Fumaric acid-chloroacetamide compound ZWH-01 and preparation method and application thereof

Abstract

The application belongs to the technical field of medicines, and discloses a novel fumaric acid-chloroacetamide compound ZWH-01 with anti-thyroid cancer activity and a preparation method of the compound. The compound has a simple synthesis route, mild and environmentally-friendly reaction conditions, and a high yield. The compound has significant growth inhibition activity on human anaplastic thyroid cancer cells 8505c cells, is obviously stronger than a clinically-applied chemotherapeutic drug cisplatin, and can inhibit the migration and invasion of 8505c cells in a concentration-dependent manner, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of medical and pharmaceutical technology, and in particular relates to a fumaric acid-chloroacetamide compound ZWH-01, its preparation method, and its use in thyroid cancer. Background Technology

[0002] Currently, the treatment of thyroid cancer mainly adopts a comprehensive management model that combines surgical resection with radioactive iodine therapy (RAI), thyroid hormone suppression therapy (TSH suppression), and targeted therapy. For differentiated thyroid cancer (DTC), surgery combined with postoperative radioactive iodine therapy can achieve good results; however, for advanced patients, especially radioactive iodine-refractory differentiated thyroid cancer (RAIR-DTC) and undifferentiated thyroid cancer (ATC), existing treatments have limited effectiveness. Therefore, the search and development of highly effective novel anti-thyroid cancer drugs, especially for undifferentiated cancer, is of significant clinical importance.

[0003] In recent years, covalent inhibitors have attracted much attention in the field of drug development and have shown broad application prospects. The U.S. Food and Drug Administration (FDA) has successively approved a number of covalent inhibitor drugs, most of which are used in the field of anti-tumor treatment. Compared with traditional non-covalent inhibitors, these drugs have significant advantages due to their unique mechanism of action: their molecular structure adopts an innovative "guided system-warhead" design, which not only achieves more precise target binding but also significantly improves the selectivity and duration of action while reducing the incidence of adverse reactions. Based on this characteristic, we have innovatively developed a novel dual-warhead covalent compound. This design further enhances the drug's targeting, anti-tumor efficacy, and duration of action through synergistic effects. Summary of the Invention

[0004] In a first aspect, the present invention provides a fumaric acid-chloroacetamide compound ZWH-01, which has the following structure:

[0005]

[0006] The chemical name of this compound is: (E)-2-chloro-N-(3-chloro-4-((1-(2-(4-(4-(4-methoxyphenyl)-4-oxobut-2-enoyl)piperazin-1-yl)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-N-(2-oxo-2-(phenethylamino)-1-(thiophen-2-yl)ethyl)acetamide

[0007] Secondly, the present invention provides the use of the aforementioned compound ZWH-01 in the preparation of drugs for the prevention and / or treatment of cancer. In some preferred embodiments, the cancer includes thyroid cancer. In some specific embodiments, the compound exhibits significant inhibitory activity against undifferentiated thyroid cancer cells 8505c.

[0008] In this embodiment, the present invention provides a novel fumaric acid-chloroacetamide bis-covalent compound ZWH-01, which is synthesized via a route with relatively mild reaction conditions and high yield. The compound was obtained through in vitro pharmacodynamic experiments (…). Figure 3 Figure 6) fully demonstrates that ZWH-01 has significant inhibitory activity against the proliferation of human undifferentiated thyroid cancer cells 8505c, IC50. 50 = 2.4 μM, significantly better than cisplatin; inhibited cell migration in a concentration- and time-dependent manner; inhibited long-term cell proliferation in a concentration-dependent manner; and inhibited cell invasion in a concentration-dependent manner.

[0009] These results indicate that compound ZWH-01 shows good potential in inhibiting highly malignant and poorly prognostic undifferentiated thyroid carcinoma, and has the prospect of being developed into a novel anti-thyroid cancer drug, particularly for the treatment of undifferentiated or refractory differentiated thyroid carcinoma. The preparation method of compound ZWH-01 is described below, which includes the following steps:

[0010] a) Compound 5 was obtained via a four-component reaction using Ugi;

[0011] b) The reaction of trans-3-(4-methoxybenzoyl)acrylic acid with a Boc monosubstituted piperazine derivative yields compound 8;

[0012] c) In the presence of trifluoroacetic acid, intermediate 8 is desubstituented with Boc to give compound 9;

[0013] d) Compound 9 undergoes an amide condensation reaction with azidoacetic acid in the presence of condensing agent TCFH to give azido compound 10;

[0014] e) Compound 10 and compound 5 undergo a copper-catalyzed click reaction to yield the target compound ZWH-01. . Attached Figure Description

[0015] Figure 1 This is the synthesis route diagram for ZWH-01.

[0016] Figure 2 Cell survival data and curves of human thyroid undifferentiated cancer cells 8505c under different drug concentrations of ZWH-01 and cisplatin.

[0017] Figure 3 shows micrographs of the scratched areas of human thyroid undifferentiated cancer cells 8505c at 0, 24, and 48 hours after treatment with different concentrations of ZWH-01, along with corresponding calculated cell migration rates. The scale of the micrographs is 200 micrometers.

[0018] Figure 4 shows micrographs of cell clone formation and bar charts of the number of clones in human thyroid undifferentiated cancer cells 8505c after treatment with different concentrations of ZWH-01.

[0019] Figure 5 shows Transwell cell migration assays, Transwell cell invasion assays, and statistical graphs of migrating and invading cell counts of human thyroid undifferentiated cancer cells 8505c after treatment with different concentrations of ZWH-01. The scale of the micrographs is 100 micrometers. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments to provide a better understanding of the technical solution. The technical and scientific terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. The basic raw materials and reagents are obtained commercially and have a purity of 97% or higher.

[0021] The room temperature described in this invention is 25-30°C. This invention provides a general and specific description of the materials and experimental methods used in the experiments.

[0022] Example 1: Synthesis and Structural Confirmation of ZWH-01

[0023] Figure 1 The synthetic route for compound ZWH-01 is illustrated below:

[0024] 1. Synthesis of Compound 2:

[0025] A suspension of compound 1, 4-amino-2-chlorophenol (500 mg, 3.48 mmol) and di-tert-butyl dicarbonate (836 mg, 3.83 mmol), dissolved in tetrahydrofuran (20 mL) was stirred at room temperature for 24 hours. The reaction mixture was concentrated under reduced pressure and extracted twice with ethyl acetate. The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was separated by silica gel column chromatography (mobile phase: ethyl acetate:petroleum ether, v / v) to give compound 2 (yellow liquid, 714 mg, 84% yield).

[0026] 2. Synthesis of compound 3:

[0027] Compound 2 (1000 mg, 4.1 mmol) was dissolved in N,N-dimethylformamide (20 mL), and potassium carbonate (851 mg, 6.2 mmol) and 3-bromopropane-1-yne (634 mg, 5.3 mmol) were added. The mixture was stirred overnight at room temperature. The reaction solution was diluted with water and extracted three times with ethyl acetate. The combined organic layers were washed once each with water and saturated sodium chloride solution, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was separated by silica gel column chromatography (mobile phase: ethyl acetate:petroleum ether, v / v) to give compound 3 (yellow liquid, 850 mg, yield 73%).

[0028] 3. Synthesis of compound 4:

[0029] Compound 3 (1000 mg, 3.55 mmol) was dissolved in dichloromethane (40 mL), followed by the addition of trifluoroacetic acid (10 mL), and the mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and an aqueous solution of NaHCO3 was added, followed by extraction three times with ethyl acetate. The separated organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give compound 4 (white solid, 480 mg, yield 74%).

[0030] 4. Synthesis of compound 5 (ML162-yne):

[0031] Compound 4 (1.82 g, 10 mmol) and 2-thiophenecarboxaldehyde (1.12 g, 10 mmol) were dissolved in methanol (25 mL), activated at 25 °C for 1 h, and then (2-isocyanoethyl)benzene (1.09 g, 8.33 mmol) and chloroacetic acid (787.45 mg, 8.33 mmol) were added. The mixture was stirred overnight at room temperature. The reaction solution was concentrated under reduced pressure, and the residue was separated by silica gel column chromatography (mobile phase: ethyl acetate:petroleum ether, v / v) to give 5 mL of compound 162-yne (white solid, 850 mg, yield 20%).

[0032] 5. Synthesis of Compound 8:

[0033] Compound 6, trans-3-(4-methoxybenzoyl)acrylic acid (977 mg, 7.3 mmol), HATU (3.1 g, 8.1 mmol), DIPEA (3.8 mL, 21.8 mmol), and compound 7, 1-Boc-piperazine (1.5 g, 8.0 mmol), were dissolved in DMF and reacted overnight at room temperature. The crude residue was purified by silica gel chromatography (0-50% EtOAC / petroleum ether) to give 1.59 g of target compound 8 as a yellow solid, with a yield of 58%.

[0034] 6. Synthesis of Compound 9:

[0035] Compound 8 (78.6 mg, 0.21 mmol) and trifluoroacetic acid (0.51 mL, 6.7 mmol) were stirred for 1.5 hours. The reaction mixture was concentrated under vacuum, redissolved in DCM, and stirred with saturated NaHCO3 solution (2 mL) for 30 minutes. The organic layer was separated, washed with brine, dried over Na2SO4, and concentrated under vacuum. The crude residue was purified by silica gel chromatography (0–12% MeOH / DCM) to give 20.8 mg of target compound 9 as a white solid, in 81% yield.

[0036] 7. Synthesis of Compound 10:

[0037] Compound 9 (200 mg, 0.38 mmol) was dissolved in acetonitrile (5 mL), and 2-azidoacetic acid (340 mg, 3.4 mmol), tetramethylchlorourea hexafluorophosphate (128.94 mg, 0.46 mmol), and N-methylimidazole (109.2 mg, 1.33 mmol) were added. The mixture was stirred overnight at room temperature. The reaction solution was concentrated under reduced pressure, and the residue was separated by silica gel column chromatography (eluting with ethyl acetate:petroleum ether in a volume ratio of 10:1) to give compound 10 (white solid, 200 mg, yield 80%).

[0038] 8. Synthesis of the final product ZWH-01:

[0039] Compound 10 (1.0 eq, 0.5 mmol) and compound 5 ML162-yne (1.0 eq, 0.5 mmol) were dissolved in tetrahydrofuran (5 mL), and copper sulfate (0.05 eq, 0.025 mmol) and sodium vitamin C (2.5 eq, 1.25 mmol) were dissolved in water (1 mL). The reaction mixture was reacted overnight at room temperature under nitrogen protection. The reaction solution was diluted with ethyl acetate (200 mL), washed once with water and once with saturated sodium chloride solution, and the organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated to dryness by rotary evaporation, and the concentrate was separated by silica gel column chromatography (eluent: dichloromethane and methanol in a volume ratio of 20:1) to obtain the target compound ZWH-01.

[0040] (E)-2-chloro-N-(3-chloro-4-((1-(2-(4-(4-(4-methoxyphenyl)-4-oxobut-2-enoyl)piperazin-1-yl)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-N-(2-oxo-2-(phenethylamino)-1-(thiophen-2-yl)ethyl)acetamide; 1H NMR (400 MHz, CDCl3) δ 7.98 (d, J = 2.1 Hz, 1H), 7.97 (d, J = 1.9 Hz, 1H), 7.91 (s, 1H), 7.81 (d, J = 5.5 Hz, 1H), 7.40(d, J = 6.5 Hz, 1H), 7.36 (s, 1H), 7.13 (d, J = 7.2 Hz, 2H), 7.09 – 7.04 (m,3H), 6.96 – 6.89 (m, 4H), 6.78 (s, 4H), 6.01 (s, 1H), 5.23 (s, 2H), 5.22 (s,2H), 3.83 (s, 3H), 3.75 (d, J = 1.8 Hz, 4H), 3.66 – 3.64 (m, 2H), 3.59 (s,2H), 3.48 (dq, J = 13.4, 6.5 Hz, 4H), 2.76 – 2.71 (m, 2H).

[0041] Example 2: Assay for Anti-cell Proliferation Activity

[0042] Experimental Methods: 3000 8505c cells were seeded into each well of a 96-well plate (100 μL serum-containing medium). Blank wells (medium only) and control wells (untreated cells) were included, and the cells were incubated at 37°C for 24 h. Complete medium containing different concentrations of ZWH-01 (concentration gradient: 1, 2, 4, 8, 16, 32 μM) or cisplatin (positive control) were added according to the experimental design, and the cells were incubated at 24°C for 48 h. 10 μL of CCK-8 reagent was added directly to each well (avoiding air bubbles), and the cells were incubated at 37°C in the dark for 2 h. The absorbance (OD) value at 450 nm was measured using a microplate reader.

[0043] Experimental results: such as Figure 2 As shown, ZWH-01 exhibits significant inhibitory activity against the proliferation of human undifferentiated thyroid cancer cells 8505c in a concentration-dependent manner. Its IC50 value was calculated. 50 The value was 2.4 μM, significantly lower than the IC50 of cisplatin, a commonly used chemotherapy drug in this cell line. 50 The value (14.8 μM) indicates that ZWH-01 has stronger in vitro antitumor activity.

[0044] Example 3 Cell Scratch Assay

[0045] Experimental method: 2×10⁻⁶ mm² of material was used in a 6-well plate. 5Cells were seeded at a high density of 100 cells / well and incubated overnight at 37°C. Observation was performed until cell confluence reached 90% or higher. Cells were vertically scraped using a 200 μL pipette tip to create scratches. Exfoliated cells were washed with PBS. Two mL of serum-free medium and two mL of serum-free medium at 0, 1.5 μM, and 3 μM drug concentrations were added, with three replicates per group. Images of the same scratch location were taken under a microscope (4× objective) at 0, 24, and 48 hours. Changes in scratch area were analyzed using ImageJ software to calculate migration rate.

[0046] Experimental results: such as Figure 3 As shown, compared with the control group, the ability of cells in the ZWH-01 treatment group to migrate to the scratch area was significantly reduced. This inhibitory effect was concentration- and time-dependent (the inhibitory effect increased with increasing drug concentration and treatment time).

[0047] Example 4: Cloning Experiment

[0048] Experimental Methods: 8505c cells were seeded at a low density (1000 cells / well) in 12-well plates and incubated at 37°C for 24 hours. A blank control group and treatment groups were then established. The blank control group received 1 ml of complete culture medium, while the treatment groups received 1 ml of complete culture medium containing 0.75 μmol / ml, 1.5 μmol / ml, and 3 μmol / ml drug concentrations, respectively. Each group had three replicates. Cell status was observed daily for 7 days. After culture, the culture medium was discarded, and cells were fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet for 15 min. The number of clones with more than 50 cells per well was counted using ImageJ software.

[0049] Experimental results: such as Figure 4 As shown, ZWH-01 significantly inhibited the colony-forming ability of 8505c cells in a concentration-dependent manner. Compared with the control group, the number and size of colonies formed were significantly reduced with increasing drug concentration, indicating that ZWH-01 can effectively inhibit the long-term proliferation and survival of tumor cells.

[0050] Example 5 Cell migration and invasion assay

[0051] Experimental Methods: 8505c cells were pretreated with drug concentrations of 0.75 μmol / ml, 1.5 μmol / ml, and 3 μmol / ml. The control group (without drug) and the three experimental groups were then digested with trypsin, centrifuged, and resuspended in serum-free medium for counting. Serum-free cell suspensions were prepared, and 2 × 10⁶ cells were seeded in each chamber. 4Cells / 200μL, three replicates per group. Lower chamber: Add 600μL of culture medium containing 10% FBS; after culturing for 24 hours, fix cells with 4% paraformaldehyde for 15 min and stain with crystal violet for 15 min. Wash with PBS, wipe away unmigrated cells from the upper chamber with cotton swabs, and photograph randomly selected fields of view under a microscope (10× objective). Count the number of cells that have migrated to the lower membrane surface using ImageJ software. For the invasion assay, thaw Matrigel at 37 ℃ beforehand and coat Transwell membranes overnight at 4 ℃. Subsequent steps are the same as for the migration assay (6×10⁶ cells per chamber). 4 (cells / 200μL).

[0052] Experimental results: such as Figure 5 As shown, ZWH-01 significantly inhibited the migration and invasion abilities of 8505c cells in a concentration-dependent manner. Compared with the control group, the number of cells that crossed the Transwell membrane in the migration experiment or the Matrigel membrane in the invasion experiment was significantly reduced in the drug-treated group. The experimental results indicate that ZWH-01 can effectively inhibit the ability of tumor cells to degrade the matrix and invade.

Claims

1. A fumaric acid-chloroacetamide compound ZWH-01, characterized by, The compound ZWH-01 has the following structure: 。 2. Use of the compound ZWH-01 according to claim 1 in the preparation of a drug for preventing and / or treating cancer, which is thyroid cancer.

3. The method for preparing compound ZWH-01 as described in claim 1, characterized in that, The preparation method comprises the following steps: 。

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