Glucagon-like peptide compounds

Abstract

Provided herein are novel compounds comprising a GLP-1 compound and a fatty acid or fatty acid derivative, the preparation of said novel compounds and uses thereof.

Description

[Technical field]

[0001] Provided herein are novel compounds comprising a GLP-1 compound and a fatty acid or fatty acid derivative, the manufacture of said novel compounds, and uses thereof. These novel compounds exhibit advantageous pharmacological efficacy. [Background technology]

[0002] Glucagon-like peptide (GLP) 1 (GLP1) agonists belong to an important class of therapeutically active compounds. GLP1 agonists are commonly used in the treatment of type 2 diabetes. Various approaches have been used to modify the structure of such glucagon-like peptide 1 (GLP1) compounds to prevent rapid biodegradation, provide a satisfactory duration of action in vivo, and improve tolerability.

[0003] For example, WO 2006 / 097537 (Novo Nordisk) describes GLP1 compounds having at least one non-natural amino acid residue modification at positions 7 and / or 8 compared to the sequence GLP-1(7-37) (SEQ ID NO: 1), in which the lysine residue at position 26 is acylated with a moiety containing at least two acidic groups.

[0004] WO 2015 / 200078 (Novartis) discloses conjugates comprising a biomolecule such as GDF15 linked to a fatty acid via a linker. The corresponding conjugates may be useful in the treatment or prevention of metabolic diseases or disorders. Summary of the Invention

[0005] Formula (i): [ka] (In the formula, R1 and R2 are independently selected from CH3, OH, CO2H, CH=CH2, and C≡CH; n and m are each an integer independently selected from 5 to 30. or a pharma- ceutically acceptable salt thereof, optionally via a linker, wherein the compound of formula (i) is covalently bonded through one of its CO2H groups.

[0006] The compounds described herein can generally act as agonists of glucagon-like peptide 1 receptor (GLP1R).Therefore, these compounds can be useful in the treatment of metabolic diseases, disorders and conditions, such as, but not limited to, diseases or disorders, including obesity, type 2 diabetes, insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia, one or more diabetic complications (including but not limited to chronic kidney disease), diabetic nephropathy, dyslipidemia, cardiovascular disease and neuropathy.The compounds can also potentially be useful in the treatment of progressive liver disease and neuropathy.

[0007] definition The term "peptide" as used herein means a compound composed of at least five amino acids linked by peptide bonds. The amino acids can be natural and unnatural amino acids. Some peptides can be composed entirely of natural amino acids. Some peptides can be composed entirely of unnatural amino acids. Some peptides can be composed of a mixture of natural and unnatural amino acids.

[0008] The term "natural" refers to a material that is found in nature and has not been manipulated by man. Similarly, "non-natural", "non-natural" and the like, as used herein, refer to a material that is not found in nature or that has been structurally modified or synthesized by man.

[0009] The term "naturally occurring" when used in connection with amino acids typically refers to the 22 conventional amino acids, such as alanine (A or Ala), cysteine ​​(C or Cys), cystine (CySS), aspartic acid (D or Asp), glutamic acid (E or Glu), phenylalanine (F or Phe), glycine (G or Gly), histidine (H or His), isoleucine (I or Ile), lysine (K or Lys), leucine (L or Leu), methionine (M or Met), asparagine (N or Asn), proline (P or Pro), 4-hydroxyproline (O or Hyp), glutamine (Q or Gln), arginine (R or Arg), serine (S or Ser), threonine (T or Thr), valine (V or Val), tryptophan (W or Trp), and tyrosine (Y or Tyr).

[0010] The terms "non-naturally occurring amino acid", "non-natural amino acid" and "unnatural amino acid" as used herein are intended to refer interchangeably to amino acid structures, whether the same or different, that cannot be produced biosynthetically in any organism using unmodified or modified genes from any organism. These include, but are not limited to, modified amino acids and / or amino acid analogs that are not one of the 22 naturally occurring amino acids listed above.

[0011] Examples of unnatural amino acids are gamma-carboxyglutamate, ornithine, phosphoserine, D-amino acids such as D-alanine and D-glutamine. Synthetic unnatural amino acids include amino acids produced by chemical synthesis, i.e. D-isomers of amino acids such as D-alanine and D-leucine, Aib (α-aminoisobutyric acid), Abu (α-aminobutyric acid), Tle (tert-butylglycine), 3-aminomethylbenzoic acid, anthranilic acid, des-amino-histidine, beta analogs of amino acids such as β-alanine, e.g. D-histidine, desamino-histidine, 2-amino-histidine, beta-hydroxy-histidine, and the like. Histidine, homohistidine, Na-acetyl-histidine, α-fluoromethyl-histidine, α-methyl-histidine, 3-pyridylalanine, 2-pyridylalanine or 4-pyridylalanine, (1-aminocyclopropyl)carboxylic acid, (1-aminocyclobutyl)carboxylic acid, (1-aminocyclopentyl)carboxylic acid, (1-aminocyclohexyl)carboxylic acid, (1-aminocycloheptyl)carboxylic acid or (1-aminocyclooctyl)carboxylic acid.

[0012] All amino acids, where no reference is made to optical isomers, are understood to refer to the L-isomers.

[0013] The term "analog" or "analog" of a peptide, as used herein, refers to a modified peptide in which one or more amino acid residues of the peptide have been replaced one or more times by another amino acid residue, and / or one or more amino acid residues have been deleted from the peptide, and / or one or more amino acid residues have been added to the peptide. Such addition or deletion of amino acid residues may occur at any position within the peptide. For example, such addition or deletion of amino acid residues may occur within the N-terminal portion of the peptide and / or at the C-terminal portion of the peptide.

[0014] The term "GLP-1" as used herein means GLP-1(7-37) (SEQ ID NO:1).

[0015] The term "GLP-1 analogue" as used herein refers to an analogue of GLP-1(7-37) as defined above, where the term "analog" is as defined above. For example, 34 ]GLP-1(7-37)Lys is a GLP-1(7-37) in which the native lysine at position 34 is replaced by arginine, and the lysine is replaced by the terminal amino acid residue, i.e., Gly. 37 This refers to a GLP-1(7-37) analogue attached to

[0016] Further embodiments Another embodiment is a compound of formula (I): [ka] (In the formula, R1 and R2 are independently selected from CH3, OH, CO2H, CH=CH2, and C≡CH; n and m are each an integer independently selected from 5 to 30; L is an optional linker and P is GLP-1 or a GLP-1 analog. or a pharma- ceutically acceptable salt thereof.

[0017] Another embodiment provides a compound of formula (I) according to the previous embodiment, or a pharma- ceutically acceptable salt thereof, wherein the GLP-1 or GLP-1 analogue (P) is linked to the optional linker (L) via an NH group.

[0018] Another embodiment is where the linker (L) is [ka] [ka] (In the formula, y is an integer selected from 1 to 36; l is 0, 1, 2, 3, 4, 5 or 6; k is 1, 2 or 3; s is 0, 1, 2 or 3; t is 0, 1, 2, 3 or 4; p is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23; ** The wavy line marked indicates the connection to the CO group of formula (I), *** The wavy line marked indicates the linkage to the group P. or a pharma- ceutically acceptable salt thereof.

[0019] Another embodiment is y is an integer selected from 1 to 36; l is 2, 3, 4 or 5; k is 1 or 2; s is 0, 1 or 2; t is 0, 1, 2 or 3; Compounds of formula (I) according to the above embodiments, or pharma- ceutically acceptable salts thereof, are provided, wherein p is 1, 2, 3, 4, 7, 11 or 23.

[0020] Another embodiment is y is an integer selected from 1 to 36; l is 2, 3, 4 or 5; k is 1 or 2; s is 0, 1 or 2; t is 0 or 1; and Compounds of formula (I) according to the above embodiments, or pharma- ceutically acceptable salts thereof, are provided, wherein p is 1, 2, 3, 4 or 11.

[0021] Another embodiment is where the linker (L) is [ka] (In the formula, y is an integer selected from 1 to 36; s is 1 and k is 1; ** The wavy line marked indicates the connection to the CO group of formula (I), *** The wavy line marked indicates the linkage to the group P. or a pharma- ceutically acceptable salt thereof.

[0022] Another embodiment is where L is: [ka] (In the formula, y is an integer selected from 1 to 36; s is 0, 1 or 2 and k is 1, 2 or 3; and ** The wavy line marked indicates the connection to the CO group of formula (I), and *** The wavy line marked indicates the linkage to the group P. or a pharma- ceutically acceptable salt thereof.

[0023] Another embodiment is where the linker (L) is [ka] (wherein y is an integer selected from 1 to 36). or a pharma- ceutically acceptable salt thereof.

[0024] Another embodiment provides a compound of formula (I) according to the previous embodiment, or a pharma- ceutically acceptable salt thereof, wherein the carbon atom of the C(O) group of said linker is linked to the nitrogen atom of the NH group of a lysine residue of GLP-1 or a GLP-1 analogue.

[0025] Another embodiment provides a compound of formula (I) according to the preceding embodiment, or a pharma- ceutically acceptable salt thereof, wherein R1 and R2 are independently selected from CH3, OH and CO2H.

[0026] Another embodiment is a compound of formula (II) [ka] (In the formula, NH-P' represents a group P (i.e., GLP-1 or a GLP-1 analog) that is linked to a linker L via an NH moiety; R1 and R2 are independently selected from CH3, OH, and CO2H; n and m are each an integer independently selected from 5 to 30; and y is an integer selected from 1 to 36. or a pharma- ceutically acceptable salt thereof.

[0027] In one embodiment, the group P (i.e., GLP-1 or a GLP-1 analogue) corresponds to P'-NH2, i.e., a P group having a free -NH2 group that is part of an amino acid side chain, and P is linked to the linker L via said -NH group.

[0028] Another embodiment provides a compound of formula (II) as defined hereinabove, wherein R1 and R2 are independently selected from CO2H and CH3.

[0029] Another embodiment provides a compound of formula (II) as defined hereinabove, wherein n and m are each integers independently selected from 5 to 20.

[0030] Another embodiment provides a compound of formula (II) as defined hereinabove, wherein n and m are integers independently selected from 10, 11, 13 and 14, respectively.

[0031] Another embodiment is R1 is CO2H and R2 is CH3; n is 10 and m is 10; R1 is CO2H and R2 is CO2H; n is 10 and m is 10; R1 is CO2H and R2 is CO2H; n is 10 and m is 11; R1 is CO2H and R2 is CO2H; n is 10 and m is 13; or The present invention provides a compound of formula (II) as defined hereinabove, wherein R1 is CO2H and R2 is CO2H; n is 10 and m is 14.

[0032] Another embodiment is a compound of formula (III) [ka] (wherein R1 is CO2H and R2 is CH3). or a pharma- ceutically acceptable salt thereof.

[0033] Another embodiment is a compound of formula (IIIa) or a compound of formula (IIIb) [ka] (wherein R1 is CO2H and R2 is CH3). or a pharma- ceutically acceptable salt thereof.

[0034] Another embodiment is a compound of formula (IV) [ka] or a pharma- ceutically acceptable salt thereof, which compound exists as a racemate or as a stereochemically enriched mixture, or *The present invention provides a compound of formula (II) as defined herein which is stereochemically pure with respect to the carbon atom marked:

[0035] Another embodiment is a compound of formula (IVa) or a compound of formula (IVb) [ka] (wherein y is an integer selected from 1 to 36). or a pharma- ceutically acceptable salt thereof.

[0036] Another embodiment provides a compound of formula (IVa) or a compound of formula (IVb) as defined herein, wherein y is an integer selected from 2 to 24.

[0037] Another embodiment provides a compound of formula (IVa) or a compound of formula (IVb) as defined herein, wherein y is an integer selected from 2, 8 and 24.

[0038] Another embodiment provides a compound of formula (IVa) or a compound of formula (IVb) as defined herein, wherein y is 2.

[0039] Another embodiment provides a compound of formula (IVa) or a compound of formula (IVb) as defined herein, wherein y is 8.

[0040] Another embodiment provides a compound of formula (IVa) or a compound of formula (IVb) as defined herein, wherein y is 24.

[0041] Another embodiment provides a compound according to any of the above defined embodiments or a pharma- ceutically acceptable salt thereof, wherein P is GLP-1(7-37): His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly (SEQ ID NO: 1), and GLP-1 analogs that contain non-natural amino acid residues at positions 7 or 8 or at positions 7 and 8, as compared to the sequence GLP-1(7-37) (SEQ ID NO:1). is selected from.

[0042] Another embodiment is where P is Xaa7-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Xaa 16 -Ser-Xaa 18 -Xaa 19 -Xaa 20 -Glu-Xaa 22 -Xaa 23 -Ala-Xaa 25 -Arg-Xaa 27 -Phe-lle-Xaa 30 -Trp-Leu-Xaa 33 -Xaa 34 -Xaa 35 -Xaa 36 -Xaa 37 (SEQ ID NO: 2) (hereinafter P * ) (In the formula, Xaa7 is His, imidazopropionyl, α-hydroxy-histidine, D-histidine, desamino-histidine, 2-amino-histidine, β-hydroxy-histidine, homohistidine, N α -Acetyl-histidine, N α -formyl-histidine, α-fluoromethyl-histidine, α-methyl-histidine, 3-pyridylalanine, 2-pyridylalanine or 4-pyridylalanine; Xaa8 is Ala, Gly, Val, Leu, Ile, Thr, Ser, Lys, Aib, (1-aminocyclopropyl)carboxylic acid, (1-aminocyclobutyl)carboxylic acid, (1-aminocyclopentyl)carboxylic acid, (1-aminocyclohexyl)carboxylic acid, (1-aminocycloheptyl)carboxylic acid, or (1-aminocyclooctyl)carboxylic acid; Xaa 16 is Val or Leu; Xaa 18 is Ser, Lys or Arg; Xaa 19 is Tyr or Gln; Xaa 20 is Leu or Met; Xaa 22 is Gly, Glu, or Aib; Xaa 23 is Gln, Glu, Lys or Arg; Xaa 25 is Ala or Val; Xaa 27 is Glu or Leu; Xaa 30 is Ala, Glu or Arg; Xaa 33 is Val or Lys; Xaa 34 is Lys, Glu, Asn, or Arg; Xaa 35 is Gly or Aib; Xaa 36 is Arg, Gly, or Lys, or is absent; and Xaa 37 is Gly, Ala, Glu, Pro, Lys or absent) or a pharma- ceutically acceptable salt thereof.

[0043] Another embodiment provides a compound according to the preceding embodiment, * In Xaa7 is His or desamino-histidine; Xaa8 is Ala, Gly, Val, Leu, Lys, or Aib; Xaa 16 is Val; Xaa 18 is Ser; Xaa 19 is Tyr; Xaa 20 is Leu; Xaa 22 is Gly, Glu, or Aib; Xaa 23 is Gln or Glu; Xaa 25 is Ala; Xaa 27 is Glu; Xaa 30 is Ala or Glu; Xaa 33 is Val; Xaa 34 is Lys or Arg; Xaa 35 is Gly or Aib; Xaa 36 is Arg, Lys or absent; and Xaa 37 is Gly or absent.

[0044] Another embodiment provides a compound according to the preceding embodiment, * In Xaa7 is His; Xaa8 is Gly or Aib; Xaa 16 is Val; Xaa 18 is Ser; Xaa 19 is Tyr; Xaa 20 is Leu; Xaa 22 is Glu or Aib; Xaa 23 is Gln or Glu; Xaa 25 is Ala; Xaa 27 is Glu; Xaa 30 is Ala; Xaa 33 is Val; Xaa 34 is Lys or Arg; Xaa 35 is Gly or Aib; Xaa 36 is Arg; and Xaa 37 is Gly.

[0045] Another embodiment provides a compound of formula (I) as defined herein, or a pharma- ceutically acceptable salt thereof, wherein P is [Aib8, Arg34]GLP-1(7-37):His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Le u-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly (SEQ ID NO: 3); and [Arg34]GLP-1(7-37):His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly (SEQ ID NO: 4) is selected from.

[0046] Another embodiment is where P is [Aib8,Arg34]GLP-1(7-37) (SEQ ID NO:3); or alternatively [ka] or a pharma- ceutically acceptable salt thereof, wherein the wavy line at the amino acid member Lys indicates the point of attachment to the linker.

[0047] Another embodiment is [ka] (wherein y is an integer selected from 1 to 36). or a pharma- ceutically acceptable salt thereof, which compound exists as a diastereomeric mixture, a stereochemically enriched mixture, or * It is stereochemically pure with respect to the carbon atom marked with

[0048] Another embodiment is a compound of formula (X) or formula (XI): [ka] (wherein y is an integer selected from 1 to 36). The present invention provides a compound of formula (I) as defined herein, or a pharma- ceutically acceptable salt thereof, which is

[0049] Another embodiment provides a compound of formula (X) or formula (XI) as defined herein, wherein y is an integer selected from 2 to 24.

[0050] Another embodiment provides a compound of formula (X) or formula (XI) as defined herein, wherein y is an integer selected from 2, 8 and 24.

[0051] Another embodiment provides a compound of formula (X) or formula (XI) as defined herein, wherein y is 2.

[0052] Another embodiment provides a compound of formula (X) or formula (XI) as defined herein, wherein y is 8.

[0053] Another embodiment provides a compound of formula (X) or formula (XI) as defined herein, wherein y is 24.

[0054] Another embodiment is [ka] (Compound 1), [ka] (Compound 2), [ka] (compound 3), [ka] (compound 4), [ka] (Compound 5), [ka] (Compound 6), [ka] (compound 7), [ka] (Compound 8), and [ka] (Compound 9) or a pharma- ceutically acceptable salt thereof.

[0055] Another embodiment is [ka] (Compound 1) or a pharma- ceutically acceptable salt thereof.

[0056] Another embodiment is [ka] (Compound 2) or a pharma- ceutically acceptable salt thereof.

[0057] Another embodiment is [ka] (Compound 3) or a pharma- ceutically acceptable salt thereof.

[0058] Another embodiment provides a pharmaceutical composition comprising a compound described herein, or a pharma- ceutically acceptable salt thereof, and one or more pharma- ceutically acceptable carriers.

[0059] Another embodiment provides a pharmaceutical composition according to the previous embodiment, wherein the compound is selected from compounds 1, 2, 3, 4, 5, 6, 7, 8 and 9.

[0060] Another embodiment provides a pharmaceutical composition according to the previous embodiment, wherein the compound is selected from compounds 1, 2 and 3.

[0061] Another embodiment provides a combination comprising a therapeutically effective amount of a compound described herein, or a pharma- ceutically acceptable salt thereof, and one or more therapeutically active agents.

[0062] Another embodiment provides a combination according to the previous embodiment, wherein the compound is selected from compounds 1, 2, 3, 4, 5, 6, 7, 8 and 9.

[0063] Another embodiment provides a combination according to the previous embodiment, wherein the compound is selected from compounds 1, 2 and 3.

[0064] Another embodiment provides a compound described herein, or a pharma- ceutically acceptable salt thereof, for use as a medicament.

[0065] Another embodiment provides a compound for use according to the preceding embodiment selected from compounds 1, 2, 3, 4, 5, 6, 7, 8 and 9.

[0066] Another embodiment provides a compound for use according to the previous embodiment, selected from compounds 1, 2 and 3.

[0067] Another embodiment provides a compound as described herein, or a pharma- ceutically acceptable salt thereof, for use in treating a disease or disorder selected from obesity, type 2 diabetes, insulin resistance, hyperinsulinemia, impaired glucose tolerance, hyperglycemia, one or more diabetic complications (including but not limited to chronic kidney disease), diabetic nephropathy, dyslipidemia, metabolic syndrome, progressive liver disease, cardiovascular disease, and neuropathy. As a non-limiting example, the neuropathy is a peripheral neuropathy (e.g., may be associated with diabetes).

[0068] Another embodiment provides a compound as described herein, or a pharma- ceutically acceptable salt thereof, for use in the treatment of a cardiovascular disease or disorder selected from hypertension, atherosclerosis, peripheral artery disease, stroke, cardiomyopathy, atrial fibrillation, heart failure (e.g., heart failure with reduced ejection fraction (HFrEF), heart failure with mid-range ejection fraction (HFmrEF), and heart failure with preserved ejection fraction (HFpEF), coronary heart disease, and arrhythmias (e.g., atrial arrhythmias and ventricular arrhythmias).

[0069] Another embodiment provides a compound for use according to the previous two embodiments, selected from compounds 1, 2, 3, 4, 5, 6, 7, 8 and 9.

[0070] Another embodiment provides a compound for use according to the previous embodiment, selected from compounds 1, 2 and 3.

[0071] Another embodiment provides a method of treating a patient in need of treatment who is sensitive to an agonist of the glucagon-like peptide 1 receptor (GLP1R), comprising administering to the patient a therapeutically effective amount of a compound as described herein, or a pharma- ceutically acceptable salt thereof.

[0072] Another embodiment provides a method of treatment according to the previous embodiment, wherein the compound is selected from compounds 1, 2, 3, 4, 5, 6, 7, 8 and 9.

[0073] Another embodiment provides a method of treatment according to the previous embodiment, wherein the compound is selected from compounds 1, 2 and 3.

[0074] Another embodiment provides a method of treatment according to the preceding embodiments, wherein the patient suffers from a disease or disorder selected from obesity, type 2 diabetes, insulin resistance, hyperinsulinemia, impaired glucose tolerance, hyperglycemia, one or more diabetic complications (including but not limited to chronic kidney disease), diabetic nephropathy, dyslipidemia, metabolic syndrome, progressive liver disease, cardiovascular disease, and neuropathy. As a non-limiting example, the neuropathy is a peripheral neuropathy (e.g., may be associated with diabetes).

[0075] Another embodiment provides a method of treatment according to the preceding embodiments, wherein the patient is suffering from a cardiovascular disease or disorder selected from hypertension, atherosclerosis, peripheral artery disease, stroke, cardiomyopathy, atrial fibrillation, heart failure (e.g., heart failure with reduced ejection fraction (HFrEF), heart failure with mid-range ejection fraction (HFmrEF) and heart failure with preserved ejection fraction (HFpEF), coronary heart disease and arrhythmias (e.g., atrial arrhythmias and ventricular arrhythmias).

[0076] Further Aspects Depending on the selection of starting materials and procedures, the compounds may exist in one of the possible stereoisomers or as mixtures thereof, such as pure optical isomers or mixtures of stereoisomers, such as racemic and diastereomeric mixtures, depending on the number of asymmetric carbon atoms. The compounds as described herein are not limited, and the compounds include all such possible stereoisomers, including racemic mixtures, diastereomeric mixtures, and optically pure forms. Optically active (R)- and (S)-stereoisomers can be prepared using chiral synthons or chiral reagents and resolved using conventional techniques. When the compounds contain double bonds, the substituents can be in the E or Z configuration. When the compounds contain disubstituted cycloalkyl, the cycloalkyl substituents can have cis- or trans-configuration. All tautomeric forms are also intended to be included.

[0077] As used herein, the term "salt" or "salts" refers to acid addition salts or base addition salts of the compounds of the present disclosure. "Salt" specifically includes "pharmaceutically acceptable salts". The term "pharmaceutically acceptable salts" refers to salts that retain the biological effectiveness and properties of the compounds of the present disclosure, which are generally not biologically or otherwise undesirable. In many cases, the compounds of the present disclosure are capable of forming acid and / or base salts due to the presence of basic nitrogen atoms, such as those found in amino and pyridine groups or other similar groups and / or acidic protons, such as those found in carboxylic acids or 5-oxo-4,5-dihydro-1,2,4-oxadiazole groups or other similar groups.

[0078] Pharmaceutically acceptable acid addition salts can be formed with inorganic and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, sulfosalicylic acid, etc.

[0079] Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, ammonium salts and metals from columns I-XII of the periodic table. In certain embodiments, salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; particularly suitable salts include ammonium, potassium, sodium, calcium, and magnesium salts. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like. Particular organic amines include isopropylamine, benzathine, cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazine, and tromethamine.

[0080] In another aspect, the compounds of the present disclosure are provided in a sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, copper, isopropylamine, benzathine, cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazine, or tromethamine salt form.

[0081] In another aspect, the compounds of the present disclosure may be in the form of acetate, ascorbate, adipate, aspartate, benzoate, besylate, bromide / hydrobromide, bicarbonate / carbonate, bisulfate / sulfate, camphorsulfonate, caprate, chloride / hydrochloride, chlortheophyllonate, citrate, ethanedisulfonate, fumarate, gluceptate, gluconate, glucuronate, glutamate, glutarate, glycolate, hippurate, hydroiodide / iodide, isethionate, lactate, lactobionate, laminate, selenium ... and n-butyl uryl sulfate, malate, maleate, malonate, mandelate, mesylate, methylsulfate, mucate, naphthoate, napsylate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate, phosphate / hydrogenphosphate / dihydrogenphosphate, polygalacturonate, propionate, sebacate, stearate, succinate, sulfosalicylate, sulfate, tartrate, tosylate, triphenylacetate, trifluoroacetate or xinafoate salt forms.

[0082] Any formula given herein is intended to represent unlabeled form of the compound as well as isotopically labeled form.Isotopically labeled compounds have the structure shown by the formula given herein, except that one or more atoms are replaced by an atom having a selected atomic weight or atomic number.Isotopes that can be incorporated into the compounds described herein include, for example, isotopes of hydrogen.

[0083] In addition, certain isotopes, particularly deuterium (i.e. 2The incorporation of H or D) may allow for certain therapeutic advantages, such as increased in vivo half-life or reduced dosing requirements or improved therapeutic index or tolerability due to greater metabolic stability. In this context, it is understood that deuterium is considered a substituent of the compounds as described herein. The concentration of deuterium can be defined by the isotopic enrichment factor. The term "isotopic enrichment factor" as used herein means the ratio between the isotopic abundance and the natural abundance of a particular isotope. When a substituent in a compound described herein is designated as deuterium, such compound has an isotopic enrichment factor for each designated deuterium atom of at least 3500 (52.5% deuterium incorporation at each designated deuterium atom), at least 4000 (60% deuterium incorporation), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium incorporation), at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium incorporation), at least 6333.3 (95% deuterium incorporation), at least 6466.7 (97% deuterium incorporation), at least 6600 (99% deuterium incorporation), or at least 6633.3 (99.5% deuterium incorporation). It should be understood that the term "isotopic enrichment factor" can be applied to any isotope in the same manner as described for deuterium.

[0084] Other examples of isotopes that may be incorporated into the compounds described herein include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, and sulfur, e.g., 3 H, 11 C. 13 C. 14 C. 15 N, 18 F, 35 Thus, for example, radioisotopes, e.g. 3 H and 14 C, or non-radioactive isotopes, e.g. 2 H and 13 It should be understood that the present invention includes any of the compounds described herein that incorporate one or more of any of the above-mentioned isotopes, including those in which C, etc. are present. Such isotopically labeled compounds may be used in metabolic studies ( 14C), reaction kinetic studies (e.g. 2 H or 3 H), detection or imaging techniques such as positron emission tomography (PET) or single photon emission computed tomography (SPECT) including drug or substrate tissue distribution assays or radiation treatment of patients. 18 F or labeled compounds may be particularly desirable for PET or SPECT studies. The isotopically labeled compounds described herein can generally be prepared by conventional techniques known to those skilled in the art or by processes similar to those described in the accompanying examples and preparations, using the appropriate isotopically labeled reagents in place of previously used non-labeled reagents.

[0085] As used herein, the term "pharmaceutical composition" refers to a compound as described herein, or a pharma- ceutically acceptable salt thereof, together with at least one pharma- ceutically acceptable carrier in a form suitable for oral or parenteral administration.

[0086] As used herein, the term "pharmaceutically acceptable carrier" refers to a substance useful in the preparation or use of a pharmaceutical composition, and includes, as would be known to one of skill in the art, for example, suitable diluents, solvents, dispersion media, surfactants, antioxidants, preservatives, isotonicity agents, buffers, emulsifiers, absorption delaying agents, salts, drug stabilizers, binders, excipients, disintegrants, lubricants, wetting agents, sweeteners, flavoring agents, dyes, and combinations thereof (see, e.g., Remington The Science and Practice of Pharmacy, 22). nd (See Ed. Pharmaceutical Press, 2013, pp. 1049-1070).

[0087] The term "therapeutically effective amount" of a compound described herein refers to an amount of a compound that elicits a biological or medical response in a subject. As a series of non-limiting examples, such a therapeutically effective amount of a compound described herein can, for example, agonize GLP1R activity, ameliorate one or more symptoms, alleviate one or more conditions, slow or delay the progression of a disease, disorder or condition, or prevent a disease, disorder or condition.

[0088] As used herein, the term "therapeutically effective amount" refers to an amount of a compound described herein that, when administered to a subject, alleviates, prevents, and / or ameliorates a condition or disorder or disease that is responsive to increasing or agonizing the activity of GLP1R, at least in part. In another embodiment, the term "therapeutically effective amount" refers to an amount of a compound described herein that, when administered to a subject, cell or tissue; or non-cellular biological material; or culture medium, at least in part increases or agonizes the activity of GLP1R; or at least in part increases or agonizes the expression of GLP1R. In another embodiment, the term "therapeutically effective amount" refers to an amount of a compound described herein that, when administered to a subject, causes an observable level of one or more desired biological or medical responses selected from, for example, lowering glucose levels (e.g., lowering blood glucose levels), increasing insulin sensitivity, improving glucose homeostasis, lowering triglyceride or cholesterol levels, weight loss, reduced food intake, and body fat mass loss (e.g., peripheral and / or visceral fat).

[0089] As used herein, the terms "patient" or "subject" are used interchangeably and refer to primates (e.g., humans, male or female; or non-human primates), dogs, rabbits, guinea pigs, pigs, rats, and mice. In certain embodiments, the subject is a primate. In other embodiments, the subject is a human.

[0090] As used herein, the terms "agonize," "agonism," and "agonizing" refer to increasing GLP1R signaling, for example, as measured by an increase in intracellular cyclic adenosine monophosphate (cAMP).

[0091] As used herein, the terms "treat," "treating," or "treatment of" any disease, disorder, or condition refers to alleviating or ameliorating the disease, disorder, or condition (i.e., slowing or arresting the onset or progression of the disease, disorder, or condition, or at least one of its clinical symptoms); or alleviating or ameliorating at least one physiological parameter or biomarker associated with the disease, disorder, or condition, including those that may not be discernible to the patient.

[0092] As used herein, the terms "prevent", "preventing" or "prevention of" any disease, disorder or condition refers to the prophylactic treatment of the disease, disorder or condition; or the delay of the onset or progression of the disease, disorder or condition.

[0093] As used herein, a subject is "in need of" a treatment if such subject would benefit biologically, medically, or in quality of life from such treatment.

[0094] As used herein, the words "a," "an," "the," and similar words used (particularly in connection with the claims) are to be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0095] All of the methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any examples or representative language (e.g., "such as") provided herein is merely to better illustrate the compositions and methods or uses provided herein and does not pose a limitation on the scope otherwise claimed.

[0096] Any asymmetric atom (e.g., carbon, etc.) of the compounds described herein may be present in racemic or enantiomerically enriched, e.g., (R)-, (S)- or (R,S)-configuration. In certain embodiments, each asymmetric atom has at least 50% enantiomeric excess in the (R)- or (S)-configuration, at least 60% enantiomeric excess, at least 70% enantiomeric excess, at least 80% enantiomeric excess, at least 90% enantiomeric excess, at least 95% enantiomeric excess, or at least 99% enantiomeric excess.

[0097] Thus, as used herein, the compounds as described herein may be in the form of one of the possible stereoisomers, rotamers, atropisomers, tautomers, or mixtures thereof, for example, as substantially pure diastereomers, enantiomers (antipodes), racemates, or mixtures thereof.

[0098] Any resulting mixture of stereoisomers can be separated into pure or substantially pure geometric or optical isomers, diastereomers, racemates, on the basis of the physical chemical differences of the constituents, for example, by chromatography and / or fractional recrystallization.

[0099] Any resulting racemic forms of the compounds or intermediates described herein can be resolved into their optical antipodes by known methods, for example by separation of their diastereomeric salts obtained with optically active acids or bases and liberating the optically active acidic or basic compounds. Thus, in particular, basic moieties can be used to resolve the compounds described herein into their optical antipodes, for example by fractional recrystallization of salts formed with optically active acids, for example tartaric acid, dibenzoyltartaric acid, diacetyltartaric acid, di-O,O'-p-toluoyltartaric acid, mandelic acid, malic acid or camphor-10-sulfonic acid. The racemic compounds or racemic intermediates described herein can also be resolved by chiral chromatography, for example by high pressure liquid chromatography (HPLC) using a chiral adsorbent.

[0100] The compounds of the present application may be prepared by one of ordinary skill in the art of organic synthesis using commercially available starting materials, compounds known in the literature, or from readily prepared intermediates, by employing standard synthetic methods and procedures that are either known to those skilled in the art or will be apparent to the skilled chemist in light of the teachings herein.

[0101] The compounds described herein can be prepared by methods known in the art of organic synthesis, as illustrated in part by the following synthetic schemes. In the following schemes, it is well understood that protecting groups are used for sensitive or reactive groups as necessary, in accordance with general principles of chemistry. Protecting groups are manipulated according to standard methods of organic synthesis, for example, as described in Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons: New York, 1999 or Protecting Groups, 3rd edition, Thieme, Stuttgart, 2004. Protecting groups are removed at a convenient stage of the compound synthesis using methods that are readily apparent to those skilled in the art.

[0102] Those skilled in the art will recognize whether a stereocenter exists in the compounds disclosed herein. Resolution of the final product, intermediate, or starting material can be effected by any suitable method known in the art. See, for example, "Stereochemistry of Organic Compounds" by EL Eliel, SH Wilen, and LN Mander (Wiley-Interscience, 1994).

[0103] The compounds described herein can be made from commercially available starting materials or synthesized using known organic, inorganic and / or enzymatic processes.

[0104] Preparation of compounds The compounds described herein can be prepared by a number of methods well known to those skilled in the art of organic synthesis. By way of example, the compounds of the present disclosure can be synthesized using the methods described below, together with synthetic methods known in the art of synthetic organic chemistry, or variations thereon as appreciated by those skilled in the art.

[0105] General synthetic procedure The compounds described herein may be prepared as detailed in the Experimental Section (Chemistry Section), for example as follows.

[0106] General scheme (I): Synthesis of fatty acid moiety, compound of formula (i): A general method for preparing compounds of formula (i) is outlined in general scheme (I). [ka] Malonic acid derivative (60) can be reacted with R1-(CH2) in the presence of a base, such as sodium hydride, potassium or cesium carbonate, sodium hydroxide, lithium diisopropylamide, sodium bis(trimethylsilyl)amide, and in the presence or absence of a solvent, such as DMF, THF, or dimethylacetamide, at temperatures around, above, or below RT. m-X to give an alkylated intermediate (61), which can then be reacted with R2-(CH2) in the presence of a base. n -X to provide the di-alkylated intermediate (62). The variables R1, R2, n, and m have the meanings as defined herein, X is a leaving group selected from halogen (e.g., Br, Cl, I), trifluoromethanesulfonyloxy, and the like, and P1 and P2 are a carboxylic acid protecting group, such as, for example, methyl, ethyl, tert-butyl, methoxybenzyl, benzyl, trimethylsilyl, t-butyldimethylsilyl, or a 2-alkyl 1,3 oxazoline.

[0107] Depending on the protecting groups P1 and P2, intermediate (62) is then reacted with a base, such as, but not limited to, NaOH, KOH or LiOH, or an acid selected from, but not limited to, TFA, HCl or BCl3, or, when protecting groups P1 and P2 are benzyl or methoxybenzyl, intermediate (62) is typically reacted with hydrogen in the presence of a catalyst, such as, but not limited to, palladium on carbon, to provide compound (65), which corresponds to the compound of formula (i) (i.e., when P1 is hydrogen).

[0108] Alternatively, intermediate (61) can be converted to CH2=CH-(CH2) in the presence of a base such as NaH, potassium or cesium carbonate, sodium hydroxide, lithium diisopropylamide, and in the presence or absence of a solvent such as DMF, THF, or dimethylacetamide. j -X, where j is 1-10 and X is as defined herein, for example, allyl bromide, to give the unsaturated di-alkylated intermediate 63, which can be separated into its R or S enantiomers by chromatography. Intermediate 63 can then be reacted with an excess, for example, two equivalents, of an alkylating agent R2-(CH2) in the presence of a solvent such as DCM or THF. n’-CH=CH2 (where n' is 5-27) and in the presence of an olefin metathesis catalyst, such as Grubbs II, to give intermediate 64, which may be reacted with hydrogen in the presence of a catalyst, such as Pd / C, in the presence of a solvent, such as THF, methanol, etc., optionally followed by a deprotection reaction, for example if P2 provided is not a benzyl group, typically as disclosed in the reaction of intermediate 62 to intermediate 65; for example with NaOH, KOH or LiOH, in methanol, ethanol or dioxane, or with an acid selected from, but not limited to, TFA, HCl or BCl3. The double bond in the side chain may also be hydrogenated after the linker is connected to the fatty acid as shown in scheme (II). The parameters j and n' together with the CH=CH group are determined by the chain length determined by n in intermediate 65, i.e. (CH2) n is selected to provide

[0109] General scheme (II): Synthesis of fatty acid moiety containing linker (L): [ka] A general method for preparing intermediate 66 by using intermediate 65 is outlined in general scheme (II). In the presence of a coupling reagent such as carbonyldiimidazole (DCC), in the presence or absence of a base such as N,N-diisopropylethylamine or K2CO3, and in the presence or absence of a solvent such as DMF, fatty acid derivative 65 can be reacted with an amino acid derivative of formula H2N-L-COOP3 (wherein P3 is hydrogen or a carboxylic acid protecting group (e.g., methyl, ethyl, tert-butyl, methoxybenzyl, benzyl, trimethylsilyl, t-butyldimethylsilyl or 2-alkyl 1,3 oxazoline), and L is a linker as described herein, except that the linker L in the amino acid derivative of formula H2N-L-COOP3 is shown with its terminal group, i.e., NH2 and COOP3), to obtain derivatized fatty acid derivative (66).

[0110] Standard peptide coupling reactions include, for example, the conversion of a carboxylic acid group to its activated form, for example using standard N-hydrosuccinimide chemistry, or by reacting the carbonate group with reagents such as triphosgene, carbonyldiimidazole, 4-nitrophenyl chloroformate or disuccinimidyl carbonate, to the corresponding pyrrolidine-2,5-dione group, for example by using reagents such as thionyl chloride or oxalyl chloride, or by converting the carbonate group to the corresponding mixed anhydride using reagents such as ClC(O)O-isobutyl, 2,4,6-trichlorobenzoyl chloride or propylphosphonic anhydride cyclic trimer (T3P), followed by reaction of the oxazolidine-2,5-dione, acid halide or mixed anhydride in the presence or absence of a base, such as a tertiary amine (e.g. triethylamine or N,N-diisopropylethylamine) or an inorganic base, for example K2CO3. Alternatively, peptide coupling reaction reagents include dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC HCl), benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP) or benzotriazol-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP) in the presence or absence of reagents such as 1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole or dimethylaminopyridine.

[0111] General scheme (III): Synthesis of compounds of formula (I): [ka] A general method for preparing compounds of formula (I) by using intermediate 66 is outlined in general scheme (III). Provided P3 is a protecting group (e.g., not hydrogen), the fatty acid derivative of formula (66) can be converted to its carboxylic acid using, for example, an acid such as HCl or p-toluenesulfonic acid in the presence or absence of a solvent such as methanol, then converted to an activated carbonate such as an NHS-ester using, for example, DCC and N-hydroxysuccinimide (NHS) in the presence or absence of a solvent such as DCM or THF, which can then be converted to GLP-1 or GLP-1 analog P with a free -NH2 group in the presence of, for example, piperidine and a solvent such as DMF or DMA. * react with variable P * and P has the meaning as defined herein (as shown in general scheme (III)).

[0112] Depending on the nature of the separation, the mixtures of enantiomers, diastereomers and cis / trans isomers resulting from the above processes can be separated into their single components by chiral salt techniques, chromatography using normal phase, reverse phase or chiral columns.

[0113] The resulting racemates of any of the compounds or intermediates of the present disclosure can be resolved into their optical antipodes by known methods, for example by separation of their diastereomeric salts obtained with optically active acids or bases, liberating the optically active acidic or basic compounds. Thus, in particular, basic moieties can be used to resolve the compounds of the present disclosure into their optical antipodes by fractional recrystallization of salts formed with optically active acids, for example tartaric acid, dibenzoyltartaric acid, diacetyltartaric acid, di-O,O'-p-toluoyltartaric acid, mandelic acid, malic acid or camphor-10-sulfonic acid. The racemates or racemic intermediates of the present disclosure can also be resolved by chiral chromatography, for example by high pressure liquid chromatography (HPLC) using a chiral adsorbent.

[0114] Pharmaceutical Compositions The pharmaceutical compositions described herein comprise a compound as described herein or a pharma- ceutically acceptable salt thereof and a pharma- ceutically acceptable carrier. In further embodiments, the compositions comprise at least two pharma- ceutically acceptable carriers, such as those described herein. The pharmaceutical compositions may be formulated for a particular route of administration, such as oral administration, parenteral administration (e.g., by injection, infusion, transdermal or topical administration) and rectal administration. Topical administration may also be suitable for inhalation or intranasal application. The pharmaceutical compositions described herein may be made in solid form (including, but not limited to, capsules, tablets, pills, granules, powders or suppositories) or in liquid form (including, but not limited to, solutions, suspensions or emulsions). Tablets may be film-coated or enteropositive-coated according to methods known in the art. Generally, the pharmaceutical compositions are tablets or gelatin capsules that contain the active ingredient together with one or more of the following: a) diluents, such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine; b) lubricants, such as silica, talcum, stearic acid, its magnesium or calcium salts and / or polyethylene glycol; For tablets, c) binders, such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone; optionally d) disintegrants, such as starch, agar, alginic acid or its sodium salt or effervescent mixtures; and e) Absorbents, colouring agents, flavouring agents and sweeteners.

[0115] Pharmaceutical compositions suitable for injectable use generally include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.

[0116] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition should be sterile and fluid to the extent that easy puncture is present. Preferred pharmaceutical formulations must be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms, such as bacteria and fungi. In general, suitable carriers can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols, for example, mannitol, amino acids, sorbitol, sodium chloride in the composition. Sustained absorption of the injectable composition can be achieved by including an agent that delays absorption, such as aluminum monostearate and gelatin in the composition. In some embodiments, a multifunctional excipient such as recombinant albumin can be incorporated into the formulation process to promote the stability of the compound from degradation or aggregation, improve solubility, aid in administration, and release of the active ingredient (BioPharm International, 2012, Vol 23, Issue 3, pp 40-44).

[0117] Certain injectable compositions are aqueous isotonic solutions or suspensions, and suppositories are advantageously prepared from fatty emulsions or suspensions. The compositions may be stabilized and / or contain adjuvants such as preservatives, stabilizing agents, wetting agents or emulsifying agents, dissolution promoters, salts for regulating osmotic pressure and / or buffers. In addition, they may also contain other therapeutically valuable substances. The compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain 0.1-75% or 1-50% of the active ingredient.

[0118] Sterile injectable solution can be prepared by incorporating active compound in the required amount in suitable solvent with one or combination of the above-listed components as required, followed by filtration sterilization.Generally, dispersion is prepared by incorporating active compound into a sterile vehicle that contains a basic dispersion medium and other components required from the above-listed ones.In the case of sterile powder for preparing sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying, which obtains powder of active ingredient + any additional desired components from the solution that has been previously sterilized by filtration.

[0119] The compounds described herein, whether in free form or in pharma- ceutically acceptable salt form, exhibit valuable pharmacological properties, e.g., as agonists of GLP1R, as shown, e.g., in the in vitro and in vivo tests provided herein, and are therefore indicated for use as research chemicals, e.g., for therapy or as tool compounds.

[0120] Usefulness List The compounds described herein may be useful in treating metabolic and related diseases, disorders and conditions selected from, for example, obesity, type 2 diabetes, insulin resistance, hyperinsulinemia, impaired glucose tolerance, hyperglycemia, one or more diabetic complications (including, but not limited to, chronic kidney disease), diabetic nephropathy, dyslipidemia, metabolic syndrome, progressive liver disease, cardiovascular disease and neuropathy. As a non-limiting example, the neuropathy is peripheral neuropathy (which may be associated with, for example, diabetes).

[0121] The progressive liver disease can be, for example, non-alcoholic fatty liver disease (FLD or NAFLD) and, for example, non-alcoholic steatohepatitis (NASH).

[0122] The cardiovascular disease may be selected from hypertension, atherosclerosis, peripheral arterial disease, stroke, cardiomyopathy, atrial fibrillation, heart failure (e.g., heart failure with reduced ejection fraction (HFrEF), heart failure with mid-range ejection fraction (HFmrEF) and heart failure with preserved ejection fraction (HFpEF), coronary heart disease and arrhythmias (e.g., atrial arrhythmias and ventricular arrhythmias).

[0123] The compounds of the present invention may be useful in treating several diseases, disorders or conditions (called comorbidities) occurring simultaneously in a subject. A comorbidity may be, for example, in a subject that is type 2 diabetes, is obese, and / or exhibits heart failure and / or NASH. For example, an obese subject may also exhibit type 2 diabetes, and / or exhibits cardiovascular disease (e.g., heart failure). Such a subject may also exhibit progressive liver disease (e.g., NASH). For example, an obese subject may also exhibit type 2 diabetes, and / or exhibits cardiovascular disease (e.g., heart failure), and / or exhibits progressive liver disease (e.g., NASH). The subject may also have high blood pressure and / or high blood cholesterol levels. The subject may also suffer from peripheral neuropathy.

[0124] As used herein, the instructions disclosed in the Utility section above may hereinafter be referred to as the "list above."

[0125] In one embodiment, the disease, disorder or condition is selected from obesity, type 2 diabetes, atherosclerosis, heart failure (particularly heart failure with preserved ejection fraction) and NASH.

[0126] In one embodiment, the disease, disorder or condition is selected from obesity, type 2 diabetes, atherosclerosis and heart failure (particularly heart failure with preserved ejection fraction).

[0127] Another aspect of the present disclosure relates to a method for treating, preventing, inhibiting or eliminating a disease or disorder in a patient associated with an alteration in GLP1R, comprising administering to a patient in need of treatment for a disease or disorder associated with an alteration in GLP1R an effective amount of a compound as described herein or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound as described herein or a pharma- ceutically acceptable salt thereof and one or more pharma- ceutically acceptable carriers.

[0128] Thus, as a further aspect, there is provided herein the use of a compound as described herein or a pharma- ceutically acceptable salt thereof in therapy. In a further embodiment, the therapy is the treatment of a disease, disorder or condition that can be treated by agonism of GLP1R. In another embodiment, the therapy is the treatment of a disease, disorder or condition selected from any of the above lists.

[0129] Thus, as a further aspect, there is provided herein a compound as described herein, or a pharma- ceutically acceptable salt thereof, for use in therapy. In a further embodiment, the therapy is treatment of a disease, disorder or condition that can be treated by agonism of GLP1R. In another embodiment, the therapy is treatment of a disease, disorder or condition selected from any of the above lists.

[0130] In another aspect, provided herein is a method for treating a disease, disorder, or condition in a patient, which is treatable by agonism of GLP1R, comprising administering a therapeutically effective amount of a compound described herein, or a pharma- ceutically acceptable salt thereof.

[0131] In another embodiment, provided herein is a method of treating a disease, disorder, or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound described herein, wherein the disease, disorder, or condition is selected from any of the lists above.

[0132] In a further aspect, provided herein is the use of a compound as described herein or a pharma- ceutically acceptable salt thereof for the manufacture of a medicament. In a further embodiment, the medicament is for the treatment of a disease that can be treated by agonism of GLP1R. In another embodiment, the disease is selected from any of the above lists.

[0133] Furthermore, the present invention provides the use of any compound described herein, or a pharma- ceutically acceptable salt thereof, for the treatment of a disease, disorder or condition selected from any of the above lists.

[0134] The term "metabolic disorder" or "metabolic disease" refers to a set of related traits including, but not limited to, obesity, impaired glucose tolerance, insulin resistance, hyperinsulinemia, excess visceral adiposity, hypertension, dyslipidemia, characterized by high triglycerides, low high density lipoprotein (HDL) cholesterol, and high low density lipoprotein (LDL) cholesterol. Subjects with metabolic diseases or disorders are at risk for developing type 2 diabetes and, for example, atherosclerosis.

[0135] The term "obesity" in human adults refers to a body mass index (BMI) of 30 or greater (Centers for Disease Control and Prevention). Such subjects may also be referred to as obese. This is referred to as Class I obesity. Class II obesity includes individuals with a BMI between 35 and 39.9, and Class III obesity refers to individuals with a BMI greater than 40. Body Mass Index (BMI) is a measure of body fat based on height and weight. The formula for calculation is BMI = weight in kilograms / height in meters. 2 In one embodiment, the obese human subject has a BMI of ≧30 or ≧35, or a BMI range of ≧35 to <40, or ≧30 to <40. An amount <40 can be, for example, 39.9. In some embodiments, the obesity is severe obesity or morbid obesity, where the human subject has a BMI of ≧40.

[0136] The term "type 2 diabetes" refers to a condition characterized by persistently high glucose levels in both fasting and fed states that results from a combination of abnormal glucose utilization and excessive glucose production, which can result either from inadequate insulin production from the pancreas or from peripheral insulin resistance.

[0137] The term "insulin resistance," as used herein, refers to a state in which abnormal amounts of insulin fail to induce the expected physiological response and fail to activate downstream pathways. In many instances, insulin above the physiological range, either endogenously produced or exogenously administered, is sufficient to induce a complete or partial biological response to induce the expected physiological response.

[0138] The term "hyperinsulinemia" refers to a condition in which excess insulin can be detected in the blood.

[0139] The term "impaired glucose tolerance" encompasses any disorder characterized by a clinical symptom or combination of clinical symptoms associated with elevated basal or postprandial glucose levels and / or elevated insulin levels or abnormal glucose-stimulated insulin release or HOMA-IR (homeostatic model assessment of insulin resistance) in a subject compared to a healthy individual. Elevated glucose and / or insulin levels may be manifested in the following diseases, disorders and conditions: obesity, metabolic syndrome, impaired glucose tolerance, type II diabetes, gestational diabetes, type I diabetes, insulin resistance, hyperinsulinemia, lipodystrophy, lipodystrophy and various MODY (maturity-onset diabetes of the young) mutations. The GLP1R agonists and compositions thereof of the present disclosure may be used, for example, to lower glucose levels in the bloodstream and / or lower insulin levels to the range found in healthy subjects, for example, to achieve and / or maintain glucose homeostasis.

[0140] The term "hyperglycemia," as used herein, refers to a condition in which an increased amount of glucose circulates in the plasma of a subject compared to a healthy individual. Hyperglycemia can be diagnosed using methods known in the art, including measuring fasting blood glucose levels as described herein.

[0141] The term "diabetic complications" refers to problems caused by persistently high blood sugar levels that damage other organs, including the kidneys, peripheral limbs, and eyes (e.g., retinopathy), or induce vascular disease and neuropathy. Vascular dysfunction can contribute to erectile dysfunction and lead to increased risk of skin infections. Diabetes also increases the risk for heart disease and bone and joint disorders. Other long-term complications of diabetes include excess risk of cancer, including hepatocellular carcinoma, endometrial cancer, breast cancer, and pancreatic cancer.

[0142] The term "diabetic nephropathy" refers to a condition resulting from diabetes, caused by damage to the blood vessels and other cells of the kidney that reduces kidney function.

[0143] The term "dyslipidemia" refers to a complex disorder of lipoprotein metabolism involving lipoprotein overproduction or abnormal metabolism. Dyslipidemia can be manifested by elevated levels of total cholesterol, low-density lipoprotein (LDL) cholesterol, and triglycerides and reduced levels of high-density lipoprotein (HDL) cholesterol in the blood.

[0144] The term "metabolic syndrome" refers to a set of risk factors that increase the risk for cardiovascular disease, including coronary artery disease, heart failure with reduced ejection fraction, heart failure with preserved ejection fraction, cerebrovascular disease, and peripheral vascular disease. These risk factors include abdominal fat, high blood sugar after fasting (at least 110 milligrams per deciliter (mg / dl)); high triglycerides in the bloodstream (at least 150 mg / dL); low HDL (less than 40 mg / dl); and blood pressure above 130 / 85 mmHg (World Health Organization).

[0145] The term "progressive liver disease" refers to the progression from a benign state of fatty liver manifested by fibrosis and cirrhosis predisposing to hepatocellular carcinoma. The progression of obesity-associated nonalcoholic fatty liver (NAFL) to NASH, fibrosis and cirrhosis is well documented.

[0146] The term "non-alcoholic fatty liver disease (FLD)", also known as NAFLD, is a condition in which excess lipids accumulate in hepatocytes, which can result from either excessive de novo lipid biosynthesis in the liver or abnormal clearance and oxidation of fatty acids. NAFLD is excluded from other causes of liver disease, including alcoholic liver disease and viral liver disease. NAFLD includes three histological entities that reflect the progression of the disease: fatty liver, hepatic steatosis, and fibrosis or cirrhosis. The most common cause of NAFLD is obesity, but NAFLD can also be seen in lean individuals. Fat accumulation can progress to inflammation accompanied by changes in hepatocyte histology, including infiltration and swelling of macrophages, called steatohepatitis, which is called non-alcoholic steatohepatitis (NASH). NASH can progress to fibrosis with interlobular bridging fibrosis or cirrhosis. As used herein, the term NASH can encompass steatohepatitis, ballooning hepatocytes, and lobular inflammation.

[0147] The term "cardiovascular disease" refers to diseases related to the heart or blood vessels.

[0148] The term "atherosclerosis" refers to a vascular disease characterized by irregularly distributed lipid deposits in the intima of large and medium-sized arteries, which cause narrowing of the arterial lumen and may eventually progress to fibrosis and calcification. The lesions are usually localized and progress slowly and intermittently. Blood flow restriction is the primary cause of most clinical symptoms and varies with the distribution and severity of the lesions.

[0149] The term "peripheral artery disease" refers to when the buildup of fatty deposits in the arteries restricts the blood supply to the lower leg muscles.

[0150] The term "stroke" refers to when the blood supply to part of the brain is cut off.

[0151] The term "cardiomyopathy" is defined as an acquired or congenital structural abnormality of the atrial or ventricular myocardium that can affect cardiac function or physiology and status.

[0152] The term "heart failure" refers to a condition in which the heart's blood pumping function is impaired, and can include heart failure with preserved ejection fraction (HFpEF), heart failure with reduced ejection fraction (HFrEF), and heart failure with intermediate ejection fraction (HFmrEF).

[0153] The term "coronary heart disease", also known as coronary artery disease, is the narrowing of the arteries that supply blood to the heart.

[0154] The term "arrhythmia" refers to abnormal heart rhythms and can include atrial arrhythmias, atrial fibrillation, and ventricular arrhythmias.

[0155] The term "neuropathy" refers to when nerves are damaged. This term includes peripheral neuropathy, which occurs when nerves in the extremities, such as the hands, feet, and arms, are damaged. Diabetes is a common cause of peripheral neuropathy.

[0156] Another embodiment relates to a compound comprising a compound of formula (I) for use in the treatment of a disease or disorder selected from obesity, type 2 diabetes, insulin resistance, hyperinsulinemia, impaired glucose tolerance, hyperglycemia, diabetic complications (including but not limited to chronic kidney disease), diabetic nephropathy, dyslipidemia, metabolic syndrome, progressive liver disease, cardiovascular disease, and neuropathy (particularly peripheral neuropathy, e.g., associated with diabetes mellitus). [ka] (Compound 1) or a pharma- ceutically acceptable salt thereof.

[0157] Another embodiment relates to a compound comprising a compound of formula (I) for use in the treatment of a disease or disorder selected from obesity, type 2 diabetes, insulin resistance, hyperinsulinemia, impaired glucose tolerance, hyperglycemia, diabetic complications (including but not limited to chronic kidney disease), diabetic nephropathy, dyslipidemia, metabolic syndrome, progressive liver disease, cardiovascular disease, and neuropathy (particularly peripheral neuropathy, e.g., associated with diabetes mellitus). [ka] (Compound 2) or a pharma- ceutically acceptable salt thereof.

[0158] Another embodiment relates to a compound comprising a compound of formula (I) for use in the treatment of a disease or disorder selected from obesity, type 2 diabetes, insulin resistance, hyperinsulinemia, impaired glucose tolerance, hyperglycemia, diabetic complications (including but not limited to chronic kidney disease), diabetic nephropathy, dyslipidemia, metabolic syndrome, progressive liver disease, cardiovascular disease, and neuropathy (particularly peripheral neuropathy, e.g., associated with diabetes mellitus). [ka] (Compound 3) or a pharma- ceutically acceptable salt thereof.

[0159] Another embodiment relates to a compound comprising a medicament for use in the treatment of a disease or disorder selected from hypertension, atherosclerosis, peripheral artery disease, stroke, cardiomyopathy, atrial fibrillation, heart failure (e.g., heart failure with reduced ejection fraction (HFrEF), heart failure with mid-range ejection fraction (HFmrEF) and heart failure with preserved ejection fraction (HFpEF), coronary heart disease and arrhythmias (e.g., atrial arrhythmias and ventricular arrhythmias), [ka] (Compound 1) or a pharma- ceutically acceptable salt thereof.

[0160] Another embodiment relates to a compound comprising a medicament for use in the treatment of a disease or disorder selected from hypertension, atherosclerosis, peripheral artery disease, stroke, cardiomyopathy, atrial fibrillation, heart failure (e.g., heart failure with reduced ejection fraction (HFrEF), heart failure with mid-range ejection fraction (HFmrEF) and heart failure with preserved ejection fraction (HFpEF), coronary heart disease and arrhythmias (e.g., atrial arrhythmias and ventricular arrhythmias), [ka] (Compound 2) or a pharma- ceutically acceptable salt thereof.

[0161] Another embodiment relates to a compound comprising a medicament for use in the treatment of a disease or disorder selected from hypertension, atherosclerosis, peripheral artery disease, stroke, cardiomyopathy, atrial fibrillation, heart failure (e.g., heart failure with reduced ejection fraction (HFrEF), heart failure with mid-range ejection fraction (HFmrEF) and heart failure with preserved ejection fraction (HFpEF), coronary heart disease and arrhythmias (e.g., atrial arrhythmias and ventricular arrhythmias), [ka] (Compound 3) or a pharma- ceutically acceptable salt thereof.

[0162] Dosage form The pharmaceutical compositions or combinations as described herein may be in unit dosage form of about 1-100 mg of active ingredient for a subject of about 50-70 kg. The therapeutically effective dose of the compound, pharmaceutical composition or combination thereof depends on the species, weight, age and individual condition of the subject, the disorder or disease being treated or its severity.

[0163] Combination Mode Any of the compounds described herein may be administered either simultaneously with, prior to, or subsequent to one or more other therapeutic agents. Any of the compounds described herein may be administered separately by the same or different routes of administration or together in the same pharmaceutical composition with other agents. The therapeutic agent may be, for example, a chemical compound, peptide, peptide conjugates and fusions, antibody, antibody fragment, or nucleic acid, which is therapeutically active or enhances therapeutic activity when administered to a subject in combination with a compound described herein.

[0164] Accordingly, in another aspect, provided herein are combinations, particularly pharmaceutical combinations, comprising a compound described herein (e.g., a therapeutically effective amount) or a pharma- ceutically acceptable salt thereof and one or more other therapeutically active agents.

[0165] In one embodiment, provided herein is a combination comprising a compound as described herein and at least one other therapeutic agent as a combined preparation for simultaneous, separate or sequential use in therapy, in one embodiment, the therapy is the treatment of a disease, disorder or condition selected from the list above.

[0166] Products provided as combined preparations include compositions that contain a compound described herein and one or more additional therapeutic agents together in the same pharmaceutical composition, or a compound described herein and the other therapeutic agent(s) in separate form, for example, in the form of a kit.

[0167] In one embodiment, provided herein is a pharmaceutical combination comprising a compound as described herein and one or more additional therapeutic agents. Optionally, the pharmaceutical combination may comprise a pharma- ceutically acceptable carrier as described above.

[0168] In one embodiment, a kit is provided herein that includes two or more separate pharmaceutical compositions, at least one of which contains a compound described herein. In one embodiment, the kit includes a means for separately holding the compositions, such as containers, separate bottles, or separate foil packets. An example of such a kit is a blister pack, such as those commonly used for packaging tablets, capsules, and the like.

[0169] The kits can be used to administer different dosage forms, e.g., oral and parenteral, to administer the separate compositions at different dosage intervals, or to titrate the separate compositions relative to each other. To aid in compliance, the kits generally include directions for administration.

[0170] In the combination therapy described herein, any compound described herein and the other therapeutic agent may be manufactured and / or formulated by the same or different manufacturers.

[0171] Additionally, any compound described herein and other therapeutic agents may be included together in a combination therapy: (i) prior to release of the combination product to the physician (e.g., in the case of a kit containing a compound described herein and other therapeutic agents); (ii) by the physician (or under the physician's guidance) immediately prior to administration; or (iii) in the patient himself / herself, e.g., during sequential administration of a compound described herein and other therapeutic agents.

[0172] Also provided herein is a combination comprising a compound as described herein and one or more additional therapeutic agents for use in a method of treating a disease, disorder, or condition selected from any of the foregoing lists.

[0173] Also provided herein is the use of a combination comprising a compound as described herein and one or more additional therapeutic agents for treating a disease, disorder or condition selected from any of the above lists.

[0174] In one embodiment, the other therapeutic agent may be selected from the following: 1. Antidiabetic drugs, such as insulin, insulin derivatives and mimetics; insulin secretagogues, such as sulfonylureas (e.g. chlorpropamide); or DPPIV (dipeptidyl peptidase IV) inhibitors, such as vildagliptin; 2. Lipid-lowering drugs, such as 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors, such as lovastatin; squalene synthase inhibitors; FXR (farnesoid X receptor) and LXR (liver X receptor) ligands; bile acid sequestrants, such as cholestyramine and colesevelam; fibrates; nicotinic acid or aspirin; 3. Anti-obesity agents, such as Orlistat; 4. Antihypertensives, for example loop diuretics, for example ethacrynic acid; angiotensin converting enzyme (ACE) inhibitors, for example benazepril; inhibitors of the Na-K-ATPase membrane pump, for example digoxin; neutral endopeptidase (NEP) inhibitors; ACE / NEP inhibitors, for example omapatrilat; angiotensin II antagonists, for example valsartan; angiotensin receptor-neprilysin inhibitors (ARNi), for example sacubitril / valsartan (LCZ696); renin inhibitors, for example ditekiren; β-adrenergic receptor blockers, for example timolol; inotropes, for example digoxin; calcium channel blockers, for example amlodipine; aldosterone receptor antagonists; or aldosterone synthase inhibitors; 5. Peroxisome proliferator-activated receptor agonists, such as fenofibrate; 6. Compounds that bind to the corticotropin releasing hormone receptor, such as urocortin 2. EXAMPLES

[0175] The present disclosure is further illustrated by the following examples and synthetic schemes, which should not be construed as limiting the disclosure in scope or spirit to the specific procedures described herein. It should be understood that the examples are provided to illustrate specific embodiments and that no limitation to the scope of the disclosure is intended thereby. It should be further understood that various other embodiments, modifications and equivalents thereof that may themselves suggest themselves to those skilled in the art may be sought without departing from the spirit of the present disclosure and / or the scope of the appended claims.

[0176] The compounds of the present disclosure can be prepared by methods known in the art of organic synthesis. It is understood that in all of the methods, protective groups for sensitive or reactive groups can be used where necessary in accordance with general principles of chemistry. Protective groups are manipulated according to standard methods of organic synthesis (TW Green and PG M Huts (1999) Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons). These groups are removed at a convenient stage of the compound synthesis using methods that are readily apparent to those skilled in the art.

[0177] Experimental Section Analytical methods, materials and instruments Reagents and solvents were used as received from commercial suppliers unless otherwise stated. Proton nuclear magnetic resonance (NMR) spectra were obtained on either a Bruker Avance spectrometer or a Varian Oxford 400 MHz spectrometer unless otherwise stated. Spectra are given in ppm (δ) and coupling constants, J, are reported in Hertz. Tetramethylsilane (TMS) was used as the internal standard. Chemical shifts are reported in ppm relative to dimethylsulfoxide (δ 2.50), methanol (δ 3.31), chloroform (δ 7.26) or other solvents, if indicated in the NMR spectral data. A small amount of dried sample (2-5 mg) is dissolved in an appropriate deuterated solvent (1 mL). Chemical names were generated using ChemBioDraw Ultra v17from CambridgeSoft.

[0178] Abbreviation: AC 50 Half-maximal compound effect concentration ACN Acetonitrile A inf Plateau value of Hill curve at high concentration Plateau value of Hill curve at low A0 concentration Aib α-aminoisobutyric acid ALS Autosampler AUC inf Area under the plasma concentration-time curve from time 0 to infinity br Broad BSA Bovine Serum Albumin BW Body Weight cAMP cyclic adenosine monophosphate cat # Catalog number CHO Chinese hamster ovary cells C max maximum plasma concentration CO2 Carbon Dioxide cynoGLP1R Cynomolgus monkey glucagon-like peptide 1 receptor d double line dd Double line double line DCC N,N'-dicyclohexylcarbodiimide DCM Dichloromethane DCU N,N'-dicyclohexylurea DEA N,N-Diethylaniline DERET Dissociation Enhanced Resonance Energy Transfer DIEA / DIPEA Diethylisopropylamine DIO Diet-Induced Obesity DMEM Dulbecco's Modified Eagle's Medium DMF N,N-Dimethylformamide DMSO Dimethyl sulfoxide DSC N,N'-Disuccinimidyl Carbonate DMA Dimethylacetamide DMAP 4-(N,N-dimethylamino)pyridine EA Enzyme Acceptor EC Effective Concentration Effective concentration of a compound that does not give an EC0 response EC 50 Effective concentration of compound giving half-maximal response EC 100 Effective concentration of compound that gives maximum (100%) response E max Efficacy: The maximum response achievable from an administered drug. EDC or EDCI N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide EDTA Ethylenediaminetetraacetic acid Ex9-39 Exendin 9-39 ELSD Evaporative Light Scattering Detector equiv equivalent ESI Electrospray Ionization EtOAc Ethyl acetate FBS Fetal Bovine Serum FI Food Intake Fmoc 9-Fluorenylmethoxycarbonyl FRET Fluorescence Resonance Energy Transfer g grams GLP1 Glucagon-like peptide 1 GLP1R Glucagon-like peptide 1 receptor GPCR G-protein coupled receptor G418 Geneticin, a selective antibiotic Grubbs II Dichloro[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene](benzylidene)(tricyclohexylphosphine)ruthenium(II) h time HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate HDF high fat diet HESI Heated Electrospray Ionization hGLP1R Human glucagon-like peptide 1 receptor HPLC High Pressure Liquid Chromatography HTRF Homogeneous time-resolved fluorescence measurement IBMX 3-isobutyl-1-methylxanthine kg Kilogram L Liter LCMS Liquid Chromatography and Mass Spectrometry MeOH Methanol MS mass spectrometry MTBE Methyl tert-butyl ether m multiplet mg milligram min mL Milliliters mmol mM millimolar concentration m / z mass-to-charge ratio nM nanomolar nmol nanomol NMP N-Methyl-2-pyrrolidinone NMR nuclear magnetic resonance NPLC Normal Phase Liquid Chromatography p quintet Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl PBS Phosphate Buffered Saline Pd / C Palladium Carbon PEG Polyethylene glycol PK Pharmacokinetics PD Pharmacodynamics ppm parts per million QC quality control QD Once a day Q3D Once every 3 days Q1W Once a week RCF Relative Centrifugal Force RPM revolutions per minute R t retention time RT room temperature rotovap rotary evaporator s Single line sc or SC subcutaneous sec second SEM Standard error of the mean SFC Supercritical Fluid Chromatography SM starting material t triple line TEA Triethylamine TFA Trifluoroacetic acid THF Tetrahydrofuran T max Time taken to reach maximum plasma concentration T 1 / 2 Half-life v / v volume / volume μg microgram μL microliter μM micromolar

[0179] Biological Assays and Data The compounds described herein were tested in the following cellular assay, which measures intracellular cAMP concentrations. cAMP is generated by activation of the GLP1R. The data obtained are shown in Tables 1-3. EC 50 E is defined as the concentration of compound that results in half the maximal response (after baseline correction). max is defined as the maximal response observed to a test compound normalized to the maximal response observed to the endogenous ligand for GLP1R (GLP1(7-36)).

[0180] Human GLP1R cAMP agonist assay The agonist activity of the compounds was determined using the GloSensor™ cAMP assay (Promega Corp.), which measures the change in intracellular concentration of cAMP following ligand activation of a GPCR. This assay uses a biosensor encoded by the pGloSensor™-22F cAMP plasmid (Promega, cat #E2301) with the cAMP binding domain fused to a mutant form of Photinus pyralis luciferase. Binding to cAMP causes a conformational change that promotes a large increase in light output, which can be measured by a luminescence detector. HEK293-SNAP-hGLP1R-GloSensors cells stably overexpressing the human GLP1 receptor (hGLP1R) and pGloSensor™-22F were seeded in CO2-independent medium (Gibco cat #18045-088 with 1.0% FBS, 2 mM L-glutamine, penicillin and streptomycin) into white 384-well poly-D-lysine coated plates (Greiner Bio One, cat #781945) and incubated overnight at 37°C, 5% CO2, humidified. The next morning, the assay was started by adding an equal volume of CO2-independent medium containing a 4% v / v dilution of GloSensor substrate (Promega, cat #E1291) to all wells. The cell plates were incubated in the dark at RT for 2 h. A Biomek i7 (Beckman Coulter) instrument was used for the liquid handling steps. To generate duplicate dose-response curves, compounds were added to the cell assay plate in a final volume of 60 μL, serially diluted 3-fold to final concentrations ranging from 100 nM to 0.03 pM in CO2-independent medium containing 0.1% BSA, 0.5 mM IBMX, and 0.4% DMSO. EC10 containing GLP1(7-36) peptide (Bachem, cat #H-6795) at a final concentration of 2 nM. 100Control wells and peptide-free EC0 control wells were tested simultaneously in the same plate using the same assay buffer as the test compound. After compound was added to the cells, the plate was incubated at RT in the dark for 12 min. Luminescence was then measured using an Envision 2104 multilabel reader (PerkinElmer) with a "TRF Light Unit, 337 nm" using the Ultra-Sensitive protocol setting "384-well US Luminescence Detector" with a 384-well emission aperture at 0.1 sec / well. GLP1(7-36)EC 100 cAMP activity was calculated as a percentage of control wells: [(sample signal-mean EC0 signal) / (mean EC 100 -Average EC0 signal) * 100. In the Helios module of the software package DAVID, EC 50 Curve fitting was performed to determine the variability. A 4-parameter logistic model with Hill slope was used: y = A inf +(A0-A inf ) / (1+(x / AC 50 ) Hill Slope ), where y is the functional response; x is the compound concentration; A0 is the minimum value (at 0 dose); A inf is the maximum value (at infinite dose); AC 50 corresponds to the bending point (i.e. A0 and A inf (The point at the halfway point of the sigmoid curve between EC 50 Values ​​are AC values ​​calculated from Helios in μM. 50 The value is expressed as E max is the maximum activity detected within the concentration range derived from the fitted curve.

[0181] Generation of HEK293-SNAP-hGLP1R cell line 327 μL of Opti-MEM medium (Gibco, cat #31985-062) was mixed with 12 μL of FuGENE® HD (Promega, cat #E2311) and incubated at RT for 5 min. Then, 8.2 μL (4 μg, 0.485 μg / μL solution) of pSNAP-hGLP1R plasmid (Cisbio, cat #PSNAP-GLP1) encoding human GLP1R fused with a SNAP tag (NCBI reference sequence: NM_002062.3) was added to the Fugene HD / Opti-MEM mix and incubated at RT for 20 min. A suspension of HEK293 cells (ATCC® CRL-1573™) was prepared at 800,000 cells / mL. Then, the plasmid / FuGene HD mixture was added to 8 mL of cells and mixed gently. 2 mL of the new mixture was added to 4 wells in a 6-well plate, and 2 mL of non-transfected cells were added to 2 wells as a control. The plate was incubated at 37°C until 100% confluent. Antibiotic selection [800 μg / mL G418 (Geneticin, Gibco, cat #10131-035)] was performed after cell trypsinization at a dilution of 2500 cells / mL. 1 mL of the cell suspension was added to 20 mL of selection medium in a 10 cm culture dish (total of 2500 cells), and in parallel, 4 mL of the diluted cell suspension was added to 20 mL of selection medium in a 10 cm culture dish (total of 10000 cells). The rest of the cells were cultured in a T150 flask. Additionally, HEK293 cells were cultured in selection medium in a T75 flask as a negative control. Finally, single clones were picked from the 10 cm culture dish and cultured until there were enough cells for gene expression analysis and HTRF cAMP assay. Clone 2 showed the greatest GLP1R-dependent cAMP response and was expanded for generation of the GloSensor stable cell line.

[0182] Generation of HEK293-SNAP-hGLP1R-GloSensor stable cell lines HEK293 cells (above) stably overexpressing SNAP-hGLP1R were seeded at a density of 3 million cells in a 10 cm dish containing 17 mL of DMEM complete growth medium (Gibco, cat #11965-092) + 10% fetal bovine serum (FBS, Gibco, cat #16140-071). The next day, the cells were transfected as follows: DNA complex was prepared as 0.020 μg / μL pGloSensor™-22F cAMP plasmid (Promega, cat #E2301; GenBank® accession number is GU174434) by adding 37 μg of plasmid DNA in 1758 μL Opti-MEM solution. Then, 112 μL of FuGENE® HD reagent was added to it by mixing carefully. After 5-10 min incubation at RT, 850 μL of complex / well was added to the cells and mixed thoroughly. After 24 h incubation at humidified 37°C, 5% CO2, the medium was removed and the cells were rinsed with PBS. Selection medium [600 μg / mL G418 and 600 μg / mL hygromycin B (Gibco, cat #10687010)] was then added. The medium was changed twice a week until no dead cells were observed. Once cell clones were visible, single cells were isolated by pipetting up and down after the addition of 10 μL of 0.05% trypsin-EDTA solution. These single cell-derived clones were then cultured in 6-well plates in selection medium (600 μg / mL G418 + 600 μg / mL hygromycin B) until enough cells were available to test for cAMP agonist response in the GloSensor luminescence assay described herein. HEK293-SNAP-hGLP1R stable cell clones that produced the desired responses to the human GLP1R cAMP agonist assay were used. These data are indicative of the relative potency of the compounds tested.

[0183] [Table 1]

[0184] Cynomolgus GLP1R cAMP agonist assay The agonist activity of the described compounds was further tested using the HTRF cAMP assay (CisBio, cat #62AM4PEC), which measures the change in intracellular concentration of cAMP following ligand activation of a GPCR. 3+ It is based on a competitive format involving a specific anti-cAMP monoclonal antibody labeled with cryptate (donor fluorophore) and cAMP (acceptor fluorophore) coupled to d2. This allows the direct characterization of compounds acting on G protein-coupled receptors in cells. Native cAMP produced by cells is detected by the anti-cAMP antibody-Eu 3+It competes with d2-labeled cAMP for binding to cryptate. HEK293-cynoGLP1R F6 cells stably overexpressing the cynomolgus monkey GLP1 receptor (cynoGLP1R) were seeded in white 384-well poly-D-lysine coated plates (Greiner Bio One, cat #781945) at 5000 cells / well in DMEP complete medium (Gibco, cat #11965-092, 10% heat inactivated FBS, 0.5mg / ml geneticin; Gibco Life Technologies, cat #10131027) and incubated overnight at 37°C, 5% CO2 in a humidified atmosphere. The assay was performed the next day. Peptides were diluted in stimulation buffer [1X HBSS (Life Technologies, cat #14065-056), 20 mM HEPES (Life Technologies, cat #15630), 0.1% BSA (Sigma, cat #A0281) and 0.5 mM IBMX]. To generate triplicate dose-response curves, 3-fold serially diluted compounds (2x concentration) were diluted in DMSO. Cells were washed with an ELx405 Select, BioTek plate washer leaving 10 μL / well of assay buffer [1X HBSS (Life Technologies, cat #14065-056), 20 mM HEPES (Life Technologies, cat #15630)]. Plates were centrifuged briefly and 10 μL of 2x diluted peptides were added per well. Plates were centrifuged briefly again and incubated at RT for 30 min. 20x d2 and Eu in lysis buffer provided with the kit were added. 3+ Cryptates were diluted. After 30 min incubation with peptide, 10 μL of diluted d2 was added per well, followed by 10 μL of diluted Eu 3+ Cryptates were added. The plates were covered with black lids and incubated for 1 h at RT, followed by a brief centrifugation. The HTRF signal was then measured in an Envision 2104 multilabel reader (Perkin Elmer) with fluorescence emission set at two different wavelengths (665 nm and 620 nm). EC 50Curve fitting was performed to determine the four standard parameters: std_crv_ac50 (standard curve AC 50 ), std_crv_a0(standard curve A0), std_crv_ainf(standard curve A inf A four-parameter logistic curve fit was performed on the section of the plate where the standard curve compounds were placed to obtain y = std_crv_ac50 (standard curve Hill slope), std_crv_hill (standard curve Hill slope). These four parameter values ​​were then used to fit the equation: y = std_crv_ac50 * Standard curve transformation was applied to each well using [(X-std_crv_a0) / (std_crv_ainf-X)]^(1 / std_crv_hill). y is the functional response; x is the compound concentration. EC values ​​for this assay in μM for all test compounds are 50 AC equivalent to value 50 A four-parameter logistic curve fitting was performed on the transformed data to obtain E max is GLP1(7-36)EC 100 : [(Sample A max -Sample A0) / (GLP1(7-36)A max -GLP1(7-36)A0)] * Expressed as a percentage of 100.

[0185] Generation of HEK293-cynoGLP1R stable cell lines The day before transfection, 1 × 10 cells were cultured in 8 mL of DMEM complete growth medium + 10% FBS in a T25 flask. 6HEK293 cells were seeded at a density of 1000 x 1000 cells. The next day, the cells were transfected as follows: DNA complex was prepared by adding 8.8 μg of pcDNA3.1(+)Neo plasmid encoding cyno GLP1R cDNA [codon optimized, GeneArt (Thermo Fisher Scientific); NCBI reference sequence: NP_001274592] in 414 μL of OptiMEM solution at 0.020 μg / μL. Then, 26 μL of FuGENE® HD reagent was added to it by mixing carefully. After 5-10 min incubation at RT, 400 μL complex / well was added to the cells and mixed thoroughly. After 48 h incubation at 37 °C and 5% CO2 with humidification, the cells were transferred to 15 cm dishes in the presence of 0.5 mg / mL geneticin. The HEK293T-cynoGLP1R stable cell clone that showed the most activity in the functional cAMP assay (clone F6) was selected for further analysis in the cyno GLP1R cAMP cellular agonist assay.

[0186] These data indicate the relative potency of the test compounds.

[0187] [Table 2]

[0188] Mouse GLP1R cAMP agonist assay Compounds were tested for cAMP agonist activity using a procedure similar to the Cynomolgus GLP1R cAMP assay (see above), except that HEK293-mGLP1R CRE-Luc (clone C3) cells stably overexpressing the mouse GLP1 receptor (mGLP1R) were used (construction below).

[0189] Generation of HEK293-mGLP1R CRE-Luc stable cell lines 3 x 10 cells in 17 mL of DMEM complete growth medium + 10% FBS in a 10 cm dish 6HEK293T CRE-Luc cells were seeded at a density of 1000 x 1000 cells. The next day, the cells were transfected as follows: DNA complex was prepared by adding 37 μg of plasmid DNA encoding mouse GLP1R cDNA (GeneCopoeia, cat #EX-Mm23901-M67; NCBI reference sequence: NM_021332.2) in 1758 μL Opti-MEM solution at 0.020 μg / μL. Then, 112 μL of FuGENE® HD Reagent was added to it by mixing carefully. After 5-10 min incubation at RT, 850 μL of DNA complex / well was added to the cells and mixed thoroughly. After 24 h incubation at 37 °C, 5% CO2 with humidification, the medium was removed; the cells were rinsed with PBS and separated. Selection medium [2 μg / mL puromycin (Corning, cat #61-385-RA) and 100 μg / mL hygromycin (Gibco, cat #10687010)] was then added. The medium was changed three times a week until no dead cells were observed. When cell clones were visible, single cells were isolated. For mouse GLP1R cAMP cellular agonist assays, the HEK293T-mGLP1R-CRE-Luc stable cell clone (clone C3) that showed the highest gene expression was used.

[0190] These data indicate the relative potency of the test compounds.

[0191] [Table 3]

[0192] Human GLP1R β-arrestin recruitment assay The PathHunter® β-arrestin assay (DiscoverX) was used to measure the extent to which agonists recruited β-arrestin. This assay uses an enzyme complementation approach to measure the binding of β-arrestin to the receptor. Two inactive portions of the β-galactosidase enzyme (called Prolink and enzyme acceptor or "EA") are tagged such that human GLP1R (hGLP1R) contains the Prolink portion and β-arrestin contains the EA portion. When β-arrestin is recruited to the receptor, the enzyme becomes active and produces light in the presence of a chemiluminescent substrate (PathHunter® Detection Kit, DiscoverX cat #93-0001). Luminescence can be measured on an appropriate detector. CHO-hGLP1R-β-arrestin cells stably overexpressing Prolink-tagged hGLP1R and EA-tagged β-arrestin were plated in white 384-well poly-D-lysine coated plates (Greiner Bio One, cat #781945) at 20 μL / well in plating reagent 2 (DiscoverX, cat #93-0563R2A) and incubated overnight at 37°C with humidified 5% CO2. The following day, agonists were prepared at 5x the final required concentration. To generate triplet dose-response curves, compounds were serially diluted 3-fold in assay buffer (HBSS, 10 mM Hepes and 0.1% BSA) and then added to the cell assay plate for a final volume of 25 μL and final maximum concentrations starting at or below 3 μM, depending on the compound. EC100 containing GLP1(7-36) peptide (Bachem, cat #H-6795) at a final concentration of 1 μM was incubated at 37°C for 1 h. 100Control wells and compound-free EC0 control wells were tested simultaneously on the same plate using the same assay buffer as the test compounds. After compound was added to the cells, the plate was incubated at 37°C, humidified with 5% CO2 for 2h. Detection reagent was then prepared (19 parts cell assay buffer, 5 parts substrate reagent 1 and 1 part substrate reagent 2, DiscoverX cat #93-0001, as recommended by the manufacturer) and 12μL per well was added to the cell assay plate. The plate was incubated for an additional hour in the dark at RT. Luminescence was then measured with an Envision 2104 multilabel reader with a "TRF Light Unit, 337nm" (Perkin Elmer) using the Ultra-Sensitive protocol setting "384-well US Luminescence Detector" with 384-well emission aperture at 0.1s / well. β-arrestin recruitment was calculated using Microsoft Excel, and GLP1(7-36)EC 100 Expressed as a percentage of control wells: [(sample signal-mean EC0 signal) / (mean EC0 of GLP1(7-36) signal 100 -Average EC0 signal)] * 100. Using GraphPad Prism to analyze EC 50 Curve fitting was performed to determine the EC ratio. A 4-parameter logistic model with Hill slope was used: Y=Bottom+(Top-Bottom) / (1+10^((Log EC 50~ X) * where Y is the functional response; X is the compound concentration; bottom is A0 or the minimum value (at 0 dose); top is A inf or maximum value (at infinite dose);EC 50 is the inflection point (i.e., A0 and A1 in the sigmoid curve). inf (The point halfway between EC 50 Values ​​were calculated in μM. max is the maximum activity detected within the concentration range derived from the fitted curve for GLP1(7-36).

[0193] Generation of CHO-hGLP1R-β-arrestin cell line 2 × 10 in 22 mL complete medium (AssayComplete Cell Culture kit107, DiscoverX, cat #92-3107G) 6 Cell / T75cm 2 PathHunter® CHO-K1-EA parental cells (DiscoverX, cat #93-0164) were seeded at flask density. The next day, a medium change was performed with 22 mL of fresh medium without antibiotics and the cells were transfected as follows: Plasmid / Fugene® HD Transfection mix was prepared in Opti-MEM medium (3:1 reagent:DNA ratio). 25 μg (34 μL) of pCMV-PK1-GLP1R plasmid [(DiscoverX pCMV PK vector bundle, cat #93-0491, with inserted sequence encoding full-length human GLP1R-NCBI reference sequence: NM_002062, synthesized by GeneArt (Thermo Fisher Scientific)] was added to 1129 μL of Opti-MEM to a total volume of 1163 μL. Then, 74 μL of FuGENE® HD Reagent was added by mixing carefully. After incubation at RT for 5-10 min, 1125 μL of the complex solution was added to the cells and incubated at 37 °C for 48 h. Subsequently, the medium was removed and 300 μg / mL hygromycin (Gibco, cat #10687010) and 500 μg / mL geneticin (Gibco, cat #10687010) were added to the cells. Selection medium containing β-antibiotics (#10131035) was added. Medium was changed every 2-3 days until no dead cells were observed. Cells were detached, resuspended at 300000 cells / mL, and strained through a 40 μm strainer. Cells were then FACS sorted into single cells using an Aria G instrument in black clear bottom poly-D-lysine coated 96 well plates in 100 μL medium. Medium was changed every 2-3 days by removing up to 80 μL and adding fresh medium containing selection antibiotics. Surviving single clones were expanded and tested. Based on the optimal signal and curve profile, single clone 1 was selected for β-arrestin assay.

[0194] [Table 4]

[0195] Data evaluated in the β-arrestin assay may inversely correlate with the gastrointestinal tolerability (reduction of nausea / vomiting) of the compounds described herein, i.e., compounds with lower activity in the β-arrestin assay may be better tolerated. [e.g., Jones et.al. Nat. Commun. 2018,9,1602]

[0196] Human GLP1R DERET internalization and recycling assay The extent to which agonists internalize or allow recycling of human GLP1R was determined based on an optimized version of a RealTime FRET-based "DERET" (Dissociation Enhanced Resonance Energy Transfer) assay. This technique relies on labeling SNAP-tagged GPCRs with SNAP-Lumi-Terbium (donor fluorophore, Cisbio, cat #SSNPTBD). Compounds are incubated with cells overexpressing the GPCR of interest in the presence of excess fluorescein (acceptor fluorophore). When the GPCR is on the cell surface, the donor signal is inactivated by the acceptor, and the donor / acceptor ratio is low. If the GPCR is internalized, the donor signal is no longer inactivated and the acceptor is no longer excited, so the donor / acceptor ratio increases. Addition of excess antagonist prevents further receptor internalization and allows the receptor to recycle back to the membrane, leading to a subsequent decrease in the donor / acceptor ratio.

[0197] HEK293-SNAP-hGLP1R-GloSensor cells (stably overexpressing SNAP-tagged hGLP1R) were seeded overnight in regular DMEM growth medium (Gibco, cat #11965-092, 10% heat-inactivated FBS, 10 mM HEPES, 1x penicillin / streptomycin, 0.5 mg / mL geneticin (Gibco, cat #10131-035) and 0.25 mg / mL hygromycin B (Invitrogen, cat #10687010) in white 384-well poly-D-lysine coated plates (Greiner Bio One, cat #781945). On the day of assay, cell medium was removed and 100 nM SNAP-Lumi-Tb reagent was added in Opti-MEM solution. Cells were incubated at 37°C for 1 h. Assay buffer [1X HBSS (10X Gibco, cat #10131-035) and 0.25 mg / mL hygromycin B (Invitrogen, cat #10687010)] was added. Cells were washed using a plate washer in 20 mM Hepes (Gibco, cat #14065-056), 20 mM Hepes (Gibco, cat #15630-080), 1 mM CaCl2 (Fluka, cat #21114-1L), 1 mM MgCl2 (Ambion, cat #AM9530G) pH 7.4 and 20 μL buffer with 0.1% BSA was added to each well. After allowing the cells to equilibrate for approximately 15 min at 37°C, 10 μl of fluorescein (sodium salt, Sigma, cat #F6377, diluted in buffer) was added to a final concentration of 25 μM. To generate triplicate dose-response curves, compounds were serially diluted 3-fold in assay buffer and then added to the cell assay plate in a final volume of 40 μL and final maximum concentrations (in some cases) starting at 3 μM or lower. EC 100A control curve of GLP1(7-36) peptide (Bachem, cat #H-6795) at a final maximum concentration of 1 μM in the same plate and assay buffer as the test compounds was included to establish the EC0 well with buffer only. Plate FRET fluorescence was measured immediately and then every 30 min using a Perkin Elmer Envision with a LANCE / DELFIA D400 single mirror, excitation filter X320 and emission filters M615_203 (donor emission) and M515 (acceptor emission). Peak internalization was reached at 120 min, at which point 10 μM (final) exendin 9-39 (Bachem, cat #H8740, a GLP1R antagonist) was added to all wells to further block agonist binding. Measurements were continued for a further 180 min to establish how well the receptor was recycled back to the membrane. Plates were kept at 37° C. between readings. Data were expressed as the ratio of donor / acceptor emission using Microsoft Excel and plotted in GraphPad Prism. EC 50 and E max To determine the GLP1(7-36)EC 100 Expressed as a percentage of control wells: [(sample signal-mean EC0 signal) / (mean EC0 of GLP1(7-36) signal 100 -Average EC0 signal)] * 100. Using GraphPad Prism to analyze EC 50 Curve fitting was performed to determine the 4-parameter logistic model with Hill slope: Y=Bottom+(Top-Bottom) / (1+10^((Log EC 50 -X) * Hill slope) where Y is the functional response; X is the compound concentration; bottom is A0 or the minimum value (at 0 dose); top is A inf or maximum value (at infinite dose);EC 50is the inflection point (i.e., A0 and A1 of the sigmoid curve) inf (The point halfway between EC 50 Values ​​were calculated in μM. max is the maximum activity detected within the concentration range derived from the fitted curve for GLP1(7-36). To determine the receptor recycling parameters, the relative E max was calculated and curve-fitted over time using a 4-parameter sigmoidal fit. Using this model, we can calculate the T 1 / 2 We also determined the maximum percentage of receptors recycled as a percentage of the amount initially internalized.

[0198] [Table 5]

[0199] [Table 6]

[0200] The slower the internalization rate and the faster the receptor recycling rate, the more receptors are retained in the membrane. Table 5 shows the receptor internalization data, and Table 6 shows the receptor recycling data. In the latter, T 1 / 2 Lower values ​​indicate a more rapid return of the receptors to the cell surface and a greater number of receptors available for interaction with the compound [see, e.g., Jones et.al. Nat. Commun. 2018, 9, 1602]. This may inversely correlate with the tolerability of the described compound, i.e., the lower the potency, the better the tolerability.

[0201] Pharmacokinetics experiments Mouse PK: For all in vivo experiments, the compounds disclosed herein were prepared in stock solutions in PBS to a concentration of 1 mg / mL of the compounds. These stock solutions were then diluted with saline to obtain the concentrations disclosed in the experiments.

[0202] For all in vivo experiments, semaglutide was purchased in a clinical formulation called OZEMPIC, which is a stock solution of semaglutide at 1.34 mg / mL. The stock solution contains the inactive substances disodium phosphate dihydrate, 1.42 mg; propylene glycol, 14.0 mg; phenol, 5.50 mg; and water for injection. The pH of OZEMPIC is approximately 7.4. This stock solution was then diluted with saline to obtain the concentrations disclosed herein.

[0203] For all compounds: To obtain pharmacokinetic parameters, compounds were administered subcutaneously (sc) at 0.24 mg / kg in saline to three C57BL / 6 mice (20-30 weeks old) fed a high fat diet (60% calories from fat) from 6 weeks of age using a dosing volume of 5 mL / kg, where the concentration of compound was 48 μg / mL. After compound administration, blood samples were collected from tail snips into EDTA-coated tubes at 0.5, 1, 3 and 6 h after dosing on day 0 and then at 24, 48, 72, 96, 168, 240, 336 and 408 h after dosing (i.e., days 1, 2, 3, 4, 7, 10, 14 and 17). Plasma fractions were obtained by centrifugation (13,000 rpm, 4 °C, 5 min); 30 μL aliquots of mouse plasma were transferred to 96-well plates for bioanalysis. Calibration standards and QC samples were prepared in blank mouse plasma (plasma from untreated mice). PK samples were diluted 2-fold in blank mouse plasma (10 μL sample + 10 μL blank mouse plasma) and extracted using a protein precipitation procedure involving the addition of 150 μL methanol containing the internal standard. Samples were vortexed and centrifuged at 4000 rpm for 15 minutes at 4° C. A 125 μL aliquot of the supernatant was transferred to a 96-well plate and 100 μL of water was added to each well and vortexed. Samples were analyzed and quantified by LC-MS / MS using the conditions outlined below.

[0204] LC / MS / MS method Mass spectrometer:Thermo QExactive HFX Liquid chromatograph: Thermo Vanquish Autosampler (ALS): Thermo Vanquish

[0205] HPLC conditions LC column: Waters Acquity UPLC Protein BEH C 4, 50 x 2.1 mm, 1.7 μm Solvent A: 100:0.1 (v:v) water:formic acid Solvent B: 100:0.1 (v:v) acetonitrile:formic acid Injection volume: 10 μL Column oven temperature: 40℃ ALS temperature: 4℃.

[0206] [Table 7]

[0207] MS conditions Ion source: HESI Polarity: Positive Auxiliary gas heater temperature: 380℃ Sheath gas flow rate: 60 Aux Gas Flow Rate: 14 Sweep gas flow rate: 3 Ion spray voltage: 3500V Capillary temperature: 320℃

[0208] [Table 8]

[0209] Cynomolgus Monkey PK: To obtain pharmacokinetic parameters, obese male cynomolgus monkeys were given a single sc dose of compound at a prescribed concentration of 30, 60 or 90 μg / mL in saline using a dose volume of 0.5 mL / kg. Monkeys were dosed in the morning before food but were not fasted. Animals were bled via the saphenous vein at defined intervals (pre-dose, 0.25, 0.5, 1, 3, 7, 24, 48, 96, 168, 240, 336 and 504 h post-dose). Blood was collected in vacutainer tubes containing K2EDTA and kept on ice until centrifugation. Plasma fractions were obtained by centrifugation at 1000-2000 RCF (typically 1300 RCF) for 10 min at 4°C. 50 μL aliquots of monkey plasma were transferred to 96-well plates for bioanalysis. Calibration standards and QC samples were prepared in blank obese cynomolgus monkey plasma (plasma from untreated obese monkeys). PK samples were diluted 2-fold with blank obese monkey plasma (10 μL sample + 10 μL blank obese monkey plasma) and extracted using a protein precipitation procedure involving the addition of 150 μL methanol containing an internal standard. Samples were vortexed and centrifuged at 4000 rpm for 15 minutes at 4° C. A 125 μL aliquot of the supernatant was transferred to a 96-well plate and 100 μL of water was added to each well and vortexed. Samples were analyzed and quantified by LC-MS / MS using the conditions outlined.

[0210] [Table 9]

[0211] The data evaluated in Tables 8 and 9 provide evidence for the superior in vivo stability of the compound against metabolic degradation compared to semaglutide.

[0212] Efficacy Studies: Acute Feeding Studies: Diet-induced obese (DIO) male mice (C57BL / 6 mice fed a high-fat diet (60% calories from fat) from 6 weeks of age) were assessed for food intake (FI) following a single SC subcutaneous (sc) administration of each test compound (e.g., Compound 1) at a formulated concentration of 24, 38, or 48 μg / mL (5 mL / kg dose volume) in saline. Males aged 24-30 weeks were used in this study. Animals were housed one per cage in a room with a normal photoperiod (lights on 6:00 am-6:00 pm, lights off at other times) under an approved IACUC protocol. The average food intake (FI) (24 h food intake measured at 3-day intervals before the start of the study) was used as a baseline. At the start of the study, food weights were recorded and test compounds were administered subcutaneously to the animals. 24 h after test compound administration, food intake weights were measured. Food intake (FI) was assessed 24 hours later in obese mice after subcutaneous administration of a single dose of compound at the indicated dosage; the data obtained are shown in Table 10. As a comparison, the effect of semaglutide was evaluated.

[0213] [Table 10]

[0214] Efficacy Testing Efficacy (food intake and weight loss) following compound (e.g., Compound 1) treatment was evaluated in diet-induced obese (DIO) male mice (C57BL / 6 mice fed a high fat diet (60% calories from fat) from 6 weeks of age). Males aged 24-30 weeks were used in this study (n=7 / group). Animals were housed one per cage in a room with a normal light cycle under an approved IACUC protocol. Mice were assigned to vehicle (saline) or treatment groups based on the average of body weight (BW) and food intake (FI) (24 h food intake measured over 3 days prior to the start of the study). At the start of the study, body weight and food weight were recorded and animals were subcutaneously dosed with vehicle or compound (12, 24, 29.6, 38 or 48 μg / ml in saline using a dosing volume of 5 ml / kg). Compounds or vehicle were given QD or, where indicated, Q3D (every 3 days). Semaglutide was administered QD. Body weight and food intake were measured daily. Compound 1 and semaglutide doses were selected based on (i) maximal efficacy assessed in separate and serial studies with published data (semaglutide) and (ii) equimolar concentrations. Doses of stereoisomers (compounds 2 and 3) were selected based on maximal efficacy of compound 1 assessed in separate studies.

[0215] Weight loss in obese mice after 18, 24 and 30 days of subcutaneous compound administration is shown in Table 11. Depending on the dose, test compounds and vehicle were administered QD or Q3D; semaglutide was administered QD; all compounds were dissolved in saline.

[0216] [Table 11]

[0217] Cynomolgus Monkey Efficacy Study: The pharmacokinetic (PK) / pharmacodynamic (PD) relationship of a novel long-acting GLP1R agonist (Compound 1) was examined in obese cynomolgus monkeys by evaluating its effects on BW and FI. Efficacy was defined as a reduction in FI and BW. Tolerability was evaluated as a reduction in and / or absence of vomiting, and retention of interest in the choice of fruits and vegetables and peanuts as rewards.

[0218] Monkeys were acclimated to the test diet (5TUR diet, 1 g pellet (TestDiet Cat #1815639-310)) for at least one week. After the acclimation period, food intake was recorded for one week prior to the first day of dosing to establish baseline food intake (FI). Prior to the first day of dosing, baseline body weight was calculated as the mean of two independent measurements obtained within a seven day period.

[0219] Ten male obese cynomolgus monkeys were given subcutaneous (sc) injections of either vehicle (n=4) or compound (n=6) (0.03 mg / kg, hence 0.06 mg / mL concentration, using a dose volume of 0.5 mL / kg). Test compounds were dissolved in saline at the indicated concentrations. Monkeys were dosed in the morning before food but were not fasted. Food intake was measured daily throughout the study. Monkeys were given pre-weighed amounts of food, 200 g / day; half of their allotment was given in the morning and the rest in the afternoon. Remaining food was weighed the following morning to determine daily FC. Body weight (BW) was measured twice weekly.

[0220] [Table 12]

[0221] [Table 13]

[0222] Compound 1 suppresses food intake and reduces body weight in obese monkeys (see Tables 12 and 13). All data are expressed as mean±SEM with n=7 / group.

[0223] Tolerability assessment: Surprisingly, the compounds described herein were found to be much better tolerated when administered to obese monkeys (assessed against semaglutide as a control drug). None of the monkeys showed any signs of vomiting after administration of compounds 1, 2, 5 or 9, whereas compound 3 caused 1 / 6 monkeys to vomit, and all monkeys administered semaglutide vomited (see Table 14).

[0224] [Table 14]

[0225] Chemistry Section A: Analysis section LCMS Methodology:

[0226] [Table 15]

[0227] [Table 16]

[0228] [Table 17]

[0229] [Table 18]

[0230] [Table 19]

[0231] [Table 20]

[0232] [Table 21]

[0233] [Table 22]

[0234] [Table 23]

[0235] [Table 24]

[0236] [Table 25]

[0237] B: Synthesis section Intermediate 1: Benzyl 11-bromoundecanoate [ka] To a mixture of 11-bromoundecanoic acid (4.60 kg, 17.3 mol, 1.1 equiv) in DCM (26.5 kg) at 0° C. was added EDCI (3.8 kg, 20.2 mol, 1.28 equiv) in portions along with DMAP (98 g, 0.8 mol, 0.05 equiv). Benzyl alcohol (1.70 kg, 15.7 mol, 1.00 equiv) was then added dropwise. After stirring at 20° C. for 4 h, water (70.0 kg) was added dropwise. The reaction mixture was then concentrated under vacuum. Heptane (23.2 kg) and 19% NaCl solution (17 kg) were added and the phases were separated. The organic phase was washed with 5% Na2CO3, 25.0 kg; 19% NaCl solution, 25.0 kg (x2), 5.2% aqueous HCl, 25.0 kg; 19% NaCl solution, 25.0 kg, water (5.0 kg) and brine (5.0 kg). The organic phase was then concentrated under vacuum at 50° C. to give intermediate 1, which was used as such for the next step. 1 H NMR(400MHz,chloroform-d)δ ppm 1.18-1.36(m,10H)1.37-1.47(m,2H)1.64(quin,J=7.33Hz,2H)1.85(dt,J=14.56,7. 06Hz,2H)2.35(t,J=7.58Hz,2H)3.40(t,J=6.88Hz,2H)5.11(s,2H)7.28-7.45(m,5H).

[0238] Intermediate 2: 1,11-Dibenzyl 11-(tert-butyl)docosane-1,11,11-tricarboxylate [ka] To a solution of benzyl tert-butyl malonate (3.0 kg, 12.0 mol, 1.0 equiv) in NMP (30 L) was added 1-iodoundecane (3.55 kg, 12.58 mol, 1.05 equiv) and Cs2CO3 (11.76 kg, 36.09 mol, 3.0 equiv) at 20°C. The resulting mixture was stirred at 20°C for 6 h, and then intermediate 1 (5.53 kg, 15.6 mol, 1.3 equiv) was added. The reaction mixture was heated to 85°C and stirred for 12 h. Then, the mixture was cooled to 20°C, and a mixture of water (30 kg) and heptane (10 kg) was added. After stirring for 30 min, the organic phase was separated and washed three times with a mixture of brine (5 kg) and MeOH (4 kg). The organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. Further purification was carried out by column chromatography: eluting with heptane / EtOAc=1 / 0 to 100 / 1 to give intermediate 2. 1 H NMR(400MHz,chloroform-d)δ ppm 0.84-0.94(m,3H)1.12(m,J=6.60Hz,4H)1.19-1.33(m,28H)1.35(s,9H)1.66(quin,J=7.40Hz ,2H)1.85(t,J=8.44Hz,4H)2.37(t,J=7.52Hz,2H)5.14(s,2H)5.16(s,2H)7.30-7.42(m,10H).

[0239] Intermediate 3: 13-(benzyloxy)-2-((benzyloxy)carbonyl)-13-oxo-2-undecyltridecanoic acid [ka] To a solution of intermediate 2 (6.0 kg, 8.8 mol, 1.0 equiv) in heptane (21 L) was added TFA (10.0 kg, 88.4 mol, 10.0 equiv) dropwise at 20±5° C. After stirring at 20±5° C. for 8 h, most of the TFA was removed under reduced pressure and the resulting residue was redissolved in heptane (42 L, 7 V) and washed with brine (42 L x 3). After phase separation, the organic phase was concentrated to give the crude product as a yellow oil. The crude product was purified by column chromatography: elution from heptane to heptane:EtOAc=10 / 1 gave intermediate 3. 1 H NMR(400MHz,chloroform-d)δ ppm 0.87-0.94(m,3H)0.94-1.05(m,2H)1.19(br.s.,14H)1.23-1.37(m,16H)1.65(quin,J=7.40Hz,2H)1 .78-1.91(m,2H)1.93-2.05(m,2H)2.37(t,J=7.52Hz,2H)5.14(s,2H)5.27(s,2H)7.31-7.44(m,10H).

[0240] The pure enantiomers of racemic intermediate 3 were separated via chiral SFC to give enantiopure intermediates 3A and 3B, which were used to prepare compounds 2 and 3, respectively. The parameters to obtain enantiopure intermediates 3A and 3B were as follows: Equipment: Thar 350 preparative SFC(SFC-18) Column: ChiralPak AD, 300×50mm ID, 10μm Mobile phase: A for CO2 and B for ethanol Gradient: B40% Flow rate: 200mL / min Back pressure: 100bar Column temperature: 38℃ Wavelength: 210nm Cycle time: approx. 3.7 min Peak 1: R enantiomer (3A) Peak 2: S enantiomer (3B) [ka] The absolute configuration of enantiomer 3A was determined by its derivative (shown below): enantiomer 3A was reacted in a first step with oxalyl chloride in DMF, followed by reaction of the resulting acid chloride with (S)-1-(4-nitrophenyl)-ethan-1-amine, which was then treated with hydrogen in the presence of Pd / C to give the structure shown below, from which single X-ray crystals were obtained. Thus, following the determination that the absolute configuration of enantiomer 3A is R, peak 2 of the enantiomeric mixture separated by chiral SFC was associated with the S-configuration and assigned to enantiomer 3B. [ka] Derivative of (R)-enantiomer 3A - conformation determined via X-ray (R)-2-(((S)-1-(4-ammoniophenyl)ethyl)carbamoyl)-2-(10-carboxydecyl)tridecanoate

[0241] Intermediate 4: 14-((benzyloxy)carbonyl)-3,15-dioxo-1-phenyl-14-undecyl-2,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76,79,82,85,88-pentacosaoxa-16-azahennonacontan-91-onic acid [ka] To a flask was added intermediate 3 (640 g, 1.03 mol), DCM (8.3 kg) and DMF (3 g). The resulting mixture was stirred at 25° C., then oxalyl chloride (170 g, 1.34 mol) was added dropwise. Stirring was continued for another 2-3 h. Concentration of the reaction mixture and solvent exchange with heptane gave a crude mixture to which 8.5 kg of DCM was added to form a solution which was used directly in the next step.

[0242] To a flask was added 1-amino-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tetracosaoxapentaheptacontan-75-oic acid (amine-PEG24-oic acid, 900 g, 0.79 mol), DCM (6.0 kg) and DIPEA (203 g) and the resulting mixture was stirred at 25 °C. Then, the acyl chloride crude solution from step 1 was added dropwise. The reaction mixture was stirred for an additional 1-2 h. Acidic resin (1.3 kg) was added and stirring was continued for 30 min. Then, the mixture was filtered. MgSO4 (1.3 kg) was added and stirring was continued for 30 min. The mixture was filtered and concentrated to provide a crude residue. The crude residue was purified over Al2O3 using a mobile phase containing MTBE, DCM, MeOH, then all the desired fractions were collected and concentrated to give intermediate 4. 1 H NMR(400MHz,chloroform-d)δ ppm 0.86-0.93(m,3H)0.93-1.04(m,2H)1.19(br.s.,15H)1.23-1.37(m,15H)1.61-1.68( m,2H)1.78(td,J=12.44,4.34Hz,2H)1.92-2.05(m,2H)2.37(t,J=7.58Hz,2H)2.62(t, J=6.05Hz,2H)3.49(dd,J=6.72,2.32Hz,2H)3.52-3.59(m,2H)3.59-3.73(m,92H)3.8 0(t,J=6.05Hz,2H)5.13(s,2H)5.18(s,2H)7.31-7.42(m,10H)8.09(t,J=5.26Hz,1H).

[0243] Intermediate 5: 77,87-Dibenzyl 1-(2,5-dioxopyrrolidin-1-yl) 76-oxo-77-undecyl-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tetracosaoxa-75-azaheptaoctacontane-1,77,87-tricarboxylate [ka] To a solution of intermediate 4 (920 g, 0.53 mol) in DCM (6.1 kg) was added TEA (11 g) and the mixture was stirred to give a clear solution. Then DSC (161 g, 0.63 mol) was added and stirring was continued for 2 h at 25° C. Acidic resin (180 g) was added and the mixture was stirred for 30 min. MgSO4 (180 g) was added and stirring was continued for 30 min. Then the mixture was filtered to give a clear pale yellow solution. Concentration under vacuum gave crude intermediate 5, which was used directly in the next step. LCMS Method A: Rt=1.5 min, [M+H3O+H] +2 =933.9.

[0244] Intermediate 6: 2-((75-((2,5-dioxopyrrolidin-1-yl)oxy)-75-oxo-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tetracosaoxapentaheptacontyl)carbamoyl)-2-undecyltridecanedioic acid [ka] A hydrogenation reactor was charged with intermediate 5 (986 g, 0.48 mol, 90% purity), THF (7.6 kg), and 10% Pd / C (110 g), followed by MgSO4 (110 g), and the resulting mixture was purged with N2, then with H2, and stirred at 25 °C for 3-24 h. After complete consumption of the starting material, additional MgSO4 (220 g) was added and stirring was continued for another 30 min. The reaction mixture was filtered. The cake was washed with 100 mL of THF, and the filtrates were combined and concentrated to give intermediate 6. 1H NMR(400MHz,chloroform-d)δ ppm 0.84-0.94(m,3H)1.17(br.s.,2H)1.21-1.39(m,30H)1.57-1.68(m,2H)1.69-1.80(m,2H)1.97-2.10(m,2H)2.34(t ,J=7.21Hz,2H)2.86(s,4H)2.92(t,J=6.48Hz,2H)3.51-3.73(m,96H)3.87(t,J=6.48Hz,2H)7.45(t,J=4.46Hz,1H).

[0245] Intermediate 7: 1-Benzyl 3-(tert-butyl) 2-undecylmalonate [ka] Benzyl tert-butyl malonate (110 g, 439 mmol, 1 equiv.) was placed in DMF (800 mL). To the resulting mixture were added 1-iodoundecane (130 g, 461 mmol, 1.05 equiv.) and K2CO3 (151 g, 1.10 mol, 2.5 equiv.). The resulting suspension was stirred at 50 °C for 12 h. The reaction mixture was then diluted with ethyl acetate (500 mL) and then poured into ice water. The combined organic phase was washed twice with brine (150 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure to give the crude residue. The crude residue was purified by column chromatography (SiO2, eluted with petroleum ether / ethyl acetate = 1 / 0 to 0 / 1) to give intermediate 7 as a colorless oil. 1 H NMR(400MHz,DMSO-d6)δ ppm 7.46-7.26(m,5H),5.27-4.98(m,2H),3.44-3.26(m,1H),1.72(br d,J=6.8Hz,2H),1.33(s,9H),1.28-1.10(m,18H),0.93-0.75(m,3H).

[0246] Intermediate 8: 1-Benzyl 3-(tert-butyl) 2-allyl-2-undecyl malonate [ka] NaH (14.5 g, 364 mmol, 60%, 1.2 equiv) was added dropwise to DMF (1230 mL) at 0 °C, followed by slow addition of intermediate 7 (123 g, 304 mmol, 1 equiv) in DMF (123 mL). The resulting mixture was stirred at 0 °C for 0.5 h, then allyl bromide (40.4 g, 334 mmol, 29 mL, 1.1 equiv) was added dropwise. The reaction mixture was stirred at 20 °C for 9.5 h, then diluted with ethyl acetate (2500 mL). The mixture was poured into ice-cold saturated NH4Cl4 (1200 mL). The organic phase was separated from the aqueous phase, and the aqueous phase was washed twice with ethyl acetate. The organic phases were then combined, washed three times with brine (500 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, eluted with petroleum ether / ethyl acetate = 1 / 0 to 98 / 2) to give Intermediate 8 as a yellow oil. 1 H NMR(400MHz,DMSO-d6)δ ppm 7.50-7.26(m,5H),5.70-5.50(m,1H),5.24-5.01(m,4H),4.99-4.74(m,1H),1.70(br s,2H),1.45-1.34(m,3H),1.27(s,9H),1.25-1.15(m,18H),0.85(br t,J=6.8Hz,3H).

[0247] Intermediates 8A and 8B: 1-benzyl 3-(tert-butyl) 2-allyl-2-undecyl malonate [ka] Intermediate 8 (159 g) was separated using SFC (column: Chiralpak IG-3 100 * Purification by LCMS Method B: Rt=1.334 min, MS(ESI) m / z[M+Na].+ =467.3. SFC: intermediate 8A (peak 1), pure enantiomer (R); SFC: intermediate 8B (peak 2), pure enantiomer (S).

[0248] Intermediate 9: Benzyldec-9-enoate [ka] To a solution of dec-9-enoic acid (70.0 g, 411 mmol, 76.0 mL, 1 equiv) in DCM (1400 mL) was added BnOH (66.6 g, 616 mmol, 64.1 mL, 1.5 equiv), DMAP (5.02 g, 41.12 mmol, 0.1 equiv), EDCI (94.5 g, 493.3 mmol, 1.2 equiv) and DIEA (63.7 g, 493 mmol, 85.9 mL, 1.2 equiv). The resulting mixture was stirred at 25 °C for 12 h. The reaction mixture was then diluted with DCM (500 mL). The resulting solution was washed twice with brine (500 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a crude residue. The crude residue was purified by column chromatography (SiO2, eluted with petroleum ether / ethyl acetate = 1 / 0 to 0 / 1) to give Intermediate 9 as a colorless oil. 1 H NMR(400MHz,DMSO-d6)δ ppm 7.46-7.29(m,5H),5.78(dd,J=10.4,16.8Hz,1H),5.13-5.05(m,2H),5.03-4.77(m,2H),2.3 8-2.26(m,2H),2.13-1.92(m,2H),1.62-1.48(m,2H),1.38-1.28(m,2H),1.28-1.15(m,6H).

[0249] Intermediate 10A: 1,11-dibenzyl 11-(tert-butyl) (R)-docos-8-ene-1,11,11-tricarboxylate [ka] Intermediate 8A (26.0 g, 58.4 mmol, 1 equiv.) and intermediate 9 (30.4 g, 116 mmol, 2 equiv.) were dissolved in CHCl (520 mL). Grubbs II (2.38 g, 3.80 mmol, 0.065 equiv.) was then added and the resulting mixture was stirred at 40° C. for 1.5 h. The reaction mixture was then concentrated to provide a crude residue. This residue was purified by column chromatography (SiO, eluted with petroleum ether / ethyl acetate=1 / 0 to 0 / 1) to give intermediate 10A as a colorless oil. LCMS Method B: Rt=1.456 min, [M+Na] + =699.6.

[0250] Intermediate 11A: (S)-13-(benzyloxy)-2-((benzyloxy)carbonyl)-13-oxo-2-undecyltridec-4-enoic acid [ka] Intermediate 10A (50.0 g, 73.8 mmol, 1 equiv) was dissolved in TFA (500 mL) and the resulting mixture was stirred at 25° C. for 30 min. The reaction mixture was then concentrated to give a crude residue, which was dissolved in ethyl acetate (500 mL) and then washed twice with saturated NaHCO3 (500 mL), washed with brine (100 mL), dried over Na2SO4, filtered and concentrated to give a crude residue. The residue was purified by column chromatography (SiO2, eluted with petroleum ether / ethyl acetate=1 / 0 to 0 / 1) to give Intermediate 11A as a colorless oil. 1 H NMR(400MHz,DMSO-d6)δ ppm 7.44-7.14(m,10H),5.41(br s,1H),5.22-4.86(m,5H),2.49-2.41(m,2H),2.37-2.28(m,2H),1.95-1.83(m,2H),1.76-1.65( m,2H),1.58-1.46(m,2H),1.34-0.98(m,25H),0.90-0.76(m,3H).LCMS method B: Rt=1.323min,[M+H] + =622.3.

[0251] Intermediate 12A: 1,11-dibenzyl 11-(2,5-dioxopyrrolidin-1-yl)(R)-docos-8-ene-1,11,11-tricarboxylate [ka] To a solution of intermediate 11A (29.0 g, 46.7 mmol, 1 equiv.) in CHCl (260 mL) and THF (29 mL) was added NHS (5.64 g, 49.0 mmol, 1.05 equiv.) and DCC (11.5 g, 56.0 mmol, 11.3 mL, 1.2 equiv.) at 25° C., and the resulting mixture was stirred at 25° C. for 5 h. Then, the reaction mixture was filtered and washed three times with CHCl (30 mL). The organic phase was concentrated to give the crude residue. The crude residue was purified by column chromatography (SiO, eluted with petroleum ether / ethyl acetate = 1 / 0 to 85 / 15) to give intermediate 12A as a pale yellow oil. 1 H NMR(400MHz,chloroform-d)δ ppm 7.37-7.20(m,10H),5.53-5.34(m,1H),5.24-5.16(m,1H),5.15-5.12(m,2H),5.16-5.12(m,2H),5.07-5.00(m,2H),2.73(br s,4H),2.66-2.53(m,2H),2.34-2.16(m,2H),1.97-1.77(m,4H),1.70-1.47( m,3H),1.37-0.99(m,26H),0.88-0.65(m,3H).LCMS method B: Rt=1.348min,[M+H] + =718.6.

[0252] Intermediate 13A: (S)-14-((benzyloxy)carbonyl)-3,15-dioxo-1-phenyl-14-undecyl-2,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76,79,82,85,88-pentacosaoxa-16-azahennonacont-11-en-91-oic acid [ka] To intermediate 12A (545 mg, 0.759 mmol) in DMF (3 mL) was added 1-amino-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tetracosaoxapentaheptacontan-75-oic acid (amine-PEG-24-acid, 1131 mg, 0.987 mmol) and DIPEA (0.199 mL, 1.139 mmol). After 16 h, the reaction was complete. Volatiles were removed and the resulting residue was purified directly on RPLC (ISCO C18 Gold 150 g column, eluted with 10-100% ACN:water gradient with 0.1% TFA). The product containing fractions were combined, frozen and lyophilized to give Intermediate 13A as a thick oil. LCMS Method H: Rt=2.93 min, [M+H] + =1750.5.

[0253] Intermediate 14A: 77,87-dibenzyl 1-(2,5-dioxopyrrolidin-1-yl)-(S)-76-oxo-77-undecyl-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tetracosaoxa-75-azaheptaoctacont-79-ene-1,77,87-tricarboxylate [ka] To intermediate 13A (183 mg, 0.105 mmol) dissolved in 5 mL of anhydrous DCM was added DSC (32.2 mg, 0.126 mmol) and DIPEA (0.027 mL, 0.157 mmol) and the resulting mixture was stirred for 16 h, after which the reaction was complete. The crude mixture was loaded directly onto a DCM-equilibrated ISCO Gold 40 gram column and purified by NPLC (eluted with 0-30% MeOH in DCM, silica). The product-containing fractions were combined and concentrated to give intermediate 14A as a thick clear oil. LCMS Method H: Rt=2.79 min, [M+H] + =1847.5.

[0254] Intermediate 15A: (S)-2-((75-((2,5-dioxopyrrolidin-1-yl)oxy)-75-oxo-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tetracosaoxapentaheptacontyl)carbamoyl)-2-undecyltridecanedioic acid [ka] Intermediate 14A (165 mg, 0.089 mmol) was dissolved in 2 mL of anhydrous THF, degassed and replaced with nitrogen three times. To this mixture was added 10% palladium on carbon (9.51 mg, 8.94 μmol), degassed and replaced with hydrogen from a balloon with magnetic stirring. After 16 h, the reaction was complete. After dilution with 5 mL of anhydrous DCM, the reaction mixture was filtered through Celite®. The palladium on carbon and Pad were washed twice with 5 mL of DCM, and all organic phases were combined and concentrated to give Intermediate 15A. LCMS Method F: Rt=3.29 min, [M+H] + =1669.5.

[0255] Intermediate 10B: 1,11-dibenzyl 11-(tert-butyl)(S)-docos-8-ene-1,11,11-tricarboxylate [ka] Intermediate 8B (36.0 g, 80.9 mmol, 1 equiv.) and Intermediate 3 (42.1 g, 161 mmol, 2 equiv.) were dissolved in CHCl (720 mL) and then Grubbs II (3.30 g, 5.26 mmol, 0.065 equiv.) was added. The resulting mixture was stirred at 40° C. for 1 h and then concentrated in vacuo to give a crude residue. The crude residue was purified by column chromatography (SiO, eluted with petroleum ether / ethyl acetate=1 / 0 to 0 / 1) to give Intermediate 10B as a colorless oil. 1H NMR(400MHz,chloroform-d)δ ppm 7.45-7.25(m,10H),5.50-5.30(m,1H),5.20-5.02(m,5H),2.48-2.40(m,1H),2.39-2.23(m,3H),1.96-1.83(m,3H),1.79-1.64(m,1H) ),1.61-1.45(m,3H),1.40-1.14(m,22H),1.13-0.94(m,3H),1.14-0.93(m,3H),0.87-0.80(m,1H).LCMS method B: RT=1.448min,[M-56+H] + =622.3.

[0256] Intermediate 11B: (R)-13-(benzyloxy)-2-((benzyloxy)carbonyl)-13-oxo-2-undecyltridec-4-enoic acid [ka] Intermediate 10B (62 g, 91.59 mmol, 1 equiv) was dissolved in TFA (620 mL) and the resulting mixture was stirred at 25° C. for 30 min. The reaction mixture was then concentrated in vacuo to give a crude residue. The crude residue was dissolved in ethyl acetate (800 mL), then washed twice with sat.NaHCO3 (200 mL) and with brine (100 mL), dried using Na2SO4, filtered and concentrated to give a crude residue. The crude residue was purified by column chromatography (SiO2, eluted with petroleum ether / ethyl acetate=1 / 0 to 0 / 1) to give Intermediate 11B as a yellow oil. 1 H NMR(400MHz,DMSO-d6)δ 13.15-12.55(m,1H),7.54-6.92(m,10H),5.54-5.33(m,1H),5.25-4.94(m,5H),2.47(br d,J=7.2Hz,1H),2.33(br t,J=7.3Hz,2H),1.97-1.83(m,2H),1.79-1.63(m,2H),1.60-1.45(m,2H),1.3 8-0.96(m,26H),0.92-0.76(m,3H).LCMS method B: RT=1.323min, MS(ESI)m / z[M+H] +=621.6.

[0257] Intermediate 12B: 1,11-dibenzyl 11-(2,5-dioxopyrrolidin-1-yl)(S)-docos-8-ene-1,11,11-tricarboxylate [ka] To a solution of intermediate 11B (27 g, 43.4 mmol, 1 equiv.) in CHCl (243 mL) and THF (27 mL) was added NHS (5.26 g, 45.6 mmol, 1.05 equiv.) and DCC (10.7 g, 52.1 mmol, 10.5 mL, 1.2 equiv.), and the resulting mixture was stirred at 25° C. for 6 h. The reaction mixture was then filtered and washed three times with CHCl (30 mL) to obtain a filtrate, which was then concentrated to obtain a crude residue. The crude residue was purified by column chromatography (SiO, eluted with petroleum ether / ethyl acetate=1 / 0 to 85 / 15) to obtain intermediate 12B as a pale yellow oil. 1 H NMR(400MHz,chloroform-d)δ 13.15-12.55(m,1H),7.54-6.92(m,10H),5.54-5.33(m,1H),5.25-4.94(m,5H),2.47(br d,J=7.2Hz,1H),2.33(br t,J=7.3Hz,2H),1.97-1.83(m,2H),1.79-1.63(m,2H),1.60-1.45(m,2H),1.38-0.96(m,26H),0.92-0.76(m,3H).LCMS method B:Rt=1.348min,[M+H] + =718.5.

[0258] Intermediate 13B: (R)-14-((benzyloxy)carbonyl)-3,15-dioxo-1-phenyl-14-undecyl-2,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76,79,82,85,88-pentacosaoxa-16-azahennonacont-11-en-91-oic acid [ka] Intermediate 12B (1.07 g, 1.49 mmol) was treated with 1-amino-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tetracosaoxapentaheptacontan-75-oic acid (Biopharm, 1.88 g, 1.64 mmol), DIPEA (390 μL, 2.236 mmol) and DMAP (18 mg, 0.05 mmol). After 16 h, the reaction was complete. Volatiles were removed and the resulting residue was purified by RPLC (ISCO C18 Gold 150 g column, eluted with a 10-100% ACN:water gradient with 0.1% TFA). The product containing fractions were combined, frozen and lyophilized to give Intermediate 13B as a thick oil. LCMS Method C: Rt=4.04 min, [M+2H] 2+ =875.8.

[0259] Intermediate 14B: 77,87-dibenzyl 1-(2,5-dioxopyrrolidin-1-yl)-(R)-76-oxo-77-undecyl-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tetracosaoxa-75-azaheptaoctacont-79-ene-1,77,87-tricarboxylate [ka] Intermediate 13B (312 mg, 0.178 mmol) was dissolved in 1.8 mL of anhydrous DCM along with 1-hydroxypyrrolidine-2,5-dione (24.63 mg, 0.214 mmol) and then treated with 1M DCC in DCM (Aldrich, 196 μL), which immediately resulted in precipitation of the dicyclohexylurea by-product. After 16 h, the reaction was complete, and the reaction mixture was then loaded directly onto a DCM-equilibrated ISCO Gold 40 gram column and purified by NPLC (eluted with 0-30% MeOH in DCM, silica). Fractions containing the product were combined and concentrated to give Intermediate 14B as a thick clear oil. LCMS Method F: Rt=4.21 min, [M+H+H2O]+ =1864.4.

[0260] Intermediate 15B: (R)-2-((75-((2,5-dioxopyrrolidin-1-yl)oxy)-75-oxo-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tetracosaoxapentaheptacontyl)carbamoyl)-2-undecyltridecanedioic acid [ka] Intermediate 14B (172 mg, 0.093 mmol) was dissolved in 1.8 mL of anhydrous THF, degassed and replaced with nitrogen three times. To this mixture was added 10% palladium on carbon (10 mg, 9.4 umol), degassed and replaced with hydrogen from a balloon with magnetic stirring. After 16 h, the reaction was complete. After dilution with 5 mL of anhydrous DCM, the reaction mixture was filtered through Celite®. The palladium on carbon and Celite-cake were washed twice with 5 mL of DCM and filtered. All organics were combined and concentrated to give Intermediate 15B. LCMS Method F: Rt=3.31 min, [M+H] + =1669.0.

[0261] Intermediate 16: 1,11-Dibenzyl 11-(2,5-dioxocyclopentyl)docosane-1,11,11-tricarboxylate [ka] To a 1000 mL 3-neck round bottom flask (fitted with a mechanical stirrer and nitrogen inlet) was added intermediate 3 (37.7 g, 60.5 mmol), DCM (360 mL, ratio: 9.0) and THF (40 mL, ratio: 1.0), followed by N-hydroxysuccinimide (7.31 g, 63.6 mmol) and DCC (14.99 g, 72.6 mmol). 5 min after addition the resulting mixture became a white suspension. The reaction mixture was stirred at RT for a total of 6 h and then filtered over a pad of Celite®. The pad was washed thoroughly with DCM (2 bed volumes). The combined organic phases were concentrated in vacuo and the crude residue was dried under high vacuum. The crude product was isolated as a white oil. To the crude product was added DCM (approximately 400 mL) and silica gel (75 g). The resulting suspension was concentrated in vacuo and the residue was dried under high vacuum for 3 h. The batch was purified via column chromatography (750 g SiO2 gel, eluting with 2% ethyl acetate / heptane to 35% ethyl acetate / heptane). The product-containing fractions were combined, concentrated in vacuo, and dried under high vacuum overnight to give Intermediate 16 as a colorless oil. 1 H NMR(400MHz,chloroform-d)δ ppm 0.86-0.93(m,3H)1.12-1.21(m,2H)1.21-1.37(m,30H)1.66(quin,J=7.40Hz,2H)1.89-2.0 7(m,4H)2.37(t,J=7.58Hz,2H)2.84(br.s.,4H)5.13(s,2H)5.25(s,2H)7.30-7.47(m,10H).

[0262] Intermediate 17: 14-((benzyloxy)carbonyl)-3,15-dioxo-1-phenyl-14-undecyl-2,19,22,25,28,31,34,37,40-nonaoxa-16-azatritetracontan-43-oic acid [ka] To a 250 mL round bottom flask (fitted with a magnetic stirrer and nitrogen inlet) was added intermediate 16 (7.0 g, 9.72 mmol) and DCM (70 mL), followed by 1-amino-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-oic acid (amino-PEG8-acid) (4.51 g, 10.21 mmol), DIPEA (4.25 mL, 24.31 mmol) and DMAP (0.119 g, 0.972 mmol). The resulting pale yellow homogeneous solution was stirred overnight at ambient temperature. The reaction mixture was then concentrated in vacuo to give a pale yellow oily residue. This residue was then diluted with ethyl acetate (150 mL) and the solution was transferred to a 500 mL separatory funnel. The solution was then washed with brine (500 mL). The resulting aqueous phase was back extracted with ethyl acetate (150 mL; then 100 mL). The combined organic phases were dried (sodium sulfate), filtered over Celite® and concentrated in vacuo. The crude product was purified via column chromatography (330 g SiO2 gel, eluted with DCM to 10% methanol / DCM). Fractions containing the main product were combined and concentrated in vacuo. The residue was dried under high vacuum overnight to give intermediate 17. LCMS Method E: Rt=1.43 min, [M+H] + =1047.0.

[0263] Intermediate 18: 29,39-Dibenzyl 1-(2,5-dioxopyrrolidin-1-yl) 28-oxo-29-undecyl-3,6,9,12,15,18,21,24-octaoxa-27-azanonatriacontane-1,29,39-tricarboxylate [ka] To a 50 mL round bottom flask containing intermediate 17 (5.51 g, 5.27 mmol) was added DCM (27.5 mL, ratio: 1.0) and THF (27.5 mL, ratio: 1.0), followed by DCC (1.412 g, 6.85 mmol) and N-hydroxysuccinimide (0.697 g, 6.06 mmol). After stirring for approximately 10 min, the resulting mixture became a thick white suspension. The reaction mixture was then stirred at ambient temperature for 3 h45 min and concentrated in vacuo to give a white paste. DCM (35 mL) was added to the mixture and the resulting white suspension was stirred for 10 min. The mixture was then filtered over a pad of Celite® and the pad was washed with cold DCM (1 bed volume). The combined filtrates were concentrated in vacuo. The residue was dried under high vacuum overnight to give intermediate 18 as a colorless oil. LCMS Method E: Rt=1.45 min, [M+H] + =1044.0.

[0264] Intermediate 19: 2-((27-((2,5-dioxopyrrolidin-1-yl)oxy)-27-oxo-3,6,9,12,15,18,21,24-octaoxaheptacosyl)carbamoyl)-2-undecyltridecanedioic acid [ka] To a 250 mL round bottom flask (fitted with a magnetic stirrer) was added intermediate 18 (6.0 g, 5.25 mmol) and THF (70 mL). To this solution was added 10% Pd / C (0.603 g, 0.567 mmol) and the reaction vessel was purged with nitrogen followed by hydrogen. The resulting mixture was then exposed to hydrogen (balloon pressure) for 3 h. The reaction vessel was purged with nitrogen and the suspension was filtered over a pad of Celite®. The pad was washed thoroughly with THF and the combined filtrate was concentrated in vacuo. The resulting residue was then dried under high vacuum overnight to give intermediate 19 as a colorless oil. LCMS Method E: Rt=0.91 min, [M+H] + =963.8.

[0265] Intermediate 20: 14-((benzyloxy)carbonyl)-3,15-dioxo-1-phenyl-14-undecyl-2,19,22-trioxa-16-azapentacosane-25-oic acid [ka] To a 250 mL round bottom flask (with magnetic stir bar) was added intermediate 16 (5.0 g, 6.94 mmol) and DCM (volume: 100 mL), followed by 3-(2-(2-aminoethoxy)ethoxy)propanoic acid (amino-PEG2-acid, 1.231 g, 6.94 mmol), DIPEA (3.03 mL, 17.36 mmol) and DMAP (0.085 g, 0.694 mmol). The resulting white suspension was stirred at ambient temperature for 22 h. LCMS showed significant NHS ester starting material remaining. The reaction mixture was then warmed to 40° C. and stirring was continued for 5.5 h. The mixture was cooled to ambient temperature and then concentrated in vacuo to give a white paste. To the mixture was added ethyl acetate (150 mL). The solution was transferred to a 500 mL separatory funnel along with brine (150 mL). The phases were separated and the aqueous phase was extracted twice with ethyl acetate (150 mL). The combined organic phases were dried (sodium sulfate), filtered over Celite® and concentrated in vacuo. The crude product was purified via column chromatography (120 g silica gel, eluting with 0.5% methanol / DCM to 60% methanol / DCM). Fractions containing the main product by TLC (5% methanol / DCM) were combined and concentrated in vacuo, and the resulting residue was dried under high vacuum overnight to give Intermediate 20 as a colorless oil. LCMS Method E: Rt=1.45 min, [M+H] + =782.8.

[0266] Intermediate 21: Dibenzyl 2-((2-(2-(3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropoxy)ethoxy)ethyl)carbamoyl)-2-undecyltridecanedioate [ka] To a 500 mL round bottom flask (fitted with a magnetic stirrer and nitrogen inlet) was added intermediate 20 (3.7 g, 4.73 mmol), DCM (19 mL, ratio: 1.0) and THF (19 mL, ratio: 1.0), followed by N-hydroxysuccinimide (0.626 g, 5.44 mmol) and DCC (1.269 g, 6.15 mmol). The resulting mixture was stirred at ambient temperature for 3 h, at which point it became a white suspension. The suspension was then filtered over Celite® and the pad was washed with DCM. The combined filtrates were concentrated in vacuo. The resulting residue was then suspended in DCM (20 mL) and the mixture was stirred at ambient temperature for 10 min, then filtered over a pad of Celite®. The pad was washed with cold DCM. The combined filtrates were concentrated in vacuo. The residue was dried under high vacuum overnight to give intermediate 21 as a pale yellow oil. LCMS method E: Rt=1.47min,[M+H] + =879.7.

[0267] Intermediate 22: 2-((2-(2-(3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropoxy)ethoxy)ethyl)carbamoyl)-2-undecyltridecanedioic acid [ka] To a 100 mL round bottom flask containing intermediate 21 (4.16 g, 4.73 mmol) was added THF (40 mL). To this solution was added 10% Pd / C (0.42 g, 3.93 mmol) and the vessel was purged with nitrogen. The reaction vessel was then purged with hydrogen and exposed to hydrogen pressure (balloon). The resulting black suspension was stirred for 4 h and then filtered over a pad of Celite®. The pad was washed with THF. The combined filtrate was concentrated in vacuo and then dried under high vacuum to give intermediate 22 as a colorless oil. LCMS Method G: Rt=1.72 min, [M+H] + =699.4.

[0268] Intermediate 23: Benzyl 11-bromoundecanoate [ka] To a 2 L round, 3-necked round-bottom flask (fitted with a mechanical stirrer, temperature probe, and nitrogen inlet) was added 11-bromoundecanoic acid (50 g, 189 mmol) and 500 mL of dichloromethane. To the resulting orange homogeneous solution was then added benzyl alcohol (23.53 mL, 226 mmol), EDCI·HCl (54.2 g, 283 mmol), and DMAP (1.152 g, 9.43 mmol). The reaction mixture was stirred overnight. TLC analysis (30% ethyl acetate in heptane) indicated consumption of 11-bromoundecanoic acid. The reaction mixture was transferred to a 2 L round-bottom flask and concentrated in vacuo. The resulting residue was diluted with 1 L of water and 800 mL of MTBE. The phases were separated and the aqueous phase was extracted twice with 600 mL of MTBE. The combined organic phase was washed with 750 mL of brine, dried over sodium sulfate, filtered over Celite®, and concentrated in vacuo. The material was dried under high vacuum for 2 h to give a pale yellow oil. The crude product was dissolved in 500 mL of DCM and 100 g silica gel was added. The mixture was concentrated in vacuo and then dried under high vacuum overnight. The residue was purified via chromatography (750 g silica column, eluting with a gradient of 1% EtOAc / heptane to 20% EtOAc / heptane). The fractions containing benzyl 11-bromoundecanoate were combined and concentrated in vacuo. The residue was dried under high vacuum for 5 h to give intermediate 23 as a colorless oil. 1 H NMR(400MHz,chloroform-d)δ 7.58-7.31(m,5H),5.14(s,2H),3.43(t,J=6.9Hz,2H),2.38(t,J=7.5Hz,2H),1 .87(p,J=7.0Hz,2H),1.73-1.61(m,2H),1.49-1.40(m,2H),1.37-1.26(m,10H).

[0269] Intermediate 24: 1,11,21-Tribenzyl 11-tert-butylhexanoic acid-1,11,11,21-tetracarboxylate [ka] To a 250 mL 3-neck round bottom flask equipped with a mechanical stirrer, temperature probe, and nitrogen inlet was added benzyl tert-butyl malonate (6 g, 23.97 mmol) and 30 mL of DMF, followed by a mixture of intermediate 23 (18.74 g, 52.7 mmol) and 60 mL of DMF. To this colorless solution was added cesium carbonate (31.2 g, 96 mmol) and the resulting suspension was stirred at ambient temperature. After stirring at ambient temperature for 5.5 h, LCMS indicated that no benzyl tert-butyl malonate was present. The reaction mixture was a mixture of mono- and di-alkylated products. Thus, the mixture was stirred for 22 h, and LCMS indicated that the mono-alkylated intermediate still remained. The reaction mixture was then heated to 40 °C and stirred for 3 h. LCMS still showed minimal progress. The mixture was cooled to 0-5 °C and 200 mL of deionized (DI) water was added in a thin line. The mixture was then warmed to ambient temperature and transferred to a 500 mL separatory funnel. The aqueous phase was extracted twice with 200 mL MTBE. The combined organic phase was washed with 200 mL brine, dried over sodium sulfate, filtered over Celite®, and concentrated in vacuo. The residue was dried under high vacuum for 2 h to give the crude colorless product, which was purified via NPLC (330 g ISCO silica column, eluting with 0.5% ethyl acetate / heptane to 30% ethyl acetate / heptane gradient). The product-containing fractions were combined and concentrated in vacuo. The residue was dried under high vacuum overnight to give intermediate 24 as a colorless oil. LCMS Method E: Rt=1.75 min, [M+H+H2O] + =821.3.

[0270] Intermediate 25: 13-(benzyloxy)-2-(11-(benzyloxy)-11-oxoundecyl)-2-((benzyloxy)carbonyl)-13-oxotridecanoic acid [ka] Intermediate 24 (17.78 g, 22.25 mmol) and 180 mL of TFA were added to a 1 L round bottom flask equipped with a magnetic stir bar and nitrogen inlet, and the resulting mixture was stirred for 45 min. When LCMS analysis showed no starting material remaining, the mixture was concentrated in vacuo to give a pale yellow oil. The resulting oil was diluted with 250 mL of toluene and then concentrated in vacuo to remove residual TFA. This final step was repeated once. The residue was dried under high vacuum over the weekend to give Intermediate 25 as a pale yellow oil, which was used directly in the next step. LCMS Method E: Rt=1.55 min, [M+H+H2O] + =760.4.

[0271] Intermediate 26: 1,11,21-Tribenzyl 11-(2,5-dioxopyrrolidin-1-yl)henicosane-1,11,11,21-tetracarboxylate [ka] To a 500 mL round bottom flask containing intermediate 25 (16.53 g, 22.25 mmol) was added 180 mL of DCM and 20 mL of THF, followed by N-hydroxysuccinimide (2.69 g, 23.36 mmol) and DCC (5.51 g, 26.7 mmol). The resulting mixture was stirred overnight after which LCMS showed complete conversion to the desired product. The resulting white suspension was filtered over a pad of Celite® and the pad was washed with two bed volumes of DCM. The combined filtrate was concentrated in vacuo to give a colorless oil, which was dried under high vacuum for 1 h to give 21.3 g of crude product. The crude product was dissolved in 250 mL of DCM and 32 g of silica gel was added. The mixture was concentrated in vacuo and then dried under high vacuum for 2 h. The residue was purified via cartridge dry loaded NPLC (330 g silica gel column, eluting with 5% ethyl acetate / heptane to 40% ethyl acetate / heptane gradient). Product-containing fractions were combined, concentrated in vacuo, and dried under high vacuum overnight to give intermediate 26 as a colorless oil. LCMS Method E: Rt=1.58 min, [M+H+H2O]+ =857.4.

[0272] Intermediate 27: 14-(11-(benzyloxy)-11-oxoundecyl)-14-((benzyloxy)carbonyl)-3,15-dioxo-1-phenyl-2,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76,79,82,85,88-pentacosaoxa-16-azahennonacontan-91-oic acid [ka] Intermediate 26 (479 mg, 0.503 mmol) was dissolved in 5.7 mL of anhydrous DCM. This solution was then treated with 1-amino 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tetracosaoxapentaheptacontan-75-oic acid (686 mg, 0.599 mmol), DIPEA (149 μL, 0.855 mmol) and DMAP (7 mg, 0.057 mmol) and stirred at room temperature for 16 h. After 16 h, the reaction was complete by LC / MS analysis and the volatiles were removed by rotary evaporation. The crude product was purified by NPLC (24 gram ISCO Gold silica column, eluted with 0-20% MeOH in DCM) to give Intermediate 27 as a thick oil. LCMS Method E: Rt=1.35 min, [M+H] + =1871.9.

[0273] Intermediate 28: 77,87-Dibenzyl 1-(2,5-dioxopyrrolidin-1-yl) 77-(11-(benzyloxy)-11-oxoundecyl)-76-oxo-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tetracosaoxa-75-azaheptaoctacontane-1,77,87-tricarboxylate [ka] Intermediate 27 (764 mg, 0.408 mmol) was treated with 1-hydroxypyrrolidine-2,5-dione (56.4 mg, 0.490 mmol) in 4 mL of DCM. To this mixture was added a solution of 1M DCC (Aldrich, 0.499 mL, 0.499 mmol) in DCM and the reaction mixture was stirred under nitrogen. After 16 h the reaction was complete. The volatiles were removed and the residue was purified by NPLC (24 gram ISCO Gold column, eluted with 0-15% MeOH in DCM). The fractions containing the product were combined and concentrated to give Intermediate 28. LCMS Method F: Rt=4.03 min, [M+2H] 2+ =985.1.

[0274] Intermediate 29: 11-((75-((2,5-dioxopyrrolidin-1-yl)oxy)-75-oxo-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tetracosaoxapentaheptacontyl)carbamoyl)henicosane-1,11,21-tricarboxylic acid [ka] Intermediate 28 (500 mg, 0.254 mmol) was dissolved in 2.5 mL of anhydrous THF with a stir bar. Degassed and replaced with nitrogen three times. Then 10% palladium on carbon (Aldrich, 27 mg, 0.025 mmol) was carefully added and the flask was degassed. The air was replaced with hydrogen from a balloon reservoir. The reaction mixture was stirred overnight for 16 h, when LC / MS showed the reaction was complete. The reaction mixture was diluted with 5 mL of anhydrous DCM and filtered through Celite®. The filtrate was concentrated to give Intermediate 29 as a thick clear oil. LCMS Method F: Rt=2.60 min, [M+2H] 2+ =849.9.

[0275] Intermediate 30: Benzyl 12-bromododecanoate [ka] 12-Bromododecanoic acid (2 g, 7.16 mmol), benzyl alcohol (1.16 g, 10.74 mmol) and EDC·HCl (2.06 g, 10.74 mmol) were combined in 24 mL of DCM. To this solution was added DMAP (44 mg, 0.358 mmol) in one portion and the resulting mixture was stirred overnight. Based on LCMS analysis, the reaction was approximately 90% complete. The reaction mixture was purified by NPLC (eluted with 0-15% EtOAc in heptane, silica). The fractions containing the product were combined and concentrated to give the desired product, Intermediate 30. 1 H NMR:(400MHz,chloroform-d)δ 7.47-7.31(m,5H),5.14(s,2H),3.43(t,J=6.9Hz,2H),2.38(t,J=7.5Hz ,2H),1.88(p,2H),1.67(p,2H),1.50-1.39(m,2H),1.35-1.26(m,12H).

[0276] Intermediate 31: 1,12,23-tribenzyl 12-(tert-butyl)tricosane-1,12,12,23-tetracarboxylate, 1,12-dibenzyl 1-(tert-butyl)dodecane-1,1,12-tricarboxylate [ka] Intermediate 30 (1 g, 2.71 mmol), benzyl tert-butyl malonate (276.4 mg, 1.104 mmol) and 60% sodium hydride in oil (97 mg, 2.43 mmol) were combined in 12 mL of anhydrous DMF and the resulting mixture was stirred overnight at RT under nitrogen atmosphere in an oven-dried round-bottom flask. After 16 h, LCMS analysis showed complete conversion to the desired product. The reaction mixture was partitioned between water and EtOAc and washed with EtOAc (2×20 mL). The organic phases were combined, washed with brine, dried over sodium sulfate, filtered and concentrated by rotary evaporation. The crude product was purified by NPLC (eluted with 0-60% EtOAc in heptane, silica, ELSD detection). Excess SM bromide eluted first immediately, the mono-alkylated product eluted second, followed by the desired product. The product-containing fractions were combined and concentrated to give intermediate 31 as a clear viscous oil. LCMS method E: Rt=1.73min, [M+H+H2O] + =845.0.

[0277] Intermediate 32: 14-(benzyloxy)-2-(12-(benzyloxy)-12-oxododecyl)-2-((benzyloxy)carbonyl)-14-oxotetradecanoic acid [ka] Intermediate 31 (650 mg, 0.786 mmol) was dissolved in DCM (7.2 mL) and treated with TFA (0.605 mL, 7.86 mmol). After 16 h, the reaction was complete as indicated by the LC / MS ELSD signal. Volatiles were removed and the resulting residue was purified by NPLC (eluted with 0-5% MeOH in DCM, silica). Fractions containing the product were combined and concentrated to give Intermediate 32 as a clear oil. LCMS Method E: Rt=1.57 min, [M+H] + +771.9.

[0278] Intermediate 33: 1,12,23-Tribenzyl 12-(2,5-dioxopyrrolidin-1-yl)tricosane-1,12,12,23-tetracarboxylate [ka] Intermediate 32 (310 mg, 0.402 mmol) was dissolved in 3.6 mL of DCM along with 1-hydroxypyrrolidine-2,5-dione (50.9 mg, 0.442 mmol) and 1M DCC in DCM (Aldrich, 422 μL, 0.422 mmol). After 15 min, precipitation of DCU by-product was observed. The reaction mixture was stirred overnight after which LC / MS showed complete conversion to product. Volatiles were partially removed and the oily product was purified by NPLC (eluted with 0-30% EtOAc in heptane, silica) to give intermediate 33. LCMS Method E: Rt=1.49 min, [M+H+H2O] + =886.5.

[0279] Intermediate 34: 15-(12-(benzyloxy)-12-oxododecyl)-15-((benzyloxy)carbonyl)-3,16-dioxo-1-phenyl-2,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74,77,80,83,86,89-pentacosaoxa-17-azadononacontan-92-oic acid [ka] Intermediate 33 (127.6 mg, 0.147 mmol) was treated with 1-amino-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tetracosaoxapentaheptacontan-75-oic acid (Biopharm, 168 mg, 0.147 mmol), DIPEA (38.5 μL, 0.220 mmol) and DMAP (1.8 mg, 0.0015 mmol). After 16 h the reaction was essentially complete. The volatiles were removed and the residue was purified by NPLC (eluted with 0-15% MeOH in DCM, silica). The fractions containing the product were combined and concentrated to give intermediate 34. LCMS method E: Rt=1.41min,[M+2H] 2+ =951.6.

[0280] Intermediate 35: 77,88-Dibenzyl 1-(2,5-dioxopyrrolidin-1-yl) 77-(12-(benzyloxy)-12-oxododecyl)-76-oxo-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tetracosaoxa-75-azaoctaoctacontane-1,77,88-tricarboxylate [ka] [ka] Intermediate 34 (194 mg, 0.102 mmol) was dissolved in 1 mL of DCM and treated with 1-hydroxypyrrolidine-2,5-dione (11.75 mg, 0.102 mmol) and 1M DCC in DCM (Aldrich, 0.107 mL, 0.107 mmol). After 15 min, precipitation of DCU was observed. After 16 h, the reaction was complete as indicated by LC / MS. Volatiles were removed to give an oily residue. This material was purified by NPLC (eluted with 0-15% MeOH in DCM, silica, ELSD detection). Fractions containing the product were combined and concentrated to give the desired product, Intermediate 35. LCMS Method E: Rt=1.40 min, [M+2H+H2O] 2+ =1008.2.

[0281] Intermediate 36: 12-((75-((2,5-dioxopyrrolidin-1-yl)oxy)-75-oxo-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tetracosaoxapentaheptacontyl)carbamoyl)tricosane-1,12,23-tricarboxylic acid [ka] Intermediate 35 (130 mg, 0.065 mmol) was dissolved in THF (2 mL) and the resulting mixture was purged with nitrogen three times. 10% palladium on carbon (36.4 mg, 0.033 mmol) was carefully added, degassed, and then replaced with hydrogen from a balloon reservoir. The reaction was complete after 16 h as shown by LC / MS analysis (ELSD detection). Purification was accomplished by NPLC (eluted with MeOH in DCM, silica, 0-20%) and fractions containing the desired product were combined and concentrated to give Intermediate 36. LCMS Method E: Rt=0.64 min, [M+2H] 2+ =864.0.

[0282] Intermediate 37: Benzyl 14-bromotetradecanoate [ka] 14-Bromotetradecanoic acid (1.00 gm, 3.25 mmol), benzyl alcohol (677 μL, 6.51 mmol) and EDC·HCl (936 mg, 4.89 mmol) were combined in DCM (11 mL). To this solution was added DMAP (19.9 mg, 0.163 mmol) in one portion and the resulting mixture was stirred overnight. After this time the reaction was complete as indicated by LC / MS analysis. Volatiles were removed and the resulting residue was purified by NPLC (eluted with 0-15% EtOAc in heptane, silica). The fractions containing the product were combined and concentrated to give the desired intermediate 37. 1 H NMR(400MHz,chloroform-d)δ 7.33-7.22(m,5H),5.04(s,2H),3.34(t,J=6.9Hz,2H),2.28(t,J=7.6Hz,2H) ,1.78(p,2H),1.58(p,J=7.3Hz,2H),1.39-1.31(m,2H),1.25-1.16(m,16H).

[0283] Intermediate 38: 1,14,27-tribenzyl 14-(tert-butyl)heptacosane-1,14,14,27-tetracarboxylate, 1,14-dibenzyl 1-(tert-butyl)tetradecane-1,1,14-tricarboxylate [ka] Intermediate 37 (713 mg, 1.793 mmol), benzyl tert-butyl malonate (187 mg, 0.747 mmol) and 60% sodium hydride in oil (65.7 mg, 1.644 mmol) were combined in anhydrous DMF (8 mL) and stirred overnight at 60° C. under nitrogen in an oven-dried round-bottom flask. After 24 h, LC / MS analysis showed the presence of mono- and bis-alkylated malonate products. The reaction mixture was treated with additional benzyl 14-bromotetradecanoate (229.2 mg, 0.57 mmol) and 60% sodium hydride in oil (45 mg, 1.13 mmol). After 16 h, the reaction was essentially complete as indicated by LC / MS. The reaction mixture was carefully partitioned between 10 mL of water and 10 mL of EtOAc. The aqueous phase was washed with 10 mL of EtOAc. The organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation. The crude product was purified by NPLC (eluted with 0-35% EtOAc in heptane, silica, ELSD detection). The excess starting bromide eluted first immediately, the mono-alkylated product eluted second, followed by the desired product. The fractions containing the product were combined and concentrated to give intermediate 38 as a clear viscous oil. LCMS Method E: Rt=1.87 min, [M+Na] + =905.7.

[0284] Intermediate 39: 16-(benzyloxy)-2-(14-(benzyloxy)-14-oxotetradecyl)-2-((benzyloxy)carbonyl)-16-oxohexadecanoic acid [ka] Intermediate 38 (290.4 mg, 0.329 mmol) was dissolved in DCM (3 mL) and then treated with TFA (0.25 mL, 3.29 mmol). After 16 h, the reaction was complete as indicated by the LC / MS ELSD signal. Volatiles were removed and the resulting residue was purified by NPLC (eluted with 0-5% MeOH in DCM, silica). Fractions containing the product were combined and concentrated to give Intermediate 39 as a clear oil. LCMS Method E: Rt=1.65 min, [M+H] + =828.1.

[0285] Intermediate 40: 1,14,27-Tribenzyl 14-(2,5-dioxopyrrolidin-1-yl)heptacosane-1,14,14,27-tetracarboxylate [ka] Intermediate 39 (170.4 mg, 0.206 mmol) was dissolved in DCM (2 mL) along with 1-hydroxypyrrolidine-2,5-dione (35.6 mg, 0.309 mmol) and 1M DCC in DCM (Aldrich, 212 μL, 0.212 mmol). After 10 min, precipitation of DCU was observed. The reaction mixture was stirred overnight at which point LC / MS showed complete conversion to product. Volatiles were partially removed and the oily product was purified by NPLC (eluted with 0-40% EtOH in heptane, silica) to give Intermediate 40. LCMS Method E: Rt=1.69 min, [M+H] + =924.4.

[0286] Intermediate 41: 17-(14-(benzyloxy)-14-oxotetradecyl)-17-((benzyloxy)carbonyl)-3,18-dioxo-1-phenyl-2,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76,79,82,85,88,91-pentacosaoxa-19-azatetranonacontan-94-oic acid [ka] Intermediate 40 (119.8 mg, 0.130 mmol) was treated with 1-amino-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tetracosaoxapentaheptacontan-75-oic acid (149 mg, 0.130 mmol), DIPEA (34.0 μL, 0.194 mmol) and DMAP (1.584 mg, 0.0013 mmol) in a solution of 1.3 mL of DCM. After 16 h, the reaction was essentially complete. The volatiles were removed and the residue was purified by NPLC (eluted with 0-65% MeOH in DCM, silica). The fractions containing the product were combined and concentrated to give intermediate 41. LCMS method E: Rt=1.56min,[M+2H] 2+ =978.9.

[0287] Intermediate 42: 77,90-Dibenzyl 1-(2,5-dioxopyrrolidin-1-yl) 77-(14-(benzyloxy)-14-oxotetradecyl)-76-oxo-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tetracosoxa-75-azanonacontane-1,77,90-tricarboxylate [ka] Intermediate 41 (144.2 mg, 0.074 mmol) was dissolved in 700 μL DCM and treated with 1-hydroxypyrrolidine-2,5-dione (12.73 mg, 0.111 mmol) and 1M DCC (dicyclohexylmethandiimine) in DCM (Aldrich, 0.077 mL, 0.077 mmol). After 15 min, precipitation of DCU was observed. After 16 h, the reaction was complete as indicated by LC / MS. Volatiles were removed to give an oily residue. This material was purified by NPLC (eluted with 0-25% MeOH in DCM, silica) with ELSD detection. Fractions containing the product were combined and concentrated to give the desired product, Intermediate 42. LCMS Method E: Rt=1.55 min, [M+2H+H2O] 2+=1036.2 /

[0288] Intermediate 43: 14-((75-((2,5-dioxopyrrolidin-1-yl)oxy)-75-oxo-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tetracosaoxapentaheptacontyl)carbamoyl)heptacosane-1,14,27-tricarboxylic acid [ka] Intermediate 42 (118.8 mg, 0.058 mmol) was dissolved in 3.75 mL of THF in a round bottom flask equipped with a stir bar. The resulting mixture was purged with nitrogen three times, then 30.8 mg (0.029 mmol) 10% Pd / C was added. It was degassed and replaced with hydrogen from a balloon. The reaction was complete in 16 h. The reaction mixture was diluted with 10 mL of DCM, filtered through Celite®, and the filtrate was concentrated to dryness. Purification was performed by NPLC (eluted with MeOH in DCM, silica, 0-20%), and the fractions containing the product were combined and concentrated to give intermediate 43. LCMS Method E: Rt=0.86 min, [M+2H] 2+ =892.0.

[0289] Intermediate 44: Benzyl 15-bromopentadecanoate [ka] 15-Bromopentadecanoic acid (1949 mg, 6.07 mmol), benzyl alcohol (984 mg, 9.10 mmol) and EDCI·HCl (1744 mg, 9.10 mmol) were combined in 24 mL of DCM. To this solution was added DMAP (37.1 mg, 0.303 mmol) in one portion and the resulting mixture was allowed to stir for 32 h. After 32 h the reaction was essentially complete. The reaction mixture was partitioned between water and DCM. The organic phase was washed with brine, dried over anhydrous sodium sulfate and filtered. The volatiles were removed and the resulting residue was purified by NPLC (eluted with 0-15% EtOAc in heptane, silica, ELSD detection). The fractions containing the product were combined and concentrated to give intermediate 44. 1 H NMR(400MHz,chloroform-d)δ 7.29-7.22(m,5H),5.02(s,2H),3.31(t,J=6.9Hz,2H),2.26(t,J=7.6Hz,2H) ,1.76(p,2H),1.54(p,J=7.3Hz,2H),1.36-1.30(m,2H),1.22-1.16(m,18H).

[0290] Intermediate 45: 1,15,29-tribenzyl 15-(tert-butyl)nonacosane-1,15,15,29-tetracarboxylate, 1,15-dibenzyl 1-(tert-butyl)pentadecane-1,1,15-tricarboxylate [ka] Intermediate 44 (1013 mg, 2.461 mmol), benzyl tert-butyl malonate (280 mg, 1.119 mmol) and 60% sodium hydride in oil (98 mg, 2.461 mmol) were combined in anhydrous DMF (5.6 mL) and stirred overnight at RT under nitrogen atmosphere in an oven-dried round-bottom flask. The reaction mixture was then carefully poured into 10 mL water and extracted three times with 10 mL EtOAc. The organic phases were combined, dried with brine and anhydrous sodium sulfate, filtered and concentrated. The product was purified by NPLC (eluted with 0-60% EtOAc in heptane, silica, ELSD detection). The fractions containing the product were combined and concentrated to give Intermediate 45 as a clear viscous oil. LCMS Method H: Rt=4.23 min, [M+H+H2O] + =928.9.

[0291] Intermediate 46: 17-(benzyloxy)-2-(15-(benzyloxy)-15-oxopentadecyl)-2-((benzyloxy)carbonyl)-17-oxoheptadecanoic acid [ka] Intermediate 45 (750 mg, 0.823 mmol) was dissolved in DCM (8.23 mL) and treated with TFA (634 μL, 8.23 ​​mmol). After 16 h the reaction was partially complete. The reaction mixture was left stirring for 1 week and was essentially complete after this time. The resulting oily residue was purified by NPLC (eluted with 0-25% EtOAc in heptane, silica) with ELSD detection. The fractions containing the product were concentrated to give intermediate 46. LCMS Method H: Rt=3.70 min, [M+H+H2O] + =873.2.

[0292] Intermediate 47: 1,15,29-tribenzyl 15-(2,5-dioxopyrrolidin-1-yl)nonacosane-1,15,15,29-tetracarboxylate [ka] Intermediate 46 (435 mg, 0.509 mmol) and 1-hydroxypyrrolidine-2,5-dione (64.4 mg, 0.560 mmol) were suspended in 5 mL of anhydrous DCM (5 mL) and 1M DCC in DCM (Aldrich, 534 μL, 0.534 mmol) was added. The resulting mixture was stirred at RT. After 15 min, a fine precipitate (ppt) formed, suggesting the formation of DCU. After 16 h, the reaction was complete as shown by LC / MS. Volatiles were removed by evaporation. The oily residue was purified by NPLC (eluted with 0-10% MeOH in DCM, silica, ELSD detection). The fractions containing the product were combined and concentrated to give intermediate 47. LCMS Method I: Rt=3.62 min, [M+H2O+H] + =970.1.

[0293] Intermediate 48: 18-(15-(benzyloxy)-15-oxopentadecyl)-18-((benzyloxy)carbonyl)-3,19-dioxo-1-phenyl-2,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74,77,80,83,86,89,92-pentacosaoxa-20-azapentanonacontan-95-oic acid [ka] Intermediate 47 (143 mg, 150 mmol) was dissolved in 1.5 mL of DCM in a 2-dram screw-cap vial along with 1-amino-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tetracosaoxapentaheptacontan-75-oic acid (198 mg, 0.173 mmol), DIPEA (48.5 μL, 0.375 mmol) and DMAP (2 mg, 0.109 mmol). The resulting mixture was stirred overnight. The volatiles were then removed and the resulting residue was purified via NPLC (eluted with 0-10% MeOH in DCM, silica). The product-containing fractions were combined and concentrated to give intermediate 48 as a clear semi-solid. LCMS method I: Rt=2.53min,[M+2H+2H2O] 2+ =1010.1.

[0294] Intermediate 49: 77,91-dibenzyl 1-(2,5-dioxopyrrolidin-1-yl) 77-(15-(benzyloxy)-15-oxopentadecyl)-76-oxo-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tetracosaoxa-75-azahennonacontane-1,77,91-tricarboxylate [ka] Intermediate 48 (210 mg, 0.106 mmol) and 1-hydroxypyrrolidine-2,5-dione (13.4 mg, 0.116 mmol) were suspended in 1 mL of anhydrous DCM while stirring in an oven-dried 10 mL round-bottom flask (RBF). To this mixture was added 1M DCC in DCM (Aldrich, 116 μL, 0.116 mmol). After 16 h, the reaction was complete as indicated by LC / MS. Volatiles were removed and the resulting residue was purified by NPLC (eluted with 0-15% MeOH in DCM, silica, ELSD detection). Fractions containing the desired product were combined and concentrated to give Intermediate 49 as a waxy solid. LCMS Method H: Rt=3.48 min, [M+H2O+2H]2+ =1049.9.

[0295] Intermediate 50: 15-((75-((2,5-dioxopyrrolidin-1-yl)oxy)-75-oxo-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tetracosaoxapentaheptacontyl)carbamoyl)nonacosane-1,15,29-tricarboxylic acid [ka] Intermediate 49 (140 mg, 0.067 mmol) was dissolved in anhydrous THF (1 mL) in a round bottom flask equipped with a stir bar and the resulting mixture was purged with N2 three times. Dry 10% Pd on carbon (7 mg, 6.73 μmol) and 20% Pd hydroxide on carbon (Aldrich) (5 mg, 6.73 umol) were then added, degassed and replaced with hydrogen from a balloon. The reaction mixture was stirred for 16 h. It was then degassed and replaced with N2. The reaction mixture was diluted with 5 mL of anhydrous DCM. After filtration through Celite®, the volatiles were removed to give Intermediate 50 as a viscous oil. LCMS Method D: Rt=1.36 min, [M+2H] 2+ =906.2.

[0296] Peptide synthesis: Standard synthesis techniques, e.g. solid phase peptide synthesis techniques as mentioned in Jose Palomo RSC Adv., 2014, 4, 32658-32672; Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 nd The GLP1 peptides may be synthesized using recombinant DNA techniques as described in Ed., Cold Spring Harbor (1989) and similar references.

[0297] GLP-1 analogue synthesis: [Fmoc-His7, Aib8, Arg34]GLP-1(7-37) [ka] Peptides were synthesized using standard Fmoc chemistry. 1) Resin preparation: 1-Chloro-2-[chloro(diphenyl)methyl]benzene (100 mmol, 1.00 equiv.) was added with Fmoc-Gly-OH (50 mol, 0.50 equiv.) and DIEA (400 mmol, 4.00 equiv.) in DCM (4.00 mL). The resulting mixture was stirred for 2 h at 25° C. under nitrogen atmosphere. Then, MeOH (100 mL) was added, and the mixture was stirred for another 30 min under nitrogen atmosphere. The resin was washed three times with DMF (500 mL). Then, 20% piperidine in DMF (500 mL) was added, and the mixture was stirred for 20 min at 25° C. under nitrogen atmosphere. The resulting mixture was filtered to obtain the resin. The resin was washed five times with DMF (500 mL), and then filtered to obtain the resin. 2) Coupling: A solution of DIEA (200 mmol, 4.00 equiv.), Fmoc-Arg(Pbf)-OH (100 mmol, 2.00 equiv.) and HBTU (95 mmol, 1.90 equiv.) in DMF (300 mL) was added to the resin and stirred for 30 min at 25° C. under nitrogen atmosphere. The resin was then washed three times with DMF (500 mL). 3) Deprotection: 20% piperidine in DMF (500 mL) was added to the resin, and the resulting mixture was stirred for 20 min under nitrogen atmosphere at 25° C. The resin was washed five times with DMF (500 mL) and filtered to obtain the resin.

[0298] The above coupling step 2 and deprotection step 3 were then repeated with additional amino acid units #3-31 to give GLP-1 or GLP-1 analogs.

[0299] [Table 26]

[0300] The coupling reaction was monitored by the ninhydrin test and the resin was washed five times with DMF.

[0301] Peptide cleavage and purification: 1) Cleavage buffer (92.5% TFA / 2.5% 3-mercaptopropionic acid / 2.5% TIS / 2.5% HO) was added to the flask containing the side-chain protected peptide at RT, followed by stirring for 2 h. 2) The precipitated peptide was washed with cold isopropyl ether. 3) The precipitated peptide was filtered and the filter cake was collected. 4) The precipitated peptide was washed two more times with isopropyl ether. 5) The crude peptide was then dried under vacuum for 2 h.

[0302] The crude peptide was purified by prep-HPLC (TFA conditions; 30 °C, A: 0.075% TFA in HO, B: elution with CHCN) and then by prep-HPLC (HOAc conditions, A: 0.5% HAc in HO, B: elution with ACN) to give [Fmoc-His7, Aib8, Arg34]GLP-1-(7-37) as a white solid.

[0303] Conjugation of fatty acid derivatives to peptides (via linkers): [ka] General procedure for peptide conjugation reactions: Method A: [Fmoc-His7, Aib8, Arg34]GLP-1-(7-37) was placed in DMA and the desired "fatty acid-linker conjugate" NHS-ester was added. The mixture was stirred at RT for 16-40 h. Once complete conversion was observed by LCMS analysis, 10 equivalents of piperidine were added and stirring was continued for 2 h to remove the Fmoc group.

[0304] The crude product was purified by HPLC (column: Waters XSelect C18 CSH 19 x 150 mm; 5 micron) and eluted with 0-100% ACN in water with 0.1% TFA modifier (30 mL / min) to give the TFA salt of the desired compound as a white fluffy solid. Residual TFA was removed by placing the compound in water with BT AG 1-XB Resin (cat#143-2446; BIO-RAD) and stirring the resulting mixture for 1 h. The mixture was then filtered and the resin was washed with acetonitrile and water. The solution was lyophilized to give the desired compound.

[0305] Method B: [Fmoc-His7, Aib8, Arg34]GLP-1-(7-37) was placed in DMF and the desired "fatty acid-linker conjugate" NHS fatty acid was added. The mixture was stirred at RT for 16-40 h. After complete conversion was observed by LCMS analysis, 10 equivalents of piperidine were added and stirring was continued for an additional 2 h to remove the Fmoc group. The crude product was diluted with 0.1 N aqueous ammonium carbonate and purified by RPLC (ISCO Gold C18 150 gram column, eluted with 10-100% ACN in water, 0.1% formic acid modifier). Pure fractions containing the desired product were combined and lyophilized to give the desired compound.

[0306] Compound 1 (diastereomeric mixture) [ka] To a solution of [Fmoc-His7, Aib8, Arg34]GLP-1-(7-37) (356.6 mg, 0.099 mmol) in anhydrous DMF (9.8 mL) was added a solution of intermediate 6 (197 mg, 0.118 mmol) in 1.5 mL of anhydrous DMF. After 16 h, the reaction had proceeded to approximately 85% conversion, so an additional 0.2 eq of intermediate 6 was added (32.9 mg in 0.5 mL anhydrous DMF). After 48 h, the reaction was complete. Fmoc removal was then initiated by the addition of piperidine (98 μL, 0.985 mmol, 10 eq). After 16 h, Fmoc removal was complete and the solvent was removed under reduced pressure. The crude product above was placed in 25 mL of 0.1 N aqueous ammonium carbonate and injected (twice) onto an ISCO RediSep Gold C18Aq 100 Gram column (cat. no. 69-2203-562) and eluted with 0-100% ACN in water as eluent with 0.1% TFA modifier. The fractions containing the product were lyophilized to give a fluffy white powder. To remove residual TFA, 700 mg of hydroxide resin (BioRad AG 1-X8) was weighed into an Eppendorf tube and rinsed with 5 x 2 mL of 1:1 ACN:H2O, 0.1% formic acid, removing the supernatant and discarding between each rinse (centrifugation to precipitate the resin). The rinsed resin was added to the product solution prepared above and shaken for 1 h. The supernatant was filtered and then rinsed with 10 mL of 1:1 ACN:H2O with 0.1% formic acid. The solution was lyophilized to give compound 1 as a white powder. LCMS Method J: Found m / z = 2474.8447 (MH2 2+ ), Rt: 1.16 min; calculated mass: 4947.6750. LCMS Method K: Found m / z = 4948.7002 (M−H) + , Rt: 2.31 min; calculated mass: 4947.6750.

[0307] Compound 2: Diastereomer 1 [ka] Compound 2 was synthesized using the general procedure for conjugation reactions, Method B, and intermediate 15A (S-enantiomer) as the starting material. LCMS Method F: Measured m / z = 1238.5 (MH4 4+ ), Rt: 2.25 min; calculated mass: 4947.6750. LCMS Method K: Measured value 4948.7002 (MH + ), Rt: 2.31 min; calculated mass: 4947.6750.

[0308] Alternative method for synthesizing compound 2: Similar to the reactions described for converting intermediate 3 to 4, then to 5, and finally to intermediate 6, an alternative method for synthesizing compound 2 starts with intermediate 3B (S-enantiomer), which is converted to 4B (=S-enantiomer of intermediate 4), then to 5B (=S-enantiomer of intermediate 5), and finally to intermediate 6B (=S-enantiomer of intermediate 6). Then, following the general procedure for conjugation reactions, intermediate 6B and [Fmoc-His7, Aib8, Arg34]GLP-1-(7-37) are reacted to give compound 2.

[0309] By using single X-ray crystallography of a derivative of the enantiomerically pure intermediate 3B, which was used as the starting material for the synthesis of compound 2, the absolute configuration in the fatty acid moiety for compound 2 was determined to be S.

[0310] Compound 3: Diastereomer 2 [ka] Compound 3 was synthesized using the general procedure for conjugation reactions, Method B, using intermediate 15B (R-enantiomer) as the starting material. LCMS Method F: Measured m / z = 1238.6 (MH4 4+ ), Rt: 2.29 min; calculated mass: 4947.6750. LCMS Method K: Measured value 4948.7002 (MH + ), Rt: 2.31 min; calculated mass: 4947.6750.

[0311] Alternative method for synthesizing compound 3: Similar to the reactions described for the conversion of intermediate 3 to 4, then to 5 and finally to intermediate 6, an alternative approach to compound 3 starts with intermediate 3A (R-enantiomer), which is converted to 4A (=R-enantiomer of intermediate 4), then to 5A (=R-enantiomer of intermediate 5) and finally to intermediate 6A (=R-enantiomer of intermediate 6). Intermediate 6A and [Fmoc-His7, Aib8, Arg34]GLP-1-(7-37) are then reacted according to the general procedure for the conjugation reaction to give compound 3.

[0312] The absolute configuration in the fatty acid moiety for compound 3 was determined to be R. This was determined by single X-ray crystallography of an enantiomerically pure derivative of intermediate 3A.

[0313] Compound 4: [ka] Compound 4 was synthesized using intermediate 19 using general procedure for conjugation reactions, Method A. LCMS Method F: Measured m / z = 1061.7 (MH4 4+ ), Rt: 2.26 min; calculated mass: 4243.2556.

[0314] Compound 5: [ka] Compound 5 was synthesized using intermediate 22 using general procedure for conjugation reactions, Method B. LCMS Method F: Measured m / z = 996.6 (MH4 4+ ), Rt: 2.34 min; calculated mass: 3979.0983.

[0315] Compound 6: [ka] Compound 6 was synthesized using intermediate 29 using general procedure for conjugation reactions, Method B. LCMS method F: Actual m / z=2490.9(MH2 2+ ), Rt: 2.09 min; calculated mass: 4977.6495. LCMS Method K: Measured value 4978.6602 (MH + ), Rt: 2.09 min; calculated mass: 4977.6495.

[0316] Compound 7: [ka] Compound 7 was synthesized using intermediate 36 using general procedure for conjugation reactions, Method B. LCMS Method F: Measured m / z = 1253.1 (MH4 4+ ), Rt: 2.13 min; Calculated mass: 5005.6805 LCMS method K: Actual measurement 5006.7100 (MH + ), Rt: 2.15min; Calculated mass: 5005.6805

[0317] Compound 8: [ka] Compound 8 was synthesized using intermediate 43 using general procedure for conjugation reactions, Method B. LCMS Method K: Measured value 5062.7700 (MH + ), Rt: 2.30 min; calculated mass: 5061.7431.

[0318] Compound 9: [ka] Compound 9 was synthesized using intermediate 50 using general procedure for conjugation reactions, Method A. LCMS Method J: Measured m / z = 1274.0 (MH4 4+), Rt: 1.18 min; calculated mass: 5089.7744.

[0319] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments specifically described herein, which equivalents are intended to be encompassed by the following claims.

[0320] Appendix 1 (Sequence Listing) GLP-1(7-37): SEQ ID NO: 1: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly SEQ ID NO: 2: Xaa7-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Xaa 16 -Ser-Xaa 18 -Xaa 19 -Xaa 20 -Glu-Xaa 22 -Xaa 23 -Ala-Xaa 25 -Arg-Xaa 27 -Phe-lle-Xaa 30 -Trp-Leu-Xaa 33 -Xaa 34 -Xaa 35 -Xaa 36 -Xaa 37 Xaa 16 is Val or Leu; Xaa 18 is Ser, Lys or Arg; Xaa 19 is Tyr or Gln Xaa 20 is Leu or Met; Xaa 22 is Gly, Glu, or Aib; Xaa 23 is Gln, Glu, Lys or Arg; Xaa 25is Ala or Val; Xaa 27 is Glu or Leu; Xaa 30 is Ala, Glu or Arg; Xaa 33 is Val or Lys; Xaa 34 is Lys, Glu, Asn, or Arg; Xaa 35 is Gly or Aib; Xaa 36 is Arg, Gly or Lys or absent; and Xaa 37 is Gly, Ala, Glu, Pro, Lys or absent. [Aib8, Arg34]GLP-1(7-37): SEQ ID NO: 3: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly [Arg34]GLP-1(7-37): SEQ ID NO: 4: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly [ka] [ka] [ka] [ka]

Claims

1. Formula (I): 【Chemistry 1】 (In the formula, R １ and R ２ are independently -CH ３ , —OH, —CO ２ H, CH=CH ２ and -C≡CH; n and m are each an integer independently selected from 5 to 30; L is a linker; and P is GLP-1 or a GLP-1 analogue. or a pharma- ceutically acceptable salt thereof.

2. 2. The compound of formula (I) according to claim 1, or a pharma- ceutically acceptable salt thereof, wherein P is bonded to L via an -NH group.

3. The compound of formula (I) according to claim 1, or a pharma- ceutically acceptable salt thereof, wherein P is bonded to L via an -NH group, said NH group being part of an amino acid side chain of P.

4. L is, 【Chemistry 2】 (In the formula, y is an integer selected from 1 to 36; s is 0, 1 or 2 and k is 1, 2 or 3; and ＊＊ indicates a link to the -C(O)- group of the compound of formula (I), and ＊＊＊ The wavy line marked indicates the connection to P.

2. The compound of formula (I) according to claim 1, selected from the group consisting of:

5. L is, 【Chemistry 3】 wherein y is an integer selected from 1 to 36.

5. The compound of formula (I) according to claim 4, which is: or a pharma- ceutically acceptable salt thereof.

6. Formula (II) 【Chemistry 4】 (In the formula, NH-P′ represents P linked to the —C(O) group of L via an NH group; R １ and R ２ are independently -CH ３ , —OH and —CO ２ H; n and m are each an integer independently selected from 5 to 30; and y is an integer selected from 1 to 36. or a pharma- ceutically acceptable salt thereof.

7. The compound of formula (I) according to claim 1, wherein R 1 is -CO 2 H and R 2 is -CH 3 , or R 1 is -CO 2 H and R 2 is -CO 2 H, or a pharma- ceutically acceptable salt thereof.

8. The compound of formula (I) of claim 1, or a pharma- ceutically acceptable salt thereof, wherein n and m are integers independently selected from 10, 11, 13 and 14, respectively.

9. R １ Ha-CO ２ H, and R ２ Ha-CH ３ n is 10 and m is 10; R １ Ha-CO ２ H, and R ２ Ha-CO ２ H; n is 10 and m is 10; R １ Ha-CO ２ H, and R ２ Ha-CO ２ H; n is 10 and m is 11; R １ Ha-CO ２ H, and R ２ Ha-CO ２ H; n is 10 and m is 13; or R １ Ha-CO ２ H, and R ２ Ha-CO ２ H; n is 10, and m is 14. The compound of formula (II) according to claim 6,

10. Formula (III) 【Chemistry 5】 (In the formula, R １ is -CO ２ H, and R ２ is -CH ３ (It is) or a pharma- ceutically acceptable salt thereof.

11. Formula (IV) 【Chemistry 6】 wherein y is an integer selected from 1 to 36. or a pharma- ceutically acceptable salt thereof, The compounds may exist as racemates or stereochemically enriched mixtures, or ＊ 11. The compound of formula (III) according to claim 10, which is stereochemically pure with respect to the carbon atom marked:

12. Compound of formula (IVa) or compound of formula (IVb) 【Chemistry 7】 wherein y is an integer selected from 1 to 36. or a pharma- ceutically acceptable salt thereof.

13. The compound of formula (IVa) or the compound of formula (IVb) according to claim 12, wherein y is an integer selected from 2 to 24.

14. 14. A compound of formula (IVa) or a compound of formula (IVb) according to claim 13, wherein y is 24.

15. P is, GLP-1 (7-37): His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu- Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly (SEQ ID NO: 1), and GLP-1 analogs that contain a non-natural amino acid residue at position 7 or position 8, or at positions 7 and 8, as compared to the sequence GLP-1(7-37).

2. The compound of claim 1, selected from the group consisting of:

16. P is, [Aib8, Arg34] GLP-1 (7-37): His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Le u-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly (SEQ ID NO: 3); and [Arg34]GLP-1(7-37): His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu -Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly (SEQ ID NO: 4) 2. The compound of formula (I) according to claim 1, selected from the group consisting of:

17. P is the following: 【Chemistry 8】 or a pharma- ceutically acceptable salt thereof, wherein the compound of formula (I) according to claim 1 is [Aib8,Arg34]GLP-1(7-37) as shown in the formula:

18. 【Chemical 9】 wherein y is an integer selected from 1 to 36. and the compound exists as a diastereomeric mixture, a stereochemically enriched mixture, or ＊ 2. A compound of formula (I) as claimed in claim 1, which is stereochemically pure with respect to the carbon atom marked: or a pharma- ceutically acceptable salt thereof.

19. 【Catalog 10】 (Compound 1), 【Chemistry 11】 (Compound 2), 【Chemistry 12】 (Compound 3), 【Chemistry 13】 (Compound 4), 【Chemistry 14】 (Compound 5), 【Chemistry 15】 (Compound 6), 【Chemistry 16】 (Compound 7), 【Chemistry 17】 (Compound 8), and 【Chemistry 18】 (Compound 9) 2. The compound of formula (I) according to claim 1, selected from the group consisting of:

20. 【Chemical 19】 20. The compound of formula (I) according to claim 19, which is: or a pharma- ceutically acceptable salt thereof.

21.

20. 20. The compound of formula (I) according to claim 19, which is: or a pharma- ceutically acceptable salt thereof.

22. 【Chemical 21】 20. The compound of formula (I) according to claim 19, which is: or a pharma- ceutically acceptable salt thereof.

23. 10. A pharmaceutical composition comprising a compound of claim 1, or a pharma- ceutically acceptable salt thereof, and one or more pharma- ceutically acceptable carriers.

24. 13. A combination comprising a therapeutically effective amount of a compound of claim 1, or a pharma- ceutically acceptable salt thereof, and one or more therapeutically active agents.

25. 25. The combination of claim 24, wherein the compound is selected from the group consisting of compounds 1, 2 and 3, or a pharma- ceutically acceptable salt thereof.

26. 11. A pharmaceutical composition for treating a patient in need of treatment who is sensitive to an agonist of the glucagon-like peptide 1 receptor (GLP1R), comprising a therapeutically effective amount of a compound according to claim 1 or a pharma- ceutical acceptable salt thereof.

27. A pharmaceutical composition for treating a disease or disorder selected from obesity, type 2 diabetes, insulin resistance, hyperinsulinemia, impaired glucose tolerance, hyperglycemia, one or more diabetic complications selected from chronic kidney disease and diabetic nephropathy, dyslipidemia, metabolic syndrome, progressive liver disease selected from NAFLD and NASH, cardiovascular disease, and peripheral neuropathy associated with diabetes, comprising a therapeutically effective amount of a compound according to claim 1 or a pharma- ceutical acceptable salt thereof.

28. 28. The pharmaceutical composition of claim 27, wherein the cardiovascular disease is selected from hypertension, atherosclerosis, peripheral artery disease, stroke, cardiomyopathy, atrial fibrillation, heart failure selected from heart failure with reduced ejection fraction (HFrEF), heart failure with intermediate ejection fraction (HFmrEF) and heart failure with preserved ejection fraction (HFpEF), coronary heart disease and arrhythmias selected from atrial arrhythmias and ventricular arrhythmias.

29. 28. The pharmaceutical composition of claim 27, wherein the compound is selected from the group consisting of compounds 1, 2 and 3, or a pharma- ceutically acceptable salt thereof.

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