CN114853810B

CN114853810B - Curcumin derivative and preparation method and application thereof

Info

Publication number: CN114853810B
Application number: CN202210494273.0A
Authority: CN
Inventors: 常聪; 陆主航; 胡俊杰; 孟燕; 郑国华
Original assignee: Hubei College of Chinese Medicine
Current assignee: Hubei College of Chinese Medicine
Priority date: 2022-05-07
Filing date: 2022-05-07
Publication date: 2023-08-01
Anticipated expiration: 2042-05-07
Also published as: CN114853810A

Abstract

The invention relates to the field of natural pharmaceutical chemistry,discloses a curcumin derivative, a preparation method and application thereof. Curcumin derivative shown in formula (I) and pharmaceutically acceptable salt thereof, wherein R is H orX is halogen; n=2 to 5. The curcumin derivative can target into tumor cells and mitochondria thereof, and induce apoptosis by influencing mitochondrial membrane potential, thereby improving anti-tumor curative effect.

Description

Curcumin derivative and preparation method and application thereof

Technical Field

The invention relates to the field of natural pharmaceutical chemistry, in particular to a curcumin derivative, a preparation method and application thereof.

Background

Tumors are one of the prominent public health problems worldwide today, and are severely threatening the health and life of humans. Common methods for treating tumors include surgery, chemotherapy, radiotherapy, immunotherapy and the like, wherein chemotherapy is a common systemic treatment method, but a nonspecific treatment method has serious toxic and side effects on normal tissues. Therefore, the development of highly selective antitumor drugs is of great significance.

Mitochondria are one of the most important organelles within a cell, involved in a variety of physiological or pathological processes. Mitochondria are the core of cellular metabolism, are the main sites for synthesizing Adenosine Triphosphate (ATP) by oxidative phosphorylation, and are involved in the processes of cell differentiation, cell information transmission, apoptosis and the like, and have the capability of regulating cell growth and cell cycle. In addition, mitochondria can also play a role in signal transduction and apoptosis by interfering with electron transfer, modulating cellular redox potential, releasing or activating apoptosis-related proteins, and the like. When mitochondrial dysfunction occurs, the permeability of the mitochondrial outer membrane changes and pro-apoptotic proteins such as Bax are activated and form multimeric pores in the mitochondrial membrane releasing cytochrome C (Cytochrome C). After entering cytoplasm, cytochrome C is combined with apoptosis protease activator-1 (APAF 1) and caspase 9 to form apoptosis bodies, and further activates caspase 3 to induce apoptosis. Research shows that features of tumor cell such as unlimited proliferation, apoptosis escape, metabolic enhancement and the like are closely related to mitochondrial dysfunction. Compared to the normal cell mitochondrial membrane potential (-160 mv), the tumor cell mitochondrial membrane potential (-220 mv) is lower and positively charged molecules are more accessible and enriched in mitochondria, thus deriving a treatment strategy for mitochondrial targeting. Triphenylphosphine (TPP) is a delocalized lipophilic cation whose good lipophilicity and electropositivity enable rapid transmembrane transport to mitochondria.

Curcumin is an electrophilic polyphenol compound extracted from Curcuma rhizome, radix Curcumae, curcumae rhizoma, etc. of Zingiberaceae. Curcumin has molecular formula of C ₂₁ H ₂₀ O ₆ The relative molecular mass was 368.39, and the appearance was orange crystalline powder. The curcumin has wide antitumor spectrum and has inhibiting effect on various cancer cells, such as liver cancer, colon cancer, breast cancer, ovarian cancer, etc. Curcumin can inhibit tumor cell formation, proliferation, and metastasis by modulating various signaling such as NF- κ B, P-gp, VEGF, COX-2, STAT3, PTEN, bcl-2, MMPs, etc. Studies have shown that the mechanism by which curcumin induces apoptosis in tumor cells is closely related to disrupting mitochondrial function. Curcumin can destroy the redox homeostasis in mitochondria by reducing internal Glutathione (GSH) and generating excessive Reactive Oxygen Species (ROS), reduce mitochondrial membrane potential, and induce cells to produce apoptosis. Curcumin is considered as an excellent candidate drug with anti-tumor activity, but clinical application is limited due to the defects of extremely low water solubility, easiness in degradation, short biological half-life, low bioavailability, lack of selectivity to mitochondria of tumor cells and the like.

In order to improve some defects of the curcumin, momekova et al prepare a mixed block micelle with a mitochondrial targeting function to load the curcumin, and research shows that the micelle can increase accumulation of the curcumin at tumor mitochondria, so that apoptosis is more effectively induced. Dan Lei and the like, the triphenyl phosphine group and the curcumin are coupled through ether linkage to prepare the mitochondrion targeting curcumin derivative, so that the uptake efficiency of tumor cells on the compound is improved. Compared with original curcumin, the novel compound has better anti-tumor curative effect. However, in these methods, the synthesis methods for the mitochondrial targeting agent and the mitochondrial targeting derivative are complicated and require many steps.

Disclosure of Invention

The invention aims to overcome the problems of multiple synthesis steps, complex preparation method and the like required by mitochondrial targeting preparations and mitochondrial targeting derivatives in the prior art, and provides a curcumin derivative and a preparation method and application thereof.

In order to achieve the above object, the first aspect of the present invention provides a curcumin derivative represented by formula (I) and pharmaceutically acceptable salts thereof,

（I）

wherein R is H orThe method comprises the steps of carrying out a first treatment on the surface of the X is halogen; n=2 to 5.

Preferably, X is Br, cl or I.

Preferably, the curcumin derivative has the following structural formula:

。

in a second aspect, the present invention provides a process for preparing the above curcumin derivative, which comprises: the curcumin and the compound shown in the formula (II) are subjected to condensation reaction in the presence of a condensing agent and a catalyst,

（Ⅱ）

wherein n=2 to 5; x is as defined above.

Preferably, the method comprises the steps of:

(1) Dissolving curcumin, a compound shown as a formula (II) and a catalyst in an organic solvent;

(2) Dissolving a condensing agent in an organic solvent;

(3) Adding the solution obtained in the step (2) into the solution obtained in the step (1) under the ice bath condition, and reacting for 1-48 h at 0-80 ℃ under the protection of inert atmosphere;

(4) Washing the solution obtained in the step (3) by sequentially using a dilute hydrochloric acid solution and a saturated sodium chloride solution, drying, and separating and purifying by silica gel column chromatography;

wherein the molar ratio of curcumin to the compound represented by the formula (II) is 1:1-2.

Preferably, the condensing agent is selected from dicyclohexylcarbodiimide and/or 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride.

Preferably, the catalyst is selected from N-hydroxysuccinimide and/or 4-dimethylaminopyridine.

The third aspect of the invention provides an antitumor pharmaceutical composition with a mitochondrial targeting function, which comprises a pharmaceutical active component and pharmaceutically acceptable auxiliary materials, wherein the pharmaceutical active component is the curcumin derivative and pharmaceutically acceptable salts thereof.

In a fourth aspect, the present invention provides the use of the curcumin derivative and pharmaceutically acceptable salts thereof in the preparation of a medicament for treating tumor diseases.

Preferably, the neoplastic disease is ovarian cancer, liver cancer or colon cancer.

The invention discloses a curcumin derivative with a mitochondrial targeting function. The curcumin derivative is a monosubstituted or disubstituted compound, is yellow solid powder at normal temperature, and is easy to dissolve in organic solvents such as methanol, dichloromethane and the like. Compared with curcumin, the curcumin derivative has the advantages of reduced melting point, improved water solubility, increased efficiency of entering mitochondria of tumor cells, obviously enhanced apoptosis induction and anti-tumor curative effect, and provides a research basis for industrial production and clinical application of the curcumin derivative. The curcumin derivative can target into tumor cells and mitochondria thereof, and induce apoptosis by influencing mitochondrial membrane potential, thereby improving anti-tumor curative effect. The curcumin derivative has the advantages of high efficiency of targeting mitochondria, good anti-tumor curative effect, simple preparation method, easy operation, convenient subsequent development and industrialization, and the like.

Drawings

FIG. 1 is a product of example 1 ¹ H-NMR chart;

FIG. 2 is a product of example 1 ¹³ C-NMR chart;

FIG. 3 is a product of example 2 ¹ H-NMR chart;

FIG. 4 is a product of example 2 ¹ H-NMR chart;

FIG. 5 is the mass spectrometry detection results of the product of example 2;

FIG. 6 is the infrared detection result in test example 3;

FIG. 7 is a graph of the mitochondrial co-localization of CUR-2T in ovarian cancer cells in test example 5, where (A) 40. Mu.M CUR, (B) 60. Mu.M CUR, (C) 40. Mu.M CUR-2T, and (D) 60. Mu.M CUR-2T;

FIG. 8 is a graph showing the results of the measurement of the effect of CUR-2T on the cell membrane potential of ovarian cancer in test example 6;

FIG. 9 is a test result of the effect of CUR-2T on Reactive Oxygen Species (ROS) levels in ovarian cancer cells in test example 7;

FIG. 10 is a test result of the CUR-2T-induced apoptosis assay of test example 8;

FIG. 11 shows the results of the test example 9 wherein CUR-2T inhibited ATP synthesis by ovarian cancer cells.

Detailed Description

The following describes specific embodiments of the present invention in detail with reference to the drawings. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.

The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.

The first aspect of the invention provides a curcumin derivative shown as a formula (I) and pharmaceutically acceptable salts thereof,

（I）

In a preferred embodiment, X is Cl, br or I.

Further preferably, X is Br.

In the present invention, n may be 2, 3, 4 or 5.

Further preferably, the curcumin derivative has the following structural formula:

the second aspect of the present invention provides a method of the above curcumin derivative, wherein the method comprises: the curcumin and the compound shown in the formula (II) are subjected to condensation reaction in the presence of a condensing agent and a catalyst,

（Ⅱ）

wherein n=2 to 5; the definition of X is the same as the previous definition.

Preferably, the method comprises the steps of:

(2) Dissolving a condensing agent in an organic solvent;

In the process according to the invention, the organic solvent used is preferably methylene chloride, chloroform or tetrahydrofuran.

In the method of the present invention, the inert atmosphere is preferably helium or nitrogen.

In the method of the present invention, in step (3), the reaction temperature may be specifically 5 ℃,10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃,40 ℃, 45 ℃, 50 ℃, 55 ℃,60 ℃, 65 ℃, 70 ℃, 75 ℃, or 80 ℃; the reaction time may be 1h, 5h, 10h, 15h, 20h, 25h, 30h, 35h, 40h, 45h or 48h.

In a preferred embodiment, the condensing agent is selected from dicyclohexylcarbodiimide and/or 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride.

In the invention, the dilute hydrochloric acid solution refers to a hydrochloric acid solution with the molar concentration of 0.01 mol/L-0.1 mol/L.

In a preferred embodiment, the catalyst is selected from N-hydroxysuccinimide and/or 4-dimethylaminopyridine.

The present invention will be described in detail by way of examples, but the scope of the present invention is not limited thereto.

Example 1

The curcumin derivative CUR-T is prepared, and has the following structure:

the specific process comprises the following steps:

(1) A clean, dry 100 mL clear colorless three-necked flask was charged with 1.00 g (2.71 mmol) curcumin, 1.24 g (2.71 mmol) 5-carboxypentyltriphenyl phosphine bromide (i.e., X is Br, n=5), 165.3 mg (1.35 mmol) 4-Dimethylaminopyridine (DMAP), and dissolved with 40 mL dichloromethane;

(2) 800.8 mg (4.06 mmol) of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) was weighed out and dissolved in 10 mL dichloromethane;

(3) Dropwise adding the solution obtained in the step (2) into the solution obtained in the step (1) under ice bath condition, and reacting for 24 hours at room temperature (25 ℃) under the protection of nitrogen;

(4) The solution obtained in step (3) was washed three times with dilute hydrochloric acid, and then with saturated sodium chloride solution, and finally the organic phase was dried over anhydrous sodium sulfate overnight and separated by silica gel column chromatography to give the curcumin derivative (787.3. 787.3 mg, yield 35.9%).

Example 2

The curcumin derivative CUR-2T is prepared, and has the following structure:

the specific preparation process comprises the following steps:

(1) A clean, dry 100 mL clear colorless three-necked flask was weighed into 1.00 g (2.71 mmol) of curcumin, 2.47 g (5.42 mmol) of 5-carboxypentyltriphenyl phosphine bromide (i.e., X is Br, n=5), 330.6 mg (2.71 mmol) of 4-dimethylaminopyridine, and dissolved with 50 mL dichloromethane;

(2) 1.60 g (8.13 mmol) of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride was weighed out and dissolved completely with 10 mL dichloromethane;

(4) The solution obtained in step (3) was washed three times with dilute hydrochloric acid, and then with saturated sodium chloride solution, and finally the organic phase was dried over anhydrous sodium sulfate overnight and separated by silica gel column chromatography to give curcumin derivative (1.06 g, yield 31.4%).

Test example 1

The product prepared in the examples was characterized by means of a nuclear magnetic resonance apparatus (Bruker, avance III 400MHz, switzerland). The detection process comprises the following steps: and (3) placing a sample to be detected in a nuclear magnetic tube, adding 0.5 mL deuterated dimethyl sulfoxide for dissolution, and detecting by taking tetramethyl siloxane (TMS) as a displacement reference substance.

In example 1 ¹ H NMR ¹³ The C NMR spectra are shown in FIGS. 1 and 2.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 9.82 (s, 1H), 8.03-7.66 (m, 15H), 7.60 (dd,J= 15.9, 3.8 Hz, 2H), 7.51 (s, 1H), 7.38-7.24 (m, 2H), 7.14 (dd,J= 25.0 and 8.2 Hz, 2H), 6.98 (d,J= 15.9 Hz, 1H), 6.83 (dd,J= 24.2 and 11.9 Hz, 2H), 6.14 (s, 1H), 3.83 (s, 6H), 3.69 - 3.58 (m, 2H), 2.54 (d,J= 6.7 Hz, 2H), 1.63 (d,J= 32.7 Hz, 6H).

¹³ C NMR (101 MHz, DMSO-d ₆ ) δ 185.37, 181.88, 171.30, 151.64, 150.17, 148.51, 142.07, 141.23, 139.43, 135.38, 135.35, 134.30, 134.15, 134.04, 130.77, 130.65, 125.10, 123.81, 123.70, 121.75, 121.58, 119.48, 118.62, 116.24, 112.48, 112.02, 101.78, 56.52, 56.22, 33.31, 29.68, 29.51, 24.11, 21.98, 20.94, 20.44.

In example 2 ¹ H NMR ¹³ The C NMR spectra are shown in FIGS. 3 and 4.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.02-7.73 (m, 30H), 7.66 (d,J= 15.8 Hz, 2H), 7.57-7.51 (m, 2H), 7.39-7.31 (m, 2H), 7.13 (d,J= 8.1 Hz, 2H), 7.04 (d,J= 15.9 Hz, 2H), 6.23 (s, 1H), 3.83 (s, 6H), 3.66 (dt,J= 14.5 and 7.1 Hz, 4H), 2.56 (t,J= 7.0 Hz, 4H), 1.63 (dd,J= 27.9, 6.4 Hz, 12H).

¹³ C NMR (101 MHz, DMSO-d ₆ ) δ 183.69, 171.30, 140.30, 135.38, 135.35, 134.16, 134.06, 130.77, 130.65, 125.14, 123.74, 121.94, 119.49, 118.64, 112.61, 56.56, 33.31, 29.68, 29.51, 24.11, 22.03, 21.99, 20.95, 20.45.

Thus, from the above characterization results, CUR-T, CUR-2T has been successfully synthesized.

Test example 2

The product obtained in example 1 was detected by a mass spectrometer (Aglient, 7250& JEOL-JMS-T100LP AccuTOF, japan), and a positive ion mode was selected.

As shown in FIG. 5, the actual measurement result was 727.28172 and the theoretical value was 727.28. Thus, from the above characterization results, it is clear that the structural formula of the product CUR-T prepared in example 2 is consistent with the expected design.

Test example 3

The products obtained in examples 1 and 2, as well as Curcumin (CUR) and 5-carboxypentyltriphenyl phosphine bromide (TPP) were tested using fourier infrared spectrometer testing (Thermo, nicolet 6700, usa). The detection process comprises the following steps: weighing a proper amount of sample to be detected, dripping the sample on a potassium bromide sheet, drying, and placing the sample in a Fourier infrared spectrometer for detection, wherein the detection range is 500-4500cm ^-1 。

The results are shown in FIG. 6, which shows that: curcumin at 1627cm ^-1 The stretching vibration absorption peak of C=C double bond appears at the position, and TPP is 1720cm ^-1 The telescopic vibration absorption of carboxyl C=O appears at the position of 690cm ^-1 The absorption of the bending vibration outside the C-H plane is single substitution of benzene ring. Example 1 (CUR-T) and example 2 (CUR-2T) at 1751cm ^-1 C=O stretching vibration absorption peak with ester bond at 1627cm ^-1 C=C double bond stretching vibration absorption appears at the position of 690cm ^-1 A benzene ring monosubstituted absorption peak appears. The hydroxyl absorption peak of CUR-2T is weaker than CUR-T because both phenolic hydroxyl groups have been esterified, leaving only the hydroxyl absorption peak in the enol-like structure. The above change in the infrared characteristic peak indicates that the compound CUR-T, CUR-2T was successfully synthesized.

Test example 4

The products prepared in examples 1 and 2 and the melting point of curcumin were examined. The detection steps are as follows: taking a sample to be detected which is dried to constant weight, grinding the sample into fine powder by an agate mortar, preparing a sample by using a glass capillary tube for melting point measurement, tightly gathering the powder on a melting end of the capillary tube through free falling, and then placing the capillary tube in a melting point instrument for detection (microcomputer melting point instrument, WRS-2, shanghai) for detection. The results are shown in Table 1.

TABLE 1

As can be seen from Table 1, the melting points of the products of examples 1 and 2 were reduced relative to curcumin, and the change in melting point indicated that the introduction of triphenyl phosphine groups into the curcumin structure reduced the melting point.

Test example 5

CUR-2T co-localization in mitochondria of ovarian cancer cells

A2780 cells in logarithmic growth phase were digested and counted, and then inoculated into a glass petri dish (35 mm) at 2X 104 cells/well, and cultured in an incubator for 24 hours to allow the cells to adhere sufficiently. CUR and CUR-2T groups were set, each with two concentrations of 40. Mu.M and 60. Mu.M, respectively, and after 4h of action, washed three times with PBS, each dish was incubated for 25min with 1mL mitochondrial dye (Mitothecker Red,60 nM), washed three times with PBS, 1mL of 4% paraformaldehyde was added to fix the cells for 10min, washed three times with PBS, and finally each dish was stained with 1mL nuclear dye Hoechst33342 (10. Mu.g/mL), stained for 10min, washed three times with PBS, and observed under a laser confocal microscope (Olympus, FV-3000, japan).

To investigate whether CUR-2T has mitochondrial targeting function, this experiment examined the distribution of CUR and CUR-2T within A2780 cells. In FIG. 7, (A) - (B) are CUR groups of 40 mu M and 60 mu M, (C) - (D) are CUR-2T groups of 40 mu M and 60 mu M respectively, 1-3 columns are blue cell nuclei, green drugs (CUR or CUR-2T) and red mitochondria respectively, and 4 columns are the superposition of three pictures of blue, green and red, wherein the superposition of red and green forms orange yellow. As can be seen from FIG. 7, when CUR and CUR-2T were incubated for 4 hours with A2780, both CUR and CUR-2T entered the cells in proportion to the amount administered, but the green fluorescence of 40. Mu.M CUR-2T was significantly stronger than 60. Mu.M CUR, indicating that CUR-2T entered the cells in greater amounts. In addition, mitochondria stained by Mitotracker Red fluoresce Red and show orange color after overlapping with green fluorescence, indicating drug entry into mitochondria. As can be seen from the figure, the orange color of 60. Mu.M CUR-2T is most pronounced, and 40. Mu.M CUR-2T times, indicating that CUR-2T can enter the mitochondria of A2780 cells and is dose dependent.

Test example 6

Effect of CUR-2T on ovarian cancer cell membrane potential

Qualitative detection: JC-1 is taken as fluorescent dye, and the influence of CUR-2T on the mitochondrial membrane potential of A2780 ovarian cancer cells is detected. A2780 ovarian cancer cells in the logarithmic growth phase were inoculated into 6-well plates at a density of 3X 104 cells/well after being digested and counted, and cultured in an incubator for 24 hours to allow the cells to adhere sufficiently. A control group, CUR group and CUR-2T group were set, wherein CUR concentration was 5. Mu.M, 10. Mu.M, 20. Mu.M, and CUR-2T concentration was 5. Mu.M, 10. Mu.M, respectively. Culture medium containing the above drugs was added to each well for further culture for 24. 24h, then the medium was discarded, washed with PBS, 1mL JC-1 dye (60. 60 nM) was added thereto, thoroughly mixed, and incubated in an incubator for 40min. After the incubation, the supernatant was discarded, washed 2 times with pre-chilled JC-1 staining buffer, and finally 2 mL medium was added and observed under a fluorescent inverted microscope (Olympus, IX73, japan).

Quantitative detection: digestion and counting of A2780 ovarian cancer cells in logarithmic growth phase followed by 3×10 ⁴ The density of each well was inoculated into a 6-well plate, and cultured in an incubator for 24 hours to allow the cells to adhere sufficiently. A blank, CUR and CUR-2T group were set with CUR concentrations of 5. Mu.M, 10. Mu.M, 20. Mu.M, and CUR-2T concentrations of 5. Mu.M, 10. Mu.M. After 24h of action, the culture medium is sucked, washed once by PBS, digested by trypsin for 4 min, centrifuged at 1200 rpm for 5min to collect the bottom cell sediment, washed once by PBS, added with 1mL JC-1 staining working solution, fully and uniformly mixed, and incubated in an incubator for 40min. After incubation, the samples were washed 2 times by centrifugation with pre-chilled JC-1 staining buffer, resuspended with 500. Mu.LPBS for each sample and detected by flow cytometry.

JC-1 dye is a fluorescent probe for detecting mitochondrial membrane potential ((Mitochondrial membrane potential, MMP, Δψm), where JC-1 aggregates to form polymers in the mitochondrial matrix in normal mitochondria, emitting orange-red fluorescence; when mitochondria are damaged, JC-1 can only exist in cytoplasm in a monomer form to generate green fluorescence when the membrane potential is reduced or lost, and FIG. 8A is a detection result of influence of a blank control group (A1-A3), a5 mu M CUR group (A4-A6), a10 mu M CUR group (A7-A9), a 20 mu M CUR group (A10-A12), a5 mu M CUR-2T group (A13-A15) and a10 mu M CUR-2T group (A16-A18) on mitochondrial membrane potential of A2780 cells, wherein the first line to the third line are respectively in bright field, red fluorescence and green fluorescence photographs, and as can be seen from a first line bright field picture of FIG. 8A, normal A2780 cells have morphological characteristics of epithelial cells, are round or polygonal, and after being treated by the 10 mu M CUR-2T, cell volume is reduced and rounded, quantity is reduced (FIG. 8A 16) -8, and the contrast cells in a red polymer form exist in a weak fluorescence form; when CUR-2T treatments at concentrations of 5. Mu.M, 10. Mu.M and 20. Mu.M were administered, a slight decrease and increase in red and green fluorescence, respectively, was observed, indicating that CUR could cause a slight decrease in MMP (FIGS. 8A 4-A12), and when CUR-2T treatments at concentrations of 5. Mu.M and 10. Mu.M were administered, a near-disappearance of red fluorescence was observed, at the same time, obvious green fluorescence appears, and the CUR-2T can target to enter mitochondria and obviously reduce MMP (FIG. 8A 13-A18) due to dose dependency. Normal MMPs are a prerequisite for maintenance of oxidative phosphorylation of mitochondria, production of adenosine triphosphate, whereas MMP decline causes a series of apoptotic cascades such as opening of permeability transition pores (Permeability transition pore, PTP), suggesting that CUR-2T might promote apoptosis of ovarian cancer cells by decreasing MMPs. FIG. 8B is a graph showing the ratio of green/red fluorescence intensities measured by flow cytometry, and the trend of the change in the results was consistent with that of the fluorescence photograph, and the two concentrations of CUR-2T were very significantly different from those of the control group. ( P <0.1 compared to control group; * P <0.01; * P <0.001; * P <0.0001; compared with CUR 20. Mu.M, # # #, P <0.0001; n=3 )

Test example 7

Effect of CUR-2T on Reactive Oxygen Species (ROS) levels in ovarian cancer cells

Qualitative detection: detecting CUR-2T pair A2780 ovum by using active oxygen detection kitEffects of ROS levels in nest cancer cells. After digestion and counting of A2780 cells in logarithmic growth phase, the cells were grown in 2X10 ⁴ Density of individual/well was seeded in 6-well plates at 37 ℃,5% co ₂ Culturing for 24h in the environment to make the cells fully adhere. A control group, a CUR group and a CUR-2T group were set, wherein the CUR concentration was 5, 10, 20. Mu.M and the CUR-2T concentration was 5, 10. Mu.M. Kit A (1:1000) was diluted with medium without Fetal Bovine Serum (FBS) to give a final concentration of dichlorofluorescein diacetate (DCFH-DA, 10 mM) of 10. Mu.M. The 6-well plate in the incubator was removed, the culture medium was aspirated, and 1mL of PBS was washed once per well. 1mL of DCFH-DA was added to each well and incubated in an incubator for 20 min. After removal, the cells were washed three times with FBS-free medium to sufficiently remove DCFH-DA that did not enter the cells. After the treatment, the mixture is placed under a laser micro confocal microscope for observation.

Quantitative detection: digestion of A2780 cells in logarithmic growth phase was followed by 2X10 ⁴ Density of individual/well was seeded in 6-well plates at 37 ℃,5% co ₂ Culturing in the environment for about 24 hours allows the cells to adhere well. A blank, CUR, and CUR-2T group were set, with CUR concentrations of 5, 10, 20. Mu.M and CUR-2T concentrations of 5, 10. Mu.M. Kit A (1:1000) was diluted with medium without Fetal Bovine Serum (FBS) to a final DCFH-DA concentration of 10. Mu.M. The 6-well plate in the incubator was removed, the culture medium was aspirated, and 1mL of PBS was washed once per well. Trypsin digestion for 4 min, centrifugation at 1200 rpm for 5min, collection of bottom cell pellet, washing with PBS once, adding 1mL DCFH-DA per sample, and incubation in incubator for 20 min. After removal, the cells were washed three times with FBS-free medium to sufficiently remove DCFH-DA that did not enter the cells. Each sample was resuspended with 500 μlpbs and examined by flow cytometry.

DCFH-DA is an oxidative stress indicator for detecting ROS content in cytoplasm and organelles (e.g., mitochondria). DCFH-DA is not fluorescent per se, has cell membrane permeability, can be hydrolyzed and deacetylated by cell esterase after entering cells to generate 2',7' -Dichlorofluorescein (DCFH), and is further rapidly oxidized to generate fluorescent product 2',7' -Dichlorofluorescein (DCF), wherein DCF can be detected by fluorescence spectrum (Ex/Em=504/529 nm). As can be seen from FIG. 9, the control groups (A1-A2) and CUR 10. Mu.M groups (A3-A4) were darker in green fluorescence, indicating lower intracellular ROS content. When treated with 5. Mu.M (A5-A6) and 10. Mu.M (A7-A7) CUR-2T, the green fluorescence intensity of these two groups increased significantly over that of CUR 10M group, indicating that CUR-2T can induce apoptosis by increasing intracellular ROS content, and is dose dependent, as compared to CUR. Fig. 9B shows the results of the flow assay, the trend of which is consistent with the results of the fluorescence photograph. ( P <0.0001; compared with CUR 10 mu M, # # #, P <0.0001; n=3 )

Test example 8

CUR-2T induces apoptosis of ovarian cancer cells

And detecting apoptosis of the A2780 ovarian cancer cells induced by CUR-2T by adopting an apoptosis detection kit and adopting a flow cytometry. A2780 cells in logarithmic growth phase were counted and then digested at 3X 10 ⁵ Density of individual/well was seeded in 6-well plates at 37 ℃,5% co ₂ The cells were allowed to adhere well by culturing in the environment at about 24℃ 24 h. A blank, CUR, and CUR-2T group were set, with a CUR concentration of 10. Mu.M and a CUR-2T concentration of 5. Mu.M and 10. Mu.M. Culture medium containing the above drugs was added to each well to continue culturing 24. 24h, cells were washed 3 times with PBS, digested with trypsin for 4 min, centrifuged at 1200 rpm for 5min to collect bottom cell pellet, resuspended in 195. Mu.L of binding solution, and stained in the dark for 15 min with 5. Mu.L of Annexin V-FITC and 10. Mu.L of PI. After the staining is completed, detection is carried out by a flow cytometer.

In normal cells, phosphatidylserine is distributed inside the cell membrane, and when the cell undergoes early apoptosis, phosphatidylserine everts outside the cell membrane, and this change occurs earlier than the apoptosis phenomena such as cell shrinkage, chromatin concentration, DNA fragmentation, and increase in cell membrane permeability. Annexin V is a phospholipid binding protein with high affinity to phosphatidylserine, and Annexin V-FITC with green fluorescence can be combined with phosphatidylserine exposed outside of early apoptosis cell membranes to serve as an indicator for early apoptosis reaction. Propidium Iodide (PI) is a nucleic acid dye that does not permeate intact cell membranes, but can permeate cell membranes of apoptotic middle and late cells and dead cells with increased membrane permeability, and stain such nuclei with red. Annexin V in combination with PI can be used to detect cell numbers at different stages of apoptosis. As shown in FIG. 10, the apoptosis rates of the control group (A1), CUR 10. Mu.M (A2), CUR-2T 5. Mu.M (A3) and CUR-2T 10. Mu.M (A4) were 4.25%, 6.27%, 7.9% and 17.48%, respectively, and the results showed that the efficiency of inducing apoptosis by CUR-2T was significantly improved and dose-dependent compared to CUR. ( P <0.001 compared to control; * P <0.0001; compared with CUR 10 mu M, #, P <0.01; # #, P <0.0001; n=3 )

Test example 9

CUR-2T inhibits the synthesis of ATP by ovarian cancer cells

The influence of CUR-2T on the synthesis of ATP in A2780 cells is detected by using a flow cytometry by adopting an ATP content detection kit. A2780 cells in logarithmic growth phase were counted and then digested at 3X 10 ⁵ Density of individual/well inoculated in 6-well plate at 37 ^o C，5% CO ₂ Culturing 24h in the environment allows the cells to adhere well. A blank, CUR, and CUR-2T group were set, with a CUR concentration of 10. Mu.M and a CUR-2T concentration of 5. Mu.M and 10. Mu.M. Culture medium containing the above drugs was added to each well for further culture 24h, cells were washed 3 times with PBS, digested with trypsin for 4 min, and cells were collected and counted. Centrifuging, removing supernatant, adding 1ml of extractive solution into each group of cells, re-suspending, and performing ice bath ultrasonic disruption for 1 min (200W, 1s interval per ultrasonic wave 2 s), adding water, stirring, and concentrating to obtain extract, adding water, and concentrating to obtain extract ^o C centrifuging at 10000 r/min for 10min, collecting supernatant, adding 500 μl chloroform per 1mL supernatant, shaking thoroughly, mixing, and adding into 4 ^o C centrifuging at 10000 r/min for 3 min, and taking the supernatant and placing on ice for testing. 20 mu L of supernatant to be detected, 128 mu L of reagent and 52 mu L of working solution are respectively added into a 96-well plate, the mixture is fully and uniformly mixed and then is immediately placed into an enzyme-labeled instrument, the absorbance value A1 at the 10 th s is measured at the position of 340nm, then the sample is placed into a 37 ℃ incubator for reaction for 3 min, the absorbance value A2 at the 3 min for 10 s is immediately measured, and the ATP generation amount is calculated according to a formula given in the kit. ATP content (mu mol/10) ⁶ cell) =0.125 Δa assay/Δa standard.

The detection of intracellular ATP content can reflect the energy metabolism state of tumor cells. Intracellular Hexokinase (HK) catalyzes the synthesis of ATP and glucose to glucose-6-phosphate, which can be further catalytically dehydrogenated by glucose-6-phosphate dehydrogenase (G6 PD) to form reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH), which is proportional to ATP content and has a characteristic absorption peak at 340nm, thus reacting ATP content. As a result, as shown in FIG. 11, after 24 hours of administration of CUR and CUR-2T-treated cells at different concentrations, the ATP content of the CUR 10. Mu.M group was not significantly changed as compared to the control group. However, CUR-2T 5. Mu.M and 10. Mu.M groups were significantly reduced compared to the placebo, CUR 10. Mu.M groups. The results show that CUR-2T can inhibit the synthesis of ATP in A2780 cells and is dose dependent, which is probably due to the fact that CUR-2T can reduce the ATP content by breaking mitochondria of A2780 cells and finally trigger apoptosis.

Test example 10

Cytotoxicity of CUR and CUR-T, CUR-2T on A2780 (human ovarian cancer cell), hepG2 (human liver cancer cell) and HCT-8 (human colon cancer cell).

The inhibition of proliferation of A2780 (human ovarian cancer cells) by CUR and CUR-T, CUR-2T was examined by MTT method. A2780 cells in the logarithmic growth phase are digested and uniformly dispersed into cell suspension, 1×104 cells are inoculated into a 96-well plate after counting and dilution, a control group and a blank group are arranged, and the cells are cultured for 24 hours to adhere to the walls. After the medium was discarded, the medium containing CUR and CUR-T, CUR-2T at different concentrations was added, respectively, and the culture was continued for 24 hours. After medium was discarded, 20. Mu.L MTT solution was added to each well for further culture for 4 hours, the supernatant was aspirated, and 150. Mu.L DMSO was added to each well to dissolve MTT-formazan crystals. The half-lethal concentrations (IC 50) of the three drugs were calculated by prism software using an enzyme-labeled instrument (BIO-RAD, xMark, USA) to measure absorbance (OD value) at 570 nm. The cell viability was calculated as follows: cell availability (%) = (OD experimental group-OD blank)/(OD control group-OD blank).

Cytotoxicity test methods of CUR and CUR-T, CUR-2T on HepG2 and HCT-8 are the same as above.

Table 2 shows the cytotoxicity results of CUR and CUR-T, CUR-2T on A2780, hepG2 and HCT-8 cells.

TABLE 2

As can be seen from Table 2, among the three tumor cells, the IC50 of CUR was 64.11.+ -. 1.09. Mu.M, 112.20.+ -. 4.32. Mu.M and 68.43.+ -. 0.69. Mu.M, respectively, the IC50 of CUR-T was 10.41.+ -. 0.55. Mu.M, 19.54.+ -. 1.05. Mu.M and 17.96.+ -. 0.72. Mu.M, respectively, and the IC50 of CUR-2T was 6.17.+ -. 0.56. Mu.M, 12.59.+ -. 0.64. Mu.M and 11.21.+ -. 0.66. Mu.M, respectively. Compared with CUR, the inhibition rate of CUR-T, CUR-2T modified by TPP on tumor cell proliferation is obviously increased. CUR-2T, which links two TPPs, is more toxic to tumor cells than CUR-T, which links one TPP. With the IC50 of CUR in three cells as a control, CUR-T was approximately 16.24%, 17.42%, 26.25% and CUR-2T was approximately 9.62%, 11.22% and 16.38% of CUR, suggesting that CUR-T and CUR-2T may induce apoptosis of three tumor cells by targeting mitochondria and causing a change in membrane potential, increasing the inhibition of cell proliferation.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.

Claims

1. A curcumin derivative and pharmaceutically acceptable salts thereof, wherein the curcumin derivative has the structural formula:

the preparation method of the curcumin derivative comprises the following steps:

(1) Dissolving curcumin, 5-carboxyamyl triphenylphosphine bromide and a catalyst in an organic solvent;

(2) Dissolving a condensing agent in an organic solvent;

(3) Adding the solution obtained in the step (2) into the solution obtained in the step (1) under the ice bath condition, and reacting for 1-24 h at 5-55 ℃ under the protection of inert atmosphere;

wherein, the mol ratio of curcumin to 5-carboxyl amyl triphenylphosphine bromide is 1:2;

the condensing agent is selected from dicyclohexylcarbodiimide and/or 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride;

the catalyst is selected from N-hydroxysuccinimide and/or 4-dimethylaminopyridine.

2. An antitumor pharmaceutical composition with a mitochondrial targeting function, which comprises a pharmaceutically active component and pharmaceutically acceptable auxiliary materials, wherein the pharmaceutically active component is the curcumin derivative and pharmaceutically acceptable salts thereof as claimed in claim 1.

3. Use of a curcumin derivative as defined in claim 1 and a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of a neoplastic disease;

the tumor disease is ovarian cancer.