CN107198774B

CN107198774B - Folic acid targeted lipid-water amphiphilic benzylidene cycloparaffinone photosensitizer, preparation method and application thereof in preparation of photosensitive drugs for photodynamic therapy

Info

Publication number: CN107198774B
Application number: CN201610150045.6A
Authority: CN
Inventors: 吴飞鹏; 方艳艳; 赵榆霞
Original assignee: Technical Institute of Physics and Chemistry of CAS
Current assignee: Technical Institute of Physics and Chemistry of CAS
Priority date: 2016-03-16
Filing date: 2016-03-16
Publication date: 2021-02-12
Anticipated expiration: 2036-03-16
Also published as: CN107198774A

Abstract

The invention discloses a folic acid targeting lipid-water amphiphilic benzylidene naphthenone photosensitizer which has specific targeting property on positive cells of a folic acid receptor, has the characteristics of good lipid-water amphipathy, simple molecular structure and easy synthesis and can realize intravenous administration. The invention also discloses a preparation method of the folic acid targeted lipid-water amphiphilic benzylidene cycloparaffin ketone photosensitizer, which has the characteristics of simple operation, mild reaction, high product purity and quantitative synthesis. The folic acid targeted lipid-water amphiphilic benzylidene cycloparaffin ketone photosensitizer has a targeting effect in photodynamic therapy and has a good application prospect in the aspect of preparing photodynamic medicaments.

Description

Folic acid targeted lipid-water amphiphilic benzylidene cycloparaffinone photosensitizer, preparation method and application thereof in preparation of photosensitive drugs for photodynamic therapy

Technical Field

The present invention relates to the field of photodynamic therapy. More particularly, relates to a folic acid targeted lipid-water amphiphilic benzylidene cycloparaffin ketone photosensitizer, a preparation method thereof and application thereof in preparing a photosensitive drug for single-photon and two-photon photodynamic therapy.

Background

Photodynamic therapy (PDT) achieves high regioselectivity of the treatment process by using light in conjunction with drugs to produce a therapeutic effect. Compared with traditional treatment means (surgery, radiotherapy and chemotherapy), PDT avoids damage to normal tissues to a large extent during treatment. The photodynamic therapy can be divided into single photon photodynamic therapy and two photon photodynamic therapy according to the difference of the number of absorbed photons when the photosensitizer is excited to transition. Because the wavelength of the light source used by the two-photon photodynamic therapy is usually twice that of the light source used by the single-photon photodynamic therapy, and the light source only occurs at the laser focus, the two-photon photodynamic therapy not only can improve the spatial selectivity of the therapy, but also can improve the tissue penetration depth of the light source, so that the two-photon photodynamic therapy is more beneficial to the precise therapy of tumor and non-tumor pathological changes. However, the current phototherapy drugs are applied to the body in a systemic or local administration mode, and the photosensitizer is also enriched in normal tissues while being taken up by the diseased tissues, so that the non-selective distribution inevitably causes damage to the normal tissues around the diseased tissues; in addition, when the photosensitizer is concentrated in a large amount in normal skin tissue, the patient is likely to induce severe skin photosensitization reaction under intense irradiation of sunlight or light. Therefore, it is necessary to improve the selectivity of photosensitizing drugs for diseased tissues.

Currently, the related studies are mainly tried from three aspects: 1) the retention capacity of the medicine in the pathological tissue is improved by utilizing the structures of liposome, microcapsule, nano particle and the like; 2) the monoclonal antibody, the polypeptide, the epidermal growth factor and other recognizable molecules are connected to serve as targeting groups to realize targeting; 3) preparation of pH-sensitive, temperature-sensitive or magnetic-field-sensitive targeting structures (Chemical Reviews,2010,110, 2795-2838).

Folic Acid (FA) is a vitamin which is necessary but not synthesized by human body, participates in one-carbon unit metabolism and de novo synthesis of purine and thymine, and plays an important role in cell division and growth and synthesis of nucleic acid, amino acid and protein. The lack of folic acid in human body can cause the abnormality of erythrocyte, the increase of immature cells, anemia and leukopenia; meanwhile, folic acid is also an indispensable nutrient for the growth and development of the fetus, and the lack of folic acid in pregnant women can cause low body weight, cleft lip and palate, heart defect and the like of the fetus during birth. Folate is taken up into cells in the human body mainly by the endocytic pathway of folate receptors. Folate Receptor (FR) is a membrane glycoprotein linked by Glycosylated Phosphatidylinositol (GPI), is a tumor-associated antigen, and has a molecular weight of 38-40 kD. Bindu Varghese et al (Molecular pharmaceuticals, 2007,4,679-685) have suggested that the expression of this receptor is highly conserved in normal tissues, but highly expressed in epithelial tissue-derived malignancies such as ovarian, cervical, renal, breast, colon, nasopharyngeal and the like.

The inventor's research team has disclosed a series of novel benzylidene cycloparaffinone photosensitizers useful for photodynamic therapy, such as chinese patent nos. CN102249940A and CN 102249939A; and articles (Journal of Medicinal Chemistry 2015,58, 7949; Organic & Biomolecular Chemistry 2011,9, 4168-. The dyes have the characteristics of simple molecular structure, easy synthesis, strong absorption in a visible light band (400-650 nm), large two-photon absorption cross section in a near infrared band (650-1000 nm), and high active oxygen quantum yield, and have great application potential in the field of two-photon photodynamic therapy. However, these photosensitizers do not actively target diseased tissues, limiting their maximum effectiveness. Therefore, further chemical or physical modification of these photosensitizers to increase their selectivity for diseased tissues is essential to fully exploit their potential in the field of photodynamic therapy.

Disclosure of Invention

The invention aims to provide a folic acid targeting lipid-water amphiphilic benzylidene naphthenone photosensitizer, which has specific targeting on positive cells of a folic acid receptor and has the characteristics of good lipid-water amphipathy, simple molecular structure, stable molecular structure and easy synthesis.

The second purpose of the invention is to provide a synthetic method of folic acid targeting lipid-water amphiphilic benzylidene naphthene ketone photosensitizer; the method has the characteristics of simple operation, mild reaction, high product purity and quantitative synthesis.

The third purpose of the invention is to provide the application of the folic acid targeting lipid-water amphiphilic benzylidene naphthene ketone photosensitizer in single photon photodynamic therapy: the photosensitizer has strong absorption in the wavelength range of 400-650 nm, can rapidly generate singlet oxygen, superoxide radical and other active oxygen species under the irradiation of a light source in the wavelength range of 400-650 nm, can be used for single photon photodynamic therapy, and can identify folic acid receptor positive tumor cells, thereby having targeting function in single photon photodynamic therapy.

The fourth purpose of the invention is to provide the application of the folic acid targeting lipid-water amphiphilic benzylidene naphthene ketone photosensitizer in two-photon photodynamic therapy; the photosensitizer has a large two-photon absorption cross section within a wavelength range of 650-1000 nm, can rapidly generate singlet oxygen, superoxide radical and other active oxygen species under the irradiation of 650-1000 nm ultrafast laser, can be used for two-photon photodynamic therapy, and can identify folic acid receptor positive tumor cells, thereby having targeting function in two-photon photodynamic therapy.

In order to achieve the first purpose, the invention adopts the following technical scheme:

the invention provides a folic acid targeted lipid-water amphiphilic benzylidene naphthenone photosensitizer which has a structural formula M as follows:

wherein: r₁Is- (C)₂H₄O)_m-R₅A group;

R₂is methyl, ethyl or- (C)₂H₄O)_n-R₆A group;

R₁and R₂May be the same or different substituent groups;

R₃is methyl, ethyl, propyl or isopropyl, preferably R₃Is methyl or ethyl;

R₄is- (CH)₂)_pA group of-CH₂CH₂(OCH₂CH₂)_q-a group;

is cyclobutanone, cyclopentanone, cyclohexanone, cycloheptanone or cyclooctanone;

the m is 3, 4, 5, 6, 7 or 8, preferably, m is 4 or 5; r₅Is methyl, ethyl, propyl or isopropyl, preferably R₅Is methyl;

n is 3, 4, 5, 6, 7 or 8, preferably n is 4 or 5; r₆Is methyl, ethyl, propyl or isopropyl, preferably R₆Is methyl;

1,2, 3, 4, 5, 6, 7 or 8, preferably p is 2, 3 or 4;

and q is 1,2, 3 or 4.

In order to achieve the second purpose, the invention adopts the following technical scheme:

the invention provides a preparation method of folic acid targeted lipid-water amphiphilic benzylidene cyclanone photosensitizer, which comprises the following steps:

1) synthesis of C6, the reaction equation is as follows:

wherein: r₃Is methyl, ethyl, propyl or isopropyl, preferably R₃Is methyl or ethyl;

the method comprises the following specific steps:

dissolving 0.01-0.2 mol of C3 in 50-250 ml of ethanol-water solution, wherein the volume percentage of ethanol in the ethanol-water solution is 30-90%; slowly adding alkaline substances with the mass 1-20 times that of C3 into the system, and continuously stirring to uniformly mix the alkaline substances; stirring the reaction mixed solution for 4-24 hours at 50-95 ℃ in a nitrogen atmosphere, neutralizing the system by using an acidic substance, and concentrating the reaction solution; adding hydrochloric acid with the mass of 3-10M and the mass of 1-15 times of that of C3 into the concentrated system, heating and refluxing for 10-48 hours in a nitrogen atmosphere, and cooling; neutralizing the final reaction liquid with an alkaline substance to be neutral, extracting with dichloromethane, drying, filtering, and removing dichloromethane by rotary evaporation to obtain an intermediate product C4;

adding phosphorus pentachloride with the molar weight 1-5 times of that of a reaction substrate into DMF with the mass 2-6 times of that of the reaction substrate under ice bath to react for 0.5-1 hour to form a Vilsmeier reagent; dropwise adding a reaction substrate C4 into the system, stirring and reacting for 2-12 hours at 70-140 ℃, then returning to normal temperature and neutralizing the reaction system with alkaline substances; extracting the reaction solution with dichloromethane, washing the extract with water, drying, filtering, and removing the extractant by rotary evaporation to obtain C5;

dissolving the intermediate product C5 in methanol with the mass 2-20 times that of the intermediate product C5, adding an aqueous solution of an alkaline substance with the molar mass 1-3 times that of the intermediate product C3578 while stirring, continuously stirring for 2-10 hours, stopping the reaction, performing rotary evaporation on the reaction solution to remove methanol, extracting a water phase with dichloromethane, washing the obtained dichloromethane extract with water to be neutral, and performing drying, filtering and rotary evaporation to remove dichloromethane to obtain a product C6;

2) synthesis of C7, the reaction equation is as follows:

R₄is- (CH)₂)_pA group of-CH₂CH₂(OCH₂CH₂)_q-a group;

1,2, 3, 4, 5, 6, 7 or 8, preferably p is 2, 3 or 4;

said q is 1,2, 3 or 4;

the method comprises the following specific steps:

dissolving C2 and C6 obtained in the step 1) in dichloromethane according to the molar weight ratio of 1: 1-2, sequentially adding a catalyst with the molar weight being 1-2 times that of C6 and an acid-binding agent with the molar weight being 1-5 times that of C6, and adding a condensing agent with the molar weight ratio of C6 being 0.8-1.5 for 3 times; after the reaction system reacts for 6-24 hours at room temperature, extracting the reaction solution by using dichloromethane, washing the extract by using water, and removing an extracting agent by drying, filtering and rotary evaporation to obtain C7;

3) synthesis of C12, the reaction equation is as follows:

wherein: r₁Is- (C)₂H₄O)_m-R₅A group;

R₂is methyl, ethyl or- (C)₂H₄O)_n-R₆A group;

R₁and R₂May be the same or different substituent groups;

R₃is methyl, ethyl, propyl or isopropyl, preferably R₃Is methyl or ethyl;

R₄is- (CH)₂)_pA group of-CH₂CH₂(OCH₂CH₂)_q-a group;

1,2, 3, 4, 5, 6, 7 or 8, preferably p is 2, 3 or 4;

said q is 1,2, 3 or 4;

the method comprises the following specific steps:

c7 and C11 obtained in the step 2) are mixed according to a molar ratio of 1: adding the mixture into a reaction container according to the proportion of 1-2.5, adding ethanol with the mass being 10-200 times of that of C11 as a solvent, adding an alkaline catalyst with the molar mass being 0.01-0.6 time of that of C11, reacting at 25-60 ℃ for 10-40 hours, removing the solvent through rotary evaporation to obtain a crude product, and separating and purifying by using a chromatographic column to obtain C12;

4) synthesis of C13, the reaction equation is as follows:

wherein: r₁Is- (C)₂H₄O)_m-R₅A group;

R₂is methyl, ethyl or- (C)₂H₄O)_n-R₆A group;

R₁and R₂May be the same or different substituent groups;

R₃is methyl, ethyl, propyl or isopropyl, preferably R₃Is methyl or ethyl;

R₄is- (CH)₂)_pA group of-CH₂CH₂(OCH₂CH₂)_q-a group;

1,2, 3, 4, 5, 6, 7 or 8, preferably p is 2, 3 or 4;

said q is 1,2, 3 or 4;

the method comprises the following specific steps:

adding 10-500 ml of dichloromethane into a reaction container, carrying out ice bath to 0-5 ℃, adding trifluoroacetic acid with the mass being 1-15 times that of C12 into the dichloromethane, and uniformly stirring the mixture; slowly dropwise adding a dichloromethane solution of C12 into the mixture, and reacting for 6-24 hours at the temperature of 0-40 ℃; removing the solvent and the excessive trifluoroacetic acid by rotary evaporation to obtain C13;

5) synthesis of M, the reaction equation is as follows:

wherein: r₁Is- (C)₂H₄O)_m-R₅A group;

R₂is methyl, ethyl or- (C)₂H₄O)_n-R₆A group;

R₁and R₂May be the same or different substituent groups;

R₃is methyl, ethyl, propyl or isopropyl, preferably R₃Is methyl or ethyl;

R₄is- (CH)₂)_pA group of-CH₂CH₂(OCH₂CH₂)_q-a group;

1,2, 3, 4, 5, 6, 7 or 8, preferably p is 2, 3 or 4;

said q is 1,2, 3 or 4;

the method comprises the following specific steps:

weighing 0.01-0.5 mmol of folic acid, namely C14, adding into a reaction vessel, mixing according to the mass ratio of C14 to dimethyl sulfoxide of 1: 50-200, and stirring to completely dissolve folic acid; adding a reactant C13 with the molar weight being 1-1.3 times of the molar weight of folic acid into a reaction system, and then sequentially adding a catalyst with the molar weight being 1-2 times of the molar weight of folic acid, an acid-binding agent with the molar weight being 1-3 times of folic acid and a condensing agent with the molar weight being 0.8-1.5 times of folic acid; after the reaction system reacts for 12-48 hours at 25-40 ℃, chloroform is used for extracting reaction liquid mixed and dissolved with saturated salt water, and the extract liquid is subjected to rotary evaporation, freeze drying and high performance liquid separation to obtain the target photosensitizer M.

Preferably, the alkaline substance is one or a mixture of more than two of sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate and potassium bicarbonate in any proportion.

Preferably, the acidic substance is one of sulfuric acid and hydrochloric acid or a mixture of the sulfuric acid and the hydrochloric acid in any proportion.

Preferably, the catalyst is one or a mixture of more than two of 1-hydroxybenzotriazole, 1-hydroxy-7-azobenzotriazol, 4-dimethylaminopyridine and N-hydroxysuccinimide in any proportion.

Preferably, the acid-binding agent is one or a mixture of more than two of N, N-diisopropylethylamine, triethylamine, pyridine and 4-dimethylaminopyridine in any proportion.

Preferably, the condensing agent is one or a mixture of more than two of dicyclohexylcarbodiimide, diisopropylcarbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate in any proportion.

Preferably, the basic catalyst is one or a combination of more of lithium hydroxide, sodium hydroxide, potassium hydroxide, anhydrous sodium carbonate, anhydrous potassium carbonate, pyridine or piperidine.

Among them, in the preparation method of the present invention, C3 is commercially available.

In the preparation method of the invention, the synthesis equation of C2 is as follows:

wherein:

R₄is- (CH)₂)_pA group of-CH₂CH₂(OCH₂CH₂)_q-a group;

1,2, 3, 4, 5, 6, 7 or 8, preferably p is 2, 3 or 4;

said q is 1,2, 3 or 4;

the method comprises the following specific steps:

reference is made to the literature, i.e.

Favre et al, entitled 6-amino carboxylic acid (6-APA) derivatives with reacting arms (Tetrahedron,2012,68, 10818) in the synthesis method, adding 10-1000 ml of dichloromethane into a single-mouth bottle, cooling in ice bath (0-5 ℃), adding a reactant C1 into the single-mouth bottle, and adding an acid binding agent with the molar weight 1-10 times of that of C1 to uniformly stir the system; dropwise adding a dichloromethane solution of di-tert-butyl dicarbonate with the molar weight of 1/3-1/10 and the molar weight of C1 into the system, and continuing to react for 12-48 hours; and extracting the final reaction solution by using dichloromethane, washing the final reaction solution for 3-6 times by using water, and removing the dichloromethane by drying, filtering and rotary evaporation to obtain C2.

In the preparation method, the synthesis steps of C11 are as follows:

synthesizing C8, wherein the reaction equation is as follows:

wherein: x is 2, 3, 4, 5, 6 or 7; r₇Is methyl, ethyl, propyl or isopropyl;

the method comprises the following specific steps:

according to the literature, namely the Synthesis method in the article entitled "Conversion of alcohols to hydrocarbons vitamin a cosystems intermediates", Synthesis,2003, vol.4, pp509-512 of Arthur Snow et al, NaOH and water with the mass 5-20 times of the NaOH are added into a three-port reactor and stirred uniformly; then, according to the molar ratio of PEG to NaOH of 1: 1-5, adding a mixed solution of PEG and Tetrahydrofuran (THF) into the NaOH aqueous solution, wherein the ratio of PEG: volume ratio of THF 1: 1-10; controlling the temperature by using an ice bath, stirring for 0.5-2 hours at 0-5 ℃, introducing nitrogen to fully remove oxygen, dissolving paratoluensulfonyl chloride with the molar weight being 0.5-1 time of the weight of PEG in THF with the volume being 1-5 times of the volume of PEG, and slowly dropwise adding the THF solution into the three-opening reaction container added with the PEG, the THF and the NaOH aqueous solution; after reacting for 2-6 hours at 0-10 ℃, removing the ice bath, and continuing to react for 4-12 hours at room temperature; extracting the reaction liquid with diethyl ether, washing diethyl ether extract with water to neutrality, drying, filtering, and rotary evaporating to remove diethyl ether to obtain p-toluenesulfonate C8 with PEG group;

② synthesizing C11, wherein the reaction equation is as follows:

wherein: r₈Is methyl, ethyl or-C₂H₄OH；

R₁Is- (C)₂H₄O)_m-R₅A group;

R₂is methyl, ethyl or- (C)₂H₄O)_n-R₆A group;

R₁and R₂May be the same or different substituent groups;

the method comprises the following specific steps:

according to The Synthesis method in Christian B.Nielsen et al entitled "Synthesis and catalysis of Water-soluble phenyl-ene-vinyl-based oxygen donors for two-photon excitation", The Journal of organic Chemistry,2005, vol.70, pp7065-7079, C9 is dissolved in 10-30 times by mass of dry, heavy distilled Tetrahydrofuran (THF), then potassium hydroxide in an amount of 1-5 times by mol The above para-aminobenzaldehyde derivative and THF in an amount of 10-30 times by mass of The C9 compound are slowly added to The mixed solution, and The mixed reaction solution is stirred under N2 atmosphere and then heated and refluxed for 1-5 hours to obtain an intermediate reaction solution. Slowly dripping an intermediate product C8 with the molar weight 1-5 times that of the C9 compound into the intermediate reaction liquid, continuously heating and refluxing for 12-48 hours, cooling, neutralizing the final reaction liquid to be neutral by using acid, extracting by using dichloromethane, drying, filtering, and removing the dichloromethane by rotary evaporation to obtain a crude product, and separating and purifying by using a chromatographic column to obtain a yellow oily liquid, namely, a benzaldehyde derivative C10 with a PEG group;

see Chinese patent application publication No. CN102249939A, derivatives of p-aminobenzaldehyde C10 and having PEG group

According to a molar ratio of 1: 1-2, adding an ethanol-water solution with the mass 10-30 times that of C10 (wherein the volume percentage of ethanol is 20-80%), adding an alkaline catalyst with the molar mass 0.01-0.6 time that of C10, reacting at 0 ℃ for 2-10 hours, removing the ice bath, reacting at room temperature for 1-5 hours, removing the solvent by filtration or rotary evaporation to obtain a crude product, and separating and purifying by using a chromatographic column to obtain C11;

wherein:

is cyclobutanone, cyclopentanone, cyclohexanone, cycloheptanone or cyclooctanone.

The folic acid targeted lipid-water amphiphilic benzylidene cycloparaffin ketone photosensitizer can identify the tumor cells with positive folic acid receptors, thereby having a targeting effect in photodynamic therapy.

The folic acid targeted lipid-water amphiphilic benzylidene cycloparaffin ketone photosensitizer has lipid-water amphiphilic property, and the hydrophilicity of the photosensitizer is increased along with the increase of the number of PEG groups.

The folic acid targeted lipid-water amphiphilic benzylidene naphthenone photosensitizer has strong single photon absorption in the wavelength range of 400-650 nm.

The folic acid targeted lipid-water amphiphilic benzylidene cycloparaffin ketone photosensitizer has strong two-photon absorption in the wavelength range of 650-1000 nm.

The folic acid targeted lipid-water amphiphilic benzylidene naphthenone photosensitizer can rapidly generate singlet oxygen, superoxide anions and other active oxygen species under the irradiation of a light source within the wavelength range of 400-650 nm, and has a good application prospect in the aspect of preparing single photon photodynamic medicaments.

The folic acid targeted lipid-water amphiphilic benzylidene cycloparaffin ketone photosensitizer can rapidly generate singlet oxygen, superoxide anions and other active oxygen species under the laser irradiation within the wavelength range of 650-1000 nm, and has good application prospect in the aspect of preparing two-photon photodynamic medicaments.

The invention has the following beneficial effects:

1. the folic acid targeted lipid-water amphiphilic benzylidene cycloparaffin ketone photosensitizer has good targeting property in photodynamic therapy.

2. The folic acid targeted lipid-water amphiphilic benzylidene naphthene ketone photosensitizer has a simple structure, small molecular weight, a determined chemical structure, and easy preparation, purification and further modification, and meets the basic requirements of clinical medication.

3. The folic acid targeted lipid-water amphiphilic benzylidene cycloparaffin ketone photosensitizer has the characteristic of lipid-water amphipathy, and the lipid-water distribution ratio can meet the use requirement of clinical photodynamic therapy.

4. The synthesis method of the folic acid targeted lipid-water amphiphilic benzylidene cycloparaffin ketone photosensitizer has the characteristics of simple operation, high product yield and high purity.

5. The folic acid targeted lipid-water amphiphilic benzylidene cycloparaffin ketone photosensitizer has high biological photodynamic activity in the wavelength ranges of 400-650 nm and 650-1000 nm, and has good application prospect in the aspect of preparing photodynamic medicaments.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 shows the absorption spectra of photosensitizer M-1 in methanol and PBS buffer in example 1.

FIG. 2 shows the rate of decrease in absorbance of DPBF in DMF with time in the presence of the photosensitizer M-1 in example 1.

FIG. 3 shows the two-photon absorption spectrum of the photosensitizer M-1 in DMF in example 1.

FIG. 4 shows the absorption spectrum of photosensitizer M-2 in methanol and PBS buffer in example 2.

FIG. 5 shows the rate of decrease in absorbance of DPBF in DMF with time in the presence of the photosensitizer M-2 in example 2.

FIG. 6 shows a two-photon absorption spectrum of the photosensitizer M-2 in DMF in example 2.

FIG. 7 shows the results of the experiment of photosensitizer M-1 used for single photon photodynamic verification of its targeting in example 1.

FIG. 8 shows the cell viability of the photosensitizer M-2 used in the two-photon photodynamic experiment in example 2.

FIG. 9 shows the results of experiments in which photosensitizer M-2 in example 2 was used to verify its targeting properties by single photon photodynamic.

FIG. 10 shows the survival rate of the photosensitizer M-1 in example 1 for two-photon photodynamic experiment.

Fig. 11 shows the results of the photosensitizer M-1 in example 1 used to verify the two-photon excitation process.

Fig. 12 shows the results of the photosensitizer M-2 used to verify the two-photon excitation process in example 2.

Detailed Description

In order to more clearly illustrate the invention, the invention is further described below with reference to preferred embodiments and the accompanying drawings. Similar parts in the figures are denoted by the same reference numerals. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.

Example 1

(I) Synthesis of C2-1, the reaction equation is as follows:

the method comprises the following specific steps:

dissolving 8.0 g (54mmol) of 2,2' - (ethylene dioxy) bis (ethylamine) in 500 ml of dichloromethane, and cooling to 0-5 ℃ in an ice bath; 6.0 g (60mmol) of triethylamine was added thereto and the system was stirred uniformly; 4.0 g (18mmol) of a dichloromethane solution of di-tert-butyl dicarbonate is dropwise added into the system, and then the reaction is continued for 12 hours; the final reaction was extracted 3 times with dichloromethane and washed 3 times with water to give 3.3 g of C2-1 (75% yield) after drying, filtration and rotary evaporation to remove dichloromethane.

(II) Synthesis of C6-1, the reaction equation is as follows:

the method comprises the following specific steps:

dissolving 17.3 g (0.1mol) of C3-1 in 50 ml of ethanol-water solution (wherein the volume percentage of ethanol is 50%); slowly adding 20 g (0.5mol) of sodium hydroxide into the system, and continuously stirring to uniformly mix the sodium hydroxide and the sodium hydroxide; stirring the reaction mixed solution for 6 hours at 60 ℃ in a nitrogen atmosphere, neutralizing the system by using hydrochloric acid, and concentrating the reaction solution; adding 20 g of 5M hydrochloric acid into the concentrated system, heating and refluxing for 12 hours in a nitrogen atmosphere, and cooling; the final reaction was neutralized to neutrality with sodium carbonate, extracted with dichloromethane, dried, filtered and rotary evaporated to remove dichloromethane to yield intermediate 21.6 g of C4-1 (98% yield).

32 g (0.15mol) of phosphorus pentachloride was added to 80 g of DMF in an ice bath and reacted for 1 hour to form Vilsmeier reagent; 21.6 g (0.09mol) of a reaction substrate C4-1 is dropwise added into the system, stirred at 140 ℃ for reaction for 2 hours, and then returned to normal temperature and neutralized by sodium bicarbonate; the reaction was extracted with dichloromethane, the extract was washed with water, dried, filtered and the extractant removed by rotary evaporation to give 24.0 g of C5-1 (99% yield).

24.0 g (0.11mol) of intermediate C5-1 was dissolved in 50 g of methanol, 4.8 g (0.12mol) of NaOH was added under stirring, the reaction was stopped by stirring for 10 hours, the methanol was removed by rotary evaporation from the reaction mixture, the aqueous phase was extracted with dichloromethane, the dichloromethane extract obtained was washed with water to neutrality, and the dichloromethane was removed by drying, filtration and rotary evaporation to obtain 20.6 g of C6-1 (yield 97%).

(III) Synthesis of C7-1, the reaction equation is as follows:

the method comprises the following specific steps:

0.50 g (2mmol) of C2-1 and 0.44 g (2mmol) of C6-1 were dissolved in 15 ml of dichloromethane, then 0.28 g (2.2mmol) of HOBT, 0.20 g of triethylamine were added in succession, and finally 0.38 g (2mmol) of EDCI were added in 3 portions; after the reaction system was reacted at room temperature for 12 hours, the reaction solution was extracted with dichloromethane, and the extract was washed with water, dried, filtered, and the extractant was removed by rotary evaporation to obtain 0.87 g of C7-1. (yield 97%).

(IV) Synthesis of C8-1, the reaction equation is as follows:

the method comprises the following specific steps:

a250 ml three-neck flask is added with 8 g (0.2mol) of NaOH and 80 ml of water, and stirred to be dissolved uniformly; adding 24.9 g (0.12mol) of tetraethyleneglycol monomethyl ether into 50 ml of THF for uniform dissolution, then adding the mixture into the three-neck flask, and uniformly mixing the mixture with NaOH solution; 22.7 g (0.12mol) of paratoluensulfonyl chloride and 40 ml of THF are uniformly mixed, then slowly added into the three-neck flask in a dropwise manner, the temperature of the reaction solution is kept not more than 10 ℃ in the dropwise adding process, and after the dropwise adding is finished, stirring reaction is continuously carried out for 6 hours, and the reaction is stopped. The reaction solution was extracted with ether three times, and the ether extract was washed with water to neutrality, dried over anhydrous magnesium sulfate, filtered, and rotary-evaporated to remove ether, to obtain 39.5 g of the corresponding p-toluenesulfonate C8-1 (yield: 91%);

(V) Synthesis of C11-1, the reaction equation is as follows:

the method comprises the following specific steps:

2.1 g (0.01mol) of 4- (N, N-bis (2-hydroxy-ethyl) amino) benzaldehyde C9-1 was dissolved in 50 g of dried and steamed THF, and a solution of 1.68 g (0.03mol) of KOH and 50 g of THF was slowly added thereto, and after completion of dropwise addition, the reaction mixture was stirred under an atmosphere of N2 for half an hour and then heated under reflux for 1 hour to obtain an intermediate reaction mixture. 10.9 g (0.03mol) of p-toluenesulfonate C8-1 was dissolved in 50 g of THF, and slowly added dropwise to the above intermediate reaction solution, and the mixture was refluxed for 48 hours, cooled, and then the final reaction solution was neutralized to neutrality with dilute hydrochloric acid, extracted with dichloromethane, and the extract was dried over anhydrous magnesium sulfate, filtered, and rotary-evaporated to remove dichloromethane to obtain a crude product, which was purified by column chromatography to obtain 4.2 g of yellow oily liquid C10-1 (yield: 71%).

3.5 g (0.006mol) of p-aminobenzaldehyde derivative C10-1 and 1 g (0.012mol) of cyclopentanone were added to a reaction vessel, 40 g of an ethanol-water solution (wherein the volume percentage of ethanol is 80%) was added, 0.03 g (0.75mmol) of sodium hydroxide was added, the reaction was carried out at 0 ℃ for 4 hours, the ice bath was removed, the reaction was carried out at room temperature for 1 hour, and the solvent was removed by filtration or rotary evaporation to obtain a crude product, which was then purified by column chromatography to obtain 1.5 g of intermediate product C11-1 (38% yield).

(VI) Synthesis of C12-1, the reaction equation is as follows:

the method comprises the following specific steps:

0.45 g (0.001mol) of C7-1 and 0.66 g (0.001mol) of C11-1 were added to a reaction vessel, followed by addition of 20 g of ethanol as a solvent and 0.02 g (0.5mmol) of NaOH as a catalyst, followed by reaction at 25 ℃ for 24 hours, removal of the solvent by rotary evaporation to give a crude product, which was purified by column chromatography to give 0.5 g of C12-1 (yield 46%).

(VII) Synthesis of C13-1, the reaction equation is as follows:

the method comprises the following specific steps:

adding 15 ml of dichloromethane into a reaction container, carrying out ice bath to 0-5 ℃, adding 0.5 g of trifluoroacetic acid into the dichloromethane, and uniformly stirring; 0.25 g (0.23mmol) of a solution of C12-1 in methylene chloride was slowly dropped thereinto and reacted at 25 ℃ for 12 hours; the solvent and excess trifluoroacetic acid were removed by rotary evaporation to give 0.21 g of C13-1 (95% yield).

(VIII) synthesizing a target photosensitizer M-1, wherein the reaction equation is as follows:

the method comprises the following specific steps:

weighing 88 mg (0.2mmol) of folic acid C14, adding into a reaction vessel, adding about 6 g of DMSO into a reaction flask, and stirring to completely dissolve folic acid; to this was added another reactant, 0.22 g (0.22mmol) of C13-1, followed by the addition of 29 mg (0.25mmol) of NHS, 4 mg (0.4mmol) of triethylamine and 42 mg (0.22mmol) of EDCI in that order; after the reaction system was reacted at 40 ℃ for 12 hours, the reaction mixture miscible with saturated saline was extracted with chloroform, and the extract was subjected to rotary evaporation, freeze-drying and high performance liquid separation to obtain 0.1 g of the objective photosensitizer M-1 (yield 47%). HR-MS (ESI) M/z [ M + H]:Calcd.for[C₇₁H₁₀₃N₁₁O₁₉]⁺1412.7348；found 1412.7384。

(IX) detecting the solubility of the target photosensitizer M-1 in a water system by using a phosphate buffer solution (PBS buffer solution for short) with the pH value of 7.4, wherein the solubility is more than 2mg/mL at 25 ℃; the target photosensitizer M-1 is respectively dissolved in methanol and PBS buffer solution, and the absorption spectrum is tested, so that the photosensitizer M-1 has a strong absorption peak in the wavelength range of 350-650 nm, as shown in figure 1.

(X) dissolving the target photosensitizer M-1 in DMF to make its absorbance at 473nm 0.1, and determining the singlet oxygen quantum yield Φ_Δ0.47) as reference, the spectrum shown in figure 2 was obtained. Indicating that the photosensitizer M-1 generates singlet oxygen under the excitation condition of 473nm laser.

(XI) formulation of the photosensitizer to 2X 10^-4DMF solution of M, 1X 10^-4M rhodamine B (Rhodamine B) is taken as a reference, an up-conversion fluorescence method is adopted to test the two-photon absorption cross section, and the result is shown in figure 3, the photosensitizer has a larger two-photon absorption cross section in the wavelength range of 720-880nm, and the photosensitizer can generate photodynamic action by using two-photon excitation.

Example 2

(I) Example 1 was repeated except that cyclopentanone was changed to cyclobutanone in the above step (V) and other conditions were not changed to obtain the objective photosensitizer M-2 (yield 20%). HR-MS (ESI) M/z [ M + H]:Calcd.for[C₇₀H₁₀₁N₁₁O₁₉]⁺1398.7191；found 1398.7229。

(II) with reference to the procedure of (IX) in example 1, it was confirmed that the solubility of M-2 was more than 2 mg/mL; and has a strong absorption peak in the wavelength range of 350-600 nm, as shown in figure 4.

(III) referring to the operation of example 1(X), it was also confirmed that the object photosensitizer M-2 can generate reactive oxygen species under 473nm laser irradiation, as shown in FIG. 5.

(IV) referring to the operation of example 1(XI), it was also confirmed that the photosensitizer has a large two-photon absorption cross section in the wavelength range of 720-880nm, and photodynamic action can be generated using two-photon excitation. See figure 6.

Example 3

(I) Referring to the procedure of (I) in example 1, C2-2 was synthesized according to the following reaction equation:

the method comprises the following specific steps:

dissolving 4.0 g (45mmol) of C1-2 in 100 ml of dichloromethane, and cooling to 0-5 ℃ in an ice bath; 5.8 g (45mmol) of N, N-Diisopropylethylamine (DIEA) was added thereto, and the system was stirred uniformly; dropwise adding a dichloromethane solution of 0.9 g (4.5mmol) of di-tert-butyl dicarbonate into the system, and continuing to react for 12 hours; the final reaction was extracted 3 times with dichloromethane and washed 3 times with water to give 0.7 g of C2-2 (80% yield) after drying, filtration and rotary evaporation to remove dichloromethane.

(II) referring to the operation of (V) in example 1, an intermediate product C11-2 was prepared by reacting C10-1 with cyclohexanone in the presence of an alkali catalyst of lithium hydroxide in a molar amount of 0.01 times that of the reactants. (yield 30%)

(III) the other steps in example 1 were repeated, referring to the procedure of (VIII) in example 1, adding a catalyst (1-hydroxybenzotriazole, HOBT) in an amount of 2 times the molar amount of folic acid, an acid-binding agent (4-dimethylaminopyridine, DMAP) in an amount of 3 times and a condensing agent Dicyclohexylcarbodiimide (DCC) in an amount of 1.5 times the molar amount, and reacting the reaction system at 25 ℃ for 48 hours to obtain the target photosensitizer M-3 (yield 30%). HR-MS (ESI) M/z [ M + H]:Calcd.for[C₇₀H₁₀₀N₁₁O₁₇]⁺1365.7220；found 1365.7221。

Example 4

(I) The procedure of (II) in example 1 was repeated to change C3-1 to N-methyl-N-cyanoethyl-4-aminobenzaldehyde to prepare intermediate C6-2. (yield 97%)

(II) referring to the operation of (III) in example 1, C2-1 and C6-2 are charged according to the molar ratio of 1:2, a catalyst 1-hydroxy-7-azobenzotriazole (HOAT) with the molar weight 2 times of that of C6-2, 5 times of acid-binding agent pyridine and 1.2 times of condensing agent 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea Hexafluorophosphate (HATU) are added, and the reaction is carried out at room temperature for 24 hours to obtain a compound C7-2. (yield 98%)

(III) the other steps in example 1 were repeated to obtain the objective photosensitizer M-4 (yield 20%). HR-MS (ESI) M/z [ M + H]:Calcd.for[C₇₀H₁₀₀N₁₁O₁₉]⁺1397.7119；found 1397.7110。

Example 5

(I) Referring to the operation of (IV) in example 1, intermediate C8-2 was prepared in a 92% yield by feeding p-toluenesulfonyl chloride and pentaethyleneglycol monomethyl ether in a molar ratio of 0.8:1 at a reaction temperature of 0 ℃ for 3 hours.

(II) referring to the procedure of (V) in example 1, intermediate C10-2 was prepared.

The method comprises the following specific steps:

3.6 g (0.02mol) of N-hydroxyethyl-4-amino-2-methylbenzaldehyde C9-2 were dissolved in 60 g of dry, redistilled THF, and a solution of 5.6 g (0.03mol) of KOH and 50 g of THF was slowly added thereto, and after completion of dropwise addition, the reaction mixture was stirred in N₂Stirring for half an hour under the atmosphere, and then heating and refluxing for 2 hours to obtain an intermediate reaction solution. 24.4 g (0.06mol) of p-toluenesulfonate C8-2 was dissolved in 50 g of THF, and slowly added dropwise to the above intermediate reaction solution, and the mixture was refluxed for 48 hours, cooled, and then the final reaction solution was neutralized to neutrality with dilute hydrochloric acid, extracted with dichloromethane, and the extract was dried over anhydrous magnesium sulfate, filtered, and rotary-evaporated to remove dichloromethane to obtain a crude product, which was purified by column chromatography to obtain 7.3 g of yellow oily liquid C10-2 (yield: 89%).

(III) the other operations in example 1 were repeated except that cyclopentanone was changed to cyclobutanone in step (V) in example 1 to obtain the objective photosensitizer M-5 (yield 15%). HR-MS (ESI) M/z [ M + H]:Calcd.for[C₆₂H₈₄N₁₁O₁₅]⁺1221.6070；found 1221.6066。

Example 6

(I) The procedure of example 1 was repeated except that cyclopentanone in step (V) of example 1 was changed to cycloheptanone, the mass of DMSO used in step (VIII) was 10 g, a catalyst (4-dimethylaminopyridine, DMAP) in a molar amount of 1.5 times that of folic acid, an acid-binding agent (a mixture of triethylamine and pyridine) in a molar amount of 1.2 times and a condensing agent (diisopropylcarbodiimide, DIC) in a molar amount of 0.9 times were sequentially added, and the reaction system was reacted at 35 ℃ for 36 hours to obtain the objective photosensitizer M-6 (yield 40%). HR-MS (ESI) M/z [ M + H]:Calcd.for[C₇₃H₁₀₆N₁₁O₁₉]⁺1439.7588；found 1439.7575。

Example 7

The photosensitizer prepared in example 3 was used in the measurement experiment of the distribution ratio of fat to water

Dissolving 10 micromole of photosensitizer in 2mL of PBS, adding 2mL of n-octanol, shaking the mixed solution for 3min, placing the solution in ultrasonic waves for 5min, and centrifuging the solution for 5min at the speed of 5000 revolutions per minute to separate two phases. Measuring the absorption spectrum of the photosensitizer in the two phases, calculating the concentration of the photosensitizer in the two phases by Lambert-beer law, wherein the distribution ratio (Log PC) of lipid-water is the concentration ratio of the photosensitizer in the two phases, and the concentration ratio is shown in Table 1.

Example 8

The photosensitizer prepared in example 4 was used in the experiment for measuring the distribution ratio of fat water by referring to the procedure in example 7, see table 1.

Example 9

The photosensitizer prepared in example 5 was used in the experiment for measuring the distribution ratio of fat water by referring to the procedure in example 7, see table 1.

Example 10

The photosensitizer prepared in example 6 was used in the experiment for measuring the distribution ratio of fat water by referring to the procedure in example 7, see table 1.

Example 11:

the photosensitizer M-1 prepared in example 1 is used for single photon photodynamic experiment to prove the targeting property of the photosensitizer

(I) MCF-7 cells with over-expressed folic acid and A549 cells with normal expressed folic acid receptor are subjected to 10⁴Inoculating the cells in a 96-well cell culture plate at a cell density of one/ml, wherein the culture solution is 5% bovine serum (FBS) in RPMI 1640 without folic acid, incubating at 37 ℃ for 24 hours, adding photosensitizer M-1 with a photosensitizer concentration of 2.5 mu M, and continuing to incubate for 8 hours;

(II) taking out the 96-well plate, and using a 515nm LED lamp (half-peak width 40nm, power 36mW, power density 10mW cm)^-2) The samples were irradiated for 15 minutes and incubated for a further 24 hours after irradiation, and the cell viability was tested using CCK-8. Control blank test ofCells incubated with photosensitizer were taken as baseline for 100% cell viability. The killing rate of the photosensitizer to two cells is calculated through a test result, and the inactivation effect of the folic acid targeted photosensitizer to the tumor cells MCF-7 over-expressed by the folic acid receptor is found to be better, so that the targeting property of the folic acid targeted photosensitizer is proved. As shown in fig. 7.

Example 12:

the photosensitizer M-2 prepared in example 2 was used in two-photon photodynamic experiments

(I) Hela cells cultured in vitro in the ratio of 1.0X 10⁴Inoculating the cells in a 96-well cell culture plate at a cell density of one/ml, wherein the culture solution is RPMI 1640 containing 5% bovine serum (FBS), incubating at 37 ℃ for 24 hours, adding a photosensitizer M-2 with the concentration of 2.5 mu M, and continuing to incubate for 8 hours;

(II) irradiating with 800nm femtosecond laser for 10 min with a light source of Ti, Sapphire regenerative amplifier (Spitfire, Spectra-Physics,1kHz, <130fs,630mW) and a spot size of about 7mm, one well at a time (well diameter of 96-well plate is 6.4 mm); after irradiation, incubation was continued for 24 hours and the cell activity was tested using CCK-8. Control blank test cells incubated without photosensitizer were taken as baseline for 100% cell viability. The killing rate of the photosensitizer to the two cells was calculated from the test results, as shown in fig. 8.

Example 13

The photosensitizer prepared in example 2 was targeted by single photon photodynamic experiments as demonstrated in example 11, with reference to the procedure described in example 11, as shown in figure 9.

Example 14

The photosensitizer prepared in example 1 was used in the two-photon photodynamic experiment with reference to the procedure in example 12. As shown in fig. 10.

Example 15

The biological activity of the photosensitizer prepared in example 3 was predicted using Molinspiration software

The chemical structure obtained from ChemDraw of the photosensitizer was entered into the window for bioactivity prediction by molinospiration software, the prediction button was clicked, the parameters of the test results were analyzed, and the degree of electronegativity was observed, see table 2.

Example 16

The photosensitizer prepared in example 4 was used in a prediction experiment of biological activity, referring to the procedure in example 15. See table 2.

Example 17

The photosensitizer prepared in example 5 was used in a prediction experiment of biological activity, see table 2, with reference to the procedure in example 15.

Example 18

The photosensitizer prepared in example 6 was used in the prediction experiment of biological activity, see table 2, with reference to the procedure in example 15.

Example 19

The photosensitizer prepared in example 1 was used to verify the two-photon excitation process

(I) The light source used was a spectroscopic physical Tsunami mode-locked Ti Sapphire femtosecond laser (720-. The up-converted fluorescence was recorded by a fiber optic spectrometer (Ocean Optics USB2000 CCD). And (5) building a light path.

(II) fixing the laser wavelength at 800nm, adjusting the incident light intensity to 50mW, 70mW, 90mW, 110mW and 130mW respectively, and collecting the up-conversion fluorescence spectra of the photosensitizer under different light intensities.

(III) calculating the fluorescence intensity by using the area of the up-conversion fluorescence spectrum, and plotting the logarithm of the fluorescence intensity to the logarithm of the incident light intensity to obtain a straight line slope, wherein the slope value is close to 2, thereby proving that the process is a two-photon absorption process. See fig. 11.

Example 20

The photosensitizer prepared in example 2 was used for a process experiment for verifying two-photon excitation, referring to the procedure in example 19. See fig. 12.

TABLE 1 fat-water partition ratio of photosensitizer obtained in examples 3, 4, 5 and 6

Photosensitizer name	Log PC
		M-3	0.98
M-4	-0.66
		M-5	0.07
M-6	0.72

TABLE 2 prediction index parameters of biological activity of photosensitizers obtained in examples 3, 4, 5 and 6

It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all embodiments may not be exhaustive, and all obvious variations or modifications may be included within the scope of the present invention.

Claims

1. The preparation method of the folic acid targeted lipid-water amphiphilic benzylidene cycloparaffinone photosensitizer is characterized in that the folic acid targeted lipid-water amphiphilic benzylidene cycloparaffinone photosensitizer has the following structural formula M:

wherein: r₁Is- (C)₂H₄O)_m-R₅A group;

R₂is methyl, ethyl or- (C)₂H₄O)_n-R₆A group;

R₁and R₂May be the same or different substituent groups;

R₃is methyl, ethyl, propyl or isopropyl;

R₄is- (CH)₂)_pA group of-CH₂CH₂(OCH₂CH₂)_q-a group;

3, 4, 5, 6, 7 or 8; r₅Is methyl, ethyl, propyl or isopropyl;

the n is 3, 4, 5, 6, 7 or 8; r₆Is methyl, ethyl, propyl or isopropyl;

1,2, 3, 4, 5, 6, 7 or 8;

said q is 1,2, 3 or 4;

the preparation method of the folic acid targeted lipid-water amphiphilic benzylidene naphthenone photosensitizer comprises the following steps:

1) synthesis of C6, the reaction equation is as follows:

the method comprises the following specific steps:

2) synthesis of C7, the reaction equation is as follows:

the method comprises the following specific steps:

dissolving C2 and C6 obtained in the step 1) in dichloromethane according to the molar weight ratio of 1: 1-2, sequentially adding a catalyst with the molar weight of 1-2 times that of C6, an acid binding agent with the molar weight of 1-5 times, and a condensing agent with the molar weight ratio of C6 of 0.8-1.5; after the reaction system reacts for 6-24 hours at room temperature, extracting the reaction solution by using dichloromethane, washing the extract by using water, and removing an extracting agent by drying, filtering and rotary evaporation to obtain C7;

3) synthesis of C12, the reaction equation is as follows:

the method comprises the following specific steps:

4) synthesis of C13, the reaction equation is as follows:

the method comprises the following specific steps:

5) synthesis of M, the reaction equation is as follows:

the method comprises the following specific steps:

2. The preparation method according to claim 1, wherein the alkaline substance is one or a mixture of two or more of sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate and potassium bicarbonate in any proportion.

3. The preparation method according to claim 1, wherein the acidic substance is one of sulfuric acid and hydrochloric acid or a mixture thereof in any proportion.

4. The preparation method according to claim 1, wherein the catalyst is one or a mixture of more than two of 1-hydroxybenzotriazole, 1-hydroxy-7-azobenzotriazol, 4-dimethylaminopyridine and N-hydroxysuccinimide in any proportion.

5. The preparation method of claim 1, wherein the acid-binding agent is one or a mixture of two or more of N, N-diisopropylethylamine, triethylamine, pyridine and 4-dimethylaminopyridine in any proportion.

6. The preparation method according to claim 1, wherein the condensing agent is one or a mixture of two or more of dicyclohexylcarbodiimide, diisopropylcarbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate in any proportion.

7. The preparation method of claim 1, wherein the basic catalyst is one or more of lithium hydroxide, sodium hydroxide, potassium hydroxide, anhydrous sodium carbonate, anhydrous potassium carbonate, pyridine and piperidine.