CN1070500C

CN1070500C - Analogs of parathyroid hormone and parathyroid hormone related peptide: synthesis and use for the treatment of osteoporosis

Info

Publication number: CN1070500C
Application number: CN94192745A
Authority: CN
Inventors: J·L·克尔斯特纳斯基; J·J·内斯特; Ｊｒ.Ｔ·Ｌ·侯; B·H·维克里; C·T·贝奇
Original assignee: Syntex Ua Inc
Current assignee: Syntex Ua Inc; Syntex USA LLC
Priority date: 1993-07-13
Filing date: 1994-01-21
Publication date: 2001-09-05
Anticipated expiration: 2014-01-21
Also published as: WO1995002610A1; CN1127000A

Abstract

Synthetic polypeptide analogs of parathyroid hormone PTH, parathyroid hormone related peptide PTHrp, and of the physiologically active truncated homologs and analogs of PTH and PTHrp, in which amino acid residues(22-31) form an amphipathic alpha-helix, said residues(22-31) selected from hydrophilic amino acids(Haa) and lipophilic amino acids(Laa) ordered in the sequence: Haa(Laa Laa Haa Haa)2 Laa and their pharmaceutically acceptable salts are useful for the prophylaxis and treatment of osteoporosis in mammals. Processes for the production of the polypeptides via solid phase and recombinant methods are provided.

Description

Parathyroid hormone analogs and parathyroid hormone-related peptide: the purposes of synthesizing and treating osteoporosis

Background of invention

A) invention field

The present invention relates to novel Rat parathyroid hormone 1-34 and the analogue of parathyroid hormone-related peptide, their solid phase synthesis and recombinant technology and they increase the purposes of sclerotin in Mammals.

B) description of correlative technology field

Osteoporosis is modal metabolic bone disease form, and can think Symptomatic, the fracture stage of bone forfeiture (osteopenia).Occur although osteoporosis can be secondary to many primary diseases, 90% case is idiopathic.Postmenopausal women especially easy idiopathic osteoporosis (after the menopause or I type osteoporosis).The height morbidity group of another spontaneous osteoporosis is people at advanced age (sex) (a senile or II type osteoporosis).Find that osteoporosis is relevant with following situation: use reflunomide, immobilization or CBR, excessive drinking, diabetes, to the chemotherapy of sexual gland virose (go-nadotoxic), blood prolactin too much, anorexia nervosa, primary and secondary amenorrhea and oophorectomize.

In various forms of osteoporosis, often to fracture, this is that the bone forfeiture reaches the result that the depleted point of mechanicalness is caused.The feature of post-menopausal osteoporosis is the fracture of wrist and backbone, and the fracture of the neck of femur principal character of senile osteoporosis seemingly.

The mechanism of bone forfeiture thinks to relate to the imbalance of bone self process in the osteoporosis.This renewal process is called as the bone remodeling process.This process is carried out in a series of various active bags.These bags seemingly are arranged on sclerotin and the certain bone surface as the bone resorption position spontaneously.Osteoclast (bone dissolving or absorptive cell) is responsible for absorbing the sclerotin of general constant big small portion.After this absorption process is scleroblast (bone forming cell) to occur, fills up the hole that osteoclast stays again by new sclerotin then.

In the grownup of health, osteoclast and osteoblastic formation speed are to make the process of bone forming and bone resorption be in equilibrium state.Yet the imbalance owing to the bone remodeling process in osteoporosis causes the speed of bone forfeiture faster than bone forming speed.All may have to a certain degree unbalance although increase with the age in most of people, at post-menopausal osteoporosis or this unbalance much serious after oophorectomize, and the age of appearance is littler.

The trial that many treatment osteoporosis aspect arranged is to slow down further bone forfeiture or more desirably make sclerotin have a net increase of length.Some medicament as oestrogenic hormon and diphosphate, can slow down the further forfeiture of sclerotin in the osteoporosis.Because bone resorption is different with osteoplastic period, as if can increase sclerotin (about 3-7%) so slow down the medicament of bone forfeiture.But this apparent growth is subjected to the restriction of time, is not progressive, and is that minimizing owing to " reinventing the space " causes.In addition, because be closely related between bone resorption and the formation, stop the treatment of bone resorption finally also can stop bone forming.

Existing people proposes, and treats with Rat parathyroid hormone 1-34 (PTH) can promote the bone turnover and cause positive calcium balance.But the human clinical trial shows that any increase of trabecular bone is offseted by the minimizing of cortical bone (cortical bone), does not therefore have the length that has a net increase of of total sclerotin.

Hefti, et al. is at Clinical Science 62, the middle report of 389-396 (1982) awards normal ash with the bPTH (1-84) of adult female rats subcutaneous dosage every day of suffering from osteoporosis or the calcareous and various bones that hPTH (1-34) can increase whole body and weighs.

Liu, et al. are at J.Bone Miner.Res.6:10, and 1071-1080 points out in (1991), can bring out nearly tibial metaphysis trabecular bone forfeiture 47% after the female rats spay of growing up, and the while is with the obvious increase of scleroblast and girder osteoclast number.Subcutaneous injection every day hPTH (1-34) thus the forfeiture that can reverse trabecular bone fully causes the quantity of trabecular bone to surpass the control group of simulated operation.Osteoblastic number increases and the number decline of osteoclast.

Hock et al. is at J.Bone Min.Res.7:1, and report among the 65-71 (1992) to healthy male rat subcutaneous injection every day hPTH (1-34) 12 days, causes the increase of the calcareous and dry weight of girder and cortical bone.Total sclerotin, trabecular bone volume, little cantilever thickness and number and scleroblast surface have all increased.

The Mammals Rat parathyroid hormone 1-34 as people's (hPTH), (bPTH) and (pTTH) of pig of ox, is the single polypeptide chain with 84 amino-acid residues, and molecular weight is about 9500.Biological activity is relevant in the N-terminal portions, and wherein residue (1-34) obviously is required minimum length.

The difference of the N-terminal fragment of the N-terminal fragment of people PTH and the hormone of ox and pig only has respectively 3 and 2 amino acid residue: hPTH (1-34) Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu1,5 10 15Asn Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp

20 25 30Val His Asn Phe (SEQ?ID?NO:1)；bPTH(1-34):Ala Val Ser Glu Ile Gln Phe Met His Asn Leu Gly Lys His Leu1 5 10 15Ser Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp

20 25 30Val His Asn Phe (SEQ?ID?NO:2)；pPTH(1-34)?:Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu1 5 10 15Ser Ser Leu Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp

20 25 30Val His Asn Phe (SEQ?ID?NO:3)．

The major function of PTH is to cause adaptive variation, to keep Ca in the extracellular fluid ²⁺Constant density.PTH acts on kidney, increases uriniferous tubules to Ca in the urine ²⁺Heavily absorption, and stimulate 25-Hydroxy cholecalciferol to be transformed into 1, the 25-dihydroxycholecalciferol.This compound is responsible for Ca ²⁺Little intestinal absorption.A unusual effect is Ca in promoting bone ²⁺Move.When PTH acts on bone, increase Ca ²⁺With phosphatic absorption.PTH stimulates the bone resorption speed of osteoclast, increases the differentiation speed of mesenchymal cell to osteoclast, and the transformation period that prolongs osteoclast.Because the prolongation effect of PTH, osteoplastic osteoblastic number also increases; Therefore, the speed that has improved the bone turnover and reinvented.As if but each osteoblastic activity is lower than normal cell.

Rosenblatt, et al. be at U.S. patent No.4, discloses by disappearance N-end (1-6) amino acid in 423,037,4,968,669 and 5,001,223 and optionally replace Phe7, Met8,18 and Gly12 and the PTH antagonist that obtains.It is reported Tyr34-NH ₂Can increase the active and stable of these compounds.

Parathyroid hormone-related peptide (PTHrp), a kind of more than 140 individual amino acid whose protein and fragment thereof can be simulated the main biological function of PTH.PTHrp can be synthetic by tumour and its hetero-organization of many humans and animals, and it can play a role in malignant hypercalcemia.The sequence of hPTHrp (1-34) is as follows: Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Phe Phe Leu His His Leu Ile Ala Glu

20 25 30Ile His Thr Ala?(SEQ?ID?NO:4)．

Sequence homology between hPTH and the hPTHrp mainly is confined to 13 N-terminal residues, and wherein 8 is identical; In the receptors bind zone of hPTH (25-34), have only 1 amino acid in hPTHrp, to guard in 10 amino acid.Comformational similarity may be to have common active reason.Cohen, et al. are at J.Biol.Chem.266:3, and 1997-2004 points out in (1991), most of sequence of PTH (1-34) and PTHrp (1-34), and especially zone (5-18) and (21-34) presents alpha-helix conformation.Simultaneously he also points out, whether this conformation is prevalent in that carboxyl terminal also has a question under the physiological condition.This secondary structure is vital for lipid interaction, acceptor interaction and/or structural stability.

We have synthesized the analogue of PTH and PTHrp so that develop therapeutical agent improvement, that be used for comprising the Mammals recovery sclerotin of suffering from osteoporosis.

Summary of the invention

The invention provides Rat parathyroid hormone 1-34 (PTH), parathyroid hormone-related peptide (PTHrp) and PTH and PTHrp and have the short homologue of physiologically active and the synthetic polypeptide analog and the salt thereof of analogue, wherein amino-acid residue (22-31) forms amphipathic alpha-helix, this residue (22-31) is selected from hydrophilic amino acid (the hy-drophilic amino acids by following series arrangement, Haa) and lipophilic amino-acid (lipophilic amino acids, Laa):

Haa(Laa?Laa?Haa?Haa)2Laa

When the concrete illustrative example of this sequence type inserts PTH, PTHrp, and PTH and PTHrp be when having the short homologue of physiologically active and analogue, the polypeptide of acquisition is effective bone remodeling agent.

On the one hand, the invention provides PTH, PTHrp and PTH and PTHrp and have the short homologue of physiologically active and analogue or its salt of analogue, wherein amino-acid residue (22-31) forms amphipathic alpha-helix, and the sequence of this residue (22-31) is selected from: a) Xaa ¹Xaa ²Leu Xaa ⁴Xaa ⁵Leu Xaa ⁷Xaa ⁶Xaa ⁹Xaa ¹⁰15 10Xaa wherein ¹And Xaa ⁴Be Glu independently of one another, Glu (OCH ₃), His, or Phe; Xaa ²Be Leu or Phe; Xaa ⁵Be Lys or His; Xaa ⁷And Xaa ¹⁰Be Leu or Ile independently of one another; Xaa ⁸Be Ala, Arg, or Glu; And Xaa ⁹Be Lys or Glu (SEQ ID NO:26); B) Xaa ¹Xaa ²Leu Xaa ⁴Arg Leu Leu Xaa ⁸Arg Leu is 15 10Xaa wherein ¹And Xaa ⁴Be Glu independently of one another, Glu (OCH ₃), His, or Phe; Xaa ²Be Leu or Phe; Xaa ⁸Be Glu, Lys, or Lys (COCH ₂PEGX) and PEGX be that molecular weight is 100-10, poly-(Ethylene Glycol Methyl ether) residue (SEQ ID NO:27) of 000; C) Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu (SEQ ID NO:28);

1 5 10d) Ser Leu Leu Ser Ser Leu Leu Ser Ser Leu (SEQ?ID?NO:29)；

1 5 10e) Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu (SEQ?ID?NO:30)．

1 5 10

On the other hand, the invention provides PTH, PTHrp with following formula structure and PTH and PTHrp and have the short homologue of physiologically active and the analogue or its salt: the Xaa of analogue ¹Xaa ²Xaa ³Xaa ⁴Xaa ⁵Xaa ⁶Xaa ⁷Leu His Xaa ¹⁰Xaa ¹¹Gly Xaa ¹³Ser Ile Gln Xaa ¹⁷Leu Xaa ¹⁹Xaa ²⁰Xaa ²¹Xaa ^22-31Xaa ³²Xaa ³³Xaa ³⁴Xaa ³⁵Xaa ³⁶Xaa ³⁷Xaa ³⁸Term, wherein: Xaa ¹Disappearance or Ala; Xaa ²Disappearance or Val; Xaa ³Disappearance or Ser; Xaa ⁴Disappearance or Glu or Glu (OCH ₃); Xaa ⁵Disappearance or His or Ala; Xaa ⁶Disappearance or Gln; Xaa ⁷Disappearance or Leu; Xaa ¹⁰And Xaa ¹⁷Be Asp or Asp (OCH independently of one another ₃); Xaa ¹¹Be Lys, Arg, or Leu; Xaa ¹³Be Lys, Arg, Tyr, Cys, Leu, Cys (CH ₂CONH (CH ₂) ₂NH (biotinyl)), Lys (7-dimethylamino-2-oxygen-2H-1-chromene-4-ethanoyl), or Lys (dihydro cinnamon acyl group); Xaa ²⁰Be Arg or Leu; Xaa ¹⁹And Xaa ²¹Be Lys independently of one another, Ala, or Arg; Xaa ^22-31Be selected from (SEQ ID NOS:26,27,28,29, or 30); Xaa ³²Be His, Pro, or Lys; Xaa ³³Disappearance, or Pro, Thr, Glu, or Ala; Xaa ³⁴Disappearance, or Pro, Arg, Met, Ala, hSer, the hSer lactone, Tyr, Leu, or 1,4-diamino butyryl lactan; Xaa ³⁵Disappearance or Pro, Glu, Ser, Ala, or Gly; Xaa ³⁶Disappearance or Ala, Arg, or Ile; Xaa ³⁷Disappearance or Arg, Trp, or 3-(2-naphthyl)-L-Ala; Xaa ³⁸Disappearance or Ala or hSer or Xaa38-42 are Thr Arg Ser Ala Trp; With Term be OR or NR ₂Wherein each R is H independently of one another, (C ₁-C ₄) alkyl or phenyl (C ₁-C ₄) alkyl; And pharmacy acceptable salt.

On the other hand, the invention provides PTH, PTHrp and PTH and PTHrp and have the short homologue of physiologically active and analogue or its salt of analogue, wherein amino-acid residue (22-31) forms amphipathic alpha-helix, and this residue (22-31) sequence is selected from: a) Glu Leu Leu Glu Lys Leu Leu Xaa Lys Leu wherein

15 10Xaa are Glu or Arg (SEQ ID NO:26); B) Glu Leu Leu Glu Arg Leu Leu Xaa Arg Leu wherein

15 10Xaa ²⁹Be Glu, Lys, or Lys (COCH ₂PEGX) and PEGX be that molecular weight is 100-10, poly-(Ethylene Glycol Methyl ether) residue (SEQ ID NO:27) of 000; C) Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu (SEQ ID NO:28);

1 5 10d) Ser Leu Leu Ser Ser Leu Leu Ser Ser Leu (SEQ?ID?NO:29)；

1 5 10e) Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu (SEQ?ID?NO:30)．

1 5 10

The pharmaceutical composition that is used to prevent or treat the osteopenia situation also is provided, it comprises that Rat parathyroid hormone 1-34 (PTH), parathyroid hormone-related peptide (PTHrp) and the PTH and the PTHrp that add significant quantity with pharmaceutically acceptable carrier and hyperostosis have the short homologue of physiologically active and the polypeptide analog and the salt thereof of analogue, wherein amino-acid residue (22-31) forms amphipathic alpha-helix, and this residue (22-31) is selected from hydrophilic amino acid (Haa) and the lipophilic amino-acid (Laa) by following series arrangement:

Haa(Laa?Laa?Haa?Haa) ₂?Laa．

The method of pharmaceutical compositions also is provided, and it comprises mixes above-claimed cpd mutually with pharmaceutically acceptable carrier.

In addition, the present invention also provides and has been used for the treatment of the method that the Mammals sclerotin reduces situation, this method comprises that adding PTH, the PTHrp of significant quantity and PTH and PTHrp to the administration hyperostosis of needs has the short homologue of physiologically active and polypeptide analog or its salt of analogue, wherein amino-acid residue (22-31) forms amphipathic alpha-helix, and this residue (22-31) is selected from hydrophilic amino acid (Haa) and the lipophilic amino-acid (Laa) by following series arrangement:

Haa(Laa?Laa?Haa?Haa) ₂?Laa

More specifically, the invention provides and be used for the treatment of the method that the Mammals sclerotin reduces situation, this method comprises that adding PTH, the PTHrp of significant quantity and PTH and PTHrp to the administration hyperostosis of needs has the short homologue of physiologically active and polypeptide analog or its salt of analogue, wherein amino-acid residue (22-31) forms amphipathic alpha-helix, this residue (22-31) sequence is selected from: SEQ ID NO:26,27,28,29 and 30.

The present invention comprises that also PTH, PTHrp and PTH and PTHrp have the short homologue of physiologically active and the polypeptide analog of analogue, or the solid phase synthesis process of the salt of polypeptide analog, wherein amino-acid residue (22-31) forms amphipathic alpha-helix, and this residue (22-31) is selected from hydrophilic amino acid (Haa) and the lipophilic amino-acid (Laa) by following series arrangement:

Haa (Laa Laa Haa Haa) ₂Laa, this method comprises in order that protected amino acid is coupled in the appropriate resin carrier, removes side chain and N ^α-blocking group, and with polypeptide from resin separately.

The present invention comprises that also PTH, PTHrp and PTH and PTHrp have short homologue and the polypeptide analog of analogue or the solid phase synthesis process of its salt of physiologically active, wherein amino-acid residue (22-31) forms amphipathic alpha-helix, this residue (22-31) sequence is selected from: SEQ ID NO:26,27,28,29 and 30.This method comprises in order that protected amino acid is coupled in the appropriate resin carrier, removes side chain and N ^α-blocking group, and with polypeptide from resin separately.

Also comprise PTH, PTHrp and PTH and PTHrp have the polypeptide analog of the short homologue of physiologically active and analogue or its salt the method that is re-combined into, wherein amino-acid residue (22-31) forms amphipathic alpha-helix, and this residue (22-31) is selected from hydrophilic amino acid (Haa) and the lipophilic amino-acid (Laa) by following series arrangement:

Haa(Laa?Laa?Haa?Haa) ₂?Laa

The present invention also comprises dna sequence dna, carrier and the plasmid that is used to be re-combined into this polypeptide analog.Particularly, the invention provides and be used to be re-combined into short homologue and the polypeptide analog of analogue or dna sequence dna, carrier and the plasmid of its salt that PTH, PTHrp and PTH and PTHrp have physiologically active, wherein amino-acid residue (22-31) forms amphipathic alpha-helix, this residue (22-31) sequence is selected from: SEQ ID NO:26,27,28,29 and 30.

The accompanying drawing summary

Fig. 1 is the dna sequence dna and the restriction enzyme site (hPTH pcr amplification figure) of the synthetic gene of coding PTHrp of the present invention (1-34) analogue.

Fig. 2 has summarized the preparation (Trp LE 18 hPTHrp (1-34) 1 design of graphics) of the plasmid that is integrated with PTHrp (1-34) analogue gene.

Fig. 3 has summarized the preparation (Trp LE 18 hPTHrp (1-34) 2 design of graphics) of the plasmid that is integrated with two copy PTHrp (1-34) analogue genes.

Fig. 4 has summarized the preparation (Trp LE 18 hPTHrp (1-34) 4 design of graphics) of the plasmid that is integrated with 4 copy PTHrp (1-34) analogue genes.

Detailed Description Of The Invention

Abbreviation and definition

Press for various common nucleotide bases and amino acid whose monocase and three-character doctrine abbreviation Pure Appl.Chem.31,639-645 (1972) and 40,277-290 (1974) In advise and also meet 37 CFR § 1.822 (55FR 18245, May 1 nineteen ninety Day) and PCT rule (WIPO Standard ST.23:Recommendation for The Presentation of Nucleotide and Amino Acid Sequences in Patent APPlications and in Published Patent Documents). Monocase As follows with the three-character doctrine abbreviation: amino acid abbreviations

Amino acid three-character doctrine monocase

Alanine Ala A

Arginine Arg R

Asparagine Asn N

L-aminobutanedioic acid Asp D

Asn+Asp Asx B

Cysteine Cys C

Glutamine Gln Q

Glutamic acid Glu E

Gln+Glu Glx Z

Glycine Gly G

Histidine H

Isoleucine Ile I

Leucine Leu L

Lysine Lys K

Methionine Met M

Phenylalanine Phe F

Proline Pro P

Serine Ser S

Threonine Thr T

Tryptophan Trp W

Tyrosine Tyr Y

Valine Val V Abbreviation represents L-amino acid, unless in addition with D-or D, L-indicates. Some natural and non-natural Amino acid such as glycine are achiral. The method for expressing of all peptide sequences all is that the N-end is amino Acid is positioned at the left side and C-terminal amino acid is positioned at the right.

Other amino acid used herein and the abbreviation of compound are as follows:

The hSer homoserine

The hSerlac homoserine lactone

The Nle nor-leucine

The residue of PEG2 Diethylene Glycol methyl ether claims methoxyl group two (ethylene oxy again

Base), CH ₃O (CH ₂CH ₂O) ₂-, (molecular weight=119)

The residue of PEG5000 poly-(Ethylene Glycol Methyl ether) claims methoxyl group to gather (ethene again

The oxygen base), CH ₃O (CH ₂CH ₂O) ₁₁₀-, (molecular-weight average

=5000)

The residue of PEGX poly-(Ethylene Glycol Methyl ether), CH ₃O

(CH ₂CH ₂O) _N-9, n=2-225, (molecular-weight average

=100-10,000)

" hydrophilic amino acid (Haa) " refers to except forming the required group of peptide bond, amino acid with at least one hydrophilic functional groups, for example arginine, amino-succinamic acid, Aspartic Acid, L-glutamic acid, glutamine, Histidine, Methionin, Serine, Threonine and homologue thereof.

" lipophilic amino-acid (Laa) " refers to uncharged, aliphatic series or aromatic amino acid, for example Isoleucine, leucine, methionine(Met), phenylalanine, tryptophane, tyrosine, Xie Ansuan and homologue thereof.

In the present invention, L-Ala is classified as " amphoteric amino acids ", and promptly it both can also can be used as lipophilic amino-acid as hydrophilic amino acid.

" PTH and PTHrp have the short homologue and the analogue of physiologically active " refer to have less than the corresponding sequence of amino acid of the total length of finding at PTH or PTHrp, but can cause the polypeptide of similar physiologic response.The PTH of brachymemma or PTHrp needn't could cause similar physiologic response with PTH or the complete homology of PTHrp.PTH (1-34) and PTHrp (1-34) are the preferred example in this group but do not get rid of other examples.

" amphipathic alpha-helix " refers to that amino acid takes the secondary structure that some polypeptide showed of alpha-helical conformation, and wherein the major axis along spiral has relative polarity and non-polar plane." Schiffer-Edmundson wheel " (M.Schiffer and the A.B.Edmundson that in interested polypeptide, exists the possibility of α-Luo Xuanjiegou can have suitable pitch to a certain extent by structure, Biophys.J.7,121 (1967)) and observe hydrophilic and lipophilic residue and whether separately be positioned at the circumscribed cylindrical opposite face of spiral and survey.Perhaps, also can for example garden dichroism or X ray diffracting data show have the alpha-helix zone in given polypeptide by the experimental evidence that obtains.The every circle of ideal alpha-helix has 3.6 amino acid, and wherein adjacent side chain is 100 degree separately.Eisenberg et al. in Nature 299:371-374 (1982) and Proc.Nat.Acad.Sci.USA 81:140-144 (1984) with hydrophobicity numerical value and spiral combination, quantitatively to determine the notion of amphipathic helix.Average hydrophobic square be defined as constituting spiral respectively form the hydrophobic vector of amino acid and.Eisen-berg (1984) is with the scale of following amino acid whose hydrophobicity as " consistence ": Ile 0.73; Phe 0.61; Val 0.54; Leu 0.53; Trp 0.37; Met 0.26; Ala 0.25; Gly 0.16; Cys 0.04; Tyr 0.02; Pro-0.07; Thr-0.18; Ser-0.26; His-0.40; Glu-0.62; Asn-0.64; Gln-0.69; Asp-0.72; Lys-1.10; Arg-1.76.

The hydrophobic square that has the desirable alpha-helix of 3.6 residues (being side chain 100 radians=(360 °/3.6) at interval) for every circle _{μ H}, can calculate with following formula:

μ H=[(Σ H _NSin δ (N-1)) 2+ (Σ H _NCos δ (N-1)) 2] ^1/2, wherein, H _NBe N _ThAmino acid whose hydrophobicity value, and and be in the sequence of frequency δ=100 ° N amino acid whose and.Hydrophobic square can be used μ _HMerchant＜the μ that obtains divided by N _HBe expressed as the average hydrophobic square of each residue.In 100 ° ± 20 ° scopes＜μ _HValue for about 0.20 or bigger just expression have amphipathic helix.For hPTHrp (22-31) and hPTH (22-31), 100 °＜μ _HValue is respectively 0.19 and 0.37.

Cornett, et al. at J.Mol.Biol., has further expanded the research of amphipathic alpha-helix among the 195:659-685 (1987), introduced " amphipathic index " as amphipathic predictor.They reach a conclusion, and it is amphipathic that half all known alpha-helix is arranged approximately, and main frequency is 97.5 ° rather than 100 °, and the residue number of every circle is more near 3.7 rather than 3.6.Although this levels of precision is interesting on science, people's such as Eisenberg basic skills has been used for enough determining whether given sequence is amphipathic, especially starts anew implementation sequence when forming amphipathic alpha-helix as people.

The amphipathic alpha-helix aminoacid sequence that replaces can be not and the given fragment sequence homology of naturally occurring polypeptide, but should have similar secondary structure under physiological status, promptly has the relative polarity and the alpha-helix of non-polar plane.Replace naturally occurring aminoacid sequence with other sequences and may produce favorable influence for physiologically active, stability or other performances of the parent polypeptide that changes.At J.L.Krstenansky, et al., FEBS Letters 242:2,409-413 (1989), and J.P.Segrest, et al.Proteins:Structure, the guidance that designs and select this sequence aspect is arranged among Function and the Genetics 8:103-117 (1990) etc.

10 amino acid whose amphipathic alpha-helixs of the present invention have following formula:

Haa (Laa Laa Haa Haa) ₂Laa wherein, Haa is selected from hydrophilic amino acid as mentioned above, and Laa is selected from above-mentioned lipophilic amino-acid.Suppose the ideal alpha-helix,

residue

1,4,5,8 and 9 are distributed on the face (A) of spiral, and within about each other 140 °, and

residue

2,3,6,7 and 10 occupy 140 ° of another side (B) of spiral.Preferably, all residues that are positioned at one side all are identical polars, and all residues that are positioned at another side all are opposite polarities, if promptly the A face all is hydrophilic, the B face all is lipophilic so, and vice versa.Those of skill in the art know, although spiral of the present invention is to use

Haa (Laa Laa Haa Haa) ₂Laa represents, but opposite sequence

Laa (Haa Haa Laa Laa) ₂Therefore Haa also meets residue distribution standard, is the describing mode that the another kind of spiral of the present invention is equal to.

L-Ala can replace hydrophilic amino acid or lipophilic amino-acid, because L-Ala can be positioned at any one side of amphipathic alpha-helix without difficulty, although Ala ₁₀Can not form amphipathic alpha-helix.Generally speaking, do not use proline(Pro), halfcystine and tyrosine; Their existence and other random errors can be tolerated in sequence, for example have hydrophilic residue at the lipophilic face, as long as all the other amino acid in the fragment meet the division of hydrophilic-lipophilic face on substantially.Be used for determining that thereby whether a certain sequence has enough amphipathic short-cut methods that can become sequence of the present invention is to calculate its average hydrophobic square as mentioned above.If the average square peak value of each residue surpasses approximately 0.20 in 100 ° ± 20 ° scopes, this sequence can form amphipathic helix and be sequence of the present invention so.

For example, for SEQ ID NO:26, Xaa=Glu, being calculated as follows of 100 ° of average hydrophobic squares of locating each residue: A.A.H _Nδ (N-1) H sinδ (N-1) H cosδ (N-1)E -.62 0 0 -.62L .53 100 .52 -.17L .53 200 -.18 -.50E -.62 300 .34 -.31K -1.1 400 -.70 -.85L .53 500 .34 -.41L .53 600 -.46 -.27E -.62 700 .21 -.58K -1.1 800 -1.08 -.19L .53 900 0 -.53

∑=0.81?Σ=-4.43

μ _H=[(0.81) ²+(-4.43) ²] ^1/2=4.50

<μ _H>=4.50/10=0.45

For this sequence, the peak value of average hydrophobic square appears at 92 °, and its value is 0.48.

When this notion is applied to Rat parathyroid hormone 1-34 and parathyroid hormone-related peptide, can suppose zone (7-16) and (22-31) in a zone or two zones have the alpha-helix secondary structure, and can be with non-homogeneous sequence displacement with similar structures tendency can loss of biological activity or the performance of induction of immunity reaction.Preferred example

On the one hand, the invention provides PTH, PTHrp and PTH and PTHrp and have the short homologue of physiologically active and analogue or its salt of analogue, wherein amino-acid residue (22-31) forms amphipathic alpha-helix, and this residue (22-31) sequence is selected from: a) Xaa ¹Xaa ²Leu Xaa ⁴Xaa ⁵Leu Xaa ⁷Xaa ⁸Xaa ⁹Xaa ¹⁰Wherein

15 10Xaa ¹And Xaa ⁴Be Glu independently of one another, Glu (OCH ₃), His, or Phe; Xaa ²Be Leu or Phe; Xaa ⁵Be Lys or His; Xaa ⁷And Xaa ¹⁰Be Leu or Ile independently of one another; Xaa ⁸Be Ala, Arg, or Glu; And Xaa ⁹Be Lys or Glu (SEQ ID NO:26); B) Xaa ¹Xaa ²Leu Xaa ⁴Arg Leu Leu Xaa ⁸Arg Leu wherein

15 10Xaa ¹And Xaa ⁴Be Glu independently of one another, Glu (OCH ₃), His, or Phe; Xaa ²Be Leu or Phe; Xaa ⁸Be Glu, Lys, or Lys (COCH ₂PEGX) and PEGX be that molecular weight is 100-10, poly-(Ethylene Glycol Methyl ether) residue (SEQ ID NO:27) of 000; C) Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu (SEQ ID NO:28);

1 5 10d) Ser Leu Leu Ser Ser Leu Leu Ser Ser Leu (SEQ?ID?NO:29)；

1 5 10e) Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu (SEQ?ID?NO:30).

1 5 10

On the other hand, the invention provides PTH, PTHrp with following formula structure and PTH and PTHrp and have the short homologue of physiologically active and the analogue or its salt: the Xaa of analogue ¹Xaa ²Xaa ³Xaa ⁴Xaa ⁵Xaa ⁶Xaa ⁷Leu His Xaa ¹⁰Xaa ¹¹Gly Xaa ¹³Ser Ile Gln Xaa ¹⁷Leu Xaa ¹⁹Xaa ²⁰Xaa ²¹Xaa ^22-31Xaa ³²Xaa ³³Xaa ³⁴Xaa ³⁵Xaa ³⁶Xaa ³⁷Xaa ³⁸Term, wherein: Xaa ¹Disappearance or Ala; Xaa ²Disappearance or Val; Xaa ³Disappearance or Ser; Xaa ⁴Disappearance or Glu or Glu (OCH ₃); Xaa ⁵Disappearance or His or Ala; Xaa ⁶Disappearance or Gln; Xaa ⁷Disappearance or Leu; Xaa ¹⁰And Xaa ¹⁷Be Asp or Asp (OCH independently of one another ₃); Xaa ¹¹Be Lys, Arg, or Leu; Xaa ¹³Be Lys, Arg, Tyr, Cys, Leu, Cys (CH ₂CONH (CH ₂) ₂NH (biotinyl)), Lys (7-dimethylamino-2-oxygen-2H-1-chromene-4-ethanoyl), or Lys (dihydro cinnamon acyl group); Xaa ²⁰Be Arg or Leu; Xaa ¹⁹And Xaa ²¹Be Lys independently of one another, Ala, or Arg; Xaa ^22-31Be selected from (SEQ ID NOS:26,27,28,29, or 30); Xaa ³²Be His, Pro, or Lys; Xaa ³³Disappearance, or Pro, Thr, Glu, or Ala; Xaa ³⁴Disappearance, or Pro, Arg, Met, Ala, hSer, the hSer lactone, Tyr, Leu, or 1,4-diamino butyryl lactan; Xaa ³⁵Disappearance or Pro, Glu, Ser, Ala, or Gly; Xaa ³⁶Disappearance or Ala, Arg, or Ile; Xaa ³⁷Disappearance or Arg, Trp, or 3-(2-naphthyl)-L-Ala; Xaa ³⁸Disappearance or Ala or hSer or Xaa ^38-42Be Thr Arg Ser Ala Trp; With Term be OR or NR ₂Wherein each R is H independently of one another, (C ₁-C ₄) alkyl or phenyl (C ₁-C ₄) alkyl; And pharmacy acceptable salt.

On the other hand, the present invention includes the polypeptide analog of the short homologue of hPTHrp shown in formula I, that have physiologically active (1-34): Ala Val Ser Glu Xaa ⁵Gln Leu Leu His Asp Xaa ¹¹Gly Xaa ¹³Ser IleGln Asp Leu Xaa ¹⁹Arg Xaa ²¹Xaa ^22-31Xaa ³²Xaa ³³Xaa ³⁴Term, wherein: Xaa ⁵Be His or Ala; Xaa ¹¹And Xaa ¹³Be Lys independently of one another, Arg, or Leu; Xaa ¹⁹And Xaa ²¹Be Ala or Arg independently of one another; Xaa ^22-31Be selected from: a) Glu Leu Leu Glu Lys Leu Leu Xaa Lys Leu wherein

15 10Xaa are Glu, Lys, or Lys (COCH ₂PEGX) and PEGX be that molecular weight is 100-10, poly-(Ethylene Glycol Methyl ether) residue (SEQ ID NO:27) of 000; C) Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu (SEQ ID NO:28);

1 5 10d) Ser Leu Leu Ser Ser Leu Leu Ser Ser Leu (SEQ?ID?NO:29)；

1 5 10e) Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu (SEQ?ID?NO:30)；

15 10Xaa ³²Be His or Lys; Xaa ³³Be Thr, Glu, or Ala; Xaa ³⁴Be Ala, hSer, Tyr, or Leu; With Term be Gly Arg Arg, lactone, OH or NR ₂, wherein each R is H or (C ₁-C ₄) alkyl; And pharmacy acceptable salt (formula I).

Another concrete aspect of the present invention comprises Xaa ^22-31Be the formula I polypeptide of (SEQ ID NO:26), these polypeptide 100 °＜μ _HSurpass 0.45.Of the present invention one more specifically the aspect comprise Xaa ^22-31Be (SEQ ID NO:26), Xaa ¹¹And Xaa ¹³All be Lys and Xaa ¹⁹And Xaa ²¹It all is the formula I polypeptide of Arg.

Representational polypeptide comprises, but is not limited to: Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Arg Lys

20 25 30Leu His Thr Ala OH (SEQ?ID?NO:5);Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys

20 25 30Leu His Thr Ala OH (SEQ?ID?NO:6)；Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys

20 25 30Leu His Thr Ala NH2 (SEQ?ID?NO:7)；Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys

20 25 30Leu His Thr hSer NH2 (SEQ?ID?NO:8)；Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys

20 25 30Leu His Thr hSerlac (SEQ?ID?NO:9)；Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys

20 25 30Leu His Thr Ala Gly Arg Arg OH (SEQ ID NO:10); With 35Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser lle1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys

20 25 30Leu Lys Glu Leu NH ₂ (SEQ?ID?NO:11).

Another aspect of the present invention comprises Xaa ^22-31Be (SEQ ID NO:26), Xaa ¹¹And Xaa ¹³All be Lys and Xaa ¹⁹And Xaa ²¹In one for Arg another is the formula I polypeptide of Ala.The representational polypeptide of this subclass comprises, but is not limited to: Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Ala Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys

20 25 30Leu His Thr Ala NH2 (SEQ ID NO:12) and Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Ala Glu Leu Leu Glu Lys Leu Leu Glu Lys

20 25 30Leu His Thr Ala NH ₂ (SEQ?ID?NO:13).

Another concrete aspect of the present invention comprises Xaa ^22-31Be (SEQ ID NO:26), Xaa ¹¹And Xaa ¹³In one for Leu another is Lys and Xaa ¹⁹And Xaa ²¹It all is the formula I polypeptide of Arg.The representational polypeptide of this subclass comprises, but is not limited to: Ala Val Ser Glu Ala Gln Leu Leu His Asp Leu Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys

20 25 30Leu His Ala Leu OH (SEQ?ID?NO:14).

Another concrete aspect of the present invention comprises Xaa ^22-31Be the formula I polypeptide of (SEQ ID NO:27), these polypeptide 100 °＜μ _HSurpass 0.50.Further aspect of the present invention comprises Xaa ^22-31Be (SEQ ID NO:27), Xaa ¹¹And Xaa ¹³All be Lys or all be Arg and Xaa ¹⁹And Xaa ²¹It all is the formula I polypeptide of Arg.The representational polypeptide of this subclass comprises, but is not limited to: Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Glu Arg

20 25 30Leu His Thr Ala OH (SEQ?ID?NO:15)；Ala Val Ser Glu His Gln Leu Leu His Asp Arg Gly Arg Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Glu Arg

20 25 30Leu His Thr Ala OH (SEQ?ID?NO:16)；Ala Val Ser Glu His Gln Leu Leu His Asp Arg Gly Arg Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Lys Arg

20 25 30Leu His Thr Ala OH (SEQ?ID?NO:17)；Ala Val Ser Glu His Gln Leu Leu His Asp Arg Gly Arg Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu

20 25Lys (COCH:PEG2) Arg Leu His Thr Ala OH (SEQ ID NO:18); With

30Ala Val Ser Glu His Gln Leu Leu His Asp Arg Gly Arg Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu

20 25Lys(COCH:PEG5000) Arg Leu His Thr Ala OH (SEQ?ID?NO:19).

30

Another concrete aspect of the present invention comprises Xaa ^22-31Be the formula I polypeptide of (SEQ ID NO:28), these polypeptide 100 °＜μ _HBe about 0.25.The representational polypeptide of this subclass comprises, but is not limited to: Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15 Gln Asp Leu Arg Arg Arg Ala Leu Ala Glu Ala Leu Ala Glu Ala

20 25 30Leu His Thr Ala NH2 (SEQ?ID?NO:20).

Another concrete aspect of the present invention comprises Xaa ^22-31Be the formula I polypeptide of (SEQ ID NO:29), these polypeptide 100 °＜μ _HBe about 0.28.The representational polypeptide of this subclass comprises, but is not limited to: Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Ser Leu Leu Ser Ser Leu Leu Ser Ser

20 25 30Leu His Thr Ala NH (SEQ?ID?NO:21).

Another concrete aspect of the present invention comprises Xaa ^22-31Be the formula I polypeptide of (SEQ ID NO:30), these polypeptide 100 °＜μ _HBe about 0.29.The representational polypeptide of this subclass comprises, but is not limited to: Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Ala Phe Tyr Asp Lys Val Ala Glu Lys

20 25 30Leu His Thr Ala NH2 (SEQ?ID?NO:22).

Another aspect of the present invention comprises the polypeptide analog of the short homologue of bPTH shown in formula II, that have physiologically active (1-34): Xaa ¹Val Ser Glu Ile Gln Xaa ⁷Xaa ⁸His Asn Leu Gly Lys His LeuXaa ¹⁵Ser Xaa ¹⁸Xaa ¹⁹Arg Xaa ²¹Xaa ^22-31His Asn Xaa ³⁴Term, wherein: Xaa ¹Be Ser or Ala; Xaa ⁷Be Leu or Phe; Xaa ⁸Be Met or Nle; Xaa ¹⁶Be Asn or Ser; Xaa ¹⁸Be Leu, Met, or Nle; Xaa ¹⁹Be Glu or Arg; Xaa ²¹Be Val or Arg; Xaa ^22-31Be selected from (SEQ ID NO:26,27,28,29 and 30); Xaa ³⁴Be Phe or Tyr; Term is OH or NR ₂, wherein each R is H or (C ₁-C ₄) alkyl; And pharmacy acceptable salt (formula II).Representational polypeptide comprises, but is not limited to: Ala Val Ser Glu Ile Gln Phe Nle His Asn Leu Gly Lys His Leu1 5 10 15Ser Ser Nle Glu Arg Val Glu Leu Leu Glu Lys Leu Leu Glu Lys

20 25 30Leu His Asn Tyr NHz (SEQ ID NO:23) and Ala Val Ser Glu Ile Gln Phe Nle His Asn Leu Gly Lys His Leu1 5 10 15Ser Ser Nle Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys

20 25 30Leu His Asn Tyr NH2 (SEQ?ID?NO:24).

In still another aspect of the invention, be surprised to find, the homologue and the analogue that are less than 34 amino acid whose PTH and PTHrp also are effective bone remodeling agent.These compounds have following general formula: Ala Val Ser Glu Xaa ⁵Gln Leu Leu His Asp Xaa ¹¹Gly Xaa ¹³Ser IleGln Asp Leu Xaa ¹⁹Arg Xaa ²¹Xaa ^22-31Xaa ³²Xaa ³³Xaa ³⁴Term, representational polypeptide comprises, but is not limited to: compound 41:AVSEHQLLHD KGKSIQDLRR RELLEKLLEKLHP-NH2 (SEQ ID NO:55) physical data m.p.142.8-166.1 ℃ [α] ²⁵ _D-53.80 (c 0.38, H ₂O) FAB (C ₁₇₃H ₂₉₅N ₅₅O ₉): [M+H] ⁺3929AAA:AsX2.0 (2) Glx5.7 (6) Ser1.8 (2)

His3.0(3)Gly1.1(1)Ala0.9(1)

Arg2.8(3)Val1.2(1)Ile0.9(1)

Leu7.4 (8) Lys4.4 (4) Pro0.9 (1) compound 42:AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LP-NH2 (SEQ ID NO:56) physical data m.p.161.0-177.0 ℃ [α] ²⁵ _D-61.97 (c 0.19, H ₂O) FAB (C ₁₆₇H ₈₈N ₅₂O ₄₈): [M+H] ⁺3792.0AAA:Asx2.2 (2) Glx5.9 (6) Serl.9 (2)

His2.1(2)Gly1.1(1)Ala1.0(1)

Arg3.0(3)Val1.1(1)Ile1.0(1)

Leu7.9(8)Lys4.3(4)Pro0.9(1)

Those of skill in the art know, can synthesize changing form of a large amount of polypeptide analogs.These change form and have the useful performance of analogue described herein, if will be in 100 ° ± 20 ° scopes the average hydrophobic square of each residue greater than about 0.20 aminoacid sequence on position (22-31).The classics of polypeptide are synthetic

Polypeptide of the present invention can use-case such as following method synthetic: J.M.Stewart and J.D.Young, Solid Phase Peptide Synthesis, 2nd ed., PierceChemical Co., Rockford, Illinois (1 984) and J.Meienhofer, Hor-monal Proteins and Peptides, Vol.2, Academic Press, NewYork, solid phase synthesis and the E.Schroder and the K.Lubke of (1973) etc., ThePeptides, Vol.l, Academic Press, New York, the solution of (1965) etc. is synthetic.

Generally, the peptide chain that relates in synthetic of these methods adds protected amino acid successively.Usually, first amino acid whose amino or carboxyl and all reactive side chains are all protected.Then the amino acid with protection is connected in inert solid carrier, perhaps is used for solution, adds by the next amino acid in the sequence of due care under the condition that is fit to the formation amido linkage then.After all required amino acid all link to each other with suitable sequence, thereby remove blocking group and solid carrier obtains thick polypeptide.This polypeptide is through desalination and purifying (preferably passing through chromatography), thus the acquisition the finished product.

A kind of preparation is that solid-phase polypeptide synthesizes less than about 40 preferred method amino acid whose, short polypeptide with physiologically active.In the method, a-amino acid (N ^α) functional group and any reactive side chain all uses acid-sensitive or alkali is quick (acid-or base-sensitive) radical protection.Blocking group should be stable forming under the condition of peptide bond, can be removed without difficulty again simultaneously and does not influence established polypeptide chain.Suitable alpha-amino group blocking group comprises; but be not limited to: uncle-butoxy carbonyl (Boc), benzyloxycarbonyl (Cbz), neighbour-chlorine benzyloxycarbonyl, phenylbenzene isopropoxy carbonyl, uncle-pentyloxy carbonyl (Amoc), iso-borneol oxygen base (isoborny-loxy) carbonyl, α; alpha-alpha-dimethyl-3,5-dimethoxy benzyloxy-carbonyl, o-nitrophenyl sulfinyl (nitrophenylsulfenyl), 2-cyano group-uncle-butoxy carbonyl, 9-fluorenyl methoxy carbonyl (Fmoc) etc., preferred tertiary-butoxy carbonyl (Boc).Suitable side chain protected group comprises; but be not limited to: ethanoyl, benzyl (Bzl), benzyloxymethyl (Bom), neighbour-bromo-benzyloxy-carbonyl, tert-butyl, tert-butyl dimetylsilyl, 2-benzyl chloride base (Cl-z), 2,6-dichloro benzyl, cyclohexyl, cyclopentyl, sec.-propyl, valeryl, tetrahydropyrans-2-base, tosyl group (Tos), trimethyl silyl and trityl.

In solid phase synthesis, the C-end amino acid at first is connected in the appropriate resin carrier.To be those be inertia and be insoluble to used medium progressively condensation reaction and the reagent in the protective reaction and reaction conditions the appropriate resin carrier.The example of commercially available resin comprises the vinylbenzene/divinylbenzene resin with the active group modification, for example copolymerization of chloromethylation (vinylbenzene-Vinylstyrene), methylolated copolymerization (vinylbenzene-Vinylstyrene) etc.Preferred benzylization, methylolated phenyl-acetamides methyl (PAM) resin.When the C-terminal of compound was acid amides, preferred resin was right-methyldiphenyl methylamino-copolymerization (vinylbenzene-Vinylstyrene) resin.

Can be by at ethanol, acetonitrile, N, in the dinethylformamide (DMF) etc. as ammonium salt, cesium salt, triethyl ammonium salt, 1,5-diazabicylo [5.4.0] 11 carbon-5-alkene, the N that tetramethyl ammonium or class are saloid (preferably being arranged in the cesium salt of DMF) ^aThe amino acid of protection, preferred Boc-amino acid, with resin 40-60 ℃ according to appointment of high temperature, be preferably about 50 ℃ of reactions about 12-72 hour down, preferable about 48 hours, and it is connected in the PAM resin.

Can be as methylene dichloride or dimethyl formamide, be preferably in the solvent of methylene dichloride and so on, by as N, the coupling reaction of N '-di-isopropyl carbodiimide (DIC)/l-hydroxybenzotriazole (HOBt) mediation, at about 10-50 ℃, be preferably about 25 ℃, reacted about 2-24 hour, be preferably about 2 hours and N ^a-Boc-amino acid is connected in benzhydrylamine resin.

The continuous coupled of protected amino-acid subsequently can be carried out with method well known in the art, typically by the automatic peptide synthesizer.After with triethylamine or similar alkali neutralization; be preferably the every kind of protection amino acid that adds about 1.5-2.5 times molar excess, and coupling reaction is to carry out under room temperature in inert, nonaqueous, polar solvent such as methylene dichloride, DMF or its compound (preferably methylene dichloride).Representational coupling agent is N; N '-dicyclohexyl carbodiimide (DCC), N; N '-di-isopropyl carbodiimide (DIC) or other carbodiimide, they can use separately or use in the presence of I-hydroxybenzotriazole (HOBt), O-acylurea, phosphofluoric acid benzotriazole-1-base-oxo three (pyrrolidyl)-Phosphonium (PyBop), N-hydroxy-succinamide, other N-hydroxyl imides or oxime.Also can use shielded amino acid whose active ester (for example right-nitrophenyl, pentafluorophenyl group etc.) or symmetric acid anhydrides.

Last at solid phase synthesis removed the peptide of full guard from resin.When the key that links to each other with resin carrier is benzyl ester type,, can carries out ammonolysis reaction and reach cracking with alkylamine or fluoro-alkyl amine for peptide with alkylamide C-end; For peptide with unsubstituted acid amides C-end, can carry out ammonolysis reaction and reach cracking with for example ammonia/methyl alcohol or ammonia/ethanol, these reactions can be between approximately-10 ℃ and 50 ℃, are preferably under about 25 ℃ about 12-24 hour, are preferably about 18 ℃.Peptide with hydroxyl C-end can go protected mode or the cracking by saponification reaction with HF cracking cut-out or with other strongly-acids.Perhaps, can remove peptide from resin, for example carry out transesterificationization, and then separate or saponification is carried out by ammonia with methyl alcohol by transesterificationization.Protected peptide can pass through the silica gel chromatography purifying.

Can use following method that the side chain protected group is removed from peptide: in the presence of methyl-phenoxide or other carbon ion scavenging agents, to handle the ammonia hydrolysis products with for example anhydrous liquid hydrogen fluoride; handle with hydrogen fluoride/pyridine mixture; handle with three (trifluoroacetyl group) boron and trifluoroacetic acid; reduce with palladium on hydrogen and the carbon or Polyvinylpyrolidone (PVP); perhaps reduce, preferably handle with liquid hydrogen fluoride and methyl-phenoxide with the sodium in the liquid ammonia.These reactions are that preferable about 0 ℃ was carried out about 15 minutes to 2 hours, and was preferably about 1.5 hours between about-10 ℃ and 10 ℃.

For the peptide on benzhydrylamine resin, scission reaction and go to protect step to merge into a step from the resin with above-mentioned liquid hydrogen fluoride and methyl-phenoxide.

Solution can desalination (for example be used BioRad AG-3 ^_Anionite-exchange resin), and peptide can be with one or more carry out a series of chromatographic step and are able to purifying in the following method: on weakly alkaline acetate form resin, carry out ion-exchange; At the copolymerization (vinylbenzene-Vinylstyrene) of non-derivative type Amberlite for example ^_Carry out hydrophobic adsorption chromatography on the XAD; Silica gel adsorption chromatography; On carboxymethyl cellulose, carry out ion-exchange chromatography; At for example Sephadex ^_Carry out distribution chromatography on the G-25; Adverse current is distributed; Or high performance liquid chromatography (HPLC), especially octyl group-or octadecyl silyl silicon oxide (ODS) bonding phase column on reverse high performance liquid chromatography.

Therefore; the present invention relates to the method for preparing polypeptide and pharmacy acceptable salt thereof on the other hand; these methods are included on the appropriate resin carrier amino acid of the protection of condensation successively; remove blocking group and resin carrier; and thereby purified product obtains PTH and PTHrp has the short homologue of physiologically active and the analogue of analogue, especially H (1-34) and PTHrp (1-34), and the amino acid that wherein is positioned at position (22-31) forms aforesaid amphipathic alpha-helix peptide sequence.Being re-combined into of polypeptide

In addition, the preparation of polypeptide of the present invention also can be by cloning and express the encoding gene of required polypeptide.In the method, preparation contains the plasmid of required dna sequence dna, is inserted into the appropriate host microorganism then, typically is bacterium such as intestinal bacteria, or yeast such as yeast saccharomyces cerevisiae.Induce host microorganism to produce the code cDNA of the polypeptide analog of the present invention of the plasmid of multiple copied and multiple copied.

At first, the synthetic gene coding of the selected PTH or PTHrp of design is assisted subsequently change with easy restriction enzyme site.Can use Mullis at U.S. patent No.4,683,195 and 4,683, the polymerase chain reaction described in 202 (PCR) comes sequence spreading.

Separate the synthetic gene of expansion then and it is connected in suitable plasmid, Trp LE plasmid for example can tandem ground in this plasmid inserts the gene of 4 copies.The preparation of Trp LE plasmid is in U.S. Patent No. 4,738,921 and European patent publication No.0212532 in description is arranged.Trp LE plasmid produces the protein that 8-10 manys times than Trp E plasmid usually.Multi-copy gene can be as expressing in intestinal bacteria or the yeast saccharomyces cerevisiae in appropriate host then.

Use herein embody carrier be the Trp LE18 Prot that contains the following units (Ile3, Pro5): the pBR322 fragment (EcoR I-BamH I) that contains ampicillin resistance gene and plasmid replication starting point; EcoR I-Sac II the fragment that contains trp promotor and trpE gene; Hiv protease (Ile3, Pro5) gene fragment (Sac II-Hind III); BGRF gene fragment (Hind III-BamH I); With from intestinal bacteria rpoc gene transcription terminator.Hiv protease and bGRF gene fragment are not vital, can be replaced by other encoding sequences if desired.

The fusion protein polymer of expressing gathers in the inclusion body of cell inner stablity, can itself and other cell proteins be separated by centrifugal.Isolating protein then is transformed into monomer PTH or PTHrp analogue, and can carry out purifying with cationic exchange and/or reverse hplc.

For those skilled in the art, other clone, expansion, expression and purification process are conspicuous.Representational method is disclosed in Maniatis, et al., and MolecularCloning, A LaboratoryManual, 2nd Ed. is among the Cold Spring HarborLaboratory (1989).Use and use

Polypeptide of the present invention can be used for prevention and treat various Mammals sclerotin forfeiture symptoms.Especially, shown that compound of the present invention can be used to prevent and treat as therapeutical agent people's osteoporosis and osteopenia.

Usually, the amount of application of polypeptide of the present invention or its salt is about 0.002-1 microgram/kg body weight every day, is preferably about 0.04-0.2 microgram/kg body weight every day.For the women of one 50 kg body weight, the activeconstituents dosage of every day is about 0.1-50 microgram, is preferably about 2.0-10 microgram.For other Mammals, for example horse, dog and ox need higher dosage.This dosage can be with the pharmaceutical compositions of routine, thus on demand by single administration, by repeatedly using or supplying medicine to reach best drug effect by controlled release forms, wherein preferably inject one or many every day.

The selection of precise dosage and composition and best dosage regimen is subjected to the influence of many factors, particularly: the pharmacological properties of selected polypeptide, the character of illness to be treated and severity and patient's physical appearance and mental acuity (mental acuity).

Representational dosage regimen comprises: oral, parenteral admin (comprising subcutaneous, intramuscular and intravenous injection), rectal administration, orally administering (comprising sublingual administration), pulmonary administration, through skin and intranasal administration.

Pharmacy acceptable salt has kept the useful biological activity of former polypeptide and has not produced toxic side effect.The example of this salt is: (a) acid salt that forms with mineral acid example hydrochloric acid, Hydrogen bromide, sulfuric acid, phosphoric acid, nitric acid etc.; The salt that forms with organic acid such as acetate, oxalic acid, tartrate, succsinic acid, toxilic acid, fumaric acid, glyconic acid, citric acid, oxysuccinic acid, xitix, phenylformic acid, Weibull, pamoic acid, alginic acid, polyglutamic acid, naphthene sulfonic acid, naphthalene disulfonic acid, polygalacturonic acid etc.; (b) with multivalent metal cation such as zinc, calcium, bismuth, barium, magnesium, aluminium, copper, cobalt, nickel, cadmium etc., or and N, the base addition salt that the organic cation that N '-dibenzyl-ethylenediamin or quadrol form forms; Or (c) (a) and combining form (b) such as Weibull zinc salt etc.

Another aspect of the present invention relates to pharmaceutical composition, and they contain polypeptide of the present invention or its pharmacy acceptable salt as active constituent and pharmaceutically acceptable, nontoxic carrier.As mentioned above, this composition of preparation can be used for parenteral admin (subcutaneous, intramuscular and intravenous injection), especially with liquid solution or suspension form; Be used for oral or orally administering, especially with tablet or capsule combinate form formula; Be used for lung and intranasal administration, especially with pulvis, nasal drop or aerosol form; And be used for rectum or percutaneous dosing.

Composition can be used with presented in unit dosage form easily, and can be prepared with any method of knowing in the pharmacy field, for example at Remington ' s PharmaceuticalSciences, 17th ed., Mack Publishing Company, Easton, PA., the method described in (1985).For the prescription of parenteral admin, can contain sterilized water or physiological saline, aklylene glycol (alkylene glycols) as propylene glycol, poly-alkylene glycol such as polyoxyethylene glycol, vegetables oil, hydrogenated naphthalene etc. as vehicle.For oral administration, prescription can increase drug effect by adding cholate or fatty acyl carnitine.The preparation that is used for intranasal administration can be solid and contain vehicle for example lactose or dextran, perhaps can be that water-based or oily solution are used with the form of nasal drop or metering spray agent.For orally administering, typical vehicle comprises sugar, calcium stearate, Magnesium Stearate, pregelatinized starch etc.

When preparing for intranasal administration; can be by adding for example increase Nasal Mucosa Absorption such as paddy ammonia cholic acid, cholic acid, taurocholate, second cholic acid (ethocholic acid), Septochol, gallodesoxycholic acid, Felacrinos, paddy ammonia Septochol, cyclodextrin of acid surface active agent; addition is about the 0.2-15 weight percent; be preferably about 0.5-4 weight percent, the best is about 2 weight percents.

Controlled release system by containing q.s effective constituent (for required release period) single-dose can be in long-time for example one be supplied with the pill taker with compound of the present invention in thoughtful 1 year.Various controlled release systems are individual layer or depot micro-capsule, depot implant, osmotic pump, vesica, capsule, liposome, transdermal patch for example, and iontophoresis device and other injection types may be used to this purpose.To be confined to need the position of active constituent be another feature of some controlled release drug administration device with using, and this is favourable for some disease of treatment.

A kind of controlled release preparation disperses polypeptide or its salt or is wrapped in slow degraded, nontoxic, nonantigenic polymkeric substance such as the copolymerization (lactic acid/oxyacetic acid), as at Kent, and Lewis, Sanders, with described in the work initiative in the United States Patent (USP) 4,675,189 of Tice like that.Also compound or better its insoluble relatively salt can be formulated in cholesterol or other lipid matrix pillers, or in the silastomer matrix implant.Other sustained release dosage, depot implant or injectable formulation are that those of skill in the art are known.Referring to for example, Sustainedand Controlled Release Drug Delivery Systems, J.R.Robinsoned., Marcel Dekker, Inc., New York, 1978, and R.W.Baker, Controlled Release of Biologically Active Agents, John Wiley﹠Sons, New York, 1987.

Following specific embodiment is the synthetic and test that is used to set forth representative compounds of the present invention, and they are not limited to scope of the present invention.In an embodiment, " m.p. " represents fusing point, and " [α] 25D " is in the time of 25 ℃, shown in specific rotation under the solvent, given concentration, " FAB " is that fast atom bombardment mass spectroscopy(FABMS) and " AAA " are amino acid analysises, is desired value in the observed value unquote wherein.Amino acid analysis carries out according to the scheme of manufacturers's suggestion on Hewlett-Packard AminoQuant Ana-lyzer.One-level amino acid is derived with Phthalyldicarboxaldehyde, and secondary amino acid is derived with Fmoc.Derivative amino is carried out the fluoroscopic examination quantitative analysis.Protected amino acid derive from Applied Biosystems Inc. (Foster City, CA).

The embodiment I adopts solid phase synthesis process, on 4-methyldiphenyl methylamine resin, with the compound 1 (SEQ ID NO:7) about the synthetic 0.5mmol of AppliedBiosystems Model 430A automatic peptide synthesizer.Alpha-amino group is protected with tertiary butyl carbonyl (Boc).The side chain protected group is as follows: to Asp, and Glu, and Ser benzyl (Bzl); To Arg tosyl group (Tos); To His benzyloxymethyl (Bom); To Lys 2-benzyl chloride base (Cl-z).Press the method for Stewart and Young (source as above), use N, N-dicyclohexyl carbodiimide (DCC)/I-hydroxybenzotriazole (HOBt) links to each other these amino acid successively.With each amino acid coupled after, the mixture in N-Methyl pyrrolidone makes the peptide acetylize with diacetyl oxide and diisopropylethylamine., in the presence of methyl-phenoxide, handled 30 minutes and 0 ℃ of processing 60 minutes with anhydrous HF (25mL) at-10 ℃, thereby with the cracking and slough the side chain protected group simultaneously from the resin of synthetic peptide.Vacuum boils off after the HF, and with anhydrous ether wash residual thing, thick peptide extracts with 10% acetate.With 10% acetic acid extraction thing freeze-drying, obtain 900 milligrams of thick products.With the reverse column chromatography of the ODS of middle pressure, adopt 22-45%CH among the 0.1%TFA ₃CN gradient purified peptide.Product is gone out 3 parts by wash-out, concentrate and freeze-drying after, obtain 130 milligrams, the white solid of purity＞98%.

Compound 1

AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTA-NH2 (SEQ ID NO:7) physical data: m.p.150-159 ℃ [α] ²⁵ _D-34.88* (c0.16, H ₂O) FAB (C ₁₇₅H ₃₀₀N ₅₆O ₅₁): [M+H]+4005.5AAA:Asp, 1.9 (2); Glu, 5.6 (6); Ser, 1.6 (2); His, 2.7 (3); Gly, 1.0 (1); Thr, 0.9 (1);

Ala,1.9(2)；Arg,2.8(3)；Val,1.0(1)；Ile,0.9(1)；Leu,7.3(8)；Lys,4.0(4).

Similarly, preparation compound 2,5-18, and 21-70, and the characteristic of definite these compounds.Be the polypeptide of hydroxyl wherein, replace the PAM resin on demand for synthetic end.

Compound 2

AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTA-OH (SEQ ID NO:6) physical data: m.p.154-170 ℃ [α] ²⁵ _D-49.35 ° of (c0.46, H ₂O) FAB (C ₁₇₅H ₃₀₁N ₅₇O ₅₀): [M+H] ⁺4005.0AAA:Asp, 2.1 (2); Glu, 5.9 (6); Ser, 1.7 (2); His, 2.9 (3); Glyl.1 (1); Thr, 1.0 (1);

Ala,1.9(2)；Arg,3.0(3)；Val,1.2(1)；Ile,1.0(1)；Leu,7.8(8)；Lys,4.2(4).

Compound 5

AVSEHQLLHDKGKSIQDLRRRELLERLLERLHTA-OH (SEQ ID NO:15) physical data: m.p.147-165 ℃ [α] ²⁵ _D49.17 ° (c0.66, H ₂O) FAB (C ₁₇₅H ₂₉₉N ₅₉O ₅₂): [M+H] ⁺4061AAA:Asp, 2.1 (2); Gly, 6.1 (6); Ser, 1.8 (2); His, 3.1 (3); Gly, 1.1 (1); Thr, 1.0 (1);

Ala,2.0(2)；Arg,5.0(5)；Val,1.0(1)；Ile,0.9(1)；Leu,7.7(8)；Lys,1.9(2).

Compound 6

AVSEHQLLHDRGRSIQDLRRRELLERLLERLHTA-OH (SEQ ID NO:16) physical data: m.p.150-170 ℃ [α] ²⁵ _D-48.65 ° of (c0.54, H ₂O) FAB (C ₁₇₅H ₂₉₉N ₆₃O ₅₂): [M+H] ⁺4118.0AAA:Asp, 2.1 (2); Glu, 6.1 (6); Ser, 1.8 (2); His, 3.2 (3); Gly, 1.2 (1); Thr, 1.0 (1);

Ala,2.0(2)；Arg,6.9(7)；Val,1.0(1)；Ile,1.0(1)；Leu,7.8(8).

Compound 7

AVSEHQLLHDRGRSIQDLRRRELLERLLKRLHTA-OH (SEQ ID NO:17) physical data: m.p.177-182 ℃ [α] ²⁵ _D-46.17 ° of (c0.14, H ₂O) FAB (C ₁₇₆H ₃₀₄N ₆₀O ₅₀): [M+H] ⁺4117AAA:Asp, 2.0 (2); Glu, 4.8 (5); Ser, 1.8 (2); His, 3.2 (3); Gly, 1.1 (1); Thr, 0.9 (1);

Ala,1.9(2)；Arg,6.7(7)；Val,1.0(1)；Ile,1.0(1)；Lys,7.7(8)；Lys,0.9(1).

Compound 8

AVSEHQLLHDKGKSIQDLRRRELLEKLLRKLHTA-OH (SEQ ID NO:5) physical data: m.p.147-165 ℃ [α] ²⁵ _D-49.17 ° of (c0.66, H ₂O) FAB (C ₁₇₆H ₃₀₅N ₅₉O ₄₉): [M+H] ⁺4033.0AAA:Asp, 2.0 (2); Glu, 4.8 (5); Ser, 1.8 (2); His, 2.7 (3); Gly, 1.1 (1); Thr, 0.9 (1);

Ala,2.0(2)；Arg,3.9(4)；Val,1.0(1)；Ile,1.0(1)；Leu,7.9(8)；Lys,4.0(4).

Compound 9

AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTAGRR-OHSEQ ID NO:10) physical data: m.p.158-160 ℃ [α] ²⁵ _D-44.76 ° of (c0.1, H ₂O) FAB (C ₁₈₉H ₃₂₆N ₆₄O ₅₅): [M] ⁺4375.0AAA:Asp, 2.0 (2); Glu, 5.9 (6); Ser, 1.7 (2); His, 2.9 (3); Gly, 2.3 (2); Thr, 1.0 (1);

Ala,1.9(2)；Arg,5.0(5)；Val,1.2(1)；Ile,1.0(1)；Leu,7.8(8)；Lys,4.3(4).

Compound 10

AVSEAQLLHDLGKSIQDLRRRELLEKLLEKLHAL-OH (SEQ ID NO:14) physical data: m.p.170-175 ℃ [α] ²⁵ _D-31.59 ° of (c0.54, H ₂O) FAB (C ¹⁷⁴H ³⁰⁰N ⁵²O ⁵¹): [M+H] ⁺3936.0AAA:Asp, 2.0 (2); Glu, 6.0 (6); Ser, 1.8 (2); His, 2.0 (2); Gly, 1.2 (1); Ala, 3.0 (3);

Arg,2.8(3)；Val,1.1(1)；Ile,1.0(1)；Leu,9.9(10)；Lys,3.0(3).

Compound 11

AVSEHQLLHDKGKSIQDLRRRELLEKLLELLKEL-NH ₂(SEQ ID NO:11) physical data: m.p.172-174 ℃ [α] ²⁵ _D-43.29 ° of (c0.2, H ₂O) FAB (C ₁₇₉H ₃₁₁N ₅₅O ₅₂): [M+H] ⁺4065.8AAA:Asp, 2.2 (2); Glu, 7.7 (7); Ser, 1.7 (2); His, 2.0 (2); Gly, 1.0 (1); Ala, 1.0 (1);

Arg,3.0(3)；Val,1.1(1)；Ile,1.0(1)；Leu,9.3(9)；Lys,5.1(5).

Compound 12 AVSEIQFXHNLGKHLSSXERVELLEKLLEKLHNY-NH ₂(X=Nle, SEQ ID NO:23) physical data: m.p.178 ℃ [α] ²⁵ _D-36.88 ° of (c0.4, H ₂O) FAB (C ₁₈₂H ₂₉₅N ₅₀O ₅₁): [M+H] ⁺4001.6AAA:Asp, 2.1 (2); Glu, 6.5 (6); Ser, 2.7 (3); His, 3.1 (3); Gly, 1.1 (1); Ala, 1.0 (1);

Arg,1.0(1)；Tyr,0.8(1)；Val,2.0(2)；Phe,1.0(1)；Ile,0.9(1)；Leu+Nle,

8.5(7+2)；Lys,3.1(3).

Compound 13 AVSEIQFXHNLGKHLSSXRRRELLEKLLEKLHNY-NH ₂(X=Nle, SEQ ID NO:24) physical data: m.p.260 ℃ [α] ²⁵ _D-37.02 ° of (c0.2, H ₂O) FAB (C ₁₈₄H ₃₀₄N ₅₆O ₄₉): [M+H] ⁺4084AAA:Asp, 2.1 (2); Glu, 5.5 (5); Ser, 2.6 (3); His, 3.1 (3); Ala, 1.0 (1); Gly, 1.1 (1);

Arg,3.2(3)；Tyr,1.0(1)；Val,1.0(1)；Phe,1.0(1)；Ile,1.0(1)；Leu,9.0(9)；

Lys,3.0(3).

Compound 14

AVSEHQLLHDKGKSIQDLRRRALAEALAEALHTA-NH ₂(SEQ ID NO:20) physical data: m.p.190-225 ℃ [α] ²⁵ _D-56.58 ° of (c0.36, H ₂O) FAB (C ₁₆₁H ₂₇₂N ₅₄O ₄₉): [M+H] ⁺3747.0AAA:Asp, 2.1 (2); Glu, 4.9 (5); Ser, 1.7 (2); His, 2.6 (3); Gly, 1.1 (1); Thr, 1.0 (1); Ala, 7.6 (7); Arg, 2.8 (3); Val, 1.2 (1); Ile, 1.0 (1); Leu, 6.6 (6); Lys, 1.9 (2).

Compound 15

AVSEHQLLHDKGKSIQDLARRELLEKLLEKLHTA-NH ₂(SEQ ID NO:12) physical data: m.p.170-180 ℃ [α] ²⁵ _D-48.19 ° of (c0.2, H ₂O) FAB (C ₁₇₂H ₂₉₃N ₅₃O ₅₁): [M+H] ⁺3919.0AAA:Asp, 2.1 (2); Glu, 6.1 (6); Ser, 1.7 (2); His, 3.1 (3); Gly, 1.1 (1); Thr, 1.0 (1);

Ala,3.0(3)；Arg,2.1(2)；Val,1.1(1)；Ile,1.0(1)；Leu,8.0(8)；Lys,4.4(4).

Compound 16

AVSEHQLLHDKGKSIQDLRRAELLEKLLEKLHTA-NH ₂(SEQ ID NO:13) physical data: m.p.190-195 ℃ [α] ²⁵ _D-50.50 ° of (c0.4, H ₂O) FAB (C ₁₇₂H ₂₉₃N ₅₃O ₅₁): [M+H] ⁺3919.0AAA:Asp, 2.1 (2); Glu, 6.0 (6); Ser, 1.8 (2); His, 3.1 (3); Gly, 1.1 (1); Thr, 1.0 (1);

Ala,3.0(3)；Arg,2.1(2)；Val,1.1(1)；Ile,1.0(1)；Leu,7.5(8)；Lys,4.2(4).

Compound 17

AVSEHQLLHDKGKSIQDLRRRSLLSSLLSSLHTA-NH ₂(SEQ ID NO:21) physical data: m.p.195-204 ℃ [α] ²⁵ _D-67.11 ° of (c0.3, H ₂O) FAB (C ₁₆₃H ₂₈₀N ₅₄O ₅₀): [M+H] ⁺3796.0AAA:Asp, 2.1 (2); Glu, 2.9 (3); Ser, 6.8 (7); His, 3.1 (3); Gly, 1.2 (1); Thr, 1.0 (1);

Ala,2.0(2)；Arg,3.0(3)；Val,1.0(1)；Ile,1.0(1)；Leu,8.2(8)；Lys,2.0(2).

Compound 18

AVSEHQLLHDKGKSIQDLRRRAFYDKVAEKLHTA-NH ₂(SEQ ID NO:22) physical data: m.p.200-207 ℃ [α] ²⁵ _D-60.26 ° of (c0.6, H ₂O) FAB (C ₁₇₄H ₂₈₄N ₅₆O ₅₀): [M+H] ⁺3960.0AAA:Asp, 2.9 (3), Glu, 3.5 (4); Ser, 1.4 (2); His, 2.6 (3); Gly, 0.9 (1); Thr, 1.0 (1);

Ala,4.0(4)；Arg,3.0(3)；Tyr,0.9(1)；Val,1.9(2)；Phe,1.1(1)；Ile,0.9(1)；

Leu,3.6(4)；Lys,4.1(4).

Compound 21

AVSEIQFLHN LGKHLSSLRR RELLEKLLEK LHNY-NH ₂(SEQ ID NO:35) physical data: m.p.148-155 ℃ [α] ²⁵ _D-45.97 (c0.26, H ₂O) FAB (C ₁₈₄H ₃₀₄N ₅₆O ₄₉): [M+H] ⁺4084AAA:Asx, 2.1 (2); Glx, 5.0 (5); Ser, 2.7 (3); His, 3.0 (3); Gly, 1.0 (1); Ala, 0.9 (1);

Arg,3.1(3)；Tyr,0.9(1)；Val,1.0(1)；Phe,0.9(1)；Ile,0.9(1)；Leu?9.3(9)；

Lys,3.2(3).

Compound 22

AVSEIQFLHN?LGKHLSSLRR?RELLEKLLEK?LHNX-NH ₂(SEQ?ID?NO:36)

(X=homoserine) physical data: m.p.69.4-128 ℃ [α] ²⁵ _D-43.93 (c0.15, H ₂O) FAB (C ₁₇₉H ₃₀₂N ₅₆O ₄₉): [M+H] ⁺4022.9AAA:Asx, 2.0 (2); Glx, 4.9 (5); Ser, 2.6 (3); His, 2.8 (3); Gly, 1.0 (1); Ala, 1.0 (1);

Arg,3.0(3)；Val,1.0(1)；Phe,1.0(1)；Ile,0.9(1)；Leu,8.8(9)；Lys,3.4(3)；

Compound 23

AVSEIQFLHN?KGKHLSSLRR?RELLEKLLEK?LHNX-NH ₂(SEQ?ID?NO:37)

(X=homoserine) physical data: m.p.87.1-142.1 ℃ [α] ²⁵ _D-52.14 (c0.41, H ₂O) FAB (C ₁₇₉H ₃₀₃N ₅₇O ₄₉): [M+H] ⁺4038 AAA:Asx, 2.1 (2); Glx, 4.9 (5); Ser, 2.7 (3); His, 2.8 (3); Gly, 1.0 (1);

hSer,1.0(1)；Ala,1.0(1)；Arg,3.0(3)；Val,1.1(1)；Phe,0.9(1)；

Ile,0.9(1)；Leu,7.9(8)；Lys,3.7(4).

Compound 24

AVSEHQLLHD KGKSIQDLKL KELLEKLLEK LHTA-NH ₂(SEQ ID NO:38) physical data: m.p.175-182 ℃ [α] ²⁵ _D-49.99 (c0.47, H ₂O) FAB (C ₁₇₅H ₂₉₉N ₄₉O ₅₁): [M+H] ⁺3906.5AAA:Asx, 2.1 (2); Glx, 6.5 (6); Ser, 1.8 (2); His, 3.1 (3); Gly, 1.1 (1); Thr, 1.0 (1);

Ala,2.1(2)；Val,1.1(1)；Ile,1.0(1)；Leu,9.1(9)；Lys,6.5(6).

Compound 25

AVSEHQLLHD KGKSIQDLRR RELLERLLER LHTA-NH ₂(SEQ ID NO:39) physical data: m.p.136.5-153.5 ℃ [α] ²⁵ _D-32.57 (c0.13, H ₂O) FAB (C ₁₇₅H ₃₀₀N ₆₀O ₅₁): [M+H] ⁺4060.8AAA:Asx, 2.2 (2); Glx, 6.2 (6); Ser, 1.8 (2); His, 3.2 (3); Gly, 1.1 (1); Thr, 1.0 (1);

Ala,2.1(2)；Arg,5.2(5)；Val,1.1(1)；Ile,1.1(1)；Leu,8.4(8)；Lys,2.2(2).

Compound 26

AVSEHQLLHD KGKSIQDLRR RELLERLLER LHTAP-OH (SEQ ID NO:40) physical data: m.p.125.8-127.2 ℃ [α] ²⁵ _D-54.62 (c0.23, H ₂O) FAB (C ₁₈₀H ₃₀₆N ₆₀O ₅₃): [M+H] ⁺4158.0AAA:Asx, 2.1 (2); Glx, 6.2 (6); Ser, 1.8 (2); His, 2.9 (3); Gly, 1.1 (1); Thr, 1.0 (1);

Ala,2.0(2)；Arg,5.1(5)；Val,1.0(1)；Ile,1.0(1)；Leu,8.0(8)；Lys,2.1(2)；

Pro,1.1(1).

Compound 27 AVSEHQLLHD KGKSIQDLRR RELLERLLER LHTAGRR-OH (SEQ ID NO:41) physical data: m.p.106-137.3 ℃ [α] ²⁵ _D-39.55 (c0.67, H ₂O) FAB (C ₁₈₉H ₃₂₆N ₆₈O ₅₅): [M+H] ⁺4430.5AAA:Asx, 2.1 (2); Glx, 5.9 (6); Ser, 1.6 (2); His, 2.7 (3); Gly, 2.2 (2); Thr, 1.0 (1);

Ala,1.8(2)；Arg,7.3(7)；Val,0.8(1)；Ile,1.0(1)；Leu,8.1(8)；Lys,2.1(2).

Compound 28 AVSEHQLLHD KGKSIQDLRR RELLERLLER LHTAGRRX-NH ₂(SEQ ID NO:42)

(X=homoserine) physical data: m.p.80 ℃ [α] ²⁵ _D-48.64 (c0.09, H ₂O) FAB (C ₁₉₃H ₃₃₄N ₇₀O ₅₆): [M+H] ⁺4530.0AAA:Asx, 2.2 (2); Glx, 6.1 (6); Ser, 1.7 (2); His, 3.0 (3); Gly, 1.9 (2);

hSer,1.0(1)；Thr,1.0(1)；Ala,2.1(2)；Arg,7.2(7)；Val,0.8(1)；

Ile,1.0(1)；Leu,8.4(8)；Lys,2.1(2).

Compound 29

AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTY-NH ₂(SEQ ID NO:43) physical data: m.p.160-172 ℃ [α] ²⁵ _D-49.85 (c0.34, H ₂O) FAB (C ₁₈₁H ₃₀₄N ₅₆O ₅₂): [M+H] ⁺4096.9AAA:Asx, 2.0 (2); Glx, 5.6 (6); Ser, 1.7 (2); His, 3.1 (3); Gly, 1.1 (1); Thr, 0.9 (1);

Ala,0.9(1)；Arg,3.0(3)；Tyr,0.9(1)；Val,1.0(1)；Ile,1.0(1)；Leu,7.7(8)；

Lys,4.4(4).

Compound 30

AVSEHQLLHD KGYSIQDLRR RELLEKLLEK LHTA-NH ₂(SEQ ID NO:44) physical data: m.p.130-171 ℃ [α] ²⁵ _D-40.65 (c0.34, H ₂O) FAB (C ₁₇₈H ₂₉₇N ₅₅O ₅₂): [M+H] ⁺4039.4AAA:Asx, 2.0 (2); Glx, 5.5 (6); Ser, 1.8 (2); His, 3.4 (3); Gly, 1.1 (1); Thr, 1.0 (1);

Ala,2.0(2)；Arg,2.9(3)；Tyr,0.8(1)；Val,1.0(1)；Ile,0.9(1)；Leu,7.9(8)；

Lys,3.4(3).

Compound 31

AVSEHQLLHD KG ℃ of SIQDLRR RELLEKLLEK LHTA-NH ₂(SEQ ID NO:45) physical data: m.p.140-160 ℃ [α] ²⁵ _D-44.48 (c0.25, H ₂O) FAB (C ₁₇₂H ₂₉₃N ₅₅O ₅₁S ₁): [M+H] ⁺3979AAA:Asx+Cys, 3.0 (2+1); Glx, 5.6 (6); Ser, 1.7 (2); His, 3.0 (3); Gly, 1.0 (1);

Thr,0.9(1)；Ala,1.9(2)；Arg,2.5(3)；Val,1.0(1)；Ile,0.9(1)；Leu,7.5(8)；

Lys,3.3(3).

Compound 32

AVSEHQLLHD?KGXSIQDLRR?RELLEKLLEK?LHTA-NH ₂(SEQ?ID?NO:46)

(X=Cys (CH ₂ONH (CH ₂) ₂NH (biotinyl))) physical data: m.p. (not measuring) [α] ²⁵ _D(not measuring) FAB (C ₁₈₆H ₃₁₆N ₅₉O ₅₄S ₂): [M+H] ⁺4306.6AAA:Asx, 2.2 (2); Glx, 6.1 (6); Ser, 1.8 (2); His, 3.8 (3); Gly, 1.0 (1); Thr, 1.0 (1);

Ala,2.0(2)；Arg,3.1(3)；Val,1.1(1)；Ile,0.9(1)；Leu,8.3(3)；Lys,3.3(3),

Compound 33

AVSEHQLLHD?KGXSIQDLRR?RELLEKLLEK?LHTA-NH ₂(SEQ?ID?NO:47)

(X=Lys (7-dimethylamino-2-oxygen-2H-1| chromene-4-ethanoyl)) physical data: m.p.135-205 ℃ 1] [α] ²⁵ _D-26.92 (c 0.104, the 50%HOAc aqueous solution) FAB (C ₁₈₈H ₃₁₁N ₅₇O ₅₄): [M+H] ⁺4233AAA:Asx, 1.9 (2); Glx, 6.3 (6); Ser, 1.7 (2); His, 3.2 (3); Gly, 1.0 (1); Thr, 1.1 (1);

Ala,2.0(2)；Arg,3.2(3)；Val,1.1(1)；Ile,0.9(1)；Leu,8.2(8)；Lys,4.5(4).

Compound 34

AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTAG-OH (SEQ ID NO:48) physical data: m.p.92.1-146.6 ℃ [α] ²⁵ _D-40.76 (c0.34, H ₂O) FAB (C ₁₇₇Hc ₃₀₂N ₅₆O ₅₃): [M+H] ⁺4062.0AAA:Asx, 2.0 (2); Glx, 5.7 (6); Ser, 1.8 (2); His, 3.0 (3); Gly, 2.2 (2); Thr, 0.9 (1);

Ala,1.9(2)；Arg,2.8(3)；Val,1.2(1)；Ile,0.9(1)；Leu,7.5(8)；Lys,4.2(4).

Compound 35

AVSXHQLLHZ?KGKSIQZLRR?RXLLXKLLXK?LHA-OH(SEQ?ID?NO:49)

(X=Glu (OCH ₃); Z=Asp (OCH ₃)) physical data: m.p. (not measuring) [α] ²⁵ _D-21.96 (c0.132, H ₂O) FAB (C ₁₈₁H ₃₁₁N ₅₅O ₅₂): [M+H] ⁺4089.0AAA:Asx, 2.1 (2); Glx, 6.3 (6); Ser, 1.8 (2); His, 3.3 (3); Gly, 1.1 (1); Thr, 1.0 (1);

Ala,2.0(2)；Arg,3.1(3)；Val,1.1(1)；Ile,0.9(1)；Leu,8.0(8)；Lys,4.2(4).

Compound 36

AVSXHQLLHZ?KGKSIQZLRR?RXLLXKLLXK?LHA-OCH ₃(SEQ?ID?NO:50)

(X=Glu (OCH ₃); Z=Asp (OZCH ₃)) physical data: m.p. (not measuring) [α] ²⁵ _D-46.80 (c0.07, H ₂O) FAB (C ₁₈₂H ₃₁₃N ₅₅O ₅₂): [M+H] ⁺4103AAA:Asx, 2.1 (2); Glx, 6.2 (6); Ser, 1.4 (2); His, 3.0 (3); Gly, 1.1 (1); Thr, 1.1 (1);

Ala,1.7(2)；Arg,3.2(3)；Val,0.6(1)；Ile,0.9(1)；Leu,8.0(8)；Lys,4.1(4).

Compound 37

AVSEHQLLHD?KGKSIQDLRR?RELLEKLLEK?LHTX-OH(SEQ?ID?NO:51)

(X=homoserine) physical data: m.p. (not measuring) [α] ²⁵ _D(not measuring) FAB (C ₁₇₆H ₃₀₁N ₅₅O ₅₃): [M+H] ⁺4035.1AAA:Asx, 2.1 (2); Glx, 5.9 (6); Ser, 2.0 (2); His, 3.1 (3); Gly, 0.8 (1);

hSer,0.8(1)；Thr,1.0(1)；Ala,1.0(1)；Arg,3.0(3)；Val,1.3(1)；

Ile,1.0(1)；Leu,8.1(8)；Lys,3.8(4).

Compound 38

AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTAP-OH (SEQ ID NO:52) physical data: m.p.152.1-186.5 ℃ [α] ²⁵ _D-55.91 (c0.33, H ₂O) FAB (C ₁₈₀H ₃₀₆N ₅₆O ₅₃): [M+H] ⁺4102.6AAA:Asx, 2.0 (2); Glx, 5.6 (6); Ser, 1.7 (2); His, 2.9 (3); Gly, 1.1 (1); Thr, 0.9 (1);

Ala,1.9(2)；Arg,2.9(3)；Val,1.2(1)；Ile,1.0(1)；Leu,7.7(8)；Lys,4.3(4).

Compound 39

AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTP-OH (SEQ ID NO:53) physical data: m.p.120-148.2 ℃ [α] ²⁵ _D-52.78 (c0.47, H ₂O) FAB (C ₁₇₇H ₃₀₁N ₅₅O ₅₂): [M+H] ⁺4031.0AAA:Asx, 2.0 (2); Glx, 5.5 (6); Ser, 1.8 (2); His, 2.9 (3); Gly, 1.0 (1); Thr, 1.0 (1);

Ala,0.9(1)；Arg,2.9(3)；Val,1.2(1)；Ile,0.9(1)；Leu,7.5(8)；Lys,3.6(3)；

Pro,0.9(1).

Compound 40

AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTP-NH ₂(SEQ ID NO:54) physical data: m.p.133.9-155.1 ℃ [α] ²⁵ _D-54.22 (c0.37, H ₂O) FAB (C ₁₇₇H ₃₀₂N ₅₆O ₅₁): [M+H] ⁺4030.7AAA:Asx, 2.0 (2); Glx, 5.6 (6); Ser, 1.9 (2); His, 2.9 (3); Gly, 1.1 (1); Thr, 0.9 (1);

Ala,0.9(1)；Arg,2.8(3)；Val,1.2(1)；Ile,1.1(1)；Leu,7.8(8)；Lys,4.2(4)；

Pro,0.9(1).

Compound 41

AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHP-NH ₂(SEQ ID NO:55) physical data: m.p.142.8-166.1 ℃ [α] ²⁵ _D-53.80 (c0.38, H ₂O) FAB (C ₁₇₃H ₂₉₅N ₅₅O ₄₉): [M+H] ⁺3929AAA:Asx, 2.0 (2); Glx, 5.7 (6); Ser, 1.8 (2); His, 3.0 (3); Gly, 1.1 (1); Ala, 0.9 (1);

Arg,2.8(3)；Val,1.2(1)；Ile,0.9(1)；Leu,7.4(8)；Lys,4.4(4)；Pro,0.9(1).

Compound 42

AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LP-NH ₂(SEQ ID NO:56) physical data: m.p.161.0-177.0 ℃ [α] ²⁵ _D-61.97 (c0.19, H ₂O) FAB (C ₁₆₇H ₂₈₈N ₅₂O ₄₈): [M+H] ⁺3792.0AAA:Asx, 2.2 (2); Glx, 5.9 (6); Ser, 1.9 (2); His, 2.1 (2); Gly, 1.1 (1); Ala, 1.0 (1);

Arg,3.0(3)；Val,1.1(1)；Ile,1.0(1)；Leu,7.9(8)；Lys,4.3(4)；Pro,0.9(1).

Compound 43 AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTRSAW-OH (SEQ ID NO:57) physical data: m.p.181-202 ℃ [α] ²⁵ _D-45.14 (c0.19, H ₂O) FAB (C ₁₉₅H ₃₂₆N ₆₂O ₅₆): [M+H] ⁺4435.2AAA:Asx, 2.0 (2); Glx, 5.8 (6); Ser, 2.8 (3); His, 2.8 (3); Gly, 1.1 (1); Thr, 0.9 (1);

Ala,1.9(2)；Arg,3.7(4)；Ile,0.9(1)；Leu,7.5(8)；Lys,4.3(4)；Trp,0.9(1).

Compound 44

AVSEHQLLHD RGRSIQDLRR RELLERLLER LHTAGRRTRSAW-OH (SEQID NO:58) physical data: m.p.130-132.2 ℃ [α] ²⁵ _D-46.66 (c0.195, H ₂O) FAB (C ₂₁₆H ₃₆₅N ₈₁O ₆₂): [M+H] ⁺5088.8AAA:Asx, 2.2 (2); Glx, 6.0 (6); Ser, 2.7 (3); His, 3.0 (3); Gly, 2.2 (2); Thr, 2.1

(2)；Ala,3.0(3)；Arg,10.5(10)；Val,0.9(1)；Ile,1.0(1)；Leu,8.2(8)；Trp,

1.0(1).

Compound 45

AVSEHQLLHD RGRSIQDLRR RELLERLLER LHTAGRRTRSAW-NH ₂(SEQ ID NO:59) physical data: m.p.158-174 ℃ [α] ²⁵ _D-43.57 (c0.53, H ₂O) FAB (C ₂₁₆H ₃₆₅N ₈₂O ₆₁): [M+H] ⁺5087.4AAA:Asx, 1.9 (2); Glx, 5.6 (6); Ser, 2.6 (3); His, 3.3 (3); Gly, 2.1 (2); Thr, 2.0 (2);

Ala,2.9(3)；Arg,10.1(10)；Val,0.9(1)；Ile,1.0(1)；Leu,8.3(8)；Trp1.1(1).

Compound 46

AVSEHQLLHD?RGXSIQDLRR?RELLERLLER?LHTAGRRTRSAW-OH(SEQ?ID?NO:60)

(X=Lys (dihydro cinnamon acyl group)) physical data: m.p.165.4-175.2 ℃ [α] ²⁵ _D-40.43 (c0.20, H ₂O) FAB (C ₂₂₅H ₃₇₄N ₈₀O ₆₂): [M+H] ⁺5191AAA:Asx, 2.1 (2), Glx, 6.3 (6); Ser, 2.8 (3); His, 3.2 (3); Gly, 2.1 (2), Thr, 2.0

(2)；Ala,3.2(3)；Arg,9.9(9)；Val,1.0(1),Ile,0.9(1)；Leu,8.6(8)；Lys,1.1

(1)；Trp,1.1(1).

Compound 47

AVSEIQFXHN?LGKHLSSXTR?SAWLRKKLQD?VHNY-NH ₂(SEQ?ID?NO:61)

(X=nor-leucine) physical data: m.p.140-160 ℃ [α] ²⁵ _D-56.88 (c0.16, H ₂O) FAB (C ₁₈₀H ₂₈₇N ₅₆O ₅₈); [M+H] ⁺3989.8AAA:Asx, 3.0 (3); Glx, 2.9 (3); Ser, 3.7 (4); His, 2.8 (3); Gly, 1.1 (1); Thr, 0.9 (1);

Ala,1.9(2)；Arg,2.0(2)；Tyr,1.0(1)；Val,1.7(2)；Phe,0.9(1)；Ile,0.9(1)；

Leu+Nle5.8(2+4)；Lys,3.4(3)；Trp,1.1(1).

Compound 48

AVSEHQLLHD KGKSJQDLRR RELLSEKLLEK LHTMA-NH ₂(SEQ ID NO:62) physical data: m.p.140-210 ℃ [α] ²⁵ _D-47.75 (c0.178, H ₂O) FAB (C ₁₈₀H ₃₀₉N ₅₇O ₅₂S ₁): [M+H] ⁺4135.0AAA:Asx, 2.3 (2); Glx, 6.6 (6); Ser, 1.4 (2); His, 3.2 (3); Gly, 1.1 (1); Thr, 1.0 (1);

Ala,2.0(2)；Arg,3.1(3)；Val,0.9(1)；Met,1.1(1)；Ile,1.0(1)；Leu,8.8(8)；

Lys,4.4(4).

Compound 49

AVSEHQLLHD?KGKSIQDLRR?RELLEKLLEK?LHTX(SEQ?ID?NO:63)

(X=1,4-diamino butyrolactam) physical data: m.p.161-181 ℃ [α] ²⁵ _D-48.38 (c0.25, H ₂O) FAB (C ₁₇₆H ₃₀₀N ₅₆O ₅₁): [M+H] ⁺4016.8AAA:Asx, 2.1 (2); Glx, 6.3 (6); Ser, 1.7 (2); His, 3.3 (3); Gly, 1.1 (1); Thr, 1.0 (1);

Ala,2.1(2)；Arg,2.9(3)；Val,0.9(1)；Ile,0.9(1)；Leu,8.0(8)；Lys,3.8(4)

Compound 50

AVSEHQLLHD KGKSIQDLRR RFFLEKLLEK LHTA-NH ₂(SEQ ID NO:64) physical data: m.p.136.5-156.8 ℃ [α] ²⁵ _D-49.89 (c0.24, H ₂O) FAB (C ₁₈₂H ₃₀₀N ₅₆O ₄₉): [M+H] ⁺4056.0AAA:Asx, 2.2 (2); Glx, 5.0 (5); Ser, 1.9 (2); His, 3.3 (3); Gly, 1.0 (1); Thr, 1.0 (1);

Ala,2.1(2)；Arg,3.1(3)；Val,1.0(1)；Phe,2.0(2)；Ile,0.9(1)；Leu,7.2(7)；

Lys,3.5(4).

Compound 51

AVSEHQLLHD KGKSIQDLRR RELLHKLLEK LHTA-NH ₂(SEQ ID NO:65) physical data: m.p.80.7-141.0 ℃ [α] ²⁵ _D-55.38 (c0.23, H ₂O) FAB (C ₁₇₆H ₃₀₀N ₅₈O ₄₉): [M+H] ⁺4012.8AAA:Asx, 2.2 (2); Glx, 4.9 (5); Ser, 1.8 (2); His, 4.3 (4); Gly, 1.1 (1); Thr, 1.0 (1):

Ala,2.0(2)；Arg,3.1(3)；Val,1.1(1)；Ile,1.0(1)；Leu,8.1(8)；Lys,3.9(4).

Compound 52

AVSEHQLLHD KGKSIQDLRR RELLEHLLEK LHTA-NH ₂(SEQ ID NO:66) physical data: m.p.134.3-157.9 ℃ [α] ²⁵ _D-50.72 (c0.45, H ₂O) FAB (C ₁₇₅H ₂₉₅N ₅₇O ₅₁): [M+H] ⁺4012.8AAA:Asx, 2.1 (2); Glx, 5.9 (6); Ser, 1.8 (2); His, 4.2 (4); Gly, 1.1 (1); Thr, 1.0 (1);

Ala,2.0(2)；Arg,3.0(3)；Val,1.1(1)；Ile,0.9(1)；Leu,8.1(8)；Lys,3.1(3).

Compound 53

AVSEHQLLHD KGKSIQDLRR RELLEKLIAK LHTA-NH ₂(SEQ ID NO:67) physical data: m.p.142.7-159.8 ℃ [α] ²⁵ _D-54.01 (c0.21, H ₂O) FAB (C ₁₇₃H ₂₉₈N ₅₆O ₄₉): [M+H] ⁺3946.0AAA:Asx, 2.2 (2); Glx, 4.9 (5); Ser, 1.8 (2); His, 3.1 (3); Gly, 1.1 (1); Thr, 1.0 (1);

Ala,3.1(3)；Arg,3.1(3):Val,1.0(1)；Ile,1.9(2)；Leu,7.0(7)；Lys,4.3(4).

Compound 54

AVSEHQLLHD KGKSIQDLRR RELLEKLLEE 1HTA-NH ₂(SEQ ID NO:68) physical data: m.p.138-185 ℃ [α] ²⁵ _D-50.17 (c0.14, H ₂O) FAB (C ₁₇₄H ₂₉₅N ₅₅O ₅₃): [M+H] ⁺4005AAA:Asx, 2.2 (2); Glx, 7.1 (7); Ser, 1.7 (2); His, 2.8 (3); Gly, 1.0 (1); Thr, 1.0 (1);

Ala,2.1(2)；Arg,3.1(3)；Val,1.1(1)；Ile,1.7(2)；Leu,7.1(7)；Lys,2.7(3).

Compound 55AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTX-NHCH ₂CH ₃(SEQ ID NO:69)

(X=homoserine) physical data: m.p. (not measuring) [α] ²⁵ _D(not measuring) FAB (C ₁₇₈H ₃₀₆N ₅₆O ₅₂): [M+H] ⁺4063.0AAA:Asx, 2.1 (2); Glx, 5.8 (6); Ser, 1.7 (2); His, 3.1 (3); Gly, 0.9 (1); Thr, 1.0 (1);

Ala,0.9(1)；Arg,3.0(3)；Val,1.1(1)；Ile,1.0(1)；Leu,8.4(8)；Lys,3.7(4)；

hSer,0.9(1).

Compound 56

AVSEHQLLHD?KGKSIQDLRR?RELLEKLLEK?LHTX-NHCH ₂CH ₂C ₆H ₅(SEQID?NO:70)

(X=homoserine) physical data: m.p. (not measuring) [α] ²⁵ _D(not measuring) FAB (C ₁₈₄H ₃₁₀N ₅₆O ₅₂): [M+H] ⁺4138.8AAA:Asx, 2.0 (2); Glx, 5.9 (6); Ser, 1.7 (2); His, 2.9 (3); Gly, 0.9 (1); Thr, 1.0 (1);

Ala,0.9(1)；Arg,3.0(3)；Val,1.0(1)；Ile,0.9(1)；Leu,8.0(8)；Lys,4.1(4)；

hSer,0.9(1).

Compound 57

Physical data: m.p.142.5-163.5 ℃ [α] ²⁵ _D-34.31 (c0.17, H ₂O) FAB (C ₁₇₅H ₂₉₈N ₅₆O ₅₀): [M+H] ⁺3986.4AAA:Asx, 1.9 (2); Glx, 5.9 (6); Ser.1.8 (2); His, 3.2 (3); Gly, 1.1 (1); Ala, 2.0 (2);

Arg,3.0(3)；Thr,1.0(1):Val,1.1(1)；1le,0.9(1)；Leu,8.0(8)；Lys,4.0(4).

Compound 58AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTRSAW-NH ₂(SEQ ID NO:72) physical data: m.p.158-163 ℃ [α] ²⁵D-46.06 (c0.17, H ₂O) FAB (C ₁₉₅H ₃₂₇N ₆₃O ₅₅): [M+H] ⁺4434.8AAA:Asx, 2.0 (2); Glx, 5.5 (6); Ser, 2.7 (3); His, 3.1 (3); Gly, 1.0 (1); Ala, 1.8 (2);

Arg,4.0(4)；Thr,0.9(1)；Val,0.9(1)；Ile,0.9(1)；Leu,7.5(8)；Lys,3.9

(4)；Trp,1.0(1).

Compound 59AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTRSAX-OH (SEQ ID NO:73)

(X=Nal (2)=3-(2-naphthyl)-L-L-Ala) physical data: m.p.156-162 ℃ [α] ²⁵ _D-44.44 (c0.189, H ₂O) FAB (C ₁₉₇H ₃₂₈N ₆₂O ₅₅): [M+H] ⁺4445.6AAA:Asx, 2.1 (2); Glx, 5.5 (6); Ser, 2.8 (3); His, 2.9 (3); Gly, 1.0 (1); Ala, 2.0 (2);

Arg,4.0(4)；Thr,0.9(1)；Val,1.0(1)；Ile,0.9(1)；Leu.7.5(8)；Lys,4.2(4)；

Nal,1.1(1).

Compound 60AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTASAW-OH (SEQ ID NO:74) physical data: m.p.159-164 ℃ [α] ²⁵ _D-50.94 (c0.29, H ₂O) FAB (C ₁₉₂H ₃₂₀N ₆₀O ₅₅): [M+H] ⁺4349.0AAA:Asx, 2.0 (2); Glx, 5.6 (6); Ser, 2.7 (3); His, 3.2 (3); Gly, 1.0 (1); Ala, 3.1 (3);

Arg,2.8(3)；Thr,1.0(1)；Val,1.1(1)；Ile,0.9(1)；Leu,7.6(8)；Lys,4.0(4)；

Trp,1.0(1).

Compound 61AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTAEIRA-OH (SEQ ID NO:75) physical data: m.p.155-210 ℃ [α] ²⁵ _D-46.15 (c0.12, H ₂O) FAB (C ₁₉₅H ₃₃₄N ₆₂O ₅₈): [M+H] ⁺4475.8AAA:Asx, 2.2 (2); Glx, 6.9 (7); Ser, 1.7 (2); His, 3.2 (3); Gly, 1.1 (1); Ala, 3.1 (3);

Arg,4.0(4)；Thr,0.9(1)；Val,1.1(1)；Ile,1.9(2)；Leu,8.1(8)；Lys,4.1(4).

Compound 62

AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTAEIR-OH (SEQ ID NO:76) physical data: m.p.186-218 ℃ [α] ²⁵ _D-52.73 (c0.265, H ₂O) FAB (C ₁₉₂H ₃₂₉N ₆₁O ₅₇): [M+H] ⁺4404.4AAA:Asx, 2.0 (2); Glx, 6.6 (7); Ser, 1.9 (2); His, 3.4 (3); Gly, 1.1 (1); Ala, 2.0 (2);

Arg,3.8(4)；Thr,1.0(1)；Val,1.1(1)；Ile,1.7(2)；Leu,7.9(8)；Lys,4.0(4).

Compound 63AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTAEI-OH (SEQ ID NO:77) physical data: m.p.169-205 ℃ [α] ²⁵ _D-50.78 (c0.51, H ₂O) FAB (C ₁₈₆H ₃₁₇N ₅₇O ₅₆): [M+H] ⁺4248.0AAA:Asx, 2.2 (2); Glx, 6.8 (7); Ser, 1.8 (2); His, 3.3 (3): Gly, 1.0 (1); Ala, 2.0 (2);

Arg,3.0(3)；Thr,1.0(1)；Val,1.0(1)；Ile,1.8(2)；Leu,7.8(8)；Lys,3.6(4).

Compound 64

AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTAE-OH (SEQ ID NO:78) physical data: m.p.199-205 ℃ [α] ²⁵ _D-52.47 (c0.41, H ₂O) FAB (C ₁₈₀H ₃₀₆N ₅₆O ₅₅): [M+H] ⁺4135.0AAA:Asx, 2.0 (2); Glx, 6.6 (7); Ser, 1.9 (2); His, 3.3 (3); Gly, 1.1 (1); Ala, 2.0 (2);

Arg,2.9(3)；Thr,1.0(1)；Val,1.1(1)；Ile,1.0(1)；Leu,8.2(8)；Lys,3.8(4).

Compound 65

AVSEIQFX ₁HN?KGKHLSSX ₁ER?VEWLRKKLQD?VHNX ₂(SEQ?ID?NO:79)

(X ₁=L-nor-leucine; X ₂=Kos oxygen acid lactone) physical data: m.p.166-176 ℃ [α] ²⁵ _D-52.22 (c0.25, H ₂O) FAB (C ₁₈₀H ₂₈₈N ₅₄O ₅₀): [M+H] ⁺4008.6AAA:Asx, 3.1 (3); Glx, 4.8 (5); Ser, 2.9 (3); His, 2.9 (3); Gly, 1.1 (1); Ala, 1.1 (1);

Arg,2.0(2)；Val,2.7(3)；Phe,1.0(1)；Ile,1.0(1)；Leu+Nle?5.9(4+2)；

Lys,2.8(3).

Compound 66

SEHQLLHD KGKSIQDLRR RELLEKLLEK LHTA-NH ₂(SEQ ID NO:80) physical data: m.p.134.2 ℃ [α] ²⁵ _D-48.12 (c0.36, H ₂O) FAB (C ₁₆₇H ₂₈₆N ₅₄O ₄₉): [M+H] ⁺3834.4AAA; Asx, 2.0 (2); Glx, 5.7 (6); Ser, 1.7 (2); His, 2.9 (3); Gly, 1.0 (1); Thr, 0.9 (1);

Ala,1.0(1)；Arg,2.8(3)；Ile,0.9(1)；Leu,7.4(8)；Lys,4.3(4).

Compound 67

LLHD KGKSIQDLRR RELLEKLLEK LHTA-NH ₂(SEQ ID NO:81) physical data: m.p.128.5-184.5 ℃ [α] ²⁵ _D-6.53 (c0.69, MeOH) FAB (C ₁₄₈H ₂₅₉N ₄₇O ₄₁): [M+H] ⁺3353AAA:Asx, 2.0 (2); Glx, 4.1 (4); Ser, 0.9 (1); His, 2.1 (2); Gly, 1.0 (1); Thr, 0.9 (1);

Ala,1.0(1)；Arg,3.0(3)；Ile,1.0(1)；Leu,8.1(8)；Lys,4.2(4).

Compound 68

LHD KGKSIQDLRR RELLEKLLEK LHTA-NH ₂(SEQ ID NO:82) physical data: m.p.165-210 ℃ [α] ²⁵ _D-36.05 (c0.12, H ₂O) FAB (C ₁₄₂H ₂₄₈N ₄₆O ₄₀): [M+H] ⁺3239.0AAA:Asx, 2.0 (2); Glx, 3.9 (4); Ser, 0.9 (1); His, 1.9 (2); Gly, 1.0 (1); Thr, 1.0 (1);

Ala,1.0(1)；Arg,2.9(3)；Ile,0.9(1)；Leu,6.8(7)；Lys,4.2(4).

Compound 69 SEHQLLHD RGRSIQDLRR RELLERLLER LHAGRRTRSAW-OH (SEQ ID NO:83) physical data: m.p.150-210 ℃ [α] ²⁵ _D-18.0 (c0.64, H ₂O) FAB (C ₂₀₈H ₃₅₁N ₇₉O ₆₀): [M+H] ⁺4918.6AAA:Asx, 2.2 (2); Glx, 6.2 (6); Ser, 2.8 (3); His, 3.1 (3); Gly, 2.2 (2); Thr, 2.2

(2)；Ala,2.2(2)；Arg,10.4(10)；Ile,1.0(1)；Leu,8.0(8)；Trp,1.1(1).

Compound 70

LLHDRGRSIQDLRR RELLERLLER LHAGRRT RSAW-OH (SEQ ID NO:84) physical data: m.p.150-210 ℃ [α] ²⁵ _D-41.70 (c0.36, H ₂O) FAB (C ₁₈₉H ₃₂₄N ₇₂O ₅₂): [M+H] ⁺4437.14AAA:Asx, 2.1 (2); Glx, 4.1 (4); Ser, 1.9 (2); His, 2.0 (2); Gly, 2.1 (2); Thr, 2.0 (2);

Ala,2.1(2)；Arg,9.7(10)；Ile,0.9(1)；Leu,7.4(8).

The embodiment II

Step preparation and purifying [Met according to the embodiment I ³⁴, Ala ³⁵] compound 1, AVSEHQLLHDKGKSIQDLRRRELLEK-LLEKLHTMA-NH ₂, (SEQ ID NO:25).And with the following method this polypeptide is transformed into homoserine lactone.The peptide (160 milligrams) of purifying is dissolved in 44% formic acid (4 milliliters).With this solution in 0 ℃ be pre-mixed, cyanogen bromide (700 milligrams) and phenol (1.6 milligrams) merges at the solution of 44% formic acid (4 milliliters).Solution stirred 2 hours at 0 ℃, at room temperature stirred then 2 hours.The formation of product HPLC (Vydac ^_C-18,300 dusts, 4.6 * 250mm, flow velocity 1.2mL/min, gradient is the 25-45% acetonitrile among the 0.1%TFA, 10 minutes) monitoring.Be reflected in 4 hours and finish.Half sample is concentrated and with preparation property RP-HPLC (Vydac ^_C-18, gradient is the 25-45% acetonitrile among the 0.1%TFA).Collect homoserine lactone peptide moiety and freeze-drying, obtain the white solid of 28 milligrams of purity＞95%, promptly compound 4.

Compound 4

AVSEHQLLHDKGKSIQDLRRRELLEK LLEKLHTX (X=hSerlac, SEQ ID NO:9) physical data: m.p.138-142 ℃ [α] ²⁵ _D-50.66 ° of (c0.1, H ₂O) FAB (C ₁₇₆H ₂₉₉N ₅₅O ₅₂): [M+H] ⁺4017.61AAA:Asp, 2.1 (2); Glu, 6.1 (6); Ser, 1.8 (2); His, 3.0 (3);

Thr,1.1(1)；Ala,1.1(1)；Arg,2.7(3)；Val,1.0(1)；Ile,

1.0(1)；Leu,8.2(8)；Lys,3.8(4)；Gly1.09(1)；hSer,

1.09(1).

The embodiment III

In order to prepare the homoserine acid amides, concentrate thick homoserine lactone (hSerlac) analog compounds 4, use 25 milliliters of saturated NH then ₃Methanol solution handle.Solution stirred 2 hours at 0 ℃, at room temperature stirred then 16 hours.Reaction HPLC (Vydac ^_C-18,300 dusts, 4.6 * 250mm, flow velocity 1.2mL/min, gradient is the 20-45% acetonitrile among the 0.1%TFA) monitoring, and in 18 hours, finish.Solution concentration is also used preparation property RP-HPLC (Vydac ^_C-18, gradient is the 20-45% acetonitrile among the 0.1%TFA) purifying.Collect homoserine amidated peptide part and freeze-drying, obtain the white solid of 30 milligrams of purity＞98%, promptly compound 3.

Compound 3AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTX-NH ₂(X=hSer, SEQ ID NO:8) physical data: m.p.138-142 ℃ [α] ²⁵ _D-45.97 ° of (c0.25, H ₂O) FAB (C ₁₇₆H ₃₀₂N ₅₆O ₅₂): [M+H] ⁺4033.9AAA:Asp, 2.1 (2); Glu, 6.1 (6); Ser, 1.6 (2); His, 2.8 (3);

Gly,0.97(1)；hSer,0.97(1)；Thi,1.0(1)；Ala,1.0(1)；

Arg,2.9(3)；Val,1.0(1)；Ile,1.0(1)；Leu,7.6(8)；Lys,

3.9(4).

The embodiment IV

According to the method for embodiment I, protected peptide-resin B ocAVS (Bzl) E (OBz) H (Bom) QLLHD (OBzl) R (Ts) GR (Ts) S (Bzl) IQD (OBz)-LR (Ts) R (Ts) E (OBz) LLE (OBzl) R (Ts) LLK (Fmoc) R (Ts) LH (Bom) T (Bzl) A-O-PAM about preparation 0.35mmol.All N ^aGroup is protected with tert-butoxycarbonyl (Boc); The side chain protected group marks.After end of synthesis, at room temperature handled peptide resin 30 minutes with the dimethyl formamide (DMF) of 50 milliliter of 20% piperidines, thereby remove fluorenyl methoxy carbonyl (Fmoc) blocking group on the Methionin.Use DMF subsequently successively, MeOH, and CH ₂Cl ₂Washing resin, and dry, obtain the peptide that 1.6 gram parts are protected.In 20 milliliters of DMF, in the presence of 0.16g (0.3mmol) phosphofluoric acid benzotriazole base-three (pyrrolidyl) microcosmic salt and 0.067g (0.525mmol) diisopropylethylamine (DIEA), with 0.44 gram (0.3mmol) methoxyl group two (vinyloxy group) acetate [PEG (2) CH ₂COOH] make the peptide of 0.8g (0.175mmol) part protection carry out acetylization reaction 5 hours at the Methionin place.After 5 hours, filter resin, use DMF, MeOH and CH then successively ₂Cl ₂Washing.Repeat acetylize step twice, until on resin, obtaining negative triketohydrindene hydrate result.With the cracking from the resin of final peptide, and as the embodiment I, remove the side chain protected group and carry out purifying.Obtain 100 milligrams of compounds 19.

Compound 19

Physical data: m.p.145-195 ℃ [α] ²⁵ _D-44.60 ° (c0.2,

H ₂O)FAB(C ₁₈₃H ₃₁₆N ₆₄O ₅₄):[M+H] ⁺4276.2AAA:Asp,2.1(2)；Glu,5.0(5)；Ser,1.6(2)；His,2.9(3)；Gly,0.9(1)；Thr,1.9

(2)；Arg,7.1(7)；Val,1.1(1)；Ile,1.0(1)；Leu,8.0(8)；Lys,0.9(1).

The embodiment V

Synthesize like that, cut and purified peptide by the embodiment IV, except using poly-(vinyloxy group) acetate [PEG (5000) CH of 2-methoxyl group ₂CO ₂H] as acetylizing agent.Peptide with the protection of 0.8g (0.175mmol) part obtains 300 milligrams of pure compounds 20.

Compound 20

Physical data: m.p.105 ℃ [α] ²⁵ _D-22.95 ° of (c0.1l, the 50%HOAc aqueous solution) AAA:Asp, 2.0 (2); Glu, 4.8 (5); Ser, 1.6 (2); His, 2.6 (3); Gly, 1.1 (1); Thr, 1.1

(1)；Arg,7.3(7)；Val,0.8(1)；Ile,0.9(1)；Leu,8.3(8)；Lys,1.1(1)；Ala,

1.8(2)．

The embodiment VI

Synthetic hPTHrp (1-34) analogue gene

The synthetic gene of design coding hPTHrp (1-34) analog compounds 4 (SEQ ID NO:9), it has as shown in Figure 1 nucleotide sequence and restriction enzyme site.Essential oligonucleotide dna synthesizer (Milligen/Biosearch), and adopt Sinha, etal., Nucleic Acid Research 12, phosphoramidate (phosphoramidite) the method preparation of 4539-4557 (1984).After going protection, thick oligonucleotide on preparation property 15% polyacrylamide gel by gel electrophoresis purifying.Position with the definite oligonucleotide of UV irradiation cuts out from gel with it then, desalination on Waters c18 Sep-pak_ post, then freeze-drying.

Amplified reaction by polymerase chain reaction is to carry out on Perkin-Elmer Cetus thermal cycler, condition is: 94 ℃ 1 minute, 50 ℃ of 2 minutes and 72 ℃ 3 minutes, " GeneAmp " DNA cloning test kit (Perkin-Elmer Cetus) that comprises the Taq polysaccharase is wherein used in totally 25 circulations.

The oligonucleotide of two kinds of overlappings of preparation, the PTH4 promptly PTH3 of 88 polymers (2 microgram) and antisense, 90 polymers (2 microgram) are as the template DNA sequence of hPTHrp (1-34) analogue gene.PTH3,(SEQ?ID?NO:31):CCTCTAGATC?TCCGCGGCGC?TAGC?ATG?GCT?GTT?TCT?GAA?CAT?CAG?45

Met?Ala?Val?Ser?Glu?His?Gln

1 5CTG?CTT?CAT?GAC?AAA?GGT?AAA?TCG?ATT?CAA?GAT?CTG?AGA?CGT?C

88Leu?Leu?His?Asp?Lys?Gly?Lys?Ser?Ile?Gln?Asp?Leu?Arg?Arg

10 15 20PTH4(SEQ?ID?NO:32):CCTCGAAGCT TATGCATCAT TATCTAGA?CAT?AGT?ATG?CAG?CTT?TTC?46

Met?Thr?His?Leu?Lys?Glu

30AAG?CAG?TTT?CTC?CAG?CAG?CTC?GCG?ACG?TCT?CAG?ATC?TTG?AAT?88Leu?Leu?Lys?Glu?Leu?Leu?Glu?Arg?Arg?Arg?Leu?Asp?Gln?Ile

25 20 15CG 90, adopt both sides primer PTHPCRl:CCTCTAGATC TCCGCGGCGC TAG (SEQ ID NO:33) and PTHPCR2:CCTCGAAGCT TATGCATCAT TATC (SEQ IDNO:34), by the whole gene of pcr amplification.The amplification DNA on the 4%NuSieve_ sepharose by the gel electrophoresis purifying.From gel, cut the band of hPTHrp (1-34) analogue gene that contains about 150 bases of synthetic, use Elu-Quik ^_(Schleicher﹠Schuell, Keene NH) isolate about 200ng DNA to glass gel DNA extraction process.

The embodiment VII

The molecular cloning of hPTHrp (1-34) 1 analogue gene

For hPTHrp (1-34) analogue gene of subclone embodiment VI, separate the DNA of 200ng amplification, carry out enzyme with restriction enzyme HinD III and Sac II then and cut.As shown in Figure 2, DNA is connected in TrpLE 18 Prot (Ile3, Pro5) plasmid cut with HinD III and Sac II enzyme of 2 micrograms.

Plasmid TrpLE 18 hPTHrp (1-34) 1 that produce contain hPTHrp (1-34) analogue gene of single copy, with this plasmid change over to active intestinal bacteria HB 101 cells (CLONTECH, Palo Alto, CA).Transformant through pcr analysis to confirm the insertion situation.Select transformed cell cloning, in 200 microliters of water, boiled 5 minutes; With two primers that are positioned at the insert both sides 2 microlitre samples are carried out PCR.On 1% sepharose, analyze the PCR product then to determine to exist hPTHrp (1-34) the gene insert of single copy.Then at automatic dna sequencer (Applied Biosystems Model 373A, Foster City, CA) on, use supplier's Dye Deoxy Terminator sequencing kit, confirm TrpLE 18 hPTHrp (1-34) 1 construction by dna sequencing.

The embodiment VIII makes up Trp LE 18 carriers that contain multiple copied hPTHrp (1-34) analogue gene

Near the beginning of hPTHrp (1-34) analogue gene sequence and end, have unique N he I and Xba I restriction site.The site that can use these two the different sequences of identification but produce identical strand sticky end is at Trp LE 18 vector construction multiple copied hPTHrp (1-34) genes.

The scheme of in Fig. 3, having summarized multiple hPTHrp (1-34) sequence that makes up the tandem arrangement.In each reaction, digest Trp LE 18 hPTHrp (1-34) 1 plasmid that 5 micrograms contain single copy gene with Bam H I+Nhe I and Xba I+Bam H I.From each digest, isolate the fragment that about 300ng contains hPTHrp (1-34) analogue gene.Mix two kinds of fragments and connection, thereby form Trp LE 18 hPTHrp (1-34) 2 plasmids.With these plasmid activity of conversion intestinal bacteria HB 101 cells.Utilize the size of PCR product on 1% sepharose that transforms to determine to exist hPTHrp (1-34) the gene insert of two copies.Confirm to contain Tr-pLE 18 hPTHrp (1-34) 2 of two copy genes then by dna sequencing.Nhe I and Xba I site are eliminated in the correct fusion meeting of two hPTHrp (1-34) gene in the joint.This makes Xba I and Nhe I be still unique in the gene both sides that tandem is arranged.

By repeating this program, make up final plasmid Trp LE 18 hPTHrp (1-34) 4 that contain 4 copy hPTHrp (1-34) genes, as shown in Figure 4.Find that by dna sequence analysis Trp LE 18 hPTHrp (1-34) 4 sequences are correct.

The embodiment IX

Expression and the purifying of Trp LE 18 hPTHrp (1-34) 4

Induce Trp LE 18 hPTHrp (1-34) 4

50 milliliters of LB substratum starting culture (J.H.Miller that will contain 50 mcg/ml penbritins and 100 mcg/ml tryptophanes, Experiments in Molecu-lar Genetics, " p.431 (1972); be incorporated herein by reference) inoculate with the Bacillus coli cells that contains Trp LE 18hPTHrp (1-34) 4 plasmids; acutely shake grow overnight at 37 ℃ then, to A ₅₅₀Be about 6.2 liters of LB substratum that are used to produce are preheated to 37 ℃, inoculate the A that obtains then with 20 milliliters of starting cultures ₅₅₀Be about 0.06.Culture grows to A under acutely shaking then ₅₅₀Be about 0.6-0.8, add 2 milliliters of solution that contain 10mg/mL indole acrylic acid (IAA).Continuation is grown 16 hours until whole A under the good condition of ventilation ₅₅₀Be about 6 (typically, being 4-10).By centrifugal concentrating cells, and then be suspended in 500mL 50mM Tris-HCl, pH7.5 is in the 0.1mM edta buffer liquid (Tris damping fluid).

Suspension Heat Systems-Ultrasonics, and the 220F type ultrasonoscope of Inc. (be equipped with 3/4 " loud speaker), carry out ultrasonication under (for fear of overheated) at full capacity 50%.

In order to determine the inductive degree, analyze whole cell with SDS-PAGE.Observe from TrpLE 18 hPTHrp (1-34) 4 constructions derive and gene product for estimating the main band of about 17,000 Dalton molecular weights.This band is corresponding to about 10% total cellular protein.

Divide isolated fusion protein

Eccentric cell I lysate is 15 minutes under about 3600 * g, makes Trp LE 18hPTHrp (1-34) 4 fusion roteins deposition; Abandoning supernatant.Settling is suspended in 100 milliliters of Tris damping fluids again (typically is 40-80A ₅₅₀/ mL)

The embodiment X

The purifying of the processing of fusion rotein and homoserine lactone hPTHrp (1-34) peptide

The methionine(Met) that will be positioned at hPTHrp (1-34) polymer fusion rotein one side with CNBr downcuts, and discharges required homoserine lactone hPTHrp (1-34) polypeptide, carries out purifying by following method then.Handle fusion rotein with CNBr

Washed TrpLE 18 hPTHrp (1-34) 4 fusion rotein settlings are suspended in (about 20mg/mL total protein in 60mL 70% formic acid again by stirring lightly; Typically will be from 1000 A ₅₀₀The raw material of unit cell is dissolved in 3 milliliters).Add several octanols, use N ₂Purge solution 20 minutes adds 5.5g CNBr then.Then, in 25 ℃ of reactions 6 hours, with isopyknic 50: 50 MeOH: H ₂O and sample mix are removed by rotary evaporation subsequently.Repeat this process 2-4 time, formic acid and CNBr are removed basically.Evaporate sample then to dry, be dissolved in again in 200 ml waters, freeze-drying stores.The purifying of homoserine lactone hPTHrp (1-34)

With CNBr cracked supernatant liquor at the 50mM KH that changes to some extent ₂PO ₄Dialysis is 24 hours among the pH6.5.In dialysis, pH maintains 6.5.After the dialysis, the high speed centrifugation disgorging.Supernatant liquor is by Gelman0.45 μ filtration unit (Acrodisc 4184) clarificationization.Cation-exchange chromatography

Initial purifying is by (cation-exchange chromatography on 21.5 * 150mm) is finished at Bio-Gel TSK-SP-5PW HPLC post.Corresponding to flow velocity is that the chromatographic condition of 8mL/min and about 12 milligrams of high purity homoserine lactone hPTHrp (1-34) peptide output is as follows:

1. use 50mM KH ₂PO ₄, pH 6.5 balance columns

2. adorn the clarifying supernatant liquor of 10mL (about 1.5 liters of nutrient solutions or 2.4g inclusion body).

3. with the 50mM KH that contains 50mM NaCl ₂PO ₄, the pH6.5 washing column is until baseline stability

4. with the 50mM KH that contains 90mM NaCl ₂PO ₄, pH6.5 wash-out post.Collected cut about 45 minutes.

5. analyze homoserine lactone hPTHrp (1-34) content in the 90mM NaCl cut with C18 HPLC, collect storage then.The reverse hplc chromatogram

(Perseptive Biosystems, Cambridge MA) carry out last purification step with anti-phase Poros R/H 4.6 * 100 mm posts.This chromatographic condition is as follows: mobile phase A: 0.1% trifluoroacetic acid (TFA)/water

B:0.1% trifluoroacetic acid (TFA)/CH ₃CN

Time flow velocity %B

0min 4ml/min 15

5.0min 4ml/min 40

5.2min 4ml/min 100

6.8min 4ml/min 100

7.0min 4ml/min 15

Homoserine lactone hPTHrp (1-34), the retention time of compound 4 is about 2.943 minutes.The peptide of purifying is about 98% with mass spectrometric determination purity.

The embodiment XI

On ovariectomized rat, substantially according to Gunness-Hey and Hock, the method for Metab.Bone Dis.5:177 181 (1984) is assessed the effect of compound of the present invention to sclerotin.

The Sprague-Dawley female rats of growing up is conformed, and both sides oophorectomize (OVX) or sham operated are carried out in grouping (n=9,10 or 12) then by weight.In operation administration in back 17 days, continue 20 days.Every day is with 2% mouse serum/brine media subcutaneous administration test compounds.

After 20 days, kill mouse, take out right femur.Femur is cut in half, and half femur (DHF) of far-end further is divided into trabecular bone (TB) and osteoderm (CB) by getting out girder.Extract calcium, measure by Calcette calcium analyser, represent with average bone calcium, unit is the mg/DHF/100g body weight.

Use two sample t-to check comparison OVX and simulation group.Compare the OVX group with unidirectional ANOVA, the LSD multiple comparisons method with Fisher compares each group with medium subsequently.

The oophorectomize meeting causes serious total deossification, mainly is the deossification of trabecular bone.Compare total calcareous low 47-54% of bone with the control group of sham operated.

Show that by statistics 80 micrograms/kg/ days bPTH (1-34) and hPTHrp (1-34) can make the calcareous obvious increase of total bone of the OVX mouse of handling, are respectively 53-95% and 18-40%; Obviously do not increase but osteoderm is calcareous.

Compound of the present invention during by administration in 80 micrograms/kg/ days, can increase the calcareous about 66-138% of total bone, increases the calcareous 87-128% of trabecular bone.For untreated OVX control group, calcareous, little cantilever thickness of osteoderm and bone volume also obviously increase.

In this is analyzed, tested following compounds: (% increases the calcareous numbering of the calcareous total bone of compound n trabecular bone (test number #), OVX) (% increases, OVX) compound 16 101-128% 88-138% compounds 23 87-102% 66-114% compounds 4 3-88-114%

In similarly studying, press and gave ovariectomized rat administration 5 in 40 micrograms/kg/ days, 10 and 20 days, obtain following result: (d) (% increases the calcareous numbering of the total bone of compound n administration fate (test number #), OVX) compound 13 20d 73-109% compounds 45 20d 79-105% compounds 41 10d 79% compound 49 1 10d 93% compound 41 5d 55% compound 42 1 5d 60%

The embodiment XII

Toxicity

In the superincumbent embodiment XI, do not observe toxic action for compound of the present invention.

Claims

1. a Rat parathyroid hormone 1-34 PTH, parathyroid hormone-related peptide PTHrp and the short homologue of PTH and PTHrp and the synthetic polypeptide analog of analogue, or its salt, it is characterized in that, the amino acid of PTH and PTHrp (22-31) forms amphipathic alpha-helix, and the sequence of this residue (22-31) is selected from:

A) Xaa ¹Xaa ²Leu Xaa ⁴Xaa ⁵Leu Xaa ⁷Xaa ⁸Xaa ⁹Xaa ¹⁰Wherein

1 5 10

Xaa ¹And Xaa ⁴Be Glu independently of one another, Glu (OCH ₃), His, or Phe; Xaa ²Be Leu or Phe; Xaa ⁵Be Lys or His; Xaa ⁷And Xaa ¹⁰Be Leu or Ile independently of one another; Xaa ⁸Be Ala, Arg, or Glu; And Xaa ⁹Be Lys or Glu (SEQ IDNO:26); Condition is that following situation can not occur simultaneously:

Xaa ¹And Xaa ⁴All be Glu;

Xaa ²Be Leu;

Xaa ⁵Be Lys;

Xaa ⁷And Xaa ¹⁰All be Leu;

Xaa ⁸Be Arg or Glu; With

Xaa ⁹Be Lys, perhaps

B) Xaa ¹Xaa ²Leu Xaa ⁴Arg Leu Leu Xaa ⁸Arg Leu wherein

1 5 10

Xaa ¹And Xaa ⁴Be Glu independently of one another, Glu (OCH3), His, or Phe; Xaa ²Be Leu or Phe; Xaa ⁸Be Glu, Lys, or Lys (COCH ₂PEGX) and PEGX be that molecular weight is 100-10, poly-(Ethylene Glycol Methyl ether) residue (SEQ ID NO:27) of 000; Condition is that following situation can not occur simultaneously:

Xaa ¹And Xaa ⁴All be Glu;

Xaa ²Be Leu.

2. polypeptide as claimed in claim 1 and pharmacy acceptable salt thereof is characterized in that it has following formula:

Xaa ¹?Xaa ²?Xaa ³?Xaa ⁴?Xaa ⁵?Xaa ⁶?Xaa ⁷?Leu?His?Xaa ¹⁰?Xaa ¹¹?GlyXaa ¹³?Ser?Ile?Gln?Xaa ¹⁷?Leu?Xaa ¹⁹?Xaa ²⁰?Xaa ²¹?Xaa ^22-31?Xaa ³²?Xaa ³³Xaa ³⁴?Xaa ³⁵?Xaa ³⁶?Xaa ³⁷?Xaa ³⁸?Term,

Wherein:

Xaa ¹Disappearance or Ala;

Xaa ²Disappearance or Val;

Xaa ³Disappearance or Ser;

Xaa ⁴Disappearance or Glu or Glu (OCH ₃);

Xaa ⁵Disappearance or His or Ala;

Xaa ⁶Disappearance or Gln;

Xaa ⁷Disappearance or Leu;

Xaa ¹⁰And Xaa ¹⁷Be Asp or Asp (OCH independently of one another ₃);

Xaa ¹¹Be Lys, Arg, or Leu;

Xaa ¹³Be Lys, Arg, Tyr, Cys, Leu, Cys (CH ₂CONH (CH ₂) ₂NH (biotinyl)), Lys (7-dimethylamino-2-oxygen-2H-1-chromene-4-ethanoyl), or Lys (dihydro cinnamon acyl group);

Xaa ²⁰Be Arg or Leu;

Xaa ¹⁹And Xaa ²¹Be Lys independently of one another, Ala, or Arg;

Xaa ^22-31As defined in claim 1;

Xaa ³²Be His, Pro, or Lys;

Xaa ³³Disappearance, or Pro, Thr, Glu, or Ala;

Xaa ³⁴Disappearance, or Pro, Arg, Met, Ala, hSer, the hSer lactone, Tyr, Leu, or 1,4-diamino butyryl lactan;

Xaa ³⁵Disappearance or Pro, Glu, Ser, Ala, or Gly;

Xaa ³⁶Disappearance or Ala, Arg, or Ile;

Xaa ³⁷Disappearance or Arg, Trp, or 3-(2-naphthyl)-L-Ala;

Xaa ³⁸Disappearance or Ala or hSer or Xaa ^38-42Be Thr Arg Ser Ala Trp;

With Term be OR or NR ₂, wherein each R is H independently of one another, (C ₁-C ₄) alkyl or phenyl (C ₁-C ₄) alkyl.

3. polypeptide as claimed in claim 2 is characterized in that Xaa ^22-31Be sequence Xaa a) and wherein in the claim 1 ¹¹And Xaa ¹³All be Lys; Xaa ¹⁹And Xaa ²¹All be Arg.

4. polypeptide as claimed in claim 3, it is characterized in that it is Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile GlnAsp Leu Arg Arg Arg Phe Phe Leu Glu Lys Leu Leu Glu Lys Leu HisThr Ala NH ₂(compound 50).

5. polypeptide as claimed in claim 3, it is characterized in that it is Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile GlnAsp Leu Arg Arg Arg Glu Leu Leu His Lys Leu Leu Glu Lys Leu HisThr Ala NH ₂(compound 51).

6. polypeptide as claimed in claim 3, it is characterized in that it is Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile GlnAsp Leu Arg Arg Arg Glu Leu Leu Glu His Leu Leu Glu Lys Leu HisThr Ala NH ₂(compound 52).

7. polypeptide as claimed in claim 3, it is characterized in that it is Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile GlnAsp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Ile Ala Lys Leu HisThr Ala NH ₂(compound 53).

8. polypeptide as claimed in claim 3, it is characterized in that it is Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile GlnAsp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Glu Ile HisThr Ala NH ₂(compound 54).

9. a pharmaceutical composition is characterized in that, it contains polypeptide as claimed in claim 1 or its salt and the pharmaceutically acceptable carrier that hyperostosis adds significant quantity.

10. a PTH, PTHrp or the short homologue of PTH and PTHrp and the polypeptide analog of analogue, or the solid phase synthesis process of its salt, wherein the amino-acid residue of PTH or PTHrp (22-31) is modified and is formed amphipathic alpha-helix, and this residue (22-31) is selected from:

A) Xaa ¹Xaa ²Leu Xaa ⁴Xaa ⁵Leu Xaa ⁷Xaa ⁸Xaa ⁹Xaa ¹⁰Wherein

1 5 10

Xaa ¹And Xaa ⁴All be Glu;

Xaa ²Be Leu;

Xaa ⁵Be Lys;

Xaa ⁷And Xaa ¹⁰All be Leu;

Xaa ⁸Be Arg or Glu; With

Xaa ⁹Be Lys, perhaps

B) Xaa ¹Xaa ²Leu Xaa ⁴Arg Leu Leu Xaa ⁸Arg Leu wherein

1 5 10

Xaa ¹And Xaa ⁴Be Glu independently of one another, Glu (OCH ₃), His, or Phe; Xaa ²Be Leu or Phe; Xaa ⁸Be Glu, Lys, or Lys (COCH ₂PEGX) and PEGX be that molecular weight is 100-10, poly-(Ethylene Glycol Methyl ether) residue (SEQ ID NO:27) of 000; Condition is that following situation can not occur simultaneously:

Xaa ¹And Xaa ⁴All be Glu;

Xaa ²Be Leu,

This method comprises in order that protected amino acid is coupled in the appropriate resin carrier, removes side chain and N ^α-blocking group, and with polypeptide cracking from the resin carrier.

11. a PTH, PTHrp or the short homologue of PTH and PTHrp and the polypeptide analog of analogue, or the preparation method of its salt, wherein the amino-acid residue of PTH or PTHrp (22-31) is modified the amphipathic alpha-helix of formation, and this residue (22-31) is selected from:

A) Xaa ¹Xaa ²Leu Xaa ⁴Xaa ⁵Leu Xaa ⁷Xaa ⁸Xaa ⁹Xaa ¹⁰Wherein

1 5 10

Xaa ¹And Xaa ⁴All be Glu;

Xaa ²Be Leu;

Xaa ⁵Be Lys;

Xaa ⁷And Xaa ¹⁰All be Leu;

Xaa ⁸Be Arg or Glu; With

Xaa ⁹Be Lys, perhaps

B) Xaa ¹Xaa ²Leu Xaa ⁴Arg Leu Leu Xaa ⁸Arg Leu wherein

1 5 10

Xaa ¹And Xaa ⁴All be Glu;

Xaa ²Be Leu,

This method comprises: the gene of expressing this polypeptide of coding.

12. the purposes of a compound as claimed in claim 1 or its pharmacy acceptable salt is characterized in that, it is used to prepare treatment Mammals sclerotin and reduces situation, especially treats the medicament of osteoporosis.