CN111690040A

CN111690040A - Polymyxin derivatives, preparation method and application thereof

Info

Publication number: CN111690040A
Application number: CN201910185485.9A
Authority: CN
Inventors: 李亚利; 戈梅; 饶敏; 夏兴; 黄亚妮; 蔡晓龙
Original assignee: Shanghai Health Creation Center For Biopharmaceutical R&d Co ltd
Current assignee: Shanghai Health Creation Center For Biopharmaceutical R&d Co ltd
Priority date: 2019-03-12
Filing date: 2019-03-12
Publication date: 2020-09-22

Abstract

The invention discloses a polymyxin derivative shown as a general formula (I) and pharmaceutically acceptable salts and solvates thereof:

Description

Polymyxin derivatives, preparation method and application thereof

Technical Field

The invention belongs to the technical field of pharmaceutical chemical synthesis, and particularly relates to a polymyxin derivative, and a preparation method and application thereof.

Background

In recent years, the number of cases of infection caused by gram-negative bacteria has increased, and many gram-negative bacteria have become resistant to a broad spectrum of antibiotics in clinical practice, and development of novel antibacterial agents against gram-negative resistant bacteria is urgent. Until now, polymyxin has a very good effect on the treatment of multidrug-resistant gram-negative bacterial infections, in particular on pseudomonas aeruginosa, acinetobacter baumannii and klebsiella pneumoniae, and thus, polymyxin is also called the last line of defense for the treatment of G-bacterial infections. Polymyxin is a group of cyclic peptide antibiotics consisting of A, B, C, D, E and the like produced by bacillus polymyxa, and finally only polymyxin B and polymyxin E (also called colistin) are clinically used due to good curative effect and relatively high safety. Polymyxin B is a sulfate preparation formed by more than 30 polypeptides, B1 and B2 are main parts of the polymyxin B, the polymyxin B is mainly administered in a parenteral form and is used for treating eye and ear diseases, most of the polymyxin B is a mesylate preparation, and the polymyxin B needs to be hydrolyzed into the polymyxin in vivo to play a role.

However, polymyxin has strong nephrotoxicity (incidence rate of 0-37%) and neurotoxicity (incidence rate of 0-7%) while sterilizing, and is dose-dependent, so that polymyxin drugs are limited to be clinically used in human in the 80 s of 20 th century and are only used as veterinary drugs. With the continuous emergence of drug resistance G-of gram-negative bacteria, people are prompted to develop polymyxin as a novel clinical drug for treating gram-negative bacteria again. Therefore, there is an urgent need to develop novel polymyxin antibiotics.

Disclosure of Invention

The inventor of the invention uses polymyxin as a starting compound and carries out chemical transformation on the polymyxin to obtain a series of improved polymyxin derivatives and pharmaceutically acceptable salts and solvates thereof. Tests prove that the polymyxin derivative has good bacteriostatic activity on gram-negative bacteria, and can be used for preparing medicaments for preventing or treating gram-negative bacteria infection.

The first object of the present invention is to provide polymyxin derivatives represented by the general formula (I) and pharmaceutically acceptable salts and solvates thereof:

wherein:

R¹is CH (CH)₃)₂Or CH₂C₆H₅；

n is 0 or 1;

when n is 0:

R²comprises the following steps:

-Y-C_7-14alkyl, -Y-C_3-10Cycloalkyl, -Y-C_2-10Heterocyclyl, -Y-C_6-12Aryl, -Y-C_5-12A heteroaryl group;

-Y-C_1-7alkyl radical C_3-10Cycloalkyl, -Y-C_1-7Alkyl radical C_2-10Heterocyclyl, -Y-C_1-7Alkyl radical C_6-12Aryl, -Y-C_1-7Alkyl radical C_5-12A heteroaryl group;

-Y-C_3-10cycloalkyl radical C_1-7Alkyl, -Y-C_2-10Heterocyclyl radical C_1-7Alkyl, -Y-C_6-12Aryl radical C_1-7Alkyl, -Y-C_5-12Heteroaryl C_1-7An alkyl group;

-Y-C_1-7alkyl-O-C_1-10Alkyl, -Y-C_1-7alkyl-O-C_3-10Cycloalkyl, -Y-C_1-7alkyl-O-C_2-10Heterocyclyl, -Y-C_1-7alkyl-O-C_6-12Aryl, -Y-C_1-7alkyl-O-C_5-12A heteroaryl group;

-Y-C_1-7alkyl-NH-C_1-10Alkyl, -Y-C_1-7alkyl-NH-C_3-10Cycloalkyl, -Y-C_1-7alkyl-NH-C_2-10Heterocyclyl, -Y-C_1-7alkyl-NH-C_6-12Aryl, -Y-C_1-7alkyl-NH-C_5-12A heteroaryl group;

y is a bond, -C (O) -;

x-is a bond, -C (O) -, -NHC (O) -, -OC (O) -, -CH₂-or-SO₂-；

R⁵Comprises the following steps:

-C_3-10cycloalkyl, -C_2-10Heterocyclyl radical, -C_6-12Aryl radical, -C_12-14Biphenyl, -C_5-12A heteroaryl group;

-C_1-10alkyl-Z-C_1-10Alkyl, -C_1-7alkyl-Z-C_3-10Cycloalkyl, -C_1-7alkyl-Z-C_2-10Heterocyclyl radical, -C_1-7alkyl-Z-C_6-12Aryl radical, -C_1-7alkyl-Z-C_12-14Biphenyl, -C_1-7alkyl-Z-C_5-12A heteroaryl group;

-C_3-10cycloalkyl-Z-C_1-7Alkyl, -C_2-10heterocyclyl-Z-C_1-7Alkyl, -C_6-12aryl-Z-C_1-7Alkyl, -C_12-14biphenyl-Z-C_1-7Alkyl, -C_5-12heteroaryl-Z-C_1-7An alkyl group;

-C_6-12-aryl-Z-C_1-10Alkyl, -C_6-12-aryl-Z-C_3-10Cycloalkyl, -C_6-12-aryl-Z-C_2-10Heterocyclyl radical, -C_6-12-aryl-Z-C_6-12Aryl radical, -C_6-12-aryl-Z-C_12-14Biphenyl, -C_6-12-aryl-Z-C_5-12A heteroaryl group;

z is a covalent bond, -C_1-3Alkyl-, -C (O) -, -NHC (O) -, -C (O) NH-, -OC (O) -, -C (O) O-, -S (O)₂)O-、 -OS(O₂)-；

R²、R⁵Each of which is optionally substituted by one or more-N (R)⁶)-L-R⁷Substituted, 0-3-OR⁸Substituted or halo substituted; r⁶represents-H, -C_1-10Alkyl, -C_3-10Cycloalkyl, -C_2-10Heterocyclyl radical, -C_6-12Aryl radical, -C_12-14Biphenyl, -C_5-12A heteroaryl group;

and R is⁷represents-H, -C_1-10Alkyl, -C_3-10Cycloalkyl, -C_2-10Heterocyclyl radical, -C_6-12Aryl radical, -C_12-14Biphenyl, -C_5-12A heteroaryl group;

R⁸represents-H, -C_1-10Alkyl, -C_3-10Cycloalkyl, -C_2-10Heterocyclyl radical, -C_6-12Aryl radical, -C_12-14Biphenylradical-C_5-12A heteroaryl group.

When n is 1:

R²is a compound of formula (I) in the formula (H),

R³is CH₂SH、CH₂CH₂SCH₃、CH₂OH、CH(CH₃)OH、CH₂CONH₂、CH₂CH₂CONH₂、 CH₂C₄H₅N₂、CH₂C₆H₅OH、CH₂C₈H₆N、CH₂CH₂CH₂CH₂NH₂、CH₂CH₂NH₂、 CH₂CH₂CH₂CH₄N₃，

R⁴Comprises the following steps:

-Y-C_1-7alkyl-NH-C_1-10Alkyl, -Y-C_1-7alkyl-NH-C_3-10Cycloalkyl, -Y-C_1-7alkyl-NH-C_2-10Heterocyclyl, -Y-C_1-7alkyl-NH-C_6-12Aryl, -Y-C_1-7alkyl-NH-C_5-12Heteroaromatic compoundsA group;

y is a bond, -C (O) -;

x-is a bond, -C (O) -, -NHC (O) -, -OC (O) -, -CH₂-or-SO₂-；

R⁵Comprises the following steps:

R²、R³、R⁴、R⁵Each of which is optionally substituted by one or more-N (R)⁶)-L-R⁷Substituted, 0-3-OR⁸Substituted or halo substituted;

R⁶represents-H, -C_1-10Alkyl, -C_3-10Cycloalkyl, -C_2-10Heterocyclyl radical, -C_6-12Aryl radical, -C_12-14Biphenyl, -C_5-12A heteroaryl group;

R⁸represents-H, -C_1-10Alkyl, -C_3-10Cycloalkyl, -C_2-10Heterocyclyl radical, -C_6-12Aryl radical, -C_12-14Biphenyl, -C_5-12A heteroaryl group.

According to a preferred technical scheme of the invention, the pharmaceutically acceptable salt is a salt formed by the polymyxin derivative shown in the general formula (I) and acid.

Preferably, the acid is selected from one or more of hydrochloric acid, hydrobromic acid, sulphuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulphonic acid, aspartic acid or glutamic acid.

Preferably, in the solvate, the solvent does not interfere with the biological activity of the solute. By way of example, the solvent may be water, ethanol or acetic acid, and the solvation method is a method well known in the art. The solvate is hydrate, ethanol compound or acetic acid compound.

The second objective of the present invention is to provide a pharmaceutical composition, which comprises a therapeutically effective amount of the polymyxin derivative or the pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

In the present invention, the pharmaceutically acceptable carrier refers to a pharmaceutical carrier that is conventional in the pharmaceutical field, such as diluents, excipients (e.g., water, etc.), binders (e.g., cellulose derivatives, gelatin, polyvinylpyrrolidone, etc.), fillers (e.g., starch, etc.), and disintegrating agents (e.g., calcium carbonate, sodium bicarbonate). In addition, other adjuvants such as flavoring agents and sweeteners can also be added to the pharmaceutical composition.

The pharmaceutical composition of the present invention may be administered to a patient in need of treatment by intravenous injection, subcutaneous injection or oral administration. For oral administration, it can be prepared into conventional solid preparations such as tablet, powder or capsule; for injection, it can be prepared into injection. The various dosage forms of the pharmaceutical composition of the invention can be prepared by conventional methods in the medical field, wherein the content of the active ingredients is 0.1-99.5% (weight ratio). In the preparation, the weight content of the compound is 0.1-99.9%, and the preferable content is 0.5-90%.

The general dosage of the above pharmaceutical composition to be administered to a patient in need of treatment can be referred to the dosage of polymyxin used in the art.

A third object of the present invention is to provide a process for the preparation of a polymyxin derivative as described above, which comprises:

the synthesis route of the polymyxin derivative shown in the general formula (I) is as follows when n is 1:

(ii) a Wherein:

R¹is CH (CH)₃)₂Or CH₂C₆H₅，

R²Is a compound of formula (I) in the formula (H),

R⁴Comprises the following steps:

y is a bond, -C (O) -;

x-is a bond, -C (O) -, -NHC (O) -, -OC (O) -, -CH₂-or-SO₂-；

R⁵Comprises the following steps:

Method (ii), when n is 0, the synthesis route of the polymyxin derivative represented by the general formula (i) is:

(ii) a Or,

wherein:

R¹is CH (CH)₃)₂Or CH₂C₆H₅；

R²Comprises the following steps:

y is a bond, -C (O) -;

x-is a bond, -C (O) -, -NHC (O) -, -OC (O) -, -CH₂-or-SO₂-；

R⁵Comprises the following steps:

R²、R⁵Each of which is optionally substituted by one or more-N (R)⁶)-L-R⁷Substituted, 0-3-OR⁸Substituted or halo substituted;

In the above method, the preparation method of the compound II is described in detail in the preparation methods of the intermediate 1 and the intermediate 3.

The fourth purpose of the invention is to provide the application of the polymyxin derivative in preparing the medicines for treating bacterial infectious diseases.

According to a preferred embodiment of the present invention, there is provided the use of a polymyxin derivative as described above for the manufacture of a medicament for the treatment of infectious diseases caused by gram-negative bacteria.

Preferably, the polymyxin derivative is applied to preparation of a medicine for preventing or treating polymyxin-resistant gram-negative bacteria infectious diseases. I.e. the gram-negative bacterium is a polymyxin resistant gram-negative bacterium, such as: acinetobacter baumannii resistant to polymyxin.

A fifth object of the present invention is to provide a method for preventing or treating gram-negative bacterial infection, comprising administering a therapeutically effective amount of the above-mentioned polymyxin derivatives and pharmaceutically acceptable salts and solvates thereof to a subject in need thereof.

Compared with the prior art, the polymyxin derivative has the beneficial effects that:

the polymyxin derivative shown as the general formula (I) and pharmaceutically acceptable salts and solvates thereof have good antibacterial activity, especially gram-negative bacteria resistance activity, have equivalent or improved antibacterial activity to gram-negative bacteria compared with the existing polymyxin E or B, and can be applied to the preparation of medicines for treating bacterial infection and gram-negative bacteria infection; compared with polymyxin E, the polymyxin E has equivalent or improved antibacterial activity on drug-resistant gram-negative bacteria, and can be applied to preparation of drugs for preventing and treating drug-resistant bacterial infection and drug-resistant gram-negative bacterial infection. Therefore, the method has important significance for the development of new antibacterial drugs.

Detailed Description

The present invention will be further described with reference to the following specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.

In the present invention, the following abbreviations have the following meanings. Undefined abbreviations have their commonly accepted meaning, unless otherwise stated, all room temperatures refer to temperatures of 20 ℃ to 30 ℃.

PMB polymyxin B

PMBN polymyxin B nonapeptide

Boc-ON 2- (tert-butyloxycarbonyloxyiimino) -2-phenylacetonitrile

PME polymyxin E

DCM dichloromethane

EDCI 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride

HOBt 1-hydroxybenzotriazole

LiOH-H₂OLithium hydroxide monohydrate

TFA trifluoroacetic acid

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

DIPEA N, N-diisopropylethylamine

MTBE methyl tert-butyl ether

PYBOP 1H-benzotriazol-1-yl-oxytripyrrolidinyl hexafluorophosphate

Minimum Inhibitory Concentration (MIC)

In the following examples, the concentrations of the elution solution and the prewash solution are in percent by volume, and the yields are molar yields.

In the following examples, HPLC monitoring conditions:

mobile phase: a.0.05% formic acid water B.0.05% formic acid acetonitrile

A chromatographic column: agilent Poroshell 120EC-C18(4.6 mm. times.150 mm, 2.7 μm)

Column temperature: 45 deg.C

Detection wavelength: 210, 240, 254, 280, 360nm

Analysis time: 50min

Flow rate: 0.7mL/min

HPLC gradient conditions are shown in table 1:

TABLE 1 HPLC gradient conditions

Time (min)	A％	B％	Flow rate (ml/min)
				0	95	5	0.7
10	85	15	0.7
				15	75	25	0.7
25	45	55	0.7
				30	5	95	0.7
50	5	95	0.7

PSN column chromatography conditions were:

PSN column chromatography conditions of (I), polymyxin B nonapeptide (intermediate 1) and polymyxin E nonapeptide (intermediate 3):

(1) column volume: 1L of the compound. The sample loading liquid is an aqueous solution of a sample to be separated, and the sample loading volume (ml) is V_{Sample loading}＝20*m_{Sample loading/g}

(2) And washing 4 times of column volume after sample loading, and collecting the column volume by parts, wherein each bottle is 0.5 time of column volume.

(3) After the completion of the water washing, the column was washed with 10% acetonitrile acid (TFA 0.05%) for 3 column volumes and collected.

(4) And after the acid washing with 10% acetonitrile, washing with 20% acetonitrile for 3 times of column volume. And (6) merging and collecting. The column was then washed with 50% glycolic acid and finally equilibrated to neutral with water.

PSN column chromatography conditions for (di), tetra- (Boc) polymyxin B nonapeptide (intermediate 2) and tetra- (Boc) polymyxin E nonapeptide (intermediate 4):

the column volume is 1L. The sample loading liquid is the aqueous solution of the sample to be separated, and the sample loading volume V_{Sample loading}＝20*m_{Sample loading/g}. After the sample is loaded, 50% methanol water is firstly used for washing 4 times of column volume, the column volume is combined and collected, then 70% methanol water is used for fractional collection, one bottle is collected every 0.5 time of column volume, the column volume is washed for 4 times, then 75% methanol water is used for elution, fractional collection is carried out, the column volume is washed for 4 times of column volume, finally 85% methanol water is used for washing for twice of column volume, and the column volume is combined and collected.

The invention provides a polymyxin derivative shown as a general formula (I) and pharmaceutically acceptable salts and solvates thereof:

the structures of specific compounds of the general formula (I) referred to in the following examples are shown in Table 2.

TABLE 2 Structure of the respective Compounds

The compounds in table 2 are prepared below by way of a number of specific examples, as examples.

EXAMPLE 1 Synthesis of intermediate 1-polymyxin B nonapeptide

(1) PMB (2.0g) was dissolved in potassium phosphate buffer (pH 7.5,25ml) with ultrasound, papain (3.0U/mg) was added with stirring at 50 ℃ and further reacted at 50 ℃ for 1h, and the reaction progress was monitored by HPLC. After the reaction is finished, quenching the reaction system by using methanol, and concentrating the supernatant in vacuum to obtain a crude polymyxin B nonapeptide product.

(2) Purifying the crude polymyxin B nonapeptide product obtained in the step (1) by using a PSN column, and concentrating eluent to obtain a white solid product, namely an intermediate 1-polymyxin B nonapeptide (PMBN) (0.5g, 0.52mmol) M/z 963.57[ M + H ] (M + H)]⁺。

The reaction process of this example is shown as follows.

EXAMPLE 2 Synthesis of intermediate 2-tetra- (Boc) polymyxin B nonapeptide

(1) Intermediate 1 polymyxin B nonapeptide prepared in example 1(1.0 g, 1.04mmol) was suspended in water (1.5ml), and 1, 4-dioxane (1) was added to the suspension.5ml) and triethylamine (1.5ml), the system was stirred for 10min under ice-bath conditions, followed by addition of Boc-ON (1.08g, 4.37mmol), stirring continued at room temperature and the progress of the reaction was monitored by HPLC. After the reaction is finished, the organic phase in the system is dried by spinning, PSN column chromatography is carried out, and white solid is obtained, namely the intermediate 2-tetra- (Boc) polymyxin B nonapeptide (0.9g, 0.66mmol) and M/z 1363.78[ M + H ]]⁺。

The reaction process of this example is shown as follows.

EXAMPLE 3 Synthesis of intermediate 3-polymyxin E nonapeptide

(1) PME (2.0g) was dissolved in potassium phosphate buffer (pH 7.5,25ml) with ultrasound, papain (3.0U/mg) was added with stirring at 50 ℃ and further reacted at 50 ℃ for 1h, and the reaction progress was monitored by HPLC. After the reaction is finished, quenching the reaction system by using methanol, and concentrating the supernatant in vacuum to obtain a crude polymyxin E nonapeptide product.

(2) Purifying the crude polymyxin E nonapeptide prepared in the step (1) by using a PSN column, and concentrating eluent to obtain a white solid product, namely an intermediate 3-polymyxin E nonapeptide (PMEN for short) (0.98g, 10.55mmol), M/z 929.58[ M + H ] (M + H)]⁺。

The reaction process of this example is shown as follows.

EXAMPLE 4 Synthesis of intermediate 4-tetra- (Boc) polymyxin E nonapeptide

Intermediate 3 polymyxin E nonapeptide prepared in example 3 (1.0g, 1.08mmol) was suspended in water (1.5ml), 1, 4-dioxane (1.5ml) and triethylamine (1.5ml) were added to the suspension, the system stirred under ice bath conditions for 10min, followed by Boc-ON (1.08g, 4.37mmol), stirring continued at room temperature and reaction progress monitored by HPLC. After the reaction is finished, the organic phase in the system is dried in a spinning way and PSN column chromatography is carried out to obtain white solid, namely the intermediate 4-4 tetra- (Boc) polymyxin E nonapeptide (0.76g, 0.57mmol), M/z 1329.79[ M + H]⁺。

The reaction process of this example is shown as follows.

Example 5N- (([1, 1' -Biphenyl)]-4-methyl) - [ 3-aminoundecanoyl]Of polymyxin B (Compound 7) Synthesis of(1) Methyl (S) -2-amino-4- ((tert-butoxycarbonyl) amino) butyrate (845mg, 3.64mmol) was dissolved in methanol (10ml), and biphenylcarboxaldehyde (797mg, 4.37mmol) and NaCNBN were added thereto₃(275mg, 4.37mmol), anhydrous ZnCl₂(496mg, 3.64mmol), the system was stirred at room temperature overnight and the reaction progress was monitored by HPLC. After the reaction is finished, EA is used for extracting a system, an organic phase is concentrated, silica gel column chromatography is carried out, petroleum ether-ethyl acetate (15:1) is used as an eluent, and a white solid product, M/z 399.22[ M + H ] is obtained]⁺Intermediate 5(876mg, 2.2 mmol).

(2) Intermediate 5(905mg, 2.27mmol) prepared in the above step was dissolved in anhydrous DCM (5ml), and 3- ((tert-butoxycarbonyl) amino) undecanoic acid (822mg, 2.73mmol), EDCI (523mg, 2.73mmol), HOBt (369mg, 2.73mmol), N, were added to the system₂Under the protection condition, the reaction is carried out at room temperature, and the reaction progress is monitored by HPLC. After the reaction is finished, the system is extracted by EA, the organic phase is concentrated and subjected to column chromatography, petroleum ether-ethyl acetate (10:1) is used as an eluent, the product is colorless transparent oily liquid, and M/z 682.44[ M + H ]]⁺Intermediate 6(1.04g, 1.53 mmol).

(3) Ultrasonically suspending the intermediate 6(2.26g, 3.31mmol) prepared in the previous step in water (10ml) and methanol (2.5ml), and adding LiOH-H into the system₂O (835mg, 19.88mmol), stirred at room temperature, and the reaction progress monitored by HPLC. After the reaction is finished, EA is extracted, the water phase is taken, citric acid solution is added into the water phase, EA is extracted, and the product is obtained by concentration, wherein the product is light yellow solid, M/z 668.42[ M + H ]]⁺Intermediate 7(1.84g, 2.76 mmol).

(4) Intermediate 2 prepared in example 2(0.6g, 0.44mmol) was dissolved in waterTo water in DCM (2ml) was added intermediate 7(294mg, 0.44mmol), EDCI (102mg, 0.53mmol), HOBt (72mg, 0.53mmol), N₂Under the protection condition, the reaction is carried out at room temperature, and the reaction progress is monitored by HPLC. After the reaction is finished, the system is extracted by EA, the organic phase is concentrated and subjected to column chromatography, petroleum ether-ethyl acetate (5:1) is used as an eluent, and the product is white solid (0.3g, 0.15mmol), M/z 2013.18[ M + H ]]⁺。

The resulting white solid (0.3g, 0.15mmol) was dissolved in DCM (1ml), TFA (171mg, 110ul, 1.5mmol) was added, the reaction was carried out at rt, the reaction progress was monitored by HPLC, after completion of the reaction, the system was dried and recrystallized from MTBE, and filtered to give the final product compound 7: n- (([1, 1' -Biphenyl)]-4-methyl) - [ 3-aminoundecanoyl]Polymyxin B, M/z1412.87[ M + H ]]⁺。

The reaction process of this example is shown as follows.

Example 6N-N-nonyl- [ 4-methylbenzenesulfonyl group]Synthesis of polymyxin B (Compound 5)

(1) Methyl (S) -2-amino-4- ((tert-butoxycarbonyl) amino) butyrate (1g, 3.84mmol) was dissolved in methanol (10ml), and nonanal (601mg, 4.23mmol), NaCNBH, and the mixture was added thereto₃(266mg, 4.23mmol), anhydrous ZnCl₂(523mg, 3.84mg), the system was stirred at room temperature overnight and the reaction progress monitored by HPLC. After the reaction is finished, EA is used for extracting a system, an organic phase is concentrated, silica gel column chromatography is carried out, petroleum ether-ethyl acetate (15:1) is used as an eluent, and a white solid product, M/z 387.28[ M + H ] is obtained]⁺Intermediate 8(1.11g, 2.88 mmol).

(2) Intermediate 8(1.11g, 2.88mmol) from the previous step was dissolved in anhydrous DCM (5ml), p-toluenesulfonyl chloride (604mg, 3.17mmol) and triethylamine (437mg, 4.32mmol) were added to the system, and the reaction was carried out at room temperature with monitoring of the progress of the reaction by HPLC. After the reaction is finished, the system is extracted by EA, the organic phase is concentrated and subjected to column chromatography, petroleum ether-ethyl acetate (10:1) is used as eluent, the product is colorless transparent oily liquid, and M/z 541.29[ M/z+H]⁺Intermediate 9(987mg, 1.82 mmol).

(3) Ultrasonically suspending the intermediate 9(987mg, 1.82mmol) in the previous step in water (10ml) and methanol (2.5ml), and adding LiOH-H into the system₂O (458mg, 10.92mmol), stirred at room temperature and the reaction progress monitored by HPLC. After the reaction is finished, EA is extracted, the water phase is taken, citric acid solution is added into the water phase, EA is extracted, and the product is obtained by concentration, wherein the product is light yellow solid, M/z 527.27[ M + H ]]⁺Intermediate 10(910mg, 1.73 mmol).

(4) Intermediate 10(1.16g, 2.2mmol) was dissolved in anhydrous DCM (2ml), and intermediate 2 prepared in example 2(0.6g, 0.44mmol), HATU (837mg, 2.2mmol), DIPEA (284mg, 2.2mmol), N, were added to the system₂Under the protection condition, the reaction is carried out at room temperature, and the reaction progress is monitored by HPLC. After the reaction is finished, the system is extracted by EA, the organic phase is concentrated and subjected to column chromatography, petroleum ether-ethyl acetate (5:1) is used as an eluent, and the product is a white solid. The resulting white solid was dissolved in DCM (1ml), TFA (0.15ml) was added, the reaction was carried out at RT, the progress of the reaction was monitored by HPLC, after the reaction was complete, the system was dried by rotary drying and recrystallized from MTBE, and the final product was filtered off, M/z 1343.78[ M + H ]]⁺. Compound 5 was obtained: N-N-nonyl- [ 4-methylbenzenesulfonyl ] compounds]Polymyxin B.

The reaction process of this example is shown as follows.

EXAMPLE 7 Synthesis of N-benzyl-N' -N-octyl polymyxin E (Compound 8)

(1) (S) -tyrosine methyl ester (5g, 22.4mmol) is dissolved in methanol (20ml), and benzaldehyde (2.6g, 24.6mmol) and NaCNBH are added into the system₃(1.55g, 24.6mmol), anhydrous ZnCl₂(3.05g, 22.4mmol), the system is stirred at room temperature overnight and the progress of the reaction is monitored by HPLC. After the reaction is finished, EA is used for extracting a system, an organic phase is concentrated, silica gel column chromatography is carried out, petroleum ether-ethyl acetate (15:1) is used as an eluent, and a white solid product, M/z 314.13[ M + H ] is obtained]⁺Is intermediate 11(5.12g，16.4mmol)。

(2) Dissolving the intermediate 11(5.12g, 16.4mmol) in anhydrous methanol (10ml), and adding n-octanal (2.3g, 18mmol) and NaCNBH into the system₃(1.13g, 18mmol), anhydrous ZnCl₂(2.24g, 16.4mmol), the system is stirred at room temperature overnight and the progress of the reaction is monitored by HPLC. After the reaction is finished, EA is used for extracting a system, an organic phase is concentrated, silica gel column chromatography is carried out, petroleum ether-ethyl acetate (15:1) is used as an eluent, and a white solid product, M/z 426.26[ M + H ] is obtained]⁺Intermediate 12(4.6g, 10.8 mmol).

(3) Ultrasonically suspending the intermediate 12(4.6g, 10.8mmol) in the previous step in water (20ml) and methanol (5ml), and adding LiOH-H into the system₂O (1.36g, 32.4mmol), stirred at room temperature and the reaction progress monitored by HPLC. After the reaction is finished, EA is extracted, the water phase is taken, citric acid solution is added into the water phase, EA is extracted, and the product is obtained by concentration, wherein the product is light yellow solid, M/z412.24[ M + H ]]⁺To obtain an intermediate 13.

(4) Intermediate 13(905mg, 2.2mmol) was dissolved in anhydrous DCM (2ml), and intermediate 4(0.6g, 0.44mmol), HATU (837mg, 2.2mmol), DIPEA (284mg, 2.2mmol), N, prepared in example 4, was added to the system₂Under the protection condition, the reaction is carried out at room temperature, and the reaction progress is monitored by HPLC. After the reaction is finished, the system is extracted by EA, the organic phase is concentrated and subjected to column chromatography, petroleum ether-ethyl acetate (5:1) is used as an eluent, and the product is a white solid. The resulting white solid was dissolved in DCM (1ml), TFA (0.15ml) was added, the reaction was carried out at RT, the progress of the reaction was monitored by HPLC, after the reaction was complete, the system was dried by rotary drying and recrystallized from MTBE, and the final product was filtered off, M/z 1294.82[ M + H ]]⁺. Compound 8 was obtained: N-benzyl-N' -N-octyl polymyxin E.

The reaction process of this example is shown as follows.

Example 8N- (N-octyl) - [ 2-chlorophenylaminoacyl]Synthesis of polymyxin E (Compound 1)

(1) Will be provided with(S) -Aspartame (5g, 28.7mmol) was dissolved in methanol (20ml), and octanal (4.05g, 31.6mmol), NaCNBH, was added to the system₃(2.0g, 31.6mmol), anhydrous ZnCl₂(3.9g, 28.7mmol), the system is stirred at room temperature overnight and the progress of the reaction is monitored by HPLC. After the reaction is finished, EA is used for extracting a system, an organic phase is concentrated, silica gel column chromatography is carried out, petroleum ether-ethyl acetate (15:1) is used as an eluent, and a white solid product, M/z 287.19[ M + H ] is obtained]⁺Intermediate 14(4.1g, 14.4 mmol).

(2) O-chloroaniline (3g, 23.6mmol) was dissolved in dry DCM (50ml), DIPEA (6.1g, 47.2mmol) was added to the system, and triphosgene (2.33g, 7.87mmol) in dichloromethane (10ml) was slowly added to the system under the same conditions, and after the addition was completed, the reaction was continued for 3 hours under ice bath conditions, and then intermediate 14(6.75g, 23.6mmol) in dichloromethane (5ml) was added to the system and allowed to react overnight at room temperature. The next day, HPLC monitors the reaction complete, spin-dry the solvent in the system, and use EA/H in the system₂O extraction, washing of the organic phase with saturated brine, drying over anhydrous sodium sulfate, filtration, concentration of the organic phase to give crude intermediate 15, chromatography of the crude product on silica gel column to give intermediate 15(8.19g, 18.6mmol), M/z 440.19[ M + H ] as a white powder]⁺。

(3) Ultrasonically suspending the intermediate 15(5g, 11.38mmol) in the previous step in water (10ml) and methanol (2.5ml), and adding LiOH-H into the system₂O (1.43g, 34.15mmol), stirred at room temperature and the reaction progress monitored by HPLC. After the reaction is finished, EA is extracted, the water phase is taken, citric acid solution is added into the water phase, EA is extracted, and the product is obtained by concentration, wherein the product is light yellow solid, M/z 426.17[ M + H ]]⁺Intermediate 16(4.35g, 10.24 mmol).

(4) Intermediate 16(935mg, 2.2mmol) was dissolved in anhydrous DCM (2ml), and intermediate 4(0.585g, 0.44mmol), HATU (837mg, 2.2mmol), DIPEA (284mg, 2.2mmol), N, were added to the system₂Under the protection condition, the reaction is carried out at room temperature, and the reaction progress is monitored by HPLC. After the reaction is finished, the system is extracted by EA, the organic phase is concentrated and subjected to column chromatography, petroleum ether-ethyl acetate (5:1) is used as an eluent, and the product is a white solid. Mixing the obtained white solidDissolved in DCM (1ml), added TFA (0.2ml), reacted at room temperature, monitored by HPLC for progress, after the reaction was complete, the system was dried by spinning and recrystallized from MTBE, filtered to give the final product, M/z 1308.75[ M + H ]]⁺. Compound 1 was obtained: n- (N-octyl) - [ 2-chlorophenylaminoacyl]Polymyxin E.

The reaction process of this example is shown as follows.

₄Example 9 Synthesis of N- (N-octyl) - (Boc) -PME

The intermediate 4-tetra- (Boc) polymyxin E nonapeptide (0.585g, 0.44mmol) was dissolved in methanol (5ml) and n-octanal (68mg, 0.53mmol), NaCNBH₃(33mg, 0.53mmol), anhydrous ZnCl₂(60mg, 0.44mmol), the system is stirred at room temperature overnight and the reaction progress is monitored by HPLC. After the reaction is finished, EA is used for extracting a system, an organic phase is concentrated, silica gel column chromatography is carried out, petroleum ether-ethyl acetate (15:1) is used as an eluent, and a white solid product intermediate 17, M/z1441.92[ M + H ] is obtained]⁺Is N- (N-octyl) - (Boc)₄-PME。

The reaction process of this example is shown as follows.

Example 10N- (N-octyl) - [ N, N-dimethylbenzoyl-]Synthesis of polymyxin E (Compound 18)

4- (dimethylamino) aniline (136mg, 1mmol) was dissolved in dry DCM (5ml), DIPEA (260mg, 2mmol) was added to the system, and triphosgene (99mg, 0.33mmol) in dichloromethane (1ml) was slowly added to the system under the conditions, after which the reaction was continued for 3h under ice-bath conditions, and then intermediate 17 (1.44g, 1mmol) in dichloromethane (5ml) was added to the system and allowed to react overnight at room temperature. The next day, HPLC monitors the reaction complete, spin-dry the solvent in the system, and use EA/H in the system₂O extraction and washing of the organic phase with saturated brineDrying with anhydrous sodium sulfate, filtering, concentrating the organic phase to obtain crude intermediate 18, and subjecting the crude intermediate to silica gel column chromatography to obtain intermediate 18(1.04g, 0.65mmol) as white powder, M/z1604.00[ M + H ]]⁺. Intermediate 18 from the previous step was dissolved in the appropriate amount of DCM (5ml), TFA (0.5ml) was added and stirred at rt for 2h, the system was directly spin dried and dried to give compound 18: n- (N-octyl) - [ N, N-dimethylbenzoyl)]Polymyxin E, M/z 1203.79[ M + H [ ]]⁺。

The reaction process of this example is shown as follows.

Example 11 Synthesis of Compound 12

(1) Intermediate 4(0.585g, 0.44mmol) was dissolved in methanol (5ml), and 2-morpholinoacetaldehyde (68mg, 0.53mmol), NaCNBH, was added to the system₃(33mg, 0.53mmol), anhydrous ZnCl₂(60mg, 0.44mmol), the system is stirred at room temperature overnight and the reaction progress is monitored by HPLC. After the reaction is finished, the EA extraction system is used, the organic phase is concentrated and subjected to silica gel column chromatography, petroleum ether-ethyl acetate (15:1) is used as an eluent, and the white solid product, namely the intermediate 19(380mg, 0.26mmol), M/z1442.88[ M + H ] is obtained]⁺Is N- (2-morpholinoethyl) - (Boc)₄-PME。

(2) Intermediate 19(380mg, 0.26mmol) from the previous step was dissolved in DCM (5ml), and N-Boc-phenylalanine (345mg, 1.3mmol), HATU (494mg, 1.3mmol), DIPEA (168mg, 1.3mmol) were added to the system, and the reaction was carried out overnight at room temperature. The next day, HPLC monitors the reaction complete, spin-dry the solvent in the system, and use EA/H in the system₂O extraction, washing the organic phase with saturated brine, drying over anhydrous sodium sulfate, filtering, concentrating the organic phase to give crude intermediate 20, and subjecting the crude intermediate to silica gel column chromatography to give intermediate 20 (264mg, 0.156mmol) as a white powder, M/z 1690.00[ M + H ], (M + H)]⁺。

(3) Dissolving the intermediate 20 obtained in the previous step in a proper amount of DCM (5ml), adding TFA (0.1ml), stirring at room temperature for 2h, directly spin-drying the system, and drying to obtain the intermediateCompound 12, M/z 1189.73[ M + H ]]⁺。

The reaction process of this example is shown as follows.

Example 12 Synthesis of Compound 15

(1) Intermediate 2(0.6g, 0.44mmol) prepared in example 2 was dissolved in methanol (5ml), and n-decanal (83mg, 0.53mmol), NaCNBH, was added to the system₃(33mg, 0.53mmol), anhydrous ZnCl₂(60mg, 0.44mmol), the system is stirred at room temperature overnight and the reaction progress is monitored by HPLC. After the reaction is finished, the EA is used for extracting a system, an organic phase is concentrated, silica gel column chromatography is carried out, petroleum ether-ethyl acetate (15:1) is used as an eluent, and a white solid product, namely an intermediate 21(463mg, 0.31mmol), M/z 1503.93[ M + H ]]⁺。

(2) Intermediate 21 from the previous step was dissolved in DCM (5ml), benzyl bromide (64mg, 0.37mmol) and potassium carbonate (86mg, 0.62mmol) were added to the system, the system was stirred at room temperature overnight, and the progress of the reaction was monitored by HPLC. After the reaction is finished, the EA extraction system is used, the organic phase is concentrated and subjected to silica gel column chromatography, petroleum ether-ethyl acetate (15:1) is used as an eluent, and the white solid product, namely the intermediate 22(420mg, 0.26mmol) M/z 1593.98[ M + H]⁺。

(3) Dissolving the intermediate 22 obtained in the previous step in an appropriate amount of DCM (5ml), adding TFA (0.1ml), stirring at room temperature for 2H, directly spin-drying the system, and drying to obtain the compound 15, M/z 1193.77[ M + H ]]⁺。

The reaction process of this example is shown as follows.

Example 13 Synthesis of Compound 21

(1) Intermediate 2(0.6g, 0.44mmol) prepared in example 2 was dissolved in methanol (5ml), and furaldehyde (51mg, 0.53mmol), NaCNBH, was added to the system₃(33mg, 0.53mmol), anhydrous ZnCl₂(60mg, 0.44mmol), the system is stirred at room temperature overnight and the reaction progress is monitored by HPLC. After the reaction was complete, the system was extracted with EA, the organic phase was concentrated and subjected to silica gel column chromatography using petroleum ether-ethyl acetate (15:1) as eluent to give intermediate 23(476mg, 0.33mmol), M/z 1443.80[ M + H ] as a white solid product]⁺。

(2) Intermediate 23 from the previous step was dissolved in DCM (5ml), and octanesulfonyl chloride (84mg, 0.4mmol) and potassium carbonate (91mg, 0.66mmol) were added to the system, and the system was stirred at room temperature overnight. The next day, HPLC monitors the reaction complete, spin-dry the solvent in the system, and use EA/H in the system₂O extraction, washing the organic phase with saturated brine, drying over anhydrous sodium sulfate, filtering, concentrating the organic phase to obtain crude intermediate 24, and subjecting the crude intermediate to silica gel column chromatography to obtain intermediate 24(374mg, 0.23mmol) M/z 1619.89[ M + H ] as white powder]⁺。

(3) Dissolving the intermediate 24 obtained in the previous step in an appropriate amount of DCM (5ml), adding TFA (0.1ml), stirring at room temperature for 2H, directly spin-drying the system, and drying to obtain the compound 21, M/z 1219.68[ M + H ]]⁺。

The reaction process of this example is shown as follows.

Example 14 preparation of Compound 22

(1) (S) -valine methyl ester hydrochloride (5g,30.0mmol) was dissolved in methanol (10ml), and octanal (5.55ml,35.1mmol) and NaCNBH were added to the system₃(2.75g,35.1mmol), the system is stirred at room temperature overnight and the progress of the reaction is monitored by HPLC. After the reaction is finished, EA is used for extracting a system, the organic phase is concentrated and is subjected to silica gel column chromatography, petroleum ether-ethyl acetate (30:1) is used as an eluent, colorless viscous liquid is obtained, and M/z 244.32[ M + H ]]+, intermediate 25(2.7g,11.09 mmol).

(2) Intermediate 25((2.5g,10.27mmol)) was dissolved in DCM (10ml), o-chlorobenzene plus acid chloride (2.2g,12.57mmol) was dissolved in the system, potassium carbonate (1.5g,10.85mmol) was added to the system, and the reaction was allowed to proceed at room temperature overnight. The next day, HPLC detects the reaction is complete, the solvent in the system is dried, the system is extracted by EA/H2O, the organic phase is washed by saturated saline solution, dried by anhydrous sodium sulfate, filtered, the organic phase is concentrated to obtain a crude product of the intermediate 26, the crude product is subjected to silica gel column chromatography to obtain the intermediate 26(2.3g,6.27mmol) which is white powder, and M/z 382.21 [ M + H ] +

(3) Ultrasonically suspending the intermediate 26(2.3g,6.27mmol) in the last step in water (5ml) and methanol (5ml), and adding LiOH-H into the system₂O (1.5g, 37.6mmol), stirred at room temperature and the reaction progress monitored by HPLC. After the reaction is finished, EA is extracted, the water phase is taken, citric acid solution is added into the water phase, EA is extracted, and the product is obtained by concentration, wherein the product is light yellow solid, M/z368.19[ M + H ]]⁺Intermediate 27(2.0g,5.44 mmol).

(4) Intermediate 27(86mg,0.226mmol) from the previous step was dissolved in anhydrous DCM (2ml) and intermediate 4(100mg, 0.075mmol), PYBOP (118mg,0.226mmol), DIPEA (40ul,0.226mmol), N, were added to the system₂Under the protection condition, the reaction is carried out at room temperature, and the reaction progress is monitored by HPLC. After the reaction is finished, the system is extracted by EA, the organic phase is concentrated and subjected to column chromatography, petroleum ether-ethyl acetate (7: 1) is used as an eluent, and the product is a white solid, namely the intermediate 28. Dissolving the obtained intermediate 28 in DCM (1ml), adding TFA (0.2ml), reacting at room temperature, monitoring the reaction progress by HPLC, after the reaction is finished, drying the system by spinning, and performing C18 column chromatography to obtain white solid powder, M/z 1278.76[ M + H ] 1278.76]⁺I.e. compound 22.

The reaction process of this example is shown as follows.

Example 15 preparation of Compound 23

(1) Decanoic acid (4.62g, 27.24mmol) was dissolved in DCM (10ml), EDCI (5.22g, 27.24mmol), HOBt (3.69g, 27.24mmol) were added to the system, stirring was carried out at room temperature for 0.5h, p-aminobenzaldehyde (3g, 24.75mmol) was added to the system, stirring was carried out at room temperature overnight, and the progress of the reaction was monitored by HPLC. After the reaction is finished, the reaction is carried outExtracting with EA, concentrating the organic phase, performing silica gel column chromatography, eluting with petroleum ether-ethyl acetate (20:1) to obtain colorless viscous liquid, M/z 276.19[ M + H ]]⁺Intermediate 29(1.45g,5.27 mmol).

(2) (S) -valine methyl ester hydrochloride (100mg,0.825mmol) was dissolved in methanol (2ml), and intermediate 29(250mg,0.908mmol), NaCNBH, was added to the system₃(56mg,0.908mmol), the system is stirred at room temperature overnight and the reaction progress is monitored by HPLC. After the reaction is finished, EA is used for extracting a system, the organic phase is concentrated and is subjected to silica gel column chromatography, petroleum ether-ethyl acetate (5:1) is used as an eluent, colorless viscous liquid is obtained, and M/z 391.29[ M + H ]]⁺Intermediate 30(285mg,0.73 mmol).

(3) Intermediate 30 from the previous step ((830mg,2.13mmol)) was dissolved in acetone (20ml), decanoyl chloride (526mg,2.77mmol) was dissolved in the system, potassium carbonate (588mg,4.26mmol) was added to the system, and the reaction was allowed to proceed at room temperature overnight. The next day, HPLC detection reaction is complete, solvent in the system is dried by spinning, and EA/H is used for the system₂O extraction, washing the organic phase with saturated brine, drying over anhydrous sodium sulfate, filtering, concentrating the organic phase to give crude intermediate 31, and subjecting the crude intermediate to silica gel column chromatography to give intermediate 31(1.09g,2.0mmol) as a white powder, M/z 545.42[ M + H ], (M + H)]⁺

(4) Ultrasonically suspending the intermediate 31(0.68g,1.25mmol) in the previous step in water (5ml) and methanol (5ml), and adding LiOH-H into the system₂O (0.157g, 3.75mmol), stirred at room temperature and the reaction progress monitored by HPLC. After the reaction is finished, EA is extracted, the water phase is taken, citric acid solution is added into the water phase, EA is extracted, and the product is obtained after concentration, wherein the product is white solid, and M/z 531.41[ M + H ]]⁺Intermediate 32(0.2g,0.377 mmol).

(5) Intermediate 32(40mg,0.077mmol) from the previous step was dissolved in anhydrous DCM (2ml) and intermediate 4(34mg, 0.025mmol) prepared in example 4, PYBOP (40mg,0.077mmol), DIPEA (14ul,0.077mmol), N, were added to the system₂Under the protection condition, the reaction is carried out at room temperature, and the reaction progress is monitored by HPLC. After the reaction is finished, the system is extracted by EA, the organic phase is concentrated and subjected to column chromatography, petroleum ether-ethyl acetate (5.5: 1) is used as eluent, and the product is obtainedThe product is a white solid, namely the intermediate 33. Dissolving the obtained intermediate 33 in DCM (1ml), adding TFA (0.2ml), reacting at room temperature, monitoring the reaction progress by HPLC, after the reaction is finished, drying the system by spinning, and performing C18 column chromatography to obtain white solid powder, M/z 1441.98[ M + H ] 1441.98]⁺I.e. compound 23.

The reaction process of this example is shown as follows.

Example 16 preparation of Compound 24

(1) β -aminopropionic acid methyl ester hydrochloride (100mg, 0.716mmol) was dissolved in DCM (2ml), triethylamine (217mg, 2.148mmol) was added to the system and ice bath was performed, bischlorobenzoyl chloride (171mg, 0.788mmol) was added to the system, reaction was performed at room temperature overnight, progress of the reaction was monitored by HPLC, after completion of the reaction, the system was spun dry and slurried in MeOH to give a white solid, M/z 284.12[ M + H ] M/z 284.12]⁺Intermediate 34(0.14g, 0.5 mmol).

(2) The intermediate 34(0.1g, 0.36mmol) in the previous step was suspended in MeOH (5ml) and water (5ml), and LiOH. H was added₂O (91mg, 2.16mmol), heated at 70 ℃ for 3h and the reaction progress monitored by HPLC. After the reaction is finished, hydrochloric acid is added into the system for acidification, a large amount of white solid, M/z 270.11[ M + H ], appears]⁺This was intermediate 35(81mg, 0.3 mmol).

(3) N' -Boc-L-2, 4-diaminobutyric acid methyl ester hydrochloride (1g,3.72mmol) was dissolved in methanol (10ml), and octanal (570mg,4.47mmol), NaCNBH, was added to the system₃(890mg,11.6mmol), the system is stirred at room temperature overnight and the reaction progress is monitored by HPLC. After the reaction is finished, EA is used for extracting a system, the organic phase is concentrated, silica gel column chromatography is carried out, petroleum ether-ethyl acetate (3:1) is used as an eluent, colorless liquid is obtained, and M/z 345.27 [ M + H ]]⁺Intermediate 36(0.88g,2.56 mmol).

(4) Intermediate 35(37mg, 0.145mmol) was added to DMF, EDCI (31mg, 0.16mmol), HOBt (22mg, 0.16mmol) and triethylamine (44mg, 0.435mmol) were added to the system, and the mixture was stirred at room temperature for 0.5 h. Intermediate 36(50mg, 0.145mmol) was added to the system, stirred at room temperature and the reaction progress monitored by HPLC. When the reaction is finished, the system uses EA/H₂O extraction, washing the organic phase with saturated brine, drying over anhydrous sodium sulfate, filtering, concentrating the organic phase to obtain crude intermediate, and subjecting the crude intermediate to silica gel column chromatography to obtain intermediate 37(0.488g,0.82mmol), M/z596.36[ M + H ] as white powder]⁺

(5) Ultrasonically suspending the intermediate 37(0.2g,0.336mmol) in the previous step in water (5ml) and methanol (5ml), and adding LiOH-H into the system₂O (42mg, 1.0mmol), stirred at room temperature and the reaction progress monitored by HPLC. After the reaction is finished, EA is extracted, the water phase is taken out, a citric acid solution is added into the water phase to acidify the system until the pH value is 2, EA is extracted, and the product is obtained by concentration, wherein the product is light yellow solid, and M/z 582.35[ M + H ]]⁺Intermediate 38(174mg,0.3 mmol).

(6) Intermediate 38(38mg,0.086mmol) was dissolved in anhydrous DCM (2ml) and intermediate 4(50mg, 0.027mmol), PYBOP (70mg,0.135mmol), DIPEA (28ul,0.162mmol), N, were added to the system₂Under the protection condition, the reaction is carried out at room temperature, and the reaction progress is monitored by HPLC. After the reaction is finished, the system is extracted by EA, the organic phase is concentrated and subjected to column chromatography, petroleum ether-ethyl acetate (5:1) is used as an eluent, and the product is a white solid, namely the intermediate 39. Dissolving the obtained intermediate 39 in DCM (1ml), adding TFA (0.2ml), reacting at room temperature, monitoring the reaction progress by HPLC, after the reaction is finished, drying the system in a rotary manner, and performing C18 column chromatography to obtain white solid powder, M/z 1392.87[ M + H ] 1392.87]⁺I.e. compound 24.

The reaction process of this example is shown as follows.

Example 17 preparation of Compound 25

(1) 4-methoxy biphenyl-4-methylTo acid (1.0g, 4.39mmol) in DCM (10ml) was added EDCI (924mg, 4.82mmol), HOBt (650mg, 4.82mmol), triethylamine (1.3g, 13.13mmol), stirred at room temperature for 0.5H, β -aminopropionic acid methyl ester hydrochloride (613mg, 4.39mmol) was added to the system under ice bath, reacted at room temperature overnight, the reaction progress was monitored by HPLC, after the reaction was completed, the system was treated with EA/H₂Extracting with water, washing the organic phase with saturated sodium chloride, drying with anhydrous sodium sulfate, filtering, concentrating the organic phase to obtain crude product of intermediate, subjecting the crude product to silica gel column chromatography (PE: EA ═ 6:1) to obtain white solid, M/z 314.13[ M + H]⁺Intermediate 40(1.1g,3.5 mmol).

(2) Intermediate 40(0.63g, 2.01mmol) was suspended in MeOH (5ml) and water (5ml), and LiOH. H was added₂O (420mg, 6.03mmol), heated at 70 ℃ for 3h and the progress of the reaction monitored by TLC. After the reaction is finished, hydrochloric acid is added into the system for acidification, a large amount of white solid, M/z 300.11[ M + H ], appears]⁺This gave intermediate 41 (0.41g, 1.37 mmol).

(3) Intermediate 41(287mg, 0.84mmol) was added to DMF, HATU (632mg, 1.07mmol) and DIPEA (874mg, 5.01mmol) were added to the system, and the mixture was stirred at room temperature for 0.5 h. Intermediate 36(500mg, 1.67mmol) was added to the system, stirred at room temperature and the reaction progress monitored by HPLC. When the reaction is finished, the system uses EA/H₂O extraction, washing the organic phase with saturated brine, drying over anhydrous sodium sulfate, filtering, concentrating the organic phase to obtain crude intermediate, and subjecting the crude intermediate to silica gel column chromatography to obtain intermediate 42(0.25g,0.4mmol), M/z 626.37[ M + H ] as white powder]⁺

(4) Ultrasonically suspending the intermediate 42(0.1g,0.16mmol) in the previous step in water (5ml) and methanol (5ml), and adding LiOH-H into the system₂O (40mg, 0.96mmol), stirred at room temperature and the reaction progress monitored by HPLC. After the reaction is finished, EA is extracted, the water phase is taken, citric acid solution is added into the water phase, EA is extracted, and the product is obtained by concentration, wherein the product is light yellow solid, M/z612.36[ M + H ]]⁺Intermediate 43(174mg,0.3 mmol).

(5) Intermediate 43(90mg,0.147mmol) was dissolved in dry DCM (5ml) and intermediate 4(65mg, 0.049mmol), PYBOP (115mg, 0) was added to the system.22mmol)，DIPEA(51ul,0.29mmol)，N₂Under the protection condition, the reaction is carried out at room temperature, and the reaction progress is monitored by HPLC. After the reaction is finished, the system is extracted by EA, the organic phase is concentrated and subjected to column chromatography, petroleum ether-ethyl acetate (5:1) is used as an eluent, and the product is a white solid, namely the intermediate 44. Dissolving the obtained intermediate 44 in DCM (1ml), adding TFA (0.2ml), reacting at room temperature, monitoring the reaction progress by HPLC, after the reaction is finished, drying the system in a rotary manner, and performing C18 column chromatography to obtain white solid powder, M/z 1422.88[ M + H ] 1422.88]⁺I.e. compound 25.

The reaction process of this example is shown as follows.

Example 18 preparation of Compound 26

(1) β -amino methyl propionate hydrochloride (200mg, 2.245mmol) is dissolved in DCM (5ml), potassium carbonate (465mg, 3.368mmol) is added into the system, ice bath is carried out, o-chlorobenzoyl chloride (432mg, 2.47mmol) is added into the system, the reaction is carried out overnight at room temperature, the reaction progress is monitored by HPLC, after the reaction is finished, the system is filtered, the filtrate is dried in a rotary mode to obtain colorless transparent liquid, M/z 242.05[ M + H ] M/z 242.05]⁺Intermediate 46(0.482g,2.0 mmol).

(2) Intermediate 46(0.25g, 1.034mmol) was suspended in MeOH (5ml) and water (1ml), and LiOH-. H was added₂O (260mg, 6.21mmol), heated at 70 ℃ for 3h and the reaction progress monitored by HPLC. After the reaction is finished, hydrochloric acid is added into the system for acidification, EA-water is used for extraction, the organic phase is concentrated and is subjected to column chromatography (DCM: MeOH is 20:1), and M/z is 228.03[ M + H ]]⁺This was intermediate 47(204mg, 0.9 mmol).

(3) Intermediate 47(100mg, 0.44mmol) was added to DCM, HATU (209mg, 0.55mmol), DIPEA (213mg, 1.65mmol) were added to the system, and the mixture was stirred at room temperature for 0.5 h. Intermediate 36(126mg, 0.366mmol) was added to the system, stirred at room temperature and the reaction progress monitored by HPLC. When the reaction is finished, the system uses EA/H₂Extracting with water, washing the organic phase with saturated sodium chloride solution, drying with anhydrous sodium sulfate, filtering, and concentrating the organic phase to obtain extractCrude intermediate, which was subjected to silica gel column chromatography to give intermediate 48(0.14g,0.25mmol), M/z 554.29[ M + H78 ] as a white powder]⁺

(4) Ultrasonically suspending the intermediate 48(0.14g,0.253mmol) in the last step in water (5ml) and methanol (1ml), and adding LiOH-H into the system₂O (32mg, 0.76mmol), stirred at room temperature and the reaction progress monitored by HPLC. After the reaction is finished, EA is extracted, the water phase is taken out, a citric acid solution is added into the water phase to acidify the system until the pH value is 2, EA is extracted, and the product is obtained by concentration, wherein the product is light yellow solid, and M/z 540.27[ M + H ]]⁺Intermediate 49(0.1g,0.185 mmol).

(5) Intermediate 49(40mg,0.072mmol) from the previous step was dissolved in anhydrous DCM (2ml), and intermediate 4(64mg, 0.048mmol), PYBOP (50mg,0.096mmol), DIPEA (25ul,0.144mmol), N, were added to the system₂Under the protection condition, the reaction is carried out at room temperature, and the reaction progress is monitored by HPLC. After the reaction is finished, the system is extracted by EA, the organic phase is concentrated and subjected to column chromatography, petroleum ether-ethyl acetate (6: 1) is used as an eluent, and the product is a white solid, namely the intermediate 50. Dissolving the obtained intermediate 50 in DCM (1ml), adding TFA (0.2ml), reacting at room temperature, monitoring the reaction progress by HPLC, after the reaction is finished, drying the system in a rotary manner, and performing C18 column chromatography to obtain white solid powder, M/z 1350.80[ M + H ] 1350.80]⁺I.e. compound 26.

The reaction process of this example is shown as follows.

Example 19 determination of the minimum concentration of bacteriostatic Activity of a Compound

The compounds are subjected to in vitro antibacterial activity measurement, and the Minimum Inhibitory Concentration (MIC) value is read, and the measurement method refers to the method provided in pharmacopoeia of the people's republic of China (2015 edition). Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae were purchased from ATCC, polymyxin B and polymyxin E were used as control drugs, and the results of comparative experiments are shown in Table 3.

MIC of the Compounds of Table 3

As can be seen from the comparative test results in Table 3, the exemplary compounds have comparable or improved bacteriostatic activity against the gram-negative bacteria Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae compared to polymyxin E and polymyxin B, and can be used for preparing medicaments for treating bacterial infections as well as gram-negative bacterial infections.

MIC and MBC assays for Compounds 22-26

(I) test materials

Drug-resistant test bacterium (strain 2): inducing drug-resistant mutant acinetobacter baumannii: ab-1 and Ab-3 (obtained by reference method based on polymyxin-induced drug-resistant bacteria of ATCC 19606: ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June2009, p.2298-2305)

Sensitive strain (2 strain): pseudomonas aeruginosa HCCB20322 (i.e., ATCC 9029), acinetobacter baumannii: HCCB20325 (clinical isolate of Huashan Hospital affiliated to the university of Compound Dan, original number aba1604)

Culture medium: LB liquid Medium (g/L): 10g of tryptone, 5g of yeast extract powder, 10g of NaCl and pH 7.4.

(II) an experimental method:

(1) preparation of sample mother liquor

5.12mg of the compound sample is precisely weighed by an analytical balance into a 2mL sterile EP tube, and 1mL of sterile water is added to obtain a mother solution with a concentration of 5.12mg/mL for later use.

(2) Preparation of sample solution (two-fold dilution)

A sterile 96-well plate was taken, 60. mu.L of 5.12mg/mL of the stock solution was added to the first well, and 30. mu.L of sterile water was added to the 2 nd to 9 th wells. Add 30. mu.L of the mixture from well 1 to well 2 and mix well. Add 30. mu.L of the mixture from well 2 to well 3 and mix well. This was repeated to well 9, 30. mu.L was discarded, and only 30. mu.L of sterile water was added to well 10 as a growth control. Thus, the sample solution to be tested with a series of gradient concentrations is obtained.

(3) Preparation of bacterial suspension

The glycerol tubes of the four types of bacteria were inoculated into 4mL of LB medium, cultured overnight with shaking at 37 ℃, and then adjusted to an OD600 of 0.4 (about 10) using fresh LB medium⁸CFU/mL), and then LB was used to dilute the stationary phase bacterial suspension 10 times (10 times)⁷CFU/mL), then inoculated in LB medium at an inoculum size of 1%, to obtain a bacterial concentration of about 10⁵CFU/mL of bacterial suspension.

(4) MIC determination and interpretation

Taking a sterile 96-well culture plate, adding 10 mu L of sample solution with different gradient concentrations prepared in the step (2) into each well, and then respectively adding 90 mu L of bacteria concentration of about 10⁵And (3) uniformly mixing bacterial suspensions of four bacteria of CFU/mL, placing in an incubator at 37 ℃, standing and culturing for 20h, and judging the result. The test concentrations of the sample solutions were 512, 256, 128, 64, 32, 16, 8, 4, 2, 1, 0 μ g/mL, 5 replicates each. And (4) observing by naked eyes after culturing for 20h, wherein the lowest sample concentration of the thalli which does not grow is MIC.

(5) Determination and interpretation of MBC

Taking a blank sterile culture plate, adding 200uL LB liquid culture medium into each hole, then sucking 2uL (or continuously diluting 100 times) of bacterial suspension in MIC determination plate holes with the concentration of more than or equal to 1/2MIC, placing the bacterial suspension in corresponding holes, placing the bacterial suspension in an incubator at 37 ℃, standing and culturing for 20h, and judging the result. The lowest sample concentration at which the thalli do not grow is MBC.

(III) results of the experiment

The results of the MIC and MBC measurements are shown in Table 4.

TABLE 4 results of MIC and MBC determination

As can be seen from Table 4, the exemplary compounds 22-26 are more potent than polymyxin E against drug-resistant test bacteria Ab-1, Ab-3, and sensitive bacteria: the HCCB20322 and the HCCB20325 have equivalent or improved bacteriostatic activity and can be applied to the preparation of medicaments for preventing and treating drug-resistant bacterial infection and drug-resistant gram-negative bacterial infection.

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Claims

1. Polymyxin derivatives of general formula (I) and pharmaceutically acceptable salts and solvates thereof:

wherein:

R¹is CH (CH)₃)₂Or CH₂C₆H₅；

n is 0 or 1;

when n is 0:

R²comprises the following steps:

y is a bond, -C (O) -;

x-is a bond, -C (O) -, -NHC (O) -, -OC (O) -, -CH₂-or-SO₂-；

R⁵Comprises the following steps:

z is a covalent bond, -C_1-3Alkyl radical-、-C(O)-、-NHC(O)-、-C(O)NH-、-OC(O)-、-C(O)O-、-S(O₂)O-、-OS(O₂)-；

When n is 1:

R²is a compound of formula (I) in the formula (H),

R³is CH₂SH、CH₂CH₂SCH₃、CH₂OH、CH(CH₃)OH、CH₂CONH₂、CH₂CH₂CONH₂、CH₂C₄H₅N₂、CH₂C₆H₅OH、CH₂C₈H₆N、CH₂CH₂CH₂CH₂NH₂、CH₂CH₂NH₂、CH₂CH₂CH₂CH₄N₃，

R⁴Comprises the following steps:

y is a bond, -C (O) -;

x-is a bond, -C (O) -, -NHC (O) -, -OC (O) -, -CH₂-or-SO₂-；

R⁵Comprises the following steps:

z is a covalent bond, -C_1-3Alkyl-, -C (O) -, -NHC (O) -, -C (O) NH-, -OC (O) -, -C (O) O-, -S (O)₂)O-、-OS(O₂)-；

2. The polymyxin derivative of claim 1, wherein the pharmaceutically acceptable salt is a salt with an acid.

3. Polymyxin derivatives according to claim 2, wherein the acid is selected from one or more of hydrochloric acid, hydrobromic acid, sulphuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulphonic acid, aspartic acid and glutamic acid.

4. Polymyxin derivatives according to claim 1, wherein the solvate is a hydrate, an ethanolate or an acetate.

5. A process for the preparation of a polymyxin derivative according to any of claims 1-4, comprising:

method (i), when n is 1, the general formula (, the synthetic route of the polymyxin derivative is:

；

wherein:

R¹is CH (CH)₃)₂Or CH₂C₆H₅，

R²Is a compound of formula (I) in the formula (H),

R⁴Comprises the following steps:

y is a bond, -C (O) -;

x-is a bond, -C (O) -, -NHC (O) -, -OC (O) -, -CH₂-or-SO₂-；

R⁵Comprises the following steps:

R⁸represents-H, -C_1-10Alkyl, -C_3-10Cycloalkyl, -C_2-10Heterocyclyl radical, -C_6-12Aryl radical, -C_12-14Biphenyl, -C_5-12A heteroaryl group;

；

or,

wherein:

R¹is CH (CH)₃)₂Or CH₂C₆H₅；

R²Comprises the following steps:

y is a bond, -C (O) -;

x-is a bond, -C (O) -, -NHC (O) -, -OC (O) -, -CH₂-or-SO₂-；

R⁵Comprises the following steps:

R²、R⁵Each of which is optionally substituted by one or more-N (R)⁶)-L-R⁷Substituted, 0-3-OR⁸Substituted or halogen substituted.

6. A pharmaceutical composition comprising a therapeutically effective amount of a polymyxin derivative of any of claims 1-4 or pharmaceutically acceptable salts and solvates thereof, and at least one pharmaceutically acceptable carrier.

7. The pharmaceutical composition of claim 6, wherein the pharmaceutically acceptable carrier is one or more of a diluent, an excipient, a binder, a filler, or a disintegrant.

8. Use of a polymyxin derivative according to any of claims 1-4 in the manufacture of a medicament for the prevention or treatment of bacterial infectious disease.

9. Use of a polymyxin derivative according to claim 8 in the preparation of a medicament for the prevention or treatment of infectious diseases caused by gram-negative bacteria.

10. Use of a polymyxin derivative according to claim 9 in the preparation of a medicament for the prevention or treatment of polymyxin resistant gram negative bacterial infectious disease.

11. The use of a polymyxin derivative according to claim 10 in the preparation of a medicament for the prevention or treatment of polymyxin-resistant acinetobacter baumannii infectious disease.