阅读说明：本技术 一种果胶-阿霉素轭合物及其中间体的制备方法 (Preparation method of pectin-adriamycin conjugate and intermediate thereof ) 是由 唐小海 陈锞 于 2020-12-09 设计创作，主要内容包括：本发明涉及一种果胶-阿霉素轭合物及其中间体的制备方法。本发明的技术方案通过对果胶-阿霉素轭合物重要中间体的合成的整体路线进行重新设计,提高了合成该中间体小分子化合物的纯度和总收率,具有收率高、后处理简便易纯化的优点；同时节约了两个昂贵的重要原料Fmoc-NH-PEG-8-COOH与阿霉素的用量,减少了其它试剂的耗费,大大降低了成本,提高效率,减少了废料的生成,利于环保,适合大规模生产。(The invention relates to a pectin-adriamycin conjugate and a preparation method of an intermediate thereof. The technical scheme of the invention improves the purity and the total yield of the intermediate micromolecule compound by redesigning the integral route of the synthesis of the important intermediate of the pectin-adriamycin conjugate, and has the advantages of high yield, simple and convenient post-treatment and easy purification; saves two expensive important raw materials of Fmoc-NH-PEG 8 The consumption of the-COOH and the adriamycin reduces the consumption of other reagents, greatly reduces the cost, improves the efficiency, reduces the generation of waste materials, is beneficial to environmental protection and is suitable for large-scale production.)

1. A method for preparing a pectin-doxorubicin conjugate intermediate, wherein the pectin-doxorubicin conjugate intermediate has a structure represented by formula (I):

b-c-d-e (I)

in the formula (I), the main structure of b is PEG_nC is an enzymatic cleavage group, a polypeptide or a polypeptide derivative, d is a self-degrading group, e is doxorubicin DOX;

the method comprises the following specific steps: 1) preparing Fmoc-b-c-d-e from Fmoc-b-c-d;

2) removing the protecting group Fmoc in the presence of non-nucleophilic or weak-nucleophilic basic reagent to obtain b-c-d-e.

2. The process according to claim 1, wherein in the formula (I), n is an integer of 1 to 20, and c is any one of a dipeptide, a dipeptide derivative, a tripeptide derivative, a tetrapeptide or a tetrapeptide derivative;

preferably, n is an integer from 6 to 12; more preferably, n is 8;

preferably, c is a dipeptide; still more preferably, Val-Ala;

preferably, d represents a self-degrading group aminobenzyloxycarbonyl; preferably, the aminobenzyloxycarbonyl group is a p-aminobenzyloxycarbonyl group, i.e., PABC.

3. The method of claim 1, wherein the Fmoc protecting group is deprotected in the presence of a non-nucleophilic or weakly nucleophilic basic reagent to yield b-c-d-e; the alkaline agent is an organic base, preferably piperidine, fatty amine, alkaloid; still more preferred is a non-nucleophilic, strongly basic reagent, more preferably DBU.

4. The preparation method according to any one of claims 1 to 3, comprising the following specific steps:

1) from Fmoc-NH-PEG₈-CO-Val-Ala-PABOH by Fmoc-NH-PEG₈Preparation of Fmoc-NH-PEG from-CO-Val-Ala-PABC-PNPO₈-CO-Val-Ala-PABC-DOX, PNPO representing p-nitrophenoxy;

2) Fmoc-NH-PEG in the Presence of DBU₈-CO-Val-Ala-PABC-DOX removing protecting group Fmoc-to obtain NH₂-PEG_n-CO-Val-Ala-PABC-DOX。

5. The method of claim 4, wherein the Fmoc-NH-PEG is prepared from₈reacting-CO-Val-Ala-PABOH with DPNP in the presence of DIEA to obtain Fmoc-NH-PEG₈-CO-Ala-PABC-PNPO, DPNP is di (p-nitrophenyl) carbonate.

6. The method of claim 5, wherein the peptide is prepared from Val-Ala-PABOH and Fmoc-NH-PEG₈Reaction of-COOH to yield Fmo-NH-PEG₈-CO-Val-Ala-PABOH。

7. The method of claim 6, wherein the protecting group Fmoc is deprotected by Fmoc-Val-Ala-PABOH in the presence of a non-nucleophilic or weakly nucleophilic basic reagent; the alkaline agent is an organic base, preferably piperidine, fatty amine, alkaloid; further preferably, Val-Ala-PABOH is obtained by removing the Fmoc-protecting group in the presence of DEA.

8. The method according to any one of claims 1 to 7, comprising the following steps:

9. the method of claim 8, wherein said Fmoc-NH-PEG is prepared by₈the-COOH structure is shown below:

10. a process for the preparation of a pectin-doxorubicin conjugate, characterized in that a compound of formula (13) is reacted with NH prepared by the process of any one of claims 1 to 9₂-PEG₈-CO-Val-Ala-PABC-DOX in the presence of an alkaline reagent to prepare a pectin-doxorubicin conjugate,

in the formula (13), a is an integer selected from 2 to 4, and m is 1 to 60 KD.

Technical Field

The invention relates to the field of medicine preparation methods, in particular to a pectin-adriamycin conjugate and a preparation method of an intermediate thereof.

Background

The 'pectin-adriamycin conjugate' targeting drug delivery system (PAC) is based on a great deal of preliminary work and literature research, pectin is selected as a carrier of a high-molecular anticancer prodrug, and the structure of the pectin is modified so that the pectin can be covalently combined with polypeptide-adriamycin to form a water-soluble pectin-adriamycin conjugate with high drug loading.

PAC is high scoreThe particle size of the subconjugate is about 200nm, and partial drugs entering a circulatory system after injection can be accumulated in tumor tissues by utilizing the enhanced permeability and retention Effect (EPR) of the tumor tissues on macromolecular substances, so that the aim of passive targeting is fulfilled. PAC is gradually phagocytized by tumor cells in tumor tissues, and water in lysosomes is used for releasing adriamycin, thereby playing a role in killing tumors. The patent application with the application number of CN201910524576.0 discloses a pectin-adriamycin conjugate, which overcomes the problem that pectin and adriamycin are difficult to dissolve in water due to direct amide bond or acylhydrazone bond, but the disclosed production process has many problems, such as low total yield, difficult post-treatment, great obstacle to scale-up production and the like. The production process adopts the steps of firstly connecting the adriamycin to Fmoc-Val-Ala-PABC (actually Fmoc-Val-Ala-PABC-PNPO) intermediate to obtain an important intermediate Fmoc-Val-Ala-PABC-DOX, and then removing the fluorenylmethoxycarbonyl protecting group to obtain the Val-Ala-PABC-DOX and Fmoc-NH-PEG₈Condensation of-COOH to Fmoc-NH-PEG₈-CO-Val-Ala-PABC-DOX. Two difficulties are encountered in this process and the process has serious drawbacks: 1) the Fmoc protecting group (Fmoc) is not easy to be amplified after the Fmoc protecting group is removed, basic amino groups after the Fmoc protecting group is removed are easy to react with adriamycin, the process is difficult to control, and the product is not easy to purify; 2) Val-Ala-PABC-DOX with Fmoc-NH-PEG₈The reaction conversion rate of the condensation of-COOH is not high, a lot of byproducts are generated, column chromatography (the existing adsorption tailing can not be solved almost by normal phase system column chromatography) is required to be adopted in the treatment and purification process, the separation is difficult, and the yield is low.

Disclosure of Invention

The invention aims to provide a preparation method of pectin-adriamycin conjugate and an intermediate thereof, which has the advantages of high yield, simple and convenient post-treatment, low production cost, environmental protection and suitability for large-scale production, aiming at the defects of the prior art.

The technical scheme adopted by the invention for realizing the purpose is as follows: a method of preparing a pectin-doxorubicin conjugate intermediate having the structure of formula (I):

b-c-d-e(I)

in the formula (I), the main structure of b is PEG_nC is an enzymatic cleavage group, a polypeptide or a polypeptide derivative, d is a self-degrading group, e is doxorubicin DOX;

the pectin-doxorubicin conjugate drug has the structure: polymer-peptide linker-doxorubicin conjugate drug a-b-c-d-e, wherein polymer a is preferably pectin;

the optimized preparation method of the peptide linker intermediate b-c-d-e of the polymer-based adriamycin conjugate drug comprises the following specific steps:

1) preparing Fmoc-b-c-d-e from Fmoc-b-c-d;

2) and (3) removing the Fmoc protecting group in the presence of a non-nucleophilic basic reagent to obtain b-c-d-e.

Further, in the formula (I), n is an integer of 1-20, c is any one of dipeptide, dipeptide derivative, tripeptide derivative, tetrapeptide or tetrapeptide derivative;

further, n is an integer of 6 to 12; more preferably, n is 8;

further, c is Val-Ala;

further, d represents, in the case of direct linkage to e, a self-degrading group aminobenzyloxycarbonyl;

further, d, when not attached to e, may represent a self-degrading group aminobenzyloxycarbonyl group attached to a protecting group; still more preferably, the aminobenzyloxycarbonyl group is a p-aminobenzyloxycarbonyl group, i.e., PABC.

Further, the non-nucleophilic basic agent is DBU;

further, b is linked to c through an amide bond via a carbonyl group; more preferably, PEG₈Through an additional carbonyl group to form an amide bond with c;

further, b is connected with Fmoc through an amido bond formed by an amino group; more preferably, PEG₈Linking with Fmoc through an additional amino group to form an amide bond;

still further, b is NH₂-PEG_n-CO-。

Further, the specific steps are as follows:

1) from Fmoc-NH-PEG₈-CO-Val-Ala-PABC either without or with Fmoc-NH-PEG₈Preparation of-NH-PEG from-CO-Val-Ala-PABC-PNPO₈-CO-Val-Ala-PABC-DOX, PNPO representing p-nitrophenoxy;

2) Fmoc-NH-PEG in the Presence of DBU₈Removing a protecting group from-CO-Val-Ala-PABC-DOX to obtain NH₂-PEG_n-CO-Val-Ala-PABC-DOX。

Further, from Fmoc-NH-PEG₈reacting-CO-Val-Ala-PABOH with DPNP in the presence of DIEA to obtain Fmoc-NH-PEG₈-CO-Ala-PABC-PNPO, DPNP is di (p-nitrophenyl) carbonate.

Further, Val-Ala-PABOH and Fmoc-NH-PEG₈Reaction of-COOH to yield Fmo-NH-PEG₈-CO-Val-Ala-PABOH。

Further, Val-Ala-PABOH was prepared by Fmoc-Val-Ala-PABOH deprotection in the presence of DEA.

Further, the specific steps are as follows:

further, the Fmoc-NH-PEG₈the-COOH structure is shown below:

the invention also provides a preparation method of the pectin-adriamycin conjugate, and the compound shown in the formula (13) and NH prepared by the preparation method₂-PEG₈-CO-Val-Ala-PABC-DOX in the presence of an alkaline reagent to prepare a pectin-doxorubicin conjugate,

in the formula (13), a is an integer selected from 2 to 4, and m is 1 to 60 KD.

The invention has the advantages and effects that:

1) the route basically solves a large number of problems existing before, the synthesis process of related steps can be feasible and controllable, firstly, the overall yield of the synthesized micromolecular compound is improved from 1.9% to 20.1% by redesigning the whole route for synthesizing the micromolecular compound, and the unsolved process problems such as adsorption tailing and the like which can not be solved by normal phase system column chromatography in the prior art are basically solved; and secondly, DBU (DBU) which is an excellent non-nucleophilic alkaline organic reagent is used for removing Fmoc (Fmoc reaction), a better effect is achieved, a great breakthrough is made in comparison with the prior art, the milligram-scale quantity obtained by one-time preparation is successfully amplified to gram-scale, and the large-scale production is facilitated.

2) The method solves the problems of low yield and difficult post-treatment in the prior process route. Saves two expensive important raw materials of Fmoc-NH-PEG₈The dosage of-COOH and adriamycin, and the consumption of other solvent reagents are reduced, the cost is greatly reduced, the efficiency is improved, the generation of waste materials is reduced, the environment is protected, and the method is closer to the production process.

Drawings

FIG. 1 is a scheme for preparing NH₂-PEG₈-CO-Val-Ala-PABC-DOX.

Detailed Description

The present invention will be further specifically described below with reference to examples, but the present invention is not limited to these examples.

Example 1

Compound 1(100g, 0.2946mol) was dissolved in 1LTHF, NHS (37.2g, 0.3231mol), DCC (86g, 0.4175mol) were added in this order, and the reaction was completed by stirring at room temperature for 4 hours. The white solid was removed by filtration, the filter cake washed with THF (100ml x 4), the filtrate was collected and the filtrate was concentrated in vacuo to give a yellow gummy crude which was slurried with 200ml petroleum ether to precipitate a white solid which was dispersed completely and filtered and the filter cake washed with petroleum ether (50ml x 4) to give 134g of the compound as a white solid in 98% yield.

Example 2

The compound Fmoc-Val-OSu (134g, 0.307mol) was dissolved in 1.35L THF, followed by the addition of L-alanine (29.2g, 0.3277mol) and NaHCO₃(27.3g，0.3277mol)、425ml H₂And O. The reaction was stirred at room temperature for 27h and monitored for completion. Vacuum concentrating to remove THF, gelatinizing, diluting with water (2L), adjusting pH to 3 with HCl solution (2mol/ml), precipitating solid, pulping overnight, vacuum filtering, and filtering with H to obtain filter cake₂Washing with O (100ml 4) gave crude S3. Crude S3 was slurried with EA: PE 1:3(1.45L) for 1h, filtered with suction and the cake was washed with EA: PE 1:3(200ml 4). 114.5g of the compound (Fmoc-Val-Ala-OH) was obtained as a white solid in a yield of 91%.

Example 3

The compound Fmoc-Val-Ala-OH (82.52g, 0.2015mol) was added to a reaction flask, 982ml DCM was added to make a suspension, 4-aminobenzyl alcohol (30.24g, 0.24555mol), EEDQ (74.27g, 0.30033mol) and finally 512ml methanol were added in sequence until the solution was clear, reacted at room temperature for 5 hours, filtered to give a white solid, which was washed with DCM (100ml x 3). The filtrate was collected, DCM was removed under reduced pressure and the remaining solid was slurried 2 times and washed with DCM (50ml x 3) to give a white solid. The two white solids were combined, washed with DCM (200 ml. times.2), filtered and dried under vacuum at ambient temperature to give 60.1g of the product as a white solid (Fmoc-Val-ALa-PABOH) in 57% yield.

Example 4

The compound Fmoc-Val-Ala-PABOH (20g, 0.03879mol) was added to a reaction flask, dissolved in 50ml DMF, 20ml DCM was added, DEA (3.9959ml, 0.03879mol) was added, and the reaction was carried out at room temperature for 2 hours. Using petroleum ether: the solution of dichloromethane (10: 1) is used for extracting the reaction liquid for 8 times to obtain a pure yellow colloid compound Val-Ala-PABCOH. The product was purified with 40ml MeOH: DCM (1: 1) was dissolved and transferred to a round bottom flask and the remaining diethylamine was removed by rotary evaporation under vacuum at 40 ℃. 10.5g of a yellow colloidal compound (Val-Ala-PABOH) was obtained in a yield of 92%.

Example 5

Weighing Fmoc-NH-PEG₈-COOH (1.738g, 2.6214mmol), dissolved in 8ml DCM, HATU (1.396g, 3.6715mmol) added, Val-Ala-PABOH (1g, 3.4088mmol) dissolved in 7ml DCM and added to the flask, DIPEA (685ul, 3.9328mmol) added after 10 minutes ice bath, stirring in ice bath for 10 minutes and then returning to room temperature for 6 hours. The reaction was filtered, washed with DCM (5ml × 3) and the filtrate was collected. The filtrate was washed with hydrochloric acid solution (30ml × 3) at PH 3, the organic layer was dried over anhydrous sodium sulfate, filtered, and the solid was washed with DCM (10ml × 3). The solvent was removed under reduced pressure at 30 ℃ and then dried in vacuum using an oil pump to obtain 2.678g of a yellow colloidal compound (Fmoc-PEG)₈-CO-Val-Ala-PABOH), yield 84%.

Example 6

Fmoc-NH-PEG₈-CO-Val-Ala-PABOH (15.66g, 0.01668mol) was dissolved in 110ml DCM and after 10 min of ice-water bath di (p-nitrophenyl) carbonate (7.3g, 0.02668mol) was added followed by DIPEA (6.54ml, 0.04169mol) and after 10 min of stirring in ice bath the reaction was allowed to return to room temperature overnight. The reaction solution was washed with aqueous sodium carbonate solution at PH 9(100ml × 3), then with aqueous hydrochloric acid solution at PH 3 (100ml × 3), the organic layer was dried over anhydrous sodium sulfate, filtered, and the solid was washed with DCM (10ml × 3). Vacuum rotary evaporation at 20 ℃ to obtain a crude product, dissolving the crude product with 40ml of DCM, dropping 400ml of DCM, and rapidly stirringIn the methyl tert-butyl ether, the first batch of solid product Fmoc-NH-PEG is obtained by filtration₈-CO-Val-Ala-PABC and mother liquor. Vacuum rotary evaporating the mother liquor at 20 ℃ to obtain a crude product, dissolving the crude product with 10ml of DCM, dripping the dissolved crude product into 100ml of methyl tert-butyl ether under rapid stirring, and filtering to obtain a second batch of solid product Fmoc-NH-PEG₈-CO-Val-Ala-PABC, and combining the two batches to obtain 14.3g of a yellow colloid product (Fmoc-NH-PEG)₈-CO-Val-Ala-PABC), yield 78%.

Example 7

Fmoc-NH-PEG₈-CO-Val-Ala-PABC (552.3mg, 0.5mmol) was dissolved in DMF 3ml in the reaction tube, DOX (doxorubicin 274.2mg, 0.5112mmol) was added after 10 minutes of ice bath (external temperature around 5 ℃),

DIPEA (196ul, 1.2822mmol) was then added and the reaction was allowed to proceed overnight in an ice bath. The reaction was poured into stirred methyl tert-butyl ether/water 1:1(30ml) to give a red gummy solid. The red gummy solid was dissolved in DMF2ml and recrystallized from methyl tert-butyl ether/water 1:1(20ml) to give crude 1. Dissolving crude product 1 with DCM 2ml, pouring into stirred methyl tert-butyl ether (20ml) for crystallization to obtain dispersed dark red solid, filtering, washing the solid with methyl tert-butyl ether (5ml × 3), rotary evaporating at normal temperature under vacuum, and drying with oil pump to obtain 505mg of dark red solid (Fmoc-NH-PEG)₈-CO-Val-Ala-PABC-DOX), yield 67%.

Example 8

Fmoc-NH-PEG₈-CO-Val-Ala-PABC-DOX (1g, 0.663mmol) was dissolved in 10ml DMF, and DBU (1, 8-diazabicycloundecen-7-ene) (364mg, 2.39mmol) was dissolved in a small amount of solvent and added to the reaction mixture for 45 seconds. The reaction was poured into methyl tert-butyl ether (100ml) to which lactic acid (480mg) was added with stirring to give crude 1 as a red gum. Crude product 1 used10ml of methanol was dissolved and poured into stirred methyl tert-butyl ether (100ml) to crystallize to give crude 2 as a red solid. Crude 2 was dissolved in DCM: MeOH ═ 4:1(10ml) and crystallized by addition to 100ml of methyl tert-butyl ether to give 900mg of red powdery solid (NH)₂-PEG₈CO-Val-Ala-PABC-DOX), yield 98%.

Example 9

Weighing a compound shown as a formula 13 (the preparation method can refer to CN201910524576.0), completely dissolving the compound in a mixed solution of DMF and DMSO, adding a compound shown as a formula 9, uniformly stirring, adding DIEA, reacting at room temperature for 24h, directly dialyzing the solution by using DMSO-water gradient, detecting by HPLC until no adriamycin and derivatives thereof exist, and freeze-drying after dialysis is finished to obtain the pectin-adriamycin conjugate.

Confirmation of pectin-doxorubicin conjugates:

¹H NMR(400MHz，DMSO-d₆)δppm:14.04(s，1H)，13.27(s，1H)，9.88-9.90(s，1H)，8.33-6.81(m,10H)，5.47-3.39(m)，1.95-0.85(m).

the pectin-doxorubicin conjugates have the following structure:

comparative example

A compound represented by formula 9 was synthesized according to the method described in CN 201910524576.0:

the specific yields are as follows:

compound numbering	2	3	4	5	6	7	8	9	Overall yield of
										Yield of	95％	94％	53％	25％	78％	66％	35％	90％	1.9％

The above embodiments are only for illustrating the technical concept and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the content of the present invention and implement the present invention accordingly, and not to limit the protection scope of the present invention and the optimization of the synthetic route accordingly. All equivalent changes or modifications made in accordance with the spirit of the present disclosure are intended to be covered by the scope of the present disclosure.