CN101407539A - Oligopeptide having marrow hemopoiesis protection function, preparation thereof, pharmaceutical composition containing the oligopeptide and use - Google Patents

Abstract

The invention discloses an oligopeptide chemical compound containing a neighboring structure of acid residues and alkaline residues, a method for synthesizing the oligopeptide chemical compound by a crossed matching strategy and medicine compounds containing the oligopeptide chemical compound. The oligopeptide chemical compound is proved to have the activity for protecting marrow hematogenesis functions through influences of oligopeptide products on GM forming and BFUE forming and influences on death rate of irradiating Gamma ray on mice, and the invention can prepare medicament for protecting marrow hematogenesis stem cells from being injured by chemotherapy medicines and radiotherapy.

Description

Protection hemopoietic function of bone marrow oligopeptides, its method for making and pharmaceutical composition and purposes

Technical field

The present invention relates to the oligopeptide compounds that a class contains acidic residues and alkaline residue adjacent structure, the preparation method of this class oligopeptide compounds; the pharmaceutical composition that contains this class oligopeptide compounds; and prepare the application that the protection marrow hemopoietic stem cells is avoided the medicine of chemotherapeutic and radiation-induced damage, belong to medical technical field.

Background technology

Present 60% to 70% tumour patient needs radiotherapy, chemotherapy; general operation back was carried out rational chemicotherapy and can in time kill " metastasis " in 2 to 4 week; effectively resist the recurrence and the transfer of tumour, and the key that can chemicotherapy adhere to is the protection of hemopoietic function of bone marrow.Yet many antineoplastic chemotherapy medicines all cause bone marrow depression in various degree, show as white corpuscle, thrombocyte decline at first, and along with chemotherapy dosage increases, red corpuscle and oxyphorase all descend when serious, even aplastic anemia may take place.No matter be radiotherapy or chemotherapy, all can when killing and wounding cancer cells, damage the hemopoietic function of patient's marrow.

Isolated a Goralatide tetrapeptide compound by name in 1977 from the tire Medulla Bovis seu Bubali, its structure is Ac-Ser-Asp-Lys-Pro-OH (abbreviating SP4 as).Discover that SP4 all can alleviate damage [Bogder, people such as AE, Int J Cancer, 1998, the 76:38-46 of the hemopoietic function of bone marrow that chemotherapeutic, gamma-rays and phototherapy cause outside animal body, in the body; Watanabe, people such as T, Exp Hematol, 1996,24:713-721; Masse, people such as A, Blood, 1998,91:441-449; Wierenga, people such as PK, Br J Haematol, 1997,99:692-698; Jackson, people such as JD, J Hematother Stem Cell Res, 2000,9:489-496].SP4 can also significantly improve the survival rate [Suzuki, people such as A, Exp Hematol, 1998,26:79-83] of raying zoografting hemopoietic stem cell.Therefore for the patient who accepts chemotherapy, radiotherapy, the partner treatment that the medicine of exploitation SP4 type is used for cancer is the research topic that merits attention.

The same with other oligopeptides, SP4 is too fast in intravital degraded of machine and the speed that is eliminated, and biologically stable is too poor, so the time of performance drug action is very short, therefore existing experimentation on animals can only can't be used for clinical to inject at least every day three times or to carry out with the mode of Micropump successive administration.SP4 is carried out structure of modification, and improving its tolerance proteasome degradation, improving biologically stable is the basic outlet that it develops into clinical application.So far, existed the design of some SP4 modification with synthetic, as changing the peptide bond in the molecule (CONH) into isosteric pseudopeptide bond (Pseudo-Peptide) successively, as CONH → CH ₂NH[Gaudron, people such as S, Stem Cells, 1999,17:100-106]; Certain residue is replaced, as Homo-Ser → Ser[Thierry, and people such as J, J Pept Sci, 2001,7:284-293] and the local Freidinger ring texture [Kumar that introduces of peptide chain, people such as S, J Org Chem, 2005,70:5946-5953] etc. form, though keeping the biologically stable that has improved SP4 on the former active basis,, academic significance only arranged because of the synthetic height that drops into, big producing feasibility is poor, and all difficulty develops into practical medicine.

Summary of the invention

The object of the present invention is to provide a kind of new oligopeptide compounds that contains acidic residues and alkaline residue adjacent structure.

Another object of the present invention is to provide a kind of method for preparing oligopeptide compounds.

A further object of the present invention is to provide a kind of pharmaceutical composition that contains one or more oligopeptide compounds.

Another purpose of the present invention is to provide the application of a kind of this compounds in preparing the medicine of protecting marrow hemopoietic stem cells to avoid chemotherapy or radiation-induced damage.

In order to finish the present invention's purpose, can adopt following technical scheme:

Compound of the present invention as general formula as (I), (II) with (III):

Wherein,

R ¹=H, Boc, Ac, Pro, RCO (R=C _1～10Alkane), Sal (salicylyl), Succinic Acid list acyl

X ¹=Ser, Tar (tartrate list acyl), Thr, Cys or des (disappearance)

X ²=Asp, Glu, Pro, Ida (iminodiethanoic acid list acyl) or des

X ³=any natural amino acid residue, β-Ala, Inp (γ-piperidine formyl) or Asp-Ser fragment

R ²=NHR[R=C _1～10Alkane or Bn], NR ₂[R=C _1～10Alkane],

N=2～5

Y=des or CH ₂,-SS-,-S-, CONH.

Preferred compound is selected from

Ac-Ser-Asp-Lys-Pro-NHEt (1)

Ac-Ser-Asp-Lys-NHEt (3)

Ac-Ser-Asp-Lys-NMe ₂ (4)

Sal-Ser-Asp-Lys-NHEt (5)

HOOC-(CH ₂) ₂-CO-Ser-Asp-Lys-NHEt (6)

CH ₃CH ₂CO-Ser-Asp-Lys-Phe-NHEt (9)

Sal-Ser-Asp-Lys-Phe-NMe ₂ (10)

HOOC-(CH ₂) ₂-CO-Ser-Asp-Lys-Phe-NMe ₂?(11)

Tar-Glu-Lys-Inp-NHMe (14)

Tar-Asp-Lys-Inp-NHMe (15)

The present invention relates to the method for preparing The compounds of this invention on the other hand; comprise following synthesis strategy; comprise the synthetic and solid phase synthesis dual mode of liquid phase, this method has been set up the strategy (Cross Mated Strategy, i.e. CMS) of Boc chemoproection mode and Fmoc chemoproection mode cross-matched.The synthesis mode that wherein relates to liquid phase synthesis mode, solid phase synthesis mode and " sequence n-1 assembling/ammonia separate+1 ".

The target compound that realization meets said structure synthesizes, and the present invention has set up a kind of with the New Policy of Boc protection with Fmoc protection dual mode cross-matched (Cross Mated).The raw material amino acid protection form that wherein relates to is: Boc-Arg (HCl)-OH, Fmoc-Asp (tBu)-OH, Fmoc-Glu (tBu)-OH, Boc-Ida-OH, Boc-Cys (Acm)-OH or Fmoc-Cys (Trt)-OH, Fmoc-Lys (Boc)-OH or Boc-Lys (Boc)-OH; Boc-Phe-OH; Boc-Pro-OH or Fmoc-Pro-OH; Boc-Tyr-OH; Fmoc-Ser-OH; Fmoc-Thr-OH, Boc-β-Ala-OH and Boc-Inp-OH.The condensing agent component is respectively the molar mixture that waits of DCC (dicyclohexylcarbodiimide) and HOBt (N-hydroxybenzotriazole), HBTU (O-benzotriazole-N, N, N ', N '-tetramethyl-urea father-in-law hexafluorophosphate) with HOBt and NMM (N-methylmorpholine) etc. molar mixture, or TBTU (O-benzotriazole-N, N, N ', N '-tetramethyl-urea father-in-law a tetrafluoro borate) wait molar mixture.The abbreviated form of other reagent and solvent is: H ₂NMe (methylamine), H ₂NEt (ethamine), H ₂NBu (n-Butyl Amine 99), TEA (triethylamine), H ₂NBn (benzylamine), HNMe ₂(dimethylamine), TFA (trifluoroacetic acid), TESi (triethyl silicon), DMF (dimethyl formamide), Py (pyridine), DCM (methylene dichloride), THF (tetrahydrofuran (THF)), HOAc (acetate).Remove the Boc reagent composition: 3N HCl/HOAc or saturated HCl/EtOAc or 50%TFA/DCM.Remove the Fmoc reagent composition: 20% piperidines/DMF.Remove Pac (benzene carbonyl methylene radical ester) composition: Zn/HOAc.Excision resin (ammonia is separated) composition: H ₂NR/H ₂O-THF (1: 1).

During the compound that the present invention relates to is synthetic; having changed that the peptide of Boc protected mode is synthetic must be through strong acid [as HF or HBr/HOAc or TFMSA (trifluoromethanesulfonic acid)] cracking excision resin, or Fmoc protected mode synthetic essential used expensive resin (as Wang resin, Rink resin etc.) and excise two kinds of resin traditional ways independently each other with the TFA cracking.Having adopted the synthesis strategy of cross-matched is solid phase carrier with the most cheap chloromethyl resin, and the amino acid of dual-purpose Boc and two kinds of protected modes of Fmoc is raw material.Wherein correctly using Boc-amino acid and Fmoc-amino acid is the key of guaranteeing the synthetic technology success or not that the present invention relates to.To this, the present invention has determined the principle of following reasonable use protected mode:

1. amino acid that only need not side chain protected (as Gly, Ala, Phe, Leu, Ile Val) can use the Boc protected mode.

2. the amino acid whose condensation of Boc-should be prior to Fmoc-amino acid.

In a single day 3. use Fmoc amino acid, can not be in condensation subsequently again with Boc amino acid (N holds last residue to introduce with Boc amino acid).

4. some side chains can protect the amino acid that also can not protect (Arg, Tyr, Ser, Thr, Trp, Asn Gln) adopts minimum protected mode (being the side chain no protective mode).

If 5. the C of object construction end contains the amino-acid residue that side chain needs protection,, then should use Fmoc amino acid to be raw material for fear of behind end of synthesis, using strong acid strategy (as HF, HBr/HOAc or TFMSA) to remove Side chain protective group.In the case, whole condensations subsequently all should be adopted Fmoc-amino acid (except residue of N end).

6. under situation 5., the Fmoc of C end can not upload (Loading) reaction with chloromethyl resin.Therefore adopt Boc-β-Ala, Boc-Abu or Boc-Aca at first realize that with chloromethyl resin loading is necessary as space arm (spacer).

7. based on above-mentioned prerequisite, all the other residues use the Fmoc amino acid form to participate in condensation without exception.

The basic line of " sequence n-1 is synthetic/ammonia separates+1 " technology that the present invention relates to is as follows:

(promptly project organization lacks the alpha-non-natural amino acid residue structure (X) that a C holds to n-1 wherein for this reason, the latter in ammonolysis reaction with n-1 sequence bonding, the benzyl of cracking simultaneously ester Linker realizes the excision of solid phase carrier, finishes with this mode as the synthetic of compound among the embodiment 2,7.The benefit of this synthetic technology be following some:

(1) when sequence is synthetic, saves the condensation and the deprotection two-step reaction of a C-terminal.

(2) be the Fmoc protected mode without all raw materials, available according to circumstances cheap Boc-amino acid replaces Fmoc-amino acid.

(3) need not be than the immobilized resin in the expensive Fmoc strategy, only with the most cheap chloromethyl resin.

(4) purity of crude product obviously is better than the product of traditional Boc/ strong acid cracking or Fmoc/TFA cracking gained

Purity.Principle wherein is that the present invention sets up " cross-matched " strategy and can in the end excise the whole protecting groups that remove fully before the resin on the target peptide; " bare " peptide one resin that generates is separated the crude product that is not contained other impurity through ammonia again, so purity is very desirable.By contrast; traditional Boc chemistry and Fmoc chemistry in the end also excise solid phase carrier the various protecting group of one-step removal the time, and plurality of impurities such as the binding substances of crude product that obtains and deprotecting regent, scavenging agent, protecting group and scavenging agent, neutral alkali mix.Crude product purity difference, separation and purification in a conventional manner more miscellaneous.

Accompanying drawing 1 is the comparison diagram of the cracking of Boc/ strong acid, Fmoc/TFA cracking and cross-matched strategy of the present invention.

The advantage of the inventive method: cross-matched strategy synthetic method, the preparation method is practicable, and is economical and practical

Further aspect of the present invention also relates to the pharmaceutical composition of The compounds of this invention as active ingredient. This medicine group Compound can be according to method preparation well known in the art. Can be by with The compounds of this invention and one or more pharmaceutically Any formulation that is suitable for human or animal's use is made in acceptable solid or liquid excipient and/or assistant agent combination. The content of The compounds of this invention in its pharmaceutical composition is generally the 0.1-95 % by weight.

The compounds of this invention or contain its pharmaceutical composition can the unit dosage form administration, method of administration can be intestines Road or non-enteron aisle, as oral, intravenous injection, intramuscular injection, hypodermic injection, nasal cavity, oral mucosa, eye, Lung and respiratory tract, skin, vagina, rectum etc.

Form of administration can be liquid dosage form, solid dosage forms or semisolid dosage form. Liquid dosage form can be solution (bag Draw together true solution and colloidal solution), emulsion (comprising o/w type, w/o type and emulsion), supensoid agent, injection (bag Draw together liquid drugs injection, powder-injection and transfusion), eye drops, nasal drop, lotion and liniment etc.; Solid dosage forms can be sheet Agent (comprising ordinary tablet, enteric coatel tablets, lozenge, dispersing tablet, chewable tablets, effervescent tablet, oral disnitegration tablet), capsule Agent (comprising hard shell capsules, soft capsule, capsulae enterosolubilis), granule, powder, micropill, dripping pill, suppository, film, Paster, the agent of gas (powder) mist, spray etc.; Semisolid dosage form can be ointment, gel, paste etc.

The compounds of this invention can make ordinary preparation, also make be sustained release preparation, controlled release preparation, targeting preparation and Various particulate delivery systems.

For The compounds of this invention is made tablet, can be extensive use of various excipient well known in the art, bag Draw together diluent, binder, wetting agent, disintegrant, lubricant, glidant. Diluent can be starch, paste Essence, sucrose, glucose, lactose, sweet mellow wine, sorbierite, xylitol, microcrystalline cellulose, calcium sulfate, phosphorus Acid hydrogen calcium, calcium carbonate etc.; Wetting agent can be water, ethanol, isopropyl alcohol etc.; Adhesive can be starch slurry, Dextrin, syrup, honey, glucose solution, microcrystalline cellulose, mucialga of arabic gummy, gelatine size, carboxymethyl fibre Tie up plain sodium, methylcellulose, hydroxypropyl methylcellulose, ethyl cellulose, acrylic resin, carbomer, Polyvinylpyrrolidone, polyethylene glycol etc.; Disintegrant can be dried starch, microcrystalline cellulose, low-substituted hydroxypropyl Base cellulose, PVPP, Ac-Di-Sol, sodium carboxymethyl starch, bicarbonate Sodium and citric acid, polyoxyethylene sorbitol fatty acid ester, dodecyl sodium sulfate etc.; Lubricant and glidant Can be talcum powder, silica, stearate, tartaric acid, atoleine, polyethylene glycol etc.

Tablet further can also be made coating tablet, for example sugar coated tablet, thin membrane coated tablet, ECT, Or double-layer tablets and multilayer tablet.

For capsule is made in the administration unit, can be with active ingredient The compounds of this invention and diluent, help stream Agent mixes, and mixture is directly placed hard shell capsules or soft capsule. Also can be with the active ingredient The compounds of this invention earlier With diluent, binder, disintegrant granulation or micropill, place again hard shell capsules or soft capsule. Be used for system Each diluent, binder, wetting agent, disintegrant, the glidant kind of standby The compounds of this invention tablet are also available Capsule in the preparation The compounds of this invention.

For The compounds of this invention is made injection, can water, ethanol, isopropyl alcohol, propane diols or they Mixture as solvent also adds an amount of this area solubilizer commonly used, cosolvent, pH adjustment agent, osmotic pressure adjusting Agent. Solubilizer or cosolvent can be poloxamer, lecithin, HP-β-CD etc.; PH adjusts agent Can be phosphate, acetate, hydrochloric acid, NaOH etc.; Osmotic pressure regulator can be sodium chloride, sweet dew Alcohol, glucose, phosphate, acetate etc. As prepare freeze drying powder injection, also can add sweet mellow wine, glucose Deng as proppant.

In addition, such as needs, also can add colouring agent, anticorrisive agent, spices, flavouring or its in the pharmaceutical preparation Its additive.

For reaching the medication purpose, strengthen result for the treatment of, medicine of the present invention or pharmaceutical composition can be with any known The medication administration.

The dosage of The compounds of this invention pharmaceutical composition is according to the character that will prevent or treat disease and serious Degree, the individual instances of patient or animal, method of administration and formulation etc. can have large-scale variation. General next Say that the suitable dose scope of the every day of The compounds of this invention is the 0.001-150mg/Kg body weight, is preferably 0.1-100mg/Kg body weight, more preferably the 1-60mg/Kg body weight most preferably is the 2-30mg/Kg body weight. Above-mentioned Dosage can a dosage unit or is divided into several dosage unit administrations, this depend on the doctor clinical experience and Comprise the dosage regimen of using other treatment means.

Compound of the present invention or composition can be taken separately, or make with other treatment medicine or symptomatic drugs merging With. When compound of the present invention and other medicine existence synergy, should adjust it according to actual conditions Dosage.

The present invention is by (1) external bone matrix stem cell growth proliferation experiment; (2) external marrow mesenchymal stem The experiment of cell colony form; (3) protection navel blood stem cell activity experiment; (4) mouse endogenous protective bone marrow suppression Experiment; (5) radiotherapy mouse survival description of test compound of the present invention have the protection marrow hemopoietic stem cells avoid The treatment of chemotherapeutic (cytarabine) and gamma-rays damage is used, and makes the cancer patient accept to tie up in chemotherapy and the radiotherapy Hold normal hematopoiesis function, provide condition for curing smoothly cancer

Embodiment

Embodiment 1Ac-Ser-Asp-Lys-Pro-NHEt's (1) is synthetic

1. the inferior phenyl methyl ketone (Pac) of Boc-Pro--resin is synthetic

Take by weighing 10g (5mmol) Pac-resin (replacing equivalent 0.5mmol/g, granularity 100-200 order, degree of crosslinking 1%), the Boc-Pro-OH of three times of (15mmol) mole numbers, the K of three times of mole numbers ₂CO ₃And the KI of 1/10th mole numbers (0.5mmol) is common and 150mL DMF is blended in the round-bottomed flask.Under 70 ℃ of airbath heating and rotating condition, reaction 24h.The reaction suspended matter is moved in the filter of band suction filtration sand plate, wash 5 times about at every turn 50mL with 60 ℃ DMF.Use following solvent filter wash (* number of times) subsequently successively: 95%EtOH * 3, DMF * 3,50%EtOH/H ₂O * 3, anhydrous EtOH * 3, anhydrous Et ₂O * 2.Drain and take out resin behind the solvent and dry in the air under the infrared light to about 50 ℃, air-dry to constant weight.Get Boc-Pro-OCH through weighing ₂-polystyrene resin 10.65g, actual weightening finish 650mg (theoretical Δ W=670mg), this step yield is 97%.

2. remove the Boc protection

Get Boc-Pro-Pac-resin 10g and 50%TFA/DCM (100mL) and be mixed in the reaction tubes of tool sand filtration plate, shake 3min.Extract acid solution, add 50%TFA/DCM solution 100ml again, shake 40min again, resin is used following solvent filter wash successively after the filtering acid solution, about at every turn 50mL, 1min:DCM * 5, anhydrous Et ₂O * 2, DMF * 3,95%EtOH * 3, DMF * 2, EtOAc * 2, anhydrous Et ₂Drain O * 2.Get the about 2mg of resin-like, put into small test tube and carry out the triketohydrindene hydrate color reaction, check free amine group.Test solution and sample resin are dark-brown as a result, show that the Boc base is removed, generate the HClPro-Pac resin.

3. Fmoc-Lys (Boc)-Pro-Pac-resin is synthetic

It is standby to take by weighing 3g HClPro-Pac-resin (1.44mmol).

The activation of carboxyl component [this example is Fmoc-Lys (Boc)-OH]: the band protecting group amino acid, HOBt and the HBTU that take by weighing three times of molar weights of amino group on the suitable resin mix with the dry DMF of 20mL, make molten entirely.The NMM that adds four times of molar weights at last, mixing is placed 2min.HClPro-Pac-mixed with resin with this activated solution and 1.44mmol amount.Room temperature is shaken and is carried out condensation reaction, needs 6h approximately.Filtering supernatant liquor, the following successively preface filter wash of resin: DMF * 3, EtOH * 3, DMF * 3, EtOH * 2, anhydrous Et are finished in reaction ₂O * 2.Get the about 2mg of resin sample and carry out the triketohydrindene hydrate color reaction, test solution and resin particle are light yellow (feminine gender) as a result, show that amino on the Pro is fully by the Lys acidifying.

4. remove the Fmoc protection

Above-mentioned peptide resin and 50% piperidines/DMF carry out removing fast for twice reaction (1min, 4min).Be intended to prevent to generate the cracking in advance that DKP causes.[annotate: except second residue of C end, the condition that other position residue removes Fmoc is 20% piperidines/DMF, twice of 3min and 20min].Resin is used following solvent filter wash successively after removing Fmoc: DMF * 3, EtOH * 3, DMF * 3, EtOH * 3, anhydrous Et ₂Drain O * 2.

5. Fmoc-Asp (tBu)-Lys (Boc)-Pro-Pac-resin is synthetic

Take by weighing Fmoc-Asp (the tBu)-OH of 4.32mmol and HOBt, the HBTU of same mole and mix with DMF, activation subsequently and condensation operation are identical with (3), and it is identical with (4) to take off Fmoc.

6. Ac-Ser-Asp (tBu)-Lys (Boc)-Pro-Pac-resin is synthetic

Condition according to (3) and (4) obtains intermediate Ser-Asp (tBu)-Lys (Boc)-Pro-Pac-resin, adds 10 times of molar weight Ac then ₂O and 2mLPy-20mLDMF mixed solution, room temperature is shaken 2h.By behind the above-mentioned conventional filter wash, sample is carried out the triketohydrindene hydrate detection and is shown that acetylize is complete.

7. remove Side chain protective group (tBu and Boc)

Above-mentioned peptide resin reacts 10min with 50%TFA/DCM earlier, extracts acid solution.Add again and contain 90%TFA-5% thio phenyl methyl ether-5%H in right amount ₂The mixed solution of O, room temperature is shaken 50min, successively resin is carried out following filter wash: DCM * 5 after the reaction, anhydrous Et ₂O * 2,95%EtOH * 3, DMF * 3,6%TEA/EtOAc * 2, DMF * 3,95%EtOH * 3, anhydrous Et ₂Drain O * 2, obtains unprotected side chain protection, N holds acetylizad tetrapeptide resin.

8. ethamine is separated the excision resin, 1 preparation

Above-mentioned Ac-Ser-Asp-Lys-Pro-Pac-resin with contain 70%H ₂The H of NEt ₂O solution and isopyknic THF mixed solution (50mL) are mixed in the reaction tubes of excellent sealing.Place room temperature (or shake) 24h, collect filtrate.Remaining resin 50%EtOH filter wash twice, merging filtrate is evaporated to dried under 50 ℃.The residue that obtains (semi-solid) mixes with 20mLEtOH and 20mL toluene, is evaporated to dried again under 50 ℃.Residue is through the anhydrous Et of 50mL ₂O fully grinds, and obtains white powdery precipitation.Collecting precipitation obtain the heavy 570mg of product 1 (theoretical amount: 740mg), overall yield 77%.Analyze through FAB-MS: 515.3 (M+H).

Embodiment 2

Synthesizing (2)

1. Fmoc-Lys (Boc)-Pac-resin is synthetic

Because the Fmoc base can not tolerate K ₂C _O3/ 60 ℃ condition, synthetic the using with Fmoc-Lys (Boc)-OH equivalent DIEA of this example replaces K ₂CO ₃, temperature changes room temperature into, and the reaction times is extended down to 35h, and all the other operations are identical with the condition of (1) among the embodiment 1.The result obtains 11.57g Fmoc-Lys (Boc)-Pac-resin, and weightening finish 1.57g (theoretical amount: 1.938g), actual recovery 81%.

2. Ac-Ser (tBu)-Asp (tBu)-Lys (Boc)-Pac-resin is synthetic

Get 2g Fmoc-Lys (Boc)-Pac-resin, obtain Acization full guard resin 2.25g according to embodiment 1 (3)～(6) condition.

3. the piperidines aminolysis excises resin, Preparation

Above-mentioned full guard three peptide resins and 8mL piperidines and 8mL THF are mixed in the sealable reaction tubes rotational response 40h in 50 ℃ of airbaths.Collect filtrate, remove TH-F under reduced pressure, surplus solution adds 100mL 1N HCl aqueous solution extraction three times again with 100mL EtOAc dilution, removes excessive piperidines.Organic layer washes twice with water, anhydrous Na ₂SO ₄Drying and concentrating under reduced pressure.Residual solids is mixed with 50mL toluene, and dried through being evaporated to again, residual solids is directly used in acidolysis and removes side chain protected.

4. remove side chain protected, the preparation of target compound 2

Above-mentioned solid and TFA/ thio phenyl methyl ether/H ₂Mix under O (90: 5: 5) solution is bathed outside frozen water and stir 1h.Remove TFA under reduced pressure under 40 ℃, residue is evaporated to dried with after 100mL EtOAc/ toluene (1: 1) mixes once more.Residue mixes with 100mL water makes most of dissolving, and the aqueous solution is removed oil-soluble impuritieses such as thio phenyl methyl ether through EtOAc extraction three times (100mL * 3) again.The water layer concentrating under reduced pressure is closely dried, residue be evaporated to again after 20mLEtOH and 30mL toluene mix absolutely dry.Residue is through anhydrous Et ₂O fully grind Powdered precipitation.Filter collecting precipitation get the heavy 323mg of product 2 (form with trifluoroacetate exists) (theoretical amount: 571mg), overall yield 56.6%.2 structure is through FAB-MS analytical proof: 458.4 (M+H).

Embodiment 3Ac-Ser-Asp-Lys-NHEt's (3) is synthetic

Synthetic method and condition are identical with embodiment 1.Obtain at last the heavy 311mg of product 3 (theoretical amount: 410mg), total recovery 75.8%, FAB-MS proves conclusively structure: 418.4 (M+H).

Embodiment 4Ac-Ser-Asp-Lys-N (Me) ₂Synthesizing (4)

According to the method for embodiment 1 obtain product 4 heavy 281mg (theoretical amount: 410mg), overall yield 68.5%, FAB-MS proves conclusively structure: 418.2 (M+H).

Embodiment 5Sal-Ser-Asp-Lys-NHEt's (5) is synthetic

According to the method for embodiment 1, prepare intermediate Ser (tBu)-Asp (tBu)-Lys (Boc)-Pac-resin earlier.Different with the Acization of front is, this example is the carboxyl component with Whitfield's ointment (Sal), and condensing agent and condition are identical with other amino acid.Subsequently remove side protection and ethamine is separated also identical with embodiment 1.Obtain at last the heavy 336mg of product 5 (theoretical amount: 409mg), total recovery 68.7%, FAB-MS:496.2 (M+H).

Embodiment 6HOOC-(CH ₂) ₂-CO-Ser-Asp-Lys-NHEt's (6) is synthetic

Replace diacetyl oxide in the compound 3 with Succinic anhydried during this synthetic final step condensation, all the other conditions are identical with embodiment 1, at last product 6 weight 308mg (theoretical amount: 461mg), total recovery 66.9%, FAB-MS:476.1 (M+H).

Embodiment 7

Synthesizing (7)

The condensation reaction condition is identical with embodiment 6.The mode that elder generation's ammonia is separated the back deprotection is identical with embodiment 2.Get product 7 heavy 281mg (theoretical amount 490mg) at last, total recovery is 57.3%, FAB-MS:502.1 (M+H).

Embodiment 8

Synthesizing (8)

1. prepare Boc-Tyr-OCH according to 1. condition among the embodiment 1 ₂-polystyrene resin.The difference is that with chloromethyl resin (substitution value is 0.53mmol/g) as solid phase carrier and Boc-Tyr-OH bonding.Wherein equal 1. identical with embodiment 1 of the amount ratio of each reactant and other condition obtains Boc-Tyr-OCH at last ₂-polystyrene resin 11.206g, (theoretical value: 1.295g), this goes on foot yield 93% to actual weightening finish 1.206g.

2. Pro-Ser (tBu)-Asp (tBu)-Lys (Boc)-Tyr-OCH ₂The preparation of-resin

Get the above-mentioned resin of 2g and at first obtain intermediate Fmoc-Pro-Ser (tBu)-Asp (tBu)-Lys (Boc)-Tyr-OCH according to 2.～5. mode among the embodiment 1 ₂-resin.The Fmoc (condition with embodiment 1 4.) that sloughs the N-end then obtains Pro-Ser (tBu)-Asp (tBu)-Lys (Boc)-Tyr-OCH ₂-resin.

Preparation

This goes on foot into ring and realizes according to intramolecularly Mannich reaction: get 37% formalin 6mL, dioxane 10mL and TFA 0.1mL and above-mentioned Pro-Ser (tBu)-Asp (tBu)-Lys (Boc)-Tyr-OCH ₂-mixed with resin, room temperature reaction 5 days is got sample and is carried out three same color reactions, and the result is light yellow (feminine gender), and the phenol ring that shows the imido grpup of N end Pro and Tyr is through CH ₂Formed Mannich alkali.

4. remove side chain protected and ammonia is separated, the preparation of compound 8

According to 8. the condition of 7. reaching among the embodiment 1 get target product 8 heavy 506mg (theoretical amount: 685mg), total recovery 73.8%, FAB-MS measurement result: 648.3 (M+H).

Embodiment 9CH ₃CH ₂CO-Ser-Asp-Lys-Phe-NHEt's (9) is synthetic

1. Boc-Phe-OCH ₂The preparation of-polystyrene resin

With reference to 1. condition among the embodiment 8, obtain target compound 11.14g, increment 1.14g (theoretical Δ W=1.21), this goes on foot yield 94%.

2. Ser (tBu)-Asp (tBu)-Lys (Boc)-Phe-OCH ₂-resins

Get 2g Boc-Phe-OCH ₂-polystyrene resin obtains this intermediate with reference to 2.～5. condition among the embodiment 1.

3. CH ₃CH ₂CO-Ser (tBu)-Asp (tBu)-Lys (Boc)-Phe-OCH ₂The preparation of-resin

Except replacing with propionic anhydride the diacetyl oxide, 6. operation is identical among all the other conditions and the embodiment 1.

4. the preparation of target product 9

Wherein relate to remove the condition that side chain protected and ethamine separates 8. identical with 7. reaching of embodiment 1, obtain at last product 9 heavy 426mg (theoretical amount: 613mg), total recovery 69.6%, FAB-MS measured value: 579.2 (M+H).

Embodiment 10Sal-Ser-Asp-Lys-Phe-NMe ₂Synthesizing (10)

1. Ser (tBu)-Asp (tBu)-Lys (Boc)-Phe-OCH ₂2. condition is identical among the preparation of-polystyrene resin and the embodiment 9.

2. N-holds the bigcatkin willow acidylate

Get the HBTU of Whitfield's ointment (Sal) 690mg (5mmol) and equimolar amount, HOBt and NMM are mixed in DMF.All the other conditions are identical with embodiment 1.

3. the preparation of target compound 10

It is 8. identical that 7. removing of relating to reach among side chain protected and diformazan aminolysis and the embodiment 1.Obtain at last the heavy 255mg of product 10 (theoretical amount: 680mg), total recovery 37.5%, FAB-MS measured value: 643.2 (M+H).

Embodiment 11HOOC-(CH ₂) ₂CO-Ser-Asp-Lys-Phe-NMe ₂Synthesizing (11)

1. Ser (tBu)-Asp (tBu)-Lys (Boc)-Phe-OCH ₂1. condition is identical among the preparation of-resin and the embodiment 10.

2. N end Succinic Acid list acidylate is carried out according to the final step condensation of embodiment 6.

3. the preparation of target compound 11 obtains the heavy 264mg of product according to 3. condition among the embodiment 10 (theoretical amount: 659mg), total recovery is 40%, the FAB-MS measured value: 623.3 (M+H).

Embodiment 12

Synthesizing (12)

This product and compound 8 are similar also to be the Mannich cyclisation product, and difference is the Lys-Asp-Ser structure of inverted sequence for centre, and the C end is the dimethyl substituted amide.Synthesis condition is identical with embodiment 8.Obtain at last the heavy 269mg of compound 12 (theoretical amount: 686mg), total recovery 39.3%, FAB-MS measured value: 648.2 (M+H).

Embodiment 13

Synthesizing (13)

1. Boc-β-Ala-OCH ₂The preparation of-polystyrene resin

With Boc-β-Ala-OH and chloromethyl resin is raw material, obtains Boc-β-Ala-OCH according to the mode of embodiment 8 ₂-polystyrene resin 10.775g, actual weightening finish 775mg (theoretical weightening finish 808mg), this goes on foot yield 96%.

2. obtain six peptide resin H-Cys (Trt)-Ser (tBu)-Asp (tBu)-Lys (Boc)-β-Ala-OCH of side chain protected according to general condensation condition among the embodiment 1 ₂-resin

3. remove side chain protected

The peptide resin 2.1g of above-mentioned band side protection (～1mmol) contain TFA-thio phenyl methyl ether-H with 20mL earlier ₂The solution hybrid reaction 40min of O (90: 5: 5).Extract and add 20mL again after the acid solution and contain TFA-Et ₃The solution hybrid reaction 30min of SiH-DCM (5: 5: 90).Press order filter wash: DCM * 5, EtOH * 3, DMF * 2, EtOH * 3 subsequently successively.

4. oxidation generates disulfide linkage

Above-mentioned six bare peptide resins and 20mL 6%TEA/DMSO hybrid reaction 38h, filter wash peptide resin routinely then.

5. methylamine is separated, 13 preparation

With 33%H ₂NMe/H ₂O solution is aminolysis reagent, and 8. operation is identical among condition and the embodiment 1.(theoretical amount: 636mg), total recovery is 77.5%, the ESI-MS analytical value: 659.4 (M+Na) to obtain product 13 heavy 493mg at last.

Embodiment 14Tar-Glu-Lys-Inp-NHMe's (14) is synthetic

1. Boc-Inp-OCH ₂The preparation of-polystyrene resin

With Boc-Inp-OH (γ-piperidine carboxylic acid) 3.65g (16mol) is raw material and chloromethyl resin 10g (5.3mmol) bonding, 1. identical among reaction conditions and the embodiment 1, at last Boc-Inp-OCH ₂-polystyrene resin 10.85g, actual weightening finish 0.850g (theoretical weightening finish 1.015g), this step yield is 83.7%.

2. the condensation condition according to embodiment 1 obtains H-Glu (tBu)-Lys (Boc)-Inp-OCH ₂-polystyrene resin.

3. Tar-Glu (tBu)-Lys (Boc)-Inp-OCH ₂The preparation of-resin

This step replaces the Ser and side protection tripeptides resin condensation of precursor structure with L-tartrate (Tar), condition and preceding together.

4. take off side protection, methylamine is separated 14 preparation

Condition is identical with front embodiment, obtain the heavy 345mg of product 14 (theoretical amount: 490mg), total recovery 70.5%, ESI-MS analyzes: 545.3 (M+H).

Embodiment 15Tar-Asp-Lys-Inp-NHMe's (15) is synthetic

Except replacing the Glu with Asp, other structure and synthesis condition were all identical with embodiment 15 during this was synthetic, and (theoretical amount: 477mg), total recovery is 75.7%, ESI-MS analysis: 531.2 (M+H) to obtain product 15 weight 361mg at last.

Embodiment 16

Synthesizing (16)

1. Boc-Aca-OCH ₂The preparation of-polystyrene resin

With Boc-Aca-OH (omega-amino-caproic acid) 3.73g (16mmol) is raw material and chloromethyl resin 10g (5.3mmol) bonding, and 1. identical among reaction conditions and the embodiment 1 obtains Boc-Aca-OCH at last ₂-resin 10.99g, actual weightening finish 0.99g (theoretical weightening finish 1.04g), this step yield is 95.1%.

Preparation

With Boc-Lys (Boc)-OH is the carboxyl component, with H-Aca-OCH ₂The condensation condition of-resin is identical with the standard conditions of embodiment 1.

Preparation

Since the amino component in this step contain two take place simultaneously acidylate-NH ₂Therefore gene respectively goes on foot carboxyl component, the condensing agent that condensation uses and all Duos one times than each routine consumption of front.Other condition is identical with the condensation circulation of embodiment 1.

4. remove the example protection, ethamine is separated, the preparation of product 16

Mode according to embodiment 1 obtains target product 16, and (theoretical amount: 927mg), total recovery is 81.1% to heavy 752mg, and ESI-MS analyzes: 1031.5 (M+H).

Pharmacological evaluation

The influence of 1 pair of external bone marrow stroma stem cell of experimental example

(1) external bone matrix stem cell growth proliferation experiment

Separate bone marrow stroma stem cell from rat femur, vitro culture goes down to posterity 5-8 generation.Choose well-grown cell and be used for experiment.Being inhibitor with Zorubicin (DOX) wherein, is given the test agent with the oligopeptides, with co-culture of cells.After 24 hours, calculate stem cell rate of increase (%) by measuring the OD value.

(2) external bone marrow stroma stem cell colony form experiment

The bone marrow stroma stem cell that inoculation is gone down to posterity mixes with DOX and oligopeptides+DOX in culture dish separately, respectively as suppressing control group and treatment group.Cultivate the situation of observation of cell colony formation (〉=50 cells are a colony) after 7 days altogether.

Test the biological activity of partial synthesis product, the results are shown in Table 1 and table 2.

The synthetic peptide of table 1 is to the influence of external bone marrow stem cell rate of increase

^*P＜0.05

The influence that the synthetic peptide of table 2 forms external bone marrow stem cell colony

^*P＜0.05； ^**P＜0.01

The above results shows that compound 3,5,6,11 and 12 injuries of avoiding Zorubicin at external protection bone marrow stroma stem cell have the significance activity.

Experimental example 2 synthetic peptides are to the protection activity of navel blood stem cell

This experiment is a chemotherapeutics with Ara-C (cytosine arabinoside), avoids the degree of Ara-C injury in order to estimate synthetic peptide protection navel blood stem cell.

A) Cord Blood Mononuclear Cell preparation: with fresh bleeding of the umbilicus (in 8 hours) and PBS by 1: 1 ratio (volume ratio) dilute, be added on carefully (volume ratio 1: 1) on the Ficoll, form layering clearly.The centrifugal 25min of 200g.The careful tunica albuginea layer of drawing is with twice of IMDM nutrient solution washing.Regulate cell concn 1 * 10 ⁶/ mL.

B) Ara-c is configured to the storage liquid of 5mg/mL.

C) storage liquid that different types of synthetic peptide is configured to 0.002M is standby.

D) get Cord Blood Mononuclear Cell 1 * 10 ⁶, add different types of synthetic peptide respectively, final concentration 2 * 10 ^-9, hatch 6h for 37 ℃.Control group and simple Ara-c group do not add synthetic peptide, and synthetic merely peptide group adds synthetic peptide (48H), final concentration 2 * 10 ^-9

F) add Ara-c in the Cord Blood Mononuclear Cell suspension, making its final concentration is 1 μ g/mL; Hatch 0.5h for 37 ℃.IMDM nutrient solution washed cell twice, 120 μ L IMDM nutrient solution re-suspended cell.Add H4434 substratum (stemcell company) 1.2mL mixing, place 37 ℃ of 5%CO ₂Cultivate in the incubator.Control group and simple synthetic peptide group do not add Ara-c, and simple Ara-c group adds Ara-c, and final concentration is 1 μ g/mL.

G) count CFU-GM respectively under the inverted microscope after 16 days, BFU-E colony number.

The partial synthesis peptide has been tested in middle to specifications 5-experiment 3., the results are shown in Table 3.

The synthetic peptide of table 3 is to the provide protection of navel blood stem cell

^*GM: the single colony forming unit of grain; ^*BFUE: burst forming unit erythroid

The above results shows that compound 8,9,13 and 14 makes the single colony forming unit of grain far away more than the Ara-C group of not treating, and near the normal group level.The BFUE numerical value of compound 10 is obviously more than the Ara-C group of not treating.

Myelosuppressive influence in 3 pairs of mouse bodies of experimental example

Experimental technique: every group of mouse is 7, sets up endoxan (CTX) separately and is chemotherapy poisoning control group, normal group and CTX+ oligopeptides (experiment) group.The mode that gives of CTX: irritate stomach 100mg/kg body weight.The oligopeptides administering mode: subcutaneous injection (is 0 time point to give CTX), establish the administration group respectively 3 times, 4 administration groups and 5 administration groups (concrete dosage sees back embodiment for details).Measure at last and respectively organize body weight, measure the marrow dna content behind the execution animal, bone marrow nucleated cell counting, peripheral blood leucocyte counting.

Middle to specifications 5-4., the mouse bone marrow cells that the partial synthesis peptide has been carried out being caused by endoxan (CTX) suppresses the activity rating of influence, the results are shown in Table 4.

The myelosuppressive provide protection that the synthetic peptide of table 4 causes CTX

1. each group all compares 2.CTX (filling stomach) 100mg/kg with positive group

3. medication (subcutaneous injection, to be 0 time point): 3 times to CTX :-24h, 0h, 24h 4 times :-24h ,-12h, 0h, 24h 5 times :-24h ,-12h, 0h, 24h, 48h

The result of table 4 proves that all apparent better protection of body weight, marrow dna content and peripheral leukocytes of mouse was active after compound 3 and 5 couples of CTX poisoned.3 significance＜0.01 of bone marrow nucleated cell counting of significance＜0.01, two kind of synthetic peptide that add the peripheral leukocytes counting of CTX group wherein.

The influence of 4 pairs of radiotherapy mouse death rates of experimental example

Experimental technique: set up the C57 mouse separately gamma-radiation group and gamma-radiation+oligopeptides treatment group.Irradiation dose is the LD70 level, oligopeptides dosage: the normal saline solution 0.5mL/ of subcutaneous injection c=25mg/L, and per 12 hours, totally 5 days.Statistics mortality ratio after two weeks.

Carried out by LD ₇₀After the irradiation of dosage gamma-radiation, the partial synthesis peptide influences the evaluation of mouse death rate, the results are shown in Table 5.

The synthetic peptide of table 5 causes the influence of mouse death rate to gamma-rays

The result shows that three kinds of synthetic peptides all can obviously improve the survival rate of mouse behind radiation exposure, and wherein compound 3 is all survived mouse.

Claims (4)

1, as general formula as (I), (II) with the compound (III):

Wherein,

R ¹=H, Boc, Ac, Pro, RCO (R=C _1～10Alkane), Sal (salicylyl), Succinic Acid list acyl

X ¹=Ser, Tar (tartrate list acyl), Thr, Cys or des (disappearance)

X ²=Asp, Glu, Pro, Ida (iminodiethanoic acid list acyl) or des

X ³=any natural amino acid residue, β-Ala, Inp (γ-piperidine formyl) or Asp-Ser fragment

R ²=NHR[R=C _1～10Alkane or Bn], NR ₂[R=C _1～10Alkane], N=2～5

Y=des or CH ₂,-SS-,-S-, CONH.

According to the compound of claim 1, it is characterized in that 2, described compound is selected from

Ac-Ser-Asp-Lys-Pro-NHEt (1)

Ac-Ser-Asp-Lys-NHEt (3)

Ac-Ser-Asp-Lys-NMe ₂ (4)

Sal-Ser-Asp-Lys-NHEt (5)

HOOC-(CH ₂) ₂-CO-Ser-Asp-Lys-NHEt (6)

CH ₃CH ₂CO-Ser-Asp-Lys-Phe-NHEt (9)

Sal-Ser-Asp-Lys-Phe-NMe ₂ (10)

HOOC-(CH ₂) ₂-CO-Ser-Asp-Lys-Phe-NMe ₂ (11)

Tar-Glu-Lys-Inp-NHMe (14)

Tar-Asp-Lys-Inp-NHMe (15)

3, a kind of pharmaceutical composition is characterized in that, comprises acceptable carrier at least one compound of claim 1-2 of effective dose and the pharmacodynamics.

4, the application of the compound described in claim 1-2 in preparing the medicine of protecting marrow hemopoietic stem cells to avoid chemotherapy or radiation-induced damage.

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