CN1085911A - Peptide compounds - Google Patents

Abstract

The invention discloses two peptide compounds, wherein two peptide chains are connected to the C of non-end amino acid by divalent abutment group-A- ₂On the atom, the C alpha atom that is connected with group-A-all is in equivalent position on each peptide chain, and each peptide chain all lacks their primary α one side chains.Base closes-A-such as claim 1 qualification.Disclosed mixing connects two peptide compounds and has irritation cell splitted activity, and especially having stimulates medullary cell to generate activity with medullary cell.

Description

Peptide compounds

The present invention relates to the application that on cell proliferation has the peptide of promoter action, also relate to novel peptide compound with special and/or general cell promoter action.

The cell of mammalian body has very big difference aspect 26S Proteasome Structure and Function, and the developmental mechanism of cytodifferentiation has become the centrostigma of many research work.People know that for the cell system with continuous renewal, its mechanism is usually directed to the storage master of multipotential stem cell, and this stem cell division also continues to provide new cell to this system.From the stem cell of " storage is main ",, become one or another kind of form very soon, and then develop into desired functioning cell although when beginning, be homologous.

Hemopoietic system in the marrow and epithelial lining and epidermis system are the examples of this stem cell system.

The operation of stem cell division or be controlled at and have very big potentiality in the treatment, and many research work continue to be devoted to explain related mechanism and the chemical messenger that works.Up to the present, by in cell generation and atomization, carrying out one section stimulation or suppressing to identify several biomolecules that in this process, have effect.Once studying medullary cell in this respect particularly well generates, the molecule that relates in its control comprises: colony stimulating factor (CSF), as granulocyte colony stimulating factor (G-CSF), scavenger cell colony stimulating factor (M-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), multispectral is colony stimulating factor (Multi-CSF; IL-3) [referring to Metcalf, Science 229:16(1985)], interior leukin (IL-11) is [referring to people such as Paul, Proc.Natl.Acad.Sci.USA87:7521(1990)], lactoferrin is [referring to people such as Broxmeyer, Blood Cells11:429(1986)], prostaglandin(PG) is [referring to people such as Pelens, Immunol.140:479(1988)], acid (H-subunit) ferritin is [referring to people such as Broxmeyer, Blood 68:1257(1986)], Interferon, rabbit (α, β and γ) [referring to people such as Pelus, people such as the same and Broxmeyer, J.Immunol.131:1300(1983)], tumor lethal factor (α and β) [referring to people such as Broxmeyer, J.Immunol.136:4487(1986)], transforming growth factor-beta is [referring to people such as Ottman, and Nrolone Phenylpropionate and statin [referring to people such as Broxmeyer, Proc.Natl.Acad.Sci.USA86:779(1989)] J.Immunol.140:2661(1988)].

Also find, blood is regulated pentapeptide (PEEDCK) and is optionally suppressed the propagation of marrow founder cell (referring to people such as Paukovits, Z.Naturforsch 37:1297(1982)], people find that also other peptides with very narrow chemical general formula have similar restraining effect (referring to EP-A-112656 and WO 90/02753) in hematopoiesis.The oxidation of this peptide monomer has caused the dimer molecule that connect by the halfcystine bridging, it is found that these dimer molecules stimulate marrow to generate [referring to people such as Laerum, Exp.Hematol 16:274(1988)].WO-A-88/03535 discloses (pEEDCK) ₂Dimer and other similar compounds.EP-A-408371 discloses other dimer peptide compounds, and wherein disulfide linkage is connected carbon or carbon/sulphur bridge replacement of selected peptide chain.Therefore, this bridge is a quite stable to hydrolysis, but itself is an inert, can not participate in acceptor-dimeric interaction.

Although we do not wish to be bound by theory, we think that this peptide compounds and the interior stroma cell of body interact now, and by other solvable factors, this stroma cell is responsible for stimulating or suppressing the division of cell.When monomeric peptide can suppress fission process or causes producing and prevent or when stoping the fissional factor, can think that then dimer induces or promote the matrix of pungency cell regulating factor to produce.Therefore, according to present opinion, stroma cell can play stimulation or the restraining effect that strengthens dimer and monomeric peptide respectively.

Irritation cell hyperplasia to the dimerization peptide compounds of level of significance is still that people need to continue in vivo.In this respect, be noted that stimulation in various degree some clinical setting is compared other situation may be more suitable, especially, be particularly important to the selective stimulating of individual cells type.

The invention provides a kind of peptide compounds, it contains two strand blood and regulates (for example suppressing hematopoiesis) peptide, this peptide is coupled together by a kind of abutment group, and this abutment group endways with each described peptide in be arranged in the non-end amino acid at equivalent position place the C alpha atom link to each other, no original α in each peptide-side chain exists, described abutment group is a kind of divalent group-A-, and wherein A is

In the formula

Each y is 0,1 or 2 independently;

Each z is 0 or 1 independently;

Each Y is O, S or NR independently,

Wherein, R represent hydrogen or with C successive organic group (as alkyl, aralkyl or aryl);

B represents carbocyclic ring or heterocycle (as 5 or 6 yuan of aromatic rings), and its heterocycle optionally contains one or two heteroatoms (for example oxygen, nitrogen or sulphur) and optionally by-OR ^A,-NR ^AR ^A,-COOR ^AOr the heterocycle that the halogen atom such as iodine, chlorine, fluorine or bromine atoms carries out once, secondary or three times replace;

Each R ^ARepresent hydrogen atom, alkyl, alkanoyl, alkoxyalkyl independently, but each group hydroxylation also wherein.

Group B is by above-mentioned arbitrary group secondary or three replacements, and this moment, every kind of substituting group needn't identically maybe needn't have identical type with other.

During radicals R is with carbon is connected organic group, it preferably contains 1-10 carbon atom, an especially 1-6 carbon atom.Alkyl can be a straight or branched, and can be by aryl (promptly generating aralkyl), alkoxyl group, hydroxyl, acyloxy, amino, amido or the carboxyl substituted of 6-10 carbon atom.Aryl comprises having one or more heteroatomic five or the hexa-member heterocycle aryl, and heteroatoms is selected from O, N or S, as furyl, imidazolyl, pyrryl, pyridyl and thienyl.Substituting group on aryl comprises C _1-6Alkyl, hydroxyl and carboxyl.Example comprises methyl, ethyl, propyl group, the tertiary butyl, amyl group, propyloic and phenmethyl.

R ^ABase preferably contains 1-6 carbon atom, an especially 1-4 carbon atom, at this moment R ^ARepresent alkyl,, alkanoyl or alkoxyalkyl.

At one preferably in the embodiment, divalent abutment group-A-is-CH ₂CH ₂BzCH ₂CH ₂-, wherein Bz represents phenyl ring, and it is randomly by-OR ^A,-NR ^AR ^A,-COOR ^AGroup or once replace or replace R for two, three times such as iodine, chlorine, fluorine or bromine atoms ^AAs above limit.

Any strand peptide with blood regulating effect all is suitable for doing the peptide of bridging of the present invention.

In other words, the invention provides some compounds like this, comprise peptide chain with following chemical formula at the Hemoregulatory peptides chain described in this compound:

R wherein ^aRepresentative

N and m represent 0 or 1 independently in the formula;

P, q, r represent 1 or 2 independently;

S represents 3 or 4;

R ¹And R ²The both is hydrogen atom or represents the oxo group together;

R ³And R ⁴The both is hydrogen atom or represents C-C together;

R ⁵Be hydrogen or acyl group;

R ⁶And R ⁷Respectively representation hydroxy or amino independently, and hydroxyl preferably;

R ⁸Represent hydrogen; C _2-6Alkyl; C _7-20Aralkyl, the one or more hydroxyls of its portability, amino or methoxyl group substituting group; Or in metabolism unsettled S-protecting group;

R ⁹Represent hydrogen or methyl;

R ¹⁰Representation hydroxy or amino, the residue of amino acid glutamine or the unitary peptide of tool N-end glutamine.

All described amino-acid residues can be D type or L type.Yet L type amino acid is more desirable.

When having N-end protecting group to exist, just as noted earlier, this base may be the acyl group with 1-20 carbon atom; the lower alkane acyl group that for example has 1-5 carbon atom; as ethanoyl, or have the aroyl or the aralkanoyl of 7-20 carbon atom, as benzoyl or phenylacetyl.

R ⁵Also can be the acyl group of deriving out by amino acid or peptide chain, particularly, R ⁵Can be by Serine deutero-acyl group, or by removing the acyl group that N continuous-terminal amino acid is obtained by any peptide that following aminoacid sequence produced:

Lys-Ile-Ile-His-Glu-Asp-Gly-Tyr-Ser

The terminal amino group of the whole peptide of chemistry formula I is preferably by carrying out acidylate with alkanoyl, aralkanoyl or aroyl and being protected.

R ⁸Be C _2-6During alkyl, it can be for example ethyl, butyl or hexyl.Work as R ⁸When being aralkyl, it can be an arylmethyl easily, as phenmethyl, diphenyl-methyl or trityl.Work as R ⁸Be in the metabolism during unsettled group, it can be the arylthio that for example has 5-10 carbon atom, as the pyridine sulfenyl, or the acyl group as limiting previously.

Compound of the present invention pentapeptide preferably in each chain, i.e. n O preferably.

R ⁹Preferably five yuan of cyclic groups in the residue, i.e. m O preferably.

Regulate under the active low or inappreciable situation at the blood of any peptide that limits by above-mentioned chemical formula I, in any case under bridge form of the present invention their equal activated cell propagation effectively.

In the dimerization peptide that is derivatized to by the peptide of describing as the chemical formula I, the bridging point of suitable chain is at R ^dThe place.

The desirable especially peptide compounds of the present invention is those compounds with chemical formula II.

R in the formula ^a, R ^b, R ^c, R ^e, R ^f, A and n define as the front ,-NH-CH-CO-base is R ^dDerivative form, this R ^dBeing connected to last consequently its original side chain of abutment-A-with a kind of like this form has not existed.

A kind of desirable especially peptide compounds of chemical formula II is

Wherein-A-is a divalent abutment group as discussed earlier.

The present invention especially is applied to stimulate medullary cell to generate on one's body the patient who suffers from the active reduction of myelopoiesis one-tenth, comprising bone marrow injury, and agranulocytosis and aplastic anemia.This comprises owing to carrying out the patient that immunosuppressant therapy (as in the bone marrow transplantation surgical operation) has a reduced bone marrow function and treat suppressing tissue reaction.

These compounds also can be used for tumour and virus disease are suppressed to promote marrow to regenerate faster after the chemotherapy of cell and the radiotherapy.

In addition, for owing to the marrow fault lacks immune response, thereby cause the patient of severe infections, these novel cpds can have special value.

Another clinical application be with EP-A-112656 or WO-A-90/02753 in disclosed corresponding monomer or similarly medullary cell generate and suppress reagent and combine and induce that high reactivity alternately occurs with the low activity peak in the medullary cell, thereby the natural circadian rhythm of increase hematopoiesis.By this way, can make the cell suppression therapy during low marrow activity, therefore reduce the danger of bone marrow injury, regenerative process is owing to the active peak that continues is excited simultaneously.

Usually, in order to apply hormesis, peptide of the present invention is can be by patient oral or to its injection, its dosage is every 70Kg body weight 0.001-100mg every day, as 1-5mg.If intravenous injection or subcutaneous injection medication, then dosage can be every 70Kg body weight 1-10mg every day, for example about 6mg, maximum ten days.Nose, part (seeing through skin) or rectal application also are feasible certainly.Reaching peptide concentration suitably in the body fluid of patient extracellular in principle is about 10 ^-13M-10 ^-15M.

According to further aspect of the present invention, it provides pharmaceutical composition, and said composition comprises one or more peptide compounds of the present invention as active ingredient, particularly has those compounds of above-mentioned chemical formula I, or it is at the salt of physical compatibility, and is equipped with pharmaceutical carrier or vehicle.Composition of the present invention can be suitable form as oral, nose medication, non-enteron aisle or rectal application.

Term used herein " medicine " comprises the application of the present invention aspect the animal doctor.

The compounds of this invention can be common medicament forms, as tablet, coated tablet, nasal spray, solution, emulsion, powder, capsule or slowly-releasing form.Can use common drug excipient and common production method when preparing the medicine of these forms.For example, a kind of active ingredient or various active component can produce tablet: thinner for example by being mixed with following known excipients, as lime carbonate, calcium phosphate or calcium lactate, disintegrating agent such as W-Gum or alginic acid can also add binding agent therein, as starch or gelatin, lubricant, as Magnesium Stearate or talcum powder, and/or sustained release dosage, as carboxyl polymethylene, carboxymethyl cellulose, acetate phosphorus benzene bis-acid potassium Mierocrystalline cellulose or polyvinylacetate.

If need, tablet can be made up of which floor.By to adopting tablet coating preparation commonly used, carry out coated production coated tablet as polyvinylpyrrolidone or shellac, gum arabic, talcum powder, titanium dioxide or sugar with the nuclear that obtains with method like the tablet class.For obtaining slowly-releasing or avoid incompatible, its nuclear also can be made up of which floor.The tablet coating also can be made up of which floor, so that obtain slowly-releasing, in this case, can use the front with regard to the said vehicle of tablet.

Also can use organ specific support system.

For example can produce injection liquid in a usual manner, for example add sanitas, as the P-hydroxy benzoate, or stablizer, as EDTA, in then solution is packed into injection phial or the ampoule.

Nasal spray can be prepared in the aqueous solution similarly, and puts in the ejecting container that aerosol propellant or outfit manual pressure device are arranged.Can produce the capsule that one or more active ingredients are housed, for example active ingredient be mixed with inert support (as lactic acid type or Sorbitol Powder), in the gelatine capsule of again mixture being packed into.

Can produce suitable suppository, as activity combination or active ingredient composition are mixed with the carrier that is usually used in this purpose, as mixing with natural fat or polyoxyethylene glycol or derivatives thereof.

The dose unit that contains The compounds of this invention is 0.1-10mg preferably, as 1-5mg chemistry formula I peptide or its salt.

The another one feature according to the present invention, it provides a kind of irritation cell splitted method, the method that especially stimulates medullary cell to generate, this method comprises the pharmaceutical composition that limits previously from significant quantity to medication person that use.

Yet the another one important application of this novel peptide compound is to produce the material of using for the immunity test technology.This peptide can with suitable polymer carrier (as albumin, polylysine or polyproline) covalent attachment so that can inject the animal (for example rabbit, Guinea's ester or goat) of generation antibody.Also can use external immunological technique.Use polymer carrier, use the antiserum(antisera) that the absorption techniques of knowing can obtain high specific.By with radioactivity ( ³H, ¹²⁵I, ¹⁴C, ³⁵S) introduce in this peptide molecule, can design radioimmunoassay, and be used for measuring different biological fluids, as the peptide in serum (blood plasma), urine and the celiolymph.

Peptide of the present invention can be synthetic with any method easily.For example narrated the unitary proper method of formation amino acid in " Synthesis of optically Active α-Amino Acids " (Robert M.Williams, Pergamon Press, 1989).Usually; during the coupled reaction in total synthesizing; being present in reaction side-chain radical in the peptide (amino, sulfenyl and/or carboxyl) will be protected, but may not be protected (amide group in hydroxyl, imidazolyl, primary amide base, the cyclic amino acids at similar pyroGlu) staying some side-chain radicals between whole synthesis phase.

Therefore, final step will be that the peptide derivant of the formula I of protecting fully or partly protecting is removed protection, and this process all constitutes another aspect of the present invention.

People such as Schoellkopf once described by two lactim ethers are carried out metal replace and then carry out alkylation prepare various amino acid (referring to, as Tetrahedron 39:2085(1983) and Topics Curr.Chem.109:65(1983)).Proved that adopting this method is useful especially to the bridge amino acid that preparation constitutes basis of the present invention.Particularly, the two-lactim ether that is derived by the Xie Ansuan glycine dipeptidase has formed the useful initial compounds of bridging reaction, and this bridging reaction can be summarized as follows:

(wherein X is the group that stays, and halogen atom for example is as bromine).

If when beginning uses the D-Xie Ansuan to generate two-lactim ether, then by this method can prepare bridging (S, S)-α, α '-diamino acid.Similarly, use the L-Xie Ansuan can generate bridging (R, R)-α, α '-diamino acid.

We also find, can prepare aryl (comprising heteroaryl) Diglycocol derivative by corresponding two-(halogen carbonyl methyl) aryl compounds.Described two (halogen carbonyl methyl) aryl compound can generate bmap acid carboxylic imines (carboximide enolate) with the chiral oxazolidinone reaction, form reagent with nitrine then, as 2,4, the reaction of 6-tri isopropyl benzenesulfonyl trinitride is to introduce azido-, then with carrying out hydrogenolysis to generate needed Diglycocol as 10% palladium/coke.

Our method is to adopt people such as Evans at J.Am.Chem.Soc.109,6881(1987) and J.Am.Chem.Soc.111,1063(1987) processing method of the description in.In addition, can find the electrophilic amino nitrogen that suits in the azodicarboxylate, in this case, corresponding two hydrazine acid are that hydrogenolysis becomes amino acid whose intermediate.

This processing method is illustrated by following response diagram.

Therefore, the present invention also provides the method for producing peptide compounds, and this method comprises makes partially or completely that the derivative of protected peptide compounds goes protection.

In addition, the present invention also provides the method for producing peptide compounds, and described method comprises:

Thereby a) will two-lactim ether carrying out metal replaces alkylation then and generates two-lactim dipeptides ether;

B) two-lactim two amidogen ether hydrolysis of step (a) are generated the α of bridging, α '-diamino acid;

C) in peptide chain, introduce remaining amino acid;

D) any shielded group is removed protection.

Two-lactim dipeptides the ether made by this technology and the sour α of bridging, α ' diamino acid constitutes another aspect of the present invention.

In case the dipeptides of bridging generates, so available routine techniques adds all the other amino acid in the peptide chain.

When setting up peptide chain, people can be to adopt usually although C-end initial step is only arranged in C-end or the beginning of N-end in principle.

Therefore, people can be at the C-end by (as the glycine) derivative of due care and the cysteine derivative reaction beginning of due care.Glycine derivative has the free alpha-amino group, and other reactants have free or activatory carboxyl and shielded amino.After the coupling, can then, optionally its intermediate be carried out N-and go protection, so that can add other N-protecteds and free or activatory amino-acid residue with for example its intermediate of chromatography purification.Till this step lasts till that always desired amino-acid sequence is finished.

Operable carboxylic acid activation substituting group comprises symmetry or blended acid anhydrides, or active ester, for example P-nitrophenyl ester, 2,4,5 trichlorophenyl esters, N-hydroxybenzotriazole ester (OBt), N-hydroxy-succinamide base ester (OSu) or pentafluorophenyl group ester (oPTF).

For example, can adopt dicyclohexyl carbodiimide (DCC) to carry out the coupling of free amine group and carboxyl.Another kind of operable coupler is N-ethoxy carbonyl-2-oxyethyl group-1,2-dihydroquinoline (EEDQ).

Generally, at low temperature, as-20 ℃ to being up to room temperature, be easily in the suitable solvent system, carry out coupled reaction in the mixture as tetrahydrofuran (THF), diox, dimethyl formamide, methylene dichloride or these solvents.

Synthesizing on the solid-phase resin carrier may be more convenient.The polystyrene of chloromethylation (using 1% divinylbenzene crosslink) is a kind of useful carrier type, and in this case, it is synthetic can be coupled to by the glycine with N-protected on the carrier and to begin at C-end place.

Following document description many suitable solid phase techniques:

Eric Atherton, Christopher J.Logem and Robert C.Sheppard, J.Chem.Soc.Perkin I, 538-46(1981); James P.Tam, Foe S.Tjoeng, and R.B, Merrifield J.Am.Chem.Soc.102,6117-27(1980); James P.Tam, Richard D.Dimarchi and R.B.Merrifield Int.J.Peptide Protein Res.16 412-25(1980); Manfred Mutler and Dieter Bellof, Helvetca Chimica Acta 672009-16(1984).

It also is possible carrying out coupled reaction in solution.

Known that amino protecting group has extensive selection, and illustrated at following reference:

Schroeder, E. and L ü bke, K., The Peptides, Vol.1 and 2, Academic Press, New York and London, 1965 and 1966; Pettit, G.R., Synthetic Peptides, Vols 1-4, Van Nostrand, Reinhold, New York 1970,1971,1975 and 1976; Houben-Weyl, Methodender Organischen Chemie, Synthese Von Peptiden, Band 15, Georg Thieme Verlag Stuttgart, NY, 1983; The Peptides Analysis, Synthesis, biology 1-7, Ed:Erhard Gross, Johannes Meienhofer, Academic Press, NY, San Fransisco, London, Solid phase peptide synthesis 2nd ed., John M.Stewart, Janis D.Young, Pierce Chemical Company.

Therefore, for example operable amine protecting group comprises such as carbobenzoxy (Z-), tert-butoxycarbonyl (Boc-), 4-methoxyl group-2,3, the protecting group of 6-Three methyl Benzene alkylsulfonyl (Mtr-) and 9-fluorenyl methoxy carbonyl (Fmoc-) and so on.Should see that when making up peptide from C-is terminal, the ammonia protecting group is present on the alpha-amino group of each new residue that adds, and need optionally to be removed before the further coupling step carrying out.For the solid phase system, a kind of especially effectively group that this interim amine is protected is the Fmoc group, and this group can be by handling and optionally remove with piperidines at organic solvent.For synthetic in solution, Boc-is a kind of more desirable blocking group, and it can ordinary method be introduced or remove.

Before for example protecting, usually need it is carried out silanization for improving amino acid or the peptide solubleness in organic solution by adding Fmoc.

Silanization and Fmoc protective reaction are summarized as follows:

For example operable carboxyl-protecting group comprises and is easy to the splitted ester group, and (OBZI), the p-nitrobenzyl (ONB) or tert-butyl (coupler tobu) and on the solid carrier, the methyl that connects as polystyrene as benzyl.

Sulfur protecting group comprises p-methoxy-benzyl (Mob), trityl (Trt) and acetylamino methyl (Acm).

People will see as what be described in detail in the above referred-to references, also having a large amount of other these class groups to exist, and use all these groups all within the scope of the present invention in the previously described method of this paper.

The method of removing amine and carboxyl-protecting group has a lot.Yet these methods should be consistent with employed synthetic schemes.Side chain protective group must be stable for removed the employed condition of interim alpha-amino group protecting group before next step coupling step.

Can be removed simultaneously with acid treatment as the amine protecting group of Boc with as the carboxyl-protecting group of toBu as trifluoroacetic acid and so on.Use the oxygenant such as iodine, can optionally remove sulphur protective material as Trt.

In the end synthesis step adopts the synthetic peptide that contains halfcystine of method described herein after removing all protecting groups that comprise sulfur protecting group.

Only list following embodiment in the explanation mode.

The HoBt=hydroxybenzotriazole

The pfp=pentafluorophenyl group

Fmoc=9 fluorenyl methoxy carbonyl

Uncle Boc=-butoxy carbonyl

The Dcc=dicyclohexyl carbodiimide

(E)-DHS=(S, S)-β, β '-diaminostilbene, 3-diethylbenzene-β, β '-dicarboxylic acid

Embodiment 1

(S, S)-α, α '-diaminostilbene, 3-benzene dipropionic acid

A) α, α '-two [(2R, 5S)-2,5-dihydro-3,6-dimethoxy-2-sec.-propyl-5-pyrazinyl]-m-dimethylbenzene

(1.6M, 9.4ml 15mmol) are added to (2R)-(-)-2 that is cooled to-78 ℃, and 5-dihydro-3 is in the solution in the 6-dimethoxy-2-isopropylpyrazine (2.8g is 15mmol) at anhydrous THF(5ml) with the solution of n-butyl potassium in hexane.This mixture stirred 15 minutes.Add α, the solution in the α '-two bromo-m-dimethylbenzene (2.0g is 7.5mmol) at anhydrous THF(5ml).Reaction mixture is risen to ambient temperature overnight.Boil off solvent, resistates is put into diethyl ether, wash this ether solvents with water, dry (MgSO ₄) and evaporation.With its product of flash chromatography method purifying (silica dioxide gel, hexane/EtOAc3: 1).Output 2.8g(80%), yellow-white, semisolid.

¹H NMR（200 MHz，CDCl ₃）：δ0.60（6H，d），0.94（6H，d，CH ₃），2.1（2H，m），3.0（4H，m），3.3（2H，m），3.66（6H，s）3.71（6H，s），4.3（2H，dd），6.8-7.1（4H，m）; ¹³C NMR（50 MHz，CDCl ₃）;δ18.0，20.6，32.6，41.5，53.2，53.4，57.9，61.3，127.5，128.1，131.9，137.1，162.5，163.7.

MS（IP??70??eV）：m/z??470（0.2），288（50），183（47），182（20），141（100），105（40）.

C ₂₆H ₃₈N ₄O ₄Calc：C 66.34;H 8.14;N 11.91;

Found：C??66.48;H??8.07;N??11.85.

B) dimethyl (S, S)-α, α '-diaminostilbene, 3-benzene-dipropionate

With α, α '-two [(2R, 5S)-2,5-dihydro-3,6-dimethoxy-2-sec.-propyl-5-pyrazinyl]-m-dimethylbenzene (2.00g, 4.25mmol) be dissolved in the methyl alcohol (10ml), add again 0.25M hydrochloric acid (68ml, 17mmol), its mixture at room temperature stirs and spends the night, with this water/methanol solution of ether extraction, ammonification is with pH regulator to 10 then.This product is extracted in the ether dry ethereal solution (MgSO ₄), filter evaporation.In the Kygelrohr device, boil off D-Xie Ansuan methyl ester (50-60 ℃/0.1 millibar).Its resistates need not repurity and just can use in next step.

Productive rate 1.10g(92%)

¹H NMR（200MHz，CDCl ₃）：δ1.4（4H，br s），2.83（2H，dd， ²J 13.5Hz， ³J 7.9Hz），3.05（2H，dd， ²J 13.5Hz， ³J 5.1Hz），3.7（2H，dd），3.71（6H，s），7.0-7.3（4H，m）.

C) (S, S)-α, α '-diaminostilbene, 3-benzene-dipropionic acid 2HCl

With dimethyl (S, S)-α, α '-diaminostilbene, (1.0g, 3.6mmol) solution in 6M hydrochloric acid (10ml) spends the night 50 ℃ of stirrings 3-benzene-dipropionate.On rotation steam (rotavapor), remove hydrochloric acid, and the white solid that obtains carries out drying in 40 ℃ in vacuum oven.

Productive rate 1.1g(95%)

¹H NMR（200MHz，H ₂O）：δ3.18（2H，dd， ²J 14.5Hz， ³J 7.4Hz），3.29（2H，dd， ²J 14.5Hz， ³J 5.7Hz），4.28（2H，dd， ³J 5.7，7.4Hz），7.1-7.4（4H，m）; ¹³C，NMR（50MHz，H ₂O）：δ39.2），57.7，131.8，132.8，133.0，137.7，173.7.

C ₁₂H ₁₈Cl ₂N ₂O ₄Calculated value: C 44.32; H 5.58; N 8.61;

Measured value: C 44.78; H 5.81; N 8.73.

D) (S, S)-α, α '-two (9-fluorenyl methoxy carbonylamino)-1,3-benzene-dipropionic acid

Will (S, S)-α, α '-diaminostilbene, (1.05g, the 3.23mmol) suspension in the mixture of hexamethyldisilazane (15ml) and trimethylchlorosilane (1.5ml) spend the night in 100 ℃ of stirrings under nitrogen 3-benzene dipropionic acid two hydrochloric acid.Its reaction mixture of infusing is clearly removed excessive silylating reagent under vacuum after cooling.Resistates is dissolved in anhydrous methylene dichloride (10ml), cools off in ice/water-bath.(its reaction mixture stirred 3 hours under nitrogen for 0.75g, the 6.76mmol) solution in anhydrous methylene chloride (5ml) to add 9-fluorenyl methoxy carbonyl chlorine.Remove methylene dichloride in rotation steam (rotavapor), resistates is dissolved in THF(8ml) and the mixture of water (1ml) in.This mixture stirred 30 minutes, then evaporation.Take out resistates and put into ethyl acetate, and wash with water several times.Organic phase is carried out drying (MgSO ₄) filter and concentrate.Add hexane and make the product crystallization.The crystal hexane wash, and with column chromatography (silicon-dioxide, hexane/ethyl acetate/acetate 3: 6: 1) its product of purifying, white solid, mpt143-144 ℃

Productive rate 1.85g(82%)

¹H NMR（200 MHz，CDCl ₃）：δ2.8-3.1（4H，m），4.0-4.4（8H，m），7.0-8.0（22H，m）; ¹³C NMR（50 MHz，CDCl ₃）;δ37.5，47.6，55.3，66.4，120.2，125.3，127.1，127.3，127.7，128.1，129.9，137.8，140.6，143.6，155.7，172.9.

The FAB-MS signal is at m/z 719.1(2), 539.1(6), 271.0(6) and 179.1(100).

Embodiment 2

(S, S)-α, α '-two (9-fluorenyl methoxy carbonylamino)-1,4-benzene dipropionic acid

A) α, α '-two [(2R, 5S)-2,5 dihydro-3,6-dimethoxy-2-sec.-propyl-5-pyrazinyl]-P-dimethylbenzene

As previously described by (2R)-(-)-(2,5-dihydro-3,6-dimethoxy-2-isopropylpyrazine (18mmol) and α, α '-two bromo-P-dimethylbenzene (9mmol) is prepared title compound.On silica dioxide gel, utilize flash chromatography method purifying crude product (CHCl ₃).Productive rate 76%.

¹H NMR（CDCl ₃）：δ0.6-0.9（2d，12H），2.05-2.20（m，2H）;3.05（d，4H），3.25（s，2H），3.60（s，6H），3.70（s，6H），6.9（s，4H）.

TLC(hexane: EtOAc; 12: 1), Rf 0.32.

HPLC(50-100% MeOH, 10 minutes), RT 7.87 minutes

B) dimethyl (S, S)-α, α '-diaminostilbene, 4-benzene dipropionate

As the front to the m-isomer described, at room temperature the product (3.4mmol) of preceding step is resolved into title compound with 0.25MHCl.

TLC（MeCN：MeOH：H ₂O，4∶1∶1），Rf 0.36

C) (S, S)-α, α '-diaminostilbene, 4-benzene dipropionic acid dihydrochloride

To as described in the m-isomer, make methyl hydrolysis of ester group in the aforementioned product as the front with dense HCl heating.

TLC（MeCN∶MeOH∶H ₂O，4∶1∶1）.

¹H NMR（D ₂O）：δ0.8-0.9（2d，6H），2.10（m，1H），3.00（m，2H9），3.45（d，1H），3.81（t，2H），7.13（s，4H）.

D) (S, S)-α, α '-two (9 fluorenyl methoxy carbonylamino)-1,4-benzene dipropionic acid

To as described in the m-isomer, prepare title compound as the front by above-mentioned amino acid and 9-fluorenyl methoxy carbonyl chlorine.

TLC(heptane: EtOAc: AcOH, 3: 6: 1), Rf0.58.

HPLC(40-70%MeCN, in 0.1%TFA, 10 minutes), RT 4.86 minutes.

¹H NMR（DMSO-d ₆）：δ2.79-3.11（m，6H），4.09-4.21（br.s，6H），7.13-7.92（m，20H）.

FAB-MS：（M+H）697.

Embodiment 3

(S, S)-α, α '-diaminostilbene, 2-benzene-dipropionic acid

A) α, α '-two [(2R, 5S)-2,5-dihydro-3,6-dimethoxy-2-sec.-propyl-5-pyrazinyl]-O-toluene

With (R)-2,5-dihydro-3 is in the 6-dimethoxy-2-isopropylpyrazine (3.50g 19.00mmol) is dissolved in anhydrous THF(8ml).With solution cooling (78 ℃), and drip BuLi(13ml under nitrogen, 19.50mmol is in hexane, 1.5M).Drip (78 ℃) electrophilic, at THF(5ml) in α, α '-two chloro-O-dimethylbenzene.Its reaction mixture is heated to ambient temperature overnight.Remove and desolvate, make elutriant flash chromatography method purifying crude product with hexane/ethyl acetate (4: 1).Light yellow crystal, 3.512g(79%).Mp.109-112℃。

¹H NMR（300MHz，CDCl ₃）δ：0.59（6H，d，J 6.7Hz，2xCH ₃），0.94（6H，d，J 6.8 Hz，2xCH ₃），2.13（2H，m，2xCH），2.95，3.45（4H，m，2xCH ₂），3.28（2H，t，J3.3Hz，2xCH），3.62（6H，s，OCH ₃），3.71（6H，s，OCH ₃），4.26（2H，m，2xCH），7.05（4H，m，4xCH）. ¹³C NMR（75MHz，CDCl ₃）δ：16.38，13.93，30.96，36.49，52.10，52.20，57.14，59.98，125.86，130.41，136.75，162.85，163.61.

C ₂₆H ₃₈N ₄O ₄Calculated value: C, 66.35; H, ε .14.

Measured value: C, 66.03; H, 8.04.

B) dimethyl (S, S)-α, α '-diaminostilbene, 2-benzene-dipropionate

0.5M HCl(20ml, 10.00mmol) with diox (20ml) adds α, α '-two [(2R, 5S)-2,5-dihydro-3,6-dimethoxy-2-sec.-propyl-5-pyrazine]-O-dimethylbenzene (1.155g, 2.45mmol) in.This mixture at room temperature stirred reach 7 hours.Under decompression, water (15ml) and chloroform (adding 15ml) condition, remove most of solvent.Remove chloroform layer, use dense NH ₃Water (pH10) transfers to alkalescence with water layer.(3 * 20ml) extract water, and the chloroform layer that is combined carries out drying (MgSO with chloroform ₄) and concentrate.Adopt bulb that bulb (bulb to bulb) distillation method is removed the Xie Ansuan methyl ester, (0.3mmHg, 50 ℃), 1.5 hours.Residue is flaxen oil, 0.55g(80%).

¹H NMR（200MHz，CDCl ₃）δ：1.63（4H，s，NH ₂），3.02（4H，m，2xCH ₂），3.71（6H，s，CCH ₃），3.74（2H，m，2xCH），7.18（4H，m，4xCH）. ¹³C NMR（50 MHz，CDCl ₃）δ：38.26，52.47，56.10，127.48，130.80，136.73，175.92.

C ₁₄H ₁₃N ₂O ₄Calculated value: C, 59.98; H, 7.19.

Measured value: C, 59.54; H, 7.04.

C) (S, S)-α, α '-diaminostilbene, 2-benzene-dipropyl 2HCl

Use 6N HCl(20ml at 50 ℃) and the processing dimethyl (S, S)-α, α '-diaminostilbene, (270mg 0.963mmol) reaches 20 hours to 2-benzene-dipropionate.Remove in a vacuum and anhydrate, add the benzo evaporation (2 * 20ml), to stay colourless solid 0.313g.

¹H NMR（300 MHz，D ₂O）δ：3.18（4H，m，2xCH ₂），4.12（2H，m，CH），4.64（6H，s，NH ₃），7.23（4H，m，4xCH）. ¹³C NMR（75MHz，D ₂O）δ：32.56，53.85，128.62，130.81，133.27，171.36.

D) (S, S)-α, α '-two (9-fluorenyl methoxy carbonylamino)-1,2-benzene-dipropionic acid

Will (S, S)-α, α '-diaminostilbene, (130mg 0.40mmol) is suspended in hexamethyldisilazane (7ml) and the TMS chlorine (0.5ml) 2-dipropionic acid dihydrochloride, and spends the night in refluxed under nitrogen.Remove in a vacuum and desolvate, resistates is dissolved in anhydrous methylene chloride (10ml).Cooling (0 ℃) its solution drips 9-fluorenyl methoxy carbonyl chlorine (260mg, 1.00mmol) solution in anhydrous methylene chloride (5ml).Remove cryostat after 1 hour, reaction mixture is stirred under nitrogen spend the night.Add 1N HCl(5ml) debris, drying.

Productive rate: 100mg(40%).

¹H NMR（200MHz，DMSO-d ₆）δ：2.49-3.54（14H，m，4xCH ₂，4xCH，2xNH），7.29-7.84（20H，m，20xCH）. ¹³C NMR（75MHz，DMSO-d ₆）δ：33.41，46.94，54.75，66.14，120.46，125.64，126.79，127.48，128.07，130.21，136.42，141.02，144.05，156.37，173.64.

Embodiment 4

(S, S)-α, α '-diamino-2,5-thiophene-dipropionic acid

A) 2,5-pair [(2R, 5S)-(-)-2,5-dihydro-3,6-dimethoxy-2-isopropylpyrazine base] methyl } thiophene

With (R)-2,5-dihydro-3 is in the 6-dimethoxy-2-isopropylpyrazine (5.00g 27.14mmol) is dissolved in anhydrous THF(15ml).Cooling solution (78 ℃) drips BuLi(19ml under nitrogen, 30.4mmol, 1.5M in hexane).After ten minutes, drip electrophilic, at THF(10ml) in 2, two (chloromethyl) thiophene of 5-(2.50g, 13.80mmol).Allow reaction mixture reach ambient temperature overnight (under nitrogen).Remove in the vacuum and desolvate, with flash chromatography method (hexane/ethyl acetate is 4: 1) purifying crude product.

Productive rate: 4.87g(75%) white crystal, 2.3%Wrong diastereomer (its available flash chromatography method or from acetonitrile recrystallization remove)

Mp.79℃（MeCN）。

C ₂₄H ₃₆N ₄O ₄S calculated value: C, 60.47, H, 7.61

Measured value: C, 60.45, H, 7.68

¹H NMR（300MHz，CDCl ₃）δ：0.63（6H，d，J6.9Hz，CH ₃），0.96（6H，d，J6.9Hz，CH ₃），2.16（2H，m，CH），3.58（2H，t，J3.5Hz，CH），3.65（6H，s，OCH ₃），3.71（6H，s，OCH ₃），4.23（2H，dd，CH），6.48（2H，s，H-3，H-4）. ¹³C NMR（75MHz，CDCl ₃）δ：16.56，18.85，31.46，34.59，51.94，52.41，56.04，60.48，125.80，137.65，161.57，164.43.

B) dimethyl (S, S)-α, α '-diamino-2,5-thiophene dipropionate

With 2,5-pair [(2R, 5S)-(-)-2,5-dihydro-3,6-dimethoxy-2-sec.-propyl-5-pyrrole thiophene base] methyl } (1.007g 2.113mmol) is suspended in diox (20ml), HCl(0.7ml to thiophene, 8.5mmol, 12M) and in the mixture of water (20ml).Under room temperature, add diethyl ether after four hours (30ml).Remove the ether phase, use dense NH ₃Water (pH10) is transferred to alkalescence with water.(3 * 30ml) extract water to use chloroform again.Organic layer drying (the MgSO that merges ₄) after, remove and desolvate.Adopt bulb to bulb (bulb to bulb) distillation method remove valine methyl ester (0.5mmHg, 50 ℃, 1.5h)

Productive rate 0.555g(92%), yellowish oil.

C ₁₂H ₁₆N ₂O ₄S calculated value: C, 50.33; H, 6.34

Measured value: C, 50.79; H, 6.53.

¹H NMR（300 MHz，CDCl ₃）δ：1.60（4H，bs，NH ₂），3.16（4H，m，CH ₂），3.70（2H，m，CH），3.73（6H，s，OCH ₃），6.68（2H，s，H-3，H-4）. ¹³C NMR（75MHz，CDCl ₃）δ：35.26，52.02，55.52，126.32，138.19，174.85.

C) (S, S)-α, α '-diamino-2,5-thiophene-dipropionic acid 2HCl

70 ℃ with 6N HCl handle dimethyl (S, S)-α, α '-diamino-2, (531mg 1.854mmol) reaches 20 hours to 5-thiophene-dipropionate.Evaporating solns, remaining water and benzene azeotropic (2 * 30ml).Pale yellow crystals, 613mg(100%)

¹H NMR（300MHz，D ₂O）δ：3.35（4H，d，J5.9，CH ₂），4.16（2H，t，J5.8Hz，CH），4.65（8H，s，NH ₃），6.78（2H，s，H-3，H-4）. ¹³C NMR（75MHz，D ₂O）δ：29.96，54.07，128.61，135.89，171.35.

D) (S, S)-α, α '-two (9-fluorenyl methoxy carbonylamino)-2,5-thiophene dipropionic acid

Will (S, S)-α, α '-diamino-2, (590mg 1.78mmol) is suspended in hexamethyldisilazane (15ml) and the TMS chlorine (1ml) 5-thiophene dipropionic acid dihydrochloride.The mixture backflow is spent the night, and with its solution decompression evaporation, residue is dissolved in the anhydrous methylene chloride (15ml).Solution is cooled to 0 ℃, under nitrogen chlorine, drips 9-fluorenylmethyloxycarbonyl chlorine (1.055g, 4.08mmol) solution in methylene dichloride (5ml).Remove cryostat after 1 hour, at room temperature in nitrogen, stir the mixture and spend the night.Remove and desolvate, and this compound is dissolved in THF(5ml) in, add 1N HCl(5ml again).Remove water after 2 hours, and (3 * 20ml) wash with chloroform.Organic layer drying (the MgSO that merges ₄) after, remove and desolvate, make eluent with methylene chloride/acetate (10: 1: 0.1) and adopt flash chromatography method purified product.Yellow crystals: 0.557g(44%).Mp.249 ℃ (decomposition).

¹H NMR（200MHz，DMSO-d ₆）δ2.90-4.20（14H，m，CH ₂x4，CHx4，NHx2），6.69（2H，s，H-3，H-4），7.20-8.00（16H，m，CHx16）. ¹³C NMR（50MHz，DMSO-d ₆）δ：21.47，31.50，109.50，120.20，121.56，127.50，129.15，137.71，139.83，143.00，172.00.

Embodiment 5

(S, S)-α, α '-diamino-suitable-1,2-cyclopropane-dipropionic acid

A) suitable-1, two (brooethyl) cyclopropane of 2-

(6.6g, (10.8g is 41.16mmol) in cold (0 ℃) suspension in acetonitrile (70ml) 41.16mmol) to add triphenylphosphine with bromine lentamente.Remove cryostat, drip suitable-1,2-two (methylol) cyclopropane (2.02g, 19.6mmol) solution in acetonitrile (5ml).Its mixture was at room temperature stirred 1 hour, and the elimination precipitation concentrates filtrate under vacuum, resistates is dissolved in the ether (70ml), filters, concentrates, and carries out vacuum distilling (12 millibars, bp135-155 ℃) again.

Productive rate: 3.463g(71%).

¹H NMR（CDCl ₃）δ：0.44（q，J＝5.8 Hz，1H）1.18（m，1H），1.66（m，2H），3.50（m，4H） ¹³C NMR（CDCl ₃）δ：16.66，22.68，33.46

B) suitable-1,2-two [(2R, 5S)-2,5-dihydro-3,6-diethoxy-2-sec.-propyl-5-pyrazinyl] methyl-cyclopropane

(8.774mmol) solution in adds (2R)-2 for 1.6N, 5.483ml at hexane with n-Butyl Lithium lentamente, 5-dihydro-3, in cold (78 ℃) solution in the 6-diethoxy-2-isopropylpyrazine (1.862g is 8.774mmol) at THF(30ml), continue to stir 30 minutes.During this period, add suitablely-1 lentamente, two (brooethyl) cyclopropane of 2-are at anhydrous THF(10ml) in solution, allow its solution reach ambient temperature overnight.Make this stopping of reaction by adding pH7 phosphate buffered saline buffer (30ml), with ether (each 30ml) this mixture of extracted twice.Organic layer drying (the MgSO that merges ₄) revaporization is to doing, product separates with flash chromatography method (hexane/ether, 8/1).

Productive rate: 0.609g, 28%.

¹H NMR（CDCl ₃）δ：-0.15（q，J＝5.85Hz，1H），0.5（m，1H），0.65（d，6H），0.99（d，6H），1.22（dt，12H），1.51（m，2H），1.92（m，2H），2.23（m，2H），3.83（m，2H），3.98-4.19（m，12H） ¹³C NMR（CDCl ₃）δ：10.51，10.90，11.08，14.37，16.55，17.03，19.02，19.12，31.41，31.59，32.54，33.31，33.70，46.78，55.83，56.15，60.26，60.38，60.52，60.55，60.68，61.03，162.87，163.03，163.26，163.38.

C) (S, S)-α, α '-diamino amino-suitable-1,2-cyclopropane dipropionate for diethyl

With HCl(1N, 3.51ml) be added to suitablely-1, (430mg is 0.876mmol) in the solution in the Zai diox (3.5ml) for methyl-cyclopropane for 2-two [(2R, 5S)-2,5-dihydro-3,6-diethoxy-2-sec.-propyl-5-pyrazine].Its mixture was at room temperature stirred 1 hour, then under vacuum, remove diox.After adding the 1.5ml strong aqua, with chloroform (each 15ml) extracted twice resistates, the extract drying (MgSO of merging ₄), the evaporation after, remove L-valine ester by vacuum distilling (55 ℃, 0.03 millibar) again.The oily resistates is made up of pure title compound.

Productive rate: 238mg, 99.7%.

¹H NMR（CDCl ₃/D ₂O）δ：-0.15（q，1H），0.68（m，1H），0.79（m，2H），1.22（t，J7.2Hz，6H），1.36（m，2H），1.78（m，2H），3.41（m，2H），4.10（q，J7.2Hz，4H）. ¹³C NMR（CDCl ₃/D ₂O）δ：10.72，11.37，11.44，13.88，33.74，54.56，54.79，60.36，60.38，175.39，175.672.

D) (S, S)-α, α '-two (9 fluorenyl methyl oxygen carbonylamino)-suitable-1,2-cyclopropane dipropionate for diethyl

At room temperature (678mg is 2.622mmol) with 1N NaHCO with Fmoc-chlorine successively ₃Solution (3ml) be added to diethyl (S, S)-α, α '-diamino amino-suitable-1, (238mg is 0.874mmol) in the solution in the Zai diox (4ml) for 2-cyclopropane-dipropionate.Stir and remove diox after 30 minutes, then extract with chloroform (30ml), CHCl is removed in evaporation ₃After resistates obtain title compound through flash chromatography method purifying (hexane/ethyl acetate, 4/1 to 2/1)

Productive rate: 483mg, 77.1%.

¹H NMR（CDCl ₃）δ：-0.10（br s，1H），0.77（br s，3H），1.28（t，6H），1.56（m，2H），2.03（m，2H），4.23（m，6H），4.41（m，6H），5.46（m，2H），7.25-7.77（m，16H） ¹³C NMR（CDCl ₃）δ：11.05，11.24，14.13，31.77，31.82，47.16，54.11，54.28，61.49，66.93，119.93，125.01，126.99，127.65，141.26，143.72，143.86，155.69，172.11，172.28.

FAB-MS：MH+717.4

E) (S, S)-α, α '-two (9-fluorenyl methyl oxygen carbonylamino)-suitable-1,2-cyclopropane-dipropionic acid

With diethyl (S, S)-α, α '-two (9-fluorenyl methoxy carbonylamino)-suitable-1,2-cyclopropane-dipropionate (497mg, 0.693mmol) the solution in the Zai diox (5ml) is with 6N HCl(3ml) is heated to 90 ℃ and reaches 26 hours.Remove diox in a vacuum, and with chloroform (each 20ml) extracted twice aqueous residue.Dry (MgSO ₄) after boil off solvent, with flash chromatography method this resistates of purifying (hexane/ethyl acetate/acetate, 10/10/1), the cut that contains this product obtains very little volume through concentrating, concentrating in the vacuum, freezing acquisition title compound,

Productive rate: 280mg, 61.2%.

¹H NMR（DMSO-d ₆/D ₂O）δ：-0.04（m，1H），0.56（m，1H），0.82（m，2H），1.41（m，1H），1.60（m，1H），1.74（m，1H），1.88（m，1H），3.97（m，2H），4.25（m，6H），7.27-7.88（m，16H）. ¹³C NMR（DMSO-d ₆/D ₂O）δ：10.88，12.35，13.13，30.15，30.59，47.02，54.84，66.02，120.43，125.63，127.44，128.02，136.68，141.04，144.10，144.15，156.43，156.49，174.00，174.12.

FAB-MS：MH+661.3

Embodiment 6

(S, S)-α, α '-diamino-suitable-1,2-tetramethylene-dipropionic acid

A) suitable-1, two (brooethyl) tetramethylene of 2-

(1.623g, (2.664g is 10.16mmol) in cold (0 ℃) suspension in acetonitrile (15ml) 10.16mmol) to be added to triphenylphosphine lentamente with bromine.Remove cryostat, drip suitable-1,2-two (methylol) tetramethylene (562mg, 4.838mmol) solution in acetonitrile (5ml).This mixture at room temperature stirred 1 hour, then removed under vacuum and desolvated, and added ether (30ml) and ground residue, removed by filter precipitation, vacuum distilling again after the concentrating filter liquor (Kugelrohr, 0.3 millibar, bp110 ℃)

Productive rate: 628mg, 53.6%.

¹H NMR（CDCl ₃）δ：1.71（s，2H），2.10（s，2H），2.82（s，2H），3.44（m，2H），3.58（m，2H） ¹³C NMR（CDCl ₃）δ：23.72，33.69，39.45

B) suitable-1,2-two [(2R, 5S)-2,5-dihydro-3,6-diethoxy-2-sec.-propyl-5-dihydro pyrazinyl] methyl-tetramethylene

(1.6N, 4.92ml 7.87mmol) slowly are added to (2R)-2 with the solution of n-Butyl Lithium in hexane, 5-dihydro-3, in cold (78 ℃) solution in the 6-dimethoxy-2-isopropylpyrazine (1.449g is 7.865mmol) at THF(30ml), restir 30 minutes.During this period, slowly add suitablely-1,2-is two-solution in (brooethyl) tetramethylene (906mg is 3.745mmol) at anhydrous THF(4ml), allow solution reach ambient temperature overnight.End this reaction by adding pH7 phosphate buffered saline buffer (30ml), use ether (each 30ml) extracted twice mixture again.Organic layer drying (the MgSO that merges ₄) back revaporization is to doing, usefulness flash chromatography method (hexane/ether, 8/1) is separated this product.

Productive rate: 517mg, 30%.

¹H NMR（CDCl ₃）δ：0.60（d，6H），0.98（d，6H），1.63（m，4H），1.85（m，4H），2.17（m，2H），2.45（m，2H），3.57-3.67（m，12H），3.80-3.94（m，4H）. ¹³C NMR（CDCl ₃）δ：16.35，16.40，19.02，24.84，25.54，31.30，31.36，33.87，34.22，34.72，34.81，51.96，52.04，52.16，54.22，54.75，60.23，60.36，162.73，163.20，164.02，164.19

C) (S, S)-α, α '-diamino amino-suitable-1,2-tetramethylene-dipropionate for diethyl

With HCl(1N, 4.61ml) add suitable-1, the two [(2R of 2-, 5S)-2,5-dihydro-3,6-diethoxy-2-sec.-propyl-5-dihydro pyrazine] (517mg is 1.152mmol) in the solution in the Zai diox (5ml) for methyl-tetramethylene, mixture at room temperature stirred 1 hour, then removed diox under the vacuum.After adding the 1.5ml strong aqua, with chloroform (each 15ml) its resistates of extracted twice, the extract drying (MgSO of merging ₄), the evaporation after remove L-valine ester by vacuum distilling (55 ℃, 0.03 millibar) again, the oily resistates is made up of pure title compound.

Productive rate: 293mg, 98.5%.

¹H NMR（CDCl ₃）δ：1.36（br s，4H），1.50（m，4H），1.74（m，2H），1.91（m，2H），2.43（m，2H），3.24（m，2H），3.59（m，6H） ¹³C NMR（CDCl ₃）δ：24.02，24.80，33.54，33.86，35.07，35.67，51.56，51.68，52.47，53.03，176.25，176.59

D) (S, S)-α, α '-two (9-fluorenyl methyl oxygen carbonylamino)-suitable-1,2-tetramethylene dipropionate for diethyl

(880mg is 3.402mmol) with 1N NaHCO with Fmoc-chlorine successively ₃Solution (3ml) at room temperature be added to diethyl (S, S)-α, α '-diamino amino-suitable-1, (293mg is 1.134mmol) in the solution in the Zai diox (4ml) for 2-tetramethylene-dipropionate.After stirring 30 minutes, remove diox, and extract, use flash chromatography purifying (hexane/ethyl acetate 1/1 to 2/1) to boil off CHCl thereafter with chloroform (30ml) ₃After the resistates that stays, obtain title compound.

Productive rate: 572mg, 71.8%.

¹H NMR（CDCl ₃）δ：1.77（m，4H），2.00（m，6H），2.25（m，2H），3.74（m，6H），4.10-4.50（m，6H），5.10-5.20（m，2H），7.27-7.77（m，16H） ¹³C NMR（CDCl ₃）δ：24.38，24.95，33.01，33.18，33.68，33.78，47.15，52.19，52.30，52.40，52.65，66.93，119.92，125.01，126.98，127.65，141.25，143.67，143.79，155.63，155.91，172.84，173.16

E) (S, S)-α, α '-two (9-fluorenyl methyl oxygen carbonylamino)-suitable-1,2-tetramethylene-dipropionic acid

Diethyl (S, S)-α, α '-two (9-fluorenyl methoxy carbonylamino)-suitable-1, and 2-tetramethylene-dipropionate (572mg, 0.874mmol) the solution in the Zai diox (5ml) is with 6N HCl(3ml) is heated to 65 ℃ and reaches 20 hours.Remove diox under the vacuum, with chloroform (each 20ml) second extraction aqueous residue, dry (MgSO ₄) after boil off solvent, use flash chromatography method (hexane/ethyl acetate/acetate, 10/10/1) purifying resistates again.The fraction post precipitation that will contain product concentrates under vacuum and obtains little volume, the freezing title compound that obtains white powder.

Productive rate: 316mg, 57.5%.

¹H NMR（DMSO-d ₆/D ₂O）δ：1.4-2.0（m，6H），2.40（m，2H），3.6（br，2H），3.82（m，2H），4.25（m，6H），7.26-7.90（m，16H） ¹³C NMR（DMSO-d ₆/D ₂O）δ：23.58，24.77，31.00，32.12，33.62，46.98，51.84，53.08，65.83，120.37，125.55，127.36，127.94，141.00，144.01，144.05，144.11，156.27，156.43，174.13，174.55.

Embodiment 7

(S, S)-α, α '-diamino-anti--1,4-hexanaphthene-oxalic acid

A) anti--1,4-two (1S, 4R)-3,6-dimethoxy-4 '-sec.-propyl-2,5-dihydro pyrazinyl)-hexanaphthene

With 1.6M n-BuLi(9.6ml, 15.3mmol) be added drop-wise to anti-form-1,4-is two [in the solution in (2R, 5S)-2,5-dihydro-3,6-dimethoxy-2-sec.-propyl-5-pyrazinyl hexanaphthene is at THF(48ml).After 1 hour, dripping 1,4-is anti--solution in the dibromo-cyclohexane (1.82g is 7.5mmol) at THF(5ml).Allow mixture reach ambient temperature overnight, and with 1M phosphate buffered saline buffer (pH=7) stopped reaction.Dilute its mixture with ether (100ml) and water (20ml), and separate stratification.With ether (2 * 150ml) extracted twice waterbearing stratums, the organic layer drying (MgSO of merging ₄), filter, concentrate, use flash chromatography method purifying (dioxide/silica gel, hexane: ether=12: 1) again.By merging product that cut obtains through MeOH(100ml) recrystallization.

Productive rate: 2.774g(6.18mmol, 82.4%)

¹H NMR（300MHz，CDCl ₃）：δ＝3.91（t，2H，J＝3.6Hz），3.86（q，2H，J＝3.0Hz），3.69（s，6H），3.68（s，6H），2.67（d sept，2H，J＝3.0，6.9Hz），1.78-1.54（m，8H），1.05（d，6H，J＝6.9Hz），0.97（m，2H），0.66（d，6H）; ¹³C NMR（75MHz，CDCl ₃）：δ＝163.23，163.15，60.30，59.95，52.24，41.30，31.40，28.91，26.17，19.03，16.38.

FAB-MS m/z 449（M ⁺+1）.

C ₂₄H ₄₀N ₄O ₄(448.60). calculated value: C, 64.26; H, 8.98; N, 12.49.

Measured value: C, 64.20; H, 9.04; N, 12.37.

B) (S, S)-α, α '-diamino-anti--1,4-hexanaphthene-oxalic acid for dimethyl

(1.00g 2.23mmol) is dissolved in the diox (17.8ml) compound that step (a) is obtained, and adds 0.5M HCl(17.8ml again, and 8.92mmol), its mixture at room temperature stirred 12 hours.With removing diox under the vacuum of bleeding, the dilute with water resistates is used ether (3 * 30ml) extractions again.With the CHCl that merges ₃Dry (the MgSO of-layer ₄), filter and concentrate, resistates is transferred in the Kugelrohr device, removes valine methyl ester at 25-50 ℃ under 0.1 holder.

Productive rate: 530mg(2.05mmol, 92%).

¹H NMR（300MHz，CDCl ₃）：δ＝3.68（9s，6H），3.26（d，2H，J＝5.1Hz），1.72-1.54（m，6H），1.26-1.19（m，2H），1.13-1.06（m，2H）; ¹³C NMR（75MHz，CDCl ₃）：δ＝175.83，59.22，51.74，41.64，29.10，27.17.

C((S, S)-α, α '-diamino-anti--1,4-hexanaphthene-oxalic acid

Solution in the above-claimed cpd (442mg is 1.71mmol) at 6N HCl(10ml) was 50 ℃ of heating 14 hours.After its mixture reached room temperature, (rotavap) boiled off volatile compound, resistates (not exclusively) soluble in water on rotation steam.Add ethanol, mixture places refrigerator overnight.Filtering-depositing is with the ether washing, dry more then.

Productive rate: 378mg(1.25mmol, 72.9%)

¹H NMR（300MHz，D ₂O，DCl）：δ＝3.63（m，2H），1.68-1.43（m，6H），1.00（dd，2H），0.89（dd，2H）. ¹³C NMR（75MHz，D ₂O/DCl）：δ＝170.77，56.96，37.30，26.90，26.71.

FAB-MS m/z 231（M ⁺-72）;

C ₁₀H ₂₂N ₂O ₄（258.31）.

D) (S, S)-α, α '-two (9-fluorenyl methyl oxygen carbonylamino)-anti--1,4-hexanaphthene-oxalic acid

With FmocCl(1.43g, 5.54mmol the solution in the) Zai diox (3ml), then be 1M NaOH(8.31ml, 8.31mmol) join the amino acid (420mg that obtains by step (c) successively, 1.38mmol) (4ml) is with in the solution in the diox (4ml), its mixture stirs and spends the night at water.(2 * 30ml) extract the slurry that obtains with ether.CHCl is used again with 6M HCl acidifying in the waterbearing stratum ₃Extract (3 * 30ml).CHCl ₃-after concentrated, use flash chromatography method purifying (silica dioxide gel, hexane: ethyl acetate: acetate=10: 10: 1) mutually

Productive rate 648mg(0.96mmol, 69.5%0.

¹H NMR（300MHz，DMSO）：δ＝7.87（d，4H，J＝7.5Hz），7.73（d，4H，J＝7.5Hz），7.41（t，4H，J＝7.5Hz），7.33（t，4H，J＝7.5Hz），4.30-4.20（m，6H），3.86（t，2H），1.75-155（m，6H），1.25-0.95（m，4H）. ¹³C（DMSO）δ＝173.60，156.64，144.29，144.17，141.10，128.02，127.45，125.77，120.48，66.05，59.35，47.10，29.14，27.90，21.51.

FAB-MS m/z 677（M ⁺+1）.

C ₄₀H ₄₀N ₂O ₈(676.76). calculated value: C, 70.99; H, 5.96; N, 4.14.

Measured value: C, 68.9; H, 6.08; N, 3.99.

Embodiment 8

(S, S)-α, α '-two (9-fluorenyl methoxy carbonylamino)-1,4-phenylene-diacetic acid

A) (S, S)-β, β '-dioxy-β, β '-two (4-phenmethyl-2-oxygen-3-oxazolidinyl)-1,4-diethylbenzene

With (4S)-4-phenmethyl-2-oxazolidone (1.086g, 6.13mmol) and triphenyl methane (indicator) (2.5mg) at anhydrous THF(10ml) in solution under argon gas in-78 ℃ of stirrings, and use 1.6M nBuLi(3.83ml, 6.13mmol) drip processing, up to orange constant.When adding nBuLi, its solution remains on-65 ℃ to-78 ℃.Be cooled to after-78 ℃, solution is with 1 again, and (0.75g 3.25mmol) handles more than 2 minutes 4-benzene diethyl acyl chlorides.With the mixture heating up to 25 that obtains ℃, and handle, stirred 30 minutes at 25 ℃ with the 10ml saturated sodium bicarbonate aqueous solution, with mixture with three parts of dichloromethane extraction.Dichloromethane extract is merged,, use the salt water washing, dry (MgSO with the washing of 5% aqueous sodium carbonate ₄) and evaporation.Crude product is used hexane: ethyl acetate=1: 2 wash-out with flash chromatography method purifying.

Productive rate: (white solid) 698mg(45%).

¹H NMR(200MHz, CDCl ₃): δ 2.77(dd, J9.4,13.3Hz, CHHPh), and 3.28(dd, J3.2,13.3Hz, CHHPh), 4.18-4.22(m, CH ₂O), 4.31(s, CH ₂CO), 4.64-4.72(m, CHN), 7.35-7.13(m, 14H, aromatic series). ¹³C NMR(50MHz, CDCl ₃): δ 37.7,41.2,55.2,66.0,126.9,128.5,129.0,132.1,134.7,152.9,

B) (S, S, S, S)-and α, α '-diazido-β, β '-dioxy-β, β '-two (4-phenmethyl-2-oxygen-3-oxazolidinyl)-1,4-diethylbenzene

With (S, S)-β, β '-dioxy-β, β '-two (4-phenmethyl-2-oxygen-3-oxazolidinyl)-1, pre-cooled (78 ℃) solution in the 4-diethylbenzene (1,4g is 2.73mmol) at anhydrous THF(25ml), join in pre-cooled (78 ℃) solution in two (TMS) acid amides potassium of 0.5M (12ml 6.01mmol) dilutes at toluene (use anhydrous THF(30ml)) by sleeve pipe.After 30 minutes, the enol phenol potassium solution of generation is by overlapping the effective anhydrous THF(15ml that is dissolved in) 2,4,6-tri isopropyl benzenesulfonyl trinitride (trisylazide) (2.02g, 6.55mmol)-78 ℃ solution-treated.After 2 minutes, (0.983g 16.98mmol) ends its reaction to ice acetic acid in this solution stirring.Slurry is heated to 25 ℃ with water-bath at once, and stirred 1.5 hours in this temperature.Allow the reaction slurry between 200ml salt solution and 250ml ethyl acetate, distribute then, water 100ml ethyl acetate washed twice.The organic extraction that merges places drying on the sodium sulfate with the washing of alkene sodium bicarbonate aqueous solution, and vacuum concentration.Crude product flash chromatography method purifying, use hexane this moment for the first time: ethyl acetate=2: 1, carry out wash-out with ethyl acetate then.This product is used flash chromatography method purifying again, uses for the first time hexane this moment: ethyl acetate=2:, use hexane then: ethyl acetate=1: 2 is carried out wash-out.

Productive rate (pale yellow glassy foam body): 698mg(43%).

¹H NMR(200MHz, CDCl ₃): δ 2.88(dd, J9.4,13.3Hz, CHHPh), and 3.41(dd, J3.2,13.3Hz, CHHPh), 4.17-4.20(m, CH ₂O), 4.64-4.73(m, CHN), 6.13(s, CHN ₃), 7.21-7.42(m, 10H, aromatic series), 7.50(s, 4H, aromatic series). ¹³C NMR(50MHz, CDCl ₃): δ 37.9,55.8,63.1,66.6,127.2,128.7,128.9,129.0,133.7,134.1,152.0,168.2.

C) (S, S)-α, α '-diazido-1,4-phenylene-diacetic acid

At 0 ℃ with LiOHH ₂O(0.222g, 5.29mmol) drips of solution in water (8ml) is added to (S, S, S, S)-α, α '-two nitrine-β, β '-dioxy-β, β '-two (4-phenmethyl-2-oxygen-3-oxazolidinyl)-1 is in the solution in the 4-diethylbenzene (0.683g is 1.15mmol) at THF(16ml).This solution stirred 1 hour at 0 ℃, added the dilute sodium carbonate aqueous solution, used ethyl acetate extraction Chu oxazolidone again.Organic extraction is with rare aqueous sodium carbonate washed twice, the aqueous extract that merges adds aqueous potassium hydrogen sulfate and carries out acidifying, use ethyl acetate (2X) to extract its product again, its solution places dry and evaporation on the sodium sulfate, and resistates need not be further purified when the step uses down.

Productive rate: 206mg(65%)

¹H NMR（200MHz，CDCl ₃/DMSO-d ₆）：δ4.93（s，CH），7.40（s，Ph）. ¹³C NMR（50MHz，CDCl ₃/DMSO-d ₆）：δ67.5，126.6，134.0，170.1.

D) (S, S)-α, α '-diaminostilbene, 4-phenylene-diacetic acid

With 10%Pd/C(70mg) join (S, S)-α, α '-two nitrine-1, (180mg is 0.65mmol) at ethanol: acetate=8: in the solution 1(12ml) for the 4-phenylene-diacetic acid.The reaction slurry that obtains is at 15psi H ₂Hydrogenation is 4 hours under the condition.React slurry then through diatomite filtration, the diatomite filter cake is with several parts of washing with alcohol.Remove in the vacuum and desolvate, again by removing acetate with the methylbenzene azeotropic distillation.In ethyl acetate, precipitate white solid, filter and vacuum-drying.Productive rate: 99mg(68%), ¹H NMR(200MHz, D ₂O): δ 5.16(s, CH), 7.48(s, aromatic substance).

E) (S, S)-α, α '-two (9-fluorenyl methoxy carbonylamino)-1,4-phenylene-diacetic acid

With α, α '-diaminostilbene, (45mg, 0.2mmol) suspension in the mixture of hexamethyldisilazane (5ml) and trimethylchlorosilane (0.5ml) spends the night in 100 ℃ of stirrings under hydrogen the 4-phenylene-diacetic acid.Reaction mixture, vacuum is removed excessive silylating reagent again.Resistates is dissolved in the anhydrous methylene dichloride (5ml) again, and cools off in ice/water-bath.Add 9-fluorenyl methoxy carbonyl chlorine (109mg, 0.42mmol) solution in anhydrous methylene chloride (2ml), this reaction mixture stirred 3 hours under argon gas, behind the steaming vibrating dichloromethane, its resistates was dissolved in THF(4ml) and the mixture of water (0.5ml) in.With mixture stirring evaporation after 30 minutes.Get resistates and be placed in the ethyl acetate, and wash with water several times.Organic phase drying (MgSO ₄) after, filter and evaporation.Crude product is used ethyl acetate: acetate=95: 5 wash-outs with flash chromatography method purifying.

Productive rate (light yellow solid): 48mg(40%).

¹H NMR（200MHz，DMSO-d ₆）：δ4.20-4.50（m，CH and CH ₂），4.88（s，CHN），7.20-8.0（m，20H，aromatics）. ¹³C NMR（50MHz，DMSO-d ₆）：δ46.8，65.5，77.7，119.9，127.0，127.8，128.1，128.5，136.7，140.5，143.7，155.9，172.8.

Embodiment 9

Synthesizing of peptide: (pGlu-Glu-Asp) ₂-[(m)-Xyl] (Lys) ₂

With synthetic this peptide of Labortec peptide synthesizer.With Fmoc-Lys(Boc) Sasrin polymkeric substance (1.0g, 0.6mmol; Bachem A.G.; Replacement 0.6mmol/g) in the 100ml reaction flask of packing into, again with Fmoc-[(m)-Xyl] (160mg, 0.23mmol), DCC(290mg, 1.4mmol) and HOBt(211mg, 1.4mmol) at DMF(20ml) and in solution join in this polymkeric substance, allow reaction carry out 9 hours, add DCC(1.4mmol) and HOBt(1.4mmol), allow reaction carry out again 2 hours, use CH then ₂Cl ₂, 30%MeOH is at CH ₂Cl ₂In solution and DMF wash this polymkeric substance.Use 10%Ac ₂The solution of O in DMF makes the free amine group alkylation (a hour 3 times on the polymkeric substance; Negative Kaiser test).

Surplus one synthetic use Fmoc-Asp(OtBu)-Opfp(1.33g, 2.3mmol), Fmoc-Glu(OtBu)-Opfp(1.35g, 2.3mmol) and pGlu-five chlorophenyl ester (0.86g 2.3mmol) adopts standard program to carry out.Each coupling step all adds HOBt(3.50mg, 2.3mmol), carries out 1 hour.Can determine the coupling performance by negative Kaiser test.With the coupling of Fmoc-amino acid after use the DMF washing copolymer, with the solution division blocking group of 20% piperidines in DMF, polymkeric substance is washed with DMF once more.After in the end coupling is finished, use MeOH/CH ₂Cl ₂And CH ₂Cl ₂Washing copolymer.The weight of polymkeric substance-peptide of doing is 1g.Use TFA: CH ₂Cl ₂From polymkeric substance divide peptide at 1: 1, the solution lyophilize, resistates is water-soluble, filters (0.45 μ) and filtrate lyophilize.For being further purified, in this product of 100mg water-soluble (0.5ml), and with 2.1 * 1.5cm Beckman Ultrasphere OSO and solution A: the 0.1%TFA aqueous solution, B:0.1%TFA MeCN: H ₂O(40: 60) being prepared property of solution HPLC.Gradient is 0-15%B, and 120 minutes, flow velocity 5ml/ branch obtained the pure peptide of 19mg (＞95%) after with collected uniform fraction lyophilize.

Embodiment 10

The solid phase synthesis of peptide

Mainly according to principle (Atherton and the Sheppard of fluorenes methoxycarbonyl (Fmoc)-polymeric amide synthetic route, Solid phase peptide synthesis:a practical approach.Oxford IRL Press at Oxforcl Clniversity Press, 1989) carry out the synthetic of solid-phase peptide, use the synthetic resins that to purchase from the market; Manual or carry out batch when synthesizing with semi-automatic instrument (Labortec Reptidl Synthesizer 5P 650), these resins are to have the unsettled (Wang of acid, J.Am.Chem.Soc., 95,1328-1333,1973) or the highly unsettled linking agent of acid (people such as Merger, Tetrahedron Letters 29,4005-4008,1988) polystyrene.With full-automatic mode (people such as Atherton, J.Chem.Soc.Chem.Commun., 1151-2,1981) the assembly peptide on stir-in resin of LKB Biolynx 4170 Automatcd Peptide Synthesizer.The synthetic resins of buying has contained desired protected C-end Fmoc-amino-acid residue.Adopt dicyclohexyl carbodiimide (DCC)/1-hydroxy benzo diazole (HOBt) (Koenig and Geigtr; Chem.Ber.103,2034-2040,1070) activation or adopt people such as coupler PyBOP(Coste; Tetrahedron Lett.; 31,205-208,1990); with the protected Fmoc-amino acid of side chain pentafluorophenyl group ester (Kisfaludy and Schoen; Synthesis, 325-327,1983) acquisition elongation chain in various degree.Lysine side-chain is amino with tertiary butyl oxygen carbonyl-protection, and the side chain carboxyl group of L-glutamic acid and aspartic acid is with uncle-butyl ester protection.

The synthetic resins that has the de-protected C-of required N-end residue carries out acidylate with the shielded diamino dicarboxylic acid of semi-normal Fmoc by DCC and HOBt.Finish the reaction excessive reagent of flush away afterwards, and then once handle peptide resin, so that the fixing carboxyl that may still keep unbound state with DCC/HOBt.After this step, use the methanol wash resin, make any carboxyl inactivation, make it can not with the amino bonded of resin-bonded.At last the excess of ammonia amount is sealed by acidylate.Synthetic as the common mode of peptide continues then.

After required sequence is finished the solid phase assembly, use the trifluoroacetic acid be added with suitable scavenging agent people such as (, Int.J.PeptidlProtein Res, 36,255-268,1990) King, utilize the side chain of following to go the protection process and from synthetic resins, divide peptide.After the evaporation, isolate peptide with ether sedimentation, and dry.Carry out purifying with preparation property reversed phase high efficiency liquid chromatography then.

Table 1

Mutual-through type structure (Pyr-Glu-Asp) ₂-bridging residue-(Lys-OH) ₂The analytical data bridge residue type of peptide ^aThe HPLC method ^bPurity ^cFAB-MS

(retention time) be [M+H] (%) ⁺

1,2-benzene dipropionate 0-30-20 (20) 98 1219.1

1,3-benzene dipropionate 0-30-20 (19) 96 1219.8

1,4-benzene dipropionate 10-40-20 (18) 95 1219

2,5-sulphur benzene dipropionate 0-30-20 (18) 98 1225.3

Suitable-1,2-cyclopropane dipropionate 0-30-20 (15) 100 1183.5

Suitable-1,2-tetramethylene dipropionate

Instead-1,4-hexanaphthene dipropionate 0-30-20 (14) 99 1197.8

1,4-phenylene-diacetic acid ester

A refers to embodiment

This method of b is to represent with the gradient of mobile phase B in A in the whole time, and for example 10-40-20 means in the gradient of 10 beginnings and the gradient of finishing with 40%B at 20 minutes.Mobile phase: A) 0.1%TFA B) 0.1%TFA is in 40%MeCN.Post: Vydac TP54, C18,0.46 * 25 μ m, 5 μ m posts, 100

The hole.Flow velocity 1ml/ branch.

C means the integration (λ=215nm) of HPLC chromatographic peak.

Table 2

Has formula (Pyr-Glu-Asp) ₂-bridging residue-(Lys-OH) ₂The amino acid analysis data of peptide

Bridging residue type ^aAsp measured value Glu measured value Lys measured value

(theory) (theory) (theory)

1,2-benzene dipropionate

1,3-benzene dipropionate

1,4-benzene dipropionate 1.0 (1) 2.16 (2) 0.93 (1)

2,5-sulphur benzene dipropionate

Suitable-1,2-cyclopropane dipropionate

Suitable-1,2-tetramethylene dipropionate

Instead-1,4-hexanaphthene dipropionate 1.0 (1) 2.12 (2) 0.81 (1)

1,4-phenylene-diacetic acid ester

A means embodiment

Claims (21)

1, the compound that contains two strand Hemoregulatory peptides, described peptide is linked together by divalent abutment group-A-, this abutment group is connected on the C alpha atom of non-end amino acid in the equivalent position of each described peptide endways, the original side chain that wherein is connected to the C alpha atom on group-A-does not have, and described divalent abutment group-A-is

In the formula

Each y is 0,1 or 2 independently;

Each z is 0 or 1 independently;

Each Y is O, S or NR independently,

The organic group that R represents hydrogen or is connected with C,

B represents carbocyclic ring or heterocycle, its heterocycle at random contain one or two heteroatoms and at random by

-OR ^A,-NR ^AR ^A,-COOR ^AOr the heterocycle that halogen atom once replaces, secondary or three times replace;

Each R ^ARepresent hydrogen atom, alkyl, alkanoyl, alkoxyalkyl independently, but also hydroxylation of each group wherein.

2, peptide compounds according to claim 1, wherein said divalent abutment group-A-is

-CH ₂CH ₂BzCH ₂CH ₂-

Wherein Bz representative is by-OR ^A,-NR ^AR ^A,-COOR ^AThe phenyl ring that group or halogen atom randomly once replace, secondary or three times replace, R ^ASuch as claim 1 definition.

3,, it is characterized in that described strand Hemoregulatory peptides has the chemical formula I according to the described peptide compounds of arbitrary claim of claim 1 or 2

R wherein ^aRepresentative

N and m represent 0 or 1 independently in the formula;

P, q and r represent 1 or 2 independently;

S represents 3 or 4;

R ¹And R ²The both is a hydrogen atom or together for epoxy group;

R ³And R ⁴The both is hydrogen atom or represents the C-C key together;

R ⁵Be hydrogen or acyl group;

R ⁶And R ⁷Representation hydroxy or amino independently, but hydroxyl preferably,

R ⁸Represent hydrogen; C _2-6Alkyl; C _7-20Aralkyl, it can be with one or more hydroxyls, amino or methoxyl group substituting group; Or in metabolism unsettled S-protecting group;

R ⁹Represent hydrogen or methyl;

R ¹⁰Representation hydroxy or amino, the residue of amino acid L-glutamic acid, or peptide with N-end glutamic acid units.

4, peptide compounds according to claim 3, wherein n represents O in the peptide chain of chemical formula I.

5, according to the described peptide compounds of arbitrary claim of claim 3 or 4, wherein m represents O in the peptide chain of chemical formula I.

6, according to the described peptide compounds of arbitrary claim of claim 3-5, wherein said abutment group-A-is connected to the amino acid R of chemical formula I ^dThe C alpha atom on.

7, the arbitrary claim according to claim 3-6 requires described peptide compounds with chemical formula II

Wherein-A-such as claim 1 qualification, R ^a, R ^b, R ^c, R ^e, R ^fWith n all such as claim 3 qualification.

8, according to the described peptide compounds of arbitrary claim of claim 1-7, its chemical formula is as follows:

Wherein-A-such as claim 1 qualification.

9, a kind of containing as the described peptide compounds of arbitrary claim of claim 1-8 and the pharmaceutical composition of pharmaceutical carrier or vehicle.

10, the described peptide compounds of arbitrary claim according to claim 1-8 is applied to the irritation cell division.

11, the described peptide compounds of arbitrary claim according to claim 1-8 is applied to stimulate medullary cell to generate or the regeneration of marrow.

12, the described peptide compounds of arbitrary claim according to claim 1-8 is used to produce irritation cell splitted medicine.

13, the application in producing the medicine that stimulates medullary cell generation or bone marrow regeneration according to claim 12.

14, according to the application of the described peptide compounds irritation cell of the arbitrary claim division aspect of claim 1-8.

15, described according to claim 14 in the application aspect generation of stimulation medullary cell or the bone marrow regeneration.

16, stimulate fissional method among the patient, described method comprises to described patient uses significant quantity composition according to claim 9.

17,, it is characterized in that stimulating marrow to generate or the division of medullary cell according to the described method of claim 16.

The method of the described peptide compounds of arbitrary claim of 18, production claim 1-8 is gone protection comprising the derivative that makes part or all of protected peptide compounds.

The method of the described peptide compounds of arbitrary claim of 19, production claim 1-8, described method comprises

A) two-lactim metal replacement is generated its alkylation the two peptide ethers of two lactims subsequently,

B) make the two peptide ether hydrolysis of two-lactim of step (a) generate the α of bridging, α '-diamino acid,

C) amino acid with remainder adds in the peptide chain,

D) make any protected group go protection.

20, the bridging α that has following chemical formula, α '-diamino acid

Wherein-A-such as claim 1 qualification.

21, the two peptide ethers of two-lactim that have following chemical formula

Wherein-A-such as claim 1 qualification.