CN113480606B - Olefin ring-forming derivative containing tryptophan polypeptide and preparation and application thereof - Google Patents

Olefin ring-forming derivative containing tryptophan polypeptide and preparation and application thereof Download PDF

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CN113480606B
CN113480606B CN202110894675.5A CN202110894675A CN113480606B CN 113480606 B CN113480606 B CN 113480606B CN 202110894675 A CN202110894675 A CN 202110894675A CN 113480606 B CN113480606 B CN 113480606B
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tryptophan
polypeptide
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olefin
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CN113480606A (en
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朱勍
王鹏
晏郑情
刘江
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Zhejiang University of Technology ZJUT
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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Abstract

The invention relates to an olefin cyclization derivative containing tryptophan polypeptide, a preparation method and application thereof, wherein the structure of the olefin cyclization derivative containing tryptophan polypeptide is shown as a formula (I). The beneficial effects of the invention are mainly as follows: the compound is prepared by directly oxidizing a Heck reaction of a tryptophan side chain C (sp 2) -H through palladium catalysis, has the advantages of simple operation process, no guide group, high site selectivity and high reaction efficiency, can be prepared by only one step, has good anti-tumor application prospect, and provides a new scheme for developing anti-tumor drugs.

Description

Olefin ring-forming derivative containing tryptophan polypeptide and preparation and application thereof
Field of the art
The invention relates to an olefin cyclization derivative containing tryptophan polypeptide, a preparation method and application thereof.
(II) background art
The polypeptide is an important bioactive molecule and has wide application in the fields of pharmaceutical chemistry, biotechnology, chemical biology and the like. Compared with linear peptides, the spike peptide has the outstanding advantages of higher binding affinity, targeting selectivity, cell permeability, proteolytic stability, and ability to modulate protein-protein interactions (PPIs), etc. In order to better understand their physiological and pharmacological functions, in particular their inhibition of intracellular PPIs, the construction of fluorescent stapling peptides has proven to be a powerful tool in the pharmaceutical chemistry field and is therefore of great interest. In recent years, several approaches based on post-modification of Trp residues have been successfully developed. Whereas traditional methods for post-modification of Trp residues: it is generally necessary to introduce a protecting group or a guiding group into the number 1 of the indole heterocycle of Trp, and then to deprotect or guide the group after finishing the modification of other sites, so as to realize the modification. Such methods have a number of steps and low reaction yields, and these problems have greatly reduced the progress of subsequent modification studies on Trp-containing polypeptides.
(III) summary of the invention
The invention aims to provide an olefin ring-forming derivative containing tryptophan polypeptide, and a preparation method and application thereof.
The technical scheme adopted by the invention is as follows:
an olefination ring derivative containing tryptophan polypeptide, the structure of which is shown as (I):
in the formula (I), AA is an amino acid residue, m is a natural number of 0-1, and n is a natural number of 2-3.
Preferably, the olefination ring derivative is one of the following formulas:
The invention also relates to a method for preparing an olefin ring-forming derivative containing tryptophan polypeptide shown in the formula (I), which comprises the following steps: taking an acrylic ester modified tryptophan-containing polypeptide shown in a formula (II) as a substrate, and stirring and reacting in a solvent at 50-100 ℃ for 12-36 hours in the presence of a catalyst and an oxidant to prepare an olefin-containing cyclization derivative of the tryptophan-containing polypeptide shown in the formula (I);
General Synthesis procedure for Cyclic peptide precursor (II)
As shown, dichloro resin (300 mg,0.3 mmol) was suspended in 5mL of dichloromethane, fmoc-AA-OH (0.9 mmol) and DIEA (154.8 mg,1.2 mmol) were then added, after 2 hours of reaction in a shaker, 300. Mu.L of methanol was added to cap for 10 minutes, and then Fmoc-AA-dichloro resin was washed 3 times with DMF. Fmoc-AA-dichloro resin was deprotected with 20% piperidine/DMF for 30 min. After completion, the H-AA-dichloro resin was washed four times with DMF. Subsequent amino acid coupling, using standard solid phase peptide synthesis procedure (SPPS), was performed until the end of the last amino acid Boc-AA-OH reaction. The polypeptide was cleaved from the dichloro resin using 25% hexafluoroisopropanol/dichloromethane for 1 hour, filtered, the resin was washed 3 times with dichloromethane, the filtrates combined and concentrated in vacuo to give the polypeptide. Finally, linear peptide (0.2 mmol), 1A (44 mg,0.2 mmol), EDCI (60 mg,0.3 mmol) and HOBT (40 mg,0.3 mmol) were dissolved in 3mL DMF using conventional liquid phase condensation reaction steps, followed by DIEA (78 mg,0.6 mmol) and stirred at room temperature for 12 hours. After the reaction was completed, 10mL of ethyl acetate and 10mL of water were added, and the organic layer was separated, washed with 5mL of 1n hydrochloric acid, 5mL of saturated sodium bicarbonate, 5mL of saturated sodium chloride solution, and dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give crude linear peptide (II), which was further separated and purified by passing through a silica gel column and preparing a silica gel plate.
The catalyst is one of the following: palladium acetate, palladium trifluoroacetate, palladium dichloride;
The oxidant is one of the following: oxygen, tert-butyl peroxybenzoate, p-benzoquinone, copper acetate and silver acetate;
The solvent is one of the following: para-xylene, N, N-dimethylformamide, acetic acid, toluene, tetrahydrofuran/acetic acid solution, 1,4 dioxane/acetic acid solution;
The ratio of the amounts of the tryptophan-containing polypeptide, the catalyst and the oxidant is 1:0.05-0.15:0.5-2.
Specifically, the volume ratio of the tetrahydrofuran/acetic acid solution or the 1, 4-dioxane/acetic acid solution is 1-5:1.
Preferably, the catalyst is palladium acetate, the oxidant is p-benzoquinone, the solvent is 1, 4-dioxane/acetic acid solution (3:1, v/v), the reaction temperature is 80 ℃, the reaction time is 24 hours, and the ratio of the amounts of tryptophan-containing polypeptide, the catalyst and the oxidant is 1:0.1:1.
The separation and purification method of the olefination derivative comprises the following steps: adding saturated NaCl aqueous solution into the reaction mixture, extracting with ethyl acetate, drying an organic layer by anhydrous sodium sulfate, filtering, and rotationally evaporating the solvent at normal temperature to obtain a crude product; and (3) performing silica gel column chromatography on the crude product, collecting the eluent, removing the solvent by decompression, and drying to obtain the tryptophan-containing polypeptide olefin ring-forming derivative shown in (I).
The invention also relates to application of the olefine derivative in preparing antitumor drugs.
Preferably, the tumor is lung cancer.
Preferably, the compound has the structure shown in the following formula:
The beneficial effects of the invention are mainly as follows: the compound is prepared by directly oxidizing a Heck reaction of a tryptophan side chain C (sp 2) -H through palladium catalysis, has the advantages of simple operation process, no guide group, high site selectivity and high reaction efficiency, can be prepared by only one step, has good anti-tumor application prospect, and provides a new scheme for developing anti-tumor drugs.
(IV) description of the drawings
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of a compound represented by the formula (I a);
FIG. 2 is a carbon spectrum of a compound of formula (I a);
FIG. 3 is a nuclear magnetic resonance hydrogen spectrum of a compound represented by the formula (I b);
FIG. 4 is a carbon spectrum of a compound of formula (I b);
FIG. 5 is an anti-tumor activity study of the cyclopeptide compound (I a).
(Fifth) detailed description of the invention
The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:
example 1: preparation of Compound (I a)
Olefin-containing tryptophan linear polypeptide as shown in formula II a (general synthesis procedure of cyclic peptide precursor (II) (0.2 mmol), p-benzoquinone (0.4 mmol), pd (OAc) 2 (0.02 mmol) was weighed into a 10 mL-specification round bottom flask, 10mL of 1,4 dioxane/acoh=3: 1 solution. The plug reaction was covered and heated to 80 degrees celsius for 24 hours. 40mL of ethyl acetate and 20mL of water were added to the reaction solution. The organic layer was washed with 20mL of 1n hydrochloric acid, 20mL of saturated sodium bicarbonate, 20mL of saturated sodium chloride solution, and dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo, and the obtained crude product was further purified by a silica gel column or a preparative silica gel plate (ethyl acetate: petroleum ether=4:1, r f =0.2), to obtain 80.7mg (yield 40%) of a pure compound represented by formula I a, whose nuclear magnetic hydrogen spectrum, carbon spectrum are shown in fig. 1 to 2.
1H NMR(600MHz,DMSO)δ11.43(s,1H),8.20(s,1H),8.15(d,J＝8.9Hz,1H),7.94(s,1H),7.89(d,J＝7.2Hz,1H),7.63(d,J＝15.9Hz,1H),7.53(d,J＝8.0Hz,1H),7.34(d,J＝8.2Hz,1H),7.22(t,J＝7.5Hz,1H),7.04(t,J＝7.6Hz,1H),6.82(d,J＝9.0Hz,1H),6.70(d,J＝15.4Hz,1H),6.40(d,J＝15.9Hz,1H),6.34(dd,J＝17.3,1.4Hz,1H),4.78–4.68(m,2H),4.55(d,J＝8.3Hz,1H),4.47–4.42(m,1H),4.32(dd,J＝13.6,7.5Hz,1H),3.79(s,1H),3.69(s,3H),3.65(d,J＝6.0Hz,1H),3.63(s,1H),3.29–3.19(m,2H),2.96(s,5H),2.84(dd,J＝15.3,5.7Hz,1H),2.46(s,3H),2.41(s,3H),1.41(s,8H),1.39(s,8H),1.38(s,7H).13C NMR(151MHz,DMSO)δ172.53,171.08,169.79,169.73,169.02,166.15,157.93,156.49,155.24,138.10,137.24,133.10,131.89,131.58,129.21,124.78,120.88,120.53,119.16,116.75,114.61,86.77,80.61,79.00,62.70,60.22,56.24,52.89,49.88,42.94,37.77,28.76,28.61,28.06,27.12,21.22,19.39,18.04,14.55,12.73.MS(ESI)m/z(relative intensity)1094.81(100)[M+H+].
Example 2: preparation of Compound (I b)
Olefin-containing tryptophan linear polypeptide as shown in formula II b (general synthesis procedure of cyclic peptide precursor (II) (0.2 mmol), p-benzoquinone (0.4 mmol), pd (OAc) 2 (0.02 mmol) was weighed into a 10 mL-specification round bottom flask, 10mL of 1,4 dioxane/acoh=3: 1 solution. The plug reaction was covered and heated to 80 degrees celsius for 24 hours. 40mL of ethyl acetate and 20mL of water were added to the reaction solution. The organic layer was washed with 20mL of 1n hydrochloric acid, 20mL of saturated sodium bicarbonate, 20mL of saturated sodium chloride solution, and dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo, and the obtained crude product was further purified by a silica gel column or a preparative silica gel plate (ethyl acetate: petroleum ether=4:1, r f =0.45), to obtain 80.7mg (yield 40%) of a pure compound represented by formula I b, whose nuclear magnetic hydrogen spectrum, carbon spectrum are shown in fig. 3 to 4.
1H NMR(600MHz,DMSO)δ11.43(s,1H),8.20(s,1H),8.15(d,J＝8.9Hz,1H),7.94(s,1H),7.89(d,J＝7.2Hz,1H),7.63(d,J＝15.9Hz,1H),7.53(d,J＝8.0Hz,1H),7.34(d,J＝8.2Hz,1H),7.22(t,J＝7.5Hz,1H),7.04(t,J＝7.6Hz,1H),6.82(d,J＝9.0Hz,1H),6.70(d,J＝15.4Hz,1H),6.40(d,J＝15.9Hz,1H),6.34(dd,J＝17.3,1.4Hz,1H),4.78–4.68(m,2H),4.55(d,J＝8.3Hz,1H),4.47–4.42(m,1H),4.32(dd,J＝13.6,7.5Hz,1H),3.79(s,1H),3.69(s,3H),3.65(d,J＝6.0Hz,1H),3.63(s,1H),3.29–3.19(m,2H),2.96(s,5H),2.84(dd,J＝15.3,5.7Hz,1H),2.46(s,3H),2.41(s,3H),1.41(s,8H),1.39(s,8H),1.38(s,7H).13C NMR(151MHz,DMSO)δ172.53,171.08,169.79,169.73,169.02,166.15,157.93,156.49,155.24,138.10,137.24,133.10,131.89,131.58,129.21,124.78,120.88,120.53,119.16,116.75,114.61,86.77,80.61,79.00,62.70,60.22,56.24,52.89,49.88,42.94,37.77,28.76,28.61,28.06,27.12,21.22,19.39,18.04,14.55,12.73.MS(ESI)m/z(relative intensity)1094.81(100)[M+H+].
Example 9: detection of antitumor Activity of Compound (I a)
Tumor cell A549 (lung cancer cell) is selected, and the MTT method is adopted to detect the proliferation activity of the anti-tumor cell. Cells were inoculated into 96-well plates containing 1640 medium of 10% fetal bovine serum at a concentration of 4000 to 5000 cells/well, and annotated on the plate cover, incubated at 5% CO 2 at 37℃for 12 hours, the cells were allowed to adhere to the 96-well plates, the drug to be tested (compound (I a) prepared in example 1) was added in a sterile operating station with a pipette gun to give five concentration gradients of 2. Mu.M, 5. Mu.M, 10. Mu.M, 20. Mu.M, 40. Mu.M, respectively, each concentration set with three parallel groups, and the 96-well plates were again incubated at 5% CO 2 at 37℃for 24 hours. Taking out the 96-well plate, adding 10 mu L of MTT kit reagent (purchased from Promega company) into each well, hatching for 4 hours at 37 ℃ in the dark under the condition of 5% CO 2, absorbing the supernatant, adding 150uL of sterile DMSO to dissolve formazan, further dissolving in an incubator at 37 ℃ for 5-10 min, and finally measuring the absorbance by using an enzyme-labeled instrument. Cell viability and cytotoxicity were thus calculated, IC 50 and IC 50% confidence interval was 22.976 ± 8.221, as seen in fig. 5, by treatment with GRAPHPAD PRISM software.
Experimental results show that the compound (I a) can target tumor cells with high expression of integrin alpha v beta 3 and has certain anti-tumor activity.

Claims (3)

1. An olefination ring derivative containing tryptophan polypeptide, the structure of which is shown as a formula (I a):
。
2. A process for preparing the tryptophan-containing polypeptide olefin derivative of claim 1, which comprises: taking an acrylic ester modified tryptophan-containing polypeptide shown in a formula (II a) as a substrate, and stirring and reacting in a solvent at 50-100 ℃ for 12-36 hours in the presence of a catalyst and an oxidant to prepare an olefin-containing cyclization derivative of the tryptophan-containing polypeptide shown in the formula (I a);
The catalyst is one of the following: palladium acetate, palladium trifluoroacetate, palladium dichloride;
The oxidant is one of the following: oxygen, tert-butyl peroxybenzoate, p-benzoquinone, copper acetate and silver acetate;
The solvent is one of the following: para-xylene, N, N-dimethylformamide, acetic acid, toluene, tetrahydrofuran/acetic acid solution, 1,4 dioxane/acetic acid solution;
The ratio of the amounts of the tryptophan-containing polypeptide, the catalyst and the oxidant is 1:0.05-0.15:0.5-2.
3. Use of an alkylated derivative according to claim 1 for the preparation of an anti-tumour agent, said tumour being lung cancer.
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CN111040020A (en) * 2018-12-28 2020-04-21 中国人民解放军军事科学院军事医学研究院 Alkene thioether staple peptide and preparation method and application thereof
CN112778403A (en) * 2021-01-04 2021-05-11 上海大学 Cyclic peptide antitumor active compound and preparation method and application thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111040020A (en) * 2018-12-28 2020-04-21 中国人民解放军军事科学院军事医学研究院 Alkene thioether staple peptide and preparation method and application thereof
CN112778403A (en) * 2021-01-04 2021-05-11 上海大学 Cyclic peptide antitumor active compound and preparation method and application thereof

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