WO2015149627A1 - Structurally modified glp-1 analogue and preparation method therefor - Google Patents

Abstract

The present invention provides a structurally modified GLP-1 analogue and a preparation method therefor, and specifically provides a derivative with a C end of Exenatide structurally modified and a preparation method therefor, as well as pharmaceutical compositions comprising same and uses thereof in treating diseases such as type II diabetes mellitus.

Description

Structurally modified GLP-1 analogue and preparation method thereof Technical field

The present invention relates to the field of therapeutic peptides, in particular to structurally modified GLP-1 analogs and methods for their preparation, and further to structurally modified exenatide derivatives, and to exenatide-derived structurally modified exenatide The preparation method of the substance.

Background technique

Exenatide (also known as exenatide or exenatide, Exenatide, or Exendin-4, trade name Byetta) is a polypeptide consisting of 39 amino acids with a molecular weight of 4186.6 and a molecular formula of C ₁₈₄ H ₂₈₂ N ₅₀ O ₆₀ S, CAS Registry Number is 141758-74-9, amino acid sequence is: His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu- Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ ; Produced and sold by Amylin Pharmaceuticals and Eli Lilly Company (Eli Lillyand Company). Exenatide has been approved by the FDA in April 2005. It is a subcutaneous injection preparation that promotes glucose-dependent insulin secretion, restores first-phase insulin secretion, inhibits glucagon secretion, and slows gastric contents. The emptying, improving the function of pancreatic beta cells, is very suitable for the treatment of type 2 diabetes, for example, to improve and control the blood glucose of patients with type 2 diabetes who are not ideal for metformin and sulfonylureas.

Exenatide is a synthetic form of the hormone exendin-4 in the saliva of the lizard Gilamonster grown in several states in the southwestern United States (J. Biol. Chem. 1990, 265, 20259-20262; J. Biol. .1992, 267, 7402-7405), is an analog of human glucagon-like peptide-1 (GLP-1), whose amino acid sequence partially overlaps with the amino acid sequence of GLP-1, and is a relatively effective GLP- A receptor agonist, also known as an incretin agonist, because exenatide mimics the glucose-regulating action of GLP-1. Unlike sulfonylureas and meglitinides, exenatide increases insulin synthesis and secretion only in the presence of glucose, reducing the risk of hypoglycemia. Some physicians also use Byetta for the treatment of insulin resistance.

Despite this, due to the ubiquitous half-life of the protein/peptide drugs, the poor physical and chemical stability, and the degradation of various proteases in the body, these drugs usually need to be injected multiple times a day. It brings a lot of pain and a lot of inconvenience. The PEGylation technology that emerged in the 1970s proved to be a more suitable technique in the field of protein/peptide delivery. However, the activity of the drug is generally reduced after the modification with PEG alone.

A range of different methods have been used to modify the structure of GLP-1 analogs to provide longer duration of action in vivo. Chinese Patent No. CN00804847.9 discloses a novel exendin agonist preparation and a method of administration thereof, in which a compound structure of exenatide and a preparation method thereof are disclosed. Chinese patent CN102532303 discloses the use of a methoxypolyethylene glycol residue conjugated to the amino group of the lysine residue in the exenatide molecule or the amino group of the N-terminal histidine residue to synthesize polyethylene glycol conjugated The method of exenatide; CN200980111088 also explicitly discloses the structure of fatty acid-PEG-exenatide, and the modification site of PEG is on the N-terminal His; WO2005028516 and WO2012035139 also disclose fatty acid-PEG-exenatide structure. Chinese patent CN101215324B discloses an exenatide short peptide mimetic peptide obtained by structurally modifying exenatide. Chinese patent CN101125207 reports PEG modification of Exendin-4. WO 99/43708 discloses GLP-1 (7-35) and GLP-1 (7-36) derivatives having a lipophilic substituent linked to a C-terminal amino acid residue. WO2013059323 A1 discloses PEG-conjugated exenatide and a process for the preparation thereof. Despite these various efforts, the diabetic population still has a huge demand for long-acting active GLP-1 analogs.

The stability of GLP-1 analogues, including stability in vivo, remains a major issue to be addressed. Therefore, despite advances in the field, the current market for Exendin-4 and some modified Exendin-4 drugs are short-lived, and there is still an urgent need to develop new exenatide derivatives, which have long-lasting action and good stability in vivo. The hypoglycemic effect is good while maintaining low toxicity and good activity.

Summary of the invention

The object of the present invention is to overcome the shortcomings of the prior art, to provide exenatide derivatives which have long half-life, good stability, and can simultaneously maintain good hypoglycemic effect and low toxicity, and at the same time, the present invention also provides exenatide derivatives. Methods of preparation, and the use of these derivatives in the manufacture of a medicament for the treatment and/or prevention of diabetes.

In order to achieve the above object, the present invention adopts the following technical solutions:

The present invention provides a compound having the structure: Compound: D-A-P-L

Wherein D is a GLP-1 analog moiety;

A is of the following structure: -NR ₁ -X-NR ₂ -,

Wherein R ₁ and R _{2 are} independently selected from the group consisting of H and C _1-6 alkyl;

X is selected from:

(1) -(CH ₂ ) _n -, wherein n is an integer from 2 to 10;

(2) -[(CH ₂ ) _n Z] _p (CH ₂ ) _n -, wherein Z is O, NR ₃ or S, n is independently selected from an integer from 1 to 10, and p is an integer from 0 to 10, wherein R ₃ is H or a C _1-6 alkyl group;

(3) an amino acid residue or an amino acid residue substituted with one or more substituents selected from the group consisting of H, C _1-6 alkyl, carbonyl and -NR ₂ R ₃ , wherein R ₂ , R ₃ independently selected from the group consisting of H and C _1-6 alkyl;

(4)-(CH ₂ ) _n C(O)Z(CH ₂ ) _n -, wherein Z is O, a single bond or -NR ₃ , n is independently selected from an integer from 1 to 10, wherein R ₃ is H or a C _1-6 alkyl group; preferably Z is O or a single bond;

with

(5) an alkylene group of C _1-10 , and H in the alkylene group is substituted by one or more of the following substituents selected from the group consisting of:

a linear or branched alkyl group of C ₁ -C ₄ , halogen, NHR ₅ , CN, carboxyl, nitro and C ₁ -C ₄ alkyl alcohol; wherein R ₅ is H or C ₁ -C ₄ straight Chain or branched alkyl group;

P is a linking moiety containing an oligoethylene glycol, and L is a lipophilic linking moiety;

Further, the carbon terminal of the terminal amino acid residue of D is linked to NR _{1 of A} through an amide bond, NR _{2 of} A is linked to P via an amide bond, and P and L are linked by an amide bond.

Further, it is preferred that P in the compound is a moiety having the following structure:

The L has the following structure:

Where m is an integer from 1 to 20; k is an integer from 6 to 20; wavy lines

Indicates a linking site; further preferably, m is 2 or 5.

Further, the A has the following structure: -NR ₁ -X-NR ₂ -,

The X is -(CH ₂ ) _n -, wherein the -(CH ₂ ) _n - represents an alkylene structure; the compound DAPL has the following structure:

Wherein R ₁ and R _{2 are} independently selected from the group consisting of H and C _1-6 alkyl groups, preferably R ₁ and R ₂ are both H; preferably n is an integer of 2 to 6, more preferably n is 2 or 4; m is an integer of 1-20, and k is an integer of 6-20.

On the other hand, the A has the following structure: -NR ₁ -X-NR ₂ -,

The X is -[(CH ₂ ) _n Z] _p (CH ₂ ) _n -,

The compound D-A-P-L preferably has the following structure:

Preferably, Z is O or NR ₃ , n is independently selected from an integer of 1 to 10, p is an integer of 0 to 10, preferably p is an integer of 2 to 5, m is an integer of 1 to 20, and k is 6 to 20. An integer; wherein R ₃ is H or a C _1-6 alkyl group, preferably R ₃ is H; further, preferably Z is O or NH, and n is 2 or 4 and p is 3.

In another aspect, the A is a structure having: -NR ₁ -X-NR ₂ -, wherein the X is an amino acid residue or an amino acid residue substituted with one or more substituents, and the amino acid The residue may be substituted with any substituent, provided that the substituent facilitates attachment of NHR ₁ and NHR ₂ to the amino acid backbone; preferably the substituent is selected from the group consisting of H, C _1-6 alkylene, C= O, NH and an alkyleneamino group and binding thereof; the amino acid residue is a natural amino acid or a non-natural amino acid residue; exemplified by a glutamic acid residue, and the glutamic acid residue is NH, C _1-6 alkylene and C=O are substituted, and NHR ₁ and NHR _{2 are} attached to the amino acid residue backbone through these substituents, resulting in A having the following structure:

Wavy

Indicates a connection site;

In another aspect, the A is a structure having the following structure: -NR ₁ -X-NR ₂ -, wherein X is -(CH ₂ ) _n C(O)Z(CH ₂ ) _n -, preferably Z is O Or a single bond, n independently selected from an integer from 1 to 10, wherein R _{3 is} selected from the group consisting of H and C _1-6 alkyl; further preferably X has the structure: -CH ₂ CH ₂ -C(O)- CH ₂ CH ₂ - or -CH ₂ CH ₂ -C(O)-NH-CH ₂ CH ₂ -; preferably A has the structure -NR ₁ -CH ₂ CH ₂ -C(O)-CH ₂ CH ₂ -NR ₂ - or -NR ₁ -CH ₂ CH ₂ -C(O)-NH-CH ₂ CH ₂ -NR ₂ -;

Preferably, the D-A-P-L has a compound having the following structure:

Among them, Z is preferably O or a single bond, and R ₁ and R ₂ are H.

In another aspect, the A is a structure having the following structure: -NR ₁ -X-NR ₂ -, the X is a C _1-10 alkylene group, preferably the H in the alkylene group is one or Substituted by a plurality of substituents selected from the group consisting of:

A linear or branched alkyl group of C ₁ -C _4, NHR _5, carboxy and C ₁ -C ₄ alkyl alcohols; wherein, R ₅ is H or a straight-chain or branched-chain C ₁ -C ₄ alkyl group; and Further preferably, the substituent is

Also, R ₅ is H; preferably A has the following structure:

Further, the compound D-A-P-L has the following structure:

D of the present invention is a GLP-1 analog moiety;

Further GLP-1 analogs are preferably: GLP-1 (7-35), GLP-1 (7-36), GLP-1 (7-37), GLP-1 (7-38), GLP-1 (7) -39), GLP-1 (7-40), GLP-1 (7-41), GLP-1 (7-42), GLP-1 (7-43), GLP-1 (7-44), GLP -1 (7-45), GLP-1 (7-46) or an analogue thereof; still more preferably the GLP-1 analogue is a peptide comprising the following amino acid sequence:

aa7-aa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-aa16-Ser-aa18-aa19-aa20-Glu-aa22-aa23-Ala-aa25-aa26-aa27-Phe-Ile-aa30-Trp- Leu-aa33-aa34-aa35-aa36-aa37-aa38-aa39-aa40-aa41-aa42-aa43-aa44-aa45-aa46;

Wherein, aa7 is L-His, D-His, 2-aminohistidine, β-histidine or α-methylhistidine;

Aa8 is Ala, Gly, Val, Leu, Ile, Lys, Aib, 1-aminocyclopropylcarboxylic acid, 1-aminocyclobutylcarboxylic acid, 1-aminocyclopentylcarboxylic acid or 1-aminocycloheptylcarboxylate Acid; aa16 is Tyr, Trp, Val or Leu; aa18 is Tyr, Ser, Lys or Arg; aa19 is Trp, Tyr or Gln; aa20 is Leu or Met; aa22 is Lys, Arg, His, Gly, Glu or Aib; Aa23 is Gln, Glu, Lys or Arg; aa25 is Ala or Val; aa26 is Lys, Glu or Arg; aa27 is Glu or Leu; aa30 is Ala, Glu or Arg; aa33 is Ile, Val or Lys; Lys, Asn, Glu or Arg; aa35 is Gly or Aib; aa36 is Lys, Gly or Arg; aa37 is His, Gly, Ala, Glu, Pro, Lys, amino or absent; aa38 is Lys, Ser, amino or not Exist; aa39 is Ser, Lys, amino or absent; aa40 is Gly, amino or absent; aa41 is Ala, amino or absent; aa42 is Pro amino or absent; aa43 is Pro amino or absent; aa44 is Pro Amino or absent; aa45 is Ser, amino or absent; aa46 is amino or absent, and when aa37, aa38, aa39, aa40aa41, aa42, aa43, aa44, aa45 or aa46 are not present, the corresponding Amino acids not exist.

Further, preferably, the GLP-1 analogue is selected from the group consisting of: Arg ³⁴ GLP-1 (7-37), Lys ³⁸ Arg ^{26, 34} GLP-1 (7-38), Lys ³⁸ Arg ^{26, 34} GLP-1 (7- ^{37) -OH, Lys 36 Arg 26,34} GLP-1 (7-36), Aib 8,22,35 GLP-1 (7-37), Aib 8,35 GLP-1 (7-37), Aib 8 ^{, 22 GLP-1 (7-37)} , Aib 8,22,35 Arg 26,34 GLP-1 (7-38), Aib 8,35 Arg 26,34 Lys 38 GLP-1 (7-38), Aib ^8,22 Arg ^26,34 Lys ³⁸ GLP-1(7-38),Aib ^8,22,35 Arg ²⁶ Lys ³⁸ GLP-1(7-38),Aib ⁸ Arg ²⁶ Lys ³⁸ GLP-1(7-38 ), Aib ^8,35 Arg ²⁶ Lys ³⁸ GLP-1(7-38), Aib ²² Arg ²⁶ Lys ³⁸ GLP-1(7-38), Aib ^8,22,35 Arg ³⁴ Lys ³⁸ GLP-1(7- ^{38), Aib 8,35 Arg 34 Lys} 38 GLP-1 (7-38), Aib 8,22,35 Ala 37 Lys 38 GLP-1 (7-38), Aib 8,35 Ala 37 Lys 38 GLP-1 ^{(7-38), Aib 8, 37} GLP- 35 Ala 37 Lys 38 GLP-1 (7-38), Aib 8,22 Ala 37 Lys 38 GLP-1 (7-38), Aib 8,22,35 Lys 1(7-38), Aib ^8,35 Lys ³⁷ GLP-1(7-38), Aib ^8,22 Lys ³⁷ GLP-1(7-38), Gly ⁸ GLP-1(7-36)-amide, Gly ⁸ GLP-1 (7-37), Val ⁸ GLP-1 (7-36)-amide, Val ⁸ GLP-1 (7-37), Val ⁸ Asp ²² GLP-1 (7-36)-amide, ^{Val 8 Asp 22 GLP-1 (} 7-37), Val 8 Glu 22 GLP-1 (7-36) - ^{Amine, Val 8 Glu 22 GLP-1} (7-37), Val 8 Lys 22 GLP-1 (7-36) - ^{amide, Val 8 Lys 22 GLP-1} (7-37), Val 8 Arg 22 GLP-1 (7-36)-amide, Val ⁸ Arg ²² GLP-1 (7-37), Val ⁸ His ²² GLP-1 (7-36)-amide, Val ⁸ His ²² GLP-1 (7-37), Val ⁸ Trp ¹⁹ Glu ²² GLP-1 (7-37), Val ⁸ Glu ²² Val ²⁵ GLP-1 (7-37), Val ⁸ Tyr ¹⁶ Glu ²² GLP-1 (7-37), Val ⁸ Trp ¹⁶ Glu ²² GLP-1 (7-37), Val ⁸ Leu ¹⁶ Glu ²² GLP-1 (7-37), Val ⁸ Glu ²² Ile ³³ GLP-1 (7-37), Val ⁸ Trp ¹⁶ Glu ²² Val ²⁵ Ile ³³ GLP -1 (7-37), Val ⁸ Trp ¹⁶ Glu ²² Ile ³³ GLP-1 (7-37), Val ⁸ Glu ²² Val ²⁵ Ile ³³ GLP-1 (7-37) and Val ⁸ Trp ¹⁶ Glu ²² Val ²⁵ GLP-1 (7-37).

Still more preferably, the GLP-1 analog is Exendin-3, Exendin-4 or a derivative thereof; most preferably the GLP-1 analog is Exendin-4.

Wherein Exendin-4 is a peptide having the following amino acid sequence:

His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp- Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ ;

Exendin-3 is a peptide having the following amino acid sequence:

His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp- Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-OH.

Taking Exendin-4 as an example, the structure of the compound of the present invention is further illustrated as follows:

Ex has the following structure:

Wavy line

Indicates a connection site;

Illustrative, for example

Indicates that it is equivalent to a compound having the following structure:

In another aspect, the invention provides a pharmaceutical composition comprising a compound D-A-P-L provided herein, and optionally a pharmaceutically acceptable carrier.

"Pharmaceutical composition" means one or more active ingredients and optionally one or more inert ingredients, as well as any product, which is obtained directly or indirectly from a combination of any two or more ingredients, Compounding or agglomeration, or dissociation from one or more components, or other types of reactions or interactions from one or more components. Accordingly, the pharmaceutical compositions of the present invention include any composition prepared by admixing a compound of the present invention and a pharmaceutically acceptable excipient (pharmaceutically acceptable carrier).

The term "excipient" refers to a diluent, adjuvant or carrier that is administered with a therapeutic agent. The pharmaceutical excipient can be a sterile liquid, such as water and oil, including those of petroleum, animal, vegetable or synthetic origin, including but not limited to peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred excipient when the pharmaceutical composition is administered orally. Saline and aqueous dextrose are preferred excipients when the pharmaceutical composition is administered intravenously. Preferably, a saline solution and an aqueous dextrose solution and a glycerol solution are used as liquid excipients for the injectable solution. Appropriate Pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, natural calcium carbonate, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, Skimmed milk powder, glycerin, propylene, ethylene glycol, water, ethanol, etc. If desired, the compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. The composition can be formulated as a suppository with conventional binders and excipients such as triglycerides. Oral formulations may contain standard excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.

Examples of suitable pharmaceutical excipients are described in "Remington's Pharmaceutical Sciences" by EW Martin, which contains a therapeutically effective amount of a therapeutic agent (preferably in purified form) and an appropriate amount of excipient, Thereby providing a form suitable for administration to a patient. The formulation should suit the mode of administration.

In another aspect, the invention relates to the use of any of the compounds provided for the manufacture of a medicament for delaying or treating the development of diabetes. The therapeutically effective amount of the Exenatide derivative provided by the present invention will depend on the route of administration, the type of subject and the physical characteristics of the particular mammal in question. These factors and their relationship to determining this amount are well known to those skilled in the medical arts. This amount and method of use can be adjusted to achieve optimal efficacy to deliver the peptide to the subject, but will depend on factors well known to those skilled in the medical arts such as body weight, diet, concomitant medication, and other factors.

In order to ensure a complete understanding of the invention, the following definitions are provided:

Glucagon-like peptide (GLP): Glucagon-like peptide (GLP) and GLP derivatives are enteric hormones, which generally stimulate insulin secretion in hyperglycemia, inhibit glucagon secretion, and promote insulin ( Biosynthesis of the original) and slowing gastric emptying and gastric acid secretion. Some GLP and GLP derivatives promote cellular uptake of glucose but do not promote insulin expression as described in U.S. Patent 5,574,008.

"Glucagon-like peptide" refers to glucagon elongation protein (exendin) and a homologous peptide derived from the proglucagon gene other than glucagon, ie, glucagon-like peptide- 1 (GLP-1), glucagon-like peptide 2 (GLP-2) HE oxynthomodulin (OXM) and their analogs and derivatives. GLP-1 and GLP-2 have the following amino acid sequences, respectively:

GLP-1 (7-36)-NH ₂ : HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR-NH ₂ ;

GLP-1 (7-37)-OH: HAEGT FTSDV SSYLE GQAAK EFIAW LVKGRG-COOH;

GLP-2: HADGS FSDEM NTILD NLAAR DFINW LIQTK ITD.

The amino acid sequence structure of native Exendin-4 is as follows:

His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp- Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ ;

The amino acid sequence structure of Exendin-3 is as follows:

His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp- Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-OH;

GLP-L and its derivatives: the known synthetic hormone glucagon is a large molecular weight precursor molecule which is subsequently cleaved under proteolysis into three peptides: glucagon, glucagon-like peptide 1 (GLP-1) and glucagon-like peptide 2 (GLP-2). Exendin-4 (exenatide) 39 amino acid peptide, about 53% homologous to GLP-1, and has insulinotropic activity. A GLP-1 analog refers to a modified peptide in which one living multiple amino acid residue of the peptide is replaced by another amino acid residue, and/or one or more amino acid residues are deleted from the peptide, And/or one of the living multiple amino acid residues is added to the peptide. Addition or deletion of an amino acid residue can occur at the N-terminus of the peptide and/or at the C-terminus of the peptide, eg Lys ³⁸ Arg ^{26, 34} GLP-1 (7-38) refers to a GLP-1 analogue, wherein Adding lysine 38, the naturally occurring lysine at positions 26 and 34 is replaced by arginine; Aib ^8,22,35 GLP-1 (7-37) refers to a GLP-1 analogue, wherein position 8 The naturally occurring alanine and the glycine at positions 22 and 35 are replaced by alpha-aminoisobutyric acid.

The exenatide, Exendin-4, and Exenatide mentioned in the present invention all refer to the same substance, and the CAS Registry Number thereof is 141758-74-9.

Linking moiety: A linking moiety refers to a fatty long-chain carboxylic acid structure or a polyethoxy-containing structure capable of reacting with an amino group on an amino acid to form an amide bond, such as palmitic acid or (2-amino)ethoxyacetic acid.

Protecting group: A protecting group is a chemical group used to protect a peptide derivative from self-reaction. Including acetyl, decylmethoxycarbonyl (Fmoc), tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), etc., the specifically protected amino acids and abbreviations and abbreviations used in the present invention are shown in Table 1:

The Ts-Cl or TsCl mentioned in the present invention means p-toluenesulfonyl chloride, EA means ethyl acetate, (Boc) ₂ O means di-tert-butyl dicarbonate, and "Boc" means t-butoxycarbonyl, "DIC" means N,N'-diisopropylcarbodiimide, "Fmoc" means fluorenylmethoxycarbonyl, "HOBT" means 1-hydroxybenzotriazole, and "OtBu" means tert-butoxy NMM refers to N-methylmorpholine, AC or HOAC refers to acetic acid, and HBTU refers to O-benzotriazol-1-yl-N,N,N',N'-tetramethylurea-hexafluorophosphate Salt, Alloc means allyloxycarbonyl, and Aib means α-aminoisobutyric acid.

Within the scope of the invention are those molecules known as derivatives of Exendin-4, which refer to all or part of the amino acid structural sequence of Exendin, which shares Exendin A substantially homologous or similarly sized GLP-1 fragment of -4; a class of polypeptide that binds to the glucagon-like peptide-1 receptor and produces a cellular signaling cascade.

Exendin or analogs thereof according to the invention include, but are not limited to, those similar polypeptides obtained by amino acid substitution, addition and deletion of the amino acid sequence of native Exendin-3 or Exendin-4.

Further, the invention relates to a process for the preparation of the provided compounds.

The general preparation method of the compound D-A-P-L provided by the invention is to separately prepare the polypeptide moiety D and the linking moiety H-A-P-L, and then couple the C-terminus of the D-terminal amino acid with the amino group of the linker moiety H-A-P-L, the specific route is as follows:

First, the GLP-1 analogue (D) can be expressed by chemical solid phase synthesis or genetically engineered host bacteria, and then purified by one or more steps. The "Fmoc strategy" is used herein, and the "Fmoc strategy" refers to a method for synthesizing a polypeptide by sequentially condensing an amino terminal Fmoc-protected amino acid in the presence of a coupling reagent by using a polymer resin as a solid phase reaction substrate. For a specific method, see Fmoc solid phase peptide synthesis: a practical approach, 2000, Oxford University Press. J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, W. H. Freeman Co. (San Francisco), 1963 and J. Meienhofer, Hormonal Proteins and Peptides, Vol. 2, p. 46, Academic Press (New York), 1973. Portions of these documents are incorporated herein by reference.

Generally, the method involves the sequential addition of one or more amino acids or appropriately protected amino acids to lengthen the peptide chain. The amino or carboxyl group of the first amino acid is normally protected by a suitable protecting group. Subsequently, the protecting group or derivatized amino acid is attached to an inert solid support, or by adding the next suitably protected amino acid having a complementary (amino or carboxyl) group in the sequence and under conditions suitable for the formation of an amide bond The solution form is utilized. The protecting group is then removed from the newly added amino acid residue and the next amino acid is added and continues as such.

When all of the expected amino acids have been joined in the appropriate order, any remaining protecting groups (and any solid phase carrier) are removed sequentially or simultaneously to yield the final polypeptide.

Second, the synthesis of side chains:

The synthesis of the linker moiety, H-A-P-L, in the compounds of the invention comprises the following steps:

1. Preparation of HNR ₁ -X-NR ₂ H can be obtained by methods known to those skilled in the art or prepared according to the methods provided herein:

(1), X is selected from -(CH ₂ ) _n -, wherein n is an integer of 2 to 10: R ₁ NH-(CH ₂ ) _n -NHR ₂ can be obtained by the following formula,

For specific methods of operation, see Organic Reactions (Hoboken, NJ, United States), 72, 1-366; 2008.

(2), X is selected from -[(CH ₂ ) _n Z] _p (CH ₂ ) _n -, and when Z is O, NH ₂ -[(CH ₂ ) ₂ O] _n (CH ₂ ) ₂ - NH ₂ can be prepared as follows:

Specific methods of operation can be found in U.S. Patents US20130224881 and WO2012170206.

(3) X is an amino acid residue or an amino acid residue substituted by one or more substituents selected from the group consisting of H, C _1-6 alkyl, carbonyl and -NR ₂ R ₃ and combinations thereof Wherein R ₂ and R _{3 are} independently selected from the group consisting of H and C _1-6 alkyl; the specific preparation method of HNR ₁ -X-NR ₂ H may be based on the specific structure of the selected amino acid residue or substituted amino acid residue. It is prepared according to the specific molecular structure according to methods known in the art.

(4) When X is -(CH ₂ ) _n C(O)Z(CH ₂ ) _n -, wherein Z is O or a single bond, n is independently selected from an integer from 1 to 10; when Z is a single bond, For the specific preparation method of NHR ₁ -(CH ₂ ) _n C(O)(CH ₂ ) _n -NHR _2, please refer to the following literature: Structure-activity relationship studies of 1-(4-chloro-2,5-dimethoxyphenyl)-3- (3-propoxypropyl)thiourea, a nonnucleoside reverse transcriptase inhibitor of human immunodeficiency virus type-1 (European Journal of Medicinal Chemistry, Volume: 46, Issue: 2, Pages: 447-467, 2011); Imidazolic H2-agonists. Synthesis and Activity of 2-(2-amino-4-imidazolyl)ethylamine dihydrochloride(2-aminohistamine) (Farmaco, Edizione Scientifica, Volume: 39, Issue: 1, Pages: 70-80, 1984), Synthesis of histamine from 2-butyne -1,4-diol (Journal of the Chemical Society, Pages: 226-8, 1952). When Z is O, a specific preparation method of NHR ₁ -(CH ₂ ) _n C(O)O(CH ₂ ) _n -NHR ₂ can be referred to WO 2002053526 A1.

(5) When X is a C _1-10 alkylene group, and H in the alkylene group is substituted with one or more of the following substituents selected from the group consisting of:

a linear or branched alkyl group of C ₁ -C ₄ , halogen, NHR ₅ , CN, carboxyl, nitro and C ₁ -C ₄ alkyl alcohol; wherein R ₅ is H or C ₁ -C ₄ straight Chain or branched alkyl; may be prepared according to specific structures by methods known in the art, or commercially available.

Illustrative, when X is

Substituted C ₅ alkylene, HAH has the following structure:

Preferably, R ₁ and R ₂ are H, which can be prepared by the method disclosed in Chemical Communications (Cambridge, United Kingdom), 49 (91), 10703-10705;

2. General preparation method of HO-P-L (Compound 10):

The preparation method of the compound represented by Formula 10 can be found in Inorganica Chimica Acta 365 (2011) 38-48; Bioorganic & Medicinal The process disclosed in Chemistry 20 (2012) 4443-4450 or The Royal Society of Chemistry 2010. Analyst, 2010, 135, 1360-1364 is prepared, and can also be obtained by the method provided in this patent.

Specifically, the following steps are included:

1) The compound of the formula 1 is reacted with TsCl in a pyridine solution to obtain a compound of the formula 2, and the compound of the formula 2 is subjected to diazotization, hydrogenation reduction, Boc protection to obtain a compound of the formula 5, and a compound of the formula 5 The compound of the formula 6 is obtained by reacting with 2-bromoacetic acid, and the compound of the formula 6 is hydrolyzed to a salt under acidic conditions to give a compound of the formula 7.

The reaction process is as follows:

2) The compound of the formula 8 is first reacted with N-hydroxysuccinimide to obtain a compound of the formula 9, and the compound of the formula 9 is further reacted with the compound of the formula 7 prepared in the step 1) to give a compound of the formula 10. , ie HOOC-PL; the reaction equation is as follows:

The compound of the formula 8 is commercially available.

Third, the preparation of H-A-P-L:

First, one NH of compound HAH was protected by Boc, then Boc-protected HAH and compound 10 were added to DMF, cooled to 0 ° C in an ice bath, then DIEA and HBTU were added, then the temperature was raised to room temperature, and the reaction progress was monitored by TLC. After the reaction is completed, the reaction liquid is poured into water, extracted with EA, and the organic phase is combined, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate and evaporated. The solid was added to ice HCl/EA (2.2 M) and stirred to remove the protecting group Boc from NH to afford H-A-P-L.

Finally, the preparation of D-A-P-L:

The fully protected polypeptide was reacted with the side chain HAPL at a molar ratio of 1.0:1.5, using DMF as a solvent, and stirred at room temperature overnight in the presence of HOAT and DIC. The reaction was monitored by TLC. After the reaction was completed, water was added and extracted with DCM. The combined organic phases were washed with water, washed with brine, dried over anhydrous sodium sulfate EDT) / 5% thioanisole, followed by precipitation with ice methyl tert-butyl ether (MTBE), washed crude product, crude product using C ₁₈ reversed phase column, flow (A: 0.1% TFA / water , B: acetonitrile) was eluted at a flow rate of 50 mL/min, and the component solution was collected and lyophilized to obtain a pure product. Product purity was determined by RP-HPLC and product structure was confirmed by electrospray ionization mass spectrometry (ES-MS) analysis.

Preparative HPLC purification of crude derivatives

Preparative HPLC was performed on Beijing Xintongtong liquid chromatography. A solution of various crude peptides was injected into a Beijing Innovation Tongheng ODS (50*150 mm) column and eluted with a gradient of acetonitrile and 0.1% TFA. The typical gradient used was 60 min acetonitrile increased from 30% to 40%, the detection wavelength was 210 nm, the flow rate was 50 ml/min, the fraction was collected in stages, and the fraction was lyophilized to obtain a purified target peptide.

HPLC analysis of the purified derivative:

A YMC ODS (4.6*150mm) column was used and eluted using the following gradient: B phase increased from 25% to 65% over 20 min; B phase increased from 65% to 95% over a period of 5 min; Phase B is stable at 95%; last 5 minutes, phase B is stable at 25%, detection wavelength is 215 nm, flow rate is 1.0 ml/min, phase A is 0.1% TFA, and phase B is acetonitrile.

Illustratively, when A is an amino acid residue or a substituted amino acid residue, for example, when the compound of the present invention has the following structure:

It is also possible to first attach A to the polypeptide, and then carry out the amide reaction of the carboxyl group of the side chain HO-PL (compound 10) with the amino group of the amino acid A modified polypeptide to obtain the desired compound, and the amino acid substituted lysine. For example, the specific method includes the following methods: (1) solid phase synthesis of D-Lys(Alloc)-OH by Fmoc method; (2) selective removal of Lys(Alloc) side chain protecting group to obtain D-Lys-NH ₂ ; (3) attaching compound 10 to the target peptide D-Lys-NH ₂ ; (4) cleavage of the resin to obtain the target compound.

Taking D as Exendin-4 and A as an amino-substituted lys residue as an example, the compound D-A-P-L provided by the present invention and a preparation method thereof are further described; the preparation method specifically includes the following steps:

1. Preparation method of amino acid modified Exendin-4:

Exendin-4 can be synthesized by Fmoc solid phase synthesis method well known to those skilled in the art, such as Fmoc-Rink MBHA Amide resin, Fmoc with 20% piperidine/DMF, HOBT/DIC coupling reagent, DMF as reaction solvent. The reaction was monitored by ninhydrin detection method, and the protected amino acid was sequentially attached to Rink MBHA Amide resin to obtain a fully protected Exendin-4 resin. The resin was cleaved using 85.2% TFA/5% phenol/5% water/5. % thioanisole / 2.5% EDT, MTBE precipitation on ice, washing, crude product purified by reverse phase HPLC, RP-HPLC The purity of the desired peptide is determined; or prepared by reference to the related methods disclosed in WO2008109079A2.

2. The modified Exendin-4 prepared in the step 1 and the HO-P-L-containing compound (the compound of the formula 10) prepared in the scheme 2 are condensed with HOBT and DIC.

The Exenatide derivative provided by the present invention is capable of significantly increasing intracellular cAMP content, and the inventors have unexpectedly discovered that the Exenatide derivative provided by the present invention retains GLP-1 compared to Exenatide. At the same time as the receptor agonist activity, the in vivo biological half-life and/or plasma half-life is significantly prolonged. Since the exenatide derivative provided by the present invention has a long half-life and good stability in vivo, the frequency of administration can be reduced, and patient compliance can be remarkably improved. Moreover, the preparation method of the Exenatide derivative provided by the invention is simple and effective, and is suitable for industrial production.

DRAWINGS

The drawings are intended to provide a further understanding of the invention, and are not intended to limit the invention.

1 to 10 show mass spectra of the compounds pp01a03, pp01b, pp01c, pp01d, pp01e, pp01f, pp01g, pp01h, pp01j, and pp01k, respectively.

Figure 11 shows a standard curve for a non-interfering protein concentration assay kit.

Figure 12 shows the Mouse/Rat cAMP Assay kit standard curve.

Figure 13 is a dose-effect relationship of each test drug to stimulate c12 production by PC12 cells.

Specific embodiment

The invention will now be further fully described by the following non-limiting examples. The preferred embodiments described herein are to be construed as illustrative only and not limiting.

Compound used in the present invention

Wherein k is an integer from 6 to 20, and a polyethylene glycol compound

Wherein m is an integer of from 1 to 6, which is commercially available by a conventional method.

Preparation of the compound of the formula 10 of Example 1

Under a nitrogen atmosphere, 200 mL of pyridine was added to a 1000 ml three-necked flask, 120 g of the compound of formula 1 (1.0 eq), and the mixture was cooled to 0 ° C, and 151.8 g of TsCl (1.0 eq) was added in portions, stirred for 1 h, and then slowly warmed to room temperature. Continue to stir for 3-4h. After the reaction is completed, the reaction solution is poured into iced dilute hydrochloric acid solution, solid is produced, extracted with EA, and the EA layer is washed once with dilute hydrochloric acid, washed with saturated sodium hydrogencarbonate, washed with saturated brine, dried over anhydrous Na ₂ SO ₄ The solvent was evaporated to give 119 g (yield:ield: petroleum ether: ethyl acetate: acetic acid = 4:1:0.1).

Add 55g (1.0eq) of the compound of formula 2 and 160mL of DMSO to a 1000mL three-necked flask, stir evenly, then add 23.52g (2.0eq) of NaN ₃ , heat to 50 ° C for 3 hours, cool to room temperature, and pour the reaction solution It was poured into 1.2 L of water, extracted with EA, and the organic phase was combined, dried over anhydrous sodium sulfate, and then concentrated to give 29.2 g.

To a 1 L hydrogenation reactor, 29 g of the compound of the formula 3, 360 mL of methanol, and 5.0 g of palladium on carbon were added, stirred, and subjected to hydrogen reaction for 3-4 hours. After the reaction was monitored by TLC, the reaction mixture was filtered, and the filtrate was concentrated to give the oil of the formula 4 23.5 g.

23.5 g (1.0 eq) of the compound of the formula 4, (Boc) ₂ O 68.6 g (2.0 eq), and 500 ml of a mixed solution of methanol / triethylamine (9 / ₁ ) were added to a 1 L three-necked flask, and the mixture was stirred and heated to reflux. After the reaction was completed for 3 hours, the reaction was completed by TLC, and then methanol (methanol) was evaporated, dissolved in water, and extracted twice with dichloromethane. The organic layer was washed with water, dried over anhydrous sodium sulfate, evaporated and evaporated to give a solid g.

To a 1000 mL three-necked flask, 34.8 g of a compound of the formula 5 (1.0 eq), 150 ml of toluene and methanol, and 58.2 g of bromoacetic acid were added, stirred, heated to 45 to 50 ° C, and then added with 33.5 g of sodium hydroxide, and reacted overnight. After the TLC monitoring reaction is completed, the reaction liquid is distilled off, water and EA are added, the aqueous phase is adjusted to pH 3, the aqueous phase is extracted with dichloromethane, the dichloromethane layer is combined, and the organic solvent is distilled off to obtain 18 g of the compound of formula 6. .

To a 250 mL three-necked flask, 18 g of the compound of formula 6, 6, 120 ml of EA/HCl (2.2 M) was added, and the reaction was monitored by TLC.

24.7 g of N-hydroxysuccinimide (HOSU), 50 g of the compound of formula 8 and 300 ml of DMF were added to a 500 mL three-necked flask, the temperature was controlled to about -10 to 0 ° C, and 44.2 g of N,N'-dicyclohexyl carbon was added. Diimine (DCC) was stirred at room temperature overnight, and the reaction was completed by TLC. Water and EA was evaporated, washed with brine, and then evaporated.

To a 100 mL three-necked flask, 1.9 g of the compound of the formula 7, 20 ml of water, 1.31 g of NaHCO _{3 was} added, stirred, and 4.13 g of the compound of the formula 9 and 20 ml of DME (ethylene glycol dimethyl ether) were added dropwise, stirred overnight, and the reaction was monitored by TLC. After completion, add water to adjust pH=3, extract three times with EA, combine EA layer and wash with sodium hydroxide aqueous solution, combine the aqueous phase, adjust pH=2, extract the aqueous phase with EA, combine the two extracted EA layers, use no The aqueous sodium sulfate was dried, the solvent was evaporated, and dried to give 1.5 g of compound of formula 10.

Preparation of the compound of the formula 13 of Example 2

Compound 12 was prepared in a similar manner to compound 9. To a 100 mL three-necked flask, 1.0 g of Compound 7, 15 ml of water, 0.7 g of NaHCO _{3 was} added, and 1.32 g of Compound 12 and 15 ml of a DME solution were added dropwise, stirred overnight, and the progress of the reaction was examined by TLC.

Post-reaction treatment:

Add water to adjust the pH to about 3, extract with EA, wash the EA layer with dilute sodium hydroxide solution, adjust the pH of the aqueous layer to about 2, add EA extraction, wash with water, wash with saturated brine, dry over anhydrous sodium sulfate, and evaporate solvent under reduced pressure. The compound 13 was obtained in an amount of about 1.2 g.

Preparation of Compound 16 of Example 3

To a 100 mL three-necked flask was added 6.3 g of HOSU, 10 g of compound 14 and 60 ml of DMF, the temperature was controlled below 0 ° C, and 11.3 g of DCC was added and stirred overnight.

Post-reaction treatment: suction filtration, addition of water to the filtrate, extraction with EA, washing with water, washing with saturated brine, dried over anhydrous sodium sulfate,

To a 100 mL three-necked flask was added 1.0 g of Compound ₇ , 15 ml of water and 0.7 g of NaHCO ₃ , and a solution of 1.5 g of Compound 15 and 15 ml of DME was added dropwise and stirred overnight.

Post-reaction treatment:

Add water to adjust the pH value to about 3, extract with EA, wash the EA layer with dilute sodium hydroxide solution, adjust the pH of the water layer to about 2, add EA extraction, wash with water, wash with saturated brine, dry with anhydrous sodium sulfate, and distill off under reduced pressure. The solvent gave 0.88 g of Compound 16.

Example 4 Compound 19, Compound 22 was prepared according to the general procedure of scheme 2.

Preparation of Compound 19

Preparation of Compound 22

Preparation of Example 5 Compound PP01a03

1) Preparation of Exendin-4(1-39)-Lys40(Alloc)-OH

The solid phase peptide synthesis of the target peptide was carried out by solid phase synthesis using Fmoc method, Fmoc-Rink MBHA Amide resin, Fmoc with 20% piperidine/DMF, HOBT/DIC with coupling reagent, DMF as reaction solvent, and reaction monitoring. The ninhydrin assay, in turn, linked the following protected amino acids to Rink MBHA Amide resin: Fmoc-Lys(Alloc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc -Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly -OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ile-OH , Fmoc-Phe-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)- OH, Fmoc-Glu(OtBu)-OH, Fmoc-Met-OH, Fmoc-Gln(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Asp(OtBu)OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, (BOC) ₂ O (using DIEA, DCM).

2) Selective removal of the Lys (Alloc) side chain protecting group:

Treatment with Pd(PPh ₃ ) ₄ in 3 equivalents of CHCl ₃ :NMM:HOAC (18:1:0.5) for 2 hours gave the target peptide Exendin-4(1-39)-Lys40-NH ₂ (PP01a03) ).

The specific experimental methods are as follows:

To a 500 ml reaction flask, 7.0 g of Fmoc-Rink MBHA Amide resin (1.2 eqiv), 50 ml of 20% piperidine was added, and the reaction was carried out for 20 min. DMF was washed 5 times for ninhydrin detection reaction, and the color was positive. 950 mg of Fmoc-Lys(Alloc)-OH (1.0 eqiv), 1.55 g of HBTU (2.0 eqiv), 567 mg of HOBT (2.0 eqiv) and 40 ml of DMF were added, the solution was completely dissolved, and 775 ul DIEA (2.0 eqiv) was slowly added dropwise at 30 After shaking for 1 h at °C, the reaction was detected by ninhydrin. The color was slightly blue. 7 ml of acetic anhydride and 7 ml of pyridine were added, and 80 ml of DCM was shaken at 30 ° C for 30 min. The reaction was detected by ninhydrin and the color was yellowish.

Post-treatment: Transfer to 500ml reaction column, DMF cleaning 3 times, add 50ml 20% piperidine DMF solution, nitrogen bubbling for 20min, DMF cleaning 5 times, ninhydrin monitoring reaction after adding 3.0eqiv amino acid 3.3eq HOBT , 3.3 eq DIC, after 1 h of reaction, the ninhydrin monitoring was negative, DMF was washed, and 20% piperidine DMF solution was added. This step is repeated to condense the subsequent amino acids in sequence until condensation to the last amino acid (as is the solid phase synthesis of the fragments of the polypeptide). Add 3 eq of Pd(PPh ₃ ) _{4 solution} of CHCl ₃ :AC:NMM (18:1:0.5) for 2 h, then wash with chloroform (6*200 ml), 20% HOAc in DCM (6*200ml) Washing with DCM (6*200ml) and DMF (6*200ml), ninhydrin monitoring positive, DMF cleaning 3 times, MeOH cleaning once, DCM cleaning once, draining. After the synthesis was completed, the cleavage reagent, free polypeptide, and HPLC purification were carried out, and the HPLC purity was 96.3%. The mass spectrum is shown in Figure 1.

Preparation of the compound of Example 6 pp01b

1) Preparation of Exendin-4(1-39)-Lys40(Alloc)-OH

The solid phase peptide synthesis of the target peptide was carried out by solid phase synthesis using Fmoc method, Fmoc-Rink MBHA Amide resin, Fmoc with 20% piperidine/DMF, HOBT/DIC with coupling reagent, DMF as reaction solvent, and reaction monitoring. The ninhydrin assay, in turn, linked the following protected amino acids to Rink MBHA Amide resin: Fmoc-Lys(Alloc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc -Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly -OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ile-OH , Fmoc-Phe-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)- OH, Fmoc-Glu(OtBu)-OH, Fmoc-Met-OH, Fmoc-Gln(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Asp(OtBu)OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, (BOC) ₂ O (using DIEA, DCM).

2) Selective removal of the Lys (Alloc) side chain protecting group:

Treatment with Pd(PPh ₃ ) ₄ in 3 equivalents of dissolved CHCl ₃ :NMM:HOAC (18:1:0.5) for 2 hours gave the target peptide Exendin-4(1-39)-Lys40-NH ₂ .

3) Attaching Compound 10 to the target peptide Exendin-4(1-39)-Lys40-NH ₂ :

4) Cracking of resin

The cracking of the resin was carried out using 82.5% TFA / 5% phenol / 5% water / 2.5% EDT / 5% thioanisole, followed by precipitation with ice methyl tert-butyl ether (MTBE) and washing. The crude product was purified by HPLC to give the title compound pp01b. The purity of the target compound was determined by RP-HPLC to be >96%.

Experimental specific steps:

To a 50 ml reaction flask was added 800 mg of Fmoc-Rink MBHA Amide resin (1.2 eqiv), 10 ml of 20% piperidine, and reacted for 20 min. DMF was washed 5 times, ninhydrin detection reaction, color positive. Then add 92mg Fmoc-Lys(Alloc)-OH (1.0eqiv), 180mg HBTU (2.0eqiv), 150mg HOBT (2.0eqiv), 5ml DMF, completely dissolved in the shock, then slowly add 200ul DIEA (2.0eqiv) and 30 After shaking for 1 h at °C, the reaction was detected by ninhydrin. The color was slightly blue. 500 ul of acetic anhydride and 500 ul of pyridine were added, and 10 ml of DCM was shaken at 30 ° C for 30 min. The reaction was detected by ninhydrin and the color was yellowish.

Post-treatment: Transfer the above reaction mixture to 50ml reaction column, DMF clean 3 times, add 10ml 20% piperidine DMF solution, nitrogen bubbling for 20min, DMF cleaning 5 times, ninhydrin monitoring reaction, add 3.0eqiv The amino acid HOBT (3.3 eq), DMF, DIC (3.3 eq), after 1 h of reaction, the ninhydrin monitoring was negative, DMF was washed, and 20% piperidine DMF solution was added. This step is repeated to condense the subsequent amino acids in sequence until condensation to the last amino acid (as is the solid phase synthesis of the fragments of the polypeptide).

Add 3 eq of Pd(PPh ₃ ) _{4 solution} of CHCl ₃ :AC:NMM (18:1:0.5) for 2 h, then wash with chloroform (6*10 ml), 20% HOAc in DCM (6*10 ml) Washing with DCM (6*10ml) and DMF (6*10ml), ninhydrin monitoring positive, adding 430mg compound 10 (3eq), reaction for 1h, ninhydrin monitoring negative, DMF cleaning 3 times, MeOH cleaning once, DCM cleaning Once, drain. After the synthesis, the cleavage reagent 82.5% TFA / 5% phenol / 5% water / 2.5% EDT / 5% thioanisole was added, followed by precipitation, washing with free methyl tert-butyl ether (MTBE), free peptide, 890 mg of the target compound pp01b, HPLC: 96%, and the mass spectrum is shown in Fig. 2.

Example 7 Preparation of a similar compound pp01b Compounds pp01c, pp01d, pp01e, pp01f, pp01g, pp01h, pp01j, pp01k were prepared.

Compound pp01c:

HPLC: 97.3%, MS [M+5H] ⁵⁺ : 933.05 (933.05*5-5=4660.25, which is close to the average molar molecular weight of pp01c of 4658.20), and the mass spectrum is shown in Figure 3;

Compound pp01d:

HPLC: 97.1%, MS [M-5H] ^5- : 936.50 (936.50*5+5=4687.5, which is close to the average molar molecular weight of pp01d 4686.26), and the mass spectrum is shown in Figure 4;

Compound pp01e:

HPLC: 96.6%, MS [M+5H] ⁵⁺ : 944.30 (944.30*5-5=4716.50, close to the average molar molecular weight of pp01e, 471.31), and the mass spectrum is shown in Figure 5;

Compound pp01f:

HPLC: 96.3%, MS [M+5H] ⁵⁺ : 955.40 (955.40*5-5=4772.00, close to the average molar molecular weight of pp01f 4770.42), the mass spectrum is shown in Figure 6;

Compound pp01g:

HPLC: 96.0%, MS [M+4H] ⁴⁺ : 1118.99 (1118.99*4-4 = 4471.96, which is close to the average molar molecular weight of pp01g of 4649.99), and the mass spectrum is shown in Figure 7;

Compound pp01h:

HPLC: 97.0%, MS [M+5H] ⁵⁺ : 925.21 (928.63*5-5=4638.15, close to the average molar molecular weight of pp01h 4636.11), and the mass spectrum is shown in Figure 8;

Compound pp01j:

HPLC: 96.0%, MS [M+4H] ⁴⁺ : 1195.54 (1199.54*4-4=4794.16, close to the average molar molecular weight of pp01j of 4790.36), and the mass spectrum is shown in Figure 9;

Compound pp01k:

HPLC: 96.1%, MS [M+4H] ⁴⁺ : 1220.61 (1220.61*4-4=4878.44, which is close to the average molar molecular weight of pp01k, 4874.52), and the mass spectrum is shown in FIG.

It will be apparent to those skilled in the art that the compound D-AA-L of the present invention, wherein D is an other GLP-1 analog of the present invention, can be obtained by a similar method when D is Exendin-4.

Test Example 1 Compound Activity Evaluation Experiment

Test sample: Compound pp01b and compound pp01c prepared according to the present invention and reference substance Exendin-4.

Tumor cell line: PC12 (rat adrenal pheochromocytoma) from the cell bank of Zhongshan University College of Science and Technology;

The 25 cm ² flasks, 24-well plates, 96-well plates used were purchased from Corning; DMEM high-sugar medium (Cat no. SH30022.01), fetal bovine serum (Cat no. SV30087), purchased from Hyclone; Serum (Cat no. 16050-122), trypsin purchased from Gibco; 3-isobutyl-1-methylxanthine (IBMX) purchased from Sigma, Cat no. I5879-100 mg; non-interfering protein concentration determination The kit (Cat no. SK3071) was purchased from Biotech (Shanghai) Co., Ltd.; the Mouse/Rat cAMP Assay kit (Cat no. KGE012) was purchased from R&D Systems.

Test equipment: Sujing ultra-clean workbench, Themro carbon dioxide incubator, BIO-RAD 680 microplate reader.

Sample processing method: Each test sample was dissolved in double distilled water at a final concentration of 1.0×10 ^-2 mol/L, and stored at 4 °C.

experiment method:

1. Culture and dosing intervention of PC12 cells: PC12 cells were cultured in 25 cm ² flasks and placed in a CO ₂ incubator (37 ° C, 95% air, 5% CO ₂ ). The medium was DMEM (pH 7.4, High sugar), adding 5% fetal bovine serum and 10% horse serum. PC12 cells with good growth state were digested with 0.25% trypsin, adjusted to a cell concentration of 1.0×10 ⁵ /ml, and seeded in 24-well plates. When the cells were grown to a density of 60-70%, they were washed twice with PBS and added. 1 ml of PBS containing 1% BSA, each of the three test drugs were divided into 5 gradients (10 ^-10 , 10 ^-9 , 10 ^-8 , 10 ^-7 , 10 ^-6 mol / L ) and IBMX (100 μmol / L) A total of 30 min incubation, 3 replicate wells per sample.

2. Extraction of intracellular cAMP: Immediately after the end of drug intervention, the cells were collected, suspended in cold PBS, adjusted to a cell concentration of 1.0×10 ⁷ /ml, and immediately added 1 volume of IN HCl to 9 volumes of cell suspension. Incubate for 10 min at room temperature and sonicate for 15 s. The cell debris was removed by centrifugation at 1000 rpm for 10 min at 4 ° C, and the supernatant was neutralized with an equal volume of 1 N NaOH of 1 N HCl. The obtained solution was a sample solution containing cAMP and stored at -20 ° C for detection.

3. Determination of total protein content of the sample

This test uses a non-interfering protein concentration determination kit. The special precipitation reagent in the kit can remove most interfering substances. The precipitated protein is mixed with the alkaline substance containing copper. The copper that is not combined with the protein is being Reducing to a monovalent value and reacting with a specific chromogenic reagent to produce a specific absorption wavelength of 480 nm, so as the concentration of the protein increases, the copper that is not bound to the protein is correspondingly reduced, so that the absorption value of the specific absorption wavelength and the protein in the solution Concentration and inverse ratio.

Steps:

1) Take 12 1.5ml centrifuge tubes, one for each two tubes, and add 0, 4, 8, 12 to the different numbered centrifuge tubes. 20, 25 ul of protein standard solution (protein standard BSA, concentration 2 mg/ml), the same number of centrifuge tubes were added to the same volume of protein standard solution.

2) Take two additional 1.5 ml centrifuge tubes and add the same volume of 10 μl of the protein sample solution to be quantified.

3) Add 0.5 ml of the precipitating reagent 1 to the above 14 tubes, vortex for 30 seconds, and let stand for 2-3 minutes at room temperature.

4) Add 0.5 ml of precipitation reagent 2 and vortex for 30 seconds.

5) Centrifuge at 12,000 RPM for 5 min, which will form a dense or invisible tight protein pellet at the bottom of the tube.

6). Remove the tube from the centrifuge, remove the supernatant as much as possible with the tip, retain the pellet, and then invert the tube onto the filter paper to allow the residue in the tube to drain.

7) Add 100 ul of copper reagent and 400 μl of double distilled water and vortex for 30 seconds until the protein pellets are completely dissolved.

8) Add 1 ml of the chromogenic reagent mixture (1 volume of chromogenic reagent B and 100 volumes of chromogenic reagent A), and mix the tubes several times by inverting.

9) Leave at room temperature for 15-20 minutes, transfer 250 μl of the mixture to each well of the corresponding 96-well microtiter plate, and measure the absorbance at 480 nm in 10-20 minutes.

10) The average value of the absorption values of the same number of centrifuge tubes is plotted on the ordinate, and the corresponding BSA protein amount is plotted on the abscissa. Curves are calculated using CurveExpert 1.3 software and the standard curve formula is calculated.

11) Find the concentration of the corresponding diluted protein sample solution based on the standard curve and the average value of the 480 nm absorption value of the protein sample solution to be quantified, thereby obtaining the original concentration of the protein sample solution.

4. Detection of cAMP in cell lysate by ELISA

The content of cAMP in the cell lysate was determined by R&D Systems' cAMP detection kit (Mouse/Rat cAMP Assay kit (Cat no. KGE012)), ELISA method, according to the instructions.

Curves were calculated using CurveExpert 1.3 software and the standard curve formula was calculated to calculate the concentration of each sample. (The sample was diluted 10 times and its actual concentration should be ×10); data processing and mapping were performed using the computer program Microsoft Excel and GraphPad Prism 5 software, and the EC50 of each test drug was calculated.

Experimental result

1 Total protein concentration in cells after each drug intervention (mg/ml)

The reliable quantitative range of the non-interfering protein concentration assay kit is 1-50 μg. Within this range, the standard BSA is concentration dependent on the absorbance value, and the fit curve is y=a+bx+cx^2, where a , b, c are constants, a=0.54543092, b=-0.028833977, c=0.00059713018, the correlation coefficient r is greater than 0.99, and the standard curve is shown in FIG. The total protein concentration of each sample was calculated from the standard curve and the 480 nm absorption value of the protein sample solution to be quantified, as shown in Table 1.

Table 1 Total protein concentration (mg/ml) of each group of samples

2. Results of intracellular cAMP concentration detection after drug intervention:

1) cAMP standard curve

Within the reliable quantitative range of the Mouse/Rat cAMP Assay kit, the standard cAMP is concentration dependent on the absorbance value, and the fit curve is y=(a+bx)^(-1/c), where a, b, c It is a constant, a=3.202598, b=0.18163734, c=1.5075207, the correlation coefficient r is greater than 0.99, and the standard curve is shown in Fig. 12.

2) Concentration of intracellular cAMP after each drug intervention

The test drugs pp01b, pp01c, and Exendin-4 were diluted to 5 gradient concentrations (10 ^-10 , 10 ^-9 , 10 ^-8 , 10 ^-7 , 10 ^-6 mol/L ) and PC12 was co-incubated with IBMX (100 μmol/L). The cells were incubated for 30 min, and the biological activities of the respective test drugs are shown in Table 2.

Table 2 Intracellular cAMP concentration (x±s) pmol/mL after each drug intervention (n=3in each group)

P is the value of the sample group compared to the control group.

It can be seen from the data in Table 2 above that the concentration of each test drug is in a concentration-dependent manner with the amount of cAMP in the range of 10 ^-10 ～10 ^-6 mol/L drug concentration. When the concentration of the test drug was in the range of 10 ^-9 to 10 ^-6 mol/L, the amount of intracellular cAMP in the sample group was significantly different from that in the blank control group (p<0.05). Under the same concentration conditions, when the concentration of the test drug increased to a certain extent, the amount of intracellular cAMP no longer increased, which may be due to the saturation of the binding of the test drug to the GLP-1 receptor of PC12 cells.

Further, the concentration of cAMP was converted into a content equivalent to total protein as shown in Table 3.

Table 3 Concentration of intracellular cAMP after each drug intervention (x±s) pmol/mg (n=3in each group)

P is the value of the sample group compared to the control group.

From the data shown in Table 3, it can be seen that the maximum amount of cAMP produced by pp01b-stimulated cells is 73.312±4.407 pmol/mg, and the maximum amount of cAMP produced by pp01c-stimulated cells is 109.021±4.387 pmol/mg, Exendin-4 stimulates cells. The maximum amount of cAMP produced was 68.629 ± 8.416 pmol/mg. The compounds provided by the present invention obtained by structural modification at the end of Exendin-4C are superior to Exendin-4 in biological activity.

3 dose-effect relationship of test drugs to stimulate c12 production in PC12 cells

The GraphPad Prism 5 software was used for mapping and calculating the half-effect dose (EC50) of the drug, as shown in FIG. The EC50 of pp01b is 5.098 nmol/L, the EC50 of pp01c is 7.210 nmol/L, and the EC50 of Exendin-4 is 5.096 nmol/L.

Test Example 2 pp01b, pp01c and pp01k hypoglycemic experiments

Taking the compounds pp01b, pp01c and pp01k provided by the present invention as an example, the present invention provides the in vivo activity of the compound:

The compounds pp01b, pp01c and pp01k provided by the present invention were used as experimental drugs, and male rats with the same physiological state were divided into 5 groups (4 in each group) with fasting for 24 hours, using exenatide and physiological saline as controls. Glucose (20 mmol/kg, body weight) was injected into the abdominal cavity of the five groups of rats, and then treated as follows:

Experimental group: injections were injected with pp01b (10nmol/kg), pp01c (10nmol/kg), and pp01k (10nmol/kg);

Control group 1: exenatide was injected (same dose as the experimental group);

Control group 2: saline was injected (volume was the same as the experimental group). After the injection, blood was collected from the tail vein at 0, 15 min, 30 min, 60 min, 240 min, and 480 min. The glucose content in the blood was measured by the glucose oxidase method (GOD method). The experimental results are shown in Table 4 below:

Table 4

From the experimental results in Table 4, exenatide achieves a stable hypoglycemic effect at about 0.5, and blood glucose is reduced by about 16% compared with physiological saline; the compounds pp01b, pp01c and pp01k provided by the present invention are also realized at about 0.5 h. The corresponding hypoglycemic effect, and at about 1.5h, reached the most significant hypoglycemic effect. Compared with the saline group, blood glucose was reduced by about 70%, and the hypoglycemic effect was maintained for more than 8 hours.

The insulin content in the blood was examined by an enzyme-linked immunosorbent kit for insulin, and the results are shown in Table 5:

table 5

It can be seen from the results of the insulin content test that the insulin content of exenatide in the blood of the rat reaches a peak after about 0.5 h, while the compounds pp01b, pp01c and pp01k all achieve the peak of insulin in the blood after about 1.5 h.

The blood of the experimental mice was taken every hour, and the content of exenatide was measured. It was determined that the half-lives of pp01b, pp01c and pp01k in the rats were significantly prolonged compared with exenatide.

The exenatide derivatives provided by the present invention have improved pharmacokinetic properties, can significantly lower blood glucose, and have comparable or superior biological activity to exenatide. Moreover, the Exenatide derivative provided by the invention significantly prolongs the half-life, and the stability in vivo is much better than that of Exenatide, and the hypoglycemic effect is more obvious.

It should be noted that the above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Those skilled in the art will be aware of the technical solutions described in the foregoing embodiments of the present invention within the spirit and principles of the present invention. Any modifications, equivalent substitutions, improvements, etc. of some of the technical features are intended to be included within the scope of the present invention.

Claims (13)

1. Compound: D-A-P-L

   Wherein D is a GLP-1 analog moiety;

   A has the following structure: -NR ₁ -X-NR ₂ -,

   Wherein R ₁ and R _{2 are} independently selected from the group consisting of H and C _1-6 alkyl;

   X is selected from:

   (1) -(CH ₂ ) _n -, wherein n is an integer from 2 to 10;

   (2) -[(CH ₂ ) _n Z] _p (CH ₂ ) _n -, wherein Z is O, NR ₃ or S, n is independently selected from an integer from 1 to 10, and p is an integer from 0 to 10, wherein R ₃ is H or a C _1-6 alkyl group;

   (3) an amino acid residue or an amino acid residue substituted with one or more substituents selected from the group consisting of H, C _1-6 alkyl, carbonyl and -NR ₂ R ₃ , wherein R ₂ , R ₃ independently selected from the group consisting of H and C _1-6 alkyl;

   (4)-(CH ₂ ) _n C(O)Z(CH ₂ ) _n -, wherein Z is O or a single bond, and n is independently selected from an integer of from 1 to 10;

   with

   (5) an alkylene group of C _1-10 , and H in the alkylene group is substituted by one or more of the following substituents selected from the group consisting of:

   

   a linear or branched alkyl group of C ₁ -C ₄ , halogen, NHR ₅ , CN, carboxyl, nitro and C ₁ -C ₄ alkyl alcohol; wherein R ₅ is H or C ₁ -C ₄ straight Chain or branched alkyl group;

   P is a linking moiety containing an oligoethylene glycol, and L is a lipophilic linking moiety;

   Further, the carbon terminal of the terminal amino acid residue of D is linked to NR _{1 of A} through an amide bond, NR _{2 of} A is linked to P via an amide bond, and P and L are linked by an amide bond.
2. The compound according to claim 1, wherein P is a moiety having the structure:

   

   L has the following structure:

   

   Where m is an integer from 1 to 20; k is an integer from 6 to 20; wavy lines Indicates the connection site.
3. The compound of claim 1 wherein A has the structure:

   
4. The compound of claim 1 having the structure:

   
5. The compound according to claim 4, wherein the GLP-1 analogue is selected from the group consisting of GLP-1 (7-35), GLP-1 (7-36), GLP-1 (7-37), and GLP-1. (7-38), GLP-1 (7-39), GLP-1 (7-40), GLP-1 (7-41), GLP-1 (7-42), GLP-1 (7-43) , GLP-1 (7-44), GLP-1 (7-45), GLP-1 (7-46) or an analogue thereof.
6. The compound according to claim 5, wherein the GLP-1 analogue is a peptide comprising the following amino acid sequence: aa7-aa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-aa16-Ser-aa18-aa19- aa20-Glu-aa22-aa23-Ala-aa25-aa26-aa27-Phe-Ile-aa30-Trp-Leu-aa33-aa34-aa35-aa36-aa37-aa38-aa39-aa40-aa41-aa42-aa43-aa44- Aa45-aa46;

   Wherein, aa7 is L-His, D-His, 2-aminohistidine, β-histidine or α-methylhistidine;

   Aa8 is Ala, Gly, Val, Leu, Ile, Lys, Aib, 1-aminocyclopropylcarboxylic acid, 1-aminocyclobutylcarboxylic acid, 1-aminocyclopentylcarboxylic acid or 1-aminocycloheptylcarboxylate Acid; aa16 is Tyr, Trp, Val or Leu; aa18 is Tyr, Ser, Lys or Arg; aa19 is Trp, Tyr or Gln; aa20 is Leu or Met; aa22 is Lys, Arg, His, Gly, Glu or Aib; Aa23 is Gln, Glu, Lys or Arg; aa25 is Ala or Val; aa26 is Lys, Glu or Arg; aa27 is Glu or Leu; aa30 is Ala, Glu or Arg; aa33 is Ile, Val or Lys; Lys, Asn, Glu or Arg; aa35 is Gly or Aib; aa36 is Lys, Gly or Arg; aa37 is His, Gly, Ala, Glu, Pro, Lys, amino or absent; aa38 is Lys, Ser, amino or not Exist; aa39 is Ser, Lys, amino or absent; aa40 is Gly, amino or absent; aa41 is Ala, amino or absent; aa42 is Pro amino or absent; aa43 is Pro amino or absent; aa44 is Pro Amino or absent; aa45 is Ser, amino or absent; aa46 is amino or absent, and when aa37, aa38, aa39, aa40, aa41, aa42, aa43, aa44, aa45 or aa46 are not present, the corresponding Amino acids not exist.
7. The compound of claim 6 wherein the GLP-1 analogue is selected from the group consisting of:

   Arg ³⁴ GLP-1 (7-37), Lys ³⁸ Arg ^{26, 34} GLP-1 (7-38), Lys ³⁸ Arg ^{26, 34} GLP-1 (7-37)-OH, Lys ³⁶ Arg ^{26, 34} GLP ^{-1 (7-36), Aib 8,22,35 GLP} -1 (7-37), Aib 8,35 GLP-1 (7-37), Aib 8,22 GLP-1 (7-37), Aib ^{8,22,35 Arg 26,34 GLP-1 (7-38} ), Aib 8,35 Arg 26,34 Lys 38 GLP-1 (7-38), Aib 8,22 Arg 26,34 Lys 38 GLP-1 (7-38), Aib ^8,22,35 Arg ²⁶ Lys ³⁸ GLP-1(7-38), Aib ⁸ Arg ²⁶ Lys ³⁸ GLP-1(7-38), Aib ^8,35 Arg ²⁶ Lys ³⁸ GLP- 1(7-38), Aib ²² Arg ²⁶ Lys ³⁸ GLP-1(7-38), Aib ^8,22,35 Arg ³⁴ Lys ³⁸ GLP-1(7-38), Aib ^8,35 Arg ³⁴ Lys ³⁸ GLP ^{-1 (7-38), Aib 8,22,35 Ala} 37 Lys 38 GLP-1 (7-38), Aib 8,35 Ala 37 Lys 38 GLP-1 (7-38), Aib 8, 35 Ala 37 Lys ³⁸ GLP-1 (7-38), Aib ⁸ , ²² Ala ³⁷ Lys ³⁸ GLP-1 (7-38), Aib ⁸ , ²² , ³⁵ Lys ³⁷ GLP-1 (7-38), Aib 8, ³⁵ Lys ³⁷ GLP-1 (7-38), Aib ^8,22 Lys ³⁷ GLP-1 (7-38), Gly ⁸ GLP-1 (7-36)-amide, Gly ⁸ GLP-1 (7-37), Val ⁸ GLP-1(7-36)-amide, Val ⁸ GLP-1 (7-37), Val ⁸ Asp ²² GLP-1 (7-36)-amide, Val ⁸ Asp ²² GLP-1 (7-37) , Val ⁸ Glu ²² GLP-1(7-36)-amide, Val ⁸ Glu ²² GLP-1 (7-37), Val ⁸ Lys ²² GLP-1(7-36)-amide, Val ⁸ Lys ²² GLP-1 (7-37), Val ⁸ Arg ²² GLP-1 (7-36)-amide, Val ⁸ Arg ²² GLP-1 (7- 37), Val ⁸ His ²² GLP-1 (7-36)-amide, Val ⁸ His ²² GLP-1 (7-37), Val ⁸ Trp ¹⁹ Glu ²² GLP-1 (7-37), Val ⁸ Glu ²² Val ²⁵ GLP-1 (7-37), Val ⁸ Tyr ¹⁶ Glu ²² GLP-1 (7-37), Val ⁸ Trp ¹⁶ Glu ²² GLP-1 (7-37), Val ⁸ Leu ¹⁶ Glu ²² GLP-1 (7-37), Val ⁸ Glu ²² Ile ³³ GLP-1 (7-37), Val ⁸ Trp ¹⁶ Glu ²² Val ²⁵ Ile ³³ GLP-1 (7-37), Val ⁸ Trp ¹⁶ Glu ²² Ile ³³ GLP- 1 (7-37), Val ⁸ Glu ²² Val ²⁵ Ile ³³ GLP-1 (7-37) and Val ⁸ Trp ¹⁶ Glu ²² Val ²⁵ GLP-1 (7-37).
8. The compound according to claim 5, wherein the GLP-1 analogue is Exendin-3, Exendin-4 or a derivative thereof.
9. The compound of claim 4 having the structure:

   

   

   Ex has the following structure:

   

   Wavy line

   

   Indicates the connection site.
10. A pharmaceutical composition comprising the compound of claim 1 and optionally a pharmaceutically acceptable carrier.
11. Use of a compound according to any one of claims 1 to 9 for the manufacture of a medicament for lowering blood glucose.
12. Use of a compound according to any one of claims 1 to 9 for the manufacture of a medicament for the treatment of type 2 diabetes.
13. Use of a compound according to any one of claims 1 to 9 for the manufacture of a medicament for reducing body weight.

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