CN111410682A - Cyclization preparation method of polymyxin B related compound - Google Patents

Abstract

The invention discloses a preparation method for the cyclization of polymyxin B related compounds, which has the advantages of simple reaction operation, convenient post-treatment and high yield, and has high reference value for the synthesis of single compounds B1, B1-I, B2 and B3 of polymyxin B. In addition, the invention provides important basis for carrying out scientific evaluation on the efficacy, pharmacological research, pharmacokinetics and the like of the polymyxin B by preparing each single component compound of the polymyxin B, facilitates the deeper research on the metabolic process and the pharmacokinetics of the polymyxin B in a human body and has better medicinal prospect.

Description

Cyclization preparation method of polymyxin B related compound

Technical Field

The invention relates to a synthesis method of polypeptide antibiotics, in particular to a cyclization preparation method of polymyxin B related compounds.

Technical Field

Polymyxins are a group of polypeptide antibiotics produced by bacillus polymyxa (bacillus polymyxa), the name english: polymyxin, Polymyxin B, consists essentially of: b1, B1-I, B2 and B3, wherein polymyxin B1 (C)₅₆H₉₈N₁₆O₁₃) And B2 (C)₅₅H₉₆N₁₆O₁₃) In many cases, the content of polymyxin B1 is the maximum, generally reaching more than 50%.

Polymyxin B has inhibitory or bactericidal effect on gram-negative bacilli such as Escherichia coli, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Acidobacterium acidophilum, Bordetella pertussis, and Bacillus dysenteriae. The traditional Chinese medicine composition is mainly used for infection caused by sensitive bacteria, urinary system infection caused by pseudomonas aeruginosa, eye, trachea, meningitis, septicemia, burn infection, skin mucosa infection and the like in clinic.

Because the polymyxin B is poor in stability, the existing polymyxin B synthesis method is prepared by adopting a fermentation method, all the polymyxin B are obtained as a mixture, and few documents report that single polymyxin B can be obtained. In patents CN201110385129 and CN201310506594, polymyxin B is reported to be prepared by fermentation, but the obtained polymyxin B is a mixture, and the yield is low, which is not suitable for large-scale production.

Disclosure of Invention

The purpose of the invention is as follows: in view of the prior art, the invention provides a preparation method for the polymyxin B related compound by cyclization.

The technical scheme is as follows: the invention discloses a cyclization preparation method of polymyxin B related compounds B1, B1-I, B2 and B3.

The preparation method of the polymyxin B related compound B1 comprises the following steps:

(1) solid-phase synthesis:

1) using CTC-Resin as a solid phase carrier, firstly coupling Fmoc-Thr (tBu) -OH to obtain Fmoc-Thr (tBu) -Resin, and removing Fmoc protection to obtain H-Thr (tBu) -Resin;

2) coupling Fmoc-Dab (Boc) -OH with H-Thr (tBu) -Resin obtained in step 1) to obtain Fmoc-Dab (Boc) -Thr (tBu) -Resin, and then condensing Fmoc-Dab (Boc) -OH, Fmoc-L eu-OH, Fmoc-D-Phe-OH, Fmoc-Dab (Boc) -OH, Fmoc-Dab (Boc) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Dab (Boc) -OH, Fmoc-Thr (Boc) -OH;

3) final coupling of S-6-methyloctanoic acid CH₃CH₂(CH₃)CH(CH₂)₄COOH, cracking to remove resin to obtain a segment I;

(2) liquid phase condensation cyclization: coupling the fragment I obtained in the step (1) to obtain a fragment II;

(3) and (3) removing side chain protection of the fragment II obtained in the step (2) under the action of trifluoroacetic acid, and purifying to obtain a polymyxin B related compound B1.

The preparation method of polymyxin B related compound B1-I comprises the step of replacing Fmoc-L eu-OH in step (1) and step 2) of the preparation method of B1 with Fmoc-Ile-OH.

The preparation method of the polymyxin B related compound B2 comprises the following steps: CH in the step (1) and the step 3) of the preparation method of B1₃CH₂(CH₃)CH(CH₂)₄COOH by CH₃(CH₃)CH(CH₂)₄COCOOH。

The preparation method of the polymyxin B related compound B3 comprises the following steps: CH in the step (1) and the step 3) of the preparation method of B1₃CH₂(CH₃)CH(CH₂)₄COOH was replaced with CH3(CH2)6 COOH.

Further, in the step (1) of the above preparation method:

step 1), coupling agent of solid phase carrier coupling amino acid is DIEA;

step 2) coupling agent is DIC + A or DIEA + B, A is HOBT or HOAT, B is HBTU, HATU, PyBOP, TBTU;

step 3) the coupling agent is DIC + A or B + A + C, wherein A is HOBT or HOAT, B is HBTU, HATU, TBTU or PyBOP, and C is DIEA or NMM;

and 3) cracking the resin by using a reagent of trifluoroethanol and dichloromethane in a volume ratio of 1: 1-1: 4, preferably 1: 2. The cleavage reagent was used in an amount 10 times the weight of CTC-Resin (w: v ═ 1:10) the corresponding volume, and the Resin was removed.

Further, in the step (2) of the preparation method, the cyclization solvent is N, N-dimethylformamide, and the coupling agent is DIC + A or DIEA + B, wherein A is HOBT, and B is HBTU, HATU, PyBop, TBTU. The reaction temperature is 20-30 ℃, and the reaction time is 1-2 hours. Among them, DIC + HOBT is preferred, and other condensing agents do not allow the linear peptide to be cyclized or the product to be relatively impure. After condensation, the reaction solution is added into water with 50 times of volume for precipitation, and is dried by an oil pump after suction filtration.

Further, in the step (3) of the preparation method, reagents used for protection in the cleavage sequence are trifluoroacetic acid, water and a triisopropylsilane solution, wherein the volume ratio of the trifluoroacetic acid is 80-90%, the volume ratio of the water is 5-10%, and the volume ratio of the triisopropylsilane is 5-10%. The preferred volume ratio of trifluoroacetic acid, water, triisopropylsilane is 90:10: 10. The reaction temperature is 10-20 ℃, and the reaction time is 1-2 hours.

And (4) after the reaction in the step (3) is finished, performing rotary evaporation and rotary drying, precipitating by using ether with the volume 10 times that of the cracking reagent, and centrifuging to obtain a crude product of the polymyxin B related compound.

Purifying the target substance in the step (3) by adopting a chromatographic column: mobile phase a phase: aqueous 0.1% TFA, mobile phase B: 0.1% TFA in acetonitrile, gradient: 15 to 45 percent.

Has the advantages that: the preparation method of the polymyxin B related compound (B1, B1-I, B2 and B3) comprises the steps of synthesizing fragment I in a solid phase, and finally cyclizing under the coupling condition in a HOBT + DIC liquid phase. Wherein, the cost of the solid phase resin is effectively reduced by using the CTC resin, and the yield is effectively improved and the cost is reduced by selecting HOBT + DIC as the best coupling reagent. In addition, through the research on each single component compound of the polymyxin B, the invention can carry out more intensive research on the metabolic process and the pharmacokinetics of the polymyxin B in a human body, so that the polymyxin B has better medicinal prospect.

Drawings

FIG. 1 is a scheme showing the synthesis of polymyxin B related compound B1 according to the present invention.

Detailed Description

The present application will be described in detail with reference to specific examples.

List of reagents used in this application:

example 1

The preparation method of polymyxin B1 by cyclization has a synthetic flow shown in figure 1, and specifically comprises the following steps:

the method comprises the following steps:

CH₃CH₂(CH₃)CH(CH₂)₄synthesis of CO-Dab (Boc) -Thr (tBu) -Dab (Boc) -Dab (Boc) -D-Phe-L eu-Dab (Boc) -Thr (tBu) -OH:

1) adding 10g of CTC-Resin with the degree of substitution of 0.4-0.6 mmol/g into a polypeptide reactor, adding 2g of Fmoc-Thr (tBu) -OH and 10ml of DIEA to react for 2 hours, then pumping out reaction liquid, and washing twice with DMF; after Fmoc protection removal, DMF washing is carried out twice, DCM washing is carried out once, and DMF washing is carried out twice;

2) then Fmoc-Dab (Boc) -OH, Fmoc-L eu-OH, Fmoc-D-Phe-OH, Fmoc-Dab (Boc) -OH, Fmoc-Dab (Boc) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Dab (Boc) -OH, CH-CH) in sequence₃CH₂(CH₃)CH(CH₂)₄COOH, the reaction time is 2 hours, the deprotection time is 0.5 hour, and the reaction end point is detected by an indantrione method; after the reaction is finished, washing with DMF twice, washing with DCM once and washing with DMF twice;

3) washing the washed resin twice with methanol, and removing the resin 2 times with 200ml of trifluoroethanol/dichloromethane, wherein the reaction stirring time is 2 hours each time; the resin was filtered off, the reaction solution was collected and spin-dried to give 6.8g of CH₃CH₂(CH₃)CH(CH₂)₄CO-Dab(Boc)-Thr(tBu)-Dab(Boc)-Dab-Dab(Boc)-D-Phe-Le u-Dab(Boc)-Dab(Boc)-Thr(tBu)-OH。

Step two:

CH₃CH₂(CH₃)CH(CH₂)₄CO-Dab(Boc)-Thr(tBu)-Dab(Boc)-Cyclo

(Dab-dab (Boc) -D-Phe-L eu-dab (Boc) -Thr (tBu) -OH) Synthesis 6.8gCH₃CH₂(CH₃)CH(CH₂)₄CO-Dab (Boc) -Thr (tBu) -Dab (Boc) -Dab (Boc) -D-Ph e-L eu-Dab (Boc) -Thr (tBu) -OH was dissolved in 20ml of DMF solution, 0.5g of HOBT and 0.5ml of DMF were added, the reaction was stirred at room temperature for 2 hours, the reaction solution was poured into 200ml of ice water, the magnetic particles were stirred for 2 hours, after suction filtration to obtain a solid, the solid was rinsed with 50ml of water 2 times, and the filter cake was suction-dried with an oil pump to obtain 5.4g of a white solid.

Step three:

CH₃CH₂(CH₃)CH(CH₂)₄CO-Dab-Thr-Dab-Cyclo

(Dab-D-Phe-L eu-Dab-Thr-OH) Synthesis:

CH3CH2(CH3)CH(CH2)4CO-Dab(Boc)-Thr(tBu)-Dab(Boc)-Cyclo

(Dab-Dab (Boc) -D-Phe-L eu-Dab (Boc) -Thr (tBu) -OH)5.4g was dissolved in 60ml of TFA/H2O/TIS (8:1:1) solution, stirred for 3 hours, dropped into 500ml of glacial ethyl ether solution, the precipitate was centrifuged, washed three times with ethyl ether, and dried under reduced pressure to give 2.2g of a white solid.

Step four:

purification of polymyxin B1:

CH3CH2(CH3)CH(CH2)4CO-Dab-Thr-Dab-Cyclo

(Dab-D-Phe-L eu-Dab-Thr-OH) 2.2g was dissolved in 10% acetonitrile and water and filtered;

purification equipment: hanbang UV3000/NP7000, chromatography column DAC-50, C18,10um, wavelength: 220. mobile phase a phase: aqueous 0.1% TFA, mobile phase B: 0.1% TFA in acetonitrile, gradient: 15% -45% for 60 minutes, to give 1.1g of polymyxin B1.

The confirmation sequence after structure confirmation is:

CH3CH2(CH3)CH(CH2)4CO-Dab-Thr-Dab-Cyclo

(Dab-Dab-D-Phe-Leu-Dab-Dab-Thr-OH)

example 2

The preparation method of polymyxin B1 by cyclization comprises the following steps:

the method comprises the following steps:

synthesis of CH3CH2(CH3) CH (CH2)4CO-Dab (Boc) -Thr (tBu) -Dab (Boc) -Dab (Boc) -D-Phe-L eu-Dab (Boc) -Thr (tBu) -OH:

1) adding 10g of CTC-Resin with the substitution degree of 0.4-0.6 mmol/g into a polypeptide reactor, adding 2g of Fmoc-Thr (tBu) -OH and 10ml of DIEA to react for 2 hours, then pumping out reaction liquid, and washing twice with DMF; after Fmoc protection removal, DMF washing is carried out twice, DCM washing is carried out once, and DMF washing is carried out twice;

2) then Fmoc-Dab (Boc) -OH, Fmoc-L eu-OH, Fmoc-D-Phe-OH, Fmoc-Dab (Boc) -OH, Fmoc-Dab (Boc) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Dab (Boc) -OH, CH3CH2(CH3) CH (CH2)4COOH are sequentially carried out, the reaction time is 2 hours, the deprotection time is 0.5 hour, the reaction endpoint is detected by an indetrione method, after the reaction is finished, DMF is washed twice, DCM is washed once, and DMF is washed twice;

3) after washing the washed resin twice with methanol, the resin was removed with 200ml of trifluoroethanol/dichloromethane 2 times with stirring for 2 hours each time, the resin was filtered off, and the reaction solution was collected and spin-dried to obtain 6.8g of CH3CH2(CH3) CH (CH2)4CO-Dab (Boc) -Thr (tBu) -Dab (Boc) -D-Phe-L eu-Dab (Boc) -Thr (tBu) -OH.

Step two:

CH3CH2(CH3)CH(CH2)4CO-Dab(Boc)-Thr(tBu)-Dab(Boc)-Cyclo

(Dab-Dab (Boc) -D-Phe-L eu-Dab (Boc) -Thr (tBu) -OH) Synthesis 6.8gCH3CH2(CH3) CH (CH2)4CO-Dab (Boc) -Thr (tBu) -Dab (Boc) -Dab (Boc) -D-Phe-L eu-Dab (Boc) -Thr (tBu) -OH was dissolved in 20ml DMF solution, 0.65g HBTU and 0.5ml DIEA were added, the reaction was stirred at room temperature for 2 hours, the reaction solution was poured into 200ml ice water, the magneton was stirred for 2 hours, after suction filtration to give a solid, it was rinsed with 50ml water for 2 times, and the filter cake was suction-dried with an oil pump to give 4.6g white solid.

Step three:

CH3CH2(CH3)CH(CH2)4CO-Dab-Thr-Dab-Cyclo

(Dab-D-Phe-L eu-Dab-Thr-OH) Synthesis:

CH3CH2(CH3)CH(CH2)4CO-Dab(Boc)-Thr(tBu)-Dab(Boc)-Cyclo

(Dab-Dab (Boc) -D-Phe-L eu-Dab (Boc) -Thr (tBu) -OH)4.6g was dissolved in 60ml TFA/H2O/TIS (8:1:1) solution, stirred for 3 hours, dropped into 500ml ethyl ice ether solution, the precipitate centrifuged, washed three times with ethyl ether, dried under reduced pressure to give 1.8g of white solid (HP L C trace, no major peak evident, and more minor peaks)

Step four:

purification of polymyxin B1:

CH3CH2(CH3)CH(CH2)4CO-Dab-Thr-Dab-Cyclo

(Dab-D-Phe-L eu-Dab-Thr-OH) 2.2g was dissolved in 10% acetonitrile and water and filtered;

purification equipment: hanbang UV3000/NP7000, chromatography column DAC-50, C18,10um, wavelength: 220. mobile phase a phase: aqueous 0.1% TFA, mobile phase B: 0.1% TFA in acetonitrile, gradient: 15% -45% for 60 minutes, to give 0.3g of polymyxin B1.

The confirmation sequence after structure confirmation is:

CH3CH2(CH3)CH(CH2)4CO-Dab-Thr-Dab-Cyclo

(Dab-Dab-D-Phe-Leu-Dab-Dab-Thr-OH)

example 3

The preparation method of polymyxin B1 by cyclization comprises the following steps:

the method comprises the following steps:

synthesis of CH3CH2(CH3) CH (CH2)4CO-Dab (Boc) -Thr (tBu) -Dab (Boc) -Dab (Boc) -D-Phe-L eu-Dab (Boc) -Thr (tBu) -OH:

1) adding 10g of CTC-Resin with the substitution degree of 0.4-0.6 mmol/g into a polypeptide reactor, adding 2g of Fmoc-Thr (tBu) -OH and 10ml of DIEA to react for 2 hours, then pumping out reaction liquid, and washing twice with DMF; after Fmoc deprotection, DMF wash twice, DCM wash once, DMF wash twice

2) Then Fmoc-Dab (Boc) -OH, Fmoc-L eu-OH, Fmoc-D-Phe-OH, Fmoc-Dab (Boc) -OH, Fmoc-Dab (Boc) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Dab (Boc) -OH, CH3CH2(CH3) CH (CH2)4COOH are sequentially carried out, the reaction time is 2 hours, the deprotection time is 0.5 hour, the reaction endpoint is detected by an indetrione method, after the reaction is finished, DMF is washed twice, DCM is washed once, and DMF is washed twice;

3) after washing the washed resin twice with methanol, the resin was removed with 200ml of trifluoroethanol/dichloromethane 2 times with stirring for 2 hours each time, the resin was filtered off, and the reaction solution was collected and spin-dried to obtain 6.8g of CH3CH2(CH3) CH (CH2)4CO-Dab (Boc) -Thr (tBu) -Dab (Boc) -D-Phe-L eu-Dab (Boc) -Thr (tBu) -OH.

Step two:

CH3CH2(CH3)CH(CH2)4CO-Dab(Boc)-Thr(tBu)-Dab(Boc)-Cyclo

(Dab-Dab (Boc) -D-Phe-L eu-Dab (Boc) -Thr (tBu) -OH) Synthesis 6.8gCH3CH2(CH3) CH (CH2)4CO-Dab (Boc) -Thr (tBu) -Dab (Boc) -Dab (Boc) -D-Phe-L eu-Dab (Boc) -Thr (tBu) -OH was dissolved in 20ml DMF solution, 0.65g HBTU and 0.5ml DIEA were added, the reaction was stirred at room temperature for 2 hours, the reaction solution was poured into 200ml ice water, the magneton was stirred for 2 hours, after suction filtration to give a solid, it was rinsed with 50ml water for 2 times, and the filter cake was suction-dried with an oil pump to give 4.8g white solid.

Step three:

CH3CH2(CH3)CH(CH2)4CO-Dab-Thr-Dab-Cyclo

(Dab-D-Phe-L eu-Dab-Thr-OH) Synthesis:

CH3CH2(CH3)CH(CH2)4CO-Dab(Boc)-Thr(tBu)-Dab(Boc)-Cyclo

(Dab-Dab (Boc) -D-Phe-L eu-Dab (Boc) -Thr (tBu) -OH)4.8g was dissolved in 60ml TFA/H2O/TIS (8:1:1) solution, stirred for 3 hours, dropped into 500ml ethyl ice ether solution, the precipitate centrifuged, washed three times with ethyl ether, dried under reduced pressure to give 2.1g white solid (HP L C trace, no major peak and more minor peaks)

Step four:

purification of polymyxin B1:

CH3CH2(CH3)CH(CH2)4CO-Dab-Thr-Dab-Cyclo

(Dab-D-Phe-L eu-Dab-Thr-OH) 2.2g was dissolved in 10% acetonitrile and water and filtered;

purification equipment: hanbang UV3000/NP7000, chromatography column DAC-50, C18,10um, wavelength: 220. mobile phase a phase: aqueous 0.1% TFA, mobile phase B: 0.1% TFA in acetonitrile, gradient: 15% -45% for 60 minutes, 0.2g of polymyxin B1 was obtained.

The confirmation sequence after structure confirmation is:

CH3CH2(CH3)CH(CH2)4CO-Dab-Thr-Dab-Cyclo

(Dab-Dab-D-Phe-Leu-Dab-Dab-Thr-OH)

example 4

A process for the cyclisation preparation of polymyxin B1-I comprising the steps of:

the method comprises the following steps:

synthesis of CH3CH2(CH3) CH (CH2)4CO-Dab (Boc) -Thr (tBu) -Dab (Boc) -Dab (Boc) -D-Phe-Ile-Dab (Boc) -Thr (tBu) -OH:

1) adding 10g of CTC-Resin with the degree of substitution of 0.4-0.6 mmol/g into a polypeptide reactor, adding 2g of Fmoc-Thr (tBu) -OH and 10ml of DIEA to react for 2 hours, then pumping out reaction liquid, and washing twice with DMF; after Fmoc deprotection, DMF wash twice, DCM wash once, DMF wash twice

2) Then Fmoc-Dab (Boc) -OH, Fmoc-Ile-OH, Fmoc-D-Phe-OH, Fmoc-Dab (Boc) -OH, Fmoc-Dab (Boc) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Dab (Boc) -OH, CH3CH2(CH3) CH (CH2)4COOH are sequentially carried out, the reaction time is 2 hours, the deprotection time is 0.5 hours, and the reaction endpoint is detected by an indetrione method; after the reaction is finished, washing with DMF twice, washing with DCM once and washing with DMF twice;

3) washing the washed resin twice with methanol, and removing the resin 2 times with 200ml of trifluoroethanol/dichloromethane, wherein the reaction stirring time is 2 hours each time; the resin was filtered off, and the reaction solution was collected and spin-dried to give 7.1g of CH3CH2(CH3) CH (CH2)4CO-Dab (Boc) -Thr (tBu) -Dab (Boc) -D-Phe-I le-Dab (Boc) -Thr (tBu) -OH.

Step two:

CH3CH2(CH3)CH(CH2)4CO-Dab(Boc)-Thr(tBu)-Dab(Boc)-Cyclo

(Synthesis of Dab-Dab (Boc) -D-Phe-Ile-Dab (Boc) -Thr (tBu) -OH): dissolving 7.1gCH3CH2(CH3) CH (CH2)4CO-Dab (Boc) -Thr (tBu) -Dab (Boc) -Dab (Boc) -D-phe-Ile-Dab (Boc) -Thr (tBu) -OH in 20ml DMF, adding 0.5g HOBT and 0.5ml DIC, and stirring at room temperature for 2 hours; the reaction solution was poured into 200ml of ice water, the magnetons were stirred for 2 hours, and after obtaining a solid by suction filtration, the solid was rinsed 2 times with 50ml of water, and the filter cake was dried by an oil pump to obtain 6.0g of a white solid.

Step three:

CH3CH2(CH3)CH(CH2)4CO-Dab-Thr-Dab-Cyclo

(Dab-D-Phe-Ile-Dab-Thr-OH) Synthesis:

CH3CH2(CH3)CH(CH2)4CO-Dab(Boc)-Thr(tBu)-Dab(Boc)-Cyclo

(Dab-Dab (Boc) -D-Phe-L eu-Dab (Boc) -Thr (tBu) -OH)6.0g was dissolved in 60ml of TFA/H2O/TIS (8:1:1) solution, stirred for 3 hours, dropped into 500ml of glacial ethyl ether solution, the precipitate was centrifuged, washed three times with ethyl ether, and dried under reduced pressure to give 2.4g of a white solid.

Step four:

purification of polymyxin B1-I:

CH3CH2(CH3)CH(CH2)4CO-Dab-Thr-Dab-Cyclo

(Dab-D-Phe-Ile-Dab-Thr-OH) 2.4g was dissolved in 10% acetonitrile and water and filtered;

purification equipment: hanbang UV3000/NP7000, chromatography column DAC-50, C18,10um, wavelength: 220. mobile phase a phase: aqueous 0.1% TFA, mobile phase B: 0.1% TFA in acetonitrile, gradient: 15% -45% for 60 minutes, to give 1.2g of polymyxin B1-I. z is a radical of

The confirmation sequence after structure confirmation is:

CH3CH2(CH3)CH(CH2)4CO-Dab-Thr-Dab-Cyclo

(Dab-Dab-D-Phe-Ile-Dab-Dab-Thr-OH)

example 5

The preparation method of polymyxin B2 by cyclization comprises the following steps:

the method comprises the following steps:

CH₃(CH₃)CH(CH₂)₄synthesis of CO-Dab (Boc) -Thr (tBu) -Dab (Boc) -Dab (Boc) -D-Phe-L eu-Dab (Boc) -Thr (tBu) -OH:

1) adding 10g of CTC-Resin with the degree of substitution of 0.4-0.6 mmol/g into a polypeptide reactor, adding 2g of Fmoc-Thr (tBu) -OH and 10ml of DIEA to react for 2 hours, then pumping out reaction liquid, and washing twice with DMF; after Fmoc deprotection, DMF wash twice, DCM wash once, DMF wash twice

2) Then Fmoc-Dab (Boc) -OH, Fmoc-L eu-OH, Fmoc-D-Phe-OH, Fmoc-Dab (Boc) -OH, Fmoc-Dab (Boc) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Dab (Boc) -OH, CH-CH) in sequence₃(CH₃)CH(CH₂)₄COCOOH, the reaction time is 2 hours, the deprotection time is 0.5 hour, and the reaction end point is detected by an indetrione method; after the reaction is finished, washing with DMF twice, washing with DCM once and washing with DMF twice;

3) washing the washed resin twice with methanol, and removing the resin 2 times with 200ml of trifluoroethanol/dichloromethane, wherein the reaction stirring time is 2 hours each time; the resin was filtered off, the reaction solution was collected and spin-dried to give 6.9g of CH₃(CH₃)CH(CH₂)₄CO-Dab(Boc)-Thr(tBu)-Dab(Boc)-Dab-Dab(Boc)-D-Phe-Leu- Dab(Boc)-Dab(Boc)-Thr(tBu)-OH。

Step two:

CH₃(CH₃)CH(CH₂)₄CO-Dab(Boc)-Thr(tBu)-Dab(Boc)-Cyclo

(Dab-Dab (Boc) -D-Phe-L eu-Dab (Boc) -Thr (tBu) -OH) Synthesis 6.9gCH₃(CH₃)CH(CH₂)₄CO-Dab (Boc) -Thr (tBu) -Dab (Boc) -D-Phe-L e u-Dab (Boc) -Thr (tBu) -OH was dissolved in 20ml of DMF solution, 0.5g of HOBT and 0.5ml of DIC were added, the reaction mixture was stirred at room temperature for 2 hours, the reaction mixture was poured into 200ml of ice water, the magnetons were stirred for 2 hours, and after suction filtration to obtain a solid, the solid was rinsed with 50ml of water for 2 times, and the cake was suction-dried by an oil pump to obtain 6.1g of a white solid.

Step three:

CH₃(CH₃)CH(CH₂)₄CO-Dab-Thr-Dab-Cyclo

(Dab-D-Phe-L eu-Dab-Thr-OH) Synthesis:

CH₃(CH₃)CH(CH₂)₄CO-Dab(Boc)-Thr(tBu)-Dab(Boc)-Cyclo

(Dab-Dab (Boc) -D-Phe-L eu-Dab (Boc) -Thr (tBu) -OH)6.1g was dissolved in 60ml of TFA/H2O/TIS (8:1:1) solution, stirred for 3 hours, dropped into 500ml of glacial ethyl ether solution, the precipitate was centrifuged, washed three times with ethyl ether, and dried under reduced pressure to give 2.3g of a white solid.

Step four:

purification of polymyxin B2:

CH₃(CH₃)CH(CH₂)₄CO-Dab-Thr-Dab-Cyclo

(Dab-D-Phe-L eu-Dab-Thr-OH) 2.4g was dissolved in 10% acetonitrile and water and filtered;

purification equipment: hanbang UV3000/NP7000, chromatography column DAC-50, C18,10um, wavelength: 220. mobile phase a phase: aqueous 0.1% TFA, mobile phase B: 0.1% TFA in acetonitrile, gradient: 15% -45% for 60 minutes, to give 1.2g of polymyxin B2.

The confirmation sequence after structure confirmation is:

CH₃(CH₃)CH(CH₂)₄CO-Dab-Thr-Dab-Cyclo

(Dab-Dab-D-Phe-Leu-Dab-Dab-Thr-OH)

example 6

The preparation method of polymyxin B3 by cyclization comprises the following steps:

the method comprises the following steps:

synthesis of CH3(CH2)6CO-Dab (Boc) -Thr (tBu) -Dab (Boc) -Dab (Boc) -D-Phe-L eu-Dab (Boc) -Thr (tBu) -OH:

1) adding 10g of CTC-Resin with the degree of substitution of 0.4-0.6 mmol/g into a polypeptide reactor, adding 2g of Fmoc-Thr (tBu) -OH and 10ml of DIEA to react for 2 hours, then pumping out reaction liquid, and washing twice with DMF; after Fmoc deprotection, DMF wash twice, DCM wash once, DMF wash twice

2) Then Fmoc-Dab (Boc) -OH, Fmoc-L eu-OH, Fmoc-D-Phe-OH, Fmoc-Dab (Boc) -OH, Fmoc-Dab (Boc) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Dab (Boc) -OH, CH3(CH2)6COCOOH, the reaction time is 2 hours, the deprotection time is 0.5 hour, the reaction endpoint is detected by an indetrione method, after the reaction is finished, DMF is washed twice, DCM is washed once, and DMF is washed twice;

3) after washing the washed resin twice with methanol, the resin was removed with 200ml of trifluoroethanol/dichloromethane 2 times with stirring for 2 hours each time, the resin was filtered off, and the reaction solution was collected and spin-dried to give 7.3g of CH3(CH2)6CO-Dab (Boc) -Thr (tBu) -Dab (Boc) -Dab (Boc) -D-Phe-L eu-Dab (Boc) -Thr (tBu) -OH.

Step two:

CH3(CH2)6CO-Dab(Boc)-Thr(tBu)-Dab(Boc)-Cyclo

(Synthesis of Dab-Dab (Boc) -D-Phe-L eu-Dab (Boc) -Thr (tBu) -OH):

will be provided with

7.3gCH3(CH2)6CO-Dab (Boc) -Thr (tBu) -Dab (Boc) -D-Phe-L eu-Dab (B oc) -Dab (Boc) -Thr (tBu) -OH was dissolved in 20ml DMF solution, 0.5g HOBT and 0.5ml DIC were added and the reaction was stirred at room temperature for 2 hours, the reaction mixture was poured into 200ml ice water and stirred with a magneton for 2 hours, after obtaining a solid by suction filtration, 50ml water was used for 2 times, and the cake was rinsed with an oil pump to obtain 6.2g white solid.

Step three:

synthesis of CH3(CH2)6CO-Dab-Thr-Dab-Cyclo (Dab-D-Phe-L eu-Dab-Thr-OH):

CH3(CH2)6CO-Dab(Boc)-Thr(tBu)-Dab(Boc)-Cyclo

(Dab-Dab (Boc) -D-Phe-L eu-Dab (Boc) -Thr (tBu) -OH)6.2g was dissolved in 60ml of TFA/H2O/TIS (8:1:1) solution, stirred for 3 hours, dropped into 500ml of glacial ethyl ether solution, the precipitate was centrifuged, washed three times with ethyl ether, and dried under reduced pressure to give 2.3g of a white solid.

Step four:

purification of polymyxin B3:

CH3(CH2)6CO-Dab-Thr-Dab-Cyclo (Dab-D-Phe-L eu-Dab-Thr-OH) 2.4g was dissolved with 10% acetonitrile and water, filtered;

purification equipment: hanbang UV3000/NP7000, chromatography column DAC-50, C18,10um, wavelength: 220. mobile phase a phase: aqueous 0.1% TFA, mobile phase B: 0.1% TFA in acetonitrile, gradient: 15% -45% for 60 minutes, to give 1.3g of polymyxin B3.

The confirmation sequence after structure confirmation is:

CH3(CH2)6CO-Dab-Thr-Dab-Cyclo(Dab-Dab-D-Phe-Leu-Dab-Dab-Thr-OH)。

Claims (10)

1. a method for preparing polymyxin B related compound B1 through cyclization, which is characterized by comprising the following steps:

(1) solid-phase synthesis:

1) using CTC-Resin as a solid phase carrier, firstly coupling Fmoc-Thr (tBu) -OH to obtain Fmoc-Thr (tBu) -Resin, and removing Fmoc protection to obtain H-Thr (tBu) -Resin;

2) coupling Fmoc-Dab (Boc) -OH with H-Thr (tBu) -Resin obtained in step 1) to obtain Fmoc-Dab (Boc) -Thr (tBu) -Resin, and then condensing Fmoc-Dab (Boc) -OH, Fmoc-L eu-OH, Fmoc-D-Phe-OH, Fmoc-Dab (Boc) -OH, Fmoc-Dab (Boc) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Dab (Boc) -OH;

3) final coupling of CH₃CH₂(CH₃)CH(CH₂)₄COOH, cracking to remove resin to obtain a segment I;

(2) liquid phase condensation cyclization: coupling the fragment I obtained in the step (1) to obtain a fragment II;

(3) and (3) removing side chain protection of the fragment II obtained in the step (2) under the action of trifluoroacetic acid, and purifying to obtain a polymyxin B related compound B1.

2. A process for the cyclic preparation of polymyxin B related compound B1-I, characterized in that Fmoc-L eu-OH in step 2) of step (1) of claim 1 is replaced with Fmoc-Ile-OH.

3. A process for the cyclic preparation of polymyxin B related compound B2, which comprises reacting CH of step 3) of step (1) of claim 1₃CH₂(CH₃)CH(CH₂)₄COOH by CH₃(CH₃)CH(CH₂)₄COCOOH。

4. A process for the cyclisation of polymyxin B related compound B3, characterised in that CH from step 3) of step (1) of claim 1 is used₃CH₂(CH₃)CH(CH₂)₄COOH was replaced with CH3(CH2)6 COOH.

5. The production method according to any one of claims 1 to 4, wherein in the step (1): step 1), coupling agent of solid phase carrier coupling amino acid is DIEA; step 2) coupling agent is DIC + A or DIEA + B, A is HOBT or HOAT, B is HBTU, HATU, PyBOP, TBTU; and step 3) the coupling agent is DIC + A or B + A + C, wherein A is HOBT or HOAT, B is HBTU, HATU, TBTU or PyBOP, and C is DIEA or NMM.

6. The production method according to any one of claims 1 to 4, wherein in the step (1): and 3) cracking the resin by using reagents of trifluoroethanol and dichloromethane in a volume ratio of 1: 1-1: 4.

7. The process according to any one of claims 1 to 4, wherein in step (2), the ring-forming solvent is N, N-dimethylformamide and the coupling agent is DIC + A or DIEA + B, wherein A is HOBT and B is HBTU, HATU, PyBop, TBTU.

8. The method according to any one of claims 1 to 4, wherein in the step (2), the reaction temperature is 20 to 30 ℃ and the reaction time is 1 to 2 hours.

9. The process according to any one of claims 1 to 4, wherein in step (3), the reagents used for the protection in the cleavage sequence are a solution of trifluoroacetic acid, water and triisopropylsilane, wherein the ratio by volume of trifluoroacetic acid is 80% to 90%, the ratio by volume of water is 5% to 10%, and the ratio by volume of triisopropylsilane is 5% to 10%.

10. The method according to any one of claims 1 to 4, wherein in the step (3), the reaction temperature is 10 to 20 ℃ and the reaction time is 1 to 2 hours; after the reaction is finished, purifying the target substance by adopting a chromatographic column: mobile phase a phase: aqueous 0.1% TFA, mobile phase B: 0.1% TFA in acetonitrile, gradient: 15 to 45 percent.

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