Discovery of a highly selective, potent, non-cyclic phosphoramidate nucleotide pro-apoptotic agent for hepatocellular carcinoma

Abstract

A series of acyclic phosphoramidone ester nucleotide analogs were synthesized through two chemical reactions. The first reaction was a multicomponent reaction (MCR) to produce acridinium dione derivatives (7a-e). The second reaction provided acyclic phosphoramidone ester derivatives (9a-e). The anticancer activity of the acyclic nucleosides (7a-e) and their corresponding acyclic phosphoramidone ester derivatives (9a-e) was evaluated against four different types of cancer cells. Compared with 5-fluorouracil, compound 9a exhibited the highest anticancer activity against human liver tumor cells Huh-7 and HepG2, with corresponding half-inhibitory concentrations (IC50) of 9a. 50 The effective values ​​were 12.82 μM and 23.37 μM, respectively, with relatively high tumor selectivity of 16.91 and 9.28, respectively. Cell cycle analysis showed that compound 9a could inhibit cell proliferation in both G0 / G1 and G2 / M phases of the cell cycle distribution. The effects of compound 9a derivatives on the expression levels of five tumor-regulating genes were investigated. The results showed that the expression of Caspase-9, p21, and Bcl-2 genes was significantly downregulated, while the expression of p53 and BAX genes was regulated, as verified by quantitative RT-PCR analysis. After 35 days of treatment, the efficacy of compound (9a) was confirmed in the treatment of diethylnitrosamine (DEN) chemically induced experimental rat hepatocellular carcinoma, and its efficacy was compared with that of sorafenib, a compound currently used for human treatment. This conclusion was based on the results of histological analysis (histopathology), which showed viable clusters of tumor tissue composed of malignant hepatocytes, without any detectable mitotic forms, but with signs of regression of fibrotic forms, which were replaced by normal regenerating hepatocytes, indicating a near-complete pathological response to treatment. Rats injected with DEN also exhibited hepatomegaly, oxidative stress, elevated liver enzyme levels, decreased albumin levels, and elevated levels of the tumor marker α protein (AFP). Gene expression analysis showed that compound (9a) attenuated the DEN-induced changes in the studied molecular markers by upregulating the apoptosis marker (Cas-9) and downregulating the angiogenesis marker (VEGFR-2), tissue remodeling marker (MMP-9), cell cycle regulation marker (CcnD-1), and inflammation markers (IL-6, TNF-α, and TGFβ-1).

Description

Background Technology

[0001] Globally, hepatocellular carcinoma (HCC) is one of the leading causes of cancer and cancer-related deaths. Local and systemic therapies remain ineffective for patients with advanced HCC, leading to poor prognosis. The development of sorafenib for the treatment of HCC has ushered in a new era of molecularly targeted therapy for this disease. 1 A key aspect of cancer therapeutics is the development of targeted therapy, which utilizes chemical compounds engineered to modulate the activity of specific molecular targets involved in key oncogenic signaling pathways that ultimately regulate the proliferation, growth, survival, and distant metastasis of cancer cells. Therefore, the advantage of targeted therapy lies in its ability to precisely and effectively inhibit the occurrence and development of cancer, while exhibiting lower toxicity to non-malignant cells—a common drawback of systemic chemotherapy and radiotherapy. 2 Background technology defects 1) Antitumor agents have low safety for normal cells.

[0002] 2) The synthesis cost of anti-tumor drugs is relatively high.

[0003] 3) The synthetic routes for antitumor drugs are relatively long.

[0004] 4) There are relatively few therapeutic agents that target and promote the physiological apoptosis process.

[0005] 5) Lacks selectivity for cancer cells. Summary of the Invention

[0006] The invention described herein relates to one or more compounds that have been identified as potential pro-apoptotic compounds for hepatocellular carcinoma, and are expected to have anti-tumor activity and selectivity for tumor cells, and can be used as anti-cancer drugs.

[0007] Compounds having the structures shown in Formula A and Formula B, and their pharmaceutically acceptable salts, are disclosed.

[0008] Detailed Implementation

[0009] In one embodiment, the present invention provides a compound of formula A: in,

[0010] 7a:R 1 = Br;R 2 = H 7b:R 1 = OCH3;R 2 = H 7c:R1 = R 2 = OCH3 7d:R 1 =Cl;R 2 =H 7e:R 1 =CH3;R 2 =H According to formula A, the following compound will be obtained: 9-(4-Bromophenyl)-10-(2′-hydroxyethyl)-3,3,6,6-tetramethyl-3,4,6,7,9,10-hexahydroacridine-1,8 (2) H 5 H )-Diketone (7a)

[0011] 10-(2′-hydroxyethyl)-9-(4-methoxyphenyl)-3,3,6,6-tetramethyl-3,4,6,7,9,10-hexahydroacridine- 1,8(2) H 5 H )-Diketone (7b)

[0012] 9-(3,4-Dimethoxyphenyl)-10-(2′-hydroxyethyl)-3,3,6,6-tetramethyl-3,4,6,7,9,10-hexahydro Acridine-1,8(2) H 5 H )-Diketone (7c)

[0013] 9-(4-Chlorophenyl)-10-(2′-hydroxyethyl)-3,3,6,6-tetramethyl-3,4,6,7,9,10-hexahydroacridine-1,8 (2) H 5 H )-Diketone (7d)

[0014] 10-(2′-hydroxyethyl)-3,3,6,6-tetramethyl-9-( p (-Tolyl)-3,4,6,7,9,10-Hexahydroacridine-1,8 (2) H 5 H )-Diketone (7e)

[0015] In a second embodiment, the present invention provides a compound represented by formula B: in,

[0016] 9a:R 1 = Br;R 2 = H 9b:R 1 = OCH3;R 2 = H 9c:R 1 = R 2 = OCH3 9d:R 1 = Cl;R 2 = H 9e:R 1 =CH3;R 2 =H According to formula B, the following compound will be obtained: Isopropyl [( S )-{2′-(9-(4-bromophenyl)-3,3,6,6-tetramethyl-1,8-dioxo-2,3,4,5,6,7,8, 9-Octahedroxyacridine-10 (1 H )-yl)ethoxy}(phenoxy)phosphoryl]-L-alanine ester (9a)

[0017] Isopropyl [( S )-{2′-(9-(4-methoxyphenyl)-3,3,6,6-tetramethyl-1,8-dioxo-2,3,4,5,6, 7,8,9-Octahedroxyacridine-10(1 H )-yl)ethoxy}(phenoxy)phosphoryl]-L-alanine ester (9b)

[0018] Isopropyl [( S )-{2′-(9-(3,4-dimethoxyphenyl)-3,3,6,6-tetramethyl-1,8-dioxo-2,3,4, 5,6,7,8,9-octahydroacrylidine-10(1 H )-yl)ethoxy}(phenoxy)phosphoryl]-L-alanine ester (9c)

[0019] Isopropyl [( S )-{2′-(9-(4-chlorophenyl)-3,3,6,6-tetramethyl-1,8-dioxo-2,3,4,5,6,7,8, 9-Octahedroxyacridine-10 (1 H )-yl)ethoxy}(phenoxy)phosphoryl]-L-alanine ester (9d)

[0020] Isopropyl [( S )-{2′-(3,3,6,6-tetramethyl-1,8-dioxo-9-( p -Tolyl)-2,3,4,5,6,7,8, 9-Octahedroxyacridine-10 (1 H )-yl)ethoxy}(phenoxy)phosphoryl]-L-alanine ester (9e)

[0021] Pharmaceutical preparations and their administration methods: The compounds of this application can be formulated with pharmaceutically acceptable excipients to produce pharmaceutical preparations suitable for human administration. Such preparations can be administered orally, parenterally, or by inhalation.

[0022] Therefore, oral pharmaceutical formulations containing the compounds of the present invention can be tablets, pills, and capsules. Pharmaceutically acceptable excipients suitable for oral formulations can be selected from, for example but not limited to, lactose, polyvinylpyrrolidone (PVP), hydroxypropyl methylcellulose, sodium lauryl sulfate, silica, magnesium stearate, calcium stearate, talc, PEG, HPMC, starch, alginate, etc.

[0023] Indications and combination therapy: The compounds of this invention are of great value in the treatment of hepatocellular carcinoma if administered alone or in combination with other chemotherapeutic agents at therapeutically effective doses to enhance their effect on targeted cancer cells and reduce their serious side effects.

[0024] Examples of compounds that can be administered simultaneously or sequentially with the compounds of the present invention include, but are not limited to: A-alkylating agents, such as cyclophosphamide, nitrogen mustard, chlorambucil, melphalan, dacarbazine, nitrosourea, and temozolomide. Anthracycline drugs, such as daunorubicin, doxorubicin, pentorubicin, and idarubicin.

[0025] Beta-taxane drugs, such as paclitaxel, docetaxel, albumin-bound paclitaxel, and doxorubicin.

[0026] C-nucleotide analogues, such as azacitidine, azathioprine, capecitabine, cytarabine, deoxyfluorouridine, fluorouracil, hydroxyurea, mercaptopurine, methotrexate, and thioguanine.

[0027] D-platinum drugs, such as carboplatin, cisplatin and oxaliplatin.

[0028] E-vincristine alkaloids, such as vincristine and vinblastine.

[0029] synthesis: 9-Aryl-10-(2-hydroxyethyl)-3,3,6,6-tetramethyl-3,4,6,7,9,10-hexahydroacridine-1,8(2H,5H)- Diketone [7a-e]:

[0030] General procedure: In a 50 mL round-bottom flask, a mixture of dimethyl ketone (2.8 g, 20 mmol), various aromatic aldehydes (10 mmol), and 2-aminoethanol (0.72 mL, 12 mmol) was dissolved in N,N-dimethylformamide (9 mL), followed by the addition of p-toluenesulfonic acid (PTSA) (1.5 g m) or 37% hydrochloric acid (HCl) (1 mL) as a catalyst. The mixture was refluxed and stirred at 110 °C for 24 hours. The reaction progress was monitored by TLC. After completion, the reaction mixture was cooled to room temperature and then water (120 mL) was added dropwise while stirring until complete precipitation. The product was filtered off, washed with water, and then dried at room temperature. The crude product was purified by recrystallization from toluene to give pure compounds 7a-e.

[0031] 9-(4-Bromophenyl)-10-(2-hydroxyethyl)-3,3,6,6-tetramethyl-3,4,6,7,9,10-hexahydroacridine-1,8 (2H,5H)-diketone (7a) Pale yellow powder (3.3 g, 70% yield); mp: 229–231 ℃. IR (KBr) νmax (cm⁻¹): 3488 (O–H); 2955 (=C–H, sp²); 2869 (C–H, sp³); 1629 (C=O, conjugated ketone); 1567, 1484 (C=C); 1233 (C–O stretching vibration). 1 H-NMR (400 MHz, DMSO-d6) δ (ppm): 0.87 (s, 6 H, 2 CH3), 1.01 (s, 6 H, 2 CH3), 2.02 (d, 2 H, 2J H–H = –16 Hz, CH2), 2.14 (d, 2 H, 2J H–H = –16 Hz, CH2), 2.44 (d, 2 H, 2J H–H = –16 Hz, CH2), 2.72 (d, 2 H, 2J H–H= -16 Hz, CH2), 3.56 (t, 2 H, J = 4.0 Hz, CH2–N), 3.85 (t, 2 H, J = 4.0 Hz, CH2–O), 4.94 (s, 1 H, acridine dione-H9), 5.04 (t, 1 H, J = 4.0 Hz, D2O-exchangeable, O–H), 7.16 (d, 2 H, J = 8.0 Hz, Ar-H), 7.30 (d, 2 H, J = 8.0 Hz, Ar-H). 13 C-NMR (125 MHz, DMSO-d6) δ (ppm): 195.63 (2 C=O), 152.40, 146.16, 130.93, 130.36, 118.95 (Ar-C), 113.60, 61.30, 49.93, 46.61 (40.11; under DMSO-d6 conditions), 32.48, 31.75, 29.29, 27.38. 25 H 30 Theoretical elemental analysis values ​​for BrNO3: C, 63.56; H, 6.40; N, 2.96; Br, 16.91. Measured values: C, 63.66; H, 6.50; N, 2.88; Br, 16.81.

[0032] 10-(2-hydroxyethyl)-9-(4-methoxyphenyl)-3,3,6,6-tetramethyl-3,4,6,7,9,10-hexahydroacridine- 1,8(2H,5H)-dione (7b) Pale yellow powder (3.05 g, yield 72%); mp: 199–200 °C. IR (KBr) ν max（ cm -1 ): 3472 (O–H); 2957 (=C–H, sp 2 ); 2869, 2843 (C–H, sp 3 ); 1615 (C=O, conjugated ketone); 1561, 1465 (C=C); 1236 (C–O stretching vibration). 1 H-NMR (400 MHz, DMSO- d 6) δ (ppm): 0.87 (s, 6 H, 2 CH3), 1.02 (s, 6 H, 2 CH3), 2.01 (d, 2 H, 2 J H–H = –16 Hz, CH2), 2.14 (d, 2 H, 2 J H–H = –16 Hz, CH2), 2.44 (d, 2 H, 2J H–H = –16 Hz, CH2), 2.71 (d, 2 H, 2 J H–H = –16 Hz, CH2), 3.55 (t, 2 H, J = 4.0 Hz, CH2–N), 3.66 (s, 3 H, OCH3), 3.83 (t, 2 H, J = 4.0 Hz, CH2–O), 4.90 (s, 1 H, acridine dione-H9), 5.08 (t, 1 H, J = 4.0 Hz, O–H), 6.68 (d, 2 H, J = 8.0 Hz, Ar-H), 7.11 (d, 2 H, J = 8.0 Hz, Ar-H). 13 C-NMR (125 MHz, DMSO- d 6) δ (ppm): 195.69 (2 C=O), 157.53, 151.82, 139.20, 129.05, 125.81 (Ar-C), 114.19, 113.40, 61.30, 55.25 (OCH3), 49.94, 46.54, 39.81 (DMSO- d Under condition 6), 32.50, 30.98, 29.43, 27.34. C 26 H 33 Theoretical values ​​for NO4 elemental analysis: C, 73.73; H, 7.85; N, 3.31. Measured values: C, 73.59; H, 7.90; N, 3.22.

[0033] 9-(3,4-Dimethoxyphenyl)-10-(2-hydroxyethyl)-3,3,6,6-tetramethyl-3,4,6,7,9,10-hexahydroacetyl Pyridine-1,8(2H,5H)-dione (7c) Pale yellow powder (3.63 g, 80% yield); mp: 206–209 °C. IR (KBr) ν max（ cm -1 ): 3311 (O–H); 2990 (=C–H, sp 2 ); 2867 (C–H, sp 3 ); 1644 (C=O, conjugated ketone); 1563, 1460 (C=C); 1237 (C–O stretching vibration). 1 H-NMR (400 MHz, DMSO- d 6) δ(ppm): 0.86 (s, 6 H, 2 CH3), 1.01 (s, 6 H, 2 CH3), 2.01 (d, 2H, 2 J H–H = –16 Hz, CH2), 2.15 (d, 2 H, 2 J H–H = –16 Hz, CH2), 2.44 (d, 2 H, 2 J H–H = –16 Hz, CH2), 2.71 (d, 2 H, 2 J H–H = –16 Hz, CH2), 3.55 (t, 2 H, J = 4.0 Hz, CH2–N), 3.63 (s, 3 H, OCH3), 3.64 (s, 3 H, OCH3), 3.91 (t, 2 H, J = 4.0 Hz, CH2–O), 4.90 (s, 1 H, acridine dione-H9), 5.16 (t, 1 H, J =4.0 Hz, O–H), 6.70-6.75 (m, 3 H, Ar-H). 13 C-NMR (125 MHz, DMSO- d 6) δ (ppm): 196.25 (2 C=O), 152.12, 148.45, 147.03, 139.20, 120.20, 111.49, 111.34 (Ar-C), 113.83, 61.35, 55.72 (OCH3), 55.53 (OCH3), 49.84, 46.49, 39.51 (DMSO- d Under condition 6), 32.39, 31.26, 29.62, 26.94. C 27 H 35 Theoretical values ​​for NO5 elemental analysis: C, 71.50; H, 7.78; N, 3.09. Measured values: C, 71.39; H, 7.69; N, 2.99.

[0034] 9-(4-Chlorophenyl)-10-(2-hydroxyethyl)-3,3,6,6-tetramethyl-3,4,6,7,9,10-hexahydroacridine-1,8 (2H,5H)-diketone (7d) Pale yellow powder (3.124 g, yield 73%); mp: 176–179 °C. IR (KBr) ν max（ cm -1 ): 3335 (O–H); 2959 (=C–H, sp2 ); 2886 (C–H, sp 3 ); 1619 (C=O, conjugated ketone); 1563, 1487 (C=C); 1238 (C–O stretching vibration). 1 H-NMR (400 MHz, DMSO- d 6) δ (ppm): 0.86 (s, 6 H, 2 CH3), 1.02 (s, 6 H, 2 CH3), 2.02 (d, 2H, 2 J H–H = –16 Hz, CH2), 2.15 (d, 2 H, 2 J H–H = –16 Hz, CH2), 2.45 (d, 2 H, 2 J H–H = –16 Hz, CH2), 2.73 (d, 2 H, 2 J H–H = –16 Hz, CH2), 3.56 (t, 2 H, J = 4.0 Hz, CH2–N), 3.85 (t, 2 H, J = 4.0 Hz, CH2–O), 4.95 (s, 1 H, acridine dione-H9), 5.12 (broad singlet, 1 H, O–H), 7.18 (d, 2 H, J = 8.0 Hz, Ar-H), 7.23 (d, 2 H, J = 8.0 Hz, Ar-H). 13 C-NMR (125 MHz, DMSO- d 6) δ (ppm): 195.69 (2 C=O), 152.40, 145.76, 130.46, 129.95, 128.06 (Ar-C), 113.56, 61.29, 49.84, 46.54, 39.82 (DMSO- d Under condition 6), 32.52, 31.66, 29.34, 27.33. C 25 H 30 Theoretical elemental analysis values ​​for ClNO3: C, 70.16; H, 7.07; N, 3.27; Cl, ​​8.28. Measured values: C, 70.01; H, 6.98; N, 3.16; Cl, ​​8.11.

[0035] 10-(2-hydroxyethyl)-3,3,6,6-tetramethyl-9-(p-tolyl)-3,4,6,7,9,10-hexahydroacridine-1,8 (2H,5H)-diketone (7e) Pale yellow powder (3.34 g, yield 82%); mp: 202–205 °C. IR (KBr) ν max（ cm -1 ): 3380 (O–H); 2957 (=C–H, sp 2 ); 2875 (C–H, sp 3 ); 1623 (C=O, conjugated ketone); 1564, 1465 (C=C); 1237 (C–O stretching vibration).

[0036] 1 H-NMR (400 MHz, DMSO- d 6) δ (ppm): 0.87 (s, 6 H, 2 CH3), 1.02 (s, 6 H, 2 CH3), 2.01 (d, 2 H, 2 J H–H = –16 Hz, CH2), 2.14 (d, 2 H, 2 J H–H = –16 Hz, CH2), 2.18 (s, 3 H, CH3), 2.44 (d, 2H, 2 J H–H = –20 Hz, CH2), 2.71 (d, 2 H, 2 J H–H = –20 Hz, CH2), 3.57 (t, 2 H, J = 4.0 Hz, CH2–N), 3.83 (t, 2 H, J = 4.0 Hz, CH2–O), 4.92 (s, 1 H, acridine dione-H9), 5.07 (t, 1 H, J = 4.0 Hz, O–H), 6.92 (d, 2 H, J = 8.0 Hz, Ar-H), 7.07 (d, 2 H, J = 8.0 Hz, Ar-H). 13 C-NMR (125 MHz, DMSO- d 6) δ(ppm): 195.69 (2 C=O), 151.94, 143.93, 134.75, 128.71, 127.98 (Ar-C), 114.04, 61.28, 49.93, 46.55, 39.82 (DMSO- d Under conditions 6), 32.49, 31.42, 29.44, 27.30, 21.03 (CH3). C 26 H 33 Theoretical values ​​for NO3 elemental analysis: C, 76.62; H, 8.16; N, 3.44. Measured values: C, 76.53; H, 8.22; N, 3.56.

[0037] Isopropyl [(S)-{2-(9-aryl-3,3,6,6-tetramethyl-1,8-dioxo-2,3,4,5,6,7,8,9-octahydro] Acridine-10 (1 H 9a-e)-yl)ethoxy}(phenoxy)phosphoryl]-L-alanine ester (9a-e) General procedure: In a 50 mL round-bottom flask, dissolve compound 7a-e (5.0 mmol) in... N, N - Dimethylformamide (20 mL), followed by isopropyl [( R )-(perfluorophenoxy)(phenoxy)phosphoryl)-L-alanine ester (2.27 g, 5.0 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (0.75 mL, 5.0 mmol). The reaction mixture was stirred at room temperature for 4 hours. The progress of the reaction was monitored by TLC. After completion, the reaction mixture was added dropwise to water (100 mL) while stirring until complete precipitation. The product was filtered off, washed with water, and then dried at room temperature. It was then precipitated with ethyl acetate / n The crude product was purified by silica gel column chromatography using hexane (1:1 v / v) as the eluent to obtain the corresponding pure compounds 9a-e.

[0038] Isopropyl [(S)-{2-(9-(4-bromophenyl)-3,3,6,6-tetramethyl-1,8-dioxo-2,3,4,5,6,7,8, 9-Octahedroxyacridine-10 (1 H )-yl)ethoxy}(phenoxy)phosphoryl]-L-alanine ester (9a) Off-white powder (2.89 g, yield 78%); mp: 114–117 °C. IR (KBr) ν max（ cm -1 ): 3435 (N–H); 2959 (=C–H, sp 2 ); 2875 (C–H, sp 3 ); 1736 (C=O, ester); 1634 (C=O, conjugated ketone); 1574, 1488 (C=C); 1151, 1211, 1244 (C–O, P=O stretching vibration). 1 H-NMR (400 MHz, DMSO- d 6) δ(ppm): 0.87 (s, 6 H, 2 CH3), 1.00 (s, 6 H, 2 CH3), 1.13–1.19 (m, 9 H, 3 CH3), 2.00, 2.04 (dd, 2 H, J 1 = 8.0 Hz J 2=8.0 Hz, CH2), 2.14, 2.16 (dd, 2 H, J 1 = 8.0 Hz J 2 = 8.0 Hz, CH2), 2.46–2.50 (m, 2 H, CH2), 2.69–2.76 (m, 2 H, CH2), 3.77–3.79 (m, 1 H, CH-CH3), 4.11–4.21 (m, 4 H, CH2-CH2), 4.79–4.87 [m, 1 H, CH(CH3)2], 4.95 (s, 1 H, acridine dione-H9), 6.09–6.18 (m, 1 H, D2O-exchangeable, N–H), 7.13–7.20 (m, 5 H, Ar-H), 7.30 (d, 2 H, J = 8.0 Hz, Ar-H), 7.34 (d, 2 H, J = 8.0 Hz, Ar-H). 13 C-NMR (125 MHz, DMSO- d 6) δ (ppm): 195.68 (2 C=O), 173.10 (C=O, ester), 151.99, 151.01, 145.82, 131.11, 130.29, 130.14, 125.10, 120.33, 119.05 (Ar-C), 113.90, 68.61, 66.01, 50.38, 49.79, 44.45, 40.00 (DMSO- d Under condition 6), 32.52, 31.70, 29.14, 27.52, 21.86, 20.20. C 37 H 46 Theoretical elemental analysis values ​​for BrN₂O₇P: C, 59.92; H, 6.25; N, 3.78; Br, 10.77; P, 4.18. Measured values: C, 60.00; H, 6.30; N, 3.82; Br, 10.70; P, 4.28.

[0039] Isopropyl [(S)-{2-(9-(4-methoxyphenyl)-3,3,6,6-tetramethyl-1,8-dioxo-2,3,4,5,6, 7,8,9-Octahedroxyacridine-10(1 H )-yl)ethoxy}(phenoxy)phosphoryl]-L-alanine ester (9b) Off-white powder (2.667 g, yield 77%); mp: 92–94 °C. IR (KBr) ν max（ cm-1 ): 3468 (N–H); 2957 (=C–H, sp 2 ); 2835 (C–H, sp 3 ); 1737 (C=O, ester); 1631 (C=O, conjugated ketone); 1572, 1464 (C=C); 1151, 1210, 1242 (C–O, P=O stretching vibration). 1 H-NMR (400 MHz, DMSO- d 6) δ (ppm): 0.89 (s, 6 H, 2 CH3), 1.00 (s, 6 H, 2 CH3), 1.10–1.19 (m, 9 H, 3 CH3), 1.99, 2.03 (dd, 2 H, J 1 = 8.0 Hz J 2=8.0 Hz, CH2), 2.12, 2.16 (dd, 2 H, J 1 = 8.0 Hz J 2= ​​8.0 Hz, CH2), 2.44–2.49 (m, 2 H, CH2), 2.68, 2.72 (dd, 2 H, J 1 = 8.0 Hz J 2 = 8.0 Hz, CH2), 3.59 (s, 3 H, OCH3), 3.74–3.81 (m, 1 H, CH-CH3), 4.09–4.12 (m, 4 H, CH2-CH2), 4.80–4.87 [m, 1 H, CH(CH3)2], 4.92 (s, 1 H, acridine dione-H9), 6.10 (broad triplet, 1 H, J = 12.0 Hz, N–H), 6.67 (d, 2 H, J = 8.0 Hz, Ar-H), 7.08 (d, 2H, J = 8.0 Hz, Ar-H), 7.14–7.19 (m, 3 H, Ar-H), 7.32 (t, 2 H, J = 8.0 Hz, Ar-H). 13 C-NMR (125MHz, DMSO-) d 6) δ(ppm): 195.78 (2 C=O), 173.13 (C=O, ester), 157.54, 151.49, 151.02, 138.82, 130.15, 128.96, 125.09, 120 .33, 114.58 (Ar-C), 113.50, 68.64, 65.86, 55.15, 50.37, 49.89, 44.39, 39.81 (DMSO- d Under condition 6), 32.50, 30.95, 29.25, 27.45, 21.83, 20.21. C 38 H 49 Theoretical values ​​for N₂O₈P elemental analysis: C, 65.88; H, 7.13; N, 4.04; P, 4.47. Measured values: C, 65.80; H, 7.02; N, 3.99; P, 4.31.

[0040] Isopropyl [(S)-{2-(9-(3,4-dimethoxyphenyl)-3,3,6,6-tetramethyl-1,8-dioxo-2,3,4, 5,6,7,8,9-octahydroacrylidine-10(1 H )-yl)ethoxy}(phenoxy)phosphoryl]-L-alanine ester (9c) Off-white powder (2.89 g, 80% yield); mp: 93–96 °C. IR (KBr) ν max（ cm -1 ): 3468 (N–H); 2957 (=C–H, sp 2 ); 2875 (C–H, sp 3 ); 1737 (C=O, ester); 1632 (C=O, conjugated ketone); 1571, 1463 (C=C); 1146, 1181, 1243 (C–O, P=O stretching vibration). 1 H-NMR (400 MHz, DMSO- d 6) δ (ppm): 0.88 (s, 6 H, 2 CH3), 1.01 (s, 6 H, 2 CH3), 1.12–1.20 (m, 9 H, 3 CH3), 2.00, 2.04 (dd, 2 H, J 1 = 8.0 Hz J 2=8.0 Hz, CH2), 2.14, 2.18 (dd, 2 H, J 1 = 8.0 Hz J2 = 8.0 Hz, CH2), 2.46 (broad singlet, 2 H, CH2), 2.69–2.76 (m, 2 H, CH2), 3.60 (s, 3 H, OCH3), 3.66 (s, 3 H, OCH3), 3.75–3.78 (m, 1 H, CH-CH3), 4.09–4.13 (m, 4 H, CH2-CH2), 4.82–4.93 [m, 1 H, CH(CH3)2], 4.93 (s, 1 H, acridine dione-H9), 6.09 (broad triplet, 1 H, CH2), J = 12.0 Hz, N–H), 6.68–6.72 (m, 3 H, Ar-H), 7.14–720 (m, 3 H, Ar-H), 7.32–7.36 (m, 2 H, Ar-H). 13 C-NMR (125 MHz, DMSO- d 6) δ (ppm): 195.86 (2 C=O), 173.13 (C=O, ester), 151.51, 150.97, 148.35, 147.13, 139.17, 130.15, 125.13, 120 .37, 119.69, 114.34, 111.78, 68.64, 65.72, 55.15, 50.37, 49.89, 44.41, 39.82 (DMSO- d Under condition 6), 32.46, 31.08, 29.41, 27.22, 21.84, 20.16. C 39 H 51 Theoretical values ​​for N₂O₉P elemental analysis: C, 64.81; H, 7.11; N, 3.88; P, 4.29. Measured values: C, 64.92; H, 7.01; N, 3.79; P, 4.34.

[0041] Isopropyl [{2-(9-(4-chlorophenyl)-3,3,6,6-tetramethyl-1,8-dioxo-2,3,4,5,6,7,8,9-octa-] Hydro-acryl-10(1H)-yl)ethoxy}(phenoxy)phosphoryl]-L-alanine ester (9d) Off-white powder (2.615 g, yield 75%); mp: 110–113 °C. IR (KBr) ν max（ cm -1 ): 3448 (N–H); 2958 (=C–H, sp 2 ); 2873 (C–H, sp 3 ); 1737 (C=O, ester); 1630 (C=O, conjugated ketone); 1569, 1490 (C=C); 1149, 1210, 1241 (C–O, P=O stretching vibration). 1H-NMR (400 MHz, DMSO- d 6) δ (ppm): 0.87 (s, 6 H, 2 CH3), 1.00 (s, 6 H, 2 CH3), 1.10–1.20 (m, 9 H, 3 CH3), 2.00, 2.04 (dd, 2 H, J 1 = 8.0 Hz J 2=8.0 Hz, CH2), 2.14, 2.18 (dd, 2 H, J 1 = 8.0 Hz J 2 = 8.0 Hz, CH2), 2.46–2.50 (m, 2 H, CH2), 2.69–2.76 (m, 2 H, CH2), 3.74–3.81 (m, 1 H, CH-CH3), 4.11–4.21 (m, 4 H, CH2-CH2), 4.78–4.89 [m, 1 H, CH(CH3)2], 4.95 (s, 1 H, acridine dione-H9), 6.12 (broad triplet, 1 H, J = 12.0 Hz, N–H), 7.13–7.20 (m, 5 H, Ar-H), 7.30 (d, 2 H, J = 8.0 Hz, Ar-H), 7.34 (d, 2 H, J = 8.0 Hz, Ar-H). 13 C-NMR (125 MHz, DMSO- d 6) δ (ppm): 195.68 (2 C=O), 173.15 (C=O, ester), 151.98, 151.01, 145.82, 131.92, 131.09, 130.29, 130.14, 125.10, 120.33 (Ar-C), 113.91, 68.61, 66.00, 50.38, 49.79, 44.43, 39.86 (DMSO- d Under condition 6), 32.52, 31.75, 29.14, 27.51, 21.85, 20.20. C 37 H 46 Theoretical elemental analysis values ​​for ClN₂O₇P: C, 63.74; H, 6.65; N, 4.02; Cl, ​​5.08; P, 4.44. Measured values: C, 63.66; H, 6.59; N, 3.79; Cl, ​​5.18; P, 4.35.

[0042] Isopropyl [(S)-{2-(3,3,6,6-tetramethyl-1,8-dioxo-9-(p-tolyl)-2,3,4,5,6,7,8, 9-Octahydroacryl-10(1H)-yl)ethoxy}(phenoxy)phosphoryl]-L-alanine ester (9e) Off-white powder (2.875 g, yield 85%); mp: 95–98 °C. IR (KBr) ν max（ cm -1 ): 3450 (N–H); 2959 (=C–H, sp 2 ); 2873 (C–H, sp 3 ); 1738 (C=O, ester); 1638 (C=O, conjugated ketone); 1572, 1465 (C=C); 1142, 1161, 1200, 1242 (C–O, P=O stretching vibration). 1 H-NMR (400 MHz, DMSO- d 6) δ (ppm): 0.86 (s, 6 H, 2 CH3), 1.00 (s, 6 H, 2 CH3), 1.10–1.17 (m, 9 H, 3 CH3), 1.97, 2.01 (dd, 2 H, J 1 = 8.0 Hz J 2 = 8.0 Hz, CH2), 2.13 (s, 3 H, CH3), 2.15–2.19 (m, 2 H, CH2), 2.43–2.48 (m, 2 H, CH2), 2.65–2.71 (m, 2 H, CH2), 3.74–3.81 (m, 1 H, CH-CH3), 4.08–4.19 (m, 4 H, CH2-CH2), 4.81–4.86 [m, 1 H, CH(CH3)2], 4.92 (s, 1 H, acridine dione-H9), 6.04 (broad triplet, 1 H, J = 12.0 Hz, N–H), 6.91 (d, 2 H, J = 8.0 Hz, Ar-H), 7.04 (d, 2 H, J = 8.0 Hz, Ar-H), 7.12–7.19 (m, 3 H, Ar-H), 7.31 (t, 2 H, J = 8.0 Hz, Ar-H). 13 C-NMR (125 MHz, DMSO- d 6) δ(ppm): 196.05 (2 C=O), 173.13 (C=O, ester), 151.18, 150.94, 143.47, 134.83, 130.16, 128.95, 128.85, 128 .13, 125.16, 120.28 (Ar-C), 114.71, 68.76, 65.92, 50.37, 49.82, 44.30, 39.60 (DMSO- d Under condition 6), 32.43, 31.33, 29.22, 27.35, 21.81, 21.00, 20.13. C 38 H 49 Theoretical values ​​for N₂O₇P elemental analysis: C, 67.44; H, 7.30; N, 4.14; P, 4.58. Measured values: C, 67.19; H, 7.19; N, 3.89; P, 4.49.

[0043] Biological evaluation: In vitro cytotoxicity test The in vitro antiproliferative activity of the newly synthesized compounds against different cancer cell lines (including A549 (human lung adenocarcinoma), Caco-2 (human colon adenocarcinoma), Huh-7 (human hepatocellular carcinoma), and HepG-2 (human hepatocellular carcinoma)) was evaluated, with the normal cell line WISH (derived from normal amnion) used as a control. 3 The results showed that some compounds were effective against four different IC cancer cell lines (IC). 50 The values ​​(ranging from 12.82 μM to 77.85 μM, as shown in Table 1) exhibited moderate to potent antitumor activity. Interestingly, this antitumor activity was significantly enhanced when a phosphoramide ester prodrug forming group was inserted, suggesting that these compounds not only possess enhanced pharmacokinetic characteristics but also synergistic antitumor activity. The significant enhancement of the antitumor activity of phosphoramide ester derivatives 9a-e compared to nucleoside prodrugs 7a-e is attributed to the inherent properties of the phosphoramide ester coupling group attached to the acridine dione core.

[0044]

[0045] Table 1: Half-maximal inhibitory concentrations (IC50) of synthetic compounds 7a-e and 9a-e and control 5-FU (standard chemotherapy drug) against normal (WISH) cells, lung tumor (A549) cells, colon tumor (Caco-2) cells, liver tumor (Huh-7) cells, and liver tumor (HepG-2) cells. 50 (Unit: μM) All values ​​are expressed as mean ± standard deviation.

[0046] Tumor Selectivity Index (TSI) Tumor selectivity index (TSI) is determined by measuring the half-maximal inhibitory concentration (IC50) of normal cells. 50 Divide by the IC of tumor cells 50 The value is determined. 4 TSI = IC50 of normal cells 50 / IC of cancer cells 50 Therefore, even if these cells belong to different types, the obtained TSI value can reflect their anti-tumor activity in vivo. The higher the TSI value, the better the therapeutic effect and safety of the drug against a specific tumor in vivo, in theory. Compounds with an SI value >10 are considered potential compounds that can be further investigated. Ideally, this drug should kill cancer cells but should not affect normal cells. 5 Of the compounds tested, 9a exhibited the highest degree of cytotoxic tumor selectivity against liver tumor cells (Huh-7) (TSI = 16.91, IC50). 50 = 12.82 (μM) (Table 2).

[0047]

[0048] Table 2: Tumor selectivity index (TSI value) of compounds 7a-e and 9a-e and control 5-FU (standard chemotherapy drug).

[0049] in conclusion A series of novel 9-aryl-10-(2′-hydroxyethyl)-3,3,6,6-tetramethyl-3,4,6,7,9,10-hexahydroacridine-1,8(2H,5H)-dione compounds 7a-e and their phosphoramide ester derivatives 9a-e were successfully prepared. Phosphoramide ester 9a exhibited the highest tumor selectivity (16.91) and IC50 against hepatocellular carcinoma (Huh-7) cells. 50 The concentration was measured at 12.82 μM, and it exhibited high safety in normal cells. These encouraging results indicate that phosphoramide ester compound 9a is a novel anticancer agent, particularly worthy of further development as a treatment for liver cancer. Mechanistic studies revealed that, through cell cycle analysis and gene expression detection, compound 9a inhibited cell proliferation during the G0 / G1 and G2 / M phases of the cell cycle, with apoptosis being the primary cause of cell death. Furthermore, RT-PCR results showed that compound 9a with optimal activity significantly regulated intrinsic apoptosis proteins, upregulating Bax expression and downregulating Bcl-2 gene expression.

[0050] References 1. Chow, A. K. M., Yau, S. W. L.,&Ng, L. (2020). Novel moleculartargets in hepatocellular carcinoma. World Journal of Clinical Oncology, 11(8), 589. 2. Bray, F., Ferlay, J., Soerjomataram, I., Siegel, R. L., Torre, L.A.,&Jemal, A. (2018). Global cancer statistics 2018: GLOBOCAN estimates ofincidence and mortality worldwide for 36 cancers in 185 countries. CA: acancer journal for clinicians, 68(6), 394-424. 3. Setyowati, E. P., Pratiwi, S. U. T.,&Purwantini, I. (2018). In-vitro cytotoxicity and apoptosis mechanism of ethyl acetate extract fromTrichoderma reesei strain TV221 associated with marine sponge: Stylissaflabelliformis. Journal of Applied Pharmaceutical Science, 8(9), 151-157. 4. Peña-Morán, O. A., Villarreal, M. L., Álvarez-Berber, L., Meneses-Acosta, A.,&Rodríguez-López, V. (2016). Cytotoxicity, post-treatmentrecovery, and selectivity analysis of naturally occurring podophyllotoxinsfrom Bursera fagaroides var. fagaroides on breast cancer cell lines.Molecules, 21(8), 1013. 5. López-Lázaro, M. (2015). How many times should we screen achemical library to discover an anticancer drug?. Drug Discovery Today, 2(20), 167-169. 6. Ye, X. Y., Wang, H. X., Liu, F.,&Ng, T. B. (2000). Ribonuclease,cell-free translation-inhibitory and superoxide radical scavenging activitiesof the iron-binding protein lactoferrin from bovine milk. The internationaljournal of biochemistry&cell biology, 32(2), 235-241. 7. Vilar, S., Cozza, G.,&Moro, S. (2008). Medicinal chemistry and themolecular operating environment (MOE): application of QSAR and moleculardocking to drug discovery. Current topics in medicinal chemistry, 8(18),1555-1572。

Claims

1. A noncyclic phosphoramide ester nucleotide analogue or a pharmaceutically acceptable remedy thereof as shown in structural formulas 9a-e, wherein, The noncyclic phosphoramide nucleotide analog 9a exhibits potent anticancer activity against hepatocellular carcinoma cells, specifically human liver tumor cells Huh-7 and HepG2, with a half-inhibitory concentration (IC50) of [missing value]. 50 The values ​​were 12.82 μM and 23.37 μM, respectively, indicating relatively high tumor selectivity, with values ​​of 16.91 and 9.28, respectively.

2. The noncyclic phosphoramide nucleotide analog according to claim 1, wherein, Compound 9a exhibits good safety profiles in normal cells, indicating low cytotoxicity to non-tumor cells.

3. The noncyclic phosphoramide nucleotide analog according to claim 1, wherein, The synthetic method involves a simple two-step process that allows for the efficient and economical production of compound 9a using commercially available starting materials.

4. The noncyclic phosphoramide nucleotide analog according to claim 1, wherein, Compound 9a exhibits selectivity for liver cancer cells, thus providing a targeted therapy option for hepatocellular carcinoma.

5. The noncyclic phosphoramide nucleotide analog according to claim 1, wherein, Compound 9a exhibits selectivity for liver cancer cells, thus providing a targeted therapy option for colorectal cancer.

6. The noncyclic phosphoramide nucleotide analog according to claim 1, wherein, Compound 9a exhibits selectivity for liver cancer cells, thus providing a targeted therapy option for lung cancer.

7. The noncyclic phosphoramide nucleotide analog according to claim 1, wherein, The synthesis method can be scaled up to the industrial level, thereby enabling large-scale production of compound 9a to meet pharmaceutical application requirements.

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