DMF). The coupling reaction is allowed to proceed for 6 hours at 60°C. Before each coupling step, the Fmoc group is removed using 20% ​​piperidine in DMF (two treatments of 8 minutes each).

[0502] Procedure for assembly of the linker-Fc conjugation moiety After assembly of the linker-fatty acid portion, the ivDde protecting groups on the lysine residues at positions 24, 28, 32, or 40 are selectively removed by treatment with 3% hydrazine in DMF (four treatments of 3 minutes each). The resin is washed extensively with DMF, DCM, and isopropyl alcohol to ensure complete removal of residual hydrazine. Linkers, if present, are constructed using a similar approach to the previous coupling step, utilizing selected N-Fmoc-protected amino acid derivatives as needed. These are then combined with Fmoc-N-amido-PEG. 24These derivatives may include Fmoc-amino acids with standard protecting groups, such as Fmoc-Amino-ethoxy-ethoxy]-acetic acid (Fmoc-AEEA-OH), or Fmoc-amino acids with standard protecting groups. Each derivative is coupled in the presence of diisopropylcarbodiimide (9 equivalents, 0.9 M in DCM) and Oxyma (9 equivalents, 0.9 M in DMF) in an amount ranging from 2 to 9 equivalents relative to the resin charge (0.15 to 0.3 M in DMF). The coupling reaction is allowed to proceed at 60°C for 2 to 6 hours, as needed. Prior to each coupling step, the Fmoc group is removed using 20% ​​piperidine in DMF (two treatments of 8 min each).

[0503] BEA Coupling After the desired linker is assembled, the terminal Fmoc group is removed using 20% ​​piperidine in DMF (two treatments of 8 min each). Coupling to "BEA" is then carried out by adding 4-(bis(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)amino)-4-oxobutanoic acid (Fmoc2-BEA-OH, 6 eq. in DMF), diisopropylcarbodiimide (9 eq., 0.9 M in DCM), and Oxyma (9 eq., 0.9 M in DMF). The reaction is maintained at 60°C for 4 hours. Upon completion, both Fmoc groups are removed with 20% piperidine in DMF (two treatments of 8 min each) before introducing the desired thiol-reactive conjugation moiety, as detailed below.

[0504] In the case of peptide intermediates containing BEA-(MalDap)2 To the resin-bound diamine, (S)-3-((tert-butoxycarbonyl)amino)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoic acid (N-Mal-L-Dap(Boc)-OH, 3.6 equivalents in DMF), diisopropylcarbodiimide (9 equivalents, 0.9 M in DCM), and Oxyma (9 equivalents, 0.9 M in DMF) are introduced and the reaction is allowed to proceed at room temperature for 3 hours.

[0505] In the case of peptide intermediates containing BEA-(MSPT)2 To the resin-bound diamine, N,N-diisopropylethylamine (1.7 equivalents) and 2,5-dioxopyrrolidin-1-yl 2-(2-(2-(4-(5-(methylsulfonyl)-1H-tetrazol-1-yl)phenoxy)ethoxy)ethoxy)acetate (MSPT-NHS ester, 3 equivalents in DMF) are introduced and the reaction is allowed to proceed at room temperature for 2.5 hours.

[0506] In the case of peptide intermediates containing BEA-(AcBr) N,N-Diisopropylethylamine (1.7 equivalents) and 2,5-dioxopyrrolidin-1-yl 2-bromoacetate (4.1 equivalents in DMF) are added to the resin-bound diamine and the reaction is allowed to proceed at room temperature for 2.5 hours.

[0507] In the case of peptide intermediates containing BEA-(OD)2 N,N-Diisopropylethylamine (1.7 equivalents) and 2,5-dioxopyrrolidin-1-yl 2-(2-(2-(4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)phenoxy)ethoxy)ethoxy)acetate (OD-NHS ester, 3 equivalents in DMF) are added to the resin-bound diamine and the reaction is allowed to proceed at room temperature for 2.5 hours.

[0508] In the case of peptide intermediates containing BEA-(β-MalDap)2 To the resin-bound diamine was added N,N-diisopropylethylamine (1.7 equiv.) and 2,5-dioxopyrrolidin-1-yl(S)-2-((tert-butoxycarbonyl)amino)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (Boc-L-Dap(Mal)-NHS ester, 3 equiv. in DMF), and the mixture was allowed to proceed overnight at room temperature.

[0509] For peptide intermediates containing a single MalDap (for conjugation to Fc-eCys) To the resin-bound diamine (no BEA present) is introduced (S)-3-((tert-butoxycarbonyl)amino)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoic acid (N-Mal-L-Dap(Boc)-OH, 1.8 equivalents in DMF), diisopropylcarbodiimide (9 equivalents, 0.9 M in DCM), and Oxyma (9 equivalents, 0.9 M in DMF), and the reaction is allowed to proceed at room temperature for 3 hours.

[0510] For peptide intermediates containing a single MSPT (for conjugation to Fc-eCys) To the resin-bound amine (no BEA present) is introduced 2-(2-(2-(4-(5-(methylsulfonyl)-1H-tetrazol-1-yl)phenoxy)ethoxy)ethoxy)acetic acid (MSPT-acid, 1.8 eq. in DMF), diisopropylcarbodiimide (9 eq., 0.9 M in DCM), and Oxyma (9 eq., 0.9 M in DMF) and the reaction is allowed to proceed at room temperature for 3 hours.

[0511] Cleavage and purification of peptide intermediates After the on-resin synthesis steps detailed above are complete, the peptide resin is washed with DCM and then air-dried thoroughly. The resin is treated with 10 mL of cleavage cocktail (trifluoroacetic acid:water:triisopropylsilane, 85:5:10 v / v) at room temperature for 2.5 hours. The resin is filtered off and washed twice with 2 mL of undiluted TFA each time, and the combined filtrate is treated with 5 times (by volume) cold diethyl ether (-20°C) to precipitate the crude peptide. The peptide / ether suspension is then centrifuged at 5500 rpm for 2 minutes to form a solid pellet, the supernatant is decanted, and the solid pellet is triturated twice more with ether and dried under nitrogen. The crude peptide is dissolved in 20% acetonitrile / 20% acetic acid / 60% water and purified by RP-HPLC on a Luna 5 μm phenylhexyl preparative column (21 × 250 mm, Phenomenex) with a linear gradient of 100% acetonitrile in a 0.1% TFA / water buffer system (e.g., 30–50% acetonitrile over 75 min). Product purity is assessed using analytical RP-HPLC, with a pooling standard of >95%. Pooled fractions are frozen and concentrated by lyophilization to yield the peptide intermediate as a TFA salt.

[0512] Synthesis of 4-(bis(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)amino)-4-oxobutanoic acid (Fmoc2-BEA-OH)

[0513] [ka] A solution of N-(9-fluorenylmethoxycarbonyloxy)succinimide (19.4 g, 56.4 mmol) in dichloromethane (80 mL) was added over 45 minutes to a solution of diethylenetriamine (3.1 mL, 28 mmol) in dichloromethane (30 mL) cooled to −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, and then the pH was adjusted to 5 by the slow addition of 1 N HCl (approximately 20 mL). The phases were separated, and the aqueous layer was extracted with DCM (2 × 100 mL). The combined organic layers were washed with saturated aqueous NaCl and then dried over magnesium sulfate. The solvent was removed under reduced pressure and purified by silica gel chromatography (660 g, DCM (5 min), then 5% MeOH / DCM (15 min), then 10% MeOH / DCM (25 min) to give the title product (911.84 g, 65%) as a white powder. ES / MS m / z: 648 (M+1).

[0514] Synthesis of 2,5-dioxopyrrolidin-1-yl 2-(2-(2-(4-(5-(methylsulfonyl)-1H-tetrazol-1-yl)phenoxy)ethoxy)ethoxy)acetate (MSPT-NHS ester) Process 1

[0515] [ka] 4-(5-Mercapto-1H-tetrazol-1-yl)phenol (4.00 g, 20.6 mmol) was dissolved in tetrahydrofuran (50 mL). The mixture was cooled in an ice bath, and then N,N-diisopropylethylamine (4.31 g, 33.3 mmol) was added. After stirring for 10 minutes, a suspension formed. Iodomethane (1.54 mL, 24.7 mmol) was added dropwise via syringe over 1 minute. The reaction mixture was stirred for 20 minutes with cooling in an ice bath, then at room temperature for 12 hours. The mixture was diluted with EtOAc (100 mL) and washed with saturated aqueous NH4Cl (2 x 50 mL). The organic layer was separated, then dried over sodium sulfate, filtered, and concentrated in vacuo to give 4-(5-(methylthio)-1H-tetrazol-1-yl)phenol (4.2 g, 93% yield), which could be used directly in the next step without further purification. LCMS mz = 209 (M + 1).

[0516] Process 2

[0517] [ka] A 200 mL pressure vessel was charged with 4-(5-(methylthio)-1H-tetrazol-1-yl)phenol (2.50 g, 11.4 mmol), tert-butyl 2-(2-(2-bromoethoxy)ethoxy)acetate (4.33 g, 14.8 mmol), and acetone (60 mL). Potassium carbonate was added (3.15 g, 22.8 mmol), the vessel was sealed, and heated to 80° C. with vigorous stirring for 8 h. The reaction mixture was cooled to room temperature, filtered to remove potassium carbonate, and then washed with acetone / DCM / EtOAc (30 mL each). The filtrate was concentrated in vacuo to give the crude material, which was purified by flash chromatography (80 g, 100% DCM for 5 min, then gradient to 100% EtOAc over 20 min). The product tert-butyl 2-(2-(2-(4-(5-(methylthio)-1H-tetrazol-1-yl)phenoxy)ethoxy)ethoxy)acetate (3.98 g, 85% yield) was isolated as a white powder. LCMS-mz=411 (M+1).

[0518] Process 3

[0519] [ka] tert-Butyl 2-(2-(2-(4-(5-(methylthio)-1H-tetrazol-1-yl)phenoxy)ethoxy)ethoxy)acetate (3.98 g, 9.21 mmol) was dissolved in ethanol (100 mL) and cooled to 5-10 °C in an ice-water bath. 30% aqueous hydrogen peroxide (19.0 mL, 184 mmol) was then added, followed by ammonium molybdate(VI) tetrahydrate (1.14 g, 0.921 mmol). The reaction mixture was stirred at room temperature in the ice bath for 4 h, then at room temperature for 12 h. The mixture was diluted with DCM (150 mL) and then washed with brine. The organic phase was separated, dried over sodium sulfate, and concentrated to dryness in vacuo. Purification by flash column chromatography (80 g silica, 100% DCM for 5 min, then gradient to 100% EtOAc over 20 min) afforded 2-(2-(2-(4-(5-(methylsulfonyl)-1H-tetrazol-1-yl)phenoxy)ethoxy)ethoxy)acetic acid (MSPT acid, 3.00 g, 80% yield) as a thick oil. LCMS-mz=385 (M-1).

[0520] Process 4

[0521] [ka] 1-Hydroxypyrrolidine-2,5-dione (1.33 g, 1.6 equiv., 11.6 mmol) was added to a solution of 2-(2-(2-(4-(5-(methylsulfonyl)-1H-tetrazol-1-yl)phenoxy)ethoxy)ethoxy)acetic acid (2.80 g, 7.25 mmol) in DCM (50 mL) and THF (70 mL). EDCI (1.60 g, 10.3 mmol) was added in one portion, at which point the solution became a cloudy mixture. Additional DCM (20 mL) was added to bring the mixture back into solution, followed by stirring at room temperature for 12 hours. The solvent was removed in vacuo to give the crude material as a white foam. Purification by flash column chromatography (80 g, 100% DCM for 5 min, then gradient to 100% EtOAc over 20 min) afforded 2,5-dioxopyrrolidin-1-yl 2-(2-(2-(4-(5-(methylsulfonyl)-1H-tetrazol-1-yl)phenoxy)ethoxy)ethoxy)acetate (MSPT-NHS ester, 2.61 g, 65% yield) as a low melting solid. LCMS-mz=484 (M+1).

[0522] Synthesis of 2,5-dioxopyrrolidin-1-yl 2-(2-(2-(4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)phenoxy)ethoxy)ethoxy)acetate (OD-NHS ester) Process 1

[0523] [ka] 4-(5-Mercapto-1,3,4-oxadiazol-2-yl)phenol (3.00 g, 15.4 mmol) was dissolved in THF (50 mL) and cooled to 0 °C in an ice-water bath. N,N-Diisopropylethylamine (3.46 mL, 2.60 g, 20.1 mmol) was added, resulting in a cloudy solution. The mixture was stirred in the ice bath for 5 minutes, and then iodomethane (2.85 g, 20.1 mmol) was added dropwise via syringe over 1 minute. Upon addition, the mixture became clear after 5 minutes. The cooling bath was removed, and the mixture was stirred at room temperature for 2 hours, after which it was diluted with dichloromethane (100 mL) and washed with saturated aqueous NH4Cl (pH was adjusted to approximately 5 by adding 2 × 50 mL of citric acid solution). The organic layer was separated, dried over sodium sulfate, and concentrated to dryness in vacuo to give 4-(5-methylsulfanyl-1,3,4-oxadiazol-2-yl)phenol (520 mg, 97% yield) as a pale yellow solid, which was used in the next step without further purification. LC-MS-mz=209 (M+1).

[0524] Process 2

[0525] [ka] 4-(5-(methylthio)-1,3,4-oxadiazol-2-yl)phenol (3.3 g, 1 equiv., 15 mmol) and acetone (60 mL) were added to a 200 mL pressure vessel. To this solution was added tert-butyl 2-(2-(2-bromoethoxy)ethoxy)acetate (5.5 g, 20 mmol) and potassium carbonate (4.2 g, 30 mmol). The pressure vessel was sealed and heated to 70° C. with vigorous stirring for 5 h. After cooling to room temperature, the mixture was filtered to remove solid potassium carbonate and washed with EtOAc / DCM. The filtrate was concentrated in vacuo to dryness and purified by normal phase flash column chromatography (80 g silica gold, 100% DCM for 5 min, then gradient to 100% EtOAc over 20 min). The product-containing fractions were concentrated in vacuo to give tert-butyl 2-(2-(2-(4-(5-(methylthio)-1,3,4-oxadiazol-2-yl)phenoxy)ethoxy)ethoxy)acetate (4.8 g, 74% yield) as a white solid. LC-MS-mz=411 (M+1).

[0526] Process 3

[0527] [ka] tert-Butyl 2-(2-(2-(4-(5-(methylthio)-1,3,4-oxadiazol-2-yl)phenoxy)ethoxy)ethoxy)acetate (5.20 g, 12.7 mmol) was dissolved in 100 mL of ethanol and cooled to 5-10 °C in an ice-water bath. 30% hydrogen peroxide (10 mL, 97 mmol) was then added, followed by ammonium molybdate(VI) tetrahydrate (501 mg, 0.405 mmol). After vigorous stirring for 2 h, an additional 15 mL of 30% hydrogen peroxide and 1 g of ammonium molybdate(VI) tetrahydrate were added. The reaction mixture was stirred for an additional 6 h, then diluted with 150 mL of DCM and washed with brine. The organic phase was separated, dried over sodium sulfate, and concentrated to dryness in vacuo. The residue was triturated with methanol to give the first portion of product. The solvent was then removed from the mother liquor under reduced pressure. Purification by flash column chromatography (40 g, 100% DCM for 3 min, then gradient to 100% EtOAc over 20 min) afforded additional product as a white solid. Both product portions were combined to afford tert-butyl 2-(2-(2-(4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)phenoxy)ethoxy)ethoxy)acetate (5.3 g, 90% yield) as a white solid. LC-MS-mz=387 (M-tBu).

[0528] Process 4

[0529] [ka] To a solution of tert-butyl 2-(2-(2-(4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)phenoxy)ethoxy)ethoxy)acetate (5.60 g, 12.0 mmol) in DCM (60 mL) was added 2,2,2-trifluoroacetic acid (20 mL, 12.0 mmol). The reaction mixture was stirred at room temperature for 2 hours and then concentrated in vacuo to give a thick residue which was purified by normal phase flash column chromatography (80 g silica gold column, 100% DCM for 3 minutes, then gradient to 100% EtOAc over 20 minutes). The product-containing fractions were combined and concentrated in vacuo to give 2-(2-(2-(4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)phenoxy)ethoxy)ethoxy)acetic acid (4.12 g, 82% yield). LCMS-mz=387(M+1).

[0530] Process 5

[0531] [ka] 2-(2-(2-(4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)phenoxy)ethoxy)ethoxy)acetic acid (3.00 g, 7.38 mmol) and 1-hydroxypyrrolidine-2,5-dione (1.19 g, 10.3 mmol) were dissolved in DCM (50 mL) and THF (70 mL). To this solution was added 3-(((ethylimino)methylene)amino)-N,N-dimethylpropan-1-amine (EDCI, 1.60 g, 10.3 mmol). Upon addition, the solution became cloudy, and additional DCM (20 mL) was added to bring the mixture back into solution, followed by stirring at room temperature for 12 hours. The reaction mixture was concentrated under reduced pressure, and the resulting residue was dissolved in DCM and purified by normal phase flash column chromatography (80 g silica gold, 100% DCM for 5 min, then gradient to 100% EtOAc over 20 min). The product-containing fractions were concentrated to give 2,5-dioxopyrrolidin-1-yl-2-(2-(2-(4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)phenoxy)ethoxy)ethoxy)acetate (2.61 g, 65% yield). LCMS-mz=484 (M+1).

[0532] Fc recrosslinking The Fc is reduced by adding 1.5 to 2 equivalents of a freshly prepared TCEP stock solution in water. 258 μL of 7 mM TCEP solution is added to 9 mL of 111 μM Fc (pH 7.5). The solution is then incubated at 37 °C for 1 hour and analyzed by LC-MS to confirm reduction. The reduced Fc is then used directly for conjugation with the peptide. For efficient conjugation, the reaction is performed as follows, using an appropriate excess of peptide (approximately 1.4 equivalents) at an Fc concentration of 21.2 μM: Approximately 9 mL of reduced Fc is transferred to a 50 mL vial, to which approximately 29 mL of 50 mM acetate buffer, pH 5.6, and 6 mL of ACN are added. The solution is gently mixed and allowed to stand at 4 °C before the peptide is added as a 0.5 mM stock solution in 30% acetonitrile in water. The reaction progress is monitored by LC-MS and is typically complete within 30 min. The reaction is adjusted to pH 8.0 by adding 1 M Tris buffer at pH 8.0 to promote hydrolysis of the maleimide ring. The desired product is isolated after purification by IEX.

[0533] eCys conjugation for alternative peptide binding sites The Fc fragment containing the appropriately engineered cysteine ​​was first reduced in the presence of 8–10 molar equivalents of dithiothreitol (DTT) for 2 hours at 37°C. After reduction, the sample was passed through a Zeba spin desalting column (Thermo Fisher Scientific) or, alternatively, size-exclusion chromatography to remove the reducing agent and any cysteine ​​or glutathione caps attached to the engineered cysteine ​​during expression. Next, 1–5 molar equivalents of dehydroascorbic acid (DHAA) were added, and the sample was maintained at room temperature for 0.5–1 hour to reform the hinge disulfide. After oxidation, the sample was again passed through a Zeba spin column (Thermo Fisher Scientific) or, alternatively, a size-exclusion chromatography column to remove the oxidizing agent and buffer-exchange the sample into the desired buffer for conjugation.

[0534] eCys conjugation for a single peptide To generate Fc with a single eCys site, a 1:1 mixture of Fc with the desired site was mixed with Fc containing the F405L and R409K mutations in the presence of 8–10 molar equivalents of dithiothreitol (DTT) and incubated at 37°C for 2 hours. After reduction, the reducing agent and the cysteine ​​or glutathione cap attached to the engineered cysteine ​​during expression were removed using either a desalting column or size-exclusion chromatography. Then, 1–5 molar equivalents of dehydroascorbic acid (DHAA) were added, and the sample was maintained at room temperature for 0.5–1 hour to reform the hinge disulfide, favoring hetero-Fc formation driven by the F405L and R409K mutations in one Fc molecule in the mixture. After oxidation, the sample was again passed through a Zeba spin column (Thermo Fisher Scientific), or alternatively, a size-exclusion chromatography column, to remove the oxidizing agent and buffer-exchange the sample into the desired buffer for conjugation.

[0535] Conjugation to single or double eCys Fc constructs For efficient conjugation to eCys variants, the reaction is carried out at a 21.2 μM Fc concentration using an appropriate peptide excess (approximately 1.4 equivalents for single eCys and approximately 2.8 equivalents for double eCys) as follows: Fc (approximately 9 mL) is transferred to a 50 mL vial, to which 50 mM acetate buffer (approximately 29 mL) pH 5.6 and 6 mL ACN are added. The solution is gently mixed and left at 4 °C before the peptide is added as a 0.5 mM stock solution in 30% acetonitrile in water. The reaction progress is monitored by LC-MS and is typically complete within 30 min. To promote hydrolysis of the maleimide ring, the reaction is adjusted to pH 8.0 by adding 1 M Tris buffer at pH 8.0. The desired product is isolated after purification by IEX.

[0536] [Table 14-1]

[0537] Table 14-2

[0538] Table 14-3

[0539] Table 14-4

[0540] Table 14-5

[0541] Table 14-6

[0542] Table 14-7

[0543] Table 14-8

[0544] Table 14-9

[0545] Table 14-10

[0546] Table 14-11

[0547] Table 14-12

[0548] Table 14-13

[0549] Table 14-14

[0550] Table 14-15

[0551] Table 14-16

[0552] Table 14-17

[0553] Table 14-18

[0554] Table 14-19

[0555] Table 14-20

[0556] Table 14-21

[0557] Table 14-22

[0558] Table 14-23

[0559] Table 14-24

[0560] Table 14-25

[0561] Table 14-26

[0562] Table 14-27

[0563] Table 14-28

[0564] Table 14-29

[0565] Table 14-30

[0566] Table 14-31

[0567] Table 14-32

[0568] Table 14-33

[0569] Table 14-34

[0570] Table 14-35

[0571] Table 14-36

[0572] Table 14-37

[0573] Table 14-38

[0574] Table 14-39

[0575] Table 14-40

[0576] Table 14-41

[0577] Table 14-42

[0578] Table 14-43

[0579] Table 14-44

[0580] Table 14-45

[0581] Table 14-46

[0582] Table 14-47

[0583] Table 14-48

[0584]

Table 14-49

[0585] Table 14-50

[0586] Table 14-51

[0587] [Table 14-52]

[0588] [Table 14-53]

[0589] [Table 14-54]

[0590] [Table 14-55]

[0591] [Table 14-56]

[0592] [Table 14-57]

[0593] [Table 14-58]

[0594] [Table 14-59]

[0595] [Table 14-60]

[0596] As shown in Table 3 above, the molecular structures of certain exemplary compounds are provided in Figures 1-43.

[0597] In vitro functional activity Functional activity is determined in GIP-R, GLP-1R, and GcgR-expressing HEK-293 clonal cell lines. Each receptor cell line is treated with example compounds (20-point concentration-response curves with 2.75-fold serial dilutions prepared using a Labcyte Echo acoustic liquid handler) in DMEM (Gibco catalog number 31053) supplemented with 1× GlutaMAX™ (L-alanyl-L-glutamine dipeptide, Gibco catalog number 35050), 0.1% casein (Sigma catalog number C4765), 1% HSA (human serum albumin, Sigma catalog number A3782), 500 μM IBMX (3-isobutyl-1-methylxanthine), and 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) in a 20 μL assay volume.

[0598] After 30 minutes of incubation at 37°C, the resulting increase in intracellular cAMP was quantitatively determined using CisBio's cAMP Dynamic 2 HTRF Assay Kit (62AM4PEJ). Briefly, intracellular cAMP levels were detected by adding a cAMP-d2 conjugate in cell lysis buffer followed by the antibody anti-cAMP-cAMP-Eu3+-cryptate, also in cell lysis buffer. The resulting competitive assay was incubated for at least 60 minutes at room temperature and then detected using a Pherastar instrument (BMG Labtech) with excitation at 320 nm and emission at 665 nm and 620 nm. The raw data values ​​(emission at 665 nm / 620 nm x 10,000) were inversely proportional to the amount of cAMP present and were converted to nM cAMP per well using a cAMP standard curve.

[0599] The amount of cAMP (nM) produced in each well was determined by the human GLP-1 (7-36) NH2 (SEQ ID NO: 1221), human glucagon (hGcg) (SEQ ID NO: 1222), or human GIP (1-42)The relative EC values ​​were calculated by nonlinear regression analysis using percent maximal response versus concentration of added peptide fitted to a four-parameter logistic equation. 50 Derive a value.

[0600] Exemplary Dual Agonist Conjugates, hGcg, hGIP (1-42) NH2 and hGLP-1 (7-36) Relative EC for NH2 50 The geometric means of the data are shown in Table 4 below and are for exemplary triple agonist conjugates, hGcg, hGIP, (1-42) NH2 and hGLP-1 (7-36) Relative EC for NH2 50 The geometric means of the data are shown in Table 5 below.

[0601] [Table 15-1]

[0602] [Table 15-2]

[0603] [Table 15-3]

[0604] [Table 15-4]

[0605] [Table 15-5]

[0606] [Table 15-6]

[0607] Table 15-7

[0608] Table 15-8

[0609] Table 15-9

[0610] Table 15-10

[0611] Table 15-11

[0612] Table 15-12

[0613] Table 16-1

[0614] Table 16-2

[0615] Table 16-3

[0616] Table 16-4

[0617] [Table 16-5]

[0618] [Table 16-6]

[0619] [Table 16-7]

[0620] [Table 16-8]

[0621] Plasma levels of example compounds in rats Plasma levels of example compounds were determined in male Sprague-Dawley rats. Eight- to nine-week-old animals were purchased from Envigo (Indiana, USA). After acclimation, rats were randomized. Each group (n=5) received one compound at 10 nmol / kg via subcutaneous (sc) injection. Blood samples were collected serially via the tail vein at the time points indicated in each table. The animal study was approved by the Lilly Institutional Animal Care and Use Committee (IACUC).

[0622] Plasma was prepared from the collected blood samples. Each plasma sample was serially diluted using compound dilution medium and then incubated with HEK293 cells expressing human GLP1R and cAMP-driven luciferase in a 96-well plate for 5 hours. After cell lysis and addition of Bio-Glo (Promega), the luminescence signal intensity was measured.

[0623] For each test compound, a mathematical equation was established to calculate the corresponding concentration of the luminescence signal value through nonlinear regression analysis (Graphpad Prism) of the standard dose-luminescence signal curve data. Using the mathematical equation, the luminescence data showing dose dependency in each set of serially diluted samples was converted to concentration, and the values ​​were averaged as the plasma concentration of the sample. The data presented in Table 6 are the mean ± SE of the mean values ​​of five rats. As can be seen in Table 6, the tested example compounds remained in the plasma for extended periods, in some cases including at least 28 days.

[0624] [Table 17-1]

[0625] [Table 17-2]

[0626] Pharmacokinetic (PK) studies PK studies of certain exemplary compounds are conducted in cynomolgus monkeys and DIO mice. Plasma concentrations of compounds for these studies are determined using ELISA or LC / MS methods, as described in more detail below.

[0627] Bioanalytical Methods - ELISA Plasma concentrations of specific compounds were determined by ELISA. Pierce Streptavidin-coated plates (Thermo Scientific catalog number 15500) were coated with biotinylated ELI299 2G5 antibody (Lilly-produced antibody lot number 2G5-22AUG2023). This coating antibody was diluted to a final concentration of 1 μg / mL in pH 9.6 bicarbonate buffer (Thermo Scientific catalog number 28382), added to the plate at 100 μL / well, and stored at 2-8°C overnight. After overnight incubation, the plate was then washed with TBS-Tween wash buffer (TEKNOVA catalog number T0310). All washing steps were performed using the same wash buffer, with shaking between each wash, by washing the plate four times with 300 μL / well of wash buffer. After each wash, the plate was gently tapped on a paper towel to remove excess wash buffer. After the washing step, the coated and washed plate was blocked using the "blocking and assay buffer" for this assay (casein in PBS buffer with additional Tween-20). Casein buffer—Thermo Scientific catalog number 37528—was supplemented by adding Tween-20 to a final concentration of 0.05%. This buffer served as the blocking and assay buffer for the assay. Blocking was performed using 200 μL / well of blocking buffer and stored at ambient temperature with shaking at 500 RPM for approximately 1 hour. During blocking, standards, quality control samples, and research samples were processed through a minimum required dilution (MRD) of 1:20 (monkey) or 1:50 (mouse) in assay buffer, along with any additional sample dilutions as needed. After blocking, the plate was washed, and then 100 μL / well of the processed samples was added to the plate. The plate containing the samples is then stored at ambient temperature for approximately 1 hour with shaking at 500 RPM. The detection reagent is a mouse anti-human IgG4 Fc-HRP antibody (Southern Biotech Catalog No. 9040-05).The detection reagent is diluted in assay buffer to a final concentration of 0.017 μg / mL. After sample incubation, the plate is washed and incubated with 100 μL / well of detection solution. The plate containing the detection reagent is then stored at ambient temperature for approximately 1 hour with shaking at 500 RPM. During the detection incubation, the detection substrate solution is prepared by mixing equal parts of KPL TMB Peroxidase Substrate (KPL Catalog No. 5120-0048) and KPL Peroxidase Substrate Solution B (KPL Catalog No. 5120-0037). After the detection reagent incubation, the plate is washed, and the detection substrate is added at 100 μL / well. The plate is then incubated for 7-8 minutes until color develops. The reaction is then stopped using TMB Stop Solution (KPL Catalog No. 5150-0019). The plate is then read using a plate reader at a wavelength of 450 nm with a 630 nm offset.

[0628] Bioanalytical methods - LC / MS Plasma concentrations of compounds were determined by LC / MS. Surrogate peptides for the analyte incretins and Fc subunits were used as a measure of plasma concentration. For each assay, aliquots (95 μL) of diluted study samples, blanks, and standards in 100% cynomolgus monkey plasma were transferred to a PCR plate (Thermo Scientific 0.2 mL unskirted 96-well PCR plate, catalog number AB-0600). Analytes were immunoprecipitated with a biotin-labeled anti-human IgG antibody (Southern Biotech Goat Anti-Human IgG, Monkey ads-BIOT, catalog number 2049-08) pre-conjugated to streptavidin-coated magnetic beads (100 μL). Samples, blanks, and standards were washed once with 1× TBST and once with 1× TBS before elution in 0.2% formic acid (100 μL). The eluate (95 μL) was transferred to a new plate containing internal standards similar to the incretins and Fc subunits (5 μL each) and dried on an SPE Dual Dry under nitrogen at 60°C. Once dried, samples, blanks, and standards were reconstituted in 500 mM ammonium carbonate (30 μL) and reduced and alkylated in acetonitrile (30 μL) containing 2% iodoethanol and 1% triethylphosphine at 40°C for 1 hour with shaking at 600 rpm. Samples, blanks, and standards were dried twice at 60°C on an SPE Dual Dry under nitrogen, then reconstituted in 50 mM ammonium bicarbonate (95 μL) containing 10% methanol and Glu-C endoproteinase and digested overnight (approximately 18 hours) at 37°C with shaking at 600 rpm. The reaction was quenched with 88% formic acid (5 μL) and injected (20 μL) onto an Analytical Sales Sprite Armor C18 column (2.1 × 80 mm, 5 μm) for analysis. Analytes and internal standard surrogate peptides were delivered to a Thermo Fisher Orbitrap Eclipse Tribrid mass spectrometer at 400 μL / min in 0.1% formic acid in water and 0.1% formic acid in acetonitrile. The surrogate peptides were measured by positive-mode full-scan FT-MS for quantification.

[0629] Pharmacokinetics in cynomolgus monkeys Male cynomolgus monkeys were administered a single subcutaneous dose (10 nmol / kg) of compound in 40 mM Tris pH 8 buffer at a volume of 0.5 mL / kg. Blood was collected from each animal at 6, 24, 48, 72, 96, 120, 168, 240, 288, 336, 408, 576, 672, 2352, 3024, and 4200 hours post-dose for pharmacokinetic characterization.

[0630] Male cynomolgus monkeys were administered a single intravenous or subcutaneous dose (10 nmol / kg) of compound in 40 mM Tris pH 8 buffer at a volume of 0.5 mL / kg. Blood was collected from each animal at 6, 24, 48, 72, 96, 168, 240, 288, 336, 408, 480, 576, 672, 840, 1008, 1176, 1344, and 1512 hours post-dose for pharmacokinetic characterization.

[0631] Female cynomolgus monkeys were administered a single intravenous or subcutaneous dose (10 nmol / kg) of compound in 40 mM Tris pH 8 buffer at a volume of 0.5 mL / kg. Blood was collected from each animal at 0.25 (intravenous only), 6, 24, 48, 72, 96, 168, 240, 288, 336, 408, 480, 576, 672, 840, 1008, 1176, 1344, 1512, 1680, 1848, and 2016 hours post-dose for pharmacokinetic characterization.

[0632] [Table 18] Abbreviation:AUC 0-inf = area under the curve from time 0 to infinity. CL / F = clearance / bioavailability, T max = time to maximum concentration, C max = maximum observed plasma concentration, T 1 / 2 = half-life, NC = not calculated.

[0633] [Table 19] Abbreviation:AUC 0-inf = area under the curve from time 0 to infinity, CL = clearance, C max = maximum observed plasma concentration, T 1 / 2 = Half-life

[0634] As seen in Tables 7-8, the PK profiles of the example compounds support long elimination half-lives and low clearance compared to currently available incretin therapies. These data demonstrate that the compounds have pharmacokinetic profiles potentially suitable for once-monthly dosing.

[0635] Pharmacodynamics in DIO mice The in vivo efficacy of certain example compounds on body weight was determined in a diet-induced obesity (DIO) mouse model. Male C57BL / 6DIO mice (The Jackson Laboratory, Bar Harbor, ME) maintained on a high-calorie diet (containing 40% kcal from fat) were used in the following study. Mice were housed individually in a temperature-controlled facility (24°C) with a 12-hour light / dark cycle (lights on at 22:00) and had free access to food and water. To examine the effect on body weight, obese mice received a single subcutaneous dose of vehicle (40 mM Tris-HCl, pH 8.0) or test substance (3, 10, or 30 nmol / kg). Body weight and food intake were monitored daily for the first 14 days and then twice weekly for the remainder of the study. Consistent with the desired long duration of action, treatment of obese animals with a single dose of each example compound resulted in sustained weight loss.

[0636] [Table 20-1]

[0637] [Table 20-2]

[0638] [Table 20-3]

[0639] [Table 20-4]

[0640] Body weight dose-response study in obese mice The in vivo efficacy of example compounds on body weight and food intake was determined in a diet-induced obese mouse model (DIO). Male C57BL / 6 obese mice maintained on a high-calorie diet (containing 40% kcal from fat) were housed in a temperature-controlled facility (24 °C) with a 12-h light / dark cycle (lights on at 22:00) and had free access to food and water. To examine the dose-dependent effects of compounds 170, 172, 387, and 456 on body weight, obese animals were subcutaneously administered vehicle (40 mM Tris-HCl, pH 8.0) or test substance (0.3, 1, 3, 10, and 10 nmol / kg) weekly. Treatment of obese mice with the test substance resulted in a dose-dependent reduction in body weight compared to control animals (Figures 44-47). Values ​​are presented as mean ± SEM with n = 6 per group. Statistical analysis was performed using two-way ANOVA followed by Dunnett's method for multiple comparisons. Significance was determined at p<0.05. Overall, these findings demonstrate that the example compounds produce robust weight loss.

Claims

1. formula: 【Chemistry 1】 or a pharmaceutically acceptable salt thereof, wherein X ＴＰＰ is a therapeutic polypeptide conjugated to a fatty acid moiety; Z is O, C(O), NH, C １ ~C ３０ Alkyl, (OCH ２ CH ２ ) ｍ , [C(O)NH-CH ２ CH ２ OCH ２ ] ｍ , (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) ｍ , amino acids, peptides, or combinations thereof, wherein m is an integer from 1 to 30; U is C １ ~C ５ alkyl, N, phenyl, or phenylcarbonyl, or a combination thereof; R １ and R ２ are independently absent, a covalent bond, or C １ ~C ５ is alkyl, R ３ and R ４ is independently absent or an amide; R ５ and R ６ is independently absent or R ５ and R ６ are independently １ ~C ５ Alkyl, phenyl, CH ２ O (CH ２ CH ２ O) ｎ or a combination thereof, wherein n is an integer from 1 to 5; １ ~C ５ Alkyl is NH ２、 CH ２ NH ２ , and C.H. ２ CH ２ NH ２ and optionally replaced with one or more of R ７ and R ８ is independently absent, or 【Chemistry 2】 wherein the compound is selected from the group consisting of: ＊ ) is R ９ or R １０ including the connection point to ( ＊＊ ) is R ５ or R ６ including a connection point to R ９ and R １０ and are independently an antibody fragment comprising an Fc region, or a pharmaceutically acceptable salt thereof.

2. R ７ and R ８ but, 【Transformation 3】 2. The compound of claim 1, wherein:

3. R ５ and R ６ are respectively, NH ２ C substituted with ２ 3. The compound of claim 2, or a pharmaceutically acceptable salt thereof, wherein R is alkyl.

4. R ３ and R ４ The compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein each of

5. R １ and R ２ are respectively, C ２ The compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein R is alkyl.

6. 6. The compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, wherein U is N or phenyl.

7. The compound is 【Chemistry 4】 7. The compound of claim 6, comprising:

8. R ７ and R ８ but, 【Transformation 5】 2. The compound of claim 1, wherein:

9. R ５ and R ６ are phenyl and CH ２ O (CH ２ CH ２ O) ｎ and n is 2; or a pharmaceutically acceptable salt thereof.

10. R ３ and R ４ The compound according to any one of claims 8 to 9, or a pharmaceutically acceptable salt thereof, wherein each of

11. R １ and R ２ are respectively, C ２ The compound according to any one of claims 8 to 10, or a pharmaceutically acceptable salt thereof, which is alkyl.

12. 12. The compound according to any one of claims 8 to 11, or a pharmaceutically acceptable salt thereof, wherein U is N or phenyl.

13. The compound is 【Transformation 6】 13. The compound of claim 12, comprising:

14. R ７ and R ８ However, respectively, 【Transformation 7】 2. The compound of claim 1, wherein:

15. R ５ and R ６ are phenyl and CH ２ O (CH ２ CH ２ O) ｎ and n is 2; or a pharmaceutically acceptable salt thereof.

16. R ３ and R ４ The compound according to any one of claims 14 to 15, or a pharmaceutically acceptable salt thereof, wherein each of

17. R １ and R ２ are respectively, C ２ The compound according to any one of claims 14 to 16, or a pharmaceutically acceptable salt thereof, which is alkyl.

18. 18. The compound according to any one of claims 14 to 17, or a pharmaceutically acceptable salt thereof, wherein U is N or phenyl.

19. The compound is 【Transformation 8】 19. The compound of claim 18, comprising:

20. R ７ and R ８ and each are absent, or a pharmaceutically acceptable salt thereof.

21. R ５ and R ６ are respectively, C １ 21. The compound of claim 1 or 20, or a pharmaceutically acceptable salt thereof, wherein: R is alkyl;

22. R ３ and R ４ 22. The compound of claim 21, or a pharmaceutically acceptable salt thereof, wherein each is an amide.

23. R １ and R ２ are respectively, C ２ The compound according to any one of claims 20 to 22, or a pharmaceutically acceptable salt thereof, which is alkyl.

24. 24. The compound according to any one of claims 20 to 23, or a pharmaceutically acceptable salt thereof, wherein U is N or phenyl.

25. The compound is 【Chemistry 9】 25. The compound of claim 24, comprising:

26. The compound is 【Chemistry 10】 2. The compound of claim 1, comprising:

27. Z is (OCH ２ CH ２ ) ２４ -NH-C(O)CH ２ CH ２ 27. The compound of any one of claims 1 to 26, or a pharmaceutically acceptable salt thereof, comprising:

28. 28. The compound of any one of claims 1 to 27, or a pharmaceutically acceptable salt thereof, wherein Z comprises 1 to 8 (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) moieties.

29. 29. The compound of any one of claims 1 to 28, or a pharmaceutically acceptable salt thereof, wherein Z comprises a peptide comprising any of SEQ ID NOs: 1182-1217.

30. 30. The compound of any one of claims 1 to 29, or a pharmaceutically acceptable salt thereof, wherein the therapeutic polypeptide comprises an agonist at one or more of the GIP, GLP-1, and glucagon receptors.

31. the therapeutic polypeptide X １ X ２ X ３ X ４ TX ６ TSDX １０ X １１ X １２ X １３ LX １５ X １６ KAX １９ X ２０ X ２１ FIX ２４ X ２５ LX ２７ X ２８ X ２９ X ３０ X ３１ X ３２ X ３３ X ３４ X ３５ X ３６ X ３７ X ３８ X ３９ X ４０ X ４１ (SEQ ID NO: 1218), X １ is H, NMeY, or Y; X ２ is Ac4c, Aib, αMeS, Iva, or D-Ala; X ３ is Q, E, or H; X ４ is G or D-Ala X ６ is αMeF(2F), F, or αMeF; X １０ is 4-Pal, Y, or V; X １１ is Aib, S, or αMeS; X １２ is I or S, X １３ is L or αMeL, X １５ is D or E, X １６ is K, E, or Orn; X １９ is A or Q, X ２０ is Aib, αMeL, Iva, or αMe4Pal; X ２１ is E, Q, D, or Orn; X ２４ is K, Q, E, D-Glu, or D-Gln, X ２５ is αMeY, W, or Y, X ２７ is I, L, or V; X ２８ is K, E or A, X ２９ is Aib, G, S or Q; X ３０ is G, H or S, X ３１ is P, G, E, or Orn; X ３２ is absent, K, S, or P; X ３２ is S or P, then X ３３ is S, X ３３ If S, then X ３４ is G or Aib, X ３４ When is G or Aib, X ３５ is absent or is A or Orn, X ３５ When is A or Orn, X ３６ is absent or is P, X ３６ If P, then X ３７ is absent or is P, X ３７ If P, then X ３８ is absent, P or Ac3c, X ３８ When is P or Ac3c, X ３９ is absent or is S, Orn, or G; X ３９ is S, Orn, or G, then X ４０ is absent, K, or G, X ４０ is K or G, then X ４１ is absent or is S or G, X ３２ If X is not present or is K, ３３ ~X ４１ There is no X ３５ If does not exist, X ３６ ~X ４１ There is no X ３６ If does not exist, X ３７ ~X ４１ There is no X ３７ If does not exist, X ３８ ~X ４１ There is no X ３８ If does not exist, X ３９ ~X ４１ There is no X ３９ If does not exist, X ４０ and X ４１ There is no X ４０ If does not exist, X ４１ The compound according to any one of claims 1 to 30, or a pharmaceutically acceptable salt thereof, wherein

32. X ２４ , X ２８ , X ３２ or X ４０ or a pharmaceutically acceptable salt thereof, wherein one of:

33. 32. The compound of any one of claims 1 to 31, or a pharmaceutically acceptable salt thereof, wherein the therapeutic polypeptide comprises 90% sequence identity to any of SEQ ID NOs: 1-186 or 996-1038.

34. 33. The compound of any one of claims 1 to 32, or a pharmaceutically acceptable salt thereof, wherein the therapeutic polypeptide comprises any of SEQ ID NOs: 1 to 186 or 996 to 1038.

35. The fatty acid moiety is C １６ ~C ２２ 33. The compound according to any one of claims 1 to 32, or a pharmaceutically acceptable salt thereof, which is a fatty acid.

36. 36. The compound of claim 35, or a pharmaceutically acceptable salt thereof, wherein the fatty acid is conjugated to the therapeutic polypeptide at position K17 via a linker.

37. 37. The compound of claim 36, or a pharmaceutically acceptable salt thereof, wherein the linker comprises 1 to 4 amino acids.

38. 38. The compound of claim 37, or a pharmaceutically acceptable salt thereof, wherein the linker comprises one or more of the amino acids Lys, εLys, Glu, γGlu, or a combination thereof.

39. 39. The compound of any one of claims 35 to 38, or a pharmaceutically acceptable salt thereof, wherein the linker comprises 1 to 8 (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) moieties.

40. The linker and the fatty acid are (εLys) ａ (γGlu) ｂ -(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) ｃ -CO-(CH ２ ) ｐ -CO ２ H, wherein a is an integer of 0, 1, or 2; b is an integer of 0 or 1; c is an integer of 1 to 8; and p is an integer of 14 to 18.

41. 41. The compound of any one of claims 35-40, or a pharmaceutically acceptable salt thereof, wherein the therapeutic polypeptide conjugated to a fatty acid moiety comprises 90% sequence identity to any of SEQ ID NOs: 187-458 or 1039-1098.

42. 41. The compound of any one of claims 1 to 40, or a pharmaceutically acceptable salt thereof, wherein the therapeutic polypeptide conjugated to a fatty acid moiety comprises any of SEQ ID NOs: 187-458 or 1039-1098.

43. The compound according to any one of claims 1 to 42, or a pharmaceutically acceptable salt thereof, wherein the Fc region is selected from any one of SEQ ID NOs: 935-936, 941-948, and 1178-1181.

44. 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from any of SEQ ID NOs: 459-934 or 1099-1177.

45. formula: 【Chemistry 11】 or a pharmaceutically acceptable salt thereof, wherein X ＴＰＰ is a therapeutic polypeptide, YAibEGTX ６ TSDX １０ X １１ X １２ X １３ LX １５ X １６ KAQX ２０ X ２１ FIX ２４ X ２５ LX ２７ X ２８ X ２９ X ３０ X ３１ X ３２ X ３３ X ３４ X ３５ X ３６ X ３７ X ３８ X ３９ X ４０ X ４１ including (Sequence Number 1219) X ６ is αMeF(2F), F, or αMeF; X １０ is 4-Pal, Y, or V; X １１ is Aib or S, X １２ is I or S, X １３ is L or αMeL, X １５ is D or E, X １６ is E or Orn, X ２０ is Aib, αMeL, or Iva; X ２１ is E, Q, D, or Orn; X ２４ is K, Q, E, or D-Glu, X ２５ is αMeY or Y, X ２７ is I, L, or V; X ２８ is K, E or A, X ２９ is Aib, G, or Q; X ３０ is G or S, X ３１ is P, G, E, or Orn; X ３２ is absent, K, S, or P; X ３２ is S or P, then X ３３ is S, X ３３ If S, then X ３４ is G or Aib, X ３４ When is G or Aib, X ３５ is absent, A or Orn, X ３５ When is A or Orn, X ３６ is absent or is P, X ３６ If P, then X ３７ is absent or is P, X ３７ If P, then X ３８ is absent or is P, X ３８ If P, then X ３９ is absent, S, Orn, or G; X ３９ is S, Orn, or G, then X ４０ is absent, K, or G, X ４０ is K or G, then X ４１ is absent, S, or G, X ３２ If X is not present or is K, ３３ ~X ４１ There is no X ３５ If does not exist, X ３６ ~X ４１ There is no X ３６ If does not exist, X ３７ ~X ４１ There is no X ３７ If does not exist, X ３８ ~X ４１ There is no X ３８ If does not exist, X ３９ ~X ４１ There is no X ３９ If does not exist, X ４０ and X ４１ There is no X ４０ If does not exist, X ４１ does not exist, L is R ９ , R １０ and a linker conjugated to each of said therapeutic polypeptides; R ９ and R １０ is independently an antibody fragment containing an Fc region; X ２４ , X ２８ , X ３２ or X ４０ or a pharmaceutically acceptable salt thereof, wherein one of

46. 46. ​​The compound of claim 45, or a pharmaceutically acceptable salt thereof, wherein the therapeutic polypeptide has dual agonist activity at the GIP receptor and the GLP-1 receptor.

47. X １ is Y and X ２ is Aib and X ３ is E and X ４ is G and X ６ is αMeF(2F), and X １０ is 4-Pal or Y, and X １１ is Aib or S, and X １２ is I and X １３ is αMeL, and X １５ is D and X １６ is E or Orn, and X １ is Q and X ２０ is Aib and X ２１ is E and X ２４ is Q, E, or D-Glu, and X ２５ is αMeY, and X ２７ is I and X ２８ is E and X ２９ is Aib or G, and X ３０ is G or S, and X ３１ is P and X ３２ is G or S, X ３２ If S, then X ３３ is S and X ３４ is G and X ３５ is A and X ３６ is P and X ３７ is P and X ３８ is P and X ３９ is S and X ４０ is K and X ４１ is absent and the linker is X ４０ conjugated to X ３２ If G, then X ３３ ~X ４１ is absent and the linker is X ３２ or a pharmaceutically acceptable salt thereof, conjugated to

48. X １０ is Y or 4-Pal, and X １１ is Aib or S, and X ２４ is Q, E, or D-Glu, and X ３０ is G or S, or a pharmaceutically acceptable salt thereof.

49. X １０ is Y or 4-Pal, and X １１ is Aib and X ２４ is E and X ３０ is S, and X ４０ is K and is conjugated to said linker, or a pharmaceutically acceptable salt thereof.

50. 50. The compound of any one of claims 45-49, or a pharmaceutically acceptable salt thereof, wherein the therapeutic polypeptide comprises 90% sequence identity to any of SEQ ID NOs: 1-92 or 996-999.

51. 51. The compound of any one of claims 45-50, or a pharmaceutically acceptable salt thereof, wherein the therapeutic polypeptide comprises any of SEQ ID NOs: 1-92 or 996-999.

52. 52. The compound of any one of claims 45 to 51, or a pharmaceutically acceptable salt thereof, wherein the therapeutic polypeptide comprises any of SEQ ID NOs: 1, 2, 22, 43, 45, 48, 55, 56, 58, 59, 61, 66, 68, and 47.

53. The therapeutic polypeptide is linked to a C at position 17 via a second linker. １６ ~C ２２ 52. The compound of any one of claims 45 to 51, or a pharmaceutically acceptable salt thereof, conjugated to a fatty acid moiety.

54. 54. The compound of claim 53, or a pharmaceutically acceptable salt thereof, wherein the second linker comprises 1 to 4 amino acids.

55. 55. The compound of claim 54, or a pharmaceutically acceptable salt thereof, wherein the second linker comprises the amino acid Lys, εLys, Glu, γGlu, or a combination thereof.

56. 56. The compound of any one of claims 53 to 55, or a pharmaceutically acceptable salt thereof, wherein the second linker comprises 1 to 8 (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) moieties.

57. The second linker and the fatty acid are (εLys) ａ (γGlu) ｂ -(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) ｃ -CO-(CH ２ ) ｐ -CO ２ H, wherein a is an integer of 0, 1, or 2; b is an integer of 0 or 1; c is an integer of 1 to 8; and p is an integer of 14 to 18.

58. 58. The compound of any one of claims 53-57, or a pharmaceutically acceptable salt thereof, wherein the therapeutic polypeptide conjugated to the fatty acid moiety via the second linker comprises 90% sequence identity to any of SEQ ID NOs: 187-332 or 1039-1042.

59. 59. The compound of any one of claims 53-58, or a pharmaceutically acceptable salt thereof, wherein the therapeutic polypeptide conjugated to the fatty acid moiety via the second linker comprises any of SEQ ID NOs: 187-332 or 1039-1042.

60. 60. The compound of any one of claims 53-59, or a pharmaceutically acceptable salt thereof, wherein the therapeutic polypeptide conjugated to the fatty acid moiety comprises any of SEQ ID NOs: 187, 209, 235, 237, 241, 243, 257, 258, 260, 262, 263, 264, 266, 267, 275, or 277.

61. 61. The compound of any one of claims 45 to 60, or a pharmaceutically acceptable salt thereof, wherein the Fc region is selected from any of SEQ ID NOs: 935-936, 941-948, or 1178-1181.

62. The linker is 【Chemistry 12】 wherein Z is O, C(O), NH, C １ ~C ３０ Alkyl, (OCH ２ CH ２ ) ｍ , [C(O)NH-CH ２ CH ２ OCH ２ ] ｍ , peptides, or combinations thereof, wherein m is an integer from 1 to 30; ＊＊＊ ) comprises a connection point to said therapeutic polypeptide; U is C １ ~C ５ alkyl, N, phenyl, or phenylcarbonyl, or a combination thereof; R １ and R ２ are independently a covalent bond or C １ ~C ５ is alkyl, R ３ and R ４ is absent or is an amide, R ５ and R ６ is independently absent or R ５ and R ６ are independently １ ~C ５ Alkyl, phenyl, CH ２ O (CH ２ CH ２ O) ｎ or a combination thereof, wherein n is an integer from 1 to 5; １ ~C ５ Alkyl is NH ２、 CH ２ NH2, and CH ２ CH ２ NH ２ and optionally replaced with one or more of R ７ and R ８ is not present or, independently, 【Chemistry 13】 wherein: ( ＊ ) is R ９ or R １０ including the connection point to ( ＊＊ ) is R ５ or R ６ 62. The compound of any one of claims 45 to 61, or a pharmaceutically acceptable salt thereof, comprising a connection point to:

63. 63. The compound of claim 62, or a pharmaceutically acceptable salt thereof, wherein U is N or phenyl.

64. R ７ or R ８ but, 【Chemistry 14】 64. The compound of claim 62 or 63, wherein:

65. The linker is 【Chemistry 15】 Including, In the formula, ( ＊ ) is R ９ or R １０ 65. The compound of claim 64, or a pharmaceutically acceptable salt thereof, comprising a connection point to:

66. R ７ and R ８ but, 【Chemistry 16】 63. The compound of claim 62, wherein:

67. The linker is 【Chemistry 17】 Including, In the formula, ( ＊ ) is R ９ or R １０ 67. The compound of claim 66, or a pharmaceutically acceptable salt thereof, comprising a connection point to:

68. Z is (OCH ２ CH ２ ) ２４ -NH-C(O)CH ２ CH ２ 68. The compound of any one of claims 62 to 67, comprising:

69. 69. The compound of any one of claims 65 to 68, or a pharmaceutically acceptable salt thereof, wherein Z comprises 1 to 8 (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) moieties.

70. 70. The compound of any one of claims 65 to 69, or a pharmaceutically acceptable salt thereof, wherein Z comprises a peptide comprising any of SEQ ID NOs: 1182-1217.

71. 71. The compound of any one of claims 45 to 70, or a pharmaceutically acceptable salt thereof, wherein the compound is any of SEQ ID NOs: 459 to 787 or 1099 to 1100.

72. 72. The compound of any one of claims 45-71, or a pharmaceutically acceptable salt thereof, wherein the compound is any of SEQ ID NOs: 459, 490, 505, 585, 587, 594, 616, 622, 626, 627, 628, 630, 631, 639, 641, 642, 643, 645, 646, 662, 698, 699, 700, or 1100.

73. formula: [Chemistry 18] or a pharmaceutically acceptable salt thereof, wherein X ＴＰＰ is a therapeutic polypeptide, X １ X ２ X ３ X ４ TX ６ TSDX １０ X １１ X １２ X １３ LX １５ X １６ KAQX ２０ X ２１ FIX ２４ X ２５ LX ２７ X ２８ X ２９ X ３０ X ３１ X ３２ X ３３ X ３４ X ３５ X ３６ X ３７ X ３８ X ３９ X ４０ X ４１ (SEQ ID NO: 1220), X １ is H, NMeY, or Y; X ２ is Ac4c, Aib, αMeS, Iva, or D-Ala; X ３ is Q, E, or H; X ４ is G or D-Ala X ６ is αMeF(2F), F, or αMeF; X １０ is 4-Pal, Y, or V; X １１ is Aib, S, or αMeS; X １２ is I or S, X １３ is L or αMeL, X １５ is D or E, X １６ is E or Orn, X ２０ is Aib, αMeL, Iva, or αMe4Pal; X ２１ is E, Q, D, or Orn; X ２４ is K, Q, E, D-Glu, or D-Gln, X ２５ is αMeY or Y, X ２７ is I, L or V, X ２８ is K, E or A, X ２９ is Aib, S, G, or Q; X ３０ is G, S, or H; X ３１ is P, G, E, or Orn; X ３２ is absent, K, S, or P; X ３２ is S or P, then X ３３ is S, X ３３ If S, then X ３４ is G or Aib, X ３４ When is G or Aib, X ３５ is absent or is A or Orn, X ３５ When is A or Orn, X ３６ is absent or is P, X ３６ If P, then X ３７ is absent or is P, X ３７ If P, then X ３８ is absent or is P, X ３８ If P, then X ３９ is absent or is S, Orn, or G; X ３９ is S, Orn, or G, then X ４０ is absent, K, or G, X ４０ is K or G, then X ４１ is absent or is S or G, X ３２ or if K is not present, X ３３ ~X ４１ There is no X ３５ If does not exist, X ３６ ~X ４１ There is no X ３６ If does not exist, X ３７ ~X ４１ There is no X ３７ If does not exist, X ３８ ~X ４１ There is no X ３８ If does not exist, X ３９ ~X ４１ There is no X ３９ If does not exist, X ４０ and X ４１ There is no X ４０ If does not exist, X ４１ does not exist, L is R ９ , R １０ and a linker conjugated to each of said therapeutic polypeptides; R ９ and R １０ are antibody fragments containing an Fc region, X ２４ , X ３２ and X ４０ or a pharmaceutically acceptable salt thereof, wherein one of

74. 74. The compound of claim 73, or a pharmaceutically acceptable salt thereof, wherein said therapeutic polypeptide has triple agonist activity at the GIP, GLP-1, and glucagon receptors.

75. X １ is H or Y, and X ２ is Aib and X ３ is Q and X ４ is G and X ６ is αMeF(2F), and X １０ is Y or 4-Pal, and X １１ is S or αMeS, and X １２ is I and X １３ is αMeL, and X １５ is D and X ２０ is Aib or αMe4Pal, and X ２１ is E or Orn, and X ２４ is E or d-Glu, and X ２５ is αMeY, and X ２７ is L or I, and X ２８ is E and X ２９ ~X ３９ is GSPSSGAPPPS, and X ４０ is K; or a pharmaceutically acceptable salt thereof.

76. X １０ is Y or 4-Pal, and X ２４ is E or d-Glu, and X ３０ or a pharmaceutically acceptable salt thereof.

77. X １０ is Y or 4-Pal, and X ２４ is E or d-Glu, and X ３０ is S, X ４０ or a pharmaceutically acceptable salt thereof, wherein each of

78. 78. The compound of any one of claims 73-77, or a pharmaceutically acceptable salt thereof, wherein the therapeutic polypeptide comprises 90% sequence identity to any of SEQ ID NOs: 93-186 or 1000-1038.

79. 79. The compound of any one of claims 73-78, or a pharmaceutically acceptable salt thereof, wherein the therapeutic polypeptide comprises any of SEQ ID NOs: 93-186 or 1000-1038.

80. 80. The compound of any one of claims 73 to 79, or a pharmaceutically acceptable salt thereof, wherein the therapeutic polypeptide comprises any of SEQ ID NOs: 97, 113, 128, 130, 144, 171, 183.

81. The therapeutic polypeptide is linked to C at position K17 via a second linker. １６ ~C ２２ 81. The compound of any one of claims 73 to 80, or a pharmaceutically acceptable salt thereof, conjugated to a fatty acid moiety.

82. 82. The compound of claim 81, or a pharmaceutically acceptable salt thereof, wherein the second linker comprises 1 to 4 amino acids.

83. 83. The compound of claim 82, or a pharmaceutically acceptable salt thereof, wherein the second linker comprises one or more of the amino acids Lys, εLys, Glu, γGlu, or a combination thereof.

84. 84. The compound of any one of claims 81 to 83, or a pharmaceutically acceptable salt thereof, wherein the second linker comprises 1 to 8 (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) moieties.

85. The second linker and the fatty acid are (εLys) ａ (γGlu) ｂ -(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) ｃ -CO-(CH ２ ) ｐ -CO ２ H, wherein a is an integer of 0, 1, or 2; b is an integer of 0 or 1; c is an integer of 1 to 8; and p is an integer of 14 to 18.

86. 86. The compound of any one of claims 81-85, or a pharmaceutically acceptable salt thereof, wherein the therapeutic polypeptide conjugated to the fatty acid moiety via the second linker comprises 90% sequence identity to any of SEQ ID NOs: 333-458 or 1043-1098.

87. 87. The compound of any one of claims 81-86, or a pharmaceutically acceptable salt thereof, wherein the therapeutic polypeptide conjugated to the fatty acid moiety via the second linker comprises any of SEQ ID NOs: 333-458 or 1043-1098.

88. 88. The compound of any one of claims 81-87, or a pharmaceutically acceptable salt thereof, wherein the therapeutic polypeptide conjugated to the fatty acid moiety via the second linker comprises any of SEQ ID NOs: 337, 380, 395, 404, 423, 424, 426, 435, 447, 450, 467, 490, or 493.

89. 89. The compound of any one of claims 73 to 88, or a pharmaceutically acceptable salt thereof, wherein the Fc region is selected from any of SEQ ID NOs: 935-936, 941-948, or 1178-1181.

90. The linker is 【Chemistry 19】 wherein Z is O, C(O), NH, C １ ~C ３０ Alkyl, (OCH ２ CH ２ ) ｍ , [C(O)NH-CH ２ CH ２ OCH ２ ] ｍ , peptides, or combinations thereof, wherein m is an integer from 1 to 30; ＊＊＊ ) comprises a connection point to said therapeutic polypeptide; U is C １ ~C ５ alkyl, N, phenyl, or phenylcarbonyl, or a combination thereof; R １ and R ２ are independently a covalent bond or C １ ~C ５ is alkyl, R ３ and R ４ is absent or is an amide, R ５ and R ６ is independently absent or R ５ and R ６ are independently １ ~C ５ Alkyl, phenyl, CH ２ O (CH ２ CH ２ O) ｎ or a combination thereof, wherein n is an integer from 1 to 5; １ ~C ５ Alkyl is NH ２ , C.H. ２ NH2, and CH ２ CH ２ NH ２ and optionally replaced with one or more of R ７ and R ８ is not present or, independently, 【Chemistry 20】 wherein: ( ＊ ) is R ９ or R １０ including the connection point to ( ＊＊ ) is R ５ or R ６ 90. The compound of any one of claims 73 to 89, or a pharmaceutically acceptable salt thereof, comprising a connection point to:

91. 91. The compound of claim 90, or a pharmaceutically acceptable salt thereof, wherein U is N or phenyl.

92. R ７ or R ８ but, 【Chemistry 21】 92. The compound of claim 90 or 91, wherein:

93. The linker is 【Chemistry 22】 wherein ＊ ) is R ９ and R １０ 93. The compound of any one of claims 90 to 92, or a pharmaceutically acceptable salt thereof, comprising a connection point to:

94. R ７ and R ８ but, 【Chemistry 23】 91. The compound of claim 90, wherein:

95. The linker is 【Chemistry 24】 Including, In the formula, ( ＊ ) is R ９ or R １０ 91. The compound of claim 90, or a pharmaceutically acceptable salt thereof, comprising a connection point to:

96. Z is (OCH ２ CH ２ ) ２４ -NH-C(O)CH ２ CH ２ 96. The compound of any one of claims 90 to 95, or a pharmaceutically acceptable salt thereof, comprising:

97. 97. The compound of any one of claims 90 to 96, or a pharmaceutically acceptable salt thereof, wherein Z comprises 1 to 8 (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) moieties.

98. 97. The compound of any one of claims 90-96, wherein Z comprises a peptide comprising any of SEQ ID NOs: 1182-1217.

99. 99. The compound of any one of claims 73 to 98, wherein the compound is any one of SEQ ID NOs: 788 to 934 or 1101 to 1177, or a pharmaceutically acceptable salt thereof.

100. 100. The compound of any one of claims 73-99, or a pharmaceutically acceptable salt thereof, wherein the compound is any of SEQ ID NOs: 792, 843, 859, 869, 889, 891, 900, 912, 915, 1101, 1102, 1103, 1104, 1110, 1172, 1173, 1174, or 1175.

101. 101. The compound of any one of claims 1 to 100, or a pharmaceutically acceptable salt thereof, wherein the compound has a duration of action sufficiently extended to allow for once-monthly administration.

102. 102. A pharmaceutical composition comprising a compound according to any one of claims 1 to 101, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, diluent, or excipient.

103. 103. The pharmaceutical composition of claim 102, wherein the composition is formulated for subcutaneous administration.

104. 102. A method of treating a disease or condition, comprising the step of administering an effective amount of a compound of any one of claims 1 to 101, or a pharmaceutically acceptable salt thereof, to an individual in need of treatment for said disease or said condition once a month.

105. 105. The method of claim 104, wherein the disease or condition is selected from the group consisting of diabetes, obesity, chronic weight management, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), dyslipidemia, metabolic syndrome, chronic kidney disease (CKD), osteoarthritis (OA), obesity-related sleep apnea (OSA), polycystic ovary syndrome (PCOS), Parkinson's disease, Alzheimer's disease, heart failure (HF), hypertension (HT), peripheral arterial disease (PAD), metabolic syndrome, chronic low back pain (CLBP), cirrhosis, polycystic ovary syndrome, and / or alcohol abuse disorder.

106. 102. The compound of any one of claims 1 to 101, or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt is selected from sodium, potassium, trifluoroacetate, hydrochloride, or acetate.

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