Polyethylene glycol block polymer containing amino acid as well as preparation method and application of polyethylene glycol block polymer

Abstract

The invention relates to the technical field of polyethylene glycol, and discloses a polyethylene glycol block polymer containing amino acid as well as a preparation method and application thereof. The polymer has a structural general formula as shown in a formula (1); m is 2-10, and n is 1-8; r is selected from one or more of H,-CH3,-CH-(CH3) 2,-CH2-CH (CH3) 2,-CH-(CH3)-CH2-CH3,-(CH2) 2-S-CH3,-CH2-C6H5,-CH2-OH,-CH2-CH3-OH,-CH2-C6H4-OH,-CH2-CONH2,-(CH2) 2-CONH2,-CH2-COOH,-(CH2) 2-COOH,-(CH2) 4-NH2,-(CH2) 3-NHC (NH) NH2 and-CH2-C3H3N2, R is selected from one or more of H,-CH3,-CH-CH (CH3) 2,-CH2-CH (CH3) 2,-CH2-CH ( # imgabs0 # wherein Fmoc is # imgabs1 #

Description

Technical Field

[0001] The present invention relates to the technical field of polyethylene glycol, and in particular to a polyethylene glycol block polymer containing amino acids, and a preparation method and application thereof. Background Art

[0002] Polyethylene glycol (PEG) is a polyether composed of ethoxy units. The number after PEG indicates the average molecular weight, and the commonly used molecular weight is 200-20000. PEG is amphiphilic and can be dissolved in aqueous media and organic solvents. In addition, PEG has excellent biocompatibility, thermal stability and low toxicity. As of 2021, the US FDA has approved more than twenty drugs containing PEG, which play an important role in helping people overcome diseases. Therefore, PEG has been widely used in the medical field, and its performance has become the gold standard for pharmaceutical excipients.

[0003] PEG is generally produced based on the anionic ring-opening polymerization of ethylene oxide. Due to the randomness of the process, the product is often a mixture of PEG homologues of various molecular weights, which leads to a series of problems in the application of biopharmaceuticals, including the difficulty in obtaining consistent composition, characterization, and loss of biological activity of PEGylated products. These shortcomings are one of the main reasons why PEGylated drugs are difficult to obtain FDA approval. In recent years, in order to solve this problem, monodisperse polyethylene glycol (M-PEG) has received increasing attention in biomedical research. That is, M-PEG is a pure compound with precise, non-continuous molecular weight (i.e., the molecular weight is a fixed value, not a range) and exact molecular configuration.

[0004] Solid phase synthesis is a method of synthesizing target compounds on a solid phase carrier. Its characteristics are that intermediates are easy to purify, it is easy to automate the synthesis operation, and by using excess condensation reagents, efficient condensation can be achieved. It is widely used in the synthesis of peptides. Jiang Zhongxing's team at Wuhan University first reported in the journal Organic & Biomolecular Chemistry (Vol. 14, p7912-7919, 2016) that M-PEG with a molecular weight of more than 10 kDa was synthesized by solid phase synthesis, opening the era of solid phase synthesis of M-PEG. However, the flexibility of M-PEG causes its shape to be often curved, possibly because the ethylene glycol units are connected by ether bonds. It is worth noting that the shape of PEG is crucial to its pharmacokinetics and pharmacodynamics in biopharmaceuticals. For example, Achim M. Goepferich reported in the journal ACS Appl Mater Interfaces (Vol. 11, p1311-1320, 2019) that in PEGylated targeted liposomes, the flexibility of the PEG chains causes the PEG chains to entangle with each other, so that the ligands are mainly distributed in the hydrophilic shell, thereby reducing the binding of the ligands on the cell membrane to the receptors on the PEG, resulting in reduced targeting.

[0005] Therefore, the synthesis of novel M-PEG is of great significance to the development of biopharmaceuticals. Summary of the invention

[0006] The purpose of the present invention is to overcome the defects of uneven number average molecular weight of PEG and bending and folding of PEG chains in the prior art, and to provide a polyethylene glycol block polymer containing amino acids and a preparation method and application thereof. The polyethylene glycol block polymer containing amino acids has a single number average molecular weight and strong rigidity, overcomes the defects of bending and folding of PEG chains in the prior art, and the preparation method is simple and efficient.

[0007] In order to achieve the above-mentioned object, the first aspect of the present invention provides a polyethylene glycol block polymer containing amino acids, wherein the polymer has a general structural formula shown in formula (1);

[0008]

[0009] Among them, Fmoc is m is 2-10, n is 1-8;

[0010] R is selected from one or more of H, -CH3, -CH-(CH3)2, -CH2-CH(CH3)2, -CH-(CH3)-CH2-CH3, -(CH2)2-S-CH3, -CH2-C6H5, -CH2-OH, -CH2-CH3-OH, -CH2-C6H4-OH, -CH2-CONH2, -(CH2)2-CONH2, -CH2-COOH, -(CH2)2-COOH, -(CH2)4-NH2, -(CH2)3-NHC(NH)NH2 and -CH2-C3H3N2.

[0011] The second aspect of the present invention provides a method for preparing a polyethylene glycol block polymer containing amino acids, wherein the preparation method comprises:

[0012] (1) at room temperature, activating the 2-chlorotrityl resin of formula (a) to obtain an activated resin; subjecting the activated resin, 9-fluorenylmethoxycarbonyl-amino-polyethylene glycol-carboxylic acid of formula (b), dichloromethane, and N,N-diisopropylethylamine to a first contact and shaking treatment, and then contacting with methanol for a second time; and then contacting with a mixture of piperidine and N,N-dimethylformamide for a third contact and shaking treatment; obtaining a compound of formula (c);

[0013]

[0014] Where m is 2-10,

[0015] (2) treating the compound represented by formula (c), 9-fluorenylmethoxycarbonyl-amino acid or 9-fluorenylmethoxycarbonyl-amino acid-(Boc, Trt, tBu, Pbf) represented by formula (d), N,N-dimethylformamide, ethyl 2-oxime cyanoacetate, N,N-diisopropylethylamine, and diisopropylcarbodiimide at room temperature for the fourth contact and shaking treatment; washing with N,N-dimethylformamide and dichloromethane in sequence, and then contacting and shaking with a mixture of piperidine and N,N-dimethylformamide for the fifth time; and then washing with N,N-dimethylformamide and dichloromethane in sequence; obtaining the compound represented by formula (e);

[0016]

[0017] wherein R1 is selected from one or more of H, -CH3, -CH-(CH3)2, -CH2-CH(CH3)2, -CH-(CH3)-CH2-CH3, -(CH2)2-S-CH3, -CH2-C6H5, -CH2-OH, -CH2-CH3-OH, -CH2-C6H4-OH, -CH2-CONH2, -(CH2)2-CONH2, -CH2-COOH, -(CH2)2-COOH, -(CH2)4-NH2, -(CH2)3-NHC(NH)NH2, and -CH2-C3H3N2;

[0018] (3) treating the compound represented by formula (e), 9-fluorenylmethoxycarbonyl-amino-polyethylene glycol-carboxylic acid represented by formula (b), N,N-dimethylformamide, ethyl 2-oxime cyanoacetate, N,N-diisopropylethylamine, and diisopropylcarbodiimide by contacting and shaking for the sixth time; washing with N,N-dimethylformamide and dichloromethane in sequence, and then contacting and shaking with a mixture of piperidine and N,N-dimethylformamide for the seventh time; and then washing with N,N-dimethylformamide and dichloromethane in sequence to obtain the compound represented by formula (f);

[0019]

[0020] (4) Repeat steps (2) and (3) to obtain a compound represented by formula (g);

[0021]

[0022] Where n is 1-8;

[0023] (5) treating the compound represented by formula (g) with trifluoroacetic acid, water and / or thioanisole by shaking for the eighth time, washing with dichloromethane, subjecting the washing liquid to silica gel chromatography, and distilling under reduced pressure to obtain the amino acid-containing polyethylene glycol block polymer represented by formula (1);

[0024]

[0025] Fmoc

[0026] The third aspect of the present invention provides a polyethylene glycol block polymer containing amino acids prepared by the aforementioned preparation method.

[0027] The fourth aspect of the present invention provides a use of the aforementioned amino acid-containing polyethylene glycol block polymer connected to the surface of a nano-drug.

[0028] Through the above technical scheme, a monodisperse polyethylene glycol block polymer containing amino acids is prepared. Each unit of the polymer is connected by an amide bond, and the charged groups (-NH2 and -COOH) of the amino acid side chains greatly enhance the rigidity of the polymer through electrostatic repulsion, which is expected to solve the problem of flexible and different lengths of PEG intertwining with each other, resulting in reduced targeting of the PEG terminal ligand. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is a schematic diagram of the particle size distribution of alanine-block PEGylated liposomes prepared in Example 9 of the present invention;

[0030] Figure 2 is a schematic diagram of the Zeta potential of alanine-block PEGylated liposomes prepared in Example 9 of the present invention;

[0031] Figure 3 is the liquid phase-mass spectrum of the alanine-block polyethylene glycol prepared in Example 1 of the present invention;

[0032] Figure 4 This is the H-NMR spectrum of the alanine-block polyethylene glycol prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0033] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0034] As mentioned above, the first aspect of the present invention provides a polyethylene glycol block polymer containing amino acids, wherein the polymer has the general structural formula shown in formula (1);

[0035]

[0036] Among them, Fmoc is m is 2-10, n is 1-8;

[0037] R is selected from one or more of H, -CH3, -CH-(CH3)2, -CH2-CH(CH3)2, -CH-(CH3)-CH2-CH3, -(CH2)2-S-CH3, -CH2-C6H5, -CH2-OH, -CH2-CH3-OH, -CH2-C6H4-OH, -CH2-CONH2, -(CH2)2-CONH2, -CH2-COOH, -(CH2)2-COOH, -(CH2)4-NH2, -(CH2)3-NHC(NH)NH2 and -CH2-C3H3N2.

[0038] The inventors of the present invention have discovered that the present invention adopts "polyethylene glycol blocks containing amino acids" so that the prepared polyethylene glycol block polymers containing amino acids have precise and non-continuous molecular weights, that is, the number average molecular weight is a fixed value rather than a range value; in addition, each unit of the polymer of the present invention is connected by an amide bond, and the charged groups (-NH2 and -COOH) of the amino acid side chains greatly enhance the rigidity of the polymer through electrostatic repulsion, so that the prepared polyethylene glycol block polymers containing amino acids overcome the defect problem of bending and folding of PEG chains in the prior art.

[0039] According to the present invention, preferably, m is 3-9 and n is 2-7; more preferably, m is 4 and n is 3.

[0040] According to the present invention, in formula (1) One or more selected from glycine group, alanine group, valine group, leucine group, isoleucine group, methionine group, phenylalanine group, serine group, threonine group, tyrosine group, asparagine group, glutamine group, aspartic acid group, glutamic acid group, lysine group, arginine group and histidine group.

[0041] According to the present invention, preferably,

[0042] In formula (1) One or more selected from alanine group, valine group, leucine group, phenylalanine group, glutamic acid group, glutamine group and lysine group.

[0043] In the present invention, it should be noted that R is selected from different substituents such that The group is selected from one or more of the above groups.

[0044] According to the present invention, the number average molecular weight of the polymer is 580-6000, preferably 1385-1613.

[0045] According to the present invention, the polymer comprises one or more of formula (2) to formula (8);

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053] The second aspect of the present invention provides a method for preparing a polyethylene glycol block polymer containing amino acids, wherein the preparation method comprises:

[0054] (1) at room temperature, activating the 2-chlorotrityl resin of formula (a) to obtain an activated resin; subjecting the activated resin, 9-fluorenylmethoxycarbonyl-amino-polyethylene glycol-carboxylic acid of formula (b), dichloromethane, and N,N-diisopropylethylamine to a first contact and shaking treatment, and then contacting with methanol for a second time; and then contacting with a mixture of piperidine and N,N-dimethylformamide for a third contact and shaking treatment; obtaining a compound of formula (c);

[0055]

[0056] Where m is 2-10,

[0057] (2) treating the compound represented by formula (c), 9-fluorenylmethoxycarbonyl-amino acid or 9-fluorenylmethoxycarbonyl-amino acid-(Boc, Trt, tBu, Pbf) represented by formula (d), N,N-dimethylformamide, ethyl 2-oxime cyanoacetate, N,N-diisopropylethylamine, and diisopropylcarbodiimide at room temperature for the fourth contact and shaking treatment; washing with N,N-dimethylformamide and dichloromethane in sequence, and then contacting and shaking with a mixture of piperidine and N,N-dimethylformamide for the fifth time; and then washing with N,N-dimethylformamide and dichloromethane in sequence; obtaining the compound represented by formula (e);

[0058]

[0059] wherein R1 is selected from one or more of H, -CH3, -CH-(CH3)2, -CH2-CH(CH3)2, -CH-(CH3)-CH2-CH3, -(CH2)2-S-CH3, -CH2-C6H5, -CH2-OH, -CH2-CH3-OH, -CH2-C6H4-OH, -CH2-CONH2, -(CH2)2-CONH2, -CH2-COOH, -(CH2)2-COOH, -(CH2)4-NH2, -(CH2)3-NHC(NH)NH2, and -CH2-C3H3N2;

[0060] (3) treating the compound represented by formula (e), 9-fluorenylmethoxycarbonyl-amino-polyethylene glycol-carboxylic acid represented by formula (b), N,N-dimethylformamide, ethyl 2-oxime cyanoacetate, N,N-diisopropylethylamine diisopropylcarbodiimide by contacting and shaking for the sixth time; washing with N,N-dimethylformamide and dichloromethane in sequence, and then contacting and shaking with a mixture of piperidine and N,N-dimethylformamide for the seventh time; and then washing with N,N-dimethylformamide and dichloromethane in sequence to obtain the compound represented by formula (f);

[0061]

[0062] (4) Repeat steps (2) and (3) to obtain a compound represented by formula (g);

[0063]

[0064] Where n is 1-8;

[0065] (5) treating the compound represented by formula (g) with trifluoroacetic acid, water and / or thioanisole by shaking for the eighth time, washing with dichloromethane, subjecting the washing liquid to silica gel chromatography, and distilling under reduced pressure to obtain the amino acid-containing polyethylene glycol block polymer represented by formula (1);

[0066]

[0067] Fmoc

[0068] In the present invention, it should be noted that: 9-fluorenylmethoxycarbonyl-amino acid-(Boc, Trt, tBu, Pbf) shown in formula (d), that is, 9-fluorenylmethoxycarbonyl-amino acid-(tert-butyloxycarbonyl, trityl, tert-butyl, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl) shown in formula (d), means that: the compound shown in formula (d) can be one or more of 9-fluorenylmethoxycarbonyl-amino acid-tert-butyloxycarbonyl, 9-fluorenylmethoxycarbonyl-amino acid-trityl, 9-fluorenylmethoxycarbonyl-amino acid-tert-butyl, and 9-fluorenylmethoxycarbonyl-amino acid-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl.

[0069] In the present invention, the "amino acid group" in the 9-fluorenylmethoxycarbonyl-amino acid in step (2) can be selected from one or more of a glycine group, an alanine group, a valine group, a leucine group, an isoleucine group, a methionine group, a phenylalanine group, a serine group, a threonine group, a tyrosine group, an asparagine group, a glutamine group, an aspartic acid group, a glutamic acid group, a lysine group, an arginine group and a histidine group; preferably, selected from one or more of alanine group, valine group, leucine group, phenylalanine group, glutamic acid group, glutamine group and lysine group.

[0070] According to the present invention, preferably, m is 3-9 and n is 2-7; more preferably, m is 4 and n is 3;

[0071] According to the present invention, in formula (1) is a natural amino acid group; preferably, in formula (1) One or more selected from a glycine group, an alanine group, a valine group, a leucine group, an isoleucine group, a methionine group, a phenylalanine group, a serine group, a threonine group, a tyrosine group, an asparagine group, a glutamine group, an aspartic acid group, a glutamic acid group, a lysine group, an arginine group and a histidine group.

[0072] More preferably, in formula (1) One or more selected from alanine group, valine group, leucine group, phenylalanine group, glutamic acid group, glutamine group and lysine group.

[0073] According to the present invention, the activation treatment comprises: washing with N,N-dimethylformamide and dichloromethane in sequence, soaking in dichloromethane and N,N-dimethylformamide for 11-13 hours, filtering and drying to obtain the activated resin.

[0074] According to the present invention, in step (1), the amount of the activated resin is 3-6 parts by weight, the amount of 9-fluorenylmethoxycarbonyl-amino-polyethylene glycol-carboxylic acid shown in formula (b) is 1.4-2.8 parts by weight, the amount of dichloromethane is 40-80 parts by weight, the amount of N,N-diisopropylethylamine is 3.8-7.6 parts by weight, the amount of methanol is 23-46 parts by weight, and the amount of the mixture of piperidine and N,N-dimethylformamide is 30-60 parts by weight.

[0075] According to the present invention, in step (2), the amount of 9-fluorenylmethoxycarbonyl-amino acid or 9-fluorenylmethoxycarbonyl-amino acid-(Boc, Trt, tBu, Pbf) shown in formula (d) is 0.6-1.2 parts by weight, the amount of N,N-dimethylformamide is 28-56 parts by weight, the amount of ethyl 2-oximecyanoacetate is 0.27-0.54 parts by weight, the amount of N,N-diisopropylethylamine is 0.25-0.5 parts by weight, the amount of diisopropylcarbodiimide is 0.24-0.48 parts by weight, and the amount of the mixture of piperidine and N,N-dimethylformamide is 30-60 parts by weight.

[0076] According to the present invention, in step (3), the amount of 9-fluorenylmethoxycarbonyl-amino-polyethylene glycol-carboxylic acid represented by formula (b) is 0.9-1.8 parts by weight, the amount of N,N-dimethylformamide is 28-56 parts by weight, the amount of ethyl 2-oximecyanoacetate is 0.27-0.54 parts by weight, the amount of N,N-diisopropylethylamine is 0.25-0.5 parts by weight, the amount of diisopropylcarbodiimide is 0.24-0.48 parts by weight, and the amount of the mixture of piperidine and N,N-dimethylformamide is 30-60 parts by weight.

[0077] According to the present invention, in step (5), the total amount of 95% trifluoroacetic acid, 2.5% water and / or 2.5% thioanisole is 30-60 parts by weight.

[0078] According to the present invention, the conditions of the silica gel chromatography include: V (dichloromethane): V (methanol) = 20:1 or V (dichloromethane): V (methanol) = 2:1.

[0079] According to the present invention, in step (1), the first contact time is 11-13 hours; the second contact time is 20-40 minutes; and the third contact time is 1-3 hours.

[0080] According to the present invention, in step (2), the fourth contacting time is 11-13 hours; the fifth contacting time is 1-3 hours.

[0081] According to the present invention, in step (3), the sixth contact time is 11-13 hours; the seventh contact time is 1-3 hours.

[0082] According to the present invention, in step (5), the eighth contacting time is 1-3 hours.

[0083] The third aspect of the present invention provides a polyethylene glycol block polymer containing amino acids prepared by the aforementioned preparation method.

[0084] The fourth aspect of the present invention provides a use of the aforementioned amino acid-containing polyethylene glycol block polymer connected to the surface of a nano-drug.

[0085] According to the present invention, the nano drug comprises liposomes and / or micelles, preferably liposomes.

[0086] The present invention will be described in detail below through examples.

[0087] In the following examples and comparative examples:

[0088] The particle size and zeta potential parameters were measured by dynamic light scattering method;

[0089] Molecular weight test method: Waters liquid chromatography-mass spectrometry.

[0090] Room temperature refers to 25°C.

[0091] All reagents used in the synthesis process were purchased from Inotech.

[0092] In the present invention, "parts" means "parts by weight".

[0093] In the present invention, conventional methods in the prior art can be used to confirm the structural unit of each intermediate and polymer, such as nuclear magnetic resonance hydrogen spectrum and the amount of raw materials added during the synthesis process.

[0094] Preparation Example 1

[0095] This preparation example is to illustrate the 9-fluorenylmethoxycarbonyl-amino-polyethylene glycol-carboxylic acid whose terminal functional groups are 9-fluorenylmethoxycarbonyl and carboxyl prepared by the present invention.

[0096] Preparation of intermediate 1a:

[0097] Take a three-necked flask, add a rotor, 10 parts by weight of tetraethylene glycol, 78 parts by weight of acetonitrile (MeCN), 20 parts by weight of triethylamine (TEA) in sequence under argon protection, add 9 parts by weight of p-toluenesulfonyl chloride (TsCl) at 0°C, and stir at room temperature for 16 hours. After the reaction is completed, concentrate under reduced pressure, extract with dichloromethane (DCM) and dilute hydrochloric acid, dry the dichloromethane with anhydrous sodium sulfate, filter and concentrate. Silica gel chromatography [V (ethyl acetate): V (petroleum ether) = 5:1]. Distill under reduced pressure to obtain intermediate 1a;

[0098]

[0099] Preparation of intermediate 1b:

[0100] Take a single-mouth bottle, add a rotor, 10 parts by weight of intermediate 1a (10 g, 28.70 mmol), 98 parts by weight of N,N-dimethylformamide (DMF), and 2.2 parts by weight of sodium azide (NaN3) in sequence under argon protection, and stir at 40°C for 12 hours. The reaction solution is filtered through diatomaceous earth and concentrated under reduced pressure to obtain intermediate 1b;

[0101]

[0102] Preparation of intermediate 1c:

[0103] Take a three-necked flask, add a rotor, 4.4 parts by weight of sodium hydroxide (NaOH), 79.5 parts by weight of dichloromethane in sequence under argon protection, and then add 6 parts by weight of intermediate 1b and 10.6 parts by weight of tert-butyl bromoacetate (BrCH2CO2) dropwise at 0°C. t Bu), stirred at room temperature for 12 hours. The system was concentrated under reduced pressure, extracted with dichloromethane and water, dried over anhydrous sodium sulfate, filtered and concentrated, and chromatographed on silica gel [V (ethyl acetate): V (petroleum ether) = 1:3]. Distilled under reduced pressure to obtain intermediate 1c;

[0104]

[0105] Preparation of intermediate 1d:

[0106] Take a single-mouth bottle, add a rotor, 6 parts by weight of intermediate 1c, 52 parts by weight of toluene, 5.6 parts by weight of triphenylphosphine (Ph3P) at 0°C, and 1.4 parts by weight of water dropwise after 2 hours. Stir at room temperature for 10 hours. After the reaction is completed, the system is concentrated under reduced pressure, extracted with ether and water, the aqueous phase is concentrated under reduced pressure, chromatographed on neutral alumina [V (dichloromethane): V (methanol) = 20:1], and distilled under reduced pressure to obtain intermediate 1d;

[0107]

[0108] Preparation of intermediate 1e:

[0109] Take a single-mouth bottle, add a rotor, 4 parts by weight of intermediate 1d, 35 parts by weight of tetrahydrofuran (THF), 3.3 parts by weight of sodium bicarbonate (NaHCO3), 5 parts by weight of water, and then add 4 parts by weight of 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) at 0°C. Stir at room temperature for 12 hours. After the reaction is completed, concentrate the system under reduced pressure, dry the dichloromethane over anhydrous sodium sulfate, filter and concentrate. Silica gel chromatography [V (ethyl acetate): V (petroleum ether) = 5:1]. Distill under reduced pressure to obtain intermediate 1e;

[0110]

[0111] Preparation of 9-fluorenylmethoxycarbonyl-amino-polyethylene glycol-carboxylic acid:

[0112] Take a three-necked flask, add a rotor, 6 parts by weight of intermediate 1e, and 80 parts by weight of dichloromethane in sequence, then add 43 parts by weight of trifluoroacetic acid and 2 parts by weight of water dropwise at 0°C, and stir at room temperature for 8 hours. After the reaction is completed, concentrate the system under reduced pressure, dry the dichloromethane over anhydrous sodium sulfate, filter and concentrate. Silica gel chromatography [V (dichloromethane): V (methanol) = 20:1]. Distill under reduced pressure to obtain 9-fluorenylmethoxycarbonyl-amino-polyethylene glycol-carboxylic acid;

[0113]

[0114] Example 1

[0115] This example is to illustrate the preparation of polyethylene glycol block polymer containing alanine.

[0116] Step 1: Take the 2-chlorotrityl resin shown in formula (a), wash it with dichloromethane (DCM), N,N-dimethylformamide (DMF), and dichloromethane in sequence, and soak it in 50% dichloromethane / 50% N,N-dimethylformamide for 12 hours. Filter and dry to obtain the activated resin. Take a three-necked flask, add 1.4 parts by weight of 9-fluorenylmethoxycarbonyl-amino-polyethylene glycol-carboxylic acid prepared in Preparation Example 1, 40 parts by weight of dichloromethane, 3.8 parts by weight of N,N-diisopropylethylamine (DIPEA), and 3 parts by weight of activated resin, shake at room temperature for 12 hours, add 23 parts by weight of methanol ((Methanol)), wash the resin with dichloromethane after 30 minutes, add 30 parts by weight of 25% piperidine (Piperidine) / 75% N,N-dimethylformamide, shake at room temperature for 2 hours, and wash the resin with N,N-dimethylformamide and dichloromethane in sequence; the following compound is obtained:

[0117]

[0118] Step 2, add 0.6 parts by weight of 9-fluorenylmethoxycarbonyl-alanine, 28 parts by weight of N,N-dimethylformamide, 0.27 parts by weight of ethyl 2-oxime cyanoacetate (Oxyma), 0.25 parts by weight of N,N-diisopropylethylamine (DIEA), 0.24 parts by weight of diisopropylcarbodiimide (DIC is the abbreviation of N,N'-diisopropylcarbodiimide), shake at room temperature for 12 hours, wash the resin with N,N-dimethylformamide and dichloromethane in turn, add 30 parts by weight of 25% piperidine / 75% N,N-dimethylformamide, shake at room temperature for 2 hours, wash the resin with N,N-dimethylformamide and dichloromethane in turn; obtain the following compound:

[0119]

[0120] Step 3, add 0.9 parts by weight of 9-fluorenylmethoxycarbonyl-amino-polyethylene glycol-carboxylic acid prepared in Preparation Example 1, 28 parts by weight of N,N-dimethylformamide, 0.27 parts by weight of ethyl 2-oxime cyanoacetate, 0.25 parts by weight of N,N-diisopropylethylamine, 0.24 parts by weight of diisopropylcarbodiimide, shake at room temperature for 12 hours, wash the resin with N,N-dimethylformamide and dichloromethane in turn, add 30 parts by weight of 25% piperidine / 75% N,N-dimethylformamide, shake at room temperature for 2 hours, wash the resin with N,N-dimethylformamide and dichloromethane in turn; obtain the following compound:

[0121]

[0122] Step 4, repeat step 2 and step 3 twice in sequence; the following compound is obtained:

[0123]

[0124] Step 5, add 30 parts by weight of 95% trifluoroacetic acid (TFA) / 5% water, shake at room temperature for 1 hour, and wash the resin with dichloromethane. The washing liquid is chromatographed on silica gel [V (dichloromethane): V (methanol) = 20:1]. Reduced pressure distillation is performed to obtain alanine-blocked polyethylene glycol, and the measured average molecular weight is shown in Table 1;

[0125]

[0126] in addition, Figure 3 is the liquid phase-mass spectrum of the alanine-block polyethylene glycol prepared in Example 1 of the present invention, from Figure 3 It can be seen that the peak with a retention time of 4.608 minutes is the alanine-block polyethylene glycol UV peak, and the theoretical value is m / z: 1385.6953. Because it carries two positive charges and two NH3, the measured value is the molecular weight after halving m / z: 667.2545 [M+2NH3] + .

[0127] Figure 4 is the H NMR spectrum of the alanine-blocked polyethylene glycol prepared in Example 1 of the present invention. Figure 4It can be seen that: the peaks with chemical shifts of 7.69, 7.54, 7.33 and 7.24 ppm correspond to the hydrogen of the benzene ring; the peak with a chemical shift of 5.51-5.43 ppm corresponds to the hydrogen of the methylene on the Fmoc group; the peak with a chemical shift of 4.55-4.41 ppm corresponds to the hydrogen of the methylene on alanine; the peak with a chemical shift of 4.33 ppm corresponds to the hydrogen of the methylene on the Fmoc group; the peaks with chemical shifts of 4.19-3.88, 3.71-3.22, 2.89 and 2.83-2.80 ppm correspond to the hydrogen of the tetraethylene glycol main chain; the peak with a chemical shift of 1.36-1.26 ppm corresponds to the methyl hydrogen on alanine. 1 H NMR(400MHz,Chloroform-d)δ7.69(d,J=7.5,0.9Hz,2H),7.54(d,J=7.4Hz,2H),7.33(t,J=8.2,7.4,1.1Hz,2H),7.24(t,J=7.5,1.2Hz,2H),5.51- 5.43(m,1H),4.55-4.41(m,3H),4.33(d,J=6.9Hz,2H),4.19-3.88(m,8H) ,3.71-3.22(m,60H),2.89(s,2H),2.83-2.80(m,2H),1.36-1.26(m,9H).

[0128] Example 2

[0129] This example is to illustrate the preparation of polyethylene glycol block polymer containing valine.

[0130] Step 1, take the 2-chlorotrityl resin shown in formula (a), wash it with dichloromethane, N,N-dimethylformamide, and dichloromethane in sequence, and soak it in 50% dichloromethane / 50% N,N-dimethylformamide for 12 hours. Filter and dry to obtain the activated resin. Take a three-necked flask, add 1.4 parts by weight of 9-fluorenylmethoxycarbonyl-amino-polyethylene glycol-carboxylic acid prepared in Preparation Example 1, 40 parts by weight of dichloromethane, 3.8 parts by weight of N,N-diisopropylethylamine, and 3 parts by weight of activated resin, shake at room temperature for 12 hours, add 23 parts by weight of methanol, wash the resin with dichloromethane after 30 minutes, add 30 parts by weight of 25% piperidine / 75% N,N-dimethylformamide, shake at room temperature for 2 hours, and wash the resin with N,N-dimethylformamide and dichloromethane in sequence; the following compound is obtained:

[0131]

[0132] Step 2, add 0.64 parts by weight of 9-fluorenylmethoxycarbonyl-valine, 28 parts by weight of N,N-dimethylformamide, 0.27 parts by weight of ethyl 2-oxime cyanoacetate, 0.25 parts by weight of N,N-diisopropylethylamine, 0.24 parts by weight of diisopropylcarbodiimide, shake at room temperature for 12 hours, wash the resin with N,N-dimethylformamide and dichloromethane in turn, add 30 parts by weight of 25% piperidine / 75% N,N-dimethylformamide, shake at room temperature for 2 hours, wash the resin with N,N-dimethylformamide and dichloromethane in turn; obtain the following compound:

[0133]

[0134] Step 3, add 0.9 parts by weight of 9-fluorenylmethoxycarbonyl-amino-polyethylene glycol-carboxylic acid prepared in Preparation Example 1, 28 parts by weight of N,N-dimethylformamide, 0.27 parts by weight of ethyl 2-oxime cyanoacetate, 0.25 parts by weight of N,N-diisopropylethylamine, 0.24 parts by weight of diisopropylcarbodiimide, shake at room temperature for 12 hours, wash the resin with N,N-dimethylformamide and dichloromethane in turn, add 30 parts by weight of 25% piperidine / 75% N,N-dimethylformamide, shake at room temperature for 2 hours, wash the resin with N,N-dimethylformamide and dichloromethane in turn; obtain the following compound:

[0135]

[0136] Step 4, repeat step 2 and step 3 twice in sequence; the following compound is obtained:

[0137]

[0138] Step 5, add 30 parts by weight of 95% trifluoroacetic acid / 5% water, shake at room temperature for 1 hour, and wash the resin with dichloromethane. The washing liquid is chromatographed on silica gel [V (dichloromethane): V (methanol) = 20:1]. Vacuum distillation is performed to obtain valine-blocked polyethylene glycol, and the measured average molecular weight is shown in Table 1;

[0139]

[0140] Example 3

[0141] This example is intended to illustrate the preparation of a polyethylene glycol block polymer containing leucine.

[0142] Step 1, take the 2-chlorotrityl resin shown in formula (a), wash it with dichloromethane, N,N-dimethylformamide, and dichloromethane in sequence, and soak it in 50% dichloromethane / 50% N,N-dimethylformamide for 12 hours. Filter and dry to obtain the activated resin. Take a three-necked flask, add 1.4 parts by weight of 9-fluorenylmethoxycarbonyl-amino-polyethylene glycol-carboxylic acid prepared in Preparation Example 1, 40 parts by weight of dichloromethane, 3.8 parts by weight of N,N-diisopropylethylamine, and 3 parts by weight of activated resin, shake at room temperature for 12 hours, add 23 parts by weight of methanol, wash the resin with dichloromethane after 30 minutes, add 30 parts by weight of 25% piperidine / 75% N,N-dimethylformamide, shake at room temperature for 2 hours, and wash the resin with N,N-dimethylformamide and dichloromethane in sequence; the following compound is obtained:

[0143]

[0144] Step 2, add 0.66 parts by weight of 9-fluorenylmethoxycarbonyl-leucine, 28 parts by weight of N,N-dimethylformamide, 0.27 parts by weight of ethyl 2-oxime cyanoacetate, 0.25 parts by weight of N,N-diisopropylethylamine, 0.24 parts by weight of diisopropylcarbodiimide, shake at room temperature for 12 hours, wash the resin with N,N-dimethylformamide and dichloromethane in turn, add 30 parts by weight of 25% piperidine / 75% N,N-dimethylformamide, shake at room temperature for 2 hours, wash the resin with N,N-dimethylformamide and dichloromethane in turn; obtain the following compound:

[0145]

[0146] Step 3, add 0.9 parts by weight of 9-fluorenylmethoxycarbonyl-amino-polyethylene glycol-carboxylic acid prepared in Preparation Example 1, 28 parts by weight of N,N-dimethylformamide, 0.27 parts by weight of ethyl 2-oxime cyanoacetate, 0.25 parts by weight of N,N-diisopropylethylamine, 0.24 parts by weight of diisopropylcarbodiimide, shake at room temperature for 12 hours, wash the resin with N,N-dimethylformamide and dichloromethane in turn, add 30 parts by weight of 25% piperidine / 75% N,N-dimethylformamide, shake at room temperature for 2 hours, wash the resin with N,N-dimethylformamide and dichloromethane in turn; obtain the following compound:

[0147]

[0148] Step 4, repeat step 2 and step 3 twice in sequence; the following compound is obtained:

[0149]

[0150] Step 5, add 30 parts by weight of 95% trifluoroacetic acid / 5% water, shake at room temperature for 1 hour, and wash the resin with dichloromethane. The washing liquid is chromatographed on silica gel [V (dichloromethane): V (methanol) = 20:1]. Vacuum distillation is performed to obtain leucine-blocked polyethylene glycol, and the measured average molecular weight is shown in Table 1;

[0151]

[0152] Example 4

[0153] This example is intended to illustrate the preparation of a polyethylene glycol block polymer containing phenylalanine.

[0154] Step 1, take the 2-chlorotrityl resin shown in formula (a), wash it with dichloromethane, N,N-dimethylformamide, and dichloromethane in sequence, and soak it in 50% dichloromethane / 50% N,N-dimethylformamide for 12 hours. Filter and dry to obtain the activated resin. Take a three-necked flask, add 1.4 parts by weight of 9-fluorenylmethoxycarbonyl-amino-polyethylene glycol-carboxylic acid prepared in Preparation Example 1, 40 parts by weight of dichloromethane, 3.8 parts by weight of N,N-diisopropylethylamine, and 3 parts by weight of activated resin, shake at room temperature for 12 hours, add 23 parts by weight of methanol, wash the resin with dichloromethane after 30 minutes, add 30 parts by weight of 25% piperidine / 75% N,N-dimethylformamide, shake at room temperature for 2 hours, and wash the resin with N,N-dimethylformamide and dichloromethane in sequence; the following compound is obtained:

[0155]

[0156] Step 2, add 0.73 parts by weight of 9-fluorenylmethoxycarbonyl-phenylalanine, 28 parts by weight of N,N-dimethylformamide, 0.27 parts by weight of ethyl 2-oxime cyanoacetate, 0.25 parts by weight of N,N-diisopropylethylamine, 0.24 parts by weight of diisopropylcarbodiimide, shake at room temperature for 12 hours, wash the resin with N,N-dimethylformamide and dichloromethane in turn, add 30 parts by weight of 25% piperidine / 75% N,N-dimethylformamide, shake at room temperature for 2 hours, wash the resin with N,N-dimethylformamide and dichloromethane in turn; obtain the following compound:

[0157]

[0158] Step 3, add 0.9 parts by weight of 9-fluorenylmethoxycarbonyl-amino-polyethylene glycol-carboxylic acid prepared in Preparation Example 1, 28 parts by weight of N,N-dimethylformamide, 0.27 parts by weight of ethyl 2-oxime cyanoacetate, 0.25 parts by weight of N,N-diisopropylethylamine, 0.24 parts by weight of diisopropylcarbodiimide, shake at room temperature for 12 hours, wash the resin with N,N-dimethylformamide and dichloromethane in turn, add 30 parts by weight of 25% piperidine / 75% N,N-dimethylformamide, shake at room temperature for 2 hours, wash the resin with N,N-dimethylformamide and dichloromethane in turn; obtain the following compound:

[0159]

[0160] Step 4, repeat step 2 and step 3 twice in sequence; the following compound is obtained:

[0161]

[0162] Step 5, add 30 parts by weight of 95% trifluoroacetic acid / 5% water, shake at room temperature for 1 hour, and wash the resin with dichloromethane. The washing liquid is chromatographed on silica gel [V (dichloromethane): V (methanol) = 20:1]. Reduced pressure distillation is performed to obtain phenylalanine-blocked polyethylene glycol, and the measured average molecular weight is shown in Table 1;

[0163]

[0164] Example 5

[0165] This example is to illustrate the preparation of polyethylene glycol block polymer containing glutamic acid.

[0166] Step 1, take the 2-chlorotrityl resin shown in formula (a), wash it with dichloromethane, N,N-dimethylformamide, and dichloromethane in sequence, and soak it in 50% dichloromethane / 50% N,N-dimethylformamide for 12 hours. Filter and dry to obtain the activated resin. Take a three-necked flask, add 1.4 parts by weight of 9-fluorenylmethoxycarbonyl-amino-polyethylene glycol-carboxylic acid prepared in Preparation Example 1, 40 parts by weight of dichloromethane, 3.8 parts by weight of N,N-diisopropylethylamine, and 3 parts by weight of activated resin, shake at room temperature for 12 hours, add 23 parts by weight of methanol, wash the resin with dichloromethane after 30 minutes, add 30 parts by weight of 25% piperidine / 75% N,N-dimethylformamide, shake at room temperature for 2 hours, and wash the resin with N,N-dimethylformamide and dichloromethane in sequence; the following compound is obtained:

[0167]

[0168] Step 2, add 0.8 parts by weight of 9-fluorenylmethoxycarbonyl-glutamic acid-tert-butyl, 28 parts by weight of N,N-dimethylformamide, 0.27 parts by weight of ethyl 2-oxime cyanoacetate, 0.25 parts by weight of N,N-diisopropylethylamine, 0.24 parts by weight of diisopropylcarbodiimide, shake at room temperature for 12 hours, wash the resin with N,N-dimethylformamide and dichloromethane in turn, add 30 parts by weight of 25% piperidine / 75% N,N-dimethylformamide, shake at room temperature for 2 hours, wash the resin with N,N-dimethylformamide and dichloromethane in turn; obtain the following compound:

[0169]

[0170] Step 3, add 0.9 parts by weight of 9-fluorenylmethoxycarbonyl-amino-polyethylene glycol-carboxylic acid prepared in Preparation Example 1, 28 parts by weight of N,N-dimethylformamide, 0.27 parts by weight of ethyl 2-oxime cyanoacetate, 0.25 parts by weight of N,N-diisopropylethylamine, 0.24 parts by weight of diisopropylcarbodiimide, shake at room temperature for 12 hours, wash the resin with N,N-dimethylformamide and dichloromethane in turn, add 30 parts by weight of 25% piperidine / 75% N,N-dimethylformamide, shake at room temperature for 2 hours, wash the resin with N,N-dimethylformamide and dichloromethane in turn; obtain the following compound:

[0171]

[0172] Step 4, repeat step 2 and step 3 twice in sequence; the following compound is obtained:

[0173]

[0174] Step 5, add 30 parts by weight of 95% trifluoroacetic acid / 2.5% water / 2.5% thioanisole, shake at room temperature for 1 hour, and wash the resin with dichloromethane. The washing liquid is chromatographed on silica gel [V (dichloromethane): V (methanol) = 20:1]. Vacuum distillation is performed to obtain glutamic acid-blocked polyethylene glycol, and the measured average molecular weight is shown in Table 1;

[0175]

[0176] Example 6

[0177] This example is to illustrate the preparation of polyethylene glycol block polymer containing glutamine.

[0178] Step 1, take the 2-chlorotrityl resin shown in formula (a), wash it with dichloromethane, N,N-dimethylformamide, and dichloromethane in sequence, and soak it in 50% dichloromethane / 50% N,N-dimethylformamide for 12 hours. Filter and dry to obtain the activated resin. Take a three-necked flask, add 1.4 parts by weight of 9-fluorenylmethoxycarbonyl-amino-polyethylene glycol-carboxylic acid prepared in Preparation Example 1, 40 parts by weight of dichloromethane, 3.8 parts by weight of N,N-diisopropylethylamine, and 3 parts by weight of activated resin, shake at room temperature for 12 hours, add 23 parts by weight of methanol, wash the resin with dichloromethane after 30 minutes, add 30 parts by weight of 25% piperidine / 75% N,N-dimethylformamide, shake at room temperature for 2 hours, and wash the resin with N,N-dimethylformamide and dichloromethane in sequence; the following compound is obtained:

[0179]

[0180] Step 2, add 1.2 parts by weight of 9-fluorenylmethoxycarbonyl-glutamic acid-trityl, 28 parts by weight of N,N-dimethylformamide, 0.27 parts by weight of ethyl 2-oxime cyanoacetate, 0.25 parts by weight of N,N-diisopropylethylamine, 0.24 parts by weight of diisopropylcarbodiimide, shake at room temperature for 12 hours, wash the resin with N,N-dimethylformamide and dichloromethane in turn, add 30 parts by weight of 25% piperidine / 75% N,N-dimethylformamide, shake at room temperature for 2 hours, wash the resin with N,N-dimethylformamide and dichloromethane in turn; obtain the following compound:

[0181]

[0182] Step 3, add 0.9 parts by weight of 9-fluorenylmethoxycarbonyl-amino-polyethylene glycol-carboxylic acid prepared in Preparation Example 1, 28 parts by weight of N,N-dimethylformamide, 0.27 parts by weight of ethyl 2-oxime cyanoacetate, 0.25 parts by weight of N,N-diisopropylethylamine, 0.24 parts by weight of diisopropylcarbodiimide, shake at room temperature for 12 hours, wash the resin with N,N-dimethylformamide and dichloromethane in turn, add 30 parts by weight of 25% piperidine / 75% N,N-dimethylformamide, shake at room temperature for 2 hours, wash the resin with N,N-dimethylformamide and dichloromethane in turn; obtain the following compound:

[0183]

[0184] Step 4, repeat step 2 and step 3 twice in sequence; the following compound is obtained:

[0185]

[0186] Step 5, add 30 parts by weight of 95% trifluoroacetic acid / 2.5% water / 2.5% thioanisole, shake at room temperature for 1 hour, and wash the resin with dichloromethane. The washing liquid is chromatographed on silica gel [V (dichloromethane): V (methanol) = 20:1]. Vacuum distillation is performed to obtain glutamine-block polyethylene glycol, and the measured average molecular weight is shown in Table 1;

[0187]

[0188] Example 7

[0189] This example is intended to illustrate the preparation of a polyethylene glycol block polymer containing lysine.

[0190] Step 1, take the 2-chlorotrityl resin shown in formula (a), wash it with dichloromethane, N,N-dimethylformamide, and dichloromethane in sequence, and soak it in 50% dichloromethane / 50% N,N-dimethylformamide for 12 hours. Filter and dry to obtain the activated resin. Take a three-necked flask, add 1.4 parts by weight of 9-fluorenylmethoxycarbonyl-amino-polyethylene glycol-carboxylic acid prepared in Preparation Example 1, 40 parts by weight of dichloromethane, 3.8 parts by weight of N,N-diisopropylethylamine, and 3 parts by weight of activated resin, shake at room temperature for 12 hours, add 23 parts by weight of methanol, wash the resin with dichloromethane after 30 minutes, add 30 parts by weight of 25% piperidine / 75% N,N-dimethylformamide, shake at room temperature for 2 hours, and wash the resin with N,N-dimethylformamide and dichloromethane in sequence; the following compound is obtained:

[0191]

[0192] Step 2, add 1.2 parts by weight of 9-fluorenylmethoxycarbonyl-lysine-tert-butyloxycarbonyl, 28 parts by weight of N,N-dimethylformamide, 0.27 parts by weight of ethyl 2-oximecyanoacetate, 0.25 parts by weight of N,N-diisopropylethylamine, 0.24 parts by weight of diisopropylcarbodiimide, shake at room temperature for 12 hours, wash the resin with N,N-dimethylformamide and dichloromethane in turn, add 30 parts by weight of 25% piperidine / 75% N,N-dimethylformamide, shake at room temperature for 2 hours, wash the resin with N,N-dimethylformamide and dichloromethane in turn; obtain the following compound:

[0193]

[0194] Step 3, add 0.9 parts by weight of 9-fluorenylmethoxycarbonyl-amino-polyethylene glycol-carboxylic acid prepared in Preparation Example 1, 28 parts by weight of N,N-dimethylformamide, 0.27 parts by weight of ethyl 2-oxime cyanoacetate, 0.25 parts by weight of N,N-diisopropylethylamine, 0.24 parts by weight of diisopropylcarbodiimide, shake at room temperature for 12 hours, wash the resin with N,N-dimethylformamide and dichloromethane in turn, add 30 parts by weight of 25% piperidine / 75% N,N-dimethylformamide, shake at room temperature for 2 hours, wash the resin with N,N-dimethylformamide and dichloromethane in turn; obtain the following compound:

[0195]

[0196] Step 4, repeat step 2 and step 3 twice in sequence; the following compound is obtained:

[0197]

[0198] Step 5, add 30 parts by weight of 95% trifluoroacetic acid / 2.5% water / 2.5% thioanisole, shake at room temperature for 1 hour, and wash the resin with dichloromethane. The washing liquid is chromatographed on silica gel [V (dichloromethane): V (methanol) = 2:1]. Reduced pressure distillation is performed to obtain lysinamide-block polyethylene glycol, and the measured average molecular weight is shown in Table 1;

[0199]

[0200] Comparative Example 1

[0201] This comparative example 1 is to illustrate the preparation of a polyethylene glycol block polymer with non-uniform molecular weight and containing alanine.

[0202] Step 1, replace the "tetraethylene glycol" in Preparation Example 1 with "polyethylene glycol with an average molecular weight of 600", and use the method of Preparation Example 1 to prepare 9-fluorenylmethoxycarbonyl-amino-polyethylene glycol 600-carboxylic acid.

[0203] Step 2: Replace "9-fluorenylmethoxycarbonyl-amino-polyethylene glycol-carboxylic acid" in Example 1 with "9-fluorenylmethoxycarbonyl-amino-polyethylene glycol 600-carboxylic acid" and prepare alanine-containing polyethylene glycol 600 block polymer using the method of Preparation Example 1. The measured average molecular weight is shown in Table 1.

[0204] Comparative Example 2

[0205] This comparative example 2 is intended to illustrate the preparation of a polymer without "amino acid blocks".

[0206] Step 1, take 2-chlorotrityl resin, wash with dichloromethane, N, N-dimethylformamide, and dichloromethane in turn, and soak in 50% dichloromethane / 50% N, N-dimethylformamide for 12 hours. Filter and dry to obtain activated resin. Take a three-necked flask, add 1.4 parts by weight of 9-fluorenylmethoxycarbonyl-amino-polyethylene glycol-carboxylic acid prepared in Preparation Example 1, 40 parts by weight of dichloromethane, 3.8 parts by weight of N, N-diisopropylethylamine, and 3 parts by weight of activated resin, shake at room temperature for 12 hours, add 23 parts by weight of methanol, wash the resin with dichloromethane after 30 minutes, add 30 parts by weight of 25% piperidine / 75% N, N-dimethylformamide, shake at room temperature for 2 hours, and wash the resin with N, N-dimethylformamide and dichloromethane in turn.

[0207]

[0208] Step 2, add 1.2 parts by weight of 9-fluorenylmethoxycarbonyl-amino-polyethylene glycol-carboxylic acid prepared in Preparation Example 1, 28 parts by weight of N,N-dimethylformamide, 0.27 parts by weight of ethyl 2-oxime cyanoacetate, 0.25 parts by weight of N,N-diisopropylethylamine, and 0.24 parts by weight of diisopropylcarbodiimide, shake at room temperature for 12 hours, wash the resin with N,N-dimethylformamide and dichloromethane in turn, add 30 parts by weight of 25% piperidine / 75% N,N-dimethylformamide, shake at room temperature for 2 hours, and wash the resin with N,N-dimethylformamide and dichloromethane in turn.

[0209]

[0210] Step 3: Repeat step 2 5 times;

[0211]

[0212] Step 4: Add 30 parts by weight of 95% trifluoroacetic acid / 2.5% water, shake at room temperature for 1 hour, and wash the resin with dichloromethane. The washing liquid is chromatographed on silica gel [V (dichloromethane): V (methanol) = 20:1]. Then, distill under reduced pressure to obtain polyethylene glycol without "amino acid block". The measured number average molecular weight is shown in Table 1.

[0213]

[0214] Table 1

[0215] project Molecular weight Example 1 Number average molecular weight 1358 Example 2 Number average molecular weight 1470 Example 3 Number average molecular weight 1510 Example 4 Number average molecular weight 1613 Example 5 Number average molecular weight 1560 Example 6 Number average molecular weight 1556 Example 7 Number average molecular weight 1557 Comparative Example 1 Average molecular weight 2982 Comparative Example 2 Number average molecular weight 1664

[0216] Using dihydroxytetraethylene glycol as the raw material, the end group is selectively modified to obtain the important reaction block in solid phase synthesis: 9-fluorenylmethoxycarbonyl-amino-polyethylene glycol-carboxylic acid. Using 9-fluorenylmethoxycarbonyl-amino-polyethylene glycol-carboxylic acid as the reaction block, amino acids are inserted into it, and alanine block polyethylene glycol, valine block polyethylene glycol, leucine block polyethylene glycol, phenylalanine block polyethylene glycol, glutamic acid block polyethylene glycol, glutamine block polyethylene glycol, and lysine block polyethylene glycol are prepared by solid phase synthesis.

[0217] In addition, the tetraethylene glycol in Preparation Example 1 was replaced with polyethylene glycol with an average molecular weight of 600, and 9-fluorenylmethoxycarbonyl-amino-polyethylene glycol 600-carboxylic acid was prepared by the method of Preparation Example 1. 9-fluorenylmethoxycarbonyl-amino-polyethylene glycol 600-carboxylic acid and alanine were used as reaction blocks to prepare the polyethylene glycol 600 block polymer containing alanine in Comparative Example 1.

[0218] In addition, polyethylene glycol without "amino acid block" of Comparative Example 2 was prepared by using 9-fluorenylmethoxycarbonyl-amino-polyethylene glycol-carboxylic acid as a reaction block without inserting amino acids.

[0219] As can be seen from the above table, the molecular weight of each embodiment is different, which is mainly caused by the different molecular weights of amino acids; the molecular weight of each embodiment is single, each unit is connected by an amide bond, and the charged groups (-NH2 and -COOH) of the amino acid side chains greatly enhance their own rigidity through electrostatic repulsion, which is expected to solve the problem that flexible and long and short PEGs are entangled with each other, and the targeting of the PEG terminal ligands is reduced. Since the molecular weight of polyethylene glycol 600 is dispersed, the molecular weight of comparative example 1 is dispersed, and its terminal modified ligands and connected to the surface of nanoparticles may cause the targeting to be reduced. Comparative example 2 does not contain amino acids, and its own rigidity cannot be enhanced by electrostatic repulsion, so there is a problem of low targeting of the terminal ligands.

[0220] Example 8

[0221] This example is to illustrate the preparation of alanine-block polyethylene glycol-distearoylphosphatidylethanolamine.

[0222] Alanine has a simple structure and will not increase the complexity of the reaction system and post-treatment. Therefore, the polymer containing alanine-block polyethylene glycol prepared in Example 1 is selected to synthesize alanine-block polyethylene glycol-distearoylphosphatidylethanolamine.

[0223] Specifically:

[0224] Take a three-necked flask, add 2.3 parts by weight of the polymer containing alanine-blocked polyethylene glycol prepared in Example 1, 30 parts by weight of dichloromethane, 0.35 parts by weight of N-hydroxysuccinimide (NHS), and 0.58 parts by weight of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride under argon protection, and stir at room temperature for 12 hours. After the reaction is completed, extract, dry and filter the organic phase, and spin dry to obtain alanine-blocked polyethylene glycol-succinimide ester.

[0225]

[0226] Take a three-necked flask, add 2.7 parts by weight of alanine-block polyethylene glycol-succinimide ester, 40 parts by weight of dichloromethane, 1.7 parts by weight of distearoylphosphatidylethanolamine (DSPE), and 0.9 parts by weight of triethylamine under argon protection, and stir at room temperature for 12 hours. Silica gel chromatography [V (dichloromethane): V (methanol) = 20: 1] is performed to obtain alanine-block polyethylene glycol-distearoylphosphatidylethanolamine.

[0227]

[0228] In addition, the method according to Example 8 is different in that:

[0229] 2.49 parts by weight of the valine-containing polyethylene glycol block polymer prepared in Example 2 was used to synthesize valine block polyethylene glycol-distearoylphosphatidylethanolamine.

[0230] 2.56 parts by weight of the leucine-containing polyethylene glycol block polymer prepared in Example 3 was used to synthesize leucine block polyethylene glycol-distearoylphosphatidylethanolamine.

[0231] 2.73 parts by weight of the polyethylene glycol block polymer containing phenylalanine prepared in Example 4 was used to synthesize phenylalanine block polyethylene glycol-distearoylphosphatidylethanolamine.

[0232] 2.64 parts by weight of the polyethylene glycol block polymer containing glutamic acid prepared in Example 5 was used to synthesize glutamic acid block polyethylene glycol-distearoylphosphatidylethanolamine.

[0233] 2.64 parts by weight of the glutamine-containing polyethylene glycol block polymer prepared in Example 6 was used to synthesize glutamine block polyethylene glycol-distearoylphosphatidylethanolamine.

[0234] 2.64 parts by weight of the lysine-containing polyethylene glycol block polymer prepared in Example 7 was used to synthesize lysine block polyethylene glycol-distearoylphosphatidylethanolamine.

[0235] 5 parts by weight of the polyethylene glycol 600 block polymer containing alanine prepared in Comparative Example 1 was used to synthesize alanine block polyethylene glycol 600-distearoylphosphatidylethanolamine.

[0236] 2.74 parts by weight of the polyethylene glycol without "amino acid block" prepared in Comparative Example 2 was used to synthesize polyethylene glycol-distearoylphosphatidylethanolamine without "amino acid block".

[0237] Example 9

[0238] This example is to illustrate the preparation of alanine-block PEGylated liposomes.

[0239] (1) The thin film dispersion method was used to prepare liposomes. First, the three lipid materials of alanine-block polyethylene glycol-distearoylphosphatidylethanolamine, egg yolk phosphatidylcholine and cholesterol prepared in Example 8 were accurately weighed in a molar ratio of 20:40:40.

[0240] (2) Then, use an appropriate amount of anhydrous ethanol to dissolve the three lipid materials respectively, and then pour the dissolved three lipid materials into the same round-bottom bottle. Ultrasonicate the round-bottom bottle in an ice-water bath to completely dissolve the contents of the round-bottom bottle.

[0241] (3) Using a rotary evaporator to evaporate the solvent in the flask under reduced pressure in the dark to remove the anhydrous ethanol, a uniform thin film is formed on the inner wall of the round-bottom flask after rotary evaporation.

[0242] (4) Anhydrous ethanol is then added to the round-bottom bottle to dissolve the film. Water-soluble drugs or dyes can be added as needed. After the contents of the bottle are completely dissolved by ultrasonication in an ice-water bath, the ethanol in the bottle is removed by rotary evaporation in a dark place to form a uniform film on the inner wall of the bottle. An appropriate amount of physiological saline is then added to dissolve the film.

[0243] (5) The reconstituted liquid is filtered through a 0.22 μm filter membrane and a 0.15 μm filter membrane respectively, and the filtering through each filter membrane is repeated three times to obtain a suspension solution containing alanine-block PEGylated liposomes.

[0244] In addition, the method of Example 9 is followed, except that the "alanine-block polyethylene glycol-distearoylphosphatidylethanolamine synthesized in Example 1" is replaced, specifically:

[0245] The residue was replaced with “valine-block polyethylene glycol-distearoylphosphatidylethanolamine synthesized in Example 2”, and as a result, a suspension solution containing alanine-block PEGylated liposomes was prepared.

[0246] The residue was replaced with “leucine-block polyethylene glycol-distearoylphosphatidylethanolamine synthesized in Example 3”, and as a result, a suspension solution containing leucine-block polyethylene glycol-distearoylphosphatidylethanolamine was prepared.

[0247] The residue was replaced with “phenylalanine-block polyethylene glycol-distearoylphosphatidylethanolamine synthesized in Example 4”, and as a result, a suspension solution containing phenylalanine-block polyethylene glycol-distearoylphosphatidylethanolamine was prepared.

[0248] The residue was replaced with “glutamic acid block polyethylene glycol-distearoylphosphatidylethanolamine synthesized in Example 5”, and as a result, a suspension solution of glutamic acid block polyethylene glycol liposomes was prepared.

[0249] The residue was replaced with "glutamine-block polyethylene glycol-distearoylphosphatidylethanolamine synthesized in Example 6", and a suspension solution of glutamine-block polyethylene glycol liposomes was prepared.

[0250] The above was replaced with "lysine-block polyethylene glycol-distearoylphosphatidylethanolamine synthesized in Example 7", and as a result, a suspension solution of lysine-block polyethylene glycol liposomes was prepared.

[0251] The mixture was replaced with "alanine-block polyethylene glycol 600-distearoylphosphatidylethanolamine synthesized in Comparative Example 1", and a suspension solution of amide-bonded PEGylated liposomes was prepared.

[0252] The PEG-distearoylphosphatidylethanolamine without "amino acid block" synthesized in Comparative Example 2 was used to replace the PEG-distearoylphosphatidylethanolamine. As a result, a suspension solution of PEGylated liposomes without "amino acid block" was prepared.

[0253] Test Example 1

[0254] Example 9 Particle size potential measurement of alanine-block PEGylated liposomes prepared

[0255] The suspension of alanine-blocked PEGylated liposomes was diluted with physiological saline, and after being diluted to an appropriate concentration, 1 mL of the suspension of liposomes was taken into a cuvette, and the average particle size and Zeta potential of the alanine-blocked PEGylated liposomes in the cuvette were measured with a laser particle size meter at room temperature. Based on the measured data, the average particle size and stability of the liposomes were analyzed to see whether they met the requirements.

[0256] The particle size and Zeta potential of the prepared alanine-block PEGylated liposomes were measured using a laser particle size analyzer.

[0257] The results are as follows Figure 1 The particle size distribution of alanine-block PEGylated liposomes prepared in Example 9 of the present invention is shown in the schematic diagram. Figure 1 It can be seen that the average particle size of liposomes is 128.9 nm.

[0258] The results are as follows Figure 2The schematic diagram of the Zeta potential of the alanine-block PEGylated liposome prepared in Example 9 of the present invention shows that the Zeta potential is -24.6 mV. From the data, it can be seen that the liposome particle size is small, the stability is high, and the system is relatively stable.

[0259] Test Example 2

[0260] According to the test method of Test Example 1, the liposomes prepared in Examples 2-7 and Comparative Examples 1-2 were tested. The results are shown in Table 2.

[0261] Table 2

[0262] project Average particle size of liposomes (nm) Zeta potential of liposomes (mV) Example 1 128.9 -24.6 Example 2 112.8 -22.2 Example 3 123.4 -25.7 Example 4 135.8 -20.5 Example 5 113.4 -101.7 Example 6 101.4 10.2 Example 7 106.5 105.4 Comparative Example 1 176.8 -20.4 Comparative Example 2 76.6 -22.7

[0263] It can be seen from Table 2 that the liposomes prepared in Examples 1-7 have a smaller particle size, high stability and a more stable system. The glutamic acid block polyethylene glycol-distearoylphosphatidylethanolamine synthesized in Example 5 has a relatively strong negatively charged carboxyl group in its structure, and the liposomes prepared therefrom have a relatively small Zeta potential. The lysine block polyethylene glycol-distearoylphosphatidylethanolamine synthesized in Example 7 has a relatively strong positively charged amino group in its structure, and the liposomes prepared therefrom have a relatively large Zeta potential. The alanine block polyethylene glycol 600-distearoylphosphatidylethanolamine synthesized in Comparative Example 1 has an uneven molecular weight, and the liposomes prepared therefrom have a larger particle size and poor stability. The PEGylated liposomes without "amino acid blocks" synthesized in Comparative Example 2 have a smaller particle size, which may be caused by the lack of amino acids in its structure.

[0264] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A polyethylene glycol block polymer containing an amino acid, characterized in that: The polymer has the general structural formula shown in formula (1); Among them, Fmoc is m is 2-10, n is 1-8; R is selected from one or more of H, -CH3, -CH-(CH3)2, -CH2-CH(CH3)2, -CH-(CH3)-CH2-CH3, -(CH2)2-S-CH3, -CH2-C6H5, -CH2-OH, -CH2-CH3-OH, -CH2-C6H4-OH, -CH2-CONH2, -(CH2)2-CONH2, -CH2-COOH, -(CH2)2-COOH, -(CH2)4-NH2, -(CH2)3-NHC(NH)NH2 and -CH2-C3H3N2.

2. The polymer according to claim 1, wherein m is 3-9, and n is 2-7; preferably, m is 4, and n is 3.

3. The polymer according to claim 1 or 2, wherein In formula (1) One or more selected from the group consisting of a glycine group, an alanine group, a valine group, a leucine group, an isoleucine group, a methionine group, a phenylalanine group, a serine group, a threonine group, a tyrosine group, an asparagine group, a glutamine group, an aspartic acid group, a glutamic acid group, a lysine group, an arginine group and a histidine group; Preferably, in formula (1) One or more selected from alanine group, valine group, leucine group, phenylalanine group, glutamic acid group, glutamine group and lysine group.

4. The polymer according to any one of claims 1 to 3, wherein The number average molecular weight of the polymer is 580-6000, preferably 1385-1613.

5. The polymer according to any one of claims 1 to 4, wherein The polymer comprises one or more of formula (2) to formula (8); 6. A method for preparing a polyethylene glycol block polymer containing amino acids, characterized in that: The preparation method comprises: (1) at room temperature, activating the 2-chlorotrityl resin of formula (a) to obtain an activated resin; subjecting the activated resin, 9-fluorenylmethoxycarbonyl-amino-polyethylene glycol-carboxylic acid of formula (b), dichloromethane, and N,N-diisopropylethylamine to a first contact and shaking treatment, and then contacting with methanol for a second time; and then contacting with a mixture of piperidine and N,N-dimethylformamide for a third contact and shaking treatment; obtaining a compound of formula (c); Wherein, m is 2-10; (2) treating the compound represented by formula (c), 9-fluorenylmethoxycarbonyl-amino acid or 9-fluorenylmethoxycarbonyl-amino acid-(Boc, Trt, tBu, Pbf) represented by formula (d), N,N-dimethylformamide, ethyl 2-oxime cyanoacetate, N,N-diisopropylethylamine, and diisopropylcarbodiimide at room temperature for the fourth contact and shaking treatment; washing with N,N-dimethylformamide and dichloromethane in sequence, and then contacting and shaking with a mixture of piperidine and N,N-dimethylformamide for the fifth time; and then washing with N,N-dimethylformamide and dichloromethane in sequence; obtaining the compound represented by formula (e); wherein R1 is selected from one or more of H, -CH3, -CH-(CH3)2, -CH2-CH(CH3)2, -CH-(CH3)-CH2-CH3, -(CH2)2-S-CH3, -CH2-C6H5, -CH2-OH, -CH2-CH3-OH, -CH2-C6H4-OH, -CH2-CONH2, -(CH2)2-CONH2, -CH2-COOH, -(CH2)2-COOH, -(CH2)4-NH2, -(CH2)3-NHC(NH)NH2, and -CH2-C3H3N2; (3) treating the compound represented by formula (e), 9-fluorenylmethoxycarbonyl-amino-polyethylene glycol-carboxylic acid represented by formula (b), N,N-dimethylformamide, ethyl 2-oxime cyanoacetate, N,N-diisopropylethylamine, and diisopropylcarbodiimide by contacting and shaking for the sixth time; washing with N,N-dimethylformamide and dichloromethane in sequence, and then contacting and shaking with a mixture of piperidine and N,N-dimethylformamide for the seventh time; and then washing with N,N-dimethylformamide and dichloromethane in sequence to obtain the compound represented by formula (f); (4) Repeat steps (2) and (3) to obtain a compound represented by formula (g); Where n is 1-8; (5) treating the compound represented by formula (g) with trifluoroacetic acid, water and / or thioanisole by shaking for the eighth time, washing with dichloromethane, subjecting the washing liquid to silica gel chromatography, and distilling under reduced pressure to obtain the amino acid-containing polyethylene glycol block polymer represented by formula (1); Fmoc 7. The preparation method according to claim 6, wherein: m is 3-9, n is 2-7; preferably, m is 4, n is 3; And / or, in formula (1) One or more selected from the group consisting of a glycine group, an alanine group, a valine group, a leucine group, an isoleucine group, a methionine group, a phenylalanine group, a serine group, a threonine group, a tyrosine group, an asparagine group, a glutamine group, an aspartic acid group, a glutamic acid group, a lysine group, an arginine group and a histidine group; Preferably, in formula (1) One or more selected from alanine group, valine group, leucine group, phenylalanine group, glutamic acid group, glutamine group and lysine group.

8. The preparation method according to claim 6, wherein: The activation treatment comprises: washing the 2-chlorotrityl resin with N,N-dimethylformamide and dichloromethane in sequence, and then soaking it in dichloromethane and N,N-dimethylformamide for 11-13 hours, filtering it, and drying it to obtain the activated resin; And / or, in step (1), the amount of the activated resin is 3-6 parts by weight, the amount of 9-fluorenylmethoxycarbonyl-amino-polyethylene glycol-carboxylic acid represented by formula (b) is 1.4-2.8 parts by weight, the amount of dichloromethane is 40-80 parts by weight, the amount of N,N-diisopropylethylamine is 3.8-7.6 parts by weight, the amount of methanol is 23-46 parts by weight, and the amount of the mixture of piperidine and N,N-dimethylformamide is 30-60 parts by weight; and / or, in step (2), the amount of 9-fluorenylmethoxycarbonyl-amino acid or 9-fluorenylmethoxycarbonyl-amino acid-(Boc, Trt, tBu, Pbf) represented by formula (d) is 0.6-1.2 parts by weight, the amount of N,N-dimethylformamide is 28-56 parts by weight, the amount of ethyl 2-oximecyanoacetate is 0.27-0.54 parts by weight, the amount of N,N-diisopropylethylamine is 0.25-0.5 parts by weight, the amount of diisopropylcarbodiimide is 0.24-0.48 parts by weight, and the amount of the mixture of piperidine and N,N-dimethylformamide is 30-60 parts by weight; and / or, in step (3), the amount of 9-fluorenylmethoxycarbonyl-amino-polyethylene glycol-carboxylic acid represented by formula (b) is 0.9-1.8 parts by weight, the amount of N,N-dimethylformamide is 28-56 parts by weight, the amount of ethyl 2-oximecyanoacetate is 0.27-0.54 parts by weight, the amount of N,N-diisopropylethylamine is 0.25-0.5 parts by weight, the amount of diisopropylcarbodiimide is 0.24-0.48 parts by weight, and the amount of the mixture of piperidine and N,N-dimethylformamide is 30-60 parts by weight; And / or, in step (5), the total amount of 95% trifluoroacetic acid, 2.5% thioanisole and / or 2.5% water is 30-60 parts by weight.

9. The preparation method according to claim 6, wherein: In step (1), the first contact time is 11-13 hours; the second contact time is 20-40 minutes; the third contact time is 1-3 hours; And / or, in step (2), the fourth contacting time is 11-13 hours; the fifth contacting time is 1-3 hours; And / or, in step (3), the sixth contact time is 11-13 hours; the seventh contact time is 1-3 hours; And / or, in step (5), the eighth contacting time is 1-3 hours.

10. A polyethylene glycol block polymer containing amino acids prepared by the preparation method according to any one of claims 6 to 9.

11. Use of the polyethylene glycol block polymer containing amino acids according to any one of claims 1 to 5 and 10 attached to the surface of a nano drug.

12. The use according to claim 11, wherein: The nano drug comprises liposomes and / or micelles, preferably liposomes.

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