GB2448895A - GLP-1 like compounds and uses thereof - Google Patents

Abstract

Compound comprising a moiety having (i) a GLP-1 like activity, (ii) a 1, 2 aminothiol moiety, and an optional linker group joining (i) and (ii) are disclosed. The 1, 2 aminothiol moiety is preferably a cysteine residue, and the GLP-1 like moiety is preferably GLP-1 (7-37) or exendin. The use of analogues of GLP-1 in such compounds is also disclosed. In a preferred embodiment, the linker is attached to a lysine residue of a GLP-1 analogue. The compound may be used in the treatment of hyperglycemia, diabetes, cardiovascular disordes or bowel disorders.

Description

-1-2448895

S

Compounds and Uses Thereof

Field of the Invention

The present invention relates to certain compounds, to processes for their preparation, compositions comprising them and methods of treatment of the human or anima' body employing them

Background

Type II diabetes (non-insulin dependent diabetes mellitus -NIDDM) is a condition characterized by a resistance to insulin action in peripheral tissues such as muscle, adipose and liver, and by a progressive failure of the ability of the islet 3-cells to secrete insulin. Because current therapies do not halt the progression of n-cell failure, virtually all NIDDM patients eventually require insulin to control blood glucose levels. The most commonly prescribed therapeutics for such patients are the sulfonylureas, a class of drugs that stimulate insulin secretion. Each year, 10-20% of the patients receiving sulfonylurea therapy fail to maintain acceptable blood glucose levels, and switch to insulin therapy.

Insulin therapy, however, is undesirable from a variety of points of view. Firstly, it has a narrow therapeutic index. This leads to poor control of blood glucose levels, since most patients and physicians err in favour of high glucose levels rather than risk hypoglycaemia and coma.

Secondly, assessment of the appropriate dosage is difficult. The factors to be taken into account include the amount of food consumed, the interval between meals, the amount of physical exercise, and the prevailing blood glucose level (the determination of which requires blood glucose monitoring).

Thirdly, administration of insulin is inconvenient because it must be given parenterally.

For these reasons, satisfactory control of blood glucose levels is frequently not achieved in patients receiving insulin therapy.

The hormone glucagon-like peptide-1 (7-37) (GLP-1) is released by intestinal L cells in response to ingested nutrients and acts to promote glucose-dependent insulin secretion ensuring efficient postprandial glucose homeostasis This hormone binds the GLP-1 receptor present on a number target tissues including the lungs, pancreatic 3 cells, brain, muscle, and gut, achieving normal glucose levels in five separate but complementary ways. The peptide is synthesised as a prohormone in which the N terminal 6 amino acids are processed to yield the biologically active GLP-1 7-37 sequence: His7-Ala8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-GlY-GlflAla -AIa-Lys26-Glu-Phe-l le-Ala-Trp-Leu-Val-Lys-Gly-Arg-G1y37 (S EQ ID NO: 1) Unfortunately, the beneficial actions of GLP-1 which give this hormone many of the desirable properties of an antidiabetic drug are short lived due to N terminal degradation by dipeptidylpeptidase IV (DPP IV) and rapid clearance by renal filtration.

DPP IV cleavage occurs between Ala8 and G1u9, completely inactivating it and resulting in a circulating half-life of less than 2 minutes for the active form of GLP-1 Various strategies have been employed to protect GLP-1 against both renal clearance and DPP-IV cleavage in efforts to produce a more stable, enduring anti-diabetic drug. They include amino acid substitutions at Ala6 and elsewhere, covalent attachment of fatty acids, and the linking of GLP-1 to albumin Modification of GLP-1 through fatty acid attachment confers protection from renal clearance and DPP-lV activity from its resulting association in the bloodstream with albumin A therapeutic candidate for treating Type II diabetes based on this modification has emerged and is currently undergoing clinical trials In addition, a number of small molecule DPP IV inhibitors are being developed for oral delivery to prolong the activity of endogenously secreted GLP-1

Prior Art

US5981488 discloses GLP-1 analogues of the formula: Phe-l le-Aia-Trp-Leu-val-Lys-GIy-Arg-R2 or a pharmacuetically accetable salt thereof, wherein: R1 is selected from the group consisting of His, D-histidine, desamino-histidine, 2-amino-histidine, j3-hydroxy-histidine, homohistidine, a-fluoromethyl-histidine, and a-methyt-histidine; X is selected from the group consisting of Met, Asp, Lys, Thr, Leu, Asn, Gin, Phe, Val, and Tyr; Y and Z are independently selected from the group consisting of Glu, Gin, -3-.

Ala, Thr, Ser, and Gly, and R2 is selected from the group consisting of NH2, and Gly-OH, provided that, if R1 is His, X is Val, Y is Glu, and Z is Glu, then R2 is NH2.

The GLP-1 analogues are stated to have an increased duration of action and resistance to DPP-lV US6620910 discloses GLP-1 analogues of the formula Z1-X1-X2-X3-Gly-Thr-Phe-Thr-Ser-X4-X5-Ser-X6-X7-X8-Glu-GIy-Gin-Ala-X9-LysX10-Xi 1 X12-Ala-X1 3-X14-Val-Lys-Gly-X1 5-Gly-Z2 wherein Z, is substituent of the terminal amino group of the peptide; Z2 is a substituent of the terminal carbonyl group of the peptide; and X, to X14 each represents, independently of the others a natural or non-natural amino acid residue, having the D or L configuration.

The compounds are stated to have agonist character in relation to tGLP1 receptors, in addition to increased metabolic stability and duration of action when compared to the natural peptide US6703365 discloses peptides of the formula R1-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Y-Gly-Gln-Ala-AlaLys -Z-Phe-l le-Ala-Trp-Leu-Val-Lys-Gly-Arg-R2 wherein R1 is selected from the group consisting of L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, 13-hydroxy-histidine, homohistidine, a-fluoromethyl-histidine, and a-methyl-histidine; X is selected from the group consisting of Ala, Gly, Val, Thr, lie, and alpha-methyl-Ala; Y is selected from the group consisting of Glu, Gin, Ala, Thr, Ser, and dy; Z is selected from the group consisting of Glu, GIn, Ala, Thr, Ser, and Gly; R2 is selected from the group consisting of NH2, and Gly-OH, providing that the compound has an isoelectric point in the range from about 6 0 to about 9 0 and further providing that when R1 is His, X is Ala, Y is Glu, and Z is Glu, R2 must be NH2 The peptides are said to have increased stability to storage and in vivo.

US6849708 discloses insulinotropic peptides comprising a fragment of GLP-1 and derivatives thereof.

A problem that remains is the provision of therapeutic agents for the treatment of diabetes.

A further problem that remains is the provision of compounds having GLP-1 like activity while having superior stability compared with naturally occurring GLP-1.

A further problem that remains is the provision of GLP-1 analogues having increased resistance to degradation by DPP-IV The present invention addresses problems of the prior art.

Summary of the Invention

According to a first aspect, there is provided a compound comprising 10) a moiety having GLP-1 like activity, ii) a 1,2-aminothiol moiety, iii) an optional linker group joining i) and ii).

According to a second aspect, there is provided a compound of the invention covalently linked to a carrier molecule ("complexed compound").

According to a third aspect, there is provided a pharmaceutical composition comprising a compound or complexed compound of the invention above together with a pharmaceutically acceptable carrier or excipient.

According to a fourth aspect, there is provided a method of treatment of a human or animal suffering from a condition selected from hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, hypertension, syndrome X, dyslipidemia, cognitive disorders, atheroschlerosis, myocardial infarction, coronary heart disease and other cardiovascular disorders, stroke, inflammatory bowel syndrome, dyspepsia and gastric ulcers, comprising administering to said mammal a compound or complexed compound of the invention or a composition comprising such a compound According to a fifth aspect, there is provided a compound or of a complexed compound of the invention or a composition comprising such a compound for the treatment of a condition selected from hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, hypertension, syndrome X, dyslipidemia, cognitive disorders, atheroschlerosis, myocardial infarction, coronary heart disease and other cardiovascular disorders, stroke, inflammatory bowel syndrome, dyspepsia and gastric ulcers.

Detailed Description of the Invention

Moiety having GLP-1 like activity The compounds of the invention comprise a moiety having GLP-1 like activity As used herein, the term "having GLP-1 like activity" refers to a moiety which in isolation (I e. not attached to the linker or aminothiol moiety) has activity of at least 10 % of that exhibited by GLP-1 (7-37) in a binding assay.

Preferably, the moiety has activity of at least 10 % of that exhibited by GLP-1 (7-37) in a binding assay. More preferably, the moiety has activity of at least 20 % of that exhibited by GLP-1 (7-37) in a binding assay. More preferably, the moiety has activity of at least 50 % of that exhibited by GLP-1 (7-37) in a binding assay. More preferably, the moiety has activity of at least 90 % of that exhibited by GLP-1 (7-37) in a binding assay. More preferably, the moiety has activity of at least equal to that exhibited by GLP-1 (7-37) in a binding assay Examples of specific moieties having GLP-1 like activity are polypeptides comprising the 1-37 amino acid sequence of GLP-1, viz, formula I: His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-SerTy r-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-l le-Ala-Trp-Leu-Val-Lys-Gly-ArgNH2 polypeptides comprising the 7-37 amino acid sequence of GLP-1, viz formula II: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-AlaAl a-Lys-Glu-Phe-l le-Ala-Trp-Leu-VaI-Lys-Gly-Arg-Gly

II

polypeptides comprising the 7-36 amino acid sequence of GLP-1, viz, formula III' His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-AlaAi a-Lys-Glu-Phe-l le-Ala-Trp-Leu-Val-Lys-Gly-Arg

S Ill

or a peptide sequence derived from formulae I, II or lii without eliminating the GLP-1 like activity Alternative moieties having GLP-1 like activity include R1-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Y-Gly-Gln-Ala-AlaLys -Z-Phe-l le-Aia-Trp-Leu-val-Lys-Gly-Arg-R2 or a pharmacuetically accetable salt thereof, wherein: R1 is selected from the group consisting of His, D-histidine, desamirio-histithne, 2-amino-histidine, 3-hydroxy-histidine, homohistidine, a-fluoromethyl-histidine, and a-methyl-histidine; X is selected from the group consisting of Met, Asp, Lys, Thr, Leu, Asn, Gin, Phe, Val, and Tyr; Y and Z are independently selected from the group consisting of Glu, Gin, Ala, Thr, Ser, and Gly, and R2 is selected from the group consisting of NH2, and Gly-; Z1-X1-X2-X3-Gly-Thr-Phe-Thr-Ser-X4-X5-Ser-X6-X7-X8-Glu-Gly-Gln-Ala-X9-LysX10-X1 1 X1 2-AIa-X1 3-X4-VaI-Lys-Gly-X1 5-Gly-Z2 wherein Z1 is substituent of the terminal amino group of the peptide; Z2 is a substituent of the terminal carbonyl group of the peptide, and X, to X14 each represents, independently of the others a natural or non-natural amino acid residue, having the D or L configuration.

R1-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Y-Gly-Gln-Ala-AiaLys -Z-Phe-l Ie-Ala-Trp-Leu-VaI-Lys-Gly-Arg-R2 wherein R1 is selected from the group consisting of L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, 3-hydroxy-histidine, homohistidine, a-fluoromethyl-histidine, and cx-methyl-histidine; X is selected from the group consisting of Ala, Gly, Val, Thr, lie, and alpha-methyl-Ala; Y is selected from the group consisting of Glu, Gin, Ala, Thr, Ser, and Gly; Z is selected from the group consisting of Glu, GIn, Ala, Thr, Ser, and Gly; R2 is selected from the group consisting of NH2, and Gly-OH; providing that the compound has an isoelectric point in the range from about 6 0 to about 9.0 and further providing that when R1 is His, X is Ala, Y is Glu, and Z is Glu, R2 must be NH2.

1,2-aminothiol moiety The free compounds of the invention comprise a 1,2 aminothiol moiety, that is a moiety of the formula (IV)

SH (IV)

Wherein R is a chemical group, preferably C1-C5 alkyl or hydrogen, or the remainder of the molecule. The remainder of the molecule may alternatively be attached at either carbon atom of the 1,2-aminothiol moiety Preferably, the 1,2-aminothiol moiety is a cysteine residue Cysteine has the formula (V)

HO

the remainder of the molecule may be attached at any chemically feasible point of the cysteine residue, for example. Re HO

(VI) (VII) (VIII) (IX) wherein Re represents the remainder of the molecule.

S

The cysteine residue is suitably of either enantiomeric form Preferably, it is of the naturally occurring (L) form.

Preferably, the compound has the formula (X) Re wherein Re represents the remainder of the molecule, and R represents H or C1-C6 alkyl, preferably H Optional linker group The optional linker group, when present, forms a covalent link to both the moiety having GLP-1 like activity and the 1,2 aminothiol moiety.

Any chemical species having two free valences is suitable. Examples of such species are i) single atoms linkers (i.e. the "link" as such contains only one atom), such as R R1 R2 GAt GAt GAt GAt (Xl) (XII) (Xlii) (XIV) Wherein R', Ri and R2 are independently selected from H and C1-C6 alkyl, and n is 0, 1 or 2, G is the moiety having GLP-1 like activity and At is the 1,2 amiriothiol moiety; ii) polyatom linkers, such as alkylene groups, polyethylene glycol (PEG) groups, diacids, diamines, aminoacids (especially a, 3, y, and), aminoalcohols, diols, poly(aminoacids), bissuifides.

S

A preferred class of linker is aminoacids. Aminoacids of any length (a, J3, y, 6 and longer) may be employed. In one embodiment, the aminoacid is an a aminoacid.

Preferably, the a aminoacid is a naturally occurring a aminoacid.

In an alternative preferred embodiment, the aminoacid is a y aminoacid. Highly preferably, the aminoacid is 4-aminobutyric acid; i.e the free compound of the invention has the structure (XV) or (XVI); 0 0 At (XV) (XV!) Very preferably, the free compound of the invention has the structure (XV).

Combination of linker and I,2-aminothiol Preferably, the linker is a y aminoacid and the 1,2-aminothiol is a cysteine residue.

Highly preferably, the free molecule of the invention has the formula (XVII)

SH GNH2

(XVII) More preferably, the free molecule of the invention has the formula (XVIII) (XVIII) Point of attachment of linker and moiety having GLP-1 like activity The linker (or the 1,2-aminothiol moiety if no linker is present) is attached to the moiety having GLP-1 like activity at any chemically appropriate site. Preferably, the

I

point of attachment is such that the free compound of the invention also possesses GLP-1 like activity.

Preferably, the linker (or the 1,2-aminothiol moiety if no linker is present) is attached to an amine group present in the moiety having GLP-1 like activity. Wherein the moiety having GLP-1 like activity comprises a lysine residue, preferably the linker is attached to the c-nitrogen of the lysine When the moiety having GLP-1 like activity is GLP-1 or a derivative thereof, the point of attachment of the linker (or the 1,2-aminothiol moiety if no linker is present) is the c-nitrogen of lysine 26 Highly preferably, the moiety having GLP-1 like activity is GLP-1 (7-37), and the point of attachment of the linker (or the 1,2-aminothiol moiety if no linker is present) is the c-nitrogen of lysine 26. In this embodiment, the free compound of the invention has the structure (XIX) HN (Linker)-(1,2-aminothiol moiety) His7----Ala2,GIu27--Gly37 (XIX) Very highly preferably, the moiety having GLP-1 like activity is GLP-1 (7-37), the linker is 4-aminobutyric acid and the point of attachment of the linker is the c-nitrogen of lysine 26. In this embodiment, the free compound of the invention has the structure (XX) rN(12aminothiof moie) His7----Ala2 Gki27--G1y37 (XX) Very highly preferably, the moiety having GLP-1 like activity is GLP-1 (7-37), the 1,2- aminothiol moiety is cysteine, and the point of attachment of the linker is the -nitrogen of lysine 26. In this embodiment, the free compound of the invention has the structure (XXI)

SH

(Linker) NH2 His7----AIa2.,.GIu27--GIy37 (XXI) The free compounds of the invention preferably have insulinotropic activity. As used herein, the term "insulinotropic activity" refers to the property of a compound to stimulate the synthesis or expression of the hormone insulin. -12-

Preferably, the free compounds of the invention have insulinotropic activity that is at least 10 % of that of GLP-1 (7-36). More preferably, the free compounds of the invention have insulinotropic activity that is at least 20 % of that of GLP-1 (7-36) More preferably, the free compounds of the invention have insulinotropic activity that is at least 50 % of that of GLP-1 (7-36). More preferably, the free compounds of the invention have insulinotropic activity that is at least 80 % of that of GLP-1 (7-36).

More preferably, the free compounds of the invention have insulinotropic activity that is at least equal to that of GLP-1 (7-36) Insulinotropic activity in this context may be measured using a suitable assay The insulinotropic property of a compound may be determined by providing that compound to animal cells, or injecting that compound into animals and monitoring the release of immunoreactive insulin (IRI) into the media or circulatory system of the animal, respectively. The presence of IRI is detected through the use of a radioimmunoassay which can specifically detect insulin. Although any radioimmunoassay capable of detecting the presence of IRI may be employed, it is preferable to use a modification of the assay method of Albano, J D. M., et al., (Acta Endocrinol. 70, 487-509 (1972)). In this modification, a phosphate/albumin buffer with a pH of 7.4 was employed. The incubation was prepared with the consecutive addition of 500 p.1 of phosphate buffer, 50 p.1 of perfusate sample or rat insulin standard in perfusate, 100 p.1 of anti-insulin antiserum (Weilcome Laboratories; 1:40,000 dilution), and 100 p.1 of [1251] insulin, giving a total volume of 750 p.1 in a 10*75mm disposable glass tube. After incubation for 2-3 days at 40 C., free insulin was separated from antibody-bound insulin by charcoal separation. The assay sensitivity was 1-2 pU/mI. In order to measure the release of IRI into the cell culture medium of cells grown in tissue culture, one preferably incorporates radioactive label into proinsulin. Although any radioactive label capable of labelling a polypeptide can be used, it is preferable to use 3H leucine in order to obtain labelling of proinsulin.

Labeling can be done for any period of time sufficient to permit the formation of a detectably labelled pool of proinsulin molecules; however, it is preferable to incubate cells in the presence of radioactive label for a 60-minute time period. Although any cell line capable of expressing insulin can be used for determining whether a compound has an insulinotropic effect, it is preferable to use rat insulinoma cells, and especially RIN-38 rat insulinoma cells. Such cells can be grown in any suitable medium, however, it is preferable to use DME medium containing 0.1% BSA and 25 mM glucose. -13-

The insulinotropic property of a compound may also be determined by pancreatic infusion. The in situ isolated perfused rat pancreas preparation was a modification of the method of Penhos, J C., et al (Diabetes 18, 733-738 (1969)). In accordance with such a method, fasted rats (preferably male Charles River strain albino rats), weighing 350-600 g, are anesthetized with an intraperitoneal injection of Amytal Sodium (Eli Lilly and Co., 160 nglkg) Renal, adrenal, gastric, and lower coloriic blood vessels are ligated. The entire intestine is resected except for about four cm of duodenum and the descending colon and rectum Therefore, only a small part of the intestine is perfused, thus minimizing possible interference by enteric substances with glucagon-like immunoreactivity. The perfusate is preferably a modified Krebs-Ringer bicarbonate buffer with 4% dextran T70 and 0 2% bovine serum albumin (fraction V), and is preferably bubbled with 95% 02 and 5% CO2. A nonpulsatile flow, four-channel roller-bearing pump (Buehler polystatic, Buehler Instruments Division, Nuclear-Chicago Corp.) is preferably used, and a switch from one perfusate source to another is preferably accomplished by switching a three-way stopcock. The manner in which perfusion is performed, modified, and analyzed preferably follows the methods of Weir, G. C., et al., (J. Clin. Investigat. 541403-1412 (1974)), which is hereby incorporated by reference DPP IV resistance Preferably, the free compounds of the invention show increased resistance to deactivation by DPP IV compared with natural GLP-1. Resistance to DPP IV deactivation is suitably measured for example by the technique described in in Green, B.D et al, Biological Chemistry, 385, 169-177(2004) Side chain modification of GLP-1 analogues The further enhancement of the utility of the free compounds of the invention as active ingredients in Type 2 diabetes therapies may depend on their incorporation into suitable extended release formulations. Such free compounds may block elimination through the urinary pathway and confer a degree of proteolytic protection on the attached peptide, thus prolonging duration of action.

While some of these delivery agents can be introduced as relatively straightforward encapsulating agents which are safe and biodegradable, a number of others which may impart desirable pharmacokinetic profiles on these compounds require covalent attachment. Amino-and thio-reactive biopolymeric ligands are among those -14- currently employed for this purpose and appear compatible for modification and delivery of our GLP-1 variants. The fragility of certain of these biopolymers in harsh chemical environments requires their attachment to therapeutic proteins and peptides post-synthetically.

If using an amine-reactive delivery agent for modification, the presence of three free amino groups in each analogue would likely yield a polymer-modified heterogeneous mixture of mono, di-, and tn-substituted derivatives The separation of these would likely be difficult and costly, and would complicate regulatory approval.

The introduction of a single, unique thiol group into the structure of these GLP-1 mimetics would simplify the introduction of a thio-reactive ligand for delivery purposs, as only one modified species would be expected to form Preferred moieties having GLP-1 like activity Preferred moieties having GLP-1 like activity include those of the formula (XXII) X4AImY4B]nZ (XXII) wherein X is optionally present and represents a substituent of the terminal carboxyl or amino group; Y is a linker group, n is an integer; m is 1 or 2; each A is an independently selected amino acid; each B is n independently selected amino acid which may be further optionally Z is optionally present and represents a substituent of the terminal amino or carboxy group; provided that the compound is not GLP-1 or a naturally occurring fragment thereof; or a prodrug or a pharmaceutically acceptable salt form thereof.

Amino acids A1 and A2 In one preferred embodiment, the moiety having GLP-1 like activity of the invention has the formula (XXIII)

K

(Xi II) Wherein X, Y, B, n and Z are as defined above, R1 and R2 represent the backbone/sidechain of the amino acids, and P is H, a substituent, or a link to R1 or R2 (e g. in the case of proline), and K represents H, H2, NH or 0.

In an alternative preferred embodiment, the moiety having GLP-1 like activity of the invention has the formula (XXIV) (XXIV) wherein X, Y, B, n and Z are as defined above, and R, and R2 represent the backbone/sidechain of the amino acids, and P is H, a substituent, or a link to R1 or R2 (e g in the case of proline) Amino acids A1 and A2 may be a, 1, y or other amino acids. Preferably, A1 and A2 are a amino acids Preferably, amino acids A1 and A2 are independently selected from groups of the formula (XXV) (XXV) wherein R3 and R4 are independently selected from hydrogen atom or an alkyl, aminoalkyl (optionally substituted on the nitrogen atom by one or two alkyl, phenyl, benzyl, cycloalkyl, optionally substituted aryloxycarbonyl, optionally substituted arylalkoxycarbonyl and/or optionally substituted alkoxycarbonyl groups), thioalkyl (optionally substituted on the sulphur atom by an alkyl, phenyl, benzyl or cycloalkyl group), hydroxyalkyl (optionally substituted on the oxygen atom by an alkyl, phenyl, benzyl or cycloalkyl group), carboxyalkyl, carbamoylalkyl, guanidinoalkyt, cycloalkyl, cycloalkylalkyl, optionally substituted fused cycloalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl or optionally substituted heteroarylalkyl group, or an imidazolyl or imidazolylalkyl group, or R3 and R4 together with the carbon atom carrying them, form a cycloalkyl or fused cycloalkyl group, P is selected from hydrogen, alkyl, or cycloalkyl, or taken with R2 or R3 together with the carbon and nitrogen atoms to which they are attached represents a mono-or bicyclic group having from 4 to 12 ring members which is saturated, partially unsaturated, or unsaturated and is optionally substituted.

Preferably, at least one of R3 and R4 is hydrogen The amino acids A1 and A2 may have the D-or L-configuration Preferably, when the compound has the formula (XXIII) above, the amino acids A1 and A2 have the L-configuration.

Preferably, when the moieties having GLP-1 like activity have the formula (XXIV) above, the amino acids A1 and A2 have the D-configuration. -17-

I

Preferably, A, is His. More preferably, A, is D-His. Preferably, A2 is Ala. More preferably, A2 is 0-Ala. More preferably, A1 is His and A2 is Ala. More preferably, A1 is D-His and A2 is D-Ala. Still more preferably, the moiety having GLP-1 like activity has the formula (XXIV), A, is D-His and A2 is D-Ala; that is the moiety having GLP-1 like activity has the formula (XXVI) çNH X [8) (XXVI) Wherein X, Y, B, n and Z have the meanings defined above Still more preferably, the N-terminus of the peptide [B] is bound covalently to the group Y. GroupX Group X, where present, is preferably selected from a hydrogen atom, alkyl, alkenyl, alkynyl, acyl, alkoxy, cycloalkyl, cycloalkoxy, thioalkyl, alkylthio, sulfoxoalkyl, haloalkyl, aryl, heteroaryl, NH2, NH(AIkyI), N(Alkyl)2, alkyieneNH2, AlkyleneNHAlkyl, Alkylene(Alkyl)2, NHAryI, N(Aryl)2 and heteroaryl In compounds of the invention of formula (Ill) above, it is preferred that group X has basic character "Basic character" in this context means that group X is capable of binding a proton in aqueous solution at pH 7 Highly preferred groups X are -(C,)alkyIene-NH(C16)alkyl, -(C,6)alkylene-N(C, 6)aIkyI2 (alkyl the same or different), -NH(C16)aikyl, -N(C,)alkyI2, and -NH2. Most preferred is -NH2.

In a preferred embodiment, the moiety having GLP-1 like activity of the invention has the formula (XXV) above and X is -NH2; that is the compound has the formula (XXVII) -18-

S

H2N [B] (XXVII) Wherein Y, B, n and Z are as defined above Group (B] Each group B is an independently selected amino acid The groups B are linked together in a conventional manner (i.e. via amide bonds) to form a peptide chain. As used herein, the groups B are numbered according to formula (XXVIII), X-A1 -A2-Y-B1 -B2-B3-B4-B5-B5-. . . -B.1 1-B-Z (XXVIII) In a preferred embodiment, the N-terminus of the group [B] is covalently bound to the group Y, and the C-terminus of the group [B] is covalently bound to the group Z where present.

In an alternative preferred embodiment, the C-terminus of the group [B] is covalently bound to the group Y, and the N-terminus of the group [B] is covalently bound to the group Z where present.

Amino acids B may be cx, 13, y or other amino acids. Preferably, B are independently selected a amino acids. The amino acids B may have the D-or L-configuration.

Preferably, amino acids B are independently selected from groups of the formula (XXIX) -19-(XXIX) wherein R5 and R6 are independently selectedfrom hydrogen atom or an alkyl, aminoalkyl (optionally substituted on the nitrogen atom by one or two alkyl, phenyl, benzyl, cycloalkyl, optionally substituted aryloxycarbonyl, optionally substituted arylalkoxycarbonyl and/or optionally substituted aikoxycarbonyl groups), thioalkyl (optionally substituted on the sulphur atom by an alkyl, phenyl, benzyl or cycloalkyl group), hydroxyalkyl (optionally substituted on the oxygen atom by an alkyl, phenyl, benzyi or cycloalkyl group), carboxyalkyl, carbamoylalkyl guanidinoalkyl, cycloalkyl, cycloalkylalkyl, optionally substituted fused cycloalkyl, optionally substituted aryl, optionally substituted aryialkyl, optionally substituted heteroaryl or optionally substituted heteroarylalkyl group, or an imidazolyl or imidazoiylalkyl group, or R5 and R5 together with the carbon atom carrying them, form a cycloalkyl or fused cycloalkyl group, Q is selected from hydrogen, alkyl, or cycloalkyl, or taken with R5 or R6 together with the carbon and nitrogen atoms to which they are attached represents a mono-or bicyclic group having from 4 to 12 ring members which is saturated, partially unsaturated, or unsaturated and is optionally substituted.

Preferably, at least one of R5 and R6 is hydrogen.

Preferably, amino acids B are independently selected from naturally occurring amino acids.

Preferably, n is less than 100. More preferably, n is less than 50. More preferably, n is less than 30. More preferably, n is 28 or 29.

Preferably, the amino acid sequence [B] is substantially homologous to GLP-1 (9- 37) or a fragment of GLP-1 having n residues. As used herein, the term substantially homologous" means that at least 80 %, preferably at least 90 % and more preferably at least 95 % of the amino acid sequence is the same For the avoidance of doubt, GLP-1 (1 to 37) has the sequence His Asp Glu Phe Glu Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gin Ala Ala Lys Glu Phe lie Ala Trp Leu Val Lys Gly Arg Gly.

GLP-1 (7 to 37) has the sequence His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gin Ala Ala Lys Glu Phe lie Ala Trp Leu Val Lys Gly Arg Gly -20 -GLP-1 (9 to 37) has the sequence Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gin Ala Ala Lys Glu Phe lie Ala Trp Leu Vat Lys Gly Arg Gly.in a preferred embodiment, B is selected from a group of the formula B, Gly Thr Phe Thr Ser B7 B8 Ser B,0 B,1 B,2 Glu Gly Gin Ala B,7 Lys B,9 B20 B2, Ala B23 B24 Vat Lys Gly B28 Gly.

wherein each of B1, B7, B8, B,0, B,1, B12, B,7, B,9, B20, B21, B23, B24 and B28 is independently selected from the definition of B above In an alternative preferred embodiment, B is selected from a group of the formula B1 Gly Thr Phe Thr Ser B7 B8 Ser B,0 B11 B,2 Glu Gly Gin Ala B,7 Lys B,9 B20 B2, Ala B23 B24 Vat Lys Giy B28 wherein each of B1, B7, B8, B,0, B11, B,2, B,7, B,9, B20, B21, B23, B24 and B28 is independently selected from the definition of B above.

In an alternative preferred embodiment, B is selected from a group having the formula B, Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu B,3 Gly Gin Ala Ala Lys B19 Phe lie Ala Trp Leu Vat Lys Gly Arg Gly wherein each of B,, B,3 and B,9 is independently selected from the definition of B above.

In an alternative preferred embodiment, [B] comprises a sequence of the formula Glu Gly Thr Phe Thr Ser Asp Vat Ser Ser Tyr Leu Glu Gly Gin Ala Ala Lys Glu Phe lie Ala Trp Leu Val Lys Gly Arg Gly or Glu Gly Thr Phe Thr Ser Asp Vat Ser Ser Tyr Leu Giu Gly Gin Ala Ala Lys Glu Phe lie Ala Trp Leu Val Lys GJy Arg more preferably, [B] has the formula Glu Gly Thr Phe Thr Ser Asp Vat Ser Ser Tyr Leu Glu Giy GIn Ala Ala Lys Glu Phe lIe Ala Trp Leu Vat Lys Gly Arg Gly or -21 -Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gin Ala Ala Lys Glu Phe lie Ala Trp Leu Vat Lys Gly Arg.

Linker Group V in a broad sense, the linker group Y is defined as a chemical moiety capable of forming a covalent bond with the C or N terminus of [B1 and the C or N terminus of A1-A2 Preferred linker groups Y are those of the formula (XXX) -F'. ..G.. H'-(XXX) wherein F' is optionally present and is selected from -NH, -NAikyl, 0, S, SO, SO2, -CH2, -CHAlkyl, -C(AIkyl)2 (each alkyl independently selected), C=O, C=NH, CN(Alkyl), an amino acid residue, and C=S; G is selected from the group comprising alkylene of 1 to 12 carbon atoms, cycloalkylene of 3 to 12 carbon atoms, carbocyclic aryl of 6 to 12 carbon atoms, mono-or bicyclic heteroaryl of 5 to 12 ring members having from 1 to 5 heteroatorns independently selected from 0, N, or S; -NH, -NAlkyl, 0, S, SO, SO2, -CH2, -CHAlkyl, -C(Alkyl)2 (each alkyl independently selected), C=O, C=NH, C=N(Alkyl), an amino acid residue, and CS, H' is optionally present and is selected from -NH, -NAlkyl, 0, S, SO, SO2, -CH2, -CHAlkyl, -C(Alkyl)2 (each alkyl independently selected), C0 CNH, CN(Aikyl), an amino acid residue, and C=S; and. represents an optional single or double bond, The group Y can also be a bond (single, double or triple). The group Y may independently feature single, double or triple bonds to amino acids A1 or A2 and B1.

The carboxy and/or amino terrnini linked to Y may be in any oxidation state.

In a preferred embodiment, the compound is of formula (II) above, and the linker group Y is selected from =N-O-, =N-NH-, N-N(C16)Aikyi and N-S-, and K is H; that is the compounds have the formula (XXXI) -22 -(XXXI) wherein X, R1, R2, P, B, n and Z are as defined above, and R7 is selected from 0, NH, N(C15)AlkyI, and S. Highly preferably, R7 is 0.

Alternatively and preferably, the group Y has the formula (XX)(Il) R10 (XXXII) wherein X' is selected from 0, NH or N(C1-C6)alkyl; Y' is selected from 0, NH or N(C1-C6)alkyl; wherein R9 and R10 are independently selected from hydrogen atom or an alkyl, aminoalkyl (optionally substituted on the nitrogen atom by one or two alkyl, phenyl, benzyl, cycloalkyl, optionally substituted aryloxycarbonyl, optionally substituted arylalkoxycarbonyl and/or optionally substituted alkoxycarbonyl groups), thioalkyl (optionally substituted on the sulphur atom by an alkyl, phenyl, benzyl or cycloalkyl group), hydroxyalkyl (optionally substituted on the oxygen atom by an alkyl, phenyl, benzyl or cycloalkyl group), carboxyalkyl, carbamoylalkyl, guanidinoalkyl, cycloalkyl, cycloalkylalkyl, optionally substituted fused cycloalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl or optionally substituted heteroarylalkyl group, or an imidazolyl or imidazolylalkyl group, or R9 and R10 together with the carbon atom carrying them, form a cycloalkyl or fused cycloalkyl group.

-23 -Preferably, Xis NH.

Preferably, Y' is NH or 0.

Preferably, R9 is H or methyl Preferably, R10 is H or methyl.

Preferably, R9 is H and R,0 is methyl, or R9 is H and R10 is methyl Alternatively, and preferably, R9 and R10 are H. Highly preferably, group Y has the formula (XXlll, XXXIV or XXXV) (.X.XllI) (XX.)(l\f) (XXV) In an alternative preferred embodiment, the compound has the formula p(XXVI) (XXXVI) wherein X, R1, R2, P, B, n and Z are as defined above.

In another preferred embodiment, the compound has the formula (XXXVII) -24 -y}rB]n (XXXVII) wherein X, R1, R2, P, B, n and Z are as defined above, and R5 is selected from 0, S, NH, and N(AIkyl).

Highly preferably, R8 is 0. In this embodiment, the compounds of the invention feature a urea group linking A2 and [BJ.

In an alternative preferred embodiment, the compound has the formula (XXXVIII)

X Al

(XXXVIII) wherein X, Y, B, n and Z are as defined above, and R2 represents the backbone/sidechain of the amino acids, P is H, a substituent, or a link to R1 or R2 (e.g in the case of proline), represents an optional single or double bond, and K represents H, H2, NH or 0.

Group Z Group Z, where present, is preferably selected from a hydrogen atom, alkyl, alkenyl, alkynyl, acyl, alkoxy, cycloalkyl, cycloalkoxy, thioaikyl, alkylthio, sulfoxoalkyl, haloalkyl, aryl, heteroaryl, NH2, NH(Alkyl), and N(Alkyl)2.

Preferred Compounds -25 -Amongst specifically preferred compounds of the invention are included the following: / NH c

N

I I In-AIa-AIa-Lys-Glu-Phe-IIe-AIa-Trp-Leu-Vai-Lys-GPy-Ar 0 0 g-Gy NHCOCH2CH2CH2NHCys (XXXIX) / NH c N N Giuiy-Thr-Phe-Thr-Ser-p-Va)-Ser-Ser-Tyr-Leu-GIu H -Gy- -Gin-Ala-Ala L o 0 Arg-NH2 -ys-GIu-Phe-lie-AIa-Trp-Leu-VaI-Lys-Gly-NHCOCH2CH2CH2NHCys (XL) / NH H2N -Gly- _ GIu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu -GIn-Aia-Ala-Lys-Giu-Phe-Iie-Ala-Trp-Leu-Vat-Lys-Giy-o = Arg-Gy NHCOCH2CH2CH2NI-iCys (XLI)

NH co

H2N H -GIn-Ala-Aia-Lys-GIu-Phe-Aia-TrLeu-VaI-Lysiy- _ Giu-Giy-Thr*Phe-Thr-Ser-Asp-VaI-Ser-Ser-Tyr-Leu-GIu -Gly-j Arg-NH2 \ NHCOCH2CH2CH2NHCys -26 -(XLII)

NH

H

H2N N GIy-Thr-Phe-Thr-Ser-AsVaI-Ser-Ser-Tyr-Leu-GIu -GIn-Aa-AJa-Lys-GIu-Phe-IIe-AIa-Trp-Leu-VaI-Lys-Gy-o o Arg-GIy NHCOCH2CH2CH2NHCys (XLIII)

NH

H

H2N N N r GIu-Gy-Thr-Phe-Thr-Ser-Asp-VaI-Ser-Ser-Tyr-Leu-GIu -Gin-Ala-Ala-Lys-Glu-Phe--He-Ala-Trp-Leu-Val-Lys-Giy-o o Arg-NH2 NHCOCH2CH2CH2NHCys (XLIV) H2N N GluIy-Thr-Phe-Thr-Ser-p-VaI-Ser-Ser-Tyr-Leu-Glu /1 N o I -Gly--Gln-Aia-Ala-Lys-Glu-Phe-lle-AJa-Trp-Leu-Vai-Lys-GIy-o o Arg-Gly NHCOCH2CH2CH2NHCys (XLV) /NH

N N

H N Glu-Gly-Thr-Phe-Thr-Ser-Asp-Vai-Ser-Ser-Tyr-Leu-Giu fl-Gy- II -Gln-AIa-Aia-Lys-GIu-Phe-lle-Ala-Trp-Leu-Vai-Lys-Gly-o Arg-NH2 NHCOCH2CH2CH2NHCys -27 -

S

(XLVI)

N

r)-NH

H

H2N N N Gu-Gty-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu- H Glu-Gly-o -Gtn-AIa-AIa-Lys-GIu-Phe-IIe-AIa-Trp-Leu-VaI-Lys-G y-Arg-Gy NHCOCH2CH2CH2NHCys (XLVI I)

N

NH

H2N N GIu-GIy-Thr-Phe-Thr-Ser-Asp-VaI-Ser-Ser-Tyr-Leu-GI H u-GIy-o = -GIn-AIa-Ata-Lys-G u-Phe-IIe-AIa-Trp-Leu-VaI-Lys-GIy -Arg-GIy NHCOCH2CH2CH2NHCys (XL VIII)

N

-NH

H2N GIu-GIy-Thr-Phe-Thr-Ser-Asp-VaI-Ser-Ser-Tyr-Leu-GIu-GIy--GIn-AIa-AIa-LysG Iu-Phe-He-AIa-Trp-Leu-VaI-Lys-GIy-Arg-GIy NHCOCH2CH2CH2NHCys (XLIX) -28 -NTh

NH H2N -

-GIn-AIa-AJaLYSGILJPheIIeAIaTr.T.LeUVaILYSGIYAGIY 0 I NHCOCH2CH2CH2NHCys (L) p

NH

H2N -GIn-AIa-AIa-Lys-GIu-Phe-Ile-AIa-TrLeu-VaI-Lys-GIy-Arg-GIy o I NHCOCH2CH2CH2NHCys (LI) NTh 1)-NH 0 0 H2N N GIu-GIy-Thr-Phe-Thr-Ser-Asp-VaI-Ser-Ser-Tyr-Leu-G!u-GIy-H -Gn-AIa-AIa-Lys-Glu-Phe-I Ie-AIa-Trp-Leu-VaI-Lys-GIy-Arg-GIy o = I NHCOCH2CH2CH2NHCys (LII)

NH

H2N JIIII

H_ U

N GIu-cIy-mr-Phe-mr-ser-Asp-vaI-ser-ser-Tyr-Leu-GIu-cty--GIn-Ata-AIa-LysGIu -Phe-IIe-AIa-Trp-Leu-VaI-Lys-GIy-Arg-GIy 0 I NHCOCH2CH2CH2NHCys -29 -

I

(LIII) H is-AIa-GIu-GIy-Thr-Phe-Thr-Se r-Asp-VaI-Ser-Ser-Tyr-Leu-GIu-GIy- -GIn-AIa-AIa-Lys-GIu-Phe-I Ie-AIa-Trp-Leu-VaI-Lys-Giy-Arg-GIy NHCOCH2CH2CH2NHCys (LIV) H is-AIa-GIu-Gly-Th r-Phe-Thr-Ser-Asp-VaI-Ser-Ser-Tyr-Leu-GIu-GIy--GIn-AIa-AIa-Lys-GIu-PheI!e -AIa-Trp-Leu-VaI-Arg-GIy-Arg-GIy NHCOCH2CH2CH2NHCys (LV) His-AIa-GIu-GIy-Thr-Phe-Thr-Ser-Asp-Vai-Ser-Ser-Tyr-Leu-GIu-GIy--GIn-AlaA Ia-Lys-GIu-Phe-I e-AIa-Trp-Leu-VaI-Lys-Gly-Arg-GIy NHCOCH2CH2CH2NHCyS (LVI) In a further alternative preferred embodiment, the compound has the formula H2N GIu-GIy-Thr-Phe-Thr-Ser-Asp-VaI-Ser-Ser-Tyr-Leu-GIu-GIy--GIn-AIa-Ata-LysG Iu-Phe-IIe-AIa-Trp-Leu-VaI-Lys-GIy-Arg-GIy NHCH2OCH2CH2CH2NH-Cys (LVII, SPO13) it being understood that in the above structures, the group COCH2CH2CH2NHCys is attached to the c amine of the Lysine residue.

Complexed compounds -30 -

S

In one aspect, the invention relates to a free compound of the invention covalently linked to a carrier ligand ("complexed compound"). Highly preferably, the carrier ligand is linked to the free compound via the aminothiol moiety.

Highly preferably, the carrier ligand in its free state (i.e. prior to attachment to the free compound) has a carbonyl group which serves as the point of attachment of the free compound. In this embodiment, the 1,2 aminothiol moiety forms a thiazolidine group with the carbonyl group of the carrier ligand, as shown in scheme 1.

Re N HR + Re Scheme 1 wherein Re represents the remainder of the molecule, R represents chemical group, preferably C1-C6 alkyl or hydrogen, and X and Y represent chemical groups.

Preferably, the carrier ligand in its free state is polymeric or oligomeric. Preferably, the complexed compound has a longer half life in vivo compared with the free compound. The complexed compound preferably has intrinsic activity, that is it has GLP-1 like activity; however, the complexed compound may lack intrinsic activity, and only be transformed to an active compound (i.e. one possessing GLP-1 like activity) when in vivo In this latter aspect, the complexed molecule behaves as a pro-drug.

Preferably, the carrier ligand is a poly-or oligosaccharide. In the invention the polysaccharide compound may be a naturally occurring poly-or oligosaccharide, a derivative of a naturally occurring poly-or oligosaccharide, for instance a poly-or oligosaccharide which has been derivatised by reaction of one or more active groups on the saccharide residues, or which has been covalently linked to a derivatising group by either end of the poly-or oligosaccharide chain or by an active group midway along the chain, or may be a naturally occurring poly-or oligosaccharide derivative, for instance comprising attached phospholipids or proteins, or derivatives of naturally occurring poly-or oligosaccharide derivatives, for instance chemically derivatised compounds, such as hydrolysed or otherwse chemically reacted derivatives The poly-or oligosaccharide portion of the compound has more than 5, preferably at least 10, and more preferably at least 20 or 50 monosaccharide -31 -residues in the polymer chainReadily available polysaccharide compounds may have up to 500 saccharide residues in total, but usually have fewer than 300 residues in the polymer chain Polyether derivatives In a preferred embodiment, the ligand is a polyether moiety As used herein, the term "polyether moiety" refers to a group containing a plurality of ether linkages.

A highly preferred polyether ligand is a PEG (polyethylene glycol ligand PEG ligands comprise at least a repeat ethylene glycol unit, , wherein n is an integer. The skilled person will understand that a variety of PEG groups are known and described in the art. The term PEG includes poly(ethylene glycol) in any of its forms, including alkoxy PEG, difunctional PEG, multiarmed PEG, forked PEG, branched PEG, pendent PEG (i.e PEG or related polymers having one or more functional groups perident to the polymer backbone), or PEG with degradable linkages therein. The polymer backbone can be linear or branched Branched polymer backbones are generally known in the art. Typically, a branched polymer has a central branch core moiety and a plurality of linear polymer chains linked to the central branch core. PEG is commonly used in branched forms that can be prepared by addition of ethylene oxide to various polyols, such as glycerol, pentaerythritol and sorbitol The central branch moiety can also be derived from several amino acids, such as lysine. The branched poly(ethylene glycol) can be represented in general form as R(-PEG-OH).sub.m in which R represents the core moiety, such as glycerol or pentaerythritol, and m represents the number of arms Multi-armed PEG molecules, such as those described in U S. Pat. No. 5,932,462, which is incorporated by reference herein in its entirety, can also be used as the polymer backbone.

Preferably, the ligand is carbonyl-containing polyether moiety; that is, a moiety comprising at least one polyether group and at least one carbonyl group. Preferably, the carbonyl group is an aldehyde group.

Preferably, the ligand is an aldehyde-containing poly(ethyleneglycol) moiety (PEG-CHO) More preferably, the ligand is of the formula (LXVIII) -32 -R3 0 CH0 I. in (LXVIII) wherein Ra is a C-C5 alkyl group, Alk is a branched or straight C1-C6 alkylene group When the ligand is an aldehyde-contairiing poly(ethyleneglycol) moiety of formula LXVIII above, the complexed compound of the invention preferably has the formula LXIX, as shown in scheme 4. a AIk

R SH (L)ll) CHO L H (LXIX) Re(.-N R Scheme 4 wherein R, Re, R, AIk and n have the meanings set out above In a preferred embodiment, the compound of the invention has the formula LXX Hcs-AIa-GIu-GIy-Thr-Phe-Thr-Ser-Asp-Vai-Ser-Ser-Tyr-Leu-Giu-Gly -Gin-Aia-Aia-Lys-GIu-Phe-Ile-Ala-Trp-Leu-Vat-Lys-Gty-ArgGly <s AIk Ra (LXX) wherein Alk, n and R have the meanings ascribed above.

-33 -

S

The complexed compounds of the invention have intnnsic GLP-1 like activity.

Alternatively, the complexed compounds of the invention do not have intrinsic activity, and behave as pro-drugs of the activity species.

Preparation of Compounds The invention also encompasses a process for the preparation of the compounds of formula (I) which may be obtained by various methods Convenient methods include solid phase sequential synthesis, synthesis and coupling of fragments in solution, enzymatic synthesis, or by employing molecular biology techniques.

The general methods of solid phase peptide synthesis have been described in "Fmoc Solid Phase Peptide Synthesis: A Practical Approach", Chan, W.C. and White, PD. eds, Chapter 3, pp 41-76, Ocford University Press, 2000; "Chemical Approaches to the Synthesis of Peptides and Proteins"; P Lloyd-Williams et al, CRC Press 1997; and "The Chemical Synthesis of Peptides", John Jones, Oxford University Press, 1994 Solid phase synthesis is for example carried out using an automatic device which executes in a repetitive and programmable manner the deprotection, coupling and washing cycles necessary for the sequential introduction of amino acids into the peptide chain The C-terminal amino acid is fixed on a resin conventionally used for the preparation of polypeptides, preferably a polystyrene cross-linked with 0.5 to 3.0% divinylbenzene and provided with activated radicals that enable the first amino acid to be fixed covalently to the resin Suitable resins are well known to those skilled in the art. Appropriate selection of the resin allows the introduction after synthesis of the C-terminal Z function.

The amino acids are then introduced one by one in the desired sequence. Each cycle of synthesis corresponding to the introduction of an amino acid comprises N-terminal deprotection of the peptide chain, successive washings designed to remove the reagents or swell the resin, coupling with activation of the amino acid and further washings Each of those operations is followed by filtration effected as a result of the presence of a sintered glass filter incorporated in the reactor in which the synthesis is beings carried out The couplings reagents used are the conventional reagents for peptide synthesis, such as dicyclohexylcarbodiimide (DCC) and hydroxybenzotriazole (HOBT) or benzotriazol-1 -yl-oxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), or also diphenyl-phosphorylazide (DPPA). Activation by the formation of mixed or symmetrical anhydrides is also possible.

Each amino acid is introduced into the reactor in an approximately 6-fold excess in relation to the degree of substitution of the resin and in an approximately equivalent amount in relation to the coupling agents. The coupling reaction may be confirmed at each stage of the synthesis by the ninhydrin reaction test described by Kaiser et al. (Anal. Biochem., 34, 595, 1970).

After assembling the peptide chain on the resin, an appropriate treatment, for example using a strong acid such as trifluoroacetic acid, or hydrofluoric acid in the presence of anisole, ethanedithiol or 2-methylindole, is used to cleave the peptide from the resin and also to free the peptide of its protecting groups. Alternatively, the group Z may be introduced at this stage, concomitant with cleavage from the resin.

Dependent on the nature of the group Z, the skilled person will be able to select suitable conditions for such a transformation Alternatively, the peptide may be cleaved from the resin with a free carboxyl group, which may subsequently be coupled to the group Z. Again, the skilled person will be able to determine suitable conditions.

The compound is then optionally purified by conventional purification techniques, especially chromatography techniques.

The 1,2 aminothiol moiety and linker group (where present) may be introduced at any appropriate stage of peptide synthesis As an example, wherein the linker group is a 4-amino butyric acid group, the 1,2 aminothiol moiety is a cysteirie residue, the moiety having GLP-1 like activity is GLP- 1 (7-37), and the point of attachment is Lys26, the free compound of the invention is suitably synthesised according to scheme 5 -35 -

S

1) Dde-Ly(Fmoc)-OH N2N Gk12r_GIy37_C___? HCTU De-HN-Lys(Fmoc)-GJu77-Iy-C zu) Fmoc-4-amino ii) 20% piperidinefDMF butync acid Dde_4N_Lrs.GIuv_GI)37_C___O Dde-HN-Lya-G1u27-G}y37-C M2N 0 0 Fn,oc 0e-1N -Lys-GIu--GC iv) 20% pipi,diiifDM Dde-MN-Lys GIu,.-Gly37---C)Boc.cysçrntyI) J) 0 DIC and HOST Cys(TntyI)HN 12N Lys GIuvGii-C H,SrAIaLYsUv-GPyi7-C vi)25/sbydrazzneinDMF rO Boys(Trt)l)HN 8ocCys(Trityl)HN H,s,.-AIa-Lys GIu-GIy, IFA, TIS, thioanisok, EDT CysHN Scheme 5 Alternatively, the peptide may be assembled in the N to C direction Suitable methodology is described in GB0505200.6, the contents of which are incorporated herein by reference.

The peptides of the present invention may also be obtained by coupling in solution selectively protected peptide fragments which may themselves be prepared either in the solid phase or in solution. The use of protecting groups and the exploitation of their differences in stability is analogous to solid phase methods with the exception of the attachment of the peptide chain to the resin The C-terminal carboxy group is protected, for example, by a methyl ester or an amide function The methods of -36-activation during coupling are likewise analogous to those employed in solid phase synthesis.

The peptides of the present invention may also be obtained using molecular biology techniques, employing nucleic acid sequences that encode those peptides Those sequences may be RNA or DNA and may be associated with control sequences and/or inserted into vectors. The latter are then transfected into host cells, for example bacteria. The preparation of the vectors and their production or expression in a host are carried out by conventional molecular biology and genetic engineering techniques The introduction of the linker group Y may be achieved as a step in the solid-phase synthesis of peptides as described above, or alternatively in solution. For example, compounds of formula (XVll) above wherein R5 is 0 are suitably prepared as shown in scheme 6.

caonyIdumdazoJe 0 X X R1 NPH -_____ ____"__________ Al X _.-JL PN IBI z Scheme 6 -37 -Peptide [B] is assembled conventionally (as described above) on solid support P' in the C to N direction. Therefore, the C-terminus of [B] is attached (optionally via a linker) to the solid support, and the N terminus is free. The peptide is reacted with carbonyldiimidazole to give derivative This is subsequently reacted with dipeptide X-A-A-NPH to give solid-bound urea. This is cleaved from the solid support (e g. in the presence of Z) to give the compound of the invention Other forms The invention also encompasses all pharmaceutically acceptable forms of compounds of formula I, including without limitation, its free form (zwittenon), and its pharmaceutically acceptable complexes, salts, solvates, hydrates, and polymorphs Salts include, without limitation, acid addition salts and base addition salts, including hemisalts.

Pharmaceutically Acceptable Salts The present invention also encompasses pharmaceutically acceptable salts of the present compounds Such salts include pharmaceutically acceptable acid addition salts, pharmaceutically acceptable metal salts, ammonium and alkylated ammonium salts.

Acid addition salts include salts of inorganic acids as well as organic acids.

Representative examples of suitable inorganic acids include hydrochloric, hydrobromic, hydriodic, phosphoric, sulfuric, nitric acids and the like.

Representative examples of suitable organic acids include formic, acetic, trichloroacetic, trifluoroacetic, propionic, behzoic, cinnamic, citric, fumaric, glycolic, lactic, maleic, malic, malonic, mandelic, oxalic, picric, pyruvic, salicylic, succinic, methane-sulfonic, ethanesulfonic, tartaric, ascorbic, pamoic, bismethylene salicylic, ethanedisulfonic, gluconic, citraconic, aspartic, stearic, palmitic, EDTA, glycolic, p-aminobenzoic, glutamic, benzenesutfonic, p-toluenesulfonic acids and the like Further examples of pharmaceutically acceptable inorganic or organic acid addition salts include the pharmaceutically acceptable salts listed in J. Pharm Sci. 1977, 66, 2, which is incorporated herein by reference.

Examples of metal salts include lithium, sodium, potassium, magnesium, calcium salts and the like Examples of ammonium and alkylated ammonium salts include -38 -

S

ammonium, methyl-, dimethyl-, tnmethyl-, ethyl-, hydroxyethyl-, diethyl-, n-butyl-, sec-butyl-, tert-butyl-, tetramethylammonium salts and the like.

Methods of Treatment In one embodiment of the invention a compound according to the invention is used in the treatment or prevention of a condition selected from hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, hypertension, syndrome X, dyslipidemia, cognitive disorders, atheroschlerosis, myocardial infarction, coronary heart disease and other cardiovascular disorders, stroke, inflammatory bowel syndrome, dyspepsia and gastric ulcers.

Combination Therapies The compounds of the present invention may be combined in use with one or more pharmacologically active substances. Preferred classes of pharmacologically active substances are antidiabetic agents, antiobesity agents, appetite regulating agents, antihypertensive agents, agents for the treatment and/or prevention of complications resulting from or associated with diabetes and agents for the treatment and/or prevention of complications and disorders resulting from or associated with obesity.

In the present context the expression "antidiabetic agent"includes compounds for the treatment and/or prophylaxis of insulin resistance and diseases wherein insulin resistance is the pathophysiological mechanism.

Examples of these pharmacologically active substances are: Insulin, GLP-1 agonists, sulphonylureas (e. g. tolbutamide, glibenclamide, glipizide and gliclazide), biguanides e. g. metformin, meglitinides, glucosidase inhibitors (e. g. acorbose), glucagon antagonists, DPP-IV (dipeptidylpeptidase-IV) inhibitors, inhibitors of hepatic enzymes involved in stimulation of gluconeogenesis and/or glycogenolysis, glucose uptake modulators, thiazolidinediones such as troglitazone and ciglitazone, compounds modifying the lipid metabolism such as antihyperlipidemic agents as HMG CoA inhibitors (statins), compounds lowering food intake, RXR agonists and agents acting on the ATP-dependent potassium channel of the micelles, e. g glibenclamide, glipizide, gliclazide and repaglinide; Cholestyramine, colestipol, clofibrate, gemfibrozil, lovastatin, pravastati n, simvastatin, probucol, dextrothyroxine, neteglinide, repaglinide; J3-blockers such as alprenolol, atenolol, timolol, pindolol, propranolol and metoprolol, ACE (angiotensin converting enzyme) inhibitors such as benazepril, capto pril, enalapril, fosinopril, lisinopril, alatriopril, quinapril and ramipril, -39 -calcium channel blockers such as nifedipine, felodipine, nicardipine, isradipine, nimodipine, diltiazem and verapamil, and cr-blockers such as doxazosin, urapidil, prazosin and terazosin; CART (cocaine am-phetamine regulated transcript) agonists, NPY (neuropeptide Y) antagonists, MC4 (melanocortin 4) agonists, orexin antagonists, TNF (tumor necrosis factor) agonists, CRF (corticotropin releasing factor) agonists, CRF BP (corticotropin releasing factor binding protein) antagonists, urocortin agonists, ss3 agonists, MSH (melanocyte-stimulating hormone) agonists, MCH (melanocyte-concentrating hormone) antagonists, CCK (cholecystokinin) agonists serotonin re-uptake inhibitors, serotonin and noradrenaline re-uptake inhibitors, mixedserotonin and noradrenergic compounds, SHT (serotonin) agonists, bombesin agorusts, galanin antagonists, growth hormone, growth hormone releasing compounds, TRH (thyreotropin releasing hormone) agonists, UCP 2 or 3 (uncoupling protein 2 or 3) modulators, leptin agonists, DA agonists (bromocriptin, doprexin), lipasefamylase inhibitors, RXR (retinoid X receptor) modulators, TRss agontsts; histamine H3 antagonists.

It should be understood that any suitable combination of the compounds according to the invention with one or more of the above-mentioned compounds and optionally one or more further pharmacologically active substances are considered to be within the scope of the present invention.

Furthermore, the compound of the invention and the additional therapeutic agent may be present in the same dosage form, or separately for sequential, separate or simultaneous administration.

Pharmaceutical Compositions The present invention also provides pharmaceutical compositions comprising a compound of the present invention in combination with a pharmaceutically acceptable carrier, diluent, or excipient. Such pharmaceutical compositions are prepared in a manner well known in the pharmaceutical art, and are administered individually or in combination with other therapeutic agents, preferably via parenteral routes. Especially preferred routes include intramuscular and subcutaneous administration Parenteral daily dosages, preferably a single, daily dose, are in the range from about 1 pg/kg to about 1,000 ig/kg of body weight, although lower or higher dosages may be administered The required dosage will depend upon the severity of the condition -40 -of the patient and upon such criteria as the patient's height, weight, sex, age, and medical history.

In making the compositions of the present invention, the active ingredient, which comprises at least one compound of the present invention, is usually mixed with an excipient or diluted by an excipient When an excipient is used as a diliient, it may be a solid, semi-solid, or liquid material which acts as a vehicle, carrier, or medium for the active ingredient.

In preparing a formulation, it may be necessary to mill the active compound to provide the appropriate particle size prior to combining with the other ingredients. If the active compound is substantially insoluble, it ordinarily is milled to particle size of less than about 200 mesh, If the active compound is substantially water soluble, the particle size is normally adjusted by milling to provide a substantially uniform distribution in the formulation, e.g., about 40 mesh.

Some examples of suitable excipients include lactose, dextrose, sucrose, trehalose, sorbitol, and mannitol. The compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures well known in the art The compositions are preferably formulated in a unit dosage form with each dosage normally containing from about 50 p.g to about 100 mg, more usually from about 1 mg to about 10 mg of the active ingredient. The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with a suitable pharmaceutical excipient.

Amongst the pharmaceutical compositions according to the invention there are those which are suitable for oral, parenteral or nasal administration, including tablets or dragees, sublingual tablets, sachets, soft gelatin capsules, suppositories, creams, ointments, derrnai gels, transdermal devices, aerOsols, drinkable and injectable ampoules The dosage varies according to the age and weight of the patient, the nature and severity of the disorder and the route of administration For the purpose of parenteral administration, compositions containing a compound of the present invention preferably are combined with distilled water and the pH is adjusted to about 6.0 to about 9 0.

Additional pharmaceutical methods may be employed to control the duration of action. Controlled release preparations may be achieved by the use of polymers to complex or absorb a compound of the present invention. The controlled delivery may be exercised by selecting appropriate macromolecules (for example, polyesters, polyamino acids, polyvinylpyrrolidone, ethylenevinyl acetate, methylcellulose, carboxymethylcellulose, and protamirie sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release.

Another possible method to control the duration of action by controlled release preparations is to incorporate a compound of the present invention into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylene vinylacetate copolymers.

Alternatively, instead of incorporating a compound into these polymeric particles, it is possible to encapsulate a compound of the present invention in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nariocapsules, or in macroemulsions Such teachings are disclosed in Remington's Pharmaceutical Sciences (1980).

The compounds of the present invention have insulinotropic activity. Thus, another aspect of the present invention provides a method for enhancing the expression of insulin comprising providing to a mammalian pancreatic B-type islet cell an effective amount of a compound of the present invention.

Also intended as pharmaceutically acceptable acid addition salts are the hydrates and other solvates which the present compounds are able to form.

Furthermore, the pharmaceutically acceptable salts comprise basic amino acid salts such as lysine, arginine and ornithine.

The acid addition salts may be obtained as the direct products of compound synthesis In the alternative, the free base may be dissolved in a suitable solvent -42 -

S

containing the appropriate acid, and the salt isolated by evaporating the solvent or otherwise separating the salt and solvent.

The compounds of the present invention may form solvates with standard low molecular weight solvents using methods well known to the person skilled in the art.

Such solvates are also contemplated as being within the scope of the present invention.

Prodrugs The invention also encompasses prodrugs of the present compounds, which on administration undergo chemical conversion by metabolic processes before becoming pharmacologically active substances. In general, such prodrugs will be functional derivatives of the compounds of the general formula (I), which are readily convertible in vivo into the required compound of the formula (I). Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in "Design of Prodrugs", ed. H. Bundgaard, Elsevier, 1985.

For example, it is well known that several forms of GLP-1 are processed in vivo from proglucagon; GLP-1 (1-37), GLP-1 (7-37) and GLP-1 (7-36) Accordingly, the person skilled in the art will understand that the present invention also embraces peptides incorporating a fragment of structure (I) above which are metabolisable in vivo to give compounds of formula (I) The invention also encompasses active metabolites of the present compounds.

Definitions Multiply Occurring Definitions Where a term occurs in a formula more than once, it is intended that its use is independent of any other occurrence in that formula; for example, the term N(alkyl)2 refers to an amine group having two independently selected alkyl substituents.

Where a group or substituent is itself defined as optionally substituted, unless otherwise specified, it has up to three substituents independently selected from the group consisting of halogen, OH, NH2, NH(alkyl), N(alkyl)2, alkyl, alkenyl, alkynyl, alkoxy, carbomoyl, carboxy, cyano, nitro, and SH.

-43 -Alkyl Alkyl, as used herein refers to an aliphatic hydrocarbon chain and includes straight and branched chains preferably of 1 to 12, more preferably 1 to 6 carbon atoms such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, sopentyl, neo-pentyl, n-hexyl, and isohexyl.

Alkylene The term alkylene as used herein refers to a divalent saturated branched or unbranched hydrocarbon chain containing from 1 to 6 carbon atoms, and includes, for example, methylene (CH2), ethylene (CH2CH2), propylene (CH2CH2CH2), 2-methylpropylene (CH2CH(CH3)CH2), hexylene ((CH2)6), and the like Alkenyl Alkeriyl, as used herein, refers to an aliphatic hydrocarbon chain having at least one double bond, and preferably one double bond, and includes straight and branched chains e, g. of 2 to 6 carbon atoms such as ethenyl, propenyl, isopropenyl,but-1-enyl, but-2-enyl, but-3-enyl, 2-methypropenyl Al kynyl Alkynyl, as used herein, refers to an aliphatic hydrocarbon chain having at least one triple bond, and preferably one triple bond, and includes straight and branched chains e. g. of 2 to 6 carbon atoms such as ethynyl, propynyl, but-1-ynyl, but-2-ynyl and but-3-ynyl. Acyl

Acyl as used herein refers to the group -C(0)alkyl or -C(=O)H, wherein alkyl is as defined above Examples of acyl groups are formyl (-C(O)H), acetyl (-C(=O)CH3), and propanoyl (-C(0)CH2CH3).

Cycloakyi Cycloalkyl, as used herein, refers to a cyclic, saturated hydrocarbon group having from 3 to 8 ring carbon atoms. Examples of cycloalkyl groups are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

Cycloalkylene The term cycloalkylene as used herein refers to a divalent saturated cyclic hydrocarbon group containing from 3 to 8 ring carbon atoms, and includes, for example, methylene cyclopropylene, cyclobutylene, cyclohexylene and the like.

Cycloalkylalkyl The term cyctoalkylalkyl as used herein refers to the group -alkylene-cycloalkyl, wherein alkylene and cycloalkyl are as defined above. Examples of cycloalkylalkyl groups include cyclopropylmethyl, 2-cyclobutylethyl, cyclopentylmethyl, cyclohexylmethyl and 4-cycloheptylbutyl, and the like, Alkoxy Alkoxy as used herein refers to the group -0-alkyl, wherein alkyl is as defined above Examples of alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n- butoxy, isobutoxy, sec-butoxy, t-butoxy, n-pentoxy, isopentoxy, neo-pentoxy, n-hexyloxy, and isohexyloxy.

Alkylthio Alkylthio as used herein refers to the group -S-alkyl, wherein alkyl is as defined above. Examples of alkylthio groups include thiomethyl, thioethyl and thiohexyl.

Cycloalkoxy Cycloalkoxy as used herein refers to the group -0-cycloalkyl, wherein cycloalkyl is as defined above. Examples of cycloalkoxy groups are cyclopropoxy, cyclobutoxy, cyclopentoxy, cyclohexyloxy, cycloheptyloxy and cyclooctyloxy.

Aminoalkyl Aminoalkyl as used herein refers to the term -alkylene-NH2, wherein alkylene is as defined above Examples of aminoalkyl groups include methylamino (-CH2NH2) and 2-ethylamino (-CH2CH2NH2) Thioalkyl Thioalkyl as used herein refers to the group -alkylene-SH, wherein alkylene is as defined above Examples of thioalkyl groups are methylthio (CH2SH) and 2-ethylthio (CH2CH2SH)

I

S u lfoxoa I kyl Sulfoxoalkyl as used herein refers to the group -S(O)-alkyl, wherein alkyl is as defined above.

Sulfonoalkyl Sulfonoalkyl as used herein refers to a the group -S(O)2-alkyl wherein alkyl is as defined above.

Halogen Halogen, halide or halo-refers to iodine, bromine, chlorine and fluorine Haloalkyl Haloalkyl as used herein refers to an alkyl group as defined above wherein at least one hydrogen atom has been replaced with a halogen atom as defined above.

Examples of haloalkyl groups include chloromethyl, dichloromethyl, trichioromethyl, fluoromethyl, difluoromethyl and trifluoromethyl Preferred haloalkyl groups are fluoroalkyl groups (i.e. haloalkyl groups containing fluorine as the only halogen).

More highly preferred haloalkyl groups are perf]uoroalkyl groups, i.e alkyl groups wherein all the hydrogen atoms are replaced with fluorine atoms Hydroxyalkyl Hydroxyalkyl, as used herein, refers to an alkyl group as defined above wherein at least one hydrogen atom is replaced by a hydroxyl group. Examples of hydroxyalkyl groups include hydroxymethyl (-CH2OH), 2-hydroxyethyl (-CH2CH2OH), and 1-hydroxyethyl (-CH(OH)CH3). Aryl

As used herein,"aryl" refers to an unsaturated aromatic carbocyclic group of from 6 to carbon atoms having a single ring (e g phenyl) or multiple condensed (fused) rings (e g, naphthyl). Preferred aryl groups include phenyl, naphthyl and the like.

Heteroaryl Heteroaryl, as used herein refers to 5 to 10 membered mono or bicyclic aromatic rings having from 1 to 3 heteroatoms selected from N, 0 and S. Monocyclic rings -46 -preferably have 5 or 6 members and bicyclic rings preferably have 8, 9 or 10 membered ring structures. Exemplary heteroaryls include pyrrolyl, furyl, thienyl, imidazolyl pyrazolyl, oxazolyl isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrazinyl pyrimidinyl, indolyl quinolyl and isoquinolyl Alkoxycarbonyl As used herein, the term alkoxycarbonyl refers to the group -C(0)-O-AIkyl, wherein alkyl is defined above Examples of alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl and propoxycarbonyl.

Aryloxycarbonyl As used herein, the term aryloxycarbonyl refers to the group -C(=O)-O-AryI wherein aryl is defined above. Examples of aryloxycarbonyl groups include phenoxycarboriyl and naphthyloxycarbonyl.

Carboxyalkyl The term carboxyalkyl as used herein refers to the group -a!kylene-CO2H, wherein alkylene is as defined above Carbamoyl The terni carbamoyl as used herein refers to the group -CONH2.

Carbamoylalkyl The term carbamoylalkyl as used herein refers to the group -alkylene-carbamoyl, wherein alkylene and carbamoyl are as defined above ArylaIky Arylalkyl as used herein refers to the group -alkylene-aryl, wherein alkylene and aryl are as defined above Examples of arylalkyl groups are benzyl (-CH2Ph) and 2-phenethyl (-CH2CH2Ph).

Arylalkoxy Arylalkoxy as used herein refers to the group -0-alkylene-aryl, wherein alkylene and aryl are as defined above, Examples of arylalkoxy groups include 2-phenylethoxy (-OCH2CH2Ph), 5-phenylpentyloxy (-O-(CH2)5Ph) and the like.

-47 -

S

Arylalkoxycarbonyl Arylalkoxycarbonyl as used herein refers to the group -C(=O)-O-alkylene-aryl, wherein alkylene and aryl are as defined above Examples of arylalkoxycarbonyl groups are benzyloxycarbonyl (-C(=O)-O-CH2Ph).

Heteroarylalkyl The term heteroarylalkyl as used herein refers to the group -alkylene-heteroaryl, wherein alkylene and heteroaryl are as defined above.

Guanidinoalkyl The term guanidinoalkyl as used herein refers to the group -alkylene-NHC(=NH)NH2 wherein alkylene is as defined above Imidazolylalkyl The term imidazolylalkyl as used herein refers to the group -alkylene-imidazole, wherein alkylene is as defined above.

Prodrugs Also contemplated within the invention are prodrugs, that is compounds capable of undergoing metabolism to give compounds of formula (I) as defined above. Suitable prodrugs are N-oxides and compounds having a quaternary nitrogen.

Leaving group The term "leaving group" as used herein refers to any moiety or atom that is susceptible to nucleophilic substitution or elimination Typically, these are atoms or moieties that when removed by nucleophilic substitution or elimination are stable in anionic form. Examples of leaving groups useful in the present invention include alkyl-or arylsulphonate groups such as tosylate, brosylate, mesylate or nosylate, or halides such as fluoride, chloride, bromide, or iodide.

Multiply occurring groups When any variable (e g. aryl, heterocycle, R7 etc.) occurs more than one time in any compound of the invention, its definition at each occurrence is independent of its definition at every other occurrence.

S

Examples

1 DPP-IV assay Both the L7,L8 and D7,D8 analogues of GLP-1 above were subject to dipeptidyl peptidase IV hydrolysis as described in Green, B.D, V.A Gault, M.H. Mooney, N. Irwin, P Harriott, B. Greer, C J Bailey, F. P.M. O'Harte, and P.R. Flatt. (2004) Degradation, receptor binding, insulin-secreting, and antihyperglycaemic actions of palmitatederivatised native and Ala8-substituted GLP-1 analogues Biological Chemistry. 385, pp 169-1 77.

Briefly, 100 -200 microgams of each peptide was dissolved in 0.5 ml of 50 mM triethanolamine buffer, pH 7.8. To this was added a 10 microlitre solution of 11 milliunits DPP-IV (Sigma) and the solutions incubated at 37 C for up to 42 hours A control peptide, GLP-1 7-22, was treated identically with DPP-lV. The incubations were terminated by addition of 5 microlitres TFA to each tube and 50 microlitre of each were injected onto a reversed phase 4 6 x 250 mm C18 HPLC column, eluting a gradient of 20% -45% acetonitrile in 0 1% TFNwater over 25 minutes After 18 hr incubation, the N terminal dipeptide of the GLP-1 7-22 sequence was completely cleaved to give the 9-22 sequence (MH-1491) as seen by HPLC and ES-MS, while the 2 GLP-1 analogues of the invention remained intact under these conditions After 42 hours, the 2 analogues slowly degraded to a product that gave a mass spectrum consistent with the mass of the 9-37 sequence, indicating longer term instability of the urea bond. This represented as much as 15% of the product by HPLC. This degradation appeared to be non-DPP-IV related, since samples without enzyme gave the same profile.

2 Synthesis of SPO12 GLP-1 27-37 (0.5 g Chemmatrix Wang resin, 0.6 mniol/g)was assembled by Fmoc solid phase chemistry as for the other analogues. Following Fmoc deprotection of Glu 27, N-ct-i -(4, 4-Dimethyl-2,6-dioxocyclohex-1 -ylidene)ethyl-N-c-Fmoc-L-Lys [Dde-Lys(Fmoc)-OH] was coupled 3 times (1.2 mmol each) with HCTU to give a negative ninhydrin The E-N-Fmoc group was removed with 20% piperidine/DMF, and Fmoc-4-Abu (aminobutyric acid) was double-coupled with HCTU to give a negative ninhydrin Boc-Cys(Trityl) (1.5 mmol) was coupled overnight using DIC and HOBT and the ninhydrin test was negative. The N-a-Dde group was removed by treating the peptidyl resin twice with 10 ml of 2% hydrazine in DMF (1 x 5 -49 -

S

mm, 1 x 8 mm) and washing with DMF (1 x 10 ml), 10 % DIPEAIDMF (1 x 10 ml), and DMF (3 x 10 ml). Fmoc -Ala 25 was then coupled to the resin, and the synthesis was continued through Glu 9 The resin was split into 2 portions (-.15 mmol each) and the first portion was coupled successively with Ala 8 and His 7 to give the native GLP-1 sequence with 4-Abu-Cys at the c amino of Lys 26. Final Fmoc deprotection was followed by cleavage with 10 ml TFA containing H20 (5%), TIS (4%), thioanisole (2.5%), and EDT(0 5%) for 1.5 hr The solution was filtered into cold ether, the precipitate washed twice with cold ether, and dried under nitrogen to give 110 mg of crude SPO12. The product was purified by RP-HPLC and gave a mass of 3543.2, the expected MW 3. Synthesis of SPO1 3 Reference is made to example 2 such that after splitting the GLP-1 resin following Glu 9 addition, 2-bromoacetic acid was double-coupled as the symmetric anhydride using DIC for 1.5 hr each, to obtain a negative riinhydrin. The resin was then coupled with Fmoc-hydrazine (0.8 mmol) and 1 mrnol DIPEA (.132 ml) in DMF overnight. After washing, quantitative Fmoc release showed the reaction was only 10% complete. The coupling was repeated twice with hydrazine hydrate (2 mmol, 20.106 ml) in 5 ml DMF, containing.67 mmol DIPEA) for 2.5 hr each After washing and piperidine deprotection, His 7 (0 6 mmol) was coupled with DIC and HOBT for only mm. to minimize the chance of double acylation. After washing, Fmoc release gave a substitution of 0.155 mmol/g. Double His acylation of the hydrazine was considered minimal with this substitution level The resin was Fmoc deprotected, washed, and the peptide cleaved with a 10 ml TFA solution containing 5% H20, 4% TIS, 2.5% thioarusole, and 0.5% EDT for 1.5 hours. The solution was filtered into cold ether, washed twice with cold ether, and dried under nitrogen. The product was HPLC purified as in example 5 ES-MS analysis gave a mass of 3544, consistent with the expected MW 4 Preparation of Fmoc-4-aminobutyrate (4-Abu) 4-Abu (5 mmol, 516 mg) was dissolved in 25 ml of 10% potassium carbonate and cooled to 4 on ice. A solution of Fmoc-O-(N-hydroxysuccinimide) (Fmoc-OSu) (6 mmol, 2 02g, dissolved in 21 ml dioxaneTHF(2:1)) was added dropwise over 10 mm THF (7 ml) was added to maintain solubility and the mixture stirred at room temp overnight Add 100 ml H20. Extract with ethyl ether(1 x 50 ml), ethyl acetate (1 x 50 -50 -ml), and cool on ice Titrate to pH 2.3 with 6 N HCI to give cloudy solution. Extract with ethyl acetate (100 mID, chloroform (50 ml), and ethyl acetate (100 ml). Add methanol (5-10 ml) to break up emulsions during extraction. Combine organic layers and wash with 0.1N HCI (1 x 100 ml), H20 (1 x 100 ml), saturated aqueous sodium chloride (1 x 50 ml), and dry over powdered magnesium sulfate. Analytical HPLC of crude Fmoc-Abu showed greater than 90% purity (monitored at 300 nm) ES-MS of HPLC peak gave expected mass of 325 daltons. The solvent was evaporated to give a whte powder, which HPLC analysis at 230 nm showed to contain residual N-hydroxysuccinimide (NHS) This was removed by suspending the crude product in a 1:lmixture of ether ethyl acetate (200 ml) and stirring overnight. After filtration and washing of the precipitate, a yield of 1.24 g ( 3.81 mmo!, 76%) was recovered.

5. Preparation of Fmoc-hydrazide Tert-butyl carbazate (Sigma-Aldrich, 1.32 g, 10 mmol), was dissolved in 10 ml H20. After addition of 10 mmol TEA (1.4 ml), a 15 ml solution of 10 mmol Fmoc-OSu (3 37g) in acetonitrile was added dropwise over 10 mm. The pH dropped to 8 4, and was maintained at 8.5-9.0 by addition of TEA. After 30 additional mm, the solution was filtered, and the filtrate added with stirring to a 100 ml aqueous solution of 20% citric acid. An oily white precipitate resulted. This was extracted with 2 x 150 ml portions of ethyl acetate. The ethyl acetate solution was washed with H20 (50 ml), saturated sodium chloride, then dried over sodium sulfate. The ethyl acetate volume was reduced, and petroleum ether was added to precipitate the product at 40 overnight. After filtration and drying, 1 95 g (5.4 mmol, 54%) was recovered ES-MS showed the presence of excess NHS The crude product was re-dissolved in 100 ml ethyl acetate, a fine white precipitate was filtered out, and the ethyl acetate solution washed with H20 (2x) and brine (1 x). After drying over sodium sulfate and evaporation, 1.64 g (4. 6 mmol, 46%) of Fmoc-NH-NH-Boc was recovered which was free of NHS This was treated with 15 ml of 23% TFA in DCM for 45-50 mm to remove the Boc group. The resulting orange solution was evaporated, triturated with DCM, evaporated, and finally evaporated from ethyl acetate. The residue was dissolved in 125 ml ethyl acetate and washed with 10% sodium bicarbonate (2 x 50 ml), H20 (50 ml), then brine (40 ml) After drying over sodium sulfate, the product was recrystallized from chloroform.petroleum ether overnight at 40 Yield -0.95 g (3.74 mmcl, 37% from starting Boc-hydrazide) Purity by HPLC (at 290nm) was >96%. ES-MS gave MH+ 255 1.

-51 -

S

Abbreviations GLP-1, glucagon-like peptide-1; DPP-IV, dipeptidyI-peptdase; mPEG, monornethoxy polyethylene glycol; CDI, carbonyldilmidazole, DMF, dimethylformamide; DIPEA, diisopropylethylamine; HMPA, 2-(4-hydroxymethyl phenoxy)acetic acid, DCM, dichioromethane; THF, tetrahydrofuran; Trityl, triphenylmethyl, MS NT, 1 -(mesitylene-2-sulphonyl)-3-nitro-IH-1,2,4-triazole; HCTU, O-( 1 H-6-Chlorobenzotriazole-1-yl)- 1,1, 3,3-tetramethyluroniumhexaflourphosphate; TFA, trifluoroacetic acid, TI S, trisopropylsiIane.

Claims (1)

1. -52 -

   1. A compound comprising i) a moiety having GLP-1 like activity; ii) a 1,2-aminothiol moiety; iii) an optional linker group joining i) and ii) 2. A compound according to claim 1 wherein the 1,2-aminothiol moiety is a cysterne residue.

   3. A compound according to claim 1 or 2 wherein the linker is an amino acid.

   4 A compound according to claim 3 wherein the linker is a aminoacid.

   5. A compound according to claim 4 wherein the linker is 4-aminobutyric acid.

   6. A compound according to any one of claims 1 to 5 of the formula (XVII) wherein C represents the moiety having GLP-1 like activity.

   7. A compound according to any precedng claim wherein the moiety having GLP- 1 like activity is a peptide or a derivative thereof.

   8 A compound according to claim 7 wherein the moiety having GLP-1 like activity is GLP-1 (1-37) or an active portion thereof.

   9. A compound according to claim 8 wherein the moiety having GLP-i like activity is GLP-1 (7-37) A compound according to any one of claims 1 to 7 wherein the moiety having GLP-1 like activity is an exendin -53 - 11. A compound according to any one of claims 1 to 7 wherein the GLP-1 like moiety has the formula X[AImY[B]rZ (XXII) wherein X is optionally present and represents a substituent of the terminal carboxyl or amino group; Y is a linker group; n is an integer; m is 1 or 2; each A is an independently selected amino acid; each B is an independently selected amino acid, and Z is optionally present and represents a substituent of the terminal amino or carboxy group; or a prodrug or a pharmaceutically acceptable salt form thereof.

   12 A compound according to claim 1 wherein [Aim is a group of the formula A1-A2.

   13. A compound according to claim 11 or 12 wherein Y is -F'. G. H'-wherein F' is optionally present and is selected from -NH, -NAlkyl, 0, S, SO, SO2, -C H2-, -CHAlky!-, -C(AIkyI)2-(each alkyl independently selected), C=O, CNH, C=N(Alkyl), an amino acid residue, and CS, G is selected from alkylene of 1 to 12 carbon atoms, cycloalkylene of 3 to 12 carbon atoms, carbocyclic aryl of 6 to 12 carbon atoms, mono-or bicyclic heteroaryl of 5 to 12 ring members having from 1 to 5 heteroatoms independently selected from 0, N, or S; -NH, -NAlkyl, 0, S, SO, SO2, -CH2, - -54 -CHAlkyl, -C(Alkyl)2 (each alkyl independently selected), C=O, CNH, C=N(Alkyl), an amino acid residue, and C=S; H' is optionally present and is selected from -NH, -NAlkyl, 0, S, SO, SO2, -CH2, -CHAlkyl, -C(Alkyl)2 (each alkyl independently selected), C=O C=NH, C=N(Atkyl), an amino acid residue, and C=S, and represents an optional single or double bond, or Y is a single, double or triple bond 14. A compound according to any one of claims 12 to 13 wherein A1 and A2 (where present) are independently

   P

   wherein R3 and R4 are independently selected from a hydrogen atom or an alkyl, aminoalkyl (optionally substituted on the nitrogen atom by one or two alkyl, phenyl, benzyl, cycloalkyl, optionally substituted aryloxycarbonyl, optionally substituted arylalkoxycarbonyl andfor optionally substituted alkoxycarbonyl groups), thioalkyl (optionally substituted on the sulphur atom by an alkyl, phenyl, benzyl or cycloalkyl group), hydroxyalkyl (optionally substituted on the oxygen atom by an alkyl, phenyl, benzyl or cycloalkyl group), carboxyalkyl, carbamoylalkyl, guanidinoalkyl, cycloalkyl, cycloalkylalkyl, optionally substituted fused cycloalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryt or optionally substituted heteroarylalkyl group, or an imidazolyl or imidazolylalkyl group, or R3 and RA together with the carbon atom carrying them, form a cycloalkyl or fused cyc!oalkyl group, and each P is independently selected from hydrogen, alkyl, or cycloalkyl, or taken with R2 or R3 together with the carbon and nitrogen atoms to which they are attached represents a mono-or bicyclic group having from 4 to 12 ring members which is saturated, partially unsaturated, or unsaturated and is -55 -t isO or 1; or a prodrug or a pharmaceuticaHy acceptable salt form thereof.

   A compound according to claim any one of claims 12 to 14 wherein A1 is His or a prodrug or a pharmaceutically acceptable salt form thereof 16. A compound according to any one of claims 12 to 15 wherein A1 is a D-amino acid or a prodrug or a pharmaceutically acceptable salt form thereof 17. A compound according to any one of claims 12 to 16 wherein A2 (when present) is Ala or a prodrug or a pharmaceutically acceptable salt form thereof 18. A compound according to any one of claims 12 to 17 wherein A2 (when present) is a D-amino acid or a prodrug or a pharmaceutically acceptable salt form thereof.

   19. A compound according to any one of claims 12 to 18 wherein the N-terminus of the group [BJ is covalently bound to the group Y, and the C-terminus of the group [B] is covalently bound to the group Z where present or a prodrug or a pharmaceutically acceptable salt form thereof.

   20. A compound according to any one of claims 12 to 19 wherein [B] is substantially homologous to GLP-1 (9-37) or a fragment of GLP-1 having n residues or a prodrug or a pharmaceutically acceptable salt form thereof.

   21. A compound according to any one of claims 12 to 20 wherein [B] has the formula Glu Gly Thr Phe Thr Ser Asp Val Ser Set Tyr Leu Glu Gly GIn Ala Ala Lys Glu Phe lie Ala Trp Leu Val Lys Gly Arg Gly or Glu Gly Thr Phe Thr Ser Asp Val Ser Set Tyr Leu Glu Gly Gin Ala Ala Lys Glu Phe lie Ala Trp Leu Val Lys Gly Arg or a prodrug or a pharmaceutically acceptable salt form thereof.

   22. A compound according to any one of claims 11 to 21 wherein the GLP-1 like moiety has the formula (II) -56 -

   K

   (XiII) wherein X, Y, B, n and Z are as defined in claim 1, and R1 and R2 represent the backbone/sidechain of the amino acids, and P is H, a substituent, or a link to R1 or R2 (e.g. in the case of proline), represents an optional single or double bond, and K represents H, H2, NH or 0 or a prodrug or a pharmaceutically acceptable salt form thereof 23. A compound according to any one of claims 11 to 21 wherein the GLP-1 like moiety has the formula (XXXVIII)

   K

   P

   N RY [B] Al (XXXVIII) wherein X, Y, B, n and Z are as defined in claim 1, and R2 represents the backbone/sidechain of the amino acids, P is H, a substituent, or a link to R1 or R2 (e g. in the case of proline),

   . represents an optional single or double bond, and K represents H, H2, NH or 0 or a prodrug or a pharmaceutically acceptable salt form thereof 24. A compound according to claim 23 wherein Y is a bond or =N-0-or a prodrug or a pharmaceutically acceptable salt form thereof...CLME: A compound according to claim 23 or 24 wherein Y is a group of the formula (XXXII) -57 -R10 (XXXII) wherein Xis selected from 0, NH or N(C1-C6)alkyl; Y' is selected from 0, NH or N(C1-C6)alkyl; wherein R9 and R10 are independently selected from hydrogen atom or an alkyl, aminoalkyl (optionally substituted on the nitrogen atom by one or two alkyl, phenyl, benzyl, cycloalkyl, optionally substituted aryloxycarbonyl, optionally substituted arylalkoxycarbonyl and/or optionally substituted alkoxycarbonyl groups), thioalkyl (optionally substituted on the sulphur atom by an alkyl, phenyf, berizyl or cycloalkyl group), hydroxyalkyl (optionally substituted on the oxygen atom by an alkyl, phenyl, benzyl or cycloalkyl group), carboxyalkyl, carbamoylalkyl, guanidinoalkyl, cycloalkyl, cycloalkylalkyl, optionally substituted fused cycloalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl or optionally substituted heteroarylalkyl group, or an imidazolyl or imidazolylalkyl group, or R9 and R10 together with the carbon atom carrying them, form a cycloalkyl or fused cycloalkyl group, or a prodrug or a pharmaceutically acceptable salt form thereof.

   26 A compound according to claim 25 wherein Y is a group of the formula (XXXIII) (X..X)(l II) or a prodrug or a pharmaceutically acceptable salt form thereof -58 - 27. A compound according to claim 25 wherein Y is a group of the formula (XX.XV) (XXXV) or a prodrug or a pharmaceutically acceptable salt form thereof 28. A compound according to claim 25 wherein Y s a group of the formula (XXXIV) (XXXIV) or a prodrug or a pharmaceutically acceptable salt form thereof.

   29 A compound according to any one of claims 22 to 28 wherein K is H or H2 or a prodrug or a pharmaceutically acceptable salt form thereof.

   A compound according to any one of claims 1 to 7 wherein the GLP-1 like moiety has the formula \\/

   NH H2N N

   H Ser-Ser-Tyr-Leu-Glu-Gly- -Gtn-AIa-AIa-Lys-GIu-Phe-De-AIa- 0 Trp-Leu-Vat-Lys-GIy-Arg-Gy -59 -or H2N Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-GIu-Gy-Gin-Ala-Ala-Lys-Glu-Phe-Ile-Ala- 0 Trp-Leu-Val-Lys-Gly-Arg-Gly or a prodrug or a pharmaceutically acceptable salt form thereof.

   31. A compound according to any one of claims 1 to 7 wherein the GLP-1 like moiety has the formula X yRi [BIOZ wherein X, Y, B, n and Z are as defined in claim 1, and R, and R2 represent the backbone/sidechain of the amino acids A, and A2, and each P is independently H, a substituent, or a link to R1 or R2 (e g in the case of proline) or a prodrug or a pharmaceutically acceptable salt form thereof.

   32 A compound according to claim 31 wherein Y is C=0 or a prodrug or a pharmaceutically acceptable salt form thereof.

   33. A compound according to claim 31 or 32 wherein X is NH2 or a prodrug or a pharmaceutically acceptable salt form thereof 34 A compound according to any one of claims 31 to 33 wherein Z is H or a prodrug or a pharmaceutically acceptable salt form thereof -60 - 35. A compound according to any one of claims 31 to 34 wherein each B is modified.

   36. A compound according to any one of claims 31 to 35 wherein at least one B is modified.

   37 A compound according to any one of claims 31 to 36 wherein at least one of the amino acids of group [B1 is modified whereby [B] has the formula Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gin Ala Ala Lys Glu Phe lie Ala Trp Leu Val Lys Gly Arg Gly or Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gin Ala Ala Lys Glu Phe lie Ala Trp Leu Val Lys Gly Arg.

   38. A compound according to any preceding claim wherein the point of attachment of the linker (or the 1,2 aminothiol moiety) is to the c amine of a lysine residue.

   39 A compound according to claim 8 or 9 wherein the point of attachment of the linker (or the 1,2 aminothiol moiety) is to the E amine of a lysine residue Lys26 A compound which is one of N o H2N N N GiuIy-Thr-Phe-Thr-Ser-Asp-Vai-Ser-Ser-Tyr-Leu-GIu-G -Gin-Aia-Aia-Lys-Giu-Phe-iIe-AIa-Trp-Leu-Val-Lys-GIy-Ar 0 = g-Gy NHcocH2cH2cI-12NHcys (XXXIX) -61 -

   N

   H2N N GIuThr-Phe-Thr-Ser-Asp-VaSer-Ser*Tyr-LeGIu -Giy- H -GIn-AIa-AIa-Lys-GIu-Phe-ile-Ala-Trp-Leu-VaI-Lys-GIy- 0 = 0 Arg-NH2 NHCOCH2CH2CH2NI-tCys (XL)

   NH

   N -Gly- _-GIu-GIy-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-LeU-GIU -GIn-Ala-AIa-Lys-Gu-Phe-IIe-Aia-Trp-Leu-Va-Lys-GIy-o o Arg-GIy NHCOCH2CH2CH2NHCys (XLI)

   NH

   H2N GIu-GIy-Thr-Phe-Thr-Ser-Asp-V3I-Ser-Ser-Tyr-Leu-GIu -Gay--GIn-AIa-AIa-Lys-GIu-Phe-fle-AIa-Trp-Le u-Va-Lys-GIy-o = o Arg-NH2 NHCOCH2CH2CH2NHCys (XLII)

   NH

   H

   H2N N N -GIy-GIu-GIy-Thr-Phe-Thr-Ser-Asp-VaI-Ser-Ser-Tyr-Leu-GIu -GIn-AIa-Aia-Lys-GIu-Phe-IIe-AJa-Trp-Leu-VaI-Lys-GIy- 0 0 AT9-GIY NHCOCH2CH2CH2NHCys (XLIII) -62 -

   NH

   H

   H2N N GIuy-Thr-Phe-Thr-Ser-Asp-VaI-Ser-Ser-Tyr-Leu-GIu

   N -Gly-

   o Arg-NH2 N HCOCH2CH2C H2 N HCys (XLIV)

   NH c

   K2N N GIu-GIy-Thr-Phe-Thr-Ser-Asp-VaI-Ser-Ser-Tyr-LeGIu -Gly--Gln-AIa-Aia-Lys-Gu-Phe-IIe-Ala-Trp-Leu-VaI-Lys-GIy-o 0 Arg-GIy NHCOCH2CH2CH2NHCys (XLV) /NH

   N

   GIuIy-Thr-Phe-Thr-Ser-Asp-VaI-Ser-Ser-Tyr-Leu-Gu H2N N -GIn-AIa-a-Lys-GIu-PhIIe-AIa-TrLeu-V&-Lys-GIy- -Gly-o Arg-NH2 NHCOCH2CI-I2CH2NHCys (XLVI) N"

   H

   H2N JNH

   N

   H Glu-Gly-o -Gh-AIa-AIa-Lys-GIu-Phe-IIe-AIa-Trp-Leu-Va-Lys-G ty-Arg-GIy NHCOCH2CH2CH2NHCys

   S

   (XL VII)

   N

   NH

   H2 N N GIu-GIy-Thr-Phe-Thr-Ser-Asp-VaI-Ser-Ser-Tyr-LeU-GI H u-GIy-o = -GIn-AIa-Ala-Lys-Gu-Phe-l Ie-AIa-Trp-Leu-VaI-Lys-Gty -Arg-GIy NHCOCH2CH2CH2NHCys (XLVIU)

   N

   NH

   H2N N G) u-GIy-Thr-Phe-Thr-Ser-Asp-VaI-Ser-Ser-Tyr-Leu-Gu-Gty--Gtn-AIa-AIa-Lys-GIuPhe-IIe-AIa-Trp-Leu-VakLys-GIy-Arg-GIy o NHCOCH2CH2CH2NHCys (XLIX) NTh

   NH

   H2N GIu-GIy-Thr-PheThrSerAspVaISeNSerTyrLeuGIuGly 0 I NHCOCH2CH2CH2NHCys (L)

   S N"

   I

   H2N GLu-GIy-Thr-Phe-Thr-Ser-Asp-Va-Ser-Ser-Tyr-Leu-GIu-GIy--GIn-Aa-Aa-Lys-GIuPhe-IIe-AIa-Trp-Leu-VaI-Lys-Gty-Arg-GIy o I NHCOCH2CH2CH2NHCys (LI) N" i-NH H2N N N r-Ser-Asp-Val-Ser-Ser-Tyr-Leu-GIu-G ly-H -GIn-AIa-AIa-Lys-GIu-Phe-IIe-AIa-Trp-Leu-Va)-Lys-Gty-Arg-GIy NHCDCH2CH2CH2NHCys (LII) H2N GIu-GIy-Thr-Phe-Thr-Ser-Asp-VaI-Ser-Ser-Tyr-Leu-GIu-GIy--GIn-AIa-AIa-LysG Iu-Phe-I Ie-AIa-Trp-Leu-Va-Lys-GIy-Arg-Giy 0 NHCOCH2CH2CH2NHCys (LIII) His-AIa-GIu-GIy-Thr-Phe-Thr-Ser-Asp-VaI-Ser-SerTyr-Leu-GIu-GIy--GIn-AIaAI a-Lys-GIu-Phe-Ue-AIa-Trp-Leu-Va-Lys-GIy-Arg-GIy NHCOCH2CH2CH2NHCys (LIV) -65 -His-Ala-GIu-GIy-Thr-Phe-Thr-Ser-Asp-VaI-Ser-Ser-Tyr-Leu-Gu-GIy--GIn-AIaA Ia-Lys-GIu-Phe-t Ie-AIa-Trp-Leu-VaI-Arg-GIy-Arg-GIy NHCOCH2CI-42CH2NHCys (LV) His-AIa-GIu-GIy-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-GIU-GIY--GIn-AIaA la-Lys-GIu-Phe-l Ie-AIa-Trp-Leu-VaI-Lys-GIy-Arg-GIy NHCOCH2CH2CH2NHCys (LVI) and H2N Glu-GIy-Thr-Phe-Thr-Ser-Asp-Va-Ser-Ser-Tyr-LeU-GU-GIY-GI n-AIa-AIa-Lys-GIu-Phe-l le-AIa-Trp-Leu-VaI-Lys-GIy-Arg -Gly NHCH2OCH2CH2CH2NH-Cys (LVII; SPO13).

   41. A complexed compound comprising a compound according to any one of ciaims 1 to 40 covalently linked to a carrier ligand.

   42. A cornplexed compound according to claim 41 wherein the carrier ligand is attached to the 1,2-aminothiol moiety to form a thiazolidine ring.

   43 A cornplexed compound according to claim 41 or 42 wherein the carrier ligand is an oiigo-or poly-saccharide.

   44. A complexed compound according to claim 41 or 42 wherein the carrier ligand is a polyether.

   -66 - 45. A complexed compound according to claim 44 wherein the carrier lgand is a PEG.

   46. A compound as claimed in any one of claims 1 to 45 for use as a pharmaceutical.

   47. A pharmaceutical composition comprising a compound as claimed in any one of claims 1 to 45 together with a pharmaceutically acceptable carrier or excipient 48 A method of treatment of a human or animal suffering from a condition selected from hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, hypertension, syndrome X, dyslipidemia, cognitive disorders, atheroschlerosis, myocardial infarction, coronary heart disease and other cardiovascular disorders, stroke, inflammatory bowel syndrome, dyspepsia and gastric ulcers, comprising administering to said mammal a compound as claimed in any one of claims 1 to 45 or a composition as claimed in claim 47.

   49. Use of a compound as claimed in any one of claims 1 to 45 or a composition as claimed in claim 47 in the preparation of a medicament for the treatment of a condition selected from hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, hypertension, syndrome X, dyslipidemia, cognitive disorders, atheroschlerosis, myocardial infarction, coronary heart disease and other cardiovascular disorders, stroke, inflammatory bowel syndrome, dyspepsia and gastric ulcers.

   50. A compound as claimed in any one of claims 1 to 45 or a composition as claimed in claim 47 in the preparation of a medicament for the treatment of a condition selected from hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, hypertension, syndrome X, dyslipidemia, cognitive disorders, atheroschlerosis, myocardial infarction, coronary heart disease and other cardiovascular disorders, stroke, inflammatory bowel syndrome, dyspepsia and gastric ulcers 51. A compound substantially as described herein with reference to the examples.

   52. A medicament substantially as described herein with reference to the

   examples