CN1951965B - Glp-1 derivatives - Google Patents

Abstract

Derivatives of GLP-1 and its analogs with lipophilic substituents have interesting pharmacological properties, in particular they have a longer action profile than GLP-1 (7-37).

Description

GLP-1 derivatives

The technical field of the invention

The present invention relates to novel derivatives of human glucagon-like peptide-1 (GLP-1) and its fragments and analogs of these fragments, which have a protracted profile of action, and also to the Methods of preparation and use.

Technical Background of the Invention

Peptides are widely used in medical practice and their importance can be expected to increase in the coming years due to the availability of recombinant DNA techniques to prepare peptides. When natural peptides or their analogs are used in therapy, they are generally found to have high clearance. In cases where it is desired to maintain high blood levels of the therapeutic agent over a longer period of time, a high clearance of the therapeutic agent is undesirable as this would require repeated dosing. Peptides with high clearance include: ACTH, corticotropin-releasing factor, angiotensin, calcitonin, insulin, glucagon, glucagon-like peptide-1, glucagon-like peptide-2 , insulin-like growth factor-1, insulin-like growth factor-2, enterogastroinhibitory peptide, growth hormone releasing factor, pituitary adenylate cyclase-activating peptide, secretin, enterogastrin, growth-stimulating hormone Inhibin, somatomodulin, somatotropin, parathyroid hormone, thrombopoietin, erythropoietin, hypothalamic releasing factor, prolactin, thyrotropin, endorphin, enkephalin, vasopressin, Oxytocin, opioids and their analogs, superoxide dismutase, interferon, asparaginase, arginase, arginine deaminase, adenosine deaminase, and ribonuclease. In some cases, suitable pharmaceutical compositions can be used to influence the release profile of the peptide, but this approach suffers from various disadvantages and is generally not applicable.

The hormones that regulate insulin secretion belong to the so-called entero-islet axis, which refers to a group of hormones released from the gastrointestinal mucosa in the presence and absorption of enteral nutrients, which promote the early release of insulin and make it stronger. The action of promoting insulin secretion, the so-called incretin action, is probably necessary for normal glucose tolerance. Many gastrointestinal hormones, including gastrin and secretin, are insulinotropic (human cholecystokinin is not), but only the glucose-dependent incretins are physiologically important. Insulin polypeptide GIP and glucagon-like peptide-1 (GLP-1). Due to its insulinotropic action, GIP attracted considerable interest among diabetologists immediately after its isolation in 1973 (1). However, a large number of studies carried out in the ensuing years clearly showed that defects in GIP secretion were not related to the pathogenesis of insulin-dependent diabetes mellitus (IDDM) or non-insulin-dependent diabetes mellitus (NIDDM) (2). It was further found that GIP, although an insulinotropic hormone, has little effect on NIDDM (2). Another incretin hormone, GLP-1, is the most potent insulinotropic substance known (3). Unlike GIP, GLP-1 is very effective in promoting insulin secretion in NIDDM patients. In addition, GLP-1 also potently inhibits glucagon secretion compared to other insulinotropic hormones (except perhaps incretin). Due to these effects, it has a remarkable hypoglycemic effect especially for NIDDM patients.

GLP-1 is a product of proglucagon (4), one of the newest members of the secretin-VIP family of peptides, but it has been and metabolism of important gastrointestinal hormones with regulatory functions (5). The glucagon gene is processed differently in the pancreas and gut. In the pancreas (9), processing leads to the formation and parallel secretion of: 1) glucagon itself, occupying positions 33-61 of proglucagon (PG); 2) a 30-amino acid (PG( 1-30)), commonly referred to as glucagon-like peptide-related pancreatic peptide, GRPP (10,11); 3) a hexapeptide corresponding to PG (64-69); 4 ) and finally the so-called major proglucagon fragment (PG(72-158)), which harbors two glucagon-like sequences (9). Glucagon appears to be the only biologically active product. In contrast, within the intestinal mucosa, glucagon is buried within a single macromolecule, whereas the two glucagon-like peptides are formed separately (8). The following products are formed and secreted in parallel: 1) Glucagon-like peptide corresponding to PG(1-69), where the glucagon sequence occupies residues No.33-61 (12); 2) GLP-1 (7-36)amide, PG(78-107)amide (13), and not PG(72-107)amide or PG(72-108)amide as originally thought, was inactive. GLP-1(7-37)(PG(78-108))(14) with equivalent biological activity was also generated with a small amount of C-terminal glycine extension; 3) intermediate peptide-2(PG(111-122)amide)( 15); and 4) GLP-2 (PG(126-158)) (15, 16). A portion of the glucagon-like peptide is further cleaved into GRPP (PG(1-30)) and oxyntomodulin (PG(33-69)) (17, 18). Among these peptides, GLP-1 has the most significant biological activity.

Since GLP-1 is secreted in parallel with glucagon-like peptide/glucagon, many studies on glucagon secretion (6, 7) can also be used to some extent for GLP-1 secretion, but GLP- 1 is more rapidly metabolized, with a half-life of 2 minutes in human plasma (19). A diet rich in carbohydrates or fats can promote secretion (20), presumably as a result of direct interaction of unabsorbed nutrients with the microvilli of open L-cells of the intestinal mucosa. There may be endocrine or neural mechanisms that promote GLP-1 secretion, but this has not been demonstrated in humans.

The incretin effect of GLP-1 has been clearly demonstrated by experiments with the GLP-1 receptor antagonist exendin 9-39 (29-31), in which exendin 9-39 surprisingly reduced oral Incretin action after glucose (21, 22). The hormone interacts directly with β-cells via the GLP-1 receptor belonging to the glucagon/VIP/calcitonin family of G-protein coupled 7-transmembrane crossing receptors (23). The importance of the GLP-1 receptor in regulating insulin secretion has been demonstrated in recent experiments in which mice were subjected to targeted disruption of the GLP-1 receptor gene. Homozygous animals with this disruption have markedly regressed glucose tolerance and rapidly onset hyperglycemia, and even heterozygous animals are intolerant to glucose (24). The signal transduction mechanism mainly involves the activation of adenylate cyclase, but there must also be an increase in intracellular Ca ²⁺ (25, 26). The action of this hormone is best described as enhancing glucose-stimulated insulin release (25), but the mechanism of coupling between glucose and GLP-1 stimulation is unknown. It may involve a calcium-induced calcium release (26, 27). As already mentioned, the [beta]-cells of diabetics have an insulinotropic effect of GLP-1. Nor is the relationship between GLP-1 and its ability to confer "glucose sensitivity" on isolated insulin-secreting cells (26, 28 ) barely responsive to glucose or GLP-1 alone, whereas is a response to the combination of the two. Equally important, however, this hormone also potently inhibits glucagon secretion (29). The mechanism is unclear but appears to be through paracrine from neighboring insulin or somatostatin cells (25). Glucagonostatic action is also glucose-dependent, so when blood glucose is lowered, the inhibitory effect is reduced. Because of this dual effect, if the concentration of GLP-1 in plasma is increased by increased secretion or by exogenous infusion, the molar ratio between insulin and glucagon in blood reaching the liver through the portal circulation will be significantly increased, whereas Glycogen production is reduced (30). As a result, blood sugar concentration decreases. Due to the glucose-dependence of insulinotropic and glucagon-inhibiting actions, the glucose-lowering effect is self-limiting and, therefore, the hormone does not cause hypoglycemia regardless of dose (31). These effects are still present in patients with diabetes (32), despite poor metabolic control and sulfonylurea attachment disorders (33), and infusion of GLP-1 in these patients at slightly supraphysiological doses may completely normalize blood glucose values . GLP-1 has also been found to lower blood glucose in type 1 diabetics without residual β-cell secretory capacity (34), illustrating the importance of the glucagon inhibitory effect.

In addition to its effects on islets, GLP-1 also has potent effects on the gastrointestinal tract. Physiological doses of GLP-1 infused significantly inhibited pentagastrin-induced and meal-induced gastric acid secretion (35,36). GLP-1 also inhibits gastric emptying rate and pancreatic enzyme secretion (36). Human ileal infusion of solutions containing sugar or lipid may have similar inhibitory effects on gastric and pancreatic secretion and motility (37, 38). Concomitantly, GLP-1 secretion is markedly stimulated, and it has been speculated that GLP-1 may be at least partly responsible for this so-called "ileal-brake" effect (38). Indeed, recent studies suggest that, physiologically, the ileal gate effect of GLP-1 may be more important than its effect on islets. Thus, GLP-1 affects gastric emptying rate in dose-response studies at least at the minimal infusion rate required to affect islet secretion (39).

GLP-1 appears to have a role in food intake. Intraventricular administration of GLP-1 severely inhibits food intake in rats (40, 42). This effect appears to be idiosyncratic. Thus, the N-terminally extended GLP-1 (PG72-107) amide is inactive, and an appropriate dose of the GLP-1 antagonist, exendin9-39, abolishes the effects of GLP-1 (41). Rapid peripheral administration of GLP-1 does not rapidly inhibit food intake in rats (41, 42). However, it is still possible that GLP-1 secreted from intestinal L-cells acts as a satiety signal.

Diabetics have not only insulinotropic effects but also gastrointestinal effects of GLP-1 (43), which may help to attenuate meal-induced glucose excursions but, more importantly, may also affect food intake . Continuous intravenous administration of GLP-1 at 4 ng/kg/min for one week has been shown to significantly improve glycemic control in NIDDM patients without significant side effects (44). Peptides are fully effective following subcutaneous administration (45), but are rapidly degraded mainly due to degradation by dipeptidyl peptidase IV-like enzymes (46, 47).

In particular, the amino acid sequence of GLP-1 is given by Schmidt et al. (Diabetologia, 28, 704-707 (1985)). Although the interesting pharmacological properties of GLP-1(7-37) and its analogs have attracted much attention in recent years, little is known about the structures of these molecules. Thorton et al. described the secondary structure of GLP-1 in micelles (Biochemistry, 33, 3532-3539 (1994)), but in normal solution, GLP-1 is considered to be a very deformable molecular. Unexpectedly, we found that derivatization of this small and highly deformable molecule yielded compounds with greatly prolonged plasma distribution profiles and retained activity.

GLP-1, GLP-1 analogs and fragments thereof are particularly effective in the treatment of type 1 and type 2 diabetes. However, high clearance limits the effectiveness of these compounds, so improvements are needed in this regard. Accordingly, it is an object of the present invention to provide derivatives of GLP-1 and its analogues which have a longer duration of action than GLP-1(7-37). It is a further object of the present invention to provide derivatives of GLP-1 and its analogues which have lower clearance than GLP-1(7-37). It is a further object of the present invention to provide a pharmaceutical composition containing the compound of the present invention, and to use the compound of the present invention for the preparation of such a composition. It is also an object of the present invention to provide a method for treating insulin-dependent and non-insulin-dependent diabetes.

references

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Summary of the invention

Human GLP-1 is a 37 amino acid residue peptide derived from preproglucagon, which is mainly synthesized in the distal ileal L-cells, pancreas and brain. Processing from preproglucagon to GLP-1(7-36)amide, GLP-1(7-37) and GLP-2 occurs primarily in L-cells. A simple system can be used to describe fragments and analogs of this peptide. Thus, for example, Gly ⁸ -GLP-1(7-37) denotes GLP formed by deleting amino acid residues Nos. 1-6 from GLP-1 and substituting Gly for the naturally occurring amino acid residue at position 8 (Ala) -1 fragment. Similarly, Lys ³⁴ (N ^ε -tetradecanoyl)-GLP-1(7-37) represents GLP-(7-37) in which the ε-amino group of the Lys residue at position 34 has been myristoylated. Where reference is made herein to C-terminally extended GLP-1 analogues, then unless otherwise stated, the amino acid residue at position 38 is Arg, and the optional amino acid residue at position 39 is also Arg (unless otherwise stated), An optional amino acid residue at position 40 is Asp (unless otherwise stated). Likewise, if the analog of the C-terminal extension extends to position 41, 42, 43, 44 or 45, the amino acid sequence of this extension is the same as the corresponding sequence in human preproglucagon unless otherwise stated.

In its broadest aspect, the invention relates to derivatives of GLP-1 and analogs thereof. The derivatives of the invention have interesting pharmacological properties, in particular they have a longer action profile than the parent peptide.

As used herein, "analogue" is used to denote a peptide in which one or more amino acid residues of the parent peptide have been substituted by another amino acid residue, and/or in which one or more amino acid residues on the parent peptide has been deleted, and/or wherein one or more amino acid residues have been added to the parent peptide. This addition can occur at the N-terminus or C-terminus of the parent peptide, or at both ends.

The term "derivative" is used herein to denote a peptide that has been chemically modified at one or more amino acid residues of the parent peptide, eg, alkylated, acylated, to form an ester or amide.

The term "GLP-1 derivative" is used herein to denote a derivative of GLP-1 or an analogue thereof. Herein, the parent peptide from which the derivative is derived is referred to in some places as the "GLP-1 portion" of the derivative.

In a preferred embodiment, the present invention relates to a GLP-1 derivative, wherein at least one amino acid residue of the parent peptide has attached a lipophilic substituent, provided that if there is only one lipophilic substituent, and this substituent is attached to the N-terminal or C-terminal amino acid residue of the parent peptide, then the substituent is an alkyl group or has an omega-carboxylic acid group.

In a preferred embodiment, the present invention relates to GLP-1 derivatives having only one lipophilic substituent.

In a preferred embodiment, the present invention relates to GLP-1 derivatives having only one lipophilic substituent, either an alkyl group or an omega-carboxylic acid group, attached to the N-terminal amino acid of the parent peptide on the residue.

In a preferred embodiment, the present invention relates to GLP-1 derivatives having only one lipophilic substituent, either an alkyl group or an omega-carboxylic acid group, attached to the C-terminal amino acid of the parent peptide on the residue.

In a preferred embodiment, the present invention relates to GLP-1 derivatives having only one lipophilic substituent which can be attached to any amino acid residue on the parent peptide which is neither N-terminal nor C-terminal.

In a preferred embodiment, the present invention relates to GLP-1 derivatives having 2 lipophilic substituents.

In a preferred embodiment, the present invention relates to GLP-1 derivatives having two lipophilic substituents, wherein one lipophilic substituent is attached to the N-terminal amino acid residue of the parent peptide and the other lipophilic substituent is attached to to the C-terminal amino acid residue of the parent peptide.

In a preferred embodiment, the present invention relates to GLP-1 derivatives having two lipophilic substituents, wherein one lipophilic substituent is attached to the N-terminal amino acid residue of the parent peptide and the other lipophilic substituent is attached to to an amino acid residue that is neither N-terminal nor C-terminal of the parent peptide.

In a preferred embodiment, the present invention relates to GLP-1 derivatives having 2 lipophilic substituents, wherein one lipophilic substituent is attached to the C-terminal amino acid residue of the parent peptide and the other lipophilic substituent is attached to to an amino acid residue that is neither N-terminal nor C-terminal of the parent peptide.

In a further preferred embodiment, the present invention relates to a derivative of GLP-1 (7-C), wherein C is selected from the group consisting of 38, 39, 40, 41, 42, 43, 44 and 45, the derivative The peptide has only one lipophilic substituent attached to the C-terminal amino acid residue of the parent peptide.

In a further preferred embodiment, the present invention relates to a GLP-1 derivative wherein the lipophilic substituent contains 4 to 40 carbon atoms, more preferably 8 to 25 carbon atoms.

In a further preferred embodiment, the present invention relates to a GLP-1 derivative wherein a lipophilic substituent is attached to an amino acid residue in such a way that a carboxyl group of the lipophilic substituent is bound to the An amino group of an amino acid residue forms an amide bond.

In a further preferred embodiment, the present invention relates to a GLP-1 derivative wherein a lipophilic substituent is attached to an amino acid residue in such a way that an amino group of the lipophilic substituent is associated with the One carboxyl group of an amino acid residue forms an amide bond.

In a further preferred embodiment, the invention relates to a GLP-1 derivative in which a lipophilic substituent is attached to the parent peptide via a spacer.

In a further preferred embodiment, the present invention relates to a GLP-1 derivative in which a lipophilic substituent (optionally via a spacer) is attached to the ε- on the amino group.

In a further preferred embodiment, the invention relates to a GLP-1 derivative in which a lipophilic substituent is passed through an unbranched alkane α with 1 to 7 methylene groups, preferably with 2 methylene groups , an omega-dicarboxy spacer that forms a bridge between an amino group of the parent peptide and an amino group of the lipophilic substituent.

In a further preferred embodiment, the present invention relates to a GLP-1 derivative in which a lipophilic substituent is attached to the parent peptide via a spacer which is an amino acid residue other than Cys group, or a dipeptide such as Gly-Lys. In this context, the expression "a dipeptide such as Gly-Lys" refers to a dipeptide in which the C-terminal amino acid residue is Lys, His or Trp, preferably Lys, in which the N-terminal amino acid residue is The residues are selected from the group consisting of Ala, Arg, Asp, Asn, Gly, Glu, Gln, Ile, Leu, Val, Phe and Pro.

In a further preferred embodiment, the present invention relates to a GLP-1 derivative in which a lipophilic substituent is attached to the parent peptide via a spacer which is an amino acid residue other than Cys group, or a dipeptide such as Gly-Lys, and wherein a carboxyl group of the parent peptide forms an amide bond with a Lys residue or an amino group in a dipeptide containing a Lys residue, the Lys residue or containing a Another amino group in the dipeptide of the Lys residue forms an amide bond with a carboxyl group in the lipophilic substituent.

In a further preferred embodiment, the present invention relates to a GLP-1 derivative in which a lipophilic substituent is attached to the parent peptide via a spacer which is an amino acid residue other than Cys , or a dipeptide such as Gly-Lys, wherein an amino group of the parent peptide forms an amide bond with the amino acid residue spacer or a carboxyl group in the dipeptide spacer, and the amino acid residue spacer Or an amino group in the dipeptide spacer forms an amide bond with a carboxyl group of the lipophilic substituent.

In a further preferred embodiment, the present invention relates to a GLP-1 derivative in which a lipophilic substituent is attached to the parent peptide via a spacer which is an amino acid residue other than Cys , or a dipeptide such as Gly-Lys, and wherein a carboxyl group of the parent peptide forms an amide bond with the amino acid residue spacer or an amino group in the dipeptide spacer, the amino acid residue spacer Or the carboxyl group in the dipeptide spacer forms an amide bond with an amino group of the lipophilic substituent.

In a further preferred embodiment, the present invention relates to a GLP-1 derivative in which a lipophilic substituent is attached to the parent peptide via a spacer which is an amino acid residue other than Cys , or a dipeptide such as Gly-Lys in which a carboxyl group of the parent peptide forms an amino group with Asp or Glu as a spacer, or an amino group in a dipeptide spacer containing an Asp or Glu residue An amide bond, a carboxyl group in the spacer forms an amide bond with an amino group in the lipophilic substituent.

In a further preferred embodiment, the invention relates to a GLP-1 derivative having a lipophilic substituent containing a partially or fully hydrogenated cyclopentanophenathrene backbone.

In a further preferred embodiment, the invention relates to a GLP-1 derivative which has a lipophilic substituent which is a linear or branched alkyl group.

In a further preferred embodiment, the invention relates to a GLP-1 derivative which has a lipophilic substituent which is an acyl group of a linear or branched fatty acid.

In a further preferred embodiment, the present invention relates to a GLP-1 derivative having a lipophilic substituent which is an acyl group selected from the group comprising _CH3 ( _CH2 ) _nCO- , wherein n is an integer of 4-38, preferably an integer of 4-24, further preferably the lipophilic substituent is selected from CH ₃ (CH ₂ ) ₆ CO-, CH ₃ (CH ₂ ) ₈ CO-, CH ₃ (CH ₂ ) ₁₀ CO-, CH ₃ (CH ₂ ) ₁₂ CO-, CH ₃ (CH ₂ ) ₁₄ CO-, CH ₃ (CH ₂ ) _l6 CO-, CH ₃ (CH ₂ ) ₁₈ CO-, CH ₃ A group of (CH ₂ ) ₂₀ CO— and CH ₃ (CH ₂ ) ₂₂ CO—.

In a further preferred embodiment, the invention relates to a GLP-1 derivative having a lipophilic substituent which is an acyl group of a linear or branched alkane α,ω-dicarboxylic acid.

In a further preferred embodiment, the present invention relates to a GLP-1 derivative having a lipophilic substituent which is an acyl group selected from the group comprising HOOC( _CH2 ) _mCO- , Wherein m is an integer of 4-38, preferably an integer of 4-24, more preferably the lipophilic substituent is selected from the group consisting of HOOC(CH ₂ ) ₁₄ CO-, HOOC(CH ₂ ) ₁₆ CO-, HOOC(CH ₂ ) ₁₈ CO-, the group of HOOC(CH ₂ ) ₂₀ CO- and HOOC(CH ₂ ) ₂₂ CO-.

In a further preferred embodiment, the present invention relates to a GLP-1 derivative having a formula CH ₃ (CH ₂ ) _p ((CH ₂ ) _q COOH)CHNH—CO(CH ₂ ) ₂ CO— The lipophilic substituent of wherein p and q are integers, and p+q is an integer of 8-33, preferably an integer of 12-28.

In a further preferred embodiment, the invention relates to a GLP-1 derivative having a lipophilic substituent having the formula _CH3 ( _CH2 ) _rCO -NHCH(COOH)( _CH2 ) _2CO- , where r is an integer of 10-24.

In a further preferred embodiment, the invention relates to a GLP-1 derivative having a lipophilic substituent having the formula _CH3 ( _CH2 ) _sCO -NHCH(( _CH2 ) _2COOH )CO- , where s is an integer of 8-24.

In a further preferred embodiment, the invention relates to a GLP-1 derivative having a lipophilic substituent having the formula COOH( _CH2 ) _tCO- , wherein t is an integer from 8 to 24.

In a further preferred embodiment, the invention relates to a GLP-1 derivative having a lipophilic substituent having the formula -NHCH(COOH)(CH ₂ ) ₄ NH-CO(CH ₂ ) _u CH ₃ , where u is an integer of 8-18.

In a further preferred embodiment, the present invention relates to a GLP-1 derivative having a compound having the formula -NHCH(COOH)(CH ₂ ) ₄ NH-COCH((CH ₂ ) ₂ COOH)NH-CO( A lipophilic substituent of CH ₂ ) _w CH ₃ , wherein w is an integer of 10-16.

In a further preferred embodiment, the present invention relates to a GLP-1 derivative having a formula -NHCH(COOH)(CH ₂ ) ₄ NH-CO(CH ₂ ) ₂ CH(COOH)NH-CO A lipophilic substituent of (CH ₂ ) _x CH ₃ , wherein x is an integer from 10-16.

In a further preferred embodiment, the present invention relates to a GLP-1 derivative having a formula -NHCH(COOH)(CH ₂ ) ₄ NH-CO(CH ₂ ) ₂ CH(COOH)NH-CO ( _CH2 ) _y is a lipophilic substituent of _CH3 , wherein y is 0 or an integer of 1-22.

In a further preferred embodiment, the invention relates to a GLP-1 derivative which has a lipophilic substituent which may be negatively charged. Such a lipophilic substituent may be, for example, a substituent having a carboxyl group.

In a further preferred embodiment, the present invention relates to a GLP-1 derivative whose parent peptide is selected from GLP-1(1-45) or analogs thereof.

In a further preferred embodiment, the present invention relates to a GLP-1 derivative, which is selected from the group consisting of GLP-1(7-35), GLP-1(7-36), GLP-1(7-36 ) amides, of the group of GLP-1(7-37), GLP-1(7-38), GLP-1(7-39), GLP-1(7-40) and GLP-1(7-41) A GLP-1 fragment or analog thereof is derived.

In a further preferred embodiment, the present invention relates to a GLP-1 analog, which is selected from the group consisting of GLP-1 (1-35), GLP-1 (1-36), GLP-1 (1-36 ) amides, of the group of GLP-1(1-37), GLP-1(1-38), GLP-1(1-39), GLP-1(1-40) and GLP-1(1-41) A GLP-1 analogue or an analogue thereof is derived.

In a further preferred embodiment, the present invention relates to a GLP-1 derivative, wherein the specified analog comprises a derivative in which a total of at most 15, preferably at most 10, amino acid residues have been replaced by any α-amino acid residue replace.

In a further preferred embodiment, the present invention relates to a derivative of GLP-1, wherein at most 15, preferably at most 10 amino acid residues of the derivative comprised by the specified analog have been encoded by any α-Amino acid residue substitution.

In a further preferred embodiment, the present invention relates to a derivative of GLP-1, wherein the indicated analog comprises a derivative in which at most 6 amino acid residues have been replaced by another α-amino acid residue which can be encoded by the genetic code base replacement.

In a further preferred embodiment, the present invention relates to a derivative of GLP-1 (A-B), wherein A is an integer of 1-7, and B is an integer of 38-45; or an analog of this derivative, which contains A lipophilic substituent attached to the C-terminal amino acid residue, and optionally contains a second lipophilic substituent attached to another amino acid residue.

In a further preferred embodiment, the parent peptide of the derivative of the present invention is selected from the following members: Arg ²⁶ -GLP-1(7-37), Arg ³⁴ -GLP-1(7-37), Lys ³⁶ -GLP- 1(7-37), Arg ^{26, 34} Lys ³⁶ -GLP-1(7-37), Arg ^{26, 34} Lys ³⁸ -GLP-1(7-38), Arg ^{26, 34} Lys ³⁹ -GLP-1( 7-39), Arg ^{26, 34} Lys ⁴⁰ -GLP-1(7-40), Arg ²⁶ Lys ³⁶ -GLP-1(7-37), Arg ³⁴ Lys ³⁶ -GLP-1(7-37), Arg ²⁶ Lys ³⁹ -GLP-1(7-39), Arg ³⁴ Lys ⁴⁰ -GLP-1(7-40), Arg ^{26, 34} Lys 36 ^{, 39} -GLP-1(7-39), Arg ^{26, 34} Lys ^36,40 -GLP-1(7-40), Gly ⁸ Arg ²⁶ -GLP-1(7-37), Gly ⁸ Arg ³⁴ -GLP-1(7-37), Gly ⁸ Lys ³⁶ -GLP-1( 7-37), ^{Gly 8} Arg ^{26, 34} Lys ³⁶ -GLP-1(7-37), Gly ⁸ Arg ^{26, 34 Lys 39 -} GLP-1(7-39), Gly ⁸ Arg 26, 34 Lys ^40- GLP-1(7-40), Gly ⁸ Arg ²⁶ Lys ³⁶ -GLP-1(7-37), Gly ⁸ Arg ³⁴ Lys ³⁶ -GLP-1(7-37), Gly ⁸ Arg ²⁶ Lys ³⁹ -GLP- 1(7-39), Gly ⁸ Arg ³⁴ Lys ⁴⁰ -GLP-1(7-40), Gly ⁸ Arg 26, 34 Lys ^{36 , 39} -GLP-1(7-39) and Gly ⁸ Arg ^{26, 34} Lys ^36,40 -GLP-1(7-40).

In a further preferred embodiment, the parent peptide of the derivative of the present invention is selected from the following members: Arg ^26,34 Lys ³⁸ -GLP-1(7-38), Arg ^26,34 Lys ³⁹ -GLP-1(7- 39), Arg ^{26, 34} Lys ⁴⁰ -GLP-1 (7-40), Arg ^{26, 34} Lys ⁴¹ -GLP-1 (7-4 1), Arg ^{26, 34} Lys ⁴² -GLP-1 (7-42 ), Arg ^{26, 34} Lys ⁴³ -GLP-1(7-43), Arg ^{26, 34} Lys ⁴⁴ -GLP-1(7-44), Arg ^{26, 34} Lys ⁴⁵ -GLP-1(7-45), Arg ^{26, 34} Lys ³⁸ -GLP-1(1-38), Arg ^{26, 34} Lys ³⁹ -GLP-1(1-39), Arg ²⁶ , 34 Lys ⁴⁰ -GLP-1(1-40), Arg ^{26 , 34} Lys ⁴¹ -GLP-1(1-41), Arg ^{26, 34} Lys ⁴² -GLP-1(1-42), Arg ^{26, 34} Lys ⁴³ -GLP-1(1-43), Arg ^{26, 34} Lys ⁴⁴ -GLP-1(1-44), Arg ^{26, 34} Lys ⁴⁵ -GLP-1(1-45), Arg ^{26, 34} Lys ³⁸ -GLP-1(2-38), Arg 26, ³⁴ Lys ³⁹ -GLP-1(2-39), Arg ^{26, 34} Lys ⁴⁰ -GLP-1(2-40), Arg ^{26, 34} Lys ⁴¹ -GLP-1(2-41), Arg 26, ³⁴ Lys ⁴² -GLP -1(2-42), Arg ^{26, 34} Lys ⁴³ -GLP-1(2-43), Arg ^{26, 34} Lys ⁴⁴ -GLP-1(2-44), Arg ^{26, 34} Lys ⁴⁵ -GLP-1 (2-45), Arg ^{26, 34} Lys ³⁸ -GLP-1 (3-38), Arg ^{26, 34} Lys ³⁹ -GLP-1 (3-39), Arg ^{26, 34} Lys ⁴⁰ -GLP-1 (3 -40), Arg ^{26, 34} Lys ⁴¹ -GLP-1 (3-41), Arg ^{26, 34} Lys ⁴² -GLP-1 (3-42), Arg ^{26, 34} Lys ⁴³ -GLP-1 (3-43 ), Arg ^{26, 34} Lys ⁴⁴ -GLP-1(3-44), Arg ^{26, 34} Lys ⁴⁵ -GLP-1(3-45), Arg ^{2 6, 34} Lys ³⁸ -GLP-1 (4-38), Arg ^{26, 34} Lys ³⁹ -GLP-1 (4-39), Arg ^26, 34 Lys ⁴⁰ -GLP-1 (4-40), Arg ^{26, 34} Lys ⁴¹ -GLP-1(4-41), Arg ^{26, 34} Lys ⁴² -GLP-1(4-42), Arg ^{26, 34} Lys ⁴³ -GLP-1(4-43), Arg ^{26, 34} Lys ⁴⁴ -GLP-1(4-44), Arg ^26, 34 Lys ^45- GLP-1(4-45), Arg ^{26, 34} Lys ³⁸ -GLP-1(5-38), Arg 26, ³⁴ Lys ^39- GLP-1(5-39), Arg ^{26, 34} Lys ⁴⁰ -GLP-1(5-40), Arg ^{26, 34} Lys ⁴¹ -GLP-1(5-41), Arg 26, ³⁴ Lys ⁴² -GLP- 1(5-42), Arg ^{26, 34} Lys ⁴³ -GLP-1(5-43), Arg ^{26, 34} Lys ⁴⁴ -GLP-1(5-44), Arg ^{26, 34} Lys ⁴⁵ -GLP-1( 5-45), Arg ^{26, 34} Lys ³⁸ -GLP-1 (6-38), Arg ^{26, 34} Lys ³⁹ -GLP-1 (6-39), Arg ^{26, 34} Lys ⁴⁰ -GLP-1 (6- 40), Arg ^{26, 34} Lys ⁴¹ -GLP-1 (6-41), Arg ^{26, 34} Lys ⁴² -GLP-1 (6-42), Arg ^{26, 34} Lys ⁴³ -GLP-1 (6-43) , Arg ^26, 34 Lys ⁴⁴ -GLP-1(6-44), Arg ^26, 34 Lys ⁴⁵ -GLP-1(6-45), Arg ²⁶ Lys ³⁸ -GLP-1(1-38), Arg ³⁴ Lys ³⁸ -GLP-1(1-38), Arg ^{26, 34} Lys ^{36, 38} -GLP-1(1-38), Arg ²⁶ Lys ³⁸ -GLP-1(7-38), Arg ³⁴ Lys ³⁸ -GLP- 1(7-38), Arg 26, 34 Lys ^{36 , 38} -GLP-1(7-38), Arg ^{26, 34} Lys ³⁸ -GLP-1(7-38), Arg ²⁶ Lys ³⁹ -GLP-1( 1-39), Arg ³⁴ Lys ³⁹ -GLP-1 (1-39), Arg ^{26, 34} Lys 36 ^{, 39} -GLP-1 ( 1-39), Arg ²⁶ Lys ³⁹ -GLP-1(7-39), Arg ³⁴ Lys ³⁹ -GLP-1(7-39) and Arg ^26,34 Lys ^36,39 -GLP-1(7-39) .

In a further preferred embodiment, the present invention relates to a GLP-1 derivative, wherein the parent peptide is selected from the following members: [Arg ²⁶ -GLP-1(7-37), Arg ³⁴ -GLP-1(7 -37), Lys ³⁶ -GLP-1(7-37), Arg ^{26, 34} Lys ³⁶ -GLP-1(7-37), Arg ²⁶ Lys ³⁶ -GLP-1(7-37), Arg ³⁴ Lys ³⁶ -GLP-1(7-37), Gly ⁸ Arg ²⁶ -GLP-1(7-37), Gly ⁸ Arg ³⁴ -GLP-1(7-37), Gly ⁸ Lys ³⁶ -GLP-1(7-37 ), Gly ⁸ Arg ^{26, 34} Lys ³⁶ -GLP-1(7-37), Gly ⁸ Arg ²⁶ Lys ³⁶ -GLP-1(7-37) and Gly ⁸ Arg ³⁴ Lys ³⁶ -GLP-1(7-37 ).

In a further preferred embodiment, the present invention relates to a GLP-1 derivative, wherein the parent peptide is selected from the following members: Arg ²⁶ Lys ³⁸ -GLP-1 (7-38), Arg ^26,34 Lys ³⁸ - GLP-1(7-38), Arg ^{26, 34} Lys ^{36, 38} -GLP-1(7-38), Gly ⁸ Arg ²⁶ Lys ³⁸ -GLP-1(7-38) and Gly ⁸ Arg 26 ^{, 34} Lys ^36,38 -GLP-1(7-38).

In a further preferred embodiment, the present invention relates to a GLP-1 derivative, wherein the parent peptide is selected from the following members: Arg ²⁶ Lys ³⁹ -GLP-1 (7-39), Arg ^{26, 34} Lys ^{36, 39} -GLP- ¹ (7-39 ⁾ , Gly8Arg26Lys39- ^GLP -1( ^7-39 ) and ^{Gly8Arg26,34Lys36,39-} GLP-1(7-39) ^.

In a further preferred embodiment, the present invention relates to a GLP-1 derivative, wherein the parent peptide is selected from the following members: Arg ³⁴ Lys ⁴⁰ -GLP-1(7-40), Arg ^{26, 34} Lys ^{36, 40} -GLP- ¹ ( ^{7-40 )} , Gly8Arg34Lys40- ^GLP -1(7-40) and ^{Gly8Arg26,34Lys36,40} -GLP-1(7-40) ^.

In a further preferred embodiment, the present invention relates to a GLP-1 derivative, wherein the parent peptide is selected from the following members:

Lys ²⁶ (N ^ε -tetradecanoyl)-GLP-1(7-37);

Lys ³⁴ (N ^ε -tetradecanoyl)-GLP-1(7-37);

Lys ^26,34 -bis(N ^ε -tetradecanoyl)-GLP-1(7-37);

Gly ⁸ Lys ²⁶ (N ^ε -tetradecanoyl)-GLP-1(7-37);

Gly ⁸ Lys ³⁴ (N ^ε -tetradecanoyl)-GLP-1(7-37);

Gly ⁸ Lys ^26,34 -bis(N ^ε -tetradecanoyl)-GLP-1(7-37);

Arg ²⁶ Lys ³⁴ (N ^ε -tetradecanoyl)-GLP-1(7-37);

Lys ²⁶ (N ^ε -tetradecanoyl)-GLP-1(7-38);

Lys ³⁴ (N ^ε -tetradecanoyl)-GLP-1(7-38);

Lys ^26,34 -bis(N ^ε -tetradecanoyl)-GLP-1(7-38);

Gly ⁸ Lys ²⁶ (N ^ε -tetradecanoyl)-GLP-1(7-38);

Gly ⁸ Lys ³⁴ (N ^ε -tetradecanoyl)-GLP-1(7-38);

Gly ⁸ Lys ^26,34 -bis(N ^ε -tetradecanoyl)-GLP-1(7-38);

Arg ²⁶ Lys ³⁴ (N ^ε -tetradecanoyl)-GLP-1(7-38);

Lys ²⁶ (N ^ε -tetradecanoyl)-GLP-1(7-39);

Lys ³⁴ (N ^ε -tetradecanoyl)-GLP-1(7-39);

Lys ^26,34 -bis(N ^ε -tetradecanoyl)-GLP-1(7-39);

Gly ⁸ Lys ²⁶ (N ^ε -tetradecanoyl)-GLP-1(7-39);

Gly ⁸ Lys ³⁴ (N ^ε -tetradecanoyl)-GLP-1(7-39);

Gly ⁸ Lys ^26,34 -bis(N ^ε -tetradecanoyl)-GLP-1(7-39);

Arg ²⁶ Lys ³⁴ (N ^ε -tetradecanoyl)-GLP-1(7-39);

Lys ²⁶ (N ^ε -tetradecanoyl)-GLP-1(7-40);

Lys ³⁴ (N ^ε -tetradecanoyl)-GLP-1(7-40);

Lys ^26,34 -bis(N ^ε -tetradecanoyl)-GLP-1(7-40);

Gly ⁸ Lys ²⁶ (N ^ε -tetradecanoyl)-GLP-1(7-40);

Gly ⁸ Lys ³⁴ (N ^ε -tetradecanoyl)-GLP-1(7-40);

Gly ⁸ Lys ^26,34 -bis(N ^ε -tetradecanoyl)-GLP-1(7-40);

Arg ²⁶ Lys ³⁴ (N ^ε -tetradecanoyl)-GLP-1(7-40);

Lys ²⁶ (N ^ε -tetradecanoyl)-GLP-1(7-36);

Lys ³⁴ (N ^ε -tetradecanoyl)-GLP-1(7-36);

Lys ^26,34 -bis(N ^ε -tetradecanoyl)-GLP-1(7-36);

Gly ⁸ Lys ²⁶ (N ^ε -tetradecanoyl)-GLP-1(7-36);

Gly ⁸ Lys ³⁴ (N ^ε -tetradecanoyl)-GLP-1(7-36);

Gly ⁸ Lys ^26,34 -bis(N ^ε -tetradecanoyl)-GLP-1(7-36);

Arg ²⁶ Lys ³⁴ (N ^ε -tetradecanoyl)-GLP-1(7-36);

Lys ²⁶ (N ^ε -tetradecanoyl)-GLP-1(7-35);

Lys ³⁴ (N ^ε -tetradecanoyl)-GLP-1(7-35);

Lys ^26,34 -bis(N ^ε -tetradecanoyl)-GLP-1(7-35);

Gly ⁸ Lys ²⁶ (N ^ε -tetradecanoyl)-GLP-1(7-35);

Gly ⁸ Lys ³⁴ (N ^ε -tetradecanoyl)-GLP-1(7-35);

Gly ⁸ Lys ^26,34 -bis(N ^ε -tetradecanoyl)-GLP-1(7-35);

Arg ²⁶ Lys ³⁴ (N ^ε -tetradecanoyl)-GLP-1(7-35);

Lys ²⁶ (N ^ε -tetradecanoyl)-GLP-1(7-36)amide;

Lys ³⁴ (N ^ε -tetradecanoyl)-GLP-1(7-36)amide;

Lys ^26,34 -bis(N ^ε -tetradecanoyl)-GLP-1(7-36)amide;

Gly ⁸ Lys ²⁶ (N ^ε -tetradecanoyl)-GLP-1(7-36)amide;

Gly ⁸ Lys ³⁴ (N ^ε -tetradecanoyl)-GLP-1(7-36)amide;

Gly ⁸ Lys ^26,34 -bis(N ^ε -tetradecanoyl)-GLP-1(7-36)amide;

Arg ²⁶ Lys ³⁴ (N ^ε -tetradecanoyl)-GLP-1(7-36) amide;

Gly ⁸ Arg ²⁶ Lys ³⁴ (N ^ε -tetradecanoyl)-GLP-1(7-37);

Lys ²⁶ (N ^ε -tetradecyl)Arg ³⁴ -GLP-1(7-37);

Gly ⁸ Lys ²⁶ (N ^ε -tetradecyl)Arg ³⁴ -GLP-1(7-37);

Arg ^{26, 34} Lys ³⁶ (N ^ε -tetradecanoyl)-GLP-1(7-37);

Gly ⁸ Arg ^26, 34 Lys ³⁶ (N ^ε -tetradecanoyl)-GLP-1(7-37);

Gly ⁸ Arg ²⁶ Lys ³⁴ (N ^ε -tetradecanoyl)-GLP-1(7-38);

Lys ²⁶ (N ^ε -tetradecyl)Arg ³⁴ -GLP-1(7-38);

Gly ⁸ Lys ²⁶ (N ^ε -tetradecanoyl) Arg ³⁴ -GLP-1(7-38);

Arg ^{26, 34} Lys ³⁶ (N ^ε -tetradecanoyl)-GLP-1(7-38);

Arg ^26,34 Lys ³⁸ (N ^ε -tetradecanoyl)-GLP-1(7-38);

Gly ⁸ Arg ^26, 34 Lys ³⁶ (N ^ε -tetradecanoyl)-GLP-1(7-38);

Gly ⁸ Arg ²⁶ Lys ³⁴ (N ^ε -tetradecanoyl)-GLP-1(7-39);

Lys ²⁶ (N ^ε -tetradecyl)Arg ³⁴ -GLP-1(7-39);

Gly ⁸ Lys ²⁶ (N ^ε -tetradecanoyl) Arg ³⁴ -GLP-1(7-39);

Arg ^{26, 34} Lys ³⁶ (N-tetradecanoyl)-GLP-1 (7-39);

Gly ⁸ Arg ^26, 34 Lys ³⁶ (N ^ε -tetradecanoyl)-GLP-1(7-39);

Gly ⁸ Arg ²⁶ Lys ³⁴ (N ^ε -tetradecanoyl)-GLP-1(7-40);

Lys ²⁶ (N ^ε -tetradecyl)Arg ³⁴ -GLP-1(7-40);

Gly ⁸ Lys ²⁶ (N ^ε -tetradecanoyl) Arg ³⁴ -GLP-1(7-40);

Arg ^{26, 34} Lys ³⁶ (N ^ε -tetradecanoyl)-GLP-1(7-40);

Gly ⁸ Arg ^26, 34 Lys ³⁶ (N ^ε -tetradecanoyl)-GLP-1(7-40);

Lys ²⁶ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-37);

Lys ³⁴ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-37);

Lys ^26,34 -bis(N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-37);

Gly ⁸ Lys ²⁶ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-37);

Gly ⁸ Lys ³⁴ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-37);

Gly ⁸ Lys ^26,34 -bis(N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-37);

Lys ²⁶ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-38);

Lys ³⁴ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-38);

Lys ^26,34 -bis(N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-38);

Gly ⁸ Lys ²⁶ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-38);

Gly ⁸ Lys ³⁴ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-38);

Gly ⁸ Lys ^26,34 -bis(N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-38);

Lys ²⁶ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-39);

Lys ³⁴ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-39);

Lys ^26,34 -bis(N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-39);

Gly ⁸ Lys ²⁶ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-39);

Gly ⁸ Lys ³⁴ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-39);

Gly ⁸ Lys ^26,34 -bis(N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-39);

Lys ²⁶ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-40);

Lys ³⁴ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-40);

Lys ^26,34 -bis(N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-40);

Gly ⁸ Lys ²⁶ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-40);

Gly ⁸ Lys ³⁴ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-40);

Gly ⁸ Lys ^26,34 -bis(N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-40);

Lys ²⁶ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-36);

Lys ³⁴ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-36);

Lys ^26,34 -bis(N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-36);

Gly ⁸ Lys ²⁶ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-36);

Gly ⁸ Lys ³⁴ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-36);

Gly ⁸ Lys ^26,34 -bis(N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-36);

Lys ²⁶ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-36 amide;

Lys ³⁴ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-36)amide;

Lys ^26,34 -bis(N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-36)amide;

Gly ⁸ Lys ²⁶ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-36)amide;

Gly ⁸ Lys ³⁴ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-36)amide;

Gly ⁸ Lys ^26,34 -bis(N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-36)amide;

Lys ²⁶ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-35);

Lys ³⁴ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-35);

Lys ^26,34 -bis N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-35);

Gly ⁸ Lys ²⁶ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-35);

Gly ⁸ Lys ³⁴ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-35);

Gly ⁸ Lys ^26,34 -bis(N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-35);

Arg ²⁶ Lys ³⁴ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-37);

Gly ⁸ Arg ²⁶ Lys ³⁴ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-37);

Lys ²⁶ (N ^ε -(ω-carboxynonadecanoyl))Arg ³⁴ -GLP-1(7-37);

Gly ⁸ Lys ²⁶ (N ^ε -(ω-carboxynonadecanoyl))Arg ³⁴ GLP-1(7-37);

Arg ^{26, 34} Lys ³⁶ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-37);

Gly ⁸ Arg ^{26, 34} Lys ³⁶ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-37);

Arg ²⁶ Lys ³⁴ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-38);

Gly ⁸ Arg ²⁶ Lys ³⁴ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-38);

Lys ²⁶ (N ^ε -(ω-carboxynonadecanoyl))Arg ³⁴ -GLP-1(7-38);

Gly ⁸ Lys ²⁶ (N ^ε -(ω-carboxynonadecanoyl))Arg ³⁴ -GLP-1(7-38);

Arg ^{26, 34} Lys ³⁶ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-38);

Arg ^{26, 34} Lys ³⁸ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-38);

Gly ⁸ Arg ^{26, 34} Lys ³⁶ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-38);

Arg ²⁶ Lys ³⁴ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-39);

Gly ⁸ Arg ²⁶ Lys ³⁴ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-39);

Lys ²⁶ (N ^ε -(ω-carboxynonadecanoyl))Arg ³⁴ -GLP-1(7-39);

Gly ⁸ Lys ²⁶ (N ^ε -(ω-carboxynonadecanoyl))Arg ³⁴ -GLP-1(7-39);

Arg ^{26, 34} Lys ³⁶ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-39);

Gly ⁸ Arg ^{26, 34} Lys ³⁶ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-39);

Arg ²⁶ Lys ³⁴ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-40);

Gly ⁸ Arg ²⁶ Lys ³⁴ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-40);

Lys ²⁶ (N ^ε -(ω-carboxynonadecanoyl))Arg ³⁴ -GLP-1(7-40);

Gly ⁸ Lys ²⁶ (N ^ε -(ω-carboxynonadecanoyl))Arg ³⁴ -GLP-1(7-40);

Arg ^{26, 34} Lys ³⁶ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-40);

Gly ⁸ Arg ^{26, 34} Lys ³⁶ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-40);

Lys ²⁶ (N ^ε -(7-deoxycholyl))-GLP-1(7-37);

Lys ³⁴ (N ^ε -(7-deoxycholyl))-GLP-1(7-37);

Lys ^26,34 -bis(N ^ε -(7-deoxycholyl))-GLP-1(7-37);

Gly ⁸ Lys ²⁶ (N ^ε -(7-deoxycholyl))-GLP-1(7-37);

Gly ⁸ Lys ³⁴ (N ^ε -(7-deoxycholyl))-GLP-1(7-37);

Gly ⁸ Lys ^26,34 -bis(N ^ε -(7-deoxycholyl))-GLP-1(7-37);

Arg ²⁶ Lys ³⁴ (N ^ε -(7-deoxycholyl))-GLP-1(7-37);

Lys ²⁶ (N ^ε -(7-deoxycholyl))-GLP-1(7-38);

Lys ³⁴ (N ^ε -(7-deoxycholyl))-GLP-1(7-38);

Lys ^26,34 -bis(N ^ε -(7-deoxycholyl))-GLP-1(7-38);

Gly ⁸ Lys ²⁶ (N ^ε -(7-deoxycholyl))-GLP-1(7-38);

Gly ⁸ Lys ³⁴ (N ^ε -(7-deoxycholyl))-GLP-1(7-38);

Gly ⁸ Lys ^26,34 -bis(N ^ε -(7-deoxycholyl))-GLP-1(7-38);

Arg ²⁶ Lys ³⁴ (N ^ε -(7-deoxycholyl))-GLP-1(7-38);

Lys ²⁶ (N ^ε -(7-deoxycholyl))-GLP-1(7-39);

Lys ³⁴ (N ^ε -(7-deoxycholyl))-GLP-1(7-39);

Lys ^26,34 -bis(N ^ε -(7-deoxycholyl))-GLP-1(7-39);

Gly ⁸ Lys ²⁶ (N ^ε -(7-deoxycholyl))-GLP-1(7-39);

Gly ⁸ Lys ³⁴ (N ^ε -(7-deoxycholyl))-GLP-1(7-39);

Gly ⁸ Lys ^26,34 -bis(N ^ε -(7-deoxycholyl))-GLP-1(7-39);

Arg ²⁶ Lys ³⁴ (N ^ε -(7-deoxycholyl))-GLP-1(7-39);

Lys ²⁶ (N ^ε -(7-deoxycholyl))-GLP-1(7-40);

Lys ³⁴ (N ^ε -(7-deoxycholyl))-GLP-1(7-40);

Lys ^26,34 -bis(N ^ε -(7-deoxycholyl))-GLP-1(7-40);

Gly ⁸ Lys ²⁶ (N ^ε -(7-deoxycholyl))-GLP-1(7-40);

Gly ⁸ Lys ³⁴ (N ^ε -(7-deoxycholyl))-GLP-1(7-40);

Gly ⁸ Lys ^26,34 -bis(N ^ε -(7-deoxycholyl))-GLP-1(7-40);

Arg ²⁶ Lys ³⁴ (N ^ε -(7-deoxycholyl))-GLP-1(7-40);

Lys ²⁶ (N ^ε -(7-deoxycholyl))-GLP-1(7-36);

Lys ³⁴ (N ^ε -(7-deoxycholyl))-GLP-1(7-36);

Lys ^26,34 -bis(N ^ε -(7-deoxycholyl))-GLP-1(7-36);

Gly ⁸ Lys ²⁶ (N ^ε -(7-deoxycholyl))-GLP-1(7-36);

Gly ⁸ Lys ³⁴ (N ^ε -(7-deoxycholyl))-GLP-1(7-36);

Gly ⁸ Lys ^26,34 -bis(N ^ε -(7-deoxycholyl))-GLP-1(7-36);

Arg ²⁶ Lys ³⁴ (N ^ε -(7-deoxycholyl))-GLP-1(7-36);

Lys ²⁶ (N ^ε -(7-deoxycholyl))-GLP-1(7-35);

Lys ³⁴ (N ^ε -(7-deoxycholyl))-GLP-1(7-35);

Lys ^26,34 -bis(N ^ε -(7-deoxycholyl))-GLP-1(7-35);

Gly ⁸ Lys ²⁶ (N ^ε -(7-deoxycholyl))-GLP-1(7-35);

Gly ⁸ Lys ³⁴ (N ^ε -(7-deoxycholyl))-GLP-1(7-35);

Gly ⁸ Lys ^26,34 -bis(N ^ε -(7-deoxycholyl))-GLP-1(7-35);

Arg ²⁶ Lys ³⁴ (N ^ε -(7-deoxycholyl))-GLP-1(7-35);

Lys ²⁶ (N ^ε -(7-deoxycholyl))-GLP-1(7-36)amide;

Lys ³⁴ (N ^ε -(7-deoxycholyl))-GLP-1(7-36)amide;

Lys ^26,34 -bis(N ^ε -(7-deoxycholyl))-GLP-1(7-36)amide;

Gly ⁸ Lys ²⁶ (N ^ε -(7-deoxycholyl))-GLP-1(7-36)amide;

Gly ⁸ Lys ³⁴ (N ^ε -(7-deoxycholyl))-GLP-1(7-36)amide;

vGly ⁸ Lys ^26,34 -bis(N ^ε -(7-deoxycholyl))-GLP-1(7-35)amide;

Arg ²⁶ Lys ³⁴ (N ^ε -(7-deoxycholyl))-GLP-1(7-35)amide;

Gly ⁸ Arg ²⁶ Lys ³⁴ (N ^ε -(7-deoxycholyl))-GLP-1(7-37);

Lys ²⁶ (N ^ε -(7-deoxycholyl))Arg ³⁴ -GLP-1(7-37);

Gly ⁸ Lys ²⁶ (N ^ε -(7-deoxycholyl))Arg ³⁴ -GLP-1(7-37);

Arg ^{26, 34} Lys ³⁶ (N ^ε -(7-deoxycholyl))-GLP-1(7-37);

Gly ⁸ Arg ^{26, 34} Lys ³⁶ (N ^ε -(7-deoxycholyl))-GLP-1(7-37);

Lys ²⁶ (N ^ε -(cholyl))-GLP-1(7-37);

Lys ³⁴ (N ^ε -(cholyl))-GLP-1(7-37);

Lys ^26,34 -bis(N ^ε -(cholyl))-GLP-1(7-37);

Gly ⁸ Lys ²⁶ (N ^ε -(cholyl))-GLP-1(7-37);

Gly ⁸ Lys ³⁴ (N ^ε -(cholyl))-GLP-1(7-37);

Gly ⁸ Lys ^26,34 -bis(N ^ε -(cholyl))-GLP-1(7-37);

Arg ²⁶ Lys ³⁴ (N ^ε -(cholyl))-GLP-1(7-37);

Gly ⁸ Arg ²⁶ Lys ³⁴ (N ^ε -(7-deoxycholyl))-GLP-1(7-38);

Lys ²⁶ (N ^ε -(7-deoxycholyl))Arg ³⁴ -GLP-1(7-38);

Gly ⁸ Lys ²⁶ (N ^ε -(7-deoxycholyl))Arg ³⁴ -GLP-1(7-38);

Arg ^{26, 34} Lys ³⁶ (N ^ε -(7-deoxycholyl))-GLP-1(7-38);

Arg ^{26, 34} Lys ³⁸ (N ^ε -(7-deoxycholyl))-GLP-1(7-38);

Gly ⁸ Arg ^{26, 34} Lys ³⁶ (N ^ε -(7-deoxycholyl))-GLP-1(7-38);

Lys ²⁶ (N ^ε -(cholyl))-GLP-1(7-38);

Lys ³⁴ (N ^ε -(cholyl))-GLP-1(7-38);

Lys ^26,34 -bis(N ^ε -(cholyl))-GLP-1(7-38);

Gly ⁸ Lys ²⁶ (N ^ε -(cholyl))-GLP-1(7-38);

Gly ⁸ Lys ³⁴ (N ^ε -(cholyl))-GLP-1(7-38);

Gly ⁸ Lys ^26,34 -bis(N ^ε -(cholyl))-GLP-1(7-38);

Arg ²⁶ Lys ³⁴ (N ^ε -(cholyl))-GLP-1(7-38);

Gly ⁸ Arg ²⁶ Lys ³⁴ (N ^ε -(7-deoxycholyl))-GLP-1(7-39);

Lys ²⁶ (N ^ε -(7-deoxycholyl))Arg ³⁴ -GLP-1(7-39);

Gly ⁸ Lys ²⁶ (N ^ε -(7-deoxycholyl))Arg ³⁴ -GLP-1(7-39);

Arg ^{26, 34} Lys ³⁶ (N ^ε -(7-deoxycholyl))-GLP-1(7-39);

Gly ⁸ Arg ^{26, 34} Lys ³⁶ (N ^ε -(7-deoxycholyl))-GLP-1(7-39);

Lys ²⁶ (N ^ε -(cholyl))-GLP-1(7-39);

Lys ³⁴ (N ^ε -(cholyl))-GLP-1(7-39);

Lys ^26,34 -bis(N ^ε -(cholyl))-GLP-1(7-39);

Gly ⁸ Lys ²⁶ (N ^ε -(cholyl))-GLP-1(7-39);

Gly ⁸ Lys ³⁴ (N ^ε -(cholyl))-GLP-1(7-39);

Gly ⁸ Lys ^26,34 -bis(N ^ε -(cholyl))-GLP-1(7-39);

Arg ²⁶ Lys ³⁴ (N ^ε -(cholyl))-GLP-1(7-39);

Gly ⁸ Arg ²⁶ Lys ³⁴ (N ^ε -(7-deoxycholyl))-GLP-1(7-40);

Lys ²⁶ (N ^ε -(7-deoxycholyl))Arg ³⁴ -GLP-1(7-40);

Gly ⁸ Lys ²⁶ (N ^ε -(7-deoxycholyl))Arg ³⁴ -GLP-1(7-40);

Arg ^{26, 34} Lys ³⁶ (N ^ε -(7-deoxycholyl))-GLP-1(7-40);

Gly ⁸ Ar ^{26, 34} Lys ³⁶ (N ^ε -(7-deoxycholyl))-GLP-1(7-40);

Lys ²⁶ (N ^ε -(cholyl))-GLP-1(7-40);

Lys ³⁴ (N ^ε -(cholyl))-GLP-1(7-40);

Lys ^26,34 -bis(N ^ε -(cholyl))-GLP-1(7-40);

Gly ⁸ Lys ²⁶ (N ^ε -(cholyl))-GLP-1(7-40);

Gly ⁸ Lys ³⁴ (N ^ε -(cholyl))-GLP-1(7-40);

Gly ⁸ Lys ^26,34 -bis(N ^ε -(cholyl))-GLP-1(7-40);

Arg ²⁶ Lys ³⁴ (N ^ε -(cholyl))-GLP-1(7-40);

Lys ²⁶ (N ^ε -(cholyl))-GLP-1(7-36);

Lys ³⁴ (N ^ε -(cholyl))-GLP-1(7-36);

Lys ^26,34 -bis(N ^ε -(cholyl))-GLP-1(7-36);

Gly ⁸ Lys ²⁶ (N ^ε -(cholyl))-GLP-1(7-36);

Gly ⁸ Lys ³⁴ (N ^ε -(cholyl))-GLP-1(7-36);

Gly ⁸ Lys ^26,34 -bis(N ^ε -(cholyl))-GLP-1(7-36);

Arg ²⁶ Lys ³⁴ (N ^ε -(cholyl))-GLP-1(7-36);

Lys ²⁶ (N ^ε -(cholyl))-GLP-1(7-35);

Lys ³⁴ (N ^ε -(cholyl))-GLP-1(7-35);

Lys ^26,34 -bis(N ^ε -(cholyl))-GLP-1(7-35);

Gly ⁸ Lys ²⁶ (N ^ε -(cholyl))-GLP-1(7-35);

Gly ⁸ Lys ³⁴ (N ^ε -(cholyl))-GLP-1(7-35);

Gly ⁸ Lys ^26,34 -bis(N ^ε -(cholyl))-GLP-1(7-35);

Arg ²⁶ Lys ³⁴ (N ^ε -(cholyl))-GLP-1(7-35);

Lys ²⁶ (N ^ε -(cholyl))-GLP-1(7-36)amide;

Lys ³⁴ (N ^ε -(cholyl))-GLP-1(7-36)amide;

Lys ^26,34 -bis(N ^ε -(cholyl))-GLP-1(7-36)amide;

Gly ⁸ Lys ²⁶ (N ^ε -(cholyl))-GLP-1(7-36)amide;

Gly ⁸ Lys ³⁴ (N ^ε -(cholyl))-GLP-1(7-36)amide;

Gly ⁸ Lys ^26,34 -bis(N ^ε -(cholyl))-GLP-1(7-36)amide;

Arg ²⁶ Lys ³⁴ (N ^ε -(cholyl))-GLP-1(7-36)amide;

Gly ⁸ Arg ²⁶ Lys ³⁴ (N ^ε -(cholyl))-GLP-1(7-37);

Lys ²⁶ (N ^ε -(cholyl))Arg ³⁴ -GLP-1(7-37);

Gly ⁸ Lys ²⁶ (N ^ε -(cholyl))Arg ³⁴ -GLP-1(7-37);

Arg ^{26, 34} Lys ³⁶ (N ^ε -(cholyl))-GLP-1(7-37);

Gly ⁸ Arg ^{26, 34} Lys ³⁶ (N ^ε -(cholyl))-GLP-1(7-37);

Lys ²⁶ (N ^ε -(lithoyl))-GLP-1(7-37);

Lys ³⁴ (N ^ε -(lithoyl))-GLP-1(7-37);

Lys ^26,34 -bis(N ^ε -(lithoyl))-GLP-1(7-37);

Gly ⁸ Lys ²⁶ (N ^ε -(lithoyl))-GLP-1(7-37);

Gly ⁸ Lys ³⁴ (N ^ε -(lithoyl))-GLP-1(7-37);

Gly ⁸ Lys ^26,34 -bis(N ^ε -(lithochyl))-GLP-1(7-37);

Arg ²⁶ Lys ³⁴ (N ^ε -(lithoyl))-GLP-1(7-37);

Gly ⁸ Arg ²⁶ Lys ³⁴ (N ^ε -(cholyl))-GLP-1(7-38);

Lys ²⁶ (N ^ε -(cholyl))Arg ³⁴ -GLP-1(7-38);

Gly ⁸ Lys ²⁶ (N ^ε -(cholyl))Arg ³⁴ -GLP-1(7-38);

Arg ^{26, 34} Lys ³⁶ (N ^ε -(cholyl))-GLP-1(7-38);

Arg ^{26, 34} Lys ³⁸ (N ^ε -(cholyl))-GLP-1(7-38);

Gly ⁸ Arg ^{26, 34} Lys ³⁶ (N ^ε -(cholyl))-GLP-1(7-38);

Lys ²⁶ (N ^ε -(lithoyl))-GLP-1(7-38);

Lys ³⁴ (N ^ε -(lithoyl))-GLP-1(7-38);

Lys ^26,34 -bis(N ^ε -(lithochyl))-GLP-1(7-38);

Gly ⁸ Lys ²⁶ (N ^ε -(lithoyl))-GLP-1(7-38);

Gly ⁸ Lys ³⁴ (N ^ε -(lithoyl))-GLP-1(7-38);

Gly ⁸ Lys ^26,34 -bis(N ^ε -(lithochyl))-GLP-1(7-38);

Arg ²⁶ Lys ³⁴ (N ^ε -(lithoyl))-GLP-1(7-38);

Gly ⁸ Arg ²⁶ Lys ³⁴ (N ^ε -(cholyl))-GLP-1(7-39);

Lys ²⁶ (N ^ε -(cholyl))Arg ³⁴ -GLP-1(7-39);

Gly ⁸ Lys ²⁶ (N ^ε -(cholyl))Arg ³⁴ -GLP-1(7-39);

Arg ^{26, 34} Lys ³⁶ (N ^ε -(cholyl))-GLP-1(7-39);

Gly ⁸ Arg ^{26, 34} Lys ³⁶ (N ^ε -(cholyl))-GLP-1(7-39);

Lys ²⁶ (N ^ε -(lithoyl))-GLP-1(7-39);

Lys ³⁴ (N ^ε -(lithoyl))-GLP-1(7-39);

Lys ^26,34 -bis(N ^ε -(lithoyl))-GLP-1(7-39);

Gly ⁸ Lys ²⁶ (N ^ε -(lithoyl))-GLP-1(7-39);

Gly ⁸ Lys ³⁴ (N ^ε -(lithoyl))-GLP-1(7-39);

Gly ⁸ Lys ^26,34 -bis(N ^ε -(lithochyl))-GLP-1(7-39);

Arg ²⁶ Lys ³⁴ (N ^ε -(lithoyl))-GLP-1(7-39);

Gly ⁸ Arg ²⁶ Lys ³⁴ (N ^ε -(cholyl))-GLP-1(7-40);

Lys ²⁶ (N ^ε -(cholyl))Arg ³⁴ -GLP-1(7-40);

Gly ⁸ Lys ²⁶ (N ^ε -(cholyl))Arg ³⁴ -GLP-1(7-40);

Arg ^{26, 34} Lys ³⁶ (N ^ε -(cholyl))-GLP-1(7-40);

Gly ⁸ Arg ^{26, 34} Lys ³⁶ (N ^ε -(cholyl))-GLP-1(7-40);

Lys ²⁶ (N ^ε -(lithoyl))-GLP-1(7-40);

Lys ³⁴ (N ^ε -(lithoyl))-GLP-1(7-40);

Lys ^26,34 -bis(N ^ε -(lithochyl))-GLP-1(7-40);

Gly ⁸ Lys ²⁶ (N ^ε -(lithoyl))-GLP-1(7-40);

Gly ⁸ Lys ³⁴ (N ^ε -(lithoyl))-GLP-1(7-40);

Gly ⁸ Lys ^26,34 -bis(N ^ε -(lithochyl))-GLP-1(7-40);

Arg ²⁶ Lys ³⁴ (N ^ε -(lithoyl))-GLP-1(7-37);

Lys ²⁶ (N ^ε -(lithoyl))-GLP-1(7-36);

Lys ³⁴ (N ^ε -(lithoyl))-GLP-1(7-36);

Lys ^26,34 -bis(N ^ε -(lithoyl))-GLP-1(7-36);

Gly ⁸ Lys ²⁶ (N ^ε -(lithoyl))-GLP-1(7-36);

Gly ⁸ Lys ³⁴ (N ^ε -(lithoyl))-GLP-1(7-36);

Gly ⁸ Lys ^26,34 -bis(N ^ε -(lithochyl))-GLP-1(7-36);

Arg ²⁶ Lys ³⁴ (N ^ε -(lithoyl))-GLP-1(7-36);

Lys ²⁶ (N ^ε -(lithoyl))-GLP-1(7-35);

Lys ³⁴ (N ^ε -(lithoyl))-GLP-1(7-35);

Lys ^26,34 -bis(N ^ε -(lithochyl))-GLP-1(7-35);

Gly ⁸ Lys ²⁶ (N ^ε -(lithoyl))-GLP-1(7-35);

Gly ⁸ Lys ³⁴ (N ^ε -(lithoyl))-GLP-1(7-35);

Gly ⁸ Lys ^26,34 -bis(N ^ε -(lithochyl))-GLP-1(7-35);

Arg ²⁶ Lys ³⁴ (N ^ε -(lithoyl))-GLP-1(7-35);

Lys ²⁶ (N ^ε -(lithoyl))-GLP-1(7-36)amide;

Lys ³⁴ (N ^ε -(lithoyl))-GLP-1(7-36)amide;

Lys ^26,34 -bis(N ^ε -(lithochyl))-GLP-1(7-36)amide;

Gly ⁸ Lys ²⁶ (N ^ε -(lithoyl))-GLP-1(7-36)amide;

Gly ⁸ Lys ³⁴ (N ^ε -(lithoyl))-GLP-1(7-36)amide;

Gly ⁸ Lys ^26,34 -bis(N ^ε -(lithochyl))-GLP-1(7-36)amide;

Arg ²⁶ Lys ³⁴ (N ^ε -(lithoyl))-GLP-1(7-36)amide;

Gly ⁸ Arg ²⁶ Lys ³⁴ (N ^ε -(lithoyl))-GLP-1(7-37);

Lys ²⁶ (N ^ε -(lithoyl))Arg ³⁴ -GLP-1(7-37);

Gly ⁸ Lys ²⁶ (N ^ε -(lithoyl))Arg ³⁴ -GLP-1(7-37);

Arg ^{26, 34} Lys ³⁶ (N ^ε -(lithoyl))-GLP-1(7-37);

Arg ^{26, 34} Lys ³⁸ (N ^ε -(lithoyl))-GLP-1(7-37);

Gly ⁸ Arg ^{26, 34} Lys ³⁶ (N ^ε -(lithochyl))-GLP-1(7-37);

Gly ⁸ Arg ²⁶ Lys ³⁴ (N ^ε -(lithoyl))-GLP-1(7-38);

Lys ²⁶ (N ^ε -(lithoyl))Arg ³⁴ -GLP-1(7-38);

Gly ⁸ Lys ²⁶ (N ^ε -(lithoyl))Arg ³⁴ GLP-1(7-38);

Arg ^{26, 34} Lys ³⁶ (N ^ε -(lithoyl))-GLP-1(7-38);

Arg ^{26, 34} Lys ³⁸ (N ^ε -(lithoyl))-GLP-1(7-38);

Gly ⁸ Arg ^{26, 34} Lys ³⁶ (N ^ε -(lithochyl))-GLP-1(7-38);

Gly ⁸ Arg ²⁶ Lys ³⁴ (N ^ε -(lithoyl))-GLP-1(7-39);

Lys ²⁶ (N ^ε -(lithoyl))Arg ³⁴ -GLP-1(7-39);

Gly ⁸ Lys ²⁶ (N ^ε -(lithoyl))Arg ³⁴ -GLP-1(7-39);

Arg ^{26, 34} Lys ³⁶ (N ^ε -(lithoyl))-GLP-1(7-39);

Gly ⁸ Arg ^{26, 34} Lys ³⁶ (N ^ε -(lithochyl))-GLP-1(7-39);

Gly ⁸ Arg ²⁶ Lys ³⁴ (N ^ε -(lithoyl))-GLP-1(7-40);

Lys ²⁶ (N ^ε -(lithoyl))Arg ³⁴ -GLP-1(7-40);

Gly ⁸ Lys ²⁶ (N ^ε -(lithoyl))Arg ³⁴ -GLP-1(7-40);

Arg ^26,34 Lys ³⁶ (N ^ε -(lithoyl))-GLP-1(7-40); and

Gly ⁸ Arg ^26,34 Lys ³⁶ (N ^ε -(lithochyl))-GLP-1(7-40).

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

In a further preferred embodiment, the invention relates to the use of a GLP-1 derivative according to the invention for the preparation of a medicament with a longer duration of action profile than GLP-1(7-37).

In a further preferred embodiment, the present invention relates to the use of the GLP-1 derivatives according to the present invention in the preparation of a medicament for the treatment of non-insulin-dependent diabetes mellitus with a longer duration of action.

In a further preferred embodiment, the present invention relates to the use of the GLP-1 derivatives according to the present invention in the preparation of a medicament for the treatment of insulin-dependent diabetes mellitus with a longer duration of action.

In a further preferred embodiment, the present invention relates to the use of a GLP-1 derivative according to the invention for the preparation of a medicament for the treatment of obesity with a longer-lasting effect.

In a further preferred embodiment, the present invention relates to a method of treating insulin-dependent or non-insulin-dependent diabetes mellitus in a patient in need of such treatment, the method comprising administering to the patient a therapeutically effective amount of GLP according to claim 1 -1 derivative and a pharmaceutically acceptable carrier.

Detailed description of the invention

In order to obtain a sufficiently long-term action profile of the GLP-1 derivative, the lipophilic substituent attached to the GLP-1 moiety preferably contains 4-40 carbon atoms, especially 8-25 carbon atoms. The lipophilic substituent may be attached to the amino group of the GLP-1 moiety through the formation of an amide bond between its carboxyl group and the amino group of the amino acid residue to which it is attached. Or alternatively, the lipophilic substituent may be attached to the amino acid residue via an amide bond formed between its amino group and the carboxyl group of said amino acid residue. Alternatively, the lipophilic substituent may be attached to the GLP-1 moiety through an ester linkage. Normally, an ester linkage can be formed by the reaction between the carboxyl group of the GLP-1 moiety and the hydroxyl group of the substituent, or by the reaction between the hydroxyl group of the GLP-1 moiety and the carboxyl group of the substituent. Alternatively, the lipophilic substituent may be an alkyl group introduced into the primary amino group of the GLP-1 moiety.

In a preferred embodiment of the invention, the lipophilic substituent is attached to the GLP-1 moiety via the formation of an amide bond between a carboxyl group of a spacer and an amino group of the GLP-1 moiety. Examples of suitable spacers are succinic acid, Lys, Glu or Asp, or a dipeptide such as Gly-Lys. When the spacer is succinic acid, one of its carboxyl groups can form an amide bond with an amino group of the amino acid residue, and its other carboxyl group can form an amide bond with the amino group of the lipophilic substituent. When the spacer is Lys, Glu or Asp, its carboxyl group can form an amide bond with the amino group of the amino acid residue, and its amino group can form an amide bond with the carboxyl group of the lipophilic substituent. When Lys is used as a spacer, an additional spacer can be inserted between the ε-amino group of Lys and the lipophilic substituent in some cases. In a preferred embodiment, this additional spacer group is succinic acid, which forms an amide bond with the ε-amino group of Lys and the amino group present in the lipophilic substituent. In another preferred embodiment, this additional spacer group is Glu or Asp, which forms one amide bond with the ε-amino group of Lys and another amide bond with the carboxyl group present in the lipophilic substituent, i.e. the The lipophilic substituent is an ^Nε -acylated lysine residue.

In another preferred embodiment of the invention, the lipophilic substituent has a group which can be negatively charged. A preferred group that can be negatively charged is carboxyl.

The parent peptide may be prepared by a method comprising culturing a host cell containing the DNA sequence encoding the polypeptide and capable of expressing the peptide in a suitable nutrient medium under conditions that permit expression of the peptide, and then extracting the peptide from the culture The resulting peptides were recovered.

The medium used for culturing the cells may be any conventional medium for culturing the host cells, such as a minimal medium or a complex medium containing appropriate supplements. Suitable media can be obtained commercially or prepared according to published procedures (eg, as described in catalogs of the American Type Culture Collection). Polypeptides produced by the cells can then be recovered from the culture medium by conventional methods, including separation of the host cells from the culture medium by centrifugation or filtration, precipitation of the protein components of the supernatant or filtrate with salts such as ammonium sulfate, according to Various chromatographic methods such as ion exchange chromatography, gel filtration chromatography, affinity chromatography, etc. are used for purification according to the type of the target peptide.

The DNA sequence encoding the parental peptide can be derived from genomic or cDNA, e.g., by preparing a genomic or cDNA library, and following standard techniques (e.g., see Sambrook, J, Fritsch, EF, and Maniatis, T, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989) use synthetic oligonucleotide probes to hybridize and screen out all or part of the DNA sequence encoding the peptide. Alternatively, by established standard methods, such as the phosphoamidite method described by Beaucage and Caruthers (Tetrahedron Letters, 22 (1981), 1859-1869), or the method described by Matthes et al. (European Molecular Biology Organization Journal, 3 (1984), 801-805) to synthesize the DNA sequence encoding the peptide. DNA sequences can also be prepared by polymerase chain reaction using specific primers as described in US 4,683,202 or Saiki et al., Science, 239 (1988), 487-491.

The DNA sequence can be inserted into any vector that facilitates the DNA recombination process, and the choice of vector will often depend on the host cell into which the vector is to be introduced. Thus, the vector may be an autonomously replicating vector, ie, a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid. Alternatively, the vector may be of a type that, when introduced into a host cell, integrates into the host cell genome and replicates with the chromosome into which it has integrated.

The vector is preferably an expression vector, in which the DNA sequence encoding the peptide is operatively linked to other segments required for transcription of the DNA (such as a promoter). The promoter can be any DNA sequence that is transcriptionally active in the host cell of choice and can be derived from a gene encoding a protein either homologous or heterologous to the host cell. Examples of promoters suitable for directing the transcription of DNA encoding the peptide of the present invention in various host cells are well known in the art, see, for example, Sambrook et al., supra.

The DNA sequence encoding the peptide may also be operably linked to appropriate terminators, polyadenylation signals, transcriptional enhancer sequences, and translational enhancer sequences, if desired. The recombinant vector of the present invention may also contain a DNA sequence that enables it to replicate in the intended host cell.

The vector may also contain a selectable marker, such as a gene whose gene product will complement a defect in the host cell, or confer resistance to drugs such as ampicillin, kanamycin, tetracycline, chloramphenicol, neomycin, hygromycin Resistance to antibiotics or methotrexate, etc.

To introduce a parent peptide of the invention into the secretory pathway of a host cell, a secretion signal sequence (also known as a leader sequence, preprosequence or presequence) can be provided in the recombinant vector. The secretory signal sequence is linked in correct reading frame to the DNA sequence encoding the peptide. A secretion signal sequence is usually located 5' to the DNA sequence encoding the peptide. The secretory signal sequence may be that normally linked to the peptide, or may be derived from a gene encoding another secreted protein.

The method for separately linking the DNA sequence encoding the peptide of the present invention, the promoter and the optional terminator and/or the secretion signal sequence, and inserting it into a suitable vector containing the information necessary for replication is known to those skilled in the art The skilled person is known (see eg Sambrook et al., supra).

The host cell into which the DNA sequence or recombinant vector will be introduced may be any cell capable of producing the peptide of the present invention, including bacteria, yeast, fungi and higher eukaryotic cells. Examples of suitable host cells known and used by those skilled in the art include, but are not limited to, Escherichia coli, Saccharomyces cerevisiae, or mammalian BHK or CHO cell lines.

International Patent Application No. WO87/06941 (The General Hospital Corporation) describes examples of compounds that can be used as GLP-1 moieties according to the present invention, which relates to a compound containing GLP-1(7-37) and its functional derivatives A peptide fragment and its use as an insulinotropic agent.

Other GLP-1 analogs are described in International Patent Application No. 90/11296 (The General Hospital corporation), which relates to peptide fragments containing GLP-1(7-36) and its functional derivatives, which have more than Insulinotropic activity of GLP-1(1-36) or GLP-1(1-37), also relating to their use as insulinotropic agents.

International Patent Application No. 91/11457 (Buckley et al.) discloses analogs of active GLP-1 peptides 7-34, 7-35, 7-36 and 7-37 which can also be used as GLP-1 moieties according to the present invention .

pharmaceutical composition

Pharmaceutical compositions containing GLP-1 derivatives according to the invention can be administered parenterally to patients in need of such treatment. Parenteral administration can be by subcutaneous, intramuscular or intravenous injection with a syringe, optionally a pen syringe. Alternatively, parenteral administration can be performed via an infusion pump. Another option is a powder or liquid composition of the GLP-1 derivative for administration in the form of a nasal or pulmonary spray. The GLP-1 derivatives of the present invention can also be administered via a transdermal route, such as through a patch for transdermal administration, and an iontophoretic patch can be selected; or via a transmucosal route, such as through buccal mucosa.

Pharmaceutical compositions containing the GLP-1 derivatives of the present invention can be prepared using conventional techniques, such as those described in Remington's Pharmaceutical Sciences, 1985 or Remington: The Science and Practice of Pharmaceuticals, 19th Edition, 1995.

Accordingly, the injectable compositions of the GLP-1 derivatives of the present invention can be prepared using conventional techniques of the pharmaceutical industry, including dissolving and mixing the components as appropriate to obtain the desired end product.

According to one method, the GLP-1 derivative is dissolved in an amount of water which is slightly less than the final volume of the prepared composition. Isotonic agents, preservatives and buffers are added as necessary, and the pH of the solution is adjusted with an acid such as hydrochloric acid, or a base such as aqueous sodium hydroxide solution as necessary. Finally, adjust the volume of the solution with water to obtain the desired concentration of the components.

Isotonic agents are, for example, sodium chloride, mannitol, glycerin.

Preservatives are, for example, phenol, m-cresol, methylparaben, benzyl alcohol.

Suitable buffers are sodium acetate and sodium phosphate.

In addition to the above ingredients, the solution containing the GLP-1 derivative of the present invention may also contain a surfactant to improve the solubility and stability of the GLP-1 derivative.

Compositions of certain peptides for nasal administration may be prepared as described in European Patent No. 272097 (Nordisk A/S) or WO 93/18785.

According to a preferred embodiment of the present invention, the composition of the GLP-1 derivative is provided in the form of a composition suitable for injectable administration. This composition can be ready-to-use injection, or a certain amount of solid composition, such as a freeze-dried product, which needs to be dissolved in a solvent before injection. The GLP-1 derivative contained in the solution for injection is not less than 2 mg/ml, preferably not less than 5 mg/ml, more preferably not less than 10 mg/ml, and preferably not more than 100 mg/ml.

The GLP-1 derivatives of the present invention can be used in the treatment of various diseases. The particular GLP-1 derivative and optimal dosage level for a patient will depend on the disease being treated and various factors, including the potency of the particular peptide derivative used and the patient's age, weight, physical activity and diet, and will also depend on Depending on the possible combination with other drugs and the severity of the condition. It is recommended that those skilled in the art determine the dosage of the GLP-1 derivative of the present invention for each patient.

In particular, it is expected that GLP-1 derivatives will be useful for the preparation of medicaments for the treatment of non-insulin-dependent diabetes mellitus and/or the treatment of obesity with a longer time action profile.

The present invention can be further illustrated by the following examples, but these examples should not be considered as limiting the scope of protection. The features disclosed in the foregoing description and the following embodiments are, individually or in any combination, important for implementing the invention in various ways.

Example

The following abbreviations for commercially available chemicals are used:

DMF: N,N-Dimethylformamide

NMP: N-methyl-2-pyrrolidone

EDPA: N-ethyl-N,N-diisopropylamine

EGTA: Ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid

GTP: Guanosine 5'-triphosphate

TFA: Trifluoroacetic acid

THF: Tetrahydrofuran

Myr-ONSu: 2,5-dioxopyrrolidin-1-yl myristate

Pal-ONSu: 2,5-dioxopyrrolidin-1-yl hexadecanoate

Ste-ONSu: 2,5-dioxopyrrolidin-1-yl octadecanoate

HOOC-(CH ₂ ) ₆ -COONSu: 2,5-dioxopyrrolidin-1-yl ω-carboxyheptanoate

HOOC-(CH ₂ ) ₁₀ -COONSu: ω-carboxyundecanoic acid 2,5-dioxopyrrolidin-1-yl ester

HOOC-(CH ₂ ) ₁₂ -COONSu: ω-carboxytridecanoic acid 2,5-dioxopyrrolidin-1-yl ester

HOOC-(CH ₂ ) ₁₄ -COONSu: ω-carboxypentadecanoic acid 2,5-dioxopyrrolidin-1-yl ester

HOOC-(CH ₂ ) ₁₆ -COONSu: 2,5-dioxopyrrolidin-1-yl ω-carboxyheptadecanoate

HOOC-(CH ₂ ) ₁₈ -COONSu: ω-carboxynonadecanoic acid 2,5-dioxopyrrolidin-1-yl ester

abbreviation:

PDMS: Plasma Desorption Mass Spectormetry

MALDI-MS: Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

HPLC: High Performance Liquid Chromatography

amu: atomic mass unit

analyze

plasma desorption mass spectrometry

Sample Preparation:

The samples were dissolved in 0.1% TFA/EtOH (1:1) to a concentration of 1 μg/μl. The sample solution (5-10 μl) was placed on a nitrocellulose target (Bio-ion AB, Uppsala, Sweden) and allowed to absorb on the surface of the target for 2 minutes. Targets were then washed with 2 x 25 μl 0.1% TFA and spin dried. Finally the nitrocellulose target is placed in the target rotator and placed into the mass spectrometer.

Mass Spectrometry

PDMS analysis was performed with a Bio-ion 20 time-of-flight device (Bio-ion Nordic AB, Uppsala, Sweden). Using an accelerating voltage of 15kV, the molecular ions formed by the bombardment of 252-Cf fission fragments on the surface of nitrocellulose are accelerated to a terminal detector. The resulting time-of-flight spectrum was corrected to a true mass spectrum using H ⁺ and NO ⁺ ions at m/z 1 and 30, respectively. Typically 1.0 x ¹⁰⁶ fissions accumulated in 15-20 min to mass spectrometry. The measured masses all correspond to isotopic average molecular masses. The accuracy of mass determination is generally better than 0.1%.

MALDI-MS

MALDI-MS analysis was performed using a Voyager RP instrument (PerSeptive Biosystems Inc., Framingham, MA) equipped with a delayed separation device operating in a linear fashion. Mass determination was performed based on an external calibration method using α-cyano-4-hydroxy-cinnamic acid as a matrix.

example 1

Synthesis of Lys ²⁶ (N ^ε -tetradecanoyl)-GLP-1(7-37)

The target compound was synthesized from GLP-1(7-37). A mixture of GLP-1(7-37) (25 mg, 7.45 μm), EDPA (26.7 mg, 208 μm), NMP (520 μl) and water (260 μl) was gently shaken at room temperature for 5 minutes. To the resulting mixture was added a solution of Myr-ONSu (2.5 mg, 7.67 μm) dissolved in NMP (62.5 μl), and the reaction mixture was gently shaken at room temperature for 5 minutes and then left for 20 minutes. A solution of Myr-ONSu (2.5 mg, 7.67 μm) dissolved in NMP (62.5 μl) was further added, and the resulting mixture was gently shaken for 5 minutes. After a total reaction time of 40 minutes, a solution prepared by adding glycine (12.5 mg, 166 μmol) dissolved in 50% aqueous ethanol (12.5 ml) was added to terminate the reaction. Use cyanopropyl (cyanopropyl) column (Zorbax 300SB-CN ) and a standard acetonitrile/TFA system, the target compound was isolated from the reaction mixture by HPLC with a yield of 1.3 mg (equivalent to 4.9% of theoretical yield). The column was heated to 65°C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The isolated product was analyzed by PDMS method, and the m/z value of the protonated molecular ion peak was found to be 3567.9±3. The resulting molecular weight was thus 3566.9 ± 3 amu (theoretical 3565.9 amu). The target compound was cleaved by Staphylococcus aureus V8 protease, followed by mass determination of the peptide fragment by PDMS to determine the acylation position (Lys26).

In addition to the target compound, two other GLP-1 derivatives were separated from the reaction mixture using the same chromatographic column and a narrower gradient (35-38% acetonitrile in 60 minutes). See Examples 2 and 3.

Example 2

Synthesis of Lys ³⁴ (N ^ε -tetradecanoyl)-GLP-1(7-37)

The title compound was isolated from the reaction mixture described in Example 1 by HPLC. The protonated molecular ion peak with m/z of 3567.7±3 was obtained by PDMS analysis. The molecular weight was thus found to be 3566.7 ± 3 amu (theoretical 3565.9 amu). The position of acylation is determined according to the fragmentation mode.

Example 3

Synthesis of Lys ^26,34 -bis(N ^ε -tetradecanoyl)-GLP-1(7-37)

The title compound was isolated from the reaction mixture described in Example 1 by HPLC. PDMS analysis yielded a protonated molecular ion peak with m/z 3778.4±3. The molecular weight was thus found to be 3777.4 ± 3 amu (theoretical 3776.1 amu).

Example 4

Synthesis of Lys ²⁶ (N ^ε -tetradecyl)Arg ³⁴ -GLP-1(7-37)

The target compound was synthesized from Arg ³⁴ -GLP-1(7-37). A mixture of Arg ³⁴ -GLP-1(7-37) (5 mg, 1.47 μm), EDPA (5.3 mg, 41.1 μm), NMP (105 μl) and water (50 μl) was gently shaken at room temperature for 5 minutes. To the resulting mixture was added a solution of Myr-ONSu (0.71 mg, 2.2 μm) dissolved in NMP (17.8 μl), and the reaction mixture was gently shaken at room temperature for 5 minutes and then left for 20 minutes. After a total reaction time of 30 minutes, a solution prepared by dissolving glycine (25 mg, 33.3 µm) in 50% aqueous ethanol (2.5 ml) was added thereto to terminate the reaction. The reaction mixture was purified by HPLC as described in Example 1. PDMS analysis yielded a protonated molecular ion peak with an m/z value of 3594.9±3. The resulting molecular weight was 3593.9 ± 3 amu (theoretical 3593.9 amu).

Example 5

Synthesis of Gly ⁸ Arg ^26, 34 Lys ³⁶ (N ^ε -tetradecanoyl)-GLP-1(7-37)

The target compound was synthesized from Gly ⁸ Arg ^26,34 Lys ³⁶ -GLP-1(7-37) purchased from QCB. A mixture of Gly ⁸ Arg ^26,34 Lys ³⁶ -GLP-1(7-37) (1.3 mg, 0.39 μm), EDPA (1.3 mg, 10 μm), NMP (125 μl) and water (30 μl) was gently shaken at room temperature for 5 minute. To the resulting mixture was added a solution of Myr-ONSu (0.14 mg, 0.44 μm) dissolved in NMP (3.6 ml), and the reaction mixture was gently shaken at room temperature for 15 minutes. A solution prepared by dissolving glycine (0.1 mg, 1.33 µm) in 50% aqueous ethanol (10 µl) was added thereto to terminate the reaction. The reaction mixture was purified by HPLC, and the title compound (60 μg, 4%) was isolated.

Example 6

Synthesis of Arg ^{26, 34} Lys ³⁶ (N ^ε -tetradecanoyl)-GLP-1(7-37)-OH

A mixture of Arg ^26,34 Lys ³⁶ -GLP-1(7-37)-OH (5.0 mg, 1.477 μmol), EDPA (5.4 mg, 41.78 μmol), NMP (105 μl) and water (50 μl) was gently shaken at room temperature 5 minutes. To the resulting mixture was added a solution of Myr-ONSu (0.721 mg, 2.215 μmol) dissolved in NMP (18 μl). The reaction mixture was gently shaken at room temperature for 5 minutes and allowed to remain at room temperature for an additional 45 minutes. A solution prepared by dissolving glycine (2.5 mg, 33.3 µmol) in 50% aqueous ethanol (250 µl) was added thereto to terminate the reaction. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax300SB-CN) and a standard acetonitrile/TFA system. The column was heated to 65°C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The target compound (1.49 mg, 28%) was isolated and the product was analyzed by PDMS method. The m/z value of the protonated molecular ion peak was found to be 3595±3. The resulting molecular weight was thus 3594 ± 3 amu (theoretical 3594 amu).

Example 7

Synthesis of Lys ^26,34 bis(N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-37)-OH

A mixture of GLP-1(7-37)-OH (70 mg, 20.85 μmol), EDPA (75.71 mg, 585.8 μmol), NMP (1.47 ml) and water (700 μl) was gently shaken at room temperature for 10 minutes. A solution of HOOC-(CH ₂ ) ₁₈ -COONSu (27.44 mg, 62.42 μmol) dissolved in NMP (686 μl) was added to the resulting mixture, and the reaction mixture was gently shaken at room temperature for 5 minutes and allowed to stand at room temperature for an additional 50 minutes . A solution prepared by dissolving glycine (34.43 mg, 458.7 µmol) in 50% aqueous ethanol (3.44 ml) was added thereto to terminate the reaction. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile/TFA system. The column was heated to 65°C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The target compound (8.6 mg, 10%) was isolated and the product was analyzed by PDMS method. The m/z value of the protonated molecular ion peak was found to be 4006±3. The resulting molecular weight was 4005 ± 3 amu (theoretical 4005 amu).

Example 8

Synthesis of Arg ^{26, 34} Lys ³⁶ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-36)-OH

A mixture of Arg ^{26, 34} Lys ³⁶ -GLP-1(7-36)-OH (5.06mg, 1.52μmol), EDPA (5.5mg, 42.58μmol), NMP (106μl) and water (100μl) was heated at room temperature Shake for 5 minutes. A solution of HOOC-(CH ₂ ) ₁₈ -COONSu (1.33 mg, 3.04 μmol) dissolved in NMP (33.2 μl) was added to the resulting mixture, and the reaction mixture was gently shaken at room temperature for 5 minutes and then left at room temperature for another 2.5 hours. A solution prepared by dissolving glycine (2.50 mg, 33.34 µmol) in 50% aqueous ethanol (250 µl) was added thereto to terminate the reaction. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile/TFA system. The column was heated to 65°C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The title compound (0.46 mg, 8%) was isolated and the product was analyzed by PDMS method. The m/z value of the protonated molecular ion peak was found to be 3652±3. This gave a molecular weight of 3651 ± 3 amu (theoretical 3651 amu).

Example 9

Synthesis of Arg ^{26, 34} Lys ³⁸ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-38)-OH

A mixture of Arg ^26,34 Lys ³⁸ -GLP-1(7-38)-OH (5.556 mg, 1.57 μmol), EDPA (5.68 mg, 43.96 μmol), NMP (116.6 μl) and water (50 μl) was heated at room temperature Shake for 10 minutes. A solution of HOOC-(CH ₂ ) ₁₈ -COONSu (1.38 mg, 3.14 μmol) dissolved in NMP (34.5 μl) was added to the resulting mixture, and the reaction mixture was gently shaken at room temperature for 5 minutes and then left at room temperature for another 2.5 hours. A solution prepared by dissolving glycine (2.5 mg, 33.3 µmol) in 50% aqueous ethanol (250 µl) was added thereto to terminate the reaction. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile/TFA system. The column was heated to 65°C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The title compound (0.7 mg, 12%) was isolated and the product was analyzed by PDMS method. The m/z value of the protonated molecular ion peak was found to be 3866±3. This gave a molecular weight of 3865 ± 3 amu (theoretical 3865 amu).

Example 10

Synthesis of Arg ³⁴ Lys ²⁶ (N ^ε -(ω-carboxynonadecanoyl))-GLP-1(7-37)-OH

A mixture of Arg ³⁴ -GLP-1(7-37)-OH (5.04 mg, 1.489 μmol), EDPA (5.39 mg, 41.70 μmol), NMP (105 μl) and water (50 μl) was gently shaken at room temperature for 10 minutes. A solution obtained by dissolving HOOC-(CH ₂ ) ₁₈ -COONSu (1.31 mg, 2.97 μmol) in NMP (32.8 μl) was added to the resulting mixture, and the reaction mixture was gently shaken at room temperature for 5 minutes and then left at room temperature for another 30 minutes . A solution prepared by dissolving glycine (2.46 mg, 32.75 µmol) in 50% ethanol aqueous solution (246 µl) was added thereto to terminate the reaction. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile/TFA system. The column was heated to 65°C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The title compound (1.2 mg, 22%) was isolated and the product was analyzed by PDMS method. The m/z value of the protonated molecular ion peak was found to be 3709±3. This gave a molecular weight of 3708 ± 3 amu (theoretical 3708 amu).

Example 11

Synthesis of Arg ³⁴ Lys ²⁶ (N ^ε -(ω-carboxyheptadecanoyl))-GLP-1(7-37)-OH

A mixture of Arg ³⁴ -GLP-1(7-37)-OH (5.8 mg, 1.714 μmol), EDPA (6.20 mg, 47.99 μmol), NMP (121.8 μl) and water (58 μl) was gently shaken at room temperature for 10 minutes. A solution of HOOC-(CH ₂ ) ₁₆ -COONSu (2.11 mg, 5.142 μmol) dissolved in NMP (52.8 μl) was added to the resulting mixture, and the reaction mixture was gently shaken at room temperature for 5 minutes and then left at room temperature for another 2 hours. A solution prepared by dissolving glycine (2.83 mg, 37.70 µmol) in 50% aqueous ethanol (283 µl) was added thereto to terminate the reaction. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile/TFA system. The column was heated to 65°C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The title compound (0.81 mg, 13%) was isolated and the product was analyzed by PDMS method. The m/z value of the protonated molecular ion peak was found to be 3681±3. This gave a molecular weight of 3680±3 amu (theoretical 3680 amu).

Example 12

Synthesis of Arg ^{26, 34} Lys ³⁶ (N ^ε -(ω-carboxyheptadecanoyl))-GLP-1(7-37)-OH

A mixture of Arg ^26,34 Lys ³⁶ -GLP-1(7-37)-OH (3.51mg, 1.036μmol), EDPA (3.75mg, 29.03μmol), NMP (73.8μl) and water (35μl) was heated at room temperature Shake for 10 minutes. A solution obtained by dissolving HOOC-(CH ₂ ) ₁₆ -COONSu (1.27 mg, 3.10 μmol) in NMP (31.8 μl) was added to the resulting mixture, and the reaction mixture was gently shaken at room temperature for 5 minutes, then left at room temperature for another 2 hours 10 minute. A solution prepared by dissolving glycine (1.71 mg, 22.79 µmol) in 50% aqueous ethanol (171 µl) was added thereto to terminate the reaction. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile/TFA system. The column was heated to 65°C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The title compound (0.8 mg, 21%) was isolated and the product was analyzed by PDMS method. The m/z value of the protonated molecular ion peak was found to be 3682±3. This gave a molecular weight of 3681 ± 3 amu (theoretical 3681 amu).

Example 13

Synthesis of Arg ^{26, 34} Lys ³⁸ (N ^ε -(ω-carboxyheptadecanoyl))-GLP-1(7-38)-OH

A mixture of Arg ^26,34 Lys ³⁸ -GLP-1(7-38)-OH (5.168 mg, 1.459 μmol), EDPA (5.28 mg, 40.85 μmol), NMP (108.6 μl) and water (51.8 μl) was incubated at room temperature Shake gently for 10 minutes. A solution of HOOC-(CH ₂ ) ₁₆ -COONSu (1.80 mg, 4.37 μmol) dissolved in NMP (45 μl) was added to the resulting mixture, and the reaction mixture was gently shaken at room temperature for 10 minutes, then left at room temperature for another 2 hours and 15 minutes . A solution prepared by dissolving glycine (2.41 mg, 32.09 µmol) in 50% ethanol aqueous solution (241 µl) was added thereto to terminate the reaction. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile/TFA system. The column was heated to 65°C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The title compound (0.8 mg, 14%) was isolated and the product was analyzed by PDMS method. The m/z value of the protonated molecular ion peak was found to be 3838±3. This gave a molecular weight of 3837 ± 3 amu (theoretical 3837 amu).

Example 14

Synthesis of Arg ^{26, 34} Lys ³⁶ (N ^ε -(ω-carboxyheptadecanoyl))-GLP-1(7-36)-OH

A mixture of Arg ^26,34 Lys ³⁶ -GLP-1(7-36)-OH (24.44 mg, 7.34 μmol), EDPA (26.56 mg, 205.52 μmol), NMP (513 μl) and water (244.4 μl) was heated at room temperature Shake for 5 minutes. A solution of HOOC-(CH ₂ ) ₁₆ -COONSu (9.06 mg, 22.02 μmol) dissolved in NMP (1.21 ml) was added to the resulting mixture, and the reaction mixture was gently shaken at room temperature for 5 minutes and then left at room temperature for another 30 minutes. A solution prepared by dissolving glycine (12.12 mg, 161.48 µmol) in 50% aqueous ethanol (1.21 ml) was added thereto to terminate the reaction. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile/TFA system. The column was heated to 65°C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The title compound (7.5 mg, 28%) was isolated and the product was analyzed by PDMS method. The m/z value of the protonated molecular ion peak was found to be 3625±3. This gave a molecular weight of 3624±3 amu (theoretical 3624 amu).

Example 15

Synthesis of Arg ^{26, 34} Lys ³⁶ (N ^ε -(ω-carboxyundecanoyl))-GLP-1(7-37)-OH

A mixture of Arg ^26,34 Lys ³⁶ -GLP-1(7-37)-OH (4.2 mg, 1.24 μmol), EDPA (4.49 mg, 34.72 μmol), NMP (88.2 μl) and water (42 μl) was heated at room temperature Shake for 10 minutes. A solution of HOOC-(CH ₂ ) ₁₀ -COONSu (1.21 mg, 3.72 μmol) dissolved in NMP (30.25 μl) was added to the resulting mixture, and the reaction mixture was gently shaken at room temperature for 5 minutes and then left at room temperature for another 40 minutes. A solution prepared by dissolving glycine (2.04 mg, 27.28 µmol) in 50% aqueous ethanol (204 µl) was added thereto to terminate the reaction. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile/TFA system. The column was heated to 65°C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The target compound (0.8 mg, 18%) was isolated and the product was analyzed by PDMS method. The m/z value of the protonated molecular ion peak was found to be 3598±3. This gave a molecular weight of 3597±3 amu (theoretical 3597 amu).

Example 16

Synthesis of Arg ^{26, 34} Lys ³⁸ (N ^ε -(ω-carboxyundecanoyl))-GLP-1(7-38)-OH

A mixture of Arg ^26,34 Lys ³⁸ -GLP-1(7-38)-OH (5.168 mg, 1.46 μmol), EDPA (5.28 mg, 40.88 μmol), NMP (108.6 μl) and water (51.7 μl) at room temperature Shake gently for 10 minutes. A solution of HOOC-(CH ₂ ) ₁₀ -COONSu (1.43 mg, 4.38 μmol) dissolved in NMP (35.8 μl) was added to the resulting mixture, and the reaction mixture was gently shaken at room temperature for 5 minutes and then left at room temperature for another 50 minutes. A solution prepared by dissolving glycine (2.41 mg, 32.12 µmol) in 50% aqueous ethanol (241 µl) was added thereto to terminate the reaction. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile/TFA system. The column was heated to 65°C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The title compound (0.85 mg, 16%) was isolated and the product was analyzed by PDMS method. The m/z value of the protonated molecular ion peak was found to be 3753±3. This gave a molecular weight of 3752±3 amu (theoretical 3752 amu).

Example 17

Synthesis of Arg ^{26, 34} bis(N ^ε -(ω-carboxyundecanoyl))-GLP-1(7-37)-OH

A mixture of GLP-1(7-37)-OH (10.0 mg, 2.98 μmol), EDPA (10.8 mg, 83.43 μmol), NMP (210 μl) and water (100 μl) was gently shaken at room temperature for 10 minutes. A solution of HOOC-(CH ₂ ) ₁₀ -COONSu (2.92 mg, 8.94 μmol) dissolved in NMP (73 μl) was added to the resulting mixture, and the reaction mixture was gently shaken at room temperature for 5 minutes and then left at room temperature for another 50 minutes. A solution prepared by dissolving glycine (4.92 mg, 65.56 µmol) in 50% aqueous ethanol (492 µl) was added thereto to terminate the reaction. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile/TFA system. The column was heated to 65°C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The title compound (1.0 mg, 9%) was isolated and the product was analyzed by PDMS method. The m/z value of the protonated molecular ion peak was found to be 3781±3. This gave a molecular weight of 3780±3 amu (theoretical 3780 amu).

Example 18

Synthesis of Arg ^{26, 34} Lys ³⁶ (N ^ε -(ω-carboxyundecanoyl))-GLP-1(7-36)-OH

A mixture of Arg ^26,34 Lys ³⁶ -GLP-1(7-36)-OH (15.04 mg, 4.52 μmol), EDPA (16.35 mg, 126.56 μmol), NMP (315.8 μl) and water (150.4 μl) was incubated at room temperature Shake gently for 10 minutes. A solution of HOOC-(CH ₂ ) ₁₀ -COONSu (4.44 mg, 13.56 μmol) dissolved in NMP (111 μl) was added to the resulting mixture, and the reaction mixture was gently shaken at room temperature for 5 minutes and then left at room temperature for another 40 minutes. A solution prepared by dissolving glycine (7.5 mg, 99.44 µmol) in 50% aqueous ethanol (250 µl) was added thereto to terminate the reaction. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile/TFA system. The column was heated to 65°C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The title compound (3.45 mg, 22%) was isolated and the product was analyzed by PDMS method. The m/z value of the protonated molecular ion peak was found to be 3540±3. This gave a molecular weight of 3539±3 amu (theoretical 3539 amu).

Example 19

Synthesis of Arg ³⁴ Lys ²⁶ (N ^ε -(ω-carboxyundecanoyl))-GLP-1(7-37)-OH

A mixture of Arg ³⁴ -GLP-1(7-37)-OH (5.87 mg, 1.73 μmol), EDPA (6.27 mg, 48.57 μmol), NMP (123.3 μl) and water (58.7 μl) was gently shaken at room temperature for 10 minutes . A solution of HOOC-(CH ₂ ) ₁₀ -COONSu (1.70 mg, 5.20 μmol) dissolved in NMP (42.5 μl) was added to the resulting mixture, and the reaction mixture was gently shaken at room temperature for 5 minutes and then left at room temperature for another 40 minutes. A solution prepared by dissolving glycine (2.86 mg, 286 µmol) in 50% ethanol aqueous solution (286 µl) was added thereto to terminate the reaction. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile/TFA system. The column was heated to 65°C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The title compound (1.27 mg, 20%) was isolated and the product was analyzed by PDMS method. The m/z value of the protonated molecular ion peak was found to be 3597±3. This gave a molecular weight of 3596±3 amu (theoretical 3596 amu).

Example 20

Synthesis of Arg ³⁴ Lys ²⁶ (N ^ε -(ω-carboxyheptanoyl))-GLP-1(7-37)-OH

A mixture of ^Arg34 -GLP-1(7-37)-OH (4.472 mg, 1.32 μmol), EDPA (4.78 mg, 36.96 μmol), NMP (94 μl) and water (44.8 μl) was gently shaken at room temperature for 5 minutes. A solution obtained by dissolving HOOC-(CH ₂ ) ₆ -COONSu (1.07 mg, 3.96 μmol) in NMP (26.8 μl) was added to the resulting mixture, and the reaction mixture was gently shaken at room temperature for 5 minutes, and then left at room temperature for 1 hour 50 minute. A solution prepared by dissolving glycine (2.18 mg, 29.04 µmol) in 50% aqueous ethanol (218 µl) was added thereto to terminate the reaction. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile/TFA system. The column was heated to 65°C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The title compound (0.5 mg, 11%) was isolated and the product was analyzed by PDMS method. The m/z value of the protonated molecular ion peak was found to be 3540±3. This gave a molecular weight of 3539±3 amu (theoretical 3539 amu).

Example 21

Synthesis of Arg ^{26, 34} Lys ³⁸ (N ^ε -(ω-carboxyheptanoyl))-GLP-1(7-38)-OH

A mixture of Arg ^26,34 Lys ³⁸ -GLP-1(7-38)-OH (5.168 mg, 1.459 μmol), EDPA (5.28 mg, 40.85 μmol), NMP (108.6 μl) and water (51.6 μl) at room temperature Shake gently for 10 minutes. A solution obtained by dissolving HOOC-(CH ₂ ) ₆ -COONSu (1.18 mg, 4.37 μmol) in NMP (29.5 μl) was added to the resulting mixture, and the reaction mixture was gently shaken at room temperature for 5 minutes, then left at room temperature for another 1 hour 50 minute. A solution prepared by dissolving glycine (2.40 mg, 32.09 µmol) in 50% aqueous ethanol (240 µl) was added thereto to terminate the reaction. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN and a standard acetonitrile/TFA system. The column was heated to 65 °C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The target compound was isolated (0.5 mg, 9%), the product was analyzed by PDMS method. It was found that the m/z value of the protonated molecular ion peak was 3697 ± 3. Thus, the molecular weight was 3695 ± 3 amu (theoretical value was 3695 amu).

Example 22

Synthesis of Arg ^{26, 34} Lys ³⁶ (N ^ε -(ω-carboxyheptanoyl))-GLP-1(7-37)-OH

A mixture of Arg ^26,34 Lys ³⁶ -GLP-1(7-37)-OH (5.00 mg, 1.47 μmol), EDPA (5.32 mg, 41.16 μmol), NMP (105 μl) and water (50 μl) was gently shaken at room temperature 5 minutes. A solution of HOOC-(CH ₂ ) ₆ -COONSu (1.19 mg, 4.41 μmol) dissolved in NMP (29.8 μl) was added to the resulting mixture, and the reaction mixture was gently shaken at room temperature for 5 minutes and then left at room temperature for another 2 hours. A solution prepared by dissolving glycine (2.42 mg, 32.34 µmol) in 50% aqueous ethanol (242 µl) was added thereto to terminate the reaction. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile/TFA system. The column was heated to 65°C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The title compound (0.78 mg, 15%) was isolated and the product was analyzed by PDMS method. The m/z value of the protonated molecular ion peak was found to be 3542±3. This gave a molecular weight of 3541 ± 3 amu (theoretical 3541 amu).

Example 23

Synthesis of Arg ^{26, 34} Lys ³⁶ (N ^ε -(ω-carboxyheptanoyl))-GLP-1(7-36)-OH

A mixture of Arg ^26,34 Lys ³⁶ -GLP-1(7-36)-OH (5.00 mg, 1.50 μmol), EDPA (5.44 mg, 42.08 μmol), NMP (210 μl) and water (50 μl) was gently shaken at room temperature 5 minutes. A solution of HOOC-(CH ₂ ) ₆ -COONSu (1.22 mg, 4.5 μmol) dissolved in NMP (30.5 μl) was added to the resulting mixture, and the reaction mixture was gently shaken at room temperature for 5 minutes and then left at room temperature for another 2 hours. A solution prepared by dissolving glycine (2.47 mg, 33.0 µmol) in 50% aqueous ethanol (247 µl) was added thereto to terminate the reaction. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300sB-CN) and a standard acetonitrile/TFA system. The column was heated to 65°C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The title compound (0.71 mg, 14%) was isolated and the product was analyzed by PDMS method. The m/z value of the protonated molecular ion peak was found to be 3484±3. This gave a molecular weight of 3483 ± 3 amu (theoretical 3483 amu).

Example 24

Synthesis of Arg ^26,34 bis(N ^ε -(ω-carboxyheptanoyl))-GLP-1(7-37)-OH

A mixture of GLP-1(7-37)-OH (10 mg, 2.5 μmol), EDPA (10.8 mg, 83.56 μmol), NMP (210 μl) and water (100 μl) was gently shaken at room temperature for 10 minutes. A solution obtained by dissolving HOOC-(CH ₂ ) ₆ -COONSu (2.42 mg, 8.92 μmol) in NMP (60.5 μl) was added to the resulting mixture, and the reaction mixture was gently shaken at room temperature for 5 minutes, then left at room temperature for another 2 hours 35 minute. A solution prepared by dissolving glycine (4.92 mg, 65.54 µmol) in 50% aqueous ethanol (492 µl) was added thereto to terminate the reaction. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile/TFA system. The column was heated to 65°C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The title compound (2.16 mg, 24%) was isolated and the product was analyzed by PDMS method. The m/z value of the protonated molecular ion peak was found to be 3669±3. The resulting molecular weight was 3668 ± 3 amu (theoretical 3668 amu).

Example 25

Synthesis of Arg ³⁴ Lys ²⁶ (N ^ε -(ω-carboxypentadecanoyl))-GLP-1(7-37)-OH

A mixture of ^Arg34 -GLP-1(7-37)-OH (4.472 mg, 1.321 μmol), EDPA (4.78 mg, 36.99 μmol), NMP (93.9 μl) and water (44.7 μl) was gently shaken at room temperature for 10 minutes . A solution of HOOC-(CH ₂ ) ₁₄ -COONSu (1.519 mg, 3.963 μmol) dissolved in NMP (38 μl) was added to the resulting mixture, and the reaction mixture was gently shaken at room temperature for 5 minutes and then left at room temperature for another 1 hour. A solution prepared by dissolving glycine (2.18 mg, 29.06 µmol) in 50% aqueous ethanol (218 µl) was added thereto to terminate the reaction. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax300SB-CN and a standard acetonitrile/TFA system. The column was heated to 65 °C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The target compound ( 0.58mg, 12%), analyze product with PDMS method. Find that the m/z value of the molecular ion peak of protonation is 3654 ± 3. Thus drawing molecular weight is 3653 ± 3amu (theoretical value is 3653amu).

Example 26

Synthesis of Arg ^{26, 34} Lys ³⁶ (N ^ε -(ω-carboxyheptanoyl))-GLP-1(7-36)-OH

A mixture of Arg ^26,34 Lys ³⁶ -GLP-1(7-36)-OH (5.00 mg, 1.50 μmol), EDPA (5.44 mg, 42.08 μmol), NMP (210 μl) and water (50 μl) was gently shaken at room temperature 5 minutes. A solution of HOOC-(CH ₂ ) ₁₄ -COONSu (1.72 mg, 4.5 μmol) dissolved in NMP (43 μl) was added to the resulting mixture, and the reaction mixture was gently shaken at room temperature for 5 minutes and then left at room temperature for another 1 hour. A solution prepared by dissolving glycine (2.48 mg, 33 µmol) in 50% aqueous ethanol (248 µl) was added thereto to terminate the reaction. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile/TFA system. The column was heated to 65°C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The title compound (0.58 mg, 11%) was isolated and the product was analyzed by PDMS method. The m/z value of the protonated molecular ion peak was found to be 3596±3. This gave a molecular weight of 3595 ± 3 amu (theoretical 3595 amu).

Example 27

Synthesis of 2,5-dioxopyrrolidin-1-yl lithocholic acid

To a mixture containing lithocholic acid (5.44g, 14.34mmol), N-hydroxysuccinimide (1.78g, 15.0mmol), anhydrous THF (120ml) and anhydrous acetonitrile (30ml) and kept at 10°C was added A solution of N,N'-dicyclohexylcarbodiimide (3.44 g, 16.67 mmol) was dissolved in anhydrous THF. The reaction mixture was stirred at ambient temperature for 16 hours, filtered and concentrated in vacuo. The residue was dissolved in dichloromethane (450ml), washed with 10% aqueous sodium carbonate (2x150ml) and water (2x150ml) and dried (magnesium sulfate). Filter and concentrate the filtrate in vacuo to give a crystalline residue. The residue was recrystallized from a mixture of dichloromethane (30ml) and n-heptane (30ml) to obtain the title compound (3.46g, 51%) as a crystalline solid.

Example 28

Synthesis of Arg ³⁴ Lys ²⁶ (N ^ε -lithocholic acid)-GLP-1(7-37)-OH

A mixture of ^Arg34- GLP-1(7-37)-OH (4.472 mg, 1.32 μmol), EDPA (4.78 mg, 36.96 μmol), NMP (94 μl) and water (44.8 μl) was gently shaken at room temperature for 10 minutes. A solution obtained by dissolving 2,5-dioxopyrrolidin-1-yl lithocholic acid (1.87 mg, 3.96 μmol) in NMP (46.8 μl) was added to the resulting mixture, and the reaction mixture was gently shaken at room temperature for 5 minutes , and then left at room temperature for another 1 hour. A solution prepared by dissolving glycine (2.18 mg, 29.04 µmol) in 50% aqueous ethanol (218 µl) was added to terminate the reaction. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile/TFA system. The column was heated to 65°C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The title compound (1.25 mg, 25%) was isolated and the product was analyzed by PDMS method. The m/z value of the protonated molecular ion peak was found to be 3744±3. This gave a molecular weight of 3743 ± 3 amu (theoretical 3743 amu).

Example 29

Synthesis of N ^α -tetradecanoyl-Glu(ONSu)-OBu ^t

To ^{a suspension of} H-Glu(OH)-OBut (2.5g, 12.3mmol), DMF (283ml) and EDPA (1.58g, 12.3mmol) was added dropwise Myr-ONSu (4.0g, 12.3mmol) dissolved in DMF (59ml) of the resulting solution. The reaction mixture was stirred at room temperature for 16 hours, then concentrated in vacuo to a total volume of 20ml. The residue was partitioned between 5% aqueous citric acid (250ml) and ethyl acetate (150ml) and the phases were separated. After concentration of the organic phase in vacuo, the residue was dissolved in DMF (40ml). The resulting solution was added dropwise to a 10% aqueous citric acid solution (300 ml) kept at 0°C. The precipitated compound was collected, washed with ice water, and dried in a vacuum oven. The dried compound was dissolved in DMF (23ml), and HONSu (1.5g, 13mmol) was added. To the resulting mixture was added a solution of N,N'-dicyclohexylcarbodiimide (2.44 g, 11.9 mmol) dissolved in dichloromethane (47 ml). The reaction mixture was stirred at room temperature for 16 hours, then the precipitated compound was filtered. The precipitate was recrystallized from n-heptane/2-propanol to obtain the title compound (3.03 g, 50%).

Example 30

Synthesis of Glu ^{22, 23 , 30 Arg 26, 34} Lys ³⁸ (N ^ε -(γ-glutamyl(N ^α -tetradecanoyl)))-GLP-1(7-38)-OH

柱体上，用5％乙腈水溶液(10ml)清洗已固定的化合物，最终通过用TFA(10ml)洗脱将其从柱中释放下来。将洗脱物真空浓缩，使用氰丙基柱(Zorbax 300SB-CN)和标准乙腈/TFA系统，通过柱层析法纯化反应混合物。将柱加热到65℃，乙腈的浓度梯度为60分钟内0-100％。分离目标化合物(0.35mg，32％)，用PDMS法分析产物。发现质子化的分子离子峰的m/z值为4012±3。因而得出分子量为4011±3amu(理论值为4011amu)。Glu ^{22, 23, 30} Arg 26 ^{, 34} Lys ³⁸ -GLP-1(7-38)-OH (1.0 mg, 0.272 μmol), EDPA (0.98 mg, 7.62 μmol), NMP (70 μl) and water (70 μl) The mixture was shaken gently for 5 min at room temperature. N ^α -tetradecanoyl-Glu(ONSu)-OBu ^t (0.41mg, 0.816μmol) prepared by the method of Example 29 was dissolved in NMP (10.4μl) and the resulting solution was added to the resulting mixture, and the reaction mixture was warmed at room temperature Shake for 5 minutes, then leave at room temperature for an additional 45 minutes. A solution prepared by dissolving glycine (0.448 mg, 5.98 µmol) in 50% aqueous ethanol (45 µl) was added thereto to terminate the reaction. A 0.5% aqueous solution of ammonium acetate (0.9ml) was added and the resulting mixture was rinsed into Varian 500mg C8 Mega Bond Elut

On the cartridge, the immobilized compound was washed with 5% acetonitrile in water (10 ml) and finally released from the column by elution with TFA (10 ml). The eluate was concentrated in vacuo and the reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile/TFA system. The column was heated to 65°C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The title compound (0.35 mg, 32%) was isolated and the product was analyzed by PDMS method. The m/z value of the protonated molecular ion peak was found to be 4012±3. This gives a molecular weight of 4011 ± 3 amu (theoretical 4011 amu).

Example 31

Synthesis of Glu ^23, 26 Arg ³⁴ Lys ³⁸ (N ^ε -(γ-glutamyl(N ^α -tetradecanoyl)))-GLP-1(7-38)-OH

柱体上，用5％乙腈水溶液(10ml)清洗已固定的化合物，最终通过用TFA(10ml)洗脱将其从柱中释放下来。将洗脱物真空浓缩，使用氰丙基柱(Zorbax 300SB-CN)和标准乙腈/TFA系统，通过柱层析法纯化反应混合物。将柱加热到65℃，乙腈的浓度梯度为60分钟内0-100％。分离出目标化合物(0.78mg，12％)，用PDMS法分析产物。发现质子化的分子离子峰的m/z值为3854±3。因而得出分子量为3853±3amu(理论值为3853amu)。A mixture of Glu ^23, 26 Arg ³⁴ Lys ³⁸ -GLP-1(7-38)-OH (6.07 mg, 1.727 μmol), EDPA (6.25 mg, 48.36 μmol), NMP (425 μl) and water (425 μl) at room temperature Shake gently for 5 minutes. N ^α -tetradecanoyl-Glu(ONSu)-OBu ^t (2.65 mg, 5.18 μmol) prepared by the method in Example 29 was dissolved in NMP (66.3 μl) to obtain a solution, added to the resulting mixture, and the reaction mixture was warmed at room temperature Shake for 5 minutes, then leave at room temperature for an additional 45 minutes. A solution prepared by dissolving glycine (2.85 mg, 38.0 µmol) in 50% aqueous ethanol (285 µl) was added thereto to terminate the reaction. A 0.5% aqueous solution of ammonium acetate (5.4ml) was added and the resulting mixture was rinsed into Varian 500mg C8 Mega Bond Elut

On the cartridge, the immobilized compound was washed with 5% acetonitrile in water (10 ml) and finally released from the column by elution with TFA (10 ml). The eluate was concentrated in vacuo and the reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile/TFA system. The column was heated to 65°C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The title compound (0.78 mg, 12%) was isolated and the product was analyzed by PDMS method. The m/z value of the protonated molecular ion peak was found to be 3854±3. This gave a molecular weight of 3853 ± 3 amu (theoretical 3853 amu).

Example 32

Synthesis of Lys ^26,34 -bis(N ^ε -(ω-carboxytridecanoyl))-GLP-1(7-37)-OH

A mixture of GLP-1(7-37)-OH (30 mg, 8.9 μmol), EDPA (32.3 mg, 250 μmol), NMP (2.1 ml) and water (2.1 ml) was gently shaken at room temperature for 5 minutes. A solution obtained by dissolving HOOC-(CH ₂ ) ₁₂ -COONSu (12.7 mg, 35.8 μmol) in NMP (318 μl) was added to the resulting mixture, and the reaction mixture was gently shaken at room temperature for 1 hour and 40 minutes. A solution prepared by dissolving glycine (3.4 mg, 44.7 µmol) in 50% aqueous ethanol (335 µl) was added thereto to terminate the reaction. , the reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile/TFA system. The column was heated to 65°C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The title compound (10 mg, 29%) was isolated and the product was analyzed by PDMS method. The m/z value of the protonated molecular ion peak was found to be 3840±3. This gave a molecular weight of 3839±3 amu (theoretical 3839 amu).

Example 33

Synthesis of Lys ^26,34 -bis(N ^ε -(γ-glutamyl(N ^α -tetradecyl))-GLP-1(7-37)-OH (NNC 90-1167)

柱体上，用0.1％TFA水溶液(3.5ml)清洗已固定的化合物，最终用70％乙腈水溶液(4ml)洗脱而将其从柱中释放下来。用0.1％TFA水溶液(300ml)将合并的洗脱物稀释。离心收集沉淀化合物，用0.1％TFA水溶液(50ml)洗涤，最终离心分离沉淀化合物。向沉淀中加入TFA(60ml)，将所得反应混合物在室温搅拌1小时30分钟。真空除去多余的TFA，将残余物倾入水(50ml)中。使用氰丙基柱(Zorbax300SB-CN)和标准乙腈/TFA系统，通过柱层析法纯化沉淀化合物。将柱加热到65℃，乙腈的浓度梯度为60分钟内0-100％。分离出目标化合物(27.3mg，8％)，用PDMS法分析产物。发现质子化的分子离子峰的m/z值为4036±3。因而得出分子量为4035±3amu(理论值为4035amu)。A mixture of GLP-1(7-37)-OH (300 mg, 79.8 μmol), EDPA (288.9 mg, 2.24 mmol), NMP (21 ml) and water (21 ml) was gently shaken at room temperature for 5 minutes. N ^α -tetradecanoyl-Glu(ONSu)-OBu ^t (163 mg, 319.3 μmol) prepared by the method of Example 29 was dissolved in NMP (4.08 ml) and the resulting solution was added to the resulting mixture, and the reaction mixture was warmed at room temperature Shake for 5 minutes, then leave at room temperature for an additional 1 hour. A solution prepared by dissolving glycine (131.8 mg, 1.76 mmol) in 50% aqueous ethanol (13.2 ml) was added thereto to terminate the reaction. A 0.5% aqueous solution of ammonium acetate (250 ml) was added and the resulting mixture was divided into 4 equal portions. Rinse each sample into Varian 500mg C8 MegaBond Elut

On the cartridge, the immobilized compound was washed with 0.1% aqueous TFA (3.5 ml) and finally eluted with 70% aqueous acetonitrile (4 ml) to release it from the column. The combined eluates were diluted with 0.1% TFA in water (300ml). The precipitated compound was collected by centrifugation, washed with 0.1% TFA aqueous solution (50 ml), and finally the precipitated compound was separated by centrifugation. TFA (60 ml) was added to the precipitate, and the resulting reaction mixture was stirred at room temperature for 1 hour and 30 minutes. Excess TFA was removed in vacuo and the residue was poured into water (50ml). The precipitated compound was purified by column chromatography using a cyanopropyl column (Zorbax300SB-CN) and a standard acetonitrile/TFA system. The column was heated to 65°C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The target compound (27.3 mg, 8%) was isolated and the product was analyzed by PDMS method. The m/z value of the protonated molecular ion peak was found to be 4036±3. This gives a molecular weight of 4035 ± 3 amu (theoretical 4035 amu).

Example 34

Synthesis of Arg ^{26, 34} Lys ³⁸ (N ^ε -(ω-carboxypentadecanoyl))-GLP-1(7-38)-OH

A mixture of Arg ^26,34 Lys ³⁸ -GLP-1(7-38)-OH (30 mg, 8.9 μmol), EDPA (32.3 mg, 250 μmol), NMP (2.1 ml) and water (2.1 ml) was gently shaken at room temperature 5 minutes. A solution obtained by dissolving HOOC-(CH ₂ ) ₁₄ -COONSu (13.7 mg, 35.8 μmol) in NMP (343 μl) was added to the resulting mixture, and the reaction mixture was gently shaken at room temperature for 1 hour. A solution prepared by dissolving glycine (3.4 mg, 44.7 µmol) in 50% aqueous ethanol (335 µl) was added thereto to terminate the reaction. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile/TFA system. The column was heated to 65°C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The target compound (4.8 mg, 14%) was isolated and the product was analyzed by PDMS method. The m/z value of the protonated molecular ion peak was found to be 3894±3. This gave a molecular weight of 3893 ± 3 amu (theoretical 3893 amu).

Example 35

Synthesis of N ^α -hexadecanoyl-Glu(ONSu)-OBu ^t

To ^{a suspension of} H-Glu(OH)-OBut (4.2g, 20.6mmol), DMF (500ml) and EDPA (2.65g, 20.6mmol) was added dropwise Pal-ONSu (7.3g, 20.6mmol) dissolved in DMF (100ml) of the resulting solution. The reaction mixture was stirred at room temperature for 64 hours, then concentrated in vacuo to a total volume of 20ml. The residue was partitioned between 10% aqueous citric acid (300ml) and ethyl acetate (250ml) and the phases were separated. After concentration of the organic phase in vacuo, the residue was dissolved in DMF (50ml). The resulting solution was added dropwise to a 10% aqueous citric acid solution (500 ml) kept at 0°C. The precipitated compound was collected, washed with ice water, and dried in a vacuum oven. The dried compound was dissolved in DMF (45ml), and HONSu (2.15g, 18.7mmol) was added. To the resulting mixture was added a solution of N,N'-dicyclohexylcarbodiimide (3.5 g, 17 mmol) dissolved in dichloromethane (67 ml). The reaction mixture was stirred at room temperature for 16 hours, then the precipitated compound was filtered. The precipitate was recrystallized from n-heptane/2-propanol to obtain the title compound (6.6 g, 72%).

Example 36

Synthesis of Lys ^26,34 -bis(N ^ε -(γ-glutamyl(N ^α -hexadecanoyl)))-GLP-1(7-37)-OH

A mixture of GLP-1(7-37)-OH (10 mg, 2.9 μmol), EDPA (10.8 mg, 83.4 μmol), NMP (0.7 ml) and water (0.7 ml) was gently shaken at room temperature for 5 minutes. N ^α -hexadecanoyl-Glu(ONSu)-OBu ^t (163 mg, 319.3 μmol) prepared by the method of Example 33 was dissolved in NMP (4.08 ml) to obtain a solution, and added thereto to the resulting mixture, and the reaction mixture was Gentle shaking at room temperature for 1 hour and 20 minutes. A solution prepared by dissolving glycine (4.9 mg, 65.6 µmol) in 50% aqueous ethanol (492 µl) was added to terminate the reaction. A 0.5% aqueous solution of ammonium acetate (9ml) was added and the resulting mixture was rinsed into a Varian 1g C8 Mega Bond Elut On the cartridge, the immobilized compound was washed with 5% aqueous acetonitrile (10 ml) and finally released from the column by elution with TFA (10 ml). The eluate was concentrated in vacuo and the residue was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile/TFA system. The column was heated to 65°C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The target compound (2.4 mg, 20%) was isolated and the product was analyzed by PDMS method. The m/z value of the protonated molecular ion peak was found to be 4092±3. This gave a molecular weight of 4091 ± 3 amu (theoretical 4091 amu).

Example 37

Synthesis of Arg ³⁴ Lys ²⁶ (N ^ε -(γ-glutamyl(N ^α -hexadecanoyl)))-GLP-1(7-37)-OH

膜柱体上，用5％乙腈水溶液(10ml)清洗已固定的化合物，最终用TFA(6ml)洗脱而将其从柱中释放下来。将洗脱物在室温放置2小时，然后真空浓缩。使用氰丙基柱(Zorbax 300SB-CN)和标准乙腈/TFA系统，通过用柱层析法纯化残余物。将柱加热到65℃，乙腈的浓度梯度为60分钟内0-100％。分离出目标化合物(0.23mg，6％)，用PDMS法分析产物。发现质子化的分子离子峰的m/z值为3752±3。因而得出分子量为3751±3amu(理论值为3751amu)。A mixture of Arg ³⁴ -GLP-1(7-37)-OH (3.7 mg, 1.1 μmol), EDPA (4.0 mg, 30.8 μmol), acetonitrile (260 μl) and water (260 μl) was gently shaken at room temperature for 5 minutes. N ^α -hexadecanoyl-Glu(ONSu)-OBu ^t (1.8 mg, 3.3 μmol) prepared by the method in Example 35 was dissolved in acetonitrile (44.2 μl) to obtain a solution, added to the resulting mixture, and the reaction mixture was heated at room temperature Gentle shaking for 1 hour 20 minutes. A solution prepared by dissolving glycine (1.8 mg, 24.2 µmol) in 50% aqueous ethanol (181 µl) was added thereto to terminate the reaction. A 0.5% aqueous solution of ammonium acetate (12 ml) and NMP (300 μL) were added and the resulting mixture was rinsed into Varian 1 g C8 Mega Bond Elut

On the membrane cartridge, the immobilized compound was washed with 5% aqueous acetonitrile (10 ml) and finally released from the column by elution with TFA (6 ml). The eluate was left at room temperature for 2 hours, then concentrated in vacuo. The residue was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile/TFA system. The column was heated to 65°C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The title compound (0.23 mg, 6%) was isolated and the product was analyzed by PDMS method. The m/z value of the protonated molecular ion peak was found to be 3752±3. This gave a molecular weight of 3751 ± 3 amu (theoretical 3751 amu).

Example 38

Synthesis of Arg ^{26, 34} Lys ³⁸ (N ^ε -(γ-glutamyl(N ^α -tetradecanoyl)))-GLP-1(7-38)-OH

A mixture of Arg ^26,34 Lys ³⁸ -GLP-1(7-38)-OH (14 mg, 4.0 μmol), EDPA (14.3 mg, 110.6 μmol), NMP (980 μl) and water (980 μl) was gently shaken at room temperature for 5 minute. N ^α -tetradecanoyl-Glu(ONSu)-OBu ^t (12.1mg, 23.7μmol) prepared by the method in Example 29 was dissolved in NMP (303μl) and the resulting solution was added to the resulting mixture, and the reaction mixture was warmed at room temperature Shake for 2 hours. A solution prepared by dissolving glycine (6.5 mg, 86.9 mmol) in 50% aqueous ethanol (652 µl) was added thereto to terminate the reaction. A 0.5% aqueous solution of ammonium acetate (50ml) was added and the resulting mixture was rinsed into Varian 1gC8 Mega Bond Elut On the cartridge, the immobilized compound was washed with 5% aqueous acetonitrile (15 ml) and finally released from the column by elution with aqueous TFA (6 ml). The eluate was left at room temperature for 1 hour and 45 minutes, then concentrated in vacuo. The residue was purified by column chromatography using a cyanopropyl column (Zorbax300SB-CN) and a standard acetonitrile/TFA system. The column was heated to 65°C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The target compound (3.9 mg, 26%) was isolated and the product was analyzed by PDMS method. The m/z value of the protonated molecular ion peak was found to be 3881±3. This gave a molecular weight of 3880±3 amu (theoretical 3880 amu).

Example 39

Synthesis of Arg ^{26, 34} Lys ³⁸ (N ^ε -(ω-carboxypentadecanoyl))-GLP-1(7-38)-OH

A mixture of Arg ^26,34 Lys ³⁸ -GLP-1(7-38)-OH (14 mg, 4.0 μmol), EDPA (14.3 mg, 111 μmol), NMP (980 μl) and water (980 μl) was gently shaken at room temperature for 5 minutes . A solution obtained by dissolving HOOC-(CH ₂ ) ₁₄ -COONSu (4.5 mg, 11.9 μmol) in NMP (114 μl) was added to the resulting mixture, and the reaction mixture was gently shaken at room temperature for 1 hour and 45 minutes. A solution obtained by dissolving HOOC-(CH ₂ ) ₁₄ -COONSu (4.0 mg, 10.4 μmol) in NMP (100 μl) was added, and the resulting mixture was further shaken gently at room temperature for 1 hour and 30 minutes. A solution prepared by dissolving glycine (1.5 mg, 19.8 µmol) in 50% aqueous ethanol (148 µl) was added thereto to terminate the reaction. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile/TFA system. The column was heated to 65°C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The target compound (3.9 mg, 26%) was isolated and the product was analyzed by PDMS method. The m/z value of the protonated molecular ion peak was found to be 3809±3. This gave a molecular weight of 3808±3 amu (theoretical 3808 amu).

Example 40

Synthesis of Arg ^{26, 34} Lys ³⁸ (N ^ε -(γ-glutamyl(N ^α -hexadecanoyl)))-GLP-1(7-38)-OH

柱体上，用5％乙腈水溶液(10ml)清洗已固定的化合物，最终用TFA(6ml)洗脱而将其从柱中释放下来。将洗脱物在室温静置1小时30分钟，然后真空浓缩。使用氰丙基柱(Zorbax 300SB-CN)和标准乙腈/TFA系统，通过柱层析法纯化残余物。将柱加热到65℃，乙腈的浓度梯度为60分钟内0-100％。分离出目标化合物(7.2mg，47％)，用PDMS法分析产物。发现质子化的分子离子峰的m/z值为3881±3。因而得出分子量为3880±3amu(理论值为3880amu)。A mixture of Arg ^{26, 34} Lys ³⁸ -GLP-1(7-38)-OH (14 mg, 4.0 μmol), EDPA (14.3 mg, 110.6 μmol), NMP (980 μl) and water (980 μl) was gently shaken at room temperature for 5 Minutes. N ^α -hexadecanoyl-Glu(ONSu)-OBu ^t (6.4 mg, 11.9 μmol) prepared by the method of Example 35 was dissolved in NMP (160 μl) gained solution, joined in the resulting mixture, and the reaction mixture was Gentle shaking at room temperature for 1 hour and 20 minutes. A solution prepared by dissolving glycine (6.5 mg, 87 mmol) in 50% aqueous ethanol (653 µl) was added thereto to terminate the reaction. A 0.5% aqueous solution of ammonium acetate (50ml) was added and the resulting mixture was rinsed into Varian 1g C8 MegaBond Elut

On the cartridge, the immobilized compound was washed with 5% acetonitrile in water (10 ml) and finally released from the column by elution with TFA (6 ml). The eluate was allowed to stand at room temperature for 1 hour and 30 minutes, then concentrated in vacuo. The residue was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile/TFA system. The column was heated to 65°C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The target compound (7.2 mg, 47%) was isolated and the product was analyzed by PDMS method. The m/z value of the protonated molecular ion peak was found to be 3881±3. This gave a molecular weight of 3880±3 amu (theoretical 3880 amu).

Example 41

Synthesis of Arg ^{18, 23, 26} , 30, 34 Lys ³⁸ (N ^ε -hexadecanoyl)-GLP-1(7-38)-OH

A mixture of Arg ^{18, 23, 26} , 30, 34 Lys ³⁸ -GLP-1(7-38)-OH (1.0 mg, 0.27 μmol), EDPA (0.34 mg, 2.7 μmol) and DMSO (600 μl) was warmed at room temperature Shake for 5 minutes. To the resulting mixture was added a solution of Pal-ONSu (0.28 mg, 0.8 µmol) dissolved in NMP (7 µl). The reaction mixture was gently shaken at room temperature for 5 minutes and then left at room temperature for 6 hours. A solution prepared by dissolving glycine (1.6 mg, 21.7 µmol) in 50% aqueous ethanol (163 µl) was added thereto to terminate the reaction. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile/TFA system. The column was heated to 65°C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The title compound (0.17 mg, 16%) was isolated and the product was analyzed by MALDI-MS. The m/z value of the protonated molecular ion peak was found to be 3961±3. The resulting molecular weight was 3960 ± 3 amu (theoretical 3960 amu).

Example 42

Synthesis of Arg ^{26, 34} Lys ³⁸ (N ^ε -(ω-carboxytridecanoyl))-GLP-1(7-38)-OH

A mixture of Arg ^26,34 Lys ³⁸ -GLP-1(7-38)-OH (14 mg, 4.0 μmol), EDPA (14.3 mg, 111 μmol), NMP (980 μl) and water (980 μl) was gently shaken at room temperature for 5 minutes . A solution obtained by dissolving HOOC-(CH ₂ ) ₁₂ -COONSu (4.2 mg, 11.9 μmol) in NMP (105 μl) was added to the resulting mixture, and the reaction mixture was gently shaken at room temperature for 1 hour and 50 minutes. A solution prepared by dissolving glycine (6.5 mg, 87 µmol) in 50% ethanol aqueous solution (652 µl) was added thereto to terminate the reaction. The reaction mixture was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile/TFA system. The column was heated to 65°C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The title compound (5.8 mg, 39%) was isolated and the product was analyzed by MALDI-MS. The m/z value of the protonated molecular ion peak was found to be 3780±3. This gave a molecular weight of 3779±3 amu (theoretical 3781 amu).

Example 43

Synthesis of Arg ³⁴ Lys ²⁶ (N ^ε -(γ-glutamyl(N ^α -tetradecanoyl)))-GLP-1(7-37)-OH

柱体上，用5％乙腈水溶液(15ml)清洗已固定的化合物，最终用TFA(6ml)洗脱而将其从柱中释放下来。将洗脱物在室温放置1小时45分钟，然后真空浓缩。使用氰丙基(cyanopropyl)柱(Zorbax 300SB-CN)和标准乙腈/TFA系统，通过柱层析法纯化残余物。将柱加热到65℃，乙腈的浓度梯度为60分钟内0-100％。分离出目标化合物(3.9mg，26％)，用MALDI-MS法分析产物。发现质子化的分子离子峰的m/z值为3723±3。因而得出分子量为3722±3amu(理论值为3723amu)。A mixture of Arg ³⁴ -GLP-1(7-37)-OH (15 mg, 4.4 μmol), EDPA (16 mg, 124 μmol), NMP (2 ml) and water (4.8 ml) was gently shaken at room temperature for 5 minutes. N ^α -tetradecanoyl-Glu(ONSu)-OBu ^t (12.1mg, 23.7μmol) prepared by the method in Example 29 was dissolved in NMP (303μl) and the resulting solution was added to the resulting mixture, and the reaction mixture was warmed at room temperature Shake for 2 hours. A solution prepared by dissolving glycine (6.5 mg, 86.9 µmol) in 50% aqueous ethanol (652 µl) was added thereto to terminate the reaction. A 0.5% aqueous solution of ammonium acetate (50ml) was added and the resulting mixture was rinsed into Varian 1g C8 Mega Bond Elut

On the cartridge, the immobilized compound was washed with 5% acetonitrile in water (15 ml) and finally released from the column by elution with TFA (6 ml). The eluate was left at room temperature for 1 hour and 45 minutes, then concentrated in vacuo. The residue was purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile/TFA system. The column was heated to 65°C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The title compound (3.9 mg, 26%) was isolated and the product was analyzed by MALDI-MS. The m/z value of the protonated molecular ion peak was found to be 3723±3. This gave a molecular weight of 3722±3 amu (theoretical 3723 amu).

Example 44

Synthesis of N ^α -octadecanoyl-Glu(ONSu)-OBu ^t

To ^{a suspension} of H-Glu(OH)-OBut (2.82g, 13.9mmol), DMF (370ml) and EDPA (1.79g, 13.9mmol) was added dropwise Ste-ONSu (5.3g, 13.9mmol) dissolved in DMF (60ml) of the resulting solution. Dichloromethane (35ml) was added and the reaction mixture was stirred at room temperature for 24 hours, then concentrated in vacuo. The residue was partitioned between 10% aqueous citric acid (330ml) and ethyl acetate (200ml) and the phases were separated. The organic phase was concentrated in vacuo and the residue was dissolved in DMF (60ml). The resulting solution was added dropwise to a 10% aqueous citric acid solution (400 ml) kept at 0°C. The precipitate was collected, washed with ice water, and dried in a vacuum oven. The dried compound was dissolved in DMF (40ml), and HONSu (1.63g, 14.2mmol) was added. A solution of DCC (2.66 g, 12.9 mmol) dissolved in dichloromethane (51 mL) was added to the reaction mixture. The resulting mixture was stirred at room temperature for 64 hours and filtered to obtain a precipitated compound. The precipitate was recrystallized from n-heptane/2-propanol to obtain the title compound (4.96 g, 68%).

Example 45

Synthesis of Arg ^{26, 34} Lys ³⁸ (N ^ε -(γ-glutamyl(N ^α -octadecanoyl)))-GLP-1(7-38)-OH

柱体上，用5％乙腈水溶液(25ml)清洗已固定的化合物，最终用TFA(25ml)洗脱而将其从柱中释放下来。将合并的洗脱物在室温放置1小时25分钟，然后真空浓缩。使用氰丙基柱(Zorbax300SB-CN)和标准乙腈/TFA系统，通过柱层析法纯化残余物。将柱加热到65℃，乙腈的浓度梯度为60分钟内0-100％。分离出目标化合物(3.6mg，11％)，用MALDI-MS法分析产物。发现质子化的分子离子峰的m/z值为3940±3。因而得出分子量为3939±3amu(理论值为3937amu)。A mixture of Arg ^26,34 -GLP-1(7-38)-OH (28mg, 7.9μmol), EDPA (28.6mg, 221.5μmol), NMP (1.96ml) and water (1.96ml) was shaken gently at room temperature for 5 minute. N ^α -octadecanoyl-Glu(ONSu)-OBu ^t (17.93g, 31.6 μmol) prepared by the method of Example 44 was dissolved in NMP (448 μl) and the resulting solution was added to the resulting mixture, and the reaction mixture was warmed at room temperature Shake for 2 hours. A solution prepared by dissolving glycine (13.1 mg, 174 µmol) in 50% aqueous ethanol (1.3 ml) was added thereto to terminate the reaction. A 0.5% aqueous solution of ammonium acetate (120 ml) was added and the resulting mixture was divided into 2 equal portions. Rinse each serving into Varian5g C8 Mega Bond Elut

On the cartridge, the immobilized compound was washed with 5% acetonitrile in water (25ml) and finally released from the column by elution with TFA (25ml). The combined eluates were left at room temperature for 1 hour and 25 minutes, then concentrated in vacuo. The residue was purified by column chromatography using a cyanopropyl column (Zorbax300SB-CN) and a standard acetonitrile/TFA system. The column was heated to 65°C and the concentration gradient of acetonitrile was 0-100% in 60 minutes. The title compound (3.6 mg, 11%) was isolated and the product was analyzed by MALDI-MS. The m/z value of the protonated molecular ion peak was found to be 3940±3. This gave a molecular weight of 3939±3 amu (theoretical 3937 amu).

biological observation

Prolonged action of GLP-1 derivatives after subcutaneous administration

The long-term effects of some of the GLP-1 derivatives of the present invention were determined by monitoring plasma concentrations of healthy pigs following subcutaneous administration of the GLP-1 derivatives by the following method. For comparison, the plasma concentration of GLP-1(7-37) was also determined following subcutaneous administration. The results are shown in Table 1. The long-term effects of other GLP-1 derivatives of the invention can be determined in this way.

Pigs (50% Duroc, 25% Yorkshire, 25% Danish Landrace, weighing about 40 kg) were fasted from the beginning of the experiment. Each pig was given per kilogram of body weight dissolved in 50μM isotonic solution (5mM phosphate, pH7.4, 0.02% Tween -20 (Merck), 45 mg/ml mannitol (depyrogenated, Novo Nordisk)) in 0.5 nmol test compound. Blood samples were drawn from the jugular catheter at the times indicated in Table 1. A 5 ml blood sample was poured into an ice-cold glass containing 175 μl of the following solution: 0.18 M EDTA, 1500 KIE/ml aprotinin (Novo Nordisk) and 3% bacitracin (Sigma), pH 7.4. Within 30 minutes, the sample was Centrifuge at 5-6000 ^* g for 10 minutes. The temperature was maintained at 4°C. The supernatant was pipetted into separate glasses and stored at -20°C until use.

The plasma concentrations of the peptides were determined by the RIA method using a monoclonal antibody specific for the N-terminal region of GLP-1(7-37). Cross-reactivity with GLP-1(1-37) and GLP-1(8-36)amide is less than 1%, with GLP-1(9-37), GLP-1(10-36)amide and GLP-1( 11-36) Amide cross-reactivity is less than 0.1%. All reactions were performed at 4°C.

The assay was performed as follows: 100 μl plasma was mixed with 271 μl 96% ethanol, mixed with a vortex mixer and centrifuged at 2600 ^* g for 30 minutes. The supernatant was decanted gently into Minisorp tubes and evaporated completely (Savant Speedvac AS290). The evaporation residue was redissolved in a solution containing 80 mM monobasic/disodium phosphate, 0.1% HSA (Orpha 20/21, Behring), 10 mM EDTA, 0.6 mM ethylmercury sodium thiosalicylate (Sigma), pH 7 .5 in the assay buffer. Dissolve the sample in a volume suitable to achieve its intended concentration and allow to dissolve for 30 minutes. To 300 µl of the sample was added 10 µl of an antibody solution made of a dilution buffer containing 40 mM sodium dihydrogen phosphate/disodium hydrogen phosphate, 0.1% HSA, 0.6 mM ethylmercury thiosalicylate, pH 7.5. Make non-specific samples by mixing 300 μl of buffer with 100 μl of dilution buffer. Individual standards were prepared from lyophilized stocks and dissolved in 300 [mu]l assay buffer. All samples were pre-incubated for 72 hours in Minisorp tubes with antibodies as described above. To this was added 200 μl of a tracer containing 6-7000 CPM dissolved in a dilution buffer, and the samples were mixed and incubated for 48 hours. To each tube was added 200 ml/l of heparin-stabilized bovine plasma in 40 mM sodium dihydrogen phosphate/disodium hydrogen phosphate, 0.6 mM sodium ethylmercury thiosalicylate, pH 7.5, and 18 g/l activated carbon (Merck ) suspension 1.5ml. The suspension was mixed and allowed to stand at 4°C for 2 hours before use. All samples were incubated at 4°C for 1 hour and then centrifuged at 3400 ^* g for 25 minutes. Immediately after centrifugation, the supernatant was gently removed and counted in a gamma-counter. Concentrations in samples were calculated from individual standard curves. The following plasma concentrations were determined as a percentage of the maximum concentration of the respective compound (n=2):

Table 1

test compound) Time after subcutaneous administration (hours) 0.75 1 2 4 6 8 10 12 twenty four GLP-(7-37) 100 9 1 Example 25 73 92 100 98 82 twenty four 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 twenty four 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 twenty two 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

) The test compound is the target compound of each example given the example number

As shown in Table 1, the GLP-1 derivatives of the present invention have a longer action profile than GLP-1(7-37) and are much more persistent in plasma than GLP-1(7-37). Table 1 also shows that the time to peak plasma concentration varies widely depending on the specific GLP-1 derivative selected.

Stimulation of cAMP formation in cell lines expressing cloned human GLP-1 receptors

To demonstrate the potency of the GLP-1 derivatives, their ability to stimulate cAMP formation in cell lines expressing the cloned human GLP-1 receptor was determined. _EC50 was calculated from dose-response curves.

Baby hamster kidney (BHK) cells expressing the human pancreatic GLP-1 receptor were used (Knudsen and Pridal, 1996, Eur. J. Pharmac., 318, 429-435). By buffering (10mmol/l Tris-HCl and 30mmol/lNaCl pH7.4, additionally containing 1mmol/l dithiothreitol, 5mg/l leupeptin (Sigma, St.Louis, MO, USA), 5 mg/l pepstatin (Sigma, St. Louis, MO, USA), 100 mg/l bacitracin (Sigma, St. Louis, MO, USA) and 16 mg/l aprotinin (Novo Nordisk A/S, Bagsvaerd , Denmark)) to prepare plasma membranes (Adelhorst et al., 1994, Journal of Biochemistry, 269, 6275). The homogenate was centrifuged over a 41 w/v% sucrose layer. The white band between the two layers was diluted in buffer and centrifuged. Plasma membranes were stored at -80°C for later use.

Assays were performed in 96-well microtiter plates in a total volume of 140 μl. The buffer used was 50mmol/l Tris-HCl, pH7.4, and 1mmol/lEGTA, 1.5mmol/lMgSO ₄ , 1.7mmol/lATP, 20mM GTP, 2mmol/l 3-isobutyl-1-methyl yellow Purines, 0.01% Tween-20 and 0.1% human serum albumin (Reinst, Behringwerke AG, Marburg, Germany). The compound whose agonist activity is to be tested is dissolved and diluted in buffer, added to the plasma membrane preparation, and the mixture is incubated at 37°C for 2 hours. Add 25 μl of 0.05 mol/l hydrochloric acid to terminate the reaction. Samples were diluted 10-fold and analyzed for cAMP by the scintillation proximity assay (RPA 538, Amersham, UK). and get the following result:

test compound) _EC50 , pM test compound) _EC50 , pM GLP-1(7-37) 61 Example 31 96 Example 45 120 Example 30 41 Example 43 twenty four 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

⁾ The test compound is the target compound of the example given the example number

Claims (24)

1. A GLP-1 derivative, wherein 1 or 2 amino acid residues of the GLP-1 peptide are linked with a lipophilic substituent, wherein the lipophilic substituent is selected from the group consisting of: i) selected from CH ₃ (CH ₂ ) ₁₀ CO—, CH ₃ (CH ₂ ) ₁₂ CO—, CH ₃ (CH ₂ ) ₁₄ CO—, CH ₃ (CH ₂ ) ₁₆ CO—, CH ₃ (CH ₂ ) ₁₈ CO-, CH ₃ (CH ₂ ) ₂₀ CO- and CH ₃ (CH ₂ ) ₂₂ CO- the acyl group of linear or branched fatty acids; ii) the acyl group of a linear or branched alkane α,ω-dicarboxylic acid having the formula HOOC(CH ₂ ) _m CO—, wherein m is 10, 12, 14 or 16; and iii) cholyl, 7-deoxycholyl or lithochyl; And if there is only one lipophilic substituent, and the substituent is attached to the N-terminal or C-terminal amino acid residue of the GLP-1 peptide, then the substituent is an alkyl group or a group with an omega-carboxyl group, Wherein, the 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, 34} Lys ³⁶ -GLP-1(7-37), Arg ^{26, 34} Lys ³⁶ -GLP-1( 7-36), Arg ^{26, 34} Lys ³⁸ -GLP-1(7-38), Arg ²⁶ Lys ³⁶ -GLP-1(7-37), Arg ³⁴ Lys ³⁶ -GLP-1(7-37), Arg ²⁶ Lys ³⁸ -GLP-1(7-38), Arg ³⁴ Lys ³⁸ -GLP-1(7-38), Arg ^{26, 34} Lys 36, ³⁸ -GLP-1(7-38), and Arg ^{26, 34} Lys ³⁸ -GLP-1(7-38). 2. The GLP-1 derivative according to claim 1, wherein the lipophilic substituent is selected from the group consisting of: HOOC(CH ₂ ) ₁₀ CO-, HOOC(CH ₂ ) ₁₂ CO-, HOOC(CH ₂ ) ₁₄ CO- and HOOC(CH ₂ ) ₁₆ CO-. 3. The GLP-1 derivative according to claim 1, wherein there is only one lipophilic substituent. 4. The GLP-1 derivative according to claim 3, wherein the lipophilic substituent is attached to the N-terminal amino acid residue. 5. The GLP-1 derivative according to claim 3, wherein the lipophilic substituent is attached to the C-terminal amino acid residue. 6. The GLP-1 derivative according to claim 3, wherein the lipophilic substituent is attached to an amino acid residue which is neither N-terminal nor C-terminal. 7. The GLP-1 derivative according to claim 1, wherein there are two lipophilic substituents. 8. The GLP-1 derivative according to claim 7, wherein one lipophilic substituent is attached to the N-terminal amino acid residue and the other lipophilic substituent is attached to the C-terminal amino acid residue. 9. The GLP-1 derivative according to claim 7, wherein one lipophilic substituent is attached to the C-terminal amino acid residue, and the other lipophilic substituent is attached to neither the N-terminal nor the C-terminal amino acid residue. at the terminal amino acid residue. 10. The GLP-1 derivative according to claim 7, wherein both lipophilic substituents are attached to an amino acid residue that is neither N-terminal nor C-terminal. 11. The GLP-1 derivative according to claim 1, wherein a lipophilic substituent is attached to an amino acid residue by forming an amide bond with its carboxyl group to the amino group of the amino acid residue. 12. The GLP-1 derivative according to claim 1, wherein a lipophilic substituent is attached to an amino acid residue by forming an amide bond with its amino group to a carboxyl group of the amino acid residue. 13. The GLP-1 derivative according to claim 1, wherein the lipophilic substituent is attached to the GLP-1 peptide through a spacer group. 14. The GLP-1 derivative according to claim 13, wherein the spacer group is an unbranched chain alkane α with 1-7 methylene groups, ω-dicarboxyl, the spacer group in the GLP-1 peptide A bridge is formed between the amino group of the lipophilic substituent and the amino group of the lipophilic substituent. 15. The GLP-1 derivative according to claim 14, wherein the α,ω-dicarboxylic group has 2 methylene groups. 16. The GLP-1 derivative according to claim 13, wherein the spacer i) is an amino acid residue other than Cys, or ii) is a dipeptide. 17. The GLP-1 derivative according to claim 16, wherein the dipeptide is Gly-Lys. 18. The GLP-1 derivative according to claim 16, wherein a carboxyl group of the GLP-1 peptide forms an amide bond with Lys or an amino group in a dipeptide containing Lys residues, and the Lys spacer group or contains Lys residues Another amino group in the dipeptide spacer of the base forms an amide bond with the carboxyl group in the lipophilic substituent. 19. The GLP-1 derivative according to claim 16, wherein an amino group of the GLP-1 peptide forms an amide bond with a carboxyl group of the amino acid residue or a dipeptide spacer, and the amino acid residue or two An amino group of the peptide spacer forms an amide bond with a carboxyl group of the lipophilic substituent. 20. The GLP-1 derivative according to claim 16, wherein a carboxyl group of the GLP-1 peptide forms an amide bond with an amino group of the amino acid residue spacer or dipeptide spacer, and the amino acid residue A carboxyl group of the radical spacer or dipeptide spacer forms an amide bond with an amino group of the lipophilic substituent. 21. The GLP-1 derivative according to claim 16, wherein a carboxyl group of the GLP-1 peptide forms an amide with an amino group of an Asp or Glu spacer, or a dipeptide spacer containing Asp or Glu residues bond, and a carboxyl group of the spacer forms an amide bond with an amino group of the lipophilic substituent. 22. A pharmaceutical composition comprising the GLP-1 derivative according to any one of claims 1-21 and a pharmaceutically acceptable excipient or carrier. 23. Use of the GLP-1 derivative according to any one of claims 1-21 in the preparation of a medicament for the treatment of obesity. 24. Use of the GLP-1 derivative according to any one of claims 1-21 in the preparation of a medicament for treating insulin-dependent or non-insulin-dependent diabetes.