Amino acid modified bodipy compounds, methods of making and uses thereof

Abstract

The present application belongs to the technical field of drug synthesis, and relates to an amino acid modified BODIPY compound, in particular to an amino acid residue or hydroxyl group substituted BODIPY compound of the following formula, a preparation method thereof and application in pharmacy. The compound of the present application is shown by experiments to have a significant inhibitory effect on tumor growth through photodynamic action. The compound has good anti-tumor activity, and can be further prepared into a photodynamic therapy drug and an anti-malignant tumor drug, and is used for treating skin cancer, prostate cancer, oral squamous cell carcinoma, cervical cancer, lung cancer, liver cancer, gastric cancer, breast cancer, colon cancer, bladder cancer and esophageal cancer and the like.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical synthesis and relates to novel amino acid-modified BODIPY compounds, their preparation methods, and applications. Specifically, it relates to a BODIPY compound containing amino acid residues or hydroxyl substitutions, its preparation method, and its application in pharmaceutical manufacturing. Background Technology

[0002] Malignant tumors have become a common disease that seriously endangers people's lives and health. According to incomplete statistics, there were approximately 19.3 million new cancer cases and 10 million cancer deaths worldwide in 2020; in my country, there were approximately 4.57 million new cases and 3 million deaths. Clinical practice shows that due to the metastatic ability of tumors in their early stages, about 50% of patients with clinically diagnosed primary tumors have already developed distant metastases. Tumor cells grow rapidly, are prone to mutation, and easily develop multidrug resistance, leading to chemotherapy failure. According to relevant statistics, more than 90% of these failures are related to multidrug resistance in tumor cells. Immunotherapy has achieved great success in recent years, but the response rate to immunotherapy in most patients is less than 30%, thus severely limiting its application. Therefore, currently available anti-tumor drugs cannot meet the treatment requirements, and there is an urgent need to develop new tumor treatment methods.

[0003] Photodynamic therapy (PDT), approved by the FDA for clinical use in 1996, is a novel cancer treatment method. Compared with traditional cancer treatments, PDT has the following significant advantages: it can selectively kill tumor cells; PDT can be used alone or in combination with chemotherapy, surgery, radiotherapy, or immunotherapy for synergistic cancer treatment; it has significant therapeutic effects with minimal invasiveness; and it does not induce drug resistance with repeated treatments. Currently, several photosensitizers for PDT have been marketed or entered clinical trials and are being used to treat skin cancer, prostate cancer, oral squamous cell carcinoma, acne, cervical cancer, lung cancer, liver cancer, gastric cancer, and esophageal cancer, with particularly significant efficacy in the treatment of skin cancer.

[0004] Photofrin, a typical representative of first-generation photosensitizers, is clinically approved for the treatment of malignant tumors. However, its clinical application suffers from serious drawbacks, including long-term skin phototoxicity, slow metabolism, poor tumor selectivity, chemical heterogeneity, low molar absorptivity, and poor bioavailability. Second-generation photosensitizers are mainly tetracyclic pyrrole-type photosensitizers (dihydroporphyrin, chlorophyll, and phthalocyanine), such as talaporfin, verteporfin, photochlor, and Pc4, which have been commercialized. Fluoroborodipyrrolemethyl fluoride (BODIPY), as a novel non-porphyrin photosensitizer, has become a research hotspot due to its excellent photostability, high molar absorptivity, rapid metabolism, high phototoxicity, very low dark toxicity, and easily modifiable chemical structure. ADPM06 is a typical representative of BODIPY photosensitizers, demonstrating potent antitumor effects, particularly achieving complete cure in animal models with no recurrence within 8 weeks of treatment. However, BODIPY photosensitizers and most tetrapyrrole-type photosensitizers have very poor water solubility and usually require the assistance of emulsifiers to achieve effective antitumor effects, thus limiting their clinical application. Although emulsifiers have been successfully used in approved drug formulations (such as Cremophor EL emulsified paclitaxel), studies have shown that their use may lead to severe allergic reactions. Therefore, extensive research has been conducted on novel water-soluble photosensitizers, and various analogs have been derivatized to increase water solubility, such as by introducing glycosyl groups, ethylene glycol chains, quaternary ammonium salts, peptides, and acidic groups. Among these, talaporfin and padeliporfin, which contain fatty acid moieties, are successful examples of clinically approved photosensitizers.

[0005]

[0006] Given that most of the currently reported BODIPY photosensitizers have poor water solubility, the inventors of this application intend to provide novel amino acid-modified BODIPY compounds, especially a class of BODIPY photosensitizers with amino acid residues or hydroxyl groups substituted. These compounds have advantages over existing compounds, such as significantly increased water solubility, enhanced photostability, increased singlet oxygen yield, increased phototoxicity, and good in vivo antitumor activity, and can be further developed into novel photosensitizers. Summary of the Invention

[0007] The purpose of this invention is to provide novel amino acid-modified BODIPY-type compounds, their preparation methods, and uses, based on the current state of the technology, particularly a class of BODIPY-type photosensitizers with amino acid residues or hydroxyl groups substituted. The amino acid residues or hydroxyl groups substituted BODIPY-type photosensitizers of this application have advantages such as significantly increased water solubility, enhanced photostability, improved singlet oxygen yield, increased phototoxicity, and good in vivo antitumor activity.

[0008] Specifically, this invention provides novel BODIPY-type photosensitizers with good antitumor activity, relating to BODIPY-type compounds with amino acid residues or hydroxyl substitutions of general formula (I) or (II):

[0009]

[0010] in:

[0011] R1 and R2 are independently selected from phenyl, C 1-6 Alkyl-substituted phenyl, C 1-6 Alkoxy-substituted phenyl, halogen-substituted phenyl;

[0012] R3 is selected from hydroxyl group, -NR4;

[0013] R4 is selected from amino acid residues.

[0014] The dashed line indicates a CC key or that the key does not exist.

[0015] Preferably, the dashed line represents a CC bond.

[0016] Preferably, R1 and R2 are selected from phenyl groups.

[0017] Preferably, R4 is selected from aspartic acid residues, glutamic acid residues, arginine residues, serine residues, threonine residues, asparagine residues, and glycine residues; more preferably, R4 is selected from aspartic acid residues and glutamic acid residues.

[0018] In this invention, the compounds have the structures of compounds 1, 2, 3, 4, 5, 6, and 7:

[0019]

[0020]

[0021]

[0022] Another object of the present invention is to provide a method for preparing the above-mentioned BODIPY compounds, particularly a method for preparing BODIPY compounds with amino acid residues or hydroxyl groups substituted.

[0023] Taking compound 1 as an example, the preparation process of the compound of the present invention is as follows:

[0024]

[0025] Taking compound 2 as an example, the preparation process of the compound of the present invention is as follows:

[0026]

[0027] Furthermore, the amino acid residues or hydroxyl-substituted BODIPY compounds of the present invention can be used to prepare photosensitizers for photodynamic antitumor activity. The tumors include skin cancer, prostate cancer, oral squamous cell carcinoma, cervical cancer, liver cancer, gastric cancer, breast cancer, lung cancer, colon cancer, bladder cancer, and esophageal cancer.

[0028] The compounds of the present invention were subjected to tumor inhibitory activity tests, and the results showed good tumor inhibitory activity. Compound 2 showed cell inhibitory activity of less than 0.1 μM against three tumor strains.

[0029] The pharmacodynamic testing methods used in this invention are methods well known to those skilled in the art.

[0030] In this invention, the B16-F10 cells, A375 cells, and HeLa cells used are commercially available to those skilled in the art.

[0031] This invention provides novel amino acid-modified BODIPY-type compounds, particularly a class of BODIPY-type photosensitizers with amino acid residues or hydroxyl groups substituted. The amino acid residues or hydroxyl groups substituted with these BODIPY-type photosensitizers exhibit advantages such as significantly increased water solubility, enhanced photostability, increased singlet oxygen yield, improved phototoxicity, and good in vivo antitumor activity. These amino acid residues or hydroxyl groups substituted with these BODIPY-type photosensitizers can treat malignant tumors including skin cancer, prostate cancer, oral squamous cell carcinoma, cervical cancer, liver cancer, gastric cancer, breast cancer, lung cancer, colon cancer, bladder cancer, and esophageal cancer. Attached Figure Description

[0032] Figure 1 Organelle localization experiment of compound 2 in B16-F10, where (A1,A2,A3) cell morphology in bright field; (B1) image after incubation with mitochondrial green fluorescent probe (250 nM); (B2) image after incubation with lysosomal green fluorescent probe (500 nM); (B3) image after incubation with endoplasmic reticulum green fluorescent probe (2 μM); (C1,C2,C3) red fluorescence image of compound 2; (D1,D2,D3) superimposed image of green fluorescence of the corresponding probe and red fluorescence of compound 2, scale bar: 10 μm.

[0033] Figure 2 Intracellular reactive oxygen species (ROS) generated by compound 2 were detected in B16-F10 cells (A) or A375 cells (B). Cells (A1, B1) were incubated in the dark with 100 nM of compound 2; cells were incubated with 10 nM (A2, B2), 20 nM (A3, B3), and 100 nM (A4, B4) of compound 2, respectively, followed by a light dose of 54 J / cm². 2The percentage of ROS-positive cells (A5, B5). *p<0.05,**p<0.01,***p<0.001.

[0034] Figure 3 Compound 2 induced apoptosis in B16-F10 cells. (A) was the control group; (B, C, D) cells were incubated with 10 nM (B), 50 nM (C), and 250 nM (D) of compound 2, respectively, followed by a light dose of 54 J / cm². 2 (E) The proportion of apoptotic and necrotic cells, **p<0.01,****p<0.0001.

[0035] Figure 4 The tumor-suppressive effect of compound 2 was evaluated in the B16-F10 xenograft model, where (A) images of ex vivo tumors (dashed lines represent cured tumors); (B) tumor volume growth curves; (C) ex vivo tumor weight; (D) mouse body weight change curves; (E) H&E staining of tumor tissues from different groups, **p<0.01, ***p<0.001, and ****p<0.0001. Detailed Implementation

[0036] Example 1: Synthesis of Compound 1

[0037] (1) Synthesis of 3-phenyl-2H-azacyclic propylene

[0038] The starting material (1,2-dibromoethyl)benzene (5.2 g, 19.9 mmol) was dissolved in DMF (30 mL), and sodium azide (3.9 g, 60 mmol) was slowly added to the solution. The reaction was carried out at room temperature for 10 h. The mixture was then extracted three times with anhydrous diethyl ether and saturated sodium carbonate solution, and the organic phases were combined and concentrated to obtain (1-azidovinyl)benzene.

[0039] Triethylenediamine (DABCO, 1.5 g, 13.4 mmol) was added to toluene (70 mL), and the mixture was heated to 110 °C. (1-Azidevinyl)benzene, dissolved in toluene, was added dropwise to the solution. The mixture was refluxed for 2 h, and toluene was removed by rotary evaporation to obtain a yellow, oily crude product, 3-phenyl-2H-azacyclic propene. The product was used directly in the next reaction without further separation.

[0040] (2) Synthesis of 2-(4-methoxyphenyl)-4-phenyl-1H-pyrrole

[0041] p-Methoxyacetophenone (1 g, 6.7 mmol) was dissolved in dry THF (30 mL), and under nitrogen protection, cooled to -78 °C. LDA (13.4 mmol) was slowly added by injection, and the reaction was allowed to proceed for 30 min. 3-Phenyl-2H-azacyclopropene (784 mg, 6.7 mmol) was dissolved in tetrahydrofuran and slowly added to the reaction system. The reaction was allowed to proceed for 2 h, followed by 1 h at room temperature. After the reaction was complete, the reaction was quenched by adding ammonium chloride aqueous solution. The mixture was extracted with dichloromethane and water, and the combined organic phases were washed with saturated saline solution, dried over anhydrous sodium sulfate, and subjected to column chromatography with dichloromethane:petroleum ether = 1:10 to give the intermediate 2-(4-methoxyphenyl)-4-phenyl-1H-pyrrole, a white crystalline solid, 939 mg, yield 56.3%. 1 H NMR (400MHz, CDCl3) δ8.27 (s, 1H), 7.45 (d, J = 7.1Hz, 2H), 7.33 (s, 2H), 7.2 4(s,2H),7.08(s,1H),7.00(s,1H),6.83(s,2H),6.59(s,1H),3.72(s,3H).

[0042] (3) Synthesis of boron difluoride chelate (Z)-2-((5-(4-methoxyphenyl)-3-phenyl-2H-pyrrole-2-phenylene)amino)-3-phenyl-4,5-dihydro-1H-benzo[g]indole-7-ol

[0043] The starting material 7-((tert-butyldimethylsilyl)oxy)-3-phenyl-4,5-dihydro-1H-benzo[g]indole (37.5 mg, 0.1 mmol) was added to glacial acetic acid (1 mL) and stirred. The temperature was lowered to 5 °C, and then sodium nitrite (6.9 mg, 0.1 mmol) was added, followed by stirring for 15 min. The color of the solution changed from colorless to brown, then to green, and finally back to brown. Then, the starting material 2-(4-methoxyphenyl)-4-phenyl-1H-pyrrole (24.9 mg, 0.1 mmol) was added, followed by acetic anhydride (0.4 mL). The reaction solution immediately turned green. The mixture was stirred at room temperature for 30 min, then heated to 80 °C and stirred for 30 min. TLC showed that the reaction was complete. After the reaction solution cooled to room temperature, ice water was added to quench the reaction. The precipitated blue dye was filtered, the filter cake was washed with water, and dried. The filter cake was subjected to neutral alumina column chromatography with dichloromethane as the eluent. The solvent was evaporated by rotary evaporation, and the product was obtained by pumping dry with an oil pump.

[0044] The above product was dissolved in dry 1,2-dichloroethane (10 mL), triethylamine (0.24 mL) was added, followed by dropwise addition of boron trifluoride diethyl ether (0.24 mL). The mixture was stirred at room temperature for 30 min, then heated to 80 °C and stirred for another 30 min. The reaction solution was cooled to room temperature, and the reaction was quenched by adding ice water. The mixture was extracted three times with dichloromethane, and the organic phases were combined, dried over anhydrous sodium sulfate, and purified by dichloromethane / petroleum ether (1 / 8) column chromatography to obtain the crude product.

[0045] The product obtained above (1.0 mmol) was dissolved in a CH2Cl2 / MeOH / H2O (20 mL / 8 mL / 1 mL) system, and K2CO3 (6.0 mmol, 829.2 mg) was added. The mixture was stirred at room temperature for 16 h, and the organic layer was removed by rotary evaporation. The pH of the aqueous phase was adjusted to 5 by adding 0.5 N HCl, and the compound precipitated out. The product was then recrystallized from dichloromethane / n-hexane (1 / 1) to obtain a copper-colored solid with a yield of 45.1%. 1 HNMR (600MHz, DMSO) δ10.91(s,1H),8.48(d,J=8.9Hz,1H),8.13(d,J=7.3Hz,2H),8.09(d,J=8.9Hz,2H),7.78(d,J=7.2Hz,2H),7.58(t,J=7.6 Hz,2H),7.51(t,J=7.4Hz,1H),7.44(t,J=7.5Hz,2H),7.40–7.33(m,2H),7.13(d,J=8.9Hz,2H),6.93–6.86(m,2H),3.88(s,3H),2.94(s,4H). 13 C NMR(151MHz,DMSO-d6)δ162.45,160.72,155.52,152.27,146.55,145.88,142.72,138.10,137.53,133.36,132.30,131.84,13 0.92,130.73,130.03,128.72,128.48,128.35,128.16,124.08,117.18,117.09,115.96,115.23,114.09,55.30,29.26,21.04.

[0046] (4) Synthesis of boron difluoride chelate of tert-butyl(Z)-2-((2-((5-(4-methoxyphenyl)-3-phenyl-2H-pyrrole-2-ylidene)amino)-3-phenyl-4,5-dihydro-1H-benzo[g]indol-7-yl)oxy)acetate

[0047] The starting material, boron difluoride chelated with (Z)-2-((5-(4-methoxyphenyl)-3-phenyl-2H-pyrrole-2-phenylene)amino)-3-phenyl-4,5-dihydro-1H-benzo[g]indole-7-ol (56.9 mmol, 0.1 mmol), was dissolved in DMF (5 mL), and K2CO3 (0.2 mmol, 27.6 mg) was added. Then, tert-butyl bromoacetate (0.3 mmol, 58.5 mg) was added dropwise, and the mixture was stirred at 80 °C for 1 h. The reaction solution was extracted three times with dichloromethane and water, and the organic layer was concentrated. The solution was then subjected to column chromatography in a dichloromethane / petroleum ether system to give the product as a copper-colored solid with a yield of 88.7%. 1 H NMR (600MHz, CDCl3) δ8.72 (d, J = 9.0 Hz, 1H), 8.10 (t, J = 7.9 Hz, 4H), 7.75 (d, J = 7. 5Hz,2H),7.52(t,J=7.5Hz,2H),7.46(t,J=7.3Hz,1H),7.42(t,J=7.4Hz,2H),7.3 9–7.35(m,1H),7.07(d,J=8.7Hz,2H),6.99(s,1H),6.94(dd,J=8.9,2.4Hz,1H), 6.88(d,J=2.0Hz,1H),4.62(s,2H),3.93(s,3H),3.03–2.89(m,4H),1.53(s,9H). 13 CNMR(151MHz,CDCl3)δ167.42,161.39,160.82,155.34,154.80,146.54, 144.90,144.71,140.82,138.48,132.92,132.29,132.05,131.99,131.2 8,130.50,129.01,128.74,128.60,128.51,128.23,124.91,120.89,117 .36,115.37,114.21,113.48,82.89,65.61,55.46,30.55,28.14,21.82.

[0048] (5) Synthesis of boron difluoride chelate (Z)-2-((2-((4-iodo-5-(4-methoxyphenyl)-3-phenyl-2H-pyrrole-2-ylidene)amino)-3-phenyl-4,5-dihydro-1H-benzo[g]indol-7-yl)oxy)acetic acid (compound 1)

[0049] Boron difluoride chelated tert-butyl(Z)-2-((2-((5-(4-methoxyphenyl)-3-phenyl-2H-pyrrole-2-ylidene)amino)-3-phenyl-4,5-dihydro-1H-benzo[g]indol-7-yl)oxy)acetate (68.3 mg, 0.1 mmol) was dissolved in a DCM / TFA (5 mL / 1 mL) system and stirred at room temperature for 5 h. The organic solvent was removed from the reaction mixture, and the pH was adjusted to 5 with potassium carbonate solution. The mixture was then extracted three times with dichloromethane and water. The combined organic layers were subjected to column chromatography in a DCM / MeOH (50 / 1) system to obtain an intermediate. The obtained intermediate (0.05 mmol) was dissolved in dichloromethane (3 mL), acetic acid (1 mL) was added, followed by NIS (0.055 mmol, 12.4 mg), and the mixture was stirred at room temperature for 40 min. The reaction solution was extracted three times with dichloromethane and sodium sulfite aqueous solution. The concentrated organic layers were combined and subjected to column chromatography using a DCM / MeOH (50 / 1) system to obtain the product. The product was then recrystallized from dichloromethane / n-hexane (1 / 1) to obtain final product 1, a copper-colored solid, with a yield of 61.2%. 1 H NMR (600MHz, DMSO-d6) δ8.39(d,J=9.1Hz,1H),7.78(d,J=7.5Hz,2H),7.75(d,J=7.4Hz,2H),7.57(d,J=8.4Hz,2H ),7.54–7.38(m,6H),7.16–7.01(m,4H),4.82(s,2H),3.88(s,3H),2.98(d,J=5.5Hz,2H),2.94(d,J=5.6Hz,2H). 13 C NMR(151MHz,DMSO-d6)δ169.36,162.85,159.97,158.63,151.50,147.30,141.31,140.14,139.21,135.00,132.45,132.07, 130.33,130.06,129.02,128.12,127.69,124.16,118.06,115.02,114.55,113.10,80.01,64.98,55.08,29.07,21.03.HRMS m / z:754.1188[M+H] + calcd for C 37 H 28 BF2IN3O4 754.1180.

[0050] Example 2: Synthesis of Boron Difluoride Chelating (Z)-(2-((2-((4-iodo-5-(4-methoxyphenyl)-3-phenyl-2H-pyrrole-2-ylidene)amino)-3-phenyl-4,5-dihydro-1H-benzo[g]indol-7-yl)oxy)acetyl)aspartic acid (Compound 2)

[0051] Boron difluoride chelated (Z)-2-((2-((4-iodo-5-(4-methoxyphenyl)-3-phenyl-2H-pyrrole-2-ylidene)amino)-3-phenyl-4,5-dihydro-1H-benzo[g]indol-7-yl)oxy)acetic acid (75.3 mg, 0.1 mmol) was dissolved in a DCM / THF (6 mL / 3 mL) system, and HATU (0.12 mmol, 45.6 mg) was added. The mixture was stirred at room temperature for 30 min. L-Asp(O t Bu₂·HCl (0.13 mmol, 36.6 mg) and DIPEA (0.13 mmol, 16.8 mg) were mixed in 5 mL of dichloromethane and added dropwise to the above reaction solution. The reaction was carried out at room temperature for 3 h. The reaction solution was washed three times with 0.1 N HCl solution, extracted with dichloromethane and water, and the organic layers were combined. The intermediate was obtained by DCM / MeOH (100 / 1) column chromatography. This intermediate was dissolved in DCM / TFA (7 mL / 1 mL) and reacted at room temperature for 9 h. The reaction solution was extracted three times with dichloromethane and water, and the organic layers were combined. The intermediate was obtained by DCM / MeOH (30 / 1) column chromatography to give final product 2, a copper-colored solid, in 55.6% yield. 1 H NMR (600MHz, DMSO-d6) δ8.40(d,J=9.0Hz,1H),7.79(d,J=7.6Hz,2H),7.76(d,J=7.4Hz,2H),7.58(d,J=8.4Hz,2H),7.52(dd,J=14.0,7.0Hz,4H),7. 48–7.40(m,2H),7.14–7.06(m,4H),4.73(s,2H),4.53(s,1H),3.88(s,3H ),2.99(d,J=5.9Hz,2H),2.95(d,J=6.6Hz,2H),2.70(s,1H),2.62(s,1H). 13C NMR(151MHz,DMSO-d6)δ172.23,171.80,166.33,162.31,160.02,158.45,151.89,147.25,141.44,140.47,139.26,134.94,134.38,132.41,13 2.08,130.36,130.07,129.04,128.14,127.70,124.06,118.42,115.18 ,114.72,113.12,80.23,66.65,55.10,48.24,29.11,28.87,21.03.HRMS m / z: 869.1454 [M+H] + calcd for C 41 H 33 BF2IN4O7 869.1450.

[0052] Example 3: Synthesis of Boron Difluoride Chelation of (Z)-(2-((2-((4-iodo-5-(4-methoxyphenyl)-3-phenyl-2H-pyrrole-2-ylidene)amino)-3-phenyl-4,5-dihydro-1H-benzo[g]indol-7-yl)oxy)acetyl)glutamic acid (Compound 3)

[0053] The synthetic method is the same as that for compound 2. Compound 3 is a copper-colored solid with a yield of 51.8%. 1 H NMR(600MHz, DMSO-d6)δ8.40(d,J=7.2Hz,1H),7.81–7.73(m,3H),7.64–7.57(m,3H),7.53–7.48(m,4H),7.47–7.41(m,2H),7.28(s,1H),7. 10(m,3H),4.74(m,2H),4.28(d,J=6.0Hz,1H),3.88(s,3H),3.04–2.88(m,4H),2.29(t,J=7.3Hz,2H),2.05–1.96(m,1H),1.90–1.82(m,1H). 13C NMR(151MHz,DMSO-d6)δ173.62,172.77,166.96,162.48,160.01,158.48,151.82,147.23,1 41.40,140.42,139.26,134.93,134.40,134.13,132.40,132.08,131.89,131.35,131.29,13 0.54,130.35,130.27,130.07,129.05,128.64,128.56,128.26,128.14,127.71,124.08,11 8.32,115.02,114.88,113.12,80.17,66.52,55.10,50.89,29.88,29.15,25.87,21.03.HRMS m / z:883.1608[M+H] + calcd for C 42 H 35 BF2IN4O7 883.1606.

[0054] Example 4: Synthesis of Boron Difluoride Chelating (Z)-2-(4-(4-iodo-2-((7-methoxy-3-phenyl-4,5-dihydro-1H-benzo[g]indol-2-yl)imino)-3-phenyl-2H-pyrrolo-5-yl)phenoxy)acetic acid (compound 4)

[0055] (1) Synthesis of boron difluoride chelate (Z)-4-(2-((7-methoxy-3-phenyl-4,5-dihydro-1H-benzo[g]indol-2-yl)imino)-3-phenyl-2H-pyrrole-5-yl)phenol

[0056] The synthetic method is the same as that used for the chelation of (Z)-2-((5-(4-methoxyphenyl)-3-phenyl-2H-pyrrole-2-phenylene)amino)-3-phenyl-4,5-dihydro-1H-benzo[g]indole-7-ol with boron difluoride. The product is a copper-colored solid with a yield of 40.6%. 1H NMR (600MHz, CDCl3) δ8.71(d,J=9.0Hz,1H),8.07(d,J=7.4Hz,2H),8.02(d,J=8.6Hz,2H),7.73(d,J=7.3Hz,2H),7.50(t,J=7.5Hz,2H),7.44(t ,J=7.4Hz,1H),7.39(t,J=7.4Hz,2H),7.34(t,J=7.2Hz,1H),6.99–6.92(m,4H),6.84(d,J=2.4Hz,1H),5.06(s,1H),3.90(s,3H),2.96(s,4H). 13 CNMR(151MHz, CDCl3)δ162.82,157.24,155.48,154.46,146.69,145.07,144.31,140.26,138.56,132.96,132.49,132.27,132.20,132 .12,131.88,131.37,130.44,128.91,128.58,128.44,128.18,125.32,119.94,116.96,115.62,114.58,113.28,55.53,30.52,21.84.

[0057] (2) Synthesis of boron difluoride chelate of tert-butyl(Z)-2-(4-(2-((7-methoxy-3-phenyl-4,5-dihydro-1H-benzo[g]indol-2-yl)imino)-3-phenyl-2H-pyrrole-5-yl)phenoxy)acetate

[0058] The synthetic method is the same as that used for the synthesis of boron difluoride chelated tert-butyl(Z)-2-((2-((5-(4-methoxyphenyl)-3-phenyl-2H-pyrrole-2-ylidene)amino)-3-phenyl-4,5-dihydro-1H-benzo[g]indol-7-yl)oxy)acetate. The compound is a copper-colored solid in yield of 85.2%. 1H NMR(600MHz, CDCl3) δ8.71(d,J=9.0Hz,1H),8.06(dd,J=8.1,3.7Hz,4H),7.73(d,J =7.2Hz,2H),7.50(t,J=7.5Hz,2H),7.44(t,J=7.4Hz,1H),7.39(t,J=7.4Hz,2H),7. 34(t,J=7.3Hz,1H),7.02(d,J=8.9Hz,2H),6.97(dd,J=9.0,2.6Hz,1H),6.94(s,1H ),6.84(d,J=2.5Hz,1H),4.61(s,2H),3.90(s,3H),3.01–2.92(m,4H),1.52(s,9H). 13 C NMR (151MHz, CDCl3) δ167.74,162.85,159.40,155.59,154.26,146.74,145.11,144.31,140.20,138.59,132.97,132.53,132.25,131.87,13 1.12,130.44,128.90,128.57,128.44,128.18,125.87,119.93,116.9 8,114.69,114.55,113.29,82.63,65.73,55.53,30.52,28.08,21.85.

[0059] (3) Synthesis of boron difluoride chelate (Z)-2-(4-(4-iodo-2-((7-methoxy-3-phenyl-4,5-dihydro-1H-benzo[g]indol-2-yl)imino)-3-phenyl-2H-pyrrole-5-yl)phenoxy)acetic acid

[0060] The synthetic method is the same as that for compound 1. Compound 4 is a copper-colored solid with a yield of 56.2%. 1 H NMR(600MHz,DMSO-d6)δ8.42(d,J=9.1Hz,1H),7.80–7.72(m,4H),7.58–7.48(m,6H),7.44(m, 2H),7.12(s,2H),7.06(d,J=8.5Hz,2H),4.80(s,2H),3.90(s,3H),2.99(s,2H),2.94(s,2H). 13C NMR(151MHz,DMSO-d6)δ170.12,164.36,159.09,158.61,151.30,147.76,147.57,141.35,140.09,139.42,135.31,132.63,132.20,130.52,1 30.42,130.24,129.23,128.31,127.87,124.83,117.96,114.69,114.5 3,113.69,80.07,64.53,55.99,29.28,21.24.HRMSm / z:754.1187[M+H] + calcd for C 37 H 28 BF2IN3O4 754.1180.

[0061] Example 5: Synthesis of compound 5

[0062] The synthetic method is the same as that for compound 2. Compound 5 is a copper-colored solid with a yield of 56.9%. 1 H NMR(600MHz,DMSO-d6)δ8.43(d,J=9.1Hz,1H),7.81–7.74(m,3H),7.65–7.60(m,2H),7.59–7.55(m,2H),7.53–7.49(m,3H),7.47–7.41(m,2H) ,7.29(s,1H),7.13(d,J=7.3Hz,3H),4.66(s,2H),4.42(s,1H),3.91(s ,3H),3.03–2.99(m,2H),2.98–2.93(m,2H),2.65(s,1H),2.58(s,1H). 13 C NMR(151MHz,DMSO-d6)δ174.16,172.09,171.77,167.21,164.27,159.03,158.3 8,151.07,147.66,147.48,141.21,139.91,139.31,135.22,132.51,132.10,13 0.40,130.29,130.12,129.54,129.10,128.19,127.74,124.95,117.82,114.56 ,114.40,113.89,79.78,66.58,55.88,55.65,48.18,29.16,28.91,21.13.HRMS m / z:869.1453[M+H] + ,calcdfor C 41 H33 BF2IN4O7 869.1450.

[0063] Example 6: Synthesis of Compound 6

[0064] The synthetic method is the same as that for compound 3. Compound 6 is a copper-colored solid with a yield of 49.3%. 1 H NMR (600MHz, DMSO-d6) δ8.44(dd,J=13.3,8.8Hz,2H),7.77(dd,J=12.4,7.7Hz,3H),7.65–7.55(m,3H),7.53–7.40(m,6H),7.12(d,J=7.7Hz,3H),4.67( q,J=14.7Hz,2H),4.35(m,1H),3.90(s,3H),3.00(d,J=6.5Hz,2H),2.96(d, J=6.8Hz,2H),2.33(t,J=7.5Hz,2H),2.10–2.04(m,1H),1.94–1.84(m,1H). 13 C NMR(151MHz,DMSO-d6)δ173.69,172.82,167.54,164.20,158.95,158.45,151 .07,147.59,147.41,141.17,139.87,139.24,135.15,134.36,132.45,132.0 1,130.34,130.24,130.06,129.04,128.13,127.69,124.80,117.78,114.51, 114.33,113.80,79.73,66.46,55.82,50.91,29.99,29.11,25.99,21.07.HRMS m / z:883.1611[M+H] + calcd for C 42 H 35 BF2IN4O7 883.1606.

[0065] Example 7: Synthesis of Compound 7

[0066] The synthetic method is the same as that for compound 2. Compound 7 is a copper-colored solid with a yield of 78.6%. HRMS m / z: 811.1523 [M+H] + calcd for C 39 H 31 BF2IN4O5 811.1516.

[0067] Example 8: Photophysical / photochemical properties of the compound, solubility in PBS, and log P test

[0068] The maximum absorption wavelength (λ) of the synthesized compounds in their respective solvents was tested. abs ), molar extinction coefficient (ε), fluorescence emission wavelength (λ) em ), fluorescence quantum yield (Φ f ), relative rate of singlet oxygen production (rel.rate), singlet oxygen yield (Φ) Δ The solubility and logP of the synthesized compounds are listed in Table 1. The data in the table show that, compared to the positive control BDP-4, the synthesized compounds exhibit maximum absorption in the near-infrared band, higher molar absorptivity, and increased fluorescence quantum yield. Using zinc phthalocyanine (ZnPc) as a reference, the relative rate (rel.rate) and singlet oxygen yield (Φ) of the compounds in N,N-dimethylformamide were tested. Δ Data showed that, except for compound 1, the photosensitizing ability of the other compounds was superior to that of the control. Compared with the control photosensitizer BDP-4, the water solubility of all compounds was significantly improved, and the logP value was significantly reduced.

[0069] The photosensitization efficiency test method is as follows:

[0070] 1) Preparation of DPBF solution

[0071] Weigh 2.70 mg of DPBF (M = 270.32), transfer to a 100 mL brown volumetric flask, dilute to volume with organic solvent, mix well, sonicate, and let stand in the dark for 30 min to prepare a 100 μM organic solution of DPBF.

[0072] 2) Preparation of compound solutions

[0073] Pipette 2 mL (10 μM compound solution) into a 10 mL volumetric flask and dilute with organic solvent to prepare a 2 μM solution.

[0074] 3) Preparation of mixed solutions

[0075] Pipette 2 mL of the prepared 100 μM DPBF solution into an EP tube protected from light by aluminum foil. Similarly, take 2 mL of the prepared 2 μM compound solution into the EP tube, mix well, and immediately test the sample absorption, recording it as 0 s.

[0076] 4) Lighting operation

[0077] The light source is a halogen lamp (25W), with an insulating water layer and a filter (>510nm) in between, and the optical power is 6.0mW / cm². 2Turn on the light source, remove the light-blocking plate, and irradiate for 10 seconds. After 10 seconds, replace the light-blocking plate and immediately measure the absorption of the sample. Repeat this process to measure the absorption of the sample after irradiation for 20 seconds, 30 seconds, 40 seconds, 50 seconds, and 60 seconds.

[0078] 5) Use the photosensitization efficiency of standard zinc phthalocyanine (ZnPc) as a reference.

[0079] Table 1. Photophysical / photochemical properties, solubility in PBS, and log P data.

[0080]

[0081] a Tested in methanol; b Test in chloroform; c The degradation rate of DPBF relative to BDP-4 in DMF; d Tested in DMF, ZnPc(Φ Δ =0.56) as a reference.

[0082] Example 9: Cell viability test

[0083] (1) Cell Culture

[0084] Cell culture conditions: Three tumor cell lines were used in the experiment: B16-F10 (mouse melanoma cells), A375 (human melanoma cells), and HeLa (human cervical cancer cells). The culture medium was DMEM (HyClone) containing 10% fetal bovine serum (FBS) and 1% penicillin (10,000 units / mL)-streptomycin (10,000 μg / mL) (HyClone). After cell resuscitation, the cells were cultured at 37°C in an incubator containing 5% CO2.

[0085] Cell and animal illumination conditions: Halogen lamps (75W) were used as the light source, and near-infrared light (λ>590nm) was obtained by filtering with a filter. A hot water insulation layer was used to remove the heat emitted by the halogen lamps.

[0086] (2) Cytotoxicity test

[0087] Cell phototoxicity was evaluated using the CCK-8 assay. Cultured cells were trypsinized and seeded into 96-well plates at a density of approximately 3000 cells / well, and cultured for 12 hours to allow for adherence. The culture medium was then removed, and different concentrations of the compound were added, with five replicates for each concentration. Incubation was performed for 16 hours. The compound-containing medium was then aspirated, fresh medium was added, and the plates were illuminated with a halogen lamp (λ>590nm, light dose 54J / cm²). 2After incubating for another 24 hours, the culture medium was aspirated, and 100 μl of 10% CCK-8 medium was added to each well of a 96-well plate. The plate was then incubated for 2 hours, and the absorbance (OD value) at 450 nm was measured using a microplate reader. Cell viability was calculated using the following formula: Cell viability = [(OD value of experimental group - OD value of zeroing well) / (OD value of control group - OD value of zeroing well)] × 100%.

[0088] The cell dark toxicity test was performed without light exposure, while the other tests used the same methods as the phototoxicity test.

[0089] (3) Specific results are shown in Table 2. The results indicate that, among the three tumor cell models tested, chemical 2 exhibited relatively better inhibitory activity than the positive control BDP-4. The compounds of this invention can be further developed into novel photodynamic antitumor photosensitizers.

[0090] Table 2. Phototoxicity and dark toxicity of the compounds in three tumor cell lines: B16-F10, A375, and HeLa.

[0091]

[0092] Example 10: Organelle Localization Experiment

[0093] The localization properties of photosensitizers determine the site of photodynamic action and influence cell death mechanisms. In photodynamic reactions, singlet oxygen produced in type II reactions is generally considered to play a major role. Due to the very short half-life (0.03 to 0.18 ms) and limited diffusion distance (<0.04 μm) of singlet oxygen, the distribution location of the photosensitizer in tumor cells is the site of its action. Therefore, to investigate the organelle localization ability of compound 2, an organelle co-localization experiment was conducted. Using a laser confocal live-cell workstation, B16-F10 cells were incubated with the compound and a commercially available organelle dye, respectively. The localization effect was determined based on the overlap of localization between the two dyes.

[0094] B16-F10 cells were seeded in culture dishes and incubated at 37°C for 12 h in an incubator containing 5% CO2. The culture medium was then removed, and fresh culture medium containing compound 2 (5 μM) was added. The cells were incubated for another 16 h. Afterward, the cells were washed three times with PBS, and then incubated for 40 min with Mito-Tracker Green (250 nM), Lyso-Tracker Green (500 nM), or ER-Tracker Green (2 μM), respectively. The cells were then washed three times with PBS, and fresh culture medium was added. Confocal localization imaging was then performed using a Leica TCS SP8 live-cell imaging workstation.

[0095] like Figure 1As shown in the figures, A1, A2, and A3 show the morphology of cells under bright field conditions; B1, B2, and B3 are green fluorescence images of cells after incubation with mitochondria, lysosomes, and endoplasmic reticulum localization probes, respectively; C1, C2, and C3 are red fluorescence images of cells after incubation with compound 2; and D1, D2, and D3 are superimposed images of the green fluorescence of the corresponding probes and the red fluorescence of the compound. The localization effects in D1, D2, and D3 show that compound 2 is distributed in mitochondria, lysosomes, and endoplasmic reticulum, indicating that it may exert photodynamic effects through multiple cell death pathways.

[0096] Example 11: Intracellular reactive oxygen species level test

[0097] In photodynamic therapy (PDT), photosensitizers kill tumor cells through reactive oxygen species (ROS) generated by type I and type II reactions. To further and more intuitively investigate the level of ROS generated in PDT, we used the commercially available ROS probe H2DCFDA to detect the intracellular ROS level of compound 2.

[0098] B16-F10 cells were incubated with different concentrations (10, 20, 100 nM) of compound 2, followed by incubation with the reactive oxygen species probe H2DCFDA (10 μM) and a light dose of 54 J / cm². 2 The control group was incubated with compound 2 (100 nM) without light, and otherwise treated the same as the experimental group. Intracellular reactive oxygen species levels were then detected using a CytoFLEX S flow cytometer (Beckman, USA). Figure 2 As shown, compared with control group A, experimental groups B, C, and D all showed significant differences. Moreover, within the tested concentration range, the generation of reactive oxygen species and the concentration of compound 2 exhibited a dose-dependent relationship.

[0099] Example 12: Apoptosis Experiment

[0100] The experiment used B16-F10 cells, which were seeded in six-well plates and incubated at 37°C for 12 hours in a 5% CO2 incubator. Afterwards, the original culture medium was aspirated, and different concentrations of compound 2 were added, followed by incubation for 16 hours, with a light dose of 54 J / cm². 2 The cells were then washed three times with PBS, added with fresh culture medium, and incubated for another 24 hours. Control group A (250 nM) was not exposed to light. Cell necrosis and apoptosis were evaluated using Annexin V-FITC and PI (Beyotime, China) double staining methods. The experimental instrument was a CytoFLEX S flow cytometer (Beckman, USA). The light dose used in the experiment was 54 J / cm². 2 The experimental results are as follows Figure 3As shown: at a concentration of 10 nM, the apoptosis rate in group B reached 10.23%; at a concentration of 50 nM, the apoptosis rate in group C reached 54.62%; at a concentration of 250 nM, the apoptosis rate in group D was 95.96%; and the apoptosis rate in control group A was only 8.80%. The data indicate that groups C and D were significantly different from the control group, and that the effect of compound 2 on cell apoptosis was concentration-dependent.

[0101] Example 13: B16-F10 mouse xenograft experiment

[0102] BDP-4 and the commercially available photosensitizer Ce6 were used as references to study the antitumor activity of compound 2 in mice. B16-F10 tumor cells were inoculated into the right flank of mice, with a cell density of 3 × 10⁶ cells per tumor. 5 On the seventh day after vaccination, patients weighing between 18-22g and with tumors measuring 80-100mm in size were selected. 3 Mice were used in the experiments. Mice were randomly divided into four groups: control group, Ce6 experimental group, BDP-4 experimental group, and compound 2 experimental group, with five mice in each group. The control group received an equal volume of physiological saline, while the experimental groups received the compound solution (2 mg / kg) via tail vein injection. Twelve hours after injection, the tumor site was exposed to light using a laser lamp (200 mW / cm²). 2 Each mouse was irradiated for 5 minutes, with a total light dose of 60 J / cm². 2 After providing light, all mice were housed in an animal room. The major axis (L) and minor axis (W) of the tumor were recorded using calipers the following day, and the mice were weighed using a balance. The tumor volume was calculated using the formula: V = 0.5 × (L × W) 2 ).

[0103] Experimental results are as follows Figure 4 As shown, Figure A shows in vitro images of tumors from each group after 14 days; Figure B shows the tumor growth curves for each group; Figure C shows the tumor weight for each group; Figure D shows the changes in mouse body weight during growth; and Figure E shows H&E staining of various tumor tissues. The tumor volume and weight in the BDP-4 and Compound 2 groups were significantly lower than those in the control and Ce6 groups, with both tumors in the Compound 2 group completely disappearing. Starting on day 6 after light exposure, tumors in the Compound 2 and ADPM06 groups began to rebound. Subsequently, with prolonged exposure, the tumor growth rate in the ADPM06 group gradually exceeded that in the Compound 2 group. There was no significant difference in body weight between the experimental and control groups. H&E staining of tumor tissues showed significant damage to the tumor tissues in the BDP-4 and Compound 2 groups, while no significant changes were observed in the tumor tissues of the control and Ce6 groups. Therefore, Compound 2 exhibits superior antitumor effects in the B16-F10 xenograft model compared to BDP-4 and the commercially available photosensitizer Ce6.

Claims

1. A BODIPY-type compound modified with amino acids, characterized in that, The compound is selected from compound 2, compound 3, compound 5 or compound 6 below: Compound 2 has the following structure: ； Compound 3 has the following structure: ； Compound 5 has the following structure: ； Compound 6 has the following structure: 。 2. Use of the amino acid-modified BODIPY compound of claim 1 in the preparation of photodynamic therapy drugs.

3. Use of the amino acid-modified BODIPY compound of claim 1 in the preparation of drugs for treating malignant tumors.

4. The use according to claim 3, characterized in that, The malignant tumors mentioned are selected from skin cancer, prostate cancer, oral squamous cell carcinoma, cervical cancer, lung cancer, liver cancer, stomach cancer, breast cancer, colon cancer, bladder cancer, and esophageal cancer.

Images