Aza-BODIPY compound as well as preparation method and application thereof

Abstract

The invention belongs to the field of medicine synthesis, and relates to a pyridine or pyridine quaternary ammonium salt modified monoiodo Aza-BODIPY compound as well as a preparation method and medical application of the pyridine or pyridine quaternary ammonium salt modified monoiodo Aza-BODIPY compound. The compound disclosed by the invention plays a significant role in inhibiting tumor growth by generating a photodynamic effect. Results show that the compound has good anti-tumor activity and can be further used for preparing novel anti-tumor drugs.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry and relates to Aza-BODIPY compounds, their preparation methods, and uses. Specifically, it relates to a pyridine or pyridine quaternary ammonium salt-substituted monoiodine Aza-BODIPY compound, its preparation method, and its medical applications. Background Technology

[0002] Malignant tumors have become a common disease that seriously endangers people's lives and health. It is estimated that there will be nearly 20 million new cancer cases and 9.7 million cancer deaths worldwide in 2022 (CA Cancer J Clin 2024, 74(3), 229-263). Due to the ability of tumors to metastasize in the early stages, about 50% of patients with clinically diagnosed primary tumors have already developed distant metastases. Tumor cells grow rapidly and are prone to mutation, easily developing multidrug resistance, which leads to chemotherapy failure. According to relevant statistics, more than 90% of these cases are related to multidrug resistance of tumor cells. Currently, the anti-tumor drugs used in clinical practice are far from meeting the requirements of treatment, and finding effective treatment methods for tumors has always been the focus of research in the medical field.

[0003] Photodynamic therapy (PDT) is a novel cancer treatment method that is gaining increasing attention. Compared with traditional cancer treatments, PDT has several 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] Selective distribution of photosensitizers in specific organelles may improve the efficacy of photodynamic therapy. Because reactive oxygen species (ROS) are highly reactive in biological systems, have short half-lives (<40 ns), and limited diffusion ranges (<20 nm), the location of photosensitizers, as the initial site of ROS generation, has a crucial impact on therapeutic efficacy. Photosensitizers specifically accumulate selectively in key organelles of cancer cells, effectively destroying these "fatal sites," and their activity may far exceed that of photosensitizers randomly distributed within tumor cells. Various important organelles in cells, such as the cell membrane, mitochondria, endoplasmic reticulum, lysosomes, and nucleus, are considered potential targets for enhancing photodynamic activity (ACS Appl. Mater. Interfaces 2021, 13(17), 19543-19571; Chem. Rev. 2021, 121(21), 13454-13619). Although various organelle-targeting photosensitizers have been developed, most are constructed by attaching external organelle-targeting groups, which is cumbersome to prepare and results in large molecular weights, making them less than ideal. Therefore, the development of novel organelle-targeting photosensitizers is of paramount importance.

[0005] Lipid droplets play a crucial role in cellular lipid and energy homeostasis and protein degradation, and are essential for lipid and energy homeostasis. Increased reactive oxygen species in lipid droplets often lead to the oxidation of polyunsaturated fatty acids, which in turn triggers cell death. Lipid droplets have become an important target organelle for enhancing photodynamic therapy (Coord. Chem. Rev. 2024, 506, 215710). Although some lipid droplet-targeting photosensitizers have been developed, most of them absorb in the ultraviolet-visible region and have limited penetration depth. Near-infrared absorption photosensitizers that specifically target lipid droplets are still scarce. In addition, although molecular lipophilicity and appropriate clogP values ​​are beneficial for targeting lipid droplets (Chem. Commun. 2022, 58(10), 1495-1509), there is currently no universally applicable design strategy, and the development of lipid droplet-targeting photosensitizers remains quite challenging.

[0006] This invention fuses the organelle-targeting pyridine or pyridine quaternary ammonium salt portion with the photosensitizer core to construct a photosensitizer that is "organelle-targeted in its own structure," thus avoiding the drawbacks of complicated preparation and large molecular weight of external organelle-targeting groups. This provides a novel organelle-targeting Aza-BODIPY-type photosensitizer with good anti-tumor effects. Summary of the Invention

[0007] The first aspect of this invention provides an Aza-BODIPY type compound having the following structure (I):

[0008]

[0009] in:

[0010] Ar 1 Ar 2 Independently selected from pyridin-2-yl or its quaternary ammonium salt, pyridin-3-yl or its quaternary ammonium salt, pyridin-4-yl or its quaternary ammonium salt, and phenyl, and Ar 1 Ar 2 Different;

[0011] R1 is independently selected from F, Cl, Br, and I;

[0012] The dashed line indicates a CC key or that the key does not exist.

[0013] In some preferred embodiments, the Aza-BODIPY compounds of the present invention have the following general formula (IA), (IB), or (IC):

[0014]

[0015] In some preferred embodiments of the present invention, Ar 1 Or Ar 2 Selected from pyridin-3-yl.

[0016] In some preferred embodiments of the present invention, Ar 1 Or Ar 2 Selected from pyridin-4-yl.

[0017] In some preferred embodiments of the present invention, R1 is I.

[0018] In some preferred embodiments of the present invention, the quaternary ammonium salt is methylpyridine quaternary ammonium salt, ethylpyridine quaternary ammonium salt or benzylpyridine quaternary ammonium salt, and its anion is bromide anion, iodide anion or trifluoromethanesulfonic acid anion.

[0019] In some preferred embodiments of the present invention, the structure of the Aza-BODIPY-type compound is as follows:

[0020]

[0021] The second aspect of the present invention provides the use of the Aza-BODIPY class compounds described in the first aspect in the preparation of photodynamic therapy drugs.

[0022] A third aspect of this invention provides the use of the Aza-BODIPY compounds described in the first aspect in the preparation of medicaments for treating malignant tumors. The malignant tumors are preferably selected from 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.

[0023] The fourth aspect of this invention provides a method for preparing the Aza-BODIPY-type compounds described in the first aspect. The preparation process of the compounds of this invention is as follows:

[0024]

[0025] The photosensitizers (10,16) decompose in solution, and the photosensitizers in parentheses cannot be obtained.

[0026] The compounds of this invention were tested for tumor suppressor activity, and the results showed good tumor suppressor activity. All photosensitizers exhibited molar-level cell suppressor activity against the HeLa tumor cell line, with the preferred photosensitizer also showing excellent cell suppressor activity against HepG2. The meta-pyridine-modified monocyclic photosensitizer 20 can target important organelle lipid droplets and exhibits excellent photodynamic effects with almost no dark toxicity. In addition, this photosensitizer has excellent cellular uptake and intracellular reactive oxygen species generation, and also has excellent in vivo antitumor effects, showing potential as a clinical photosensitizer.

[0027] The pharmacodynamic testing methods used in this invention are methods well known to those skilled in the art.

[0028] In this invention, the HeLa cells and HepG2 cells used are commercially available to those skilled in the art. Attached Figure Description

[0029] Figure 1 Chemical stability test results of the compounds of this invention: change of normalized absorbance in DMSO over time ( Figure 1 A- Figure 1 B); Normalized absorbance in PBS over time ( Figure 1 C- Figure 1 D). Figure 2 Photostability test results of the compounds of this invention: normalized absorbance in DMSO as a function of illumination time ( Figure 2 A- Figure 2 B); Normalized absorbance in PBS as a function of illumination time ( Figure 2 C- Figure 2 D). Figure 3 The fluorescence of BDP-15 and 20 (5 μM) in HeLa cells as a function of incubation time.

[0030] Figure 4 HeLa cell photosensitizer uptake: Untreated ( Figure 4 A) BDP-15 Figure 4 B) and 20 ( Figure 4 C) Incubate for 3 hours; intracellular fluorescence intensity at different incubation times ( Figure 4 D).

[0031] Figure 5 Intracellular reactive oxygen species production in HeLa cells under different concentrations of BDP-15 (A1–5) and 20 (B1–5) light conditions.

[0032] Figure 6 Intracellular reactive oxygen species production in HeLa cells under light-protected conditions with different concentrations of BDP-15 and 20.

[0033] Figure 7 Colocalization experiments of 20 with Mito-Tracker Green, ER-Tracker Green, Lyso-Tracker Green, and BODIPY 493 / 503. Fluorescence of Mito-Tracker Green (A1), ER-Tracker Green (B1), Lyso-Tracker Green (C1), and BODIPY 493 / 503 (D1); red fluorescence of the corresponding group 20 (A2–D2); overlay of organelle-targeting dyes and 20 (A3–D3); fluorescence intensity changes in the underlined regions in A3–D3 (A4–D4).

[0034] Figure 8 Nude mice in vivo imaging at different times (A); fluorescence images of various organs at different times (B); fluorescence values ​​of various organs at different times (C).

[0035] Figure 9 The tumor-suppressive effect of photosensitizer 20 was evaluated in a nude mouse HeLa xenograft tumor model. Images of ex vivo tumors (A); tumor volume growth curve (B); ex vivo tumor weight (C); nude mouse body weight change curve (D). Figure 10 H&E staining images of tumors and major organ sections from mice in different treatment groups. Detailed Implementation

[0036] Example 1: Synthesis of Compound 7

[0037] 1a (111 mg, 0.20 mmol) (Chem. Eur. J. 2022, 28(56), e202201503) was dissolved in DCM (12 mL) and AcOH (4 mL), and N-iodosuccinimide (NIS) (71 mg, 0.32 mmol) was slowly added, followed by stirring at room temperature for 3 h. The reaction was quenched with excess Na2SO3 (aq), extracted with DCM, dried over anhydrous sodium sulfate, evaporated to dryness, and purified by silica gel column chromatography to give 7, a green solid, 40 mg, yield 29%. 1H NMR (600MHz, CDCl3) δ8.74(d,J=4.8Hz,2H),8.13(d,J=8.9Hz,2H),8.00(d,J=6.8Hz,2H),7.75(d,J=5.5Hz,2H),7.67(d,J=8 .6Hz,2H),7.43(tt,J=14.2,7.0Hz,3H),7.21(s,1H),7.02(t,J=8.3Hz,2H),6.99(d,J=8.9Hz,2H),3.89(s,3H),3.87(s,3H). 13 C NMR (151MHz, CDCl3) δ164.28,163.79,161.09,155.02,149.54,147.91,146.80,142.82,140.73,139.84,132. 98,132.57,131.32,130.58,129.42,128.93,125.09,124.05,122.78,121.06,114.87,113.53,55.76,55.42. 19 F NMR (376MHz, CDCl3) δ-133.36 (dd, J=60.0, 30.3Hz).ESI-HRMS (m / z): [M+H] + Calcd.for[C 33 H 25 BF2IN4O2] + :685.1078,found685.1084.

[0038] Example 2: Synthesis of compound 8

[0039] The synthesis method follows the synthetic route in 7. Using 2a (111 mg, 0.20 mmol) (Chem.Eur.J.2022,28(56),e202201503) and NIS (71 mg, 0.32 mmol) as raw materials, product 8 was obtained, a dark green solid, 20 mg, with a yield of 15%. 1 H NMR (600MHz, CDCl3) δ9.08(d,J=1.7Hz,1H),8.67(d,J=3.6Hz,1H),8.12(t,J=8.9Hz,3H),7.98(dd,J=6.4,2.8Hz,2H),7 .70(d,J=8.6Hz,2H),7.42(m,4H),7.17(s,1H),7.02(t,J=9.3Hz,2H),6.99(d,J=8.9Hz,2H),3.90(s,3H),3.88(s,3H). 13C NMR (151MHz, CDCl3) δ163.54,163.46,161.16,155.56,151.25,149.50,147.65,146.54,143.25,140.57,137.95,132. 77,132.57,131.48,130.38,129.38,129.16,128.93,124.11,123.00,122.83,120.78,114.80,113.55,55.72,55.42. 19 F NMR (376MHz, CDCl3) δ-133.33 (dd, J=60.2, 30.0Hz).ESI-HRMS (m / z): [M+H] + Calcd.for[C 33 H 25 BF2IN4O2] + :685.1078,found 685.1084.

[0040] Example 3: Synthesis of compound 11

[0041] 8 (68 mg, 0.10 mmol) was dispersed in CH3CN (10 mL), MeI (1 mL) was added, the tube was sealed, and the reaction was carried out at 50 °C for 4 h. After the reaction was complete, the solution was evaporated to dryness, PhMe was added, and the mixture was sonicated and filtered to obtain 11.68 mg, with a yield of 82%. 1 H NMR (600MHz, DMSO-d6) δ9.49(s,1H),9.05(d,J=5.9Hz,1H),8.98(d,J=8.1Hz,1H),8.35(t,J=7.5Hz,3H),8.14(d,J=6.7Hz ,2H),8.08(s,1H),7.58–7.50(m,5H),7.22(d,J=8.9Hz,2H),7.12(d,J=8.6Hz,2H),4.47(s,3H),3.92(s,3H),3.87(s,3H). 13 C NMR(151MHz,DMSO-d6)δ165.99,164.72,160.46,152.13,148.61,146.63,145.58,145.26,144.97,141.15,134.14,132.59 ,132.41,132.14,130.82,130.58,129.49,128.95,127.14,124.20,123.53,121.24,115.24,113.50,56.11,55.34,48.28. 19F NMR(376MHz,DMSO-d6)δ-131.72(dd,J=61.7,29.7Hz).ESI-HRMS(m / z):[M] + Calcd.for[C 34 H 27 BF2IN4O2] + :699.1240,found699.1255.

[0042] Example 4: Synthesis of compound 13

[0043] The synthesis method follows the synthetic route in section 7. Using 13a (116 mg, 0.20 mmol) (Chem.Eur.J.2022,28(56),e202201503) and NIS (71 mg, 0.32 mmol) as raw materials, product 13 was obtained, a dark green solid, 116 mg, with a yield of 82%. 1 HNMR(600MHz,CD2Cl2)δ8.64(s,2H),8.53(d,J=8.6Hz,1H),7.78(d,J=6.0Hz,2H),7.68(d,J=7.0Hz,2H),7.56(s,2H),7.49(s ,2H),7.44(d,J=6.7Hz,1H),7.07(d,J=6.8Hz,2H),6.94(d,J=8.7Hz,1H),6.89(s,1H),3.91(s,3H),3.90(s,3H),2.97(s,4H). 13 C NMR (151MHz, CD2Cl2) δ164.67,161.27,158.43,154.25,150.15,147.71,147.08,143.25,142.92,138.92,136.70,136.54,133.46 ,133.39,133.24,132.91,131.03,129.13,128.26,124.91,124.57,119.21,114.99,114.30,113.55,56.17,55.75,30.49,22.34. 19 F NMR (376MHz, CDCl3) δ-135.33 (dd, J=61.5, 30.5Hz).ESI-HRMS (m / z): [M+Na] + Calcd.for[C 35 H 26 BF2IN4NaO2] + :733.1054,found 733.1060.

[0044] Example 5: Synthesis of compound 14

[0045] The synthesis method follows the synthetic route in section 7. Using 14a (116 mg, 0.20 mmol) (Chem.Eur.J.2022,28(56),e202201503) and NIS (71 mg, 0.32 mmol) as raw materials, product 14 was obtained, a dark green solid, 100 mg, with a yield of 70%. 1 HNMR (600MHz, CDCl3) δ8.83 (s, 1H), 8.59 (d, J = 9.1Hz, 2H), 8.03 (dt, J = 7.9, 1.8 Hz,1H),7.81–7.75(m,2H),7.71(d,J=8.7Hz,2H),7.46(t,J=7.5Hz,2H),7.41( t,J=7.4Hz,1H),7.33(dd,J=7.8,4.9Hz,1H),7.05(t,J=5.8Hz,2H),6.92(dd,J =9.0,2.6Hz,1H),6.83(d,J=2.6Hz,1H),3.91(s,3H),3.89(s,3H),2.96(s,4H). 13 C NMR (151MHz, CDCl3) δ164.05,160.85,158.04,154.23,150.32,149.61,147.51,146.03,143.08,142.79,137.71,136.23,134.57,133.39 ,132.93,132.65,130.80,130.07,128.79,127.95,127.66,124.63,1 23.31,119.20,114.87,113.92,113.42,55.78,55.40,30.39,22.12. 19 F NMR (376MHz, CDCl3) δ-135.30 (dd, J=61.9, 30.8Hz).ESI-HRMS (m / z): [M+H] + Calcd.for[C 35 H 27 BF2IN4O2] + :711.1234,found 711.1244.

[0046] Example 6: Synthesis of compound 17

[0047] The synthesis method follows the synthetic route of 11. Using 14 (68 mg, 0.10 mmol) as the starting material, product 17 was obtained, a dark green solid, 70 mg, with a yield of 82%. 1H NMR (600MHz, DMSO-d6) δ9.30(s,1H),9.01(d,J=5.7Hz,1H),8.88(d,J=7.8Hz,1H),8.40(d,J=9.0Hz,1H),8.32–8.26(m,1H),7.82(d,J=7.4Hz,2 H),7.60(d,J=8.3Hz,2H),7.56(t,J=7.6Hz,2H),7.46(t,J=7.2Hz,1H), 7.17–7.10(m,4H),4.39(s,3H),3.91(s,3H),3.89(s,3H),3.03(s,4H). 13 C NMR(151MHz,DMSO-d6)δ164.42,160.55,156.79,154.42,147.54,146.20,145.49,145.25,145.09,142.52,142.39,137.51,132.29,130.9 6,130.42,130.25,128.90,128.83,128.21,128.12,127.33,123.80, 117.84,114.91,114.76,113.39,56.07,55.34,48.37,28.93,20.81. 19 F NMR (376MHz, DMSO) δ-133.79 (dd, J=62.5, 29.9Hz). 19 F NMR(376MHz,DMSO-d6)δ-133.79(dd,J=62.5,29.9Hz).ESI-HRMS(m / z):[M] + Calcd.for[C 36 H 29 BF2IN4O2] + :725.1396,found 725.1398.

[0048] Example 7: Synthesis of compound 19

[0049] The synthesis method follows the synthetic route in section 7. Using 19a (116 mg, 0.20 mmol) (Chem.Eur.J.2022,28(56),e202201503) and NIS (71 mg, 0.32 mmol) as raw materials, product 19 was obtained as a black solid, 128 mg, with a yield of 90%. 1H NMR (400MHz, CDCl3) δ8.65(d,J=9.0Hz,3H),7.73(d,J=5.0Hz,2H),7.65(dd,J=11.3,5.3Hz,4H),7.47–7.41(m,3H),7.0 5(d,J=8.7Hz,2H),6.93(dd,J=9.0,2.6Hz,1H),6.84(d,J=2.4Hz,1H),3.91(s,3H),3.90(s,3H),2.98(t,J=6.4Hz,4H). 13 C NMR (151MHz, CDCl3) δ164.74,160.88,160.64,151.84,149.39,148.91,147.06,141.99,141.01,140.71,136.83,135.15,134 .18,132.60,130.89,130.32,129.51,128.50,125.02,124.67,118.91,114.93,114.10,113.43,55.86,55.39,30.43,22.22. 19 F NMR (376MHz, CDCl3) δ-135.80 (dd, J=61.7, 30.2Hz).ESI-HRMS (m / z): [M+H] + Calcd.for[C 35 H 27 BF2IN4O2] + :711.1234,found711.1241.

[0050] Example 8: Synthesis of compound 20

[0051] The synthesis method follows the synthetic route in section 7. Using 20a (116 mg, 0.20 mmol) (Chem.Eur.J.2022,28(56),e202201503) and NIS (71 mg, 0.32 mmol) as raw materials, product 20 was obtained, a dark green solid, 114 mg, with a yield of 81%. 1HNMR(600MHz, CDCl3) δ9.03(d,J=2.0Hz,1H),8.64(d,J=9.1Hz,1H),8.60(dd,J=4.8,1. 5Hz,1H),8.09(dt,J=7.9,1.8Hz,1H),7.69(d,J=8.6Hz,2H),7.65–7.60(m,2H),7.42(dd d,J=11.0,9.6,5.8Hz,3H),7.35(dd,J=7.8,4.9Hz,1H),7.05(d,J=8.7Hz,2H),6.92(dd ,J=9.0,2.6Hz,1H),6.83(d,J=2.5Hz,1H),3.91(s,3H),3.89(s,3H),3.00–2.91(m,4H). 13 C NMR (151MHz, CDCl3) δ164.46,160.68,160.11,152.24,151.18,148.95,148.65,146.77,142.31,140.54,137.86,137.53,134.82,133 .93,132.59,130.98,130.30,129.44,129.37,128.52,124.70,122.74,119.04,114.88,113.97,113.43,55.82,55.39,30.45,22.17. 19 F NMR (376MHz, CDCl3) δ-135.56 (dd, J=61.7, 30.6Hz).ESI-HRMS (m / z): [M+H] + Calcd.for[C 35 H 27 BF2IN4O2] + :711.1234,found 711.1243.

[0052] Example 9: Synthesis of compound 21

[0053] The synthesis method follows the synthetic route in section 7. Using 21a (116 mg, 0.20 mmol) (Chem.Eur.J.2022,28(56),e202201503) and NIS (71 mg, 0.32 mmol) as raw materials, product 21 was obtained, a dark green solid, 80 mg, with a yield of 56%. 1HNMR (600MHz, CDCl3) δ8.78(d,J=4.7Hz,1H),8.63(d,J=9.0Hz,1H),7.87(d,J=7.9Hz,1H),7.74–7.64(m,5H),7.46–7.39(m,3H),7.27(dd,J=6. 3,3.6Hz,1H),7.04(d,J=8.7Hz,2H),6.92(dd,J=9.0,2.5Hz,1H),6.82(d,J=2.4Hz,1H),3.90(s,3H),3.87(s,3H),2.95(td,J=10.1,6.3Hz,4H). 13 C NMR (151MHz, CDCl3) δ164.36,160.60,159.96,152.83,152.06,149.15,148.50,146.62,142.75,140.37,138.66,135.65,134.66,133 .88,132.68,131.25,130.49,129.20,128.36,126.78,124.88,122.72,119.14,114.89,113.91,113.36,55.80,55.36,30.44,22.17. 19 F NMR (376MHz, CDCl3) δ-135.74 (dd, J=61.9, 30.3Hz).ESI-HRMS (m / z): [M+H] + Calcd.for[C 35 H 27 BF2IN4O2] + :711.1234,found 711.1239.

[0054] Example 10: Synthesis of compound 22

[0055] The synthesis method follows the synthetic route of 11. Using 19 (71 mg, 0.10 mmol) as the starting material, product 22 was obtained, a dark green solid, 60 mg, with a yield of 70%. 1H NMR(600MHz,DMSO-d6)δ9.02(d,J=6.8Hz,2H),8.53–8.49(m,1H),8.48(d,J=6 .5Hz,2H),7.80(d,J=7.6Hz,2H),7.61(t,J=7.6Hz,2H),7.53(t,J=7.4Hz,1H), 7.50(d,J=8.6Hz,2H),7.20(s,1H),7.17(d,J=9.1Hz,1H),7.11(d,J=8.7Hz,2 H), 4.34 (s, 3H), 3.95 (s, 3H), 3.87 (s, 3H), 3.06 (d, J = 5.3Hz, 2H), 3.01 (s, 2H). 13 C NMR(151MHz,DMSO-d6)δ166.22,163.38,160.04,150.30,150.23,149.03,148.42,144.61,140.89,140.29,137.99,134.04,132.2 2,130.18,129.82,129.75,129.48,128.62,127.38,124.07,117.24,115.36,115.01,113.38,56.39,55.26,47.47,29.21,21.43. 19 F NMR(376MHz,DMSO-d6)δ-134.31(dd,J=61.3,28.7Hz).ESI-HRMS(m / z):[M] + Calcd.for[C 36 H 29 BF2IN4O2] + :725.1396,found 725.1403.

[0056] Example 11: Synthesis of compound 23

[0057] The synthesis method follows the synthetic route in 11. Using 20 (71 mg, 0.10 mmol) as the starting material, product 23 was obtained, a dark green solid, 74 mg, with a yield of 87%. 1H NMR (600MHz, DMSO-d6) δ9.41(s,1H),8.97(d,J=6.0Hz,1H),8.93(d,J=8.2Hz,1H),8.50(d, J=9.2Hz,1H),8.29(dd,J=8.0,6.2Hz,1H),7.79(d,J=7.3Hz,2H),7.58(t,J=7.6Hz,2H),7. 52(dd,J=12.9,8.0Hz,3H),7.19(d,J=2.2Hz,1H),7.16(dd,J=9.1,2.5Hz,1H),7.12(d,J=8 .7Hz,2H),4.43(s,3H),3.94(s,3H),3.88(s,3H),3.08–3.02(m,2H),2.98(t,J=6.3Hz,2H). 13 C NMR(151MHz,DMSO-d6)δ165.79,162.38,160.13,149.71,149.58,149.23,145.41,145.20,144.46,140.50,140.19,137.56,133.71,132.7 6,132.17,130.13,130.05,129.72,129.67,128.57,127.05,123.99, 117.37,115.18,114.95,113.44,56.32,55.30,48.28,29.25,21.34. 19 F NMR(376MHz, DMSO-d6)δ-134.08(dd,J=61.5,28.4Hz).ESI-HRMS(m / z):[M] + Calcd.for[C 36 H 29 BF2IN4O2] + :725.1396,found725.1400.

[0058] Example 12: Synthesis of compound 24

[0059] The synthesis method follows the synthetic route of 11. Using 21 (71 mg, 0.10 mmol) as the starting material, product 24 was obtained, a dark green solid, 53 mg, with a yield of 62%. 1H NMR (600MHz, DMSO-d6) δ9.24(d,J=6.0Hz,1H),8.77(t,J=7.8Hz,1H),8.54(d,J=9.3Hz, 1H),8.38(d,J=7.8Hz,1H),8.26–8.22(m,1H),7.63–7.60(m,2H),7.58(d,J=8.6Hz,2H) ,7.47–7.43(m,3H),7.21(t,J=4.3Hz,1H),7.19(dd,J=9.1,2.5Hz,1H),7.11(t,J=7.5H z,2H),4.35(s,3H),3.95(s,3H),3.87(s,3H),3.05(t,J=6.6Hz,2H),3.00–2.95(m,2H). 13 C NMR(151MHz,DMSO-d6)δ166.43,164.05,160.12,150.44,150.29,148.65,147.85,147.49,145.24,140.85,140.15,138.30,134.23,134.17,1 32.25,132.14,130.05,129.73,129.68,128.40,127.49,126.96,123.6 2,117.19,115.49,115.05,113.45,56.44,55.29,47.27,29.22,21.39. 19 F NMR(376MHz,DMSO-d6)δ-132.82to-133.80(m),-134.32to-135.11(m).ESI-HRMS(m / z):[M] + Calcd.for[C 36 H 29 BF2IN4O2] + :725.1396,found725.1395.

[0060] Example 13: Photophysical properties and stability test of photosensitizer

[0061] The synthesized photosensitizer was tested to determine its maximum absorption wavelength (λ) in the corresponding solvent. abs ), molar extinction coefficient (ε), fluorescence emission wavelength (λ) em ), fluorescence quantum yield (Φ), relative rate of singlet oxygen production (rel.rate), and singlet oxygen production (Φ) Δ The corresponding data is listed in Table 1.

[0062] The photosensitization efficiency test method is as follows:

[0063] A 75W halogen lamp, a 590nm cutoff filter, and cooling water were used as the light source. 1 ml of the photosensitizer (1 μM) in DMSO solution was mixed with 1 ml of DPBF (100 μM) in DMSO solution. The solution was then irradiated with light passing through the cutoff filter (λ>590nm), and the change in the maximum absorption of DPBF (411nm) over time was monitored. Φ △ The test was conducted according to the method reported in the literature, and the calculation formula is as follows:

[0064] Φ △(s) =(K s / K ZnPc )·(A ZnPc / A s )·Φ △(ZnPc)

[0065] Where K s and K ZnPc These are the DPBF photobleaching rates of the photosensitizer and ZnPc under illumination conditions, respectively; A s and A ZnPc Φ represents the area of ​​the absorption peaks of the photosensitizer and ZnPc in the near-infrared (590-800 nm) region, respectively. △(ZnPc) The singlet oxygen quantum yield of ZnPc in the DMSO system is given. Additionally, the relative DPBF photobleaching rate of the photosensitizer was tested in a PBS system (containing 1% DMSO and 1% emulsifier) ​​with MB as a reference.

[0066] The data in the table show that the pyridine-modified photosensitizer retains the excellent photophysical properties of the lead photosensitizers BDP-4 and BDP-15, such as certain near-infrared fluorescence, strong near-infrared absorption, and high photosensitization efficiency, demonstrating its potential as a highly efficient photosensitizer. However, after modification with pyridine quaternary ammonium salt, the absorption of the photosensitizer is weakened and the photosensitization efficiency is reduced, especially in the PBS system.

[0067] Table 1. Spectral data of pyridine or pyridine quaternary ammonium salt photosensitizers in DMSO and PBS (containing 1% DMSO and 1% emulsifier).

[0068]

[0069]

[0070] a. Tested in DMSO; b. Tested in PBS; c. Tested in DMSO with ZnPc(Φ) Δ =0.40) is the reference test. d DPBF degradation rate relative to MB in PBS.

[0071] Stability is crucial for the efficacy of photosensitizers. Only with good stability can a photosensitizer ensure strong absorption of light energy in vitro and in vivo, transferring energy to oxygen to generate highly reactive singlet oxygen, thereby producing a highly efficient photodynamic effect. Therefore, we tested the chemical and photostable stability of the organelle-targeted modified Aza-BODIPY photosensitizer. Specifically, the photosensitizer (10 μM) was dispersed in DMSO or PBS (containing 1% DMSO and 1% emulsifier) ​​systems, and the change in absorption over time under light-protected conditions was tested. The results were normalized and plotted to obtain the chemical stability in different solvent systems. Figure 1 Similarly, with halogen lamps (90mW / cm²) 2 The sample was irradiated with light (λ>590nm), and the change in absorption with irradiation time was tested to obtain photostability. Figure 2 ).

[0072] The photosensitizers provided by this invention exhibit similar chemical stability in DMSO to the lead Aza-BODIPY photosensitizers BDP-4 and BDP-15, retaining over 85% of their initial absorbance after 6 days of storage in the dark at room temperature. However, the non-cyclic pyridine photosensitizers (7 and 8) show slightly worse stability in PBS than other photosensitizers (especially 7). Furthermore, while some pyridine quaternary ammonium salt Aza-BODIPY photosensitizers (e.g., 23 and 24) are relatively stable in DMSO, all pyridine quaternary ammonium salt Aza-BODIPY photosensitizers exhibit stability issues and rapid degradation in PBS. Pyridine photosensitizers 19 and 20 show better stability than pyridine photosensitizer 21. Regarding photostability, all pyridine Aza-BODIPY photosensitizers demonstrate good photostability in both DMSO and PBS, while the pyridine quaternary ammonium salt Aza-BODIPY photosensitizers show poorer stability under light, particularly in PBS.

[0073] In summary, pyridine photosensitizers retain the excellent properties of lead photosensitizers (BDP-4 and BDP-15), such as strong near-infrared absorption, high photosensitization efficiency, and high stability. However, pyridine quaternary ammonium salt photosensitizers show a decrease in molar absorptivity, photosensitization efficiency, and stability, especially in PBS, which may negatively impact their photodynamic effects.

[0074] Example 14: Cell viability test

[0075] The cytotoxicity of the photosensitizer was tested in HeLa cell lines according to the methods reported in the literature. Specifically, the photosensitizer (2 μmmol) was dissolved in 4 mL of THF, and 30 μL of polyoxyethylene castor oil (CrEL) / 1,2-propylene glycol (10:3, v / v) and 200 μL of DMSO were added, followed by sonication for 10 min. THF was removed by rotary evaporation, and 3.8 mL of PBS was added, followed by sonication for 10 min to obtain the photosensitizer stock solution (500 μM). This stock solution was diluted to D++ to obtain D++ solutions containing different concentrations of photosensitizer.

[0076] Add 100 μL of HeLa cell suspension (30,000 / mL, 3,000 / well) to a 96-well cell culture plate and incubate for 12 h to allow cell adhesion. Remove the cell culture medium, add 100 μL of D++ containing different concentrations of photosensitizer, and incubate in the dark for 3 h. Irradiate the cells for 10 min (90 mw, λ>590 nm) at a photometric concentration of 54 J / cm². 2 The cells were then incubated in the dark for 24 hours. The cell culture medium was removed, and 100 μL of cell culture medium containing CCK-8 was added. After incubation at 37°C in the dark for 2 hours, the absorbance at 450 nm was measured using a microplate reader. The inhibition of cell proliferation by the photosensitizer under light conditions was calculated using the following formula: Cell viability = [(Experimental group OD value - Zero-adjustment well OD value) / (Control group OD value - Zero-adjustment well OD value)] × 100%. The light exposure was removed, while other operations remained unchanged, to obtain the dark toxicity of the photosensitizer.

[0077] As shown in Table 1, all pyridine Aza-BODIPY photosensitizers of this invention exhibit superior phototoxicity against HeLa cells compared to BDP-4 / BDP-15. 50 It is distributed between 6.1 and 50.1 nM and has low dark toxicity (IC50). 50 ≥17.1 μM). In comparison, although pyridine quaternary ammonium salt photosensitizers also exhibit strong photodynamic activity (IC50, ≥17.1 μM). 50 =6.0-214.3nM), but dark toxicity is significantly increased (IC50). 50 =1.2-11.2 μM). Among them, the pyridine-modified monocyclic Aza-BODIPY photosensitizers (13, 14, and 19-21) not only exhibit excellent photodynamic activity but also good safety. Furthermore, these photosensitizers still show high photosensitivity (IC50) in HepG2 cells. 50 =6.9-35.8nM), and excellent security (IC). 50 >50 μM). Notably, the meta-pyridine-modified monocyclic Aza-BODIPY photosensitizer 20 exhibited the strongest photosensitivity (IC50 for HeLa and HepG2). 50The toxicities are 10.4 and 6.9 nM respectively, and the dark toxicity is negligible, with a dark / phototoxicity ratio greater than 4800.

[0078] Table 2. HeLa cell activity of pyridine or pyridine quaternary ammonium salt modified Aza-Aza-BODIPY photosensitizer

[0079]

[0080]

[0081] Table 3. HepG2 cell viability of pyridine-modified mono-cyclic Aza-Aza-BODIPY photosensitizer

[0082]

[0083] The structure of Ce6 compounds is as follows:

[0084]

[0085] The compound structure of BDP-4 is as follows:

[0086]

[0087] The compound structure of BDP-15 is as follows:

[0088]

[0089] Example 13: Cell Uptake Experiment

[0090] Cellular uptake of photosensitizers significantly impacts their photodynamic activity. To investigate the internal mechanism of high photodynamic activity in HeLa cells, laser confocal microscopy was used to study uptake at different time points, with BDP-15 as a reference compound. Specifically, HeLa cells were seeded onto 35mm glass-bottomed culture dishes and incubated for 12 hours to allow cell adhesion. The cell culture medium was removed, and 1 ml of D++ containing different photosensitizers (5 μM) was added. Cells were incubated in the dark for 0 min, 30 min, 60 min, 120 min, and 180 min, respectively. After incubation, the cell culture medium was removed, the cells were washed twice with PBS, and fresh D++ was added. Laser confocal microscopy was used to excite the cells at 633 nm and detect the signal in the 650-750 nm wavelength range for fluorescence imaging, obtaining the uptake of different photosensitizers by HeLa cells over time.

[0091] Given the similar photophysical properties of 20 and BDP-15, intracellular photosensitizer concentrations were compared using intracellular fluorescence intensity as an indicator. Figure 3As shown, these photosensitizers exhibited time-dependent cellular uptake, with intracellular fluorescence intensity increasing over time. Furthermore, after the same incubation time, the intracellular fluorescence intensity of 20 was significantly higher than that of BDP-15. After 3 hours of incubation, HeLa cells treated with BDP-15 showed only a weak red signal, while HeLa cells treated with 20 showed bright intracellular fluorescence, with an intensity approximately 6 times that of BDP-15, indicating that the uptake of 20 in HeLa cells was significantly enhanced compared to BDP-15. Figure 4 ).

[0092] Example 15: Intracellular reactive oxygen species level test

[0093] During photodynamic therapy, photosensitizers generate reactive oxygen species (ROS) that kill tumor cells. The level of ROS generation by the photosensitizer in HeLa cells was investigated using 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA). This ROS fluorescent probe itself is non-fluorescent and has good membrane permeability. After entering the cell, it is hydrolyzed by intracellular esterases to 2',7'-dichlorodihydrofluorescein, and then rapidly oxidized by ROS to 2',7'-dichlorofluorescein, emitting green fluorescence. The more ROS generated, the stronger the fluorescence. Specifically, HeLa cells were pre-incubated with different concentrations of photosensitizer, followed by incubation in cell culture medium containing H2DCFDA. Intracellular fluorescence signals were detected using laser confocal microscopy after light or dark treatment. Specifically, HeLa cells were seeded into 96-well transparent black cell culture plates and incubated for 12 hours to allow cell adhesion. Then, 100 μL of cell culture medium containing different concentrations of photosensitizer (0, 25, 50, 100, 200 nM) was added and incubated in the dark for 2 hours. Remove the cell culture medium, wash twice with cell culture medium to remove the photosensitizer, add 100 μL of cell culture medium containing H2DCFDA (10 μM), and incubate in the dark for 30 min. Remove the cell culture medium, wash twice with cell culture medium to remove any H2DCFDA that has not entered the cells, add fresh cell culture medium, and irradiate for 10 min (90 mw, λ>590 nm) at a photometric concentration of 54 J / cm². 2 The cells were fluorescently imaged using a laser confocal microscope at 488 nm to detect signals in the 515-545 nm band, thus obtaining the reactive oxygen species production levels in HeLa cells at different concentrations of photosensitizer.

[0094] like Figure 5As shown, under illumination, the green fluorescence increased with increasing concentrations of 20 and BDP-15 (25-200 nM), indicating that both photosensitizers generate reactive oxygen species (ROS) in a concentration-dependent manner. Cells treated with the more efficient meta-pyridine photosensitizer 20 showed brighter intracellular green fluorescence than those treated with BDP-15, indicating higher efficiency in ROS generation. However, under dark conditions, even at high concentrations (200 nM), the green fluorescence produced by both photosensitizers was quite weak, indicating lower ROS generation under light-protected conditions. Figure 6 ).

[0095] Example 16: Organelle Localization Experiment

[0096] During photodynamic therapy, photosensitizers generate reactive oxygen species (ROS) in the physiological environment with short half-lives (<40 ns) and limited diffusion ranges (<20 nm). The intracellular distribution of photosensitizers plays a crucial role in their cytotoxic activity. Organelle-targeted photosensitizers can effectively attack the "fatal sites" of cancer cells, and their killing power may be greater than that of photosensitizers randomly distributed in tumor cells. Photosensitizers targeting various important eukaryotic organelles, such as mitochondria, lysosomes, and endoplasmic reticulum, may be beneficial in improving therapeutic efficacy. To investigate the distribution of 20 in HeLa cells, organelle co-localization experiments were conducted.

[0097] HeLa cells were seeded onto 35mm glass-bottomed culture dishes and incubated in a cell culture incubator for 12 hours to allow cell adhesion. The cell culture medium was removed, and 1 ml of D++ containing different photosensitizers (5 μM) was added. The cells were incubated in the dark for 2 hours. The cell culture medium was then removed, and the cells were washed twice with PBS. 1 ml of D++ containing mitochondrial-targeting Mito-Tracker Green (250 nM), endoplasmic reticulum-targeting ER-Tracker Green (2 μM), lysosome-targeting Lyso-Tracker Green (160 nM), and lipid droplet-targeting BODIPY 493 / 503 (2 μM, prepared by the applicant's research group) was added, and the cells were incubated in the dark for 30 minutes. The cell culture medium was then removed, and the cells were washed twice with PBS. Fresh D++ was added. Confocal imaging of the cells was performed using a laser confocal microscope equipped with 633nm and 488nm lasers. Excitation with the 488nm laser was used to detect the fluorescence signal in the 500-560nm band, obtaining the fluorescence signal of each organelle-targeting dye. Excitation with a 633nm laser and detection of signals in the 650-750nm band yielded the photosensitizer fluorescence signal.

[0098] like Figure 7As shown in AC, both the colocalization images and luminescence intensity distributions indicate low overlap between 20 and commercially available endoplasmic reticulum (ER-Tracker Green), mitochondrial (Mito-Tracker Green), or lysosomal (Lyso-Tracker Green) targeting probes, with Pearson coefficients all below 0.10, indicating limited distribution of 20 in these organelles. However, the Pearson coefficient for 20's overlap with lipid droplet-targeted BODIPY 493 / 503 is 0.90, indicating its specific accumulation in lipid droplets. Figure 7 D). 20 The good lipid droplet targeting ability may reasonably explain why its cell activity is superior to that of BDP-15.

[0099] The compound structure of BODIPY 493 / 503 is as follows:

[0100]

[0101] Example 17: Small Animal In vivo Imaging and Ex vivo Organ Imaging Experiments

[0102] Nude mice weighing between 18 and 22 grams were used in the experiment and inoculated with HeLa tumor cell lines at a cell density of 5 × 10⁻⁶ cells / year. 6 When the tumor reaches a size of 80-100mm 3 Used in experiments. After a tail vein injection of 20 μmol / kg, the sample was analyzed at different time points (λ) using an IVIS imaging system. ex / em Mice were imaged using a method with a resolution of 680 / 720 nm. Simultaneously, mice were euthanized, and their major organs, skin, and tumors were extracted for imaging. The distribution of 20 in the major organs, skin, and tumors was quantified by the average gray value of the corresponding regions.

[0103] Following administration, 20 rapidly spread throughout the mouse body and distributed in various organs, reaching peak fluorescence intensity approximately 1 hour later, and then gradually disappeared over 24 hours. Figure 8 Although 20 is mainly distributed in the liver, lungs, spleen, and kidneys, fluorescence imaging showed some accumulation in tumors within 3 hours. Therefore, the interval between drug administration and light exposure was set at 1 hour. Notably, 20 has a lower distribution in the skin, which helps reduce the side effect of photosensitivity.

[0104] Example 18: Nude Mouse HeLa Xenograft Tumor Experiment

[0105] BDP-15 and the commercially available photosensitizer Ce6 were used as references to study the antitumor activity of compound 20 in nude mice. HeLa tumor cell lines were inoculated into the right dorsum of nude mice, with a cell density of 5 × 10⁶ cells per tumor. 6On the seventh day after vaccination, patients weighing between 18-22g and with tumors measuring 80-100mm in size were selected. 3 Nude mice were used in the experiment. The mice were randomly divided into four groups: control, Ce6, BDP-15, and 20, with five mice in each group. The Ce6, BDP-15, and 20 groups were injected with the corresponding photosensitizer (2.5 μmol / kg) via tail vein, while the control group received an equal volume of saline. One hour after injection, the tumor sites in all groups were treated with a fixed wavelength of 660 nm and a power density of 100 mW / cm². 2 Laser irradiation for 5 minutes (total energy 30 J / cm²) 2 The long axis (L) and short axis (W) of the tumor were measured every other day, and the tumor volume was calculated using the formula V = 0.5 × (L × W²). Simultaneously, changes in mouse body weight were monitored.

[0106] like Figure 9 As shown in AC, the tumor volume in the control group increased rapidly within 24 days. Although Ce6 is considered a potent porphyrin photosensitizer, it was almost unable to inhibit tumor growth under mild photodynamic therapy conditions. In contrast, the lead Aza-BODIPY photosensitizer BDP-15 slowed tumor growth after photodynamic therapy. However, due to the extremely low light dose (30 J / cm²), the tumor growth was significantly reduced. 2 Furthermore, with only a single irradiation, the tumor gradually increased in size after photodynamic therapy. Encouragingly, compound 20 of this invention exhibited a stronger anti-tumor effect, and at the end of the experiment, the tumor completely disappeared in one of the five mice. In addition, photosensitizer 20 showed low toxicity, and the mice steadily increased their body weight during the experiment. Figure 9 D), and no obvious pathological changes were observed in the major organs after hematoxylin-eosin (H&E) staining. Figure 10 Furthermore, H&E staining showed that, compared with the control group and Ce6, 20 and BDP-15 caused significant damage to the tumor, further demonstrating the excellent photodynamic therapy effect of these Aza-BODIPY photosensitizers.

[0107] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Aza-BODIPY type compounds, said compounds having the following structure (I): in: Ar 1 Ar 2 Independently selected from pyridin-2-yl or its quaternary ammonium salt, pyridin-3-yl or its quaternary ammonium salt, pyridin-4-yl or its quaternary ammonium salt, and phenyl, and Ar 1 Ar 2 Different; R1 is independently selected from F, Cl, Br, and I; The dashed line indicates a CC key or that the key does not exist.

2. The Aza-BODIPY compound according to claim 1, characterized in that, The compound has the following general formula (IA), (IB), or (IC):

3. The Aza-BODIPY compound according to any one of claims 1-2, Ar 1 Or Ar 2 Selected from pyridin-3-yl.

4. The Aza-BODIPY compound according to any one of claims 1-2, Ar 1 Or Ar 2 Selected from pyridin-4-yl.

5. The Aza-BODIPY compound according to any one of claims 1-4, wherein R1 is I.

6. The Aza-BODIPY compound according to any one of claims 1-5, wherein the quaternary ammonium salt is a methylpyridine quaternary ammonium salt, an ethylpyridine quaternary ammonium salt, or a benzylpyridine quaternary ammonium salt, and its anion is a bromide anion, an iodide anion, or a trifluoromethanesulfonic acid anion.

7. The Aza-BODIPY compound according to any one of claims 1-6, characterized in that, Selected from the following compounds:

8. Use of the Aza-BODIPY compound according to any one of claims 1-7 in the preparation of photodynamic therapy drugs.

9. Use of the Aza-BODIPY compound according to any one of claims 1-7 in the preparation of a medicament for treating malignant tumors.

10. The use according to claim 9, 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.

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