CN118852182A - A porphyrin-chrysin conjugate and its synthesis method and application - Google Patents
A porphyrin-chrysin conjugate and its synthesis method and application Download PDFInfo
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Abstract
本发明公开了一种类卟啉‑白杨素偶合物及其合成方法和应用，属于药物合成技术领域。将类卟啉(如焦脱镁叶绿酸a)与白杨素通过酯化反应或碳碳双键加成反应进行偶联，得到具有光动力和化疗双重生物活性的类卟啉‑白杨素偶合物，可用于制备抗肿瘤药物。
The invention discloses a porphyrin-like chrysin conjugate and a synthesis method and application thereof, belonging to the technical field of drug synthesis. A porphyrin-like chrysin conjugate is coupled with a porphyrin-like chrysin through an esterification reaction or a carbon-carbon double bond addition reaction to obtain a porphyrin-like chrysin conjugate having both photodynamic and chemotherapeutic biological activities, which can be used to prepare anti-tumor drugs.
Description
Technical Field
The invention relates to a porphyrin-like chrysin conjugate and a synthesis method thereof, and also relates to application of the porphyrin-like chrysin conjugate in preparation of an anti-tumor agent for photodynamic therapy combined chemotherapy, belonging to the technical field of drug synthesis.
Background
Cancer remains a major cause of abnormal death in humans in the world, and malignant tumors are severely threatening the life and health of humans. Although the current surgical excision, radiation therapy, chemotherapy and immunotherapy have certain effects, the traditional Chinese medicine composition also has the problems of large traumas, large toxic and side effects, easy recurrence of tumors and the like.
Photodynamic therapy (photodynamic therapy, PDT) is an emerging high-efficiency treatment modality that uses lasers to intersystem cross and rapidly form triplet excited states of photosensitizers enriched in lesions, transferring energy to ground-state oxygen (3O2) to produce cytotoxic singlet oxygen (1O2). Therefore, the upper PDT has the advantages of low toxicity, small wound, good selectivity and the like. 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.
The photodynamic therapy-chemotherapy combined anti-tumor can exert the dual effects of photodynamic therapy and chemotherapy under the illumination, enhance the killing capacity of cancer cells, weaken the side effect of chemotherapy drugs on human bodies, and have great significance and value in research.
Chrysin is a flavonoid compound and has strong anti-tumor effect on breast cancer, cervical cancer, glioblastoma, esophageal cancer, colon cancer, prostatic cancer and the like in-vitro researches. However, the chemotherapy drugs such as chrysin generally have the problems of low bioavailability or large toxic and side effects in use. The porphyrin compound has a macrocyclic conjugated structure, is the most common photosensitizer, and has the advantages of easy accumulation in tumor focus, low cytotoxicity, strong absorption around a near infrared window, high singlet oxygen production rate and the like.
Chinese patent (CN 109912607A) discloses a novel porphyrin-chrysin derivative, which takes chemically synthesized hydroxy porphyrin as a matrix, dibromoalkane with different carbon atoms as a linking chain, and coupling chrysin phenolic hydroxyl and porphyrin phenolic hydroxyl in an ether bond mode to obtain the novel anti-tumor compound, but the porphyrin compound contains a symmetrical skeleton structure, the absorption wavelength of the porphyrin compound in a Q section is relatively smaller, and the porphyrin compound is mainly coupled in a bonding mode with good stability, so that a carrier and an anti-tumor drug are difficult to be released in cancer cell degradation.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide a porphyrin-like chrysin conjugate, which takes porphyrin-like with photosensitive property as a carrier, chrysin with anticancer activity as a chemotherapeutic drug, and the conjugate of the porphyrin-like chrysin and chrysin can be absorbed in visible light of 400-750 nm and can be used as an anticancer drug combined with photodynamic therapy.
The second purpose of the invention is to provide a novel synthesis method of porphyrin-like-chrysin conjugate, which has the advantages of wide source of porphyrin-like adopted, mild condition, high coupling efficiency, high yield of target conjugate and contribution to mass production.
The third object of the invention is to provide an application of porphyrin-like chrysin conjugate, which has double biological activities of photodynamic and chemotherapy and can be applied as an anti-tumor drug.
The fourth object of the invention is to provide a preparation method of porphyrin-like material, which simplifies the obtaining process of pyropheophorbide a as raw material of porphyrin-like material, improves yield, and can reduce the synthesis cost of porphyrin-like chrysin conjugate.
In order to achieve the technical aim, the invention provides a porphyrin-like chrysin conjugate, which has the following characteristics that
Formula 1, formula 2, formula 3 or formula 4:
Wherein,
R 1 and R 3 are selected fromR is selected fromOr C 1～C10 alkyl; r 2、R8 and R 9 are independently selected from H,Or C 1～C10 alkyl;
R 4 and R 5 are selected from H or R 6 and R 7 are independently selected fromR 10、R11、R12 and R 13 are independently selected from H orAnd each of the formulae 1 to 4 includes at least one
As a preferred embodiment, the porphyrin-like chrysin conjugate has the following structural formula:
The invention also provides a synthesis method of the porphyrin-chrysin-like conjugate, which comprises the following steps:
coupling carboxyl porphyrin-like and phenolic hydroxyl of chrysin through esterification reaction to obtain the product; or alternatively
Coupling vinyl porphyrin-like and phenolic hydroxyl of chrysin through indirect addition reaction to obtain the coupling agent; or alternatively
Coupling carboxyl and vinyl porphyrin-like compound with phenolic hydroxyl of chrysin through esterification and/or indirect addition reaction;
the porphyrin-like compound has a structure of formula 5, formula 6, formula 7, or formula 8:
Wherein,
R 15 and R 16 are selected fromR 0 is selected from alkyl of C 1～C10;
R 14、R17 and R 18 are independently selected from H or C 1～C10 alkyl.
As a preferred embodiment, the esterification reaction conditions are: in the presence of DCM as solvent, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and/or N, N' -dicyclohexyl carbodiimide as condensing agent and 4-dimethylaminopyridine and/or 1-hydroxybenzotriazole as catalyst, and reacting at 0-25 deg.C for 12-24 hr. The solvent may also be selected from chloroform and Tetrahydrofuran (THF), but less effective than Dichloromethane (DCM). A preferred catalyst is 4-Dimethylaminopyridine (DMAP). The preferred reaction temperature is 15 to 25 ℃.
As a preferable scheme, the ratio of carboxyl content in carboxyl-containing porphyrin-like or carboxyl-containing and vinyl-containing porphyrin-like to the amounts of condensing agent, chrysin and catalyst is 1 (1.5-2): 0.5, and the feeding ratio of the condensing agent and chrysin is the same.
As a preferred scheme, the indirect addition reaction is carried out by: the porphyrin containing vinyl or porphyrin containing carboxyl and vinyl is first added with hydrogen bromide and then nucleophilic substituted with chrysin.
As a preferred embodiment, the conditions of the addition reaction are: the mixed solution of hydrogen bromide and acetic acid is used as an addition reagent to react for 8 to 16 hours at the temperature of between 0 and 25 ℃. The mixed solution of the hydrogen bromide and the acetic acid is 33 percent mixed solution of the hydrogen bromide and the acetic acid, and the mass concentration of the hydrogen bromide is 33 percent.
As a preferred embodiment, the nucleophilic substitution reaction conditions are: k 2CO3 is used as an alkali reagent, 18-crown ether-6 is used as a cosolvent, and the reaction is carried out for 8 to 16 hours at the temperature of 0 to 25 ℃.
The invention also provides application of the porphyrin-chrysin-like conjugate in preparing an antitumor agent for photodynamic therapy combined chemotherapy. The invention takes the esterification coupling product PPA-C of pyropheophorbide a (PPA) and chrysin (Chrysin) as an example to verify the photodynamic and chemotherapeutical dual anti-tumor effect of the pyropheophorbide on melanoma.
The invention also provides a preparation method of the porphyrin-like product, which comprises the steps of mixing silkworm excrement mud with an acid degradation solvent for acidolysis reaction, and pouring the reaction mixed solution into saturated saline water after acetic acid is recovered by reduced pressure distillation after acidolysis reaction is completed, so as to precipitate a porphyrin-like crude product; the acid degradation solvent consists of concentrated hydrochloric acid and acetic acid according to the volume ratio of 1:8-10; the liquid-solid ratio of the acid degradation solvent to the silkworm excrement mud is 2.5-5 mL/1 g. The acidolysis reaction is carried out at room temperature, and the reaction time is 8-16 hours.
The porphyrin-like compound can be derived from degradation products of chlorophyll a, for example, the porphyrin-like compound is obtained by acid degradation and extraction of silkworm excrement mud containing chlorophyll a as a raw material:
the acid degradation route of chlorophyll a is as follows:
Compound 1 and compound 2 are prepared from chlorophyll a by acid degradation, usually using diethyl ether hydrochloride, usually 1-5 times the amount of diethyl ether (reference: chemical study of chlorophyll a degradation process in silkworm excrement, yao Jianzhong et al, chinese herbal medicine, 1999, (08): 568-571).
According to the invention, acetic acid is used for replacing diethyl ether, the proportion of hydrochloric acid used is greatly reduced (the volume ratio of hydrochloric acid to acetic acid is 1:8-10), most acetic acid can be recovered through reduced pressure rotary evaporation, and 10 times of cold saturated saline water is added into the residual acetic acid and hydrochloric acid, so that a chlorophyll a acid degradation crude product can be separated out.
Porphyrin-like compounds of the present invention are coupled to chrysin by esterification (illustrated by pyropheophorbide a):
According to the invention, EDCI is used as a dehydration condensing agent, DMAP is used as a nucleophilic catalyst, pyro-magnesium chlorophyllin a and chrysin are subjected to esterification coupling in DCM, the condition is mild, and as chrysin is insoluble in DCM, the byproducts of direct reaction of chrysin and the dehydration condensing agent are reduced, and the esterification coupling product with higher purity is obtained.
Porphyrin-like compounds are addition coupled with chrysin (illustrated by pyropheophorbide a methyl ester):
According to the invention, the addition coupling product of the chlorophyll a derivative and the chrysin is prepared by a two-step method, and the reaction is carried out at room temperature, so that the elimination reaction of the brominated porphyrin intermediate under alkaline conditions is reduced, the chrysin nucleophilic substitution product and the hydrolysis byproduct are obtained, and the raw material conversion rate is improved.
The molecular structure of the part of porphyrin-chrysin-like conjugate prepared by the invention is as follows:
Porphyrins of the present invention include carboxyl or vinyl groups such as chlorophyll-a acid-degrading derivatives, heme derivatives, and synthetic porphyrin compounds, including but not limited to the following:
Compared with the prior art, the technical scheme of the invention has the beneficial technical effects that:
The porphyrin-like-chrysin conjugate provided by the invention takes porphyrin-like with photosensitive property as a carrier, chrysin with anticancer activity as a chemotherapeutic drug, particularly the conjugate of the porphyrin-chrysin and the chrysin can be absorbed in visible light of 400-750 nm and can be used as an anticancer drug combined with photodynamic therapy, meanwhile, the porphyrin-like-chrysin conjugate can be bonded with chrysin by utilizing ester groups with biological activity, so that the porphyrin-like-chrysin conjugate has good stability outside cancer cells, and is easy to degrade and release an antitumor drug in the cancer cells.
The synthesis method of the porphyrin-like chrysin conjugate provided by the invention has the advantages of wide raw material sources, mild conditions, high coupling efficiency and high yield of the target conjugate, and is beneficial to large-scale production.
The synthesis method of the porphyrin-like raw material provided by the invention is simple, the yield is high, and the raw material cost of the porphyrin-like-chrysin conjugate is greatly reduced.
Drawings
FIG. 1 is a PPIX-C2 mass spectrometry detection (ESI +).
FIG. 2 is a TCPP-C4 mass spectrometry detection map (ESI +).
FIG. 3 is a TCPP-C3 mass spectrometry detection map (ESI +).
FIG. 4 is a TCPP-C2 mass spectrometry detection map (ESI +).
FIG. 5 is a mass spectrometric detection diagram (ESI +) of TCPP-C2 and TCPP-C1.
FIG. 6 is a schematic illustration of photodynamic and chemotherapeutic combination anti-tumor of porphyrin-like chrysin conjugates.
FIG. 7 is an ultraviolet visible light absorption spectrum of porphyrin-like chrysin conjugate in DCM.
FIG. 8 is an infrared spectrum of PPA and CHRYSIN with PPA-C.
FIG. 9 is a photograph showing the anti-melanoma activity test (cell live/dead staining analysis) of PPA-C and a control thereof.
FIG. 10 is an ultraviolet-visible absorption spectrum of PPA, NPC, PPA-C anti-melanoma test sample (PBS solution).
Detailed Description
In order to make the solution of the present invention clearer, technical advantages of the solution of the present invention are further highlighted, the following description is provided with reference to specific embodiments.
The chlorophyll-a derivative used in the present invention is prepared by the following method.
The methyl pyropheophorbide a and the hexyl ether derivatives thereof are prepared from silkworm excrement mud.
Example 1
Preparation of crude drug pyropheophorbide a (PPA):
Taking 50g of silkworm excrement mud, adding 160mL of acetic acid for dissolution, adding 20mL of concentrated hydrochloric acid under ice bath, continuously stirring at room temperature for more than 12h, carrying out suction filtration, decompressing filtrate to evaporate most of acetic acid, adding 150mL of cold saturated saline water, standing for 1h, filtering, drying a filter cake, and obtaining a crude product according to the volume ratio of dichloromethane, wherein the volume ratio of methanol=10: 1 as developing agent, obtaining pyropheophorbide a (PPA) 489mg by silica gel column chromatography, detecting purity by High Performance Liquid Chromatography (HPLC) 83%, obtaining 178mg of purple rod-like crystal with metallic luster by ethanol-petroleum ether recrystallization, and detecting purity by High Performance Liquid Chromatography (HPLC) >95%.
PPA molecular structure characterization ：1H-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-CH),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).
MS(ESI+)m/z:535.27[M+H]+(100％)。
Example 2
Preparation of crude drug pyropheophorbide a methyl ester (MPPA):
50g of silkworm excrement mud is taken, 200mL of methanol is added for dissolution, 5mL of concentrated sulfuric acid is added under ice bath while stirring, stirring is continued for more than 12 hours at room temperature, most of methanol is distilled off under reduced pressure, 100mL of ultrapure water is added, the pH is regulated to 4-6 by using a concentrated NaOH solution, diatomite is used for filtration assistance, after a filter cake is dried, dichloromethane is used for washing, and filtrate is distilled under reduced pressure to obtain black sticky matter. Dissolving with a small amount of ethyl acetate, adding petroleum ether 10 times of ultrasonic treatment for 1-5min, and suction filtering to obtain black solid with metallic luster 4.5g (containing methyl pheophorbide a and methyl pyropheophorbide a).
Dissolving the crude product with 80mL of pyridine, protecting with argon, heating and refluxing for 8h, removing methoxycarbonyl (-COO CH 3) at position 33, evaporating pyridine under reduced pressure, performing ultrasonic treatment with dilute hydrochloric acid with pH=1 for 1min, extracting with dichloromethane, washing organic phase with water, performing rotary evaporation under reduced pressure to obtain 3.8g of pyropheophorbide a methyl ester crude product, and detecting content by High Performance Liquid Chromatography (HPLC) to 13.82%. Ethyl acetate in volume ratio: petroleum ether = 1:2 is developing agent silica gel column chromatography to obtain refined pyropheophorbide a methyl ester 504mg. High Performance Liquid Chromatography (HPLC) assay purity 91.7%.
MPPA molecular structure characterization ：1H-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(dd,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).
MS(ESI+)m/z:549.29[M+H]+(100％)。
Example 3
Preparation of crude drug pyropheophorbide a-hexyl ether (HPPA):
Taking 100mg of pyropheophorbide a (PPA), dissolving the pyropheophorbide a (PPA) with 10mL of anhydrous dichloromethane, adding 1mL of 33% HBr-CH 3 COOH solution, carrying out ice bath at 0-15 ℃ for reaction for 12h, decompressing, steaming to remove solvent and acetic acid, drying by argon, adding 10mL of anhydrous dichloromethane for redissolving, and adding 0.5mL of n-hexylether. Reacting at room temperature (15-20 ℃) for 12h, decompressing and removing the solvent, dissolving with ethanol containing a small amount of NaOH for 1-5 min, adding excessive petroleum ether to separate out HPPA sodium salt, dissolving in water, and regulating pH to obtain the HPPA crude product. Dichloromethane: methanol=15: 1 silica gel column chromatography to obtain pyropheophorbide a-hexyl ether 58mg. High Performance Liquid Chromatography (HPLC) assay purity 93.9%.
Characterization of HPPA molecular Structure ：1H-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).
MS(ESI+)m/z:637.38[M+H]+(100％)。
Preparation of chlorophyll a derivative and chrysin conjugate:
Example 4
Preparation of the esterification coupling product (PPA-C) of pyropheophorbide a (PPA) and chrysin (chrysin):
Into a 25mL flask, PPA53.4mg (0.1 mmol), chrysin 38.1mg (0.15 mmol), EDCI 28.8mg (0.15 mmol), DMAP 6.1mg (0.05 mmol) and 10mL of anhydrous Dichloromethane (DCM) were added and the reaction was carried out at room temperature in the dark for about 24h. The solvent was distilled off under reduced pressure, washed with 1% dilute hydrochloric acid and then with ultrapure water, extracted with dichloromethane, and the organic phase was removed by rotary evaporation under reduced pressure to obtain crude PPA-C esterification product, which was prepared by mixing the following components in a volume ratio of developing solvent: 1 silica gel column chromatography to obtain 66.3mg of PPA-C esterified product with 85% yield.
PPA-C molecular structure characterization ：1H-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,37-CH3),1.66-1.70(t,30-CH3),0.42(s,1H,21-NH),-1.74(s,1H,23-NH).
MS(ESI+)m/z:771.32[M+H]+(100％)。
By comparing the infrared absorption spectrum of the raw materials PPA and CHRYSIN with the esterified coupling product (PPA-C) thereof; the carboxyl O-H stretching vibration peak (3293 cm -1) and Chrysin (CHRYSIN) 7-position phenolic hydroxyl O-H stretching vibration peak (2632 cm -1) of the raw material pyropheophorbide a (PPA-C) disappear in the coupling product, and the rest characteristic peaks are basically matched.
Example 5
Preparation of the coupling product of pyropheophorbide a-hexyl ether (HPPA) and chrysin (chrysin) (HPPA-C):
Into a 25mL round bottom flask was added 63.7mg (0.1 mmol) HPPA, chrysin 38.1mg (0.15 mmol), EDCI 38.4mg (0.2 mmol), DMAP 6.1mg (0.05 mmol), and 8mL of anhydrous Dichloromethane (DCM) was added and after ultrasonic dispersion, the reaction was carried out at room temperature in the dark for about 24h. The solvent was distilled off under reduced pressure, washed with 1% dilute hydrochloric acid and then with ultrapure water, extracted with dichloromethane, and the organic phase was removed by rotary evaporation under reduced pressure to give crude HPPA-C esterified product, in a volume ratio of developing solvent to dichloromethane of methanol=30: 1 silica gel column chromatography gave 82mg of HPPA-C esterified product in 92% yield.
Characterization of HPPA-C molecular Structure ：1H-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),
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).
MS(ESI+)m/z:873.42[M+H]+(100％)。
Example 6
Preparation of pyropheophorbide a methyl ester (MPPA) and chrysin (chrysin) indirect addition coupling product (MPPA-C):
MPPA 54.9mg (0.1 mmol) was taken and dissolved in 5mL of anhydrous dichloromethane, and then added
0.5ML of 33% HBr-CH 3 COOH solution, carrying out reaction for 12 hours at the temperature of 0-15 ℃ in an ice bath, evaporating solvent and acetic acid under reduced pressure, blowing dry by argon, adding 50.8mg (0.2 mmol) of chrysin, 200mg of newly baked anhydrous K 2CO3, 200mg of 18-crown ether-6 and 10mL of anhydrous tetrahydrofuran alkane for redissolving, carrying out reaction for 12 hours at room temperature (15-20 ℃), removing the solvent under reduced pressure, carrying out suction filtration after dissolving by using dichloromethane, washing the filtrate by using dilute hydrochloric acid with the pH value of 1 and ultrapure water, and carrying out rotary evaporation on an organic phase to remove the solvent to obtain purple black solid, wherein the volume ratio of petroleum ether: acetone=3: 1 silica gel column chromatography to obtain pyropheophorbide a methyl ester (MPPA) and chrysin (chrysin) indirect addition coupling product (MPPA-chrysin) 26.1mg with yield of 32.5%.
Characterization of MPPA-C molecular structure:
1H-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-CH2),3.64(s,3H,38-CH3),3.58(s,3H,32-CH3),3.46(d,3H,29-CH3),3.33(s,3H,25),2.48-2.57(m,H,36-CH2),2.19-2.27(m,H,365-CH2),2.34-2.36(dd,2H,35-CH2),1.76-1.81(q,3H,39-CH3),1.68-1.72(m,3H,31-CH3),-1.85(s,1H,23-NH).
MS (ESI +)m/z:803.34.42[M+H]+ (100%) (MPPA-C and by-product mass spectrometry detection map).
Chrysin (chrysin) can also be coupled with protoporphyrin (PPIX) and tetra-carboxyphenyl porphyrin (TCPP) by esterification to give bifunctional products including, but not limited to, the following compounds.
Example 7
Preparation of an esterified conjugate of a polycarboxy porphyrin compound and chrysin:
56.3mg (0.1 mmol) of protoporphyrin (PPIX), 40.6mg (0.2 mmol) of N, N' -Dicyclohexylcarbodiimide (DCC) were taken, dissolved by ultrasound with 25mL of tetrahydrofuran, activated for 1h at room temperature, 6mg (0.05 mmol) of 4-Dimethylaminopyridine (DMAP), 50.8mg (0.2 mmol) of chrysin and a little pyridine were added, and after continuing the reaction for 12h, PPIX-C2 (FIG. 1) was detectable by mass spectrometry. The same method can be used for carrying out esterification coupling on tetracarboxyl phenyl porphyrin and chrysin, and different coupling products are detected by mass spectrum (figures 2-5).
Monocarboxylic porphyrin compounds, particularly chlorophyll a acid degradation products, such as pyropheophorbide a, are coupled with chrysin by Steglich esterification, EDCI is selected as condensing agent, DMAP is catalyst, residual EDCI and DMAP can be removed by dilute hydrochloric acid and water washing, and the chrysin is insoluble in DCM, so that excess chrysin can be separated by DCM. The PPA-C and PA-C obtained by the invention have the yield of HPPA-C higher than 80% and the purity higher than 90%.
Polycarboxyporphyrin compounds require the selection of a solvent such as THF which is still relatively large because of poor solubility in DCM, and require the selection of DCC as a condensing agent and DMAP catalysis and pyridine as a base because EDCI is insoluble in THF, thereby improving esterification efficiency.
The porphyrin compound vinyl and chrysin are subjected to indirect addition reaction, and intermediate bromoporphyrin is easy to hydrolyze, and elimination reaction is easy to occur in the presence of organic base such as triethylamine at the temperature higher than 30 ℃, so that the water content and the temperature of the raw materials and the solvent have great influence on the yield and purity. The raw materials used in the invention are all dried for 48 hours by phosphorus pentoxide under vacuum, the solvent is anhydrous dichloromethane and anhydrous tetrahydrofuran, newly baked K 2CO3 is selected as alkali, 18-crown ether-6 is taken as cosolvent of alkali, and the indirect addition product of MPPA and chrysin is obtained after reaction for more than 12 hours at room temperature.
Example 8
Evaluation of anti-tumor cell Activity of porphyrin-chrysin conjugate Using PPA-C as an example:
The PPA-C anti-melanoma cell activity experiments are arranged into 10 groups of 3 parallel experiments. The numbers 1 to 5 are illumination groups, and the numbers 6 to 10 do not illuminate. Wherein NPC represents a co-assembled nanoparticle of PPA and Chrysin.
| Numbering device | Medicine name | Conditions (conditions) |
| 1 | Blank control | Illumination (660 nm,0.1W/cm 2) |
| 2 | PPA | Illumination (660 nm,0.1W/cm 2) |
| 3 | Chrysin | Illumination (660 nm,0.1W/cm 2) |
| 4 | PPA-C | Illumination (660 nm,0.1W/cm 2) |
| 5 | NPC | Illumination (660 nm,0.1W/cm 2) |
| 6 | Blank control | Dark black |
| 7 | PPA | Dark black |
| 8 | Chrysin | Dark black |
| 9 | PPA-C | Dark black |
| 10 | NPC | Dark black |
PPA, chrysin, PPA-C and NPC of the medicine to be tested are configured according to the following method:
PPA: 1mgPPA was weighed and dissolved in 1mL of LDMSO, the DMSO solution of 10 μLPPA was diluted with 10: 10mLPBS, and finally 100 μ LPBS diluted sample was added to 1mL of melanoma-containing medium to a final concentration of 0.19 μmol/L.
And (3) the same principle:
chrysin:0.5mg/1 mM DS, 10. Mu.l/10 mM BS, 100. Mu.l/1 mL medium, 0.18. Mu. Mol/L.
PPA-C:1.5mg/1 mM DS, 10. Mu.L/10 mM BS, 100. Mu.L/1 mL medium, 0.19. Mu. Mol/L.
Preparation of NPC:
1mg Chrysin is weighed and dissolved in 2ml of THF, 400 mu L is dripped into 10ml of 0.1M NaOH aqueous solution, 1mg PPA is weighed and dissolved with 1ml of THF, 400 mu L is dripped into 10ml of 0.1M NaOH aqueous solution, the pH is adjusted to 6-7 by 1M hydrochloric acid under vigorous stirring, stirring is continued for 4h, and THF is removed by argon bubbling, thus obtaining NPC. After 250. Mu. LNPC was diluted with 10mLPBS, the 100. Mu. LPBS diluted sample was finally added to 1mL of melanoma-containing medium at a final concentration of 0.19. Mu. Mol/L.
In vitro anti-melanoma Activity assay of PPA-C:
First, melanoma cells were seeded into 24-well plates at a density of 5×10 3 cells/well and the plates were incubated for 24 hours. Then, the medium in each well was removed, 1mL of fresh medium and 100. Mu.L of the sample to be tested were added, and incubated for 4 hours. The substrate was irradiated with 660nm laser (0.1W/cm 2) for 5 minutes. Incubating for 3 hours, discarding the culture medium, digesting the cells with 100-150 mu L of pancreatin, collecting and centrifuging, washing off residual pancreatin with PBS, adding CALCEIN AM/PI detection working solution according to the instructions for use to stain live and dead cells (green-labeled live cells, red-labeled dead cells), incubating for 30 minutes in the absence of light and imaging the melanoma cells using an Olympus inverted fluorescence microscope.
The experimental results are shown in FIG. 9, and the chrysin has certain in vitro anti-melanoma activity under illumination or not at the same concentration (0.19 mu mol/L), and the activity under illumination is slightly strong. PPA shows its photodynamic anti-melanoma activity only under light, NPC is more potent than Chrysin and PPA, PPA-C shows very potent anti-melanoma activity under light and is much higher than NPC and PPA under equivalent conditions. The ultraviolet and visible light absorption spectrum analysis of the PPA, NPC and PPA-C to-be-detected samples is obtained, a part of the PPA DMSO solution is dispersed into the PBS buffer solution to form nano particles by self-assembly, and the Q section (650-750) of the PBS ultraviolet and visible light region has two absorption peaks which belong to the visible light absorption peaks of free PPA and PPA nano particles respectively. Whereas PPA-C, due to the absence of carboxyl groups, has poor self-assembly ability and exists mainly in the form of single molecules before entering cells. After entering the cell, PPA-C may be hydrolyzed by intracellular esterases, thereby releasing chrysin, which allows PPA-C to show a certain activity also in the absence of light. And PPA and NPC have limited number of cells due to the mutual encapsulation of molecules, so that the photodynamic activity of the PPA and NPC under illumination is weaker than that of PPA-C.
Therefore, the carboxyl porphyrin and chrysin esterified conjugate prepared by the invention has practical application value in the aspect of photodynamic therapy anti-tumor.
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