Preparation method of antibiotic polymyxin

Abstract

The invention provides a preparation method of antibiotic polymyxin, and relates to the field of preparation of polymyxin compounds. According to the preparation method, Fmoc-Leu-CTCResin is adopted as an initial raw material, protected amino acids are sequentially coupled from a carbon terminal to a nitrogen terminal by using a solid-phase synthesis method to obtain fragments, then allyloxycarbonyl protecting groups on Dab (Alloc) are removed, and then the protected amino acids are continuously and sequentially coupled to obtain resin with branched chains; the preparation method comprises the following steps: firstly preparing a CTCresinin segment, then splitting the CTCresinin segment from the resin by using a low-concentration acid, then carrying out liquid-phase cyclization, cutting and purifying to obtain a final product with the purity of more than 99.0% and the total yield of more than 35%; according to the method, a solid-phase synthesis fragment is adopted, a carbon terminal starts from Leu, the problem of subsequent cyclization racemization can be effectively avoided, the process for removing an allyloxycarbonyl protecting group on Dab (Alloc) is simple, the purity and yield of the obtained product are relatively high, and the yield and purity of the structure (1) and antibiotic polymyxin are further improved.

Description

Technical Field

[0001] The present invention relates to the field of preparation of polymyxin compounds, and particularly to a method for preparing the antibiotic polymyxin. Background Art

[0002] Polymyxin B nonapeptide (PMBN) is a cationic cyclic peptide derived from the naturally occurring peptide polymyxin B by enzymatic treatment, and can increase the permeability of the outer membrane of Gram-negative bacteria to hydrophobic antibiotics by binding to bacterial lipopolysaccharide (LPS). Compared with polymyxin B, polymyxin B nonapeptide has less toxicity and lacks bactericidal activity, but still has the ability to disrupt the outer membrane of Gram-negative bacteria. The effectiveness of polymyxin B nonapeptide as an anti-endotoxin agent is evaluated by detecting its ability to block the enhanced release of toxic oxygen free radicals induced by lipopolysaccharide in human neutrophils.

[0003] In vivo, at doses of 1.5 and 3.0 mg / kg, polymyxin B nonapeptide did not exhibit the neuromuscular blockade, neurotoxicity or nephrotoxicity effects observed with polymyxin B sulfate. Both polymyxin B and polymyxin B nonapeptide inhibited lipopolysaccharide-induced neutrophil activation in a concentration-dependent manner, but the effectiveness of the parent compound polymyxin B was 63 times that of the weight. However, when these two compounds were added after the start of lipopolysaccharide-neutrophil incubation, their inhibitory activity decreased rapidly.

[0004] The toxicity of polymyxin B nonapeptide is lower than that of polymyxin B, and at the tested doses, it lacks the neurotoxicity and nephrotoxicity of the parent compound. Polymyxin B nonapeptide retains the anti-endotoxin activity of polymyxin B, but the potency is much lower. Given the high specificity and drug resistance of this type of drug, there is an interest in developing polymyxins with improved therapeutic indices and effects. Therefore, the development of derivatives such as synthetic polymyxin B nonapeptide is of great significance.

[0005] In the prior art, in the process of synthesizing polymyxin B nonapeptide, there are problems of low yield and high by-products, such as the β-elimination of the Thr(tBu) side chain at the carbon terminus to generate α-aminocrotonic acid derivatives; or catalytic hydrogenation for deprotection, with harsh experimental conditions and high requirements for equipment. Summary of the Invention

[0006] In order to solve the problems of low yield, high by-products or harsh reaction conditions in the preparation process of polymyxin B nonapeptide in the prior art, the present application provides a method for preparing the antibiotic polymyxin.

[0007] A method for preparing an antibiotic polymyxin:

[0008] Preparation method of antibiotic polymyxin, which obtains antibiotic polymyxin Thr-Dab-Cyclo(Dab-Dab-DPhe-Leu-Dab-Dab-Thr) by successively carrying out cyclization reaction and deprotection cleavage reaction on structure (1);

[0009] The structure of the said structure (1) is as follows: ;

[0010] Among them, the groups A1, A2, A3, A4, and A5 are amino protecting groups, and the groups B1 and B2 are carboxyl protecting groups.

[0011] Furthermore, the groups A1, A2, A3, A4, and A5 are each any one of Pht, Tos, Fmoc, Boc, Cbz, Bn, PMB, Alloc, and Mtt; the groups B1 and B2 are each any one of tBu, CTCResin, and WangResin.

[0012] Furthermore, A1, A2, A3, A4, and A5 are all Boc, and B1 and B2 are both tBu;

[0013] The structure of the said structure (1) is as follows: .

[0014] Furthermore, cleave the CTC Resin group in S1:

[0015] Use the CTCResin group in the cleavage resin Boc-Thr(tBu)-Dab(Boc)-Dab(Thr(tBu)-Dab(Boc)-Dab(Boc))-Dab(Boc)-DPhe-Leu-CTCResin containing TFA to obtain structure (1), then wash and lyophilize;

[0016] S2 liquid-phase cyclization:

[0017] 1) After activating the structural formula (1) Boc-Thr(tBu)-Dab(Boc)-Dab(Thr(tBu)-Dab(Boc)-Dab(Boc))-Dab(Boc)-DPhe-Leu-OH with HOOBt and / or EDC·HCl, carry out intramolecular amidation reaction;

[0018] 2) After the reaction is completed, obtain the crude product of Boc-Thr(tBu)-Dab(Boc)-Cyclo(Dab-Dab(Boc)-DPhe-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)) by rotary evaporation, separation, and washing;

[0019] S3 Deprotection and cleavage:

[0020] Use solution F to cleave the protecting groups in Boc-Thr(tBu)-Dab(Boc)-Cyclo(Dab-Dab(Boc)-DPhe-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)). After removing the cleavage solution by rotary evaporation, separating, centrifuging, and drying, the crude product of Thr-Dab-Cyclo(Dab-Dab-Thr-Dab-Dab-DPhe-Leu-Dab-Dab-Thr) is obtained;

[0021] S4 Preparation and purification of the crude product:

[0022] Dissolve the crude product obtained in step S3 in acetonitrile / water, filter it through a mixed cellulose ester membrane with a pore size of less than 1 μm, and then purify it using a chromatographic column to obtain the antibiotic polymyxin.

[0023] The process of further preferably cleaving the CTC Resin group in S1 is as follows:

[0024] Use 0.5 - 2% TFA / DCM to cleave the CTCResin group in the resin Boc-Thr(tBu)-Dab(Boc)-Dab(Thr(tBu)-Dab(Boc)-Dab(Boc))-Dab(Boc)-DPhe-Leu-CTCResin to obtain structure (1), then wash and lyophilize;

[0025] The preparation process of further preferably S2 liquid-phase cyclization is as follows:

[0026] 1) Dissolve 13 - 15 g of the structural formula (1) Boc-Thr(tBu)-Dab(Boc)-Dab(Thr(tBu)-Dab(Boc)-Dab(Boc))-Dab(Boc)-DPhe-Leu-OH and 2.5 - 3.5 g of HOOBt in DCM, add 3 - 4 g of EDC·HCl,

[0027] Subsequently, add 3 - 4 mL of DIPEA and react at room temperature for 10 - 15 h;

[0028] 2) After the reaction is completed, remove DCM by rotary evaporation, separate and wash to obtain the crude product of Boc-Thr(tBu)-Dab(Boc)-Cyclo(Dab-Dab(Boc)-DPhe-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu));

[0029] In the preparation and purification of the crude product in S4, it is further preferably filtered through a mixed cellulose ester membrane with a pore size of 0.35 - 0.5 μm.

[0030] Furthermore, the purity of the antibiotic polymyxin obtained by the preparation method of the present application is > 99.0%, and the total yield is > 35%.

[0031] Furthermore, the preparation process of the structure (1) includes the following steps:

[0032] Remove the A6 protecting group from the structural formula (2) A1-Thr(B1)-Dab(A2)-Dab(A6)-Dab(A3)-DPhe-Leu-B2, and then successively carry out coupling reactions with Fmoc-Thr(tBu)-OH, Fmoc-Dab(Boc)-OH, and Fmoc-Dab(Boc)-OH. Then, remove the B2 protecting group to obtain the structural formula (1).

[0033] Furthermore, the A6 group is Alloc, the A1, A2, and A3 are all Boc, the B1 is tBu, and the B2 is CTCResin.

[0034] Furthermore, the preparation process of the structure (1) includes the following preparation process:

[0035] Solid-phase synthesis

[0036] 1) Sufficient swelling of the resin

[0037] Soak and swell Fmoc-Leu-CTCResin with an organic solvent;

[0038] 2) Removal of the Fmoc protecting group

[0039] Remove the Fmoc group in the swollen Fmoc-Leu-CTCResin under an inert gas atmosphere;

[0040] 3) Linking amino acids

[0041] After activating the C-terminal amino acid Fmoc-DPhe-OH with HOBt and / or DIC, add it to the resin obtained in step 2) for coupling. After the coupling is completed, wash and drain;

[0042] 4) Repeat steps 2-4 to successively link amino acids. For the amino acid polypeptide sequence, successively link Fmoc-Dab(Boc)-OH, Fmoc-Dab(Alloc)-OH, Fmoc-Dab(Boc)-OH, and Boc-Thr(tBu)-OH to obtain the structural formula (2) Boc-Thr(tBu) -Dab(Boc) -Dab(Alloc)-Dab(Boc)-DPhe-Leu-CTCResin.

[0043] Further preferably, the preparation process of the structure (1) includes the following steps:

[0044] Solid-phase synthesis

[0045] 1) Sufficient swelling of the resin

[0046] Soak and swell Fmoc-Leu-CTCResin with DMF;

[0047] 2) Removal of the Fmoc protecting group

[0048] Remove the Fmoc group in the swollen Fmoc-Leu-CTCResin with 20% Pip / DMF under nitrogen;

[0049] 3) Opaque dark blue after Kaiser test;

[0050] 4) Coupling of amino acids

[0051] Weigh 2.5 - 3.5 times the amount of the C-terminal amino acid Fmoc-DPhe-OH and 2.5 - 3.5 times the amount of HOBt, dissolve them in 250 mL of DMF, cool down to -5 °C to -15 °C, add 2.5 - 3.5 times the amount of DIC for activation, then add it to the resin, couple for 1 - 2 h. After the Kaiser test shows colorless, wash and drain;

[0052] 5) Repeat steps 2 - 4 to couple amino acids in sequence

[0053] Repeat steps 2 - 4. According to the polypeptide sequence, couple Fmoc-Dab(Boc)-OH, Fmoc-Dab(Alloc)-OH, Fmoc-Dab(Boc)-OH, Boc-Thr(tBu)-OH in sequence to obtain the structural formula (2) Boc-Thr(tBu)-Dab(Boc)-Dab(Alloc)-Dab(Boc)-DPhe-Leu-CTCResin.

[0054] Furthermore, the A6 group is Mtt, the A1, A2, and A3 are all Boc, the B1 is tBu, and the B2 is CTCResin; cut the Mtt group in the presence of AcOH, TFE, and DOC;

[0055] The structural formula (2) is Boc-Thr(tBu)-Dab(Boc)-Dab(Mtt)-Dab(Boc)-DPhe-Leu-CTCResin.

[0056] Furthermore, the A6 group is Boc, the A1, A2, and A3 are all Cbz, the B1 is tBu, and the B2 is CTCResin; the Boc group is cleaved using a TFA solution;

[0057] The structural formula (2) is Cbz-Thr(tBu)-Dab(Cbz)-Dab(Boc)-Dab(Cbz)-DPhe-Leu-CTCResin.

[0058] Beneficial effects:

[0059] 1. The preparation method of the present application uses Fmoc-Leu-CTCResin as the starting material, and uses the solid-phase synthesis method to sequentially couple the protected amino acids from the carbon terminus to the nitrogen terminus to obtain a fragment. Then, the allyloxycarbonyl protecting group on Dab(Alloc) is removed, and then the protected amino acids are sequentially coupled to obtain a resin with a branched chain. Then, the CTCresin fragment is cleaved from the resin using a lower concentration of acid, and then liquid-phase cyclization is carried out. After cleavage and purification, the final product is obtained, with a purity > 99.0% and a total yield > 35%; this method uses solid-phase synthesis of the fragment, starting from Leu at the carbon terminus, which can effectively avoid the problem of subsequent cyclization racemization, and the process of removing the allyloxycarbonyl protecting group on Dab(Alloc) is simple, and the obtained product has high purity and yield, thereby improving the yield and purity of structure (1) and the antibiotic polymyxin.

[0060] 2. Preferably, the A1, A2, and A3 groups and the cleavage conditions are selected to improve the yield and purity of the substance of structure (1), thereby improving the yield and purity of the antibiotic polymyxin. Description of the drawings

[0061] Figure 1 is the preparation flow chart of the antibiotic polymyxin in Example 1;

[0062] Figure 2 is the structural formula of the antibiotic polymyxin;

[0063] Figure 3 is the chromatogram of the structural formula (1) obtained in Example 1;

[0064] Figure 4 is the mass spectrum of the structural formula (1) obtained in Example 1;

[0065] Figure 5 is the chromatogram of the antibiotic polymyxin obtained by using the method of Example 1;

[0066] Figure 6 is the preparation flow chart of the antibiotic polymyxin in Comparative Example 1;

[0067] Figure 7It is a process flow diagram for preparing the antibiotic polymyxin in Comparative Example 2. Detailed implementation manners

[0068] To make the technical solution of the present invention clearer, the following further describes the present invention in detail with reference to the accompanying drawings and specific embodiments.

[0069] Example 1. A method for preparing the antibiotic polymyxin, and the preparation process is as Figure 1 , and the structural formula of the antibiotic polymyxin Thr-Dab-Cyclo(Dab-Dab-DPhe-Leu-Dab-Dab-Thr) is as Figure 2 .

[0070] Specifically, it includes the following preparation process:

[0071] Preparation of the structural formula (2) A1-Thr(B1)-Dab(A2)-Dab(A6)-Dab(A3)-DPhe-Leu-B2:

[0072] Among them, the A6 group is Alloc, A1, A2, and A3 are all Boc, B1 is tBu, and B2 is ‌CTC Resin.

[0073] T1: Solid-phase synthesis step

[0074] 1) Sufficient swelling of the resin

[0075] Weigh 14.6 g of Fmoc-Leu-CTCResin with a substitution degree of 0.687 mol / g, put it into a polypeptide solid-phase reactor, and then add 250 mL of DMF for soaking and swelling for 1 h.

[0076] 2) Removal of the Fmoc protecting group

[0077] Vacuum filter off the DMF, and then wash it three times with 250 mL of DMF solution. Drain the DMF, add 200 mL of 20% Pip / DMF, purge with nitrogen for deprotection for 0.5 h, and wash it 5 times with 300 mL of DMF.

[0078] 3) Kaiser detection

[0079] Preparation of the detection reagent 1) Kaiser detection solution A: 20% ethanol + 80% phenol; 2) Kaiser detection solution B: redistilled pyridine; 3) Kaiser detection solution C: 5% ninhydrin ethanol solution. Detection operation: Take a small amount of resin in a detection tube, wash it twice with ethanol, add two drops of Kaiser detection solution A, Kaiser detection solution B, and Kaiser detection solution C each, heat it to 110 degrees for 2 minutes, and observe that the color of the resin is opaque dark blue.

[0080] Color development result: After removing the Fmoc protecting group in solid-phase synthesis and detecting the resin, an opaque dark blue color will be observed. If the amino acids in solid-phase synthesis are completely coupled and there are no free amino groups, the detection at this time shows yellow or colorless.

[0081] 4) Connect amino acids

[0082] Weigh 3 equivalents of the C-terminal amino acid Fmoc-DPhe-OH and 3 equivalents of HOBt, dissolve them in 250 mL of DMF, cool down to -10 °C, add 3 equivalents of DIC to activate for 10 min, then add it to the resin, and couple at room temperature for 1.5 h. After Kaiser detection, it is colorless. After the coupling is completed, add 250 mL of DMF to wash three times and drain.

[0083] 5) Repeat steps 2 - 4 to connect amino acids in sequence

[0084] Repeat steps 2 - 4. According to the polypeptide sequence, connect Fmoc-Dab(Boc)-OH, Fmoc-Dab(Alloc)-OH, Fmoc-Dab(Boc)-OH, Boc-Thr(tBu)-OH in sequence to obtain the structure (2) Boc-Thr(tBu)-Dab(Boc)-Dab(Alloc)-Dab(Boc)-DPhe-Leu-CTCResin.

[0085] 6) Remove allyloxycarbonyl (Alloc)

[0086] First, wash the resin twice with 200 mL of dichloromethane. Weigh 0.1 equivalent of tetrakis(triphenylphosphine)palladium catalyst in a conical flask, add 250 mL of dichloromethane to dissolve it, add this solution to the resin, react under nitrogen bubbling, then add 12 equivalents of phenylsilane and 2 equivalents of HOBt, react at room temperature for 2 h, drain the resin, and then wash it five times with 250 mL of dichloromethane. After Kaiser detection, it is dark blue.

[0087] 7) Repeat steps 2 - 4 to connect amino acids in sequence

[0088] Repeat steps 2 - 4. Using the method of DIC and HOBT, connect Fmoc-Thr(tBu)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Boc)-OH in sequence according to the polypeptide sequence. After removing Fmoc, obtain Boc-Thr(tBu)-Dab(Boc)-Dab(Thr(tBu)-Dab(Boc)-Dab(Boc))-Dab(Boc)-DPhe-Leu-CTCResin.

[0089] 8) Wash the resin

[0090] Add 300 mL of methanol and wash three times. Finally, dry it under vacuum to obtain 28.81 g of Boc-Thr(tBu)-Dab(Boc)-Dab(Thr(tBu)-Dab(Boc)-Dab(Boc))-Dab(Boc)-DPhe-Leu-CTCResin.

[0091] T2: Liquid-phase synthesis step

[0092] Preparation of the antibiotic polymyxin:

[0093] S1 Cleavage of the CTCResin group:

[0094] 1) Disperse and suspend 28.81 g of the resin in 300 mL of 1% TFA / DCM and shake it on a shaker at room temperature for 1 h.

[0095] 2) Filter the resin suspension. Wash the resin 3 times with 100 mL of DCM. Add 100 mL of water to the filtrate, stir, and then remove DCM by rotary evaporation under reduced pressure. After lyophilizing the aqueous phase, obtain 14.8 g of the crude product of structure (1) Boc-Thr(tBu)-Dab(Boc)-Dab(Thr(tBu)-Dab(Boc)-Dab(Boc))-Dab(Boc)-DPhe-Leu-OH. The chromatogram is as Figure 3 , and the mass spectrum is as Figure 4 .

[0096] 3) Yield: 92.9%

[0097] 4) Purity: 61.9%

[0098] 5) Mass spectrum: 1593.94(1594.36(M+1), 797.98(M+2 / 2))

[0099] S2 Liquid-phase cyclization:

[0100] 1) Dissolve 14.8 g of Boc-Thr(tBu)-Dab(Boc)-Dab(Thr(tBu)-Dab(Boc)-Dab(Boc))-Dab(Boc)-DPhe-Leu-OH and 3.03 g of HOOBt in 9280 mL of DCM. Add 3.56 g of EDC·HCl, and then add 3.2 mL of DIPEA. React at room temperature for 12 h.

[0101] 2) After the reaction was completed, DCM was removed by rotary evaporation until the volume reached 200 mL. 100 mL of 5% phosphoric acid aqueous solution was added, and the organic phase was separated by layering. The organic phase was washed successively with water, saturated sodium bicarbonate aqueous solution, water, and saturated brine. DCM was removed by rotary evaporation to obtain 14 g of the crude product of the antibiotic polymyxin Boc-Thr(tBu)-Dab(Boc)-Cyclo(Dab-Dab(Boc)-DPhe-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)).

[0102] 3) Yield: 95.9%

[0103] 4) Mass spectrometry: 1575.93 (1576.21 (M+1)).

[0104] S3 Deprotection and cleavage:

[0105] 1) Dissolve 14 g of Boc-Thr(tBu)-Dab(Boc)-Cyclo(Dab-Dab(Boc)-DPhe-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)) in 140 mL of Solution F (95% TFA + 2.5% TIS + 2.5% water), and stir at room temperature for 2 h.

[0106] 2) Remove the cleavage solution by rotary evaporation, add 600 mL of methyl tert-butyl ether to precipitate the product, centrifuge to separate the precipitate, and dry the precipitate in a vacuum dryer to obtain 9.63 g of the crude product of Thr-Dab-Cyclo(Dab-Dab-Thr-Dab-Dab-DPhe-Leu-Dab-Dab-Thr).

[0107] 3) Purity: 60.7%;

[0108] 4) Mass spectrometry: 963.13 (482.33 (M+2 / 2));

[0109] S4 Preparation and purification of the crude product:

[0110] 1) Sample pretreatment: Dissolve the sample in 10% acetonitrile / water and filter through a 0.45 μm mixed cellulose ester membrane.

[0111] 2) Purification preparation method:

[0112] Chromatographic column: Huapu C18 10 μm 100A 50*450 mm (P06)

[0113] Mobile phase A: 0.1% TFA aqueous solution

[0114] Mobile phase B: Acetonitrile

[0115] Sample loading flow rate: 60 ml / min Elution flow rate: 60 ml / min Detection wavelength: 220 nm

[0116] Elution gradient: 11 - 41% for 60 minutes

[0117] After preparation and purification, the product was collected and lyophilized to obtain 6026343.62 g

[0118] Purity: 99.6%;

[0119] Mass spectrometry: 963.1 (482.28 (M+2 / 2)) (MS: 602634)

[0120] The chromatogram is as Figure 5 ; <�

[0121] Total yield: 37.6%.

[0122] Example 2, a method for preparing the antibiotic polymyxin, which is different from Example 1 in that the groups A1, A2, A3, and A6 in the structural formula (2) are different, the A6 group is Mtt, and A1, A2, and A3 are all Boc; the preparation process of the structural formula (2) is different, and the specific differences in the preparation process are in steps 5) and 6);

[0123] Specifically as follows:

[0124] 5) Repeat the steps of 2 - 4 to sequentially connect amino acids

[0125] Repeat the steps of 2 - 4, and according to the polypeptide sequence, sequentially connect Fmoc - Dab(Boc) - OH, Fmoc - Dab(Mtt) - OH, Fmoc - Dab(Boc) - OH, Boc - Thr(tBu) - OH to obtain the structural formula (2) Boc - Thr(tBu) - Dab(Boc) - Dab(Mtt) - Dab(Boc) - DPhe - Leu - CTCResin.

[0126] 6) Remove the Mtt group

[0127] Place 10 g of the structural formula (2) Boc - Thr(tBu) - Dab(Boc) - Dab(Mtt) - Dab(Boc) - DPhe - Leu - CTCResin resin in a reaction vessel, then add 200 mL of a mixed solvent (specifically a composition of AcOH:TFE:DCM with a volume ratio of 1:1:8), stir and react at room temperature (25 °C) for 10 minutes; then filter to remove the reaction solution and wash the resin with DCM; repeat the above process 3 times, with a total reaction time of about 30 minutes, wash the resin 3 times with DCM to remove by - products, and the Kaiser test shows a dark blue color.

[0128] Example 3, a method for preparing the antibiotic polymyxin, which is different from Example 1 in that the groups A1, A2, A3, and A6 in structural formula (2) are different, and the preparation process of structural formula (2) is different.

[0129] The A6 group is Boc, and A1, A2, and A3 are all Cbz; the Boc group is cleaved under the condition of TFA solution;

[0130] The structural formula (2) is Cbz-Thr(tBu)-Dab(Cbz)-Dab(Boc)-Dab(Cbz)-DPhe-Leu-CTCResin.

[0131] The specific preparation processes of steps 5) and 6) are as follows:

[0132] 5) Repeat the steps of 2-4 to sequentially connect the amino acids

[0133] Repeat the steps of 2-4, and sequentially connect Fmoc-Dab(Cbz)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Cbz)-OH, and Cbz-Thr(tBu)-OH according to the polypeptide sequence to obtain the structural formula (2) Cbz-Thr(tBu)-Dab(Cbz)- Dab(Boc)-Dab(Cbz)-DPhe-Leu-CTCResin.

[0134] 6) Remove the Boc group

[0135] Place 10 g of the structural formula (2) Cbz-Thr(tBu)-Dab(Cbz)- Dab(Boc)-Dab(Cbz)-DPhe-Leu-CTCResin resin in a reaction vessel, then add 150 mL of a mixed solvent (30% TFA / DCM solution by volume), stir and react at room temperature (25 °C) for 10 minutes; then filter to remove the reaction solution, and wash the resin with DCM; repeat the above process 3 times, with a total reaction time of about 30 minutes, wash the resin 3 times with DCM to remove by-products, and the Kaiser test shows a dark blue color.

[0136] The yields and purities in the preparation methods of Example 1 and Example 3 are shown in Table 1.

[0137] Table 1. List of yields and purities in the preparation methods of Example 1 and Example 3

[0138]

[0139] The experimental data in Table 1 show that when the A6 group uses the Alloc group and the experimental conditions for removing the Alloc group are adopted, fewer by-products are generated and the purity is higher.

[0140] Comparative Example 1, Preparation method of antibiotic polymyxin, the preparation process is as follows Figure 6 , specifically including the following preparation steps:

[0141] S1 Resin pretreatment:

[0142] Swell 10 g of Fmoc-Dab-Thr(tBu)OtBu resin with 100 mL of DCM for 60 minutes, filter by suction, and then wash with 100 mL of DMF three times.

[0143] S2 Coupling reaction:

[0144] Weigh 3 equivalents of Fmoc-Dab-Thr(tBu)OtBu and dissolve it in 100 mL of DMF, add 6 equivalents of DIEA, and activate for 5 minutes; then add it to 1 equivalent (8 mmol) of resin (Benzyloxychlorotormate resin), and react at room temperature for 2 hours under a nitrogen atmosphere; confirm the completion of the reaction by ninhydrin test, and wash three times with 10 mL of DMF solvent.

[0145] S3 Fmoc deprotection:

[0146] Add 100 mL of 20% piperidine / DMF solution to the reactant in step S2, shake at room temperature for 10 minutes, and filter by suction; then repeat the operation, add 100 mL of 20% piperidine / DMF solution, shake at room temperature for 10 minutes, and filter by suction; then wash three times with 100 mL of DMF solvent to obtain the resin.

[0147] S4 Polypeptide chain extension (7 coupling cycles):

[0148] Take 3 equivalents (24 mmol) of Fmoc-AA-OH and dissolve it in 100 mL of DMF; then add 3 equivalents of HBTU, 3 equivalents of HOBt, and 6 equivalents of DIEA, and activate for 5 minutes to obtain an activation solution; add the activation solution to the resin obtained in the previous step, and react at room temperature (25 °C) for 1 hour; confirm the completion by ninhydrin test, and wash three times with 10 mL of DMF solvent; then perform Fmoc deprotection, and the specific operation is as in S3.

[0149] The order of Fmoc-AA-OH amino acids coupled in sequence is Fmoc-Dab(Cbz)-OH, Fmoc-Leu-OH, Fmoc-dPhe-OH, Fmoc-Dab(Cbz)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Cbz)-OH, Fmoc-Thr(tBu)-OH.

[0150] Deprotection of the tBu protecting group of Thr(tBu) and the Boc group of Dab(Boc):

[0151] Prepare 100 mL of 1% TFA / DCM solution and add it to the resin obtained in step S4. React at room temperature (25°C) for 30 minutes, then perform suction filtration. Subsequently, wash with 100 mL of DCM and 100 mL of DMF successively, washing 3 times each.

[0152] S6 Cyclization reaction:

[0153] Swell 1 equivalent (8 mmol) of the resin obtained in step S5 in DMF, add HATU (5 equivalents), HOAt (5 equivalents), and DIEA (10 equivalents), and activate for 1 hour. After the reaction is completed, rotary evaporate to remove DCM until the volume reaches 200 mL. Add 100 mL of 5% aqueous phosphoric acid solution, separate the organic phase by liquid-liquid extraction. Wash the organic phase successively with water, saturated sodium bicarbonate aqueous solution, water, and saturated brine, and then rotary evaporate to remove DCM.

[0154] S7 Resin cleavage:

[0155] Use 20% piperidine to deprotect the N-terminal Fmoc group, with the specific operation as in step S3. Then, take 100 mL of the cleavage solution (TFA / TMSBr / H2O volume ratio 94:5:1), add it to the resin obtained in step S7, and react at room temperature (25°C) for 2 hours. Rotary evaporate to remove the cleavage solution, add 100 mL of methyl tert-butyl ether to precipitate the product, centrifuge to separate the precipitate, and dry the precipitate in a vacuum dryer to obtain the crude product of Thr-Dab-Cyclo(Dab-Dab-Thr-Dab-Dab-DPhe-Leu-Dab -Dab-Thr).

[0156] S8 Preparation and purification of the crude product:

[0157] 1) Sample pretreatment: Dissolve the sample in 10% acetonitrile / water and filter through a 0.45 μm mixed cellulose ester membrane.

[0158] 2) Purification preparation method:

[0159] Chromatographic column: Huapu C18 10μm 100A 50*450mm (P06)

[0160] Mobile phase A: 0.1% aqueous TFA solution

[0161] Mobile phase B: Acetonitrile

[0162] Sample loading flow rate: 60 ml / min Elution flow rate: 60 ml / min Detection wavelength: 220 nm

[0163] Elution gradient: 11 - 41% over 60 minutes;

[0164] After purification, the product is collected and freeze-dried to obtain the antibiotic polymyxin.

[0165] Comparative Example 2, a method for preparing the antibiotic polymyxin, the preparation process is as Figure 7 , specifically including the following preparation process:

[0166] S1 Resin pretreatment:

[0167] 1 equivalent (8 mmol) of Fmoc-Thr(tBu)-2-Chlorotrityl resin is swollen in 100 mL of DCM for 60 minutes, filtered by suction, and then washed 3 times with 100 mL of DMF.

[0168] S2 Fmoc deprotection:

[0169] 100 mL of 20% piperidine / DMF solution is added to the resin after the S1 step treatment, shaken at room temperature for 10 minutes, and filtered by suction; then the operation is repeated, 100 mL of 20% piperidine / DMF solution is added, shaken at room temperature for 10 minutes, and filtered by suction; then washed 3 times with 100 mL of DMF solvent to obtain the resin with Fmoc removed.

[0170] S3 Coupling - 8 Cycles:

[0171] 3 equivalents (24 mmol) of Fmoc-AA-OH are dissolved in 100 mL of DMF; then 3 equivalents of HBTU, 3 equivalents of HOBt, and 6 equivalents of DIEA are added, activated for 5 minutes to obtain an activation solution; the activation solution is added to the resin obtained in the previous step, and reacted at room temperature of 25 °C for 1 hour; ninhydrin test is used to confirm completion, and washed 3 times with 10 mL of DMF solvent; then Fmoc deprotection is carried out, and the specific operation is as in S2; after 8 cycles of coupling, HO-Thr(tBu)-Dab(tBoc)-Dab(Mtt)-Dab(tBoc)-dPhe-Leu-Dab(tBoc)-Dab(tBoc)-Thr(tBu)-resin is obtained.

[0172] The order of the successively coupled Fmoc-AA-OH amino acids is Fmoc-Dab(Boc), Fmoc-Dab(Boc), Fmoc-Leu, Fmoc-dPhe, Fmoc-Dab(Boc), Fmoc-Dab(Mtt), Fmoc-Dab(Boc), Fmoc-Thr(tBu).

[0173] S4 Boc protection:

[0174] To the resin obtained in S3, add 100 mL of DMF, Boc2O (5 equivalents), and DIEA (10 equivalents), and react for 1 hour; then wash the resin with DMF three times to remove unreacted reagents, obtaining Boc-Thr(tBu)-Dab(Boc)-Dab(Mtt)-Dab(Boc)-dPhe-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-resin.

[0175] S5 Mtt Deprotection:

[0176] To the resin obtained in S4, add 100 mL of AcOH / TFE / DCM (1:1:8) solution and react for 30 minutes; repeat the operation and react for another 30 minutes to ensure complete removal of the Mtt group; then wash the resin with DCM three times to obtain Boc-Thr(tBu)-Dab(Boc)-Dab(NH2)-Dab(Boc)-dPhe-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-resin.

[0177] S6 Cyclization Reaction:

[0178] To the resin obtained in step S5, add PyBOP (4 equivalents), HOBt (4 equivalents), DIEA (8 equivalents), and 10 mL of DMF, and react for 3 hours. After the reaction, wash the resin with DMF three times to remove unreacted reagents, obtaining the product: Boc-Thr(tBu)-Dab(Boc)-Cyclo(Dab-Dab(Boc)-dPhe-Leu-Dab(Boc)-Dab(Boc) -Thr(tBu))-resin.

[0179] S7 Resin Cleavage and Boc Deprotection

[0180] To the resin obtained in step S6, add 100 mL of TFA / DCM (95:5) solution and react at room temperature (25 °C) for 2 hours; rotary evaporate to remove the TFA / DCM (95:5) solution, add 100 mL of methyl tert-butyl ether to precipitate the product, centrifuge to separate the precipitate, and dry the precipitate in a vacuum dryer to obtain the crude product of Thr-Dab-Cyclo(Dab-Dab-Thr-Dab-Dab-DPhe-Leu-Dab -Dab-Thr).

[0181] S8 Preparation and Purification of Crude Product:

[0182] 1) Sample Pretreatment: Dissolve the sample in 10% acetonitrile / water and filter through a 0.45 μm mixed fiber membrane.

[0183] 2) Purification Preparation Method:

[0184] Chromatographic column: Huapu C18 10μm 100A 50*450mm (P06)

[0185] Mobile phase A: 0.1% aqueous TFA solution

[0186] Mobile phase B: Acetonitrile

[0187] Sample loading flow rate: 60 ml / min Elution flow rate: 60 ml / min Detection wavelength: 220 nm

[0188] Elution gradient: 11 - 41% for 60 minutes;

[0189] After preparation and purification, the product is collected and freeze-dried to obtain the antibiotic polymyxin.

[0190] The yields and purities in the preparation methods of Comparative Example 1 and Comparative Example 2 are shown in Table 2.

[0191] Table 2. List of yields and purities in the preparation methods of Comparative Example 1 and Comparative Example 2

[0192]

[0193] In the preparation method of Comparative Example 1, cyclization with HATU on the resin will produce a by-product of tetramethylguanidine-capped Dab amino group, and the synthesis of the dipeptide Fmoc-Dab(resin)-Thr(tBu)-OtBu is laborious in terms of process, resulting in lower purity and yield of the resin obtained after deprotecting S5.

[0194] In the preparation method of Comparative Example 2, in the S3 coupling - 8 Cycles step, a by-product of tetramethylguanidine-capped Dab amino group is likely to appear, resulting in lower purity and yield of the resin obtained.

[0195] Compared with the prior art Comparative Example 1 and Comparative Example 2, the present application provides a structure (1) in which a branched chain -Thr-Dab-Dab is connected to the third amino acid Dab starting from the N-terminus. The internal cyclization yield of structure (1) is 92.8 - 95.9%, while the cyclization yield of the amino acid structure with a linear structure in Comparative Example 1 and Comparative Example 2 is 72.3 - 75.9%. Using the structure (1) of the present application has higher yield and product purity, and has excellent technical effects; it may be because the active sites on the two branched chains connected to the third amino acid Dab in the structure (1) of the present application are more likely to come into contact, with higher reaction activity and fewer side reactions, and thus have higher cyclization yield, total yield and higher product purity.

[0196] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent for the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention shall be subject to the appended claims.

Claims

1. A method for preparing the antibiotic polymyxin, characterized in that, Through the cyclization reaction and deprotection cleavage reaction of structure (1) in sequence, the antibiotic polymyxin Thr-Dab-Cyclo(Dab-Dab-DPhe-Leu-Dab-Dab-Thr) is obtained; The structure of the said structure (1) is as follows: ； Wherein, the groups A1, A2, A3, A4, and A5 are amino protecting groups, and the groups B1 and B2 are carboxyl protecting groups. Wherein the sequence of structural formula (1) is shown in SEQ ID NO.1, and the sequence of the antibiotic polymyxin is shown in SEQ ID NO.

2.

2. The preparation method of the antibiotic polymyxin according to claim 1, characterized in that, The said groups A1, A2, A3, A4, and A5 are each any one of Pht, Tos, Fmoc, Boc, Cbz, Bn, PMB, Alloc, and Mtt; the said groups B1 and B2 are each any one of tBu, CTCResin, and WangResin.

3. The preparation method of the antibiotic polymyxin according to claim 1, wherein, The said A1, A2, A3, A4, and A5 are all Boc, and the said B1 and B2 are both tBu; The structure of the said structure (1) is as follows: 。 4. The preparation method of the antibiotic polymyxin according to claim 3, characterized in that, It includes the following preparation process: S1 Cleave the CTC Resin group; S2 Liquid-phase cyclization; S3 Deprotection cleavage; S4 Preparation and purification of the crude product.

5. The preparation method of the antibiotic polymyxin according to claim 4, characterized in that, The purity of the obtained antibiotic polymyxin is >99.0%, and the total yield is >35%.

6. The preparation method of the antibiotic polymyxin according to claim 1, characterized in that, The preparation process of the said structure (1) includes the following steps: Remove the A6 protecting group from the structural formula (2) A1-Thr(B1)-Dab(A2)-Dab(A6)-Dab(A3)-DPhe-Leu-B2, and then successively carry out coupling reactions with Fmoc-Thr(tBu)-OH, Fmoc-Dab(Boc)-OH, and Fmoc-Dab(Boc)-OH, and then remove the B2 protecting group to obtain the structural formula (1), wherein the sequence of the structural formula (2) is shown in SEQ ID NO.

3.

7. The preparation method of the antibiotic polymyxin according to claim 6, wherein, The said A6 group is Alloc, the said A1, A2, and A3 are all Boc, the said B1 is tBu, and the said B2 is CTC resin.

8. The preparation method of the antibiotic polymyxin according to claim 7, characterized in that, The preparation process of the said structure (1) includes the following preparation process: Solid-phase synthesis 1) Sufficient swelling of the resin; 2) Removal of the Fmoc protecting group; 3) Linking amino acids; 4) Repeat steps 2-4.

9. The preparation method of the antibiotic polymyxin according to claim 6, characterized in that, The said A6 group is Mtt, the said A1, A2, and A3 are all Boc, the said B1 is tBu, and the said B2 is CTCResin; cleave the Mtt group under the condition of the presence of AcOH, TFE, and DOC; The said structural formula (2) is Boc-Thr(tBu)-Dab(Boc)-Dab(Mtt)-Dab(Boc)-DPhe-Leu-CTCResin.

10. The preparation method of the antibiotic polymyxin according to claim 6, characterized in that, The said A6 group is Boc, the said A1, A2, and A3 are all Cbz, the said B1 is tBu, and the said B2 is CTCResin; use TFA solution to cleave the Boc group; The said structural formula (2) is Cbz-Thr(tBu)-Dab(Cbz)-Dab(Boc)-Dab(Cbz)-DPhe-Leu-CTCResin.

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