Phenanthroimidazole derivative as well as preparation method and application thereof

Abstract

The invention provides a phenanthroimidazole derivative as well as a preparation method and application thereof, and belongs to the technical field of medicine synthesis. The phenanthroimidazole derivative provided by the invention has chemical structures as shown in a formula I and a formula II. A rigid planar skeleton of the phenanthroimidazole derivative is utilized, a substituent group is introduced, the targeting capacity is improved by enhancing pi-pi accumulation and hydrophobic interaction with HDAC1 active sites, meanwhile, lipophilicity is optimized to enhance the transmembrane transport efficiency, and the obtained phenanthroimidazole derivative can be used as an HDAC1 inhibitor when being used for preparing antitumor drugs. Formulae I and II

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine synthesis, and particularly relates to a phenanthroimidazole derivative and a preparation method and application thereof. BACKGROUND

[0002] GBM overexpresses histone deacetylase 1 (HDAC1), which drives tumor growth by epigenetic dysregulation of histone acetylation, increases invasiveness, treatment resistance and leads to poor clinical outcomes. Preclinical studies have shown that HDAC1 activates epithelial-mesenchymal transition (EMT) and inhibits tumor suppressor networks to promote tumorigenesis. Selective HDAC1 inhibitor RGFP109 reduces malignant behavior by restoring acetylation-dependent transcriptional homeostasis. Next-generation multi-target drugs (such as DDI199, Panobinostat) target HDAC1 / 6 subtypes and metabolic regulators (MAO-B / BChE), inhibit tumor initiating cell populations and overcome chemotherapy resistance in orthotopic models to cope with the inherent heterogeneity and adaptive resistance of GBM. In addition, HDAC1 inhibitors have immunomodulatory effects by changing the tumor microenvironment. SAHA-mediated HDAC1 inhibition disrupts c-Myc / CCL1-dependent Treg recruitment and increases CD8+ T cell infiltration by blocking the PD-L1 axis, improving survival in immunocompetent GBM models. The existing HDAC inhibitor Abexinostat mainly targets HDAC1 (Ki: 7nM), which can optimize central nervous system (CNS) formulations and improve blood-brain barrier penetration; and a common HDACi, MTX110 (soluble Panobinostat), delivered using cyclodextrin, can reduce tumor burden and prolong survival (median OS: 12-13 months) in recurrent GBM cohorts. These studies show that HDAC1 inhibitors show potential therapeutic effects in glioblastoma, so exploring new HDAC inhibitors has broad application prospects.

[0003] Heterocyclic compounds are the cornerstone of medicinal chemistry, among which benzimidazoles are particularly notable for their unique bicyclic structure resulting from the fusion of a benzene ring and an imidazole ring. Some benzimidazole derivatives have been identified as inhibitors of histone lysine methyltransferase DOT1L. DOT1L plays a key role in the development of hematological malignancies such as acute myeloid leukemia; in addition, nicotinate phosphoribosyltransferase (NAPRT) plays a key role in nicotinamide adenine dinucleotide (NAD) biosynthesis, and NAD metabolic disorders are associated with various cancers, neurodegenerative diseases and metabolic diseases. Studies have found that benzimidazole compounds with appropriate substitutions can be effective modulators of NAPRT. Of course, benzimidazole derivatives also play an important role in other aspects. For the global parasitic disease schistosomiasis, benzimidazole analogs have been found to have activity against Schistosoma mansoni adult worms. Preliminary mechanism studies have shown that these compounds may act by inhibiting heme formation, providing a new direction and target for the development of new anti-schistosome drugs; in the face of the increasingly serious problem of drug-resistant malaria, molecular hybridization and metal incorporation strategies have been applied to the development of benzimidazole derivatives. The existing technology "Ferrocenyl Quinoline-Benzimidazole Hybrids" shows significant potential in combating drug-resistant malaria through a multi-stage strategy, which demonstrates the effectiveness of structural modification in overcoming drug resistance. Therefore, benzimidazoles have great potential and broad application prospects in drug development and will undoubtedly be full of hope in the future field of drug innovation. SUMMARY

[0004] The object of the present application is to provide a phenanthroimidazole derivative, a preparation method and application thereof. The phenanthroimidazole derivative provided by the present application can be used as an HDAC1 inhibitor in the preparation of an anti-tumor drug.

[0005] In order to achieve the object of the present application, the present application provides the following technical solutions. A phenanthroimidazole derivative has any one of the following chemical structures: Formula I, Formula II; In Formula I and Formula II, R is ; In R of Formula I, R1, R2 and R3 are each independently selected from one or more of an alkyl group with 1-5 carbon atoms, an alkinyl group with 2-5 carbon atoms, a substituted alkinyl group with 2-5 carbon atoms, iodine, fluorine and dioxolane; each of R1, R2, R3 in R of formula II is independently selected from one or more of hydrogen, alkyl group with carbon number of 1-5, substituted alkyl group with carbon number of 1-5, alkyne group with carbon number of 2-7, substituted alkyne group with carbon number of 2-7, halogen and dioxolane; n is 2-16.

[0006] Preferably, the alkyl group with carbon number of 1-5 in formula I is selected from methyl, ethyl or propyl; the alkyne group with carbon number of 2-5 is ethynyl; the substituted alkyne group with carbon number of 2-5 is trimethylsilyl ethynyl or phenyl ethynyl.

[0007] Preferably, the alkyl group with carbon number of 1-5 in formula II is selected from methyl, ethyl or propyl; the substituted alkyl group with carbon number of 1-5 is monofluoromethyl or trifluoromethyl; the alkyne group with carbon number of 2-5 is ethynyl; the substituted alkyne group with carbon number of 2-5 is trimethylsilyl ethynyl or phenyl ethynyl; the halogen is selected from fluorine, chlorine, bromine or iodine.

[0008] Preferably, the chemical structure is shown in any one of the following structural formulae: , , , , , , , , , , , , , , , , , , , , , , , , , , .

[0009] The present application also provides a preparation method of the phenanthroimidazole derivative described in the above technical solution, and the preparation method of the phenanthroimidazole derivative shown in formula I comprises: The phenanthroimidazole derivative shown in formula I is prepared by mixing 1,10-phenanthroline-5,6-dione, R-formaldehyde, ammonium acetate and acetic acid. The preparation method of the phenanthroimidazole derivative shown in formula II comprises: The phenanthroimidazole derivative shown in formula I is reacted with Br(CH) n X and K2CO3 to obtain the phenanthroimidazole derivative shown in formula II. The Br(CH) n X in X is H or Br, and n is 2-16.

[0010] Preferably, the temperature for preparing the phenanthroimidazole derivative is 80-120 DEG C, and the time is 1-8 h.

[0011] Preferably, the preparation of the phenanthroimidazole derivative is carried out under microwave irradiation, and the time of microwave irradiation is 5-30 min.

[0012] The application further provides the use of the phenanthroimidazole derivative prepared by the preparation method in the preparation of an anti-tumor drug.

[0013] The application further provides the use of the phenanthroimidazole derivative prepared by the preparation method in the preparation of an HDAC-targeting anti-tumor drug.

[0014] The application provides a phenanthroimidazole derivative, which has a chemical structure shown in any one of formula I or formula II. BRIEF DESCRIPTION OF DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the application or the prior art, the drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative effort on the basis of these drawings.

[0016] Figure 1Inhibitory effect of the iodophenanthroimidazole derivative obtained in Example 4 on proliferation of brain glioma cells; Figure 2 Inhibitory effect of the iodophenanthroimidazole derivative obtained in Example 4 on proliferation and metastasis of U87-MG glioblastoma cells in a zebrafish glioblastoma orthotopic model; Figure 3 Inhibitory effect of the iodophenanthroimidazole derivative obtained in Example 4 as an HDAC1 inhibitor. DETAILED DESCRIPTION

[0017] The present application provides a phenanthroimidazole derivative having a chemical structure as shown in Formula I or Formula II: Formula I, Formula II; Formula I or Formula II, R is ; In Formula I, R1, R2, and R3 are each independently selected from one or more of alkyl having 1-5 carbon atoms, alkynyl having 2-5 carbon atoms, substituted alkynyl having 2-5 carbon atoms, iodine, fluorine, and dioxolane; In Formula II, R1, R2, and R3 are each independently selected from one or more of hydrogen, alkyl having 1-5 carbon atoms, substituted alkyl having 1-5 carbon atoms, alkynyl having 2-7 carbon atoms, substituted alkynyl having 2-7 carbon atoms, halogen, and dioxolane.

[0018] In the present application, all raw materials for preparation are commercially available products well known to those skilled in the art or are prepared by using preparation methods well known to those skilled in the art, unless otherwise specified.

[0019] In the present application, in Formula I or Formula II, R is a substituted phenyl group; R1, R2, and R3 in the substituted phenyl group are selected from one or more of alkyl having 1-5 carbon atoms, substituted alkyl having 1-5 carbon atoms, alkynyl having 2-5 carbon atoms, substituted alkynyl having 2-5 carbon atoms, halogen, and dioxolane; in specific embodiments, it can be one or more of methyl, monofluoromethyl, trifluoromethyl, ethynyl, trimethylsilyl ethynyl, phenylacetylenyl, fluorine, chlorine, bromine, and iodine; when there are multiple substituents, they can be ortho-substituted, para-substituted, or meta-substituted.

[0020] In the present application, n in Formula II is 2-16, and in specific embodiments, it can be 2, 3, 4, 6, 8, or 10.

[0021] In the present application, the phenanthroimidazole derivative has a chemical structure as shown below: , , , 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 .

[0022] The application further provides a preparation method of the phenanthroimidazole derivative. The phenanthroimidazole derivative shown in the structure of formula I is obtained by mixing 1,10-phenanthroline-5,6-dione, R-formaldehyde, ammonium acetate and acetic acid and then performing reaction. The preparation method of the phenanthroimidazole derivative shown in the structure of formula II comprises the following steps. The phenanthroimidazole derivative shown in the structure of formula II is obtained by mixing the phenanthroimidazole derivative shown in the structure of formula I, Br(CH) n X and K2CO3 and then performing reaction. n X and K2CO3 and then performing reaction.

[0023] In the application, the temperature during preparation of the phenanthroimidazole derivative is 80-120℃, and in specific embodiments, can be 100 or 110℃; the time is 1-8h, and in specific embodiments, can be 5 or 6h; the preparation of the phenanthroimidazole derivative is performed under microwave irradiation; and the time of microwave irradiation is 5-30min.

[0024] The application further provides application of the phenanthroimidazole derivative or the phenanthroimidazole derivative prepared by the preparation method in preparation of an HDAC-targeting PROTACs antitumor drug.

[0025] In order to further illustrate the application, the phenanthroimidazole derivative, the preparation method and the application thereof provided by the application are described in detail below in combination with the drawings and examples, but they should not be understood as limiting the protection scope of the application.

[0026] Example 1 The following compound was prepared according to the procedure described in Example 1, except that 2-methylbenzaldehyde was replaced by 4-methylbenzaldehyde; ESI-MS (in DMSO, m / z): 312.14, [M+H] A mixture of 1,10-phenanthroline-5,6-dione (315.00 mg, 1.50 mmol), 2- methylbenzaldehyde (238.78 mg, 2.25 mmol), ammonium acetate (4.50 g, 51.9 mmol) and glacial acetic acid (20 mL) was placed in a 30 mL quartz microwave reaction tube equipped with a magnetic stir bar. After the solid was completely dissolved with stirring, the reaction mixture was subjected to microwave irradiation at 100 °C for 20 min. The resulting solution was transferred to a beaker, diluted with distilled water (20 mL), and the pH was adjusted to 7.0 with concentrated ammonia water at room temperature. The yellow crude product was isolated by suction filtration under reduced pressure and dried. The crude product was dissolved in ethanol and purified on a silica gel (60-100 mesh) column with ethanol as the eluent. The target fractions were collected, concentrated under reduced pressure and dried to give a yellow solid in 83.2% yield.

[0027] ESI-MS (in DMSO, m / z): 312.14, [M+H] + .

[0028] Example 2 Prepared according to the procedure described in Example 1, except that 2-methylbenzaldehyde was replaced by 4-methylbenzaldehyde; ESI-MS (in DMSO, m / z): 312.14, [M+H] + .

[0029] Example 3 Prepared according to the procedure described in Example 1, except that 2-methylbenzaldehyde was replaced by 6-bromopiperonal; ESI-MS (in DMSO, m / z): 418.00, [M+H] + .

[0030] 1 H NMR (500 MHz, DMSO) δ 9.05 (d, J = 3.5 Hz, 2H), 8.87 (ddd, J = 14.2, 8.1, 1.4 Hz, 2H), 7.48 (s, 1H), 7.42 (d, J = 2.0 Hz, 2H), 7.28 (s, 1H), 6.22(d, J = 4.8 Hz, 2H). 13C NMR (126 MHz, DMSO) δ 153.93 (s), 150.00 (s), 149.87 (s), 148.58 (s), 147.74 (s), 144.05 (s), 135.52 (s), 130.09 (s), 127.85 (s), 125.88 (s), 123.95 (s), 123.70 (s), 121.15 (s), 113.93 (s), 113.74 (s), 113.51 (s), 111.97 (s), 107.91 (s), 103.79 (s), 103.14 (s).

[0031] Example 4 Prepared according to the procedure described in Example 1, except that 2- methylbenzaldehyde was replaced by 4-iodobenzaldehyde; ESI-MS (in DMSO, m / z): 422.23, [M+H] + .

[0032] 1 H NMR (600 MHz, DMSO) δ 9.05 – 9.02 (m, 2H), 8.89 (dd, J = 8.0, 1.1Hz, 2H), 8.08 – 8.05 (m, 2H), 8.02 – 7.98 (m, 2H), 7.83 (dd, J = 17.3, 3.8 Hz,2H). 13 C NMR (151 MHz, DMSO) δ 150.13 (s), 148.38 (s), 144.03 (s), 138.32 (s), 130.13 (s), 129.92 (s), 128.48 (s).

[0033] Example 5 Prepared according to the procedure described in Example 4, except that 4- methylbenzaldehyde was replaced by 3-iodobenzaldehyde.

[0034] Example 6 Prepared according to the procedure described in Example 4, except that 4- methylbenzaldehyde was replaced by 2-iodobenzaldehyde. Example 7 Prepared according to the procedure described in Example 1, except that 2-methylbenzaldehyde was replaced by 2,3-difluorobenzaldehyde; ESI-MS (in DMSO, m / z): 332.31, [M+H] + .

[0035] Example 8 Prepared according to the procedure described in Example 1, except that 2-methylbenzaldehyde was replaced by 4-(phenylethynyl)benzaldehyde; denoted as L0627; ESI-MS (in DMSO, m / z): 396.14, [M+H] + .

[0036] Example 9 Prepared according to the procedure described in Example 1, except that 2-methylbenzaldehyde was replaced by 2-alkynylbenzaldehyde; denoted as L0628; ESI-MS (in DMSO, m / z): 320.35, [M+H] + .

[0037] Example 10 Prepared according to the procedure described in Example 1, except that 2-methylbenzaldehyde was replaced by 3-alkynylbenzaldehyde; denoted as L0629; ESI-MS (in DMSO, m / z): 320.35, [M+H] + .

[0038] Example 11 Prepared according to the procedure described in Example 1, except that 2-methylbenzaldehyde was replaced by phenylacetylene aldehyde; denoted as L0630; ESI-MS (in DMSO, m / z): 320.35, [M+H] + .

[0039] Example 12 Prepared according to the procedure described in Example 1, except that 2-methylbenzaldehyde was replaced by 4-((trimethylsilyl)ethynyl)benzaldehyde; denoted as L0631; ESI-MS (in DMSO, m / z): 392.54, [M+H] + .

[0040] Example 13 The phenanthroimidazole derivative obtained in Example 3 was further used to prepare alkyl chain modified phenanthroimidazole derivatives, the reaction formula is shown below, and the specific steps are as follows: ; ; The phenanthroimidazole derivative obtained in Example 3 (200 mg, 0.478 mmol), anhydrous potassium carbonate (2 g, 14.47 mmol) and 10 mL of DMF were weighed; stirred for 10 min at room temperature, then 1,5-dibromopentane (2.170 g, 9.56 mmol) was added; transferred to a microwave reactor and reacted at 60°C for 30 min; after the reaction was completed, it was filtered under suction and rotary evaporated to obtain a crude product; eluted with dichloromethane and ethanol, and the light blue fluorescent band was collected to obtain the target product with a yield of 79%; ESI-MS (in CH2Cl2, m / z): 569.00, [M+H] + .

[0041] 1 H NMR (500 MHz, CDCl3) δ 9.29 (dd, J = 4.5, 1.7 Hz, 2H), 9.23 (dd, J =4.4, 1.4 Hz, 1H), 9.14 (dd, J = 8.1, 1.7 Hz, 1H), 8.62 (dd, J = 8.4, 1.6 Hz, 2H),7.81 (dt, J = 8.3, 4.1 Hz, 1H), 7.21 (s, 1H), 7.08 (s, 2H), 6.15 (s, 2H), 4.49(d, J = 132.9 Hz, 2H), 3.67 (t, J = 6.4 Hz, 2H), 1.83 (dp, J = 61.8, 7.0 Hz,4H),1.60 (dt, J = 15.8, 6.7 Hz, 2H). 13C NMR (126 MHz, CDCl3) δ 152.08 (s), 150.38(s), 136.20 (s), 132.02 (s), 128.64 (s), 124.38 (s), 124.10 (s), 123.82 (s),123.36 (s), 120.13 (s), 116.06 (s), 113.12 (s), 112.01 (s), 102.66 (s), 62.55(s), 46.68 (s), 33.75 (s), 32.98 (s), 32.52 (s), 31.62 (s), 28.90 (s), 25.05(s), 24.46 (s)。

[0042] Example 14 Prepared according to the procedure described in Example 11, except that 1,5- dibromopentane was replaced by 1,6-dibromohexane, in 83% yield; ESI-MS (in CH2Cl2, m / z): 583.02, [M+H] + .

[0043] 1 H NMR (500 MHz, CDCl3) δ 9.19 (ddd, J = 8.2, 4.3, 1.6 Hz, 2H), 9.04(dd, J = 8.1, 1.8 Hz, 1H), 8.56 (dd, J = 8.4, 1.6 Hz, 1H), 7.72 (dt, J = 8.5, 4.7Hz, 2H), 7.20 (s, 1H), 7.06 (s, 1H), 6.14 (s, 2H), 4.46 (d, J = 133.8 Hz, 2H),3.33 (t, J = 6.6 Hz, 2H), 1.77 (q, J = 6.9 Hz, 2H), 1.34 (dp, J = 21.9, 7.0, 6.4Hz, 4H). 13C NMR (126 MHz, CDCl3) δ 151.69 (s), 150.20 (s), 148.99 (s), 147.89(s), 147.77 (s), 144.96 (s), 144.23 (s), 136.58 (s), 130.39 (s), 128.13 (s),124.51 (s), 124.19 (s), 124.13 (s), 123.58 (s), 122.62 (s), 119.95 (s),116.15 (s), 113.08 (s), 112.04 (s), 102.58 (s), 46.63 (s), 33.42 (s), 32.21(s), 29.61 (s), 27.47 (s), 25.67 (s)..

[0044] Example 15 Prepared according to the procedure described in Example 11, except that 1,5- dibromopentane was replaced by 1,8-dibromooctane, in 85% yield; ESI-MS (in CH2Cl2, m / z): 611.05, [M+H] + .

[0045] 1 H NMR (500 MHz, CDCl3) δ 9.19 (ddd, J = 8.1, 4.3, 1.7 Hz, 2H), 9.05(dd, J = 8.1, 1.8 Hz, 1H), 8.57 (dd, J = 8.4, 1.7 Hz, 1H), 7.72 (dt, J = 8.4, 4.4Hz, 2H), 7.20 (s, 1H), 7.07 (s, 1H), 6.13 (s, 2H), 4.44 (d, J = 137.5 Hz, 2H),3.36 (t, J = 6.7 Hz, 2H), 2.02 – 1.72 (m, 4H), 1.39 – 1.17 (m, 8H). 13C NMR (126 MHz, CDC13) δ 151.70 (s), 150.14 (s), 148.96 (s), 147.86 (s), 147.71 (s), 144.95 (s), 144.23 (s), 136.56 (s), 130.39 (s), 128.20 (s), 124.60 (s), 124.18 (s), 123.56 (s), 122.59 (s), 119.99 (s), 116.18 (s), 113.05 (s), 112.09 (s), 102.54 (s), 46.81 (s), 33.87 (s), 32.55 (s), 29.74 (s), 28.71 (s), 28.38 (s), 27.89 (s), 26.36 (s).

[0046] Example 16 Prepared according to the procedure described in Example 11, except that 1,5- dibromopentane was replaced by 1,9-dibromooctane, in 80% yield; ESI-MS (in CH2Cl2, m / z): 625.06, [M+H] + .

[0047] 1 H NMR (500 MHz, CDC13) δ 9.18 (ddd, J = 8.2, 4.3, 1.8 Hz, 2H), 9.04 (dd, J = 8.2, 1.8 Hz, 1H), 8.57 (dd, J = 8.4, 1.7 Hz, 1H), 7.72 (dt, J = 9.6, 4.0 Hz, 2H), 7.20 (d, J = 1.7 Hz, 1H), 7.07 (d, J = 1.6 Hz, 1H), 6.13 (s, 2H), 4.44 (d, J = 138.6 Hz, 2H), 3.37 (td, J = 6.8, 1.6 Hz, 2H), 1.96 - 1.75 (m, 4H), 1.38 - 1.16 (m, 10H). 13C NMR (126 MHz, CDCl3) δ 151.70 (s), 150.13 (s), 148.97 (s),147.86 (s), 147.70 (s), 144.96 (s), 144.24 (s), 136.55 (s), 130.37 (s),128.22 (s), 124.63 (s), 124.17 (s), 123.55 (s), 122.58 (s), 120.00 (s),116.18 (s), 113.04 (s), 112.11 (s), 102.53 (s), 46.84 (s), 33.92 (s), 32.66(s), 29.73 (s), 29.06 (s), 28.78 (s), 28.52 (s), 27.97 (s), 26.44 (s)。

[0048] Example 17 Prepared according to the procedure described in Example 11, except that 1,5- dibromopentane was replaced by 1,10-dibromodecane, in 83% yield; ESI-MS (in CH2Cl2, m / z): 639.08, [M+H] + .

[0049] 1 H NMR (500 MHz, CDCl3) δ 9.18 (ddd, J = 7.9, 4.3, 1.7 Hz, 2H), 9.05 (dd, J = 8.1, 1.8 Hz, 1H), 8.57 (dd, J = 8.4, 1.6 Hz, 1H), 7.72 (ddd, J = 8.1, 4.3,3.1 Hz, 2H), 7.20 (s, 1H), 7.07 (s, 1H), 6.13 (s, 2H), 4.44 (d, J = 138.1 Hz,2H), 3.38 (t, J = 6.8 Hz, 2H), 1.93 – 1.74 (m, 4H), 1.42 – 1.16 (m, 12H). 13C NMR (126 MHz, CDC13) δ 151.70 (s), 150.13 (s), 148.95 (s), 147.85 (s), 147.70 (s), 144.94 (s), 144.22 (s), 136.54 (s), 130.39 (s), 128.24 (s), 124.62 (s), 124.21 (s), 124.18 (s), 123.55 (s), 122.58 (s), 120.01 (s), 116.18 (s), 113.03 (s), 112.11 (s), 102.53 (s), 46.85 (s), 34.01 (s), 32.69 (s), 29.76 (s), 29.22 (s), 29.15 (s), 28.86 (s), 28.62 (s), 28.06 (s), 26.47 (s).

[0050] Example 18 Prepared according to the procedure described in Example 11, except that 1,5- dibromopentane was replaced by 1,12-dibromododecane, in 78% yield; ESI-MS (in CH2Cl2, m / z): 667.11, [M+H] + .

[0051] 1 H NMR (500 MHz, CDC13) δ 9.18 (ddd, J = 7.6, 4.3, 1.7 Hz, 2H), 9.05 (dd, J = 8.1, 1.8 Hz, 1H), 8.57 (dd, J = 8.4, 1.7 Hz, 1H), 7.72 (ddd, J = 8.2, 4.3, 2.5 Hz, 2H), 7.20 (s, 1H), 7.06 (s, 1H), 6.13 (s, 2H), 4.43 (d, J = 138.4 Hz, 2H), 1.94 - 1.74 (m, 4H), 1.51 - 1.07 (m, 18H). 13C NMR (126 MHz, CDCl3) δ 151.70 (s), 150.13 (s), 148.92 (s), 147.84 (s), 147.70 (s), 144.91 (s), 144.19 (s), 136.51 (s), 130.42 (s), 128.26 (s), 124.62 (s), 124.21 (s), 124.19 (s), 123.56 (s), 122.59 (s), 120.02 (s), 116.17 (s), 113.02 (s), 112.10 (s), 102.52 (s), 46.86 (s), 34.06 (s), 32.77 (s), 29.77 (s), 29.40 (s), 29.37 (s), 29.26 (s), 28.91 (s), 28.70 (s), 28.11 (s), 26.49 (s).

[0052] Example 19 The phenanthroimidazole derivative is specifically as follows: Compound L02 (0.676 mmol, 200 mg), the structural formula of , was weighed in a 30 mL quartz tube, 12 mL DMF was added, and L02 was stirred to completely dissolve, then calcined K2CO3 (2 g, 14.47 mmol) was added, stirred for 10 min, and then excess 1,4-dibromobutane (1.6 mL) was slowly added. Microwave reaction at 60°C for 30 min, and the reaction was completed after cooling to room temperature. Filtration and rotary evaporation to obtain a crude product. Purification: the crude product was eluted with a mixture of dichloromethane and ethanol as a gradient eluent, and the yield was 75%, labeled as L02-BBr.

[0053] ESI-MS (in CH3CN, m / z): 431, [M+H] + . 1 H NMR (500 MHz, DMSO- d 6) δ 9.79 (d, J = 6.0 Hz, 1H), 9.57 (d, J = 3.8 Hz, 1H), 9.25 - 9.17 (m, 4H), 8.49 (s, 1H), 8.36 (d, J = 6.5 Hz, 2H), 8.14 (s, 1H), 7.67 (d, J= 6.7 Hz, 2H), 7.62 (d, J = 6.7Hz, 1H), 5.95 (s, 2H), 3.67 (s, 2H), 2.21 (d, J = 49.7 Hz, 2H), 2.07 (d, J = 34.5Hz, 2H). 13 C NMR (126 MHz, DMSO- d 6) δ 153.56-152.82 (m), 149.42 (s), 148.13(s), 140.17 (s), 137.94 (s), 134.54 (s), 131.75 (s), 131.06 (s), 129.70 (s),129.53 (s), 127.19 (s), 125.76 (s), 125.23 (s), 63.41 (s), 34.86 (s), 30.30(s), 29.69 (s). Example 20 The phenanthroimidazole derivative is specifically as follows: labeled as L271-BBr; Prepared according to the method described in Example 19, except that L02 was replaced with the compound L271 obtained in Example 1, in a yield of 71%, labeled as L271-BBr.

[0054] ESI-MS (in CH3CN, m / z): 445.10, [M+H] + . 1 H NMR (600 MHz, CDCl3) δ 9.20(td, J = 4.5, 1.5 Hz, 2H), 9.08 (dt, J = 8.1, 1.5 Hz, 1H), 8.59 (dt, J = 8.4, 1.3Hz, 1H), 7.74 (ddd, J = 8.0, 4.4, 3.4 Hz, 2H), 7.50 (t, J = 7.5 Hz, 2H), 7.47 –7.37 (m, 2H), 4.51 – 4.37 (m, 2H), 3.24 (t, J = 6.4 Hz, 2H), 2.31 (s, 3H), 2.06(p,J = 7.5 Hz, 2H), 1.83 – 1.71 (m, 2H). 13 C NMR (151 MHz, CDCl3) δ 153.22 (d, J =2.8 Hz), 148.97, 147.83, 138.37, 136.74, 130.82, 130.36, 129.62, 128.09,126.22, 124.20, 123.98, 123.61, 122.72, 119.91, 45.53, 32.12, 29.24, 28.44,19.90。

[0055] Example 21 The phenanthroimidazole derivative is specifically as follows: labeled as L273-BBr; Prepared according to the method described in Example 19, except that L02 was replaced with the compound L273 obtained in Example 2, in a yield of 76%, labeled as L273-BBr.

[0056] ESI-MS (in CH3CN, m / z): 445.10, [M+H] + . 1 H NMR (600 MHz, DMSO) δ 9.74(dt, J = 8.8, 2.8 Hz, 1H), 9.54 (dd, J = 5.9, 1.6 Hz, 1H), 9.27 – 9.10 (m, 2H),8.47 (dd, J = 8.1, 5.9 Hz, 1H), 8.21 (dd, J = 8.2, 2.1 Hz, 2H), 8.11 (dt, J = 8.2,4.1 Hz, 1H), 7.47 (d, J = 7.8 Hz, 2H), 4.22 (t, J = 6.6 Hz, 2H), 3.66 (d, J = 6.6Hz, 2H), 2.44 (s, 3H), 2.24 (ddd, J = 15.1, 8.7, 6.1 Hz, 2H), 2.10 (dp, J = 14.0,7.3, 6.8 Hz, 2H).13 C NMR (151 MHz, DMSO) δ 153.31, 149.32, 148.04, 147.93, 140.95, 140.10, 139.99, 137.83, 137.79, 134.38, 131.63, 130.24, 129.13, 127.08, 126.76, 125.71, 125.15, 125.09, 63.38, 34.87, 30.28, 29.68, 21.55, 19.12, 14.03.

[0057] Example 22 The phenanthroimidazole derivative is specifically as follows: Prepared according to the method described in Example 19, except that L02 was replaced by L043, yield: 81%, labeled as L043-BBr.

[0058] ESI-MS (in CH3CN, m / z): 450.80, [M+H] + . 1 H NMR (500 MHz, DMSO- d 6) δ 9.70 (s, 1H), δ 9.55 (dd, J = 5.9, 1.2 Hz, 1H), 9.21 (dd, J = 4.2, 1.8 Hz, 1H), 9.13 (s, 1H), 8.46 (s, 1H), 8.35 (d, J = 3.4 Hz, 2H), 8.12 - 8.06 (m, 1H), 7.51(t, J = 8.8 Hz, 2H), 5.95 - 5.86 (m, 2H), 3.67 (t, J = 6.6 Hz, 2H), 2.30 - 2.17(m, 2H), 2.16 - 2.03 (m, 2H). 13 C NMR (126 MHz, DMSO- d6) δ 164.86 (s), 162.89(s), 149.41 (s), 148.09 (s), 140.06 (s), 137.78 (s), 134.40 (s), 131.65 (s),126.11 (s), 125.69 (s), 125.19 (s), 116.87 (s), 116.70 (s), 63.38 (s), 34.84(s), 30.29 (s), 29.70 (s)。

[0059] Example 23 Phenanthroimidazole derivatives are specifically as follows: Prepared according to the procedure described in Example 19, except that L02 was replaced by L053, yield: 79%, labeled as L053-BBr.

[0060] ESI-MS (in CH3CN, m / z): 466.70, [M+H] + . 1 H NMR (500 MHz, DMSO- d 6) δ9.75 (d, J = 6.9 Hz, 1H), 9.55 (d, J = 4.5 Hz, 1H), 9.25 (dd, J = 4.3, 1.8 Hz, 1H),9.16 (dd, J = 8.3, 1.8 Hz, 1H), 8.49 (d, J = 6.2 Hz, 1H), 8.39 - 8.26 (m,2H),8.14 (d, J = 4.0 Hz, 1H), 7.78 - 7.70 (m, 2H), 6.06 - 5.83 (m, 2H), 3.67 (t, J =6.6 Hz, 2H), 2.30 - 2.19 (m, 2H), 2.14 - 1.98 (m, 2H). 13 C NMR (126 MHz, DMSO- d6) δ 152.08 (s), 149.51 (s), 148.23 (s), 140.16 (s), 138.01 (s), 135.70 (s), 134.64 (s), 131.68 (s), 129.85 (s), 128.81 (s), 128.38 (s), 125.81 (s), 125.29 (s), 63.45 (s), 34.85 (s), 30.29 (s), 29.67 (s).

[0061] Example 24 The phenanthroimidazole derivative is specifically as follows: Prepared according to the method described in Example 19, except that L02 was replaced by L063, yield: 77%, labeled as L063-BBr.

[0062] ESI-MS (in CH3CN, m / z): 510.70, [M+H] + . 1 H NMR (500 MHz, DMSO- d 6) δ 9.76 (d, J = 7.0 Hz, 1H), 9.57 (d, J = 4.6 Hz, 1H), 9.26 (dd, J = 4.3, 1.8 Hz, 1H), 9.17 (dd, J = 8.3, 1.6 Hz, 1H), 8.50 (s, 1H), 8.27-8.25 (m, 2H), 8.14 (d, J = 3.9Hz, 1H), 7.90-7.87 (m, 2H), 5.96-5.92 (m, 2H), 3.67 (t, J = 6.6 Hz, 2H), 2.30-2.18 (m,2H), 2.14-2.05 (m,2H). 13 C NMR (126 MHz, DMSO- d6) δ 152.31 (s), 149.53 (s), 148.24 (s), 140.16 (s), 138.03 (s), 134.65 (s), 132.76 (s), 131.71 (s), 128.99 (s), 128.72 (s), 125.81 (s), 125.31 (s), 124.51 (s), 63.43 (s), 34.86 (s), 30.30 (s), 29.69 (s).

[0063] Example 25 The phenanthroimidazole derivative is specifically as follows: Prepared according to the method described in Example 19, except that L02 was replaced by L081, in a yield of 82%, labeled as L081-BBr.

[0064] ESI-MS (in CH3CN, m / z): 499.07, [M+H] + . 1 H NMR (600 MHz, CDCl3) δ 9.21 (ddd, J = 10.0, 4.3, 1.8 Hz, 2H), 9.04 (dt, J = 8.1, 1.7 Hz, 1H), 8.57 (dd, J =8.4, 1.6 Hz, 1H), 7.94 (dd, J = 7.8, 1.6 Hz, 1H), 7.82 – 7.72 (m, 4H), 7.70(dd, J = 7.3, 1.7 Hz, 1H), 4.39 (d, J = 204.3 Hz, 2H), 3.30 (t, J = 6.5 Hz, 2H),2.02 (s, 2H). 13 C NMR (151 MHz, CDCl3) δ 149.89, 149.11, 148.04, 136.70,132.67, 132.09, 130.79, 130.49, 128.17, 127.09, 124.15, 123.68, 122.75,119.78, 46.15, 32.05, 29.38, 28.26.

[0065] Example 26 Phenanthroimidazole derivatives are specifically as follows: Prepared according to the procedure described in Example 19, except that L02 was replaced by L082, yield: 74%, labeled as L082-BBr.

[0066] ESI-MS (in CH3CN, m / z): 499.07, [M+H] + . 999.14 ([2M+H] + ), 1 H NMR (600MHz, DMSO) δ 9.73 (t, J = 7.9 Hz, 1H), 9.57 (d, J = 5.5 Hz, 1H), 9.24 – 9.19 (m,1H), 9.14 – 9.10 (m, 1H), 8.62 – 8.57 (m, 2H), 8.48 (dd, J = 8.3, 5.8 Hz, 1H),8.11 (dd, J = 8.2, 4.3 Hz, 1H), 7.96 (d, J = 7.8 Hz, 1H), 7.90 (t, J = 7.7 Hz, 1H),5.90 (t, J = 7.6 Hz, 2H), 3.66 (t, J = 6.6 Hz, 2H), 2.23 (q, J = 8.1 Hz, 2H), 2.10(q, J = 6.9 Hz, 2H). 13 C NMR (151 MHz, DMSO) δ 151.37, 149.61, 148.29, 140.19,138.03, 134.67, 131.70, 131.01, 130.42, 129.12, 127.37, 125.76, 125.33,123.34, 65.50, 64.26, 63.40, 60.71, 34.86, 30.46, 30.28, 28.67, 19.12, 14.02.

[0067] Example 27 Prepared according to the procedure described in Example 19, except that L02 was replaced by L083, yield: 83%, labeled as L083-BBr.

[0068] ESI-MS (in CH3CN, m / z): 499.07, [M+H] + . 1 H NMR (600 MHz, DMSO) δ 9.70(d, J = 8.1 Hz, 1H), 9.56 (dd, J = 5.9, 1.5 Hz, 1H), 9.20 (dd, J = 4.2, 1.8 Hz,1H), 9.11 (d, J = 8.3 Hz, 1H), 8.47 (d, J = 8.1 Hz, 3H), 8.08 (dd, J = 8.2, 4.2 Hz,1H), 8.02 (d, J = 8.2 Hz, 2H), 5.93 – 5.82 (m, 2H), 3.66 (t, J = 6.6 Hz, 2H),2.23 (ddd, J = 15.1, 8.7, 6.0 Hz, 2H), 2.09 (p, J = 6.8 Hz, 2H). 13 C NMR (151 MHz,DMSO) δ 151.35, 149.63, 148.31, 140.13, 138.01, 134.66, 133.09, 131.73,127.68, 126.64, 125.77, 123.62, 63.38, 34.85, 30.27, 29.67.

[0069] Test Example 1 Inhibition of tumor cell growth by iodophenanthroimidazole derivatives of Examples 4-6 (MTT colorimetric method) The proliferation inhibitory effect of halophenanthroimidazole derivatives of Examples 4-6 on tumor cell lines was evaluated using the MTT colorimetric method, and the specific steps were as follows: The cells (5×10 3HepG2 (hepatocellular carcinoma), MDA-MB-231 (metastatic breast cancer), U87-MG (glioblastoma), A549 (non-small cell lung cancer) and QSG-7701 (normal hepatocyte) were seeded in 96-well cell culture plates (n = 3) and pre-incubated for 24 h at 37 °C under 5% CO2 to allow cell attachment. Subsequently, gradient concentrations of the test compounds (0.1-100 mM concentration range) were added and incubation was continued for 72 h. Incubation was terminated by adding 20 pL MTT working solution (5 mg / mL in sterile PBS) per well and incubating for 4 h at 37 °C in the dark. The culture supernatant was gently aspirated and the purple crystals were dissolved in 150 pL DMSO and shaken on a microshaker for 10 min to ensure complete dissolution. The absorbance was measured using a Thermo Scientific Multiskan GO microplate reader at a wavelength of 570 nm. All experiments were independently repeated three times and the half maximal inhibitory concentration (IC50) and its 95% confidence interval were calculated using a four-parameter logistic regression model fitted with GraphPad Prism 8.02. 50

[0070] The structure-activity relationship between the modification of ortho-, meta- and para-iodo substituents in the iodophenanthrimidazole derivatives obtained in Examples 4-6 and their anti-tumor activity was investigated. The 72 h cytotoxicity of the three iodophenanthrimidazole derivatives against HepG2 (hepatocellular carcinoma), MDA-MB-231 (metastatic breast cancer), U87-MG (glioblastoma), A549 (non-small cell lung cancer) and QSG-7701 (normal hepatocyte) was evaluated by the MTT method described above. The experimental results are shown in Table 1.

[0071] Table 1 Results of the inhibitory effect of the iodophenanthrimidazole derivatives obtained in Examples 4-6 on different human cancer cell lines and normal cell lines

[0072] The IC 50 values in Table 1 are expressed as the mean ± standard deviation of three independent experiments; the safety index (SI) = IC 50 (QSG-7701) / IC 50 (U87-MG).

[0073] As can be seen from the results in Table 1, the position of the iodine substitution significantly affects the activity and selectivity of the compounds. The para-substituted iodophenanthrimidazole derivative showed the strongest inhibitory effect on U87-MG glioblastoma cells (IC 50 = 0.23 ± 0.10 mM), which was 11.2 times and 1.5 times more active than the ortho-substituted (IC 50 = 2.58 ± 0.04 mM) and meta-substituted (IC 50 = 0.35 ± 0.02 mM) derivatives, respectively. The para-substituted iodophenanthrimidazole derivative also showed lower toxicity against QSG-7701 normal hepatocytes (IC 50 ​= 0.80 ± 0.04 μM), the calculated safety index (SI = 3.39) indicates that it has a good therapeutic window. The results confirm that the steric hindrance of the iodine substituent directly affects the molecular targeting potency, especially for glioblastoma, which provides a key basis for the development of anti-tumor lead compounds based on structural optimization.

[0074] Test Example 2 The alkynyl-substituted phenanthroimidazole derivatives of Examples 8-12 were tested for their inhibition of tumor cell growth according to the method described in Test Example 1, and the results are shown below.

[0075] Table 2 Inhibition of different human cancer cell lines and normal cell lines by the alkynyl-substituted phenanthroimidazole derivatives of Examples 8-12

[0076] Note: Safe Index = IC 50 (QSG-7701) / IC 50 (HepG2) Test Example 3 The alkane chain-modified phenanthroimidazole derivatives of Examples 19-27 were tested for their inhibition of tumor cell growth according to the method described in Test Example 1, and the results are shown below.

[0077] Table 3 Inhibition of different cancer cell lines and normal cell lines by the alkane chain-modified phenanthroimidazole derivatives of Examples 19-27

[0078] Note: Safe Index = IC 50 (QSG-7701) / IC 50 (HepG2).

[0079] Test Example 4 Inhibition of brain glioma cell proliferation by the iodinated phenanthroimidazole derivative of Example 4 (EdU method) was tested according to the procedure shown below; the results are shown in Figure 1 .

[0080] Figure 1 Inhibition of brain glioma cell proliferation by the iodinated phenanthroimidazole derivative of Example 4; wherein (A) is the visualization result of the EdU incorporation experiment, the proliferating cells (red fluorescence) and the Hoechst 33342-stained nuclei (blue) after treatment with the iodinated phenanthroimidazole derivative of Example 4 (0, 0.05, 0.10, and 0.20 μM) for 72 hours, with a scale bar of 25 μm; (B) is a flow cytometry profile showing the cell cycle distribution (G0 / G1, S, G2 / M phase) under the indicated treatments; (C) is a quantitative analysis of the ratio of EdU positive cells relative to the control; (D) is a column chart quantification of the proportion of each phase of the cell cycle (n=3 independent experiments); (E) is a Western blot analysis of cell cycle regulators (Cyclin B1, Cyclin A2, CDK1, CDK2), with GAPDH as a loading control. U87-MG glioblastoma cells were treated with different concentrations of the iodophenalenyl imidazole derivative obtained in Example 4 (0, 0.05, 0.10 and 0.20 μΜ) for 72 hours.

[0081] Figure 1 EdU detection of the results obtained used the BeyoClick™ EdU Cell Proliferation Kit (C0078S, Beyotime). Protein expression levels were quantified using ImageJ software and normalized to GAPDH. Data are expressed as the mean ± standard error of the mean (SEM) of three independent experiments. P <0.01, * P <0.001.

[0082] U87-MG glioblastoma cells in the logarithmic growth phase were collected at a concentration of 5 x 10 4Cells were seeded at a density of 1 x 104cells / well in 6-well plates. After incubation in a humidified atmosphere (37 °C, 5% CO2) using a Heracell™ 150i incubator (Thermo Fisher Scientific, MA, USA) for 24 h, adherent cells were exposed to gradient concentrations of iodophenalene imidazole derivatives obtained from Example 4 (0, 0.05, 0.10 and 0.20 μΜ) for 72 h. After treatment, cells were washed with ice-cold PBS three times and fixed with 4% paraformaldehyde for 20 min at 25 °C. Permeabilization was performed using 0.3% Triton X-100 (Solarbio, Beijing, China) for 10 min. EdU incorporation detection was performed according to the manufacturer’s protocol of the BeyoClick™ EdU Cell Proliferation Kit (C0078S, Beyotime), and 500 μL of reaction mixture was added per well, followed by incubation at 37 °C for 30 min in the dark. Nuclear counterstaining was performed using 1 μg / mL DAPI in PBS for 30 min at room temperature. Fluorescence imaging was performed on a Leica DMI8 inverted microscope (Leica Microsystems, Wetzlar, Germany). EdU-positive cells were quantitatively analyzed using ImageJ, and the calculation formula was as follows: EdU positive rate (%) = [EdU + Cells / DAPI + Cells] x 100%. Five randomly selected fields were analyzed per well under a 20x objective.

[0083] As one of the most aggressive malignancies of the central nervous system, glioblastoma is characterized by abnormal proliferation, infiltrative growth, and high frequency of recurrence after treatment. Histone deacetylase 1 (HDAC1), as a core molecule of epigenetic regulation, participates in the proliferation regulation and apoptosis escape mechanism of tumor cells by dynamically regulating the acetylation level of histone. This test example explores whether the iodophenalene imidazole derivatives obtained from Example 4 inhibit the proliferation of glioblastoma U87-MG cells by targeting HDAC1 and elucidates its potential molecular mechanism.

[0084] The tumor inhibitory effect of the iodophenanthroimidazole derivative obtained in Example 4 was evaluated using an EdU (5-ethynyl-2'-deoxyuridine) cell proliferation assay system. This method is based on the principle that EdU competes with thymidine for incorporation into newly synthesized DNA strands, and specific fluorescent labeling of nascent DNA is achieved through a Click reaction. After U87-MG glioblastoma cells were exposed to 0, 0.05, 0.10, and 0.20 μM of the iodophenanthroimidazole derivative obtained in Example 4 for 72 hours, it was observed that the percentage of EdU-positive cells (red fluorescence) decreased to 77.61 ± 3.28% in the 0.05 μM treatment group, and significantly decreased to 40.26 ± 0.38% in the 0.10 μM group, compared to the control group P <0.001) (p < 0.001) Figure 1 A and 1C). In contrast, almost no EdU-positive signal was detected in the 0.20 μM group. Notably, the high concentration group (0.20 μM) was accompanied by a significant increase in the intensity and number of nuclear staining of DAPI-labeled cells, indicating that the cells may have undergone significant DNA damage.

[0085] To elucidate the potential mechanism by which the iodophenanthroimidazole derivative obtained in Example 4 inhibits tumor cell proliferation, the effect of the iodophenanthroimidazole derivative obtained in Example 4 on the cell cycle progression of U87-MG glioblastoma cells was analyzed using flow cytometry. U87-MG glioblastoma cells were seeded at a density of 5 x 105cells / well in a 6-well plate and exposed to gradient concentrations of the iodophenanthroimidazole derivative obtained in Example 4 (0, 0.05, 0.10, and 0.20 μM) in a humidified 5% CO2incubator at 37°C for 72 hours. To analyze cell cycle arrest, the cells were fixed with 70% ethanol at 4°C overnight. The fixed cells were washed with PBS and stained with propidium iodide (PI) for 5 minutes in the dark. Then, data were collected and analyzed using an Epics XL-MCL flow cytometer (Beckman Coulter, Miami, FL, USA). 4 To analyze cell cycle arrest, the cells were fixed with 70% ethanol at 4°C overnight. The fixed cells were washed with PBS and stained with propidium iodide (PI) for 5 minutes in the dark. Then, data were collected and analyzed using an Epics XL-MCL flow cytometer (Beckman Coulter, Miami, FL, USA).

[0086] As shown in Figure 1 B and 1D, the cell cycle distribution showed a biphasic regulatory feature after treatment with the iodophenanthroimidazole derivative obtained in Example 4 (0, 0.05, 0.10, and 0.20 μM) for 72 hours. The proportion of cells in the G2 / M phase significantly increased from 22.55 ± 0.38% in the control group to 57.42 ± 0.62% in the 0.10 μM treatment group P<0.001, consistent with the typical G2 / M phase arrest pattern, is a biological marker of DNA damage checkpoint activation. In contrast, cells in the 0.20 μM treatment group showed an increase in the proportion of S phase cells from 15.71 ± 0.55% to 27.93 ± 0.91% (…). P The concentration <0.01 indicates that high concentrations may trigger S-phase arrest by interfering with DNA replication. Notably, this biphasic arrest effect is consistent with the characteristic mode of action of HDAC1 inhibitors: low concentrations trigger the silencing of mitosis-related genes (such as the Cyclin B1 / CDK1 complex) by interfering with histone acetylation; while high concentrations may lead to excessive chromatin relaxation, which may exacerbate replication fork instability, associated with inhibition of the Cyclin A2 / CDK2 pathway.

[0087] To verify the above mechanism, the expression levels of key cycle regulatory proteins (such as...) were detected by Western blot. Figure 1 E). Quantitative analysis showed that the expression of G2 / M phase regulators Cyclin B1 and CDK1 in the 0.10 μM treatment group decreased to 48.2±5.1% and 33.5±4.3%, respectively. P <0.001), which is highly consistent with its phenotype of inducing G2 / M phase arrest. Conversely, in the 0.20 μM treatment group, the expression of the core regulatory proteins Cyclin A2 and CDK2 in S phase decreased to 48.4±3.8% and 37.6±4.2%, respectively. P <0.001, providing a molecular-level explanation for the S-phase prolongation phenomenon. Notably, p53 and p21 expression were upregulated by 2.3-fold in the 0.20 μM treatment group ( P <0.01), indicating that HDAC1 inhibition may enhance the cell cycle arrest effect by activating the p53 / p21 signaling axis. These results demonstrate that the iodophenanthrimidazole derivative obtained in Example 4 can effectively block the cell cycle progression of glioblastoma cells in vitro, and its potency is comparable to that of the clinically commonly used HDAC inhibitor vorinostat (IC50). 50 = 10 μM) is comparable, suggesting its potential HDAC1 targeting inhibition properties.

[0088] Test Example 5 Evaluation of the in vivo antitumor activity of the iodophenanthreneimidazole derivative obtained in Example 4; The specific steps are as follows; the results are as follows: Figure 2 As shown.

[0089] Figure 2 The effect of the iodophenanthreneimidazole derivative obtained in Example 4 on inhibiting the proliferation and metastasis of U87-MG glioblastoma cells in a zebrafish glioblastoma orthotopic model; (A) is the growth and metastasis of U87-MG glioblastoma cells in zebrafish orthotopic model in the absence or presence of the iodinated phenanthroimidazole derivative obtained in Example 4; (B) is the head tumor xenograft proliferation ratio (n = 6 / group); (C) is the head tumor xenograft area ratio (n = 6 / group); Figure 2 All data represent mean ± standard error (SEM), statistical significance was assessed by two-way ANOVA combined with Sidak's multiple comparison test P <0.01, * P <0.001, ns- not significant compared to control group).

[0090] Zebrafish (Danio rerio) has become a highly efficient in vivo model for preclinical evaluation of anti-tumor drugs due to its embryonic transparency, high-throughput screening characteristics, and high conservation with human tumor microenvironment. In this test example, the in vivo anti-tumor activity of the iodinated phenanthroimidazole derivative obtained in Example 4 was evaluated by constructing a zebrafish model of orthotopic transplantation of U87-MG glioblastoma cells, and its synergistic mechanism was elucidated by in vitro experiments. The WPI Nanoject III microinjection system precisely located the Dil fluorescently labeled U87-MG glioblastoma single cell suspension (1 x 10 7 Cell / mL) in the mesencephalic ventricle of zebrafish embryos 48 hours after embryonic development (48 hpf) to establish an orthotopic glioblastoma model. Zebrafish embryos successfully constructed the model were exposed to culture water containing gradient concentrations of the iodinated phenanthroimidazole derivative obtained in Example 4 (0, 12.5, 25 and 50 μM) containing 0.003% PTU to inhibit pigmentation, and were continuously treated at 37°C for 48 hours.

[0091] Dynamic images of tumors were taken every 24 hours using a fluorescence inverted microscope Figure 2 A). The results showed that the iodinated phenanthroimidazole derivative obtained in Example 4 exhibited a significant anti-tumor effect in the in vivo environment, with a 52% reduction in tumor fluorescence intensity in the 50 μM concentration group compared to the control group P <0.001), and the tumor area was reduced to 12% P<0.001), exhibiting significant dose-dependent inhibition of proliferation. Simultaneously, it significantly inhibited the invasive spread of tumor cells along fli1a:EGFP-labeled blood vessels, indicating its metastasis-blocking effect through targeted regulation of the tumor microenvironment. Notably, at concentrations below 50 μM, this compound showed no significant inhibitory effect on zebrafish development, and no obvious developmental abnormalities were observed. This model, combining embryonic transparency (real-time tracking) and the conservation of vascular interactions, provides crucial evidence with both high spatiotemporal resolution and pathological relevance for the clinical translation of the iodophenimidazol derivative obtained in Example 4. The in vivo activity of the iodophenimidazol derivative obtained in Example 4 forms a closed-loop mechanism with previously discovered cell cycle arrest (G2 / M phase), DNA damage, and activation of the mitophagy pathway. This finding further validates the synergistic inhibitory pattern of the iodophenimidazol derivative obtained in Example 4 on glioblastoma progression and highlights its significant clinical translational potential as a candidate for targeted therapy of glioblastoma.

[0092] Test Example 6 The iodophenanthreneimidazole derivative obtained in Example 4 was used as an HDAC1 inhibitor; The specific steps are as follows; the results are as follows: Figure 3 As shown.

[0093] Figure 3 The inhibitory effect of the iodophenanthreneimidazole derivative obtained in Example 4 as an HDAC1 inhibitor is shown. (A) Molecular docking analysis of the iodophenanthreneimidazole derivative obtained in Example 4 binding to the HDAC1 active site; (B) and (C) are 2D ligand interaction diagrams of the iodophenanthreneimidazole derivatives obtained in Example 4 at binding site 1 (B) and site 2 (C); (DF) is the molecular dynamics simulation of the iodophenanthreneimidazole derivative complex obtained in HDAC1-Example 4, (D) is the binding energy curve, (E) is the number of hydrogen bonds, and (F) is the RMSD evolution of the complex during the 100 ns simulation. (G) is the ITC binding isotherm of the iodophenanthreneimidazole derivative (20 μM) obtained in Example 4 titrated into HDAC1 (1 μM) (Tris-HCl buffer, pH 7.4, 298K). (H) is the UV-Vis absorption spectrum analysis of HDAC1 titrated with different concentration gradients into the iodophenanthreneimidazole derivative (20 μM) obtained in Example 4; (I) CETSA thermogram analysis, Western blot analysis of HDAC1 in U87-MG cell lysates that were not treated with iodophenanthreneimidazole derivative (0.4 μM) obtained in Example 4 after heating at different temperatures (GAPDH loading control). (J) To achieve dose-dependent inhibition of HDAC1 expression, U87-MG cells were treated with the iodophenanthreneimidazole derivatives obtained in Example 4 (0, 0.05, 0.10, and 0.20 μM) for 72 hours. The HDAC1 level was quantified by Western blotting (GAPDH control). Treatment with 0.20 μM of the iodophenanthreneimidazole derivatives obtained in Example 4 for 72 hours significantly downregulated the expression of HDAC1 in U87-MG glioblastoma cells. HDAC1 is abnormally highly expressed in glioblastoma and drives cell proliferation, invasion, and epithelial-mesenchymal transition (EMT); inhibiting its activity significantly reduces tumor migration. In this test case, the iodophenanthreneimidazole derivative obtained in Example 4 was used as an HDAC1 inhibitor. Molecular docking results showed that the iodophenanthreneimidazole derivative obtained in Example 4 effectively bound HDAC1 through dual binding sites (binding energies: -7.75 and -7.74 kcal / mol; inhibition constants KI: 2.1 and 2.11 μM). Figure 3 A). Key interactions include hydrophobic interactions and hydrogen bonds between binding site 1 and residues such as Tyr23 and Gln26, supplemented by anionic-π interactions with Glu98 / Asp99 / Asp104 ( Figure 3 B); and the cation-π interaction and multi-residue hydrophobic network at binding site 2 with Arg169 / His375 (B); Figure 3 C). This dual-site binding mode occupies Zn... 2+ The catalytic pocket blocks the substrate channel, and the metastable pocket enhances stability, thereby significantly inhibiting the deacetylation activity of HDAC1. Compared with the classic inhibitor AR-42, the iodine substituent of the iodophenimazole derivative optimizes the hydrophobic interaction with Phe150 / Phe205 and exhibits a more complex interaction mode. Molecular dynamics simulations further revealed the dynamic interaction between the iodophenimazole derivative and HDAC1 under physiological conditions. Neglecting solvation effects, the van der Waals forces (VDW, including hydrophobic interactions) and electrostatic interactions (ELE) between the ligand and receptor in the iodophenimazole derivative-HDAC1 complex obtained in Example 4 were calculated. Figure 3 D). The binding energy (the sum of VDW and ELE, representing the binding energy under non-solvent conditions) was analyzed during molecular dynamics simulations. The results showed that VDW and ELE gradually stabilized and eventually reached equilibrium during the simulation, directly indicating that the interaction between the iodophenanthreneimidazole derivative and the HDAC1 protein tended to stabilize, forming a stable binding mode. Simulations at 100 ns showed convergence of the root mean square deviation (RMSD) of the complex system, indicating the existence of a stable binding conformation (…). Figure 3 E) One to three hydrogen bonds are dynamically maintained between the two, and the binding free energy is continuously optimized. Changes in the number of secondary structures of residues in the key binding domain indicate a conformational regulation effect. Figure 3 F). These results confirm the reliability of the combined model of docking prediction in dynamic environments.

[0094] Complementary ITC analysis confirmed the high-affinity, exothermic binding (Kd = 104 nM); Figure 3 The stoichiometric ratio (n=1.877) indicates that each HDAC1 monomer binds approximately two ligand molecules. A strong negative enthalpy change (ΔH = -91.24 kJ / mol) dominates the Gibbs free energy (ΔG = -39.2 kJ / mol, 298 K), accounting for over 95% of the total binding energy, suggesting the presence of specific intermolecular interactions, such as I···Asp104 halogen bonds. Conversely, an unfavorable entropy change (ΔS = -172.3 J·mol) -1 K -1 This reflects significant conformational ordering, including the rigidification of the HDAC1 catalytic pocket and the immobilization of the ligand moiety. This significant enthalpy-driven characteristic (ΔH > TΔS) confirms directional interactions, consistent with molecular dynamics simulations.

[0095] Furthermore, UV-Vis titration studies revealed a concentration-dependent binding of the iodophenanthreneimidazole derivative obtained in Example 4 to HDAC1, characterized by a gradual increase in protein concentration (…). Figure 3 A significant hypochromic effect (28.49%) was observed at 276 nm in HDAC1. The spectral attenuation saturated at concentrations exceeding 2 μM, indicating complete occupancy of the binding sites and a near 1:2 stoichiometric ratio (ligand: HDAC1 monomer). This hypochromic effect reflects the π→π transition of benzimidazole. The electronic perturbation of the transitions and the restricted aromatic vibrations induced by the stacking interaction between the iodophenanthreneimidazole core and the hydrophobic subsites of HDAC1 (Phe150 / His178). This convergence, coupled with the absence of peak broadening or shift and a sharp isoabsorption point at 276 nm, validates that the binding-driven electronic recombination occurs through a conformational selection mechanism. Furthermore, the stoichiometry derived by ITC (n=1.877) is consistent with the observed 2 μM saturation threshold, indicating the presence of concerted ligand binding behavior. In summary, these orthogonal methods demonstrate that high-affinity HDAC1 binding exhibits dual targeting through concerted binding (proven by saturation kinetics and stoichiometry), a hydrophobic pocket (hypochromic effect), and a catalytic zinc site (enthalpy-dominant), as well as a conformational selection mechanism.

[0096] Furthermore, the target binding of the iodophenanthrollimidazole derivative obtained in Example 4 in cells was verified using cellular thermal shift assay (CETSA). HDAC1 in control cells was completely denatured at 50°C, while cells treated with the iodophenanthrollimidazole derivative obtained in Example 4 exhibited significantly enhanced thermal stability, with protein conformation still detectable at 60°C, and complete denaturation delayed until 65°C Figure 3 I. This ligand-induced thermal shift (ΔT>10°C) confirmed the formation of specific iodophenanthrollimidazole derivative-HDAC1 complex in living cells. This result directly demonstrated the target binding ability of the iodophenanthrollimidazole derivative under physiological conditions through the conformational stabilization of HDAC1. Consistent with the behavior of the target bound to the ligand, these data provided key evidence for target occupancy in cells. Notably, the expression of HDAC1 in U87-MG glioblastoma cells was significantly down-regulated by treatment with 0.20 μM of the iodophenanthrollimidazole derivative obtained in Example 4 for 72 hours ​ J), further supporting its function as a novel HDAC1 inhibitor, acting through effective stabilization of the active pocket.

[0097] In the present application, a strategy combining calculation and experiment was adopted to verify the three iodophenanthrollimidazole derivatives obtained in Examples 4-6 as potent HDAC1 inhibitors. Comprehensive analysis of molecular docking, molecular dynamics simulation and isothermal titration calorimetry (ITC) revealed their stable binding to the catalytic site (binding energy: -7.75 and -7.74 kcal / mol), which was mediated by the key halogen bond (iodine-Asp104) and phenanthrollimidazole skeleton-Zn 2+ coordination, and confirmed the high-affinity binding (Kd=1.04×10 -7 M) at sub-micromolar level. In addition, cellular thermal shift assay (CETSA) confirmed their target binding in cells. In vitro experiments demonstrated their potent inhibitory effect on the proliferation of U87-MG glioblastoma cells (IC 50 =0.23 μM) and their ability to induce G2 / M phase cell cycle arrest. The zebrafish model in vivo further verified their efficient blood-brain barrier penetration, inhibition of orthotopic tumor growth, and blocking of metastasis, laying a foundation for clinical translation.

[0098] At the level of cell cycle, the compound induced p21-mediated G2 / M phase arrest at low concentrations, and S phase arrest at high concentrations.

[0099] In summary, the present application demonstrates multifaceted scientific and clinical significance. It elucidated the iodophenanthrollimidazole derivative binding to HDAC1 through synergistic halogen bond (iodine-Asp104) and metal ion coordination (phenanthrollimidazole-Zn 2+) molecular mechanism of HDAC1 inhibition, establishing a new structural paradigm for the design of epigenetic-targeted therapeutics. Furthermore, we demonstrate that chemotherapy resistance in glioblastoma can be overcome by cell cycle arrest. This lead compound exhibits triple therapeutic advantages, including sub-micromolar anti-tumor potency (IC 50 = 0.23 μΜ), significant blood-brain barrier penetration, and anti-metastatic activity. These properties address two key clinical challenges in glioblastoma treatment: inefficient central nervous system drug delivery and uncontrolled recurrence / metastasis. Upon optimization of selectivity and safety, this drug is poised for clinical translation as the first halogen-bonding-based HDAC1-targeted therapy.

[0100] Although the above embodiments have been described in great detail, it should be understood that the application is not limited to the embodiments but only to the claims. Other embodiments can be derived from the embodiments by those skilled in the art without departing from the scope of the present application.

Claims

1. A phenanthroimidazole derivative, characterized in that, having any one of the chemical structures shown as follows: Formula I, Formula II; In formula I, formula II, R is ; each of R1, R2, R3 in R of formula I is independently selected from one or more of alkyl having 1-5 carbon atoms, alkenyl having 2-5 carbon atoms, substituted alkenyl having 2-5 carbon atoms, iodine, fluorine and dioxolane; each of R1, R2, R3 in R of formula II is independently selected from one or more of hydrogen, alkyl having 1-5 carbon atoms, substituted alkyl having 1-5 carbon atoms, alkenyl having 2-7 carbon atoms, substituted alkenyl having 2-7 carbon atoms, halogen and dioxolane; n is 2-16.

2. The phenanthroimidazole derivative according to claim 1, characterized in that the alkyl having 1-5 carbon atoms in formula I is selected from methyl, ethyl or propyl; the alkenyl having 2-5 carbon atoms is ethynyl; the substituted alkenyl having 2-5 carbon atoms is trimethylsilyl ethynyl or phenyl ethynyl.

3. The phenanthroimidazole derivative according to claim 1, characterized in that the alkyl having 1-5 carbon atoms in formula II is selected from methyl, ethyl or propyl; the substituted alkyl having 1-5 carbon atoms is monofluoromethyl or trifluoromethyl; the alkenyl having 2-5 carbon atoms is ethynyl; the substituted alkenyl having 2-5 carbon atoms is trimethylsilyl ethynyl or phenyl ethynyl; the halogen is selected from fluorine, chlorine, bromine or iodine.

4. The phenanthroimidazole derivative according to any one of claims 1 to 3, characterized in that having any one of the chemical structures shown as follows: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 5. A process for the preparation of a phenanthroimidazole derivative according to any one of claims 1 to 4, characterized in that, The preparation method of the phenanthroimidazole derivative shown in formula I comprises: mixing 1,10-phenanthroline-5,6-dione, R-formaldehyde, ammonium acetate and acetic acid and then reacting to obtain the phenanthroimidazole derivative shown in formula I; The preparation method of the phenanthroimidazole derivative shown in formula II comprises: The phenanthroimidazole derivative of the structure shown in formula I, Br(CH) n After mixing X and K2CO3, the reaction is carried out to obtain the phenanthroimidazole derivative of the structure shown in formula II; Br(CH) n X is H or Br, n = 2-16.

6. The preparation method according to claim 5, characterized in that, The temperature during the preparation of the phenanthroimidazole derivative is 80-120℃, and the time is 1-8h.

7. The preparation method according to claim 5, characterized in that, The preparation of the phenanthroimidazole derivative is carried out under microwave irradiation, and the time of the microwave irradiation is 5-30min.

8. The use of the phenanthroimidazole derivative of any one of claims 1-4 or the phenanthroimidazole derivative prepared by the preparation method of any one of claims 5-7 in the preparation of an antitumor drug.

9. The use of the phenanthroimidazole derivative of any one of claims 1-4 or the phenanthroimidazole derivative prepared by the preparation method of any one of claims 5-7 in the preparation of an HDAC-targeting antitumor drug.

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