A dihydroxanthene / cyanoselenoisoflavone compound and its preparation method and application

Abstract

The present invention discloses a xanthene / cyanoselenoisoflavone compound having a structure represented by the general formula I: wherein R is one of halogen, OH, OMe, NH2, NMe2, and NPh2. Upon illumination, the xanthene / cyanoselenoisoflavone compound can generate reactive oxygen molecules, including superoxide anions and singlet oxygen, and can simultaneously produce both Type I and Type II photodynamic effects.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a novel dihydroxanthene / cyanoselenoisoflavone compound and a preparation method and application thereof. Background Art

[0002] In recent years, photodynamic therapy (PDT), as an innovative treatment method, has attracted widespread attention and application in the medical field. Photodynamic therapy utilizes specialized compounds—photosensitizers—combined with light of a specific wavelength to exert its therapeutic effects. Upon photoactivation, the photosensitizer is activated from the ground singlet state (S0) to the excited singlet state (Sn), and then undergoes intersystem crossing migration to the excited triplet state (Tn), where it generates reactive oxygen species (ROS) through two pathways. Type I photosensitizers do not require oxygen. The excited state participates in an electron transfer process, directly transferring energy to target cells or tissues, triggering the production of ROS (superoxide anions and hydroxyl radicals), which in turn kill the target cells or tissues. Type II photosensitizers, on the other hand, require oxygen. After the triplet excited state is formed, energy is transferred to surrounding oxygen molecules, generating highly reactive singlet oxygen, which in turn produces the photodynamic therapeutic effect. Compared to traditional treatments, PDT offers the advantages of high efficiency, painlessness, non-invasiveness, and minimal side effects, providing patients with more options and more effective treatment options. Summary of the Invention

[0003] The purpose of the present invention is to solve the defects in the prior art and provide a novel dihydroxanthene / cyanoselenoisoflavone compound having effective photodynamic therapy activity.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] A xanthene / cyanoselenoisoflavone compound, characterized in that the xanthene / cyanoselenoisoflavone compound has a structure shown in general formula I:

[0006]

[0007] Wherein, R is one of halogen, OH, OMe, NH2, NMe2, and NPh2.

[0008] The present invention also provides a method for preparing the above-mentioned compound: dissolving chemical 1 and compound 2 in anhydrous ethanol, adding 2 to 5 drops of a catalyst, and carrying out a condensation reaction to obtain the dihydroxanthene / cyanoselenoisoflavone compound;

[0009] The structural formula of the compound 1 is:

[0010] The structural formula of the compound 2 is:

[0011] The catalyst is one of acetic acid and piperidine or a mixture of the two.

[0012] More specifically, the preparation method of the dihydroxanthene / cyanoselenoisoflavone compound of the present invention is as follows:

[0013] Compound 1 and compound 2 were dissolved in anhydrous ethanol, 3 drops of acetic acid and 2 drops of piperidine were added as catalysts, and the mixture was refluxed at 80°C for 12 hours. After the reaction was completed, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain the compound of formula I.

[0014] More preferably, the compound codes of the above general formula I and their corresponding compound names are as follows:

[0015] I1: (E)-2-(2-(2-(6-hydroxy-2,3-dihydro-1H-xanthen-4-yl)vinyl)-4H-selenochromen-4-ylidene)malononitrile;

[0016] I2: (E)-2-(2-(2-(6-methoxy-2,3-dihydro-1H-xanthen-4-yl)vinyl)-4H-selenochromen-4-ylidene)malononitrile;

[0017] I3: (E)-2-(2-(2-(6-bromo-2,3-dihydro-1H-xanthen-4-yl)vinyl)-4H-selenochromen-4-ylidene)malononitrile;

[0018] I4: (E)-2-(2-(2-(6-Diphenylamino-2,3-dihydro-1H-xanthen-4-yl)vinyl)-4H-selenochromen-4-ylidene)malononitrile.

[0019] The present invention also provides the use of the aforementioned xanthene / cyanoselenoisoflavone compound in preparing a photodynamic therapy drug. Furthermore, the xanthene / cyanoselenoisoflavone compound exhibits cytotoxicity and photodynamic therapy activity in tumor cells, and its use in preparing a tumor therapy drug.

[0020] Among them, the above-mentioned tumors include colon cancer, liver cancer, lung cancer, breast cancer and cervical cancer.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] This invention utilizes the unique photochemical properties of the conjugated backbone of the dihydroxanthene ring structure. By combining the three rings into a single structure, the photosensitizer's structural stability is enhanced, helping to ensure its effectiveness in photodynamic therapy. Furthermore, the photosensitizer's light absorption efficiency is enhanced. Due to the interaction between the rings and the conjugated structure, light energy is more efficiently absorbed and converted into the desired energy, exerting a photodynamic therapeutic effect.

[0023] At the same time, the strong electron-withdrawing group of the 2-(4H-methylene-4-ylidene)malononitrile group of the selenium atom is coupled with the dihydroxanthene structure (electron donor) to form a donor-acceptor (DA) skeleton, which has more excellent photodynamic properties.

[0024] The novel xanthene / cyanoselenoisoflavone compounds of the present invention generate reactive oxygen species, including superoxide anions and singlet oxygen, upon illumination, resulting in both Type I and Type II photodynamic effects. Furthermore, testing of their cytotoxic and photodynamic therapeutic activities on multiple tumor cells revealed that the compounds of the present invention can exert synergistic anti-tumor effects combining chemotherapy and photodynamic therapy, while also significantly inhibiting tumor cell proliferation under both normoxia and hypoxia. This demonstrates that the compounds of the present invention are oxygen-independent in their therapeutic effects on solid tumors and effectively inhibit tumor cell proliferation. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The synthetic route of the dihydroxanthene / cyanoselenoisoflavone compound of the present invention is as follows;

[0026] Figure 2 The fluorescence emission spectra of active oxygen generated by compounds I1 and I2 of the present invention are shown in FIG. 1 , where the abscissa represents wavelength and the ordinate represents fluorescence intensity;

[0027] Figure 3 The fluorescence emission spectra of superoxide anions generated by compounds I1 and I2 of the present invention are shown in FIG. 1 , where the abscissa represents wavelength and the ordinate represents fluorescence intensity.

[0028] Figure 4 The figure is the ultraviolet absorption spectrum of singlet oxygen produced by compounds I1 and I2 of the present invention, where the abscissa is the wavelength and the ordinate is the absorbance value;

[0029] Figure 5 : is the fluorescence emission spectrum of active oxygen (A) and superoxide anion (B) generated by compound I3 of the present invention, the abscissa is wavelength and the ordinate is fluorescence intensity;

[0030] Figure 6 It is a fluorescence emission spectrum of active oxygen (A) and superoxide anion (B) generated by compound I4 of the present invention, with the abscissa being the wavelength and the ordinate being the fluorescence intensity. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings so that those skilled in the art can better understand the advantages and features of the present invention and thus more clearly define the scope of protection of the present invention. The embodiments described in the present invention are only a part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention.

[0032] The preparation method of the dihydroxanthene / cyanoselenoisoflavone compound of the present invention is as follows: Compound 1 and 2-(2-methyl-4H-selenochromen-4-ylidene)malononitrile (Compound 2) are dissolved in anhydrous ethanol, 2 to 5 drops of acetic acid and / or piperidine are added as a catalyst, and a novel dihydroxanthene / cyanoselenoisoflavone compound I is obtained through a condensation reaction; the synthetic route is as follows: Figure 1 As shown in the figure, R is one of halogen, OH, OMe, NH2, NMe2, and NPh2.

[0033] Example 1: Preparation of (E)-2-(2-(2-(6-hydroxy-2,3-dihydro-1H-xanthen-4-yl)vinyl)-4H-selenochromen-4-ylidene)malononitrile (I1)

[0034] 6-Hydroxy-2,3-dihydro-1H-xanthen-4-carbaldehyde (1a, 500 mg, 2.52 mmol) was dissolved in 5 mL of anhydrous ethanol, and compound 2 (610.2 mg, 2.52 mmol) was added. Three drops of acetic acid and two drops of piperidine were then added as catalysts. The mixture was refluxed at 80°C for 12 hours. After completion of the reaction, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound I1 in a 65% yield.

[0035] The spectrum data of compound I1 is: 1 H NMR(400MHz,DMSO-d6)δ8.89(m,1H,ArH),8.37(m,1H,ArH),8.07(m,1H,ArH),7.89(m,1H,ArH),7.79(m,1H,ArH),7.75-7.64(m,2H,2Ar H),7.59(m,1H,ArH),7.36(m,1H,CH=),7.05(m,1H,CH=),6.78(m,1H,CH=),4.60(s,1H,OH),2.75(m,4H,2CH2),1.95-1.78(m,2H,CH2). 13CNMR(101MHz, CDCl3)δ203.8,179.00,167.0,161.5,157.4,145.1,136.3,135.4,133.43,131.9,131. 7,128.4,127.7,124.8,123.7,122.1,115.7,107.7,100.4,91.0,83.8,83.6,62.0,49.0,20.9,17.6.

[0036] 1 H NMR(400MHz,DMSO-d6)δ8.89(m,1H,ArH),8.50(m,1H,ArH),8.12-8.04(m,1H,ArH),7.79(m,1H,ArH),7.74-7.65(m,2H,2ArH),7.59(m,1H,Ar H),7.36(m,1H,CH=),7.21(m,1H,CH=),7.08-7.02(m,1H,CH=),6.79(m,1H,CH=),4.60(s,1H,OH),2.76(m,4H,2CH2),1.91-1.81(m,2H,CH2). 13 C NMR(101MHz,DMSO-d6)δ179.7,169.6,167.1,161.7,160.0,154.2,140.4,138.6,136.1,132.8,132.2,12 9.5,128.9,128.5,126.6,123.0,120.0,116.0,114.1,105.2,102.4,70.3,66.8,52.5,50.4,36.1,33.7.

[0037] Example 2: Preparation of (E)-2-(2-(2-(6-methoxy-2,3-dihydro-1H-xanthen-4-yl)vinyl)-4H-selenochromen-4-ylidene)malononitrile (I2)

[0038] Referring to the synthesis method of compound (I1) in Example 1, compound 1a was replaced by 6-methoxy-2,3-dihydro-1H-xanthen-4-carbaldehyde (1b) to obtain compound I2 with a yield of 65%.

[0039] The spectrum data of compound I2 is: 1H NMR(400MHz,DMSO-d6)δ8.92(m,1H,ArH),8.38(m,1H,ArH),8.09(m,1H,ArH),7.91(m,1H,ArH),7.82(m,1H,ArH),7.73-7.67(m,2H,2ArH),7.62 (m,1H,ArH),7.38(d,J=6.8Hz,1H,CH=),7.05(m,1H,CH=),6.78(m,1H,CH=),3.86(s,1H,CH3),2.72-2.81(m,4H,2CH2),1.92-1.84(m,2H,CH2).

[0040] Example 3: Preparation of (E)-2-(2-(2-(6-bromo-2,3-dihydro-1H-xanthen-4-yl)vinyl)-4H-selenochromen-4-ylidene)malononitrile (I3)

[0041] Referring to the synthesis method of (I1) in Example 1, compound 1a was replaced by compound 6-bromo-2,3-dihydro-1H-xanthen-4-carbaldehyde (1c) to obtain compound I3 with a yield of 71%.

[0042] The spectral data of compound I3 are: 1 H NMR(400MHz,DMSO-d6)δ8.94(m,1H,ArH),8.50(m,1H,ArH),8.16-8.11(m,1H,ArH),7.85(m,1H,ArH),7.76-7.68(m,2H,2ArH),7.63(m,1H,ArH), 7.36(m,1H,CH=),7.21(m,1H,CH=),7.08-7.02(m,1H,CH=),6.79(m,1H,CH=),3.87(s,1H,CH3),2.71-2.78(m,4H,2CH2),1.91-1.81(m,2H,CH2).

[0043] Example 4: Preparation of (E)-2-(2-(2-(6-diphenylamino-2,3-dihydro-1H-xanthen-4-yl)vinyl)-4H-selenochromen-4-ylidene)malononitrile (I4)

[0044] Referring to the synthesis method of (I1) in Example 1, compound 6-diphenylamino-2,3-dihydro-1H-xanthen-4-carbaldehyde (1d) was used instead of compound 1a to obtain compound I4 with a yield of 66%.

[0045] The spectral data of compound I4 are: 1 H NMR(400MHz,DMSO-d6)δ8.91(m,1H,ArH),8.35(m,1H,ArH),8.06(m,1H,ArH), 7.88(m,1H,ArH),7.78(m,1H,ArH),7.71-7.65(m,2H,2ArH),7.57-7.52(m,3H ,ArH),7.37(d,J=6.8Hz,1H,CH=),7.26-7.18(m,4H,4ArH),7.08-6.99(m,5H, CH=,4ArH),6.76(m,1H,CH=),2.70-2.78(m,4H,2CH2),1.91-1.85(m,2H,CH2).

[0046] Example 5: Testing the Ability of the Novel Xanthene / Cyanoselenoisoflavone Compounds of the Present Invention to Generate Active Oxygen Species

[0047] Fluorescence spectroscopy was used to detect the ability of the present compound to generate active oxygen species, using 2,7-dichlorodihydrofluorescein (DCFH) as an active oxygen scavenger. The specific method was to mix the present compound with a solution of the DCFH scavenger and then irradiate with laser for a certain period of time. The present compound was detected at 520nm (100mW / cm 2 ) After laser irradiation, the changes in DCFH fluorescence intensity were detected.

[0048] The results show that ( Figure 2 、 Figure 5 、 Figure 6 ), the fluorescence intensity of compounds I1, I2, I3 and I4 at around 525 nm increased with the increase of illumination time, indicating that the compounds of the present invention can effectively generate reactive oxygen species after illumination and have the potential for photodynamic therapy.

[0049] Example 6: Testing the Superoxide Anion Generation Capacity of the Novel Xanthene / Cyanoselenoisoflavone Compounds of the Present Invention

[0050] Fluorescence spectroscopy was used to detect the ability of the compound of the present invention to generate superoxide anions. Dihydroethidium (DHE) was used as a superoxide anion scavenger. The specific method was to mix the compound of the present invention and a solution of the scavenger DHE, and then irradiate the solution with laser for a certain period of time. The compound of the present invention was detected at 520nm (100mW / cm 2 ) After laser irradiation, the changes in DHE fluorescence intensity were detected.

[0051] The results show that ( Figure 3 、 Figure 5 、 Figure 6), the maximum fluorescence intensity of compounds I1, I2, I3 and I4 increased with the increase of illumination time, indicating that the compounds of the present invention can effectively generate a large amount of superoxide anions after illumination and can play the role of type I photodynamic therapy.

[0052] Example 7: Testing the Singlet Oxygen Generation Capacity of the Novel Xanthene / Cyanoselenoisoflavone Compounds of the Present Invention

[0053] The ability of the compound of the present invention to generate singlet oxygen was tested by ultraviolet spectroscopy, with 1,3-diphenylbenzofuran (DPBF) as a singlet oxygen scavenger. The specific method was to mix the compound of the present invention and a solution of the scavenger DPBF, and then irradiate with laser for a certain period of time. The compound of the present invention was detected at 520nm (100mW / cm 2 ) After laser irradiation, the changes in DPBF absorbance were detected.

[0054] The results show that ( Figure 4 ), the absorbance values ​​of compounds I1 and I2 at around 415 nm decreased with the increase of illumination time, and the maximum ultraviolet absorption value of compound I2 decreased more significantly. At the same time, compound I2 had a higher singlet oxygen quantum yield of 0.75, which proved that the compound of the present invention can effectively generate singlet oxygen after illumination and can play the role of type II photodynamic therapy.

[0055] Example 8: Activity test of the novel dihydroxanthene / cyanoselenoflavone compound of the present invention in different tumor cells

[0056] The cytotoxicity and photodynamic therapy activity of the compounds of the present invention against human lung cancer A549 cell line, human colon cancer HT29 cell line, human cervical cancer Hela cell line, mouse breast cancer 4T1 cell line and human liver cancer HepG2 cell line were evaluated by using the MTT colorimetric method in vitro toxicity test. First, cells in a good exponential growth phase were taken and prepared into a solution containing 2×10 4 ~4×10 4 The suspension of cells was inoculated into a 96-well plate, 180 μL per well, and cultured in a constant temperature CO2 incubator for 24 hours. Dark group: The compound of the present invention was dissolved in DMSO and diluted with PBS, and cultured for 24 hours. Light group: The compound of the present invention was dissolved in DMSO and diluted with PBS and incubated for 2 hours, and then irradiated with 520 nm (200 mW / cm 2 ) for 10 minutes and then incubated for 24 hours. Furthermore, a 96-well plate seeded with cells was placed in a transparent hypoxic box and, after administration, protected by nitrogen, to test the photodynamic activity of the compounds of the present invention under hypoxic conditions. Finally, MTT was added to the 96-well plate and reacted in an incubator for 4 hours. The absorbance of each well was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) and the cell proliferation inhibition rate was calculated.

[0057] The compounds of the present invention were subjected to a series of anti-proliferative activity tests on tumor cells. The experimental results show (Table 1 and Table 2) that under normoxic conditions, compounds I1, I2, I3 and I4 of the present invention have certain inhibitory activity against the proliferation of HepG2, A549, HT29 and 4T1 tumor cells; under hypoxic conditions, compounds I1, I2, I3 and I4 have certain inhibitory activity against the proliferation of A549, HT29 and Hela tumor cells, and the inhibitory activity on tumor cells is significantly enhanced after illumination, indicating that the compounds of the present invention have oxygen-independent photodynamic therapy properties and exert significant chemo / photodynamic therapy effects on tumor cells.

[0058] Table 1 Inhibitory rate of some compounds of the present invention on tumor cells under normoxic conditions (25 μM)

[0059]

[0060] ND: Not tested.

[0061] Table 2 Inhibitory rate of some compounds of the present invention on tumor cells under hypoxic conditions (25 μM)

[0062]

[0063] ND: Not tested.

[0064] The embodiments of the present invention are described in detail above, but the contents described are only preferred embodiments of the present invention and should not be considered to limit the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. Use of a dihydroxanthene / cyanoselenoflavone compound in the preparation of a drug for photodynamic therapy of tumors, wherein the tumor is non-small cell lung cancer, colon cancer, cervical cancer, breast cancer, or liver cancer; the dihydroxanthene / cyanoselenoflavone compound has the structure shown in Formula I: ， in, R is one of Br, OH, OMe, and NPh2.

Images