A porphyrin-eugenol derivative, a preparation method and use thereof

Abstract

The application discloses a porphyrin-eugenol derivative with antitumor activity, uses DPBF to detect the yield of singlet oxygen, and in-vitro antitumor activity test shows that the compound has good activity. The porphyrin-coumarin compound can realize the synergistic treatment of phototherapy and chemotherapy, in addition, the insertion of metal Zn in the porphyrin can also enhance the antitumor activity of the compound. Thus, the compound can be used as an active ingredient of an antitumor drug, and has good development and application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicinal chemistry, in particular to a porphyrin-eugenol derivative, a preparation method and uses thereof. BACKGROUND

[0002] Photodynamic therapy (PDT) has been discovered more than 100 years ago and has become a widely studied therapy for the treatment of cancer and various non-malignant diseases including infections. So far, this therapy has been successfully used to treat a variety of malignancies. The basic elements of PDT can be divided into: photosensitizer (PS), specific excitation light, molecular oxygen. Photosensitizer is irradiated by light of appropriate energy wavelength to form an excited singlet state, and then converted into a long-lived excited triplet state. This triplet state can undergo photochemical reactions in the presence of oxygen, transferring energy to the surrounding oxygen molecules to form reactive oxygen species (ROS), thus killing cancer cells, pathogenic microorganisms and unwanted tissues. Compared with more traditional cancer therapies such as surgery, radiotherapy and drug therapy, PDT has the advantages of non-invasiveness, less side effects, etc., and is attractive in the field of tumor treatment research.

[0003] With the development of PDT, the preparation of photosensitizers, especially porphyrin-based photosensitizers, has made remarkable progress. Most photosensitizers used for cancer treatment have a macrocyclic skeleton based on porphyrin. The main advantages of porphyrin compounds in photodynamic studies include: 1) stability of aromatic compounds; 2) effective absorption of visible light; 3) high active oxygen yield; 4) easy functional modification and structural diversity; 5) long triplet state lifetime and low dark toxicity. Photofri, the first photosensitizer drug approved by the US FDA, is a classic porphyrin structure drug.

[0004] Li et al. (Chinese Chemical Letters, 2007, 18(11): 1331-1334) designed and synthesized a new potential targeted anticancer drug based on porphyrin and 5-fluorouracil, and evaluated the in vitro anticancer activity against human hepatoma cell SMCC-7721 by MTT method. The preliminary results showed that the anticancer activity of the coupling compound was more than 2 times that of 5-fluorouracil. Porphyrin had no killing effect on tumor cells under the condition of no light, indicating that the coupling compound actually improved the targeting of 5-fluorouracil to tumor tissue, thereby enhancing the anticancer activity. S. Weimin et al. (Bioorg Med Chem, 2008, 16(10): 5665-5671) coupled 5-fluorouracil / Boc-L-phenylalanine with aminophthalocyanine to synthesize a series of porphyrin compounds containing 5-fluorouracil / L-phenylalanine. The in vitro anticancer activity study results showed that the introduction of 5-fluorouracil and L-phenylalanine could significantly improve the phototoxicity of porphyrin. The high selectivity of porphyrin compounds to tumor tissue broadens the prospects for the study of anticancer drugs linked to porphyrin compounds.

[0005] The present inventors have conducted a large amount of research on porphyrin compounds in the early stage, for example, porphyrin is linked to chrysin and amino acid to synthesize a series of new PDT compounds with good anticancer activity. However, the previous work mainly focused on linking porphyrin to anticancer drugs to enhance the anticancer activity of the drugs, and there was little research on how to improve the O2 generation ability of porphyrin compounds to enhance the anticancer activity. 1 O2 generation ability to enhance the anticancer activity.

[0006] Eugenol is a phenylpropionic acid derivative, which is a bioactive component in clove. It is a pale yellow viscous oil at room temperature. The anticancer activity of eugenol on cancer cell lines and animal models is currently being studied, and the molecular mechanism of eugenol-induced apoptosis of melanoma, skin tumor, leukemia and mast cells has been confirmed. Eugenol is considered a promising candidate drug in in vivo and in vitro studies.

[0007] Li et al. (Thorac Cancer, 2018.9(1):p. 25-29) observed the chemotherapeutic activity of eugenol on human lung cancer, and found that low concentrations of eugenol can inhibit the migration and invasion of cancer cells, hinder the sustainability of lung cancer cells, and prevent metastasis. At a larger concentration (1000M), eugenol showed a fatal effect on normal cells and lung cancer cells. Moreover, the expression levels of phospho-AKT and matrix metalloproteinase (MMP-2) in lung cancer cells were reduced after eugenol treatment. MMPs are a key family of zinc enzymes responsible for degrading extracellular matrix components closely related to tumor metastasis. In various diseases including lung cancer, excessive MMP expression can contribute to the onset of the tissue destruction process. Petrocelli et al. studied the anti-cancer effect of eugenol on NCM-460 cells (epithelial colon) and on colorectal cancer (CRC) cell lines, and determined that eugenol has clear anti-tumor activity, selectively targeting transformed colon cells. After 72 hours of treatment, eugenol activated cell death, necrosis, and cell cycle slowing in Caco-2 and SW-620 cells, but not in NCM-460 cells. In recent years, a number of studies have shown that eugenol has anti-cancer activity, not only inhibiting cancer cells, but also inhibiting stromal cells in the tumor microenvironment, and has gradually become a research hotspot for many scholars.

[0008] Porphyrin compounds are a popular drug for research, have good research prospects and anti-tumor potential, and the research on the anti-cancer activity of eugenol has gradually attracted widespread attention. The present application designs and synthesizes a new type of porphyrin-eugenol derivative, which combines eugenol with porphyrin, utilizes the tumor targeting effect of porphyrin and the ability to generate singlet oxygen, and adds the anti-cancer activity of eugenol, to be able to exert the anti-tumor effect of the drug. While having good anti-tumor activity, it also has smaller toxic side effects. SUMMARY

[0009] The technical problem to be solved by the present application is to provide a porphyrin-eugenol derivative that can specifically inhibit tumor cells without killing activity on normal human cells.

[0010] On the basis of the previous work, in order to obtain a porphyrin-eugenol derivative with targeted inhibition, a large amount of work has been carried out, a series of porphyrin-eugenol derivatives have been designed and synthesized, a compound that can better bind to EGFR kinase has been screened, and factors such as the toxicity of the compound to the cell cycle, apoptosis, cell migration and human normal vascular endothelial cells have been considered, and finally the compound of the present application has been screened.

[0011] The first aspect of the present application provides a compound represented by formula I or formula II and a pharmaceutically acceptable salt thereof, which has the following structure:

[0012]

[0013] wherein n is selected from an integer from 1 to 8;

[0014] R1is selected from H, halogen, hydroxyl, nitro, CN, C1-6alkyl, C2-C6alkenyl, C2-C6alkynyl, C6-10aryl, C2-C10heteroaryl;

[0015] R is selected from H, halogen, hydroxyl, nitro, CN, C1-6alkyl, C2-C6alkenyl, C2-C6alkynyl;

[0016] M is selected from Zn, Ni, Mn.

[0017] Preferably, n is selected from 1, 2, 3, 4 or 5.

[0018] Preferably, R1is selected from H, halogen, hydroxyl, nitro, CN, C1-6alkyl; more preferably, R1is selected from H or Cl;

[0019] Preferably, R is selected from H, halogen, hydroxyl, nitro, CN, C1-6alkyl; more preferably, R is selected from H or methyl;

[0020] Preferably, M is selected from Zn.

[0021] Preferably, the compound of the present application has the following structure:

[0022]

[0023] Another aspect of the present application provides a method for preparing the compound of formula I, which has the following synthetic route:

[0024]

[0025] wherein n, R1, R are defined as described above.

[0026] The specific reaction steps are as follows:

[0027] In an organic solvent, a base and a porphyrin derivative 1 are added, heated and stirred, and a eugenol derivative 2 is added to the reaction system until the reaction is complete, and a porphyrin-eugenol derivative of formula I is obtained after post-treatment.

[0028] Preferably, the molar ratio of the porphyrin derivative 1 to the eugenol derivative 2 is 1: (1-1.5), preferably 1:1-1.2, more preferably 1:1.2.

[0029] Preferably, the base is selected from potassium hydroxide, triethylamine or potassium carbonate, more preferably triethylamine.

[0030] Preferably, the organic solvent is selected from dichloromethane, chloroform, tetrahydrofuran, DMSO, DMF, methanol, ethanol, isopropanol; more preferably dichloromethane.

[0031] Another aspect of the present application provides a method for preparing a compound of formula II, the synthetic route of which is as follows:

[0032]

[0033] wherein n, R1, R and M are as defined above.

[0034] The specific reaction steps are as follows:

[0035] The compound of formula I is dissolved in an organic solvent, and a metal salt of M is added, and the reaction is refluxed, and TLC is monitored until the reaction is complete, and after-treatment is performed to obtain the porphyrin-eugenol derivative of formula II.

[0036] Preferably, the molar ratio of the compound of formula I to the metal salt of M is 1:3-7, preferably 1:5.

[0037] Another aspect of the present application provides a pharmaceutical composition comprising a compound of formula I or II or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0038] Another aspect of the present application relates to the use of a compound of formula I or II and a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same in the preparation of an anticancer drug.

[0039] Preferably, the cancer is selected from lung cancer or liver cancer; in particular, human non-small cell lung cancer cells A549 and human liver cancer cells HepG2.

[0040] Compared with the prior art, the present application has the following beneficial effects:

[0041] (1) The present application provides a new class of porphyrin-coumarin derivatives with anticancer activity, which widens the scope of existing anticancer compounds and can be used as a lead compound for further optimization.

[0042] (2) The compound of the present application uses a porphyrin molecule as a carrier and takes advantage of its tumor tissue aggregation effect to target tumor cells, thereby reducing the killing side effects on normal cells.

[0043] (3) The porphyrin-coumarin compound of the present application can achieve synergistic treatment of phototherapy and chemotherapy, in addition to which, the insertion of metal Zn in the porphyrin can also enhance the anti-tumor activity of the compound.

[0044] Definitions:

[0045] In certain embodiments, the pharmaceutically acceptable form is a pharmaceutically acceptable salt, which are well known in the art. Examples of pharmaceutically acceptable salts are salts of acids such as hydrochloric, hydrobromic, phosphoric, sulfuric, perchloric, acetic, oxalic, maleic, tartaric, citric, succinic or malonic, acetic, propionic, glycolic, pyruvic, oxalic, lactic, trifluoroacetic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, salicylic, etc., with the compound.

[0046] A "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. The BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 Trend graph of the change in fluorescence intensity of porphyrin compounds 4 and 6a-6e with light irradiation time.

[0048] Figure 2 Trend graph of the change in fluorescence intensity of porphyrin compounds 5 and 7a-7e with light irradiation time.

[0049] Figure 3 Trend graph of the change in fluorescence intensity of porphyrin compounds 4 and 8a-8e with light irradiation time.

[0050] Figure 4 Trend graph of the change in fluorescence intensity of porphyrin compounds 5 and 9a-9e with light irradiation time. DETAILED DESCRIPTION

[0051] The present application is specifically explained below by way of examples. In the present application, the following examples are to better illustrate the present application, and are not intended to limit the scope of the present application. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0052] Example 1 Synthesis of eugenol derivatives

[0053]

[0054] Take 154 μL (1 mmol) of eugenol, 691 mg (5 mmol) of K2CO3, 166 mg (1 mmol) of KI in a 100 mL round-bottom flask, add 10 mL of DMF as solvent, dissolve thoroughly, stir at 60°C for 20 min, then slowly add 0.33 mL (3 mmol) of ethyl bromoacetate / 0.39 mL (3 mmol) of 2-bromopropionic acid ethyl ester / 0.43 mL (3 mmol) of 4-bromobutyric acid ethyl ester / 0.47 mL (3 mmol) of 5-bromovaleric acid ethyl ester / 0.53 mL (3 mmol) of 6-bromohexanoic acid ethyl ester to the reaction system, monitor by TLC spotting, until the reaction is complete, stop heating, cool, pour the reaction liquid into a separatory funnel, extract with dichloromethane, wash with water, release the lower organic layer, dry over anhydrous Na2SO4, rotary evaporate under reduced pressure, to obtain a light yellow oily liquid. Then add the yellow liquid to a round-bottom flask, dissolve in a suitable amount of methanol, adjust the pH to 10-11, hydrolyze at 60°C for 1.5 h, end the reaction, stand, add 10% HCl to the reaction system to adjust the pH to 2-3, rotary evaporate under reduced pressure, precipitate white solid,

[0055] Water is added to filter, the filter cake is washed with a suitable amount of deionized water, to obtain eugenol derivatives 3a-3e. Yield: 75-90%

[0056] 3a: 1 H NMR (500 MHz, Chloroform-d) δ 6.87 (d, J = 8.0 Hz, 1H), 6.76-6.73 (m, 2H), 5.99-5.89 (m, 1H), 5.12-5.05 (m, 2H), 4.65 (s, 2H), 3.89 (s, 3H), 3.35 (d, J = 6.6 Hz, 2H).

[0057] 3b: 1 H NMR (500 MHz, DMSO-d6) δ 12.11 (s, 1H), 6.80 (d, J = 2.0 Hz, 1H), 6.73 (d, J = 8.2 Hz, 1H), 6.65 (d, J = 8.1 Hz, 1H), 5.97-5.91 (m, 1H), 5.10-5.00 (m, 2H), 4.69-4.65 (m, 1H), 3.75 (s, 3H), 3.28 (d, J = 6.5 Hz, 2H), 1.47 (d, J = 6.7 Hz, 3H).

[0058] 3c: 1H NMR (500 MHz, DMSO-d6) δ 12.16 (s, 1H), 6.85 (d, J = 8.1 Hz, 1H), 6.77 (s, 1H), 6.66 (d, J = 8.1 Hz, 1H), 5.99 - 5.87 (m, 1H), 5.10 - 4.99 (m, 2H), 3.91 (t, J = 6.5 Hz, 2H), 3.73 (s, 3H), 3.28 (d, J = 6.8 Hz, 2H), 2.37 (t, J = 7.3 Hz, 2H), 1.92 - 1.87 (m, 2H).

[0059] 3d: 1 H NMR (500 MHz, DMSO-d6) δ 12.06 (s, 1H), 6.89 - 6.62 (m, 3H), 6.03 - 5.84 (m, 1H), 5.08 - 4.99 (m, 2H), 3.90 (t, J = 6.3 Hz, 2H), 3.73 (s, 3H) 3.29 (d, J = 7.0 Hz, 2H), 2.35 -

[0060] 2.22 (m, 2H), 1.78 - 1.61 (m, 4H).

[0061] 3e: 1 H NMR (500 MHz, DMSO-d6) δ 11.99 (s, 1H), 6.86 (d, J = 8.1 Hz, 1H), 6.77 (d, J = 1.9 Hz, 1H), 6.67 (dd, J = 8.2, 1.9 Hz, 1H), 6.00 - 5.88 (m, 1H), 5.10 - 4.99 (m, 2H), 3.89 (t, J = 6.5 Hz, 2H), 3.73 (s, 3H), 3.29 (d, J = 6.7 Hz, 2H), 2.23 (t, J = 7.3 Hz, 2H), 1.74 - 1.64 (m, 2H), 1.61 - 1.51 (m, 2H), 1.46 - 1.36 (m, 2H).

[0062] Synthesis of Example 2 parent compound porphyrin

[0063]

[0064] Accurately weigh 6.13 mL (60 mmol) of benzaldehyde and 2.48 g (20 mmol) of p-hydroxybenzaldehyde into a 500 mL three-necked flask, add 120 mL of propionic acid as solvent, stir at 135°C until the system reaches a micro-boiling state, slowly drop 5.2 mL (80 mmol) of freshly distilled pyrrole through a constant pressure funnel, control the time to be about 30 min, after the drop of pyrrole is completed, reflux for 1 h. Stop the reaction, hot rotary evaporation of 2 / 3 propionic acid under reduced pressure, add 100 mL of anhydrous ethanol, put it in the refrigerator at 4°C for 24 h, reduce pressure and filter, dry, get black purple solid crude product, then purify it through a silica gel column with developing agent (dichloromethane: n-hexane = 2:1), collect the second color band, rotary evaporation under reduced pressure, get porphyrin compound 4. Yield: 8%

[0065] 4: 1 H NMR (500 MHz, Chloroform-d) δ 8.86 (d, J = 12.2 Hz, 8H), 8.25 - 8.21 (m, 6H), 8.06 (d, J = 8.3 Hz, 2H), 7.80 - 7.73 (m, 9H), 7.16 (d, J = 8.3 Hz, 2H), -2.76 (s, 2H).

[0066]

[0067] Accurately weigh 6.13 mL (60 mmol) of benzaldehyde and 2.48 g (20 mmol) of p-hydroxybenzaldehyde into a 500 mL three-necked flask, add 120 mL of propionic acid as solvent, stir at 135°C until the system reaches a micro-boiling state, slowly drop 5.2 mL (80 mmol) of freshly distilled pyrrole through a constant pressure funnel, control the time to be about 30 min, after the drop of pyrrole is completed, reflux for 1 h. Stop the reaction, hot rotary evaporation of 2 / 3 propionic acid under reduced pressure, add 100 mL of anhydrous ethanol, put it in the refrigerator at 4°C for 24 h, reduce pressure and filter, dry, get black purple solid crude product, then purify it through a silica gel column with developing agent (dichloromethane: n-hexane = 2:1), collect the second color band, rotary evaporation under reduced pressure, get porphyrin compound 4. Yield: 8%

[0068] 5: 1 H NMR (500 MHz, Chloroform-d) δ 8.92 - 8.80 (m, 8H), 8.14 (d, J = 7.8 Hz, 6H), 8.06 (d, J = 8.6 Hz, 2H), 7.75 (d, J = 8.4 Hz, 6H), 7.22 (d, J = 8.6 Hz, 2H), -2.84 (s, 2H).

[0069] Example 3 Synthesis of porphyrin-eugenol derivatives

[0070]

[0071] Accurately weigh 44.4 mg (2 mmol) of 3a / 47.2 mg (2 mmol) of 3b / 50.0 mg (2 mmol) of 3c / 52.8 mg (2 mmol) of 3d / 55.6 mg (2 mmol) of 3e eugenol derivatives in a 50 mL single-necked flask, dissolve in an appropriate amount of dichloromethane, add 1 mL of thionyl chloride, 3 drops of DMF as catalyst, react for 3 h, remove the solvent by rotary evaporation under reduced pressure, redissolve in 15 mL of dichloromethane, rotary evaporate again under reduced pressure, dissolve in an appropriate amount of dichloromethane, and reserve. Take another round-bottom flask, add 0.63 g of compound 4 or 0.73 g of compound 5, 416 μL (5 mmol) of triethylamine, and dissolve in 30 mL of dichloromethane. Slowly add the above-mentioned dichloromethane solution to the reaction system, reflux at 40°C for 30 min, after the reaction is completed, rotary evaporate under reduced pressure, and obtain the crude product. Purify the crude product by silica gel column chromatography with developing agent (dichloromethane: n-hexane: ethyl acetate = 20:10:1), collect the second color band, and rotary evaporate under reduced pressure to obtain compounds 6a-6e and compounds 7a-7e.

[0072] Dissolve the prepared compounds 6a-6e and compounds 7a-7e in an appropriate amount of dichloromethane, and add 100 mg of zinc acetate dihydrate to it. Stir at 40°C for 1 h, after the reaction is completed, wash the metal salt in the reaction system with an appropriate amount of deionized water, remove water with anhydrous Na2SO4, and rotary evaporate under reduced pressure to obtain purple-red crystals, which are the target compounds 8a-8e and 9a-9e.

[0073] In this application, 20 eugenol-porphyrin compounds are synthesized, and the structures are confirmed by nuclear magnetic resonance hydrogen spectrum and mass spectrum.

[0074] 6a: Yield: 65.3%, 1 HNMR (500 MHz, Chloroform-d) δ 8.85 (s, 8H), 8.22 (d, J = 6.4 Hz, 8H), 7.81-7.74 (m, 9H), 7.46 (d, J = 8.0 Hz, 2H), 7.10 (d, J = 8.2 Hz, 1H), 6.85-6.80 (m, 2H), 6.05-5.95 (m, 1H), 5.15-5.08 (m, 4H), 3.97 (s, 3H), 3.39 (d, J = 6.7 Hz, 2H), -2.80 (s, 2H). Purity: 98.74%, HRMS: C 56 H 43 N4O4 for [M+H] + , calculated 835.3284, found 835.3245.

[0075] 6b: Yield: 59.5%,1 H NMR (500 MHz, Chloroform-d) δ 8.87 (s, 8H), 8.24 (d, J = 6.7 Hz, 8H), 7.80 - 7.75 (m, 9H), 7.48 (d, J = 7.8 Hz, 2H), 7.12 (d, J = 8.1 Hz, 1H), 6.86 - 6.81 (m, 2H), 6.05 - 5.97 (m, 1H), 5.16 - 5.08 (m, 3H), 3.98 (s, 3H), 3.41 (d, J = 6.7 Hz, 2H), 1.98 (d, J = 6.8 Hz, 3H), -2.77 (s, 2H). Purity: 96.02%, HRMS: C 57 H 45 N4O4 for [M+H] + , calculated 849.3440, found 849.3498.

[0076] 6c: Yield: 68.4%, 1 H NMR (500 MHz, Chloroform-d) δ 8.89 - 8.88 (m, 8H), 8.24 (d, J = 7.0 Hz, 8H), 7.82 - 7.76 (m, 9H), 7.52 (d, J = 7.9 Hz, 2H), 6.94 (d, J = 7.9 Hz, 1H), 6.79 - 6.77 (m, 2H), 6.04 - 5.96 (m, 1H), 5.14 - 5.07 (m, 2H), 4.25 (t, J = 6.1 Hz, 2H), 3.94 (s, 3H), 3.38 (d, J = 6.7 Hz, 2H), 3.03 (t, J = 7.3 Hz, 2H), 2.45 - 2.40 (m, 2H), -2.76 (s, 2H). Purity: 99.33%, HRMS: C 58 H 47 N4O4 for [M+H] + , calculated 863.3596, found 863.3578.

[0077] 6d: Yield: 62.7%, 1H NMR (500 MHz, Chloroform-d) δ 8.87-8.86 (m, 8H), 8.23 (d, J = 7.1 Hz, 8H), 7.81-7.74 (m, 9H), 7.49 (d, J = 7.5 Hz, 2H), 6.89 (d, J = 8.4 Hz, 1H), 6.76-6.75 (m, 2H), 6.02-5.94 (m, 1H), 5.12-5.05 (m, 2H), 4.18-4.14 (m, 2H), 3.91 (s, 3H), 3.36 (d, J = 6.7 Hz, 2H), 2.86 (t, J = 6.8 Hz, 2H), 2.14-2.08 (m, 4H), -2.78 (s, 2H). Purity: 98.89%, HRMS: C 59 H 49 N4O4 for [M+H] + , calculated 877.3752, found 877.3777.

[0078] 6e: Yield: 64.0%, 1 H NMR (500 MHz, Chloroform-d) δ 8.87-8.86 (m, 8H), 8.22 (d, J = 7.2 Hz, 8H), 7.81-7.74 (m, 9H), 7.49 (d, J = 7.5 Hz, 2H), 6.90-6.83 (m, 2H), 6.74-6.73 (m, 1H), 6.02-5.92 (m, 1H), 5.11-5.04 (m, 2H), 4.11-4.07 (m, 2H), 3.89 (s, 3H), 3.35 (d, J = 6.7 Hz, 2H), 2.80 (s, 2H), 2.03-1.97 (m, 4H), 1.75-1.73 (m, 2H), -2.78 (s, 2H). Purity: 99.34%, HRMS: C 60 H 51 N4O4 for [M+H] + , calculated 891.3908, found 891.3882.

[0079] 7a: Yield: 74.3%, 1H NMR (500 MHz, Chloroform-d) δ 8.86 - 8.84 (m, 8H), 8.20 (d, J = 8.1 Hz, 2H), 8.13 (d, J = 7.8 Hz, 6H), 7.75 (d, J = 8.2 Hz, 6H), 7.53 (d, J = 8.1 Hz, 2H), 7.06 (d, J = 8.2 Hz, 1H), 6.85 - 6.80 (m, 2H), 6.04 - 5.95 (m, 1H), 5.14 - 5.09 (m, 4H), 3.98 (s, 3H), 3.40 (d, J = 6.8 Hz, 2H), -2.86 (s, 2H). Purity: 91.48%, HRMS: C 56 H 40 Cl3N4O4 for [M+H] + , calculated 937.2115, found 937.2093.

[0080] 7b: Yield: 42.6%, 1 H NMR (500 MHz, Chloroform-d) δ 8.86 - 8.84 (m, 8H), 8.19 (d, J = 8.0 Hz, 2H), 8.13 (d, J = 7.9 Hz, 6H), 7.75 (d, J = 7.8 Hz, 6H), 7.47 (d, J = 7.9 Hz, 2H), 7.10 (d, J = 8.1 Hz, 1H), 6.85 - 6.80 (m, 2H), 6.05 - 5.96 (m, 1H), 5.15 - 5.08 (m, 3H), 3.97 (s, 3H), 3.40 (d, J = 6.6 Hz, 2H), 1.97 (d, J = 6.9 Hz, 3H), -2.86 (s, 2H). Purity: 92.21%, HRMS:

[0081] C 57 H 42 Cl3N4O4 for [M+H] + , calculated 951.2271, found 951.2366.

[0082] 7c: Yield: 41.5%, 1H NMR (500 MHz, Chloroform-d) δ 8.89-8.84 (m, 8H), 8.20 (d, J = 8.0 Hz, 2H), 8.14 (d, J = 8.0 Hz, 6H), 7.75 (d, J = 8.2 Hz, 6H), 7.51 (d, J = 7.9 Hz, 2H), 6.94 (d, J = 8.2 Hz, 1H), 6.78-6.76 (m, 2H), 6.03-5.94 (m, 1H), 5.13-5.07 (m, 2H), 4.25 (t, J = 6.2

[0083] Hz, 2H), 3.93 (s, 3H), 3.37 (d, J = 6.6 Hz, 2H), 3.02 (t, J = 7.3 Hz, 2H), 2.44-2.39 (m, 2H),

[0084] -2.85 (s, 2H). Purity: 97.47%, HRMS: C 58 H 44 Cl3N4O4 for [M+H] + , calculated 965.2427, found 965.2505.

[0085] 7d: Yield: 43.5%, 1 H NMR (500 MHz, Chloroform-d) δ 8.89-8.84 (m, 8H), 8.20 (d, J = 8.4 Hz, 2H), 8.14 (d, J = 8.0 Hz, 6H), 7.75 (d, J = 8.2 Hz, 6H), 7.50 (d, J = 8.4 Hz, 2H), 6.90 (d, J = 8.5 Hz, 1H), 6.77-6.75 (m, 2H), 6.02-5.94 (m, 1H), 5.12-5.04 (m, 2H), 4.16 (t, J = 5.8 Hz, 2H), 3.91 (s, 3H), 3.36 (d, J = 6.7 Hz, 2H), 2.89-2.83 (m, 2H), 2.14-2.07 (m, 4H), -2.85 (s, 2H). Purity: 97.79%, HRMS: C 59 H 46 Cl3N4O4 for [M+H] + , calculated 979.2583, found 979.2535.

[0086] 7e: Yield: 44.3%, 1H NMR (500 MHz, Chloroform-d) δ 8.84 (s, 8H), 8.21 (d, J = 8.0 Hz, 2H), 8.14 (d, J = 7.8 Hz, 6H), 7.75 (d, J = 7.8 Hz, 6H), 7.50 (d, J = 7.8 Hz, 2H), 6.88 - 6.84 (m, 2H), 6.74 (s, 1H), 6.01 - 5.92 (m, 1H), 5.10 - 5.06 (m, 2H), 4.11 - 4.07 (m, 2H), 3.89 (s, 3H), 3.34 (d, J = 6.7 Hz, 2H), 2.80 (t, J = 8.0 Hz, 2H), 2.03 - 1.97 (m, 4H), 1.78 - 1.71 (m, 2H), -2.85 (s, 2H). Purity: 99.14%, HRMS: C 60 H 48 Cl3N4O4 for [M+H] + , calculated 993.2739, found 993.2746.

[0087] 8a: Yield: 89.9%, 1 H NMR (500 MHz, Chloroform-d) δ 8.95 (s, 8H), 8.22 (d, J = 7.2 Hz, 8H), 7.80 - 7.73 (m, 9H), 7.50 (d, J = 8.0 Hz, 2H), 7.04 (d, J = 8.0 Hz, 1H), 6.84 - 6.80 (m, 2H), 6.04 - 5.95 (m, 1H), 5.14 - 5.08 (m, 4H), 3.97 (s, 3H), 3.39 (d, J = 6.8 Hz, 2H). Purity: 97.70%, HRMS: C 56 H 41 N4O4Zn for [M+H] + , calculated 897.2419, found 897.2414.

[0088] 8b: Yield: 91.0%, 1H NMR (500 MHz, Chloroform-d) δ 8.95 (s, 8H), 8.22 (d, J = 6.8 Hz, 8H), 7.80 - 7.73 (m, 9H), 7.45 (d, J = 8.6 Hz, 2H), 7.10 (d, J = 8.1 Hz, 1H), 6.85 - 6.78 (m, 2H), 6.04 - 5.94 (m, 1H), 5.13 - 5.08 (m, 3H), 3.96 (s, 3H), 3.39 (d, J = 6.8 Hz, 2H), 1.95 (d, J = 6.9 Hz, 3H). Purity: 98.82%, HRMS: C 57 H 43 N4O4Zn for [M+H] + , calculated 911.2575, found 911.2551.

[0089] 8c: Yield: 92.3%, 1 H NMR (500 MHz, Chloroform-d) δ 8.96 - 8.95 (m, 8H), 8.22 (d, J = 6.0 Hz, 8H), 7.80 - 7.74 (m, 9H), 7.49 (d, J = 7.9 Hz, 2H), 6.93 (d, J = 8.3 Hz, 1H), 6.77 - 6.75 (m, 2H), 6.02 - 5.94 (m, 1H), 5.12 - 5.06 (m, 2H), 4.23 (t, J = 6.3 Hz, 2H), 3.91 (s, 3H), 3.36 (d, J = 6.8 Hz, 2H), 3.00 (t, J = 7.3 Hz, 2H), 2.43 - 2.38 (m, 2H). Purity: 99.72%, HRMS:

[0090] C 58 H 45 N4O4Zn for [M+H] + , calculated 925.2731, found 925.2690.

[0091] 8d: Yield: 94.1%, 1H NMR (500 MHz, Chloroform-d) δ 8.97 - 8.95 (m, 8H), 8.22 (d, J = 6.5 Hz, 8H), 7.80 - 7.73 (m, 9H), 7.47 (d, J = 8.4 Hz, 2H), 6.88 (d, J = 8.3 Hz, 1H), 6.75 - 6.74 (m, 2H), 6.01 - 5.93 (m, 1H), 5.12 - 5.03 (m, 2H), 4.18 - 4.12 (m, 2H), 3.90 (s, 3H), 3.35 (d, J = 6.7 Hz, 2H), 2.85 (t, J = 6.8 Hz, 2H), 2.12 - 2.07 (m, 4H). Purity: 99.30%, HRMS:

[0092] C 59 H 47 N4O4Zn for [M+H] + , calculated 939.2887, found 939.2863.

[0093] 8e: Yield: 86.4%, 1 H NMR (500 MHz, Chloroform-d) δ 8.96 - 8.95 (m, 8H), 8.22 (d, J = 6.1 Hz, 8H), 7.80 - 7.73 (m, 9H), 7.48 (d, J = 8.7 Hz, 2H), 6.86 (d, J = 8.2 Hz, 1H), 6.74 - 6.73 (m, 2H), 6.00 - 5.92 (m, 1H), 5.10 - 5.04 (m, 2H), 4.09 (t, J = 6.6 Hz, 2H), 3.88 (s, 3H), 3.34 (d, J = 6.7 Hz, 2H), 2.79 (t, J = 7.4 Hz, 2H), 2.02 - 1.96 (m, 4H), 1.77 - 1.72 (m, 2H).

[0094] Purity: 99.57%, HRMS: C 60 H 49 N4O4Zn for [M+H] + , calculated 953.3043, found 953.3129.

[0095] 9a: Yield: 85.0%, 1H NMR (500 MHz, Chloroform-d) δ 8.96-8.94 (m, 8H), 8.20 (d, J = 8.0 Hz, 2H), 8.14 (d, J = 7.9 Hz, 6H), 7.75 (d, J = 7.9 Hz, 6H), 7.52 (d, J = 8.1 Hz, 2H), 7.04 (d, J = 8.0 Hz, 1H), 6.84-6.78 (m, 2H), 6.04-5.96 (m, 1H), 5.15-5.07 (m, 4H), 3.97 (s, 3H), 3.39 (d, J = 6.7 Hz, 2H). Purity: 94.43%, HRMS: C 56 H 38 Cl3N4O4Zn for [M+H] + , calculated 1013.1406, found 1013.1512.

[0096] 999.1250, found 999.1365.

[0097] 9b: Yield: 93.2%, Purity: 97.33%, HRMS: C 1 H NMR (500 MHz, Chloroform-d) δ 8.96-8.94 (m, 8H), 8.20 (d, J = 8.0 Hz, 2H), 8.14 (d, J = 7.9 Hz, 6H), 7.75 (d, J = 7.9 Hz, 6H), 7.52 (d, J = 8.1 Hz, 2H), 7.04 (d, J = 8.0 Hz, 1H), 6.84-6.78 (m, 2H), 6.04-5.96 (m, 1H), 5.15-5.07 (m, 4H), 3.97 (s, 3H), 3.39 (d, J = 6.7 Hz, 2H). Purity: 94.43%, HRMS: C 57 H 40 Cl3N4O4Zn for [M+H] + , calculated 1013.1406, found 1013.1512.

[0098] 9c: Yield: 93.2%, Purity: 97.33%, HRMS: C 1H NMR (500 MHz, Chloroform-d) δ 8.98 - 8.94 (m, 8H), 8.20 (d, J = 8.3 Hz, 2H), 8.14 (d, J = 8.2 Hz, 6H), 7.75 (d, J = 8.2 Hz, 6H), 7.48 (d, J = 8.3 Hz, 2H), 6.88 (d, J = 8.3 Hz, 1H), 6.75 - 6.74 (m, 2H), 6.01 - 5.94 (m, 1H), 5.11 - 5.06 (m, 2H), 4.17 - 4.11 (m, 2H), 3.90 (s, 3H), 3.35 (d, J = 6.8 Hz, 2H), 2.88 - 2.81 (m, 2H), 2.14 - 2.07 (m, 4H). Purity: 99.49%, HRMS: C 58 H 42 Cl3N4O4Zn for [M+H] + , calculated 1041.1718, found 1041.1700.

[0099] 9d: Yield: 87.8%, 1 H NMR (500 MHz, Chloroform-d) δ 8.98 - 8.94 (m, 8H), 8.20 (d, J = 8.3 Hz, 2H), 8.14 (d, J = 8.2 Hz, 6H), 7.75 (d, J = 8.2 Hz, 6H), 7.48 (d, J = 8.3 Hz, 2H), 6.88 (d, J = 8.3 Hz, 1H), 6.75 - 6.74 (m, 2H), 6.01 - 5.94 (m, 1H), 5.11 - 5.06 (m, 2H), 4.17 - 4.11 (m, 2H), 3.90 (s, 3H), 3.35 (d, J = 6.8 Hz, 2H), 2.88 - 2.81 (m, 2H), 2.14 - 2.07 (m, 4H). Purity: 99.49%, HRMS: C

[0100] Purity: 99.49%, HRMS: C 59 H 44 Cl3N4O4Zn for [M+H] + , calculated 1041.1718, found 1041.1700.

[0101] 9e: Yield: 93.8%, 1H NMR (500 MHz, Chloroform-d) δ 8.98 - 8.94 (m, 8H), 8.20 (d, J = 7.9 Hz, 2H), 8.14 (d, J = 8.2 Hz, 6H), 7.75 (d, J = 7.8 Hz, 6H), 7.49 (d, J = 8.1 Hz, 2H), 6.86 (d, J = 8.7 Hz, 1H), 6.74 - 6.73 (m, 2H), 6.00 - 5.92 (m, 1H), 5.10 - 5.03 (m, 2H), 4.09 (t, J = 6.7 Hz, 2H), 3.88 (s, 3H), 3.34 (d, J = 6.7 Hz, 2H), 2.79 (t, J = 7.5 Hz, 2H), 2.02 - 1.96 (m, 4H), 1.77 - 1.70 (m, 2H). Purity: 99.64%, HRMS: C 60 H 46 Cl3N4O4Zn for [M+H] + , calculated 1055.1874, found 1055.1819.

[0102] Example 4 Singlet oxygen detection

[0103] 1,3-Diphenylbenzofuran (DPBF) is a kind of singlet oxygen detection probe, which can detect the rate and yield of singlet oxygen generated by porphyrin compounds. The structure of DPBF is shown below, which will react with singlet oxygen when singlet oxygen exists, generating dibenzoylbenzene (DBB), which does not have absorption, causing the change of the structure of DPBF and the change of the fluorescence signal. Therefore, the fluorescence intensity of DPBF near 463 nm can be used to judge the generation ability of singlet oxygen, and further to evaluate the effect of photodynamic therapy. The reaction mechanism of DPBF and 1O2 is as follows:

[0104]

[0105] DPBF solution preparation: 1.35 mg of DPBF was accurately weighed and dissolved in 1 ml of chloroform as a solvent, diluted to 250 ml, and prepared into a solution with a concentration of 20 μmol / L, and stored in the dark for use.

[0106] Standard sample solution preparation: an appropriate amount of standard compound was weighed, dissolved in chloroform as a solvent, gradient diluted, and prepared into a solution with a concentration of 8 μmol / L, and stored in the dark for use.

[0107] Test compound solution preparation: an appropriate amount of different test compounds was weighed, dissolved in chloroform as a solvent, gradient diluted, and prepared into a solution with a concentration of 8 μmol / L, and stored in the dark for use.

[0108] After the solution is prepared, equal volumes of DPBF solution, standard solution and test solution are taken, and the DPBF solution is mixed with the standard solution and the test solution, respectively. The initial absorbance of each group is measured using a fluorescence spectrophotometer, and then a single laser is irradiated at 418 nm to generate singlet oxygen. Then, the fluorescence intensity of DPBF at 463 nm is measured every 10 seconds. Since DPBF itself will also experience a decrease in absorbance over time, the fluorescence of DPBF is also measured by diluting the concentration of DPBF to 10 μmol / L. The test results are shown in the accompanying drawings Figures 1-4

[0109] Considering that DPBF itself is affected by the set light stimulus, a DPBF blank test was conducted, and it was found that DPBF also undergoes some decomposition, but still maintains a high intensity. Subsequently, eugenol raw material and DPBF were mixed and detected, and it was found that the addition of eugenol had no effect on the fluorescence of DPBF. However, after the addition of the synthesized eugenol-porphyrin derivative, the fluorescence of DPBF changed significantly, with a significant decrease in intensity. The size of the change in DPBF fluorescence represents the generation ability of singlet oxygen. The lower the fluorescence intensity, the stronger the generation ability of singlet oxygen, and the faster the downward trend, indicating a faster generation rate.

[0110] From the graph, it can be found that both the porphyrin parent and the target compound have good singlet oxygen generation ability. The generation ability of singlet oxygen of the porphyrin parent and the eugenol-porphyrin derivative is not much different, indicating that the bond of eugenol has no effect on the conjugated system of porphyrin. Overall, compared to the 6, 7 series of compounds, the initial downward trend of the 8, 9 series of compounds after metal chelation is slightly larger than that of the 6, 7 series, indicating that the generation ability of singlet oxygen is slightly stronger than that of the unmetalized compounds.

[0111] Example 5: In vitro anti-tumor activity test

[0112] Human hepatoma cells HepG2 and human non-small cell lung cancer cells A549 were obtained from the Cell Resource Center of Shanghai Life Sciences Research Institute, Chinese Academy of Sciences.

[0113] 5.1 Preparation of reagents

[0114] Preparation of PBS solution: Pour the PBS phosphate buffer into a clean 1 L beaker, and use a graduated cylinder to measure distilled water to the 1 L mark. Use a sterilized glass bottle to portion it, and then transfer it to a high-pressure sterilization pot at 140°C for sterilization. The sterilized PBS solution is stored in a 4°C refrigerator for later use.

[0115] ​Preparation of MTT probe solution: 50 mg of MTT powder was accurately weighed and dissolved in 10 ml of sterilized PBS solution. The solution was dissolved under ultraviolet irradiation in an ultraclean workbench and filtered with a 0.22 μm filter to remove bacteria. The solution was then sealed and stored in a refrigerator at -20°C in the dark.

[0116] Preparation of culture medium: fetal bovine serum and DMEM were mixed at a ratio of 1:9, and 1% of the mixture of penicillin and streptomycin was added to the mixture. The mixture was mixed uniformly and sealed with a sealing film for storage.

[0117] 5.2 Cell resuscitation and culture

[0118] The frozen A549 cell (non-small cell lung cancer) / HepG2 cell (human liver cancer cell) tube was taken out from a -80°C refrigerator, and 2 / 3 of the frozen tube was immersed in warm water at about 37°C. The frozen tube was gently shaken to fully thaw the tumor cells. After the cells were thawed, they were transferred to an ultraviolet irradiated ultraclean workbench, and the cells were transferred to a 15 mL centrifuge tube with a pipette. After centrifugation for five minutes, the supernatant was removed. 1-2 mL of culture medium was added to the cell precipitate, and the cells were dispersed uniformly by blowing. 3 mL of culture medium was added to a new cell culture bottle, and the cell suspension was transferred to the cell culture bottle with a pipette. Then, the cell culture bottle was placed in a constant temperature cell incubator at 5% CO2 and 37°C for culture.

[0119] When the cells were observed to grow in the culture bottle under a microscope, the original culture medium was discarded, 2-3 mL of PBS solution was aspirated for washing, and the washing was repeated three times. The PBS solution was discarded, and the residual PBS solution was aspirated with a pipette. Then, 1 mL of trypsin was aspirated and digested for 30 seconds (observed under a microscope). 2 mL of culture medium was quickly added to the culture bottle, and the cell suspension was prepared by blowing with a pipette. The cell suspension was transferred to a centrifuge tube and centrifuged again. The supernatant was discarded, and 3 mL of culture medium was added to mix the cell suspension. 3 mL of culture medium was added to two cell culture bottles, and the cell suspension obtained above was transferred to the two culture bottles, respectively. The mixture was placed in a cell culture incubator for culture.

[0120] When cells are observed to have confluently grown in the culture flask under a microscope, discard the original culture medium, wash with 2-3 mL of PBS solution, repeating the washing process three times. Discard the PBS solution, remove any remaining PBS solution with a pipette, then add 1 mL of trypsin and digest for 30 seconds (until cells become rounded under the microscope). Quickly add 2 mL of culture medium to the culture flask, pipette to create a cell suspension, transfer to a centrifuge tube, centrifuge again, discard the supernatant, and add 3 mL of culture medium to mix well to form a cell suspension. In a separate sample well, add 13 mL of culture medium, then add 200 μL of the cell suspension and mix well with a pipette. In a 96-well plate, add 200 μL of PBS solution to each well around the perimeter, and add 100 μL of cell-containing culture medium to the remaining wells. Incubate in a cell culture incubator for 24 hours.

[0121] 5.3 Cell drug administration and OD value determination

[0122] Remove the 96-well plate and discard the culture medium. The target compound concentrations were set at 128 μmol / L, 64 μmol / L, 32 μmol / L, and 16 μmol / L, with three replicates for each concentration. Add 150 μL of drug-containing culture medium to each well. Two control groups were set up: a blank group (culture medium group) and a negative control group (culture medium containing DMAC), with six replicates for each group. Label plates A (light group) and B (dark group), and then incubate them. After 24 hours, remove plates A and B. Irradiate plate A with near-infrared light for 10 minutes, while plate B is not irradiated. After illumination, add 20 μL of freshly prepared MTT solution to each replicate well and continue incubating for 4 hours. Then remove plates A and B, discard the culture medium, add 150 μL of DMSO to each replicate well, shake on a shaker for 10 minutes, and measure the absorbance (OD value) at 490 nm using a microplate reader. The IC50 values ​​of the compounds were calculated using GraphPad Prism 6.0 software, with each experiment performed in triplicate. The IC50 result is calculated as mean ± standard deviation. The test results are as follows:

[0123]

[0124]

[0125] As can be seen from the above, most of the porphyrin-eugenol derivatives of this invention exhibit stronger inhibitory effects on HepG2 and A549 cells than the eugenol raw material, porphyrin parent compounds 4 and 5. The metallized target compounds are generally more active than the free-base porphyrin compounds, indicating that the combination of eugenol and porphyrin provides a good synergistic anti-tumor effect. Under light irradiation, compounds 9a and 9c show significantly stronger effects on HepG2 cells, and compounds 8a, 8c, and 9a show significantly stronger effects on A549 cells, demonstrating good photodynamic therapy.

Claims

1. A porphyrin-eugenol derivative, characterized in that, The porphyrin-eugenol derivative has the following structure:

2. A pharmaceutical composition comprising the porphyrin-eugenol derivative or a pharmaceutically acceptable salt thereof according to claim 1, and a pharmaceutically acceptable carrier.

3. Use of the porphyrin-eugenol derivative or a pharmaceutically acceptable salt thereof according to claim 1 or the pharmaceutical composition according to claim 2 for the manufacture of a medicament for the treatment of cancer selected from lung cancer or liver cancer.

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