HK1121375A - Method of conjugating aminothiol containing molecules to a polymer - Google Patents

Description

Method for conjugating molecules containing an aminothiol to a carrier

This application claims priority to U.S. application No. (not yet assigned) on 23/2006 and claims benefit to U.S. provisional application No. 60/646,685 on 24/1/2005, which is incorporated herein by reference.

Background

Recent advances in biotechnology have made possible the large-scale production of biomolecules, such as therapeutic proteins, peptides, antibodies and antibody fragments, all of which are currently produced on a large scale, enabling widespread use of such biomolecules. Unfortunately, the proteolytic degradation of biomolecules is fast, the circulating half-life is short, the solubility is low, the immunogenicity during production, storage or administration is not stable, or after administration often limits their use. As interest in the therapeutic and/or diagnostic use of administering biomolecules has increased, various approaches to overcoming these deficiencies have been investigated.

One of the methods that has been widely studied is: proteins and other potential therapeutic agents are modified by covalent attachment to a carrier such as polyethylene glycol (hereinafter "PEG") (see, e.g., Abuchowski, A. et al, J.biol.chem.252 (11): 3579-. The use of PEG groups attached to proteins or polypeptides (hereinafter "PEGylation") to solve or ameliorate the numerous problems of protein or peptide drugs has been well documented in the literature (see, e.g., Francis et al, International Journal of Hematology, 68: 1-18 (1998); Abuchowski, A. et al, (1977); Chapman, A. et al, adv. drug Del. Rev.54, 531. 545 (2002)); and Roberts, m.j. et al, Advanced Drug delivery reviews, 54: 459-476(2002)).

In short, a carrier derivative having an active group at one or both ends is generally used in order to covalently link (hereinafter referred to as "conjugation") the carrier to an active agent such as a protein, a peptide, a polysaccharide, a polynucleotide, a lipid, or an organic molecule. The reactive groups are selected according to the type of reactive groups available on the molecule to be coupled to the carrier. For example, the following documents provide functionalized polymers: WO96/41813 and J.Pharmaceut.Sci.87, 1446-1449 (1998)). When the carrier is PEG, activated PEG derivatives suitable for reaction with nucleophilic centers of biomolecules (e.g., lysine, cysteine and like residues of proteins or peptides) include PEG-aldehyde, mixed anhydrides, N-hydroxysuccinimide esters, carbonylimidazolidine (carboylimidazolide) and chlorocyanurate. Each of these methods is known to have advantages and disadvantages (Harris, J.M., Herati, R.S., Polym Prepr (am. chem. Soc., div. Polyin. chem), 32 (1): 154-. Some of the more common problems associated with conjugation using known methods include the generation of reactive impurities, labile bonds, side reactions, and/or lack of selectivity for substituents. Furthermore, these problems also imply the complexity of isolating and purifying the desired biologically active conjugate. Sometimes, different amounts of isomers are produced. Such variability presents a potential problem of batch-to-batch reproducibility, the biggest problem being the inability to reproduce biological activity.

Activated Carrier derivatives with thiol-selective functionalities such as maleimide, vinylsulfone, iodoacetamide, thiol and disulfide (Zalipsky, S.Bioconjug. chem.6, 150-165 (1995); Greenwald, R.B. et al, bit.Rev.Ther.Drug Carrier Syst.17, 101-161 (2000); 25 Herman, S.et al, Macromol. chem.Phys.195, 203-209(1994)) are particularly suitable for coupling to cysteine side chains of proteins or peptides. However, these agents are also not without drawbacks, especially when the aim is to develop carrier-conjugated biomolecules for therapeutic use. For example, the PEG maleimide-thiol conjugates initially formed are (R) -and (S) -chiral mixtures. The formation of mixtures makes the development of pegylated biomolecules more complex at multiple levels. For example, one of the enantiomers may have problems with unwanted activity or insecurity compared to the other. Another disadvantage of the PEG maleimide-thiol conjugation method is that: the initially formed adduct is susceptible to rearrangement to thiomorpholine.

There is also a need for conjugates that reproducibly produce two or more linked active agents. In some cases, administration of these "multimeric" complexes containing more than one active agent linked to the same carrier molecule may result in additive and/or synergistic benefits. For example, a complex containing two or more identical binding peptides or polypeptides has a significantly increased affinity for the bound ligand or active site compared to the monomeric polypeptide. Alternatively, a composite consisting of the following (1) and (2) is particularly advantageous: (1) biologically active proteins that exert their effects at specific sites in the body and (2) molecules that target the complex to specific sites. Unfortunately, the above limitations are further compounded by the use of the methods of the present invention to generate vectors conjugated with more than one biologically active or biofunctional molecule. For example, an attempt to conjugate two biologically active molecules to one bivalent PEG-maleimide would result in a different number of 16 separate entities. The method of the present invention is used to generate PEG conjugated to a total of 4 bioactive molecules, for example by using tetravalent PEG-maleimide, so that there may be 256 potential separate attachment sites between PEG and bioactive molecule, and so on. The technical challenges and the deficiencies of existing tools to quantify these individual entities can greatly hinder, or even completely prevent, the development of such biomolecules.

Thus, there is a clear need for a process for preparing active agent conjugates in high yield and purity. Ideally, such conjugates are stable to hydrolysis, require relatively small amounts of reactants to prepare, and can be readily purified using procedures that maintain the integrity of the support or support segment (i.e., under mild reaction conditions) and/or maintain the desired biological activity. The present invention provides novel reagents, methods and conjugates which solve the above-mentioned problems at the state of the art and provide numerous advantages over the prior art.

Summary of The Invention

The present invention relates to carrier derivatives comprising at least one carrier segment having a1, 2-aminothiol (1, 2-aminothiol) or 1, 3-aminothiol selective terminus. The carrier derivatives of the present invention are useful for coupling to molecules comprising 1, 2-aminothiol or 1, 3-aminothiol moieties. One embodiment of the present invention relates to the attachment of one or more active agents to water soluble polymers, including but not limited to PEG.

The present invention provides methods for the preparation of the carrier derivatives of the invention and methods for using the carrier derivatives to prepare novel active agent conjugates.

One aspect of the present invention is directed to a compound having the structure:

wherein:

a is a saturated, partially saturated or unsaturated 2, 3, 4, 5 or 6 atom bridging group containing 0, 1,2 or 3 heteroatoms selected from O, N and S, with the remaining bridging atoms being carbon atoms;

E¹n, O or C;

E²is N or C;

g is a single bond, a double bond, C, N, O, B, S, Si, P, Se or Te;

andeach of which is a single bond,andone may also be a double bond; and when G is C or N, the compound,andone may also be a double bond; and when G is a single bond or a double bond,andall are absent;

L¹is divalent C_1-6Alkyl orC_1-6Heteroalkyl, each of which is substituted with 0, 1,2 or 3 substituents selected from: F. cl, Br, I, OR^a、NR^aR^aAnd an oxo group;

m is independently in each occurrence 0 or 1;

o is 0, 1,2, 3, 4 or 5;

R¹is H, C_1-6Alkyl, phenyl or benzyl, any of which groups is substituted with 0, 1,2 or 3 groups selected from: halogen, cyano, nitro, oxo, -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 radical 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 radical NR^aR^aand-NR^aC_2-6Alkyl OR^aAnd is further substituted with 0, 1,2, 3, 4, 5 or 6 atoms selected from: F. br, Cl and I;

R²as a carrier, R³Is a biologically active compound; or R³As a carrier, R²Is a biologically active compound;

R^ain each case independently H or R^b；

R^bIn each case independently of one another is phenyl, benzyl or C_1-6Alkyl, said phenyl, benzyl and C_1-6Alkyl is substituted with 0, 1,2, or 3 substituents selected from: halogen, C_1-4Alkyl radical, C_1-3Haloalkyl, -OC_1-4Alkyl, OH, -NH₂、-NHC_1-4Alkyl and-N (C)_1-4Alkyl) C_1-4An alkyl group; and

R^cin each case independently from halogen, C_1-4Alkyl radical, C_1-3Haloalkyl, -OC_1-4Alkyl, OH, -NH₂、-NHC_1-4Alkyl and-N (C)_1-4Alkyl) C_1-4An alkyl group.

Another aspect of the invention relates to a compound having the structure:

wherein:

a is a saturated, partially saturated or unsaturated 2, 3, 4, 5 or 6 atom bridging group containing 0, 1,2 or 3 heteroatoms selected from O, N and S, with the remaining bridging atoms being carbon atoms;

E¹n, O or C;

E²is N or C;

g is a single bond, a double bond, C, N, O, B, S, Si, P, Se or Te;

andeach of which is a single bond,andone may also be a double bond; and when G is C or N, the compound,andone may also be a double bond; and when G is a single bond or a double bond,andall are absent;

L¹is divalent C_1-6Alkyl or C_1-6Heteroalkyl, each of which is substituted with 0, 1,2 or 3 substituents selected from: F. cl, Br, I, OR^a、NR^aR^aAnd an oxo group;

m is independently in each occurrence 0 or 1;

n is greater than or equal to 1;

o is 0, 1,2, 3, 4 or 5;

R¹is H, C_1-6Alkyl, phenyl or benzyl, any of which groups is selected by0, 1,2 or 3 toThe following groups: halogen, cyano, nitro, oxo, -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 radical 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 radical NR^aR^aand-NR^aC_2-6Alkyl OR^aAnd is further substituted with 0, 1,2, 3, 4, 5 or 6 atoms selected from: F. br, Cl and I;

R²as a carrier, R³Is a biologically active compound; or R³As a carrier, R²Is a biologically active compound;

R^ain each case independently H or R^b；

R^bIn each case independently of one another is phenyl, benzyl or C_1-6Alkyl, said phenyl, benzyl and C_1-6Alkyl is substituted with 0, 1,2, or 3 substituents selected from: halogen, C_1-4Alkyl radical, C_1-3Haloalkyl, -OC_1-4Alkyl, OH, -NH₂、-NHC_1-4Alkyl and-N (C)_1-4Alkyl) C_1-4An alkyl group; and

R^cin each case independently from halogen, C_1-4Alkyl radical, C_1-3Haloalkyl, -OC_1-4Alkyl, OH, -NH₂、-NHC_1-4Alkyl and-N (C)_1-4Alkyl) C_1-4An alkyl group.

In another embodiment and in combination with the above and below embodiments, a is a saturated, partially saturated or unsaturated 2, 3, 4, 5 or 6 atom bridging group containing 1,2 or 3 heteroatoms selected from O, N and S, with the remaining bridging atoms being carbon atoms.

In another embodiment, and in combination with the above and below embodiments, a is a bridging group of 2, 3, 4, 5 or 6 carbon atoms which is saturated, partially saturated or unsaturated.

In another embodiment, in combination with the above and below embodiments, n is 1.

In another embodiment, in combination with the above and below embodiments, n is 2.

In another embodiment, and in combination with the above and below embodiments, n is 3.

In another embodiment, in combination with the above and below embodiments, n is 4.

In another embodiment, and in combination with the above and below embodiments, n is 5.

In another embodiment, and in combination with the above and below embodiments, n is 6.

In another embodiment, and in combination with the above and below embodiments, n is 7.

In another embodiment, and in combination with the above and below embodiments, n is 8.

In another embodiment, and in combination with the above and below embodiments, a is an unsaturated bridging group of 4 carbon atoms; e²Is C; g is a double bond.

In another embodiment andin combination with the above and the following embodiments, G is a single or double bond, andandall are absent.

In another embodiment and in combination with the above and below embodiments, G is C, N, O, B, S, Si, P, Se, or Te.

In another embodiment in combination with the above and below embodiments,andeach is a single bond.

In another embodiment, and in combination with the above and below embodiments, G is C or N; andandone is a double bond.

In another embodiment, in combination with the above and below embodiments, R²As a carrier, R³Are biologically active compounds.

In another embodiment, in combination with the above and below embodiments, R³As a carrier, R²Are biologically active compounds.

In another embodiment, in combination with the above and below embodiments, R³Selected from poly (alkylene oxide), poly (vinylpyrrolidone), poly (vinyl alcohol), polyoxoalkoxyOxazoline, poly (acryloylmorpholine-), poly (oxyethylated polyol), poly (ethylene glycol), carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, amino acid homopolymers, polypropylene oxide, copolymers of ethylene/propylene glycol, ethylene/maleic anhydride copolymers, amino acid copolymers, copolymers of PEG and amino acids, polypropylene oxide/ethylene oxide copolymers and polyethylene glycol/thiomalic acid copolymers; or any combination thereof.

In another embodiment, in combination with the above and below embodiments, R³Is PEG.

In another embodiment, where the above and below embodiments are combined, n is 1,2, 3, 4, 5,6, 7, 8, 9, or 10.

In another embodiment, in combination with the above and below embodiments, R³Is branched PEG, and n is 2, 3, 4, 5,6, 7, 8, 9 or 10.

In another embodiment, in combination with the above and below embodiments, R²Is a B1 peptide antagonist.

In another embodiment, in combination with the above and below embodiments, R²Is a polypeptide selected from SEQ ID NO: 5-26 and 42-62, wherein the peptide is modified to have an N-terminal cysteine residue.

Another aspect of the present invention relates to a process for the preparation of a compound of claim 1, comprising the steps of:

A) make R²-(C(＝O))_mCH(NH₂)CH₂(CH₂)_mSH reaction with the following Compounds

Or

B) Make R²-[(C(＝O))_mCH(NH₂)CH₂(CH₂)_mSH]_nWith the following compounds:

wherein J is carbonyl or a protected form thereof.

Another aspect of the present invention relates to a process for the preparation of a compound of claim 1, comprising the steps of:

A) make R²-(C(＝O))_mCH(NH₂)CH₂(CH₂)_mSH reacts with the following compounds:

or

B) Make R²-[(C(＝O))_mCH(NH₂)CH₂(CH₂)_mSH]_nWith the following compounds:

wherein J is carbonyl or a protected form thereof.

In another embodiment and in combination with the above and below embodiments, J is selected from C (═ O), C (OCH)₂CH₂O)、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)₂And C (NR)^aR^b)₂。

In another embodiment, in combination with the above and below embodiments, the reaction is carried out at a pH between 2 and 7.

In another embodiment, in combination with the above and below embodiments, the reaction is carried out at a pH between 3 and 5.

Another aspect of the invention relates to a compound having the structure:

wherein:

a is a saturated, partially saturated or unsaturated 2, 3, 4, 5 or 6 atom bridging group containing 0, 1,2 or 3 heteroatoms selected from O, N and S, with the remaining bridging atoms being carbon atoms;

E¹n, O or C;

E²is N or C;

g is a single bond, a double bond, C, N, O, B, S, Si, P, Se or Te;

andeach of which is a single bond,andone may also be a double bond; and when G is C or N, the compound,andone may also be a double bond; and when G is a single bond or a double bond,andall are absent;

j is carbonyl or a protected form thereof;

L¹is divalent C_1-12Alkyl or C_1-12Heteroalkyl, each of which is substituted with 0, 1,2 or 3 substituents selected from: F. cl, Br, I, OR^a、NR^aR^aAnd an oxo group;

m is independently in each occurrence 0 or 1;

n is 1,2, 3, 4, 5,6, 7, 8, 9 or 10;

o is 0, 1,2, 3, 4 or 5;

R¹is H, C_1-6Alkyl, phenyl or benzyl, any of which groups is substituted with 0, 1,2 or 3 groups selected from: halogen, cyano, nitro, oxo, -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 radical 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 radical NR^aR^aand-NR^aC_2-6Alkyl OR^aAnd is further substituted with 0, 1,2, 3, 4, 5 or 6 atoms selected from: F. br, Cl and I;

R³as a biologically active compound or carrier;

R^ain each case independently H or R^b；

R^bIn each case independently of one another is phenyl, benzyl or C_1-6Alkyl, said phenyl, benzyl and C_1-6Alkyl is substituted with 0, 1,2, or 3 substituents selected from: halogen, C_1-4Alkyl radical, C_1-3Haloalkyl, -OC_1-4Alkyl, OH, -NH₂、-NHC_1-4Alkyl and-N (C)_1-4Alkyl) C_1-4An alkyl group;

R^cin each case independently from halogen, C_1-4Alkyl radical, C_1-3Haloalkyl, -OC_1-4Alkyl, OH, -NH₂、-NHC_1-4Alkyl and-N (C)_1-4Alkyl) C_1-4An alkyl group; and

x is C (═ O), Y is NH; or X is NH and Y is C (═ O).

In another embodiment, in combination with the above and below embodiments, n is 1.

In another embodiment, in combination with the above and below embodiments, n is 2.

In another embodiment, and in combination with the above and below embodiments, n is 3.

In another embodiment, in combination with the above and below embodiments, n is 4.

In another embodiment, and in combination with the above and below embodiments, n is 5.

In another embodiment, and in combination with the above and below embodiments, n is 6.

In another embodiment, and in combination with the above and below embodiments, n is 7.

In another embodiment, and in combination with the above and below embodiments, n is 8.

In another embodiment and in combination with the above and below embodiments, a is a saturated, partially saturated or unsaturated 2, 3, 4, 5 or 6 atom bridging group containing 1,2 or 3 heteroatoms selected from O, N and S, with the remaining bridging atoms being carbon atoms.

In another embodiment, and in combination with the above and below embodiments, a is a bridging group of 2, 3, 4, 5 or 6 carbon atoms which is saturated, partially saturated or unsaturated.

In another embodiment, and in combination with the above and below embodiments, a is an unsaturated bridging group of 4 carbon atoms; e²Is C; and G is a double bond.

In another embodiment, and in combination with the above and below embodiments, G is a single or double bond,andall are absent.

In another embodiment and in combination with the above and below embodiments, G is C, N, O, B, S, Si, P, Se, or Te.

In another embodiment in combination with the above and below embodiments,andeach is a single bond.

In another embodiment, and in combination with the above and below embodiments, G is C or N; whileAndone is a double bond.

In another embodiment, in combination with the above and below embodiments, R³Are biologically active compounds.

In another embodiment, in combination with the above and below embodiments, R³Is a carrier.

In another embodiment, in combination with the above and below embodiments, R³Selected from the group consisting of poly (alkylene oxides), poly (vinylpyrrolidone), poly (vinyl alcohol), polyoxazoline, poly (acryloylmorpholine-), poly (oxyethylated polyol), poly (ethylene glycol), carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, amino acid homopolymers, polypropylene oxide, ethylene/propylene glycol copolymers, ethylene/maleic anhydride copolymers, amino acid copolymers, PEG and amino acid copolymers, polypropylene oxide/ethylene oxide copolymers, and polyethylene glycol/thiomalic acid copolymers; or any combination thereof.

In another embodiment, in combination with one anotherEmbodiments, R³Is PEG.

Another aspect of the present invention relates to a process for the preparation of a compound as described above, comprising the steps of: make (Y-L)²)_nR³With the following compounds:

wherein: l is²In each case independently C_1-6Alkyl or C_1-6Heteroalkyl, each of which is substituted with 0, 1,2, 3 or 4 substituents selected from: F. cl, Br, I, OR^a、NR^aR^aAnd an oxo group;

x is a nucleophile and Y is an electrophile; or X is an electrophile and Y is a nucleophile.

In another embodiment of the invention, the nucleophile is selected from NH₂And OH; and the electrophile is selected from CH₂Halogen, CH₂SO₂OR^b、C(＝O)NR^aR^bAnd C (═ O) OR^b。

Another aspect of the invention relates to a method of treating pain and/or inflammation comprising administering to a patient in need thereof a therapeutically effective amount of a compound as described above.

Another aspect of the invention relates to a pharmaceutical composition comprising a compound as described above and a pharmaceutically acceptable carrier or diluent.

Another aspect of the invention relates to a process for the preparation of a medicament comprising a compound as described above.

Another aspect of the invention relates to a method for the preparation of a medicament for the treatment of pain and/or inflammation, said medicament comprising a compound as described above.

One aspect of the present invention relates to a compound having the structure:

wherein:

a is selected from i)2 carbon atoms, or sp³Hybridisation or sp²Hybridization (substituted or unsubstituted), wherein the two carbon atoms can be cyclic or acyclic, and connect the two carboxyl groups of the electrophile, or ii)3 atoms selected from carbon (substituted or unsubstituted, cyclic or acyclic), nitrogen (substituted or unsubstituted, cyclic or acyclic), or oxygen (cyclic or acyclic); and

b is selected from i)2 carbon atoms, or sp³Hybridisation or sp²Hybridization (substituted or unsubstituted) wherein the two carbon atoms may be cyclic or acyclic and connect the two carboxyl groups of the electrophile, or ii)3 atoms selected from carbon (substituted or unsubstituted, cyclic or acyclic), nitrogen (substituted or unsubstituted, cyclic or acyclic) or oxygen (cyclic or acyclic).

In one embodiment, in combination with the above and below embodiments, R¹Is H, C_1-6Alkyl, phenyl or benzyl, any of which groups is substituted with 0, 1,2 or 3 groups selected from: halogen, cyano, nitro, oxo, -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 radical 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 radical NR^aR^aand-NR^aC_2-6Alkyl OR^aAnd is further substituted with 0, 1,2, 3, 4, 5 or 6 atoms selected from: F. br, Cl and I;

in one embodiment, in combination with the above and below embodiments, R³Selected from the group consisting of poly (alkylene oxides), poly (vinylpyrrolidone), poly (vinyl alcohol), polyoxazoline, poly (acryloylmorpholine-), poly (oxyethylated polyol), poly (ethylene glycol), carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, amino acid homopolymers, polypropylene oxide, ethylene/propylene glycol copolymers, ethylene/maleic anhydride copolymers, amino acid copolymers, PEG and amino acid copolymers, polypropylene oxide/ethylene oxide copolymers, and polyethylene glycol/thiomalic acid copolymers; or any combination thereof.

In another embodiment and in combination with the above and below embodiments, the carrier segment is a poly (ethylene oxide).

In another embodiment and in combination with the above and below embodiments, the carrier is a linear structure.

In another embodiment and in combination with the above and below embodiments, the carrier is PEG.

In another embodiment, and in combination with the above and below embodiments, the polycyclic N, S-heterocycle is (9bS) (9bH) -2, 3-dihydrothiazolo [2, 3-a ]]Isoindol-5-ones, R²Is a protein orPeptide, R³Is PEG.

In another embodiment, in combination with the above and below embodiments, R²Is a B1 peptide antagonist.

In another embodiment and in combination with the above and below embodiments, the B1 peptide antagonist is a peptide selected from the group consisting of SEQ ID NOs: 5-26 and 42-62, wherein the peptide is modified to have an N-terminal cysteine residue.

In another embodiment, in combination with the above and below embodiments, the carrier is a branched structure having two or more water-soluble segments.

In another embodiment and in combination with the above and below embodiments, the carrier is a branched PEG (bpeg) with more than two PEG segments, i.e. a bifurcated PEG (fpeg).

In another embodiment, and in combination with the above and below embodiments, the polycyclic N, S-heterocycle is (9bS) (9bH) -2, 3-dihydrothiazolo [2, 3-a ]]Isoindol-5-ones, R²Is a protein or peptide.

In another embodiment, and in combination with the above and below embodiments, the bPEG has 3-8 polymer segments- (bPEG)_3-8。

In another embodiment, and in combination with the above and below embodiments, at least one of the segments of the bPEG has a terminus that is activated by an amine (C- [ (bPEG)_3-8]-(NH₂)_1-8)。

In another embodiment, and in combination with the above and below embodiments, the bPEG has 4 polymer segments (C- [ (bPEG)₄]-(NH₂)_1-4) And wherein at least one of said segments has an amine-activated terminus.

In another embodiment and in combination with the above and below embodiments, at least 50% of the segments have ends that are activated by an amine.

In another embodiment and in combination with the above and below embodiments, at least one of the polymer segments is end-capped.

In another embodiment, and in combination with the above and below embodiments, the PEG has a nominal average molecular weight of about 200 daltons to about 100,000 daltons.

In another embodiment, and in combination with the above and below embodiments, the PEG has a nominal average molecular weight of about 5,000 daltons to about 60,000 daltons.

In another embodiment, and in combination with the above and below embodiments, the PEG has a nominal average molecular weight of about 10,000 daltons to about 40,000 daltons.

In another embodiment, in combination with the above and below embodiments, R²In all cases, B1 peptide antagonists.

In another embodiment and in combination with the above and below embodiments, the B1 peptide antagonist is selected from the group consisting of SEQ ID NOs: 27-35 and 38-62.

In another embodiment, in combination with the above and below embodiments, R²In one case a B1 peptide antagonist.

In another embodiment, in combination with the above and below embodiments, R²In 2 of 4 cases, B1 peptide antagonists.

In another embodiment, in combination with the above and below embodiments, R²In 3 of 4 cases, B1 peptide antagonists.

In another embodiment and in combination with the above and below embodiments, the B1 peptide antagonists are each independently selected from the group consisting of SEQ ID NOs: 27-34 and 38-62.

In another embodiment, in combination with the above and below embodiments, R²In at least one instance, is an agent that is not a B1 peptide antagonist.

Another aspect of the invention relates to a pharmaceutical composition comprising any of the above compounds and a pharmaceutically acceptable excipient.

Another aspect of the invention relates to the administration of a pharmaceutical composition comprising any of the above compounds, together with pharmaceutically acceptable excipients, said administration being parenteral, transmucosal or transdermal.

In another embodiment, combined with the above and the following embodiments, the transmucosal means oral, nasal, pulmonary, vaginal or rectal.

In another embodiment, combined with the above and below embodiments, the parenteral means intraarterial, intravenous, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, intraocular, intraorbital, or intracranial.

In another embodiment and in combination with the above and below embodiments, the administering is oral administration.

In another embodiment and in combination with the above embodiment, the polypeptide or peptide comprises a Tat-inhibitory polypeptide comprising R-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-X- (biotin) -Cys-NH₂And biologically and pharmaceutically acceptable salts, stereoisomers, optical isomers and geometric isomers thereof, including retro-inverso analogs (if any), and pharmaceutically acceptable salts and solvates thereof, wherein R comprises a carboxylic acid or acetyl residue; x is a Cys residue.

In another embodiment and in combination with the above and below embodiments, the polypeptide or peptide comprising an aminothiol compound comprises an amino acid sequence selected from the group consisting of: N-acetyl-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Cys- (biotin) -Cys-NH₂(SEQ ID NO: 64), N-acetyl-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Lys- (biotin) -Cys-NH₂(SEQ ID NO: 65), N-acetyl-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-D-Cys- (biotin) -Cys-NH₂(SEQ ID NO: 66), N-acetyl-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-D-Lys- (biotin) -Cys-NH₂(SEQ ID NO: 67), N-acetyl-Gln-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-D-Lys- (biotin) -Cys-NH₂(SEQ ID NO: 68), N-acetyl-Arg-Lys-Lys-Arg-Arg-Pro-Arg-Arg-Arg-Cys- (biotin) -Cys-NH₂(SEQ ID NO: 69), N-acetyl-DCys-DLys- (biotin) -DArg-DArg-DArg-DGln-DArg-DArg-DLys-DLys-DArg-NH₂Or a biologically and pharmaceutically acceptable salt thereof.

In another embodiment and in combination with the previous and subsequent embodiments, the carrier is selected from the group consisting of poly (ethylene glycol), carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, amino acid homopolymer, polypropylene oxide, ethylene glycol/propylene glycol copolymer, ethylene/maleic anhydride copolymer, amino acid copolymer, PEG and amino acid copolymer, polypropylene oxide/ethylene oxide copolymer, and PEG/thiomalic acid copolymer, or any combination thereof.

In another embodiment, and in combination with the above and below embodiments, the polymer has a molecular weight of about 100 daltons to about 200,000 daltons.

In another embodiment, and in combination with the above and below embodiments, the molecular weight of the polymer is from about 2,000 daltons to about 50,000 daltons.

In another embodiment, and in combination with the above and below embodiments, the spacer is from about 100 daltons to about 10,000 daltons.

In another embodiment, and in combination with the above and below embodiments, the spacer is from about 300 daltons to about 5,000 daltons.

Another aspect of the invention relates to a process for the preparation of a1, 2-aminothiol or 1, 3-aminothiol selective carrier derivative, which process comprises the steps of:

(a) providing a vector comprising at least one vector segment having the formula:

Y-R³

wherein Y is a nucleophile or electrophile, R₃Is a carrier.

(b) Reacting the carrier derivative with a molecule comprising a1, 2-aminothiol or 1, 3-aminothiol selective moiety having the formula:

wherein A is i)2 carbon atoms, or sp³Hybridisation or sp²Hybridization (substituted or unsubstituted), wherein the two carbon atoms can be cyclic or acyclic, and connect the two carboxyl groups of the electrophile, or ii)3 atoms selected from carbon (substituted or unsubstituted, cyclic or acyclic), nitrogen (substituted or unsubstituted, cyclic or acyclic), or oxygen (cyclic or acyclic); wherein R is¹Selected from H and electron withdrawing groups; wherein R is²Alkyl; wherein X is an electrophile when Y is a nucleophile, or a nucleophile when Y is an electrophile.

In another embodiment, in combination with the above and below embodiments, a has the structure of formula:

in another embodiment, and in combination with the above and below embodiments, a is acyclic.

In another embodiment and in combination with the above and below embodiments, F is carbon and D is selected from the group consisting of i) carbon ii) oxygen and iii) nitrogen.

In another embodiment and in combination with the above and below embodiments, D is carbon, E is selected from carbon substituted with X, nitrogen substituted with X, oxygen, sulfur, silicon substituted with X, boron substituted with X, a bond, phosphorus substituted with X; or ii) oxygen, E is selected from carbon, nitrogen, silicon, boron and a bond; or iii) nitrogen, E is selected from carbon, nitrogen, oxygen, silicon, sulfur, boron and chemical bonds.

In another embodiment, in combination with the above and below embodiments, a has the structure of formula:

in another embodiment and in combination with the above and below embodiments, F is carbon and D is selected from the group consisting of i) carbon, ii) oxygen, and iii) nitrogen.

In another embodiment and in combination with the above and below embodiments, Y is an acid.

In another embodiment and in combination with the above and below embodiments, Y is an amine.

In another embodiment and in combination with the above and below embodiments, Y is a primary amine.

In another embodiment, and in combination with the above and below embodiments, greater than 95% of Y is covalently attached to the 1, 2-aminothiol or 1, 3-aminothiol selective moiety.

In another embodiment, in combination with the above and below embodiments, at least one of said R₃Selected from H, alkyl, C₁-C₁₀Linear alkyl, poly (alkylene oxide), poly (vinylpyrrolidone), poly (vinyl alcohol), polyoxazoline, poly- (acryloylmorpholine-), poly (oxyethylated polyol), and poly (ethylene oxide).

In another embodiment and in combination with the above and below embodiments, the support has a branched, bifurcated, or multi-armed structure.

In another embodiment combined with the above and below embodiments, at least R³Is PEG.

In another embodiment, and in combination with the above and below embodiments, the carrier has a nominal average molecular weight of from about 200 daltons to about 100,000 daltons.

In another embodiment, and in combination with the above and below embodiments, the method further comprises a first step of purifying the support such that > 95% of the segments have amine-activated ends.

In another embodiment and in combination with the above and below embodiments, the purification step comprises chromatographic separation or chemical separation.

In another embodiment and in combination with the above and below embodiments, the purification step comprises cation exchange chromatography.

In another embodiment and in combination with the above and below embodiments, the nucleophile is selected from a secondary amine, a hydroxyl group, an imino group, or a thiol.

In another embodiment and in combination with the above and below embodiments, the electrophile is an activated ester.

In another embodiment and in combination with the above and below embodiments, the activated ester is selected from the group consisting of N-hydroxysuccinimide group, succinimide group, N-hydroxybenzotriazolyl group, perfluorophenyl group, alkylated moieties (e.g., chloroalkanes, bromoalkanes, iodoalkanes), activated alcohols (e.g., methanesulfonyl-, trifluoromethanesulfonyl-, p-toluenesulfonyl-, trichloroiminoacetate), and in situ activated alcohols such as triphenylphosphonium ether.

In another embodiment, and in combination with the above and below embodiments, Y is selected from the group consisting of alkoxy, substituted alkoxy, alkenyloxy, substituted alkenyloxy, alkynyloxy, substituted alkynyloxy, aryloxy, and substituted aryloxy.

In another embodiment, and in combination with the above and below embodiments, the PEG has a nominal average molecular weight of about 5,000 daltons to about 60,000 daltons.

In another embodiment, and in combination with the above and below embodiments, the PEG has a nominal average molecular weight of about 10,000 daltons to about 40,000 daltons.

Another aspect of the present invention relates to a process for preparing a subject composition (matter), the process comprising the steps of:

(a) providing a vector comprising at least one vector segment having the formula:

Y-R³

wherein Y is a nucleophile or electrophile, R₃Is a carrier.

(b) Reacting the carrier derivative with a molecule comprising a1, 2-aminothiol or 1, 3-aminothiol selective moiety having the formula:

wherein A is i)2 carbon atoms, or sp³Hybridisation or sp²Hybridization (substituted or unsubstituted), wherein the two carbon atoms can be cyclic or acyclic, and connect the two carboxyl groups of the electrophile, or ii)3 atoms selected from carbon (substituted or unsubstituted, cyclic or acyclic), nitrogen (substituted or unsubstituted, cyclic or acyclic), or oxygen (cyclic or acyclic); wherein R is¹Selected from H and electron withdrawing groups; wherein X is an electrophile when Y is a nucleophile, or a nucleophile when Y is an electrophile; and

(c) reacting the main product from steps (a) and (b) with an active agent or substrate comprising a1, 2-aminothiol or a1, 3-aminothiol.

In another embodiment and in combination with the above and below embodiments, the active agent is a polypeptide or peptide.

In another embodiment and in combination with the above and below embodiments, the peptide is a B1 peptide antagonist.

In another embodiment and in combination with the above and below embodiments, the peptide is a peptide selected from the group consisting of SEQ id nos: 27-35 and 38-41.

In another embodiment and in combination with the above and below embodiments, the peptide is selected from the group consisting of SEQ id nos: 11-26 and 43-46, and further comprises a cysteine at the N-terminus of the peptide.

In another embodiment and in combination with the above or below embodiments, the 1, 2-aminothiol or 1, 3-aminothiol selective moiety is a1, 2-formyl ester or a1, 3-formyl ester.

In another embodiment and in combination with the above and below embodiments, the electrophile is an acid.

In another embodiment, and in combination with the above and below embodiments, the nucleophile is an amine.

In another embodiment and in combination with the above and below embodiments, the electrophile is a primary amine.

In another embodiment and in combination with the above embodiment, the carrier segment is selected from the group consisting of poly (alkylene oxide), poly (vinylpyrrolidone), poly (vinyl alcohol), polyoxazoline, poly (acryloylmorpholine-), poly (oxyethylated polyol), poly (ethylene glycol), carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, amino acid homopolymer, polypropylene oxide, ethylene glycol/propylene glycol copolymer, ethylene/maleic anhydride copolymer, amino acid copolymer, PEG and amino acid copolymer, polypropylene oxide/ethylene oxide copolymer, and polyethylene glycol/thiomalic acid copolymer; or any combination thereof.

In another embodiment, in combination with the above and below embodiments, the¹³C NMR measurement of contents of¹³Activated end of C, or by absence¹³Other suitable methods exist for the activation of the ends of carbons, more than 95% of which are covalently bound to a1, 2-aminothiol or 1, 3-aminothiol selective moiety.

In another embodiment and in combination with the above and below embodiments, the carrier segment is a poly (ethylene oxide).

In another embodiment and in combination with the above and below embodiments, the carrier segment is polyethylene glycol (PEG).

In another embodiment, and in combination with the above and below embodiments, the PEG has a linear, branched (bPEG), branched (fPEG), or multi-arm structure.

In another embodiment and in combination with the above and below embodiments, the branched PEG has 3-8 polymer segments (C- [ bPEG)_3-8])。

In another embodiment and in combination with the above and below embodiments, at least one of the segments has a terminus activated by an amine (C- [ bPEG)_3-8]-(NH₂)_1-8)。

In another embodiment and in combination with the above and below embodiments, the bPEG has 4 polymer segments (C- [ bPEG)₄]-(NH₂)_1-4) Wherein at least one of said segments has a terminus activated by an amine.

In another embodiment and in combination with the above and below embodiments, at least 50% of the ends of the segments are activated by an amine.

In another embodiment and in combination with the above and below embodiments, at least one of the polymer segments is end-capped.

In another embodiment, and in combination with the above and below embodiments, the PEG has a nominal average molecular weight of about 200 daltons to about 100,000 daltons.

In another embodiment, and in combination with the above and below embodiments, the method further comprises a first step of purifying the amine-activated support such that > 95% of the segments have amine-activated ends.

In another embodiment and in combination with the above and below embodiments, the purifying step comprises chromatographic separation or chemical separation.

In another embodiment and in combination with the above and below embodiments, the purification step comprises cation exchange chromatography.

In another embodiment and in combination with the above and below embodiments, the nucleophile is selected from a secondary amine, a hydroxyl group, an imino group, or a thiol.

In another embodiment and in combination with the above and below embodiments, the electrophile is an activated ester.

In another embodiment and in combination with the above and below embodiments, the activated ester is selected from the group consisting of N-hydroxysuccinimide group, succinimide group, N-hydroxybenzotriazolyl group, perfluorophenyl group, alkylated moieties (e.g., chloroalkanes, bromoalkanes, iodoalkanes), activated alcohols (e.g., methanesulfonyl-, trifluoromethanesulfonyl-, p-toluenesulfonyl-, trichloroiminoacetate), and in situ activated alcohols such as triphenylphosphonium ether.

In another embodiment and in combination with the above and below embodiments, the end capping group (cap) comprises a chemical group selected from the group consisting of: alkoxy, substituted alkoxy, alkenyloxy, substituted alkenyloxy, alkynyloxy, substituted alkynyloxy, aryloxy, and substituted aryloxy.

In another embodiment and in combination with the above and below embodiments, the end-capping group further comprises a radioactive group, a magnetic group, a colorimetric group, or a fluorescent group.

In another embodiment, and in combination with the above and below embodiments, the PEG has a nominal average molecular weight of about 5,000 daltons to about 60,000 daltons.

In another embodiment, and in combination with the above and below embodiments, the PEG has a nominal average molecular weight of about 10,000 daltons to about 40,000 daltons.

In another embodiment and in combination with the above or below embodiments, the polypeptide or peptide is selected from the group consisting of a biological transporter, receptor, binding or targeting ligand of any moiety capable of binding to a cell surface component, including, but not limited to, vitamins (e.g., biotin, folic acid, pantothenic acid, B-6, B-12), sugars (e.g., glucose, N-acetylglucosamine), chemokines (e.g., RANTES, IL-2, OPG), peptide (or non-peptide) vectors (e.g., Tat, fMLF, transmembrane peptide (pentrat), VEGF [ a glycoprotein ], transferrin), retro-inverso peptides (e.g., TAT), membrane fusion peptides (e.g., gp41, VEGF [ a glycoprotein ]), lipids (or phospholipids) (e.g., myristic acid, stearic acid), sense (or antisense) oligonucleotides (e.g., aptamers containing 5- (1-pentyl) -2' -deoxyuridine), aptamers, and mixtures thereof, Enzymes (e.g., neuraminidase), toxins, antibodies (or antibody fragments) (e.g., CD4[ target helper T cells ], CD44[ target ovarian cancer cells ]), antigens (or epitopes) (e.g., influenza virus hemagglutinin), peptide ligands, hormones (e.g., estrogen, progesterone, LHRH, ACTH, growth hormone), adhesion molecules (e.g., lectin, ICAM), and analogs of any of the above molecules.

In another embodiment, and in combination with the above and below embodiments, the active agent comprises a1, 2-aminothiol group (1, 2-aminothiol group) or a1, 3-aminothiol group or is derivatized with a1, 2-aminothiol group or a1, 3-aminothiol group.

Another aspect of the invention relates to a method for identifying a compound suitable for therapeutic or diagnostic use, said compound being free of components having an adverse effect on the biological activity of the peptide or protein component of said compound, said method comprising preparing a compound of the invention and screening said compound for the biological activity of the therapeutic and/or diagnostic moiety contained therein.

One embodiment of the present invention is a process for the preparation of a1, 2-aminothiol or 1, 3-aminothiol selective derivative of a carrier, which process comprises the steps of:

(a) providing a support having at least one support segment, at least one end of the support segment being activated by a nucleophile or electrophile; and

(b) reacting the polymer with a molecule comprising a1, 2-aminothiol or 1, 3-aminothiol selective moiety, or protected form thereof, as defined by the following general formula I to form a covalent linkage:

formula I

Thereby forming a carrier derivative comprising a1, 2-aminothiol or a1, 3-aminothiol selective terminus or protected form thereof, wherein A is i)2 carbon atoms, or sp³Hybridisation or sp²Hybridization (substituted or unsubstituted) wherein the two carbon atoms may be cyclic or acyclic and connect the two carboxyl groups of the electrophile, or ii)3 atoms selected from carbon (substituted or unsubstituted, cyclic or acyclic), nitrogen (substituted or unsubstituted, cyclic or acyclic) or oxygen (cyclic or acyclic).

Another embodiment of the present invention is a method of preparing the subject composition, the method comprising the steps of:

(a) providing a support having at least one support segment that is activated by a nucleophile or electrophile;

(b) reacting said support with a reagent comprising a1, 2-aminothiol or 1, 3-aminothiol selective moiety or protected form thereof as defined in formula I, wherein A is I)2 carbon atoms, or sp³Hybridisation or sp²Hybridization (substituted or unsubstituted), wherein the two carbon atoms can be cyclic or acyclic, and connect the two carboxyl groups of the electrophile, or ii)3 atoms selected from carbon (substituted or unsubstituted, cyclic or acyclic), nitrogen (substituted or unsubstituted, cyclic or acyclic), or oxygen (cyclic or acyclic); and

(c) reacting the main product of steps (a) and (b) with a reactive agent comprising 1, 2-aminothiol or 1, 3-aminothiol. A general description of such a process is given in scheme 1 below:

reaction scheme 1

R₁H, alkyl, ethynyl; r₂Alkyl, R₃H, alkyl, polymer, biologically active substance. A is 2 or 3 carbon atoms; b ═ 2 or 3 atoms;

x and Y are two groups capable of forming a covalent linkage, i.e. X ═ electrophile and Y ═ nucleophile

The general reaction shown above (scheme 1) is particularly advantageous when the support is a multivalent support comprising multiple activated support segments. In such cases, the methods of the invention are effective to produce high yields and relatively pure conjugates that are functionalized (as defined herein) on virtually every suitable activating support segment of the polymer. In one embodiment, multiple agents may be conjugated to a single branched carrier. In one non-limiting example, the present invention provides a biocompatible water-soluble polymer having multiple branches conjugated to a peptide antagonist.

In accordance with features and principles of the present invention, various agents can be operatively conjugated to an activating support through appropriate reactive groups on the agent. Such agents include, but are not limited to, bioactive or diagnostic agents.

In another embodiment of the invention and in combination with the above and below embodiments, the agent may be a small molecule compound having pharmacological activity. Alternatively, the agent may be a retro-inverted or optimized form of a biologically active peptide which has the same or similar biological activity as the original form, but has other desirable characteristics, such as reduced sensitivity to enzymatic attack or metabolic enzymes. More specifically, the agent may include, but is not limited to, an antibody or antibody fragment. Reagents containing the appropriate 1, 2-aminothiol or 1, 3-aminothiol group may be of synthetic origin or naturally occurring in the particular reagent. Thus, the agent may have or be modified to have a1, 2-or 1, 3-group, or may be conjugated to a compound having a1, 2-amino thiol group or a1, 3-amino thiol group, such as a biologically active agent containing a modified peptide or cysteine.

An exemplary aspect of the invention includes a method of preparing a carrier-conjugated B1 peptide antagonist, including but not limited to carrier-conjugated B1 peptide antagonists described in: co-pending U.S. application serial No. 10/972,236 (filed on day 10/21 2004), which was published as U.S. patent application publication No. 2005/0215470 (on day 9/29 2005) (hereinafter "U.S. application '236'").

It is another object of the present invention to provide a pharmaceutical composition comprising an excipient carrier material having dispersed therein at least one carrier-conjugation agent of the invention.

It is another object of the present invention to provide methods of treating B1 mediated diseases, conditions, or disorders, comprising administering a pharmaceutically effective amount of a composition comprising an excipient and at least one of the carrier-conjugated B1 peptide antagonist of the present invention or one of the carrier-conjugated B1 peptide antagonists prepared using the agents and methods of the present invention.

The novel carrier-conjugated B1 peptide antagonists and carrier-conjugated B1 peptide antagonists of the present invention, produced using the reagents and methods of the present invention, are useful in the treatment or prevention of a broad spectrum of B1-mediated diseases, conditions or disorders, including but not limited to cancer and the diseases, conditions or disorders disclosed in U.S. application '236', including but not limited to inflammatory and neuropathic-induced inflammatory and chronic pain states, septic shock, arthritis, osteoarthritis, angina, cancer, asthma, allergic rhinitis, and migraine.

The present vector-conjugated B1 peptide antagonists or vector-conjugated B1 peptides produced using the present agents and methods are useful for treating or preventing the diseases and disorders described above and below by formulating them into compositions with suitable pharmaceutical carrier materials known in the art and administering an effective amount of the compositions to a patient, e.g., a human (or other mammal), in need thereof.

These and other aspects of the invention will be apparent upon consideration of the following drawings and detailed description of the invention.

Detailed Description

The headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including but not limited to patents, patent applications, articles, books, and clusters of articles, are hereby incorporated by reference for any purpose. In the event that a term defined by one or more of the referenced documents conflicts with a term defined herein, the present application controls.

Definition of

Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and the production and identification of antibodies or antibody fragments. The foregoing techniques and methods may be generally practiced according to conventional methods well known in the art and in various general and specific references that are cited and discussed herein. See, e.g., Molecular Cloning, Sambrook et al: a Laboratory Manual (2 nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). Unless otherwise indicated, the nomenclature used in the laboratory procedures and techniques of analytical chemistry, synthetic organic chemistry, and medicinal chemistry described herein is well known and commonly employed in the art. Standard techniques are available for chemical synthesis, peptide synthesis, chemical analysis, chemical purification, pharmaceutical preparation, formulation, administration and treatment of patients.

In this application, the use of the singular includes the plural unless otherwise stated. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including" as well as other forms is not limiting.

Natural amino acid residues are discussed in three ways: the amino acids are fully named, according to the standard 3-letter code or standard single letter code shown in the following table.

In certain embodiments, one or more non-canonical amino acids may be incorporated into the polypeptide. The term "non-conventional amino acid" refers to any amino acid that is not one of the 20 conventional amino acids. The term "non-naturally occurring amino acid" refers to an amino acid not found in nature. Non-naturally occurring amino acids are a group of unconventional amino acids. Non-conventional amino acids include, but are not limited to, stereoisomers of 20 conventional amino acids (e.g., D-amino acids), non-natural amino acids such as α -, α -disubstituted amino acids, N-alkyl amino acids, lactic acid, homoserine, homocysteine, 4-hydroxyproline, γ -carboxyglutamic acid, ε -N, N, N-trimethyllysine, ε -N-acetyl lysine, O-phosphoserine, N-acetyl serine, N-formyl methionine, 3-methylhistidine, 5-hydroxylysine, σ -N-methylarginine, and other similar amino acids and imino acids known in the art (e.g., 4-hydroxyproline). In the polypeptide symbols used herein, the left-hand orientation is the amino-terminal orientation and the right-hand orientation is the carboxy-terminal orientation, according to standard usage and convention. Unless otherwise indicated, the designation of natural or unnatural amino acids herein includes both D-and L-isomers of the amino acids. Other abbreviations for certain unnatural amino acids used herein are the same as those described in the following references: U.S. Pat. No. 5,834,431, PCT publication No. WO98/07746 and Neugebauer et al (2002). In addition, the abbreviations "Dab" and "D-Dab" refer to the L-and D-isomers of the unnatural amino acid D-2-aminobutyric acid, respectively. The abbreviations "3 ' -Pal" and "D-3 ' -Pal" refer to the L-and D-isomers, respectively, of the unnatural amino acid 3 ' -pyridylalanine. Also, the abbreviation "Igl" includes both "Igla" and "Iglb" (α - (1-indanyl) glycine and α - (2-indanyl) glycine, respectively). Similarly, "D-Igl" includes both "D-Igla" and "D-Iglb" (D-isomers of α - (1-indanyl) glycine and α - (2-indanyl) glycine, respectively). Preferably, when used herein, Igl is Iglb and D-Igl is D-Iglb.

Various other abbreviations used in this application are listed below:

ACN, MeCN-acetonitrile

APCI MS-atmospheric pressure chemical ionization mass spectrum

AgNO₃-silver nitrate (I)

AIBN-2, 2' -azobis (2-methylpropanenitrile)

BBr₃Boron tribromide

t-BDMS-Cl-tert-butyldiethylsilyl chloride

CCl₄-carbon tetrachloride

Cs₂CO₃-cesium carbonate

CHCl₃-chloroform

CH₂Cl₂DCM-dichloromethane

CuBr-cupric bromide

CuI-copper iodide

DIBAL-diisobutylaluminum hydride

DIC-1, 3-diisopropylcarbodiimide

DIEA，(iPr)₂Net

DIPEA，Hunigs

Base-diisopropylethylamine

DCE-dichloroethane

DCM-N-hydroxysuccinimide

DME-dimethoxyethane

DMF-dimethylformamide

DMAP-4-dimethylaminopyridine

DMSO-dimethyl sulfoxide

DSS-trimethylsilyl-2-silapentane-5-sulfonate-d 6,

sodium salt

EDC-1- (3-dimethylaminopropyl) -3-ethylcarbodiimide

Et₂O-Ether

EtOAc-ethyl acetate

FBS-fetal bovine serum

FTMS-Fourier transform mass spectrum

G, gm, G-gram

h, hr-hour

H₂-hydrogen gas

HATU-hexafluorophosphate O- (7-azabenzotriazol-1-yl) -

N, N, N ', N' -tetramethyluronium

HBr-hydrobromic acid

HCl-hydrochloric acid

HOBt-1-hydroxybenzotriazole hydrate

HPLC-high pressure liquid chromatography

HRMS-high resolution Mass Spectrometry

IPA, i-PrOH-isopropyl alcohol

K₂CO₃-Potassium carbonate

KI-potassium iodide

LiCl-lithium chloride

LiOH-lithium hydroxide

MgSO₄Magnesium sulfate

MeOH-methanol

MW-molecular weight

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

N₂-nitrogen gas

NaCNBH₃-Cyanoborohydride sodium salt

NaHCO₃Sodium bicarbonate

NaH-sodium hydride

NaOCH₃Sodium methoxide

NaOH-sodium hydroxide

Na₂SO₄-sodium sulphate

NBS-N-bromosuccinimide

NH₄Cl-ammonium chloride

NH₄OH-ammonium hydroxide

NMP-N-methylpyrrolidone

P(t-bu)₃-tri (tert-butyl) phosphine

PBS-phosphate buffered saline solution

RT, RT-Room temperature

TBAF-tetra-n-butylammonium fluoride

TBTU-tetrafluoroboric acid O-benzotriazol-1-yl-N, N, N', N-

Tetramethyluronium salts

TEA，Et₃N-Triethylamine

TFA-trifluoroacetic acid

THF-tetrahydrofuran

As used in accordance with this specification, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

the term "active agent" is meant to include any therapeutic, bioactive and/or diagnostic agent. The term "B1" refers to the bradykinin B1 receptor (see Judith M Hall, A reviews of BK receptors (BK receptor review); Pharmac. Ther., 56: 131-190 (1992)). Unless otherwise indicated, B1 or bradykinin B1 receptor refers to the human bradykinin B1 receptor (hB 1). Preferred hB1 is the wild-type receptor. More preferred hB1 is the bradykinin receptor described in GenBank accession No. AJ 238044.

The compounds of the invention may generally have several asymmetric centers, and are generally described in the form of a racemic mixture. The present invention includes racemic mixtures, partially racemic mixtures and individual enantiomers and diastereomers.

Unless otherwise indicated, the following definitions apply to the terms in the specification and claims:

“C_α-βalkyl "refers to a branched, cyclic or straight chain or any combination of these three containing at least α and up to β carbon atoms, where α and β represent integers.The alkyl groups described in this subsection may also contain one or two double or triple bonds. C_1-6Examples of alkyl groups include, but are not limited to, the following groups:

“C_α-βheteroalkyl "means C with any of the carbon atoms in the alkyl replaced with O, N or S_α-βAn alkyl group. C_1-6Examples of heteroalkyl groups include, but are not limited to, the following:

"leaving group" generally refers to a group that is easily displaced by a nucleophile, such as an amine, thiol, or alcohol nucleophile. Such leaving groups are well known in the art. Examples of such leaving groups include, but are not limited to, N-hydroxysuccinimide, N-hydroxybenzotriazole, halides, triflates, tosylates, and the like. Preferred leaving groups are as indicated herein, where appropriate. "protecting group" generally refers to groups well known in the art for protecting a selected reactive group (e.g., carboxyl, amino, hydroxyl, sulfhydryl, etc.) from undesired reactions (e.g., nucleophilic, electrophilic, oxidative, reductive, etc.). Where appropriate, preferred protecting groups are as shown herein. Examples of amino protecting groups include, but are not limited to, aralkyl, substituted aralkyl, cycloalkenylalkyl and substituted cycloalkenylalkyl, allyl, substituted allyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, silyl, and the like. Examples of aralkyl groups include, but are not limited to, benzyl, ortho-methylbenzyl, trityl, and benzhydryl (which may be optionally substituted with halogen, alkyl, alkoxy, hydroxy, nitro, acylamino, acyl, and the like), and salts (e.g., phosphonium salts and ammonium salts). Examples of aryl groups include phenyl, naphthyl, indanyl, anthracenyl, 9- (9-phenylfluorenyl), phenanthrenyl, durenyl, and the like. Examples of cycloalkenylalkyl or substituted cycloalkenylalkyl (preferably having 6 to 10 carbon atoms) include, but are not limited to, cyclohexenylmethyl and the like. Suitable acyl, alkoxycarbonyl and aralkoxycarbonyl groups include benzyloxycarbonyl, tert-butoxycarbonyl, isobutoxycarbonyl, benzoyl, substituted benzoyl, butyryl, acetyl, trifluoroacetyl, trichloroacetyl, phthaloyl and the like. Mixtures of protecting groups can be used to protect the same amino group, for example primary amino groups can be protected with aralkyl and aralkoxycarbonyl groups. Amino protecting groups may also form heterocycles with the nitrogen to which they are attached, such as 1, 2-bis (methylene) benzene, phthalimide, succinimidyl, maleimido, and the like, and wherein these heterocyclic groups may also contain adjacent aryl and cycloalkyl rings. In addition, the heterocyclic group may be mono-, di-or tri-substituted, such as a nitrophenylphthalimide group. Amino groups can also be protected from unwanted reactions, such as oxidation, by the formation of addition salts (e.g., hydrochloride, tosylate, trifluoroacetate). Many amino protecting groups are also suitable for protecting carboxyl, hydroxyl and thiol groups. Such as an aralkyl group. Alkyl groups are also suitable groups for protecting hydroxyl and mercapto groups, for example tert-butyl.

A silyl protecting group is a silicon atom optionally substituted with one or more alkyl, aryl and aralkyl groups. Suitable silyl protecting groups include, but are not limited to, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, dimethylphenylsilyl, 1, 2-bis (dimethylsilyl) benzene, 1, 2-bis (dimethylsilyl) ethane, and diphenylmethylsilyl. Silylation of the amino group provides a mono-or di-silylamino group. Silylation of the aminoalcohol compounds can produce N, O-trimethylsilyl derivatives. Silyl functional groups can be readily removed from silyl ether functional groups by treatment with, for example, a metal hydroxide or ammonium fluoride reagent in a separate reaction step, or by treatment with, for example, a metal hydroxide or ammonium fluoride reagent in situ during reaction with an alcohol group. Suitable silylating agents are, for example, trimethylsilyl chloride, tert-butyl-dimethylsilyl chloride, phenyldimethylsilyl chloride, diphenylmethylsilyl chloride or their combination products with imidazole or DMF. Amine silylation and deprotection methods are well known to those skilled in the art. Methods for preparing these amine derivatives from the corresponding amino acids, amino acid amides or amino acid esters are well known to those skilled in the art of organic chemistry, including amino acid/amino acid ester or amino alcohol chemistry.

The protecting group is removed under conditions that do not affect the rest of the molecule. These methods are well known in the art and include acid hydrolysis, hydrogenolysis, and the like. A preferred method involves deprotecting the protecting group, such as by hydrogenolysis using palladium on carbon in a suitable solvent system such as an alcohol, acetic acid or mixture thereof to remove the benzyloxycarbonyl group. The tert-butoxycarbonyl protecting group may be removed using an inorganic or organic acid such as HCl or trifluoroacetic acid in a suitable solvent system such as dioxane or dichloromethane. The resulting amino salt can be easily neutralized to give the free amine. The carboxyl protecting group may be removed under hydrolysis and hydrogenolysis conditions well known to those skilled in the art, such as methyl, ethyl, benzyl, t-butyl, 4-methoxyphenylmethyl, and the like.

It should be noted that the compounds of the present invention may contain groups that exist in tautomeric forms, such as cyclic and acyclic amidino and guanidino groups, heteroatom-substituted heteroaryl groups (Y' ═ O, S, NR), and the like, see the following examples:

although a form is named, described, shown, and/or claimed herein, all tautomeric forms are intended to be encompassed by such name, description, display, and/or claim.

Prodrugs of the compounds of the invention are also included in the invention. Prodrugs are active or inactive compounds that, after administration to a patient, may be chemically modified by in vivo physiological effects such as hydrolysis, metabolism, and the like, into the compounds of the present invention. The preparation of prodrugs and the applicability and techniques for their use are well known to those skilled in the art. A general discussion of Prodrugs related to esters is found in Svensson and Tunekdrug Metabolism Reviews 165(1988) and Bundgaard Design of Prodrugs, Elsevier (1985). Examples of masked carboxylate anions include various esters, such as alkyl esters (e.g., methyl, ethyl), cycloalkyl esters (e.g., cyclohexyl), arylalkyl esters (e.g., benzyl, p-methoxybenzyl) and alkylcarbonyloxyalkyl esters (e.g., pivaloyloxymethyl). The amine is masked as an arylcarbonyloxymethyl substituted derivative which can be cleaved by an in vivo esterase to release the free drug and formaldehyde (Bungaard j. med. chem.2503 (1989)). And, a drug having an acidic NH group (e.g., imidazole, imide, indole, etc.) masked with an N-acyloxymethyl group (Bundgaard Design of Prodrugs, Elsevier (1985)). The hydroxyl groups are masked to esters and ethers. EP 039,051(Sloan and Little, 4/11/81) discloses mannich base hydroxamic acid prodrugs, methods for their preparation and use.

The specification and claims contain a list of categories using the phrases "selected from … … and … …" and "is … … or … …" (sometimes referred to as markush elements). When such terms are used in this application, they are meant to include all groups or any individual member or subclass thereof, unless otherwise indicated. The use of this term is for shorthand purposes only and is not to be taken in any way as a limitation to the removal of elements or subclasses from view-by-view.

The term "diagnostic agent" is meant to include any compound, composition or particle that can be used in a method for detecting the presence or absence of, determining the amount of and/or imaging a particular substance in vivo or in vitro.

The term "isolated polynucleotide" as used herein shall mean a polynucleotide of genomic, cDNA, or synthetic origin, or a combination thereof, from which an "isolated polynucleotide" (1) does not associate with all or part of the polynucleotide, wherein the "isolated polynucleotide" is present in nature, (2) is linked to a polynucleotide and which is not linked to the polynucleotide in nature, or (3) is not present in nature as part of a larger sequence.

The term "polymer" refers to a compound consisting of repeating non-peptide structural units. In certain embodiments of the invention, the carrier may be a water soluble polymer, such as PEG and methoxypolyethylene glycol (mPEG).

The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer herein to a polymer of nucleotides at least 10 bases in length. In certain embodiments, a base can include at least one ribonucleotide, a deoxyribonucleotide, and modified forms of both nucleotides. The term includes single-stranded and double-stranded forms of DNA.

The term "naturally occurring nucleotide" includes deoxyribonucleotides and ribonucleotides. Deoxyribonucleotides include, but are not limited to, adenosine, guanine, cytosine and thymidine. Ribonucleotides include, but are not limited to, adenosine, cytosine (cytosine), thymidine (thymine), and uracil (uracil). The term "modified nucleotide" includes, but is not limited to, nucleotides having modified or substituted sugar groups and the like. The term "polynucleotide linkage" includes, but is not limited to, polynucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate (phosphoroselenoate), phosphorodiselenoate (phosphorodiselenoate), phosphoroanilothioate, phosphororaniladate, phosphoroamidate (phosphoroamidate), and the like. See, e.g., LaPlanche et al, nucleic acids res.14: 9081 (1986); stec et al, J.Am.chem.Soc.106: 6077 (1984); stein et al, nucleic acids res.16: 3209 (1988); zon et al, Anti-Cancer Drug Design 6: 539 (1991); zon et al, Oligonucleotides and Analogues: a Practical Approach, pp 87-108 (F. Eckstein, Oxford University Press, Oxford England (1991)); stec et al, U.S. patent No. 5,151,510; uhlmann and Peyman Chemical Reviews 90: 543(1990). In certain embodiments, the polynucleotide may comprise a detectable label.

The term "purified" when used with respect to polypeptides, peptides or proteins shall mean that the polypeptides, peptides and proteins are substantially free of cellular components, i.e., contain less than about 50%, preferably less than about 70%, more preferably less than about 90% of cellular components with which the molecule of interest is naturally associated. Methods for the purification of polypeptides, peptides and proteins are well known in the art.

The terms "polypeptide", "peptide" and "protein" all refer to a polymer of two or more amino acids linked to each other by peptide bonds or modified peptide bonds, i.e., a peptide isostere molecule. The term applies to amino acid polymers containing naturally occurring amino acids as well as to amino acid polymers that: wherein one or more amino acid residues is a non-naturally occurring amino acid or a chemical analog of a naturally occurring amino acid. A polypeptide, peptide, or protein may contain one or more amino acid residues that have been modified by one or more natural processes (e.g., post-translational processing, such as glycosylation, acetylation, phosphorylation, etc.), and/or by one or more chemical modification techniques known in the art.

A "fragment" of a reference polypeptide refers to a contiguous stretch of amino acids of any portion of the reference polypeptide. The fragment length can be any length less than the reference polypeptide.

All polypeptide, peptide and protein sequences are written according to well-known conventions, with the N-terminal amino acid residue on the left and the C-terminal on the right. As used herein, the term "N-terminus" refers to the free alpha-amino group of an amino acid of a peptide, while the term "C-terminus" refers to the free alpha-carboxylic acid terminus of an amino acid in polypeptides, peptides and proteins.

The term "selective" as used herein refers to a chemical reaction between an active agent and a carrier, or activating a carrier refers to a chemical reaction that proceeds in a defined and known manner such that i) other functional groups including but not limited to free amines, guanidines, hydroxyls, and carboxylic acids need not be protected, and ii) the desired conjugate constitutes at least 50% of the reaction product.

A "variant" of a reference polypeptide refers to a polypeptide having one or more amino acid substitutions, deletions, or insertions relative to the reference polypeptide. In certain embodiments, a variant of a reference polypeptide has an altered post-translational modification site (i.e., glycosylation site). In certain embodiments, both the reference polypeptide and the variant of the reference polypeptide are specific binding agents. In certain embodiments, the reference polypeptide and the variant of the reference polypeptide are both antibodies.

Variants of the reference polypeptide include, but are not limited to, cysteine variants. In certain embodiments, cysteine variants include variants in which one or more cysteine residues in a reference polypeptide are substituted with one or more non-cysteine residues; and/or a variant in which one or more non-cysteine residues in the reference polypeptide are substituted with one or more cysteine residues. In certain embodiments, the cysteine variant has more cysteine residues than the native protein.

"derivative" of a reference polypeptide refers to: polypeptide: (1) one or more amino acid residues in a reference polypeptide have one or more modifications; and/or (2) wherein one or more peptide bonds are replaced with one or more non-peptide bonds; and/or (3) wherein the N-terminus and/or C-terminus are modified; and/or (4) wherein the side chain is modified. Certain exemplary modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, biotinylation, covalent attachment to a flavin, covalent attachment to a heme moiety, covalent attachment to a nucleotide or nucleotide derivative, covalent attachment to a lipid or lipid derivative, covalent attachment to phosphoinositide, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, t-RNA mediated addition of amino acids to proteins (e.g., arginylation), and ubiquitination. In certain embodiments, both the reference polypeptide and a derivative of the reference polypeptide are specific binding agents. In certain embodiments, the reference polypeptide and the derivative of the reference polypeptide are both antibodies. Polypeptides include, but are not limited to, amino acid sequences modified by natural processes (e.g., post-translational processing) or chemical modification techniques well known to those skilled in the art. In certain embodiments, the modification may occur anywhere on the polypeptide, including the peptide backbone, the amino acid side chains, and the amino or carboxyl termini. In certain such embodiments, the same or different degrees of modification may be present at several sites in a given polypeptide. In certain embodiments, a given polypeptide contains various modifications, such as deletions, additions and/or substitutions of one or more amino acids in the native sequence. In certain embodiments, the polypeptide may be branched and/or cyclic. Cyclic, branched and branched cyclic polypeptides may result from post-translational natural processing (including but not limited to paninisation) or may be prepared synthetically.

The term "bioactive" refers to an agent described as being capable of exerting and/or inducing a biological effect on an interaction with a biomolecule or biological system (e.g., a polypeptide, a cell, or an organism) in vitro or in vivo. Methods of demonstrating biological activity include in vitro bioassays, many of which are well known in the art. Bioactive agents include, but are not limited to, therapeutic agents. Included within the meaning of the term "therapeutic agent" are any substance, composition or particle that can be used for any therapeutic purpose, such as a method for treating a disease in a patient. Thus, a therapeutic agent includes any compound or material that can be used to treat (including prevent, alleviate, relieve or cure) any pathological condition of a patient, including but not limited to a condition, affliction, disorder, disease, disorder, lesion, wound, or injury. Non-limiting examples of therapeutic agents include drugs, vitamins such as biotin, pantothenic acid, vitamin B6, and vitamin B12, nutrients, nucleic acids such as antisense oligonucleotides and short interfering rna (sirna) molecules, amino acids, polypeptides, peptides, retro-inverso (RI) peptides, and formyl-methionyl peptides, enzymes, hormones, growth factors, chemokines, antibodies and fragments thereof, enzyme cofactors, steroids, carbohydrates, lipids, organics such as heparin, metal-containing agents, receptor agonists, receptor antagonists, binding proteins, receptors or receptor portions, extracellular matrix proteins, cell surface molecules, adhesion molecules, antigens, haptens, targeting groups, and chelators. All receptors referred to include all forms of the receptor, as long as more than one form is present.

Other non-limiting examples of therapeutic agents include insulin, anti-HIV peptides (e.g., Tat inhibitors, see below), growth hormones, interferons, immunoglobulins, parathyroid hormone, calcitonin, enkephalins, endorphins, pharmaceuticals, cytotoxic agents, chemotherapeutic agents, radiotherapeutic agents, proteins, natural or synthetic peptides (including oligo-and polypeptides), vitamins, steroids, and genetic material (including nucleosides, nucleotides, oligonucleotides, polynucleotides, and plasmids). Among them, drugs are preferred. Examples of drugs include: antiulcer agents, such as cimetidine, famotidine, ranitidine, roxatidine acetate, pantoprazole, omeprazole, lansoprazole or sucralfate; enteric relaxants or prokinetic agents (prokinetics), such as propantheline bromide, camirofene (acylacryphenine), dicyclomine, scopolamine butylbromide, mebeverine, cisapride, oxybutynin, methylpiperoxate, triptavine, metoclopramide, clidinium bromide, iprodione or oxyphenium bromide; enzymes or carminative agents (carminative), such as pancreatin, papain, pepsin or amylase; hepatobiliary agents, for example chenodeoxycholic acid, ursodeoxycholic acid, L-ornithine or silymarin; antihypertensives such as clonidine, methyldopa sodium nitroprusside, terazosin, doxazosin, (DI) hydralazine or prazosin; beta-blockers such as esmolol, celiprolol, atenolol, labetalol (labetalol), propranolol, metoprolol, carvedilol, sotalol, oxyprenolol or bisoprolol; calcium channel blockers such as felodipine, nitrendipine, nifedipine, benidipine, verapamil, amlodipine or lacidipine; angiotensin converting enzyme (ace) inhibitors, e.g. enalapril, lisinopril, ramipril, perindopril, benazepril or captoprilA pril; angiotensin II inhibitors, such as losartan potassium; potassium channel activators such as nicorandil; diuretics and antidiuretic agents, such as hydrochlorothiazide, xipamide, bumetanide, amiloride, spironolactone, indapamide, triamterene, chloropapamide, furosemide or chlorthalidone; antianginal agents, e.g. isosorbide dinitrate, oxicarbide, isosorbide 5-mononitrate, diltiazemButantetranitrate, trimetazidine, ridafluozine, pentantritrol, nitroglycerin or delazine(ii) a Coagulant agents, e.g. conjugated estrogens, diosmin, vitamin K₃(menaphhthone), menadione, haemocoagulase, haemostasis (cyclemine), rutin-flavonoids or adrenaline monosemicarbazone (adrenochromamone rbazone); anticoagulant, antithrombotic, or antiplatelet agents, such as ticlopidine, warfarin, streptokinase, phenindione, rtpa, urokinase, vasopressin, nitrocoumarin (nicournanone), heparin, low molecular weight heparin, mucopolysaccharide polysulfate, or dipyridamole; antiarrhythmic agents, such as quinidine, propiram, procainamide, lignocaine, mexiletine, amiodarone, adenosylpropafenone; drugs for heart failure and shock, such as mephenbutamine, digoxin dopamine, dobutamine or norepinephrine, vasodilators, such as isoxsuprine, nicoxanthinol tenol, bucinnine hydrochloride (nylidrinin hcl), pentoxifylline (pentitein) or cyclamate; cardiac glycosides, such as deacetyl-hairy glycoside (deslaneseide), digitoxin, digoxin or digitonin; penicillins, such as benzylpenicillin, procaine penicillin (G), benzathine penicillin (G), phenoxymethylpenicillin, penicillin G/V, ampicillin, carbenicillin, piperacillin, ampicillin, cloxacillin or amoxicillin; quinolones or fluoroquinolones, e.g. nalidixic acid, pefloxacin, ofloxacin, sparfloxacin, norfloxacin, cycloidsPropifloxacin, lomefloxacin, cephalosporins, such as ceftizoxime, cefuroxime, cefixime, cefotaxime, cefaclor, ceftriaxone sodium, cefadroxil, cephalexin, cefazolin, ceftazidime or cefoperazone (cefoperazone); sulfonamides, such as sulfanilamide, sulfamethoxazole, sulfadimethoxine (sulfomedimehtoxine), cotifamole, sulfamethoxazole, trimethoprim, aminoglycosides, such as gentamicin, tobramycin, neomycin, amikacin, sisomicin, kanamycin, netilmicin, polymyxin (e.g., polymyxin-b), colistin sulfate; chloramphenicol; tetracyclines, such as tetracycline, doxycycline, minocycline, demeclocycline, oxytetracycline; macrolides such as erythromycin, clarithromycin, vancomycin, lincomycin, azithromycin, spiramycin, roxithromycin, clindamycin, cefpirome, teicoplanin (vancomycin a 2); antiviral agents, such as abacavir, lamivudine, acyclovir, amantadine, interferon, ribavirin, stavudine (stavudine), lamivudine or zidovudine (AZT); antimalarial agents such as quinine, proguanil, chloroquine, primaquine, anodiaquine, artemether, artesunate, mefloquine, pyrimethamine, arteether, quinacrine; antituberculosis drugs, such as cycloserine, capreomycin, ethionamide, prothiocyanimide, rifampin, isoniazid, pyrazinamide, ethambutol; ethambutol, streptomycin, pyrazinamide; anthelmintic and anti-infective agents, such as piperazine, niclosamide, pyrantel pamoate, levamisole, diethylcarbamazine, tetramisole, albendazole, praziquantel, sodium antimony gluconate or menbendazole; anti-leprosy agents, such as dapsone or clofazimine; anti-anaerobe, antiprotozoal, or anti-amoebic agents, such as tinidazole, metronidazole, dichloronitate furoate, secnidazole, hydroxyquinolone, dehydroimidyl, ornidazole (omidazole), furazolidone; antifungal agents, such as fluconazole, ketoconazole, hamycin, terbinafine, econazole, amphotericin-B, nystatin, clotrimazole, griseofulvin, miconazole or itraconazole; a vitamin; respiratory stimulants such as doxorfin hydrochloride; antiasthmatic agents, e.g. isoproterenol, albuterolAlcohol (albuterol), metaproterenol, ephedrine, terbutaline sulfate, salmeterol, aminophylline, thermophylline, beclomethasone propionate, or fluticasone propionate; antiallergic agents such as terfenadine, astemizole, loratadine, clemastine, difindine maleate, fexofenadine hydrochloride, hydroxyzine, chlorpheniramine, azatadine maleate, methdiline, pheniramine maleate, diphenhydramine or cetrizine; skeletal muscle relaxants, such as tizanidine, methocarbamol, carisoprodol, pentazamide (valethamate), baclofen, clomeptazinone or chlorzoxazone; smooth muscle relaxants such as oxfenammonium bromide, propantheline bromide, diclomine, scopolamine butylbromide, mebeverine, drotaverine, clidinium bromide, isopropylammonium or camirofene dihydrochloride; non-steroidal anti-inflammatory drugs such as naproxen, mefenamic acid, nimesulide, diclofenac, tenoxicam, ibuprofen, meloxicam, aspirin, flurbiprofen, ketoprofen, ketorolac, phenylbutazone, oxybutyzone, indomethacin, or piroxicam; antineoplastic agents, such as nitrogen mustard compounds (e.g., cyclophosphamide, trofosfamide, iofosfamide, melphalan, or chlorambucil), aziridines (e.g., thioeta), N-nitrosourea derivatives (e.g., carmustine, lomustine, or nimustine), platinum compounds (e.g., spiroplatinum, cisplatin, and carboplatin), procarbazine, dacarbazine, methotrexate, adriamycin, mitomycin, ansamycin, cytarabine, arabinodenine, mercaptopolylysine, vinetine, busulfan, chlorambucil, melphalan (e.g., PAM, L-PAM, or melphalan), mercaptopurine, mitotane, procarbazine hydrochloride, actinomycin D, daunorubicin hydrochloride, doxorubicin hydrochloride, epirubicin, plicamycin (mithramycin), mitoxantrone, bleomycin sulfate, aminoglutethimide, estramustine, sodium phosphate, and mechlorethamine, Flutamide, leuprolide acetate, megestrol acetate, tamoxifen citrate, testolactone, trilostane, amsacrine (m-AMSA), asparaginase (L-asparaginase), Erwina (Erwina) asparaginase, etoposide (VP-16), interferons (including but not limited to interferon a-2a, interferon a-2b), teniposide (VM-26), vinblastine sulfate (VLB), vincristine sulfate, vindesineOctanes, paclitaxel (Taxol), methotrexate, doxorubicin, arabinosyl, hydroxyurea; folic acid antagonists (e.g., aminopterin, methotrexate), antagonists of purine and pyrimidine bases (e.g., mercaptopurine, thioguanine, fluorouracil, or cytarabine); narcotics, opioids or sedatives, such as tincture of opium camphor, codeine, morphine, opium, amobarbital, sodium amobarbital, alprenol, sodium barbital, chloral hydrate, ethopronol, norethisterone, flurazepam hydrochloride, glutethimide, levomepromazine hydrochloride, meperidone (methyprylon), midazolam hydrochloride, paraformaldehyde, pentobarbital, secobarbital, sodium secobarbital, tabebitutal, temazepam or triazolam; local or systemic anesthetics such as bupivacaine, chloroprocaine, etidocaine, lidocaine, mepivacaine, procaine or tetracaine, droperidol, etomidate, fentanyl citrate-droperidol, ketamine hydrochloride, methohexital sodium or thiopental; neuromuscular blockers such as atracurium mesylate, gosalium iodide, bifluoreneammonium bromide, iododiformin, pancuronium bromide, succinylcholine chloride, tubocurarine chloride or vecuronium bromide; or a therapeutic agent for the hormonal system, such as growth hormone, melanocyte stimulating hormone, estradiol, beclomethasone propionate, betamethasone, cortisone acetate, dexamethasone, flunisolide, hydrocortisone, methylprednisolone, paramethasone acetate, prednisolone, prednisone, triamcinolone, fludrocortisone acetate, adenosine deaminase, amprenavir, albumin, hyaluronidase, interferon alpha-N3, palonosetron hydrochloride, human antihemophilic factor, human blood coagulation factor IX, alefacept, amphotericin B, testosterone, bivalirudin, darbepoetin alfa, tazarotene, bevacizumab, morphine sulfate, interferon beta-1 a, coagulation factor IX, interferon beta-1B, tositumomab and I-131 tositumomab, antihemophilic factor, human growth hormone (e.g., sumatripin), botulinum toxin type A, exartende, exatidone, Alemtuzumab, hyaluronic acid, acritumomab, arabinocerebrosidase, beta-glucocerebrosidase, imiglucerase, Tadalafil, cloofarabine, codeine-sulfonated divinylbenzene-styrene copolymer, chlorpheniramine-sulfonated divinylbenzene-styrene copolymer, Haemophilus BConjugates [ meningococcal conjugates ]]Collagen, rattlesnake polyvalent immune Fab, daptomycin, hyaluronidase, CMV immunoglobulin IV, daunorubicin, cytarabine, doxorubicin hydrochloride, epinastine hydrochloride, leuprolide, labyrine, Emtricitabine, etanercept, hepatitis B antigen, epoietin alfa, cetuximab, estradiol, clindamycin, gemifloxacin mesylate, urofollitropin, influenza virus antigen, dexmethylphenidate hydrochloride, follitropin beta, teriparatide, calcitonin, frovatriptan succinate, enfuvirtide, nitric acid, human somatropin (somatropipin), imatinib mesylate, glucagon, metformin hydrochloride, follicle stimulating hormone alpha, dulcitol, adefovir dipivoxil, trastuzumab, hydroxyethyl starch, insulin analogue and insulin analogue, von Willebrand (Willebrand), Willebrand factor, amand (amadine), anti-amadine, amantadine hydrochloride, heparin, Interferon-alpha complex, bone morphogenetic protein-2, eptifibatide, interferon-alpha, timolol, palifermin, anakinra, insulin glargine, granulocyte macrophage colony stimulating factor, cladribine, calcium fosamprenavir, eszopiclone, luteinizing hormone alpha, betamethasone, OspA lipoprotein, pegaptanib, methylphenidate, methylaminolevulinic acid (methyaminolevulinic acid), mitomycin, gemumab, ozomicin, botulinum toxin type B, human hepatitis B immunoglobulin, gaulsfase, memantine hydrochloride, vitamin B12, cetirizine, PEG-filgrastim (peggrastim), olgrelide, interleukin filgrastim, technetium [99mTc ] 99mTc]fanolesomab, mitoxantrone, insulin aspart, factor VIIa, clobetasol propionate, L-asparaginase, dinil interleukin-toxin linker, amlexanox, nitrendipine, Moluomab-CD 3, human chorionic gonadotropin, BCG antigen, Aliverine A acid, diphtheria (diphtheria), PEG-interferon alpha-2 a, porphin sodium, gonadotropin releasing hormone antagonist, repaglinide, pneumococcal 7-valent conjugate, ziconotide, ciprofloxacin hydrochloride, indium In 111 Carlo mab pentapeptide, human methionine, modafinil, alpha-streptokinase, Lysima SM-153, omeprazole, Efaluzumab, ribavirin and Lexidun (Lexidronam)Interferon alpha, lepirudin, gelatinous becaplamine (gel becaplmin), infliximab, treprostinil sodium, sevelamer HCl, abciximab, reteplase, Rh0 immunoglobulin, rituximab, interferon alpha-2 a, trospium chloride, fluoxetine hydrochloride, synthetic porcine pancreatic secretin, cinacalcet HCl, basiliximab, pevisomant, pramlintide acetate, palivizumab, osemivir phosphate, erlotinib (OSI Pharmaceuticals, inc. and Genentech), bexarotene, antithymocyte globulin, thyrotropin alpha, thyroglobulin (Tg), tenecteplase, influenza, trioxygoxin, diphtheria toxoid and acellular pertussis antigens, diarsenized arsenic, trientine, nateglinide, tiprexinib, irvir, tiprezovir, tiprexinavir, tenofovir, ritonavir, rituximab, and acellular pertussis antigens, Fumivir, interferon alpha-n 1, Rho [ D ]]Immunoglobulin, bromfenac sodium, rifaximin (rifaximin), drotrecoginalfa, Omalizumab, sodium oxybate, miglustat, omeprazole, daclizumab, ibritumomab, zonisamide, loteprednol etabonate, tobramycin, bromhexine, carboxymethcysteine or clavulanic acid, docosanol, acetaminophen, interferon gamma-1 b, alteplase, and technetium Tc-99 apcitide.

The active agent attached to the carrier in the conjugates of the invention has or is modified to have a1, 2-aminothiol or 1, 3-aminothiol moiety or group of formula I capable of reacting with a carrier derivative through its complementary functional groups as described herein. Examples of active 1, 2-aminothiols are found in the amino acid cysteine.

Many proteins do not have free cysteines (cysteines not involved in disulfide bond formation) or any other active 1, 2-or 1, 3-amino thiol groups. In addition, cysteine 1, 2-aminothiol may not be suitable for attachment to polymers, as 1, 2-aminothiol is necessary for biological activity. In addition, proteins must fold into a certain conformation in order to be active. In the active conformation, the cysteine 1, 2-aminothiol is inaccessible because it is buried inside the protein. Furthermore, even cysteine 1, 2-aminothiols that are accessible and not essential for activity may be in a position that is not suitable for attachment to a polymer. Amino acids that are not essential for activity are referred to as "non-essential". Non-essential cysteines may be at unsuitable conjugation positions because the position of the cysteine relative to the active site inactivates the polypeptide after conjugation to the carrier.

Like proteins, many other biologically active molecules also have an active 1, 2-aminothiol or 1, 3-aminothiol, since they are not suitable for conjugation to a specific carrier or do not contain an active 1, 2-aminothiol or 1, 3-aminothiol group, for reasons similar to those described above. Thus, the present invention includes the introduction of a reactive 1, 2-aminothiol group or 1, 3-aminothiol group, if necessary, into a biologically active agent, which can be conjugated to a carrier derivative of the present invention. Examples of bioactive agents containing a thioamide moiety can be found in U.S. patent application serial No. 09/621,109. Such compounds include, but are not limited to UC 781; r82150; HBY 097; trovirdine (troviridine); s2720; UC38 and 2 ', 3 ' -dideoxy-3 ' -fluoro-4-thiothymidine.

Reactive thiol or thioamide groups can be introduced by chemical methods well known in the art. Chemical modifications may be applied to polypeptide or non-peptide molecules and include the introduction of thiols into the molecule alone or as part of a larger group such as a cysteine residue. Cysteine may also be chemically reduced using, for example, DTT, to produce free cysteine in the polypeptide.

A polypeptide modified to contain an amino acid residue at a position not present in the native protein before modification is referred to as a "mutein". To produce cysteine muteins, the N-terminal non-essential amino acid may be substituted with cysteine. Mutation of the N-terminal lysine to cysteine is also suitable, since lysine residues are often visible on the surface of the protein in the active conformation. In addition, in the possible mutation position selection, the technicians in this field can use the polypeptide binding site or active site of any known information. Cysteine muteins can also be produced by those skilled in the art using well-known recombinant DNA techniques. Nucleic acids encoding the native polypeptide can also be altered by standard site-directed mutagenesis methods to encode the mutant protein. Examples of standard mutagenesis techniques are found in Kunkel, T.A., Proc.Nat.Acad.Sci., Vol.82, p.488-382 (1985) and Kunkel, T.A., et al, Methods enzymol, Vol.154, p.367-382 (1987).

Potential sites for the introduction of non-native cysteines include the glycosylation site and the N-terminus of the polypeptide. In these examples, the glycosyl donor can contain either a1, 2-aminothiol or a1, 3-aminothiol. One skilled in the art can link the sugar group to a serine or threonine on the active agent.

Alternatively, nucleic acids encoding the muteins can be chemically synthesized by techniques well known in the art. A DNA synthesizer can be used, which is commercially available, for example from applied biosystems (Foster City, Calif.). The nucleic acid encoding the desired mutein can be expressed in various expression systems, such as animal, insect and bacterial systems. After the desired mutant protein is produced, the technicians in this field can be used for the mutant protein biological assay and comparison of mutant protein and natural polypeptide activity. Conjugates formed with the muteins can be particularly useful even when the relative activity of the muteins is reduced. For example, the conjugates have increased solubility, decreased antigenicity, or decreased immunogenicity relative to the unconjugated molecule, or have decreased clearance times in biological systems.

"polypeptide" and "protein" are used synonymously herein to refer to any compound that is substantially a protein in nature. However, the polypeptide group may contain some non-peptide elements. For example, glycosylated polypeptides or synthetically modified proteins are included in this definition.

The terms "effective amount" and "therapeutically effective amount" as used herein when used in reference to a biologically active agent, such as a peptide, carrier-conjugated peptide or PEG-conjugated peptide, refer to an amount or dose sufficient to achieve the desired result. For carrier-conjugated B1 peptides and/or PEG-conjugated peptide B1 antagonists, the desired result may be, for example, a desired reduction in inflammation and/or pain, or to support a visible decrease in the level of one or more biological activities of B1. More specifically, a therapeutically effective amount is an amount of a biologically active agent that is sufficient to reduce, inhibit or prevent one or more clinically defined pathological processes associated with the condition (e.g., inflammation or pain) in a patient being treated with the agent in vivo over a period of time. The effective amount may vary from biological agent to biological agent and will depend upon various factors and conditions associated with the patient to be treated and the severity of the disease. For example, when the biologically active conjugate is administered in vivo, factors such as the age, weight, and health of the patient, as well as dose response curves and toxicity data obtained in preclinical animal studies, are considered. If the biologically active conjugate is contacted with cells in vitro, one will also design various preclinical in vitro studies to evaluate parameters such as uptake, half-life, dose, toxicity, etc. Determination of an effective or therapeutically effective amount of a given active agent is well within the ability of those skilled in the art.

The term "pharmacologically active" means that the substance being described is determined to have an activity that affects a medical parameter or a disease state (e.g., pain). With respect to the carrier-conjugated B1 peptides of the present invention, the term generally refers to B1-induced or B1-mediated diseases, disorders, or abnormal medical conditions, and more particularly, to antagonism of inflammation or pain.

The terms "antagonist," "inhibitor," and "inverse agonist" (see, e.g., rianne a.f. de Ligt et al, British Journal of Pharmacology 2000, 130, 131) refer to molecules that block, prevent, reduce, diminish, or otherwise interfere to some extent with the biological activity of the associated protein of interest. A preferred "B1 peptide antagonist" of the invention is a molecule that binds to and inhibits B1, the IC of which is determined in vitro by the activity of B1₅₀Below 500 nM. More preferred B1 peptide antagonists of the invention are receptor binding molecules with Ki's of less than 100nM that inhibit B1-mediated functions (e.g., calcium flux) and IC's in vitro assays for B1 activity₅₀Less than 100 nM. Most preferred B1 peptide antagonists of the invention are molecules that bind to and inhibit B1, active at B1In an in vitro assay, its Ki is less than 10nM, IC₅₀Under 10 nM. Furthermore, the molecule can prevent, ameliorate or eliminate pain or inflammation as determined in at least one generally accepted animal model of pain in vivo; and/or inhibiting biochemical attacks in an animal model of edema, inflammation, or pain in vivo.

In addition, physiologically acceptable salts of the peptides or conjugate peptides of the invention are also included herein. The terms "physiologically acceptable salt" and "pharmacologically acceptable salt" as used herein, are used interchangeably and are meant to include any salt which is known or later discovered to be pharmaceutically acceptable, i.e., useful in the treatment of a warm-blooded animal. Some specific examples are: an acetate salt; hydrohalic acid salts, such as hydrochloride and hydrobromide salts; a sulfate salt; a citrate salt; a tartrate salt; a glycolate; an oxalate salt; inorganic and organic acid salts including, but not limited to, hydrochloride, hydrobromide, sulfate, phosphate, methanesulfonate, ethanesulfonate, malate, acetate, oxalate, tartrate, citrate, lactate, fumarate, succinate, maleate, salicylate, benzoate, phenylacetate, mandelate and the like. When the compounds of the present invention contain an acidic functional group, such as a carboxyl group, suitable pharmaceutically acceptable cation pairs for the carboxyl group are well known to those skilled in the art and include alkali metal, alkaline earth metal, ammonium, quaternary ammonium cations, and the like. Further examples of "pharmacologically acceptable salts" are found below and in Berge et al, j.pharm.sci.66: 1(1977).

"protecting group" generally refers to groups well known in the art for protecting a selected reactive group (e.g., carboxyl, amino, hydroxyl, sulfhydryl, etc.) from undesired reactions (e.g., nucleophilic, electrophilic, oxidative, reductive, etc.) reactions. Where appropriate, preferred protecting groups are as shown herein. Examples of amino protecting groups include, but are not limited to, arylalkyl-, substituted arylalkyl-, cycloalkenylalkyl-, and substituted cycloalkenylalkyl-, allyl-, substituted allyl-, acyl-, alkoxycarbonyl-, arylalkoxycarbonyl-, silyl-, and the like. Examples of arylalkyl-include, but are not limited to, benzyl-, o-methylbenzyl-, trityl-, and benzhydryl- (which may be optionally substituted with halogen, alkyl-, alkoxy-, hydroxy-, nitro-, acylamino-, acyl-, and the like) and salts (e.g., phosphonium and ammonium salts). Examples of aryl groups include phenyl-, naphthyl-, indanyl-, anthracenyl-, 9- (9-phenylfluorenyl) -, phenanthryl-, durenyl-, and the like. Examples of cycloalkenylalkyl-or substituted cycloalkenylalkyl- (preferably having 6 to 10 carbon atoms) include, but are not limited to, cyclohexenyl-, methyl-, and the like. Suitable acyl-, alkoxycarbonyl-, and aralkoxycarbonyl-groups include benzyloxycarbonyl-, tert-butoxycarbonyl-, isobutoxycarbonyl-, benzoyl-, substituted benzoyl-, butyryl-, acetyl-, trifluoroacetyl-, trichloroacetyl-, phthaloyl-, and the like. Mixed protecting groups may be used to protect the same amino group, for example primary amino groups may be protected with aralkyl and aralkoxycarbonyl groups. Amino protecting groups may also form heterocycles with the nitrogen to which they are attached, such as 1, 2-bis (methylene) benzene, phthalimide-, succinimidyl-, maleimido-, and the like, and wherein these heterocyclic groups may also contain adjacent aryl-and cycloalkyl-rings. In addition, the heterocyclic group may be mono-, di-or tri-substituted, such as, for example, a nitrophenylphthalimido group. Amino groups can also be protected from unwanted reactions, such as oxidation, by the formation of addition salts (e.g., hydrochloride, tosylate, trifluoroacetate). Many amino protecting groups are also suitable for protecting carboxy-, hydroxy-and mercapto-. Such as aralkyl-. Alkyl groups are also suitable groups for protecting hydroxy-and mercapto-, for example tert-butyl.

The silyl-protecting group is a silicon atom optionally substituted with one or more alkyl, aryl and aralkyl groups. Suitable silyl protecting groups include, but are not limited to, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, dimethylphenylsilyl, 1, 2-bis (dimethylsilyl) benzene, 1, 2-bis (dimethylsilyl) ethane, and diphenylmethylsilyl. Silylation of the amino group provides a mono-or di-silylamino group. Silylation of the aminoalcohol compounds can produce N, O-trimethylsilyl derivatives. Silyl functional groups can be readily removed from silyl ether functional groups by treatment with, for example, a metal hydroxide or ammonium fluoride reagent in a separate reaction step, or by treatment with, for example, a metal hydroxide or ammonium fluoride reagent in situ during reaction with an alcohol group. Suitable silylating agents are, for example, trimethylsilyl chloride, tert-butyl-dimethylsilyl chloride, phenyldimethylsilyl chloride, diphenylmethylsilyl chloride or their combination products with imidazole or DMF. Amine silylation and deprotection methods are well known to those skilled in the art. Methods for preparing these amine derivatives from the corresponding amino acids, amino acid amides or amino acid esters are well known to those skilled in the art of organic chemistry, including amino acid/amino acid ester or amino alcohol chemistry.

The protecting group is removed without affecting the rest of the molecule. These methods are well known in the art and include acid hydrolysis, hydrogenolysis, and the like. Preferred methods include deprotecting the benzyloxycarbonyl group by hydrogenolysis, for example, using palladium on carbon in a suitable solvent system such as an alcohol, acetic acid, or mixture thereof. The tert-butoxy-carbonyl protecting group can be removed using an inorganic or organic acid such as HCl or trifluoroacetic acid in a suitable solvent system such as dioxane or dichloromethane. The resulting amino salt can be readily neutralized to give the free amine. The carboxyl protecting group may be removed under hydrolysis and hydrogenolysis conditions well known to those skilled in the art, such as methyl, ethyl, benzyl, t-butyl, 4-methoxyphenylmethyl, and the like. For a more extensive use of protecting Groups see Theodora W.Green and Peter G.M.Wuts (1999), "Protective Groups in Organic Synthesis", 3 rd edition, Wiley, New York, N.Y..

The present invention is based on the identification of a novel chemical approach that provides novel carrier derivatives as excellent 1, 2-aminothiol or 1, 3-aminothiol selective agents for conjugation to unprotected targets (e.g., polypeptides, peptides or organic compounds) that have or are modified to have a1, 2-aminothiol or 1, 3-aminothiol group. The excellent specific reaction regioselectivity forms covalent bonds between the carrier derivative and the 1, 2-aminothiol or 1, 3-aminothiol moiety of the targeted active agent. The reaction proceeds almost completely under very mild conditions.

Although the synthesis of proteins by chemoselective reaction of cysteine-containing fragments with aldehyde-containing fragments 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.; Fruchar, J.S.; Grandjean, C.; Gras-Masse, H.J.Org.m.2001, 66, 4153), the chemical ligation methods described herein have not been used as a method for conjugating peptides, proteins, or organic compounds to carriers.

In one embodiment, the present invention relies on the unique ability of 1, 2-aminothiol or 1, 3-aminothiol to react chemoselectively with aldehydes to form thiazolines. Once formed, the thiazoline nitrogen kinetically tends to form an amide bond. This is accomplished by placing the 5-or 6-atom of the ester carbonyl group removed from the thiazoline nitrogen. In addition, the novel chemical reactions of the present invention generally produce a major product, making the desired conjugates easy to purify, analyze and characterize.

The novel chemical reagents and methods of the invention are particularly effective in strategies for producing multiple peptide carrier conjugates. For example, the reagents and methods of the invention are useful for the efficient conjugation of a B1 peptide antagonist containing 4 cysteines to a branched multivalent PEG polymer. The reagents and methods described herein are effective to produce a desired plurality of peptide PEG conjugates in high yield and purity. Various multiple peptide PEG conjugates exhibit increased activity (hB1 Ki 100pm, in some cases), significantly longer circulation half-life, and reduced PEG loading compared to peptide conjugates with a single peptide on each carrier, which allow for acceptable dosing regimens that significantly provide greater exposure and prolonged efficacy in vivo. The carrier-conjugated B1 peptides provide significant therapeutic advantages over known unconjugated B1 peptide antagonists and are useful for treating and/or preventing B1-mediated diseases, conditions, or disorders, including but not limited to inflammation and pain.

The use of the novel activated carrier derivatives of the present invention in the methods of the present invention brings numerous surprising and unexpected advantages over previously known polymer conjugation methods, particularly with respect to multivalent polymer conjugation strategies (see, e.g., PCT publication No. WO95/06058, U.S. patent application publication No. US 2003/0040127).

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Bradykinin B1 receptor binding peptides contemplated for use for conjugation to a carrier and in the manner described herein include, but are not limited to, the novel B1 binding peptide antagonists disclosed herein and B1 peptide antagonists known in the art, including but not limited to any of the peptides disclosed in any of the following publications (each of which is incorporated herein by reference in its entirety): regoli et al, bradykinirectors and the anti-agonist (bradykinin receptor and its antagonist), eur.j.of pharma, 348: 1-10 (1998); neugebauer, W, et al, Kinin B₁Recepttorsanthonists with multi-enzymatic resistance properties (kinins B with multi-enzyme resistance properties)₁Receptor antagonist) can.j.physiol.pharmacol, 80: 287-292 (2002); stewart, j.m., et al, bradykin antagonists: presentation progression and future promoters immunopharmacology (bradykinin antagonist: current progression and future prospects), 43: 155-161 (1999); stewart, j.m., et al, Metabolism-resistant bradykin antagnosts: development and Applications (metabolic resistant bradykinin antagonist: Development and application), biol. 37-41 (2001); PCT publication Nos. WO98/07746 and WO 2005042027; U.S. Pat. nos. 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.

A "functionalizing agent" of the present invention is an agent suitable for functionalizing a support of the present invention.

"functionalization reactions" are reactions according to the present invention which functionalize the support. The functionalization reaction may consist of one or more steps.

The term "vector" as used herein refers to a molecule that: for active agents, it can delay degradation, increase half-life, reduce toxicity, reduce immunogenicity, and/or increase biological activity. Vectors for use in the present invention are known in the art and include, but are not limited to, Fc regions, polyethylene glycol, and dextran. Various vectors can be found, for example, in U.S. patent No. 6,660,843, published PCT application nos. WO 99/25044 and WO98/07746, Langer, r., "Biomaterials in Drug Delivery," 33acc. chem. res.94 (2000); and Langer, R., "Tissue Engineering," 1MOL.THER.12(2000), Haisch, A. et al, Tissue Engineering of Human Cartilage Tissue, 44HNO 624 (1996); ershov, I.A., et al, Polymer Biocompatible X-Ray Contract Hydrogel, 2MED.TEKH.37 (1994); polous, I.M. et al, Use of A biocompatible antibacterial Polymer Film, 134VESTN.KHIR.IM.II GREK.55 (1985). Other examples of carriers include N-vinylpyrrolidone-methyl methacrylate copolymers, possibly together with polyamide-6 (Buron, F. et al, Biocompatible carbohydrate encapsulating Polymer, 16CLIN. MATER.217(1994)), poly (DL-lactide-co-glycolide) (isoboe, M. et al, Bone macromolecular Protein Encapsulated with a biodegradable Polymer, 32J. BIOMED. MATER.RES.433(1996)), a mixture of methyl methacrylate and 2-hydroxyethyl methacrylate in a ratio of 70: 30 (Bar, F.W. et al, New Biocompatable Polymer Surface Coating, 52J. BIOMM. MATER.RES.193(2000)), 2-methacryloyloxyethyl phosphorylcholine phosphate (optionally with polyurethane, Isaki, Y. et al, Semiik-methacrylate copolymer, 52J. 2001. MATER.52 (2000)), a high Alginate content Alginate Polymer such as Calcium Alginate, 52J. polyK. Alginate, K. RTM. et al, Calcium Alginate, K.5, K. E. Alginate, K. E. M. 5, M. E. M. E. Alginate, M. E. 20. Alginate, E. Alginate, P. E. Alginate, P. E. 20, P. E. J. et al, Surface Characterization of ports, Biocompatible Protein Polymer Thin Films, 22BIOMATERIALS 1289 (2001); see Raudino, a. et al, Binding of Lipid vehicles, 231j. cold. interfacial sci.66(2000)), polyvinylpyrrolidone, polymethylene glycol, polyhydroxypropylene glycol, polypropylene glycol and oxides, polymethylpropylene glycol, poly-hydroxypropylene oxide, linear and branched polypropylene glycols, polyethylene and polypropylene glycols and their monomethyl ethers, monocetyl ethers, mono-n-butyl ethers, mono-tert-butyl ethers and monooleyl ethers, esters of poly-alkylene glycols with carboxylic acids and dehydration condensation products of polyalkylene glycols with amines, and other polyalkylene oxides and glycols, poly (vinylpyrrolidone), polyvinyl alcohol, poly (vinyl acetate), copolymer poly (vinyl acetate-co-vinyl alcohol), polyvinyl oxazolidone, poly (vinylmethyl-oxazolidone) and poly (vinylmethyl ether), poly (acrylic acid), Poly (methacrylic acid), polyhydroxyethyl-methacrylate, poly (acrylamide) and poly (methacrylamide), poly NN-dimethylacrylamide, poly (N-isopropylacrylamide), poly (N-acetamidoacrylamide) and poly (N-acetamidomethacrylamide) and other N-substituted derivatives of these amides.

PEG is a water-soluble, non-immunogenic biocompatible material. When used as a carrier, PEG typically confers useful properties to the attached agent including increased solubility, increased circulating half-life in the bloodstream, resistance to proteases and nucleases, reduced immunogenicity, and the like. The high molecular weight of the PEG allows the final conjugate to be easily separated from excess unconjugated peptide and other minor impurities. Thus, the PEG conjugates are stable and convenient for use in diagnostic assays when stored under controlled conditions. Although the polyether backbone of PEG is relatively chemically inert, the primary hydroxyl groups at both ends are reactive and can be used to attach actives directly. These hydroxyl groups are typically converted to more reactive functional groups for conjugation purposes.

The terms "activating support derivative", "activating support", "functionalized support derivative" and "functionalized support" are used interchangeably herein and refer to a support having a reactive group at the end of at least one support segment. Likewise, the terms "activating support segment" and "functionalizing support segment" are used interchangeably herein to refer to a support segment having a terminal reactive group.

PEG is a water-soluble, non-immunogenic biocompatible material. When used as a carrier, PEG typically confers useful properties to the attached agent including increased solubility, increased circulating half-life in the bloodstream, resistance to proteases and nucleases, reduced immunogenicity, and the like. The high molecular weight of the PEG allows the final conjugate to be easily separated from excess unconjugated peptide and other minor impurities. Thus, the PEG conjugates are stable and convenient for use in diagnostic assays when stored under controlled conditions. Although the polyether backbone of PEG is relatively chemically inert, the primary hydroxyl groups at both ends are reactive and can be used to attach actives directly. These hydroxyl groups are typically converted to more reactive functional groups (i.e., "activated") for conjugation purposes.

The terms "carrier-conjugated active agent" and "conjugated active agent" are used interchangeably herein and refer to a conjugate comprising at least one active agent and a carrier comprising at least one carrier segment covalently attached to the active agent itself or to a linker, including but not limited to a peptide-based or non-peptide-based linker (e.g., an aromatic linker) covalently bound to the active agent.

In certain embodiments of the invention, a "carrier-conjugated peptide" or "conjugated peptide" refers to a conjugate comprising a peptide and a carrier, wherein the peptide has or is modified to have an N-terminal cysteine, and the carrier comprises a carrier segment covalently linked to the N-terminal cysteine residue of at least one peptide. In other embodiments, the conjugates comprise at least one peptide and a carrier comprising at least one carrier segment, wherein the carrier segment is covalently bound to a non-peptidyl linker (including but not limited to an aromatic linker) which is covalently bound to a residue of the peptide.

In certain embodiments of the invention, a "PEG-conjugated peptide" refers to a conjugate comprising at least one peptide having or modified to have an N-terminal cysteine and PEG comprising a PEG segment covalently bound to the N-terminal cysteine residue of the at least one peptide. In other embodiments, the conjugate comprises at least one peptide and a PEG comprising at least one PEG segment, wherein the PEG segment is covalently attached to a non-peptidyl linker (including but not limited to an aromatic linker) which is covalently attached to a residue of the at least one peptide.

In another embodiment and in combination with the above and below embodiments, the conjugate peptide comprises a peptide sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 11-23 and 43-46 and further modifying the carrier segment to which the N-terminal cysteine residue of the peptide having an N-terminal cysteine is covalently bound.

In certain embodiments of the invention, the carrier has a nominal average molecular weight in the range of about 100 daltons to about 200,000 daltons, or a nominal average molecular weight in the range of about 100 daltons to about 100,000 daltons, or a nominal average molecular weight in the range of about 5,000 daltons to about 100,000 daltons, or a nominal average molecular weight in the range of about 10,000 daltons to about 60,000 daltons, or a nominal average molecular weight in the range of about 10,000 daltons to about 40,000 daltons, or a nominal average molecular weight in the range of about 20,000 daltons to about 40,000 daltons.

The active group on the activating support can be any number of moieties that can participate in a reaction that links the components of the desired conjugate together without significant deleterious consequences. Non-limiting examples include acids, esters, thiols, amines or primary amines, but these are merely illustrative of the invention. Importantly, the covalent bond formed between the carrier or carrier segment and any given active agent conjugated thereto should be relatively stable.

Typically, the activating support is linear and therefore can have at most only two functional groups (i.e., one at each end). It is clear that this limits the number of conjugations to only two. In some cases, carriers having multiple reactive groups are preferred for attaching multiple active agents to the same carrier molecule. The methods of the present invention are very helpful in designing conjugation strategies that provide relatively precise numbers of functional groups on the desired multivalent support.

In particular embodiments of the invention, the carrier may be a multivalent carrier molecule, including but not limited to linear carriers activated at both ends, branched carriers with more than one activated carrier segment, and branched carriers with more than one activated carrier segment. In certain embodiments of the invention, the carrier may be a multivalent PEG, including but not limited to a linear carrier with both ends activated, a bifurcated PEG (fpeg) with more than one activated carrier segment, and a branched PEG (bpeg) with more than one activated carrier segment.

In a specific embodiment of the invention, an amine-derivatized support or a support comprising a plurality of support segments, at least one of which is amine-derivatized, is reacted with a1, 2-or 1, 3-formyl ester to produce a support conjugate of the invention.

The specific embodiments described herein do not limit the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

Examples

General purpose experiment

NMR: proton MR of PEG-containing molecules is seen in PEG singlet (3.7ppm, D relative to DSS)₂O)。¹³C NMR spectra were seen on PEG singlet (72.0ppm, D relative to DSS)₂O)。

FTMS data was obtained operating on a Bruker Q-FTMS at 7 tesla. The instrument was externally calibrated with PEG300/600 solution using standard France's equations. The error between the calculated mass and the measured value of each calibration ion is less than 1.0 ppm. For each spectrum, 512k data points were collected and the run-length (86Da mass cutoff) was measured at 1.25 MHz. The time domain data is not processed before the number-mode Fourier transform is performed.

GC-MS data were recorded using Hewlett-Packard GC-Ms, with the following parameters:

column: j and W DB-XLB capillary columns, 30M by 0.25mm by 0.50. mu.M, PN 1221236.

The method comprises the following steps:

injector parameters: the temperature of the injector is 250 ℃; the split ratio is 50: 1; helium flow rate 1 ml/min.

GC parameters: the starting temperature is 80 ℃; 0-2 minutes, keeping at 80 ℃; slowly heating to 200 ℃ for 2-14 minutes; held at 200 ℃ for 5 minutes. And then the balance is 0.5 min.

The mass transfer temperature was 280 ℃.

Mass spectrum parameters: scanning from 50-550amu, the EI voltage was 2376.5 mV.

The method 2 comprises the following steps:

injector parameters: the temperature of the injector is 250 ℃; the split ratio is 50: 1; helium flow rate 1 ml/min.

GC parameters: the starting temperature was 140 ℃; 0-2 minutes, keeping at 140 ℃; slowly rising to 320 ℃ for 2-11 minutes; held at 320 ℃ for 1 minute. And then the balance is 0.5 min.

The mass transfer temperature was 280 ℃.

GC parameters: scanning from 50-550amu, the EI voltage is 2376.5mV

The method 3 comprises the following steps:

injector parameters: the temperature of the injector is 250 ℃; the split ratio is 50: 1; helium flow rate 1 ml/min.

GC parameters: the starting temperature was 70 ℃; 0-2 minutes; slowly increasing the temperature to 90 ℃ at a speed of 10 ℃ per minute; slowly increasing the temperature to 320 ℃ at a speed of 20 ℃ per minute; held at 320 ℃ for 4.5 minutes. And then the balance is 0.5 min.

The mass transfer temperature was 280 ℃.

Mass spectrum parameters: scanning from 50-550amu, the EI voltage is 2376.5mV

Peptides were synthesized using standard FMOC strategies as described by Stewart and Young (1984) "Solid Phase Peptide Synthesis". Those skilled in the art of peptide synthesis are able to synthesize the peptides by either manual or automated solid phase methods.

Peptide content was determined by HPLC and chemiluminescence detection (CLND):

solvent system: a ═ 0.04% TFA/water, B ═ 0.04% TFA/90% methanol.

Column: jupiter C1830050X 2.0mm column, 5 μm particle size.

CLND: antek 8060, oven temperature 1048 ℃, detector operated at high sensitivity and attenuation 1.

HPLC: HP1100LC, diode array Detector

Gradient: 10% B to 100% B, keeping for 2min within 10min, and balancing for 4 min.

Flow rate and split flow: the total flow rate was 0.3ml/min and was split by a tee at a ratio of about 2: 1 between CLND and waste.

Preparative reverse phase HPLC:

the system comprises the following steps: two Agilent series 1100prep pumps, Agilent series 1100prep autoinjectors, Rheodyne manual injectors with 5-20ml sample rings, Agilent series 1100 multi-wavelength detectors (set at 215nm and 254nm) and Agilent series 1100 automatic flow collectors.

Software: agilent Chemstation.

Solvent system:

1: a 10mM ammonium formate/water (pH 3.75); and B is acetonitrile.

2: a ═ 0.1% acetic acid/water, B ═ 0.1% acetic acid/acetonitrile.

3: a 10mM ammonium bicarbonate (pH 10)/water; and B is acetonitrile.

Column:

1: waters Xterra Prep C18 MS (packaged by Vydac/The Separations Group), 50mm X300 mm (PN PA0000-050730), 10 μm particle size, spherical.

2：30×100mm Waters Xterra Prep C18 OBD，100Pore size, 5 μm particle size, spherical, PN 186001942.

Gradiometer:

1

time (min)	％B	Flow rate (ml/min)
			0	25	20
4	25	20
			5	25	100
25	55	100
			35	55	100
35.1	100	100
			49.9	100	100
50	25	100
			60	25	100
60.1 end

2

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

24.9	55	35
			24.95	100	35
29.9	100	35
			29.95	25	35
40 end of

3

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

4

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

Preparative cation exchange LC:

system and software: same as in preparative HPLC.

Solvent:

1: 10mM boric acid/5: 40: 55 MeOH-acetonitrile-water; b ═ a +0.2M KCl.

Column:

1: tosoh Bioscience TSKGel SP-5PW-HR, PN 43382, 20 μm particles were loaded onto a 50X 250mm glass column (Hodge Bioseparations Ltd. P/N ═ TAC50/250S 2-SR-1). The length of the detection bed is 180 mm.

2：21.5×150mm TSK Gel SP-5PW，PN 07575。

Gradiometer:

1

time (min)	％B	Flow rate (ml/min)
			0	0	10
2	0	10
			5	0	30
25	0	30
			25.1	20	30
80	80	30
			80.1	100	30
110	100	30
			110.1	0	30
130 end of

2

Time (min)	％B	Flow rate (ml/min)
			0	0	10
5	0	10
			30	100	10
45	100	10
			45.05	0	10
End of 60

Experimental part

Scheme 1

Reagents and conditions: a) BBr₃，-78℃，CH₂Cl₂；b)TBDMSCl，DMF，DIPEA，rt；c)CH₂Cl₂Carbonyl diimidazole, rt; d) CH (CH)₃OH，DCE，MW，100℃，2min.；e)NBS，AIBN，CCl₄Refluxing; f) AgNO₃，H₂O, i-PrOH, rt, then TBAF, DCM; g) 2-Bromoacetic acid benzyl ester, K₂CO₃Acetone, 0 ℃; h)2, 6-di-tert-butylpyridine, 1, 2-bis (trimethylsiloxy) ethane, trimethylsilyl trifluoromethanesulfonate, 2-pyridylcarbinol, CH₂Cl₂，0℃；i)H₂Pd/C, EtOAc; j) n-hydroxysuccinimide, PS-carbodiimide (Argonaut technologies), EtOAc.

4-hydroxy-2-methylbenzoic acid (2). To a 250ml flame-dried 3-necked round bottom flask was added 4-methoxy-2-methylbenzoic acid (1) (5.0g, 30.08mmol) and CH₂Cl₂(80 ml). The reaction was cooled to-78 ℃ and pure BBr was added dropwise via addition funnel₃(5.7ml, 60.17mmol) was processed. The reaction was stirred at-78 ℃ for 30min. The solution was warmed to-15 ℃ and stirred for 4h (-15 ℃ to-10 ℃). The cooling bath was removed. The reaction was stirred at room temperature for 20 h. The solution was cooled to 0 ℃ and quenched with diethyl ether (15ml) and water (15ml) (note: water caused a strong reaction; water was added dropwise). The biphasic mixture was extracted with EtOAc (3X 100 ml). The combined organic layers were over MgSO₄Dried, filtered and concentrated in vacuo. The crude product is passed through SiO₂Chromatographic purification (300g SiO₂70: 30 Hexane-propanone, R_f0.31) to yield the title compound. APCI MS (m/z): 151.12 (M-H); c₈H₈O₃The calculated value of (a): 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- (tert-butyldimethylsilyloxy) -2-methylbenzoic acid (3). To a stirred solution of 4-hydroxy-2-methylbenzoic acid (2) (4.2g, 27.60mmol) in DMF (20ml) was added t-BDMSCl (10.2g, 67.63mmol) and stirred for 15 min. Dropping anhydrous i-Pr through a liquid adding funnel₂NEt (14.0ml, 80.05mmol) and stirred at room temperature for 20 h. 1MH for reactants₃PO₄(7ml) quench until final pH 3-4. The solution was extracted with hexane (4X 100 ml). Incorporated by referenceThe organic layer was MgSO₄Dried, filtered and concentrated in vacuo. SiO for crude product₂Chromatographic purification (300g SiO₂90: 9: 1 Hexane-propanone-AcOH, R_f0.28) to yield the title compound. APCI MS (m/z): 267.15(M + H); c₁₄H₂₂O₃Calculated value of Si: 266.13.¹HNMR (300MHz, chloroform-d) delta 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- (tert-butyldimethylsilyloxy) -2-methylphenyl) (1H-imidazolyl-1-yl) methanone (4). 4- (tert-butyldimethylsilyloxy) -2-methylbenzoic acid (3) (5.8g, 21.77mmol) was dissolved in CH₂Cl₂(50ml) in N₂Then treated with 1, 1' -carbonyldiimidazole (4.2g, 26.12mmol) at room temperature for 20 h. CH for solution₂Cl₂(50ml) dilution. The organic layer was washed with water (2X 50ml), brine (2X 30ml) and MgSO₄Drying, filtration and concentration in vacuo afforded the title compound (70: 29: 1 hexane-acetone-NEt)₃，R_f＝0.14)。APCI MS(m/z)：317.15(M+H)；C₁₇H₂₄N₂O₃Calculated value of Si: 316.47.¹h NMR (300MHz, chloroform-d) δ ppm 0.25(s, 6H)1.00(s, 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- (tert-butyldimethylsilyloxy) -2-methylbenzoic acid¹³C-methyl ester (5). To an oven-dried 20ml conventional Smith synthesis tube was added (4- (tert-butyldimethylsilyloxy) -2-methylphenyl) (1H-imidazol-1-yl) methanone (4) (5.5g, 17.38mmol), DCE (10ml), and,¹³CH₃OH (Cambridge Isotrope Laboratory, 2.2ml, 52.13mmol) and DBU (0.8ml, 5.21 mmol). The tube was sealed and heated with a Smith synthesizer at 100 ℃ for 2min with a microwave. The reaction was concentrated in vacuo. SiO for crude product₂Chromatographic purification (300 gSiO)₂95: 5 Hexane-propanone, R_f0.65) to yield the title compound. APCI MS (m/z): 282.5(M + H); c₁₄ ¹³CH₂₄O₃Calculated value of Si: 281.15.¹h NMR (300MHz, chloroform-d) δ ppm 0.22(s, 6H)0.98(s, 9H)2.56(s, 3H)3.85(d, J ═ 146.75Hz, 3H)6.62 to 6.74(m, 2H)7.86(d, J ═ 8.85Hz, 1H).

4- (tert-butyldimethylsilyloxy) -2- (dibromomethyl) benzoic acid¹³C-methyl ester (6). To 4- (tert-butyldimethylsilyloxy) -2-methylbenzoic acid¹³CCl of C-methyl ester (5) (4.0g, 14.21mmol)₄To a stirred solution (50ml) were added N-bromosuccinimide (7.6g, 42.64mmol) and 2, 2' -azobisisobutyronitrile (2.3g, 14.21 mmol). In N₂The reaction was heated to reflux (83 ℃ C.) for 18 h. The reaction was cooled to room temperature and filtered. The solvent was removed by vacuum filtration. SiO for crude product₂Chromatographic purification (300g SiO₂90: 10 Hexane-propanone, R_f0.78) to yield the title compound. APCI MS (m/z): 440.2(M + H); c₁₄ ¹³CH₂₂O₃Calculated value of Si: 439.22.¹h NMR (300MHz, chloroform-d) δ ppm 0.28(s, 6H)1.01(s, 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-formyl-4-hydroxybenzoic acid¹³C-methyl ester (7). To 4- (tert-butyldimethylsilyloxy) -2- (dibromomethyl) benzoic acid¹³A stirred solution of C-methyl ester (6) (5.0g, 11.38mmol) in i-PrOH (60ml) was added to a solution of silver nitrate (3.86g, 22.77mmol) in water (6 ml). The resulting mixture is in N₂Stirring for 20 h. The reaction was filtered and the filtrate was concentrated in vacuo. The residue is dissolved in CH₂Cl₂Over MgSO₄Dried, filtered and treated with 1M tetra-n-butylammonium fluoride in THF (6.6ml, 22.77 mmol). In N₂After 3h, the reaction was concentrated in vacuo. SiO for crude product₂Chromatographic purification (120g SiO₂80: 20 Hexane-propanone, R_f0.33) to yield the title compound. APCI MS (m/z): 182.2(M + H); c₈ ¹³CH₈O₄The calculated value of (a): 181.05.¹HNMR (300MHz, chloroform-d) δ ppm 3.95(d, J-147.50 Hz, 3H)7.09(dd, 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-oxoethoxy) -2-formylbenzoic acid¹³C-methyl ester (8). 2-formyl-4-hydroxybenzoic acid¹³C-methyl ester (7) (1.55g, 8.56mmol) was dissolved in acetone (20ml) and cooled to 0 ℃. Benzyl 2-bromoacetate (1.9ml, 11.97mmol) and potassium carbonate (1.4g, 10.27mmol) were added. Reactant is in N₂Stirring was continued for 18h at 0 ℃. The reaction was quenched with water (5ml) and the solvent removed in vacuo. The residue was partitioned between EtOAc (100ml) and water (40 ml). The layers were separated and the organic layer was washed with water (2X 20ml), brine (1X 20ml) and MgSO₄Dried, filtered and concentrated in vacuo. SiO for crude product₂Chromatographic purification (120g SiO₂85: 15 Hexane-propanone, R_f0.35) to yield the title compound. APCI MS (m/z): 330.1(M + H); c₁₇ ¹³CH₁₆O₆The calculated value of (a): 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-oxoethoxy) -2- (1, 3-dioxolan-2-yl) benzoic acid¹³C-methyl ester (9). Reacting 4- (2- (benzyloxy) -2-oxoethoxy) -2-formylbenzoic acid¹³C-methyl ester (8) (2.17g, 6.6mmol) dissolved in CH₂Cl₂(20ml) and cooled to 0 ℃.2, 6-di-tert-butylpyridine (0.150ml, 0.66mmol), 1, 2-bis (trimethylsiloxy) ethane (2.4ml, 9.88mmol) and trimethylsilyl triflate (0.180ml, 0.98mmol) were added. Reactant is in N₂Stirring was continued for 18h at 0 ℃. The solution was quenched with 2-pyridylcarbinol (0.127ml, 1.32 mmol). The solvent was removed in vacuo. SiO for crude product₂Chromatographic purification (120g SiO₂80: 20 Hexane-propanone, R_f0.22) to yield the title compound. APCI MS (m/z): 374.1(M + H); c₁₉ ¹³CH₂₀O₇The calculated value of (a): 373.12.¹h NMR (300MHz, chloroform-d) δ ppm 3.88(d, J ═ 147.12Hz, 3H)3.97-4.06(m, J ═ m2.26Hz，4H)4.73(s，2H)5.24(s，2H)6.65(s，1H)6.88(dd，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- (1, 3-dioxolan-2-yl) -4-, (¹³C-methoxycarbonyl) phenoxy) acetic acid (10). To 4- (2- (benzyloxy) -2-oxoethoxy) -2- (1, 3-dioxolan-2-yl) benzoic acid¹³A stirred solution of C-methyl ester (9) (1.72g, 4.6mmol) in EtOAc (25ml) was added 10% on target carbon (170 mg). The solution was degassed with 3 evacuation/nitrogen fill cycles. After the last evacuation, H from the balloon is used₂To backfill for the last evacuation. The reaction is at room temperature in H₂Stirring for 3 h. The reaction was filtered through a pad of celite. The solvent was removed from the filtrate in vacuo to give the title compound (60: 40 hexane-acetone, R)_f＝0.11)。APCI MS(m/z)：284.3(M+H)；C₁₂ ¹³CH₁₄O₇The calculated value of (a): 283.08.¹h NMR (300MHz, chloroform-d) δ ppm 3.89(d, J ═ 147.12Hz, 3H)4.06(s, 4H)4.75(s, 2H)6.65(s, 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- (Succinimidyloxy) -2-oxoethoxy) -2- (1, 3-dioxolan-2-yl) benzoic acid¹³C-methyl ester (11). To 2- (3- (1, 3-dioxolan-2-yl) -4-, (¹³C-methoxycarbonyl) phenoxy) acetic acid (10) (1.21g, 4.27mmol) in EtOAc (20ml) was added 1-hydroxypyrrolidine-2, 5-dione (0.74g, 6.41mmol) and PS-carbodiimide (Argonun technology, 1.29mmol/g) (4.6g, 5.98 mmol). The reaction was sealed and stirred at room temperature for 20 h. The solution was filtered through a medium pore sintered glass funnel. N introduction through sintered glass₂The resin was stirred with EtOAc (20mL) for 10 min. The EtOAc was filtered and combined with the original filtrate. The resin was washed a second time using the same protocol. The combined filtrates were concentrated in vacuo. SiO for crude product₂Chromatographic purification (120g SiO₂70: 29: 1 Hexane-propanone-AcOH, R_f0.14) to yield the title compound. APCI MS (m/z): 381.2(M + H); c₁₆ ¹³CH₁₇NO₉Is calculated byThe value: 380.09.¹h NMR (300MHz, chloroform-d) δ ppm2.87(s, 4H)3.89(d, J ═ 147.12Hz, 3H)4.02-4.10(m, J ═ 1.70Hz, 4H)5.04(s, 2H)6.67(s, 1H)6.95(dd, J ═ 8.67, 2.83Hz, 1H)7.35(d, J ═ 2.83Hz, 1H)7.95(d, J ═ 8.67Hz, 1H).

Reagents and conditions: a) n-BuLi, then methyl chloroformate; b) toluene, 170 ℃; c) bromoacetic acid benzyl ester, K₂CO₃Acetone; d) h₂，Pd/C，EtOAc。

4, 4-diethoxybutyn-2-ynoic acid methyl ester (13). A solution of diethoxypropyne (Aldrich, 10.93g, 85.3mmol) in diethylene glycol dimethyl ether (100ml) was added to N₂Then cooled to-30 ℃. N-butyllithium (81.0mmol) was added dropwise over 5 min. The reaction was incubated for 6 h. The anion formed was led via a conduit to a solution of methyl chloroformate (6.5ml, 84.1mmol) in 50ml of diethylene glycol dimethyl ether while maintaining the solution in N₂Next, the mixture was stirred overhead in a dry ice/acetone bath (overhead still). The reaction was allowed to warm to room temperature overnight. The solid was removed by filtration through an alumina pad (100g basic alumina, washed with 200ml diethyl ether). The solution was concentrated well by rotary evaporation (bath temperature 35 ℃). The solid was removed by filtration through an alumina pad (washed with 500ml of diethyl ether, 100g of basic alumina). The solution was concentrated well by rotary evaporation (bath temperature 35 ℃). The product was purified by distillation (fractions boiling at 57-60 ℃ C. under 1mm Hg) to give the title compound.¹H NMR (300MHz, chloroform-d) delta 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: the method comprises the following steps: 4.22min (EIMS (M/z) ═ 141 (M-OEt); C₇H₉O₃ ⁺The calculated value of (a): 141).

Methyl 2- (diethoxymethyl) -4-hydroxybenzoate (16). To an oven-dried 5ml Conical Smith tube was added methyl 4, 4-diethoxybutyn-2-ynoate (0.25g, 1.3mmol) (13)) (E) - (4-methoxybut-1, 3-dien-2-yloxy) trimethylsilane (0.52ml, 2.7mmol) (12), 4- (3, 5-di-tert-butyl-4-hydroxybenzyl) -2, 6-di-tert-butylphenol (0.11g, 0.27mmol) and toluene (4 ml). The synthesis tube was sealed and heated at 170 ℃ for 20 h. The reaction was cooled to room temperature, transferred to a round bottom flask and treated with 1M tetra-n-butylammonium fluoride in THF (0.78ml, 2.7 mmol). The solution was sealed and stirred at room temperature for 3 h. The solvent was removed in vacuo. SiO for crude product₂Chromatographic purification (40g SiO₂80: 20 Hexane-propanone, R_f0.42) to yield the title compound. APCI MS (m/z): 255.2(M + H); c₁₃H₁₈O₅The calculated value of (a): 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, 3H)5.59(s, 1H)6.26(s, 1H)6.80(dd, J ═ 8.57, 2.73Hz, 1H)7.30(d, J ═ 2.64Hz, 1H)7.82(d, J ═ 8.67Hz, 1H).

4- (2- (benzyloxy) -2-oxoethoxy) -2- (diethoxymethyl) benzoic acid methyl ester (17). To a0 deg.C stirred solution of methyl 2- (diethoxymethyl) -4-hydroxybenzoate (0.5g, 2mmol) (16) in acetone (15ml) was added benzyl 2-bromoacetate (0.4ml, 3mmol) and potassium carbonate (0.3g, 2 mmol). Solution in N₂Stirring was continued for 20h at 0 ℃. The solution was quenched with water (5ml) and the solvent was concentrated in vacuo. The residue was partitioned between EtOAc (75ml) and water (30 ml). The layers were separated and the organic layer was washed with water (2X 20ml), brine (1X 20ml) and MgSO₄Dried, filtered and concentrated in vacuo. SiO for crude product₂Chromatographic purification (40g SiO₂85: 15 Hexane-propanone, R_f0.35) to yield the title compound. APCI MS (m/z): 255.2 (M-EtOH). C₂₂H₂₆O₇The calculated value of (a): 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, 3H)4.73(s, 2H)5.24(s, 2H)6.22(s, 1H)6.86(dd, J ═ 8.67, 2.64Hz, 1H)7.35(s, 6H)7.83(d, J ═ 8.67, 1H).

2- (3- (diethoxymethyl) -4- (methoxycarbonyl) phenoxy) acetic acid (18). To 4- (2- (benzyloxy) -2-oxoethoxy) -2- (2-methoxy-ethoxy) -2- (bDiethoxymethyl) benzoic acid methyl ester (0.65g, 1.6mmol) (17) to a stirred solution in EtOAc (15mL) was added palladium (0.052g, 0.48 mmol). The solution was degassed with 3 evacuation/nitrogen fill cycles. After the last evacuation, H from the balloon is used₂To backfill for the last evacuation. The reaction is at room temperature in H₂Stirring for 3 h. The reaction was filtered through a pad of celite. The solvent was removed in vacuo to give the title compound (70: 29: 1 hexane-acetone-AcOH, R)_f＝0.21)。APCI MS(m/z)：311.1(M-H)。C₁₅H₁₉O₇The calculated value of (a): 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, 3H)4.74(s, 2H)6.24(s, 1H)6.90(dd, J ═ 8.67, 2.83Hz, 1H)7.36(d, J ═ 2.64Hz, 1H)7.86(d, J ═ 8.67Hz, 1H).

4- (N- (Succinimidyloxy) -2-oxoethoxy) -2- (1, 3-dioxolan-2-yl) benzoic acid methyl ester (19). To a stirred solution of 2- (3- (diethoxymethyl) -4- (methoxycarbonyl) phenoxy) acetic acid (450mg, 1.44mmol) (18) in EtOAc (15ml) was added N-hydroxysuccinimide (248mg, 2.16mmol) and PS-carbodiimide (Argonaunt Technology, 1.29mmol/g) (1.5g, 2.02 mmol). The reaction was sealed and stirred at room temperature for 20 h. The solution was filtered through a medium pore sintered glass funnel. N introduction through sintered glass₂The resin was stirred with EtOAc (20mL) for 10 min. The EtOAc was filtered and combined with the original filtrate. The resin was washed a second time using the same protocol. The combined filtrates were concentrated in vacuo. SiO for crude product₂Chromatographic purification (40g SiO₂80: 19: 1 Hexane-propanone-AcOH, R_f0.38) to yield the title compound. APCI MS (m/z): 364.23(M + H-OEt). C₁₇H₁₈NO₈ ⁺The calculated value of (a): 364.1.¹h NMR (300MHz, chloroform-d) ppm 1.23(t, J ═ 7.16Hz, 6H)2.87(s, 4H)3.54-3.76(m, 4H)3.87(s, 3H)5.03(s, 2H)6.23(s, 1H)6.91(dd, J ═ 8.67, 2.83Hz, 1H)7.40(d, J ═ 2.64Hz, 1H)7.86(d, J ═ 8.67Hz, 1H).

Scheme 3

Reagents and conditions: a) acetonitrile, 25 ℃; b) DCl, D₂O。

Tetrakis- [ omega- (4-aza-5-oxo-7-oxa-7- ((3- (2, 4-dioxolanyl) -4-) (¹³C-methoxy) carbonyl) benzene) heptane) -2.5kD polyoxyethylene]Methane 22. PTE-100PA (NOFcorp, 547mg,. about.52. mu. mol) was dissolved in 2.5ml of anhydrous acetonitrile and treated with succinate 11(100mg, 260. mu. mol, 5 equivalents). The reaction was heated to 40 ℃ for 7 h. The reaction was cooled to room temperature and treated with 10mM ammonium formate (10 ml). The solution was loaded onto column 1 and eluted according to the solvent system 1/gradient 1 as specified in the preparative reverse phase HPLC part of the general experiment. Bands eluting at 27.4-28.8 minutes were separated and concentrated in vacuo to remove acetonitrile. 2560g of the aqueous solution were filtered through a 0.22 μm centrifugal filter (National Scientific, PN 66064-466) and the filtrate was then lyophilized. The solid was dissolved in 5ml of D₂And O, and freeze-drying to obtain the 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, (CH) 3.₂CH₂O)_n)3.90(d，J＝149.07Hz，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)7.97(d，J＝8.61Hz，1H)8.40(s，1H)。¹³C NMR (101MHz, deuterium oxide) delta ppm55.08(s, 4C), 72.00(s, 5C).

Tetrakis- [ omega- (4-aza-5-oxo-7-oxa-7- ((3- (2, 4-dioxolanyl) -4-) (¹³C-methoxy) carbonyl) benzene) heptane) -5.0kD polyoxyethylene]Methane 23. PTE-200PA (NOFcorp, 1.55g,. about.64. mu. mol; proof of analysis: 83% tetrafunctional), succinate 11(147mg,. about.386. mu. mol) and 5ml acetonitrile were heated to 40 ℃ for 4 h. Acetonitrile was removed and 5ml of 0.1% acetic acid was added. The solution was heated to 35 ℃ to aid dissolution. The solution was loaded onto column 1 (jacket and solvent heated to 35 ℃) in accordance with the solvent system 2-Elution was performed in gradient 1. The bands eluted from 22.8-26min were concentrated in vacuo and dried at 35 ℃ under reduced pressure (1mm Hg). The residue is dissolved in 10ml of D₂And O, and freeze-drying to obtain the product. Warp beam¹H NMR analysis gave a 6: 1 mixture of solids of 23 and 25;¹h NMR was 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, (CH, 2H) 3.70(s), (CH, g, H, g₂CH₂O)_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) delta ppm55.08(s, 9.98C)72.00(s, 2.84C).

Tetra- [ omega- (4-aza-5-oxo-7-oxa-7- ((3-formyl-4-) (¹³C-methoxy) carbonyl) benzene) heptane) -2.5kD polyoxyethylene]Methane 24. PEG reagent 22(439mg, 38.3. mu. mol) was dissolved in 5ml D₂O, cooled to 0 ℃ and degassed by 4 evacuation/nitrogen fill cycles. DCl/D was added at 85mM₂O solution (360. mu.l, 0.2 eq/acetal). The cooling bath was removed and the reaction was stirred at room temperature for 24 h. After 24h, an additional portion of DCl (360. mu.l) was added. The reaction was stirred for 63 h. The lyophilized aqueous solution was dissolved in 2ml of D₂And (4) in O. The solution was filtered through a 0.1 μm centrifuge filter (Micron Bioseparations, PN UFC40W00) and lyophilized to give the 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, (CH), (oh) 3.36 (r, H, g, H₂CH₂O)_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)。

Tetra- [ omega- (4-aza-5-oxo-7-oxa-7- ((3-formyl-4-) (¹³C-methoxy) carbonyl) benzene) heptane) -5.0kD polyoxyethylene]Methane 25. PEG reagent 23(840mg, 39. mu. mol) was dissolved in 10ml H₂O, cooled to 0 ℃ and treated with 85mM DCl/D₂O (183. mu.l, 15.6. mu. mol, 0.1 eq/acetal). After 4.5 days, the reaction was lyophilized and dissolved in 10ml D₂85mM combined used for ODCl/D₂O (183. mu.l, 15.6. mu. mol) was treated at room temperature for 1 day. The solution was lyophilized to give the 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, (CH), (oh) 3.36₂CH₂O)_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)。

Scheme 4

Reagents and conditions: 600mM LiCl, pH 2.5-6 ascorbic acid buffer.

Tetrakis- [ omega- (4-aza-5-oxo-7-oxa-7- (((3 ' R, 9 ' bS) -3 ' - (carbonyl (HN-GGGGGKKRP(Hyp) G (Cpg) S (D-Tic) (Cpg) -OH)) -2 ', 3 ' -dihydrothiazolo [2 ', 3 ' -a ])]Isoindol-5 ' (9 ' bH) -on-8 ' -yl)) heptane) -2.5kD polyoxyethylene]Methane 27. Peptide 26(1.12g, PPL laboratories) was dissolved in 1.8ml D₂O, D with 0.25ml of 0.50M sodium ascorbate/4.8M LiCl₂O treatment and then cooling to 0 ℃. The solution was degassed by 3 evacuation/nitrogen fill cycles. The pH was adjusted to 6.1 with 1M LiOH under nitrogen and degassed by 3 evacuation/nitrogen fill cycles. The peptide concentration was measured by HPLC using chemiluminescence nitrogen detection (CLND) calibrated for caffeine at 114.4mM as described in the general experimental section. PEG reagent 24(400mg, 35.4. mu. mol) was dissolved in 2.5ml D₂O, D with 0.25ml of 0.50M sodium ascorbate/4.8M LiCl in succession₂D of O and 0.25ml of 0.55 ascorbic acid/4.86M LiCl₂And (4) O treatment. Peptide 26(1.4ml, 159.4. mu. mol) was then added. The pD of the assay solution was 5.1. The reaction was stirred under nitrogen for 3 days. The solution was loaded onto column 1 and eluted using the solvent system 2/gradient 1 as specified in the preparative reverse phase HPLC section of the general experiment. The bands eluted at 12.2-15.4 min were concentrated in vacuo to remove acetonitrile (bath temperature 35 ℃) and lyophilized. Residue ofThe material was further purified using cation exchange column 1 and eluted using the solvent system 1/gradient 1 specified in the preparative ion exchange section of the general experiment. The band eluted at 41.2-58.2min was concentrated to dryness by rotary evaporation (bath temperature 35 ℃). The residue was dissolved in 10ml water, placed in 3500MWCO dialysis membrane (Pierce, PN65035) and dialyzed against deionized water (3X 500ml, 1-2h per cycle). The dialyzed solution was lyophilized to give the product. CLND: 29.3 percent; theoretical value: 36.3 percent. NMR resonance identification peaks were selected for ligation chemistry:¹h NMR (400MHz, deuterium oxide) δ ppm 4.85(dd, J ═ 14.87Hz, 1H)4.98(t, J ═ 7.04Hz, 1H)5.01 to 5.07(m, 1H)5.17(t, J ═ 5.48Hz, 1H)5.30 to 5.40(m, 1H)6.19(s, 1H)7.13 to 7.36(m, 1H)7.80(d, J ═ 8.61Hz, 1H).

Tetrakis- [ omega- (4-aza-5-oxo-7-oxa-7- (((3 ' R, 9 ' bS) -3 ' - (carbonyl (HN-GGGGGKKRP(Hyp) G (Cpg) S (D-Tic) (Cpg) -OH)) -2 ', 3 ' -dihydrothiazolo [2 ', 3 ' -a ])]Isoindol-5 ' (9 ' bH) -on-8 ' -yl)) heptane) -5.0kD polyoxyethylene]Methane 28. PEG reagent 28(99.4mg, 4.66. mu. mol) was dissolved in 2ml D₂O, D with 0.5ml of 0.50M sodium ascorbate/4.8M LiCl₂And (4) O treatment. The pD was determined to be 4.3. To this solution was added peptide 26 (peptide content 72%, 47.4mg, 21.7. mu. mol). The reaction was stirred at room temperature for 18h under nitrogen and then heated to 45 ℃ for 2 h. The solution was loaded onto column 2 and eluted using the solvent system 2/gradient 2 as specified in the preparative reverse phase HPLC section of the general experiment. The bands eluting over 10.5-12 minutes were collected and concentrated in vacuo to 2ml (bath temperature 34 ℃). The solution was loaded onto cation exchange column 2 and eluted using solvent system 1/gradient 2 as specified in the preparative cation exchange LC section of the general experiment. The band eluted at 20-24 min was concentrated in vacuo (bath temperature 35 ℃) and dialyzed against 500ml of deionized water using 10K MWCO Slide-a-lyzer (Pierce, PN 66810). The water was replaced in portions of 2h, 10h and 2h with fresh 500ml portions. 2560g of the dialyzed solution was filtered through a 0.22 μm centrifugal filter (National Scientific, PN 66064-466) and the filtrate was lyophilized to give the title compound. CLND: peptide content 22.4%; theoretical value: 23.2 percent. This sample was used for detailed structural characterization.

Detailed structural analysis of conjugate 28:

NMR experiments.

NMR experiments were performed in 3mm tubes using 5mm reverse direction detection cryoprobes on a Bruker drx-600 spectrometer.

Determination of chemical Displacement values

According to 2D TOCSY (100ms DIPSI-2 mixing time) and 2D¹³C-¹H HMBC(ⁿJ_CHThe proton chemical shift value of 28 (FIG. 1) was measured at 60ms deposition (evolution) and n is 1 to 4. Only the resonances of the major rotamer (trans) are listed in table 2. The minor rotamer is derived from the hindered C-terminus and proline amide bond rotation.

FIG. 1. conjugate 28 with defined resonance.

Of FIG. 2.28¹H NMR Spectrum (D)₂O, 298K), whose HOD signal and PEG signal are attenuated by a spin-diffusion filter (spin-diffusion filter) and a weak presaturation (presaturation), respectively.

Table 1 proton chemical shift values of fig. 2, PEG singlet was set at δ 3.55ppm.

Residue order (residue order) is PEG → cp²As shown in scheme 1.

Correlation of PEG resonance with peptide.

A triple bond,¹H-¹³The C-correlation spectrum was used to determine the location of pegylation. Phenoxyacetamide methylene (PEG)_αFIG. 3) was used as starting point (4.58ppm, 600MHz, Table 3). The observed association pathway is PEG_α(4.59ppm) to C₈(162.2ppm) to H₆(7.67ppm) to C₅(173.2ppm) to H₃(4.85ppm) to C_3’(172.7ppm) to Gly_5-α·(3.89 ppm). Observed C₅(173.2ppm) with H_9bThe correlation of (6.06ppm) supported the formation of a central B ring. Likewise, H₃(4.85ppm) to C_9b(67.2ppm) to H_2RThe associated order of (3.73ppm) supports the formation of the A ring. H_2RSignal representation and C_3’Support the approach of the A-ring to gly of the peptide₅。

FIG. 3.PEG resonance is associated with 13C and 1H NMR of the N-terminal glycine of peptide 26 via the (9bS) -2, 3-dihydrothiazolo [2, 3-a ] isoindol-5 (9bH) -one ring.

H₁Determination of the relevant stereoisomeric chemistry (FIG. 3)

1) Determined according to 2D NOESY experiments (500ms mixing time) and short (100ps) MD runs, residue H₁Related stereoisomeric chemistry with H₄Are in trans with each other. The calculated distances for the cis and trans diastereomers are shown in table 2, and the measured distances are given in terms of 2D NOESY. In particular, predicting the trans configuration of H₁-H₄Distance of 4.1The cis diastereomer is significantly shorter (3.1)). Measured distance 4.4Consistent with the prediction of the trans diastereomer.

TABLE 2D NOE of (9bS) -2, 3-dihydrothiazolo [2, 3-a ] isoindol-5 (9bH) -one ring gives and averages MD interprotonic distances (FIG. 3)

2: atom H of cis-and trans-diastereomer₄-C₄-N-C₁And H₁-C₁-N-C₄The predicted dihedral angles formed are shown in Table 3. From these angles, H is derived₃-C_9bAnd H_9b-C₃3-bond coupling constant. H₃-C_9bAnd H_9b-C₃The observed couplings at 8 and 0Hz correspond to the predicted trans diastereomer shown in figure 1, respectively. In addition, a correlation was seen in HMBC 2D experiments.

Predicted dihedral angles and 3-bond C-H couplings of Table 3.28. The atomic labels are defined in figure 1.

	Cis-preproMeasuring	Prediction of trans form	Experiment of
				θ(H3C3N4C9b)3J(H3，C9b)	-680Hz	1528Hz	--8Hz
θ(H9bC9bN4C3)3J(H9b，C3)	860Hz	870	--0Hz
				H3-C9b	Without cross peaks	Cross peak	Cross peak
H9b-C3	Without cross peaks	Without cross peaks	Without cross peaks

3. Molecular mechanics calculations indicated that the trans diastereomer had a lower enthalpy of 5.5kcal/mol than the cis diastereomer (figure 4). Gly₅The measured distance of the carbonyl group 5 of (a) to the amide NH is 2.1This supports the presence of intramolecular hydrogen bonds.

FIG. 4 molecular mechanics calculations for the trans and cis diastereomers.

Conjugates 28 were analyzed using a Bruker Q-FTMS system equipped with a 7-T superconducting magnet. The individual ions were separated using a front-end quadrupole. Ions were trapped in FTMS cells using "gas-assisted dynamic trapping". From 4: 1MeOH-H₂O solution, the solution was electrosprayed at a flow rate of 0.5 uL/min. For IRMPD dissociation experiments Synrad CO was turned on₂Laser energy is 15% for laser 200 ms. Ions were detected with an acquisition bandwidth of 900kHz using direct mode detection, and 512K data points were collected. Before the transformation of the number-mode Fourier is performed, the time-domain data is summed and zero-filled (zero-filled). The instrument was externally calibrated with an Agilent tuningmix. In this experiment (fig. 5), a full deconvoluted spectrum (full deconvoluted spectra) was obtained that represents the heterogeneity of the polymer. Has 420 repeats- (CH)₂CH₂The dispersed isomers of the O) -units were captured by FT-MS cells and irradiated with an IR laser (fig. 6). This caused ion dissociation, resulting in 4 subfragments, each spaced 1478.6742 amu. These data are consistent with the presence of 4 peptides per polymer and the dissociation occurring between glycine 5 and the newly formed tricyclic system (fig. 7).

FIG. 5: deconvolved FT-MS spectra

FIG. 6: ion isolation (n ═ 420) and IRMPD dissociation.

FIG. 7: and IRMPD segment assignment.

Scheme 5

Reagents and conditions: a) methanol-water, 100mM L-ascorbic acid, 20mM sodium L-ascorbate.

TABLE 7 native ligation with 2-formyl ester.

(3 ' R, 9 ' bS) -3 ' - (carbonyl (HN-GGGGGKKRP(Hyp) G (Cpg) S (D-Tic) (Cpg) -OH)) -2 ', 3 ' -dihydrothiazolo [2 ', 3 ' -a ]]Isoindol-5 '(9' bH) -one 32. Peptide 26(116mg, peptide content 72%, 52.8. mu. mol) was dissolved in 4.0ml 100mM L-ascorbic acid/20 mM sodium L-ascorbate. Methyl 2-formylbenzoate (10.4mg, 63.3. mu. mol) was added, followed by 400. mu.L of MeOH. The reaction was stirred for 50 h. The solution was loaded onto column 2 and eluted according to the solvent system 2/gradient 3 specified in the preparative reverse phase HPLC part of the general experiment. The bands eluted at 14-15 min were concentrated in vacuo, acetonitrile was removed, and the product was lyophilized. Peptide content was 56% by CLND. Selection of 26¹H NMR resonance (assignment to protons), see fig. 8.¹H NMR (400MHz, deuterium oxide) delta ppm 4.96 (H)₃，t，J＝7.43Hz，1H)6.19(H₉，s，1H)7.15-7.28(D-Tic，m，4H)7.61(H₆，t，J＝7.43Hz，1H)7.64(H₈，d，J＝8.61Hz，1H)7.72(H₇，t，J＝7.04Hz，1H)7.79(H₅，d，J＝7.82Hz，1H)。APCI MS(m/z)848.8971(M+2，z＝2)；C₇₈H₁₁₅N₂₁O₂₀Calculated value of S (z ═ 2): 848.909.

peptides 33-36 were synthesized using the method described for 33. The mass spectral data are shown in table 7.

The determined resonance of fig. 8.32.

Detailed structural analysis of peptide 32:

NMR experiment

Combinations of 2D Cosy45, 2D Noesy (phase sense, 25 and 40 ℃), 2D¹H/¹³CHSQC、2D¹H/¹³C HMBC was measured by 1H NMR spectroscopy at 600MHz using a 5mm reverse broadband probe. For H₃To obtain H₉Stereochemistry of (A), H₃From L-cysteine. In particular, H₉And H_(2S)nOe (40 ℃ C.) was observed therebetween. Proton pair H_(2R)Is obtained from 2D Cosy45 experiments. This same resonance (H)_(2R)) Shows that the reaction solution has high reaction efficiency on H in a 2DNOESY experiment at 40 DEG C₃The association of (a). In summary, NMR experiments supported H₉And H₃Trans relationship to thiazoline ring plane (figure 8).

Scheme 6

Reagents and conditions: a) the content of the CDI is measured by the CDI,¹³C-MeOH，DBU；b)NBS，AIBN。

5-bromo-2-methylbenzoic acid¹³C methyl ester (38). To a stirred solution of 5-bromo-2-methylbenzoic acid (37) (25g, 116mmol) in 100ml of anhydrous DCM was added 1, 1' -carbonyldiimidazole (21g, 128 mmol). The solution was stirred for 3.5 h. Transferring the solution into a pressure vessel¹³CH₃OH and DBU treatment. Solution H₂O(2×20ml)、5％NaHCO₃(2X 20ml) washed and the organic layer was MgSO₄And (5) drying. The solvent was removed in vacuo to give the product.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).

5-bromo-2- (dibromomethyl) benzoic acid¹³C-methyl ester (39). To 38(5.6g 24mmol) of CCl₄To the stirred solution were added N-bromosuccinimide (13.0g, 73mmol) and 2, 2' -azobisisobutyronitrile (4.0g, 24 mmol). The solution was refluxed until TLC showed the starting material was consumed. The mixture was purified by flash chromatography using a Biotage 40+ silica gel column packed with a gradient of 0-10% EtOAc in hexane (R of 39)_f0.4 in 1: 9 EtOAc/hexanes) to give the title compound.¹H NMR (300MHz, chloroform-d) δ ppm 3.95(d, J-147.91 Hz, 3H)7.52(dd, J-8.48, 2.05Hz, 1H)7.78(d, J-8.48 Hz, 1H)8.00(s, 1H)8.30(d, J-1.90 Hz, 1H).

Scheme 7

Reagents and conditions: a) NaH, PS-DIEA; b) methanol-water, 100mM L-ascorbic acid, 20mM sodium L-ascorbate.

5-bromo-2- (3-butylthiazolidin-2-yl) benzoic acid¹³C-methyl ester (41). To a stirred solution of 2- (butylamino) ethanethiol (40) (621.5mg, 5mmol) in 20ml THF was added PS-triphenylphosphine (Argonaut Technologies, 2.1030g, 5 mmol). The reaction was stirred for 30 minutes and filtered through a medium pore sintered glass funnelAnd (3) solution. N introduction through sintered glass₂The resin was stirred with THF (20ml) for 10 min. The THF was filtered and combined with the original filtrate. The resin was washed 2 nd time using the same protocol. The combined filtrates were cooled to 0 ℃. Sodium hydride (0.06ml, 3mmol) and 5-bromo-2- (dibromomethyl) benzoic acid were added¹³C-methyl ester (39) (897.0mg, 2mmol) and PS-DIEA (Argonaut Technologies, 1.2429g, 5mmol) were stirred at room temperature for 2 days. The reaction was refluxed overnight, cooled to room temperature and stirred for 10 days. The solution was filtered, concentrated in vacuo and purified by reverse phase chromatography (column 1, solvent system 3, gradient 4). The bands eluting over 26-27 minutes were concentrated in vacuo to afford the title compound. APCI MS (m/z): 359.0(M + H); c₁₄ ¹³CH₂₁ ⁷⁹BrNO₂Calculated value of S: 359.04.APCI MS (m/z): 361.0(M + H); c₁₄ ¹³CH₂₁ ⁸¹BrNO₂Calculated value of S: 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' -dihydrothiazolo [2 ', 3' -a ]]Isoindol-5 '(9' bH) -one 42. Prepared as described for 32. HR FTMS (m/z): 887.8612(M +2, z ═ 2); c₇₈H₁₁₄ ⁷⁹BrN₂₁O₂₀Calculated value of S (z ═ 2): 887.8650, respectively; 888.8509(M +2, z ═ 2); c₇₈H₁₁₄ ⁸¹BrN₂₁O₂₀Calculated value of S (z ═ 2): 888.8650.

scheme 8

Reagents and conditions: a) PS-carbodiimide, pentafluorophenol; b) PEG reagent 21, Hunig base; c) d₂O, 100mM LiCl, D of 50mM deuterated ascorbic acid with 1M NaOD₂Basified to pD 3.7.

Methyl 2- (diethoxymethyl) -4- (2-oxo-2-pentafluorophenoxyethoxy) benzoate (43). Evacuating and backfilling N₂A50 ml RB flask was charged with 132mg of washed/dried 10% palladium on carbon (0.12mmol Pd) and 4ml of anhydrous THF. By careful evacuation (minimizing bumping) and backfilling with N₂For a total of 3 cycles, the mixture was degassed. A solution of methyl 4- (2- (benzyloxy) -2-oxoethoxy) -2- (diethoxymethyl) benzoate (0.500g, 1.24mmol) (17) in dry THF (3ml) was added to the slurry, the vial was washed with 1ml THF and transferred to the RB flask, evacuated/backfilled with nitrogen for 3 cycles. After the last evacuation, with H from the balloon₂To refill the evacuated RB flask. Reaction at room temperature, H₂Stirring was continued for 4hr at which time GC/MS (method 3) detection indicated completion of the reaction (17, 17.4min, m/z 358.1, C)₁₉ ¹³CH₂₁O₆ ⁺Calculated value of 358.1, M-OEt; 18, 14.26min, m/z 268.1, C₁₂ ¹³CH₁₅O₆ ⁺Calculated value of 268.1, M-OEt). The solution was filtered through a pad of celite using a sintered glass vacuum filter into a 50ml RB flask already charged with PS-carbodiimide suspended in 15ml anhydrous THF (Argonaut Technologies, Inc, 2.4g, 3.1 mmol). The celite was washed three times with THF (3ml) and combined with the filtrate/PS-carbodiimide. Heterogeneous mixture in N₂Stirring was continued for 20min and treated with pentafluorophenol (456mg, 2.48mmol) in THF. Reaction mixture in N₂Stirring for 16hr, at which time the reaction was detected to be complete by GC/MS (method 3) (18, 14.26 min; 43, 15.6 min; m/z 434.1, C)₁₈ ¹³CF₅H₁₅O₆ ⁺Calculated value of 434.1, M-OEt). The mixture was filtered through a medium fritted funnel into a tar-coated 50ml RB flask. Then 10ml THF was added to the resin with N₂Mix gently. Combining the filtrates, removing the solvent, and passing the product throughVacuum drying to obtain the product. EI MS m/z 434.1, C₁₈ ¹³CF₅H₁₅O₆ ⁺Calculated value of 434.1, M-OEt).

Compound 44 evacuation and backfill of N₂A50 ml RB flask was charged with 20K tetraaminoPEG (21, 440mg, 22. mu. mol) and 3ml anhydrous acetonitrile. By careful evacuation (minimizing bumping) and backfilling with N₂For a total of 3 cycles, the mixture was degassed. To the solution was added Hunig's base (0.172mmol, 30. mu.l) and a solution of methyl 2- (diethoxymethyl) -4- (2-oxo-2-pentafluorophenoxyethoxy) benzoate (43, 0.128mmol) in dry acetonitrile (1ml +1ml for rinsing). Molecular sieves (powder form, 4)Pore size, 100mg) was added to the mixture. The solution was degassed by evacuating/backfilling with nitrogen for 3 cycles. Reacting the mixture with N₂Stirring at 40 deg.C for 24 hr. The mixture was filtered through a medium fritted funnel into a 50ml RB flask already charged with Si-bound piperazine (silica inc., 171mg, 0.15mmol) and Si-bound carbonate (silica inc., 0.3mmol, 434mg) and washed with 10ml acetonitrile. The combined filtrates are under N₂Stirring at 40 deg.C for 15 hr. The mixture was then filtered through a medium fritted funnel into a tar-coated 50ml RB flask, which was then washed with 10ml acetonitrile. The solvent was removed and the product was dried under vacuum to give 44.¹³C NMR(D₂O, partial structure): δ 170.14, 72.00.

Compound 45 was synthesized according to the procedure described for 28. The reaction is at pD 3.7, D₂In O. In particular, with D₂O preparation of 100mM LiCl and 50mM deuterated ascorbic acid (from D)₂O lyophilized to give, 3 cycles) a solution. pD was adjusted to 3.7 with 1M NaOD. To this solution was added the PEG reagent 44 and the peptide 26. The reaction was stirred at room temperature for 13h and worked up as described at 28. FT-MSMS confirmed a structure similar to 28, but for¹³C becomes 1amu higher.

Example (b): in vivo antinociceptive Activity of Polymer-conjugated anti-B1 peptides in rat and monkey pain models

A.Neuropathic pain model in rats. Male Sprague-Dawley rats (200g) were anesthetized with isoflurane inhalant following the procedure first described by Kim and Chung (Experimental model for peripheral neuropathy caused by segmental spinal nerve ligation, rat experimental model for peripheral neuropathy, Pain 50: 355) 363, (1992)), and then the L5 and L6 levels of the left lumbar spinal nerve were firmly ligated (wire number 4-0) distal to the dorsal root ganglion and prior to the sciatic nerve entrance. The incision was closed and the rats were allowed to recover. This method causes mechanical (tactile) allodynia in the left hind paw as assessed by observing and recording the pressure at which the affected paw (ipsilateral to the site of nerve injury) retreats from a graduated stimulus (von Frey filaments range from 4.0-148.1mN) applied perpendicularly to the plantar surface of the paw (between the pads) through a wire mesh viewing cage. Paw Withdrawal Thresholds (PWT) were determined by sequentially increasing and decreasing stimulation intensity and analyzing the withdrawal data using a Dixon non-parametric test, see Chaplan, s.r. et al (quantitative assessment of paw tactile allodynia in rats) j.neurosci.meth, 53: 55-63 (1994).

Normal rats and control surgical rats (isolated nerves but not ligated) withstood a pressure of at least 148.1mN (equivalent to 15g) without response. Spinal nerve ligated rats responded when only 4.0mN (equivalent to 0.41g) pressure was applied to the affected paw. Rats were included in the study only when they did not exhibit motor dysfunction (e.g. paw drag or drop) and their PWT was less than 39.2mN (equivalent to 4.0 g). At least 7 days post-surgery, rats were treated by s.c. injection of either the test peptide or test vehicle-conjugated peptide (typically at screening doses of about 1mg/kg and about 60mg/kg, respectively) or control diluent (PBS), after which PWT was measured daily for 7 days.

B.Rat CFA inflammatory pain model. Male Sprague-Dawley rats (200g) were anesthetized with isoflurane inhalation and injected in the left hind paw with 0.15ml of Complete Freund's Adjuvant (CFA).This method results in mechanical (tactile) allodynia of the left hind paw as assessed by observing and recording the pressure at which the affected paw is withdrawn from a graduated stimulus (von Frey wires range from 4.0 to 148.1mN) applied perpendicularly to the plantar surface of the paw (between the pads) using a wire mesh viewing cage. PWT was determined by sequentially increasing and decreasing the stimulus intensity and analyzing the regression data using the Dixon nonparametric test, see Chaplan et al (1994). Rats were included in the study only when they did not exhibit motor dysfunction (e.g. paw drag or drop) or skin breakdown and their PWT was less than 39.2mN (equivalent to 4.0 g). At least 7 days after CFA injection, rats were treated by s.c. injection of one test polymer-conjugated peptide (typically screening dose about 60mg/kg) or control solution (PBS), after which PWT was determined daily for 7 days. The average Paw Withdrawal Threshold (PWT) can be converted to the maximum possible effect (% MPE) using the following equation: % MPE ═ 100 (PWT of treated rats-PWT of control rats)/(PWT of 15-control rats). Thus, the 15g (148.1mN) cutoff corresponds to 100% MPE and the control reaction to 0% MPE.

Preferred polymer-conjugated peptides of the invention are capable of producing antinociceptive effects at screening doses of about 1mg/kg and about 60mg/kg, respectively, in relation to PD.

B.Green monkey LPS inflammation model. The effect of polymer-conjugated peptides as inhibitors of B1 activity can be evaluated in male green monkeys (Cercopathiecus aethiops St kits) locally challenged with B1 agonist essentially as described by debois and Horlick (British journal of pharmacology.132: 327-335(2002), which is incorporated herein by reference in its entirety).

To determine whether the PEG-conjugated peptide antagonists of the invention inhibit B1-induced edema, the following study was conducted on male green monkeys (cercopithecus aethiops St Kitts; Caribbean scripts ltd. experimental farm (St Kitts, West industries)). Animals weighing 6.0 ± 0.5kg (n ═ 67) were anesthetized (50mg ketamine kg)^-1) And a single intravenous injection of LPS (90. mu.g kg) through the saphenous vein^-1) Or saline (1ml) for pretreatment.

1. Inflammation study

Kinin-induced edema can be assessed by the ventral skinfold test (Sciberras et al, 1987). Briefly, anesthetized monkeys were injected with captopril (1mg kg)^-130min before the test). A single subcutaneous injection of dKD, BK or vehicle (2mM amatadine/100. mu.l ringer's lactate) was given to the abdomen and the increase in skin fold thickness was monitored with calibrated calipers for 30-45 min. The results are expressed as the difference in skin fold thickness before and after subcutaneous injection. Captopril and amatadine are useful in reducing the degradation of kinins at the carboxy-and amino-termini.

Antagonist SCHILD assay

The dose-response relationship of dKD (1-100nmol) induced edema was determined 24h after LPS in the presence or absence of different concentrations of PEG-peptide antagonist. BK (30nmol) was used as a positive control.

Antagonist time course

The time course of antagonist inhibition was determined at 4h, 24h, 48h, 72h and/or 96h after a single bolus administration. BK (30nmol) was used as a positive control.

Medicine

Ketamine hydrochloride, LPS, amatadine, and captopril are available from Sigma company (MO, u.s.a.). All peptides are available from Phoenix Pharmaceuticals (CA, U.S.A.).

Statistics of

Values are expressed as mean ± mean standard error (s.e.mean). In the edema study, the pre-injection skin fold thickness was subtracted from the post-subcutaneous challenge value. The Delta Graph 4.0 software is applied on the apple brand computer to obtain curve fitting and EC₅₀The calculated value of (a). Data were compared by two-sided anova, followed by unpaired, single tailed student t-test and correction with Bonferroni. p < 0.05 was considered statistically significant.

In the edema formation test, LPS was administered to green monkeys, andincrease its sensitivity from zero level to B₁Levels of receptor agonist. In contrast, for B₂The response of the receptor agonist BK is not affected.

Example (b): pharmacokinetic study in rats

The various peptides or conjugated peptides (in aqueous medium) were given as bolus injections to male Sprague-Dawley rats by intravenous (iv) or subcutaneous (sc) routes. At various time points (e.g., 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 post-injection), blood samples are collected and added to tubes containing heparin. Plasma is removed from the pelleted cells after centrifugation, and may be frozen or processed immediately. The target compounds in plasma are quantified by analyte-specific LC-MS/MS or ELISA methods. Different standard pharmacokinetic parameters such as Clearance (CL), apparent clearance (CL/F), volume of distribution (Vss), Mean Residence Time (MRT), area under the curve (AUC) and terminal half-life (t) can be calculated by non-compartmental methods (non-comparative methods)_1/2)。

Sequence listing

<110> AmmGen Ltd (Amgen Inc.)

<120> method for conjugating an aminothiol-containing molecule to a carrier

<130>A-982

<140> PCT/US not determined

<141>2006-01-24

<150>60/646,685

<151>2005-01-24

<150> not determined

<151>2006-01-23

<160>62

<170>PatentIn version 3.2

<210>1

<211>9

<212>PRT

<213> human

<400>1

Arg Pro Pro Gly Phe Ser Pro Phe Arg

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<212>PRT

<213> human

<400>2

Lys Arg Pro Pro Gly Phe Ser Pro Phe Arg

1 5 10

<210>3

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> synthetically prepared

<400>3

Met Lys Arg Pro Pro Gly Phe Ser Pro Phe Arg

1 5 10

<210>4

<211>8

<212>PRT

<213> human

<400>4

Arg Pro Pro Gly Phe Ser Pro Phe

1 5

<210>5

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> synthetically prepared

<400>5

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> Xaa at position 1 defined as the D isomer of arginine (DArg)

<220>

<221> other features

<222>(4)..(4)

<223> Xaa at position 4 defined as trans-4-hydroxy-proline (Hyp)

<220>

<221> other features

<222>(6)..(6)

<223> Xaa at position 6 defined as beta- (2-thienyl) -alanine (Thi)

<220>

<221> other features

<222>(8)..(8)

<223> Xaa at position 8 defined as the D isomer of 1,2, 3, 4-tetrahydroisoquinoline-3-carboxylic acid (Dtic)

<220>

<221> other features

<222>(9)..(9)

<223> Xaa at position 9 defined as octahydroindole-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)

Xaa at position <223>1 is defined as DArg

<220>

<221> other features

<222>(4)..(4)

Xaa at position <223>4 is defined as Hyp

<220>

<221> other features

<222>(6)..(6)

Xaa at position <223>6 is defined as Thi

<220>

<221> other features

<222>(8)..(8)

Xaa at position <223>8 is defined as Dtic

<220>

<221> other features

<222>(9)..(9)

Xaa at position <223>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)

Xaa at position <223>1 is defined as DArg

<220>

<221> other features

<222>(4)..(4)

Xaa at position <223>4 is defined as Hyp

<220>

<221> other features

<222>(6)..(6)

Xaa at position <223>6 is defined as Thi

<220>

<221> other features

<222>(8)..(8)

<223> Xaa at position 8 defined as the D isomer of 4S-D-prolyl ether (DHype)

<220>

<221> other features

<222>(9)..(9)

Xaa at position <223>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> Xaa at position 7 defined as L-phenylalanine N-methyl ester (MePhe)

<220>

<221> other features

<222>(9)..(9)

<223> Xaa at position 9 defined as the D isomer of β -2-naphthyl-alanine (D- β -NaI)

<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

<220>

<221> other features

<222>(1)..(1)

Xaa at position <223>1 is defined as DArg

<220>

<221> other features

<222>(4)..(4)

Xaa at position <223>4 is defined as Hyp

<220>

<221> other features

<222>(6)..(6)

<223> Xaa at position 6 defined as 2-indanyl glycine (Igl)

<220>

<221> other features

<222>(8)..(8)

<223> Xaa at position 8 defined as the D isomer of 2-indanyl glycine (DIgl)

<220>

<221> other features

<222>(9)..(9)

Xaa at position <223>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)

Xaa at position <223>5 is defined as Hyp

<220>

<221> other features

<222>(7)..(7)

Xaa at position <223>7 is defined as Igl

<220>

<221> other features

<222>(9)..(9)

Xaa at position <223>9 is defined as DIgl

<220>

<221> other features

<222>(10)..(10)

Xaa at position <223>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)

Xaa at position <223>5 is defined as Hyp

<220>

<221> other features

<222>(7)..(7)

<223> Xaa at position 7 defined as cyclopentylglycine (Cpg)

<220>

<221> other features

<222>(9)..(9)

Xaa at position <223>9 is defined as Dtic

<220>

<221> other features

<222>(10)..(10)

Xaa at position <223>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)

Xaa at position <223>1 is defined as DArg

<220>

<221> other features

<222>(4)..(4)

Xaa at position <223>4 is defined as Hyp

<220>

<221> other features

<222>(6)..(6)

Xaa at position <223>6 is defined as Igl

<220>

<221> other features

<222>(8)..(8)

<223> Xaa at position 8 defined as D isomer of pentafluorophenylalanine (Df5f)

<220>

<221> other features

<222>(9)..(9)

Xaa at position <223>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)

Xaa at position <223>1 is defined as DOrn

<220>

<221> other features

<222>(5)..(5)

Xaa at position <223>5 is defined as Hyp

<220>

<221> other features

<222>(7)..(7)

Xaa at position <223>7 is defined as Cpg

<220>

<221> other features

<222>(9)..(9)

Xaa at position <223>9 is defined as Dtic

<220>

<221> other features

<222>(10)..(10)

Xaa at position <223>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)

Xaa at position <223>1 is defined as DOrn

<220>

<221> other features

<222>(5)..(5)

<223> Xaa at position 5 defined as thiazolidine-4-carboxylic acid (Thz)

<220>

<221> other features

<222>(7)..(7)

Xaa at position <223>7 is defined as Cpg

<220>

<221> other features

<222>(9)..(9)

Xaa at position <223>9 is defined as Dtic

<220>

<221> other features

<222>(10)..(10)

Xaa at position <223>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

<220>

<223> synthetically prepared

<220>

<221> other features

<222>(1)..(1)

<223> Xaa at position 1 defined as beta-3-pyridyl-alanine (3Pa1)

<220>

<221> other features

<222>(5)..(5)

Xaa at position <223>5 is defined as Hyp

<220>

<221> other features

<222>(7)..(7)

Xaa at position <223>7 is defined as Cpg

<220>

<221> other features

<222>(9)..(9)

Xaa at position <223>9 is defined as Dtic

<220>

<221> other features

<222>(10)..(10)

Xaa at position <223>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> Xaa at position 1 defined as D-4' pyridylalanine (4Pa1)

<220>

<221> other features

<222>(5)..(5)

Xaa at position <223>5 is defined as Hyp

<220>

<221> other features

<222>(7)..(7)

Xaa at position <223>7 is defined as Cpg

<220>

<221> other features

<222>(9)..(9)

Xaa at position <223>9 is defined as Dtic

<220>

<221> other features

<222>(10)..(10)

Xaa at position <223>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> Xaa at position 1 defined as cyclohexylalanine (Cha)

<220>

<221> other features

<222>(4)..(4)

Xaa at position <223>4 is defined as Hyp

<220>

<221> other features

<222>(6)..(6)

Xaa at position <223>6 is defined as Cpg

<220>

<221> other features

<222>(8)..(8)

Xaa at position <223>8 is defined as Dtic

<220>

<221> other features

<222>(9)..(9)

Xaa at position <223>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> Xaa at position 1 defined as beta-2-naphthyl-alanine (2Nal)

<220>

<221> other features

<222>(4)..(4)

Xaa at position <223>4 is defined as Hyp

<220>

<221> other features

<222>(6)..(6)

Xaa at position <223>6 is defined as Cpg

<220>

<221> other features

<222>(8)..(8)

Xaa at position <223>8 is defined as Dtic

<220>

<221> other features

<222>(9)..(9)

Xaa at position <223>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)

Xaa at position <223>4 is defined as Hyp

<220>

<221> other features

<222>(6)..(6)

Xaa at position <223>6 is defined as Cpg

<220>

<221> other features

<222>(8)..(8)

Xaa at position <223>8 is defined as Dtic

<220>

<221> other features

<222>(9)..(9)

Xaa at position <223>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> Xaa at position 1 defined as the D isomer of lysine (DLys)

<220>

<221> other features

<222>(5)..(5)

Xaa at position <223>5 is defined as Hyp

<220>

<221> other features

<222>(7)..(7)

Xaa at position <223>7 is defined as Cpg

<220>

<221> other features

<222>(9)..(9)

Xaa at position <223>9 is defined as Dtic

<220>

<221> other features

<222>(10)..(10)

Xaa at position <223>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)

Xaa at position <223>2 is defined as DOrn

<220>

<221> other features

<222>(5)..(5)

Xaa at position <223>5 is defined as Hyp

<220>

<221> other features

<222>(7)..(7)

Xaa at position <223>7 is defined as Cpg

<220>

<221> other features

<222>(9)..(9)

Xaa at position <223>9 is defined as Dtic

<220>

<221> other features

<222>(10)..(10)

Xaa at position <223>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)

Xaa at position <223>2 is defined as Cha

<220>

<221> other features

<222>(5)..(5)

Xaa at position <223>5 is defined as Hyp

<220>

<221> other features

<222>(7)..(7)

Xaa at position <223>7 is defined as Cpg

<220>

<221> other features

<222>(9)..(9)

Xaa at position <223>9 is defined as Dtic

<220>

<221> other features

<222>(10)..(10)

Xaa at position <223>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> Xaa at position 2 defined as S-aminobutyric acid (Abu)

<220>

<221> other features

<222>(5)..(5)

Xaa at position <223>5 is defined as Hyp

<220>

<221> other features

<222>(7)..(7)

Xaa at position <223>7 is defined as Cpg

<220>

<221> other features

<222>(9)..(9)

Xaa at position <223>9 is defined as Dtic

<220>

<221> other features

<222>(10)..(10)

Xaa at position <223>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)

Xaa at position <223>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)

Xaa at position <223>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)

Xaa at position <223>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)

Xaa at position <223>9 is defined as Dtic

<220>

<221> other features

<222>(10)..(10)

Xaa at position <223>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)

Xaa at position <223>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)

Xaa at position <223>13 is defined as MePhe

<220>

<221> other features

<222>(15)..(15)

Xaa at position <223>15 is defined as D-beta-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)

Xaa at position <223>11 is defined as Hyp

<220>

<221> other features

<222>(13)..(13)

Xaa at position <223>13 is defined as Cpg

<220>

<221> other features

<222>(15)..(15)

Xaa at position <223>15 is defined as Dtic

<220>

<221> other features

<222>(16)..(16)

Xaa at position <223>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)

Xaa at position <223>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)

Xaa at position <223>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)

Xaa at position <223>16 is defined as Dtic

<220>

<221> other features

<222>(17)..(17)

Xaa at position <223>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)

Xaa at position <223>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)

Xaa at position <223>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)

Xaa at position <223>16 is defined as Dtic

<220>

<221> other features

<222>(17)..(17)

Xaa at position <223>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)

Xaa at position <223>5 is defined as Hyp

<220>

<221> other features

<222>(7)..(7)

Xaa at position <223>7 is defined as Cpg

<220>

<221> other features

<222>(9)..(9)

Xaa at position <223>9 is defined as Dtic

<220>

<221> other features

<222>(10)..(10)

Xaa at position <223>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)

Xaa at position <223>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)

Xaa at position <223>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)

Xaa at position <223>10 is defined as Dtic

<220>

<221> other features

<222>(11)..(11)

Xaa at position <223>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)

Xaa at position <223>7 is defined as DOrn

<220>

<221> other features

<222>(11)..(11)

Xaa at position <223>11 is defined as Hyp

<220>

<221> other features

<222>(13)..(13)

Xaa at position <223>13 is defined as Cpg

<220>

<221> other features

<222>(15)..(15)

Xaa at position <223>15 is defined as Dtic

<220>

<221> other features

<222>(16)..(16)

Xaa at position <223>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)

Xaa at position <223>7 is defined as DOrn

<220>

<221> other features

<222>(11)..(11)

Xaa at position <223>11 is defined as Thz

<220>

<221> other features

<222>(13)..(13)

Xaa at position <223>13 is defined as Cpg

<220>

<221> other features

<222>(15)..(15)

Xaa at position <223>15 is defined as Dtic

<220>

<221> other features

<222>(16)..(16)

Xaa at position <223>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)

Xaa at position <223>8 is defined as DOrn

<220>

<221> other features

<222>(11)..(11)

Xaa at position <223>11 is defined as Hyp

<220>

<221> other features

<222>(13)..(13)

Xaa at position <223>13 is defined as Cpg

<220>

<221> other features

<222>(15)..(15)

Xaa at position <223>15 is defined as Dtic

<220>

<221> other features

<222>(16)..(16)

Xaa at position <223>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> Xaa at position 1 defined as D isomer of D-2-aminobutyric acid (D-Dab)

<220>

<221> other features

<222>(5)..(5)

Xaa at position <223>5 is defined as Hyp

<220>

<221> other features

<222>(7)..(7)

Xaa at position <223>7 is defined as Cpg

<220>

<221> other features

<222>(9)..(9)

Xaa at position <223>9 is defined as Dtic

<220>

<221> other features

<222>(10)..(10)

Xaa at position <223>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)

Xaa at position <223>1 is defined as D-Arg

<220>

<221> other features

<222>(5)..(5)

Xaa at position <223>5 is defined as Hyp

<220>

<221> other features

<222>(7)..(7)

Xaa at position <223>7 is defined as Cpg

<220>

<221> other features

<222>(9)..(9)

Xaa at position <223>9 is defined as Dtic

<220>

<221> other features

<222>(10)..(10)

Xaa at position <223>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)

Xaa at position <223>1 is defined as DOrn

<220>

<221> other features

<222>(5)..(5)

Xaa at position <223>5 is defined as Hyp

<220>

<221> other features

<222>(7)..(7)

Xaa at position <223>7 is defined as Cpg

<220>

<221> other features

<222>(9)..(9)

Xaa at position <223>9 is defined as Dtic

<220>

<221> other features

<222>(10)..(10)

Xaa at position <223>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)

Xaa at position <223>1 is defined as the D isomer of Arg

<220>

<221> other features

<222>(5)..(5)

Xaa at position <223>5 is defined as the D isomer of Arg

<220>

<221> other features

<222>(7)..(7)

Xaa at position <223>7 is defined as Cpg

<220>

<221> other features

<222>(9)..(9)

Xaa at position <223>9 is defined as Dtic

<220>

<221> other features

<222>(10)..(10)

Xaa at position <223>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> Xaa at position 1 defined as the D isomer of 3 '-pyridylalanine (D-3' Pa1)

<220>

<221> other features

<222>(5)..(5)

Xaa at position <223>5 is defined as Hyp

<220>

<221> other features

<222>(7)..(7)

Xaa at position <223>7 is defined as Cpg

<220>

<221> other features

<222>(9)..(9)

Xaa at position <223>9 is defined as Dtic

<220>

<221> other features

<222>(10)..(10)

Xaa at position <223>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)

Xaa at position <223>1 is defined as D-3' Pa1

<220>

<221> other features

<222>(5)..(5)

Xaa at position <223>5 is defined as Hyp

<220>

<221> other features

<222>(7)..(7)

Xaa at position <223>7 is defined as Cpg

<220>

<221> other features

<222>(9)..(9)

Xaa at position <223>9 is defined as Dtic

<220>

<221> other features

<222>(10)..(10)

Xaa at position <223>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)

Xaa at position <223>1 is defined as D-Lys

<220>

<221> other features

<222>(2)..(2)

Xaa at position <223>2 is defined as D-2-Nal

<220>

<221> other features

<222>(5)..(5)

Xaa at position <223>5 is defined as Hyp

<220>

<221> other features

<222>(7)..(7)

Xaa at position <223>7 is defined as Cpg

<220>

<221> other features

<222>(9)..(9)

Xaa at position <223>9 is defined as Dtic

<220>

<221> other features

<222>(10)..(10)

Xaa at position <223>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> Xaa at position 2 defined as the D isomer of b-2 naphthyl-alanine (D-2-Nal)

<220>

<221> other features

<222>(5)..(5)

Xaa at position <223>5 is defined as Hyp

<220>

<221> other features

<222>(7)..(7)

Xaa at position <223>7 is defined as Cpg

<220>

<221> other features

<222>(9)..(9)

Xaa at position <223>9 is defined as Dtic

<220>

<221> other features

<222>(10)..(10)

Xaa at position <223>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)

Xaa at position <223>1 is defined as DOrn

<220>

<221> other features

<222>(3)..(3)

Xaa at position <223>3 is defined as Oic

<220>

<221> other features

<222>(6)..(6)

Xaa at position <223>6 is defined as Me-Phe

<220>

<221> other features

<222>(8)..(8)

Xaa at position <223>8 is defined as D-beta-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)

Xaa at position <223>1 is defined as DOrn

<220>

<221> other features

<222>(3)..(3)

Xaa at position <223>3 is defined as Oic

<220>

<221> other features

<222>(6)..(6)

Xaa at position <223>6 is defined as Me-Phe

<220>

<221> other features

<222>(8)..(8)

Xaa at position <223>8 is defined as D-beta-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)

Xaa at position <223>1 is defined as DOrn

<220>

<221> other features

<222>(4)..(4)

Xaa at position <223>4 is defined as Oic

<220>

<221> other features

<222>(7)..(7)

Xaa at position <223>7 is defined as Me-Phe

<220>

<221> other features

<222>(9)..(9)

Xaa at position <223>9 is defined as D beta-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)

Xaa at position <223>1 is defined as DOrn

<220>

<221> other features

<222>(4)..(4)

Xaa at position <223>4 is defined as Oic

<220>

<221> other features

<222>(7)..(7)

Xaa at position <223>7 is defined as Me-Phe

<220>

<221> other features

<222>(9)..(9)

Xaa at position <223>9 is defined as D-beta-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)

Xaa at position <223>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)

Xaa at position <223>8 is defined as D-beta-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)

Xaa at position <223>1 is defined as ornithine (Orn)

<220>

<221> other features

<222>(3)..(3)

Xaa at position <223>3 is defined as Oic

<220>

<221> other features

<222>(6)..(6)

Xaa at position <223>6 is defined as Me-Phe

<220>

<221> other features

<222>(8)..(8)

Xaa at position <223>8 is defined as D-beta-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)

Xaa at position <223>1 is defined as Orn

<220>

<221> other features

<222>(3)..(3)

Xaa at position <223>3 is defined as Oic

<220>

<221> other features

<222>(6)..(6)

Xaa at position <223>6 is defined as Me-Phe

<220>

<221> other features

<222>(8)..(8)

Xaa at position <223>8 is defined as D-beta-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)

Xaa at position <223>3 is defined as Oic

<220>

<221> other features

<222>(6)..(6)

Xaa at position <223>6 is defined as Me-Phe

<220>

<221> other features

<222>(8)..(8)

Xaa at position <223>8 is defined as D-beta-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)

Xaa at position <223>3 is defined as Oic

<220>

<221> other features

<222>(6)..(6)

Xaa at position <223>6 is defined as Me-Phe

<220>

<221> other features

<222>(8)..(8)

Xaa at position <223>8 is defined as D-beta 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)

Xaa at position <223>1 is defined as D-Dab

<220>

<221> other features

<222>(5)..(5)

Xaa at position <223>5 is defined as Hyp

<220>

<221> other features

<222>(7)..(7)

Xaa at position <223>7 is defined as Cpg

<220>

<221> other features

<222>(9)..(9)

Xaa at position <223>9 is defined as Dtic

<220>

<221> other features

<222>(10)..(10)

Xaa at position <223>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)

Xaa at position <223>1 is defined as DOrn

<220>

<221> other features

<222>(5)..(5)

Xaa at position <223>5 is defined as Hyp

<220>

<221> other features

<222>(7)..(7)

Xaa at position <223>7 is defined as Cpg

<220>

<221> other features

<222>(9)..(9)

Xaa at position <223>9 is defined as Dtic

<220>

<221> other features

<222>(10)..(10)

Xaa at position <223>10 is defined as Cpg

<400>62

Xaa Leu Arg Pro Xaa Gly Xaa Ser Xaa Xaa

1 5 10

Claims (43)

1. A compound having the structure or a pharmaceutically acceptable salt or hydrate thereof,

wherein:

a is a saturated, partially saturated or unsaturated 2, 3, 4, 5 or 6 atom bridging group containing 0, 1,2 or 3 heteroatoms selected from O, N and S, with the remaining bridging atoms being carbon atoms;

E¹n, O or C;

E²is N or C;

g is a single bond, a double bond, C, N, O, B, S, Si, P, Se or Te;

andeach of which is a single bond,andone may also be a double bond; and when G is C or N, the compound,andone may also be a double bond; and when G is a single bond or a double bond,andall are absent;

L¹is divalent C_1-6Alkyl or C_1-6Heteroalkyl, each of which is substituted with 0, 1,2 or 3 substituents selected from: F. cl, Br, I, OR^a、NR^aR^aAnd an oxo group;

m is independently in each occurrence 0 or 1;

n is greater than or equal to 1;

o is 0, 1,2, 3, 4 or 5;

R¹is H, C_1-6Alkyl, phenyl or benzyl, any of which groups is substituted with 0, 1,2 or 3 groups selected from: halogen, cyano, nitro, oxo, -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 radical 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 radical NR^aR^aand-NR^aC_2-6Alkyl OR^aAnd is further substituted with 0, 1,2, 3, 4, 5 or 6 atoms selected from: F. br, Cl and I;

R²as a carrier, R³Is a biologically active compound; or R³As a carrier, R²Is a biologically active compound;

R^ain each case independently H or R^b；

R^bIn each case independently of one another is phenyl, benzyl or C_1-6Alkyl, said phenyl, benzyl and C_1-6Alkyl is substituted with 0, 1,2, or 3 substituents selected from: halogen, C_1-4Alkyl radical, C_1-3Haloalkyl, -OC_1-4Alkyl, OH, -NH₂、-NHC_1-4Alkyl and-N (C)_1-4Alkyl) C_1-4An alkyl group; and

R^cin each case independently from halogen, C_1-4Alkyl radical, C_1-3Haloalkyl, -OC_1-4Alkyl, OH, -NH₂、-NHC_1-4Alkyl and-N (C)_1-4Alkyl) C_1-4An alkyl group.

2. The compound of claim 1, having the following general structure:

3. the compound of claim 1, having the following general structure:

4.a compound according to claim 3 wherein a is a saturated, partially saturated or unsaturated 2, 3, 4, 5 or 6 atom bridging group containing 1,2 or 3 heteroatoms selected from O, N and S, with the remaining bridging atoms being carbon atoms.

5. A compound according to claim 3 wherein a is a bridging group of 2, 3, 4, 5 or 6 carbon atoms which is saturated, partially saturated or unsaturated.

6. The compound of claim 3, wherein:

a is an unsaturated bridging group of 4 carbon atoms;

E²is C; and

g is a double bond.

7. The compound of claim 1 wherein G is a single or double bond,andall are absent.

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

9. The compound of claim 1, whereinAndeach is a single bond.

10. The compound of claim 1, wherein:

g is C or N;

andone is a double bond.

11. The compound of claim 1, wherein R²As a carrier, R³Are biologically active compounds.

12. The method of claim 1Compound (I) wherein R³As a carrier, R²Are biologically active compounds.

13. The compound of claim 1, wherein R³Selected from the group consisting of poly (alkylene oxides), poly (vinylpyrrolidone), poly (vinyl alcohol), polyoxazoline, poly (acryloylmorpholine-), poly (oxyethylated polyol), poly (ethylene glycol), carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, amino acid homopolymers, polypropylene oxide, ethylene/propylene glycol copolymers, ethylene/maleic anhydride copolymers, amino acid copolymers, PEG and amino acid copolymers, polypropylene oxide/ethylene oxide copolymers, and polyethylene glycol/thiomalic acid copolymers; or any combination thereof.

14. The compound of claim 1, wherein R³Is PEG.

15. The compound of claim 1, wherein R²Is a B1 peptide antagonist.

16. The compound of claim 1, wherein R²Is a polypeptide selected from SEQ ID NO: 5-26 and 42-62, wherein the peptide is modified to have an N-terminal cysteine residue.

17. A process for the preparation of a compound according to claim 1, which process comprises the steps of:

A) make R²-(C(＝O))_mCH(NH₂)CH₂(CH₂)_mSH reacts with the following compounds:

or

B) Make R²-[(C(＝O))_mCH(NH₂)CH₂(CH₂)_mSH]_nWith the following compounds:

wherein J is carbonyl or a protected form thereof.

18. A process for the preparation of a compound according to claim 1, which process comprises the steps of:

A) make R²-(C(＝O))_mCH(NH₂)CH₂(CH₂)_mSH reacts with the following compounds:

or

B) Make R²-[(C(＝O))_mCH(NH₂)CH₂(CH₂)_mSH]_nWith the following compounds:

wherein J is carbonyl or a protected form thereof.

19. The method of claim 17, wherein J is selected from C (═ O), C (OCH)₂CH₂O)、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)₂And C (NR)^aR^b)₂。

20. The method of claim 17, wherein the reaction is carried out at a pH between 2 and 7.

21. The method of claim 17, wherein the reaction is carried out at a pH between 3 and 5.

22. The method of claim 18, wherein J is selected from C (═ O), C (OCH)₂CH₂O)、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)₂And C (NR)^aR^b)₂。

23. The method of claim 18, wherein the reaction is carried out at a pH between 2 and 7.

24. The method of claim 18, wherein the reaction is carried out at a pH between 3 and 5.

25. A compound having the structure:

wherein:

a is a saturated, partially saturated or unsaturated 2, 3, 4, 5 or 6 atom bridging group containing 0, 1,2 or 3 heteroatoms selected from O, N and S, with the remaining bridging atoms being carbon atoms;

E¹n, O or C;

E²is N or C;

g is a single bond, a double bond, C, N, O, B, S, Si, P, Se or Te;

andeach of which is a single bond,andone may also be a double bond; and when G is C or N, the compound,andone may also be a double bond; and when G is a single bond or a double bond,andall are absent;

j is carbonyl or a protected form thereof;

L¹is divalent C_1-12Alkyl or C_1-12Heteroalkyl, each of which is substituted with 0, 1,2 or 3 substituents selected from: F. cl, Br, I, OR^a、NR^aR_aAnd an oxo group;

m is independently in each occurrence 0 or 1;

n is 1,2, 3, 4, 5,6, 7, 8, 9 or 10;

o is 0, 1,2, 3, 4 or 5;

R¹is H, C_1-6Alkyl, phenyl or benzyl, any of which groups is substituted with 0, 1,2 or 3 groups selected from: halogen, cyano, nitro, oxo, -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 radical 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 radical NR^aR^aand-NR^aC_2-6Alkyl OR^aAnd is further substituted with 0, 1,2, 3, 4, 5 or 6 atoms selected from: F. br, Cl and I;

R³as a biologically active compound or carrier;

R^ain each case independently H or R^b；

R^bIn each case independently of one another is phenyl, benzyl or C_1-6Alkyl, said phenyl, benzyl and C_1-6Alkyl is substituted with 0, 1,2, or 3 substituents selected from: halogen, C_1-4Alkyl radical, C_1-3Haloalkyl, -OC_1-4Alkyl, OH, -NH₂、-NHC_1-4Alkyl and-N (C)_1-4Alkyl) C_1-4An alkyl group;

R^cin each case independently from halogen, C_1-4Alkyl radical, C_1-3Haloalkyl, -OC_1-4Alkyl, OH, -NH₂、-NHC_1-4Alkyl and-N (C)_1-4Alkyl) C_1-4An alkyl group; and

x is C (═ O), Y is NH; or X is NH and Y is C (═ O).

26. The compound of claim 25, having the following general structure:

27. the compound of claim 25, having the following general structure:

28. the compound of claim 27, wherein a is a saturated, partially saturated or unsaturated 2, 3, 4, 5 or 6 atom bridging group containing 1,2 or 3 heteroatoms selected from O, N and S, with the remaining bridging atoms being carbon atoms.

29. The compound of claim 27, wherein a is a bridging group of 2, 3, 4, 5, or 6 carbon atoms that is saturated, partially saturated, or unsaturated.

30. The compound of claim 27, wherein:

a is an unsaturated bridging group of 4 carbon atoms;

E²is C; and

g is a double bond.

31. The compound of claim 25 wherein G is a single or double bond, andandall are absent.

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

33. The compound of claim 25, whereinAndeach is a single bond.

34. The compound of claim 25, wherein:

g is C or N; and

andone is a double bond.

35. The compound of claim 25, wherein R³Are biologically active compounds.

36. The compound of claim 25, wherein R³Is a carrier.

37. The compound of claim 25, wherein R³Selected from the group consisting of poly (alkylene oxides), poly (vinylpyrrolidone), poly (vinyl alcohol), polyoxazoline, poly (acryloylmorpholine-), poly (oxyethylated polyol), poly (ethylene glycol), carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, amino acid homopolymers, polypropylene oxide, ethylene/propylene glycol copolymers, ethylene/maleic anhydride copolymers, amino acid copolymers, PEG and amino acid copolymers, polypropylene oxide/ethylene oxide copolymers, and polyethylene glycol/thiomalic acid copolymers; or any combination thereof.

38. The compound of claim 25, wherein R³Is PEG.

39. A process for the preparation of a compound according to claim 25, which process comprises the steps of: make (Y-L)²)_n-R³With the following compounds:

wherein: l is²In each case independently C_1-6Alkyl or C_1-6Heteroalkyl, each of which is substituted with 0, 1,2, 3 or 4 substituents selected from: F. cl, Br, I, OR^a、NR^aR^aAnd an oxo group;

x is a nucleophile and Y is an electrophile; or X is an electrophile and Y is a nucleophile.

40. The method of claim 39, wherein:

the nucleophilic reagent is selected from SH and NH₂And OH; and

the electrophile is selected from CH₂Halogen, CH₂SO₂OR^bC (═ O) O (succinimide), C (═ O) O (perfluoroalkyl), C (═ O) O (CH)₂CN) and C (═ O) O (C)₆F₅)。

41. A method of treating pain and/or inflammation comprising administering to a patient in need thereof a therapeutically effective amount of a compound of claim 1.

42. A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable carrier or diluent.

43. A process for the preparation of a medicament comprising a compound of claim 1.