Use of a class of 1-pentyl-tetrahydro-1h-cyclopenta[1,2-a]phenanthrenes in the treatment of acute myeloid leukemia

Abstract

The application belongs to the technical field of pharmaceutical chemistry, and particularly relates to application of 1-pentyl-tetradecahydro-1H-cyclopenta[1,2-a]phenanthrene in treatment of acute myeloid leukemia. The application provides 1-pentyl-tetradecahydro-1H-cyclopenta[1,2-a]phenanthrene compounds with a novel skeleton. Experimental verification shows that the compounds have excellent inhibitory activity on AML cancer cells, and the mechanism is clear: the compounds can directly inhibit the activity of AML cancer cells, inhibit the proliferation of cancer cells by blocking the cell cycle process, and can specifically induce AML cancer cell apoptosis, thereby realizing the AML treatment effect. The application provides a new active compound candidate for the targeted treatment of acute myeloid leukemia, and lays an important foundation for the research and development of related drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry technology, specifically relating to the application of a class of 1-pentyl-tetradecano-1H-cyclopenta[1,2-a]phenanthrene in the treatment of acute myeloid leukemia. Background Technology

[0002] Acute myeloid leukemia (AML) is a malignant clonal disease originating from hematopoietic stem cells, belonging to the category of acute leukemia. The disease is characterized by the malignant proliferation of myeloid blast cells in the bone marrow, suppressing normal hematopoietic function and leading to symptoms such as anemia, infection, and bleeding. Clinically, AML is characterized by rapid disease progression and extremely poor prognosis, making clinical treatment very challenging. Without timely intervention, the average survival time is only about 3 months, and even after treatment, there is still a high risk of relapse. Therefore, exploring effective treatment strategies is of significant practical importance in alleviating the pressure on medical and health care.

[0003] Traditional chemotherapy is the cornerstone of acute myeloid leukemia (AML) treatment. The "7+3" regimen, consisting of anthracyclines combined with cytarabine, is the gold standard in clinical treatment. Other classic chemotherapy drugs include homoharringtonine and aclarubicin. This type of regimen is suitable for most treatment-naïve AML patients. Its core mechanism of action is broad-spectrum killing of rapidly proliferating leukemia cells, which can rapidly reduce the tumor burden in the patient, laying the foundation for subsequent treatment.

[0004] However, current chemotherapy drugs used in clinical practice have significant shortcomings: First, their targeting is poor. While killing leukemia cells, they easily damage bone marrow hematopoietic stem cells and normal tissue cells, leading to significant toxic side effects such as bone marrow suppression, infection, and organ damage. Second, innovation in drug structure has reached a bottleneck. Existing chemotherapy drugs are mostly concentrated in categories such as nucleoside analogs (e.g., cytarabine) and anthracyclines (e.g., daunorubicin and idarubicin). Their core nucleus structures tend to be fixed, and it is difficult to overcome the core bottleneck of tumor drug resistance and toxic side effects through simple structural modifications, which greatly limits the further improvement of their clinical treatment effects.

[0005] Based on this, developing AML inhibitors with novel core frameworks can fundamentally overcome the technical limitations of the solidified core structure of existing chemotherapy drugs and targeted drugs, becoming a key research and development direction for solving AML chemotherapy resistance, reducing toxic side effects, and improving treatment efficacy. Summary of the Invention

[0006] To overcome the shortcomings of the prior art, this invention provides a class of 1-pentyl-tetradecano-1H-cyclopenta[1,2-a]phenanthrene compounds. These compounds can inhibit the growth of leukemia cells by inhibiting the activity of ATG4B protein. Among them, compounds FGPU-8 and FGPU-13 have superior inhibitory activity compared to cytarabine, a first-line drug for acute myeloid leukemia. This demonstrates that these compounds are promising for use in the preparation of drugs to inhibit acute myeloid leukemia and have good development prospects and broad application potential.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention provides a 1-pentyl-tetradecano-1H-cyclopentano[1,2-a]phenanthrene compound, the structure of which is shown in formula (I): ; In the formula, R1 is selected from hydroxyl, carbonyl, phenyl, methyl acetate, methyl benzoate; R2 is selected from hydrogen, methyl, isopropane; R3 is selected from hydrogen, hydroxyl, carbonyl; R4 is selected from hydrogen, hydroxyl, carbonyl; R5 is selected from isobutane, 2-methylhexane, propionic acid, propane, formic acid, 2-methylbut-3-enoic acid, 2-methylprop-1,2-diol, 2,2-dimethylazacyclopropane; R6 is selected from hydrogen, hydroxyl.

[0008] Preferably, the 1-pentyl-tetradecano-1H-cyclopentano[1,2-a]phenanthrene compound is selected from at least one of compounds FGPU-1 to 17: .

[0009] The second aspect of the present invention also provides the use of the 1-pentyl-tetradecano-1H-cyclopento[1,2-a]phenanthrene compounds described in the first aspect in the preparation of anti-acute myeloid leukemia drugs and / or inhibitors that inhibit the growth of acute myeloid leukemia cells.

[0010] Experimental verification shows that these compounds possess excellent inhibitory activity against AML cancer cells, and their mechanism of action is clear: by inhibiting the activity of ATG4B protein, they suppress the growth of leukemia cells, thereby achieving a therapeutic effect against AML. This invention provides novel active compound candidates for targeted therapy of acute myeloid leukemia and lays an important foundation for the development of related drugs.

[0011] Preferably, the 1-pentyl-tetradecano-1H-cyclopentano[1,2-a]phenanthrene compound is selected from compound FGPU-8 or compound FGPU-13: .

[0012] A third aspect of the present invention also provides a pharmaceutical composition for treating acute myeloid leukemia, wherein the pharmaceutical composition uses the 1-pentyl-tetradecano-1H-cyclopento[1,2-a]phenanthrene compound described in the first aspect as the main active ingredient.

[0013] Preferably, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0014] More preferably, the excipients include at least one of the following: solubilizer, emulsifier, colorant, binder, disintegrant, lubricant, wetting agent, osmotic pressure regulator, stabilizer, flow aid, flavoring agent, preservative, coating material, pH adjuster, absorbent, and antioxidant.

[0015] Preferably, the dosage form of the pharmaceutical composition includes an oral formulation, an injectable formulation, an inhaled formulation, or a transdermal formulation.

[0016] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a class of AML inhibitors with novel core skeletons, specifically 1-pentyl-tetradecano-1H-cyclopento[1,2-a]phenanthrene compounds, aiming to fundamentally solve the technical challenge of the rigid core structure of existing chemotherapy drugs and targeted drugs. These novel skeleton inhibitors hold promise for development into anti-AML small molecule drugs with entirely new skeleton structures, effectively overcoming the bottleneck of existing drug skeleton innovation. Representative compounds FGPU-8 and FGPU-13 exhibit significantly superior in vitro activity compared to cytarabine, a first-line AML treatment, fully demonstrating the promising development prospects and broad application potential of the 1-pentyl-tetradecano-1H-cyclopento[1,2-a]phenanthrene compounds described in this invention in the field of AML treatment. Attached Figure Description

[0017] Figure 1 The inhibitory effect of compound FGPU1-17 on HL-60 cells is shown in the figure. Figure 2 The inhibitory effects of different concentrations of compounds (FGPU-5, FGPU-8, FGPU-9, FGPU-13, FGPU-17) on HL-60 cells after treatment for 48h / 72h are shown in the figure.

[0018] Figure 3 To verify using SPR experiments that compound FGPU1-17 can bind to ATG4B; Figure 4 To detect the inhibitory effect of 10 μM compound FGPU1-17 on ATG4B enzyme activity by FRET method. Detailed Implementation

[0019] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0020] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0021] This invention provides a class of 1-pentyl-tetradecano-1H-cyclopentano[1,2-a]phenanthrene compounds, the structure of which is shown in formula (I): ; In the formula, R1 is selected from hydroxyl, carbonyl, phenyl, methyl acetate, methyl benzoate; R2 is selected from hydrogen, methyl, isopropane; R3 is selected from hydrogen, hydroxyl, carbonyl; R4 is selected from hydrogen, hydroxyl, carbonyl; R5 is selected from isobutane, 2-methylhexane, propionic acid, propane, formic acid, 2-methylbut-3-enoic acid, 2-methylprop-1,2-diol, 2,2-dimethylazacyclopropane; R6 is selected from hydrogen, hydroxyl.

[0022] Preferably, the above-mentioned 1-pentyl-tetradecano-1H-cyclopentano[1,2-a]phenanthrene compounds are selected from at least one of compounds FGPU1-17: .

[0023] Further research revealed that the 1-pentyl-tetradecano-1H-cyclopento[1,2-a]phenanthrene compounds can inhibit the growth of leukemia cells by inhibiting the activity of ATG4B protein, thus confirming that the above-mentioned 1-pentyl-tetradecano-1H-cyclopento[1,2-a]phenanthrene compounds have the potential to be used in the preparation of drugs to inhibit acute myeloid leukemia.

[0024] To fully and clearly present the technical solution and significant advantages of the present invention, the present invention will be described in detail below with reference to specific embodiments.

[0025] Example 1: Inhibitory effect of 1-pentyl-tetradecano-1H-cyclopentano[1,2-a]phenanthrene compounds (FGPU1-17) on the proliferation of HL-60 cell line (1) Test methods Acute myeloid leukemia HL-60 cells were cultured to the logarithmic growth phase (viability >95%), and the test compound (FGPU1-17) and the positive control (cytarabine) were diluted to the required working concentration (10 μM) with complete medium (RPMI-1640 + 10% FBS + 1% P / S).

[0026] Press 2×10 4 HL-60 cells were seeded into 96-well plates, with 100 µL of cell suspension added to each well. Experimental wells, a control group, and a blank control group were also included. The blank control group received only 100 µL of complete culture medium for subsequent background subtraction. The 96-well plates were pre-cultured at 37°C with 5% CO2 for 4–6 h, followed by drug treatment: the test compound was prepared to twice its final concentration (10 μM) using complete culture medium, and 100 µL of this drug-containing medium was added to each experimental well. The control group received 100 µL of complete culture medium containing an equal volume of DMSO per well, ensuring a final volume of 200 µL for each group. After the drug was added, the 96-well plate was returned to the incubator and cultured for another 48 h. After the culture was completed, the plate was removed, 20 μL of CCK-8 solution was added to each well, and the plate was incubated again for 1 h. After the incubation was completed, the plate was removed, and the absorbance (OD value) of each well was measured at 450 nm using a microplate reader. The OD value of each group after subtracting the background of the blank group was calculated, and the cell viability was calculated.

[0027] Calculate the OD value after subtracting blanks: ； Calculate cell viability: .

[0028] (2) Test results like Figure 1 As shown, compounds FGPU-5, FGPU-8, FGPU-13, and FGPU-17 exhibited good inhibitory activity against HL-60 cells at 10 μM. Among them, compounds FGPU-8 and FGPU-13 showed better inhibitory activity against HL-60 cells at 10 μM than cytarabine, a first-line drug for acute myeloid leukemia.

[0029] Example 2: Inhibitory effect of different concentrations (0.1-100 μM) of FGPU compounds on the proliferation of leukemia cell lines Based on Example 1, compounds (FGPU-5, FGPU-8, FGPU-9, FGPU-13, FGPU-17) were selected as examples to verify the inhibitory effects of different concentrations (0.1-100 μM) of the compounds on the proliferation of leukemia cell lines: HL-60 cells were cultured to the logarithmic growth phase (cell viability > 95%), and the test compound and positive control were diluted to the required working concentration (0.1-100 μM) using complete culture medium; 1×10 4 HL-60 cells were seeded into 96-well plates at a density of cells / well, with 100 μL of cell suspension added to each well. Experimental wells, control wells, and blank wells were set up simultaneously. The blank wells were filled with only complete culture medium for subsequent background value subtraction.

[0030] After pre-culturing the 96-well plates in a 37℃, 5% CO2 incubator for 4 h, drug treatment was performed: the drug was prepared into a gradient solution with a final concentration of 2 times using complete culture medium. 100 μL of drug-containing culture medium was added to each well of the plated plates, while the control group received an equal volume of complete culture medium containing DMSO, ensuring that the final volume of each group was consistent. After drug addition, the 96-well plates were returned to the incubator and cultured for 48 h and 72 h, respectively. After the culture was completed, the plates were removed, and 20 μL of CCK-8 solution was added to each well. The plates were then incubated again for 1 h. After incubation, the absorbance (OD value) of each well was measured at 450 nm using a microplate reader. The OD value after subtracting the background of the control group was calculated first, and then the cell viability was calculated accordingly.

[0031] like Figure 2 As shown, compounds FGPU-8 and FGPU-13 at different concentrations exhibited better inhibitory activity against HL-60 cells than cytarabine.

[0032] Example 3: SPR technology detection of the binding of 10 μM FGPU compounds (FGPU1-17) to ATG4B ATG4B is abnormally highly expressed in acute myeloid leukemia (AML) cells, and its expression level is significantly correlated with the malignancy and poor prognosis of AML patients (Wang Z, Fu Y, Lin S, et al. Non-canonical roleof ATG4B in PRMT1-mediated DNA repair and leukemia progression[J]. Autophagy,2025, 21(12): 3425-3427.). Therefore, this experiment used a Biacore 8K instrument (Cytiva) with surface plasmon resonance (SPR) to detect the binding affinity of compounds to ATG4B. The specific experimental procedure is as follows: ATG4B (TargetMol, catalog number TMPH-04256) was immobilized on a CM5 sensor chip at 25°C using a standard amine coupling method. The buffer used in the experiment was 10 mM phosphate buffer (PBST pH 7.4) containing 150 mM NaCl and 0.005% Tween 20. A reference channel was also included, which was activated and blocked only to eliminate interference from non-specific binding of the compound to the chip surface.

[0033] The immobilized response value of ATG4B was approximately 15,000 RU. Subsequently, 10 μM compound FGPU1-17 containing 0.2% DMSO was sequentially injected into the channel to evaluate its binding response value (RU) at a flow rate of 30 µL / min. After each set of sample analysis, the channel was flushed with 50% DMSO to remove any remaining sample and ensure the accuracy of subsequent experimental results.

[0034] The results are as follows Figure 3As shown in the literature (Kudo, Y., Endo, S., Fujita, M., Ota, A.,Kamatari, YO, Tanaka, Y., Ishikawa, T., Ikeda, H., Okada, T., Toyooka, N.,Fujimoto, N., Matsunaga, T., & Ikari, A. (2022). Discovery and Structure-Based Optimization of Novel Atg4B Inhibitors for the Treatment of Castration-Resistant Prostate Cancer. Journal of medicinal chemistry, 65(6), 4878-4892.), the ATG4B positive control inhibitor Atg4B-IN-2 (TargetMol, catalog number T60967) showed a response value of 18.31 (RU) for binding to ATG4B at a concentration of 10 μM; while FGPU at a concentration of 10 μM... Most of the compounds in the series can specifically bind to ATG4B. Among them, FGPU-2, FGPU-4, FGPU-5, FGPU-8, FGPU-9, FGPU-13 and FGPU-17 (all at a concentration of 10 μM) showed better binding response values ​​(RU) to ATG4B than the positive control inhibitor Atg4B-IN-2.

[0035] Example 4: Inhibitory activity of 10 μM FGPU compound (FGPU1-17) on ATG4B enzyme activity Using fluorescence resonance energy transfer (FRET) technology, the fluorescently labeled substrate FRET-GATE-16 (prepared according to the method in the "Protein expression and purification" section on page 410 of the reference "Li, M., Chen, X., Ye, QZ, Vogt, A., & Yin, XM (2012). A high-throughput FRET-based assay for determination of Atg4 activity. Autophagy, 8(3), 401–412.") was used as the detection target to evaluate the inhibitory effect of the compound on ATG4B enzyme activity: if the compound can inhibit ATG4B enzyme activity, it will reduce substrate cleavage and decrease the fluorescence signal enhancement rate. The activity of the inhibitor can be evaluated by quantifying this difference.

[0036] The experiment was conducted using a 100 μL reaction system, including 70 μL PBS reaction buffer, 10 μL of the test compound (FGPU1-17), 10 μL of recombinant ATG4B protein, and 10 μL of FRET-GATE-16. Before preparing the system, 1.25 μg / mL of recombinant ATG4B protein was incubated at 37°C for 30 min. FRET-GATE-16 was diluted to 50 μg / mL, and the 10 mM stock solution of the compound was serially diluted to a final concentration of 10 μM.

[0037] Two control groups were set up to subtract interference and calibrate data, ensuring the accuracy of the results: the blank control well contained 80 μL of reaction buffer + 10 μL of substrate + 1 μL of DMSO, used to subtract background fluorescence. The compound-free well contained 70 μL of reaction buffer + 10 μL of enzyme + 10 μL of substrate + 1 μL of DMSO, representing the compound-free, enzyme-substrate-only complete digestion group.

[0038] After mixing the reaction system, place the reaction plate in a 37 ℃ microplate reader for 30 min. Measure the fluorescence values ​​at 527 nm and 477 nm at the end of the reaction. The entire experiment should be independently repeated at least three times to ensure data reproducibility. The formula for the inhibitory effect of the compound on ATG4B is: Inhibition rate = (RFU) max -RFU compound ) / ( RFU max -RFU min ) 100%. (RFU:527 / 477;RFU max : Blank group; RFU compound Compound group; RFU min (No compounds, only enzymes and substrates).

[0039] like Figure 4 As shown in the literature, the ATG4B positive control inhibitor Atg4B-IN-2 inhibited ATG4B enzyme activity by 76.15±3.38% at a concentration of 10 μM; while FFGPU-4, FGPU-5, FGPU-8, FGPU-9, FGPU-13 and FGPU-17 (all at a concentration of 10 μM) inhibited ATG4B enzyme activity by more than 50%, indicating that these compounds have significant inhibitory effects on ATG4B.

[0040] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A 1-pentyl-tetradecano-1H-cyclopentano[1,2-a]phenanthrene compound, characterized in that, The structure of the 1-pentyl-tetradecano-1H-cyclopentano[1,2-a]phenanthrene compounds is shown in formula (I): ； In the formula, R1 is selected from hydroxyl, carbonyl, phenyl, methyl acetate, methyl benzoate; R2 is selected from hydrogen, methyl, isopropane; R3 is selected from hydrogen, hydroxyl, carbonyl; R4 is selected from hydrogen, hydroxyl, carbonyl; R5 is selected from isobutane, 2-methylhexane, propionic acid, propane, formic acid, 2-methylbut-3-enoic acid, 2-methylprop-1,2-diol, 2,2-dimethylazacyclopropane; R6 is selected from hydrogen, hydroxyl.

2. A 1-pentyl-tetradecano-1H-cyclopentano[1,2-a]phenanthrene compound according to claim 1, characterized in that, The 1-pentyl-tetradecano-1H-cyclopentano[1,2-a]phenanthrene compound is selected from at least one of compounds FGPU-1 to 17: 。 3. The use of the 1-pentyl-tetradecano-1H-cyclopento[1,2-a]phenanthrene compound as described in claim 1 or 2 in the preparation of anti-acute myeloid leukemia drugs and / or inhibitors that inhibit the growth of acute myeloid leukemia cells.

4. The application according to claim 3, characterized in that, The 1-pentyl-tetradecano-1H-cyclopentano[1,2-a]phenanthrene compound is selected from compound FGPU-8 or compound FGPU-13: 。 5. A pharmaceutical composition for treating acute myeloid leukemia, characterized in that, The pharmaceutical composition uses the 1-pentyl-tetradecano-1H-cyclopenta[1,2-a]phenanthrene compound as described in claim 1 or 2 as the main active ingredient.

6. The pharmaceutical composition for treating acute myeloid leukemia according to claim 5, characterized in that, The pharmaceutical composition also includes pharmaceutically acceptable excipients.

7. The pharmaceutical composition for treating acute myeloid leukemia according to claim 6, characterized in that, The excipients include at least one of the following: solubilizer, emulsifier, colorant, binder, disintegrant, lubricant, wetting agent, osmotic pressure regulator, stabilizer, flow aid, flavoring agent, preservative, coating material, pH adjuster, absorbent, and antioxidant.

8. The pharmaceutical composition for treating acute myeloid leukemia according to claim 6, characterized in that, The dosage forms of the pharmaceutical composition include oral formulations, injectable formulations, inhaled formulations, or transdermal formulations.

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