CN101316625A

CN101316625A - Neuropeptide-2 receptor-agonists

Info

Publication number: CN101316625A
Application number: CNA2006800443770A
Authority: CN
Inventors: 卡琳·康德-纳普; 瓦利德·丹霍; 乔治·埃利希; 纳德·佛投赫; 戴维·C·弗里; 瓦吉哈·卡恩; 阿尼希·孔卡; 克里斯蒂娜·马莎·龙迪诺内; 约瑟夫·斯威斯托克; 丽贝卡·安妮·陶布; 吉斐逊·W·蒂勒比
Original assignee: F Hoffmann La Roche AG
Current assignee: F Hoffmann La Roche AG
Priority date: 2005-12-07
Filing date: 2006-11-27
Publication date: 2008-12-03
Also published as: ZA200804919B; UA90039C2

Abstract

Provided herein are neuropeptide-2 receptor agonists of the formula (I): as well as pharmaceutically acceptable salts, derivatives and fragments thereof, wherein the substituents are as those disclosed in the specification. These compounds, and the pharmaceutical compositions containing them, are useful for the treatment of diseases such as, for example, obesity and diabetes.

Description

Neuropeptide-2 receptor-agonists

The present invention relates to PYY _3-36The truncate analog.Described analog is the agonist of neuropeptide-2 receptor-and is used for the treatment of metabolic disease and disease, as for example obesity, and type 2 diabetes mellitus, metabolism syndrome, insulin resistance and dyslipidemia.Neuropeptide-2 receptor-agonists of the present invention is suc as formula shown in (I):

Wherein:

X is 4-oxo-6-(1-piperazinyl)-3 (4H)-quinazoline-acetic acid (Pqa),

Y is H, and acyl moiety replaces or unsubstituted alkyl, replaces or unsubstituted low alkyl group, replaces or unsubstituted aryl, replaces or unsubstituted heteroaryl, replaces or unsubstituted alkoxyl polyalkylene glycol moiety, PEG _m-SSA, PEG _m-β-SBA, PEG _m-SPA or PEG _m-BTC,

Y ' is H, polyalkylene glycol moiety, PEG _m-SSA, PEG _m-β-SBA, PEG _m-SPA or PEG _m-BTC,

R ₁Be Ile, Ala, (D) Ile, N-methyl Ile, Aib, 1-1Aic, 2-2 Aic, Ach or Acp,

R ₂Be Lys, Ala, (D) Lys, NMelys, Nle or (Lys-Gly),

R ₃Be Arg, Ala, (D) Arg, N-methyl Arg, Phe, 3,4,5-trifluoro Phe or 2,3,4,5,6-five fluorine Phe,

R ₄Be His, Ala, (D) His, N-methyl His, 4-MeOApc, 3-Pal or 4-Pal,

R ₅Be Tyr, Ala, (D) Tyr, N-methyl Tyr, Trp, Tic, Bip, Dip, (1) Nal, (2) Nal, 3,4,5-trifluoro Phe or 2,3,4,5,6-five fluorine Phe,

R ₆Be Leu, Ala, (D) Leu or N-methyl Leu,

R ₇Be Asn, Ala or (D) Asn,

R ₈Be Leu or Trp,

R ₉Be Val, Ala, (D) Val or N-methyl Val,

R ₁₀Be Thr, Ala or N-methyl Thr,

R ₁₁Be Arg, (D) Arg or N-methyl Arg,

R ₁₂Be Gln or Ala,

R ₁₃Be Arg, (D) Arg or N-methyl Arg,

R ₁₄Be Tyr, (D) Tyr or N-methyl Tyr, modification-Tyr, Phe, modification-Phe, Cha, (1) Nal, (2) Nal, C-Alpha-Methyl Tyr or Trp and

PEG _mBe 1-60KDa,

Or its pharmaceutical salts.

Incorporate the document of this paper all references into this paper clearly as a reference hereby.

Metabolic disease and disease are the serious health problems that developed country has extensively recognized, it has reached popular level in the U.S..For example, about the research of obesity, the U.S. population above 50% is considered to overweight according to recently, surpasses 25% and is diagnosed as obesity clinically and has sizable risk to develop into heart disease, type 2 diabetes mellitus and some cancer.These epidemic diseases become the significant burden of health care system, surpass 70,000,000,000 dollars plan bariatrician expense because only just have in the U.S. every year.The strategy of treatment of obesity comprises the minimizing food intake and increases energy expenditure.

Neuropeptide tyrosine (NPY) is a kind of neurotransmitter of 36 amino acid peptides, is to have shown the neurotransmitter/neurohormonal pancreas polypeptide classification that is present among periphery and the central nervous system.NPY is one of known the most effective appetizer and is presented in the food intake of regulating the animal that comprises the people and has important function.

Cloned 6 kinds of neuropeptide Y receptors (NPY), Y1-, Y2-, Y3-, Y4 and Y5-and Y6-hypotype, it belongs to the 7-transmembrane receptor (GPCR) of rhodopsin sample G albumen coupling.NPY Y2 receptor (Y2R) is a kind of 381 amino acid whose receptors, and it passes through G _iSuppress the activation of adenyl cyclase, show low homology simultaneously with other known npy receptor.Between rat and people Y2 receptor, there is high conservative, has 98% aminoacid homogeneity.

The Y2R receptor extensively is distributed among rodent and people's the central nervous system.At hypothalamus, Y2 mRNA is positioned at arcuate nucleus, in NPO and the dorsomedial nucleus.In human brain, Y2R is main Y receptor subtype.In arcuate nucleus, surpass 80% NPY neuron coexpression Y2R mRNA.The application demonstration of Y2-selective agonist has reduced external release from the hypothalamus section, and the non-peptide antagonists BIIE0246 of Y2 increases NPY release.These find to support the effect of Y2R as presynaptic autoreceptor, and this autoreceptor is regulated the adjusting that NPY discharges and therefore can relate to feed.(Kaga T etc., Peptides (peptide) 22:501-506 (2001) and King PJ etc., Eur J Pharmacol (European pharmacology's magazine) 396:R1-3 (2000)).

Peptide YY _3-36(PYY _3-36) be 34 amino acid whose linear peptides, it has neuropeptide Y 2 (NPY2R) agonist activity.(IC) or intraperitoneal (IP) injection PYY in the arc nuclear have been shown _3-36Reduced the feed in the rat, and reduced weight increase as chronic treatment.Intravenous (IV) infusion (0.8pmol/kg/min) PYY _3-36In 24 hours, reduced obesity and normal volunteer's food intake in 90 minutes.These find to show that the PYY system can be treatment target (Batterham RL etc., Nature (nature) 418:650-654 (2002) of bariatrician; Batterham RL etc., New Engl J Med (New England's medical magazine) 349:941-948 (2003)).In addition, the Cys of PYY ²-(D) Cys ²⁷-cyclisation form, wherein residue 5-24 is substituted by the methene chain of 5-8 carbon length, shows the activation of small intestinal pyy receptor, reduces being confirmed as the electric current of the voltage clamp mucosa prepared product by jejunum in rats.(Krstenansky, etc. at Peptides .J.Smith and J.Rivier edit ESCOM. Leiden, 136-137 page or leaf among the Proceedings of theTwelfth American Peptide Symposium (peptide, the 12 U.S.'s peptide symposium)).

In addition, about superoxide dismutase (Somack, R is etc., (1991) free radical research communication (Free Rad Res Commun) 12-13:553-562; U.S. Patent number 5,283,317 and 5,468,478) and the protein of other type for example cytokine (Saifer, M G P is etc., (1997) PolymPreprints 38:576-577; Sherman, M R, Deng, (1997) at J M Harris, Deng, (Eds.), Poly (ethylene glycol) Chemistry and Biological Applications.ACSSymposium Series 680 (PEG chemistry and biologic applications .ACS symposium series 680) (155-169 page or leaf) Washington: American Chemical Society (american chemical association)), with Polyethylene Glycol or gather (oxygen ethylene) (all being called PEG) covalent modification protein.

In addition, nearest data show stomach bypass patient has early stage and significant the increasing of PYY level, and it may partly be responsible for early stage glycemic control and long term weight is kept, and the importance of this peptide in metabolic disease pathology is described.Other known action of PYY comprises: the stomach emptying of minimizing and the gastrointestinal transmission of delay, the glycemic control after meal that it is responsible for improving.The indication of hyperglycemia such as HbA _1CUse PYY with fructosamine periphery in the type 2 diabetes mellitus animal model _3-36The dependent minimizing of back show dose.Therefore, these results show PYY _3-36Or medicinal relevant agonist can provide the long-term treatment method at blood glucose and Weight control (Korner etc., J Clin Endocrinol Metabol (clinical endocrine metabolism magazine) 90:359-365 (2005); Chan JL etc., obesity (obesity) 14:194-198 (2006); Stratis C etc., Obes Surg (bariatric surgeries) 16:752-758 (2006); Borg CM etc., Br J Surg 93:210-215 (2006); With Pittner RA etc., Int J Obes (obesity international magazine) 28:963-971 (2004)).

Yet need the PYY analog of new transformation, it has low-molecular-weight, has identical simultaneously or better at Y1, the effectiveness and the selectivity of Y4 and Y5 receptor, pharmacokinetics character and pharmacological property.Preferably need to have the chemical compound of those longer active duratioies that exist than prior art.The analog that also needs the PEGization of PYY, thus proteinic half-life and the immunogenicity of minimizing in the experimenter of the such agonist of needs for example increased.

Fig. 1 shows the HPLC chromatogram of the reactant mixture that comprises chemical compound of the present invention (embodiment 34).

Fig. 2 shows the HPLC chromatogram of purifying compounds of the present invention (embodiment 34).

Fig. 3 shows the MALDI-TOF spectrum of chemical compound of the present invention (embodiment 34).

Fig. 4 shows the HPLC chromatogram of the reactant mixture of another kind of chemical compound of the present invention (embodiment 35).

Fig. 5 shows the HPLC chromatogram of the chemical compound (embodiment 35) of purification of the present invention.

Fig. 6 shows the MALDI-TOF spectrum of The compounds of this invention (embodiment 35).

Fig. 7 shows the HPLC chromatogram of the reactant mixture of The compounds of this invention (embodiment 36).

Fig. 8 shows the HPLC chromatogram of the chemical compound (embodiment 36) of purification of the present invention.

Fig. 9 shows the MALDI-TOF spectrum of the another kind of chemical compound of the present invention (embodiment 36).

Figure 10 shows the HPLC chromatogram of the reactant mixture of chemical compound of the present invention (embodiment 37).

Figure 11 shows the HPLC chromatogram of the chemical compound (embodiment 37) of purification of the present invention.

Figure 12 shows the MALDI-TOF spectrum of chemical compound of the present invention (embodiment 37).

Figure 13 shows the HPLC chromatogram of the reactant mixture of another kind of chemical compound of the present invention (embodiment 38).

Figure 14 shows the HPLC chromatogram of the chemical compound (embodiment 38) of purification of the present invention.

Figure 15 shows the MALDI-TOF spectrum of chemical compound of the present invention (embodiment 38).

Figure 16 shows the HPLC chromatogram of the reactant mixture of chemical compound of the present invention (embodiment 39).

Figure 17 shows the HPLC chromatogram of the chemical compound (embodiment 39) of purification of the present invention.

Figure 18 shows the MALDI-TOF spectrum of another kind of chemical compound of the present invention (embodiment 39).

The HPLC chromatogram that comprises the reactant mixture of chemical compound of the present invention (embodiment 41) before Figure 19 is presented at and protects.

Figure 20 shows the HPLC chromatogram of the de-protected reactant mixture of inclusion compound of the present invention (embodiment 41).

Figure 21 shows the HPLC chromatogram of the chemical compound (embodiment 41) of purification of the present invention.

Figure 22 shows the MALDI-TOF spectrum of chemical compound of the present invention (embodiment 41).

Figure 23 is presented at the influence of obesity (DIO) the rat Central Asia chronic administration chemical compound (embodiment 41) of male diet induced to body weight.

Figure 24 shows chemical compound (embodiment 41) acute effects to oral glucose tolerance test (OGTT) in female db/db mice.

Figure 25 is presented at the influence of the inferior chronic administration of chemical compound in the female db/db mice (embodiment 41) to basic blood glucose (A) and oral glucose tolerance test (B).

Although to those any methods similar or of equal value as herein described, device and material can be used for enforcement of the present invention or test, describe preferable methods now, device and material.

Write all peptide sequences that this paper mentions according to general stipulator, N terminal amino acid on the left side wherein, and the C terminal amino acid is on the right, unless otherwise noted.Short-term indication peptide bond between two amino acid residues.When aminoacid had isomeric forms, it was amino acid whose L shaped formula, unless otherwise specifically indicated.In order to describe convenience of the present invention, use various amino acid whose routines and unconventional abbreviation.These abbreviations are that those skilled in the art are familiar with, but list below for clear.

The Asp=D=aspartic acid; The Ala=A=alanine; The Arg=R=arginine; The Asn=N=agedoite; The Gly=G=glycine; Glu=E=glutamic acid; The Gln=Q=glutamine; The His=H=histidine; The Ile=I=isoleucine; The Leu=L=leucine; Lys=K=lysine; The Met=M=methionine; The Phe=F=phenylalanine; The Pro=P=proline; The Ser=S=serine; The Thr=T=threonine; The Trp=W=tryptophan; Tyr=Y=tyrosine; The Cys=C=cysteine; With the Val=V=valine; The Nle=nor-leucine.

For convenience, following abbreviation or symbol are used to represent part used in the present invention, reagent etc.

The Ac acetyl group;

The Aib α-An Jiyidingsuan;

1-1-Aic 1-aminoidan-1-carboxylic acid;

2-2-Aic 2-aminoidan-2-carboxylic acid;

Ach alpha-amido cyclohexane extraction-carboxylic acid;

Acp alpha-amido cycloheptane-carboxylic acid;

Tic alpha-amido-1,2,3,4, tetrahydroisoquinoline-3-carboxylic acid;

3-Pal alpha-amido-3-pyridine radicals alanine-carboxylic acid;

4-Pal alpha-amido-4-pyridine radicals alanine-carboxylic acid;

4-MeO-Apc 1-amino-4-(4-methoxyphenyl)-cyclohexane extraction-1-carboxylic acid;

Bip 4-phenyl-phenylalanine-carboxylic acid;

Dip 3,3-two phenylalanine-carboxylic acid;

Pqa 4-oxo-6-(1-piperazinyl)-3 (4H)-quinazoline-acetic acid

(CAS 889958-08-1)；

3,4,5, F3-Phe 3,4,5-trifluoro-benzene alanine;

2,3,4,5,6, F5-

Phe

2,3,4,5,6-phenyl-pentafluoride alanine;

The Cha Cyclohexylalanine;

(1) Nal 1-naphthyl alanine;

(2) Nal 2-naphthyl alanine;

Fmoc 9-fluorenyl methoxy carbonyl;

Mtt 4-methyl trityl;

2Pip 2-propyloxy phenyl base ester;

Pmc 2,2,5,7,8-pentamethyl benzo dihydropyran-6-sulfonyl;

CH ₂Cl ₂Dichloromethane;

The A2O acetic anhydride;

CH ₃The CN acetonitrile;

The DMAc dimethyl acetylamide;

The DMF dimethyl formamide;

DIPEA N, the N-diisopropylethylamine;

The TFA trifluoroacetic acid;

HOBT N-hydroxybenzotriazole;

DIC N, N '-DIC;

BOP benzotriazole-1-base oxygen base-three-(dimethylamino) phosphorus-

Hexafluorophosphate;

HBTU 2-(1H-benzotriazole-1-yl)-1,1,3, the 3-tetramethylurea-

Hexafluorophosphate;

NMP 1-methyl 2-Pyrolidenone;

SSA succinimido butanimide;

β-SBA succinimido β-butanoic acid;

The SPA succinyl phosphorons amino propyl acid;

BTC benzotriazole carbonic ester;

The substance assistant laser desorpted ionized flight time of MALDI-TOF;

The FAB-MS fast atom bombardment mass spectroscopy;

ES-MS electrospray mass spectrum;

PEG _m-SSA PEG _m-CH ₂CH ₂NHCOCH ₂CH ₂CO-；

PEG _m-β-SBA PEG _m-CH(CH ₃)CH ₂CO-；

PEG _m-SPA PEG _m-CH ₂CH ₂CO-；

PEG _m-BTC PEG _m-CO-; And

PEG _mIt is polyalkylene glycol moiety greater than about 1KDa.

When being used for this paper, term " alkyl " refers to side chain or straight chain, ring-type or non-annularity, and saturated or undersaturated alkyl, it can be substituted or not be substituted.When being ring-type, alkyl is C preferably ₃-C ₁₂, more preferably be C ₅To C ₁₀, more preferably be C ₅To C ₇When being non-annularity, described alkyl is C preferably ₁To C ₁₀, more preferably be C ₁To C ₆, more preferably be methyl, ethyl, propyl group (n-pro-pyl or isopropyl), butyl (normal-butyl, the isobutyl group or the tert-butyl group) or amyl group (comprising n-pentyl and isopentyl) they more preferably are methyl.Therefore, be appreciated that term " alkyl " comprises alkyl (side chain or straight chain) when being used for this paper, the alkyl of replacement (side chain or straight chain), the alkynyl (side chain or straight chain) that replaces, cycloalkyl, the cycloalkyl of replacement, cycloalkenyl group, the cycloalkenyl group of replacement, the cycloalkynyl radical of cycloalkynyl radical and replacement.Preferred alkyl is a non-annularity and saturated.

When being used for this paper, term " low alkyl group " refers to side chain or straight chain, ring-type or non-annularity, and saturated or undersaturated alkyl, wherein said cyclic low-grade alkyl is C ₅, C ₆Or C ₇, and wherein said non-annularity low alkyl group is C ₁, C ₂, C ₃Or C ₄, and preferably be selected from methyl, ethyl, propyl group (n-pro-pyl or isopropyl) or butyl (normal-butyl, the isobutyl group or the tert-butyl group).Therefore, should be understood that term " low alkyl group " when being used for this paper, comprise low alkyl group (side chain or straight chain), low-grade alkenyl (side chain or straight chain), low-grade alkynyl (side chain or straight chain), ring low alkyl group and ring low-grade alkenyl.Preferred low alkyl group is acyclic and saturated.

When being used for this paper, term " acyl group " refers to the alkyl that randomly replaces, cycloalkyl; heterocyclic radical, aryl or heteroaryl, it is via the carbonyl combination; and comprise group such as acetyl group, propiono, benzoyl; 3-pyridine radicals carbonyl, 2-morpholino carbonyl, 4-maloyl group; 4-fluorobenzene formoxyl; 2-naphthoyl base, 2-phenyl acetyl, 2-methoxyl group acetyl group etc.

When being used for this paper, term " aryl " refers to replace or unsubstituted carbocyclic aromatic group, as phenyl or naphthyl.

Term " heteroaryl " individually or with other moiety combinations, the monocycle or the bicyclo-atomic group that refer to 5-12 annular atoms, it has at least one and comprises and be selected from N, O, with of S, the aromatic rings of two or three ring hetero atoms, and remaining annular atoms is C, and condition is that the junction point of heteroaryl will be on aromatic rings.One or two ring carbon atom of heteroaryl can be by carbonyl substituted.

Alkyl, aryl and heteroaryl can be that replace or unsubstituted.When replacing, there are 1 to 3 substituent group, preferred 1 substituent group to exist usually.Substituent group can comprise: carbon-containing group such as alkyl, aryl, aryl alkyl (for example replace and unsubstituted phenyl, replace and unsubstituted benzyl); Halogen atom and the group such as the haloalkyl (for example, trifluoromethyl) that contain halogen atom; Oxy radical is as alcohol (for example hydroxyl, hydroxyalkyl, aryl (hydroxyl) alkyl), alkoxyl, aryloxy group, alkoxyalkyl, aryloxy alkyl, acyl group, acid is (for example, carboxyl, carboxyalkyl), acid derivative such as ester (for example, alkoxy carbonyl, alkoxy carbonyl alkyl, the alkyl-carbonyl oxygen base, alkyl-carbonyl oxygen base alkyl), amide is (for example, amino carbonyl, one-or two-alkyl amino-carbonyl, the amino carbonyl alkyl, one-or two-alkyl amino alkyl carbonyl, aromatic yl aminocarbonyl), carbamate (alkoxycarbonyl amino for example, aryloxy carbonyl amino, amino carbonyl oxygen base, one-or two-alkyl amino carbonyl oxy, aromatic yl aminocarbonyl oxygen base) and urea (for example-or two-alkyl amino-carbonyl amino or aromatic yl aminocarbonyl amino); Nitrogen-containing group such as amine (for example amino, one-or two-alkyl amino, aminoalkyl, one-or two-alkyl amino alkyl), azide, nitrile (for example cyano group, cyano group alkyl), nitro; Sulfur-containing group such as thio-alcohol, thioether, sulfoxide and sulfone (alkylthio group for example, alkyl sulphinyl, alkyl sulphonyl, alkylthio alkyl, the alkyl sulphinyl alkyl, the alkyl sulphonyl alkyl, arylthio, aryl sulfonyl kia, aryl sulfonyl, arylthio alkyl, aryl sulfonyl kia alkyl, aryl sulfonyl alkyl); With contain one or more, a preferred heteroatomic heterocyclic group (thienyl for example, furyl, pyrrole radicals, imidazole radicals, pyrazolyl, thiazolyl, isothiazolyl ， oxazolyl ， oxadiazole base, thiadiazolyl group, aziridinyl, azetidinyl, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, tetrahydrofuran base, pyranose, pyrans ketone group, pyridine radicals, pyrazinyl, pyridazinyl, piperidyl, six hydrogen azepines

Base, piperazinyl (peperazinyl), morpholinyl, thia naphthyl (thianaphthyl), benzofuranyl, isobenzofuran-base, indyl, hydroxyindole base, isoindolyl, indazolyl, indolinyl, 7-azaindolyl, benzopyranyl, coumarin base, isocoumarinyl, quinolyl, isoquinolyl, naphthridinyl, the cinnolines base, quinazolyl, pyridopyridine base benzoxazinyl, quinoxalinyl, chromenyl, chromanyl, the isochroman base, 2 base and carbolinyl (carbolinyl)).

Described low alkyl group can be that replace or unsubstituted, preferably unsubstituted.When replacing, there is 1 to 3 substituent group usually, preferred 1 substituent group.Substituent group comprises the substituent group group listed above except alkyl, aryl and aryl alkyl.

As used herein, term " alkoxyl " is meant that alkyl-O-and " alkanoyl " are meant alkyl-CO-.Alkoxy substituent or the substituent group group that contains alkoxyl can be replaced by one or more alkyl.

As used herein, term " halogen " is meant fluorine, chlorine, bromine or iodine group, preferred fluorine, chlorine or bromine group, and fluorine or chlorine group more preferably.

Pharmaceutical salts is meant the conventional acid addition salts of the biological effectiveness of freeze mode I chemical compound and character or base addition salts and can be formed by the acid of non-toxicity organic or inorganic or the organic or inorganic alkali that are fit to.The example of acid-addition salts comprise by mineral acid deutero-those and from organic acid deutero-those, described mineral acid example hydrochloric acid, hydrobromic acid, hydroiodic acid, sulphuric acid, sulfamic acid, phosphoric acid and nitric acid, described organic acid such as acetic acid, p-methyl benzenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, maleic acid, lactic acid, fumaric acid etc.The example of base addition salts comprises from ammonium hydroxide, potassium hydroxide, sodium hydroxide, and quaternary ammonium hydroxide, as for example Tetramethylammonium hydroxide deutero-those.With medicinal compound (being medicine) chemical modification is that salt is technology well known in the art, and it is used to attempt to improve physics or the chemical stability that relates to chemical compound, hygroscopicity for example, mobile or deliquescent character.See, for example H.Ansel etc., Pharmaceutical Dosage Forms and Drug Delivery Systems (pharmaceutical dosage form and drug delivery system) (the 6th edition .1995) the 196th and 1456-1457 page or leaf.

" medicinal ester " refers to have the conventional esterification compound of the formula I of carboxylic group, and the biological effectiveness of the chemical compound that described ester can freeze mode I and character and cracking in vivo (in vivo) are corresponding active carboxylic acid.Those that cleaved in vivo (being hydrolyzed in this case) replaced by low alkyl group for the example of the ester group of corresponding carboxylic acid is wherein cleaved hydrogen, described low alkyl group is randomly replaced, for example by heterocycle, replacements such as cycloalkyl.The example of the lower alkyl esters that replaces be wherein low alkyl group by pyrrolidine, piperidines, morpholine, those that N methyl piperazine etc. replace.Cleaved in vivo group can be for example ethyl, morpholino ethyl and diethylamino ethyl.Related to the present invention ,-CONH ₂Also be considered to ester, because-NH ₂Cleaved in vivo and alternative to form corresponding carboxylic acid by hydroxyl.

Term " modification-Tyr " refer to adorned by any way tyrosine, preferably by 1-3 substituent group, preferably 1-2 substituent group replaces modification, and described substituent group is independently selected from the group of being made up of rudimentary-alkyl and halogen.Preferred modification-Tyr is methyl-tyrosine, C-Alpha-Methyl-tyrosine (C-α-Me-Tyr) preferably, 3-iodo-tyrosine ((3-I) Y), 3,5-two fluoro-tyrosine ((3,5 two F) Y), 2,6-two fluoro-tyrosine ((2,6 two F) Y) and 2,6-dimethyl-tyrosine ((2,6 two Me) Y).Another preferably modifies-Tyr be between position-tyrosine ((m-) Y).Particularly preferred modification-Tyr is those that occur in the concrete below example.

Term " modification-Phe " refers to wherein adorned by any way phenylalanine, preferably modify with 1-4 substituent replacement, described substituent group is independently selected from the group of being made up of following: rudimentary-alkyl, hydroxyl-rudimentary-alkyl, fluoro-is rudimentary-alkyl, rudimentary-alkoxyl, amino and halogen.Preferred modification-Phe is 4-methoxyl group-phenylalanine (F (4-O-CH ₃)), 4-amino-phenylalanine ((4-NH ₂) Phe), 4-fluoro-phenylalanine ((4-F) Phe), 4-hydroxymethyl-phenylalanine ((4-CH ₂OH) 4-trifluoromethyl-phenylalanine ((4-CF Phe), ₃) Phe), 3-fluoro-phenylalanine ((3-F) Phe), 2,3,4,5,6-five fluoro-phenylalanine ((2,3,4,5,6-five-F) Phe) and 3,4-dichloro-benzenes alanine ((3,4 two-Cl) Phe).Particularly preferred modification-Tyr is those that occur in the specific embodiment below.

Term " hydroxyl-rudimentary-alkyl " refers to low alkyl group as defined above, and it is replaced by hydroxyl, is preferably replaced by hydroxymethyl.

Term " fluoro-rudimentary-alkyl " refers to rudimentary as defined above-alkyl, and it is by fluorine list or polysubstituted.Fluoro-is rudimentary-and the example of alkyl is CFH for example ₂, CF ₂H, CF ₃, CF ₃CH ₂, CF ₃(CH ₂) ₂, (CF ₃) ₂CH and CF ₂H-CF ₂, CF preferably ₃

Particularly, the present invention relates to the neuropeptide-2 receptor-agonists of formula (I):

Wherein:

X is 4-oxo-6-(1-piperazinyl)-3 (4H)-quinazoline-acetic acid (Pqa),

R ₁Be Ile, Ala, (D) Ile, N-methyl Ile, Aib, 1-1 Aic, 2-2 Aic, Ach or Acp,

R ₂Be Lys, Ala, (D) Lys, NMelys, Nle or (Lys-Gly),

R ₄Be His, Ala, (D) His, N-methyl His, 4-MeOApc, 3-Pal or 4-Pal,

R ₆Be Leu, Ala, (D) Leu or N-methyl Leu,

R ₇Be Asn, Ala or (D) Asn,

R ₈Be Leu or Trp,

R ₉Be Val, Ala, (D) Val or N-methyl Val,

R ₁₀Be Thr, Ala or N-methyl Thr,

R ₁₁Be Arg, (D) Arg or N-methyl Arg,

R ₁₂Be Gln or Ala,

R ₁₃Be Arg, (D) Arg or N-methyl Arg,

R ₁₄Be Tyr, (D) Tyr or N-methyl Tyr, modification-Tyr, Phe, modification-Phe, Cha, (1) Nal, (2) Nal, C-Alpha-Methyl Tyr or Trp, and

PEG _mBe 1 to 60KDa,

Or its pharmaceutical salts.

The chemical compound of formula (I) is preferred separately and its pharmaceutical salts is preferred separately, and the chemical compound of its Chinese style (I) is particularly preferred.

Preferred as mentioned above neuropeptide-2 receptor-agonists is to be characterised in that those of formula (Ia):

Y-R ₁-R ₂-X-R ₃-R ₄-R ₅-R ₆-R ₇-R ₈-R ₉-R ₁₀-R ₁₁-R ₁₂-R ₁₃-R ₁₄-NH ₂(Ia)

Wherein:

X is N-piperazine-1-base-4 (3H)-quinazolinone-3-acetic acid (Pqa),

Y is H, and acyl moiety replaces or unsubstituted alkyl, replaces or unsubstituted low alkyl group, replaces or unsubstituted aryl, replace or unsubstituted alkoxyl, and polyalkylene glycol moiety, PEG-SSA, PEG-β-SBA, PEG-SPA or PEG-BTC,

R ₁Be Ile, Ala, (D) Ile, N-methyl Ile, Aib, 1-1 Aic, 2-2 Aic, Ach or Acp,

R ₂Be Lys, Ala, (D) Lys, NMelys, Nle or (Lys-Gly),

R ₄Be His, Ala, (D) His, N-methyl His, 4-MeOApc, 3-Pal or 4-Pal,

R ₆Be Leu, Ala, (D) Leu or N-methyl Leu,

R ₇Be Asn, Ala or (D) Asn,

R ₈Be Leu or Trp,

R ₉Be Val, Ala, (D) Val or N-methyl Val,

R ₁₀Be Thr, Ala or N-methyl Thr,

R ₁₁Be Arg, (D) Arg or N-methyl Arg,

R ₁₂Be Gln or Ala,

R ₁₃Be Arg, (D) Arg or N-methyl Arg, and

R ₁₄Be Tyr, (D) Tyr or N-methyl Tyr, modification-Tyr, Phe, modification-Phe or Trp,

Or its pharmaceutical salts.

The preferred embodiment of the invention relates to neuropeptide-2 receptor-agonists as defined above, wherein R ₂By Y ' replacement, and Y ' is H, polyalkylene glycol moiety, PEG _m-SSA, PEG _m-β-SBA, PEG _m-SPA or PEG _m-BTC.More preferably, Y ' is a polyalkylene glycol moiety, PEG _m-SSA, PEG _m-β-SBA, PEG _m-SPA or PEG _m-BTC.

Another embodiment preferred of the present invention relates to neuropeptide-2 receptor-agonists as defined above, wherein

Y is H or acyl moiety, and

Y ' is a polyalkylene glycol moiety, PEG _m-SSA, PEG _m-β-SBA, PEG _m-SPA or PEG _m-BTC.More preferably, Y ' is PEG _m-SSA or PEG _m-SPA.

Preferably, Y is an acyl moiety.In another preferred embodiment, Y can be H.In addition, preferably Y ' can be H.

In neuropeptide-2 receptor-agonists as defined above, PEG preferably _mBe 20 to 40KDa.Preferably, PEG _mBe 30KDa.Preferably, polyalkylene glycol moiety has the molecular weight (weight) of 1-60KDa, and more preferably 20 to 40KDa, the more preferably molecular weight of 30KDa.

In neuropeptide-2 receptor-agonists as defined above, preferably, R ¹Be Ile.In addition, R preferably ²Be Lys or Nle.In addition, R preferably ³Be Arg.In addition, R preferably ⁴Be His.In addition, R preferably ⁵Be Tyr.In addition, R preferably ⁶Be Leu.In addition, R preferably ⁷Be Asn.In addition, R preferably ⁸Be Leu or Trp.In addition, preferably R9 is Val.In addition, R preferably ¹⁰Be Thr.In addition, R preferably ¹¹Be Arg.In addition, R preferably ¹²Gln.In addition, R preferably ¹³Be Arg or (N-methyl) Arg.

In addition, R preferably ₁₄Be Tyr, (D) Tyr or N-methyl Tyr, modification-Tyr, Phe, modification-Phe or Trp.Preferably, R ¹⁴Be Y, (m-) Y, (3-I) Y, (3,5 two F) Y, (2,6 two F) Y, (2,6 two Me) Y, F (4-O-CH ₃), F, (4-NH ₂) Phe, (4-F) Phe, (4-CH ₂OH) Phe, (4-CF ₃) Phe, (3-F) Phe, (2,3,4,5,6 five F) Phe, (3,4 two Cl) Phe, Cha, W, (1) Nal, (2) Nal or C-α-Me-Tyr.More preferably, R ¹⁴Be Tyr or (2,6 two F) Tyr.

Preferred as mentioned above neuropeptide-2 receptor-agonists is to be selected from by those of the following group of forming:

IK-Pqa-RHYLNLVTRQRY，

IK-Pqa-RHYLNLVTRQ (N-methyl) RY,

IK-Pqa-RHYLNLVTRQ (N-methyl) R (m-) Y,

IK-Pqa-RHYLNLVTRQ (N-methyl) R (3-I) Y,

IK-Pqa-RHYLNLVTRQ (N-methyl) R (3,5 two F) Y,

IK-Pqa-RHYLNLVTRQ (N-methyl) R (2,6 two F) Y,

IK-Pqa-RHYLNLVTRQ (N-methyl) R (2,6 two Me) Y,

IK-Pqa-RHYLNLVTRQ (N-methyl) RF (4-O-CH ₃),

IK-Pqa-RHYLNLVTRQ (N-methyl) RF,

IK-Pqa-RHYLNLVTRQ (N-methyl) R (4-NH ₂) Phe,

IK-Pqa-RHYLNLVTRQ (N-methyl) R (4-F) Phe,

IK-Pqa-RHYLNLVTRQ (N-methyl) R (4-CH ₂OH) Phe,

IK-Pqa-RHYLNLVTRQ (N-methyl) R (4-CF ₃) Phe,

IK-Pqa-RHYLNLVTRQ (N-methyl) R (3-F) Phe,

IK-Pqa-RHYLNLVTRQ (N-methyl) R (2,3,4,5,6 five F) Phe,

IK-Pqa-RHYLNLVTRQ (N-methyl) R (3,4 two Cl) Phe,

IK-Pqa-RHYLNLVTRQ (N-methyl) RCha,

IK-Pqa-RHYLNLVTRQ (N-methyl) RW,

IK-Pqa-RHYLNLVTRQ (N-methyl) R (1) Nal,

IK-Pqa-RHYLNLVTRQ (N-methyl) R (2) Nal,

IK-Pqa-RHYLNLVTRQR-C-α-Me-Tyr，

IK-Pqa-RHYLNWVTRQ (N-methyl) RY,

INle-Pqa-RHYLNWVTRQ (N-methyl) RY,

Ac-IK-Pqa-RHYLNWVTRQ (N-methyl) R (2,6 two F) Y,

Ac-IK-Pqa-RHYLNWVTRQ (N-methyl) RY,

Amyl group-IK-Pqa-RHYLNWVTRQ (N-methyl) RY,

Pivaloyl group-IK-Pqa-RHYLNWVTRQ (N-methyl) RY,

Cyclohexyl-IK-Pqa-RHYLNWVTRQ (N-methyl) RY,

Benzoyl-IK-Pqa-RHYLNWVTRQ (N-methyl) RY,

Adamantyl-IK-Pqa-RHYLNWVTRQ (N-methyl) RY,

(PEG 30,000 SPA) IK-Pqa-RHYLNWVTRQ (N-methyl) RY,

(PEG40,000 BTC)-IK-Pqa-RHYLNWVTRQ (N-methyl RY,

(PEG30,000)-SSA-INle-Pqa-RHYLNWVTRQ (N-methyl) RY,

(PEG30,000)-β-SBA-INle-Pqa-RHYLNWVTRQ (N-methyl) RY,

Ac-Ile-Lys (PEG30,000 SPA)-Pqa-RHYLNWVTRQ (N-methyl) RY,

Ac-Ile-Lys (PEG30,000 SSA)-Pqa-RHYLNWVTRQ (N-methyl) RY and

IK (PEG30,000 SSA)-Pqa-RHYLNWVTRQ (N-methyl) RY,

Or its pharmaceutical salts.

Other aforesaid preferred neuropeptide-2 receptor-agonists is to be selected from by those of the following group of forming:

Ac-Ile-Lys (PEG30,000 SPA)-Pqa-RHYLNWVTRQ (N-methyl) RY,

Ac-Ile-Lys (PEG30,000 SSA)-Pqa-RHYLNWVTRQ (N-methyl) RY and

IK (PEG30,000 SSA)-Pqa-RHYLNWVTRQ (N-methyl) RY,

Or its pharmaceutical salts.

Preferred aforesaid chemical compound, it does not exist as the pharmaceutical salts form.Above-mentioned each individuation compound of mentioning constitutes embodiment preferred respectively.

Particularly preferred neuropeptide-2 receptor-agonists as defined above is so a kind of, and wherein said agonist is Ac-IK-Pqa-RHYLNWVTRQ (N-methyl) R (2-6 two F) Y.

Another kind of particularly preferred neuropeptide-2 receptor-agonists as defined above is so a kind of, and wherein said agonist is Ac-IK-Pqa-RHYLNWVTRQ (N-methyl) RY.

Another kind of particularly preferred neuropeptide-2 receptor-agonists as defined above is so a kind of, and wherein said agonist is (PEG 30,000)-SPA-IK-Pqa-RHYLNWVTRQ (N-methyl) RY.

Another kind of particularly preferred neuropeptide-2 receptor-agonists as defined above is so a kind of, and wherein said agonist is (PEG30,000)-SSA-INle-Pqa-RHYLNWVTRQ (N-methyl) RY.

Another kind of particularly preferred neuropeptide-2 receptor-agonists as defined above is so a kind of, and wherein said agonist is Ac-Ile-Lys (PEG30,000 SSA)-Pqa-RHYLNWVTRQ (N-methyl) RY.

Another kind of particularly preferred neuropeptide-2 receptor-agonists as defined above is so a kind of, and wherein said agonist is H-Ile-Lys (PEG30,000 SSA)-Pqa-RHYLNWVTRQ (N-methyl) RY.

Aforesaid neuropeptide-2 receptor-agonists is not a PEGization, and promptly wherein Y and Y ' are not polyalkylene glycol moieties, PEG _m-SSA, PEG _m-β-SBA, PEG _m-SPA or PEG _m-BTC, it also can be as the intermediate of preparation chemical compound, and wherein one or two among Y and the Y ' is polyalkylene glycol moiety, PEG _m-SSA, PEG _m-β-SBA, PEG _m-SPA or PEG _m-BTC.Can introduce such group Y and Y ' by well known to a person skilled in the art standard method.

Preferably, aforesaid neuropeptide-2 receptor-agonists is not selected from the group of being made up of following:

IK-Pqa-RHYLNLVTRQRY，

Ac-IK-Pqa-RHYLNLVTRQRY，

IK-Pqa-RHYLNLVTRARY，

IK-Pqa-RHYLNLVARQRY，

IK-Pqa-RHYLNLATRQRY，

IK-Pqa-RHYLALVTRQRY，

IK-Pqa-RHYANLVTRQRY，

IK-Pqa-RHALNLVTRQRY，

IK-Pqa-RAYLNLVTRQRY，

IK-Pqa-AHYLNLVTRQRY，

IA-Pqa-RHYLNLVTRQRY，

Ac-IA-Pqa-RHYLNLVTRQRY，

AK-Pqa-RHYLNLVTRQRY，

IK-Pqa-RHYLNLVTRQR(D)Y，

IK-Pqa-RHYLNLVTRQ(D)RY，

IK-Pqa-RHYLNLVT(D)RQY，

IK-Pqa-RHYLNL(D)VTRQRY，

IK-Pqa-RHYL(D)NLVTRQRY，

IK-Pqa-RHY(D)LNLVTRQRY，

IK-Pqa-RH(D)YLNLVTRQRY，

IK-Pqa-R(D)HYLNLVTRQRY，

IK-Pqa-(D)RHYLNLVTRQRY，

I(D)K-Pqa-RHYLNLVTRQRY，

(D)IK-Pqa-RHYLNLVTRQRY，

IK-Pqa-RHYLNLVTRQR (N-methyl) Y,

IK-Pqa-RHYLNLVTRQ (N-methyl) RY,

IK-Pqa-RHYLNLVT (N-methyl) RQRY,

IK-Pqa-RHYLNLV (N-methyl) TRQRY,

IK-Pqa-RHYLNL (N-methyl) VTRQRY,

IK-Pqa-RHY (N-methyl) LNLVTRQRY,

IK-Pqa-RH (N-methyl) YLNLVTRQRY,

IK-Pqa-R (N-methyl) HYLNLVTRQRY,

IK-Pqa-(N-methyl) RHYLNLVTRQRY,

I (N-methyl) K-Pqa-RHYLNLVTRQRY,

(N-methyl) IK-Pqa-RHYLNLVTRQRY,

INle-Pqa-RHYLNLVTRQRY，

Ac-INle-Pqa-RHYLNLVTRQRY，

Ac-INle-Pqa-FHYLNLVTRQRY，

IK-Pqa-RHWLNLVTRQRY，

IK-Pqa-AHWLNLVTRQRY，

Ac-INle-Pqa-RHYLNLVTRQR(D)Y，

Ac-INle-Pqa-RHYLNLVTRQR (N-methyl) Y,

Ac-INle-Pqa-RHTicLNLVTRQRY，

Ac-INle-Pqa-RHBipLNLVTRQRY，

Ac-INle-Pqa-RHDipLNLVTRQRY，

Ac-INle-Pqa-RH(1)NalLNLVTRQRY，

Ac-INle-Pqa-RH(2)NalLNLVTRQRY，

Ac-INle-Pqa-RH (3.4,5 trifluoro Phe) LNLVTRQRY,

Ac-INle-Pqa-RH (2,3.4,5,6 five fluorine Phe) LNLVTRQRY,

Ac-INle-Pqa-R(4-MeOApc)YLNLVTRQRY，

Ac-INle-Pqa-R(3-Pal)YLNLVTRQRY，

Ac-INle-Pqa-R(4-Pal)YLNLVTRQRY，

Ac-INle-Pqa-(3,4,5 trifluoro Phe) HYLNLVTRQRY,

Ac-INle-Pqa-(2,3,4,5,6 five fluorine Phe) HYLNLVTRQRY,

Ac-Aib-Nle-Pqa-RHYLNLVTRQRT，

Ac1-1-Aic-Nle-Pqa-RHYLNLVTRQRT，

Ac-2-2Aic-Nle-Pqa-RHYLNLVTRQRT，

Ac-Ach-Nle-Pqa-RHYLNLVTRQRT，

Ac-Acp-Nle-Pq a-RHYLNLVTRQRT，

H-INle-Pqa-RHYLNLVTRQRY，

(PEG-10,000) INle-Pqa-RHYLNLVTRQRY and

(PEG-30,000)INle-Pqa-RHYLNLVTRQRY.

Chemical compound of the present invention still is favourable, because for example they are PYY _3-36Clipped form.For example, should not only promote the easier synthetic and purification of chemical compound by short peptide, and improve and reduced fabrication schedule and expense.In addition, chemical compound of the present invention will be preferred and the Y2 acceptor interaction, and not with homologous receptor such as NPY Y1, Y4 and Y5 interaction.Undesirable excitement or antagonism side reaction are minimized thus.

Chemical compound of the present invention is preferably used for treating metabolic disease and disease.Described metabolic disease and disease comprise, obesity for example, diabetes, type 2 diabetes mellitus preferably, metabolism syndrome (being also referred to as syndrome X), insulin resistance, dyslipidemia, impaired fasting glucose (IFG) (impaired fastingglucose) and impaired glucose tolerance.

Therefore, the invention still further relates to and comprise the pharmaceutical composition of neuropeptide-2 receptor-agonists and pharmaceutical carrier and/or adjuvant as defined above.

Equally, the present invention includes aforesaid neuropeptide-2 receptor-agonists, it is as therapeutic active substance, especially as the treatment of diseases active substance that is used for the treatment of and/or prevents to regulate, particularly as being used for the treatment of and/or prevent obesity type 2 diabetes mellitus by neuropeptide-2 receptor-agonists, metabolism syndrome, insulin resistance, dyslipidemia, the therapeutic active substance of impaired fasting glucose (IFG) and impaired glucose tolerance.

In another preferred embodiment, the present invention relates to be used for the treatment of and/or disease that prophylactic treatment is regulated by neuropeptide-2 receptor-agonists, especially for therapeutic and/or prophylactic treatment metabolic disease or disease, especially obesity, type 2 diabetes mellitus, metabolism syndrome, insulin resistance, dyslipidemia, the method for impaired fasting glucose (IFG) and impaired glucose tolerance, described method comprise neuropeptide-2 receptor-agonists as defined above are applied to the human or animal.In aforesaid preferable methods, described neuropeptide-2 receptor-agonists once-a-day is applied to described patient.Preferably, described neuropeptide-2 receptor-agonists once was applied to described patient in per three days.More preferably, described neuropeptide-2 receptor-agonists is applied to described patient weekly.Preferably, described neuropeptide-2 receptor-agonists is applied to described patient in the following manner: oral, intranasal, intravenous, subcutaneous, parenteral, transdermal, intraperitoneal or rectum or by sucking.Preferably, described neuropeptide-2 receptor-agonists intranasal administration.Further preferably, described neuropeptide-2 receptor-agonists passes through subcutaneous administration.In said method, described neuropeptide-2 receptor-agonists is preferably used with about dosage of 0.001 to about 100mg.

The present invention also comprises the disease that being used for the treatment of property of neuropeptide-2 receptor-agonists and/or prophylactic treatment are as defined above regulated by neuropeptide-2 receptor-agonists, especially for therapeutic and/or prophylactic treatment obesity, type 2 diabetes mellitus, metabolism syndrome, insulin resistance, dyslipidemia, the application of impaired fasting glucose (IFG) and impaired glucose tolerance.

The invention still further relates to the application that aforesaid neuropeptide-2 receptor-agonists is used to prepare medicine, the disease that described being used for the treatment of property of medicine and/or prophylactic treatment are regulated by neuropeptide-2 receptor-agonists, especially for therapeutic and/or prophylactic treatment obesity, type 2 diabetes mellitus, metabolism syndrome, insulin resistance, dyslipidemia, impaired fasting glucose (IFG) and impaired glucose tolerance.Described medicine comprises aforesaid neuropeptide-2 receptor-agonists.

Should be appreciated that to the invention is not restricted to specific embodiments of the present invention as herein described, because can carry out the change of specific embodiments and still fall in the scope of appended claim.It is also understood that used term is that to be not intended in order to describe specific embodiments be restrictive.In fact, scope of the present invention will be determined by appended claim.

The other information that relates to the example of the ester that is used for the delivering drugs chemical compound and application can be available from Design of Prodrugs (prodrug design) .Bundgaard H.ed. (Elsevier, 1985). also see, H.Ansel etc., Pharmaceutical Dosage Forms and Drug Delivery Systems (pharmaceutical dosage form and drug delivery system) (the 6th edition .1995), the 108-109 page or leaf; Krogsgaard-Larsen, etc., Textbook of Drug Design and Development (textbook of drug design and exploitation) (second edition .1996), 152-191 page or leaf.

Representative compounds of the present invention can be easily synthetic by any known conventional method that is used for formation peptide bond between aminoacid.These conventional methods comprise for example any solution phase method, the condensation between free uncle's carboxylic group of the free alpha-amido group of this method permission aminoacid or its residue (its carboxyl and other reactive group are protected) and another aminoacid or its residue (its amino or other reactive group is protected).

These conventional methods that are used for synthetic new compound of the present invention comprise for example any solid phase method of peptide synthesis.In the method, the synthetic of noval chemical compound can followingly carry out: according to the solid phase general theory of law, order is attached to every next of amino acid needed residue on the peptide chain of growth.These methods are disclosed in for example Merrifield, R.B., american chemical association (J.Amer.Chem.Soc.) 85,2149-2154 (1963); Barany etc., The Peptides, Analysis, Synthesis and Biology (peptide, analyze synthetic and biology), Vol.2, Gross, E. and Meienhofer, J., among Eds. academic press (Academic Press) 1-284 (1980), it is by with reference to being incorporated into this.

Chemistry of peptides is synthetic commonly to be protected the reactive side chain group of various amino acid moieties with suitable protecting group, and this will prevent to take place at the chemical reaction in this site, finally be removed until protecting group.Usually also commonly when this entity reacts on carboxyl, the α amino group on aminoacid or its fragment is protected, then selective removal α amino protecting group makes and can carry out subsequent reaction in this site.Although disclose specific protecting group, should be appreciated that each aminoacid can be used for each amino acid whose protecting group protection by routine in solution is combined to about solid-phase synthesis.

α amino can be selected from following suitable protecting group protection: the protecting group of aromatic amine Ethyl formate type, as allyloxycarbonyl, benzyloxycarbonyl group (Z) and the benzyloxycarbonyl group that replaces, as to the benzyloxycarbonylchloride base, to the nitro benzyloxycarbonyl group, to bromo-benzyloxycarbonyl, to xenyl-different third oxygen carbonyl, 9-fluorenylmethyloxycarbonyl (Fmoc) and to methoxyl group benzyloxy carbonyl (Moz); The protecting group of aliphatic amino Ethyl formate type, as tertbutyloxycarbonyl (Boc), diisopropyl methoxycarbonyl group, the different third oxygen carbonyl, and allyloxycarbonyl.Here, Fmoc most preferably is used for the α amido protecting.

Guanidine radicals can be by being selected from the protection of following due care base: nitro, p-toluenesulfonyl (Tos), (Z) pentamethyl color alkane sulfonyl (Pmc); 4-methoxyl group-2,3,6;-trimethylbenzene sulfonyl (Mtr) (Pmc), and (Mtr) most preferably is used for arginine (Arg).

The epsilon-amino group can be by being selected from the protection of following due care base: 2-benzyloxycarbonylchloride base (2-Cl-Z), and 2-bromo-benzyloxycarbonyl (2-Br-Z)-and uncle-butoxy carbonyl (Boc).For (Lys), Boc is most preferred.

Hydroxyl (OH) can be protected by being selected from following suitable protecting group: benzyl (Bzl), 2,6 dichloro benzyls (2,6 two Cl-Bzl), and the tert-butyl group (t-Bu) for (Tyr), (Ser) and (Thr), are most preferred (tBu).

β-and γ-amide group can protect by being selected from following suitable protecting group: 4-methyl trityl (Mtt); 2,4,6-trimethoxy benzyl (Tmob); 4,4-dimethoxy dityl pair-(4-methoxyphenyl)-methyl (Dod) and trityl (Trt).For (Asn) and (Gln), Trt is most preferred.

Indolyl radical can be protected by being selected from following suitable protecting group: formoxyl (For), mesityl-2-sulfonyl (Mts) and tertbutyloxycarbonyl (Boc).For (Trp), Boc is most preferred.

Imidazole radicals can be protected by being selected from following suitable protecting group: benzyl (Bzl) ,-tertbutyloxycarbonyl (Boc), and trityl (Trt).For (His), Trt is most preferred.

Aminoacid Pqa's is synthetic by (J.Med.Chem (medical chemistry magazine) .1996,39,4583-4591) described carrying out such as J.Hutchinson.The Fmoc-Pqa derivant is available from NeoMPS, company. and (Santiago CA).

All solvents, isopropyl alcohol (iPrOH), dichloromethane (CH ₂Cl ₂), dimethyl formamide (DMF) and N-methylpyrroline ketone (NMP) are available from taking snow (Fisher) or Burdick ﹠amp; Jackson and use under not distilling in addition.Trifluoroacetic acid is purchased from halocarbon (Halocarbon) or Fluka and not being further purified down and uses.

DIC (DIC) and diisopropylethylamine (DIPEA) are available from Fluka or A De Ritchie (Aldrich) and use under not being further purified.Hydroxybenzotriazole (HOBT), dimethyl sulfide (DMS) and 1,2-ethylene dithiol phenol (EDT) is available from SIGMA chemical company (Sigma Chemical Co.) and be not further purified use down.The aminoacid of protection is L configuration and being purchased from Bachem normally, or Neosystem.The purity of these reagent confirms by thin layer chromatography, NMR and fusing point before use.Benzhydrylamine resin (BHA) is the copolymer of styrene-1% divinylbenzene (100-200 or 200-400 order) available from Bachem or Advanced Chemtech.The total nitrogen content of these resins is generally 0.3-1.2meq/g.

In preferred embodiments, by Merrifield, (american chemical association (J.Amer.Chem.Soc.), 85,2149 (1963)) method of general description uses solid phase synthesis to prepare peptide, but as discussed previously, can use the chemosynthesis that other is equal to known in the art.Solid phase synthesis is terminal from the C-of peptide, and the a-amino acid of protecting is coupled to suitable resin.This raw material can be connected on right-benzyloxy benzyl alcohol (Wang) resin by the aminoacid with alpha-amido-protection by ester bond; or by the Fmoc-joint; as to ((R; S)-and α-(1-(9H-fluorenes-9-yl)-methoxymethylamide base)-2, the 4-dimethoxy-benzyl)-amido link between the phenoxyacetic acid (Rink joint) is connected to benzhydryl amine (BHA) resin and prepares.The preparation of hydroxymethyl resin is well known in the art.Fmoc-joint-bha resin support is commercially available and usually uses when the C-end has unsubstituted amide when the required peptide that is synthesized.

Typically, use the Fmoc protection form of aminoacid or analogies,, aminoacid or analogies are coupled on Fmoc-joint-bha resin with normal aminoacid of 2-5 and suitable coupling agent.After coupling, can washing resin and dry under vacuum.The load of aminoacid on resin can be by determining to the amino acid analysis of the Fmoc-amino-acid resin of aliquot or by UV assay determination Fmoc group.Any unreacted amino can add medicated cap by resin is reacted with acetic anhydride in dichloromethane and diisopropylethylamine.

Resin is carried out several repetitive cycling so that amino acid sequence is added.Under alkali condition, remove the amino Fmoc protecting group of α.Piperidines in DMF, piperazine or morpholine (20-40%v/v) can be used for this purpose.Preferred 40% piperidines that uses among the DMF.

After removing the α amino protecting group, with the aminoacid of protection subsequently with desired sequence progressively coupling to obtain intermediate, the peptide-resin of protection.It is well known in the art being used for the link coupled activating reagent of aminoacid in the solid phase synthesis of peptide.For example, being used for this synthetic suitable reagent is benzotriazole-1-base oxygen base-three-(dimethylamino) Phosphonium hexafluorophosphate (BOP), bromo-three-pyrrolidino-Phosphonium hexafluorophosphates (PyBroP), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate (HBTU), and DIC (DIC).Here preferred HBTU and DIC.Can use other activator that Barany and Merrifield describe (at The Peptides (peptide), volume 2, J.Meienhofer, ed., academic press (Academic Press) is in 1979, the 1-284 pages or leaves).Can be with all ingredients as 1 hydroxybenzotriazole (HOBT), N-hydroxy-succinamide (HOSu) and 3,4-dihydro-3-hydroxyl-4-oxo-1,2,3-phentriazine (HOOBT) adds in the coupling mixture, so that the optimization synthesis cycle.Here preferred HOBT.

In order to prepare N end acetyl derivative, carry out acetylation by being used in 20% anhydride among the DMF and 5% DIEA process resin binding peptide.For other N end acylation, use and carried out acylation 30 minutes with the corresponding carboxylic acid of DIC/HOBT in-situ activation.

The scheme that is used for typical synthesis cycle is as follows:

Scheme 1

The step reagent time

1 DMF 2 * 30 seconds

2 20% piperidines/DMF 1 minute

3 20% piperidines/DMF 15 minutes

4 DMF 2 * 30 seconds

5 iPrOH 2 * 30 seconds

6 DMF 3 * 30 seconds

7 couplings 60 minutes-18 hours.

8 DMF 2 * 30 seconds

9 iPrOH 1 * 30 second

10 DMF 1 * 30 second

11 CH ₂Cl ₂2 * 30 seconds

To be used for all washings and link coupled solvent measures to the volume of 10-20ml/g resin.Run through synthetic coupling reaction and monitor, with the degree of determining to finish (Kaiser etc., analytical biochemistry (Anal.Biochem.) 34,595-598 (1970)) by Kaiser Ninhydrin test.Observe slow reaction kinetics for Fmoc-Arg (Pmc) with for the coupling of sterically hindered acid and secondary amine.Any incomplete coupling reaction or with the activated amino acid coupling again of prepared fresh, perhaps add medicated cap by handling peptide resin as mentioned above with acetic anhydride.With the peptide-resin of assembling fully dry in a vacuum a few hours.

For most compounds, remove blocking groups and cleavage of peptide from the resin.For example, every gram resin is with 100 μ L ethylene dithiol phenol, 100 μ L dimethyl sulfide, and 300 μ L methyl phenyl ethers anisoles and 9.5mL trifluoroacetic acid were at room temperature handled peptide resin 180 minutes.Or alternatively, every gram resin was at room temperature handled peptide resin 180 minutes with 1.0mL tri isopropyl silane and 9.5mL trifluoroacetic acid.With the resin elimination, filtrate is precipitated in sudden cold ether.Centrifugal and the decantation ether layer with precipitate.With the Et of residue with two or three volumes ₂The O washing is also centrifugal again.Crude product is dry under vacuum.

Preferably, in Shimadzu LC-8A system, carry out the purification of rough peptide by the high performance liquid chromatography (HPLC) on anti-phase C-18 post (50 * 250mm.300 A, 10-15 μ m).With the 0.1AcOH/H of peptide in minimum volume ₂O or CH3CH/H ₂Be expelled in the post among the O.Gradient elution begins with the 2%B buffer usually, 2%-70%B in 70 minutes, (buffer A: 0.1%TFA/H ₂O, buffer B: 0.1%TFA/CH ₃CN), flow velocity 50ml/min.Carrying out UV under 220/280nm detects.Separate the purity that contains the fraction of product and judge them on Shimadzu LC-10AT analytical system, (4.6 * 50mm), flow velocity is 2ml/min., 10 minutes inside gradient (2-70%) (buffer A: 0.1%TFA/H wherein to use anti-phase Ace C18 post ₂O, buffer B: 0.1%TFA/CH ₃CN)).To be judged as highly purified fraction merges and lyophilizing.

The purity of final products is checked by the analysis HPLC on aforesaid reversed-phase column.The purity of all products is according to being judged as about 95-99%.All finished products also carry out fast atom bombardment mass spectroscopy (FAB-MS) or electrospray mass spectrum (ES-MS).All products have produced the parent M+H ion in tolerance interval of expection.

Chemical compound of the present invention can provide with the form of pharmaceutical salts.The example of preferred salt is those and the salt that forms with mineral acid that forms with pharmaceutically acceptable organic acid and polymeric acid, described organic acid such as acetic acid, lactic acid, maleic acid, citric acid, malic acid, ascorbic acid, succinic acid, benzoic acid, salicylic acid, methanesulfonic acid, toluenesulfonic acid, trifluoroacetic acid or pamoic acid, described polymeric acid such as tannic acid or carboxymethyl cellulose, described mineral acid such as halogen acids are (for example, hydrochloric acid), sulphuric acid, or phosphoric acid etc.Can use the method for any acquisition pharmaceutical salts well known by persons skilled in the art.

In the enforcement of the inventive method, with the combination of the peptide any of the present invention of effective dose or any peptide of the present invention or its pharmaceutical salts via any routine with acceptable method known in the art, separately or combined administration.Use and can for example carry out once a day, per three days once or weekly.Therefore chemical compound or compositions can be used in the following manner: oral (for example oral cavity), the Sublingual, parenteral (for example, intramuscular, intravenous, or subcutaneous), rectum (for example, by suppository or lotion), transdermal (for example, the skin electroporation) or by sucking (for example passing through aerosol) and, comprising tablet and suspensoid with the form of solid, liquid or gas dosage form.Administration can be treated continuously or arbitrarily treat with single dose and carry out with single unit dosage form.Therapeutic combination can also be oil emulsion or with the form of lipophilic salts such as the bonded dispersion of pamoic acid, or be used for the form of the Biodegradable sustained-release compositions of subcutaneous or intramuscular administration.

Therefore, when the special needs of alleviating of symptom or may be critical the time, implement method of the present invention.Alternatively, method of the present invention is as continuously or prophylactic treatment and effectively implementing.

The useful pharmaceutical carrier that is used to prepare compositions thus can be a solid, liquid or gas; Therefore, compositions can be taked tablet, pill, capsule, suppository, powder, the preparation of enteric coating or other protection (for example be combined on ion-exchanger resin or be packaged in fat-albumen vesicle), slow releasing preparation, solution, suspensoid, elixir, forms such as aerosol.Carrier can be selected from various oils, comprises those of oil, animal, plant or synthetic source, Oleum Arachidis hypogaeae semen for example, soybean oil, mineral oil, Semen Sesami wet goods.Water, saline, glucose solution and dihydroxylic alcohols are preferred liquid-carriers, and particularly (when oozing with blood etc.) is used for injection.For example, the preparation that is used for intravenous administration comprises the aseptic aqueous solution of active component, and it is prepared as follows: solid active agent is dissolved in the water, produces aqueous solution, and make solution aseptic.Suitable pharmaceutical excipient comprises starch, cellulose, Talcum, glucose, lactose, Talcum, gelatin, Fructus Hordei Germinatus, rice, flour, Chalk, Silicon stone, magnesium stearate, sodium stearate, glyceryl monostearate, sodium chloride, dried defatted milk powder, glycerol, propylene glycol, water, ethanol etc.Compositions can add the conventional medicine additive, as antiseptic, and stabilizing agent, wetting agent or emulsifying agent are used to regulate the salt of osmotic pressure, buffer agent etc.Suitable pharmaceutical carrier and their preparation are described in the Remington ' of E.W.Martin s Pharmaceutical Sciences (Remington's Pharmaceutical Science).These compositionss under any circumstance will contain the reactive compound and the appropriate carriers of effective dose, so that preparation is used for suitably delivering medicine to receiver's appropriate dosage forms.

The dosage of chemical compound of the present invention depends on many factors, as for example, and administering mode, experimenter's age and body weight and experimenter's to be treated disease, and determined by the doctor in charge or veterinary the most at last.The amount of the reactive compound of being determined by the doctor in charge or veterinary like this is called " effective dose " in this article and in the claims.For example, the dosage that is used for intranasal administration typically about 0.001 to the scope of about 0.1mg/kg body weight.In the people, based on the preferred subcutaneous dosage of peptide content from about 0.001mg to about 100mg; Preferably arrive about 15mg from about 0.1mg.For API, its scope from about 0.015mg to about 100mg; Preferably from about 1mg to about 100mg.

To further describe the present invention in the following embodiment that lists now, it is intended to not limit the scope of the invention as just illustrating.

Embodiment

The preparation of reagent

MPEG _30k -methanesulfonates

At the equipment magnetic stirring apparatus, mPEG 30 kDa (available from Japanese oils and fats (Nippon Oil and Fat)) of 100g (3.34mmol) and the toluene of 500mL are filled in Dean-Rodney Stark grabber in the 1L round-bottomed flask of reflux condenser and argon (or nitrogen) import bubbler.Removing 250mL toluene by distillation comes the PEG solution of azeotropic drying in toluene also then with described solution cool to room temperature.In described solution, add the anhydrous methylene chloride of 200mL, and described solution is cooled to 0-5 ℃ and use syringe dropwise to add by rubber septum the mesyl chloride of 0.67mL (4.84mmol) triethylamine and 0.33mL (4.34mmol).Described mixture was stirred 2 hours and followed stirred overnight at room temperature under argon at about 4 ℃.

Described mixture is concentrated on rotary evaporimeter and filter to remove salt by coarse glass dust.(note: the temperature rising glass powder solidifies to prevent solution in filter process).Cold isopropanol by adding about 1800ml and diethyl ether (30: 70, v/v) precipitate described product.Collect described product and at room temperature in a vacuum dried overnight to obtain the white solid of 90g (90%).

Step 2.mPEG _30k -amine

With the 2L round-bottomed flask of equipment magnetic stirring apparatus and argon import bubbler fill with 90g (3mmol) the mPEG 30 kDa methanesulfonates 2 of preparation in the above and the ammonium hydroxide aqueous solution of 1600mL (30%, v/v).The ammonium chloride that in this solution, adds 160g.Described solution is carefully heated up to dissolve all PEG methanesulfonates.With the solution stirring that obtains 48 hours, discharge excessive gas to prevent pressure accumulated in reaction bulb by bubbler in room temperature simultaneously.

After reaction finishes, add the sodium chloride of 160g (10wt%) and with the dichloromethane extraction of described mixture with 3 * 200mL=1200mL.With the organic extract that merges about 1 hour, filter and on rotary evaporimeter, concentrate by anhydrous sodium sulfate drying.With described product by adding the cold diethyl ether precipitation of 1800mL, filter and room temperature under vacuum dried overnight to obtain 85g (94%) 3 as white solid.

Step 3.mPEG _30k The acid of-butanimide

The 1L round-bottomed flask of assembling magnetic stirring apparatus and argon import bubbler is filled with the mPEG 30 kDa amine 3 of 60g (2.00mmol) and the anhydrous acetonitrile of 500mL.Described solution is cooled to about 4 ℃, then will will use the adding funnel slowly to add at the 2g in the 50mL anhydrous acetonitrile (20.00mmol) succinic anhydride.Under argon gas stream, with reactant mixture in stirred overnight at room temperature.

After reaction finishes, extremely dry by rotary evaporimeter evaporating solvent.Then, described product is dissolved in the 400mL water.The pH of described solution is adjusted to 7.0 and stirred 1 hour with 1M NaOH solution, keeps pH simultaneously 7.0.In this solution, add 40g (10wt.%) sodium chloride and regulate pH to～4.2 with 6N HCl solution.With 200,100, the dichloromethane of 50mL=350mL extracts with the aqueous mixture that obtains.With the organic extract that merges about 1 hour by anhydrous sodium sulfate drying.Filter sodium sulfate and concentrated filtrate on rotary evaporimeter.Precipitated product in the cold diethyl ether of 1L.Collect product and room temperature under vacuum, be dried spend the night with obtain as white solid 56g (93%) 4.

Step 4.mPEG _30k -succinimido butanimide

The round-bottomed flask that assembles the 500-mL of magnetic stirring apparatus and argon import bubbler is filled with the mPEG 30 kDa butanimides acid 4 of 56g (1.87mmol) and the anhydrous methylene chloride of 500mL.In this solution, slowly add 1 of the N-hydroxy-succinamide of 0.24g (2.05mmol) and 0.46g (2.24mmol), the 3-dicyclohexylcarbodiimide.With described reactant mixture under argon gas stream in stirred overnight at room temperature.

Reaction is evaporated to drying with mixture after finishing on rotary evaporimeter.Then, described product is dissolved in the dry toluene of 200mL and with described solution and filters by pre-warm coarse glass dust, described glass dust is equipped with Celite pad.Cold anhydrous isopropyl alcohol by adding 1200mL and diethyl ether (30: 70, v/v) come precipitated product.Collect product and room temperature under vacuum dried overnight with obtain as white solid 20g (80%) 5.

The preparation of preferred compound

Embodiment 1

Preparation Fmoc-joint-bha resin

With benzhydryl amine co polystyrene-1% divinyl benzene crosslinked resin (10.0g, 9.3m equivalent (mequiv), 100-200 ASTM order, high chemical technology (Advanced ChemTech)) swelling at 100mL CH ₂Cl ₂In, filter and continuous each CH with 100ml ₂Cl ₂, 6%DIPEA/CH ₂Cl ₂(twice), CH ₂Cl ₂(twice) washing.Resin is used in 100mL 25%DMF/CH ₂Cl ₂In right-((R, S)-α-(1-(9H-fluorenes-9-yl)-methoxymethylamide base)-2, the 4-dimethoxy-benzyl)-phenoxyacetic acid (Fmoc-joint) (7.01g, 13.0mmol), N-hydroxybenzotriazole (2.16g, 16.0mmol) and diisopropyl-carbodiimide (2.04ml 13.0mmol) at room temperature handles 24 hours.With resin filter and continuous each CH with 100ml ₂Cl ₂(twice), isopropyl alcohol (twice), DMF, and CH ₂Cl ₂(three times) washing.It is negative that Kaiser Ninhydrin analyzes.Resin is dry under vacuum, Fmoc-joint-bha resin of acquisition 16.12g.This resin of a part (3.5mg) is carried out Fmoc deprotection and quantitatively UV analysis, and it shows that load is 0.56mmol/g.

Embodiment 2

Use fluorenyl methyl oxygen base carbonyl (Fmoc) chemical method, by the scheme of the synthetic peptide of the 433A of applying biological system synthesizer

For (Foster City, the peptide of 0.25mmol scale CA) is synthetic, uses FastMoc 0.25mmol circulation, uses resin sampling or non-resin sampling, the 41mL reaction vessel by applying biological system (Applied Biosystem) 433A synthesizer.With Fmoc-amino-acid resin and 2.1g NMP, the 0.45M HOBT/HBTU in DMF of 2g and 2M DIEA dissolving are transferred in the reaction vessel then.The basic FastMoc coupling of the expression circulation with " BADEIFD, ", wherein module of each letter representation (such as by applying biological system (Applied Biosystem) definition).For example:

B represents to be used for the module of Fmoc deprotection, uses 20% piperidines/NMP and relevant washing, and reads 30 minutes (perhaps UV monitor or electrical conductivity); A represents with aminoacid in 0.45 M HBTU/HOBt and the 2.0 M DIEA activation cylinder and uses N ₂The blended module of bubbling; D is illustrated in the module of NMP washing resin in the reaction vessel; E represents activated amino acid is transferred to the module that is used for link coupled reaction vessel; I represents 10 minutes of the reaction vessel modules of waiting for period, wherein vortex by spells; Represent the module that is used to clean cylinder, about 10 minutes of coupling and drains reaction vessel with F.Coupling typically increases module " I " by one or many and extends.For example, carry out double couple crosslinking by performing a programme " BADEIIADEIFD ".Other module can be provided, for example be used for the c and " C " that be used for adding medicated cap of washed with dichloromethane with acetic anhydride.Independent module also is modifiable, for example by changing the timing of various functions, as transfer time, so that the solvent that change is transferred or the amount of reagent.Above circulation typically is used for aminoacid of coupling.Yet for synthetic tetrapeptide, repetitive cycling and linking up.For example; BADEIIADEIFD is used for first aminoacid of coupling, then second aminoacid of BADEIIADEIFD coupling, then the 3rd aminoacid of BADEIIADEIFD coupling; then the 4th aminoacid of BADEIIADEIFD coupling is followed BIDDcc and is used for final deprotection and washing.

Embodiment 3

H-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Gl u-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Le u-Val-The-Arg-Gln-Arg-Tyr-NH ₂(PYY _3-36) preparation

Use the Fmoc chemistry, synthetic above-mentioned peptide on applying biological system (Applied Biosystem) 433A synthesizer.Use embodiment 2 described modules, synthesizer is programmed for double couple crosslinking.Synthesize on the 0.25mmol scale and carry out, use Fmoc-joint-bha resin from embodiment 1 (450mg, 0.25mmol).When end of synthesis, with in the reaction vessel of resin transfer to the agitator in order to cracking.At room temperature use 13.5mL 97%TFA/3%H2O and 1.5mL tri isopropyl silane, with peptide cracking 180 minutes from the resin.Deprotection solution is added the cold ET of 100mL ₂Among the O, with 1mL TFA and the cold ET of 30mL ₂The O washing is with precipitation of peptides.Peptide is centrifugal in 2 * 50mL polypropylene tube.To be incorporated in from the precipitation of single pipe in the single pipe, and use cold ET ₂O washing 3 times, and dry down in indoor vacuum (house vacuum) in exsiccator.

With the preparation HPLC purification of crude material by on Pursuit C18-post (250 * 50mm, 10 μ m granularities), and in 90 minutes with 2-70%B (buffer A: 0.1%TFA/H2O; Buffer B: 0.1%TFA/CH3CN) linear gradient elution, flow velocity 60mL/min, and 220/280nm detects.Collect fraction and pass through to analyze the HPLC inspection.To contain the fraction merging of pure products and lyophilizing and obtain the white amorphous powder of 151mg (15%).(ES)+-LCMS m/e value of calculation (" calcd ") C ₁₈0H ₂₇₉N ₅₃O ₅₄4049.55, measured value 4050.40.

Embodiment 4

H-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Ar g-Tyr-NH ₂Preparation

Will (450mg 0.25mmol) carries out solid phase synthesis and obtain the white amorphous powder of 48mg (9%) according to the method purification among the embodiment 3 from Fmoc-joint-bha resin of embodiment 1.(ES)+-LCMS m/e value of calculation C ₉₈H ₁₅₅N ₃₃O ₂₁2131.53 measured value 2130.56.

Embodiment 5

H-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe) Arg-Tyr-NH ₂Preparation

Will (450mg 0.25mmol) carries out solid phase synthesis (N-methyl Arg is inserted described sequence 35) and according to the method purification among the embodiment 3, obtain the white amorphous powder of 32mg (6%) from Fmoc-joint-bha resin of embodiment 1.(ES)+-LCMS m/e value of calculation C ₉₉H ₁₅₅N ₃₃O ₂₁2143.56 measured value 2143.50.

Embodiment 6

H-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe) Arg-m-Tyr-NH ₂Preparation

Will from Fmoc-joint-bha resin of embodiment 1 (450mg, 0.25mmol) carry out solid phase synthesis and according to the method purification among the embodiment 3 to produce the white amorphous powder of 38.5mg (7%).(ES)+-LCMS m/e value of calculation C99H155N33O21 2143.5477 measured values 2143.50.

Embodiment 7

H-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe) Arg-3-iodo-Tyr-NH ₂Preparation

Will from Fmoc-joint-bha resin of embodiment 1 (450mg, 0.25mmol) carry out solid phase synthesis and according to the method purification among the embodiment 3 to produce the white amorphous powder of 41mg (7%).(ES)+-LCMS m/e value of calculation C99H154IN33O21 2269.44 measured values 2269.20.

Embodiment 8

H-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe) Arg-3,5 two F-Tyr-NH ₂Preparation

Will from Fmoc-joint-bha resin of embodiment 1 (450mg, 0.25mmol) carry out solid phase synthesis and according to the method purification among the embodiment 3 to produce the white amorphous powder of 28mg (5%).(ES)+-LCMS m/e value of calculation C99H153F2N33O21 2179.52 measured values 2179.46.

Embodiment 9

H-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe) Arg-2,6 two F-Tyr-NH ₂Preparation

Will from Fmoc-joint-bha resin of embodiment 1 (450mg, 0.25mmol) carry out solid phase synthesis and according to the method purification among the embodiment 3 to produce the white amorphous powder of 49.3mg (9%).(ES)+-LCMS m/e value of calculation C99H153F2N33O21 2179.53 measured values 2179.50.

Embodiment 10

H-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe) Arg-2,6 two Me-Tyr-NH ₂Preparation

Will from Fmoc-joint-bha resin of embodiment 1 (450mg, 0.25mmol) carry out solid phase synthesis and according to the method purification among the embodiment 3 to produce the white amorphous powder of 13.5mg (3%).(ES)+-LCMS m/e value of calculation C101H159N33O21 2171.60 measured values 2171.40.

Embodiment 11

H-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe) Arg-4 methoxyl group-Phe-NH ₂Preparation

Will from Fmoc-joint-bha resin of embodiment 1 (450mg, 0.25mmol) carry out solid phase synthesis and according to the method purification among the embodiment 3 to produce the white amorphous powder of 72mg (13%).(ES)+-LCMS m/e value of calculation C100H157N33O21 2157.57 measured values 2157.58.

Embodiment 12

H-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe) Arg-Phe-NH ₂Preparation

Will from Fmoc-joint-bha resin of embodiment 1 (450mg, 0.25mmol) carry out solid phase synthesis and according to the method purification among the embodiment 3 to produce the white amorphous powder of 85.3mg (16%).(ES)+-LCMS m/e value of calculation C99H155N33O20 2127.55 measured values 2127.53.

Embodiment 13

H-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe) Arg-4 amino-Phe-NH ₂Preparation

Will from Fmoc-joint-bha resin of embodiment 1 (450mg, 0.25mmol) carry out solid phase synthesis and according to the method purification among the embodiment 3 to produce the white amorphous powder of 51.4mg (10%).(ES)+-LCMS m/e value of calculation C99H156N34O20 2142.56 measured values 2142.55.

Embodiment 14

H-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe) Arg-4F-Phe-NH ₂Preparation

Will from Fmoc-joint-bha resin of embodiment 1 (450mg, 0.25mmol) carry out solid phase synthesis and according to the method purification among the embodiment 3 to produce the white amorphous powder of 35mg (7%).(ES)+-LCMS m/e value of calculation C99H154FN33O20 2145.54 measured values 2145.51.

Embodiment 15

H-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe) Arg-4-(CH2OH)-Phe-NH ₂Preparation

Will from Fmoc-joint-bha resin of embodiment 1 (450mg, 0.25mmol) carry out solid phase synthesis and according to the method purification among the embodiment 3 to produce the white amorphous powder of 24mg (4%).(ES)+-LCMS m/e value of calculation C100H1571N33O21 2157.57 measured values 2157.56.

Embodiment 16

H-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe) Arg-4-trifluoromethyl-Phe-NH ₂Preparation

Will from Fmoc-joint-bha resin of embodiment 1 (450mg, 0.25mmol) carry out solid phase synthesis and according to the method purification among the embodiment 3 to produce the white amorphous powder of 81mg (15%).(ES)+-LCMS m/e value of calculation C100H154F3N33O20 2195.54 measured values 2195.51.

Embodiment 17

H-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe) Arg-3 fluoro-Phe-NH ₂Preparation

Will from Fmoc-joint-bha resin of embodiment 1 (450mg, 0.25mmol) carry out solid phase synthesis and according to the method purification among the embodiment 3 to produce the white amorphous powder of 84mg (16%).(ES)+-LCMS m/e value of calculation C99H154FN33O20 2145.54 measured values 2145.53.

Embodiment 18

H-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe) Arg-2,3,4,5,, 6 five fluoro-Phe-NH ₂Preparation

Will from Fmoc-joint-bha resin of embodiment 1 (450mg, 0.25mmol) carry out solid phase synthesis and according to the method purification among the embodiment 3 to produce the white amorphous powder of 89mg (16%).(ES)+-LCMS m/e value of calculation C99H150FN33O20 2217.50 measured values 2217.48.

Embodiment 19

H-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe) Arg-3,4-two chloro-Phe-NH ₂Preparation

Will from Fmoc-joint-bha resin of embodiment 1 (450mg, 0.25mmol) carry out solid phase synthesis and according to the method purification among the embodiment 3 to produce the white amorphous powder of 46mg (8%).(ES)+-LCMS m/e value of calculation C99H153C12N33O20 2196.44 measured values 2196.41.

Embodiment 20

H-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe) Arg-Cha-NH ₂Preparation

Will from Fmoc-joint-bha resin of embodiment 1 (450mg, 0.25mmol) carry out solid phase synthesis and according to the method purification among the embodiment 3 to produce the white amorphous powder of 49mg (9%).(ES)+-LCMS m/e value of calculation C106H162N34O21 2248.69 measured values 2248.71.

Embodiment 21

H-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe) Arg-Trp-NH ₂Preparation

Will from Fmoc-joint-bha resin of embodiment 1 (450mg, 0.25mmol) carry out solid phase synthesis and according to the method purification among the embodiment 3 to produce the white amorphous powder of 57mg (10%).(ES)+-LCMS m/e value of calculation C108H157N35O21 2281.68 measured values 2281.67.

Embodiment 22

H-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe) Arg-1-Nal-NH ₂Preparation

Will from Fmoc-joint-bha resin of embodiment 1 (450mg, 0.25mmol) carry out solid phase synthesis and according to the method purification among the embodiment 3 to produce the white amorphous powder of 45mg (8%).(ES)+-LCMS m/e value of calculation C103H157N33O20 2177.61 measured values 2177.59.

Embodiment 23

H-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-(NMe) Arg-2-Nal-NH ₂Preparation

Will from Fmoc-joint-bha resin of embodiment 1 (450mg, 0.25mmol) carry out solid phase synthesis and according to the method purification among the embodiment 3 to produce the white amorphous powder of 43mg (8%).(ES)+-LCMS m/e value of calculation C103H157N33O20 2177.60 measured values 2177.58.

Embodiment 24

H-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Ar g-C-Alpha-Methyl Tyr--NH ₂Preparation

Will from Fmoc-joint-bha resin of embodiment 1 (450mg, 0.25mmol) carry out solid phase synthesis and according to the method purification among the embodiment 3 to produce the white amorphous powder of 35.1mg (7%).(ES)+-LCMS m/e value of calculation C99H155N33O21 2143.55 measured values 2143.56.

Embodiment 25

H-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe) Arg-Tyr-NH ₂Preparation

Will from Fmoc-joint-bha resin of embodiment 1 (450mg, 0.25mmol) carry out solid phase synthesis and according to the method purification among the embodiment 3 to produce the white amorphous powder of 130mg (23%).(ES)+-LCMS m/e value of calculation C104H154N34O21 2216.60 measured values 2216.62.

Embodiment 26

H-Ile-Nle-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe) Arg-Tyr-NH ₂Preparation

Will from Fmoc-joint-bha resin of embodiment 1 (450mg, 0.25mmol) carry out solid phase synthesis and according to the method purification among the embodiment 3 to produce the white amorphous powder of 84mg (15%).(ES)+-LCMS m/e value of calculation C104H153N33O21 2201.59 measured values 2201.56.

Embodiment 27

Ac-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe) Arg-2, the preparation of 6-F2-Tyr-NH2

Will from Fmoc-joint-bha resin of embodiment 1 (450mg, 0.25mmol) carry out solid phase synthesis and according to the method purification among the embodiment 3 to produce the white amorphous powder of 24mg (4%).(ES)+-LCMS m/e value of calculation C106H154F2N34O22 2099.49 measured values 2100.3

Embodiment 28

Ac-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe) Arg-Tyr-NH ₂Preparation

Will from Fmoc-joint-bha resin of embodiment 1 (450mg, 0.25mmol) carry out solid phase synthesis and according to the method purification among the embodiment 3 to produce the white amorphous powder of 68mg (12%).(ES)+-LCMS m/e value of calculation C106H156N34O22 2258.64 measured values 2258.61

Embodiment 29

Valeryl (Pentoyl)-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe) Arg-Tyr-NH ₂Preparation

Will from Fmoc-joint-bha resin of embodiment 1 (450mg, 0.25mmol) carry out solid phase synthesis and according to the method purification among the embodiment 3 to produce the white amorphous powder of 67mg (12%).(ES)+-LCMS m/e value of calculation C109H162N34O22 2300.72 measured values 2300.69.

Embodiment 30

Pivaloyl group-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe) Arg-Tyr-NH ₂Preparation

Will from Fmoc-joint-bha resin of embodiment 1 (450mg, 0.25mmol) carry out solid phase synthesis and according to the method purification among the embodiment 3 to produce the white amorphous powder of 6mg (1%).(ES)+-LCMS m/e value of calculation C109H162F2N34O22 2300.72 measured values 2300.68.

Embodiment 31

Cyclohexyl acetyl group-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe) Arg-Tyr-NH ₂Preparation

Will from Fmoc-joint-bha resin of embodiment 1 (450mg, 0.25mmol) carry out solid phase synthesis and according to the method purification among the embodiment 3 to produce the white amorphous powder of 15mg (3%).(ES)+-LCMS m/e value of calculation C112H166N34O22 2340.79 measured values 2340.81.

Embodiment 32

Benzoyl-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe) Arg-Tyr-NH ₂Preparation

Will from Fmoc-joint-bha resin of embodiment 1 (450mg, 0.25mmol) carry out solid phase synthesis and according to the method purification among the embodiment 3 to produce the white amorphous powder of 23mg (4%).(ES)+-LCMS m/e value of calculation C111H158N34O22 2320.71 measured values 2320.68.

Embodiment 33

Diamantane (obsolete) acyl group (Adamantoyl)-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe) Arg-Tyr-NH ₂Preparation

Will from Fmoc-joint-bha resin of embodiment 1 (450mg, 0.25mmol) carry out solid phase synthesis and according to the method purification among the embodiment 3 to produce the white amorphous powder of 29mg (5%).(ES)+-LCMS m/e value of calculation C116H170N34O22 2392.86 measured values 2392.89.

Analytical method for embodiment 34-39 and 41

Use following reversed-phase HPLC/UV methods analyst test and contrast goods:

Autosampler Alliance Waters 2690 separation modules (Alliance Waters 2690

Separation Module)

Volume injected 10 μ L

The injection temperature room temperature

Detector Waters 996 photodiode array detectors

Detector wavelength 280nm

Post Agilent (Agilent) Eclipse XDB-C8,5 microns, 150mm * 4.6mm

i.d.PN：99367-906

25 ℃ of column temperatures

Flow velocity 1.0mL/ minute (～1000psi)

Mobile phase A comprises the water of 0.05%TFA

Mobile phase B comprises the acetonitrile of 0.05%TFA

About 30 minutes of running time

The about 0.2-0.5mg/ml of sample separation

The diluent deionized water

Eluent gradient condition 1 (RP-HPLC1):

Eluent gradient condition 2 (RP-HPLC2):

Embodiment 34

(PEG-30,000) CH ₂CH ₂CO) Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe) Arg-Tyr-NH ₂And Ile ((PEG-30,000) CH ₂CH ₂CO) (ε) Lys-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe) Arg-Tyr-NH ₂The preparation of mixture

N=～675 wherein

With

N=～675 wherein

Analytical method	The result
Analytical method	The result	The RP-HPLC1-rxn mixture	69.8% transforms
The RP-HPLC1-purification	10.1 minute retention time	The RP-HPLC1-rxn mixture	69.8% transforms
The RP-HPLC1-purification	10.1 minute retention time	MALDI-TOF MS	Average quality=33.9kDa

Weigh up from the 25mg peptide of embodiment 25 and be dissolved in the 50mM borate, in pH 7.5 buffer.Weighing 500mg 30kDa PEG-succinyl phosphorons amino propyl acid (available from Nektar) is to obtain 2: 1PEG: the peptide mol ratio also adds dissolved peptide with it.Reactant mixture at room temperature stirred spend the night, afterwards at 20mM NaOAc, dilution is 10 times in pH 4.5 buffer, by the cation-exchange chromatography purification on SP-agarose gel (Sepharose) FF.Fig. 1 is the HPLC chromatogram of reactant mixture.Reaction produces 69.8% 30kDa peptide.

Mono-pegylated PYY peptide is used step NaCl gradient elution.Typically, required mono-pegylated peptide is with 250mM NaCl eluting.The PEG-PYY-sample peptide of eluting uses the film of 10kDa MW cutoff to concentrate in Amicon ultrafiltration chamber.Use 10 times of PBS diafiltrations once then.

Submit the concentrated peptide of embodiment 34 to analysis, measure and be stored in-20 ℃.Fig. 2 is the HPLC chromatogram (RT=10.1min) of the 30kDa PEG-PYY peptide of purification.The purity of 30kDa peptide is measured as＞and 97%.Fig. 3 is a chart, and expression is carried out the MALDI-TOF of 30kDa PEG-PYY peptide to confirm molecular weight.

The combination of method is used for determining the PEG decorating site.These comprise MALDI TOF MS, reversed-phase HPLC, proteolytic digestion and N-end order-checking (Ai Deman (Edman)).Result from these analyses shows that most of PEG connects by the epsilon-amino of the lysine on (R2) position of peptide.

Embodiment 35

((PEG-40,000) CO) Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe) Arg-Tyr-NH ₂And (ε) Lys-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe) Arg-Tyr-NH of Ile ((PEG-40,000) CO) ₂The preparation of mixture

N=～900 wherein

With

N=～900 wherein

Analytical method	The result
Analytical method	The result	The RP-HPLC1-rxn mixture	60.4% transforms
The RP-HPLC1-purification	10.1 minute retention time	The RP-HPLC1-rxn mixture	60.4% transforms
The RP-HPLC1-purification	10.1 minute retention time	MALDI-TOF MS	Average quality=41.9kDa

Weigh up from the 25mg peptide of embodiment 25 and be dissolved in the 50mM borate, in pH 8.0 buffer.Weighing 319mg 40kDa PEG-benzotriazole carbonate is to obtain 0.8: 1 PEG: the peptide mol ratio also adds dissolved peptide with it.Reactant mixture was at room temperature stirred 1 hour, and afterwards at 20mMNaOAc, dilution is 10 times in pH 4.5 buffer, by the cation-exchange chromatography purification on SP-agarose gel FF.Fig. 4 is the HPLC chromatogram of reactant mixture.Reaction produces 60.4% 40kDa peptide.

Submit the concentrated peptide of embodiment 35 to analysis, measure and be stored in-20 ℃.Fig. 5 is the HPLC chromatogram (RT=10.1min) of the 40kDa PEG-PYY peptide of purification.The purity of 40kDa peptide is measured as＞and 90%.Fig. 6 is a chart, and expression is carried out the MALDI-TOF of 40kDa PEG-PYY peptide to confirm molecular weight.

Embodiment 36

(PEG-30,000) CH ₂CH ₂NHCOCH ₂CH ₂CO) Ile-Nle-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe) Arg-Tyr-NH ₂Preparation

N=～675 wherein

Analytical method	The result
Analytical method	The result	The RP-HPLC1-rxn mixture	94.1% transforms
The RP-HPLC1-purification	10.4 minute retention time	The RP-HPLC1-rxn mixture	94.1% transforms
The RP-HPLC1-purification	10.4 minute retention time	MALDI-TOF MS	Average quality=34.2kDa

Weigh up from the 13mg peptide of embodiment 26 and be dissolved in the 50mM borate, in pH 8.0 buffer.Weighing 624mg 30kDa PEG-succinimido butanimide is to obtain 4: 1 PEG: the peptide mol ratio also adds dissolved peptide with it.Reactant mixture was at room temperature stirred 2 hours, and afterwards at 20mM NaOAc, dilution is 10 times in pH 4.5 buffer, by the cation-exchange chromatography purification on SP-agarose gel FF (Amersham).Fig. 7 is the HPLC chromatogram of reactant mixture.Reaction produces 94.1% 30kDa peptide.

Mono-pegylated PYY peptide is used step NaCl gradient elution.Typically, required mono-pegylated peptide is with 250mM NaCl eluting.The PEG-PYY-sample peptide of eluting uses the film of 10kDa MW cutoff to concentrate in Amicon ultrafiltration chamber.Use 10 times of PBS diafiltrations once then.To concentrate peptide and submit analysis to, measure and be stored in-20 ℃.Fig. 8 is the HPLC chromatogram (10.4min) of the 30kDa PEG-PYY peptide of purification.The purity of 30kDa peptide is measured as＞and 90%.Fig. 9 represents to carry out the MALDI-TOF of 30kDa PEG-PYY peptide to confirm molecular weight.

Embodiment 37

(PEG-30,000) CH (CH ₃) CH ₂CO) Ile-Nle-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe) Arg-Tyr-NH ₂Preparation

N=～675 wherein

Analytical method	The result
Analytical method	The result	The RP-HPLC1-rxn mixture	93.4% transforms
The RP-HPLC1-purification	10.5 minute retention time	The RP-HPLC1-rxn mixture	93.4% transforms
The RP-HPLC1-purification	10.5 minute retention time	MALDI-TOF MS	Average quality=34.6kDa

Weigh up from the 1.25mg peptide of embodiment 26 and be dissolved in the 50mM borate, in pH 8.0 buffer.Weighing 62mg 30kDa PEG-succinimido β-SBA is to obtain 4: 1 PEG: the peptide mol ratio also adds dissolved peptide with it.Reactant mixture was at room temperature stirred 2 hours, and afterwards at 20mM NaOAc, dilution is 10 times in pH 4.5 buffer, by the cation-exchange chromatography purification on SP-agarose gel FF (Amersham).Figure 10 is the HPLC chromatogram of reactant mixture.Reaction produces 93.4% 30kDa peptide.

Mono-pegylated PYY peptide is used step NaCl gradient elution.Typically, required mono-pegylated peptide is with 250mM NaCl eluting.The PEG-PYY-sample peptide of eluting uses the film of 10kDa MW cutoff to concentrate in Amicon ultrafiltration chamber.Use 10 times of PBS diafiltrations once then.To concentrate peptide and submit analysis to, measure and be stored in-20 ℃.Figure 11 is the HPLC chromatogram (10.5min) of the 30kDa PEG-PYY peptide of purification.The purity of 30kDa peptide is measured as＞and 90%.Figure 12 represents to carry out the MALDI-TOF of 30kDa PEG-PYY peptide to confirm molecular weight.

Embodiment 38

Ac-Ile (PEG-30,000) CH ₂CH ₂CO (ε) .Lys-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe) Arg-Tyr-NH ₂Preparation

N=～675 wherein

Analytical method	The result
Analytical method	The result	The RP-HPLC1-rxn mixture	83.3% transforms
The RP-HPLC1-purification	9.5 minute retention time	The RP-HPLC1-rxn mixture	83.3% transforms
The RP-HPLC1-purification	9.5 minute retention time	MALDI-TOF MS	Average quality=34.6kDa

Weigh up from the 100mg peptide of embodiment 28 and be dissolved in the 50mM borate, in pH 8.0 buffer.Weighing 1.8g 30kDa PEG-butanimide propanoic acid (available from Nektar) is to obtain 1.5: 1PEG: the peptide mol ratio also adds dissolved peptide with it.Reactant mixture at room temperature stirred spend the night, afterwards at 20mM NaOAc, dilution is 10 times in pH 4.5 buffer, by the cation-exchange chromatography purification on SP-agarose gel FF.Figure 13 is the HPLC chromatogram of reactant mixture.Reaction produces 83.3% 30kDa peptide.

Submit the concentrated peptide of embodiment 38 to analysis, measure and be stored in-20 ℃.Figure 14 is the HPLC chromatogram (RT=9.5min) of the 30kDa PEG-PYY peptide of purification.The purity of 30kDa peptide is measured as＞and 95%.Figure 15 represents to carry out the MALDI-TOF of 30kDa PEG-PYY peptide to confirm molecular weight.

Embodiment 39

Ac-Ile ((PEG-30,000) CH ₂CH ₂NHCOCH ₂CH ₂CO) (ε) .Lys-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe) Arg-Tyr-NH ₂Preparation

N=～675 wherein

Analytical method	The result
Analytical method	The result	The RP-HPLC1-rxn mixture	81.4% transforms
The RP-HPLC1-purification	9.5 minute retention time	The RP-HPLC1-rxn mixture	81.4% transforms
The RP-HPLC1-purification	9.5 minute retention time	MALDI-TOF MS	Average quality=33.6kDa

Weigh up from the 100mg peptide of embodiment 28 and be dissolved in the 50mM borate, in pH 8.0 buffer.Weighing 3.6g 30kDa PEG-succinimido butanimide is to obtain 3: 1 PEG: the peptide mol ratio also adds dissolved peptide with it.Reactant mixture at room temperature stirred spend the night, afterwards at 20mM NaOAc, dilution is 10 times in pH 4.5 buffer, by the cation-exchange chromatography purification on SP-agarose gel FF.Figure 16 is the HPLC chromatogram of reactant mixture.Reaction produces 81.4% 30kDa peptide.

Submit the concentrated peptide of embodiment 39 to analysis, measure and be stored in-20 ℃.Figure 17 is the HPLC chromatogram (RT=9.5min) of the 30kDa PEG-PYY peptide of purification.The purity of 30kDa peptide is measured as＞and 97%.Figure 18 is that expression is carried out the MALDI-TOF of 30kDa PEG-PYY peptide to confirm the figure of molecular weight.

Embodiment 40

The Arg-Tyr-NH of Fmoc-Ile-Lys-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe) ₂Preparation

It is synthetic and carry out purification by following method that 55.0g Fmoc-joint BHA@0.45mm/g (available from AnaSpec Inc. catalog number (Cat.No.) 408/452-5055) is carried out solid-phase peptide: with the aminoacid of Fmoc protection by 25% excessive DIC/HOBt coupling (55.0gX0.45mm/gX1.25eqv=31.0mm).All 20% piperidines that are used among the DMF that go to protect carry out 2 times with about 10ml/g, each 10 minutes (the volume increase with peptide-resin is adjusted).After going protection, peptide resin is washed 4 times with DMF (20 volume).

All couplings with～40mL DIC (～6eqv) and 4.5g HOBt (1.25eqv) carry out.After the coupling,, use CH with resin solution sampling～.25ml ₂Cl ₂Wash, and finish by the 1,2,3-indantrione monohydrate verification.After being coupled to NMe-Arg and Pqa, with resin CH ₂Cl ₂Washing, and drop in 2%Chlorinal and 3 among the DMAc with 3 drop in 2% acetaldehyde verification (do not change, indicating does not have secondary amine, indicates incomplete coupling from indigo plant to the black pearl) among the DMAc in yellow solution.If judge that coupling is incomplete, DIEA is added coupling solution and continues 1 hour again.When finishing, resin is washed 4 times with DMF (20 volume).

Fmoc-Tyr(But)-OH：14.0g.Fmoc-NMeArg(Mtr)-OH：20.0g.Fmoc-Gln(Trt)-OH：18.5g.Fmoc-Arg(Pbf)-OH：20.0g.Fmoc-Thr(But)-OH：18.0g.Fmoc-Val-OH：10.5g.Fmoc-Trp-OH：13.0.Fmoc-Asn(Trt)-OH：18.0g.Fmoc-Leu-OH 11.0g，Fmoc-Tyr(But)-OH 14.0g，Fmoc-His(Trt)-OH 20.0g，Fmoc-Arg(Pbf)-OH 20.0g，Fmoc-Pqa-OH 15.4g，Fmoc-Lys(Boc)-OH 14.1g，Fmoc-Ile-OH 11.0g.

Peptide resin with DMF washing 3 times, is used CH ₂Cl ₂Wash 3 times, and with MeOH washing 3 times, dry to obtain the 115.38g peptide resin under bleeding.Resin is divided into 6X19.25g batch to be used for the TFA cracking.

Peptide resin 8.0mL 1: 1: 1DTE with 19.25g; Methyl phenyl ethers anisole; THIOANISOLE, 8.0mLiPr ₃SiH, 8.0mL H2O and 200mL TFA/ cracking 6 hours (6:30AM to 13:30PM) precipitate at 2.0L cold (20 ℃) Et2O subsequently.By centrifugal, also use cold Et at 8 * 50mL polypropylene centrifuge tube collecting precipitation thing ₂O washing 3 times.Then with precipitate in indoor vacuum dried overnight to obtain the rough peptide of 6.5g.After going protection for 6 times, the total amount of rough peptide is 38.71g.

In Shimadzu LC-8A system, carry out the purification of rough peptide by the high performance liquid chromatography (HPLC) on anti-phase Pursuit C-18 post (50 * 250mm.300A °, 10 μ m).With the rough peptide of 38.71g purification in 36preps.At every turn will the rough peptide of about 1.1g be dissolved in minimum water and the acetonitrile and be injected in the post.Gradient elution begins with the 20%B buffer usually, 20%-70%B in 70 minutes, (buffer A: 0.1%TFA/H ₂O, buffer B: 0.1%TFA/CH ₃CN), flow velocity 50ml/min.Carrying out UV under 220/280nm detects.Separate the purity that contains the fraction of product and on the ShimadzuLC-10AT analytical system, judge them, wherein use anti-phase Pursuit C18 post (4.6 * 50mm), flow velocity is 2.5ml/min., 10 minutes inside gradient (20-70%) (buffer A: 0.1%TFA/H ₂O, buffer B: 0.1%TFA/CH ₃CN)).To be judged as highly purified fraction merging and lyophilizing to produce white amorphous powder.Will from the lyophilized products of 36preps merge and once more lyophilizing to produce the pure peptide (12.6%) of 8.233g.On reversed-phase column, check the purity of end-product and described purity to be about 95-99% by analyzing HPLC once more as mentioned above.(ES)+-LCMS m/e value of calculation C119H164N34O232438.85, measured value 2438.84

Embodiment 41

Ile ((PEG-30,000) CH ₂CH ₂NHCOCH ₂CH ₂CO) (ε) Lys-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe) Arg-Tyr-NH ₂Preparation

n＝～675

Analytical method	The result
Analytical method	The result	RP-HPLC2	PEG-peptide 89.5% gross production rate of 93.8% conversion-protection-de-protected PEG-peptide
The RP-HPLC2-purification	10.1 minute retention time	RP-HPLC2
The RP-HPLC2-purification	10.1 minute retention time	MALDI-TOF MS	Average quality=35kDa

Weigh up from the 1.8g peptide of embodiment 40 and be dissolved in the 50mM borate, in the pH7.5 buffer.Weighing 37.5g 30kDa PEG-succinimido butanimide is to obtain about 2: 1PEG: the peptide mol ratio also adds dissolved peptide with it.Reactant mixture was at room temperature stirred 2 hours.The peptide of PEGization was gone protection in the reactant mixture in 1 hour by add piperidines (20%) in room temperature.Described reactant mixture placed on ice and carry out acidify by slowly adding glacial acetic acid (20%).Then, at 20mM NaOAc, dilution is 10 times in pH 4.5 buffer, by the cation-exchange chromatography purification on SP-agarose gel FF with reactant mixture.Figure 19 is the HPLC chromatogram of reactant mixture.The peptide of the PEGization of the protection of reaction generation 93.8%.Figure 20 is the HPLC chromatograph of de-protected reactant mixture, and wherein the total output of the de-protected peptide of PEGization is 89.5%.

Mono-pegylated PYY peptide is used step NaCl gradient elution.Typically, required mono-pegylated peptide is with 175mM NaCl eluting.The PEG-PYY-sample peptide of eluting uses the film of 10kDa MW cutoff to concentrate in Amicon ultrafiltration chamber.Use 10 times of PBS diafiltrations once then.

To concentrate peptide and submit analysis to, measure and be stored in-20 ℃.Figure 21 is the HPLC chromatogram (RT=10.1min) of the 30kDaPEG-PYY peptide of purification.The purity of 30kDa peptide is measured as＞and 95%.Figure 22 is that expression is carried out the MALDI-TOF of 30kDa PEG-PYY peptide to confirm the figure of molecular weight.

Embodiment 42

Calcium current goes out to measure

The HEK-293 cell with G albumen chimera G α qi9 stable transfection, is selected transfection with further choose NPY2 receptor and G418 antibiotic of hygromycin-B resistant gene.After in hygromycin-B and G418, selecting, measure single clone they to the reaction of PYY.Cells transfected (HEK293/hNPY2R) is cultivated in the DMEM culture medium that is supplemented with 10% hyclone, 50 μ g/ml hygromycin-B, 2mM glutamine, 100U/ml penicillin, 100 μ g/ml streptomycins and 250 μ g/ml G418.Cell is counted with trypsin-EDTA results and use ViaCount reagent.The cell suspension volume is adjusted to 4.8 * 10 with complete growth medium ⁵Cell/ml.With 25 μ L aliquots be assigned to 384 holes poly--black/transparent microtest plate (Falcon) of D lysine bag quilt in, with microtest plate at 37 ℃ of CO ₂Place in the incubator and spend the night.

Be dissolved in by content (Express Kit) and prepare sample loading buffer (calcium-3 is measured test kit, molecular device (Molecular Devices)) in 1000ml Hank ' the s balanced salt solution (HBSS) of the probenecid that contains 20mM HEPES and 5mM a tubule.The aliquot of 25 μ L dilution dyestuff is assigned in the cell flat board, then with flat board 37 ℃ of following incubations 1 hour.

Between incubation period, in HBSS (20mM HEPES)/0.05%BSA/1%DMSO, prepare test compounds, and transfer in the 384 hole flat boards at FLIPR with the 3.5X desired concn

(FLIPR, fluorescence imaging flat bed reader are the registered trade marks of molecular device company (Molecular Devices Corp.)) are gone up and are used.

Behind incubation, with cell and chemical compound dull and stereotyped both put to FLIPR

And pass through FLIPR

The chemical compound of 20 μ L dilution is transferred to Tissue Culture Plate.In mensuration, whole 384 holes from Tissue Culture Plate obtained the fluorescence reading automatically in per 1.5 seconds.Obtain 5 readings to set up stable baseline, then with 20 μ L samples rapidly (30 μ L/ second) and add each hole of Tissue Culture Plate simultaneously.Before sample adds, during and continuous monitoring fluorescence elapsed time of 100 seconds altogether afterwards.Be determined at the response (raising of peak fluorescence) that adds in each hole, back.Will be before ligand stimulation from the initial fluorescence reading in every hole as zero base line value from the data in that hole.Response Table is shown the peak response % of positive control.

Embodiment 43

Ring-type AMP measures

In the present embodiment, use following material: 384-hole flat board; Tropix cAMP-screening reagent box; (applying biological system (Applied Biosystems), catalog number (Cat.No.) T1504); Forskolin (Calbiochem catalog number (Cat.No.) 344270); Cell: HEK293/hNPY2R cell; Inoculation medium: DMEM/F12w/o phenol red (Gibco catalog number (Cat.No.) 1133032); 10% heat-deactivation FBS (Gibco catalog number (Cat.No.) 10082-147); 1% penicillin/streptomycin (Gibco catalog number (Cat.No.) 15140-122); 500mg/mlG418 (Geneticin, Gibco catalog number (Cat.No.) 11811-031).

Use many dispersers with the HEK293/hNPY2R cell with 10 ⁴The density of cells/well is seeded in the 384 hole flat boards and with described plate and is incubated overnight at 37 ℃.Second day, in this experiment, use to reach the cell that 75-85% converges.Make culture medium and reagent be warmed up to room temperature.Before the preparation diluent, make the Y2 receptors ligand in dimethyl sulfoxine (DMSO, SIGMA, catalog number (Cat.No.) D2650) and the storage liquid of contrast be warming up to 32 ℃, 5-10 minute.Use incubation culture medium [the DMEM/F12 culture medium that comprises 0.5mM 3-isobutyl-1-methylxanthine (IBMX, Calbiochem, catalog number (Cat.No.) 410957) and 0.5mg/ml BSA (SIGMA, catalog number (Cat.No.) A8806)] preparation diluent.The final DMSO in the incubation culture medium and the concentration of forskolin are respectively 1.1% and 5 μ M.

Remove incubation culture medium (the 50 μ l/ hole) displacement that inoculation medium is also used the Y2-receptors ligand (quadruplicate/concentration) that comprises various concentration by slight reversing 384 hole flat boards on napkin.With described plate room temperature incubation 30 minutes.After date when 30 minutes processing abandons the incubation culture medium and replaces with the mensuration lysis buffer (providing in the Tropix test kit) in 50 μ l/ holes.By coming cell lysis in 45 minutes at 37 ℃ of incubation plates.Lysate (20 μ l) is transferred in the antibody panel (384-hole) of the pre-bag quilt of in the Tropix test kit, supplying.AP conjugate (10 μ l) and anti--cAMP antibody (20 μ l) added in each hole and with described plate on shaking machine room temperature incubation 1 hour.Drain with described plate washing 5 times and with described plate with lavation buffer solution (70 μ l/ hole/washing).Add CSPD/ sapphire (Saphire)-II RTU substrate/reinforcing agent solution (30 μ l/ hole) and room temperature incubation 45 minutes.Use photometer (VICTOR-V), measure the signal (1 second/hole) in each hole.

The external selectivity neuropeptide-2 receptor-of compound exhibits of the present invention activity is as going out to measure (FLIPR at calcium current

Embodiment 42) and ring-type AMP measures, and (embodiment 43) are middle to be confirmed.With the in vitro results of embodiment 3-39 and 41, EC ₅₀Be summarised in following table 1 illustrated:

Table 1

Embodiment	Sequence	Y2R EC50 (nM) FLIPR	Y2R EC50 (nM) cAMP	Y1R EC50 (nM) FLIPR	Y4REC50(nM)FLIPR	Y5R EC50 (nM) FLIPR
Embodiment	Sequence	Y2R EC50 (nM) FLIPR	Y2R EC50 (nM) cAMP	Y1R EC50 (nM) FLIPR	Y4REC50(nM)FLIPR	Y5R EC50 (nM) FLIPR	3	IKPEAPGEDASPEELNRYY ASLRHYLNLVTRQRY (3-36)	0.12	0.033	63	3238	265
4	IK-Pqa-RHYLNLVTRQRY	0.28	0.047	57	＞5000	1887	3	IKPEAPGEDASPEELNRYY ASLRHYLNLVTRQRY (3-36)	0.12	0.033	63	3238	265
4	IK-Pqa-RHYLNLVTRQRY	0.28	0.047	57	＞5000	1887	5	IK-Pqa-RHYLNLVTRQ (N-methyl) RY	2.3	0.42	＞5000	＞5000	＞5000
6	IK-Pqa-RHYLNLVTRQ (N-methyl) R (m-) Y	3.32	1.5	＞5000	＞5000	＞5000	5	IK-Pqa-RHYLNLVTRQ (N-methyl) RY	2.3	0.42	＞5000	＞5000	＞5000
6	IK-Pqa-RHYLNLVTRQ (N-methyl) R (m-) Y	3.32	1.5	＞5000	＞5000	＞5000	7	IK-Pqa-RHYLNLVTRQ (N-methyl) R (3-I) Y	1.15	0.31	173	＞5000	＞5000
8	IK-Pqa-RHYLNLVTRQ (N-methyl) R (3-5 two F) Y	0.15	0.36	＞5000	＞5000	＞5000	7	IK-Pqa-RHYLNLVTRQ (N-methyl) R (3-I) Y	1.15	0.31	173	＞5000	＞5000

9	IK-Pqa-RHYLNLVTRQ (N-methyl) R (2-6 two F) Y	0.11	0.19	303	＞5000	＞5000
9	IK-Pqa-RHYLNLVTRQ (N-methyl) R (2-6 two F) Y	0.11	0.19	303	＞5000	＞5000	10	IK-Pqa-RHYLNLVTRQ (N-methyl) R (2-6 two Me) Y	0.28	0.67	762	＞5000	＞5000
11	IK-Pqa-RHYLNLVTRQ (N-methyl) RF (O-CH3)	0.74	0.55	189	＞5000	＞5000	10	IK-Pqa-RHYLNLVTRQ (N-methyl) R (2-6 two Me) Y	0.28	0.67	762	＞5000	＞5000
11	IK-Pqa-RHYLNLVTRQ (N-methyl) RF (O-CH3)	0.74	0.55	189	＞5000	＞5000	12	IK-Pqa-RHYLNLVTRQ (N-methyl) RF	1.54	0.69	422	＞5000	＞5000
13	IK-Pqa-RHYLNLVTRQ (N-methyl) R (4-NH2) Phe	11.4	0.31	＞5000	＞5000	＞5000	12	IK-Pqa-RHYLNLVTRQ (N-methyl) RF	1.54	0.69	422	＞5000	＞5000
13	IK-Pqa-RHYLNLVTRQ (N-methyl) R (4-NH2) Phe	11.4	0.31	＞5000	＞5000	＞5000	14	IK-Pqa-RHYLNLVTRQ (N-methyl) R (4-F) Phe	0.45	0.96	＞5000	＞5000	2259
15	IK-Pqa-RHYLNLVTRQ (N-methyl) R (4-CH2OH) Phe	0.46	0.45	＞5000	＞5000	＞5000	14	IK-Pqa-RHYLNLVTRQ (N-methyl) R (4-F) Phe	0.45	0.96	＞5000	＞5000	2259
15	IK-Pqa-RHYLNLVTRQ (N-methyl) R (4-CH2OH) Phe	0.46	0.45	＞5000	＞5000	＞5000	16	IK-Pqa-RHYLNLVTRQ (N-methyl) R (4-CF3) Phe	6.13	3.55	3268	＞5000	729
17	IK-Pqa-RHYLNLVTRQ (N-methyl) R (3-F) Phe	0.635	0.75	＞5000	＞5000	＞5000	16	IK-Pqa-RHYLNLVTRQ (N-methyl) R (4-CF3) Phe	6.13	3.55	3268	＞5000	729
17	IK-Pqa-RHYLNLVTRQ (N-methyl) R (3-F) Phe	0.635	0.75	＞5000	＞5000	＞5000	18	IK-Pqa-RHYLNLVTRQ (N-methyl) R (2,3.4,5,6-five-F) Phe	11.9	2.5	＞5000	＞5000	＞5000
19	IK-Pqa-RHYLNLVTRQ (N-methyl) R (3.4-two Cl) Phe	4.03	1.47	＞5000	＞5000	＞5000	18	IK-Pqa-RHYLNLVTRQ (N-methyl) R (2,3.4,5,6-five-F) Phe	11.9	2.5	＞5000	＞5000	＞5000
19	IK-Pqa-RHYLNLVTRQ (N-methyl) R (3.4-two Cl) Phe	4.03	1.47	＞5000	＞5000	＞5000	20	IK-Pqa-RHYLNLVTRQ (N-methyl) RCha	0.498	0.5	＞5000	＞5000	352

21	IK-Pqa-RHYLNLVTRQ (N-methyl) RW	0.454	1.06	＞5000	＞5000	＞5000
21	IK-Pqa-RHYLNLVTRQ (N-methyl) RW	0.454	1.06	＞5000	＞5000	＞5000	22	IK-Pqa-RHYLNLVTRQ (N-methyl) R (1) Nal	2.73	1.14	＞5000	＞5000	4772
23	IK-Pqa-RHYLNLVTRQ (N-methyl) R (2) Nal	4.11	2.4	＞5000	＞5000	2162	22	IK-Pqa-RHYLNLVTRQ (N-methyl) R (1) Nal	2.73	1.14	＞5000	＞5000	4772
23	IK-Pqa-RHYLNLVTRQ (N-methyl) R (2) Nal	4.11	2.4	＞5000	＞5000	2162	24	IK-Pqa-RHYLNLVTRQR-C- α-Me-Tyr	5.44	1.35	3259	＞5000
25	IK-Pqa-RHYLNWVTRQ (N-methyl) RY	0.44	0.25	298	＞5000	＞5000	24	IK-Pqa-RHYLNLVTRQR-C- α-Me-Tyr	5.44	1.35	3259	＞5000
25	IK-Pqa-RHYLNWVTRQ (N-methyl) RY	0.44	0.25	298	＞5000	＞5000	26	INle-Pqa-RHYLNWVTRQ (N-methyl) RY	8.1	0.108	＞5000	＞5000	＞5000
27	Ac-IK-Pqa-RHYLNWVTRQ (N-methyl) R (2-6 two F) Y	1.44	0.07	＞5000	＞5000	5812	26	INle-Pqa-RHYLNWVTRQ (N-methyl) RY	8.1	0.108	＞5000	＞5000	＞5000
27	Ac-IK-Pqa-RHYLNWVTRQ (N-methyl) R (2-6 two F) Y	1.44	0.07	＞5000	＞5000	5812	28	Ac-IK-Pqa-RHYLNWVTRQ (N-methyl) RY	0.458	0.18	＞5000	＞5000	＞5000
29	Amyl group-IK-Pqa-RHYLNWVTRQ (N-methyl) RY	0.873	0.51	＞5000	＞5000	＞5000	28	Ac-IK-Pqa-RHYLNWVTRQ (N-methyl) RY	0.458	0.18	＞5000	＞5000	＞5000
29	Amyl group-IK-Pqa-RHYLNWVTRQ (N-methyl) RY	0.873	0.51	＞5000	＞5000	＞5000	30	Pivaloyl group-IK-Pqa-RHYLNWVTRQ (N-methyl) RY	1.1	0.26	＞5000	＞5000	＞5000

31	Cyclohexyl-IK-Pqa-RHYLNWVTRQ (N-methyl) RY	1.67	1.37	＞5000	＞5000	＞5000
31	Cyclohexyl-IK-Pqa-RHYLNWVTRQ (N-methyl) RY	1.67	1.37	＞5000	＞5000	＞5000	32	Benzoyl-IK-Pqa-RHYLNWVTRQ (N-methyl) RY	0.79	0.66	＞5000	＞5000	＞5000
33	Adamantyl-IK-Pqa-RHYLNWVTRQ (N-methyl) RY	2.33	2.9	＞5000	＞5000	＞5000	32	Benzoyl-IK-Pqa-RHYLNWVTRQ (N-methyl) RY	0.79	0.66	＞5000	＞5000	＞5000
33	Adamantyl-IK-Pqa-RHYLNWVTRQ (N-methyl) RY	2.33	2.9	＞5000	＞5000	＞5000	34	(PEG 30,000 SPA) IK-Pqa-RHYLNWVTR Q (N-methyl) RY	37.9	18	＞5000	＞5000	＞5000
35	(PEG40,000 BTC)-IK-Pqa-RHYLNWVTR Q (N-methyl RY	590	14.7	＞5000	＞5000	＞5000	34	(PEG 30,000 SPA) IK-Pqa-RHYLNWVTR Q (N-methyl) RY	37.9	18	＞5000	＞5000	＞5000
35	(PEG40,000 BTC)-IK-Pqa-RHYLNWVTR Q (N-methyl RY	590	14.7	＞5000	＞5000	＞5000	36	(PEG30,000)-SSA-INle-Pqa-RHYLNWVTRQ (N-methyl) RY	289	7.8	＞5000	＞5000	＞5000
37	(PEG30,000)-β-SBA-INle-Pq a-RHYLNWVTRQ (N-methyl) RY	239	22.4	＞5000	＞5000	＞5000	36	(PEG30,000)-SSA-INle-Pqa-RHYLNWVTRQ (N-methyl) RY	289	7.8	＞5000	＞5000	＞5000
37	(PEG30,000)-β-SBA-INle-Pq a-RHYLNWVTRQ (N-methyl) RY	239	22.4	＞5000	＞5000	＞5000	38	Ac-Ile-Lys (PEG30,000 SPA)-Pqa-RHYLNWVTRQ (N-methyl) RY	549	24.4	＞5000	＞5000	＞5000

39	Ac-Ile-Lys (PEG30,000 SSA)-Pqa-RHYLNWVTRQ (N-methyl) RY	1078	20.7	＞5000	＞5000	＞5000
39	Ac-Ile-Lys (PEG30,000 SSA)-Pqa-RHYLNWVTRQ (N-methyl) RY	1078	20.7	＞5000	＞5000	＞5000	41	IK (PEG30,000 SSA)-Pqa-RHYLNWVTRQ (N-methyl) RY	13.5	9.8	＞5000	＞5000	＞5000

Embodiment 44

Chronic DIO rat studies

Male Sprague Dawley rat (7 week age) is available from charles (Charles) river laboratory (U.S.) and have illumination in 12 hours: in the environment that the temperature and humidity of 12 hours dark cycles is controlled at indoor cultivation.Rat is arbitrarily got the higher fatty acid standard diet (HFD of food in whole research; 60% meals kilocalorie is a fat, research diet (Research Diets) D12492) and water.After 7 weeks on the HFD, make rat according to the body weight sorting and raise in cages separately.The administration rat, afterwards when dark cycle begins the chemical compound of administration excipient (s.c.) or embodiment 41 (1,5 and 10mg/kg, s.c.), per two days 1 time, carried out for 3 weeks (N=6-8 rat/group).The natural law record body weight of in Figure 23, pointing out.

Data analysis:

All data that show are mean+/-standard error (s.e.m.).Use unidirectional ANOVA to carry out the statistics assessment of data, carry out the statistics significant difference of Dunnett ' s check subsequently to determine between excipient and drug treating group, to exist.When P＜0.05, think that difference is significant on the statistics.(GraphPad Prism) carries out data analysis with GraphPad software.

The result:

After date when handling in 3 weeks is compared with the animal that excipient is handled, and (5 and 10mg/kg, q48hr s.c.) induces the remarkable minimizing (Figure 23) of weight increase to chronic the using of embodiment 41 chemical compounds in male DIO rat.

Acute db/db mice study

When accepting, female db/db mice (C57BL/KsJ-Lep ^Db/db, Jackson laboratory, the U.S.) and be 6 ages in week.The mice indoor cultivation had illumination in 12 hours: in the environment of the temperature and humidity control of 12 hours dark cycles, and get food food (Purina standard diet for rodents 5008) and water arbitrarily.In research preceding 4 days, mice (12 age in week) is taken a blood sample in advance, and be chosen in the narrow fasting blood glucose level scope those and study so that the transmutability between excipient contrast and drug treating group minimizes.Before oral glucose tolerance test (N=10 mice/group) 28 hours, to mice use excipient (s.c.) or embodiment 41 chemical compound (0.3,1 and 10mg/kg, s.c.).After fasting in 6 hours, collect blood sample to determine baseline value (t=0 minute) from the tail folder.Carry out glucose measurement then to mice tube feed oral glucose pill (1g/kg), and at routine interval (t=30,60 and 120 minutes) the other blood sample of collection.For the influence of the chemical compound of analyzing embodiment 41, calculate at the blood glucose (fasting glucose) of each time point and the antipode of baseline to oral glucose tolerance.Use trapezoid method to determine area under a curve (AUC _{0-120 minute}).

Data analysis:

All data that show are mean+/-standard error (s.d.).Use unidirectional ANOVA to carry out the statistics assessment of data, carry out the statistics significant difference of Dunnett ' s check subsequently to determine between excipient and drug treating group, to exist.When P＜0.05, think significant on the difference foot statistics.(GraphPad Prism) carries out data analysis with GraphPad software.

The result:

Excite in response to oral glucose, and the acute administration of the chemical compound of embodiment 41 (1 and 10mg/kg, s.c.) obviously reduce glucose excursion (Figure 24) for female db/db mice.

Chronic db/db mice study

When accepting, female db/db mice (C57BL/KsJ-Lep ^Db/db, Jackson laboratory, the U.S.) and be 6 ages in week.The mice indoor cultivation had illumination in 12 hours: in the environment of the temperature and humidity control of 12 hours dark cycles, and arbitrarily get food food (Purina standard diet for rodents 5008) and water.Preceding 4 days of drug treating mice (9 age in week) is taken a blood sample in advance, and be chosen in the narrow fasting blood glucose level scope those and study so that the transmutability between excipient contrast and drug treating group minimizes.Per two greatly once to the chemical compound of mice administration excipient (s.c.) or embodiment 41 (1,3 and 10mg/kg, s.c.), carried out for 3 weeks (N=10 mice/group).Carry out baseline fasting (2-6 hour) blood glucose measurement once in a week.At the 20th day of research, after fasting in 6 hours, carry out oral glucose tolerance test.Collect blood sample to determine baseline value (t=0 minute) from the tail folder.Then, with oral glucose pill (1g/kg) tube feed mice, and collect other blood sample (t=30,60 and 120 minutes) at interval with routine and carry out glucose measurement.For the effect of the chemical compound of analyzing embodiment 41, calculate the antipode of blood glucose (fasting glucose, t=0 minute) and baseline at each time point to oral glucose tolerance.Use trapezoid method to determine area under a curve (AUC _{0-120 minute}).

Data analysis:

All data that show are mean+/-standard error (s.d.).Use unidirectional ANOVA to carry out the statistics assessment of data, carry out the statistics significant difference of Dunnett ' s check subsequently to determine between excipient and drug treating group, to exist.When P＜0.05, think that difference is significant on the statistics.(GraphPad Prism) carries out data analysis with GraphPad software.

The result:

In 3 all processing processes in period, the animal of handling with respect to excipient, (1,3 and 10mg/kg, q48hr s.c.) chronicly is applied to female db/db mice and reduces basic blood sugar level (the 8th, 15 and 21 day) (Figure 25 A) with the chemical compound of embodiment 41.As shown in Figure 25 B, at the 20th day, excite in response to oral glucose, (all 3 and 10mg/kg, q48hr s.c.) significantly reduces the glucose excursion to the chemical compound of embodiment 41.

Should be appreciated that the present invention is not limited to above-described specific embodiments of the present invention, because can carry out the change of specific embodiments and still fall in the scope of appended claim.

Embodiment A

Can prepare the film coating tablet that contains following component in a usual manner:

CompositionEvery

Nuclear:

The chemical compound 10.0mg 200.0mg of formula (I)

Microcrystalline Cellulose 23.5mg 43.5mg

Lactis Anhydrous 60.0mg 70.0mg

Polyvinylpyrrolidone K30 12.5mg 15.0mg

Primojel 12.5mg 17.0mg

Magnesium stearate 1.5mg 4.5mg

(nuclear is heavy) 120.0mg 350.0mg

Film coating:

Hydroxypropyl emthylcellulose 3.5mg 7.0mg

Polyethylene glycol 6000 0.8mg 1.6mg

Talcum 1.3mg 2.6mg

Ferrum oxide (Iron oxyde) (Huang) 0.8mg 1.6mg

Titanium dioxide 0.8mg 1.6mg

The screening active component, mixed with microcrystalline Cellulose, the aqueous solution of mixture and polyvinylpyrrolidone is granulated.Granule and primojel and magnesium stearate is mixed, and compacting obtains 120 or the core of 350mg respectively.With core with above-mentioned film-coated aqueous solution/suspension coating.

Embodiment B

Can prepare the capsule that contains following ingredients in a usual manner:

Composition Every capsule

Formula (I) chemical compound 25.0mg

Lactose 150.0mg

Corn starch 20.0mg

Talcum 5.0mg

Sieve each component and mix and be filled in the 2# capsule.

Embodiment C

Injection can have following composition:

The chemical compound 3.0mg of formula (I)

PEG400 150.0mg

Acetic acid adds to pH 5.0 in right amount

The injection water adds to 1.0ml

Active component is dissolved in PEG400 and water for injection (part) mixture.By acetic acid with pH regulator to 5.0.Water by adding residual volume with volume-adjustment to 1.0ml.Solution is filtered, use in the bottle that suitably exceeds the quata to pack into and sterilization.

Embodiment D

Can prepare the Perle that contains following ingredients in a usual manner:

Capsule 's content

Formula (I) chemical compound 5.0mg

Cera Flava 8.0mg

Oil with hydrogenated soybean 8.0mg

Partially hydrogenated vegetable oil 34.0mg

Soybean oil 110.0mg

Capsule 's content weight 165.0mg

Gelatine capsule

Gelatin 75.0mg

Glycerol 85% 32.0mg

Karion 83 8.0mg (dry)

Titanium dioxide 0.4mg

Iron oxide yellow 1.1mg

Active component is dissolved in the warm molten mass of other composition, mixture is filled in the Perle of appropriate size.Handle the Perle of filling according to usual method.

Embodiment E

Can prepare the sachet that contains following ingredients in a usual manner:

Formula (I) chemical compound 50.0mg

Lactose, fine powder 1015.0mg

Microcrystalline Cellulose (AVICEL PH 102) 1400.0mg

Sodium carboxymethyl cellulose 14.0mg

Polyvinylpyrrolidone K30 10.0mg

Magnesium stearate 10.0mg

Flavouring additive 1.0mg

Active component and lactose, microcrystalline Cellulose and sodium carboxymethyl cellulose is mixed, granulate with the mixture of polyvinylpyrrolidone in water.With granule and magnesium stearate with flavouring additive is mixed and the sachet of packing into.

Sequence table

＜110〉Hoffman-Laluoai Ltd

＜120〉neuropeptide-2 receptor-agonists

<130>23353

<140>

<141>

<150>60/748,071

<151>2005-12-07

<160>41

<170>PatentIn Ver.3.3

<210>1

<211>34

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<400>1

Ile Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu Asn

1 5 10 15

Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln

20 25 30

Arg Tyr

<210>2

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<400>2

Ile Lys Xaa Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr

1 5 10 15

<210>3

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<400>3

Ile Lys Xaa Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr

1 5 10 15

<210>4

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<220>

<221>MOD_RES

<222>(15)

<223>m-Tyr

<400>4

Ile Lys Xaa Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr

1 5 10 15

<210>5

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<220>

<221>MOD_RES

<222>(15)

＜223〉3-iodo-Tyr

<400>5

Ile Lys Xaa Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr

1 5 10 15

<210>6

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<220>

<221>MOD_RES

<222>(15)

＜223〉3,5 two F-Tyr

<400>6

Ile Lys Xaa Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr

1 5 10 15

<210>7

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<220>

<221>MOD_RES

<222>(15)

＜223〉2,6 two F-Tyr

<400>7

Ile Lys Xaa Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr

1 5 10 15

<210>8

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<220>

<221>MOD_RES

<222>(15)

<223>2，6-Me-Tyr

<400>8

Ile Lys Xaa Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr

1 5 10 15

<210>9

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<220>

<221>MOD_RES

<222>(15)

＜223〉4-methoxyl group-Phe

<400>9

Ile Lys Xaa Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Phe

1 5 10 15

<210>10

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<400>10

Ile Lys Xaa Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Phe

1 5 10 15

<210>11

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<220>

<221>MOD_RES

<222>(15)

＜223〉4-amino-Phe

<400>11

Ile Lys Xaa Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Phe

1 5 10 15

<210>12

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<220>

<221>MOD_RES

<222>(15)

<223>4 F-Phe

<400>12

Ile Lys Xaa Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Phe

1 5 10 15

<210>13

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<220>

<221>MOD_RES

<222>(15)

<223>4(CH2OH)-Phe

<400>13

Ile Lys Xaa Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Phe

1 5 10 15

<210>14

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<220>

<221>MOD_RES

<222>(15)

＜223〉4-trifluoromethyl-Phe

<400>14

Ile Lys Xaa Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Phe

1 5 10 15

<210>15

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<220>

<221>MOD_RES

<222>(15)

<223>3F-Phe

<400>15

Ile Lys Xaa Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Phe

1 5 10 15

<210>16

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<220>

<221>MOD_RES

<222>(15)

＜223〉2,3,4,5,6 five F-Phe

<400>16

Ile Lys Xaa Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Phe

1 5 10 15

<210>17

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<220>

<221>MOD_RES

<222>(15)

＜223〉3,4 two chloro-Phe

<400>17

Ile Lys Xaa Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Phe

1 5 10 15

<210>18

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<220>

<221>MOD_RES

<222>(15)

<223>Cha

<400>18

Ile Lys Xaa Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Xaa

1 5 10 15

<210>19

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<400>19

Ile Lys Xaa Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Trp

1 5 10 15

<210>20

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<220>

<221>MOD_RES

<222>(15)

<223>1-Nal

<400>20

Ile Lys Xaa Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Xaa

1 5 10 15

<210>21

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<220>

<221>MOD_RES

<222>(15)

<223>2-Nal

<400>21

Ile Lys Xaa Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Xaa

1 5 10 15

<210>22

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(15)

＜223〉(C-Alpha-Methyl)-Tyr

<400>22

Ile Lys Xaa Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr

1 5 10 15

<210>23

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<400>23

Ile Lys Xaa Arg His Tyr Leu Asn Trp Val Thr Arg Gln Arg Tyr

1 5 10 15

<210>24

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(2)

<223>Nle

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<400>24

Ile Xaa Xaa Arg His Tyr Leu Asn Trp Val Thr Arg Gln Arg Tyr

1 5 10 15

<210>25

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(1)

<223>Ac-Ile

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<220>

<221>MOD_RES

<222>(15)

＜223〉2,6 two fluoro-Tyr

<400>25

Ile Lys Xaa Arg His Tyr Leu Asn Trp Val Thr Arg Gln Arg Tyr

1 5 10 15

<210>26

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(1)

<223>Ac-Ile

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<400>26

Ile Lys Xaa Arg His Tyr Leu Asn Trp Val Thr Arg Gln Arg Tyr

1 5 10 15

<210>27

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(1)

＜223〉valeryl-Ile

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<400>27

Ile Lys Xaa Arg His Tyr Leu Asn Trp Val Thr Arg Gln Arg Tyr

1 5 10 15

<210>28

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(1)

＜223〉pivaloyl group-Ile

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<400>28

Ile Lys Xaa Arg His Tyr Leu Asn Trp Val Thr Arg Gln Arg Tyr

1 5 10 15

<210>29

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(1)

＜223〉cyclohexyl acetyl group-Ile

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<400>29

Ile Lys Xaa Arg His Tyr Leu Asn Trp Val Thr Arg Gln Arg Tyr

1 5 10 15

<210>30

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(1)

＜223〉benzoyl-Ile

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<400>30

Ile Lys Xaa Arg His Tyr Leu Asn Trp Val Thr Arg Gln Arg Tyr

1 5 10 15

<210>31

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(1)

＜223〉diamantane (obsolete) acyl group-Ile

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<400>31

Ile Lys Xaa Arg His Tyr Leu Asn Trp Val Thr Arg Gln Arg Tyr

1 5 10 15

<210>32

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<400>32

Ile Lys Xaa Arg His Tyr Leu Asn Trp Val Thr Arg Gln Arg Tyr

1 5 10 15

<210>33

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(2)

<223>Lys(PEG-30,000SPA)

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<400>33

Ile Lys Xaa Arg His Tyr Leu Asn Trp Val Thr Arg Gln Arg Tyr

1 5 10 15

<210>34

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<400>34

Ile Lys Xaa Arg His Tyr Leu Asn Trp Val Thr Arg Gln Arg Tyr

1 5 10 15

<210>35

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(2)

<223>Lys(PEG-40,000BTC)

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<400>35

Ile Lys Xaa Arg His Tyr Leu Asn Trp Val Thr Arg Gln Arg Tyr

1 5 10 15

<210>36

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(2)

<223>Nle

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<400>36

Ile Xaa Xaa Arg His Tyr Leu Asn Trp Val Thr Arg Gln Arg Tyr

1 5 10 15

<210>37

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(2)

<223>Nle

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<400>37

Ile Xaa Xaa Arg His Tyr Leu Asn Trp Val Thr Arg Gln Arg Tyr

1 5 10 15

<210>38

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(1)

<223>Ac-Ile

<220>

<221>MOD_RES

<222>(2)

<223>Lys(PEG-30,000SPA)

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<400>38

Ile Lys Xaa Arg His Tyr Leu Asn Trp Val Thr Arg Gln Arg Tyr

1 5 10 15

<210>39

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(1)

<223>Ac-Ile

<220>

<221>MOD_RES

<222>(2)

<223>Lys(PEG-30,000SSA)

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<400>39

Ile Lys Xaa Arg His Tyr Leu Asn Trp Val Thr Arg Gln Arg Tyr

1 5 10 15

<210>40

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(1)

<223>Fmoc-Ile

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<400>40

Ile Lys Xaa Arg His Tyr Leu Asn Trp Val Thr Arg Gln Arg Tyr

1 5 10 15

<210>41

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic

Peptide

<220>

<221>MOD_RES

<222>(2)

<223>Lys(PEG-30,000SSA)

<220>

<221>MOD_RES

<222>(3)

<223>Pqa

<220>

<221>MOD_RES

<222>(14)

<223>(NMe)Arg

<400>41

Ile Lys Xaa Arg His Tyr Leu Asn Trp Val Thr Arg Gln Arg Tyr

1 5 10 15

Claims

1. the neuropeptide-2 receptor-agonists of formula (I),

Wherein:

X is 4-oxo-6-(1-piperazinyl)-3 (4H)-quinazoline-acetic acid (Pqa),

R ₁Be Ile, Ala, (D) Ile, N-methyl Ile, Aib, 1-1Aic, 2-2Aic, Ach or Acp,

R ₂Be Lys, Ala, (D) Lys, NMelys, Nle or (Lys-Gly),

R ₄Be His, Ala, (D) His, N-methyl His, 4-MeOApc, 3-Pal or 4-Pal,

R ₆Be Leu, Ala, (D) Leu or N-methyl Leu,

R ₇Be Asn, Ala or (D) Asn,

R ₈Be Leu or Trp,

R ₉Be Val, Ala, (D) Val or N-methyl Val,

R ₁₀Be Thr, Ala or N-methyl Thr,

R ₁₁Be Arg, (D) Arg or N-methyl Arg,

R ₁₂Be Gln or Ala,

R ₁₃Be Arg, (D) Arg or N-methyl Arg,

PEG _mBe 1 to 60KDa,

Or its pharmaceutical salts.

2. according to the neuropeptide-2 receptor-agonists of claim 1, it is characterized in that formula (Ia)

Wherein:

X is N-piperazine-1-base-4 (3H)-quinazolinone-3-acetic acid (Pqa),

Y is H, and acyl moiety replaces or unsubstituted alkyl, replaces or unsubstituted low alkyl group, replaces or unsubstituted aryl, replace or unsubstituted alkoxyl, and polyalkylene glycol moiety, PEG-SSA,

PEG-β-SBA, PEG-SPA or PEG-BTC,

R ₁Be Ile, Ala, (D) Ile, N-methyl Ile, Aib, 1-1Aic, 2-2Aic, Ach or Acp,

R ₂Be Lys, Ala, (D) Lys, NMelys, Nle or (Lys-Gly),

R ₄Be His, Ala, (D) His, N-methyl His, 4-MeOApc, 3-Pal or 4-Pal,

R ₆Be Leu, Ala, (D) Leu or N-methyl Leu,

R ₇Be Asn, Ala or (D) Asn,

R ₈Be Leu or Trp,

R ₉Be Val, Ala, (D) Val or N-methyl Val,

R ₁₀Be Thr, Ala or N-methyl Thr,

R ₁₁Be Arg, (D) Arg or N-methyl Arg,

R ₁₂Be Gln or Ala,

R ₁₃Be Arg, (D) Arg or N-methyl Arg, and

R ₁₄Be Tyr, (D) Tyr or N-methyl Tyr, modification-Tyr, Phe, modification-Phe or Trp, or its pharmaceutical salts.

3. according to the neuropeptide-2 receptor-agonists of claim 2, R wherein ₂By Y ' replacement, and Y ' is H, polyalkylene glycol moiety, PEG _m-SSA, PEG _m-β-SBA, PEG _m-SPA or PEG _m-BTC.

4. according to claim 1 or 3 each neuropeptide-2 receptor-agonists, wherein Y ' is a polyalkylene glycol moiety, PEG _m-SSA, PEG _m-β-SBA, PEG _m-SPA or PEG _m-BTC.

5. according to each neuropeptide-2 receptor-agonists of claim 1-4, wherein:

Y is H or acyl moiety, and

Y ' is a polyalkylene glycol moiety, PEG _m-SSA, PEG _m-β-SBA, PEG _m-SPA or PEG _m-BTC.

6. according to each neuropeptide-2 receptor-agonists among the claim 1-5, wherein Y is an acyl moiety.

7. according to each neuropeptide-2 receptor-agonists among the claim 1-6, wherein Y is H.

8. according to claim 1 or 3 to 7 each neuropeptide-2 receptor-agonists, wherein Y ' is H.

9. according to each neuropeptide-2 receptor-agonists of claim 1-8, wherein R ¹Be Ile, R ²Be Lys or Nle, R ³Be Arg, R ⁴Be His, R ⁵Be Tyr, R ⁶Be Leu, R ⁷Be Asn, R ⁸Be Leu or Trp, R ⁹Be Val, R ¹⁰Be Thr, R ¹¹Be Arg, R ¹²Gln, R ¹³Be Arg or (N-methyl) Arg, R ¹⁴Be Y, (m-) Y, (3-I) Y, (3,5 two F) Y, (2,6 two F) Y, (2,6 two Me) Y, F (4-O-CH ₃), F, (4-NH ₂) Phe, (4-F) Phe, (4-CH ₂OH) Phe, (4-CF ₃) Phe, (3-F) Phe, (2,3,4,5,6 five F) Phe, (3,4 two Cl) Phe, Cha, W, (1) Nal, (2) Nal or C-α-Me-Tyr.

10. according to each neuropeptide-2 receptor-agonists of claim 1-9, wherein, R ¹⁴Be Tyr or (2,6 two F) Tyr.

11. according to each neuropeptide-2 receptor-agonists of claim 1-10, wherein PEG _mBe 20 to 40KDa.

12. according to each neuropeptide-2 receptor-agonists of claim 1-11, wherein PEG _mBe 30KDa.

13. according to each neuropeptide-2 receptor-agonists of claim 1-12, it is selected from the group of being made up of following:

IK-Pqa-RHYLNLVTRQRY，

IK-Pqa-RHYLNLVTRQ (N-methyl) RY,

IK-Pqa-RHYLNLVTRQ (N-methyl) R (m-) Y,

IK-Pqa-RHYLNLVTRQ (N-methyl) R (3-I) Y,

IK-Pqa-RHYLNLVTRQ (N-methyl) R (3,5 two F) Y,

IK-Pqa-RHYLNLVTRQ (N-methyl) R (2,6 two F) Y,

IK-Pqa-RHYLNLVTRQ (N-methyl) R (2,6 two Me) Y,

IK-Pqa-RHYLNLVTRQ (N-methyl) RF (4-O-CH ₃),

IK-Pqa-RHYLNLVTRQ (N-methyl) RF,

IK-Pqa-RHYLNLVTRQ (N-methyl) R (4-NH ₂) Phe,

IK-Pqa-RHYLNLVTRQ (N-methyl) R (4-F) Phe,

IK-Pqa-RHYLNLVTRQ (N-methyl) R (4-CH ₂OH) Phe,

IK-Pqa-RHYLNLVTRQ (N-methyl) R (4-CF ₃) Phe,

IK-Pqa-RHYLNLVTRQ (N-methyl) R (3-F) Phe,

IK-Pqa-RHYLNLVTRQ (N-methyl) R (2,3,4,5,6 five F) Phe,

IK-Pqa-RHYLNLVTRQ (N-methyl) R (3,4 two Cl) Phe,

IK-Pqa-RHYLNLVTRQ (N-methyl) RCha,

IK-Pqa-RHYLNLVTRQ (N-methyl) RW,

IK-Pqa-RHYLNLVTRQ (N-methyl) R (1) Nal,

IK-Pqa-RHYLNLVTRQ (N-methyl) R (2) Nal,

IK-Pqa-RHYLNLVTRQR-C-α-Me-Tyr，

IK-Pqa-RHYLNWVTRQ (N-methyl) RY,

INle-Pqa-RHYLNWVTRQ (N-methyl) RY,

Ac-IK-Pqa-RHYLNWVTRQ (N-methyl) R (2,6 two F) Y,

Ac-IK-Pqa-RHYLNWVTRQ (N-methyl) RY,

Amyl group-IK-Pqa-RHYLNWVTRQ (N-methyl) RY,

Pivaloyl group-IK-Pqa-RHYLNWVTRQ (N-methyl) RY,

Cyclohexyl-IK-Pqa-RHYLNWVTRQ (N-methyl) RY,

Benzoyl-IK-Pqa-RHYLNWVTRQ (N-methyl) RY,

Adamantyl-IK-Pqa-RHYLNWVTRQ (N-methyl) RY,

(PEG 30,000SPA) IK-Pqa-RHYLNWVTRQ (N-methyl) RY,

(PEG40,000BTC)-IK-Pqa-RHYLNWVTRQ (N-methyl) lRY,

(PEG30,000)-SSA-INle-Pqa-RHYLNWVTRQ (N-methyl) RY,

(PEG30,000)-β-SBA-INle-Pqa-RHYLNWVTRQ (N-methyl) RY,

Ac-Ile-Lys (PEG30,000SPA)-Pqa-RHYLNWVTRQ (N-methyl) RY,

Ac-Ile-Lys (PEG30,000SSA)-Pqa-RHYLNWVTRQ (N-methyl) RY and

IK (PEG30,000S SA)-Pqa-RHYLNWVTRQ (N-methyl) RY,

Or its pharmaceutical salts.

14., be selected from the group of forming by following according to each neuropeptide-2 receptor-agonists of claim 1-13:

Ac-Ile-Lys (PEG30,000SPA)-Pqa-RHYLNWVTRQ (N-methyl) RY,

Ac-Ile-Lys (PEG30,000SSA)-Pqa-RHYLNWVTRQ (N-methyl) RY and

IK (PEG30,000SSA)-Pqa-RHYLNWVTRQ (N-methyl) RY,

Or its pharmaceutical salts.

15. according to each neuropeptide-2 receptor-agonists of claim 1-14, wherein said agonist is Ac-IK-Pqa-RHYLNWVTRQ (N-methyl) R (2-6 two F) Y.

16. according to each neuropeptide-2 receptor-agonists of claim 1-14, wherein said agonist is Ac-IK-Pqa-RHYLNWVTRQ (N-methyl) RY.

17. according to each neuropeptide-2 receptor-agonists of claim 1-14, wherein said agonist is (PEG 30,000)-SPA-IK-Pqa-RHYLNWVTRQ (N-methyl) RY.

18. according to each neuropeptide-2 receptor-agonists of claim 1-14, wherein said agonist is (PEG30,000)-SSA-INle-Pqa-RHYLNWVTRQ (N-methyl) RY.

19. according to each neuropeptide-2 receptor-agonists of claim 1-14, wherein said agonist be Ac-Ile-Lys (PEG30,000SSA)-Pqa-RHYLNWVTRQ (N-methyl) RY.

20. according to each neuropeptide-2 receptor-agonists of claim 1-14, wherein said agonist be H-Ile-Lys (PEG30,000SSA)-Pqa-RHYLNWVTRQ (N-methyl) RY.

21. a pharmaceutical composition, it comprises each neuropeptide-2 receptor-agonists and pharmaceutical carrier and/or the adjuvant according to claim 1-20.

22. according to each neuropeptide-2 receptor-agonists of claim 1-20, it is as the therapeutic active substance.

23. according to each neuropeptide-2 receptor-agonists of claim 1-20, the treatment of diseases active substance that it is used for the treatment of and/or prevents to be regulated by neuropeptide-2 receptor-agonists.

24. comprising, disease, particularly therapeutic that therapeutic and/or prophylactic treatment are regulated by neuropeptide-2 agonist and/or the method for prophylactic treatment metabolic disease or disease, described method will be applied to the human or animal according to each neuropeptide-2 receptor-agonists of claim 1-20.

25. according to claim 1-20 each being used for the treatment of property of neuropeptide-2 receptor-agonists and/or the application of the prophylactic treatment disease of regulating by neuropeptide-2 receptor-agonists.

26. according to each being used for the treatment of property of neuropeptide-2 receptor-agonists and/or prophylactic treatment obesity of claim 1-20, type 2 diabetes mellitus, metabolism syndrome, insulin resistance, dyslipidemia, the application of impaired fasting glucose (IFG) and impaired glucose tolerance.

27. according to the application that each neuropeptide-2 receptor-agonists of claim 1-20 is used to prepare medicine, the disease that described being used for the treatment of property of medicine and/or prophylactic treatment are regulated by neuropeptide-2 receptor-agonists.

28. according to the application that each neuropeptide-2 receptor-agonists of claim 1-20 is used to prepare medicine, described being used for the treatment of property of medicine and/or prophylactic treatment obesity, type 2 diabetes mellitus, metabolism syndrome, insulin resistance, dyslipidemia, impaired fasting glucose (IFG) and impaired glucose tolerance.

29. the present invention as defined above.