Homoharringtonine derivative as well as preparation method and application thereof

Abstract

The invention discloses a homoharringtonine derivative. The homoharringtonine derivative has the characteristics of good tumor treatment effect, better safety and smaller toxic and side effects.

Description

Technical Field

[0001] The present invention belongs to the field of natural medicines and medicinal chemistry, and relates to homoharringtonine derivatives, methods for preparing these compounds, compositions containing these compounds, and their uses in the preparation of anti-tumor drugs. Background Art

[0002] Homoharringtonine (HHT) exists in plants of the genus Cephalotaxus. In 1963, Paudler et al. first isolated cephaloyaxine from Cephalotaxus drupacea Sieb. et Zucc and Cephalotaxus fortunei Hook.f. In 1969, Powell et al. first isolated homoharringtonine from Cephalotaxus harringtonia var. drupacea, determined its structure as (2R)-2-hydroxy-2-(4-hydroxy-4-methylpentyl)succinic acid (methyl)(cephalotaxine) ester, and found that it had obvious activity against murine lymphocytic leukemia P388. In September 1969, at the first "Anti-tumor Drug Research Conference" held in Shijiazhuang, China, considering that Cephalotaxus is mainly produced in China, a national research collaboration group for anti-tumor drugs of Cephalotaxus was established. In September 1977, after formal appraisal at the national conference, it was proved that homoharringtonine had significant curative effects on human non-lymphatic system leukemia, and it became a new anti-leukemia drug first recommended for clinical application in China. In the 1990s, it was listed as one of the 47 commonly used anti-cancer drugs by the Ministry of Health and was included in the "Chinese Pharmacopoeia" in the 1990 edition.

[0003] Although homoharringtonine has achieved good results in the clinical treatment of leukemia, and preliminary research results on its biological activity show that it also has high inhibitory activity against a variety of solid tumors, homoharringtonine still has strong toxic and side effects. The main toxic and side effects are the toxic and side effects on the heart, digestive system and hematopoietic cells, and allergic reactions and allergic reactions are also relatively common. Hypotension and hypoglycemia are also relatively common. The main symptoms of the cardiac toxicity of HHT are sinus tachycardia, atrial or ventricular premature contractions, and the electrocardiogram shows myocardial ischemia manifestations such as S-T segment changes and T wave flattening. Gallop rhythm, atrioventricular block, bundle branch block and atrial fibrillation also occasionally occur. Common digestive tract reactions such as anorexia, nausea and vomiting, and occasional liver function damage are also seen.

[0004] It can be seen that the prior art still needs a compound with better anti-tumor treatment effect, milder toxic and side effects and better safety. Summary of the Invention

[0005] The object of the present invention is to provide a homoharringtonine derivative with better safety and smaller toxic and side effects while having a good anti-tumor treatment effect.

[0006] The present invention is achieved through the following technical solutions:

[0007] A homoharringtonine derivative of formula I or its isomer, or its pharmaceutically acceptable salt, or its prodrug molecule

[0008]

[0009] Use of the derivative or its isomer, or its pharmaceutically acceptable salt, or its prodrug molecule according to the present invention in the preparation of anti-tumor agents.

[0010] The tumor according to the present invention is one or more of melanoma, prostate cancer, pancreatic cancer, leukemia, esophageal cancer, multiple myeloma, lymphoma, nasopharyngeal carcinoma, clear cell renal carcinoma, glioma, osteosarcoma, liver cancer, endometrial cancer, basal cell carcinoma, cervical cancer, laryngeal cancer, colorectal cancer, gastric cancer, ovarian cancer.

[0011] The derivative or its isomer, or its pharmaceutically acceptable salt, or its prodrug molecule according to the present invention and a pharmaceutically acceptable carrier or excipient form a pharmaceutical composition.

[0012] The pharmaceutical composition according to the present invention is suitable for oral administration or parenteral administration.

[0013] Use of the pharmaceutical composition according to the present invention in the preparation of anti-tumor agents.

[0014] The present invention also provides a method for preparing the derivative of formula I or its isomer, or its pharmaceutically acceptable salt, or its prodrug molecule: reacting homoharringtonine with an alcohol having C1-C 12 to form formula II under the catalysis of sodium hydride Then reacting formula II with acetic anhydride, acetyl chloride or acetic acid to form formula I.

[0015] The homoharringtonine derivative provided by the present invention has better safety and smaller toxic and side effects while having good anti-tumor effects. Detailed implementation mode

[0016] Experimental materials

[0017] Homoharringtonine (HHT): Han Suyuan batch number HHRT20230106-2

[0018] Sodium cyanide (NaH): damas-beta, 013474116

[0019] Heptanol: Bidepharm, CRY700

[0020] Acetic anhydride: Chengdu Kelong Chemical Co., Ltd.

[0021] A375, Bxpc-3, K562, Z-138, CNE2Z, 786-O, U87, MG63, Ishikawa, PE / CA-PJ34, Hela, MKN-1, A2780, MCF-7, A549, SW579, SW780 cells were purchased from Nanjing Kebai Biotechnology Co., Ltd.;

[0022] DU145 cells were purchased from Ningbo Taisituo Biotechnology Co., Ltd.;

[0023] OE-19 cells were purchased from Wuhan Punosai Life Science Co., Ltd.;

[0024] RMPI-8226 and HCT-15 cells were purchased from Beina Biology;

[0025] HepG2 and TU686 cells were purchased from Shanghai Jining Biotechnology Co., Ltd.;

[0026] Homoharringtonine (HHT) (Hansuyuan Biotechnology, Lot. No. HZH3Y20220325-3)

[0027] Trypsin (Gibco, Lot. No. 2764663), FBS (Sciencell, Lot. No. 37850)

[0028] DMEM medium (Gibco, Lot. No. 2323071)

[0029] 1640 medium (Gibco, Lot. No. 2572135)

[0030] F12K medium (Ningbo Taisituo Biology, Lot. No. 2023080301)

[0031] IMDM medium (Gibco, Lot. No. 2702966)

[0032] MCF complete medium (Nanjing Kebai Biotechnology, Lot. No. CBFB23101707)

[0033] Penicillin-streptomycin double antibody (Gibco, Lot. No. 2588019)

[0034] CCK8 detection kit (Beyotime, Lot. No. 112921220223), Chengdu Kelong Chemical Co., Ltd.

[0035] Experimental instruments

[0036] Thermostatic heating magnetic stirrer with heat collection (Zhengzhou Saitelis Biotechnology Co., Ltd., DF101S)

[0037] Fluorescence cell analyzer (Countstar, Rigel S2)

[0038] Multifunctional microplate reader (Tecan, Spark)

[0039] Electronic balance (Sartorius, SECURA225D - 1CN)

[0040] Laminar flow hood (ESCO, SW - CJ - 2FD)

[0041] Biological microscope (Olympus, CKX53SF)

[0042] Centrifuge (Shanghai Medical Instrument, Model 0406 - 1)

[0043] CO2 incubator (Thermo Scientific, 371)

[0044] Preparation of the compound of Example 1

[0045] Synthesis of the compound of Formula II: Homoharringtonine (500 mg, 0.92 mmol) was added to a 100 mL three - necked flask, then 60% NaH (11 mg, 0.27 mmol) was added, and then 5 ml of heptanol was added. The mixture was stirred at room temperature for 1.5 hours. The reaction was stopped, and 1N dilute hydrochloric acid was added to adjust the pH to 5. Then it was extracted twice with EA, and the organic phase was collected. Ammonia water was added to the aqueous phase to adjust the pH to 8, and it was extracted five times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, concentrated to obtain a crude product, and purified by silica gel column chromatography (DCM:MeOH = 15:1) to obtain 280 mg of a colorless transparent liquid. That is, the compound of Formula II. m / z 630.30 [M + H]+

[0046] 1 HNMR(400MHz,CDCl3)δ6.62(s,1H),6.60(s,1H),6.01(d,J = 8Hz,1H),5.90(m,2H),5.03(s,1H),4.2(m,1H),4.04 - 3.96(m,1H),3.94 - 3.87(m,2H),3.77(s,3H),3.59 - 3.41(m,2H),3.31 - 3.18(m,1H),2.99 - 2.86(m,2H),2.57 - 2.46(m,1H),2.42 - 2.25(m,2H),2.17 - 2.06(m,1H),2.05 - 1.91(m,3H),1.68 - 1.49(m,5H),1.43 - 1.23(m,12H),1.18 - 1.07(m,6H),0.92 - 0.82(m,3H).

[0047] The structure of the compound of Formula II is as follows:

[0048]

[0049] Synthesis of the compound of Formula I: Add the control compound 1 (240 mg, 0.38 mmol) into a 100 ml three-necked flask, then add 4 ml of acetic anhydride, stir and react at 80 °C for 24 hours. Stop the reaction, concentrate most of the solvent under reduced pressure, adjust the pH to 8 with saturated sodium bicarbonate, extract three times with dichloromethane, combine the organic phases, dry over anhydrous sodium sulfate, and concentrate to obtain the crude product. Purify by silica gel column chromatography (DCM:MeOH = 10:1) to obtain 120 mg of a colorless transparent liquid, and further obtain 62 mg of the compound of Formula I by HPLC preparative separation. m / z 672.43 [M+H]+

[0050] 1 HNMR (400 MHz, CDCl3) δ 6.62 (s, 1H), 6.60 (s, 1H), 6.01 (d, J = 8 Hz, 1H), 5.90 (m, 2H), 5.03 (s, 1H), 4.2 (m, 1H), 4.04 - 3.96 (m, 1H), 3.94 - 3.87 (m, 2H), 3.77 (s, 3H), 3.59 - 3.40 (m, 2H), 3.31 - 3.18 (m, 1H), 2.99 - 2.86 (m, 2H), 2.57 - 2.46 (m, 1H), 2.42 - 2.25 (m, 2H), 2.17 - 2.06 (m, 1H), 2.05 - 1.91 (m, 6H), 1.68 - 1.49 (m, 4H), 1.43 - 1.36 (m, 7H), 1.35 - 1.23 (m, 11H), 0.92 - 0.82 (m, 3H).

[0051] The structure of the compound of Formula I is as follows:

[0052]

[0053] Synthesis of the compound of Formula III: Add homoharringtonine (500 mg, 0.92 mmol) into a 100 ml three-necked flask, then add 5 ml of acetic anhydride, stir and react at 90 °C for 14 hours. Stop the reaction, concentrate most of the solvent under reduced pressure, adjust the pH to 8 with saturated sodium bicarbonate, extract three times with dichloromethane, combine the organic phases, dry over anhydrous sodium sulfate, and concentrate to obtain the crude product. Purify by silica gel column chromatography (DCM:MeOH = 10:1) to obtain 240 mg of a colorless transparent liquid, and further obtain 100 mg of the compound of Formula III by HPLC preparative separation. m / z 588.2 [M+H]+

[0054] 1HNMR(400MHz,CDCl3)δ6.63(s,1H),6.59(s,1H),6.01(d,J=8Hz,1H),5.90(m,2H),5.04(s,1H),3.85(d,J=8Hz,1H),3.74(s,3H),3.57(s,3H),3.39 - 3.25(m,3H),3.25 - 3.12(m,1H),2.82(m,2H),2.47(m,1H),2.34 - 2.17(m,2H),2.13 - 2.00(m,1H),2.0 - 1.83(m,6H),1.64(m,2H),1.45 - 1.34(m,9H),1.19 - 1.08(m,1H).

[0055] The compound of Formula III is as follows:

[0056]

[0057] Example 2 In Vitro Antitumor Activity

[0058] 1. Experimental Procedures

[0059] 1) Collect cells in the logarithmic growth phase, count them, resuspend the cells with complete medium, adjust the cell concentration to an appropriate concentration, inoculate a 96-well plate, and add 100 μl of cell suspension to each well. The cells are incubated in an incubator at 37 °C, 100% relative humidity, and 5% CO2 for 24 h.

[0060] 2) Dilute the compound to be tested with the medium to the required working solution concentration, add 100 μl of the corresponding medium containing the drug to each well, and set up a negative control group at the same time. The final concentration of the compound starts from 1000 nM and is diluted in a 3-fold serial dilution with 9 concentration points, and the test is performed in duplicate.

[0061] 3) The cells are incubated in an incubator at 37 °C, 100% relative humidity, and 5% CO2 for 72 h respectively.

[0062] 4) Aspirate the medium containing the drug, add 10% CCK-8 detection solution to the 96-well plate, and incubate it in an incubator at 37 °C, 100% relative humidity, and 5% CO2 for 1 - 4 h.

[0063] 5) After gently shaking, measure the absorbance at a wavelength of 450 nm on an enzyme-linked immunosorbent assay (ELISA) reader, calculate the proliferation inhibition rate, and the cell proliferation inhibition rate = (OD value of the negative control group - OD value of the experimental group) / (OD value of the experimental group - OD value of the blank group). Use the software Graphpad Prism and adopt a four-parameter Logistic regression model to fit the IC50 curve and calculate the IC50 value.

[0064] 2. Experimental Results

[0065] The experimental results are shown in Table 1, and the results show that the compound of Formula I has curative effects on melanoma, prostate cancer, pancreatic cancer, leukemia, esophageal cancer, multiple myeloma, lymphoma, nasopharyngeal carcinoma, renal clear cell carcinoma, glioma, osteosarcoma, liver cancer, endometrial cancer, basal cell carcinoma, cervical cancer, laryngeal cancer, colorectal cancer, gastric cancer, and ovarian cancer.

[0066] Table 1

[0067]

[0068]

[0069] Example 3 Canine Safety Experiment

[0070] 1. Experimental Materials

[0071] 1.1 Experimental Reagents

[0072] 5% Glucose Injection (Sichuan Kelun Pharmaceutical Co., Ltd., U22090504)

[0073] Normal Saline (Qingdao Hi-Tech Industrial Park Haibo Biotechnology Co., Ltd., 20230404)

[0074] Homoharringtonine (HHT) Injection (Hangzhou Minsheng, batch number 2208257, content 1mg / mL)

[0075] 1.2 Experimental Instruments

[0076] Electronic Table Scale (Zhejiang Xingheng Electronics Co., Ltd., TCS-300)

[0077] Blood Pressure Monitor (Contec Medical Systems (Qinhuangdao) Co., Ltd., 08AVET)

[0078] Infusion and Injection Pump (Shanghai Land Medical Instrument Co., Ltd., LD-P2020II)

[0079] Low Temperature High-Speed Centrifuge (Sichuan Shuke Instrument Co., Ltd., TDL-6)

[0080] 2. Preparation of Administration Solution

[0081] HHT Marketed Solution (Hangzhou Minsheng, batch number 2208257, content 1mg / mL): Dilute it to an appropriate concentration with 5% Glucose Injection for injection according to the animal weight before use.

[0082] Solutions of Compounds of Formula I, Formula II, and Formula III: Appropriate amount of API was taken and dissolved in 20 mL of dichloromethane. Approximately 3% of HS15 was added and dissolved. After dissolution, the organic reagent was removed by rotary evaporation under reduced pressure at 40 °C. 0.9% NaCl solution was added for dissolution, and it was filtered through a 0.22 μm PVDF membrane to obtain a dosing preparation with a concentration of approximately 1 mg / mL. Before use, it was diluted to an appropriate concentration with 0.9% NaCl solution according to the animal body weight.

[0083] 3. Experimental Methods

[0084] Five adult male Beagle dogs were selected. After 5 - 7 days of adaptive feeding, they were continuously administered for 1 hour by intravenous bolus injection using an infusion pump, and then the animal status was observed for 3 - 5 hours. At the same time, the blood pressure and heart rate of the dogs were continuously measured and recorded within 4 - 6 hours. Blood of the dogs was collected at different time points during the experiment. The anticoagulant was EDTA - 2K, and it was centrifuged at 5600 r / min for 10 min. The upper plasma was taken to detect the drug concentration. The grouping, dosing dose, and dosing volume are shown in Table 2 below.

[0085] Table 2

[0086]

[0087] 4. Experimental Results

[0088] The experimental results are shown in Table 3. The results show that the safety of the Compound of Formula I is better than that of Compound HHT, the Compound of Formula III, and the Compound of Formula II. There are no arrhythmia, tachycardia, hematochezia, hemolysis, diarrhea, or vomiting, and no obvious abnormalities are seen in the systolic and diastolic blood pressures. Overall, it shows outstanding advantages in terms of safety.

[0089] Table 3

[0090]

[0091]

[0092] A heart rate exceeding 160 beats / min is defined as tachycardia.

Claims

1. A homoharringtonine derivative of formula I or an isomer thereof, or a pharmaceutically acceptable salt thereof, or a prodrug molecule thereof 2. Use of the derivative or an isomer thereof, or a pharmaceutically acceptable salt thereof, or a prodrug molecule thereof according to claim 1 in the preparation of an anti-tumor agent 3. The use according to claim 2, wherein The tumor is one or more of melanoma, prostate cancer, pancreatic cancer, leukemia, esophageal cancer, multiple myeloma, lymphoma, nasopharyngeal carcinoma, clear cell renal carcinoma, glioma, osteosarcoma, liver cancer, endometrial cancer, basal cell carcinoma, cervical cancer, laryngeal cancer, colorectal cancer, gastric cancer, ovarian cancer 4. A pharmaceutical composition comprising the derivative or an isomer thereof, or a pharmaceutically acceptable salt thereof, or a prodrug molecule thereof according to claim 1 and a pharmaceutically acceptable carrier or excipient 5. The pharmaceutical composition according to claim 4, wherein The pharmaceutical composition is suitable for oral administration or parenteral administration 6. The pharmaceutical composition according to claim 4 or 5, characterized in that, Use of the pharmaceutical composition in the preparation of an anti-tumor agent 7. A method for preparing a derivative of formula I or an isomer thereof, or a pharmaceutically acceptable salt thereof, or a prodrug molecule thereof, characterized in that, Homoharringtonine is reacted with an alcohol having 1 to 12 carbon atoms in the presence of sodium hydride to produce formula II React formula II with acetic anhydride, acetyl chloride or acetic acid to produce formula I.

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