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CN116655729A - BPA oligomer and targeting compound thereof, preparation method and application - Google Patents

BPA oligomer and targeting compound thereof, preparation method and application Download PDF

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CN116655729A
CN116655729A CN202310595357.8A CN202310595357A CN116655729A CN 116655729 A CN116655729 A CN 116655729A CN 202310595357 A CN202310595357 A CN 202310595357A CN 116655729 A CN116655729 A CN 116655729A
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bpa
ome
boc
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oligomer
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陈河如
蔡绍晖
杜军
王可
刘志军
曾燕聪
徐俊
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Chongqing Gaoboron Biotechnology Co ltd
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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Abstract

The invention discloses a BPA oligomer and a targeting compound thereof, a preparation method and application thereof, belonging to the fields of pharmaceutical chemistry and radiation medicine. The boron uptake and the T/N ratio of the BPA oligomer and the targeting compound thereof in tumor cells are superior to those of a positive control drug BPA; its permeability to cells is superior to that of BPA. The BPA oligomer targeting compound is capable of cleaving by fapα to release the corresponding BPA oligomer; the preparation method of the BPA oligomer and the targeting compound thereof has the characteristics of mild reaction conditions, simple experimental steps, high yield, high product purity, economy, practicability and the like.

Description

BPA oligomer and targeting compound thereof, preparation method and application
Technical Field
The invention relates to the fields of pharmaceutical chemistry and radiation medicine, in particular to a BPA oligomer for cancer boron neutron treatment, a targeting compound thereof and preparation thereof.
Background
Boron neutron capture therapy (boron neutron capture therapy, BNCT) is a fission reaction based on boron neutron capture. Non-radioactive isotopes 10 B atom is absorbed by low energy<0.5 eV) neutron (thermal neutron) split into two heavy particles 4 He (He) 7 Li), these particles release energy over a short distance (< 10 μm). This distance corresponds exactly to the diameter of a single cell. Based on this, if it is possible to 10 B is selectively delivered and enriched in malignant tumor cells, thus realizing the selective killing of tumor cells and simultaneously protecting normal tissues from damage. BNCT is a novel radiotherapy technique that is a binary therapy based on nuclear trapping and fission reactions. Compared with the traditional radiotherapy and chemotherapy, the method has the advantages of obvious low side effect, high selectivity and high efficiency. However, the technology not only needs high-quality neutron beams, but also needs high-targeting boron-containing drugs, and the high-quality neutron beams and the high-targeting boron-containing drugs are closely matched and indispensible, so that cell-level accurate targeted killing of tumors can be realized under the condition of both aspects. Currently, there has been a great deal of progress in the development of BNCT neutron beam equipment [ science fiction 2022, 67 (14), 1471-1478]The method comprises the steps of carrying out a first treatment on the surface of the In the aspect of boron medicine, only mercaptododecaboron disodium salt (BSH) and L-p-dihydroxyboro-phenylalanine (BPA) which are approved by the FDA for clinical application have the problem of insufficient tumor targeting.For BPA, the preparation also has the problems of small boron loading amount, narrow clinical indications (only suitable for glioma, melanoma and other few tumors), large clinical application dosage and the like. Therefore, the clinical and practical high-targeting boron drug still cannot meet the requirements of BNCT treatment.
Structurally, BPA belongs to an unnatural amino acid, whose uptake in vivo is likely to be transported by L-amino acid transporter 1 (L-amino acid tansporter, LAT 1). The research shows that the absorption process of amino acid consumes more energy, the carrier is easy to saturate and absorb slowly, and the absorption competition among amino acid exists; the oligopeptide, especially dipeptide and tripeptide, has low consumption energy, faster transport speed, less saturation of carrier, and lower osmotic pressure. Thus, converting BPA into oligomers, it is possible to increase the accumulation and selectivity of boron-containing compounds in tumor cells, while increasing the ability to penetrate the Blood Brain Barrier (BBB).
In recent years, a great deal of research shows that fibroblast activation protein alpha (FAPalpha) is a tumor interstitial antigen molecule which is specifically and highly expressed by tumor-related fibroblasts, and has important promotion effect on the occurrence and development of tumors. FApα has specific endopeptidase activity and is capable of selectively hydrolyzing N-terminally blocked glycylproline dipeptide sequences, such as substrates carrying Z-Gly-Pro (Z-GP) dipeptide. Therefore, the selection of fibroblast activation protein α (fΑpα) as a target may also be an effective strategy to increase the accumulation and selectivity of boron in tumor cells.
Disclosure of Invention
In order to improve the tumor targeting, BBB penetration and active absorption capacity of L-p-dihydroxyboryl phenylalanine (BPA), the invention aims to provide a BPA oligomer, a targeting compound and a preparation method thereof.
Another object of the present invention is to provide the use of the above-described BPA oligomers and their targeting compounds in the treatment of cancer by boron neutron capture.
In order to achieve the above purpose, the invention adopts the following specific scheme:
a BPA oligomer and a targeting compound thereof have the structure shown in formulas I and II,
wherein BPA is (S) -2-amino-3- [4- ] 10 B) Dihydroxyborane-based phenyl]Propionic acid; n=0, 1,2; Z-GP is benzyloxycarbonyl glycyl prolyl, which has the following structure:
preferably, the BPA oligomer and the targeting compound thereof include the following specific compounds:
the preparation method of the BPA oligomer compound comprises the following steps:
(1-1) preparation of N-Boc-BPA-OH: weighing N-tert-butoxycarbonyl-4-iodo-L-phenylalanine in a reaction flask, and sequentially adding tributyl borate at room temperature 10 B(O n Bu) 3 ]NaH, bis (2-dimethylaminoethyl) ether, and the reaction flask was placed in an ice bath. Slowly adding a metal organic reagent under the protection of nitrogen, removing an ice bath after the dropwise addition, stirring for 16 hours at room temperature, adding ice water under the ice bath condition, stirring for 10 minutes, quenching, adding methyl tertiary butyl ether, regulating the pH value to 3 by using concentrated hydrochloric acid, extracting by using ethyl acetate for two times, concentrating under reduced pressure to remove an organic solvent, adding water into the concentrate, regulating the pH value to 12 by using a 1mol/L NaOH aqueous solution, washing a water layer by using n-butanol for two times, regulating the pH value to 3 by using concentrated hydrochloric acid, stirring to separate out white precipitate, pulping and washing by using dichloromethane, filtering and drying to obtain the product.
The synthetic route is as follows:
in the above-mentioned steps, the step of,N-Boc-4-iodo-L-phenylalanine and tributyl borate 10 B(O n Bu) 3 ]The molar ratio of NaH, bis (2-dimethylaminoethyl) ether and metal organic reagent is 1:1.0-5.0, 1:1.0-3.0, 1:1.0-10.0 and 1:1.0-10.0 respectively;
the metal organic reagent is any one of isopropyl magnesium chloride and isopropyl magnesium chloride lithium chloride.
(1-2) preparation of N-Boc-BPA-OMe: N-Boc-4-dihydroxyboron-L-phenylalanine (N-Boc-BPA-OH) is weighed and dissolved in DMF, inorganic base is added at room temperature, methyl iodide is added dropwise after stirring for 30min, and stirring reaction is continued for 24 hours after the dropwise addition is completed. After the reaction, water was added, extraction was performed with ethyl acetate, and the organic layers were combined, washed with saturated sodium bicarbonate and saturated brine, respectively, and dried Na 2 SO 4 Drying, rotary evaporating to remove the organic solvent, and purifying the residue by silica gel column chromatography to obtain the target product.
The synthetic route is as follows:
in the step, the feeding mole ratio of the N-Boc-4-dihydroxyboron-L-phenylalanine to the inorganic base to the methyl iodide is 1:1.0-5.0 and 1:1.0-5.0 respectively; the inorganic base is any one of potassium bicarbonate, sodium bicarbonate and lithium bicarbonate.
(1-3) preparation of H-BPA-OMe: N-Boc-BPA-OMe is weighed, an acidic reagent is added, and the mixture is stirred at room temperature for reaction for 3 to 5 hours. And after the reaction is finished, removing the solvent by rotary evaporation, adding petroleum ether for pulping, and carrying out vacuum drying to obtain the product.
The synthesis steps are as follows:
in the step, the feeding molar ratio of the N-Boc-BPA-OMe to the acidic reagent is 1:4.0-10.0; the acidic reagent is any one of trifluoroacetic acid and 4mol/L HCl methanol solution.
(1-4)N-Boc-(BPA) n Preparation of OMe (n=2, 3, 4): weighing N-Boc-BPA-OH, condensing agent and organic alkali, dissolving in a proper amount of organic solvent, stirring for 30min, and adding H- (BPA) in batches m OMe (m=1, 2, 3), and after the addition, the reaction was stirred at room temperature for 12 hours. After the reaction is finished, adding an organic solvent, washing an organic phase by saturated sodium bicarbonate and water, washing by saturated saline water and anhydrous Na 2 SO 4 Drying, rotary evaporating to remove the organic solvent, and purifying the residue by silica gel column chromatography to obtain the target product.
The synthetic route is as follows:
in the above steps, N-Boc-BPA-OH is reacted with condensing agent, organic base, H- (BPA) m The molar ratio of the feed of OMe (m=1, 2, 3) is 1:1.1-1.5, 1:2.0-5.0, 1.1-1.5:1.0 respectively; the condensing agent is any one of 2- (7-oxo-benzotriazole) -N, N, N ', N ' -tetramethyl urea Hexafluorophosphate (HATU), ethyl chloroformate, 1-ethyl- (3-dimethyl aminopropyl) carbodiimide hydrochloride (EDCI), N, N ' -diisopropyl carbodiimide (DIC), benzotriazol-1-yl-oxy-tripyrrolidinylphosphine hexafluorophosphate (PyBOP), tetramethyl chlorourea hexafluorophosphate (TCFH); the organic base is any one of N, N-Diisopropylethylamine (DIPEA) and N-methylimidazole (NMI).
(1-5)H-(BPA) n Preparation of OMe (n=2, 3, 4): weighing the compound N-Boc- (BPA) n OMe (n=2, 3, 4), adding acid reagent, stirring at room temperature to react for 3-5 hr, rotating and steaming to eliminate solvent, adding petroleum ether to pulp, and vacuum drying to obtain the product.
The synthetic route is as follows:
in the above step, the compound N-Boc- (BPA) n -OMe (n=2, 3, 4) to acidic reagent feed molar ratio of 1.0:5.0-10.0; the acid is trifluoroacetic acid (TFA),4mol/L of HCl methanol solution.
(1-6) BPA oligomer [ (BPA) 2 ～(BPA) 4 ]Is prepared from the following steps: weighing compound H- (BPA) n OMe (n=2, 3, 4), adding alkaline reagent, stirring at room temperature for 1-4 hr, TLC tracking and monitoring until hydrolysis is completed, adding 1M HCl solution to regulate pH value to 6, precipitating white solid, filtering, washing and vacuum drying to obtain target product.
The synthetic route is as follows:
in the above step, compound H- (BPA) n -OMe (n=2, 3, 4) to alkaline agent feed molar ratio 1.0:5.0-15.0; the alkaline agent may be any aqueous solution of NaOH, KOH, liOH.
The preparation method of the BPA oligomer targeting compound comprises the following steps:
(2-1) preparation of N-benzyloxycarbonylglycinyl proline methyl ester (Z-GP-OMe): N-Boc-L-BPA-OH was replaced with N-Z-Gly-OH, H- (BPA) m OMe is prepared as described in step (1-4) using H-Pro-OMe.
The synthetic route is as follows:
(2-2) preparation of N-benzyloxycarbonylglycinyl proline (Z-GP-OH): the hydrolysis substrate was replaced with N-benzyloxycarbonylglycinyl proline methyl ester (Z-GP-OMe) and prepared as described in steps (1-6).
The synthetic route is as follows:
(2-3) Z-GP-BPA oligomer methyl ester [ Z-GP- (BPA) n -OMe,n＝2,3,4]Is prepared from the following steps: the N-Boc-L-BPA-OH was replaced with Z-GP-OH as described in step (1-4)The method is used for preparing the medicine. The synthetic route is as follows:
(2-4) targeting Compounds of BPA oligomer [ Z-GP- (BPA) n -OH,n＝2,3,4]Is prepared from the following steps: replacement of hydrolysis substrate with Z-GP-BPA oligomer methyl ester [ Z-GP- (BPA) n -OMe,n＝2,3,4]Prepared according to the method described in step (1-6). The synthetic route is as follows:
the invention detects cytotoxicity of serial BPA oligomers and targeting compounds thereof to Human Umbilical Vein Endothelial Cells (HUVEC), human normal liver cells (LO 2) and human liver cancer cells (HepG 2) by using an MTT method, and discovers that the compounds have no toxicity or extremely low toxicity in the concentration range of 500 mu M.
The invention utilizes ICP-MS method to detect the uptake of series BPA oligomer and targeting compound thereof in Human Umbilical Vein Endothelial Cells (HUVEC), human liver cancer cells (HepG 2), FAP alpha transfected human liver cancer cells (HepG 2/FAP alpha), human breast cancer cells (MDA-MB-231) and human glioma cells (U87) and T/N ratio (uptake of tumor cell boron/uptake of normal cell boron). As a result, it was found that the uptake of boron in the cells under investigation was greater than 10 for both the series of BPA oligomers and their targeting compounds 9 B/cell, T/N>3, a step of; the BPA oligomers and their targeting compounds exhibit superior boron uptake characteristics over BPA for different tumor cells compared to BPA.
The invention utilizes a shake flask method and an HPLC technology to detect the lipophilic and hydrophilic coefficients of the series of BPA oligomers and the targeting compounds thereof. As a result, it was found that all BPA oligomers and their targeting compounds exhibit a lipophilic hydrophilic character over BPA.
As a preferred embodiment of the use according to the invention, the targeting compound of the BPA oligomer is a specific hydrolysis substrate for tumor stromal fibroblast activation protease alpha (fapα).
Compared with the prior art, the invention has the following beneficial effects:
(1) The compounds shown in the general formulas I and II and the synthesis method thereof are reported for the first time. The synthesis method has the characteristics of mild reaction conditions, simple experimental steps, high yield, high product purity, economy, practicability and the like;
(2) Compared with BPA, the BPA oligomer and the targeting compound thereof have higher T/N ratio and boron loading in tumor cells;
(3) Compared with BPA, the BPA oligomer and the targeting compound thereof have better oleophilic and hydrophilic characteristics.
Drawings
FIG. 1 shows the boron loading of BPA oligomer and its targeting compound in human hepatoma cell HepG-2 and FAP alpha transfected human hepatoma cell HepG-2.
FIG. 2 is a T/N ratio of BPA oligomer and its targeting compound in human hepatoma cell HepG-2 and FAP alpha transfected human hepatoma cell HepG-2 to human umbilical vein endothelial cell HUVEC.
FIG. 3 is a graph of F.alpha.P.alpha.versus the enzymatic hydrolysis rate of compound Z-GP- (BPA) 2.
FIG. 4 is a graph of F.alpha.P.alpha.versus the enzymatic hydrolysis rate of compound Z-GP- (BPA) 3.
FIG. 5 is a graph of F.alpha.P.alpha.versus the enzymatic hydrolysis rate of compound Z-GP- (BPA) 4.
Detailed Description
The present invention is further illustrated by the following examples, which are not intended to limit the invention in any way.
EXAMPLE 1 preparation of N-Boc-BPA-OH
1.17g (3.0 mmol) of N-t-butoxycarbonyl-4-iodo-L-phenylalanine was weighed into a 100ml three-necked flask and tributyl borate [ was added successively at room temperature 10 B(O n Bu) 3 ]2.14g (9.3 mmol), naH 0.24g (60%) (6.0 mmol) and bis (2-dimethylaminoethyl) ether 3.85g (24.0 mmol) were slowly added under nitrogen protection in ice bath with 14mL (24.0 mmol) of isopropyl magnesium chloride (1.7M in THF) under ice bath conditions, after the addition of 30min was completed, after the ice bath was removed, stirred at room temperature for 16h, and under ice bath conditions with ice water added and stirred for 10minQuenching, adding methyl tertiary butyl ether, regulating the pH value to 3 by using concentrated hydrochloric acid, extracting twice, separating an organic layer, adding an aqueous solution of NaOH into the organic layer, regulating the pH value to 12, washing twice by using n-butanol after extracting a water layer, regulating the pH value to 3 by using concentrated hydrochloric acid, stirring to separate out white precipitate, filtering, and vacuum-drying to obtain 653.0mg of white solid, wherein the yield is 70.4%. 1 H NMR(400MHz,DMSO-d 6 )δ7.96(s,2H),7.71(d,J＝6.6Hz,2H),7.21(d,J＝6.5Hz,2H),7.05(d,J＝7.7Hz,1H),4.12(s,1H),3.03(d,J＝12.4Hz,1H),2.94-2.73(m,1H),1.32(s,9H)； 13 C NMR(101MHz,DMSO-d 6 )δ174.09,155.91,140.39,134.51,132.34,128.62,78.56,55.50,36.96,28.61。
EXAMPLE 2.H preparation of BPA-OMe hydrochloride
Synthesized through two steps.
Preparation of N-Boc-BPA-OMe: N-Boc-BPA-OH 2g (6.5 mmol) was weighed into a 25mL round bottom flask and dissolved in 10mL DMF and KHCO was added at room temperature 3 1.3g, stirring for half an hour, adding 0.8mL of methyl iodide, stirring at room temperature for 24 hours, adding 20mL of ethyl acetate after the reaction, washing the organic layers with saturated brine (3X 10 mL), 1M hydrochloric acid (3X 10 mL) and saturated sodium bicarbonate (3X 10 mL) in sequence, combining the organic phases, concentrating, purifying by column chromatography (DCM/MeOH=30/l, R f =0.36), dried in vacuo to give 1.786g of a white solid in 85.4% yield. 1 H NMR(400MHz,CDCl 3 )δ7.93(s,2H),7.52(d,J＝8.0Hz,2H),7.21(d,J＝7.8Hz,2H),5.04(d,J＝7.4Hz,1H),4.64(d,J＝7.0Hz,1H),3.75(s,3H),3.14(ddd,J＝31.9,13.8,5.9Hz,2H),1.44(s,9H)； 13 CNMR(101MHz,CDCl 3 )δ169.45,161.86,136.90,136.44,135.51,128.58,84.08,54.27,53.27,36.33,24.71。
Preparation of H-BPA-OMe hydrochloride: 0.97g (3.0 mmol) of N-Boc-BPA-OMe was weighed into a 25mL round bottom flask, 4mL of 4M hydrochloric acid methanol solution was added, stirred at room temperature for 4h, concentrated under reduced pressure, and dried under vacuum to give 0.762g of white solid with a yield of 97.9%. 1 H NMR(400MHz,DMSO-d 6 )δ8.84(br,2H),8.04(s,2H),7.77(d,J＝7.5Hz,2H),7.20(d,J＝7.6Hz,2H),4.34-4.07(s,1H),3.59(s,3H),3.32-2.97(m,2H)； 13 C NMR(101MHz,DMSO-d 6 )δ169.64,137.00,134.96,133.25,128.92,105.12,53.77,53.03,36.27。
Example 3.Z preparation of GP-OH
Synthesized through two steps.
Preparation of Z-GP-OMe: l-proline methyl ester hydrochloride 0.497g (3.0 mmol) and N-benzyloxycarbonglycine 0.628g (3.0 mmol) were weighed into a 50mL reaction flask, 20mL of acetonitrile was added and stirred for dissolution, NMI 0.739g (9.0 mmol) and TCFH 287.6mg (3.0 mmol) were added at room temperature, and the reaction was stirred at room temperature for 4 hours. After TLC monitoring the end of the reaction, the solvent was removed by spinning, after 20mL of DCM was added, the organic layers were washed successively with distilled water (3X 10 mL), 1M hydrochloric acid (3X 10 mL), saturated sodium bicarbonate (3X 10 mL), the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by column chromatography (DCM/MeOH=20/l, R f =0.28) to give 902.0mg of a colorless oily liquid in a yield of 93.6%. 1 H NMR(400MHz,CDCl 3 )δ7.27(m,5H),5.87(s,1H),5.03(s,2H),4.42(d,J＝7.6Hz,1H),3.92(dd,J＝10.1,3.8Hz,2H),3.62(s,1H),3.55-3.27(m,2H),2.07(m,1H),1.91(m,3H)； 13 C NMR(101MHz,CDCl 3 )δ172.36,167.19,156.35,136.54,128.41,127.95,127.87,66.67,58.84,52.24,45.83,43.26,28.91,24.57。
Preparation of Z-GP-OH: 0.96g (3.0 mmol) of Z-GP-OMe was dissolved in 10ml of THF, 10ml of 1M NaOH solution was added and stirred at room temperature for 12 hours, TLC was used to monitor completion of the substrate reaction, 1M HCl solution was added to adjust pH to 2, THF was removed by concentration under reduced pressure, white solid was precipitated, filtered, purified by column chromatography (DCM/MeOH=10/l, R f =0.31), and dried under vacuum to give 0.823g of a white solid with a yield of 95.4%. 1 H NMR(300MHz,DMSO-d 6 )δ12.64(s,1H),7.37(m,5H),5.05(s,2H),4.25(dd,J＝8.7,3.2Hz,1H),3.84(ddd,J＝33.3,17.2,6.0Hz,2H),3.60-3.28(m,2H),2.13(m,1H),1.91(m,3H)； 13 C NMR(75MHz,DMSO-d 6 )δ173.82,167.61,156.94,137.59,128.87,128.24,128.14,65.97,59.06,45.90,43.08,29.07,24.83。
EXAMPLE 4 BPA oligomer [ (BPA) 2 ～(BPA) 4 ]Is prepared from
BPA dimer [ (BPA) 2 ]Is prepared from
Synthesized in 3 steps.
4.1.1.N-Boc-(BPA) 2 Preparation of OMe: 0.831g (3.0 mmol) of H-BPA-OMe hydrochloride and 0.99g (3.0 mmol) of N-Boc-BPA-OH were weighed into a 50ml reaction flask, 15mL of dry redistilled DCM was added, 1.5mL of DMF was dissolved, placed in an ice bath, 1.255g (3.3 mmol) of HATU was slowly added, 4.5mL of DIPEA (9.0 mmol) was added after 5min, and after 30mm stirring, the mixture was moved to room temperature and stirred for 12H. After completion of the TLC monitoring, DCM 10mL was added to the reaction mixture, the organic layers were washed successively with distilled water (3X 10 mL), 1M hydrochloric acid (3X 10 mL), saturated sodium bicarbonate (3X 10 mL), the organic phases were combined, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography (PE/EA=2/1, V/V, R) f =0.23) was separated and purified by gradient elution to obtain 1.279g of a white solid with a yield of 82.9%. 1 H NMR(400MHz,DMSO-d 6 )δ8.31(d,J＝7.2Hz,1H),7.96(s,2H),7.93(s,2H),7.70(dd,J＝13.5,7.4Hz,4H),7.20(d,J＝6.9Hz,4H),6.84(d,J＝8.5Hz,1H),4.52(m,1H),4.20(m,1H),3.59(s,3H),3.13-2.79(m,3H),2.69(m,1H),1.29(s,9H)； 13 C NMR(101MHz,DMSO-d 6 )δ172.34,172.25,155.57,140.45,139.26,134.66(2),134.38(2),128.69(2),128.64(2),103.48,103.23,78.53,60.23,53.92,52.32,37.88,37.27,28.59(3)。
4.1.2.H-(BPA) 2 Preparation of OMe hydrochloride: N-Boc- (BPA) was weighed out 2 OMe 1.028g (2.0 mmol) in a 25mL round bottom flask, 3mL of 4M methanolic hydrochloric acid was added, stirred at room temperature for 4h, concentrated under reduced pressure, and purified by column chromatography (DCM/MeOH=5/l, V/V, R f =0.11) was dried under vacuum to give 0.893g of a white solid in 99.1% yield. 1 H NMR(400MHz,DMSO-d 6 )δ9.22(br,2H),8.33(m,1H),8.14(s,4H),7.72(m,4H),7.23(dd,J＝11.8,7.0Hz,4H),4.54(m,1H),4.06(m,1H),3.59(s,3H),3.15(m,1H),3.00(m,3H)； 13 C NMR(101MHz,DMSO-d 6 )δ171.62,168.63,139.10,137.06,134.79(2),134.73(2),129.14(2),128.66(2),103.96,103.82,54.32,53.51,52.54,37.26,37.17。
BPA dimer [ (BPA) 1.3 2 ]Is prepared from the following steps: weighing H- (BPA) 2 OMe hydrochloride 0.45g (1.0 mmol), adding 1M NaOH solution 6ml, stirring at room temperature for 1 hr, TLC monitoring substrate hydrolysis, adding 1M HCl solution to adjust pH to 6, precipitating white solid, filtering, washing, and vacuumAir-drying gave 0.342g of a white solid in 85.5% yield. 1 H NMR(400MHz,CD 3 OD)δ8.92(br,2H),8.31(m,1H),7.76(m,4H),7.69(dd,J＝20.6,7.3Hz,4H),7.22(t,J＝7.8Hz,4H),4.50(m,1H),3.71(m,1H),3.22(m,1H),3.08(m,2H),2.74(m,1H)； 13 CNMR(101MHz,CD 3 OD)δ178.48,176.53,140.47,139.03,134.14(2),133.70(2),128.65(2),128.50(2),111.12,107.52,55.84,55.69,48.64,39.77；TOF-MS ES(m/z):calcd.for C 18 H 21 10 B 2 N 2 O 7 ([M-H] - ) 397.1613, found:397.1615. All data confirm that the compound is a BPA dimer [ (BPA) 2 ]The structure is as follows:
BPA trimer [ (BPA) 3 ]Is prepared from
Synthesized in 3 steps.
4.2.1.N-Boc-(BPA) 3 Preparation of OMe: 0.831g (3.0 mmol) of H-BPA-OMe hydrochloride was replaced by H- (BPA) 2 1.344g (3.0 mmol) of OMe hydrochloride, prepared in accordance with the method of step 4.1.1. 1.753g of white solid was obtained in a yield of 83.2%. 1 H NMR(400MHz,DMSO-d 6 )δ8.33(m,2H),7.98(s,2H),7.94(m,4H),7.73(dd,J＝13.5,7.4Hz,6H),7.23(d,J＝6.9Hz,6H),6.85(m,1H),4.55(m,2H),4.23(m,1H),3.61(s,3H),3.15-2.81(m,4H),2.72(m,2H),1.31(s,9H)； 13 C NMR(101MHz,DMSO-d 6 )δ172.35,172.27,172.23,155.57,140.45,140.31,139.27,134.71(3),134.42(3),128.65(3),128.62(3),103.51,103.35,103.27,78.56,60.21,59.16,56.75,52.18,37.79,37.56,36.92,28.57(3)。
4.2.2.H-(BPA) 3 Preparation of OMe hydrochloride: N-Boc- (BPA) was weighed out 3 1.405g (2.0 mmol) of OMe was prepared according to the method of step 4.1.2. 1.20g of white solid was obtained in 99.3% yield. 1 H NMR(400MHz,DMSO-d 6 )δ9.22(br,2H),8.31(m,2H),8.11(m,6H),7.73(m,6H),7.25(m,6H),4.85(m,1H),4.57(m,1H),4.06(m,1H),3.63(s,3H),3.19(m,2H),3.03(m,4H)； 13 C NMR(101MHz,DMSO-d 6 )δ171.62,170.93,169.12,139.11,138.63,137.11,134.76(3),134.75(3),129.15(3),128.67(3),103.96,103.82,103.53,55.23,54.35,53.56,52.52,38.65,37.25,37.18。
BPA trimer [ (BPA) 2.3 3 ]Is prepared from the following steps: weighing H- (BPA) 3 0.64g (1.0 mmol) of OMe hydrochloride, prepared in accordance with the method of step 4.1.3. 0.50g of white solid was obtained in a yield of 84.6%. 1 H NMR(400MHz,CD 3 OD)δ8.93(br,2H),8.34(m,2H),7.75(m,6H),7.69(m,6H),7.22(m,6H),4.91(m,1H),4.72(m,1H),3.87(m,1H),3.23(m,2H),3.11(m,2H),2.76(m,2H)； 13 C NMR(101MHz,CD 3 OD)δ178.45,176.51,176.37,140.52,140.34,139.15,134.15(3),133.72(3),128.64(3),128.51(3),111.15,107.53,107.45,57.32,55.84,55.72,48.65,39.82,39.36；TOF-MS ES(m/z):calcd.for C 27 H 31 10 B 3 N 3 O 10 ([M-H] - ) 587.2403, found:587.2405. All data confirm that the compound is a BPA trimer [ (BPA) 3 ]The structure is as follows:
BPA tetramer [ (BPA) 4 ]Is prepared from
Synthesized in 3 steps.
4.3.1.N-Boc-(BPA) 4 Preparation of OMe: 0.831g (3.0 mmol) of H-BPA-OMe hydrochloride was replaced by H- (BPA) 3 1.873g (3.0 mmol) of OMe hydrochloride, prepared as in step 4.1.1. 2.184g of a white solid was obtained in a yield of 81.6%. 1 H NMR(400MHz,DMSO-d 6 )δ8.31(m,3H),7.95(m,4H),7.93(m,4H),7.76(m,8H),7.25(m,8H),6.93(m,1H),4.63(m,3H),4.27(m,1H),3.63(s,3H),3.17-2.83(m,5H),2.73(m,3H),1.31(s,9H)； 13 C NMR(101MHz,DMSO-d 6 )δ172.46,172.31,172.27,171.93,155.59,140.47,140.33,139.44,139.27,134.68(4),134.43(4),128.67(4),128.65(4),103.52,103.36(2),103.25,78.58,60.13,59.17,58.67,56.74,52.16,37.83,37.56,37.33,36.94,28.55(3)。
4.3.2.H-(BPA) 4 Preparation of OMe hydrochloride: weighing N-Boc- (B)PA) 4 OMe 1.785g (2.0 mmol), prepared according to the method of step 4.1.2. 1.562g of white solid was obtained in 98.6% yield. 1 H NMR(400MHz,DMSO-d 6 )δ9.18(br,2H),8.34(m,3H),8.05(m,8H),7.75(m,8H),7.21(m,8H),4.87(m,1H),4.83(m,1H),4.56(m,1H),4.02(m,1H),3.65(s,3H),3.23(m,3H),3.05(m,5H)； 13 C NMR(101MHz,DMSO-d 6 )δ171.73,171.61,170.95,169.21,139.13,138.65,137.11,136.58,134.69(4),134.73(4),129.12(4),128.65(4),103.94,103.78,103.68,103.46,55.67,55.34,54.32,53.54,52.31,38.72,37.37,37.25,36.83。
BPA tetramer [ (BPA) 3.3.3.BPA tetramer 4 ]Is prepared from the following steps: weighing H- (BPA) 4 0.83g (1.0 mmol) of OMe hydrochloride, prepared in accordance with the method of step 4.1.3. 663.9mg of a white solid was obtained in a yield of 85.3%. 1 H NMR(400MHz,CD 3 OD)δ8.89(br,2H),8.36(m,3H),7.79(m,8H),7.65(m,8H),7.19(m,8H),4.94(m,1H),4.92(m,1H),4.74(m,1H),3.93(m,1H),3.25(m,3H),3.13(m,2H),2.75(m,3H)； 13 C NMR(101MHz,CD 3 OD)δ178.52,176.51,176.37,176.15,140.61,140.26,139.54,139.21,134.17(4),133.75(4),128.62(4),128.53(4),110.85,109.32,106.54,106.18,58.21,57.36,55.78,55.66,48.33,39.73,39.28,36.76；TOF-MS ES(m/z):calcd.for C 36 H 41 10 B 4 N 4 O 13 ([M-H] - ) 777.3193, found:777.3187. All data confirm that the compound is a BPA tetramer [ (BPA) 4 ]The structure is as follows:
EXAMPLE 5 BPA oligomer targeting Compound [ Z-GP- (BPA) 2 ～Z-GP-(BPA) 4 ]Is prepared from
BPA dimer targeting compound [ Z-GP- (BPA) 2 ]Is prepared from
Synthesized in 2 steps.
5.1.1.Z-GP-(BPA) 2 Preparation of OMe: 0.613g (2.0 mmol) of the compound Z-GP-OH was weighed into a 50mL reaction flask, dissolved in 10mL of distilled DCM and 1mL of DMF was added dropwise, and placed in an ice bath, and HAT was slowly addedU0.76 g (2.2 mmol), 3ml DIPEA (6.0 mmol) after 5min and H- (BPA) after 5min 2 OMe hydrochloride 0.9g (2.0 mmol), stirred for 30mm and then brought to room temperature for stirring for 12h. After completion of the TLC monitoring, 10mL of DCM was added to the reaction mixture, the organic layers were washed successively with distilled water (3X 10 mL), 1M hydrochloric acid (3X 10 mL), saturated sodium bicarbonate (3X 10 mL), the organic phases were combined, dried over anhydrous sodium sulfate, and chromatographed on silica gel (DCM/MeOH=20/1, V/V, R) f =0.23) was separated and purified by gradient elution to obtain 1.163g of a white solid with a yield of 83.0%. 1 H NMR(400MHz,DMSO-d 6 )δ8.22(m,2H),7.95(m,1H),7.92(s,4H),7.77-7.63(m,4H),7.33(m,5H),7.18(m,4H),5.03(s,2H),4.90(m,1H),4.51(m,1H),4.43(m,1H),3.85(m,1H),3.53(s,3H),3.45(m,2H),3.18(m,2H),3.00(m,3H),2.78(m,1H),1.62(m,2H)； 13 C NMR(101MHz,DMSO-d 6 )δ172.11,171.54,171.43,168.43,156.95,139.32,137.57,137.42,134.62(2),134.38(2),128.78,128.68,128.59(2),128.50(2),128.24,128.15,128.11,105.24,104.92,65.96,60.22,56.81,56.73,54.29,53.85,52.19,43.29,37.24,36.85,29.17,24.46。
5.1.2.Z-GP-(BPA) 2 Is prepared from the following steps: weighing Z-GP- (BPA) 2 OMe 0.70g (1.0 mmol), adding 1mol/LNaOH solution 6ml, stirring at room temperature for 1 hr, TLC monitoring substrate hydrolysis, adding 1mol/L HCl solution to adjust pH to 2, precipitating precipitate, filtering, purifying by column chromatography (DCM/MeOH=9/L, V/V, R) f =0.21) was dried under vacuum to give 0.523g of a white solid in 75.9% yield. 1 H NMR(400MHz,DMSO-d 6 )δ8.36(m,2H),7.96(m,1H),7.93(s,4H),7.67(d,J＝7.4Hz,4H),7.33(m,5H),7.17(d,J＝7.5Hz,4H),5.02(s,2H),4.93(m,1H),4.74(m,1H),4.45(m,1H),3.84(s,2H),3.47(m,2H),3.19(m,2H),2.96(m,3H),2.78(m,1H),1.62(m,2H)； 13 C NMR(101MHz,DMSO-d 6 )δ172.17,171.48,171.32,168.57,156.93,139.35,137.54,137.52,134.71(2),134.42(2),128.82,128.73,128.61(2),128.53(2),128.22,128.16,128.13,105.25,104.93,65.97,60.23,56.84,56.75,54.32,53.86,43.33,37.37,36.83,29.22,24.51；TOF-MS ES-(m/z):calcd.for.C 33 H 37 10 B 2 N 4 O 11 ([M-H] - ) 685.2723, found:685.2726. All data confirm that the compound is BPDimer targeting compound [ Z-GP- (BPA) 2 ]The structure is as follows:
BPA trimer targeting Compounds [ Z-GP- (BPA) 3 ]Is prepared from
Synthesized in 2 steps.
5.2.1.Z-GP-(BPA) 3 Preparation of OMe: h- (BPA) 2 OMe hydrochloride 0.9g (2.0 mmol) was replaced by H- (BPA) 3 1.276g (2.0 mmol) of OMe hydrochloride, prepared according to the method of step 5.1.1. 1.467g of white solid was obtained in 82.4% yield. 1 H NMR(400MHz,DMSO-d 6 )δ8.24(m,3H),7.95(m,1H),7.91(s,6H),7.78-7.65(m,6H),7.33(m,5H),7.19(m,6H),5.05(s,2H),4.91(m,2H),4.53(m,1H),4.41(m,1H),3.87(m,1H),3.61(s,3H),3.47(m,2H),3.19(m,3H),3.05(m,3H),2.83(m,2H),1.62(m,2H)； 13 CNMR(101MHz,DMSO-d 6 )δ172.13,171.57,171.46,171.34,168.62,156.68,139.37,138.74,137.62,137.53,134.62(3),134.38(3),128.92,128.75,128.59(3),128.50(3),127.67,127.26,127.18,105.25,104.93,104.68,66.87,59.36,58.41,56.83,56.72,54.83,52.16,42.41,37.44(2),36.87,29.52,24.36。
5.2.2.Z-GP-(BPA) 3 Is prepared from the following steps: weighing Z-GP- (BPA) 3 OMe 0.89g (1.0 mmol), prepared as described in experimental procedure 5.1.2. 0.671g of white solid was obtained in 76.6% yield. 1 H NMR(400MHz,DMSO-d 6 )δ8.32(m,3H),7.94(m,1H),7.91(s,6H),7.73(d,J＝7.3Hz,6H),7.33(m,5H),7.19(d,J＝7.4Hz,6H),5.04(s,2H),4.92(m,2H),4.73(m,1H),4.45(m,1H),3.84(s,2H),3.49(m,2H),3.21(m,3H),2.95(m,3H),2.81(m,2H),1.63(m,2H)； 13 C NMR(101MHz,DMSO-d 6 )δ172.19,171.52,171.37,171.25,168.64,156.89,139.28,137.43(2),137.31,134.68(3),134.35(3),128.79,128.68,128.57(3),128.45(3),128.31,128.22,128.15,105.26,104.92(2),66.72,60.12,58.76,58.32,56.83,54.84,43.35,37.41(2),36.58,29.54,24.33；TOF-MS ES-(m/z):calcd.for.C 42 H 47 10 B 3 N 5 O 14 ([M-H] - )875.3513, found:875.3517. All data confirm that this compound is a BPA trimer targeting compound [ Z-GP- (BPA) 3 ]The structure is as follows:
BPA tetramer targeting compound [ Z-GP- (BPA) 3 ]Is prepared from
Synthesized in 2 steps.
5.3.1.Z-GP-(BPA) 4 Preparation of OMe: h- (BPA) 2 OMe hydrochloride 0.9g (2.0 mmol) was replaced by H- (BPA) 4 1.657g (2.0 mmol) of OMe hydrochloride, prepared as in step 5.1.1. 1.467g of white solid was obtained in a yield of 81.8%. 1 H NMR(400MHz,DMSO-d 6 )δ8.32(m,4H),7.97(m,1H),7.93(s,8H),7.79-7.67(m,8H),7.32(m,5H),7.21(m,8H),5.06(s,2H),4.92(m,3H),4.56(m,1H),4.42(m,1H),3.84(m,2H),3.65(s,3H),3.43(m,2H),3.16(m,4H),3.03(m,3H),2.85(m,3H),1.64(m,2H)； 13 CNMR(101MHz,DMSO-d 6 )δ172.15,171.61(2),171.43,171.32,168.91,156.34,139.41,138.72(2),137.58,137.47,134.61(4),134.36(4),128.89,128.72,128.58(4),128.46(4),127.65,127.23,127.14,105.24,104.92,104.65(2),66.86,59.34,58.46(2),56.81,56.69,54.85,52.18,42.36,37.43(2),36.85(2),29.48,24.33。
5.3.2.Z-GP-(BPA) 4 Is prepared from the following steps: weighing Z-GP- (BPA) 4 OMe 1.08g (1.0 mmol) prepared as described in experimental procedure 5.1.2. 0.834g of white solid was obtained in 78.2% yield. 1 H NMR(400MHz,DMSO-d 6 )δ8.34(m,4H),7.98(m,1H),7.93(s,8H),7.74(d,J＝7.4Hz,8H),7.33(m,5H),7.21(d,J＝7.3Hz,8H),5.05(s,2H),4.93(m,3H),4.71(m,1H),4.43(m,1H),3.86(s,2H),3.48(m,2H),3.26(m,4H),2.96(m,4H),2.83(m,2H),1.65(m,2H)； 13 C NMR(101MHz,DMSO-d 6 )δ172.21,171.46,171.35(2),171.23,168.55,156.82,139.23,137.47(2),137.36(2),134.64(4),134.32(4),128.81,128.73,128.54(4),128.42(4),128.35,128.23,128.17,105.24(2),104.89(2),66.83,60.21,58.82,58.31(2),56.85,54.86,42.33,37.45(3),36.54,29.52,24.28；TOF-MS ES-(m/z):calcd.for C 51 H 57 10 B 4 N 6 O 17 ([M-H] - ) 1065.4303, found:1065.4301. All data confirm that this compound is a BPA tetramer targeting compound [ Z-GP- (BPA) 4 ]The structure is as follows:
EXAMPLE 6 BPA oligomer and targeting Compounds thereof cytotoxicity
The cell culture solution containing the peptide bovine serum was incubated at 37℃for 24 hours. Subculturing human normal liver cell LO2 cells with tissue culture medium to obtain 1×10 5 Each/mL of the cell suspension was inoculated into a 96-well cell culture plate (100. Mu.l/well), and cultured in a carbon dioxide incubator at 37℃for 24 hours. After the equal cells are grown by adherence, removing the supernatant, adding tissue culture media of a control group (without adding drugs) and a test group (adding drugs) for incubation and culture, wherein the concentration of the drugs in the cell culture fluid is 500 mu mol/L, and placing the cell culture fluid in a carbon dioxide incubator at 37 ℃ for continuous culture. After incubation for 2 days, the cells were removed. MTT solution was added for further culture for 4 hours. The cell incubation was removed, DMSO was then added, and shaking was performed for 10min, and finally the absorbance was measured with a microplate reader at a wavelength of 570nm, and the relative proliferation degree (RGR) of the cells was calculated according to the formula from the absorbance. The relative proliferation of cells was calculated as:
the results are shown in Table 1. It can be seen that the RGR of the series of BPA oligomers and their targeting compounds after 2 days incubation with human normal hepatocytes LO2 cells was nearly 100%, indicating that these compounds were not toxic or very low in human normal hepatocytes at a concentration of 500. Mu. Mol/L.
Table 1 relative proliferation of cells (RGR) as measured by MTT colorimetry
EXAMPLE 7 boron uptake characterization of series BPA oligomers and their targeting compounds in cells
The boron uptake characteristics of the series of BPA oligomers and the targeting compounds thereof in Human Umbilical Vein Endothelial Cells (HUVEC), human liver cancer cells HepG2, FAP alpha transfected human liver cancer cells HepG2 (HepG 2/FAP alpha), human breast cancer cells MDA-MB-231 and human glioma cells U87 are determined by adopting an ICP-MS method.
The specific process comprises the following steps: adding appropriate amount of RPMI 1640 culture solution (fetal bovine serum 10% and penicillin 100U/ml) into tumor cells in logarithmic growth phase, and adjusting cell concentration to 1×10 5 Each ml was inoculated into 6-well plates, and 100. Mu.l of tumor cell suspension was inoculated per well. After 24h of culture, the prepared test drug (no drug is added in the control group) is added to make the final concentration of the drug be 100 mu M, and 5% CO is added 2 After incubation in incubator at 37℃for 24h, the medium in the dishes was removed with a pipette, washed with PBS (3X 1 mL), 0.5mL pancreatin was added to the dishes, followed by CO at 37℃constant temperature 2 Culturing in an incubator for 3min, observing whether cells shrink and round under a microscope, rapidly adding 0.6ml of culture medium to stop digestion after the shrinkage and round, lightly blowing the adherent cells to enable the cells to fall off from the bottom of a culture dish, transferring the cell suspension into a centrifuge tube, diluting, and counting by a blood cell counting plate to calculate the number of cells in each hole. The cell suspension was centrifuged, the supernatant was removed, and PBS was added to wash (3X 1 mL). Adding 500 mu L of concentrated nitric acid into the washed cell-containing centrifuge tube for digestion; the boron content in the cells was measured by ICP-MS and the T/N ratio was calculated. The definition of the T/N ratio is: boron uptake in tumor cells/boron loading in HUVEC cells.
The results are shown in tables 2 to 5. It can be seen that in the four tumor cells examined, the boron loading of all BPA oligomers and their targeting compounds was greater than 10 9 The T/N ratio of each B/cell is more than 3.0, and meets the precondition of serving as a boron delivery agent. All BPA oligomers and their targeting compounds exhibited boron uptake characteristics superior to the positive control BPA; in tumor cells not transfected with fapα, BAP oligomers all showed superior boron uptake over the corresponding targeting compoundsFeatures; however, in fapα transfected HepG-2 cells, the BPA oligomer targeting compounds all exhibited higher boron loading and T/N ratios than the corresponding BPA oligomers.
TABLE 2 boron uptake characteristics of series BPA oligomers and targeting compounds thereof in human liver cancer HepG2 cells
a The T/N ratio is defined as the boron uptake of the human hepatoma cell HepG 2/the boron uptake of the human umbilical vein endothelial cell HUVEC
TABLE 3 boron uptake characterization of series BPA oligomers and their targeting compounds in FAP alpha human liver cancer transfected HepG2 cells
a The T/N ratio is defined as the boron uptake of human hepatoma cell HepG2 transfected with FAP alpha/the boron uptake of human umbilical vein endothelial cell HUVEC
Table 4 boron uptake characteristics of series BPA oligomers and targeting compounds thereof in human breast cancer cells MDA-MB-231
a The T/N ratio is defined as the boron uptake of human breast cancer cells MDA-MB-231/the boron uptake of human umbilical vein endothelial cells HUVEC
Table 5 boron uptake characteristics of series BPA oligomers and targeting compounds thereof in human glioma cells U87
a The T/N ratio is defined as the boron uptake of human glioma cells U87/the boron uptake of human umbilical vein endothelial cells HUVEC
EXAMPLE 8 lipophilic hydrophilic character of series of BPA oligomers and targeting Compounds thereof
The experimental method comprises the following steps: a) And establishing a detection standard curve of each sample to be detected. Dissolving a series of BPA oligomers and targeting compounds thereof in an enzymolysis buffer (50 mM Tris-HCl,1.0M NaCl,pH 7.4) respectively, setting 5 concentration gradients of 0.012725, 0.006363, 0.003181, 0.001591 and 0.000795 mug/ml respectively, drawing a standard curve by taking the peak area as an ordinate and the drug concentration as an abscissa, and repeating the experiment for 3 times; b) Preparing n-octanol saturated by aqueous solution and aqueous solution saturated by n-octanol, mixing n-octanol and water in equal proportion, stirring for 24 hours to mutually saturate two phases, standing for layering, separating the two phases, and respectively preserving for later use; c) Accurately weighing 10mg of a sample to be measured, adding water solution saturated n-octanol into a 10mL volumetric flask to dissolve the sample, diluting to a scale, accurately sucking 8mL of the sample to a 25mL volumetric flask from the scale, and adding 8mL of water solution saturated with n-octanol. After shaking a 25mL volumetric flask for 3 hours, standing to separate out a water phase and an oil phase, filtering by a 0.22 mu m filter membrane, sampling 10 mu l each time, measuring the peak area by using HPLC, taking the peak area into a standard curve equation, and calculating the content of a sample to be detected in each phase. The oil-water distribution coefficient calculation formula:
experimental results: the results are shown in Table 6. As can be seen, the positive control drug BPA has a LogP value of-1.72+/-0.12 which is less than 1, and shows that the BPA has high solubility in water, low passive diffusion and permeation, poor absorption and brain permeability and very high renal clearance. The LogP values of the compounds are all between 1 and 3, and the compounds have good lipophilic and hydrophilic characteristics, have better permeability than BPA, are combined with metabolic enzymes to be increased, and have high metabolic degree.
TABLE 6 lipophilic hydrophilic coefficients (LogP) of the series of BPA oligomers and their targeting compounds
EXAMPLE 9 Targeted Release Property investigation experiments of BPA oligomer targeting Compounds
The experimental method comprises the following steps: HPLC chromatographic conditions are as follows: high performance liquid chromatograph Agilent 1200; chromatographic column Cosmosil C18 reverse phase chromatographic column (4.6X250 mm,5 μm); mobile phase (0 min,55% methanol and 45% water (containing 2mM ammonium formate), 10min,65% methanol and 35% water (containing 2mM ammonium formate), 15min,75% methanol and 25% water (containing 2mM ammonium formate), 30min,85% methanol and 15% water (containing 2mM ammonium formate), 40min,85% methanol and 15% water (containing 2mM ammonium formate), flow rate 1mL/min, detection wavelength 254nm, and sample injection amount 2. Mu.L.
Establishment of Z-GP- (BPA) n The (n=2, 3, 4) test standard curve method is as follows: Z-GP- (BPA) 2 、Z-GP-(BPA) 3 And Z-GP- (BPA) 4 Dissolving in enzymolysis buffer solution (50 mM Tris-HCl,1.0M NaCl,pH 7.4), setting 5 concentration gradients of 0.012725, 0.006363, 0.003181, 0.001591, 0.000795 μg/ml, drawing standard curve with peak area as ordinate and drug concentration as abscissa, repeating the experiment for 3 times, and collecting Z-GP- (BPA) with final concentration of 30mM n (n=2, 3, 4) were incubated in rhF a pα buffer containing 5 μg/ml and 10 μg/ml, respectively, at 37 ℃, and at time points of incubation 0, 4, 8, 12, 16, 24h, the supernatants were assayed for free biaryl hydrazines by HPLC.
The enzymolysis rate is calculated according to the following formula:
wherein, [ S ]] 0 Is the initial concentration of the substrate, [ S ]] t The concentration of substrate at time t is indicated.
Experimental results: the enzymolysis experimental result shows that F A P alpha can catalyze Z-GP- (BPA) n (n=2, 3, 4) hydrolysis release (BPA) n (n=2, 3, 4), and the enzymatic hydrolysis rate is positively correlated with the enzyme concentration (fig. 3 to 5). FΑpα is an activating protease secreted by tumor-associated fibroblasts and plays an important role in the development and progression of tumors. FΑpα is highly specifically expressed on the surface of tumor-associated fibroblasts in more than 90% of cancer species in humans.Z-GP-(BPA) n (n=2, 3, 4) can be hydrolyzed by fΑpα, indicating that it can be enzymatically released (BPA) in tumor tissue n (n＝2,3,4)。
The protection of the present invention is not limited to the above embodiments. Variations and advantages that would occur to one skilled in the art are included in the invention without departing from the spirit and scope of the inventive concept, and the scope of the invention is defined by the appended claims.

Claims (10)

1. A BPA oligomer and a targeting compound thereof have the structure shown in formulas I and II,
wherein BPA is (S) -2-amino-3- [4- ] 10 B) Dihydroxyborane-based phenyl]Propionic acid; n=0, 1,2; Z-GP is benzyloxycarbonyl glycyl prolyl, which has the following structure:
2. the BPA oligomer and targeting compound thereof as claimed in claim 1, characterised in that it comprises the following specific compounds:
3. the method for preparing the BPA oligomer and the targeting compound thereof according to claim 1 or 2, characterized in that the method for preparing the BPA oligomer comprises the following steps:
(1) Preparation of N-Boc-BPA-OH: weighing N-tert-butoxycarbonyl-4-iodo-L-phenylalanine in a reaction flask, and sequentially adding tributyl borate at room temperature 10 B(O n Bu) 3 、NaH、Bis (2-dimethylaminoethyl) ether, and placing the reaction flask in an ice bath; slowly adding a metal organic reagent under the protection of nitrogen, removing an ice bath after the dropwise addition is finished, stirring for 16 hours at room temperature, adding ice water under the ice bath condition, stirring for 10 minutes, quenching, adding methyl tertiary butyl ether, regulating the pH value to 3 by using concentrated hydrochloric acid, extracting by using ethyl acetate for two times, concentrating under reduced pressure to remove an organic solvent, adding water into the concentrate, regulating the pH value to 12 by using a 1mol/L NaOH aqueous solution, washing a water layer by using n-butanol for two times, regulating the pH value to 3 by using concentrated hydrochloric acid, stirring to precipitate white precipitate, pulping and washing by using dichloromethane, filtering and drying to obtain a product;
the synthetic route is as follows:
(2) Preparation of N-Boc-BPA-OMe: weighing N-Boc-L-BPA-OH, dissolving in DMF, adding inorganic base at room temperature, stirring for 30min, dropwise adding methyl iodide, stirring for 24 hr, adding water, extracting with ethyl acetate, mixing organic layers, washing with saturated sodium bicarbonate and saturated saline solution, and anhydrous Na 2 SO 4 Drying, rotary steaming to remove the organic solvent, and purifying the residue by silica gel column chromatography to obtain a target product;
the synthetic route is as follows:
(3) Preparation of H-BPA-OMe: weighing N-Boc-BPA-OMe, adding an acidic reagent, stirring at room temperature for reaction for 3-5 hours, removing a solvent by rotary evaporation, adding petroleum ether for pulping, and vacuum drying to obtain a product;
the synthesis steps are as follows:
(4)N-Boc-(BPA) n preparation of OMe (n=2, 3, 4): weighing N-Boc-L-BPA-OH, condensing agent and organic alkali, dissolving in a proper amount of organic solvent, stirring for 30min, and adding H- (BPA) in batches m OMe (m=1, 2, 3), stirring at room temperature for reaction for 12 hr after the material addition, adding organic solvent, washing the organic phase with saturated sodium bicarbonate and water, washing with saturated saline solution, and anhydrous Na 2 SO 4 Drying, rotary steaming to remove the organic solvent, and purifying the residue by silica gel column chromatography to obtain a target product;
the synthetic route is as follows:
(5)H-(BPA) n preparation of OMe (n=2, 3, 4): weighing the compound N-Boc- (BPA) n -OMe (n=2, 3, 4), adding an acidic reagent, stirring at room temperature for reaction for 3-5 hours, removing the solvent by rotary evaporation, adding petroleum ether for pulping, and vacuum drying to obtain a product;
the synthetic route is as follows:
(6) BPA oligomer (BPA) 2 ～(BPA) 4 Is prepared from the following steps: weighing compound H- (BPA) n OMe (n=2, 3, 4), adding alkaline reagent, stirring at room temperature for 1-4 hours, TLC tracking and monitoring until hydrolysis is complete, adding 1M HCl solution to adjust pH value to 6, precipitating white solid, filtering, washing, and vacuum drying to obtain target product;
the synthetic route is as follows:
4. a method of preparing a targeting compound for BPA oligomers as claimed in claim 3, comprising the steps of:
(1) Preparation of N-benzyloxycarbon glycyl proline methyl ester, Z-GP-OMe: N-Boc-L-BPA-OH was replaced with N-Z-Gly-OH, H- (BPA) m -OMe is replaced by H-Pro-OMe, prepared according to the method of step (4) of claim 3;
the synthetic route is as follows:
(2) Preparation of N-benzyloxycarbon glycyl proline, Z-GP-OH: replacement of the hydrolysis substrate with N-benzyloxycarbonylglycine methyl ester, Z-GP-OMe, prepared according to the method of step (6) of claim 3; the synthetic route is as follows:
(3) Z-GP-BPA oligomer methyl ester, Z-GP- (BPA) n Preparation of OMe (n=2, 3, 4): replacing N-Boc-L-BPA-OH with Z-GP-OH, prepared according to the method of claim 3, step (4); the synthetic route is as follows:
(4) Targeting compounds of BPA oligomers, Z-GP- (BPA) n Preparation of-OH (n=2, 3, 4): replacement of hydrolysis substrate with Z-GP-BPA oligomer methyl ester, Z-GP- (BPA) n OMe (n=2, 3, 4), prepared according to the method of step (6) of claim 3; the synthetic route is as follows:
5. a method of preparation according to claim 3, characterized in that:
in the step (1), N-tert-butoxycarbonyl-4-iodo-L-phenylalanine and tributyl borate are 10 B(O n Bu) 3 The molar ratio of NaH, bis (2-dimethylaminoethyl) ether and metal organic reagent is 1:1.0-5.0, 1:1.0-3.0, 1:1.0-10.0 and 1:1.0-10.0 respectively; the metal organic reagent is any one of isopropyl magnesium chloride and isopropyl magnesium chloride lithium chloride;
in the step (2), the feeding mole ratio of the N-Boc-BPA-OH to the inorganic base to the methyl iodide is respectively 1:1.0-5.0 and 1:1.0-5.0; the inorganic base is any one of potassium bicarbonate, sodium bicarbonate and lithium bicarbonate;
in the step (3), the feeding mole ratio of the N-Boc-BPA-OMe to the acidic reagent is 1:4.0-10.0; the acidic reagent is any one of trifluoroacetic acid and 4mol/L HCl methanol solution;
in step (4), N-Boc-BPA-OH is reacted with a condensing agent, an organic base, H- (BPA) m The molar ratio of the feed of OMe (m=1, 2, 3) is 1:1.1-1.5, 1:2.0-5.0, 1.1-1.5:1.0 respectively; the condensing agent is any one of 2- (7-oxybenzotriazol) -N, N, N ', N ' -tetramethyl urea hexafluorophosphate, ethyl chloroformate, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, N, N ' -diisopropylcarbodiimide, benzotriazol-1-yl-oxy-tripyrrolidinyl phosphate and tetramethyl chlorourea hexafluorophosphate; the organic base is any one of N, N-diisopropylethylamine and N-methylimidazole;
in step (5), compound N-Boc- (BPA) n -OMe (n=2, 3, 4) to acidic reagent feed molar ratio of 1.0:5.0-10.0; the acidic reagent is any one of trifluoroacetic acid and 4mol/L HCl methanol solution;
in step (6), compound H- (BPA) n -OMe (n=2, 3, 4) to alkaline agent feed molar ratio 1.0:5.0-15.0; the alkaline reagent is any one of aqueous solutions of NaOH, KOH, liOH.
6. Use of the BPA oligomer as claimed in any of claims 1-2 and its targeting compounds and their physiologically acceptable salts for the preparation of a boron delivery agent for use in boron neutron capture therapy for the treatment of tumors.
7. The use according to claim 6, characterized in that: the tumor is brain tumor, head and neck tumor, liver cancer, breast cancer, glioma, lung cancer, colon cancer, cervical cancer or prostate cancer.
8. The use according to claim 6, characterized in that: the physiologically acceptable salts are tartrate, sulfate, hydrochloride and sodium salt.
9. A boron delivery agent for use in boron neutron capture therapy for the treatment of tumors, comprising the BPA oligomer and targeting compound thereof as defined in any one of claims 1-2, or the BPA oligomer and targeting compound thereof as defined in any one of claims 3-5.
10. The boron delivery agent of claim 9, further comprising a physiologically acceptable salt.
CN202310595357.8A 2023-05-25 2023-05-25 BPA oligomer and targeting compound thereof, preparation method and application Pending CN116655729A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118078995A (en) * 2024-04-26 2024-05-28 东莞市人民医院 Dual-targeting high-water-solubility micromolecular boron drug as well as preparation method and application thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5392319A (en) * 1992-12-22 1995-02-21 Eggers & Associates, Inc. Accelerator-based neutron irradiation
CN106631957A (en) * 2016-12-07 2017-05-10 广州药本君安医药科技股份有限公司 Antitumor compound targeting FAP-alpha enzyme and preparation method and application thereof
CN115417889A (en) * 2022-06-15 2022-12-02 苏州大学 L-4-dihydroxyborophenylalanine-N-carboxylic acid internal anhydride monomer and polyamino acid as well as preparation method and application thereof
WO2023009172A1 (en) * 2021-07-30 2023-02-02 TAE Life Sciences Borylated amino acid compositions comprising bts and bts(ome) for use in boron neutron capture therapy and methods thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5392319A (en) * 1992-12-22 1995-02-21 Eggers & Associates, Inc. Accelerator-based neutron irradiation
CN106631957A (en) * 2016-12-07 2017-05-10 广州药本君安医药科技股份有限公司 Antitumor compound targeting FAP-alpha enzyme and preparation method and application thereof
WO2023009172A1 (en) * 2021-07-30 2023-02-02 TAE Life Sciences Borylated amino acid compositions comprising bts and bts(ome) for use in boron neutron capture therapy and methods thereof
CN115417889A (en) * 2022-06-15 2022-12-02 苏州大学 L-4-dihydroxyborophenylalanine-N-carboxylic acid internal anhydride monomer and polyamino acid as well as preparation method and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118078995A (en) * 2024-04-26 2024-05-28 东莞市人民医院 Dual-targeting high-water-solubility micromolecular boron drug as well as preparation method and application thereof
CN118078995B (en) * 2024-04-26 2024-08-23 东莞市人民医院 Dual-targeting high-water-solubility micromolecular boron drug as well as preparation method and application thereof

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