Framework structure rearrangement derivative of euphorbia factor L1 and application thereof in pharmacy

Abstract

The invention belongs to the technical field of medicinal chemistry, relates to a skeleton rearrangement product of an euphorbia factor L1 and application of the skeleton rearrangement product in pharmacy, and particularly relates to a skeleton structure rearrangement derivative of the euphorbia factor L1 and application of the skeleton structure rearrangement derivative in preparation of anti-tumor multidrug resistance drugs. According to the present invention, the skeleton isomerization reaction is performed on EFL1 by using p-TsOH to generate the derivative compound 1 having the 5 / 7 / 7 / 4 tetracyclic combined skeleton rearrangement, and the test results show that the derivative compound 1 has significantly good anti-tumor multidrug resistance activity compared to the raw material EFL1, and can be further used for preparing the anti-tumor multidrug resistance drug. The skeleton compound disclosed by the invention has important drug development value for resisting tumor multidrug resistance activity. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry technology, and relates to a skeleton rearrangement product of Euphorbia factor L1 and its use in pharmaceutical manufacturing, and specifically relates to a skeleton rearrangement derivative of Euphorbia factor L1 and its use in preparing anti-tumor multidrug resistance drugs. The present invention uses an organic strong acid to perform skeleton isomerization conversion on Euphorbia factor L1 to prepare a skeleton rearrangement derivative with anti-tumor multidrug resistance activity. Background Art

[0002] The prior art discloses that Euphorbia factor L1 (EFL1) belongs to the diterpene type compound of the euphorbia pulex, has a rich content, has a 5 / 11 / 3 bicyclic macrocyclic skeleton structure, and the hydroxyl groups at the C-3 / 5 / 15 positions form esters, and has obvious anti-tumor multidrug resistance (MDR). Structure-activity relationship studies have shown that the difference in the substituents on the parent nucleus can significantly change the MDR activity of this type of compound.

[0003] A study disclosed that the diterpene compounds of caper were first isolated from Euphorbia lathyris L. (Tetrahedron Letters, 1971, 18: 1325-1328). Subsequently, various caper diterpene derivatives were isolated from other plants, such as 5 caper diterpene alcohol ester derivatives with the ability to induce tumor cell apoptosis and anti-tumor multidrug resistance were isolated from Euphorbia lagascae (Planta Medica, 2005, 72: 162-168). At present, the structural modification of caper diterpenoid compounds mainly focuses on the hydrolysis of side chain ester bonds and the acylation of side chain hydroxyl groups. Some anti-tumor multidrug resistance derivatives with better activity have also been obtained (Bioorganic & Medicinal Chemistry, 2014, 22: 6392-6400). However, studies have shown that the effect of its 5 / 11 / 3 tricyclic skeleton structure on its activity is still unclear. In order to discover more active caper diterpenoid derivatives, it is necessary for technical personnel in the industry to modify its macrocyclic skeleton.

[0004] Studies have shown that EFL1 can generate 5 / 7 / 7 / 4 tetracyclic skeleton derivatives with acetoxy and acetamide connected at C-11 and other skeleton isomers under the action of boron trifluoride ether (BF3·Et2O) in ethyl acetate (Fitoterapia, 2019, 133: 212-8) and acetonitrile (Fitoterapia, 2020, 146: 104710) as solvents. EFL1 can also generate 5 / 7 / 7 / 4 tetracyclic skeleton derivatives with formyloxy and methoxy connected at C-11 under the action of formic acid [Organic Letters, 2001, 3(11): 1609-12] and H2SO4 / ]MeOH [Tetrahedron, 1975, 31(4): 305-309]. It can be seen that EFL1 is prone to skeletal isomerization under the action of organic acid or Lewis acid to generate a 5 / 7 / 7 / 4 tetracyclic complex.

[0005] The backbone derivatives of the drug can be connected to different groups at the C-11 position under different reagent and solvent conditions; however, the literature has not evaluated the anti-tumor MDR activity, and the effect of backbone isomerization on its activity is still unclear.

[0006] A study reported that the Jolkinol subtype of the diterpene compounds of the euphorbia cerifera can undergo skeletal isomerization reaction when reacted at room temperature for 24 hours with dichloromethane (CH2Cl2) as solvent under the action of p-toluenesulfonic acid (p-TsOH) [Journal of Natural Products, 2013, 76(6): 1039-46]. Based on the current state of the art, the inventors of the present application intend to provide a skeletal rearrangement product of Euphorbia factor L1 and its use in pharmaceutical preparation, specifically a skeletal rearrangement derivative of Euphorbia factor L1 and its use in the preparation of anti-tumor multidrug resistance. Summary of the invention

[0007] The purpose of the present invention is to provide a method and product for modifying the skeleton structure of EFL1 based on the current state of the prior art, and specifically to provide a skeleton structure rearrangement derivative of Euphorbia factor L1 and its use in preparing anti-tumor multidrug resistance drugs.

[0008] In the present invention, the skeleton of EFL1 is modified by organic acid p-TsOH to obtain a 5 / 7 / 7 / 4 tetracyclic fusion skeleton isomerization product, and the anti-tumor MDR activity of this type of skeleton compound is confirmed.

[0009] The present invention uses p-TsOH to modify the skeleton structure of EFL1 to obtain compound 1. The reaction is shown below. After identification, the structure is 11-p-methyl-phenyl-sulfonyloxy-unnaturalane.

[0010]

[0011] The compound 1 of the present invention is prepared by the following synthesis method, which comprises the steps of:

[0012] EFL1 (80 mg, 0.145 mmol) was dissolved in anhydrous CH2Cl2, p-TsOH (80 mg, 32 equiv) was added, and the mixture was stirred at room temperature for 24 h. After the reaction was completed, saturated aqueous NaHCO3 solution was added to quench the reaction, and CH2Cl2 was added for extraction. The organic layer was washed with saturated brine, dehydrated with anhydrous magnesium sulfate, and filtered. The filtrate was concentrated under reduced pressure and separated by silica gel chromatography (petroleum ether-ethyl acetate, 15:1-5:1) and semi-preparative HPLC to give compound 1 (14 mg, yield 13%).

[0013] The present invention conducted an anti-tumor multidrug resistance (MDR) activity experiment of compound 1, wherein:

[0014] The cytotoxic activity of compound 1 was evaluated, and the results showed that the inhibition rate of compound 1 on adriamycin-resistant human breast cancer cells (MCF-7 / ADM) was 5.1% at 50 μM, indicating that compound 1 had no cytotoxic activity on adriamycin-resistant human breast cancer cells; the present invention further evaluated its anti-MCF-7 / ADM cell MDR activity.

[0015] Using MCF-7 / ADM as a model, RPMI-1640 culture medium was used to obtain cells in a good logarithmic growth phase to prepare a cell suspension, which was divided into an ADM group, a test sample group, a positive control group, a negative control group, and a blank control group, and cultured in a cell culture incubator for 24 hours; the compound was subjected to an MDR reversal test, and the average cell inhibition rate of each concentration was linearly regressed with the logarithm of the ADM concentration (GraphPad Prism V9.0.0) to obtain a standard curve of the logarithm of the test sample concentration versus the cell inhibition rate, and the 1C value of ADM on the cells was calculated based on the 50% cell inhibition rate. 50 The MDR reversal fold (RF) of the drug is calculated according to the following formula. An RF value greater than 1 indicates that the compound has an MDR reversal effect. The larger the RF value, the greater the reversal effect.

[0016] RF=IC 50 (ADM) / IC 50 (ADM + sample to be tested)

[0017] The results showed that the RF value of the compound 1 was 151.33, while the RF value of EFL1 was 32.92, and the RF value of the positive drug verapamil was 13.33, indicating that the anti-tumor MDR activity of the compound 1 was significantly greater than that of EFL1 and verapamil.

[0018] The present invention provides a skeleton structure rearranged derivative of Euphorbia factor L1 and its use in preparing anti-tumor multidrug resistance. The present invention uses p-TsOH to perform a skeleton isomerization reaction on EFL1 to generate a derivative (1) with a 5 / 7 / 7 / 4 tetracyclic skeleton rearrangement, which has significantly better anti-tumor multidrug resistance activity than the raw material EFL1 and can be used to prepare anti-tumor multidrug resistance drugs. The skeleton compound of the present invention has important anti-tumor multidrug resistance activity significance. DETAILED DESCRIPTION

[0019] Example 1 Preparation of Compound 1

[0020] EFL1 (80 mg, 0.145 mmol) was dissolved in anhydrous CH2Cl2, p-TsOH (80 mg, 3.2 equiv) was added, and the mixture was stirred at room temperature for 24 h. After the reaction was complete, a saturated aqueous solution of NaHCO3 was added to quench the reaction, and CH2Cl2 was added for extraction. The organic layer was washed with saturated saline, dehydrated with anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The concentrate was dissolved with a small amount of CH2Cl2, mixed with silica gel, separated by silica gel column chromatography, and eluted with petroleum ether-ethyl acetate (15:1-5:1) to obtain the raw material EFL1 and the crude product of compound 1. 1 was purified by semi-preparative HPLC to obtain pure compound 1 (14 mg, yield 13%).

[0021] Semi-preparative HPLC chromatographic conditions are as follows:

[0022] Chromatographic column: ODS-A (10.0×250 mm, 5 μm, YMC); flow rate: 2.5 mL / min; column temperature: 25°C;

[0023] DAD detection wavelength: 230nm and 280nm; mobile phase: isocratic elution of acetonitrile-water system. Mobile phase: 97% acetonitrile; retention time: 18min.

[0024] The sample solution was spotted on a silica gel thin layer plate, developed by the ascending method of petroleum ether-ethyl acetate (5:1), and after the solvent was taken out and dried, it was sprayed with a 10% sulfuric acid-ethanol solution and heated for color development. The sample spots on the silica gel thin layer plate were brick red-brown spots, among which the Rf value of the product 1 was about 0.1; the Rf value of the raw material EFL1 was about 0.2. The NMR structural identification data of compound 1 are shown in Table 1.

[0025] Table 1. NMR data of compound 1 (600 MHz, CDCl3).

[0026] No.C C H No.C C H 1 41.1 1.68m;3.28m 19 17.1 1.16s 2 35.8 2.17m 20 22.0 0.51s 3 78.9 5.50t(4.2) 8’ 171.5 4 49.1 2.53dd(9.8,4.0) 7’ 41.4 1.69m;3.28m 5 67.1 6.09d(9.8) 1’ 133.8 6 75.6 2’，6’ 129.6 7.29d(7.4) 7 24.1 1.58m;1.70m 3’，5’ 128.7 7.82d(8.2) 8 50.8 1.60m;1.69m 4’ 127.4 7.26m 9 38.8 1.22m 5-OAc 169.7 10 41.0 21.0 1.97s 11 80.4 3.82d(8.3) 15-OAc 170.1 12 47.4 3.27m 22.2 2.20s 13 48.4 11-OR 14 203.8 1”’ 144.9 15 92.2 2”’，6”’ 129.9 7.35d(8.0) 16 14.7 0.73d(6.9) 3”’，5”’ 128.9 7.31d(7.2) 17 40.4 1.40m;1.63m 4”’ 132.8 18 27.3 1.11s <![CDATA[4”’-CH3]]> 21.8 2.44s

[0027] Example 2 Antitumor multidrug resistance (MDR) activity test of compound 1

[0028] The cytotoxic activity of compound 1 was evaluated, and the results showed that its inhibition rate against doxorubicin-resistant human breast cancer cells (MCF-7 / ADM) was 5.1% at 50 μM, indicating that compound 1 had no cytotoxic activity against these cells. Its anti-MCF-7 / ADM cell MDR activity was further evaluated.

[0029] MCF-7 / ADM was used as a model, and the cell culture medium was RPMI-1640 medium containing 10% fetal bovine serum. Cells in good logarithmic growth phase were taken, digested with trypsin, and the culture medium was blown to make the adherent cells fall off. The cell suspension was counted and inoculated in a 96-well plate, with a cell number of 1×10 per well. 4 The cell suspension volume was 180 μL per well.

[0030] The ADM group, the test sample group, the positive control group, the negative control group, and the blank control group, each with 3 replicate wells, were placed in a cell culture incubator for routine culture for 24 hours.

[0031] The stock solution of the sample to be tested was diluted with complete culture medium containing 10% FBS to a drug-containing working solution with a concentration of 400 μM (final concentration of 20 μM); the ADM stock solution was diluted stepwise to a series of ADM working solutions of 3000, 1000, 333.3, 111.1, 37.02 and 12.35 μM (final concentrations of 150, 50, 16.7, 5.56, 1.85 and 0.62 μM); the Verapamil (Ver) stock solution was diluted to a Verapamil working solution with a concentration of 200 μM (final concentration of 10 μM).

[0032] After 24 hours, the cells adhered to the wall. Each well of the ADM group was added with 10 μL of complete culture medium and 10 μL of ADM working solution with a series of concentration gradients; each well of the sample group to be tested was added with 10 μL of different sample working solutions and 10 μL of ADM working solutions with a series of concentration gradients; each well of the positive control group was added with 10 μL of verapamil working solution and 10 μL of ADM working solution with a series of concentration gradients. The complete culture solution containing 0.1% DMSO was used as the negative control group, and the wells without inoculated cells and only containing complete culture medium were used as the blank control group. The 96-well plate was placed in an incubator at 37°C, 5% CO2, and saturated humidity for 48 hours of sterile culture. 20 μL of MTT solution was added to each well, and the culture was continued for 4 hours, and the old culture solution was discarded. 150 μL of DMSO was added to each well, and the plate was shaken on a plate shaker for 10 min to fully dissolve the purple crystals. The light absorption value (OD value) of each well was measured in an ELISA reader with a detection wavelength of 570 nm. The average cell inhibition rate of each concentration was taken and the logarithm of the ADM concentration was used for linear regression (GraphPad Prism V9.0.0) to obtain a standard curve of the logarithm of the concentration of the sample to be tested versus the cell inhibition rate. The IC value of ADM on cells was calculated based on the 50% cell inhibition rate. 50 The MTT experiment was repeated 3 times, and the IC 50 The results were expressed as mean ± variance, and the MDR reversal fold (RF) of the drug was calculated according to the following formula. If the RF value is greater than 1, it means that the compound has an MDR reversal effect. The larger the RF value, the greater the reversal effect.

[0033] RF=IC 50 (ADM) / IC 50 (ADM + sample to be tested)

[0034] The results showed that the RF value of the compound 1 was 151.33, while the RF value of EFL1 was 32.92, and the RF value of the positive drug verapamil was 13.33, indicating that the anti-tumor MDR activity of the compound 1 of the present invention was significantly greater than that of EFL1 and verapamil. The compound 1 of the present invention can be further used to prepare anti-tumor multidrug resistance drugs.

Claims

1. The skeleton structure rearranged derivatives of Euphorbia factor L1, It is characterized in that The derivative is a compound 1 having a structure of formula 1, which is 11-p-methyl-phenyl-sulfonyloxy-unnaturalane, Wherein, R is phenylacetyl and R1 is p-toluenesulfonyloxy.

2. A method for preparing a skeletal rearrangement derivative of Euphorbia factor L1 according to claim 1, It is characterized in that The skeleton of EFL1 was modified by using organic acid p-TsOH according to the reaction shown below to obtain the skeleton structure rearranged derivative compound 1 of the 5 / 7 / 7 / 4 tetracyclic symmetric skeleton isomerization product Euphorbia factor L1.

3. The preparation method according to claim 2, It is characterized in that The compound 1 is prepared by the following synthesis method, which comprises the steps: Dissolve EFL1 in anhydrous CH 2 C1 2 Add p-TsOH and stir at room temperature for 24 h. After the reaction is complete, add saturated NaHCO 3 The reaction was quenched with aqueous solution and CH 2 Cl 2 Extraction, washing the organic layer with saturated brine, dehydration with anhydrous magnesium sulfate, After filtration, the filtrate was concentrated under reduced pressure and separated by silica gel column chromatography (petroleum ether-ethyl acetate, 15:1-5:1) and semi-preparative HPLC to obtain compound 1.

4. Use of the skeleton-rearranged derivative of Euphorbia factor L1 according to claim 1 in the preparation of anti-tumor multidrug resistance drugs or pharmaceutical compositions.

5. The use according to claim 4, It is characterized in that The skeleton structure rearranged derivative compound 1 of the Euphorbia factor L1 has an MDR reversal effect, and its RF value is 151.33.

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