Preparation method of antibiotic polymyxin

Abstract

The present invention provides a method for preparing the antibiotic polymyxin, and relates to the field of polymyxin compound preparation. The preparation method of the present application uses Fmoc-Leu-CTCResin as a starting material, and utilizes a solid-phase synthesis method to sequentially couple protected amino acids from the carbon end to the nitrogen end to obtain a fragment, then removes the allyloxycarbonyl protecting group on Dab(Alloc), and then continues to sequentially couple protected amino acids to obtain a resin with a branched chain; then, the CTCresin fragment is cleaved from the resin with a relatively low concentration of acid, and then liquid-phase cyclization is performed, and after cleavage and purification, the final product is obtained with a purity greater than 99.0% and a total yield greater than 35%; this method uses solid-phase synthesis of the fragment, starting from Leu at the carbon end, 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 a high purity and yield, thereby improving the yield and purity of structure (1) and the antibiotic polymyxin.

Description

Technical Field

[0001] The present invention relates to the field of preparation of polymyxin compounds, in particular 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. It can increase the permeability of the outer membrane of Gram-negative bacteria to hydrophobic antibiotics by binding to bacterial lipopolysaccharide (LPS). Compared to polymyxin B, polymyxin B nonapeptide is less toxic and lacks bactericidal activity, yet it still has the ability to disrupt the outer membrane of Gram-negative bacteria. The effectiveness of polymyxin B nonapeptide as an antiendotoxin agent was evaluated by testing its ability to block LPS-induced enhancement of toxic oxygen radical release in human neutrophils.

[0003] In vivo, polymyxin B nonapeptide did not exhibit the neuromuscular blocking, neurotoxic, or nephrotoxic effects observed with polymyxin B sulfate at doses of 1.5 and 3.0 mg / kg. Both polymyxin B and polymyxin B nonapeptide inhibited lipopolysaccharide-induced neutrophil priming in a concentration-dependent manner, but the parent compound, polymyxin B, was 63-fold more potent by weight. However, when both compounds were added after the start of the lipopolysaccharide-neutrophil incubation, their inhibitory activity rapidly decreased.

[0004] Polymyxin B nonapeptides are less toxic than polymyxin B and, at the doses tested, lack the neurotoxicity and nephrotoxicity of the parent compound. Polymyxin B nonapeptides retain the antiendotoxin activity of polymyxin B, but with much lower potency. Given the high specificity and resistance to this class of drugs, there is interest in developing polymyxins with improved therapeutic profiles and effects. Therefore, the development of synthetic derivatives such as polymyxin B nonapeptides is of great interest.

[0005] In the existing technology, the process for synthesizing polymyxin B nonapeptide has problems such as low yield and high by-product content, such as β-elimination of the Thr (tBu) side chain at the carbon end to generate α-aminocrotonic acid derivatives; or catalytic hydrogenation for deprotection, which has 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 preparation method of the antibiotic polymyxin.

[0007] A preparation method of the antibiotic polymyxin:

[0008] The preparation method of the antibiotic polymyxin comprises the following steps: sequentially subjecting the structure (1) to a cyclization reaction and a deprotection cleavage reaction to obtain the antibiotic polymyxin Thr-Dab-Cyclo (Dab-Dab-DPhe-Leu-Dab-Dab-Thr);

[0009] The structure (1) is as follows:

[0010] ;

[0011] Among them, A1, A2, A3, A4, and A5 groups are amino protecting groups, and B1 and B2 groups are carboxyl protecting groups.

[0012] Furthermore, the A1, A2, A3, A4, and A5 groups are any one of Pht, Tos, Fmoc, Boc, Cbz, Bn, PMB, Alloc, and Mtt; and the B1 and B2 groups are any one of tBu, CTCResin, and WangResin.

[0013] Further, A1, A2, A3, A4, and A5 are all Boc, and B1 and B2 are both tBu;

[0014] The structure (1) is as follows:

[0015] .

[0016] Furthermore, S1 cleaves the CTC Resin group:

[0017] The CTCResin group in the resin Boc-Thr(tBu)-Dab(Boc)-Dab(Thr(tBu)-Dab(Boc) -Dab(Boc))-Dab(Boc)-DPhe-Leu-CTCResin was cleaved using TFA to obtain structure (1), which was then washed and lyophilized;

[0018] S2 liquid phase cyclization:

[0019] 1) Boc-Thr(tBu)-Dab(Boc)-Dab(Thr(tBu)-Dab(Boc)-Dab(Boc))-Dab(Boc)-DPhe-Leu-OH of the structural formula (1) is activated with HOOBt and / or EDC·HCl to undergo intramolecular amide reaction;

[0020] 2) After the reaction is completed, the crude product of Boc-Thr(tBu)-Dab(Boc)-Cyclo(Dab-Dab(Boc)-DPhe-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)) is obtained by rotary evaporation, separation, and washing;

[0021] S3 deprotection cleavage:

[0022] The protecting group in Boc-Thr(tBu)-Dab(Boc)-Cyclo(Dab-Dab(Boc)-DPhe-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)) was cleaved using F solution, and the cleavage solution was removed by rotary evaporation. The crude product of Thr-Dab-Cyclo(Dab-Dab-Thr-Dab-Dab-DPhe-Leu-Dab-Dab-Thr) was obtained after separation, centrifugation, and drying.

[0023] Preparation and purification of crude S4:

[0024] The crude product obtained in step S3 was dissolved in acetonitrile / water, filtered through a mixed fiber membrane with a diameter less than 1 μm, and then purified using a chromatographic column to obtain the antibiotic polymyxin.

[0025] The process of further preferably cutting the CTC Resin group by S1 is as follows:

[0026] The CTCResin group in the resin Boc-Thr(tBu)-Dab(Boc)-Dab(Thr(tBu)-Dab(Boc) -Dab(Boc))-Dab(Boc)-DPhe-Leu-CTCResin was cleaved using 0.5-2% TFA / DCM to obtain structure (1), which was then washed and lyophilized;

[0027] The preparation process of further preferred S2 liquid phase cyclization is as follows:

[0028] 1) Dissolve 13-15 g of the compound of 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,

[0029] Then, 3-4 mL of DIPEA was added and the reaction was allowed to proceed at room temperature for 10-15 h;

[0030] 2) After the reaction is completed, DCM is removed by rotary evaporation, and the crude product of Boc-Thr(tBu)-Dab(Boc)-Cyclo(Dab-Dab(Boc)-DPhe-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)) is obtained by separation and washing;

[0031] It is further preferred that 0.35-0.5 μm mixed fiber membrane filtration be used in the preparation and purification of the S4 crude product.

[0032] Furthermore, the antibiotic polymyxin obtained by the preparation method of the present application has a purity of >99.0% and a total yield of >35%.

[0033] Furthermore, the preparation process of the structure (1) includes the following steps:

[0034] Structural formula (2): A1-Thr(B1)-Dab(A2)-Dab(A6)-Dab(A3)-DPhe-Leu-B2 is subjected to the removal of the A6 protecting group, followed by sequential reverse coupling reactions with Fmoc-Thr(tBu)-OH, Fmoc-Dab(Boc)-OH, and Fmoc-Dab(Boc)-OH, followed by the removal of the B2 protecting group to obtain structural formula (1).

[0035] Furthermore, the A6 group is Alloc, A1, A2, and A3 are all Boc, B1 is tBu, and B2 is CTCResin.

[0036] Furthermore, the preparation process of the structure (1) includes the following preparation process:

[0037] Solid-phase synthesis

[0038] 1) Full swelling of the resin

[0039] Fmoc-Leu-CTCResin was soaked and swollen in an organic solvent;

[0040] 2) Removal of the Fmoc protecting group

[0041] The Fmoc group in the swollen Fmoc-Leu-CTCResin was removed in an inert gas atmosphere;

[0042] 3) Connecting amino acids

[0043] The C-terminal amino acid Fmoc-DPhe-OH is activated with HOBt and / or DIC, and then added to the resin obtained in step 2) to undergo coupling. After the coupling is completed, the resin is washed and dried;

[0044] 4) Repeat steps 2-4 to sequentially connect amino acids. The amino acid polypeptide sequence is sequentially connected to 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.

[0045] Further preferably, the preparation process of the structure (1) includes the following preparation process:

[0046] Solid-phase synthesis

[0047] 1) Full swelling of the resin

[0048] Fmoc-Leu-CTCResin was soaked and swollen in DMF;

[0049] 2) Removal of the Fmoc protecting group

[0050] The Fmoc group in the swollen Fmoc-Leu-CTCResin was removed using 20% ​​Pip / DMF under nitrogen conditions;

[0051] 3) After Kaiser test, it is opaque dark blue;

[0052] 4) Connecting amino acids

[0053] Weigh 2.5-3.5 times the amount of C-terminal amino acid Fmoc-DPhe-OH and 2.5-3.5 times the amount of HOBt and dissolve them in 250 mL of DMF. Cool to -5 to -15 degrees Celsius. Add 2.5-3.5 times the amount of DIC for activation and then add to the resin. Coupling for 1-2 hours. Kaiser assay shows colorless. After coupling, wash and drain.

[0054] 5) Repeat steps 2-4 to connect amino acids in sequence

[0055] Repeat steps 2-4 and connect Fmoc-Dab(Boc)-OH, Fmoc-Dab(Alloc)-OH, Fmoc-Dab(Boc)-OH, and Boc-Thr(tBu)-OH in sequence according to the peptide sequence to obtain the structural formula (2) Boc-Thr(tBu)-Dab(Boc)-Dab(Alloc)-Dab(Boc)-DPhe-Leu-CTCResin.

[0056] Furthermore, the A6 group is Mtt, the A1, A2, and A3 are all Boc, the B1 is tBu, and the B2 is CTCResin; the Mtt group is cleaved in the presence of AcOH, TFE, and DOC;

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

[0058] 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;

[0059] The structural formula (2) is Cbz-Thr(tBu)-Dab(Cbz)-Dab(Boc)-Dab(Cbz)-DPhe-Leu-CTCResin.

[0060] Beneficial effects:

[0061] 1. The preparation method of the present application uses Fmoc-Leu-CTCResin as a starting material, and utilizes a solid-phase synthesis method to sequentially couple protected amino acids from the carbon end to the nitrogen end to obtain a fragment, then removes the allyloxycarbonyl protecting group on Dab (Alloc), and then continues to sequentially couple protected amino acids to obtain a resin with a branched chain; then, the CTCresin fragment is cleaved from the resin with a relatively low concentration of acid, and then liquid-phase cyclization is performed, and the final product is obtained after cleavage and purification, with a purity of >99.0% and a total yield of >35%; this method uses solid-phase synthesis of fragments, starting from Leu at the carbon end, 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 purity and yield of the obtained product are high, thereby improving the yield and purity of structure (1) and the antibiotic polymyxin.

[0062] 2. Optimize the A1, A2, A3 groups and cleavage conditions to improve the yield and purity of the substance of structure (1), thereby improving the yield and purity of the antibiotic polymyxin. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 is a flow chart for the preparation of the antibiotic polymyxin in Example 1;

[0064] Figure 2 is the structural formula of the antibiotic polymyxin;

[0065] Figure 3 is the chromatogram of structural formula (1) obtained in Example 1;

[0066] Figure 4 is the mass spectrum of structural formula (1) obtained in Example 1;

[0067] Figure 5 is a chromatogram of the antibiotic polymyxin obtained using the method of Example 1;

[0068] Figure 6 This is a flow chart for the preparation of the antibiotic polymyxin in Comparative Example 1;

[0069] Figure 7 This is a flow chart for the preparation of the antibiotic polymyxin in Comparative Example 2. DETAILED DESCRIPTION

[0070] In order to make the technical solution of the present invention clearer, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0071] Example 1, preparation method of antibiotic polymyxin, preparation process as follows Figure 1 The structural formula of the antibiotic polymyxin Thr-Dab-Cyclo (Dab-Dab-DPhe-Leu-Dab-Dab-Thr) is as follows Figure 2 .

[0072] The specific preparation process includes the following:

[0073] Preparation of structural formula (2) A1-Thr(B1)-Dab(A2)-Dab(A6)-Dab(A3)-DPhe-Leu-B2:

[0074] Among them, the A6 group is Alloc, A1, A2, and A3 are all Boc, B1 is tBu, and B2 is CTC Resin.

[0075] T1: Solid-phase synthesis step

[0076] 1) Full swelling of the resin

[0077] 14.6 g of Fmoc-Leu-CTCResin with a degree of substitution of 0.687 mol / g was weighed and placed in a peptide solid phase reactor, and then 250 mL of DMF was added for soaking and swelling for 1 hour.

[0078] 2) Removal of the Fmoc protecting group

[0079] The DMF was removed by vacuum filtration and then washed three times with 250 mL of DMF solution. The DMF was drained and 200 mL of 20% Pip / DMF was added. Deprotection was performed under nitrogen for 0.5 h and the product was washed five times with 300 mL of DMF.

[0080] 3) Kaiser test

[0081] Preparation of test reagents: 1) Kaiser Test Solution A: 20% ethanol + 80% phenol; 2) Kaiser Test Solution B: redistilled pyridine; 3) Kaiser Test Solution C: 5% ninhydrin in ethanol. Test procedure: Place a small amount of resin in a test tube and wash twice with ethanol. Then add two drops each of Kaiser Test Solution A, Kaiser Test Solution B, and Kaiser Test Solution C. Heat to 110°C for 2 minutes. Observe the resin until it turns an opaque dark blue.

[0082] Colorimetric results: After removing the Fmoc protecting group during solid-phase synthesis, the resin will be detected as an opaque dark blue. If the amino acid is fully coupled during solid-phase synthesis and there are no free amino groups, the detection result will be yellow or colorless.

[0083] 4) Connecting amino acids

[0084] Weigh 3 times the amount of C-terminal amino acid Fmoc-DPhe-OH and 3 times the amount of HOBt and dissolve them in 250 mL DMF. Cool to -10 degrees. Add 3 times the amount of DIC to activate for 10 minutes and then add to the resin. Couple at room temperature for 1.5 hours. Kaiser assay shows that the product is colorless. After coupling, add 250 mL DMF to wash three times and dry.

[0085] 5) Repeat steps 2-4 to connect amino acids in sequence

[0086] Repeat steps 2-4 and connect Fmoc-Dab(Boc)-OH, Fmoc-Dab(Alloc)-OH, Fmoc-Dab(Boc)-OH, and Boc-Thr(tBu)-OH in sequence according to the peptide sequence to obtain the structural formula (2) Boc-Thr(tBu)-Dab(Boc)-Dab(Alloc)-Dab(Boc)-DPhe-Leu-CTCResin.

[0087] 6) Removal of allyloxycarbonyl (Alloc)

[0088] The resin was first washed twice with 200 mL of dichloromethane. 0.1 times the amount of tetrakistriphenylphosphine palladium catalyst was weighed into a conical flask and dissolved in 250 mL of dichloromethane. The solution was added to the resin and reacted with nitrogen. Then 12 times the amount of phenylsilane and 2 times the amount of HOBt were added and reacted at room temperature for 2 hours. After the resin was dried, it was washed five times with 250 mL of dichloromethane. The Kaiser test showed a dark blue color.

[0089] 7) Repeat steps 2-4 to connect amino acids in sequence

[0090] Repeat steps 2-4 and use the DIC and HOBT methods to sequentially connect Fmoc-Thr(tBu)-OH, Fmoc-Dab(Boc)-OH, and Fmoc-Dab(Boc)-OH according to the peptide sequence. After removing Fmoc, Boc-Thr(tBu)-Dab(Boc) -Dab(Thr(tBu)-Dab(Boc)-Dab(Boc))-Dab(Boc)-DPhe-Leu-CTCResin was obtained.

[0091] 8) Resin washing

[0092] The mixture was washed three times with 300 mL of methanol and finally dried 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.

[0093] T2: Liquid phase synthesis step

[0094] Preparation of the antibiotic polymyxin:

[0095] S1 cleavage of CTCResin group:

[0096] 1) Disperse and suspend 28.81 g of resin in 300 mL of 1% TFA / DCM and shake at room temperature for 1 h.

[0097] 2) The resin suspension was filtered and the resin was washed three times with 100 mL of DCM. 100 mL of water was added to the filtrate and the DCM was removed by rotary evaporation under reduced pressure after stirring. The aqueous phase was lyophilized to obtain 14.8 g of a crude product of the structure (1) Boc-Thr(tBu)-Dab(Boc) -Dab(Thr(tBu)-Dab(Boc)-Dab(Boc))-Dab(Boc)-DPhe-Leu-OH. The chromatogram is shown in FIG. Figure 3 , mass spectrum as Figure 4 .

[0098] 3) Yield: 92.9%

[0099] 4) Purity: 61.9%

[0100] 5) Mass spectrum: 1593.94 (1594.36 (M+1), 797.98 (M+2 / 2))

[0101] S2 liquid phase cyclization:

[0102] 1) 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 were dissolved in 9280 mL of DCM. 3.56 g of EDC·HCl was added, followed by 3.2 mL of DIPEA, and the mixture was reacted at room temperature for 12 h.

[0103] 2) After the reaction was complete, the DCM was removed by rotary evaporation to a volume of 200 mL. 100 mL of 5% aqueous phosphoric acid was added, and the organic phase was separated and washed sequentially with water, saturated aqueous sodium bicarbonate, water, and saturated brine. The DCM was removed by rotary evaporation to obtain 14 g of crude product of the antibiotic polymyxin Boc-Thr(tBu)-Dab(Boc)-Cyclo(Dab-Dab(Boc)-DPhe-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)).

[0104] 3) Yield: 95.9%

[0105] 4) Mass spectrum: 1575.93 (1576.21 (M+1)).

[0106] S3 deprotection cleavage:

[0107] 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 liquid F (95% TFA + 2.5% TIS + 2.5% water) and stir at room temperature for 2 h.

[0108] 2) The cutting solution was removed by rotary evaporation, and 600 mL of tertiary methyl ether was added to precipitate the product. The precipitate was separated by centrifugation, and the precipitate was dried in a vacuum desiccator to obtain 9.63 g of crude Thr-Dab-Cyclo (Dab-Dab-Thr-Dab-Dab-DPhe-Leu-Dab-Dab-Thr).

[0109] 3) Purity: 60.7%;

[0110] 4) Mass spectrum: 963.13 (482.33 (M+2 / 2));

[0111] Preparation and purification of crude S4:

[0112] 1) Sample pretreatment: The sample was dissolved in 10% acetonitrile / water and filtered through a 0.45 μm mixed fiber membrane.

[0113] 2) Purification preparation method:

[0114] Chromatographic column: Huapu C18 10μm 100A 50*450mm (P06)

[0115] Mobile phase A: 0.1% TFA in water

[0116] Mobile phase B: acetonitrile

[0117] Loading flow rate: 60ml / min Elution flow rate: 60ml / min Detection wavelength: 220nm

[0118] Elution gradient: 11-41% in 60 minutes

[0119] After preparation and purification, the product was collected and freeze-dried to obtain 6026343.62g

[0120] Purity: 99.6%;

[0121] Mass spectrum: 963.1 (482.28 (M+2 / 2)) (MS: 602634)

[0122] Chromatogram Figure 5 ;

[0123] Total yield: 37.6%.

[0124] Example 2, a method for preparing the antibiotic polymyxin, differs from Example 1 in that the A1, A2, A3, and A6 groups in structural formula (2) are different, the A6 group is Mtt, and A1, A2, and A3 are all Boc; the preparation process of structural formula (2) is different, and the specific preparation process differs in steps 5) and 6);

[0125] The details are as follows:

[0126] 5) Repeat steps 2-4 to connect amino acids in sequence

[0127] Repeat steps 2-4 and connect Fmoc-Dab(Boc)-OH, Fmoc-Dab(Mtt)-OH, Fmoc-Dab(Boc)-OH, and Boc-Thr(tBu)-OH in sequence according to the peptide sequence to obtain the structural formula (2) Boc-Thr(tBu)-Dab(Boc) -Dab(Mtt)-Dab(Boc)-DPhe-Leu-CTCResin.

[0128] 6) Removal of Mtt group

[0129] 10 g of Boc-Thr(tBu)-Dab(Boc)-Dab(Mtt)-Dab(Boc)-DPhe-Leu-CTCResin resin of structural formula (2) was placed in a reaction vessel, followed by the addition of 200 mL of a mixed solvent (specifically a composition of AcOH: TFE: DCM with a volume ratio of 1:1:8), and the reaction was stirred at room temperature (25 °C) for 10 minutes; the reaction solution was then filtered to remove the resin, and the resin was washed with DCM; the above process was repeated 3 times, with a total reaction time of approximately 30 minutes. The resin was washed 3 times with DCM to remove by-products, and the Kaiser assay showed a dark blue color.

[0130] Example 3, a method for preparing the antibiotic polymyxin, differs from Example 1 in that the A1, A2, A3, and A6 groups in structural formula (2) are different, and the preparation process of structural formula (2) is different.

[0131] The A6 group is Boc, and A1, A2, and A3 are all Cbz; the Boc group is cleaved under TFA solution conditions;

[0132] The structural formula (2) is Cbz-Thr(tBu)-Dab(Cbz)-Dab(Boc)-Dab(Cbz)-DPhe-Leu-CTCResin.

[0133] The specific preparation process of step 5) and step 6) is as follows:

[0134] 5) Repeat steps 2-4 to connect amino acids in sequence

[0135] Repeat steps 2-4 and connect Fmoc-Dab(Cbz)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Cbz)-OH, and Cbz-Thr(tBu)-OH in sequence according to the peptide sequence to obtain the structural formula (2) Cbz-Thr(tBu)-Dab(Cbz)-Dab(Boc)-Dab(Cbz)-DPhe-Leu-CTCResin.

[0136] 6) Removal of Boc group

[0137] 10 g of Cbz-Thr(tBu)-Dab(Cbz)-Dab(Boc)-Dab(Cbz)-DPhe-Leu-CTCResin resin of structural formula (2) was placed in a reaction vessel, followed by the addition of 150 mL of a mixed solvent (30% TFA / DCM solution by volume) and the reaction was stirred at room temperature (25°C) for 10 minutes. The reaction solution was then filtered and the resin was washed with DCM. The above process was repeated three times for a total reaction time of approximately 30 minutes. The resin was washed three times with DCM to remove by-products. The Kaiser assay indicated a dark blue color.

[0138] The yield and purity of the preparation methods of Example 1 and Example 3 are shown in Table 1.

[0139] Table 1. Yield and purity list of the preparation methods of Example 1 and Example 3

[0140]

[0141] The experimental data in Table 1 show that A6 using the Alloc group and the experimental conditions of removing the Alloc group produce fewer by-products and higher purity.

[0142] Comparative Example 1, a method for preparing the antibiotic polymyxin, the preparation process is as follows Figure 6 , specifically comprising the following preparation steps:

[0143] S1 resin pretreatment:

[0144] 10 g of Fmoc-Dab-Thr(tBu)OtBu resin was swollen with 100 mL of DCM for 60 minutes, filtered with suction, and then washed three times with 100 mL of DMF.

[0145] S2 coupling reaction:

[0146] 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 1 equivalent (8 mmol) of Benzyloxychlorotormate resin and react at room temperature under nitrogen for 2 hours. Confirm the reaction is complete using the ninhydrin test and wash three times with 10 mL of DMF solvent.

[0147] S3 Fmoc deprotection:

[0148] To the reactant in step S2, add 100 mL of 20% piperidine / DMF solution, shake at room temperature for 10 minutes, and filter. Repeat the process by adding 100 mL of 20% piperidine / DMF solution, shake at room temperature for 10 minutes, and filter. Then, wash three times with 100 mL of DMF solvent to obtain a resin.

[0149] S4 polypeptide chain extension (7 coupling cycles):

[0150] 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 at 25°C for 1 hour. Confirm the complete reaction with the ninhydrin test and wash with 10 mL of DMF solvent three times. Then perform Fmoc deprotection, as described in S3.

[0151] The order of the Fmoc-AA-OH amino acids coupled sequentially was Fmoc-Dab(Cbz)-OH, Fmoc-Leu-OH, Fmoc-dPhe-OH, Fmoc-Dab(Cbz)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Cbz)-OH, and Fmoc-Thr(tBu)-OH.

[0152] S5 removes the tBu protecting group of Thr(tBu) and the Boc group of Dab(Boc):

[0153] Prepare 1% TFA / DCM solution, take 100 mL; add it to the resin obtained in step S4, react at room temperature 25°C for 30 minutes, filter; then wash with 100 mL DCM and 100 mL DMF, three times.

[0154] S6 cyclization reaction:

[0155] 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 complete, remove DCM by rotary evaporation to a volume of 200 mL. Add 100 mL of 5% aqueous phosphoric acid, separate the organic phase by layers, and wash the organic phase in sequence with water, saturated aqueous sodium bicarbonate, water, and saturated brine, and remove DCM by rotary evaporation.

[0156] S7 resin cutting:

[0157] The N-terminal Fmoc group was removed using 20% ​​piperidine, as in step S3. 100 mL of cutting solution (TFA / TMSBr / H2O, volume ratio 94:5:1) was then added to the resin obtained in step S7, and the mixture was reacted at room temperature at 25°C for 2 hours. The cutting solution was removed by rotary evaporation, and 100 mL of tertiary methyl ether was added to precipitate the product. The precipitate was separated by centrifugation and dried in a vacuum desiccator to obtain a crude Thr-Dab-Cyclo (Dab-Dab-Thr-Dab-Dab-DPhe-Leu-Dab -Dab-Thr).

[0158] Preparation and purification of crude S8:

[0159] 1) Sample pretreatment: The sample was dissolved in 10% acetonitrile / water and filtered through a 0.45 μm mixed fiber membrane.

[0160] 2) Purification preparation method:

[0161] Chromatographic column: Huapu C18 10μm 100A 50*450mm (P06)

[0162] Mobile phase A: 0.1% TFA in water

[0163] Mobile phase B: acetonitrile

[0164] Loading flow rate: 60ml / min Elution flow rate: 60ml / min Detection wavelength: 220nm

[0165] Elution gradient: 11-41% in 60 min;

[0166] After preparation and purification, the product is collected and freeze-dried to obtain the antibiotic polymyxin.

[0167] Comparative Example 2, a method for preparing the antibiotic polymyxin, the preparation process is as follows Figure 7 , specifically including the following preparation process:

[0168] S1 resin pretreatment:

[0169] One equivalent (8 mmol) of Fmoc-Thr(tBu)-2-Chlorotrityl resin was swollen in 100 mL of DCM for 60 minutes, filtered, and then washed three times with 100 mL of DMF.

[0170] S2 Fmoc deprotection:

[0171] To the resin treated in step S1, 100 mL of 20% piperidine / DMF solution was added, the mixture was shaken at room temperature for 10 minutes, and filtered. The procedure was then repeated, with 100 mL of 20% piperidine / DMF solution added, the mixture was shaken at room temperature for 10 minutes, and filtered. The mixture was then washed three times with 100 mL of DMF solvent to obtain the Fmoc-free resin.

[0172] S3 coupling-8 Cycles:

[0173] 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, activate for 5 minutes, and obtain an activated solution; add the activated solution to the resin obtained in the previous step and react at room temperature at 25°C for 1 hour; confirm the completeness with the ninhydrin test and wash with 10 mL of DMF solvent 3 times; then perform Fmoc deprotection, the specific operation is as in S2; after 8 cycles of coupling, obtain HO-Thr(tBu)-Dab(tBoc)-Dab(Mtt)-Dab(tBoc)-dPhe-Leu-Dab(tBoc)-Dab(tBoc)-Thr(tBu)-resin.

[0174] The order of the Fmoc-AA-OH amino acids coupled sequentially is Fmoc-Dab(Boc), Fmoc-Dab(Boc), Fmoc-Leu, Fmoc-dPhe, Fmoc-Dab(Boc), Fmoc-Dab(Mtt), Fmoc-Dab(Boc), and Fmoc-Thr(tBu).

[0175] S4 Boc protection:

[0176] To the resin obtained in S3, 100 mL of DMF, Boc2O (5 equivalents), and DIEA (10 equivalents) were added and reacted for 1 hour. The resin was then washed three times with DMF to remove unreacted reagents to obtain Boc-Thr(tBu)-Dab(Boc)-Dab(Mtt)-Dab(Boc)-dPhe-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-resin.

[0177] S5 Mtt deprotection:

[0178] To the resin obtained in S4, 100 mL of AcOH / TFE / DCM (1:1:8) solution was added and the reaction was allowed to proceed for 30 minutes. The operation was repeated for 30 minutes to ensure complete removal of the Mtt group. The resin was then washed three times with DCM to obtain Boc-Thr(tBu)-Dab(Boc)-Dab(NH2)-Dab(Boc)-dPhe-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-resin.

[0179] S6 cyclization reaction:

[0180] To the resin obtained in step S5, PyBOP (4 equivalents), HOBt (4 equivalents), DIEA (8 equivalents) and 10 mL of DMF were added and reacted for 3 hours. After the reaction, the resin was washed three times with DMF to remove unreacted reagents to obtain the product: Boc-Thr(tBu)-Dab(Boc)-Cyclo(Dab-Dab(Boc)-dPhe-Leu-Dab(Boc)-Dab(Boc) -Thr(tBu))-resin.

[0181] S7 resin cleavage and Boc deprotection

[0182] To the resin obtained in step S6, 100 mL of TFA / DCM (95:5) solution was added, and the reaction was carried out at room temperature at 25°C for 2 hours; the TFA / DCM (95:5) solution was removed by rotary evaporation, and 100 mL of tert-methyl ether was added to precipitate the product. The precipitate was separated by centrifugation, and the precipitate was dried in a vacuum desiccator to obtain a crude Thr-Dab-Cyclo (Dab-Dab-Thr-Dab-Dab-DPhe-Leu-Dab -Dab-Thr) product.

[0183] Preparation and purification of crude S8:

[0184] 1) Sample pretreatment: The sample was dissolved in 10% acetonitrile / water and filtered through a 0.45 μm mixed fiber membrane.

[0185] 2) Purification preparation method:

[0186] Chromatographic column: Huapu C18 10μm 100A 50*450mm (P06)

[0187] Mobile phase A: 0.1% TFA in water

[0188] Mobile phase B: acetonitrile

[0189] Loading flow rate: 60ml / min Elution flow rate: 60ml / min Detection wavelength: 220nm

[0190] Elution gradient: 11-41% in 60 min;

[0191] After preparation and purification, the product is collected and freeze-dried to obtain the antibiotic polymyxin.

[0192] The yield and purity of the preparation methods of Comparative Example 1 and Comparative Example 2 are shown in Table 2.

[0193] Table 2. Yield and purity list of the preparation methods of Comparative Example 1 and Comparative Example 2

[0194]

[0195] In the preparation method of Comparative Example 1, HATU is used for cyclization on the resin, resulting in the formation of a byproduct in which the Dab amino group is blocked by tetramethylguanidine. Furthermore, the synthesis of the Fmoc-Dab(resin)-Thr(tBu)-OtBu dipeptide is technically laborious, resulting in low purity and yield of the resin obtained after removal of the protecting group of S5.

[0196] In the preparation method of Comparative Example 2, in the S3 coupling-8 Cycles step, a byproduct of tetramethylguanidine blocking the Dab amino group is easily produced, resulting in low purity and yield of the obtained resin.

[0197] Compared with the prior art comparative examples 1 and 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 the structure (1) is 92.8-95.9%, while the cyclization yield of the amino acid structure with a straight chain structure in comparative examples 1 and 2 is 72.3-75.9%. The structure (1) of the present application has a higher yield and product purity, and has excellent technical effects; this may be because the active sites on the two branches connected to the third amino acid Dab in the structure (1) of the present application are easier to contact, the reaction activity is higher, and the side reactions are less, thereby having a higher cyclization yield, total yield and higher product purity.

[0198] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

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

2.

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

3. The method for preparing the antibiotic polymyxin according to claim 1, characterized in that: Said A1, A2, A3, A4, and A5 are all Boc, and said B1 and B2 are both tBu; The structure (1) is as follows: 。 4. The method for preparing the antibiotic polymyxin according to claim 3, characterized in that: The preparation process includes the following: S1 cuts the CTC Resin group; S2 liquid phase cyclization; S3 deprotection cleavage; S4 crude product was prepared and purified.

5. The method for preparing the antibiotic polymyxin according to claim 4, characterized in that: The obtained antibiotic polymyxin has a purity of >99.0% and a total yield of >35%.

6. The method for preparing the antibiotic polymyxin according to claim 1, characterized in that: The preparation process of the structure (1) comprises the following steps: Structural formula (2) A1-Thr(B1)-Dab(A2)-Dab(A6)-Dab(A3)-DPhe-Leu-B2 is subjected to the removal of the A6 protecting group, followed by sequential reverse coupling reactions with Fmoc-Thr(tBu)-OH, Fmoc-Dab(Boc)-OH, and Fmoc-Dab(Boc)-OH, followed by the removal of the B2 protecting group to obtain structural formula (1), wherein the sequence of structural formula (2) is SEQ ID NO.

3.

7. The method for preparing the antibiotic polymyxin according to claim 6, characterized in that: The A6 protecting group is Alloc, A1, A2, and A3 are all Boc, B1 is tBu, and B2 is CTCResin.

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

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

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

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