Photosensitizer of merocyanine structure based on twisted molecular skeleton as well as preparation method and application of photosensitizer

Abstract

The invention relates to a photosensitizer based on a merocyanine structure of a twisted molecular skeleton, the molecular skeleton of the photosensitizer is free of heavy atoms, the photosensitizer has excellent active oxygen generation capability and high killing effect on hypoxic tumor cells, and the photosensitizer belongs to a Type I photosensitizer. The problem that in the prior art, heavy atoms such as I need to be introduced into a molecular skeleton of a merocyanine photosensitizer with high reactive oxygen generation capacity, and consequently dark toxicity is high is solved, and the defect that in the prior art, a Type I photosensitizer with the structure easy to modify lacks is overcome.

Description

Technical Field

[0001] The invention belongs to the technical field of photodynamic therapy and photosensitizers, and specifically relates to a photosensitizer based on a merocyanine structure with a twisted molecular skeleton, and a preparation method and application thereof. Background Art

[0002] Photodynamic therapy (PDT) is a disease treatment program that uses light to excite photosensitizers to produce cytotoxic substances, thereby killing diseased cells and achieving therapeutic effects. It has the characteristics of good targeting, low resistance to drugs, and low systemic side effects. Its basic principle is: when endogenous or exogenous photosensitizers in biological tissues are irradiated with light of corresponding wavelengths (ultraviolet light, visible light or near-infrared light), they absorb photon energy and transition from the ground state to the excited state. The photosensitizers in the excited state are very unstable and quickly release energy through physical or chemical deexcitation processes and return to the ground state. The deexcitation process can generate a large amount of reactive oxygen (including singlet oxygen, superoxide anion free radicals and hydroxyl free radicals, etc.). These reactive oxygen species can undergo redox reactions with biological macromolecules such as proteins and nucleic acids, damage cells and tissues, and ultimately achieve "in situ" killing of target tissues.

[0003] Photosensitizers are the research focus in the field of PDT. According to the different mechanisms of generating reactive oxygen species, photosensitizers can be divided into Type-I photosensitizers (chemical deexcitation, reactive oxygen species are superoxide anion free radicals and hydroxyl free radicals, etc.) and Type-II photosensitizers (physical deexcitation, reactive oxygen species are singlet oxygen). Currently reported photosensitizers are mainly Type-II photosensitizers, which consume a lot of oxygen during the PDT process, but the rapid growth of solid tumors will lead to insufficient oxygen supply inside the tumor, thus restricting the therapeutic effect of Type-II photosensitizers. Type-I photosensitizers have low dependence on oxygen and are expected to break the shackles of hypoxia in deep tumors, and have received widespread attention in recent years.

[0004] There are relatively few types of Type-Ⅰ photosensitizers that can be used in practice, and some photosensitizers introduce heavy atoms into their molecular skeletons to increase the probability of intersystem crossing, thereby increasing the quantum yield of reactive oxygen species. However, the introduction of heavy atoms will increase the dark toxicity of cells, which is not conducive to the application of clinical photodynamic therapy. In previous studies, Liu Shiyang et al. prepared a series of heavy-atom-free thiadiazole [3,4-g] quinoxaline Type-Ⅰ photosensitizers by introducing a strategy of distorting the molecular skeleton structure and achieved good PDT effects on hypoxic tumor cells (European Journal of Medicinal Chemistry, 2024, 265, 116059), but the synthesis of such photosensitizers is very difficult.

[0005] Shuang Zeng et al. reported a viscosity-sensitive semi-anthocyanidin dye that can be used for photodynamic therapy by activating cell apoptosis pathways (Angew. Chem. Int. Ed. 2024, 63, e202316487). Its chemical structure is as follows:

[0006]

[0007] The compound reported in the literature has two heavy atoms I connected to the benzene ring in order to have a suitable quantum yield. Although the presence of heavy atoms I can improve the quantum yield of reactive oxygen species, it will increase the dark toxicity of the compound. When the concentration of the compound is 30 μM, the cell survival rate has dropped to 80%. The biosafety of this compound is reduced, which may become an obstacle when used for PDT treatment. Therefore, it is of great practical significance to continue to explore and screen new Type-Ⅰ photosensitizers with high biosafety, adjustable structure and easy synthesis. Summary of the invention

[0008] In order to solve the problem that Type-I photosensitizers in the prior art are less studied and their photosensitivity is not satisfactory, the present invention provides a photosensitizer based on a merocyanine structure with a twisted molecular skeleton, which can generate peroxyhydroxyl radicals under light and can effectively kill hypoxic tumor cells, and has a simple structure, strong modifiability, and is easy to prepare. To achieve the above purpose, the present invention adopts the following technical solutions:

[0009] A photosensitizer based on a merocyanine structure with a twisted molecular skeleton, wherein the photosensitizer is a compound having the following general formula (I):

[0010]

[0011] R 1 represent

[0012] A 1 express

[0013] R 2 , R 3 , R 4 , R 5 Each independently represents hydrogen, hydroxyl, halogen, nitro, C1-3 alkyl; the anion X- is selected from Cl - Br - ,I - ; It represents the bond connecting the substituent to the molecular skeleton.

[0014] The twisted skeleton merocyanine structure of formula (I) provided by the present invention has no heavy atoms on the molecular skeleton and has excellent active oxygen generation capacity, which solves the problem of high dark toxicity caused by the introduction of heavy atoms such as I on the molecular skeleton of merocyanine photosensitizers with high active oxygen generation capacity in the prior art. The inventor believes that the main reason is that the molecular skeleton The possible reason is that the tertiary amine group with strong electron-donating ability at the fourth position of the benzene ring can enhance the charge transfer within the molecule and reduce the triplet energy gap; the hydroxyl group at the second position of the benzene ring can increase the steric hindrance of the benzene ring, effectively avoiding the non-radiative transition caused by the free rotation of the double bond of the merocyanine photosensitizer. These two groups in the structure can convert the traditional Type-Ⅱ merocyanine photosensitizer into a Type-Ⅰ photosensitizer.

[0015] R1 is a relatively large steric hindrance group, so as to obtain a distorted molecular skeleton structure, which has the potential to bring high quantum yield. The relatively large steric hindrance group R1 is preferably This type of compound has obvious Type I photosensitization activity, especially A 1 for hour.

[0016] It should be noted that in the specific application process, those skilled in the art can also selectively design the structures of R1, R2, R3, R4, R5 and A1, A2, A3, A4, A5 according to the requirements of the specific performance of the molecule, which is also within the scope of protection of the present invention.

[0017] Furthermore, the photosensitizer is selected from one of the following compounds:

[0018]

[0019]

[0020] The present invention also provides a method for preparing the above-mentioned photosensitizer, comprising the following steps:

[0021] (S1) The compound of the general formula P1 reacts with the compound of the general formula P2 to obtain an intermediate product of the general formula P3.

[0022]

[0023] (S2) Under an inert atmosphere, the intermediate product P3 reacts with a compound of the general formula P4 to obtain a compound of the general formula I.

[0024]

[0025] Furthermore, in step (S1), the reaction medium is a polar aprotic solvent, including but not limited to one or more of acetonitrile, N,N-dimethylformamide, and dimethyl sulfoxide; preferably, the molar ratio of the compound of the general formula P1 to the compound of P2 is 1-2:1-2; preferably, the reaction temperature is 60-100°C, and the reaction time is 10-20 hours.

[0026] Furthermore, in step (S2), the reaction medium is a polar protic solvent, including but not limited to one or more of methanol, ethanol, and glacial acetic acid; preferably, the molar ratio of the compound of general formula P3 to the compound of general formula P4 is 1-2:1-2; preferably, the reaction temperature is 30-90°C, and the reaction time is 10-20h.

[0027] For R 1 for When, the preparation method comprises the following steps:

[0028] (L1) reacting the compound of formula P1 with 4-(halomethyl)benzoic acid to obtain intermediate P3-1;

[0029]

[0030] (L2) intermediate P3-1 reacts with compound P4 to obtain intermediate T1-1;

[0031]

[0032] (L3) Intermediate T1-1 and Compound A with an amino group 1 -H reaction to obtain product T1,

[0033]

[0034] A 1 express

[0035] Further, in step (L1), the molar ratio of compound P1 to 4-(halomethyl)benzoic acid is 1-1.5:1, and the reaction medium is selected from one or more of acetonitrile, N,N-dimethylformamide, and dimethyl sulfoxide; preferably, the reaction temperature is 60-100°C, and the reaction time is 10-20 hours; in step (L2), the molar ratio of compound P3-1 to compound P4 is 1.2-1.7:1, and the reaction medium is selected from one or more of methanol, ethanol, and glacial acetic acid; preferably, the reaction temperature is 30-90°C, and the reaction time is 10-20 hours; in step (L2), T1-1 and compound A with an amino group 1 The molar ratio of -H is 1:1-1.2; preferably, the reaction temperature is 20-40°C and the reaction time is 2-8 hours.

[0036] The present invention also provides the use of the photosensitizer in preparing anti-tumor photodynamic drugs.

[0037] Furthermore, in the application of preparing anti-tumor photodynamic drugs, the light source is a laser or LED light source; preferably, the wavelength of the light source is 450-750nm, the illumination time is 0.5-60min, and the illumination intensity is 5-1000mWcm -2 Preferably, the concentration of the photosensitizer is 0.001 to 30 μg mL -1 The art knows how to adjust the wavelength, intensity, illumination time and amount of photosensitizer according to actual conditions.

[0038] The beneficial effects of the present invention are as follows:

[0039] The present invention discloses a photosensitizer having a merocyanine structure with a twisted molecular skeleton. The photosensitizer introduces a large steric hindering group into the molecular skeleton to obtain a twisted molecular skeleton structure, thereby having the following advantages:

[0040] (1) The photosensitizer prepared by the present invention can generate a large amount of reactive oxygen species under anaerobic conditions and only generate a small amount of singlet oxygen. It exhibits good peroxyhydroxyl radical generation ability in methanol and possesses the characteristics of a Type-I photosensitizer.

[0041] (2) The photosensitizer prepared by the present invention has good light stability and can be continuously irradiated by laser for more than 30 minutes.

[0042] (3) The photosensitizer prepared by the present invention has good water solubility and does not require nanoparticle coating.

[0043] (4) The photosensitizer prepared by the present invention can destroy the coenzyme balance in cells.

[0044] (5) The photosensitizer prepared by the present invention has excellent biosafety in a dark environment and has a highly effective tumor cell killing effect under light conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is the paramagnetic signal of the peroxy hydroxyl radical generated by T1-a prepared in Example 1 under light irradiation captured by DMPO.

[0046] Figure 2 It is the paramagnetic signal of the peroxy hydroxyl radical generated by T1-b prepared in Example 2 under light irradiation captured by DMPO.

[0047] Figure 3 This is a picture showing the change in absorbance at the maximum absorption wavelength of T1-a prepared in Example 1 as the illumination time increases.

[0048] Figure 4 This is a picture showing the change in absorbance at the maximum absorption wavelength of T1-b obtained in Example 2 as the illumination time increases.

[0049] Figure 5 The T1-a prepared in Example 1 catalyzes the conversion of reduced coenzyme NADP into oxidized coenzyme NAD + Ultraviolet spectrum monitoring picture.

[0050] Figure 6 The T1-b prepared in Example 2 catalyzes the conversion of reduced coenzyme NADP into oxidized coenzyme NAD + Ultraviolet spectrum monitoring picture.

[0051] Figure 7 The active oxygen generation capabilities of T1-a prepared in Example 1 and T1-b prepared in Example 2 are shown.

[0052] Figure 8 The results show that T1-a prepared in Example 1 has a dark toxicity to normal cells and tumor cells.

[0053] Fig. 9 The phototoxicity of T1-a prepared in Example 1 to tumor cells under normoxic and hypoxic conditions is shown.

[0054] Fig.10 The results show that T1-b prepared in Example 2 has a dark toxicity to normal cells and tumor cells.

[0055] Fig.11 The phototoxicity of T1-b prepared in Example 2 to tumor cells under normoxic and hypoxic conditions is shown. DETAILED DESCRIPTION

[0056] In order to more clearly illustrate the present invention, the present invention is further described below in conjunction with preferred embodiments and drawings. It should be clear that the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0057] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0058] Example 1

[0059] Preparation of Compounds of Formula T1-a

[0060] (1) Under ice bath conditions, commercially available 1,1,2-trimethyl-1H-benzo[e]indole (5.0 g, 23.9 mmol) and 20 ml of acetonitrile were added to a 50 ml round-bottom flask. After stirring for 15 min, 4-bromomethylbenzoic acid (3.8 g, 17.7 mmol) was slowly added to the system. The temperature was raised to 80°C and refluxed for 12 h. The reaction system was slowly cooled to room temperature, and n-hexane was added. The intermediate P3-1 was obtained by recrystallization. 1 H NMR (400 MHz, DMSO-d 6 )δ8.43(d,J=8.4Hz,1H),8.21(dd,J=14.4,8.5Hz,2H),7.97(t,J=8.6Hz,3H),7.82(t,J=7.7 Hz, 1H), 7.74 (t, J = 7.6 Hz, 1H), 7.56 (d, J = 8.0 Hz, 2H), 6.10 (s, 2H), 3.10 (s, 3H), 1.87 (s, 6H).

[0061]

[0062] (2) Under argon protection, P3-1 (3 g, 8.7 mmol) and 15 ml of ethanol were added to a 50 ml round-bottom flask. After stirring for 20 min, commercially available 4-(dimethylamino)-2-methoxybenzaldehyde (0.9 g, 5.5 mmol) was slowly added to the system, and the temperature was raised to 80 ° C and refluxed for 12 h. The reaction system was slowly cooled to room temperature and filtered to obtain a crude product. Recrystallization was performed using methanol and ether to obtain compound T1-1. 1 H NMR (400 MHz, DMSO-d 6 )δ11.22(s,1H),8.61(d,J=15.1Hz,1H),8.41(d,J=8.6Hz,1H),8.13(t,J=8.1Hz,2H ),8.00-7.90(m,3H),7.81(d,J=8.9Hz,1H),7.75(ddd,J=8.4,6.9,1.4Hz,1H),7.62 (ddd,J=8.1,6.8,1.0Hz,1H),7.46(d,J=8.3Hz,2H),7.29(d,J=15.5Hz,1H),6.57(d d,J=9.3,2.4Hz,1H),6.21(d,J=2.4Hz,1H),5.92(s,2H),3.15(s,6H),2.06(s,6H).

[0063]

[0064] (3) Under argon protection, T1-1 (0.2 g, 0.41 mmol), commercially available 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.2 g, 0.5 mmol), 0.1 ml N,N-diisopropylethylamine and 5 ml N,N-dimethylformamide were added to a 50 ml round-bottom flask and stirred at room temperature for 35 min. Then, N-toluenesulfonylethylenediamine (0.1 g, 0.47 mmol) was slowly added under stirring, and the reaction progress was monitored by TLC. After reacting at room temperature for 3.5 h, the reaction solution was poured into 100 ml of deionized water, and solid precipitated. The solid was collected by suction filtration and washed with deionized water several times to obtain a solid powder. The obtained solid powder was separated and purified by column chromatography (filler was 200-300 mesh silica gel) (methanol: dichloromethane = 1:40) to obtain compound T1-b. 1 H NMR (400 MHz, DMSO-d 6 )δ8.44(t,J=5.7Hz,1H),7.93(d,J=8.6Hz,1H),7.76(dd,J=15.3,8.1Hz,4H),7.64(t,J=9.2Hz,3H),7.42(d, J=7.9Hz,3H),7.33(d,J=7.9Hz,2H),7.20(t,J=7.5Hz,1H),6.93(dd,J=14.2,9.3Hz,2H),6.66(d,J=8.6Hz,1 H),6.19(d,J=8.7Hz,1H),5.95(d,J=2.5Hz,1H),5.58(d,J=10.1Hz,1H),4.52(d,J=17.0Hz,1H),4.34(d,J=1 7.1Hz,1H),3.27(d,J=6.4Hz,2H),2.86(t,J=6.8Hz,2H),2.80(s,6H),2.32(s,3H),1.59(s,3H),1.36(s,3H). 13 C NMR (101 MHz, DMSO-d 6 )δ166.54,155.67,152.41,145.59,143.29,142.97,138.12,130.79,130.06,129.78,129.26,128.91,128.02,127.92,126.97,1 26.88,121.94,121.59,113.27,110.89,107.92,106.06,104.96,98.05,53.49,47.25,42.47,24.67,21.80,21.40.HR-MS(MALDL TOF):m / z[M] +cacld for C 41 H 43 N 4 O 4 S + 687.29995; found[M] + 687.29975.

[0065]

[0066] Example 2

[0067] Preparation of Compounds of Formula T1-b

[0068] Step 1 and step 2 are the same as in Example 1, and step 3 is as follows: Under argon protection, T1-1 (0.2 g, 0.41 mmol), commercially purchased 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.2 g, 0.5 mmol), 0.1 ml N,N-diisopropylethylamine and 5 ml N,N-dimethylformamide were added to a 50 ml round-bottom flask, and stirred at room temperature for 35 min. Then, N-(2-aminoethyl)morpholine (60.0 mg, 0.46 mmol) was slowly added under stirring, and the reaction progress was monitored by TLC. After reacting at room temperature for 3.5 h, the reaction solution was poured into 100 ml of deionized water, and solid precipitated. The solid was collected by suction filtration and washed with deionized water several times to obtain a solid powder. The obtained solid powder was separated and purified by column chromatography (filler is 200-300 mesh silica gel) (methanol: dichloromethane = 1:20) to obtain compound T1-a. 1H NMR (400MHz, DMSO-d6) δ8.34(t,J=5.7Hz,1H),7.93(d,J=8.6Hz,1H),7.77(t,J=6.8Hz,3H) ,7.63(d,J=8.6Hz,1H),7.42(dd,J=13.4,7.6Hz,3H),7.20(t,J=7.5Hz,1H),6.93(dd,J=14 .0,9.3Hz,2H),6.66(d,J=8.6Hz,1H),6.20(dd,J=8.6,2.4Hz,1H),5.95(d,J=2.4Hz,1H),5 .58(d,J=10.1Hz,1H),4.52(d,J=17.0Hz,1H),4.35(d,J=17.0Hz,1H),3.55(t,J=4.6Hz,4H) ,3.36(d,J=6.5Hz,2H),2.81(s,6H),2.43(t,J=6.9Hz,2H),2.39(d,J=5.1Hz,4H),1.60(s, 3H),1.37(s,3H).13CNMR(101MHz,DMSO-d6)δ155.68,152.42,145.59,143.16,133.73,130 .79,129.78,128.92,128.03,127.87,126.94,121.94,121.59,113.28,110.90,107.93,10 6.07,104.96,98.06,66.68,57.86,53.78,53.49,47.24,36.99,24.68,21.81.HR-MS(MALDL TOF):m / z[M] + cacldfor C 38 H 43 N4 O 3 + 603.33297; found[M] + 603.33279.

[0069]

[0070] Example 3 Test of photosensitizer active oxygen generation ability

[0071] The photochemical capture method was used to detect the generation of active oxygen in deionized water by photosensitizer. DCFH was selected as the capture agent. DCFH itself has no fluorescence and can be oxidized by active oxygen species to DCF with bright green fluorescence. Methylene blue (MB) was used as the reference. In order to avoid the influence of the internal filter effect of the instrument, the absorption of the photosensitizer at the excitation wavelength in each mixed solution was kept at 0.1OD during the test, and the concentration of DCFH in each mixed solution was kept consistent (100μM). The sample solution was constantly stirred during the illumination process to keep the solution saturated with air. A 577nm laser was used to irradiate the sample to be tested, and the emission spectrum of the solution was tested at fixed intervals. The illumination time was used as the horizontal axis, and the fluorescence peak area of ​​DCF in the mixed solution of the photosensitizer and DCFH was plotted against the enhancement value of the illumination time as the vertical axis. The results are shown in Figure 7 As shown, compared with MB, the active oxygen production capacity of photosensitizer T1-a is better than that of MB, and photosensitizer T1-b also has good active oxygen production capacity.

[0072] Example 4 Singlet oxygen quantum yield test of photosensitizer

[0073] The photochemical capture method was used to determine the relative singlet oxygen quantum yield of the photosensitizer in water, using ABDA as the capture agent and methylene blue (MB) as the reference. To avoid the influence of the internal filter effect of the instrument, the absorption of the photosensitizer at the excitation wavelength of each mixed solution during the test needs to be maintained at 0.1OD, and the absorption of ABDA in each mixed solution should be kept consistent (the absorption value at 400nm is maintained at 1.0OD). The sample solution is continuously stirred during the illumination process to keep the air saturated in the solution. A 577nm laser is used to irradiate the sample to be tested, and the absorption spectrum of the mixed solution is tested at fixed intervals. The illumination time is used as the horizontal axis, and the decrease in the absorbance of the mixed solution of each photosensitizer and ABDA at 400nm over time is used as the vertical axis to draw a graph, and the slope of the linear correlation between the two is fitted, and the relative singlet oxygen quantum yield of the photosensitizer is quantitatively calculated according to the following formula:

[0074]

[0075] Wherein, K is the slope of the linear correlation between the absorbance decrease value of the mixed solution of ABDA and each photosensitizer at 400nm and the illumination time, s is the photosensitizer to be tested, r is the reference photosensitizer, is the singlet oxygen quantum yield of the reference photosensitizer, is the singlet oxygen quantum yield of the reference photosensitizer to be measured. The test results are shown in Table 1:

[0076] Table 1 Singlet oxygen data of photosensitizer

[0077]

[0078] As can be seen from Table 1, under 577nm laser irradiation, the singlet oxygen quantum yields of the series of photosensitizers in water are 0.14 and 0.02, respectively, indicating that photosensitizer T1-a can only produce a small amount of singlet oxygen, while T1-b hardly produces singlet oxygen.

[0079] Example 5 Testing of Active Oxygen Free Radicals of Photosensitizer

[0080] This example uses the photochemical capture method to measure the electron spin resonance signal of the photosensitizer in the dimethyl sulfoxide solution. 30 μL of the methanol solution (1 mM) of T1-a prepared in Example 1 and T1-b prepared in Example 2 and 10 μL of the methanol solution (0.2 M) of DMPO were mixed, the sample was loaded using a capillary tube, and the electron spin resonance instrument was used for testing. The EPR signal was first measured under dark conditions, and then the EPR signal under light conditions was tested. The results are as follows: Figure 1 , Figure 2 As shown in the figure, both photosensitizer T1-a and photosensitizer T1-b can generate obvious peroxyhydroxyl radical signals in the methanol system, which is the characteristic of Type-Ⅰ photosensitizer. Combined with the above test results, T1-a is a Type-Ⅰ / Ⅱ photosensitizer, and T1-b is a pure Type-Ⅰ photosensitizer.

[0081] Example 6 Photosensitizer T1-a photostability test

[0082] 2 ml of methanol solution of T1-a prepared in Example 1 and T1-b prepared in Example 2 were placed in a cuvette with a length, width and height of 1×1×3.5 cm respectively. -2 The photostability performance was tested under light conditions, and the absorbance at the maximum absorption wavelength was taken, such as Figure 3 , Figure 4 As shown, after 30 minutes of laser irradiation, the absorbance at the maximum absorption wavelength remained basically unchanged, indicating that these two photosensitizers have good photostability.

[0083] Example 7

[0084] Photosensitizers T1-a and T1-b catalyze the conversion of reduced coenzyme NADP into oxidized coenzyme NAD + Test

[0085] 2 ml aqueous solution of T1-a (1 mM, 20 μL) prepared in Example 1, T1-b (1 mM, 20 μL) prepared in Example 2, and coenzyme NADP (1 mM, 200 μL) were placed in a cuvette with a length, width, and height of 1×1×3.5 cm, respectively. -2 Under light conditions, it catalyzes the conversion of reduced coenzyme NADP into oxidized coenzyme NAD + Test. Figure 5 , Figure 6 As shown in the figure, the absorbance of the reduced coenzyme NADP at its 339nm characteristic absorption peak decreases with the increase of illumination time, while the oxidized coenzyme NAD + The absorbance at its 260nm characteristic absorption peak increases with the increase of illumination time, which means that T1-a and T1-b can catalyze the transformation of coenzymes. Coenzyme NADP plays an important role in organisms, and many biochemical reactions in cells require the catalysis of coenzyme NADP. The experimental results show that T1-a and T1-b are expected to break the coenzyme cycle in tumor cells, destroy the homeostasis of tumor cells, and kill tumor cells efficiently.

[0086] Example 8

[0087] Comparison of the dark toxicity, normoxic phototoxicity and hypoxic phototoxicity of photosensitizer T1-a and photosensitizer T1-b on normal cells L929 and cancer cells MDA-MB-231:

[0088] L929 cells and MDA-MB-231 cells (2000 cells / well) were seeded in 96-well plates and incubated at 37°C in a humidified environment containing 5% CO. 2 Cultured in an incubator for 24 hours. The culture medium was replaced with complete culture medium (100 μL / well) containing different concentrations (0, 2 μM, 4 μM, 6 μM, 8 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM) of T1-a prepared in Example 1 and T1-b prepared in Example 2, and the culture medium was discarded and washed twice with PBS buffer. The cells were stained with CCK8 solution, and the absorbance of the solution in each well at a wavelength of 450 nm was measured with an enzyme-linked immunosorbent assay to obtain the dark toxicity effect of the cells at different concentrations of photosensitizer. Figure 8 , Fig.10As shown, T1-a and T1-b exhibited excellent biosafety. When the concentration increased to 100 μM, the cell survival rate remained above 80%.

[0089] The phototoxicity test operation process was the same as the dark toxicity test operation process before incubation with the photosensitizer. The cell culture medium was replaced with complete culture medium (100 μL / well) containing different concentrations (0, 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM) of T1-a prepared in Example 1 and T1-b prepared in Example 2 and continued to incubate. After incubation for 4 h, a 577 nm laser (30 mW cm -2 ) for 10 min and incubate for 24 h. Then CCK8 staining was performed and the absorbance of the solution in each well at 450 nm was measured using an enzyme-linked immunosorbent assay to obtain the phototoxic effect of the cells at different concentrations of the photosensitizer. Fig. 9 , Fig.11 As shown in the figure, after mathematical processing of the cell data, the IC50 of T1-a was 0.512 μM, and the IC of T1-b was 50 0.933μM (IC 50 represents the drug concentration at which the cell viability drops to 50%).

[0090] The tumor site consumes a lot of oxygen, which leads to the limited effect of common Type-II photosensitizers during treatment due to insufficient oxygen supply. The photodynamic therapy effect of T1-a and T1-b under hypoxic conditions was further evaluated. The operation process of hypoxic phototoxicity test was the same as that of phototoxicity test before laser irradiation. The cells incubated with photosensitizer were treated with hypoxia and a 577nm laser (30mW cm -2 ) for 10 min and incubate for 24 h. Then CCK8 staining was performed and the absorbance of the solution in each well at 450 nm was measured using an enzyme-linked immunosorbent assay to obtain the hypoxic phototoxicity effect of cells at different concentrations of photosensitizer. Fig. 9 , Fig.11 As shown in Figure 2, the photodynamic therapy effects of these two photosensitizers under hypoxia are not much different from those under normoxic conditions. After mathematical processing of the cell data, the IC of T1-a was obtained. 50 The IC value of T1-b is 0.588 μM. 50 It is 0.830 μM. This shows that the photodynamic therapy Type-Ⅰ mechanism plays a major role in killing tumor cells by photosensitizers.

[0091] In summary, the photosensitizers T1-a and T1-b in the examples of this patent have excellent biosafety while maintaining high efficiency in killing normoxic and hypoxic cancer cells, which is significantly improved compared with the compounds reported in the literature. When the photosensitizer concentration is increased to 100 μM, the cell survival rate is still maintained at more than 80%.

[0092] Example 9

[0093] We also prepared compounds with other substituents in A1 and tested their IC 50 and IC in hypoxic state 50 , and dark toxicity.

[0094] The preparation method is basically the same as that of Example 1, except that in step (3), N-toluenesulfonylethylenediamine is replaced by equimolar amounts of 1-methylsulfonylazetidine-3-amine, N-(2-aminoethyl)ethanesulfonamide, N-(2-aminoethyl)benzenesulfonamide, and N-(2-aminoethyl)-4-fluorobenzenesulfonamide, respectively, to obtain compounds T1-c, T1-d, T1-e, and T1-f, respectively. IC values ​​of the above compounds in the normoxic state 50 and IC in hypoxic state 50 , and dark toxicity are listed in Table 2 below:

[0095]

[0096] 1 H NMR (400 MHz, DMSO-d 6 )δ9.74(s,1H),8.17(dt,J=7.7,1.6 Hz,1H),7.97(ddd,J=9.2,7.6,1.5 Hz,2H),7.82(m,3H),7.64(d,J=7.5 Hz,1H),7.57(m,1H),7.50(m,2H),7.39(m,J=7.5,1.5 Hz,1H),7.29(m,3H),6.44(d,J=1.5 Hz,1H),6.34(dd,J=7.5,1.5Hz,1H),5.68(t,J=1.0 Hz,2H),4.18(m,J=9.9,7.0 Hz,1H),3.61(m,4H),2.87(s,4H),2.82(s,2H),1.52(s,4H). 13 C NMR (101 MHz, DMSO-d 6)δ171.73,167.39,157.23,152.46,142.43,137.87,134.38,133.98,133.18,132.58,130.17,129.56,129.24,128.06,128.05,128.01,127.99,126.89,124.98,117.53,115.04,113.20,112.61,106.52,56.77,52.65,44.99,44.78,41.31,40.45,26.37.HR-MS(MALDL TOF):m / z[M] + cacld for C 36 H 39 N 4 O 4 S + 623.26865;found[M] + 623.26837。

[0097]

[0098] 1H NMR(400 MHz,DMSO-d 6 )δ9.74(s,1H),8.17(m,J=7.7,1.7 Hz,1H),8.04(t,J=7.0 Hz,1H),7.97(ddd,J=9.2,7.6,1.5 Hz,2H),7.82-7.76(m,2H),7.64(d,J=7.5 Hz,1H),7.57-7.50(m,1H),7.50-7.43(m,2H),7.39(m,J=7.5,1.5 Hz,1H),7.36-7.29(m,3H),6.65(t,J=9.4 Hz,1H),6.44(d,J=1.5 Hz,1H),6.34(dd,J=7.5,1.5 Hz,1H),5.68(t,J=1.0Hz,2H),3.56(q,J=7.1 Hz,2H),3.16(q,J=8.0 Hz,2H),3.00(m,J=9.4,7.1 Hz,2H),2.87(s,4H),1.52(s,4H),1.31(t,J=8.0 Hz,3H).13C NMR(101 MHz,DMSO-d 6)δ171.73,167.67,157.14,152.46,142.43,137.87,134.35,134.03,133.18,132.58,130.17,129.51,129.11,128.06,128.05,128.01,127.96,126.89,124.98,117.53,115.04,113.20,112.61,106.52,56.77,52.65,46.00,44.11,41.31,39.47,26.37,9.71.HR-MS(MALDLTOF):m / z[M] + cacld for C 36 H 41 N 4 O 4 S + 625.28430;found[M] + 625.28421。

[0099]

[0100] 1 H NMR(400MHz,DMSO-d 6 )δ9.74(s,1H),8.13(m,J=7.5,1.5Hz,1H),8.03(t,J=7.1Hz,1H),7.98(dd,J=7.5,1.4Hz,1H),7.94(dd,J=7.5,1.4Hz,1H),7.82-7.76(m,2H),7.72(m,2H),7.56(m,2H),7.50(m,1H),7.41(m,4H),7.35(m,2H),7.29(m,3H),6.44(d,J=1.5Hz,1H),6.34(dd,J=7.5,1.5Hz,1H),5.68(t,J=1.0Hz,2H),3.58(q,J=7.1Hz,2H),3.25(m,J=10.2,7.0Hz,2H),2.87(s,4H),1.52(s,4H). 13 C NMR(101MHz,DMSO-d 6)δ171.73,167.67,157.14,152.46,142.12,139.84,137.87,134.43,134.03,133.18,132.62,131.27,129.51,129.39,129.11,128.63,128.38,128.01,127.91,127.19,127.03,125.32,115.25,113.71,113.20,112.61,106.52,56.77,52.65,43.30,41.31,40.32,26.37.HR-MS(MALDLTOF):m / z[M] + cacld for C 40 H 41 N 4 O 4 S + 673.28430;found[M] + 673.28427。

[0101]

[0102] 1 H NMR(400MHz,DMSO-d 6 )δ9.99(s,1H),8.13(m,J=7.5,1.5Hz,1H),8.03(t,J=7.1Hz,1H),7.98(dd,J=7.5,1.4Hz,1H),7.94(dd,J=7.5,1.4Hz,1H),7.82(m,2H),7.76(m,2H),7.53(m,J=7.5,1.5Hz,1H),7.53-7.43(m,3H),7.42-7.35(m,2H),7.35-7.26(m,5H),6.76(d,J=1.5Hz,1H),6.17(dd,J=7.5,1.5Hz,1H),5.72(q,J=2.7Hz,1H),5.68(t,J=1.0Hz,2H),3.58(q,J=7.1Hz,2H),3.25(m,J=10.1,7.1Hz,2H),2.91(d,J=2.7Hz,3H),1.52(s,5H). 13 C NMR(101MHz,DMSO-d 6)δ171.73,167.67,158.69,150.87,142.12,137.87,134.43,134.03,1 33.18,132.62,129.92,129.39,129.11,128.35,128.29,128.01,127.9 1,127.19,127.03,125.32,117.12,116.96,115.25,114.52,114.06,113.20,105.89,56.77,52.65,43.30,40.32,28.16,26.37.HR-MS(MALDL TOF):m / z[M] + cacld for C 40 H 40 FN 4 O 4 S + 691.27488; found[M] + 691.27473.

[0103] Table 2 Photosensitizer performance

[0104]

[0105]

[0106]

Claims

1. A photosensitizer based on a merocyanine structure with a distorted molecular skeleton, characterized in that: The photosensitizer is a compound having the following general formula (I): R1 stands for A1 means R2, R3, R4, R5 each independently represent hydrogen, hydroxyl, halogen, nitro, C1-3 alkyl; anion X- is selected from Cl - Br - ,I - ; It represents the bond connecting the substituent to the molecular skeleton.

2. The photosensitizer according to claim 1, characterized in that R1 is 3. The photosensitizer according to claim 1, characterized in that The photosensitizer is selected from one of the following compounds:

4. The method for preparing the photosensitizer according to any one of claims 1 to 3, characterized in that: The steps include: (S1) reacting a compound of the general formula P1 with a compound of the general formula P2 to obtain an intermediate product of the general formula P3; (S2) Under an inert atmosphere, the intermediate product P3 reacts with a compound of the general formula P4 to obtain a compound of the general formula I; 5. The preparation method according to claim 4, characterized in that: In step (S1), the reaction medium is a polar aprotic solvent, including but not limited to one or more of acetonitrile, N,N-dimethylformamide, and dimethyl sulfoxide; preferably, the molar ratio of the compound of the general formula P1 to the compound of the general formula P2 is 1-2:1-2; preferably, the reaction temperature is 60-100°C, and the reaction time is 10-20 hours.

6. The preparation method according to claim 4, characterized in that: In step (S2), the reaction medium is a polar protic solvent, including but not limited to one or more of methanol, ethanol, and glacial acetic acid; preferably, the molar ratio of the compound of general formula P3 to the compound of general formula P4 is 1-2:1-2; preferably, the reaction temperature is 30-90°C and the reaction time is 10-20h.

7. The method for preparing the photosensitizer according to claim 2 or 3, characterized in that: The following steps are involved: (L1) reacting the compound of formula P1 with 4-(halomethyl)benzoic acid to obtain intermediate P3-1; (L2) intermediate P3-1 reacts with compound P4 to obtain intermediate T1-1; (L3) The intermediate T1-1 reacts with a compound A1-H having an amino group to obtain a product T1.

8. The preparation method according to claim 7, characterized in that: In step (L1), the molar ratio of P1 compound to 4-(halomethyl)benzoic acid is 1-1.5:1, and the reaction medium is selected from one or more of acetonitrile, N,N-dimethylformamide, and dimethyl sulfoxide; preferably, the reaction temperature is 60-100°C, and the reaction time is 10-20 hours; in step (L2), the molar ratio of P3-1 to compound P4 is 1.2-1.7:1, and the reaction medium is selected from one or more of methanol, ethanol, and glacial acetic acid; preferably, the reaction temperature is 30-90°C, and the reaction time is 10-20 hours; in step (L2), the molar ratio of T1-1 to compound A1-H having an amino group is 1:1-1.2; preferably, the reaction temperature is 20-40°C, and the reaction time is 2-8 hours.

9. Use of the photosensitizer according to any one of claims 1 to 3 in the preparation of anti-tumor photodynamic drugs.

10. The use according to claim 9, characterized in that The light source is a laser or LED light source; preferably, the wavelength of the light source is 450-750nm, the illumination time is 0.5-60min, and the illumination intensity is 5-1000mW cm -2 Preferably, the concentration of the photosensitizer is 0.001 to 30 μg mL -1 .

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