Glucagon and GLP-1 co-agonist compounds

Abstract

The present invention relates to glucagon and GLP-1 co-agonist compounds. The present invention provides glucagon and GLP-1 co-agonist compounds useful in treating type II diabetes, obesity, non-alcoholic fatty liver disease (NAFLD) and / or non-alcoholic steatohepatitis (NASH).

Description

[0001] This application is a divisional application of Chinese patent application 201680036771.3 "Glucagon and GLP-1 synergistic agonist compounds" with a filing date of June 16, 2016. Technical Field

[0002] The present invention belongs to the field of medicine. More specifically, the present invention belongs to the field of treating diabetes and obesity, and relates to compounds that stimulate both the glucagon (Gcg) receptor and the glucagon-like peptide-1 (GLP-1) receptor. Specifically provided are oxyntomodulin / glucagon analogs having amino acid modifications introduced for regulating the activity of both the Gcg receptor and the GLP-1 receptor. Background Art

[0003] Over the past decades, the prevalence of diabetes has continued to rise. Type II diabetes (T2D) is the most common form of diabetes, which contributes to about 90% of all diabetes. T2D is characterized by high blood sugar levels caused by insulin resistance. The current standard for treating T2D includes dieting and exercise and available oral or injectable hypoglycemic drugs. Nevertheless, many patients suffering from T2D are still not fully controlled. Uncontrolled diabetes leads to a variety of conditions, which affect the incidence and mortality of patients. The leading cause of death in diabetic patients is cardiovascular complications. One of the main risk factors for type II diabetes is obesity. Most T2D patients (~90%) are overweight or obese. It has been recorded that the reduction in body obesity will lead to improvements in the secondary complications of obesity-related diseases including hyperglycemia and cardiovascular events. Therefore, for better disease management, effective treatment is needed in terms of blood sugar control and weight reduction.

[0004] Many peptides derived from preproglucagon and its analogs have been proposed as therapeutic approaches for treating T2D and obesity, particularly Gcg, GLP-1, and oxyntomodulin (OXM). Preproglucagon is a 158 amino acid precursor polypeptide that is differentiated in tissues to form a variety of structurally related proglucagon-derived peptides, including Gcg, GLP-1, glucagon-like peptide-2 (GLP-2), and oxyntomodulin (OXM). These molecules are involved in a wide variety of physiological functions, including blood glucose homeostasis, insulin secretion, gastric emptying and intestinal development, and food intake regulation.

[0005] Gcg is a 29 amino acid peptide that corresponds to amino acids 53 to 81 of preproglucagon. OXM is a 37 amino acid peptide and consists of the complete 29 amino acid sequence of Gcg with an octapeptide carboxyl-terminal extension (amino acids 82 to 89 of preproglucagon and is referred to as "insertion peptide 1" or IP-1). The major biologically active fragment of GLP-1 (GLP-1 7-36) is produced as a 30 amino acid C-terminally amidated peptide corresponding to amino acids 98 to 127 of preproglucagon.

[0006] Gcg helps maintain blood sugar levels by binding to Gcg receptors on liver cells, which causes the liver to release glucose stored in the form of glycogen through glycogenolysis. As these stores are depleted, Gcg stimulates the liver to synthesize additional glucose through gluconeogenesis. This glucose is released into the bloodstream, avoiding the development of hypoglycemia.

[0007] GLP-1 has different biological activities compared to Gcg. Its effects include stimulating insulin synthesis and secretion, inhibiting Gcg secretion and inhibiting food intake. GLP-1 has been shown to reduce hyperglycemia in diabetes. A variety of GLP-1 agonists have been approved for the treatment of T2D in humans, including exenatide, liraglutide, lixisenatide, albiglutide and dulaglutide. Such GLP-1 agonists are effective in blood sugar control, with favorable effects on body weight without the risk of hypoglycemia. However, due to dose-dependent gastrointestinal side effects, weight loss is moderate.

[0008] OXM is released from L cells in the small intestine with GLP-1 in proportion to nutrient intake. OXM activates both Gcg and GLP-1 receptors, and has slightly higher efficacy for Gcg receptors than GLP-1 receptors. It has lower efficacy for each receptor compared with natural Gcg and GLP-1. Human Gcg is also able to activate both receptors, although there is a stronger preference for Gcg receptors than for GLP-1 receptors. GLP-1 cannot activate Gcg receptors. OXM is involved in the regulation of food intake and body weight. It has been shown in people to suppress appetite and inhibit food intake. In a 4-week study of overweight and obese subjects, subcutaneous administration of OXM three times a day before meals produced a weight loss of 2.3 kg compared to 0.5 kg in the placebo group. In this trial, the most common side effects associated with GLP-1-based treatments (such as exenatide and liraglutide), i.e., nausea, were less frequent. In another shorter study, OXM showed reduced caloric intake and increased energy expenditure associated with activity in overweight and obese subjects.

[0009] These data suggest that OXM has the potential to be a well-tolerated antidiabetic / obesity drug. However, OXM presents many challenges for development as a commercially viable therapeutic agent. Endogenous OXM is rapidly degraded in vivo by dipeptidyl peptidase IV and other peptidases, and is subject to rapid renal clearance due to its small size. Therefore, it is desirable to identify peptides that activate Gcg and GLP-1 receptors and have improved metabolic stability and reduced clearance.

[0010] In the prior art, OXM peptides with amino acid substitutions to improve stability and with additional modifications, such as PEGylation or lipidation to slow clearance, are disclosed. Other peptides are also described to bind to and activate both the Gcg receptor and the GLP-1 receptor, and suppress weight gain (see, for example, WO 2011 / 075393 A2 and WO 2012 / 177444 A2).

[0011] In addition to the availability of multiple peptides that agonize both Gcg and GLP-1 receptors, there is a need for more potent, stable, long-acting and / or well-tolerated compounds having an optimized ratio of Gcg receptor (Gcg-R) / GLP-1 receptor (GLP-1-R) activity such that the efficacy and insulinotropic activity of the compound provide effective treatment for diabetes, preferably T2D and related conditions. Specifically, there is a need for compounds with a balanced ratio of Gcg-R / GLP-1-R co-agonist activity for weight loss. And, there is a need to provide compounds with a balanced ratio of Gcg-R / GLP-1-R co-agonist activity that supports potential daily, biweekly, weekly or monthly dosing in humans. Accordingly, the present invention seeks to provide effective treatments for diabetes, obesity, non-alcoholic fatty liver disease (NAFLD) and / or non-alcoholic steatohepatitis (NASH). Summary of the invention

[0012] In one aspect, the present invention provides a compound of the following formula or a pharmaceutically acceptable salt thereof:

[0013] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Phe-Val-Glu-Trp-Leu-Leu-Xaa28-Gly-Gly-Pro-Ser-Ser-Gly

[0014] in

[0015] Xaa2 is Aib;

[0016] Xaa28 is Glu or Ser;

[0017] The Lys at position 20 is chemically modified by conjugating the ε-amino group of the Lys side chain to a C14-C24 fatty acid via (i) a direct bond or (ii) a linker between the Lys at position 20 and the C14-C24 fatty acid; and

[0018] The C-terminal amino acid is optionally amidated (SEQ ID NO: 2).

[0019] In another aspect, the present invention provides a compound of the following formula or a pharmaceutically acceptable salt thereof:

[0020] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly

[0021] in

[0022] Xaa2 is Aib;

[0023] The Lys at position 20 is chemically modified by conjugating the ε-amino group of the Lys side chain to a C14-C24 fatty acid via (i) a direct bond or (ii) a linker between the Lys at position 20 and the C14-C24 fatty acid; and

[0024] The C-terminal amino acid is optionally amidated (SEQ ID NO: 3).

[0025] In yet another aspect, the present invention provides a compound of the following formula or a pharmaceutically acceptable salt thereof:

[0026] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Phe-Val-Glu-Trp-Leu-Leu-Ser-Gly-Gly-Pro-Ser-Ser-Gly

[0027] in

[0028] Xaa2 is Aib;

[0029] The Lys at position 20 is chemically modified by conjugating the ε-amino group of the Lys side chain to a C14-C24 fatty acid via (i) a direct bond or (ii) a linker between the Lys at position 20 and the C14-C24 fatty acid; and

[0030] The C-terminal amino acid is optionally amidated (SEQ ID NO: 4).

[0031] In a preferred aspect of the compound of the present invention or a pharmaceutically acceptable salt thereof, Lys at position 20 is chemically modified by conjugation with a C14-C24 fatty acid via a linker between Lys at position 20 and the C14-C24 fatty acid.

[0032] Further preferably, the linker is selected from:

[0033] (a) aminopolyethylene glycol carboxylates of formula I:

[0034] H-{NH-CH2-CH2-[O-CH2-CH2] m -O-(CH2) p -CO} n -OH (I)

[0035] wherein m is any integer from 1 to 12, n is any integer from 1 to 12, and p is 1 or 2;

[0036] (b) an amino acid selected from the group consisting of arginine (Arg), asparagine (Asn), aspartic acid (Asp), glutamine (Gln), glutamic acid (Glu), histidine (His), lysine (Lys), serine (Ser), threonine (Thr), citrulline (Cit), ornithine (Orn), sarcosine (Sar), glycine (Gly), γ-aminobutyric acid (γ-Abu) and γ-glutamic acid (γ-Glu);

[0037] (c) a dipeptide selected from the group consisting of Ala-Ala, β-Ala-β-Ala, Glu-Glu, Gly-Gly, Leu-Leu, Ser-Ser, Thr-Thr, γ-Glu-γ-Glu, Glu-γ-Glu, γ-Glu-Glu, γ-Abu-γ-Abu, 6-aminohexanoic acid-6-aminohexanoic acid, 5-aminopentanoic acid-5-aminopentanoic acid, 7-aminoheptanoic acid-7-aminoheptanoic acid, and 8-aminooctanoic acid-8-aminooctanoic acid;

[0038] (d) Selected from Ala-Ala-Ala, β-Ala-β-Ala-β-Ala, Glu-Glu-Glu, γ-Glu-γ-Glu-γ-Glu, Glu-γ-Glu-γ-Glu, γ-Glu-γ-Glu-Glu, γ-Glu-Glu-γ-Glu, Gly-Gly-Gly, Gly-Gly-Ser , Ser-Gly-Gly, Gly-Ser-Gly, Gly-Gly-Glu, Glu-Gly-Gly, Gly-Glu-Gly, Gly-Gly-γ-Glu, γ-Glu-Gly-Gly, Gly-γ-Glu-Gly, Leu-Leu-Leu and γ-Abu-γ-Abu-γ-Abu tripeptides;

[0039] (e) selected from (Gly-Gly-Ser) q (Gly-Gly-Gly-Ser) r and (Gly-Gly-Gly-Gly-Ser) r 、(6-aminocaproic acid) s , (5-aminovaleric acid) s 、(7-aminoheptanoic acid) s and (8-aminooctanoic acid) s A polypeptide wherein q is any integer from 2 to 5, r is any integer from 1 to 3, and s is any integer from 4 to 15; and

[0040] (f) a conjugated linker, wherein the aminopolyethylene glycol carboxylate of formula I as defined in (a) is conjugated to:

[0041] (i) an amino acid selected from the group consisting of Arg, Asn, Asp, Gln, Glu, His, Lys, Ser, Thr, Cit, Orn, Sar, Gly, γ-Abu and γ-Glu;

[0042] (ii) a dipeptide selected from the group consisting of Ala-Ala, β-Ala-β-Ala, Glu-Glu, Gly-Gly, Leu-Leu, Ser-Ser, Thr-Thr, γ-Glu-γ-Glu, Glu-γ-Glu, γ-Glu-Glu, γ-Abu-γ-Abu, 6-aminohexanoic acid-6-aminohexanoic acid, 5-aminopentanoic acid-5-aminopentanoic acid, 7-aminoheptanoic acid-7-aminoheptanoic acid, and 8-aminooctanoic acid-8-aminooctanoic acid;

[0043] (iii) Selected from Ala-Ala-Ala, β-Ala-β-Ala-β-Ala, Glu-Glu-Glu, γ-Glu-γ-Glu-γ-Glu, Glu-γ-Glu-γ-Glu, γ-Glu-γ-Glu-Glu, γ-Glu-Glu-γ-Glu, Gly-Gly-Gly, Gly-Gly-Se or

[0044] (iv) selected from (Gly-Gly-Ser) q (Gly-Gly-Gly-Ser) r and (Gly-Gly-Gly-Gly-Ser) r 、(6-aminocaproic acid) s , (5-aminovaleric acid) s 、(7-aminoheptanoic acid) s and (8-aminooctanoic acid) s A polypeptide wherein q is any integer from 2 to 5, r is any integer from 1 to 3, and s is any integer from 4 to 15.

[0045] In a preferred aspect of the compound of the present invention or a pharmaceutically acceptable salt thereof, the linker is an aminopolyethylene glycol carboxylate of formula I:

[0046] H-{NH-CH2-CH2-[O-CH2-CH2] m -O-(CH2) p -CO} n -OH (I)

[0047] wherein m is any integer from 1 to 12, n is any integer from 1 to 12, and p is 1 or 2.

[0048] Preferably, for the aminopolyethylene glycol carboxylates of formula I, n is 1, 2, 3, 4, 5 or 6, and m is 1, and p is 1.

[0049] Further preferably, for the aminopolyethylene glycol carboxylate of formula I, n is 2, m is 1, and p is 1.

[0050] In a further preferred aspect of the compound of the present invention or a pharmaceutically acceptable salt thereof, the linker is an amino acid selected from Arg, Asn, Asp, Gln, Glu, His, Lys, Ser, Thr, Cit, Orn, Sar, Gly, γ-Abu and γ-Glu.

[0051] Preferably, the amino acid is γ-Glu.

[0052] In a further preferred aspect of the compound of the present invention or a pharmaceutically acceptable salt thereof, the linker is a dipeptide selected from Ala-Ala, β-Ala-β-Ala, Glu-Glu, Gly-Gly, Leu-Leu, Ser-Ser, Thr-Thr, γ-Glu-γ-Glu, Glu-γ-Glu, γ-Glu-Glu, γ-Abu-γ-Abu, 6-aminohexanoic acid-6-aminohexanoic acid, 5-aminopentanoic acid-5-aminopentanoic acid, 7-aminoheptanoic acid-7-aminoheptanoic acid and 8-aminooctanoic acid-8-aminooctanoic acid.

[0053] Preferably, the dipeptide is γ-Glu-γ-Glu.

[0054] In a still further preferred aspect of the compounds of the invention or pharmaceutically acceptable salts thereof, the linker is a tripeptide selected from Ala-Ala-Ala, β-Ala-β-Ala-β-Ala, Glu-Glu-Glu, γ-Glu-γ-Glu-γ-Glu, Glu-γ-Glu-γ-Glu, γ-Glu-γ-Glu-Glu, γ-Glu-Glu-γ-Glu, Gly-Gly-Gly, Gly-Gly-Ser, Ser-Gly-Gly, Gly-Ser-Gly, Gly-Gly-Glu, Glu-Gly-Gly, Gly-Glu-Gly, Gly-Gly-γ-Glu, γ-Glu-Gly-Gly, Gly-γ-Glu-Gly, Leu-Leu-Leu and γ-Abu-γ-Abu-γ-Abu.

[0055] In a further preferred aspect of the compounds of the present invention or their pharmaceutically acceptable salts, the linker is selected from (Gly-Gly-Ser) q (Gly-Gly-Gly-Ser) r and (Gly-Gly-Gly-Gly-Ser) r 、(6-aminocaproic acid) s , (5-aminovaleric acid) s 、(7-aminoheptanoic acid) s and (8-aminooctanoic acid) sA polypeptide wherein q is any integer from 2 to 5, r is any integer from 1 to 3, and s is any integer from 4 to 15.

[0056] In a further preferred aspect of the compounds of the present invention or pharmaceutically acceptable salts thereof, the linker is a conjugated linker wherein the aminopolyethylene glycol carboxylate of formula I

[0057] H-{NH-CH2-CH2-[O-CH2-CH2] m -O-(CH2) p -CO} n -OH (I)

[0058] wherein m is any integer from 1 to 12, n is any integer from 1 to 12, and p is 1 or 2;

[0059] Conjugated with:

[0060] (i) an amino acid selected from the group consisting of Arg, Asn, Asp, Gln, Glu, His, Lys, Ser, Thr, Cit, Orn, Sar, Gly, γ-Abu and γ-Glu;

[0061] (ii) a dipeptide selected from the group consisting of Ala-Ala, β-Ala-β-Ala, Glu-Glu, Gly-Gly, Leu-Leu, Ser-Ser, Thr-Thr, γ-Glu-γ-Glu, Glu-γ-Glu, γ-Glu-Glu, γ-Abu-γ-Abu, 6-aminohexanoic acid-6-aminohexanoic acid, 5-aminopentanoic acid-5-aminopentanoic acid, 7-aminoheptanoic acid-7-aminoheptanoic acid, and 8-aminooctanoic acid-8-aminooctanoic acid;

[0062] (iii) Selected from Ala-Ala-Ala, β-Ala-β-Ala-β-Ala, Glu-Glu-Glu, γ-Glu-γ-Glu-γ-Glu, Glu-γ-Glu-γ-Gl u, γ-Glu-γ-Glu-Glu, γ-Glu-Glu-γ-Glu, Gly-Gly-Gly, Gly-Gly-Ser, Ser-Gly-Gly, Gly-Ser-Gly, or the tripeptides Gly-Gly-Glu, Glu-Gly-Gly, Gly-Glu-Gly, Gly-Gly-γ-Glu, γ-Glu-Gly-Gly, Gly-γ-Glu-Gly, Leu-Leu-Leu, and γ-Abu-γ-Abu-γ-Abu; or

[0063] (iv) selected from (Gly-Gly-Ser) q (Gly-Gly-Gly-Ser)r and (Gly-Gly-Gly-Gly-Ser) r 、(6-aminocaproic acid) s , (5-aminovaleric acid) s 、(7-aminoheptanoic acid) s and (8-aminooctanoic acid) s A polypeptide wherein q is any integer from 2 to 5, r is any integer from 1 to 3, and s is any integer from 4 to 15.

[0064] Preferably, for the aminopolyethylene glycol carboxylates of formula I, n is 1, 2, 3, 4, 5 or 6, and m is 1, and p is 1.

[0065] Further preferably, for the aminopolyethylene glycol carboxylate of formula I, n is 2, m is 1, and p is 1.

[0066] Still further preferably, the amino acid is γ-Glu.

[0067] Still further preferably, the dipeptide is γ-Glu-γ-Glu.

[0068] In a preferred embodiment of the compound of the present invention, the linker is ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu) t , where t is 1 or 2.

[0069] Preferably, t is 1.

[0070] More preferably, t is 2.

[0071] In a still further preferred aspect of the compounds of the present invention or pharmaceutically acceptable salts thereof, the C14-C24 fatty acid is a saturated monobasic acid or a saturated dibasic acid.

[0072] Preferably, the fatty acid is selected from myristic acid (tetradecanoic acid) (C14 monoacid), tetradecanedioic acid (C14 diacid), palmitic acid (hexadecanoic acid) (C16 monoacid), hexadecanedioic acid (C16 diacid), heptadecanoic acid (heptadecanoic acid) (C17 monoacid), heptadecanedioic acid (C17 diacid), stearic acid (octadecanoic acid) (C18 monoacid), octadecanedioic acid (C18 diacid), nonadecanoic acid (nonadecanoic acid) (C19 monoacid), decadecanedioic acid (C19 diacid), octadecanoic acid (octadecanoic acid) (C18 monoacid), octadecanedioic acid (C18 diacid), Saturated monoacids or saturated diacids of nonacanedioic acid (C19 diacid), arachidic acid (eicosanoic acid) (C20 monoacid), eicosanoic acid (C20 diacid), heneicosanoic acid (heneicosanoic acid) (C21 monoacid), heneicosanoic acid (C21 diacid), behenic acid (docosanoic acid) (C22), docosanoic acid (C22 diacid), tetracosanoic acid (tetracosanoic acid) (C24 monoacid) and tetracosanoic acid (C24 diacid).

[0073] Still further preferably, the C14-C24 fatty acid is myristic acid.

[0074] Still further preferably, the C14-C24 fatty acid is tetradecanedioic acid.

[0075] Still further preferably, the C14-C24 fatty acid is palmitic acid.

[0076] Still further preferably, the C14-C24 fatty acid is hexadecanedioic acid.

[0077] Still further preferably, the C14-C24 fatty acid is stearic acid.

[0078] Still further preferably, the C14-C24 fatty acid is octadecanedioic acid.

[0079] Still further preferably, the C14-C24 fatty acid is nonadecanedioic acid.

[0080] Still further preferably, the C14-C24 fatty acid is arachidic acid.

[0081] Still further preferably, the C14-C24 fatty acid is eicosanedioic acid.

[0082] Still further preferably, the C14-C24 fatty acid is docosandioic acid.

[0083] In a still further preferred aspect of the compound of the present invention or a pharmaceutically acceptable salt thereof, the C-terminal amino acid is amidated.

[0084] In a further aspect, the present invention provides a compound of the following formula or a pharmaceutically acceptable salt thereof:

[0085] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly

[0086] wherein Xaa2 is Aib;

[0087] Lys at position 20 is synthesized by reacting the ε-amino group of the Lys side chain with ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2) 16 Chemically modified by CO2H conjugation; and

[0088] The C-terminal amino acid was amidated (SEQ ID NO: 5).

[0089] In a further aspect, the present invention provides a compound of the following formula or a pharmaceutically acceptable salt thereof:

[0090] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly

[0091] wherein Xaa2 is Aib;

[0092] Lys at position 20 is synthesized by reacting the ε-amino group of the Lys side chain with ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2) 18 Chemically modified by CO2H conjugation; and

[0093] The C-terminal amino acid was amidated (SEQ ID NO: 6).

[0094] In a further aspect, the present invention provides a compound of the following formula or a pharmaceutically acceptable salt thereof:

[0095] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Phe-Val-Glu-Trp-Leu-Leu-Ser-Gly-Gly-Pro-Ser-Ser-Gly

[0096] wherein Xaa2 is Aib;

[0097] Lys at position 20 is synthesized by reacting the ε-amino group of the Lys side chain with ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2) 16 Chemically modified by CO2H conjugation; and

[0098] The C-terminal amino acid was amidated (SEQ ID NO: 7).

[0099] In a further aspect, the present invention provides a compound of the following formula or a pharmaceutically acceptable salt thereof:

[0100] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Phe-Val-Glu-Trp-Leu-Leu-Ser-Gly-Gly-Pro-Ser-Ser-Gly

[0101] wherein Xaa2 is Aib;

[0102] Lys at position 20 is synthesized by reacting the ε-amino group of the Lys side chain with ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2) 18 Chemically modified by CO2H conjugation; and

[0103] The C-terminal amino acid was amidated (SEQ ID NO: 8).

[0104] In a further aspect, the present invention provides a pharmaceutical composition comprising a compound of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, diluent or excipient.

[0105] In a further aspect, the present invention provides a pharmaceutical composition comprising a compound of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, diluent or excipient and other therapeutic ingredients.

[0106] In a still further aspect, the present invention provides a method of treating type II diabetes in a subject in need thereof, comprising administering to the subject an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof.

[0107] In a still further aspect, the present invention provides a method for treating obesity in a subject in need thereof, comprising administering to the subject an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof.

[0108] In a still further aspect, the present invention provides a method of treating non-alcoholic fatty liver disease (NAFLD) in a subject in need thereof, comprising administering to the subject an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof.

[0109] In a still further aspect, the present invention provides a method of treating nonalcoholic steatohepatitis (NASH) in a subject in need thereof, comprising administering to the subject an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof.

[0110] In a still further aspect, the present invention provides a method of inducing non-therapeutic weight loss in a subject, comprising administering to the subject an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof.

[0111] In a yet further aspect, the invention provides a compound of the invention for use in therapy.

[0112] In a still further aspect, the present invention provides the use of a compound of the present invention in the treatment of type II diabetes.

[0113] In a still further aspect, the present invention provides the use of a compound of the present invention in the treatment of obesity.

[0114] In a still further aspect, the present invention provides the use of a compound of the present invention in the treatment of non-alcoholic fatty liver disease (NAFLD).

[0115] In a still further aspect, the present invention provides the use of a compound of the present invention in the treatment of non-alcoholic steatohepatitis (NASH).

[0116] In a still further aspect, the present invention provides the use of a compound of the present invention in the preparation of a medicament for the treatment of type II diabetes.

[0117] In a still further aspect, the present invention provides the use of a compound of the present invention in the preparation of a medicament for the treatment of obesity.

[0118] In a still further aspect, the present invention provides the use of a compound of the present invention in the preparation of a medicament for the treatment of non-alcoholic fatty liver disease (NAFLD).

[0119] In a still further aspect, the present invention provides the use of a compound of the present invention in the preparation of a medicament for the treatment of non-alcoholic steatohepatitis (NASH).

[0120] In a still further aspect, the present invention provides an intermediate compound of the following formula or a pharmaceutically acceptable salt thereof:

[0121] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Phe-Val-Glu-Trp-Leu-Leu-Xaa28-Gly-Gly-Pro-Ser-Ser-Gly

[0122] wherein Xaa2 is Aib;

[0123] Xaa28 is Glu or Ser (SEQ ID NO: 9); and

[0124] The C-terminal amino acid is optionally amidated.

[0125] Preferably, the present invention provides an intermediate compound of the following formula or a pharmaceutically acceptable salt thereof:

[0126] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly

[0127] Xaa2 is Aib; and

[0128] The C-terminal amino acid is optionally amidated (SEQ ID NO: 10).

[0129] More preferably, the present invention provides an intermediate compound of the following formula or a pharmaceutically acceptable salt thereof:

[0130] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Phe-Val-Glu-Trp-Leu-Leu-Ser-Gly-Gly-Pro-Ser-Ser-Gly

[0131] Xaa2 is Aib; and

[0132] The C-terminal amino acid is optionally amidated (SEQ ID NO: 11).

[0133] In a still further aspect, the present invention provides a process for preparing a compound of the following formula or a pharmaceutically acceptable salt thereof:

[0134] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Phe-Val-Glu-Trp-Leu-Leu-Xaa28-Gly-Gly-Pro-Ser-Ser-Gly

[0135] Xaa2 is Aib; and

[0136] Xaa28 is Glu or Ser;

[0137] The Lys at position 20 is chemically modified by conjugating the ε-amino group of the Lys side chain to a C14-C24 fatty acid via (i) a direct bond or (ii) a linker between the Lys at position 20 and the C14-C24 fatty acid; and

[0138] The C-terminal amino acid is optionally amidated (SEQ ID NO: 2),

[0139] The method comprises the following steps:

[0140] (i) modifying the intermediate compound of the following formula by conjugating the ε-amino group of the Lys side chain at position 20 of the intermediate compound with a C14-C24 fatty acid, optionally via a linker:

[0141] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Phe-Val-Glu-Trp-Leu-Leu-Xaa28-Gly-Gly-Pro-Ser-Ser-Gly

[0142] wherein Xaa2 is Aib;

[0143] Xaa28 is Glu or Ser; and

[0144] The C-terminal amino acid is optionally amidated (SEQ ID NO: 9).

[0145] Preferably, the Lys at position 20 of the intermediate compound is chemically modified by conjugation with a C14-C24 fatty acid via a linker between the Lys at position 20 and the C14-C24 fatty acid.

[0146] In a still further aspect, the present invention provides a compound produced by the above method.

[0147] The compounds of the present invention are capable of binding to and activating both the GLP-1 receptor and the Gcg receptor. The compounds of the present invention are capable of inducing a reduction in food intake in overweight and obese subjects. The compounds of the present invention have the potential to provide a weight loss effect superior to that of wild-type human OXM.

[0148] The compounds of the invention may improve glucose tolerance and lipid metabolism in subjects suffering from T2D and / or related metabolic disorders and may be more effective than wild-type human OXM.

[0149] A particular advantage of the compounds of the present invention is that the frequency of side effects, such as nausea, commonly associated with GLP-1 therapy (e.g., exenatide and liraglutide) can be reduced or eliminated. The compounds of the present invention can therefore have reduced side effects compared to GLP-1 therapy.

[0150] The compounds of the present invention comprise polypeptides conjugated to fatty acids. Fatty acids, through their albumin binding motifs, can improve the pharmacokinetics of peptides by extending plasma half-life and reducing clearance. Although the compounds of the present invention can be expected to exhibit an improved pharmacokinetic profile relative to wild-type human OXM, the magnitude of the improvement is unpredictable. The inventors have found that the length, composition and position of the fatty acids and optional linkers in the compounds of the present invention result in compounds having a desired pharmacokinetic profile that supports daily, biweekly, weekly or monthly dosing.

[0151] In addition to the improved pharmacokinetic profile, the inventors have discovered that the length, composition and position of the fatty acid and optional linker are critical for optimizing the Gcg-R / GLP-1-R co-agonist activity ratio.

[0152] Wild-type human OXM has full potency and efficacy at human GLP-1-R and human Gcg-R. The amino acid sequence of wild-type human OXM is provided below:

[0153] His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala (SEQ ID NO: 1).

[0154] Certain compounds of the invention have a balanced Gcg-R / GLP-1-R co-agonist activity ratio. Balanced Gcg and GLP-1 activity as used herein refers to compounds with affinity for Gcg and GLP-1 receptors that are close to 1:1 in an in vitro binding assay, such as 1:1 GLP-1 / Gcg, 2:1 GLP-1 / Gcg, 3:2 GLP-1 / Gcg, 1:2 GLP-1 / Gcg, or 2:3 GLP-1 / Gcg. Studies conducted by the inventors have shown that the length, composition, and position of the fatty acids are critical to achieving a balanced Gcg-R / GLP-1-R co-agonist activity ratio that is a characteristic of the compounds of the invention, as well as affecting the plasma half-life, physical stability, solubility, and in vivo stability of the compounds of the invention.

[0155] Although conjugating peptides to fatty acids has advantages in terms of improved pharmacokinetic profiles and / or balanced Gcg-R / GLP-1-R co-agonist activity ratios, it is also expected that the compounds may lose activity due to potential interference with the binding interface of the Gcg receptor or the GLP-1 receptor. However, it has been found that conjugation of the lysine residue at position 20 to fatty acids maintains in vitro and in vivo activity at both receptors to a higher degree than conjugation of amino acids at other positions to fatty acids.

[0156] In addition, the claimed compounds are capable of increasing the efficacy of Gcg-R and / or GLP-1-R relative to wild-type human OXM amino acid substitutions, thereby compensating for the loss of efficacy due to conjugation to fatty acids while maintaining an appropriate ratio of Gcg-R / GLP-1-R co-agonist activity. It is important to note that substitution of one amino acid residue in a particular protein may affect the characteristics of the protein as a whole, and the overall effect may be beneficial or detrimental to pharmacological efficacy and / or pharmaceutical stability. Certain amino acid substitutions may increase efficacy but have a deleterious effect on molecular stability, and vice versa. The compounds of the present invention include S2Aib, S16E, R17K, R18K, Q20K, D21E, Q24E, M27L, N28E or N28S and T29G relative to wild-type human OXM (SEQ ID NO: 1). In addition, the C-terminal sequence of OXM, KRNRNNIA, has been replaced by the GPSSG C-terminal sequence.

[0157] The S2Aib substitution protects the peptide from degradation by peptidases, especially dipeptidyl peptidases. The S16E, R17K, R18K and Q20K substitutions can improve the efficacy of the compounds of the invention in in vitro tests and in vivo animal models. The D21E and Q24E substitutions can improve the stability of the compounds of the invention and modulate in vitro activity. The M27L substitution can protect the peptide from oxidation by methionine residues. The N28E substitution can improve the solubility of the compound comprising the substitution. The N28S substitution can also improve the solubility of the compound comprising the substitution, but not to the same extent as the N28E substitution. However, the solubility of the compound comprising the N28S substitution can be improved by selecting an appropriate linker. The substitution of the asparagine residue at position 28 avoids the possibility of deamidation occurring at this position.

[0158] Removal of residues of the C-terminal sequence of OXM, KRNRNNIA, can improve solubility, which can be attributed to the removal of arginine residues. The present inventors evaluated (i) compounds without a C-terminal sequence, (ii) compounds with a GPSSG C-terminal sequence, and (iii) compounds with a GPSSGAPPPS C-terminal sequence. It was unexpectedly found that certain compounds with a GPSSG C-terminal sequence exhibited improved in vivo efficacy in animal models relative to wild-type human OXM, compounds without a C-terminal sequence, and compounds with a GPSSGAPPPS C-terminal sequence. The GPSSG C-terminal sequence also improves the stability and solubility of the compounds according to the present invention relative to wild-type human OXM and compounds without a C-terminal sequence.

[0159] The compounds of the present invention thus contain amino acid substitutions which, individually or collectively, not only improve efficacy but also provide improved physical stability and solubility characteristics and increased in vivo stability.

[0160] In some aspects of the compounds of the invention, the C14-C24 fatty acid is conjugated to the ε-amino group of lysine via a linker, wherein the linker is selected from:

[0161] (a) aminopolyethylene glycol carboxylates of formula I:

[0162] H-{NH-CH2-CH2-[O-CH2-CH2] m -O-(CH2) p -CO} n -OH (I)

[0163] wherein m is any integer from 1 to 12, n is any integer from 1 to 12, and p is 1 or 2;

[0164] (b) an amino acid selected from the group consisting of Arg, Asn, Asp, Gln, Glu, His, Lys, Pro, Ser, Thr, Cit, Orn, Sar, Gly, γ-Abu and γ-Glu;

[0165] (c) a dipeptide selected from the group consisting of Ala-Ala, β-Ala-β-Ala, Glu-Glu, Gly-Gly, Leu-Leu, Pro-Pro, Ser-Ser, Thr-Thr, γ-Glu-γ-Glu, Glu-γ-Glu, γ-Glu-Glu, γ-Abu-γ-Abu, 6-aminohexanoic acid-6-aminohexanoic acid, 5-aminopentanoic acid-5-aminopentanoic acid, 7-aminoheptanoic acid-7-aminoheptanoic acid, and 8-aminooctanoic acid-8-aminooctanoic acid;

[0166] (d) Selected from Ala-Ala-Ala, β-Ala-β-Ala-β-Ala, Glu-Glu-Glu, γ-Glu-γ-Glu-γ-Glu, Glu-γ-Glu-γ-Glu, γ-Glu-γ-Glu-Glu, γ-Glu-Glu-γ-Glu, Gly-Gl y-Gly, Gly-Gly-Ser, Ser-Gly-Gly, Gly-Ser-Gly, Gly-Gly-Glu, Glu-Gly-Gly, Gly-Glu-Gly, Gly-Gly-γ-Glu, γ-Glu-Gly-Gly, Gly-γ-Glu-Gly, tripeptides Leu-Leu-Leu, Pro-Pro-Pro and γ-Abu-γ-Abu-γ-Abu;

[0167] (e) selected from (Gly-Gly-Ser) q (Gly-Gly-Gly-Ser) r and (Gly-Gly-Gly-Gly-Ser) r 、(6-aminocaproic acid) s , (5-aminovaleric acid) s 、(7-aminoheptanoic acid) s and (8-aminooctanoic acid) s A polypeptide wherein q is any integer from 2 to 5, r is any integer from 1 to 3, and s is any integer from 4 to 15; and

[0168] (f) a conjugated linker, wherein the aminopolyethylene glycol carboxylate of formula I as defined in (a) is conjugated to:

[0169] (i) an amino acid selected from the group consisting of Arg, Asn, Asp, Gln, Glu, His, Lys, Pro, Ser, Thr, Cit, Orn, Sar, Gly, γ-Abu and γ-Glu;

[0170] (ii) a dipeptide selected from the group consisting of Ala-Ala, β-Ala-β-Ala, Glu-Glu, Gly-Gly, Leu-Leu, Pro-Pro, Ser-Ser, Thr-Thr, γ-Glu-γ-Glu, Glu-γ-Glu, γ-Glu-Glu, γ-Abu-γ-Abu, 6-aminohexanoic acid-6-aminohexanoic acid, 5-aminopentanoic acid-5-aminopentanoic acid, 7-aminoheptanoic acid-7-aminoheptanoic acid, and 8-aminooctanoic acid-8-aminooctanoic acid;

[0171] (iii) Selected from Ala-Ala-Ala, β-Ala-β-Ala-β-Ala, Glu-Glu-Glu, γ-Glu-γ-Glu-γ-Glu, Glu-γ-Glu-γ-Glu, γ-Glu-γ-Glu-Glu, γ-Glu-Glu-γ-Glu, Gly-G ly-Gly, Gly-Gly-Ser, Ser-Gly-Gly, Gly-Ser-Gly, Gly-Gly-Glu, Glu-Gly-Gly, Gly-Glu-Gly, Gly-Gly-γ-Glu, γ-Glu-Gly-Gly, Gly-γ-Glu-Gly, Leu-Leu-Leu, Pro-Pro-Pro and γ-Abu-γ-Abu-γ-Abu tripeptides; or

[0172] (iv) selected from (Gly-Gly-Ser) q (Gly-Gly-Gly-Ser) r and (Gly-Gly-Gly-Gly-Ser) r 、(6-aminocaproic acid) s , (5-aminovaleric acid) s 、(7-aminoheptanoic acid) s and (8-aminooctanoic acid) s A polypeptide wherein q is any integer from 2 to 5, r is any integer from 1 to 3, and s is any integer from 4 to 15.

[0173] In a preferred aspect of the compounds of the invention, the linker is an aminopolyethylene glycol carboxylate of formula I or a conjugated linker in which an aminopolyethylene glycol carboxylate of formula I is conjugated to an amino acid, dipeptide, tripeptide or polypeptide as defined above, wherein n is 1, 2, 3, 4, 5 or 6, m is 1, and p is 1.

[0174] In a more preferred aspect of the compounds of the invention, the linker is an aminopolyethylene glycol carboxylate of formula I or a conjugated linker in which an aminopolyethylene glycol carboxylate of formula I is conjugated to an amino acid, dipeptide, tripeptide or polypeptide as defined above, wherein n is 2, m is 1, and p is 1.

[0175] The aminopolyethylene glycol carboxylate linker of formula I or the conjugated linker in which the aminopolyethylene glycol carboxylate of formula I is conjugated to an amino acid, dipeptide, tripeptide or polypeptide as defined above comprises a conjugated linker having the structure [-O-CH2-CH2-] m A small polyethylene glycol moiety (PEG) of , wherein m is an integer between 1 and 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12). Such small PEGs are referred to herein as "mini-PEGs." In a preferred aspect, the mini-PEG has a structure of Formula I:

[0176] H-{NH-CH2-CH2-[O-CH2-CH2] m -O-(CH2) p -CO} n -OH (I)

[0177] wherein m is any integer from 1 to 12, n is any integer from 1 to 12, and p is 1 or 2. Preferably, the micro-PEG has the structure of Formula I, wherein n is 1, 2, 3, 4, 5 or 6, m is 1, and p is 1. Further preferably, the micro-PEG has the structure of Formula I, wherein n is 1, m is 1, and p is 1. Suitable reagents for acylation of amino acids with micro-PEGs are commercially available from suppliers such as Peptides International (Louisville, KY) and ChemPep, Inc. (Wellington, FL). In addition, suitable techniques for acylation of amino acids with micro-PEGs are described herein (see Examples 1-4).

[0178] The micro-PEG of formula I is a functionalized micro-PEG comprising an amine functional group and a carboxyl functional group. The carboxyl functional group reacts with the ε-amino group of the lysine side chain to form an amide bond. The amine functional group reacts with the carboxyl group of the fatty acid. The lysine at position 20 of the peptide of SEQ ID NO: 2 is thus conjugated to a C14-C24 fatty acid via the micro-PEG of formula I.

[0179] Alternatively, when the micro-PEG of formula I is part of a conjugation linker (i.e., the micro-PEG of formula I is conjugated to an amino acid, dipeptide, tripeptide or polypeptide as defined above), the amine functional group of the micro-PEG of formula I reacts with a functional group of the amino acid, dipeptide, tripeptide or polypeptide. Another functional group of the amino acid, dipeptide, tripeptide or polypeptide reacts with a carboxyl group of a fatty acid. The lysine at position 20 of the peptide of SEQ ID NO: 2 is thereby conjugated to a C14-C24 fatty acid via a conjugation linker as defined above.

[0180] The hydrophilicity of the mini-PEG of formula I serves to increase the solubility of the compounds of the invention comprising a linker comprising an aminopolyethylene glycol carboxylate of formula I or a conjugated linker in which an aminopolyethylene glycol carboxylate of formula I is conjugated to a defined amino acid, dipeptide, tripeptide or polypeptide.

[0181] Preferred linkers comprising mini-PEGs of Formula I include, but are not limited to, ([2-(2-aminoethoxy)-ethoxy]-acetyl) 2 and 8-amino-3,6-dioxaoctanoic acid.

[0182] The linker can be a single amino acid between the ε-amino group of the lysine side chain and the C14-C24 fatty acid. In some preferred aspects, the amino acid is a hydrophilic amino acid. Suitable amino acids include Arg, Asn, Asp, Gln, Glu, His, Lys, Pro, Ser, Thr, Cit, Orn, Sar, Gly, γ-Abu and γ-Glu.

[0183] In a more preferred aspect, the amino acid is γ-Glu.

[0184] Optionally, the linker is a dipeptide selected from Ala-Ala, β-Ala-β-Ala, Glu-Glu, Gly-Gly, Leu-Leu, Pro-Pro, Ser-Ser, Thr-Thr, γ-Glu-γ-Glu, Glu-γ-Glu, γ-Glu-Glu, γ-Abu-γ-Abu, 6-aminohexanoic acid-6-aminohexanoic acid, 5-aminopentanoic acid-5-aminopentanoic acid, 7-aminoheptanoic acid-7-aminoheptanoic acid and 8-aminooctanoic acid-8-aminooctanoic acid.

[0185] In a further preferred optional aspect, each amino acid of the dipeptide may be the same or different from another amino acid of the dipeptide and may be independently selected from Ala, β-Ala, Glu, Gly, Leu, Pro, Ser, Thr, γ-Glu, γ-Abu, 6-aminohexanoic acid, 5-aminopentanoic acid, 7-aminoheptanoic acid and 8-aminooctanoic acid.

[0186] In a more preferred aspect, the linker is γ-Glu-γ-Glu.

[0187] In some aspects, the linker is a tripeptide, wherein the amino acids of the tripeptide are independently selected from: Ala, β-Ala, Glu, Gly, Leu, Pro, Ser, Thr, γ-aminobutyric acid (γ-Abu), γ-glutamic acid (γ-Glu), 6-aminohexanoic acid, 5-aminopentanoic acid, 7-aminoheptanoic acid, and 8-aminooctanoic acid.

[0188] In a preferred aspect, the linker is selected from Ala-Ala-Ala, β-Ala-β-Ala-β-Ala, Glu-Glu-Glu, γ-Glu-γ-Glu-γ-Glu, Glu-γ-Glu-γ-Glu, γ-Glu-γ-Glu-Glu, γ-Glu-Glu-γ-Glu, GI y-Gly-Gly, Gly-Gly-Ser, Ser-Gly-Gly, Gly-Ser-Gly, Gly-Gly-Glu, Glu-Gly-Gly, Gly-Glu-Gly, Gly-Gly-γ-Glu, γ-Glu-Gly-Gly, Gly-γ-Glu-Gly, Tripeptides Leu-Leu-Leu, Pro-Pro-Pro and γ-Abu-γ-Abu-γ-Abu.

[0189] In some aspects, the linker is selected from (Gly-Gly-Ser) q (Gly-Gly-Gly-Ser) r and (Gly-Gly-Gly-Gly-Ser) r 、(6-aminocaproic acid) s , (5-aminovaleric acid) s 、(7-aminoheptanoic acid) s and (8-aminooctanoic acid) s A polypeptide wherein q is any integer from 2 to 5, r is any integer from 1 to 3, and s is any integer from 4 to 15.

[0190] In a preferred aspect, the linker is a conjugation linker wherein the aminopolyethylene glycol carboxylate of Formula I:

[0191] H-{NH-CH2-CH2-[O-CH2-CH2] m -O-(CH2) p -CO} n -OH (I)

[0192] wherein m is any integer from 1 to 12, n is any integer from 1 to 12, and p is 1 or 2;

[0193] The linker is conjugated to an amino acid, a dipeptide, a tripeptide or a polypeptide as defined above.

[0194] In a preferred aspect, the aminopolyethylene glycol carboxylate conjugated to the linker is ([2-(2-aminoethoxy)-ethoxy]-acetyl) 2 or 8-amino-3,6-dioxaoctanoic acid.

[0195] In a more preferred aspect, the linker comprises ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu)t (Also known as (AEEA) 2- -(γ-Glu) t ), wherein t is 1 or 2. The fatty acid and the gamma-glutamic acid in the linker act as albumin binders and provide the potential to generate long-acting compounds in vivo. In the most preferred aspect, the compounds of the present invention comprise a lysine side chain ε-amino group linked to ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu) t -CO-(CH2) d The lysine at position 20 is chemically modified by conjugation with -CO2H, wherein t is 1 or 2, and d is 16 or 18.

[0196] As shown in the chemical structures of Examples 1 to 4, the [2-(2-amino-ethoxy)-ethoxy]-acetyl group of the first unit is connected to the ε-amino group of the lysine side chain. Then, the [2-(2-amino-ethoxy)-ethoxy]-acetyl group of the second unit is connected to the [2-(2-amino-ethoxy)-ethoxy]-acetyl amino group of the first unit. Then, the γ-Glu of the first unit is connected to the [2-(2-amino-ethoxy)-ethoxy]-acetyl amino group of the second unit through the γ-carboxyl group of the side chain. When t=2, the γ-Glu of the second unit is connected to the α-amino group of the γ-Glu of the first unit through the γ-carboxyl group of the side chain. Finally, the fatty acid is connected to the α-amino group of the γ-Glu of the first (t=1) or second (t=2) unit.

[0197] When the linker is an amino acid, dipeptide, tripeptide or polypeptide as defined above, it is preferred that the amino acid or at least one of the amino acids of the dipeptide, tripeptide or polypeptide is a hydrophilic amino acid.

[0198] Similarly, when the linker is a conjugated linker in which an aminopolyethylene glycol carboxylate of Formula I is conjugated to an amino acid, dipeptide, tripeptide or polypeptide as defined above,

[0199] H-{NH-CH2-CH2-[O-CH2-CH2] m -O-(CH2) p -CO} n -OH (II)

[0200] wherein m is any integer from 1 to 12, n is any integer from 1 to 12, and p is 1 or 2;

[0201] It is preferred that the amino acid / amino acid of the dipeptide / tripeptide / polypeptide / at least one amino acid is a hydrophilic amino acid.

[0202] Suitable amino acids include, but are not limited to, Arg, Asn, Asp, Gln, Glu, His, Lys, Pro, Ser, Thr, Cit, Orn, Sar, Gly, γ-Abu, and γ-Glu.

[0203] The present inventors have discovered that the presence of one or more hydrophilic amino acids in the linker compensates for the loss of solubility that would normally be expected to result from amino acid substitutions in the peptide of SEQ ID NO: 1. For example, in a peptide wherein the linker comprises ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu) t , wherein t is 1 or 2, Xaa28 of the peptide of SEQ ID NO: 1 can be serine or glutamic acid. The selection of glutamic acid at position 28 of the peptide of SEQ ID NO: 2 improves the solubility of such compounds. The selection of serine at position 28 of the peptide of SEQ ID NO: 2 can be expected to reduce the solubility of such compounds relative to those compounds that differ only in having a glutamic acid residue at position 28. However, the second γ-Glu amino acid in the above-mentioned linker (i.e., t is 2) compensates for such an expected reduction in solubility.

[0204] The compounds of the present invention utilize C14-C24 fatty acids chemically conjugated to the epsilon-amino group of a lysine side chain, either by a direct bond or by a linker. The term "C14-C24 fatty acid" as used herein refers to a carboxylic acid having between 14 and 24 carbon atoms. C14-C24 fatty acids suitable for use herein may be saturated monobasic acids or saturated dibasic acids. By "saturated" is meant that the fatty acid does not contain a carbon-carbon double or triple bond.

[0205] Examples of specific saturated C14-C24 fatty acids suitable for the compounds and uses disclosed herein include, but are not limited to: myristic acid (tetradecanoic acid) (C14 monoacid), tetradecanedioic acid (C14 diacid), palmitic acid (hexadecanoic acid) (C16 monoacid), hexadecanedioic acid (C16 diacid), heptadecanoic acid (heptadecanoic acid) (C17 monoacid), heptadecanedioic acid (C17 diacid), stearic acid (octadecanoic acid) (C18 monoacid), octadecanedioic acid (C18 diacid), nonadecanoic acid (Nonadecanoic acid) (C19 monoacid), nonadecanedioic acid (C19 diacid), arachidic acid (eicosanoic acid) (C20 monoacid), eicosandioic acid (C20 diacid), heneicosanoic acid (heneicosandioic acid) (C21 monoacid), heneicosandioic acid (C21 diacid), behenic acid (docosanic acid) (C22), docosandioic acid (C22 diacid), tetracosanoic acid (tetracosanoic acid) (C24 monoacid), tetracosandioic acid (C24 diacid), including branched and substituted derivatives thereof.

[0206] In a preferred aspect of the compounds of the present invention, the C14-C24 fatty acid is selected from saturated C14 monoacid, saturated C14 diacid, saturated C16 monoacid, saturated C16 diacid, saturated C18 monoacid, saturated C18 diacid, saturated C19 diacid, saturated C20 monoacid, saturated C20 diacid, saturated C22 diacid, and branched and substituted derivatives thereof.

[0207] In a more preferred aspect of the compounds of the present invention, the C14-C24 fatty acid is selected from the group consisting of myristic acid, tetradecanedioic acid, palmitic acid, hexadecanedioic acid, stearic acid, octadecanedioic acid, nonadecanedioic acid, arachidic acid, eicosanedioic acid and docosanedioic acid.

[0208] Preferably, the C14-C24 fatty acid is octadecanedioic acid or eicosanedioic acid.

[0209] The inventors have found that the position of the fatty acid is critical in achieving a compound with a desired Gcg-R / GLP-1-R co-agonist activity ratio. The length and composition of the fatty acid affect the plasma half-life of the compound, the efficacy of the compound in an in vivo animal model, and also affect the solubility and stability of the compound. The peptide defined in SEQ ID NO: 2 and the C14-C24 saturated fatty mono- or di-acids result in compounds that exhibit the desired plasma half-life, the desired efficacy in an in vivo animal model, and also possess the desired solubility and stability characteristics. Myristic acid, tetradecanedioic acid, palmitic acid, hexadecanedioic acid, stearic acid, octadecanedioic acid, nonadecanedioic acid, arachidic acid, eicosanedioic acid, and docosanedioic acid are particularly preferred fatty acids.

[0210] In particular, conjugation of the lysine residue at position 20 of the peptide defined in SEQ ID NO: 2 with octadecanedioic acid or eicosanedioic acid produces compounds that: (i) are capable of achieving a desired Gcg-R / GLP-1-R co-agonist activity ratio; (ii) are capable of improving efficacy in in vivo animal models; and / or (iii) are capable of improving physical stability and solubility characteristics.

[0211] The compounds of the present invention are preferably formulated as pharmaceutical compositions for administration by a parenteral route (eg, subcutaneously, intravenously, intraperitoneally, intramuscularly or transdermally).

[0212] The compounds of the present invention are usually administered parenterally. Parenteral administration includes, for example, systemic administration, such as by intramuscular, intravenous, subcutaneous, intradermal or intraperitoneal injection. The preferred route of administration is subcutaneous injection. The compounds of the present invention are combined with acceptable pharmaceutical carriers, diluents or excipients as part of a pharmaceutical composition for the treatment of type II diabetes, obesity, NAFLD and / or NASH and administered to the subject. The pharmaceutical composition can be a solution or a suspension, such as one in which the compound of the present invention is complexed with a divalent metal cation, such as zinc. The compounds of the present invention can also be formulated as solid preparations, such as by lyophilization or spray drying, which are then reconstituted in a suitable diluent solvent before administration. Suitable pharmaceutical carriers may contain inert ingredients that do not interact with peptides or peptide derivatives. Pharmaceutical carriers suitable for parenteral administration include, for example, sterile water, physiological saline, bacteriostatic saline (saline containing about 0.9% mg / ml of benzyl alcohol), phosphate buffered saline, Hank's solution, Ringer's lactate, etc. Some examples of suitable excipients include lactose, dextrose, sucrose, trehalose, sorbitol, and mannitol, and preservatives such as phenol and m-cresol.

[0213] Standard pharmaceutical formulation techniques may be employed, such as those described in Remington: Science and Practice of Pharmacy (DB Troy ed., 21st ed., Lippincott, Williams & Wilkins, 2006). The compounds of the invention may alternatively be formulated for buccal, oral, transdermal, nasal or pulmonary administration.

[0214] The compounds of the present invention can react with any number of inorganic and organic acids to form pharmaceutically acceptable acid addition salts. Pharmaceutically acceptable salts and conventional methodologies for preparing them are well known in the art. See, for example, P. Stahl et al., Handbook of Pharmaceutical Salts: Properties, Selection and Use, 2nd Revised Edition (Wiley-VCH, 2011). Pharmaceutically acceptable salts of the present invention include trifluoroacetate, hydrochloride and acetate.

[0215] The compounds of the present invention can be used to treat diabetes, particularly type II diabetes. Other subjects that may benefit from treatment using the compounds of the present invention include: those with impaired glucose tolerance or impaired fasting glucose, subjects whose weight is about 25% or more above normal weight for the subject's height and size, subjects with one or two parents suffering from type II diabetes, subjects who have suffered from gestational diabetes, and subjects suffering from metabolic disorders, such as those caused by reduced endogenous insulin secretion. The compounds of the present invention can be used to prevent subjects with impaired glucose tolerance from developing type II diabetes, prevent pancreatic β-cell degeneration, induce β-cell proliferation, improve β-cell function, activate dormant β-cells, promote cell differentiation into β-cells, stimulate β-cell replication, and inhibit β-cell apoptosis. In the methods of the present invention, other disorders and conditions that can be treated or prevented using the compounds of the present invention include: adult-onset diabetes (MODY) (Herman et al., Diabetes 43:40, 1994); latent autoimmune diabetes in adults (LADA) (Zimmet et al., Diabetes Med. 11:299, 1994); impaired glucose tolerance (IGT) (Expert Committee on Classification of Diabetes Mellitus, Diabetes Care 22 (Supp. 1):S5, 1999); impaired fasting glucose (IFG) (Charles et al., Diabetes 40:796, 1991); gestational diabetes (Metzger, Diabetes, 40:197, 1991); metabolic syndrome X, dyslipidemia, hyperglycemia, hyperinsulinemia, hypertriglyceridemia and insulin resistance.

[0216] The compounds of the present invention can also be used in the methods of the present invention to treat secondary diabetes (Expert Committee on Classification of Diabetes Mellitus, Diabetes Care 22 (Supp. 1): S5, 1999). Such secondary causes include glucocorticoid excess, growth hormone excess, pheochromocytoma, and drug-induced diabetes. Drugs that may induce diabetes include, but are not limited to, pyridoxine, niacin, glucocorticoids, phenytoin, thyroid hormones, beta-adrenergic drugs, alpha-interferon, and drugs used to treat HIV infection.

[0217] The compounds of the present invention may be effective in suppressing food intake and treating obesity.

[0218] An "effective amount" of a compound of the invention is an amount that produces the desired therapeutic and / or preventive effect when administered to a subject without causing unacceptable side effects. "Desired therapeutic effect" includes one or more of the following: 1) improvement of one or more symptoms associated with a disease or condition; 2) delaying the onset of symptoms associated with a disease or condition; 3) increasing life expectancy compared to no treatment; and 4) a higher quality of life compared to no treatment. For example, an "effective amount" of a compound of the invention for the treatment of T2D is an amount that produces better control of blood glucose concentrations compared to no treatment, thereby delaying the onset of diabetic complications, such as retinopathy, neuropathy, or nephropathy. An "effective amount" of a compound of the invention for the prevention of type II diabetes, for example, in a subject with impaired glucose tolerance or impaired fasting glucose, is an amount that delays the onset of elevated blood glucose levels compared to no treatment, the elevated blood glucose levels requiring treatment with antihyperglycemic drugs, such as thioureas, thiazolidinediones, insulin, and / or biguanides.

[0219] The "effective amount" of the compound of the invention administered to a subject will also depend on the type and severity of the disease and the characteristics of the subject, such as overall health, age, sex, weight, and tolerance to drugs. The dosage of the compound of the invention effective for normalizing blood glucose in a subject will depend on a number of factors, including but not limited to the sex, weight and age of the subject, the severity of the inability to regulate blood glucose, the route of administration and bioavailability, the pharmacokinetic profile of the peptide, efficacy, and formulation.

[0220] Some compounds of the present invention are generally effective across a wide range of dosages. For example, the dosage for weekly administration can fall within the range of about 0.05 to about 30 mg per person per week. Some compounds of the present invention can be administered daily. In addition, some compounds of the present invention can be administered every two weeks, weekly or monthly.

[0221] A "subject" is a mammal, preferably a human, but may also be an animal, including companion animals (eg, dogs, cats, etc.), livestock (eg, cows, sheep, pigs, horses, etc.), and laboratory animals (eg, rats, mice, guinea pigs, etc.).

[0222] As used herein, the term "treat" or "treating" includes inhibiting, slowing, stopping or reversing the course or severity of an existing symptom or disorder.

[0223] The term "plasma half-life" refers to the time required for half of the compound of interest to be cleared from the plasma. An alternative term used is "exclusion half-life". The term "extended" or "longer" used in the context of plasma half-life or exclusion half-life means that there is a significant improvement in the half-life of a compound of the invention relative to a reference molecule (e.g., a non-fatty acid conjugated form of the peptide, wild-type human OXM or semaglutide) as determined under comparable conditions.

[0224] Clearance is a measure of the body's ability to eliminate a drug from the circulation. As clearance decreases due to, for example, modifications to the drug, one would expect half-life to increase. However, this reciprocal relationship is accurate only if there is no change in the profile volume. The useful terminal log half-life (T 1 / 2 The approximate relationship between clearance (C) and profile volume (V) is given by the following formula: 1 / 2 ≈ 0.693 (V / C). The clearance rate does not indicate how much drug is removed, but rather the volume of biological fluid, such as blood or plasma, that will be completely free of drug due to elimination. The clearance rate is expressed as volume per unit time.

[0225] As used herein, "hydrophilic" refers to the property of easily absorbing moisture and having a strong polar group that easily interacts with water.

[0226] As used herein, "semaglutide" refers to a chemically synthesized GLP-1 analog having a peptide backbone and overall compound structure found in CAS Registry Number 910463-68-2.

[0227] The amino acid sequences of the present invention contain the standard single-letter or three-letter codes for the 20 naturally occurring amino acids. In addition, "Aib" is α-aminoisobutyric acid, "Abu" is aminobutyric acid, "Orn" is ornithine, "Cit" is citrulline, and "Sar" is sarcosine.

[0228] As used herein, the term "C-terminal amino acid" refers to the last amino acid in a peptide sequence that contains a free carboxyl group. The C-terminal amino acid of the compounds of the present invention is Gly at position 34. DETAILED DESCRIPTION

[0229] The present invention also encompasses novel intermediates and methods useful for synthesizing the compounds of the present invention or their pharmaceutically acceptable salts. Intermediates and compounds of the present invention can be prepared by a variety of procedures known in the art. In particular, the method using chemical synthesis is described in the following examples. The specific synthesis steps for each approach can be combined in different ways to prepare the compounds of the present invention or their salts. Reagents and starting materials are readily available to those of ordinary skill in the art. It is to be understood that these embodiments are not intended to limit the scope of the present invention in any way.

[0230] Example 1

[0231] HXaa2QGTFTSDYSKYLDEKKAKEFVEWLLEGGPSSG

[0232] wherein Xaa2 is Aib;

[0233] The K at position 20 was synthesized by using ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)1-CO-(CH2) 16 -CO2H is conjugated to the ε-amino group of the K side chain and thus chemically modified; and

[0234] The C-terminal amino acid was amidated to a C-terminal primary amide (SEQ ID NO: 5).

[0235]

[0236] The figure above illustrates the structure of the compound of SEQ ID NO: 5 (hereinafter referred to as "Compound 1") using standard single-letter amino acid codes except for residues Aib2 and K20, in which the structures of these amino acids have been expanded.

[0237] The peptide component of Compound 1 was synthesized by automated solid phase synthesis using Fmoc / t-Bu chemistry on a Symphony 12-channel multiplex peptide synthesizer (Protein Technologies, Inc. Tucson, AZ).

[0238] The synthetic resin consisted of 1% DVB cross-linked polystyrene (Fmoc-Rink-MBHALow Loading resin, 100-200 mesh, EMD Millipore, Temecula, CA) at a degree of substitution of 0.3-0.4 meq / g. Standard side chain protecting agents were as follows: tert-butyloxycarbonyl (Boc) for Trp and Lys; tert-butyl ester (OtBu) for Asp and Glu; tBu for Ser, Thr and Tyr; and triphenylmethyl (Trt) for Gln; N-α-Fmoc-N-ε-4-methyltriphenylmethyl-L-lysine (Fmoc-Lys(Mtt)-OH) was used for lysine at position 20 of SEQ ID NO: 3, and N α ,N (im) -DiBoc-L-histidine (Boc-His(Boc)-OH) was used for the histidine at position 1. The Fmoc group was removed prior to each coupling step (2 x 7 minutes) using 20% ​​piperidine in dimethylformamide (DMF). All standard amino acid couplings used equal molar ratios of Fmoc amino acids (EMD Millipore, Temecula, CA), diisopropylcarbodiimide (DIC) (Sigma-Aldrich, St. Louis, MO), and ethyl 2-oximecyanoacetate (Oxyma Pure, Iris Biotech, Marktredwitz, Germany) at a 9-fold molar excess relative to the theoretical peptide loading and at a final concentration of 0.18 M in DMF for 1 hour.

[0239] Two exceptions were: the glutamine residue at position 3 of SEQ ID NO: 5, which was double coupled (2 x 1 hour); and the histidine residue at position 1 of SEQ ID NO: 5, which was coupled for 18 hours using 1-hydroxy-7-azabenzotriazole (HOAt) instead of ethyl 2-oximecyanoacetate in a 6-fold molar excess. After the completion of the synthesis of the linear peptide, the resin was transferred to a disposable glass 25 mL polypropylene syringe (Torviq, Niles, MI) equipped with a polytetrafluoroethylene (PTFE) piston (Biotage, Charlotte, NC), and the 4-methyltriphenylmethyl (Mtt) protecting group on the lysine at position 20 of SEQ ID NO: 5 was selectively removed from the peptide resin using three treatments with 20% hexafluoroisopropanol (Oakwood Chemicals, West Columbia, SC) in DCM (2 x 10 minutes and 1 x 45 minutes) to expose the ε-amine of the free lysine at position 20 and make it available for further reaction.

[0240] The attachment of the subsequent fatty acid-linker moiety was accomplished by performing a sequential coupling of two [2-(2-(Fmoc-amino)ethoxy)ethoxy]acetic acid (Fmoc-AEEA-OH) (ChemPep, Inc. Wellington, FL; three-fold excess of amino acid (AA): 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyrimidinium-3-oxide hexafluorophosphate (HATU): N,N-diisopropylethylamine (DIPEA) [1:1:5 mol / mol], 3 hours coupling time), followed by Fmoc-glutamic acid α-tert-butyl ester (Fmoc-Glu-OtBu) (Ark Pharm, Inc. Libertyville IL, 3-fold excess of AA:HATU:DIPEA [1:1:5 mol / mol], 3 hours coupling time). In each case, the Fmoc moiety was removed as described above. Finally, mono-OtBu-octadecandioic acid (WuXi AppTec, Shanghai, China) was coupled to the resin using a 3-fold excess of acid:HATU:DIPEA (1:1:5 mol / mol) over 18 h.

[0241] After completing the synthesis, the peptide resin was washed with dichloromethane (DCM), diethyl ether, and air dried thoroughly by applying vacuum suction to the syringe for 5 minutes. The dried resin was treated at room temperature for 2 hours with shear mixture (trifluoroacetic acid (TFA): anisole: water: triisopropylsilane, 88:5:5:2 v / v), thereby releasing the peptide from the solid support and removing all side chain protecting groups. The resin was filtered out, washed twice with pure TFA, and the filtrate combined was treated with cold diethyl ether, thereby precipitating the crude peptide. The peptide / ether suspension was then centrifuged at 4000 rpm, to form a solid precipitate, the supernatant was decanted, and the solid precipitate was finely ground with ether for additional two times, and dried in a vacuum. The crude peptide was dissolved in 20% acetonitrile / water, and purified by RP-HPLC on a C8 preparative column (Luna 21 x 250 mm, Phenomenex, Torrance, CA) with a linear gradient of acetonitrile and water, using three different buffer systems:

[0242] 1) 0.1 M ammonium acetate in water, pH 5.0;

[0243] 2) 0.1% TFA in water; and

[0244] 3) 5% acetic acid in water.

[0245] The final master product pool was then lyophilized to produce lyophilized peptide acetate.

[0246] In the synthesis carried out essentially as described above, the purity of Compound 1 was assessed using analytical RP-HPLC and was found to be >97%.

[0247] Molecular weight was determined by analytical electrospray MS. The molecular weight of Compound 1 was calculated to be 4535.0 Daltons, while the observed deconvoluted average molecular weight was determined to be 4535.0 Daltons, and the following ions were observed: 1512.3 (M+3H), 1134.3 (M+4H), 908 (M+5H).

[0248] Example 2

[0249] HXaa2QGTFTSDYSKYLDEKKAKEFVEWLLEGGPSSG

[0250] wherein Xaa2 is Aib;

[0251] The K at position 20 was synthesized by using ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)1-CO-(CH 2 ) 18 -CO2H is conjugated to the ε-amino group of the K side chain and thus chemically modified; and

[0252] The C-terminal amino acid was amidated to a C-terminal primary amide (SEQ ID NO: 6).

[0253]

[0254] The figure above illustrates the structure of the compound of SEQ ID NO: 6 (hereinafter referred to as "Compound 2") using standard single-letter amino acid codes except for residues Aib2 and K20, in which the structures of these amino acids have been expanded.

[0255] Compound 2 was synthesized as in Example 1, except that mono-OtBu-eicosanedioic acid (WuXi AppTec, Shanghai, China) was coupled to the resin using a 3-fold excess of AA:HATU:DIPEA (1:1:5 mol / mol) over 18 h instead of mono-OtBu-octadecandioic acid as in Example 1.

[0256] The molecular weight of compound 2 was calculated to be 4563.1 Daltons, while the observed deconvoluted average molecular weight was determined to be 4562.9 Daltons, and the following ions were observed: 1521.7 (M+3H), 1141.3 (M+4H), 913.5 (M+5H).

[0257] Example 3

[0258] HXaa2QGTFTSDYSKYLDEKKAKEFVEWLLSGGPSSG

[0259] wherein Xaa2 is Aib;

[0260] The K at position 20 was synthesized by using ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)2-CO-(CH2) 16 -CO2H is conjugated to the ε-amino group of the K side chain and thus chemically modified; and

[0261] The C-terminal amino acid was amidated to a C-terminal primary amide (SEQ ID NO: 7).

[0262]

[0263] The figure above illustrates the structure of the compound of SEQ ID NO: 7 (hereinafter referred to as "Compound 3") using standard single-letter amino acid codes except for residues Aib2 and K20, in which the structures of these amino acids have been expanded.

[0264] Compound 3 was synthesized as in Example 1, except that an additional Fmoc-Glu-OtBu moiety was added during the linker synthesis cycle.

[0265] The molecular weight of compound 3 was calculated to be 4622.1 Daltons, while the observed deconvoluted average molecular weight was determined to be 4621.9 Daltons, and the following ions were observed: 1541.3 (M+3H), 1156.2 (M+4H), 925.2 (M+5H).

[0266] Example 4

[0267] HXaa2QGTFTSDYSKYLDEKKAKEFVEWLLSGGPSSG

[0268] wherein Xaa2 is Aib;

[0269] The K at position 20 was synthesized by using ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)2-CO-(CH2) 18 -CO2H is conjugated to the ε-amino group of the K side chain and thus chemically modified; and

[0270] The C-terminal amino acid was amidated to a C-terminal primary amide (SEQ ID NO: 8).

[0271]

[0272] The figure above illustrates the structure of the compound of SEQ ID NO: 8 (hereinafter referred to as "Compound 4") using standard single-letter amino acid codes except for residues Aib2 and K20, in which the structures of these amino acids have been expanded.

[0273] Compound 4 was synthesized as in Example 1, except that mono-OtBu-eicosanedioic acid (WuXi AppTec, Shanghai, China) was coupled to the resin using a 3-fold excess of AA:HATU:DIPEA (1:1:5 mol / mol) over 18 hours instead of mono-OtBu-octadecandioic acid as used in Example 1. In addition, an additional amino acid Fmoc-Glu-OtBu moiety was added in the linker synthesis cycle.

[0274] The molecular weight of the peptide was calculated to be 4650.1 Daltons, and the observed deconvoluted average molecular weight was determined to be 4650.1 Daltons, and the following ions were observed: 1550.7 (M+3H), 1163.3 (M+4H), 930.8 (M+5H).

[0275] Physical characteristics

[0276] Viscosity

[0277] The viscosity of the compounds according to the invention was measured in a Rheosense mVroc viscometer with the following settings:

[0278] (a) Syringe size: 500 μL syringe

[0279] (b) Flow rate: 100 μL / min

[0280] (c) Average temperature: 25℃

[0281] (d) Shear rate: 1934 s -1

[0282] Weigh dry powder (compound), dissolve in water as turbid precipitate, and titrate to about pH8.0 with 1N NaOH.Sonicate the solution, and shake by hand until the peptide is in solution.The sample is sterile filtered (0.22 μm PVDF filter).Then, the sample is evaluated by UV-Vis for stock solution concentration.The solution is diluted to a final concentration of about 10 mg / mL peptide by weight in 10 mM Tris + 3 mg / mL meta-cresol at pH 8.0 using 3x meta-cresol in 10 mM Tris pH 8.0 buffer.Just before viscosity analysis, the sample is filtered through a 0.22 μm filter.Before and after analysis, remove 25 μL of sample to verify concentration by RP-HPLC.

[0283] Before and after analyzing each sample, water and buffer control samples were measured. Between the analysis of each sample, the instrument was washed with buffer (3x). The sample was loaded into a separate syringe and analyzed. The first measurement was not included in the final calculation, thereby allowing the system to balance. The samples were then analyzed in triplicate (n=3).

[0284] The viscosity of compounds 1-4 was measured essentially as described in this assay. The viscosity data for compounds 1-4 are summarized in Table 1.

[0285] Table 1: Viscosity data of compounds 1-4

[0286] sample 500 μL Syringe Viscosity (cP or mPa-s) Buffer control 0.99 Compound 1 1.06 Compound 2 1.04 Compound 3 1.03 Compound 4 1.05

[0287] Solubility

[0288] The solubility of the compounds of the present invention was measured in Agilent 1100 HPLC, Agilent 1200 HPLC and Nanodrop 2000. The following HPLC columns were used:

[0289] (a) RP-HPLC: Waters Symmetry Shield C18, 3.6 um, 4.6 x 100 mm

[0290] (b) HPLC-SEC: Tosoh Biosciences, TSK2000SWXL 7.8 cm x 30 mm

[0291] All peptide concentrations were made to be 10 mg / mL in the following:

[0292] (a) 10 mM Tris pH 8.0 + 3 mg / mL m-cresol

[0293] (b) 10 mM Tris pH 8.0 + 3 mg / mL m-cresol + 150 mM NaCl

[0294] (c) 10 mM Tris pH 8.0 + 3 mg / mL m-cresol + 0.02% Tween-20

[0295] (d) PBS pH 7.4

[0296] 5mL of 10 mg / mL peptide dissolved in 10 mM Tris at pH 8.0 was concentrated to about 20 mg / mL using Amicon-ultra 3kDaMWCO equipment. The solution was filtered using Millivex 0.22 μM filter (PVDF membrane) and the final concentration was measured by NanoDrop spectrometer. Using this stock solution, 3x meta-cresol, 10x NaCl and 100x Tween-20 stock solutions were used to prepare the above-mentioned final conditions. Also 10 mg / mL PBS solutions were prepared by directly dissolving with 5 mg / mL and using Amicon-ultra 3kDaMWCO equipment to concentrate.

[0297] Each solution was placed in a refrigerator at 4°C for 1 week, the concentration was evaluated by RP-HPLC, and the HMW species formation was evaluated by HPLC-SEC. The analysis was completed at T-0 week and T-1 week.

[0298] The solubility of compounds 1-4 was measured essentially as described in this assay. The solubility data for compounds 1-4 are summarized in Table 2(a)-(d).

[0299] Table 2(a): Solubility data of compound 1

[0300]

[0301] Table 2(b): Solubility data of compound 2

[0302]

[0303] Table 2(c): Solubility data of compound 3

[0304]

[0305] Table 2(d): Solubility data of compound 4

[0306]

[0307] In vitro function

[0308] Binding affinity of compounds 1-4 to recombinant human Gcg receptor (hGcg-R) and human GLP-1 receptor (hGLP-1-R)

[0309] The equilibrium dissociation constants (K) of compounds 1-4 were determined by radioligand competition binding assays using scintillation proximity assay (SPA) and membranes prepared from 293HEK stably transfected cells overexpressing hGcg-R or hGLP-1-R. i ). The experimental procedures and results are described below.

[0310] hGLP-1R binding assay

[0311] The GLP-1 receptor binding assay used cloned hGLP-1-R isolated from 293HEK cells overexpressing recombinant hGLP-1R (Graziano MP, Hey PJ, Borkowski D, Chicchi GG, Strader CD, Biochem Biophys Res Commun. 196(1): 141-6, 1993). The hGLP-1R cDNA was subcloned into the expression plasmid phD (Trans-activated expression of fully gamma-carboxylated recombinant human protein C, an antithrombotic factor. Grinnell, BW, Berg, DT, Walls, J. and Yan, SBBio / Technology 5: 1189-1192, 1987). The plasmid DNA was transfected into 293HEK cells and selected with 200 μg / mL hygromycin.

[0312] Crude plasma membranes were prepared using cells from adherent cultures. Cells were lysed on ice in hypotonic buffer containing 50 mM Tris HCl, pH 7.5 and Roche Complete™ protease inhibitors with EDTA. The cell suspension was disrupted by 25 strokes using a glass Potter-Elvehjem homogenizer equipped with a Teflon® grinding rod. The homogenate was centrifuged at 1100 x g for 10 min at 4 °C. The supernatant was collected and stored on ice while the pellet was resuspended in hypotonic buffer and rehomogenized. The mixture was centrifuged at 1100 x g for 10 min. The second supernatant was combined with the first supernatant and centrifuged at 35000 x g for 1 h at 4 °C. The membrane pellet was resuspended in homogenization buffer containing protease inhibitors, snap frozen in liquid nitrogen, and stored in aliquots in a -80 °C freezer until use.

[0313] GLP-1 was radioiodinated by the I-125-lactoperoxidase procedure and purified by reverse phase HPLC on a Perkin-Elmer (NEX308). The specific activity was 2200 Ci / mmol. Due to the high propanol content in the I-125 GLP-1 material, K was determined by homologous competition rather than saturation binding. D OK. D was estimated to be 1.24 nM and was used to calculate the K for all tested compounds. i value.

[0314] Receptor binding assays were performed using the scintillation proximity assay (SPA) format with wheat germ agglutinin (WGA) beads (PerkinElmer). The binding buffer contained 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4, 2.5 mM CaCl2, 1 mM MgCl2, 0.1% (w / v) bacitracin (Affymetrix), 0.003% (w / v) polyoxyethylene sorbitan monolaurate (TWEEN®-20), and EDTA-free Roche Complete™ protease inhibitors. GLP-1 was dissolved in DMSO at 0.339 mg / mL (0.1 mM) and stored frozen at -20°C in 100 μL aliquots. Within one hour, the GLP-1 aliquots were diluted and used in the binding assay. The peptide analogs were dissolved in dimethyl sulfoxide (DMSO) and serially diluted 3-fold in 100% DMSO. Next, 5 μL of serially diluted compounds or DMSO were transferred to a Corning® 3632 clear bottom assay plate containing 45 μL of assay binding buffer or unlabeled GLP-1 control (non-specific binding (NSB) at 0.25 μM final concentration). Next, 50 μL of hGLP-1R membrane (0.5 μg / well), 50 μL of I-125 GLP-1 (0.15 nM final concentration), and 50 μL of WGA beads (150 μg / well) were added, the plate was sealed and mixed on a plate shaker (setting 6) for 1 minute. After a 12-hour rest period at room temperature, the plate was read using a PerkinElmer Trilux MicroBeta® scintillation counter.

[0315] Results were calculated as percent specific I-125-GLP-1 binding in the presence of compound. The absolute IC of the compound was derived by nonlinear regression of the percent specific binding of I-125-GLP-1 versus the added compound. 50 Concentration. IC 50 The concentration was converted to K using the Cheng-Prusoff formula i(Cheng, Y., Prusoff, WH, Biochem. Pharmacol. 22, 3099-3108, (1973)).

[0316] K of compounds 1-4, human Gcg and human GLP-1(7-36)NH2 under hGLP-1-R i The results are shown in Table 3 below. The number of replicates (n) is indicated in parentheses. The (>) limit indicates that the % inhibition rate did not reach 50%, and the calculated K i is obtained using the highest concentration tested. n = 1 / n indicates that no average was calculated when all values ​​have a > sign and the value shown is the highest calculated value.

[0317] Table 3: K values ​​of compounds 1-4, human Gcg and human GLP-1(7-36)NH2 under hGLP-1-R i

[0318] Compound <![CDATA[K i ,nM ± SEM,(n)]]> Compound 1 23.0 ± 5.5(n=6) Compound 2 28.6 ± 4.8(n=4) Compound 3 8.68 ± 1.60(n=4) Compound 4 20.2 ± 8.2(n=4) PeopleGcg >4940(n=1 / 2) <![CDATA[Human GLP-1(7-36)NH2]]> 0.75 ± 0.056(n=67)

[0319] hGcg-R binding assay

[0320] The Gcg receptor binding assay utilized cloned hGcg-R isolated from 293HEK cells overexpressing recombinant hGcg-R (Lok, S et al., Gene 140 (2), 203-209 (1994)). The hGcg-R cDNA was subcloned into the expression plasmid phD (Trans-activated expression of fully gamma-carboxylated recombinant human protein C, an antithrombotic factor. Grinnell, BW et al., Bio / Technology 5:1189-1192 (1987)). The plasmid DNA was transfected into 293HEK cells and selected with 200 μg / mL hygromycin.

[0321] Crude plasma membranes were prepared using cells from adherent cultures. Cells were lysed on ice in hypotonic buffer containing 50 mM Tris HCl, pH 7.5 and Roche Complete™ protease inhibitors with EDTA. The cell suspension was disrupted by 25 strokes using a glass Potter-Elvehjem homogenizer equipped with a Teflon® grinding rod. The homogenate was centrifuged at 1100 x g for 10 min at 4 °C. The supernatant was collected and stored on ice while the pellet was resuspended in hypotonic buffer and rehomogenized. The mixture was centrifuged at 1100 x g for 10 min. The second supernatant was combined with the first supernatant and centrifuged at 35000 x g for 1 h at 4 °C. The membrane pellet was resuspended in homogenization buffer containing protease inhibitors, snap frozen in liquid nitrogen, and stored in aliquots in a -80 °C freezer until use.

[0322] Gcg was radioiodinated by the I-125-lactoperoxidase procedure and purified by reverse phase HPLC on a Perkin-Elmer (NEX207). The specific activity was 2200 Ci / mmol. Due to the high propanol content in the I-125 Gcg material, K was performed by homologous competition rather than saturation binding. D OK. D was estimated to be 3.92 nM and was used to calculate the K for all tested compounds. i value.

[0323] Receptor binding assays were performed using the scintillation proximity assay (SPA) format with wheat germ agglutinin (WGA) beads (PerkinElmer). The binding buffer contained 25 mM HEPES, pH 7.4, 2.5 mM CaCl2, 1 mM MgCl2, 0.1% (w / v) bacitracin (Affymetrix), 0.003% (w / v) polyoxyethylene sorbitan monolaurate (TWEEN®-20), and EDTA-free Roche Complete™ protease inhibitors. Gcg was dissolved in DMSO at 3.48 mg / mL (1 mM) and stored frozen at -20°C in 100 μL aliquots. Within one hour, the Gcg aliquots were diluted and used in the binding assay. The peptide analogs were dissolved in DMSO and serially diluted 3-fold in 100% DMSO. Next, 5 μL of serially diluted compounds or DMSO were transferred to Corning® 3632 clear bottom assay plates containing 45 μL of assay binding buffer or unlabeled Gcg control (NSB at 1 μM final concentration). Next, 50 μL of hGcg-R membrane (0.5 μg / well), 50 μL of I-125 Gcg (0.15 nM final reaction concentration), and 50 μL of WGA beads (150 μg / well) were added, the plate was sealed and mixed on a plate shaker (setting 6) for 1 minute. After a 12-hour rest period at room temperature, the plate was read using a PerkinElmer Trilux MicroBeta® scintillation counter.

[0324] Results were calculated as percent specific I-125-Gcg binding in the presence of compound. The absolute IC of the compound was derived by nonlinear regression of the percent specific binding of I-125-Gcg versus the added compound. 50 Concentration. IC 50 The concentration was converted to K using the Cheng-Prusoff formula i (Cheng, Y., Prusoff, WH, Biochem. Pharmacol. 22, 3099-3108, (1973)). K of compounds 1-4, human Gcg and human GLP-1(7-36)NH2 under hGcg-R i The results are shown in Table 4 below. The number of replicates (n) is indicated in parentheses. The (>) limit indicates that the % inhibition rate did not reach 50%, and the calculated K i Obtained using the highest concentration tested. n=1 / 2 indicates that no average was calculated when all values ​​have a > sign and the resulting value shown is the highest calculated value.

[0325] Table 4: K values ​​of compounds 1-4, human Gcg and human GLP-1(7-36)NH2 under hGcg-R i

[0326] Compound <![CDATA[K i ,nM ± SEM,(n)]]> Compound 1 14.6 ± 4.4(n=6) Compound 2 17.7 ± 5.0(n=4) Compound 3 37.3 ± 4.2(n=4) Compound 4 27.4 ± 3.3(n=4) PeopleGcg 2.49 ± 0.24(n=73) <![CDATA[Human GLP-1(7-36)NH2]]> >2420(n=1 / 2)

[0327] Functional hGLP-1-R and hGcg-R assays

[0328] Functional activity was determined in HEK-293 cloned cell lines expressing hGLP-1-R and hGcg-R. The experimental procedures and results are described below.

[0329] Each receptor-overexpressing cell line was treated with peptide in an assay volume of 40 μL in DMEM (Dulbecco's Modified Eagle's Medium, Gibco Cat# 31053) supplemented with 1X GlutaMAX TM (L-alanine-L-glutamine dipeptide in 0.85% NaCl, Gibco Cat# 35050), 0.25% FBS (dialyzed fetal bovine serum, Gibco Cat# 26400), 0.05% Fraction V BSA (bovine albumin fraction V, Gibco Cat# 15260), 250 μM IBMX (3-isobutyl-1-methylxanthine) and 20 mM HEPES [N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid), HyClone Cat# SH30237.01]. After 60 minutes incubation at room temperature, the resulting increase in intracellular cAMP (3',5'-cyclic adenosine monophosphate) was quantified using the CisBio cAMP Dynamic 2 HTRF kit (62AM4PEJ). cAMP-d2 conjugate (20 μl) was added to the cell lysis buffer, followed by the addition of antibody anti-cAMP-Eu 3+ -cryptate (20 μl) to detect intracellular cAMP levels. The resulting competition assay was incubated at room temperature for at least 60 minutes and then detected using a PerkinElmer Envision® instrument with excitation at 320 nm and emission at 665 nm and 620 nm. Envision units (emission at 665 nm / 620 nm * 10,000) are inversely proportional to the amount of cAMP present and are converted to nM cAMP per well using a cAMP standard curve. The amount of cAMP produced in each well (nM) was converted to a percentage of the maximum response observed with 10 nM human GLP-1 (7-36) NH2 or 10 nM human Gcg.

[0330] Relative EC 50Values ​​and percent maximum values ​​(Emax) were derived by nonlinear regression analysis using the percent maximum response versus the concentration of added peptide, fit to a four-parameter logistic equation (Genedata Screener®).

[0331] The functional data of compounds 1-4, human GLP-1(7-36)NH2, human Gcg and wild-type human OXM are shown in Table 5 below. 50 The average values ​​are expressed as geometric mean ± standard error of the mean (SEM), with the number of replicates (n) indicated in parentheses. max The average values ​​are expressed as arithmetic mean ± standard error. ND indicates no agonist activity was detected. All values ​​shown are three (3) significant figures.

[0332] Table 5: Functional efficacy (EC 50 ) and efficacy (E max )

[0333]

[0334] Functional activation of rat GLP-1-R in the insulinoma cell line INS1 832-3

[0335] The functional activity of compounds 1-4 on endogenous GLP-1 receptor-stimulated cAMP production was determined using rat pancreatic β-cell line, INS1 832-3 cells. Cells were maintained in RPMI 1640 medium (HyClone, Cat# SH30027) supplemented with 10% fetal bovine serum, 10 mM HEPES, 1 mM sodium pyruvate, 2 mM L-glutamine, 50 μM 2-mercaptoethanol, and 100 U / ml penicillin / 100 μg / ml streptomycin at 37°C in a 5% CO2 incubator and passaged twice a week.

[0336] The assay was performed by detaching cells from the culture flask using a non-enzymatic cell detachment agent and pelleting them by centrifugation at 1000 rpm for 5 minutes at room temperature. The cell pellet was resuspended in Earle's balanced salt solution (EBSS) supplemented with 11.2 mM glucose and 0.1% BSA. 640 μl of cell suspension at 50 μl / ml was placed in a 96-well half-volume black microplate (Costar 3875) and incubated for 2 hours at 37°C, 5% CO2 incubator to recover and starve. Serial dilutions of test compounds were prepared in 100% DMSO at 100X final test concentration and further diluted 20 times in EBSS supplemented with 11.2 mM glucose, 0.1% BSA and 1.25 mM IBMX (Sigma I-7816). After 2 hours of starvation, cells were treated with compounds by adding 10 μl of 5X compound dilutions to cell microplates (n = 2) and incubated for 30 minutes at 37°C, 5% CO2 incubator.

[0337] cAMP concentration was measured using the HTRF cAMP assay kit (Cisbio): cAMP-d2 conjugate (20 μl) in cell lysis buffer was added to the cells in the microplate wells, followed by the addition of the antibody anti-cAMP-Eu also in cell lysis buffer. 3+ -cryptate (20 μl). The resulting competition assay was incubated at room temperature for at least 60 minutes and then detected using a PerkinElmer Envision® instrument with excitation at 320 nm and emission at 665 nm and 620 nm. Envision units (emission at 665 nm / 620 nm*10,000) are inversely proportional to the amount of cAMP present and are converted to nM cAMP per well using a cAMP standard curve.

[0338] The concentration of cAMP in each well (nM) was calculated using the cAMP standard curve and converted to a percentage of the maximum response observed with native GLP-1 peptide at 300 nM for the curve fit.

[0339] Relative EC 50 Value and percentage maximum (%E max ) were derived by nonlinear regression analysis using the percent maximal response relative to the concentration of added peptide, fitted to a four-parameter logistic equation (GraphPad Prism (version 6.05) software). The assay was performed in duplicate microplates. The number of replicates (n) is indicated in parentheses.

[0340] EC of wild-type human OXM, semaglutide, and compounds 1 and 2 50 and % E max The calculations are essentially as described above. The EC 50 and % E max The data are provided in Table 6. Further, compounds 1 and 2 increased cAMP production in a dose-dependent manner (data not shown).

[0341] Table 6: EC values ​​of wild-type human OXM, semaglutide, and compounds 1 and 2 at rat GLP-1-R in the insulinoma cell line INS1 832-3 50

[0342] Compound <![CDATA[EC 50 (nM)]]> <![CDATA[E max (%)]]> Wild-type human OXM 7.3(n=1) 121.1 Semaglutide 5.8(n=1) 107.2 Compound 1 26.1(n=1) 112.3 Compound 2 62(n=1) 104.5

[0343] Functional activation of hGcg-R in primary human hepatocytes

[0344] Human primary hepatocytes were used to determine the functional activity of the compounds in stimulating cAMP production at the endogenous Gcg receptor.

[0345] The vials of human primary hepatocytes were frozen in a liquid nitrogen tank. Upon removal, the vials were immediately thawed in a water bath with a temperature of 37° C. Then, the cell suspension was transferred to 50 ml CHRM (Gibco / Life Technologies cat# CM7000 Cryopreserved Hepatocyte Recovery Medium).

[0346] The cell suspension was centrifuged at 1,000 xg for 10 min. After CHRM was removed by aspiration, the cell pellet was resuspended in 5 ml of plating medium. Plating medium was prepared by adding full CM3000 Supplement Packs to 500 ml of Williams medium (Gibco / LifeTechnologies) and sterile filtered through a 0.22 μm membrane.

[0347] The cell density was counted on a hemacytometer by adding 100 μl of the cell suspension to 100 μl of Trypan Blue (HyClone Trypan Blue 0.04%, catalog number SV30084.01). The cell suspension was further diluted to 0.8 X 10 6 65 ml of plating medium was added to each well of a collagen-coated 96-well microplate (Corning BioCoat, catalog number 354649, Lot# 22314033). Then 65 ml of cell suspension was added to each well of the collagen-coated 96-well microplate to a final cell density of 50,000 cells / well. The cell microplate was incubated at 37°C, 5% CO 2 Incubate in an incubator for 3 to 4 hours.

[0348] After 3-4 hours of incubation, the medium was aspirated and replaced with 100 ml of maintenance medium. Maintenance medium was prepared by adding full CM4000 SupplementPacks to 500 ml of Williams medium (Gibco / Life Technologies) and sterile filtered through a 0.22 μm membrane. In preparation for the cAMP assay, the cell microplate was returned to a 37°C, 5% CO2 incubator overnight.

[0349] In preparation for the assay, compounds 1 and 2 and wild-type human OXM were subjected to 3-fold serial dilutions at 10 concentrations in compound assay buffer (HBSS containing 20 mM HEPES and 1% heat-inactivated FBS).

[0350] The cell microplate was removed from the incubator and the medium was removed by gentle aspiration without disturbing the cell monolayer. The cells were treated by adding 40 μl of cell assay buffer and 40 μl of test solution (i.e., compound 1, compound 2 or wild-type human OXM diluted in compound assay buffer) to the cell microplate and incubated at room temperature with gentle aspiration for 1 hour.

[0351] cAMP concentration was measured using the HTRF cAMP assay kit (Cisbio): cAMP-d2 conjugate (40 μl) in cell lysis buffer was added to the cells in the microplate wells, followed by the addition of the antibody anti-cAMP-Eu also in cell lysis buffer. 3+ -cryptate (40 μl). The resulting competition assay was incubated at room temperature for at least 60 minutes and then detected using a PerkinElmer Envision® instrument with excitation at 320 nm and emission at 665 nm and 620 nm. Envision units (emission at 665 nm / 620 nm*10,000) are inversely proportional to the amount of cAMP present and are converted to nM cAMP per well using a cAMP standard curve.

[0352] The concentration of cAMP in each well (nM) was used for the cAMP standard curve and converted to a percentage of the maximum response observed with the Gcg analog conjugated to a saturated C18 fatty acid (diacid) for curve fitting.

[0353] Relative EC 50 Value and percentage maximum (%E max ) were derived by nonlinear regression analysis using the percent maximal response versus the concentration of added peptide, fitted to a four-parameter logistic equation (GraphPad Prism (version 6.05) software).

[0354] EC of compounds 1 and 2 and wild-type human OXM 50 and % E max The calculations are essentially as described above. The EC 50 and % E max The data are provided in Table 7. Further, compounds 1 and 2 increased cAMP production in a dose-dependent manner (data not shown). The number of replicates (n) is indicated in parentheses.

[0355] Table 7: EC of Compounds 1 and 2 and wild-type human OXM on hGcg-R in human primary hepatocytes 50

[0356] Compound <![CDATA[EC 50 (nM)]]> <![CDATA[E max (%)]]> Wild-type human OXM 29.8(n=1) 90.0 Compound 1 99.9(n=1) 100.0 Compound 2 159.4(n=1) 101.2

[0357] Pharmacokinetics

[0358] Pharmacokinetics in cynomolgus monkeys

[0359] The in vivo pharmacokinetic properties of the compounds of the present invention were demonstrated using cynomolgus monkeys.

[0360] Compounds were administered by a single intravenous or subcutaneous dose of 50 nmole / kg or 250 nmole / kg. Blood was collected from each animal at 4, 8, 12, 24, 48, 72, 96, 120, 144, 168, 192, 240, 288, 208, 480, 576 and 672 hours after dosing.

[0361] The plasma concentration of the compound was determined by LC / MS. In brief, acetonitrile was used to extract the compound of the present invention from 100% monkey plasma (25 μl). Two separate layers were formed by centrifugation, wherein the compound was located in the liquid layer. The supernatant of 80 μl aliquots was transferred to a 96-well microplate and diluted with 150 μl of water and 25 μl of formic acid. The diluted sample (10 μl) was injected into a Supelco Analytical Discovery BIO Wide Pore C5-3, 5 cm X 1 mm, 3 μm column. The column effluent was introduced into a Thermo Q-Exactive mass spectrometer for detection and quantification.

[0362] In an experiment performed essentially as described for the assay, cynomolgus monkeys were administered a single subcutaneous (50 nmole / kg) dose of Compound 1 in 40 mM Tris HCl (pH 8.0) in a volume of 0.20 mL / kg. Blood was collected from each animal at 2 (IV only), 7, 12, 24, 48, 72, 96, 120, 168, 192, 240, 336, 480, 576, and 672 hours after dosing.

[0363] Other cynomolgus monkeys were administered a single intravenous (50 nmole / kg) or subcutaneous (50 or 250 nmole / kg) dose of Compound 2 in 40 mM Tris HCl (pH 8.0) in a volume of 0.20 mL / kg. Blood was collected from each animal at 2 (IV only), 7, 12, 24, 48, 72, 96, 120, 168, 192, 240, 336, 480, 576, and 672 hours after dosing.

[0364] The data for Compound 1 are provided in Table 9, and the data for Compound 2 are provided in Table 10.

[0365] Compound 1 reached mean maximum plasma concentrations approximately 12 hours after a 50 nmol / kg subcutaneous dose. The mean half-life was 57 hours, and the mean clearance was 2.16 mL / hour / kg (Table 8).

[0366] Compound 2 reached mean maximum plasma concentrations approximately 24 hours after a 50 nmol / kg subcutaneous dose. The mean half-life was 122 hours, and the mean clearance was 0.55 mL / hour / kg (Table 9).

[0367] Table 8: Individual and mean pharmacokinetic parameters following a single 50 nmol / kg subcutaneous dose of Compound 1 in male cynomolgus monkeys

[0368]

[0369] Abbreviation: AUC 0-inf = area under the curve from 0 to infinity, CL / F = clearance / bioavailability, T max = time to reach maximum concentration, C max = maximum plasma concentration, T 1 / 2 = half-life.

[0370] Table 9: Individual or mean pharmacokinetic parameters of Compound 2 after a single intravenous or subcutaneous administration to male cynomolgus monkeys

[0371]

[0372] Abbreviation: AUC 0-inf = area under the curve from 0 to infinity, CL = clearance, CL / F = clearance / bioavailability, T max = time to reach maximum concentration, C0 = concentration extended to time 0 hours, C max = maximum plasma concentration, T 1 / 2 = half-life, NA = not applicable.

[0373] In vivo studies

[0374] Oral glucose tolerance test (OGTT) in DIO mice

[0375] The (DIO) mouse model of diet-induced obesity is a model of insulin resistance. In this study, male DIO mice (C57BL / 6) from Taconic Biosciences were used at five to six months of age. Animals were raised in a temperature-controlled (24°C) facility with a 12-hour day / night cycle (lights on at 06:00), and free access to food and water. The adaptation period to the facility was two weeks. The day before the study, animals were randomly divided based on their body weight. In the afternoon of the same day, animals were fasted in a clean cage, and vehicle (40 mM Tris-HCl, pH 8.0) or test substance was administered by subcutaneous injection. The next morning, 16 hours after the injection of peptide, fasting body weight was obtained to calculate glucose dosage. Blood samples were collected to measure zero-time glucose. Then, glucose (2 g / kg) was orally fed to animals. Two glucose readings were obtained by glucometer 15, 30, 60 and 120 minutes after oral glucose. The mean value of two glucose readings was reported at each time point, and the area under the curve was calculated. Statistics were analyzed using analysis of variance with Dunnett's comparisons by JMP 6; significance was indicated at p ≤ 0.05 relative to vehicle.

[0376] In experiments performed essentially as described in this assay, Compound 2 showed a dose-dependent decrease in glucose over the course of the tolerance test, with glucose AUC being reduced at all three doses tested, 1, 3 and 10 nmol / kg (Table 10).

[0377] Table 10: Glucose AUC in response to OGTT (2 g / kg) in male DIO mice treated with Compound 2 and Semaglutide

[0378]

[0379] % Vehicle was calculated as 100 x (value calculated for compound group / value calculated for vehicle group)

[0380] * p ≤ 0.05.

[0381] OGTT in streptozotocin (STZ)-treated DIO mice

[0382] The mouse model treated by STZ is a model of early diabetes. In this study, male DIO mice (C57BL / 6) from five to six months of age from Taconic Biosciences were used. Animals were raised in temperature-controlled (24°C) facilities with a 12-hour day / night cycle (lights on at 06:00), and free access to food and water. After adapting to the facility for two weeks, mice were injected with 50mg / kg of STZ intraperitoneally on Tuesday and Friday. After injection for two weeks, animals with blood sugar levels between 180-300mg / dL at 09:00 were selected for OGTT research. The day before the study, animals were randomly divided based on their body weight and blood sugar levels. Before removing food overnight (16:00), animals were treated with vehicle or test substance by subcutaneous injection. At 08:00 the next morning, after 16 hours of injection of compound, blood samples were collected to measure zero-time glucose. Animals were orally administered glucose (2 g / kg). Glucose was measured 15, 30, 60 and 120 minutes after oral glucose challenge. Statistics were analyzed using analysis of variance with Dunnett's comparisons by JMP 6. Significance was indicated at p ≤ 0.05 relative to vehicle.

[0383] In experiments performed essentially as described in this assay, Compound 2 demonstrated a dose-dependent reduction in glucose excursions during the tolerance test. Glucose AUC was reduced at all three doses tested, 1, 3 and 10 nmol / kg (Table 11).

[0384] Table 11: Glucose AUC in response to OGTT (2 g / kg) in male STZ mice treated with Compound 2 and Semaglutide

[0385]

[0386] % Vehicle was calculated as 100 x (value calculated for compound group / value calculated for vehicle group)

[0387] * p ≤ 0.05.

[0388] Glucose control in DIO mice

[0389] In this study, male DIO mice (C57BL / 6) from Taconic Biosciences were used at five to six months of age. Animals were raised in a temperature-controlled (24°C) facility with a 12-hour day / night cycle (lights on at 06:00), and free access to food and water. After two weeks of adaptation to the facility, mice were randomly divided into treatment groups (n=7 / group) based on their body weight and blood sugar. Mice were injected subcutaneously with vehicle or compound (25 nmol / kg) once. Blood sugar was monitored 2 and 8 hours after injection, and then monitored once a day for 4 days at 08:00. OGTT was performed 44 and 78 hours after the injection of peptide. Statistics were analyzed using analysis of variance using Dunnett's comparisons by JMP 6. Significance was expressed with p ≤ 0.05 relative to vehicle.

[0390] In experiments performed essentially as described in this assay, Compound 2 and Compound 4 treated mice had glucose lower than vehicle controls up to 96 hours post-injection. When an OGTT was performed, Compound 2 and Compound 4 treated mice had lower glucose excursions at both time points following oral glucose challenge.

[0391] Compound 1 and Compound 3 reduced blood glucose for up to 72 hours (Table 12). Compound 1 and Compound 3 treated mice had lower sugar excursions 44 hours after oral glucose challenge following peptide injection.

[0392] Table 12: Blood glucose measured at 2, 8, 24, 48, 72 and 96 hours after injection in male DIO mice

[0393]

[0394] Table 13: Glucose excursions during OGTT 44 and 78 hours after compound injection

[0395]

[0396] % Vehicle was calculated as 100 x (value calculated for compound group / value calculated for vehicle group)

[0397] * p ≤ 0.05.

[0398] Long-term treatment in DIO mice

[0399] Effects on food intake and body weight / fat were evaluated in DIO mice. In this study, five to six month old DIO mice (C57BL / 6) from Taconic Biosciences were used. Animals were housed in a temperature-controlled (24°C) facility with a 12-hour day / night cycle (lights on at 06:00) and free access to food and water. Mice were adapted to the facility for two weeks. Fat mass was measured by nuclear magnetic resonance (NMR) using an Echo Medical System (Houston, TX) instrument one day before the start of the study. Mice were randomly divided into treatment groups (N=7 / group) based on body weight and fat mass, so that each group had a similar starting average body weight and fat mass. The vehicle (40mM Tris-HCl, pH 8.0), test compound or positive control semaglutide was administered to any mouse at 8-10am every three days for 15 days by subcutaneous (SC) injection. SC injections were performed on days 1, 4, 7, 10 and 13. Body weight and food intake were measured immediately before each injection throughout the study. The percent change in body weight was calculated as follows:

[0400] 100 x (Final weight of the animal – Initial weight of the animal)

[0401] Initial weight of the animal

[0402] At the end of the study, fat mass was measured again by NMR. Statistics were analyzed using analysis of variance with Dunnett's comparisons by JMP 6. Significance was indicated at p ≤ 0.05 relative to vehicle.

[0403] In experiments performed essentially as described in this assay, compounds 1-4 reduced food intake and body weight / body fat as shown in Table 14 below:

[0404] Table 14: % Body Weight Change and % Body Fat Change in DIO Mice

[0405]

[0406] * p ≤ 0.05.

[0407] Acute treatment in DIO mice

[0408] In order to study the metabolic pathways involved in the treatment of the compounds of the present invention that are independent of weight loss, compounds were acutely tested in DIO mice (C57BL / 6). The mice used were three to four months old and were on a high-fat diet for at least 4 weeks. The animals were raised in a temperature-controlled (24°C) facility with a 12-hour day / night cycle (lights on at 06:00) and free access to food and water. The vehicle or test compound was administered to the mice at 16:00 a day before the study day by subcutaneous injection. The animals were sacrificed 16 hours later to collect blood by cardiac puncture. Statistics were analyzed using an analysis of variance using Dunnett's comparisons for JMP 6. Significance was expressed with p ≤ 0.05 relative to the vehicle.

[0409] In experiments performed essentially as described in this assay, compounds 1-3 reduced serum cholesterol and PCSK9 levels and increased FGF-21 levels, as shown in Table 15. In contrast, treatment with semaglutide did not reduce serum cholesterol and PCSK9 levels and increase FGF-21 levels. Food intake was reduced to similar levels in all treatment groups, which may indicate that changes in cholesterol, PCSK9 and FGF-21 are independent of food intake.

[0410] Table 15: Acute effects on PCSK9, FGF-21 and cholesterol levels.

[0411]

[0412] % Vehicle was calculated as 100 x (value calculated for compound group / value calculated for vehicle group)

[0413] * p ≤ 0.05.

[0414] Effects on energy expenditure in DIO mice

[0415] In this study, male DIO mice (C57BL / 6) weighing 45-50 g at seven to eight months of age were used to evaluate the effects of the compounds of the present invention on energy metabolism. The animals were housed in a temperature-controlled (24°C) facility with a 12-hour day / night cycle (lights on at 22:00), and food (TD95217) (Teklad) and water were freely available. After acclimation to the facility for 2 weeks, the mice were randomly divided into treatment groups (N=6 / group) based on body weight so that each group had a similar starting average body weight. The animals were placed in a PhenoMaster / LabMaster calorimeter (TSE Systems, Chesterfield, MO) for 8 days to adapt. The vehicle (40 mM Tris HCl buffer at pH 8.0, 10 ml / kg), the test substance (15 nmol / kg) or semaglutide (60 nmol / kg) was subcutaneously administered to DIO mice bred arbitrarily every three days 30-90 minutes before the start of the night cycle for 15 days.

[0416] Heat and respiratory quotient (RER) were measured using an open circuit calorimeter system by indirect calorimetry. RER is the ratio of the volume of CO2 produced (Vco2) to the volume of O2 consumed (Vo2). Heat was calculated using the lean body mass considered. Energy expenditure was kcal / kg / 3 days and was expressed as mean ± SEM for 6 mice per group. Statistical significance was evaluated by two-way ANOVA followed by Tukey's multiple comparison test.

[0417] In experiments performed essentially as described in this assay, mice treated with Compounds 1 and 2 increased their metabolic rate from week 2 onwards, and the effect was maintained throughout the treatment period, as shown in Table 16. However, semaglutide had no effect on metabolic rate. This increase in metabolic rate can be attributed to the additional weight loss observed with the administration of Compounds 1 and 2 compared to the administration of semaglutide.

[0418] Table 16: Effect of long-term treatment with Compound 1, Compound 2 or Semaglutide on metabolic rate in DIO mice

[0419]

[0420] * p ≤ 0.05 relative to carrier

[0421] ** p ≤ 0.05 vs. semaglutide.

[0422] Amino acid sequence

[0423] SEQ ID NO:1 (human OXM)

[0424] His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala

[0425] (HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA)

[0426] SEQ ID NO:2 (artificial sequence)

[0427] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Phe-Val-Glu-Trp-Leu-Leu-Xaa28-Gly-Gly-Pro-Ser-Ser-Gly

[0428] (HXaa2QGTFTSDYSKYLDEKKAKEFVEWLLXaa28GGPSSG)

[0429] wherein Xaa2 is Aib;

[0430] Xaa28 is Glu (E) or Ser (S);

[0431] The Lys at position 20 is chemically modified by conjugating the ε-amino group of the Lys side chain to a C14-C24 fatty acid via (i) a direct bond or (ii) a linker between the Lys at position 20 and the C14-C24 fatty acid; and

[0432] The C-terminal amino acid is optionally amidated.

[0433] SEQ ID NO:3 (artificial sequence)

[0434] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly

[0435] (HXaa2QGTFTSDYSKYLDEKKAKEFVEWLLEGGPSSG)

[0436] wherein Xaa2 is Aib;

[0437] The Lys at position 20 is chemically modified by conjugating the ε-amino group of the Lys side chain to a C14-C24 fatty acid via (i) a direct bond or (ii) a linker between the Lys at position 20 and the C14-C24 fatty acid; and

[0438] The C-terminal amino acid is optionally amidated.

[0439] SEQ ID NO:4 (artificial sequence)

[0440] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Ph e-Val-Glu-Trp-Leu-Leu-Ser-Gly-Gly-Pro-Ser-Ser-Gly(HXaa2QGTFTSDYSKYLDEKKAKEFVEWLLSGGPSSG)

[0441] wherein Xaa2 is Aib;

[0442] The Lys at position 20 is chemically modified by conjugating the ε-amino group of the Lys side chain to a C14-C24 fatty acid via (i) a direct bond or (ii) a linker between the Lys at position 20 and the C14-C24 fatty acid; and

[0443] The C-terminal amino acid is optionally amidated.

[0444] SEQ ID NO:5 (artificial sequence)

[0445] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly

[0446] (HXaa2QGTFTSDYSKYLDEKKAKEFVEWLLEGGPSSG)

[0447] wherein Xaa2 is Aib;

[0448] Lys at position 20 is synthesized by reacting the ε-amino group of the Lys side chain with ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2) 16Chemically modified by CO2H conjugation; and

[0449] The C-terminal amino acid is amidated.

[0450] SEQ ID NO:6 (artificial sequence)

[0451] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly

[0452] (HXaa2QGTFTSDYSKYLDEKKAKEFVEWLLEGGPSSG)

[0453] wherein Xaa2 is Aib;

[0454] Lys at position 20 is synthesized by reacting the ε-amino group of the Lys side chain with ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2) 18 Chemically modified by CO2H conjugation; and

[0455] The C-terminal amino acid is amidated.

[0456] SEQ ID NO:7 (artificial sequence)

[0457] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Phe-Val-Glu-Trp-Leu-Leu-Ser-Gly-Gly-Pro-Ser-Ser-Gly

[0458] (HXaa2QGTFTSDYSKYLDEKKAKEFVEWLLSGGPSSG)

[0459] wherein Xaa2 is Aib;

[0460] Lys at position 20 is synthesized by reacting the ε-amino group of the Lys side chain with ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2) 16 Chemically modified by CO2H conjugation; and

[0461] The C-terminal amino acid is amidated.

[0462] SEQ ID NO:8 (artificial sequence)

[0463] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Phe-Val-Glu-Trp-Leu-Leu-Ser-Gly-Gly-Pro-Ser-Ser-Gly

[0464] (HXaa2QGTFTSDYSKYLDEKKAKEFVEWLLSGGPSSG)

[0465] wherein Xaa2 is Aib;

[0466] Lys at position 20 is synthesized by reacting the ε-amino group of the Lys side chain with ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2) 18 Chemically modified by CO2H conjugation; and

[0467] The C-terminal amino acid is amidated.

[0468] SEQ ID NO:9 (artificial sequence)

[0469] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Phe-Val-Glu-Trp-Leu-Leu-Xaa28-Gly-Gly-Pro-Ser-Ser-Gly

[0470] (HXaa2QGTFTSDYSKYLDEKKAKEFVEWLLXaa28GGPSSG)

[0471] wherein Xaa2 is Aib;

[0472] Xaa28 is Glu(E) or Ser(S); and

[0473] The C-terminal amino acid is optionally amidated.

[0474] SEQ ID NO:10 (artificial sequence)

[0475] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly

[0476] (HXaa2QGTFTSDYSKYLDEKKAKEFVEWLLEGGPSSG)

[0477] wherein Xaa2 is Aib; and

[0478] The C-terminal amino acid is optionally amidated.

[0479] SEQ ID NO:11 (artificial sequence)

[0480] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Phe-Val-Glu-Trp-Leu-Leu-Ser-Gly-Gly-Pro-Ser-Ser-Gly

[0481] (HXaa2QGTFTSDYSKYLDEKKAKEFVEWLLSGGPSSG)

[0482] wherein Xaa2 is Aib; and

[0483] The C-terminal amino acid is optionally amidated. Sequence Listing <110> Eli Lilly and Company <120> Glucagon and GLP-1 co-agonist compounds <130> X-20637 <150> 62 / 182847 <151> 2015-06-22 <160> 11 <170> PatentIn version 3.5 <210> 1 <211> 37 <212> PRT <213> Homo sapiens <400> 1 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Lys Arg Asn 20 25 30 Arg Asn Asn Ile Ala 35 <210> 2 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MISC_FEATURE <222> (2) <223> Xaa at position 2 is a non-naturally occurring amino acid 2-Aminoisobutyric acid <220> <221> MOD_RES <222> (2) <223> Aib <220> <221> MOD_RES <222> (20)..(20) <223> The Lys at position 20 is chemically modified by conjugating the ε-amino group of the Lys side chain to a C14-C24 fatty acid via (i) a direct bond or (ii) a linker between the Lys at position 20 and the C14-C24 fatty acid. <220> <221> MISC_FEATURE <222> (28)..(28) <223> Xaa at position 28 is Glu or Ser <220> <221> MOD_RES <222> (34)..(34) <223> The Gly at position 34 is optionally amidated <400> 2 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Lys Ala Lys Glu Phe Val Glu Trp Leu Leu Xaa Gly Gly Pro Ser 20 25 30 Ser Gly <210> 3 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MISC_FEATURE <222> (2) <223> Xaa at position 2 is a non-naturally occurring amino acid 2-Aminoisobutyric acid <220> <221> MOD_RES <222> (2) <223> Aib <220> <221> MOD_RES <222> (20)..(20) <223> Lys at position 20 is chemically modified by conjugating the ε-amino group of the Lys side chain to a C14-C24 fatty acid via (i) a direct bond or (ii) a linker between Lys at position 20 and the C14-C24 fatty acid. <220> <221> MOD_RES <222> (34)..(34) <223> The Gly at position 34 is optionally amidated <400> 3 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Lys Ala Lys Glu Phe Val Glu Trp Leu Leu Glu Gly Gly Pro Ser 20 25 30 Ser Gly <210> 4 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MISC_FEATURE <222> (2) <223> Xaa at position 2 is a non-naturally occurring amino acid 2-Aminoisobutyric acid <220> <221> MOD_RES <222> (2) <223> Aib <220> <221> MOD_RES <222> (20)..(20) <223> Lys at position 20 is chemically modified by conjugating the ε-amino group of the Lys side chain to a C14-C24 fatty acid via (i) a direct bond or (ii) a linker between Lys at position 20 and the C14-C24 fatty acid. <220> <221> MOD_RES <222> (34)..(34) <223> The Gly at position 34 is optionally amidated <400> 4 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Lys Ala Lys Glu Phe Val Glu Trp Leu Leu Ser Gly Gly Pro Ser 20 25 30 Ser Gly <210> 5 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MISC_FEATURE <222> (2) <223> Xaa at position 2 is a non-naturally occurring amino acid 2-Aminoisobutyric acid <220> <221> MOD_RES <222> (2) <223> Aib <220> <221> MOD_RES <222> (20)..(20) <223> Lys at position 20 binds to the ε-amino group of the Lys side chain. ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(y-Glu)-CO-(CH2)16CO2H conjugation to chemical modification <220> <221> MOD_RES <222> (34)..(34) <223> Gly at position 34 is amidated <400> 5 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Lys Ala Lys Glu Phe Val Glu Trp Leu Leu Glu Gly Gly Pro Ser 20 25 30 Ser Gly <210> 6 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MISC_FEATURE <222> (2) <223> Xaa at position 2 is a non-naturally occurring amino acid 2-Aminoisobutyric acid <220> <221> MOD_RES <222> (2) <223> Aib <220> <221> MOD_RES <222> (20)..(20) <223> Lys at position 20 binds to the ε-amino group of the Lys side chain. ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(y-Glu)-CO-(CH2)18CO2H conjugation to chemical modification <220> <221> MOD_RES <222> (34)..(34) <223> Gly at position 34 is amidated <400> 6 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Lys Ala Lys Glu Phe Val Glu Trp Leu Leu Glu Gly Gly Pro Ser 20 25 30 Ser Gly <210> 7 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MISC_FEATURE <222> (2) <223> Xaa at position 2 is a non-naturally occurring amino acid 2-Aminoisobutyric acid <220> <221> MOD_RES <222> (2) <223> Aib <220> <221> MOD_RES <222> (20)..(20) <223> Lys at position 20 binds to the ε-amino group of the Lys side chain. ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(y-Glu)-CO-(CH2)16CO2H conjugation to chemical modification <220> <221> MOD_RES <222> (34)..(34) <223> Gly at position 34 is amidated <400> 7 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Lys Ala Lys Glu Phe Val Glu Trp Leu Leu Ser Gly Gly Pro Ser 20 25 30 Ser Gly <210> 8 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MISC_FEATURE <222> (2) <223> Xaa at position 2 is a non-naturally occurring amino acid 2-Aminoisobutyric acid <220> <221> MOD_RES <222> (2) <223> Aib <220> <221> MOD_RES <222> (20)..(20) <223> Lys at position 20 binds to the ε-amino group of the Lys side chain. ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(y-Glu)-CO-(CH2)18CO2H conjugation to chemical modification <220> <221> MOD_RES <222> (34)..(34) <223> Gly at position 34 is amidated <400> 8 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Lys Ala Lys Glu Phe Val Glu Trp Leu Leu Ser Gly Gly Pro Ser 20 25 30 Ser Gly <210> 9 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MISC_FEATURE <222> (2) <223> Xaa at position 2 is a non-naturally occurring amino acid 2-Aminoisobutyric acid <220> <221> MOD_RES <222> (2) <223> Aib <220> <221> MISC_FEATURE <222> (28)..(28) <223> Xaa at position 28 is Glu or Ser <220> <221> MOD_RES <222> (34)..(34) <223> The Gly at position 34 is optionally amidated <400> 9 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Lys Ala Lys Glu Phe Val Glu Trp Leu Leu Xaa Gly Gly Pro Ser 20 25 30 Ser Gly <210> 10 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MISC_FEATURE <222> (2) <223> Xaa at position 2 is a non-naturally occurring amino acid 2-Aminoisobutyric acid <220> <221> MOD_RES <222> (2) <223> Aib <220> <221> MOD_RES <222> (34)..(34) <223> The Gly at position 34 is optionally amidated <400> 10 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Lys Ala Lys Glu Phe Val Glu Trp Leu Leu Glu Gly Gly Pro Ser 20 25 30 Ser Gly <210> 11 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MISC_FEATURE <222> (2) <223> Xaa at position 2 is a non-naturally occurring amino acid 2-Aminoisobutyric acid <220> <221> MOD_RES <222> (2) <223> Aib <220> <221> MOD_RES <222> (34)..(34) <223> The Gly at position 34 is optionally amidated <400> 11 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Lys Ala Lys Glu Phe Val Glu Trp Leu Leu Ser Gly Gly Pro Ser 20 25 30 Ser Gly

Claims

1. A compound or a pharmaceutically acceptable salt thereof, wherein the compound has the following formula: His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly wherein Xaa2 is Aib; Lys at position 20 is synthesized by reacting the ε-amino group of the Lys side chain with ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2) 16 Chemically modified by CO2H conjugation; and The C-terminal amino acid is amidated.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the C-terminal amino acid is amidated to form a C-terminal primary amide.

3. A compound or a pharmaceutically acceptable salt thereof, wherein the compound has the formula: His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Phe-Val-Glu-Trp-Leu-Leu-Ser-Gly-Gly-Pro-Ser-Ser-Gly wherein Xaa2 is Aib; Lys at position 20 is synthesized by reacting the ε-amino group of the Lys side chain with ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu)2-CO-(CH2) 16 Chemically modified by CO2H conjugation; and The C-terminal amino acid is amidated. The compound according to claim 3 or a pharmaceutically acceptable salt thereof, wherein the C-terminal amino acid is amidated to a C-terminal primary amide.

5. A compound or a pharmaceutically acceptable salt thereof, wherein the compound has the formula: His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly wherein Xaa2 is Aib; Lys at position 20 is synthesized by reacting the ε-amino group of the Lys side chain with ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2) 18 Chemically modified by CO2H conjugation; and The C-terminal amino acid is amidated. The compound according to claim 5 or a pharmaceutically acceptable salt thereof, wherein the C-terminal amino acid is amidated to a C-terminal primary amide.

7. A compound or a pharmaceutically acceptable salt thereof, wherein the compound has the formula: His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Phe-Val-Glu-Trp-Leu-Leu-Ser-Gly-Gly-Pro-Ser-Ser-Gly wherein Xaa2 is Aib; Lys at position 20 is synthesized by reacting the ε-amino group of the Lys side chain with ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu)2-CO-(CH2) 18 Chemically modified by CO2H conjugation; and The C-terminal amino acid is amidated.

8. The compound according to claim 7 or a pharmaceutically acceptable salt thereof, wherein the C-terminal amino acid is amidated to a C-terminal primary amide.

9. A pharmaceutical composition comprising the compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

10. Use of a compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for use in a method of treating type II diabetes in a subject in need thereof.

11. Use of a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for use in a method of treating obesity in a subject in need thereof.

12. Use of a compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for use in a method of treating non-alcoholic fatty liver disease (NAFLD) in a subject in need thereof.

13. Use of a compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for use in a method of treating non-alcoholic steatohepatitis (NASH) in a subject in need thereof.

14. Use of a compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for use in a method of inducing weight loss in a subject in need thereof.

15. A method for preparing a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, the method comprising the steps of: (i) modifying the intermediate compound of the following formula by conjugating the ε-amino group of the Lys side chain at position 20 of the intermediate compound with a C14-C24 fatty acid via a linker: His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Phe-Val-Glu-Trp-Leu-Leu-Xaa28-Gly-Gly-Pro-Ser-Ser-Gly in Xaa2 is Aib; Xaa28 is Glu or Ser; and The C-terminal amino acid is amidated.

16. The method of claim 15, wherein the C-terminal amino acid of the intermediate compound is amidated to a C-terminal primary amide.

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