CN1740198A - GLP-1 derivative - Google Patents

Abstract

Derivatives of GLP-1 and analogues thereof having a lipophilic substituent have interesting pharmacological properties, in particular they have a more protracted profile of action than GLP-1(7-37).

Description

GLP-1 derivative

Technical field that the present invention belongs to

The present invention relates to human glucagon-like-peptide-1 (GLP-1) and its new derivatives of the analog of segment and these segments, they have the effect curves (protracted profile ofaction) of longer time, further relate to the preparation and application of these substances.

The technical background of invention

Peptide is widely used in medical practice, due to that can use DNA recombinant technique peptide, it is therefore contemplated that its importance will also will increase within following some years.When using native peptides or its analog in the treatment, generally it is found that they have high clearance rate.In the case where needing to make therapeutic agent to keep its high blood level in a period of the longer time, the high clearance rate of therapeutic agent is unfavorable, because this will need repeat administration.There is the peptide of high clearance rate for instance that ACTH, corticotropin-releasing factor gives, angiotensin, calcitonin, insulin, glucagon, glucagon-like-peptide-1, glucagon-like-peptide-2, insulin-like growth factor-i, insulin-like growth factor-2, gastrointestinal inhibitory peptide, somatotropin releasing factor, hypophysis adenylate cyclase activating peptide, secretin, enterogastrone (enterogastrin), somatostatin, somatomedin, somatotropin, parathormone, thrombopoietin, erythropoietin(EPO), hypothalamic releasing factor, prolactin, thyroid-stimulating hormone, endorphin, enkephalins, vasopressin, oxytocins, opioid and the like, superoxide dismutase, interferon, asparaginase, arginase, arginine deaminase , adenosine deaminase and ribalgilase.In some cases, the release profiles of peptide can be influenced using suitable pharmaceutical composition, but there are various shortcomings for this method, usually not applicable.

The hormone for adjusting insulin secretion belongs to so-called enteroinsular axis, refers to the presence with enteral nutrients and absorbs from one group of hormone that gastrointestinal mucosa discharges, it promotes the early stage of insulin to discharge and is allowed to enhance.Promote the effect of insulin secretion, i.e., so-called duodenin effect, is perhaps required for normal glucose tolerance.Many gastrointestinal hormones including gastrin and secretin have pancreotropic hormone effect (cholecystokinin of people do not have pancreotropic hormone effect), but physiologically it is important, can lead to duodenin effect only have glucose-dependent-insulinotropic polypeptide GIP and glucagon-like-peptide-1 (GLP-1).Since the pancreotropic hormone of GIP acts on, it causes sizable interest after being separated (1) in 1973 in diabetes scholar immediately.However, the numerous studies carried out in subsequent some years clearly illustrate, the pathogenic factor of GIP hyposecretion and insulin-dependent diabetes mellitus (IDDM) or adult-onset diabetes (NIDDM) is simultaneously irrelevant (2).And then it has also been found that, though GIP is a kind of insulinotropic hormone, its (2) nearly unavailable for NIDDM.Another incretin hormone GLP-1 is known most effective pancreotropic hormone substance (3).Different from GIP, GLP-1 is highly effective in terms of the insulin secretion for promoting patient NIDDM.In addition, GLP-1 also effectively inhibits glucagon secretion compared with other insulinotropic hormones (perhaps in addition to secretin).Due to these effects, it has significant blood sugar decreasing effect particularly with patient NIDDM.

GLP-1 is a kind of product (4) of Proglucagon (proglucagon), it is one of newest member in secretin-VIP race peptide, but it has been identified it is a kind of important gastrointestinal hormone (5) for glucose metabolism and gastrointestinal secretion and metabolism with regulatory function.Glucagon gene is different with enteral processing method in pancreas.In pancreas (9), processing, which is resulted in, secretes following substance with parallel: 1) glucagon itself occupies the position 33-61 of Proglucagon (PG)；2) one kind has the N- terminal peptide of 30 amino acid (PG (1-30)), the normally referred to as relevant pancreas peptide of glucagon-like peptide, GRPP (10,11)；3) a kind of hexapeptide for corresponding to PG (64-69)；4) last there are also so-called main Proglucagon segments (PG (72-158)), wherein there are two glucagon sequence (9) for hiding.Glucagon seemingly unique bioactive product.In contrast, being that glucagon is buried in a macromolecular, and two glucagon-like peptides are then (8) being respectively formed in intestinal mucosa.Following product is formed and is secreted in parallel: 1) corresponding to the glucagon-like peptide of PG (1-69), wherein glucagon sequence occupies No.33-61 residue (12)；2) GLP-1 (7-36) amide, i.e. PG (78-107) amide (13), rather than PG (72-107) amide or PG (72-108) amide being initially considered that, it is inactive.Also the GLP-1 (7-37) (PG (78-108)) (14) with same bioactivity that a small amount of C- terminal glycine extends is generated；3) intermediate peptide -2 (PG (111-122) amide) (15)；With 4) GLP-2 (PG (126-158)) (15,16).A part of glucagon-like peptide is further cut into GRPP (PG (1-30)) and oxyntomodulin (PG (33-69)) (17,18).In these peptides, GLP-1 has most significant bioactivity.

It is secreted since GLP-1 is parallel with glucagon-like peptide/enteroglucagon, therefore to many researchs (6 of enteroglucagon secretion, 7) it can also be used for GLP-1 secretion to some extent, but GLP-1 metabolism is faster, the half-life period in human plasma is 2 minutes (19).Diet rich in carbohydrate or fat can promote to secrete (20), thus it is speculated that this is the result of the microvillus direct interaction of unabsorbed nutrient and intestinal mucosa style of opening L- cell.There may be the endocrine for promoting GLP-1 secretion or neuromechanisms, but have not been demonstrated in human body.

The duodenin effect (29-31) of GLP-1 has been clearly illustrated with the experiment that GLP-1 receptor antagonist exendin 9-39 is carried out, in this experiment, exendin 9-39 shockingly reduces the duodenin effect (21,22) after Oral Administration in Rats glucose.Hormone passes through glucagon/VIP/ calcitonin family GLP-1 receptor and beta cell direct interaction (23) of receptor via the 7- cross-film for belonging to G- protein coupling.It is nearest the experiment proves that importance of the GLP-1 receptor in terms of adjusting insulin secretion, in this experiment, the orientation for having carried out GLP-1 acceptor gene to mouse is destroyed.The hyperglycemia or even Heterozygous animals that homozygous animal with this destruction has the glucose tolerance significantly degenerated and quickly occurs can not be resistant to glucose (24).Signal transduction mechanism mainly includes adenyl cyclase activation, but must also have intracellular Ca²⁺Raising (25,26).The functions of hormones is best described as that the insulin releasing effect (25) of glucose stimulation can be enhanced, but does not know still and couple mechanism between glucose and GLP-1 stimulation.It may include the calcium release (26,27) that a calcium induces.As already mentioned, the beta cell of diabetes patient has the pancreotropic hormone of GLP-1 to act on.Do not know that GLP-1 assigns the insulin secretory cell (26 separated with it yet, 28) relationship between the ability of " glucose irritability ", this isolated insulin secretory cell hardly reacts to individual glucose or GLP-1, but reacts to the combination of the two.However, of equal importance, which also effectively inhibits glucagon secretion (29).Its mechanism is not clear, but seemingly passes through adjacent insulin or the paracrine (25) of somatostatin cell.Pressing down glucagon effect (glucagonostatic action) is also glucose dependency, so depression effect reduces when blood glucose reduces.Due to this double effect, if increasing the GLP-1 concentration in blood plasma by increasing secretion or by exogenous implantation, reaching the molar ratio in the blood of liver between insulin and glucagon by portal circulation will dramatically increase, and glycogen production quantity reduces (30).As a result, blood sugar concentration reduces.Since pancreotropic hormone acts on and press down the glucose dependency of glucagon effect, the effect that glucose reduces is self limiting, and therefore, regardless of dosage, which does not lead to hypoglycemia (31).Having melituric patient, there are still these effects (32), in spite of the weak attached sexual dysfunction of Metabolism control and sulfonylurea (33), may make the complete normalization of blood glucose value slightly above the GLP-1 of physiological dose to these patients implantation.It has been found that GLP-1 can also reduce blood glucose (34) in the type-1 diabetes mellitus people without remaining beta cell secretion capacity, this illustrates the importance of suppression glucagon effect.

In addition to it is for the effect of pancreas islet, GLP-1, which also has gastrointestinal tract, to be had a strong effect.The GLP-1 for inculcating physiological dose can significantly inhibit gastric acid secretion (35,36) pentagastrin induction and that diet induces.GLP-1 also inhibits gastric emptying rate and pancreatin to secrete (36).Solution of people's ileum implantation containing sugar or rouge may have similar depression effect (37,38) to stomach, pancreas secretion and motility.Concomitantly, GLP-1, which secretes, is significantly stimulated, and has speculated the reason in part for GLP-1 may be at least this so-called " ileum lock " (ileal-brake) effect (38).In fact, it has recently been demonstrated that physiologically, the ileum lock effect of GLP-1 may be more important to the effect of pancreas islet than it.Therefore, in dose response research, at least under the minimum infusion rate needed for influencing islet secretion, GLP-1 will affect gastric emptying rate (39).

GLP-1 seems there is effect to food intake.The intraventricular administration of GLP-1 extremely inhibits the food intake (40,42) of rat.This effect seems very special.Therefore, GLP-1 (PG72-107) amide of the end N- extension is inactive, and the GLP-1 antagonist of suitable dosage, exendin9-39, can eliminate the effect (41) of GLP-1.GLP-1 is quickly peripherally administered will not rapidly to inhibit rat chow to take in (41,42).However, the GLP-1 secreted from intestines L- cell is it is possible to as a satiety signal.

Diabetes patient not only has pancreotropic hormone effect, and play the role of GLP-1 to gastrointestinal tract (43), this potentially contributes to glucose bias caused by weakening diet, but, it is often more important that, it is also possible to influence food intake.GLP-1 by administration one week in 4ng/kg/min continuous intravenous infusion, the glucemia for having proven to significantly improve patient NIDDM control and without apparent side effect (44).Peptide is fully effective (45) after subcutaneous administration, but mainly due to the degradation of DPP IV sample enzyme, peptide meeting fast degradation (46,47).

Especially Schmidt etc. gives the amino acid sequence (diabetology (Diabetologia), 28,704-707 (1985)) of GLP-1.Although the interesting pharmacological property of GLP-1 (7-37) and the like has caused very big concern in recent years, the structure about these molecules is known little.Thorton etc. describes secondary structure (biochemistry, 33,3532-3539 (1994)) of the GLP-1 in micella (micelle), but in normal solution, GLP-1 is considered as a kind of very easily-deformable molecule.Unexpectedly, it has been found that the compound that this smaller and very easily-deformable molecule is generated through derivatization, blood plasma distribution curve greatly prolong and still remain activity.

GLP-1, GLP-1 analog and their segment are especially highly effective in 1 type for the treatment of and diabetes B.However high clearance rate limits the validity of these compounds, therefore also needs to improve in this respect.Correspondingly, it is an object of the present invention to provide the derivative of GLP-1 and its analog, these derivatives have the effect curves than GLP-1 (7-37) longer time.It is of the invention further objective is that providing the derivative of GLP-1 and the like, these derivatives have clearance rate more lower than GLP-1 (7-37).It is of the invention further objective is that provide a kind of pharmaceutical composition containing the compounds of this invention, and prepare this composition using the compound of the present invention.The purpose of the present invention also includes providing a kind of method for treating insulin-dependent and adult-onset diabetes.

Bibliography

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Brief summary of the invention

People GLP-1 is the peptide of 37 amino acid residues, it originates from preceding Proglucagon, and preceding Proglucagon mainly synthesizes in distal ileum L- cell, pancreas and brain.It is occurred mainly in L- cell by the process that preceding glucagon original processing generates GLP-1 (7-36) amide, GLP-1 (7-37) and GLP-2.The segment and the like that can describe this peptide with a simple system.Thus, for example Gly⁸- GLP-1 (7-37) indicates to miss out amino acid residue Nos.1-6 from GLP-1, and replaces the GLP-1 segment formed after naturally occurring amino acid residue with Gly at position 8 (Ala).Similarly, Lys³⁴(N^εMyristoyl base)-GLP-1 (7-37) represents the epsilon-amino in 34 Lys residues by the GLP- (7-37) of myristoyl base.When the GLP-1 analog that the end C- referred to herein extends, then unless otherwise stated, it is Arg in 38 amino acid residues, is also Arg (unless otherwise indicated) in 39 optional amino acid residues, is Asp (unless otherwise indicated) in 40 optional amino acid residues.Equally, if the analog that the end C- extends extends to position 41,42,43,44 or 45, unless otherwise stated, the amino acid sequence of this extension is as the corresponding sequence before people in Proglucagon.

At widest aspect, the present invention relates to the derivatives of GLP-1 and the like.Derivative of the invention has interesting pharmacological property, and especially they are with the effect curves than the parent peptide longer time.

Herein, " analog " is for representing a kind of peptide, wherein one or more amino acid residues of parent peptide are replaced by another amino acid residue, and/or wherein one or more amino acid residues on parent peptide are lacked, and/or are wherein added to one or more amino acid residues to parent peptide.This addition can occur in the end N- or the end C- of parent peptide, or occur at both ends.

A kind of peptide is indicated with term " derivative " herein, which has occurred chemical modification in one or more amino acid residues of parent peptide, such as is alkylated, and is acylated, forms ester or amide.

GLP-1 or the derivative of its analog are indicated with term " GLP-1 derivative " herein.Herein, the parent peptide of derivative derivative is referred to as " part GLP-1 " of derivative in some places.

In a preferred embodiment as described in claim 1, the present invention relates to a kind of GLP-1 derivatives, wherein at least one amino acid residue of parent peptide is connected to a lipophilic substituent, condition is if only one lipophilic substituent, and this substituent group is connected on the end N- or C- terminal amino acid residue of parent peptide, then this substituent group is an alkyl or has a ω-carboxylic acid group.

In another preferred embodiment as claimed in claim 2, the present invention relates to the GLP-1 derivatives of only one lipophilic substituent.

In another preferred embodiment as claimed in claim 3, the present invention relates to the GLP-1 derivative of only one lipophilic substituent, which is alkyl or has a ω-carboxylic acid group, and is connected on the -terminal amino acid residue of parent peptide.

In another preferred embodiment as claimed in claim 4, the present invention relates to the GLP-1 derivative of only one lipophilic substituent, which is alkyl or has a ω-carboxylic acid group, and is connected on the C- terminal amino acid residue of parent peptide.

In another preferred embodiment as claimed in claim 5, the present invention relates to the GLP-1 derivative of only one lipophilic substituent, which be may be coupled on parent peptide on any amino acid residue of the non-end N- nor the end C-.

In another preferred embodiment as claimed in claim 6, the present invention relates to the GLP-1 derivatives for having 2 lipophilic substituents.

In another preferred embodiment as claimed in claim 7, the present invention relates to the GLP-1 derivatives for having 2 lipophilic substituents, one of lipophilic substituent is connected on the -terminal amino acid residue of parent peptide, and another lipophilic substituent is connected on the C- terminal amino acid residue of parent peptide.

In another preferred embodiment as claimed in claim 8, the present invention relates to the GLP-1 derivatives for having 2 lipophilic substituents, one of lipophilic substituent is connected on the -terminal amino acid residue of parent peptide, another lipophilic substituent be connected to parent peptide neither on the amino acid residue of the end N- nor the end C-.

In another preferred embodiment as claimed in claim 9, the present invention relates to the GLP-1 derivatives for having 2 lipophilic substituents, one of lipophilic substituent is connected on the C- terminal amino acid residue of parent peptide, another lipophilic substituent be connected to parent peptide neither on the amino acid residue of the end N- nor the end C-.

In further preferred embodiment as claimed in claim 10, the present invention relates to a kind of GLP-1 (7-C) derivatives, wherein C is selected from one group containing 38,39,40,41,42,43,44 and 45, which only has the lipophilic substituent on the C- terminal amino acid residue that one is connected to parent peptide.

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivatives, and wherein lipophilic substituent contains 4 to 40 carbon atoms, further preferably 8-25 carbon atom.

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivatives, one of lipophilic substituent is attached in some way on an amino acid residue, so that a carboxyl of the lipophilic substituent and an amino of the amino acid residue be made to form amido bond.

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivatives, one of lipophilic substituent is attached in some way on an amino acid residue, so that an amino of the lipophilic substituent and a carboxyl of the amino acid residue be made to form amido bond.

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative, one of lipophilic substituent is connected on parent peptide by a spacer group.

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative, one of lipophilic substituent (optionally by a spacer group) is connected on the epsilon-amino group of a Lys residue contained by parent peptide.

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivatives, one of lipophilic substituent by one have 1-7 methylene, preferably have 2 methylene regardless of branched paraffin α, ω-dicarboxyl spacer group and be connected on parent peptide, this spacer group forms a bridge between an amino of parent peptide and an amino of the lipophilic substituent.

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivatives, one of lipophilic substituent is connected on parent peptide by a spacer group, this spacer group is an amino acid residue or a kind of dipeptides of such as Gly-Lys in addition to Cys.Herein, a kind of " dipeptides of such as Gly-Lys " this expression way refers to such a dipeptides, C- terminal amino acid residue therein is Lys, His or Trp, it is preferred that Lys, -terminal amino acid residue therein is selected from the group containing Ala, Arg, Asp, Asn, Gly, Glu, Gln, Ile, Leu, Val, Phe and Pro.

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivatives, one of lipophilic substituent is connected on parent peptide by a spacer group, this spacer group is an amino acid residue in addition to Cys, or a kind of dipeptides of such as Gly-Lys, and wherein an amino in a carboxyl of parent peptide and Lys residue or dipeptides containing a Lys residue forms an amido bond, and the carboxyl in another amino and the lipophilic substituent in the Lys residue or dipeptides containing a Lys residue forms an amido bond.

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivatives, one of lipophilic substituent is connected on parent peptide by a spacer group, this spacer group is an amino acid residue in addition to Cys, or a kind of dipeptides of such as Gly-Lys, wherein a carboxyl in an amino of parent peptide and the amino acid residue spacer group or dipeptides spacer group forms an amido bond, and a carboxyl of the amino and lipophilic substituent in the amino acid residue spacer group or dipeptides spacer group forms an amido bond.

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivatives, one of lipophilic substituent is connected on parent peptide by a spacer group, this spacer group is an amino acid residue in addition to Cys, or a kind of dipeptides of such as Gly-Lys, and wherein an amino in a carboxyl of parent peptide and the amino acid residue spacer group or dipeptides spacer group forms an amido bond, an amino one amido bond of formation of the amino acid residue spacer group or carboxyl and lipophilic substituent in dipeptides spacer group.

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivatives, one of lipophilic substituent is connected on parent peptide by a spacer group, this spacer group is an amino acid residue in addition to Cys, or a kind of dipeptides of such as Gly-Lys, and wherein a carboxyl of parent peptide forms an amido bond with as an amino in the Asp or Glu of spacer group or the dipeptides spacer group containing an Asp or Glu residue, and the amino in a carboxyl and the lipophilic substituent in the spacer group forms an amido bond.

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative with a lipophilic substituent, which contains luxuriant and rich with fragrance (cyclopentanophenathrene) skeleton of ring penta of a partly or completely perhydrogenating.

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivatives, it has the lipophilic substituent that one is straight chain or branched alkyl groups.

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivatives, it has the lipophilic substituent for the acyl group that one is straight chain or branched fatty acid.

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative for having a lipophilic substituent, which is selected from containing CH₃(CH₂)_nOne acyl group of the group of CO-, wherein n is the integer of 4-38, preferably the integer of 4-24, and the further preferred lipophilic substituent is selected from containing CH₃(CH₂)₆CO-, CH₃(CH₂)₈CO-, CH₃(CH₂)₁₀CO-, CH₃(CH₂)₁₂CO-, CH₃(CH₂)₁₄CO-, CH₃(CH₂)₁₆CO-, CH₃(CH₂)₁₈CO-, CH₃(CH₂)₂₀CO- and CH₃(CH₂)₂₂The group of CO-.

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative with a lipophilic substituent, which is an acyl group of straight chain or branch's alkane alpha, omega-dicarboxylic acid.

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative for having a lipophilic substituent, which is selected from (the CH containing HOOC₂)_mOne acyl group of the group of CO-, wherein m is the integer of 4-38, preferably the integer of 4-24, and the further preferred lipophilic substituent is selected from the (CH containing HOOC₂)₁₄CO-, HOOC (CH₂)₁₆CO-, HOOC (CH₂)₁₈CO-, HOOC (CH₂)₂₀CO- and HOOC (CH₂)₂₂The group of CO-.

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivatives, it has one to have formula CH₃(CH₂)_p((CH₂)_qCOOH)CHNH-CO(CH₂)₂The lipophilic substituent of CO-, wherein p and q is integer, and p+q is the integer of 8-33, preferably the integer of 12-28.

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivatives, it has one to have formula CH₃(CH₂)_rCO-NHCH(COOH)(CH₂)₂The lipophilic substituent of CO-, wherein r is the integer of 10-24.

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivatives, it has one to have formula CH₃(CH₂)_sCO-NHCH((CH₂)₂COOH) the lipophilic substituent of CO-, wherein s is the integer of 8-24.

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivatives, it has one to have formula COOH (CH₂)_tThe lipophilic substituent of CO-, wherein t is the integer of 8-24.

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivatives, it has one to have formula-NHCH (COOH) (CH₂)₄NH-CO(CH₂)_uCH₃Lipophilic substituent, wherein u is the integer of 8-18.

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivatives, it has one to have formula-NHCH (COOH) (CH₂)₄NH-COCH((CH₂)₂COOH)NH-CO(CH₂)_wCH₃Lipophilic substituent, wherein w is the integer of 10-16.

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivatives, it has one to have formula-NHCH (COOH) (CH₂)₄NH-CO(CH₂)₂CH(COOH)NH-CO(CH₂)_xCH₃Lipophilic substituent, wherein x is the integer of 10-16.

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivatives, it has one to have formula-NHCH (COOH) (CH₂)₄NH-CO(CH₂)₂CH(COOH)NH-CO(CH₂)_yCH₃Lipophilic substituent, wherein y is the integer of 0 or 1-22.

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivatives, it has a lipophilic substituent that can be negatively charged.Such lipophilic substituent for example can be the substituent group of a carboxyl.

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative, parent peptide is selected from GLP-1 (1-45) or its analog.

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivatives, it is by being selected from (7-35) containing GLP-1, GLP-1 (7-36), GLP-1 (7-36) amide, GLP-1 (7-37), GLP-1 (7-38), one GLP-1 segment of the group of GLP-1 (7-39), GLP-1 (7-40) and GLP-1 (7-41) or its analog derivative obtain.

In a further preferred embodiment, the present invention relates to a kind of GLP-1 analogs, it is by being selected from (1-35) containing GLP-1, GLP-1 (1-36), GLP-1 (1-36) amide, GLP-1 (1-37), GLP-1 (1-38), the GLP-1 analog of the group of GLP-1 (1-39), GLP-1 (1-40) and GLP-1 (1-41) or its analog derivative obtain.

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivatives, wherein total at most 15, preferably up to 10 amino acid residues are replaced by any a-amino acid residue in the derivative that specified analog includes.

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivatives, wherein at most 15, preferably up to 10 amino acid residues can be replaced by any by the a-amino acid residue of genetic code encoding in the derivative that specified analog includes.

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivatives, wherein at most 6 amino acid residues can be replaced by another by the a-amino acid residue of genetic code encoding in the derivative that specified analog includes.

In a further preferred embodiment, the present invention relates to a kind of GLP-1 (A-B) derivatives, and wherein A is the integer of 1-7, and B is the integer of 38-45；Or the analog of this derivative, wherein the lipophilic substituent being connected on C- terminal amino acid residue containing one, and optionally containing second lipophilic substituent being connected on another amino acid residue.

In a further preferred embodiment, the parent peptide of derivative of the present invention is selected from following member: Arg²⁶- GLP-1 (7-37), Arg³⁴- GLP-1 (7-37), Lys³⁶- GLP-1 (7-37), Arg^26,34Lys³⁶- GLP-1 (7-37), Arg^26,34Lys³⁸- GLP-1 (7-38), Arg^26,34Lys³⁹- GLP-1 (7-39), Arg^26,34Lys⁴⁰- GLP-1 (7-40), Arg²⁶Lys³⁶- GLP-1 (7-37), Arg³⁴Lys³⁶- GLP-1 (7-37), Arg²⁶Lys³⁹- GLP-1 (7-39), Arg³⁴Lys⁴⁰- GLP-1 (7-40), Arg^26,34Lys^36,39- GLP-1 (7-39), Arg^26,34Lys^36,40- GLP-1 (7-40), Gly⁸Arg²⁶- GLP-1 (7-37), Gly⁸Arg³⁴- GLP-1 (7-37), Gly⁸Lys³⁶- GLP-1 (7-37), Gly⁸Arg^26,34Lys³⁶- GLP-1 (7-37), Gly⁸Arg^26,34Lys³⁹- GLP-1 (7-39), Gly⁸Arg^26,34Lys⁴⁰- GLP-1 (7-40), Gly⁸Arg²⁶Lys³⁶- GLP-1 (7-37), Gly⁸Arg³⁴Lys³⁶- GLP-1 (7-37), Gly⁸Arg²⁶Lys³⁹- GLP-1 (7-39), Gly⁸Arg³⁴Lys⁴⁰- GLP-1 (7-40), Gly⁸Arg^26,34Lys^36,39- GLP-1 (7-39) and Gly⁸Arg^26,34Lys^36,40-GLP-1(7-40)。

In a further preferred embodiment, the parent peptide of derivative of the present invention is selected from following member: Arg^26,34Lys³⁸- GLP-1 (7-38), Arg^26,34Lys³⁹- GLP-1 (7-39), Arg^26,34Lys⁴⁰- GLP-1 (7-40), Arg^26,34Lys⁴¹- GLP-1 (7-41), Arg^26,34Lys⁴²- GLP-1 (7-42), Arg^26,34Lys⁴³- GLP-1 (7-43), Arg^26,34Lys⁴⁴- GLP-1 (7-44), Arg^26,34Lys⁴⁵- GLP-1 (7-45), Arg^26,34Lys³⁸- GLP-1 (1-38), Arg^26,34Lys³⁹- GLP-1 (1-39), Arg^26,34Lys⁴⁰- GLP-1 (1-40), Arg^26,34Lys⁴¹- GLP-1 (1-41), Arg^26,34Lys⁴²- GLP-1 (1-42), Arg26,34Lys⁴³- GLP-1 (1-43), Arg^26,34Lys⁴⁴- GLP-1 (1-44), Arg^26,34Lys⁴⁵- GLP-1 (1-45), Arg^26,34Lys³⁸- GLP-1 (2-38), Arg^26,34Lys³⁹- GLP-1 (2-39), Arg^26,34Lys⁴⁰- GLP-1 (2-40), Arg^26,34Lys⁴¹- GLP-1 (2-41), Arg^26,34Lys⁴²- GLP-1 (2-42), Arg^26,34Lys⁴³- GLP-1 (2-43), Arg^26,34Lys⁴⁴- GLP-1 (2-44), Arg^26,34Lys⁴⁵- GLP-1 (2-45), Arg^26,34Lys³⁸- GLP-1 (3-38), Arg^26,34Lys³⁹- GLP-1 (3-39), Arg^26,34Lys⁴⁰- GLP-1 (3-40), Arg^26,34Lys⁴¹- GLP-1 (3-41), Arg^26,34Lys⁴²- GLP-1 (3-42), Arg^26,34Lys⁴³- GLP-1 (3-43), Arg^26,34Lys⁴⁴- GLP-1 (3-44), Arg^26,34Lys⁴⁵- GLP-1 (3-45), Arg^26,34Lys³⁸- GLP-1 (4-38), Arg^26,34Lys³⁹- GLP-1 (4-39), Arg^26,34Lys⁴⁰- GLP-1 (4-40), Arg^26,34Lys⁴¹- GLP-1 (4-41), Arg^26,34Lys⁴²- GLP-1 (4-42), Arg^26,34Lys⁴³- GLP-1 (4-43), Arg^26,34Lys⁴⁴- GLP-1 (4-44), Arg^26,34Lys⁴⁵- GLP-1 (4-45), Arg^26,34Lys³⁸- GLP-1 (5-38), Arg^26,34Lys³⁹- GLP-1 (5-39), Arg^26,34Lys⁴⁰- GLP-1 (5-40), Arg^26,34Lys⁴¹- GLP-1 (5-41), Arg^26,34Lys⁴²- GLP-1 (5-42), Arg^26,34Lys⁴³- GLP-1 (5-43), Arg^26,34Lys⁴⁴- GLP-1 (5-44), Arg^26,34Lys⁴⁵- GLP-1 (5-45), Arg^26,34Lys³⁸- GLP-1 (6-38), Arg^26,34Lys³⁹- GLP-1 (6-39), Arg^26,34Lys⁴⁰- GLP-1 (6-40), Arg^26,34Lys⁴¹- GLP-1 (6-41), Arg^26,34Lys⁴²- GLP-1 (6-42), Arg^26,34Lys⁴³- GLP-1 (6-43), Arg^26,34Lys⁴⁴- GLP-1 (6-44), Arg^26,34Lys⁴⁵- GLP-1 (6-45), Arg²⁶Lys³⁸- GLP-1 (1-38), Arg³⁴Lys³⁸- GLP-1 (1-38), Arg^26,34Lys^36,38- GLP-1 (1-38), Arg²⁶Lys³⁸- GLP-1 (7-38), Arg³⁴Lys³⁸- GLP-1 (7-38), Arg^26,34Lys^36,38- GLP-1 (7-38), Arg^26,34Lys³⁸- GLP-1 (7-38), Arg²⁶Lys³⁹- GLP-1 (1-39), Arg³⁴Lys³⁹- GLP-1 (1-39), Arg^26,34Lys^36,39- GLP-1 (1-39), Arg²⁶Lys³⁹- GLP-1 (7-39), Arg³⁴Lys³⁹- GLP-1 (7-39) and Arg^26,34Lys^36,39-GLP-1(7-39).

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative, parent peptide therein is selected from following member: [Arg²⁶- GLP-1 (7-37), Arg³⁴- GLP-1 (7-37), Lys³⁶- GLP-1 (7-37), Arg^26,34Lys³⁶- GLP-1 (7-37), Arg²⁶Lys³⁶- GLP-1 (7-37), Arg³⁴Lys³⁶- GLP-1 (7-37), Gly⁸Arg²⁶- GLP-1 (7-37), Gly⁸Arg³⁴- GLP-1 (7-37), Gly⁸Lys³⁶- GLP-1 (7-37), Gly⁸Arg²⁶, 34Lys³⁶- GLP-1 (7-37), Gly⁸Arg²⁶Lys³⁶- GLP-1 (7-37) and Gly⁸Arg³⁴Lys³⁶-GLP-1(7-37).

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative, parent peptide therein is selected from following member: Arg²⁶Lys³⁸- GLP-1 (7-38), Arg^26,34Lys³⁸- GLP-1 (7-38), Arg^26,34Lys^36,38- GLP-1 (7-38), Gly⁸Arg²⁶Lys³⁸- GLP-1 (7-38) and Gly⁸Arg^26,34Lys^36,38-GLP-1(7-38).

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative, parent peptide therein is selected from following member: Arg²⁶Lys³⁹- GLP-1 (7-39), Arg^26,34Lys^36,39- GLP-1 (7-39), Gly⁸Arg²⁶Lys³⁹- GLP-1 (7-39) and Gly⁸Arg^26,34Lys^36,39-GLP-1(7-39).

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative, parent peptide therein is selected from following member: Arg³⁴Lys⁴⁰- GLP-1 (7-40), Arg^26,34Lys^36,40- GLP-1 (7-40), Gly⁸Arg³⁴Lys⁴⁰- GLP-1 (7-40) and Gly⁸Arg²⁶, 34Lys^36,40-GLP-1(7-40).

In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative, parent peptide therein is selected from following member:

Lys²⁶(N^εMyristoyl base)-GLP-1 (7-37)；

Lys³⁴(N^εMyristoyl base)-GLP-1 (7-37)；

Lys^26,34Bis- (N^εMyristoyl base)-GLP-1 (7-37)；

Gly⁸Lys²⁶(N^εMyristoyl base)-GLP-1 (7-37)；

Gly⁸Lys³⁴(N^εMyristoyl base)-GLP-1 (7-37)；

Gly⁸Lys^26,34Bis- (N^εMyristoyl base)-GLP-1 (7-37)；

Arg²⁶Lys³⁴(N^εMyristoyl base)-GLP-1 (7-37)；

Lys²⁶(N^εMyristoyl base)-GLP-1 (7-38)；

Lys³⁴(N^εMyristoyl base)-GLP-1 (7-38)；

Lys^26,34Bis- (N^εMyristoyl base)-GLP-1 (7-38)；

Gly⁸Lys²⁶(N^εMyristoyl base)-GLP-1 (7-38)；

Gly⁸Lys³⁴(N^εMyristoyl base)-GLP-1 (7-38)；

Gly⁸Lys^26,34Bis- (N^εMyristoyl base)-GLP-1 (7-38)；

Arg²⁶Lys³⁴(N^εMyristoyl base)-GLP-1 (7-38)；

Lys²⁶(N^εMyristoyl base)-GLP-1 (7-39)；

Lys³⁴(N^εMyristoyl base)-GLP-1 (7-39)；

Lys^26,34Bis- (N^εMyristoyl base)-GLP-1 (7-39)；

Gly⁸Lys²⁶(N^εMyristoyl base)-GLP-1 (7-39)；

Gly⁸Lys³⁴(N^εMyristoyl base)-GLP-1 (7-39)；

Gly⁸Lys^26,34Bis- (N^εMyristoyl base)-GLP-1 (7-39)；

Arg²⁶Lys³⁴(N^εMyristoyl base)-GLP-1 (7-39)；

Lys²⁶(N^εMyristoyl base)-GLP-1 (7-40)；

Lys³⁴(N^εMyristoyl base)-GLP-1 (7-40)；

Lys^26,34Bis- (N^εMyristoyl base)-GLP-1 (7-40)；

Gly⁸Lys²⁶(N^εMyristoyl base)-GLP-1 (7-40)；

Gly⁸Lys³⁴(N^εMyristoyl base)-GLP-1 (7-40)；

Gly⁸Lys^26,34Bis- (N^εMyristoyl base)-GLP-1 (7-40)；

Arg²⁶Lys³⁴(N^εMyristoyl base)-GLP-1 (7-40)；

Lys²⁶(N^εMyristoyl base)-GLP-1 (7-36)；

Lys³⁴(N^εMyristoyl base)-GLP-1 (7-36)；

Lys^26,34Bis- (N^εMyristoyl base)-GLP-1 (7-36)；

Gly⁸Lys²⁶(N^εMyristoyl base)-GLP-1 (7-36)；

Gly⁸Lys³⁴(N^εMyristoyl base)-GLP-1 (7-36)；

Gly⁸Lys^26,34Bis- (N^εMyristoyl base)-GLP-1 (7-36)；

Arg²⁶Lys³⁴(N^εMyristoyl base)-GLP-1 (7-36)；

Lys²⁶(N^εMyristoyl base)-GLP-1 (7-35)；

Lys³⁴(N^εMyristoyl base)-GLP-1 (7-35)；

Lys^26,34Bis- (N^εMyristoyl base)-GLP-1 (7-35)；

Gly⁸Lys²⁶(N^εMyristoyl base)-GLP-1 (7-35)；

Gly⁸Lys³⁴(N^εMyristoyl base)-GLP-1 (7-35)；

Gly⁸Lys^26,34Bis- (N^εMyristoyl base)-GLP-1 (7-35)；

Arg²⁶Lys³⁴(N^εMyristoyl base)-GLP-1 (7-35)；

Lys²⁶(N^εMyristoyl base)-GLP-1 (7-36) amide；

Lys³⁴(N^εMyristoyl base)-GLP-1 (7-36) amide；

Lys^26,34Bis- (N^εMyristoyl base)-GLP-1 (7-36) amide；

Gly⁸Lys²⁶(N^εMyristoyl base)-GLP-1 (7-36) amide；

Gly⁸Lys³⁴(N^εMyristoyl base)-GLP-1 (7-36) amide；

Gly⁸Lys^26,34Bis- (N^εMyristoyl base)-GLP-1 (7-36) amide；

Arg²⁶Lys³⁴(N^εMyristoyl base)-GLP-1 (7-36) amide；

Gly⁸Arg²⁶Lys³⁴(N^εMyristoyl base)-GLP-1 (7-37)；

Lys²⁶(N^εMyristoyl base) Arg³⁴-GLP-1(7-37)；

Gly⁸Lys²⁶(N^εMyristoyl base) Arg³⁴-GLP-1(7-37)；

Arg^26,34Lys³⁶(N^εMyristoyl base)-GLP-1 (7-37)；

Gly⁸Arg^26,34Lys³⁶(N^εMyristoyl base)-GLP-1 (7-37)；

Gly⁸Arg²⁶Lys³⁴(N^εMyristoyl base)-GLP-1 (7-38)；

Lys²⁶(N^εMyristoyl base) Arg³⁴-GLP-1(7-38)；

Gly⁸Lys²⁶(N^εMyristoyl base) Arg³⁴-GLP-1(7-38)；

Arg^26,34Lys³⁶(N^εMyristoyl base)-GLP-1 (7-38)；

Arg^26,34Lys³⁸(N^εMyristoyl base)-GLP-1 (7-38)；

Gly⁸Arg^26,34Lys³⁶(N^εMyristoyl base)-GLP-1 (7-38)；

Gly⁸Arg²⁶Lys³⁴(N^εMyristoyl base)-GLP-1 (7-39)；

Lys²⁶(N^εMyristoyl base) Arg³⁴-GLP-1(7-39)；

Gly⁸Lys²⁶(N^εMyristoyl base) Arg³⁴-GLP-1(7-39)；

Arg^26,34Lys³⁶(N- myristoyl base)-GLP-1 (7-39)；

Gly⁸Arg^26,34Lys³⁶(N^εMyristoyl base)-GLP-1 (7-39)；

Gly⁸Arg²⁶Lys³⁴(N^εMyristoyl base)-GLP-1 (7-40)；

Lys²⁶(N^εMyristoyl base) Arg³⁴-GLP-1(7-40)；

Gly⁸Lys²⁶(N^εMyristoyl base) Arg³⁴-GLP-1(7-40)；

Arg^26,34Lys³⁶(N^εMyristoyl base)-GLP-1 (7-40)；

Gly⁸Arg^26,34Lys³⁶(N^εMyristoyl base)-GLP-1 (7-40)；

Lys²⁶(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-37)；

Lys³⁴(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-37)；

Lys^26,34Bis- (N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-37)；

Gly⁸Lys²⁶(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-37)；

Gly⁸Lys³⁴(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-37)；

Gly⁸Lys^26,34Bis- (N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-37)；

Lys²⁶(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-38)；

Lys³⁴(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-38)；

Lys^26,34Bis- (N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-38)；

Gly⁸Lys²⁶(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-38)；

Gly⁸Lys³⁴(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-38)；

Gly⁸Lys^26,34Bis- (N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-38)；

Lys²⁶(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-39)；

Lys³⁴(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-39)；

Lys^26,34Bis- (N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-39)；

Gly⁸Lys²⁶(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-39)；

Gly⁸Lys³⁴(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-39)；

Gly⁸Lys^26,34Bis- (N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-39)；

Lys²⁶(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-40)；

Lys³⁴(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-40)；

Lys^26,34Bis- (N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-40)；

Gly⁸Lys²⁶(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-40)；

Gly⁸Lys³⁴(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-40)；

Gly⁸Lys^26,34Bis- (N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-40)；

Lys²⁶(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-36)；

Lys³⁴(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-36)；

Lys^26,34Bis- (N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-36)；

Gly⁸Lys²⁶(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-36)；

Gly⁸Lys³⁴(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-36)；

Gly⁸Lys^26,34Bis- (N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-36)；

Lys²⁶(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-36) amide；

Lys³⁴(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-36) amide；

Lys^26,34Bis- (N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-36) amide；

Gly⁸Lys²⁶(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-36) amide；

Gly⁸Lys³⁴(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-36) amide；

Gly⁸Lys^26,34Bis- (N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-36) amide；

Lys²⁶(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-35)；

Lys³⁴(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-35)；

Lys^26,34Double N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-35)；

Gly⁸Lys²⁶(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-35)；

Gly⁸Lys³⁴(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-35)；

Gly⁸Lys^26,34Bis- (N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-35)；

Arg²⁶Lys³⁴(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-37)；

Gly⁸Arg²⁶Lys³⁴(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-37)；

Lys²⁶(N^ε(19 acyl group of ω-carboxyl)) Arg³⁴-GLP-1(7-37)；

Gly⁸Lys²⁶(N^ε(19 acyl group of ω-carboxyl)) Arg³⁴-GLP-1(7-37)；

Arg^26,34Lys³⁶(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-37)；

Gly⁸Arg^26,34Lys³⁶(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-37)；

Arg²⁶Lys³⁴(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-38)；

Gly⁸Arg²⁶Lys³⁴(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-38)；

Lys²⁶(N^ε(19 acyl group of ω-carboxyl)) Arg³⁴-GLP-1(7-38)；

Gly⁸Lys²⁶(N^ε(19 acyl group of ω-carboxyl)) Arg³⁴-GLP-1(7-38)；

Arg^26,34Lys³⁶(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-38)；

Arg^26,34Lys³⁸(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-38)；

Gly⁸Arg^26,34Lys³⁶(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-38)；

Arg²⁶Lys³⁴(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-39)；

Gly⁸Arg²⁶Lys³⁴(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-39)；

Lys²⁶(N^ε(19 acyl group of ω-carboxyl)) Arg³⁴-GLP-1(7-39)；

Gly⁸Lys²⁶(N^ε(19 acyl group of ω-carboxyl)) Arg³⁴-GLP-1(7-39)；

Arg^26,34Lys³⁶(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-39)；

Gly⁸Arg^26,34Lys³⁶(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-39)；

Arg²⁶Lys³⁴(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-40)；

Gly⁸Arg²⁶Lys³⁴(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-40)；

Lys²⁶(N^ε(19 acyl group of ω-carboxyl)) Arg³⁴-GLP-1(7-40)；

Gly⁸Lys²⁶(N^ε(19 acyl group of ω-carboxyl)) Arg³⁴-GLP-1(7-40)；

Arg^26,34Lys³⁶(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-40)；

Gly⁸Arg^26,34Lys³⁶(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-40)；

Lys²⁶(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-37)；

Lys³⁴(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-37)；

Lys^26,34Bis- (N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-37)；

Gly⁸Lys²⁶(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-37)；

Gly⁸Lys³⁴(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-37)；

Gly⁸Lys^26,34Bis- (N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-37)；

Arg²⁶Lys³⁴(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-37)；

Lys²⁶(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-38)；

Lys³⁴(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-38)；

Lys^26,34Bis- (N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-38)；

Gly⁸Lys²⁶(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-38)；

Gly⁸Lys³⁴(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-38)；

Gly⁸Lys^26,34Bis- (N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-38)；

Arg²⁶Lys³⁴(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-38)；

Lys²⁶(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-39)；

Lys³⁴(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-39)；

Lys^26,34Bis- (N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-39)；

Gly⁸Lys²⁶(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-39)；

Gly⁸Lys³⁴(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-39)；

Gly⁸Lys^26,34Bis- (N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-39)；

Arg²⁶Lys³⁴(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-39)；

Lys²⁶(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-40)；

Lys³⁴(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-40)；

Lys^26,34Bis- (N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-40)；

Gly⁸Lys²⁶(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-40)；

Gly⁸Lys³⁴(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-40)；

Gly⁸Lys^26,34Bis- (N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-40)；

Arg²⁶Lys³⁴(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-40)；

Lys²⁶(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-36)；

Lys³⁴(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-36)；

Lys^26,34Bis- (N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-36)；

Gly⁸Lys²⁶(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-36)；

Gly⁸Lys³⁴(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-36)；

Gly⁸Lys^26,34Bis- (N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-36)；

Arg²⁶Lys³⁴(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-36)；

Lys²⁶(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-35)；

Lys³⁴(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-35)；

Lys^26,34Bis- (N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-35)；

Gly⁸Lys²⁶(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-35)；

Gly⁸Lys³⁴(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-35)；

Gly⁸Lys^26,34Bis- (N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-35)；

Arg²⁶Lys³⁴(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-35)；

Lys²⁶(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-36) amide；

Lys³⁴(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-36) amide；

Lys^26,34Bis- (N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-36) amide；

Gly⁸Lys²⁶(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-36) amide；

Gly⁸Lys³⁴(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-36) amide；

Gly⁸Lys^26,34Bis- (N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-35) amide；

Arg²⁶Lys³⁴(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-35) amide；

Gly⁸Arg²⁶Lys³⁴(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-37)；

Lys²⁶(N^ε(7- deoxidation gallbladder acyl group)) Arg³⁴-GLP-1(7-37)；

Gly⁸Lys²⁶(N^ε(7- deoxidation gallbladder acyl group)) Arg³⁴-GLP-1(7-37)；

Arg^26,34Lys³⁶(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-37)；

Gly⁸Arg^26,34Lys³⁶(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-37)；

Lys²⁶(N^ε(gallbladder acyl group))-GLP-1 (7-37)；

Lys³⁴(N^ε(gallbladder acyl group))-GLP-1 (7-37)；

Lys^26,34Bis- (N^ε(gallbladder acyl group))-GLP-1 (7-37)；

Gly⁸Lys²⁶(N^ε(gallbladder acyl group))-GLP-1 (7-37)；

Gly⁸Lys³⁴(N^ε(gallbladder acyl group))-GLP-1 (7-37)；

Gly⁸Lys^26,34Bis- (N^ε(gallbladder acyl group))-GLP-1 (7-37)；

Arg²⁶Lys³⁴(N^ε(gallbladder acyl group))-GLP-1 (7-37)；

Gly⁸Arg²⁶Lys³⁴(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-38)；

Lys²⁶(N^ε(7- deoxidation gallbladder acyl group)) Arg³⁴-GLP-1(7-38)；

Gly⁸Lys²⁶(N^ε(7- deoxidation gallbladder acyl group)) Arg³⁴-GLP-1(7-38)；

Arg^26,34Lys³⁶(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-38)；

Arg^26,34Lys³⁸(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-38)；

Gly⁸Arg^26,34Lys³⁶(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-38)；

Lys²⁶(N^ε(gallbladder acyl group))-GLP-1 (7-38)；

Lys³⁴(N^ε(gallbladder acyl group))-GLP-1 (7-38)；

Lys^26,34Bis- (N^ε(gallbladder acyl group))-GLP-1 (7-38)；

Gly⁸Lys²⁶(N^ε(gallbladder acyl group))-GLP-1 (7-38)；

Gly⁸Lys³⁴(N^ε(gallbladder acyl group))-GLP-1 (7-38)；

Gly⁸Lys^26,34Bis- (N^ε(gallbladder acyl group))-GLP-1 (7-38)；

Arg²⁶Lys³⁴(N^ε(gallbladder acyl group))-GLP-1 (7-38)；

Gly⁸Arg²⁶Lys³⁴(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-39)；

Lys²⁶(N^ε(7- deoxidation gallbladder acyl group)) Arg³⁴-GLP-1(7-39)；

Gly⁸Lys²⁶(N^ε(7- deoxidation gallbladder acyl group)) Arg³⁴-GLP-1(7-39)；

Arg^26,34Lys³⁶(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-39)；

Gly⁸Arg^26,34Lys³⁶(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-39)；

Lys²⁶(N^ε(gallbladder acyl group))-GLP-1 (7-39)；

Lys³⁴(N^ε(gallbladder acyl group))-GLP-1 (7-39)；

Lys^26,34Bis- (N^ε(gallbladder acyl group))-GLP-1 (7-39)；

Gly⁸Lys²⁶(N^ε(gallbladder acyl group))-GLP-1 (7-39)；

Gly⁸Lys³⁴(N^ε(gallbladder acyl group))-GLP-1 (7-39)；

Gly⁸Lys^26,34Bis- (N^ε(gallbladder acyl group))-GLP-1 (7-39)；

Arg²⁶Lys³⁴(N^ε(gallbladder acyl group))-GLP-1 (7-39)；

Gly⁸Arg²⁶Lys³⁴(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-40)；

Lys²⁶(N^ε(7- deoxidation gallbladder acyl group)) Arg³⁴-GLP-1(7-40)；

Gly⁸Lys²⁶(N^ε(7- deoxidation gallbladder acyl group)) Arg³⁴-GLP-1(7-40)；

Arg^26,34Lys³⁶(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-40)；

Gly⁸Arg^26,34Lys³⁶(N^ε(7- deoxidation gallbladder acyl group))-GLP-1 (7-40)；

Lys²⁶(N^ε(gallbladder acyl group))-GLP-1 (7-40)；

Lys³⁴(N^ε(gallbladder acyl group))-GLP-1 (7-40)；

Lys^26,34Bis- (N^ε(gallbladder acyl group))-GLP-1 (7-40)；

Gly⁸Lys²⁶(N^ε(gallbladder acyl group))-GLP-1 (7-40)；

Gly⁸Lys³⁴(N^ε(gallbladder acyl group))-GLP-1 (7-40)；

Gly⁸Lys^26,34Bis- (N^ε(gallbladder acyl group))-GLP-1 (7-40)；

Arg²⁶Lys³⁴(N^ε(gallbladder acyl group))-GLP-1 (7-40)；

Lys²⁶(N^ε(gallbladder acyl group))-GLP-1 (7-36)；

Lys³⁴(N^ε(gallbladder acyl group))-GLP-1 (7-36)；

Lys^26,34Bis- (N^ε(gallbladder acyl group))-GLP-1 (7-36)；

Gly⁸Lys²⁶(N^ε(gallbladder acyl group))-GLP-1 (7-36)；

Gly⁸Lys³⁴(N^ε(gallbladder acyl group))-GLP-1 (7-36)；

Gly⁸Lys^26,34Bis- (N^ε(gallbladder acyl group))-GLP-1 (7-36)；

Arg²⁶Lys³⁴(N^ε(gallbladder acyl group))-GLP-1 (7-36)；

Lys²⁶(N^ε(gallbladder acyl group))-GLP-1 (7-35)；

Lys³⁴(N^ε(gallbladder acyl group))-GLP-1 (7-35)；

Lys^26,34Bis- (N^ε(gallbladder acyl group))-GLP-1 (7-35)；

Gly⁸Lys²⁶(N^ε(gallbladder acyl group))-GLP-1 (7-35)；

Gly⁸Lys³⁴(N^ε(gallbladder acyl group))-GLP-1 (7-35)；

Gly⁸Lys^26,34Bis- (N^ε(gallbladder acyl group))-GLP-1 (7-35)；

Arg²⁶Lys³⁴(N^ε(gallbladder acyl group))-GLP-1 (7-35)；

Lys²⁶(N^ε(gallbladder acyl group))-GLP-1 (7-36) amide；

Lys³⁴(N^ε(gallbladder acyl group))-GLP-1 (7-36) amide；

Lys^26,34Bis- (N^ε(gallbladder acyl group))-GLP-1 (7-36) amide；

Gly⁸Lys²⁶(N^ε(gallbladder acyl group))-GLP-1 (7-36) amide；

Gly⁸Lys³⁴(N^ε(gallbladder acyl group))-GLP-1 (7-36) amide；

Gly⁸Lys^26,34Bis- (N^ε(gallbladder acyl group))-GLP-1 (7-36) amide；

Arg²⁶Lys³⁴(N^ε(gallbladder acyl group))-GLP-1 (7-36) amide；

Gly⁸Arg²⁶Lys³⁴(N^ε(gallbladder acyl group))-GLP-1 (7-37)；

Lys²⁶(N^ε(gallbladder acyl group)) Arg³⁴-GLP-1(7-37)；

Gly⁸Lys²⁶(N^ε(gallbladder acyl group)) Arg³⁴-GLP-1(7-37)；

Arg^26,34Lys³⁶(N^ε(gallbladder acyl group))-GLP-1 (7-37)；

Gly⁸Arg^26,34Lys³⁶(N^ε(gallbladder acyl group))-GLP-1 (7-37)；

Lys²⁶(N^ε(stone gallbladder acyl group))-GLP-1 (7-37)；

Lys³⁴(N^ε(stone gallbladder acyl group))-GLP-1 (7-37)；

Lys^26,34Bis- (N^ε(stone gallbladder acyl group))-GLP-1 (7-37)；

Gly⁸Lys²⁶(N^ε(stone gallbladder acyl group))-GLP-1 (7-37)；

Gly⁸Lys³⁴(N^ε(stone gallbladder acyl group))-GLP-1 (7-37)；

Gly⁸Lys^26,34Bis- (N^ε(stone gallbladder acyl group))-GLP-1 (7-37)；

Arg²⁶Lys³⁴(N^ε(stone gallbladder acyl group))-GLP-1 (7-37)；

Gly⁸Arg²⁶Lys³⁴(N^ε(gallbladder acyl group))-GLP-1 (7-38)；

Lys²⁶(N^ε(gallbladder acyl group)) Arg³⁴-GLP-1(7-38)；

Gly⁸Lys²⁶(N^ε(gallbladder acyl group)) Arg³⁴-GLP-1(7-38)；

Arg^26,34Lys³⁶(N^ε(gallbladder acyl group))-GLP-1 (7-38)；

Arg^26,34Lys³⁸(N^ε(gallbladder acyl group))-GLP-1 (7-38)；

Gly⁸Arg^26,34Lys³⁶(N^ε(gallbladder acyl group))-GLP-1 (7-38)；

Lys²⁶(N^ε(stone gallbladder acyl group))-GLP-1 (7-38)；

Lys³⁴(N^ε(stone gallbladder acyl group))-GLP-1 (7-38)；

Lys^26,34Bis- (N^ε(stone gallbladder acyl group))-GLP-1 (7-38)；

Gly⁸Lys²⁶(N^ε(stone gallbladder acyl group))-GLP-1 (7-38)；

Gly⁸Lys³⁴(N^ε(stone gallbladder acyl group))-GLP-1 (7-38)；

Gly⁸Lys^26,34Bis- (N^ε(stone gallbladder acyl group))-GLP-1 (7-38)；

Arg²⁶Lys³⁴(N^ε(stone gallbladder acyl group))-GLP-1 (7-38)；

Gly⁸Arg²⁶Lys³⁴(N^ε(gallbladder acyl group))-GLP-1 (7-39)；

Lys²⁶(N^ε(gallbladder acyl group)) Arg³⁴-GLP-1(7-39)；

Gly⁸Lys²⁶(N^ε(gallbladder acyl group)) Arg³⁴-GLP-1(7-39)；

Arg^26,34Lys³⁶(N^ε(gallbladder acyl group))-GLP-1 (7-39)；

Gly⁸Arg^26,34Lys³⁶(N^ε(gallbladder acyl group))-GLP-1 (7-39)；

Lys²⁶(N^ε(stone gallbladder acyl group))-GLP-1 (7-39)；

Lys³⁴(N^ε(stone gallbladder acyl group))-GLP-1 (7-39)；

Lys^26,34Bis- (N^ε(stone gallbladder acyl group))-GLP-1 (7-39)；

Gly⁸Lys²⁶(N^ε(stone gallbladder acyl group))-GLP-1 (7-39)；

Gly⁸Lys³⁴(N^ε(stone gallbladder acyl group))-GLP-1 (7-39)；

Gly⁸Lys^26,34Bis- (N^ε(stone gallbladder acyl group))-GLP-1 (7-39)；

Arg²⁶Lys³⁴(N^ε(stone gallbladder acyl group))-GLP-1 (7-39)；

Gly⁸Arg²⁶Lys³⁴(N^ε(gallbladder acyl group))-GLP-1 (7-40)；

Lys²⁶(N^ε(gallbladder acyl group)) Arg³⁴-GLP-1(7-40)；

Gly⁸Lys²⁶(N^ε(gallbladder acyl group)) Arg³⁴-GLP-1(7-40)；

Arg^26,34Lys³⁶(N^ε(gallbladder acyl group))-GLP-1 (7-40)；

Gly⁸Arg^26,34Lys³⁶(N^ε(gallbladder acyl group))-GLP-1 (7-40)；

Lys²⁶(N^ε(stone gallbladder acyl group))-GLP-1 (7-40)；

Lys³⁴(N^ε(stone gallbladder acyl group))-GLP-1 (7-40)；

Lys^26,34Bis- (N^ε(stone gallbladder acyl group))-GLP-1 (7-40)；

Gly⁸Lys²⁶(N^ε(stone gallbladder acyl group))-GLP-1 (7-40)；

Gly⁸Lys³⁴(N^ε(stone gallbladder acyl group))-GLP-1 (7-40)；

Gly⁸Lys^26,34Bis- (N^ε(stone gallbladder acyl group))-GLP-1 (7-40)；

Arg²⁶Lys³⁴(N^ε(stone gallbladder acyl group))-GLP-1 (7-37)；

Lys²⁶(N^ε(stone gallbladder acyl group))-GLP-1 (7-36)；

Lys³⁴(N^ε(stone gallbladder acyl group))-GLP-1 (7-36)；

Lys^26,34Bis- (N^ε(stone gallbladder acyl group))-GLP-1 (7-36)；

Gly⁸Lys²⁶(N^ε(stone gallbladder acyl group))-GLP-1 (7-36)；

Gly⁸Lys³⁴(N^ε(stone gallbladder acyl group))-GLP-1 (7-36)；

Gly⁸Lys^26,34Bis- (N^ε(stone gallbladder acyl group))-GLP-1 (7-36)；

Arg²⁶Lys³⁴(N^ε(stone gallbladder acyl group))-GLP-1 (7-36)；

Lys²⁶(N^ε(stone gallbladder acyl group))-GLP-1 (7-35)；

Lys³⁴(N^ε(stone gallbladder acyl group))-GLP-1 (7-35)；

Lys^26,34Bis- (N^ε(stone gallbladder acyl group))-GLP-1 (7-35)；

Gly⁸Lys²⁶(N^ε(stone gallbladder acyl group))-GLP-1 (7-35)；

Gly⁸Lys³⁴(N^ε(stone gallbladder acyl group))-GLP-1 (7-35)；

Gly⁸Lys^26,34Bis- (N^ε(stone gallbladder acyl group))-GLP-1 (7-35)；

Arg²⁶Lys³⁴(N^ε(stone gallbladder acyl group))-GLP-1 (7-35)；

Lys²⁶(N^ε(stone gallbladder acyl group))-GLP-1 (7-36) amide；

Lys³⁴(N^ε(stone gallbladder acyl group))-GLP-1 (7-36) amide；

Lys^26,34Bis- (N^ε(stone gallbladder acyl group))-GLP-1 (7-36) amide；

Gly⁸Lys²⁶(N^ε(stone gallbladder acyl group))-GLP-1 (7-36) amide；

Gly⁸Lys³⁴(N^ε(stone gallbladder acyl group))-GLP-1 (7-36) amide；

Gly⁸Lys^26,34Bis- (N^ε(stone gallbladder acyl group))-GLP-1 (7-36) amide；

Arg²⁶Lys³⁴(N^ε(stone gallbladder acyl group))-GLP-1 (7-36) amide；

Gly⁸Arg²⁶Lys³⁴(N^ε(stone gallbladder acyl group))-GLP-l (7-37)；

Lys²⁶(N^ε(stone gallbladder acyl group)) Arg³⁴-GLP-1(7-37)；

Gly⁸Lys²⁶(N^ε(stone gallbladder acyl group)) Arg³⁴-GLP-1(7-37)；

Arg^26,34Lys³⁶(N^ε(stone gallbladder acyl group))-GLP-1 (7-37)；

Arg^26,34Lys³⁸(N^ε(stone gallbladder acyl group))-GLP-1 (7-37)；

Gly⁸Arg^26,34Lys³⁶(N^ε(stone gallbladder acyl group))-GLP-1 (7-37)；

Gly⁸Arg²⁶Lys³⁴(N^ε(stone gallbladder acyl group))-GLP-1 (7-38)；

Lys²⁶(N^ε(stone gallbladder acyl group)) Arg³⁴-GLP-1(7-38)；

Gly⁸Lys²⁶(N^ε(stone gallbladder acyl group)) Arg³⁴-GLP-1(7-38)；

Arg^26,34Lys³⁶(N^ε(stone gallbladder acyl group))-GLP-1 (7-38)；

Arg^26,34Lys³⁸(N^ε(stone gallbladder acyl group))-GLP-1 (7-38)；

Gly⁸Arg^26,34Lys³⁶(N^ε(stone gallbladder acyl group))-GLP-1 (7-38)；

Gly⁸Arg²⁶Lys³⁴(N^ε(stone gallbladder acyl group))-GLP-1 (7-39)；

Lys²⁶(N^ε(stone gallbladder acyl group)) Arg³⁴-GLP-1(7-39)；

Gly⁸Lys²⁶(N^ε(stone gallbladder acyl group)) Arg³⁴-GLP-1(7-39)；

Arg^26,34Lys³⁶(N^ε(stone gallbladder acyl group))-GLP-1 (7-39)；

Gly⁸Arg^26,34Lys³⁶(N^ε(stone gallbladder acyl group))-GLP-1 (7-39)；

Gly⁸Arg²⁶Lys³⁴(N^ε(stone gallbladder acyl group))-GLP-1 (7-40)；

Lys²⁶(N^ε(stone gallbladder acyl group)) Arg³⁴-GLP-1(7-40)；

Gly⁸Lys²⁶(N^ε(stone gallbladder acyl group)) Arg³⁴-GLP-1(7-40)；

Arg^26,34Lys³⁶(N^ε(stone gallbladder acyl group))-GLP-1 (7-40)；With

Gly⁸Arg^26,34Lys³⁶(N^ε(stone gallbladder acyl group))-GLP-1 (7-40)

In a further preferred embodiment, the present invention relates to contain a kind of GLP-1 derivative and a kind of pharmaceutical composition of pharmaceutically acceptable carrier.

In a further preferred embodiment, the purposes the present invention relates to GLP-1 derivative according to the present invention in the medicament that preparation has than the effect curves of GLP-1 (7-37) longer time.

In a further preferred embodiment, the purposes the present invention relates to GLP-1 derivative according to the present invention in the medicament for treating adult-onset diabetes that preparation has the function and effect of longer time.

In a further preferred embodiment, the purposes the present invention relates to GLP-1 derivative according to the present invention in the medicament for treating insulin-dependent diabetes mellitus that preparation has the function and effect of longer time.

In a further preferred embodiment, the purposes the present invention relates to GLP-1 derivative according to the present invention in the medicament for treating obesity that preparation has the function and effect of longer time.

In a further preferred embodiment, the present invention relates to the method for treating insulin-dependent or adult-onset diabetes in the patient for needing this treatment, this method includes dose a patient with the GLP-1 derivative described in claim 1 and pharmaceutically acceptable carrier of therapeutically effective amount.

Detailed description of the present invention

To obtain the sufficiently prolonged effect curves of GLP-1 derivative, the lipophilic substituent for being connected to the part GLP-1 preferably comprises 4-40 carbon atom, especially 8-25 carbon atom.The lipophilic substituent can form amido bond by the amino of its carboxyl and the amino acid residue being attached thereto and be connected on the amino of the part GLP-1.Or selectively, which can form amido bond by the carboxyl of its amino and the amino acid residue and be connected on the amino acid residue.Alternatively, which can be connected to the part GLP-1 by ester bond.Normally, ester bond can be formed by reacting between the carboxyl of the part GLP-1 and the hydroxyl of the substituent group, or by reacting between the hydroxyl of the part GLP-1 and the carboxyl of substituent group.Alternatively, which can be alkyl, it is introduced into the primary amino group of the part GLP-1.

In a preferred embodiment of the invention, which forms amido bond by a carboxyl of a spacer group and an amino of the part GLP-1, is thus connected to the part GLP-1.The example of suitable spacer group has the dipeptides of succinic acid, Lys, Glu or Asp or a kind of such as Gly-Lys.When spacer group is succinic acid, its carboxyl can form amido bond with an amino of amino acid residue, and its another carboxyl can form amido bond with the amino of the lipophilic substituent.When spacer group is Lys, Glu or Asp, carboxyl can form amido bond with the amino of amino acid residue, and its amino can form amido bond with the carboxyl of lipophilic substituent.When using Lys as spacer group, other spacer group can be inserted between the epsilon-amino of Lys and the lipophilic substituent in some cases.In a preferred embodiment, this other spacer group is succinic acid, and amino present in the epsilon-amino and lipophilic substituent of it and Lys forms amido bond.In another preferred embodiment, this other spacer group is Glu or Asp, it forms another amido bond with the carboxyl occurred in epsilon-amino formation one amido bond, with lipophilic substituent of Lys, i.e., the lipophilic substituent is a N^εThe lysine residue being acylated.

In another preferred embodiment of the invention, the lipophilic substituent have one can be with electronegative group.It is a kind of preferably can negatively charged group be carboxyl.

A kind of method it can prepare parent peptide in this way, under conditions of this method is included in peptide is allowed to express, in suitable nutrient medium, then culture recycles the peptide of generation containing encoding the DNA sequence dna of the polypeptide and can express the host cell of the peptide from culture.

It can be any conventional medium for cultivating the host cell, the complex medium such as minimal medium or containing suitable additive for cultivating the culture medium of cell.It can be by being commercially available suitable culture medium, or the culture medium (method in such as American type culture collection catalogue introduced) suitable according to the preparation of published preparation method.It may then pass through conventional method and recycle the polypeptide generated by the cell from culture medium, these methods include that host cell is separated from culture medium by centrifugation or filtering, with the protein component in salt such as ammonium sulfate precipitation supernatant or filtrate, selecting various chromatography methods such as ion-exchange chromatography, gel permeation chromatography, affinity chromatography according to the type of purpose peptide etc. is purified.

The DNA sequence dna for encoding parent peptide can derive from genome or cDNA, such as by preparing genome or cDNA library, and (such as according to standard technique, see Sambrook, J, Fritsch, EF and Maniatis, T, molecular cloning: experimental implementation guide, Cold Spring HarborLaboratory Press, New York, 1989) using the oligonucleotide probe of synthesis, hybridized and filter out all or part of DNA sequence dna for encoding the peptide.Established standard method can also be passed through, phosphamide (phosphoamidite) method (tetrahedron flash report as described in Beaucage and Caruthers, 22 (1981), 1859-1869), or method (European Molecular Biology Organization's magazine described in Matthes etc., 3 (1984), 801-805) synthesize the DNA sequence dna of the encoded peptide.Also specific primer, such as US4 be can be used, 683,202 or Saiki etc. is in " science ", 239 (1988), described in 487-491, prepares DNA sequence dna by polymerase chain reaction.

DNA sequence dna can be inserted into any carrier for being convenient for DNA regrouping process, and the selection of carrier is frequently depend upon the host cell that the carrier will be introduced into.Therefore, carrier can be a kind of autonomous replicating vector, i.e., as carrier existing for an extrachromosomal entity, replicates independent of chromosome replication, such as plasmid.Alternatively, carrier can be such a type, when being introduced into host cell, it be would be integrated into host cell gene group, and be replicated together with the chromosome that it is integrated into.

Carrier is preferably a kind of expression vector, and the DNA sequence dna of peptide described in interior coding is effectively connected with other sections (such as promoter) needed for DNA transcription.Promoter can be any DNA sequence dna for having transcriptional activity in the host cell of selection, it can be from coding host cell homology or the gene of heterologous protein.It is well known that being suitable for the guidance in a variety of host cells encodes the promoter example that the DNA of peptide of the present invention is transcribed, referring to such as Sambrook etc., ibidem.

When needing, the DNA sequence dna for encoding the peptide can also effectively be connect with suitable terminator, polyadenylation signal, transcription enhancer sequences and translational enhancer sequence.Recombinant vector of the invention can also contain the DNA sequence dna that it can be made to replicate in purpose host cell.

Carrier can also contain a selected marker, such as a gene, the defect that its gene product will make up in host cell, or the resistance to drug such as ampicillin, kanamycins, tetracycline, chloramphenicol, neomycin, hygromycin or methotrexate etc. can be assigned.

For the secretory pathway that parent peptide of the invention is introduced to host cell, a secretory signal sequence (also referred to as leader sequence, preceding former sequence or presequence) can be provided in recombinant vector.Secretory signal sequence is connect with correct frame with the DNA sequence dna for encoding the peptide.Secretory signal sequence is usually located at the 5 ' sides for encoding the DNA sequence dna of the peptide.Secretory signal sequence can be the secretory signal sequence normally connecting with the peptide, or can be derived from the gene for encoding another secretory protein.

For being separately connected the DNA sequence dna for encoding peptide of the present invention, promoter and selectable terminator and/or secretory signal sequence, and insert it into the method in the suitable carrier containing information necessary to replicating, it is known to those skilled in the art that (referring to such as Sambrook etc., ibidem).

The host cell for importing DNA sequence dna or recombinant vector can be to any cell that can generate peptide of the present invention, including bacterium, yeast, fungi and higher eukaryotic cell.The example for the suitable host cell that those skilled in the art know and use is as follows, but is not limited to these: Escherichia coli, saccharomyces cerevisiae or mammal BHK or CHO cell line.

International patent application No.WO87/06941 (The General HospitalCorporation) describes the example that may be used as the compound of the part GLP-1 according to the present invention, and this application is related to a kind of peptide fragment for contain GLP-1 (7-37) and its functional derivatives and its purposes as pancreotropic hormone agent.

International patent application No.90/11296 (The General Hospital orporation) describes other GLP-1 analogs, this application is related to the peptide fragment containing GLP-1 (7-36) and its functional derivatives, these peptide fragments have the insulinotropic activity more than GLP-1 (1-36) or GLP-1 (1-37), further relate to their purposes as pancreotropic hormone agent.

International patent application No.91/11457 (Buckley etc.) discloses the analog of active GLP-1 peptide 7-34,7-35,7-36 and 7-37, they can also serve as the part GLP-1 according to the present invention.

Pharmaceutical composition

Pharmaceutical composition according to the present invention containing GLP-1 derivative can be with parenteral administration in the patient for needing this treatment.Syringe can be used, parenteral is selectively carried out by subcutaneous, intramuscular or intravenous injection with pen-type injector.Furthermore it is possible to carry out parenteral by infusion pump.In addition selection is a kind of GLP-1 derivative powdery applied for nose or lung Sprayable or liquid composition.GLP-1 derivative of the invention is also an option that ion-transmission patch may be selected such as through patch cutaneous penetration in transdermal routes；Or be administered through transmucosal route, such as penetrate buccal mucosa.

Routine techniques can be used, such as " pharmaceutical science " of Remington, 1985 or Remington: pharmaceutical science and practice, 19 editions, method described in 1995, preparation contains the pharmaceutical composition of GLP-1 derivative of the present invention.

Therefore, the composition for injection of GLP-1 derivative of the present invention can be prepared with the routine techniques of pharmaceuticals industry, these technologies include dissolving and mixing each component suitably to obtain required final product.

According to a kind of method, GLP-1 derivative is dissolved in a certain amount of water, wherein the amount of water is slightly smaller than the final volume of prepared composition.Isotonic agent, preservative and buffer are added as needed, and adjusts the pH value of solution with acid such as hydrochloric acid or alkali such as sodium hydrate aqueous solution when needed.Finally, water use regulation liquor capacity obtains required concentration of component.

Isotonic agent for example has sodium chloride, mannitol, glycerol.

Preservative for example has phenol, metacresol, methyl parabens, benzylalcohol.

Suitable buffer such as sodium acetate and sodium phosphate.

In addition to the aforementioned ingredients, the solution containing GLP-1 derivative of the present invention can also improve the dissolubility and stability of GLP-1 derivative containing surfactant.

It can be by composition of the preparation for certain peptides of nasal-cavity administration described in European patent No.272097 (Nordisk A/S) or WO93/18785.

According to a preferred embodiment of the invention, the composition forms for being suitable for drug administration by injection provide the composition of GLP-1 derivative.This composition is also possible to a certain amount of solid-state composition either ready-to-use injection, such as a kind of freeze-dried products, needs to be dissolved in solvent before the injection.Contained GLP-1 derivative is no less than 2mg/ml, preferably no less than 5mg/ml, more preferably no less than 10mg/ml, and preferably no more than 100mg/ml in this injection solution.

GLP-1 derivative of the invention can be used for treating various diseases.Specific GLP-1 derivative and optimal dose level used in patient will depend on disease to be treated and various factors, age, weight, physical activity and the diet of efficiency and patient including specific peptide derivant used, additionally depending on may be with the drug combination of other medicines and the severity of the state of an illness.It is recommended that those skilled in the art determines the dosage of GLP-1 derivative of the present invention for each patient.

Specifically, it is contemplated that GLP-1 derivative will be useful for treating adult-onset diabetes and/or treating the drug of obesity for prepare the effect curves with the longer time.

The present invention can be further illustrated with following embodiment, but it is not considered that these embodiments are the restrictions to protection scope.Feature disclosed in the description and following embodiment of front respectively or in any combination, for realizing that the present invention is important in various ways.

Embodiment

Use the following abbreviation of commercially available chemicals:

DMF:N, dinethylformamide

NMP:N- N-methyl-2-2-pyrrolidone N

EDPA:N- ethyl-N, N- diisopropylamine

EGTA: ethylene glycol two (beta-amino ether)-N, N, N ', N '-tetraacethyl

GTP: guanosine 5 '-triphosphoric acid

TFA: trifluoroacetic acid

THF: tetrahydrofuran

Myr-ONSu: tetradecanoic acid 2,5- dioxo pyrrolidin -1- base ester

Pal-ONSu: hexadecanoic acid 2,5- dioxo pyrrolidin -1- base ester

Ste-ONSu: octadecanoid acid 2,5- dioxo pyrrolidin -1- base ester

HOOC-(CH₂)₆- COONSu: ω-carboxyl enanthic acid 2,5- dioxo pyrrolidin -1- base ester

HOOC-(CH₂)₁₀- COONSu: ω-carboxyl hendecanoic acid 2,5- dioxo pyrrolidin -1- base ester

HOOC-(CH₂)₁₂- COONSu: ω-carboxyl tridecanoic acid 2,5- dioxo pyrrolidin -1- base ester

HOOC-(CH₂)₁₄- COONSu: ω-carboxyl pentadecanoic acid 2,5- dioxo pyrrolidin -1- base ester

HOOC-(CH₂)₁₆- COONSu: ω-carboxyl Heptadecanoic acide 2,5- dioxo pyrrolidin -1- base ester

HOOC-(CH₂)₁₈- COONSu: ω-carboxyl nonadecylic acid 2,5- dioxo pyrrolidin -1- base ester

Abbreviation:

PDMS: the attached mass spectroscopy of plasma desorption (Plasma Desorption MassSpectormetry)

MALDI-MS: the laser desorption of Matrix-assisted is attached/ionization mass spectrometry

HPLC: high performance liquid chromatography (HPLC)

Amu: atomic mass unit

Analysis

The attached mass spectroscopy of plasma desorption

Sample preparation:

Sample is dissolved in 0.1%TFA/EtOH (1: 1), its concentration is made to reach 1 μ g/ μ l.Sample solution (5-10 μ l) is placed on nitrocellulose target (Bio-ion AB, Uppsala, Sweden) and makes it at Surface absorption 2 minutes of target.Then target is cleaned and rotarily dried with 2 × 25 μ l 0.1%TFA.Finally nitrocellulose target is placed in target rotator and is put into mass spectrograph.

Mass spectral analysis

PDMS analysis is carried out with 20 time of flight arrangement of Bio-ion (Bio-ion Nordic AB, Uppsala, Sweden).Using the acceleration voltage of 15kV, the molecular ion formed by 252-Cf fission segment bombardment nitrocellulose surface is made to accelerate one terminal detector of directive.H is used respectively in m/z 1 and 30⁺And NO⁺The time of flight spectrum of generation is corrected to a kind of real mass spectrum by ion.General 1.0 × 10 accumulated in 15-20 minutes⁶Secondary fissure becomes to mass spectrum.Measured quality both corresponds to isotope average molecular mass.The accuracy of quality determination is generally better than 0.1%.

MALDI-MS

MALDI-MS analysis is carried out using a kind of Voyager RP instrument (PerSeptive Biosystems Inc., Framingham, MA) for being equipped with delay separator and working in a linear fashion.Use alpha-cyano -4- Hydroxy-cinnamic acid as matrix, and quality determination is carried out based on external calibration method.

Example 1

Lys²⁶(N^εMyristoyl base)-GLP-1 (7-37) synthesis

It synthesizes to obtain target compound by GLP-1 (7-37).By GLP-1 (7-37) (25mg, 7.45 μm), the mixture of EDPA (26.7mg, 208 μm), NMP (520 μ l) and water (260 μ l) mildly shakes 5 minutes in room temperature.Myr-ONSu (2.5mg, 7.67 μm) is added into gained mixture and is dissolved in the solution that NMP (62.5 μ l) is obtained, reaction mixture is mildly shaken 5 minutes in room temperature, is then placed 20 minutes.Another be added Myr-ONSu (2.5mg, 7.67 μm) is dissolved in the solution that NMP (62.5 μ l) is obtained, and gained mixture is mildly shaken 5 minutes.After 40 minutes total reaction times, glycine (12.5mg, 166 μm of ol) are added thereto and are dissolved in solution termination reaction obtained in 50% ethanol water (12.5ml).Using cyanogen propyl (cyanopropyl) column (Zorbax 300SB-CN) and standard acetonitrile/TFA system, by HPLC method, isolating target compound, yield are 1.3mg (be equivalent to theoretical yield 4.9%) from reaction mixture.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.The product gone out with PDMS method Analyze & separate, the m/z value at discovery protonated molecular ion peak are 3567.9 ± 3.Therefore the molecular weight obtained is 3566.9 ± 3amu (theoretical value 3565.9amu).Enzymatic hydrolysis cutting is carried out to target compound by staphylococcus aureus V8 protease, the quality determination of peptide fragment is then carried out by PDMS, so that it is determined that being acylated position (Lys26).

In addition to target compound, with same chromatographic column and narrower gradient (acetonitrile 35-38% in 60 minutes) from reaction mixture isolated other two kinds of GLP-1 derivatives, see example 2 and 3.

Example 2

Lys³⁴(N^εMyristoyl base)-GLP-1 (7-37) synthesis

By HPLC method from reaction mixture described in example 1 isolated target compound.It analyzes to obtain the protonated molecular ion peak that m/z is 3567.7 ± 3 through PDMS.Consequently found that molecular weight is 3566.7 ± 3amu (theoretical value 3565.9amu).It is determined according to fracture mode and is acylated position.

Example 3

Lys^26,34Bis- (N^εMyristoyl base)-GLP-1 (7-37) synthesis

By HPLC method from reaction mixture described in example 1 isolated target compound.PDMS analyzes to obtain the protonated molecular ion peak that m/z is 3778.4 ± 3.Consequently found that molecular weight is 3777.4 ± 3amu (theoretical value 3776.1amu).

Example 4

Lys²⁶(N^εMyristoyl base) Arg³⁴The synthesis of-GLP-1 (7-37)

By Arg³⁴- GLP-1 (7-37) synthesis obtains target compound.By Arg³⁴The mixture of-GLP-1 (7-37) (5mg, 1.47 μm), EDPA (5.3mg, 41.1 μm), NMP (105 μ l) and water (50 μ l) mildly shakes 5 minutes in room temperature.Myr-ONSu (0.71mg, 2.2 μm) is added into gained mixture and is dissolved in the solution that NMP (17.8 μ l) is obtained, reaction mixture is mildly shaken 5 minutes in room temperature, is then placed 20 minutes.After 30 minutes total reaction times, glycine (25mg, 33.3 μm) is added thereto and is dissolved in solution termination reaction obtained in 50% ethanol water (2.5ml).Reaction mixture is purified through HPLC method by described in example 1.PDMS analyzes to obtain the protonated molecular ion peak that m/z value is 3594.9 ± 3.The molecular weight obtained is 3593.9 ± 3amu (theoretical value 3593.9amu).

Example 5

Gly⁸Arg^26,34Lys³⁶(N^εMyristoyl base)-GLP-1 (7-37) synthesis

By the Gly for being purchased from QCB⁸Arg^26,34Lys³⁶- GLP-1 (7-37) synthesis obtains target compound.By Gly⁸Arg^26,34Lys³⁶The mixture of-GLP-1 (7-37) (1.3mg, 0.39 μm), EDPA (1.3mg, 10 μm), NMP (125 μ l) and water (30 μ l) mildly shakes 5 minutes in room temperature.Myr-ONSu (0.14mg, 0.44 μm) is added into gained mixture and is dissolved in the solution that NMP (3.6ml) is obtained, reaction mixture is mildly shaken 15 minutes in room temperature.Glycine (0.1mg, 1.33 μm) is added thereto and is dissolved in solution termination reaction obtained in 50% ethanol water (10 μ l).Purify reaction mixture with HPLC method, isolates target compound (60 μ g, 4%).

Example 6

Arg^26,34Lys³⁶(N^εMyristoyl base)-GLP-1 (7-37)-OH synthesis

By Arg^26,34Lys³⁶- GLP-1 (7-37)-OH (5.0mg, 1.477 μm of ol), EDPA (5.4mg, 41.78 μm of ol), the mixture of NMP (105 μ l) and water (50 μ l) mildly shake 5 minutes in room temperature.Myr-ONSu (0.721mg, 2.215 μm of ol) are added into gained mixture and are dissolved in the solution that NMP (18 μ l) is obtained.Reaction mixture is mildly shaken 5 minutes in room temperature, and keeps it in addition 45 minutes in room temperature.Glycine (2.5mg, 33.3 μm of ol) are added thereto and are dissolved in solution termination reaction obtained in 50% ethanol water (250 μ l).Using cyanogen propyl column (Zorbax300SB-CN) and standard acetonitrile/TFA system, reaction mixture is purified by column chromatography.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Isolating target compound (1.49mg, 28%) then analyzes product with PDMS method.It was found that the m/z value of the molecular ion peak of protonation is 3595 ± 3.Therefore the molecular weight obtained is 3594 ± 3amu (theoretical value 3594amu).

Example 7

Lys^26,34Bis- (N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-37)-OH synthesis

By GLP-1 (7-37)-OH (70mg, 20.85 μm of ol), EDPA (75.71mg, 585.8 μm of ol), the mixture of NMP (1.47ml) and water (700 μ l) mildly shakes 10 minutes in room temperature.HOOC- (CH is added into gained mixture₂)₁₈- COONSu (27.44mg, 62.42 μm of ol) is dissolved in the solution that NMP (686 μ l) is obtained, and reaction mixture is mildly shaken 5 minutes in room temperature, and keep it in addition 50 minutes in room temperature.Glycine (34.43mg, 458.7 μm of ol) are added thereto and are dissolved in solution termination reaction obtained in 50% ethanol water (3.44ml).Using cyanogen propyl column (Zorbax 300SB-CN) and standard acetonitrile/TFA system, reaction mixture is purified by column chromatography.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Isolating target compound (8.6mg, 10%) then analyzes product with PDMS method.It was found that the m/z value of the molecular ion peak of protonation is 4006 ± 3.The molecular weight thus obtained is 4005 ± 3amu (theoretical value 4005amu).

Example 8

Arg^26,34Lys³⁶(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-36)-OH synthesis

By Arg^26,34Lys³⁶- GLP-1 (7-36)-OH (5.06mg, 1.52 μm of ol), EDPA (5.5mg, 42.58 μm of ol), the mixture of NMP (106 μ l) and water (100 μ l) mildly shake 5 minutes in room temperature.HOOC- (CH is added into gained mixture₂)₁₈- COONSu (1.33mg, 3.04 μm of ol) is dissolved in the solution that NMP (33.2 μ l) is obtained, and reaction mixture is mildly shaken 5 minutes in room temperature, is then placed again 2.5 hours in room temperature.Glycine (2.50mg, 33.34 μm of ol) are added thereto and are dissolved in solution termination reaction obtained in 50% ethanol water (250 μ l).Using cyanogen propyl column (Zorbax 300SB-CN) and standard acetonitrile/TFA system, reaction mixture is purified by column chromatography.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Target compound (0.46mg, 8%) is isolated, analyzes product with PDMS method.It was found that the m/z value of the molecular ion peak of protonation is 3652 ± 3.Thus show that molecular weight is 3651 ± 3amu (theoretical value 3651amu).

Example 9

Arg^26,34Lys³⁸(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-38)-OH synthesis

By Arg^26,34Lys³⁸- GLP-1 (7-38)-OH (5.556mg, 1.57 μm of ol), EDPA (5.68mg, 43.96 μm of ol), NMP (116.6 μ l) and water (50 μ l) mixture mildly shaken 10 minutes in room temperature.HOOC- (CH is added into gained mixture₂)₁₈- COONSu (1.38mg, 3.14 μm of ol) is dissolved in the solution that NMP (34.5 μ l) is obtained, and reaction mixture is mildly shaken 5 minutes in room temperature, is then placed again 2.5 hours in room temperature.Glycine (2.5mg, 33.3 μm of ol) are added thereto and are dissolved in 50% ethanol water (250 μ l) solution obtained to terminate reaction.Using cyanogen propyl column (Zorbax 300SB-CN) and standard acetonitrile/TFA system, reaction mixture is purified by column chromatography.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Target compound (0.7mg, 12%) is isolated, analyzes product with PDMS method.It was found that the m/z value of the molecular ion peak of protonation is 3866 ± 3.Thus show that molecular weight is 3865 ± 3amu (theoretical value 3865amu).

Example 10

Arg³⁴Lys²⁶(N^ε(19 acyl group of ω-carboxyl))-GLP-1 (7-37)-OH synthesis

By Arg³⁴- GLP-1 (7-37)-OH (5.04mg, 1.489 μm of ol), EDPA (5.39mg, 41.70 μm of ol), NMP (105 μ l) and water (50 μ l) mixture mildly shaken 10 minutes in room temperature.HOOC- (CH is added into gained mixture₂)₁₈- COONSu (1.31mg, 2.97 μm of ol) is dissolved in solution obtained from NMP (32.8 μ l), and reaction mixture is mildly shaken 5 minutes in room temperature, is then placed again 30 minutes in room temperature.Glycine (2.46mg, 32.75 μm of ol) are added thereto and are dissolved in 50% ethanol water (246 μ l) solution obtained to terminate reaction.Using cyanogen propyl column (Zorbax 300SB-CN) and standard acetonitrile/TFA system, reaction mixture is purified by column chromatography.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Target compound (1.2mg, 22%) is isolated, analyzes product with PDMS method.It was found that the m/z value of the molecular ion peak of protonation is 3709 ± 3.Thus show that molecular weight is 3708 ± 3amu (theoretical value 3708amu).

Example 11

Arg³⁴Lys²⁶(N^ε(ω-carboxyl heptadecanoyl base))-GLP-1 (7-37)-OH synthesis

By Arg³⁴- GLP-1 (7-37)-OH (5.8mg, 1.714 μm of ol), EDPA (6.20mg, 47.99 μm of ol), NMP (121.8 μ l) and water (58 μ l) mixture mildly shaken 10 minutes in room temperature.HOOC- (CH is added into gained mixture₂)₁₆- COONSu (2.11mg, 5.142 μm of ol) is dissolved in the solution that NMP (52.8 μ l) is obtained, and reaction mixture is mildly shaken 5 minutes in room temperature, is then placed again 2 hours in room temperature.Glycine (2.83mg, 37.70 μm of ol) are added thereto and are dissolved in 50% ethanol water (283 μ l) solution obtained to terminate reaction.Using cyanogen propyl column (Zorbax 300SB-CN) and standard acetonitrile/TFA system, reaction mixture is purified by column chromatography.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Target compound (0.81mg, 13%) is isolated, analyzes product with PDMS method.It was found that the m/z value of the molecular ion peak of protonation is 3681 ± 3.Thus show that molecular weight is 3680 ± 3amu (theoretical value 3680amu).

Example 12

Arg^26,34Lys³⁶(N^ε(ω-carboxyl heptadecanoyl base))-GLP-1 (7-37)-OH synthesis

By Arg^26,34Lys³⁶- GLP-1 (7-37)-OH (3.51mg, 1.036 μm of ol), EDPA (3.75mg, 29.03 μm of ol), NMP (73.8 μ l) and water (35 μ l) mixture mildly shaken 10 minutes in room temperature.HOOC- (CH is added into gained mixture₂)₁₆- COONSu (1.27mg, 3.10 μm of ol) is dissolved in the solution that NMP (31.8 μ l) is obtained, and reaction mixture is mildly shaken 5 minutes in room temperature, is then placed again 10 minutes 2 hours in room temperature.Glycine (1.71mg, 22.79 μm of ol) are added thereto and are dissolved in 50% ethanol water (171 μ l) solution obtained to terminate reaction.Reaction mixture is purified by column chromatography using cyanogen propyl column (Zorbax 300SB-CN) and standard acetonitrile/TFA system.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Target compound (0.8mg, 21%) is isolated, analyzes product with PDMS method.It was found that the m/z value of the molecular ion peak of protonation is 3682 ± 3.Thus show that molecular weight is 3681 ± 3amu (theoretical value 3681amu).

Example 13

Arg^26,34Lys³⁸(N^ε(ω-carboxyl heptadecanoyl base))-GLP-1 (7-38)-OH synthesis

By Arg^26,34Lys³⁸- GLP-1 (7-38)-OH (5.168mg, 1.459 μm of ol), EDPA (5.28mg, 40.85 μm of ol), NMP (108.6 μ l) and water (51.8 μ l) mixture mildly shaken 10 minutes in room temperature.HOOC- (CH is added into gained mixture₂)₁₆- COONSu (1.80mg, 4.37 μm of ol) is dissolved in the solution that NMP (45 μ l) is obtained, and reaction mixture is mildly shaken 10 minutes in room temperature, is then placed again 15 minutes 2 hours in room temperature.Glycine (2.41mg, 32.09 μm of ol) are added thereto and are dissolved in 50% ethanol water (241 μ l) solution obtained to terminate reaction.Using cyanogen propyl column (Zorbax 300SB-CN) and standard acetonitrile/TFA system, reaction mixture is purified by column chromatography.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Target compound (0.8mg, 14%) is isolated, analyzes product with PDMS method.It was found that the m/z value of the molecular ion peak of protonation is 3838 ± 3.Thus show that molecular weight is 3837 ± 3amu (theoretical value 3837amu).

Example 14

Arg^26,34Lys³⁶(N^ε(ω-carboxyl heptadecanoyl base))-GLP-1 (7-36)-OH synthesis

By Arg^26,34Lys³⁶- GLP-1 (7-36)-OH (24.44mg, 7.34 μm of ol), EDPA (26.56mg, 205.52 μm of ol), NMP (513 μ l) and water (244.4 μ l) mixture mildly shaken 5 minutes in room temperature.HOOC- (CH is added into gained mixture₂)₁₆- COONSu (9.06mg, 22.02 μm of ol) is dissolved in the solution that NMP (1.21ml) is obtained, and reaction mixture is mildly shaken 5 minutes in room temperature, is then placed again 30 minutes in room temperature.Glycine (12.12mg, 161.48 μm of ol) are added thereto and are dissolved in 50% ethanol water (1.21ml) solution obtained to terminate reaction.Using cyanogen propyl column (Zorbax 300SB-CN) and standard acetonitrile/TFA system, reaction mixture is purified by column chromatography.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Target compound (7.5mg, 28%) is isolated, analyzes product with PDMS method.It was found that the m/z value of the molecular ion peak of protonation is 3625 ± 3.Thus show that molecular weight is 3624 ± 3amu (theoretical value 3624amu).

Example 15

Arg^26,34Lys³⁶(N^ε(ω-carboxyl undecanoyl base))-GLP-1 (7-37)-OH synthesis

By Arg^26,34Lys³⁶- GLP-1 (7-37)-OH (4.2mg, 1.24 μm of ol), EDPA (4.49mg, 34.72 μm of ol), NMP (88.2 μ l) and water (42 μ l) mixture mildly shaken 10 minutes in room temperature.HOOC- (CH is added into gained mixture₂)₁₀- COONSu (1.21mg, 3.72 μm of ol) is dissolved in the solution that NMP (30.25 μ l) is obtained, and reaction mixture is mildly shaken 5 minutes in room temperature, is then placed again 40 minutes in room temperature.Glycine (2.04mg, 27.28 μm of ol) are added thereto and are dissolved in 50% ethanol water (204 μ l) solution obtained to terminate reaction.Using cyanogen propyl column (Zorbax 300SB-CN) and standard acetonitrile/TFA system, reaction mixture is purified by column chromatography.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Target compound (0.8mg, 18%) is isolated, analyzes product with PDMS method.It was found that the m/z value of the molecular ion peak of protonation is 3598 ± 3.Thus show that molecular weight is 3597 ± 3amu (theoretical value 3597amu).

Example 16

Arg^26,34Lys³⁸(N^ε(ω-carboxyl undecanoyl base))-GLP-1 (7-38)-OH synthesis

By Arg^26,34Lys³⁸- GLP-1 (7-38)-OH (5.168mg, 1.46 μm of ol), EDPA (5.28mg, 40.88 μm of ol), NMP (108.6 μ l) and water (51.7 μ l) mixture mildly shaken 10 minutes in room temperature.HOOC- (CH is added into gained mixture₂)₁₀- COONSu (1.43mg, 4.38 μm of ol) is dissolved in the solution that NMP (35.8 μ l) is obtained, and reaction mixture is mildly shaken 5 minutes in room temperature, is then placed again 50 minutes in room temperature.Glycine (2.41mg, 32.12 μm of ol) are added thereto and are dissolved in 50% ethanol water (241 μ l) solution obtained to terminate reaction.Using cyanogen propyl column (Zorbax 300SB-CN) and standard acetonitrile/TFA system, reaction mixture is purified by column chromatography.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Target compound (0.85mg, 16%) is isolated, analyzes product with PDMS method.It was found that the m/z value of the molecular ion peak of protonation is 3753 ± 3.Thus show that molecular weight is 3752 ± 3amu (theoretical value 3752amu).

Example 17

Arg^26,34Bis- (N^ε(ω-carboxyl undecanoyl base))-GLP-1 (7-37)-OH synthesis

The mixture of GLP-1 (7-37)-OH (10.0mg, 2.98 μm of ol), EDPA (10.8mg, 83.43 μm of ol), NMP (210 μ l) and water (100 μ l) is mildly shaken 10 minutes in room temperature.HOOC- (CH is added into gained mixture₂)₁₀- COONSu (2.92mg, 8.94 μm of ol) is dissolved in the solution that NMP (73 μ l) is obtained, and reaction mixture is mildly shaken 5 minutes in room temperature, is then placed again 50 minutes in room temperature.Glycine (4.92mg, 65.56 μm of ol) are added thereto and are dissolved in 50% ethanol water (492 μ l) solution obtained to terminate reaction.Using cyanogen propyl column (Zorbax 300SB-CN) and standard acetonitrile/TFA system, reaction mixture is purified by column chromatography.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Target compound (1.0mg, 9%) is isolated, analyzes product with PDMS method.It was found that the m/z value of the molecular ion peak of protonation is 3781 ± 3.Thus show that molecular weight is 3780 ± 3amu (theoretical value 3780amu).

Example 18

Arg^26,34Lys³⁶(N^ε(ω-carboxyl undecanoyl base))-GLP-1 (7-36)-OH synthesis

By Arg^26,34Lys³⁶- GLP-1 (7-36)-OH (15.04mg, 4.52 μm of ol), EDPA (16.35mg, 126.56 μm of ol), NMP (315.8 μ l) and water (150.4 μ l) mixture mildly shaken 10 minutes in room temperature.HOOC- (CH is added into gained mixture₂)₁₀- COONSu (4.44mg, 13.56 μm of ol) is dissolved in the solution that NMP (111 μ l) is obtained, and reaction mixture is mildly shaken 5 minutes in room temperature, is then placed again 40 minutes in room temperature.Glycine (7.5mg, 99.44 μm of ol) are added thereto and are dissolved in 50% ethanol water (250 μ l) solution obtained to terminate reaction.Using cyanogen propyl column (Zorbax 300SB-CN) and standard acetonitrile/TFA system, reaction mixture is purified by column chromatography.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Target compound (3.45mg, 22%) is isolated, analyzes product with PDMS method.It was found that the m/z value of the molecular ion peak of protonation is 3540 ± 3.Thus show that molecular weight is 3539 ± 3amu (theoretical value 3539amu).

Example 19

Arg³⁴Lys²⁶(N^ε(ω-carboxyl undecanoyl base))-GLP-1 (7-37)-OH synthesis

By Arg³⁴- GLP-1 (7-37)-OH (5.87mg, 1.73 μm of ol), EDPA (6.27mg, 48.57 μm of ol), NMP (123.3 μ l) and water (58.7 μ l) mixture mildly shaken 10 minutes in room temperature.HOOC- (CH is added into gained mixture₂)₁₀- COONSu (1.70mg, 5.20 μm of ol) is dissolved in the solution that NMP (42.5 μ l) is obtained, and reaction mixture is mildly shaken 5 minutes in room temperature, is then placed again 40 minutes in room temperature.Glycine (2.86mg, 286 μm of ol) are added thereto and are dissolved in 50% ethanol water (286 μ l) solution obtained to terminate reaction.Using cyanogen propyl column (Zorbax 300SB-CN) and standard acetonitrile/TFA system, reaction mixture is purified by column chromatography.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Target compound (1.27mg, 20%) is isolated, analyzes product with PDMS method.It was found that the m/z value of the molecular ion peak of protonation is 3597 ± 3.Thus show that molecular weight is 3596 ± 3amu (theoretical value 3596amu).

Example 20

Arg³⁴Lys²⁶(N^ε(ω-carboxyl heptanoyl group))-GLP-1 (7-37)-OH synthesis

By Arg³⁴- GLP-1 (7-37)-OH (4.472mg, 1.32 μm of ol), EDPA (4.78mg, 36.96 μm of ol), NMP (94 μ l) and water (44.8 μ l) mixture mildly shaken 5 minutes in room temperature.HOOC- (CH is added into gained mixture₂)₆- COONSu (1.07mg, 3.96 μm of ol) is dissolved in the solution that NMP (26.8 μ l) is obtained, and reaction mixture is mildly shaken 5 minutes in room temperature, is then placed again 50 minutes 1 hour in room temperature.Glycine (2.18mg, 29.04 μm of ol) are added thereto and are dissolved in 50% ethanol water (218 μ l) solution obtained to terminate reaction.Using cyanogen propyl column (Zorbax 300SB-CN) and standard acetonitrile/TFA system, reaction mixture is purified by column chromatography.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Target compound (0.5mg, 11%) is isolated, analyzes product with PDMS method.It was found that the m/z value of the molecular ion peak of protonation is 3540 ± 3.Thus show that molecular weight is 3539 ± 3amu (theoretical value 3539amu).

Example 21

Arg^26,34Lys³⁸(N^ε(ω-carboxyl heptanoyl group))-GLP-1 (7-38)-OH synthesis

By Arg^26,34Lys³⁸- GLP-1 (7-38)-OH (5.168mg, 1.459 μm of ol), EDPA (5.28mg, 40.85 μm of ol), NMP (108.6 μ l) and water (51.6 μ l) mixture mildly shaken 10 minutes in room temperature.HOOC- (CH is added into gained mixture₂)₆- COONSu (1.18mg, 4.37 μm of ol) is dissolved in the solution that NMP (29.5 μ l) is obtained, and reaction mixture is mildly shaken 5 minutes in room temperature, is then placed again 50 minutes 1 hour in room temperature.Glycine (2.40mg, 32.09 μm of ol) are added thereto and are dissolved in 50% ethanol water (240 μ l) solution obtained to terminate reaction.Using cyanogen propyl column (Zorbax 300SB-CN) and standard acetonitrile/TFA system, reaction mixture is purified by column chromatography.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Target compound (0.5mg, 9%) is isolated, analyzes product with PDMS method.It was found that the m/z value of the molecular ion peak of protonation is 3697 ± 3.Thus show that molecular weight is 3695 ± 3amu (theoretical value 3695amu).

Example 22

Arg^26,34Lys³⁶(N^ε(ω-carboxyl heptanoyl group))-GLP-1 (7-37)-OH synthesis

By Arg^26,34Lys³⁶- GLP-1 (7-37)-OH (5.00mg, 1.47 μm of ol), EDPA (5.32mg, 41.16 μm of ol), NMP (105 μ l) and water (50 μ l) mixture mildly shaken 5 minutes in room temperature.HOOC- (CH is added into gained mixture₂)₆- COONSu (1.19mg, 4.41 μm of ol) is dissolved in the solution that NMP (29.8 μ l) is obtained, and reaction mixture is mildly shaken 5 minutes in room temperature, is then placed again 2 hours in room temperature.Glycine (2.42mg, 32.34 μm of ol) are added thereto and are dissolved in 50% ethanol water (242 μ l) solution obtained to terminate reaction.Using cyanogen propyl column (Zorbax 300SB-CN) and standard acetonitrile/TFA system, reaction mixture is purified by column chromatography.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Target compound (0.78mg, 15%) is isolated, analyzes product with PDMS method.It was found that the m/z value of the molecular ion peak of protonation is 3542 ± 3.Thus show that molecular weight is 3541 ± 3amu (theoretical value 3541amu).

Example 23

Arg^26,34Lys³⁶(N^ε(ω-carboxyl heptanoyl group))-GLP-1 (7-36)-OH synthesis

By Arg^26,34Lys³⁶- GLP-1 (7-36)-OH (5.00mg, 1.50 μm of ol), EDPA (5.44mg, 42.08 μm of ol), NMP (210 μ l) and water (50 μ l) mixture mildly shaken 5 minutes in room temperature.HOOC- (CH is added into gained mixture₂)₆- COONSu (1.22mg, 4.5 μm of ol) is dissolved in the solution that NMP (30.5 μ l) is obtained, and reaction mixture is mildly shaken 5 minutes in room temperature, is then placed again 2 hours in room temperature.Glycine (2.47mg, 33.0 μm of ol) are added thereto and are dissolved in 50% ethanol water (247 μ l) solution obtained to terminate reaction.Using cyanogen propyl column (Zorbax 300SB-CN) and standard acetonitrile/TFA system, reaction mixture is purified by column chromatography.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Target compound (0.71mg, 14%) is isolated, analyzes product with PDMS method.It was found that the m/z value of the molecular ion peak of protonation is 3484 ± 3.Thus show that molecular weight is 3483 ± 3amu (theoretical value 3483amu).

Example 24

Arg^26,34Bis- (N^ε(ω-carboxyl heptanoyl group))-GLP-1 (7-37)-OH synthesis

The mixture of GLP-1 (7-37)-OH (10mg, 2.5 μm of ol), EDPA (10.8mg, 83.56 μm of ol), NMP (210 μ l) and water (100 μ l) is mildly shaken 10 minutes in room temperature.HOOC- (CH is added into gained mixture₂)₆- COONSu (2.42mg, 8.92 μm of ol) is dissolved in the solution that NMP (60.5 μ l) is obtained, and reaction mixture is mildly shaken 5 minutes in room temperature, is then placed again 35 minutes 2 hours in room temperature.Glycine (4.92mg, 65.54 μm of ol) are added thereto and are dissolved in 50% ethanol water (492 μ l) solution obtained to terminate reaction.Using cyanogen propyl column (Zorbax 300SB-CN) and standard acetonitrile/TFA system, reaction mixture is purified by column chromatography.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Isolating target compound (2.16mg, 24%) analyzes product with PDMS method.It was found that the m/z value of the molecular ion peak of protonation is 3669 ± 3.The molecular weight obtained is 3668 ± 3amu (theoretical value 3668amu).

Example 25

Arg³⁴Lys²⁶(N^ε(ω-carboxyl pentadecanoyl base))-GLP-1 (7-37)-OH synthesis

By Arg³⁴- GLP-1 (7-37)-OH (4.472mg, 1.321 μm of ol), EDPA (4.78mg, 36.99 μm of ol), NMP (93.9 μ l) and water (44.7 μ l) mixture mildly shaken 10 minutes in room temperature.HOOC- (CH is added into gained mixture₂)₁₄- COONSu (1.519mg, 3.963 μm of ol) is dissolved in the solution that NMP (38 μ l) is obtained, and reaction mixture is mildly shaken 5 minutes in room temperature, is then placed again 1 hour in room temperature.Glycine (2.18mg, 29.06 μm of ol) are added thereto and are dissolved in 50% ethanol water (218 μ l) solution obtained to terminate reaction.Using cyanogen propyl column (Zorbax300SB-CN) and standard acetonitrile/TFA system, reaction mixture is purified by column chromatography.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Target compound (0.58mg, 12%) is isolated, analyzes product with PDMS method.It was found that the m/z value of the molecular ion peak of protonation is 3654 ± 3.Thus show that molecular weight is 3653 ± 3amu (theoretical value 3653amu).

Example 26

Arg^26,34Lys³⁶(N^ε(ω-carboxyl heptanoyl group))-GLP-1 (7-36)-OH synthesis

By Arg^26,34Lys³⁶- GLP-1 (7-36)-OH (5.00mg, 1.50 μm of ol), EDPA (5.44mg, 42.08 μm of ol), NMP (210 μ l) and water (50 μ l) mixture mildly shaken 5 minutes in room temperature.HOOC- (CH is added into gained mixture₂)₁₄- COONSu (1.72mg, 4.5 μm of ol) is dissolved in the solution that NMP (43 μ l) is obtained, and reaction mixture is mildly shaken 5 minutes in room temperature, is then placed again 1 hour in room temperature.Glycine (2.48mg, 33 μm of ol) are added thereto and are dissolved in 50% ethanol water (248 μ l) solution obtained to terminate reaction.Using cyanogen propyl column (Zorbax 300SB-CN) and standard acetonitrile/TFA system, reaction mixture is purified by column chromatography.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Target compound (0.58mg, 11%) is isolated, analyzes product with PDMS method.It was found that the m/z value of the molecular ion peak of protonation is 3596 ± 3.Thus show that molecular weight is 3595 ± 3amu (theoretical value 3595amu).

Example 27

Lithocholic acid 2, the synthesis of 5- dioxo pyrrolidin -1- base ester

To contain lithocholic acid (5.44g, 14.34mmol), n-hydroxysuccinimide (1.78g, 15.0mmol), it anhydrous THF (120ml) and anhydrous acetonitrile (30ml) and is maintained in 10 DEG C of mixture N is added, N '-dicyclohexylcarbodiimide (3.44g, 16.67mmol) is dissolved in solution made from anhydrous THF.Reaction mixture stirs 16 hours at ambient temperature, is filtered and concentrated in vacuo.Residue is dissolved in methylene chloride (450ml), is washed with 10% aqueous sodium carbonate (2 × 150ml) and water (2 × 150ml), and is dry (magnesium sulfate).Filtering, is concentrated to get crystalline state residue for filter vacuum.From residue is recrystallized in the mixture of methylene chloride (30ml) and normal heptane (30ml), target compound (3.46g, 51%) is obtained with crystalline solid forms.

Example 28

Arg³⁴Lys²⁶(N^εLithocholic acid base)-GLP-1 (7-37)-OH synthesis

By Arg³⁴- GLP-1 (7-37)-OH (4.472mg, 1.32 μm of ol), EDPA (4.78mg, 36.96 μm of ol), NMP (94 μ l) and water (44.8 μ l) mixture mildly shaken 10 minutes in room temperature.It is added into gained mixture by lithocholic acid 2,5- dioxo pyrrolidin -1- base ester (1.87mg, 3.96 μm of ol) it is dissolved in the solution that NMP (46.8 μ l) is obtained, reaction mixture is mildly shaken 5 minutes in room temperature, is then placed again 1 hour in room temperature.Glycine (2.18mg, 29.04 μm of ol) are added and are dissolved in 50% ethanol water (218 μ l) solution obtained to terminate reaction.Using cyanogen propyl column (Zorbax 300SB-CN) and standard acetonitrile/TFA system, reaction mixture is purified by column chromatography.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Target compound (1.25mg, 25%) is isolated, analyzes product with PDMS method.It was found that the m/z value of the molecular ion peak of protonation is 3744 ± 3.Thus show that molecular weight is 3743 ± 3amu (theoretical value 3743amu).

Example 29

N^αMyristoyl base-Glu (ONSu)-OBu^tSynthesis

To H-Glu (OH)-OBu^tMyr-ONSu (4.0g, 12.3mmol) is added dropwise in the suspension of (2.5g, 12.3mmol), DMF (283ml) and EDPA (1.58g, 12.3mmol) and is dissolved in solution made from DMF (59ml).Reaction mixture is stirred at room temperature 16 hours, being then concentrated in vacuo to total volume is 20ml.Residue is distributed between 5% aqueous citric acid solution (250ml) and ethyl acetate (150ml), separates two-phase.After organic phase vacuum concentration, residue is dissolved in DMF (40ml).Acquired solution is added dropwise in 10% aqueous citric acid solution (300ml) for being maintained at 0 DEG C.Compound precipitates are collected, and are washed with ice water, are dried in a vacuum drying oven.Compound after drying is dissolved in DMF (23ml), and HONSu (1.5g, 13mmol) is added.N is added into gained mixture, N '-dicyclohexylcarbodiimide (2.44g, 11.9mmol) is dissolved in solution made from methylene chloride (47ml).Reaction mixture is stirred at room temperature 16 hours, then filters compound precipitates.Precipitating is recrystallized to give target compound (3.03g, 50%) from normal heptane/2- propyl alcohol.

Example 30

Glu^22,23,30Arg^26,34Lys³⁸(N^ε(γ-glutamyl (N^αMyristoyl base))) synthesis of-GLP-1 (7-38)-OH

By Glu^22,23,30Arg^26,34Lys³⁸- GLP-1 (7-38)-OH (1.0mg, 0.272 μm of ol), EDPA (0.98mg, 7.62 μm of ol), NMP (70 μ l) and water (70 μ l) mixture mildly shaken 5 minutes in room temperature.It will be by N made from 29 method of example^αMyristoyl base-Glu (ONSu)-OBu^t(0.41mg, 0.816 μm of ol) is dissolved in NMP (10.4 μ l) acquired solution, is added in gained mixture, reaction mixture is mildly shaken 5 minutes in room temperature, is then placed again 45 minutes in room temperature.Glycine (0.448mg, 5.98 μm of ol) are added thereto and are dissolved in 50% ethanol water (45 μ l) solution obtained to terminate reaction.0.5% aqueous solution (0.9ml) of ammonium acetate is added, gained mixture is flushed to Varian 500mg C8Mega Bond ElutOn cylinder, the compound fixed is cleaned with 5% acetonitrile solution (10ml), releases it from column eventually by being eluted with TFA (10ml).Eluate is concentrated in vacuo, using cyanogen propyl column (Zorbax 300SB-CN) and standard acetonitrile/TFA system, reaction mixture is purified by column chromatography.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Isolating target compound (0.35mg, 32%) analyzes product with PDMS method.It was found that the m/z value of the molecular ion peak of protonation is 4012 ± 3.Thus show that molecular weight is 4011 ± 3amu (theoretical value 4011amu).

Example 31

Glu^23,26Arg³⁴Lys³⁸(N^ε(γ-glutamyl (N^αMyristoyl base))) synthesis of-GLP-1 (7-38)-OH

By Glu^23,26Arg³⁴Lys³⁸- GLP-1 (7-38)-OH (6.07mg, 1.727 μm of ol), EDPA (6.25mg, 48.36 μm of ol), NMP (425 μ l) and water (425 μ l) mixture mildly shaken 5 minutes in room temperature.It will be by N made from 29 method of example^αMyristoyl base-Glu (ONSu)-OBu^t(2.65mg, 5.18 μm of ol) are dissolved in NMP (66.3 μ l) acquired solution, are added in gained mixture, reaction mixture is mildly shaken 5 minutes in room temperature, is then placed again 45 minutes in room temperature.Glycine (2.85mg, 38.0 μm of ol) are added thereto and are dissolved in 50% ethanol water (285 μ l) solution obtained to terminate reaction.0.5% aqueous solution (5.4ml) of ammonium acetate is added, gained mixture is flushed to Varian 500mg C8Mega Bond ElutOn cylinder, the compound fixed is cleaned with 5% acetonitrile solution (10ml), releases it from column eventually by being eluted with TFA (10ml).Eluate is concentrated in vacuo, using cyanogen propyl column (Zorbax 300SB-CN) and standard acetonitrile/TFA system, reaction mixture is purified by column chromatography.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Target compound (0.78mg, 12%) is isolated, analyzes product with PDMS method.It was found that the m/z value of the molecular ion peak of protonation is 3854 ± 3.Thus show that molecular weight is 3853 ± 3amu (theoretical value 3853amu).

Example 32

Lys^26,34Bis- (N^ε(ω-carboxyl tridecanoyl base))-GLP-1 (7-37)-OH synthesis

The mixture of GLP-l (7-37)-OH (30mg, 8.9 μm of ol), EDPA (32.3mg, 250 μm of ol), NMP (2.1ml) and water (2.1ml) is mildly shaken 5 minutes in room temperature.By HOOC- (CH₂)₁₂- COONSu (12.7mg, 35.8 μm of ol) is dissolved in NMP (318 μ l) acquired solution, is added in gained mixture, reaction mixture is mildly shaken 40 minutes 1 hour in room temperature.Glycine (3.4mg, 44.7 μm of ol) are added thereto and are dissolved in 50% ethanol water (335 μ l) solution obtained to terminate reaction., reaction mixture is purified by column chromatography using cyanogen propyl column (Zorbax 300SB-CN) and standard acetonitrile/TFA system.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Target compound (10mg, 29%) is isolated, analyzes product with PDMS method.It was found that the m/z value of the molecular ion peak of protonation is 3840 ± 3.Thus show that molecular weight is 3839 ± 3amu (theoretical value 3839amu).

Example 33

Lys^26,34Bis- (N^ε(γ-glutamyl (N^αMyristoyl base))) synthesis (NNC 90-1167) of-GLP-1 (7-37)-OH

The mixture of GLP-1 (7-37)-OH (300mg, 79.8 μm of ol), EDPA (288.9mg, 2.24mmol), NMP (21ml) and water (21ml) is mildly shaken 5 minutes in room temperature.It will be by N made from 29 method of example^αMyristoyl base-Glu (ONSu)-OBu^t(163mg, 319.3 μm of ol) are dissolved in NMP (4.08ml) acquired solution, are added in gained mixture, reaction mixture is mildly shaken 5 minutes in room temperature, is then placed again 1 hour in room temperature.Glycine (131.8mg, 1.76mmol) is added thereto and is dissolved in 50% ethanol water (13.2ml) solution obtained to terminate reaction.0.5% aqueous solution (250ml) of ammonium acetate is added, gained mixture is divided into 4 equal portions.By every part of sample wash to Varian 500mg C8MegaBond ElutOn cylinder, the compound fixed is cleaned with 0.1%TFA aqueous solution (3.5ml), it is final to be released it from column with 70% acetonitrile solution (4ml) elution.Combined eluate is diluted with 0.1%TFA aqueous solution (300ml).Compound precipitates are collected by centrifugation, is washed with 0.1%TFA aqueous solution (50ml), is finally centrifugated compound precipitates.TFA (60ml) is added into precipitating, gained reaction mixture is stirred at room temperature 30 minutes 1 hour.Extra TFA is removed in vacuum, residue is poured into water (50ml).Using cyanogen propyl column (Zorbax300SB-CN) and standard acetonitrile/TFA system, compound precipitates are purified by column chromatography.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Target compound (27.3mg, 8%) is isolated, analyzes product with PDMS method.It was found that the m/z value of the molecular ion peak of protonation is 4036 ± 3.Thus show that molecular weight is 4035 ± 3amu (theoretical value 4035amu).

Example 34

Arg^26,34Lys³⁸(N^ε(ω-carboxyl pentadecanoyl base))-GLP-1 (7-38)-OH synthesis

By Arg^26,34Lys³⁸- GLP-1 (7-38)-OH (30mg, 8.9 μm of ol), EDPA (32.3mg, 250 μm of ol), NMP (2.1ml) and water (2.1ml) mixture mildly shaken 5 minutes in room temperature.By HOOC- (CH₂)₁₄- COONSu (13.7mg, 35.8 μm of ol) is dissolved in NMP (343 μ l) acquired solution, is added in gained mixture, reaction mixture is mildly shaken 1 hour in room temperature.Glycine (3.4mg, 44.7 μm of ol) are added thereto and are dissolved in 50% ethanol water (335 μ l) solution obtained to terminate reaction.Using cyanogen propyl column (Zorbax 300SB-CN) and standard acetonitrile/TFA system, reaction mixture is purified by column chromatography.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Target compound (4.8mg, 14%) is isolated, analyzes product with PDMS method.It was found that the m/z value of the molecular ion peak of protonation is 3894 ± 3.Thus show that molecular weight is 3893 ± 3amu (theoretical value 3893amu).

Example 35

N^αHexadecanoyl group-Glu (ONSu)-OBu^tSynthesis

To H-Glu (OH)-OBu^tPal-ONSu (7.3g, 20.6mmol) is added dropwise in the suspension of (4.2g, 20.6mmol), DMF (500ml) and EDPA (2.65g, 20.6mmol) and is dissolved in solution made from DMF (100ml).Reaction mixture is stirred at room temperature 64 hours, being then concentrated in vacuo to total volume is 20ml.Residue is distributed between 10% aqueous citric acid solution (300ml) and ethyl acetate (250ml), separates two-phase.After organic phase vacuum concentration, residue is dissolved in DMF (50ml).Acquired solution is added dropwise in 10% aqueous citric acid solution (500ml) for being maintained at 0 DEG C.Compound precipitates are collected, and are washed with ice water, are dried in a vacuum drying oven.Compound after drying is dissolved in DMF (45ml), and HONSu (2.15g, 18.7mmol) is added.N is added into gained mixture, N '-dicyclohexylcarbodiimide (3.5g, 17mmol) is dissolved in solution made from methylene chloride (67ml).Reaction mixture is stirred at room temperature 16 hours, then filters compound precipitates.Precipitating is recrystallized to give target compound (6.6g, 72%) from normal heptane/2- propyl alcohol.

Example 36

Lys^26,34Bis- (N^ε(γ-glutamyl (N^αHexadecanoyl group))) synthesis of-GLP-1 (7-37)-OH

The mixture of GLP-1 (7-37)-OH (10mg, 2.9 μm of ol), EDPA (10.8mg, 83.4 μm of ol), NMP (0.7ml) and water (0.7ml) is mildly shaken 5 minutes in room temperature.It will be by N made from 33 method of example^αHexadecanoyl group-Glu (ONSu)-OBu^t(163mg, 319.3 μm of ol) are dissolved in NMP (4.08ml) acquired solution, are added in gained mixture thereto, reaction mixture is mildly shaken 20 minutes 1 hour in room temperature.Glycine (4.9mg, 65.6 μm of ol) are added and are dissolved in 50% ethanol water (492 μ l) solution obtained to terminate reaction.0.5% aqueous solution (9ml) of ammonium acetate is added, gained mixture is flushed to a Varian 1g C8Mega Bond ElutOn cylinder, the compound fixed is cleaned with 5% acetonitrile solution (10ml), is finally eluted with TFA (10ml) and releases it from column.It is concentrated in vacuo eluate and residue is purified by column chromatography using cyanogen propyl column (Zorbax 300SB-CN) and standard acetonitrile/TFA system.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Target compound (2.4mg, 20%) is isolated, analyzes product with PDMS method.It was found that the m/z value of the molecular ion peak of protonation is 4092 ± 3.Thus show that molecular weight is 4091 ± 3amu (theoretical value 4091amu).

Example 37

Arg³⁴Lys²⁶(N^ε(γ-glutamyl (N^αHexadecanoyl group))) synthesis of-GLP-1 (7-37)-OH

By Arg³⁴- GLP-1 (7-37)-OH (3.7mg, 1.1 μm of ol), EDPA (4.0mg, 30.8 μm of ol), acetonitrile (260 μ l) and water (260 μ l) mixture mildly shaken 5 minutes in room temperature.It will be by N made from 35 method of example^αHexadecanoyl group-Glu (ONSu)-OBu^t(1.8mg, 3.3 μm of ol) are dissolved in acetonitrile (44.2 μ l) acquired solution, are added in gained mixture, reaction mixture is mildly shaken 20 minutes 1 hour in room temperature.Glycine (1.8mg, 24.2 μm of ol) are added thereto and are dissolved in 50% ethanol water (181 μ l) solution obtained to terminate reaction.0.5% aqueous solution (12ml) and NMP (300 μ L) of ammonium acetate is added, gained mixture is flushed to Varian 1g C8Mega Bond ElutOn membrane cartridge, the compound fixed is cleaned with 5% acetonitrile solution (10ml), is finally eluted with TFA (6ml) and releases it from column.Eluate is being placed at room temperature for 2 hours, is then being concentrated in vacuo.Using cyanogen propyl column (Zorbax 300SB-CN) and standard acetonitrile/TFA system, by purifying residue with column chromatography.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Target compound (0.23mg, 6%) is isolated, analyzes product with PDMS method.It was found that the m/z value of the molecular ion peak of protonation is 3752 ± 3.Thus show that molecular weight is 3751 ± 3amu (theoretical value 3751amu).

Example 38

Arg^26,34Lys³⁸(N^ε(γ-glutamyl (N^αMyristoyl base))) synthesis of-GLP-1 (7-38)-OH

By Arg^26,34Lys³⁸- GLP-1 (7-38)-OH (14mg, 4.0 μm of ol), EDPA (14.3mg, 110.6 μm of ol), NMP (980 μ l) and water (980 μ l) mixture mildly shaken 5 minutes in room temperature.It will be by N made from 29 method of example^αMyristoyl base-Glu (ONSu)-OBu^t(12.1mg, 23.7 μm of ol) are dissolved in NMP (303 μ l) acquired solution, are added in gained mixture, reaction mixture is mildly shaken 2 hours in room temperature.Glycine (6.5mg, 86.9mmol) is added thereto and is dissolved in 50% ethanol water (652 μ l) solution obtained to terminate reaction.0.5% aqueous solution (50ml) of ammonium acetate is added, gained mixture is flushed to Varian 1gC8 Mega Bond ElutOn cylinder, the compound fixed is cleaned with 5% acetonitrile solution (15ml), it is final to be released it from column with TFA aqueous solution (6ml) elution.Eluate is being placed at room temperature for 45 minutes 1 hour, is then being concentrated in vacuo.Using cyanogen propyl column (Zorbax300SB-CN) and standard acetonitrile/rFA system, residue is purified by column chromatography.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Target compound (3.9mg, 26%) is isolated, analyzes product with PDMS method.It was found that the m/z value of the molecular ion peak of protonation is 3881 ± 3.Thus show that molecular weight is 3880 ± 3amu (theoretical value 3880amu).

Example 39

Arg^26,34Lys³⁸(N^ε(ω-carboxyl pentadecanoyl base))-GLP-1 (7-38)-OH synthesis

By Arg^26,34Lys³⁸- GLP-1 (7-38)-OH (14mg, 4.0 μm of ol), EDPA (14.3mg, 111 μm of ol), NMP (980 μ l) and water (980 μ l) mixture mildly shaken 5 minutes in room temperature.By HOOC- (CH₂)₁₄- COONSu (4.5mg, 11.9 μm of ol) is dissolved in NMP (114 μ l) acquired solution, is added in gained mixture, reaction mixture is mildly shaken 45 minutes 1 hour in room temperature.It is another that HOOC- (CH is added₂)₁₄- COONSu (4.0mg, 10.4 μm of ol) is dissolved in NMP (100 μ l) acquired solution, and gained mixture is mildly shaken 30 minutes 1 hour again in room temperature.Glycine (1.5mg, 19.8 μm of ol) are added thereto and are dissolved in 50% ethanol water (148 μ l) solution obtained to terminate reaction.Using cyanogen propyl column (Zorbax 300SB-CN) and standard acetonitrile/TFA system, by purifying reaction mixture with column chromatography.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Target compound (3.9mg, 26%) is isolated, analyzes product with PDMS method.It was found that the m/z value of the molecular ion peak of protonation is 3809 ± 3.Thus show that molecular weight is 3808 ± 3amu (theoretical value 3808amu).  

Example 40

Arg^26,34Lys³⁸(N^ε(γ-glutamyl (N^αHexadecanoyl group))) synthesis of-GLP-1 (7-38)-OH

By Arg^26,34Lys³⁸- GLP-1 (7-38)-OH (14mg, 4.0 μm of ol), EDPA (14.3mg, 110.6 μm of ol), NMP (980 μ l) and water (980 μ l) mixture mildly shaken 5 minutes in room temperature.It will be by N made from 35 method of example^αHexadecanoyl group-Glu (ONSu)-OBu^t(6.4mg, 11.9 μm of ol) are dissolved in NMP (160 μ l) acquired solution, are added in gained mixture, reaction mixture is mildly shaken 20 minutes 1 hour in room temperature.Glycine (6.5mg, 87mmol) is added thereto and is dissolved in 50% ethanol water (653 μ l) solution obtained to terminate reaction.0.5% aqueous solution (50ml) of ammonium acetate is added, gained mixture is flushed to Varian 1g C8 MegaBond ElutOn cylinder, the compound fixed is cleaned with 5% acetonitrile solution (10ml), is finally eluted with TFA (6ml) and releases it from column.Eluate is being stored at room temperature 30 minutes 1 hour, is then being concentrated in vacuo.Using cyanogen propyl column (Zorbax 300SB-CN) and standard acetonitrile/TFA system, residue is purified by column chromatography.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Target compound (7.2mg, 47%) is isolated, analyzes product with PDMS method.It was found that the m/z value of the molecular ion peak of protonation is 3881 ± 3.Thus show that molecular weight is 3880 ± 3amu (theoretical value 3880amu).

Example 41

Arg^{18,23,26,30,34}Lys³⁸(N^εHexadecanoyl group)-GLP-1 (7-38)-OH synthesis

By Arg^{18,23,26,30,34}Lys³⁸The mixture of-GLP-1 (7-38)-OH (1.0mg, 0.27 μm of ol), EDPA (0.34mg, 2.7 μm of ol) and DMSO (600 μ l) mildly shake 5 minutes in room temperature.Pal-ONSu (0.28mg, 0.8 μm of ol) is added into gained mixture and is dissolved in solution made from NMP (7 μ l).Reaction mixture is mildly shaken 5 minutes in room temperature, is then being placed at room temperature for 6 hours.Glycine (1.6mg, 21.7 μm of ol) are added thereto and are dissolved in 50% ethanol water (163 μ l) solution obtained to terminate reaction.Using cyanogen propyl column (Zorbax 300SB-CN) and standard acetonitrile/TFA system, reaction mixture is purified by column chromatography.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Target compound (0.17mg, 16%) is isolated, analyzes product with MALDI-MS method.It was found that the m/z value of the molecular ion peak of protonation is 3961 ± 3.The molecular weight thus obtained is 3960 ± 3amu (theoretical value 3960amu).

Example 42

Arg^26,34Lys³⁸(N^ε(ω-carboxyl tridecanoyl base))-GLP-1 (7-38)-OH synthesis

By Arg^26,34Lys³⁸- GLP-1 (7-38)-OH (14mg, 4.0 μm of ol), EDPA (14.3mg, 111 μm of ol), NMP (980 μ l) and water (980 μ l) mixture mildly shaken 5 minutes in room temperature.By HOOC- (CH₂)₁₂- COONSu (4.2mg, 11.9 μm of ol) is dissolved in NMP (105 μ l) acquired solution, is added in gained mixture, reaction mixture is mildly shaken 50 minutes 1 hour in room temperature.Glycine (6.5mg, 87 μm of ol) are added thereto and are dissolved in 50% ethanol water (652 μ l) solution obtained to terminate reaction.Using cyanogen propyl column (Zorbax 300SB-CN) and standard acetonitrile/TFA system, reaction mixture is purified by column chromatography.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Target compound (5.8mg, 39%) is isolated, analyzes product with MALDI-MS method.It was found that the m/z value of the molecular ion peak of protonation is 3780 ± 3.Thus show that molecular weight is 3779 ± 3amu (theoretical value 3781amu).

Example 43

Arg³⁴Lys²⁶(N^ε(γ-glutamyl (N^αMyristoyl base))) synthesis of-GLP-1 (7-37)-OH

By Arg³⁴- GLP-1 (7-37)-OH (15mg, 4.4 μm of ol), EDPA (16mg, 124 μm of ol), NMP (2ml) and water (4.8ml) mixture mildly shaken 5 minutes in room temperature.It will be by N made from 29 method of example^αMyristoyl base-Glu (ONSu)-OBu^t(12.1mg, 23.7 μm of ol) are dissolved in NMP (303 μ l) acquired solution, are added in gained mixture, reaction mixture is mildly shaken 2 hours in room temperature.Glycine (6.5mg, 86.9 μm of ol) are added thereto and are dissolved in 50% ethanol water (652 μ l) solution obtained to terminate reaction.(gained mixture is flushed to Varian 1g C8Mega Bond Elut to 0.5% aqueous solution of addition ammonium acetate by 50mDOn cylinder, the compound fixed is cleaned with 5% acetonitrile solution (15ml), is finally eluted with TFA (6ml) and releases it from column.Eluate is being placed at room temperature for 45 minutes 1 hour, is then being concentrated in vacuo.Using cyanogen propyl (cyanopropyl) column (Zorbax 300SB-CN) and standard acetonitrile/TFA system, residue is purified by column chromatography.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Target compound (3.9mg, 26%) is isolated, analyzes product with MALDI-MS method.It was found that the m/z value of the molecular ion peak of protonation is 3723 ± 3.Thus show that molecular weight is 3722 ± 3amu (theoretical value 3723amu).

Example 44

N^αOctadecanoyl-Glu (ONSu)-OBu^tSynthesis

To H-Glu (OH)-OBu^tSte-ONSu (5.3g, 13.9mmol) is added dropwise in the suspension of (2.82g, 13.9mmol), DMF (370ml) and EDPA (1.79g, 13.9mmol) and is dissolved in the solution that DMF (60ml) is obtained.It is added methylene chloride (35ml), reaction mixture is stirred at room temperature 24 hours, is then concentrated in vacuo.It distributes residue between 10% aqueous citric acid solution (330ml) and ethyl acetate (200ml), separates two-phase.Organic phase is concentrated in vacuo, residue is made to be dissolved in DMF (60ml).Acquired solution is added drop-wise in 10% aqueous citric acid solution (400ml) for being maintained at 0 DEG C.Sediment is collected, and is washed with ice water, it is dry in vacuum oven.Compound after drying is dissolved in DMF (40ml), and HONSu (1.63g, 14.2mmol) is added.The solution that DCC (2.66g, 12.9mmol) is dissolved in methylene chloride (51ml) formation is added into reaction mixture.Gained mixture is stirred at room temperature 64 hours, compound precipitates are obtained by filtration.It will be deposited in normal heptane/2- propyl alcohol and be recrystallized to give target compound (4.96g, 68%).

Example 45

Arg^26,34Lys³⁸(N^ε(γ-glutamyl (N^αOctadecanoyl))) synthesis of-GLP-1 (7-38)-OH

By Arg^26,34- GLP-1 (7-38)-OH (28mg, 7.9 μm of ol), EDPA (28.6mg, 221.5 μm of ol), NMP (1.96ml) and water (1.96ml) mixture mildly shaken 5 minutes in room temperature.It will be by N made from 44 method of example^αOctadecanoyl-Glu (ONSu)-OBu^t(17.93g, 31.6 μm of ol) are dissolved in NMP (448 μ l) acquired solution, are added in gained mixture, reaction mixture is mildly shaken 2 hours in room temperature.Glycine (13.1mg, 174 μm of ol) are added thereto and are dissolved in 50% ethanol water (1.3ml) solution obtained to terminate reaction.0.5% aqueous solution (120ml) of ammonium acetate is added, gained mixture is divided into 2 equal portions.Varian5g C8Mega Bond Elut is flushed to by every partOn cylinder, the compound fixed is cleaned with 5% acetonitrile solution (25ml), is finally eluted with TFA (25ml) and releases it from column.Combined eluate is being placed at room temperature for 25 minutes 1 hour, is then being concentrated in vacuo.Using cyanogen propyl column (Zorbax300SB-CN) and standard acetonitrile/TFA system, residue is purified by column chromatography.Column is heated to 65 DEG C, the concentration gradient of acetonitrile is 0-100% in 60 minutes.Target compound (3.6mg, 11%) is isolated, analyzes product with MALDI-MS method.It was found that the m/z value of the molecular ion peak of protonation is 3940 ± 3.Thus show that molecular weight is 3939 ± 3amu (theoretical value 3937amu).

Biology Observation

Long duration of action after GLP-1 derivative subcutaneous administration

Using following methods, the concentration after health pig subcutaneous administration GLP-1 derivative in its blood plasma is monitored, to measure the long duration of action of some GLP-1 derivatives of the invention.In order to be compared, also to subcutaneous administration GLP-1 (7-37), its concentration in blood plasma is determined afterwards.As a result shown in table 1.The long duration of action of the other GLP-1 derivatives of the present invention can according to said method be measured.

Pig even if (50%Duroc, 25%Yorkshire, 25%DanishLandrace weigh about 40kg) fasting since experiment.Every pig is given to be dissolved in 50 μM of isotonic solution (5mM phosphate, pH7.4,0.02% tween by per kilogram of body weight- 20 (Merck), 45mg/ml mannitol (remove pyrogen removal, Novo Nordisk)) 0.5nmol test compound.The time shown in table 1 extracts blood sample from jugular vein conduit.5ml blood sample is poured into the ice-cold glass containing the 175 following solution of μ l: 0.18M EDTA, 1500KIE/ml aprotinin (Novo Nordisk) and 3% bacitracin (Sigma), pH7.4.In 30 minutes, by sample in 5-6000^*G is centrifuged 10 minutes.Keep the temperature at 4 DEG C.Supernatant is drawn in different glasses with suction pipe, stores in one 20 DEG C until using.

There is the monoclonal antibody of specificity using the N- terminal region to GLP-1 (7-37), the plasma concentration of all peptides is measured by RIA method.Cross reaction with GLP-1 (1-37) and GLP-1 (8-36) amide is less than 1%, and the cross reaction with GLP-1 (9-37), GLP-1 (10-36) amide and GLP-1 (11-36) amide is less than 0.1%.In 4 DEG C of progress total overall reactions.

It is measured as follows: 100 μ l blood plasma is mixed with 271 μ l, 96% ethyl alcohol, mixed with turbine mixer and 2600^*G is centrifuged 30 minutes.Supernatant is gently poured into Minisorp test tube, and evaporating completely (Savant Speedvac AS290).Evaporation residue is re-dissolved in containing 80mM sodium dihydrogen phosphate/disodium hydrogen phosphate, 0.1%HSA (Orpha 20/21, Behring), 10mM EDTA, 0.6mM thiomersalate (Sigma), in the measurement buffer of pH7.5.Sample is dissolved in the volume for being suitable for reaching its predicted concentration, and is made it dissolve 30 minutes.100 μ l are added into 300 μ l samples by containing 40mM sodium dihydrogen phosphate/disodium hydrogen phosphate, 0.1%HSA, 0.6mM thiomersalate, pH7.5 dilution buffer made of antibody-solutions.300 μ l buffers and 100 μ l dilution buffers are mixed and made into non-specific sample.Each standard is made from freeze-drying reserve object, is dissolved in 300 μ l measurement buffer.All samples in Minisorp pipe with antibody preincubation as described above 72 hours.The 200 μ l of tracer containing 6-7000CPM being dissolved in dilution buffer is added thereto, sample is mixed and keeps the temperature 48 hours.The stable ox blood of the heparin being added in 40mM sodium dihydrogen phosphate/disodium hydrogen phosphate, 0.6mM thiomersalate, pH7.5 in every pipe starches the suspension 1.5ml of 200ml/l and 18g/l activated carbon (Merck).Before use, suspension is mixed and places it 2 hours at 4 DEG C.All samples are kept the temperature 1 hour at 4 DEG C, then 3400^*G is centrifuged 25 minutes.Supernatant is gently taken out after centrifugation immediately and is counted in one γ-counter.The concentration in sample is calculated from each standard curve.Following plasma concentration is measured, they are the percentage (n=2) of the maximum concentration of each compound:

Table 1

Test compound)	Time (hour) after subcutaneous administration
										0.75	1	2	4	6	8	10	12	24
	GLP-(7-37)		100	9	1
Example 25										73	92	100	98	82	24	16	16	16
	Example 17	76	71	91	100	84	68	30											9
Example 43											39	71	93	100	91	59	50	17
	Example 37		26	38	97	100	71	81	80										45
Example 11										24	47	59	71	100	94	100		94
	Example 12	36	54	65	94	80	100	85											93
Example 32										55	53	90	83	88	70	98	100	100
	Example 14	18	25	32	47	98	83	97											100
Example 13										15	22	38	59	97	85	100		76
	Example 38	60	53	100	66	48	39	25	29										0
Example 39										38	100	70	47	33	33	18	27	14
	Example 40	47	19	50	100	51	56	34	14										0
Example 34										19	32	44	84	59	66	83	84	100

^.)Test is each example target compound for providing example number with compound

As shown in table 1, GLP-1 derivative of the invention has the effect curves than GLP-1 (7-37) longer time, and in blood plasma far persistently than GLP-1 (7-37).Table 1 also shows that selected specific GLP-1 derivative is different, and the time change range that they reach Cmax in blood plasma is very big.

The stimulation that cAMP in the cell line of people's GLP-1 receptor of expression cloning is formed

For the efficiency for confirming GLP-1 derivative, the ability that they are formed in the cell line internal stimulus cAMP of people's GLP-1 receptor of expression cloning is determined.EC is calculated from dose-response curve₅₀。

Use baby hamster kidney (BHK) cell (Knudsen and Pridal, 1996, European pharmacology magazine, 318,429-435) of expression human pancreas GLP-1 receptor.By in buffer (10mmol/l Tris-HCl and 30mmol/l NaCl pH7.4, in addition also contain 1mmol/l dithiothreitol (DTT), 5mg/l leupeptin (Sigma, St.Louis, MO, USA), 5mg/l pepstatin (Sigma, St.Louis, MO, USA), 100mg/l bacitracin (Sigma, St.Louis, MO, USA) and 16mg/l aprotinin (Novo Nordisk A/S, Bagsvaerd, Denmark)) in homogenate prepare plasma membrane (Adelhorst etc., 1994, journal of biological chemistry, 269,6275).Homogenate is centrifuged on the sucrose layer top 41w/v%.White ribbon between two layers is diluted and is centrifuged in buffer.Plasma membrane be stored in -80 DEG C it is spare.

It is measured in 96 hole microtiter plates with 140 μ l of total volume.Buffer used is 50mmol/l Tris-HCl, pH7.4, is additionally added 1mmol/l EGTA, 1.5mmol/lMgSO₄, 1.7mmol/l ATP, 20mM GTP, 2mmol/l 3-isobutyl-1-methylxanthine, 0.01% Tween-20 and 0.1% human serum albumins (Reinst, BehringwerkeAG, Marburg, Germany).The compound for being intended to survey its agonist activity is dissolved and is diluted in buffer, is added in plasma membrane preparation, and mixture is kept the temperature 2 hours at 37 DEG C.25 μ l0.05mol/l hydrochloric acid are added and terminate reaction.CAMP is analyzed with scintillation proximity assay (scintillation proximity assay) (RPA 538, Amersham, UK) after sample is diluted 10 times.Obtain following result:

Test compound.)	EC50, pM	Test compound.)	EC50, pM
				GLP-1(7-37)	61	Example 31	96
Example 45	120	Example 30	41
				Example 43	24	Example 26	8.8
Example 40	55	Example 25	99
				Example 39	5.1	Example 19	79
Example 38	54	Example 16	3.5
				Example 37	60

^.)Test is the target compound for providing the example of example number with compound

Claims (48)

1. a kind of GLP-1 derivative, wherein at least one amino acid residue of parent peptide is connected with a lipophilic substituent, and if only one lipophilic substituent, and the substituent group is connected on the end N- or C- terminal amino acid residue of parent peptide, then the substituent group is alkyl or with ω-carboxyl group.

2. GLP-1 derivative according to claim 1, only one of them lipophilic substituent.

3. GLP-1 derivative according to claim 2, lipophilic substituent therein are connected on -terminal amino acid residue.

4. GLP-1 derivative according to claim 2, lipophilic substituent therein are connected on C- terminal amino acid residue.

5. GLP-1 derivative according to claim 2, lipophilic substituent therein is connected to neither on an amino acid residue of the end N- nor the end C-.

6. GLP-1 derivative according to claim 1, wherein there are two lipophilic substituents.

7. GLP-1 derivative according to claim 6, one of lipophilic substituent is connected on -terminal amino acid residue, and another lipophilic substituent is connected on C- terminal amino acid residue.

8. GLP-1 derivative according to claim 6, one of lipophilic substituent are connected on C- terminal amino acid residue, and another lipophilic substituent is connected to neither on an amino acid residue of the end N- nor the end C-.

9. GLP-1 derivative according to claim 6, two of them lipophilic substituent is all connected to neither on the amino acid residue of the end N- nor the end C-.

The derivative of 10.GLP-1 (7-C), wherein C is selected from the group containing 38,39,40,41,42,43,44 and 45, which only has the lipophilic substituent being connected on C- terminal amino acid residue.

11. lipophilic substituent therein contains 4-40 carbon atom, particularly preferred 8-25 carbon atom according to GLP-1 derivative described in any of the above-described claim.

12. one of lipophilic substituent forms an amido bond with the amino of its carboxyl and an amino acid residue according to GLP-1 derivative described in any of the above-described claim, it is thus connected on the amino acid residue.

13. GLP-1 derivative described in any one of -11 according to claim 1, one of lipophilic substituent forms amido bond with the carboxyl of its amino and an amino acid residue, is thus connected on the amino acid residue.

14. wherein lipophilic substituent is connected on parent peptide by a spacer group according to GLP-1 derivative described in any of the above-described claim.

15. GLP-1 derivative according to claim 14, spacer group therein be have 1-7 methylene, preferably have 2 methylene regardless of branched paraffin α, ω-dicarboxyl, the spacer group form a bridge between the amino of parent peptide and the amino of lipophilic substituent.

16. GLP-1 derivative according to claim 14, spacer group therein is an amino acid residue or a kind of dipeptides of such as Gly-Lys in addition to Cys.

17. GLP-1 derivative according to claim 16, wherein an amino in a carboxyl of parent peptide and Lys or dipeptides containing Lys residue forms amido bond, and the carboxyl in another amino and lipophilic substituent in Lys spacer group or dipeptides spacer group containing Lys residue forms an amido bond.

18. GLP-1 derivative according to claim 16, wherein a carboxyl of an amino of parent peptide and the amino acid residue or dipeptides spacer group forms an amido bond, and a carboxyl of an amino of the amino acid residue or dipeptides spacer group and the lipophilic substituent forms an amido bond.

19. GLP-1 derivative according to claim 16, wherein an amino of a carboxyl of parent peptide and the amino acid residue spacer group or dipeptides spacer group forms an amido bond, and an amino of a carboxyl of the amino acid residue spacer group or dipeptides spacer group and the lipophilic substituent forms an amido bond.

20. GLP-1 derivative according to claim 16, wherein a carboxyl of parent peptide and Asp or Glu spacer group, or an amino of the dipeptides spacer group containing Asp or Glu residue forms an amido bond, and an amino of a carboxyl of the spacer group and the lipophilic substituent forms an amido bond.

21. according to GLP-1 derivative described in any of the above-described claim, lipophilic substituent therein contains the luxuriant and rich with fragrance skeleton of ring penta of a partly or completely perhydrogenating.

22. GLP-1 derivative described in any one of -20 according to claim 1, lipophilic substituent therein is the alkyl of a straight chain or branch.

23. GLP-1 derivative described in any one of -20 according to claim 1, lipophilic substituent therein is the acyl group of the fatty acid of straight chain or branch.

24. GLP-1 derivative according to claim 23, acyl group therein, which is selected from, contains CH₃(CH₂)_nThe group of CO-, wherein n is 4-38, is preferably selected from following member: CH₃(CH₂)₆CO-, CH₃(CH₂)₈CO-, CH₃(CH₂)₁₀CO-, CH₃(CH₂)₁₂CO-, CH₃(CH₂)₁₄CO-, CH₃(CH₂)₁₆CO-, CH₃(CH₂)₁₈CO-, CH₃(CH₂)₂₀CO- and CH₃(CH₂)₂₂CO-。

25. GLP-1 derivative described in any one of -20 according to claim 1, lipophilic substituent therein is the acyl group of the alkane alpha, omega-dicarboxylic acid of straight chain or branch.

26. GLP-1 derivative according to claim 25, acyl group therein is selected from the (CH containing HOOC₂)_mThe group of CO-, wherein m is 4-38, preferably 4-24, further preferably selected from the group containing following members: HOOC (CH₂)₁₄CO-, HOOC (CH₂)₁₆CO-, HOOC (CH₂)₁₈CO-, HOOC (CH₂)₂₀CO- and HOOC (CH₂)₂₂CO-。

27. GLP-1 derivative described in any one of -20 according to claim 1, lipophilic substituent therein is with formula CH₃(CH₂)_p((CH₂)_qCOOH)CHNH-CO(CH₂)₂A group of CO-, wherein p and q is integer, and p+q is the integer of 8-33, preferably the integer of 12-28.

28. GLP-1 derivative described in any one of -20 according to claim 1, lipophilic substituent therein is with formula CH₃(CH₂)_rCO-NHCH(COOH)(CH₂)₂A group of CO-, wherein r is the integer of 10-24.

29. GLP-1 derivative described in any one of -20 according to claim 1, lipophilic substituent therein is with formula CH₃(CH₂)_sCO-NHCH((CH₂)₂COOH) a group of CO-, wherein s is the integer of 8-24.

30. GLP-1 derivative described in any one of -20 according to claim 1, lipophilic substituent therein is with formula-NHCH (COOH) (CH₂)₄NH-CO(CH₂)_uCH₃A group, wherein u is the integer of 8-18.

31. GLP-1 derivative described in any one of -20 according to claim 1, lipophilic substituent therein is with formula-NHCH (COOH) (CH₂)₄NH-COCH((CH₂)₂COOH)NH-CO(CH₂)_wCH₃A group, wherein w is the integer of 10-16.

32. GLP-1 derivative described in any one of -20 according to claim 1, lipophilic substituent therein is with formula-NHCH (COOH) (CH₂)₄NH-CO(CH₂)₂CH(COOH)NH-CO(CH₂)_xCH₃A group, wherein x is the integer of 10-16.

33. GLP-1 derivative described in any one of -20 according to claim 1, lipophilic substituent therein is with formula-NHCH (COOH) (CH₂)₄NH-CO(CH₂)₂CH(COOH)NH-CO(CH₂)_yCH₃A group, wherein y is the integer of 0 or 1-22.

34. GLP-1 derivative described in any one of -33 according to claim 1, wherein parent peptide is selected from the group containing GLP-1 (1-45) or its analog or their segment.

35. GLP-1 described in any one of -33 (A-B) derivative according to claim 1, wherein A is the integer of 1-7, B is the integer of 38-45, or its analog, the analog has a lipophilic substituent to be connected on C- terminal amino acid residue, and optionally, there is another lipophilic substituent to be connected on another amino acid residue.

36. GLP-1 derivative according to claim 34, wherein parent peptide is selected from (7-35) containing GLP-1, GLP-1 (7-36), GLP-1 (7-36) amide, GLP-1 (7-37), the group of GLP-1 (7-38), GLP-1 (7-39), GLP-1 (7-40) and GLP-1 (7-41) and the like.

37. GLP-1 derivative according to claim 34, wherein parent peptide is selected from (1-35) containing GLP-1, GLP-1 (1-36), GLP-1 (1-36) amide, GLP-1 (1-37), GLP-1 (1-38), GLP-1 (1-39), the group of GLP-1 (1-40), GLP-1 (1-41) and its analog.

38. specified analog therein contains sum and is no more than the derivative that 15, preferably up to 10 amino acid residues have been replaced by any a-amino acid residue according to GLP-1 derivative described in any of the above-described claim.

39. specified analog therein contains the derivative that total at most 15, preferably up to 10 amino acid residues can have been replaced by any a-amino acid residue of genetic code encoding according to GLP-1 derivative described in any of the above-described claim.

40. according to GLP-1 derivative described in any of the above-described claim, wherein specified analog contains total at most 6 amino acid residues by any derivative that can be replaced by the a-amino acid residue of genetic code encoding.

41. wherein parent peptide is selected from: Arg according to GLP-1 derivative described in any of the above embodiments²⁶- GLP-1 (7-37), Arg³⁴- GLP-1 (7-37), Lys³⁶- GLP-1 (7-37), Arg^26,34Lys³⁶- GLP-1 (7-37), Arg^26,34Lys³⁸- GLP-1 (7-38), Arg^26,34Lys³⁹- GLP-1 (7-39), Arg^26,34Lys⁴⁰- GLP-1 (7-40), Arg²⁶Lys³⁶- GLP-1 (7-37), Arg³⁴Lys³⁶- GLP-1 (7-37), Arg²⁶Lys³⁹- GLP-1 (7-39), Arg³⁴Lys⁴⁰- GLP-1 (7-40), Arg^26,34Lys^36,39- GLP-1 (7-39), Arg^26,34Lys^36,40GLP-1 (7-40), Gly⁸Arg²⁶- GLP-1 (7-37), Gly⁸Arg³⁴- GLP-1 (7-37), Gly⁸Lys³⁶- GLP-1 (7-37), Gly8Arg^26,34Lys³⁶- GLP-1 (7-37), Gly⁸Arg^26,34Lys³⁹- GLP-1 (7-39), Gly⁸Arg^26,34Lys⁴⁰- GLP-1 (7-40), Gly⁸Arg²⁶Lys³⁶- GLP-1 (7-37), Gly⁸Arg³⁴Lys³⁶- GLP-1 (7-37), Gly⁸Arg²⁶Lys³⁹- GLP-1 (7-39), Gly⁸Arg³⁴Lys⁴⁰- GLP-1 (7-40), Gly⁸Arg^26,34Lys^36,39- GLP-1 (7-39) and Gly⁸Arg^26,34Lys^36,40-GLP-1(7-40)。

42. GLP-1 derivative described in any one of -40 according to claim 1, wherein parent peptide is selected from:

Arg^26,34Lys³⁸- GLP-1 (7-38), Arg^26,34Lys³⁹- GLP-1 (7-39), Arg^26,34Lys⁴⁰- GLP-1 (7-40), Arg^26,34Lys⁴¹- GLP-1 (7-41), Arg^26,34Lys⁴²- GLP-1 (7-42), Arg^26,34Lys⁴³- GLP-1 (7-43), Arg^26,34Lys⁴⁴- GLP-1 (7-44), Arg^26,34Lys⁴⁵- GLP-1 (7-45), Arg^26,34Lys³⁸- GLP-1 (1-38), Arg^26,34Lys³⁹- GLP-1 (1-39), Arg^26,34Lys⁴⁰- GLP-1 (1-40), Arg^26,34Lys⁴¹- GLP-1 (1-41), Arg^26,34Lys⁴²- GLP-1 (1-42), Arg^26,34Lys⁴³- GLP-1 (1-43), Arg^26,34Lys⁴⁴- GLP-1 (1-44), Arg^26,34Lys⁴⁵- GLP-1 (1-45), Arg^26,34Lys³⁸- GLP-1 (2-38), Arg^26,34Lys³⁹- GLP-1 (2-39), Arg^26,34Lys⁴⁰- GLP-1 (2-40), Arg^26,34Lys⁴¹- GLP-1 (2-41), Arg^26,34Lys⁴²- GLP-1 (2-42), Arg^26,34Lys⁴³- GLP-1 (2-43), Arg^26,34Lys⁴⁴- GLP-1 (2-44), Arg^26,34Lys⁴⁵- GLP-1 (2-45), Arg^26,34Lys³⁸- GLP-1 (3-38), Arg^26,34Lys³⁹- GLP-1 (3-39), Arg^26,34Lys⁴⁰- GLP-1 (3-40), Arg^26,34Lys⁴¹- GLP-1 (3-41), Arg^26,34Lys⁴²- GLP-1 (3-42), Arg^26,34Lys⁴³- GLP-1 (3-43), Arg^26,34Lys⁴⁴- GLP-1 (3-44), Arg^26,34Lys⁴⁵- GLP-1 (3-45), Arg^26,34Lys³⁸- GLP-1 (4-38), Arg^26,34Lys³⁹- GLP-1 (4-39), Arg^26,34Lys⁴⁰- GLP-1 (4-40), Arg^26,34Lys⁴¹- GLP-1 (4-41), Arg^26,34Lys⁴²- GLP-1 (4-42), Arg^26,34 Lys⁴³- GLP-1 (4-43), Arg^26,34Lys⁴⁴- GLP-1 (4-44), Arg^26,34Lys⁴⁵- GLP-1 (4-45), Arg^26,34Lys³⁸- GLP-1 (5-38), Arg^26,34Lys³⁹- GLP-1 (5-39), Arg^26,34Lys⁴⁰- GLP-1 (5-40), Arg^26,34Lys⁴¹- GLP-1 (5-41), Arg^26,34Lys⁴²- GLP-1 (5-42), Arg^26,34Lys⁴³- GLP-1 (5-43), Arg^26,34Lys⁴⁴- GLP-1 (5-44), Arg^26,34Lys⁴⁵- GLP-1 (5-45), Arg^26,34Lys³⁸- GLP-1 (6-38), Arg^26,34Lys³⁹- GLP-1 (6-39), Arg^26,34Lys⁴⁰- GLP-1 (6-40), Arg^26,34Lys⁴¹- GLP-1 (6-41), Arg^26,34Lys⁴²- GLP-1 (6-42), Arg^26,34Lys⁴³- GLP-1 (6-43), Arg^26,34Lys⁴⁴- GLP-1 (6-44), Arg^26,34Lys⁴⁵- GLP-1 (6-45), Arg²⁶Lys³⁸- GLP-1 (1-38), Arg³⁴Lys³⁸- GLP-1 (1-38), Arg^26,34Lys^36,38- GLP-1 (1-38), Arg²⁶Lys³⁸- GLP-1 (7-38), Arg³⁴Lys³⁸- GLP-1 (7-38), Arg^26,34Lys^36,38- GLP-1 (7-38), Arg^26,34Lys³⁸- GLP-1 (7-38), Arg²⁶Lys³⁹- GLP-1 (1-39), Arg³⁴Lys³⁹- GLP-1 (1-39), Arg^26,34Lys³⁶, 39-GLP-1 (1-39), Arg²⁶Lys³⁹- GLP-1 (7-39), Arg³⁴Lys³⁹- GLP-1 (7-39) and Arg^26,34Lys^36,39-GLP-1(7-39)

43. a kind of pharmaceutical composition contains GLP-1 derivative of the invention and pharmaceutically acceptable excipient or carrier.

44. the purposes of GLP-1 derivative of the invention in the drug that preparation has than the effect curves of GLP-1 (7-37) longer time.

45. the purposes of GLP-1 derivative of the invention in the drug for preparing the effect curves with the longer time, can be used for treating adult-onset diabetes.

46. the purposes of GLP-1 derivative of the invention in the drug for preparing the effect curves with the longer time, can be used for treating insulin-dependent diabetes mellitus.

47. the purposes of GLP-1 derivative of the invention in the drug for preparing the effect curves with the longer time, can be used for treating obesity.

48. a kind of method for treating insulin-dependent or adult-onset diabetes in the patient for needing such treatment, GLP-1 derivative according to claim 1 and pharmaceutically acceptable carrier including the therapeutically effective amount that doses a patient with.