阅读说明：本技术 一类内质网靶向激活型光敏剂的制备方法及应用 (Preparation method and application of endoplasmic reticulum targeted activation type photosensitizer ) 是由 李昌华 杨凤 翟文豪 于 2021-01-18 设计创作，主要内容包括：本发明涉及一类激活型内质网靶向光敏剂的制备方法及其应用,该类靶向激活型光敏剂首次被合成。式(Ⅰ)所示通式的化合物为该类内质网靶向激活型光敏剂激活后的结构通式,可由半花菁母核与3,5-二溴-4-羟基苯甲醛和3,5-二碘-4-羟基苯甲醛进行Knoevenagel反应得到,式(Ⅱ)所示通式的化合物为该类内质网靶向激活型光敏剂的结构通式,本发明创造性地在靶向激活型光敏剂式(Ⅱ)上预留官能团(R),并首次合成了过氧化氢刺激响应的内质网靶向光敏剂。本发明提供的一类靶向激活型光敏剂。解决了现有光敏剂选择性差、无法可视化、生理条件下可用范围小、光动力效果有限等技术问题。(The invention relates to a preparation method and application of an activated endoplasmic reticulum targeted photosensitizer, wherein the targeted activated photosensitizer is synthesized for the first time. The compound with the general formula shown in the formula (I) is a structural general formula of the activated endoplasmic reticulum targeted activation type photosensitizer, can be obtained by carrying out Knoevenagel reaction on hemicyanine mother nucleus, 3, 5-dibromo-4-hydroxybenzaldehyde and 3, 5-diiodo-4-hydroxybenzaldehyde, is a structural general formula of the endoplasmic reticulum targeted activation type photosensitizer, creatively reserves a functional group (R) on the targeted activation type photosensitizer formula (II), and synthesizes the endoplasmic reticulum targeted photosensitizer with hydrogen peroxide stimulation response for the first time. The invention provides a targeted activation type photosensitizer. The photosensitizer solves the technical problems of poor selectivity, incapability of visualization, small available range under physiological conditions, limited photodynamic effect and the like of the existing photosensitizer.)

1. An endoplasmic reticulum targeted photosensitizer and endoplasmic reticulum targeted activation type photosensitizer have structural formulas (shown in figure 11) shown as formula (I) and (II), wherein X is I or Br, and R is

2. The endoplasmic reticulum targeted photosensitizer according to claim 1, characterized in that mother nucleus is creatively modified to connect Br or I to the mother nucleus to obtain endoplasmic reticulum targeted photosensitizer molecule, the synthetic route is shown in figure 3.

3. The photosensitizer of claim 1, wherein the hydrogen atom in formula (i) is substituted with a functional group responsive to hydrogen peroxide to obtain a class of photosensitizer with targeting activation of endoplasmic reticulum, and the synthetic route is shown in fig. 4.

4. The method for preparing the photosensitizer according to claim 2, wherein piperidine is added only to the Knoevenagel reaction.

5. The method for preparing an endoplasmic reticulum targeted activation type photosensitizer according to claim 3, wherein a solvent of Knoevenagel reaction is acetonitrile, the temperature is 0 ℃, and the volume ratio of acetic acid to piperidine is 1: 2.

6. the endoplasmic reticulum targeted photosensitizer according to claim 2, characterized in that halogen atoms (Br, I) are introduced, on one hand, pKa of phenolic hydroxyl group of the photosensitizer can be effectively reduced, so that the activated photosensitizer can be used in a wider pH range, on the other hand, excited state electrons can be favorably subjected to interstitial crossing, and the photodynamic effect is improved.

7. The endoplasmic reticulum-targeted photosensitizer according to claim 2, characterized by strong photodynamic to allow effective photodynamic therapy.

8. The endoplasmic reticulum targeted activation type photosensitizer according to claim 3, wherein there is substantially no fluorescence and photodynamic under visible light excitation, and the activated photosensitizer has red fluorescence and photodynamic under visible light excitation, so that visible photodynamic therapy can be performed.

9. The endoplasmic reticulum targeted activation type photosensitizer according to claim 3, wherein the positively charged nitrogen atom and the halogen are key parts for targeting endoplasmic reticulum, and when the contrast is carried out by using a commercial endoplasmic reticulum probe, the fluorescence of the two is highly coincident, the Pearson coefficient is as high as 95%, which indicates that the activated photosensitizer is very good in endoplasmic reticulum targeting.

10. The method of claim 3, wherein the substitution or transformation (e.g., R is replaced by another stimuli-responsive group) is equivalent to the substitution or transformation.

Technical Field

The invention relates to the technical field of photodynamic therapy, in particular to an endoplasmic reticulum targeted activation type photosensitizer, a preparation method and application thereof.

Background

The photodynamic therapy is a novel photoactivated and nondestructive treatment mode, and the photosensitizer is excited by an effective light source to convert oxygen in cells into cytotoxic singlet oxygen so as to kill tumor cells and achieve the effect of treating tumors. The endoplasmic reticulum plays a very important role in the synthesis of cellular proteins. On one hand, the endoplasmic reticulum is used as a membrane structure for connecting the cell nucleus, the cytoplasm and the cell membrane, and plays an important role in the process of material transportation; on the other hand, the endoplasmic reticulum is also a key site for protein synthesis in cells, the folding structure of proteins is important for the physiological functions of cells, and the misfolding of proteins often causes cell death, so the endoplasmic reticulum also becomes an important therapeutic target in clinical application. However, most of the endoplasmic reticulum-targeted photosensitizers require the attachment of an endoplasmic reticulum-targeted group, which complicates the structure and synthesis of the photosensitizer molecule, and thus it is necessary to design a simple endoplasmic reticulum-targeted activated photosensitizer.

In the conventional photodynamic therapy, a photosensitizer drug is generally delivered to a tumor site, but the delivery mode not only enables the drug to accumulate at a diseased site, but also enables the drug to accumulate at a normal tissue site, so that the normal tissue is damaged, and the photosensitizer can be activated to overcome the defect. Combines the endoplasmic reticulum targeting and the biological factor activated photodynamic therapy, gives full play to the advantages of the endoplasmic reticulum targeting and the biological factor activated photodynamic therapy, generates singlet oxygen under the irradiation of an external light source, causes the dysfunction of the endoplasmic reticulum, leads the error folding of protein to cause the death of tumor cells, and further achieves the treatment purpose. Not only improves the curative effect of the medicine on the tumor part, but also reduces the damage to the normal tissue part, and is a more accurate treatment mode.

Disclosure of Invention

The invention provides a preparation method and application of an endoplasmic reticulum targeted activation type photosensitizer, and solves the technical problems that the existing photosensitizer is poor in selectivity, cannot be visualized, is small in available range under physiological conditions, is limited in photodynamic effect and the like.

The invention provides an endoplasmic reticulum targeted activation type photosensitizer which has a structural formula shown as a formula (I) shown in the attached figure 11.

In the formula (I), X is Br or I.

In the formula (II), X is Br or I, R is

The invention takes the structural formula in the formula (I) as a mother nucleus, creatively transforms the mother nucleus, and Br or I is connected to the mother nucleus to obtain endoplasmic reticulum targeted photosensitizer molecules, wherein the specific structure is shown in figure 1, and the synthesis steps are shown in figure 3.

The invention provides a preparation method of an endoplasmic reticulum targeted activation type photosensitizer, which is characterized in that a formula (I) is further transformed to obtain the endoplasmic reticulum targeted activation type photosensitizer, the specific structure is shown in figure 2, and the specific synthesis steps are shown in figure 4.

Preferably, the solvent for the Knoevenagel reaction in formula (I) is ethanol and the temperature is 85 ℃ under reflux.

Preferably, the solvent for the Knoevenagel reaction in formula (ii) is acetonitrile, the temperature is 0 ℃, and the volume ratio of acetic acid to piperidine is 1: 2.

preferably, the photosensitizer molecule is of the activated type^ERPS_2IHP can be completely converted into photosensitizer molecules in a system of acetonitrile and PBS (v/v,1/1) within 60min under the stimulation of hydrogen peroxide^ERPS_2ISee fig. 5.

Preferably, halogen (Br, I) atoms are introduced, so that on one hand, the pKa of phenolic hydroxyl groups of the photosensitizer can be effectively reduced, the activated photosensitizer can be used in a wider pH range (see figure 6), and on the other hand, excited-state electrons can be favorably subjected to interstitial crossing, and the photodynamic effect is improved.

As a preference, the first and second liquid crystal compositions are,^ERPS_2Ihas strong photodynamic power, and can effectively perform photodynamic therapy, as shown in figure 7.

As a preference, the first and second liquid crystal compositions are,^ERPS_2I-HP is substantially free of fluorescence under visible excitation, and after activation^ERPT_2IUnder the excitation of visible light, the red fluorescence exists, and visual photodynamic therapy can be carried out, as shown in figure 8.

Preferably, the nitrogen atom and the halogen atom are positively chargedThe fact that the fluorescence is highly coincident and the Pearson coefficient is as high as 95% shows that the activated endoplasmic reticulum is a key part of the endoplasmic reticulum targeting, and the contrast is carried out by using a commercial endoplasmic reticulum probe^ERPS_2ITargeting to the endoplasmic reticulum was very good, and the results are shown in FIG. 9.

In order to further explore the killing effect of the photosensitizer on patient cells, MTT method is used for researching^ERPS_2IThe toxicity to HeLa cells under light conditions is shown in FIG. 10, and under light conditions,^ERPS_2Isinglet oxygen is produced in the endoplasmic reticulum of the cell, killing the patient's cells.

The above description is only a preferred embodiment of the present invention, and all equivalent substitutions or alterations (e.g., substitution of R for other stimuli-responsive groups) made in accordance with the present invention are within the scope of the present invention.

Drawings

Figure 1 shows the structural formula of the endoplasmic reticulum-targeted photosensitizer.

Figure 2 shows the structural formula of the endoplasmic reticulum targeted activated photosensitizer.

FIG. 3 illustrates a synthetic route to endoplasmic reticulum-targeted photosensitizer.

FIG. 4 shows a synthetic route for an endoplasmic reticulum-targeted activated photosensitizer.

FIG. 5 generation I photosensitizers^ERPS_2IHP as an example, UV absorption under hydrogen peroxide activation.

FIG. 6 illustrates^ERPS_2IUV absorption at 585nm under different pH conditions.

FIG. 7 illustrates^ERPS_2IPhotodynamic using QDPBF as indicator.

FIG. 8 illustrates^ERPS_2I-fluorescence intensity of HP before and after hydrogen peroxide activation.

FIG. 9 illustrates^ERPS_2ISpecific localization in patient cells (compared to commercial endoplasmic reticulum probes).

FIG. 10 illustrates^ERPS_2IKilling of patient cells (MTT).

The structural general formulas of the endoplasmic reticulum targeted photosensitizer and the endoplasmic reticulum targeted activated photosensitizer are shown in figure 11.

Detailed Description

The present invention is further explained and illustrated with reference to the drawings (taking X ═ I as an example) and the specific embodiments, and based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without inventive efforts shall fall within the scope of the present invention.

The first embodiment is as follows: synthesis of^ERPS_2IThe synthetic route is shown in figure 3

1) Dissolving compound 1(0.5g,2.1mmol) and 3, 5-diiodo-4-hydroxybenzaldehyde (0.6g,2.52mmol) in 10mL of ethanol, adding 2.4mL of piperidine, heating and refluxing the reaction solution under the protection of argon for 12 hours, cooling to room temperature, removing the solvent by rotary evaporation, and performing column chromatography purification by using dichloromethane and methanol as eluent to obtain a pure purple compound^ERPS_2I(0.83g,66.6％)。¹H NMR(400MHz,DMSO-d₆)δ(ppm)8.76(s,1H),8.40(s,1H),8.26(d,J＝8.6Hz,1H),8.15–8.02(m,3H),7.78(dd,J＝8.9,1.2Hz,1H),7.68(dd,J＝8.5,6.9Hz,1H),7.54(t,J＝7.5Hz,1H),6.82(d,J＝14.8Hz,1H),4.47(q,J＝7.1Hz,2H),1.94(d,J＝1.2Hz,6H),1.43–1.30(m,3H).¹³C NMR(100MHz,DMSO-d₆)δ(ppm)176.09,171.52,148.84,138.87,134.11,131.56,130.35,129.89,127.75,127.37,125.03,122.51,122.21,111.60,98.14,51.15,40.20,26.57,12.87.MS 593.9787.

Example two: synthesis of^ERPS_2I-HP, the synthetic route is shown in figure 4

1) 3, 5-diiodo-4-hydroxybenzaldehyde (3.78g,10.1mmol,1.5eq) was dissolved in 30mL acetonitrile, potassium carbonate (1.85g,13.4mmol,2eq) was added and stirred at room temperature for 30min, followed by addition of(2g,6.7mmol,1 eq.) the reaction was stirred at 85 ℃ under reflux for 12 h, cooled to room temperature, the solvent removed by rotary evaporation and column chromatography using petroleum ether and dichloromethane as eluents gave the product as a white solid (3g, 75.8%).¹H NMR(400MHz,CDCl₃)δ(ppm)9.83(d,J＝2.7Hz,1H),8.30(d,J＝2.7Hz,2H),7.88(dd,J＝8.0,2.5Hz,2H),7.63(dd,J＝7.9,2.5Hz,2H),5.10(d,J＝2.5Hz,2H),1.36(d,J＝2.7Hz,12H).¹³C NMR(100MHz,CDCl₃)δ(ppm)188.14,162.249,141.36,138.56,135.568,135.06,127.63,91.95,83.97,74.65,24.99.

2) Compound 1(0.2g,0.84mmol,1eq) and compound(0.74g,1.26mmol,1.5eq) is dissolved in 30mL acetonitrile, then 100uL of acetic acid is added dropwise, 200uL of piperidine is added, the mixture is stirred for 6h at 0 ℃ under the protection of argon, the solvent is removed by low-temperature rotary evaporation, and column chromatography is carried out by taking dichloromethane and methanol as eluent, so as to obtain yellow solid^ERPS_2I-HP(0.27g,39.7％)。¹H NMR(400MHz,DMSO-d6)δ8.85(s,2H),8.48–8.40(m,2H),8.23(d,J＝8.2Hz,1H),8.17(d,J＝9.0Hz,1H),7.85–7.80(m,1H),7.79–7.72(m,4H),7.64(d,J＝7.7Hz,2H),5.04(s,2H),4.93(q,J＝7.1Hz,2H),2.02(s,6H),1.54(t,J＝7.1Hz,3H),1.29(s,12H).¹³C NMR(100MHz,DMSO-d6)δ(ppm)182.003,159.955,148.81,141.19,139.19,139.10,137.97,134.63,134.52,133.37,131.21,130.06,128.52,127.49,127.45,127.447,123.30,113.34,113.26,93.32,83.74,73.87,54.10,42.86,25.19,24.68,14.23.

The above description is only a preferred embodiment of the present invention, and it should be noted that various modifications made to the embodiments by a researcher using the technical field of the present invention without departing from the technical principle of the present invention should be regarded as the protection scope of the present invention.