CN110903353B

CN110903353B - Method for preparing bremelanotide by solid-liquid phase combination

Info

Publication number: CN110903353B
Application number: CN201911337614.8A
Authority: CN
Inventors: 陈丰; 张萌; 金晶; 陈辉; 顾海东
Original assignee: Suzhou University of Science and Technology
Current assignee: Suzhou University of Science and Technology
Priority date: 2019-12-23
Filing date: 2019-12-23
Publication date: 2021-07-20
Anticipated expiration: 2039-12-23
Also published as: CN110903353A

Abstract

The invention discloses a method for preparing a bremelanotide by solid-liquid phase combination. The invention relates to a method for preparing a buminuo peptide, which synthesizes an AC-Nle-Asp-O-2-Phipr dipeptide fragment by a liquid phase method, a main chain and a side chain of Lys are respectively coupled and prolonged by peptide sequences to obtain Fmoc-His (Boc) -D-Phe-Arg (pbf) -Trp (Boc) -Lys (AC-Nle-Asp-O-2-Phipr) -royal resin, the main chain and the side chain are cyclized after cutting off a protecting group, the head and tail folding and cyclization are avoided, the problem of large folding and cyclization steric hindrance is solved, and the influence of the cyclized peptide on the non-cyclized peptide is reduced to the minimum due to the closer cyclization sites. In addition, alkali is not adopted for hydrolysis, and corresponding impurities are not generated. The synthetic method of the bremelanotide has the advantages of high purity and low cost, and is suitable for large-scale production.

Description

Method for preparing bremelanotide by solid-liquid phase combination

Technical Field

The invention relates to a method for preparing a bremelanotide by solid-liquid phase combination, belonging to the technical field of polypeptide synthesis.

Background

Buminuo peptide, british name Bremelanotide, was originally developed by university of arizona with a target of action of MC3R/MC4R, structurally a peptide analog, for the treatment of pre-menopausal female sexual desire disorders; the Paladin company gained right to develop the compound in 2007, followed by development, with clinical research focused in North America; NDA applications were filed 3.2018 to the US FDA, and 6.2019 were approved by the FDA and marketed 21. The brennol peptide consists of 7 amino acids and has the same core sequence as alpha-melanocyte stimulating hormone, the receptor of which plays an important role in appetite and sexual arousal. The peptide sequence is AC-Nle- [ Asp-His-D-Phe-Arg-Trp-Lys ] cycle-OH, and the structural formula is as follows:

the buminuo peptide is a cyclic peptide consisting of 7 amino acids, and the technical difficulty of synthesis is cyclization. The existing synthesis technology of the buminuo peptide can be divided into two types according to a ring forming method, wherein one type is ring forming in a liquid phase, and the other type is ring forming on resin. In a liquid phase, a large amount of intermolecular coupling products are easily generated by cyclization, and side reactions such as dipeptide, tripeptide and the like are generated to influence the yield; in order to inhibit the generation of side reactions, the reaction concentration must be reduced, a large amount of waste water is generated, and the environment is not protected; and the operation is troublesome. Therefore, the research of the solid phase cyclization method is more worthy of exploration.

At present, the synthesis method of the buminuolide peptide is mainly a solid-phase synthesis method. The solid phase ring formation is mainly to remove protection after the main chain is coupled, and the peptide chain is connected end to end, but the peptide chain is difficult to fold to end due to too large steric hindrance, so that the efficiency is not high, and the main reasons for causing the large steric hindrance comprise the following two aspects: (1) various protective group structures on the peptide chain lead the peptide chain to have a rigid structure and be difficult to fold back to form a ring; (2) after cyclization, the cyclic peptide has a larger spatial structure on the resin, making it difficult to fold the acyclic peptide back into a ring. The patent CN200610086841.4 adopts a step-by-step synthesis method, after the synthesis of peptide resin is finished, protecting groups of Asp and Lys are selectively removed, then, the resin is cyclized, and finally, HF is used for cracking, the method cannot be used in a large scale due to the severe corrosivity of HF, and the yield of refined peptide disclosed by the embodiment is 30-35%; patent CN200710048824.6 also adopts a selective deprotection method, but the selected deprotection method can remove the Asp and Lys side chain protecting groups and also remove the His protecting group Trt, and the yield of the crude peptide disclosed by the embodiment is 80%; in the solid-phase synthesis method of patent CN200910131598.7 buminuo peptide, p-hydroxybenzoic acid is connected to RinkAmideResin, then common amino acid raw materials are adopted for gradual synthesis, TFA is used for cracking after the synthesis is finished, cyclization is carried out under the liquid-phase condition, however intermolecular coupling is easy to occur when liquid-phase cyclization is carried out, polymers such as dimer, trimer and the like are generated, and the synthesis yield is seriously reduced; in patent 201610070195.6, HO- (CH2) n-COOH is used as a linker for connecting a solid phase carrier and a target peptide, and cyclization is carried out on resin.

Disclosure of Invention

In order to solve the technical problems, the invention provides a method for preparing the buminotide by solid-liquid phase combination, which synthesizes an AC-Nle-Asp-O-2-Phiprr dipeptide fragment by a liquid phase method, respectively carries out coupling extension peptide sequences on a main chain and a side chain of Lys to obtain Fmoc-His (Boc) -D-Phe-Arg (pbf) -Trp (Boc) -Lys (AC-Nle-Asp-O-2-Phiprr) -royal resin, carries out cyclization on the main chain and the side chain after cutting off a protecting group, avoids head-tail folding cyclization, solves the problem of large folding cyclization steric hindrance, and reduces the influence of the cyclized peptide on the non-cyclized peptide to the minimum due to closer cyclization sites.

The first purpose of the invention is to provide a method for preparing a buminuo peptide by solid-liquid phase combination, which comprises the following steps:

(1) synthesizing a dipeptide fragment AC-Nle-Asp-O-2-Phipr by a liquid phase synthesis method;

(2) sequentially coupling Lys, Trp, Arg, D-Phe and His protecting group amino acid on solid phase carrier resin in a coupling system by a solid phase synthesis method to obtain linear protecting peptide resin;

(3) selectively removing a protecting group on a Lys side chain, and coupling the dipeptide fragment AC-Nle-Asp-O-2-Phipr prepared in the step (1) on the Lys side chain;

(4) sequentially removing a protecting group O-2-Phipr on an Arg side chain and a protecting group Fmoc on His, and cyclizing under a coupling system;

(5) cracking, purifying and freeze-drying to obtain the buminuo peptide.

Further, the amino acids of Lys, Trp, Arg, D-Phe, and His protecting group are, in order, Fmoc-Lys (R1) -OH, Fmoc-Trp (Boc) -OH, Fmoc-Arg (pbf) -OH, Fmoc-D-Phe-OH, and Fmoc-His (Boc) -OH.

Further, the solid phase carrier resin is Wang resin, and the substitution degree of the Wang resin is 0.05-0.90 mmol/g.

Further, the protecting group R1 on the Lys side chain is IVDde or Dde.

Further, the coupling system comprises a condensing agent and a reaction solvent; the condensing agent is one or more of DIC, HOBt, PyBOP, HOBt, DIEA, HATU, HOBt and DIEA; the reaction solvent is one or more of DMF, DCM, NMP and DMSO.

Further, in the step (1), the liquid phase synthesis method for synthesizing the dipeptide fragment AC-Nle-Asp-O-2-Phipr specifically comprises the following steps: synthesizing AC-Nle-OSu and H-Asp-O-2-Phipr, and mixing AC-Nle-OSu, H-Asp-O-2-Phipr and Na₂CO₃Dissolving the dipeptide fragment into a mixed solution of THF and water, and reacting for 10-20 hours at 20-30 ℃ to prepare the dipeptide fragment AC-Nle-Asp-O-2-Phipr.

Further, the removing condition of the protecting group on the Lys side chain is to adopt hydrazine hydrate/DMF solution with the volume concentration of 1-5% for processing for 4-8 min, and repeat for 2-3 times.

Further, the removing condition of the protecting group O-2-Phipr on the Arg side chain is that TAF/DMF solution with the volume concentration of 0.8-2% is adopted for processing for 4-8 min, and the process is repeated for 2-3 times.

Further, the removing condition of the protecting group Fmoc on the His is to adopt a mixed solution of DMF and pyridine with the volume ratio of 3-5: 1 to treat for 4-8 min and repeat for 2-3 times.

Further, the cleavage reagent for cleavage is TFA, thioanisole, TIS, EDT and H₂O, wherein the volume ratio is as follows: TFA-thioanisole-TIS-EDT-H₂O＝90～95:1～5:1～5:1～5:1～5。

The invention has the beneficial effects that:

the invention relates to a method for preparing a buminuo peptide by solid-liquid combination, which synthesizes an AC-Nle-Asp-O-2-Phipr dipeptide fragment by a liquid phase method, and respectively carries out coupling extension peptide sequences on a main chain and a side chain of Lys to obtain Fmoc-His (Boc) -D-Phe-Arg (pbf) -Trp (Boc) -Lys (AC-Nle-Asp-O-2-Phipr) -royal resin. In addition, alkali is not adopted for hydrolysis, and corresponding impurities are not generated. The synthetic method of the bremelanotide has the advantages of high purity and low cost, and is suitable for large-scale production.

Drawings

FIG. 1 is a mass spectrum of a brennol peptide of the present invention.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

The abbreviations used in the present invention have the following meanings:

fmoc: fluorenylmethoxycarbonyl Otbu: tert-butoxy BOC tert-butoxycarbonyl

HOBt 1-hydroxybenzotriazole DIC N, N-diisopropylcarbodiimide

DMF N, N-dimethylformamide TFA trifluoroacetic acid

PyBop: benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate

Lys: lysine

His: histidine

Asp: aspartic acid

Nle: novel leucine

Trp: tryptophan

Arg: arginine

Phe: phenylalanine

Boc: tert-butyloxycarbonyl radical

Pbf: 2,2, 4, 6, 7-pentamethyldihydrobenzofuran-5-sulfonyl

DMF: n, N-dimethylformamide Piperdine: hexahydropyridine

O-2-Phipr: o-2-phenylisopropyl group

And (5) OAll: carboxyallyl esters

Dde: 1- (4, 4-dimethyl-2, 6-dioxocyclohexylidene) ethyl

IVDde: 1- (4, 4-dimethyl-2, 6-dioxocyclohexylmethylene) -3-methylbutyl

TFA: trifluoroacetic acid HAC: acetic acid

PyBop: benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate

Trt: 2-Chlorotriphenyl DCM, dichloromethane THF, tetrahydrofuran EDT: ethanedithiol

DIEA: diisopropylethylamine

HATU: 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate

DMSO, DMSO: dimethyl sulfoxide

HOSu: n-hydroxysuccinimide

Example 1: synthesis of AC-Nle-OSu activated ester

173.21g of AC-Nle-OH (1.0mol) and 138.10g of HOSu (1.2mol) are weighed and added into 2000ml of DMF, 247.59g of DCC (1.2mol) are added into an ice-water bath for reaction for 1 hour, the temperature is raised to room temperature for reaction for 3 hours, reaction liquid is filtered, mother liquid is dried in a spinning mode, DCM is added for dissolution, filtration is carried out, glacial ethanol is recrystallized for 3 times, filtration is carried out, a solid oil pump is used for drying, and 240.57gAC-Nle-OSu activated ester is obtained, wherein the yield is 89%.

Example 2: synthesis of H-Asp-O-2-Phipr

395.41g of Fmoc-Asp (OAll) -OH (1.0mol) were weighed out, added to 3000ml of methylene chloride, 561.16g of 2-phenylpropan-2-yl 2,2, 2-trichloroiminoacetate (2.0mol) dissolved in 5000ml of n-hexane were added dropwise in ice bath, and reacted at room temperature for 16 hours. 198.65g of 2-phenylpropan-2-yl chloroformate (1.0mol) dissolved in 2000ml of methylene chloride was added dropwise in an ice bath, and the reaction was carried out at room temperature for 16 hours. The solution was concentrated. 2000ml of THF were added to dissolve it, and 60g of Pd (PPh) were added₃)4(0.05mol) and 244mL PhSiH3(2.0mol) were reacted at room temperature for 2 hours. The solution was concentrated. 4000ml of dichloromethane and 1000ml of diisopropylethylamine. The reaction was carried out at room temperature for 0.5 hour. The solution was concentrated. The glacial ethanol is recrystallized for 3 times, filtered, and the solid oil is pumped to dryness to obtain 201.02g H-Asp-O-2-Phipr with the yield of 80 percent.

Example 3: synthesis of AC-Nle-Asp-O-2-Phipr

125.76g H-Asp-O-2-Phipr (0.5mol), 135.15g AC-Nle-OSu (0.5mol) and 79.50g Na were weighed out₂CO₃(0.75mol) was dissolved in a mixture of 500ml of water and 500ml of THF, reacted overnight at room temperature, and diluted with 10% hydrochloric acidThe pH was adjusted to 7, THF was removed by rotary evaporation, and then the pH was adjusted to 3. A large amount of white precipitate was obtained and filtered. The resulting white precipitate was recrystallized from glacial ethanol, the resulting solid was recrystallized from dioxane hydrochloride solution with stirring for 2 hours and the resulting solid oil was pumped dry to 172.55g of AC-Nle-Asp-O-2-Phipr, yield 85%.

Example 4: synthesis of Fmoc-Lys (Dde) -Wang resin having degree of substitution of 0.60mmol/g

Weighing 20g of queen resin with the substitution degree of 1.0mmol/g, adding the queen resin into a solid-phase reaction column, washing the queen resin with DMF for 1 time, swelling the resin with DMF for 30 minutes, taking 5.33g of Fmoc-Lys (Dde) -OH (100mmol) and 13.51g of HOBt (100mmol), dissolving the dissolved resin with DMF, adding 12.62g of DIC (100mmol) into the reaction column filled with the resin after ice-water bath for activation, adding the obtained mixture into the reaction column filled with the resin after 5 minutes, adding 1.22g of DMAP (10mmol), washing the obtained mixture with DMF for 3 times after 2 hours of reaction, washing the obtained mixture with DCM for 3 times, blocking the obtained mixture with 200ml of acetic anhydride and pyridine with the volume ratio of 1:1 overnight, and shrinking and drying the methanol to obtain Fmoc-Lys (Dde) -queen resin, wherein the detection substitution degree is 0.60 mmol/g.

Example 5: synthesis of Fmoc-Lys (Dde) -Wang resin having degree of substitution of 0.90mmol/g

Weighing 20g of queen resin with the substitution degree of 1.25mmol/g, adding the queen resin into a solid-phase reaction column, washing the queen resin with DMF for 1 time, swelling the resin with DMF for 30 minutes, taking 6.66g of Fmoc-Lys (Dde) -OH (125mmol) and 16.89g of HOBt (125mmol), dissolving the dissolved resin with DMF, adding 15.78g of DIC (125mmol) into the reaction column filled with the resin after ice-water bath for activation, adding the activated resin into the reaction column filled with the resin after 5 minutes, adding 1.53g of DMAP (12.5mmol), washing the reaction column with DMF for 2 hours for 3 times, washing DCM for 3 times, blocking the reaction column with 200ml of acetic anhydride and pyridine with the volume ratio of 1:1 overnight, shrinking and drying the methanol to obtain Fmoc-Lys (Dde) -queen resin, and detecting the substitution degree of 0.90 mmol/g.

Example 6: synthesis of Fmoc-His (Boc) -D-Phe-Arg (pbf) -Trp (Boc) -Lys (Dde) -Wang resin

Weighing 10.50g (10mmol) of Fmoc-Lys (Dde) -Wang resin with the degree of substitution of 0.60mmol/g, adding the resin into a solid-phase reaction column, washing the resin with DMF for 1 time, swelling the Fmoc-Lys (Dde) -Wang resin with DMF for 30 minutes, removing Fmoc protection by using a mixed solution of DMF and pyridine with the volume ratio of 4:1, then washing the resin with DMF for 6 times, weighing 15.80g of Fmoc-Trp (Boc) -OH (30mmol) and 4.05g of HOBt (30mmol), adding a mixed solution of DCM and DMF with the volume ratio of 1:1, adding 3.79g of DIC (30mmol) into an ice water bath for activation, adding the activated resin into the reaction column filled with the resin, reacting for 2 hours at room temperature, judging the reaction end point by an indetrione method, and indicating that the reaction is complete if the resin is colorless and transparent; and if the resin is developed, the reaction is not complete, the reaction needs to be carried out for 1 hour, and the judgment standard is suitable for judging the reaction end point by the detection of an indetrione method in the subsequent amino acid coupling. Repeating the steps of Fmoc protection removal and coupling by adding corresponding amino acid, and sequentially completing the coupling of Fmoc-Arg (pbf) -OH, Fmoc-D-Phe-OH and Fmoc-His (Boc) -OH to obtain Fmoc-His (Boc) -D-Phe-Arg (pbf) -Trp (Boc) -Lys (Dde) -Queen resin.

Example 7: removal of side chain protecting group Dde from Fmoc-His (Boc) -D-Phe-Arg (pbf) -Trp (Boc) -Lys (Dde) -Queen resin

The resin was removed twice with 3% hydrazine hydrate in DMF for about 5 minutes, and then washed five times alternately with DCM and DMF to give Fmoc-His (Boc) -D-Phe-Arg (pbf) -Trp (Boc) -Lys-Wang resin.

Example 8: coupling dipeptide fragment AC-Nle-Asp-O-2-Phipr on Lys side chain in Fmoc-His (Boc) -D-Phe-Arg (pbf) -Trp (Boc) -Lys-Wang resin

Weighing 12.18g of AC-Nle-Asp-O-2-Phiprr (30mmol) and 4.05g of HOBt (30mmol), adding a mixed solution of DCM and DMF with the volume ratio of 1:1, adding 3.79g of DIC (30mmol) for activation in an ice water bath, adding the activated mixture into the reaction column filled with the resin of the example 7, reacting for 2 hours at room temperature, detecting by an indantrione method to judge the reaction end point, and if the resin is colorless and transparent, indicating that the reaction is complete; the resin is developed, the reaction is not complete, the reaction needs to be carried out for 1 hour, and the judgment standard is suitable for judging the reaction end point by the indetrione method in the subsequent amino acid coupling, so that Fmoc-His (Boc) -D-Phe-Arg (pbf) -Trp (Boc) -Lys (AC-Nle-Asp-O-2-Phiprr) -Wang resin is obtained.

Example 9: removal of side chain protecting group O-2-Phipr from Fmoc-His (Boc) -D-Phe-Arg (pbf) -Trp (Boc) -Lys (AC-Nle-Asp-O-2-Phipr) -Queen resin

The resin of example 8 above was removed twice with 1% TAF in DCM for about 5 minutes, and then washed five times alternately with DCM and DMF to give Fmoc-His (Boc) -D-Phe-Arg (pbf) -Trp (Boc) -Lys (AC-Nle-Asp-OH) -Queen resin.

Example 10: Fmoc-His (Boc) -D-Phe-Arg (pbf) -Trp (Boc) -Lys (AC-Nle-Asp-OH) -Queen resin side chain protecting group Fmoc removal

The resin of example 9 was removed twice with a mixture of DMF and pyridine at a volume ratio of 4:1 for about 5 minutes, and then the resin was washed with DCM and DMF five times alternately to obtain H-His (Boc) -D-Phe-Arg (pbf) -Trp (Boc) -Lys (AC-Nle-Asp-OH) -Queen resin.

Example 11: synthesis of AC-Nle- [ Asp-His (Boc) -D-Phe-Arg (pbf) -Trp (Boc) -Lys ] cycle-Wang resin

To the obtained H-His (Boc) -D-Phe-Arg (pbf) -Trp (Boc) -Lys (AC-Nle-Asp-OH) -Queen resin prepared in example 10, 100ml of DMF, 11.38 g of HBTU (30mmol) and 3.4 ml of NMM (30mmol) were added in this order, followed by shaking reaction for 1 hour, detection of ninhydrin on a small amount of the resin, followed by extraction of the reaction solution, washing of the resin with DMF for 3 times with shaking, washing of the resin with methanol for 3 times, and vacuum drying for 8 hours to obtain AC-Nle- [ Asp-His (Boc) -D-Phe-Arg (pbf) -Trp (Boc) -Lys ] cycle-Queen resin.

Example 12: cracking

To AC-Nle- [ Asp-His (Boc) -D-Phe-Arg (pbf) -Trp (Boc) -Lys prepared in example 11]Adding 200ml of cracking reagent into cycle-queen resin, reacting at 25 deg.C for 3H, vacuum filtering, adding filtrate into 10 times volume of precooled ether for precipitation and centrifugation, washing the precipitate with ether for four times, and vacuum drying to obtain the buminuo peptide, wherein the cracking reagent is TFA, benzyl sulfide, TIS, EDT and H₂O, wherein the volume ratio is as follows: TFA-thioanisole-TIS-EDT-H₂O-90: 5:2:2: 1. 10.10g of white bumetanide solid is obtained, the purity of the crude peptide is 97.8 percent, and the yield of the crude peptide is 98.2 percent.

Example 13: purification of

The solid bremelanotide from example 12 was dissolved in 1L of water/acetonitrile at a volume ratio of 95:5 and filtered through a 0.45 μm filter for further use. C18 column with inner diameter of 100mm, mobile phase A of 0.1% TFA/water, mobile phase B of 0.1% TFA/acetonitrile, loading 500 ml/needle, flow rate of 300ml/min, detection wavelength of 210 nm. Gradient elution, and circulating sample injection before and after peaks to obtain refined peptide solution with central control analysis purity of more than 99.2%, transferring salt with acetic acid/acetonitrile/water system, and lyophilizing to obtain refined peptide of butonopeptide 8.71g, purity of 99.7%, and total yield of 85%.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Claims

1. A synthetic method for preparing a brennol peptide by solid-liquid phase combination is characterized by comprising the following steps:

(4) sequentially removing a protecting group O-2-Phipr on an Asp side chain and a protecting group Fmoc on His, and cyclizing under a coupling system;

(5) cracking, purifying and freeze-drying to obtain the buminuo peptide;

the solid phase carrier resin is Wang resin, and the substitution degree of the Wang resin is 0.05-0.90 mmol/g.

2. The method of synthesis according to claim 1, wherein the Lys, Trp, Arg, D-Phe, and His protecting group amino acids are, in order, Fmoc-Lys (R1) -OH, Fmoc-Trp (boc) -OH, Fmoc-Arg (pbf) -OH, Fmoc-D-Phe-OH, and Fmoc-His (boc) -OH; wherein R1 is IVDde or Dde.

3. The method of synthesis of claim 1, wherein the coupling system comprises a condensing agent and a reaction solvent; the condensing agent is one or more of DIC, HOBt, PyBOP, DIEA, HATU and DIEA; the reaction solvent is one or more of DMF, DCM, NMP and DMSO.

4. The method of claim 1, wherein the step (1) of synthesizing the dipeptide fragment AC-Nle-Asp-O-2-Phipr by liquid phase synthesis comprises: synthesizing AC-Nle-OSu and H-Asp-O-2-Phipr, and mixing AC-Nle-OSu, H-Asp-O-2-Phipr and Na₂CO₃Dissolving the dipeptide fragment into a mixed solution of THF and water, and reacting for 10-20 hours at 20-30 ℃ to prepare the dipeptide fragment AC-Nle-Asp-O-2-Phipr.

5. The synthetic method according to claim 1, wherein the removing condition of the protecting group on the Lys side chain is to treat with 1-5% by volume hydrazine hydrate/DMF solution for 4-8 min, and repeat for 2-3 times.

6. The synthetic method according to claim 1, wherein the removing condition of the protecting group O-2-Phipr on the Asp side chain is to treat with 0.8-2% by volume TAF/DMF solution for 4-8 min, and repeat 2-3 times.

7. The synthesis method of claim 1, wherein the removing condition of the Fmoc protecting group on His is to treat the mixture of DMF and pyridine at a volume ratio of 3-5: 1 for 4-8 min, and repeat the treatment for 2-3 times.

8. The method of claim 1, wherein the cleavage reagent used for the cleavage is TFA, thioanisole, TIS, EDT, and H₂O, wherein the volume ratio is as follows: TFA-thioanisole-TIS-EDT-H₂O＝90～95:1～5:1～5:1～5:1～5。