Aza GLP-1-based therapeutic analogues

Abstract

This invention provides, inter alia, for an aza GLP-l-based therapeutic analogue wherein at least one peptide bond of a GLP-1-based therapeutic peptide has been replaced with an azapeptide or azatide bond.

Description

AZA GLP-1-BASED THERAPEUTIC ANALOGUES RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 609,981, filed on December 14, 2023, the entire contents of which are herein incorporated by reference in its entirety. INCORPORATION BY REFERENCE

[0001] All publications cited in this specification as well as the publications cited in said publications are hereby incorporated by reference. The discussion of these publications herein is intended merely to summarize the assertions made by applicant and no admission is made that any publication constitutes prior art. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (9081014_Sequence12Dec2024.xml; Size: 54000 bytes; and Date of Creation: December 12, 2024) is herein incorporated by reference in its entirety. FIELD OF THE INVENTION

[0003] This disclosure relates to analogues of incretin peptides (i.e., glucagon-like peptide-1 (“GLP-1”), glucose-dependent insulinotropic polypeptide (“GIP”) and glucagon), the analogues containing at least one azapeptide bond (“-NH-NR-C(O)-”). The analogues of incretin peptides of the invention encompass novel and inventive aza GLP-1-based therapeutic analogues; pharmaceutical compositions comprising aza GLP-1-based therapeutic analogues; and uses of aza GLP-1-based therapeutic analogues for the treatment of diseases. As defined herein, a “GLP- 1-based therapeutic peptide” is a GLP-1 receptor agonist peptide, a dual-acting GLP-1 and a GIP receptor agonist peptide, or a triple acting GLP-1, GIP, and glucagon receptor agonist peptide. An “aza GLP-1-based therapeutic analogue” is a GLP-1 receptor agonist peptide, a dual-acting GLP-1 and GIP receptor agonist peptide, or a triple acting GLP-1, GIP, and glucagon receptor agonist peptide in which at least one peptide bond (“-NH-NR-C(O)-”) has been replaced with an azapeptide bond (“-NH-NR-C(O)-)”).BACKGROUND OF THE INVENTION

[0004] Peptides are made up of chains of amino acids from 2 to 200 residues long, linked together by peptide (i.e., amide) bonds. Peptides play a critical role in a variety of biological processes, including inter- and intracellular signaling, as well as in the regulation of physiological functions.

[0005] The field of peptide therapeutics began with the first medical application of insulin in 1922. Since then, peptide-based drugs have become increasingly popular in the pharmaceutical industry because they possess several advantages over small molecules, including, for example, larger surface area for specific target recognition, potential for enhanced efficacy and increased selectivity / specificity, and lower toxicity. These features make peptide-based drugs an attractive option for treating an array of conditions including but not limited to, cancer, autoimmune diseases, and neurodegenerative disorders.

[0006] As of today, there are more than 80 peptide-based drugs on the global market, and hundreds of peptide-based compounds are in various stages of preclinical testing and clinical development.

[0007] However, as active pharmaceuticals, peptides have certain limitations. These limitations include their short biological half-lives (seconds to minutes); which are due in part to rapid degradation by proteolytic enzymes. Thus, despite their advantages, many potential therapeutic peptides have not been approved or have been discontinued by the FDA often because of their poor physicochemical characteristics and / or short biological half-lives.

[0008] To circumvent their shortcomings, researchers have developed chemically modified peptides known as peptidomimetics. These compounds mimic the three-dimensional structure and biological activities of natural peptides, while being engineered to possess improved pharmacokinetic properties (e.g., longer biological half-lives).

[0009] GLP-1 is one of therapeutically important peptides. It is a 37-amino acid incretin hormone derived from proglucagon metabolism that plays important roles in promoting glucose- dependent insulin secretion and inhibiting glucagon release. GLP-1 lowers blood sugar levels indiabetic patients (with cumulative lowering effects on HbA1c levels in these patients). GLP-1 was also found to promote satiety, resulting in reduced food and water intake and, consequently, a lower body weight. GLP-1 also has been found to have cardio- and neuroprotective effects, decrease inflammation and apoptosis, and to have implications for memory and learning. See, e.g., US 10,335,462 and T.D. Miller, et al., “Glucagon-like peptide 1”, Molecular Metabolism, 30, 72-130 (2019). Although GLP-1 has shown promising results, due to rapid enzymatic degradation by dipeptidylpeptidase-4 (DPP-IV) and other proteases in the blood and gut, GLP-1 has a short half-life of less than five minutes, limiting its therapeutic efficacy, routes of administration and application.

[0010] A class of compounds that has shown promise is GLP-1 receptor agonists peptides. Compounds from this class include, for example, albiglutide, dulaglutide, liraglutide, semaglutide, exenatide, and lixisenatide. W. Peng, et al., “Novel Insights into the Roles and Mechanisms of GLP-1 Receptor Agonists against Aging-Related Diseases”, Aging and Disease, 13(2), pp.468-490 (2022). A.M. Kastreboff and R.F. Kushner, “New Frontiers in Obesity Treatment GLP-1 and Nascent Nutrient-Stimulated Hormone-Based Therapeutics”, Annu. Rev. Med., 74, pp. 125-39 (2023). In view of their popularity in treating diabetes and weight loss, GLP-1 receptor agonist peptides had an estimated market of approximately USD $4.01 billion in 2022.

[0011] The approach to extend the half-lives of GLP-1 receptor agonists peptides followed two main strategies: 1) modifying the amino acid sequence by alkylating to protect the peptide from cleavage; and 2) attaching a lipid moiety to enhance its binding to serum proteins and reduce its renal clearance. See, e.g., US 8,129,343 and US 8,536,122. For example, in semaglutide, the first FDA-approved oral GLP-1 receptor agonist, the alanine at position 8 was replaced with an unnatural amino acid (Aib) to prevent DPP-IV degradation. Additionally, a lipid chain was added to lysine at position 26 to optimize receptor binding. The structure of semaglutide is depicted in Fig. 2C. While this was an improvement, semaglutide is still susceptible to proteolytic degradation in the gastrointestinal tract.

[0012] In addition to GLP-1, there is also a second incretin peptide: GIP. GIP is a 42-amino acid gastrointestinal regulatory peptide that plays a physiological role in glucose homeostasis bystimulating insulin secretion from pancreatic beta cells in the presence of glucose and protecting pancreatic beta cells.

[0013] In healthy individuals, the ingestion of food result in the release of both GLP-1 and GIP, as well as pancreatic beta cell hormones (insulin and amylin). GLP-1 and amylin exert an inhibitory effect on gastric emptying, glucagon release, and appetite. Following the absorption of food, GLP-1 and GIP elicit insulin secretion, otherwise known as the incretin effect. The secretion and metabolism of GIP, GLP-1 and glucagon is summarized in Fig. 1. The roles of GLP-1 and GIP in maintaining glucose homeostasis is summarized in Fig. 2B. In diabetes, these steps are disrupted.

[0014] In addition to GLP-1 and GIP, there is also a third incretin hormone: glucagon. Glucagon is a 29-amino acid peptide produced by the pancreas. When bound to glucagon receptor, glucagon signals the liver to release glucose leading to an increase in blood glucose.

[0015] The amino acid sequences of GIP, GLP-1, and glucagon are depicted in Fig. 2A.

[0016] Azapeptides are a class of peptidomimetics, where one or more α-carbons in the peptide backbone are replaced by a trivalent nitrogen atom.

[0018] Since the first synthesis of azaVal3 angiotensin II in 1963, azapeptides have shown biological effects on several therapeutic targets. Several azapeptide derivatives have been discovered and advanced as therapeutic agents. For example, azapeptides have been used as enzyme inhibitors, receptor ligands (antagonist and agonist), and stabilizers for triple-helical collagen peptides. In 1989, the aza-Gly peptide analog, Goserelin (Zoladex®), was approved by the FDA for the treatment of prostate and breast cancers. Later, the non-native side chain azapeptide analog, Atazanavir (Reyataz®), was approved by the FDA in 2003 for the treatment of HIV infection. By definition, azapeptides comprise at least one aza-amino acid.

[0017] Unlike natural amino acids, aza-amino acids are not stable and are easily decarboxylated, making traditional peptide synthesis strategies unsuitable for efficient synthesis of azapeptides.

[0018] Traditional azapeptide synthetic strategy via activation of N'-alkyl-N-protected- hydrazines is depicted in Fig. 2D.

[0019] Despite limited successes and their potential as therapeutic agents for an unlimited array of disease, and conditions and, in addition to their potential use as diagnostic reagents, azapeptides use has been limited, mainly due to the additional difficulties and intricacies of azapeptide peptide synthesis.

[0020] The search for analogues of incretin peptides, including therapeutic GLP-1 receptor agonists, that are resistant to enzymatic attack and degradation, and have improved physio- chemical properties is ongoing. SUMMARY OF THE INVENTION

[0019] This invention provides analogues of incretin peptides (e.g., GLP-1-based therapeutic peptides) and their salts. The analogues of incretin peptides differ from incretin peptides in that the analogues of incretin peptides contain at least one bond (“-NH-NR-C(O)-”), which is a result of a substitution of an amino acid of an incretin peptide with an aza-amino acid, which incretin peptides do not have. In other words, the analogues of incretin peptides differ from incretin peptides in that at least one “-CR- “of the incretin peptide backbone is replaced with “-NR-“ in the corresponding analogue of the incretin peptide, wherein R is a side chain radical of a natural or unnatural amino acid that is optionally conjugated to (i) a lipid (e.g., a fatty acid) with an albumin binding motif, directly or through a linker or (ii) a different compound, at one or more position(s), including, e.g., position 26.

[0020] In addition to containing an aza-amino acid, the analogues of incretin peptides may further structurally differ from incretin peptides by containing, at a corresponding position, (i) at least one unnatural amino acid (e.g., 2-aminoisobutyric acid (“Aib”)) instead of a natural amino acid of the incretin peptide and / or (ii) a natural amino acid that is different from a natural amino acid.

[0021] Despite these structural changes, as compared to the incretin peptides, the analogues of incretin peptides retain and / or have an improved efficacy at respective incretin receptors, including, e.g., GLP-1 receptors.

[0022] At the same time, the analogues of incretin peptides are more resistant to degradation by serum and gastric proteases than the incretin peptides. This is because, in the analogues of incretin peptides, the at least one azapeptide bond and / or the at least one unnatural amino acidand / or the different natural amino acid is located at a serum and / or digestive protease cleavage site(s) of the analogues of incretin peptides. The at least one azapeptide bond, the at least one unnatural amino acid and the at least one different amino acid therefore make the analogues of incretin peptides more resistant to degradation by serum and / or digestive proteases than the incretin peptide(s), and, at the same time, allow the analogues of incretin peptides to retain and / or have an improved efficacy at respective incretin receptors, including, e.g., GLP-1 receptors, and / or longer biological half-lives.

[0023] The at least one azapeptide bond and / or the at least one unnatural amino acid and / or the different natural amino acid may further allow the analogues of incretin peptides (i) to have improved physicochemical properties, such as, e.g., improved water solubility, of the analogues of incretin peptides and / or (ii) be formulated in alternative dosage forms, such as, e.g., oral pharmaceutical dosage forms and / or (iii) to be administered at a lower dose (due, e.g., to an increased potency) and / or at a lower frequency (due, e.g., to a longer biological half-life).

[0024] The analogues of incretin peptides and their salts include: (i) aza GLP-1-based therapeutic peptide analogues in which at least one peptide bond of a GLP-1-based therapeutic peptide has been replaced with an azapeptide bond, and (ii) aza GLP-1-based therapeutic peptide analogues in which (a) at least one peptide bond of the GLP-1-based therapeutic peptide has been replaced with an azapeptide bond and (b) at least one amino acid of the GLP-1-based therapeutic peptide has been replaced with an unnatural amino acid or a different amino acid. In an embodiment, in the aza GLP-1-based therapeutic peptide analogues, the azapeptide bond may be located between the first and second and / or second and third residues of aza GLP-1-based therapeutic peptide analogues, which is the site cleaved by DPP-IV, and the at least one unnatural amino acid, and the different amino acid may be the first and / or second residue(s) of the aza GLP-1-based therapeutic peptide analogues, which are at the site cleaved by DPP-IV.

[0025] The invention is directed in part to analogues of incretin peptides and aza GLP-1-based therapeutic peptide analogues that are selected from a group consisting of compounds of SEQ ID Nos. 4 to 25 and pharmaceutically acceptable salts thereof.

[0026] In an embodiment, the present invention provides a compound of Formula:X1X2X3GX4X5X6X7X8X9X10X11X12EGX13X14AX15X16X17X18X19X20X21X22X23X24X25X26X27- X28X29X30X31X32X33X34X35, wherein X1 is azaH, azaA, AzaG, azaY, azaF, F, Y, A, G, or H; X2 is azaA, azaG, aza-D, D, A, Aib, G, or A; X3is azaE, E, Q, or azaQ; X4is azaT, azaS, T or S; X5is azaF or F; X6is azaT or T; X7 is azaS or S; X8 is azaD or D; X9 is azaV, azaY, Y or V; X10 is azaS or S; X11 is azaS, azaI, azaL, azaV, azaF, I, L, V, F, or S; X12 is azaY, Y, Aib, L, or azaL; X13 is azaG, azaK, K or G; X14 is azaQ, azaE, azaI, I, K, azaK, Q, or E; X15is azaA, A, azaE, E, AzaQ, or Q; X16is azaK, K or Aib; X17 is azaE, azaA, A or E; X18 is azaF or F; X19 is azaI, azaV, V, or I; X20 is azaA, azaQ, Q, E, azaE, or A; X21 is AzaW, W, Y, or azaY; X22 is azaL, azaV, L, azaL, V, or azaV; X23 is azaV, azaI, L, aza L, I, or V; X24is azaK, azaE, azaR, K, E or R; X25is azaG or G; X26is azaG, azaR, G or R; X27is absent or is azaG, G, azaP, or P; X28is absent or S; X29is absent or S; X30is absent or G; X31 is absent or A; X32 is absent or P; X33 is absent or P; X34 is absent or P; X35 is absent or S, and wherein X35, if present, is optionally amidated as a C-terminal primary amide (SEQ ID NO: 4); wherein at least one of X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, X21, X22, X23, X24, X25, X26, and X27 is an aza-amino acid; or a pharmaceutically acceptable salt thereof.

[0018] In an embodiment, the present invention provides a compound of SEQ ID NO: 4, wherein at least one of X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, X21, X22, X23, X24, X25, X26, and X27 is an aza-amino acid; or a pharmaceutically acceptable salt thereof, and the compound is optionally conjugated to a lipid (e.g., a fatty acid) with an albumin binding motif, directly or through a linker.

[0019] In an embodiment, the present invention provides a compound of SEQ ID NO: 4, wherein at least one of X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, X21, X22, X23, X24, X25, X26, and X27is an aza-amino acid; or a pharmaceutically acceptable salt thereof, and the compound is conjugated to a lipid (e.g., a fatty acid) with an albumin binding motif, directly or through a linker.

[0020] In an embodiment, the present invention provides a compound of SEQ ID NO: 4, wherein at least one of X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, X21, X22, X23, X24, X25, X26, and X27 is an aza-amino acid; or a pharmaceuticallyacceptable salt thereof, and the compound is conjugated to a lipid (e.g., a fatty acid) at position 26.

[0021] In an embodiment, the present invention provides an aza GLP-1-based therapeutic analogue or a salt thereof of a GLP-1- based therapeutic peptide, wherein (i) at least one peptide bond of the GLP-1- based therapeutic peptide has been replaced with an azapeptide bond, (ii) at least one amino acid of the GLP-1- based therapeutic peptide has been replaced with an unnatural amino acid or a different amino acid, and the aza GLP-1-based therapeutic analogue is a compound of SEQ ID NO: 4, and wherein the aza GLP-1-based therapeutic analogue is optionally conjugated to (i) a lipid (e.g., a fatty acid) with an albumin binding motif, directly or through a linker, or (ii) a different compound, at one or more position(s), including, e.g., position 26.

[0022] In an embodiment, the present invention provides an aza GLP-1-based therapeutic analogue or a salt thereof of a GLP-1- based therapeutic peptide, wherein (i) at least one peptide bond of the GLP-1- based therapeutic peptide has been replaced with an azapeptide bond, (ii) at least one amino acid of the GLP-1- based therapeutic peptide has been replaced with an unnatural amino acid or a different amino acid, and (iii) the aza GLP-1-based therapeutic analogue is a compound of SEQ ID NO: 4, and wherein aza GLP-1-based therapeutic analogue is conjugated to (i) a lipid (e.g., a fatty acid) with an albumin binding motif, directly or through a linker.

[0023] In an embodiment, the present invention provides a compound of Formula: X1X2X3GX4X5X6X7X8X9X10X11X12EGX13X14AX15X16X17X18X19X20X21X22X23X24X25X26X27, wherein X1is azaH, azaA, AzaG, azaY, azaF, F, Y, A, G, or H; X2is azaA, azaG, aza-D, D, A, Aib, G, or A; X3 is azaE or E; X4 is azaT, azaS, T or S; X5 is azaF or F; X6 is azaT or T; X7 is azaS or S; X8 is azaD or D; X9 is azaV, azaY, Y or V; X10 is azaS or S; X11 is azaS, azaI, azaL, azaV, azaF, I, L, V, F, or S; X12is azaY, Aib, or Y; X13is azaG, azaK, K or G; X14is azaQ, azaE, azaI, I, Q or E; X15 is azaA, azaE, E, or A; X16 is azaK or K; X17 is azaE, azaA, A or E; X18 is azaF or F; X19 is azaI, azaV, V, or I; X20 is azaA, azaQ, Q, or A; X21 is AzaW or W; X22 is azaL, azaV, L or V; X23is azaV, azaI, I or V; X24is azaK, azaE, azaR, K, E or R; X25is azaG or G; X26is azaG, azaR, G or R; X27is azaG or G (SEQ ID NO: 5); wherein at least one of X1, X2, X3, X4,X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, X21, X22, X23, X24, X25, X26, and X27 is an aza-amino acid; or a pharmaceutically acceptable salt thereof.

[0024] In an embodiment, the present invention provides a compound of Formula: X1X2X3GX4X5X6X7X8X9X10X11X12EGX13X14AX15X16X17X18X19X20X21X22X23X24X25X26, wherein X1is azaH, azaA, AzaG, azaY, azaF, F, Y, A, G, or H; X2is azaA, azaG, aza-D, D, A, Aib, G, or A; X3 is azaE or E; X4 is azaT, azaS, T or S; X5 is azaF or F; X6 is azaT or T; X7 is azaS or S; X8 is azaD or D; X9 is azaV, azaY, Y or V; X10 is azaS or S; X11 is azaS, azaI, azaL, azaV, azaF, I, L, V, F, or S; X12is azaY, Aib, or Y; X13is azaG, azaK, K or G; X14is azaQ, azaE, azaI, I, Q or E; X15 is azaA, azaE, E, or A; X16 is azaK or K; X17 is azaE, azaA, A or E; X18 is azaF or F; X19 is azaI, azaV, V, or I; X20 is azaA, azaQ, Q, or A; X21 is AzaW or W; X22 is azaL, azaV, L or V; X23is azaV, azaI, I or V; X24is azaK, azaE, azaR, K, E or R; X25is azaG or G; X26is azaG, azaR, G or R (SEQ ID NO: 6); wherein at least one of X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, X21, X22, X23, X24, X25, and X26 is an aza- amino acid; or a pharmaceutically acceptable salt thereof.

[0025] In an embodiment, the present invention provides a compound of Formula: X1X2X3GX4X5TSX6VSSX7LEGX8AAX9EX10IAX11X12VX13GX14G, wherein X1 is azaH or H; X2 is azaA, Aib, or A; X3 is azaE or E; X4 is T or S; X5 is azaF or F; X6 is azaD or D; X7is azaY, K, or Y; X8is Q or E; X9is azaK or K; X10is azaF or F; X11is azaW, W or K; X12 is L or V; X13 is K, R or E; and X14 is R or G (SEQ ID NO: 7); wherein at least one of X1, X2, X3, X5, X6, X7, X9, X10, and X11 is an aza-amino acid; or a pharmaceutically acceptable salt thereof.

[0026] In an embodiment, the present invention provides a compound of Formula: HXEGTFTSDVSSYLEGQAAKEFIAWLVRGRG, wherein X is Aib (SEQ ID NO: 8); or a pharmaceutically acceptable salt thereof.

[0027] In an embodiment, the present invention provides a compound of Formula: HX1X2GTFTSDVSSYLEGQAAKEFIAWLVRGRG, wherein X1 is Aib and X2 is azaE (SEQ ID NO: 9); or a pharmaceutically acceptable salt thereof.

[0028] In an embodiment, the present invention provides a compound of Formula: HXEGTFTSDVSSYLEGQAAKEFIAWLVKGRG,wherein X is azaA (SEQ ID NO: 10); or a pharmaceutically acceptable salt thereof.

[0018] In an embodiment, the present invention provides a compound of Formula: HXEGTFTSDVSSYLEGQAAKEFIAWLVKGRG, wherein X is azaA (SEQ ID NO: 10); or a pharmaceutically acceptable salt thereof, wherein the compound is conjugated to a lipid at position 26.

[0019] In an embodiment, the present invention provides a compound of Formula: HXEGTFTSDVSSYLEGQAAKEFIAWLVKGRG, wherein X is azaA (SEQ ID NO: 11); or a pharmaceutically acceptable salt thereof.

[0020] In an embodiment, the present invention provides a compound of Formula: HAXGTFTSDVSSYLEGQAAKEFIAWLVKGRG, wherein X is azaE (SEQ ID NO: 12); or a pharmaceutically acceptable salt thereof.

[0021] In an embodiment, the present invention provides a compound of Formula: HAXGTFTSDVSSYLEGQAAKEFIAWLVRGRG, wherein X is azaE (SEQ ID NO: 13); or a pharmaceutically acceptable salt thereof.

[0022] In an embodiment, the present invention provides a compound of Formula: X1AEGTFTSDVSSYLEGQAAKEFIAWLVKGRG, wherein X1is azaH (SEQ ID NO: 14); or a pharmaceutically acceptable salt thereof.

[0023] In an embodiment, the present invention provides a compound of Formula: X1X2X3GTFTSDVSSYLEGQAAKEFIAWLVRGRG, wherein X1is azaH; X2is Aib, and X3is azaE (SEQ ID NO: 15); or a pharmaceutically acceptable salt thereof.

[0024] In an embodiment, the present invention provides a compound of Formula: X1X2EGTFTSDVSSYLEGQAAKEFIAWLVRGRG, wherein X1is azaH, and X2is Aib (SEQ ID NO: 16); or a pharmaceutically acceptable salt thereof.

[0025] In an embodiment, the present invention provides a compound of Formula: X1X2EGTFTSDVSSYLEGQAAKEFIAWLVRGRG,wherein X1is azaH, and X2is azaA (SEQ ID NO: 17); or a pharmaceutically acceptable salt thereof.

[0026] In an embodiment, the present invention provides a compound of Formula: XAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG, wherein X is azaH (SEQ ID NO: 18); or a pharmaceutically acceptable salt thereof.

[0027] In an embodiment, the present invention provides a compound of Formula: X1AX2GTFTSDVSSYLEGQAAKEFIAWLVRGRG, wherein X1is azaH, and X2is AzaE (SEQ ID NO: 19); or a pharmaceutically acceptable salt thereof.

[0028] In an embodiment, the present invention provides a compound of Formula: HX1EGTFTSX2VSSX3LEGQAAKEX4IAX5LVKGRG, wherein X1 is AzaA; X2 is AzaD; X3 is azaY; X4 is azaF, and X5 is azaW (SEQ ID NO: 20); or a pharmaceutically acceptable salt thereof.

[0029] In an embodiment, the present invention provides a compound of Formula: HX1X2GTFTSDVSSYLEGQAAKEFIAWLVRGRG, wherein X1 is Aib; and X2 is azaE (SEQ ID NO: 21); or a pharmaceutically acceptable salt thereof.

[0030] In an embodiment, the present invention provides a compound of Formula: X1X2X3GX4X5TSX6VSSX7LEGX8AAX9EX10IAX11X12VX13GX14, wherein X1 is azaH or H; X2 is azaA, Aib, or A; X3 is azaE or E; X4 is T or S; X5 is azaF or F; X6 is azaD or D; X7is azaY, K, or Y; X8is Q or E; X9is azaK or K; X10is azaF or F; X11is azaW, W or K; X12 is L or V; X13 is K, R or E; and X14 is R or G (SEQ ID NO: 22); wherein at least one of X1, X2, X3, X5, X6, X7, X9, X10, and X11, is an aza-amino acid; or a pharmaceutically acceptable salt thereof.

[0031] In an embodiment, the present invention provides a compound of Formula: HX1EGSX2TSDVSSKLEGEAAX3EX4IAKVVEGG, wherein X1 is azaA; X2 is azaF; X3 is azaK; and X4 is azaF (SEQ ID NO: 23); or a pharmaceutically acceptable salt thereof.

[0032] In an embodiment, the present invention provides a compound of Formula: X1X2X3GX4X5TSX6VSIX7LDKX8AQX9AX10IEX11X12LX13GX14PSSGAPPPS, wherein X1 is azaH, H, Y, or azaY; X2 is azaA, Aib, or A; X3 is azaE, E, Q, or azaQ; X4 is T or S; X5is azaF or F; X6is azaD or D; X7is azaY, K, L, azaL, azaK, azaA, or Y; X8is Q, E or K; X9 is azaK, K or Aib; X10 is azaF or F; X11 is azaW, W, Y or K; X12 is L or V; X13 is K, R or E; and X14 is R or G; wherein S at position 39 is optionally amidated as a C-terminal primary amide; ); wherein at least one of X1, X2, X3, X5, X6, X7, X9, X10, and X11, is an aza-amino acid (SEQ ID NO: 24); or a pharmaceutically acceptable salt thereof.

[0033] In an embodiment, the present invention provides a compound of Formula: X1X2X3GX4X5TSX6VSIX7LDKX8AQX9AFIEYLLEGFPSSGAPPPS, wherein X1is Y or azaY; X2is Aib; X3is Q or azaQ; X4is T; X5is azaF or F; X6is azaD or D; X7 is L or azaL; X8 is K or azaK; X9 is Aib; or a pharmaceutically acceptable salt thereof.

[0034] Compounds of SEQ ID Nos. 4 to 25 may also optionally be conjugated to a lipid (e.g., a fatty acid) with an albumin binding motif, directly or through a linker. In one embodiment, compounds of SEQ ID NOs. 4 to 25 are conjugated to a lipid (e.g., a fatty acid) with an albumin binding motif, directly or through a linker. For example, in one embodiment, a compound of SEQ ID NO. 10 is conjugated to a lipid (e.g., a fatty acid) with an albumin binding motif, directly or through a linker.

[0035] In one embodiment, compounds of SEQ ID Nos. 4 to 25 are conjugated to a lipid at position 26.

[0036] In one embodiment, compounds of SEQ ID NOs. 4 to 25 are conjugated to a lipid (e.g., a fatty acid) with an albumin binding motif, directly or through a linker.

[0037] Generally, compounds of SEQ ID Nos. 4 to 25 are at least three (3) times more resistant to in-vivo degradation by DPP-IV than GLP-1, based on comparison of in-vivo plasma half- lives. In certain embodiments, the compounds of SEQ ID Nos. 4 to 25 are at least 10, 20 or 30 times more resistant to degradation by DPP-IV than GLP-1.

[0038] The compounds of SEQ ID Nos. 4 to 25 have biological half-lives greater than about 24 hours. For example, a biological half-live of a compound selected from a group consisting of SEQ ID NOs: 4 to 25, or a pharmaceutically acceptable salt thereof, may be about 48 hours,about 72 hours, about 96 hours, about 120 hours, about 144 hours, about 168 hours, about 192 hours, about 216 hours, or about 240 hours.

[0039] To further increase resistance of degradation of compounds of SEQ ID Nos. 4 to 25 by degradation by DPP-IV and provide a longer biological half-lives, compounds of SEQ ID Nos. 4 to 25 may be chemically modified to conjugate (i) a lipid (e.g., a fatty acid) with an albumin binding motif, directly or through a linker, or (ii) a different compound, at one or more positions of the compounds of SEQ ID Nos. 4 to 25. For example, compounds of SEQ ID Nos. 4 to 25 may be chemically modified at position 17 though conjugation to (AEEA)-gamma-Glu-C20 diacid and / or at position 20 by conjugation to (N-ε-(γ-Glu(N-α-hexadecanoyl) and / or at position 26 through conjugation to ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)1-CO-(CH2)18-CO2H and / or conjugation to palmitic acid with a glutamic acid spacer.

[0040] In one embodiment, compounds of SEQ ID Nos. 4 to 25 are chemically modified at position 17 though conjugation to (AEEA)-gamma-Glu-C20 diacid and / or at position 20 by conjugation to (N-ε-(γ-Glu(N-α-hexadecanoyl).

[0041] In one embodiment, compounds of SEQ ID Nos. 4 to 25 are chemically modified at position 26 through conjugation to ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)1-CO-(CH2)18- CO2H.

[0042] In one embodiment, compounds of SEQ ID Nos. 4 to 25 are chemically modified at position 26 through conjugation to palmitic acid with a glutamic acid spacer.

[0043] The chemical modification may increase binding of compounds of SEQ ID Nos. 4 to 25 to a plasma protein (e.g., albumin) and may, therefore, provide for a longer biological half-life and allow for a less frequent administration of the compounds of SEQ ID Nos. 4 to 25.

[0044] Generally, compounds of SEQ ID Nos. 4 to 25 are also more resistant to degradation by gastric peptidases than GLP-1, which may, e.g., allow for oral administration of the compounds of SEQ ID Nos. 4 to 25.

[0045] Further, in certain embodiments, the compounds of SEQ ID Nos. 4 to 25 are formulated with one or more pharmaceutical excipients into a formulation that is resistant to degradation by gastric peptidases. The resistance to degradation by gastric peptidases may, e.g., be provided by(i) an enteric coating coated over the solid dosage form comprising a compound according to any one of SEQ ID Nos. 4 to 25 and / or (ii) use of a pH-dependent polymer in a formulation comprising a compound according to any one of SEQ ID Nos. 4 to 25 and / or (iii) microencapsulation of a compound according to any one of SEQ ID Nos. 4 to 25 in a protective shell and / or (iv) using a lipid-based carrier (e.g., a liposome).

[0046] Compounds of SEQ ID Nos. 4 to 25 are capable of modulating a GLP-1 receptor and / or a GIP receptor and / or a glucagon receptor. In certain embodiments, compounds of SEQ ID Nos. 4 to 25 modulate the GLP-1 receptor. In certain embodiments, compounds of SEQ ID Nos. 4 to 25 modulate the GLP-1 receptor and the GIP receptor. In certain embodiments, compounds of SEQ ID Nos. 4 to 25 modulate the GLP-1 receptor, the GIP receptor and the glucagon receptor.

[0047] Compounds of SEQ ID Nos. 4 to 25 may therefore be used to modulate effect(s) of GLP-1 and / or GIP and / or Glucagon in humans and non-human subjects.

[0048] In an embodiment, compounds of SEQ ID Nos. 4 to 25 act as agonists on human GLP- 1 receptor (hGLP-1R) and have an EC50to hGLP-1R in a nanomolar or, preferably, in a picomolor range. For example, in certain embodiments, compounds of SEQ ID Nos. 4 to 25 have an EC50 to human GLP-1 receptor (hGLP-1R) of from about 5 pM to about 990 nM, from about 10 pM to about 800 pM or from about 20 pM to about 800 pM.

[0049] In another embodiment, this invention provides for a pharmaceutical composition comprising (i) a therapeutically effective amount of an aza GLP-1-based therapeutic analogue wherein at least one peptide bond of a GLP-1 based therapeutic peptide has been replaced with an azapeptide bond, and (ii) at least one pharmaceutically acceptable excipient (e.g., a pharmaceutically acceptable carrier). Starting from the N-terminal end of the GLP-1 based therapeutic peptide, the at least one peptide bond that is replaced may, e.g., be the 1st, 2nd, 6th, 7th, 9th, 10th, 11th, 12, 13th, 17th, 20th, 22nd, 23rd, 25th, 26th, 28, and / or 30thpeptide bond of the GLP-1 based therapeutic peptide. The replacement of the 1stand / or 2ndpeptide bond may, e.g., make the aza GLP-1-based therapeutic analogue more resistant to degradation by DPP-IV. The replacement of the 9th, 12th, 13th, 22nd, 23rd, and 25thpeptide bond may make the aza GLP-1- based therapeutic analogue more resistant to degradation by neprilysin. The replacement of the 6th, 7th, 9th, 10th, 11th, 12th, 13th, 17th, 20th, 22nd, 23rd, 25th, 26th, 28th, and 30thpeptide bond maymake the aza GLP-1-based therapeutic analogue more resistant to degradation by digestive proteases including, e.g., chymotrypsin, pepsin, elastase, and trypsin.

[0050] In one embodiment, the invention provides for a pharmaceutical composition comprising (i) a therapeutically effective amount of an analogue of an incretin peptide or a pharmaceutically acceptable salt thereof, the analogue of an incretin peptide comprising an azapeptide bond, which the incretin peptide does not have and (ii) at least one pharmaceutically excipient (e.g., a pharmaceutically acceptable carrier).

[0051] In an additional embodiment, the invention provides for a pharmaceutical composition comprising (i) a therapeutically effective amount of an analogue of an incretin peptide or a pharmaceutically acceptable salt thereof, the analogue of an incretin peptide comprising (a) an azapeptide bond, which the incretin peptide does not have and (b) an unnatural amino acid, which the incretin peptide does not have and / or (c) an amino acid that is different from an amino acid at a corresponding position of the incretin peptide, and (ii) at least one pharmaceutically excipient (e.g., a pharmaceutically acceptable carrier).

[0052] In an additional embodiment, the invention provides for a pharmaceutical composition comprising (i) a therapeutically effective amount of an aza GLP-1-based therapeutic peptide analogue of a GLP-1-based therapeutic peptide or a pharmaceutically acceptable salt thereof, the aza GLP-1-based therapeutic peptide analogue comprising an azapeptide bond, which the GLP- 1-based therapeutic peptide does not have, and (ii) at least one pharmaceutically excipient (e.g., a pharmaceutically acceptable carrier).

[0053] In an additional embodiment, the invention provides for a pharmaceutical composition comprising (i) a therapeutically effective amount of an aza GLP-1-based therapeutic peptide analogue of a GLP-1-based therapeutic peptide or a pharmaceutically acceptable salt thereof, the aza GLP-1-based therapeutic peptide analogue comprising (a) an azapeptide bond, which the GLP-1-based therapeutic peptide does not have and (b) an unnatural amino acid, which the GLP- 1-based therapeutic peptide does not have, and / or (c) an amino acid that is different from an amino acid at a corresponding position of the GLP-1-based therapeutic peptide, and (ii) at least one pharmaceutically excipient (e.g., a pharmaceutically acceptable carrier).

[0054] In an additional embodiment, the invention provides for a pharmaceutical composition comprising: (i) a therapeutically effective amount of a compound of SEQ ID NO: 4, and wherein the compound is optionally conjugated to (i) a lipid (e.g., a fatty acid) with an albumin binding motif, directly or through a linker, and (ii) a permeation enhancer. The permeation enhancer may, e.g., render the composition suitable for oral administration.

[0055] Another embodiment provides for a method of treating a disease state for which GLP- 1-based therapeutic peptide is indicated by administering of an effective amount of an incretin peptide analogue in which at least one peptide bond of the incretin peptide has been replaced with an azapeptide bond to a human or non-human patient in need thereof.

[0056] In yet another embodiment, this invention provides for method for treating a disease state, for which a GLP-1-based therapeutic peptide is indicated for a human or non-human patient, which comprises administering a therapeutically effective amount of an aza GLP-1- based therapeutic analogue as provided for in the embodiments above to a human or non-human patient.

[0057] Non-limiting examples of specific disease state that can be treated by the methods of the invention include hyperglycemia, impaired glucose tolerance, pre-diabetes, non-insulin dependent diabetes (e.g., type-2 diabetes), obesity, cardiovascular diseases (e.g., hypertension), dyslipidemias, neurodegenerative diseases, non-alcoholic fatty liver disease, metabolic syndrome, oxidative stress, acute inflammation, and chronic inflammation. The aza GLP-1-based therapeutic analogue may also be used for chronic weight management, reduction of hunger and inducing satiety.

[0058] In an embodiment, the invention is directed in part to a method of treatment of pre- diabetes comprising administering to a human in need thereof a therapeutically effective amount of a compound selected from a group consisting of compounds of SEQ ID Nos. 4 to 25 and pharmaceutically acceptable salts thereof. The method of treatment of pre-diabetes includes administration of the compound selected from a group consisting of compounds of SEQ ID Nos. 4 to 25 alone or in a combination with a therapeutically effective amount of a drug used in the treatment or prevention of diabetes. The drug used in the treatment or prevention of diabetes may, e.g., be selected from a group consisting of metformin, thiazolidinediones, sulfonylureas,dipeptidyl peptidase 4 inhibitors, and sodium glucose co-transporters, and may be administered simultaneously, separately, or sequentially with the compound selected from a group consisting of compounds of SEQ ID Nos. 4 to 25, and via the same or a different route of administration.

[0059] The invention is also directed in part to a method of treatment of impaired glucose tolerance comprising administering to a human in need thereof a therapeutically effective amount of a compound selected from a group consisting of compounds of SEQ ID Nos. 4 to 25 and pharmaceutically acceptable salts thereof. The method of treatment of impaired glucose tolerance includes administration of the compound selected from a group consisting of compounds of SEQ ID Nos. 4 to 25 alone or in a combination with a therapeutically effective amount of a drug used in the treatment or prevention of diabetes. The drug used in the treatment or prevention of diabetes may, e.g., be selected from a group consisting of metformin, thiazolidinediones, sulfonylureas, dipeptidyl peptidase 4 inhibitors, and sodium glucose co-transporters, and may be administered simultaneously, separately, or sequentially with the compound selected from a group consisting of compounds of SEQ ID Nos. 4 to 25, and via the same or a different route of administration.

[0060] The invention is also directed in part to a method of treatment of hyperglycemia comprising administering to a human in need thereof a therapeutically effective amount of a compound selected from a group consisting of compounds of SEQ ID Nos. 4 to 25 and pharmaceutically acceptable salts thereof. The method of treatment of hyperglycemia includes administration of the compound selected from a group consisting of compounds of SEQ ID Nos. 4 to 25 alone or in a combination with a therapeutically effective amount of a drug used in the treatment or prevention of diabetes. The drug used in the treatment or prevention of diabetes may, e.g., be selected from a group consisting of metformin, thiazolidinediones, sulfonylureas, dipeptidyl peptidase 4 inhibitors, and sodium glucose co-transporters, and may be administered simultaneously, separately, or sequentially with the compound selected from a group consisting of compounds of SEQ ID Nos. 4 to, and via the same or a different route of administration.

[0061] The invention is also directed in part to a method of treatment of non-insulin dependent diabetes (e.g., type-2 diabetes) comprising administering to a human in need thereof a therapeutically effective amount of a compound selected from a group consisting of compounds of SEQ ID Nos. 4 to 25 and pharmaceutically acceptable salts thereof. The methods of treatmentof non-insulin dependent diabetes include administration of the compound selected from a group consisting of compounds of SEQ ID Nos. 4 to 25 alone or in a combination with a therapeutically effective amount of a drug used in the treatment or prevention of diabetes. The drug used in the treatment or prevention of diabetes may, e.g., be selected from a group consisting of metformin, thiazolidinediones, sulfonylureas, dipeptidyl peptidase 4 inhibitors, and sodium glucose co-transporters, and may be administered simultaneously, separately, or sequentially with the compound selected from a group consisting of compounds of SEQ ID Nos. 4 to 25 and pharmaceutically acceptable salts thereof, and via the same or a different route of administration.

[0062] The invention is also directed in part to a method of treatment of obesity comprising administering to a human in need thereof a therapeutically effective amount of a compound selected from a group consisting of compounds of SEQ ID Nos. 4 to 25 and pharmaceutically acceptable salts thereof. The method of treatment of obesity includes administration of the compound selected from a group consisting of compounds of SEQ ID Nos. 4 to 25 alone or in a combination with a therapeutically effective amount of an additional drug used in the treatment obesity, and via the same or a different route of administration.

[0063] The invention is also directed in part to a method of treatment of a cardiovascular disease comprising administering to a human in need thereof a therapeutically effective amount of a compound selected from a group consisting of compounds of SEQ ID Nos. 4 to 25 and pharmaceutically acceptable salts thereof. The cardiovascular diseases that could be treated with the methods of the present invention include, e.g., hypertension and congestive heart failure. The method of treatment of the cardiovascular disease includes administration of the compound selected from a group consisting of compounds of SEQ ID Nos. 4 to 25 alone or in combination with a therapeutically effective amount of an additional drug used in the treatment of cardiovascular disease, and via the same or a different route of administration.

[0064] The invention is also directed in part to a method of treatment of a dyslipidemia comprising administering to a human in need thereof a therapeutically effective amount of a compound selected from a group consisting of compounds of SEQ ID Nos. 4 to 25 and pharmaceutically acceptable salts thereof. The method of treatment of the dyslipidemia includes administration of the compound selected from a group consisting of compounds of SEQ ID Nos.4 to 25 alone or in combination with a therapeutically effective amount of an additional drug used in the treatment of dyslipidemia, and via the same or a different route of administration.

[0065] The invention is also directed in part to a method of treatment of non-alcoholic fatty liver disease comprising administering to a human in need thereof a therapeutically effective amount of a compound selected from a group consisting of compounds of SEQ ID Nos. 4 to 25 and pharmaceutically acceptable salts thereof. The method of treatment of non-alcoholic fatty liver disease includes administration of the compound selected from a group consisting of compounds of SEQ ID Nos. 4 to 25 alone or in a combination with a therapeutically effective amount of an additional drug used in the treatment of non-alcoholic fatty liver disease, and via the same or a different route of administration.

[0066] The invention is also directed in part to a method of treatment of a neurodegenerative disorder comprising administering to a human in need thereof a therapeutically effective amount of a compound selected from a group consisting of compounds of SEQ ID Nos. 4 to 25 and pharmaceutically acceptable salts thereof. The neurodegenerative disorders that could be treated by the methods of the present invention include, e.g., tauopathies, alpha-synucleopathies and dementias. For examples Alzheimer’s disease, Parkinson’s disease, dementia with Lewy bodies, frontotemporal dementia, chronic traumatic encephalopathy, Prion's disease, Down Syndrome, Huntington's disease, multiple sclerosis, and Amyloid Lateral Sclerosis may be treated by the methods of the present invention. The method of treatment of neurodegenerative disorder includes administration of the compound selected from a group consisting of compounds of SEQ ID Nos. 4 to 25 alone or in a combination with a therapeutically effective amount of an additional drug used in the treatment of neurodegenerative disorder, and via the same or a different route of administration.

[0067] The invention is also directed in part to a method of treatment of metabolic syndrome comprising administering to a human in need thereof a therapeutically effective amount of a compound selected from a group consisting of compounds of SEQ ID Nos. 4 to 25 and pharmaceutically acceptable salts thereof. The method of treatment of metabolic syndrome includes administration of the compound selected from a group consisting of compounds of SEQ ID Nos. 4 to 25 alone or in a combination with a therapeutically effective amount of anadditional drug used in the treatment of metabolic syndrome, and via the same or a different route of administration.

[0068] The invention is also directed in part to a method of treatment of chronic inflammation comprising administering to a human in need thereof a therapeutically effective amount of a compound selected from a group consisting of compounds of SEQ ID Nos. 4 to 25 and pharmaceutically acceptable salts thereof. The method of treatment of chronic inflammation includes administration of the compound selected from a group consisting of compounds of SEQ ID Nos. 4 to 25 alone or in a combination with a therapeutically effective amount of an additional drug used in the treatment of chronic inflammation, and via the same or a different route of administration.

[0069] The invention is also directed in part to a method of treatment of acute inflammation comprising administering to a human in need thereof a therapeutically effective amount of a compound selected from a group consisting of compounds of SEQ ID Nos. 4 to 25 and pharmaceutically acceptable salts thereof. The method of treatment of acute inflammation includes administration of the compound selected from a group consisting of compounds of SEQ ID Nos. 4 to 25 alone or in a combination with a therapeutically effective amount of an additional drug used in the treatment of acute inflammation, and via the same or a different route of administration.

[0070] In the methods of the invention, compounds of SEQ ID Nos. 4 to 25 may be administered orally or parenterally. Parenteral administration in accordance with the invention includes subcutaneous administration of compounds selected from the group consisting of compounds of SEQ ID Nos. 4 to 25 and their pharmaceutically acceptable salts thereof.

[0071] The subcutaneous administration of compounds of SEQ ID Nos. 4 to 25 may, e.g., be daily, weekly, monthly, bi-monthly, every three months, every six months, or once-a-year.

[0072] In an embodiment, the subcutaneous dose of the compound selected from a group consisting of compounds of SEQ ID Nos. 4 to 25 may, e.g., from about 0.1 mg to about 100 mg, from about 0.25 mg to about 50 mg, or from about 0.25 mg to about 15 mg.

[0073] The oral administration of the compounds of the invention compounds of SEQ ID Nos. 4 to 25 may, e.g., be one or more times a day.

[0074] Any one of the compounds of SEQ ID Nos. 4 to 25 and their salts may be administered in the methods of treatment of the invention. For example, compound of SEQ ID No. 10 or a salt thereof may be administered in any of the methods of treatment mentioned above.

[0075] In the methods of treatment of the invention, compounds of SEQ ID Nos. 4 to 25 may be administered alone or in a pharmaceutical composition comprising (i) a compound selected from a group consisting of compounds of SEQ ID Nos. 4 to 25 and pharmaceutically acceptable salt(s) and (ii) one or more pharmaceutically acceptable excipient(s).

[0076] It is noted that in this disclosure and particularly in the claims and / or paragraphs, terms such as “comprises”, “comprised”, “comprising”, and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of’ and “consists essentially of’ have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

[0077] As used above, and throughout the specification, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0078] The term “about” in the present specification means a value within 15% (±15%) of the value recited immediately after the term “about,” including the value equal to the upper limit (i.e., +15%) and the value equal to the lower limit (i.e., −15%) of this range. For example, the phrase “about 100” encompasses any numeric value that is between 85 and 115, including 85 and 115.

[0079] An “azapeptide” means a peptide in which all α-carbons are replaced by nitrogen trivalent atoms.

[0080] An “α-nitrogen” means a nitrogen atom bonded to a carbonyl group in an azapeptide or an azatide (“azatide” means two consequent aza-amino acids). The carbon atom next to the α- nitrogen is called the β-carbon.

[0081] An “aza-amino acid” is defined as an amino acid where the chiral α-carbon atom is replaced by a nitrogen atom.

[0082] The following table shows the definitions of the abbreviations of natural, unnatural and aza-amino acids: Abbreviation Definition G Glycine A Alanine I Isoleucine L Leucine V Valine C Cysteine M Methionine S Serine T Threonine N Asparagine Q Glutamine F Phenylalanine W Tryptophan P Proline D Aspartic acid E Glutamic acid Y Tyrosine R ArginineK Lysine H Histidine Aib 2-aminoisobutyric acid aG Aza Glycine aA Aza Alanine aI Aza Isoleucine aL Aza Leucine aV Aza Valine aC Aza Cysteine aM Aza Methionine aS Aza Serine aT Aza Threonine aN Aza Asparagine aQ Aza Glutamine aF Aza Phenylalanine aW Aza Tryptophan aP Aza Proline aD Aza Aspartic acid aE Aza Glutamic acid aY Aza Tyrosine aR Aza ArginineaK Aza Lysine aH Aza Histidine

[0083] An “aza analogue” or an “azapeptide” means a compound which differs from a peptide that it is an analogue of in that one or more α-carbon atoms of the peptide have been replaced by a nitrogen atom with or without additional structural modification(s) to the side chain(s) of the amino acid residues of the peptide. The one or more α-carbon atoms of the peptide may, e.g., be at the N-termini of the peptide (i.e., the first residue of the peptide), at the second residue of the peptide, the C-termini of the peptide (i.e., the last residue of the peptide), the residue covalently bound to the C-termini of the peptide, and / or at another residue of the peptide (e.g., at the site of hydrolysis of the peptide). Despite having a backbone different from the peptide, the aza analogue preserves, extends and / or improves functional activity of the peptide. The aza analogue is often more resistant to degradation than the peptide and / or has an improved therapeutic activity than the peptide and / or has an improved selectivity for a biological receptor than the peptide and / or improved affinity to a biological receptor and / or reversed activity at a biological receptor (agonistic activity instead of antagonist activity or antagonistic activity instead of agonistic activity).

[0084] An “amine” in the process of the invention may, e.g., be an amino ester, an ester of an amino acid, an amino ester of an aza-amino acid, a peptide, or an aza-peptide, an amino acid, an aza-amino acid, provided that, if the amino ester, the ester of an amino acid, the amino ester of the aza-amino acid, the peptide, the aza-peptide, the amino acid, or the aza-amino acid contains a group selected from amino, amide, guanidino N, carboxyl, sulfhydryl, carboxyl, hydroxyl, indole, imidazole phenol, the group is protected with a protecting group selected from tert- butoxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), or 2-(3,5-dimethoxyphenyl)propan- 2-yloxycarbonyl (Ddz), phthalimide (Phth), carboxybenzyl (Cbz), 2,2,4,6,7-pentamethyl- dihydrobenzofuran-5-sulfonyl (Pbf), trityl or triphenylmethyl (Trt), t-butyl ester (OtBu), t-butyl ether (tBu), allyloxycarbonyl (Aloc), methoxytrimethylbenzene sulfonyl (Mtr), 4,4- dimethyloxybenzhydryl (Mbh), 2,2,5,7,8-pentamethyl-chroman-6-sulfonyl chloride (Pmc), 2,4,6-trimethoxybenzyl (Tmob), allyl ester (OAT), acetamidomethyl (Acm), and the like. The amino ester may, e.g., be t-butyl, p-methoxy benzyl ester, glycine ethyl ester, etc.

[0085] The term “protected” as it is used herein means that one or more group(s) (e.g., -OH) in an amino acid, an aza-amino acid, a peptide, an azapeptide, or a compound is protected with a protecting group (e.g., Phth, Boc, Ddz, etc.). Unless otherwise indicated, the term “protecting group” or “protective group,” when used to refer to part of a molecule subjected to a chemical reaction, means a chemical moiety that is not reactive under the conditions of that chemical reaction, and which may be removed to provide a moiety that is reactive under those conditions. Protecting groups include, for example, nitrogen protecting groups and hydroxy-protecting groups. Examples of protective groups include, e.g., benzyl, diphenylmethyl, trityl, Cbz, Boc, Fmoc, methoxycarbonyl, ethoxycarbonyl, Phth, Ddz, as well as other protective groups known to those skilled in the art.

[0086] “Effective amount” or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result. Such results may include, but are not limited to, the treatment of a disease or condition as determined by any means suitable in the art.

[0087] The abbreviation “N-Phth” means “N-phthalimidyl.”

[0088] The abbreviation “Boc” means “tert-butoxycarbonyl.

[0089] The abbreviation “Fmoc” means “9-fluorenylmethoxycarbonyl.”

[0090] The abbreviation “Ddz” means “2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl.”

[0091] The abbreviation “HOBt” means “1-OH-Benzotriazole.”

[0092] The abbreviation “Cbz” means “carboxybenzyl.”

[0093] The abbreviation “Pbf” means “2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl.”

[0094] In peptide chemistry, “deprotection” refers to a process of removing the protecting groups (e.g., phthaloyl, Boc, Cbz, Fmoc, etc.) by a chemical agent. For example, Boc protecting group could be removed under acidic conditions (e.g., 4 M HCl, or neat trifluoroacetic acidTFA); Fmoc protecting group could be removed under basic conditions when pH is higher than 12 (20% piperidine / DMF or DCM); and Phthaloyl group can be cleaved, e.g., under basic conditions or by the use of hydrazine.

[0095] These and embodiments are disclosed or are obvious from and encompassed by the preceding Summary of the Invention and the following Detailed Description. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings.

[0097] Fig. 1 summarizes the secretion and metabolism of GIP, GLP-1, and glucagon.

[0098] Fig. 2A summarizes the role of GLP-1 and GIP in glucose homeostasis.

[0099] Fig. 2B depicts the amino acid sequences of GLP-1 (SEQ ID NO: 1), GIP (SEQ ID NO. 2) and glucagon (SEQ ID NO. 3).

[0100] Fig. 2C depicts the structure of semaglutide.

[0101] Fig. 2D depicts traditional azapeptide synthetic strategies-activation of N'-alkyl-N- protected-hydrazines.

[0102] Fig. 3A and 3B describe the synthesis of GLP-1 (7-37) (SEQ ID NO: 5) (1) and the synthesis of azaA8GLP-1(7-37) (SEQ ID NO: 10) (2).

[0103] Fig. 4 describes the synthesis of azaE9GLP-1(7-37) (SEQ ID NO: 12) (3).

[0104] Fig. 5 describes the synthesis of azaH-Aib dipeptide (31).

[0105] Fig. 6 describes the synthesis of azaH-azaA diazatide (41).

[0106] Fig. 7 describes the synthesis of [R34]-GLP-1(7-37) (6).

[0107] Fig. 8 describes the synthesis of [R34, Aib8]-GLP-1(7-37) (7).

[0108] Fig. 9 describes the synthesis of [R34, azaHis7]-GLP-1(7-37) (8).

[0109] Fig. 10 describes the synthesis of [R34, azaAla8]-GLP-1(7-37) (9).

[0110] Fig. 11 describes the synthesis of [R34, azaGlu9, Aib8]-GLP-1(7-37) (10).

[0111] Fig. 12 describes the synthesis of [R34, azaHis7, Aib8]-GLP-1(7-37) (11).

[0112] Fig. 13 describes the synthesis of [R34, azaAla8, azaHis7]-GLP-1(7-37) (12).

[0113] Fig. 14 describes the synthesis of [R34, azaGlu9, azaHis7]-GLP-1(7-37) (13).

[0114] Fig. 15 depicts the results of Example 13.

[0115] Fig. 16A depicts mean plasma concentrations of GLP-1 obtained in Example 14.

[0116] Fig. 16B depicts mean plasma concentration of ([R34, Aib8]-GLP-1(7-37)) (7) obtained in Example 14.

[0117] Fig. 16C depicts mean plasma concentration of [R34, azaHis7]-GLP-1(7-37)) (8) obtained in Example 14.

[0118] Fig. 16D depicts mean plasma concentration of [R34, azaAla8]-GLP-1(7-37) (9) obtained in Example 14.

[0119] Fig. 17 depicts strategies for synthesizing aza-GLP-1 analogues.

[0120] Fig. 18 summarizes thiocarbazate-based azapeptide synthetic platform.

[0121] Fig. 19 depicts serum and digestive protease cleavages cites of GLP-1.

[0122] Fig. 20 depicts the results of Example 15. DETAILED DESCRIPTION OF THE INVENTION

[0123] Azapeptides are a class of peptidomimetics, where one or more α-carbons in the peptide backbone are replaced by a trivalent nitrogen atom. This relatively minor modification not only results in a more rigid trigonal or achiral geometry, but also increases the resistance of the aza- peptide bond (-NH-NR-C(O)-) to enzymatic hydrolysis when compared to the common peptidebond (-NH-CHR-C(O)-). This replacement eliminates chirality at the α-position, decreases the electrophilicity of the carbonyl group, and provides a semicarbazide with two adjacent nitrogen atoms. Further, incorporating aza-amino acids into a peptide sequence increases the number of intramolecular hydrogen bonds and stabilizes β-turns and polyproline-type II helices.

[0124] Incretin peptides, including GLP-1, are degraded by serum and digestive proteases. Figure 19 depicts serum and digestive protease cleavages cites of GLP-1.

[0125] In one embodiment, this invention provides for GLP-1-based therapeutic peptide analogues and their salts in which at least one peptide bond of a GLP-1-based therapeutic peptide (e.g., at a protease cleavage site) has been replaced with an azapeptide bond.

[0126] In one embodiment of the aza GLP-1-based therapeutic analogue, pharmaceutical compositions, and methods provided for in the embodiments above, the aza GLP-1-based therapeutic peptide analogue is a GLP-1-based therapeutic peptide that has two peptide bonds which have been replaced with azapeptide bonds. In another embodiment, the two peptide bonds are an N-terminal His-Ala or an N-terminal His-Gly.

[0127] In alternative embodiments, the GLP-1-based therapeutic analogue is a GLP-1-based therapeutic peptide that has three peptide bonds which have been replaced with azapeptide bonds.

[0128] In alternative embodiments, the GLP-1-based therapeutic analogue is a GLP-1-based therapeutic peptide that has four or 5 peptide bonds which have been replaced with azapeptide bonds.

[0129] In one embodiment in the analogues, pharmaceutical compositions, and methods provided for in the embodiments above, GLP-1 based therapeutic peptide is a GLP-1 receptor agonist peptide. In alternative embodiment, the GLP-1 based therapeutic peptide is a dual-acting GLP-1 and GIP receptor agonists peptide. In yet another embodiment, the GLP-1 based therapeutic peptide is a triple acting GLP-1, GIP, and glucagon receptor agonist peptide.

[0130] GLP-1 receptor agonist peptides, dual-acting GLP-1 and GIP receptor agonist peptides, and triple acting GLP-1, GIP, and glucagon receptor agonist peptides that may serve as target peptides are known in art (see, e.g., A. M. Jastreboff et al., Annu. Rev. Med., “New Frontiers inObesity Treatment: GLP-1 and Nascent Nutrient-Stimulated Hormone-Based Therapeutics”, 74, 125-139 (2023), W. Peng, et al., Aging and Disease, “Novel Insights into the Roles and Mechanisms of GLP-1 Receptor Agonists against Aging-Related Diseases”, 13(2), 468-490 (2022); T.D. Miller, et al., Molecular Metabolism, 30, 72-130 (2019), B.A. Smith, et al., Ther. Adv. Endocrinal. Metab., 12, 1-15 (2021).

[0131] Non-limiting examples of GLP-1 receptor agonist peptides include liraglutide, danuglipron, LY3502970 (Lilly), lixisenatide, dulaglutide, and exenatide. Other examples include injectable or oral semaglutide.

[0132] Non-limiting examples of dual-acting GLP-1 and GIP receptor agonist peptides include tirzepatide.

[0133] Non-limiting examples of a triple acting GLP-1, GIP, and glucagon receptor agonist peptide is retatrutide.

[0134] In some embodiments, the aza GLP-1-based therapeutic analogue is azaA8GLP-1(7- 37), azaE9GLP-1(7-37), azaHis7, Abi8, Arg34GLP-1(7-37), or azaHis7, azaAla8, Arg34GLP-1 (7- 37).

[0135] In other embodiments, the aza GLP-1-based therapeutic analogue is azaA8,R34GLP- 1(7-37) or azaH7,azaE9,R34GLP-1(7-37).

[0136] In other embodiments, the aza GLP-1-based therapeutic analogue is azaA8GLP-1(7- 37).

[0137] In some embodiments, the pharmaceutical composition is an oral dosage form.

[0138] In yet another embodiment, the pharmaceutical composition is an oral liquid dosage form.

[0139] In yet other embodiments, the pharmaceutical composition is a buccal dosage form, a sublingual dosage form, a nasal dosage form, an inhaler, a nebulizer, a topical dosage form, a transdermal dosage form, or a suppository.

[0140] In another embodiment, the pharmaceutical composition is a parenteral dosage form.

[0141] In yet another embodiment, the pharmaceutical composition is a subcutaneous dosage form.

[0142] In some embodiments, the pharmaceutical composition is such that the aqueous solubility of the aza GLP-1-therapeutic peptide analogue is greater than about 1 mg / ml. In other embodiments, the aqueous solubility of the aza GLP-1-therapeutic peptide analogue is greater than about 5 mg / ml.

[0143] In other embodiments, the pharmaceutical composition is such that the aza GLP-1- therapeutic peptide analogue is stable for greater than 60 minutes in plasma and / or simulated stomach acid. In some embodiments, the pharmaceutical composition is such that the aza GLP-1- therapeutic peptide analogue is stable for greater than 24 hours in plasma and / or simulated stomach acid.

[0144] The pharmaceutical compositions provided for herein may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[0145] The compositions of the invention may consist of the active ingredient alone, in a form suitable for administration to a (human) subject or patient, or the composition may comprise at least one active ingredient and one or more pharmaceutically acceptable excipients.

[0146] In one embodiment, the compositions of the invention are formulated using one or more pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers that are useful, include, but are not limited to, glycerol, water, saline, ethanol, and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey). The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.

[0147] Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, vaginal, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and / or aromatic substances and the like. They may also be combined were desired with other active agents.

[0148] As used herein, “additional ingredients” include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional ingredients” that may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Genaro, ed. (1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.), which is incorporated herein by reference.

[0149] The composition of the invention may comprise a preservative from about 0.005% to 2.0% by total weight of the composition. The preservative is used to prevent spoilage in the case of exposure to contaminants in the environment. Examples of preservatives useful in accordance with the invention included but are not limited to those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea and combinations thereof.

[0150] The composition may include an antioxidant and a chelating agent that inhibits the degradation of the compound. Examples of antioxidants for some compounds are BHT, BHA, alpha-tocopherol and ascorbic acid in the preferred range of about 0.01% to 0.3% and more preferably BHT in the range of 0.03% to 0.1% by weight by total weight of the composition. Preferably, the chelating agent is present in an amount of from 0.01% to 0.5% by weight by total weight of the composition. Chelating agents include edetate salts (e.g., disodium edetate) and citric acid in the weight range of about 0.01% to 0.20% and more preferably in the range of0.02% to 0.10% by weight by total weight of the composition. The chelating agent is useful for chelating metal ions in the composition that may be detrimental to the shelf life of the formulation. While BHT and disodium edetate are the particularly preferred antioxidant and chelating agent respectively for some compounds, other suitable and equivalent antioxidants and chelating agents may be substituted therefore as would be known to those skilled in the art.

[0151] Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water, and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents. Oily suspensions may further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose. Known dispersing or wetting agents include, but are not limited to, naturally occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin, and acacia. Known preservatives include, but are not limited to, methyl, ethyl, or n- propyl para hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol.

[0152] Liquid solutions of the active ingredient in aqueous or oily solvents may be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent. As used herein, an “oily” liquid is one which comprises a carbon-containing liquid molecule and which exhibits a less polarcharacter than water. Liquid solutions of the pharmaceutical composition of the invention may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water, and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.

[0153] Powdered and granular formulations of a pharmaceutical preparation of the invention may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations.

[0154] Controlled- or sustained-release formulations of a composition of the invention may be made using conventional technology, in addition to the disclosure set forth elsewhere herein. In some cases, the dosage forms to be used can be provided as slow or controlled-release of one or more active ingredients therein using, for example, hydropropylmethylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, or microspheres or a combination thereof to provide the desired release profile in varying proportions. Suitable controlled-release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the compositions of the invention.

[0155] For oral administration, particularly suitable are tablets, dragees, liquids, drops, capsules, caplets and gelcaps. Other formulations suitable for oral administration include, but are not limited to, a powdered or granular formulation, an aqueous or oily suspension, an aqueous or oily solution, a paste, a gel, toothpaste, a mouthwash, a coating, an oral rinse, or an emulsion. The compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more inert, non-toxic pharmaceutically excipients. Such excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agentssuch as magnesium stearate. The oral compositions of the invention in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents; fillers; lubricants; disintegrates; or wetting agents.

[0156] Tablets may be non-coated, or they may be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the active ingredient. By way of example, a material such as glyceryl monostearate or glyceryl distearate may be used to coat tablets. Further by way of example, tablets may be coated using methods described in U.S. Pat. Nos. 4,256,108; 4,160,452; and U.S. Pat. No. 4,265,874 to form osmotically controlled release tablets. Tablets may further comprise a sweetening agent, a flavoring agent, a coloring agent, a preservative, or some combination of these in order to provide for pharmaceutically elegant and palatable preparation. For oral administration, if desired, the tablets may be coated using suitable methods and coating materials such as OPADRY® film coating systems available from Colorcon, West Point, Pa. (e.g., OPADRY® OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type and OPADRY® White, 32K18400).

[0157] Hard capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such hard capsules comprise the active ingredient, and may further comprise additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin. Soft gelatin capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such soft capsules comprise the active ingredient, which may be mixed with water or an oil medium such as peanut oil, liquid paraffin, or olive oil.

[0158] Liquid preparation for oral administration may be in the form of solutions, syrups, or suspensions. The liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or ethyl alcohol); and preservatives (e.g., methyl or propyl para-hydroxy benzoates or sorbic acid). Liquid formulations of a pharmaceutical composition of the invention which are suitable for oral administration may be prepared, packaged, and soldeither in liquid form or in the form of a dry product intended for reconstitution with water or another suitable vehicle prior to use.

[0159] A tablet comprising the active ingredient may, for example, be made by compressing or molding the active ingredient, optionally with one or more additional ingredients. Compressed tablets may be prepared by compressing, in a suitable device, the active ingredient in a free- flowing form such as a powder or granular preparation, optionally mixed with one or more of a binder, a lubricant, an excipient, a surface-active agent, and a dispersing agent. Molded tablets may be made by molding, in a suitable device, a mixture of the active ingredient, a pharmaceutically acceptable carrier, and at least sufficient liquid to moisten the mixture. Pharmaceutically acceptable excipients used in the manufacture of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, binding agents, and lubricating agents. Known dispersing agents include, but are not limited to, potato starch and sodium starch glycollate. Known surface-active agents include, but are not limited to, sodium lauryl sulphate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate. Known granulating and disintegrating agents include, but are not limited to, corn starch and alginic acid. Known binding agents include, but are not limited to, gelatin, acacia, pre- gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Known lubricating agents include, but are not limited to, magnesium stearate, stearic acid, silica, and talc.

[0160] As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, intraocular, intravitreal, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, intratumoral, and kidney dialytic infusion techniques.

[0161] Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen free water) prior to parenteral administration of the reconstituted composition.

[0162] A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for topical administration. There are several advantages to delivering compounds, including drugs or other therapeutic agents, into the skin (dermal drug delivery) or into the body through the skin (transdermal drug delivery). Transdermal compound delivery offers an attractive alternative to injections and oral medications.

[0163] Additional dosage forms of this invention include dosage forms as described in U.S. Pat. Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389; 5,582,837 and 5,007,790. Additional dosage forms of this invention also include dosage forms as described in U.S. Patent Applications Nos. 20030147952, 20030104062, 20030104053, 20030044466, 20030039688, and 20020051820. Additional dosage forms of this invention also include dosage forms as described in PCT Applications Nos. WO 03 / 35041, WO 03 / 35040, WO 03 / 35029, WO 03 / 35177, WO 03 / 35039, WO 02 / 96404, WO 02 / 32416, WO 01 / 97783, WO 01 / 56544, WO 01 / 32217, WO 98 / 55107, WO 98 / 11879, WO 97 / 47285, WO 93 / 18755, and WO 90 / 11757.

[0164] A therapeutically effective amount of the active agent will be administered in a suitable pharmaceutical composition for the treatment and / or prevention and / or inhibition of the specific disease state. Diseases that may be treated and / or prevented and / or inhibited using the pharmaceutical composition of the present invention include the various disease states for whichthe parent, non-azapeptide containing GLP-1-based therapeutic peptide is indicated. These disease states include, for example, type-2 diabetes, non-insulin dependent diabetes, impaired glucose tolerance, hyperglycemia, chronic weight management, obesity, inducing satiety, chronic inflammation, etc. Determining the effective amount is with the skill level of the artisan.

[0165] The compounds of the invention can be used in the form of salts derived from inorganic or organic acids, which include pharmaceutically acceptable salts. For clarity, the term “pharmaceutically acceptable salt[s]” as used herein generally refers to salts prepared from pharmaceutically acceptable acids or bases including inorganic acids and bases and organic acids and bases. Suitable pharmaceutically acceptable base addition salts include, e.g., metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from lysine, N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. Suitable non-toxic acids include inorganic and organic acids such as acetic, alginic, anthranilic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethenesulfonic, formic, fumaric, furoic, galacturonic, gluconic, glucuronic, glutamic, glycolic, hydrobromic-, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phenylacetic, phosphoric, propionic, salicylic, stearic, succinic, sulfanilic, sulfuric, tartaric acid, and p-toluenesulfonic acid. Specific acids include, e.g., hydrochloric, hydrobromic, phosphoric, sulfuric, and methanesulfonic acids. Examples of specific salts include, e.g., hydrochloride and mesylate salts. Others are well-known in the art. See, e.g., Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing, Easton Pa.: 1990) and Remington: The Science and Practice of Pharmacy, 19th ed. (Mack Publishing, Easton Pa.: 1995). The preparation and use of acid addition salts, carboxylate salts, amino acid addition salts, and zwitterion salts of compounds of the present invention may also be considered pharmaceutically acceptable if they are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, are commensurate with a reasonable benefit / risk ratio, and are effective for their intended use. Such salts may also include various solvates and hydrates of the compound of the present invention.

[0166] Certain compounds of the present invention may be isotopically labelled, e.g., with various isotopes of carbon, fluorine, or iodine, as applicable when the compound in questioncontains at least one such atom. In preferred embodiments, methods of diagnosis of the present invention comprise administration of such an isotopically labelled compound.

[0167] Certain compounds of the present invention may exist as stereoisomers wherein, asymmetric or chiral centers are present. These stereoisomers are “R” or “S” depending on the configuration of substituents around the chiral carbon atom. The terms “R” and “S” used herein are configurations as defined in IUPAC 1974 Recommendations for. Section E, Fundamental Stereochemistry, in Pure Appl. Chem., 1976, 45: 13-30. The invention contemplates various stereoisomers and mixtures thereof and these are specifically included within the scope of this invention. Stereoisomers include enantiomers and diastereomers, and mixtures of enantiomers or diastereomers. Individual stereoisomers of compounds of the invention may be prepared synthetically from commercially available starting materials which contain asymmetric or chiral centers or by preparation of racemic mixtures followed by resolution well known to those of ordinary skill in the art. These methods of resolution are exemplified by (1) attachment of a mixture of enantiomers to a chiral auxiliary, separation of the resulting mixture of diastereomers by recrystallization or chromatography and optional liberation of the optically pure product from the auxiliary as described in Furniss, Hannaford, Smith, and Tatchell, “Vogel's Textbook of Practical Organic Chemistry”, 5th edition (1989), Longman Scientific & Technical, Essex CM20 2JE, England, or (2) direct separation of the mixture of optical enantiomers on chiral chromatographic columns or (3) fractional recrystallization methods.

[0168] Certain compounds of the present invention may exist as cis or trans isomers, wherein substituents on a ring may attach in such a manner that they are on the same side of the ring (cis) relative to each other, or on opposite sides of the ring relative to each other (trans). Such methods are well known to those of ordinary skill in the art and may include separation of isomers by recrystallization or chromatography. It should be understood that the compounds of the invention may possess tautomeric forms, as well as geometric isomers, and that these also constitute an aspect of the invention.

[0169] The term “solid-phase peptide synthesis” or “SSPS” means a method in which molecules (e.g., amino acids, aza-amino acids, etc.) are covalently bound on a solid support material and synthesized step-by-step in a single reaction vessel utilizing selective protecting group chemistry. In this method, building blocks are typically protected at all reactive functionalgroups. The order of functional group reactions can be controlled by the order of deprotection. For example, in an aza-peptide synthesis, an amino-protected amino acid or an amino-protected aza-amino acid is bound to a solid phase material (e.g., low cross-linked polystyrene beads), forming a covalent bond between the carbonyl group and the resin, e.g., an amido or an ester bond. Then the amino group is deprotected and reacted with the carbonyl group of the next amino-protected amino acid or amino-protected aza-amino acid. This cycle is repeated to form the desired peptide or aza-peptide chain. After all reactions are complete, the synthesized peptide or aza-peptide is cleaved from the bead.

[0170] The terms “solution phase synthesis” and “liquid phase synthesis” means a method in which molecules (e.g., amino acids, aza-amino acids, etc.) are synthesized in a solution without being covalently bound on a solid support material.

[0171] The term “synthon” means a synthetic building block.

[0172] The term “ambient temperature” means 18-28°C. Synthetic Methods for Preparing Analogues of Incretin Peptides and aza GLP-1-based Therapeutic Analogues

[0173] Analogues of incretin peptide and aza GLP-1-based therapeutic analogues are made by a substitution of a natural amino acid of an incretin peptide at a cleavage site of the GLP peptide with an aza-amino acid, an unnatural peptide and or a different natural peptide. Exemplary strategies for making analogues of incretin peptide aza GLP-1 analogues are outlined in Figure 17.

[0174] Typically, the synthesis of azapeptides involves a combination of hydrazine chemistry and traditional peptide synthesis in the presence of a carbonyl group source. The most common strategy for incorporating aza-amino acid residues into peptide sequences is to prepare N'- alkylated hydrazine derivatives, activate them using an appropriate carbonyl donating reagent, and couple the activated intermediate to the growing resin-bound peptide (Fig. 2D). See, e.g., K.F. Cheng, et al., “A History of Synthetic Milestones and Key Examples”, Curr. Med. Chem., 29, 6336-6358 (2022) and D. Boeglin, et al., “Aza-Amino Acid Scanning of Secondary Structure Suited for Solid-Phase Peptide Synthesis with Fmoc Chemistry and Aza-Amino Acids with Heteroatomic Side Chains”, J. Comb. Chem. 7, 864-878 (2005).

[0175] Various reagents, such as p-nitrophenyl chloroformate, bis(2,4-dinitrophenyl) carbonate, carbonyl-diimidazole (“CDI”) and 1,1'-carbonyl-di-(1,2,4-triazole) (“CDT”), have been used as carbonyl donors to activate hydrazines. Despite the active nature of resulting intermediates like nitrophenylcarbazates and imidazolides, their exceptional stability prevents effective coupling with resin-bound peptides. Other coupling reagents, such as bis(pentafluorophenyl) carbonate or N,N'-disuccinimidyl carbonate (“DSC”), have faced similar issues related to their reduced reactivity. Consequently, these intermediates suffer from complicated side reactions, poor reaction yields, and / or prolonged reaction times.

[0176] Until recently, the activation of N-(Fmoc)-N'-alkyl hydrazines with phosgene is the most effective protocol for incorporating aza motifs into peptide chains. This approach generally provides azapeptides in good yields but using phosgene-based reagents can create challenges in terms of stability and toxicity. These reagents must be freshly prepared and require specialist organic chemists to perform the reactions. Moreover, during the synthesis, side products like hydantoin, oxadiazalone, or symmetric urea may form, which can reduce the yield.

[0177] A more recent approach to synthesizing azapeptides utilizes a thiocarbazate platform, which is summarized Figure 18. This approach is described in more detail in US 11,440,881 B2 and US 2022 / 0306577 A1, which provide for thiosemicarbazates and their use in azapeptide synthesis. See also, A. Altiti, et al., “Thiocarbazate Building Blocks Enable the Construction of Azapeptides for Rapid Development of Therapeutic Candidates”, Nature Communications 13:7127 (2022).

[0178] Other classes of synthons that can be used in azapetide synthesis are known. For example, US 10,919,882 B2 and US 11,414,505 B2 provide for O-benzotriazole and imidazole synthons that can be used in the preparation of azapeptides. Dimers that may be used as synthons are provided for in WO 2021 / 226431 A1. Other synthons are provided for in US 2020 / 0354404 A1. Peptidomimetic small molecules modeled after HMGB1 antagonist tetramer peptides that have been stabilized with at least one azapeptide bond are provided for in US 11,471,507 B2, US 2020 / 0000908 A1, US 2020 / 0354418 A1, and US 11,414,505 B2. U.S. Application Serial No 63 / 609,975, entitled “Sulfoxide and Sulfone Derivatives of Thiocarbazates as Synthons for Azapeptides and Processes of Using the Same” to Yousef Al-Abed and International PCT Application entitled “Sulfoxide and Sulfone Derivatives of Thiocarbazates as Synthons forAzapeptides and Processes of Using the Same” filed concurrently herewith, provides for another synthetic route to making azapeptides analogues of peptide molecules.

[0179] The synthons and synthetic procedures that can be used to prepare a specific aza GLP-1 analogue are described, for example, in US 10,919,882 B2, US 11,414,505 B2, US 2020 / 0354404 A1, WO 2021 / 226431 A1, U.S. Application Serial No. 63 / 609,975, entitled “Sulfoxide and Sulfone Derivatives of Thiocarbazates as Synthons for Azapeptides and Processes of Using the Same” to Yousef Al-Abed, and International PCT Application entitled “Sulfoxide and Sulfone Derivatives of Thiocarbazates as Synthons for Azapeptides and Processes of Using the Same” filed concurrently herewith.

[0180] Certain embodiments of the present invention will now be illustrated by the following Examples, which are given for illustration purposes only and are not intended to limit the invention in any way and using the general method for synthesis and isolation of GLP-1 and its aza analogues described below.

[0181] Additional Embodiments

[0182] Additional embodiments of the invention are further described by the following numbered paragraphs:

[0183] 1. An aza GLP-1-based therapeutic analogue or a salt thereof of a GLP-1- based therapeutic peptide, wherein (i) at least one peptide bond of the GLP-1- based therapeutic peptide has been replaced with an azapeptide bond, and / or (ii) at least one amino acid of the GLP-1- based therapeutic peptide has been replaced with an unnatural amino acid or a different amino acid, and / or (iii) the aza GLP-1-based therapeutic analogue is a compound of SEQ ID NO: 4, the aza GLP-1-based therapeutic analogue is optionally conjugated to (i) a lipid (e.g., a fatty acid) with an albumin binding motif, directly or through a linker, or (ii) a different compound, at one or more position(s).

[0184] 2. The aza GLP-1-based therapeutic analogue according to paragraph 1, wherein two peptide bonds of a GLP-1-based therapeutic peptide have been replaced with azapeptide bonds.

[0185] 3. The aza GLP-1-based therapeutic analogue according to paragraph 2, wherein the two peptide bonds are an N-terminal His-Ala or an N-terminal His-Gly.

[0186] 4. The aza GLP-1-based therapeutic analogue according to paragraph 1, wherein three peptide bonds of a GLP-1-based therapeutic peptide have been replaced with azapeptide bonds.

[0187] 5. The aza GLP-1-based therapeutic analogue according to paragraph 1, wherein four or five peptide bonds of a GLP-1-based therapeutic peptide have been replaced with azapeptide bonds.

[0188] 6. The aza GLP-1-based therapeutic analogue according to any one of paragraphs 1- 5, wherein the GLP-1 based therapeutic peptide is a GLP-1 receptor agonist.

[0189] 7. The aza GLP-1-based therapeutic analogue according to paragraph 6, wherein the GLP-1-based therapeutic peptide selected from the group consisting of liraglutide, semaglutide, danuglipron, LY3502970, lixisenatide, dulaglutide, tirzepatide, and exenatide.

[0190] 8. The GLP-1-based therapeutic receptor analogue according to paragraph 6, wherein the GLP-1-based therapeutic peptide is semaglutide, and at least one amino acid of semaglutide has been replaced with an unnatural amino acid or a different amino acid.

[0191] 9. An aza GLP-1 based therapeutic receptor analogue, which is azaA8GLP-1(7-37), azaE9GLP-1(7-37), azaHis7, Abi8, Arg34GLP-1(7-37), or azaHis7, azaAla8, Arg34GLP-1 (7-37).

[0192] 10. The aza GLP-1 based therapeutic receptor analogue and according to paragraph 9, which is azaA8GLP-1(7-37).

[0193] 11. The aza GLP-1-based therapeutic analogue according to any one of paragraphs 1- 5, wherein the GLP-1 based therapeutic peptide is a dual-acting GLP-1 and GIP receptor agonists peptide.

[0194] 12. The aza GLP-1-based therapeutic analogue according to paragraph 11, wherein the GLP-1 based therapeutic peptide is tirzepatide.

[0195] 13. The aza GLP-1-based therapeutic analogue according to any one of paragraphs 1- 5, wherein the GLP-1 based therapeutic peptide is a triple acting GLP-1, GIP, and glucagon receptor agonist peptide.

[0196] 14. The aza GLP-1-based therapeutic analogue according to claim 13, wherein the GLP-1-based therapeutic peptide is retatrutide.

[0197] 15. An aza GLP-1-based therapeutic analogue, which is a compound selected from a group consisting of compounds of SEQ ID NOs: 5 to 25.

[0198] 16. A pharmaceutical composition comprising (i) a therapeutically effective amount of the compound of paragraph 1, and (ii) at least one pharmaceutically acceptable excipient (e.g., a pharmaceutically acceptable carrier).

[0199] 17. The pharmaceutical composition according to paragraph 16, wherein two peptide bonds of a GLP-1 based therapeutic peptide have been replaced with azapeptide bonds

[0200] 18. The pharmaceutical composition according to paragraph 16, wherein three peptide bonds of a GLP-1 based therapeutic peptide have been replaced with azapeptide bonds.

[0201] 19. The pharmaceutical composition according to paragraph 16, wherein four or five peptide bonds of a GLP-1 based therapeutic peptide have been replaced with azapeptide bonds.

[0202] 20. The pharmaceutical composition according to any one of paragraphs 16-19, wherein the aza GLP-1-based therapeutic peptide is a GLP-1 receptor agonist.

[0203] 21. The pharmaceutical composition according to paragraph 22, wherein the GLP-1- based therapeutic peptide is selected from the group consisting of liraglutide, semaglutide, danuglipron, LY3502970 (Lilly), lixisenatide, dulaglutide, tirzepatide, and exenatide.

[0204] 22. The pharmaceutical composition according to paragraph 21, wherein the GLP-1- based therapeutic peptide is semaglutide.

[0205] 23. An aza GLP-1-based therapeutic receptor analogue, which is azaA8GLP-1(7-37), azaE9GLP-1(7-37), azaHis7, Abi8, Arg34GLP-1(7-37), or azaHis7, azaAla8, Arg34GLP-1 (7-37).

[0206] 24. The pharmaceutical composition according to any one of paragraph 16-19, wherein the aza GLP-1-based therapeutic peptide is a dual-acting GLP-1 and GIP receptor agonists peptide.

[0207] 25. The pharmaceutical composition according to paragraph 24, wherein the dual- acting GLP-1 and GIP receptor agonists peptide is tirzepatide.

[0208] 26. The pharmaceutical composition according to any one of paragraphs 16-19, wherein the aza GLP-1-based therapeutic peptide is a triple acting GLP-1, GIP, and glucagon receptor agonist peptide.

[0209] 27. The pharmaceutical composition according to paragraph 26, wherein the triple acting GLP-1, GIP, and glucagon receptor agonist peptide is retatrutide.

[0210] 28. The pharmaceutical composition according to paragraph 16, wherein the pharmaceutical composition is an oral dosage form.

[0211] 29. The pharmaceutical composition according to paragraph 16 wherein the aza GLP- 1-based therapeutic analogue has an aqueous solubility of greater than 1 mg / ml.

[0212] 30. The pharmaceutical composition according to paragraph 16, wherein the aza GLP-1-based therapeutic analogue has an aqueous solubility of greater than 5 mg / ml.

[0213] 31. The pharmaceutical composition according to paragraph 16, wherein the aza GLP-1-based therapeutic analogue is stable for greater than 60 minutes in plasma or simulated stomach acid.

[0214] 32. The pharmaceutical composition according to paragraph 16, wherein the pharmaceutical composition is a dosage form selected from the group consisting of a parenteral dosage form, a buccal dosage form, a sublingual dosage form, a nasal dosage form, an inhaler, a nebulizer, a topical dosage form, a transdermal dosage form, and a suppository.

[0215] 33. A pharmaceutical composition comprising (i) a compound selected from a group consisting of compounds of SEQ ID Nos: 5 to 25 and salts thereof, compound selected from a group consisting of compounds of SEQ ID Nos: 5 to 25 and salts thereof optionally conjugated to (i) a lipid (e.g., a fatty acid) with an albumin binding motif, directly or through a linker, or adifferent compound, at one or more position(s); and (ii) one or more pharmaceutically acceptable excipient(s).

[0216] 34. A method for treating a disease state, for which a GLP-1-based therapeutic receptor agonist peptide is indicated for a human or non-human patient in need of said treatment, which comprises administering a therapeutically effective amount of an aza GLP-1-based therapeutic analogue according to paragraph 1 to a human or non-human patient.

[0217] 35. The method according to paragraph 34, wherein the disease state is type-2 diabetes, non-insulin dependent diabetes, impaired glucose tolerance, hyperglycemia, a cardiovascular disease, a dyslipidemia, a neurodegenerative disorder, obesity, acute inflammation, or chronic inflammation.

[0218] 36. The method according to paragraph 35, wherein the aza GLP-1-based therapeutic analogue is administered subcutaneously.

[0219] 37. The method according to paragraph 35, wherein the aza GLP-1-based therapeutic analogue is administered orally.

[0220] 38. The aza GLP-1-based therapeutic analogue of paragraph 1, which is conjugated to (i) a lipid (e.g., a fatty acid) with an albumin binding motif, directly or through a linker, or (ii) a different compound, at one or more position(s).

[0221] 38. The aza GLP-1-based therapeutic analogue of paragraph 15, which is conjugated to (i) a lipid (e.g., a fatty acid) with an albumin binding motif, directly or through a linker, or (ii) a different compound, at one or more position(s).

[0222] Example 1 General method for synthesis and isolation of GLP-1 and its aza analogues

[0223] Solid-phase peptide syntheses were executed using Tribute® Peptide synthesizer from Gyros Protein Technologies, Inc. The machine is fully automated with two independent reaction vessels with polytetrafluoroethylene frits, five solvent positions and 101 amino acid positions. Standard protocol is described sequentially as below:1. Swelling: The Wang resin (loaded with the first Fmoc-protected amino acid) was swelled twice successively for 20 min in DMF, each swelling step was followed by drainage and drying step. 2. Fmoc Cleavage: the protected amino acid / or peptide was shaken for 2.0 min with 20% piperidine solution in DMF to remove the Fmoc group. The process was repeated twice, followed by several washing steps with DMF (3-5 times). 3. Amino acid coupling: 5 equivalents of the next acylating component (Fmoc- protected amino acid), 5 equivalents of HCTU (coupling reagent), and 10 equivalents of N- methylmorpholine (base) were used to add the next amino acid in the sequence. This step is fully automated and was run according to the software installed on the Tribute® synthesizer. The amino acid including coupling reagent was delivered to the reaction vessel from the specified loading position upon dissolution. Then the base was added as 0.4 M solution in DMF, the total volume of solvent was adjusted to give 0.2 M solution. The coupling time was limited to 15 min shaking followed by drainage then washing steps. Step 2 and step 3 were repeated until the desired peptide sequence was achieved. 4. Washing: repeated washing steps were performed after each cleavage or coupling event using DMF as solvent (5 times). At the final coupling or cleavage steps additional washing with DCM was performed (5 times) to remove any trace of DMF. Then was followed by a drying step. 5. Cleavage from the Resin: 5.0 mL of a freshly made solution of TFA / H2O / TIPS (95:2.5:2.5. v / v / v) was cooled down to 0 °C and added at 0 °C to a 0.3 mmol of resin. The mixture was shaken at room temperature for 2 h, filtered, and the remaining resin was further washed with a 0.5-1.0 mL of TFA / H2O) (95:5 v / v) solution. The filtrate was precipitated by adding 10 mL of ether. Upon centrifugation, the resulting solid was dissolved in a 1:1 solution of CH3CN: H2O. The resulting solution was lyophilized.

[0224] Purification: Purification of the peptidomimetics were performed on a preparative HPLC purification system (Waters Prep 150 LC system combining 2545 Binary Gradient Module using XSelect Peptide CSH C18 OBD Prep Column, 130Å, 5 µm, 19 mm X 150 mm. Chromatography was performed at ambient temperature with a flow rate of 18mL / min with a linear gradient from water (0.1% FA): CH3CN (0.1% FA)[95:5] to water (0.1% FA): CH3CN(0.1% TFA) [5:95] in 12 minutes, monitored by 2998 Photodiode Array (PDA) Detector UV at 254 nm and / or 215 nM.

[0225] Integration of the aza-amino acid in SPPS: Wang Resin® (based on 0.1 mmol) was swelled twice in DMF (2.5 mL) for 10 min each time, followed by treatment (twice) with a 20% solution of piperidine in DMF (2.5 mL) for 2 min to cleave the Fmoc group. After successive washes (5 times) with DMF (2.5 mL) for 30 seconds, followed by drainage. The Fmoc-aza- amino acid-OBt was delivered into the resin with 2.5mL DMF only (No base). The mixture was shaken for 2 h to 20h, followed by 5 cycles of DMF washing and 5 cycles of DCM washing and then drying. A small amount of the resin was cleaved using a freshly made solution of TFA / H2O (95:5 v / v), and the resulting peptide was analyzed by HPLC.

[0226] Coupling of the aza-amino acids on the peptidyl chain (aza-peptide bond formation): Coupling to the aza-amino acid using amino acid and COMU as coupling agent. After cleavage (3 cycles with 20% piperidine in DMF) of the Fmoc group from the peptidyl chain, a solution of the designated amino acid (1.0 mmol) mixed with COMU (1.0 mmol) in 0.4 M solution of NMM in DMF (2.5 mL, 1.0 mmol) was added to the resin (0.1 mmol). The resulting suspension was shaken for 1 h at rt, followed by drainage and 5 times washing with DMF. The cycle was repeated once or twice depending on the conversion rate, which was established based on the HPLC analysis after a cleavage of small amount of the resin.

[0227] Synthesis of GLP-1(7-37)-azaA8(Cmpd 2) and GLP-1(7-37)-azaE9(Cmpd 3)

[0228] Using the processes described above, GLP-1 (7-37) (1), GLP-1(7-37)-azaA8(2), and GLP-1(7-37)-azaE9(3) were prepared. Figs. 3A and 3B describe the synthesis of GLP-1 (7-37) (1) and the synthesis of GLP-1(7-37)-azaA8(2). Fig. 4 describes the synthesis of GLP-1(7-37)- azaE9(3). The HOBt aza-amino acid intermediates (synthons) used in the synthesis are described, for example, in US 10,919,882 B2 and US 11,414,505 B2. Table 1 describes the reaction time, purity, and yield for the syntheses. Table 1Compd Total RT Crude Crude Isolated Amount Purity Rxn (mins) purity yield yield (mg) Time GLP-1(7-37) 33h 7.46 51.0% 45.7% 32.8% 25 > 90% (1) GLP-1(7-37)- azaA850h 7.36 60.1% 47.7% 23.9% 10 > 90% (2) GLP-1(7-37)- azaE950h 7.44 56.2% 47.0% 27.5% 23 > 90% (3)

[0229] Example 2 Synthesis of azaHis7, Abi8, Arg34GLP-1(26-37) (Cmpd 4) 1. Fmoc-AzaHIS (DNP)Aib-OH (Cmpd 31) OUsing the processes described in Fig. 5, the Fmoc-AzaHIS (DNP)Aib-OH dimer building block (azaH-Aib dipeptide (31)) was synthesized. 2. azaHis7, Abi8, Arg34GLP-1(26-37) (Cmpd 4) Starting with NH₂-EGTFTSDVSSYLEGQAAKEFIAWLVRGRG-Resin, solid- phase synthesis was used to incorporate Fmoc-AzaHIS (DNP)Aib-OH into the peptide according to the following reaction scheme using know reaction conditions: NH₂-EGTFTSDVSSYLEGQAAKEFIAWLVRGRG-ResinSPPS NH₂-azaHisAibEGTFTSDVSSYLEGQAAKEFIAWLVRGRG-COOH O FmocHN OHScheme 1: Synthesis of azaHis7, Abi8, Arg34GLP-1(7-37)

[0230] Example 3 Synthesis of azaHis7, azaAla8, Arg34GLP-1 (7-37) (Cmpd 5) 1. Fmoc-azaHis (DNP)azaAla-OBt (Cmpd 41) Using the processes described in Fig. 6, the Fmoc-azaHis (DNP)azaAla-OBt dimer building block (azaH-azaA diazatide (41)) was synthesized. 2. azaHis7, azaAla8, Arg34GLP-1 (7-37) (Cmpd 5) Starting with NH₂-EGTFTSDVSSYLEGQAAKEFIAWLVRGRG-Resin, solid- phase synthesis was used to incorporate Fmoc-azaHis (DNP)azaAla-OBt into the peptide according to the following reaction scheme using know reaction conditions: NH₂-EGTFTSDVSSYLEGQAAKEFIAWLVRGRG-ResinSPPS NH₂-azaHisazaAlaEGTFTSDVSSYLEGQAAKEFIAWLVRGRG-COOHO FmocHNN OBtScheme 2: azaAla8, Arg34GLP-1 (7-37)

[0231] Example 4 Fully automatic synthesis of [R34]-GLP-1(7-37) (Cmpd 6)

[0232] Using the processes described in Fig. 7, [R34]-GLP-1(7-37) was synthesized.

[0233] Example 5 Fully automatic synthesis of [R34, Aib8]-GLP-1(7-37) (Cmpd 7)

[0234] Using the processes described in Fig. 8, [R34, Aib8]-GLP-1(7-37) was synthesized.

[0235] Example 6 Fully automatic synthesis of [R34, azaHis7]-GLP-1(7-37) (Cmpd 8)

[0236] Using the processes described in Fig. 9, [R34, azaHis7]-GLP-1(7-37) was synthesized.

[0237] Example 7 Fully automatic synthesis of [R34, azaAla8]-GLP-1(7-37) (Cmpd 9)

[0238] Using the processes described in Fig. 10, [R34, azaAla8]-GLP-1(7-37) was synthesized.

[0239] Example 8 Fully automatic synthesis of [R34, azaGlu9, Aib8]-GLP-1(7-37) (Cmpd 10)

[0240] Using the processes described in Fig. 11, [R34, azaGlu9, Aib8]-GLP-1(7-37) was synthesized.

[0241] Example 9: Fully automatic synthesis of [R34, azaHis7, Aib8]-GLP-1(7-37) (NH2-azaH-Aib- EGTFTSDVSSYLEGQAAKEFIAWLVRGRG-OH) (Cmpd 11)

[0242] Using the processes described in Fig. 5, azaH-Aib dipeptide (31) was synthesized.

[0243] Then, using the processes described in Fig. 12, [R34, azaHis7, Aib8]-GLP-1(7-37) was synthesized.

[0244] Example 10 Fully automatic synthesis of [R34, azaAla8, azaHis7]-GLP-1(7-37) (Cmpd 12)

[0245] Using the processes described in Fig. 6, azaH-azaA diazatide (41) was synthesized.

[0246] Using the processes described in Fig. 13, [R34, azaAla8, azaHis7]-GLP-1(7-37) was then synthesized.

[0247] Example 11 Fully automatic synthesis of [R34, azaGlu9, azaHis7]-GLP-1(7-37) (Cmpd 13)

[0248] Using the processes described in Fig. 14, [R34, azaGlu9, azaHis7]-GLP-1(7-37) was synthesized.Example 12 Ex-vivo and In-vitro DPP-IV stability testing

[0249] . The following peptide or azapeptides were either prepared in the examples above or prepared using a similar procedure and their ex-vivo DPP-IV stability was measured. These results are summarized in Table 2 below

[0250] The results are summarized in Table 1. Table2: Percentage Remaining Intact Peptide Upon Incubation with DPP-IV (AUC at specified time-point by RP-HPLC) Ex-vivo t = 24 t = 48 DPP-IV Peptide / Azapeptide t = 0 t = 1 hr t = 4 hr t = 8 hr hr hr stability (t ½) GLP-1(7-37)-R34(6) 92% 9.2% <5% - - - ~ 18 min GLP-1(7-37)-R34, 8 100% 100% 100% 100% >95% >95% >24 hr azaA (9) GLP-1(7-37)-R34, 7 100% 100% 100% 100% >96% - >24 hr azaH (14) GLP-1(7-37)-R34, 98100% 93% 89% >80% >80% - >24hr azaE , Aib (15) GLP-1(7-37)-R34, Aib8100% 76.2% 72.3% 40.3% 20.6% 10.5% ~ 6 hr (7)GLP-1(7-37)-R34, 100% 100% 100% 100% >97% >97% >24 hr azaA8,azaHis7(5) GLP-1(7-37)-R34, Aib8100% 100% 100% 100% >96% >96% >24 hr ,azaHis7(16) GLP-1(7-37)-R34, 9 100% 100% 100% 100% >95% - >24hr azaE ,azaHis7(17)

[0251] In-vitro DDP-IV stabilities of azapeptides prepared in Examples 4 to 11 were tested, and the t1 / 2(h) were determined and summarized in Table 3 below. Table 3 (In vitro DPP-IV Stability t1 / 2 (h)) Peptide / Azapeptide Sequence In vitro DPP-IV stability t1 / 2 (h) [R34]-GLP-1(7-37) NH2-HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG-OH (SEQ ID 0.3 (6) NO: 26) [R34, Aib8]-GLP- NH2-HAibEGTFTSDVSSYLEGQAAKEFIAWLVRGRG-OH (SEQ 6 1(7-37) (7) ID NO: 8) [R34, azaHis7]- NH2-azaH-AEGTFTSDVSSYLEGQAAKEFIAWLVRGRG-OH (SEQ>24 GLP-1(7-37) (8) ID NO: 18)[R34, azaAla8]- NH2-H-azaA-EGTFTSDVSSYLEGQAAKEFIAWLVRGRG-OH >24 GLP-1(7-37) (9) (SEQ ID NO: 11) [R34, azaGlu9, NH2-H-Aib-azaE-GTFTSDVSSYLEGQAAKEFIAWLVRGRG-OH >24 Aib8]-GLP-1(7-37) (SEQ ID NO: 13) (10) [R34, azaHis7, NH2-azaH-Aib-EGTFTSDVSSYLEGQAAKEFIAWLVRGRG-OH >24 Aib8]-GLP-1(7-37) (SEQ ID NO: 16) (11) [R34, azaAla8, NH2-azaH-azaA-EGTFTSDVSSYLEGQAAKEFIAWLVRGRG-OH >24 azaHis7]-GLP-1(7- (SEQ ID NO: 17) 37) (12) [R34, azaGlu9, NH2-azaH-A-azaE-GTFTSDVSSYLEGQAAKEFIAWLVRGRG-OH >24 azaHis7]-GLP-1(7- (SEQ ID NO: 19) 37) (13)

[0252] Example 13 In vitro potency testing (EC50)

[0253] The in vitro functional receptor assay of azapepeptides prepared in Examples 4 to 11 was carried out by the cloned human GLP-1 receptor overexpressed in baby hamster kidney (BHK) cells.

[0254] The reporter gene assay (CRE- luc) utilized BHK-21 cells overexpressing the hGLP- 1R. For these experiments, the compounds were tested in four technical pseudo-replicates.

[0255] The combined results from three independent experiments are depicted in Fig. 15 as EC50 values with the respective 95% confidence interval and are summarized in Table 4. Table 4 Cmpd Peptide / azapeptide Potency (EC50, pM) 6 [R34]-GLP-1(7-37) 11.3 7 [R34, Aib8]-GLP-1(7-37) 8.77 8 [R34, azaHis7]-GLP-1(7-37) 728 9 [R34, azaAla8]-GLP-1(7-37) 22.2 [R34, azaGlu9, Aib8]-GLP-1(7- 10 344 37) [R34, azaHis7, Aib8]-GLP-1(7- 11 303 37) [R34, azaAla8, azaHis7]-GLP- 12 487 1(7-37) [R34, azaGlu9, azaHis7]-GLP- 13 >10 nM 1(7-37)

[0256] Example 14 In vivo testing

[0257] GLP-1 (Cmpd 1), the peptide of Example 5, and azapeptides of Examples 6 and 7 were administered to mice (1mg / kg, IV). The mean plasma concentrations provided by the administrations are depicted in Figs. 16A (GLP-1) (1), 16B ([R34, Aib8]-GLP-1(7-37)) (7), 16C ([R34, azaHis7]-GLP-1(7-37)) (8), and 16D ([R34, azaAla8]-GLP-1(7-37)) (9).

[0258] In vivo plasma half-lives in mice of GLP-1, the peptide of Example 5 and azapeptides of Examples 6 and 7 (1mg / kg, IV) are summarized in Table 5. Table 5 i.v. administration Compound Peptide / azapeptide (n=3) t 1 / 2 (h) 1 GLP-1(7-37) 0.07 7 [R34, Aib8]-GLP-1(7-37) 0.23 8 [R34, azaHis7]-GLP-1(7-37) 2.49 9 [R34, azaAla8]-GLP-1(7-37) 0.50

[0259] The in-vivo plasma half-lives of the unnatural amino acid-containing peptide of Example 5 (Aib) and the azapeptides of Examples 6 and 7 are significantly longer than the in- vivo plasma half-live of GLP-1(7-37). The in-vivo half-lives of the azapeptides of Examples 6 and 7 (compounds 8 and 9 respectively) were significantly longer than the in-vivo half-life of the peptide of Example 5. The in-vivo half-life of the azapeptide of Example 6 (8) was about 10 times longer than the in-vivo half-life of the unnaturally amino acid-containing peptide ofExample 5. The in-vivo half-life of the azapeptide of Example 7 (9) was about 2 times longer than the in-vivo half-life of the peptide of Example 5. Example 15 In Vivo Effect of [R34, azaAla8]-GLP-1(7-37) (9) on Glucose Levels

[0260] The effect on glucose levels in lean mice of azaAla8-GLP-1 analogue (9) were compared against the semaglutide root peptide (7) to demonstrate the effectiveness of the inventive azapeptides to treat type-2 diabetes and obesity when compared to a semaglutide analogue. Specifically, glucoregulatory effects of azaAla8-GLP-1 analogue (9) as compared with controls in lean male mice (C57BL / 6 mice, 21 weeks) via standard oral glucose tolerance tests performed 2 hours after test article administration were assessed. The results are shown in Fig. 20.

[0261] Compared with vehicle control, both semaglutide root peptide (7), which has been modified to contain the unnatural amino acid Aib, and the azaAla8analogue (9) significantly reduced blood glucose levels when given at equivalent concentration. At timepoints 60-120 minutes of the oral glucose tolerance test, the azaAla8analogue (9) reduced blood glucose levels to a greater extent than the semaglutide root peptide (7). These findings indicate that the azaAla8- analogue (9) is biologically active and therapeutically efficacious.

[0262] The extended reduction in glucose levels as compared with semaglutide root peptide (7), suggests that azaAla8analogue (9) may provide extended benefits possibly through its longer blood half-life. Furthermore, with additional modifications (lipidation), the AzaAla8analogue (9) could be used as an alternative to semaglutide or other GLP-1 agonists.

[0263] While illustrative embodiments of the disclosure have been described and illustrated above, it should be understood that these are exemplary of the disclosure and are not intended to be limiting. Additions, deletions, substitutions, and other modifications can be made without departing from the spirit or scope of the disclosure. Accordingly, the disclosure is not to be considered as limited by the foregoing description.

Claims

What is claimed is:

1. An aza GLP-1-based therapeutic analogue or a salt thereof of a GLP-1- based therapeutic peptide, wherein (i) at least one peptide bond of the GLP-1- based therapeutic peptide has been replaced with an azapeptide bond, (ii) at least one amino acid of the GLP-1- based therapeutic peptide has been replaced with an unnatural amino acid or a different amino acid, and (iii) the aza GLP-1-based therapeutic analogue is a compound of SEQ ID NO: 4, and wherein aza GLP-1-based therapeutic analogue is optionally conjugated to (i) a lipid with an albumin binding motif, directly or through a linker.

2. The aza GLP-1-based therapeutic analogue according to claim 1, wherein two peptide bonds of a GLP-1-based therapeutic peptide have been replaced with azapeptide bonds.

3. The aza GLP-1-based therapeutic analogue according to claim 2, wherein the two peptide bonds are an N-terminal His-Ala or an N-terminal His-Gly.

4. The aza GLP-1-based therapeutic analogue according to claim 1, wherein three peptide bonds of a GLP-1-based therapeutic peptide have been replaced with azapeptide bonds.

5. The aza GLP-1-based therapeutic analogue according to claim 1, wherein four or five peptide bonds of a GLP-1-based therapeutic peptide have been replaced with azapeptide bonds.

6. The aza GLP-1-based therapeutic analogue according to any one of claims 1-5, wherein the GLP-1 based therapeutic peptide is a GLP-1 receptor agonist.

7. The aza GLP-1-based therapeutic analogue according to claim 6, wherein the GLP-1- based therapeutic peptide is selected from the group consisting of liraglutide, semaglutide, danuglipron, LY3502970, lixisenatide, dulaglutide, tirzepatide, and exenatide.

8. The GLP-1-based therapeutic receptor analogue according to claim 6, wherein the GLP- 1-based therapeutic peptide is semaglutide.

9. An aza GLP-1 based therapeutic receptor analogue, which is azaA8GLP-1(7-37), azaE9GLP-1(7-37), azaHis7, Abi8, Arg34GLP-1(7-37), or azaHis7, azaAla8, Arg34GLP-1 (7-37).

10. The aza GLP-1 based therapeutic receptor analogue of claim 9, which is azaA8GLP-1(7- 37).

11. The aza GLP-1-based therapeutic analogue according to any one of claims 1-5, wherein the GLP-1 based therapeutic peptide is a dual-acting GLP-1 and GIP receptor agonists peptide.

12. The aza GLP-1-based therapeutic analogue according to claim 11, wherein the GLP-1 based therapeutic peptide is tirzepatide.

13. The aza GLP-1-based therapeutic analogue according to any one of claims 1-5, wherein the GLP-1 based therapeutic peptide is a triple acting GLP-1, GIP, and glucagon receptor agonist peptide.

14. The aza GLP-1-based therapeutic analogue according to claim 13, wherein the GLP-1- based therapeutic peptide is retatrutide.

15. An aza GLP-1-based therapeutic analogue which is a compound selected from a group consisting of compounds of SEQ ID NOs: 5 to 25 and salts thereof, the compound selected from a group consisting of compounds of SEQ ID NOs: 5 to 25 and salts thereof is optionally conjugated to (i) a lipid with an albumin binding motif, directly or through a linker, or (ii) a different compound, at one or more position(s).

16. A compound of SEQ ID NO: 10 or a salt thereof, which is optionally conjugated to (i) a lipid with an albumin binding motif, directly or through a linker, or (ii) a different compound, at one or more position(s).

17. A pharmaceutical composition comprising (i) a therapeutically effective amount of the compound of claim 1, and (ii) at least one pharmaceutically acceptable carrier.

18. A pharmaceutical composition comprising (i) a therapeutically effective amount of the compound of claim 15, and (ii) at least one pharmaceutically acceptable carrier.

19. The pharmaceutical composition according to any one of claims 15-18, wherein the aza GLP-1-based therapeutic peptide is a GLP-1 receptor agonist.

20. The pharmaceutical composition according to claim 19, wherein the GLP-1-based therapeutic peptide is selected from the group consisting of liraglutide, semaglutide, danuglipron, LY3502970 (Lilly), lixisenatide, dulaglutide, tirzepatide, and exenatide.

21. The pharmaceutical composition according to claim 20, wherein the GLP-1-based therapeutic peptide is semaglutide.

22. The pharmaceutical composition according to claim 17, wherein the aza GLP-1-based therapeutic receptor analogue is azaA8GLP-1(7-37), azaE9GLP-1(7-37), azaHis7, Abi8, Arg34GLP-1(7-37), or azaHis7, azaAla8, Arg34GLP-1 (7-37).

23. The pharmaceutical composition according to any one of claims 17 or 18, wherein the aza GLP-1-based therapeutic peptide is a dual-acting GLP-1 and GIP receptor agonists peptide.

24. The pharmaceutical composition according to claims 23, wherein the dual-acting GLP-1 and GIP receptor agonists peptide is tirzepatide.

25. The pharmaceutical composition according to claim 17, wherein the aza GLP-1-based therapeutic peptide is a triple acting GLP-1, GIP, and glucagon receptor agonist peptide.

26. The pharmaceutical composition according to claim 25, wherein the triple acting GLP-1, GIP, and glucagon receptor agonist peptide is retatrutide.

27. The pharmaceutical composition according to claim 17, wherein the pharmaceutical composition is an oral dosage form.

28. The pharmaceutical composition according to claim 27 wherein the aza GLP-1-based therapeutic analogue has an aqueous solubility of greater than 1 mg / ml.

29. The pharmaceutical composition according to claim 15, wherein the aza GLP-1-based therapeutic analogue has an aqueous solubility of greater than 5 mg / ml.

30. The pharmaceutical composition according to claim 15, wherein the aza GLP-1-based therapeutic analogue is stable for greater than 60 minutes in plasma or simulated stomach acid.

31. The pharmaceutical composition according to claim 17, wherein the pharmaceutical composition is a dosage form selected from the group consisting of a parenteral dosage form, abuccal dosage form, a sublingual dosage form, a nasal dosage form, an inhaler, a nebulizer, a topical dosage form, a transdermal dosage form, and a suppository.

32. The pharmaceutical composition according to claim 15, wherein the aza GLP-1-based therapeutic analogue is a compound selected from a group consisting of compounds of SEQ ID Nos: 5 to 25.

33. A method for treating a disease state, for which a GLP-1-based therapeutic receptor agonist peptide is indicated for a human or non-human patient in need of said treatment, which comprises administering a therapeutically effective amount of an aza GLP-1-based therapeutic analogue according to claim 1 or to a human or non-human patient.

34. The method according to claim 33, wherein the disease state is type-2 diabetes, non- insulin dependent diabetes, impaired glucose tolerance, hyperglycemia, a cardiovascular disease, a dyslipidemia, a neurodegenerative disorder, obesity, acute inflammation, or chronic inflammation.

35. The method according to claim 34, wherein the aza GLP-1-based therapeutic analogue is administered subcutaneously.

36. The method according to claim 34, wherein the aza GLP-1-based therapeutic analogue is administered orally.

37. The aza GLP-1-based therapeutic analogue of claim 1, which is conjugated to (i) a lipid with an albumin binding motif, directly or through a linker, or (ii) a different compound, at one or more position(s).

38. The aza GLP-1-based therapeutic analogue of claim 15, which is conjugated to (i) a lipid with an albumin binding motif, directly or through a linker, or (ii) a different compound, at one or more position(s).

39. The aza GLP-1-based therapeutic analogue of claim 16, which is conjugated to (i) a lipid with an albumin binding motif, directly or through a linker, or (ii) a different compound, at one or more position(s).

40. The aza GLP-1-based therapeutic analogue of claim 1, which is conjugated to ([2-(2- Amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)1-CO-(CH2)18-CO2H at position 26.

41. The aza GLP-1-based therapeutic analogue of claim 15, which is conjugated to ([2-(2- Amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)1-CO-(CH2)18-CO2H at position 26.

42. The aza GLP-1-based therapeutic analogue of claim 16, which is conjugated to ([2-(2- Amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)1-CO-(CH2)18-CO2H at position 26.

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