CN101237892A

CN101237892A - Method of conjugating aminothiol containing molecules to vehicles

Info

Publication number: CN101237892A
Application number: CNA2006800094360A
Authority: CN
Inventors: D·C·德阿米科; B·C·小阿斯库
Original assignee: Amgen Inc
Current assignee: Amgen Inc
Priority date: 2005-01-24
Filing date: 2006-01-24
Publication date: 2008-08-06
Also published as: ZA200706945B

Abstract

The present invention relates to a novel chemical process that provides novel vehicle derivatives that are exceptional 1,2- or 1,3-aminothiol specific reagents for conjugation to unprotected targeted compounds (e.g., polypeptides, peptides, or organic compounds) having or modified to have a 1,2- or 1,3 aminothiol group. The invention further relates to the methods of using novel water-soluble polymer derivatives and conjugates thereof.

Description

The method that molecule containing amineothiot is conjugated with carrier

This application claims the priority of U.S. Application No. filed in 23 days January in 2006 (not yet transferring the possession of), and the rights and interests of U.S. Provisional Application No. 60/646,685 filed in 24 days January in 2005 are required, the document is incorporated herein by reference.

Background of invention

Latest developments in terms of biotechnology make it possible the large-scale production of biomolecule, and the biomolecule such as human cytokines, peptide, antibody and antibody fragment can be prepared on a large scale at present, enable such biomolecule extensive use.Regrettably, the proteolytic degradation of biomolecule is fast, circulating half-life is short, solubility is low, production, storage or administration process in it is unstable or administration after immunogenicity, can usually limit its application.Because to giving the treatment of biomolecule and/or the interest of diagnostic uses increasingly rising, have studied to overcome the various methods of these defects.

One of widely studied method is exactly：By with carrier such as polyethylene glycol (hereinafter referred to as " PEG ") be covalently attached and modifying protein and other potential curatives (for example, see Abuchowski, A. etc., J.Biol.Chem.252 (11)：3579-3586(1977)；Davis, S. etc., Clin.Exp.Immunol., 46：649-652(1981)；With U.S. Patent Application Publication No. 20040132664).The method of (hereinafter referred to as " PEGylation ") is connected with protein or polypeptide using PEG group to solve or improve protein or numerous problems of peptide medicine have been documented (see, for example, Francis etc. in the literature, International Journal of Hematology, 68：1-18(1998)；Abuchowski, A. etc., (1977)；Chapman, A., Adv.Drug Del.Rev.54,531-545 (2002))；And Roberts, M.J. etc., Advanced Drug DeliveryReviews, 54：459-476(2002)).

In brief, the support derivatives of the active group in one or both ends are generally used in, to make carrier and protein, peptide, polysaccharide, polynucleotides, lipid or the agent of organic molecule isoreactivity be covalently attached (hereinafter referred to as " conjugated (conjugation) ").According to by the type of available active group on the molecule with carrier conjugation, to select active group.For example, it is exactly functionalized polymeric that documents below is provided：WO96/41813 and J.Pharmaceut.Sci.87,1446-1449 (1998)).When carrier is PEG, suitable for including PEG- aldehyde, mixed acid anhydride, N-hydroxy-succinamide ester, carbonylic imidazole alkane (carbonylimadazolide) and chlorocyanurate with the activation PEG derivatives that biomolecule (such as lysine, cysteine and the similar residue of protein or peptide) nucleophilic center is reacted.These known methods respectively have advantage and disadvantage (Harris, J.M., Herati, R.S., Polym Prepr. (Am.Chem.Soc., Div.Polyin.Chem), 32 (1)：154-155(1991)；Herman, S. etc., Macromol.Chem.Phys., 195：203-209(1994)；And Roberts, M.J. etc., Advanced Drug Delivery Reviews, 54：459-476(2002)).Some the related more conventional problems being conjugated with known method include producing the selectivity of reaction impurities, unstable key, side reaction and/or shortage to substituent.In addition, these problems also mean to separate and purify the complexity of required biologically active conjugates.Sometimes, the isomers of varying number can be produced.Such variability brings the potential problems of repeatability between batches, and wherein greatest problem is exactly to cause bioactivity not reproduce.

Activated carrier derivative with thiol-selective functional group is particularly suitable for the cysteine side chain coupling with protein or peptide, the functional group such as maleimide, vinyl sulfone(RemzaolHuo Xingranliaohuoxingjituan), iodoacetamide, mercaptan and disulphide (Zalipsky, S.Bioconjug.Chem.6,150-165 (1995)；Crit.Rev.Ther.Drug the Carrier Syst.17,101-161 such as Greenwald, R.B. (2000)；25 Herman, S. etc., Macromol.Chem.Phys.195,203-209 (1994)).However, these reagents are also not without at least one drawback, especially when aiming at exploitation carrier-conjugating biomolecules for therapeutical uses.For example, the PEG maleimides-mercaptan conjugate originally formed is (R)-and (S)-chiral mixture.Mixture is formed such that PEGylation biomolecule is developed in multiple levels to be become more sophisticated.For example, the problems such as one in enantiomer may have undesired active or dangerous compared with another.The further drawback of PEG maleimides-mercaptan conjugation methods is exactly：The adduct originally formed is easy to be rearranged into thiomorpholine ketone.

It is also required to produce to repeatability the conjugate of the activating agent of two or more connections.In some cases, these " polymer " compounds for containing the more than one activating agent being connected with identical carrier molecule are given, can cause to be added and/or synergistic benefits.For example, compared with monomer polypeptide, the compound containing two or more identical combination peptide or polypeptide is dramatically increased to the part or the compatibility of avtive spot that are combined.Or, the compound being made up of following (1) and (2) is particularly advantageous：(1) privileged site can play the biological activity protein of its effect and (2) and compound can be targetted to the molecule of privileged site in vivo.Regrettably, the method for the present invention is used to produce the carrier with more than one bioactivity or Biofunctional molecular conjugate, more increases above-mentioned limitation.For example, the attempt that two bioactive molecules and a divalence PEG- maleimide are conjugated, it will produce 16 kinds of single entities of varying number.For example by using tetravalence PEG- maleimides, the PEG conjugated with 4 bioactive molecules altogether is produced with the method for the present invention, as a result there may be 256 potential individually connection sites, etc. between PEG and bioactive molecule.Want to quantify these corpus separatums, in general difficult, even not possible with, technical problem, existing instrument deficiency all may greatly hinder, even prevent the exploitation of this kind of biomolecule completely in addition sometimes.

Therefore, the method for having clearly a need for preparing active agent conjugates with high-purity in high yield at present.Preferably, such conjugate is stable to hydrolysis, needing the reactant of relatively small amount can prepare, and the technique of bioactivity is easy to purifying needed for use can keep carrier or carrier segment integrality (carrying out under mild reaction conditions) and/or keep.The present invention provides new reagent, method and conjugate, can solve in above mentioned problem present on state of the art and provide many advantages for prior art.

Summary of the invention

The present invention relates to support derivatives, there is the carrier segment of 1,2- amineothiots (1,2-aminothiol) or the selective end of 1,3- amineothiot comprising at least one.The support derivatives of the present invention can be used for and the molecule coupling labeled comprising 1,2- amineothiots or 1,3- amineothiot part.One embodiment of the invention is related to one or more activating agents and the connection of water-soluble polymer (including but is not limited to PEG).

The method that the present invention is provided the preparation method of support derivatives of the present invention and new active agent conjugates are prepared using support derivatives.

An aspect of of the present present invention is related to the compound of having structure or its pharmaceutically acceptable salt or hydrate：

Wherein：

A is the bridged group of saturation, fractional saturation or undersaturated 2,3,4,5 or 6 atoms, the hetero atom that it contains 0,1,2 or 3 are selected from O, N and S, and remaining bridge atom is carbon atom；

E¹For N, O or C；

E²For N or C；

G is singly-bound, double bond, C, N, O, B, S, Si, P, Se or Te；

WithRespectively singly-bound,

With

One of can also be double bond；And when G is C or N,

With

One of can also be double bond；And when G is singly-bound or double bond,

With

All it is not present；

L¹For divalence C_1-6Alkyl or C_1-6Miscellaneous alkyl, each group is by 0,1,2 or 3 replace selected from following substituent：F、Cl、Br、I、OR^a、NR^aR^aAnd oxo base；

M independently is 0 or 1 in all cases；

O is 0,1,2,3,4 or 5；

R¹For H, C_1-6Any one in alkyl, phenyl or benzyl, the group is by 0,1,2 or 3 be selected from following substituent group：Halogen, cyano group, nitro, oxo base ,-C (=O) R^b,-C (=O) OR^b,-C (=O) NR^aR^a,-C (=NR^a)NR^aR^a、-OR^a,-OC (=O) R^b,-OC (=O) NR^aR^a,-OC (=O) N (R^a) S (=O)₂R^b、-OC_2-6Alkyl NR^aR^a、-OC_2-6Alkyl OR^a、-SR^a,-S (=O) R^b,-S (=O)₂R^b,-S (=O)₂NR^aR^a,-S (=O)₂N(R^a) C (=O) R^b,-S (=O)₂N(R^a) C (=O) OR^b,-S (=O)₂N(R^a) C (=O) NR^aR^a、-NR^aR^a、-N(R^a) C (=O) R^b、-N(R^a) C (=O) OR^b、-N(R^a) C (=O) NR^aR^a、-N(R^a) C (=NR^a)NR^aR^a、-N(R^a) S (=O)₂R^b、-N(R^a) S (=O)₂NR^aR^a、-NR^aC_2-6Alkyl NR^aR^aWith-NR^aC_2-6Alkyl OR^a, and also by 0,1,2,3,4,5 or 6 replace selected from following atom：F, Br, Cl and I；

R²For carrier, R³For bioactive compound；Or R³For carrier, R²For bioactive compound；

R^aH or R independently all is in all cases^b；

R^bPhenyl, benzyl or C independently all are in all cases_1-6Alkyl, the phenyl, benzyl and C_1-6Alkyl is by 0,1,2 or 3 replace selected from following substituent：Halogen, C_1-4Alkyl, C_1-3Haloalkyl ,-OC_1-4Alkyl, OH ,-NH₂、-NHC_1-4Alkyl and-N (C_1-4Alkyl) C_1-4Alkyl；With

R^cHalogen, C are all independently selected from all cases_1-4Alkyl, C_1-3Haloalkyl ,-OC_1-4Alkyl, OH ,-NH₂、-NHC_1-4Alkyl and-N (C_1-4Alkyl) C_1-4Alkyl.

Another aspect of the present invention is related to the compound of having structure or its pharmaceutically acceptable salt or hydrate：

Wherein：

E¹For N, O or C；

E²For N or C；

G is singly-bound, double bond, C, N, O, B, S, Si, P, Se or Te；

With

Respectively singly-bound,With

One of can also be double bond；And when G is C or N,With

One of can also be double bond；And when G is singly-bound or double bond,

With

All it is not present；

M independently is 0 or 1 in all cases；

N is more than or equal to 1；

O is 0,1,2,3,4 or 5；

R^aH or R independently all is in all cases^b；

In another embodiment and combine embodiment up and down, A is the bridged group of saturation, fractional saturation or undersaturated 2,3,4,5 or 6 atoms, the hetero atom that it contains 1,2 or 3 are selected from O, N and S, and remaining bridge atom is carbon atom.

In another embodiment and combine embodiment up and down, A is the bridged group of saturation, fractional saturation or undersaturated 2,3,4,5 or 6 carbon atoms.

In another embodiment and with reference to embodiment up and down, n is 1.

In another embodiment and with reference to embodiment up and down, n is 2.

In another embodiment and with reference to embodiment up and down, n is 3.

In another embodiment and with reference to embodiment up and down, n is 4.

In another embodiment and with reference to embodiment up and down, n is 5.

In another embodiment and with reference to embodiment up and down, n is 6.

In another embodiment and with reference to embodiment up and down, n is 7.

In another embodiment and with reference to embodiment up and down, n is 8.

In another embodiment and with reference to embodiment up and down, A is the bridged group of undersaturated 4 carbon atoms；E²For C；G is double bond.

In another embodiment and combine up and down embodiment, G be singly-bound or double bond, and

With

All it is not present.

In another embodiment and with reference to embodiment up and down, G is C, N, O, B, S, Si, P, Se or Te.

In another embodiment and with reference to embodiment up and down,

With

Respectively singly-bound.

In another embodiment and with reference to embodiment up and down, G is C or N；With

With

One of be double bond.

In another embodiment and with reference to embodiment, R up and down²For carrier, R³For bioactive compound.

In another embodiment and with reference to embodiment, R up and down³For carrier, R²For bioactive compound.

In another embodiment and with reference to embodiment, R up and down³Selected from poly- (alkylene oxide), PVP, poly- (vinyl alcohol), polyoxazoline, poly- (acryloyl morpholine -), poly- (oxygen ethylization polyalcohol), PEG, carboxymethyl cellulose, glucan, polyvinyl alcohol, polyvinylpyrrolidone, poly- 1, 3- dioxolane, poly- 1, 3, 6- trioxanes, homopolymer of amino acids, PPOX, the copolymer of ethylene glycol/propylene glycol, ethene/copolymer-maleic anhydride, amino acid copolymer, PEG and amino acid copolymer, PPOX/ethylene oxide copolymer and polyethylene glycol/thiomalic acid copolymer；Or their any combination.

In another embodiment and with reference to embodiment, R up and down³For PEG.

In another embodiment and with reference to embodiment up and down, n is 1,2,3,4,5,6,7,8,9 or 10.

In another embodiment and with reference to embodiment, R up and down³For side chain PEG, n is 2,3,4,5,6,7,8,9 or 10.

In another embodiment and with reference to embodiment, R up and down²For B1 peptide antagonists.

In another embodiment and with reference to embodiment, R up and down²For selected from SEQ ID NO：5-26 and 42-62 B1 peptide antagonists, wherein the peptide has N- ends cysteine residues through modification.

Another aspect of the present invention is related to the preparation method of the compound of claim 1, and this method comprises the following steps：

A R) is made²- (C (=O))_mCH(NH₂)CH₂(CH₂)_mSH reacts with following compound

Or

B R) is made²- [(C (=O))_mCH(NH₂)CH₂(CH₂)_mSH]_nReacted with following compound：

Wherein J be carbonyl or or its by forms of protection.

A R) is made²- (C (=O))_mCH(NH₂)CH₂(CH₂)_mSH reacts with following compound：

Or

Wherein J be carbonyl or or its by forms of protection.

In another embodiment and with reference to embodiment up and down, J is selected from C (=O), C (OCH₂CH₂O)、C(N(R^a)CH₂CH₂N(R^a))、C(N(R^a)CH₂CH₂O)、C(N(R^a)CH₂CH₂S)、C(OCH₂CH₂CH₂O)、C(N(R^a)CH₂CH₂CH₂N(R^a))、C(N(R^a)CH₂CH₂CH₂O)、C(N(R^a)CH₂CH₂CH₂S)、C(OR^b)₂、C(SR^b)₂With C (NR^aR^b)₂。

In another embodiment and with reference to embodiment up and down, the reaction is carried out in pH between 2 and 7.

In another embodiment and with reference to embodiment up and down, the reaction is carried out in pH between 3 and 5.

Another aspect of the present invention is related to the compound with having structure：

Wherein：

E¹For N, O or C；

E²For N or C；

G is singly-bound, double bond, C, N, O, B, S, Si, P, Se or Te；

With

Respectively singly-bound,

With

One of can also be double bond；And when G is C or N,With

One of can also be double bond；And when G is singly-bound or double bond,

With

All it is not present；

J be carbonyl or or its by forms of protection；

L¹For divalence C_1-12Alkyl or C_1-12Miscellaneous alkyl, each group is by 0,1,2 or 3 replace selected from following substituent：F、Cl、Br、I、OR^a、NR^aR^aAnd oxo base；

M independently is 0 or 1 in all cases；

N is 1,2,3,4,5,6,7,8,9 or 10；

O is 0,1,2,3,4 or 5；

R³For bioactive compound or carrier；

R^aH or R independently all is in all cases^b；

R^bPhenyl, benzyl or C independently all are in all cases_1-6Alkyl, the phenyl, benzyl and C_1-6Alkyl is by 0,1,2 or 3 replace selected from following substituent：Halogen, C_1-4Alkyl, C_1-3Haloalkyl ,-OC_1-4Alkyl, OH ,-NH₂、-NHC_1-4Alkyl and-N (C_1-4Alkyl) C_1-4Alkyl；

R^cHalogen, C are all independently selected from all cases_1-4Alkyl, C_1-3Haloalkyl ,-OC_1-4Alkyl, OH ,-NH₂、-NHC_1-4Alkyl and-N (C_1-4Alkyl) C_1-4Alkyl；With

X is C (=O), and Y is NH；Or X is NH, Y is C (=O).

In another embodiment and with reference to embodiment up and down, n is 1.

In another embodiment and with reference to embodiment up and down, n is 2.

In another embodiment and with reference to embodiment up and down, n is 3.

In another embodiment and with reference to embodiment up and down, n is 4.

In another embodiment and with reference to embodiment up and down, n is 5.

In another embodiment and with reference to embodiment up and down, n is 6.

In another embodiment and with reference to embodiment up and down, n is 7.

In another embodiment and with reference to embodiment up and down, n is 8.

In another embodiment and with reference to embodiment up and down, A is the bridged group of undersaturated 4 carbon atoms；E²For C；And G is double bond.

In another embodiment and combine up and down embodiment, G be singly-bound or double bond,

With

All it is not present.

In another embodiment and with reference to embodiment up and down,

With

Respectively singly-bound.

In another embodiment and with reference to embodiment up and down, G is C or N；And

With

One of be double bond.

In another embodiment and with reference to embodiment, R up and down³For bioactive compound.

In another embodiment and with reference to embodiment, R up and down³For carrier.

In another embodiment and with reference to embodiment, R up and down³Selected from poly- (alkylene oxide), PVP, poly- (vinyl alcohol), polyoxazoline, poly- (acryloyl morpholine -), poly- (oxygen ethylization polyalcohol), PEG, carboxymethyl cellulose, glucan, polyvinyl alcohol, polyvinylpyrrolidone, poly- 1, 3- dioxolane, poly- 1, 3, 6- trioxanes, homopolymer of amino acids, PPOX, ethylene glycol/propylene glycol copolymers, ethene/copolymer-maleic anhydride, amino acid copolymer, PEG and amino acid copolymer, PPOX/ethylene oxide copolymer and polyethylene glycol/thiomalic acid copolymer；Or their any combination.

In another embodiment and with reference to embodiment, R up and down³For PEG.

Another aspect of the present invention is related to the preparation method of compound as described above, and this method comprises the following steps：Make (Y-L²)_nR³Reacted with following compound：

Wherein：L²C independently all is in all cases_1-6Alkyl or C_1-6Miscellaneous alkyl, each group is by 0,1,2,3 or 4 replace selected from following substituent：F、Cl、Br、I、OR^a、NR^aR^aAnd oxo base；

X is nucleopilic reagent, and Y is electrophilic reagent；Or X is electrophilic reagent, Y is nucleopilic reagent.

In another embodiment of the present invention, nucleopilic reagent is selected from NH₂And OH；And electrophilic reagent is selected from CH₂Halogen, CH₂SO₂OR^b, C (=O) NR^aR^bWith C (=O) OR^b。

Another aspect of the present invention is related to the method for treating pain and/or inflammation, and this method includes the compound as described above for giving bacterium in need.

Another aspect of the present invention is related to pharmaceutical composition, and said composition includes compound as described above and pharmaceutically acceptable carrier or diluent.

Another aspect of the present invention is related to the preparation method of medicine, and the medicine includes compound as described above.

Another aspect of the present invention is related to the preparation method for the medicine for treating pain and/or inflammation, and the medicine includes compound as described above.

An aspect of of the present present invention is related to the compound of having structure or its any pharmaceutically acceptable salt or hydrate：

Wherein：

A is selected from i) 2 carbon atoms, or sp³Hydridization or sp²Hydridization (substitution is unsubstituted), wherein the two carbon atoms can be loop section or acyclic portion, and connect two carboxyls of electrophilic reagent, or ii) 3 atoms, selected from carbon, (substitution is unsubstituted, loop section or acyclic portion), nitrogen (substitution or unsubstituted, loop section or acyclic portion) or oxygen (loop section or acyclic portion)；With

B is selected from i) 2 carbon atoms, or sp³Hydridization or sp²Hydridization (substitution is unsubstituted), wherein the two carbon atoms can be loop section or acyclic portion, and connect two carboxyls of electrophilic reagent, or ii) 3 atoms, selected from carbon, (substitution is unsubstituted, loop section or acyclic portion), nitrogen (substitution or unsubstituted, loop section or acyclic portion) or oxygen (loop section or acyclic portion).

In one embodiment and with reference to embodiment, R up and down¹For H, C_1-6Any one in alkyl, phenyl or benzyl, the group is by 0,1,2 or 3 be selected from following substituent group：Halogen, cyano group, nitro, oxo base ,-C (=O) R^b,-C (=O) OR^b,-C (=O) NR^aR^a,-C (=NR^a)NR^aR^a、-OR^a,-OC (=O) R^b,-OC (=O) NR^aR^a,-OC (=O) N (R^a) S (=O)₂R^b、-OC_2-6Alkyl NR^aR^a、-OC_2-6Alkyl OR^a、-SR^a,-S (=O) R^b,-S (=O)₂R^b,-S (=O)₂NR^aR^a,-S (=O)₂N(R^a) C (=O) R^b,-S (=O)₂N(R^a) C (=O) OR^b,-S (=O)₂N(R^a) C (=O) NR^aR^a、-NR^aR^a、-N(R^a) C (=O) R^b、-N(R^a) C (=O) OR^b、-N(R^a) C (=O) NR^aR^a、-N(R^a) C (=NR^a)NR^aR^a、-N(R^a) S (=O)₂R^b、-N(R^a) S (=O)₂NR^aR^a、-NR^aC_2-6Alkyl NR^aR^aWith-NR^aC_2-6Alkyl OR^a, and also by 0,1,2,3,4,5 or 6 replace selected from following atom：F, Br, Cl and I；

In one embodiment and with reference to embodiment, R up and down³Selected from poly- (alkylene oxide), PVP, poly- (vinyl alcohol), polyoxazoline, poly- (acryloyl morpholine -), poly- (oxygen ethylization polyalcohol), PEG, carboxymethyl cellulose, glucan, polyvinyl alcohol, polyvinylpyrrolidone, poly- 1, 3- dioxolane, poly- 1, 3, 6- trioxanes, homopolymer of amino acids, PPOX, ethylene glycol/propylene glycol copolymers, ethene/copolymer-maleic anhydride, amino acid copolymer, PEG and amino acid copolymer, PPOX/ethylene oxide copolymer and polyethylene glycol/thiomalic acid copolymer；Or their any combination.

In another embodiment and with reference to embodiment up and down, the carrier segment is poly- (oxirane).

In another embodiment and with reference to embodiment up and down, the carrier is linear chain structure.

In another embodiment and with reference to embodiment up and down, the carrier is PEG.

In another embodiment and with reference to embodiment up and down, the polycyclic N, S- heterocycles are (9bS) (9bH) -2,3- thiazolines simultaneously [2,3-a] iso-indoles -5- ketone, R²For protein or peptide, R³For PEG.

In another embodiment and with reference to embodiment up and down, B1 peptide antagonists are to be selected from SEQ ID NO：5-26 and 42-62 peptide, wherein the peptide has N- ends cysteine residues through modification.

In another embodiment and with reference to embodiment up and down, the carrier is the bifurcation structure i.e. branched structure respectively with the water-soluble section of two or more.

In another embodiment and with reference to embodiment up and down, the carrier is the bifurcated PEG (fPEG) of the side chain PEG (bPEG) i.e. with two or more PEG sections.

In another embodiment and with reference to embodiment up and down, the polycyclic N, S- heterocycles are (9bS) (9bH) -2,3- thiazolines simultaneously [2,3-a] iso-indoles -5- ketone, R²For protein or peptide.

In another embodiment and with reference to embodiment up and down, the bPEG has 3-8 polymer section-(bPEG)_3-8。

In another embodiment and with reference to embodiment up and down, at least one described section of the bPEG has the end (C- [(bPEG) activated by amine_3-8]-(NH₂)_1-8)。

In another embodiment and with reference to embodiment up and down, the bPEG has 4 polymer section (C- [(bPEG)₄]-(NH₂)_1-4), and section described in wherein at least one has the end activated by amine.

In another embodiment and with reference to embodiment up and down, at least 50% section has the end activated by amine.

In another embodiment and with reference to embodiment up and down, at least one described polymer section is end-blocking.

In another embodiment and with reference to embodiment up and down, the nominal average molecular weight of the PEG is about 200 dalton to about 100,000 dalton.

In another embodiment and with reference to embodiment up and down, the nominal average molecular weight of the PEG is about 5,000 dalton to about 60,000 dalton.

In another embodiment and with reference to embodiment up and down, the nominal average molecular weight of the PEG is about 10,000 dalton to about 40,000 dalton.

In another embodiment and with reference to embodiment, R up and down²All it is B1 peptide antagonists in all cases.

In another embodiment and with reference to embodiment up and down, the B1 peptide antagonists are selected from SEQ ID NO：27-35 and 38-62.

In another embodiment and with reference to embodiment, R up and down²It is B1 peptide antagonists in one case.

In another embodiment and with reference to embodiment, R up and down²It is B1 peptide antagonists under 2 kinds in 4 kinds of situations.

In another embodiment and with reference to embodiment, R up and down²It is B1 peptide antagonists under 3 kinds in 4 kinds of situations.

In another embodiment and with reference to embodiment up and down, the B1 peptide antagonists are each independently selected from SEQ ID NO：27-34 and 38-62.

In another embodiment and with reference to embodiment, R up and down²Activating agent in the case of at least one for not B1 peptide antagonists.

Another aspect of the present invention is related to pharmaceutical composition, and said composition includes any of above compound and pharmaceutical excipient.

Another aspect of the present invention is related to the administration comprising any of above compound and pharmaceutical excipient pharmaceutical composition, and the administration is parenteral, transmucosal or percutaneous dosing.

In another embodiment and combine embodiment up and down, the transmucosal refer to orally, nasal cavity, lung, vagina or rectum.

In another embodiment and combine embodiment up and down, it is described parenteral to refer in intra-arterial, intravenous, intramuscular, intracutaneous, subcutaneous, intraperitoneal, intra-ventricle, intraocular, socket of the eye or encephalic.

In another embodiment and with reference to embodiment up and down, the administration is oral administration.

In another embodiment and with reference to embodiment up and down, the polypeptide or peptide include Tat- inhibitory polypeptides, including R-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-X- (biotin)-Cys-NH₂Amino acid sequence (SEQ ID NO：63) and its biologically with pharmaceutically acceptable salt, stereoisomer, optical isomer and geometric isomer; including inverse-inversion analog (retro inverso analogue) (if such isomers) and its pharmaceutically acceptable salt and solvate, wherein R includes carboxylic acid or acetyl group residue；X is Cys residues.

In another embodiment and with reference to embodiment up and down, the polypeptide or peptide comprising aminothiol compound include being selected from following amino acid sequence：N- acetyl group-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Cys- (biotin)-Cys-NH₂(SEQ ID NO：64), N- acetyl group-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Lys- (biotin)-Cys-NH₂(SEQ ID NO：65), N- acetyl group-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-D-Cys- (biotin)-Cys-NH₂(SEQ ID NO：66), N- acetyl group-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-D-Lys- (biotin)-Cys-NH₂(SEQ IDNO：67), N- acetyl group-Gln-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-D-Lys- (biotin)-Cys-NH₂(SEQ ID NO：68), N- acetyl group-Arg-Lys-Lys-Arg-Arg-Pro-Arg-Arg-Arg-Cys- (biotin)-Cys-NH₂(SEQ ID NO：69), N- acetyl group-DCys-DLys- (biotin)-DArg-DArg-DArg-DGln-DArg-DArg-DLys-DLys-DArg-NH₂Or its biology and pharmaceutically acceptable salt.

In another embodiment and with reference to embodiment up and down, the carrier is selected from PEG, carboxymethyl cellulose, glucan, polyvinyl alcohol, polyvinylpyrrolidone, poly- 1,3- dioxolane, poly- 1,3,6- trioxanes, homopolymer of amino acids, PPOX, ethylene glycol/propylene glycol copolymers, ethene/copolymer-maleic anhydride, amino acid copolymer, PEG and amino acid copolymer, PPOX/ethylene oxide copolymer and PEG/ thiomalic acid copolymers, or their any combination.

In another embodiment and with reference to embodiment up and down, the molecular weight of the polymer is about 100 dalton to about 200,000 dalton.

In another embodiment and with reference to embodiment up and down, the molecular weight of the polymer is about 2,000 dalton to about 50,000 dalton.

In another embodiment and with reference to embodiment up and down, the interval base is about 100 dalton to about 10,000 dalton.

In another embodiment and with reference to embodiment up and down, the interval base is about 300 dalton to about 5,000 dalton.

Another aspect of the present invention is related to the preparation method of 1,2- amineothiots or the selective support derivatives of 1,3- amineothiot, and this method comprises the following steps：

(a) carrier for including at least one carrier segment with following formula is provided：

Y-R³

Wherein Y is nucleopilic reagent or electrophilic reagent, R₃For carrier.

(b) make the support derivatives with comprising 1, the 2- amineothiots with following formula or 1,3- amineothiot selectivity part or its reacted by the molecule of forms of protection, formed be covalently attached：

Wherein A is i) 2 carbon atoms, or sp³Hydridization or sp²Hydridization (substitution is unsubstituted), wherein the two carbon atoms can be loop section or acyclic portion, and connect two carboxyls of electrophilic reagent, or ii) 3 atoms, selected from carbon, (substitution is unsubstituted, loop section or acyclic portion), nitrogen (substitution or unsubstituted, loop section or acyclic portion) or oxygen (loop section or acyclic portion)；Wherein R¹Selected from H and electron withdraw group；Wherein R²=alkyl；Wherein when Y is nucleopilic reagent, X is electrophilic reagent, or when Y is electrophilic reagent, X is nucleopilic reagent.

In another embodiment and with reference to embodiment up and down, A has the structure of following formula：

In another embodiment and with reference to embodiment up and down, A is non-annularity.

In another embodiment and embodiment up and down is combined, F is carbon, and D is selected from i) carbon ii) oxygen and iii) nitrogen.

In another embodiment and with reference to embodiment up and down, D is carbon, and E is selected from carbon, the nitrogen replaced by X, oxygen, sulphur, the silicon replaced by X, the boron replaced by X, chemical bond, the phosphorus replaced by X replaced by X；Or ii) oxygen, E is selected from carbon, nitrogen, silicon, boron and chemical bond；Or iii) nitrogen, E is selected from carbon, nitrogen, oxygen, silicon, sulphur, boron and chemical bond.

In another embodiment and embodiment up and down is combined, F is carbon, and D is selected from i) carbon, ii) oxygen and iii) nitrogen.

In another embodiment and with reference to embodiment up and down, Y is acid.

In another embodiment and with reference to embodiment up and down, Y is amine.

In another embodiment and with reference to embodiment up and down, Y is primary amine.

In another embodiment and with reference to embodiment up and down, more than the 95% selective some covalent connection of Y and 1,2- amineothiot or 1,3- amineothiot.

In another embodiment and with reference to embodiment, at least one described R up and down₃Selected from H, alkyl, C₁-C₁₀Straight chained alkyl, poly- (alkylene oxide), PVP, poly- (vinyl alcohol), polyoxazolines, poly- (acryloyl morpholine -), poly- (oxygen ethylization polyalcohol) and poly- (oxirane).

In another embodiment and with reference to embodiment up and down, the carrier has side chain, bifurcated or many arm configurations.

In another embodiment and with reference to embodiment, at least R up and down³For PEG.

In another embodiment and with reference to embodiment up and down, the nominal average molecular weight of the carrier is about 200 dalton to about 100,000 dalton.

In another embodiment and embodiment up and down is combined, methods described also includes the first step for purifying the carrier so that ＞ 95% section has the end activated by amine.

In another embodiment and with reference to embodiment up and down, the purification step includes chromatographic isolation or Chemical Decomposition.

In another embodiment and with reference to embodiment up and down, the purification step includes cation-exchange chromatography.

In another embodiment and with reference to embodiment up and down, the nucleopilic reagent is selected from secondary amine, hydroxyl, imino group or mercaptan.

In another embodiment and with reference to embodiment up and down, the electrophilic reagent is Acibenzolar.

In another embodiment and combine embodiment up and down, the Acibenzolar be selected from n-hydroxysuccinimide base, succinimido, N- hydroxybenzotriazoles base, perfluoro phenyl, allcylating moiety (such as chloralkane, brominated alkanes, alkane iodide), activation alcohol (for example mesyl-, trifyl-, p-toluenesulfonyl-, trichlorine imino-acetic acid ester) and in-situ activation alcohol such as triphenyl phosphonium ether.

In another embodiment and embodiment up and down is combined, Y is selected from alkoxy, substituted alkoxy, alkenyloxy group, substitution alkenyloxy group, alkynyloxy group, substitution alkynyloxy group, aryloxy group and substituted aryloxy.

Another aspect of the present invention is related to theme composition (composition of matter) preparation method, and this method comprises the following steps：

Y-R³

Wherein Y is nucleopilic reagent or electrophilic reagent, R₃For carrier.

Wherein A is i) 2 carbon atoms, or sp³Hydridization or sp²Hydridization (substitution is unsubstituted), wherein the two carbon atoms can be loop section or acyclic portion, and connect two carboxyls of electrophilic reagent, or ii) 3 atoms, selected from carbon, (substitution is unsubstituted, loop section or acyclic portion), nitrogen (substitution or unsubstituted, loop section or acyclic portion) or oxygen (loop section or acyclic portion)；Wherein R¹Selected from H and electron withdraw group；Wherein when Y is nucleopilic reagent, X is electrophilic reagent, or when Y is electrophilic reagent, X is nucleopilic reagent；With

(c) by the primary product from step (a) and (b) and activating agent or substrate reactions comprising 1,2- amineothiots or 1,3- amineothiot.

In another embodiment and with reference to embodiment up and down, the activating agent is polypeptide or peptide.

In another embodiment and with reference to embodiment up and down, peptide is B1 peptide antagonists.

In another embodiment and with reference to embodiment up and down, the peptide is selected from SEQ IDNO：27-35 and 38-41 peptide.

In another embodiment and with reference to embodiment up and down, the peptide is selected from SEQ IDNO：11-26 and 43-46, and also include cysteine at the N- ends of the peptide.

In another embodiment and embodiment up and down is combined, 1, the 2- amineothiots or 1,3- amineothiot selectivity part are 1,2- formyls base ester or 1,3- formyl base ester.

In another embodiment and with reference to embodiment up and down, the electrophilic reagent is acid.

In another embodiment and with reference to embodiment up and down, the nucleopilic reagent is amine.

In another embodiment and with reference to embodiment up and down, the electrophilic reagent is primary amine.

In another embodiment and with reference to embodiment up and down, the carrier segment is selected from poly- (alkylene oxide), PVP, poly- (vinyl alcohol), polyoxazoline, poly- (acryloyl morpholine -), poly- (oxygen ethylization polyalcohol), PEG, carboxymethyl cellulose, glucan, polyvinyl alcohol, polyvinylpyrrolidone, poly- 1, 3- dioxolane, poly- 1, 3, 6- trioxanes, homopolymer of amino acids, PPOX, ethylene glycol/propylene glycol copolymers, ethene/copolymer-maleic anhydride, amino acid copolymer, PEG and amino acid copolymer, PPOX/ethylene oxide copolymer and polyethylene glycol/thiomalic acid copolymer；Or their any combination.

In another embodiment and with reference to embodiment up and down, warp¹³C NMR, which are determined, to be contained¹³C activated terminus, or pass through nothing¹³Other existing appropriate methods of the activated terminus of carbon are measured, and more than the 95% selective some covalent of the activated terminus and 1,2- amineothiot or 1,3- amineothiot is combined.

In another embodiment and with reference to embodiment up and down, the carrier segment is polyethylene glycol (PEG).

In another embodiment and with reference to embodiment up and down, the PEG has straight chain, side chain (bPEG), bifurcated (fPEG) or many arm configurations.

In another embodiment and with reference to embodiment up and down, the side chain PEG has 3-8 polymer section (C- [bPEG_3-8])。

In another embodiment and with reference to embodiment up and down, at least one described section has the end (C- [bPEG activated by amine_3-8]-(NH₂)_1-8)。

In another embodiment and with reference to embodiment up and down, the bPEG has 4 polymer section (C- [bPEG₄]-(NH₂)_1-4), section described in wherein at least one has the end activated by amine.

In another embodiment and with reference to embodiment up and down, the end of at least 50% section is activated by amine.

In another embodiment and embodiment up and down is combined, methods described also includes the first step for purifying the amine activated carrier so that ＞ 95% section has the end activated by amine.

In another embodiment and with reference to embodiment up and down, the end-capping group (cap) includes being selected from following chemical group：Alkoxy, substituted alkoxy, alkenyloxy group, substitution alkenyloxy group, alkynyloxy group, substitution alkynyloxy group, aryloxy group and substituted aryloxy.

In another embodiment and with reference to embodiment up and down, the end-capping group also includes radioactive group, magnetic group, colorimetric group or fluorophor.

In another embodiment and with reference to embodiment up and down, the polypeptide or peptide are selected from any portion of biological transporter that can be combined with cell surface constituent, acceptor, with reference to or targeting ligand, including but not limited to vitamin (such as biotin, folic acid, pantothenic acid, B-6, B-12), sugared (such as glucose, N- acetyl glucosamines), chemotactic factor (CF) (such as RANTES, IL-2, OPG), peptide (or non-peptide) carrier (such as Tat, fMLF, cell-penetrating peptide (penetratin), VEGF [a kind of glycoprotein], transferrins), inverse-inversion peptide (such as RI TAT), film fusogenic peptide (such as gp41, VEGF [a kind of glycoprotein]), lipid (or phosphatide) (such as myristic acid, stearic acid), there is adopted (or antisense) oligonucleotides (such as containing the fit of 5- (1- amyl groups) -2 '-BrdU), enzyme (such as neuraminidase), toxin, antibody (or antibody fragment) (such as CD4 [targeting helper cell], CD44 [targeting ovarian cancer cell]), antigen (or epitope) (such as influenza virus hemagglutinin), peptide ligand, hormone (such as estrogen, progesterone, LHRH, ACTH, growth hormone), adhesion molecule (such as agglutinin, ICAM) and any of above molecule analog.

In another embodiment and embodiment up and down is combined, the activating agent has 1,2- amineothiots base or 1,3- amineothiot base comprising 1,2- amineothiots base (1,2-aminothiol group) or 1,3- amineothiot base or through derivative.

Another aspect of the present invention is related to the authentication method of the compound suitable for treatment or diagnostic uses, the component being had adverse effect to the bioactivity of the peptide or protein matter component of the compound is free of in the compound, methods described includes preparing the compound of the present invention and screens the bioactivity of contained treatment and/or diagnosis of partial in the compound.

One embodiment of the invention is 1, the 2- amineothiots of carrier or the preparation method of 1,3- amineothiot selective derivatization thing, and this method comprises the following steps：

(a) carrier with least one carrier segment is provided, at least one end of the carrier segment is activated by nucleopilic reagent or electrophilic reagent；With

(b) make the polymer with comprising 1,2- amineothiots defined in below general formula I or 1,3- amineothiot selectivity part or its reacted by the molecule of forms of protection, formed be covalently attached：

Formulas I

So as to formed comprising the selective end of 1,2- amineothiots or 1,3- amineothiot or its by the support derivatives of forms of protection, wherein A is i) 2 carbon atoms, or sp³Hydridization or sp²Hydridization (substitution is unsubstituted), wherein the two carbon atoms can be loop section or acyclic portion, and connect two carboxyls of electrophilic reagent, or ii) 3 atoms, selected from carbon, (substitution is unsubstituted, loop section or acyclic portion), nitrogen (substitution or unsubstituted, loop section or acyclic portion) or oxygen (loop section or acyclic portion).

Another embodiment of the invention is the preparation method of theme composition, and this method comprises the following steps：

(a) carrier with least one carrier segment is provided, the carrier segment is activated by nucleopilic reagent or electrophilic reagent；

(b) make the carrier with comprising 1,2- amineothiots defined in formula I or 1,3- amineothiot selectivity part or its reacted by the reagent of forms of protection, formed be covalently attached, wherein A be i) 2 carbon atoms, or sp³Hydridization or sp²Hydridization (substitution is unsubstituted), wherein the two carbon atoms can be loop section or acyclic portion, and connect two carboxyls of electrophilic reagent, or ii) 3 atoms, selected from carbon, (substitution is unsubstituted, loop section or acyclic portion), nitrogen (substitution or unsubstituted, loop section or acyclic portion) or oxygen (loop section or acyclic portion)；With

(c) by step (a) and (b) primary product and the active agent response comprising 1,2- amineothiots or 1,3- amineothiot.Following reaction process 1 is shown in the general description of this kind of method：

Reaction process 1

R₁=H, alkyl, acetenyl；R₂=alkyl, R₃=H, alkyl, polymer, bioactive substance.A=2 or 3 carbon atom；B=2 or 3 atom；

X and Y, which is two, can form the group of covalent attachment, i.e. X=electrophilic reagents, and Y=nucleopilic reagents

When carrier is the multivalent carrier comprising multiple activated carrier sections, the generality reaction shown in the above (reaction process 1) is especially advantageous.In this case, method of the invention can effectively produce in high yield with relatively pure conjugate, the conjugate functionalised (as defined herein) on each suitable activated carrier section of reality of polymer.In one embodiment, multiple reagents can be conjugated with single branch chain carrier.In a non-limiting examples, the present invention provides the water-soluble polymer of the bio-compatible with the highly branched chain being conjugated with peptide antagonists.

According to the feature and principle of the present invention, various reagents can be effectively conjugated by the suitable reactive group on reagent and activated carrier.This kind of reagent includes but is not limited to bioactivator or diagnostic reagent.

In another embodiment of the present invention and with reference to embodiment up and down, reagent can be the micromolecular compound with pharmacological activity.Or, reagent can be the inverse-inversion form or optimization form of biologically active peptide, and it has same or analogous bioactivity with primitive form, but with other desired features, for example, enzyme is attacked or the sensitiveness of metabolic enzyme is reduced.More particularly, reagent may include but be not limited to antibody or antibody fragment.Reagent comprising suitable 1,2- amineothiots base or 1,3- amineothiot base can be that synthesis source is either naturally occurring in particular agent.Therefore, reagent can have or have 1,2- or 1,3- group through modification, or can be conjugated with the compound with 1,2- amineothiots base or 1,3- amineothiot base, bioactivator of the compound for example containing modified peptides or cysteine.

The exemplary aspect of the present invention includes the preparation method of carrier-conjugated B1 peptide antagonists, and the carrier-conjugated B1 peptide antagonists include but is not limited to carrier-conjugated B1 peptide antagonists described in documents below：Pending US Application Serial No 10/972,236 (application on October 21st, 2004), it is published as U.S. Patent Application Publication No. 2005/0215470 (in September in 2005 29 days) (hereinafter referred to as " U. S. application ' 236 " ').

It is a further object to provide pharmaceutical composition, said composition includes excipient carrier material, wherein being dispersed with least one carrier-conjugation reagents of the invention.

It is a further object to provide the treatment method of the disease of B1 mediations, illness or obstacle, this method includes giving the composition of medicinal effective dose, a kind of carrier that said composition is prepared comprising excipient and at least one carrier of the invention-conjugated B1 peptide antagonists or with reagent of the present invention and method-conjugated B1 peptide antagonists.

The new support of the present invention produced with reagent of the present invention and method-conjugated B1 peptide antagonists and carrier-conjugated B1 peptide antagonists, disease, illness or obstacle available for the B1 mediations for treating or preventing wide spectrum, disease, illness or obstacle including but not limited to disclosed in cancer and U. S. application ' 236 ', inflammation and chronic pain conditions, septic shock, arthritis, osteoarthritis, angina pectoris, cancer, asthma, allergic rhinitis and antimigraine including but not limited to caused by inflammation and neuropathy.

Carrier of the present invention-conjugated B1 peptide antagonists or the carrier-conjugated B1 peptides produced with reagent of the present invention and method, available for the disease and illness treated or prevented described in context, i.e. by the way that they are configured into composition together with suitable pharmaceutical carriers material known in the art and patient in need, such as people (or other mammals) is given by the composition of effective dose.

In view of the following drawings and detailed description of the invention, the aspects of the invention and other side would is that obviously.

Detailed description of the invention

Subhead used herein is only used for the purpose of tissue, is not construed as the limitation to the theme.The All Files or file part quoted in the application, including but not limited to patent, patent application, paper, books and treatises, it is all incorporated herein by reference, for any purpose.In the case where term defined in one or more reference documents and the definition of the term of the application conflict, it is defined by the application.

Definition

Standard technique can be used for the generation and identification of recombinant DNA, oligonucleotide synthesis and antibody or antibody fragment.Generally can be as cited in conventional method well-known in the art and the application and the various general and specific bibliography that discusses, implementation aforementioned techniques and method.See, for example, the Molecular such as Sambrook Cloning：A Laboratory Manual (second edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)).Unless otherwise stated, the nomenclature used in analytical chemistry as described herein, the laboratory method of synthetic organic chemistry and medical chemistry and technology is all it is well known in the art that simultaneously generally use.Standard technique can be used for chemical synthesis, peptide symthesis, chemical analysis, chemical purification, medicine preparation, preparation, administration and patient treated.

In this application, unless otherwise stated, including plural number using odd number.In this application, "and/or" is referred to using "or", unless otherwise indicated.In addition, the use of term " comprising " and other forms not being restricted.

Native amino acid residues are discussed by following three kinds of modes：Amino acid full name, encode according to the alpha code of standard 3 shown in following table or Standard single-letter.

In certain embodiments, one or more unconventional amino acids can be incorporated into polypeptide.Term " unconventional amino acid " refers to any amino acid of not one of 20 kinds of conventional amino acids.Term " non-naturally occurring amino acid " refers to the amino acid for not having to find in nature.Non-naturally occurring amino acid is one group of unconventional amino acid.Unconventional amino acid includes but is not limited to the stereoisomer (such as D- amino acid) of 20 kinds of conventional amino acids, alpha-non-natural amino acid such as α-, α-disubstituted amino acid, N- alkyl amino acids, lactic acid, homoserine, homocysteine, 4- hydroxy-prolines, Gla, ε-N, N, N- trimethyl lysines, ε-N- acetyllysines, O- phosphoserines, N- acetyl-serines, N- formyl methionines, 3-Methyl histidine, 5- oxylysines, σ-N- methylarginines and other similar amino acid and imino acid (such as 4- hydroxy-prolines) known in the art.According to normal usage and convention, in polypeptide symbol used herein, left hand direction is aminoterminal direction, and right-hand direction is carboxyl extreme direction.Unless otherwise stated, this paper natural or alpha-non-natural amino acid title includes the D- types and L-type isomers of amino acid simultaneously.Other abbreviations of some alpha-non-natural amino acids used herein are identical with described in documents below：(2002) such as U.S. Patent No. 5,834,431, PCT Publication WO 98/07746 and Neugebauer.In addition, abbreviation " Dab " and " D-Dab " refer respectively to the L-type and D- type isomers of alpha-non-natural amino acid D-2- aminobutyric acids.Abbreviation " 3 '-Pal " and " D-3 '-Pal " refer respectively to the L-type and D- type isomers of alpha-non-natural amino acid 3 '-pyriylalanine.Also, abbreviation " Igl " is while including " Igla " and " Iglb " (being respectively α-(1- indanyls) glycine and α-(2- indanyls) glycine).Equally, " D-Igl " is while including " D-Igla " and " D-Iglb " (being respectively the D- types isomers of α-(1- indanyls) glycine and α-(2- indanyls) glycine).It is preferred that when used herein, Igl is Iglb, D-Igl is D-Iglb.

Various other abbreviations used herein are listed as follows：

ACN, MeCN-acetonitrile

APCI MS-APCI mass spectrum

AgNO₃- silver nitrate (I)

AIBN -2,2 '-azo two (2- methyl propionitrile)

BBr₃- Boron tribromide

T-BDMS-Cl-tert-butyl group diethylsilane base chlorine

CCl₄- carbon tetrachloride

Cs₂CO₃- cesium carbonate

CHCl₃- chloroform

CH₂Cl₂, DCM-dichloromethane

CuBr-copper bromide

CuI-cupric iodide

DIBAL-diisobutyl aluminium hydride

DIC -1,3- DICs

DIEA, (iPr)₂Net

DIPEA, Hunigs

Base-diisopropylethylamine

DCE-dichloroethanes

DCM-n-hydroxysuccinimide

DME-dimethoxy-ethane

DMF-dimethylformamide

DMAP-DMAP

DMSO-dimethyl sulfoxide

DSS-trimethyl silyl -2- silicon pentane -5- sulphonic acid ester-d6,

Sodium salt

EDC -1- (3- dimethylamino-propyls) -3- ethyl carbodiimides

Et₂O-ether

EtOAc-ethyl acetate

FBS-hyclone

FTMS-Fourier Transform Mass Spctrometry

G, gm, g-gram

H, hr-hour

H₂- hydrogen

HATU-hexafluorophosphoric acid O- (7- azepine benzos triazol-1-yl)-

N, N, N ', N '-tetramethylurea

HBr-hydrobromic acid

HCl-hydrochloric acid

HOBt-I-hydroxybenzotriazole hydrate

HPLC-high pressure liquid chromatography

HRMS-high resolution mass spec

IPA, i-PrOH-isopropanol

K₂CO₃- potassium carbonate

KI-KI

LiCl-lithium chloride

LiOH-lithium hydroxide

MgSO₄- magnesium sulfate

MeOH-methanol

MW-molecular weight

MWCO-Molecular weight cut-off value (molecular weight cut-off)

N₂- nitrogen

NaCNBH₃- sodium cyanoborohydride

NaHCO₃- sodium acid carbonate

NaH-sodium hydride

NaOCH₃- sodium methoxide

NaOH-sodium hydroxide

Na₂SO₄- sodium sulphate

NBS-N-bromosuccinimide

NH₄Cl-ammonium chloride

NH₄OH-ammonium hydroxide

NMP -1-METHYLPYRROLIDONE

P(t-bu)₃- three (tert-butyl group) phosphines

PBS-phosphate buffered saline solution

RT, rt-room temperature

TBAF-tetra-n-butyl ammonium fluoride

TBTU-tetrafluoro boric acid O- BTA -1- bases-N, N, N ', N ' -

Tetramethylurea

TEA, Et₃N-triethylamine

TFA-trifluoroacetic acid

THF-tetrahydrofuran

According to used in this specification, unless otherwise stated, following term should be understood to following meanings：

The implication of term " activating agent " includes any therapeutic agent, bioactivator and/or diagnostic reagent.Term " B1 " refers to bradykinin b 1 receptor (referring to Judith M Hall, A reviewof BK receptors (BK acceptors summary) .Pharmac.Ther., 56：131-190(1992)).Unless otherwise stated, B1 or bradykinin b 1 receptor refer to people's bradykinin b 1 receptor (hB1).It is preferred that hB1 be wild-type receptor.Preferred hB1 is the bradykinin receptor described by GenBank searching numbers AJ238044.

The compound of the present invention could generally have some asymmetric centers, generally be described in the form of racemic mixtures.The present invention includes racemic mixture, partial racemic compound and single enantiomer and diastereomer.

Unless otherwise stated, the term defined below being applied in the specification and claims：

“C_α-βAlkyl " refers to the alkyl that side chain, ring-type or straight chain or this three class comprising at least α and at most β carbon atom are combined, and wherein α and β represent integer.Alkyl described in this trifle can also contain one or two double or triple bonds.C_1-6The example of alkyl includes but is not limited to following groups：

“C_α-βMiscellaneous alkyl " refers to the C that any carbon atom in alkyl is replaced by O, N or S_α-βAlkyl.C_1-6The example of miscellaneous alkyl includes but is not limited to following groups：

" leaving group " is typically referred to easily by the group of nucleopilic reagent (such as amine, mercaptan or alcohol nucleopilic reagent replace).Such leaving group is well-known in the art.The example of this kind of leaving group includes but is not limited to n-hydroxysuccinimide, N- hydroxybenzotriazoles, halide, triflate, tosylate etc..When appropriate, leaving group preferably is as shown here." protection group " typically refers to well-known in the art for protecting selected active group (such as carboxyl, amino, hydroxyl, sulfydryl) in order to avoid undergoing the group of undesired reaction (such as nucleophilic, electrophilic, oxidation, reduction).When appropriate, protection group preferably is as shown here.The example of amino protecting group includes but is not limited to aralkyl, substituted aralkyl, cycloalkenyl alkyl and substituted cycloalkenyl alkyl, pi-allyl, substituted allyl, acyl group, alkoxy carbonyl, aromatic alkoxy carbonyl, silicyl etc..The example of aralkyl includes but is not limited to benzyl, adjacent methyl-benzyl, trityl and benzhydryl, and (it can be optionally by following substituent group：Halogen, alkyl, alkoxy, hydroxyl, nitro, acyl amino, acyl group etc.) and salt (Li such as phosphonium salt and ammonium salt).The example of aryl includes phenyl, naphthyl, indanyl, anthryl, 9- (9- phenylfluorenyls), phenanthryl, durene base etc..The example of cycloalkenyl alkyl or substituted cycloalkenyl alkyl (preferably with 6-10 carbon atom) includes but is not limited to cyclohexenyl methyl etc..Suitable acyl group, alkoxy carbonyl and aromatic alkoxy carbonyl include benzyloxycarbonyl, tert-butoxycarbonyl, isobutoxy carbonyl, benzoyl, substituted benzoyl, bytyry, acetyl group, trifluoroacetyl group, tribromo-acetyl base, phthalyl etc..The mixture of protection group can be used for protecting same amino, and such as primary amino radical can be protected with aralkyl and aromatic alkoxy carbonyl.The nitrogen that amino protecting group can be also connected with them constitutes heterocycle; such as 1; 2- double (methylene) benzene, phthalimidyl, succinimido, dimaleoyl iminos etc., and wherein these heterocyclic groups can also include adjacent aryl and cycloalkyl ring.In addition, heterocyclic radical can be one, two or trisubstituted, such as nitro phthalimidyl.Amino can be also protected by forming addition salts (such as hydrochloride, toluene fulfonate, trifluoroacetate), to exempt from undesired reaction, such as oxidation reaction.Many amino protecting groups are also suitable for protection carboxyl, hydroxyl and sulfydryl.Such as aralkyl.Alkyl is also the proper group for protecting hydroxyl and sulfydryl, such as tert-butyl group.

Silyl-protecting groups are the silicon atoms optionally replaced by one or more alkyl, aryl and aralkyl.Suitable silyl-protecting groups include but is not limited to trimethyl silyl, triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl, dimethylphenylsilyl, 1; double (dimetylsilyl) benzene of 2-, double (dimetylsilyl) ethane of 1,2- and diphenylmethylsilyl.The silylated offer one-of amino or two-silicyl amino.The silylated of alkamine compound produces N, N, O- trimethylsilane radical derivatives.By using such as metal hydroxides or ammonium fluoride agent treatment in single reactions steps, or with carrying out in-situ treatment with such as metal hydroxides or fluorination ammonium reagent in alcohol radical course of reaction, silyl functional group easily can be sloughed from silyl ether functional group.Suitable monosilane agent is such as trimethylsilyl chloride, t-butyl-dimethylsilyl chlorine, phenyldimethylsilyl chlorine, diphenylmethylsilyl chlorine or their combination products with imidazoles or DMF.Silylated and silyl-protecting groups the deprotection method of amine is all well known to the skilled person.The method for preparing these amine derivatives from corresponding amino acid, amino acid amide or amino-acid ester is that organic chemistry (including amino acid/amino-acid ester or amino alcohol chemistry) art personnel are well-known.

Protection group is divested under conditions of the remainder of molecule is not influenceed.These methods are well-known in the art, including acid hydrolysis, hydrogenolysis etc..It is preferred that method include slough protection group, such as by using palladium on carbon, in suitable solvent system such as alcohol, acetic acid or its mixture, benzyloxycarbonyl is sloughed through hydrogenolysis.Using inorganic acid or organic acids such as HCl or trifluoroacetic acid, in suitable solvent system such as dioxane or dichloromethane, Boc protecting group is sloughed.Gained amide can be easily neutralized, and obtain unhindered amina., can be with decarboxylize protection group, such as methyl, ethyl, benzyl, the tert-butyl group, 4- methoxyphenyl methyls under hydrolysis well known to the skilled person and hydrogenolytic conditions.

It should be noted that the compound of the present invention can contain the group existed with tautomeric forms, such as the heteroaryl (Y '=O, S, NR) that ring-type and non-annularity amidino groups and guanidine radicals, hetero atom replace, the example seen below：

Although naming, describing herein, showing and/or a kind of form is claimed, all tautomeric forms are included among such title, description, display and/or claim certainly.

The pro-drug of the compounds of this invention is also included in the present invention.Pro-drug is active or inactive compound, and the pro-drug can be acted on such as hydrolysis, metabolism by body physiological after patient is given and turn into the compound of the present invention through chemical modification.The applicability and technology that prepare and its use of pro-drug are well known to the skilled person.General discuss of pro-drug about being related to esters can be found in Svensson and TunekDrug Metabolism Reviews 165 (1988) and Bundgaard Design of Prodrugs, Elsevier (1985).The example for the carboxylate anion sheltered includes various esters, such as Arrcostab (such as methyl esters, ethyl ester), cycloalkyl ester (such as cyclohexyl ester), aralkyl ester (such as benzyl ester, to methoxy benzyl ester) and alkyl carbonyl oxy Arrcostab (such as pivaloyloxymethyl).Amine is masked into the methyl substituted derivative of aryl-carbonyl oxygen, and it can be cut by internal esterase and discharge free drug and formaldehyde (Bungaard J.Med.Chem.2503 (1989)).Also, the medicine (Bundgaard Design of Prodrugs, Elsevier (1985)) of the group (such as imidazoles, imidodicarbonic diamide, indoles) containing acidity NH is sheltered with N- pivaloyloxymethyls.Hydroxyl is masked into ester and ether.EP 039,051 (Sloan and Little, 4/11/81) discloses Mannich base hydroxamic acid pro-drug, preparation method and use.

Contain the Category List using " be selected from ... and ... " and " be ... or ... " term (being sometimes referred to Markush key element) in the specification and claims.When such term is used for the application, unless otherwise stated, referring to include whole groups or its any single member or its any group.The use of the term must not be considered as the limitation to optionally removing each key element or group in any way just to the purpose write a Chinese character in simplified form.

The implication of term " diagnostic reagent " includes can be used for detection predetermined substance presence or absence in vivo or in vitro, any compound, composition or the particle of the method for determining content of predetermined substance and/or radiography being carried out to predetermined substance.

Term " polynucleotides of separation " used herein should refer to the polynucleotides or combinations thereof of genome, cDNA or synthesis source, by its source, " polynucleotides of separation " (1) does not associate with all or part of polynucleotides, wherein described " polynucleotides of separation " are present in nature, (2) it is connected with polynucleotides, and it is not connected in nature with the polynucleotides, or (3) are not intended as the part of bigger sequence in nature and existed.

Term " polymer " " refers to the compound being made up of the non-peptide construction unit repeated.In certain embodiments of the invention, carrier can be water-soluble polymer, such as PEG and methoxy poly (ethylene glycol) (mPEG).

Term " polynucleotides " and " oligonucleotides " are used interchangeably, and in this article refer to the polymer that length is at least the nucleotides of 10 bases.In certain embodiments, base may include the modified forms of at least one ribonucleotide, deoxyribonucleotide and both nucleotides.The term includes DNA single-stranded and double chain form.

Term " naturally occurring nucleotides " includes deoxyribonucleotide and ribonucleotide.Deoxyribonucleotide includes but is not limited to adenosine, guanine (guanine), cytimidine (cytosine) and thymidine (thymidine).Ribonucleotide includes but is not limited to adenosine, cytimidine (cytosine), thymidine (thymidine) and uracil (uracil).Term " modified nucleotide " includes but is not limited to the nucleotides for modifying or replacing glycosyl etc..Term " polynucleotides key " includes but is not limited to the polynucleotides key such as thiophosphate, phosphorodithioate, phosphoroselenoate (phosphoroselenoate), two phosphoroselenoates (phosphorodiselenoate), phosphoroanilothioate, phoshoraniladate, phosphoramidate (phosphoroamidate).See, for example, LaPlanche etc., Nucl.Acids Res.14：9081(1986)；Stec etc., J.Am.Chem.Soc.106：6077(1984)；Stein etc., Nucl.Acids Res.16：3209(1988)；Zon etc., Anti-Cancer Drug Design 6：539(1991)；Zon etc., Oligonucleotidesand Analogues：A Practical Approach, the 87-108 pages (F.Eckstein writes, Oxford University Press, Oxford England (1991))；Stec etc., U.S. Patent No. 5,151,510；Uhlmann and Peyman Chemical Reviews 90：543(1990).In certain embodiments, polynucleotides can include detection mark.

Term " purifying " is when for polypeptide, peptide or protein matter, it is substantially free of cellular component that polypeptide, peptide and protein, which should be referred to, contain the cellular component less than about 50%, preferably less than about 70%, more preferably less than about 90%, the target molecule naturally associates with the cellular component.Polypeptide, peptide and protein purification method are well-known in the art.

The polymer that term " polypeptide ", " peptide " and " protein " is connected to each other all referring to two or more amino acid by peptide bond or modification peptide bond, i.e. the structure molecule such as peptide.The term is applied to amino acid polymer and such amino acid polymer containing naturally occurring amino acid：Wherein one or more amino acid residues are the chemical analogs of non-naturally occurring amino acid or naturally occurring amino acid.Polypeptide, peptide or protein matter can contain by one or more natural processes (such as post translational processing; such as glycosylation, acetylation, phosphorylation) and one or more amino acid residues of modification, and/or the one or more amino acid residues modified by one or more chemical modification technologies known in the art.

Reference polypeptide " fragment " refers to any portion of one section of continuous amino acid sequence of reference polypeptide.Fragment length can be less than any length of reference polypeptide.

All polypeptides, peptide and protein sequence are all write according to known convention, and wherein n terminal amino acid residue is on the left side, and C- ends are on the right.Term " N- ends " used herein refers to the free alpha-amido of the amino acid of peptide, and term " C- ends " refers to free α-carboxylic acid end of polypeptide, peptide and Amino Acids in Proteins.

Term " selectivity " used herein refers to the chemical reaction between activating agent and carrier; or activated carrier refers to the chemical reaction to limit and known way is carried out; so that other functional groups including i) including but is not limited to unhindered amina, amine, guanidine, hydroxyl and carboxylic acid are without be protected, and ii) needed for conjugate account at least the 50% of reaction product.

" variant " of reference polypeptide refers to for reference polypeptide, with one or more 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, missing or the polypeptide inserted.In certain embodiments, the variant of reference polypeptide has the posttranslational modification site (i.e. glycosylation site) changed.In certain embodiments, the variant of reference polypeptide and reference polypeptide is all specific-binding agent.In certain embodiments, the variant of reference polypeptide and reference polypeptide is all antibody.

The variant of reference polypeptide includes but is not limited to Cysteine variants.In certain embodiments, Cysteine variants include the variant that one or more of reference polypeptide cysteine residues are replaced by one or more non-cysteine residues；And/or the variant that the non-cysteine residues of one or more of reference polypeptide are replaced by one or more cysteine residues.In certain embodiments, Cysteine variants have more cysteine residues than native protein.

" derivative " of reference polypeptide refers to：Polypeptide：(1) there are one or more modifications in one or more of reference polypeptide amino acid residue；And/or (2) wherein one or more peptide bonds are replaced by one or more non-peptide bonds；And/or (3) wherein N- ends and/or C- ends is modified；And/or (4) wherein side chain is modified.Some example sex modifications include but is not limited to acetylation, it is acylated, ADP- ribosylation, amidatioon, biotinylation, it is covalently attached with flavine, it is covalently attached with heme moiety, it is connected with nucleotides or nucleotide derivative, covalent, it is covalently attached with lipid or lipid derivate, it is covalently attached with phosphoinositide, crosslinking, cyclisation, form disulfide bond, demethylation, form covalent cross-linking, form cystine, form pyroglutamic acid, formylated, γ-carboxylated, glycosylation, form GPI grapplings, hydroxylating, iodate, methylate, myristoylation (myristoylation), oxidation, proteolysis are processed, phosphorylation, prenylation, racemization, selenium is acylated, sulphation, the amino acid of t-RNA mediations is added on protein (such as arginyl) and ubiquitination.In certain embodiments, the derivative of reference polypeptide and reference polypeptide is all specific-binding agent.In certain embodiments, the derivative of reference polypeptide and reference polypeptide is all antibody.Polypeptide includes but is not limited to the amino acid sequence modified by natural process (such as post translational processing) or chemical modification technology well known to the skilled person.In certain embodiments, from anywhere in modification can occur on polypeptide, including peptide backbone, amino acid side chain and aminoterminal or c-terminus.In some such embodiments, there can be same or different degrees of modification at some positions of given polypeptide.In certain embodiments, given polypeptide contains a variety of modifications, such as the missing of one or more amino acid, addition and/or substitution in native sequences.In certain embodiments, polypeptide can be side chain and/or ring-type.Ring-type, side chain and branched circular polypeptide may be from natural process after translation (including but is not limited to ubiquitination) or can be prepared with synthetic method.

Term " bioactivity " refers to that the interaction that described reagent can be in vitro or in vivo pair with biomolecule or biosystem (such as polypeptide, cell or organism) plays and/or induced biological effect.The many that proving the method for bioactivity is included in vitro bioassay, these methods is all well-known in the art.Bioactivator includes but is not limited to curative.The implication of term " curative " includes any material, composition or the particle available for any therapeutical uses, such as method for treating patient disease.Therefore, curative includes can be used in any compound or material of any pathological state (including but is not limited to illness, pain, illness, disease, obstacle, lesion, wound or damage) for the treatment of (including prevent, alleviate, palliate the agonizing sufferings or cure) patient.The non-limiting examples of curative include medicine, vitamin such as biotin, pantothenic acid, vitamin B6 and vitamin B12, nutrient, nucleic acid such as ASON and short interfering rna (siRNA) molecule, amino acid, polypeptide, peptide, inverse-inversion (RI) peptide and formyl-methionyl peptide, enzyme, hormone, growth factor, chemotactic factor (CF), antibody and its fragment, enzyme cofactor, steroid, carbohydrate, lipid, organic matter such as heparin, reagent containing metal, receptor stimulating agent, receptor antagonist, associated proteins, acceptor or acceptor portion, extracellular matrix protein, cell surface molecule, adhesion molecule, antigen, haptens, target group and chelating agent.The acceptor related to includes the acceptor of form of ownership, as long as more than one form is present.

Other non-limiting examples of curative include insulin, AntiHIV1 RT activity peptide (for example Tat inhibitor, sees below), growth hormone, interferon, immunoglobulin, parathyroid hormone, calcitonin, enkephalins, endorphin, medicine, pharmaceuticals, cytotoxic agent, chemotherapeutic, radiotherapy medicine, protein, natural or synthetic peptide (including oligopeptides and polypeptide), vitamin, steroidal and inhereditary material (including nucleosides, nucleotides, oligonucleotides, polynucleotides and plasmid).Wherein preferred agents.The example of medicine includes：Anti-ulcer agent, such as Cimetidine, famotidine, ranitidine, acetic acid Roxatidine, Pantoprazole, Omeprazole, Lansoprazole or ulcerlmin；Intestines relaxation medicine or dynamics-promoting medicine (prokinetics), such as propantheline bromide, camylofin (acamylophenine), bentyl, scopolamine butylbromide, mebeverine, Cisapride, oxybutynin, methyl Pipenzolate Bromide, song Ta Weilin, Metoclopramide, clidinium bromide, isopropamide bromide or Oxyphenonium Bromide；Enzyme or carminative (carminative), such as pancreatin, papain, pepsin or amylase；Liver and gall preparation, such as chenodeoxycholic acid, urso, L-Orn or silymarin；Antihypertensive, such as clonidine, ethyldopa sodium nitroprussiate, Terazosin, Doxazosin, (DI) hydrolazine or prazosin；Beta-Blocking agent, such as esmolol, celiprolol, atenolol, labetalol (labetolol), Propranolol, metoprolol, Carvedilol, Sotalol, oxyprenolol or bisoprolol；Calcium channel blocker, such as felodipine, nitrendipine, nifedipine, Benidipine, Verapamil, Amlodipine or lacidipine；Angiotensin converting enzyme (ace) inhibitor, such as enalapril, lisinopril, Ramipril, Perindopril, benazepil or captopril；Angiotensin II inhibitor, such as Losartan Potassium；Potassium channel activators, such as nicorandil；Diuretics and antidiuretic, such as Hydrochioro, Xipamide, bumetanide, amiloride, spirolactone, indapamide, triamterene, clopamide, frusemide or chlorthalidone；Antianginal drug, such as ISDN (isoscorbide dinitrate), Oxyfedrine, 5- Isosorbide Mononitrates, your sulphur

, erythrityl tetranitrate, Trimetazidine, Lidoflazine, pentaerythrityl tetranitrate, nitroglycerin or ground draws

；Coagulant, such as conjugated estrogens, diosmin, vitamin K₃(menaphthone), menadione, haemocoagulase, etamcylate (cyclonamine), rutin-flavonoids or adrenaline clearance permit semicarbazones (adrenochromemonosemicarbazone)；Anticoagulation, antithrombotic or antiplatelet drug, such as ticlopidine, warfarin, streptokinase, phenindione, rtpa, urokinase, pitressin, acenocoumarin (nicournalone), heparin, low molecular weight heparin, mucopolysaccharide polysulfate or Dipyridamole；Antiarrhymic, such as quinindium, disopyramide, procainamide, lignocaine (lidocaine), mexiletine, amiodarone (arniodarone), adenosine Propafenone；For heart failure and the medicine of shock, such as mephentermine, digoxin dopamine, dobutamine or norepinephrine, vasodilator agent, such as Isoxsuprine, Buddhist nun can Xanthones replace promise, nylidrin hydrochloride (nylidrinHCl), PTX (Pentifylline) or cyclandelate；Cardiac glycoside, such as Deslanoside (deslaneside), foxalin, digoxin or digitalin；Penicillins, such as benzylpenicillin, procaine penicillin (G), tardocillin (G), phenoxymethylpenicillin, benzyl penicillin/V, Bacampicillin, Carbenicillin, Piperacillin, ampicillin, Cloxacillin or Amoxicillin；Quinolones or fluoroquinolones, such as acidum nalidixicum, Pefloxacin, Ofloxacin, Sparfloxacin, Norfloxacin, Ciprofloxacin, Lomefloxacin, cephalosporins, such as Ceftizoxime, cefuroxime, Cefixime, CTX, Cefaclor, Ceftriaxone Sodium, cefadroxil, cefalexin, Cefazolin, cefaloridine, cefotaxime or cefoperazone (ceforperazone)；Sulfamido, such as sulfanilamide (SN), sulfamoxole, sulfadimethoxine (sulphadimehtoxine), cotrifamole, SMZco, TMP, aminoglycoside, such as gentamicin, TOB, neomycin, amikacin, SISO, kanamycins, Netilmicin, polymyxins (such as polymyxins-b), colistin sulfate；Chloramphenicol；Tetracyclines, such as tetracycline, Doxycycline, minocycline, demeclocycline, terramycin；Macrolides, such as erythromycin, CLA, vancomycin, lincomycin, azithromycin, spiramvcin, ROX, clindamycin, Cefpirome, teicoplanin (Teicoplanin Targocin a2)；Antiviral agent, such as Abacavir, Lamivudine, ACV, amantadine, interferon, Ribavirin, stavudine (stavurdine), Lamivudine or Zidovudine (AZT)；Antimalarial, such as quinine, chloroguanide, chloroquine, primaquine, anodiaquine, Artemether, Artesunate, Mefloquine, pyrimethamine, arteether, mepacrine；Anti-tubercular drug, such as seromycin, capreomycin, 2-ethylisonicotinthionamide, protionamide, Rimactazid, pyrazinamide, ethambutol；Ethambutol, streptomysin, pyrazinamide；Anthelmintic and anti-infectious agent, such as piperazine, niclosamidum, Pyrantel Pamoate, levamisol, diethylcarbamazine, tetramisole, albendazole, praziquantel, stibii natrii gluconas or menbendazole；Antileprotic, such as dapsone or Clofazimine；Antianaerobic-microbacterial drug, antiprotozoal agent or amebicide, such as Tinidazole, metronidazole, diloxanide furoate, secnidazole, hydroxy quinolone, Dehydroemetine, Ornidazole (omidazole), furazolidone；Antifungal, such as Fluconazole, ketoconazole, Hamycin, Terbinafine, econazole, amphotericin-B, nystatin, clotrimazole, griseofulvin, Miconazole or Itraconazole；Vitamin；Respiratory stimulant, such as doxapram hydrochloride；Antiasthmatic, such as isoprel, salbutamol (albuterol), orciprenaline, ephedrine, bricalin, salmeterol, aminophylline, therophylline, beclomeasone propionate or fluticasone propionate；Antiallergic, such as RMI 9918, astemizole, Loratadine, clemastine, dimethindene maleate, fexofenadine hydrochloride, hydroxyzine, chlorphenamine, azatadine maleate, first ground piperazine, pheniramine maleate, diphenhydramine or cetrizine；Skeletal muscle relaxant, such as Tizanidine, methocarbamol, carisoprodol, penta husky ammonium (valethamate), Baclofen, Chlormezanone or Chlorzoxazone；Smooth muscle relaxant, such as Oxyphenonium Bromide, propantheline bromide, diclomine, scopolamine butylbromide, mebeverine, Drotaverine, clidinium bromide, isopropyl ammonium or camylofin dihydrochloride；NSAIDs, such as naproxen, mefenamic acid, aulin, Diclofenac, tenoxicam, brufen, Meloxicam, aspirin, Flurbiprofen, Ketoprofen, ketoprolac, phenylbutazone, Oxyphenbutazone, Indomethacin or piroxicam；Antineoplastic, such as mustard compound (such as endoxan, Trofosfamide, iofosfamide, melphalan or Chlorambucil), ethylene imine (such as thioepa), N- nitroso ureas derivative (such as BCNU, lomustine or Nimustine), platinum compounds (such as Spiroplatin, cis-platinum and carboplatin), procarbazine, Dacarbazine, methotrexate (MTX), adriamycin, mitomycin, pacify Sha's rhzomorph, cytarabine, arabinosyl adenine, sulfydryl polylysine, vineristine, busulfan, Chlorambucil, melphalan (such as PAM, L-PAM or melphalan), purinethol, mitotane, procarbazine hydrochloride, actinomycin D, daunorubicin hydrochloride, doxorubicin hydrochloride, epirubicin, plicamycin (mithramycin), mitoxantrone, bleomycin, Bleomycin Sulphate, aminoglutethimide, estramustine phosphate sodium, Flutamide, leuprolide acetate, megestrol acetate, TAMOXIFEN CITRATE, Testolactone, Trilostane, amsacrine (m-AMSA), asparaginase (L-ASP), Erwinia (Erwina) asparaginase, Etoposide (VP-16), interferon (includes but is not limited to interferon a-2a, Interferon Alfa-2b), Teniposide (VM-26), vinblastine sulfate (VLB), vincristine sulphate, eldisine, taxol (Taxol), methotrexate (MTX), adriamycin, aralino, hydroxycarbamide；The antagonist (such as purinethol, thioguanine, fluorouracil or cytarabine) of antifol (such as aminopterin, methotrexate (MTX)), purine and pyrimidine bases；Arcotic, opiates medicine or sedative, such as paregoric, codeine, morphine, opium, amytal, amobarbital sodium, aprobarbital, butobarbital sodium, chloraldurate, Ethchlorvynol, ethinamate, flurazepam hydrochloride, glutethimide, levomepromazine hydrochloride, Methyprylon (methyprylon), midazolam hydrochloride, paraldehyde, amobarbital, barbose, talbutal, Temazepam or triazolam；Locally or systemically arcotic, such as Bupivacaine, chloroprocanine, Etidocaine, lidocaine, mepivacaine, procaine or totokaine, droperidol, Etomidate, citric acid fentanyl-droperidol, ketalar, methohexital sodium or thiobarbiturate；Neuromuscular blocking agent, such as methanesulfonic acid atracurium, Gallamine Triethiodide, second fluorenes bromine ammonium, dimethyl tubocurarine iodide, Pancuronium Bromide, Choline Chloride Succinate, tubocurarine chloride or Vecuronium Bromide；Or the curative for Hormone system,Such as growth hormone,Melanocyte-stimulating hormone(MSH),Estradiol,Beclomeasone propionate,Betamethasone,Cortisone acetate,Dexamethasone,Flunisolide,Hydrocortisone,Methylprednisolone,Paramethasone acetate,Prednisolone,Metacortandracin,Fluoxyprednisolone,Fludrocortisone acetate,Adenosine deaminase,APV,Albumin,Hyaluronidase,Interferon-' alpha '-N3,Palonosetron hydrochloride,Human antihemophilic factor,Human factor IX,alefacept,Amphotericin B,Testosterone,Bivalirudin,darbepoetin alfa,Tazarotene,Bevacizumab,Morphine sulfate,Interferon beta-1a,Plasma thromboplastin component,Interferon beta-1b,Tositumomab and I-131 tositumomabs,Antihemophilic factor,Human growth hormone (HGH) (such as sumatropin),Botulinum toxin type A,exenatide,Alemtuzumab,Hyaluronic acid,acritumomab,Alglucerase,β-glucocerebrosidase,Imiglucerase,Tadalafil,clofarabine,Codeine polistirex,Chlorphenamine-sulfonated divinyl benzene-ethylene benzene copolymer,Haemophilus B conjugates [meningococcal conjugate],Collagen,Rattle snake multivalent immunogen Fab,Daptomycin,Hyaluronidase,CMV immune globulin IVs,Daunorubicin,Cytarabine,Doxorubicin hydrochloride,Epinastine hydrochloride,Leuproside,Rasburicase,Emtricitabine,Etanercept,Hepatitis B antigen,epoietin alfa,Cetuximab,Estradiol,Clindamycin,Factive,Urofollitropin,Influenza antigen,dexmethylphenidatehydrochloride,Follitropin beta,Teriparatide,Calcitonin,Butanedioic acid frovatriptan,enfuvirtide,Nitric acid is sowed,Human somatotropin (somatropin),Imatinib mesylate,Glucagons,Metformin hydrochloride,Follicle-stimulating hormone (FSH) α,Doxercalciferol,Adefovir dipivoxil,Trastuzumab,HES,Insulin and insulin analog,Feng's von Willebrand (von Willebrand) factor,Adalimumab (adalimumab),Perflexane,Mecasermin,Interferon alfacon-1,Bone morphogenesis protein-2,Eptifibatide,Alpha-interferon,Timolol,palifermin,Anakinra,Insulin glargine,Granulocyte macrophage colony stimulating factor,Carat Qu Bin,Fosamprenavir calcium,eszopiclone,Lutropin alfa,Betamethasone,OspA lipoprotein,pegaptanib,Methylphenidate,Methylamino ketone valeric acid (methylaminoleyulinate),Mitomycin,Gemtuzumab,Ozogamicin,Botulinum toxin type B,Human hepatitis b immune globulin,galsulfase,Memantine,Vitamin B12,Nesiritide,PEG- Filgrastims (pegfilgrastim),Oprelvekin,Filgrastim,Technetium [99mTc] fanolesomab,Mitoxantrone,Insulin aspart,Proconvertin a,Clobetasol propionate,L-ASP,Denileukin diftitox,Amlexanox,Nitisinone,Muromonab-CD3,Human chorionic's glandular hormone,BCG vaccine antigen,Alitretinoin,Diphtheria (diphtheria),PEG- Intederon Alpha-2as,Porfimer Sodium,Antagonists of gonadotropin-releasing hormone,Repaglinide,Pneumococcus 7- valency conjugates,Ziconotide,Ciprofloxacin Hydrochloride,The capromab pendetides of indium In 111,Somatrem,Modafinil,Dornase Alfa,Carry out western certainly southern (lexidronam) samarium SM-153,Omeprazole,Efalizumab,Ribavirin and alpha interferon,Lepirudin,Gel Becaplermin (gel becaplermin),Infliximab,treprostinil sodium,Sevelamer hydrochloride,Abciximab,Reteplase,Rh0 immunoglobulins,Rituximab,Intederon Alpha-2a,Trospium chloride,Fluoxetine hydrochloride,Synthesize Sekretolin,cinacalcet HCl,Basiliximab,Pegvisomant,Pramlintide acetate,Palivizumab,Phosphoric acid Ao Sai meter Wei,Erlotinib (OSI Pharmaceuticals,Inc. and Genentech),Bexarotene,Bexarotene,ATG,Thyrotropic hormone α,Thyroglobulin (Tg),TNK,Influenza,Diphtheria toxoid,Tetanus toxoid and acellular pertussis antigen,Arsenic trioxide,emtricitabine,Natalizumab,bortezomib,Iloprost,Azacitidine,Nai Feinawei,Tenofovir disoproxil fumarate,Vistide,Verteporfin,Fomivirsen,Interferon alfa-n1,Rho [D] immunoglobulin,Bromfenac sodium,Rifaximin (rifaximin),drotrecoginalfa,Omalizumab,Sodium hydroxybutyrate,miglustat,Omeprazole,Daclizumab,Ibritumomab tiuxetan,Zonisamide,Loteprednol etabonate,TOB,Bromhexine,Carbocisteine or clavulanic acid,Tadenan,Paracetamol,Gamma interferon 1-b,Alteplase and technetium Tc-99 apcitide.

The activating agent being connected with the carrier in conjugate of the present invention has or had through modification being capable of 1, the 2- amineothiots or 1,3- amineothiot part or Formulas I group as described herein that reacted by its complementary functional groups and support derivatives.The example of activity 1,2- amineothiots is visible in amino acid cysteine.

Many protein do not have free cysteine (cysteine for being not involved in being formed disulfide bond) or any other active 1,2- amineothiots base or 1,3- amineothiot base.In addition, cysteine 1,2- amineothiots may be not suitable for being connected with polymer, because necessary to 1,2- amineothiot is bioactivity.In addition, protein must be folded into certain conformation just it is active.In activity conformation, what 1, the 2- amineothiots of cysteine were inaccessible, because it is embedded in protein interior.In addition, it is even come-at-able and and it is nonactive necessary to cysteine 1,2- amineothiots, which are likely to be at, is not suitable for the position that is connected with polymer.And nonactive necessary amino acid is referred to as " nonessential ".Nonessential cysteine is likely to be at unsuitable conjugated position, because the position of cysteine can inactivate polypeptide for active site after being conjugated with carrier.

As protein, many other bioactive molecules also active 1,2- amineothiots or 1,3- amineothiot, because similar to above-mentioned reason, they are unsuitable for conjugated or without activity 1,2- amineothiots base or 1,3- amineothiot base with specific support.Therefore, the present invention includes activity 1,2- amineothiots base or 1,3- amineothiot base are introduced into bioactivator if necessary, and it can be conjugated with the support derivatives of the present invention.The example of bioactivator containing thioamide moiety can be found in U.S. Patent Application Serial number 09/621,109.This kind of compound includes but is not limited to UC781；R82150；HBY097；Trovirdine (troviridine)；S2720；UC38 and 2 ', 3 '-dideoxy -3 '-fluoro- thio thymidines of 4-.

Active mercapto or thioamides base can be chemically incorporated into by well-known in the art.Chemical modification can be used for polypeptide or non-peptide molecule, and introduce molecule including the part by mercaptan individually or as more macoradical such as cysteine residues.Such as DTT can also be used, cysteine is reduced by chemical method, free cysteine is produced in polypeptide.

It is referred to as " mutain " through modifying the polypeptide containing amino acid residue on non-existent position in native protein before modification.In order to produce cysteine mutein, N- ends nonessential amino acid can be replaced by cysteine.N- ends lysine mutation is that cysteine is also suitable, because lysine residue is often visible in the protein surface of activity conformation.In addition, in the selection of possible mutated site, those skilled in the art can utilize any Given information about polypeptide binding site or active site.Well-known recombinant DNA technology can also be used in those skilled in the art, produces cysteine mutein.Also the nucleic acid for encoding natural polypeptides can be changed by Standard site-directed mutagenesis method, make its encoding mutant albumen.The example of Standard mutagenesis techniques is referring to Kunkel, T.A., Proc.Nat.Acad.Sci., volume 82, the 488-492 pages (1985) and Kunkel, T.A. etc., Methods Enzymol., volume 154, the 367-382 pages (1987).

Introducing the potential site of non-natural cysteine includes the glycosylation site and N-terminal of polypeptide.In these examples, glycosyl donor can contain 1,2- amineothiots or 1,3- amineothiot.Glycosyl can be connected by those skilled in the art with the serine or threonine on activating agent.

Or, can be by the nucleic acid of technology chemical synthesis coding mutain well-known in the art.DNA synthesizer can be used, the instrument is commercially available, such as from AppliedBiosystems (Foster City, CA).The nucleic acid of mutain can be expressed in the various expression systems such as animal, insect and bacterial system needed for coding.After mutain needed for producing, those skilled in the art can carry out biologicall test to mutain and compare the activity of mutain and natural polypeptides.Even when the relative activity of mutain declines, the conjugate that mutain is formed can be particularly useful.For example, for not conjugated molecule, solubility increase, antigenicity reduction or the immunogenicity reduction of conjugate, or checkout time shortens in biosystem.

" polypeptide " and " protein " is used herein as synonym, refers to be substantially protein-based any compound in nature.However, peptide group can contain some non-peptide elements.For example, the protein of glycosylated polypeptides or synthetic modification is included in this definition.

Term " effective dose " used herein and " therapeutically effective amount " refer to the quantity or dosage of result needed for being enough to obtain when for describing that peptide is conjugated in bioactivator such as peptide, carrier-conjugated peptide or PEG-.For peptide B1 antagonists are conjugated in carrier-conjugated B1 peptides and/or PEG-, required result can be the required mitigation in such as inflammation and/or pain, or support the visible decline of B1 one or more biologically active levels.More particularly, therapeutically effective amount is the amount of the bioactivator for the pathologic process clinically defined for being enough to reduce, suppress or prevent the one or more and illness (such as inflammation or pain) of the patient through reagent interior therapeutic related within a period of time.Effective dose can be because of biological agent it is different and different, also depend on the order of severity of the related various factors of patient to be treated and situation and disease.For example, when giving biologically active conjugates in vivo, age, body weight and the health status of patient, and in preclinical animal research the factor such as the dose-effect curve that obtains and toxicity data all among consideration.If biologically active conjugates be in vitro with cells contacting, people will also design various preclinical in vitro studies to evaluate the parameters such as intake, half-life period, dosage, toxicity.The determination of the effective dose or therapeutically effective amount of given activating agent is the thing within the limit of power of those skilled in the art.

Term " pharmacological activity " refers to that described material is determined to have the activity of influence medical parameter or morbid state (such as pain).For carrier-conjugated B1 peptides of the present invention, the term typically refers to B1 inductions or B1 mediations disease, obstacle or abnormal medical illness, more particularly, refers to the antagonism of inflammation or pain.

Term " antagonist ", " inhibitor " and " inverse agonist " is (for example, see RianneA.F.de Ligt etc., British Journal of Pharmacology 2000,130,131) molecule for blocking, prevent, reduce, reducing or disturbing to a certain extent the bioactivity of the target protein of correlation is referred to.It is preferred that the present invention " B1 peptide antagonists " be that can combine and suppress B1 molecule, B1 activity external test in, its IC₅₀In below 500nM.Preferred B1 peptide antagonists of the present invention are the molecules of energy bind receptor, and its Ki is in below 100nM, and the molecule can suppress the function (such as calcium flux) of B1 mediations, its IC in the external test of B1 activity₅₀Less than 100nM.Most preferred B1 peptide antagonists of the present invention are to combine and suppress B1 molecule, in the external test of B1 activity, and its Ki is less than 10nM, IC₅₀In below 10nM.In addition, being measured at least one generally accepted internal animal models of pain, the molecule can prevent, improve or eliminate pain or inflammation；And/or biochemical attack can be suppressed in the animal model of oedema, inflammation or pain in vivo.

In addition, the peptide of the present invention or the physiologically acceptable salt of conjugated peptide are also included within herein.Term " physiologically acceptable salt " used herein and " pharmacologically acceptable salt " are used interchangeably, and are referred to include known or are then discovered that any salt as pharmaceutically acceptable (treatment that can be used to warm-blooded animal).Some specific examples are：Acetate；Halogen acid salt, such as hydrochloride and hydrobromate；Sulfate；Citrate；Tartrate；Oxyacetate；Oxalates；Inorganic acid salt and acylate, including but not limited to hydrochloride, hydrobromate, sulfate, phosphate, mesylate, esilate, malate, acetate, oxalates, tartrate, citrate, lactate, fumarate, succinate, maleate, salicylate, benzoate, phenylacetate, mandelate etc..When the compound of the present invention includes acidic functionality such as carboxyl, the suitable pharmaceutically acceptable cation of carboxyl is to being well known to the skilled person, including alkali metal, alkaline-earth metal, ammonium, quaternary ammonium cation etc..Other examples of " pharmacologically acceptable salt " see below with Berge etc., J.Pharm.Sci.66：1(1977).

" protection group " typically refers to well-known in the art for protecting selected active group (such as carboxyl, amino, hydroxyl, sulfydryl) in order to avoid undergoing the group of undesired reaction (being reacted such as nucleophilic, electrophilic, oxidation, reduction).When appropriate, protection group preferably is as shown here.The example of amino protecting group include but is not limited to aryl alkyl-, substituted aryl alkyl-, cycloalkenyl alkyl-and substituted cycloalkenyl-alkyl-, pi-allyl-, substituted allyl-, acyl group-, alkoxy carbonyl-, aryl-alkoxy carbonyl-, silicyl-etc..Aryl alkyl-example include but is not limited to benzyl-, adjacent methyl-benzyl-, trityl-and benzhydryl-(it can be optionally by following substituent group：Halogen, alkyl-, alkoxy-, hydroxyl-, nitro-, acyl amino-, acyl group-etc.) and salt (Li such as phosphonium salt and ammonium salt).The example of aryl include phenyl-, naphthyl-, indanyl-, anthryl-, 9- (9- phenylfluorenyls)-, phenanthryl-, durene base-etc..Cycloalkenyl alkyl-or replace cycloalkenyl alkyl-(preferably with 6-10 carbon atom) example include but is not limited to cyclohexenyl group-, methyl-etc..Suitable acyl group-, alkoxy carbonyl-and aromatic alkoxy carbonyl-include benzyloxycarbonyl-, tert-butoxycarbonyl-, isobutoxy carbonyl-, benzoyl-, substituted benzoyl-, bytyry-, acetyl group-, trifluoroacetyl group-, tribromo-acetyl base-, phthalyl-etc..Hybrid protection base can be used for protecting same amino, and such as primary amino radical can be protected with aralkyl and aromatic alkoxy carbonyl.The nitrogen that amino protecting group can be also connected with them constitutes heterocycle; such as 1; double (methylene) benzene of 2-, phthalimidyl-, succinimido-, dimaleoyl imino-etc., and wherein these heterocyclic groups can also comprising adjacent aryl-and cycloalkyl-ring.In addition, heterocyclic radical can be one, two or trisubstituted, for example nitro phthalimidyl-.Amino can be also protected by forming addition salts (such as hydrochloride, toluene fulfonate, trifluoroacetate), to exempt from undesired reaction, such as oxidation reaction.Many amino protecting groups be also suitable for protection carboxyl-, hydroxyl-and sulfydryl-.For example aralkyl-.Alkyl be also for protect hydroxyl-and sulfydryl-proper group, such as tert-butyl group.

Silicyl-protection group is the silicon atom optionally replaced by one or more alkyl, aryl and aralkyl.Suitable silyl-protecting groups include but is not limited to trimethyl silyl, triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl, dimethylphenylsilyl, 1; double (dimetylsilyl) benzene of 2-, double (dimetylsilyl) ethane of 1,2- and diphenylmethylsilyl.The silylated offer one-of amino or two-silicyl amino.The silylated of alkamine compound produces N, N, O- trimethylsilane radical derivatives.By using such as metal hydroxides or ammonium fluoride agent treatment in single reactions steps, or with carrying out in-situ treatment with such as metal hydroxides or fluorination ammonium reagent in alcohol radical course of reaction, silyl functional group easily can be sloughed from silyl ether functional group.Suitable monosilane agent is such as trimethylsilyl chloride, t-butyl-dimethylsilyl chlorine, phenyldimethylsilyl chlorine, diphenylmethylsilyl chlorine or their combination products with imidazoles or DMF.Silylated and silyl-protecting groups the deprotection method of amine is all well known to the skilled person.The method for preparing these amine derivatives from corresponding amino acid, amino acid amide or amino-acid ester is that organic chemistry (including amino acid/amino-acid ester or amino alcohol chemistry) art personnel are well-known.

Protection group is taken off under conditions of the remainder of molecule is not influenceed.These methods are well-known in the art, including acid hydrolysis, hydrogenolysis etc..It is preferred that method include for example by using palladium on carbon, in suitable solvent system such as alcohol, acetic acid or its mixture, slough the methods such as benzyloxycarbonyl through hydrogenolysis and slough protection group.Using inorganic acid or organic acids such as HCl or trifluoroacetic acid, in suitable solvent system such as dioxane or dichloromethane, t-butoxy-carbonyl protection group is sloughed.Gained amide can be easily neutralized, unhindered amina is obtained., can be with decarboxylize protection group, such as methyl, ethyl, benzyl, the tert-butyl group, 4- methoxyphenyl methyls under hydrolysis well known to the skilled person and hydrogenolytic conditions.The broader applications of protection group are referring to Theodora W.Green and Peter G.M.Wuts (1999), " Protective Groups in Organic Synthesis ", the 3rd edition, Wiley, New York, N.Y..

The present invention is the identification based on new chemical process; methods described provides and is used as excellent 1; 2- amineothiots or 1; the new support derivative of 3- amineothiot selective reagents; the selective reagent is used for having or having 1 through modification; the unprotected object (such as polypeptide, peptide or organic compound) of 2- amineothiots base or 1,3- amineothiot base is conjugated.The covalent bond formed to excellent specific reaction regioselectivity between support derivatives and 1, the 2- amineothiots of target activity agent or 1,3- amineothiot part.Reaction can almost be carried out completely under conditions of as mild as a dove.

Although reacting synthetic protein by the chemo-selective of the fragment containing cysteine and the fragment containing aldehyde has been described (Liu, C.-F.；Tam, J.P.J.Am.Chem.Soc.1994,116 .4149.Liu, C.-F.；Rao, C.；Tam, J.P.J.Am.Chem.Soc.1996,118 .307；Tam, J.P.；Miao, Z.J.Am.Chem.Soc.1999,121 .9013.Melnyk, O.；Fruchart, J.-S.；Grandjean, C.；Gras-Masse, H.J.Org.Chem.2001,66,4153), but chemical connection process as described herein is not yet used as making peptide, protein or organic compound and the conjugated method of carrier.

In one embodiment, the present invention carries out chemo-selective reaction to form the unique ability of thiazoline dependent on 1,2- amineothiots or 1,3- amineothiot with aldehyde.Once being formed, thiazoline nitrogen is kinetically tending to form amido link.This is ester carbonyl group 5- or the 6- atom removed by disposing from thiazoline nitrogen and completed.In addition, the new chemical reaction of the present invention generally produces a kind of primary product, required conjugate is set to be easy to purify, analyze and characterize.

The new chemical reagent and method of the present invention is especially effective in terms of the strategy of multiple peptide carrier conjugates (multi-peptide vehicle conjugate) is produced.For example, the reagent and method of the present invention are used to the B1 peptide antagonists containing 4 cysteines being effectively conjugated to side chain multivalence PEG polymer.Reagent as described herein and method are with multiple peptide PEG conjugates needed for effectively being produced with high-purity in high yield.Compared with the peptide conjugate with single peptide on each carrier, various multiple peptide PEG conjugates show activity increase (hB1 Ki=100pm, in some cases), circulating half-life significantly extends, PEG carrying capacity declines, and these allow substantially provide the acceptable dosage regimen of effect of bigger exposure and extension in vivo.For known not conjugated B1 peptide antagonists, carrier-conjugated B1 peptides provide greatly treatment advantage, available for disease, illness or the obstacle for treating and/or preventing B1 to mediate, including but not limited to inflammation and pain.

For previously known polymeric conjugation method, the use of new activated carrier derivative of the present invention in the methods of the invention brings numerous wondrous and unexpected advantage, more particularly to multivalence polymeric conjugation strategy (see, for example, PCT Publication WO95/06058, U.S. Patent Application Publication No. US 2003/0040127).

It should be known that term used herein is only used for describing the purpose of specific embodiment, limitation is not construed as, because the scope of the present invention is defined solely by the appended claims.

In view of for the purpose being conjugated with carrier and including but is not limited to new B1 combinations peptide antagonists disclosed herein and B1 peptide antagonists known in the art according to the bradykinin b 1 receptor binding peptide of mode described herein, any peptide included but is not limited to disclosed in any following publication (it is fully incorporated herein each via reference)：Regoli etc., Bradykininreceptors and their antagonists (bradykinin receptor and its antagonist) .Eur.J.ofPharma., 348：1-10(1998)；Neugebauer, W. etc., Kinin B₁Receptorsantagonists with multi-enzymatic resistance properties (have the kassinin kinin B of multienzyme resistance trait₁Receptor antagonist) .Can.J.Physiol.Pharmacol., 80：287-292(2002)；Stewart, J.M. etc., Bradykinin antagonists：Present progress andfuture prospects.Immunopharmacology (Anetic kinin antagons：Current progress and following prospect), 43：155-161(1999)；Stewart, J.M. etc., Metabolism-ResistantBradykinin antagonists：Development and Applications (metabolic resistance Anetic kinin antagons：Exploitation and application) .Biol.Chem., 382：37-41(2001)；PCT Publication WO98/07746 and WO 2005042027；U.S. Patent number 4,693,993,4,801,613,4,923,963,5,648,336,5,834,431,5,849,863,5,935,932,5,648,333,5,385,889,5,444,048 and 5,541,286.

" functionalized reagent " of the present invention applies to make the reagent of carrier functionalization of the present invention.

" functionalization " is to make the reaction of carrier functionalization according to the present invention.Functionalization can be made up of one or more steps.

Term " carrier " used herein refers to such molecule：For activating agent, it can delay degraded, increase half-life period, reduce toxicity, reduction immunogenicity and/or increase bioactivity.Carrier used in the present invention is known in the art, including but not limited to Fc areas, polyethylene glycol and glucan.Various carriers can be found in such as U.S. Patent No. 6,660,843, the PCT Application No. WO 99/25044 and WO 98/07746, Langer, R. that have announced, " Biomaterials in Drug Delivery, " 33ACC.CHEM.RES.94 (2000)；And Langer, R., " Tissue Engineering, " 1MOL.THER.12 (2000), Haisch, A. etc., Tissue Engineering of Human Cartilage Tissue, 44HNO 624 (1996)；Ershov, I.A. etc., Polymer Biocompatible X-Ray Contract Hydrogel, 2MED.TEKH.37 (1994)；Polous, I.M. etc., Use of A BiocompatibleAntimicrobial Polymer Film, 134VESTN.KHIR.IM.II GREK.55 (1985).Other examples of carrier include N- vinylpyrrolidones-methylmethacrylate copolymer,Perhaps (the Buron of polyamide -6 is also added,F. etc.,Biocompatable OsteoconductivePolymer,16CLIN.MATER.217(1994)),Poly- (DL- lactide-co-glycolides) (Isobe,M. etc.,Bone Morphogenic Protein Encapsulated with aBiodegradable and Biocompatible Polymer,32J.BIOMED.MATER.RES.433(1996)),Methyl methacrylate: HEMA is the mixture (Bar of 70: 30 ratios,F.W. etc.,New Biocompatable Polymer Surface Coating,52J.BIOMED.MATER.RES.193(2000)),(it is optionally with polyurethane for 2- methylacryoyloxyethyls phosphocholine,Iwasaki,Y. etc.,Semi-Interpenetrating PolymerNetworks...,52J.BIOMED.MATER.RES.701(2000)),Calcium alginate (high guluronic acid (guluronic acid) alginates of such as purifying,Becker,T.A. etc.,Calcium Alginate Gel,54J.BIOMED.MATER.RES.76(2001)),Protein polymer (such as Buchko,C.J. etc.,Surface Characterization of Porous,Biocompatible Protein Polymer Thin Films,22BIOMATERIALS 1289(2001)；Referring to Raudino, A. etc., Binding of Lipid Vescicles..., 231J.COLLOID.INTERFACE SCI.66 (2000)), polyvinylpyrrolidone, poly- Propylene Glycol, poly- hydroxyl-propane diols, polypropylene glycol and oxide, poly- methyl propanediol, poly- hydroxyl expoxy propane, straight chain and side chain polypropylene glycol, polyethylene glycol and polypropylene glycol and its monomethyl ether, one cetyl ether, one n-butyl ether, one tert-butyl group-ether and an oleyl ether, the ester and PAG of poly- aklylene glycol and carboxylic acid and the dehydrating condensation product and other polyalkylene oxides and PAG of amine, PVP, polyvinyl alcohol, poly- (vinyl acetate), copolymer is poly- (vinyl acetate -co- vinyl alcohol), Ju Yi Xi oxazolidones, it is poly- (oxazolidone of ethylene methyl -) and poly- (vinyl methyl ether), poly- (acrylic acid), poly- (methacrylic acid), poly-hydroxyethyl-methacrylate, it is poly- (acrylamide) and poly- (Methacrylamide), poly- NN- DMAAs), NIPA, poly- (N- acetamidoacrylamides) and poly- (other N- substitutive derivatives of N- acetamidomethacrylamides and these acid amides.

PEG is water miscible, non-immunogenic biocompatible materials.When used as supports, PEG generally give to connected reagent useful properties include solubility increase, the circulating half-life increase in blood flow, the resistance to protease and nuclease, immunogenicity decline etc..The PEG of macromolecule makes final conjugate be easy to separate with excessive not conjugated peptide and other a small amount of impurity.Therefore, when storing under controlled conditions, PEG conjugates are stable and are easy to be used for diagnostic assay.Although PEG polyether backbone is relative inertness in chemistry, the primary hydroxyl at its two ends is active, available for being directly connected to active material.These hydroxyls are generally converted to the higher functional group of activity, for purpose to be conjugated.

Term " activated carrier derivative ", " activated carrier ", " functionalised supports' derivative " and " functionalised supports " is used interchangeably herein, and refers to the carrier in the active group in end of at least one carrier segment.Equally, term " activated carrier section " and " functionalised supports' section " are used interchangeably herein, and refer to the carrier segment with one end active group.

PEG is water miscible, non-immunogenic biocompatible materials.When used as supports, PEG generally give to connected reagent useful properties include solubility increase, the circulating half-life increase in blood flow, the resistance to protease and nuclease, immunogenicity decline etc..The PEG of macromolecule makes final conjugate be easy to separate with excessive not conjugated peptide and other a small amount of impurity.Therefore, when storing under controlled conditions, PEG conjugates are stable and are easy to be used for diagnostic assay.Although PEG polyether backbone is relative inertness in chemistry, the primary hydroxyl at its two ends is active, available for being directly connected to active material.These hydroxyls are generally converted to the higher functional group's (" activating ") of activity, for purpose to be conjugated.

Term " carrier-conjugated activating agent " and " conjugated activating agent " are used interchangeably herein, refer to the conjugate comprising at least one activating agent and carrier, the carrier segment that the carrier is covalently attached in itself or with joint comprising at least one with activating agent, the joint includes but is not limited to peptidyl or non-peptidyl linker joint (such as aromatics joint), the joint and activating agent covalent bond.

In certain embodiments of the invention, " carrier-conjugated peptide " or " conjugated peptide " refer to the conjugate comprising peptide and carrier, wherein peptide has or has N- ends cysteine through modification, and carrier includes the carrier segment being covalently attached with the N- ends cysteine residues of at least one peptide.In other embodiments, conjugate includes at least one peptide and the carrier comprising at least one carrier segment, wherein carrier segment and non-peptidyl linker joint (including but is not limited to aromatics joint) covalent bond, and non-peptidyl linker joint is combined with the residue covalent of peptide.

In certain embodiments of the invention, " peptide is conjugated in PEG- " refers to the conjugate comprising at least one peptide and PEG, wherein peptide has or has N- ends cysteine through modification, and PEG includes the covalently bound PEG sections of N- ends cysteine residues with least one peptide.In other embodiments, conjugate includes at least one peptide and includes the PEG of at least one PEG section, wherein PEG sections and non-peptidyl linker joint (including but is not limited to aromatics joint) covalent bond, non-peptidyl linker joint is combined with the residue covalent of at least one peptide.

In another embodiment and combine up and down embodiment, be conjugated peptide include carrier, the carrier include with selected from SEQ ID NO：11-23 and 43-46 and the further covalently bound carrier segment of N- ends cysteine residues of peptide of the modification with N- ends cysteine.

In certain embodiments of the invention, the scope of the nominal average molecular weight of carrier is about 100 dalton to about 200, 000 dalton, or the scope of nominal average molecular weight is about 100 dalton to about 100, 000 dalton, or the scope of nominal average molecular weight is about 5, 000 dalton is to about 100, 000 dalton, or the scope of nominal average molecular weight is about 10, 000 dalton is to about 60, 000 dalton, or the scope of nominal average molecular weight is about 10, 000 dalton is to about 40, 000 dalton, or the scope of nominal average molecular weight is about 20, 000 dalton is to about 40, 000 dalton.

Active group on activated carrier can be can participate in linking together each component of required conjugate without substantially detrimental consequences reaction any amount of part.Non-limiting examples include acid, ester, mercaptan, amine or primary amine, but these are merely to the explanation present invention.Importantly, the covalent bond formed between carrier or carrier segment and any specified activity agent conjugated with it should be metastable.

Generally, activated carrier is straight chain, therefore is at most only capable of with Liang Ge functional groups (i.e. one end one).It will be apparent that conjugated quantity is limited to only two by this.In some instances it is preferred to the carrier with multiple active groups, for multiple actives to be connected with identical carrier molecule.The method of the present invention is remarkably contributing to design conjugation strategy, and the strategy provides relatively accurate number of functional groups on required multivalent carrier.

In specific embodiments of the present invention, carrier can be multivalent carrier molecule, including but not limited to straight chain carrier, the bifurcated carrier with more than one activated carrier section and the branch chain carrier with more than one activated carrier section of two ends activation.In certain embodiments of the invention, carrier can be multivalence PEG, including but not limited to straight chain carrier, the bifurcated PEG (fPEG) with more than one activated carrier section and the side chain PEG (bPEG) with more than one activated carrier section of two ends activation.

In the specific embodiment of the present invention, with carrier derived from amine or comprising multiple carrier segments and at least one is reacted with carrier and 1,2- derived from amine or 1,3- formyl base ester, carrier conjugates of the invention are produced.

Specific embodiment as described herein is not limit the scope of the invention.Really, as described above and accompanying drawing, various modifications of the invention in addition to described herein would is that obviously to those skilled in the art.Such modification is fallen under the scope of the hereto appended.

Embodiment

General experimental

NMR：The proton MR of the molecule containing PEG is referring to PEG unimodal (3.7ppm, the D relative to DSS₂O)。¹³C H NMR spectroscopies are referring to PEG unimodal (72.0ppm, the D relative to DSS₂O)。

Operated on Bruker Q-FTMS, at 7tesla, obtain FTMS data.Instrument carries out extrinsic calibration with PEG300/600 solution, uses standard Francel equations.The calculating quality of each demarcation ion and the error of measured value are less than 1.0ppm.For each spectrum, 512k data points are collected, sweep length (86Da mass cutoff) is detected with 1.25MHz.Man-hour numeric field data is not added with before magnitude mode Fourier conversion is carried out.

GC-MS data Hewlett-Packard GC-Ms, are recorded according to following parameter：

Post：J and W DB-XLB capillary columns, 30m × 0.25mm × 0.50 μM, PN1221236.

Method 1：

Syringe parameter：Injector temperature=250 DEG C；50: 1 split ratios；Helium flow speed=1ml/min.

GC parameters：Initial temperature=80 DEG C；0-2 minutes, it is maintained at 80 DEG C；It is to slowly warm up to 200 DEG C within 2-14 minutes；It is maintained at 200 DEG C up to 5 minutes.Releveling 0.5min.

Mass spectrum transmits temperature=280 DEG C.

Mass spectrometry parameters：From 50-550amu scannings, EI voltages=2376.5mV.

Method 2：

GC parameters：Initial temperature=140 DEG C；0-2 minutes, it is maintained at 140 DEG C；It is slowly increased to 320 DEG C within 2-11 minutes；It is maintained at 320 DEG C up to 1 minute.Releveling 0.5min.

Mass spectrum transmits temperature=280 DEG C.

GC parameters：From 50-550amu scannings, EI voltages=2376.5mV

Method 3：

GC parameters：Initial temperature=70 DEG C；0-2 minutes；90 DEG C are slowly increased to 10 DEG C per minute of speed；320 DEG C are slowly increased to 20 DEG C per minute of speed；It is maintained at 320 DEG C up to 4.5 minutes.Releveling 0.5min.

Mass spectrum transmits temperature=280 DEG C.

Mass spectrometry parameters：From 50-550amu scannings, EI voltages=2376.5mV

According to Stewart and Young (1984), " Solid Phase Peptide Synthesis " are described, with the tactful synthetic peptides of standard FMOC.The chemist in peptide symthesis field can synthesize the peptide by manually or automatically solid phase method.

By HPLC and chemiluminescence detection (CLND), peptide content is determined：

Solvent system：A=0.04%TFA/ water, B=0.04%TFA/90% methanol.

Post：Jupiter C18 30050 × 2.0mm posts, 5 μm of particle diameters.

CLND：Antek 8060,1048 DEG C of oven temperature, detector is run at high sensitivity and decay 1.

HPLC：HP1100LC, PDAD

Gradient：10%B to 100%B, in 10min, keeps 2min, releveling 4min.

Flow velocity and shunting：Overall flow rate is 0.3ml/min, and it is shunted in CLND and salvage department about 2: 1 ratio with three-way pipe.

Preparative reversed-phase HPLC：

System：Two Agilent series 1100prep pumps, Agilent series 1100prep automatic injectors, Rheodyne hand guns simultaneously carry 5-20ml sample loops, Agilent 1100 multiwavelength detectors of series (being set in 215nm and 254nm) and the serial 1100 automatic fraction collectors of Agilent.

Software：Agilent Chemstation.

Solvent system：

1：A=10mM ammonium formates/water (pH=3.75)；B=acetonitriles.

2：A=0.1% acetic acid/waters, B=0.1% acetic acid/acetonitrile.

3：A=10mM ammonium hydrogen carbonate (pH 10)/water；B=acetonitriles.

Post：

1：Waters Xterra Prep C18 MS (are packed) by Vydac/The Separations Group, and 50mm × 300mm (PN PA0000-050730), 10 μm of particle diameters are spherical.

2：30 × 100mm Waters Xterra Prep C18 OBD, 100

Aperture, 5 μm of particle diameters, spherical, PN 186001942.

Gradient table：

1

Time (min)	%B	Flow velocity (ml/min)
Time (min)	%B	Flow velocity (ml/min)	0	25	20
4	25	20	0	25	20
4	25	20	5	25	100
25	55	100	5	25	100
25	55	100	35	55	100
35.1	100	100	35	55	100
35.1	100	100	49.9	100	100
50	25	100	49.9	100	100
50	25	100	60	25	100
60.1 terminate			60	25	100

2

Time (min)	%B	Flow velocity (ml/min)
Time (min)	%B	Flow velocity (ml/min)	0	25	35
5	25	35	0	25	35
5	25	35	20	55	35

24.9	55	35
24.9	55	35	24.95	100	35
29.9	100	35	24.95	100	35
29.9	100	35	29.95	25	35
40 terminate			29.95	25	35

3

Time (min)	%B	Flow velocity (ml/min)
Time (min)	%B	Flow velocity (ml/min)	0	10	35
5	10	35	0	10	35
5	10	35	20	25	35
24.9	25	35	20	25	35
24.9	25	35	24.95	100	35
29.9	100	35	24.95	100	35
29.9	100	35	29.95	10	35
40 terminate			29.95	10	35

4

Time (min)	%B	Flow velocity (ml/min)
Time (min)	%B	Flow velocity (ml/min)	0	70	20
4	70	20	0	70	20
4	70	20	5	70	100
25	100	100	5	70	100
25	100	100	35	100	100
49.9	100	100	35	100	100
49.9	100	100	50	70	100
60	70	100	50	70	100
60	70	100	60.1 terminate

Preparative cation exchange LC：

System and software：It is identical with preparation HPLC.

Solvent：

1：A=10mM boric acid/5: 40: 55MeOH- acetonitrile-water；B=A+0.2M KCl.

Post：

1：43382,20 μm of particles of Tosoh Bioscience TSKGel SP-5PW-HR, PN load 50 × 250mm glass columns (Hodge Bioseparations Ltd.P/N=TAC50/250S2-SR-1).Detect bed length=180mm.

2：21.5 × 150mm TSK Gel SP-5PW, PN 07575.

Gradient table：

1

Time (min)	%B	Flow velocity (ml/min)
Time (min)	%B	Flow velocity (ml/min)	0	0	10
2	0	10	0	0	10
2	0	10	5	0	30
25	0	30	5	0	30
25	0	30	25.1	20	30
80	80	30	25.1	20	30
80	80	30	80.1	100	30
110	100	30	80.1	100	30
110	100	30	110.1	0	30
130 terminate			110.1	0	30

2

Time (min)	%B	Flow velocity (ml/min)
Time (min)	%B	Flow velocity (ml/min)	0	0	10
5	0	10	0	0	10
5	0	10	30	100	10
45	100	10	30	100	10
45	100	10	45.05	0	10
60 terminate			45.05	0	10

Experimental section

Flow 1

Reagent and condition：a)BBr₃, -78 DEG C, CH₂Cl₂；B) TBDMSCl, DMF, DIPEA, rt；c)CH₂Cl₂, carbonyl dimidazoles, rt；d)CH₃OH, DCE, MW, 100 DEG C, 2min.；E) NBS, AIBN, CCl₄, backflow；f)AgNO₃, H₂O, i-PrOH, rt, then TBAF, DCM；G) 2- benzyl acetate bromides, K₂CO₃, acetone, 0 DEG C；H) 2,6- di-tert-butyl pyridines, 1,2- double (trimethylsiloxy) ethane, trimethylsilyl triflate, 2- pyridylcarbinols, CH₂Cl₂, 0 DEG C；i)H₂, Pd/C, EtOAc；J) n-hydroxysuccinimide, PS- carbodiimides (Argonaut technologies), EtOAc.

4- hydroxy-2-methylbenzoic acids (2).4- methoxyl group -2- methyl benzoic acids (1) (5.0g, 30.08mmol) and CH are added into flame-dried 3 neck round-bottom flasks of 250ml₂Cl₂(80ml).Reactant is cooled to -78 DEG C, and pure BBr is added dropwise by charging hopper₃(5.7ml, 60.17mmol) is handled.Reactant stirs 30min at -78 DEG C.Solution temperature is risen to -15 DEG C and 4h (- 15 DEG C to -10 DEG C) is stirred.Remove cooling bath.20h is stirred at room temperature in reactant.Solution is cooled to 0 DEG C, (noted with ether (15ml) and water (15ml)：Water induces a fierce reaction；Water is added dropwise) quenching.Two-phase mixture is extracted with EtOAc (3 × 100ml).The organic layer of merging is through MgSO₄Dry, filter and be concentrated in vacuo.Crude product passes through SiO₂Chromatogram purification (300g SiO₂, 70: 30 hexane-acetones, R_f=0.31), obtain title compound.APCI MS(m/z)：151.12(M-H)；C₈H₈O₃Calculated value：152.15.¹H NMR (300MHz, chloroform-d) δ ppm2.55 (s, 3H) 6.29-6.78 (m, 2H) 7.90 (d, J=9.42Hz, 1H).

4- (t-butyldimethylsilyloxy base) -2- methyl benzoic acids (3).T-BDMSCl (10.2g, 67.63mmol) is added into DMF (20ml) agitating solution of 4- hydroxy-2-methylbenzoic acids (2) (4.2g, 27.60mmol) and 15min is stirred.Anhydrous i-Pr is added dropwise by addition funnel₂NEt (14.0ml, 80.05mmol), and 20h is stirred at room temperature.Reactant 1MH₃PO₄(7ml) is quenched, until end pH is 3-4.Solution is extracted with hexane (4 × 100ml).The organic layer of merging is through MgSO₄Dry, filter and be concentrated in vacuo.Crude product SiO₂Chromatogram purification (300g SiO₂, 90: 9: 1 hexane-acetone-AcOH, R_f=0.28), obtain title compound.APCI MS(m/z)：267.15(M+H)；C₁₄H₂₂O₃Si calculated value：266.13.¹HNMR (300MHz, chloroform-d) δ ppm 0.24 (s, 6H) 0.99 (s, 9H) 2.61 (s, 3H) 6.61-6.78 (m, 2H) 7.92-8.06 (m, 1H).

(4- (t-butyldimethylsilyloxy base) -2- aminomethyl phenyls) (1H- imidazole radicals -1- bases) ketone (4).4- (t-butyldimethylsilyloxy base) -2- methyl benzoic acids (3) (5.8g, 21.77mmol) are dissolved in CH₂Cl₂(50ml), in N₂, at room temperature with 1,1 '-carbonyl dimidazoles (4.2g, 26.12mmol) handle 20h.Solution CH₂Cl₂(50ml) dilutes.Organic layer is washed with water (2 × 50ml), salt solution (2 × 30ml), through MgSO₄Dry, filter and be concentrated in vacuo, obtain title compound (70: 29: 1 hexane-acetone-NEt₃, R_f=0.14).APCI MS(m/z)：317.15(M+H)；C₁₇H₂₄N₂O₃Si calculated value：316.47.¹(the s of H NMR (300MHz, chloroform-d) δ ppm 0.25 (s, 6H) 1.00,9H) 2.39 (s, 3H) 6.75 (dd, J=8.38,2.17Hz, 1H) 6.81 (d, J=1.88Hz, 1H) 7.13 (s, 1H) 7.33 (d, J=8.29Hz, 1H) 7.47 (s, 1H) 7.92 (s, 1H).

4- (t-butyldimethylsilyloxy base) -2- methyl benzoic acids¹³C- methyl esters (5).Into the 20ml Conical Smith composite tubes of oven dried add (4- (t-butyldimethylsilyloxy base) -2- aminomethyl phenyls) (1H- imidazole radicals -1- bases) ketone (4) (5.5g, 17.38mmol), DCE (10ml),¹³CH₃OH (Cambridge Isotope Laboratory, 2.2ml, 52.13mmol) and DBU (0.8ml, 5.21mmol).2min is heated by the synthesis seal of tube and with Smith synthesizers in 100 DEG C of microwaves.Reactant is through being concentrated in vacuo.Crude product SiO₂Chromatogram purification (300gSiO₂, 95: 5 hexane-acetones, R_f=0.65), obtain title compound.APCI MS(m/z)：282.5(M+H)；C₁₄ ¹³CH₂₄O₃Si calculated value：281.15.¹(the s of H NMR (300MHz, chloroform-d) δ ppm 0.22 (s, 6H) 0.98 (s, 9H) 2.56,3H) 3.85 (d, J=146.75Hz, 3H) 6.62-6.74 (m, 2H) 7.86 (d, J=8.85Hz, 1H).

4- (t-butyldimethylsilyloxy base) -2- (two bromomethyls) benzoic acid¹³C- methyl esters (6).To 4- (t-butyldimethylsilyloxy base) -2- methyl benzoic acids¹³The CCl of C- methyl esters (5) (4.0g, 14.21mmol)₄N-bromosuccinimide (7.6g, 42.64mmol) and 2,2 '-azodiisobutyronitrile (2.3g, 14.21mmol) are added in (50ml) agitating solution.In N₂It is middle that reactant is heated to reflux (83 DEG C) 18h.Reactant is cooled to room temperature and filtered.Vacuum filter removes solvent.Crude product SiO₂Chromatogram purification (300g SiO₂, 90: 10 hexane-acetones, R_f=0.78), obtain title compound.APCI MS(m/z)：440.2(M+H)；C₁₄ ¹³CH₂₂O₃Si calculated value：439.22.¹(the s of H NMR (300MHz, chloroform-d) δ ppm 0.28 (s, 6H) 1.01,9H) 3.90 (d, J=147.31Hz, 3H) 6.80 (dd, J=8.67,2.45Hz, 1H) 7.58 (d, J=2.45Hz, 1H) 7.83 (d, J=8.67Hz, 1H) 8.10 (s, 1H).

2- formoxyls -4-HBA¹³C- methyl esters (7).To 4- (t-butyldimethylsilyloxy base) -2- (two bromomethyls) benzoic acid¹³Water (6ml) solution of silver nitrate (3.86g, 22.77mmol) is added in i-PrOH (60ml) agitating solution of C- methyl esters (6) (5.0g, 11.38mmol).Gained mixture is in N₂Lower stirring 20h.Reactant is filtered, filtrate is through being concentrated in vacuo.Residue is dissolved in CH₂Cl₂, through MgSO₄Dry, filter and handled with the THF (6.6ml, 22.77mmol) of 1M tetra-n-butyl ammonium fluorides.In N₂Middle to cross after 3h, reactant is through being concentrated in vacuo.Crude product SiO₂Chromatogram purification (120g SiO₂, 80: 20 hexane-acetones, R_f=0.33), obtain title compound.APCI MS(m/z)：182.2(M+H)；C₈ ¹³CH₈O₄Calculated value：181.05.¹(the dd of HNMR (300MHz, chloroform-d) δ ppm 3.95 (d, J=147.50Hz, 3H) 7.09, J=8.57,2.73Hz, 1H) 7.40 (d, J=2.83Hz, 1H) 7.98 (d, J=8.48Hz, 1H) 10.69 (s, 1H).

4- (2- (benzyloxy) -2- oxoethoxies) -2- formylbenzoates¹³C- methyl esters (8).By 2- formoxyls -4-HBA¹³C- methyl esters (7) (1.55g, 8.56mmol) is dissolved in acetone (20ml) and is cooled to 0 DEG C.Add 2- benzyl acetate bromides (1.9ml, 11.97mmol) and potassium carbonate (1.4g, 10.27mmol).Reactant is in N₂Under in 0 DEG C stir 18h.Reactant is quenched with water (5ml), and solvent is removed in vacuum.Residue is distributed between EtOAc (100ml) and water (40ml).Each layer is separated, organic layer is washed with water (2 × 20ml), salt solution (1 × 20ml), through MgSO₄Dry, filter and be concentrated in vacuo.Crude product SiO₂Chromatogram purification (120g SiO₂, 85: 15 hexane-acetones, R_f=0.35), obtain title compound.APCI MS(m/z)：330.1(M+H)；C₁₇ ¹³CH₁₆O₆Calculated value：329.09.¹H NMR (300MHz, chloroform-d) δ ppm 3.95 (d, J=147.69Hz, 3H) 4.77 (s, 2H) 5.25 (s, 2H) 7.15 (dd, J=8.67,2.64Hz, 1H) 7.32-7.43 (m, 6H) 7.98 (d, J=8.67Hz, 1H) 10.68 (s, 1H).

4- (2- (benzyloxy) -2- oxoethoxies) -2- (DOX -2- bases) benzoic acid¹³C- methyl esters (9).By 4- (2- (benzyloxy) -2- oxoethoxies) -2- formylbenzoates¹³C- methyl esters (8) (2.17g, 6.6mmol) is dissolved in CH₂Cl₂(20ml) and it is cooled to 0 DEG C.Add 2, double (trimethylsiloxy) ethane (2.4ml of 6- di-t-butyls pyridine (0.150ml, 0.66mmol), 1,2-, 9.88mmol) and trimethylsilyl triflate (0.180ml, 0.98mmol).Reactant is in N₂Under in 0 DEG C stir 18h.Solution is quenched with 2- pyridylcarbinols (0.127ml, 1.32mmol).Solvent is removed in vacuum.Crude product SiO₂Chromatogram purification (120g SiO₂, 80: 20 hexane-acetones, R_f=0.22), obtain title compound.APCI MS(m/z)：374.1(M+H)；C₁₉ ¹³CH₂₀O₇Calculated value：373.12.¹H NMR (300MHz, chloroform-d) δ ppm 3.88 (d, J=147.12Hz, the 3H) (s of 3.97-4.06 (m, J=2.26Hz, 4H) 4.73, the 2H) (dd of 5.24 (s, 2H) 6.65 (s, 1H) 6.88, J=8.67,2.83Hz, 1H) 7.30 (d, J=2.83Hz, 1H) 7.35 (s, 5H) 7.91 (d, J=8.67Hz, 1H).

2- (3- (DOX -2- bases) -4- (¹³C- methoxycarbonyls) phenoxy group) acetic acid (10).To 4- (2- (benzyloxy) -2- oxoethoxies) -2- (DOX -2- bases) benzoic acid¹³10% is added in EtOAc (25ml) agitating solution of C- methyl esters (9) (1.72g, 4.6mmol) and drapes over one's shoulders target carbon (170mg).Solution is deaerated with 3 evacuations/nitrogen filling circulation.After last time is evacuated, with the H from balloon₂Evacuated to backfill last time.Reactant is at room temperature in H₂Middle stirring 3h.Reactant is filtered by Celite pad.Solvent is removed in vacuum from filtrate, title compound (60: 40 hexane-acetones, R are obtained_f=0.11).APCI MS(m/z)：284.3(M+H)；C₁₂ ¹³CH₁₄O₇Calculated value：283.08.¹H NMR (300MHz, chloroform-d) δ ppm 3.89 (d, J=147.12Hz, the 3H) (s of 4.06 (s, 4H) 4.75 (s, 2H) 6.65,1H) 6.92 (dd, J=8.76,2.73Hz, 1H) 7.34 (d, J=2.83Hz, 1H) 7.94 (d, J=8.67Hz, 1H).

4- (N- (succinimide epoxide) -2- oxoethoxies) -2- (DOX -2- bases) benzoic acid¹³C- methyl esters (11).To 2- (3- (DOX -2- bases) -4- (¹³C- methoxycarbonyls) phenoxy group) acetic acid (10) (1.21g, 1- hydroxyl pyrrolidines -2 are added in EtOAc (20ml) solution 4.27mmol), 5- diketone (0.74g, 6.41mmol) with PS- carbodiimides (ArgonuntTechnology, 1.29mmol/g) (4.6g, 5.98mmol).Reactant is sealed and 20h is stirred at room temperature.The sintered glass funnel filtering of solution intermediate pore.N is passed through by sintered glass₂10min, resin is stirred together with EtOAc (20ml).EtOAc is filtered, is merged with initial filtrate.Resin is washed for the second time with same scheme.The filtrate of merging is through being concentrated in vacuo.Crude product SiO₂Chromatogram purification (120g SiO₂, 70: 29: 1 hexane-acetone-AcOH, R_f=0.14), obtain title compound.APCI MS(m/z)：381.2(M+H)；C₁₆ ¹³CH₁₇NO₉Calculated value：380.09.¹(the d of H NMR (300MHz, chloroform-d) δ ppm2.87 (s, 4H) 3.89, J=147.12Hz, 3H) 4.02-4.10 (m, J=1.70Hz, the 4H) (dd of 5.04 (s, 2H) 6.67 (s, 1H) 6.95, J=8.67,2.83Hz, 1H) 7.35 (d, J=2.83Hz, 1H) 7.95 (d, J=8.67Hz, 1H).

Reagent and condition：A) n-BuLi, then methylchloroformate；B) toluene, 170 DEG C；C) benzyl acetate bromide, K₂CO₃, acetone；d)H₂, Pd/C, EtOAc.

4,4- diethoxy butyl- 2- ynoic acid methyl esters (13).By diethylene glycol (DEG)-dimethyl ether (100ml) solution of diethoxy propine (Aldrich, 10.93g, 85.3mmol) in N₂Under be cooled to -30 DEG C.N-BuLi (81.0mmol) is added dropwise in 5min.Reactant is incubated 6h.The anion formed is passed through in 50ml diethylene glycol (DEG)s-dimethyl ether solution of methylchloroformate (6.5ml, 84.1mmol) through conduit, while in N₂Under, the top stirring (overhead stir) in dry ice/acetone batch.Reactant is warmed to room temperature overnight.Solid is filtered to remove by aluminum oxide pad (100g alkali aluminas are cleaned with 200ml ether).(bath temperature=35 DEG C) are fully concentrated solution by rotary evaporation.Solid is filtered to remove by aluminum oxide pad (being cleaned with 500ml ether, 100g alkali aluminas).(bath temperature=35 DEG C) are fully concentrated solution by rotary evaporation.Product obtains title compound by distillation purifying (cut seethes with excitement under 57-60 DEG C, 1mm Hg).¹H NMR (300MHz, chloroform-d) δ ppm：1.24 (d, J=14.32Hz, 6H) 3.56-3.68 (m, 2H) 3.68-3.83 (m, 2H) 3.79 (s, 3H) 5.36 (s, 6H).GC-MS：Method 1：4.22min (EI MS (m/z)=141 (M-OEt)；C₇H₉O₃ ⁺Calculated value：141).

2- (diethoxymethyl) -4-HBA methyl esters (16).4 are added into the 5mlConical Smith composite tubes of oven dried, 4- diethoxy butyl- 2- ynoic acid methyl esters (0.25g, 1.3mmol) (13), (E)-(4- methoxyl groups butyl- 1,3- diene -2- bases epoxide) trimethyl silane (0.52ml, 2.7mmol) (12), 4- (3,5- di-tert-butyl-4-hydroxyl benzyls) -2,6- DI-tert-butylphenol compounds (0.11g, 0.27mmol) and toluene (4ml).20h is heated by the synthesis seal of tube and at 170 DEG C.Reactant is cooled to room temperature, is transferred in round-bottomed flask, is handled with the THF (0.78ml, 2.7mmol) of 1M tetra-n-butyl ammonium fluorides.Solution is sealed, 3h is stirred at room temperature.Solvent is removed in vacuum.Crude product SiO₂Chromatogram purification (40g SiO₂, 80: 20 hexane-acetones, R_f=0.42), obtain title compound.APCI MS(m/z)：255.2(M+H)；C₁₃H₁₈O₅Calculated value：254.12.¹H NMR (300MHz, chloroform-d) δ ppm 1.23 (t, J=7.06Hz, 6H) 3.53-3.66 (m, 2H) 3.66-3.78 (m, 2H) 3.87 (s, the 3H) (dd of 5.59 (s, 1H) 6.26 (s, 1H) 6.80, J=8.57,2.73Hz, 1H) 7.30 (d, J=2.64Hz, 1H) 7.82 (d, J=8.67Hz, 1H).

4- (2- (benzyloxy) -2- oxoethoxies) -2- (diethoxymethyl) methyl benzoate (17).To 0 DEG C of 2- (diethoxymethyl) -4-HBA methyl esters (0.5g, 2- benzyl acetate bromides (0.4ml 2mmol) is added in acetone (15ml) agitating solution of (16), 3mmol) with potassium carbonate (0.3g, 2mmol).Solution is in N₂Under in 0 DEG C stir 20h.Solution is quenched with water (5ml), is concentrated in vacuo solvent.Residue is distributed between EtOAc (75ml) and water (30ml).Each layer is separated, organic layer is washed with water (2 × 20ml), salt solution (1 × 20ml), through MgSO₄Dry, filter and be concentrated in vacuo.Crude product SiO₂Chromatogram purification (40g SiO₂, 85: 15 hexane-acetones, R_f=0.35), obtain title compound.APCI MS(m/z)：255.2(M-EtOH).C₂₂H₂₆O₇Calculated value：402.17.¹H NMR (300MHz, chloroform-d) δ ppm 1.21 (t, J=6.97Hz, 6H) 3.53-3.60 (m, 2H) 3.62-3.74 (m, 2H) 3.87 (s, the 3H) (s of 4.73 (s, 2H) 5.24 (s, 2H) 6.22,1H) 6.86 (dd, J=8.67,2.64Hz, 1H) 7.35 (s, 6H) 7.83 (d, J=8.67Hz, 1H).

2- (3- (diethoxymethyl) -4- (methoxycarbonyl) phenoxy group) acetic acid (18).To 4- (2- (benzyloxy) -2- oxoethoxies) -2- (diethoxymethyl) methyl benzoates (0.65g, 1.6mmol) palladium (0.052g, 0.48mmol) is added in EtOAc (15ml) agitating solution of (17).Solution is deaerated with 3 evacuations/nitrogen filling circulation.After last time is evacuated, with the H from balloon₂Evacuated to backfill last time.Reactant is at room temperature in H₂Middle stirring 3h.Reactant is filtered by Celite pad.Solvent is removed in vacuum, title compound (70: 29: 1 hexane-acetone-AcOH, R are obtained_f=0.21).APCI MS(m/z)：311.1(M-H).C₁₅H₁₉O₇Calculated value：311.1.¹H NMR (300MHz, chloroform-d) δ ppm 1.22 (t, J=6.97Hz, 6H) 3.51-3.64 (m, 2H) 3.64-3.78 (m, 2H) 3.88 (s, the 3H) (dd of 4.74 (s, 2H) 6.24 (s, 1H) 6.90, J=8.67,2.83Hz, 1H) 7.36 (d, J=2.64Hz, 1H) 7.86 (d, J=8.67Hz, 1H).

4- (N- (succinimide epoxide) -2- oxoethoxies) -2- (DOX -2- bases) methyl benzoate (19).To 2- (3- (diethoxymethyl) -4- (methoxycarbonyl) phenoxy group) acetic acid (450mg, n-hydroxysuccinimide (248mg 1.44mmol) is added in EtOAc (15ml) agitating solution of (18), 2.16mmol) with PS- carbodiimides (Argonaunt Technology, 1.29mmol/g) (1.5g, 2.02mmol).Reactant is sealed and 20h is stirred at room temperature.The sintered glass funnel filtering of solution intermediate pore.N is passed through by sintered glass₂10min, resin is stirred together with EtOAc (20ml).EtOAc is filtered, is merged with initial filtrate.Resin is washed for the second time with same scheme.The filtrate of merging is through being concentrated in vacuo.Crude product SiO₂Chromatogram purification (40g SiO₂, 80: 19: 1 hexane-acetone-AcOH, R_f=0.38), obtain title compound.APCI MS(m/z)：364.23(M+H-OEt).C₁₇H₁₈NO₈ ⁺Calculated value：364.1.¹H NMR (300MHz, chloroform-d) ppm 1.23 (t, J=7.16Hz, the 6H) (s of 2.87 (s, 4H) 3.54-3.76 (m, 4H) 3.87, the 3H) (dd of 5.03 (s, 2H) 6.23 (s, 1H) 6.91, J=8.67,2.83Hz, 1H) 7.40 (d, J=2.64Hz, 1H) 7.86 (d, J=8.67Hz, 1H).

Flow 3

Reagent and condition：A) acetonitrile, 25 DEG C；B) DCl, D₂O。

Four-[ω-(4- azepine -5- oxo -7- oxa-s -7- ((3- (2,4- dioxolyl) -4- (¹³C- methoxyl groups) carbonyl) benzene) heptane) -2.5kD polyoxyethylene] methane 22.PTE-100PA (NOFcorp, 547mg ,~52 μm of ol) is dissolved in 2.5ml anhydrous acetonitriles, handled with succinate 11 (100mg, 260 μm of ol, 5 equivalents).Reactant is heated to 40 DEG C of 7h.Reactant is cooled to room temperature, handled with 10mM ammonium formates (10ml).Solution is loaded to post 1, eluted according to the gradient table 1 of solvent system 1/ specified by the preparative reversed-phase HPLC part of General experimental.The spectra separating eluted for 27.4-28.8 minutes is come out, is concentrated in vacuo to remove acetonitrile.The 2560g aqueous solution is filtered by 0.22 μm of centrifugal filter (National Scientific, PN 66064-466), filtrate is then freezed.Solid is dissolved in 5ml D₂O is simultaneously freezed, and obtains product.¹H NMR (400MHz, deuterium oxide) δ ppm 1.78 (p, J=6.65Hz, 2H) 3.35 (t, J=6.46Hz, 2H) 3.45 (t, J=6.26Hz, 2H) 3.48-3.52 (m, 2H) 3.70 (s, (CH₂CH₂O)_n) 3.90 (d, J=149.07Hz, 3H) 4.09-4.16 (m, the 4H) (dd of 4.71 (s, 2H) 6.51 (s, 1H) 7.12, J=8.61,2.74Hz, 1H) 7.31 (d, J=2.74Hz, 1H) 7.97 (d, J=8.61Hz, 1H) 8.40 (s, 1H).¹³C NMR (101MHz, deuterium oxide) δ ppm55.08 (s, 4C), 72.00 (s, 5C).

Four-[ω-(4- azepine -5- oxo -7- oxa-s -7- ((3- (2,4- dioxolyl) -4- (¹³C- methoxyl groups) carbonyl) benzene) heptane) -5.0kD polyoxyethylene] methane 23.By PTE-200PA (NOFcorp, 1.55g ,~64 μm of ol；Analysis certificate：83% tetrafunctional), succinate 11 (147mg, 386 μm of ol) and 5ml acetonitriles be heated to 40 DEG C of 4h.Acetonitrile is removed, the acetic acid of 5ml 0.1% is added.Solution is heated to 35 DEG C with hydrotropy.Solution is loaded to post 1 (column sleeve and solvent are heated to 35 DEG C), eluted according to the gradient table 1 of solvent system 2/ specified by the preparative reversed-phase HPLC part of General experimental.The bands of a spectrum eluted from 22.8-26min are dried under reduced pressure (1mm Hg) through being concentrated in vacuo at 35 DEG C.Residue is dissolved in 10ml D₂O is simultaneously freezed, and obtains product.Through¹H NMR are determined, and gained solid is 23 and 25 6: 1 mixtures；¹H NMR are used for 23.¹H NMR (400MHz, deuterium oxide) δ ppm 1.78 (p, J=6.06Hz, 2H) 3.35 (t, J=6.65Hz, 2H) 3.45 (t, J=6.26Hz, 2H) 3.50 (s, 2H) 3.70 (s, (CH₂CH₂O)_n) 3.90 (d, J=147.12Hz, 3H) 4.09-4.16 (m, 4H) 4.71 (s, 2H) 6.51 (s, 1H) 7.12 (dd, J=8.61,2.74Hz, 1H) 7.31 (d, J=2.74Hz, 1H) 7.98 (d, J=9.00Hz, 1H).¹³C NMR (101MHz, deuterium oxide) δ ppm 55.08 (s, 9.98C) 72.00 (s, 2.84C).

Four-[ω-(4- azepine -5- oxo -7- oxa-s -7- ((3- formoxyls -4- (¹³C- methoxyl groups) carbonyl) benzene) heptane) -2.5kD polyoxyethylene] methane 24.PEG reagents 22 (439mg, 38.3 μm of ol) are dissolved in 5ml D₂O, is cooled to 0 DEG C and is deaerated by 4 evacuations/nitrogen filling circulation.Add 85mM DCl/D₂O solution (360 μ l, 0.2 equivalent/acetal).Cooling bath is removed, 24h is stirred at room temperature in reactant.After 24h, another part DCl (360 μ l) is added.Reactant is stirred into 63h.2ml D are re-dissolved in after the lyophilized aqueous solution₂In O.Solution is filtered and freezed by 0.1 μm of centrifugal filter (Micron Bioseparations, PN UFC40W00), obtains product.¹H NMR (400MHz, deuterium oxide) δ ppm 1.80 (p, J=6.10Hz, 2H) 3.36 (t, J=6.46Hz, 2H) 3.45-3.54 (m, 4H) 3.70 (s, (CH₂CH₂O)_n) 3.96 (d, J=148.68Hz, 3H) 4.72 (s, 2H), 7.32 (d, J=9.00Hz, 1H) 7.38 (s, 1H) 8.01 (d, J=8.61Hz, 1H) 8.26 (s, 1H) 10.42 (s, 1H).

Four-[ω-(4- azepine -5- oxo -7- oxa-s -7- ((3- formoxyls -4- (¹³C- methoxyl groups) carbonyl) benzene) heptane) -5.0kD polyoxyethylene] methane 25.PEG reagents 23 (840mg, 39 μm of ol) are dissolved in 10ml H₂O, is cooled to 0 DEG C and uses 85mM DCl/D₂O (183 μ l, 15.6 μm of ol, 0.1 equivalent/acetal) processing.After 4.5 days, reactant is freezed, 10ml D are dissolved in₂O simultaneously uses 85mM DCl/D₂O (183 μ l, 15.6 μm of ol) is handled 1 day at room temperature.Solutions in Freeze-drying, obtains product.¹H NMR (400MHz, deuterium oxide) δ ppm 1.79 (p, J=6.31Hz, 2H) 3.36 (t, J=6.65Hz, 2H) 3.43-3.55 (m, 4H) 3.70 (s, (CH₂CH₂O)_n) 3.96 (d, J=148.68Hz, 3H) 4.74 (s, 2H), 7.34 (dd, J=8.61,2.74Hz, 1H) 7.42 (d, J=2.74Hz, 1H) 8.03 (d, J=8.61Hz, 1H) 10.44 (s, 1H).

Flow 4

Reagent and condition：600mM LiCl, pH 2.5-6 ascorbate buffers.

Four-[ω-(4- azepine -5- oxo -7- oxa-s -7- (((3 ' R, 9 ' bS) -3 '-(carbonyl (HN-GGGGGKKRP (Hyp) G (Cpg) S (D-Tic) (Cpg)-OH)) -2 ', 3 '-thiazoline simultaneously [2 ', 3 '-a] iso-indoles -5 ' (9 ' bH) -one -8 '-yl)) heptane) -2.5kD polyoxyethylene] methane 27.Peptide 26 (1.12g, PPL laboratories) is dissolved in 1.8ml D₂O, with 0.25ml 0.50M sodium ascorbates/4.8M LiCl D₂O processing, is subsequently cooled to 0 DEG C.Solution is deaerated by 3 evacuations/nitrogen filling circulation.Under a nitrogen, pH is adjusted to 6.1 with 1M LiOH, then deaerated by 3 evacuations/nitrogen filling circulation.According to described in General Experimentation Section, using HPLC, (CLND) is detected with the chemiluminescence nitrogen demarcated for caffeine, measured peptide concentration is 114.4mM.PEG reagents 24 (400mg, 35.4 μm of ol) are dissolved in 2.5ml D₂O, successively with 0.25ml 0.50M sodium ascorbates/4.8M LiCl D₂0.55 ascorbic acid of O and 0.25ml/4.86M LiCl D₂O processing.Then peptide 26 (1.4ml, 159.4 μm of ol) is added.The pD for determining solution is 5.1.Reactant is stirred 3 days under a nitrogen.Solution is loaded to post 1, eluted with the gradient table 1 of solvent system 2/ specified by the preparative reversed-phase HPLC part of General experimental.The bands of a spectrum that 12.2-15.4 minutes are eluted are through being concentrated in vacuo removing acetonitrile (bath temperature=35 DEG C) and freezing.Residue is further purified with cation exchange column 1, is eluted with the gradient table 1 of solvent system 1/ specified by the preparative ion exchange moieties of General experimental.The bands of a spectrum that 41.2-58.2min is eluted are concentrated to dryness (bath temperature=35 DEG C) with rotary evaporation.Residue is dissolved in 10ml water, is put into 3500MWCO dialysis membranes (Pierce, PN65035), is dialysed for deionized water (3 × 500ml, each cycle 1-2h).The lyophilized solution dialysed, obtains product.CLND：29.3%；Theoretical value：36.3%.Selection NMR resonance diagnostic peaks are used to connect chemistry：¹H NMR (400MHz, deuterium oxide) δ ppm 4.85 (dd, J=14.87Hz, 1H) 4.98 (t, J=7.04Hz, 1H) 5.01-5.07 (m, 1H) 5.17 (t, J=5.48Hz, 1H) 6.19 (s of 5.30-5.40 (m, 1H), 1H) 7.13-7.36 (m, 1H) 7.80 (d, J=8.61Hz, 1H).

Four-[ω-(4- azepine -5- oxo -7- oxa-s -7- (((3 ' R, 9 ' bS) -3 '-(carbonyl (HN-GGGGGKKRP (Hyp) G (Cpg) S (D-Tic) (Cpg)-OH)) -2 ', 3 '-thiazoline simultaneously [2 ', 3 '-a] iso-indoles -5 ' (9 ' bH) -one -8 '-yl)) heptane) -5.0kD polyoxyethylene] methane 28.PEG reagents 28 (99.4mg, 4.66 μm of ol) are dissolved in 2ml D₂O, then with 0.5ml 0.50M sodium ascorbates/4.8M LiCl D₂O processing.It is 4.3 to determine its pD.Peptide 26 (peptide content 72%, 47.4mg, 21.7 μm ol) is added into the solution.Under nitrogen atmosphere, 18h is stirred at room temperature in reactant, is then heated to 45 DEG C and reaches 2h.Solution is loaded on post 2, eluted with the gradient table 2 of solvent system 2/ specified by the preparative reversed-phase HPLC part of General experimental.The bands of a spectrum eluted for 10.5-12 minutes are collected, 2ml (bath temperature=34 DEG C) is concentrated in vacuo to.Solution is loaded on cation exchange column 2, eluted with the gradient table 2 of solvent system 1/ specified by the preparative cation exchange LC parts of General experimental.The bands of a spectrum that 20-24 minutes are eluted are dialysed through being concentrated in vacuo (bath temperature=35 DEG C) with 10K MWCO Slide-a-lyzer (Pierce, PN=66810) for 500ml deionized waters.Water is changed in 2h, 10h and 2h with fresh 500ml parts in batches.The solution that 2560g dialysed is filtered by 0.22 μm of centrifugal filter (National Scientific, PN 66064-466), is freezed filtrate, is obtained title compound.CLND：Peptide content 22.4%；Theoretical value：23.2%.The sample is characterized for detailed construction.

The detailed construction analysis of conjugate 28：

NMR is tested.

NMR experiments are carried out in 3mm pipes, and 5mm inverse detection cryoprobes are used on Bruker drx-600 spectrometers.

Chemical displacement value is determined

According to 2D TOCSY (100ms DIPSI-2 incorporation times) and 2D¹³C-¹H HMBC(ⁿJ_CH60ms separate out (evolution), n=1-4) measure 28 chemical shift of proton value (Fig. 1).The resonance of major rotomer (trans) is only listed in table 2.Minor rotamer rotates from the C- ends being obstructed and prolinamide key.

Fig. 1 have the conjugate 28 for determining resonance.

Fig. 2 .28's¹H H NMR spectroscopies (D₂O, 298K), its HOD signal and PEG signals are weakened by AB-crosslinked polymer filter (spin-diffusion filter) and weak presaturation (presaturation) respectively.

The Fig. 2 of table 1. chemical shift of proton value, PEG is unimodal to be arranged on δ 3.55ppm..

Residue order (residue order) is PEG → cp², as shown in flow 1.

The correlation that PEG resonates with peptide.

Three keys,¹H-¹³C association power spectrums are used for the position for determining PEGylation.Phenoxy-acetamide methylene (PEG_α, Fig. 3) and it is used as starting point (4.58ppm, 600MHz, table 3).Observed association approach is PEG_α(4.59ppm) arrives C₈(162.2ppm) arrives H₆(7.67ppm) arrives C₅(173.2ppm) arrives H₃(4.85ppm) arrives C_3’(172.7ppm) arrives Gly_5-α·(3.89ppm).Observed C₅(173.2ppm) and H_9bThe formation of the association Support center B rings of (6.06ppm).Equally, H₃(4.85ppm) arrives C_9b(67.2ppm) arrives H_2RThe association order of (3.73ppm) supports the formation of A rings.H_2RSignal is represented and C_3’Association, this support A rings close to peptide gly₅。

Fig. 3 .PEG resonate to be associated with the N- ends glycine of peptide 26 by 13C with the 1H NMR of (9bS) -2,3- thiazolines simultaneously [2,3-a] iso-indoles -5 (9bH) -one ring.

H₁The determination (Fig. 3) of related stereochemistry

1) (500ms incorporation times) after measured, is tested according to 2D NOESY and short (100ps) MD is run, residue H₁Related stereochemistry and H₄It is trans each other.The calculating distance of cis and trans diastereomer is shown in Table 2, and provides measured distance according to 2D NOESY.Specifically, the H of anti-configuration is predicted₁-H₄Distance is 4.1

And cis-diastereomer then appreciably shorter (3.1

).Measured distance 4.4

It is consistent with prediction anti diastereomers.

The 2D NOE of table 2. (9bS) -2,3- thiazolines simultaneously [2,3-a] iso-indoles -5 (9bH) -one ring are drawn and distance (Fig. 3) between average MD protons

2：The atom H of cis and trans diastereomer₄-C₄-N-C₁And H₁-C₁-N-C₄The prediction dihedral angle of formation is shown in Table 3.According to these angles, H is drawn₃-C_9bAnd H_9b-C₃3- key coupling constants.H₃-C_9bAnd H_9b-C₃The prediction anti diastereomers shown in Fig. 1 are corresponded with the coupling observed by 8 and 0Hz.In addition, the visible association in HMBC 2D experiments.

Prediction dihedral angle and 3- keys the C-H coupling of table 3.28.Atomic tag is limited by Fig. 1.

	Cis prediction	Trans prediction	Experiment
	Cis prediction	Trans prediction	Experiment	θ(H₃C₃N₄C_9b) ³J(H₃, C_9b)	-68 0Hz	152 8Hz	-- 8Hz
θ(H_9bC_9bN₄C₃) ³J(H_9b, C₃)	86 0Hz	87 0	-- 0Hz	θ(H₃C₃N₄C_9b) ³J(H₃, C_9b)	-68 0Hz	152 8Hz	-- 8Hz
θ(H_9bC_9bN₄C₃) ³J(H_9b, C₃)	86 0Hz	87 0	-- 0Hz	H₃-C_9b	Without intersection peak	Intersect peak	Intersect peak
H_9b-C₃	Without intersection peak	Without intersection peak	Without intersection peak	H₃-C_9b	Without intersection peak	Intersect peak	Intersect peak

3. molecular mechanics and dynamics show the enthalpy of anti diastereomers 5.5kcal/mol lower than cis-diastereomer (Fig. 4).Gly₅The measured distance of carbonyl 5 and amide NH be 2.1

This supports the presence of intramolecular hydrogen bond.

Trans and cis-diastereomer the molecular mechanics and dynamics of Fig. 4.

With the Bruker Q-FTMS systems for being equipped with 7-T superconducting magnets, conjugate 28 is analyzed.Each ion is separated with leading portion quadrupole.With " gas auxiliary Dynamical capture (gas-assisted dynamictrapping) " by ion trap into FTMS sulculuses.From 4: 1MeOH-H₂O solution, electron spray is carried out with 0.5uL/min flow velocity by solution.Dissociate and test for IRMPD, open Synrad CO₂Laser 200ms, laser can be 15%.Detected with Direct Model, by bandwidth 900kHz detection ions are obtained, collect 512K data points.Before magnitude mode Fourier conversion is carried out, time domain data is summed up and zero filling (zero-filled).Instrument carries out extrinsic calibration with Agilent tuningmix.In this experiment (Fig. 5), obtained representing that the full width of polymer heterogeneity is deconvoluted spectrum (full deconvoluted spectra).One kind has 420 repetition-(CH₂CH₂O the Dispersed heterogeneous body of)-unit is captured by FT-MS sulculuses, and irradiates (Fig. 6) with IR laser.This causes ionic dissociation, obtains 4 sub-pieces, is each spaced 1478.6742amu.There are 4 peptides in these data and each polymer and dissociation occurs to be consistent (Fig. 7) between glycine 5 and three ring systems newly formed.

Fig. 5：The FT-MS that deconvolutes is composed

Fig. 6：Ion isolation (n=420) and IRMPD dissociation.

Fig. 7：IRMPD fragment assignment.

Flow 5

Reagent and condition：A) methanol-water, 100mM L-AAs, 20mM L-AA sodium.

The natural connection of 2- formyl base esters of table 7..

(3 ' R, 9 ' bS) -3 '-(carbonyl (HN-GGGGGKKRP (Hyp) G (Cpg) S (D-Tic) (Cpg)-OH)) -2 ', 3 '-thiazoline simultaneously [2 ', 3 '-a] iso-indoles -5 ' (9 ' bH) -one 32.Peptide 26 (116mg, peptide content 72%, 52.8 μm of ol) is dissolved in 4.0ml 100mM L-AAs/20mM L-AA sodium.2- acyl radical methyl benzoates (10.4mg, 63.3 μm of ol) are added, 400 μ L MeOH are added.Reactant is stirred into 50h.Solution is loaded to post 2, eluted according to the gradient table 3 of solvent system 2/ specified by the preparative reversed-phase HPLC part of General experimental.The bands of a spectrum that 14-15 minutes are eluted are concentrated in vacuo, and remove acetonitrile, are freezed, are obtained product.It is 56% to measure peptide content through CLND.26 it is selected¹H NMR resonate (distributing to proton), see Fig. 8.¹(the H of H NMR (400MHz, deuterium oxide) δ ppm 4.96₃, t, J=7.43Hz, 1H) and 6.19 (H₉, s, 1H) and 7.61 (H of 7.15-7.28 (D-Tic, m, 4H)₆, t, J=7.43Hz, 1H) and 7.64 (H₈, d, J=8.61Hz, 1H) and 7.72 (H₇, t, J=7.04Hz, 1H) and 7.79 (H₅, d, J=7.82Hz, 1H).APCI MS (m/z) 848.8971 (M+2, z=2)；C₇₈H₁₁₅N₂₁O₂₀S (z=2) calculated value：848.909.

Using the methods described for 33, synthetic peptide 33-36.Mass spectrometric data is shown in Table 7.

Fig. 8 .32 determination resonance.

The detailed construction analysis of peptide 32：

NMR is tested

Joint 2D Cosy45,2D Noesy (phase sensitve, 25 and 40 DEG C), 2D¹H/¹³CHSQC、2D¹H/¹³C HMBC, in 600MHz, using the reverse broadband probes of 5mm, carry out the measure of 1H H NMR spectroscopies.For H₃Obtain H₉Spatial chemistry value, H₃From Cys.Specifically, H₉With H_(2S)Between observe nOe (40 DEG C).Paired proton H_(2R)Be derived from 2D Cosy45 experiment.This same resonance (H_(2R)) show to H in 40 DEG C of 2DNOESY experiments₃Association.In a word, H is supported in NMR experiments₉And H₃To the cis relationship (Fig. 8) of thiazoline ring plane.

Flow 6

Reagent and condition：A) CDI,¹³C-MeOH, DBU；B) NBS, AIBN.

The bromo- 2- methyl benzoic acids of 5-¹³C methyl esters (38).1,1 '-carbonyl dimidazoles (21g, 128mmol) are added into the anhydrous DCM agitating solutions of 100ml of the bromo- 2- methyl benzoic acids (37) (25g, 116mmol) of 5-.Solution is stirred into 3.5h.Solution is moved in pressure vessel, used¹³CH₃OH and DBU processing.Solution H₂O (2 × 20ml), 5%NaHCO₃(2 × 20ml) is washed, and organic layer is through MgSO₄Dry.Solvent is removed in vacuum, product is obtained.¹H NMR (300MHz, chloroform-d) δ ppm 2.59 (s, 3H) 3.89 (d, J=147.12Hz, 3H) 7.39 (dd, J=8.29,1.51Hz, 1H) 7.42 (s, 1H) 7.79 (d, J=8.29Hz, 1H).

The bromo- 2- of 5- (two bromomethyls) benzoic acid¹³C- methyl esters (39).To 38 (5.6g 24mmol) CCl₄N-bromosuccinimide (13.0g, 73mmol) and 2,2 '-azodiisobutyronitrile (4.0g, 24mmol) are added in agitating solution.Solution is flowed back, until showing that raw material is consumed through TLC detections.Mixture purified by flash chromatography, packs silicagel column, with 0-10%EtOAc/ hexane gradients (39 R using Biotage 40+_f=0.4, in 1: 9EtOAc/ hexane), obtain title compound.¹(the dd of H NMR (300MHz, chloroform-d) δ ppm 3.95 (d, J=147.91Hz, 3H) 7.52, J=8.48,2.05Hz, 1H) 7.78 (d, J=8.48Hz, 1H) 8.00 (s, 1H) 8.30 (d, J=1.90Hz, 1H).

Flow 7

Reagent and condition：A) NaH, PS-DIEA；B) methanol-water, 100mM L-AAs, 20mM L-AA sodium.

The bromo- 2- of 5- (3- butyl thiazolidine -2-yl) benzoic acid¹³C- methyl esters (41).PS- triphenyl phasphines (Argonaut Technologies, 2.1030g, 5mmol) are added into the 20ml THF agitating solutions of 2- (fourth amino) ethyl mercaptan (40) (621.5mg, 5mmol).Reactant is stirred 30 minutes, with intermediate pore sintered glass funnel filtering solution.N is passed through by sintered glass₂10min, resin and THF (20ml) are stirred together.THF is filtered, is merged with initial filtrate.With the 2nd washing resin of same scheme.The filtrate of merging is cooled to 0 DEG C.Add sodium hydride (0.06ml, 3mmol), the bromo- 2- of 5- (two bromomethyls) benzoic acid¹³C- methyl esters (39) (897.0mg, 2mmol) and PS-DIEA (Argonaut Technologies, 1.2429g, 5mmol), are stirred at room temperature 2 days.Reactant backflow is stayed overnight, and is cooled to room temperature and is stirred 10 days.Filtering solution, is concentrated in vacuo, with Reverse phase chromatography (post 1, solvent system 3, gradient table 4).The bands of a spectrum of elution in 26-27 minutes are concentrated in vacuo, title compound is obtained.APCI MS(m/z)：359.0(M+H)；C₁₄ ¹³CH₂₁ ⁷⁹BrNO₂S calculated value：359.04.APCI MS(m/z)：361.0(M+H)；C₁₄ ¹³CH₂₁ ⁸¹BrNO₂S calculated value：361.04.¹H NMR (300MHz, chloroform-d) δ ppm 0.91 (t, J=7.25Hz, 3H) 1.31-1.43 (m, J=11.30Hz, 2H) 1.46-1.59 (m, J=7.72Hz, 2H) 2.38-2.69 (m, J=12.06Hz, 2H) 2.85-3.01 (m, J=6.03Hz, 2H) 3.07-3.27 (m, J=11.21,6.12Hz, 2H) 3.91 (d, J=147.50Hz, 2H) 5.88 (s, 1H) 7.41 (dd, J=8.29,1.88Hz, 1H) 7.68 (d, J=8.29Hz, 1H) 8.01 (s, 1H).

(3 ' R, 9 ' bS) -7 '-bromo- 3 '-(carbonyl (HN-GGGGGKKRP (Hyp) G (Cpg) S (D-Tic) (Cpg)-OH)) -2 ', 3 '-thiazoline simultaneously [2 ', 3 '-a] iso-indoles -5 ' (9 ' bH) -one 42.Prepared according to for the method described in 32.HR FTMS(m/z)：(887.8612 M+2, z=2)；C₇₈H₁₁₄ ⁷⁹BrN₂₁O₂₀S (z=2) calculated value：887.8650；(888.8509 M+2, z=2)；C₇₈H₁₁₄ ⁸¹BrN₂₁O₂₀S (z=2) calculated value：888.8650.

Flow 8

Reagent and condition：A) PS- carbodiimides, Pentafluorophenol；B) PEG reagents 21, H ü nig alkali；c)D₂O, 100mM LiCl, 50mM deuterate ascorbic acid 1M NaOD D₂O is basified to pD 3.7.

2- (diethoxymethyl) -4- (2- oxo -2- phenyl-pentafluoride epoxides ethyoxyl) methyl benzoate (43).To evacuating and backfill N₂50ml RB flasks in add 132mg washing/dryings 10% palladium on carbon (0.12mmol Pd) and the anhydrous THF of 4ml.By carefully evacuating and (reducing bumping as far as possible) and backfill N₂Totally 3 circulations, make mixture deaerate.By 4- (2- (benzyloxy) -2- oxoethoxies) -2- (diethoxymethyl) methyl benzoates (0.500g, 1.24mmol) anhydrous THF (3ml) solution of (17) is added in slurries, bottle is washed with 1ml THF again, and move into RB flasks, totally 3 circulations of evacuation/backfill nitrogen.After last time is evacuated, with the H from balloon₂To backfill the RB flasks vacuumized.Reactant is in room temperature, H₂Lower stirring 4hr, now shows that reaction completes (17,17.4min, m/z=358.1, C through GC/MS (method 3) detections₁₉ ¹³CH₂₁O₆ ⁺Calculated value=358.1, M-OEt；18,14.26min, m/z=268.1, C₁₂ ¹³CH₁₅O₆ ⁺Calculated value=268.1, M-OEt).By Celite pad, solution is filled into 50ml RB flasks with sintered glass vacuum filter, has been loaded with being suspended in the anhydrous THF of 15ml PS- carbodiimides (Argonaut Technologies, Inc, 2.4g, 3.1mmol) in the flask.Diatomite is washed in three times with THF (3ml), and it is merged with filtrate/PS- carbodiimides.Inhomogenous mixture is in N₂Lower stirring 20min, with the THF processing of Pentafluorophenol (456mg, 2.48mmol).Reactant mixture is in N₂Middle stirring 16hr, now completes (18,14.26min through GC/MS (method 3) detection reactions；43,15.6min, m/z=434.1, C₁₈ ¹³CF₅H₁₅O₆ ⁺Calculated value=434.1, M-OEt).Mixture is filled into tarred 50ml RB flasks by medium sinter funnel.Then 10ml THF are added in resin, use N₂It is gently mixed mixing.Merging filtrate, removes solvent, and product is vacuum dried, obtains product.EI MS m/z=434.1, C₁₈ ¹³CF₅H₁₅O₆ ⁺Calculated value=434.1, M-OEt).

Compound 44. is to evacuating and backfill N₂50ml RB flasks in add 20K tetraminos PEG (21,440mg, 22 μm of ol) and 3ml anhydrous acetonitriles.By carefully evacuating and (reducing bumping as far as possible) and backfill N₂Totally 3 circulations, make mixture deaerate.H ü nig alkali (0.172mmol is added into solution, 30 μ l), add anhydrous acetonitrile (1ml+1ml is used for rinse) solution of 2- (diethoxymethyl) -4- (2- oxo -2- phenyl-pentafluoride epoxides ethyoxyl) methyl benzoate (43,0.128mmol).Molecular sieve (it is powdered, 4Aperture, 100mg) it is added in mixture.Solution is through evacuation/backfill nitrogen totally 3 circulating degasifications.By reactant in N₂Under in 40 DEG C stir 24hr.Mixture is filled into 50ml RB flasks by medium sinter funnel, piperazine (the Silicycle Inc. with reference to Si are had been loaded with the flask, 171mg, 0.15mmol) and with reference to Si carbonate (Silicycle Inc., 0.3mmol, 434mg), then with 10ml acetonitriles wash.The filtrate of merging is in N₂Under in 40 DEG C stir 15hr.Then mixture is filled into by medium sinter funnel in the 50ml RB flasks for scribbling tar, then washed with 10ml acetonitriles.Solvent is removed, product is vacuum dried, obtains 44.¹³C NMR(D₂O, part-structure)：δ 170.14,72.00.

According to for 28 methods describeds, compound 45 is synthesized.Reaction is in pD 3.7, D₂Carried out in O.Specifically, D is used₂O prepares 100mM LiCl and 50mM deuterate ascorbic acid (from D₂O is lyophilized and obtains, 3 circulations) solution.PD is adjusted to 3.7 with 1M NaOD.PEG reagents 44 and peptide 26 are added into the solution.13h is stirred at room temperature in reactant, is post-processed according to described in 28.Structure is confirmed similar to 28 through FT-MSMS, but because¹³C and become high 1amu.

Embodiment：Polymer-internal anti-nociceptive effects activity of the anti-B1 peptides in rat and monkey pain model is conjugated

A.Rat characteristic of disease pain model.According to first as Kim and Chung (Anexperimental model for peripheral neuropathy produced by segmentalspinal nerve ligation in the rat (rat experiment model of the peripheral neurophaty caused by segmental Spinal nerve ligation) .Pain 50：355-363, (1992) method described in), with isoflurane inhalation anaesthetised male Sprague-Dawley rats (200g), then away from DRGs and prior to sciatic nerve porch, the left spinal nerves of L5 and L6 levels are firmly ligatured (4-0 silk threads).Close incisions, allow rat to recover.This method causes mechanical (tactile) allodynia of left back pawl, this be observed and recorded by woven wire observation cage involvement claw (with nerve damage portion homonymy) from the vertical fractional stimulus (Frey scopes of von are 4.0-148.1mN) for imposing on claw toe face (between palmula) shrink back when pressure evaluated.Pass through sequential increase and reduction stimulus intensity, and analyze data of shrinking back using Dixon non-parametric tests, determine claw withdrawal threshold (PWT), Dixon non-parametric tests are referring to Chaplan, S.R. (Quantitativeassessment of tactile allodynia in the rat paw (quantitative assessment of rats paw tactile allodynia) .J.Neurosci.Meth, 53 are waited：55-63(1994)).

Normal rat and control operated rats (separation is neural but does not ligature) withstand at least 148.1mN (equivalent to 15g) pressure without reaction.Spinal nerve ligated rats will react when only 4.0mN (equivalent to 0.41g) pressure imposes on involvement claw.Only when rat does not show dyskinesia (such as claw towing or sagging) and their PWT is less than 39.2mN (equivalent to 4.0g), just including under study for action.Postoperative at least 7 days, a test peptides or test carrier-conjugated peptide (generally screening dosage about 1mg/kg and about 60mg/kg respectively) are injected by s.c. or control dilution (PBS) is treated to rat, PWT, totally 7 days are hereafter determined daily.

B.Rat CFA inflammatory pain models.With isoflurane inhalation anaesthetised male Sprague-Dawley rats (200g), left hind paw complete Freund's adjuvant (CFA) 0.15ml.This method causes mechanical (tactile) allodynia of left back pawl, this be observed and recorded by woven wire observation cage involvement claw from the vertical fractional stimulus (von Frey silk thread scopes are 4.0-148.1mN) for imposing on claw toe face (between palmula) shrink back when pressure evaluated.Data of shrinking back are analyzed by sequential increase and reduction stimulus intensity, and using Dixon non-parametric tests, PWT are determined, Dixon non-parametric tests are referring to Chaplan etc. (1994).Only when rat does not show dyskinesia (such as claw towing or sagging) or skin injury and their PWT is less than 39.2mN (equivalent to 4.0g), just including under study for action.At least 7 days after CFA injections, by s.c. injections, once experiment polymer-conjugated peptide (generally screening dosage about 60mg/kg) or contrast solution (PBS) are treated to rat, and PWT, totally 7 days are hereafter determined daily.Average claw withdrawal threshold (PWT) can be converted into maximum possible effect (%MPE) with following equation：%MPE=100* (PWT of the PWT- control rats for the treatment of rat)/(PWT of 15- control rats).Therefore, 15g (148.1mN) cutoffs are equivalent to 100%MPE, and control reaction is equivalent to 0%MPE.

Preferred polymers of the present invention-conjugated peptide can produce anti-nociceptive effects effect, be respectively that about 1mg/kg and about 60mg/kg have PD relations in screening dosage.

B.Grivet LPS inflammatory models.Basically according to deBlois and Horlick (BritishJournal of Pharmacology.132：327-335 (2002), the document is fully incorporated herein by quoting) it is described, polymer-conjugated peptide can be evaluated in the male grivet (Cercopithaecus aethiops St Kitts) that local assault is carried out with B1 activators as the effect of B1 activities inhibitor.

In order to determine that the edema whether peptide antagonists suppress B1 inductions is conjugated in the PEG- of the present invention, to male grivet (Cercopithaecus aethiops St Kitts；Caribbean Primates Ltd.Experimental farm (St Kitts, West Indies)) carry out following researchs.Anesthesia (50mg ketamines kg is implemented for 6.0 ± 0.5kg animal (n=67) to body weight^-1), and by injecting LPS (90 μ g kg in saphena single dose intravenous^-1) or salt solution (1ml) pre-processed.

1. inflammation research

The edema (Sciberras etc., 1987) that can be induced by veutro skin fold test evaluation kassinin kinin.In brief, monkey injection captopril (the 1mg kg to anesthesia^-1, 30min before experiment).Single subcutaneous injection dKD, BK or solvent (2mM amastatins/100 μ l ringer lactates) are given in belly, with the slide calliper rule of demarcation to skinfold increase monitoring 30-45min.As a result it is expressed as being subcutaneously injected the difference of front and rear skinfold.Captopril and amastatin can be used for reducing degraded of the kassinin kinin in c-terminus and aminoterminal.

Antagonist SCHILD is analyzed

The 24h after LPS, in the PEG- peptide antagonists existence or non-existences of various concentrations, can determine the dose-response relationship of the edema of dKD (1-100nmol) inductions.BK (30nmol) can be used as positive control.

Antagonist time course

4h, 24h, 48h, 72h and/or 96h after single bolus administration, determine the time course that antagonist suppresses.BK (30nmol) can be used as positive control.

Medicine

Ketalar, LPS, amastatin and captopril are purchased from Sigma companies (MO, U.S.A.).All peptides are all available from Phoenix Pharmaceuticals companies (CA, U.S.A.).

Statistics

Numerical value is expressed as average value ± average and misses (s.e.mean).In oedema research, the skinfold before injection is subtracted with the numerical value after subcutaneous challenge.On apple computer and with the softwares of Delta Graph 4.0, curve matching and EC can obtain₅₀Calculated value.By bilateral variance analysis, then examined and corrected with Bonferroni by paired, not single tail student t, data are compared.P ＜ 0.05 are considered to have significance,statistical.

In oedema formation experiment, grivet is given by LPS, its sensitivity can be made to increase to from zero level to B₁The level of receptor stimulating agent.By contrast, to B₂Receptor stimulating agent BK reaction is then unaffected.

Embodiment：Pharmacokinetics in rats is studied

By intravenous (iv) or subcutaneous (sc) approach, male Sprague-Dawley rat is given through injecting by various peptides or conjugated peptide (in an aqueous medium).Different time points (such as 0,15,30min. and/or 1,2,4,6,8,10,12,18,24,30,36,42,48,60,72,84,96,120,240 and/or 320 hours after injecting), collection blood sample is added in the test tube equipped with heparin.Blood plasma is taken out after centrifugation from sedimentation cell, can freeze and also process immediately.Target compound in blood plasma is quantitative determined by analyte specificity LC-MS/MS or ELISA method.Non- chamber method (non-compartmental method) can be passed through, calculate different standard pharmacokinetic parameters, such as clearance rate (CL), apparent clearance rate (CL/F), distribution volume (Vss), mean residence time (MRT), TG-AUC (AUC) and Terminal half-life (t_1/2)。

Sequence table

<110>Amgen Inc (Amgen Inc.)

<120>The method that molecule containing amineothiot is conjugated with carrier

<130>A-982

<140>PCT/US is not determined

<141>2006-01-24

<150>60/646,685

<151>2005-01-24

<150>Do not determine

<151>2006-01-23

<160>62

<170>PatentIn version 3.2

<210>1

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<213>People

<400>1

Arg Pro Pro Gly Phe Ser Pro Phe Arg

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Lys Arg Pro Pro Gly Phe Ser Pro Phe Arg

1 5 10

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<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

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Met Lys Arg Pro Pro Gly Phe Ser Pro Phe Arg

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<212>PRT

<213>People

<400>4

Arg Pro Pro Gly Phe Ser Pro Phe

1 5

<210>5

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<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

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Arg Pro Pro Gly Phe Ser Pro Leu

1 5

<210>6

<211>9

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<400>6

Lys Arg Pro Pro Gly Phe Ser Pro Leu

1 5

<210>7

<211>10

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(1)..(1)

<223>The Xaa of 1 is defined as arginic D isomers (DArg)

<220>

<221>Other features

<222>(4)..(4)

<223>The Xaa of 4 is defined as trans -4- hydroxy-prolines (Hyp)

<220>

<221>Other features

<222>(6)..(6)

<223>The Xaa of 6 is defined as β-(2- thienyls)-alanine (Thi)

<220>

<221>Other features

<222>(8)..(8)

<223>The Xaa of 8 is defined as the D isomers (Dtic) of 1,2,3,4- tetrahydroisoquinoline -3- formic acid

<220>

<221>Other features

<222>(9)..(9)

<223>The Xaa of 9 is defined as octahydro indole-2-carboxylic acid (Oic)

<400>7

Xaa Arg Pro Xaa Gly Xaa Ser Xaa Xaa Arg

1 5 10

<210>8

<211>9

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(1)..(1)

<223>The Xaa of 1 is defined as DArg

<220>

<221>Other features

<222>(4)..(4)

<223>The Xaa of 4 is defined as Hyp

<220>

<221>Other features

<222>(6)..(6)

<223>The Xaa of 6 is defined as Thi

<220>

<221>Other features

<222>(8)..(8)

<223>The Xaa of 8 is defined as Dtic

<220>

<221>Other features

<222>(9)..(9)

<223>The Xaa of 9 is defined as Oic

<400>8

Xaa Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5

<210>9

<211>10

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(1)..(1)

<223>The Xaa of 1 is defined as DArg

<220>

<221>Other features

<222>(4)..(4)

<223>The Xaa of 4 is defined as Hyp

<220>

<221>Other features

<222>(6)..(6)

<223>The Xaa of 6 is defined as Thi

<220>

<221>Other features

<222>(8)..(8)

<223>The Xaa of 8 is defined as the D isomers (DHype) of 4S-D- prolyls-prolyl ether

<220>

<221>Other features

<222>(9)..(9)

<223>The Xaa of 9 is defined as Oic

<400>9

XaaArg Pro Xaa Gly Xaa Ser Xaa Xaa Arg

1 5 10

<210>10

<211>10

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>MOD_RES

<222>(1)..(1)

<223>Acetylation

<220>

<221>Other features

<222>(7)..(7)

<223>The Xaa of 7 is defined as L- phenylalanine N- methyl esters (MePhe)

<220>

<221>Other features

<222>(9)..(9)

<223>The Xaa of 9 is defined as the D isomers (D- β-NaI) of β -2- naphthyls-alanine

<400>10

Leu Leu Arg Pro Pro Gly Xaa Ser Xaa Ile

1 5 10

<210>11

<211>10

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

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<221>Other features

<222>(1)..(1)

<223>The Xaa of 1 is defined as DArg

<220>

<221>Other features

<222>(4)..(4)

<223>The Xaa of 4 is defined as Hyp

<220>

<221>Other features

<222>(6)..(6)

<223>The Xaa of 6 is defined as 2- indanyls glycine (Igl)

<220>

<221>Other features

<222>(8)..(8)

<223>The Xaa of 8 is defined as the D isomers (DIgl) of 2- indanyl glycine

<220>

<221>Other features

<222>(9)..(9)

<223>The Xaa of 9 is defined as Oic

<400>11

Xaa Arg Pro Xaa Gly Xaa Ser Xaa Xaa Arg

1 5 10

<210>12

<211>10

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(5)..(5)

<223>The Xaa of 5 is defined as Hyp

<220>

<221>Other features

<222>(7)..(7)

<223>The Xaa of 7 is defined as Igl

<220>

<221>Other features

<222>(9)..(9)

<223>The Xaa of 9 is defined as DIgl

<220>

<221>Other features

<222>(10)..(10)

<223>The Xaa of 10 is defined as Oic

<400>12

Lys Lys Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>13

<211>10

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(5)..(5)

<223>The Xaa of 5 is defined as Hyp

<220>

<221>Other features

<222>(7)..(7)

<223>The Xaa of 7 is defined as cyclopentylglycine (Cpg)

<220>

<221>Other features

<222>(9)..(9)

<223>The Xaa of 9 is defined as Dtic

<220>

<221>Other features

<222>(10)..(10)

<223>The Xaa of 10 is defined as Cpg

<400>13

Lys LysArg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>14

<211>10

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(1)..(1)

<223>The Xaa of 1 is defined as DArg

<220>

<221>Other features

<222>(4)..(4)

<223>The Xaa of 4 is defined as Hyp

<220>

<221>Other features

<222>(6)..(6)

<223>The Xaa of 6 is defined as Igl

<220>

<221>Other features

<222>(8)..(8)

<223>The Xaa of 8 is defined as the D isomers (Df5f) of pentafluorophenyl group alanine

<220>

<221>Other features

<222>(9)..(9)

<223>The Xaa of 9 is defined as Igl

<400>14

Xaa Arg Pro Xaa Gly Xaa Ser Xaa Xaa Arg

1 5 10

<210>15

<211>10

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(1)..(1)

<223>The Xaa of 1 is defined as DOrn

<220>

<221>Other features

<222>(5)..(5)

<223>The Xaa of 5 is defined as Hyp

<220>

<221>Other features

<222>(7)..(7)

<223>The Xaa of 7 is defined as Cpg

<220>

<221>Other features

<222>(9)..(9)

<223>The Xaa of 9 is defined as Dtic

<220>

<221>Other features

<222>(10)..(10)

<223>The Xaa of 10 is defined as Cpg

<400>15

Xaa Leu Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>16

<211>10

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(1)..(1)

<223>The Xaa of 1 is defined as DOrn

<220>

<221>Other features

<222>(5)..(5)

<223>The Xaa of 5 is defined as thiazolidine -4- formic acid (Thz)

<220>

<221>Other features

<222>(7)..(7)

<223>The Xaa of 7 is defined as Cpg

<220>

<221>Other features

<222>(9)..(9)

<223>The Xaa of 9 is defined as Dtic

<220>

<221>Other features

<222>(10)..(10)

<223>The Xaa of 10 is defined as Cpg

<400>16

Xaa Leu Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>17

<211>10

<212>PRT

<213>Artificial sequence

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<223>Synthetically prepared

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<222>(1)..(1)

<223>The Xaa of 1 is defined as β -3- pyridine radicals-alanine (3Pa1)

<220>

<221>Other features

<222>(5)..(5)

<223>The Xaa of 5 is defined as Hyp

<220>

<221>Other features

<222>(7)..(7)

<223>The Xaa of 7 is defined as Cpg

<220>

<221>Other features

<222>(9)..(9)

<223>The Xaa of 9 is defined as Dtic

<220>

<221>Other features

<222>(10)..(10)

<223>The Xaa of 10 is defined as Cpg

<400>17

Xaa Leu Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>18

<211>10

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(1)..(1)

<223>The Xaa of 1 is defined as D-4 ' pyriylalanines (4Pa1)

<220>

<221>Other features

<222>(5)..(5)

<223>The Xaa of 5 is defined as Hyp

<220>

<221>Other features

<222>(7)..(7)

<223>The Xaa of 7 is defined as Cpg

<220>

<221>Other features

<222>(9)..(9)

<223>The Xaa of 9 is defined as Dtic

<220>

<221>Other features

<222>(10)..(10)

<223>The Xaa of 10 is defined as Cpg

<400>18

Xaa Leu Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>19

<211>9

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(1)..(1)

<223>The Xaa of 1 is defined as Cyclohexylalanine (Cha)

<220>

<221>Other features

<222>(4)..(4)

<223>The Xaa of 4 is defined as Hyp

<220>

<221>Other features

<222>(6)..(6)

<223>The Xaa of 6 is defined as Cpg

<220>

<221>Other features

<222>(8)..(8)

<223>The Xaa of 8 is defined as Dtic

<220>

<221>Other features

<222>(9)..(9)

<223>The Xaa of 9 is defined as Cpg

<400>19

Xaa Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5

<210>20

<211>9

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(1)..(1)

<223>The Xaa of 1 is defined as β -2- naphthyls-alanine (2Nal)

<220>

<221>Other features

<222>(4)..(4)

<223>The Xaa of 4 is defined as Hyp

<220>

<221>Other features

<222>(6)..(6)

<223>The Xaa of 6 is defined as Cpg

<220>

<221>Other features

<222>(8)..(8)

<223>The Xaa of 8 is defined as Dtic

<220>

<221>Other features

<222>(9)..(9)

<223>The Xaa of 9 is defined as Cpg

<400>20

Xaa Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5

<210>21

<211>9

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(4)..(4)

<223>The Xaa of 4 is defined as Hyp

<220>

<221>Other features

<222>(6)..(6)

<223>The Xaa of 6 is defined as Cpg

<220>

<221>Other features

<222>(8)..(8)

<223>The Xaa of 8 is defined as Dtic

<220>

<221>Other features

<222>(9)..(9)

<223>The Xaa of 9 is defined as Cpg

<400>21

Leu Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5

<210>22

<211>10

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(1)..(1)

<223>The Xaa of 1 is defined as the D isomers (DLys) of lysine

<220>

<221>Other features

<222>(5)..(5)

<223>The Xaa of 5 is defined as Hyp

<220>

<221>Other features

<222>(7)..(7)

<223>The Xaa of 7 is defined as Cpg

<220>

<221>Other features

<222>(9)..(9)

<223>The Xaa of 9 is defined as Dtic

<220>

<221>Other features

<222>(10)..(10)

<223>The Xaa of 10 is defined as Cpg

<400>22

Xaa Leu Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>23

<211>10

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(2)..(2)

<223>The Xaa of 2 is defined as DOrn

<220>

<221>Other features

<222>(5)..(5)

<223>The Xaa of 5 is defined as Hyp

<220>

<221>Other features

<222>(7)..(7)

<223>The Xaa of 7 is defined as Cpg

<220>

<221>Other features

<222>(9)..(9)

<223>The Xaa of 9 is defined as Dtic

<220>

<221>Other features

<222>(10)..(10)

<223>The Xaa of 10 is defined as Cpg

<400>23

Leu Xaa Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>24

<211>10

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(2)..(2)

<223>The Xaa of 2 is defined as Cha

<220>

<221>Other features

<222>(5)..(5)

<223>The Xaa of 5 is defined as Hyp

<220>

<221>Other features

<222>(7)..(7)

<223>The Xaa of 7 is defined as Cpg

<220>

<221>Other features

<222>(9)..(9)

<223>The Xaa of 9 is defined as Dtic

<220>

<221>Other features

<222>(10)..(10)

<223>The Xaa of 10 is defined as Cpg

<400>24

Leu Xaa Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>25

<211>10

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(2)..(2)

<223>The Xaa of 2 is defined as S- aminobutyric acids (Abu)

<220>

<221>Other features

<222>(5)..(5)

<223>The Xaa of 5 is defined as Hyp

<220>

<221>Other features

<222>(7)..(7)

<223>The Xaa of 7 is defined as Cpg

<220>

<221>Other features

<222>(9)..(9)

<223>The Xaa of 9 is defined as Dtic

<220>

<221>Other features

<222>(10)..(10)

<223>The Xaa of 10 is defined as Cpg

<400>25

Leu Xaa Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>26

<211>11

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(2)..(2)

<223>The Xaa of 2 is defined as 2Nal

<220>

<221>Other features

<222>(3)..(3)

<223>Xaa can be any naturally occurring amino acid

<220>

<221>Other features

<222>(5)..(5)

<223>The Xaa of 5 is defined as Hyp

<220>

<221>Other features

<222>(6)..(6)

<223>Xaa can be any naturally occurring amino acid

<220>

<221>Other features

<222>(7)..(7)

<223>The Xaa of 7 is defined as Cpg

<220>

<221>Other features

<222>(8)..(8)

<223>Xaa can be any naturally occurring amino acid

<220>

<221>Other features

<222>(9)..(9)

<223>The Xaa of 9 is defined as Dtic

<220>

<221>Other features

<222>(10)..(10)

<223>The Xaa of 10 is defined as Cpg

<220>

<221>Other features

<222>(11)..(11)

<223>Xaa can be any naturally occurring amino acid

<400>26

Leu Xaa Xaa Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>27

<211>13

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<400>27

Cys Gly Gly Gly Lys Arg Pro Pro Gly Phe Ser Pro Leu

1 5 10

<210>28

<211>15

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<400>28

Cys Gly Gly Gly Gly Gly Lys Arg Pro Pro Gly Phe Ser Pro Leu

1 5 10 15

<210>29

<211>15

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<400>29

Cys Gly Gly Gly Gly Gly Lys Lys Arg Pro Gly Phe Ser Pro Leu

1 5 10 15

<210>30

<211>17

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<400>30

Cys Gly Gly Gly Gly Gly Lys Arg Lys Arg Pro Pro Gly Phe Ser Pro

1 5 10 15

Leu

<210>31

<211>12

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(3)..(3)

<223>The Xaa of 3 is defined as CH2-CH2-CH2-CH2-CH2-CH2

<400>31

Cys Gly Xaa Lys Arg Pro Pro Gly Phe Ser Pro Leu

1 5 10

<210>32

<211>16

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(13)..(13)

<223>The Xaa of 13 is defined as MePhe

<220>

<221>Other features

<222>(15)..(15)

<223>The Xaa of 15 is defined as D- β-NaI

<400>32

Cys Gly Gly Gly Gly Gly Leu Leu Arg Pro Pro Gly Xaa Ser Xaa Ile

1 5 10 15

<210>33

<211>16

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(11)..(11)

<223>The Xaa of 11 is defined as Hyp

<220>

<221>Other features

<222>(13)..(13)

<223>The Xaa of 13 is defined as Cpg

<220>

<221>Other features

<222>(15)..(15)

<223>The Xaa of 15 is defined as Dtic

<220>

<221>Other features

<222>(16)..(16)

<223>The Xaa of 16 is defined as Cpg

<400>33

Cys Gly Gly Gly Gly Gly Lys Lys Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10 15

<210>34

<211>18

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(12)..(12)

<223>The Xaa of 12 is defined as Hyp

<220>

<221>Other features

<222>(13)..(13)

<223>Xaa can be any naturally occurring amino acid

<220>

<221>Other features

<222>(14)..(14)

<223>The Xaa of 14 is defined as Cpg

<220>

<221>Other features

<222>(15)..(15)

<223>Xaa can be any naturally occurring amino acid

<220>

<221>Other features

<222>(16)..(16)

<223>The Xaa of 16 is defined as Dtic

<220>

<221>Other features

<222>(17)..(17)

<223>The Xaa of 17 is defined as Cpg

<220>

<221>Other features

<222>(18)..(18)

<223>Xaa can be any naturally occurring amino acid

<400>34

Cys Gly Gly Gly Gly Gly Gly Gly Lys Lys Arg Pro Xaa Gly Xaa Ser

1 5 10 15

Xaa Xaa

<210>35

<211>16

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>MOD_RES

<222>(1)..(1)

<223>Acetylation

<220>

<221>Other features

<222>(11)..(11)

<223>Xaa can be any naturally occurring amino acid

<220>

<221>Other features

<222>(12)..(12)

<223>The Xaa of 12 is defined as Hyp

<220>

<221>Other features

<222>(13)..(13)

<223>Xaa can be any naturally occurring amino acid

<220>

<221>Other features

<222>(14)..(14)

<223>The Xaa of 14 is defined as Cpg

<220>

<221>Other features

<222>(15)..(15)

<223>Xaa can be any naturally occurring amino acid

<220>

<221>Other features

<222>(16)..(16)

<223>The Xaa of 16 is defined as Dtic

<220>

<221>Other features

<222>(17)..(17)

<223>The Xaa of 17 is defined as Cpg

<400>35

Cys Gly Gly Gly Gly Gly Lys Lys Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10 15

<210>36

<211>10

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(5)..(5)

<223>The Xaa of 5 is defined as Hyp

<220>

<221>Other features

<222>(7)..(7)

<223>The Xaa of 7 is defined as Cpg

<220>

<221>Other features

<222>(9)..(9)

<223>The Xaa of 9 is defined as Dtic

<220>

<221>Other features

<222>(10)..(10)

<223>The Xaa of 10 is defined as Cpg

<400>36

Lys Lys Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>37

<211>10

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>MOD_RES

<222>(1)..(1)

<223>Acetylation

<220>

<221>Other features

<222>(5)..(5)

<223>Xaa can be any naturally occurring amino acid

<220>

<221>Other features

<222>(6)..(6)

<223>The Xaa of 6 is defined as Hyp

<220>

<221>Other features

<222>(7)..(7)

<223>Xaa can be any naturally occurring amino acid

<220>

<221>Other features

<222>(8)..(8)

<223>The Xaa of 8 is defined as Cpg

<220>

<221>Other features

<222>(9)..(9)

<223>Xaa can be any naturally occurring amino acid

<220>

<221>Other features

<222>(10)..(10)

<223>The Xaa of 10 is defined as Dtic

<220>

<221>Other features

<222>(11)..(11)

<223>The Xaa of 11 is defined as Cpg

<400>37

Lys Lys Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>38

<211>10

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<400>38

Cys Lys Arg Pro Pro Gly Phe Ser Pro Leu

1 5 10

<210>39

<211>16

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(7)..(7)

<223>The Xaa of 7 is defined as DOrn

<220>

<221>Other features

<222>(11)..(11)

<223>The Xaa of 11 is defined as Hyp

<220>

<221>Other features

<222>(13)..(13)

<223>The Xaa of 13 is defined as Cpg

<220>

<221>Other features

<222>(15)..(15)

<223>The Xaa of 15 is defined as Dtic

<220>

<221>Other features

<222>(16)..(16)

<223>The Xaa of 16 is defined as Cpg

<400>39

Cys Gly Gly Gly Gly Gly Xaa Leu Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10 15

<210>40

<211>16

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(7)..(7)

<223>The Xaa of 7 is defined as DOrn

<220>

<221>Other features

<222>(11)..(11)

<223>The Xaa of 11 is defined as Thz

<220>

<221>Other features

<222>(13)..(13)

<223>The Xaa of 13 is defined as Cpg

<220>

<221>Other features

<222>(15)..(15)

<223>The Xaa of 15 is defined as Dtic

<220>

<221>Other features

<222>(16)..(16)

<223>The Xaa of 16 is defined as Cpg

<400>40

Cys Gly Gly Gly Gly Gly Xaa Leu Arg Pro XaaGly Xaa Ser Xaa Xaa

1 5 10 15

<210>41

<211>16

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(8)..(8)

<223>The Xaa of 8 is defined as DOrn

<220>

<221>Other features

<222>(11)..(11)

<223>The Xaa of 11 is defined as Hyp

<220>

<221>Other features

<222>(13)..(13)

<223>The Xaa of 13 is defined as Cpg

<220>

<221>Other features

<222>(15)..(15)

<223>The Xaa of 15 is defined as Dtic

<220>

<221>Other features

<222>(16)..(16)

<223>The Xaa of 16 is defined as Cpg

<400>41

Cys Gly Gly Gly Gly Gly Leu XaaArg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10 15

<210>42

<211>15

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<400>42

Gly Gly Gly Gly Gly Lys Lys Arg Pro Pro Gly Phe Ser Pro Leu

1 5 10 15

<210>43

<211>10

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(1)..(1)

<223>The Xaa of 1 is defined as the D isomers (D-Dab) of D-2- aminobutyric acids

<220>

<221>Other features

<222>(5)..(5)

<223>The Xaa of 5 is defined as Hyp

<220>

<221>Other features

<222>(7)..(7)

<223>The Xaa of 7 is defined as Cpg

<220>

<221>Other features

<222>(9)..(9)

<223>The Xaa of 9 is defined as Dtic

<220>

<221>Other features

<222>(10)..(10)

<223>The Xaa of 10 is defined as Cpg

<400>43

Xaa Lys Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>44

<211>10

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(1)..(1)

<223>The Xaa of 1 is defined as D-Arg

<220>

<221>Other features

<222>(5)..(5)

<223>The Xaa of 5 is defined as Hyp

<220>

<221>Other features

<222>(7)..(7)

<223>The Xaa of 7 is defined as Cpg

<220>

<221>Other features

<222>(9)..(9)

<223>The Xaa of 9 is defined as Dtic

<220>

<221>Other features

<222>(10)..(10)

<223>The Xaa of 10 is defined as Cpg

<400>44

XaaLeu Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>45

<211>10

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(1)..(1)

<223>The Xaa of 1 is defined as DOrn

<220>

<221>Other features

<222>(5)..(5)

<223>The Xaa of 5 is defined as Hyp

<220>

<221>Other features

<222>(7)..(7)

<223>The Xaa of 7 is defined as Cpg

<220>

<221>Other features

<222>(9)..(9)

<223>The Xaa of 9 is defined as Dtic

<220>

<221>Other features

<222>(10)..(10)

<223>The Xaa of 10 is defined as Cpg

<400>45

Xaa Leu Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>46

<211>10

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(1)..(1)

<223>The Xaa of 1 is defined as Arg D isomers

<220>

<221>Other features

<222>(5)..(5)

<223>The Xaa of 5 is defined as Arg D isomers

<220>

<221>Other features

<222>(7)..(7)

<223>The Xaa of 7 is defined as Cpg

<220>

<221>Other features

<222>(9)..(9)

<223>The Xaa of 9 is defined as Dtic

<220>

<221>Other features

<222>(10)..(10)

<223>The Xaa of 10 is defined as Cpg

<400>46

Xaa Leu Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>47

<211>10

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(1)..(1)

<223>The Xaa of 1 is defined as the D isomers (D-3 ' Pa1) of 3 '-pyriylalanine

<220>

<221>Other features

<222>(5)..(5)

<223>The Xaa of 5 is defined as Hyp

<220>

<221>Other features

<222>(7)..(7)

<223>The Xaa of 7 is defined as Cpg

<220>

<221>Other features

<222>(9)..(9)

<223>The Xaa of 9 is defined as Dtic

<220>

<221>Other features

<222>(10)..(10)

<223>The Xaa of 10 is defined as Cpg

<400>47

Xaa Leu Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>48

<211>10

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>MOD_RES

<222>(1)..(1)

<223>Acetylation

<220>

<221>Other features

<222>(1)..(1)

<223>The Xaa of 1 is defined as D-3 ' Pa1

<220>

<221>Other features

<222>(5)..(5)

<223>The Xaa of 5 is defined as Hyp

<220>

<221>Other features

<222>(7)..(7)

<223>The Xaa of 7 is defined as Cpg

<220>

<221>Other features

<222>(9)..(9)

<223>The Xaa of 9 is defined as Dtic

<220>

<221>Other features

<222>(10)..(10)

<223>The Xaa of 10 is defined as Cpg

<400>48

Xaa Leu Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>49

<211>10

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(1)..(1)

<223>The Xaa of 1 is defined as D-Lys

<220>

<221>Other features

<222>(2)..(2)

<223>The Xaa of 2 is defined as D-2-Nal

<220>

<221>Other features

<222>(5)..(5)

<223>The Xaa of 5 is defined as Hyp

<220>

<221>Other features

<222>(7)..(7)

<223>The Xaa of 7 is defined as Cpg

<220>

<221>Other features

<222>(9)..(9)

<223>The Xaa of 9 is defined as Dtic

<220>

<221>Other features

<222>(10)..(10)

<223>The Xaa of 10 is defined as Cpg

<400>49

Xaa Xaa Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>50

<211>10

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(2)..(2)

<223>The Xaa of 2 is defined as the D isomers (D-2-Nal) of b-2 naphthyls-alanine

<220>

<221>Other features

<222>(5)..(5)

<223>The Xaa of 5 is defined as Hyp

<220>

<221>Other features

<222>(7)..(7)

<223>The Xaa of 7 is defined as Cpg

<220>

<221>Other features

<222>(9)..(9)

<223>The Xaa of 9 is defined as Dtic

<220>

<221>Other features

<222>(10)..(10)

<223>The Xaa of 10 is defined as Cpg

<400>50

Leu Xaa Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>51

<211>9

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(1)..(1)

<223>The Xaa of 1 is defined as DOrn

<220>

<221>Other features

<222>(3)..(3)

<223>The Xaa of 3 is defined as Oic

<220>

<221>Other features

<222>(6)..(6)

<223>The Xaa of 6 is defined as Me-Phe

<220>

<221>Other features

<222>(8)..(8)

<223>The Xaa of 8 is defined as D- β-NaI

<400>51

Xaa Arg Xaa Pro Gly Xaa Ser Xaa Ile

1 5

<210>52

<211>9

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>MOD RES

<222>(1)..(1)

<223>Acetylation

<220>

<221>Other features

<222>(1)..(1)

<223>The Xaa of 1 is defined as DOrn

<220>

<221>Other features

<222>(3)..(3)

<223>The Xaa of 3 is defined as Oic

<220>

<221>Other features

<222>(6)..(6)

<223>The Xaa of 6 is defined as Me-Phe

<220>

<221>Other features

<222>(8)..(8)

<223>The Xaa of 8 is defined as D- β-NaI

<400>52

Xaa Arg Xaa Pro Gly Xaa Ser Xaa Ile

1 5

<210>53

<211>10

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(1)..(1)

<223>The Xaa of 1 is defined as DOrn

<220>

<221>Other features

<222>(4)..(4)

<223>The Xaa of 4 is defined as Oic

<220>

<221>Other features

<222>(7)..(7)

<223>The Xaa of 7 is defined as Me-Phe

<220>

<221>Other features

<222>(9)..(9)

<223>The Xaa of 9 is defined as D β-NaI

<400>53

Xaa Leu Arg Xaa Pro Gly Xaa Ser Xaa Ile

1 5 10

<210>54

<211>10

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>MOD_RES

<222>(1)..(1)

<223>Acetylation

<220>

<221>Other features

<222>(1)..(1)

<223>The Xaa of 1 is defined as DOrn

<220>

<221>Other features

<222>(4)..(4)

<223>The Xaa of 4 is defined as Oic

<220>

<221>Other features

<222>(7)..(7)

<223>The Xaa of 7 is defined as Me-Phe

<220>

<221>Other features

<222>(9)..(9)

<223>The Xaa of 9 is defined as D- β-NaI

<400>54

Xaa Leu Arg Xaa Pro Gly Xaa Ser Xaa Ile

1 5 10

<210>55

<211>9

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(8)..(8)

<223>The Xaa of 8 is defined as Me-Phe

<400>55

Leu Arg Pro Pro Gly Phe Ser Xaa Ile

1 5

<210>56

<211>9

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>MOD_RES

<222>(1)..(1)

<223>Acetylation

<220>

<221>Other features

<222>(8)..(8)

<223>The Xaa of 8 is defined as D- β-NaI

<400>56

Leu Arg Pro Pro Gly Phe Ser Xaa Ile

1 5

<210>57

<211>9

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(1)..(1)

<223>The Xaa of 1 is defined as ornithine (Orn)

<220>

<221>Other features

<222>(3)..(3)

<223>The Xaa of 3 is defined as Oic

<220>

<221>Other features

<222>(6)..(6)

<223>The Xaa of 6 is defined as Me-Phe

<220>

<221>Other features

<222>(8)..(8)

<223>The Xaa of 8 is defined as D- β-NaI

<400>57

Xaa Arg Xaa Pro Gly Xaa Ser Xaa Ile

1 5

<210>58

<211>9

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>MOD_RES

<222>(1)..(1)

<223>Acetylation

<220>

<221>Other features

<222>(1)..(1)

<223>The Xaa of 1 is defined as Orn

<220>

<221>Other features

<222>(3)..(3)

<223>The Xaa of 3 is defined as Oic

<220>

<221>Other features

<222>(6)..(6)

<223>The Xaa of 6 is defined as Me-Phe

<220>

<221>Other features

<222>(8)..(8)

<223>The Xaa of 8 is defined as D- β-NaI

<400>58

Xaa Arg Xaa Pro Gly Xaa Ser Xaa Ile

1 5

<210>59

<211>9

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>Other features

<222>(3)..(3)

<223>The Xaa of 3 is defined as Oic

<220>

<221>Other features

<222>(6)..(6)

<223>The Xaa of 6 is defined as Me-Phe

<220>

<221>Other features

<222>(8)..(8)

<223>The Xaa of 8 is defined as D- β-NaI

<400>59

Leu Arg Xaa Pro Gly Xaa Ser Xaa Ile

1 5

<210>60

<211>9

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>MOD_RES

<222>(1)..(1)

<223>Acetylation

<220>

<221>Other features

<222>(3)..(3)

<223>The Xaa of 3 is defined as Oic

<220>

<221>Other features

<222>(6)..(6)

<223>The Xaa of 6 is defined as Me-Phe

<220>

<221>Other features

<222>(8)..(8)

<223>The Xaa of 8 is defined as D- β NaI

<400>60

Leu Arg Xaa Pro Gly Xaa Ser Xaa Ile

1 5

<210>61

<211>10

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>MOD_RES

<222>(1)..(1)

<223>Acetylation

<220>

<221>Other features

<222>(1)..(1)

<223>The Xaa of 1 is defined as D-Dab

<220>

<221>Other features

<222>(5)..(5)

<223>The Xaa of 5 is defined as Hyp

<220>

<221>Other features

<222>(7)..(7)

<223>The Xaa of 7 is defined as Cpg

<220>

<221>Other features

<222>(9)..(9)

<223>The Xaa of 9 is defined as Dtic

<220>

<221>Other features

<222>(10)..(10)

<223>The Xaa of 10 is defined as Cpg

<400>61

Xaa Leu Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

<210>62

<211>10

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetically prepared

<220>

<221>MOD_RES

<222>(1)..(1)

<223>Acetylation

<220>

<221>Other features

<222>(1)..(1)

<223>The Xaa of 1 is defined as DOrn

<220>

<221>Other features

<222>(5)..(5)

<223>The Xaa of 5 is defined as Hyp

<220>

<221>Other features

<222>(7)..(7)

<223>The Xaa of 7 is defined as Cpg

<220>

<221>Other features

<222>(9)..(9)

<223>The Xaa of 9 is defined as Dtic

<220>

<221>Other features

<222>(10)..(10)

<223>The Xaa of 10 is defined as Cpg

<400>62

Xaa Leu Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

Claims

1. a kind of compound or its pharmaceutically acceptable salt or hydrate with having structure,

Wherein：

E¹For N, O or C；

E²For N or C；

G is singly-bound, double bond, C, N, O, B, S, Si, P, Se or Te；

With

Respectively singly-bound,

With

One of can also be double bond；And when G is C or N,

With

One of can also be double bond；And when G is singly-bound or double bond,

With

All it is not present；

M independently is 0 or 1 in all cases；

N is more than or equal to 1；

O is 0,1,2,3,4 or 5；

R^aH or R independently all is in all cases^b；

2. the compound of claim 1, the compound has general formula structure：

3. the compound of claim 1, the compound has general formula structure：

4. the compound of claim 3, wherein A are the bridged group of saturation, fractional saturation or undersaturated 2,3,4,5 or 6 atoms, the hetero atom that it contains 1,2 or 3 are selected from O, N and S, and remaining bridge atom is carbon atom.

5. the compound of claim 3, wherein A are the bridged group of saturation, fractional saturation or undersaturated 2,3,4,5 or 6 carbon atoms.

6. the compound of claim 3, wherein：

A is the bridged group of undersaturated 4 carbon atoms；

E²For C；With

G is double bond.

7. the compound of claim 1, wherein G are singly-bound or double bond,

WithAll it is not present.

8. the compound of claim 1, wherein G are C, N, O, B, S, Si, P, Se or Te.

9. the compound of claim 1, wherein

With

Respectively singly-bound.

10. the compound of claim 1, wherein：

G is C or N；

WithOne of be double bond.

11. the compound of claim 1, wherein R²For carrier, R³For bioactive compound.

12. the compound of claim 1, wherein R³For carrier, R²For bioactive compound.

13. the compound of claim 1, wherein R³Selected from poly- (alkylene oxide), PVP, poly- (vinyl alcohol), polyoxazoline, poly- (acryloyl morpholine -), poly- (oxygen ethylization polyalcohol), PEG, carboxymethyl cellulose, glucan, polyvinyl alcohol, polyvinylpyrrolidone, poly- 1, 3- dioxolane, poly- 1, 3, 6- trioxanes, homopolymer of amino acids, PPOX, ethylene glycol/propylene glycol copolymers, ethene/copolymer-maleic anhydride, amino acid copolymer, PEG and amino acid copolymer, PPOX/ethylene oxide copolymer and polyethylene glycol/thiomalic acid copolymer；Or their any combination.

14. the compound of claim 1, wherein R³For PEG.

15. the compound of claim 1, wherein R²For B1 peptide antagonists.

16. the compound of claim 1, wherein R²For selected from SEQ ID NO：5-26 and 42-62 B1 peptide antagonists, wherein the peptide has N- ends cysteine residues through modification.

17. the preparation method of the compound of claim 1, this method comprises the following steps：

Or

Wherein J be carbonyl or or its by forms of protection.

18. the preparation method of the compound of claim 1, this method comprises the following steps：

Or

Wherein J be carbonyl or or its by forms of protection.

19. the method for claim 17, wherein J are selected from C (=O), C (OCH₂CH₂O)、C(N(R^a)CH₂CH₂N(R^a))、C(N(R^a)CH₂CH₂O)、C(N(R^a)CH₂CH₂S)、C(OCH₂CH₂CH₂O)、C(N(R^a)CH₂CH₂CH₂N(R^a))、C(N(R^a)CH₂CH₂CH₂O)、C(N(R^a)CH₂CH₂CH₂S)、C(OR^b)₂、C(SR^b)₂With C (NR^aR^b)₂。

20. the method for claim 17, wherein the reaction is carried out in pH between 2 and 7.

21. the method for claim 17, wherein the reaction is carried out in pH between 3 and 5.

22. the method for claim 18, wherein J are selected from C (=O), C (OCH₂CH₂O)、C(N(R^a)CH₂CH₂N(R^a))、C(N(R^a)CH₂CH₂O)、C(N(R^a)CH₂CH₂S)、C(OCH₂CH₂CH₂O)、C(N(R^a)CH₂CH₂CH₂N(R^a))、C(N(R^a)CH₂CH₂CH₂O)、C(N(R^a)CH₂CH₂CH₂S)、C(OR^b)₂、C(SR^b)₂With C (NR^aR^b)₂。

23. the method for claim 18, wherein the reaction is carried out in pH between 2 and 7.

24. the method for claim 18, wherein the reaction is carried out in pH between 3 and 5.

25. a kind of compound with having structure：

Wherein：

E¹For N, O or C；

E²For N or C；

G is singly-bound, double bond, C, N, O, B, S, Si, P, Se or Te；

WithRespectively singly-bound,

With

One of can also be double bond；And when G is C or N,

With

One of can also be double bond；And when G is singly-bound or double bond,

WithAll it is not present；

J be carbonyl or or its by forms of protection；

L¹For divalence C_1-12Alkyl or C_1-12Miscellaneous alkyl, each group is by 0,1,2 or 3 replace selected from following substituent：F、Cl、Br、I、OR^a、NR^aR_aAnd oxo base；

M independently is 0 or 1 in all cases；

N is 1,2,3,4,5,6,7,8,9 or 10；

O is 0,1,2,3,4 or 5；

R³For bioactive compound or carrier；

R^aH or R independently all is in all cases^b；

X is C (=O), and Y is NH；Or X is NH, Y is C (=O).

26. the compound of claim 25, the compound has general formula structure：

27. the compound of claim 25, the compound has general formula structure：

28. the compound of claim 27, wherein A are the bridged group of saturation, fractional saturation or undersaturated 2,3,4,5 or 6 atoms, the hetero atom that it contains 1,2 or 3 are selected from O, N and S, and remaining bridge atom is carbon atom.

29. the compound of claim 27, wherein A are the bridged group of saturation, fractional saturation or undersaturated 2,3,4,5 or 6 carbon atoms.

30. the compound of claim 27, wherein：

A is the bridged group of undersaturated 4 carbon atoms；

E²For C；With

G is double bond.

31. the compound of claim 25, wherein G are singly-bound or double bond, and

With

All it is not present.

32. the compound of claim 25, wherein G are C, N, O, B, S, Si, P, Se or Te.

33. the compound of claim 25, wherein

With

Respectively singly-bound.

34. the compound of claim 25, wherein：

G is C or N；With

With

One of be double bond.

35. the compound of claim 25, wherein R³For bioactive compound.

36. the compound of claim 25, wherein R³For carrier.

37. the compound of claim 25, wherein R³Selected from poly- (alkylene oxide), PVP, poly- (vinyl alcohol), polyoxazoline, poly- (acryloyl morpholine -), poly- (oxygen ethylization polyalcohol), PEG, carboxymethyl cellulose, glucan, polyvinyl alcohol, polyvinylpyrrolidone, poly- 1, 3- dioxolane, poly- 1, 3, 6- trioxanes, homopolymer of amino acids, PPOX, ethylene glycol/propylene glycol copolymers, ethene/copolymer-maleic anhydride, amino acid copolymer, PEG and amino acid copolymer, PPOX/ethylene oxide copolymer and polyethylene glycol/thiomalic acid copolymer；Or their any combination.

38. the compound of claim 25, wherein R³For PEG.

39. the preparation method of the compound of claim 25, this method comprises the following steps：Make (Y-L²)_n-R³Reacted with following compound：

40. the method for claim 39, wherein：

The nucleopilic reagent is selected from SH, NH₂And OH；With

The electrophilic reagent is selected from CH₂Halogen, CH₂SO₂OR^b, C (=O) O (succinimide), C (=O) O (perfluoroalkyl), C (=O) O (CH₂) and C (=O) O (C CN₆F₅)。

41. a kind of method for treating pain and/or inflammation, this method includes the compound for giving the claim 1 of bacterium in need.

42. a kind of pharmaceutical composition, compound of the said composition comprising claim 1 and pharmaceutically acceptable carrier or diluent.

43. the preparation method of the medicine of the compound comprising claim 1.