CN1951965B - Glp-1 derivatives - Google Patents
Glp-1 derivatives Download PDFInfo
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- CN1951965B CN1951965B CN2006101108983A CN200610110898A CN1951965B CN 1951965 B CN1951965 B CN 1951965B CN 2006101108983 A CN2006101108983 A CN 2006101108983A CN 200610110898 A CN200610110898 A CN 200610110898A CN 1951965 B CN1951965 B CN 1951965B
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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
Technical field that the present invention belongs to
The present invention relates to the novel derivative of human glucagon-like-peptide-1 (GLP-1) and fragment thereof and these segmental analogues, they have the effect curves (protracted profile ofaction) of longer time, also relate to the preparation and the using method of these materials.
The technical background of invention
Peptide is widely used in medical practice, owing to can prepare peptide with the DNA recombinant technology, can estimate that therefore its importance will not increase yet in serving year.When in treatment, using native peptides or its analogue, find that usually they have high clearance rate.Needs make therapeutical agent the longer time during in the maintenance its high blood levels situation under, the high clearance rate of therapeutical agent is unfavorable, because this will need repeat administration.There is the peptide of high clearance rate for example to have: ACTH, corticotropin releasing factor(CRF), angiotonin, thyrocalcitonin, Regular Insulin, hyperglycemic-glycogenolytic factor, glucagon-like-peptide-1, glucagon-like-peptide-2, insulin-like growth factor-i, rhIGF-1-2, gastric inhibitory polypeptide, somatotropin releasing factor, pituitary adenylate cyclase activation peptide, secretin, Ileogastrone (enterogastrin), somatostatin, somatomedin, somatotropin, parathyroid hormone, thrombopoietin, erythropoietin, hypothalamic releasing factor, prolactin antagonist, thyrotropin, endorphin, enkephalin, beta-hypophamine, pitocin, opioid and analogue thereof, superoxide-dismutase, Interferon, rabbit, asparaginase, arginase, arginine desaminase, adenosine deaminase and rnase.In some cases, can use the appropriate drug composition influencing the release profiles of peptide, but there are various weak points in this method, generally is inapplicable.
The hormone of regulating insulin secretion belongs to so-called enteroinsular axis, refers to along with the existence of enteral nutrients and absorption and one group of hormone discharging from gastrointestinal mucosa, and it impels the early stage release of Regular Insulin and make it and strengthens.Promote the effect of insulin secretion, promptly so-called incretin effect perhaps is essential for normal glucose tolerance.The many gastrointestinal hormone `s that comprise gastrin and secretin have pancreotropic hormone effect (people's cholecystokinin does not have the pancreotropic hormone effect), but important on the physiology, can cause the incretin effect glucose-dependent-insulinotropic polypeptide GIP and glucagon-like-peptide-1 (GLP-1) only arranged.Because the pancreotropic hormone effect of GIP, it causes sizable interest immediately in the diabetes scholar after obtaining separating (1) in 1973.Yet the big quantity research that carries out in more subsequently year clearly illustrates, the pathogenic factor of GIP hyposecretion and insulin-dependent diabetes (IDDM) or non-insulin-dependent diabetes mellitus (NIDDM) (NIDDM) and irrelevant (2).And then find that also though GIP is a kind of hGLP-1, it is for NIDDM nearly unavailable (2).Another kind of incretin hormone GLP-1 is the most effective known pancreotropic hormone material (3).Be different from GIP, GLP-1 is very effective aspect the insulin secretion that promotes patient NIDDM.In addition, compare with other hGLP-1 (perhaps except that secretin), GLP-1 is glucagon suppression secretion effectively also.Because these effects, it especially has significant blood sugar decreasing effect for patient NIDDM.
GLP-1 is a kind of product (4) of Proglucagon (proglucagon), it is one of up-to-date member in secretin-VIP family peptide, but it to be identified be a kind of important gastrointestinal hormone ` (5) that has regulatory function for glucose metabolism and gastrointestinal secretion and metabolism.The hyperglycemic-glycogenolytic factor gene is different with processing mode in the intestines at pancreas.In pancreas (9), processing causes forming and the following material of parallel secretion: 1) hyperglycemic-glycogenolytic factor self occupies the 33-61 position of Proglucagon (PG); 2) a kind of N-terminal peptide that 30 amino acid (PG (1-30)) arranged is referred to as the relevant pancreas peptide of glucagon-like peptide, GRPP (10,11) usually; 3) a kind of six peptides corresponding to PG (64-69); 4) also have so-called main Proglucagon fragment (PG (72-158)) at last, wherein keep two glucagon sequences (9).The bioactive product that hyperglycemic-glycogenolytic factor is seemingly unique.By comparison, in intestinal mucosa, but be that hyperglycemic-glycogenolytic factor is buried in the macromole, two glucagon-like peptides then are (8) that form respectively.Following product is parallel formation justacrine: 1) corresponding to the glucagon-like peptide of PG (1-69), wherein the hyperglycemic-glycogenolytic factor sequence occupies No.33-61 residue (12); 2) GLP-1 (7-36) acid amides, i.e. PG (78-107) acid amides (13), rather than the PG that thinks at first (72-107) acid amides or PG (72-108) acid amides, it is inactive.What generate also that the terminal glycine of a small amount of C-extends has an equal bioactive GLP-1 (7-37) (PG (78-108)) (14); 3) intermediate peptide-2 (PG (111-122) acid amides) (15); With 4) GLP-2 (PG (126-158)) (15,16).The part of glucagon-like peptide further cuts into GRPP (PG (1-30)) and oxyntomodulin (PG (33-69)) (17,18).In these peptides, GLP-1 has the most significant biological activity.
Because the parallel secretion of GLP-1 with glucagon-like peptide/enteroglucagon, therefore to the many researchs (6 of enteroglucagon excretory, 7) also can be used for the GLP-1 secretion on some degree, but the GLP-1 metabolism is faster, its transformation period in human plasma is 2 minutes (19).The meals that are rich in carbohydrate or fat can promote secretion (20), infer that this is the result of the microvillus direct interaction of unabsorbed nutrient and the opening L-cell of intestinal mucosa.May exist to promote GLP-1 excretory internal secretion or neuromechanism, but in human body, also not be confirmed.
The experiment of carrying out with GLP-1 receptor antagonist exendin 9-39 has clearly illustrated the incretin effect (29-31) of GLP-1, and in this experiment, exendin 9-39 shockingly reduces the incretin effect (21,22) behind the rat oral glucose.Hormone is striden GLP-1 acceptor and the beta cell direct interaction (23) that film passes through the hyperglycemic-glycogenolytic factor/VIP/ thyrocalcitonin family of acceptor via the 7-that belongs to the G-protein coupling.The nearest GLP-1 acceptor that experimental results show that, has carried out the orientation of GLP-1 acceptor gene and has destroyed in this experiment in the importance of regulating aspect the insulin secretion to mouse.Have this destructive hyperglycemia that animal has the glucose tolerance of remarkable degeneration and occur fast of isozygotying, even the heterozygosis animal can not tolerate glucose (24).Signal transduction mechanism mainly comprises the adenylate cyclase enzyme activation, but Ca in the cell also must be arranged
2+Rising (25,26).This functions of hormones is described as strengthening the Regular Insulin release action (25) that glucose stimulates best, but does not still know the online reason of coupling between glucose and the GLP-1 stimulation.It may comprise that the calcium that a calcium brings out discharges (26,27).As already mentioned, diabetics's beta cell has the pancreotropic hormone effect of GLP-1.Do not know that GLP-1 gives isolating insulin secretory cell (26 with it yet, 28) relation between the ability of " glucose irritability ", this isolating insulin secretory cell reacts to independent glucose or GLP-1 hardly, but the two combination is reacted.Yet of equal importance is that this hormone is glucagon suppression secretion (29) effectively also.Its mechanism is not clear, but seemingly passes through the paracrine (25) of adjacent Regular Insulin or somatostatin cell.Pressing down hyperglycemic-glycogenolytic factor effect (glucagonostatic action) also is that glucose is dependent, so when blood sugar reduced, retarding effect promptly reduced.Because this double effect, if by the increase secretion or by the exogenous GLP-1 concentration that increases in the blood plasma of inculcating, reach by portal system that the mol ratio between the Regular Insulin and hyperglycemic-glycogenolytic factor will significantly increase in the blood of liver, and the glycogen growing amount reduces (30).As a result, blood sugar concentration reduces.Because pancreotropic hormone effect and the glucose dependency that presses down the hyperglycemic-glycogenolytic factor effect, the effect that glucose reduces is a self limiting, and therefore, regardless of dosage, this hormone does not all cause hypoglycemia (31).There is melituric patient still to have these effects (32),, inculcates the GLP-1 that exceeds physiological dose a little for these patients and may make the complete normalizing of blood glucose value although weak metabolism control and the attached sexual dysfunction of sulfonylurea (33) are arranged.Found that GLP-1 also can lowering blood glucose (34) in the type i diabetes people of no remaining beta cell secretion capacity, this has illustrated the importance that presses down the hyperglycemic-glycogenolytic factor effect.
Except that its effect for pancreas islet, GLP-1 also has strong effect to gi tract.Inculcate the GLP-1 of physiological dose, can significantly suppress pentagastrin gastric acid secretion (35,36) that bring out and that meals bring out.GLP-1 also suppresses gastric emptying rate and pancreatin secretion (36).People's ileum is inculcated the solution that contains sugar or fat may have similar retarding effect (37,38) with mobility to stomach, pancreas secretion.Concomitantly, the GLP-1 secretion obtains significant stimulation, and has inferred that GLP-1 may be this so-called " ileum lock " (ileal-brake) partly cause of effect (38) at least.In fact, nearest studies show that, on the physiology, the ileum lock effect of GLP-1 may be more important than its effect to pancreas islet.Therefore, in dose response research, influencing under the required minimum infusion rate of islet secretion at least, GLP-1 can influence gastric emptying rate (39).
As if GLP-1 have effect to ingestion of food.Administration extremely suppresses the food intake (40,42) of rat in the ventricle of GLP-1.It is very special that this effect seems.Therefore, terminal GLP-1 (PG72-107) acid amides that extends of N-is a non-activity, and the suitable GLP-1 antagonist of dosage, and exendin9-39 can eliminate the effect (41) of GLP-1.GLP-1 is peripherally administered fast can not to suppress rat food intake (41,42) apace.Yet, still might be as a satiety signal from the GLP-1 of intestines L-emiocytosis.
The diabetics not only has the pancreotropic hormone effect, and have GLP-1 to GI effect (43), this has and helps to weaken the glucose skew that meals cause, but, the more important thing is, also may influence food intake.GLP-1 presses 4ng/kg/min and continues one week of intravenous administration, has confirmed significantly to have improved patient's NIDDM glucemia control and does not have significant side effects (44).Peptide is (45) in full force and effect behind subcutaneous administration, but mainly due to the Degradation of DPP IV sample enzyme, peptide is degraded (46,47) fast.
Especially Schmidt etc. has provided the aminoacid sequence (diabetology (Diabetologia), 28,704-707 (1985)) of GLP-1.Though the interesting pharmacological property of GLP-1 (7-37) and analogue thereof has caused very big concern in recent years, knows little about the structure of these molecules.Thorton etc. have described the secondary structure (biological chemistry, 33,3532-3539 (1994)) of GLP-1 in micella (micelle), but in normal solution, GLP-1 is considered to a kind of very yielding molecule.Unexpectedly, we find the compound that this less and very yielding molecule produces through derivatize, and its blood plasma distribution curve prolongs greatly and still kept activity.
GLP-1, GLP-1 analogue and their fragment are especially very effective in treatment 1 type and diabetes B.Yet high clearance rate has limited the validity of these compounds, therefore also needs in this respect to improve.Correspondingly, an object of the present invention is to provide the derivative of GLP-1 and its analogue, these derivatives have the effect curves of longer time than GLP-1 (7-37).Further purpose of the present invention provides the derivative of GLP-1 and analogue thereof, and these derivatives have the lower clearance rate than GLP-1 (7-37).Further purpose of the present invention provides and a kind ofly contains the pharmaceutical composition of The compounds of this invention and use this composition of compound of the present invention.A kind of method for the treatment of insulin-dependent and non-insulin-dependent diabetes mellitus (NIDDM) that provides also is provided purpose of the present invention.
Reference
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Brief summary of the invention
People GLP-1 is the peptide of one 37 amino-acid residue, and it rises and is derived from preceding Proglucagon, and preceding Proglucagon is mainly synthetic in far-end ileum L-cell, pancreas and brain.The process that produces GLP-1 (7-36) acid amides, GLP-1 (7-37) and GLP-2 by preceding Proglucagon processing mainly occurs in the L-cell.Available one simple system is described the fragment and the analogue of this peptide.Therefore, Gly for example
8-GLP-1 (7-37) expression lacks amino-acid residue Nos.1-6 from GLP-1, and replaces the GLP-1 fragment that forms behind the naturally occurring amino-acid residue with Gly at position 8 (Ala).Similarly, Lys
34(N
ε-myristoyl)-GLP-1 (7-37) representative at the epsilon-amino of 34 Lys residue by the GLP-of myristoylization (7-37).When mentioning the GLP-1 analogue of the terminal extension of C-herein, then unless otherwise indicated, at 38 amino-acid residues all are Arg, also are Arg (except as otherwise noted) at 39 optional amino acid residues, are Asp (except as otherwise noted) at 40 optional amino acid residues.Equally, if the terminal analogue that extends of C-extends to position 41,42,43,44 or 45, then unless otherwise indicated, the aminoacid sequence of this extension is all the same with corresponding sequence in the preceding Proglucagon of people.
Aspect the most widely, the present invention relates to the derivative of GLP-1 and analogue thereof.Derivative of the present invention has interesting pharmacological property, and especially they have than the parent peptide effect curves of longer time.
In this article, " analogue " is used to represent a kind of peptide, wherein one or more amino-acid residue of parent peptide is replaced by another amino-acid residue, and/or wherein one or more amino-acid residue on the parent peptide is lacked, and/or has wherein added one or more amino-acid residue to parent peptide.This interpolation can occur in the N-end or the C-end of parent peptide, or all takes place at two ends.
Use term " derivative " to represent a kind of peptide in this article, chemically modified has taken place at one or more amino-acid residue of parent peptide in this peptide, and alkylation has for example taken place, and acylations has formed ester or acid amides.
Use the derivative of term " GLP-1 derivative " expression GLP-1 or its analogue in this article.In this article, the derive parent peptide of derivative is called as " the GLP-1 part " of derivative in some places.
In a preferred embodiment, the present invention relates to a kind of GLP-1 derivative, wherein at least one amino-acid residue of parent peptide has connected a lipophilic substituent, condition is if having only a lipophilic substituent, and this substituting group is connected on the N-end or C-terminal amino acid residue of parent peptide, and this substituting group is an alkyl or a ω-hydroxy-acid group is arranged so.
In a preferred embodiment, the present invention relates to have only the GLP-1 derivative of a lipophilic substituent.
In a preferred embodiment, the present invention relates to only have the GLP-1 derivative of a lipophilic substituent, this substituting group is alkyl or a ω-hydroxy-acid group is arranged, and is connected on the-terminal amino acid residue of parent peptide.
In a preferred embodiment, the present invention relates to only have the GLP-1 derivative of a lipophilic substituent, this substituting group is alkyl or a ω-hydroxy-acid group is arranged, and is connected on the C-terminal amino acid residue of parent peptide.
In a preferred embodiment, the present invention relates to only have the GLP-1 derivative of a lipophilic substituent, this substituting group can be connected on arbitrary amino-acid residue of the also non-C-end of non-N-end on the parent peptide.
In a preferred embodiment, the present invention relates to the GLP-1 derivative of 2 lipophilic substituents.
In a preferred embodiment, the present invention relates to the GLP-1 derivative of 2 lipophilic substituents, one of them 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 a preferred embodiment, the present invention relates to the GLP-1 derivative of 2 lipophilic substituents, one of them 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 also non-C-end of N-end.
In a preferred embodiment, the present invention relates to the GLP-1 derivative of 2 lipophilic substituents, one of them 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 also non-C-end of N-end.
In a further preferred embodiment, the present invention relates to a kind of GLP-1 (7-C) derivative, wherein C is selected from and contains 38,39,40,41,42,43,44 and 45 one group, and this derivative only has a lipophilic substituent on the C-terminal amino acid residue that is connected to parent peptide.
In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative, wherein lipophilic substituent contains 4 to 40 carbon atoms, more preferably contains 8-25 carbon atom.
In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative, one of them lipophilic substituent is attached in some way on the amino-acid residue, thereby makes a carboxyl of this lipophilic substituent and an amino amido linkage that forms of this amino-acid residue.
In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative, one of them lipophilic substituent is attached in some way on the amino-acid residue, thereby makes an amino of this lipophilic substituent and a carboxyl of this amino-acid residue form amido linkage.
In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative, one of them lipophilic substituent is connected on the parent peptide by a spacer groups.
In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative, one of them lipophilic substituent (randomly by a spacer groups) is connected on the epsilon-amino group of a contained Lys residue of parent peptide.
In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative, one of them lipophilic substituent by one 1-7 methylene radical arranged, preferably have 2 methylene radical be regardless of branched paraffin α, ω-dicarboxyl spacer groups and being connected on the parent peptide, this at interval group between an amino of an amino of parent peptide and this lipophilic substituent, form a bridge.
In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative, one of them lipophilic substituent is connected on the parent peptide by a spacer groups, and this interval group is an amino-acid residue except that Cys, or a kind of dipeptides as Gly-Lys.In this article, a kind of dipeptides " a kind of dipeptides as Gly-Lys " this phraseology refers to, C-terminal amino acid residue wherein is Lys, His or Trp, preferred Lys,-terminal amino acid residue wherein is selected from the group that contains 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 derivative, one of them lipophilic substituent is connected on the parent peptide by a spacer groups, this interval group is an amino-acid residue except that Cys, or a kind of dipeptides as Gly-Lys, and wherein parent peptide a carboxyl and a Lys residue or contain that in the dipeptides of a Lys residue one is amino to form an amido linkage, this Lys residue or contain in the dipeptides of a Lys residue another amino with this lipophilic substituent in a carboxyl form an amido linkage.
In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative, one of them lipophilic substituent is connected on the parent peptide by a spacer groups, this interval group is an amino-acid residue except that Cys, or a kind of dipeptides as Gly-Lys, wherein a carboxyl in parent peptide amino and this amino-acid residue spacer groups or the dipeptides spacer groups forms an amido linkage, and amido linkage of a carboxyl formation of amino in this amino-acid residue spacer groups or the dipeptides spacer groups and lipophilic substituent.
In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative, one of them lipophilic substituent is connected on the parent peptide by a spacer groups, this interval group is an amino-acid residue except that Cys, or a kind of dipeptides as Gly-Lys, an and amino amido linkage, amido linkage of an amino formation of the carboxyl in this amino-acid residue spacer groups or the dipeptides spacer groups and lipophilic substituent of forming of in parent peptide carboxyl and this amino-acid residue spacer groups or the dipeptides spacer groups wherein.
In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative, one of them lipophilic substituent is connected on the parent peptide by a spacer groups, this interval group is an amino-acid residue except that Cys, or a kind of dipeptides as Gly-Lys, and wherein parent peptide carboxyl and Asp or Glu as spacer groups, or contain an amino amido linkage, amido linkage of an amino formation in a carboxyl in this spacer groups and this lipophilic substituent of forming in the dipeptides spacer groups of an Asp or Glu residue.
In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative with a lipophilic substituent, this lipophilic substituent contains partially or completely hydrogenant ring penta phenanthrene (cyclopentanophenathrene) skeleton.
In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative, it has a lipophilic substituent for straight chain or branch's alkyl.
In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative, it has a lipophilic substituent for the acyl group of straight chain or branched fatty acid.
In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative that a lipophilic substituent is arranged, this lipophilic substituent is to be selected to contain CH
3(CH
2)
nAn acyl group of the group of CO-, wherein n is the integer of 4-38, the integer of preferred 4-24, further preferred this lipophilic substituent is to be selected to contain CH
3(CH
2)
6CO-, CH
3(CH
2)
8CO-, CH
3(CH
2)
10CO-, CH
3(CH
2)
12CO-, CH
3(CH
2)
14CO-, CH
3(CH
2)
L6CO-, CH
3(CH
2)
18CO-, CH
3(CH
2)
20CO-and CH
3(CH
2)
22The group of CO-.
In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative with a lipophilic substituent, this lipophilic substituent 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 that a lipophilic substituent is arranged, this lipophilic substituent is to be selected to contain HOOC (CH
2)
mAn acyl group of the group of CO-, wherein m is the integer of 4-38, the integer of preferred 4-24, further preferred this lipophilic substituent is selected from and contains HOOC (CH
2)
14CO-, HOOC (CH
2)
16CO-, HOOC (CH
2)
18CO-, HOOC (CH
2)
20CO-and HOOC (CH
2)
22The group of CO-.
In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative, it has one to have formula CH
3(CH
2)
p((CH
2)
qCOOH) CHNH-CO (CH
2)
2The lipophilic substituent of CO-, wherein p and q are integers, 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 derivative, it has one to have formula CH
3(CH
2)
rCO-NHCH (COOH) (CH
2)
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 derivative, it has one to have formula CH
3(CH
2)
sCO-NHCH ((CH
2)
2COOH) 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 derivative, it has one to have formula COOH (CH
2)
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 derivative, it has one to have formula-NHCH (COOH) (CH
2)
4NH-CO (CH
2)
uCH
3Lipophilic substituent, wherein u is the integer of 8-18.
In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative, it has one to have formula-NHCH (COOH) (CH
2)
4NH-COCH ((CH
2)
2COOH) NH-CO (CH
2)
wCH
3Lipophilic substituent, wherein w is the integer of 10-16.
In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative, it has one to have formula-NHCH (COOH) (CH
2)
4NH-CO (CH
2)
2CH (COOH) NH-CO (CH
2)
xCH
3Lipophilic substituent, wherein x is the integer of 10-16.
In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative, it has one to have formula-NHCH (COOH) (CH
2)
4NH-CO (CH
2)
2CH (COOH) NH-CO (CH
2)
yCH
3Lipophilic substituent, wherein y is 0 or the integer of 1-22.
In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative, it have one can be electronegative lipophilic substituent.Such lipophilic substituent for example can be the substituting group that a carboxyl is arranged.
In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative, its parent peptide is selected from GLP-1 (1-45) or its analogue.
In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative, it contains GLP-1 (7-35) by being selected from, GLP-1 (7-36), GLP-1 (7-36) acid amides, GLP-1 (7-37), GLP-1 (7-38), GLP-1 (7-39), a GLP-1 fragment of the group of GLP-1 (7-40) and GLP-1 (7-41) or its analogue are derived and are obtained.
In a further preferred embodiment, the present invention relates to a kind of GLP-1 analogue, it contains GLP-1 (1-35) by being selected from, GLP-1 (1-36), GLP-1 (1-36) acid amides, GLP-1 (1-37), GLP-1 (1-38), GLP-1 (1-39), the GLP-1 analogue of the group of GLP-1 (1-40) and GLP-1 (1-41) or its analogue are derived and are obtained.
In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative, 15 at the most of sums in the derivative that wherein specified analogue comprises, preferably 10 amino-acid residues are changed by any a-amino acid residue at the most.
In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative, 15 at the most, preferred 10 amino-acid residues at the most can be changed by the a-amino acid residue of genetic code coding by any in the derivative that wherein specified analogue comprises.
In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative, 6 amino-acid residues can be by the a-amino acid residue replacing of genetic code coding by another at the most in the derivative that wherein specified analogue comprises.
In a further preferred embodiment, the present invention relates to a kind of GLP-1 (A-B) derivative, wherein A is the integer of 1-7, B is the integer of 38-45; Or the analogue of this derivative, wherein contain a lipophilic substituent that is connected on the C-terminal amino acid residue, and randomly contain second lipophilic substituent that is 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: Arg
26-GLP-1 (7-37), Arg
34-GLP-1 (7-37), Lys
36-GLP-1 (7-37), Arg
26,34Lys
36-GLP-1 (7-37), Arg
26,34Lys
38-GLP-1 (7-38), Arg
26,34Lys
39-GLP-1 (7-39), Arg
26,34Lys
40-GLP-1 (7-40), Arg
26Lys
36-GLP-1 (7-37), Arg
34Lys
36-GLP-1 (7-37), Arg
26Lys
39-GLP-1 (7-39), Arg
34Lys
40-GLP-1 (7-40), Arg
26,34Lys
36,39-GLP-1 (7-39), Arg
26,34Lys
36,40-GLP-1 (7-40), Gly
8Arg
26-GLP-1 (7-37), Gly
8Arg
34-GLP-1 (7-37), Gly
8Lys
36-GLP-1 (7-37), Gly
8Arg
26,34Lys
36-GLP-1 (7-37), Gly
8Arg
26,34Lys
39-GLP-1 (7-39), Gly
8Arg
26,34Lys
40-GLP-1 (7-40), Gly
8Arg
26Lys
36-GLP-1 (7-37), Gly
8Arg
34Lys
36-GLP-1 (7-37), Gly
8Arg
26Lys
39-GLP-1 (7-39), Gly
8Arg
34Lys
40-GLP-1 (7-40), Gly
8Arg
26,34Lys
36,39-GLP-1 (7-39) and Gly
8Arg
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
26,34Lys
38-GLP-1 (7-38), Arg
26,34Lys
39-GLP-1 (7-39), Arg
26,34Lys
40-GLP-1 (7-40), Arg
26,34Lys
41-GLP-1 (7-4 1), Arg
26,34Lys
42-GLP-1 (7-42), Arg
26,34Lys
43-GLP-1 (7-43), Arg
26,34Lys
44-GLP-1 (7-44), Arg
26,34Lys
45-GLP-1 (7-45), Arg
26,34Lys
38-GLP-1 (1-38), Arg
26,34Lys
39-GLP-1 (1-39), Arg
26,34Lys
40-GLP-1 (1-40), Arg
26,34Lys
41-GLP-1 (1-41), Arg
26,34Lys
42-GLP-1 (1-42), Arg
26,34Lys
43-GLP-1 (1-43), Arg
26,34Lys
44-GLP-1 (1-44), Arg
26,34Lys
45-GLP-1 (1-45), Arg
26,34Lys
38-GLP-1 (2-38), Arg
26,34Lys
39-GLP-1 (2-39), Arg
26,34Lys
40-GLP-1 (2-40), Arg
26,34Lys
41-GLP-1 (2-41), Arg
26,34Lys
42-GLP-1 (2-42), Arg
26,34Lys
43-GLP-1 (2-43), Arg
26,34Lys
44-GLP-1 (2-44), Arg
26,34Lys
45-GLP-1 (2-45), Arg
26,34Lys
38-GLP-1 (3-38), Arg
26,34Lys
39-GLP-1 (3-39), Arg
26,34Lys
40-GLP-1 (3-40), Arg
26, 34Lys
41-GLP-1 (3-41), Arg
26,34Lys
42-GLP-1 (3-42), Arg
26,34Lys
43-GLP-1 (3-43), Arg
26,34Lys
44-GLP-1 (3-44), Arg
26,34Lys
45-GLP-1 (3-45), Arg
26,34Lys
38-GLP-1 (4-38), Arg
26,34Lys
39-GLP-1 (4-39), Arg
26,34Lys
40-GLP-1 (4-40), Arg
26,34Lys
41-GLP-1 (4-41), Arg
26,34Lys
42-GLP-1 (4-42), Arg
26,34Lys
43-GLP-1 (4-43), Arg
26,34Lys
44-GLP-1 (4-44), Arg
26,34Lys
45-GLP-1 (4-45), Arg
26,34Lys
38-GLP-1 (5-38), Arg
26,34Lys
39-GLP-1 (5-39), Arg
26,34Lys
40-GLP-1 (5-40), Arg
26,34Lys
41-GLP-1 (5-41), Arg
26,34Lys
42-GLP-1 (5-42), Arg
26,34Lys
43-GLP-1 (5-43), Arg
26,34Lys
44-GLP-1 (5-44), Arg
26,34Lys
45-GLP-1 (5-45), Arg
26,34Lys
38-GLP-1 (6-38), Arg
26,34Lys
39-GLP-1 (6-39), Arg
26,34Lys
40-GLP-1 (6-40), Arg
26,34Lys
41-GLP-1 (6-41), Arg
26,34Lys
42-GLP-1 (6-42), Arg
26,34Lys
43-GLP-1 (6-43), Arg
26,34Lys
44-GLP-1 (6-44), Arg
26,34Lys
45-GLP-1 (6-45), Arg
26Lys
38-GLP-1 (1-38), Arg
34Lys
38-GLP-1 (1-38), Arg
26,34Lys
36,38-GLP-1 (1-38), Arg
26Lys
38-GLP-1 (7-38), Arg
34Lys
38-GLP-1 (7-38), Arg
26,34Lys
36,38-GLP-1 (7-38), Arg
26,34Lys
38-GLP-1 (7-38), Arg
26Lys
39-GLP-1 (1-39), Arg
34Lys
39-GLP-1 (1-39), Arg
26,34Lys
36,39-GLP-1 (1-39), Arg
26Lys
39-GLP-1 (7-39), Arg
34Lys
39-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 wherein is selected from following member: [Arg
26-GLP-1 (7-37), Arg
34-GLP-1 (7-37), Lys
36-GLP-1 (7-37), Arg
26,34Lys
36-GLP-1 (7-37), Arg
26Lys
36-GLP-1 (7-37), Arg
34Lys
36-GLP-1 (7-37), Gly
8Arg
26-GLP-1 (7-37), Gly
8Arg
34-GLP-1 (7-37), Gly
8Lys
36-GLP-1 (7-37), Gly
8Arg
26,34Lys
36-GLP-1 (7-37), Gly
8Arg
26Lys
36-GLP-1 (7-37) and Gly
8Arg
34Lys
36-GLP-1 (7-37).
In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative, parent peptide wherein is selected from following member: Arg
26Lys
38-GLP-1 (7-38), Arg
26,34Lys
38-GLP-1 (7-38), Arg
26,34Lys
36,38-GLP-1 (7-38), Gly
8Arg
26Lys
38-GLP-1 (7-38) and Gly
8Arg
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 wherein is selected from following member: Arg
26Lys
39-GLP-1 (7-39), Arg
26,34Lys
36,39-GLP-1 (7-39), Gly
8Arg
26Lys
39-GLP-1 (7-39) and Gly
8Arg
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 wherein is selected from following member: Arg
34Lys
40-GLP-1 (7-40), Arg
26,34Lys
36,40-GLP-1 (7-40), Gly
8Arg
34Lys
40-GLP-1 (7-40) and Gly
8Arg
26,34Lys
36,40-GLP-1 (7-40).
In a further preferred embodiment, the present invention relates to a kind of GLP-1 derivative, parent peptide wherein is selected from following member:
Lys
26(N
ε-myristoyl)-GLP-1 (7-37);
Lys
34(N
ε-myristoyl)-GLP-1 (7-37);
Lys
26,34-two (N
ε-myristoyl)-GLP-1 (7-37);
Gly
8Lys
26(N
ε-myristoyl)-GLP-1 (7-37);
Gly
8Lys
34(N
ε-myristoyl)-GLP-1 (7-37);
Gly
8Lys
26,34-two (N
ε-myristoyl)-GLP-1 (7-37);
Arg
26Lys
34(N
ε-myristoyl)-GLP-1 (7-37);
Lys
26(N
ε-myristoyl)-GLP-1 (7-38);
Lys
34(N
ε-myristoyl)-GLP-1 (7-38);
Lys
26,34-two (N
ε-myristoyl)-GLP-1 (7-38);
Gly
8Lys
26(N
ε-myristoyl)-GLP-1 (7-38);
Gly
8Lys
34(N
ε-myristoyl)-GLP-1 (7-38);
Gly
8Lys
26,34-two (N
ε-myristoyl)-GLP-1 (7-38);
Arg
26Lys
34(N
ε-myristoyl)-GLP-1 (7-38);
Lys
26(N
ε-myristoyl)-GLP-1 (7-39);
Lys
34(N
ε-myristoyl)-GLP-1 (7-39);
Lys
26,34-two (N
ε-myristoyl)-GLP-1 (7-39);
Gly
8Lys
26(N
ε-myristoyl)-GLP-1 (7-39);
Gly
8Lys
34(N
ε-myristoyl)-GLP-1 (7-39);
Gly
8Lys
26,34-two (N
ε-myristoyl)-GLP-1 (7-39);
Arg
26Lys
34(N
ε-myristoyl)-GLP-1 (7-39);
Lys
26(N
ε-myristoyl)-GLP-1 (7-40);
Lys
34(N
ε-myristoyl)-GLP-1 (7-40);
Lys
26,34-two (N
ε-myristoyl)-GLP-1 (7-40);
Gly
8Lys
26(N
ε-myristoyl)-GLP-1 (7-40);
Gly
8Lys
34(N
ε-myristoyl)-GLP-1 (7-40);
Gly
8Lys
26,34-two (N
ε-myristoyl)-GLP-1 (7-40);
Arg
26Lys
34(N
ε-myristoyl)-GLP-1 (7-40);
Lys
26(N
ε-myristoyl)-GLP-1 (7-36);
Lys
34(N
ε-myristoyl)-GLP-1 (7-36);
Lys
26,34-two (N
ε-myristoyl)-GLP-1 (7-36);
Gly
8Lys
26(N
ε-myristoyl)-GLP-1 (7-36);
Gly
8Lys
34(N
ε-myristoyl)-GLP-1 (7-36);
Gly
8Lys
26,34-two (N
ε-myristoyl)-GLP-1 (7-36);
Arg
26Lys
34(N
ε-myristoyl)-GLP-1 (7-36);
Lys
26(N
ε-myristoyl)-GLP-1 (7-35);
Lys
34(N
ε-myristoyl)-GLP-1 (7-35);
Lys
26,34-two (N
ε-myristoyl)-GLP-1 (7-35);
Gly
8Lys
26(N
ε-myristoyl)-GLP-1 (7-35);
Gly
8Lys
34(N
ε-myristoyl)-GLP-1 (7-35);
Gly
8Lys
26,34-two (N
ε-myristoyl)-GLP-1 (7-35);
Arg
26Lys
34(N
ε-myristoyl)-GLP-1 (7-35);
Lys
26(N
ε-myristoyl)-GLP-1 (7-36) acid amides;
Lys
34(N
ε-myristoyl)-GLP-1 (7-36) acid amides;
Lys
26,34-two (N
ε-myristoyl)-GLP-1 (7-36) acid amides;
Gly
8Lys
26(N
ε-myristoyl)-GLP-1 (7-36) acid amides;
Gly
8Lys
34(N
ε-myristoyl)-GLP-1 (7-36) acid amides;
Gly
8Lys
26,34-two (N
ε-myristoyl)-GLP-1 (7-36) acid amides;
Arg
26Lys
34(N
ε-myristoyl)-GLP-1 (7-36) acid amides;
Gly
8Arg
26Lys
34(N
ε-myristoyl)-GLP-1 (7-37);
Lys
26(N
ε-myristoyl) Arg
34-GLP-1 (7-37);
Gly
8Lys
26(N
ε-myristoyl) Arg
34-GLP-1 (7-37);
Arg
26,34Lys
36(N
ε-myristoyl)-GLP-1 (7-37);
Gly
8Arg
26,34Lys
36(N
ε-myristoyl)-GLP-1 (7-37);
Gly
8Arg
26Lys
34(N
ε-myristoyl)-GLP-1 (7-38);
Lys
26(N
ε-myristoyl) Arg
34-GLP-1 (7-38);
Gly
8Lys
26(N
ε-myristoyl) Arg
34-GLP-1 (7-38);
Arg
26,34Lys
36(N
ε-myristoyl)-GLP-1 (7-38);
Arg
26,34Lys
38(N
ε-myristoyl)-GLP-1 (7-38);
Gly
8Arg
26,34Lys
36(N
ε-myristoyl)-GLP-1 (7-38);
Gly
8Arg
26Lys
34(N
ε-myristoyl)-GLP-1 (7-39);
Lys
26(N
ε-myristoyl) Arg
34-GLP-1 (7-39);
Gly
8Lys
26(N
ε-myristoyl) Arg
34-GLP-1 (7-39);
Arg
26,34Lys
36(N-myristoyl)-GLP-1 (7-39);
Gly
8Arg
26,34Lys
36(N
ε-myristoyl)-GLP-1 (7-39);
Gly
8Arg
26Lys
34(N
ε-myristoyl)-GLP-1 (7-40);
Lys
26(N
ε-myristoyl) Arg
34-GLP-1 (7-40);
Gly
8Lys
26(N
ε-myristoyl) Arg
34-GLP-1 (7-40);
Arg
26,34Lys
36(N
ε-myristoyl)-GLP-1 (7-40);
Gly
8Arg
26,34Lys
36(N
ε-myristoyl)-GLP-1 (7-40);
Lys
26(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-37);
Lys
34(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-37);
Lys
26,34-two (N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-37);
Gly
8Lys
26(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-37);
Gly
8Lys
34(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-37);
Gly
8Lys
26,34-two (N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-37);
Lys
26(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-38);
Lys
34(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-38);
Lys
26,34-two (N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-38);
Gly
8Lys
26(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-38);
Gly
8Lys
34(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-38);
Gly
8Lys
26,34-two (N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-38);
Lys
26(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-39);
Lys
34(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-39);
Lys
26,34-two (N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-39);
Gly
8Lys
26(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-39);
Gly
8Lys
34(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-39);
Gly
8Lys
26,34-two (N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-39);
Lys
26(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-40);
Lys
34(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-40);
Lys
26,34-two (N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-40);
Gly
8Lys
26(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-40);
Gly
8Lys
34(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-40);
Gly
8Lys
26,34-two (N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-40);
Lys
26(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-36);
Lys
34(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-36);
Lys
26,34-two (N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-36);
Gly
8Lys
26(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-36);
Gly
8Lys
34(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-36);
Gly
8Lys
26,34-two (N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-36);
Lys
26(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-36 acid amides;
Lys
34(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-36) acid amides;
Lys
26,34-two (N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-36) acid amides;
Gly
8Lys
26(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-36) acid amides;
Gly
8Lys
34(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-36) acid amides;
Gly
8Lys
26,34-two (N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-36) acid amides;
Lys
26(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-35);
Lys
34(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-35);
Lys
26,34-two N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-35);
Gly
8Lys
26(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-35);
Gly
8Lys
34(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-35);
Gly
8Lys
26,34-two (N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-35);
Arg
26Lys
34(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-37);
Gly
8Arg
26Lys
34(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-37);
Lys
26(N
ε-(ω-carboxyl 19 acyl groups)) Arg
34-GLP-1 (7-37);
Gly
8Lys
26(N
ε-(ω-carboxyl 19 acyl groups)) Arg
34GLP-1 (7-37);
Arg
26,34Lys
36(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-37);
Gly
8Arg
26,34Lys
36(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-37);
Arg
26Lys
34(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-38);
Gly
8Arg
26Lys
34(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-38);
Lys
26(N
ε-(ω-carboxyl 19 acyl groups)) Arg
34-GLP-1 (7-38);
Gly
8Lys
26(N
ε-(ω-carboxyl 19 acyl groups)) Arg
34-GLP-1 (7-38);
Arg
26,34Lys
36(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-38);
Arg
26,34Lys
38(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-38);
Gly
8Arg
26,34Lys
36(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-38);
Arg
26Lys
34(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-39);
Gly
8Arg
26Lys
34(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-39);
Lys
26(N
ε-(ω-carboxyl 19 acyl groups)) Arg
34-GLP-1 (7-39);
Gly
8Lys
26(N
ε-(ω-carboxyl 19 acyl groups)) Arg
34-GLP-1 (7-39);
Arg
26,34Lys
36(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-39);
Gly
8Arg
26,34Lys
36(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-39);
Arg
26Lys
34(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-40);
Gly
8Arg
26Lys
34(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-40);
Lys
26(N
ε-(ω-carboxyl 19 acyl groups)) Arg
34-GLP-1 (7-40);
Gly
8Lys
26(N
ε-(ω-carboxyl 19 acyl groups)) Arg
34-GLP-1 (7-40);
Arg
26,34Lys
36(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-40);
Gly
8Arg
26,34Lys
36(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-40);
Lys
26(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-37);
Lys
34(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-37);
Lys
26,34-two (N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-37);
Gly
8Lys
26(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-37);
Gly
8Lys
34(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-37);
Gly
8Lys
26,34-two (N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-37);
Arg
26Lys
34(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-37);
Lys
26(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-38);
Lys
34(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-38);
Lys
26,34-two (N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-38);
Gly
8Lys
26(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-38);
Gly
8Lys
34(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-38);
Gly
8Lys
26,34-two (N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-38);
Arg
26Lys
34(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-38);
Lys
26(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-39);
Lys
34(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-39);
Lys
26,34-two (N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-39);
Gly
8Lys
26(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-39);
Gly
8Lys
34(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-39);
Gly
8Lys
26,34-two (N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-39);
Arg
26Lys
34(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-39);
Lys
26(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-40);
Lys
34(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-40);
Lys
26,34-two (N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-40);
Gly
8Lys
26(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-40);
Gly
8Lys
34(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-40);
Gly
8Lys
26,34-two (N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-40);
Arg
26Lys
34(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-40);
Lys
26(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-36);
Lys
34(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-36);
Lys
26,34-two (N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-36);
Gly
8Lys
26(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-36);
Gly
8Lys
34(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-36);
Gly
8Lys
26,34-two (N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-36);
Arg
26Lys
34(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-36);
Lys
26(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-35);
Lys
34(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-35);
Lys
26,34-two (N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-35);
Gly
8Lys
26(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-35);
Gly
8Lys
34(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-35);
Gly
8Lys
26,34-two (N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-35);
Arg
26Lys
34(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-35);
Lys
26(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-36) acid amides;
Lys
34(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-36) acid amides;
Lys
26,34-two (N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-36) acid amides;
Gly
8Lys
26(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-36) acid amides;
Gly
8Lys
34(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-36) acid amides;
VGly
8Lys
26,34-two (N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-35) acid amides;
Arg
26Lys
34(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-35) acid amides;
Gly
8Arg
26Lys
34(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-37);
Lys
26(N
ε-(7-deoxidation courage acyl group)) Arg
34-GLP-1 (7-37);
Gly
8Lys
26(N
ε-(7-deoxidation courage acyl group)) Arg
34-GLP-1 (7-37);
Arg
26,34Lys
36(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-37);
Gly
8Arg
26,34Lys
36(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-37);
Lys
26(N
ε-(courage acyl group))-GLP-1 (7-37);
Lys
34(N
ε-(courage acyl group))-GLP-1 (7-37);
Lys
26,34-two (N
ε-(courage acyl group))-GLP-1 (7-37);
Gly
8Lys
26(N
ε-(courage acyl group))-GLP-1 (7-37);
Gly
8Lys
34(N
ε-(courage acyl group))-GLP-1 (7-37);
Gly
8Lys
26,34-two (N
ε-(courage acyl group))-GLP-1 (7-37);
Arg
26Lys
34(N
ε-(courage acyl group))-GLP-1 (7-37);
Gly
8Arg
26Lys
34(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-38);
Lys
26(N
ε-(7-deoxidation courage acyl group)) Arg
34-GLP-1 (7-38);
Gly
8Lys
26(N
ε-(7-deoxidation courage acyl group)) Arg
34-GLP-1 (7-38);
Arg
26,34Lys
36(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-38);
Arg
26,34Lys
38(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-38);
Gly
8Arg
26,34Lys
36(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-38);
Lys
26(N
ε-(courage acyl group))-GLP-1 (7-38);
Lys
34(N
ε-(courage acyl group))-GLP-1 (7-38);
Lys
26,34-two (N
ε-(courage acyl group))-GLP-1 (7-38);
Gly
8Lys
26(N
ε-(courage acyl group))-GLP-1 (7-38);
Gly
8Lys
34(N
ε-(courage acyl group))-GLP-1 (7-38);
Gly
8Lys
26,34-two (N
ε-(courage acyl group))-GLP-1 (7-38);
Arg
26Lys
34(N
ε-(courage acyl group))-GLP-1 (7-38);
Gly
8Arg
26Lys
34(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-39);
Lys
26(N
ε-(7-deoxidation courage acyl group)) Arg
34-GLP-1 (7-39);
Gly
8Lys
26(N
ε-(7-deoxidation courage acyl group)) Arg
34-GLP-1 (7-39);
Arg
26,34Lys
36(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-39);
Gly
8Arg
26,34Lys
36(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-39);
Lys
26(N
ε-(courage acyl group))-GLP-1 (7-39);
Lys
34(N
ε-(courage acyl group))-GLP-1 (7-39);
Lys
26,34-two (N
ε-(courage acyl group))-GLP-1 (7-39);
Gly
8Lys
26(N
ε-(courage acyl group))-GLP-1 (7-39);
Gly
8Lys
34(N
ε-(courage acyl group))-GLP-1 (7-39);
Gly
8Lys
26,34-two (N
ε-(courage acyl group))-GLP-1 (7-39);
Arg
26Lys
34(N
ε-(courage acyl group))-GLP-1 (7-39);
Gly
8Arg
26Lys
34(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-40);
Lys
26(N
ε-(7-deoxidation courage acyl group)) Arg
34-GLP-1 (7-40);
Gly
8Lys
26(N
ε-(7-deoxidation courage acyl group)) Arg
34-GLP-1 (7-40);
Arg
26,34Lys
36(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-40);
Gly
8Ar
26,34Lys
36(N
ε-(7-deoxidation courage acyl group))-GLP-1 (7-40);
Lys
26(N
ε-(courage acyl group))-GLP-1 (7-40);
Lys
34(N
ε-(courage acyl group))-GLP-1 (7-40);
Lys
26,34-two (N
ε-(courage acyl group))-GLP-1 (7-40);
Gly
8Lys
26(N
ε-(courage acyl group))-GLP-1 (7-40);
Gly
8Lys
34(N
ε-(courage acyl group))-GLP-1 (7-40);
Gly
8Lys
26,34-two (N
ε-(courage acyl group))-GLP-1 (7-40);
Arg
26Lys
34(N
ε-(courage acyl group))-GLP-1 (7-40);
Lys
26(N
ε-(courage acyl group))-GLP-1 (7-36);
Lys
34(N
ε-(courage acyl group))-GLP-1 (7-36);
Lys
26,34-two (N
ε-(courage acyl group))-GLP-1 (7-36);
Gly
8Lys
26(N
ε-(courage acyl group))-GLP-1 (7-36);
Gly
8Lys
34(N
ε-(courage acyl group))-GLP-1 (7-36);
Gly
8Lys
26,34-two (N
ε-(courage acyl group))-GLP-1 (7-36);
Arg
26Lys
34(N
ε-(courage acyl group))-GLP-1 (7-36);
Lys
26(N
ε-(courage acyl group))-GLP-1 (7-35);
Lys
34(N
ε-(courage acyl group))-GLP-1 (7-35);
Lys
26,34-two (N
ε-(courage acyl group))-GLP-1 (7-35);
Gly
8Lys
26(N
ε-(courage acyl group))-GLP-1 (7-35);
Gly
8Lys
34(N
ε-(courage acyl group))-GLP-1 (7-35);
Gly
8Lys
26,34-two (N
ε-(courage acyl group))-GLP-1 (7-35);
Arg
26Lys
34(N
ε-(courage acyl group))-GLP-1 (7-35);
Lys
26(N
ε-(courage acyl group))-GLP-1 (7-36) acid amides;
Lys
34(N
ε-(courage acyl group))-GLP-1 (7-36) acid amides;
Lys
26,34-two (N
ε-(courage acyl group))-GLP-1 (7-36) acid amides;
Gly
8Lys
26(N
ε-(courage acyl group))-GLP-1 (7-36) acid amides;
Gly
8Lys
34(N
ε-(courage acyl group))-GLP-1 (7-36) acid amides;
Gly
8Lys
26,34-two (N
ε-(courage acyl group))-GLP-1 (7-36) acid amides;
Arg
26Lys
34(N
ε-(courage acyl group))-GLP-1 (7-36) acid amides;
Gly
8Arg
26Lys
34(N
ε-(courage acyl group))-GLP-1 (7-37);
Lys
26(N
ε-(courage acyl group)) Arg
34-GLP-1 (7-37);
Gly
8Lys
26(N
ε-(courage acyl group)) Arg
34-GLP-1 (7-37);
Arg
26,34Lys
36(N
ε-(courage acyl group))-GLP-1 (7-37);
Gly
8Arg
26,34Lys
36(N
ε-(courage acyl group))-GLP-1 (7-37);
Lys
26(N
ε-(stone courage acyl group))-GLP-1 (7-37);
Lys
34(N
ε-(stone courage acyl group))-GLP-1 (7-37);
Lys
26,34-two (N
ε-(stone courage acyl group))-GLP-1 (7-37);
Gly
8Lys
26(N
ε-(stone courage acyl group))-GLP-1 (7-37);
Gly
8Lys
34(N
ε-(stone courage acyl group))-GLP-1 (7-37);
Gly
8Lys
26,34-two (N
ε-(stone courage acyl group))-GLP-1 (7-37);
Arg
26Lys
34(N
ε-(stone courage acyl group))-GLP-1 (7-37);
Gly
8Arg
26Lys
34(N
ε-(courage acyl group))-GLP-1 (7-38);
Lys
26(N
ε-(courage acyl group)) Arg
34-GLP-1 (7-38);
Gly
8Lys
26(N
ε-(courage acyl group)) Arg
34-GLP-1 (7-38);
Arg
26,34Lys
36(N
ε-(courage acyl group))-GLP-1 (7-38);
Arg
26,34Lys
38(N
ε-(courage acyl group))-GLP-1 (7-38);
Gly
8Arg
26,34Lys
36(N
ε-(courage acyl group))-GLP-1 (7-38);
Lys
26(N
ε-(stone courage acyl group))-GLP-1 (7-38);
Lys
34(N
ε-(stone courage acyl group))-GLP-1 (7-38);
Lys
26,34-two (N
ε-(stone courage acyl group))-GLP-1 (7-38);
Gly
8Lys
26(N
ε-(stone courage acyl group))-GLP-1 (7-38);
Gly
8Lys
34(N
ε-(stone courage acyl group))-GLP-1 (7-38);
Gly
8Lys
26,34-two (N
ε-(stone courage acyl group))-GLP-1 (7-38);
Arg
26Lys
34(N
ε-(stone courage acyl group))-GLP-1 (7-38);
Gly
8Arg
26Lys
34(N
ε-(courage acyl group))-GLP-1 (7-39);
Lys
26(N
ε-(courage acyl group)) Arg
34-GLP-1 (7-39);
Gly
8Lys
26(N
ε-(courage acyl group)) Arg
34-GLP-1 (7-39);
Arg
26,34Lys
36(N
ε-(courage acyl group))-GLP-1 (7-39);
Gly
8Arg
26,34Lys
36(N
ε-(courage acyl group))-GLP-1 (7-39);
Lys
26(N
ε-(stone courage acyl group))-GLP-1 (7-39);
Lys
34(N
ε-(stone courage acyl group))-GLP-1 (7-39);
Lys
26,34-two (N
ε-(stone courage acyl group))-GLP-1 (7-39);
Gly
8Lys
26(N
ε-(stone courage acyl group))-GLP-1 (7-39);
Gly
8Lys
34(N
ε-(stone courage acyl group))-GLP-1 (7-39);
Gly
8Lys
26,34-two (N
ε-(stone courage acyl group))-GLP-1 (7-39);
Arg
26Lys
34(N
ε-(stone courage acyl group))-GLP-1 (7-39);
Gly
8Arg
26Lys
34(N
ε-(courage acyl group))-GLP-1 (7-40);
Lys
26(N
ε-(courage acyl group)) Arg
34-GLP-1 (7-40);
Gly
8Lys
26(N
ε-(courage acyl group)) Arg
34-GLP-1 (7-40);
Arg
26,34Lys
36(N
ε-(courage acyl group))-GLP-1 (7-40);
Gly
8Arg
26,34Lys
36(N
ε-(courage acyl group))-GLP-1 (7-40);
Lys
26(N
ε-(stone courage acyl group))-GLP-1 (7-40);
Lys
34(N
ε-(stone courage acyl group))-GLP-1 (7-40);
Lys
26,34-two (N
ε-(stone courage acyl group))-GLP-1 (7-40);
Gly
8Lys
26(N
ε-(stone courage acyl group))-GLP-1 (7-40);
Gly
8Lys
34(N
ε-(stone courage acyl group))-GLP-1 (7-40);
Gly
8Lys
26,34-two (N
ε-(stone courage acyl group))-GLP-1 (7-40);
Arg
26Lys
34(N
ε-(stone courage acyl group))-GLP-1 (7-37);
Lys
26(N
ε-(stone courage acyl group))-GLP-1 (7-36);
Lys
34(N
ε-(stone courage acyl group))-GLP-1 (7-36);
Lys
26,34-two (N
ε-(stone courage acyl group))-GLP-1 (7-36);
Gly
8Lys
26(N
ε-(stone courage acyl group))-GLP-1 (7-36);
Gly
8Lys
34(N
ε-(stone courage acyl group))-GLP-1 (7-36);
Gly
8Lys
26,34-two (N
ε-(stone courage acyl group))-GLP-1 (7-36);
Arg
26Lys
34(N
ε-(stone courage acyl group))-GLP-1 (7-36);
Lys
26(N
ε-(stone courage acyl group))-GLP-1 (7-35);
Lys
34(N
ε-(stone courage acyl group))-GLP-1 (7-35);
Lys
26,34-two (N
ε-(stone courage acyl group))-GLP-1 (7-35);
Gly
8Lys
26(N
ε-(stone courage acyl group))-GLP-1 (7-35);
Gly
8Lys
34(N
ε-(stone courage acyl group))-GLP-1 (7-35);
Gly
8Lys
26,34-two (N
ε-(stone courage acyl group))-GLP-1 (7-35);
Arg
26Lys
34(N
ε-(stone courage acyl group))-GLP-1 (7-35);
Lys
26(N
ε-(stone courage acyl group))-GLP-1 (7-36) acid amides;
Lys
34(N
ε-(stone courage acyl group))-GLP-1 (7-36) acid amides;
Lys
26,34-two (N
ε-(stone courage acyl group))-GLP-1 (7-36) acid amides;
Gly
8Lys
26(N
ε-(stone courage acyl group))-GLP-1 (7-36) acid amides;
Gly
8Lys
34(N
ε-(stone courage acyl group))-GLP-1 (7-36) acid amides;
Gly
8Lys
26,34-two (N
ε-(stone courage acyl group))-GLP-1 (7-36) acid amides;
Arg
26Lys
34(N
ε-(stone courage acyl group))-GLP-1 (7-36) acid amides;
Gly
8Arg
26Lys
34(N
ε-(stone courage acyl group))-GLP-1 (7-37);
Lys
26(N
ε-(stone courage acyl group)) Arg
34-GLP-1 (7-37);
Gly
8Lys
26(N
ε-(stone courage acyl group)) Arg
34-GLP-1 (7-37);
Arg
26,34Lys
36(N
ε-(stone courage acyl group))-GLP-1 (7-37);
Arg
26,34Lys
38(N
ε-(stone courage acyl group))-GLP-1 (7-37);
Gly
8Arg
26,34Lys
36(N
ε-(stone courage acyl group))-GLP-1 (7-37);
Gly
8Arg
26Lys
34(N
ε-(stone courage acyl group))-GLP-1 (7-38);
Lys
26(N
ε-(stone courage acyl group)) Arg
34-GLP-1 (7-38);
Gly
8Lys
26(N
ε-(stone courage acyl group)) Arg
34GLP-1 (7-38);
Arg
26,34Lys
36(N
ε-(stone courage acyl group))-GLP-1 (7-38);
Arg
26,34Lys
38(N
ε-(stone courage acyl group))-GLP-1 (7-38);
Gly
8Arg
26,34Lys
36(N
ε-(stone courage acyl group))-GLP-1 (7-38);
Gly
8Arg
26Lys
34(N
ε-(stone courage acyl group))-GLP-1 (7-39);
Lys
26(N
ε-(stone courage acyl group)) Arg
34-GLP-1 (7-39);
Gly
8Lys
26(N
ε-(stone courage acyl group)) Arg
34-GLP-1 (7-39);
Arg
26,34Lys
36(N
ε-(stone courage acyl group))-GLP-1 (7-39);
Gly
8Arg
26,34Lys
36(N
ε-(stone courage acyl group))-GLP-1 (7-39);
Gly
8Arg
26Lys
34(N
ε-(stone courage acyl group))-GLP-1 (7-40);
Lys
26(N
ε-(stone courage acyl group)) Arg
34-GLP-1 (7-40);
Gly
8Lys
26(N
ε-(stone courage acyl group)) Arg
34-GLP-1 (7-40);
Arg
26,34Lys
36(N
ε-(stone courage acyl group))-GLP-1 (7-40); With
Gly
8Arg
26,34Lys
36(N
ε-(stone courage acyl group))-GLP-1 (7-40).
In a further preferred embodiment, the present invention relates to contain the pharmaceutical composition of a kind of GLP-1 derivative and a kind of pharmaceutics acceptable carrier.
In a further preferred embodiment, the present invention relates to GLP-1 derivative according to the present invention and have than the purposes in the medicament of GLP-1 (7-37) effect curves for more time in preparation.
In a further preferred embodiment, the present invention relates to GLP-1 derivative according to the present invention and have purposes in the medicament that is used for the treatment of non-insulin-dependent diabetes mellitus (NIDDM) of action effect of longer time in preparation.
In a further preferred embodiment, the present invention relates to GLP-1 derivative according to the present invention and have purposes in the medicament that is used for the treatment of insulin-dependent diabetes of action effect of longer time in preparation.
In a further preferred embodiment, the present invention relates to GLP-1 derivative according to the present invention and have purposes in the medicament that is used for the treatment of obesity of action effect of longer time in preparation.
In a further preferred embodiment, the present invention relates to treat in the patient of this treatment of needs the method for insulin-dependent or non-insulin-dependent diabetes mellitus (NIDDM), this method comprises the described GLP-1 derivative of claim 1 and the pharmaceutics acceptable carrier of the treatment significant quantity that doses a patient with.
Detailed description of the present invention
For obtaining GLP-1 derivative effect curves fully for a long time, the lipophilic substituent that is connected to the GLP-1 part preferably contains 4-40 carbon atom, an especially 8-25 carbon atom.This lipophilic substituent can form amido linkage and be connected on the GLP-1 amino partly by the amino of its carboxyl and the amino-acid residue that is attached thereto.Or selectively, this lipophilic substituent can be connected on this amino-acid residue by carboxyl formation amido linkage amino by it and described amino-acid residue.Alternatively, this lipophilic substituent can be connected to the GLP-1 part by ester bond.Normally, can pass through the carboxyl of GLP-1 part and the reaction between this substituent hydroxyl, or form ester bond by the hydroxyl of GLP-1 part and the reaction between the substituent carboxyl.Alternatively, this lipophilic substituent can be an alkyl, and it is introduced in the primary amino of GLP-1 part.
In a preferred embodiment of the invention, this lipophilic substituent is by a carboxyl of a spacer groups and an amino formation amido linkage of GLP-1 part, thereby is connected to the GLP-1 part.The example of suitable spacer groups has succsinic acid, Lys, Glu or Asp, or a kind of dipeptides as Gly-Lys.When spacer groups was succsinic acid, its carboxyl can aminoly with of amino-acid residue form amido linkage, and its another carboxyl can form amido linkage with the amino of this lipophilic substituent.When spacer groups was Lys, Glu or Asp, its carboxyl can form amido linkage with the amino of amino-acid residue, and its amino can form amido linkage with the carboxyl of lipophilic substituent.When with Lys during, can between the epsilon-amino of Lys and this lipophilic substituent, insert other spacer groups in some cases as spacer groups.In a preferred embodiment, this other spacer groups is a succsinic acid, and the amino that exists in the epsilon-amino of it and Lys and the lipophilic substituent forms amido linkage.In another preferred embodiment, this other spacer groups is Glu or Asp, and the epsilon-amino of it and Lys forms an amido linkage, forms another amido linkage with the carboxyl that occurs in the lipophilic substituent, and promptly this lipophilic substituent is a N
εThe lysine residue of-acylations.
In another preferred embodiment of the present invention, this lipophilic substituent have one can electronegative group.A kind of can electronegative group be carboxyl preferably.
Can prepare parent peptide by a kind of like this method, this method is included under the condition that allows the peptide expression, in the appropriate nutrition substratum, cultivate dna sequence dna that contains this polypeptide of encoding and the host cell that can express this peptide, from culture, reclaim the peptide that produces then.
Being used for the substratum of culturing cell can be any conventional substratum that is used to cultivate this host cell, as minimum medium or contain the complex medium of suitable additive.Can be by the commercially available suitable culture base that obtains, or prepare suitable culture base (as the method for introducing in the American type culture collection catalogue) according to disclosed method for making.Can from substratum, reclaim the polypeptide that produces by this cell by ordinary method then, these methods comprise by centrifugal or filter and separate host cell from substratum, with the protein component in salt such as ammonium sulfate precipitation supernatant liquor or the filtrate, select for use various chromatography methods such as ion exchange chromatography, gel permeation chromatography, affinity chromatography etc. to carry out purifying according to the kind of purpose peptide.
The dna sequence dna of coding parent peptide can derive from genome or cDNA, as by preparation genome or cDNA library, and according to standard technique (as, see Sambrook, J, Fritsch, EF and Maniatis, T, molecular cloning: experimental implementation guide, Cold Spring HarborLaboratory Press, New York, 1989) use the synthetic oligonucleotide probe, hybridize and filter out all or part of dna sequence dna of this peptide of coding.Also can be by the standard method of having set up, as Beaucage and the described phosphamide of Caruthers (phosphoamidite) method (tetrahedron wall bulletin, 22 (1981), 1859-1869), or described method (EMBO's magazine such as Matthes, 3 (1984), the 801-805) dna sequence dna of synthetic this encoded peptide.Also can use specific primer, as US4,683,202 or Saiki etc. in " science ", 239 (1988), 487-491 is described, prepares dna sequence dna by polymerase chain reaction.
Dna sequence dna can be inserted any being convenient to and carry out in the carrier of DNA regrouping process, and the host cell that this carrier will be introduced into is usually depended in the selection of carrier.Therefore, carrier can be a kind of self-replicating type carrier, and promptly as the carrier of the outer entity existence of a karyomit(e), it duplicates and does not rely on chromosome duplication, as plasmid.Perhaps, carrier can be such one type, and when being introduced into host cell, it will be incorporated in the host cell gene group, and duplicate with the karyomit(e) that it was integrated into.
Preferably a kind of expression vector of carrier, the dna sequence dna of the described peptide of encoding in it is transcribed required other section (as promotor) with this DNA and is effectively linked to each other.Promotor can be any dna sequence dna that transcriptional activity is arranged in the host cell of selecting for use, and it can derive from the gene of coding host cell homology or heterologous protein.The promotor example that the well known DNA that is suitable for instructing code book invention peptide in multiple host cell transcribes, referring to as Sambrook etc., the source is the same.
When needing, the dna sequence dna of this peptide of encoding also can effectively be connected with suitable terminator, polyadenylation signal, transcriptional enhancer sequence and translational enhancer sequence.Recombinant vectors of the present invention can also contain the dna sequence dna that it is duplicated in the purpose host cell.
Carrier can also contain a selective marker, as a gene, its gene product will remedy a defective in the host cell, perhaps can give the resistance to medicine such as penbritin, kantlex, tsiklomitsin, paraxin, Xin Meisu, Totomycin or methotrexate etc.
For parent peptide of the present invention being introduced the Secretory Pathway of host cell, can in recombinant vectors, provide a secretory signal sequence (also being referred to as leader sequence, preceding former sequence or presequence).Secretory signal sequence is connected with the dna sequence dna of correct frame with this peptide of coding.Secretory signal sequence is usually located at 5 ' side of the dna sequence dna of this peptide of coding.Secretory signal sequence can be the secretory signal sequence that normally is connected with this peptide, maybe can come from the gene of the another kind of secretory protein of coding.
Be used for connecting respectively dna sequence dna, promotor and the selectable terminator and/or the secretory signal sequence of code book invention peptide, and be inserted into the suitable method that contains in the carrier that duplicates information necessary, it is known to those skilled in the art that (referring to as Sambrook etc., the source is the same).
With the host cell that imports dna sequence dna or recombinant vectors can be any cell that can produce peptide of the present invention, comprises bacterium, yeast, fungi and higher eukaryotic cell.Those skilled in the art know and the example of the suitable host cells used as follows, but be not limited to these: intestinal bacteria, yeast saccharomyces cerevisiae, or Mammals BHK or Chinese hamster ovary celI system.
International patent application No.WO87/06941 (The General HospitalCorporation) has put down in writing can be as the example of GLP-1 compound partly according to the present invention, and this application relates to the peptide fragment and its purposes as the pancreotropic hormone agent of a kind of GLP-1 of containing (7-37) and functional derivatives thereof.
International patent application No.90/11296 (The General Hospital orporation) has put down in writing other GLP-1 analogue, this application relates to the peptide fragment that contains GLP-1 (7-36) and functional derivatives thereof, these peptide fragment have the insulinotropic activity above GLP-1 (1-36) or GLP-1 (1-37), also relate to their purposes as the pancreotropic hormone agent.
International patent application No.91/11457 (Buckley etc.) discloses active GLP-1 peptide 7-34,7-35, and the analogue of 7-36 and 7-37, they also can be used as the GLP-1 part according to the present invention.
Pharmaceutical composition
The pharmaceutical composition that contains the GLP-1 derivative according to the present invention can parenteral be applied to the patient of this treatment of needs.Can use syringe, selectively carry out parenteral admin by subcutaneous, intramuscular or intravenous injection with pen-type injector.In addition, can carry out parenteral admin by infusion pump.Selection in addition is a kind of GLP-1 derivative powdery or liquid composition that nose or lung Sprayable are used that be used for.GLP-1 derivative of the present invention can also be selected the transdermal route administration, as through the patch transdermal administration, can select ion to penetrate patch; Or through saturating mucosal route administration, as seeing through buccal mucosa.
Can use routine techniques, as " pharmaceutical science " of Remington, 1985 or Remington: pharmaceutical science and practice, 19 editions, 1995 described methods, preparation contains the pharmaceutical composition of GLP-1 derivative of the present invention.
Therefore, can prepare the composition for injection of GLP-1 derivative of the present invention with the routine techniques of pharmaceutical industry, these technology comprise suitably dissolves and mixes each component to obtain required final product.
According to a kind of method, the 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.Add isotonic agent, sanitas and buffer reagent on demand, and when needed with the pH value of sour example hydrochloric acid or alkali such as aqueous sodium hydroxide solution regulator solution.At last, water regulator solution volume obtains required concentration of component.
Isotonic agent for example has sodium-chlor, mannitol, glycerine.
Sanitas for example has phenol, meta-cresol, methyl p-Hydroxybenzoate, benzylalcohol.
Suitable buffer reagent such as sodium acetate and sodium phosphate.
Except that mentioned component, the solution that contains GLP-1 derivative of the present invention also can contain solvability and the stability of tensio-active agent to improve the GLP-1 derivative.
The composition that can be used for some peptide of intranasal administration by European patent No.272097 (Nordisk A/S) or the described preparation of WO93/18785.
The composition of GLP-1 derivative is provided with the composition forms that is suitable for drug administration by injection according to a preferred embodiment of the invention.This composition both can be ready-to-use injection liquid, also can be a certain amount of solid-state composition, as a kind of freeze-dried products, need before injection it be dissolved in the solvent.Contained GLP-1 derivative is no less than 2mg/ml in this injection solution, preferably is no less than 5mg/ml, more preferably is no less than 10mg/ml, and preferred no more than 100mg/ml.
GLP-1 derivative of the present invention can be used for treating various diseases.Used concrete GLP-1 derivative and the optimal dose level of patient will depend on disease to be treated and various factors, comprise the usefulness of used concrete peptide derivant and patient's age, body weight, physical activity and diet, also depend on may with the drug combination of other medicines and the severity of the state of an illness.Suggestion those skilled in the art determines the dosage of GLP-1 derivative of the present invention at each patient.
Specifically, estimate that the GLP-1 derivative will be useful for the medicine that is used for the treatment of non-insulin-dependent diabetes mellitus (NIDDM) and/or treatment of obesity that preparation has the effect curves of longer time.
Can further specify the present invention with following embodiment, but should not think that these embodiment are the qualifications to protection domain.The disclosed characteristics of the description of front and following examples respectively or arbitrary combination ground, the present invention is important for accomplished in various ways.
Embodiment
Use the following abbreviation of commercially available chemical preparations:
DMF:N, dinethylformamide
The NMP:N-N-methyl-2-2-pyrrolidone N-
EDPA:N-ethyl-N, the N-Diisopropylamine
EGTA: ethylene glycol bisthioglycolate (beta-amino ether)-N, N, N ', N '-tetraacethyl
GTP: guanosine 5 '-triphosphoric acid
TFA: trifluoroacetic acid
THF: tetrahydrofuran (THF)
Myr-ONSu: tetradecanoic acid 2,5-dioxo tetramethyleneimine-1-base ester
Pal-ONSu: hexadecanoic acid 2,5-dioxo tetramethyleneimine-1-base ester
Ste-ONSu: octadecanoic acid 2,5-dioxo tetramethyleneimine-1-base ester
HOOC-(CH
2)
6-COONSu: ω-carboxyl enanthic acid 2,5-dioxo tetramethyleneimine-1-base ester
HOOC-(CH
2)
10-COONSu: ω-carboxyl undecanoic acid 2,5-dioxo tetramethyleneimine-1-base ester
HOOC-(CH
2)
12-COONSu: ω-carboxyl tridecanoic acid 2,5-dioxo tetramethyleneimine-1-base ester
HOOC-(CH
2)
14-COONSu: ω-carboxyl pentadecylic acid 2,5-dioxo tetramethyleneimine-1-base ester
HOOC-(CH
2)
16-COONSu: ω-carboxyl margaric acid 2,5-dioxo tetramethyleneimine-1-base ester
HOOC-(CH
2)
18-COONSu: ω-carboxyl nondecylic acid 2,5-dioxo tetramethyleneimine-1-base ester
Abbreviation:
PDMS: the attached mass spectrometry of plasma desorption (Plasma Desorption MassSpectormetry)
MALDI-MS: the auxiliary laser desorption of matrix is attached/the MALDI-MS assay method
HPLC: high performance liquid chromatography (HPLC)
Amu: atomic mass unit
Analyze
The attached mass spectrometry of plasma desorption
Specimen preparation:
Sample is dissolved in 0.1%TFA/EtOH (1: 1), makes its concentration reach 1 μ g/ μ l.Place nitrocellulose target (Bio-ion AB, Uppsala, Sweden) to go up and make its surface to absorb 2 minutes sample solution (5-10 μ l) at target.Use 2 * 25 μ l 0.1%TFA that target is cleaned and Rotary drying subsequently.Place the nitrocellulose target in the target turner at last and put into mass spectrograph.
Mass spectroscopy
Carrying out PDMS with Bio-ion 20 times of flight arrangement (Bio-ion Nordic AB, Uppsala, Sweden) analyzes.Use the acceleration voltage of 15kV, make the molion that forms by 252-Cf fission fragment bombardment nitrocellulose surface add fast direction one terminal detector.Use H respectively at m/z 1 and 30
+And NO
+Ion is proofreaied and correct the time of flight spectrum that produces and is a kind of real mass spectrum.Generally be accumulated in 1.0 * 10 in 15-20 minute
6The secondary fissure mass spectrum that becomes.Measured quality is all corresponding to the isotropic substance average molecular mass.The tolerance range of quality determination generally is better than 0.1%.
MALDI-MS
Use a kind of being equipped with to postpone tripping device and (PerSeptive Biosystems Inc., Framingham MA) carry out MALDI-MS and analyze with the Voyager RP instrument of linear mode work.Use alpha-cyano-4-hydroxyl-styracin as matrix, and carry out quality determination based on the external calibration method.
Example 1
Lys
26(N
ε-myristoyl)-GLP-1 (7-37) synthetic
By the synthetic target compound that obtains of GLP-1 (7-37).With GLP-1 (7-37) (25mg, 7.45 μ m), EDPA (26.7mg, 208 μ m), the mixture of NMP (520 μ l) and water (260 μ l) shook 5 minutes in the room temperature gentleness.In the gained mixture, add Myr-ONSu (2.5mg, 7.67 μ m) and be dissolved in the solution that NMP (62.5 μ l) obtains, reaction mixture was shaken 5 minutes in the room temperature gentleness, placed then 20 minutes.Other adds Myr-ONSu (2.5mg, 7.67 μ m) and is dissolved in the solution that NMP (62.5 μ l) obtains, and gained mixture gentleness was shaken 5 minutes.Behind 40 minutes total reaction time, to wherein adding glycine (12.5mg, 166 μ mol) be dissolved in the solution termination reaction that makes in 50% aqueous ethanolic solution (12.5ml). use cyanogen propyl group (cyanopropyl) post (Zorbax 300SB-CN) and standard acetonitrile/TFA system, by HPLC method separate targets compound from reaction mixture, output is 1.3mg (be equivalent to theoretical yield 4.9%).Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.With the product that PDMS method analytical separation goes out, find that the m/z value at protonated molecular ion peak is 3567.9 ± 3.Therefore the molecular weight that draws is 3566.9 ± 3amu (theoretical value is 3565.9amu).By streptococcus aureus V8 proteolytic enzyme target compound is carried out the enzymolysis cutting, carry out the quality determination of peptide fragment subsequently by PDMS, thereby determine acylations position (Lys26).
Except that target compound, from reaction mixture, separate to obtain other two kinds of GLP-1 derivatives with narrower gradient (acetonitrile 35-38% in 60 minutes) with same chromatography column, see example 2 and 3.
Example 2
Lys
34(N
ε-myristoyl)-GLP-1 (7-37) synthetic
Separate from example 1 described reaction mixture by the HPLC method and to obtain target compound.Analyze through PDMS that to obtain m/z be 3567.7 ± 3 protonated molecular ion peak.Therefore find that molecular weight is 3566.7 ± 3amu (theoretical value is 3565.9amu).Determine the acylations position according to fracture mode.
Example 3
Lys
26,34-two (N
ε-myristoyl)-GLP-1 (7-37) synthetic
Separate from example 1 described reaction mixture by the HPLC method and to obtain target compound.PDMS analyzes and to obtain m/z is 3778.4 ± 3 protonated molecular ion peak.Therefore find that molecular weight is 3777.4 ± 3amu (theoretical value is 3776.1amu).
Example 4
Lys
26(N
ε-myristoyl) Arg
34-GLP-1's (7-37) is synthetic
By Arg
34The synthetic target compound that obtains of-GLP-1 (7-37).With Arg
34-GLP-1 (7-37) (5mg, 1.47 μ m), EDPA (5.3mg, 41.1 μ m), the mixture of NMP (105 μ l) and water (50 μ l) shook 5 minutes in the room temperature gentleness.In the gained mixture, add Myr-ONSu (0.71mg, 2.2 μ m) and be dissolved in the solution that NMP (17.8 μ l) obtains, reaction mixture was shaken 5 minutes in the room temperature gentleness, placed then 20 minutes.Behind 30 minutes total reaction time, be dissolved in the solution termination reaction that makes in 50% aqueous ethanolic solution (2.5ml) to wherein adding glycine (25mg, 33.3 μ m).Described by example 1 through HPLC method purification reaction mixture.PDMS analyzes and to obtain the m/z value is 3594.9 ± 3 protonated molecular ion peak.The molecular weight that draws is 3593.9 ± 3amu (theoretical value is 3593.9amu).
Example 5
Gly
8Arg
26,34Lys
36(N
ε-myristoyl)-GLP-1 (7-37) synthetic
By Gly available from QCB
8Arg
26,34Lys
36The synthetic target compound that obtains of-GLP-1 (7-37).With Gly
8Arg
26,34Lys
36-GLP-1 (7-37) (1.3mg, 0.39 μ m), EDPA (1.3mg, 10 μ m), the mixture of NMP (125 μ l) and water (30 μ l) shook 5 minutes in the room temperature gentleness.In the gained mixture, add Myr-ONSu (0.14mg, 0.44 μ m) and be dissolved in the solution that NMP (3.6ml) obtains, reaction mixture was shaken 15 minutes in the room temperature gentleness.Be dissolved in the solution termination reaction that makes in 50% aqueous ethanolic solution (10 μ l) to wherein adding glycine (0.1mg, 1.33 μ m).With HPLC method purification reaction mixture, isolate target compound (60 μ g, 4%).
Example 6
Arg
26,34Lys
36(N
ε-myristoyl)-GLP-1 (7-37)-OH synthetic
With Arg
26,34Lys
36-GLP-1 (7-37)-OH (5.0mg, 1.477 μ mol), EDPA (5.4mg, 41.78 μ mol), the mixture of NMP (105 μ l) and water (50 μ l) shook 5 minutes in the room temperature gentleness.In the gained mixture, add Myr-ONSu (0.721mg, 2.215 μ mol) and be dissolved in the solution that NMP (18 μ l) obtains.Reaction mixture is shaken 5 minutes in the room temperature gentleness, and it was kept 45 minutes in addition in room temperature.Be dissolved in the solution termination reaction that makes in 50% aqueous ethanolic solution (250 μ l) to wherein adding glycine (2.5mg, 33.3 μ mol).Use cyanogen propyl group post (Zorbax300SB-CN) and standard acetonitrile/TFA system, by the column chromatography purification reaction mixture.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.Separate targets compound (1.49mg, 28%) is used PDMS method assay products then.The m/z value of finding protonated molecular ion peak is 3595 ± 3.Therefore the molecular weight that draws is 3594 ± 3amu (theoretical value is 3594amu).
Example 7
Lys
26,34Two (N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-37)-OH synthetic
With GLP-1 (7-37)-OH (70mg, 20.85 μ mol), EDPA (75.71mg, 585.8 μ mol), the mixture of NMP (1.47ml) and water (700 μ l) shook 10 minutes in the room temperature gentleness.In the gained mixture, add HOOC-(CH
2)
18-COONSu (27.44mg, 62.42 μ mol) is dissolved in the solution that NMP (686 μ l) obtains, and reaction mixture is shaken 5 minutes in the room temperature gentleness, and it was kept 50 minutes in addition in room temperature.Be dissolved in the solution termination reaction that makes in 50% aqueous ethanolic solution (3.44ml) to wherein adding glycine (34.43mg, 458.7 μ mol).Use cyanogen propyl group post (Zorbax 300SB-CN) and standard acetonitrile/TFA system, by the column chromatography purification reaction mixture.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.Separate targets compound (8.6mg, 10%) is used PDMS method assay products then.The m/z value of finding protonated molecular ion peak is 4006 ± 3.Thereby the molecular weight that draws is 4005 ± 3amu (theoretical value is 4005amu).
Example 8
Arg
26,34Lys
36(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-36)-OH synthetic
With Arg
26,34Lys
36-GLP-1 (7-36)-OH (5.06mg, l.52 μ mol), EDPA (5.5mg, 42.58 μ mol), the mixture of NMP (106 μ l) and water (100 μ l) shook 5 minutes in the room temperature gentleness.In the gained mixture, add HOOC-(CH
2)
18-COONSu (1.33mg, 3.04 μ mol) is dissolved in the solution that NMP (33.2 μ l) obtains, and reaction mixture was shaken 5 minutes in the room temperature gentleness, places 2.5 hours in room temperature then again.Be dissolved in the solution termination reaction that makes in 50% aqueous ethanolic solution (250 μ l) to wherein adding glycine (2.50mg, 33.34 μ mol).Use cyanogen propyl group post (Zorbax 300SB-CN) and standard acetonitrile/TFA system, by the column chromatography purification reaction mixture.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.Isolate target compound (0.46mg, 8%), with PDMS method assay products.The m/z value of finding protonated molecular ion peak is 3652 ± 3.Thereby to draw molecular weight be 3651 ± 3amu (theoretical value is 3651amu).
Example 9
Arg
26,34Lys
38(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-38)-OH synthetic
With Arg
26,34Lys
38The mixture of-GLP-1 (7-38)-OH (5.556mg, 1.57 μ mol), EDPA (5.68mg, 43.96 μ mol), NMP (116.6 μ l) and water (50 μ l) shook 10 minutes in the room temperature gentleness.In the gained mixture, add HOOC-(CH
2)
18-COONSu (1.38mg, 3.14 μ mol) is dissolved in the solution that NMP (34.5 μ l) obtains, and reaction mixture was shaken 5 minutes in the room temperature gentleness, places 2.5 hours in room temperature then again.Be dissolved in the solution that makes in 50% aqueous ethanolic solution (250 μ l) with termination reaction to wherein adding glycine (2.5mg, 33.3 μ mol).Use cyanogen propyl group post (Zorbax 300SB-CN) and standard acetonitrile/TFA system, by the column chromatography purification reaction mixture.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.Isolate target compound (0.7mg, 12%), with PDMS method assay products.The m/z value of finding protonated molecular ion peak is 3866 ± 3.Thereby to draw molecular weight be 3865 ± 3amu (theoretical value is 3865amu).
Example 10
Arg
34Lys
26(N
ε-(ω-carboxyl 19 acyl groups))-GLP-1 (7-37)-OH synthetic
With Arg
34The mixture of-GLP-1 (7-37)-OH (5.04mg, 1.489 μ mol), EDPA (5.39mg, 41.70 μ mol), NMP (105 μ l) and water (50 μ l) shook 10 minutes in the room temperature gentleness.In the gained mixture, add HOOC-(CH
2)
18-COONSu (1.31mg, 2.97 μ mol) is dissolved in NMP (32.8 μ l) and the solution that obtains, and reaction mixture was shaken 5 minutes in the room temperature gentleness, places 30 minutes in room temperature then again.Be dissolved in the solution that makes in 50% aqueous ethanolic solution (246 μ l) with termination reaction to wherein adding glycine (2.46mg, 32.75 μ mol).Use cyanogen propyl group post (Zorbax 300SB-CN) and standard acetonitrile/TFA system, by the column chromatography purification reaction mixture.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.Isolate target compound (1.2mg, 22%), with PDMS method assay products.The m/z value of finding protonated molecular ion peak is 3709 ± 3.Thereby to draw molecular weight be 3708 ± 3amu (theoretical value is 3708amu).
Example 11
Arg
34Lys
26(N
ε-(ω-carboxyl heptadecanoyl base))-GLP-1 (7-37)-OH synthetic
With Arg
34The mixture of-GLP-1 (7-37)-OH (5.8mg, 1.714 μ mol), EDPA (6.20mg, 47.99 μ mol), NMP (121.8 μ l) and water (58 μ l) shook 10 minutes in the room temperature gentleness.In the gained mixture, add HOOC-(CH
2)
16-COONSu (2.11mg, 5.142 μ mol) is dissolved in the solution that NMP (52.8 μ l) obtains, and reaction mixture was shaken 5 minutes in the room temperature gentleness, places 2 hours in room temperature then again.Be dissolved in the solution that makes in 50% aqueous ethanolic solution (283 μ l) with termination reaction to wherein adding glycine (2.83mg, 37.70 μ mol).Use cyanogen propyl group post (Zorbax 300SB-CN) and standard acetonitrile/TFA system, by the column chromatography purification reaction mixture.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.Isolate target compound (0.81mg, 13%), with PDMS method assay products.The m/z value of finding protonated molecular ion peak is 3681 ± 3.Thereby to draw molecular weight be 3680 ± 3amu (theoretical value is 3680amu).
Example 12
Arg
26,34Lys
36(N
ε-(ω-carboxyl heptadecanoyl base))-GLP-1 (7-37)-OH synthetic
With Arg
26,34Lys
36The mixture of-GLP-1 (7-37)-OH (3.51mg, 1.036 μ mol), EDPA (3.75mg, 29.03 μ mol), NMP (73.8 μ l) and water (35 μ l) shook 10 minutes in the room temperature gentleness.In the gained mixture, add HOOC-(CH
2)
16-COONSu (1.27mg, 3.10 μ mol) is dissolved in the solution that NMP (31.8 μ l) obtains, and reaction mixture was shaken 5 minutes in the room temperature gentleness, places 2 hours 10 minutes in room temperature then again.Be dissolved in the solution that makes in 50% aqueous ethanolic solution (171 μ l) with termination reaction to wherein adding glycine (1.71mg, 22.79 μ mol).Use cyanogen propyl group post (Zorbax 300SB-CN) and standard acetonitrile/TFA system by the column chromatography purification reaction mixture.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.Isolate target compound (0.8mg, 21%), with PDMS method assay products.The m/z value of finding protonated molecular ion peak is 3682 ± 3.Thereby to draw molecular weight be 3681 ± 3amu (theoretical value is 3681amu).
Example 13
Arg
26,34Lys
38(N
ε-(ω-carboxyl heptadecanoyl base))-GLP-1 (7-38)-OH synthetic
With Arg
26,34Lys
38The mixture of-GLP-1 (7-38)-OH (5.168mg, 1.459 μ mol), EDPA (5.28mg, 40.85 μ mol), NMP (108.6 μ l) and water (51.8 μ l) shook 10 minutes in the room temperature gentleness.In the gained mixture, add HOOC-(CH
2)
16-COONSu (1.80mg, 4.37 μ mol) is dissolved in the solution that NMP (45 μ l) obtains, and reaction mixture was shaken 10 minutes in the room temperature gentleness, places 2 hours 15 minutes in room temperature then again.Be dissolved in the solution that makes in 50% aqueous ethanolic solution (241 μ l) with termination reaction to wherein adding glycine (2.41mg, 32.09 μ mol).Use cyanogen propyl group post (Zorbax 300SB-CN) and standard acetonitrile/TFA system, by the column chromatography purification reaction mixture.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.Isolate target compound (0.8mg, 14%), with PDMS method assay products.The m/z value of finding protonated molecular ion peak is 3838 ± 3.Thereby to draw molecular weight be 3837 ± 3amu (theoretical value is 3837amu).
Example 14
Arg
26,34Lys
36(N
ε-(ω-carboxyl heptadecanoyl base))-GLP-1 (7-36)-OH synthetic
With Arg
26,34Lys
36The mixture of-GLP-1 (7-36)-OH (24.44mg, 7.34 μ mol), EDPA (26.56mg, 205.52 μ mol), NMP (513 μ l) and water (244.4 μ l) shook 5 minutes in the room temperature gentleness.In the gained mixture, add HOOC-(CH
2)
16-COONSu (9.06mg, 22.02 μ mol) is dissolved in the solution that NMP (1.21ml) obtains, and reaction mixture was shaken 5 minutes in the room temperature gentleness, places 30 minutes in room temperature then again.Be dissolved in the solution that makes in 50% aqueous ethanolic solution (1.21ml) with termination reaction to wherein adding glycine (12.12mg, 161.48 μ mol).Use cyanogen propyl group post (Zorbax 300SB-CN) and standard acetonitrile/TFA system, by the column chromatography purification reaction mixture.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.Isolate target compound (7.5mg, 28%), with PDMS method assay products.The m/z value of finding protonated molecular ion peak is 3625 ± 3.Thereby to draw molecular weight be 3624 ± 3amu (theoretical value is 3624amu).
Example 15
Arg
26,34Lys
36(N
ε-(ω-carboxyl undecanoyl))-GLP-1 (7-37)-OH synthetic
With Arg
26,34Lys
36The mixture of-GLP-1 (7-37)-OH (4.2mg, 1.24 μ mol), EDPA (4.49mg, 34.72 μ mol), NMP (88.2 μ l) and water (42 μ l) shook 10 minutes in the room temperature gentleness.In the gained mixture, add HOOC-(CH
2)
10-COONSu (1.21mg, 3.72 μ mol) is dissolved in the solution that NMP (30.25 μ l) obtains, and reaction mixture was shaken 5 minutes in the room temperature gentleness, places 40 minutes in room temperature then again.Be dissolved in the solution that makes in 50% aqueous ethanolic solution (204 μ l) with termination reaction to wherein adding glycine (2.04mg, 27.28 μ mol).Use cyanogen propyl group post (Zorbax 300SB-CN) and standard acetonitrile/TFA system, by the column chromatography purification reaction mixture.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.Isolate target compound (0.8mg, 18%), with PDMS method assay products.The m/z value of finding protonated molecular ion peak is 3598 ± 3.Thereby to draw molecular weight be 3597 ± 3amu (theoretical value is 3597amu).
Example 16
Arg
26,34Lys
38(N
ε-(ω-carboxyl undecanoyl))-GLP-1 (7-38)-OH synthetic
With Arg
26,34Lys
38The mixture of-GLP-1 (7-38)-OH (5.168mg, 1.46 μ mol), EDPA (5.28mg, 40.88 μ mol), NMP (108.6 μ l) and water (51.7 μ l) shook 10 minutes in the room temperature gentleness.In the gained mixture, add HOOC-(CH
2)
10-COONSu (1.43mg, 4.38 μ mol) is dissolved in the solution that NMP (35.8 μ l) obtains, and reaction mixture was shaken 5 minutes in the room temperature gentleness, places 50 minutes in room temperature then again.Be dissolved in the solution that makes in 50% aqueous ethanolic solution (241 μ l) with termination reaction to wherein adding glycine (2.41mg, 32.12 μ mol).Use cyanogen propyl group post (Zorbax 300SB-CN) and standard acetonitrile/TFA system, by the column chromatography purification reaction mixture.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.Isolate target compound (0.85mg, 16%), with PDMS method assay products.The m/z value of finding protonated molecular ion peak is 3753 ± 3.Thereby to draw molecular weight be 3752 ± 3amu (theoretical value is 3752amu).
Example 17
Arg
26,34Two (N
ε-(ω-carboxyl undecanoyl))-GLP-1 (7-37)-OH synthetic
The mixture of GLP-1 (7-37)-OH (10.0mg, 2.98 μ mol), EDPA (10.8mg, 83.43 μ mol), NMP (210 μ l) and water (100 μ l) was shaken 10 minutes in the room temperature gentleness.In the gained mixture, add HOOC-(CH
2)
10-COONSu (2.92mg, 8.94 μ mol) is dissolved in the solution that NMP (73 μ l) obtains, and reaction mixture was shaken 5 minutes in the room temperature gentleness, places 50 minutes in room temperature then again.Be dissolved in the solution that makes in 50% aqueous ethanolic solution (492 μ l) with termination reaction to wherein adding glycine (4.92mg, 65.56 μ mol).Use cyanogen propyl group post (Zorbax 300SB-CN) and standard acetonitrile/TFA system, by the column chromatography purification reaction mixture.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.Isolate target compound (1.0mg, 9%), with PDMS method assay products.The m/z value of finding protonated molecular ion peak is 3781 ± 3.Thereby to draw molecular weight be 3780 ± 3amu (theoretical value is 3780amu).
Example 18
Arg
26,34Lys
36(N
ε-(ω-carboxyl undecanoyl))-GLP-1 (7-36)-OH synthetic
With Arg
26,34Lys
36The mixture of-GLP-1 (7-36)-OH (15.04mg, 4.52 μ mol), EDPA (16.35mg, 126.56 μ mol), NMP (315.8 μ l) and water (150.4 μ l) shook 10 minutes in the room temperature gentleness.In the gained mixture, add HOOC-(CH
2)
10-COONSu (4.44mg, 13.56 μ mol) is dissolved in the solution that NMP (111 μ l) obtains, and reaction mixture was shaken 5 minutes in the room temperature gentleness, places 40 minutes in room temperature then again.Be dissolved in the solution that makes in 50% aqueous ethanolic solution (250 μ l) with termination reaction to wherein adding glycine (7.5mg, 99.44 μ mol).Use cyanogen propyl group post (Zorbax 300SB-CN) and standard acetonitrile/TFA system, by the column chromatography purification reaction mixture.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.Isolate target compound (3.45mg, 22%), with PDMS method assay products.The m/z value of finding protonated molecular ion peak is 3540 ± 3.Thereby to draw molecular weight be 3539 ± 3amu (theoretical value is 3539amu).
Example 19
Arg
34Lys
26(N
ε-(ω-carboxyl undecanoyl))-GLP-1 (7-37)-OH synthetic
With Arg
34The mixture of-GLP-1 (7-37)-OH (5.87mg, 1.73 μ mol), EDPA (6.27mg, 48.57 μ mol), NMP (123.3 μ l) and water (58.7 μ l) shook 10 minutes in the room temperature gentleness.In the gained mixture, add HOOC-(CH
2)
10-COONSu (1.70mg, 5.20 μ mol) is dissolved in the solution that NMP (42.5 μ l) obtains, and reaction mixture was shaken 5 minutes in the room temperature gentleness, places 40 minutes in room temperature then again.Be dissolved in the solution that makes in 50% aqueous ethanolic solution (286 μ l) with termination reaction to wherein adding glycine (2.86mg, 286 μ mol).Use cyanogen propyl group post (Zorbax 300SB-CN) and standard acetonitrile/TFA system, by the column chromatography purification reaction mixture.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.Isolate target compound (1.27mg, 20%), with PDMS method assay products.The m/z value of finding protonated molecular ion peak is 3597 ± 3.Thereby to draw molecular weight be 3596 ± 3amu (theoretical value is 3596amu).
Example 20
Arg
34Lys
26(N
ε-(ω-carboxyl oenanthyl))-GLP-1 (7-37)-OH synthetic
With Arg
34The mixture of-GLP-1 (7-37)-OH (4.472mg, 1.32 μ mol), EDPA (4.78mg, 36.96 μ mol), NMP (94 μ l) and water (44.8 μ l) shook 5 minutes in the room temperature gentleness.In the gained mixture, add HOOC-(CH
2)
6-COONSu (1.07mg, 3.96 μ mol) is dissolved in the solution that NMP (26.8 μ l) obtains, and reaction mixture was shaken 5 minutes in the room temperature gentleness, places 1 hour 50 minutes in room temperature then again.Be dissolved in the solution that makes in 50% aqueous ethanolic solution (218 μ l) with termination reaction to wherein adding glycine (2.18mg, 29.04 μ mol).Use cyanogen propyl group post (Zorbax 300SB-CN) and standard acetonitrile/TFA system, by the column chromatography purification reaction mixture.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.Isolate target compound (0.5mg, 11%), with PDMS method assay products.The m/z value of finding protonated molecular ion peak is 3540 ± 3.Thereby to draw molecular weight be 3539 ± 3amu (theoretical value is 3539amu).
Example 21
Arg
26,34Lys
38(N
ε-(ω-carboxyl oenanthyl))-GLP-1 (7-38)-OH synthetic
With Arg
26,34Lys
38The mixture of-GLP-1 (7-38)-OH (5.168mg, 1.459 μ mol), EDPA (5.28mg, 40.85 μ mol), NMP (108.6 μ l) and water (51.6 μ l) shook 10 minutes in the room temperature gentleness.In the gained mixture, add HOOC-(CH
2)
6-COONSu (1.18mg, 4.37 μ mol) is dissolved in the solution that NMP (29.5 μ l) obtains, and reaction mixture was shaken 5 minutes in the room temperature gentleness, places 1 hour 50 minutes in room temperature then again.Be dissolved in the solution that makes in 50% aqueous ethanolic solution (240 μ l) with termination reaction to wherein adding glycine (2.40mg, 32.09 μ mol).(Zorbax 300SB-CN and standard acetonitrile/TFA system are by the column chromatography purification reaction mixture to use cyanogen propyl group post.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.Isolate target compound (0.5mg, 9%), with PDMS method assay products.The m/z value of finding protonated molecular ion peak is 3697 ± 3.Thereby to draw molecular weight be 3695 ± 3amu (theoretical value is 3695amu).
Example 22
Arg
26,34Lys
36(N
ε-(ω-carboxyl oenanthyl))-GLP-1 (7-37)-OH synthetic
With Arg
26,34Lys
36The mixture of-GLP-1 (7-37)-OH (5.00mg, 1.47 μ mol), EDPA (5.32mg, 41.16 μ mol), NMP (105 μ l) and water (50 μ l) shook 5 minutes in the room temperature gentleness.In the gained mixture, add HOOC-(CH
2)
6-COONSu (1.19mg, 4.41 μ mol) is dissolved in the solution that NMP (29.8 μ l) obtains, and reaction mixture was shaken 5 minutes in the room temperature gentleness, places 2 hours in room temperature then again.Be dissolved in the solution that makes in 50% aqueous ethanolic solution (242 μ l) with termination reaction to wherein adding glycine (2.42mg, 32.34 μ mol).Use cyanogen propyl group post (Zorbax 300SB-CN) and standard acetonitrile/TFA system, by the column chromatography purification reaction mixture.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.Isolate target compound (0.78mg, 15%), with PDMS method assay products.The m/z value of finding protonated molecular ion peak is 3542 ± 3.Thereby to draw molecular weight be 3541 ± 3amu (theoretical value is 3541amu).
Example 23
Arg
26,34Lys
36(N
ε-(ω-carboxyl oenanthyl))-GLP-1 (7-36)-OH synthetic
With Arg
26,34Lys
36The mixture of-GLP-1 (7-36)-OH (5.00mg, 1.50 μ mol), EDPA (5.44mg, 42.08 μ mol), NMP (210 μ l) and water (50 μ l) shook 5 minutes in the room temperature gentleness.In the gained mixture, add HOOC-(CH
2)
6-COONSu (1.22mg, 4.5 μ mol) is dissolved in the solution that NMP (30.5 μ l) obtains, and reaction mixture was shaken 5 minutes in the room temperature gentleness, places 2 hours in room temperature then again.Be dissolved in the solution that makes in 50% aqueous ethanolic solution (247 μ l) with termination reaction to wherein adding glycine (2.47mg, 33.0 μ mol).Use cyanogen propyl group post (Zorbax 300sB-CN) and standard acetonitrile/TFA system, by the column chromatography purification reaction mixture.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.Isolate target compound (0.71mg, 14%), with PDMS method assay products.The m/z value of finding protonated molecular ion peak is 3484 ± 3.Thereby to draw molecular weight be 3483 ± 3amu (theoretical value is 3483amu).
Example 24
Arg
26,34Two (N
ε-(ω-carboxyl oenanthyl))-GLP-1 (7-37)-OH synthetic
The mixture of GLP-1 (7-37)-OH (10mg, 2.5 μ mol), EDPA (10.8mg, 83.56 μ mol), NMP (210 μ l) and water (100 μ l) was shaken 10 minutes in the room temperature gentleness.In the gained mixture, add HOOC-(CH
2)
6-COONSu (2.42mg, 8.92 μ mol) is dissolved in the solution that NMP (60.5 μ l) obtains, and reaction mixture was shaken 5 minutes in the room temperature gentleness, places 2 hours 35 minutes in room temperature then again.Be dissolved in the solution that makes in 50% aqueous ethanolic solution (492 μ l) with termination reaction to wherein adding glycine (4.92mg, 65.54 μ mol).Use cyanogen propyl group post (Zorbax 300SB-CN) and standard acetonitrile/TFA system, by the column chromatography purification reaction mixture.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.Separate targets compound (2.16mg, 24%) is with PDMS method assay products.The m/z value of finding protonated molecular ion peak is 3669 ± 3.The molecular weight that draws is 3668 ± 3amu (theoretical value is 3668amu).
Example 25
Arg
34Lys
26(N
ε-(ω-carboxyl pentadecanoyl))-GLP-1 (7-37)-OH synthetic
With Arg
34The mixture of-GLP-1 (7-37)-OH (4.472mg, 1.321 μ mol), EDPA (4.78mg, 36.99 μ mol), NMP (93.9 μ l) and water (44.7 μ l) shook 10 minutes in the room temperature gentleness.In the gained mixture, add HOOC-(CH
2)
14-COONSu (1.519mg, 3.963 μ mol) is dissolved in the solution that NMP (38 μ l) obtains, and reaction mixture was shaken 5 minutes in the room temperature gentleness, places 1 hour in room temperature then again.Be dissolved in the solution that makes in 50% aqueous ethanolic solution (218 μ l) with termination reaction to wherein adding glycine (2.18mg, 29.06 μ mol).(Zorbax300SB-CN and standard acetonitrile/TFA system are by the column chromatography purification reaction mixture to use cyanogen propyl group post.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.Isolate target compound (0.58mg, 12%), with PDMS method assay products.The m/z value of finding protonated molecular ion peak is 3654 ± 3.Thereby to draw molecular weight be 3653 ± 3amu (theoretical value is 3653amu).
Example 26
Arg
26,34Lys
36(N
ε-(ω-carboxyl oenanthyl))-GLP-1 (7-36)-OH synthetic
With Arg
26,34Lys
36The mixture of-GLP-1 (7-36)-OH (5.00mg, 1.50 μ mol), EDPA (5.44mg, 42.08 μ mol), NMP (210 μ l) and water (50 μ l) shook 5 minutes in the room temperature gentleness.In the gained mixture, add HOOC-(CH
2)
14-COONSu (1.72mg, 4.5 μ mol) is dissolved in the solution that NMP (43 μ l) obtains, and reaction mixture was shaken 5 minutes in the room temperature gentleness, places 1 hour in room temperature then again.Be dissolved in the solution that makes in 50% aqueous ethanolic solution (248 μ l) with termination reaction to wherein adding glycine (2.48mg, 33 μ mol).Use cyanogen propyl group post (Zorbax 300SB-CN) and standard acetonitrile/TFA system, by the column chromatography purification reaction mixture.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.Isolate target compound (0.58mg, 11%), with PDMS method assay products.The m/z value of finding protonated molecular ion peak is 3596 ± 3.Thereby to draw molecular weight be 3595 ± 3amu (theoretical value is 3595amu).
Example 27
Lithocholic acid 2,5-dioxo tetramethyleneimine-1-base ester synthetic
To containing lithocholic acid (5.44g, 14.34mmol), N-hydroxy-succinamide (1.78g, 15.0mmol), anhydrous THF (120ml) and anhydrous acetonitrile (30ml) and remain in 10 ℃ the mixture and add N, (3.44g 16.67mmol) is dissolved in the solution that anhydrous THF makes to N '-dicyclohexylcarbodiimide.Reaction mixture stirred 16 hours at ambient temperature, filtered and vacuum concentration.Resistates is dissolved in the methylene dichloride (450ml), with 10% aqueous sodium carbonate (2 * 150ml) and water (2 * 150ml) washings, and dry (sal epsom).Filter, the filtrate vacuum concentration is obtained the crystalline state resistates.Recrystallization resistates from the mixture of methylene dichloride (30ml) and normal heptane (30ml) obtains target compound (3.46g, 51%) with crystalline solid forms.
Example 28
Arg
34Lys
26(N
ε-lithocholic acid base)-GLP-1 (7-37)-OH synthetic
With Arg
34The mixture of-GLP-1 (7-37)-OH (4.472mg, 1.32 μ mol), EDPA (4.78mg, 36.96 μ mol), NMP (94 μ l) and water (44.8 μ l) shook 10 minutes in the room temperature gentleness.Add in the gained mixture by lithocholic acid 2,5-dioxo tetramethyleneimine-1-base ester (1.87mg, 3.96 μ mol) is dissolved in the solution that NMP (46.8 μ l) obtains, and reaction mixture was shaken 5 minutes in the room temperature gentleness, places 1 hour in room temperature then again.Add glycine (2.18mg, 29.04 μ mol) and be dissolved in the solution that makes in 50% aqueous ethanolic solution (218 μ l) with termination reaction.Use cyanogen propyl group post (Zorbax 300SB-CN) and standard acetonitrile/TFA system, by the column chromatography purification reaction mixture.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is that 0-100%. isolates target compound (1.25mg, 25%) in 60 minutes, with PDMS method assay products.The m/z value of finding protonated molecular ion peak is 3744 ± 3.Thereby to draw molecular weight be 3743 ± 3amu (theoretical value is 3743amu).
Example 29
N
α-myristoyl-Glu (ONSu)-OBu
tSynthetic
To H-Glu (OH)-OBu
t(2.5g, 12.3mmol), (1.58g, (4.0g 12.3mmol) is dissolved in the solution that DMF (59ml) makes to drip Myr-ONSu in suspension 12.3mmol) for DMF (283ml) and EDPA.Stirring at room 16 hours, vacuum concentration to cumulative volume was 20ml then with reaction mixture.Resistates is distributed between 5% aqueous citric acid solution (250ml) and ethyl acetate (150ml), separate two-phase.Behind the organic phase vacuum concentration, resistates is dissolved among the DMF (40ml).The gained drips of solution is added in 10% aqueous citric acid solution (300ml) that remains on 0 ℃.The collecting precipitation compound, and with the frozen water washing, dry in vacuum drying oven.Dried compound is dissolved in DMF (23ml), and adding HONSu (1.5g, 13mmol).Add N in the gained mixture, (2.44g 11.9mmol) is dissolved in the solution that methylene dichloride (47ml) makes to N '-dicyclohexylcarbodiimide.Reaction mixture stirring at room 16 hours, will be precipitated compound then and filter.To precipitate that recrystallization obtains target compound (3.03g, 50%) from normal heptane/2-propyl alcohol.
Example 30
Glu
22,23,30Arg
26,34Lys
38(N
ε-(γ-Gu Anxianji (N
α-myristoyl)))-GLP-1 (7-38)-OH synthetic
With Glu
22,23,30Arg
26,34Lys
38The mixture of-GLP-1 (7-38)-OH (1.0mg, 0.272 μ mol), EDPA (0.98mg, 7.62 μ mol), NMP (70 μ l) and water (70 μ l) shook 5 minutes in the room temperature gentleness.The N that will make by example 29 methods
α-myristoyl-Glu (ONSu)-OBu
t(0.41mg, 0.816 μ mol) is dissolved in NMP (10.4 μ l) gained solution, adds in the gained mixture, and reaction mixture was shaken 5 minutes in the room temperature gentleness, places 45 minutes in room temperature then again.Be dissolved in the solution that makes in 50% aqueous ethanolic solution (45 μ l) with termination reaction to wherein adding glycine (0.448mg, 5.98 μ mol).Add 0.5% aqueous solution (0.9ml) of ammonium acetate, the gained mixture is flushed to Varian 500mg C8Mega Bond Elut
On the cylinder, clean fixed compound with 5% acetonitrile solution (10ml), final by it being discharged from post with TFA (10ml) wash-out.With the eluate vacuum concentration, use cyanogen propyl group post (Zorbax 300SB-CN) and standard acetonitrile/TFA system, by the column chromatography purification reaction mixture.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.Separate targets compound (0.35mg, 32%) is with PDMS method assay products.The m/z value of finding protonated molecular ion peak is 4012 ± 3.Thereby to draw molecular weight be 4011 ± 3amu (theoretical value is 4011amu).
Example 31
Glu
23,26Arg
34Lys
38(N
ε-(γ-Gu Anxianji (N
α-myristoyl)))-GLP-1 (7-38)-OH synthetic
With Glu
23,26Arg
34Lys
38The mixture of-GLP-1 (7-38)-OH (6.07mg, 1.727 μ mol), EDPA (6.25mg, 48.36 μ mol), NMP (425 μ l) and water (425 μ l) shook 5 minutes in the room temperature gentleness.The N that will make by example 29 methods
α-myristoyl-Glu (ONSu)-OBu
t(2.65mg, 5.18 μ mol) are dissolved in NMP (66.3 μ l) gained solution, add in the gained mixture, and reaction mixture was shaken 5 minutes in the room temperature gentleness, place 45 minutes in room temperature then again.Be dissolved in the solution that makes in 50% aqueous ethanolic solution (285 μ l) with termination reaction to wherein adding glycine (2.85mg, 38.0 μ mol).Add 0.5% aqueous solution (5.4ml) of ammonium acetate, the gained mixture is flushed to Varian 500mg C8 Mega Bond Elut
On the cylinder, clean fixed compound with 5% acetonitrile solution (10ml), final by it being discharged from post with TFA (10ml) wash-out.With the eluate vacuum concentration, use cyanogen propyl group post (Zorbax 300SB-CN) and standard acetonitrile/TFA system, by the column chromatography purification reaction mixture.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.Isolate target compound (0.78mg, 12%), with PDMS method assay products.The m/z value of finding protonated molecular ion peak is 3854 ± 3.Thereby to draw molecular weight be 3853 ± 3amu (theoretical value is 3853amu).
Example 32
Lys
26,34-two (N
ε-(ω-carboxyl tridecanoyl))-GLP-1 (7-37)-OH synthetic
The mixture of GLP-1 (7-37)-OH (30mg, 8.9 μ mol), EDPA (32.3mg, 250 μ mol), NMP (2.1ml) and water (2.1ml) was shaken 5 minutes in the room temperature gentleness.With HOOC-(CH
2)
12-COONSu (12.7mg, 35.8 μ mol) is dissolved in NMP (318 μ l) gained solution, joins in the gained mixture, and reaction mixture was shaken 1 hour 40 minutes in the room temperature gentleness.Be dissolved in the solution that makes in 50% aqueous ethanolic solution (335 μ l) with termination reaction to wherein adding glycine (3.4mg, 44.7 μ mol)., use cyanogen propyl group post (Zorbax 300SB-CN) and standard acetonitrile/TFA system by the column chromatography purification reaction mixture.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.Isolate target compound (10mg, 29%), with PDMS method assay products.The m/z value of finding protonated molecular ion peak is 3840 ± 3.Thereby to draw molecular weight be 3839 ± 3amu (theoretical value is 3839amu).
Example 33
Lys
26,34-two (N
ε-(γ-Gu Anxianji (N
α-myristoyl)))-synthetic (the NNC 90-1167) of GLP-1 (7-37)-OH
With GLP-1 (7-37)-OH (300mg, 79.8 μ mol), EDPA (288.9mg, 2.24mmol), the mixture of NMP (21ml) and water (21ml) shook 5 minutes in the room temperature gentleness.The N that will make by example 29 methods
α-myristoyl-Glu (ONSu)-OBu
t(163mg, 319.3 μ mol) are dissolved in NMP (4.08ml) gained solution, join in the gained mixture, and reaction mixture was shaken 5 minutes in the room temperature gentleness, place 1 hour in room temperature then again.(131.8mg 1.76mmol) is dissolved in the solution that makes in 50% aqueous ethanolic solution (13.2ml) with termination reaction to wherein adding glycine.Add 0.5% aqueous solution (250ml) of ammonium acetate, the gained mixture is divided into 4 equal portions.Every duplicate samples is flushed to Varian 500mg C8 MegaBond Elut
On the cylinder, clean fixed compound, finally use 70% acetonitrile solution (4ml) wash-out and it is discharged from post with the 0.1%TFA aqueous solution (3.5ml).With the 0.1%TFA aqueous solution (300ml) eluate that merges is diluted.The centrifugal collecting precipitation compound, with the 0.1%TFA aqueous solution (50ml) washing, final centrifugation precipitation compound.In precipitation, add TFA (60ml), with the gained reaction mixture stirring at room 1 hour 30 minutes.Vacuum is removed unnecessary TFA, in resistates impouring water (50ml).Use cyanogen propyl group post (Zorbax300SB-CN) and standard acetonitrile/TFA system, by column chromatography purification precipitation compound.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.Isolate target compound (27.3mg, 8%), with PDMS method assay products.The m/z value of finding protonated molecular ion peak is 4036 ± 3.Thereby to draw molecular weight be 4035 ± 3amu (theoretical value is 4035amu).
Example 34
Arg
26,34Lys
38(N
ε-(ω-carboxyl pentadecanoyl))-GLP-1 (7-38)-OH synthetic
With Arg
26,34Lys
38The mixture of-GLP-1 (7-38)-OH (30mg, 8.9 μ mol), EDPA (32.3mg, 250 μ mol), NMP (2.1ml) and water (2.1ml) shook 5 minutes in the room temperature gentleness.With HOOC-(CH
2)
14-COONSu (13.7mg, 35.8 μ mol) is dissolved in NMP (343 μ l) gained solution, joins in the gained mixture, and reaction mixture was shaken 1 hour in the room temperature gentleness.Be dissolved in the solution that makes in 50% aqueous ethanolic solution (335 μ l) with termination reaction to wherein adding glycine (3.4mg, 44.7 μ mol).Use cyanogen propyl group post (Zorbax 300SB-CN) and standard acetonitrile/TFA system, by the column chromatography purification reaction mixture.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.Isolate target compound (4.8mg, 14%), with PDMS method assay products.The m/z value of finding protonated molecular ion peak is 3894 ± 3.Thereby to draw molecular weight be 3893 ± 3amu (theoretical value is 3893amu).
Example 35
N
α-palmitoyl-Glu (ONSu)-OBu
tSynthetic
To H-Glu (OH)-OBu
t(4.2g, 20.6mmol), (2.65g, (7.3g 20.6mmol) is dissolved in the solution that DMF (100ml) makes to drip Pal-ONSu in suspension 20.6mmol) for DMF (500ml) and EDPA.Stirring at room 64 hours, vacuum concentration to cumulative volume was 20ml then with reaction mixture.Resistates is distributed between 10% aqueous citric acid solution (300ml) and ethyl acetate (250ml), separate two-phase.Behind the organic phase vacuum concentration, resistates is dissolved in DMF (50ml).The gained drips of solution is added in 10% aqueous citric acid solution (500ml) that remains on 0 ℃.The collecting precipitation compound, and with the frozen water washing, dry in vacuum drying oven.Dried compound is dissolved in DMF (45ml), and adding HONSu (2.15g, 18.7mmol).Add N in the gained mixture, (3.5g 17mmol) is dissolved in the solution that methylene dichloride (67ml) makes to N '-dicyclohexylcarbodiimide.Reaction mixture stirring at room 16 hours, will be precipitated compound then and filter.To precipitate that recrystallization obtains target compound (6.6g, 72%) from normal heptane/2-propyl alcohol.
Example 36
Lys
26,34-two (N
ε-(γ-Gu Anxianji (N
α-palmitoyl)))-GLP-1 (7-37)-OH synthetic
The mixture of GLP-1 (7-37)-OH (10mg, 2.9 μ mol), EDPA (10.8mg, 83.4 μ mol), NMP (0.7ml) and water (0.7ml) was shaken 5 minutes in the room temperature gentleness.The N that will make by example 33 methods
α-palmitoyl-Glu (ONSu)-OBu
t(163mg, 319.3 μ mol) are dissolved in NMP (4.08ml) gained solution, in wherein joining the gained mixture, reaction mixture are shaken 1 hour 20 minutes in the room temperature gentleness.Add glycine (4.9mg, 65.6 μ mol) and be dissolved in the solution that makes in 50% aqueous ethanolic solution (492 μ l) with termination reaction.Add 0.5% aqueous solution (9ml) of ammonium acetate, the gained mixture is flushed to a Varian 1g C8Mega Bond Elut
On the cylinder, clean fixed compound, finally use TFA (10ml) wash-out and it is discharged from post with 5% acetonitrile solution (10ml).The vacuum concentration eluate uses cyanogen propyl group post (Zorbax 300SB-CN) and standard acetonitrile/TFA system, by the column chromatography purification resistates.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.Isolate target compound (2.4mg, 20%), with PDMS method assay products.The m/z value of finding protonated molecular ion peak is 4092 ± 3.Thereby to draw molecular weight be 4091 ± 3amu (theoretical value is 4091amu).
Example 37
Arg
34Lys
26(N
ε-(γ-Gu Anxianji (N
α-palmitoyl)))-GLP-1 (7-37)-OH synthetic
With Arg
34The mixture of-GLP-1 (7-37)-OH (3.7mg, 1.1 μ mol), EDPA (4.0mg, 30.8 μ mol), acetonitrile (260 μ l) and water (260 μ l) shook 5 minutes in the room temperature gentleness.The N that will make by example 35 methods
α-palmitoyl-Glu (ONSu)-OBu
t(1.8mg, 3.3 μ mol) are dissolved in acetonitrile (44.2 μ l) gained solution, join in the gained mixture, and reaction mixture was shaken 1 hour 20 minutes in the room temperature gentleness.Be dissolved in the solution that makes in 50% aqueous ethanolic solution (181 μ l) with termination reaction to wherein adding glycine (1.8mg, 24.2 μ mol).Add 0.5% aqueous solution (12ml) and the NMP (300 μ L) of ammonium acetate, the gained mixture is flushed to Varian 1g C8Mega Bond Elut
On the membrane cartridge, clean fixed compound, finally use TFA (6ml) wash-out and it is discharged from post with 5% acetonitrile solution (10ml).Eluate was placed 2 hours in room temperature, then vacuum concentration.Use cyanogen propyl group post (Zorbax 300SB-CN) and standard acetonitrile/TFA system, by using the column chromatography purification resistates.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.Isolate target compound (0.23mg, 6%), with PDMS method assay products.The m/z value of finding protonated molecular ion peak is 3752 ± 3.Thereby to draw molecular weight be 3751 ± 3amu (theoretical value is 3751amu).
Example 38
Arg
26,34Lys
38(N
ε-(γ-Gu Anxianji (N
α-myristoyl)))-GLP-1 (7-38)-OH synthetic
With Arg
26,34Lys
38The mixture of-GLP-1 (7-38)-OH (14mg, 4.0 μ mol), EDPA (14.3mg, 110.6 μ mol), NMP (980 μ l) and water (980 μ l) shook 5 minutes in the room temperature gentleness.The N that will make by example 29 methods
α-myristoyl-Glu (ONSu)-OBu
t(12.1mg, 23.7 μ mol) are dissolved in NMP (303 μ l) gained solution, join in the gained mixture, and reaction mixture was shaken 2 hours in the room temperature gentleness.(6.5mg 86.9mmol) is dissolved in the solution that makes in 50% aqueous ethanolic solution (652 μ l) with termination reaction to wherein adding glycine.Add 0.5% aqueous solution (50ml) of ammonium acetate, the gained mixture is flushed to Varian 1gC8 Mega Bond Elut
On the cylinder, clean fixed compound, finally use the TFA aqueous solution (6ml) wash-out and it is discharged from post with 5% acetonitrile solution (15ml).Eluate was placed 1 hour 45 minutes in room temperature, then vacuum concentration.Use cyanogen propyl group post (Zorbax300SB-CN) and standard acetonitrile/TFA system, by the column chromatography purification resistates.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.Isolate target compound (3.9mg, 26%), with PDMS method assay products.The m/z value of finding protonated molecular ion peak is 3881 ± 3.Thereby to draw molecular weight be 3880 ± 3amu (theoretical value is 3880amu).
Example 39
Arg
26,34Lys
38(N
ε-(ω-carboxyl pentadecanoyl))-GLP-1 (7-38)-OH synthetic
With Arg
26,34Lys
38The mixture of-GLP-1 (7-38)-OH (14mg, 4.0 μ mol), EDPA (14.3mg, 111 μ mol), NMP (980 μ l) and water (980 μ l) shook 5 minutes in the room temperature gentleness.With HOOC-(CH
2)
14-COONSu (4.5mg, 11.9 μ mol) is dissolved in NMP (114 μ l) gained solution, joins in the gained mixture, and reaction mixture was shaken 1 hour 45 minutes in the room temperature gentleness.Other adds HOOC-(CH
2)
14-COONSu (4.0mg, 10.4 μ mol) is dissolved in NMP (100 μ l) gained solution, with the gained mixture room temperature again gentleness shook 1 hour 30 minutes.Be dissolved in the solution that makes in 50% aqueous ethanolic solution (148 μ l) with termination reaction to wherein adding glycine (1.5mg, 19.8 μ mol).Use cyanogen propyl group post (Zorbax 300SB-CN) and standard acetonitrile/TFA system, by using the column chromatography purification reaction mixture.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.Isolate target compound (3.9mg, 26%), with PDMS method assay products.The m/z value of finding protonated molecular ion peak is 3809 ± 3.Thereby to draw molecular weight be 3808 ± 3amu (theoretical value is 3808amu).
Example 40
Arg
26,34Lys
38(N
ε-(γ-Gu Anxianji (N
α-palmitoyl)))-GLP-1 (7-38)-OH synthetic
With Arg
26,34Lys
38The mixture of-GLP-1 (7-38)-OH (14mg, 4.0 μ mol), EDPA (14.3mg, 110.6 μ mol), NMP (980 μ l) and water (980 μ l) shook 5 minutes in the room temperature gentleness. the N that will make by example 35 methods
α-palmitoyl-Glu (ONSu)-OBu
t(6.4mg, 11.9 μ mol) are dissolved in NMP (160 μ l) gained solution, join in the gained mixture, and reaction mixture was shaken 1 hour 20 minutes in the room temperature gentleness.(6.5mg 87mmol) is dissolved in the solution that makes in 50% aqueous ethanolic solution (653 μ l) with termination reaction to wherein adding glycine.Add 0.5% aqueous solution (50ml) of ammonium acetate, the gained mixture is flushed to Varian 1g C8 MegaBond Elut
On the cylinder, clean fixed compound, finally use TFA (6ml) wash-out and it is discharged from post with 5% acetonitrile solution (10ml).Eluate was left standstill 1 hour 30 minutes in room temperature, then vacuum concentration.Use cyanogen propyl group post (Zorbax 300SB-CN) and standard acetonitrile/TFA system, by the column chromatography purification resistates.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.Isolate target compound (7.2mg, 47%), with PDMS method assay products.The m/z value of finding protonated molecular ion peak is 3881 ± 3.Thereby to draw molecular weight be 3880 ± 3amu (theoretical value is 3880amu).
Example 41
Arg
18,23,26,30,34Lys
38(N
ε-palmitoyl)-GLP-1 (7-38)-OH synthetic
With Arg
18,23,26,30,34Lys
38The mixture of-GLP-1 (7-38)-OH (1.0mg, 0.27 μ mol), EDPA (0.34mg, 2.7 μ mol) and DMSO (600 μ l) shook 5 minutes in the room temperature gentleness.In the gained mixture, add Pal-ONSu (0.28mg, 0.8 μ mol) and be dissolved in the solution that NMP (7 μ l) makes.Reaction mixture was shaken 5 minutes in the room temperature gentleness, placed 6 hours in room temperature then.Be dissolved in the solution that makes in 50% aqueous ethanolic solution (163 μ l) with termination reaction to wherein adding glycine (1.6mg, 21.7 μ mol).Use cyanogen propyl group post (Zorbax 300SB-CN) and standard acetonitrile/TFA system, by the column chromatography purification reaction mixture.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.Isolate target compound (0.17mg, 16%), with MALDI-MS method assay products.The m/z value of finding protonated molecular ion peak is 3961 ± 3.Thereby the molecular weight that draws is 3960 ± 3amu (theoretical value is 3960amu).
Example 42
Arg
26,34Lys
38(N
ε-(ω-carboxyl tridecanoyl))-GLP-1 (7-38)-OH synthetic
With Arg
26,34Lys
38The mixture of-GLP-1 (7-38)-OH (14mg, 4.0 μ mol), EDPA (14.3mg, 111 μ mol), NMP (980 μ l) and water (980 μ l) shook 5 minutes in the room temperature gentleness.With HOOC-(CH
2)
12-COONSu (4.2mg, 11.9 μ mol) is dissolved in NMP (105 μ l) gained solution, joins in the gained mixture, and reaction mixture was shaken 1 hour 50 minutes in the room temperature gentleness.Be dissolved in the solution that makes in 50% aqueous ethanolic solution (652 μ l) with termination reaction to wherein adding glycine (6.5mg, 87 μ mol).Use cyanogen propyl group post (Zorbax 300SB-CN) and standard acetonitrile/TFA system, by the column chromatography purification reaction mixture.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.Isolate target compound (5.8mg, 39%), with MALDI-MS method assay products.The m/z value of finding protonated molecular ion peak is 3780 ± 3.Thereby to draw molecular weight be 3779 ± 3amu (theoretical value is 3781amu).
Example 43
Arg
34Lys
26(N
ε-(γ-Gu Anxianji (N
α-myristoyl)))-GLP-1 (7-37)-OH synthetic
With Arg
34The mixture of-GLP-1 (7-37)-OH (15mg, 4.4 μ mol), EDPA (16mg, 124 μ mol), NMP (2ml) and water (4.8ml) shook 5 minutes in the room temperature gentleness.The N that will make by example 29 methods
α-myristoyl-Glu (ONSu)-OBu
t(12.1mg, 23.7 μ mol) are dissolved in NMP (303 μ l) gained solution, join in the gained mixture, and reaction mixture was shaken 2 hours in the room temperature gentleness.Be dissolved in the solution that makes in 50% aqueous ethanolic solution (652 μ l) with termination reaction to wherein adding glycine (6.5mg, 86.9 μ mol).Add 0.5% aqueous solution (50ml) of ammonium acetate, the gained mixture is flushed to Varian 1g C8 Mega Bond Elut
On the cylinder, clean fixed compound, finally use TFA (6ml) wash-out and it is discharged from post with 5% acetonitrile solution (15ml).Eluate was placed 1 hour 45 minutes in room temperature, then vacuum concentration.Use cyanogen propyl group (cyanopropyl) post (Zorbax 300SB-CN) and standard acetonitrile/TFA system, by the column chromatography purification resistates.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.Isolate target compound (3.9mg, 26%), with MALDI-MS method assay products.The m/z value of finding protonated molecular ion peak is 3723 ± 3.Thereby to draw molecular weight be 3722 ± 3amu (theoretical value is 3723amu).
Example 44
N
α-stearoyl-Glu (ONSu)-OBu
tSynthetic
To H-Glu (OH)-OBu
t(2.82g, 13.9mmol), (1.79g, (5.3g 13.9mmol) is dissolved in the solution that DMF (60ml) obtains to drip Ste-ONSu in suspension 13.9mmol) for DMF (370ml) and EDPA.Add methylene dichloride (35ml), with reaction mixture at stirring at room 24 hours, vacuum concentration then.Resistates is distributed between 10% aqueous citric acid solution (330ml) and ethyl acetate (200ml), separate two-phase.With the organic phase vacuum concentration, make resistates be dissolved in DMF (60ml).The gained drips of solution is added in 10% aqueous citric acid solution (400ml) that remains on 0 ℃.The collecting precipitation thing, and with the frozen water washing, at the vacuum drying oven inner drying.Dried compound is dissolved in DMF (40ml), and adding HONSu (1.63g, 14.2mmol).(2.66g 12.9mmol) is dissolved in the solution that methylene dichloride (51ml) forms to add DCC in reaction mixture.The gained mixture stirring at room 64 hours, is filtered and obtains precipitating compound.To be deposited in that recrystallization obtains target compound (4.96g, 68%) in normal heptane/2-propyl alcohol.
Example 45
Arg
26,34Lys
38(N
ε-(γ-Gu Anxianji (N
α-stearoyl)))-GLP-1 (7-38)-OH synthetic
With Arg
26,34The mixture of-GLP-1 (7-38)-OH (28mg, 7.9 μ mol), EDPA (28.6mg, 221.5 μ mol), NMP (1.96ml) and water (1.96ml) shook 5 minutes in the room temperature gentleness.The N that will make by example 44 methods
α-stearoyl-Glu (ONSu)-OBu
t(17.93g, 31.6 μ mol) are dissolved in NMP (448 μ l) gained solution, join in the gained mixture, and reaction mixture was shaken 2 hours in the room temperature gentleness.Be dissolved in the solution that makes in 50% aqueous ethanolic solution (1.3ml) with termination reaction to wherein adding glycine (13.1mg, 174 μ mol).Add 0.5% aqueous solution (120ml) of ammonium acetate, the gained mixture is divided into 2 equal portions.Be flushed to Varian5g C8 Mega Bond Elut with every part
On the cylinder, clean fixed compound, finally use TFA (25ml) wash-out and it is discharged from post with 5% acetonitrile solution (25ml).The eluate that merges was placed 1 hour 25 minutes in room temperature, then vacuum concentration.Use cyanogen propyl group post (Zorbax300SB-CN) and standard acetonitrile/TFA system, by the column chromatography purification resistates.Post is heated to 65 ℃, and the concentration gradient of acetonitrile is 0-100% in 60 minutes.Isolate target compound (3.6mg, 11%), with MALDI-MS method assay products.The m/z value of finding protonated molecular ion peak is 3940 ± 3.Thereby to draw molecular weight be 3939 ± 3amu (theoretical value is 3937amu).
Biology is observed
Long duration of action behind the GLP-1 derivative subcutaneous administration
Adopt following method, the concentration behind the monitoring health pig subcutaneous administration GLP-1 derivative in its blood plasma, thus measure the long duration of action of GLP-1 derivatives more of the present invention.In order to compare, also its concentration in blood plasma of subcutaneous administration GLP-1 (7-37) back is measured.The results are shown in table 1.Can according to said method measure the long duration of action of other GLP-1 derivative of the present invention.
Even from the fasting of experiment beginning pig (50%Duroc, 25%Yorkshire, 25%DanishLandrace, heavily about 40kg).Every pig gives to be dissolved in 50 μ M isotonic solution (5mM phosphoric acid salt, pH7.4,0.02% tweens by per kilogram of body weight
-20 (Merck), 45mg/ml mannitol (removing pyrogen, Novo Nordisk)) 0.5nmol test compounds.Take a blood sample from the jugular vein conduit in the time shown in the table 1.The 5ml blood sample is poured in the ice-cold glass cylinder that contains the following solution of 175 μ l: 0.18M EDTA, 1500KIE/ml presses down enzyme peptide (Novo Nordisk) and 3% bacitracin (Sigma), pH7.4.In 30 minutes, with sample at 5-6000
*Centrifugal 10 minutes of g.Temperature is remained on 4 ℃.With suction pipe supernatant liquor is drawn in the different glass cylinderes, stores in one 20 ℃ until use.
Utilization has specific monoclonal antibody to the N-stub area of GLP-1 (7-37), measures the plasma concentration of all peptides by the RIA method.With the cross reaction of GLP-1 (1-37) and GLP-1 (8-36) acid amides less than 1%, with the cross reaction of GLP-1 (9-37), GLP-1 (10-36) acid amides and GLP-1 (11-36) acid amides less than 0.1%.Carry out total overall reaction at 4 ℃.
Following mensuration: 100 μ l blood plasma are mixed with 271 μ l96% ethanol, with the turbine mixer mixing and 2600
*Centrifugal 30 minutes of g.Supernatant liquor is poured in the Minisorp test tube gently, and evaporation (Savant Speedvac AS290) fully.Evaporation residue is dissolved in again contains 80mM SODIUM PHOSPHATE, MONOBASIC/Sodium phosphate dibasic, 0.1%HSA (Orpha 20/21, and Behring), 10mM EDTA, 0.6mM thiomersal(ate) (Sigma) is in the mensuration damping fluid of pH7.5.Sample is dissolved in is suitable for reaching in the volume of its predicted concentration, and make its dissolving 30 minutes.In 300 μ l samples, add 10 μ l by containing 40mM SODIUM PHOSPHATE, MONOBASIC/Sodium phosphate dibasic, 0.1%HSA, the antibody-solutions that the dilution buffer liquid of 0.6mM thiomersal(ate), pH7.5 is made.300 μ l damping fluids and 100 μ l dilution buffer liquid are mixed and made into non-specific sample.Stock thing from lyophilize and make each standard, it is dissolved in 300 μ l measures in the damping fluid.All samples all in the Minisorp pipe with aforesaid antibody pre-incubation 72 hours.To wherein adding the tracer agent that the contains 6-7000CPM 200 μ l be dissolved in the dilution buffer liquid, with sample mix and be incubated 48 hours.In every pipe, be added in 40mM SODIUM PHOSPHATE, MONOBASIC/Sodium phosphate dibasic, 0.6 mM thiomersal(ate), the suspension 1.5ml of Ox blood plasma 200ml/l that the heparin among the pH7.5 is stable and 18g/l activated carbon (Merck).Before the use, suspension is mixed and it was placed 2 hours at 4 ℃.All samples is incubated 1 hour at 4 ℃, then 3400
*Centrifugal 25 minutes of g.Take out supernatant liquor after centrifugal immediately gently and in one γ-counter inside counting.Concentration from each standard substance curve calculation sample.Record following plasma concentration, they are the percentage ratio (n=2) of the peak concentration of each compound:
Table 1
| Test compounds) | Time behind the subcutaneous administration (hour) | ||||||||
| 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 to provide each routine target compound of routine number with compound
As shown in table 1, GLP-1 derivative of the present invention has the effect curves than GLP-1 (7-37) longer time, and is lasting far than GLP-1 (7-37) in blood plasma.Table 1 also shows, selected specific GLP-1 derivative difference, and the time variation range that they reach peak concentration in the blood plasma is very big.
The hormesis that cAMP in the clone of the people GLP-1 acceptor of cloning by expression is formed
Be to confirm the usefulness of GLP-1 derivative, measured the ability that they form at the clone internal stimulus cAMP of the people GLP-1 of cloning by expression acceptor.Calculate EC from dose-response curve
50
Young hamster kidney (BHK) cell of use expressing human pancreas GLP-1 acceptor (Knudsen and Pridal, 1996, European pharmacology magazine, 318,429-435).By at damping fluid (10mmol/l Tris-HCl and 30mmol/lNaCl pH7.4, also contain the 1mmol/l dithiothreitol (DTT) in addition, 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 press down that homogenate prepares plasma membrane (Adelhorst etc. in the enzyme peptide (Novo Nordisk A/S, Bagsvaerd, Denmark)), 1994, journal of biological chemistry, 269,6275).Homogenate is centrifugal on 41w/v% sucrose layer top.White ribbon between two-layer is diluted also centrifugal in damping fluid.Plasma membrane be kept at-80 ℃ standby.
In 96 hole microtiter plates, measure with cumulative volume 140 μ l.Used damping fluid is 50mmol/l Tris-HCl, and pH7.4 also adds 1mmol/lEGTA, 1.5mmol/lMgSO
4, 1.7mmol/lATP, 20mM GTP, 2mmol/l 3-isobutyl-1-methylxanthine, 0.01% tween 20 and 0.1% human serum albumin (Reinst, BehringwerkeAG, Marburg, Germany).Desire surveyed the compound dissolution of its agonist activity and be diluted in the damping fluid, join in the plasma membrane preparation, with mixture 37 ℃ of insulations 2 hours.Add 25 μ l0.05mol/l hydrochloric acid termination reactions.With (RPA 538, and Amersham UK) analyzes cAMP with scintillation proximity assay (scintillation proximity assay) behind 10 times of the diluted samples.Obtain following result:
| Test compounds) | EC 50，pM | Test compounds) | EC 50，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 that provides routine number example with compound
Claims (24)
1. GLP-1 derivative, wherein 1 of the GLP-1 peptide or 2 amino-acid residues are connected with a lipophilic substituent, and wherein this lipophilic substituent is selected from following:
I) be selected from CH
3(CH
2)
10CO-, CH
3(CH
2)
12CO-, CH
3(CH
2)
14CO-, CH
3(CH
2)
16CO-, CH
3(CH
2)
18CO-, CH
3(CH
2)
20CO-and CH
3(CH
2)
22The straight chain of CO-or the acyl group of branched fatty acid;
Ii) has formula HOOC (CH
2)
mThe acyl group of the straight chain of CO-or branch's alkane alpha, omega-dicarboxylic acid, wherein m is 10,12,14 or 16; With
Iii) courage acyl group, 7-deoxidation courage acyl group or stone courage acyl group;
And if have only a lipophilic substituent, and this substituting group is connected on the N-end or C-terminal amino acid residue of GLP-1 peptide, this substituting group is alkyl or the group with a ω-carboxyl so,
Wherein, described GLP-1 peptide is selected from GLP-1 (7-36), GLP-1 (7-37), GLP-1 (7-38), Arg
26-GLP-1 (7-37), Arg
34-GLP-1 (7-37), Lys
36-GLP-1 (7-37), Arg
26,34Lys
36-GLP-1 (7-37), Arg
26,34Lys
36-GLP-1 (7-36), Arg
26,34Lys
38-GLP-1 (7-38), Arg
26Lys
36-GLP-1 (7-37), Arg
34Lys
36-GLP-1 (7-37), Arg
26Lys
38-GLP-1 (7-38), Arg
34Lys
38-GLP-1 (7-38), Arg
26,34Lys
36,38-GLP-1 (7-38), and Arg
26,34Lys
38-GLP-1 (7-38).
2. GLP-1 derivative according to claim 1, lipophilic substituent wherein is selected from: HOOC (CH
2)
10CO-, HOOC (CH
2)
12CO-, HOOC (CH
2)
14CO-and HOOC (CH
2)
16CO-.
3. GLP-1 derivative according to claim 1 wherein has only a lipophilic substituent.
4. GLP-1 derivative according to claim 3, lipophilic substituent wherein is connected on the-terminal amino acid residue.
5. GLP-1 derivative according to claim 3, lipophilic substituent wherein is connected on the C-terminal amino acid residue.
6. GLP-1 derivative according to claim 3, lipophilic substituent wherein are connected to neither on the amino-acid residue of the also non-C-end of N-end.
7. GLP-1 derivative according to claim 1 wherein has two lipophilic substituents.
8. GLP-1 derivative according to claim 7, one of them lipophilic substituent is connected on the-terminal amino acid residue, and another lipophilic substituent is connected on the C-terminal amino acid residue.
9. GLP-1 derivative according to claim 7, one of them lipophilic substituent is connected on the C-terminal amino acid residue, and another lipophilic substituent is connected to neither on the amino-acid residue of the also non-C-end of N-end.
10. GLP-1 derivative according to claim 7, wherein two lipophilic substituents all are connected to neither on the amino-acid residue of the also non-C-end of N-end.
11. GLP-1 derivative according to claim 1, one of them lipophilic substituent forms an amido linkage with the amino of its carboxyl and an amino-acid residue, thereby is connected on this amino-acid residue.
12. GLP-1 derivative according to claim 1, the amino carboxyl with an amino-acid residue with it of one of them lipophilic substituent forms amido linkage, thereby is connected on this amino-acid residue.
13. GLP-1 derivative according to claim 1, wherein lipophilic substituent is connected on the GLP-1 peptide by a spacer groups.
14. GLP-1 derivative according to claim 13, spacer groups wherein be 1-7 methylene radical arranged be regardless of branched paraffin α, ω-dicarboxyl, this spacer groups forms a bridge between the amino of the amino of GLP-1 peptide and lipophilic substituent.
15. GLP-1 derivative according to claim 14, α wherein, ω-dicarboxyl have 2 methylene radical.
16. GLP-1 derivative according to claim 13, spacer groups i wherein) be an amino-acid residue except that Cys, or ii) be a kind of dipeptides.
17. GLP-1 derivative according to claim 16, dipeptides wherein is Gly-Lys.
18. GLP-1 derivative according to claim 16, wherein the GLP-1 peptide a carboxyl and a Lys or contain that in the dipeptides of Lys residue one is amino to form amido linkage, and the Lys spacer groups or contain in the dipeptides spacer groups of Lys residue another amino with lipophilic substituent in carboxyl form an amido linkage.
19. GLP-1 derivative according to claim 16, wherein a carboxyl of the GLP-1 peptide amino and described amino-acid residue or dipeptides spacer groups forms an amido linkage, and a carboxyl of amino of this amino-acid residue or dipeptides spacer groups and described lipophilic substituent forms an amido linkage.
20. GLP-1 derivative according to claim 16, wherein one of the GLP-1 peptide carboxyl and described amino-acid residue spacer groups or dipeptides spacer groups aminoly forms an amido linkage, and amido linkage of an amino formation of a carboxyl of this amino-acid residue spacer groups or dipeptides spacer groups and described lipophilic substituent.
21. GLP-1 derivative according to claim 16, wherein the GLP-1 peptide carboxyl and Asp or Glu spacer groups, the amido linkage of an amino formation that perhaps contains the dipeptides spacer groups of Asp or Glu residue, and amido linkage of an amino formation of a carboxyl of this spacer groups and described lipophilic substituent.
22. a pharmaceutical composition, it contains each described GLP-1 derivative and acceptable vehicle of pharmacology or carrier among the claim 1-21.
23. each described GLP-1 derivative is used for the treatment of purposes in the medicine of obesity in preparation among the claim 1-21.
24. each described GLP-1 derivative is used for the treatment of purposes in the medicine of insulin-dependent or non-insulin-dependent diabetes mellitus (NIDDM) in preparation among the claim 1-21.
Applications Claiming Priority (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DK0931/96 | 1996-08-30 | ||
| DK93196 | 1996-08-30 | ||
| DK1259/96 | 1996-11-08 | ||
| DK125996 | 1996-11-08 | ||
| DK1470/96 | 1996-12-20 | ||
| DK147096 | 1996-12-20 |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB971984131A Division CN1271086C (en) | 1996-08-30 | 1997-08-22 | GLP-1 derivs. |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1951965A CN1951965A (en) | 2007-04-25 |
| CN1951965B true CN1951965B (en) | 2011-07-20 |
Family
ID=27635700
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2006101108983A Expired - Lifetime CN1951965B (en) | 1996-08-30 | 1997-08-22 | Glp-1 derivatives |
| CNB2005101075881A Expired - Lifetime CN100569798C (en) | 1996-08-30 | 1997-08-22 | The GLP-1 derivative |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2005101075881A Expired - Lifetime CN100569798C (en) | 1996-08-30 | 1997-08-22 | The GLP-1 derivative |
Country Status (3)
| Country | Link |
|---|---|
| CN (2) | CN1951965B (en) |
| MA (1) | MA24129A1 (en) |
| ZA (2) | ZA977791B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012016419A1 (en) * | 2010-08-06 | 2012-02-09 | 浙江贝达药业有限公司 | Glp-1 derivatives and uses thereof |
| WO2017075505A2 (en) * | 2015-10-28 | 2017-05-04 | Tufts University | Novel polypeptides with improved proteolytic stability, and methods of preparing and using same |
| CN111518009B (en) * | 2019-02-01 | 2023-06-23 | 鲁南制药集团股份有限公司 | Fatty acid derivative and synthesis method thereof |
| CN109836368B (en) * | 2019-03-14 | 2020-12-08 | 美药星（南京）制药有限公司 | Preparation method of high-purity liraglutide side chain |
| CN115322794A (en) | 2020-01-11 | 2022-11-11 | 北京质肽生物医药科技有限公司 | Conjugates of fusion proteins of GLP-1 and FGF21 |
| CN116730902B (en) * | 2023-08-07 | 2023-11-21 | 杭州湃肽生化科技有限公司 | Method for synthesizing liraglutide |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5512549A (en) * | 1994-10-18 | 1996-04-30 | Eli Lilly And Company | Glucagon-like insulinotropic peptide analogs, compositions, and methods of use |
| WO2006040128A1 (en) * | 2004-10-12 | 2006-04-20 | Zimmer Gmbh | Pva hydrogel |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB9409496D0 (en) * | 1994-05-12 | 1994-06-29 | London Health Ass | Method for improving glycaemic control in diabetes |
-
1997
- 1997-01-29 MA MA24480A patent/MA24129A1/en unknown
- 1997-08-22 CN CN2006101108983A patent/CN1951965B/en not_active Expired - Lifetime
- 1997-08-22 CN CNB2005101075881A patent/CN100569798C/en not_active Expired - Lifetime
- 1997-08-29 ZA ZA9707791A patent/ZA977791B/en unknown
- 1997-09-01 ZA ZA9707828A patent/ZA977828B/en unknown
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5512549A (en) * | 1994-10-18 | 1996-04-30 | Eli Lilly And Company | Glucagon-like insulinotropic peptide analogs, compositions, and methods of use |
| WO2006040128A1 (en) * | 2004-10-12 | 2006-04-20 | Zimmer Gmbh | Pva hydrogel |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1951965A (en) | 2007-04-25 |
| MA24129A1 (en) | 1997-10-01 |
| CN100569798C (en) | 2009-12-16 |
| CN1740198A (en) | 2006-03-01 |
| ZA977791B (en) | 1998-03-02 |
| ZA977828B (en) | 1998-03-02 |
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