A carboxyl porphyrin-curcumin ester conjugate and its preparation method and application

Abstract

This invention discloses a carboxyl porphyrin-curcumin esterified conjugate, its preparation method, and its application, belonging to the fields of organic synthesis and biomedicine. The carboxyl porphyrin-curcumin esterified conjugate is obtained by esterifying carboxyl-containing porphyrin-like photosensitizers, such as pyrophyllofoetida a, protoporphyrin, and tetracarboxyphenylporphyrin, with curcumin, which has anticancer activity. It possesses both photodynamic and chemotherapeutic anticancer activities and can be used as an antitumor drug.

Description

Technical Field

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

[0002] Cancer, also known as malignant tumor, is a disease that, despite significant advancements in medical technology and the availability of treatments such as traditional surgery, chemotherapy, and radiotherapy, as well as targeted therapy and photodynamic therapy, still has a high incidence and mortality rate.

[0003] Photodynamic therapy (PDT), a novel cancer treatment technique, uses specific wavelengths to irradiate the lesion site. This activates photosensitizing drugs that selectively accumulate in the lesion tissue, generating highly bioactive singlet oxygen. This oxygen produces cytotoxicity, killing diseased cells, inducing tumor cell apoptosis, and destroying the lesion. Compared to traditional therapies, PDT offers precise and effective treatment with fewer side effects.

[0004] Porphyrins, as effective photosensitizers, whether chlorophyll a derivatives, hematoporphyrin derivatives, or various chemically synthesized porphyrin photosensitizers, play a very important role in photodynamic therapy.

[0005] Curcumin, a natural phenol, possesses anti-cancer therapeutic capabilities, and both curcumin and other curcumin derivatives have been designated as safe products by the U.S. Food and Drug Administration (FDA). Due to its broad biological targets and minimal side effects, curcumin has achieved therapeutic goals in treating metabolic diseases, immune-related diseases, and cancer. However, its poor bioavailability and solubility limit its clinical application.

[0006] However, to date, there have been no reports of constructing novel antitumor drugs by coupling porphyrin compounds with curcumin. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the first objective of this invention is to provide a carboxylated porphyrin-curcumin esterified conjugate that uses porphyrin as a targeted photosensitizer and carries curcumin with anticancer activity. This not only solves the technical problems of poor curcumin bioavailability and insufficient accumulation in tumor tissue, but also enhances the photodynamic therapy of porphyrin, giving it dual photodynamic and chemotherapeutic effects.

[0008] The second objective of this invention is to provide a method for preparing carboxylated porphyrin-curcumin esterified conjugates, which is simple, has low raw material costs, and is conducive to mass production.

[0009] The third objective of this invention is to provide a carboxyl porphyrin-curcumin ester conjugate that, when used as an antitumor drug, can exert both photodynamic and chemotherapeutic effects.

[0010] To achieve the above-mentioned technical objectives, the present invention provides a carboxyl porphyrin-curcumin esterified conjugate having a chemical structure of formula 1, 2, 3, 4, 5, or 6:

[0011]

[0012]

[0013] Where R is selected from R1, R2, R3, and R4 are independently selected from H or And at least one of R1, R2, R3, and R4 is

[0014] The carboxylated porphyrin-curcumin esterified conjugate of the present invention couples carboxylated porphyrin, which has the properties of a targeted photosensitizer, with curcumin, which has chemotherapeutic effects, through an ester bond. On the one hand, the ester bond is biodegradable, and when the conjugate reaches the target cells, it is easy to release the anticancer drug in a timely manner. On the other hand, the conjugate formed by the two has an absorption peak in the Q segment of the ultraviolet-visible light region, which is beneficial for photodynamic therapy of cancer. Thirdly, curcumin has poor utilization and is difficult to accumulate in tumor tissue. The use of carboxylated porphyrin as a carrier can solve the above-mentioned problems of curcumin.

[0015] The present invention also provides a method for preparing a carboxyl porphyrin-curcumin esterified conjugate, wherein the method involves coupling a carboxyl-containing porphyrin with the phenolic hydroxyl group of curcumin via a Steglich esterification reaction to obtain the conjugate.

[0016] The carboxyl-containing porphyrins have chemical structures of formula 7, 8, 9, 10, 11, or 12:

[0017]

[0018]

[0019]

[0020] As a preferred embodiment, the Steglich esterification reaction is carried out under the following conditions: using 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride as a condensing agent and 4-dimethylaminopyridine as a catalyst, the reaction is carried out at 15–25°C for 12–36 h.

[0021] As a preferred embodiment, the Steglich esterification reaction is carried out using chloroform solvent or a mixture of tetrahydrofuran and chloroform solvent.

[0022] As a preferred embodiment, the relative amount of the condensing agent added is 2 to 3 times the carboxyl content in the carboxyl-containing porphyrin.

[0023] As a preferred embodiment, the relative amount of the catalyst added is 0.4 to 0.6 times the carboxyl content in the carboxyl-containing porphyrin.

[0024] As a preferred embodiment, the relative amount of curcumin added is 2 to 3 times the carboxyl content in the carboxyl-containing porphyrin.

[0025] This invention also provides an application of a carboxyl porphyrin-curcumin esterified conjugate for the preparation of photodynamic therapy combined with chemotherapy antitumor agents.

[0026] The carboxyl-containing porphyrins of this invention can be monocarboxyl porphyrins, such as pyrophyllophosphochlorophyll a and its ether derivatives, which are widely available and easily obtained from organisms. They can also be polycarboxyl porphyrin compounds, such as protoporphyrins, and artificially synthesized tetracarboxyphenyl porphyrins. Specifically, examples include chlorophyll a acid degradation products and their ether derivatives, heme derivatives, and artificially synthesized carboxyl-containing porphyrin compounds, including but not limited to the following compounds:

[0027]

[0028] The present invention provides a method for synthesizing carboxyl porphyrin-curcumin esterified conjugates: the carboxyl group in the porphyrin molecule is coupled to the phenolic hydroxyl group in the curcumin molecule via a Steglich esterification reaction, the reaction formula of which is as follows (taking PPA as an example):

[0029]

[0030] The purification and separation method of the Steglich esterification reaction product of the present invention: purification can be carried out by rapid column chromatography, with the volume ratio of developing solvent being: dichloromethane:methanol = 30-50:1. EDCI and DMAP in the product can be washed away by dilute hydrochloric acid and ultrapure water, and residual curcumin can be washed away by ethanol.

[0031] The synthetic routes for monocarboxyl-containing porphyrin-like conjugates and polycarboxyl-containing porphyrin-like conjugates with curcumin are as follows:

[0032]

[0033] The specific steps for preparing the carboxyl-containing porphyrin-curcumin ester conjugate of the present invention are as follows:

[0034] A carboxyl-containing porphyrin-like substance was dissolved or ultrasonically dispersed in chloroform. Then, 2.5 times the amount of the carboxyl group in EDCI, 0.5 times the amount of DMAP, and 2.5 times the amount of curcumin were added. The mixture was reacted in a one-pot manner at room temperature in the dark with stirring for 24 hours. After the reaction, methanol or ethanol was added to remove the residual active intermediate. The solvent was evaporated, and the crude product was washed with dilute hydrochloric acid and water to remove residual EDCI and DMAP. Excess curcumin was washed away with ethanol. The filter cake was dried and then subjected to silica gel column chromatography with dichloromethane:methanol = 35:1 (v / v). The chromatographic band with red fluorescence was collected, yielding the high-purity esterified coupling product of porphyrin and curcumin.

[0035] This invention selects chloroform as a solvent and EDCI as a dehydrating condensing agent. Although polycarboxyporphyrin compounds, such as protoporphyrin, are insoluble in chloroform, a one-pot method can still yield 35%–50% yields of mono- and di-substituted esterification products of carboxyporphyrin with curcumin, and the products are easy to separate and purify. Because EDCI is insoluble in tetrahydrofuran (THF), if N,N'-dicyclohexylcarboimide (DCC) and THF are used in combination, the phenolic hydroxyl group has poor nucleophilic attack ability. After the carboxyl group is rapidly activated by DCC, the intermediate easily rearranges and loses its activity at room temperature, resulting in a complex reaction system. In particular, the esterification of polycarboxyporphyrin compounds with curcumin does not yield ideal products, and unreacted DCC is difficult to remove completely, increasing the difficulty of separation and purification.

[0036] The Steglich esterification reaction principle of carboxyl-containing porphyrin compounds and curcumin under different condensing agents (EDCI and DCC) is as follows:

[0037]

[0038] This invention uses carboxyl-containing porphyrins as targeted photosensitizers and curcumin as a chemotherapeutic drug, and couples them together via an esterification reaction. The resulting compound has an absorption peak in the Q-segment of the ultraviolet-visible light region and can be used as a drug for photodynamic therapy of cancer.

[0039] The molecular structure of the most representative carboxyl porphyrin-curcumin esterified conjugate of this invention is as follows:

[0040]

[0041] Compared with existing technologies, the beneficial technical effects of the present invention are as follows:

[0042] The carboxylated porphyrin-curcumin ester conjugate of the present invention uses porphyrin as a targeted photosensitizer and carries curcumin with anticancer activity. It can not only solve the technical problems of poor bioavailability of curcumin and insufficient accumulation in tumor tissue, but also enhance the photodynamic therapy of porphyrin, so that it has the dual effects of photodynamic therapy and chemotherapy.

[0043] The carboxyl porphyrin-curcumin esterified conjugate of the present invention has biodegradable ester bonds. When the conjugate reaches the target cell, it can easily release anticancer drugs in a timely manner, thereby improving the utilization rate of curcumin.

[0044] The method for preparing carboxylated porphyrin-curcumin esterified conjugates of the present invention is simple, has low raw material costs, and is conducive to mass production.

[0045] The carboxyl porphyrin-curcumin ester conjugate of the present invention can exert a dual effect of photodynamic therapy and chemotherapy when used as an anti-tumor drug. Attached Figure Description

[0046] Figure 1 The 1H NMR spectrum (1H-NMR 400MHz, CDCl3) of PPA-Curcumin, an esterified conjugate of pyrophyllite a (PPA) and curcumin.

[0047] Figure 2 PPA-Curcumin mass spectrometry detection map (MSI) + ).

[0048] Figure 3 Comparison of infrared absorption spectra of PPA, Curcumin, and PPA-Curcumin.

[0049] Figure 4 Mass spectrometry (MSI) of the esterification coupling product of protoporphyrin (PPIX) and curcumin. + ).

[0050] Figure 5 The 1H NMR spectrum (1H-NMR 400MHz, CDCl3) of the esterification product of protoporphyrin and curcumin (compound IIPPIX-CE) is shown.

[0051] Figure 6 PPIX-CE pure mass spectrometry (MSI) + ).

[0052] Figure 7 The images show the UV-Vis absorption spectra of several typical carboxyl porphyrin-curcumin esterified conjugates of this invention.

[0053] Figure 8 Fluorescence imaging for ROS detection of PPA-Curcumin and its reference standard.

[0054] Figure 9 A spectrum of PPA-Curcumin and its control for anti-melanoma activity assay (cell live / dead staining analysis). Detailed Implementation

[0055] To make the present invention more specific and clear, representative specific embodiments are selected below for further explanation.

[0056] Example 1

[0057] Preparation of esterified conjugates of monocarboxylated porphyrin and curcumin:

[0058] Preparation of the esterified conjugate of pyrophyllophospholipid a (PPA) and curcumin (PPA-Curcumin):

[0059] After purging the reaction flask with argon, 54 mg (0.1 mmol) of PPA, 92 mg (0.25 mmol) of curcumin, 48 mg (0.25 mmol) of EDCI, and 6 mg (0.05 mmol) of DMAP were added, along with 10 mL of chloroform. The reaction was carried out at room temperature in the dark for approximately 24 hours. The solvent was evaporated under reduced pressure to obtain the crude product. The crude product was washed with 1% dilute hydrochloric acid and distilled water to remove residual DMAP and EDCI, and then washed with ethanol to remove unreacted curcumin. After drying the filter cake, silica gel column chromatography was performed using dichloromethane:methanol at a volume ratio of 35:1 to obtain 56.2 mg of the PPA-Curcumin esterified product, with a yield of 63.5%.

[0060] PPA-Curcumin molecular structure characterization: ¹H-NMR (400MHz, CDCl₃, ppm), Figure 1 )δ9.48 (s, 1H), 9.38 (s, 1H), 8.58 (s, 1H), 7.97-8.04 (dd, 1H), 7.54-7.60 (m, 2H), 7.03-7.11 (m, 4H), 6.87-6 .93(m, 2H), 6.48(m, 2H), 6.27-6.31(dd, 1H), 6.16-6.19(dd, 1H), 5.80(s, 1H), 5.10-5.32(q, 2H), 4.57-4.5 9 (m, H), 4.42-4.45 (m, H), 3.93 (s, 3H), 3.80 (s, 3H), 3.68-3.71 (q, 2H), 3.66 (s, 3H), 3.41 (s, 3H), 3.24 (s, 3 H), 2.86-2.88(m, 2H), 2.42-2.63(m, 2H), 1.85-1.87(d, 3H), 1.68-1.72(t, 3H), 0.50(s, 1H), -1.67(s, 1H).

[0061] MS(ESI + m / z: 885.39 [M+H] + (100%) Figure 2 ).

[0062] Comparison of infrared absorption spectra of PPA, Curcumin, and PPA-Curcumin ( Figure 3 )

[0063] Example 2

[0064] Preparation of esterified conjugates of polycarboxylated porphyrin and curcumin:

[0065] Preparation of mono- and di-substituted esterification products of protoporphyrin (PPIX) and curcumin:

[0066] After purging the reaction flask with nitrogen, add 56.3 mg (0.1 mmol) of PPIX, 48 mg (0.25 mmol) of EDCI, 15 mL of chloroform, 5 mL of tetrahydrofuran, and 6 mg (0.05 mmol) of DMAP. Sonicate until dispersed, and continue the reaction at room temperature in the dark for approximately 24 hours. When the reaction system is no longer turbid, add 5 mL of ethanol and continue the reaction for about 30 minutes. Remove the solvent under reduced pressure, dissolve in a small amount of dichloromethane, add 10 times the volume of petroleum ether, shake, and filter to obtain the crude product. Wash the crude product with 1% dilute hydrochloric acid and distilled water to remove residual DMAP and EDCI, and then wash with ethanol to remove unreacted curcumin. Mass spectrometry analysis of the obtained crude product showed (…). Figure 4 The main compounds obtained were compound I (PPIX-CC) and compound II (PPIX-CE), whose molecular structures are shown below.

[0067]

[0068] The crude product was subjected to silica gel column chromatography with a dichloromethane:methanol volume ratio of 35:1. The red fluorescent bands were collected to obtain the main esterification product PPIX-CE, which accounted for about 60% (25 mg), while the esterification product PPIX-CC accounted for only 40% (17 mg).

[0069] Characterization of the molecular structure of the main product PPIX-CE:

[0070] 1H-NMR (400MHz, CDCl3, ppm, Figure 5)δ10.19(s, 1H), 10.13(d, 1H), 10.05(d, 1H), 10.05(s, 1H), 8.2-8.27(dd, 2H), 7.43 -7.54(m, 2H), 7.03-7.05(d, 1H), 6.97(s, H), 6.89(s, H), 6.85-6.89(t, 2H), 6.69-6. 71(m,H),6.30-6.40(m,4H),6.12-6.15(d,1H),5.7-5.79(d,1H),5.27-5.29(t,1H), 4.45-4.49(t, 2H), 4.29-4.33(t, 2H), 4.00-4.05(q, 2H), 3.87(s, 3H), 3.77-3.82(q,

[0071] H), 3.65-3.67(t, 6H), 3.56-3.62(dd, 6H), 3.50-3.54(t, 2H), 3.35(s, 3H), 3.13-3.16(t, 2H), 1.92-1.95(t, 3H), -3.68(s, 2H).

[0072] MS(ESI + m / z: 941.42 [M+H] + (100%) Figure 6 ).

[0073] Application Examples

[0074] Taking PPA-Curcumin as an example, the antitumor cell activity of the porphyrin-curcumin conjugate of this invention was evaluated. This experiment on the anti-melanoma B16 cell activity of PPA-Curcumin consisted of three groups: a blank control group, raw material PPA, and product PPA-Curcumin. Each group had three parallel experiments, with both illumination and non-illumination conditions. The illumination condition was 670-680 nm using a 96-well LED light panel (power 0.3 W / cm²). 2 The irradiation time was 5 minutes, and the concentration of the drug to be tested was 1 μM / L.

[0075]

[0076] Example 3

[0077] PPA-Curcumin Reactive Oxygen Species (ROS) Detection:

[0078] (1) Cultured melanoma B16 cells were seeded into 24-well confocal imaging plates (two plates in total, one with illumination and one without illumination), at a density of 5 × 10⁶ cells / well. 4One cell per well, 500 μL per well, three parallel experimental groups, and incubate the plate for 12 hours until the cells adhere.

[0079] (2) Remove the culture medium from each well, then add 475 μL of fresh culture medium, and then add 25 μL of medicine according to the number. Mix well and continue incubation for 24 hours.

[0080] (3) Discard the cell culture medium, add 1 mL of LCFH-DA working solution (10 μM / L), incubate in the dark for 20 minutes, remove the staining solution, and wash with PBS.

[0081] (4) The illumination group uses a 670-680nm laser (0.3W / cm²). 2 ) irradiate for 5 minutes.

[0082] (5) Finally, confocal fluorescence microscopy was used for imaging and qualitative analysis.

[0083] Example 4

[0084] In vitro activity assay of PPA-Curcumin against melanoma B16 (cell live / dead staining analysis):

[0085] (1) Cultured melanoma B16 cells were seeded into 24-well plates (confocal imaging plates) (two plates in total, one for illumination and one for non-illumination), at a density of 5 × 10⁶ cells / well. 4 1 cell / well, 500 μL per well, and incubate for 12 hours.

[0086] (2) Remove the culture medium from each well, add 1 mL of fresh culture medium and 25 μL of the sample to be tested (concentration 20 μM / L), and continue to incubate for 24 hours.

[0087] (3) Illumination group uses 670-680nm LED light panel (0.3W / cm²). 2 Irradiate for 5 minutes and incubate for 4 hours.

[0088] (4) Perform staining according to the instructions of the Calcein-AM / PI live / dead cell double staining kit.

[0089] (5) Use confocal microscopy to perform live / dead imaging of melanoma B16 cells (green indicates live cells / red indicates dead cells).

[0090] Discussion of the experimental results of PPA-Curcumin's anti-melanoma B16 cell activity:

[0091] By detecting the reactive oxygen species (ROS) and in vitro anti-melanoma B16 cell activity of PPA-Curcumin and PPA under 670–680 nm laser irradiation, it was found that under the same conditions, both PPA-Curcumin (an esterification conjugate of PPA and curcumin) and the raw material PPA exhibited strong ROS yields. Although PPA-Curcumin retains a reducing phenolic hydroxyl group of curcumin, the introduction of curcumin, which inhibits tumor cell proliferation and metastasis, did not significantly reduce the ROS yield. Cell viability assays showed that both PPA-Curcumin and PPA possessed strong anti-melanoma photodynamic activity. Since photodynamic therapy can kill a large number of tumor cells in a short time, and curcumin's tumor inhibition is time- and dose-dependent, curcumin can play an adjunctive role in PPA photodynamic anti-tumor therapy.

[0092] Therefore, the porphyrin-curcumin esterified coupling compound prepared by the present invention has practical application value in photodynamic therapy for anti-tumor purposes.

Claims

1. A carboxyl-based porphyrin-curcumin esterified conjugate, characterized in that: Having a chemical structure of formula 1, formula 2, formula 3, formula 4, formula 5, or formula 6: Where R is selected from R1, R2, R3, and R4 are independently selected from H or And at least one of R1, R2, R3, and R4 is 2. The method for preparing a carboxyl porphyrin-curcumin esterified coupling compound according to claim 1, characterized in that: The carboxyl-containing porphyrin is coupled to the phenolic hydroxyl group of curcumin via a Steglich esterification reaction to obtain the product; the carboxyl-containing porphyrin has a chemical structure of formula 7, 8, 9, 10, 11 or 12.

3. The method for preparing a carboxyl porphyrin-curcumin esterified coupling compound according to claim 2, characterized in that: The Steglich esterification reaction was carried out under the following conditions: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was used as a condensing agent, 4-dimethylaminopyridine was used as a catalyst, and the reaction was carried out at 15-25°C for 12-36 h.

4. The method for preparing a carboxyl porphyrin-curcumin esterified coupling compound according to claim 3, characterized in that: The Steglich esterification reaction is carried out using chloroform solvent or a mixture of tetrahydrofuran and chloroform solvent.

5. The method for preparing a carboxyl porphyrin-curcumin esterified coupling compound according to claim 3, characterized in that: The relative amount of the condensing agent added is 2 to 3 times the carboxyl content in the carboxyl-containing porphyrin; The relative amount of the catalyst added is 0.4 to 0.6 times the carboxyl content in the carboxyl-containing porphyrin.

6. The application of the carboxyl porphyrin-curcumin esterified coupling compound according to claim 1, characterized in that: Used to prepare antitumor agents for photodynamic therapy combined with chemotherapy.

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