A porphyrin-chrysin conjugate and its synthesis method and application

Abstract

This invention discloses a porphyrin-caprylin conjugate, its synthesis method, and its applications, belonging to the field of pharmaceutical synthesis technology. Porphyrin-like compounds (such as pyrophyllophospholipid a) are coupled with caprylin via esterification or carbon-carbon double bond addition reactions to obtain a porphyrin-caprylin conjugate with both photodynamic and chemotherapeutic biological activities, which can be used to prepare antitumor drugs.

Description

Technical Field

[0001] This invention relates to a porphyrin-caprylin conjugate and its synthesis method, and also to the application of the porphyrin-caprylin conjugate in the preparation of photodynamic therapy combined with chemotherapy antitumor agents, belonging to the field of drug synthesis technology. Background Technology

[0002] Cancer remains a leading cause of unnatural death worldwide, and malignant tumors pose a serious threat to human life and health. While surgical resection, radiotherapy, chemotherapy, and immunotherapy have achieved some success, they also present numerous challenges, including significant trauma, severe side effects, and a high risk of tumor recurrence.

[0003] Photodynamic therapy (PDT), as a novel and highly effective treatment, utilizes lasers to induce intersystemic crossover of photosensitizers enriched at the lesion site, rapidly forming their triplet excited state and transferring energy to ground-state oxygen. 3 O2) to produce singlet oxygen, which is cytotoxic. 1 O2). Therefore, PDT has advantages such as low toxicity, minimal trauma, and good selectivity. PDT has been applied to the treatment of skin cancer, squamous cell carcinoma, prostate cancer, breast cancer, cervical cancer, lung cancer, and many other cancers.

[0004] Photodynamic therapy combined with chemotherapy for anti-tumor treatment can exert the dual effects of photodynamic therapy and chemotherapy under light irradiation, enhancing its ability to kill cancer cells and reducing the side effects of chemotherapy drugs on the human body. This has great significance and value in research.

[0005] Juglansine, a flavonoid compound, has shown potent antitumor effects against breast cancer, cervical cancer, malignant glioma, esophageal cancer, colon cancer, and prostate cancer in in vitro studies. However, chemotherapeutic drugs like juglansine often suffer from low bioavailability or significant toxic side effects. Porphyrin compounds, with their macrocyclic conjugated structure, are the most common photosensitizers. Furthermore, porphyrin photosensitizers possess advantages such as easy accumulation in tumor lesions, low dark cytotoxicity, strong absorption around the near-infrared window, and high singlet oxygen production rate.

[0006] Chinese patent (CN109912607A) discloses a novel type of porphyrin-salicylate derivative. This patent uses chemically synthesized hydroxyporphyrin as the parent compound and dibromoalkanes with different carbon numbers as linking chains to couple salicylate phenolic hydroxyl groups and porphyrin phenolic hydroxyl groups in the form of ether bonds to obtain a novel type of antitumor compound. However, this type of porphyrin compound contains a symmetrical skeleton structure, its absorption wavelength in the Q band is relatively small, and it is mainly coupled through a bond with good stability, making it difficult to release the carrier and antitumor drugs during cancer cell degradation. Summary of the Invention

[0007] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a porphyrin-caprylin conjugate, which uses a porphyrin with photosensitizing properties as a carrier and caprylin with anticancer activity as a chemotherapeutic drug. The conjugate absorbs visible light in the range of 400-750 nm and can be used as an anticancer drug combining photodynamic therapy and chemotherapy.

[0008] The second objective of this invention is to provide a novel synthetic method for porphyrin-aspergillin conjugates, which utilizes widely available porphyrins, operates under mild conditions, exhibits high coupling efficiency, and yields high-quality target conjugates, thus facilitating large-scale production.

[0009] A third objective of this invention is to provide an application of a porphyrin-salicylate conjugate that possesses both photodynamic and chemotherapeutic biological activities and can be used as an antitumor drug.

[0010] The fourth objective of this invention is to provide a method for preparing porphyrin-like substances, which simplifies the process of obtaining the porphyrin-like raw material pyrophylloxera a and improves the yield, thereby reducing the synthesis cost of porphyrin-ascorbic acid couplings.

[0011] To achieve the above-mentioned technical objectives, the present invention provides a porphyrin-salicylic acid conjugate, which has the following properties:

[0012] Structural formulas of Equation 1, Equation 2, Equation 3, or Equation 4:

[0013]

[0014]

[0015] in,

[0016] R1 and R3 are selected from R is selected from Or C1~C 10 Alkyl groups; R2, R8, and R9 are independently selected from H, Or C1~C 10 Alkyl groups;

[0017] R4 and R5 are selected from H or R6 and R7 are selected independently. R 10 R 11 R 12 and R 13 Independently selected from H or And each of Equations 1 to 4 contains at least one

[0018] As a preferred option, the porphyrin-salicylate coupling compound has the following structural formula:

[0019]

[0020] This invention also provides a method for synthesizing a porphyrin-salicylic acid conjugate, the method being:

[0021] The carboxyl-containing porphyrin is coupled to the phenolic hydroxyl group of juglansine via an esterification reaction to obtain the product; or,

[0022] The vinyl-containing porphyrin is coupled to the phenolic hydroxyl group of succinin via an indirect addition reaction to obtain the product; or,

[0023] The product is obtained by coupling a porphyrin containing carboxyl and vinyl groups with the phenolic hydroxyl groups of apigenin via esterification and / or indirect addition reactions.

[0024] The porphyrin-like structures have the structures of formula 5, formula 6, formula 7 or formula 8:

[0025]

[0026]

[0027] in,

[0028] R 15 and R 16 Selected from R0 is selected from C1 to C2. 10 Alkyl groups;

[0029] R 14 R 17 and R 18 Independently selected from H or C1~C 10 Alkyl groups.

[0030] As a preferred embodiment, the esterification reaction is carried out under the following conditions: using DCM as the solvent, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and / or N,N'-dicyclohexylcarbodiimide as condensing agents, and 4-dimethylaminopyridine and / or 1-hydroxybenzotriazole as catalysts, at 0–25°C for 12–24 hours. Chloroform and tetrahydrofuran (THF) can also be used as solvents, but their effectiveness is inferior to that of dichloromethane (DCM). The preferred catalyst is 4-dimethylaminopyridine (DMAP). The preferred reaction temperature is 15–25°C.

[0031] As a preferred embodiment, the ratio of carboxyl group content to the molar amount of condensing agent, succinic acid and catalyst in the carboxyl-containing porphyrin or carboxyl and vinyl-containing porphyrin is 1:(1.5-2):(1.5-2):0.5, and the feeding ratio of condensing agent and succinic acid is the same.

[0032] As a preferred embodiment, the indirect addition reaction process is as follows: a vinyl-containing porphyrin or a carboxyl and vinyl-containing porphyrin first undergoes an addition reaction with hydrogen bromide, and then undergoes a nucleophilic substitution reaction with salicumin.

[0033] As a preferred embodiment, the addition reaction is performed under the following conditions: using a hydrogen bromide-acetic acid mixed solution as the addition reagent, and reacting at 0–25°C for 8–16 hours. The hydrogen bromide-acetic acid mixed solution is a 33% hydrogen bromide-acetic acid mixed solution, with a hydrogen bromide mass concentration of 33%.

[0034] As a preferred embodiment, the nucleophilic substitution reaction is performed under the following conditions: using K2CO3 as the base reagent and 18-crown ether-6 as the co-solvent, at 0–25°C for 8–16 hours.

[0035] This invention also provides an application of a porphyrin-chrysin conjugate for preparing an antitumor agent combining photodynamic therapy and chemotherapy. Using PPA-C, an esterification conjugate of pyrophyllite a (PPA) and chrysin, as an example, this invention verifies its dual antitumor effects against melanoma through photodynamic and chemotherapeutic interactions.

[0036] This invention also provides a method for preparing a porphyrin-like substance. The method involves mixing silkworm excrement with an acid-degrading solvent for an acid hydrolysis reaction. After the acid hydrolysis reaction is complete, the reaction mixture is distilled under reduced pressure to recover acetic acid, and then poured into saturated brine to precipitate crude porphyrin-like product. The acid-degrading solvent consists of concentrated hydrochloric acid and acetic acid in a volume ratio of 1:8-10. The liquid-to-solid ratio of the acid-degrading solvent to the silkworm excrement is 2.5-5 mL:1 g. The acid hydrolysis reaction is carried out at room temperature for 8-16 hours.

[0037] The porphyrin-like substances involved in this invention can be derived from the degradation products of chlorophyll a, for example, obtained by acid degradation extraction from silkworm excrement containing chlorophyll a.

[0038] The acid degradation pathway of chlorophyll a is as follows:

[0039]

[0040] Compound 1 and Compound 2 are usually prepared by acid degradation of chlorophyll a using the hydrochloric acid-ether method. The amount of hydrochloric acid used is usually 1 to 5 times that of the ether (Reference: "Chemical Study on the Degradation Process of Chlorophyll a in Silkworm Excrement", Yao Jianzhong et al., Chinese Traditional and Herbal Drugs, 1999, (08): 568-571).

[0041] In this invention, acetic acid is used instead of diethyl ether, and the proportion of hydrochloric acid used is greatly reduced (the volume ratio of hydrochloric acid to acetic acid is 1:8-10). Most of the acetic acid can be recovered by rotary evaporation under reduced pressure. The remaining acetic acid and hydrochloric acid are added to 10 times the amount of cold saturated brine to precipitate the crude product of chlorophyll α acid degradation.

[0042] The porphyrin-like compounds of the present invention are coupled with leucine via an esterification reaction (using pyrophyllite a as an example for illustration):

[0043]

[0044] This invention uses EDCI as a dehydrating condensing agent and DMAP as a nucleophilic catalyst to esterify and couple pyrophyllite a with leucine in DCM. The conditions are mild, and because leucine is insoluble in DCM, this reduces the byproducts of the direct reaction between leucine and the dehydrating condensing agent, resulting in a higher purity esterified coupling product.

[0045] Addition coupling of porphyrins with eugenol (using methyl pyromethoxymethyl chlorophyllin a as an example):

[0046]

[0047] This invention prepares the addition coupling product of chlorophyll a derivative and apigenin through a two-step method. The reaction is carried out at room temperature, which reduces the elimination reaction of the bromoporphyrin intermediate under alkaline conditions, thereby obtaining the nucleophilic substitution product of apigenin and hydrolysis by-products, and improving the conversion rate of raw materials.

[0048] The molecular structures of some porphyrin-salicylic acid conjugates prepared in this invention are as follows:

[0049]

[0050] The porphyrins of the present invention contain carboxyl or vinyl groups, such as chlorophyll α acid degradation derivatives, heme derivatives, and synthetically produced porphyrin compounds, including but not limited to the following compounds:

[0051]

[0052] Compared with existing technologies, the beneficial technical effects of the present invention are as follows:

[0053] The porphyrin-caprylin conjugate provided by this invention uses a porphyrin-caprylin with photosensitizing properties as a carrier and caprylin with anticancer activity as a chemotherapeutic drug. In particular, the conjugate absorbs visible light in the range of 400-750 nm, and can be used as an anticancer drug combining photodynamic therapy and chemotherapy. At the same time, the porphyrin-caprylin conjugate can be bonded by biologically active ester groups, which makes it stable outside cancer cells and easy to degrade and release antitumor drugs inside cancer cells.

[0054] The method for synthesizing porphyrin-salicylic acid conjugates provided by this invention has a wide range of raw material sources, mild conditions, high coupling efficiency, and high yield of the target conjugate, which is conducive to large-scale production.

[0055] The method for synthesizing porphyrin-like raw materials provided by this invention is simple, has a high yield, and greatly reduces the raw material cost of porphyrin-salicylic acid couplings. Attached Figure Description

[0056] Figure 1 PPIX-C2 mass spectrometry detection map (ESI) + ).

[0057] Figure 2 TCPP-C4 mass spectrometry detection map (ESI) + ).

[0058] Figure 3 TCPP-C3 mass spectrometry detection map (ESI) + ).

[0059] Figure 4 TCPP-C2 mass spectrometry detection map (ESI) + ).

[0060] Figure 5 For TCPP-C2 and TCPP-C1 mass spectrometry detection maps (ESI) + ).

[0061] Figure 6 This is a schematic diagram illustrating the combined photodynamic therapy and chemotherapy for antitumor effects of porphyrin-salicylate conjugates.

[0062] Figure 7 The UV-Vis absorption spectrum of the porphyrin-salicylic acid conjugate in DCM is shown.

[0063] Figure 8 Infrared spectra of PPA and CHRYSIN with PPA-C.

[0064] Figure 9 For the assay of the anti-melanoma activity of PPA-C and its control (cell live / dead staining analysis).

[0065] Figure 10 The UV-Vis absorption spectra of the PPA, NPC, and PPA-C anti-melanoma samples (PBS solution) are shown. Detailed Implementation

[0066] To make the present invention clearer and to highlight its technical advantages, the following detailed description is provided in conjunction with specific embodiments.

[0067] The chlorophyll a derivative used in this invention was prepared in-house using the following method.

[0068] Methyl pyromethoxyphylla a and its hexyl ether derivatives were prepared using silkworm excrement mud as raw material.

[0069] Example 1

[0070] Preparation of the active pharmaceutical ingredient pyromethesin a (PPA):

[0071] Take 50g of silkworm excrement mud, dissolve it in 160mL of acetic acid, add 20mL of concentrated hydrochloric acid under ice bath, continue stirring at room temperature for more than 12 hours, filter, distill off most of the acetic acid from the filtrate under reduced pressure, add 150mL of cold saturated saline, let stand for 1 hour, filter, dry the filter cake, and use dichloromethane:methanol = 10:1 (v / v) as the developing solvent to obtain 489mg of pyrophyllophospholipid a (PPA) by silica gel column chromatography. The purity was determined to be 83% by high performance liquid chromatography (HPLC). Recrystallization from ethanol-petroleum ether yielded 178mg of purple rod-shaped crystals with a metallic luster. The purity was determined to be >95% by high performance liquid chromatography (HPLC).

[0072] PPA molecular structure characterization: 1 H-NMR (400MHz, DMSO-d6, ppm) δ12.08 (s, 1H, 37-OH), 9.66 (s, 1H, 10-CH), 9.40 (s, 1H, 5-CH), 8.88 (s, 1H, 20-CH), 8.16-8.23 (dd, 1H, 26- CH), 6.35-6.4(dd, 1H, 27-CH), 6.18-6.22(dd, 1H, 27'-CH), 5.09-5.24(q, 2H, 33-CH2), 4.56-4.61(m, 1H, 18-CH), 4.30-4.33(m, 1H, 17-C H), 3.64-3.69(q, 2H, 29-CH2), 3.59(s, 3H, 31-CH3), 3.43(s, 3H, 28-CH3), 3.19(s, 3H, 25-CH3), 2.53-2.60(m, 2H, 35-CH2), 2.29-2.35( m, H, 34-CH), 2.07-2.15 (m, H, 34'-CH), 1.77-1.79 (d, 3H, 38-CH3), 1.59-1.63 (t, 3H, 30-CH3), 0.21 (s, 1H, 21-NH), -2.00 (s, 1H, 23-NH).

[0073] MS(ESI + m / z: 535.27 [M+H] + (100%).

[0074] Example 2

[0075] Preparation of the active pharmaceutical ingredient methyl pyromethoxyphylla (MPPA):

[0076] Take 50g of silkworm excrement mud, dissolve it in 200mL of methanol, and add 5mL of concentrated sulfuric acid while stirring under ice bath conditions. Continue stirring at room temperature for more than 12 hours. Distill off most of the methanol under reduced pressure, add 100mL of ultrapure water, adjust the pH to 4-6 with concentrated NaOH solution, use diatomaceous earth as a filter aid, dry the filter cake, wash it with dichloromethane, and distill the filtrate under reduced pressure to obtain a black viscous substance. Dissolve it in a small amount of ethyl acetate, add 10 times the amount of petroleum ether, sonicate for 1-5 minutes, and filter to obtain 4.5g of a black solid with a metallic luster (containing methyl pheophylate a and methyl pyropheophylate a).

[0077] The crude product was dissolved in 80 mL of pyridine under argon protection and heated to reflux for 8 h to remove the methoxycarbonyl group (-COOCH3) at position 33. The pyridine was then distilled off under reduced pressure, sonicated with dilute hydrochloric acid (pH=1) for 1 min, extracted with dichloromethane, washed with water, and then rotary evaporated under reduced pressure to obtain 3.8 g of crude pyromethoxyphylla α-methyl ester. The purity was determined to be 13.82% by high-performance liquid chromatography (HPLC). Further purification using silica gel column chromatography with ethyl acetate:petroleum ether (v / v) = 1:2 yielded 504 mg of purified pyromethoxyphylla α-methyl ester. The purity was determined to be 91.7% by HPLC.

[0078] MPPA molecular structure characterization: 1 H-NMR (400MHz, CDCl3, ppm) δ9.52 (s, 1H, 10-CH), 9.40 (s, 1H, 5-CH), 8.56 (s, 1H, 20-CH), 7.97-8.05 (dd, 1H, 26-CH), 6.26-6.31 (d d, 1H, 27-CH), 6.16-6.19 (dd, 1H, 27'-CH), 5.09-5.29 (q, 2H, 33-CH2), 4.46-4.52 (m, 1H, 18-CH), 4.29-4.31 (m, 1H, 17-CH), 3.68- 3.73 (q, 2H, 29-CH2), 3.68 (s, 3H, 37-OCH3), 3.61 (s, 3H, 31-CH3), 3.41 (s, 3H, 28-CH3), 3.21 (s, 3H, 25-CH3), 2.53-2.72 (m, 2H, 35 -CH2), 2.25-2.36 (m, H, 34-CH2), 1.80-1.82 (d, 3H, 38-CH3), 1.68-1.72 (t, 3H, 30-CH3), 0.46 (s, 1H, 21-NH), -1.68 (s, 1H, 23-NH).

[0079] MS(ESI + m / z: 549.29 [M+H] + (100%).

[0080] Example 3

[0081] Preparation of the active pharmaceutical ingredient pyromethesin α-hexyl ether (HPPA):

[0082] 100 mg of pyrophyllin a (PPA) was dissolved in 10 mL of anhydrous dichloromethane, and then 1 mL of 33% HBr-CH3COOH solution was added. The mixture was reacted in an ice bath at 0-15℃ for 12 h. The solvent and acetic acid were removed by vacuum distillation, and the mixture was dried under argon gas. The solution was then redissolved in 10 mL of anhydrous dichloromethane, and 0.5 mL of n-hexyl ether was added. The mixture was reacted at room temperature (15-20℃) for 12 h. The solvent was removed by vacuum distillation, and the solution was dissolved in ethanol containing a small amount of NaOH and sonicated for 1-5 min. Excess petroleum ether was added to precipitate the sodium HPPA salt. After water dissolution, the pH was adjusted to obtain crude HPPA. 58 mg of pure pyrophyllin a-hexyl ether was obtained by silica gel column chromatography with a dichloromethane:methanol ratio of 15:1. The purity was determined to be 93.9% by high-performance liquid chromatography (HPLC).

[0083] HPPA molecular structure characterization: 1 H-NMR (400MHz, CDCl3, ppm) δ9.76 (s, 1H, 10-CH), 9.48 (s, 1H, 5-CH), 8.50 (s, 1H, 20-CH), 5.86-5.91 (q, 1H, 26-CH), 5.09-5.29 (q, 2H, 33-CH2), 4.45-4.48(m, 1H, 18-CH), 4.30-4.32(m, 1H, 17-CH), 3.68-3.72(q, 2H, 29-CH2), 3.65(s, 3H, 37-OCH3), 3.61(s, 3H, 31-CH3), 3.55-3.59 (t, 2H, 38-CH2), 3.36 (s, 3H, 28-CH3), 3.25 (s, 3H, 25-CH3), 2.57-2.70 (m, 2H, 35-CH2), 2.295-2.35 (m, H, 34-CH2), 2.09-2. 11 (d, 3H, 27-CH3), 1.28-1.71 (d, 37-CH3, 30-CH3, 39-CH2, 40-CH2, 41-CH2, 42-CH2), 0.74-0.78 (t, 3H, 43-CH3), -1.68 (s, 1H, 23-NH).

[0084] MS(ESI + m / z: 637.38 [M+H] + (100%).

[0085] Preparation of chlorophyll a derivative conjugate with aspergillin:

[0086]

[0087] Example 4

[0088] Preparation of the esterification coupling product (PPA-C) of pyrophyllite a (PPA) and chrysin:

[0089] In a 25 mL flask, 53.4 mg (0.1 mmol) of PPA, 38.1 mg (0.15 mmol) of succinate, 28.8 mg (0.15 mmol) of EDCI, and 6.1 mg (0.05 mmol) of DMAP were added, followed by 10 mL of anhydrous dichloromethane (DCM). The mixture was reacted at room temperature in the dark for approximately 24 hours. The solvent was removed by vacuum distillation, followed by washing with 1% dilute hydrochloric acid and ultrapure water. The mixture was then extracted with dichloromethane, and the organic phase was removed by rotary evaporation under reduced pressure to obtain crude PPA-C esterification product. This product was then subjected to silica gel column chromatography with a dichloromethane:methanol volume ratio of 20:1 to yield 66.3 mg of PPA-C esterification product, representing a yield of 85%.

[0090] PPA-C molecular structure characterization: 1 H-NMR (400MHz, CDCl3, ppm) δ12.46 (s, 1H, -OH), 9.44 (s, 1H, 10-CH), 9.36 (s, 1H, 5-CH), 8.59 (s, 1H, 20-CH), 7.98-8.05 (dd, 1H, 26-CH), 7.77 (s, 1H ), 7.75(s, 1H), 7.50-7.57(m, 3H), 6.60(s, 1H), 6.28-6.33(dd, 1H, 27-CH2), 6.17-6.22(dd, 1H, 27'-CH2), 6.13(dd, 1H), 5.13-5.34(q, 2H, 33-CH2 ), 4.55-4.60 (m, H, 18-CH), 4.42-4.44 (m, H, 17-CH), 3.66 (s, 3H, 31-CH3), 3.61-3.65 (q, 2H, 29-CH2), 3.43 (s, 3H, 28-CH3), 3.20 (s, 3H, 25-CH3), 2 .58-2.79(m,2H,35-CH2),2.44-2.51(m,2H,34-CH2),1.84-1.86(d,3H,3 7-CH3), 1.66-1.70 (t, 30-CH3), 0.42 (s, 1H, 21-NH), -1.74 (s, 1H, 23-NH).

[0091] MS(ESI + m / z: 771.32 [M+H] + (100%).

[0092] By comparing the infrared absorption spectra of the raw material PPA and CHRYSIN with their esterification coupling product (PPA-C), it can be seen that the stretching vibration peak of the carboxyl OH group of the raw material pyrophyllic acid a (PPA) is at 3293 cm⁻¹. -1 The stretching vibration peak of the phenolic hydroxyl group OH at position 7 of chrysin (2632 cm⁻¹) and chrysin (2632 cm⁻¹) -1 The peaks disappeared in the coupling product (PPA-C), while the remaining characteristic peaks were basically consistent.

[0093] Example 5

[0094] Preparation of the esterification coupling product (HPPA-C) of pyromethoxyphylla α-hexyl ether (HPPA) and chrysin:

[0095] 63.7 mg (0.1 mmol) of HPPA, 38.1 mg (0.15 mmol) of salicylic acid, 38.4 mg (0.2 mmol) of EDCI, and 6.1 mg (0.05 mmol) of DMAP were added to a 25 mL round-bottom flask. 8 mL of anhydrous dichloromethane (DCM) was added, and the mixture was ultrasonically dispersed and reacted at room temperature in the dark for approximately 24 h. The solvent was removed by vacuum distillation, followed by washing with 1% dilute hydrochloric acid and ultrapure water. The mixture was then extracted with dichloromethane, and the organic phase was removed by rotary evaporation under reduced pressure to obtain crude HPPA-C esterification product. This product was then subjected to silica gel column chromatography with a dichloromethane:methanol volume ratio of 30:1 to obtain 82 mg of HPPA-C esterification product, with a yield of 92%.

[0096] HPPA-C molecular structure characterization: 1 H-NMR (400MHz, CDCl3, ppm) δ12.52 (s, 1H, -OH), 9.75 (s, 1H, 10-CH), 9.45 (s, 1H, 5-CH), 8.55 (s, 1H, 20-CH),

[0097] 7.77(s, 1H), 7.75(s, 1H), 7.46-7.55(m, 3H), 6.58(s, 1H), 6.38-6.39( d, 1H), 6.15 (dd, 1H), 5.89-5.94 (q, H, 26-CH), 5.12-5.32 (q, 2H, 33-CH2 ), 4.53-4.38 (m, H, 17-CH), 4.40-4.43 (m, H, 18-CH), 3.67-3.71 (q, 2H, 29-CH2), 3.64 (s, 3H, 31-CH3), 3.59-3.62 (t, 2H, 38-CH2), 3.39 (s, 3H, 28 -CH3), 3.24 (s, 3H, 25-CH3), 2.68-2.78 (m, 2H, 35-CH2), 2.39-2.60 (m, H, 34-CH2), 2.11-2.13 (d, 3H, 27-CH3), 1.84-1.86 (d, 37-CH3), 1.72-1. 76 (m, 39-CH2), 1.66-1.70 (t, 30-CH3), 1.35-1.41 (m, 6H, 40, 41, 42-CH2), 0.76-0.79 (t, 3H, 43-CH3), 0.42 (s, 1H, 21-NH), -1.75 (s, 1H, 23-NH).

[0098] MS(ESI + m / z: 873.42 [M+H] + (100%).

[0099] Example 6

[0100] Preparation of the indirect addition coupling product (MPPA-C) of methyl pyrophyllin a (MPPA) and chrysin:

[0101] Dissolve 54.9 mg (0.1 mmol) of MPPA in 5 mL of anhydrous dichloromethane, then add...

[0102] 0.5 mL of 33% HBr-CH3COOH solution was added, and the mixture was reacted in an ice bath at 0-15 °C for 12 h. The solvent and acetic acid were removed by vacuum distillation, and the mixture was dried under argon gas. 50.8 mg (0.2 mmol) of chrysin, 200 mg of freshly calcined anhydrous K2CO3, 200 mg of 18-crown ether-6, and 10 mL of anhydrous tetrahydrofuran were added and redissolved. The mixture was reacted at room temperature (15-20 °C) for 12 h. The solvent was removed by vacuum distillation, and the solid was dissolved in dichloromethane and filtered. The filtrate was washed with dilute hydrochloric acid at pH 1 and ultrapure water. The solvent was removed by rotary evaporation of the organic phase to obtain a purplish-black solid. The solid was purified by silica gel column chromatography with a volume ratio of petroleum ether:acetone = 3:1 to obtain 26.1 mg of the indirect addition coupling product (MPPA-chrysin) of methyl pyrophyllin a (MPPA) and chrysin (MPPA-chrysin), with a yield of 32.5%.

[0103] MPPA-C molecular structure characterization:

[0104] 1 H-NMR (400MHz, CDCl3, ppm) δ12.56 (s, 0.5H, -OH), 12.54 (s, 0.5H, -OH), 9.66 (s, 0.5H, 10-CH), 9.65 (s, 0.5H, 10-CH), 9.50 (s, 0.5H, 5-CH), 9.49 (s, 0.5H, 5-CH), 8 .55(s, 1H, 20-CH), 7.61(s, 1H), 7.59(s, 1H), 7.31-7.41(m, 3H), 6.83-6.87(m, 1H , 26-CH), 6.72-6.73 (d, 1H), 6.64-6.65 (t, 1H), 6.41 (d, H), 5.07-5.27 (q, 2H, 34- CH2), 4.42-4.48 (m, H, 17-CH), 4.26-4.28 (m, H, 18-CH), 3.67-3.73 (q, 2H, 30-CH 2), 3.64 (s, 3H, 38-CH3), 3.58 (s, 3H, 32-CH3), 3.46 (d, 3H, 29-CH3), 3.33 (s, 3H, 2 5), 2.48-2.57 (m, H, 36-CH2), 2.19-2.27 (m, H, 365-CH2), 2.34-2.36 (dd, 2H, 35-C H2), 1.76-1.81 (q, 3H, 39-CH3), 1.68-1.72 (m, 3H, 31-CH3), -1.85 (s, 1H, 23-NH).

[0105] MS(ESI + m / z:803.34.42[M+H] + (100%) (MPPA-C and by-product mass spectrometry).

[0106] Chrysin can also be coupled with protoporphyrin (PPIX) and tetracarboxyphenylporphyrin (TCPP) via esterification to obtain bifunctional products (including but not limited to the following compounds).

[0107]

[0108] Example 7

[0109] Preparation of polycarboxylated porphyrin compounds and esterified succinate conjugates:

[0110] 56.3 mg (0.1 mmol) of protoporphyrin (PPIX) and 40.6 mg (0.2 mmol) of N,N'-dicyclohexylcarboimide (DCC) were dissolved in 25 mL of tetrahydrofuran by sonication. After activation at room temperature for 1 h, 6 mg (0.05 mmol) of 4-dimethylaminopyridine (DMAP), 50.8 mg (0.2 mmol) of jujube extract, and a small amount of pyridine were added. After continuing the reaction for 12 h, PPIX-C2 could be detected by mass spectrometry. Figure 1 The same method can be used to esterify and couple tetracarboxyphenylporphyrin with succinin, and different coupling products were detected by mass spectrometry. Figures 2-5 ).

[0111] Monocarboxylated porphyrin compounds, especially chlorophyll a acid degradation products such as pyrophyllin a, are coupled with apigenin via a Steglich esterification reaction. EDCI is selected as the condensing agent and DMAP as the catalyst. Residual EDCI and DMAP can be removed by washing with dilute hydrochloric acid and water. Since apigenin is insoluble in DCM, excess apigenin can be separated by DCM. The yields of PPA-C, PA-C, and HPPA-C obtained by this invention are all higher than 80%, and the purity is higher than 90%.

[0112] Polycarboxylated porphyrin compounds have poor solubility in DCM, so a solvent with a larger solubility, such as THF, is required. Since EDCI is insoluble in THF, DCC is required as a condensing agent, DMAP is used as a catalyst, and pyridine is used as a base to improve esterification efficiency.

[0113] The indirect addition reaction of porphyrin compounds (ethylene) with apigenin is highly sensitive to the hydrolysis of the intermediate bromoporphyrin and the elimination reaction that easily occurs in the presence of organic bases such as triethylamine at temperatures above 30°C. Therefore, the water content of the raw materials and solvents, as well as the temperature, significantly affect the yield and purity. In this invention, the raw materials were dried under vacuum with phosphorus pentoxide for 48 hours. The solvents were anhydrous dichloromethane and anhydrous tetrahydrofuran. Freshly roasted K₂CO₃ was used as the base, and 18-crown ether-6 was used as a co-solvent. The reaction was carried out at room temperature for more than 12 hours to obtain the indirect addition product of MPPA and apigenin.

[0114] Example 8

[0115] The antitumor cell activity of the porphyrin-salicylic acid conjugate was evaluated using PPA-C as an example:

[0116] This experiment on the anti-melanoma cell activity of PPA-C consisted of 10 groups, with 3 parallel experiments in each group. Groups 1-5 were under light exposure, while groups 6-10 were not exposed to light. NPC represents the co-assembled nanoparticles of PPA and Chrysin.

[0117] serial number Drug Name condition 1 Blank control <![CDATA[Light irradiation (660 nm, 0.1 W / cm 2 )]]> 2 PPA <![CDATA[Light irradiation (660 nm, 0.1 W / cm 2 )]]> 3 Chrysin <![CDATA[Light illumination (660 nm, 0.1 W / cm 2 )]]> 4 PPA-C <![CDATA[Light irradiation (660 nm, 0.1 W / cm 2 )]]> 5 NPC <![CDATA[Light illumination (660 nm, 0.1 W / cm 2 )]]> 6 Blank control Dark 7 PPA Dark 8 Chrysin Dark 9 PPA-C Dark 10 NPC Dark

[0118] The test drugs PPA, Chrysin, PPA-C, and NPC are prepared according to the following methods:

[0119] PPA: Weigh 1 mg PPA and dissolve it in 1 mL DMSO. Take the above 10 μL PPA DMSO solution and dilute it with 10 mL PBS. Finally, add 100 μL of the diluted sample in PBS to 1 mL of culture medium containing melanoma. The final concentration is 0.19 μmol / L.

[0120] Similarly:

[0121] Chrysin: 0.5 mg / 1 mL DMSO, 10 μl / 10 mL PBS, 100 μL / 1 mL culture medium, 0.18 μmol / L.

[0122] PPA-C: 1.5 mg / 1 mL DMSO, 10 μ L / 10 mL PBS, 100 μ L / 1 mL culture medium, 0.19 μmol / L.

[0123] NPC preparation:

[0124] Weigh 1 mg of Chrysin, dissolve it in 2 mL of THF, and add 400 μL of the solution to 10 mL of 0.1 M NaOH aqueous solution. Weigh 1 mg of PPA, dissolve it in 1 mL of THF, and add 400 μL of the solution to the same 10 mL of 0.1 M NaOH aqueous solution. Adjust the pH to 6-7 with 1 M hydrochloric acid while stirring vigorously, and continue stirring for 4 hours. Remove the THF by argon bubbling to obtain NPC. Dilute 250 μL of NPC with 10 mL of PBS, and finally add 100 μL of the diluted PBS sample to 1 mL of melanoma-containing culture medium, resulting in a final concentration of 0.19 μmol / L.

[0125] In vitro anti-melanoma activity assay of PPA-C:

[0126] First, melanoma cells were seeded into 24-well plates at a density of 5 × 10⁶ cells / well. 3Cells / well were added, and the plate was incubated for 24 hours. Then, the culture medium in each well was removed, 1 mL of fresh culture medium and 100 μL of the sample to be tested were added, and the plate was incubated for 4 hours. The plate was then incubated with a 660 nm laser (0.1 W / cm²). 2 Irradiate for 5 minutes. Incubate for 3 hours, discard the culture medium, digest the cells with 100-150 μL of trypsin, collect and centrifuge, wash away residual trypsin with PBS, add Calcein AM / PI detection working solution according to the instructions for live and dead cell staining (green for live cells, red for dead cells), incubate in the dark for 30 minutes, and image the melanoma cells using an Olympus inverted fluorescence microscope.

[0127] Experimental results are as follows Figure 9 As shown, at the same concentration (0.19 μmol / L), chrysin exhibited certain in vitro anti-melanoma activity regardless of whether it was exposed to light, with slightly stronger activity under light. PPA only showed its photodynamic anti-melanoma activity under light. NPC showed stronger activity than Chrysin and PPA, while PPA-C showed very strong anti-melanoma activity under light, significantly higher than NPC and PPA under the same conditions. Combined with the UV-Vis absorption spectral analysis of the PPA, NPC, and PPA-C samples, it was found that some PPA dispersed in DMSO solution and PBS buffer solution self-assembled into nanoparticles. Two absorption peaks were observed in the Q band (650–750 nm) of the UV-Vis region of PBS, which were attributed to free PPA and the visible light absorption peaks of PPA nanoparticles, respectively. PPA-C, lacking a carboxyl group, had poorer self-assembly ability and mainly existed as a single molecule before entering cells. After entering the cell, PPA-C may be hydrolyzed by intracellular esterases, releasing chrysin, which gives PPA-C some activity even in the absence of light. However, due to the mutual encapsulation of molecules, the amount of PPA and NPC that enter the cell is limited, resulting in weaker photodynamic activity under light compared to PPA-C.

[0128] Therefore, the carboxyporphyrin-salicylate esterified coupling compound prepared by the present invention has practical application value in photodynamic therapy for anti-tumor purposes.

Claims

1. A porphyrin-ascorbic acid conjugate, characterized in that: It has the structural formula of Equation 1: Formula 1 in, R1 is selected from or R is selected from Or C1~C 10 Alkyl groups; R2 is selected from H, Or C1~C 10 Alkyl groups; And Equation 1 contains at least one .

2. The porphyrin-salicylic acid conjugate according to claim 1, characterized in that: It has the following structural formula: , or .

3. The method for synthesizing a porphyrin-ascorbic acid conjugate according to claim 1 or 2, characterized in that: The carboxyl-containing porphyrin is coupled to the phenolic hydroxyl group of apigenin via an esterification reaction to obtain the product. or, The vinyl-containing porphyrin is coupled to the phenolic hydroxyl group of succinin via an indirect addition reaction to obtain the product. or, The product is obtained by coupling a porphyrin containing carboxyl and vinyl groups with the phenolic hydroxyl groups of apigenin via esterification and / or indirect addition reactions. The porphyrin-like structure has the structure of formula 5: Formula 5 in, R 15 Selected from or R0 is selected from C1~C 10 Alkyl groups; R 14 Selected from H or C1~C 10 Alkyl groups; and R 15 Selected from At that time, R 14 Cannot be selected from C1~C at the same time 10 Alkyl groups.

4. The method for synthesizing a porphyrin-ascorbic acid conjugate according to claim 3, characterized in that: The esterification reaction conditions are as follows: using DCM as solvent, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and / or N,N'-dicyclohexylcarbodiimide as condensing agents, and 4-dimethylaminopyridine and / or 1-hydroxybenzotriazole as catalysts, the reaction is carried out at 0~25℃ for 12~24 hours.

5. The method for synthesizing a porphyrin-ascorbic acid conjugate according to claim 4, characterized in that: The ratio of carboxyl group content to the molar amount of condensing agent, succinic acid and catalyst in carboxyl-containing porphyrins or carboxyl and vinyl-containing porphyrins is 1:(1.5~2):(1.5~2):(0.4~0.6), and the feeding ratio of condensing agent and succinic acid is the same.

6. The method for synthesizing a porphyrin-ascorbic acid conjugate according to claim 3, characterized in that: The indirect addition reaction process is as follows: the vinyl-containing porphyrin or the carboxyl and vinyl-containing porphyrin first undergoes an addition reaction with hydrogen bromide, and then undergoes a nucleophilic substitution reaction with salicylic acid.

7. The method for synthesizing a porphyrin-ascorbic acid conjugate according to claim 6, characterized in that: The conditions for the addition reaction are: using a hydrogen bromide-acetic acid mixed solution as the addition reagent, and reacting at 0~25℃ for 8~16 hours.

8. The method for synthesizing a porphyrin-ascorbic acid conjugate according to claim 6, characterized in that: The nucleophilic substitution reaction was carried out under the following conditions: using K2CO3 as the base reagent and 18-crown ether-6 as the co-solvent, and reacting at 0-25°C for 8-16 hours.

9. The application of the porphyrin-salicylate coupling compound according to claim 1 or 2, characterized in that: Used to prepare antitumor agents for photodynamic therapy combined with chemotherapy.

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