CN114106079B - CDDO borate prodrug, preparation method and medical application thereof - Google Patents

CDDO borate prodrug, preparation method and medical application thereof Download PDF

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CN114106079B
CN114106079B CN202111363298.9A CN202111363298A CN114106079B CN 114106079 B CN114106079 B CN 114106079B CN 202111363298 A CN202111363298 A CN 202111363298A CN 114106079 B CN114106079 B CN 114106079B
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borate
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CN114106079A (en
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陈莉
王俊杰
李娜
张雪玲
张咏清
闻铭举
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China Pharmaceutical University
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    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
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    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

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Abstract

The invention discloses a CDDO borate prodrug with a structure shown in a formula I, wherein n=an integer of 1-10; x= -O-, -S-, -NH-, -COO-, -CONH-, -OCO-; y= -O-, -S-, -NH-, -COO-, -NHCOO-, -OCOO-. The CDDO borate ester prodrug of the invention has low sulfhydryl binding capacity in the low ROS environment of normal cells, and releases active principle drug with selectivity in the tumor microenvironment. Pharmacological research shows that the CDDO borate prodrug has obviously better proliferation inhibition effect on various tumor cells than CDDO, and has lower toxicity on normal cells and good selectivity compared with a positive drug CDDO-Me.

Description

CDDO borate prodrug, preparation method and medical application thereof
Technical Field
The invention belongs to the field of pharmaceutical chemistry, and particularly relates to a CDDO borate prodrug, a preparation method and medical application thereof.
Background
Oleanolic Acid (OA) is a pentacyclic triterpene natural product widely found in plants, and has weak antitumor, antiinflammatory, and liver protecting activities. Research workers are aiming at research modification of OA, a series of derivatives with obviously improved anti-tumor activity are obtained, wherein the derivatives comprise CDDO, CDDO-Me (Bardoxolone methyl), CDDO-Im and the like, and the most intensive research is CDDO-Me.
CDDO-Me is a star molecule in CDDO derivatives that has been introduced several times into clinical trials for the treatment of pancreatic cancer, melanin, etc., but is forced to terminate due to some non-negligible clinical responses. Fortunately, the European Union has previously granted eligibility for orphan drugs for CDDO-Me to treat Alport syndrome. The compound has the value of patent medicine. Thus, it would still be of value to find CDDO derivatives with selective low toxicity.
Disclosure of Invention
The present invention aims to provide a class of CDDO boronate prodrugs. The inventor introduces benzene ring-containing pinacol phenylboronate into CDDO to obtain borate prodrugs with low sulfhydryl binding capacity in normal cell low ROS environment and release active principle in tumor microenvironment and selectivity: the level of ROS in tumor cells is about 100 times that of normal cells, and specific uptake of anti-tumor compounds with ROS-responsive masking groups can be achieved. The pinacol phenylborate is taken as an ROS sensitive group, can be degraded by high-concentration ROS, and releases a reactive fragment; and the benzene ring-containing pinacol phenylborate is a weak polar segment, so that the pantethenyl binding capacity of the CDDO derivative can be reduced, the selectivity is improved, and the toxicity is reduced.
The invention aims at realizing the following technical scheme:
CDDO borate prodrugs of formula I:
wherein n represents a carbon chain length, n=an integer of 1 to 10;
x is selected from the group consisting of-O-, -S-, -NH-, -COO--CONH-/>-OCO-/>
Y is selected from the group consisting of-O-, -S-, -NH-, -COO--NHCOO-/>-OCOO-/>
Preferably, n=an integer from 1 to 5; x is selected from-O-, -COO-; y is selected from-O-, -NHCOO-.
Most preferably, n=an integer from 3 to 5; x is selected from-O-, -COO-; y is selected from-O-, -NHCOO-.
Specifically, the CDDO borate prodrugs of the present invention are selected from the following compounds:
it is another object of the present invention to provide a method for preparing a CDDO borate prodrug.
The preparation method of the CDDO borate prodrug comprises the following synthetic routes:
wherein X, Y, n is as previously described.
Comprising the following steps: reacting an intermediate containing a borate shielding group shown in a formula II with CDDO in the presence of an alkaline reagent and a catalyst by taking DMF as a solvent to obtain a CDDO borate prodrug; wherein, the molar ratio of the intermediate containing the borate shielding group to CDDO is 1.5:1; the alkaline reagent is potassium carbonate, and the molar ratio of the alkaline reagent to CDDO is 2-4:1, preferably 4:1; the catalyst is potassium iodide, and the molar ratio of the catalyst to CDDO is 0.3:1; the reaction temperature was 80 ℃.
Another object of the present invention is to provide CDDO derivatives of the structure shown in formula iii, which are prodrugs of CDDO borate esters:
wherein n=an integer of 1 to 10;
X＝-O-、-S-、-NH-、-COO-、-CONH-、-OCO-；
Y＝-O-、-S-、-NH-、-COO-、-NHCOO-、-OCOO-。
preferably, n=an integer from 1 to 5; x is selected from-O-, -COO-; y is selected from-O-, -NHCOO-.
Most preferably, n=an integer from 3 to 5; x is selected from-O-, -COO-; y is selected from-O-, -NHCOO-.
Specifically, the CDDO derivatives are selected from the following compounds:
the invention also aims to provide the medical application of the CDDO borate prodrug or the CDDO derivatives in preparing antitumor drugs.
The tumor is common malignant tumors such as breast cancer, gastric cancer, liver cancer, melanoma, non-small cell lung cancer and the like.
A pharmaceutical composition is prepared from CDDO borate prodrug or CDDO derivatives as effective components, and pharmaceutically acceptable carrier.
The dosage forms comprise tablets, capsules, dripping pills, granules, powder, troches, aqueous or oily suspending agents, injections, patches and nano-preparations.
The invention has the beneficial effects that:
the CDDO borate prodrug has the advantages of novel structure, mild reaction conditions in the preparation method, low toxicity of the used reagent, readily available raw materials, convenient post-treatment and higher yield.
Pharmacological research shows that the CDDO borate prodrug has obviously better proliferation inhibition effect on various tumor cells than CDDO, and has lower toxicity on normal cells and good selectivity compared with a positive drug CDDO-Me. Therefore, the CDDO borate prodrug of the invention is hopeful to become a new anti-tumor candidate drug and deserves intensive research.
Drawings
FIG. 1 shows the selectivity of CDDO borate ester prodrugs.
FIG. 2 shows the results of an in vitro release test of Compound I-2 (hydrogen peroxide concentration 100 times the drug concentration).
FIG. 3 shows the results of an in vitro release test of Compound I-2 (hydrogen peroxide concentration 40 times the drug concentration).
FIG. 4 shows the results of in vitro thiol binding assay of CDDO.
FIG. 5 shows the results of in vitro thiol binding assay of CDDO-Me.
FIG. 6 shows the results of in vitro thiol binding assays for Compound I-2.
Fig. 7 shows the results of in vitro thiol binding assay for compound I x-2.
Detailed Description
The following describes the essential aspects of the invention in connection with examples which are illustrative and therefore not intended to limit the scope of the invention.
Example 1
Synthesis of Compound 2-1
4-Bromomethylphenylboronic acid pinacol ester (Compound 1, 490mg,1.65 mmol) and parahydroxybenzoic acid (207 mg,1.5 mmol) were dissolved in 2ml of acetoneIn which K is added 2 CO 3 (622 mg,4.5 mmol) under nitrogen, stirring overnight at 70 ℃; suction filtration, spin-drying and flash column chromatography on silica gel (eluent PE: EA=50:1V/V) afforded compound 2-1 as a white crystalline powder (558 mg, 80% yield).
1 H NMR(300M Hz，DMSO,TMS):δ12.71(s,-COOH),7.90(d,2H,J＝7.5、1.5Hz),7.78(d,2H,J＝7.5、1.5Hz),7.28(d,2H,J＝7.5、1.5Hz),7.11(d,2H,J＝7.5、1.5Hz),5.18(s,2H),1.2(s,12H)； 13 C NMR(300M Hz，DMSO,TMS):δ24.7,70.8,88.1,114.2,122.5,127.1,130.4,131.3,133.6,136.7,164.2,169.3；ESI-MS(m/z):355.16[M+H] + 。
Example 2
Synthesis of Compound 3-1
Compound 2-1 (355 mg,1 mmol) and 1,3 dibromopropane (4 ml,4 mmol) were dissolved in 2ml of acetone, and 2ml of triethylamine was added thereto and stirred under nitrogen for 4 hours; suction filtration, spin-drying and flash column chromatography on silica gel (eluent PE: EA=10:1V/V) afforded compound 3-1 as a white solid (300 mg, 50% yield).
1 H NMR(300M Hz，DMSO,TMS):δ7.94(d,2H,J＝7.5、1.5Hz),7.78(d,2H,J＝7.5、1.5Hz),7.28(d,2H,J＝7.5、1.5Hz),7.01(d,2H,J＝7.5、1.5Hz),5.14(s,2H,J＝7.1Hz),4.3(t,2H,J＝7.1Hz),3.5(t,2H,J＝7.1Hz),2.17(t,2H,J＝7.1Hz),1.2(s,12H)； 13 C NMR(300M Hz，DMSO,TMS):δ165.9,163.3,136.7,133.6,130.9,130.4,127.1,122.4,114.2,88.1,70.8,63.2,32.8,29.2,24.7；ESI-MS(m/z):475.14[M+H] + 。
Synthesis of Compound 3-2
Compound 2-1 (355 mg,1 mmol) and 1,4 dibromobutane (4 ml,4 mmol) were dissolved in 2ml of acetone, and 2ml of triethylamine was added thereto and stirred under nitrogen for 4 hours; suction filtration, spin-drying and flash column chromatography on silica gel (eluent PE: EA=10:1V/V) afforded compound 3-2 as a white solid (340 mg, 56% yield).
1 H NMR(300M Hz，DMSO,TMS):δ7.94(d,2H,J＝7.5、1.5Hz),7.78(d,2H,J＝7.5、1.5Hz),7.28(d,2H,J＝7.5、1.5Hz),7.01(d,2H,J＝7.5、1.5Hz),5.14(s,2H,J＝7.1Hz),4.32(t,2H,J＝7.1Hz),3.52(t,2H,J＝7.1Hz),1.82(t,2H,J＝7.1Hz),1.79(t,2H,7.1Hz),1.2(s,12H)； 13 C NMR(300M Hz，DMSO,TMS):δ165.9,163.3,136.7,133.6,130.9,130.4,127.1,122.4,114.2,88.1,70.8,63.8,29.7,28.8,27.3,24.7；ESI-MS(m/z):489.14[M+H] + 。
Synthesis of Compound 3-3
Compound 2-1 (355 mg,1 mmol) and 1,5 dibromopentane (4 ml,4 mmol) were dissolved in 2ml of acetone, 2ml of triethylamine was added thereto, and the mixture was stirred under nitrogen for 4 hours; suction filtration, spin-drying and flash column chromatography on silica gel (eluent PE: EA=10:1V/V) afforded compound 3-3 as a white solid (350 mg, 47% yield).
1 H NMR(300M Hz，DMSO,TMS):δ7.94(d,2H,J＝7.5、1.5Hz),7.78(d,2H,J＝7.5、1.5Hz),7.28(d,2H,J＝7.5、1.5Hz),7.01(d,2H,J＝7.5、1.5Hz),5.14(s,2H,J＝7.1Hz),4.33(t,2H,J＝7.1Hz),3.52(t,2H,J＝7.1Hz),1.82(t,2H,J＝7.1Hz),1.78(t,2H,7.1Hz),1.29(t,2H,7.1Hz),1.2(s,12H)； 13 C NMR(300M Hz，DMSO,TMS):δ165.9,163.3,136.7,133.6,130.9,130.4,127.1,122.4,114.2,88.1,70.8,64.8,33.7,32.3,27.9,24.7,24.2；ESI-MS(m/z):503.15[M+H] + 。
Example 3
Synthesis of Compound I-1
Compound 3-1 (7193 mg,1.5 mmol), CDDO (491 mg,1 mmol) were dissolved in 10ml DMF, potassium carbonate (552 mg,4 mmol), KI (50 mg,0.3 mmol) were added, stirring overnight at 80℃under nitrogen, the reaction was checked by TLC, diluted with ethyl acetate, washed with saturated brine, the organic layer was dried over anhydrous sodium sulfate, and purified by flash column chromatography (eluent PE: EA=3:1V/V) to give compound I-1 as a pale yellow powder (120 mg, yield 10%).
1 H NMR(300M Hz，DMSO,TMS):δ7.94(d,2H,J＝7.5、1.5Hz),7.78(d,2H,J＝7.5、1.5Hz),7.28(d,2H,J＝7.5、1.5Hz),7.20(s,1H),7.01(d,2H,J＝7.5、1.5Hz),5.67(s,1H),5.14(s,2H),4.30(t,2H,J＝7.1Hz),4.06(t,2H,J＝7.1Hz),2.15(m,2H,J＝7.1Hz),1.06～2.09(m,20H),1.43(s,3H),1.24(s,6H),1.20(s,12H),0.89(s,3H),0.87(s,6H)； 13 C NMR(300M Hz，DMSO,TMS):δ203.3,203.1,180.1,174.4,165.9,163.3,159.5,136.7,133.6,133.6,130.9,130.9,130.4,127.1,127.1,124,122.4,115.8,114.2,114.2,111.6,88.1,88.1,70.8,62,61,56.7,49.4,47.2,41.4,39.6,39.2,38.6,36.4,34.1,33.7,32.2,32,31.7,31.7,31.4,31,28.5,27.6,26.1,24.7,24.7,24.7,24.7,21.4,21.4,18.8,17.4,16.7；ESI-MS(m/z):886.50[M+H] + 。
Synthesis of Compound I-2
Compound 3-2 (740 mg,1.5 mmol), CDDO (491 mg,1 mmol) were dissolved in 10ml DMF, potassium carbonate (552 mg,4 mmol), KI (50 mg,0.3 mmol) were added, stirred overnight at 80℃under nitrogen, the reaction was checked by TLC and ended, diluted with ethyl acetate, washed with saturated brine, the organic layer was dried over anhydrous sodium sulfate and purified by flash column chromatography (eluent PE: EA=3:1V/V) to give compound I-2 as a white powder (130 m, yield 10%).
1 H NMR(300M Hz，DMSO,TMS):δ7.94(d,2H,J＝7.5、1.5Hz),7.78(d,2H,J＝7.5、1.5Hz),7.28(d,2H,J＝7.5、1.5Hz),7.20(s,1H),7.01(d,2H,J＝7.5、1.5Hz),5.67(s,1H),5.14(s,2H),4.30(t,2H,J＝7.1Hz),4.06(t,2H,J＝7.1Hz),1.06～2.09(m,24H),1.43(s,3H),1.24(s,6H),1.20(s,12H),0.89(s,3H),0.87(s,6H)； 13 C NMR(300M Hz，DMSO,TMS):δ203.3,203.1,180.1,174.4,165.9,163.3,159.5,136.7,133.6,133.6,130.9,130.9,130.4,127.1,127.1,124,122.4,115.8,114.2,114.2,111.6,88.1,88.1,70.8,62,61,56.7,49.4,47.2,41.4,39.6,39.2,38.6,36.4,34.1,33.7,32.2,32,31.7,31.7,31.4,31,28.5,27.6,25.1,25.1,24.7,24.7,24.7,24.7,21.4,21.4,18.8,17.4,16.7；ESI-MS(m/z):900.97[M+H] + 。
Synthesis of Compound I-3
Compound 3-3 (760 mg,1.5 mmol), CDDO (491 mg,1 mmol) were dissolved in 10ml DMF, potassium carbonate (552 mg,4 mmol), KI (50 mg,0.3 mmol) were added, stirring overnight at 80℃under nitrogen, the reaction was checked by TLC and ended, diluted with ethyl acetate, washed with saturated brine, the organic layer was dried over anhydrous sodium sulfate and purified by flash column chromatography (eluent PE: EA=3:1V/V) to give compound I-3 as a white powder (180 mg, 15% yield).
1 H NMR(300M Hz，DMSO,TMS):δ7.94(d,2H,J＝7.5、1.5Hz),7.78(d,2H,J＝7.5、1.5Hz),7.28(d,2H,J＝7.5、1.5Hz),7.20(s,1H),7.01(d,2H,J＝7.5、1.5Hz),5.67(s,1H),5.14(s,2H),4.30(t,2H,J＝7.1Hz),4.06(t,2H,J＝7.1Hz),1.06～2.09(m,26H),1.43(s,3H),1.24(s,6H),1.20(s,12H),0.89(s,3H),0.87(s,6H)； 13 C NMR(300M Hz，DMSO,TMS):δ203.3,203.1,180.1,174.4,165.9,163.3,159.5,136.7,133.6,133.6,130.9,130.9,130.4,127.1,127.1,124,122.4,115.8,114.2,114.2,111.6,88.1,88.1,70.8,65.8,64.8,56.7,49.4,47.2,41.4,39.6,39.2,38.6,36.4,34.1,33.7,32.2,32,31.7,31.7,31.4,31,28.6,28.6,25.1,25.1,24.7,24.7,24.7,24.7,22.0,21.4,21.4,18.8,17.4,16.7；ESI-MS(m/z):914.53[M+H] + 。
Example 4
Synthesis of Compound 2-2
4-bromomethylphenylboronic acid pinacol ester (compound 1, 234mg,1 mmol) and CDI (243 mg,1.5 mmol) were dissolved in 10ml DCM and stirred at room temperature for 4 hours to complete the reaction; adding 3 times of DCM, washing with water, drying with anhydrous sodium sulfate, suction filtering, and spin-drying to obtain white viscous powder (328 mg, yield 100%); ESI-MS (m/z): 329.16[ M+H ]] + 。
The product of the previous step (328 mg,1 mmol) and p-aminophenol (165 mg,1.5 mmol) were dissolved in anhydrous THF, nitrogen-protected, reacted for 12h, and dried by spin to give compound 2-2 as a white crystalline powder (180 mg, 50% yield); ESI-MS (m/z): 370.13[ M+H ]] + 。
Example 5
Synthesis of Compounds 3-4
Compound 2-2 (370 mg,1 mmol) and 1,3 dibromopropane (4 ml,4 mmol) were dissolved in 2ml of acetone, potassium carbonate (552 mg,4 mmol) was added, and stirred under nitrogen for 4h; suction filtration, spin drying, flash column chromatography on silica gel (eluent PE: EA=10:1V/V) afforded Compound 3-4 as a white solid (300 mg, 70% yield); ESI-MS (m/z): 490.13[ M+H ]] + 。
Synthesis of Compound 3-5
Compound 2-2 (370 mg,1 mmol) and 1,4 dibromobutane (4 ml,4 mmol) were dissolved in 2ml of acetone, potassium carbonate (552 mg,4 mmol) was added thereto, and the mixture was stirred under nitrogen for 4 hours; suction filtration, spin drying, flash column chromatography on silica gel (eluent PE: EA=10:1V/V) afforded Compound 3-5 as a white solid (320 mg, 65% yield); ESI-MS (m/z): 504.23[ M+H ]] + 。
Synthesis of Compounds 3-6
Compound 2-2 (370 mg,1 mmol) and 1,5 dibromopentane (4 ml,4 mmol) were dissolved in 2ml of acetone, potassium carbonate (552 mg,4 mmol) was added, and stirred under nitrogen for 4h; suction filtration, spin drying, flash column chromatography on silica gel (eluent PE: EA=10:1V/V) afforded compound 3-6 as a white solid (270 mg, 45% yield); ESI-MS (m/z): 518.26[ M+H ]] + 。
Example 6
Synthesis of Compound I-4
Compound 3-4 (730 mg,1.5 mmol), CDDO (491 mg,1 mmol) was dissolved in 10ml DMF and potassium carbonate (552 mg,4 mmol), KI (50 mg,0.3 mmol) was added. Stirring overnight at 80 ℃ under the protection of nitrogen; after the completion of the reaction by TLC examination, it was diluted with ethyl acetate, washed with saturated brine, and the organic layer was dried over anhydrous sodium sulfate, and purified by flash column chromatography on silica gel (eluent: PE: ea=3:1v/V) to give compound I-4 as a white powder (180 mg, yield 15%).
1 H NMR(300M Hz，DMSO,TMS):δ9.71(s,1H),7.78(d,2H,J＝7.5、1.5Hz),7.43(s,2H,J＝7.5、1.5Hz),7.28(d,2H,J＝7.5、1.5Hz),7.20(s,1H),6.84(d,2H,J＝7.5、1.5Hz),5.77(s,1H),4.65(s,2H),4.29(t,2H,J＝7.1Hz),4.06(t,2H,J＝7.1Hz),1.43(s,3H),1.24(s,9H),1.20(s,12H),0.89(s,3H),0.87(s,6H),1.14～2.11(m,19H)； 13 C NMR(300M Hz，DMSO,TMS):δ203.3,203.1,180.1,174.4,159.5,155,153.8,136.1,133.6,133.6,130.4,129.6,127.1,127.1,124,122.2,122.2,115.8,114.6,114.6,111.6,88.1,88.1,66.8,64.9,62.1,56.7,49.4,47.2,41.4,39.6,39.2,38.6,36.4,34.1,33.7,32.2,32,31.7,31.7,31.4,31,28.5,28.3,26.1,24.7,24.7,24.7,24.7,21.4,21.4,18.8,17.4,16.7；ESI-MS(m/z):901.51[M+H] + 。
Synthesis of Compound I-5
Compound 3-5 (760 mg,1.5 mmol), CDDO (491 mg,1 mmol) was dissolved in 10ml DMF, potassium carbonate (552 mg,4 mmol), KI (50 mg,0.3 mmol) was added, and stirred overnight at 80℃under nitrogen protection; after the completion of the reaction by TLC examination, the reaction was diluted with ethyl acetate, washed with saturated brine, and the organic layer was dried over anhydrous sodium sulfate, followed by purification by flash column chromatography on silica gel (eluent PE: ea=3:1v/V) to give compound I-5 as a white powder (150 m, yield 11%).
1 H NMR(300M Hz，DMSO,TMS):δ9.71(s,1H),7.78(d,2H,J＝7.5、1.5Hz),7.43(s,2H,J＝7.5、1.5Hz),7.28(d,2H,J＝7.5、1.5Hz),7.20(s,1H),6.84(d,2H,J＝7.5、1.5Hz),5.77(s,1H),4.65(s,2H),4.10(t,2H,J＝7.1Hz),4.06(t,2H,J＝7.1Hz),1.43(s,3H),1.24(s,9H),1.20(s,12H),0.89(s,3H),0.87(s,6H),1.14～2.11(m,21H)； 13 C NMR(300M Hz，DMSO,TMS):δ203.3,203.1,180.1,174.4,159.5,155,153.8,136.1,133.6,133.6,130.4,129.6,127.1,127.1,124,122.2,122.2,115.8,114.6,114.6,111.6,88.1,88.1,68.4,65.8,56.7,49.4,47.2,41.4,39.6,39.2,38.6,36.4,34.1,33.7,32.2,32,31.7,31.7,31.4,31,28.5,28.3,26.1,27.7,25.2,24.7,24.7,24.7,24.7,21.4,21.4,18.8,17.4,16.7；ESI-MS(m/z):915.53[M+H] + 。
Synthesis of Compound I-6
Compound 3-6 (79mg, 1.5 mmol), CDDO (491 mg,1 mmol) was dissolved in 10ml DMF, potassium carbonate (552 mg,4 mmol), KI (50 mg,0.3 mmol) was added, and stirred overnight at 80℃under nitrogen protection; after the completion of the reaction by TLC examination, the reaction was diluted with ethyl acetate, washed with saturated brine, and the organic layer was dried over anhydrous sodium sulfate, followed by purification by flash column chromatography on silica gel (eluent PE: ea=3:1v/V) to give compound I-6 as a white powder (200 mg, yield 30%).
1 H NMR(300M Hz，DMSO,TMS):δ9.71(s,1H),7.78(d,2H,J＝7.5、1.5Hz),7.43(s,2H,J＝7.5、1.5Hz),7.28(d,2H,J＝7.5、1.5Hz),7.20(s,1H),6.84(d,2H,J＝7.5、1.5Hz),5.77(s,1H),4.65(s,2H),4.10(t,2H,J＝7.1Hz),4.06(t,2H,J＝7.1Hz),1.43(s,3H),1.24(s,9H),1.20(s,12H),0.89(s,3H),0.87(s,6H),1.14～2.11(m,23H)； 13 C NMR(300M Hz，DMSO,TMS):δ203.3,203.1,180.1,174.4,159.5,155,153.8,136.1,133.6,133.6,130.4,129.6,127.1,127.1,124,122.2,122.2,115.8,114.6,114.6,111.6,88.1,88.1,68.7,65.8,56.7,49.4,47.2,41.4,39.6,39.2,38.6,36.4,34.1,33.7,32.2,32,31.7,31.7,31.4,31,29.3,28.9,28.3,26.1,27.7,25.2,24.7,24.7,24.7,24.7,22.1,21.4,21.4,18.8,17.4,16.7；ESI-MS(m/z):929.54[M+H] + 。
Example 7
Original drug I * Synthesis of-2
Parahydroxybenzoic acid (690 mg,5 mmol) and 1,4 dibromobutane (4 ml,4 mmol) were dissolved in 2ml acetone, 2ml triethylamine was added thereto, and the mixture was stirred under nitrogen for 4 hours; suction filtration, spin-drying and flash column chromatography on silica gel (eluent PE: EA=10:1V/V) afforded a white powder (620 mg, 56% yield). ESI-MS (m/z): 272.13[ M+H ]] + 。
The product of the previous step (408 mg,1.5 mmol), CDDO (491 mg,1 mmol) was dissolved in 10ml DMF and potassium carbonate (552 mg,4 mmol), KI (50 mg,0.3 mmol) was added and stirred overnight at 80℃under nitrogen protection; after the completion of the reaction by TLC, the reaction was diluted with ethyl acetate, washed with saturated brine, and the organic layer was dried over anhydrous sodium sulfate, followed by purification by flash column chromatography on silica gel (eluent: PE: EA=3:1V/V) to give Compound I * -2, pale yellow powder (160 mg, 18% yield).
1 H NMR(300M Hz，DMSO,TMS):δ9.88(s,1H),8.02(s,1H),7.95(d,2H,J＝8、1.5Hz),6.91(d,2H,J＝8、1.5Hz),5.95(s,1H),4.27(m,4H,J＝7.1Hz),1.42(s,3H),1.26(s,12H),0.92(s,9H),1.01～2.09(m,18H)； 13 C NMR(300M Hz，DMSO,TMS):δ199.22,196.67,177.70,168.86,166.40,165.85,160.37,131.92(2C),123.92,122.40,115.30(3C),114.53,64.11,64.05,49.73,47.63,47.14,45.79,45.02,42.54,42.09,35.77,34.42,33.25,32.82,31.61,31.50,30.66,27.97,26.99,26.92,26.54,25.59,25.46,24.66,23.05,22.56,21.57(2C),18.19；ESI-MS(m/z):684.7[M+H] + 。
Example 8
In vitro anti-tumor Activity Studies
The compound of the invention is tested for anti-tumor activity by adopting a tetramethyl azoazole blue colorimetric method (MTT method), and CDDO-Me (Bardoxolone methyl) and CDDO (Bardoxolone) are selected as positive control medicines.
Instrument: ultra clean bench (SW-CJ-1 FD, AIRTECH, sujingtai), constant temperature CO 2 Incubator (3111, thermo, U.S.), inverted biological microscope (IX 71, olympus, japan), enzyme-linked immunosorbent assay (Model 680, bio-rad, U.S.), plate shaker (Kylin-bell lab Instruments), autoclave (YXO.SG41.280, shanghai line), centrifuge (Sigma).
Reagent: DMEM medium (Gibco), fetal bovine serum (Gibco), trypsin (Sigma), DMSO (Sigma).
Cell lines: human breast cancer cell line MCF-7, human breast cancer cell line MDA-MB-231, human normal breast cell line MCF-10A, human gastric cancer cell line BGC-823, human gastric mucosa cell line GES-1, human liver cancer cell line HepG2 and normal liver cell line L-02 (all provided by Jiangsu Kaiki Biotechnology Co., ltd.).
The method comprises the following steps:
preparing a complete culture medium: 50ml of fetal bovine serum is added into 450ml of DMEM culture medium to prepare a complete culture medium, and the complete culture medium is placed in a refrigerator at 4 ℃ for standby. The media mentioned below are all such complete media.
Resuscitate the frozen cell line, place it in medium at constant temperature 37 deg.C and CO 2 Culturing in incubator, changing culture medium once every day, adding 1ml of 0.25% trypsin digestion solution when it is in exponential growth phase and good state, digesting for 1-2 min, stopping digestion when observing the round and shrinkage of adherent cells under microscope, collecting cells, adding culture medium to prepare single cell suspension, and counting cells at a ratio of 5×10 per well 4 Cell suspension required for counting number of individual cells and total number of wellsAmount of liquid, cell suspension was inoculated into 96-well cell culture plate, 100. Mu.l/well, 96-well plate was sealed with PBS buffer around, and placed at 37℃under CO 2 Culturing in an incubator for 24 hours. Preparing a compound to be tested by using a DMEM complete culture medium, culturing cells for 24 hours, adding the compound to be tested into a 96-well plate to ensure that the final concentration is 10 mu M/well, setting up 3 compound wells, and continuously culturing for 48 hours. At the end of the incubation, MTT reagent was added to the 96-well plate at a final concentration of 10. Mu.l/well and incubation was continued for 4h. The medium in the wells was aspirated, 100. Mu.l DMSO was added to each well and the plate shaken for 10min. The enzyme-linked immunosorbent assay (ELISA) detects the absorbance of each hole at the wavelength of 570nm, and calculates the inhibition rate of the compound to cells according to the following formula, wherein the average value of the 3 primary screening results is the final inhibition rate.
Cell inhibition ratio = [ (blank OD value-dosing OD value)/blank OD value ] ×100%
Test compound is further subjected to concentration gradient screening, and IC (integrated circuit) of test compound is calculated 50 Values (Graphpad software calculation), 3 replicates were the final IC for the tested compounds 50 Values, results are shown in table 1.
TABLE 1 inhibition of different cell lines by Compounds
SI values were calculated according to Table 1 (i.e., IC of compounds against normal cells and tumor cells 50 The ratio of values), table 2 and fig. 1 were made based on SI values.
Table 2. Selectivity results for cddo boronate prodrugs
From table 1 and fig. 1, it can be derived that:
(1) Compared with the positive control drug CDDO-Me, the CDDO borate ester prodrug has different degrees of attenuation while maintaining similar anti-tumor activity as the positive drug.
(2) When X= -OCO-, Y= -O-, the selectivity to tumor cells is higher.
(3) When the carbon chain n=3 to 5, the compound activity decreases as the carbon chain increases.
(4) The selectivity of the compound I-2 is most prominent while the activity is maintained.
In conclusion, the CDDO borate prodrugs have certain proliferation inhibition effect on the 3 tumor cell lines, reduce the toxicity to normal cells, meet the design purpose of improving the selectivity and reducing the toxicity, and are hopeful to become new antitumor candidate drugs and deserve intensive research.
Example 9
In vitro Release test
According to background studies, it was demonstrated that when the hydrogen peroxide concentration was 100 times the drug concentration, the compound could be dissociated in a predicted manner. LC-MS technology is used to determine the presence of hydrogen peroxide (H 2 O 2 ) In this case, the intended drug substance can be released. And from this the concentration of peroxide required for prodrug release and the duration of action are determined. And selecting the compound I-2 as a detection object.
Instrument: high performance liquid chromatography (SPD-20A, shimadzu, japan), LC-MS (LCMS-2020, shimadzu, japan), pipette (Eppendorf, germany), chromatographic flask (1.5 ml, agilent, USA), constant temperature CO 2 Incubator (3111, thermo, U.S.).
Reagent: acetonitrile, methanol, purified water, 30% aqueous hydrogen peroxide (all available from Nanjing chemical Co., ltd.).
Preparing a liquid to be tested: test solution 1: 5mg of Compound I-2 was weighed and dissolved in 555.86. Mu.L of methanol (chromatographic purity) to prepare a 10mmol/L solution; test solution 2: 5.1. Mu.L of 30% hydrogen peroxide solution was pipetted and dissolved in 494.9. Mu.L of purified water to make 100mmol/L hydrogen peroxide solution (as prepared); test solution 3: mu.L of 30% hydrogen peroxide solution was pipetted into a pipette and dissolved in 4994.9. Mu.L of purified water to prepare 10mmol/L hydrogen peroxide solution (as prepared).
The method comprises the following steps:
measuring 20 μl of sample 1 with a pipette, adding into 200 μl of sample 2, metering volume to 500 μl with PBS buffer, and filtering with 0.45 μm mixed membrane to obtain compound I-2:H 2 O 2 Sample solution 1 with molar ratio=1:100. Incubation was performed for 0.5 hours at 37℃in an incubator, and samples were taken.
Instrument parameters:
elution mode: isocratic elution; mobile phase (methanol: water=95:5V/V); sample introduction time: 18 minutes; molecular scan range: 100-1300.
The experimental results are shown in FIG. 2.
The second method is as follows:
(1) 45. Mu.L of the sample solution 1 and 45. Mu.L of the sample solution 3 were sucked up by a pipette, the volume was adjusted to 500. Mu.L with acetonitrile, and the mixture was filtered through a 0.45 μm mixed membrane to obtain a sample solution 2 (Compound I-2:H) 2 O 2 Molar ratio = 1:1). Incubate in an incubator at 37℃for 2 hours.
(2) 45. Mu.L of the sample solution 1 and 45. Mu.L of the sample solution 2 were sucked by a pipette, the volume was adjusted to 500. Mu.L with acetonitrile, and the mixture was filtered through a 0.45 μm mixed membrane to obtain a sample solution 3 (Compound I-2:H) 2 O 2 Molar ratio = 1:10). Incubate in an incubator at 37℃for 2 hours.
(3) 45. Mu.L of the sample solution 1 and 180. Mu.L of the sample solution 2 were sucked up by a pipette, the volume was adjusted to 500. Mu.L with acetonitrile, and the mixture was filtered through a 0.45 μm mixed membrane to obtain a sample solution 4 (Compound I-2:H) 2 O 2 Molar ratio = 1:40). Incubate in an incubator at 37℃for 2 hours.
Instrument parameters:
elution mode: isocratic elution; mobile phase (acetonitrile: water=95:5V/V); sample introduction time: for 16 minutes.
The experimental results are shown in FIG. 3.
From fig. 2 and 3, it can be derived that:
(1) Compound I-2 (M/z= 898.7) can be dissociated into the desired prodrug molecule I at a hydrogen peroxide concentration of 100 times the drug concentration * -2 (M/z= 684.7), the description being in accordance with the design and in accordance with the definition of prodrug.
(2) When the concentration of the hydrogen peroxide compound is the same, the release of the raw drug is almost not generated within 2 hours. As the concentration of hydrogen peroxide increases,the prodrug release ratio gradually increases when I-2:H 2 O 2 At molar ratio = 1:40, the prodrug is released completely for 2 hours.
Therefore, the prodrug can be directionally released under the influence of high-concentration ROS in the tumor microenvironment, so that the design aims of attenuation and targeting are achieved.
Example 10
In vitro thiol binding assay
According to previous studies, the thiol binding ability of the A ring of CDDO can be effectively reduced when the 27 th carboxyl of CDDO is occupied. In vitro thiol binding experiments were performed to demonstrate the difference in thiol binding capacity between the prodrug before and after release, thus demonstrating the source of prodrug selectivity. Adopts prodrug I-2 and original drug I * -2, CDDO-Me as test subjects.
Instrument: UV-1800 (Shimadzu, japan), constant temperature CO 2 Incubator (3111, thermo, usa), pipette (Eppendorf, germany).
Reagent: dimethyl sulfoxide, PEG-200, PBS, triton X-100 (all available from Nanjing Chemicals Co., ltd.).
Composition of stock solution:
a. assay buffer: 1mol EDTA was dissolved in PBS at a final concentration of 1mmol/L;
dtt stock: 100mg of DTT (dithiothreitol) was dissolved in 855. Mu.L of PEG-200 at a final concentration of 758mmol/L;
cddo stock: 5mgCDDO was dissolved in 494 μLPEG-200 at a final concentration of 20mmol/L;
cddo-Me stock: 5mgCDDO-Me was dissolved in 494. Mu.L of PEG-200 at a final concentration of 20mmol/L;
e.I-2 stock: 5mg of Compound I-2 was dissolved in 494. Mu.L of PEG-200 at a final concentration of 20mmol/L;
f.I * -2 storage solution: 5mg of Compound I * -2 was dissolved in 494 μl PEG-200 at a final concentration of 20mmol/L;
preparing a liquid to be tested:
blank1: 2700. Mu.L assay buffer, 3. Mu.L Tris-100, 99. Mu.L DTT stock, was fixed to 3ml with PBS.
Test1: 2700. Mu.L assay buffer, 3. Mu.L Triton X-100, 99. Mu.L CDDO stock, and PBS was used to volume 3ml.
Test2: 2700. Mu.L assay buffer, 3. Mu.L Triton X-100, 99. Mu.L CDDO-Me stock, and PBS was used to volume 3ml.
Test3: 2700. Mu.L assay buffer, 3. Mu.L Triton X-100, 99. Mu.L LI-2 stock, and PBS was used to volume 3ml.
Test4: 2700. Mu.L assay buffer, 3. Mu.L Triton X-100, 99. Mu. L I * -2 stock solution, fixed volume to 3ml with PBS.
The method comprises the following steps:
the machine was zeroed with Blank1 as a control solution. Ultraviolet absorption spectrum tests are respectively carried out by using a Test1, a Test2, a Test3 and a Test4, and the absorption wavelength is 250-350 nm. Immediately after the corresponding absorbance spectrum was obtained, 99. Mu.L of DTT stock solution was added to the solution and the measurement was repeated. And obtaining a composite spectrum. The results are shown in FIGS. 4, 5, 6, and 7.
From fig. 4, 5, 6, 7, it can be derived that:
(1) CDDO, CDDO-Me, I under the action of the same concentration of DTT * -2 all showed different degrees of absorption peak at 288nm, with the CDDO peak enhancement most pronounced, followed by I * -2, finally CDDO-Me.
(2) None of the compound I-2 showed thiol binding (no peak change at 288 nm) under the action of 37 equivalents of the strong thiol binding reagent DTT. The thiol binding capacity of compound I-2 was far lower than CDDO and CDDO-Me, explaining the selective origin of the compounds.
Compound I * The sulfhydryl binding capacity of the-2 is stronger than that of CDDO-Me, and the released original drug has quite high tumor killing effect in tumors.

Claims (8)

1. CDDO borate prodrugs of formula I:
wherein n=an integer of 3 to 5;
X＝-O-、-OCO-；
Y＝-O-、-NHCOO-。
a cddo boronate prodrug characterized by: a compound selected from the group consisting of:
3. a method of preparing a CDDO borate prodrug as claimed in claim 1, wherein: the synthetic route is as follows:
wherein n=an integer of 3 to 5;
X＝-O-、-OCO-；
Y＝-O-、-NHCOO-；
comprising the following steps: reacting an intermediate containing a borate shielding group shown in a formula II with CDDO in the presence of an alkaline reagent and a catalyst by taking DMF as a solvent to obtain a CDDO borate prodrug; wherein, the molar ratio of the intermediate containing the borate shielding group to CDDO is 1.5:1; the alkaline reagent is potassium carbonate, and the molar ratio of the alkaline reagent to CDDO is 2-4:1; the catalyst is potassium iodide, and the molar ratio of the catalyst to CDDO is 0.3:1; the reaction temperature was 80 ℃.
4. CDDO derivatives with the structure shown in formula III:
wherein n=an integer of 3 to 5;
X＝-O-、-OCO-；
Y＝-O-、-NHCOO-。
CDDO derivatives, characterized in that: a compound selected from the group consisting of:
6. use of a CDDO borate ester prodrug as claimed in any of claims 1 to 2 or a CDDO class derivative as claimed in any of claims 4 to 5 for the manufacture of an anti-tumour medicament.
7. Use according to claim 6, characterized in that: the tumor is breast cancer, gastric cancer, liver cancer, melanoma and non-small cell lung cancer.
8. A pharmaceutical composition characterized by: the preparation is prepared from the CDDO borate prodrug of any one of claims 1-2 or the CDDO derivative of any one of claims 4-5 as an active ingredient and a pharmaceutically acceptable carrier.
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