阅读说明：本技术 一种多肽化合物的制备方法 (Preparation method of polypeptide compound ) 是由 付晓平 周海波 黄波 袁瑜 胡西 姜士礼 孟广鹏 于 2021-08-23 设计创作，主要内容包括：本发明提供了一种多肽化合物的制备方法,包括以下步骤：将M-1所示化合物和M-2所示化合物进行缩合反应,然后脱除保护基得到化合物I-1。本发明通过引入er值大于97/3的手性化合物M-2-2,再结合其他一些关键点的工艺优化,目标化合物I-1的合成收率大大提高,粗品收率高达80％以上,纯化拆分的难度、周期以及成本都大大降低,成功实现了1.5公斤级原料药粗品的放大纯化。(The invention provides a preparation method of a polypeptide compound, which comprises the following steps: carrying out condensation reaction on the compound shown by M-1 and the compound shown by M-2, and then removing a protecting group to obtain the compound I-1. According to the invention, by introducing the chiral compound M-2-2 with the er value of more than 97/3 and combining with process optimization of other key points, the synthesis yield of the target compound I-1 is greatly improved, the yield of crude products reaches more than 80%, the difficulty, the period and the cost of purification and resolution are greatly reduced, and the amplification and purification of crude products of crude drugs of 1.5 kg grade are successfully realized.)

1. A method for preparing a polypeptide compound, comprising the steps of:

carrying out condensation reaction on a compound shown as M-1 and a compound shown as M-2, and then removing a protecting group to obtain a compound I-1;

2. the preparation method according to claim 1, comprising the following steps:

1) carrying out liquid phase condensation reaction on the M-1 and the M-2 to obtain an intermediate M-3;

2) removing all protecting groups from the intermediate M-3 under an acidic condition, and purifying a crude product by HPLC to obtain a compound I-1;

3. the method of claim 2, wherein the liquid phase condensation reaction comprises a condensation agent selected from one or more of the following combinations:

a) HBTU, HOBt and DIEA;

b) HBTU, Cl-HOBt and DIEA;

c) DIC and HOBt;

d) DIC and Cl-HOBt;

e) PyBOP, HOBt and DIEA;

f) PyBOP, Cl-HOBt and DIEA;

g) DIC, HOSu and DIEA.

4. The method according to claim 1, wherein the compound represented by M-1 is prepared by the following method:

by utilizing a polypeptide solid-phase synthesis method, protected D-Lys, D-Phe and D-Leu are taken as raw materials, the tetrapeptide D-Phe-D-Phe-D-Leu-D-Lys is coupled to a solid-phase carrier from a C end to an N section in sequence, and after the solid-phase carrier is removed, a tetrapeptide intermediate M-1 is obtained.

5. The method according to claim 4, wherein the condensation reagent used for the coupling is selected from one or more of the following combinations:

a) HBTU, HOBt and DIEA;

b) HBTU, Cl-HOBt and DIEA;

c) DIC and HOBt;

d) DIC and Cl-HOBt;

e) PyBOP, HOBt and DIEA;

f) PyBOP, Cl-HOBt and DIEA.

6. The method according to claim 4, wherein the solid support is 2-chlorotriphenylmethyl chloride resin or Wang resin.

7. The method of claim 4, wherein said protected D-Lys is Fmoc-D-Lys (Boc) -OH;

the protected D-Phe is Fmoc-D-Phe-OH or Boc-D-Phe-OH;

the protected D-Leu is Fmoc-D-Leu-OH.

8. The preparation method according to claim 4, wherein the lysis reagent for removing the solid phase carrier is trifluoroethanol.

9. The method according to claim 1, wherein the compound represented by M-2 is prepared by the following method:

1) carrying out asymmetric hydroboration reaction on the compound S-5 and a chiral boron reagent S-6 to obtain an intermediate M-2-1, and then carrying out esterification reaction to obtain M-2-2;

2) removing the Boc protecting group of the intermediate M-2-2 under an acidic condition to obtain an intermediate M-2;

10. the method according to claim 9, wherein the temperature of the asymmetric hydroboration reaction is from-40 ℃ to-20 ℃.

11. The method of claim 9, wherein the er value of M-2 "2 is greater than 97/3.

Technical Field

The invention relates to the technical field of pharmacy, in particular to a preparation method of a polypeptide compound.

Background

Opioid receptors are a major class of G protein-coupled receptors and are the binding targets for endogenous opioid peptides as well as opioid drugs. Opioid receptors, which are activated to regulate the immune and endocrine systems of the nervous system, are the most powerful and commonly used central analgesics. Endogenous opioid peptides are naturally occurring opioid active substances in mammals, and currently known endogenous opioid peptides are broadly classified into several classes, namely enkephalins, endorphins, dynorphins and neorphins. Its corresponding opioid receptors, i.e., μ, δ and κ receptors, are present in the central nervous system. Mu receptor has the strongest analgesic activity and the strongest addiction, and is the main reason for generating side effects. The delta receptor has small addiction and also has unobvious analgesic effect. Kappa receptor (KOR) analgesic activity is intermediate between the first two. The polypeptide KOR agonist can exert analgesic effect in periphery without entering into center, and has no adverse side effects such as respiratory depression and constipation, and lower addiction, thus having potential of drug addiction treatment.

Patent CN111233974B reports a series of novel KOR agonists with excellent agonistic activity, including compounds with the structure shown in formula I-1. The main chain of the polypeptide compound is synthesized by adopting a solid-liquid phase combination mode, so that the synthesis efficiency can be greatly improved; however, this patent uses racemic pyrrole-3-boronic acid pinacol ester as key material for liquid phase condensation (enantiomerically pure material is not available) such that the ratio of the final synthesized target compound to its isomer is 1: 1 (the polarity difference between the two is very small), not only the synthesis yield is greatly reduced, but also great challenge is brought to the separation and purification of batches above gram level, repeated and continuous recovery and re-preparation are needed, the purification period is long, the solvent consumption is large, the waste liquid amount is great, and the large-scale production is not facilitated.

Therefore, a new method based on chiral synthesis is urgently needed to be developed so as to greatly improve the proportion of target isomers, reduce the purification difficulty of target compounds and improve the total yield of target products, thereby solving the problems of long purification period, high cost and difficult process amplification in the prior art.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a method for preparing a polypeptide compound, which can prepare a compound represented by formula I-1 with high purity.

In order to achieve the above object, the present invention provides a method for preparing a polypeptide compound, comprising the steps of:

carrying out condensation reaction on a compound shown as M-1 and a compound shown as M-2, and then removing a protecting group to obtain a compound I-1;

preferably, the preparation method specifically comprises the following steps:

1) carrying out liquid phase condensation reaction on the M-1 and the M-2 to obtain an intermediate M-3;

2) removing all protecting groups from the intermediate M-3 under an acidic condition, and purifying a crude product by HPLC to obtain a compound I-1;

preferably, the condensation reagent of the liquid phase condensation reaction is selected from one or more of the following combinations:

a) HBTU, HOBt and DIEA;

b) HBTU, Cl-HOBt and DIEA;

c) DIC and HOBt;

d) DIC and Cl-HOBt;

e) PyBOP, HOBt and DIEA;

f) PyBOP, Cl-HOBt and DIEA;

g) DIC, HOSu and DIEA.

More preferably g) DIC, HOSu and DIEA.

In the present invention, the deprotecting reagent for removing all protecting groups in the step 2) is preferably trifluoroacetic acid. The trifluoroacetic acid is preferably dichloromethane solution with the volume concentration of 20-80%.

In the preferred mode of the invention, the compound represented by M-1 is prepared according to the following method:

by utilizing a polypeptide solid-phase synthesis method, protected D-Lys, D-Phe and D-Leu are taken as raw materials, the tetrapeptide D-Phe-D-Phe-D-Leu-D-Lys is coupled to a solid-phase carrier from a C end to an N section in sequence, and after the solid-phase carrier is removed, a tetrapeptide intermediate M-1 is obtained.

In a preferred embodiment of the invention, the protected D-Lys is Fmoc-D-Lys (Boc) -OH.

Preferably, the protected D-Phe is Fmoc-D-Phe-OH or Boc-D-Phe-OH.

Preferably, the protected D-Leu is Fmoc-D-Leu-OH.

Preferably, the preparation of the compound represented by M-1 comprises the following steps:

1) fixing the compound S-1 on a solid phase carrier to obtain R-1, and then removing the Fmoc protecting group to obtain a compound R-1-a fixed on the solid phase carrier;

2) coupling protected amino acids S-2, S-3 and S-4 to R-1-a sequentially through the following reaction sequences to obtain a compound R-4 fixed by a solid phase carrier, and finally cracking the solid phase carrier to obtain an intermediate M-1;

preferably, the condensation reagent used for the coupling is selected from one or more of the following combinations:

a) HBTU, HOBt and DIEA;

b) HBTU, Cl-HOBt and DIEA;

c) DIC and HOBt;

d) DIC and Cl-HOBt;

e) PyBOP, HOBt and DIEA;

f) PyBOP, Cl-HOBt and DIEA.

In the invention, the solid phase carrier is preferably 2-chloro triphenyl methyl chloride resin or Wang resin. The substitution degree of the 2-chloro-triphenylmethyl chloride resin is preferably 0.1-1.6 mmol/g; the degree of substitution of the Wang resin is preferably from 0.1 to 1.6 mmol/g. 2-Chlorotriphenylmethyl chloride resin is preferably used.

The deprotection reagent for removing the Fmoc protecting group is preferably piperidine or piperazine. The piperidine or piperazine is preferably a DMF solution with a volume concentration of 10-50%.

In the invention, the preferable cracking reagent for removing the solid phase carrier is trifluoroethanol. The trifluoroethanol is preferably dichloromethane solution or water solution with the volume concentration of 10-95%.

In the invention, the compound shown as M-2 is prepared according to the following method:

1) carrying out asymmetric hydroboration reaction on the compound S-5 and a chiral boron reagent S-6 to obtain an intermediate M-2-1, and then carrying out esterification reaction to obtain M-2-2;

2) removing the Boc protecting group of the intermediate M-2-2 under an acidic condition to obtain an intermediate M-2;

the invention adopts a chiral boron reagent S-6 for the synthesis of a key boron-containing intermediate M-2-2. By means of asymmetric hydroboration derivatization, the obtained M-2-2 has high optical purity, the er value is greater than 97/3, and kilogram-level amplification can be realized; the purification method is simple, the 4-step reaction only needs one simple rapid column chromatography, and the total yield reaches more than 60 percent.

In the invention, the temperature of the asymmetric hydroboration reaction is preferably-40 ℃ to-20 ℃.

In a preferred embodiment of the present invention, the solvent for the esterification reaction is selected from ethyl acetate, isopropyl acetate or tert-butyl acetate.

In the preferred embodiment of the present invention, the deprotection reagent for removing Boc protecting group is selected from trifluoroacetic acid or hydrochloric acid; more preferably trifluoroacetic acid. The trifluoroacetic acid is preferably dichloromethane solution with volume concentration of 20-80%; the hydrochloric acid is preferably an aqueous solution of hydrochloric acid, a dioxane solution or an ethyl acetate solution.

Compared with the prior art, the invention provides a preparation method of a compound, which comprises the following steps:

and (3) mixing the compound shown by M-1 and the compound shown by M-2, carrying out condensation reaction, and then removing a protecting group to obtain the compound I-1. By introducing the chiral compound M-2-2 with the er value of more than 97/3, the synthesis yield of the target compound I-1 is greatly improved, the yield of crude products is up to more than 80%, the difficulty, the period and the cost of purification and resolution are greatly reduced, and the amplification and purification of the crude product of the crude drug of 1.5 kilogram level are successfully realized.

Furthermore, the invention further improves the yield and purity of the target compound I-1 by combining with the optimization of other key process conditions, such as condensing agents, types and equivalent of lysis solution, reaction temperature, reaction time and the like.

Detailed Description

In order to further illustrate the present invention, the following examples are given to describe in detail the preparation of the compounds provided by the present invention.

The main experimental equipment information used in the examples is shown in table 1 below:

TABLE 1

Serial number	Device name	Specification of equipment	Manufacturer of the product
				1	Polypeptide synthesizer	20L	Jian bang Hainan
2	Polypeptide settling kettle	40L	Jian bang Hainan
				3	Polypeptide cracking instrument	10L	Jian bang Hainan
4	High-capacity refrigerated centrifuge	2L	/
				5	CS-Prep industrial preparative chromatography system	DAC200	Hanbang Jiangsu
6	Vacuum freeze drier	Pilot2-4M	Medicine for curing rheumatism

The abbreviations used in the present invention have the following meanings as shown in Table 2 below:

TABLE 2

EXAMPLE 1 preparation of intermediate M-1

Step (1) preparation of intermediate Resin Fmoc-D-Lys (Boc) -2-CTC Resin (R-1)

To a 20L peptide synthesizer was added 2-chlorotriphenylmethyl chloride resin (1219.3g), followed by DCM (12.2L) and swollen for 10 min. Fmoc-D-Lys (Boc) -OH (1238.7g, 2647mmol) was weighed out and dissolved well in DCM (6L) and DIEA (650mL, 3960mmol) was added. Adding the reaction solution into 20L of resin, fully and uniformly stirring, and reacting for 2h at 25 ℃. MeOH (260mL, 10273mmol) and DIEA (650mL, 3960mmol) were added and reacted for 0.5h capping, the solution was drained and the resin was washed with 6X 6L DMF to complete the reaction to give the desired resin R-1. A portion of the resin was deprotected with piperidine/DMF solution (V/V ═ 1/3) and the Fmoc content was determined by uv spectrophotometry, calculating the degree of substitution of resin R-1 to 1.08 mmol/g.

Step (2) preparation of intermediate Resin H-D-Lys (Boc) -2-CTC Resin (R-1-a)

Treating the resin obtained in step (1) with piperidine/DMF solution (V/V ═ 1/3) for 10min, and draining the solution; additional 4L piperidine/DMF (V/V. 1/3L) was added and the treatment continued for 10 min. The solution was drained, the resin was washed with 6X 6L DMF and was purple as detected by Kaiser Test, deprotection was complete, and the solution was aspirated off to give the target resin R-1-a.

Step (3) preparation of intermediate Resin Fmoc-D-Leu-D-Lys (Boc) -2-CTC Resin (R-2)

S-2(932.4g, 2641mmol), HOBT (357.7g, 2650mmol) and HBTU (1009.8g, 2664mmol) were dissolved in DMF (4L), incubated at 0-5 ℃ and then DIEA (650mL, 3960mmol) was added and activated with stirring for 15 min. Adding the reaction solution into the resin obtained in the step (2), reacting for 1.5h at 25 ℃, draining the solution, washing the resin with 6X 6L DMF, detecting by Kaiser Test to be yellow, and completely reacting to obtain the target resin R-2.

Step (4) preparation of intermediate Resin H-D-Leu-D-Lys (Boc) -2-CTC Resin (R-2-a)

Treating the resin obtained in step (3) with piperidine/DMF solution (V/V ═ 1/3) for 10min, and draining the solution; additional 4L piperidine/DMF (V/V. 1/3L) was added and the treatment continued for 10 min. The solution was drained, the resin was washed with 6X 6L DMF and was purple as detected by Kaiser Test, deprotection was complete, and the solution was aspirated off to give the target resin R-2-a.

Step (5) preparation of intermediate Resin Fmoc-D-Phe-D-Leu-D-Lys (Boc) -2-CTC Resin (R-3)

S-3(1024.3g, 2647mmol), HOBT (358.4g, 2655mmol) and HBTU (1002.4g, 2645mmol) were dissolved in DMF (4L), incubated at 0-5 ℃ and then DIEA (650mL, 3960mmol) was added and activated with stirring for 15 min. And (3) adding the reaction solution into the resin obtained in the step (4), reacting for 1.5h at 25 ℃, draining the solution, washing the resin with 6X 6L of DMF, detecting by Kaiser Test to be yellow, and completely reacting to obtain the target resin R-3.

Step (6) preparation of intermediate Resin H-D-Phe-D-Leu-D-Lys (Boc) -2-CTC Resin (R-3-a)

Treating the resin obtained in step (3) with piperidine/DMF solution (V/V ═ 1/3) for 10min, and draining the solution; additional 4L piperidine/DMF (V/V. 1/3L) was added and the treatment continued for 10 min. The solution was drained, the resin was washed with 6X 6L DMF and was purple as detected by Kaiser Test, deprotection was complete, and the solution was aspirated off to give the target resin R-3-a.

Step (7) preparation of intermediate Resin Boc-D-Phe-D-Phe-D-Leu-D-Lys (Boc) -2-CTC Resin (R-4)

S-4(700.1g, 2642mmol), HOBT (360.3g, 360.3g mmol) and DIC (410mL, 2640mmol) were dissolved in DMF (4L), incubated at 0-5 ℃ and activated with stirring for 15 min. Adding the reaction solution into the resin obtained in the step (6), reacting for 1.5h at 25 ℃, draining the solution, washing the resin with 6X 6L DMF, detecting by Kaiser Test to show yellow, and completely reacting. The resin was washed with 6X 6L MeOH, then dried at 33 deg.C in vacuo to constant weight to give 2270.3g of the title resin R-4 in 95% yield.

Step (8) preparation of intermediate Boc-D-Phe-D-Phe-D-Leu-D-Lys (Boc) -OH (M-1)

2260.2g of resin R-4 was put into a 20L reactor, 15L of a lysate (25 vol% trifluoroethanol in dichloromethane) was added, and the reaction was stirred at room temperature for 2 hours. The mixture was transferred to a 10L peptide cleavage apparatus and filtered with suction, the filtrate was concentrated, and the filter cake was washed with 3X 3L of methylene chloride. The filtrate was concentrated to dryness and the resulting white solid was dried under vacuum at 34 ℃ to constant weight to finally obtain 1015.9g of intermediate M-1 as an off-white solid in 93.7% yield.

ESI-MS(m/z):754.5(M+H)⁺。

HPLC purity:97.23％(220nm)。

EXAMPLE 2 preparation of intermediate M-2

Step (1) preparing chiral boron reagent (-) -Ipc on site₂BH

Slowly and dropwise adding (+) -alpha-pinene (705mL) into a tetrahydrofuran solution (2.0mol/L, 1.1L) of borane dimethyl sulfide complex, keeping the temperature at-5 ℃, and continuing to perform heat preservation reaction for 5 hours after the addition. Decompressing at-5 ℃ to remove the solvent and the free dimethyl sulfide, then supplementing anhydrous tetrahydrofuran (450mL) and (+) -alpha-pinene (70mL), and continuing to perform heat preservation reaction for 3 days to obtain chiral boron reagent (-) -Ipc₂The BH solution is directly used for next hydroboration reaction.

Step (2) preparation of intermediate M-2-1

And (2) cooling the reaction liquid in the step (1) to-25 to-20 ℃, adding a tetrahydrofuran (450mL) solution of N-tert-butoxycarbonyl-pyrroline (250.2g), and keeping the temperature for 2 days after the addition. Slowly dripping methanol (500ml) to quench the reaction, and keeping the temperature at minus 10-0 ℃ to continue the reaction for 2 hours. The colorless oil obtained by concentration under reduced pressure was dissolved in 4L of methanol and 4L of potassium carbonate solution (0.25g/mL), extracted with 3X 3L of petroleum ether, and separated. The petroleum ether phase was washed with 4L of methanol and 4L of potassium carbonate solution (0.25g/mL) and separated. And (3) carrying out back extraction on the combined water phases by using 3X 3L petroleum ether to further remove impurities, and then acidifying the obtained product to a pH value of 1-2 by using dilute hydrochloric acid. After adding sodium chloride to saturation, extracting with 3X 3L ethyl acetate, washing the combined organic phases with saturated brine and drying over anhydrous sodium sulfate, filtering and concentrating to obtain 527.8g of intermediate M-2-1 as yellow oil which is directly used for the next esterification reaction.

Step (3) preparation of intermediate M-2-2

The yellow oily substance in step (2) was dissolved in 1L of isopropyl acetate, and 230g of pinacol was added and reacted at room temperature for 2 hours. The reaction solution was concentrated and purified by flash silica gel column chromatography to give 450g of intermediate M-2-2 as a colorless oil. The total yield of the three steps is 51 percent (based on N-Boc-pyrroline).

¹H NMR(400MHz,CDCl₃)δ3.54-3.49(m,2H),3.24-3.18(m,2H),2.04-1.95(m,1H),1.81-1.70(m,1H),1.62-1.50(m,1H),1.48(s,9H),1.22(s,12H).

LCMS：m/z＝298.1；er＝97.2/2.8。

HPLC purity：97.82％(220nm)。

Step (4) preparation of intermediate M-2

M2-2(438.6g, 1477mmol) was dissolved in dichloromethane (2.2L) and 1.1L TFA was added slowly and the reaction stirred for 1 h. The reaction was concentrated to dryness, diluted with DCM (1.5L), and slowly added DIEA in ice bath to adjust to pH 8 to obtain a solution of intermediate M-2, which was used directly in the next liquid phase condensation reaction.

EXAMPLE 3 preparation of intermediate M-3

M-1(929.3g, 1232mmol) and HOSu (184.2g, 1602mmol) were charged into a 50L reactor, and dissolved in 13L of dichloromethane. Reducing the temperature to 10 ℃, adding a dichloromethane solution (500mL) of DIC (247mL), and reacting for 2h at the temperature of 5-10 ℃ to obtain the activated ester of M-1. Then DIEA (609mL, 3692mmol) and the M-2 solution in example 2 are added in sequence, and the temperature is raised to 20-25 ℃ for reaction for 1 h. 5L of a 1mol/L hydrochloric acid solution was added, stirred and separated, the aqueous phase was washed with dichloromethane (600mL), and the combined organic phases were washed successively with 3X 10L of water and 10L of saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to give 1478.7g of intermediate M-3 as a dark brown liquid-solid mixture.

EXAMPLE 4 preparation of the title compound I-1([ (S) -1- (D-phenylalanyl-D-leucyl-D-lysyl) pyrrolidin-3-yl ] boronic acid hydrochloride)

M-3(1477.9g, 1232mmol) was dissolved in 4.5L of dichloromethane, and 1.1L of trifluoroacetic acid was slowly added thereto to react at room temperature for 1.5 h. The reaction solution was concentrated to give an oil, which was diluted with trifluoroacetic acid and slowly added to a 50L settling tank containing 40L of methyl t-butyl ether. The precipitated colloidal solid was subjected to refrigerated centrifugation, and the obtained solid was washed with 3X 15L of methyl t-butyl group, concentrated and vacuum-dried at 35 ℃ to constant weight to obtain 1140.6g of crude I-1 as a white solid with a crude yield of 80.5%. And purifying the crude product by HPLC liquid phase preparation and transferring salt to obtain 397g of a finished product of the target compound I-1 with the purity of more than 99 percent and the dr of 99.81/0.19, wherein the total yield is 48 percent.

LCMS：m/z＝651.4(M+H)⁺；

HPLC purity：99.50％(220nm)。

¹H NMR(400MHz,DMSO-d₆)δ9.08–9.04(m,1H),8.51–8.49(m,1H),8.31–8.01(m,7H),7.77(s,2H),7.44–7.36(m,2H),7.34–7.17(m,8H),4.66–4.55(m,1H),4.50–4.41(m,1H),4.38–4.32(m,1H),4.05–4.01(m,1H),3.63–3.59(m,1H),3.55–3.51(m,1H),3.34–3.27(m,1H),3.26–3.16(m,1H),3.15–3.01(m,2H),3.00–2.94(m,1H),2.88–2.82(m,1H),2.80–2.66(m,2H),2.06–1.86(m,1H),1.80–1.42(m,9H),1.41–1.24(m,2H),0.92–0.87(m,6H).

The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.