Mitochondrial targeting biguanide derivative, preparation method and application

Abstract

The invention belongs to the technical field of anti-cancer drugs, and particularly relates to a mitochondria-targeted biguanide derivative, a preparation method and application of the biguanide derivative. Wherein n is 2-5, n1 is 2-5, and n2 is 3-5; the anti-tumor effect is improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of anticancer drugs, and particularly relates to a mitochondria-targeted biguanide derivative, a preparation method and application. BACKGROUND

[0002] At present, cancer treatment mainly relies on drug treatment, surgery, radiotherapy and biological therapy, but the chemotherapeutic drugs represented by cisplatin generally have problems of high toxicity and strong drug resistance, and it is urgent to develop new anticancer drugs with high activity and high biological safety.

[0003] In recent years, targeted therapy as an important strategy of precision medicine has made significant progress. In particular, the development of targeted drugs for mitochondria has become a new direction of anti-tumor research. Mitochondria, as the center of energy metabolism and the hub of apoptosis regulation of cells, its structural and functional abnormalities are closely related to tumor occurrence and development. Studies have found that the mitochondrial membrane potential (MMP) of cancer cells is 40-80mV higher than that of normal cells, which provides a theoretical basis for selective targeting.

[0004] Biguanide compounds have been proven to have significant anti-tumor activity, and their mechanism of action is closely related to mitochondrial targeting. Studies have shown that metformin can selectively inhibit the activity of mitochondrial complex I of tumor cells, interfere with the energy metabolism process, and exert anti-tumor effect by inducing mitochondria-dependent apoptosis. Compared with metformin, phenformin shows stronger mitochondrial inhibitory activity and anti-tumor effect, but also has problems of large dose and high toxicity, so it is urgent to improve the anti-tumor activity of biguanide compounds and reduce toxicity. Because the biguanide structure itself has certain cationic properties, it can be targeted to accumulate in tumor cells with higher mitochondrial membrane potential, which provides an important inspiration for the development of new mitochondrial-targeted anti-tumor drugs. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a mitochondria-targeted biguanide derivative, a preparation method and application, and to improve its anti-tumor effect.

[0006] The present application provides a mitochondria-targeted biguanide derivative, and the structure of the biguanide derivative is: 、 or ; wherein n is 2-5, n1 is 2-5, and n2 is 3-5.

[0007] Preferably, n is 3-5, n1 is 2-5, and n2 is 3-5.

[0008] Preferably, n is 3, n1 is 3, and n2 is 3-4.

[0009] Preferably, n is 3 and n1 is 3.

[0010] This invention provides a method for preparing the biguanide derivative, comprising: adding compound 2 to an aqueous solution of compound 1 under stirring; adding concentrated hydrochloric acid; refluxing at 70-90°C; obtaining a crude product by chromatography; and purifying the crude product to obtain the biguanide derivative; wherein compound 1 is... Compound 2 is Or compound 1 is Compound 2 is Or compound 1 is Compound 2 is .

[0011] This invention provides an application of the aforementioned biguanide derivative, which is used to prepare a drug for treating cancer. Preferably, the cancer is bladder cancer.

[0012] The beneficial effects of this invention are that the biguanide derivatives of this invention can effectively accumulate in mitochondria, effectively inhibit the proliferation of bladder cancer cells through a dual mechanism of inducing ferroptosis and apoptosis, and have good safety in normal bladder cells. They have the potential to be anti-tumor therapeutic drugs and have good application prospects. Attached Figure Description

[0013] Figure 1 The results of this study investigated the effect of compound J6 on intracellular reactive oxygen species levels in J82 cells.

[0014] Figure 2 The results of this study investigated the effect of compound J6 on intracellular lipid peroxide levels in J82 cells.

[0015] Figure 3 The results of this study investigated the effect of compound J6 on the expression levels of ferroptosis-related proteins in J82 cells.

[0016] Figure 4 The results of this study investigated the effect of compound J6 on apoptosis levels in J82 cells. Detailed Implementation

[0017] To facilitate understanding of the technical solution of the present invention, the technical solution of the present invention will be clearly and completely described below through embodiments.

[0018] The present invention will be further described below with reference to specific examples and accompanying drawings, but the embodiments given are not intended to limit the present invention. The purpose of providing these embodiments is to make the technical solution of the present invention more thorough and comprehensive. Unless otherwise specified, all ingredients or materials involved in the methods described below are commercially available. Unless otherwise specified, all related experimental methods are conventional methods existing in the technical field.

[0019] Example 1: Preparation method of compound J1, the reaction route is as follows:

[0020] The steps are as follows: 3-Aminobenzyl alcohol (1.6 g, 1 equivalent) and dicyandiamide (1.3 g, 0.5 equivalent) were completely dissolved in water (3.6 mL) to prepare a homogeneous solution. Then, concentrated hydrochloric acid was added dropwise with stirring, and the reaction was carried out at 80 °C for 2 h, with the reaction process monitored by TLC. After the reaction was completed, the biguanide derivative (2.4 g, yield 89.2%) was obtained by silica gel column chromatography.

[0021] Triphenylphosphine (2.1 g, 1 equivalent) and 3-bromopropionic acid (2.0 g, 1.5 equivalent) were completely dissolved in acetonitrile (15 mL) to prepare a homogeneous solution. The reaction mixture was then refluxed at 80 °C for 24 hours. After the reaction was completed, the solution was purified by column chromatography to obtain dicarboxyethyltriphenylphosphine bromide (3 g, yield 62.5%).

[0022] Biguanide derivative (2.0 g, 1.3 equivalents) was dissolved in water (6 mL). Dicarboxyethyltriphenylphosphine bromide (3 g, 1 equivalent) was added under stirring. Concentrated hydrochloric acid (3 mL) was added dropwise under stirring. The mixture was refluxed at 80 °C for 4 h. The crude product was obtained by silica gel column chromatography. The crude product was further purified by reversed-phase silica gel column chromatography (C-18 bonded silica gel) with methanol-water as eluent. The target fractions were combined by TLC (thin-layer chromatography) monitoring and concentrated under reduced pressure to obtain high-purity product J1 (0.376 g pale yellow oil, yield: 8.6%).

[0023] Data for compound J1: 1 H NMR (600 MHz, DMSO- d 6) δ 7.92 – 7.89 (m, 3H), 7.85 –7.81 (m, 6H), 7.77 (td, J = 7.8, 3.5 Hz, 6H), 7.40 (d, J = 13.2 Hz, 4H), 7.32(d, J = 6.7 Hz, 2H), 7.29 – 7.21 (m, 2H), 7.18 (s, 1H), 6.99 (d, J = 7.5 Hz,1H), 4.99 (s, 2H), 3.94 – 3.86 (m, 2H), 2.80 – 2.71 (m, 2H). 13 C NMR (150 MHz, DMSO- d 6) δ 170.08 (d,J =18.0 Hz, 1C), 158.67, 154.05, 137.69, 136.34, 135.13(d, J =3.0 Hz, 3C), 133.79 (d, J =10.5 Hz, 6C), 130.35 (d, J =12.0 Hz, 6C), 129.09,124.09, 121.56, 121.20, 117.97 (d, J =87.0 Hz, 3C), 66.17, 26.81, 16.86 (d, J =54.0 Hz, 1C). HRMS (ESI) (m / z) [M-Br] + calcd for C 30 H 31 N5O2P + , 524.2210; found524.2209.

[0024] Example 2: Preparation of compound J2 Replacing 3-bromo-propionic acid in Example 1 with 2.2 g (0.0131 mol) of 4-bromobutyric acid, while keeping other conditions unchanged, yielded compound J2 (0.342 g, yield: 7.9%). The reaction route is as follows:

[0025] Data for compound J2: 1 H NMR (600 MHz, DMSO- d 6) δ 7.92 – 7.89 (m, 3H), 7.84 –7.81 (m, 6H), 7.77 (dt, J = 8.0, 4.0 Hz, 6H), 7.40 (d, J = 19.9 Hz, 4H), 7.33(s, 2H), 7.27 (t, J = 7.8 Hz, 2H), 7.19 (s, 1H), 6.99 (d, J = 7.5 Hz, 1H), 5.06 (s, 2H), 3.67 – 3.61 (m, 2H), 2.64 (t, J = 7.0 Hz, 2H), 1.81 – 1.76 (m,2H). 13 C NMR (150 MHz, DMSO- d6) δ 172.70, 161.54, 155.85, 143.60, 139.10,135.46 (d, J =3.0 Hz, 3C), 134.05 (d, J =9.0 Hz, 6C), 130.77 (d, J =13.5 Hz, 6C),128.71, 121.80, 119.78, 119.33, 118.79 (d, J =85.5 Hz, 3C), 63.20, 33.80 (d, J =18.0 Hz, 1C), 20.24 (d, J =51.0 Hz, 1C), 18.15 (d, J =3.0 Hz, 1C). HRMS (ESI) (m / z) [M-Br] + calcd for C 31 H 33 N5O2P + , 538.2366; found 538.2366.

[0026] Example 3: Preparation of compound J3 Replacing 3-bromo-propionic acid in Example 1 with 2.35 g (0.0131 mol) of 5-bromopentanoic acid, while keeping other conditions unchanged, yielded compound J3 (0.390 g, yield: 9.1%). The reaction route is as follows:

[0027] Data for compound J3: 1 H NMR (600 MHz, DMSO- d6) δ 7.89 (tt, J = 7.2, 1.7 Hz, 3H), 7.80 (ddd, J = 12.5, 8.4, 1.4 Hz, 6H), 7.76 (td, J = 7.8, 3.5 Hz, 6H), 7.41 (s, 5H), 7.31 (t, J = 1.9 Hz, 1H), 7.25 (t, J = 7.9 Hz, 1H), 7.17 (s,2H), 6.96 (dd, J = 7.7, 1.6 Hz, 1H), 5.01 (s, 2H), 3.67 (d, J = 2.4 Hz, 2H), 2.45 (t, J = 7.5 Hz, 2H), 1.76 (q, J = 7.5 Hz, 2H), 1.58 (d, J = 7.9 Hz, 2H). 13 C NMR (150 MHz, DMSO- d 6) δ 172.31, 161.25, 155.17, 138.99, 136.68, 134.92(d, J =3.0 Hz, 3C), 133.59 (d, J =9.0 Hz, 6C), 130.26 (d, J =13.5 Hz, 6C), 128.71,122.33, 120.17, 119.56, 118.51 (d, J =84.0 Hz, 3C), 65.13, 32.50, 25.18 (d, J =18.0 Hz, 1C), 21.20 (d, J =4.5 Hz, 1C), 19.96 (d, J =51.0 Hz, 1C). HRMS (ESI) (m / z) [M-Br] + calcd for C 32 H 35 N5O2P + , 552.2523; found 552.2527.

[0028] Example 4: Preparation of compound J4 Replacing 3-bromo-propionic acid in Example 1 with 2.54 g (0.0131 mol) of 6-bromohexanoic acid, while keeping other conditions unchanged, yielded compound J4 (0.272 g, yield: 6.4%). The reaction route is as follows:

[0029] Data for compound J4: 1 H NMR (600 MHz, DMSO- d 6) δ 7.90 (ddp, J = 7.2, 5.3,1.7 Hz, 3H), 7.85–7.72 (m, 12H), 7.36 (d, J = 60.1 Hz, 5H), 7.28–7.19 (m,3H), 7.17–7.05 (m, 1H), 6.98 (dd, J = 7.5, 4.9 Hz, 1H), 5.02 (s, 1H), 4.45 (d, J = 4.7 Hz, 1H), 3.64–3.56 (m, 2H), 2.43–2.16 (m, 2H), 1.53 (dddd, J =23.4, 17.9, 13.7, 7.8 Hz, 6H). 13 C NMR (150 MHz, DMSO- d 6) δ 172.55, 161.01,155.19, 143.16, 136.72, 134.89 (d, J=3.0 Hz, 3C), 133.61 (d, J=9.0 Hz, 6C), 130.23 (d, J=12.0 Hz, 6C), 128.71, 128.28, 122.28, 121.37, 118.57 (d, J=85.5Hz, 3C), 65.12, 32.99 (d, J=33.0 Hz, 1C), 29.24 (dd, J=4.5, 16.5 Hz, 1C),23.57, 21.49 (t, J=4.5 Hz, 1C), 20.11 (dd, J=7.5, 51.0 Hz, 1C). HRMS (ESI)(m / z) [M-Br] + calcd for C 33 H 37 N5O2P + , 566.2679; found 566.2692.

[0030] Example 5: Preparation of compound J5 Replacing 2.1 g (0.0153 mol) of 3-aminophenylethanol in Example 1 with the 3-aminophenylethanol, while keeping other conditions unchanged, yielded compound J5 (0.389 g, yield: 8.7%). The reaction route is as follows:

[0031] Data for compound J5: 1 H NMR (600 MHz, DMSO- d 6) δ 7.90 (tt, J = 7.3, 1.3 Hz,3H), 7.82 (ddd, J = 12.7, 8.5, 1.4 Hz, 6H), 7.79 – 7.75 (m, 6H), 7.37 (s,4H), 7.32 – 7.28 (m, 1H), 7.17 (dd, J = 15.8, 7.7 Hz, 4H), 6.88 (dt, J = 7.7,1.2 Hz, 1H), 4.13 (t, J = 7.1 Hz, 2H), 3.86 (tdd, J = 9.2, 7.1, 5.4 Hz, 2H), 2.78 (t, J = 6.9 Hz, 2H), 2.72 – 2.66 (m, 2H). 13 C NMR (150 MHz, DMSO- d 6) δ170.10 (d, J=16.5 Hz, 1C), 161.14, 155.29, 138.89, 138.12, 135.07 (d, J=3.0Hz, 3C), 133.74 (d, J=10.5 Hz, 6C), 130.29 (d, HRMS (ESI) (m / z) [M-Br] + calcd for C 31 H 33 N5O2P + , 538.2366;found 538.2375.

[0032] Example 6: Preparation of compound J6 Replacing 2.1 g (0.0153 mol) of 3-aminophenylethanol in Example 1 and replacing 2.2 g (0.0131 mol) of 4-bromobutyric acid in Example 1 with 4-bromopropionic acid, under the same conditions, yielded compound J6 (0.398 g, yield: 9.3%). The reaction route is as follows:

[0033] Data for compound J6: 1 H NMR (600 MHz, DMSO- d 6) δ 7.96 – 7.87 (m, 3H), 7.82(ddd, J = 12.6, 8.5, 1.4 Hz, 6H), 7.78 (ddd, J = 8.2, 7.3, 3.6 Hz, 6H), 7.38(s, 4H), 7.28 (ddd, J = 8.2, 2.3, 1.1 Hz, 1H), 7.21 – 7.11 (m, 4H), 6.88 (dt,J = 7.7, 1.2 Hz, 1H), 4.21 (t, J = 6.7 Hz, 2H), 3.66 – 3.59 (m, 2H), 2.83 (t,J = 6.7 Hz, 2H), 2.58 (t, J = 7.1 Hz, 2H), 1.74 (h, J = 7.4 Hz, 2H). 13 C NMR (150 MHz, DMSO-) d 6) δ 171.72, 161.15, 155.30, 138.82, 138.46, 135.00 (d, J=3.0Hz, 3C), 133.60 (d, J=10.5 Hz, 6C), 130.31 (d, J=13.5 Hz, 6C), 128.55,123.68, 120.95, 118.90, 118.31 (d, J=85.5 Hz, 3C), 64.56, 34.23, 33.47 (d, J=18.0 Hz, 1C), 19.79 (d, J=51.0 Hz, 1C), 17.64 (d, J=3.0 Hz, 1C). HRMS (ESI)(m / z) [M-Br] + calcd for C 32 H 35 N5O2P + , 552.2523; found 552.2518.

[0034] Example 7: Preparation of compound J7 Replacing 2.1 g (0.0153 mol) of 3-aminophenylethanol in Example 1 and replacing 2.35 g (0.0131 mol) of 5-bromopentanoic acid in Example 1 with 5-bromo-propionic acid, under the same conditions, yielded compound J7 (0.385 g, yield: 8.8%). The reaction route is as follows:

[0035] Data for compound J7: 1 H NMR (600 MHz, DMSO- d 6) δ 7.94 – 7.86 (m, 3H), 7.81(ddd, J = 12.5, 8.4, 1.4 Hz, 6H), 7.76 (ddd, J = 8.2, 7.2, 3.6 Hz, 6H), 7.39(s, 4H), 7.32 (ddd, J = 8.1, 2.2, 1.1 Hz, 1H), 7.21 – 7.14 (m, 4H), 6.88 (dt,J = 7.6, 1.3 Hz, 1H), 4.15 (t, J = 6.8 Hz, 2H), 3.72 – 3.60 (m, 2H), 2.77 (t,J = 6.7 Hz, 2H), 2.38 (t, J = 7.4 Hz, 2H), 1.71 (p, J = 7.4 Hz, 2H), 1.61 –1.50 (m, 2H). 13 C NMR (150 MHz, DMSO- d 6) δ 172.92, 161.64, 155.76, 139.30,138.91, 135.37 (d, J=3.0 Hz, 3C), 134.07 (d, J=10.5 Hz, 6C), 130.71 (d, J=12.0 Hz, 6C), 129.05, 124.06, 121.36, 119.29, 118.99 (d, J=85.5 Hz, 3C), 64.72, 34.79, 32.92, 25.58 (d, J=18.0 Hz, 1C), 21.61 (d, J=4.5 Hz, 1C), 20.42(d, J=51.0 Hz, 1C). HRMS (ESI) (m / z) [M-Br] + calcd for C 33 H 37 N5O2P + , 566.2679;found 566.2691.

[0036] Example 8: Preparation of compound J8 Replacing 2.1 g (0.0153 mol) of 3-aminophenylethanol in Example 1 and replacing 2.54 g (0.0131 mol) of 6-bromohexanoic acid in Example 1 with 6-bromo-propionic acid, under the same conditions, yielded compound J8 (0.325 g, yield: 7.5%). The reaction route is as follows:

[0037] Data for compound J8: 1 H NMR (600 MHz, DMSO- d 6) δ 7.90 (ddd, J = 7.2, 6.3,1.7 Hz, 3H), 7.82 – 7.78 (m, 6H), 7.78 – 7.74 (m, 6H), 7.34 (s, 4H), 7.27(dt, J = 8.1, 1.4 Hz, 1H), 7.21 – 7.15 (m, 2H), 7.12 (s, 2H), 6.90 (dt, J =7.6, 1.3 Hz, 1H), 4.17 (t, J = 6.9 Hz, 2H), 3.61 – 3.56 (m, 2H), 2.81 (t, J =6.9 Hz, 2H), 2.24 (t, J = 7.2 Hz, 2H), 1.52 (p, J = 7.2 Hz, 4H), 1.47 – 1.42(m, 2H). 13 C NMR (150 MHz, DMSO- d 6) δ 173.16, 161.60, 155.74, 139.22, 138.91,135.36 (d, J=3.0 Hz, 3C), 134.08 (d, J=10.5 Hz, 6C), 130.71 (d, J=12.0 Hz, 6C), 129.05, 124.23, 121.54, 119.50, 119.02 (d, J=85.5 Hz, 3C), 64.64, 34.82,33.54, 29.65 (d, J=18.0 Hz, 1C), 24.02, 21.98 (d, J=4.5 Hz, 1C), 20.59 (d, J=49.5 Hz, 1C). HRMS (ESI) (m / z) [M-Br] + calcd for C 34 H 39 N5O2P +, 580.2836; found580.2833.

[0038] Example 9: Preparation of compound D1 Replacing 3-aminophenylethanol (0.0153 mol) with 2.1 g (4-aminophenylethanol) in Example 1, while keeping other conditions unchanged, yielded compound J12 (0.371 g, yield: 8.3%). The reaction route is as follows:

[0039] Data for compound D1: 1 H NMR (600 MHz, DMSO- d 6) δ 7.91 (dd, J = 8.4, 6.6 Hz, 3H), 7.85 – 7.74 (m, 12H), 7.36 – 7.17 (m, 6H), 7.14 (d, J = 8.1 Hz, 2H), 7.06 (s, 2H), 4.13 (t, J = 6.8 Hz, 2H), 3.88 – 3.78 (m, 2H), 2.78 (t, J = 6.8Hz, 2H), 2.69 – 2.61 (m, 2H). 13 C NMR (150 MHz, DMSO- d 6) δ 169.96 (d, J =16.5 Hz,1C), 160.91, 155.16, 136.84, 134.97 (d, J =3.0 Hz, 3C), 133.59 (d, J =9.0 Hz,6C), 132.66, 130.17 (d, J =12.0 Hz, 6C), 128.91 (2×), 121.00 (2×), 117.81 (d, J =85.5 Hz, 3C), 65.32, 33.37, 26.58 (d, J =1.5 Hz, 1C), 16.63 (d, J =54.0 Hz,1C). HRMS (ESI) (m / z) [M-Br] + calcd for C 31 H 33 N5O2P + , 538.2366; found 538.2374.

[0040] Example 10: Preparation of compound D2 Replacing 2.1 g (0.0153 mol) of 4-aminophenylethanol with 3-aminophenylethanol in Example 1, and replacing 2.2 g (0.0131 mol) of 4-bromobutyric acid with 3-bromopropionic acid in Example 1, under the same conditions, yielded compound D2 (0.407 g, yield: 9.2%). The reaction route is as follows:

[0041] Data for compound D2: 1 H NMR (600 MHz, DMSO- d 6) δ 7.91 (dtd, J = 7.2, 3.4,1.7 Hz, 3H), 7.84 – 7.76 (m, 12H), 7.40 (s, 4H), 7.27 (d, J = 8.5 Hz, 2H), 7.16 (s, 2H), 7.10 (d, J = 8.5 Hz, 2H), 4.19 (t, J = 6.8 Hz, 2H), 3.64 – 3.58 (m, 2H), 2.80 (t, J = 6.8 Hz, 2H), 2.53 (t, J = 7.1 Hz, 2H), 1.78 – 1.67 (m,2H). 13 C NMR (150 MHz, DMSO- d 6) δ 171.87, 161.35, 155.56, 137.28, 135.21 (d, J =3.0 Hz, 3C), 133.76 (d, J =9.0 Hz, 6C), 132.91, 130.51 (d, J =13.5 Hz, 6C),129.10 (2×), 120.93 (2×), 118.47 (d, J =85.5 Hz, 3C), 64.95, 55.13, 33.80 (d, J =3.0 Hz, 1C), 19.93 (d, J =51.0 Hz, 1C), 17.84 (d, J =3.0 Hz, 1C). HRMS (ESI)(m / z) [M-Br] + calcd for C 32 H 35 N5O2P +, 552.2523; found 552.2531.

[0042] Example 11: Preparation of compound D3 Replacing 2.1 g (0.0153 mol) of 4-aminophenylethanol with 3-aminophenylethanol in Example 1, and replacing 2.35 g (0.0131 mol) of 5-bromopentanoic acid with 3-bromo-propionic acid in Example 1, under the same conditions, yielded compound D3 (0.390 g, yield: 8.9%). The reaction route is as follows:

[0043] Data for compound D3: 1 H NMR (600 MHz, DMSO- d 6) δ 7.89 (ddt, J = 8.9, 4.9,1.8 Hz, 3H), 7.83 – 7.74 (m, 12H), 7.34 (s, 4H), 7.31 – 7.25 (m, 2H), 7.12 –7.11 (m, 2H), 6.21 (t, J = 6.0 Hz, 1H), 6.08 (t, J = 5.6 Hz, 1H), 4.14 (t, J= 6.9 Hz, 2H), 3.68 – 3.55 (m, 2H), 2.76 (t, J = 6.9 Hz, 2H), 2.35 (t, J =7.4 Hz, 2H), 1.76 – 1.67 (m, 4H). 13 C NMR (150 MHz, DMSO- d 6) δ 172.48, 161.15,155.39, 137.06, 134.95 (d, J =3.0 Hz, 3C), 133.60 (d, J =12.0 Hz, 6C), 132.79,130.29 (d, J =12.0 Hz, 6C), 128.95 (2×), 120.92 (2×), 118.51 (d, J =85.5 Hz,3C), 64.45, 41.98, 36.30, 34.12, 25.24, 15.68. HRMS (ESI) (m / z) [M-Br] + calcdfor C 33 H 37 N5O2P +, 566.2679; found 566.2690.

[0044] Example 12: Preparation of compound D4 Replacing 2.1 g (0.0153 mol) of 4-aminophenylethanol with 3-aminophenylethanol in Example 1, and replacing 2.54 g (0.0131 mol) of 6-bromohexanoic acid with 3-bromo-propionic acid in Example 1, under the same conditions, yielded compound D4 (0.388 g, yield: 9.0%). The reaction route is as follows:

[0045] Data for compound D4: 1 H NMR (600 MHz, DMSO- d 6) δ 7.91 – 7.88 (m, 3H), 7.81 –7.76 (m, 12H), 7.37 – 7.23 (m, 6H), 7.18 – 7.12 (m, 2H), 7.05 (s, 2H), 4.15(t, J = 7.0 Hz, 2H), 3.57 (ddt, J = 14.0, 7.3, 4.3 Hz, 2H), 2.80 (t, J = 7.0Hz, 2H), 2.23 (t, J = 7.2 Hz, 2H), 1.54 – 1.50 (m, 4H), 1.48 – 1.44 (m, 2H). 13 C NMR (150 MHz, DMSO- d 6) δ 172.66, 160.97, 155.28, 136.82, 134.92 (d, J =4.5Hz, 3C), 133.62 (d, J =10.5 Hz, 6C), 133.06, 130.25 (d, J =12.0 Hz, 6C), 129.02(2×), 121.26 (2×), 118.55 (d, J =84.0 Hz, 3C), 64.36, 33.77, 33.07 (d, J =4.5Hz, 1C), 29.21 (d, J =16.5 Hz, 1C), 23.55, 21.50 (d, J =4.5 Hz, 1C), 20.12 (d, J=49.5 Hz, 1C). HRMS (ESI) (m / z) [M-Br] + calcd for C 34 H 39 N5O2P + , 580.2836; found580.2849.

[0046] Example 13: Preparation of compound D5 Replacing 2.1 g (0.0153 mol) of 4-aminophenylethanol with 3.0 g (0.012 mol) of 10-bromodecanoic acid with 3.0 g (0.012 mol) of 10-bromodecanoic acid, under the same conditions, yielded compound D5 (0.256 g, yield: 6.1%). The reaction route is as follows:

[0047] Data for compound D5: 1 H NMR (600 MHz, DMSO- d 6) δ 7.89 (td, J = 7.2, 1.8 Hz,3H), 7.85 – 7.71 (m, 12H), 7.39 (s, 4H), 7.30 (d, J = 8.2 Hz, 2H), 7.17 –7.08 (m, 4H), 4.17 (t, J = 6.8 Hz, 2H), 3.60 (ddd, J = 15.9, 11.0, 6.6 Hz,2H), 2.80 (t, J = 6.8 Hz, 2H), 2.23 (t, J = 7.3 Hz, 2H), 1.60 – 1.36 (m, 6H),1.33 – 1.09 (m, 8H). 13 C NMR (150 MHz, DMSO- d 6) δ 172.87, 161.18, 155.39,137.10, 134.89 (d, J =3.0 Hz, 3C), 133.61 (d, J =10.5 Hz, 6C), 132.80, 130.25 (d, J =12.0 Hz, 6C), 128.95 (2×), 120.77 (2×), 118.63 (d, J =85.5 Hz, 3C), 64.31,33.78, 33.49, 29.77 (d, J=16.5 Hz, 1C), 28.50 (2×), 28.33, 28.04, 24.39,21.76 (d, J =4.5 Hz, 1C), 20.18 (d, J =48.0 Hz, 1C). HRMS (ESI) (m / z) [M-Br] + calcd for C 38 H 47 N5O2P + , 636.3462; found 636.3482.

[0048] Example 14: Preparation of compound L1 Replacing 2.1 g (0.0153 mol) of 2-aminophenylethanol with 3-aminophenylethanol in Example 1, and replacing 2.2 g (0.0131 mol) of 4-bromobutyric acid with 3-bromopropionic acid in Example 1, under the same conditions, yielded compound L1 (0.053 g, yield: 1.2%). The reaction route is as follows:

[0049] Data for compound L1: 1 H NMR (600 MHz, DMSO- d 6) δ 7.91 (td, J = 7.3, 1.8 Hz, 3H), 7.82 (dd, J = 12.6, 7.7 Hz, 6H), 7.78 (td, J = 7.8, 3.6 Hz, 6H), 7.35 –7.29 (m, 2H), 7.23 (d, J = 7.3 Hz, 1H), 7.20 – 7.09 (m, 6H), 7.05 (d, J = 7.6Hz, 1H), 4.22 (t, J = 6.9 Hz, 2H), 3.62 – 3.58 (m, 2H), 2.96 (d, J = 8.6 Hz, 2H), 2.55 (t, J = 7.0 Hz, 2H), 1.78 – 1.70 (m, 2H). 13 C NMR (150 MHz, DMSO- d 6) δ171.70, 160.81, 157.10, 136.18, 134.99 (d,J =3.0 Hz, 3C), 133.59 (d, J =10.5 Hz,6C), 132.35, 130.30 (d, J =12.0 Hz, 6C), 130.02, 126.83, 126.30, 125.46, 118.29(d, J =85.5 Hz, 3C), 63.79, 33.52 (d, J =19.5 Hz, 1C), 30.07, 19.80 (d, J =51.0 Hz,1C), 17.65. HRMS (ESI) (m / z) [M-Br] + calcd for C 32 H 35 N5O2P + , 552.2523; found552.2533.

[0050] Example 15: Preparation of compound L2 Replacing 2.1 g (0.0153 mol) of 2-aminophenylethanol with 3-aminophenylethanol in Example 1, and replacing 2.35 g (0.0131 mol) of 5-bromopentanoic acid with 3-bromo-propionic acid in Example 1, under the same conditions, yielded compound L2 (0.0612 g, yield: 1.4%). The reaction route is as follows:

[0051] Data for compound L2: 1 H NMR (600 MHz, DMSO- d 6) δ 7.90 (tq, J = 6.8, 1.8 Hz,3H), 7.84 – 7.73 (m, 12H), 7.33 (d, J = 7.9 Hz, 1H), 7.27 (s, 1H), 7.23 –7.17 (m, 2H), 7.11 (d, J = 26.3 Hz, 4H), 4.16 (t, J = 6.9 Hz, 2H), 3.65 –3.58 (m, 2H), 2.90 (t, J = 6.8 Hz, 2H), 2.40 – 2.35 (m, 2H), 1.74 – 1.67 (m,2H), 1.56 (t, J = 7.9 Hz, 2H). 13 C NMR (150 MHz, DMSO- d6) δ 172.63, 160.89,157.29, 136.23, 135.07 (d, J =3.0 Hz, 3C), 133.73 (d, J =10.5 Hz, 6C), 132.70,130.40 (d, J =12.0 Hz, 6C), 130.13, 127.01, 126.49, 125.79, 118.64 (d, J =85.5Hz, 3C), 63.61, 32.53, 30.20, 25.26 (d, J =16.5 Hz, 1C), 21.28 (d, J =4.5 Hz, 1C), 20.10 (d, J =16.5 Hz, 1C). HRMS (ESI) (m / z) [M-Br] + calcd for C 33 H 37 N5O2P + ,566.2679; found 566.2695.

[0052] Example 16: Preparation of compound L3 2.1 g (0.0153 mol) of 2-aminophenylethanol was used to replace 3-aminophenylethanol in Example 1, and 2.54 g (0.0131 mol) of 6-bromohexanoic acid was used to replace 3-bromopropionic acid in Example 1, while other conditions remained unchanged, to obtain compound L3 (0.0954 g, yield: 2.2%).

[0053]

[0054] Data for compound L3: 1 H NMR (600 MHz, DMSO- d 6) δ 7.91 – 7.88 (m, 3H), 7.83 –7.76 (m, 12H), 7.26 (d, J = 46.0 Hz, 6H), 7.20 – 7.03 (m, 4H), 4.17 (t, J =7.0 Hz, 2H), 3.63 – 3.59 (m, 2H), 2.94 (d, J = 7.7 Hz, 2H), 2.26 – 2.21 (m,2H), 1.53 – 1.45 (m, 6H). 13 C NMR (150 MHz, DMSO-d 6) δ 172.71, 160.86, 157.13,136.38, 134.89 (d, J =3.0 Hz, 3C), 133.63 (d, J =10.5 Hz, 6C), 132.14, 130.25 (d, J =12.0 Hz, 6C), 129.97, 126.80, 126.21, 125.20, 118.57 (d, J =85.5 Hz, 3C),63.40, 33.14, 30.20, 29.17 (d, J =16.5 Hz, 1C), 23.61 (d, J =3.0 Hz, 1C), 21.54(d, J =4.5 Hz, 1C), 20.15 (d, J =49.5 Hz, 1C). HRMS (ESI) (m / z) [M-Br] + calcd forC 34 H 39 N5O2P + , 580.2836; found 580.2852.

[0055] This embodiment also provides the application of the biguanide-triphenylphosphine bromide cationic compound prepared by the aforementioned method.

[0056] Experimental Example 1 The MTT assay was used to detect the antitumor activity and toxicity of each compound, with metformin and phenformin as positive controls. Human lung cancer cell line A549, human ovarian cancer cell lines OVCAR3 and SKOV3, human bladder cancer cell lines J82 and T24, and normal human bladder cancer cell line SV-HUC-1 in logarithmic growth phase were seeded into 96-well plates at a density of 7000 cells per well. The seeded plates were incubated overnight. Different concentrations of the compound (0, 0.01, 0.1, 1, 10, 100 μM) were then used to treat the 96-well plates. After treatment, the plates were returned to the incubator and cultured for 72 hours. The absorbance (OD) value (wavelength 490 nm) was recorded. Cell inhibition rate (%) = [(OD value of blank control - OD value of drug treatment) / OD value of blank control group] * 100%. The compounds of this invention showed significant inhibitory effects on various cancer cells, as shown in Table 1.

[0057] Table 1

[0058] The structural formula of compound 3a is: The structural formula of compound 3b is: The structural formula of compound 3c is: The structural formula of compound 3d is: .

[0059] By adopting the above technical solution, the biguanide-triphenylphosphine bromide cationic compound synthesized in this invention has an inhibitory effect on a variety of cancer cells. Among them, compound J6 has the strongest activity in bladder cancer cells J82, and its activity is greatly enhanced compared with biguanide derivatives.

[0060] Experimental Example 2: Detection of ferroptosis induced by compound J6 in cancer cells (1) Detection of intracellular reactive oxygen species levels Cells were loaded at 3 × 10 5 The cells were seeded at a density of 1 / 2 well in 6-well plates and incubated overnight at 37°C with 5% CO2. After complete cell attachment, J6 drug at concentrations of 0, 2, 4, and 8 μM was added for 48 hours. The cell culture medium was aspirated, and the cells were gently washed 1-2 times with PBS. An appropriate amount of DCFH-DA working solution was added. After incubation, the staining solution was aspirated, and the cells were washed 3 times with PBS to remove any unadsorbed dye. 2 mL of PBS was added, and the stained cells were observed under a fluorescence microscope. Figure 1 As shown, after treatment with different concentrations of J6 for 48 hours, the fluorescence intensity in J82 cells increased in a concentration-dependent manner, indicating that J6 treatment can significantly increase the intracellular ROS level.

[0061] (2) Detection of intracellular lipid peroxides Cells were loaded at 3 × 10 5 The cells were seeded at a density of [number] wells in 6-well plates and incubated overnight at 37°C with 5% CO2. After cell attachment, different concentration gradients (0, 2, 4, 8 μM) of J6 were added for 48 h. The culture medium was discarded, and the cells were washed twice with PBS. 1 mL of BODIPY 581 / 591 C11 staining working solution was added to each well, and the cells were incubated at 37°C with 5% CO2 in the dark for 20 min. After incubation, the supernatant was aspirated, and the cells were washed twice with PBS. 2 mL of PBS was added, and the cells were observed using a fluorescence microscope. Figure 2 As shown, the green fluorescence signal in the cells treated with compound J6 was significantly enhanced, while the red fluorescence signal was weakened, indicating that the level of lipid peroxides in J82 cells was significantly increased after treatment with compound J6.

[0062] (3) Detection of expression levels of ferroptosis-related proteins Experimental procedure: J82 cells were cultured at a concentration of 2.5 × 10⁻⁶.5 Cells were seeded at a density of 0.5 μM in 6-well plates and treated with J6 at 0, 2, 4, and 8 μM, with PBS used as a control. Protein extracts were loaded and electrophoresed on SDS-PAGE, then transferred to a polyvinylidene fluoride (PVDF) membrane. The blot was blocked with 5% milk and incubated overnight at 4°C with GPX4, GAPDH, SLC7A11, and NRF2. After washing with TBST for 30 min, the blot was incubated with secondary antibody for 1 h. After washing again with PBS, the blot was observed using a ChemiDoc system (Tanon 4600, Shanghai, China). Finally, the gray values ​​of the bands in the resulting images were measured using ImageJ software for protein quantification.

[0063] Test results are as per the instruction manual. Figure 3 As shown, compound J6 can significantly downregulate the expression of NRF2, SLC7A11 and GPX4 in a dose-dependent manner.

[0064] The above results indicate that compound J6 disrupts the intracellular antioxidant defense system by affecting the NRF2 / SLC7A11 / GPX4 pathway, leading to the accumulation of lipid peroxides and an imbalance in redox balance, ultimately inducing ferroptosis.

[0065] Experimental Example 3: Detection of apoptosis induced by compound J6 in cancer cells Cells were loaded at 3 × 10 5 The cells were seeded at a density of / well in 6-well plates and incubated overnight at 37°C with 5% CO2. After complete cell adhesion, the cells were treated with 0, 2, 4, and 8 μM compound J6 for 48 h. Cells were then digested with EDTA-free trypsin (37°C, 1-2 min), gently detached by blowing, and the digestion was stopped by adding serum-containing medium. The cells were washed twice with PBS (1000 rpm, 5 min), the PBS was discarded, and 100 μL of 1× Binding Buffer was added. 5 μL of Annexin V-FITC / 5 μL PI solution was added to each well, and the cells were incubated on ice in the dark for 30 minutes. After incubation, the cells were immediately analyzed by flow cytometry. Results are as follows: Figure 4 As shown, after treatment with 8 μM compound J6 for 48 h, approximately 47.5% of the cells exhibited late apoptosis, and approximately 5.02% of the cells exhibited early apoptosis, indicating that compound J6 can significantly induce apoptosis in bladder cancer cells.

[0066] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of protection of this application is limited to these examples; within the framework of this application, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of different aspects of one or more embodiments of this application as described above, which are not provided in detail for the sake of brevity.

[0067] One or more embodiments in this application are intended to cover all such substitutions, modifications, and variations that fall within the broad scope of this application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of one or more embodiments in this application should be included within the protection scope of this application.

Claims

1. A mitochondrial-targeting biguanide derivative, characterized in that, The structure of the biguanide derivative is as follows: , or ; Where n is 2-5, n1 is 2-5, and n2 is 3-5.

2. The biguanide derivative as described in claim 1, characterized in that, n is 3-5, n1 is 2-5, and n2 is 3-5.

3. The biguanide derivative as described in claim 1, characterized in that, n is 3, n1 is 3, and n2 is 3-4.

4. The biguanide derivative as described in claim 1, characterized in that, n is 3, and n1 is 3.

5. A method for preparing a biguanide derivative as described in any one of claims 1-4, characterized in that, An aqueous solution of compound 1 was mixed with compound 2 under stirring, and concentrated hydrochloric acid was added. The mixture was refluxed at 70-90°C, and the crude product was obtained by chromatography. The crude product was purified to obtain the biguanide derivative. Compound 1 is... Compound 2 is Or compound 1 is Compound 2 is Or compound 1 is Compound 2 is .

6. An application of a biguanide derivative as described in any one of claims 1-4, characterized in that, The biguanide derivatives are used to prepare drugs for treating cancer.

7. The application as described in claim 6, characterized in that, The cancer in question is bladder cancer.

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