Acylated glucagon analogs

Abstract

The present application relates to acylated glucagon analogs. The present invention provides materials and methods for the treatment of obesity and overweight, diabetes, and other related metabolic diseases. In particular, the present invention provides a variety of novel acylated glucagon analog peptides that are effective in such methods. The peptide can mediate the action of human glucagon by having improved selectivity for the GLP-1 receptor as compared to the human glucagon.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201480056639.X and invention name “Acylated Glucagon Analogs”. The original application is the PCT international application PCT / EP2014 / 072293 filed on October 17, 2014, which entered the Chinese national phase on April 14, 2016. Technical Field

[0002] The present invention relates to acylated glucagon analogs and their medical use, for example, in the treatment of obesity and overweight, diabetes and other metabolic diseases. Background Art

[0003] Pre-proglucagon is a 158 amino acid precursor polypeptide that is differentially processed in tissues to form a variety of structurally related proglucagon-derived peptides, including glucagon (Glu), glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), and oxyntomodulin (OXM). These molecules are involved in a variety of physiological functions, including glucose homeostasis, insulin secretion, gastric emptying and intestinal growth, and food intake regulation.

[0004] Glucagon is a 29 amino acid peptide that corresponds to amino acids 53 to 81 of preproglucagon. Oxyntomodulin (OXM) is a 37 amino acid peptide that contains the entire 29 amino acid sequence of glucagon and an octapeptide carboxyl-terminal extension (amino acids 82 to 89 of preproglucagon, referred to as "intervening peptide 1" or IP-1). The major biologically active fragment of GLP-1 is produced as a 30 amino acid C-terminal amidated peptide corresponding to amino acids 98 to 127 of preproglucagon.

[0005] Glucagon helps maintain glucose levels in the blood by binding to glucagon receptors on liver cells and causing the liver to release glucose stored in the form of glycogen via glycogenolysis. As these stores become depleted, glucagon stimulates the liver to synthesize additional glucose via gluconeogenesis. This glucose is released into the bloodstream, preventing hypoglycemia from occurring.

[0006] GLP-1 reduces elevated blood glucose levels by increasing glucose-stimulated insulin secretion and promotes weight loss primarily by reducing food intake.

[0007] OXM is released into the blood in response to food intake and is proportional to the caloric content of the meal. OXM has been shown to suppress appetite and inhibit food intake in humans (Cohen et al., Journal of Endocrinology and Metabolism, 88, 4696-4701, 2003; WO 2003 / 022304). In addition to those appetite suppressing effects similar to GLP-1, OXM must also affect body weight through other mechanisms, because rats treated with oxyntomodulin show less weight gain compared to pair-fed rats (Bloom, Endocrinology 2004, 145, 2687). Treatment of obese rodents with OXM also improves their glucose tolerance (Parlevliet et al., Am J Physiol Endocrinol Metab, 294, E142-7, 2008) and inhibits weight gain (WO 2003 / 022304).

[0008] OXM activates both the glucagon receptor and the GLP-1 receptor, having twice the potency for the glucagon receptor than the GLP-1 receptor, but having lower potency than native glucagon and GLP-1 for their respective receptors. Human glucagon is also able to activate both receptors, but has a stronger preference for the glucagon receptor than the GLP-1 receptor. On the other hand, GLP-1 cannot activate the glucagon receptor. The mechanism of action of oxyntomodulin is not well understood. In particular, it is not known whether some of the extrahepatic effects of the hormone are mediated through the GLP-1 receptor and the glucagon receptor, or through one or more receptors that have not yet been identified.

[0009] Other peptides have been shown to bind to and activate both the glucagon receptor and the GLP-1 receptor (Hjort et al., Journal of Biological Chemistry, 269, 30121-30124, 1994), and inhibit weight gain and reduce food intake (see, e.g., WO 2006 / 134340, WO 2007 / 100535, WO 2008 / 10101, WO 2008 / 152403, WO 2009 / 155257, WO 2009 / 155258、WO2010 / 070252、WO2010 / 070253、WO2010 / 070255、WO2010 / 070251、W O2011 / 006497, WO2011 / 160630, WO2011 / 160633, WO2013 / 092703, WO2014 / 041195.

[0010] Obesity is a growing global health problem that is associated with a variety of diseases, particularly cardiovascular disease (CVD), type 2 diabetes, obstructive sleep apnea, certain types of cancer, and osteoarthritis. As a result, obesity has been found to reduce life expectancy. According to a 2005 forecast by the World Health Organization, 400 million adults (age > 15) worldwide are classified as obese. In the United States, obesity is currently considered the second leading cause of preventable death after smoking.

[0011] The rise in obesity is driving the rise in diabetes, with approximately 90% of people with type 2 diabetes being classified as obese. Worldwide, 246 million people have diabetes, and 380 million are expected to have the condition by 2025. Many also have additional cardiovascular risk factors, including high / abnormal LDL and triglycerides and low HDL. Summary of the invention

[0012] In a first aspect, the present invention provides a compound having the formula:

[0013] R 1 -P 1 -P 2 -R 2

[0014] in,

[0015] R 1 H, C 1-4 Alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;

[0016] R 2 OH or NH2;

[0017] P 1 is a peptide having the following sequence:

[0018] H-X2-X3-GTFTSDYSKYLDSΨAAHDFVEWLLSA

[0019] in:

[0020] X2 is selected from the group consisting of Aib, Ala, D-Ala, Ser, N-Me-Ser, Ac3c, Ac4c and Ac5c;

[0021] X3 is selected from Gln and His;

[0022] P 2Absent or a sequence of 1 to 20 amino acid units, said amino acid units being independently selected from the group consisting of Ala, Leu, Ser, Thr, Tyr, Cys, Glu, Lys, Arg, Dbu, Dpr and Orn;

[0023] or a pharmaceutically acceptable salt or solvate thereof;

[0024] Ψ is a residue of Lys, Arg, Orn or Cys, wherein the side chain is identical to a residue of the formula -Z 2 -Z 1 Substituent conjugation;

[0025] -Z 1 A polar group at one end of the chain and a Z at the end of the chain away from the polar group 2 The fatty chain of the linker-X-,

[0026] wherein the polar group comprises a carboxylic acid or a carboxylic acid bioisostere, a phosphonic acid or a sulfonic acid group;

[0027] and -X- is a bond, -CO-, -SO- or -SO2-;

[0028] -Z 2 - is a spacer of the following formula:

[0029]

[0030] in:

[0031] Each Y is independently -NH, -NR, -S or -O, wherein R is an alkyl group, a protecting group or a spacer Z. 2 The other parts of form the bond;

[0032] Each X is independently a bond, Co, SO- or SO2-;

[0033] The premise is that when Y is -S, X is a bond;

[0034] Each V is independently a divalent organic moiety linking Y and X;

[0035] and n is 1 to 10;

[0036] or a pharmaceutically acceptable salt or solvate thereof.

[0037] P 1 Can have the following sequence:

[0038] H-Aib-QGTFTSDYSKYLDSΨAAHDFVEWLLSA

[0039] For example, H-Aib-QGTFTSDYSKYLDS-K([15-carboxy-pentadecanoyl]-isoGlu)-AAHDFVEWLLSA.

[0040] The compounds of the present invention may be:

[0041] HH-Aib-QGTFTSDYSKYLDSΨAAHDFVEWLLSA-NH2

[0042] For example, HH-Aib-QGTFTSDYSKYLDS-K([15-carboxy-pentadecanoyl]-isoGlu)-AAHDFVEWLLSA-NH2.

[0043] In a second aspect, the present invention provides a compound having the formula:

[0044] R 1 -P 1 -P 2 -R 2

[0045] in,

[0046] R 1 H, C 1-4 Alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;

[0047] R 2 OH or NH2;

[0048] P 1 is a peptide having the following sequence:

[0049] His-X2-X3-GTFTSDYSKYL-X15-X16-X17-X18-A-X20-DFI-X24-WLE-X28-A

[0050] in:

[0051] X2 is selected from Aib, Ac3c, Ac4c and Ac5c;

[0052] X3 is selected from Gln and His;

[0053] X15 is selected from Asp and Glu;

[0054] X16 is selected from Glu and Ψ;

[0055] X17 is selected from Arg and Ψ;

[0056] X18 is selected from Ala and Arg;

[0057] X20 is selected from Lys and His;

[0058] X24 is selected from Glu and Ψ;

[0059] X28 is selected from Ser and Ψ;

[0060] And P 2 Absent or a sequence of 1 to 20 amino acid units, said amino acid units being independently selected from the group consisting of Ala, Leu, Ser, Thr, Tyr, Cys, Glu, Lys, Arg, Dbu, Dpr and Orn;

[0061] Wherein the compound contains one and only one Ψ

[0062] and wherein the Ψ is a residue of Lys, Arg, Orn or Cys, wherein the side chain is adjacent to a residue of formula -Z 2 Z 1 Substituent conjugation;

[0063] -Z 1 A polar group at one end of the chain and a Z at the end of the chain away from the polar group 2 The aliphatic chain of the linker -X-,

[0064] wherein the polar group comprises a carboxylic acid or a carboxylic acid bioisostere, a phosphonic acid or a sulfonic acid group;

[0065] and -X- is a bond, -CO-, -SO- or -SO2-;

[0066] -Z 2 - is a spacer of the formula:

[0067]

[0068] in:

[0069] Each Y is independently -NH, -NR, -S or -O, wherein R is an alkyl group, a protecting group or a spacer Z. 2 The other parts of form the bond;

[0070] Each X is independently a bond, CO, SO- or SO2-;

[0071] The premise is that when Y is -S, X is a bond;

[0072] Each V is independently a divalent organic moiety linking Y and X;

[0073] and n is 1 to 10;

[0074] or a pharmaceutically acceptable salt or solvate thereof.

[0075] In some embodiments of the second aspect:

[0076] X2 is selected from Aib and Ac4c;

[0077] X3 is Gln;

[0078] X15 is selected from Asp and Glu;

[0079] XI6 is Ψ;

[0080] X17 is Arg;

[0081] X18 is Ala;

[0082] X20 is selected from Lys and His;

[0083] X24 is Glu;

[0084] X28 is Ser.

[0085] Available combinations of residues include the following:

[0086] X2 is Ac4c and X20 is Lys;

[0087] X2 is Aib and X20 is His.

[0088] Alternatively or additionally, it is contemplated that if X15 is E, then X2 is Aib; or if X2 is Ac4c, then X15 is D.

[0089] Of particular interest is the substituent Z 2 Z 1 Including [17-carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3 and [17-carboxy-heptadecanoyl]-isoGlu-GSGSGG.

[0090] P 1 May have a sequence selected from the following:

[0091] H-Aib-QGTFTSDYSKYLDΨRAAKDFIEWLESA

[0092] H-Aib-QGTFTSDYSKYLDΨRAAKDFIEWLESA

[0093] H-Aib-QGTFTSDYSKYLEΨRAAKDFIEWLESA

[0094] H-Ac4c-QGTFTSDYSKYLDΨRAAKDFIEWLESA AND

[0095] H-Aib-QGTFTSDYSKYLEΨRAAHDFIEWLESA

[0096] For example, selected from:

[0097] H-Aib-QGTFTSDYSKYLD-K([17-Carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3)-RAAKDFIEWLESA

[0098] H-Aib-QGTFTSDYSKYLD-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-RAAKDFIEWLESA

[0099] H-Aib-QGTFTSDYSKYLE-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-RAAKDFIEWLESA

[0100] H-Ac4c-QGTFTSDYSKYLD-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-RAAKDFIEWLESA and

[0101] H-Aib-QGTFTSDYSKYLD-K([17-Carboxy-heptadecanoyl]-Glu-Peg3-Peg3)-

[0102] RAAHDFIEWLESA.

[0103] The compounds of the present invention may be selected from:

[0104] HH-Aib-QGTFTSDYSKYLDΨRAAKDFIEWLESA-NH2

[0105] HH-Aib-QGTFTSDYSKYLDΨRAAKDFIEWLESA-NH2

[0106] HH-Aib-QGTFTSDYSKYLEΨRAAKDFIEWLESA-NH2

[0107] HH-Ac4c-QGTFTSDYSKYLDΨRAAKDFIEWLESA-NH2 and

[0108] HH-Aib-QGTFTSDYSKYLEΨRAAHDFIEWLESA-NH2.

[0109] For example, selected from

[0110] HH-Aib-QGTFTSDYSKYLD-K([17-Carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3)-RAAKDFIEWLESA-NH2

[0111] HH-Aib-QGTFTSDYSKYLD-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-RAAKDFIEWLESA-NH2

[0112] HH-Aib-QGTFTSDYSKYLE-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-RAAKDFIEWLESA-NH2

[0113] HH-Ac4c-QGTFTSDYSKYLD-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-RAAKDFIEWLESA-NH2 and

[0114] HH-Aib-QGTFTSDYSKYLE-K([17-Carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3)-RAAHDFIEWLESA-NH2.

[0115] In some alternative embodiments of the second aspect:

[0116] X2 is selected from Aib and Ac4c;

[0117] X3 is selected from Gln and His;

[0118] X15 is Asp;

[0119] X16 is Glu;

[0120] X17 is selected from Arg and Ψ;

[0121] X18 is selected from Ala and Arg;

[0122] X20 is Lys;

[0123] X24 is selected from Glu and Ψ;

[0124] X28 is selected from Ser and Ψ.

[0125] In some embodiments, when X28 is Ψ, X2 is Ac4c.

[0126] In some embodiments, when X3 is His, X2 is Ac4c and X17 is Ψ.

[0127] In some embodiments, when X17 is Ψ, Z 2 Z 1 It is [17-carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3 or [17-carboxy-heptadecanoyl]-isoGlu.

[0128] In some embodiments, when X24 or X28 is Ψ, Z 2 Z 1 It is [17-carboxy-heptadecanoyl]-isoGlu-GSGSGG.

[0129] P 1 May have a sequence selected from the following:

[0130] H-Aib-QGTFTSDYSKYLDEΨAAKDFIEWLESA

[0131] H-Ac4c-QGTFTSDYSKYLDEΨRAKDFIEWLESA

[0132] H-Ac4c-HGTFTSDYSKYLDEΨRAKDFIEWLESA

[0133] H-Ac4c-QGTFTSDYSKYLDEΨAAKDFIEWLESA

[0134] H-Ac4c-QGTFTSDYSKYLDEΨRAKDFIEWLESA

[0135] H-Aib-QGTFTSDYSKYLDERAAKDFIΨWLESA

[0136] H-Ac4c-QGTFTSDYSKYLDERAAKDFIΨWLESA

[0137] H-Ac4c-QGTFTSDYSKYLDERRAKDFIΨWLESA

[0138] H-Ac4c-QGTFTSDYSKYLDERAAKDFIEWLEΨAAND

[0139] H-Ac4c-QGTFTSDYSKYLDERRAKDFIEWLEΨA.

[0140] For example, selected from:

[0141] H-Aib-QGTFTSDYSKYLDE-K([17-Carboxy-heptadecanoyl]-isoGlu)-AAKDFIEWLESA

[0142] H-Ac4c-QGTFTSDYSKYLDE-K([17-Carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3)-RAKDFIEWLESA

[0143] H-Ac4c-HGTFTSDYSKYLDE-K([17-Carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3)-RAKDFIEWLESA

[0144] H-Ac4c-QGTFTSDYSKYLDE-K([17-Carboxy-heptadecanoyl]-isoGlu)-AAKDFIEWLESA

[0145] H-Ac4c-QGTFTSDYSKYLDE-K([17-Carboxy-heptadecanoyl]-isoGlu)-RAKDFIEWLESA

[0146] H-Aib-QGTFTSDYSKYLDERAAKDFI-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA

[0147] H-Ac4c-QGTFTSDYSKYLDERAAKDFI-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-'WLESA

[0148] H-Ac4c-QGTFTSDYSKYLDERRAKDFI-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA

[0149] H-Ac4c-QGTFTSDYSKYLDERAAKDFIEWLE-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-A and

[0150] HH-Ac4c-QGTFTSDYSKYLDERRAKDFiEWLE-K1([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-A-NH2.

[0151] The compounds of the present invention may be selected from:

[0152] HH-Aib-QGTFTSDYSKYLDEΨAAKDFIEWLESA-NH2

[0153] HH-Ac4c-QGTFTSDYSKYLDEΨRAKDFIEWLESA-NH2

[0154] HH-Ac4c-HGTFTSDYSKYLDEΨRAKDFIEWLESA-NH2

[0155] HH-Ac4c-QGTFTSDYSKYLDEΨAAKDFIEWLESA-NH2

[0156] HH-Ac4c-QGTFTSDYSKYLDEΨRAKDFIEWLESA-NH2

[0157] HH-Aib-QGTFTSDYSKYLDERAAKDFIΨWLESA-NH2

[0158] HH-Ac4c-QGTFTSDYSKYLDERAAKDFIΨWLESA-NH2

[0159] HH-Ac4c-QGTFTSDYSKYLDERRAKDFIΨWLESA-NH2

[0160] HH-Ac4c-QGTFTSDYSKYLDERAAKDFIEWLEΨA-NH2 and

[0161] HH-Ac4c-QGTFTSDYSKYLDERRAKDFIEWLEΨA-NH2.

[0162] For example, selected from:

[0163] HH-Aib-QGTFTSDYSKYLDE-K([17-Carboxy-heptadecanoyl]-isoGlu)-AAKDFIEWLESA-NH2

[0164] HH-Ac4c-QGTFTSDYSKYLDE-K([17-Carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3)-RAKDFIEWLESA-NH2

[0165] HH-Ac4c-HGTFTSDYSKYLDE-K([17-Carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3)-RAKDFIEWLESA-NH2

[0166] HH-Ac4c-QGTFTSDYSKYLDE-K([17-Carboxy-heptadecanoyl]-isoGlu)-AAKDFIEWLESA-NH2

[0167] HH-Ac4c-QGTFTSDYSKYLDE-K([17-Carboxy-heptadecanoyl]-isoGlu)-RAKDFIEWLESA-NH2

[0168] HH-Aib-QGTFTSDYSKYLDERAAKDFI-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA-NH2

[0169] HH-Ac4c-QGTFTSDYSKYLDERAAKDFI-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA-NH2

[0170] HH-Ac4c-QGTFTSDYSKYLDERRAKDFI-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA-NH2

[0171] HH-Ac4c-QGTFTSDYSKYLDERAAKDFIEWLE-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-A-NH2 and

[0172] HH-Ac4c-QGTFTSDYSKYLDERRAKDFIEWLE-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-A-NH2.

[0173] For the avoidance of doubt, in all aspects of the invention, those positions not expressly designated as being allowed for variability are fixed and therefore may contain only the designated residues.

[0174] In all aspects, the compounds of the invention comprise a residue Ψ, ie, a residue selected from the group consisting of Lys, Arg, Orn and Cys, wherein the side chain is conjugated to the substituent -Z 2 -Z 1 - conjugation, as described in more detail below.

[0175] The substituent is conjugated to a functional group at the end of the side chain distal to the α-carbon. The general ability of the Lys, Arg, Orn, or Cys side chain to participate in interactions (e.g., intramolecular and intermolecular interactions) mediated by the functional group may therefore be reduced or completely eliminated by the presence of the substituent. Thus, the overall properties of the compound may be relatively insensitive to changes in the actual amino acid present as the residue Ψ. Thus, it is believed that any of the residues Lys, Arg, Orn, and Cys may be present at any position that allows Ψ. However, in certain embodiments, it may be advantageous that the amino acid component of Ψ is Lys.

[0176] In some embodiments, -Z 1 An acyl group of the formula:

[0177] AB-Alk-(CO)-

[0178] Or a sulfonyl group of the formula:

[0179] AB-Alk-(SO2)-;

[0180] A is -COOH or a carboxylic acid bioisostere;

[0181] B is a bond, C6 arylene or C6 arylene-O-;

[0182] Alk is a saturated or unsaturated fatty chain having a length of 6 to 18 carbon atoms, optionally selected from fluorine, C 1-4 Alkyl, trifluoromethyl, hydroxymethyl, amino, hydroxyl, C 1-4 substituted with one or more substituents selected from alkoxy, oxo and carboxyl;

[0183] -Z 2 -for-S A -、-S A -S B -or-S B -S A -;

[0184] -S A - is a single amino acid residue selected from the group consisting of γ-Glu, α-Glu, α-Asp, β-Asp, Ala, β-Ala (3-aminopropionic acid) and Gaba (4-aminobutyric acid);

[0185] -S B - is a linker of the following general formula:

[0186]

[0187] Where n is 1 to 10 and each P u Independently selected from P u i and P u iii ;

[0188] Each P u i are independently natural or unnatural amino acid residues; and

[0189] Each P u iii are independently residues of the following formula:

[0190]

[0191] wherein m is 0 to 5 and p is 1, 3, 4 or 5.

[0192] In any aspect of the present invention, R 1 Can be selected from H and C 1-4 Alkyl (eg, methyl).

[0193] The compounds of the present invention are glucagon analog peptides. References to glucagon analog peptides herein should be interpreted as references to the compounds of the present invention or to the peptide P as the context requires. 1 or P 1 -p 2 Unless the context indicates otherwise or is excluded, reference to a compound of the invention should be taken to include any pharmaceutically acceptable salt (eg acetate or chloride salt) or solvate thereof.

[0194] The present invention provides compositions comprising a compound as defined herein (including a pharmaceutically acceptable salt or solvate thereof, as described) mixed with a carrier. In some preferred embodiments, the composition is a pharmaceutical composition and the carrier is a pharmaceutically acceptable carrier. The glucagon analog peptide can be in the form of a pharmaceutically acceptable salt of a glucagon analog.

[0195] The compounds described herein are particularly useful for preventing weight gain or promoting weight loss. "Prevention" means inhibiting or reducing weight gain when compared to no treatment, but does not necessarily mean that weight gain stops completely. The peptides can cause a decrease in food intake and / or an increase in energy expenditure, resulting in the observed effects on body weight. Independent of their effects on body weight, the compounds of the invention can have a beneficial effect on blood sugar control and / or on circulating cholesterol levels, being able to reduce circulating LDL levels and increase the HDL / LDL ratio. Therefore, the compounds of the invention can be used to directly or indirectly treat any condition caused by or characterized by excess body weight, such as treating and / or preventing obesity, morbid obesity, obesity-related inflammation, obesity-related gallbladder disease, and obesity-induced sleep apnea. They can also be used to prevent conditions caused by or characterized by inappropriate glucose control or dyslipidemia (e.g., elevated LDL levels or decreased HDL / LDL ratios), diabetes (especially type 2 diabetes), metabolic syndrome, hypertension, atherogenic dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral arterial disease, stroke, or microvascular disease. Their effects on these conditions may be caused by or related to their effects on body weight, or may be independent of them.

[0196] The present invention also provides compounds of the invention for use in a method of medical treatment, in particular for use in a method of treating the above-mentioned disorders.

[0197] The present invention also provides the use of the compound of the present invention in preparing medicines for treating the above-mentioned diseases.

[0198] The compounds of the invention may be administered as part of a combination therapy with an agent used to treat diabetes, obesity, dyslipidemia, or hypertension.

[0199] In such cases, the two active agents may be administered together or separately, and as part of the same pharmaceutical formulation or as different formulations.

[0200] Thus, the compounds of the invention may be used in combination with antidiabetic agents including, but not limited to, biguanides (e.g., metformin), sulfonylureas, meglitinides or glinides (e.g., nateglinide), DPP-IV inhibitors, SGLT2 inhibitors, glitazones, insulin or insulin analogs. Examples of insulin analogs include, but are not limited to, Lantus TM 、Novorapid TM , Humalog TM 、Novomix TM 、Actraphane HM TM , Levemir TM and Apidra TM .

[0201] The compounds may be further used in combination with anti-obesity agents including, but not limited to, glucagon-like peptide receptor 1 agonists, peptide YY or its analogs, cannabinoid receptor 1 antagonists, esterase inhibitors, melanocortin receptor 4 agonists or melanin concentrating hormone receptor 1 antagonists, phentermine (alone or in combination with topiramate), combinations of norepinephrine / dopamine reuptake inhibitors and opioid receptor antagonists (e.g., a combination of bupropion and naltrexone), or serotonergic agents (e.g., lorcaserin).

[0202] The compounds may also be used in combination with antihypertensive agents including, but not limited to, angiotensin converting enzyme inhibitors, angiotensin II receptor blockers, diuretics, beta-blockers, or calcium channel blockers.

[0203] The compounds may be used in combination with anti-dyslipidemic agents including, but not limited to, statins, fibrates, niacins, or cholesterol absorption inhibitors.

[0204] Therefore, the present invention also provides a composition or therapeutic kit comprising a compound of the present invention and an antidiabetic agent, an antiobesity agent, an antihypertensive agent or an anti-dyslipidemic agent as described above. Such a composition or therapeutic kit is also provided for use in a medical treatment method, especially for the treatment of the above-mentioned conditions.

[0205] The compounds of the present invention may also be prepared by synthetic chemistry. Accordingly, the present invention provides methods for synthesizing the compounds of the present invention.

[0206] The present invention may also be carried out by a combination of recombinant and synthetic methods. The method may include expressing the precursor peptide sequence, optionally purifying the compound so produced, and adding or modifying one or more amino acids to produce a compound of the present invention or a compound comprising the amino acid sequence P 1 or P 1 -P 2 The modification step may comprise the introduction of an Orn residue (eg, by modification of a precursor residue) and / or the introduction of a substituent Z2Z1 at the position of the residue Ψ.

[0207] The precursor peptide can be expressed from a nucleic acid encoding the precursor peptide in a cell or a cell-free system comprising such nucleic acid. DETAILED DESCRIPTION

[0208] Throughout this specification, the conventional one-letter and three-letter codes for naturally occurring amino acids are used, as well as the generally accepted abbreviations for other amino acids, such as Aib (α-aminoisobutyric acid), Orn (ornithine), Dbu (2,4-diaminobutyric acid), Dpr (2,3-diaminopropionic acid), Ac3c (1-amino-cyclopropanecarboxylic acid), Ac4c (1-amino-cyclobutanecarboxylic acid) and Ac5c (1-amino-cyclopentanecarboxylic acid).

[0209] Ac3c, Ac4c and Ac5c have similar structures and are interchangeable to some extent, although Ac4c may be preferred.

[0210] Glucagon is a 29 amino acid peptide that corresponds to amino acids 53 to 81 of preproglucagon and has the following sequence: His-Ser-Gln-G; y-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. Oxyntomodulin (OXM) is a 37 amino acid peptide that contains the entire 29 amino acid sequence of glucagon and an octapeptide carboxyl-terminal extension (amino acids 82 to 89 of preproglucagon, having the sequence Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala, and is referred to as "intervening peptide 1" or IP-1; thus the full-length sequence of human oxyntomodulin is 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-A; a). The major biologically active fragment of GLP-1 is produced as a 30 amino acid C-terminally amidated peptide corresponding to amino acids 98 to 127 of preproglucagon.

[0211] Thus, the term "native glucagon" refers to native human glucagon having the following sequence: H-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-OH.

[0212] It can be considered that the sequence P of the compound of the present invention 1 The amino acids within are numbered consecutively from 1 to 29 in the conventional N-terminal to C-terminal direction. Thus, reference to P 1 References to "positions" within should be construed as references to positions within native human glucagon and other molecules.

[0213] The compounds of the present invention may contain a C-terminal peptide sequence P of 1 to 20 amino acids. 2 , for example to stabilize the conformation and / or secondary structure of the glucagon analog peptide, and / or to make the glucagon analog peptide more resistant to enzymatic hydrolysis, for example as described in WO99 / 46283.

[0214] If present, P 2represents a peptide sequence having 1 to 20 amino acid residues, such as 1 to 15, more preferably 1 to 10, in particular 1 to 7 amino acid residues, such as 1, 2, 3, 4, 5, 6 or 7 amino acid residues, for example 6 amino acid residues. 2 Each amino acid residue in can be independently selected from: Ala, Leu, Ser, Thr, Tyr, Cys, Glu, Lys, Arg, Dbu (2,4-diaminobutyric acid), Dpr (2,3-diaminopropionic acid) and Orn (ornithine). Preferably, the amino acid residue is selected from: Ser, Thr, Tyr, Glu, Lys, Arg, Dbu, Dpr and Orn, more preferably only selected from Glu, Lys and Cys. The above amino acids may have a D- or L-configuration, and in certain embodiments, they have an L-configuration. A particularly preferred sequence P 2 is a sequence of 4, 5, 6 or 7 consecutive lysine residues (i.e., Lys3, Lys4, Lys5, Lys6 or Lys7), and in particular 5 or 6 consecutive lysine residues. 2 Other exemplary sequences are shown in WO 01 / 04156. Alternatively, sequence P 2 The C-terminal residue of may be a Cys residue. This may facilitate modification of the compound (e.g., PEGylation, or conjugation with albumin). In such embodiments, the sequence P 2 It may, for example, be only 1 amino acid long (ie, P 2 =Cys), or may be 2, 3, 4, 5, 6 or even more amino acids long. 1 and the terminal Cys residue as a spacer.

[0215] Peptide sequence P 2 It has no more than 25% sequence identity with the corresponding sequence of the IP-1 portion of human OXM, which has the sequence Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala.

[0216] The "percent (%) amino acid sequence identity" of a given peptide or polypeptide sequence relative to another polypeptide sequence (e.g., IP-1) is calculated as the percentage of amino acid residues in a given peptide sequence that are identical to the corresponding amino acid residues in the corresponding sequence of another polypeptide when the two are aligned with each other (gaps may be introduced as appropriate for optimal alignment). Percent identity values ​​can be determined using WU-BLAST-2 (Altschul et al., Methods in Enzymology, 266: 460-480 (1996)). WU-BLAST-2 uses several search parameters, most of which are set to default values. Adjustable parameters are set to the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11. The value of amino acid sequence identity % is determined by dividing the number of identical residues in the match determined by WU-BLAST-2 by the total number of residues in the reference sequence (ignoring gaps introduced into the reference sequence by WU-BLAST-2 to maximize the alignment score) and multiplying by 100.

[0217] Therefore, when P 2 When optimally aligned with the 8 amino acids of IP-1, it has no more than 2 amino acids that are identical to the corresponding amino acids of IP-1.

[0218] In certain embodiments, P 2 Does not exist.

[0219] Ψ is a Lys, Arg, Orn or Cys residue, the side chain of which is in contact with the substituent Z 2 -Z 1 Without wishing to be bound by any particular theory, it is believed that the substituent binds to plasma proteins (e.g., albumin) in the bloodstream, thereby protecting the compound of the invention from enzymatic degradation, thereby increasing the half-life of the compound. It may also modulate the potency of the compound, for example, at the glucagon receptor and / or the GLP-1 receptor.

[0220] Group Z 1

[0221] Z 1 is a fatty chain having 2 The linker (herein referred to as -X-) and the 2 The polar group at the end of the chain of the linker. -X- can be, for example, a bond, an acyl group (-CO-), a sulfinyl group (-SO-) or a sulfonyl group (-SO2-), and the linker is located at the ω position relative to the polar group, that is, at the end of the chain away from the polar group.

[0222] Preferably, the polar group is an acidic or weakly acidic group, such as a carboxylic acid or a carboxylic acid bioisostere, a phosphonate or a sulfonate. The pK of the polar group in water is a It may be between -2 and 12, more preferably between 1 and 7, and more preferably between 3 and 6. The pK of some preferred polar groups is a Between 4 and 5.

[0223] The polar group preferably comprises a carboxylic acid or a carboxylic acid bioisostere. Suitable carboxylic acid bioisosteres are known in the art. Preferably, the bioisostere has a proton with a pKa similar to that of the corresponding carboxylic acid. Examples of suitable bioisosteres may include, but are not limited to, tetrazole, acylsulfonyl, acylhydroxylamine, and squaric acid derivatives, as shown below (--- indicates a point of attachment):

[0224]

[0225] R is, for example, Me, CF3

[0226] The polar group can be a group of formula AB-, wherein A is a carboxylic acid (-COOH) or a carboxylic acid bioisostere, a phosphonic acid (-P(O)(OH)2) or a sulfonic acid (-SO2OH) group, and B is a bond or linker between A and a fatty chain. In some embodiments, the polar group is -COOH, that is, A is -COOH and B is a bond.

[0227] When B is a linker, it can be a cycloalkylene group, a heterocycloalkylene group, a C6 arylene group or a C 5-6 Heteroarylene or C6 arylene-O- or C 5-6 Heteroarylene-O-.

[0228] When B is phenylene, it can be selected, for example, from: 1,2-phenylene, 1,3-phenylene, 1,4-phenylene, preferably 1,4-phenylene (so that AB- is a 4-benzoic acid substituent or a 4-benzoic acid bioisostere). When B is phenylene-O-, it can be selected, for example, from: 1,2-phenylene-O-, 1,3-phenylene-O-, 1,4-phenylene-O-, preferably 1,4-phenylene-O-. Each phenylene of B can be optionally selected from fluorine, methyl, trifluoromethyl, amino, hydroxyl and C 1-4 It is understood that the identity and position of the substituents can be selected to subtly change the pK of the polar group. aSuitable inductively or mesomerically electron withdrawing or donating groups and their positional effects are known in the art. In some embodiments, B may be C 5-6 Heteroarylene, for example, pyridylene or thiofuranylene, and may be optionally substituted as indicated.

[0229] For example, in some embodiments, AB- can be selected from:

[0230]

[0231] Preferably, A is -COOH. In some preferred polar groups, A is carboxylic acid and B is C6 arylene-O-.

[0232] As used herein, a fatty chain refers to a moiety comprising a chain of carbon atoms, the carbon atoms being primarily substituted with hydrogen or hydrogen-like atoms, such as a hydrocarbon chain. Such fatty chains are often referred to as lipophilic, but it is understood that substitutions can alter the lipophilic character of the entire molecule.

[0233] The fatty chain can be aliphatic. It can be fully saturated or can contain one or more double bonds or triple bonds. If present, each double bond can be an E or Z configuration. The fatty chain can also have one or more cycloalkylene or heterocyclylene moieties in its length, and can have one or more arylene or heteroarylene moieties in its length as an alternative or supplement. For example, the fatty chain can incorporate a phenylene or piperazinyl moiety in its length, for example, as shown below (wherein, --- represents the connection point in the chain).

[0234]

[0235] The fatty chain can be derived from a fatty acid, for example, it can be derived from a medium-chain fatty acid (MCFA) having an aliphatic tail of 6 to 12 carbon atoms, a long-chain fatty acid (LCFA) having an aliphatic tail of 13 to 21 carbon atoms, or a very long chain fatty acid (LCFA) having an aliphatic tail of 22 or more carbon atoms. Examples of straight-chain saturated fatty acids from which suitable fatty chains can be derived include tridecanoic acid (tridecanoic acid), myristic acid (tetradecanoic acid), pentadecanoic acid (pentadecanoic acid), palmitic acid (hexadecanoic acid) and heptadecanoic acid (heptadecanoic acid). Examples of straight-chain unsaturated fatty acids from which suitable fatty chains can be derived include myristoleic acid, palmitoleic acid, hexadecenoic acid (sapienic acid) and oleic acid.

[0236] The fatty chain can be connected to Z by an amide bond, a sulfenamide bond, a sulfonamide bond, or by an ester bond, or by an ether bond, a thioether bond, or an amine bond. 2 Therefore, the fatty chain can have a Z at the ω position (i.e., away from the polar group). 2 Preferably, the fatty chain has an acyl (-CO-) group at a position away from the polar group and is bonded to Z via an amide or ester bond. 2 connect.

[0237] In some embodiments, Z 1 A group of the formula:

[0238] AB-Alk-X-

[0239] Wherein, AB- is a polar group as defined above, X is a bond, an acyl group (-CO-), a sulfinyl group (-SO-) or a sulfonyl group (-SO2-), and Alk is an aliphatic chain which may be optionally substituted with one or more substituents. The length of the aliphatic chain is preferably 6 to 18 carbon atoms (e.g., C 6-8 alkylene), more preferably 8 to 18 carbon atoms in length (e.g., C 8-18 alkylene), more preferably, 12 to 16 carbon atoms in length (e.g., C 12-16 Preferably, Alk is saturated, i.e., preferably Alk is an alkylene group.

[0240] In some embodiments, Z 1 An acyl group of the formula:

[0241] AB-Alk-(CO)-

[0242] or a sulfonyl group of the formula:

[0243] AB-Alk-(SO2)-.

[0244] The optional substituents on the aliphatic chain may be independently selected from fluorine, C 1-4 Alkyl (preferably methyl), trifluoromethyl, hydroxymethyl, amino, hydroxy, C 1-4 The optional substituents are alkoxy (preferably methoxy), oxo and carboxyl, and may be independently located at any point along the chain. In some embodiments, the optional substituents are each selected from fluoro, methyl and hydroxy. When more than one substituent is present, the substituents may be the same or different. Preferably, the number of substituents is 0 to 3; more preferably, the aliphatic chain is unsubstituted.

[0245] Preferably, Z 1 An acyl group of the formula:

[0246] AB-Alkylene-(CO)-

[0247] wherein A and B are as defined above.

[0248] In some embodiments, Z 1 for:

[0249] 4-CarboxyphenoxynonanoylHOOC-C6H4-O-(CH2)8(CO)-.

[0250] Some preferred Z 1 Derived from the formula HOOC-(CH2) 12-18 -COOH, preferably, a long-chain saturated α, ω-dicarboxylic acid having an even number of carbon atoms D in the aliphatic chain. 1 Can be but not limited to:

[0251] 13-Carboxytridecanoyl HOOC-(CH2) 12 -(CO)-

[0252] 15-Carboxypentadecanoyl HOOC-(CH2) 14 -(CO)-; or

[0253] 17-Carboxyheptadecanoyl HOOC-(CH2) 16 -(CO)-.

[0254] The carboxylic acid group may be substituted with a bioisostere as described in detail herein.

[0255] Group Z 2

[0256] Z 2 Is Z 1 A spacer attached to the side chain of the amino acid component of Ψ. In its most general case, Z 2 is a spacer bound at one end by Y and at the other end by X, wherein Y may be a nitrogen, oxygen or sulfur atom, and wherein X may be a bond or an acyl group (-CO-), sulfinyl group (-SO-) or sulfonyl group (-SO2-). Thus, Z 2 It can be a spacer of the following formula (--- indicates a connection point):

[0257]

[0258] in:

[0259] Y may be -NH, -NR, -S or -O, wherein R may be an alkyl group, a protecting group or may form a bond to another portion of the spacer, with the remaining valences forming bonds to Z. 1 Keys;

[0260] X can be a bond, CO-, SO- or SO2-, with the remaining valences forming a bond to the side chain of the amino acid component of Ψ;

[0261] V is a divalent organic moiety connecting Y and X;

[0262] and n can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Wherein, when n is 2 or greater, Y, V and X are independent of each other Y, V and X.

[0263] Therefore, according to Z 1 and the properties of Y and X on the side chain and the corresponding connecting groups, Z 2 Can be combined on each side by amide, sulfenamide, sulfonamide or ester bond or by amino, ether, thioether bond.Preferably, when Y is -S, X is a bond.When n is 2 or greater, each V can also be combined with adjacent V by said bond.Preferably, the bond is amide, ester or sulfonamide, most preferably amide.Therefore, in some embodiments, each Y is -NH or -NR and each X is CO- or SO2-.

[0264] In some embodiments, Z 2 Formula-S A -、-S B -、-S A -S B -or-S B -S A - spacer, where S A and S B As defined below.

[0265] In some embodiments, Z 2 Selected from: -S A -or-S B -S A -, i.e., [side chain] -Z 2 Z 1 For [side chain]-S A -Z 1 or [Sidechain]-S B -S A -Z 1 .

[0266] Group S A

[0267] S A It may be a single amino acid residue or a residue of an amino acid derivative, in particular an amino acid derivative residue having a sulfinyl or sulfonyl group replacing the carboxyl moiety at the C-terminus. As an alternative or in addition, a single amino acid residue may have an oxygen atom or a sulfur atom replacing the nitrogen atom at the N-terminus. Preferably, S AA single amino acid residue.

[0268] In some embodiments, amino acid can be selected from γ-Glu, α-Glu, α-Asp, β-Asp, Ala, β-Ala (3-aminopropionic acid) and Gaba (4-aminobutyric acid). It should be understood that amino acid can be D-type or L-type or racemic or enantiomer enriched mixture. In some embodiments, amino acid is L-amino acid. In some embodiments, amino acid is D-amino acid.

[0269] In some preferred embodiments, S A Has a carboxylic acid substituent, of which γ-Glu, α-Glu, α-Asp and β-Asp and their sulfinyl and sulfonyl derivatives are preferred. Thus, in some embodiments, the amino acid residue is:

[0270]

[0271] Wherein, -X- is -CO-, -SO-, -SO2-, preferably -CO-, and a is 1 or 2, preferably 2. In some embodiments, the carboxylic acid is an ester, and the amino acid residue is:

[0272]

[0273] wherein -X- is -CO-, -SO-, -SO2-, preferably -CO-, and a is 1 or 2, preferably 2, and R is C 1-4 Preferably, R is C 1-4 The alkyl group is preferably a methyl group or an ethyl group, and more preferably an ethyl group.

[0274] Preferably, S A It is γ-Glu.

[0275] Group S B

[0276] S B The following general joints may be used:

[0277]

[0278] Where P u is a polymer unit, and n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Linker S B One end of Z is -NH, -NR, -S or -O, where R can be an alkyl group, a protecting group or can form a bond with other parts of the polymer unit; and the other end is a bond or CO-, SO- or SO2-. 1 , S A and the nature of Y and X and the corresponding linking groups on Lys, each polymer unit Pu Bonding on each side may be via an amide, sulfenamide, sulfonamide or ester bond or via an amino, ether or thioether bond.

[0279] In some embodiments, each P u Can be independently a unit of the formula:

[0280]

[0281] in:

[0282] Y can be -NH, -NR, -S or -O, wherein R can be an alkyl group, a protecting group or can form a bond with other parts of the spacer, and the remaining valence forms a bond with Z. 1 Keys;

[0283] X can be a bond, CO-, SO- or SO2-, with the remaining valences forming bonds to Lys; and

[0284] and V is a divalent organic moiety connecting Y and X.

[0285] In some embodiments, V is the α-carbon of a natural or unnatural amino acid, ie, V is -CHRA A , where R AA is an amino acid side chain; or V is an optionally substituted C 1-6 Alkylene, or V, is a chain comprising one or more ethylene glycol units arranged in series, also known as a PEG chain, such as -CH2CH2-(OCH2CH2) m -O-(CH2) p -, wherein m is 0, 1, 2, 3, 4 or 5, and p is 1, 2, 3, 4 or 5; when X is CO-, p is preferably 1, 3, 4 or 5. Optional alkylene substituents include fluorine, methyl, hydroxy, hydroxymethyl and amino.

[0286] Preferred P u Units include:

[0287] (i) Single amino acid residue: P u i ;

[0288] (ii) Dipeptide residue: P u ii ;as well as

[0289] (iii) Amino-(PEG) m -Carboxylic acid residue: P u iii ,

[0290] and can exist in any combination or order. B May contain one or more P in any orderu i , P u ii and P u iii , or may contain only one or more P u i , P u ii and P u iii Unit, or one or more selected from P u i and P u ii , P u i or P u iii or P u ii and P u iii unit.

[0291] (i).P u i Single amino acid residue

[0292] Each P u i P may be independently selected from any natural or unnatural amino acid residue, and for example may be selected from: Gly, Pro, Ala, Val, Leu, Ile, Met, Cys, Phe, Tyr, Trp, His, Lys, Arg, Gln, Asn, α-Glu, γ-Glu, Asp, Ser, Thr, Gaba, Aib, β-Ala, 5-aminocarbocyloyl, 6-aminohexanoyl, 7-aminoheptanoyl, 8-aminooctanoyl, 9-aminononanoyl and 10-aminodecanoyl. Preferably, P u i The amino acid residue is selected from the group consisting of Gly, Ser, Ala, Thr and Cys, more preferably selected from the group consisting of Gly and Ser.

[0293] In some embodiments, S B For-(P u i ) n -, wherein n is 1 to 8, more preferably 5 to 7, and most preferably 6. In some preferred embodiments, S B For-(Pu i ) n -, n is 6, each Pu i Independently selected from Gly or Ser, the preferred sequence is -Gly-Ser-Gly-Ser-Gly-Gly-.

[0294] (ii).P u ii Dipeptide residue

[0295] P u ii Can be independently selected from any dipeptide residue comprising two natural or non-natural amino acid residues bound by an amide bond. u ii The dipeptide residues include: Gly-Gly, Gly-Ser, Ser-Gly, Gly-Ala, Ala-Gly and Ala-Ala, more preferably Gly-Ser and Gly-Gly.

[0296] In some embodiments, S B For-(P u ii ) n -, wherein n is 2 to 4, more preferably 3, and each P u ii In some preferred embodiments, S B For-(Pu ii ) n -, n is 3, each Pu ii Independently selected from Gly-Ser and Gly-Gly, the preferred sequence is -(Gly-Ser)-(Gly-Ser)-(Gly-Gly).

[0297] P u i and P u ii The amino acid with a stereocenter can be racemic, enantiomerically enriched or enantiomerically pure. In some embodiments, the or each amino acid is independently an L-amino acid. In some embodiments, the or each amino acid is independently a D-amino acid.

[0298] (iii).P u iii Amino-(PEG) m -Carboxylic acid residue

[0299] Each P u iii may independently be a residue of the following general formula:

[0300]

[0301] wherein m is 0, 1, 2, 3, 4 or 5, preferably 1 or 2, and p is 1, 3, 4 or 5, preferably 1.

[0302] In some embodiments, m is 1 and p is 1, that is, P u iii It is the residue of 8-amino-3,6-dioxaoctanoic acid (also known as {2-[2-aminoethoxy]ethoxy}acetic acid and H2N-PEG3-COOH). This residue is referred to herein as -PEG3-.

[0303] In some embodiments, m is 2 and p is 1, that is, P u iii It is the residue of 11-amino-3,6,9-trioxaundecanoic acid (also known as H2N-PEG4-COOH). This residue is referred to herein as -PEG4-.

[0304] In some embodiments, S B For-(P u iii ) n -, wherein n is 1 to 3, more preferably 2.

[0305] In some preferred embodiments, S B Selected from -PEG3-PEG3- and -PEG4-PEG4-.

[0306] Preferred -Z 2 -Z 1

[0307] It should be understood that the above preferences can be independently combined to produce a preferred -Z 2 -Z 1 combination.

[0308] Some preferred -Z 2 -Z 1 The combinations are as follows (in each case, --- indicates the point of attachment of the side chain of the amino acid component of Ψ):

[0309] (i) [17-Carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3

[0310]

[0311] (ii) [17-Carboxy-heptadecanoyl]-isoGlu

[0312]

[0313] (iii) [13-Carboxy-tridecanoyl]-isoGlu-Peg3-Peg3

[0314]

[0315] (iv) [Carboxyphenoxynonanoyl]-isoGlu-Peg3-Peg3

[0316]

[0317] (v) [13-Carboxy-tridecanoyl]-isoGlu-Peg4-Peg4

[0318]

[0319] (vi) [17-Carboxy-heptadecanoyl]-Peg3-Peg3-isoGlu

[0320]

[0321] (vii) [17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG

[0322]

[0323] (viii) [17-Carboxy-heptadecanoyl]-AA-Peg3-Peg3

[0324]

[0325] Think in Z 1 The presence of polar groups at the ends of the peptides can improve the pharmacokinetic properties of the compound, for example, by increasing the half-life and / or mean residence time and reducing the clearance rate. The linker may also contribute to these pharmacokinetic properties. Linkers comprising more than one amino acid unit (or a portion of similar size) can improve pharmacokinetic properties compared to those consisting of only one amino acid unit, etc. These properties can allow the compound to be administered less frequently than an equivalent compound having the same peptide backbone without modification or with different modifications (e.g., lacking polar groups and / or having a substituent of an aliphatic fatty chain with a shorter linker portion).

[0326] Without wishing to be bound by any particular theory, the inventors have found that, particularly when longer linkers are included, 1 Polar or charged groups at the end may be able to participate in undesirable intramolecular interactions of the free N-terminus of the molecule, which may compromise the beneficial effects of the polar group on pharmacokinetics. It is believed that the peptide backbone of the compounds described herein adopts a relatively well-defined helical secondary structure, and therefore, the ability of the polar group to participate in such interactions may depend on its position within the molecule. When located toward the C-terminus, interaction with the N-terminus may be relatively unlikely. However, the inventors unexpectedly discovered that substituents can be located at residues 16 and 17 of the molecule without compromising the pharmacokinetic benefits obtained.

[0327] The term "conjugate" is used herein to describe the physical linkage of one identifiable chemical moiety to another, as well as the structural relationship between such moieties. It should not be considered to imply any particular method of synthesis.

[0328] The skilled reader is aware of suitable techniques for carrying out coupling reactions using, for example, the general synthetic methods set forth in "Comprehensive Organic Transformations, A Guide to Functional Group Preparations", 2nd edition, Larock, RC; Wiley-VCH: New York, 1999. Such transformations may be carried out at any suitable stage during the synthesis.

[0329] Peptide synthesis

[0330] The compounds of the present invention can be prepared by standard synthetic methods, recombinant expression systems or any other prior art methods. Thus, glucagon analogs can be synthesized by a variety of methods, including, for example, a method comprising the following steps:

[0331] (a) synthesizing peptides stepwise or by fragment assembly using solid phase or liquid phase methods, and isolating and purifying the final peptide product; or

[0332] (b) expressing the precursor peptide sequence from a nucleic acid construct encoding the precursor peptide, recovering the expression product, and modifying the precursor peptide to produce a compound of the present invention.

[0333] The expression is generally carried out from a nucleic acid encoding the precursor peptide, which can be carried out in a cell comprising such nucleic acid or in a cell-free expression system.

[0334] Preferably, the analogs of the invention are synthesized by solid phase or liquid phase peptide synthesis. In this regard, see WO 98 / 11125, and Fields, GB et al., 2002, "Principles and practice of solid-phase peptide synthesis". In: Synthetic Peptides (2nd edition), etc. and the examples herein.

[0335] For recombinant expression, the nucleic acid fragment encoding the precursor peptide will usually be inserted into a suitable vector to form a clone or expression vector. Depending on the application and type, the carrier can be a plasmid, phage, cosmid, minichromosome or viral form, and the naked DNA of transient expression only in some cells is also an important carrier. Preferred clones and expression vectors (plasmid vectors) can replicate autonomously, thereby making it possible to use the high-level expression or high-level replication high copy number for subsequent cloning.

[0336] In general, the expression vector comprises the following features in the 5'→3' direction and operably connected: a promoter driving the expression of the nucleic acid fragment of the present invention, a nucleic acid sequence encoding a leader peptide that can be secreted (secreted into the extracellular phase or, when appropriate, secreted into the periplasm), a nucleic acid fragment encoding a precursor peptide, and a nucleic acid sequence encoding a terminator. It may comprise additional features, such as a selection marker and an origin of replication. When the expression vector in the production strain or cell line is manipulated, it may be preferred that the vector be able to be integrated into the host cell genome. Those skilled in the art are very familiar with suitable vectors and can design vectors according to their specific needs.

[0337] The vector of the present invention is used to transform host cells to produce the precursor peptide. These transformed cells can be cultured cells or cell lines used to amplify nucleic acid fragments and vectors and / or to recombinantly produce the precursor peptide.

[0338] Preferred transformed cells are microorganisms, such as bacteria (e.g., species of Escherichia (e.g., Escherichia coli), Bacillus (e.g., Bacillus subtilis), Salmonella or Mycobacterium (preferably non-pathogenic, such as Mycobacterium bovis (M. bovis) BCG)), yeast (e.g., Saccharomyces cerevisiae and Pichia pastoris) and protozoa. Alternatively, the transformed cell may be from a multicellular organism, i.e., it may be a fungal cell, an insect cell, an algae cell, a plant cell or an animal cell (e.g., a mammalian cell). For the purpose of cloning and / or optimizing expression, preferably the transformed cell is capable of replicating the nucleic acid fragment of the invention. Cells expressing the nucleic acid fragment can be used for small-scale or large-scale preparation of the peptides of the invention.

[0339] When transformed cells are used to produce a precursor peptide, it is convenient but not essential that the expression product is secreted into the culture medium.

[0340] Effectiveness

[0341] The binding of related compounds to the GLP-1 receptor or the glucagon (Glu) receptor can be used as an indication of agonist activity, but it is generally preferred to use a bioassay that measures the intracellular signaling produced by the binding of the compound to the relevant receptor. For example, the activation of the glucagon receptor by a glucagon agonist stimulates the formation of cellular cyclic AMP (cAMP). Similarly, the activation of the GLP-1 receptor by a GLP-1 agonist stimulates the formation of cellular cAMP. Therefore, the production of cAMP in suitable cells expressing one of these two receptors can be used to monitor the activity of the relevant receptors. Therefore, a pair of suitable cell types (each cell type expresses one receptor and does not express another receptor) can be used to measure the agonist activity of these two types of receptors.

[0342] Those skilled in the art will be aware of suitable assay formats, and examples are provided below. The GLP-1 receptor and / or the glucagon receptor may have the receptor sequences described in the examples. For example, the assay may use the human glucagon receptor (glucagon-R) with original accession number GI: 4503947 and / or the human glucagon-like peptide 1 receptor (GLP-1R) with original accession number GI: 166795283 (where the sequence of the precursor protein is concerned, it will be apparent that the mature protein without the signal sequence may be used for the assay).

[0343] EC can be used 50 The EC value is a numerical measure of the potency of an agonist at a given receptor. 50 The EC value is a measure of the concentration of a compound required to achieve half of its maximal activity in a particular assay. Thus, for example, in a particular assay, the EC 50 [GLP-1] EC ratio of glucagon 50 Compounds with lower [GLP-1] can be considered to have higher agonist potency at the GLP-1 receptor compared to glucagon.

[0344] The compounds described herein are generally GluGLP-1 dual agonists as determined by the observation that they stimulate cAMP formation at both the glucagon receptor and the GLP-1 receptor. The activation of each receptor can be measured in separate assays and then compared to each other.

[0345] By targeting the EC of a given compound to the GLP-1 receptor 50 Value (EC 50 [GLP-1-R]) and EC targeting the glucagon receptor 50 Value (EC 50 [glucagon R]) compared to the relative GLP-1R selectivity can be calculated as follows:

[0346] Relative GLP-1R selectivity [compound] = (EC 50 [GLP-1R]) / (EC 50 [glucagon-R]).

[0347] The term “EC 50 ” represents half maximal effective concentration, usually for a specific receptor or level for a specific marker of receptor function, and may also refer to inhibitory or antagonistic activity, depending on the specific biochemical context.

[0348] Without wishing to be bound by any particular theory, the relative selectivity of a compound can allow a direct comparison of its effect on the GLP-1 receptor or the glucagon receptor with its effect on the other receptor. For example, the higher the relative GLP-1 selectivity of a compound, the more potent the compound may be at the GLP-1 receptor compared to the glucagon receptor. A typical result is a comparison of glucagon and GLP-1 receptor from the same species, such as human glucagon and GLP-1 receptor or mouse glucagon and GLP-1 receptor.

[0349] The compounds of the invention may have a higher relative GLP-1R selectivity than human glucagon because, for a particular level of glucagon-R agonist activity, the compounds may exhibit a higher level of GLP-1R agonist activity than glucagon (i.e., be more potent at the GLP-1 receptor). It should be understood that the absolute potency of a particular compound at the glucagon receptor and the GLP-1 receptor may be higher, lower, or about the same as native human glucagon, as long as the appropriate relative GLP-1R selectivity is achieved.

[0350] However, the compounds of the present invention may have a lower EC than human glucagon. 50 [GLP-1R]. The compound may have a lower EC than glucagon 50 [GLP-1R], while maintaining its EC 50 [Glucagon-R] and EC of human glucagon 50 [Glucagon-R] is less than 10 times higher, less than 5 times higher, or less than 2 times higher than that of humans.

[0351] The EC of the compounds of the present invention 50 [Glucagon-R] may be less than the EC of human glucagon 50 The EC of the compound is 2 times that of [glucagon-R]. 50 [Glucagon-R] may be less than the EC of human glucagon 50 [glucagon-R], and its EC 50 [GLP-1R] is smaller, less than half the size of human glucagon, less than one-fifth the size of human glucagon, or less than one-tenth the size of human glucagon.

[0352] The relative GLP-1R selectivity of the compound may be between 0.05 and 20. For example, the relative selectivity of the compound may be between 0.05-0.20, 0.1-0.30, 0.2-0.5, 0.3-0.7, or 0.5-1.0; 1.0-2.0, 1.5-3.0, 2.0-4.0, or 2.5-5.0; or 0.05-20, 0.075-15, 0.1-10, 0.15-5, 0.75-2.5, or 0.9-1.1.

[0353] In certain embodiments, it may be desirable for any given compound to have an EC for both the glucagon-R and the GLP-1R (e.g., for the human glucagon receptor and the GLP-1 receptor). 50 All are less than 1nM.

[0354] Therapeutic Uses

[0355] As will be discussed below, the compounds of the present invention may provide attractive treatment and / or prevention options for obesity and metabolic diseases, including diabetes, among others.

[0356] Diabetes mellitus includes a class of metabolic diseases characterized by hyperglycemia caused by defects in insulin secretion, insulin action, or both. Acute signs of diabetes mellitus include excessive urine production, resulting compensatory thirst and increased fluid intake, blurred vision, unexplained weight loss, lethargy, and changes in energy metabolism. Long-term hyperglycemia in diabetes mellitus is associated with long-term damage, dysfunction, and failure of multiple organs (especially eyes, kidneys, nerves, heart, and blood vessels). Diabetes mellitus is divided into type 1 diabetes, type 2 diabetes, and gestational diabetes according to pathogenic characteristics.

[0357] Type 1 diabetes accounts for 5-10% of all diabetes cases and is caused by autoimmune destruction of the insulin-secreting pancreatic beta cells.

[0358] Type 2 diabetes accounts for 90-95% of diabetes cases and is caused by a complex set of metabolic disturbances. Type 2 diabetes is the result of insufficient endogenous insulin production to keep blood glucose levels below the diagnostic threshold.

[0359] Gestational diabetes refers to any degree of glucose intolerance identified during pregnancy.

[0360] Prediabetes includes impaired fasting glucose and impaired glucose tolerance, which refer to those states that occur when blood glucose levels are elevated but still below the levels for an established clinical diagnosis of diabetes.

[0361] A large proportion of patients with type 2 diabetes and prediabetes are at increased risk of morbidity and mortality due to the high prevalence of additional metabolic risk factors, including abdominal obesity (excess fat tissue around the abdominal organs), atherogenic dyslipidemia (a lipid disorder including high triglycerides, low HDL cholesterol, and / or high LDL cholesterol that promotes plaque accumulation in artery walls), elevated blood pressure (hypertension), a prothrombotic state (e.g., high fibrinogen or plasminogen activator inhibitor-1 in the blood), and a proinflammatory state (e.g., elevated levels of C-reactive protein in the blood).

[0362] Obesity, in turn, leads to an increased risk of prediabetes, type 2 diabetes, and conditions such as certain types of cancer, obstructive sleep apnea, and gallbladder disease.

[0363] Dyslipidemia is associated with an increased risk of cardiovascular disease. High-density lipoprotein (HDL) is of clinical importance because of the negative correlation between plasma HDL concentration and the risk of atherosclerotic disease. Most of the cholesterol stored in atherosclerotic plaques is derived from LDL, so high concentrations of low-density lipoprotein (LDL) are closely associated with atherosclerosis. The HDL / LDL ratio is a clinical risk indicator for atherosclerosis, especially coronary atherosclerosis.

[0364] Metabolic syndrome is characterized by a group of metabolic risk factors in an individual. It includes abdominal obesity (excess fat tissue around abdominal organs), atherogenic dyslipidemia (disordered blood lipids including high triglycerides, low HDL cholesterol and / or high LDL cholesterol, which promotes plaque buildup in artery walls), elevated blood pressure (hypertension), insulin resistance and glucose intolerance, a prothrombotic state (e.g., the presence of high fibrinogen or plasminogen activator inhibitor-1 in the blood), and a proinflammatory state (e.g., elevated C-reactive protein in the blood).

[0365] Individuals with metabolic syndrome are at increased risk for coronary heart disease and other diseases associated with other clinical manifestations of atherosclerosis (e.g., stroke and peripheral vascular disease). In the case of this syndrome, the major underlying risk appears to be abdominal obesity.

[0366] Without wishing to be bound by any particular theory, it is believed that the compounds of the present invention act as dual-effect agonists of both human glucagon receptors and human GLP1 receptors, abbreviated herein as dual-effect GluGLP-1 agonists. The dual-effect agonists can combine the effects of glucagon on, for example, lipid metabolism with the effects of GLP-1 on, for example, blood sugar levels and food intake. Therefore, it can accelerate the removal of excess adipose tissue, induce sustainable weight loss, and improve blood sugar control. Dual-effect GluGLP-1 agonists can also reduce cardiovascular risk factors, such as high cholesterol, high LDL cholesterol, or low HDL / LDL cholesterol ratios.

[0367] Therefore, the compounds of the present invention can be used as medicaments in subjects in need thereof, for preventing weight gain, promoting weight loss, reducing excess weight or treating obesity (e.g., by controlling appetite, eating, food intake, calorie intake and / or energy expenditure), including morbid obesity and related diseases and health conditions, including but not limited to obesity-related inflammation, obesity-related gallbladder disease and obesity-induced sleep apnea. The compounds of the present invention can also be used to treat conditions caused by or associated with impaired blood sugar control in subjects in need thereof, including metabolic syndrome, insulin resistance, glucose intolerance, prediabetes, elevated fasting blood sugar, type 2 diabetes, hypertension, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral arterial disease and stroke. Some of these conditions are associated with obesity. However, the effects of the compounds of the present invention on these conditions may be mediated in whole or in part by the effects on body weight, or may not depend on the effects on body weight.

[0368] The synergistic effects of dual-acting GluGLP-1 agonists may also lead to a reduction in cardiovascular risk factors (eg, high cholesterol and LDL) that may be completely independent of the effects on body weight.

[0369] Accordingly, the present invention provides the use of a compound of the invention for treating the above-mentioned conditions in a subject in need thereof.

[0370] The present invention also provides compounds according to the invention for use in a method of medical treatment, in particular for use in a method of treating the above-mentioned disorders.

[0371] In a preferred aspect, the compounds can be used to treat diabetes, especially type 2 diabetes.

[0372] In one embodiment, the invention encompasses the use of a compound for treating diabetes, particularly type 2 diabetes, in a subject in need thereof.

[0373] In a less preferred aspect, the compounds are useful for preventing weight gain or promoting weight loss.

[0374] In a specific embodiment, the invention encompasses the use of a compound for preventing weight gain or promoting weight loss in an individual in need thereof.

[0375] In a specific embodiment, the invention includes the use of a compound in a method for treating a condition caused by or characterized by excess body weight in an individual in need thereof, such as treating and / or preventing obesity, morbid obesity, preoperative morbid obesity, obesity-related inflammation, obesity-related gallbladder disease, obesity-induced sleep apnea, prediabetes, diabetes (particularly type 2 diabetes), hypertension, atherogenic dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral arterial disease, stroke or microvascular disease.

[0376] In another aspect, the compounds can be used in a method of lowering circulating LDL levels and / or increasing the HDL / LDL ratio.

[0377] In a specific embodiment, the invention includes the use of a compound in a method of lowering circulating LDL levels and / or increasing the HDL / LDL ratio in a subject in need thereof.

[0378] In another aspect, the compounds are useful in methods of lowering circulating triglyceride levels.

[0379] Pharmaceutical composition

[0380] The compounds of the present invention can be formulated into pharmaceutical compositions for storage or administration. Such compositions generally contain a therapeutically effective amount of the compounds of the present invention in a suitable form in a pharmaceutically acceptable carrier.

[0381] The therapeutically effective amount of the compounds of the invention will depend on the route of administration, the type of mammal being treated, and the physical characteristics of the specific mammal being considered. These factors and their relationships that determine the amount are well known to those skilled in the art of medicine. The amount and method of administration can be adjusted to achieve optimal efficacy and can depend on factors well known to those skilled in the art of medicine, such as body weight, diet, concurrent medication, and the like. The optimal dose size and dosing regimen for humans can be guided by the results obtained by the present invention and can be verified in appropriately designed clinical trials. The compounds of the invention are particularly useful for human treatment.

[0382] Effective doses and treatment regimens can be determined by conventional methods, starting with low doses in experimental animals, then monitoring the effects while increasing the dose, and also systematically changing the dosage regimen. When determining the optimal dose for a given subject, clinicians may consider a variety of factors. Such considerations are known to those skilled in the art.

[0383] The term "pharmaceutically acceptable carrier" includes any standard pharmaceutical carrier. Pharmaceutically acceptable carriers for therapeutic use are well known in the pharmaceutical field and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro ed. 1985). For example, sterile saline and phosphate buffered saline at weakly acidic or physiological pH can be used. The pH buffer can be phosphate, citrate, acetate, tris (hydroxymethyl) aminomethane (TRIS), N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS), ammonium bicarbonate, diethanolamine, histidine (which is often a preferred buffer), arginine, lysine or acetate or a mixture thereof. The term also encompasses any reagent listed in the U.S. Pharmacopoeia for animals (including humans).

[0384] The term "pharmaceutically acceptable salt" refers to a salt of any of the compounds of the present invention. Salts include pharmaceutically acceptable salts such as acid addition salts and basic salts. Examples of acid addition salts include hydrochlorides, citrates and acetates. Examples of basic salts include those wherein the cation is selected from alkali metal ions (such as sodium ions and potassium ions), alkaline earth metal ions (such as calcium ions) and ammonium ions. + N(R 3 )3(R 4 ) wherein R 3 and R 4 independently represents an optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Examples of other pharmaceutically acceptable salts are described in "Remington's Pharmaceutical Sciences", 17th edition. Alfonso R. Gennaro (ed.), Mark Publishing Company, Easton, PA, USA, 1985 and later editions, and in Encyclopaedia of Pharmaceutical Technology.

[0385] "Treatment" refers to a method for obtaining a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, reduction of disease severity, stabilization (i.e., non-exacerbation) of the disease state, delay or slowing of disease progression, improvement or alleviation of the disease state, and elimination (partially or completely), whether detectable or undetectable. "Treatment" may also refer to a prolonged survival period compared to the expected survival when not receiving treatment. "Treatment" is an intervention for the purpose of preventing the occurrence of a disease or altering the pathological condition of a disease. Therefore, in certain embodiments, "treatment" refers to both therapeutic treatment and preventive or protective means. Subjects in need of treatment include subjects who are already ill and subjects to be prevented from occurring. "Treatment" refers to the inhibition or alleviation of an increase in a pathological condition or symptom (e.g., weight gain, hyperglycemia) compared to untreated conditions, and does not necessarily indicate the complete termination of the associated condition.

[0386] The pharmaceutical composition can be a unit dosage form. In such a form, the composition is divided into unit doses containing an appropriate amount of active ingredient. The unit dosage form can be a packaged preparation, and the packaging contains an independent amount of preparations, such as boxed tablets, capsules, and powders in vials or ampoules. The unit dosage form itself can also be a capsule, a cachet, or a tablet, or can be in the form of any of these packages in an appropriate number. It can be provided in a single-dose injection form, for example in the form of an injection pen. In certain embodiments, the packaging form includes a label or plug-in with instructions for use. The composition can be formulated to be suitable for any suitable route and mode of administration. Pharmaceutically acceptable carriers or diluents include those used in preparations suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal and transdermal) administration. The preparation can be conveniently provided in a unit dosage form and can be prepared by any method known in the pharmaceutical field.

[0387] Subcutaneous or transdermal administration may be particularly suitable for the compounds described herein.

[0388] The compositions of the present invention can also be mixed or attached to drug carriers, drug delivery systems and advanced drug delivery systems through, for example, covalent, hydrophobic and electrostatic interactions to further improve the stability of the compound, improve bioavailability, improve solubility, reduce adverse effects, achieve chronotherapy known to those skilled in the art, and improve patient compliance, or any combination thereof. Examples of carriers, drug delivery systems and advanced drug delivery systems include, but are not limited to, polymers such as cellulose and derivatives; polysaccharides such as dextran and derivatives, starch and derivatives; polyvinyl alcohol, acrylic and methacrylic polymers, polylactic and polyglycolic acids and block copolymers thereof, polyethylene glycol; carrier proteins such as albumin; gels such as thermogelling systems, such as block copolymer systems well known to those skilled in the art; micelles, liposomes, microspheres, nanoparticles, liquid crystals and dispersions thereof, L2 phases well known to those skilled in the art of phase behavior in lipid-water systems and dispersions thereof, polymer micelles, multiple emulsions, self-emulsifying emulsions, self-microemulsifying emulsions, cyclodextrins and derivatives thereof, and dendrimers.

[0389] Combination therapy

[0390] The compounds or compositions of the invention may be administered as part of a combination therapy with agents for the treatment of obesity, hyperglycemia, dyslipidemia, and diabetes.

[0391] In such cases, the two active agents may be administered simultaneously or separately as part of the same pharmaceutical formulation or as separate formulations.

[0392] Thus, the compounds or compositions of the present invention may also be used in combination with anti-obesity agents, including but not limited to glucagon-like peptide receptor 1 agonists, peptide YY or its analogs, cannabinoid receptor 1 antagonists, lipase inhibitors, melanocortin receptor 4 agonists or melanin concentrating hormone receptor 1 antagonists, phentermine (alone or in combination with topiramate), combinations of norepinephrine / dopamine reuptake inhibitors and opioid receptor antagonists (e.g., a combination of bupropion and naltrexone), or serotonergic agents (e.g., lorcaserin).

[0393] The compounds or compositions of the present invention may also be used in combination with antihypertensive agents including, but not limited to, angiotensin converting enzyme inhibitors, angiotensin II receptor blockers, diuretics, beta blockers, or calcium channel blockers.

[0394] The compounds or compositions of the present invention may also be used in combination with anti-dyslipidemic agents including, but not limited to, statins, fibrates, niacins, or cholesterol absorption inhibitors.

[0395] In addition, the compound or composition of the present invention can also be used in combination with an antidiabetic drug, and the antidiabetic drug includes but is not limited to biguanides (e.g., metformin), sulfonylureas, meglitinides or glinides (e.g., nateglinide), DPP-IV inhibitors, SGLT2 inhibitors, glitazones, different GLP-1 agonists, insulin or insulin analogs. In a preferred embodiment, the compound or its salt is used in combination with insulin or insulin analogs, DPP-IV inhibitors, sulfonylureas or metformin (particularly sulfonylureas or metformin) to achieve sufficient blood sugar control. The example of insulin analogs includes but is not limited to Lantus, Novorapid, Humalog, Novomix and Actraphane HM, Levemir and Apidra.

[0396] Example

[0397] Example 1: General Synthesis of Glucagon Analogs

[0398] Solid phase peptide synthesis (SPPS) was performed in NMP on a polystyrene resin (TentaGel S Ram) using a standard Fmoc strategy on a microwave-assisted synthesizer. HATU was used as a coupling agent and DIPEA as a base. Piperidine (20% in NMP) was used for deprotection. Pseudoprolines were used when appropriate: Fmoc-Phe-Thr(psiMe,Mepro)-OH and Fmoc-Asp-Ser(psiMe,Mepro)-OH (purchased from NovaBiochem).

[0399] The following abbreviations are used:

[0400] Boc: tert-butyloxycarbonyl

[0401] ivDde: 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)3-methyl-butyl

[0402] Dde: 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-ethyl

[0403] DCM: dichloromethane

[0404] DMF: N,N-dimethylformamide

[0405] DIPEA: diisopropylethylamine

[0406] EDT: 1,2-ethanedithiol

[0407] EtOH: ethanol

[0408] Et2O: ether

[0409] HATU: N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanium hexafluorophosphate N-oxide

[0410] MeCN: Acetonitrile

[0411] NMP: N-methylpyrrolidone

[0412] TFA: trifluoroacetic acid

[0413] TIS: Triisopropylsilane

[0414] Cutting:

[0415] The crude peptide was cleaved from the resin by treatment with 95 / 2.5 / 2.5% (v / v) TFA / TIS / water for 2 hours at room temperature (rt). Most of the TFA was removed under reduced pressure, the crude peptide was precipitated and washed with ether and dried to constant weight at ambient temperature.

[0416] The following compounds were synthesized:

[0417] 1.HH-Aib-QGTFTSDYSKYLDS-K([15-carboxy-pentadecanoyl]-isoGlu)-AAHDFVEWLLSA-NH2

[0418] 2. HH-Aib-QGTFTSDYSKYLD-K([17-Carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3)-RAAKDFIEWLESA-NH2

[0419] 3.HH-Aib-QGTFTSDYSKYLDERAAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA-NH2

[0420] 4.HH-Ac4c-QGTFTSDYSKYLDE-K([17-Carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3)-RAKDFIEWLESA-NH2

[0421] 5. HH-Aib-QGTFTSDYSKYLD-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-RAAKDFIEWLESA-NH2

[0422] 6. HH-Aib-QGTFTSDYSKYLE-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-RAAKDFIEWLESA-NH2

[0423] 7. HH-Ac4c-QGTFTSDYSKYLD-K ([17-carboxy-heptadecanoyl]-isoGlu-

[0424] GSGSGG)-RAAKDFIEWLESA-NH2

[0425] 8. HH-Aib-QGTFTSDYSKYLE-K ([17-carboxy-heptadecanoyl]-isoGlu-

[0426] Peg3-Peg3)-RAAHDFIEWLESA-NH2

[0427] 9.HH-Ac4c-HGTFTSDYSKYLDE-K([17-Carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3)-RAKDFIEWLESA-NH2

[0428] 10.HH-Aib-QGTFTSDYSKYLDE-K([17-carboxy-heptadecanoyl]-isoGlu)-AAKDFIEWLESA-NH2

[0429] 11.HH-Ac4c-QGTFTSDYSKYLDE-K([17-carboxy-heptadecanoyl]-isoGlu)-AAKDFIEWLESA-NH2

[0430] 12.HH-Ac4c-QGTFTSDYSKYLDE-K([17-carboxy-heptadecanoyl]-isoGlu)-RAKDFIEWLESA-NH2

[0431] 13.HH-Ac4c-QGTFTSDYSKYLDERAAKDFI-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA-NH2

[0432] 14.HH-Ac4c-QGTFTSDYSKYLDERAAKDFI-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA-NH2

[0433] 15.HH-Ac4c-QGTFTSDYSKYLDERAAKDFIEWLE-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-A-NH2

[0434] 16.HH-Ac4c-QGTFTSDYSKYLDERRAKDFIEWLE-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-A-NH2

[0435] The acylated GLP-1 analog semaglutide was also synthesized and has the following structure: HH-[2-methyl-Ala]-EGTFTSDVSSYLEGQAA-K(17-carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3)EFIAWLVRGRG-OH.

[0436] Example 2: Glucagon Receptor and GLP-1 Receptor Potency Assays

[0437] cDNA encoding human glucagon receptor (Glucagon-R) (original accession number P47871) or human glucagon-like peptide 1 receptor (GLP-1R) (original accession number P43220) was synthesized and cloned into a mammalian expression vector containing a Zeocin resistance marker.

[0438] Mammalian expression vectors encoding glucagon-R or GLP-1-R were transfected into Chinese hamster ovary (CHO) cells using the Attractene method. Stable expression clones were obtained after limiting dilution of cells resistant to selection pressure by Zeocin selection (250 μg / mL). Cell clones expressing glucagon-R and GLP-1-R were picked, proliferated and tested in the following glucagon-R and GLP-1-R potency assays. One clone expressing glucagon-R and one clone expressing GLP-1-R were selected for compound profiling.

[0439] 24 hours prior to the assay, CHO cells expressing human glucagon-R or human GLP-1-R were seeded at 30,000 cells / well in 96-well microtiter plates and cultured in 100 μl growth medium. On the day of analysis, the medium was removed and the cells were washed once with 200 μl assay buffer (Krebs-Ringer buffer, KRBH). The buffer was removed and the cells were incubated in 10 μl KRBH (KRBH + 10 mM HEPES, 5 mM NaHCO3, 0.1% (v / v) BSA) and 0.1 mM IBMX in deionized water containing increasing concentrations of the test peptides at room temperature for 15 minutes. The reaction was terminated by the addition of lysis buffer (0.1% w / v BSA, 5 mM HEPES, 0.3% v / v Tween-20). After the cells were lysed at room temperature for 10 minutes, the lysates were transferred to 384-well plates and the AlphaScreen TM cAMP Functional Assay Kit. After incubation for 1 hour at room temperature in the dark, the beads were analyzed using the AlphaScreen TM cAMP levels were measured using the cAMP Functional Assay Kit according to the manufacturer's instructions. EC was calculated using computer-assisted curve fitting. 50 And the relative potency compared to the reference compounds (glucagon and GLP-1). The GLP-1 / glucagon ratio was calculated as defined above. See Table 1.

[0440] Table 1

[0441]

[0442] Example 3: Agonist activity at endogenous GLP-1 receptor

[0443] The agonist activity of the test compounds on the endogenous GLP-1 receptor was determined using a mouse insulinoma cell line. Intracellular cAMP was used as an indicator of receptor activation.

[0444] The cells were cultured in 384-well plates at a density of 10,000 cells / well for 24 hours. The medium was removed and 10 μL of KRBH buffer (NaCl 130 mM, KCl 3.6 mM, NaH2PO4 0.5 mM, MgSO4 0.5 mM, CaCl2 1.5 mM) containing the test compound or GLP-1 (increasing concentrations from 0.1 pM to 100 nM) or solvent control (0.1% (v / v) DMSO) was added to each well at 26°C for 15 minutes.

[0445] Intracellular cAMP levels were measured using the AlphaScreen cAMP Functional Assay Kit (Perkin Elmer). Measurements were performed using Envision (PerkinElmer) according to the manufacturer's recommendations.

[0446] The results were converted to cAMP concentrations using a cAMP standard curve prepared in KRBH buffer containing 0.1% (v / v) DMSO. The resulting cAMP curve was plotted as absolute cAMP concentration (nM) versus log (test compound concentration) and analyzed using the curve fitting program XLfit.

[0447] The calculated parameters describing both potency and agonist activity of each test compound at the endogenous GLP-1 receptor were:

[0448] pEC50 (negative logarithm of EC50, the concentration that causes a half-maximal increase in cAMP levels, reflecting the potency of the test compound);

[0449] Percent Control (CTL%) (% cAMP increase for each test compound concentration, normalized to the maximal cAMP response induced by GLP-1 (100% CTL)). See Table 2.

[0450] Table 2.

[0451] Compound EC50[nM] 1 0.60nM 2 0.69nM 3 0.15nM 4 0.40nM 5 0.65nM 6 0.54nM 7 0.47nM 8 0.36nM 9 0.84nM 10 0.60nM 11 0.72nM 12 0.81nM 13 0.37nM 14 0.38nM 15 0.25nM 16 0.34nM

[0452] Example 4: Agonist activity at endogenous glucagon receptor

[0453] The agonist activity of the test compound on the endogenous glucagon receptor is determined by measuring its effect on the glycogen synthesis rate in primary rat hepatocytes. After activation of the glucagon receptor, it is expected that the glycogen synthesis rate will be inhibited. The glycogen synthesis rate is determined by counting the amount of radiolabeled glucose incorporated into the cellular glycogen reserves over a defined period of time.

[0454] Primary rat hepatocytes were cultured at a density of 40,000 cells / well in 24-well plates at 37°C and 5% CO2 for 24 hours.

[0455] The medium was discarded and the cells were washed with PBS. Then 180 μL of KRBH-based buffer containing 0.1% BSA and 22.5 mM glucose was added to the wells, followed by the test compound and 40 μCi / ml D-[U14C]glucose (20 μL each). The incubation was continued for 3 hours.

[0456] At the end of the incubation period, the incubation buffer was aspirated and the cells were washed once with ice-cold PBS before being lysed by incubation with 100 μL 1 mol / l NaOH for 30 minutes at room temperature.

[0457] The cell lysate was transferred to a 96-well filter plate and the glycogen was precipitated by incubating the filter plate at 4°C for 120 minutes and then washing the filter plate 4 times with ice-cold ethanol (70%). The resulting precipitate was filtered to dryness and the incorporated 14 C-amount of glucose.

[0458] Wells with vehicle control (0.1% (v / v) DMSO in KRBH buffer) were included as reference for uninhibited glycogen synthesis (100% CTL). 14 Wells containing 5% (C) glucose served as controls for nonspecific background signal (subtracted from all values). Endogenous glucagon peptide was used as a positive control.

[0459] All treatments were performed at least in duplicate.

[0460] The calculated parameters describing both the potency and agonist activity of each test compound at the endogenous glucagon receptor were pEC50 and CTL%.

[0461] CTL % was determined by calculating the percentage of CPM / well in the presence of test compound compared to CPM / well of vehicle control after subtracting background CPM / well:

[0462] [CPM / well (baseline) - CPM / well (sample)] ＊ 100 / [CPM / well(baseline)-CPM / well(baseline)]

[0463] Activators of the glucagon receptor will result in inhibition of the glycogen synthesis rate and give CTL% values ​​between 0% CTL (complete inhibition) and 100% CTL (no observable inhibition).

[0464] The resulting activity curves were plotted as absolute counts (unit: cpm / sample) versus log(test compound concentration) and analyzed using the curve fitting program XLfit.

[0465] pEC50 (negative logarithm of EC50) reflects the potency of the test compound.

[0466] Table 3.

[0467] Compound EC50[nM] 1 0.85nM 2 0.11nM 3 0.94nM 4 179nM 5 0.21nM 6 0.80nM 7 0.34nM 8 0.29nM 9 0.11nM 10 1.53nM 11 0.95nM 12 0.45nM 13 0.43nM 14 0.19nM 15 3.63nM 16 0.19nM

[0468] The term EC used in relation to GLP-1R activation 50and pEC 50 Can be considered equally as an IC for glycogen synthesis 50 and pIC 50 .

[0469] Example 5: Evaluation of pharmacokinetic parameters

[0470] Pharmacokinetic parameters of the test compounds were determined after intravenous administration to Han / Wister rats. For comparative purposes, the acylated GLP-1 analog semaglutide was also tested.

[0471] Male Wistar rats were obtained from Charles River (Germany) and weighed approximately 180 to 210 g when arriving at the test facility. The rats were kept in IV type European standard rat cages with a light cycle of 12 hours of darkness and 12 hours of light. During the study, the rats were housed in III type standard rat cages. Diet Altromin 1324 (Altromin, Germany) and water were used arbitrarily during the entire experiment. Animals were housed in the test facility for at least 4 days to ensure appropriate environmental adaptation.

[0472] The compounds were first dissolved in 0.1% ammonia water to a nominal concentration of 2 mg / mL and then diluted to the desired dosing strength (10 μM) in sterile PBS (pH 7.4) containing 25 mM phosphate buffer. Intravenous injections corresponding to 20 nmol / kg were given via the lateral tail vein.

[0473] Blood samples (200 μl) were collected from the periorbital plexus at 0.08, 0.25, 0.5, 1, 2, 4, 8, 24, 32 and 48 hours after administration into K3EDTA tubes and centrifuged at 4°C for 5 minutes within 20 minutes of sampling. Plasma samples (>100 μL) were transferred to 96-well PCR plates, immediately frozen and kept at -20°C until the plasma concentration of each GLP-1-glucagon compound was analyzed by LC-MS / MS. TM Individual plasma concentration-time profiles were analyzed using a non-compartmental approach using the ELISA software version 3.2 (UNILOG IT Services) and the resulting pharmacokinetic parameters were determined. See Table 4.

[0474] Table 4.

[0475]

[0476] The contents of the claims originally disclosed in the parent case of this application are incorporated herein as part of this application:

[0477] 1. A compound having the formula:

[0478] R 1 -P 1 -P 2 -R 2

[0479] in,

[0480] R 1 H, C 1-4 Alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;

[0481] R 2 OH or NH2;

[0482] P 1 is a peptide having the following sequence:

[0483] H-X2-X3-GTFTSDYSKYLDSΨAAHDFVEWLLSA

[0484] in:

[0485] X2 is selected from the group consisting of Aib, Ala, D-Ala, Ser, N-Me-Ser, Ac3c, Ac4c and Ac5c;

[0486] X3 is selected from Gln and His;

[0487] P 2 Absent or a sequence of 1 to 20 amino acid units, said amino acid units being independently selected from the group consisting of Ala, Leu, Ser, Thr, Tyr, Cys, Glu, Lys, Arg, Dbu, Dpr and Orn;

[0488] or a pharmaceutically acceptable salt or solvate thereof;

[0489] Ψ is a residue of Lys, Arg, Orn or Cys, wherein the side chain is identical to a residue of the formula -Z 2 -Z 1 Substituent conjugation;

[0490] -Z 1 A polar group at one end of the chain and a Z at the end of the chain away from the polar group 2 The aliphatic chain of the linker -X-,

[0491] wherein the polar group comprises a carboxylic acid or a carboxylic acid bioisostere, a phosphonic acid or a sulfonic acid group;

[0492] and -X- is a bond, -CO-, -SO- or -SO2-;

[0493] -Z 2- is a spacer of the formula:

[0494]

[0495] in:

[0496] Each Y is independently -NH, -NR, -S or -O, wherein R is an alkyl group, a protecting group or a spacer Z. 2 The other parts of form the bond;

[0497] Each X is independently a bond, CO-, SO- or SO2-;

[0498] The premise is that when Y is -S, X is a bond;

[0499] Each V is independently a divalent organic moiety linking Y and X;

[0500] and n is 1 to 10;

[0501] or a pharmaceutically acceptable salt or solvate thereof.

[0502] 2. The compound according to item 1, wherein P 1 With the following sequence:

[0503] H-Aib-QGTFTSDYSKYLDSΨAAHDFVEWLLSA.

[0504] 3. The compound according to item 2, which is:

[0505] HH-Aib-QGTFTSDYSKYLDSΨAAHDFVEWLLSA-NH2.

[0506] 4. Compounds having the formula:

[0507] R 1 -P 1 -P 2 -R 2

[0508] in,

[0509] R 1 H, C 1-4 Alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;

[0510] R 2 OH or NH2;

[0511] P 1 is a peptide having the following sequence:

[0512] His-X2-X3-GTFTSDYSKYL-X15-X16-X17-X18-A-X20-DFI-X24-WLE-X28-A

[0513] in:

[0514] X2 is selected from Aib, Ac3c, Ac4c and Ac5c;

[0515] X3 is selected from Gln and His;

[0516] X15 is selected from Asp and Glu;

[0517] X16 is selected from Glu and Ψ;

[0518] X17 is selected from Arg and Ψ;

[0519] X18 is selected from Ala and Arg;

[0520] X20 is selected from Lys and His;

[0521] X24 is selected from Glu and Ψ;

[0522] X28 is selected from Ser and Ψ;

[0523] And P 2 Absent or a sequence of 1 to 20 amino acid units, said amino acid units being independently selected from the group consisting of Ala, Leu, Ser, Thr, Tyr, Cys, Glu, Lys, Arg, Dbu, Dpr and Orn;

[0524] Wherein the compound contains one and only one Ψ

[0525] and wherein the Ψ is a residue of Lys, Arg, Orn or Cys, wherein the side chain is adjacent to a residue of formula -Z 2 Z 1 Substituent conjugation;

[0526] -Z 1 A polar group at one end of the chain and a Z at the end of the chain away from the polar group 2 The aliphatic chain of the linker -X-,

[0527] wherein the polar group comprises a carboxylic acid or a carboxylic acid bioisostere, a phosphonic acid or a sulfonic acid group;

[0528] and -X- is a bond, -CO-, -SO- or -SO2-;

[0529] -Z 2 - is a spacer of the formula:

[0530]

[0531] in:

[0532] Each Y is independently -NH, -NR, -S or -O, wherein R is an alkyl group, a protecting group or a spacer Z. 2 The other parts of form the bond;

[0533] Each X is independently a bond, CO-, SO- or SO2-;

[0534] The premise is that when Y is -S, X is a bond;

[0535] Each V is independently a divalent organic moiety linking Y and X;

[0536] and n is 1 to 10;

[0537] or a pharmaceutically acceptable salt or solvate thereof.

[0538] 5. The compound according to item 4, wherein:

[0539] X2 is selected from Aib and Ac4c;

[0540] X3 is Gln;

[0541] X15 is selected from Asp and Glu;

[0542] XI6 is Ψ;

[0543] X17 is Arg;

[0544] X18 is Ala;

[0545] X20 is selected from Lys and His;

[0546] X24 is Glu;

[0547] X28 is Ser.

[0548] 6. The compound according to item 4 or item 5, wherein:

[0549] X2 is Ac4c and X20 is Lys;

[0550] X2 is Aib and X20 is His.

[0551] 7. A compound according to any one of items 4 to 6, wherein:

[0552] If X15 is E, then X2 is Aib; or

[0553] If X2 is Ac4c, then X15 is D.

[0554] 8. A compound according to any one of items 4 to 7, wherein P 1Having a sequence selected from the group consisting of:

[0555] H-Aib-QGTFTSDYSKYLDΨRAAKDFIEWLESA

[0556] H-Aib-QGTFTSDYSKYLDΨRAAKDFIEWLESA

[0557] H-Aib-QGTFTSDYSKYLEΨRAAKDFIEWLESA

[0558] H-Ac4c-QGTFTSDYSKYLDΨRAAKDFIEWLESA AND

[0559] H-Aib-QGTFTSDYSKYLEΨRAAHDFIEWLESA.

[0560] 9. The compound according to item 8, which is selected from:

[0561] HH-Aib-QGTFTSDYSKYLDΨRAAKDFIEWLESA-NH2

[0562] HH-Aib-QGTFTSDYSKYLDΨRAAKDFIEWLESA-NH2

[0563] HH-Aib-QGTFTSDYSKYLEΨRAAKDFIEWLESA-NH2

[0564] HH-Ac4c-QGTFTSDYSKYLDΨRAAKDFIEWLESA-NH2 and

[0565] HH-Aib-QGTFTSDYSKYLEΨRAAHDFIEWLESA-NH2.

[0566] 10. The compound according to item 4, wherein:

[0567] X2 is selected from Aib and Ac4c;

[0568] X3 is selected from Gln and His;

[0569] X15 is Asp;

[0570] X16 is Glu;

[0571] X17 is selected from Arg and Ψ;

[0572] X18 is selected from Ala and Arg;

[0573] X20 is Lys;

[0574] X24 is selected from Glu and Ψ;

[0575] X28 is selected from Ser and Ψ.

[0576] 11. The compound according to item 10, wherein when X28 is Ψ, X2 is Ac4c.

[0577] 12. The compound according to item 10, wherein, when X3 is His, X2 is Ac4c and X17 is Ψ.

[0578] 13. The compound according to item 10, wherein P 1 Having a sequence selected from the group consisting of:

[0579] H-Aib-QGTFTSDYSKYLDEΨAAKDFIEWLESA

[0580] H-Ac4c-QGTFTSDYSKYLDEΨRAKDFIEWLESA

[0581] H-Ac4c-HGTFTSDYSKYLDEΨRAKDFIEWLESA

[0582] H-Ac4c-QGTFTSDYSKYLDEΨAAKDFIEWLESA

[0583] H-Ac4c-QGTFTSDYSKYLDEΨRAKDFIEWLESA

[0584] H-Aib-QGTFTSDYSKYLDERAAKDFIΨWLESA

[0585] H-Ac4c-QGTFTSDYSKYLDERAAKDFIΨWLESA

[0586] H-Ac4c-QGTFTSDYSKYLDERRAKDFIΨWLESA

[0587] H-Ac4c-QGTFTSDYSKYLDERAAKDFIEWLEΨAAND

[0588] H-Ac4c-QGTFTSDYSKYLDERRAKDFIEWLEΨA.

[0589] 14. A compound according to item 13, which is selected from:

[0590] HH-Aib-QGTFTSDYSKYLDEΨAAKDFIEWLESA-NH2

[0591] HH-Ac4c-QGTFTSDYSKYLDEΨRAKDFIEWLESA-NH2

[0592] HH-Ac4c-HGTFTSDYSKYLDEΨRAKDFIEWLESA-NH2

[0593] HH-Ac4c-QGTFTSDYSKYLDEΨAAKDFIEWLESA-NH2

[0594] HH-Ac4c-QGTFTSDYSKYLDEΨRAKDFIEWLESA-NH2

[0595] HH-Aib-QGTFTSDYSKYLDERAAKDFIΨWLESA-NH2

[0596] HH-Ac4c-QGTFTSDYSKYLDERAAKDFIΨWLESA-NH2

[0597] HH-Ac4c-QGTFTSDYSKYLDERRAKDFIΨWLESA-NH2

[0598] HH-Ac4c-QGTFTSDYSKYLDERAAKDFIEWLEΨA-NH2 and

[0599] HH-Ac4c-QGTFTSDYSKYLDERRAKDFIEWLEΨA-NH2.

[0600] 15. A compound according to any one of items 1 to 14, wherein -Z 1 An acyl group of the formula:

[0601] AB-Alk-(CO)-

[0602] or a sulfonyl group of the formula:

[0603] AB-Alk-(SO2)-;

[0604] A is -COOH or a carboxylic acid bioisostere;

[0605] B is a bond, C6 arylene or C6 arylene-O-;

[0606] Alk is a saturated or unsaturated fatty chain having a length of 6 to 18 carbon atoms, which is optionally selected from fluorine, C 1-4 Alkyl, trifluoromethyl, hydroxymethyl, amino, hydroxyl, C 1-4 Alkoxy, oxo and carboxyl are substituted with one or more substituents;

[0607] -Z2 -for-S A -、-S A -S B -or-S B -S A -;

[0608] -S A - is a single amino acid residue selected from the group consisting of γ-Glu, α-Glu, α-Asp, β-Asp, Ala, β-Ala (3-aminopropionic acid) and Gaba (4-aminobutyric acid);

[0609] -S B - is a linker of the following general formula:

[0610]

[0611] Where n is 1 to 10 and each P u Independently selected from P u i and P u iii ;

[0612] Each P u i are independently natural or non-natural amino acid residues; and

[0613] Each P u iii are independently residues of the following formula:

[0614]

[0615] wherein m is 0 to 5 and p is 1, 3, 4 or 5.

[0616] 16. A compound according to any one of items 1 to 14, wherein Z 1 -Z 2 Selected from:

[0617] (i) [17-Carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3;

[0618] (ii) [17-carboxy-heptadecanoyl]-isoGlu;

[0619] (iii) [13-Carboxy-tridecanoyl]-isoGlu-Peg3-Peg3;

[0620] (iv) [Carboxyphenoxynonanoyl]-isoGlu-Peg3-Peg3;

[0621] (v) [13-Carboxy-tridecanoyl]-isoGlu-Peg4-Peg4;

[0622] (vi) [17-Carboxy-heptadecanoyl]-Peg3-Peg3-isoGlu;

[0623] (vii) [17-carboxy-heptadecanoyl]-isoGlu-GSGSGG; and

[0624] (viii) [17-Carboxy-heptadecanoyl]-AA-Peg3-Peg3.

[0625] 17. A compound according to item 1, wherein P1 has the following sequence:

[0626] H-Aib-QGTFTSDYSKYLDS-K([15-Carboxy-pentadecanoyl]-isoGlu)-AAHDFVEWLLSA.

[0627] 18. The compound according to item 17, which is:

[0628] HH-Aib-QGTFTSDYSKYLDS-K([15-Carboxy-pentadecanoyl]-isoGlu)-AAHDFVEWLLSA-NH2.

[0629] 19. A compound according to any one of items 4 to 7, wherein Z 2 -Z 1 It is [17-carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3 or [17-carboxy-heptadecanoyl]-isoGlu-GSGSGG.

[0630] 20. The compound according to item 10 or item 12, wherein when X17 is Ψ, Z 2 Z 1 It is [17-carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3 or [17-carboxy-heptadecanoyl]-isoGlu.

[0631] 21. The compound according to item 10 or item 11, wherein when X24 or X28 is Ψ, Z 2 Z 1 It is [17-carboxy-heptadecanoyl]-isoGlu-GSGSGG.

[0632] 22. The compound according to item 8, wherein P 1 Having a sequence selected from the group consisting of:

[0633] H-Aib-QGTFTSDYSKYLD-K([17-Carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3)-RAAKDFIEWLESA

[0634] H-Aib-QGTFTSDYSKYLD-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-RAAKDFIEWLESA

[0635] H-Aib-QGTFTSDYSKYLE-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-RAAKDFIEWLESA

[0636] H-Ac4c-QGTFTSDYSKYLD-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-RAAKDFIEWLESA and

[0637] H-Aib-QGTFTSDYSKYLD-K([17-Carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3)-RAAHDFIEWLESA.

[0638] 23. A compound according to item 22, selected from:

[0639] HH-Aib-QGTFTSDYSKYLD-K([17-Carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3)-RAAKDFIEWLESA-NH2

[0640] HH-Aib-QGTFTSDYSKYLD-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-RAAKDFIEWLESA-NH2

[0641] HH-Aib-QGTFTSDYSKYLE-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-RAAKDFIEWLESA-NH2

[0642] HH-Ac4c-QGTFTSDYSKYLD-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-RAAKDFIEWLESA-NH2 and

[0643] HH-Aib-QGTFTSDYSKYLE-K([17-Carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3)-RAAHDFIEWLESA-NH2.

[0644] 24. A compound according to item 13, wherein P 1 Having a sequence selected from the group consisting of:

[0645] H-Aib-QGTFTSDYSKYLDE-K([17-Carboxy-heptadecanoyl]-isoGlu)-AAKDFIEWLESA

[0646] H-Ac4c-QGTFTSDYSKYLDE-K([17-Carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3)-RAKDFIEWLESA

[0647] H-Ac4c-HGTFTSDYSKYLDE-K([17-Carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3)-RAKDFIEWLESA

[0648] H-Ac4c-QGTFTSDYSKYLDE-K([17-Carboxy-heptadecanoyl]-isoGlu)-AAKDFIEWLESA

[0649] H-Ac4c-QGTFTSDYSKYLDE-K([17-Carboxy-heptadecanoyl]-isoGlu)-RAKDFIEWLESA

[0650] H-Aib-QGTFTSDYSKYLDERAAKDFI-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA

[0651] H-Ac4c-QGTFTSDYSKYLDERAAKDFI-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA

[0652] H-Ac4c-QGTFTSDYSKYLDERRAKDFI-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA

[0653] H-Ac4c-QGTFTSDYSKYLDERAAKDFIEWLE-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-A and

[0654] HH-Ac4c-QGTFTSDYSKYLDERRAKDFIEWLE-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-A-NH2.

[0655] 25. A compound according to item 24, selected from:

[0656] HH-Aib-QGTFTSDYSKYLDE-K([17-Carboxy-heptadecanoyl]-isoGlu)-AAKDFIEWLESA-NH2

[0657] HH-Ac4c-QGTFTSDYSKYLDE-K([17-Carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3)-RAKDFIEWLESA-NH2

[0658] HH-Ac4c-HGTFTSDYSKYLDE-K([17-Carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3)-RAKDFIEWLESA-NH2

[0659] HH-Ac4c-QGTFTSDYSKYLDE-K([17-Carboxy-heptadecanoyl]-isoGlu)-AAKDFIEWLESA-NH2

[0660] HH-Ac4c-QGTFTSDYSKYLDE-K([17-Carboxy-heptadecanoyl]-isoGlu)-RAKDFIEWLESA-NH2

[0661] HH-Aib-QGTFTSDYSKYLDERAAKDFI-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA-NH2

[0662] HH-Ac4c-QGTFTSDYSKYLDERAAKDFI-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA-NH2

[0663] HH-Ac4c-QGTFTSDYSKYLDERRAKDFI-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA-NH2

[0664] HH-Ac4c-QGTFTSDYSKYLDERAAKDFIEWLE-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-A-NH2 and

[0665] HH-Ac4c-QGTFTSDYSKYLDERRAKDFIEWLE-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-A-NH2.

[0666] 26. A composition comprising a compound according to any one of the preceding items in admixture with a carrier.

[0667] 27. The composition according to item 26, wherein the composition is a pharmaceutical composition and the carrier is a pharmaceutically acceptable carrier.

[0668] 28. A compound according to any one of items 1 to 25 for use in a method of medical treatment.

[0669] 29. A compound according to any one of items 1 to 25 for use in a method of preventing weight gain or promoting weight loss in an individual in need thereof.

[0670] 30. A compound according to any one of items 1 to 25 for use in a method of lowering circulating LDL levels and / or increasing the HDL / LDL ratio in a subject in need thereof.

[0671] 31. A compound according to any one of items 1 to 25 for use in a method of treating a condition caused by or characterised by excess body weight.

[0672] 32. A compound according to any one of items 1 to 25 for use in a method for preventing or treating obesity, morbid obesity, preoperative morbid obesity, obesity-related inflammation, obesity-related gallbladder disease, obesity-induced sleep apnea, diabetes, metabolic syndrome, hypertension, atherosclerotic dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral arterial disease, stroke or microvascular disease.

[0673] 33. A compound for use according to any one of items 29 to 32, wherein the compound is administered as part of a combination therapy with an agent for the treatment of diabetes, obesity, dyslipidemia or hypertension.

[0674] 34. A compound for use according to claim 33, wherein the agent for treating diabetes is a biguanide (e.g., metformin), a sulfonylurea, a meglitinide or a glinide (e.g., nateglinide), a DPP-IV inhibitor, an SGLT2 inhibitor, a glitazone, a different GLP-1 agonist, insulin or an insulin analog.

[0675] 35. A compound for use according to claim 33, wherein the agent for treating obesity is a glucagon-like peptide receptor 1 agonist, a peptide YY receptor agonist or an analog thereof, a cannabinoid receptor 1 antagonist, a lipase inhibitor, a melanocortin receptor 4 agonist, a melanin-concentrating hormone receptor 1 antagonist, phentermine, a combination of a norepinephrine / dopamine reuptake inhibitor and an opioid receptor antagonist (e.g., a combination of phentermine and topiramate), a combination of bupropion and naltrexone, or a serotonergic agent.

[0676] 36. The compound for use according to item 33, wherein the agent for treating hypertension is an angiotensin converting enzyme inhibitor, angiotensin II receptor blocker, a diuretic, a beta-blocker or a calcium channel blocker.

[0677] 37. A compound for use according to item 33, wherein the agent for treating dyslipidemia is a statin, a fibrate, a nicotinic acid and / or a cholesterol absorption inhibitor.

[0678] 38. A therapeutic kit comprising a compound according to any one of items 1 to 25 or a composition according to item 26 or 27.

[0679] 39. A method for synthesizing a compound according to any one of items 1 to 25.

[0680] 40. A method for producing a compound according to any one of items 1 to 25, the method comprising expressing a precursor peptide sequence from a nucleic acid construct encoding the precursor peptide, recovering the expression product, and modifying the precursor peptide to produce a compound according to any one of items 1 to 25.

[0681] 41. A method according to claim 40, comprising modifying the precursor peptide to introduce a substituent at residue Ψ.

Claims

1. A compound having the formula: R 1 -P 1 -P 2 -R 2 in, R 1 H, C 1-4 Alkyl, acetyl, formyl, benzoyl or trifluoroacetyl; R 2 OH or NH2; P 1 is a peptide having the following sequence: H-X2-X3-GTFTSDYSKYLDSΨAAHDFVEWLLSA in: X2 is selected from the group consisting of Aib, Ala, D-Ala, Ser, N-Me-Ser, Ac3c, Ac4c and Ac5c; X3 is selected from Gln and His; P 2 Absent or a sequence of 1 to 20 amino acid units, said amino acid units being independently selected from the group consisting of Ala, Leu, Ser, Thr, Tyr, Cys, Glu, Lys, Arg, Dbu, Dpr and Orn; or a pharmaceutically acceptable salt or solvate thereof; Ψ is a residue of Lys, Arg, Orn or Cys, wherein the side chain is identical to a residue of the formula -Z 2 -Z 1 Substituent conjugation; -Z 1 A polar group at one end of the chain and a Z at the end of the chain away from the polar group 2 The aliphatic chain of the linker -X-, wherein the polar group comprises a carboxylic acid or a carboxylic acid bioisostere, a phosphonic acid or a sulfonic acid group; and -X- is a bond, -CO-, -SO- or -SO2-; -Z 2 - is a spacer of the formula: in: Each Y is independently -NH, -NR, -S or -O, wherein R is an alkyl group, a protecting group or a spacer Z. 2 The other parts of form the bond; Each X is independently a bond, CO-, SO- or SO2-; The premise is that when Y is -S, X is a bond; Each V is independently a divalent organic moiety linking Y and X; and n is 1 to 10; or a pharmaceutically acceptable salt or solvate thereof.

2. A composition comprising a compound or a pharmaceutically acceptable salt thereof in admixture with a carrier or diluent suitable for subcutaneous administration, wherein the compound has the formula: R 1 -P 1 -P 2 -R 2 in, R 1 H, C 1-4 Alkyl, acetyl, formyl, benzoyl or trifluoroacetyl; R 2 OH or NH2; P 1 is a peptide having the following sequence: His-X2-X3-GTFTSDYSKYL-X15-X16-X17-X1 8-A-X20-DFI-X24-WLE-X28-A in: X2 is selected from Aib, Ac3c, Ac4c and Ac5c; X3 is selected from Gln and His; X15 is selected from Asp and Glu; X16 is selected from Glu and Ψ; X17 is selected from Arg and Ψ; X18 is selected from Ala and Arg; X20 is selected from Lys and His; X24 is selected from Glu and Ψ; X28 is selected from Ser and Ψ; And P 2 does not exist; Wherein the compound contains one and only one Ψ And wherein the Ψ is a residue of Lys, Arg, Orn or Cys, wherein the side chain is conjugated to a substituent having the formula [17-carboxy-heptadecanoyl]-isoGlu-GSGSGG.

3. The composition according to claim 2, wherein: X2 is selected from Aib and Ac4c; X3 is Gln; X15 is selected from Asp and Glu; XI6 is Ψ; X17 is Arg; X18 is Ala; X20 is selected from Lys and His; X24 is Glu; X28 is Ser.

4. The composition according to claim 3, wherein P 1 Having a sequence selected from the group consisting of: H-Aib-QGTFTSDYSKYLDΨRAAKDFIEWLESA H-Aib-QGTFTSDYSKYLDΨRAAKDFIEWLESA H-Aib-QGTFTSDYSKYLEΨRAAKDFIEWLESA H-Ac4c-QGTFTSDYSKYLDΨRAAKDFIEWLESA AND H-Aib-QGTFTSDYSKYLEΨRAAHDFIEWLESA.

5. The composition according to claim 2, wherein: X2 is selected from Aib and Ac4c; X3 is selected from Gln and His; X15 is Asp; X16 is Glu; X17 is selected from Arg and Ψ; X18 is selected from Ala and Arg; X20 is Lys; X24 is selected from Glu and Ψ; X28 is selected from Ser and Ψ.

6. The composition according to claim 5, wherein P 1 Having a sequence selected from the group consisting of: H-Aib-QGTFTSDYSKYLDEΨAAKDFIEWLESA H-Ac4c-QGTFTSDYSKYLDEΨRAKDFIEWLESA H-Ac4c-HGTFTSDYSKYLDEΨRAKDFIEWLESA H-Ac4c-QGTFTSDYSKYLDEΨAAKDFIEWLESA H-Ac4c-QGTFTSDYSKYLDEΨRAKDFIEWLESA H-Aib-QGTFTSDYSKYLDERAAKDFIΨWLESA H-Ac4c-QGTFTSDYSKYLDERAAKDFIΨWLESA H-Ac4c-QGTFTSDYSKYLDERRAKDFIψWLESA H-Ac 4 c-QGTFTSDYSKYLDERAAKDFIEWLEΨAAND H-Ac 4 c-QGTFTSDYSKYLDERRAKDFIEWLEΨA.

7. A composition comprising a compound or a pharmaceutically acceptable salt thereof in admixture with a carrier or diluent suitable for subcutaneous administration, Wherein the compound is selected from: HH-Aib-QGTFTSDYSKYLDERAAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA-NH2; HH-Aib-QGTFTSDYSKYLD-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-RAAKDFIEWLESA-NH2; HH-Aib-QGTFTSDYSKYLE-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-RAAKDFIEWLESA-NH2; HH-Ac4c-QGTFTSDYSKYLD-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-RAAKDFIEWLESA-NH2; HH-Ac4c-QGTFTSDYSKYLDERAAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA-NH2; HH-Ac4c-QGTFTSDYSKYLDERRAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA-NH2; HH-Ac4c-QGTFTSDYSKYLDERAAKDFIEWLE-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-A-NH2; and HH-Ac4c-QGTFTSDYSKYLDERRAKDFIEWLE-K([17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG)-A-NH2.

8. Use of the composition according to any one of claims 2 to 7 in the preparation of a medicament for: (i) preventing weight gain or promoting weight loss in individuals in need thereof; (ii) reducing circulating LDL levels and / or increasing the HDL / LDL ratio in an individual in need thereof; (iii) reducing circulating LDL levels and / or increasing the HDL / LDL ratio in an individual in need thereof; (iv) treatment of conditions caused by or characterized by excess body weight; (v) preventing or treating a condition selected from the group consisting of obesity, obesity-related inflammation, obesity-related gallbladder disease, obesity-induced sleep apnea, diabetes, metabolic syndrome, hypertension, atherosclerotic dyslipidemia, coronary heart disease, peripheral arterial disease, and microvascular disease.

9. The use according to claim 8, wherein the composition is administered as part of a combination therapy with an agent for the treatment of diabetes, obesity, dyslipidemia or hypertension.

10. A therapeutic kit comprising a composition according to any one of claims 2 to 8.

Images