Spirocyclic acridinium cyanine compounds, preparation method thereof, photosensitizer and application

Abstract

The application discloses a kind of spiro acridine cyanines compound and preparation method, photosensitizer and application thereof.The compound has the structure of formula I, has excellent near-infrared absorption and fluorescence emission properties, can be used for cancer cell near-infrared I region and II region fluorescence imaging, also has excellent anti-tumor activity and photoactivated active oxygen production activity, can be used for synergistic photodynamic therapy, acts on lesion by a variety of mechanisms, and has wide application prospect.The compound preparation method adopts one-pot method, simple operation, and reaction substrate is strong in universality, easy to expand compound structure.

Description

Technical Field

[0001] This invention relates to a spirocyclic acridine cyanine compound, its preparation method, photosensitizer, and applications, and particularly to a spirocyclic acridine cyanine compound with near-infrared absorption and fluorescence emission properties, its preparation method, photosensitizer, and applications. Background Technology

[0002] Cyanide dyes (such as trimethylcyanine, pentamethylcyanine, and heptamethylcyanine) are a class of fluorescent dyes with polymethyl-bridged chemical structures. They possess characteristics such as high molar extinction coefficients, high fluorescence quantum yields, and tunable absorption and emission spectra, and are currently widely used in research in fields such as biology and analytical sensing. However, traditional cyanide dyes have relatively simple structures, and their synthesis usually requires strong acid / base catalysis and the participation of condensing agents. Furthermore, traditional cyanide dyes have insufficient killing power against tumor cells, often requiring complex group modifications followed by phototherapy (photodynamic therapy and photothermal therapy) to achieve the desired tumor cell killing effect. Summary of the Invention

[0003] Objectives of the Invention: The first objective of this invention is to provide a spirocyclic acridine cyanine compound with strong light-harvesting ability and high fluorescence brightness; the second objective is to provide a method for preparing the compound; the third objective is to provide a photosensitizer prepared from the compound; and the fourth objective is to provide an application of the compound and the photosensitizer thereof.

[0004] Technical solution: The spirocyclic acridine cyanine compound of the present invention has the structure of Formula I:

[0005]

[0006] R1 is selected from C1-C8 alkyl groups, phenyl groups, and benzyl groups that are substituted with hydrogen, halogen, amino, or carboxyl groups.

[0007] R2 and R3 are selected from hydrogen, halogen, C1-C8 alkyl, C1-C8 haloalkyl, C1-C8 alkylamino, amino-substituted C1-C8 alkyl, C1-C8 alkoxy, C1-C8 haloalkoxy, C1-C8 alkylthio, C1-C8 alkanoyl, C1-C8 amide, C1-C8 alkylsilyl, nitro, cyano, and amino.

[0008] Y -Selected from fluoride ions, chloride ions, bromide ions, iodide ions, tetrafluoroborate anions, trifluoromethanesulfonate anions, hexafluorophosphate anions, perchlorate anions, sulfate anions, phosphate anions, methanesulfonate anions, benzenesulfonate anions, p-toluenesulfonate anions, naphthalenesulfonate anions, citrate anions, lactate anions, pyruvate anions, acetate anions, trifluoroacetate anions, maleate anions, succinate anions, fumarate anions, and salicylate anions.

[0009] Acridines are a class of nitrogen-containing heterocyclic organic compounds and important chemical and pharmaceutical intermediates, playing a vital role in scientific research, chemical industry, and biomedicine. Acridine derivatives also possess various pharmacological activities such as antibacterial and anticancer activity. Current research on acridines mainly focuses on modifying their parent compounds. In addition, a few reports document the conjugation of acridines with other dye precursors for cancer cell imaging or tumor therapy. Research on acridine dimers or trimers is relatively limited.

[0010] Preferably, in the structure:

[0011] R1 is selected from C1-C8 alkyl, phenyl, and benzyl groups.

[0012] Further preferably, in the structure:

[0013] R1 is selected from methyl, ethyl, hexyl, and phenyl.

[0014] Preferably, in the structure:

[0015] R2 and R3 are selected from hydrogen, fluorine, chlorine, bromine, methyl, ethyl, isopropyl, tert-butyl, difluoromethyl, trifluoromethyl, methylamino, ethylamino, aminomethyl, aminoethyl, methoxy, ethoxy, isopropoxy, tert-butoxy, difluoromethoxy, trifluoromethoxy, methylthio, ethylthio, acetyl, acetamido, trimethylsilyl, nitro, cyano, and amino.

[0016] Further optimization is that when R2 and R3 are the same, both are hydrogen; when R2 and R3 are different, either one is hydrogen.

[0017] Further optimization is that when R2 and R3 are different, R3 is hydrogen.

[0018] Further optimization involves R2 and R3 located at the β and γ positions of the N atom in the acridine ring system, specifically selected from...

[0019] Preferably, in the structure:

[0020] Y -Selected from fluoride ions, chloride ions, bromide ions, iodide ions, tetrafluoroborate anions, trifluoromethanesulfonate anions, hexafluorophosphate anions, perchlorate anions, sulfate anions, and phosphate anions.

[0021] Preferably, the spirocyclic acridine cyanine compound is selected from any of the following compounds:

[0022]

[0023] The method for preparing the spirocyclic acridine cyanine compound of the present invention involves a closed-loop reaction of three molecules of a 9,10-disubstituted acridine onium salt in an organic base and solvent to obtain the compound.

[0024]

[0025] R1, R2, R3, Y - As defined in claim 1, preferably R1 is methyl, ethyl, hexyl or phenyl, R2 and R3 are hydrogen or bromine, and Y - The ions are iodide ions, tetrafluoroborate anions, or trifluoromethanesulfonate anions, and more preferably the compounds are Me-PinCy, Et-PinCy, Hexyl-PinCy, Ph-PinCy, or Br-PinCy.

[0026] The preferred reaction solvent is acetonitrile, the organic base is trimethylamine, the reaction temperature is 145℃, and the reaction must be carried out in the dark.

[0027] This invention uses a one-pot method to directly prepare the spirocyclic acridine cyanine compounds, eliminating the need for condensing agents, catalysts, and other components. It has a wide range of applicable reaction substrates and strong applicability.

[0028] The photosensitizer described in this invention is prepared from the aforementioned spirocyclic acridine cyanine compound.

[0029] The spirocyclic acridine cyanine compounds and their photosensitizers described in this invention are used in the preparation of photodynamic therapy drugs and tumor cell imaging reagents.

[0030] Preferably, the drug is an anti-tumor drug.

[0031] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0032] The compounds designed in this invention possess excellent near-infrared absorption and fluorescence emission properties, making them suitable for near-infrared I and II region fluorescence imaging within cancer cells. They also exhibit excellent antitumor activity and photoactivated reactive oxygen species (ROS) generation activity, enabling synergistic photodynamic therapy. Targeting lesions through multiple mechanisms, these compounds show broad application prospects. The compound preparation method employs a one-pot process, which is simple to operate, has broad substrate versatility, and facilitates compound structural expansion. Attached Figure Description

[0033] Figure 1 The normalized absorption / fluorescence spectrum (808 nm photoexcitation) of the compound Me-PinCy prepared in Example 1 of this invention in methanol;

[0034] Figure 2 The 1H NMR 400M NMR spectrum of the compound Me-PinCy prepared in Example 1 of this invention;

[0035] Figure 3 This is a high-resolution mass spectrum of the compound Me-PinCy prepared in Example 1 of this invention;

[0036] Figure 4 The normalized absorption / fluorescence spectrum (808 nm photoexcitation) of the compound Et-PinCy prepared in Example 2 of this invention in methanol;

[0037] Figure 5 The 1H NMR 400M NMR spectrum of the compound Et-PinCy prepared in Example 2 of this invention;

[0038] Figure 6 This is a high-resolution mass spectrum of the compound Et-PinCy prepared in Example 2 of the present invention;

[0039] Figure 7 The normalized absorption / fluorescence spectrum (808 nm photoexcitation) of the compound Hexyl-PinCy prepared in Example 3 of this invention in methanol;

[0040] Figure 8 The 1H NMR 400M NMR spectrum of the compound Hexyl-PinCy prepared in Example 3 of this invention;

[0041] Figure 9 This is a high-resolution mass spectrum of the compound Hexyl-PinCy prepared in Example 3 of this invention;

[0042] Figure 10 The normalized absorption / fluorescence spectrum (808 nm photoexcitation) of the compound Ph-PinCy prepared in Example 4 of this invention in methanol;

[0043] Figure 11 The 1H NMR 400M NMR spectrum of the compound Ph-PinCy prepared in Example 4 of this invention;

[0044] Figure 12 This is a high-resolution mass spectrum of the compound Ph-PinCy prepared in Example 4 of this invention;

[0045] Figure 13The normalized absorption / fluorescence spectrum (808 nm photoexcitation) of the compound Br-PinCy prepared in Example 5 of this invention in methanol;

[0046] Figure 14 The compound Br-PinCy prepared in Example 5 of this invention 1 HNMR 400M NMR;

[0047] Figure 15 This is a high-resolution mass spectrum of the compound Br-PinCy prepared in Example 5 of the present invention;

[0048] Figure 16 The normalized absorption / fluorescence spectrum (808 nm photoexcitation) of compound ADCy3 prepared in Example 6 of this invention in methanol;

[0049] Figure 17 The fluorescence intensity values ​​of the solutions after mixing with commercial reactive oxygen species (ROS) probe DCFH are shown as the fluorescence intensity values ​​over time.

[0050] Figure 18 Images obtained by observing cell status using a laser confocal multidimensional fluorescence imaging system after incubating HeLa cells with spirocyclic acridinecyanine dyes Me-PinCy, Br-PinCy, and conventional linear acridinecyanine dye ADCy3.

[0051] Figure 19 Images obtained by observing cell state using a laser confocal multidimensional fluorescence imaging system after co-incubating spirocyclic acridinecyanine dyes Me-PinCy and Br-PinCy with conventional linear acridinecyanine dye ADCy3 and mitochondrial fluorescent probes.

[0052] Figure 20 The effect of different concentrations of the spirocyclic acridine cyanine dye Me-PinCy on the viability of HeLa cells under dark conditions;

[0053] Figure 21 300mW / cm 2 Effects of different concentrations of spirocyclic acridine cyanine dye Me-PinCy on HeLa cell viability after 5 min of 808 nm light irradiation;

[0054] Figure 22 The effect of different concentrations of spirocyclic acridine cyanine dye Br-PinCy on the viability of HeLa cells under dark conditions;

[0055] Figure 23 300mW / cm 2Effects of different concentrations of spirocyclic acridine cyanine dye Br-PinCy on HeLa cell viability after 5 min of 808 nm light irradiation;

[0056] Figure 24 The effect of different concentrations of spirocyclic acridine cyanine dye ADCy3 on the viability of HeLa cells under dark conditions;

[0057] Figure 25 300mW / cm 2 Effects of different concentrations of spirocyclic acridine cyanine dye ADCy3 on the viability of HeLa cells after irradiation with 808 nm light for 5 min. Detailed Implementation

[0058] The technical solution of the present invention will be further described below with reference to the embodiments.

[0059] Example 1: Preparation of the spirocyclic acridine cyanine dye Me-PinCy

[0060]

[0061] 5.2 mg of 9,10-dimethylacridinium iodide was added to a 15 mL sealed pressure-resistant tube, followed by 2 mL of acetonitrile and 15.5 μL of trimethylamine (2.0 M THF solution). The mixture was reacted at 145 °C in the dark for 12 h, then cooled to room temperature. After the solvent was evaporated, the mixture was separated by preparative HPLC (eluent ratio: water:acetonitrile = 95:5, gradient increased to water:acetonitrile = 5:95). The eluent with a high absorption peak at 798 nm was collected, the solvent was evaporated, and the solid was dried to obtain a black solid. The reaction yield was 65.9%.

[0062] 1 H NMR (400MHz, CDCl3) δ (ppm) 8.54 (d, J = 13.7Hz, 1H), 8.43 (d, J = 8.0Hz, 1H), 7.87 (q, J = 7.3 ,6.8Hz,4H),7.78(d,J=8.6Hz,2H),7.63(dd,J=20.6,8.0Hz,3H),7.45(t,J=7.2Hz,1H),7 .37(dd,J=8.2,5.7Hz,3H),7.29(d,J=7.7Hz,1H),7.20(d,J=7.7Hz,3H),7.14(d,J=13.6 Hz,2H),6.95-6.89(m,2H),6.74(t,J=7.3Hz,2H),4.15(s,3H),4.07(s,3H),3.74(s,3H).

[0063] 13C NMR (101MHz, CDCl3) δ (ppm) 157.62, 152.74, 150.78, 143.64, 142.90, 140.61, 137.55, 136.07, 134.88, 134.61, 128.71, 127.72, 127. 29,126.80,126.27,124.55,124.28,123.08,121.29,120.10,120.00,117.61,117.14,116.02,113.52,111.47,36.55,34.50,33.83.

[0064] HRMS(ESI)m / z calcd.[M+H] + The measured molecular weight was 616.2747; the measured molecular weight was 616.2736.

[0065] The normalized absorption / fluorescence spectrum of Me-PinCy in methanol (808 nm photoexcitation) is shown below. Figure 1 .

[0066] Example 2: Preparation of the spirocyclic acridine cyanine dye Et-PinCy

[0067]

[0068] 5.2 mg of 9-methyl-10-ethylacridinium iodide was added to a 15 mL sealed pressure-resistant tube, followed by 2 mL of acetonitrile and 14.9 μL of trimethylamine (2.0 M THF solution). The mixture was reacted at 145 °C in the dark for 12 h, then cooled to room temperature. After the solvent was evaporated, the mixture was separated by preparative HPLC (eluent ratio: water:acetonitrile = 95:5, gradient increased to water:acetonitrile = 5:95). The eluent with a high absorption peak at 798 nm was collected, the solvent was evaporated, and the solid was dried to obtain a black solid. The reaction yield was 22.7%.

[0069] 1H NMR (400MHz, CDCl3) δ (ppm) 8.52-8.29 (m, 2H), 7.94-7.78 (m, 5H), 7.77-7.67 (m, 2H), 7.63 (dd, J= 8.5,7.2Hz,1H),7.58-7.46(m,1H),7.39(ddd,J=18.6,12.6,8.0Hz,4H),7.29(s,1H),7.25-7.15( m,4H),7.11(d,J=7.2Hz,1H),6.82(dd,J=7.8,1.5Hz,2H),6.69(ddd,J=7.9,6.6,1.5Hz,2H),4.7 3(d,J=7.1Hz,2H), 4.61(q,J=7.1Hz,2H), 4.29(q,J=7.7,7.0Hz,2H), 1.64(td,J=7.1,4.6Hz,9H).

[0070] 13 C NMR(101MHz,CDCl3)δ(ppm)159.22,147.15,143.72,139.64,138.83,138.73,13 6.74,135.39,131.50,131.06,130.15,129.94,129.15,128.93,128.19,127.52 ,126.76,125.29,125.15,124.89,124.36,123.31,122.95,122.81,121.16,120.18,119.37,116.96,116.18,113.20,42.43,37.20,32.06,23.37,15.53,14.25.

[0071] HRMS(ESI)m / z calcd.[M+H] + The measured molecular weight was 658.3217; the measured molecular weight was 658.3191.

[0072] The normalized absorption / fluorescence spectrum of Et-PinCy in methanol (808 nm photoexcitation) is shown below. Figure 4 .

[0073] Example 3: Preparation of the spirocyclic acridine cyanine dye Hexyl-PinCy

[0074]

[0075] 5.0 mg of 9-methyl-10-hexylacridinium tetrafluoroborate was added to a 15 mL sealed pressure-resistant tube, followed by 2 mL of acetonitrile and 13.7 μL of trimethylamine (2.0 M THF solution). The mixture was reacted at 145 °C in the dark for 12 h, then cooled to room temperature. After the solvent was evaporated, the mixture was separated by preparative HPLC (eluent ratio: water:acetonitrile = 95:5, gradient increased to water:acetonitrile = 5:95). The eluent with a high absorption peak at 798 nm was collected, the solvent was evaporated, and the solid was dried to obtain a black solid. The reaction yield was 16.8%.

[0076] 1 H NMR (400MHz, CDCl3) δ (ppm) 8.29 (d, J = 8.1Hz, 2H), 8.00-7.82 (m, 5H), 7.77 (d, J = 9.0Hz, 2H), 7.7 1(d,J=8.4Hz,1H),7.65-7.55(m,1H),7.53-7.36(m,4H),7.36-7.28(m,1H),7.24-7.11(m,4H), 7.08(d,J=7.2Hz,1H),6.80(d,J=6.3Hz,1H),6.68(t,J=7.4Hz,1H),4.96-4.31(m,4H),4.12(d, J=9.0Hz,2H),2.11-1.92(m,6H),1.71-1.56(m,6H),1.48-1.34(m,12H),0.93(t,J=7.2Hz,9H).

[0077] 13 C NMR (101MHz, CDCl3) δ (ppm) 160.57, 153.82, 150.84, 143.98, 143.24, 140.25, 138.91, 137. 15,135.84,135.27,134.31,129.80,128.06,124.88,124.39,124.28,124.21,123.05,123. 00,122.75,121.01,116.25,115.56,113.76,111.55,49.13,48.97,47.01,31.72,31.55,31.50,27.80,27.54,27.49,26.76,26.61,26.08,22.79,22.61,22.58,15.06,14.09,14.01.

[0078] HRMS(ESI)m / z calcd.[M+H] + The measured molecular weight was 826.5095; the measured molecular weight was 826.5049.

[0079] The normalized absorption / fluorescence spectrum of Hexyl-PinCy in methanol (808 nm photoexcitation) is shown below. Figure 7 .

[0080] Example 4: Preparation of the spirocyclic acridine cyanine dye Ph-PinCy

[0081]

[0082] 5.7 mg of 9-methyl-10-phenylacridinium iodide was added to a 15 mL sealed pressure-resistant tube, followed by 2 mL of acetonitrile and 14.35 μL of trimethylamine (2.0 M THF solution). The mixture was reacted at 145 °C in the dark for 12 h, then cooled to room temperature. After the solvent was evaporated, the mixture was separated by preparative HPLC (eluent ratio: water:acetonitrile = 95:5, gradient increased to water:acetonitrile = 5:95). The eluent with a high absorption peak at 798 nm was collected, the solvent was evaporated, and the solid was dried to obtain a black solid. The reaction yield was 51.2%.

[0083] 1 H NMR (400MHz, CDCl3) δ (ppm) 8.69 (d, J = 13.9Hz, 1H), 8.44 (d, J = 8.4Hz, 1H), 7.97 (d, J = 7.1Hz, 2H),7.88-7.68(m,12H),7.62(t,J=7.7Hz,2H),7.52(d,J=1.5Hz,1H),7.50(d,J=1.6Hz,2H), 7.49-7.46(m,2H),7.46-7.38(m,3H),7.38-7.33(m,2H),7.12-7.00(m,5H),6.97(dd,J=7.8 ,1.6Hz,2H),6.74(td,J=7.5,1.2Hz,2H),6.57(d,J=8.4Hz,1H),6.50(dd,J=8.4,1.2Hz,2H).

[0084] 13C NMR (101MHz, CDCl3) δ (ppm) 160.18,158.93,154.55,149.66,144.08,143.75,143.65,14 1.05,140.74,140.10,138.02,137.81,137.52,135.31,135.17,133.77,131.67,131.46 ,131.03,130.38,129.31,128.97,128.73,128.31,127.95,127.76,127.43,126.37,125.09,124.56,123.33,122.52,121.59,119.34,118.85,118.62,118.31,115.44,112.72.

[0085] HRMS(ESI)m / z calcd.[M+H] + :802.3217; The measured molecular weight is 802.3176.

[0086] The normalized absorption / fluorescence spectrum of Ph-PinCy in methanol (808 nm photoexcitation) is shown below. Figure 10 .

[0087] Example 5: Preparation of spirocyclic acridine cyanine dye Br-PinCy

[0088]

[0089] 5.4 mg of 2-bromo-9,10-dimethylacridonium trifluoromethanesulfonate was added to a 15 mL sealed pressure-resistant tube, followed by 2 mL of acetonitrile and 12.4 μL of trimethylamine (2.0 M THF solution). The mixture was reacted at 145 °C in the dark for 12 h, then cooled to room temperature. After the solvent was evaporated, the mixture was separated by preparative HPLC (eluent ratio: water:acetonitrile = 95:5, gradient increased to water:acetonitrile = 5:95). The eluent with a high absorption peak at 798 nm was collected, the solvent was evaporated, and the solid was dried to obtain a black solid. The reaction yield was 42.4%.

[0090] 1H NMR (400MHz, CDCl3) δ (ppm) 8.58 (d, J = 2.2Hz, 1H), 8.47 (d, J = 13.9Hz, 1H), 7.99 (t, J = 9.6Hz, 4H), 7.84 (t, J = 8.5Hz,3H),7.69(dd,J=17.8,8.8Hz,2H),7.61(t,J=7.6Hz,1H),7.52-7.44(m,2H),7.37(dd,J=8.9,2.3Hz,1 H),7.31(td,J=7.7,6.9,1.6Hz,1H),7.19(d,J=7.2Hz,1H),7.11(d,J=8.9Hz,1H),6.99(d,J=13.9Hz,1H),6 .88(d,J=2.3Hz,1H),6.82(dd,J=7.8,1.6Hz,1H),6.79-6.72(m,1H),4.17(s,3H),4.12(s,3H),3.74(s,3H).

[0091] 13 C NMR(101MHz,CDCl3)δ(ppm)157.37,150.96,149.66,142.64,142.02,141.28,141.16,139.93,1 39.75,139.13,137.79,137.60,137.41,136.72,135.62,131.94,130.02,129.88,129.38,128. 88,128.10,127.89,127.46,127.04,125.45,125.34,124.96,123.03,121.94,121.29,120.15,119.31,118.45,118.37,118.25,116.45,115.59,114.06,113.51,112.39,36.97,35.03,34.27.

[0092] HRMS(ESI)m / z calcd.[M+H] + :850.0063; the measured molecular weight is 850.0042.

[0093] The normalized absorption / fluorescence spectrum of Br-PinCy in methanol (808 nm photoexcitation) is shown below. Figure 13 .

[0094] Example 6: Preparation of conventional linear acridine cyanine dye ADCy3

[0095]

[0096] (1) 100.0 mg of 9,10-dimethylacridinium iodide, 10 mL of acetic anhydride, and 148.9 μL of triethyl orthoformate were added sequentially to a 100 mL round-bottom flask. After degassing with Ar for 30 min, the mixture was reacted at 80 °C in the dark for 3 h. After the reaction was completed, the mixture was cooled to room temperature to obtain a green solution. Anhydrous diethyl ether was added to precipitate the solid, and the precipitate was separated by rapid column chromatography. The solvent was evaporated and the solid was dried to obtain a green solid. The reaction yield was 68.3%.

[0097] 1 H NMR (400MHz, CDCl3) δ (ppm) 8.22 (d, J = 8.2Hz, 4H), 7.87 (q, J = 9.2, 8.1Hz, 9H), 7.60 (d, J = 13.1Hz, 2H), 7.45 (ddd, J = 8.1, 5.9, 2.0Hz, 4H), 4.26 (s, 6H).

[0098] 13 C NMR (101MHz, CDCl3) δ (ppm) 133.36, 127.12, 124.29, 122.11, 116.48, 29.69.

[0099] HRMS(ESI)m / z calcd.[M+H] + :425.2012; The measured molecular weight is 425.1984.

[0100] The normalized absorption / fluorescence spectrum of ADCy3 in methanol (808 nm photoexcitation) is shown below. Figure 16 .

[0101] Example 7: Evaluation of the photophysical properties of spirocyclic acridine cyanine dyes

[0102] 1. Experimental Methods

[0103] (1) The absorption spectra of five spirocyclic acridine cyanine dyes and conventional linear acridine cyanine dyes in methanol were tested using a UV-Vis spectrometer (Shimadzu UV-2600), and their corresponding molar extinction coefficients were calculated. At the same time, their fluorescence emission spectra in methanol (808 nm photoexcitation) were tested using a continuous laser-excited steady-state transient fluorescence spectrometer (Edinburgh FLS980) to determine the emission wavelength of the maximum absorption of the above cyanine dyes in methanol.

[0104] (2) A series of cyanine dye solutions with absorbance values ​​between 0.02 and 0.10 at 808 nm were prepared using dichloromethane as a solvent. Their fluorescence spectra in the range of 815–1200 nm were measured under 808 nm light excitation, with IR-26 as a reference (Φ...). F=0.5%, 1,2-dichloroethane), plot the integrated area of ​​the fluorescence spectrum in the range of 815-1200 nm against the absorbance value, calculate the fitting equation for each curve, and calculate the near-infrared fluorescence quantum yield of the above five spirocyclic acridinecyanine dyes and conventional linear acridinecyanine dyes using the following formula:

[0105]

[0106] Where sample is the sample to be tested, slope is the slope of the fitted equation after plotting the integral area of ​​the sample against the absorbance value, and n is the refractive index of the corresponding solvent.

[0107] 2. Experimental Results

[0108] Table 1 shows the photophysical properties of the five spirocyclic acridine cyanine dyes mentioned above.

[0109] Table 1 Summary of photophysical properties of PinCy and ADCy3

[0110]

[0111] Note: a This indicates that the test solvent is methanol;

[0112] b This indicates that the test solvent is dichloromethane, with IR-26 as the reference (Φ F =0.5%, 1,2-dichloroethane;

[0113] Brightness is the product of molar extinction coefficient and fluorescence quantum yield.

[0114] Table 1 shows that spirocyclic acridinecyanine dyes have high molar extinction coefficients, and changes in N-substituents lead to a significant increase in near-infrared fluorescence quantum yield. Compared to conventional linear acridinecyanine dyes, spirocyclic acridinecyanine dyes exhibit higher brightness and can be used for near-infrared I and II region fluorescence imaging in cancer cells.

[0115] Example 8: Spirocyclic acridine cyanine dyes for in vitro photoactivated generation of reactive oxygen species (ROS)

[0116] 1. Experimental Methods

[0117] Prepare PBS solutions containing spirocyclic acridinecyanine dyes Me-PinCy (2 μM), Ph-PinCy (2 μM), Br-PinCy (2 μM), conventional linear acridinecyanine dyes ADCy3 (2 μM), and DCFH (40 μM), respectively, using 300 mW / cm² PBS. 2Immediately after irradiating the solution with 808 nm light, the changes in fluorescence spectrum under 488 nm excitation were recorded using a fluorescence spectrophotometer (Hitachi F-7000). The total irradiation time was 300 s, with measurements taken at intervals of 10 s, 20 s, or 30 s. The changes in fluorescence intensity at 525 nm were recorded from the initial time, and the average value was taken after repeating the process three times.

[0118] 2. Experimental Results

[0119] from Figure 17 It can be seen that spirocyclic acridine dyes have a superior photoactivated ROS generation capability compared to conventional linear acridine dyes, therefore spirocyclic acridine dyes are expected to be used in near-infrared photodynamic therapy.

[0120] Example 9: Spirocyclic acridine cyanine dyes for near-infrared fluorescence imaging of cancer cells

[0121] 1. Experimental Methods

[0122] Human cervical cancer HeLa cells were incubated with spirocyclic acridine cyanine dyes Me-PinCy (1 μM), Br-PinCy (1 μM), and conventional linear acridine cyanine dye ADCy3 (1 μM) for 2 h at 37 °C. After incubation, the cells were washed with PBS, and complete culture medium was added. Cell imaging experiments were performed using a laser confocal multidimensional fluorescence imaging system (ISS Inc., Italy), with an excitation wavelength of 780 nm and a receiving wavelength of 820–1050 nm.

[0123] 2. Experimental Results

[0124] from Figure 18 It can be seen that spirocyclic acridinecyanine dyes Me-PinCy and Br-PinCy, along with conventional linear acridinecyanine dyes, can all be used for near-infrared fluorescence imaging of cancer cells, with Me-PinCy showing better results.

[0125] Example 10: Spirocyclic acridine cyanine dyes for spatially controlled near-infrared fluorescence imaging in cancer cells

[0126] 1. Experimental Methods

[0127] Human cervical cancer HeLa cells were incubated with spirocyclic acridinecyanine dyes Me-PinCy (0.1 μM), Br-PinCy (0.5 μM), and conventional linear acridinecyanine dye ADCy3 (0.5 μM) at 37 °C for 1.5 h. After incubation, the cells were washed with PBS, and then incubated with a mitochondrial green fluorescent probe (0.1 μM) for 30 min at 37 °C. After incubation, the cells were washed with PBS, and culture medium was added. Cell imaging experiments were performed using a laser confocal multidimensional fluorescence imaging system (ISS Inc., Italy). The excitation wavelength of the dyes was 780 nm, and the receiving wavelength was 820–1050 nm. The excitation wavelength of the mitochondrial green fluorescent probe was 488 nm, and the receiving wavelength was 502–547 nm.

[0128] 2. Experimental Results

[0129] from Figure 19 It can be seen that the near-infrared fluorescence produced by spirocyclic acridinecyanine dyes Me-PinCy and Br-PinCy and conventional linear acridinecyanine dyes is mainly localized in mitochondria of cells, with corresponding Pearson correlation coefficients of 0.93, 0.88 and 0.83, respectively.

[0130] Example 11: Spirocyclic acridine cyanine dyes used in in vitro cell anticancer experiments

[0131] 1. Experimental Methods

[0132] Human cervical cancer HeLa cells were incubated with different concentrations of spirocyclic acridinecyanine dyes Me-PinCy and Br-PinCy, and the conventional linear acridinecyanine dye ADCy3 for 24 h at 37 °C. After incubation, 100 μL of MTT complete medium containing 0.1 mg / mL was added, and incubation continued for 4 h. The medium was then removed, and 100 μL of LDMSO was added. Cell viability was measured using a microplate reader at 565 nm and 720 nm absorbance to calculate cell viability.

[0133] 2. Experimental Results

[0134] from Figure 20 , Figure 22 and Figure 24 It can be seen that the spirocyclic acridinecyanine dyes Me-PinCy and Br-PinCy, as well as the conventional linear acridinecyanine dye ADCy3, all exhibited significant cytotoxicity, IC50... 50 The concentrations were 643 nM, 4.15 μM, and 3.29 μM, respectively (i.e., 0.48 μg / mL, 4.16 μg / mL, and 1.82 μg / mL). The experimental results indicate that spirocyclic acridine cyanine dyes can be used for anticancer therapy.

[0135] Example 12: Spirocyclic acridine cyanine dyes for synergistic photodynamic therapy

[0136] 1. Experimental Methods

[0137] Different concentrations of spirocyclic acridinecyanine dyes Me-PinCy and Br-PinCy, and the conventional linear acridinecyanine dye ADCy3 were incubated with human cervical cancer HeLa cells. After 2 hours of incubation, 300 mW / cm² of [a specific treatment / treatment method] was applied. 2 After irradiation with 808nm light for 5 min, the cells were incubated for 24 h at 37℃. After incubation, 100 μL of MTT complete medium containing 0.1 mg / mL was added, and the cells were incubated for another 4 h. The medium was then removed, and 100 μL of LDMSO was added. Cell viability was measured using a microplate reader at 565 nm and 720 nm absorbance to calculate the viability.

[0138] 2. Experimental Results

[0139] from Figure 21 , Figure 23 and Figure 25 It can be seen that the conventional linear acridine cyanine dye ADCy3 did not exhibit significant phototoxicity, while the spirocyclic acridine cyanine dyes Me-PinCy and Br-PinCy showed significantly enhanced phototoxicity, IC 50 The concentrations were 57.5 nM and 196 nM (i.e., 42.8 ng / mL and 197 ng / mL), respectively, corresponding to phototoxicity indices (PI) of 11.2 and 21.1. The experimental results indicate that spirocyclic acridine cyanine dyes can be used to synergistically enhance the antitumor therapeutic effect through photodynamic therapy.

Claims

1. A spirocyclic acridinecyanine compound, characterized in that, It has the structure of Formula I: I， R1 is selected from methyl, ethyl, hexyl, and phenyl. R2 and R3 are selected from hydrogen and bromine, and satisfy any one of the following conditions: When R2 and R3 are the same, they are both hydrogen. When R2 and R3 are not the same, either one is hydrogen; Y - Selected from iodide ions, tetrafluoroborate anions, and trifluoromethanesulfonate anions.

2. The spirocyclic acridinecyanine compound according to claim 1, characterized in that, In the structure: When R2 and R3 are different, R3 is hydrogen.

3. The spirocyclic acridinecyanine compound according to claim 2, characterized in that, In the structure: R2 and R3 can be located in any of the following positions within the acridine ring system: 。 4. The spirocyclic acridinecyanine compound according to claim 1, characterized in that, Compounds selected from any of the following: 。 5. A method for preparing the spirocyclic acridine cyanine compound according to claim 1, characterized in that, The compound was obtained by a closed-loop reaction of three molecules of 9,10-disubstituted acridine onium salt in an organic base and solvent: ， The solvent is acetonitrile, the organic base is trimethylamine, and the reaction conditions are 145℃ in the dark; R1, R2, R3, Y - The definition is as described in claim 1.

6. A photosensitizer, characterized in that, It is prepared from the spirocyclic acridine cyanine compound of claim 1.

7. The use of a spirocyclic acridine cyanine compound as described in claim 1 or a photosensitizer as described in claim 6 in the preparation of photodynamic therapy drugs and tumor cell imaging agents.

8. The application according to claim 7, characterized in that, The drug in question is an anti-tumor drug.

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