EA044242B1 - PEPTIDE COMPOUND - Google Patents

Description

Field of technology

The invention relates to a new peptide compound having an activating effect on GLP-1 receptors and GIP receptors, and to the use of the peptide compound as a medicine.

State of the art

Both glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are peptides called incretins. GLP-1 and GIP are secreted from small intestinal L cells and K cells, respectively.

GLP-1 acts through GLP-1 receptors and is known to have glucose-dependent insulinotropic and nutritional suppressive effects. On the other hand, GIP is known to have glucose-dependent insulinotropic effects via GIP receptors, although its effect on feeding activity is unclear.

It has been described that co-administration of the GLP-1 receptor agonist liraglutide and the GIP receptor agonist N-Ac-GIP enhances the glucose tolerance-enhancing effect and the weight-lowering effect to a greater extent than administration of liraglutide alone (Non-Patent Document 1). It has also been described that the GLP-1 receptor/GIP receptor co-agonist peptide exhibits greater hypoglycemic and weight-lowering effects than the GLP-1 receptor agonist alone (Patent Document 1).

Attempts have also been made to search for peptides having GLP-1 receptor/GIP receptor co-agonist activity or glucagon/GLP-1 receptor/GIP receptor triple agonist activity and to develop these peptides as anti-obesity drugs or therapeutic drugs for diabetes based on structures of natural glucagon, GIP or GLP-1 (patent documents 1 to 8). However, none of the documents describes the peptide compound of the present invention.

List of links

Patent document

Patent document 1 WO 2010/011439.

Patent document 2 WO 2010/148089.

Patent document 3 WO 2011/119657.

Patent document 4 WO 2012/088379.

Patent document 5 WO 2012/167744.

Patent document 6 WO 2013/164483.

Patent document 7 WO 2013/192129.

Patent document 8 WO 2013/192130.

Non-patent document

Non-Patent Document 1 Clinical Science 121, 107-117 (2011).

The essence of the invention

Technical problem

The invention is aimed at obtaining a new peptide compound having high GLP-1 receptor/GIP receptor coagonist activity, and suitable as a means for the prevention or treatment of obesity, etc.

The solution of the problem

The inventors conducted extensive studies regarding a new peptide compound having higher GLP-1 receptor/GIP receptor coagonist activity and useful as an agent for the prevention or treatment of obesity and the like, and found that the peptide compound containing a partial sequence , corresponding to formula (I) presented below, etc. has higher GLP1 receptor/GIP receptor coagonist activity, which led to the implementation of the present invention.

Thus, the present invention relates to the following:

[1] a peptide containing a partial sequence corresponding to formula (I):

P ¹ -Tyr-Aib-Glu-Gly-Thr-cxMePhe-Thr-Ser-Asp-Tyr-All-A12-A13Leu-Asp-A16-A17-Ala-Gln-A20-Glu-Phe-Val-Lys-Trp -Leu-Leu-Lys-A29 (SEQ ID NO: 1) where P ¹ represents a group corresponding to the formula:

- R ^a1 ,

- CO-RA ¹ ,

- CO-OR ^A1 ,

- CO-CORA ¹ ,

- SO-RA ¹ ,

- SO2-RA ¹ ,

- SO2-ORA ¹ ,

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-CO-NR ^a2 R ^a3 ,

-SO ₂ -NR ^A2 R ^A3 , or

-C(=NR ^a1 ) -nr ^A2 r ^A3 , wherein each of R ^a1 , Ra ² and Ra ³ independently represents a hydrogen atom, an optionally substituted hydrocarbon group or an optionally substituted heterocyclic group;

All represents Aib or Ala;

A12 is Ala, Ile, Lys, Phe or Rua (4);

And 13 is Aib, Cha, Leu, aMePhe or α-MeTyr;

And 16 is Lys or Ser;

And 17 is Gln or Ile;

And 20 represents Ala or Ser; And

A 29 is Gln or Gly, or a salt thereof (hereinafter sometimes abbreviated as compound (I));

[2] the peptide according to [l] or a salt thereof, where P ¹ represents a hydrogen atom;

[3] the peptide according to [l] or a salt thereof, where A11 represents Aib;

[4] the peptide according to [l] or a salt thereof, where A12 represents Ile;

[5] the peptide according to [l] or a salt thereof, where A13 represents Aib;

[6] the peptide according to [l] or a salt thereof, where A16 represents Lys;

[7] the peptide according to [l] or a salt thereof, where A17 represents Gln;

[8] the peptide according to [l] or a salt thereof, where A20 represents Ala;

[9] the peptide according to [l] or a salt thereof, where A29 represents Gly;

[10] the peptide according to [l] or a salt thereof, having an amino acid sequence corresponding to Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys-, on the C-terminal side of A29;

[ll] peptide according to [l] or its salt, where

P ¹ represents a hydrogen atom;

All represents Aib;

A12 represents Ile;

A13 represents Aib;

A16 represents Lys;

A17 is Gln;

A20 represents Ala;

A29 represents Gly; and a peptide having the amino acid sequence corresponding to Gly-Pro-Ser-Ser-GlyAla-Pro-Pro-Pro-Ser-Lys- on the C-terminal side of A29;

[ 12 ] H-Tyr-Aib-Glu-Gly-Thr-otMePhe-Thr-Ser-Asp-Tyr-Aib-LysTyr-Leu-Asp-Lys-Gln-Ala-Gln-Ala-Glu-Phe-Vai-Lys -Trp-Leu-LeuLys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys-NH2 or its salt;

[ 13 ] H-Tyr-Aib-Glu-Gly-Thr-otMePhe-Thr-Ser-Asp-Tyr-Aib-IleAib-Leu-Asp-Lys-Gln-Ala-Gln-Ala-Glu-Phe-Vai-Lys -Trp-Leu-LeuLys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys-NH2 or its salt;

[ 14 ] H-Tyr-Aib-Glu-Gly-Thr-otMePhe-Thr-Ser-Asp-Tyr-Aib-LysTyr-Leu-Asp-Lys-Gln-Ala-Gln-Gln-Glu-Phe-Vai-Lys -Trp-Leu-LeuLys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys-NH2 or its salt;

[15] H-Tyr-Aib-Glu-Gly-Thr-otMePhe-Thr-Ser-Asp-Tyr-Aib-Lys-

Tyr-Leu-Asp-Lys-Gln-Ala-Gln-Gln-Glu-Phe-Vai-Lys-Trp-Leu-LeuLys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro- Ser-NH2 or its salt;

[16] a medicinal product containing the peptide according to [l] or a salt thereof;

[17] the drug according to [16], which is an activator of the GLP-l receptor and the GIP receptor;

[18] a medicinal product according to [16], which is an agent for the prevention or treatment of obesity or diabetes;

[19] a method for preventing or treating obesity or diabetes in a mammal, comprising administering an effective amount of the peptide of [l] or a salt thereof to the mammal;

[20] a method of activating the GLP-l receptor and the GIP receptor in a mammal, comprising administering an effective amount of the peptide of [l] or a salt thereof to the mammal;

[21] use of the peptide according to [l] or a salt thereof for the manufacture of an agent for the prevention or treatment of obesity or diabetes;

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[22] the peptide according to [1] or a salt thereof for use in the prevention or treatment of obesity or diabetes.

Advantageous effects of the invention

Compound (I) has improved GLP-1 receptor/GIP receptor coagonist activity and exhibits significant nutritional suppression and weight loss effects in vivo. In addition, compound (I) has a low risk of hyperglycemic action and is also useful for the treatment of obesity associated with diabetes and the like due to its low glucagon receptor agonist activity. Moreover, compound (I) is excellent in solubility and also has the advantage that the compound can be easily formulated as a drug.

Detailed Description of the Invention

The definition of each substituent used herein is described in detail below. Unless otherwise specified, each substituent is defined as follows.

In the present specification, examples of the halogen atom include fluorine, chlorine, bromine and iodine.

In the present specification, examples of the C _1-6 alkyl group include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl, 1,1-dimethylbutyl , 2,2-dimethylbutyl, 3,3-dimethylbutyl and 2-ethylbutyl.

In the present specification, examples of the optionally halogenated _C1-6 alkyl group include a _C1-6 alkyl group optionally having 1-7, preferably 1-5, halogen atoms. Specific examples thereof include methyl, chloromethyl, difluoromethyl, trichloromethyl, trifluoromethyl, ethyl, 2-bromoethyl, 2,2,2-trifluoroethyl, tetrafluoroethyl, pentafluoroethyl, propyl, 2,2-difluoropropyl, 3,3,3-trifluoropropyl, isopropyl, butyl, 4, 4,4-trifluorobutyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 5,5,5-trifluoropentyl, hexyl and 6,6,6-trifluorohexyl.

In the present specification, examples of the C _2-6 alkenyl group include ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 3-methyl-2-butenyl, 1-pentenyl , 2-pentenyl, 3-pentenyl, 4-pentenyl, 4-methyl-3-pentenyl, 1-hexenyl, 3-hexenyl and 5-hexenyl.

In the present specification, examples of the C _2-6 alkynyl group include ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1hexynyl, 2 -hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl and 4-methyl-2-pentynyl.

In the present specification, examples of the C ₃ - ₁₀ cycloalkyl group include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, bicyclo[3.2.1]octyl and adamantyl.

In the present specification, examples of the optionally halogenated C _3-10 cycloalkyl group include a C _3-10 cycloalkyl group optionally having 1-7, preferably 1-5, halogen atoms. Specific examples thereof include cyclopropyl, 2,2-difluorocyclopropyl, 2,3-difluorocyclopropyl, cyclobutyl, difluorocyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

In the present specification, examples of the C ₃ - ₁₀ cycloalkenyl group include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl and cyclooctenyl.

As used herein, examples of the C _6-14 aryl group include phenyl, 1-naphthyl, 2-naphthyl, 1-anthryl, 2-anthryl and 9-anthryl.

In the present specification, examples of the C _7-16 aralkyl group include benzyl, phenethyl, naphthylmethyl and phenylpropyl.

In the present specification, examples of the _C1-6 alkoxy group include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, t-butoxy, pentyloxy and hexyloxy.

In the present specification, examples of the optionally halogenated C ₁ - ₆ alkoxy group include a C _1-6 alkoxy group optionally having 1 to 7, preferably 1 to 5, halogen atoms. Specific examples thereof include methoxy, difluoromethoxy, trifluoromethoxy, ethoxy, 2,2,2-trifluoroethoxy, propoxy, isopropoxy, butoxy, 4,4,4-trifluorobutoxy, isobutoxy, sec-butoxy, pentyloxy and hexyloxy.

As used herein, examples of C ₃ - ₁₀ cycloalkyloxy groups include cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, cycloheptyloxy and cyclooctyloxy.

As used herein, examples of C ₁ - ₆ alkylthio groups include methylthio, ethylthio, propylthio, isopropylthio, butylthio, sec-butylthio, t-butylthio, pentylthio and hexylthio.

In the present specification, examples of the optionally halogenated _C1-6 alkylthio group include a _C1-6 alkylthio group optionally having 1 to 7, preferably 1 to 5, halogen atoms. Specific examples thereof include methylthio, difluoromethylthio, trifluoromethylthio, ethylthio, propylthio, isopropylthio, butylthio, 4,4,4-trifluorobutylthio, pentylthio and hexylthio.

In the present specification, examples of the C _1-6 alkylcarbonyl group include acetyl, propanoyl, butanoyl, 2-methylpropanoyl, pentanoyl, 3-methylbutanoyl, 2-methylbutanoyl, 2,2-dimethylpropanoyl, hexanoyl and heptanoyl.

In the present specification, examples of an optionally halogenated C _1-6 alkylcarbonyl group include a C _1-6 alkylcarbonyl group optionally having 1 to 7, preferably 1 to 5, halogen atoms. Specific examples thereof include acetyl, chloroacetyl, trifluoroacetyl, trichloroacetyl, propanoyl, butanoyl, pentanoyl and hexanoyl.

In the present specification, examples of the C _1-6 alkoxycarbonyl group include methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl, sec-butoxycarbonyl, t-butoxycarbonyl, pentyloxycarbonyl and hexyloxycarbonyl.

In the present specification, examples of the C _6-14 arylcarbonyl group include benzoyl, 1-naphthoyl and 2-naphthoyl.

In the present specification, examples of the C _7-16 aralkylcarbonyl group include phenylacetyl and phenylpropionyl.

In the present specification, examples of the 5-14 membered aromatic heterocyclylcarbonyl group include nicotinoyl, isonicotinoyl, thenoyl and furoyl.

In the present specification, examples of the 3-14 membered non-aromatic heterocyclylcarbonyl group include morpholinylcarbonyl, piperidinylcarbonyl and pyrrolidinylcarbonyl.

In the present specification, examples of the mono- or di-C _1-6 alkylcarbamoyl group include methylcarbamoyl, ethylcarbamoyl, dimethylcarbamoyl, diethylcarbamoyl and K-ethyl-K-methylcarbamoyl.

In the present specification, examples of the mono- or di-C _7-16 aralkylcarbamoyl group include benzylcarbamoyl and phenethylcarbamoyl.

In the present specification, examples of the C _1-6 alkylsulfonyl group include methylsulfonyl, ethylsulfonyl, propylsulfonyl, isopropylsulfonyl, butylsulfonyl, sec-butylsulfonyl and tert-butylsulfonyl.

In the present specification, examples of the optionally halogenated C _1-6 alkylsulfonyl group include a C _1-6 alkylsulfonyl group optionally having 1 to 7, preferably 1 to 5, halogen atoms. Specific examples thereof include methylsulfonyl, difluoromethylsulfonyl, trifluoromethylsulfonyl, ethylsulfonyl, propylsulfonyl, isopropylsulfonyl, butylsulfonyl, 4,4,4-trifluorobutylsulfonyl, pentylsulfonyl and hexylsulfonyl.

In the present specification, examples of the C _6-14 arylsulfonyl group include phenylsulfonyl, 1naphthylsulfonyl and 2-naphthylsulfonyl.

In this description, examples of the deputy include the halogen atom, cyanogroup, nitro -group, an optionally replaced hydrocarbon group, an optionally replaced heterocyclic group, an acynogroup, optionally replaced by a carbamoil group, an optionally detected thiocarbamol group, not necessarily noticeable sulfamine group, not necessarily noticeable sulfamoil group not necessarily replaced by the hydroxygoup, optional a substituted sulfanyl (SH) group and an optionally substituted silyl group.

In the present specification, examples of the hydrocarbon group (including the hydrocarbon group of an optionally substituted hydrocarbon group) include a C _1-6 alkyl group, a C _2-6 alkenyl group, a C _2-6 alkynyl group, a C _3-10 cycloalkyl group, a C _3-10 cycloalkenyl group , a C ₆₁₄ aryl group and a C _7-16 aralkyl group.

As used herein, examples of an optionally substituted hydrocarbon group include a hydrocarbon group optionally having substituent(s) selected from the following substituent group A.

Group of substituents A:

(1) halogen atom, (2) nitro group, (3) cyano group, (4) oxo group, (5) hydroxy group, (6) optionally halogenated C _1-6 alkoxy group, (7) C _6-14 aryloxy group (e.g. phenoxy, naphthoxy), (8) C _7-16 aralkyloxy group (e.g. benzyloxy), (9) 5-14 membered aromatic heterocyclyloxy group (e.g. pyridyloxy), (10) 3-14 membered non-aromatic heterocyclyloxy group (e.g. morpholinyloxy, piperidinyloxy) , (11) C _1-6 alkylcarbonyloxy group (e.g. acetoxy, propanoyloxy), (12) C6-14 arylcarbonyloxy group (e.g. benzoyloxy, 1-naphthoyloxy, 2-naphthoyloxy), (13) C _1-6 alkoxycarbonyloxy group (e.g. methoxycarbonyloxy, ethoxycarbonyloxy, propoxycarbonyloxy, butoxycarbonyloxy), (14) mono- or di-C _1-6 alkylcarbamoyloxy group (e.g. methylcarbamoyloxy, ethylcarbamoyloxy, dimethylcarbamoyloxy, diethylcarbamoyloxy), (15) C _6-14 arylcarbamoyloxy group (e.g. phenylcarbamoyloxy, naphthylcarbamoyloxy), (16 ) 5-14 membered aromatic heterocyclylcarbonyloxy group (for example, nicotinoyloxy), (17) 3-14 membered non-aromatic heterocyclylcarbonyloxy group (for example, morpholinylcar-

-4 044242 Boniloxy, Piperidinilcarbonox), (18) not necessarily halogened with _1-6 alkylsulfoniloxygrand (for example, methyl sulfonylox, triformethyl sulfonylox), (19) C _6-14 aryphoniloxigrusp, not necessarily detected with _1-6 alcohol group (for example, phenyl sulovalox sulfonilox) _. optionally halogenated C _1-6 alkylcarbonyl group, (26) C _6-14 arylcarbonyl group, (27) 5-14 membered aromatic heterocyclylcarbonyl group, (28) 3-14 membered non-aromatic heterocyclylcarbonyl group, (29) C _1-6 alkoxycarbonyl group, (30) C _6-14 aryloxycarbonyl group (e.g. phenyloxycarbonyl, 1-naphthyloxycarbonyl, 2-naphthyloxycarbonyl), (31) C _7-16 aralkyloxycarbonyl group (e.g. benzyloxycarbonyl, phenethyloxycarbonyl), (32) carbamoyl group, (33) thiocarbamoyl group, (34) mono- or di- _C1-6 alkylcarbamoyl group, (35) _C6.i4 arylcarbamoyl group (e.g. phenylcarbamoyl), (36) 5-14 membered aromatic heterocyclylcarbamoyl group (e.g. pyridylcarbamoyl, thienylcarbamoyl ), (37) a 3-14 membered non-aromatic heterocyclylcarbamoyl group (e.g., morpholinylcarbamoyl, piperidinylcarbamoyl), (38) an optionally halogenated _C1-6 alkylsulfonyl group, (39) a _C6-14 arylsulfonyl group, (40) 5-14- member aromatic heterocyclylsulfonyl group (e.g. pyridylsulfonyl, thienylsulfonyl), (41) optionally halogenated C _1-6 alkylsulfinyl group, (42) C _6-14 arylsulfinyl group (e.g. phenylsulfinyl, 1-naphthylsulfinyl, 2-naphthylsulfinyl), (43) 5- 14-membered aromatic heterocyclylsulfinyl group (e.g. pyridylsulfinyl, thienylsulfinyl), (44) amino group, (45) mono- or di-C _1-6 alkylamino group (e.g. methylamino, ethylamino, propylamino, isopropylamino, butylamino, dimethylamino, diethylamino, dipropylamino , dibutylamino, K-ethyl-Kmethylamino), (46) mono- or di-C _6-14 arylamino group (for example, phenylamino), (47) 5-14-membered aromatic heterocyclylamino group (for example, pyridylamino), (48) C _{7 -16} aralkylamino group (e.g. benzylamino), (49) formylamino group, (50) C _1-6 alkylcarbonylamino group (e.g. acetylamino, propanoylamino, butanoylamino), (51) (C _1-6 alkyl)(C _1-6 alkylcarbonyl) amino group (e.g. K-acetyl-K-methylamino), (52) C6-14 arylcarbonylamino group (e.g. phenylcarbonylamino, naphthylcarbonylamino), (53) C _1-6 alkoxycarbonylamino group (e.g. methoxycarbonylamino, ethoxycarbonylamino, propoxycarbonylamino, butoxycarbonylamino, tert-butoxycarbonylamino ), (54) C _7-16 aralkyloxycarbonylamino group (e.g. benzyloxycarbonylamino), (55) C _1-6 alkylsulfonylamino group (e.g. methylsulfonylamino, ethylsulfonylamino), (56) C _6-14 arylsulfonylamino group, optionally substituted with a C _1-6 alkyl group (e.g. phenylsulfonylamino, toluenesulfonylamino), (57) optionally halogenated _C1-6 alkyl group, (58) _C2-6 alkenyl group, (59) _C2-6 alkynyl group, (60) _C3-10 cycloalkyl group, (61) C _3-10 cycloalkenyl group and (62) C _6-14 aryl group.

The number of the above-mentioned substituents in the optionally substituted hydrocarbon group is, for example, from 1 to 5, preferably from 1 to 3. When the number of substituents is two or more, the corresponding substituents may be the same or may be different.

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In the present specification, examples of the heterocyclic group (including the heterocyclic group of an optionally substituted heterocyclic group) include (i) an aromatic heterocyclic group, (ii) a non-aromatic heterocyclic group, and (iii) a 7-10 membered bridged heterocyclic group, each of which contains as constituents a ring atoms other than carbon atoms, 1-4 heteroatoms selected from a nitrogen atom, a sulfur atom and an oxygen atom.

In the present specification, examples of an aromatic heterocyclic group (including a 5 to 14 membered aromatic heterocyclic group) include a 5 to 14 membered (preferably 5 to 10 membered) aromatic heterocyclic group containing as ring constituent atoms other than carbon atoms, 1 to 4 heteroatoms selected from a nitrogen atom, a sulfur atom and an oxygen atom.

Preferred examples of the aromatic heterocyclic group include 5- or 6-membered monocyclic aromatic heterocyclic groups such as thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, 1,2,4-oxadiazolyl , 1,3,4-oxadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, triazolyl, tetrazolyl, triazinyl and the like; and 8-14 membered fused polycyclic (preferably bi- or tricyclic) aromatic heterocyclic groups such as benzothiophenyl, benzofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotriazolyl, imidazopyridinyl, thienopyridinyl, furopyridinyl, pyrrolopyridinyl, pyrazolopyr idinyl, oxazolopyridinyl, thiazolopyridinyl , imidazopyrimidinyl, imidazopyrimidinyl, thienopyrimidinyl, furopyrimidinyl, pyrrolopyrimidinyl, pyrazolopyrimidinyl, oxazolopyrimidinyl, thiazolopyrimidinyl, pyrazolotriazinyl, naphtho[2,3-b]thienyl, phenoxathiinyl, indolyl, isoindolyl, 1Nindazolyl, purinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl , cinnolinyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, etc.

In the present specification, examples of a non-aromatic heterocyclic group (including a 3-14 membered non-aromatic heterocyclic group) include a 3-14 membered (preferably 410-membered) non-aromatic heterocyclic group containing as ring constituent atoms other than carbon atom, 1-4 heteroatoms selected from a nitrogen atom, a sulfur atom and an oxygen atom.

Preferred examples of the non-aromatic heterocyclic group include 3-8 membered monocyclic non-aromatic heterocyclic groups such as aziridinyl, oxiranyl, thiiranyl, azetidinyl, oxetanyl, thiethanyl, tetrahydrothienyl, tetrahydrofuranyl, pyrrolinyl, pyrrolidinyl, imidazolinyl, imidazolidinyl, oxazolinyl, oxazolidinyl , pyrazolinil, pyrazolidinil, thiazolinyl, thiazolidinyl, tetrahydroisothiazolyl, tetrahydrooxazolyl, tetrahydroisooxazolyl, piperidinyl, piperazinyl, tetrahydropyridinyl, dihydropyridinyl, dihydrothiopyranyl, tetrahydropyrimidinyl, tetrahydropyridazinyl, dihydropyranyl, tetrahydropyranyl, tetrahydrothiopyran yl, morpholinil, thiomorpholinil, azepanil, diazepanil, azepinil, oxepanil, azocanil, diazocanil, etc. ; and 9-14 membered fused polycyclic (preferably bi- or tricyclic) non-aromatic heterocyclic groups such as dihydrobenzofuranyl, dihydrobenzimidazolyl, dihydrobenzoxazolyl, dihydrobenzothiazolyl, dihydrobenzisothiazolyl, dihydronaphtho[2,3-b]thienyl, tetrahydroisoquinolyl, tetrahydroquinolyl, 4 N-quinolizinyl, indolinyl , isoindolinyl, tetrahydrothieno[2,3-c]pyridinyl, tetrahydrobenzazepinyl, tetrahydroquinoxalinyl, tetrahydrophenanthridinyl, hexahydrophenothiazinyl, hexahydrophenoxazinyl, tetrahydrophthalazinyl, tetrahydronaphthyridinyl, tetrahydroquinazolinyl, tetrahydrocinnolinyl, tetrahydrocarbazole yl, tetrahydro-b-carbolinyl, tetrahydroacridinyl, tetrahydrophenazinyl, tetrahydrothioxanthenyl, octahydroisoquinolyl, etc. .

In the present specification, preferred examples of the 7-10 membered bridged heterocyclic group include quinuclidinyl and 7-azabicyclo[2.2.1]heptanyl.

As used herein, examples of the nitrogen-containing heterocyclic group include a heterocyclic group containing at least one nitrogen atom as a ring-constituting atom.

In the present specification, examples of an optionally substituted heterocyclic group include a heterocyclic group optionally having substituent(s) selected from the above-mentioned substituent group A.

The number of substituents on the optionally substituted heterocyclic group is, for example, from 1 to 3. When the number of substituents is two or more, the corresponding substituents may be the same or may be different.

In the present specification, examples of the acyl group include a formyl group, a carboxy group, a carbamoyl group, a thiocarbamoyl group, a sulfin group, a sulfo group, a sulfamoyl group, a phosphono group, each of which optionally has 1 or 2 substituents selected from C _1-6 alkyl group, C _2-6 alkenyl group, C ₃ cycloalkyl group, C ₃ cycloalkenyl group, C ₆ -14 aryl group, C ₇ 16 aralkyl group, 5-14 membered aromatic heterocyclic group and 3-14 membered non-aromatic heterocyclic group, each of which has

1-3 substituents selected from a halogen atom, an optionally halogenated _C1-6 alkoxy group, a hydroxy group, a nitro group, a cyano group, an amino group and a carbamoyl group.

Examples of the acyl group also include a hydrocarbon-sulfonyl group, a heterocyclylsulfonyl group, a hydrocarbon-sulfinyl group, and a heterocyclylsulfinyl group.

In the present specification, a hydrocarbon-sulfonyl group means a sulfonyl group linked to a hydrocarbon group, a heterocyclylsulfonyl group means a sulfonyl group linked to a heterocyclic group, a hydrocarbon-sulfinyl group means a sulfinyl group linked to a hydrocarbon group, and a heterocyclylsulfinyl group means a sulfinyl group linked to a heterocyclic group.

Preferable examples of the acyl group include a formyl group, a carboxy group, a C1-6 alkylcarbonyl group, a C2-6 alkenylcarbonyl group (eg, crotonoyl), a C3 _{- i0} cycloalkylcarbonyl group (for example, cyclobutanecarbonyl, cyclopentanecarbonyl, cyclohexanecarbonyl, cycloheptanecarbonyl), C3 _- i ₀ cycloalkenylcarbonyl group (e.g. 2cyclohexenecarbonyl), _{C6_14} arylcarbonyl group _{, C7_16} aralkylcarbonyl group, 5-14 membered aromatic heterocyclylcarbonyl group, 3-14 membered non-aromatic heterocyclylcarbonyl group, C1-6 alkoxycarbonyl group, _{C6_14} aryloxycarbonyl group yl group ₍ e.g. phenyloxycarbonyl, naphthyloxycarbonyl), _{C7_16} aralkyloxycarbonyl group (e.g. benzyloxycarbonyl, phenethyloxycarbonyl), carbamoyl group, mono- or di- _{C1_6} alkylcarbamoyl group, mono- or di- _{C2_6} alkenylcarbamoyl group (e.g. diallylcarbamoyl), _mono -or di-C ₃ .1o cycloalkylcarbamoyl group (for example, cyclopropylcarbamoyl), mono- or diCb-1 ₄ arylcarbamoyl group (for example, phenylcarbamoyl), mono- or di-C7_ ₁₆ aralkylcarbamoyl group, 5-14-membered aromatic heterocyclylcarbamoyl group ( e.g. pyridylcarbamoyl), thiocarbamoyl group, mono- or di- _{C1_6} alkylthiocarbamoyl group (e.g. methylthiocarbamoyl, K-ethyl-K-methylthiocarbamoyl), mono- or di- _C2-6 alkenylthiocarbamoyl group (e.g. diallylthiocarbamoyl), mono- or a di-C _{3_10} cycloalkylthiocarbamoyl group (for example, cyclopropylthiocarbamoyl, cyclohexylthiocarbamoyl), a mono- or di-C _6-14 arylthiocarbamoyl group (for example, phenylthiocarbamoyl), a mono- or di-C _7-16 aralkylthiocarbamoyl group (for example, _{benzylthiocarbamoyl} , phenethylthiocarbamoyl), 5-14 membered aromatic heterocyclylthiocarbamoyl group (e.g. pyridylthiocarbamoyl), sulfino group, C _1-6 alkylsulfinyl group (e.g. methylsulfinyl, ethylsulfinyl), sulfo group, C _1-6 alkylsulfonyl group, C _6-14 arylsulfonyl group, phosphono group and a mono- or di-C _1-6 alkylphosphono group (eg, dimethylphosphono, diethylphosphono, diisopropylphosphono, dibutylphosphono).

In the present specification, examples of an optionally substituted amino group include an amino group optionally having 1 or 2 substituents selected from a C _1-6 alkyl group, a C _2-6 alkenyl group, a C _3-10 cycloalkyl group, a C _6-14 aryl group, a C _{7- 16} aralkyl group, C _1-6 alkylcarbonyl group, C _6-14 arylcarbonyl group, C ₇ - ₁₆ aralkylcarbonyl group, 5-14 membered aromatic heterocyclylcarbonyl group, 3-14 membered non-aromatic heterocyclylcarbonyl group, C _1-6 alkoxycarbonyl group, a 5-14 membered aromatic heterocyclic group, a carbamoyl group, a mono- or di- _{C1_6} alkylcarbamoyl group, a mono- or di- _{C7_16} aralkylcarbamoyl group, a C1-6 alkylsulfonyl group and a _C6-14 arylsulfonyl _group , each optionally has from 1 to 3 substituents selected from the group of substituents A.

Preferred examples of the optionally substituted amino group include an amino group, a mono- or di-(optionally halogenated C _1-6 alkyl) amino group (for example, methylamino, trifluoromethylamino, dimethylamino, ethylamino, diethylamino, propylamino, dibutylamino), a mono- or di-C _{2 6} alkenylamino group (for example, diallylamino), mono - or di-C ₃ - ₁₀ cycloalkylamino group (for example, cyclopropylamino, cyclohexylamino), mono - or di-C ₆ - ₁₄ arylamino group (for example, phenylamino), mono - or di-C ₇ - ₁₆ aralkylamino group (e.g. benzylamino, dibenzylamino), mono- or di(optionally halogenated _{C1_6} alkyl)carbonylamino group (e.g. acetylamino, propionylamino), mono- or di- _{C6_14} arylcarbonylamino group (e.g. _benzoylamino ), mono- or di-C ₇ _ ₁₆ aralkylcarbonylamino group (for example, benzylcarbonylamino), mono- or di-5-14-membered aromatic heterocyclylcarbonylamino group (for example, nicotinoylamino, isonicotinoylamino), mono- or di-3-14-membered non-aromatic heterocyclylcarbonylamino group (for example, piperidinylcarbonylamino), mono- or di-C ₁ _ ₆ alkoxycarbonylamino group (for example, tert-butoxycarbonylamino), 5-14-membered aromatic heterocyclylamino group (for example, pyridylamino), carbamoylamino group, (mono- or di-C1- ₆ alkylcarbamoyl) amino group (for example, methylcarbamoylamino), (mono- or di-C ₇ _ ₁₆ aralkylcarbamoyl) amino group (for example, benzylcarbamoylamino), C _1-6 alkylsulfonylamino group (for example, methylsulfonylamino, ethylsulfonylamino), C _{6 14} arylsulfonylamino group (for example, phenylsulfonylamino), (C ₁ _ ₆ alkyl)(C ₁ _ ₆ alkylcarbonyl) amino group (for example, K-acetyl-K-methylamino) and (C ₁ _ ₆ alkyl)(C ₆ _ ₁₄ arylcarbonyl) amino group (for example, K-benzoyl-K-methylamino).

In the present specification, examples of an optionally substituted carbamoyl group include

- 7 044242 carbamoyl group, optionally having 1 or 2 substituents selected from a C _1-6 alkyl group, a C _2-6 alkenyl group, a C _3-10 cycloalkyl group, a C _6-14 aryl group, a C _7-16 aralkyl group, C _1-6 alkylcarbonyl group, C _6-14 arylcarbonyl group, C _7-16 aralkylcarbonyl group, 5-14-membered aromatic heterocyclylcarbonyl group, 3-14-membered non-aromatic heterocyclylcarbonyl group, C _1-6 alkoxycarbonyl group, 5-14- a member of an aromatic heterocyclic group, a carbamoyl group, a mono- or di-C _1-6 alkylcarbamoyl group and a mono- or di-C _7-16 aralkylcarbamoyl group, each of which optionally has 1-3 substituents selected from the group of substituents A.

Preferable examples of the optionally substituted carbamoyl group include a carbamoyl group, a mono- or di-C _1-6 alkylcarbamoyl group, a mono- or di-C _2-6 alkenylcarbamoyl group (for example, diallylcarbamoyl), a mono- or di-C _3-10 cycloalkylcarbamoyl group (e.g. cyclopropylcarbamoyl, cyclohexylcarbamoyl), mono- or di-C _6-14 arylcarbamoyl group (e.g. phenylcarbamoyl), mono- or di-C _7-16 aralkylcarbamoyl group, mono- or di-C _1-6 alkylcarbonylcarbamoyl group (e.g. acetylcarbamoyl , propionylcarbamoyl), a mono or di-C _6-14 arylcarbonylcarbamoyl group (eg benzoylcarbamoyl) and a 5-14 membered aromatic heterocyclylcarbamoyl group (eg pyridylcarbamoyl).

In the present specification, examples of an optionally substituted thiocarbamoyl group include a thiocarbamoyl group optionally having 1 or 2 substituents selected from a C _1-6 alkyl group, a C _2-6 alkenyl group, a C _3-10 cycloalkyl group, a C _6-14 aryl group, C _7-16 aralkyl group, C _1-6 alkylcarbonyl group, C _6-14 arylcarbonyl group, C _7-16 aralkylcarbonyl group, 5-14 membered aromatic heterocyclylcarbonyl group, 3-14 membered non-aromatic heterocyclylcarbonyl group, C1-6 alkoxycarbonyl group , a 5-14 membered aromatic heterocyclic group, a carbamoyl group, a mono- or di-C _1-6 alkylcarbamoyl group and a mono- or di-C _7-16 aralkylcarbamoyl group, each of which optionally has 1-3 substituents selected from the group deputies A.

Preferred examples of the optionally substituted thiocarbamoyl group include a thiocarbamoyl group, a mono- or di-C _1-6 alkylthiocarbamoyl group (for example, methylthiocarbamoyl, ethylthiocarbamoyl, dimethylthiocarbamoyl, diethylthiocarbamoyl, K-ethyl-K-methylthiocarbamoyl), mono- or di-C _2-6 alkenylthiocarbamoyl group (eg diallylthiocarbamoyl), mono- or di-C _3-10 cycloalkylthiocarbamoyl group (eg cyclopropylthiocarbamoyl, cyclohexylthiocarbamoyl), mono- or di-C _6-14 arylthiocarbamoyl group (eg phenylthiocarbamoyl), mono- or di-C ₇₁₆ aralkylthiocarbamoyl group (e.g. benzylthiocarbamoyl, phenethylthiocarbamoyl), mono- or di-C _1-6 alkylcarbonylthiocarbamoyl group (e.g. acetylthiocarbamoyl, propionylthiocarbamoyl), mono- or di-C _6-14 arylcarbonylthiocarbamoyl group (e.g. benzoylthiocarbamoyl ) and 514-member an aromatic heterocyclylthiocarbamoyl group (eg pyridylthiocarbamoyl).

In the present specification, examples of an optionally substituted sulfamoyl group include a sulfamoyl group optionally having 1 or 2 substituents selected from a C _1-6 alkyl group, a C _2-6 alkenyl group, a C _3-10 cycloalkyl group, a C _6-14 aryl group, a C _7-16 aralkyl group, C _1-6 alkylcarbonyl group, C _6-14 arylcarbonyl group, C _7-16 aralkylcarbonyl group, 5-14 membered aromatic heterocyclylcarbonyl group, 3-14 membered non-aromatic heterocyclylcarbonyl group, C _1-6 alkoxycarbonyl group group, a 5-14 membered aromatic heterocyclic group, a carbamoyl group, a mono- or di-C _1-6 alkylcarbamoyl group and a mono- or di-C _7-16 aralkylcarbamoyl group, each of which optionally has 1-3 substituents selected from groups of deputies A.

Preferred examples of the optionally substituted sulfamoyl group include a sulfamoyl group, a mono- or di- _C1-6 alkylsulfamoyl group (for example, methylsulfamoyl, ethylsulfamoyl, dimethylsulfamoyl, diethylsulfamoyl, K-ethyl-K-methylsulfamoyl), a mono- or _diC2-6 alkenylsulfamoyl group (e.g. diallylsulfamoyl), mono- or di-C _3-10 cycloalkylsulfamoyl group (e.g. cyclopropylsulfamoyl, cyclohexylsulfamoyl), mono- or diC _6-14 arylsulfamoyl group (e.g. phenylsulfamoyl), mono- or di-C _7-16 aralkylsulfamoyl group group (e.g. benzylsulfamoyl, phenethylsulfamoyl), mono- or di-C _1-6 alkylcarbonylsulfamoyl group (e.g. acetylsulfamoyl, propynylsulfamoyl), mono- or di-C _6-14 arylcarbonylsulfamoyl group (e.g. benzoylsulfamoyl) and 5-14 membered an aromatic heterocyclylsulfamoyl group (eg pyridylsulfamoyl).

In the present specification, examples of an optionally substituted hydroxy group include a hydroxyl group optionally having a substituent selected from a C _1-6 alkyl group, a C _2-6 alkenyl group, a C _3-10 cycloalkyl group, a C _6-14 aryl group, a C _7-16 aralkyl group. group, C _1-6 alkylcarbonyl group, C _6-14 arylcarbonyl group, C _7-16 aralkylcarbonyl group, 5-14 membered aromatic heterocyclylcarbonyl group, 3-14 membered non-aromatic heterocyclylcarbonyl group, C1-6 alkoxycarbonyl group, 5-14 membered an aromatic heterocyclic group, a carbamoyl group, a mono- or di-C _1-6 alkylcarbamoyl group, a mono- or di-C ₇₁₆ aralkylcarbamoyl group, a C _1-6 alkylsulfonyl group and a C _6-14 arylsulfonyl group,

- 8 044242 each of which optionally has 1-3 substituents selected from the group of substituents A.

Preferred examples of the optionally substituted hydroxy group include hydroxy group, C ₁ . ₆ αalkoxy group, C ₂ . ₆ alkenyloxy group (for example, allyloxy, 2-butenyloxy, 2-pentenyloxy, 3hexenyloxy), C _3-10 cycloalkyloxy group (for example, cyclohexyloxy), C _6-14 αryloxy group (for example, phenoxy, naphthyloxy), C _7-16 aralkyloxy group (for example, benzyloxy, phenethyloxy), C1. ₆ alkylcarbonyloxy group (e.g. acetyloxy, propionyloxy, butyryloxy, isobutyryloxy, pivaloyloxy), C _6-14 arylcarbonyloxy group (e.g. benzoyloxy), C _7-16 aralkylcarbonyloxy group (e.g. benzylcarbonyloxy), 5-14 membered aromatic heterocyclylcarbonyloxy group (e.g. nicotinoyloxy) , a 3-14 membered non-aromatic heterocyclylcarbonyloxy group (eg piperidinylcarbonyloxy), C ₁ . ₆ alkoxycarbonyloxy group (eg tert-butoxycarbonyloxy), 514-membered aromatic heterocyclyloxy group (eg pyridyloxy), carbamoyloxy group, C ₁ . ₆ αlkylcarbamoyloxy group (for example, methylcarbamoyloxy), C ₇ - ₁₆ aralkylcarbamoyloxy group (for example, benzylcarbamoyloxy), C ₁ . ₆ alkylsulfonyloxy group (eg methylsulfonyloxy, ethylsulfonyloxy) and C ₆ . ₁₄ arylsulfonyloxy group (for example, phenylsulfonyloxy).

In the present specification, examples of an optionally substituted sulfanyl group include a sulfanyl group optionally having a substituent selected from a C1-6 alkyl group, a _C26 alkenyl group, a _{C3_10} cycloalkyl group, _{a C6_14} aryl group, _{a C7_16} aralkyl group _, C1-6 alkylcarbonyl group, _C6 . ₁₄ arylcarbonyl group and a 5-14 membered aromatic heterocyclic group, each of which has 1-3 substituents selected from substituent group A and a halogenated sulfanyl group.

Preferred examples of the optionally substituted sulfanyl group include the sulfanyl (-SH) group, C ₁ . ₆ alkylthio group, C ₂ . ₆ alkenylthio group (for example, allylthio, 2-butenylthio, 2-pentenylthio, 3-hexenylthio), C ₃ . ₁₀ cycloalkylthio group (eg cyclohexylthio), C _{6 14} arylthio group (eg phenylthio, naphthylthio), C ₇ . ₁ baralkylthio group (eg benzylthio, phenethylthio), C ₁ . ₆ alkylcarbonylthio group (eg acetylthio, propionylthio, butyrylthio, isobutyrylthio, pivaloylthio), C ₆ . _{a 14} arylcarbonylthio group (eg benzoylthio), a 5-14 membered aromatic heterocyclylthio group (eg pyridylthio) and a halogenated thio group (eg pentafluorothio).

In the present specification, examples of an optionally substituted silyl group include a silyl group optionally having 1 to 3 substituents selected from a C1-6 alkyl group, a C2-6 alkenyl group, a _{C3_10 cycloalkyl} group, _{a C6_14} aryl group, and a _{C7_ 16} aralkyl group, each of which optionally has 1-3 substituents selected from the group of substituents A.

Preferred examples of the optionally substituted silyl group include a tri-C1_ ₆ alkylsilyl group (eg, trimethylsilyl, t-butyl(dimethyl)silyl).

The definition of each symbol in formula (I) is described in detail below.

P ¹ represents a group corresponding to the formula:

- R ^a1 ,

- CO-R ^a1 ,

- CO-OR ^a1 ,

- CO-COR ^a1 ,

- SO-R ^a1 ,

- SO2-R ^a1 ,

- SO2-OR ^a1 ,

- CO-NR ^A2 R ^A3 ,

- SO ₂ -NR ^A2 R ^A3 , or

- C(=NR ^A1 )-NR ^A2 R ^A3 , wherein each of R ^A1 , R ^A2 and R ^A3 independently represents a hydrogen atom, an optionally substituted hydrocarbon group or an optionally substituted heterocyclic group.

P ¹ preferably represents a hydrogen atom.

A 11 represents Aib or Ala.

Preferably A11 is Aib.

A12 represents Ala, Ile, Lys, Phe or Rua (4).

Preferably A12 is Ile.

In an alternative embodiment, preferably A12 is Lys.

And 13 is Aib, Cha, Leu, aMePhe or aMeTyr.

Preferably A13 is Aib.

A16 is Lys or Ser. Preferably A16 is Lys.

A17 is Gln or Ile. Preferably A17 is Gln.

A20 represents Ala or Ser. Preferably A20 is Ala.

A29 is Gln or Gly. Preferably A29 is Gly.

Compound (I) may have an additional peptide sequence at the C-terminal side (C-terminal sequence) of A29 in the partial sequence corresponding to formula (I).

- 9 044242

In this case, the length of the C-terminal sequence is not particularly limited, and is preferably 1 to 11 amino acid residues, more preferably 6 to 11 amino acid residues.

Examples of C-terminal sequence may include:

(i) an amino acid sequence corresponding to Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-ProSer-Lys (SEQ ID NO: 2), (ii) an amino acid sequence derived from the sequence according to the above (i) by deletion, substitution (preferably a conservative substitution) or insertion of 111, preferably 1-5 amino acids, and (iii) a partial sequence containing at least 1 amino acid residue (consecutive), preferably 6 consecutive amino acid residues, from the N-terminus from the sequence according to (i) or (ii) above.

Specifically used as the C-terminal sequence are (1) Gly-, (2) Gly-Pro-, (3) Gly-Pro-Ser-, (4) Gly-Pro-Ser-Ser- (SEQ ID NO: 3) , (5) Gly-Pro-Ser-Ser-Gly- (SEQ ID NO: 4), (6) Gly-Pro-Ser-Ser-Gly-Ala- (SEQ ID NO: 5), (7) Gly- Pro-Ser-Ser-Gly-Ala-Pro- (SEQ ID NO: 6), (8) Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro- (SEQ ID NO: 7), (9) Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro- (SEQ ID NO: 8), (10) Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser- ( SEQ ID NO: 9), (11) Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys- (SEQ ID NO: 2), etc.

The C-terminal sequence is preferably Gly-Pro-Ser-Ser-Gly-Ala-ProPro-Pro-Ser- or Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys-.

Particularly preferably, the C-terminal sequence is Gly-Pro-Ser-SerGly-Ala-Pro-Pro-Pro-Ser-Lys-.

Compound (I) having the above-mentioned C-terminal sequence has high solubility.

Also, compound (I) having the above-mentioned C-terminal sequence has a high activating effect of GLP-1 receptor and GIP receptor in vivo.

Preferable examples of compound (I) include the following peptide or a salt thereof.

Compound A.

Compound (I), where

P ¹ represents a hydrogen atom;

A11 represents Aib;

A12 represents Ile;

A13 represents Aib;

A16 represents Lys;

A17 is Gln;

A20 represents Ala;

A29 represents Gly; and a peptide having the amino acid sequence corresponding to Gly-Pro-Ser-Ser-GlyAla-Pro-Pro-Pro-Ser-Lys-, on the C-terminal side of A29.

Compound (I) can be produced by an essentially known peptide synthesis method. The peptide synthesis method may be any of, for example, a solid phase synthesis method and a liquid phase synthesis method. Therefore, the peptide in question can be produced by repeated condensation of a partial peptide or amino acid that may constitute compound (I) and the remainder (which may consist of two or more amino acids) according to the desired sequence. When a product having the desired sequence has a protecting group, the peptide in question can be prepared by removing the protecting group. Examples of the condensation method and the deprotection method are known and include the methods described in the following (1)(5).

- 10 044242 (1) M. Bodanszky and M.A. Ondetti: Peptide Synthesis, Interscience Publishers, New York (1966) (2) Schroeder and Luebke: The Peptide, Academic Press, New

York (1965) (3) Nobuo Izumiya, et al.: Peptide Gosei-no-Kiso to Jikken (Basics and experiments of peptide synthesis), published by Maruzen Co. (1975) (4) Haruaki Yajima and Shunpei Sakakibara: Seikagaku Jikken Koza (Biochemical Experiment) 1, Tanpakushitsu no Kagaku (Chemistry of Proteins) IV, 205 (1977) (5) Haruaki Yajima, ed. : Zoku lyakuhin no Kaihatsu (A sequel to Development of Pharmaceuticals), Vol. 14, Peptide Synthesis, published by Hirokawa Shoten

After the reaction, compound (I) can be purified and isolated using conventional purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, etc., and combinations thereof. When the peptide obtained by the above method is in free form, it can be converted into a suitable salt in a known manner; in contrast, when the peptide is obtained in salt form, the salt can be converted to the free form or another salt in a known manner.

The starting compound may also be a salt. Examples of such a salt include the salts illustrated as the salts of compound (I) mentioned below.

To condense the protected amino acid or peptide, various activation reagents suitable for peptide synthesis can be used, which are particularly preferably trisphosphonium salts, tetramethyluronium salts, carbodiimides, and the like. Examples of trisphosphonium salt include benzotriazol-1-yloxytris(pyrrolizino)phosphonium hexafluorophosphate (PyBOP), bromotris(pyrrolizino)phosphonium hexafluorophosphate (PyBroP), 7-azabenzotriazol-1 yloxytris(pyrrolizino)phosphonium hexafluorophosphate (PyAOP), examples of tetramethyluronium salt include 2-(1H- benzotriazol-1 -yl)-1,1,3,3-hexafluorophosphate (HBTU), 2-(7-azabenzotriazol-1 -yl)-1,1,3,3hexafluorophosphate (HATU), 2-(1H-benzotriazole -1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 2-(5-norbornane-2,3-dicarboxyimide)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU), O-(Nsuccimidyl )-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU), and examples of carbodiimide include DCC, N,N'-diisopropylcarbodiimide (DIPCDI), N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI-HCl) and so on. For condensation using them, it is preferred to add a racemization inhibitor (eg HONB, HOBt, HOAt, HOOBt, etc.). The solvent to be used for condensation may be suitably selected from solvents known to be useful for the peptide condensation reaction. For example, acid amides such as anhydrous and hydrated N,N-dimethylformamide, N,N-dimethylacetamide, Nmethylpyrrolidone and the like, halogenated hydrocarbons such as methylene chloride, chloroform and the like, alcohols such as such as trifluoroethanol, phenol, etc., sulfoxides such as dimethyl sulfoxide, etc., tertiary amines such as pyridine, etc., ethers such as dioxane, tetrahydrofuran, etc., nitriles such as such as acetonitrile, propionitrile and the like, esters such as methyl acetate, ethyl acetate and the like, a suitable mixture thereof and the like. The reaction temperature is suitably selected from a range known to be suitable for peptide coupling reactions and is typically selected from a range of about -20 to 50°C. The activated derivative is typically used in an excess of approximately 1.5 to 6 times. In phase synthesis, when the ninhydrin reaction test reveals that condensation is insufficient, sufficient condensation can be achieved by repeating the condensation reaction without removing the protecting groups. If condensation is still insufficient even after repeating the reaction, unreacted amino acids can be acylated with acetic anhydride, acetylimidazole, or the like. to avoid influencing subsequent reactions.

Examples of protecting groups for the amino groups of the parent amino acid include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc, trityl and the like.

Examples of the carboxyl protecting group for the parent amino acid include allyl, 2-adamantyl, 4-nitrobenzyl, 4-methoxybenzyl, 4-chlorobenzyl, phenacyl and benzyloxycarbonylhydrazide, tert-butoxycarbonylhydrazide, tritylhydrazide and the like, in addition to the above-mentioned C _1-6 alkyl group, C _3-10 cycloalkyl group, C _7-14 aralkyl group.

The hydroxyl group of serine or threonine can be protected, for example, by esterification or etherification. Examples of groups suitable for esterification include lower ( _C2-4 )

- 11 044242 alkanoyl groups such as an acetyl group and the like, aroyl groups such as a benzoyl group and the like, etc., and a group derived from an organic acid and the like. Moreover, examples of the group suitable for esterification include benzyl, tetrahydropyranyl, tert-butyl (Bu ^t ), trityl (Trt), and

etc.

Examples of the protecting group for the phenolic hydroxyl group of tyrosine include Bzl, 2,6dichlorobenzyl, 2-nitrobenzyl, Br-Z, tert-butyl and the like.

Examples of the protecting group for histidine imidazole include Tos, 4-methoxy-2,3,6trimethylbenzenesulfonyl (Mtr), DNP, Bom, Bum, Boc, Trt, Fmoc and the like.

Examples of the protecting group for the guanidino group of arginine include Tos, Z, 4-methoxy-2,3,6trimethylbenzenesulfonyl (Mtr), p-methoxybenzenesulfonyl (MBS), 2,2,5,7,8-pentamethylchroman-6sulfonyl (Pmc), mesitylene- 2-sulfonyl (Mts), 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), Boc, Z, NO ₂ , etc.

Examples of the protecting group for the amino group of the lysine side chain include Z, Cl-Z, trifluoroacetyl, Boc, Fmoc, Trt, Mtr, 4,4-dimethyl-2,6-dioxocyclohexylideneyl (Dde) and the like.

Examples of the protecting group for indolyl tryptophan include formyl (For), Z, Boc, Mts, Mtr and the like.

Examples of the protecting group for asparagine and glutamine include Trt, xanthyl (Xan), 4,4'dimethoxybenzhydryl (Mbh), 2,4,6-trimethoxybenzyl (Tmob) and the like.

Examples of activated carboxyl groups in the starting material include the corresponding acid anhydride, azide, active esters [alcohol ester (e.g., pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, Nhydroxysuccinimide, 1 -hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt)] etc. Examples of an activated amino group in the starting material include the corresponding phosphorus amide.

Examples of the protecting group removal method include catalytic reduction under a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon; acid treatment using anhydrous hydrofluoric acid, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetate, trimethylsilyl bromide (TMSBr), trimethylsilyltrifluoromethanesulfonate, tetrafluoroboric acid, tris(trifluoro)boric acid, boron tribromide, or a mixture of solutions thereof; base treatment using diisopropylethylamine, triethylamine, piperidine, piperazine and the like; and reduction with sodium in liquid ammonia, etc. The elimination reaction by treatment with the acid described above is usually carried out at a temperature of -20 to 40°C; acid treatment is effectively carried out by adding a cation trap such as anisole, phenol, thioanisole, meta-cresol and para-cresol; dimethyl sulfide, 1,4-butanedithiol, 1,2-ethanedithiol, etc. Also, the 2,4dinitrophenyl group used as the protecting group of histidine imidazole is removed by treatment with thiophenol; the formyl group used as a protecting group for indole tryptophan is removed by deprotection by acid treatment in the presence of 1,2-ethanedithiol, 1,4-butanedithiol and the like, and by alkaline treatment with dilute sodium hydroxide, dilute ammonia and the like.

Protecting a functional group that should not be involved in the reaction of the starting material and the protecting group, elimination of the protecting group, activation of the functional group involved in the reaction, etc. can be suitably selected from known protecting groups and known agents.

In the method for producing a peptide amide, it is prepared by solid phase synthesis using an amide synthesis resin, or the α-carboxyl group of the C-terminal amino acid is amidated and the peptide chain is extended to the desired chain length in the direction of the amino group, and then a peptide in which only the protecting group has been removed is obtained. for the N-terminal α-amino group of the peptide chain, and a peptide in which only the protecting group of the C-terminal carboxyl group of the peptide chain has been removed, and both peptides are condensed in the mixed solvent described above. For a detailed description of the condensation reaction, the same as described above applies. After purification of the protected peptide obtained by condensation, all protecting groups can be removed in the manner described above to obtain the desired crude polypeptide. By purifying this crude peptide using various conventional purification methods and lyophilizing the bulk fraction, the desired peptide amide can be obtained.

When compound (I) is present as a configuration isomer such as enantiomer, diastereomer, etc., conformer, etc., they are also covered by compound (I) and each of them can be isolated by methods essentially known or described above separation and purification methods, if necessary. In addition, when compound (I) is in racemate form, it can be separated into S and R forms by conventional optical separation methods.

When compound (I) includes stereoisomers, both isomers alone and mixtures of each isomer are covered by compound (I).

- 12 044242

Compound (I) can be chemically modified in a manner known per se using polyethylene glycol. For example, the chemically modified compound (I) can be obtained by conjugating polyethylene glycol with a Cys residue, an Asp residue, a Glu residue, a Lys residue and the like.

compounds (I).

Compound (I) modified with polyethylene glycol (PEG) causes, for example, the effects of stimulating biological activity, prolonging blood circulation time, reducing immunogenicity, increasing solubility and increasing resistance to metabolism of a therapeutically and diagnostically important peptide.

The molecular weight of PEG is not particularly limited and is generally from about 1 to about 1000, preferably from about 10 to about 100, more preferably from about 20 to about 60 kilodaltons.

A method well known in the art can be used as a method for modifying compound (I) with PEG, and, for example, the methods described below can be used.

(1) A PEGylation reagent having an active ester (eg, SUNBRIGHT MEGC30TS (trade name), NOF Corp.) is coupled to the amino group of compound (I).

(2) A PEGylation reagent having an aldehyde (eg, SUNBRIGHT ME-300AL (trade name), NOF Corp.) is bonded to the amino group of compound (I).

(3) A divalent cross-linking reagent (for example, GMBS (Dojindo Laboratories), EMCS (Dojindo Laboratories), KMUS (Dojindo Laboratories), SMCC (Pierce)) is coupled to the compound (I), to which a PEGylation reagent having a thiol group is then coupled (eg SUNBRIGHT ME300-SH (trade name), NOF Corp.).

(4) A thiol group is introduced into the compound (I) through an SH introducing agent (for example, a D-cysteine residue, an L-cysteine residue, Trot's reagent), and this thiol group is reacted with a PEGylation reagent having a maleimide group (for example, SUNBRIGHT ME-300MA (trade name), NOF Corp.).

(5) A thiol group is introduced into the compound (I) using an SH introducing agent (for example, a D-cysteine residue, an L-cysteine residue, Troth's reagent), and this thiol group is reacted with a PEGylation reagent having an iodoacetamide group (for example , SUNBRIGHT ME-300IA (trade name), NOF Corp.).

(6) An ω-aminocarboxylic acid or an α-amino acid is introduced as a linker with the N-terminal amino group of the compound (I), and the amino group originating from this linker is reacted with a PEGylation reagent having an active ester (for example, SUNBRIGHT MEGC- 30TS (trade name), NOF Corp.).

(7) An ω-aminocarboxylic acid or an α-amino acid is introduced as a linker with the N-terminal amino group of compound (I), and the amino group originating from this linker is reacted with a PEGylation reagent having an aldehyde group (for example, SUNBRIGHT ME-300AL ( trade name), NOF Corp.).

In addition, compound (I) may be a solvate (eg, hydrate) or non-solvate (eg, non-hydrate).

Compound (I) may be labeled with an isotope (eg, ³ H, ¹⁴ C, ³⁵ S, ¹²⁵ I) and the like.

Moreover, compound (I) may be a deuterium-converted form where 1H is converted to ^2H (D).

Compound (I) labeled or isotope-substituted can be used, for example, as a radioactive tracer (PET tracer) for use in positron emission tomography (PET), and is useful in the field of medical diagnosis and the like.

For the peptides mentioned herein, the left end is the N-terminus (amino-terminus) and the right end is the C-terminus (carboxy-terminus) according to conventional peptide notation. The C-terminus of the peptide can be any of an amide (-CONH2), a carboxyl group (-COOH), a carboxylate ( ^-COO- ), an alkylamide (-CONHRa) and an ester (-COORa). In particular, amide (-CONH2) is preferred.

Compound (I) may be in the form of a salt. Examples of such a salt include metal salts, ammonium salts, organic base salts, inorganic acid salts, organic acid salts, basic or acidic amino acid salts, and the like.

Preferable examples of the metal salt include alkali metal salts such as sodium salt, potassium salt and the like; alkaline earth metal salts such as calcium salt, magnesium salt, barium salt and the like; aluminum salt, etc.

Preferable examples of the organic base salt include salts with trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N-dibenzylethylenediamine and the like.

Preferable examples of salt with inorganic acid include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid

- 13 044242, etc.

Preferable examples of the organic acid salt include those with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p- toluenesulfonic acid, etc.

Preferable examples of the basic amino acid salt include salts with arginine, lysine, ornithine and the like. Preferable examples of the acidic amino acid salt include salts with aspartic acid, glutamic acid and the like.

Among the above-mentioned salts, a pharmaceutically acceptable salt is preferred. For example, when the compound has an acidic functional group, an inorganic salt such as an alkali metal salt (for example, a sodium salt, a potassium salt, etc.), an alkaline earth metal salt (for example, a calcium salt, a magnesium salt, a barium salt, etc.) is preferred. etc.), ammonium salt, etc., and when the compound has a basic functional group, for example, a salt with an inorganic acid such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid is preferred , phosphoric acid and the like, or a salt with an organic acid such as acetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, ptoluenesulfonic acid and the like .

Compound (I) may be in the form of a prodrug.

Prodrug means a compound that is converted to compound (I) due to reaction with an enzyme, stomach acid, etc. under physiological conditions in a living organism, i.e. a compound that is converted to compound (I) by oxidation, reduction, hydrolysis, etc. depending on the enzyme; a compound that is converted to compound (I) by hydrolysis, etc. through stomach acid, etc.

Examples of the prodrug of compound (I) include a compound where the amino group of compound (I) is acylated, alkylated or phosphorylated (for example, a compound where the amino group of compound (I) is eicosanoylated, alanylated, pentylaminocarbonylated, (5-methyl-2-oxo-1,3- dioxolen-4yl)methoxycarbonylated, tetrahydrofuranylated, pyrrolidylmethylated, pivaloyloxymethylated or tert-butylated, etc.); a compound where the hydroxy group of compound (I) is acylated, alkylated, phosphorylated or borated (for example, a compound where the hydroxy group of compound (I) is acetylated, palmitoylated, propanoylated, pivaloylated, succinylated, fumarylated, alanylated or dimethylaminomethylcarbonylated); a compound where the carboxy group of compound (I) is esterified or amidated (for example, a compound where the carboxy group of compound (I) is esterified with O-alkyl, esterified with phenyl, esterified with carboxymethyl, esterified with dimethylaminomethyl, esterified with pivaloyloxymethyl, esterified with ethoxycarbonyloxyethyl, esterified with phthalidyl, esterified with (5-methyl -2oxo-1,3-dioxolen-4-yl)methyl, esterified with cyclohexyloxycarbonylethyl or methylamidated), etc. Among others, a compound where the carboxy group of compound (I) is esterified with O-alkyl such as methyl, ethyl, tert-butyl and the like is preferably used. These compounds can be prepared from compound (I) in a manner known per se.

A prodrug of compound (I) may also be a prodrug that is converted to compound (I) under physiological conditions, such as the prodrugs described in IYAKUHIN no KAIHATSU (Development of Pharmaceuticals), Vol.7, Design of Molecules, p.163-198 , published by HIROKAWA SHOTEN (1990).

As used herein, the prodrug may form a salt. Examples of such a salt include the salts illustrated as a salt of compound (I).

Compound (I) may be a crystal. Crystals having the form of a single crystal or a mixture of multiple crystal forms are also included in compound (I). Crystals can be obtained by crystallizing compound (I) by a crystallization method known per se.

In addition, compound (I) may be a pharmaceutically acceptable co-crystal or a salt of a co-crystal. As used herein, a cocrystal or cocrystal salt means a crystalline substance consisting of two or more specific substances that are solid at room temperature, each of which has different physical properties (for example, structure, melting point, heat of fusion, hygroscopicity, solubility, stability and etc.). The cocrystal or cocrystal salt can be obtained by a cocrystallization method known per se.

The crystal of compound (I) has improved physicochemical properties (e.g. melting point, solubility, stability) and biological properties (e.g. pharmacokinetics (absorption, distribution, metabolism, excretion), expression efficiency), and thus it is extremely useful as a medicine.

Compound (I) and its prodrug (hereinafter sometimes abbreviated as the compound of the present invention) have an activating effect on GLP-1 receptors and GIP receptors.

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The compound of the present invention has a high activating effect on GLP-1 receptors and GIP receptors, in particular in vivo.

GLP-1 and GIP are gut hormones called incretins, and they have the effect of stimulating insulin secretion from the pancreas. Since incretin is closely associated with glucose metabolism, a compound having an activating effect on GLP-1 receptors and GIP receptors is useful for the prevention or treatment of symptoms associated with disorders of glucose metabolism, including obesity.

Thus, the compound of the present invention has a nutritional activity suppressing effect, a weight gain inhibiting effect, and the like.

In addition, the compound of the present invention has increased solubility. The solubility of the compound of the present invention in water is preferably 1 or higher, more preferably 10 mg/ml or higher.

The compound of the present invention can be used as an activator of GLP-1 receptor and GIP receptor (GLP-1 receptor/GIP receptor coagonist).

As used herein, a GLP-1 receptor and GIP receptor activator (GLP-1 receptor/GIP receptor coagonist) means an agent having both a GLP-1 receptor activating action (GLP-1 receptor activating action) and a GIP receptor activating action (GLP-1 receptor activating action). GIP receptor agonist). In particular, GLP-1 receptor and GIP receptor activator (GLP1 receptor/GIP receptor coagonist) means an agent wherein the EC ₅₀ for the GLP-1 receptor and the EC ₅₀ for the GIP receptor is from 1:20 to 20:1, preferably from 1:5 to 5:1.

The compound of the present invention has a low glucagon receptor activating effect (glucagon receptor agonist), and thus has a low inherent hyperglycemic effect. The EC50 of the compound of the present invention towards the glucagon receptor is 1/1000 or lower, preferably 1/10000 or lower, compared with the EC50 of the compound of the present invention towards the GLP-1 receptor or the GIP receptor.

The compound of the present invention has low toxicity (e.g., acute toxicity, chronic toxicity, genetic toxicity, reproductive toxicity, cardiac toxicity, carcinogenicity), exhibits few side effects, and can be safely administered to a mammal (e.g., human, bovine, horse, dog, cat, monkey, mouse, rat) as a means for the prevention or treatment of various diseases mentioned below, etc.

The compound of the present invention can be used as an agent for the treatment or prevention of various diseases, including obesity, through the above-mentioned activating effect on GLP-1 receptors and GIP receptors. The compound of the present invention can be used as an agent for the prevention or treatment of, for example, symptomatic obesity, obesity based on simple obesity, a disease state or disease due to obesity, nutritional disorders, diabetes (for example, type 1 diabetes, type 2 diabetes, gestational diabetes , diabetes in obesity), hyperlipidemia (eg, hypertriglyceridemia, hypercholesterolemia, high LDL cholesterolemia, low HDL cholesterolemia, postprandial hyperlipemia), hypertension, heart failure, complications of diabetes [eg, neuropathy, nephropathy, retinopathy, diabetic cardiomyopathy, cataract , macroangiopathy, osteopenia, hyperosmolar diabetic coma, infectious disease (eg, respiratory infection, urinary tract infection, gastrointestinal infection, soft tissue skin infection, lower extremity infection), diabetic gangrene, xerostomia, hypoacusia, cerebrovascular disorder, peripheral circulatory disorder] , metabolic syndrome (disease conditions having 3 or more symptoms selected from hypertriglyceride (TG)emia, low-HDL cholesterol (HDL-C)emia, hypertension, abdominal obesity and reduced glucose tolerance), sarcopenia, etc.

Examples of symptomatic obesity include endocrine obesity (eg, Cushing's syndrome, hypothyroidism, insulinoma, type II diabetes with obesity, pseudohypoparathyroidism, hypogonadism), central obesity (eg, hypothalamic obesity, frontal lobe syndrome, Kleine-Levin syndrome), hereditary obesity (eg, Prader syndrome) -Willi syndrome, Lawrence-Moon-Biedl syndrome), drug-induced obesity (eg, obesity induced by steroids, phenothiazine, insulin, sulfonylurea (SU), β-blocker), etc.

Examples of a disease state or disease associated with obesity include impaired glucose tolerance, diabetes (particularly type 2 diabetes, obesity-associated diabetes), lipid metabolism disorder (synonymous with the above-mentioned hyperlipidemia), hypertension, heart failure, hyperuricemia, gout, fatty acids. liver infiltration (including non-alcoholic steptohepatitis), coronary heart disease (myocardial infarction, angina pectoris), cerebral infarction (cerebral thrombosis, transient cerebral ischemic attack), bone/joint disease (osteoarthritis of the knee, osteoarthritis of the hip, deforming spondylitis, lumbago), apnea syndrome in sleep/Pickwick's syndrome, menstrual disorder (menstrual irregularities, menstruation and cycle disorders, amenorrhea, abnormal menstrual symptoms), metabolic syndrome, etc.

New diagnostic criteria were reported by The Japan Diabetes Society in 1999 on the topic of diagnostic criteria for diabetes.

According to this report, diabetes refers to a condition that satisfies any of a fasting blood glucose level (venous blood plasma glucose concentration) of 126 mg/dL or more, a 2-hour value (venous blood plasma glucose concentration) of 200 mg/dL. dl or more in an oral glucose tolerance test with 75 g glucose (75 g OGTT) and an irregular blood glucose level (venous blood plasma glucose concentration) of 200 mg/dL or more. Also a condition that does not apply to the diabetes mentioned above and is not a condition exhibiting a fasting blood glucose level (venous blood plasma glucose concentration) less than 110 mg/dL or a 2-hour value (venous blood plasma glucose concentration) less than 140 mg/dL in an oral glucose tolerance test with 75 g glucose (75 g OGTT) (normal type) is called borderline type.

Moreover, new diagnostic criteria were reported by the American Diabetes Association (ADA) in 1997 and the WHO (WHO) in 1998 on the topic of diagnostic criteria for diabetes.

According to these reports, diabetes refers to a condition that satisfies a fasting blood glucose level (venous blood plasma glucose concentration) of 126 mg/dL or more and a 2-hour value (venous blood plasma glucose concentration) of 200 mg/dL or more at oral glucose tolerance test with 75 glucose.

According to the reports mentioned above, impaired glucose tolerance refers to a condition that satisfies a fasting blood glucose level (venous blood plasma glucose concentration) of less than 126 mg/dL and a 2-hour value (venous blood plasma glucose concentration) of 140 or more and less than 200 mg/dL on an oral glucose tolerance test with 75 g of glucose. According to the ADA, a condition exhibiting a fasting blood glucose level (venous blood plasma glucose concentration) of 110 or more and less than 126 mg/dL is called IFG (impaired fasting glucose). On the other hand, as reported by WHO, the IFG (impaired fasting glucose) condition exhibiting a 2-hour value (venous blood plasma glucose concentration) of less than 140 mg/dL in an oral glucose tolerance test with 75 g of glucose is called IFG ( impaired fasting glucose).

The compound of the present invention is also used as an agent for the prevention or treatment of diabetes defined in accordance with the above-mentioned new diagnostic criteria, borderline diabetes, impaired glucose tolerance, IFG (impaired fasting glucose) and IFG (impaired fasting glucose). Moreover, the compounds of the present invention can prevent the progression of borderline type, impaired glucose tolerance, IFG (impaired fasting glucose) or IFG (impaired fasting glucose) to diabetes.

The compound of the present invention has a weight gain inhibitory effect, and as such can be used as a weight gain inhibitor for mammals. The mammal that is the subject of use of the compound of the present invention may be a mammal in which it is desirable to avoid weight gain. The mammal may be a mammal having a genetic risk for weight gain, or it may be a mammal suffering from a lifestyle disease such as diabetes, hypertension and/or hyperlipidemia. Weight gain may be explained by excessive food intake or an unbalanced diet, or may represent weight gain due to concomitant drug use (eg, insulin sensitizers with PPARy agonist-like effects such as troglitazone, rosiglitazone, englitazone, ciglitazone, pioglitazone and so on.). Alternatively, the weight gain may represent an increase in weight until obesity is achieved or may represent an increase in weight in obese patients. Herein, obesity is defined as a body mass index (BMI: body weight (kg)+[height(m)] ² ) of 25 or more (according to the criteria of the Japan Society for the Study of Obesity) for Japanese and as a BMI of 30 or more (according to WHO criteria) for Westerners.

The compound of the present invention is also useful as an agent for the prevention or treatment of metabolic syndrome. The incidence of cardiovascular disease is quite high in patients with metabolic syndrome compared to patients with one lifestyle-related disease. Thus, preventing or treating metabolic syndrome is extremely important for preventing cardiovascular disease.

Diagnostic criteria for metabolic syndrome were announced by WHO in 1999 and NCEP in 2001. According to the WHO diagnostic criteria, an individual having hyperinsulinemia or impaired glucose tolerance as a prerequisite and two or more of visceral obesity, dyslipidemia (high level TG or low HDL) and hypertension are diagnosed as having metabolic syndrome (World Health Organization: Definition,

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Diagnosis and Classification of Diabetes Mellitus and Its Complications. Part I: Diagnosis and Classification of Diabetes Mellitus, World Health Organization, Geneva, 1999). According to the Adult Treatment Panel III diagnostic criteria from the National Cholesterol Education Program in the USA, an individual having three or more of visceral obesity, hypertriglyceridemia, low HDL cholesterolemia, hypertension and impaired glucose tolerance is diagnosed as having metabolic syndrome (National Cholesterol Education Program: Executive Summary of the Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adults Treatment Panel III). The Journal of the American Medical Association, Vol. 285, 2486-2497, 2001).

The compound of the present invention can also be used as an agent for the prevention or treatment of, for example, osteoporosis, cachexia (for example, cancer cachexia, tuberculosis cachexia, diabetic cachexia, cachexia associated with a blood disorder, cachexia associated with an endocrine disease, cachexia infection-associated or acquired immunodeficiency syndrome cachexia), fatty liver, polycystic ovary syndrome, kidney disease (eg, chronic renal failure, diabetic nephropathy, glomerulonephritis, glomerulosclerosis, nephrotic syndrome, hypertensive nephrosclerosis, end-stage renal disease) , muscular dystrophy, myocardial infarction, angina pectoris, cerebrovascular disorder (eg, cerebral infarction, stroke), Alzheimer's disease, Parkinson's disease, anxiety, dementia, insulin resistance syndrome, syndrome X, hyperinsulinemia, paresthesia caused by hyperinsulinemia, acute or chronic diarrhea, inflammatory disease (eg, chronic rheumatoid arthritis, spondylitis deformans, arthritis deformans, lumbago, gout, post-operative or post-traumatic inflammation, bloating, neuralgia, laryngopharyngitis, cystitis, hepatitis (including non-alcoholic steatohepatitis), pneumonia, pancreatitis, enteritis, inflammatory bowel disease ( including inflammatory disease of the colon), ulcerative colitis, gastric mucosal damage (including aspirin-induced gastric mucosal damage), small intestinal mucosal damage, malabsorption, testicular dysfunction, visceral obesity syndrome and sarcopenia.

Moreover, the compound of the present invention can also be used as an agent for the prevention or treatment of various malignant tumors (in particular, breast cancer (for example, invasive ductal breast cancer, non-invasive ductal breast cancer, inflammatory breast cancer, etc. .), prostate cancer (for example, hormone-dependent prostate cancer, hormone-independent prostate cancer, etc.), pancreatic cancer (for example, pancreatic duct cancer, etc.), stomach cancer (for example, papillary adenocarcinoma, mucinous adenocarcinoma, glandular squamous cell carcinoma, etc.), lung cancer (eg, non-small cell lung cancer, small cell lung cancer, malignant mesothelioma, etc.), colon cancer (eg, gastrointestinal stromal tumor and etc.), rectal cancer (eg, gastrointestinal stromal tumor, etc.), colorectal cancer (eg, familial colorectal cancer, hereditary nonpolyposis colorectal cancer, gastrointestinal stromal tumor, etc.), malignant tumor of the small intestine (for example, non-Hodgkin's lymphoma, gastrointestinal stromal tumor, etc.), esophageal cancer, duodenal cancer, tongue cancer, pharyngeal cancer (for example, nasopharyngeal cancer, cancer oropharynx, hypopharyngeal cancer, etc.), salivary gland cancer, brain tumor (eg, pineal astrocytoma, pinocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, etc.), neurilemmoma, liver cancer (eg, primary liver cancer , extrahepatic bile duct cancer, etc.), kidney cancer (for example, renal cell carcinoma, transitional cell carcinoma of the renal pelvis and urethra, etc.), bile duct cancer, endometrial cancer, cervical cancer, ovarian cancer ( e.g., epithelial ovarian cancer, extragonadal germinoma, ovarian germ cell tumor, ovarian tumor of low malignant potential, etc.), bladder cancer, urethral cancer, skin cancer (eg, intraocular (ocular) melanoma, Merkel cell carcinoma, etc. ..), hemangioma, malignant lymphoma, malignant melanoma, thyroid cancer (eg, medullary thyroid cancer, etc.), parathyroid cancer, nasal cancer, sinus cancer, bone tumor (eg, osteosarcoma, Ewing's tumor , uterine sarcoma, soft tissue sarcoma, etc.), angiofibroma, retinal sarcoma, penile cancer, testicular cancer, pediatric solid tumor (eg, Wilms tumor, pediatric kidney tumor, etc.), Kaposi's sarcoma, Kaposi's sarcoma , caused by AIDS, maxillary sinus tumor, fibrous histiocytoma, leiomyosarcoma, rhabdomyosarcoma, leukemia (eg, acute myeloid leukemia, acute lymphoblastic leukemia, etc.), etc.).

The compound of the present invention can also be used for secondary prevention or suppression of the progression of various diseases mentioned above (eg, cardiovascular events such as myocardial infarction, etc.). In addition, the compound of the present invention is also useful as a nutritional suppressant and a weight gain inhibitor. The compound of the present invention can also be used in combination

- 17 044242 tion with diet therapy (for example, diet therapy for diabetes) and physical therapy.

The drug containing the compound of the present invention exhibits low toxicity and is prepared using the compound of the present invention alone or mixed with a pharmacologically acceptable carrier according to an essentially known method (for example, the method described in Japanese Pharmacopoeia) commonly used in as methods of producing pharmaceuticals, and are safely administered orally or perineternally (e.g., administered topically, rectally, intravenously) as a pharmaceutical preparation, for example, tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, orally disintegrating tablets), powders, granules, capsules (including soft capsules, microcapsules), liquids, lozenges, syrups, emulsions, suspensions, injectables (e.g. subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, etc.), topical preparations (e.g. transnasal preparations, dermal preparations, ointments), suppositories (eg rectal suppositories, vaginal suppositories), granules, nasal preparations, pulmonary preparations (inhaled preparations), transfusion preparations, etc.

These drugs may be controlled release drugs, such as immediate release drugs, sustained release drugs, and the like. (eg sustained release microcapsule).

The content of the compound of the present invention in the pharmaceutical preparation is approximately 0.01 approximately 100% by weight of the total preparation.

The above-mentioned pharmaceutically acceptable carrier can be illustrated by various organic or inorganic carrier materials that are commonly used as preparation materials, such as an excipient, a lubricant, a binder and a disintegrant for solid preparations; or a solvent, solubilizing agent, suspending agent, isotonicizing agent, buffering agent, soothing agent, or the like. for liquid preparations. In addition, if necessary, general additives such as a preservative, antioxidant, coloring agent, sweetener, adsorbent, wetting agent and the like can also be used. accordingly in suitable quantities.

Examples of the excipient include lactose, sucrose, D-mannitol, starch, corn starch, crystalline cellulose, light anhydrous silicic acid and the like.

Examples of the lubricant include magnesium stearate, calcium stearate, talc, colloidal silicon dioxide and the like.

Examples of the binder include crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, starch, sucrose, gelatin, methylcellulose, sodium carboxymethylcellulose and the like.

Examples of the disintegrant include starch, carboxymethylcellulose, calcium carboxymethylcellulose, sodium carboxymethylstarch, L-hydroxypropylcellulose and the like.

Examples of the solvent include water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil, olive oil and the like.

Examples of the solubilizing agent include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like.

Examples of the suspending agent include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, lauryl aminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glycerol monostearate and the like; hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose and the like.

Examples of the isotonicizing agent include glucose, D-sorbitol, sodium chloride, glycerin, D-mannitol and the like.

Examples of the buffer agent include buffer solutions such as phosphates, acetates, carbonates, citrates and the like.

Examples of the sedative include benzyl alcohol and the like.

Examples of the preservative include parahydroxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.

Examples of the antioxidant include sulfites, ascorbic acid, α-tocopherol and the like.

Examples of the colorant include water-soluble coal tar food colors (for example, food colors such as Food Red No. 2 and No. 3, Food Yellow No. 4 and No. 5, Food Blue No. 1 and No. 2, and etc.), water-insoluble varnish-forming dyes (for example, aluminum salts of the above-mentioned water-soluble coal tar food dyes), natural dyes (for example, β-carotene, chlorophyll, iron oxide), etc.

Examples of the sweetener include sodium saccharinate, dipotassium glycyrrhizinate, aspartame, stevia and the like.

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Examples of adsorbent agents include porous starch, calcium silicate (trade name: Florite RE), magnesium aluminum metasilicate (trade name: Neusilin) and light anhydrous silicic acid (trade name: Sylysia).

Examples of the wetting agent include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene lauryl ether.

In the process of preparing an oral drug, a coating can be used, if necessary, to mask taste, enteric dissolution properties, or prolonged action.

Examples of a coating base for coating applications include a sugar coating base, an aqueous film coating base, an enteric film coating base, and a sustained release film coating base.

Sucrose is used as the base of the sugar coating. Moreover, one or more types selected from talc, precipitated calcium carbonate, gelatin, gum arabic, pullulan, carnauba wax, and the like. can be used in combination.

Examples of the aqueous film coating base include cellulose polymers such as hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, methylhydroxyethylcellulose, etc.; synthetic polymers such as polyvinyl acetal diethylaminoacetate, aminoalkyl methacrylate copolymer E [Eudragit E (trade name)], polyvinylpyrrolidone, etc.; and polysaccharides such as pullulan, etc.

Examples of the enteric film coating base include cellulose polymers such as hydroxypropyl methyl cellulose phthalate, hydroxypropyl methyl cellulose acetate succinate, carboxymethyl ethyl cellulose, cellulose acetate phthalate, etc.; acrylic polymers such as methacrylic acid copolymer L [Eudragit L (trade name)], methacrylic acid copolymer LD [Eudragit L-30D55 (trade name)], methacrylic acid copolymer S [Eudragit S (trade name)], etc. ; and naturally occurring substances such as shellac, etc.

Examples of the coating base in the form of a sustained release film include cellulose polymers such as ethylcellulose, etc.; and acrylic polymers such as aminoalkyl methacrylate copolymer RS [Eudragit RS (trade name)], ethyl acrylate-methyl methacrylate copolymer suspension [Eudragit NE (trade name)], etc.

The above-mentioned coating bases can be used after mixing two or more types thereof in appropriate proportions. For the coating, for example, a light blocking agent such as titanium oxide, iron oxide and the like can be used.

The dosage of the compound of the present invention is suitably selected depending on the subject of administration, symptom, route of administration and the like. For example, when the compound of the present invention is administered orally to an obese or diabetic patient (body weight 60 kg), the daily dose of the compound of the present invention is from about 0.1 to 100, preferably from about 1.0 to 50, more preferably from about 1 ,0 to 20 mg. When the compound of the present invention is administered parenterally to an obese or diabetic patient (body weight 60 kg), the daily dose of the compound of the present invention is from about 0.01 to 30, preferably from about 0.1 to 20, more preferably from about 0.5 up to 10 mg. These amounts can be administered in about 1 to several parts per day.

The compound of the present invention can be administered, for example, every day (once a day, twice a day, 3 times a day, 4 times a day, 5 times a day, 6 times a day), every 2 days, every 3 days, once every 4 days, once every 5 days, once every 6 days, once a week, twice a week, once every two weeks, once every 3 weeks, once a month, once every 2 months, once every 3 months , once every 4 months, once every 5 months or once every 6 months.

The compound of the present invention can be used in combination with another drug that does not adversely affect the compound of the present invention, for the purpose of, for example, accelerating the action (treating the effect of obesity, diabetes, etc.) of the compound of the present invention, reducing the dose compounds of the present invention, etc.

Examples of a drug that can be used in combination with a compound of the present invention (hereinafter sometimes abbreviated as a concomitant drug) include anti-obesity drugs, diabetes drugs, diabetes complications drugs, hyperlipidemia drugs, antihypertensive drugs, diuretics, chemotherapeutic agents, immunotherapy agents, anti-inflammatory drugs, antithrombotic drugs, osteoporosis drugs, vitamins, anti-dementia drugs, erectile dysfunction drugs, therapeutic drugs for frequent urination or urinary incontinence, drugs for dysuria, etc. Specific examples of concomitant medication include those mentioned below.

Examples of antiobesity agents include monoamine reuptake inhibitors (eg, phentermine, sibutamine, mazindol, fluoxetine, tesofensine), serotonin 2C receptor agonists (eg, lorcaserin), serotonin 6 receptor antagonists, histamine H3 receptor modulator, GABA modulator (eg, topiramate) , neuropeptide Y antagonists (for example, wellneperit), antagonists

- 19 044242 cannabinoid receptors (for example, rimonabant, taranabant), ghrelin antagonists, ghrelin receptor antagonists, ghrelin acylation enzyme inhibitors, opioid receptor antagonists (for example, GSK-1521498), orexin receptor antagonists, melanocortin 4 receptor agonists, Πβ-hydroxy dehydrogenase inhibitors teroids ( e.g., AZD-4017), pancreatic lipase inhibitors (e.g., orlistat, cetilistat), β3-agonists (e.g., N-5984), diacylglycerol acyltransferase 1 (DGAT1) inhibitors, acetyl-CoA carboxylase (ACC) inhibitors, stearoyl enzyme inhibitors -CoA desaturases, microsomal triglyceride transporter protein inhibitors (eg, R-256918), Na-glucose cotransporter inhibitors (eg, JNJ-28431754, remogliflozin), NFk inhibitors (eg, HE-3286), PPAR agonists (eg, GFT -505, DRF-11605), phosphotyrosine phosphatase inhibitors (eg, sodium vanadate, trodusquemine), GPR119 agonists (eg, PSN-821, MBX-2982, APD597), glucokinase activators (eg, AZD-1656), leptin, leptin derivatives (eg, metroleptin), CNTF (ciliary neurotrophic factor), BDNF (brain-derived neurotrophic factor), cholecystokinin agonists, amylin drugs (eg, pramlitinide, AC-2307), neuropeptide Y agonists (eg, PYY3-36, PYY3 derivatives -36, obineptide, TM-30339, TM-30335), oxyntomodulin preparations: FGF21 preparations (for example, animal FGF21 preparations extracted from the pancreas of cows or pigs; human FGF21 preparations genetically synthesized using Escherichia coli or yeast; fragments or derivatives of FGF21), anorexigenic agent (for example, P-57), etc.

In the context of the present invention, for example, insulin preparations (for example, animal insulin preparations extracted from the pancreas of cows and pigs; human insulin preparations genetically synthesized using Escherichia coli or yeast; insulin zinc; protamine) may be mentioned as a medicine for diabetes. insulin zinc; insulin fragment or derivative (eg, INS-1), oral insulin preparation), insulin sensitizers (eg, pioglitazone or a salt thereof (preferably hydrochloride), rosiglitazone or a salt thereof (preferably maleate), metaglidasen, AMG-131 , balaglitazone, MBX-2044, rivoglitazone, aleglitazar, chiglitazar, lobeglitazone, PLX-204, PN-2034, GFT-505, THR-0921, compound described in WO 007/013694, WO 2007/018314, WO 2008/093639 or WO 2008/099794), α-glucosidase inhibitors (e.g. voglibose, acarbose, miglitol, emiglitate), biguanides (e.g. metformin, buformin or its salt (e.g. hydrochloride, fumarate, succinate)), insulin secretagogues (e.g. sulfonylurea (eg tolbutamide, glibenclamide, gliclazide, chlorpropamide, tolazamide, acetohexamide, glycopyramide, glimepiride, glipizide, glibuzol), repaglinide, nateglinide, mitiglinide or their calcium salt hydrate), dipeptidyl peptidase IV inhibitors (eg alogliptin or its salt (preferably benzoate ), valdagliptin, sitagliptin, saxagliptin, BI1356, GRC8200, MP-513, PF-00734200, PHX1149, SK-0403, ALS2-0426, TA-6666, TS-021, KRP-104, trelagliptin or its salt (preferably succinate) ), β3 agonists (eg N-5984), GPR40 agonists (eg Fasiglifam or its hydrate, compound described in WO 2004/041266, WO 2004/106276, WO 2005/063729, WO 2005/063725, WO 2005/087710 , WO 2005/095338, WO 2007/013689 or WO 2008/001931), SGLT2 (sodium-glucose cotransporter 2) inhibitors (e.g. depagliflozin, AVE2268, TS-033, YM543, TA-7284, remogliflozin, ASP1941), SGLT inhibitors 1 , 11β-hydroxysteroid dehydrogenase inhibitors (eg, BVT3498, INCB-13739), adiponectin or its agonist, IKK inhibitors (eg, AS-2868), leptin resistance agents, somatostatin receptor agonists, glucokinase activators (eg, pyragliatin, AZD1656 , AZD6370, TTP-355, compound described in WO 006/112549, WO 007/028135, WO 008/047821, WO 008/050821, WO 008/136428 or WO 008/156757), GPR119 agonists (e.g. PSN82 1, MVH -2982, APD597), FGF21, FGF analog, ACC2 inhibitors, etc.

Aldose reductase inhibitor (e.g., tolrestat, epalrestat, zopolrestat, fidarestat, CT-112, ranirestat (AS-3201), lidorestat), neurotrophic factor, and neurotrophic factor-enhancing agents (e.g., NGF, NT-3, BDNF, neutrotrophic factor production/secretion stimulating agent described in WO 01/14372 (e.g. 4-(4-chlorophenyl)-2-(2-methyl-1-imidazolyl)-5-[3-( 2-methylphenoxy)propyl]oxazole), compound described in WO 2004/039365), PKC inhibitors (e.g. ruboxistaurine mesylate), AGE inhibitors (e.g. ALT946, N-phenacylthiaozolium bromide (ALT766), EXO-226, pyridorine, pyridoxamine ), GABA receptor agonists (eg, gabapentin, pregabalin), serotonin-norepinephrine reuptake inhibitors (eg, duloxetine), sodium channel inhibitors (eg, lacosamide), active oxygen scavengers (eg, thioctic acid), cerebral vasodilators (eg, tiapuride, mexiletine), somatostatin receptor agonists (for example, BIM23190), apoptosis signal-regulating kinase-1 (ASK-1) inhibitors, etc.

As a drug for hyperlipidemia, HMG-COA reductase inhibitors (for example, pravastatin, simvastatin, lovastatin, atorvastatin, fluvastatin, rosuvastatin, pitavastatin or its salt (for example, sodium salt, calcium salt)), squalene synthase inhibitors (for example, compound described in WO 97/10224, for example N-[[(3R,5S)-1-(3-acetoxy-2,2dimethylpropyl)-7-chloro-5-(2,3-dimethoxyphenyl)-2-oxo -1,2,3,5-tetrahydro-4,1-benzoxazepine-3

- 20 044242 yl]acetyl]piperidine-4-acetic acid), fibrate compounds (for example, bezafibrate, clofibrate, symfibrate, clinofibrate), anion exchange resin (for example, cholestyramine), probucol, nicotinic acid-based drugs (for example, Nicomol , niceritrol, niaspan), ethylicosapentate, phytosterol (eg, soy sterol, gamma-oryzanol (γ-oryzanol)), cholesterol absorption inhibitors (eg, zechia), CETP inhibitors (eg, dalcetrapib, anacetrapib), ω-3 fatty acid preparations (for example, ethyl esters of ω-3-fatty acids 90 (ethyl esters of ω-3-acids 90)), etc.

Examples of the antihypertensive agent include angiotensin -converging enzyme inhibitors (for example, captopril, enalapril, delilee, etc.), Angiotensin II antagonists (for example, Candesartan Tsiglesert, Lausartan, Lozartan Kaliy, Evlsartan, Telmisartan, Irbesartan, Taso -Setzerstan, O -Setsenstan, O -Setsenstan. Lmesartan, olmesartan medoxomil, azilsartan, azilsartan medoxomil, etc.), calcium antagonists (for example, manidipine, nifedipine, amlodipine, efonidipine, nicardipine, cilnipidine, etc.), β-blockers (for example, metoprolol, atenolol, propranolol, carvedilol , pindolol, etc.), clonidine, etc.

As a diuretic, for example, xanthine derivatives (for example, theobromine sodium salicylate, theobromine calcium salicylate, etc.), thiazide drugs (for example, ethiazide, cyclopanthiazide, trichloromethiazide, hydrochlorothiazide, hydroflumethiazide, benzylhydrochlorothiazide, penfluthiazide, polythiazide, methyclothiazide) may be mentioned. eid etc.), antialdosterone drugs (for example, spironolactone, triamterene, etc.), carbonic anhydrase inhibitors (for example, acetazolamide, etc.), chlorobenzenesulfonamide drugs (for example, chlorthalidone, mefruzide, indapamide, etc. ), azosemide, isosorbitol, ethacrynic acid, pyretanide, bumetanide, furosemide, etc.

Examples of a chemotherapeutic agent include alkylating agents (e.g., cyclophosphamide, ifosfamide), antimetabolites (e.g., methotrexate, 5-fluorouracil), anticancer agents (e.g., mitomycin, adriamycin), herbal anticancer agents (e.g., vincristine, vindesine, taxol), cisplatin, carboplatin, etoposide, etc. Among others, the 5-fluorouracil derivative furtulon or neofurtulon and the like are preferred.

Examples of immunotherapeutic agents include microbial or bacterial components (eg, muramyl dipeptide derivative, picibanil), polysaccharides having immune system enhancing activity (eg, lentinan, sisophyran, crestin), genetically engineered cytokines (eg, interferon, interleukin (IL)) , colony-stimulating factors (eg, granulocyte colony-stimulating factor, erythropoietin), etc. Among others, interleukins such as IL-1, IL-2, IL-12 and the like are preferred.

Examples of the anti-inflammatory drug include non-steroidal anti-inflammatory drugs such as aspirin, acetaminophen, indomethacin and the like.

As an antithrombotic agent, for example, heparin (for example, heparin sodium, heparin calcium, enoxaparin sodium, dalteparin sodium), warfarin (for example, warfarin potassium), antithrombin drugs (for example, aragatroban, dabigatran), FXa inhibitors (for example, , rivaroxaban, apixaban, edoxaban, YM150, a compound described in WO 02/06234, WO 2004/048363, WO 2005/030740, WO 2005/058823 or WO 2005/113504), thrombolytic agents (eg urokinase, tisokinase, alte plaza, nateplase, monteplase, pamiteplase), platelet aggregation inhibitors (for example, ticlopidine hydrochloride, clopidogrel, prasurgel, E5555, SHC530348, cilostazol, ethylicosapentate, beraprost sodium, sarprogrelate hydrochloride), etc.

Examples of the osteoporosis drug include alfacalcidol, calcitriol, elcatonin, salmon calcitonin, estriol, ipriflavone, pamidronate disodium, alendronate sodium hydrate, incadronate disodium, risedronate disodium and the like.

Examples of the vitamin include vitamin B1, vitamin _B12 and the like.

Examples of the anti-dementia drug include tacrine, donepezil, rivastigmine, galantamine and the like.

Examples of drugs against erectile dysfunction include apomorphine, sildenafil citrate and the like.

Examples of the therapeutic drug for urinary frequency or urinary incontinence include flavoxate hydrochloride, oxybutyrine hydrochloride, propivevrine hydrochloride and the like.

Examples of the drug for dysuria include acetylcholinesterase inhibitors (eg, distigmine) and the like.

Moreover, also in combination with a compound of the present invention, a drug that has been proven to have cachexia mitigation effects, either in animal models or clinically, can be used, i.e. cyclooxygenase inhibitor (eg, indomethacin), progesterone derivative (eg, magestrol acetate), glucocorticoid (eg, dexamethasone), metoclopramide drug, tetrahydrocannabinol drug, fat metabolism enhancer (eg, eicosapentaenoic acid), growth hormone, IGF -1 or an antibody against cachexia-inducing factor TNF-α, LIF, IL-6 or oncostatin M, etc.

- 21 044242

Alternatively, a glycation inhibitor (eg, ALT-711), a nerve regeneration drug (eg, Y-128, VX853, prosaptide), an antidepressant (eg, desipramine, amitriptyline, imipramine), an antiepileptic, may be used in combination with a compound of the present invention. drug (eg, lamotrigine, trileptal, keppra, zonegran, pregabalin, harkoride, carbamazepine), antiarrhythmic drug (eg, mexiletine), acetylcholine receptor ligand (eg, ABT-594), endothelin receptor antagonist (eg, ABT-627) , monoamine uptake inhibitor (eg, tramadol), narcotic analgesic (eg, morphine), GABA receptor agonist (eg, gabapentin, gabapentin MR drug), α2 receptor agonist (eg, clonidine), topical analgesic (eg, capsaicin), antianxiety drug (eg, benzothiazepine), phosphodiesterase inhibitor (eg, sildenafil), dopamine receptor agonist (eg, apomorphine), midazolam, ketoconazole, etc.

The time of administration of the compound of the present invention and the concomitant drug is not limited, and they can be administered to an individual simultaneously or intermittently.

Examples of this route of administration include the following:

(1) administration of one preparation obtained by simultaneous treatment of a compound of the present invention and a concomitant drug, (2) simultaneous administration of two types of preparations of a compound of the present invention and a concomitant drug that were obtained separately by the same route of administration, ( 3) administering two types of preparations of the compound of the present invention and the concomitant drug, which were obtained separately, by one administration route, (4) simultaneous administration of two types of preparations of the compound of the present invention and the concomitant drug, which were obtained separately, by different routes of administration, (5) administration of two types of preparations of the compound of the present invention and the concomitant drug, which are obtained separately, by different routes of administration with a break (for example, administration in the order of the compound of the present invention and the concomitant drug, or in reverse order), and etc.

The dose of the concomitant drug can be appropriately determined based on the dose used in clinical situations. The mixing ratio of the compound of the present invention and the concomitant drug can be suitably determined depending on the subject of administration, symptom, route of administration, target disease, combination and the like. When the subject of administration is a human, for example, the concomitant drug can be used in an amount of 0.01 to 100 parts by weight. relative to 1 part by weight compounds of the present invention.

By combining a compound of the present invention and a concomitant drug:

(1) the dose of the compound of the present invention or the concomitant drug can be reduced compared with the administration of the compound of the present invention or the concomitant drug, (2) the drug to be used in combination with the compound of the present invention can be selected depending on the condition of the patients (mild , severe, etc.), (3) the treatment period can be extended by selecting a concomitant drug having an action and mechanism different from the action and mechanism of the compound of the present invention, (4) the effect of continuous treatment can be achieved by selecting a concomitant a drug having an action and mechanism different from the action and mechanism of the compound of the present invention, (5) a synergistic effect can be achieved by combining the use of the compound of the present invention and a concomitant drug, etc.

Examples

The abbreviations used in this description mean the following (Table 1-1 and Table 1-2). The hyphen in terms such as α-MePhe and the like as described herein may be omitted, and the case of absence has the same meaning.

- 22 044242

Table 1-1

Aad 2-Aminoadipic acid Abu 2-aminobutyric acid Abz (2) 2-aminobenzoic acid Ac acetyl Acp 6-aminocaproic acid Acpc 1-Aminocyclopropanecarboxylic acid Adc(12) 12-Aminododecanoic acid Aib α-aminoisobutyric acid Aipe 3-aminobutyric acid Ala(4Pip) 4-piperidinylalanine Ala(cPr) cyclopropylalanine Alb Albizziin 2-amino-3-ureidopropionic acid Amb z(4) 4-aminomethylbenzoyl Aoc(8) 8-aminocaprylic acid Arg(Me) Νω-methylarginine Asn(Me) Νω-methylasparagine Aze (2) azetidine-2-carboxylic acid Aze (3) azetidine-3-carboxylic acid CC(Acp) 6-carboxypentylcarbamoyl CC(β-Ala) 2-carboxyethylcarbamoyl CC (GABA) 3-carboxypropylcarbamoyl CC(Gly) carboxymethylcarbamoyl CC(Leu) [(1S)-1-carboxy-3-methylbutyl]carbamoyl CC(Ser) [(1S)-1-carboxy-2-hydroxyethyl]carbamoyl CC(Tyr) [(1S)-1-carboxy-2-(4hydroxyphenyl)ethyl]carbamoyl Cha cyclohexylalanine cisHyp cis-4-hydroxyproline Cit citrulline cPrCO cyclopropanecarbonyl Dab 2,4-diaminobutyric acid Dap 2,3-diaminopropionic acid GABA γ-aminobutyric acid Gly(cPr) cyclopropylglycine Gly- Ψΐ (E)CH=CH]-Leu the -CONH- bond between Gly and Leu is replaced by an (E)-type alkene Har homoarginine homoLeu homoleucine Hse homoserine Hyp trans-4-hydroxyproline Iva isovaline Leu(Me) γ-methylleucine Lys(Ac) Νε-acetyllysine Lys(Hexyl) Νε-hexylysine Lys(Me) Νε-methyllysine Lys(Me2) Νε, ε-dimethyllysine

- 23 044242

Table 1-2

Lys[Hexadecanoyl -(PEG2)] - rnr i^ ^cm · N(2- N-(2-hydroxyethyl)glycine hydroxyethyl)Gly N(iBu)Gly N-isobutylglycine Nai (1) 1-naphthylalanine Nai (2) 2-naphthylalanine Nar Mogarginin Nie Norleucine NmeAla Να-methylalanine NmeSer Να-methylserine NmePhe Να-methylphenylalanine Nva Norvaline Orn ornithine PEG2 A,,xJ NH ho P Y Phe(2,6-Me ₂ )lat 2,6-dimethylphenylalanine Phe (2F) 2-fluorophenylalanine Phe(2Me) 2-methylphenylalanine Phe(3F) 3-fluorophenylalanine Phe(3Me) 3-methylphenylalanine Phe(4C1) 4-chlorophenylalanine Phe (4F) 4-fluorophenylalanine Phe(4Me) 4-methylphenylalanine Phe( _4NH2 ) 4-aminophenylalanine Phg phenylglycine Pic (2) 2-piperidinecarboxylic acid Pic (4) 4-piperidinecarboxylic acid Pya(2) 2-pyridylalanine Pya(3) 3-pyridylalanine Pya(4) 4-pyridylalanine 4-PyCO 4-pyridylcarbonyl Ser (Me) 0—methylserine Thp(4) tetrahydro-2H-pyran-4-yl Thr (Me) 0-methylthreonine threo-PhSer threo-3-phenylserine 1,2,3,4-tetrahydroisoquinoline-3- Tic carboxylic acid Tyr (2F) 2-fluorotyrosine Tyr (3F) 3-fluorotyrosine Tyr(Me) 0-methyltyrosine Z benzyloxycarbonyl a-MePhe a-methylphenylalanine a-MePro α-methylproline β-Ala β-alanine β-HOala β-homoalanine

In the description, when bases, amino acids, etc. indicated by their codes, they are based on generally accepted codes in accordance with the IUPAC-IUB Commission on Biochemical Nomenclature or on known codes in the field, examples of which are presented below. For amino acids that may have an optical isomer, the L form is present unless otherwise indicated (eg, Ala is the L form of Ala). In addition, D- means the D-form (eg, D-Ala is the D-form of Ala), and DL- means the racemate of the D-form and the L-form (eg, DL-Ala is the DL-racemate of Ala).

TFA: trifluoroacetic acid;

Gly or G: glycine;

Ala or A: alanine;

- 24 044242

Val or V:valine;

Leu or L: leucine;

Ile or I: isoleucine;

Ser or S: serine;

Thr or T: threonine;

Cys or C: cysteine;

Met or M: methionine;

Glu or E: glutamic acid;

Asp or D: aspartic acid;

Lys or K: lysine;

Arg or R: arginine;

His or H: histidine;

Phe or F: phenylalanine;

Tyr or Y: tyrosine;

Tip or W: tryptophan;

Pro or P: proline;

Asn or N: asparagine;

Gln or Q: glutamine;

pGlu: pyroglutamic acid;

α-MeTyr: α-methyltyrosine.

The present invention is explained in detail below using the following reference examples, examples, test examples and composition examples, which are only embodiments and should not be construed as limiting. Moreover, the present invention may be modified without departing from the scope of the invention.

The term room temperature in the examples below indicates a range typically from about 10 to about 35°C. As for %, the yield is the yield in mol/mol%, the solvent used for chromatography is represented in % by volume, and otherwise the % is wt%.

THF: tetrahydrofuran;

DMF: N,N-dimethylformamide;

WSC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride;

HOBt: 1-hydroxybenzotriazole monohydrate.

Reference example 1.

Synthesis of H-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)Gly-Ala- Pro-Pro-Pro-Ser(tBu)-Lys(Boc)-amide resin Sieber (SEQ ID NO: 10).

Sieber amide resin (0.69 meq/g, 362 mg) was added to the reaction tube, which was then loaded into the peptide synthesis apparatus. Amino acids were sequentially condensed according to the Fmoc/DCC/HOBt protocol. In the last step, the N-terminal Fmoc group was removed. After condensation was completed, the resin was washed with MeOH and dried under reduced pressure. The result was 1025 mg (0.244 meq/g) of the protected peptide resin of interest.

Reference example 2.

Synthesis of H-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)Gl·y- Ala-Pro-Pro-Pro-Ser(tBu)-amide resin Sieber (SEQ ID NO: 11).

Sieber amide resin (0.69 meq/g, 362 mg) was added to the reaction tube, which was then loaded into the peptide synthesis apparatus. Amino acids were sequentially condensed according to the Fmoc/DCC/HOBt protocol. In the last step, the N-terminal Fmoc group was removed. After condensation was completed, the resin was washed with MeOH and dried under reduced pressure. The result was 1331 mg (0.188 meq/g) of the protected peptide resin of interest.

Reference example 3.

Synthesis of H-Ala-Gln(Trt)-Ala-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-ProSer(tBu)-Ser (tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc)-amide resin Sieber (SEQ ID NO: 12).

Sieber amide resin (0.61 meq/g, 410 mg) was added to the reaction tube, which was then loaded into the peptide synthesis apparatus. Amino acids were sequentially condensed according to the Fmoc/DCC/HOBt protocol. A double coupling reaction was carried out to introduce Ala at the 18th and 20th positions and Gln(Trt) at the 19th position. In the last step, the N-terminal Fmoc group was removed. After condensation was completed, the resin was washed with MeOH and dried under reduced pressure. The result was 1110 mg (0.225 meq/g) of the protected peptide resin of interest.

Example 1.

Synthesis of H-Tyr-Aib-Glu-Gly-Thr-aMePhe-Thr-Ser-Asp-Tyr-Ala-Ile-Aib-Leu-Asp-Lys-Gln-Ala-Gln-AlaGlu-Phe-Val-Lys-Trp -Leu-Leu-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys-NH2 (SEQ ID NO: 13).

- 25 044242

H-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-AlaPro- Sieber Pro-Pro-Ser(tBu)-Lys(Boc)-amide resin (0.244 meq/g, 41.0 mg) prepared according to Reference Example 1 was weighed into a reaction tube and swollen with DMF. After removing the DMF by filtration, Fmoc-Ala-OH (31.1 mg), 0.5 M OxymaPure in DMF (200 μL), and diisopropylcarbodiimide (15.9 μL) were added sequentially to the resin, and then the mixture was shaken for 1.5 h The reaction solution was filtered, and then the resin was washed with DMF 6 times. After confirmation of Kaiser test negativity, 20% piperidine in DMF was added and the mixture was shaken for 1 min. The solution was filtered and then 20% piperidine in DMF was added again and the mixture was shaken for 20 minutes. The solution was filtered and the resin was washed 10 times with DMF. This cycle of condensation of an amino acid with Fmoc - removal of the Fmoc protecting group was repeated for the sequential condensation of Gln(Trt), Ala, Gln(Trt), Lys(Boc), Asp(OtBu), Leu, Aib, Ile*, Ala, Tyr(tBu) , Asp(OtBu), Ser(tBu), Thr(tBu), aMePhe, Thr(tBu)*, Gly, Glu(OtBu), Aib and Tyr(tBu) (*: overnight reaction). The resin was washed with MeOH and then dried under reduced pressure to obtain 83 mg of H-Tyr(tBu)-AibGlu(OtBu)-Gly-Thr(tBu)-aMePhe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Tyr(tBu)-Ala-Ile-Aib-Leu-Asp(OtBu)Lys(Boc)-Gln(Trt)-Ala-Gln(Trt)-Ala-Glu(OtBu)-Phe-Val-Lys(Boc)- Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-ProSer(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc)-amide resin Sieber.

To 83 mg of the resulting resin, 1 ml of a mixture of TFA: m-cresol: thioanisole: ethanedhiol: H ₂ O: triisopropylsilane (80: 5: 5: 5: 2.5: 2.5) was added and the mixture was stirred for 1.5 h Diethyl ether was added to the reaction solution to ensure precipitation, and the precipitate was washed by repeating the action of removing the supernatant after centrifugation three times. The precipitate was extracted with 50% aqueous acetic acid and the resin was removed by filtration, followed by preparative HPLC using a Daisopak SP-100-5-ODS-P column (250x20 mm ID) [solution A: 0.1% TFA-water, solution B : acetonitrile containing 0.1% TFA, flow rate 8 ml/min, A/B: linear concentration gradient elution 65/35-55/45 (60 min)]. Fractions containing the desired product were collected and lyophilized to obtain 22.5 mg of white powder.

Mass spectrometry, (M+H)+ 4267.4 (calculated: 4267.2);

HPLC elution time: 7.1 min;

elution conditions:

Column: Merck Chromolith Performance RP-18e (100x4.6 mm I.D.);

eluent: using solution A: 0.1% TFA-water, solution B: acetonitrile containing 0.1% TFA, A/B: elution with a linear concentration gradient 95/5-35/65 (10 min);

flow rate: 3.0 ml/min.

Example 2.

Synthesis of H-Tyr-Aib-Glu-Gly-Thr-aMePhe-Thr-Ser-Asp-Tyr-Aib-Ile-Aib-Leu-Asp-Lys-Gln-Ala-Gln-AlaGlu-Phe-Val-Lys-Trp -Leu-Leu-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys-NH ₂ (SEQ ID NO: 14).

H-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-AlaPro- Sieber Pro-Pro-Ser(tBu)-Lys(Boc)-amide resin (0.244 meq/g, 41.0 mg) prepared according to Reference Example 1 was weighed into a reaction tube and swollen with DMF. After removing the DMF by filtration, Fmoc-Ala-OH (31.1 mg), 0.5 M Oxymapure in DMF (200 µl) and diisopropylcarbodiimide (15.9 µl) were added sequentially to the resin, and then the mixture was shaken for 1.5 h The reaction solution was filtered, and then the resin was washed with DMF 6 times. After confirmation of Kaiser test negativity, 20% piperidine in DMF was added and the mixture was shaken for 1 min. The solution was filtered and then 20% piperidine in DMF was added again and the mixture was shaken for 20 minutes. The solution was filtered and the resin was washed 10 times with DMF. This cycle of condensation of an amino acid with Fmoc - removal of the Fmoc protecting group was repeated for the sequential condensation of Gln(Trt), Ala, Gln(Trt), Lys (Boc), Asp (OtBu), Leu, Aib, Ile*, Aib, Tyr(tBu) , Asp(OtBu), Ser(tBu), Thr(tBu), aMePhe, Thr(tBu)*, Gly, Glu(OtBu), Aib and Tyr(tBu) (*: overnight reaction). The resin was washed with MeOH and then dried under reduced pressure to obtain 84.5 mg of HTyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-aMePhe-Thr(tBu)-Ser(tBu)-Asp(OtBu )-Tyr(tBu)-Aib-Ile-Aib-LeuAsp(OtBu)-Lys(Boc)-Gln(Trt)-Ala-Gln(Trt)-Ala-Glu(OtBu)-Phe-Val-Lys(Boc) -Trp(Boc)-Leu-Leu-Lys(Boc)Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc)- Sieber amide resin.

To 84.5 mg of the resulting resin, 1 ml of a mixture of TFA: m-cresol: thioanisole: ethanedhiol: H ₂ O: triisopropylsilane (80: 5: 5: 5: 2.5: 2.5) was added, and the mixture was stirred for 1 5 h. Diethyl ether was added to the reaction solution to ensure precipitation, and the precipitate was washed by repeating the action of removing the supernatant after centrifugation three times. The precipitate was extracted with 50% aqueous acetic acid and the resin was removed by filtration, followed by preparative HPLC using a Daisopak SP-100-5-ODS-P column (250x20 mm ID) [solution A: 0.1% TFA-water, solution B : acetonitrile containing 0.1% TFA, flow rate 8 ml/min, A/B: linear concentration gradient elution 65/35-55/45 (60 min)]. Fractions containing the desired product were collected and lyophilized to obtain 21.5 mg of white powder.

Mass spectrometry, (M+H)+ 4281.5 (calculated: 4281.2);

- 26 044242 HPLC elution time: 7.2 min;

elution conditions:

Column: Merck Chromolith Performance RP-18e (100x4.6 mm I.D.);

eluent: using solution A: 0.1% TFA-water, solution B: acetonitrile containing 0.1% TFA, A/B: elution with a linear concentration gradient 95/5-35/65 (10 min);

flow rate: 3.0 ml/min.

Example 3.

Synthesis of H-Tyr-Aib-Glu-Gly-Thr-aMePhe-Thr-Ser-Asp-Tyr-Ala-Ile-Aib-Leu-Asp-Lys-Gln-Ala-Gln-SerGlu-Phe-Val-Lys-Trp -Leu-Leu-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys-NH ₂ (SEQ ID NO: 15).

H-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-AlaPro- Sieber Pro-Pro-Ser(tBu)-Lys(Boc)-amide resin (0.244 meq/g, 41.0 mg) prepared according to Reference Example 1 was weighed into a reaction tube and swollen with DMF. After removing the DMF by filtration, Fmoc-Ser(tBu)-OH (38.3 mg), 0.5 M Oxymapure in DMF (200 μL), and diisopropylcarbodiimide (15.9 μL) were added sequentially to the resin, and then the mixture was shaken for 1 .5 hours. The reaction solution was filtered, and then the resin was washed 6 times with DMF. After confirmation of Kaiser test negativity, 20% piperidine in DMF was added and the mixture was shaken for 1 min. The solution was filtered and then 20% piperidine in DMF was added again and the mixture was shaken for 20 minutes. The solution was filtered and the resin was washed 10 times with DMF. This cycle of condensation of an amino acid with Fmoc - removal of the Fmoc protecting group was repeated for the sequential condensation of Gln(Trt), Ala, Gln(Trt), Lys(Boc), Asp(OtBu), Leu, Aib, Ile*, Ala, Tyr(tBu) , Asp(OtBu), Ser(tBu), Thr(tBu), aMePhe, Thr(tBu)*, Gly, Glu(OtBu), Aib and Tyr(tBu) (*: overnight reaction). The resin was washed with MeOH and then dried under reduced pressure to obtain 73.3 mg of HTyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-aMePhe-Thr(tBu)-Ser(tBu)-Asp(OtBu )-Tyr(tBu)-Ala-Ile-Aib-LeuAsp(OtBu)-Lys(Boc)-Gln(Trt)-Ala-Gln(Trt)-Ser(tBu)-Glu(OtBu)-Phe-Val-Lys (Boc)-Trp(Boc)-Leu-LeuLys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc )-amide resin Sieber.

To 73.3 mg of the resulting resin, 1 ml of a mixture of TFA: m-cresol: thioanisole: ethanedhiol: H ₂ O: triisopropylsilane (80: 5: 5: 5: 2.5: 2.5) was added, and the mixture was stirred for 1 5 h. Diethyl ether was added to the reaction solution to ensure precipitation, and the precipitate was washed by repeating the action of removing the supernatant after centrifugation three times. The precipitate was extracted with 50% aqueous acetic acid and the resin was removed by filtration, followed by preparative HPLC using a Daisopak SP-100-5-ODS-P column (250x20 mm ID) [solution A: 0.1% TFAwater, solution B: acetonitrile , containing 0.1% TFA, flow rate 8 ml/min, A/B: linear concentration gradient elution 65/35-55/45 (60 min)]. Fractions containing the desired product were collected and lyophilized to obtain 17.1 mg of white powder.

Mass spectrometry, (M+H)+ 4283.6 (calculated: 4283.2);

HPLC elution time: 7.1 min;

elution conditions:

Column: Merck Chromolith Performance RP-18e (100x4.6 mm I.D.);

eluent: using solution A: 0.1% TFA-water, solution B: acetonitrile containing 0.1% TFA, A/B: elution with a linear concentration gradient 95/5-35/65 (10 min);

flow rate: 3.0 ml/min.

Example 4.

Synthesis of H-Tyr-Aib-Glu-Gly-Thr-aMePhe-Thr-Ser-Asp-Tyr-Aib-Ile-Aib-Leu-Asp-Lys-Gln-Ala-Gln-SerGlu-Phe-Val-Lys-Trp -Leu-Leu-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys-NH ₂ (SEQ ID NO: 16).

H-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-AlaPro- Sieber Pro-Pro-Ser(tBu)-Lys(Boc)-amide resin (0.244 meq/g, 41.0 mg) prepared according to Reference Example 1 was weighed into a reaction tube and swelled with DMF. After removing the DMF by filtration, Fmoc-Ser(tBu)-OH (38.3 mg), 0.5 M Oxymapure in DMF (200 μL), and diisopropylcarbodiimide (15.9 μL) were added sequentially to the resin, and then the mixture was shaken for 1 .5 hours. The reaction solution was filtered, and then the resin was washed 6 times with DMF. After confirmation of Kaiser test negativity, 20% piperidine in DMF was added and the mixture was shaken for 1 min. The solution was filtered and then 20% piperidine in DMF was added again and the mixture was shaken for 20 minutes. The solution was filtered and the resin was washed 10 times with DMF. This cycle of condensation of an amino acid with Fmoc - removal of the Fmoc protecting group was repeated for the sequential condensation of Gln(Trt), Ala, Gln(Trt), Lys(Boc), Asp(OtBu), Leu, Aib, Ile*, Aib, Tyr(tBu) , Asp(OtBu), Ser(tBu), Thr(tBu), aMePhe, Thr(tBu)*, Gly, Glu(OtBu), Aib and Tyr(tBu) (*: overnight reaction). The resin was washed with MeOH and then dried under reduced pressure to obtain 63 mg of HTyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-aMePhe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Tyr(tBu)-Aib-Ile-Aib-LeuAsp(OtBu)-Lys(Boc)-Gln(Trt)-Ala-Gln(Trt)-Ser(tBu)-Glu(OtBu)-Phe-Val-Lys(Boc )-Trp(Boc)-Leu-LeuLys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc)- Sieber amide resin.

To 63 mg of the resulting resin was added 1 ml of a mixture of TFA: m-cresol: thioanisole: ethanedhiol: H2O: three

- 27 044242 isopropylsilane (80:5:5:5:2.5:2.5) and the mixture was stirred for 1.5 hours. Diethyl ether was added to the reaction solution to allow precipitation and the precipitate was washed by repeating the removal action three times supernatant after centrifugation. The precipitate was extracted with 50% aqueous acetic acid and the resin was removed by filtration, followed by preparative HPLC using a Daisopak SP-100-5-ODS-P column (250x20 mm I.D.) [solution A: 0.1% TFA-water, solution B : acetonitrile containing 0.1% TFA, flow rate 8 ml/min, A/B: linear concentration gradient elution 64/36-54/46 (60 min)]. Fractions containing the desired product were collected and lyophilized to obtain 14.7 mg of white powder.

Mass spectrometry, (M+H)+ 4297.8 (calculated: 4297.2);

HPLC elution time: 7.2 min;

elution conditions:

Column: Merck Chromolith Performance RP-18e (100x4.6 mm I.D.);

eluent: using solution A: 0.1% TFA-water, solution B: acetonitrile containing 0.1% TFA, A/B: elution with a linear concentration gradient 95/5 - 35/65 (10 min);

flow rate: 3.0 ml/min.

Example 5.

Synthesis of H-Tyr-Aib-Glu-Gly-Thr-aMePhe-Thr-Ser-Asp-Tyr-Ala-Ile-Aib-Leu-Asp-Lys-Gln-Ala-Gln-AlaGlu-Phe-Val-Lys-Trp -Leu-Leu-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ (SEQ ID NO: 17).

H-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-AlaPro- Sieber Pro-Pro-Ser(tBu)-amide resin (0.188 meq/g, 53.2 mg) prepared according to Reference Example 2 was weighed into a reaction tube and swollen with DMF. After removing the DMF by filtration, Fmoc-Ala-OH (31.1 mg), 0.5 M Oxymapure in DMF (200 µl) and diisopropylcarbodiimide (15.9 µl) were added sequentially to the resin, and then the mixture was shaken for 1.5 h The reaction solution was filtered, and then the resin was washed with DMF 6 times. After confirmation of Kaiser test negativity, 20% piperidine in DMF was added and the mixture was shaken for 1 min. The solution was filtered and then 20% piperidine in DMF was added again and the mixture was shaken for 20 minutes. The solution was filtered and the resin was washed 10 times with DMF. This cycle of condensation of an amino acid with Fmoc - removal of the Fmoc protecting group was repeated for the sequential condensation of Gln(Trt), Ala, Gln(Trt), Lys(Boc), Asp(OtBu), Leu, Aib, Ile*, Ala, Tyr(tBu) , Asp(OtBu), Ser(tBu), Thr(tBu), aMePhe, Thr(tBu)*, Gly, Glu(OtBu), Aib and Tyr(tBu) (*: overnight reaction). The resin was washed with MeOH and then dried under reduced pressure to obtain 97 mg of H-Tyr(tBu)-AibGlu(OtBu)-Gly-Thr(tBu)-aMePhe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Tyr(tBu)-Ala-Ile-Aib-Leu-Asp(OtBu)Lys(Boc)-Gln(Trt)-Ala-Gln(Trt)-Ala-Glu(OtBu)-Phe-Val-Lys(Boc)- Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-ProSer(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-amide resin Sieber.

To 97 mg of the resulting resin, 1 ml of a mixture of TFA: m-cresol: thioanisole: ethanedhiol: H ₂ O: triisopropylsilane (80: 5: 5: 5: 2.5: 2.5) was added and the mixture was stirred for 1.5 h Diethyl ether was added to the reaction solution to ensure precipitation, and the precipitate was washed by repeating the action of removing the supernatant after centrifugation three times. The precipitate was extracted with 50% aqueous acetic acid and the resin was removed by filtration, followed by preparative HPLC using a Daisopak SP-100-5-ODS-P column (250x20 mm ID) [solution A: 0.1% TFAwater, solution B: acetonitrile , containing 0.1% TFA, flow rate 8 ml/min, A/B: linear concentration gradient elution 63/37-53/47 (60 min)]. Fractions containing the desired product were collected and lyophilized to obtain 25.2 mg of white powder.

Mass spectrometry, (M+H)+ 4139.2 (calculated: 4139.1);

HPLC elution time: 7.3 min;

elution conditions:

Column: Merck Chromolith Performance RP-18e (100x4.6 mm I.D.);

eluent: using solution A: 0.1% TFA-water, solution B: acetonitrile containing 0.1% TFA, A/B: elution with a linear concentration gradient 95/5-35/65 (10 min);

flow rate: 3.0 ml/min.

Example 6.

Synthesis of H-Tyr-Aib-Glu-Gly-Thr-aMePhe-Thr-Ser-Asp-Tyr-Aib-Ile-Aib-Leu-Asp-Lys-Gln-Ala-Gln-AlaGlu-Phe-Val-Lys-Trp -Leu-Leu-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ (SEQ ID NO: 18).

H-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-AlaPro- Sieber Pro-Pro-Ser(tBu)-amide resin (0.188 meq/g, 53.2 mg) prepared according to Reference Example 2 was weighed into a reaction tube and swollen with DMF. After removing the DMF by filtration, Fmoc-Ala-OH (31.1 mg), 0.5 M Oxymapure in DMF (200 µl) and diisopropylcarbodiimide (15.9 µl) were added sequentially to the resin, and then the mixture was shaken for 1.5 h The reaction solution was filtered, and then the resin was washed with DMF 6 times. After confirmation of Kaiser test negativity, 20% piperidine in DMF was added and the mixture was shaken for 1 min. The solution was filtered, and then a 20% solution of piperidine in DMF was added again, and the mixture

- 28 044242 shaken for 20 minutes. The solution was filtered and the resin was washed 10 times with DMF. This cycle of condensation of an amino acid with Fmoc - removal of the Fmoc protecting group was repeated for the sequential condensation of Gln(Trt), Ala, Gln(Trt), Lys(Boc), Asp(OtBu), Leu, Aib, Ile*, Aib, Tyr(tBu) , Asp(OtBu), Ser(tBu), Thr(tBu), aMePhe, Thr(tBu)*, Gly, Glu(OtBu), Aib and Tyr(tBu) (*: overnight reaction). The resin was washed with MeOH and then dried under reduced pressure to obtain 78.1 mg of H-Tyr(tBu)-AibGlu(OtBu)-Gly-Thr(tBu)-aMePhe-Thr(tBu)-Ser(tBu)-Asp(OtBu )-Tyr(tBu)-Aib-ne-Aib-Leu-Asp(OtBu)Lys(Boc)-Gln(Trt)-Ala-Gln(Trt)-Ala-Glu(OtBu)-Phe-Val-Lys(Boc )-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-ProSer(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Sieber amide resin.

To 78.1 mg of the resulting resin, 1 ml of a mixture of TFA: m-cresol: thioanisole: ethanedhiol: H ₂ O: triisopropylsilane (80: 5: 5: 5: 2.5: 2.5) was added and the mixture was stirred for 1. 5 hours. Diethyl ether was added to the reaction solution to allow precipitation, and the precipitate was washed by repeating the action of removing the supernatant after centrifugation three times. The precipitate was extracted with 50% aqueous acetic acid and the resin was removed by filtration, followed by preparative HPLC using a Daisopak SP-100-5-ODS-P column (250x20 mm ID) [solution A: 0.1% TFA-water, solution B : acetonitrile containing 0.1% TFA, flow rate 8 ml/min, A/B: linear concentration gradient elution 63/37-53/47 (60 min)]. Fractions containing the desired product were collected and lyophilized to obtain 18.4 mg of white powder.

Mass spectrometry, (M+H)+ 4152.9 (calculated: 4153.1);

HPLC elution time: 7.4 min;

elution conditions:

Column: Merck Chromolith Performance RP-18e (100x4.6 mm I.D.);

eluent: using solution A: 0.1% TFA-water, solution B: acetonitrile containing 0.1% TFA, A/B: elution with a linear concentration gradient 95/5-35/65 (10 min);

flow rate: 3.0 ml/min.

Example 7.

Synthesis of H-Tyr-Aib-Glu-Gly-Thr-aMePhe-Thr-Ser-Asp-Tyr-Ala-Ile-Aib-Leu-Asp-Lys-Gln-Ala-Gln-SerGlu-Phe-Val-Lys-Trp -Leu-Leu-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ (SEQ ID NO: 19).

H-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-AlaPro- Sieber Pro-Pro-Ser(tBu)-amide resin (0.188 meq/g, 53.2 mg) prepared according to Reference Example 2 was weighed into a reaction tube and swollen with DMF. After removing the DMF by filtration, Fmoc-Ser(tBu)-OH (38.3 mg), 0.5 M Oxymapure in DMF (200 μL), and diisopropylcarbodiimide (15.9 μL, 0.1 mmol) were added sequentially to the resin, followed by the mixture was shaken for 1.5 hours. The reaction solution was filtered, and then the resin was washed 6 times with DMF. After confirmation of Kaiser test negativity, 20% piperidine in DMF was added and the mixture was shaken for 1 min. The solution was filtered and then 20% piperidine in DMF was added again and the mixture was shaken for 20 minutes. The solution was filtered and the resin was washed 10 times with DMF. This cycle of condensation of an amino acid with Fmoc - removal of the Fmoc protecting group was repeated for the sequential condensation of Gln(Trt), Ala, Gln(Trt), Lys (Boc), Asp (OtBu), Leu, Aib, Ile*, Ala, Tyr(tBu) , Asp(OtBu), Ser(tBu), Thr(tBu), aMePhe, Thr(tBu)*, Gly, Glu(OtBu), Aib and Tyr(tBu) (*: overnight reaction). The resin was washed with MeOH and then dried under reduced pressure to obtain 87 mg of HTyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-aMePhe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Tyr(tBu)-Ala-Ile-Aib-LeuAsp(OtBu)-Lys(Boc)-Gln(Trt)-Ala-Gln(Trt)-Ser(tBu)-Glu(OtBu)-Phe-Val-Lys(Boc )-Trp(Boc)-Leu-LeuLys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Sieber amide resin.

To 87 mg of the resulting resin, 1 ml of a mixture of TFA: m-cresol: thioanisole: ethanedhiol: H2O: triisopropylsilane (80:5:5:5:2.5:2.5) was added, and the mixture was stirred for 1.5 hours. Diethyl ether was added to the reaction solution to ensure precipitation, and the precipitate was washed by repeating the action of removing the supernatant after centrifugation three times. The precipitate was extracted with 50% aqueous acetic acid and the resin was removed by filtration, followed by preparative HPLC using a Daisopak SP-100-5-ODS-P column (250x20 mm I.D.) [solution A: 0.1% TFA-water, solution B : acetonitrile containing 0.1% TFA, flow rate 8 ml/min, A/B: linear concentration gradient elution 65/35-55/45 (60 min)]. Fractions containing the desired product were collected and lyophilized to obtain 17 mg of white powder.

Mass spectrometry, (M+H)+ 4154.8 (calculated: 4155.1);

HPLC elution time: 7.3 min;

elution conditions:

Column: Merck Chromolith Performance RP-18e (100x4.6 mm I.D.);

eluent: using Solution A: 0.1% TFA-water, solution B: acetonitrile containing 0.1% TFA, A/B: 95/5-35/65 linear concentration gradient elution (10 min);

flow rate: 3.0 ml/min.

Example 8.

Synthesis of H-Tyr-Aib-Glu-Gly-Thr-aMePhe-Thr-Ser-Asp-Tyr-Aib-Ile-Aib-Leu-Asp-Lys-Gln-Ala-Gln-Ser- 29 044242

Glu-Phe-Val-Lys-TIp-Leu-Leu-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2 (SEQ ID NO: 20).

H-Glu(OtBu)-Phe-Val-Lys(Boc)-TIp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-AlaPro- Sieber Pro-Pro-Ser(tBu)-amide resin (0.188 meq/g, 53.2 mg) prepared according to Reference Example 2 was weighed into a reaction tube and swelled with DMF. After removing the DMF by filtration, Fmoc-Ser(tBu)-OH (38.3 mg), 0.5 M Oxymapure in DMF (200 μL), and diisopropylcarbodiimide (15.9 μL) were added sequentially to the resin, and then the mixture was shaken for 1 .5 hours. The reaction solution was filtered, and then the resin was washed 6 times with DMF. After confirmation of Kaiser test negativity, 20% piperidine in DMF was added and the mixture was shaken for 1 min. The solution was filtered and then 20% piperidine in DMF was added again and the mixture was shaken for 20 minutes. The solution was filtered and the resin was washed 10 times with DMF. This cycle of condensation of an amino acid with Fmoc - removal of the Fmoc protecting group was repeated for the sequential condensation of Gln(Trt), Ala, Gln(Trt), Lys(Boc), Asp(OtBu), Leu, Aib, Ile*, Aib, Tyr(tBu) , Asp(OtBu), Ser(tBu), Thr(tBu), aMePhe, Thr(tBu)*, Gly, Glu(OtBu), Aib and Tyr(tBu) (*: overnight reaction). The resin was washed with MeOH and then dried under reduced pressure to obtain 76.8 mg of H-Tyr(tBu)-AibGlu(OtBu)-Gly-Thr(tBu)-aMePhe-Thr(tBu)-Ser(tBu)-Asp(OtBu )-Tyr(tBu)-Aib-Ile-Aib-Leu-Asp(OtBu)Lys(Boc)-Gln(Trt)-Ala-Gln(Trt)-Ser(tBu)-Glu(OtBu)-Phe-Val- Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-GlyPro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-αmide resin Sieber.

To 76.8 mg of the resulting resin, 1 ml of a mixture of TFA: m-cresol: thioanisole: ethanedhiol: H ₂ O: triisopropylsilane (80: 5: 5: 5: 2.5: 2.5) was added, and the mixture was stirred for 1 5 h. Diethyl ether was added to the reaction solution to ensure precipitation, and the precipitate was washed by repeating the action of removing the supernatant after centrifugation three times. The precipitate was extracted with 50% aqueous acetic acid and the resin was removed by filtration, followed by preparative HPLC using a Daisopak SP-100-5-ODS-P column (250x20 mm ID) [solution A: 0.1% TFA-water, solution B : acetonitrile containing 0.1% TFA, flow rate 8 ml/min, A/B: linear concentration gradient elution 63/37-53/47 (60 min)]. Fractions containing the desired product were collected and lyophilized to obtain 16.6 mg of white powder.

Mass spectrometry, (M+H)+ 4169.2 (calculated: 4169.1);

HPLC elution time: 7.4 min;

elution conditions:

Column: Merck Chromolith Performance RP-18e (100x4.6 mm I.D.);

eluent: using solution A: 0.1% TFA-water, solution B: acetonitrile containing 0.1% TFA, A/B: elution with a linear concentration gradient 95/5-35/65 (10 min);

flow rate: 3.0 ml/min.

Example 9.

Synthesis of H-Tyr-Aib-Glu-Gly-Thr-aMePhe-Thr-Ser-Asp-Tyr-Aib-Lys-Aib-Leu-Asp-Lys-Gln-Ala-Gln-AlaGlu-Phe-VaLLys-Tip-Leu -Leu-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys-NH2 (SEQ ID NO: 21).

H-Ala-Gln(Trt)-Ala-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)Ser (tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc)-Sieber amide resin (0.225 meq/g, 44.4 mg) prepared according to Reference Example 3 was weighed into a reaction tube and subjected to swelling with DMF. After removing the DMF by filtration, Fmoc-Gln(Trt)OH (61.1 mg), 0.5 M Oxymapure in DMF (200 μL), and diisopropylcarbodiimide (15.9 μL) were added sequentially to the resin, and then the mixture was shaken for 1. 5 hours. The reaction solution was filtered, and then the resin was washed 6 times with DMF. After confirmation of Kaiser test negativity, 20% piperidine in DMF was added and the mixture was shaken for 1 min. The solution was filtered and then 20% piperidine in DMF was added again and the mixture was shaken for 20 minutes. The solution was filtered and the resin was washed 10 times with DMF. This cycle of condensation of an amino acid with Fmoc - removal of the Fmoc protecting group was repeated for the sequential condensation of Lys(Boc), Asp(OtBu), Leu, Aib, Lys(Boc), Aib, Tyr(tBu), Asp(OtBu), Ser(tBu) , Thr(tBu), aMePhe, Thr(tBu)*, Gly, Glu(OtBu), Aib and Tyr(tBu) (*: overnight reaction). The resin was washed with MeOH and then dried under reduced pressure to obtain 72.4 mg of HTyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-aMePhe-Thr(tBu)-Ser(tBu)-Asp(OtBu )-Tyr(tBu)-Aib-Lys(Boc)-AibLeu-Asp(OtBu)-Lys(Boc)-Gln(Trt)-Ala-Gln(Trt)-Ala-Glu(OtBu)-Phe-Val-Lys (Boc)-Trp(Boc)-Leu-LeuLys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc )-amide resin Sieber.

To 72.4 mg of the resulting resin, 1 ml of a mixture of TFA: m-cresol: thioanisole: ethanedhiol: H ₂ O: triisopropylsilane (80: 5: 5: 5: 2.5: 2.5) was added, and the mixture was stirred for 1 5 h. Diethyl ether was added to the reaction solution to ensure precipitation, and the precipitate was washed by repeating the action of removing the supernatant after centrifugation three times. The precipitate was extracted with 50% aqueous acetic acid and the resin was removed by filtration, followed by preparative HPLC using a Daisopak SP-100-5-ODS-P column (250x20 mm ID) [solution A: 0.1% TFA-water, solution B : acetonitrile containing 0.1% TFA, flow rate 8 ml/min, A/B: linear concentration gradient elution 67/33-57/43 (60 min)]. Fractions containing the desired product were collected and lyophilized to obtain 13.9 mg of white powder.

- 30 044242

Mass spectrometry, (M+H)+ 4295.8 (calculated: 4296.2);

HPLC elution time: 6.9 min;

elution conditions:

Column: Merck Chromolith Performance RP-18e (100x4.6 mm I.D.);

eluent: using solution A: 0.1% TFA-water, solution B: acetonitrile containing 0.1% TFA, A/B: elution with a linear concentration gradient 95/5-35/65 (10 min);

flow rate: 3.0 ml/min.

Example 10.

Synthesis of H-Tyr-Aib-Glu-Gly-Thr-aMePhe-Thr-Ser-Asp-Tyr-Aib-Ala-Aib-Leu-Asp-Lys-Gln-Ala-Gln-AlaGlu-Phe-Val-Lys-Trp -Leu-Leu-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys-NH2 (SEQ ID NO: 22).

H-Ala-Gln(Trt)-Ala-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)Ser (tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc)-Sieber amide resin (0.225 meq/g, 44.4 mg) prepared according to Reference Example 3 was weighed into a reaction tube and subjected to swelling with DMF.

After removing the DMF by filtration, Fmoc-Gln(Trt)-OH (61.1 mg), 0.5 M Oxymapure in DMF (200 μL), and diisopropylcarbodiimide (15.9 μL) were added sequentially to the resin, and then the mixture was shaken for 1 .5 hours. The reaction solution was filtered, and then the resin was washed 6 times with DMF. After confirmation of Kaiser test negativity, 20% piperidine in DMF was added and the mixture was shaken for 1 min. The solution was filtered and then 20% piperidine in DMF was added again and the mixture was shaken for 20 minutes. The solution was filtered and the resin was washed 10 times with DMF. This cycle of condensation of an amino acid with Fmoc - removal of the Fmoc protecting group was repeated for the sequential condensation of Lys(Boc), Asp(OtBu), Leu, Aib, Ala, Aib, Tyr(tBu), Asp(OtBu), Ser(tBu), Thr( tBu), aMePhe, Thr(tBu)*, Gly, Glu(OtBu), Aib and Tyr(tBu) (*: overnight reaction). The resin was washed with MeOH and then dried under reduced pressure to obtain 86.1 mg of H-Tyr(tBu)-Aib-Glu(OtBu)Gly-Thr(tBu)-aMePhe-Thr(tBu)-Ser(tBu)-Asp( OtBu)-Tyr(tBu)-Aib-Ala-Aib-Leu-Asp(OtBu)-Lys(Boc)Gln(Trt)-Ala-Gln(Trt)-Ala-Glu(OtBu)-Phe-Val-Lys( Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc )-amide resin Sieber.

To 86.1 mg of the resulting resin, 1 ml of a mixture of TFA: m-cresol: thioanisole: ethanedhiol: H2O: triisopropylsilane (80:5:5:5:2.5:2.5) was added, and the mixture was stirred for 1.5 h. Diethyl ether was added to the reaction solution to ensure precipitation, and the precipitate was washed by repeating the action of removing the supernatant after centrifugation three times. The precipitate was extracted with 50% aqueous acetic acid and the resin was removed by filtration, followed by preparative HPLC using a Daisopak SP-100-5-ODS-P column (250x20 mm I.D.) [solution A: 0.1% TFA-water, solution B : acetonitrile containing 0.1% TFA, flow rate 8 ml/min, A/B: 66/34-56/44 linear concentration gradient elution (60 min)]. Fractions containing the desired product were collected and lyophilized to obtain 16.2 mg of white powder.

Mass spectrometry, (M+H)+ 4238.6 (calculated: 4239.2);

HPLC elution time: 7.1 min;

elution conditions:

Column: Merck Chromolith Performance RP-18e (100x4.6 mm I.D.);

eluent: using solution A: 0.1% TFA-water, solution B: acetonitrile containing 0.1% TFA, A/B: elution with a linear concentration gradient 95/5-35/65 (10 min);

flow rate: 3.0 ml/min.

Example 11.

Synthesis of H-Tyr-Aib-Glu-Gly-Thr-aMePhe-Thr-Ser-Asp-Tyr-Aib-Phe-Aib-Leu-Asp-Lys-Gln-Ala-Gln-AlaGlu-Phe-Val-Lys-Trp -Leu-Leu-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys-NH2 (SEQ ID NO: 23).

H-Ala-Gln(Trt)-Ala-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)Ser (tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc)-Sieber amide resin (0.225 meq/g, 44.4 mg) prepared according to Reference Example 3 was weighed into a reaction tube and subjected to swelling with DMF. After removing the DMF by filtration, Fmoc-Gln(Trt)OH (61.1 mg), 0.5 M Oxymapure in DMF (200 μL), and diisopropylcarbodiimide (15.9 μL) were added sequentially to the resin, and then the mixture was shaken for 1. 5 hours. The reaction solution was filtered, and then the resin was washed 6 times with DMF. After confirmation of Kaiser test negativity, 20% piperidine in DMF was added and the mixture was shaken for 1 min. The solution was filtered and then 20% piperidine in DMF was added again and the mixture was shaken for 20 minutes. The solution was filtered and the resin was washed 10 times with DMF. This cycle of condensation of an amino acid with Fmoc - removal of the Fmoc protecting group was repeated for the sequential condensation of Lys(Boc), Asp(OtBu), Leu, Aib, Phe, Aib, Tyr(tBu), Asp(OtBu), Ser(tBu), Thr( tBu), aMePhe, Thr(tBu)*, Gly, Glu(OtBu), Aib and Tyr(tBu) (*: overnight reaction). The resin was washed with MeOH and then dried under reduced pressure to obtain 76.4 mg of H-Tyr(tBu)-AibGlu(OtBu)-Gly-Thr(tBu)-aMePhe-Thr(tBu)-Ser(tBu)-Asp(OtBu )-Tyr(tBu)-Aib-Phe-Aib-Leu-Asp(OtBu)Lys(Boc)-Gln(Trt)-Ala-Gln(Trt)-Ala-Glu(OtBu)-Phe-Val-Lys(Boc )-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro

- 31 044242

Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc)-Sieber amide resin.

To 76.4 mg of the resulting resin, 1 ml of a mixture of TFA: m-cresol: thioanisole: ethanedhiol: H2O: triisopropylsilane (80:5:5:5:2.5:2.5) was added, and the mixture was stirred for 1.5 h. Diethyl ether was added to the reaction solution to ensure precipitation, and the precipitate was washed by repeating the action of removing the supernatant after centrifugation three times. The precipitate was extracted with 50% aqueous acetic acid and the resin was removed by filtration, after which preparative HPLC was carried out using a Daisopak SP-100-5-ODS-P column (250x20 mm I.D.) [Solution A: 0.1% TFA-water, solution B : acetonitrile containing 0.1% TFA, flow rate 8 ml/min, A/B: linear concentration gradient elution 65/35-55/45 (60 min)]. Fractions containing the desired product were collected and lyophilized to obtain 4.7 mg of white powder.

Mass spectrometry, (M+H)+ 4315.1 (calculated: 4315.2);

HPLC elution time: 7.2 min;

elution conditions:

Column: Merck Chromolith Performance RP-18e (100x4.6 mm I.D.);

eluent: using solution A: 0.1% TFA-water, solution B: acetonitrile containing 0.1% TFA, A/B: elution with a linear concentration gradient 95/5-35/65 (10 min);

flow rate: 3.0 ml/min.

Example 12.

Synthesis of H-Tyr-Aib-Glu-Gly-Thr-αMePhe-Thr-Ser-Asp-Tyr-Aib-Pua(4)-Aib-Leu-Asp-Lys-Gln-Ala-GlnAla-Glu-Phe-Val- Lys-Trp-Leu-Leu-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys-NH2 (SEQ ID NO: 24).

H-Ala-Gln(Trt)-Ala-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)Ser (tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc)-Sieber amide resin (0.225 meq/g, 44.4 mg) prepared according to Reference Example 3 was weighed into a reaction tube and subjected to swelling with DMF. After removing the DMF by filtration, Fmoc-Gln(Trt)OH (61.1 mg), 0.5 M Oxymapure in DMF (200 μL), and diisopropylcarbodiimide (15.9 μL) were added sequentially to the resin, and then the mixture was shaken for 1. 5 hours. The reaction solution was filtered, and then the resin was washed 6 times with DMF. After confirmation of Kaiser test negativity, 20% piperidine in DMF was added and the mixture was shaken for 1 min. The solution was filtered and then 20% piperidine in DMF was added again and the mixture was shaken for 20 minutes. The solution was filtered and the resin was washed 10 times with DMF. This cycle of condensation of an amino acid with Fmoc - removal of the Fmoc protecting group was repeated for the sequential condensation of Lys(Boc), Asp(OtBu), Leu, Aib, Pya(4)*, Aib, Tyr(tBu), Asp(OtBu), Ser(tBu ), Thr(tBu), aMePhe, Thr(tBu)**, Gly, Glu(OtBu), Aib and Tyr(tBu) (*: 17.4 µL DIPEA added during condensation; **: reaction overnight) . The resin was washed with MeOH and then dried under reduced pressure to obtain 78.4 mg of H-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-aMePheThr(tBu)-Ser(tBu)-Asp(OtBu )-Tyr(tBu)-Aib-Pya(4)-Aib-Leu-Asp(OtBu)-Lys(Boc)-Gln(Trt)-Ala-Gln(Trt)-AlaGlu(OtBu)-Phe-Val-Lys (Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-ProPro-Ser(tBu)-Lys(Boc )-amide resin Sieber.

To 78.4 mg of the resulting resin, 1 ml of a mixture of TFA: m-cresol: thioanisole: ethanedhiol: H ₂ O: triisopropylsilane (80: 5: 5: 5: 2.5: 2.5) was added, and the mixture was stirred for 1 5 h. Diethyl ether was added to the reaction solution to ensure precipitation, and the precipitate was washed by repeating the action of removing the supernatant after centrifugation three times. The precipitate was extracted with 50% aqueous acetic acid and the resin was removed by filtration, after which preparative HPLC was carried out using a Daisopak SP-100-5-ODS-P column (250x20 mm LD.) [solution A: 0.1% TFA-water, solution B: acetonitrile containing 0.1% TFA, flow rate 8 ml/min, A/B: linear concentration gradient elution 67/33-57/43 (60 min)]. Fractions containing the desired product were collected and lyophilized to obtain 15.2 mg of white powder.

Mass spectrometry, (M+H)+ 4316.2 (calculated: 4316.2);

HPLC elution time: 6.9 min;

Column: Merck Chromolith Performance RP-18e (100x4.6 mm I.D.);

eluent: using solution A: 0.1% TFA-water, solution B: acetonitrile containing 0.1% TFA, A/B: elution with a linear concentration gradient 95/5-35/65 (10 min);

flow rate: 3.0 ml/min.

Example 13.

Synthesis of H-Tyr-Aib-Glu-Gly-Thr-aMePhe-Thr-Ser-Asp-Tyr-Aib-Ile-aMePhe-Leu-Asp-Lys-Gln-Ala-GlnAla-Glu-Phe-Val-Lys-Trp -Leu-Leu-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys-NH ₂ (SEQ ID NO: 25).

H-Ala-Gln(Trt)-Ala-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)Ser (tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc)-Sieber amide resin (0.225 meq/g, 44.4 mg) prepared according to Reference Example 3 was weighed into a reaction tube and subjected to swelling with DMF. After DMF was removed by filtration, Fmoc-Gln(Trt) was sequentially added to the resin.

- 32 044242

OH (61.1 mg), 0.5 M Oxymapure in DMF (200 µl) and diisopropylcarbodiimide (15.9 µl), and then the mixture was shaken for 1.5 hours. The reaction solution was filtered and then the resin was washed 6 times with DMF . After confirmation of Kaiser test negativity, 20% piperidine in DMF was added and the mixture was shaken for 1 min. The solution was filtered and then 20% piperidine in DMF was added again and the mixture was shaken for 20 minutes. The solution was filtered and the resin was washed 10 times with DMF. This cycle of condensation of an amino acid with Fmoc - removal of the Fmoc protecting group was repeated for the sequential condensation of Lys(Boc), Asp(OtBu), Leu, aMePhe, Ile, Aib, Tyr(tBu), Asp(OtBu), Ser(tBu), Thr( tBu), aMePhe, Thr(tBu)*, Gly, Glu(OtBu), Aib and Tyr(tBu) (*: overnight reaction). The resin was washed with MeOH and then dried under reduced pressure to obtain 69.1 mg of HTyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-aMePhe-Thr(tBu)-Ser(tBu)-Asp(OtBu )-Tyr(tBu)-Aib-Ile-aMePhe-LeuAsp(OtBu)-Lys(Boc)-Gln(Trt)-Ala-Gln(Trt)-Ala-Glu(OtBu)-Phe-Val-Lys(Boc) -Trp(Boc)-Leu-Leu-Lys(Boc)Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc)- Sieber amide resin.

To 69.1 mg of the resulting resin, 1 ml of a mixture of TFA: m-cresol: thioanisole: ethanedhiol: H ₂ O: triisopropylsilane (80: 5: 5: 5: 2.5: 2.5) was added, and the mixture was stirred for 1 5 h. Diethyl ether was added to the reaction solution to ensure precipitation, and the precipitate was washed by repeating the action of removing the supernatant after centrifugation three times. The precipitate was extracted with 50% aqueous acetic acid and the resin was removed by filtration, followed by preparative HPLC using a Daisopak SP-100-5-ODS-P column (250x20 mm ID) [solution A: 0.1% TFA-water, solution B : acetonitrile containing 0.1% TFA, flow rate 8 ml/min, A/B: linear concentration gradient elution 64/36-54/46 (60 min)]. Fractions containing the desired product were collected and lyophilized to obtain 14.4 mg of white powder.

Mass spectrometry, (M+H)+ 4356.9 (calculated: 4357.3);

HPLC elution time: 7.4 min;

elution conditions:

Column: Merck Chromolith Performance RP-18e (100x4.6 mm I.D.);

eluent: using solution A: 0.1% TFA-water, solution B: acetonitrile containing 0.1% TFA, A/B: elution with a linear concentration gradient 95/5-35/65 (10 min);

flow rate: 3.0 ml/min.

Example 14.

Synthesis of H-Tyr-Aib-Glu-Gly-Thr-aMePhe-Thr-Ser-Asp-Tyr-Aib-Ile-Cha-Leu-Asp-Lys-Gln-Ala-Gln-AlaGlu-Phe-Val-Lys-Trp -Leu-Leu-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys-NH ₂ (SEQ ID NO: 26).

H-Ala-Gln(Trt)-Ala-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)Ser (tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc)-Sieber amide resin (0.225 meq/g, 44.4 mg) prepared according to Reference Example 3 was weighed into a reaction tube and subjected to swelling with DMF. After removing the DMF by filtration, Fmoc-Gln(Trt)OH (61.1 mg), 0.5 M Oxymapure in DMF (200 μL), and diisopropylcarbodiimide (15.9 μL) were added sequentially to the resin, and then the mixture was shaken for 1. 5 hours. The reaction solution was filtered, and then the resin was washed 6 times with DMF. After confirmation of Kaiser test negativity, 20% piperidine in DMF was added and the mixture was shaken for 1 min. The solution was filtered and then 20% piperidine in DMF was added again and the mixture was shaken for 20 minutes. The solution was filtered and the resin was washed 10 times with DMF. This cycle of condensation of an amino acid with Fmoc - removal of the Fmoc protecting group was repeated for the sequential condensation of Lys(Boc), Asp(OtBu), Leu, Cha, Ile, Aib, Tyr(tBu), Asp(OtBu), Ser(tBu), Thr( tBu), aMePhe, Thr(tBu)*, Gly, Glu(OtBu), Aib and Tyr(tBu) (*: overnight reaction). The resin was washed with MeOH and then dried under reduced pressure to obtain 71.5 mg of H-Tyr(tBu)-AibGlu(OtBu)-Gly-Thr(tBu)-aMePhe-Thr(tBu)-Ser(tBu)-Asp(OtBu )-Tyr(tBu)-Aib-Ile-Cha-Leu-Asp(OtBu)Lys(Boc)-Gln(Trt)-Ala-Gln(Trt)-Ala-Glu(OtBu)-Phe-Val-Lys(Boc )-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-ProSer(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc)- Sieber amide resin.

To 71.5 mg of the resulting resin, 1 ml of a mixture of TFA: m-cresol: thioanisole: ethanedhiol: H2O: triisopropylsilane (80:5:5:5:2.5:2.5) was added, and the mixture was stirred for 1.5 h. Diethyl ether was added to the reaction solution to ensure precipitation, and the precipitate was washed by repeating the action of removing the supernatant after centrifugation three times. The precipitate was extracted with 50% aqueous acetic acid and the resin was removed by filtration, followed by preparative HPLC using a Daisopak SP-100-5-ODS-P column (250x20 mm I.D.) [solution A: 0.1% TFA-water, solution B : acetonitrile containing 0.1% TFA, flow rate 8 ml/min, A/B: linear concentration gradient elution 63/37-53/47 (60 min)]. Fractions containing the desired product were collected and lyophilized to obtain 16 mg of white powder.

Mass spectrometry, (M+H)+ 4348.7 (calculated: 4349.3);

HPLC elution time: 7.5 min;

elution conditions:

Column: Merck Chromolith Performance RP-18e (100x4.6 mm I.D.);

eluent: use solution A: 0.1% TFA-water, solution B: acetonitrile containing 0.1%

- 33 044242

TFA, A/B: linear concentration gradient elution 95/5-35/65 (10 min);

flow rate: 3.0 ml/min.

Example 15.

Synthesis of H-Tyr-Aib-Glu-Gly-Thr-aMePhe-Thr-Ser-Asp-Tyr-Aib-Lys-aMePhe-Leu-Asp-Lys-Gln-Ala-GlnAla-Glu-Phe-Val-Lys-Trp -Leu-Leu-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys-NH2 (SEQ ID NO: 27).

H-Ala-Gln(Trt)-Ala-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)Ser (tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc)-Sieber amide resin (0.225 meq/g, 44.4 mg) prepared according to Reference Example 3 was weighed into a reaction tube and subjected to swelling with DMF. After removing the DMF by filtration, Fmoc-Gln(Trt)OH (61.1 mg), 0.5 M Oxymapure in DMF (200 μL), and diisopropylcarbodiimide (15.9 μL) were added sequentially to the resin, and then the mixture was shaken for 1. 5 hours. The reaction solution was filtered, and then the resin was washed 6 times with DMF. After confirmation of Kaiser test negativity, 20% piperidine in DMF was added and the mixture was shaken for 1 min. The solution was filtered and then 20% piperidine in DMF was added again and the mixture was shaken for 20 minutes. The solution was filtered and the resin was washed 10 times with DMF. This cycle of condensation of an amino acid with Fmoc - removal of the Fmoc protecting group was repeated for the sequential condensation of Lys(Boc), Asp(OtBu), Leu, aMePhe, Lys(Boc), Aib, Tyr(tBu), Asp(OtBu), Ser(tBu) , Thr(tBu), aMePhe, Thr(tBu)*, Gly, Glu(OtBu), Aib and Tyr(tBu) (*: overnight reaction). The resin was washed with MeOH and then dried under reduced pressure to obtain 77.9 mg of HTyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-aMePhe-Thr(tBu)-Ser(tBu)-Asp(OtBu )-Tyr(tBu)-Aib-Lys(Boc)aMePhe-Leu-Asp(OtBu)-Lys(Boc)-Gln(Trt)-Ala-Gln(Trt)-Ala-Glu(OtBu)-Phe-Val- Lys(Boc)-Trp(Boc)-LeuLeu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys( Boc)-amide resin Sieber.

To 77.9 mg of the resulting resin, 1 ml of a mixture of TFA: m-cresol: thioanisole: ethanedhiol: H ₂ O: triisopropylsilane (80: 5: 5: 5: 2.5: 2.5) was added, and the mixture was stirred for 1 5 h. Diethyl ether was added to the reaction solution to ensure precipitation, and the precipitate was washed by repeating the action of removing the supernatant after centrifugation three times. The precipitate was extracted with 50% aqueous acetic acid and the resin was removed by filtration, followed by preparative HPLC using a Daisopak SP-100-5-ODS-P column (250x20 mm ID) [solution A: 0.1% TFA-water, solution B : acetonitrile containing 0.1% TFA, flow rate 8 ml/min, A/B: linear concentration gradient elution 65/35-55/45 (60 min)]. Fractions containing the desired product were collected and lyophilized to obtain 13.4 mg of white powder.

Mass spectrometry, (M+H)+ 4371.7 (calculated: 4372.3;) HPLC elution time: 7.1 min;

elution conditions:

Column: Merck Chromolith Performance RP-18e (100x4.6 mm I.D.);

eluent: using solution A: 0.1% TFA-water, solution B: acetonitrile containing 0.1% TFA, A/B: elution with a linear concentration gradient 95/5-35/65 (10 min);

flow rate: 3.0 ml/min.

Example 16.

Synthesis of H-Tyr-Aib-Glu-Gly-Thr-aMePhe-Thr-Ser-Asp-Tyr-Aib-Lys-Cha-Leu-Asp-Lys-Gln-Ala-Gln-AlaGlu-Phe-Val-Lys-Trp -Leu-Leu-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys-NH ₂ (SEQ ID NO: 28).

H-Ala-Gln(Trt)-Ala-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)Ser (tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc)-Sieber amide resin (0.225 meq/g, 44.4 mg) prepared according to Reference Example 3 was weighed into a reaction tube and subjected to swelling with DMF. After removing the DMF by filtration, Fmoc-Gln(Trt)OH (61.1 mg), 0.5 M Oxymapure in DMF (200 μL), and diisopropylcarbodiimide (15.9 μL) were added sequentially to the resin, and then the mixture was shaken for 1. 5 hours. The reaction solution was filtered, and then the resin was washed 6 times with DMF. After confirmation of Kaiser test negativity, 20% piperidine in DMF was added and the mixture was shaken for 1 min. The solution was filtered and then 20% piperidine in DMF was added again and the mixture was shaken for 20 minutes. The solution was filtered and the resin was washed 10 times with DMF. This cycle of condensation of an amino acid with Fmoc - removal of the Fmoc protecting group was repeated for the sequential condensation of Lys(Boc), Asp(OtBu), Leu, Cha, Lys(Boc), Aib, Tyr(tBu), Asp(OtBu), Ser(tBu) , Thr(tBu), aMePhe, Thr(tBu)*, Gly, Glu(OtBu), Aib and Tyr(tBu) (*: overnight reaction). The resin was washed with MeOH and then dried under reduced pressure to obtain 75.1 mg of HTyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-aMePhe-Thr(tBu)-Ser(tBu)-Asp(OtBu )-Tyr(tBu)-Aib-Lys(Boc)-ChaLeu-Asp(OtBu)-Lys(Boc)-Gln(Trt)-Ala-Gln(Trt)-Ala-Glu(OtBu)-Phe-Val-Lys (Boc)-Trp(Boc)-Leu-LeuLys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc )-amide resin Sieber.

To 75.1 mg of the resulting resin, 1 ml of a mixture of TFA: m-cresol: thioanisole: ethanedhiol: H ₂ O: triisopropylsilane (80: 5: 5: 5: 2.5: 2.5) was added, and the mixture was stirred for 1 5 h. Diethyl ether was added to the reaction solution to ensure precipitation, and the precipitate was washed by repeating the action of removing the supernatant after centrifugation three times. The precipitate was extracted with 50% aqueous

- 34 044242 with a solution of acetic acid and the resin was removed by filtration, after which preparative

HPLC using a Daisopak SP-100-5-ODS-P column (250x20 mm I.D.) [solution A: 0.1% TFA-water, solution B: acetonitrile containing 0.1% TFA, flow rate 8 ml/min, A/B: linear concentration gradient elution 65/35-55/45 (60 min)]. Fractions containing the desired product were collected and lyophilized to obtain 14.7 mg of white powder.

Mass spectrometry, (M+H)+ 4364.7 (calculated: 4364.3);

HPLC elution time: 7.1 min;

elution conditions:

Column: Merck Chromolith Performance RP-18e (100x4.6 mm I.D.);

eluent: using solution A: 0.1% TFA-water, solution B: acetonitrile containing 0.1% TFA, A/B: elution with a linear concentration gradient 95/5-35/65 (10 min);

flow rate: 3.0 ml/min.

Example 17.

Synthesis of H-Tyr-Aib-Glu-Gly-Thr-aMePhe-Thr-Ser-Asp-Tyr-Aib-Ile-aMeTyr-Leu-Asp-Lys-Gln-Ala-GlnAla-Glu-Phe-Val-Lys-Trp -Leu-Leu-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys-NH ₂ (SEQ ID NO: 29).

H-Ala-Gln(Trt)-Ala-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)Ser (tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc)-Sieber amide resin (0.225 meq/g, 44.4 mg) prepared according to Reference Example 3 was weighed into a reaction tube and subjected to swelling with DMF. After removing the DMF by filtration, Fmoc-Gln(Trt)OH (61.1 mg), 0.5 M Oxymapure in DMF (200 μL), and diisopropylcarbodiimide (15.9 μL) were added sequentially to the resin, and then the mixture was shaken for 1. 5 hours. The reaction solution was filtered, and then the resin was washed 6 times with DMF. After confirmation of Kaiser test negativity, 20% piperidine in DMF was added and the mixture was shaken for 1 min. The solution was filtered and then 20% piperidine in DMF was added again and the mixture was shaken for 20 minutes. The solution was filtered and the resin was washed 10 times with DMF. This cycle of condensation of an amino acid with Fmoc - removal of the Fmoc protecting group was repeated for the sequential condensation of Lys(Boc), Asp(OtBu), Leu, aMeTyr, Ile, Aib, Tyr(tBu), Asp(OtBu), Ser(tBu), Thr( tBu), aMePhe, Thr(tBu)*, Gly, Glu(OtBu), Aib and Tyr(tBu) (*: overnight reaction). The resin was washed with MeOH and then dried under reduced pressure to obtain 56.2 mg of HTyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-aMePhe-Thr(tBu)-Ser(tBu)-Asp(OtBu )-Tyr(tBu)-Aib-Ile-aMeTyr-LeuAsp(OtBu)-Lys(Boc)-Gln(Trt)-Ala-Gln(Trt)-Ala-Glu(OtBu)-Phe-Val-Lys(Boc) -Trp(Boc)-Leu-Leu-Lys(Boc)Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc)- Sieber amide resin.

To 56.2 mg of the resulting resin, 1 ml of a mixture of TFA: m-cresol: thioanisole: ethanedhiol: H2O: triisopropylsilane (80:5:5:5:2.5:2.5) was added, and the mixture was stirred for 1.5 h. Diethyl ether was added to the reaction solution to ensure precipitation, and the precipitate was washed by repeating the action of removing the supernatant after centrifugation three times. The precipitate was extracted with 50% aqueous acetic acid and the resin was removed by filtration, followed by preparative HPLC using a Daisopak SP-100-5-ODS-P column (250x20 mm I.D.) [solution A: 0.1% TFA-water, solution B : acetonitrile containing 0.1% TFA, flow rate 8 ml/min, A/B: linear concentration gradient elution 65/35-55/45 (60 min)]. Fractions containing the desired product were collected and lyophilized to obtain 1.4 mg of white powder.

Mass spectrometry, (M+H)+ 4373.8 (calculated: 4373.2);

HPLC elution time: 7.2 min;

elution conditions:

Column: Merck Chromolith Performance RP-18e (100x4.6 mm I.D.);

eluent: using solution A: 0.1% TFA-water, solution B: acetonitrile containing 0.1% TFA, A/B: elution with a linear concentration gradient 95/5-35/65 (10 min);

flow rate: 3.0 ml/min.

Example 18.

Synthesis of Ac-Tyr-Aib-Glu-Gly-Thr-aMePhe-Thr-Ser-Asp-Tyr-Aib-Ile-Aib-Leu-Asp-Lys-Gln-Ala-Gln-AlaGlu-Phe-Val-Lys-Trp -Leu-Leu-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys-NH ₂ (SEQ ID NO: 30).

H-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-AlaPro- Sieber Pro-Pro-Ser(tBu)-Lys(Boc)-amide resin (0.253 meq/g, 39.5 mg) prepared using a similar approach to Reference Example 1 was weighed into a reaction tube and swelled with DMF . After removing the DMF by filtration, Fmoc-Ala-OH (31.1 mg), 0.5 M Oxymapure in DMF (200 µl) and diisopropylcarbodiimide (15.9 µl) were added sequentially to the resin, and then the mixture was shaken for 1.5 h The reaction solution was filtered, and then the resin was washed with DMF 6 times. After confirmation of Kaiser test negativity, 20% piperidine in DMF was added and the mixture was shaken for 1 min. The solution was filtered and then 20% piperidine in DMF was added again and the mixture was shaken for 20 minutes. The solution was filtered and the resin was washed 10 times with DMF.

- 35 044242

This cycle of condensation of an amino acid with Fmoc - removal of the Fmoc protecting group was repeated for the sequential condensation of Gln(Trt), Ala, Gln(Trt), Lys(Boc), Asp(OtBu), Leu, Aib, Ile*, Aib, Tyr(tBu) , Asp(OtBu), Ser(tBu), Thr(tBu), aMePhe, Thr(tBu)*, Gly, Glu(OtBu), Aib and Tyr(tBu) (*: overnight reaction). Next, the N-terminal group of Fmoc was removed by treatment with piperidine, and then DMF (200 µl), DIPEA (17.4 µl) and Ac ₂ O (9.4 µl) were successively added to the resin and the mixture was shaken for 90 minutes. The reaction solution was filtered, and then the resin was washed with DMF 6 times. After confirming the negative Kaiser test, the resin was washed with MeOH and dried under reduced pressure to obtain 43 mg Ac-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-αMePhe-Thr(tBu)-Ser(tBu )-Asp(OtBu)-Tyr(tBu)Aib-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Gln(Trt)-Ala-Gln(Trt)-Ala-Glu(OtBu)-Phe- Val-Lys(Boc)-Trp(Boc)-LeuLeu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)- Lys(Boc)-amide resin Sieber.

To 43 mg of the resulting resin, 1 ml of a mixture of TFA: m-cresol: thioanisole: ethanedhiol: H2O: triisopropylsilane (80:5:5:5:2.5:2.5) was added, and the mixture was stirred for 1.5 hours. Diethyl ether was added to the reaction solution to ensure precipitation, and the precipitate was washed by repeating the action of removing the supernatant after centrifugation three times. The precipitate was extracted with 50% aqueous acetic acid and the resin was removed by filtration, followed by preparative HPLC using a Daisopak SP-100-5-ODS-P column (250x20 mm I.D.) [solution A: 0.1% TFA-water, solution B : acetonitrile containing 0.1% TFA, flow rate 8 ml/min, A/B: linear concentration gradient elution 62/38-52/48 (60 min)]. Fractions containing the desired product were collected and lyophilized to obtain 7.8 mg of white powder.

Mass spectrometry, (M+H)+ 4323.4 (calculated: 4323.2);

HPLC elution time: 7.5 min;

elution conditions:

Column: Merck Chromolith Performance RP-18e (100x4.6 mm I.D.);

eluent: using solution A: 0.1% TFA-water, solution B: acetonitrile containing 0.1% TFA, A/B: elution with a linear concentration gradient 95/5-35/65 (10 min);

flow rate: 3.0 ml/min.

Example 19.

Synthesis of benzoyl-Tyr-Aib-Glu-Gly-Thr-aMePhe-Thr-Ser-Asp-Tyr-Aib-Ile-Aib-Leu-Asp-Lys-Gln-AlaGln-Ala-Glu-Phe-Val-Lys-Trp -Leu-Leu-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys-NH ₂ (SEQ ID NO: 31).

H-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-AlaPro- Sieber Pro-Pro-Ser(tBu)-Lys(Boc)-amide resin (0.253 meq/g, 39.5 mg) prepared according to Reference Example 1 was weighed into a reaction tube and swollen with DMF. After removing the DMF by filtration, Fmoc-Ala-OH (31.1 mg), 0.5 M Oxymapure in DMF (200 µl) and diisopropylcarbodiimide (15.9 µl) were added sequentially to the resin, and then the mixture was shaken for 1.5 h The reaction solution was filtered, and then the resin was washed with DMF 6 times. After confirmation of Kaiser test negativity, 20% piperidine in DMF was added and the mixture was shaken for 1 min. The solution was filtered and then 20% piperidine in DMF was added again and the mixture was shaken for 20 minutes. The solution was filtered and the resin was washed 10 times with DMF. This cycle of condensation of an amino acid with Fmoc - removal of the Fmoc protecting group was repeated for the sequential condensation of Gln(Trt), Ala, Gln(Trt), Lys(Boc), Asp(OtBu), Leu, Aib, Ile*, Aib, Tyr(tBu) , Asp(OtBu), Ser(tBu), Thr(tBu), aMePhe, Thr(tBu)*, Gly, Glu(OtBu), Aib and Tyr(tBu) (*: overnight reaction). Next, the N-terminal group of Fmoc was removed by treatment with piperidine, and then benzoic acid (12.2 mg), 0.5 M Oxymapure in DMF (200 μl) and diisopropylcarbodiimide (15.9 μl) were successively added to the resin and the mixture was shaken overnight . The reaction solution was filtered, and then the resin was washed with DMF 6 times. After confirming the negative Kaiser test, the resin was washed with MeOH and then dried under reduced pressure to obtain 54.8 mg of benzoyl-Tyr(tBu)-Aib-Glu(OtBu)-Gl·y-Thr(tBu)aMePhe-Thr(tBu )-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Aib-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Gln(Trt)-Ala-Gln(Trt)Ala-Glu( OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-ProPro-Pro- Ser(tBu)-Lys(Boc)-amide resin Sieber.

To 54.8 mg of the resulting resin, 1 ml of a mixture of TFA: m-cresol: thioanisole: ethanedhiol: H ₂ O: triisopropylsilane (80: 5: 5: 5: 2.5: 2.5) was added, and the mixture was stirred for 1 5 h. Diethyl ether was added to the reaction solution to ensure precipitation, and the precipitate was washed by repeating the action of removing the supernatant after centrifugation three times. The precipitate was extracted with 50% aqueous acetic acid and the resin was removed by filtration, followed by preparative HPLC using a Daisopak SP-100-5-ODS-P column (250x20 mm ID) [solution A: 0.1% TFA-water, solution B : acetonitrile containing 0.1% TFA, flow rate 8 ml/min, A/B: linear concentration gradient elution 60/40-50/50 (60 min)]. Fractions containing the desired product were collected and lyophilized to obtain 10.1 mg of white powder.

Mass spectrometry, (M+H)+ 4385.2 (calculated: 4385.2);

HPLC elution time: 7.7 min;

- 36 044242 elution conditions:

Column: Merck Chromolith Performance RP-18e (100x4.6 mm I.D.);

eluent: using solution A: 0.1% TFA-water, solution B: acetonitrile containing 0.1% TFA, A/B: elution with a linear concentration gradient 95/5-35/65 (10 min);

flow rate: 3.0 ml/min.

Example 20.

Synthesis of 4PyCO-Tyr-Aib-Glu-Gly-Thr-aMePhe-Thr-Ser-Asp-Tyr-Aib-[le-Aib-Leu-Asp-Lys-Gln-Ala-GlnAla-Glu-Phe-Val-Lys- Trp-Leu-Leu-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys-NH ₂ (SEQ ID NO: 32).

H-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-AlaPro- Sieber Pro-Pro-Ser(tBu)-Lys(Boc)-amide resin (0.253 meq/g, 39.5 mg) prepared according to Reference Example 1 was weighed into a reaction tube and swollen with DMF. After removing the DMF by filtration, Fmoc-Ala-OH (31.1 mg), 0.5 M Oxymapure in DMF (200 µl) and diisopropylcarbodiimide (15.9 µl) were added sequentially to the resin, and then the mixture was shaken for 1.5 h The reaction solution was filtered, and then the resin was washed with DMF 6 times. After confirmation of Kaiser test negativity, 20% piperidine in DMF was added and the mixture was shaken for 1 min. The solution was filtered and then 20% piperidine in DMF was added again and the mixture was shaken for 20 minutes. The solution was filtered and the resin was washed 10 times with DMF. This cycle of condensation of an amino acid with Fmoc - removal of the Fmoc protecting group was repeated for the sequential condensation of Gln(Trt), Ala, Gln(Trt), Lys (Boc), Asp (OtBu), Leu, Aib, Ile*, Aib, Tyr(tBu) , Asp(OtBu), Ser(tBu), Thr(tBu), aMePhe, Thr(tBu)*, Gly, Glu(OtBu), Aib and Tyr(tBu) (*: overnight reaction). Next, the N-terminal group of Fmoc was removed by treatment with piperidine, and then 4-pyridinecarboxylic acid (12.3 mg), 0.5 M Oxymapure in DMF (200 μl) and diisopropylcarbodiimide (15.9 μl) were sequentially added to the resin and the mixture was shaken during the night. The reaction solution was filtered, and then the resin was washed with DMF 6 times. After confirming the negative Kaiser test, the resin was washed with MeOH and then dried under reduced pressure to obtain 62.9 mg of 4PyCO-Tyr(tBu)-AibGlu(OtBu)-Gly-Thr(tBu)-aMePhe-Thr(tBu)-Ser (tBu)-Asp(OtBu)-Tyr(tBu)-Aib-Ile-Aib-Leu-Asp(OtBu)Lys(Boc)-Gln(Trt)-Ala-Gln(Trt)-Ala-Glu(OtBu)- Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-ProSer(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu )-Lys(Boc)-amide resin Sieber.

To 62.9 mg of the resulting resin, 1 ml of a mixture of TFA: m-cresol: thioanisole: ethanedhiol: H2O: triisopropylsilane (80:5:5:5:2.5:2.5) was added, and the mixture was stirred for 1.5 h. Diethyl ether was added to the reaction solution to ensure precipitation, and the precipitate was washed by repeating the action of removing the supernatant after centrifugation three times. The precipitate was extracted with 50% aqueous acetic acid and the resin was removed by filtration, followed by preparative HPLC using a Daisopak SP-100-5-ODS-P column (250x20 mm I.D.) [solution A: 0.1% TFA-water, solution B : acetonitrile containing 0.1% TFA, flow rate 8 ml/min, A/B: linear concentration gradient elution 62/38-52/48 (60 min)]. Fractions containing the desired product were collected and lyophilized to obtain 13.3 mg of white powder.

Mass spectrometry, (M+H)+ 4386.2 (calculated: 4386.2);

HPLC elution time: 7.3 min;

elution conditions:

Column: Merck Chromolith Performance RP-18e (100x4.6 mm I.D.);

eluent: using solution A: 0.1% TFA-water, solution B: acetonitrile containing 0.1% TFA, A/B: elution with a linear concentration gradient 95/5-35/65 (10 min);

flow rate: 3.0 ml/min.

Example 21.

Synthesis of cPrCO-Tyr-Arb-Glu-Gly-Thr-aMePhe-Thr-Ser-Asp-Tyr-Aib-Ile-Aib-Leu-Asp-Lys-Gln-Ala-GlnAla-Glu-Phe-Val-Lys-Trp -Leu-Leu-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys-NH ₂ (SEQ ID NO: 33).

H-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-AlaPro- Sieber Pro-Pro-Ser(tBu)-Lys(Boc)-amide resin (0.253 meq/g, 39.5 mg) prepared according to Reference Example 1 was weighed into a reaction tube and swollen with DMF.

After removing the DMF by filtration, Fmoc-Ala-OH (31.1 mg), 0.5 M Oxymapure in DMF (200 µl) and diisopropylcarbodiimide (15.9 µl) were added sequentially to the resin, and then the mixture was shaken for 1.5 h The reaction solution was filtered, and then the resin was washed with DMF 6 times. After confirmation of Kaiser test negativity, 20% piperidine in DMF was added and the mixture was shaken for 1 min. The solution was filtered and then 20% piperidine in DMF was added again and the mixture was shaken for 20 minutes. The solution was filtered and the resin was washed 10 times with DMF. This cycle of condensation of an amino acid with Fmoc - removal of the Fmoc protecting group was repeated for the sequential condensation of Gln(Trt), Ala, Gln(Trt), Lys(Boc), Asp(OtBu), Leu, Aib, Ile*, Aib, Tyr(tBu) , Asp(OtBu), Ser(tBu), Thr(tBu), aMePhe, Thr(tBu)*, Gly, Glu(OtBu), Aib and Tyr(tBu) (*: reaction within

- 37 044242 nights). Next, the N-terminal group of Fmoc was removed by treatment with piperidine, and then cyclopropanecarboxylic acid (8.6 mg), 0.5 M Oxymapure in DMF (200 μl) and diisopropylcarbodiimide (15.9 μl) were sequentially added to the resin and the mixture was shaken for nights. The reaction solution was filtered, and then the resin was washed with DMF 6 times. After confirming the negative Kaiser test, the resin was washed with MeOH and then dried under reduced pressure to obtain 61.8 mg cPrCO-Tyr(tBu)-AibGlu(OtBu)-Gly-Thr(tBu)-aMePhe-Thr(tBu)-Ser (tBu)-Asp(OtBu)-Tyr(tBu)-Aib-Ile-Aib-Leu-Asp(OtBu)Lys(Boc)-Gln(Trt)-Ala-Gln(Trt)-Ala-Glu(OtBu)- Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-ProSer(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu )-Lys(Boc)-amide resin Sieber.

To 61.8 mg of the resulting resin, 1 ml of a mixture of TFA: m-cresol: thioanisole: ethanedhiol: H ₂ O: triisopropylsilane (80: 5: 5: 5: 2.5: 2.5) was added, and the mixture was stirred for 1 5 h. Diethyl ether was added to the reaction solution to ensure precipitation, and the precipitate was washed by repeating the action of removing the supernatant after centrifugation three times. The precipitate was extracted with 50% aqueous acetic acid and the resin was removed by filtration, followed by preparative HPLC using a Daisopak SP-100-5-ODS-P column (250x20 mm ID) [solution A: 0.1% TFA-water, solution B : acetonitrile containing 0.1% TFA, flow rate 8 ml/min, A/B: linear concentration gradient elution 61/39-51/49 (60 min)]. Fractions containing the desired product were collected and lyophilized to obtain 11.7 mg of white powder.

Mass spectrometry, (M+H)+ 4349.2 (calculated: 4349.2);

HPLC elution time: 7.6 min;

elution conditions:

Column: Merck Chromolith Performance RP-18e (100x4.6 mm I.D.);

eluent: using solution A: 0.1% TFA-water, solution B: acetonitrile containing 0.1% TFA, A/B: elution with a linear concentration gradient 95/5-35/65 (10 min);

flow rate: 3.0 ml/min.

Example 22.

Synthesis of amidino-Tyr-Aib-Glu-Gl·y-Thr-αMePhe-Thr-Ser-Asp-Tyr-Aib-[le-Aib-Leu-Asp-Lys-Gln-AlaGln-Ala-Glu-Phe-Val- Lys-Trp-Leu-Leu-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys-NH ₂ (SEQ ID NO: 34).

H-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-AlaPro- Sieber Pro-Pro-Ser(tBu)-Lys(Boc)-amide resin (0.253 meq/g, 39.5 mg) prepared according to Reference Example 1 was weighed into a reaction tube and swollen with DMF. After removing the DMF by filtration, Fmoc-Ala-OH (31.1 mg), 0.5 M Oxymapure in DMF (200 µl) and diisopropylcarbodiimide (15.9 µl) were added sequentially to the resin, and then the mixture was shaken for 1.5 h The reaction solution was filtered, and then the resin was washed with DMF 6 times. After confirmation of Kaiser test negativity, 20% piperidine in DMF was added and the mixture was shaken for 1 min. The solution was filtered and then 20% piperidine in DMF was added again and the mixture was shaken for 20 minutes. The solution was filtered and the resin was washed 10 times with DMF. This cycle of condensation of an amino acid with Fmoc - removal of the Fmoc protecting group was repeated for the sequential condensation of Gln(Trt), Ala, Gln(Trt), Lys(Boc), Asp(OtBu), Leu, Aib, Ile*, Aib, Tyr(tBu) , Asp(OtBu), Ser(tBu), Thr(tBu), aMePhe, Thr(tBu)*, Gly, Glu(OtBu), Aib and Tyr(tBu) (*: overnight reaction). Next, the N-terminal group of Fmoc was removed by treatment with piperidine, and then DMF (200 μl), N,N'-bis(tert-butoxycarbonyl)-1H-pyrazolo-1-carboxamidine (31 mg) and DIPEA (17, 4 µl), and the mixture was shaken overnight. The reaction solution was filtered, and then DMF (200 µl), N,N'-bis(tert-butoxycarbonyl)-1H-pyrazolo-1carboxamidine (31 mg) and DIPEA (17.4 µl) were successively added to the resin and the mixture was shaken again during the night. The reaction solution was filtered, and then the resin was washed with DMF 6 times. After confirming the negative Kaiser test, the resin was washed with MeOH and then dried under reduced pressure to obtain 54.7 mg of BocNHC(=NBoc)-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-aMePhe- Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-AibIle-Aib-Leu-Asp(OtBu)-Lys(Boc)-Gln(Trt)-Ala-Gln(Trt)-Ala- Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-LeuLys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro- Pro-Ser(tBu)-Lys(Boc)-amide resin Sieber.

To 54.7 mg of the resulting resin, 1 ml of a mixture of TFA: m-cresol: thioanisole: ethanedhiol: H ₂ O: triisopropylsilane (80: 5: 5: 5: 2.5: 2.5) was added, and the mixture was stirred for 1 5 h. Diethyl ether was added to the reaction solution to ensure precipitation, and the precipitate was washed by repeating the action of removing the supernatant after centrifugation three times. The precipitate was extracted with 50% aqueous acetic acid and the resin was removed by filtration, followed by preparative HPLC using a Daisopak SP-100-5-ODS-P column (250x20 mm ID) [solution A: 0.1% TFA-water, solution B : acetonitrile containing 0.1% TFA, flow rate 8 ml/min, A/B: linear concentration gradient elution 62/38-52/48 (60 min)]. Fractions containing the desired product were collected and lyophilized to obtain 10.7 mg of white powder.

Mass spectrometry, (M+H)+ 4323.2 (calculated: 4323.2);

HPLC elution time: 7.3 min;

- 38 044242 elution conditions:

Column: Merck Chromolith Performance RP-18e (100x4.6 mm I.D.);

eluent: using solution A: 0.1% TFA-water, solution B: acetonitrile containing 0.1% TFA, A/B: elution with a linear concentration gradient 95/5-35/65 (10 min);

flow rate: 3.0 ml/min.

Example 23.

Synthesis of H-NMeTyr-Aib-Glu-Gly-Thr-aMePhe-Thr-Ser-Asp-Tyr-Aib-Ile-Aib-Leu-Asp-Lys-Gln-Ala-GlnAla-Glu-Phe-Val-Lys-Trp -Leu-Leu-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys-NH ₂ (SEQ ID NO: 35).

H-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-AlaPro- Sieber Pro-Pro-Ser(tBu)-Lys(Boc)-amide resin (0.253 meq/g, 39.5 mg) prepared according to Reference Example 1 was weighed into a reaction tube and swollen with DMF. After removing the DMF by filtration, Fmoc-Ala-OH (31.1 mg), 0.5 M Oxymapure in DMF (200 µl) and diisopropylcarbodiimide (15.9 µl) were added sequentially to the resin, and then the mixture was shaken for 1.5 h The reaction solution was filtered, and then the resin was washed with DMF 6 times. After confirmation of Kaiser test negativity, 20% piperidine in DMF was added and the mixture was shaken for 1 min. The solution was filtered and then 20% piperidine in DMF was added again and the mixture was shaken for 20 minutes. The solution was filtered and the resin was washed 10 times with DMF. This cycle of condensation of an amino acid with Fmoc - removal of the Fmoc protecting group was repeated for the sequential condensation of Gln(Trt), Ala, Gln(Trt), Lys (Boc), Asp(OtBu), Leu, Aib, Ile*, Aib, Tyr(tBu) , Asp(OtBu), Ser(tBu), Thr(tBu), aMePhe, Thr(tBu)*, Gly, Glu(OtBu), Aib and NMeTyr(tBu) (*: overnight reaction). The resin was washed with MeOH and then dried under reduced pressure to obtain 66.2 mg of HNMeTyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-aMePhe-Thr(tBu)-Ser(tBu)-Asp(OtBu )-Tyr(tBu)-Aib-Ile-Aib-LeuAsp(OtBu)-Lys(Boc)-Gln(Trt)-Ala-Gln(Trt)-Ala-Glu(OtBu)-Phe-Val-Lys(Boc) -Trp(Boc)-Leu-Leu-Lys(Boc)Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc)- Sieber amide resin.

To 66.2 mg of the resulting resin, 1 ml of a mixture of TFA: m-cresol: thioanisole: ethanedhiol: H ₂ O: triisopropylsilane (80: 5: 5: 5: 2.5: 2.5) was added, and the mixture was stirred for 1 5 h. Diethyl ether was added to the reaction solution to ensure precipitation, and the precipitate was washed by repeating the action of removing the supernatant after centrifugation three times. The precipitate was extracted with 50% aqueous acetic acid and the resin was removed by filtration, followed by preparative HPLC using a Daisopak SP-100-5-ODS-P column (250x20 mm ID) [solution A: 0.1% TFA-water, solution B : acetonitrile containing 0.1% TFA, flow rate 8 ml/min, A/B: linear concentration gradient elution 63/37-53/47 (60 min)]. Fractions containing the desired product were collected and lyophilized to obtain 9 mg of white powder.

Mass spectrometry, (M+H)+ 4295.1 (calculated: 4295.2);

HPLC elution time: 7.2 min;

elution conditions:

Column: Merck Chromolith Performance RP-18e (100x4.6 mm I.D.);

eluent: using solution A: 0.1% TFA-water, solution B: acetonitrile containing 0.1% TFA, A/B: elution with a linear concentration gradient 95/5-35/65 (10 min);

flow rate: 3.0 ml/min.

Example 24.

Synthesis of H-Tyr-Aib-Glu-Gly-Thr-aMePhe-Thr-Ser-Asp-Tyr-Aib-Lys-Tyr-Leu-Asp-Lys-Gln-Ala-Gln-AlaGlu-Phe-Val-Lys-Trp -Leu-Leu-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys-NH ₂ (SEQ ID NO: 36).

H-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-AlaPro- Sieber Pro-Pro-Ser(tBu)-Lys(Boc)-amide resin (0.244 meq/g, 41.0 mg) prepared according to Reference Example 1 was weighed into a reaction tube and swelled with DMF. After removing the DMF by filtration, Fmoc-Ala-OH (31.1 mg), 0.5 M Oxymapure in DMF (200 µl) and diisopropylcarbodiimide (15.9 µl) were added sequentially to the resin, and then the mixture was shaken for 1.5 h The reaction solution was filtered, and then the resin was washed with DMF 6 times. After confirmation of Kaiser test negativity, 20% piperidine in DMF was added and the mixture was shaken for 1 min. The solution was filtered and then 20% piperidine in DMF was added again and the mixture was shaken for 20 minutes. The solution was filtered and the resin was washed 10 times with DMF. This cycle of condensation of an amino acid with Fmoc - removal of the Fmoc protecting group was repeated for the sequential condensation of Gln(Trt), Ala, Gln(Trt), Lys(Boc), Asp(OtBu), Leu, Tyr(tBu), Lys(Boc)*, Aib, Tyr(tBu), Asp(OtBu), Ser(tBu), Thr(tBu), aMePhe, Thr(tBu)*, Gly, Glu(OtBu), Aib and Tyr(tBu) (*: overnight reaction ). The resin was washed with MeOH and then dried under reduced pressure to obtain 66.8 mg of HTyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-aMePhe-Thr(tBu)-Ser(tBu)-Asp(OtBu )-Tyr(tBu)-Aib-Lys(Boc)Tyr(tBu)-Leu-Asp(OtBu)-Lys(Boc)-Gln(Trt)-Ala-Gln(Trt)-Ala-Glu(OtBu)-Phe -Val-Lys(Boc)-Trp(Boc)-LeuLeu-Lys (Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu) -Lys(Boc)-amide resin Sieber.

- 39 044242

To 66.8 mg of the resulting resin, 1 ml of TFA: m-cresol: thioanisole: ethanedhiol: H ₂ O: triisopropylsilane (80: 5: 5: 5: 2.5: 2.5) was added and the mixture was stirred for 1 5 hours. Diethyl ether was added to the reaction solution to obtain a precipitate, and the precipitate was washed by repeating the action of removing the supernatant after centrifugation three times. The precipitate was extracted with 50% aqueous acetic acid and the resin was removed by filtration, followed by preparative HPLC using a Daisopak SP-100-5-ODS-P column (250x20 mm ID) [solution A: 0.1% TFA water, solution B: acetonitrile , containing 0.1% TFA, flow rate 8 ml/min, A/B: linear concentration gradient elution 66/34-56/44 (60 min)]. Fractions containing the desired product were collected and lyophilized to obtain 16.1 mg of white powder.

Mass spectrometry, (M+H)+ 4374.6 (calculated: 4374.2);

HPLC elution time: 6.9 min;

elution conditions:

Column: Merck Chromolith Performance RP-18e (100x4.6 mm I.D.);

eluent: using solution A: 0.1% TFA-water, solution B: acetonitrile containing 0.1% TFA, A/B: elution with a linear concentration gradient 95/5 - 35/65 (10 min);

flow rate: 3.0 ml/min.

Example 25.

Synthesis of H-Tyr-Aib-Glu-Gly-Thr-aMePhe-Thr-Ser-Asp-Tyr-Aib-Lys-Tyr-Leu-Asp-Lys-Gln-Ala-Gln-GlnGlu-Phe-Val-Lys-Trp -Leu-Leu-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys-NH2 (SEQ ID NO: 37).

H-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-AlaPro- Sieber Pro-Pro-Ser(tBu)-Lys(Boc)-amide resin (0.244 meq/g, 41.0 mg) prepared according to Reference Example 1 was weighed into a reaction tube and swollen with DMF. After removing the DMF by filtration, Fmoc-Gln(Trt)-OH (61.1 mg), 0.5 M Oxymapure in DMF (200 μL), and diisopropylcarbodiimide (15.9 μL) were added sequentially to the resin, and then the mixture was shaken for 1 .5 hours. The reaction solution was filtered, and then the resin was washed 6 times with DMF. After confirmation of Kaiser test negativity, 20% piperidine in DMF was added and the mixture was shaken for 1 min. The solution was filtered and then 20% piperidine in DMF was added again and the mixture was shaken for 20 minutes. The solution was filtered and the resin was washed 10 times with DMF. This cycle of condensation of an amino acid with Fmoc - removal of the Fmoc protecting group was repeated for the sequential condensation of Gln(Trt), Ala, Gln(Trt), Lys(Boc), Asp(OtBu), Leu, Tyr(tBu), Lys(Boc)*, Aib, Tyr(tBu), Asp(OtBu), Ser(tBu), Thr(tBu), aMePhe, Thr(tBu)*, Gly, Glu(OtBu), Aib and Tyr(tBu) (*: overnight reaction ). The resin was washed with MeOH and then dried under reduced pressure to obtain 55.0 mg of H-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-aMePhe-Thr(tBu)-Ser(tBu)-Asp (OtBu)-Tyr(tBu)-Aib-Lys(Boc)Tyr(tBu)-Leu-Asp(OtBu)-Lys(Boc)-Gln(Trt)-Ala-Gln(Trt)-Gln(Trt)-Glu (OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro- Pro-Ser(tBu)-Lys(Boc)-amide resin Sieber.

To 55.0 mg of the resulting resin, 1 ml of a mixture of TFA: mcresol: thioanisole: ethanedhiol: H ₂ O: triisopropylsilane (80: 5: 5: 5: 2.5: 2.5) was added and the mixture was stirred for 1.5 h Diethyl ether was added to the reaction solution to obtain a precipitate, and the precipitate was washed by repeating the action of removing the supernatant after centrifugation three times. The precipitate was extracted with 50% aqueous acetic acid and the resin was removed by filtration, followed by preparative HPLC using a Daisopak SP-100-5-ODS-P column (250x20 mm ID) [solution A: 0.1% TFA-water, solution B : acetonitrile containing 0.1% TFA, flow rate 8 ml/min, A/B: linear concentration gradient elution 66/34-56/44 (60 min)]. Fractions containing the desired product were collected and lyophilized to obtain 13.7 mg of white powder.

Mass spectrometry, (M+H)+ 4431.5 (calculated: 4431.3);

HPLC elution time: 6.9 min;

elution conditions:

Column: Merck Chromolith Performance RP-18e (100x4.6 mm I.D.);

eluent: using solution A: 0.1% TFA-water, solution B: acetonitrile containing 0.1% TFA, A/B: elution with a linear concentration gradient 95/5-35/65 (10 min);

flow rate: 3.0 ml/min.

Example 26.

Synthesis of H-Tyr-Aib-Glu-Gly-Thr-aMePhe-Thr-Ser-Asp-Tyr-Aib-Lys-Tyr-Leu-Asp-Lys-Gln-Ala-Gln-GlnGlu-Phe-Val-Lys-Trp -Leu-Leu-Lys-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ (SEQ ID NO: 38).

H-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-AlaPro- Sieber Pro-Pro-Ser(tBu)-amide resin (0.188 meq/g, 53.2 mg) prepared according to Reference Example 2 was weighed into a reaction tube and swollen with DMF. After removing the DMF by filtration, Fmoc-Gln(Trt)-OH (61.1 mg), 0.5 M Oxymapure in DMF (200 μL), and diisopropylcarbodiimide (15.9 μL) were added sequentially to the resin, and then the mixture was shaken for 1 .5 hours. The reaction solution was filtered, and then the resin was washed 6 times with DMF. After confirmation

- 40 044242 negativity in the Kaiser test, a 20% solution of piperidine in DMF was added and the mixture was shaken for 1 minute. The solution was filtered and then 20% piperidine in DMF was added again and the mixture was shaken for 20 minutes. The solution was filtered and the resin was washed 10 times with DMF. This cycle of condensation of an amino acid with Fmoc - removal of the Fmoc protecting group was repeated for the sequential condensation of Gln(Trt), Ala, Gln(Trt), Lys(Boc), Asp(OtBu), Leu, Tyr(tBu), Lys(Boc)*, Aib, Tyr(tBu), Asp(OtBu), Ser(tBu), Thr(tBu), aMePhe, Thr(tBu)*, Gly, Glu(OtBu), Aib and Tyr(tBu) (*: overnight reaction ). The resin was washed with MeOH and then dried under reduced pressure to obtain 95.4 mg of HTyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-aMePhe-Thr(tBu)-Ser(tBu)-Asp(OtBu )-Tyr(tBu)-Aib-Lys(Boc)Tyr(tBu)-Leu-Asp(OtBu)-Lys(Boc)-Gln(Trt)-Ala-Gln(Trt)-Gln(Trt)-Glu(OtBu )-Phe-Val-Lys(Boc)-Trp(Boc)Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro- Sieber Ser(tBu)-amide resin.

To 95.4 mg of the resulting resin, 1 ml of a mixture of TFA: m-cresol: thioanisole: ethanedhiol: H ₂ O: triisopropylsilane (80: 5: 5: 5: 2.5: 2.5) was added and the mixture was stirred for 1. 5 hours. Diethyl ether was added to the reaction solution to obtain a precipitate, and the precipitate was washed by repeating the action of removing the supernatant after centrifugation three times. The precipitate was extracted with 50% aqueous acetic acid and the resin was removed by filtration, followed by preparative HPLC using a Daisopak SP-100-5-ODS-P column (250x20 mm ID) [solution A: 0.1% TFA water, solution B: acetonitrile , containing 0.1% TFA, flow rate 8 ml/min, A/B: 61/39-51/49 linear concentration gradient elution (60 min)]. Fractions containing the desired product were collected and lyophilized to obtain 20.0 mg of white powder.

Mass spectrometry, (M+H)+ 4303.0 (calculated: 4303.2);

HPLC elution time: 7.1 min;

elution conditions:

Column: Merck Chromolith Performance RP-18e (100x4.6 mm I.D.);

eluent: using solution A: 0.1% TFA-water, solution B: acetonitrile containing 0.1% TFA, A/B: elution with a linear concentration gradient 95/5-35/65 (10 min);

flow rate: 3.0 ml/min.

Test example 1.

Evaluation of agonist activity against human GIPR, human GLP-1R and human glucagon R using increased intracellular cAMP concentration as an indicator (1) Construction of an expression plasmid for the human GIPR gene.

The human GIPR gene, having a sequence identical to that of GenBank accession no. U39231, was cloned into the pMSRa-neo vector to produce hGIPR/pMSRa-neo.

(2) Construction of a cell expressing a reporter plasmid.

A luciferase reporter gene having an upstream cAMP response sequence was introduced into Cho-Kι cells to generate CRE-LUC/CHO-K1 cells.

(3) Construction of reporter plasmid.

Four copies of the cAMP response sequence and the zeocin resistance gene were inserted into the basic vector pGL3(R2.2) (Promega) to construct the Cre-luc(Zeo) reporter plasmid.

(4) Introducing the human GIPR gene into a CRE-LUC/CHO-K1 cell and obtaining an expression cell.

Plasmid hGIPR/pMSRa-neo, obtained according to (1), was introduced into CRE-LUC/CHO-K1 cells, obtained according to (2), to obtain transformants. Next, luciferase expression was induced in the cell line, i.e. hGIPR/CRE-LUC/CHO-K1 cells were selected from the resulting transformants by adding GIP.

(5) Construction of an expression plasmid for the human GLP-1R gene.

The human GLP-1R gene, having a sequence identical to that of GenBank accession no. NM_002062, was cloned into the pIRESneo3 vector to produce hGLP-1/pIRESneo3.

(6) Introducing the human GLP-1R gene and a reporter plasmid into a CHO-K1 cell and obtaining an expressing cell.

Cre-luc(Zeo), obtained according to (3), and the hGLP-1/pIRESneo3 plasmid, obtained according to (5), were introduced into CHO-K1 cells to obtain transformants. Next, luciferase expression was induced in the cell line, i.e. hGLP-1R/CRE-luc/CHO-K1 cells were selected from the resulting transformants by adding GLP-1.

(7) Construction of an expression plasmid for the human glucagon R gene.

The human glucagon R gene, having a sequence identical to that of GenBank accession no. NM_000160, was cloned into the pMSRa-neo vector to produce hGlucagonR/pMSRa-neo.

(8) Introducing the human glucagon R gene into CRE-LUC/CHO-K1 cells and obtaining an expression cell.

Plasmid hGlucagonR/pMSRa-neo, obtained according to (7), was introduced into CRE-LUC/CHO-K1 cells, obtained according to (2), to obtain transformants. Next, expression of luciferase was induced in the cell line, i.e. hGlucagonR/CRE-LUC/CHO-K1 cells were selected from the resulting transformants by adding glucagon.

(9) Reporter analysis.

hGIPR/CRE-LUC/CHO-K1 cells were inoculated at a cell density of 25 μl/well (5 x 10 ⁴ cells/well) into a 384-well white plate (Corning) and cultured overnight in Ham F12 medium containing 10% fetal bovine serum. , 100 U/ml penicillin and 100 µg/ml streptomycin in a CO ₂ incubator at 37°C. The medium containing the test compound was added to the cells at a concentration of 5 μl/well and the resulting cells were incubated for 4 hours in a CO ₂ incubator at 37°C to achieve a final concentration of 1 μM. PicaGene LT7.5 (Toyo Ink Co., Ltd.) was added to them at a concentration of 30 μl/well, and the mixture was shaken under light-protected conditions. After 30 min, luciferase activity was measured using an Envision plate reader (PerkinElmer). Luciferase activity in the presence of 10 nM GIP was determined to be 100%, and luciferase activity when DMSO was added instead of the test compound was determined to be 0%. GIPR agonist activity was calculated using the increase in intracellular cAMP concentration as an index. The results are presented in table. 2.

GLP-1R agonist activity was assayed as described above using hGLP-1R/CRE-luc/CHO-K1 cells. Luciferase activity in the presence of 10 nM GLP-1 was determined to be 100%, and luciferase activity resulting from the addition of DMSO instead of the test compound was determined to be 0%. GLP-1R agonist activity was calculated using the increase in intracellular cAMP concentration as an indicator. The results are presented in table. 2.

Glucagon R agonist activity was assayed as described above using hGlucagonR/CRE-LUC/CHO-K1 cells. Luciferase activity in the presence of 10 nM GLP-1 was determined to be 100%, and luciferase activity resulting from the addition of DMSO instead of the test compound was determined to be 0%. GLP-1R agonist activity was calculated using the increase in intracellular cAMP concentration as an indicator. The results are presented in table. 2.

As shown in table. 2, the compound of the present invention has an improved effect of activating GLP-1 receptors and GIP receptors. In addition, the compound of the present invention has a low glucagon receptor activating effect.

table 2

Example Agonist activity (ECso) hGIPR hGLP-lR hGCGR 1 8,IE-12 1.2E-11 >1.0E-06 2 6.8E-12 1.6E-11 >1.0E-06 3 7,IE-12 1.3E-11 >1.0E-06 4 4.9E-12 9.9E-12 >1.0E-06 5 5.5E-12 7.2E-12 >1.0E-06 6 7,ZE-12 1.6E-11 >1.0E-06 7 6.9E-12 8.8E-12 >1.0E-06 8 8,OE-12 1.4E-11 >1.0E-06 9 8,OE-12 1.5E-11 >1.0E-06 10 5.4E-12 8.3E-12 >1.0E-06 eleven 1.5E-11 2.2E-11 >1.0E-06 12 7.2E-12 1.4E-11 >1.0E-06 13 7.2E-12 1,IE-11 >1.0E-06 14 1.3E-11 2,IE-11 >1.0E-06 15 1.3E-11 1.5E-11 >1.0E-06 16 9,IE-12 1.5E-11 >1.0E-06 17 1.4E-11 1.8E-11 >1.0E-06 18 2.7E-11 1.3E-10 >1.0E-06 19 2.4E-11 3.7E-11 >1.0E-06 20 2.6E-11 1.3E-10 >1.0E-06 21 2.4E-11 5.5E-11 >1.0E-06 22 1.9E-11 3.5E-11 >1.0E-06 23 1.6E-11 1.6E-11 >1.6E-11 24 9.9E-12 1,IE-11 >1.0E-06 25 1.3E-11 1.4E-11 >1.0E-06 26 1.3E-11 1.5E-11 >1.0E-06

Test example 2.

Test of continuous subcutaneous administration for 2 days.

The food suppression activity of the test compound was tested in the manner described below.

The test compound was dissolved in a solvent (50% DMSO) to allow delayed

- 42 044242 release at a dose of 10 nmol/kg/day and the solution was filled with an Alzet Pump (DURECT Corporation, model: 1003D). The pump thus filled with the injection solution was immersed in saline solution for activation and then used. The pump was introduced in the following way. Each 8–9 week old male C57BL/6J mouse (20–26°C, allowed food and water ad libitum; 12 h light–12 h dark cycle) was anesthetized; their upper back skin was incised and the above mentioned pump was placed subcutaneously; the incision was sutured. After weighing, this mouse was returned to its housing cage (housed separately) and given pre-weighed food; Feed consumption was measured 2 days after the start of administration. Feed intake was calculated by subtracting the amount of feed remaining from the amount of feed dispensed on the day of administration. When feed intake in the vehicle-only control group was considered to be a 0% inhibition level, the food suppression activity of each test compound was assessed based on the cumulative feed intake after the start of administration for 2 days. The feed intake suppression rate (%) for the test compound was determined as (control feed intake - test compound feed intake)/Control feed intake x 100.

As shown in table. 3, the compound of the present invention has an improved feed suppression effect.

Table 3

Example Feed suppression (%) 1 42, 68 2 64, 80 3 46, 49 4 57, 01 5 60, 99 6 65.20 8 48.34 9 45, 23 10 62, 04 eleven 49, 99 12 55, 07 13 57, 12 14 47.47 15 58, 80 16 54.24 17 55, 42 19 34.79 22 35, 51 23 52, 62 24 54, 18 25 53, 12 26 53, 06

Test example 3.

Test of continuous subcutaneous administration for 2 weeks in DIO mice.

The anti-obesity activity of the test compound was tested in the manner described below.

Diet-induced obesity (DIO) mice were generated by feeding male C57BL/6J mice a high-fat diet (D12451: Research Diets, Inc.). The test compound was dissolved in a vehicle (50% DMSO) for sustained release at a dose of 1 nmol/kg/day and the solution was filled into an Alzet Pump (DURECT Corporation, model: 1002). The pump thus filled with the injection solution was immersed in saline solution for activation and then used. The pump was introduced in the following way. Each male DIO-C57BL/6J mouse, 35–37 weeks of age (20–26°C, allowed food and water ad libitum; 12 h light/12 h dark cycle) was anesthetized; their upper back skin was incised and the above mentioned pump was placed subcutaneously; the incision was sutured. After weighing, this mouse was returned to its housing cage (housed separately) and given pre-weighed food; body weight was measured every 1-3 days after the start of administration. The anti-obesity activity of each test compound was assessed based on the rate of weight loss 2 weeks after the start of administration, with the rate of weight loss in the vehicle-only control group being taken as 0%.

As shown in table. 4, the compound of the present invention has excellent active

- 43 044242 stu against obesity.

Table 4

Example Change in body weight 2 -16, 3 24 -11.1 25 -11.2 26 -16.2

Test example 4.

Solubility test.

The solubility of the test compound was assessed by the method described below.

An accurate measurement was made for approximately 2 mg of test compound. Solvents (10 µl, 20 µl or 40 µl) with different pH values (Britton-Robinson buffer (pH 3, 5, 7, 9)) were added to each test compound at 25°C and solubility was examined visually.

As shown in table. 5, all tested compounds demonstrated high solubility at each pH value.

Table 5

Example Solubility (mg/ml) pH 3 pH 5 pH 7 pH 9 2 >100 >100 >50 >50 24 >200 >200 >200 >200 25 >200 >200 >200 >200 26 >200 >200 >200 >200

Compilation example 1

(1) Compound of Example 1 10.0 mg (2) Lactose 70.0 mg (3) Corn starch 50.0 mg (4) Soluble starch 7.0 mg (5) Magnesium stearate 3.0 mg

The compound of Example 1 (10.0 mg) and magnesium stearate (3.0 mg) were granulated with aqueous soluble starch (0.07 ml) (7.0 mg soluble starch), dried and mixed with lactose (70.0 mg) and corn starch (50.0 mg). The mixture is compressed to obtain a tablet.

Compilation example 2

(1) Compound of Example 1 5.0 mg (2) Sodium chloride 20.0 mg (3) Distilled water up to a total amount of 2 ml

The compound of Example 1 (5.0 mg) and sodium chloride (20.0 mg) were dissolved in distilled water and water was added to a total amount of 2.0 ml. The solution is filtered and filled into a 2 ml ampoule under aseptic conditions. The ampoule is sterilized and tightly closed to obtain a solution for injection.

Industrial applicability

The compound of the present invention has excellent GLP-1 receptor/GIP receptor coagonist activity and is useful as a medicament for the prevention or treatment of various GLP-1 receptor/GIP receptor associated diseases, such as obesity.

All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

Free text sequence list.

SEQ ID NO: 1: artificial sequence (synthetic peptide (formula (I)));

SEQ ID NO: 2: artificial sequence (synthetic peptide (C-terminal sequence));

SEQ ID NO: 3: artificial sequence (synthetic peptide (C-terminal sequence));

SEQ ID NO: 4: artificial sequence (synthetic peptide (C-terminal sequence));

SEQ ID NO: 5: artificial sequence (synthetic peptide (C-terminal sequence));

SEQ ID NO: 6: artificial sequence (synthetic peptide (C-terminal sequence));

SEQ ID NO: 7: artificial sequence (synthetic peptide (C-terminal sequence));

SEQ ID NO: 8: artificial sequence (synthetic peptide (C-terminal sequence));

-

Claims (6)

SEQ ID NO: 9: artificial sequence (synthetic peptide (C-terminal sequence)); SEQ ID NO: 10: artificial sequence (synthetic peptide (Reference Example 1)); SEQ ID NO: 11: artificial sequence (synthetic peptide (Reference Example 2)); SEQ ID NO: 12: artificial sequence (synthetic peptide (Reference Example 3)); SEQ ID NO: 13: artificial sequence (synthetic peptide (example 1)); SEQ ID NO: 14: artificial sequence (synthetic peptide (example 2)); SEQ ID NO: 15: artificial sequence (synthetic peptide (example 3)); SEQ ID NO: 16: artificial sequence (synthetic peptide (example 4)); SEQ ID NO: 17: artificial sequence (synthetic peptide (example 5)); SEQ ID NO: 18: artificial sequence (synthetic peptide (example 6)); SEQ ID NO: 19: artificial sequence (synthetic peptide (example 7)); SEQ ID NO: 20: artificial sequence (synthetic peptide (example 8)); SEQ ID NO: 21: artificial sequence (synthetic peptide (example 9)); SEQ ID NO: 22: artificial sequence (synthetic peptide (example 10)); SEQ ID NO: 23: artificial sequence (synthetic peptide (example 11)); SEQ ID NO: 24: artificial sequence (synthetic peptide (example 12)); SEQ ID NO: 25: artificial sequence (synthetic peptide (example 13)); SEQ ID NO: 26: artificial sequence (synthetic peptide (example 14)); SEQ ID NO: 27: artificial sequence (synthetic peptide (example 15)); SEQ ID NO: 28: artificial sequence (synthetic peptide (example 16)); SEQ ID NO: 29: artificial sequence (synthetic peptide (example 17)); SEQ ID NO: 30: artificial sequence (synthetic peptide (example 18)); SEQ ID NO: 31: artificial sequence (synthetic peptide (example 19)); SEQ ID NO: 32: artificial sequence (synthetic peptide (example 20)); SEQ ID NO: 33: artificial sequence (synthetic peptide (example 21)); SEQ ID NO: 34: artificial sequence (synthetic peptide (example 22)); SEQ ID NO: 35: artificial sequence (synthetic peptide (example 23)); SEQ ID NO: 36: artificial sequence (synthetic peptide (example 24)); SEQ ID NO: 37: artificial sequence (synthetic peptide (example 25)); SEQ ID NO: 38: artificial sequence (synthetic peptide (example 26)). CLAIM 1. A peptide or a pharmaceutically acceptable salt thereof selected from H-Tyr-Aib-Glu-Gly-Thr-ocMePhe-Thr-Ser-Asp-Tyr-Aib-Lys-Tyr-LeuAsp-Lys-Gln-Ala-Gln-Ala-Glu-Phe-Val-Lys-Trp- Leu-Leu-Lys-Gly-GlyPro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys-NH ₂ , H-Tyr-Aib-Glu-Gly-Thr-ocMePhe-Thr-Ser-Asp-Tyr-Aib-Lys-Tyr-LeuAsp-Lys-Gln-Ala-Gln-Gln-Glu-Phe-Val-Lys-Trp- Leu-Leu-Lys-Gly-GlyPro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys-NH ₂ , and H-Tyr-Aib-Glu-Gly-Thr-ocMePhe-Thr-Ser-Asp-Tyr-Aib-Lys-Tyr-LeuAsp-Lys-Gln-Ala-Gln-Gln-Glu-Phe-Val-Lys-Trp- Leu-Leu-Lys-Gly-GlyPro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ . 2. A medicinal product for the prevention or treatment of obesity or diabetes in a mammal, containing a peptide or a pharmaceutically acceptable salt thereof according to claim 1. 3. A method for preventing or treating obesity or diabetes in a mammal, comprising administering an effective amount of the peptide or a pharmaceutically acceptable salt thereof according to claim 1. 4. A method of activating the GLP-1 receptor and the GIP receptor in a mammal, comprising administering an effective amount of the peptide or a pharmaceutically acceptable salt thereof according to claim 1. 5. Use of the peptide or a pharmaceutically acceptable salt thereof according to claim 1 for the preparation of a medicament for the prevention or treatment of obesity or diabetes in a mammal. - 45 044242 6. Use of the peptide or a pharmaceutically acceptable salt thereof according to claim 1 for the prevention or treatment of obesity or diabetes in a mammal. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky lane, 2