CN116457002A - GLP-1, GIP and glucagon receptor triple agonists - Google Patents

GLP-1, GIP and glucagon receptor triple agonists Download PDF

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CN116457002A
CN116457002A CN202180073945.4A CN202180073945A CN116457002A CN 116457002 A CN116457002 A CN 116457002A CN 202180073945 A CN202180073945 A CN 202180073945A CN 116457002 A CN116457002 A CN 116457002A
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group
amino
aib
carboxy
compound
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P·J·克奈尔
B·P·芬南
何艳涛
R·迪玛奇
F·刘
B·普雷姆德吉
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Novo Nordisk AS
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Priority claimed from PCT/EP2021/080089 external-priority patent/WO2022090447A1/en
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Abstract

The present invention relates to peptides and derivatives thereof which are triple GLP-1/GIP/glucagon receptor agonists, and their medical use in the treatment and/or prevention of obesity, diabetes and/or liver diseases is described.

Description

GLP-1, GIP and glucagon receptor triple agonists
Technical Field
The present invention relates to novel compounds which are agonists of the glucagon-like peptide 1 receptor (GLP-1R), the glucose-dependent insulinotropic polypeptide receptor (GIPR) and the glucagon receptor (GcgR) having a prolonged spectrum of action.
Incorporation by reference of the sequence listing
The present application is filed with a sequence listing in electronic form. The entire contents of this sequence listing are incorporated herein by reference. A sequence table named "200078WO01 sequence listing_ST25" was 37,918 bytes, created at 2021, 10, 21.
Background
Glucagon-like peptide 1 (GLP-1) is an enteroendocrine cell derived hormone and is one of two important endogenous physiological incretins. GLP-1 improves glycemic control by stimulating glucose-dependent insulin secretion in response to nutrients (glucose), inhibits glucagon secretion by pancreatic alpha cells, slows gastric emptying, and induces weight loss primarily by reducing food intake. Glucose-dependent insulinotropic polypeptide (GIP) is another important incretin that improves glycemic control by stimulating insulin secretion in response to nutrients (fat, glucose). Furthermore, GIP appears to improve plasma lipid distribution and stimulate calcium accumulation in bone. GIP analogs induce weight loss and improve glycemic control by superposition/synergy with GLP-1 analogs in dual administration and as single molecule co-agonists (Finan et al, sci Transl Med,2013,5 (209): 2090151; frias et al, cell Metab,2017,26 (2): 343-352; coskun et al, mol Metab,2018,18:3-14; frias et al, lancet,2018,392 (10160):2180-2193), and thus represent suitable candidates for amplifying GLP-1-based pharmacology.
Glucagon (Gcg) enhances hepatic glucose production by promoting glycogenolysis and gluconeogenesis, thereby preventing the occurrence of hypoglycemia. In addition, glucagon has been shown to stimulate lipolysis, increase energy expenditure, and reduce hepatic lipoprotein production. Glucagon has been shown to provide additional weight loss when combined with GLP-1 (Evers et al, J Med Chem 2017,60 (10): 4293-4303; henderson et al Diabetes Obes Metab 2016,18 (12): 1176-1190; day et al, nat Chem Biol 2009,5 (10): 749-757) compared to the effect achieved by GLP-1 alone. This appears to be driven by the combination of the anorexia/satiety effect of GLP-1 with the thermogenic and lipolytic effects of glucagon. As shown by clinical studies of two GLP-1/Gcg receptor co-agonists, insulinotropic and insulin sensitisation of GLP-1 can counteract the potential diabetogenic potential of chronic glucagon action (Tillner et al, diabetes Obes Metab,2019,21 (1): 120-128; parker et al, J Clin Endocrinol Metab,2019,105 (3): 803-820). However, the ratio of GLP-1 activity to glucagon activity must be carefully controlled to maintain glycemic control, thereby limiting the weight loss efficacy that can be achieved.
When combined with both GLP-1 and GIP, the glucagon limited therapeutic window can be alleviated, both GLP-1 and GIP can acutely stimulate glucose-dependent insulin secretion and chronically enhance insulin sensitivity. This dual incretin activity is predicted based on mouse pharmacology to allow more aggressive glucagon pharmacology, pushing additional efficacy with less propensity for diabetes. In addition, the effect of glucagon on energy expenditure is neither counteracted by GLP-1 nor GIP, which in turn provide their own anorexic effects. Thus, triple agonist peptides are expected to provide glucose control with a higher fat mass reduction than can be achieved using GLP-1/GIP or GLP-1/Gcg co-agonism (Finan et al, nat. Med.,2015,21 (1): 27-36).
GLP-1/GIP/Gcg receptor triple agonists and their potential medical uses are described in several patent applications. Long acting triple agonists suitable for once weekly administration in humans are described in WO 2019/125929, WO 2019/125938 and WO 2015/067716. Co-administration of a long acting glucagon analog with a long acting GLP-1/GIP co-agonist is described in WO 2017/009236. Triple agonists intended to be extended by conjugation to an Fc fragment are described in US 2019/0218269 and US 2019/0153060. Exendin-4 derivatives with triple agonistic activity and prolongation suitable for once daily administration in humans are described in WO2017/009236, WO 2015/155141, WO 2014/096145, WO 2014/096148 and WO 2014/096150. However, no triple agonist product has been marketed until now.
Disclosure of Invention
The present invention relates to GLP-1/GIP/Gcg receptor triple agonists comprising a peptide and a substituent, wherein the substituent is covalently attached to the peptide at a position selected from the group consisting of positions 16, 17 and 21, e.g. the substituent is attached through the epsilon amino group of a lysine selected from the group consisting of positions 16, 17 and 21 of the peptide. The substituent extends the half-life of the triple agonist and comprises a di-fatty acid containing extension A-and a linker B-C-. The invention also relates to a pharmaceutical composition comprising such a triple agonist and pharmaceutically acceptable excipients, and the medical use of the triple agonist.
In one aspect, the present invention relates to a GLP-1/GIP/Gcg receptor triple agonist comprising a peptide and a substituent, wherein the amino acid sequence of the peptide is:
X 1 X 2 X 3 GTFTSDYSX 12 X 13 LX 15 KX 17 X 18 AX 20 X 21 FVX 24 WLLX 28 GGPSS GAPPPS (SEQ ID NO: 45), wherein
X 1 Is H or Y
X 2 Is Aib
X 3 Is H or Q
X 12 K, I or Y
X 13 Is Y or L
X 15 Is D or E
X 17 Q, R or K
X 18 A, Y or K
X 20 Aib, Q, E, R or H
X 21 Is E or K
X 24 Is N or Q
X 28 Is A or E;
wherein the peptide has a C-terminal acid or amide; and is also provided with
Wherein the substituent is covalently linked to an epsilon amino group of K at a position selected from the group consisting of positions 16, 17 and 21;
or a pharmaceutically acceptable salt thereof.
Additionally or alternatively, in a second aspect, the invention relates to a pharmaceutical composition comprising a GLP-1/GIP/Gcg receptor triple agonist and optionally at least one pharmaceutically acceptable excipient.
Additionally or alternatively, in another aspect, the invention relates to a GLP-1/GIP/Gcg receptor triple agonist as described herein for use as a medicament.
Additionally or alternatively, in another aspect, the invention relates to a GLP-1/GIP/Gcg receptor triple agonist as described herein for use in the prevention and/or treatment of diabetes, obesity and/or liver diseases.
Drawings
FIG. 1 shows the change in body weight over time in the maximal efficacy study in DIO mice, in which vehicle (- ≡ -); compound No. 21 (- · -); compound No. 30 (- Δ -); compound No. 33 (-, t-). DIO mice received subcutaneous doses once daily according to the titration schedule described in table 4.
Fig. 2 shows cumulative food intake in the maximal efficacy study in DIO mice, in which vehicle (- ≡ -); compound No. 21 (- · -); compound No. 30 (- Δ -); compound No. 33 (-, t-). DIO mice received subcutaneous doses once daily according to the titration schedule described in table 4.
Detailed Description
The present invention relates to compounds that are agonists of each of GLP-1, GIP and glucagon receptors.
Hereinafter, greek letters may be represented by their symbols or corresponding written names, for example: α=alpha; beta = beta; epsilon = epsilon; gamma = gamma; omega = omega; etc. Furthermore, the greek letter μmay also be denoted by "u", e.g. μl=ul or μm=um.
Terms that are presented in the singular form generally include the plural unless otherwise indicated in this specification.
Triple agonists, pharmaceutical compositions, and uses thereof are also described herein, wherein open terms such as "comprising" and "including" may be replaced with closed terms such as "consisting of.
GLP-1/GIP/glucagon receptor triple agonists
The present invention relates to compounds that are agonists of the GLP-1 receptor, the GIP receptor and the glucagon receptor. Each of these agonists may also be referred to as "GLP-1/GIP/Gcg receptor triple agonists" or "triple agonists". Receptor agonists as described herein are compounds that bind to the receptor and elicit a response to the natural ligand (see, e.g., "Principles of Biochemistry", AL Lehninger, DL Nelson, MM Cox, second edition, worth Publishers,1993, page 763).
In some embodiments, the GLP-1/GIP/Gcg triple agonist is a compound that binds to each of the three GLP-1R, GIPR and GcgR receptors and elicits a response at each receptor, i.e., a compound capable of activating each of the GLP-1R, GIPR and GcgR receptors.
The term "compound" is used herein to denote a molecular entity, and thus "compound" may have different structural elements in addition to the smallest element defined for each compound or group of compounds. Thus, the compound may be a peptide or a derivative thereof, as long as the compound comprises defined structural and/or functional elements.
The term "compound" is also intended to encompass pharmaceutically relevant forms thereof, i.e., a compound as defined herein or a pharmaceutically acceptable salt, amide or ester thereof.
The term "peptide" refers to a sequence of two or more amino acids. "peptide" may also include amino acid extensions at the N-terminal and/or C-terminal positions and/or truncations at the N-terminal and/or C-terminal positions. Typically, amino acid residues may be represented by their full name, their single letter code, and/or their three letter code. These three ways are completely equivalent.
Amino acids are molecules containing amino and carboxylic acid groups and optionally containing one or more additional groups often referred to as side chains.
The term "amino acid" includes proteinogenic (or natural) amino acids (20 of which are standard amino acids) as well as non-proteinogenic (or non-natural) amino acids. Protein-type amino acids are amino acids that are naturally incorporated into proteins. Standard amino acids are amino acids encoded by the genetic code. Non-protein amino acids are either not present in the protein or are not produced by standard cellular mechanisms (e.g., they may have undergone post-translational modification). Non-limiting examples of non-protein amino acids are Aib (alpha-aminoisobutyric acid or 2-aminoisobutyric acid), norleucine (Nle), norvaline, and the D-isomer of protein amino acids.
Hereinafter, each amino acid of a peptide for which optical isomer is not specified should be understood to mean the L-isomer (unless otherwise specified).
GLP-1/GIP/Gcg triple agonists described herein comprise or consist of a peptide and a substituent. In some embodiments, the peptide is a synthetic peptide generated to optimize activity at GLP-1, GIP and Gcg receptors. As demonstrated in the examples herein, compounds have been identified that have receptor activity at each of the GLP-1 receptor, the GIP receptor, and the Gcg receptor.
Triple agonists described herein also exhibit extended half-lives obtained by including substituents of fatty acids.
In some embodiments, the carboxyl terminus of the triple agonist has a carboxylic acid group (-COOH), also known as the C-terminal acid. In some embodiments, the carboxy terminus of the triple agonist may optionally comprise an amide group (C (=o) -NH) 2 ) Also known as C-terminal amides, which are naturally occurring in certain proteins with-NH 2 Instead of modification of-OH, such as seen with native exendin-4.
Peptides
GLP-1/GIP/Gcg receptor triple agonists described herein comprise a peptide and a substituent, wherein the substituent is attached to the peptide backbone through an amino acid residue, e.g., through a functional group on an amino acid side chain. In some embodiments, the triple agonists described herein consist of a peptide and a substituent.
In some embodiments, the amino acid sequence of the peptide is:
X 1 X 2 X 3 GTFTSDYSX 12 X 13 LX 15 KX 17 X 18 AX 20 X 21 FVX 24 WLLX 28 GGPSSGAPPPS (SEQ ID NO: 45), wherein
X 1 Is H or Y
X 2 Is Aib
X 3 Is H or Q
X 12 K, I or Y
X 13 Is Y or L
X 15 Is D or E
X 17 Q, R or K
X 18 A, Y or K
X 20 Aib, Q, E, R or H
X 21 Is E or K
X 24 Is N or Q
X 28 Is A or E;
wherein the peptide has a C-terminal acid or amide.
In some embodiments, the amino acid sequence of the peptide is:
HX 2 HGTFTSDYSX 12 X 13 LX 15 KX 17 X 18 AX 20 EFVX 24 WLLX 28 GGPSSGAPPPS (SEQ ID NO: 47), wherein
X 2 Is Aib
X 12 Is I or Y
X 13 Is Y or L
X 15 Is D or E
X 17 Is Q or K
X 18 Is A or Y
X 20 Is Aib
X 24 Is N or Q
X 28 Is A or E.
In some embodiments, the amino acid sequence of the peptide is:
YX 2 QGTFTSDYSX 12 X 13 LX 15 KX 17 X 18 AX 20 EFVX 24 WLLX 28 GGPSSGAPPPS (SEQ ID NO: 50), wherein
X 2 Is Aib
X 12 Is I or Y
X 13 Is Y or L
X 15 Is D or E
X 17 Is Q or K
X 18 Is A or Y
X 20 Is Aib
X 24 Is N or Q
X 28 Is A or E.
In some embodiments, X 1 X 2 X 3 Selected from HAibH and YAibQ. In some embodiments, X 1 X 2 X 3 Is HAibH. In some embodiments, X 1 X 2 X 3 Is YAibQ.
In some embodiments, X 12 Is I or Y. In some embodiments, X 15 Is D. In some embodiments, X 15 E is defined as E. In some embodiments, SX 12 X 13 LX 15 Selected from SYYLE (SEQ ID NO: 54), SILLE (SEQ ID NO: 55), SYLLE (SEQ ID NO: 56), SIYLE (SEQ ID NO: 57) and SIYLD (SEQ ID NO: 58). In some embodiments, SX 12 X 13 LX 15 Selected from SYYLE, SILLE, SIYLE and SIYLD. In some embodiments, SX 12 X 13 LX 15 Selected from SYYLE and SILLE.
In some embodiments, KX 17 X 18 AX 20 Selected from KQAAAib (SEQ ID NO: 59), KKAAAib (SEQ ID NO: 60), KQYAAib (SEQ ID NO: 61) and KKYAAib (SEQ ID NO: 62). In some embodiments, KX 17 X 18 AX 20 KQAAAib.
In some embodiments, X 24 Is N. In some embodiments, X 24 Q.
In some embodiments, X 28 Is A. In some embodiments, X 28 E is defined as E.
In some embodiments, the peptide is selected from any one of SEQ ID NOs 1-33. In some embodiments, the amino acid sequence of the peptide is selected from the group consisting of SEQ ID NOs 1, 11, 13-14, 17-18, 20 and 26. In some embodiments, the amino acid sequence of the peptide is selected from the group consisting of SEQ ID NOs 11, 14 and 26.
In some embodiments, the peptide has a C-terminal acid or amide. In some embodiments, the peptide has a C-terminal acid. In some embodiments, the peptide has a C-terminal amide.
Derivatives and their use as inhibitors of viral infection
In some embodiments, the GLP-1/GIP/Gcg receptor triple agonist comprises a substituent covalently attached to a peptide. Such compounds may also be referred to as derivatives of peptides, as they are obtained by covalently attaching substituents to the peptide backbone (e.g., through functional groups on amino acid side chains). In some embodiments, the GLP-1/GIP/Gcg receptor triple agonist consists of a peptide and a substituent.
One aspect of the present invention relates to a compound comprising a peptide and a substituent, wherein the amino acid sequence of the peptide is:
X 1 X 2 X 3 GTFTSDYSX 12 X 13 LX 15 KX 17 X 18 AX 20 X 21 FVX 24 WLLX 28 GGPSSGAPPPS (SEQ ID NO: 45), wherein
X 1 Is H or Y
X 2 Is Aib
X 3 Is H or Q
X 12 K, I or Y
X 13 Is Y or L
X 15 Is D or E
X 17 Q, R or K
X 18 A, Y or K
X 20 Aib, Q, E, R or H
X 21 Is E or K
X 24 Is N or Q
X 28 Is A or E;
wherein the peptide has a C-terminal acid or amide; and is also provided with
Wherein the substituent is covalently linked to an epsilon amino group of K at a position selected from the group consisting of positions 16, 17 and 21; or a pharmaceutically acceptable salt thereof.
In further embodiments, the peptide may be defined as described above.
Substituent group
In some embodiments, the substituent is covalently linked to the peptide backbone of a triple agonist as described herein, for example through the epsilon-amino group of a lysine (K) residue. In some embodiments, the substituents are covalently linked through an epsilon-amino group of lysine (K) at a position selected from the group consisting of positions 16, 17, and 21 of the backbone. In a preferred embodiment, the substituents are covalently linked through the epsilon-amino group of lysine (K) at position 16.
In one aspect, the substituents are capable of forming a non-covalent complex with a protein such as albumin or serum albumin, thereby promoting circulation of the triple agonist in the blood stream, and also have the effect of extending the duration of action of the triple agonist, since the complex of triple agonist with albumin is only slowly removed by renal clearance.
In one aspect, a substituent is covalently attached to the lysine residue by acylation, i.e., via an amide bond formed between the carboxylic acid group of the substituent and the epsilon-amino group of lysine in the peptide backbone. In some embodiments, the amino group of the lysine is coupled to the aldehyde of the substituent by reductive amination.
In some embodiments, the substituent is A-B-C-, which comprises an extender (A-) and a linker element (B-C-). In some embodiments, the extender is a fatty acid and is the terminal portion of the substituent responsible for extending the half-life of the compound.
In some embodiments, the extension a "is a fatty acid, such as a fatty diacid that may be defined by chemical formula 1:
chemical formula 1: HOOC- (CH) 2 ) p -CO-*
Wherein p is an integer in the range of 8-20. In some embodiments, a-is formula 1 and p is an integer in the range of 14-20, such as 16 or 18. Chemical formula 1 may also be referred to as C (n+2) diacid.
In some embodiments, the substituents further comprise a linker element B-C-, wherein B-is selected from formula 2 or formula 3:
chemical formula 2: * -NH-CH (COOH) - (CH) 2 ) 2 -CO-*
Chemical formula 2 may also be referred to as γGlu, which may also be described as
Chemical formula 2a:and includes both R-and S-forms. In some embodiments, formula 2 is the S-isomer of γglu.
Chemical formula 3: * -NH-CH 2 -(C 6 H 10 )-CO-NH-CH(COOH)-(CH 2 ) 2 Co—, which can also be described as formula 3a or formula 3b:
chemical formula 3a:
chemical formula 3b:
formula 3 may also be referred to as Trx-yglu, where Trx refers to tranexamic acid or trans-4- (aminomethyl) cyclohexanecarboxylic acid, where formula 3 encompasses the (1, 2), (1, 3) and (1, 4) forms, and formulas 3a and 3b designate the (1, 4) form.
In some embodiments, the substituents further comprise a linker element that may be defined by B-C-, wherein C-is selected from formula 4 or formula 5:
chemical formula 4: * - [ NH- ((CH) 2 ) 2 -O) 2 -CH 2 -CO] 2 It may also be referred to as Ado-
Ado, consisting of two 8-amino-3, 6-dioxaoctanoic acid moieties linked by amide linkages, can also be described as:
chemical formula 4a:
chemical formula 5: * - [ NH- (CH) 2 ) 4 -CH(NH 2 )-CO] 2 It may also be referred to as epsilon Lys-
Epsilon Lys, consisting of two epsilon lysine moieties linked by an amide linkage, can also be described as:
chemical formula 5a:and includes both R-and S-forms. In one embodiment, formula 5 is the S-isomer of two epsilon Lys moieties.
The symbols represent the points of attachment and A-, B-and C-are connected to each other in the order indicated by amide bonds.
In some embodiments, the substituents A-B-C-are selected from:
Chemical formula 6:
chemical formula 7:
chemical formula 8:；
chemical formula 9:；
chemical formula 10:
chemical formula 11:
chemical formula 12:
and chemical formula 13:
in some embodiments, the substituents are selected from formula 6, formula 7, formula 12, and formula 13. In certain embodiments, the substituents A-B-C-are selected from formula 12 or formula 13.
Pharmaceutically acceptable salts
In some embodiments, the triple agonists described herein are in the form of a pharmaceutically acceptable salt.
For example, salts are formed by chemical reactions between bases and acids, such as: 2NH 3 +H 2 SO 4 →(NH 4 ) 2 SO 4 。
The salt may be a basic salt, an acid salt, or it may not be both (i.e., a neutral salt). In water, the basic salt produces hydroxide ions and the acid salt produces hydronium ions.
In some embodiments, the salts of the triple agonists of the present invention are formed by adding cations or anions between the anions or cationic groups, respectively. These groups may be located in the peptide moiety, and/or in the side chain of the triple agonist.
Non-limiting examples of anionic groups of the triple agonists described herein include free carboxyl groups in the side chains and free carboxyl groups in the peptide moieties. In some embodiments, the peptide moiety includes a C-terminal free carboxylic acid group. In some embodiments, the peptide moiety includes a free carboxyl group at an internal acidic amino acid residue, such as Asp and Glu. In some embodiments, the triple agonists described herein do not include a free carboxyl group.
Non-limiting examples of cationic groups in the peptide moiety include the free amino group at the N-terminus, and the free amino group of internal basic amino acid residues such as His, arg, and Lys. In some embodiments, the triple agonists described herein do not include an N-terminal free amino group.
In a particular embodiment, the triple agonist is in the form of a pharmaceutically acceptable salt.
Functional properties
In a first aspect, the triple agonist as described herein is an agonist for all receptors of GLP-1R, GIPR and GcgR as tested by in vitro potency as described in example 2 herein. Thus, the triple agonists described herein may activate each of GLP-1R, GIPR and GcgR.
Additionally or alternatively, in a second functional aspect, the triple agonist has an in vivo effect on body weight, food intake and glucose tolerance.
Additionally or alternatively, in a third functional aspect, the triple agonist produces improved pharmacokinetic properties compared to the natural hormones GLP-1, GIP and Gcg.
Bioactivity-in vitro potency
In this section, the term "potency" is used interchangeably with "biological activity" and "activity".
According to a first functional aspect, a triple agonist as described herein is effective on GLP-1R, GIPR and GcgR. The peptides described herein are also effective against GLP-1R, GIPR and GcgR. In some embodiments, a triple agonist as described herein has an effective in vitro effect of activating hGLP-1R, hGIPR and hGcgR.
In some embodiments, potency is determined by in vitro potency, i.e., the expression of a triple agonist described herein (or a peptide described herein) in each of GLP-1R, GIPR and GcgR in a functional receptor assay. In some embodiments, the functional receptor assay determines the in vitro potency of a triple agonist (or peptide described herein) described herein, respectively, on each of hGLP-1R, hGIPR and hGcgR.
The term "half maximal effective concentration" (also referred to as "EC 50 ") refers to the concentration at which a response halfway between the baseline and maximum is induced with reference to the dose response curve. EC (EC) 50 The efficacy of the compound was measured and represented by the concentration at which 50% of its maximum effect was observed.
Thus, the in vitro potency of a triple agonist as described herein can be determined by assaying EC as follows 50 To determine. EC (EC) 50 The lower the value, the better the efficacy. To further characterize such compounds, it may also be desirable to consider the in vitro potency of the natural hormones relative to each receptor, namely hGLP-1 (7-37) (SEQ ID NO: 35), hGIP (SEQ ID NO: 36) and hGcg (SEQ ID NO: 37).
For example, in vitro efficacy can be determined in media containing membranes expressing the appropriate receptor, and/or in assays using whole cells expressing the appropriate receptor.
For example, the functional response of human GLP-1, GIP or Gcg receptor may be measured in a reporter assay, e.g., in a stably transfected BHK cell line expressing human or mouse GLP-1, GIP or Gcg receptor and containing CAMP Response Element (CRE) DNA coupled to a promoter and a firefly luciferase (CRE luciferase) gene. When cAMP is produced due to activation of GLP-1, GIP or Gcg receptors, this in turn results in expression of the luciferase. Luciferase may be determined by the addition of luciferin, which is converted by the enzyme to oxidized luciferin and produces bioluminescence, which is measured as a reporting indicator of in vitro efficacy. One non-limiting example of such an assay is described in example 2 described herein.
In some embodiments, a triple agonist as described herein is capable of activating hGLP-1R, hGIPR and hGcgR. In some embodiments, a triple agonist as described herein is capable of activating hGLP-1 in vitroR, hGIPR and hGcgR, EC in CRE luciferase reporter assays as described in example 2 herein 50 Less than 500pM, such as less than 100pM, such as less than 50pM.
In some embodiments, the triple agonists as described herein have in vitro potency against hGLP-1R, hGIPR and hGcgR, as determined using the method of example 2, corresponding to an EC of equal to or less than 200pM, more preferably less than 75pM, or most preferably less than 50pM 50 。
In one embodiment, in the human GLP-1, GIP and Gcg receptor assays described in example 2, EC 50 From 0.5 to 500pM, such as from 0.5 to 100pM, or such as from 0.5 to 50pM.
Biological Activity-pharmacodynamic Studies in DIO mice
According to a second functional aspect, a triple agonist as described herein is biologically active in vivo, as determined by any technique known in the art in any suitable animal model and in clinical trials.
Diet-induced obesity (DIO) mice are one example of a suitable animal model, and the effects on body weight, food intake, and glucose tolerance can be assessed during sub-chronic dosing of the model. The effect of a triple agonist as described herein on body weight, food intake and glucose tolerance can be determined in such mice, for example, as described in example 4 herein.
In some embodiments, the triple agonists as described herein exhibit the ability to reduce body weight, food intake, and improve glucose tolerance in DIO mice, as described in example 4.
In some embodiments, a triple agonist as described herein reduces body weight in DIO mice.
In some embodiments, a triple agonist as described herein reduces food intake in DIO mice.
In some embodiments, a triple agonist as described herein improves glucose tolerance in DIO mice.
In some embodiments, a triple agonist as described herein reduces body weight by at least 10% after administering 1nmol/kg of the compound once daily for 13 days in DIO mice, or by at least 25% after administering 3nmol/kg of the compound once daily for 13 days. In some embodiments, a triple agonist as described herein reduces food intake by at least 20% compared to vehicle after administering 1nmol/kg of the compound once daily for 13 days in DIO mice, or by at least 50% compared to vehicle after administering 3nmol/kg of the compound once daily for 13 days. In some embodiments, the triple agonist improves glucose tolerance by at least 30% compared to vehicle after 13 days of administration of 1nmol/kg or 3nmol/kg of the compound once daily in DIO mice, as measured in IPGTT (intraperitoneal glucose tolerance test).
Pharmacokinetic profile-in vivo half-life in minipigs
According to a third functional aspect, a triple agonist as described herein has improved pharmacokinetic properties, e.g. an extended terminal half-life, e.g. compared to the natural hormones GLP-1, GIP and Gcg. The extension of the terminal half-life means that the compound in question is cleared more slowly from the body. For the triple agonists described herein, this results in an extended duration of pharmacological action (entils).
The pharmacokinetic properties of a triple agonist as described herein can be suitably determined in vivo in Pharmacokinetic (PK) studies. Such studies were conducted to evaluate how the drug compound is absorbed, distributed and eliminated in vivo over time, and how these processes affect the concentration of the triple agonist in vivo.
In the discovery and preclinical stages of drug development, PK studies can be performed using animal models such as mice, rats, monkeys, dogs, or pigs. Any such model can be used to test the PK properties of the triple agonists described herein.
In such studies, single doses of the drug are typically administered intravenously (i.v.), subcutaneously (s.c.), or orally (p.o.) to animals in the form of related formulations. Blood samples were withdrawn at predetermined time points after administration and analyzed for drug concentration by a related quantitative determination. Based on these measurements, the time-plasma concentration curves of the compounds under study are plotted and the data are subjected to so-called non-compartmental pharmacokinetic analysis.
Terminal half-life is an important parameter, as long half-life indicates that less frequent dosing of the compound is possible. In some embodiments, the terminal half-life is the half-life in a minipig following intravenous administration (t 1/2 ) For example, as described in example 3 herein.
In some embodiments, the triple agonist as described herein has a terminal half-life in a minipig of at least 30 hours, preferably at least 40 hours, even more preferably at least 60 hours, as measured after intravenous administration.
Production process
The triple agonists described herein or fragments thereof may be produced by classical peptide synthesis, e.g., solid phase peptide synthesis using t-Boc or Fmoc chemistry, or other established techniques, see, e.g., greene and Wuts, "Protective Groups in Organic Synthesis", john Wiley&Sons,1999，Florencio Zaragoza"Organic Synthesis on solid Phase", wiley-VCH Verlag GmbH,2000, and "Fmoc Solid Phase Peptide Synthesis", oxford University Press,2000, by W.C. Chan and P.D. white. In some embodiments, described herein are methods for preparing a triple agonist. In some embodiments, the method for preparing a triple agonist described herein comprises a solid phase peptide synthesis step.
In addition or alternatively, a triple agonist as described herein may be produced by recombinant methods, i.e., by culturing a host cell containing a DNA sequence encoding a triple agonist peptide sequence and capable of expressing the peptide in a suitable nutrient medium under conditions that allow expression of the peptide. Non-limiting examples of host cells suitable for expressing the peptide are: coli (Escherichia coli), saccharomyces cerevisiae (Saccharomyces cerevisiae), and mammalian BHK or CHO cell lines.
For example, triple agonists described herein comprising unnatural amino acids can be produced as described in the "general methods of preparation" in the experimental section. Or see, e.g., hodgson et al, "The synthesis of peptides and proteins containing non-natural amino acids", chemical Society Reviews, volume 33, phase 7 (2004), pages 422-430.
For example, a triple agonist described herein comprising a substituent may be produced as described in the "general methods of preparation" in the experimental section. In some embodiments, the substituents are constructed as part of the solid phase peptide synthesis, or are constructed separately and linked via lysine residues after solid phase peptide synthesis.
Specific examples of methods for preparing a variety of triple agonists as described herein are provided below.
Pharmaceutical composition
Pharmaceutical compositions comprising a triple agonist as described herein, or a pharmaceutically acceptable salt thereof, and optionally one or more pharmaceutically acceptable excipients, may be prepared as known in the art.
The term "adjuvant" broadly refers to any component other than the active therapeutic ingredient. The auxiliary materials can be inert substances, inactive substances and/or non-pharmaceutically active substances.
Adjuvants may be used for various purposes, for example as carriers, vehicles, diluents, tablet auxiliaries and/or for improving the administration and/or absorption of the active substances.
The formulation of pharmaceutically active ingredients with various adjuvants is known in the art, see for example remington: the Science and Practice of Pharmacy (e.g. 19 th edition (1995) and any subsequent versions).
In some embodiments, the pharmaceutical composition comprising a triple agonist described herein is a liquid formulation, such as an aqueous formulation.
Liquid formulations suitable for injection, for example, may be prepared by conventional techniques in the pharmaceutical industry, including dissolving and mixing the ingredients as appropriate to obtain the desired end product.
In some embodiments, the triple agonists as described herein are dissolved in a suitable buffer at a suitable pH, thus minimizing or avoiding precipitation. For example, the composition for injection may be sterilized by sterile filtration.
Pharmaceutical indications
Another aspect of the invention relates to a GLP-1/GIP/Gcg receptor triple agonist as described herein for use as a medicament.
In a particular aspect of the invention, a GLP-1/GIP/Gcg receptor triple agonist as described herein may be used in the medical treatment of:
(i) For example, preventing and/or treating eating disorders such as obesity by reducing food intake, increasing energy expenditure, reducing body weight, suppressing appetite, inducing satiety; treating or preventing binge eating disorder, bulimia nervosa, and/or obesity induced by administration of antipsychotics or steroids; reducing gastric motility; delaying gastric emptying; increase physical activity; and/or preventing and/or treating co-morbidities of obesity, such as osteoarthritis and/or urinary incontinence;
(ii) Weight maintenance after successful weight loss (whether drug induced or diet and exercise induced) -i.e., preventing weight gain after successful weight loss.
(iii) Preventing and/or treating all forms of diabetes, such as hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin dependent diabetes mellitus, MODY (maturity onset diabetes of the young), gestational diabetes, and/or for reducing HbA1C;
(iv) Delaying or preventing the progression of diabetes, such as the progression of type 2 diabetes, delaying the progression of Impaired Glucose Tolerance (IGT) to type 2 diabetes requiring insulin, delaying or preventing insulin resistance, and/or delaying the progression of type 2 diabetes without insulin to type 2 diabetes requiring insulin;
(v) Preventing and/or treating liver disorders such as liver steatosis, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), liver inflammation or fatty liver.
In some embodiments, a triple agonist as described herein is used to prevent and/or treat obesity. In some embodiments, a triple agonist as described herein is used to prevent and/or treat type 2 diabetes. In some embodiments, a triple agonist as described herein is used to prevent and/or treat non-alcoholic steatohepatitis (NASH).
In some embodiments, a triple agonist as described herein is associated with a method of preventing and/or treating obesity. In some embodiments, a triple agonist as described herein is associated with a method of preventing and/or treating type 2 diabetes. In some embodiments, the triple agonists as described herein are associated with methods of preventing and/or treating non-alcoholic steatohepatitis (NASH).
In some embodiments, a triple agonist as described herein is associated with a method of weight management. In some embodiments, the triple agonists as described herein are associated with a method for reducing appetite. In some embodiments, a triple agonist as described herein is associated with a method for reducing food intake. In some embodiments, a triple agonist as described herein is associated with a method of preventing or treating overweight in a subject.
Particular embodiments
The invention is further described by the following non-limiting examples:
1. a GLP-1/GIP/Gcg receptor triple agonist comprising a peptide and a substituent, wherein the amino acid sequence of the peptide is:
X 1 X 2 X 3 GTFTSDYSX 12 X 13 LX 15 KX 17 X 18 AX 20 X 21 FVX 24 WLLX 28 GGPSSGAPPPS (SEQ ID NO: 45), wherein
X 1 Is H or Y
X 2 Is Aib
X 3 Is H or Q
X 12 K, I or Y
X 13 Is Y or L
X 15 Is D or E
X 17 Q, R or K
X 18 A, Y or K
X 20 Aib, Q, E, R or H
X 21 Is E or K
X 24 Is N or Q
X 28 Is A or E;
wherein the peptide has a C-terminal acid or amide; and is also provided with
Wherein the substituent is covalently linked to an epsilon amino group of K at a position selected from the group consisting of positions 16, 17 and 21;
or a pharmaceutically acceptable salt thereof.
2. The GLP-1/GIP/Gcg receptor triple agonist according to embodiment 1, wherein the amino acid sequence of the peptide is:
X 1 X 2 X 3 GTFTSDYSX 12 X 13 LX 15 KX 17 X 18 AX 20 X 21 FVX 24 WLLX 28 GGPSSGAPPPS (SEQ ID NO: 46), wherein
X 1 Is H or Y
X 2 Is Aib
X 3 Is H or Q
X 12 Is I or Y
X 13 Is Y or L
X 15 Is D or E
X 17 Q, R or K
X 18 A, Y or K
X 20 Aib, Q, E, R or H
X 21 Is E or K
X 24 Is N or Q
X 28 Is A or E;
wherein the peptide has a C-terminal acid or amide; and is also provided with
Wherein the substituent is covalently linked to an epsilon amino group of K at one of the positions selected from the group consisting of positions 16, 17 and 21;
or a pharmaceutically acceptable salt thereof.
3. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding embodiments, wherein X 1 X 2 X 3 Selected from HAibH and YAibQ.
4. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding embodiments, wherein X 1 X 2 X 3 Is HAibH.
5. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding embodiments, wherein X 1 X 2 X 3 Is YAibQ.
6. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding embodiments, wherein X 12 Is I.
7. The GLP-1/GIP/Gcg receptor triple agonist of any one of embodiments 1-5, wherein X 12 Y.
8. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding embodiments, wherein X 13 Y.
9. The GLP-1/GIP/Gcg receptor triple agonist of any one of embodiments 1-7, wherein X 13 Is L.
10. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding embodiments, wherein X 15 Is D.
11. The GLP-1/GIP/Gcg receptor triple agonist of any one of embodiments 1-9, wherein X 15 E is defined as E.
12. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding embodiments, wherein SX 12 X 13 LX 15 Selected from SYYLE, SILLE, SYLLE, SIYLE and SIYLD.
13. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding embodiments, wherein SX 12 X 13 LX 15 Selected from SYYLE, SILLE, SIYLE and SIYLD.
14. The GLP-1/GIP/Gcg receptor triple agonist of any one of embodiments 1-9 or 11-13, wherein SX 12 X 13 LX 15 Selected from SYYLE and SILLE.
15. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding embodiments, wherein X 17 K is the number.
16. The GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 1-14, wherein X 17 Q.
17. The GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 1-14, wherein X 17 R is R.
18. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding embodiments, wherein X 18 Is A.
19. The GLP-1/GIP/Gcg receptor triple agonist of any one of embodiments 1-17, wherein X 18 Y.
20. The GLP-1/GIP/Gcg receptor triple agonist of any one of embodiments 1-17, wherein X 18 K is the number.
21. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding embodiments, wherein X 20 Is Aib.
22. The GLP-1/GIP/Gcg receptor triple agonist of any one of embodiments 1-20, wherein X 20 Q.
23. The GLP-1/GIP/Gcg receptor triple agonist of any one of embodiments 1-20, wherein X 20 E is defined as E.
24. The GLP-1/GIP/Gcg receptor triple agonist of any one of embodiments 1-20, wherein X 20 R is R.
25. The GLP-1/GIP/Gcg receptor triple agonist of any one of embodiments 1-20, wherein X 20 H.
26. The GLP-1/GIP/Gcg receptor triple agonist of any one of embodiments 1-16, 18-19 or 21, wherein KX 17 X 18 AX 20 Selected from KQAAAib, KKAAAib, KQYAAib and KKYAAib.
27. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding embodiments, wherein X 21 E is defined as E.
28. The GLP-1/GIP/Gcg receptor triple agonist of any one of embodiments 1-26, wherein X 21 K is the number.
29. According to the foregoing embodimentThe GLP-1/GIP/Gcg receptor triple agonist of any one of the regimens, wherein X 24 Q.
30. The GLP-1/GIP/Gcg receptor triple agonist of any one of embodiments 1-28, wherein X 24 Is N.
31. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding embodiments, wherein X 28 E is defined as E.
32. The GLP-1/GIP/Gcg receptor triple agonist of any one of embodiments 1-30, wherein X 28 Is A.
33. The GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 1-4, 6-16, 18-19, 21, 26-27 or 29-32, wherein the amino acid sequence of the peptide is:
HX 2 HGTFTSDYSX 12 X 13 LX 15 KX 17 X 18 AX 20 EFVX 24 WLLX 28 GGPSSGAPPPS (SEQ ID NO: 47), wherein
X 2 Is Aib
X 12 Is I or Y
X 13 Is Y or L
X 15 Is D or E
X 17 Is Q or K
X 18 Is A or Y
X 20 Is Aib
X 24 Is N or Q
X 28 Is A or E.
34. The GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 1-4, 6-9, 11-16, 18-19, 21, 26-27 or 29-33, wherein the amino acid sequence of the peptide is:
HX 2 HGTFTSDYSX 12 X 13 LEKX 17 X 18 AX 20 EFVX 24 WLLX 28 GGPSSGAPPPS (SEQ ID NO: 48), wherein
X 2 Is Aib
X 12 Is I or Y
X 13 Is Y or L
X 17 Is Q or K
X 18 Is A or Y
X 20 Is Aib
X 24 Is N or Q
X 28 Is A or E.
35. The GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 1-4, 6-9, 11-16, 18-19, 21, 26-27, 29, 31 or 33-34, wherein the amino acid sequence of the peptide is:
HX 2 HGTFTSDYSX 12 X 13 LEKX 17 X 18 AX 20 EFVQWLLEGGPSSGAPP
PS (SEQ ID NO: 49), wherein
X 2 Is Aib
X 12 Is I or Y
X 13 Is Y or L
X 17 Is Q or K
X 18 Is A or Y
X 20 Is Aib.
36. The GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 1-3, 5-16, 18-19, 21, 26-27 or 29-32, wherein the amino acid sequence of the peptide is:
YX 2 QGTFTSDYSX 12 X 13 LX 15 KX 17 X 18 AX 20 EFVX 24 WLLX 28 GGPSSGAPPPS (SEQ ID NO: 50), wherein
X 2 Is Aib
X 12 Is I or Y
X 13 Is Y or L
X 15 Is D or E
X 17 Is Q or K
X 18 Is A or Y
X 20 Is Aib
X 24 Is N or Q
X 28 Is A or E.
37. The GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 1-3, 5-9, 11-16, 18, 21, 26-27, 29, 31 or 36, wherein the amino acid sequence of the peptide is:
YX 2 QGTFTSDYSX 12 X 13 LEKX 17 AAX 20 EFVQWLLEGGPSSGAPPPS
(SEQ ID NO: 51), wherein
X 2 Is Aib
X 12 Is I or Y
X 13 Is Y or L
X 17 Is Q or K
X 20 Is Aib.
38. The GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 1-3, 5-10, 12-13, 15-16, 18, 21, 26-27, 30, 32 or 36, wherein the amino acid sequence of the peptide is:
YX 2 QGTFTSDYSX 12 X 13 LDKX 17 AAX 20 EFVNWLLAGGPSSGAPPP
S (SEQ ID NO: 52), wherein
X 2 Is Aib
X 12 Is I or Y
X 13 Is Y or L
X 17 Is Q or K
X 20 Is Aib.
39. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding embodiments, wherein the peptide has a C-terminal amide.
40. The GLP-1/GIP/Gcg receptor triple agonist of any one of embodiments 1-38, wherein the peptide has a C-terminal acid.
41. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding embodiments, wherein the substituent is A-B-C-, wherein
A-is chemical formula 1
Chemical formula 1: HOOC- (CH) 2 ) p -CO-*
p is an integer in the range of 16-20;
b-is selected from chemical formula 2 or chemical formula 3:
chemical formula 2: * -NH-CH (COOH) - (CH) 2 ) 2 -CO-*
Chemical formula 3: * -NH-CH 2 -(C 6 H 10 )-CO-NH-CH(COOH)-(CH 2 ) 2 -CO-*；
C-is selected from formula 4 or formula 5:
chemical formula 4: * - [ NH- ((CH) 2 ) 2 -O) 2 -CH 2 -CO] 2 -*
Chemical formula 5: * - [ NH- (CH) 2 ) 4 -CH(NH 2 )-CO] 2 -*
Wherein represents the point of attachment and A-, B-, and C-are linked to each other in the order indicated by amide bonds.
42. The GLP-1/GIP/Gcg receptor triple agonist of embodiment 41 wherein A-is chemical formula 1 and p is 16 or 18.
43. The GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 41-42, wherein B-is formula 2.
44. The GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 41-42, wherein B-is formula 3.
45. The GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 41-42 or 44, wherein B-is formula 3a or formula 3B.
46. The GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 41-45, wherein C-is formula 4.
47. The GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 41-45, wherein C-is formula 5.
48. The GLP-1/GIP/Gcg receptor triple agonist of any one of embodiments 41-43 or 46, wherein a-is formula 1 and p is 18; b-is chemical formula 2; and C-is chemical formula 4.
49. The GLP-1/GIP/Gcg receptor triple agonist of any one of embodiments 41-43 or 47, wherein a-is formula 1 and p is 18; b-is chemical formula 2; and C-is chemical formula 5.
50. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding embodiments, wherein the substituent a-B-C-is selected from formula 6, formula 7, formula 8, formula 9, formula 10, formula 11, formula 12 and formula 13.
51. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding embodiments, wherein the substituents a-B-C-are selected from formula 12 and formula 13.
52. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding embodiments, wherein the substituent is covalently attached to the epsilon amino group of K at position 16.
53. The GLP-1/GIP/Gcg receptor triple agonist of any one of embodiments 1-15 or 18-51, wherein X 17 K, and the substituent is covalently linked to the epsilon-amino group of the K at position 17.
54. The GLP-1/GIP/Gcg receptor triple agonist of any one of embodiments 1-26, 28-32 or 39-51, wherein X 21 K, and the substituent is covalently linked to the epsilon-amino group of the K at position 21.
55. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding embodiments, wherein the triple agonist is selected from compounds No. 1-57.
56. The GLP-1/GIP/Gcg receptor triple agonist of any one of embodiments 1-52 or 55, wherein the triple agonist is selected from compounds nos. 1, 5-6 and 12-57.
57. The GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 1-51, 53 or 55, wherein the triple agonist is selected from compounds nos. 2 and 9-10.
58. The GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 1-51 or 54-55, wherein the triple agonist is selected from compounds nos. 3-4, 7-8 and 11.
59. The GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 1-52 or 55-56, wherein the triple agonist is compound No. 26.
60. The GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 1-52 or 55-56, wherein the triple agonist is compound No. 43.
61. The GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 1-52 or 55-56, wherein the triple agonist is compound No. 44.
62. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding embodiments, wherein the triple agonist is an agonist against hGLP-1R, hGIPR and hGcgR.
63. The GLP-1/GIP/glucagon receptor triple agonist of any one of the preceding embodiments, wherein the triple agonist is capable of activating hGLP-1R, hGIPR and hGcgR.
64. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding embodiments, wherein the triple agonist is capable of activating hGLP-1R, hGIPR and hGcgR in assays employing whole cells expressing each of the following three receptors.
65. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding embodiments, wherein the triple agonist is capable of activating hGLP-1R, hGIPR and hGcgR in vitro in a CRE luciferase assay, e.g. in example 2 described herein.
66. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding embodiments, which is an agonist against hGLP-1R, EC when measured in vitro in the absence of HSA, as determined in example 2 described herein 50 Not more than 50pM, for example not more than 10pM.
67. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding embodiments, which is an agonist against hGIPR, EC when measured in vitro in the absence of HSA, as determined in example 2 described herein 50 No more than 50pM, such as no more than 20pM.
68. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding embodiments, which is an agonist against hGcgR, EC when measured in vitro in the absence of HSA, as determined in example 2 described herein 50 No more than 500pM, such as no more than 250pM, such as no more than 50pM.
69. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding embodiments, which is an agonist to all receptors for hGLP-1R, hGIPR and hGcgR, EC when measured in vitro in the absence of HSA, as determined in example 2 described herein 50 Not more than 500pM, for example not more than 100pM.
70. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding embodiments, having improved pharmacokinetic properties.
71. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding embodiments, which has an extended half-life.
72. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding embodiments, which has an improved half-life when measured in a minipig, as determined in example 3 described herein.
73. The GLP-1/GIP/Gcg receptor triple agonist according to any of the preceding embodiments, which has a half-life in a mini-pig of at least 30 hours, such as at least 40 hours, or such as at least 60 hours, as determined in example 3 described herein.
74. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding embodiments, as determined in DIO mice, as in example 4 described herein, having a food intake reducing effect in vivo.
75. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding embodiments, as determined in DIO mice, as in example 4 described herein, having an effect of inducing weight loss in vivo.
76. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding embodiments, as determined in DIO mice, as in example 4 described herein, having an effect of improving glucose tolerance in vivo.
77. A pharmaceutical composition comprising a triple agonist according to any of the preceding embodiments.
78. The pharmaceutical composition according to embodiment 77, optionally comprising at least one pharmaceutically acceptable excipient.
79. The pharmaceutical composition according to embodiments 77-78, wherein the triple agonist is selected from compounds No. 1-57.
80. The pharmaceutical composition according to embodiments 77-79, wherein the triple agonist is compound No. 26.
81. The pharmaceutical composition according to embodiments 77-79, wherein the triple agonist is compound No. 43.
82. The pharmaceutical composition according to embodiments 77-79, wherein the triple agonist is compound No. 44.
83. The pharmaceutical composition according to any one of embodiments 77-82 for use as a medicament.
84. The GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 1-76 for use as a medicament.
85. The GLP-1/GIP/Gcg receptor triple agonist of any one of embodiments 1-76 for use in the treatment of:
(i) For example, preventing and/or treating eating disorders such as obesity by reducing food intake, increasing energy expenditure, reducing body weight, suppressing appetite, inducing satiety; treating or preventing binge eating disorder, bulimia nervosa, and/or obesity induced by administration of antipsychotics or steroids; reducing gastric motility; delaying gastric emptying; increase physical activity; and/or preventing and/or treating co-morbidities of obesity, such as osteoarthritis and/or urinary incontinence;
(ii) Weight maintenance after successful weight loss (whether drug induced or diet and exercise induced) -i.e., preventing weight gain after successful weight loss.
(iii) Preventing and/or treating all forms of diabetes, such as hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin dependent diabetes mellitus, MODY (maturity onset diabetes of the young), gestational diabetes, and/or for reducing HbA1C;
(iv) Delaying or preventing the progression of diabetes, such as the progression of type 2 diabetes, delaying the progression of Impaired Glucose Tolerance (IGT) to type 2 diabetes requiring insulin, delaying or preventing insulin resistance, and/or delaying the progression of type 2 diabetes without insulin to type 2 diabetes requiring insulin;
(v) Preventing and/or treating liver disorders such as liver steatosis, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), liver inflammation or fatty liver.
86. The GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 1-76 for use in the prevention and/or treatment of all forms of diabetes, such as hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin dependent diabetes, MODY (maturity onset diabetes), gestational diabetes, and/or for reducing HbA1C.
87. The GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 1-76 for use in the prevention and/or treatment of eating disorders, such as obesity, e.g. by decreasing food intake, increasing energy expenditure, reducing body weight, suppressing appetite, inducing satiety; treating or preventing binge eating disorder, bulimia nervosa, and/or obesity induced by administration of antipsychotics or steroids; reducing gastric motility; delaying gastric emptying; increase physical activity; and/or preventing and/or treating co-morbidities of obesity, such as osteoarthritis and/or urinary incontinence.
88. The GLP-1/GIP/Gcg receptor triple agonist of any one of embodiments 1-76 for use in weight maintenance after successful weight loss (whether drug-induced or diet-and exercise-induced) -i.e., preventing weight gain after successful weight loss.
89. The GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 1 to 76 for use in the prevention and/or treatment of a liver disorder, such as liver steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver inflammation or fatty liver.
90. The GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 1-76 for use in weight management, treatment and/or prevention of obesity and obesity-related disorders.
91. The GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 1 to 76 for use in the treatment and/or prevention of all forms of diabetes, such as type 2 diabetes, and diabetes related disorders.
92. Use of a GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 1-76 in the manufacture of a medicament for
(i) For example, preventing and/or treating eating disorders such as obesity by reducing food intake, increasing energy expenditure, reducing body weight, suppressing appetite, inducing satiety; treating or preventing binge eating disorder, bulimia nervosa, and/or obesity induced by administration of antipsychotics or steroids; reducing gastric motility; delaying gastric emptying; increase physical activity; and/or preventing and/or treating co-morbidities of obesity, such as osteoarthritis and/or urinary incontinence;
(ii) Weight maintenance after successful weight loss (whether drug induced or diet and exercise induced) -i.e., preventing weight gain after successful weight loss.
(iii) Preventing and/or treating all forms of diabetes, such as hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin dependent diabetes mellitus, MODY (maturity onset diabetes of the young), gestational diabetes, and/or for reducing HbA1C;
(iv) Delaying or preventing the progression of diabetes, such as the progression of type 2 diabetes, delaying the progression of Impaired Glucose Tolerance (IGT) to type 2 diabetes requiring insulin, delaying or preventing insulin resistance, and/or delaying the progression of type 2 diabetes without insulin to type 2 diabetes requiring insulin;
(v) Preventing and/or treating liver disorders such as liver steatosis, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), liver inflammation or fatty liver.
93. The use of a GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 1-76 for the manufacture of a medicament for the prevention and/or treatment of all forms of diabetes, such as hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin dependent diabetes, MODY (maturity onset diabetes of the young), gestational diabetes, and/or for the reduction of HbA1C.
94. Use of the GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 1-76 for the manufacture of a medicament for the prevention and/or treatment of a eating disorder, such as obesity, e.g. by reducing food intake, increasing energy expenditure, reducing body weight, suppressing appetite, inducing satiety; treating or preventing binge eating disorder, bulimia nervosa, and/or obesity induced by administration of antipsychotics or steroids; reducing gastric motility; delaying gastric emptying; increase physical activity; and/or preventing and/or treating co-morbidities of obesity, such as osteoarthritis and/or urinary incontinence.
95. Use of a GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 1-76 in the manufacture of a medicament for weight maintenance after successful weight loss (whether drug-induced or diet and exercise-induced) -i.e. for preventing weight gain after successful weight loss.
96. The use of a GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 1-76 in the manufacture of a medicament for the prevention and/or treatment of a liver disorder, such as liver steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver inflammation or fatty liver.
97. The use of a GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 1-76 in the manufacture of a medicament for weight management, treatment and/or prevention of obesity and obesity-related disorders.
98. The use of a GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 1-76 in the manufacture of a medicament for the treatment and/or prevention of all forms of diabetes, such as type 2 diabetes, and diabetes related disorders.
99. For example, preventing and/or treating eating disorders such as obesity by reducing food intake, increasing energy expenditure, reducing body weight, suppressing appetite, inducing satiety; treating or preventing binge eating disorder, bulimia nervosa, and/or obesity induced by administration of antipsychotics or steroids; reducing gastric motility; delaying gastric emptying; increase physical activity; and/or a method of preventing and/or treating co-diseases of obesity, such as osteoarthritis and/or urinary incontinence, comprising administering a pharmaceutically active amount of a GLP-1/GIP/Gcg receptor triple agonist according to any of embodiments 1-76.
100. A method of maintaining body weight after successful weight loss (whether drug-induced or diet and exercise-induced) -i.e., preventing weight gain after successful weight loss-comprising administering a pharmaceutically active amount of a GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 1-76.
101. Preventing and/or treating all forms of diabetes, such as hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin dependent diabetes mellitus, MODY (maturity onset diabetes of the young), gestational diabetes, and/or a method for reducing HbA1C, comprising administering a pharmaceutically active amount of a GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 1-76.
102. A method of delaying or preventing the progression of diabetes, such as the progression of type 2 diabetes, delaying the progression of Impaired Glucose Tolerance (IGT) to type 2 diabetes requiring insulin, delaying or preventing insulin resistance, and/or delaying the progression of type 2 diabetes without insulin to type 2 diabetes requiring insulin, the method comprising administering a pharmaceutically active amount of a GLP-1/GIP/Gcg receptor triple agonist according to any of embodiments 1-76.
103. A method of preventing and/or treating a liver disorder, such as liver steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver inflammation or fatty liver, comprising administering a pharmaceutically active amount of a GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 1-76.
104. A method of weight management, treatment and/or prevention of obesity and obesity-related disorders comprising administering a pharmaceutically active amount of a GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 1-76.
105. A method of treating and/or preventing all forms of diabetes, e.g. type 2 diabetes, and conditions associated with diabetes, the method comprising administering a pharmaceutically active amount of a GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 1-76.
106. A method of preparing a GLP-1/GIP/Gcg receptor triple agonist according to any one of embodiments 1-76.
Examples
This experimental section begins with a list of abbreviations followed by a section that includes the general methodology for synthesizing and characterizing the triple agonists described herein. The examples follow in relation to the preparation of specific triple agonists and selected comparison compounds, and finally in relation to the activity and properties of the triple agonists.
The examples serve to illustrate the invention.
List of abbreviations
The following alphabetically listed abbreviations are used hereinafter:
ac: acetyl group
Ado: 8-amino-3, 6-dioxaoctanoic acid
Aib: alpha-amino isobutyric acid (or alpha-amino isobutyric acid)
amu: atomic mass units
API-ES: atmospheric pressure ionization-electrospray
AUC: area under curve
BHK: baby hamster kidney
Boc: boc-group
BW: weight of body
Cl-HOBt: 6-chloro-1-hydroxybenzotriazole
CRE: cAMP response element
DCM: dichloromethane (dichloromethane)
DIC: diisopropylcarbodiimide
DIPEA: n, N-diisopropylethylamine
DMEM: dulbecco's modified Eagle's Medium
DPBS: dulbecco's phosphate buffered saline
EDTA: ethylenediamine tetraacetic acid
ELISA: enzyme-linked immunosorbent assay equiv: molar equivalent
FBS: fetal bovine serum
Fmoc: 9-fluorenylmethoxycarbonyl
Gcg: glucagon
GcgR: glucagon receptor
GIP: glucose-dependent insulinotropic polypeptides
GIPR: glucose-dependent insulinotropic polypeptide receptor GLP-1: glucagon-like peptide 1GLP-1R: glucagon-like peptide 1 receptor HEPES:4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid HFIP:1, 3-hexafluoro-2-propanol or hexafluoroisopropanol hGcgR: human glucagon receptor hGIPR: human glucose-dependent insulinotropic polypeptide receptor hGLP-1R: human glucagon-like peptide 1 receptor HPLC: high performance liquid chromatography
i.p.: intraperitoneal administration
IPGTT: intraperitoneal glucose tolerance test
V. the following: intravenous injection
LCMS: liquid chromatography mass spectrometry
MeCN: acetonitrile
mM: millimolar concentration of
mmol: millimoles (milli)
min: minute (min)
Mtt: 4-Methyltrityl radical
MW: molecular weight
NMP: 1-methyl-pyrrolidin-2-one
OtBu: tert-butyl ester
OxymaCyano-hydroxy imino-acetic acid ethyl ester
Pbf:2, 4,6, 7-pentamethyldihydrobenzofuran-5-sulfonyl
PBS: phosphate buffered saline
PK: pharmacokinetics of
pM: picomolar concentration
rpm: revolutions per minute
Rt: retention time
SEM: standard error of average value
SPPS: solid phase peptide synthesis
tBu: tert-butyl group
TFA: trifluoroacetic acid
TIS: triisopropylsilane
Trt: triphenylmethyl or trityl
Trx: tranexamic acid
TSTU: general preparation method of O- (N-succinimidyl) -1, 3-tetramethyluronium tetrafluoroborate
The solid phase peptide synthesis method (SPPS method, including amino acid deprotection method, method of cleaving peptide from resin, and purification method thereof), and method of detecting and characterizing the resulting peptide (LCMS method) are described below.
The resin used to prepare the C-terminal peptide Amide is an H-Rink Amide-ChemMatrix resin (e.g., loaded with 0.5 mmol/g) or Rink Amide AM polystyrene resin (e.g., loaded with 0.6 mmol/g) or PAL Amide AM resin (e.g., loaded with 0.6 mmol/g). The resin used to prepare the C-terminal peptide acid is Wang-polystyrene resin preloaded with the appropriate C-terminal Fmoc protected amino acid (e.g., loaded with 0.6 mmol/g). Unless specifically stated otherwise, the Fmoc protected amino acid derivatives used are recommended standards: fmoc-Ala-OH, fmoc-Arg (Pbf) -OH, fmoc-Asn (Trt) -OH, fmoc-Asp (OtBu) -OH, fmoc-Cys (Trt) -OH, fmoc-Gln (Trt) -OH, fmoc-Glu (OtBu) -OH, fmoc-Gly-OH, fmoc-His (Trt) -OH, fmoc-Ile-OH, fmoc-Leu-OH, fmoc-Lys (Boc) -OH, fmoc-Met-OH, fmoc-Phe-OH, fmoc-Pro-OH, fmoc-Ser (tBu) -OH, fmoc-Cys (Trt) -OH, fmoc-Trp (Boc) -OH, fmoc-Tyr (tBu) -OH, fmoc-Ile-OH, fmoc-Leu-OH, fmoc-Lys (Fmoc-OH), and the like, provided, for example, by AAPPTEC, anaspec, bachem, chemImpex, iris Biotech, midwest, gyros Protein Technologies or Novabiochem. In the absence of any other description, the natural L-form of amino acid is used. The N-terminal amino acid is protected at the α -amino group with Boc (e.g., boc-Tyr (tBu) -OH for peptides with Tyr at the N-terminus, or Boc-His (Trt) -OH for peptides with His at the N-terminus).
In the case of modular albumin binding moiety ligation using SPPS, suitably protected building blocks such as, but not limited to, fmoc-8-amino-3, 6-dioxaoctanoic acid (Fmoc-Ado-OH), fmoc-tranexamic acid (Fmoc-Trx-OH), boc-Lys (Fmoc) -OH, fmoc-Glu-OtBu, mono-tert-butyl octadecanedioate, mono-tert-butyl nonadecanedioate, mono-tert-butyl eicosanedioate or mono-tert-butyl tetradecanedioate are used. All the operations described below were carried out on a synthetic scale ranging from 0.1 to 0.5 mmol.
1. Synthesis of resin-bound protected peptide backbones
The method comprises the following steps: SPPS_A
SPPS was performed on a Protein Technologies SymphonyX solid phase peptide synthesizer using Fmoc-based chemistry with minor modifications using the protocol provided by the manufacturer. The mixing was performed by bubbling nitrogen from time to time. The step assembly was performed using the following steps: 1) Pre-swelling the resin in DMF; 2) Fmoc deprotection was performed by treatment with 20% (v/v) piperidine in DMF twice for 10min each; 3) Washing with DMF to remove piperidine; 4) Fmoc-amino acids (4-12 equiv) and Oxyma were added as 0.3-0.6M solutions in DMF respectively(4-12 equiv), followed by adding DIC (4-12 equiv) as a 0.6-1.2M solution in DMF, optionally adding DMF to reduce the final concentration of the various components if necessary, then mixing for 0.5-4h to effect Fmoc-amino acid coupling; 5) Washing with DMF to remove excess reagent; 6) The final wash was performed with DCM at the completion of the assembly. Some amino acids, such as but not limited to sterically hindered amino acids (e.g., aib), are coupled for an extended reaction time (e.g., 4 h) to ensure reaction completion.
The method comprises the following steps: SPPS_B
Protected peptidyl resin was synthesized on a Applied Biosystems A solid phase peptide synthesizer according to the Fmoc strategy using the general Fmoc protocol provided by the manufacturer. Mixing was performed by vortexing and bubbling nitrogen from time to time. The step assembly was completed using the following steps: 1) Fmoc-amino acids were activated by dissolving solid Fmoc acid (10 equiv) in Cl-HOBt (10 equiv) as a 1M solution in NMP, then adding DIC (10 equiv) as a 1M solution in NMP, then mixing simultaneously with steps 2-3; 2) Fmoc deprotection was performed by treatment with 20% (v/v) piperidine in NMP for 3min once followed by 15min for the second treatment; 3) Washing with NMP to remove piperidine; 4) Adding activated Fmoc-amino acid solution to the resin, and mixing for 45-90min; 5) Washing with NMP to remove excess reagent; 6) The final wash was performed with DCM at the completion of the assembly. The standard protected amino acid derivatives listed above are provided in pre-weighed cartridges (e.g., from Midwest Biotech), rather than the standard derivatives being manually weighed. Some amino acids, such as but not limited to, amino acids following a sterically hindered amino acid (e.g., aib) are "double coupled" to ensure completion of the reaction, which means draining the resin after the first coupling (e.g., 45 min), adding more reagents (Fmoc-amino acid, DIC, cl-HOBt), and allowing the mixture to react again (e.g., 45 min).
2. Attachment of substituents to protected peptide backbones bound to resins
The method comprises the following steps: SC_A
The N-. Epsilon. -lysine protecting group was removed by washing the resin with 30% (v/v) HFIP in DCM twice, 45min each treatment, followed by washing with DCM and DMF. Acylation was performed on a Protein Technologies SymphonyX solid phase peptide synthesizer using the protocol described in method SPPS_A, with stepwise addition of building blocks such as, but not limited to, boc-Lys (Fmoc) -OH, fmoc-8-amino-3, 6-dioxaoctanoic acid, fmoc-tranexamic acid, fmoc-Glu-OtBu, mono-tert-butyl octadecanedioate and mono-tert-butyl eicosanedioate.
The method comprises the following steps: SC_B
The N-. Epsilon. -lysine protecting group was removed by washing the resin with 30% (v/v) HFIP in DCM twice, 45min each treatment, followed by washing with DCM and DMF. Acylation was performed on a Applied Biosystems 431A solid phase peptide synthesizer using the protocol described in method SPPS_B, using stepwise addition of building blocks such as, but not limited to, boc-Lys (Fmoc) -OH, fmoc-8-amino-3, 6-dioxaoctanoic acid, fmoc-tranexamic acid, fmoc-Glu-OtBu, octadecanedioic acid mono-tert-butyl ester and icosanedioic acid mono-tert-butyl ester.
The method comprises the following steps: SC_C
The N-. Epsilon. -lysine protecting group was removed by washing the resin three times with 75:2.5:22.5v/v HFIP/TIS/DCM, 30min each treatment followed by washing with DCM and DMF. Separately, 2-chlorotrityl chloride resin (e.g., loaded with 1.1 mmol/g) was transferred to a sinter separating funnel and swollen for 30min in DCM, then drained and shaken with 10% DIPEA (v/v) in DCM for 10min. The resin was then drained and immediately treated with the first Fmoc-protected substituent building block (1.0 equiv) and DIPEA (2 equiv) in DCM and shaken overnight at room temperature. Methanol is then added to the mixture to block unreacted sites of the (cap) resin. The resin was washed with DCM, DMF and diethyl ether and then dried under vacuum. Subsequently, the substituents are assembled in a stepwise manner as described in method spps_a. The protected substituents were cleaved from the resin by three treatments with 20% HFIP (v/v) in DCM for 10min each, and the cleavage mixture was concentrated in vacuo and dried in vacuo to yield the protected substituents, which were used without further manipulation. The protected substituents were dissolved in anhydrous THF at a concentration of 0.1M, followed by treatment with TSTU (1.5 equiv) and DIPEA (3 equiv). The mixture was stirred at room temperature for two hours, filtered, and diluted five times the volume with ethyl acetate. The organic extract was washed with an equal volume of 0.1M aqueous HCl and then with an equal volume of water. The organic layer was then dried over MgSO 4 Dried, and the volatile solvents were removed in vacuo. The activated substituent (1.5 equiv) was used directly as a 0.1M solution in DMF containing DIPEA (3 equiv) and incubated with the peptide-based resin bearing free lysine epsilon-amino groups for 16 hours. The resin was then washed with DMF and DCM.
3. Cleavage and purification of resin-bound peptides
After completion of the side chain synthesis, the peptidyl resin was washed with DCM and dried, then treated with TFA/water/TIS (95:2.5:2.5 v/v/v) for about 2-3h, followed by precipitation with diethyl ether. The precipitate was washed with diethyl ether, dissolved in a suitable solvent (e.g. 2:1 water/MeCN) and allowed to stand until all the unstable adducts had resolved. Purification is carried out by reverse phase preparative HPLC (e.g. a Waters2545 binary gradient module, waters 2489 UV/visible detector, waters fraction collector III; or e.g. Waters DeltaPrep 4000) on a suitable column containing, for example, C8-or C18-silica gel. The separation of impurities and elution of the product was accomplished using a gradient of MeCN gradually increasing in water containing 0.1% TFA. The structure and purity of the relevant fractions were checked by analytical LCMS. Fractions containing the pure desired product were combined and lyophilized to give the peptide TFA salt as a white solid.
4. Salt exchange from TFA to sodium salt:
the lyophilized, purified peptide is dissolved to 3-20mg/mL in a suitable aqueous buffer such as, but not limited to, 4:1 water/MeCN, 0.2M sodium acetate, or 50mM HEPES buffer pH 7.4. If necessary, the pH of the solution is adjusted with aqueous NaOH to achieve complete dissolution. The buffer containing the peptide was subjected to salt exchange using a Sep-Pak C18 column (0.5-5 g). The column was equilibrated first with isopropanol, then with MeCN, then with water. The peptide solution was applied to the column and the flow-through was reapplied to ensure complete peptide retention. With water, then with a solvent such as, but not limited to, naHCO 3 NaOAc or Na 2 HPO 4 The column is washed with a buffer solution (e.g., pH 7.5). The column was then washed with water and the peptide eluted with 50-80% (v/v) MeCN in water. The peptide-containing eluate was freeze-dried to give the peptide sodium salt as a white solid, which was used as such.
Universal detection and characterization method
LCMS method:
the method comprises the following steps: lcms_a
Lcms_a was performed on an apparatus consisting of an Agilent 1260 affinity series HPLC system and Agilent Technologies 6120Quadrupole MS. The eluent is defined as: a:0.05% (v/v) TFA in water; b: 0.05% (v/v) TFA in 9:1 (v/v) MeCN/water. Analysis was performed at column temperature of 37 ℃ by injecting the appropriate volume of sample onto the column and eluting with a gradient of a and B. Column: phenomenex Kinetex C8,2.6 μm, 4.6X75 mm. Gradient run time: linear 20-100% B in 10min, flowThe speed was 1.0mL/min. And (3) detection: a diode array detector set at 214nm. MS ionization mode: API-ES, positive polarity. MS scan mass range: 500-2000amu. The most abundant isotope per m/z is reported.
The method comprises the following steps: lcms_b
LCMS_B was performed on a device consisting of a Waters acquisition H-grade UPLC system and Xex G2-XS QTof MS. The eluent is defined as: a:0.1% (v/v) aqueous formic acid; b: 0.1% (v/v) formic acid in MeCN; c:0.1% (v/v) TFA in water. Analysis was performed at column temperature of 40 ℃ by injecting the appropriate volume of sample onto the column and eluting with a gradient of a and B. Column: waters Acquity BEH, C-18,1.7 μm, 2.1X10 mm. Gradient run time: linear 5-95% B in 4.0min in the presence of constant 5% C at a flow rate of 0.4mL/min. MS ionization mode: ES, positive polarity. MS scan mass range: 50-4000amu. The most abundant isotope per m/z is reported.
Example 1: synthesis of Compounds
Compounds are described below using single letter amino acid codes, except for Aib. The substituents are included after the lysine (K) residue to which they are attached.
Compound No. 1
Y-Aib-QGTFTSDYSIYLD-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (17-carboxyheptadecylamido) butanoyl [ (2- [ (4S))]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-QAA-Aib-EFVNWLLAGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 1, C-terminal amide
Substituents: chemical formula 6 linked via the epsilon-amino group of K16
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4821.3DaLCMS_B: rt=3.4 min; actual measurement [ M+3H] 3+ 1607.1，[M+4H] 4+ 1205.6。
Compound No. 2
Y-Aib-QGTFTSDYSIYLDK-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (17-carboxyheptadecylamido) butanoyl [ (2- [ (4S))]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-AA-Aib-EFVNWLLAGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO 2, C-terminal amide
Substituents: chemical formula 6 linked via the epsilon-amino group of K17
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4821.4Da
Lcms_b: rt=3.0 min; actual measurement [ M+2H] 2+ 2411.8，[M+3H] 3+ 1607.8，
[M+4H] 4+ 1206.1。
Compound No. 3
Y-Aib-QGTFTSDYSIYLDKQAa-Aib-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (17-carboxyheptadecylamino) butanoyl [ [2- [2- [ (4S) -4-carboxy-heptadecylamino ] butanoyl ]]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-FVNWLLAGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO 3, C-terminal amide
Substituents: chemical formula 6 linked via the epsilon-amino group of K21
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4820.4DaLCMS_B: rt=3.0 min; actual measurement [ M+3H] 3+ 1606.8，[M+4H] 4+ 1205.6。
Compound No. 4
Y-Aib-QGTFTSDYSIYLDKQAa-Aib-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (17-carboxyheptadecylamino) butanoyl [ [2- [2- [ (4S) -4-carboxy-heptadecylamino ] butanoyl ]]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-FVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 4, C-terminal amide
Substituents: chemical formula 6 linked via the epsilon-amino group of K21
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4892.5DaLCMS_A: rt=6.1 min; actual measurement [ M+3H] 3+ 1631.5，[M+4H] 4+ 1224.0。
Compound No. 5
Y-Aib-QGTFTSDYSIYLD-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (19-carboxynona-carbo-noylamino) butanoyl ]]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-QAA-Aib-EFVNWLLAGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 1, C-terminal amide
Substituents: chemical formula 12 linked via the epsilon-amino group of K16
The synthesis method comprises the following steps: SPPS_A; SC_C
Calculated molecular weight (average): 4849.4Da
Lcms_b: rt=3.5 min; actual measurement [ M+2H] 2+ 2424.7，[M+3H] 3+ 1616.5，
[M+4H] 4+ 1212.6。
Compound No. 6
Y-Aib-QGTFTSDYSIYLD-K [2- [2- [2- [ [ (4S) -4-carboxy-4- [ [4- [ (19-carboxy-nineteen carbonylamino) methyl ] 2- [ [2- [ (4S) -4- ] methyl ]]Cyclohexane carbonyl ]Amino group]Butyryl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-QAA-Aib-EFVNWLLAGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 1, C-terminal amide
Substituents: chemical formula 10 via epsilon-amino linkage of K16
The synthesis method comprises the following steps: SPPS_A; SC_C
Calculated molecular weight (average): 4988.6Da
Lcms_b: rt=3.6 min; actual measurement [ M+2H] 2+ 2495.1，[M+3H] 3+ 1663.4，
[M+4H] 4+ 1247.8。
Compound No. 7
Y-Aib-QGTFTSDYSIYLDKQAa-Aib-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (19-carboxynineteen carbonylamino) butanoyl ]]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-FVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 4, C-terminal amide
Substituents: chemical formula 12 via epsilon-amino linkage of K21
The synthesis method comprises the following steps: SPPS_A; SC_C
Calculated molecular weight (average): 4920.5DaLCMS_B: rt=3.2 min; actual measurement [ M+2H] 2+ 2461.3，[M+3H] 3+ 1640.9。
Compound No. 8
Y-Aib-QGTFTSDYSIYLDKQAa-Aib-K [2- [2- [2- [ [ (4S) -4-carboxy-4- [ [4- [ (19-carboxynonacarbonamido) methyl ] 2- [2- [ [ [ (4S) -4-carboxy-4- [ [ amino ] methyl ] carbonyl ] amino ] carbonyl group]Cyclohexane carbonyl]Amino group]Butyryl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-FVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 4, C-terminal amide
Substituents: chemical formula 10 via epsilon-amino linkage of K21
The synthesis method comprises the following steps: SPPS_A; SC_C
Calculated molecular weight (average): 5059.7DaLCMS_B: rt=3.3 min; actual measurement [ M+3H] 3+ 1686.5，[M+4H] 4+ 1265.4。
Compound No. 9
Y-Aib-QGTFTSDYSIYLDK-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (19-carboxynona-carbo-noylamino) butanoyl ]]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-AA-Aib-EFVNWLLAGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO 2, C-terminal amide
Substituents: chemical formula 12 via epsilon-amino linkage of K17
The synthesis method comprises the following steps: SPPS_A; SC_C
Calculated molecular weight (average): 4849.4Da
Lcms_b: rt=3.2 min; actual measurement [ M+H] + 4850.0[M+2H] 2+ 2426.1，
[M+3H] 3+ 1617.6。
Compound No. 10
Y-Aib-QGTFTSDYSIYLDK-K [2- [2- [2- [ [ (4S) -4-carboxy-4- [ [4- [ (19-carboxy-nineteen carbonylamino) methyl ] 2- [ [2- [ (4S) -4- ] methyl ]]Cyclohexane carbonyl]Amino group]Butyryl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-AA-Aib-EFVNWLLAGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO 2, C-terminal amide
Substituents: chemical formula 10 via epsilon-amino linkage of K17
The synthesis method comprises the following steps: SPPS_A; SC_C
Calculated molecular weight (average): 4988.6Da
Lcms_b: rt=3.3 min; actual measurement [ M+H] + 4988.0，[M+2H] 2+ 2495.4，
[M+3H] 3+ 1663.6。
Compound 11
Y-Aib-QGTFTSDYSIYLEKQAa-Aib-K [2- [2- [2- [ [ (4S) -4-carboxy-4- [ [4- [ (19-carboxynonacarbonamido) methyl ] 2- [2- [ [ (4S) -4-carboxy-4- [ [ amino ] methyl ] carbonyl ] amino ] methyl]Cyclohexane carbonyl ]Amino group]Butyryl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-FVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO 5, C-terminal amide
Substituents: chemical formula 10 via epsilon-amino linkage of K21
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 5073.7DaLCMS_A: rt=6.6 min; actual measurement [ M+3H] 3+ 1692.0，[M+4H] 4+ 1269.4。
Compound No. 12
Y-Aib-QGTFTSDYSIYLE-K [2- [2- [2- [ [ (4S) -4-carboxy-4- [ [4- [ (19-carboxy-nineteen carbonylamino) methyl ] 2- [ [2- [ (4S) -4- ] methyl ]]Cyclohexane carbonyl]Amino group]Butyryl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-QAA-Aib-EFVNWLLAGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 6, C-terminal amide
Substituents: chemical formula 10 via epsilon-amino linkage of K16
The synthesis method comprises the following steps: SPPS_A; SC_B
Calculated molecular weight (average): 5002.6DaLcms_a: rt=6.9 min; actual measurement [ M+3H] 3+ 1668.4，[M+4H] 4+ 1251.5。
Compound No. 13
Y-Aib-QGTFTSDYSIYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- [ [4- [ (19-carboxynona-carbo-noylamino) methyl)]Cyclohexane carbonyl]Amino group]Butyryl group]Amino group]Caproyl group]Amino group]Caproyl group]-QAA-Aib-EFVNWLLAGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 6, C-terminal amide
Substituents: chemical formula 11 via epsilon-amino linkage of K16
The synthesis method comprises the following steps: SPPS_A; SC_B
Calculated molecular weight (average): 4968.7DaLCMS_A: rt=6.3 min; actual measurement [ M+3H] 3+ 1657.0，[M+4H] 4+ 1243.1。
Compound No. 14
Y-Aib-QGTFTSDYSIYLE-K [2- [2- [2- [ [ (4S) -4-carboxy-4- [ [4- [ (19-carboxy-nineteen carbonylamino) methyl ] 2- [ [2- [ (4S) -4- ] methyl ]]Cyclohexane carbonyl]Amino group]Butyryl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 7, C-terminal amide
Substituents: chemical formula 10 via epsilon-amino linkage of K16
The synthesis method comprises the following steps: SPPS_A; SC_B
Calculated molecular weight (average): 5102.7DaLCMS_A: rt=7.0 min; actual measurement [ M+3H] 3+ 1701.7，[M+4H] 4+ 1276.4。
Compound No. 15
Y-Aib-QGTFTSDYSIYLE-K [2- [2- [2- [ [ (4S) -4-carboxy- ]4- (19-carboxynona-carbonylamino) butanoyl]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-QAA-Aib-EFVNWLLAGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 6, C-terminal amide
Substituents: chemical formula 12 linked via the epsilon-amino group of K16
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4863.4DaLCMS_A: rt=6.9 min; actual measurement [ M+3H] 3+ 1621.7，[M+4H] 4+ 1216.7。
Compound No. 16
Y-Aib-QGTFTSDYSIYLE-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (19-carboxynona-carbo-noylamino) butanoyl ]]Amino group]Ethoxy group ]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 7, C-terminal amide
Substituents: chemical formula 12 linked via the epsilon-amino group of K16
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4935.5DaLCMS_A: rt=7.0 min; actual measurement [ M+3H] 3+ 1645.7，[M+4H] 4+ 1234.6。
Compound No. 17
Y-Aib-QGTFTSDYSIYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2
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Peptide backbone: SEQ ID NO. 7, C-terminal amide
Substituents: chemical formula 13 linked via the epsilon-amino group of K16
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4901.5DaLCMS_A: rt=6.2 min; actual measurement [ M+3H] 3+ 1634.5，[M+4H] 4+ 1226.7。
Compound No. 18
Y-Aib-HGTFTSDYSIYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 8, C-terminal amide
Substituents: chemical formula 13 linked via the epsilon-amino group of K16
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4910.5DaLCMS_A: rt=6.1 min; actual measurement [ M+3H] 3+ 1637.5，[M+4H] 4+ 1228.4。
Compound No. 19
H-Aib-QGTFTSDYSIYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl ]Amino group]Caproyl group]Amino group]Caproyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO 9, C-terminal amide
Substituents: chemical formula 13 linked via the epsilon-amino group of K16
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated asMolecular weight (average): 4875.5DaLCMS_A: rt=7.0 min; actual measurement [ M+3H] 3+ 1625.8，[M+4H] 4+ 1219.7。
Compound No. 20
Y-Aib-QGTFTSDYSIYLE-K [2- [2- [2- [ [ (4S) -4-carboxy-4- [ [4- [ (19-carboxy-nineteen carbonylamino) methyl ] 2- [ [2- [ (4S) -4- ] methyl ]]Cyclohexane carbonyl]Amino group]Butyryl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-QAAQEFVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 10, C-terminal amide
Substituents: chemical formula 10 via epsilon-amino linkage of K16
The synthesis method comprises the following steps: SPPS_B; SC_B
Calculated molecular weight (average): 5117.7DaLCMS_A: rt=6.8 min; actual measurement [ M+3H] 3+ 1706.8，[M+4H] 4+ 1280.3。
Compound 21
H-Aib-HGTFTSDYSIYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- [ [4- [ (19-carboxynona-carbo-noylamino) methyl)]Cyclohexane carbonyl]Amino group]Butyryl group]Amino group]Caproyl group]Amino group]Caproyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 11, C-terminal amide
Substituents: chemical formula 11 via epsilon-amino linkage of K16
The synthesis method comprises the following steps: SPPS_A; SC_B
Calculated molecular weight (average): 5023.7DaLCMS_A: rt=6.1 min; actual measurement [ M+3H ] 3+ 1675.3，[M+4H] 4+ 1256.7。
Compound No. 22
H-Aib-HGTFTSDYSIYLE-K[(2S)-2-amino-6- [ [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 11, C-terminal amide
Substituents: chemical formula 13 linked via the epsilon-amino group of K16
The synthesis method comprises the following steps: SPPS_A; SC_B
Calculated molecular weight (average): 4884.5DaLCMS_A: rt=6.0 min; actual measurement [ M+3H] 3+ 1628.8，[M+4H] 4+ 1222.0。
Compound No. 23
H-Aib-HGTFTSDYSIYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- [ [4- [ (19-carboxynona-carbo-noylamino) methyl)]Cyclohexane carbonyl]Amino group]Butyryl group]Amino group]Caproyl group]Amino group]Caproyl group]-QAAQEFVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 12, C-terminal amide
Substituents: chemical formula 11 via epsilon-amino linkage of K16
The synthesis method comprises the following steps: SPPS_B; SC_B
Calculated molecular weight (average): 5066.7DaLCMS_A: rt=6.0 min; actual measurement [ M+3H] 3+ 1689.7，[M+4H] 4+ 1267.5。
Compound No. 24
H-Aib-HGTFTSDYSIYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-QYA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 13, C-terminal amide
Substituents: chemical formula 13 linked via the epsilon-amino group of K16
The synthesis method comprises the following steps: SPPS_A; SC_B
Calculated molecular weight (average): 4976.6DaLCMS_A: rt=6.0 min; actual measurement [ M+3H] 3+ 1659.8，[M+4H] 4+ 1244.9。
Compound No. 25
H-Aib-HGTFTSDYSYYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- [ [4- [ (19-carboxynona-carbo-noylamino) methyl)]Cyclohexane carbonyl]Amino group]Butyryl group]Amino group]Caproyl group]Amino group]Caproyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2
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Peptide backbone: SEQ ID NO. 14, C-terminal amide
Substituents: chemical formula 11 via epsilon-amino linkage of K16
The synthesis method comprises the following steps: SPPS_A; SC_B
Calculated molecular weight (average): 5073.7DaLCMS_A: rt=6.0 min; actual measurement [ M+3H] 3+ 1692.0，[M+4H] 4+ 1269.1。
Compound No. 26
H-Aib-HGTFTSDYSYYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 14, C-terminal amide
Substituents: chemical formula 13 linked via the epsilon-amino group of K16
The synthesis method comprises the following steps: SPPS_A; SC_B
Calculated molecular weight (average): 4934.5DaLCMS_A: rt=5.9 min; actual measurement [ M+3H] 3+ 1645.5，[M+4H] 4+ 1234.5。
Compound No. 27
H-Aib-HGTFTSDYSIYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-QAAEEFVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 15, C-terminal amide
Substituents: chemical formula 13 linked via the epsilon-amino group of K16
The synthesis method comprises the following steps: SPPS_A; SC_B
Calculated molecular weight (average): 4928.5DaLCMS_A: rt=5.9 min; actual measurement [ M+3H] 3+ 1643.6，[M+4H] 4+ 1232.9。
Compound No. 28
H-Aib-HGTFTSDYSIYLD-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 16, C-terminal amide
Substituents: chemical formula 13 linked via the epsilon-amino group of K16
The synthesis method comprises the following steps: SPPS_A; SC_B
Calculated molecular weight (average): 4870.5DaLCMS_A: rt=5.9 min; actual measurement [ M+3H] 3+ 1624.4，[M+4H] 4+ 1218.5。
Compound No. 29
H-Aib-HGTFTSDYSYYLE-K [ (2S) -2-amino-6- [ [ (2S) -2-amino-6- [ (4S) -4-carboxy-liePhenyl-4- (19-carboxynona-carbonamido) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-QYA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 17, C-terminal amide
Substituents: chemical formula 13 linked via the epsilon-amino group of K16
The synthesis method comprises the following steps: SPPS_B; SC_B
Calculated molecular weight (average): 5026.6DaLCMS_A: rt=5.8 min; actual measurement [ M+3H] 3+ 1676.2，[M+4H] 4+ 1257.5。
Compound No. 30
H-Aib-HGTFTSDYSIYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl ]Amino group]Caproyl group]Amino group]Caproyl group]-KYA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 18, C-terminal amide
Substituents: chemical formula 13 linked via the epsilon-amino group of K16
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4976.6DaLCMS_B: rt=2.7 min; actual measurement [ M+3H] 3+ 1658.7，[M+4H] 4+ 1244.3。
Compound No. 31
H-Aib-HGTFTSDYSYYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-KAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 19, C-terminal amide
Substituents: chemical formula 13 linked via the epsilon-amino group of K16
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4934.6DaLCMS_B: rt=2.7 min; actual measurement [ M+3H] 3+ 1644.8，[M+4H] 4+ 1233.8。
Compound No. 32
H-Aib-HGTFTSDYSYYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (17-carboxyheptadecylamido) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 14, C-terminal amide
Substituents: formula 7 via epsilon-amino linkage of K16
The synthesis method comprises the following steps: SPPS_B; SC_B
Calculated molecular weight (average): 4906.5DaLCMS_A: rt=5.6 min; actual measurement [ M+3H] 3+ 1636.4，[M+4H] 4+ 1227.3。
Compound No. 33
H-Aib-HGTFTSDYSIYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl ]Amino group]Caproyl group]Amino group]Caproyl group]-KAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 20, C-terminal amide
Substituents: chemical formula 13 linked via the epsilon-amino group of K16
The synthesis method comprises the following steps: SPPS_B; SC_B
Calculated molecular weight (average): 4884.5DaLCMS_A: rt=5.7 min;actual measurement [ M+3H] 3+ 1629.0，[M+4H] 4+ 1222.0。
Compound 34
H-Aib-HGTFTSDYSIYLE-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (19-carboxynona-carbo-noylamino) butanoyl ]]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-KAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 20, C-terminal amide
Substituents: chemical formula 12 linked via the epsilon-amino group of K16
The synthesis method comprises the following steps: SPPS_B; SC_B
Calculated molecular weight (average): 4918.5DaLCMS_A: rt=6.5 min; actual measurement [ M+3H] 3+ 1640.3，[M+4H] 4+ 1230.4。
Compound No. 35
H-Aib-HGTFTSDYSIYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-QKA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 21, C-terminal amide
Substituents: chemical formula 13 linked via the epsilon-amino group of K16
The synthesis method comprises the following steps: SPPS_B; SC_B
Calculated molecular weight (average): 4941.6DaLCMS_A: rt=5.6 min; actual measurement [ M+3H] 3+ 1648.0，[M+4H] 4+ 1236.2。
Compound No. 36
H-Aib-HGTFTSDYSKYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl ]Amino group]Caproyl group]Amino group]Caproyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 22, C-terminal amide
Substituents: chemical formula 13 linked via the epsilon-amino group of K16
The synthesis method comprises the following steps: SPPS_B; SC_B
Calculated molecular weight (average): 4899.5DaLCMS_A: rt=5.6 min; actual measurement [ M+3H] 3+ 1633.8，[M+4H] 4+ 1225.7。
Compound No. 37
H-Aib-HGTFTSDYSIYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-RAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 23, C-terminal amide
Substituents: chemical formula 13 linked via the epsilon-amino group of K16
The synthesis method comprises the following steps: SPPS_B; SC_A
Calculated molecular weight (average): 4912.6DaLCMS_A: rt=5.8 min; actual measurement [ M+3H] 3+ 1636.2，[M+4H] 4+ 1229.0。
Compound No. 38
H-Aib-HGTFTSDYSIYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (17-carboxyheptadecylamido) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-KYA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 18, C-terminal amide
Substituents: formula 7 via epsilon-amino linkage of K16
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4948.6DaLCMS_A: rt=5.5 min; actual measurement [ M+3H] 3+ 1650.2，[M+4H] 4+ 1238.0。
Compound 39
H-Aib-HGTFTSDYSIYLE-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (19-carboxynona-carbo-noylamino) butanoyl ] ]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-KYA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 18, C-terminal amide
Substituents: chemical formula 12 linked via the epsilon-amino group of K16
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 5010.6DaLCMS_A: rt=6.6 min; actual measurement [ M+3H] 3+ 1670.9，[M+4H] 4+ 1253.7。
Compound No. 40
H-Aib-HGTFTSDYSYYLE-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (19-carboxynona-carbo-noylamino) butanoyl ]]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 14, C-terminal amide
Substituents: chemical formula 12 linked via the epsilon-amino group of K16
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4968.5DaLCMS_A: rt=6.5 min; actual measurement [ M+3H] 3+ 1656.9，[M+4H] 4+ 1242.9。
Compound No. 41
H-Aib-HGTFTSDYSILLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-KYA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 24, C-terminal amide
Substituents: chemical formula 13 linked via the epsilon-amino group of K16
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4926.6DaLCMS_A: rt=5.8 min; actual measurement [ M+3H ] 3+ 1643.0，[M+4H] 4+ 1232.5。
Compound No. 42
H-Aib-HGTFTSDYSYLLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 25, C-terminal amide
Substituents: chemical formula 13 linked via the epsilon-amino group of K16
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4884.5DaLCMS_A: rt=5.9 min; actual measurement [ M+3H] 3+ 1628.9，[M+4H] 4+ 1222.0。
Compound No. 43
H-Aib-HGTFTSDYSILLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 26, C-terminal amide
Substituents: chemical formula 13 linked via the epsilon-amino group of K16
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4834.5DaLCMS_A: rt=6.0 min; actual measurement [ M+3H] 3+ 1612.3，[M+4H] 4+ 1209.5。
Compound 44
H-Aib-HGTFTSDYSIYLE-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (19-carboxynona-carbo-noylamino) butanoyl ]]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 11, C-terminal amide
Substituents: chemical formula 12 linked via the epsilon-amino group of K16
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4918.5DaLCMS_A: rt=6.7 min; actual measurement [ M+3H] 3+ 1640.0，[M+4H] 4+ 1230.3。
Compound No. 45
H-Aib-HGTFTSDYSILLE-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (19-carboxynona-carbo-noylamino) butanoyl ]]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 26, C-terminal amide
Substituents: chemical formula 12 linked via the epsilon-amino group of K16
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4868.5DaLCMS_A: rt=6.7 min; actual measurement [ M+3H] 3+ 1623.4，[M+4H] 4+ 1217.9。
Compound No. 46
Y-Aib-QGTFTSDYSILLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 27, C-terminal amide
Substituents: chemical formula 13 linked via the epsilon-amino group of K16
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4851.5DaLCMS_A: rt=6.3 min; actual measurement [ M+3H] 3+ 1617.8，[M+4H] 4+ 1213.6。
Compound 47
Y-Aib-QGTFTSDYSYYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 28, C-terminal amide
Substituents: chemical formula 13 linked via the epsilon-amino group of K16
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4951.5 dalms_a: rt=6.2 min; actual measurement [ M+3H] 3+ 1651.1，[M+4H] 4+ 1238.7。
Compound 48
Y-Aib-QGTFTSDYSYLLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 29, C-terminal amide
Substituents: chemical formula 13 linked via the epsilon-amino group of K16
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4901.5DaLCMS_A: rt=6.3 min; actual measurement [ M+3H] 3+ 1634.5，[M+4H] 4+ 1226.3。
Compound No. 49
H-Aib-HGTFTSDYSILLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-QAAEEFVQWLLEGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 30, C-terminal amide
Substituents: chemical formula 13 linked via the epsilon-amino group of K16
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4878.5DaLCMS_A: rt=5.9 min; actual measurement [ M+3H] 3+ 1626.9，[M+4H] 4+ 1220.3。
Compound No. 50
H-Aib-HGTFTSDYSILLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl ] amino ] hexanoyl ] -QAAEEFVQWLLEGGPSSGAPPPS-OH
Peptide backbone: SEQ ID NO. 30, C-terminal acid
Substituents: chemical formula 13 linked via the epsilon-amino group of K16
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4879.5DaLCMS_A: rt=6.0 min; actual measurement [ M+3H] 3+ 1627.0，[M+4H] 4+ 1220.8。
Compound No. 51
H-Aib-HGTFTSDYSILLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (17-carboxyheptadecylamino) butanoyl ] amino ] hexanoyl ] -QAAEEFVQWLLEGGPSSGAPPPS-OH
Peptide backbone: SEQ ID NO. 30, C-terminal acid
Substituents: formula 7 via epsilon-amino linkage of K16
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4851.4DaLCMS_A: rt=5.7 min; actual measurement [ M+3H] 3+ 1617.8，[M+4H] 4+ 1213.7。
Compound 52
H-Aib-HGTFTSDYSILLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (17-carboxyheptadecylamino) butanoyl ] amino ] hexanoyl ] -QAAQEFVQWLLEGGPSSGAPPPS-OH
Peptide backbone: SEQ ID NO. 31, C-terminal acid
Substituents: formula 7 via epsilon-amino linkage of K16
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4850.4DaLCMS_A: rt=5.6 min; actual measurement [ M+3H] 3+ 1617.5，[M+4H] 4+ 1213.4。
Compound No. 53
H-Aib-HGTFTSDYSILLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl ] amino ] hexanoyl ] -QAAQEFVQWLLEGGPSSGAPPPS-OH
Peptide backbone: SEQ ID NO. 31, C-terminal acid
Substituents: chemical formula 13 linked via the epsilon-amino group of K16
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4878.5DaLCMS_A: rt=5.9 min; actual measurement [ M+3H] 3+ 1626.8，[M+4H] 4+ 1220.3。
Compound 54
H-Aib-HGTFTSDYSILLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (17-carboxyheptadecylamino) butanoyl ] amino ] hexanoyl ] -QAAREFVQWLLEGGPSSGAPPPS-OH
Peptide backbone: SEQ ID NO. 32, C-terminal acid
Substituents: formula 7 via epsilon-amino linkage of K16
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4878.5DaLCMS_A: rt=5.4 min; actual measurement [ M+3H] 3+ 1626.8，[M+4H] 4+ 1220.4。
Compound 55
H-Aib-HGTFTSDYSILLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl ] amino ] hexanoyl ] -QAAREFVQWLLEGGPSSGAPPPS-OH
Peptide backbone: SEQ ID NO. 32, C-terminal acid
Substituents: chemical formula 13 linked via the epsilon-amino group of K16
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4878.5DaLCMS_A: rt=5.6 min; actual measurement [ M+3H] 3+ 1636.2，[M+4H] 4+ 1227.4。
Compound 56
H-Aib-HGTFTSDYSILLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (17-carboxyheptadecylamino) butanoyl ] amino ] hexanoyl ] -QAAHEFVQWLLEGGPSSGAPPPS-OH
Peptide backbone: SEQ ID NO 33, C-terminal acid
Substituents: formula 7 via epsilon-amino linkage of K16
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4859.5DaLCMS_A: rt=5.3 min; actual measurement [ M+3H] 3+ 1620.4，[M+4H] 4+ 1215.7。
Compound 57
H-Aib-HGTFTSDYSILLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl ] amino ] hexanoyl ] -QAAHEFVQWLLEGGPSSGAPPPS-OH
Peptide backbone: SEQ ID NO 33, C-terminal acid
Substituents: chemical formula 13 linked via the epsilon-amino group of K16
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4887.5DaLCMS_A: rt=5.6 min; actual measurement [ M+3H] 3+ 1629.8，[M+4H] 4+ 1222.7. Structure of standard and comparative compounds:
natural human GLP-1 (7-37), SEQ ID NO. 35
Native human GIP, SEQ ID NO. 36
Natural human glucagon, SEQ ID NO 37
Native mouse GIP, SEQ ID NO 34
Compound 58
Y-Aib-QGTFTSDYSIYLDKQAa-Aib-EFVNWLLAGGPSSGAPPPS-K [ hexadecanoyl ]]-NH 2
Peptide backbone: SEQ ID NO. 53, C-terminal amide
Substituents: hexadecanoyl linked via the epsilon-amino group of K40
The synthesis method comprises the following steps: see US9062124 example 18, seq ID NO 124
Calculated molecular weight (average): 4472.1Da
Lcms_a: rt=7.1 min; actual measurement [ M+3H] 3+ 1491.4，[M+4H] 4+ 1118.9。
Compound No. 59
Y-Aib-QGTFTSDYSIYLDKQAa-Aib-EFVNWLLAGGPSSGAPPPS-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (17-carboxyheptadecylamido) butanoyl ]]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-NH 2
Peptide backbone: SEQ ID NO. 53, C-terminal amide
Substituents: chemical formula 6 via epsilon-amino linkage of K40
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4949.5DaLCMS_A: rt=6.1 min; actual measurement [ M+3H] 3+ 1650.4，[M+4H] 4+ 1238.2。
Compound No. 60
Y-Aib-QGTFTSDYSIYLDKQAA-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (17-carboxyheptadecylamido) butanoyl [ (2- [ (4S))]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]--EFVNWLLAGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 38, C-terminal amide
Substituents: chemical formula 6 linked via the epsilon-amino group of K20
The synthesis method comprises the following steps: SPPS_A; SC_B
Calculated molecular weight (average): 4864.4DaLCMS_A: rt=6.1 min; actual measurement [ M+3H] 3+ 1622.1，[M+4H] 4+ 1216.8。
Compound 61
Y-Aib-QGTFTSDYS-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (17-carboxyheptadecylamido) butan-2- [ [2- [ (4S) -4-carboxy-heptadecylamido ] amino ] butan-eAcyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group ]Acetyl group]-YLDKQAA-Aib-EFVNWLLAGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO 39, C-terminal amide
Substituents: chemical formula 6 linked via the epsilon-amino group of K12
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4836.4DaLCMS_A: rt=5.9 min; actual measurement [ M+3H] 3+ 1612.7，[M+4H] 4+ 1210.0。
Compound No. 62
Y-Aib-QGTFTSDYSI-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (17-carboxyheptadecylamido) butanoyl [ (2- [ (4S))]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-LDKQAA-Aib-EFVNWLLAGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 40, C-terminal amide
Substituents: chemical formula 6 linked via the epsilon-amino group of K13
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4786.3DaLCMS_A: rt=6.0 min; actual measurement [ M+3H] 3+ 1596.2，[M+4H] 4+ 1197.4。
Compound No. 63
Y-Aib-QGTFTSDYSIY-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (17-carboxyheptadecylamido) butanoyl [ (2- [ (4S))]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-DKQAA-Aib-EFVNWLLAGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 41, C-terminal amide
Substituents: chemical formula 6 linked via the epsilon-amino group of K14
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4836.4DaLCMS_A: rt=5.9 min; actual measurement [ M+3H] 3+ 1613.0，[M+4H] 4+ 1209.9。
Compound 64
Y-Aib-QGTFTSDYSIYLDKQAa-Aib-EFV-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (17-carboxyheptadecylamido) butanoyl ] ]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-WLLAGGPSSGAPPPS-NH 2
Peptide backbone: SEQ ID NO. 42, C-terminal amide
Substituents: chemical formula 6 linked via the epsilon-amino group of K24
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4835.4DaLCMS_A: rt=6.2 min; actual measurement [ M+3H] 3+ 1612.6，[M+4H] 4+ 1209.6。
Compound No. 65
Y-Aib-QGTFTSDYSIYLDKQAa-Aib-EFVNWLLA-K [2- [2- [ [ (4S) -4-carboxy-4- (17-carboxyheptadecanoylamino) butanoyl [ [2- [2- [2- [ [ (4S) -4-carboxy-heptadecanoylamino)]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-GPSSGAPPPS-NH 2
Peptide backbone: 43, C-terminal amide of SEQ ID NO
Substituents: chemical formula 6 via epsilon-amino linkage of K29
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4892.5DaLCMS_A: rt=5.8 min; actual measurement [ M+3H] 3+ 1631.5，[M+4H] 4+ 1223.9。
Compound 66
Y-Aib-QGTFTSDYSIYLDKQAa-Aib-EFVNWLLAGGPSSG-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (17-carboxyheptadecylamido) butanoyl ]]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-PPPS-NH 2
Peptide backbone: SEQ ID NO. 44, C-terminal amide
Substituents: chemical formula 6 linked via the epsilon-amino group of K35
The synthesis method comprises the following steps: SPPS_A; SC_A
Calculated molecular weight (average): 4878.4DaLCMS_A: rt=6.2 min; actual measurement [ M+3H ] 3+ 1626.8，[M+4H] 4+ 1220.5。
Example 2: in vitro functional efficacy (CRE luciferase; whole cell)
The purpose of this example is to test compounds for their functional activity or potency at human and mouse GLP-1, GIP and Gcg receptors in vitro. In vitro functional efficacy is a measure of target receptor activation in whole cell assays. The efficacy of the compounds listed in example 1 was determined as follows. Human GLP-1 (7-37) (SEQ ID NO: 35) (identical to mouse GLP-1 (7-37)), human GIP (SEQ ID NO: 36) and human glucagon (SEQ ID NO: 37) (identical to mouse glucagon) were included in the appropriate assays for comparison.
Principle of
In vitro functional efficacy was determined by measuring the response of target receptors in a reporter gene assay in a separate cell line. The assay was performed in a stably transfected BHK cell line expressing one of the following G protein-coupled receptors: human GLP-1 receptor, human GIP receptor, human Gcg receptor, mouse GLP-1 receptor, mouse GIP receptor or mouse Gcg receptor; and wherein each cell line contains CAMP Response Element (CRE) DNA coupled to a promoter and a firefly luciferase (CRE luciferase) gene. When the respective receptor is activated, it leads to the production of cAMP, which in turn leads to the expression of luciferase protein. When the assay incubation is complete, a luciferase substrate (luciferin) is added, resulting in enzymatic conversion of luciferin to oxidized luciferin and the generation of bioluminescence. The luminescence is measured as a readout of the assay.
Cell culture and preparation
The cell line used in these assays was BHK cells with BHKTS13 as the parent cell line. The cell lines are derived from clones containing CRE luciferase elements and are established by further transfection with the respective receptors to obtain the relevant cell lines. The following cell lines were used:
measurement Cell lines
GLP-1 receptor assay BHK CRE luc2P hGLP-1R
GIP receptor assay BHK CRE luc2P hGIPR
Glucagon receptor assay BHK CRE luc2P hGcgR
Cells were incubated in 5% CO in cell culture medium 2 Culturing at 37deg.C. Cells were aliquoted and stored in liquid nitrogen. The cells were maintained in continuous culture and immediately prior to each assayAnd (5) inoculating on a day.
Material
The following chemicals were used in this assay: pluronic F-68 10% (Gibco 2404), 10% fetal bovine serum (FBS; invitrogen 16140-071), egg white ovalbumin (Sigma A5503), phenol red free DMEM (Gibco 21063-029), DMEM (Gibco 12430-054), 1M Hepes (Gibco 15630), glutamax100x (Gibco 35050), G418 (Invitrogen 10131-027), hygromycin (Invitrogen 10687-010) and steadinite plus (Perkinelmer 6016757).
Buffer solution
GLP-1R and GcgR cell culture media consisted of DMEM medium containing 10% FBS, 500 μg/mL G418 and 300 μg/mL hygromycin. The GIPR cell culture medium consisted of DMEM medium containing 10% FBS, 400. Mu.g/mL G418 and 300. Mu.g/mL hygromycin. The assay buffer consisted of phenol red free DMEM, 10mM Hepes, 1 XGlutamax, 1% ovalbumin and 0.1% Pluronic F-68. The assay buffer is mixed with an equal volume of test compound 1:1 in the assay buffer to give the final assay concentration.
Procedure
1) Cells were plated at 5000 cells/well and incubated overnight in assay plates.
2) Cells were washed once in DPBS.
3) Stock solutions of test compound and reference compound at concentrations ranging from 100 to 300 μm were diluted 1:150 in assay buffer. The compounds were then diluted 1:10 in column 1 of a 96 deep well dilution plate, and then starting from this row, a 3.5-fold, 12-point dilution curve was generated.
4) Assay buffer (50 μl aliquots) was added to each well of the assay plate.
5) An aliquot of 50 μl of compound or blank was transferred from the dilution plate to the assay plate containing assay buffer.
6) The assay plate was incubated at 5% CO 2 Incubate in incubator at 37℃for 3h.
7) Cells were washed once with DPBS.
8) To each well of the assay plate, 100 μl aliquots of DPBS were added.
9) To each well of the assay plate, 100. Mu.l aliquots of the stearyleite plus reagent (photosensitive) were added.
10 Each assay plate was covered with aluminum foil to avoid light and shaken at 250RPM for 30min at room temperature.
11 Reading each assay plate in a microtiter plate reader.
Calculation and results
Data from the microtiter plate reader were first regressed in Excel to calculate x-axis, logarithmic scale concentrations from the stock concentrations of the individual test compounds and the dilutions of the assay. The data was then transferred to GraphPad Prism software for mapping and statistical analysis. The software performs nonlinear regression (log (agonist) versus response). EC calculated with this software and reported in pM 50 The values are shown in tables 1 and 2 below. A minimum of two replicates were measured for each sample. The reported values are the average of the repeated measurements.
Table 1: triple agonists altering the position of the substituents were functionally potent against human GLP-1R, GIPR and GcgR.
nd=not determined.
Compound 58 is a known (US 9062124 example 18, seq ID NO 124) and potent triple agonist with a hexadecyl moiety linked to the epsilon-amino group of Lys 40. Replacement of the substituent at Lys40 with substituent chemical formula 6 (i.e., compound 59) is expected to yield an equivalent compound with a longer half-life. However, the above results indicate that compound 59 has lower potency against all three receptors, especially for GIPR and GcgR. Thus, a change in substituents from fatty mono-acids to fatty di-acids does not achieve similar efficacy. The results in table 1 show that substitution of lysine epsilon-amine with a fatty diacid-based substituent is preferred when lysine is at position 16, 17 or 21 of the peptide backbone, while a significant decrease in potency at one or more receptors is observed when C18 diacid-based substituent chemical formula 6 is at any of positions 12, 13, 14, 20, 24, 29, 35 or 40. Table 2. Functional efficacy of triple agonists of the present invention (wherein the substituents are present at positions 16, 17 or 21) against human GLP-1R, GIPR and GcgR.
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nd=not determined.
The results in table 2 show that the compounds of the invention exhibit potent functional activation of human GLP-1R, human GIPR and human GcgR, obtained by binding the peptide backbone to substituents at positions 16, 17 or 21.
Example 3: pharmacokinetic studies in minipigs
The purpose of this example is to determine the in vivo half-life of the triple agonists described herein after intravenous administration to minipigs, i.e. their prolongation of time in vivo, and hence of their duration of action. This was done in Pharmacokinetic (PK) studies that determine the terminal half-life of the triple agonist in question. The terminal half-life is generally the length of time it takes to halve a certain plasma concentration, measured after an initial distribution phase.
Study of
Females used in the studyPig from Ellgaard +.>Minitis (Dalmose, denmark) at about 7-14 months of age and weighing about 16-35kg. Mini-pigs were housed individually and were restricted to feeding SDS mini-pig diet once daily (Special Diets Services, essex, UK).
After 3 weeks of acclimation, two permanent central venous catheters were implanted into the vena cava caudalis of each animal. Animals were allowed to recover for 1 week after surgery and then used for repeated pharmacokinetic studies with appropriate washout periods between successive administrations.
Animals were fasted for approximately 18 hours prior to dosing and 0 to 4 hours post dosing, but were given ad libitum throughout the time period.
The sodium salt of the triple agonist of example 1 was dissolved to a concentration of 20-40nmol/mL in pH 7.4 buffer containing 0.025% (v/v) polysorbate 20, 10mM sodium phosphate, 250mM glycerol. The triple agonist is injected intravenously through one catheter (the volume typically corresponds to 1.5-2nmol/kg, e.g. 0.1 mL/kg) and blood samples are collected at predetermined time points for up to 14 days after administration (preferably through another catheter). Blood samples (e.g., 0.8 mL) were collected in 8mM EDTA buffer and then centrifuged at 1942g for 10 minutes at 4 ℃.
Sampling and analysis
Plasma was pipetted into a micro tube on dry ice and kept at-20 ℃ until the plasma concentration of the triple agonist was analyzed using ELISA or similar antibody-based assay or LCMS. Each plasma concentration-time curve was analyzed by a non-compartmental model in Phoenix WinNonLin ver.6.4 (Pharsight inc., mountain View, CA, USA) and the resulting terminal half-life (harmonic mean) was determined.
Results
Table 3: terminal half-life measured after intravenous administration in minipigs
The triple agonists described herein have a very long half-life compared to the natural hormone tested. The half-lives of hGLP-1 and hGIP, measured in humans, are reported to be about 2-4min and 5-7min, respectively (Meier et al, diabetes,2004,53 (3): 654-662). Glucagon has been reported to have a half-life of between 5 and 30 minutes, depending on the route of administration (Pontirili et al Eur J Clin Pharmacol 1993, 45:555-558).
Example 4: pharmacodynamic studies in diet-induced obese (DIO) mice
The purpose of this example was to evaluate the in vivo effect of selected triple agonists on pharmacodynamic parameters in Diet Induced Obese (DIO) mice. Animals were treated once daily by subcutaneous injection of a liquid formulation of the triple agonist to be tested to assess the effects on body weight, food intake and glucose tolerance.
Animal and diet
C57BL/6J male mice were purchased from Jackson Laboratories, approximately 8 weeks old. Mice were housed in groups and fed a high fat, high sugar diet from Research Diets (D12331). Mice were maintained on this diet for 12 weeks prior to starting the pharmacological study. Mice weighing more than 50 grams were considered to have Diet Induced Obesity (DIO) and were included in pharmacological studies. Mice were exposed to a controlled 12h:12h light-dark cycle at ambient room temperature (22 ℃) and food and water were obtained ad libitum. The study was approved by the institutional animal care and use committee of cincinnati university and was conducted according to its guidelines.
Administration and formulation
All compounds in this study were formulated in the following buffers: 50mM phosphate; 70mM sodium chloride; 0.05% Tween 80, pH 7.4. The dosing solution was formulated in glass vials and stored at 2-8 ℃. The dosing solution was warmed to room temperature prior to dosing and returned to 2-8 ℃ after dosing.
DIO mice were grouped (n=8 per group) to minimize statistical differences in mean and standard deviation of fat mass and body weight between groups. Animals were grouped to receive the following treatments: a vehicle or GLP-1/GIP/Gcg receptor triple agonist as described herein, wherein the vehicle is 50mM phosphate, 70mM sodium chloride; 0.05% (v/v) Tween-80, pH 7.4. The test compound is dissolved in vehicle to a stock concentration of 100 μm and then diluted 50 to 200 times in vehicle to achieve the desired dosing solution concentration. For each treatment day, animals are given subcutaneously once a day in the morning, with a volume of dosing solution of 2-5 μl per gram of body weight as necessary to achieve the desired dose (e.g. 0.3nmol/kg, 1.0nmol/kg or 3.0 nmol/kg).
Body weight and food intake
Body Weight (BW) and food intake were measured daily immediately prior to dosing. The percent change in body weight of each mouse was calculated separately based on the initial body weight prior to the first injection.
IPGTT (intraperitoneal glucose tolerance test)
On the day of glucose tolerance test, animals were fasted for 4h. The food was removed and the animals transferred to new cages. Animals can drink water but cannot eat food. Tail blood glucose levels were measured and mice were injected (t=0) with an intraperitoneal (i.p.) glucose load of 2g/kg (200 mg/ml glucose solution, dose volume 10 ml/kg). Tail blood glucose levels were measured 0, 15, 30, 60, 90, 120 minutes after intraperitoneal injection of glucose load. Animals stratified during IPGTT such that, for example, two mice from group 1 are dosed, followed by two mice from groups 2, 3, 4, followed by treatment of the next two mice from groups 1, 2, 3, etc. This allows the "time of day" to be equally allocated among all groups.
Results:
in one study DIO mice received a subcutaneous dose of one of the following for up to 60 days per day: vehicle, compound 21, compound 30 or compound 33. Based on the titration schedule included in table 4, the dose of each compound was titrated upward to maximum efficacy. Mice that reached weight normalization (defined as body weight of 22.5g or less) were automatically removed from the study. The results are shown in Table 5 and FIGS. 1-2. All triple agonist compounds tested resulted in significantly greater weight and food intake reductions than vehicle. At a time point prior to day 42 (see fig. 1), treatment with compound No. 21 resulted in similar food intake reduction but less weight loss compared to compound No. 30 and compound No. 33. This can be explained by the fact that: when studied using the protocol described in example 2, except using mouse-specific receptors, compound No. 21 has similar GLP-1R and GIPR in vitro potency, but reduced GcgR in vitro potency, compared to compound No. 30 and compound No. 33. In vitro potency against mouse GLP-1R, mouse GIPR and mouse GcgR are shown in Table 6.
Table 4: dose titration schedule for DIO mouse study described in table 5.
Table 5: effects on food intake and body weight in DIO mice treated with vehicle or GLP-1/GIP/Gcg triple agonist for up to 60 days per day according to the dose titration schedule shown in table 4. Also included was the last day any mice received treatment in the group before being removed from the study.
Results are expressed as mean ± SEM, n=1-2 (food intake) or n=5-8 (body weight). Table 6: the selected triple agonists of the invention were directed against the functional efficacy of mouse specific GLP-1R, GIPR and GcgR.
nd=not determined.
Another mouse study with compounds 26, 43 and 44 showed that DIO mice had better food intake reduction and weight loss than vehicle after daily subcutaneous administration over 13 days. The results are shown in table 7. Compound 44 showed a dose-dependent effect on food intake reduction and weight loss and improved glucose tolerance compared to vehicle at doses of 1.0nmol/kg and 3.0 nmol/kg. This suggests that triple agonists may increase weight loss efficacy without adversely affecting glucose tolerance.
Table 7: effects on food intake, body weight and glucose tolerance in DIO mice treated daily with indicated doses of vehicle or GLP-1/GIP/Gcg triple agonist.
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Results are expressed as mean ± SEM, n=2 (food intake) or n=7-8 (body weight, IPGTT). iAUC = area under the curve minus baseline. nd=not determined.
While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those skilled in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true scope of the invention.

Claims (15)

1. A GLP-1/GIP/Gcg receptor triple agonist comprising a peptide and a substituent, wherein the amino acid sequence of the peptide is:
X 1 X 2 X 3 GTFTSDYSX 12 X 13 LX 15 KX 17 X 18 AX 20 X 21 FVX 24 WLLX 28 GGPSSGAPPPS (SEQ ID NO: 45), wherein
X 1 Is H or Y
X 2 Is Aib
X 3 Is H or Q
X 12 K, I or Y
X 13 Is Y or L
X 15 Is D or E
X 17 Q, R or K
X 18 A, Y or K
X 20 Aib, Q, E, R or H
X 21 Is E or K
X 24 Is N or Q
X 28 Is A or E;
wherein the peptide has a C-terminal acid or amide; and is also provided with
Wherein the substituent is covalently linked to an epsilon amino group of K at a position selected from the group consisting of positions 16, 17 and 21;
or a pharmaceutically acceptable salt thereof.
2. The GLP-1/GIP/Gcg receptor triple agonist according to claim 1, wherein the substituent is covalently linked to the epsilon amino group of K at position 16.
3. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding claims, wherein X 1 X 2 X 3 Selected from HAibH and YAibQ.
4. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding claims, wherein SX 12 X 13 LX 15 Selected from SYYLE (SEQ ID NO: 54), SILLE (SEQ ID NO: 55), SYLLE (SEQ ID NO: 56), SIYLE (SEQ ID NO: 57) and SIYLD (SEQ ID NO: 58).
5. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding claims, wherein KX 17 X 18 AX 20 Selected from KQAAAib (SEQ ID NO: 59), KKAAAAib (SEQ ID NO: 60), KQYAAib (SEQ ID NO: 61) and KKYAAib (SEQ ID NO: 62).
6. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding claims, wherein the amino acid sequence of the peptide is:
HX 2 HGTFTSDYSX 12 X 13 LX 15 KX 17 X 18 AX 20 EFVX 24 WLLX 28 GGPSSGAPPPS (SEQ ID NO: 47), wherein
X 2 Is Aib
X 12 Is I or Y
X 13 Is Y or L
X 15 Is D or E
X 17 Is Q or K
X 18 Is A or Y
X 20 Is Aib
X 24 Is N or Q
X 28 Is A or E.
7. The GLP-1/GIP/Gcg receptor triple agonist according to any one of claims 1-5, wherein the amino acid sequence of the peptide is:
YX 2 QGTFTSDYSX 12 X 13 LX 15 KX 17 X 18 AX 20 EFVX 24 WLLX 28 GGPSSGAPPPS (SEQ ID NO: 50), wherein
X 2 Is Aib
X 12 Is I or Y
X 13 Is Y or L
X 15 Is D or E
X 17 Is Q or K
X 18 Is A or Y
X 20 Is Aib
X 24 Is N or Q
X 28 Is A or E.
8. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding claims, wherein the substituent is a-B-C-, wherein
A-is chemical formula 1
Chemical formula 1: HOOC- (CH) 2 ) p -CO-*
p is an integer in the range of 16-20;
b-is selected from chemical formula 2 or chemical formula 3:
chemical formula 2: * -NH-CH (COOH) - (CH) 2 ) 2 -CO-*
Chemical formula 3: * -NH-CH 2 -(C 6 H 10 )-CO-NH-CH(COOH)-(CH 2 ) 2 -CO-; and is also provided with
C-is selected from formula 4 or formula 5:
chemical formula 4: * - [ NH- ((CH) 2 ) 2 -O) 2 -CH 2 -CO] 2 -*
Chemical formula 5: * - [ NH- (CH) 2 ) 4 -CH(NH 2 )-CO] 2 -*
Wherein represents the point of attachment and A, B and C are attached to each other by an amide bond in the order shown.
9. The GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding claims, wherein the substituents a-B-C-are selected from the group consisting of:
chemical formula 6:；
chemical formula 7:；
chemical formula 8:；
chemical formula 9:；
chemical formula 10:
chemical formula 11:
chemical formula 12:
and chemical formula 13:
10. the GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding claims, wherein the triple agonist is selected from the group consisting of:
Y-Aib-QGTFTSDYSIYLD-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (17-carboxyheptadecylamido) butanoyl [ (2- [ (4S))]Amino group]Ethoxy group]Ethoxy group ]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-QAA-Aib-EFVNWLLAGGPSSGAPPPS-NH 2 (compound No. 1):
；
Y-Aib-QGTFTSDYSIYLDK-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (17-carboxyheptadecylamido) butanoyl [ (2- [ (4S))]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-AA-Aib-EFVNWLLAGGPSSGAPPPS-NH 2 (compound No. 2):
；
Y-Aib-QGTFTSDYSIYLDKQAa-Aib-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (17-carboxyheptadecylamino) butanoyl [ [2- [2- [ (4S) -4-carboxy-heptadecylamino ] butanoyl ]]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-FVNWLLAGGPSSGAPPPS-NH 2 (compound No. 3):
Y-Aib-QGTFTSDYSIYLDKQAa-Aib-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (17-carboxyheptadecylamino) butanoyl [ [2- [2- [ (4S) -4-carboxy-heptadecylamino ] butanoyl ]]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-FVQWLLEGGPSSGAPPPS-NH 2 (compound No. 4):
Y-Aib-QGTFTSDYSIYLD-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (19-carboxynona-carbo-noylamino) butanoyl ]]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-QAA-Aib-EFVNWLLAGGPSSGAPPPS-NH 2 (compound No. 5):
Y-Aib-QGTFTSDYSIYLD-K [2- [2- [2- [ [ (4S) -4-carboxy-4- [ [4- [ (19-carboxy-nineteen carbonylamino) methyl ] 2- [ [2- [ (4S) -4- ] methyl ]]Cyclohexane carbonyl]Amino group]Butyryl group]Amino group]Ethoxy group]Ethoxy group ]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-QAA-Aib-EFVNWLLAGGPSSGAPPPS-NH 2 (compound No. 6):
Y-Aib-QGTFTSDYSIYLDKQAa-Aib-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (19-carboxynineteen carbonylamino) butanoyl ]]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-FVQWLLEGGPSSGAPPPS-NH 2 (compound No. 7):
；
Y-Aib-QGTFTSDYSIYLDKQAa-Aib-K [2- [2- [2- [ [ (4S) -4-carboxy-4- [ [4- [ (19-carboxynonacarbonamido) methyl ] 2- [2- [ [ [ (4S) -4-carboxy-4- [ [ amino ] methyl ] carbonyl ] amino ] carbonyl group]Cyclohexane carbonyl]Amino group]Butyryl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-FVQWLLEGGPSSGAPPPS-NH 2 (compound No. 8):
；
Y-Aib-QGTFTSDYSIYLDK-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (19-carboxynona-carbo-noylamino) butanoyl ]]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-AA-Aib-EFVNWLLAGGPSSGAPPPS-NH 2 (compound No. 9):
；
Y-Aib-QGTFTSDYSIYLDK-K [2- [2- [2- [ [ (4S) -4-carboxy-4- [ [4- [ (19-carboxy-nineteen carbonylamino) methyl ] 2- [ [2- [ (4S) -4- ] methyl ]]Cyclohexane carbonyl]Amino group]Butyryl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-AA-Aib-EFVNWLLAGGPSSGAPPPS-NH 2 (compound No. 10):
；
Y-Aib-QGTFTSDYSIYLEKQAa-Aib-K [2- [2- [2- [ [ (4S) -4-carboxy-4- [ [4- [ (19-carboxynonacarbonamido) methyl ] 2- [2- [ [ (4S) -4-carboxy-4- [ [ amino ] methyl ] carbonyl ] amino ] methyl ]Cyclohexane carbonyl]Amino group]Butyryl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-FVQWLLEGGPSSGAPPPS-NH 2 (compound No. 11):
Y-Aib-QGTFTSDYSIYLE-K [2- [2- [2- [ [ (4S) -4-carboxy-4- [ [4- [ (19-carboxy-nineteen carbonylamino) methyl ] 2- [ [2- [ (4S) -4- ] methyl ]]Cyclohexane carbonyl]Amino group]Butyryl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-QAA-Aib-EFVNWLLAGGPSSGAPPPS-NH 2 (compound No. 12):
；
Y-Aib-QGTFTSDYSIYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- [ [4- [ (19-carboxynona-carbo-noylamino) methyl)]Cyclohexane carbonyl]Amino group]Butyryl group]Amino group]Caproyl group]Amino group]Caproyl group]-QAA-Aib-EFVNWLLAGGPSSGAPPPS-NH 2 (compound No. 13):
；
Y-Aib-QGTFTSDYSIYLE-K [2- [2- [2- [ [ (4S) -4-carboxy-4- [ [4- [ (19-carboxy-nineteen carbonylamino) methyl ] 2- [ [2- [ (4S) -4- ] methyl ]]Cyclohexane carbonyl]Amino group]Butyryl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2 (compound No. 14):
；
Y-Aib-QGTFTSDYSIYLE-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (19-carboxynona-carbo-noylamino) butanoyl ]]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-QAA-Aib-EFVNWLLAGGPSSGAPPPS-NH 2 (compound No. 15):
；
Y-Aib-QGTFTSDYSIYLE-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (19-carboxynona-carbo-noylamino) butanoyl ] ]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2 (compound No. 16):
；
Y-Aib-QGTFTSDYSIYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2 (compound No. 17):
；
Y-Aib-HGTFTSDYSIYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl-sBase group]Amino group]Caproyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2 (compound No. 18):
；
H-Aib-QGTFTSDYSIYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2 (compound No. 19):
Y-Aib-QGTFTSDYSIYLE-K [2- [2- [ [ (4S) -4-carboxy-4- [ [4- [ (19-carboxynonacarbonamido) methyl ] cyclohexanecarbonyl ] amino ] butyryl ] amino ] ethoxy ] acetyl ] - ]
QAAQEFVQWLLEGGPSSGAPPPS-NH 2 (compound No. 20):
H-Aib-HGTFTSDYSIYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- [ [4- [ (19-carboxynona-carbo-noylamino) methyl)]Cyclohexane carbonyl]Amino group]Butyryl group]Amino group]Caproyl group]Amino group ]Caproyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2 (compound No. 21):
；
H-Aib-HGTFTSDYSIYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2 (compound No. 22):
；
H-Aib-HGTFTSDYSIYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- [ [4- [ (19-carboxynona-carbo-noylamino) methyl)]Cyclohexane carbonyl]Amino group]Butyryl group]Amino group]Caproyl group]Amino group]Caproyl group]-QAAQEFVQWLLEGGPSSGAPPPS-NH 2 (compound No. 23):
H-Aib-HGTFTSDYSIYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-QYA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2 (compound No. 24):
H-Aib-HGTFTSDYSYYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- [ [4- [ (19-carboxynona-carbo-noylamino) methyl)]Cyclohexane carbonyl]Amino group]Butyryl group]Amino group]Caproyl group]Amino group]Caproyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2 (compound No. 25):
H-Aib-HGTFTSDYSYYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2 (compound No. 26):
H-Aib-HGTFTSDYSIYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl ]Amino group]Caproyl group]Amino group]Caproyl group]-QAAEEFVQWLLEGGPSSGAPPPS-NH 2 (compound No. 27):
H-Aib-HGTFTSDYSIYLD-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2 (compound No. 28):
；
H-Aib-HGTFTSDYSYYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-QYA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2 (compound No. 29):
；
H-Aib-HGTFTSDYSIYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-KYA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2 (compound No. 30):
；
H-Aib-HGTFTSDYSYYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-KAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2 (compound No. 31):
；
H-Aib-HGTFTSDYSYYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (17-carboxyheptadecylamido) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2 (compound No. 32):
；
H-Aib-HGTFTSDYSIYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl ]Amino group]Caproyl group]Amino group]Caproyl group]-KAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2 (compound No. 33):
；
H-Aib-HGTFTSDYSIYLE-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (19-carboxynona-carbo-noylamino) butanoyl ]]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-KAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2 (compound No. 34):
；
H-Aib-HGTFTSDYSIYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-QKA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2 (compound No. 35):
；
H-Aib-HGTFTSDYSKYLE-K [ (2S) -2-amino-6-)[ [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynona-carbonylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2 (compound No. 36):
；
H-Aib-HGTFTSDYSIYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-RAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2 (compound No. 37):
；/>
H-Aib-HGTFTSDYSIYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (17-carboxyheptadecylamido) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-KYA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2 (compound No. 38):
；
H-Aib-HGTFTSDYSIYLE-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (19-carboxynona-carbo-noylamino) butanoyl ] ]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-KYA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2 (compound 39):
；
H-Aib-HGTFTSDYSYYLE-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (19-carboxynona-carbo-noylamino) butanoyl ]]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2 (compound No. 40):
；
H-Aib-HGTFTSDYSILLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-KYA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2 (compound No. 41):
；
H-Aib-HGTFTSDYSYLLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2 (compound No. 42):
；
H-Aib-HGTFTSDYSILLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2 (compound No. 43):
；
H-Aib-HGTFTSDYSIYLE-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (19-carboxynona-carbo-noylamino) butanoyl ]]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2 (compound 44):
；
H-Aib-HGTFTSDYSILLE-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (19-carboxyten) Nine-carbonyl amino) butyryl]Amino group]Ethoxy group]Ethoxy group]Acetyl group]Amino group]Ethoxy group]Ethoxy group]Acetyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2 (compound No. 45):
；
Y-Aib-QGTFTSDYSILLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2 (compound No. 46):
；/>
Y-Aib-QGTFTSDYSYYLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2 (compound No. 47):
Y-Aib-QGTFTSDYSYLLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-QAA-Aib-EFVQWLLEGGPSSGAPPPS-NH 2 (compound 48):
H-Aib-HGTFTSDYSILLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl]Amino group]Caproyl group]Amino group]Caproyl group]-QAAEEFVQWLLEGGPSSGAPPPS-NH 2 (compound No. 49):
H-Aib-HGTFTSDYSILLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonacarbonamido) butanoyl ] amino ] hexanoyl ] -QAAEEFVQWLLEGGPSSGAPPPS-OH (Compound No. 50):
H-Aib-HGTFTSDYSILLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (17-carboxyheptadecanoylamino) butanoyl ] amino ] hexanoyl ] -QAAEEFVQWLLEGGPSSGAPPPS-OH (Compound 51):
H-Aib-HGTFTSDYSILLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (17-carboxyheptadecanoylamino) butanoyl ] amino ] hexanoyl ] -QAAQEFVQWLLEGGPSSGAPPPS-OH (Compound No. 52):
H-Aib-HGTFTSDYSILLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonacarbonamido) butanoyl ] amino ] hexanoyl ] -QAAQEFVQWLLEGGPSSGAPPPS-OH (Compound No. 53):
H-Aib-HGTFTSDYSILLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (17-carboxyheptadecanoylamino) butanoyl ] amino ] hexanoyl ] -QAAREFVQWLLEGGPSSGAPPPS-OH (Compound No. 54):
H-Aib-HGTFTSDYSILLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonacarbonamido) butanoyl ] amino ] hexanoyl ] -QAAREFVQWLLEGGPSSGAPPPS-OH (Compound No. 55):
H-Aib-HGTFTSDYSILLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (17-carboxyheptadecanoylamino) butanoyl ] amino ] hexanoyl ] -QAAHEFVQWLLEGGPSSGAPPPS-OH (Compound 56):
H-Aib-HGTFTSDYSILLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (19-carboxynonacarbonamido) butanoyl ] amino ] hexanoyl ] -QAAHEFVQWLLEGGPSSGAPPPS-OH (Compound No. 57):
11. A pharmaceutical composition comprising a GLP-1/GIP/Gcg receptor triple agonist according to any one of the preceding claims, and optionally, at least one pharmaceutically acceptable excipient.
12. The GLP-1/GIP/Gcg receptor triple agonist according to any one of claims 1-10 for use as a medicament.
13. The GLP-1/GIP/Gcg receptor triple agonist according to any one of claims 1-10 for use in weight management, treatment and/or prevention of obesity and obesity related disorders.
14. The GLP-1/GIP/Gcg receptor triple agonist according to any one of claims 1-10 for use in the treatment and/or prevention of all forms of diabetes, such as type 2 diabetes, and diabetes related disorders.
15. The GLP-1/GIP/Gcg receptor triple agonist according to any one of claims 1-10 for use in the treatment and/or prevention of liver disorders such as liver steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver inflammation or fatty liver.
CN202180073945.4A 2020-10-30 2021-10-29 GLP-1, GIP and glucagon receptor triple agonists Pending CN116457002A (en)

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US63/107,622 2020-10-30
EP20213053.0 2020-12-10
PCT/EP2021/080089 WO2022090447A1 (en) 2020-10-30 2021-10-29 Glp-1, gip and glucagon receptor triple agonists

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