CN101591340B - 5,10,15,20-Tetrakis-(5-morpholinopentyl)-chlorin and its preparation and application in the field of medical pesticides - Google Patents

Abstract

The present invention relates to a kind of 5,10,15,20-tetrakis-(5-morpholinopentyl)-chlorin, whose chemical structural formula is shown in formula (I):

Its preparation: take pyrrole and 6-chloro-1-hexanal as raw materials, under the action of Lewis acid, add tetrachlorobenzoquinone for oxidation, organically, and obtain important intermediate 5, 10, 15, 20-tetra-(5 -chloropentyl)-porphine; then carry out substitution reaction of the intermediate with different amine compounds, and the residue is eluted by silica gel column chromatography; then carry out reduction reaction with reducing agent, remove solvent, separate, elute, Instantly. The compound has good fat solubility and water solubility, so that it has high selective absorption in tumor cells, and is an ideal photosensitizer; the synthetic route is simple to operate, low in cost, friendly to the environment, and has good economic benefits and Application prospect.

Description

5,10,15,20-Tetrakis-(5-morpholinopentyl)-chlorin and its preparation and application in the field of medical pesticides

technical field

The invention belongs to the field of alkylporphyrin compounds and their preparation and application, in particular to a 5,10,15,20-tetrakis-(5-morpholinopentyl)-chlorphin and its preparation and application in medicine Applications in the field of pesticides.

Background technique

Photodynamic therapy is one of the most promising new technologies developed in the past 20 years. Since entering clinical research in the 1970s, breakthroughs have been made in the treatment of tumors. At present, photodynamic therapy is not only limited to the treatment of malignant tumors, but also shows good prospects in the treatment of many other diseases.

In photodynamic therapy, photosensitizers play a decisive role as energy carriers and reaction bridges. The first-generation photosensitizer is represented by the first photosensitizer photofrin II, which was launched in the Netherlands in 1993. It is a mixture of complex hematoporphyrin derivatives, and its indication is tumor; the second-generation photosensitizer is based on porphyrin II. These compounds have a clear chemical structure, high purity, good photothermal stability, and strong absorption in the red light region. At the same time, the chemical modification of the porphyrin ring can adjust the photosensitizer. The hydrophobic partition coefficient is conducive to the absorption and accumulation of photosensitizers in diseased tissues, and is an ideal photosensitizer. Another focus of the development of second-generation photosensitizers is chlorin compounds. This class of compounds mainly includes chlorophylls and bacterial porphins, which are the products of the reduction of the double bond on a pyrrole ring in the porphyrin structure. This kind of compound has good photophysical properties, long absorption wavelength and strong absorption in the visible region. From a theoretical point of view, this type of compound exhibits properties suitable for PDT drugs and has great potential to be developed into PDT drugs. The chlorin-based photodynamic drugs currently on the market mainly include Temoporfin and Talaporfin, which are widely used in the treatment of various tumor diseases, and their application prospects are very promising.

Human beings have used light as a medical technology for thousands of years, but it was not until the end of the 1970s that PDT developed significantly. At present, PDT has been widely used in tumors (such as lung cancer, skin cancer, esophageal cancer, bladder cancer, head and neck cancer, etc.), macular degeneration, actinic keratosis, port wine stains, psoriasis, and rheumatoid joints. The treatment of inflammation, restenosis after angioplasty and other diseases, the application prospect is very promising.

Contents of the invention

The technical problem to be solved by this invention is to provide a kind of 5,10,15,20-tetrakis-(5-morpholinopentyl)-chlorphine and its preparation and application in the field of medical and agricultural chemicals, the compound has good Fat-soluble and water-soluble, it has high selective absorption in tumor cells, and is an ideal photosensitizer; the synthesis route is simple to operate, low in cost, friendly to the environment, and has good economic benefits and application prospects.

The chemical reaction formula of the present invention is as follows:

A kind of 5,10,15,20-tetrakis-(5-morpholinopentyl)-chlorin of the present invention, its chemical structural formula is as shown in formula (I):

A kind of 5,10,15,20-tetrakis-(5-morpholinopentyl)-chlorine preparation method of the present invention comprises:

(1) With pyrrole and 6-chloro-1-hexanal in molar ratio 1: 1～1.2 as raw material, under the action of Lewis acid, stir in organic solvent, the ratio of pyrrole and organic solvent is 1mmol: 15～20ml, Then add tetrachlorobenzoquinone with a molar ratio of 1 to 0.5:1.2 to pyrrole for oxidation, and after refluxing the organic solvent at 45°C for 1h to 2h, the important intermediate 5,10,15,20-tetrakis-(5-chloropentyl base)-porphine;

(2) In an organic solvent medium, 5,10,15,20-tetrakis-(5-chloropentyl)-porphine and morpholine are carried out in a ratio of 1mmol:5～25ml in the presence of potassium salt and catalyst Substitution reaction, the reaction temperature is the reflux temperature of the organic solvent, reflux for 1 to 2 hours, remove the solvent, and the residue is eluted by silica gel column chromatography to obtain 5,10,15,20-tetrakis-(5-morpholinopentyl) - porphine;

(3) Mix 5, 10, 15, 20-tetrakis-(5-morpholinopentyl)-porphine with a reducing agent in a molar ratio of 1:1.5 to 10, heat to reflux (temperature 110°C to 120°C), Reaction 8～10h, after reaction finishes, cooling, add and the ratio of 5,10,15,20-tetrakis-(5-morpholinopentyl)-porphine is 50～100ml: 1mmol of water and 100～200ml: 1mmol ethyl acetate, then heated to 100~105°C, refluxed for 1h, cooled again, removed the solvent, separated by climbing plate, and eluted through the column to obtain 5,10,15,20-tetrakis-(5-morpholinopentyl )-chlorin.

The Lewis acid in the step (1) is BF ₃ ·Et ₂ O, propionic acid or montmorillonite, and the molar ratio of the amount of Lewis acid to pyrrole is 1.2:1; preferably montmorillonite k10;

The organic solvent in the step (1) is a mixed solution of dichloromethane, chloroform or dichloromethane and 1% to 8% ethanol; preferably dichloromethane;

The organic solvent in the step (2) is acetonitrile, the amount of which is 0-120ml of acetonitrile: 1mmol of 5,10,15,20-tetrakis-(5-chloropentyl)-porphine;

The potassium salt and the catalyst in the step (2) are K ₂ CO ₃ and KI respectively, the molar ratio of K ₂ CO ₃ and KI is 1:1, and 5,10,15,20-tetra-(5-chloro Amyl)-porphine and K ₂ CO The molar ratio is 1 _: 5～16;

For the elution in the steps (2) and (3), the eluent used is selected from one or more mixed solvents in sherwood oil, ethyl acetate, methylene chloride, methanol;

The reducing agent in the step (3) is selected from one or more mixtures of p-toluenesulfonyl hydrazide, pyridine, hydrazine hydrate, and borane;

The reducing agent in the step (3) is added in batches during the reduction reaction, and the number of additions is controlled at 4 to 5 times.

The 5,10,15,20-tetrakis-(5-morpholinopentyl)-chlorin of the present invention is mainly used in the preparation of drugs for treating tumor diseases and as a photoactivated insecticide.

Through the photodynamic _test of this synthetic photosensitizer on colon cancer cells, it was found that the photosensitizer can significantly inhibit the proliferation of colon cancer cells when there is light; Dynamic experiments found that this type of photosensitizer can significantly inhibit mouse S ₁₈₀ sarcoma, has a significant killing effect on tumors, and has the prospect of becoming a photodynamic antitumor drug; through the photodynamic activation toxicity test of Drosophila Drosophila, it was found that this type of photosensitizer Compounds are a class of better photoactivated insecticides.

Beneficial effect

The 5,10,15,20-tetra-(5-morpholinopentyl)-chlorin has a long and strong absorption wavelength in the visible light region, which increases the penetrating ability, and has a 5,10 , 15, 20 positions introduce polar groups, which improves its fat solubility and water solubility, which is conducive to the photosensitizer being transported to the lesion through the blood, and also conducive to the photosensitizer to penetrate the cell wall, so that it has a high degree of activity in tumor cells. The selective absorption is an ideal photosensitizer.

Description of drawings

Fig. 1 is the chemical structural formula of Temoporfin;

Fig. 2 is the chemical structural formula of Talaporfin;

Fig. 3 is the chemical structural formula of 5,10,15,20-tetra-(5-morpholinyl)-chlorin;

Fig. 4 is the nuclear magnetic resonance figure of 5,10,15,20-tetra-(5-morpholinopentyl)-chlorin;

Fig. 5 is an ultraviolet absorption diagram of 5, 10, 15, 20-tetrakis-(5-morpholopentyl)-chlorin.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Example 1

(1) Synthesis of 5,10,15,20-tetrakis-(5-chloropentyl)-porphine (5,10,15,20-tetrakis(5-chloropentyl)-21H,23H-Porphine)

Weigh 12g of montmorillonite k10 into a 500mL three-neck flask and put it into an oven for activation at 120°C for 3 hours, then take it out, set up a reflux device, fill it with nitrogen, and wrap the bottle wall with aluminum foil to avoid light. Add 350 mL of dichloromethane and 10 mL of dichloromethane dissolved in 6-chloro-1-hexanal (2.87 g, 21 mmol), and stir for half an hour. With thorough stirring, pyrrole (1.52 mL, 22 mmol) was slowly added dropwise at room temperature, and stirring was continued for 1 hour. Then chloranil (3.87g, 15.75mmmol) was added and refluxed at 45°C for 1 hour. The solid powder was removed by filtration, and the filter cake was washed with ethyl acetate until it was colorless. The filtrate was spin-dried, and ethyl acetate and petroleum ether were passed through the column to obtain 1.02 g of 5,10,15,20-tetrakis-(5-chloropentyl)-porphine with a yield of 27%.

HNMR (δ, CDCl ₃ , ppm): -2.80(s, 2H), 1.86～1.89(m, 16H), 2.46～2.47(m, 8H), 3.49～3.53(m, 8H), 4.87(t, J =8.96Hz, 8H), 9.37(s, 8H), the NMR data of this compound are consistent with the literature value (ChemistryLetters, 1993, 22(1): 117-120.).

(2) Synthesis of 5,10,15,20-tetrakis-(5-morpholinopentyl)-porphine (5,10,15,20-tetrakis(5-morpholinopentyl)-21H,23H-Porphine)

5,10,15,20-tetrakis-(5-chloropentyl)-porphine (2g, 2.84mmol) was dissolved in 40mL morpholine, and added to a 100mL three-necked flask equipped with a reflux device and filled with nitrogen protection, wherein Potassium iodide (2.66g, 16mmol) and potassium carbonate (2.21g, 16mmol) had been added into the three-necked flask, heated to reflux for 1 hour, and the reaction was almost complete. Cool, add 200 mL of ethyl acetate for extraction, wash with water several times, and then wash with saturated brine several times, collect the organic phase, add anhydrous magnesium sulfate to dry, filter, and rotary evaporate. Column chromatography (dichloromethane:methanol 20:1) yielded 1.26 g of compound 5,10,15,20-tetrakis-(5-morpholinopentyl)-porphine, with a yield of 49%.

MS(ESI):931.8(M+1).

HNMR (δ, CDCl ₃ , ppm): -2.74(s, 2H), 1.63～1.72(m, 16H), 2.29～2.36(m, 24H), 2.45(m, 8H), 3.64(t, J=4.16 Hz, 16H), 4.86 (t, J=7.52Hz, 8H), 9.38 (s, 8H).

UV/vis: λ _max (CH ₂ Cl ₂ ) nm: 421, 530, 568, 672.

(3) 5,10,15,20-tetrakis-(5-morpholinopentyl)-chlorin (5,10,15,20-tetrakis(5-morpholinopentyl)-7,8-dihydro-21H, 23H-Porphine) synthesis:

In a 250mL three-necked flask, potassium carbonate (653mg), 5,10,15,20-tetrakis-(5-morpholinopentyl)-porphine (0.655g, 0.7mmol), p-toluenesulfonyl hydrazide ( 212mg, 1.13mmol), set up a reflux device, fill with nitrogen protection, then add pyridine (32mL), and heat to reflux. Additional p-toluenesulfonyl hydrazide (212 mg, 1.13 mmol) and pyridine (1 mL) were added every 2, 4, 6, and 8 hours, and the reaction was monitored by TLC. The reaction is basically over, stop heating and cooling. Water (50 mL) and ethyl acetate (100 mL) were added, heated to 100° C., and refluxed for one hour. After cooling, add ethyl acetate for extraction, wash with water several times, and wash with saturated brine, collect the organic phase, add anhydrous magnesium sulfate to dry, filter, and rotary evaporate. Since the polarity of the product and the reactant is similar, the method of climbing a large plate is adopted for separation, and then the crude product is passed through the column (dichloromethane:methanol 20:1) to obtain 5,10,15,20-tetrakis-(5-morpholinopentane Base)-chlorin 178mg, yield 27%.

MS(ESI):933.5(M+1).

HNMR (δ, CDCl ₃ , ppm): -1.80(s, 2H), 2.20～2.34(m, 40H), 3.59～3.62(m, 24H), 3.73(s, 4H), 4.04(m, 4H ¹ ) , 4.53(s, 4H), 8.66(s, 2H), 8.98(s, 2H), 9.04(s, 2H)

UV/visλ _max (CH ₂ Cl ₂ ) nm: 419, 549, 563, 614, 657.

Example 2

1. Photodynamic anti-proliferation experiment of photosensitizers on colon cancer SW480 cells

(1) Tested cells: colon cancer cells SW480

(2) Test drug: photosensitizer, hematoporphyrin derivative HpD (produced by Beijing Institute of Pharmaceutical Industry).

(3) Light source: MTZ-1 pulse laser cancer treatment machine; SD2490 laser power measuring instrument.

Photodynamic anti-tumor cell proliferation experiment: cells in the logarithmic growth phase were digested with trypsin, and the complete medium was resuspended into a cell suspension, which was then inoculated in a 96-well plate, 100 μl per well, placed at 37 Cultivate in a 5% CO ₂ incubator, add five different photosensitizers at the same concentration after 24 hours; replace with fresh medium after 48 hours, and then light (XD-635AB photodynamic PDT laser therapy instrument, power 15mW/cm ² , wavelength 630mm, irradiate the cells for 20min, and the light dose is 18J/cm ² ); MTT detection was carried out at 72h. 10 μl of 5 mg/ml MTT was added 4 hours before the termination of the culture, and after the culture solution was discarded, 100 μl of DMSO was added to terminate the reaction, and the OD value was detected with a microplate reader at 570 nm. Experiments were repeated three times. The experimental results are shown in Table 1. It was found that the four photosensitizers all had anti-proliferative effects on colon cancer cells.

The IC50 values of HpD and photosensitizer 1 were 1.572 and 0.16 μmol/L, respectively.

Table 1 Photosensitizer 1 inhibits the proliferation of SW480 colon cancer cells

2. Photodynamic therapy experiment of photosensitizer on mouse S ₁₈₀ sarcoma

(1) Test animals: Outbred Kunming strain mice with an average weight of 18-24 g, S180 sarcoma mice (provided by the Institute of Materia Medica, Chinese Academy of Sciences)

(2) Tested drug: photosensitizer 1, under aseptic conditions, the above-mentioned drug was dissolved in a minimum amount of Tween-80 in normal saline and diluted to 0.5 mg/mL solution for later use, hematoporphyrin derivative HpD (Beijing Pharmaceutical Industry Co., Ltd. produced by the institute).

(3) Light source: MTZ-1 pulse laser cancer treatment machine; SD2490 laser power measuring instrument.

Photodynamic injury experiment of mouse S180 sarcoma: S180 sarcoma was inoculated subcutaneously in the anterior chest of mice under aseptic conditions. Litters of the same sex were randomly divided into 8 mice in each group. The mice were injected intraperitoneally. The drug solution was used as a blank control ^. Vapor-dye laser (wavelength 630mm) irradiated the tumor for 20 minutes (light dose 150J/cm ² ); 5 days after irradiation, the mice were sacrificed, the tumor was peeled off, weighed, and the inhibition rate was compared with that of the control group.

式中，T：给药组平均瘤重；C：对照组平均瘤重

In the formula, T: the average tumor weight of the treatment group; C: the average tumor weight of the control group

The experimental results are shown in Table 2. Photosensitizer 1 has obvious inhibitory effect on tumors.

The inhibitory effect of table 2 photosensitizer 1 on tumor

compound Dosage (mg/Kg) Light dose (J/cm ² ) Number of test mice (only) Tumor weight (mg) Inhibition rate(%) Photosensitizer 1 10 150 8 25.6±22.2 89.6 ^* HpD 10 150 8 24.4±17.1 90.1 ^* Blank 10 150 8 246.4±99.9 --

*P<0.05 compared with blank control

3. Photodynamic effect experiment of photosensitizer on Drosophila fruit fly

Select Drosophila fruit fly test worms within one week of eclosion, anesthetize them into groups (regardless of sex) with ether, and put them into culture tubes containing different concentrations of test drug samples (0.5, 1.0, 2.0 g/L), 20 in each tube , and seal the lid tightly with nylon gauze. Each treatment was repeated 3 times. Place the culture tube in a dark place, feed in simulated darkness for 48 hours, then transfer it to the basal medium, and after a period of uniform illumination at 20 cm under incandescent lamps with different light intensities (the temperature of the tube wall does not exceed 35 degrees when illuminated), transfer to The number of deaths was observed regularly in the dark, and the death rate was calculated.

The experimental results are shown in Table 3, and it was found that photosensitizer 1 exhibited better photoactivated insecticidal activity.

Table 3 photosensitizer 1 to the photoactivation toxicity experiment of Drosophila Drosophila

Claims (9)

1. one kind 5,10,15,20-four-(5-drewamine amyl)-chlorin, its chemical structural formula is suc as formula shown in (I):

2. one kind 5,10,15, the preparation method of 20-four-(5-drewamine amyl)-chlorin comprises:

(1) pyrroles and the 6-chloro-1-hexanal with mol ratio 1: 1～1.2 is raw material, under Lewis acid effect, in organic solvent, stir, the pyrroles is 1mmol: 15～20ml with the ratio of organic solvent, adding is that 1～0.5: 1.2 tetrachlorobenzoquinones carry out oxidation with pyrroles's mol ratio then, behind organic solvent backflow 1h～2h, make important intermediate 5,10,15,20-four-(5-chlorine amyl group)-porphines, wherein, Lewis acid is BF ₃Et ₂O, propionic acid or polynite;

(2) in organic solvent medium, with 5,10,15,20-four-(5-chlorine amyl group)-porphines and morpholine carry out substitution reaction in the ratio of 1mmol: 5～25ml in the presence of sylvite and catalyzer, temperature of reaction is the reflux temperature of organic solvent, and the 1～2h that refluxes removes and desolvates, resistates makes 5,10 through the silica gel column chromatography wash-out, 15,20-four-(5-drewamine amyl)-porphines, wherein, catalyzer is KI;

(3) with 5,10,15,20-four-(5-drewamine amyl)-porphines and reductive agent 1: 1.5 in molar ratio～10 mixed, in 110 ℃～120 ℃ reflux, reaction 8～10h, after reaction finished, cooling added and 5,10,15, the ratio of 20-four-(5-drewamine amyl)-porphines is water and the 100～200ml of 50～100ml: 1mmol: the ethyl acetate of 1mmol, reheat to 100 ℃～105 ℃, backflow 1h, cooling once more removes and desolvates, climb board-like separation, cross the post wash-out, promptly get 5,10,15,20-four-(5-drewamine amyl)-chlorin, wherein, reductive agent is selected from p-toluene sulfonyl hydrazide, pyridine, hydrazine hydrate, the mixture of one or more in the borine.

3. according to claim 2 a kind of 5,10,15, the preparation method of 20-four-(5-drewamine amyl)-chlorin is characterized in that: the consumption of the Lewis acid in the described step (1) and pyrroles's mol ratio are 1.2: 1.

4. according to claim 2 a kind of 5,10,15, the preparation method of 20-four-(5-drewamine amyl)-chlorin is characterized in that: the organic solvent in the described step (1) is methylene dichloride, chloroform or methylene dichloride and 1%～8% alcoholic acid mixing solutions.

5. according to claim 2 a kind of 5,10,15, the preparation method of 20-four-(5-drewamine amyl)-chlorin is characterized in that: the organic solvent in the described step (2) is an acetonitrile, and its consumption is 5 of the acetonitrile of 0～120ml: 1mmol, 10,15,20-four-(5-chlorine amyl group)-porphines.

6. according to claim 2 a kind of 5,10,15, the preparation method of 20-four-(5-drewamine amyl)-chlorin is characterized in that: the sylvite in the described step (2) is K ₂CO ₃, K ₂CO ₃With the mol ratio of KI be 1: 1, and 5,10,15,20-four-(5-chlorine amyl group)-porphines and K ₂CO ₃Mol ratio be 1: 5～16.

7. according to claim 2 a kind of 5,10,15, the preparation method of 20-four-(5-drewamine amyl)-chlorin, it is characterized in that: the wash-out in described step (2) and (3), used elutriant are selected from one or more the mixed solvent in sherwood oil, ethyl acetate, methylene dichloride, the methyl alcohol.

8. according to claim 2 a kind of 5,10,15, the preparation method of 20-four-(5-drewamine amyl)-chlorin, it is characterized in that: the reductive agent in the described step (3) adds with the batch-wise form in the reduction reaction process, and the number of times of interpolation is controlled at 4～5 times.

9. one kind 5,10,15,20-four-(5-drewamine amyl)-chlorin is at preparation treatment tumour class disease medicament and as the application of photoactivation sterilant.

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