Halimane type diterpenoid compound as well as preparation method and application thereof

Abstract

The invention discloses a halimane type diterpenoid compound as well as a preparation method and application of the halimane type diterpenoid compound. The halimane type diterpenoid compound is extracted and separated from roots of ossein crassifolius, and has a structural formula as shown in any one of formulas (I)-(XII): # imgabs0 #. The halimane type diterpenoid compound provided by the invention has an obvious inhibition effect on HT22 cell ferroptosis induced by erastin, does not show cytotoxicity, can be applied to preparation of ferroptosis inhibitors, and has a better application prospect; relates to the technical field of compounds and medicines.

Description

Technical Field

[0001] The present invention relates to the technical field of compounds and medicines, and in particular to a halimane-type diterpenoid compound, a preparation method and application thereof. Background Art

[0002] Ferroptosis is an iron-dependent, programmed cell death, a novel type of cell death distinct from apoptosis, pyroptosis, necrosis, and autophagy. Ferroptosis is essentially caused by excessive intracellular lipid peroxidation and the metabolic disturbance of its product, lipid hydroperoxides (LOOHs). Catalyzed by iron, LOOHs are produced in large quantities, disrupting the intracellular redox balance, attacking biomacromolecules, and ultimately initiating cell death. These LOOHs are key factors in ferroptosis, and inhibiting their formation can inhibit cell death. Intracellular lipid peroxidation and LOOHs metabolic disturbances are primarily linked to intracellular iron metabolism, the GSH-GPX4 pathway, the CoQ10-dependent pathway, and the NRF2-AREs pathway. Research has shown that ferroptosis is a major cause of organ damage in ischemia-reperfusion and brain damage in neurological diseases. Currently, there are no effective clinical treatments for these diseases. Inhibiting ferroptosis offers new insights into their prevention and treatment.

[0003] In the more than a decade since the discovery of ferroptosis, new ferroptosis regulators and regulatory factors have been reported, and research on the regulatory mechanisms of ferroptosis has made significant progress. However, the types of ferroptosis regulators discovered so far are limited, and our understanding of the regulatory mechanisms of ferroptosis is still incomplete. Therefore, the development of new and specific ferroptosis regulators will not only provide lead compounds for drug development of ferroptosis-related diseases, but also facilitate the discovery of new ferroptosis regulators and deepen our understanding of the mechanisms of ferroptosis and its relationship to disease.

[0004] Croton crassifolius Geisel, a plant of the Euphorbiaceae family, is also known as earth agarwood, chicken foot incense, bone-clearing herb, rolling dragon, and golden maple. It is found in Guangdong, Guangxi, and Fujian provinces of my country. As a traditional Chinese medicine, Croton crassifolius Geisel's roots are used primarily for treating epigastric pain, rheumatic pain, dysmenorrhea, sore throat, traumatic injuries, and snake and insect bites. The compounds in Croton crassifolius Geisel are primarily diterpenes, followed by sesquiterpenes, triterpenes, and steroids. Modern pharmacological studies have shown that monomeric compounds extracted from Croton crassifolius Geisel exhibit anti-tumor, anti-inflammatory, analgesic, antibacterial, and antiviral effects. Summary of the Invention

[0005] The present invention aims to discover novel and specific ferroptosis inhibitors and provides a halimane-type diterpenoid compound. The halimane-type diterpenoid compound provided by the present invention has a significant inhibitory effect on erastin-induced ferroptosis in HT22 cells without showing cytotoxicity. It can be used to prepare ferroptosis inhibitors and has good application prospects.

[0006] Another object of the present invention is to provide a method for preparing the halimane-type diterpenoid compound.

[0007] Another object of the present invention is to provide the use of the above-mentioned halimane-type diterpenoid compound in the preparation of ferroptosis inhibitors.

[0008] Another object of the present invention is to provide a ferroptosis inhibitor.

[0009] In order to achieve the above-mentioned object of the present invention, the present invention provides the following technical solutions:

[0010] A halimane-type diterpenoid compound having a structural formula as shown in any one of formulas (I) to (XII):

[0011]

[0012] The preparation method of the above-mentioned halimane-type diterpenoid compound comprises the following steps:

[0013] S1: drying, crushing, extracting and concentrating the roots of the Chinese herb, and obtaining a crude extract;

[0014] S2: suspending the crude extract with water, extracting, concentrating the extract under reduced pressure, eluting through a normal phase chromatography column and a reverse phase chromatography column, and then separating through chromatography and high performance liquid chromatography to obtain the halimane-type diterpenoid compound.

[0015] Preferably, ethanol solution is used for extraction in S1.

[0016] More preferably, the volume fraction of the ethanol solution in S1 is 85% to 95%.

[0017] Preferably, the number of extractions in S1 is 3 to 4 times, and the time for a single extraction is 2 to 5 days.

[0018] Preferably, ethyl acetate is used for extraction in S2.

[0019] Preferably, the normal phase chromatography column is a silica gel chromatography column; the reverse phase chromatography column is a C18 reverse phase chromatography column or a dextran gel column.

[0020] Preferably, the eluent for elution in S2 is a petroleum ether / ethyl acetate mixed solution; the elution gradient is: the volume ratio of petroleum ether / ethyl acetate is 0→40min: 1:0→100:1, 40→80min: 100:1→50:1, 80→120min: 50:1→20:1, 120→160min: 20:1→9:1, 160→200min: 9:1→8:2, 200→240min: 8:2→7:3, 240→280min: 7:3→6:4, 280→320min: 6:4→1:1.

[0021] Application of the above-mentioned halimane-type diterpenoid compounds in the preparation of ferroptosis inhibitors.

[0022] A drug for inhibiting ferroptosis, comprising one or more of the above-mentioned halimane-type diterpenoid compounds, pharmaceutically acceptable carriers or excipients; the mass fraction of the diterpenoid compounds, pharmaceutically acceptable carriers or excipients in the drug is 0.1% to 99%.

[0023] The drug for inhibiting ferroptosis of the present invention can be prepared into various existing pharmaceutical preparation types.

[0024] Preferably, the dosage form of the drug is injection, tablet or capsule.

[0025] Compared with the prior art, the present invention has the following advantages and effects:

[0026] The halimane-type diterpenoid compounds provided by the present invention have a significant inhibitory effect on erastin-induced ferroptosis in HT22 cells. In particular, the halimane-type diterpenoid compounds represented by formula (I) and formula (II) exhibit significant inhibitory activity against ferroptosis in HT22 cells induced by erastin or RSL3, EC 50 The results are 4.3 μM and 6.8 μM respectively. It is inferred that the halimane-type diterpenoid compounds extracted from chicken bone incense provided in this application can be used to prepare ferroptosis inhibitors and have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The graph shows the results of the halimane-type diterpenoid compounds represented by formula (I) to (XII) inhibiting ferroptosis of HT22 cells induced by erastin at a concentration of 10 μM;

[0028] Figure 2 The dose-effect curve of the halimane-type diterpenoid compound represented by formula (I) in inhibiting erastin or RSL3-induced ferroptosis in HT22 cells;

[0029] Figure 3 This is a graph showing the level of intracellular lipid peroxidation inhibited by the halimane-type diterpenoid compound represented by formula (I);

[0030] Wherein: A: Confocal imaging results of intracellular lipid peroxidation levels in HT22 cells; B: Flow cytometry analysis results; Scale bar 20 μm; C: Quantification of intracellular lipid peroxidation levels in Figure A; D: Quantification of intracellular lipid peroxidation levels in Figure B;

[0031] Figure 4 This is a graph showing the inhibitory effect of the halimane-type diterpenoid compound represented by formula (I) on the production of intracellular ROS.

[0032] Wherein: A: Confocal imaging results of ROS in HT22 cells; B: Flow cytometry analysis results; Scale bar 20 μm; C: Quantification of intracellular ROS levels in Figure A; D: Quantification of intracellular ROS levels in Figure B;

[0033] Figure 5 The results are the test results of the specific ferroptosis inhibitory rate of the halimane-type diterpenoid compound represented by formula (I);

[0034] Figure 6 The results of the antioxidant activity test of the halimane-type diterpenoid compound represented by formula (I) against DPPH are shown;

[0035] A: Fluorescence intensity change of oxidative BODIPY-C11; B: Absorbance change of DPPH at 517 nm;

[0036] Figure 7 This is a graph showing the chelation of iron ions by the halimane-type diterpenoid compound represented by formula (I);

[0037] A: DFO and halimane-type diterpenoid compounds represented by formula (I) in Fe 2+ UV-vis absorption spectrum in the presence of DFO; B: Iron chelating ability of DFO and halimane-type diterpenoid compounds represented by formula (I) in Ferrozine-iron chelation experiment. DETAILED DESCRIPTION

[0038] The present invention is further explained below with reference to the examples and accompanying drawings, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0039] Unless otherwise specified, all reagents and materials used in the present invention are commercially available.

[0040] Example 1

[0041] The halimane-type diterpenoid compounds represented by formulas (I) to (XII) are prepared from the chicken bone incense.

[0042] Weigh the root of 5.0 kg of chicken bone incense, clean it, dry it, crush it into small pieces, and extract it with 40 L of 95% ethanol aqueous solution at room temperature for 3-4 times, each time for 3 days; the extract is concentrated under reduced pressure at 0.09 MPa (12 h / time) to obtain 700 g of crude extract; the extract is suspended in water, extracted with ethyl acetate 5 times, and concentrated under reduced pressure (0.09 MPa) to obtain ethyl acetate part (370 g), which is chromatographed on a 200-300 mesh silica gel column and eluted with petroleum ether / ethyl acetate according to a gradient. The gradient elution used a petroleum ether / ethyl acetate ratio of 1:0 to 100:1 for 0 to 40 min, 100:1 to 50:1 for 40 to 80 min, 50:1 to 20:1 for 80 to 120 min, 20:1 to 9:1 for 120 to 160 min, 9:1 to 8:2 for 160 to 200 min, 8:2 to 7:3 for 200 to 240 min, 7:3 to 6:4 for 240 to 280 min, and 6:4 to 1:1 for 280 to 320 min. The product was analyzed on silica gel GF254 thin layer plates using 10% sulfuric acid-ethanol for color development. Fractions of similar polarity were pooled and concentrated into five fractions (A to E).

[0043] Fraction B was recrystallized from ethyl acetate to obtain a halimane-type diterpenoid compound (17 mg) represented by formula (I), which was designated as compound JGX-11.

[0044] Fraction C (43 g) was purified by reverse phase RP-C18 column (methanol / water, 30%-100%, v / v) to obtain five components (C1-C5). Fraction C2 (26 g) was purified by gradient elution with petroleum ether / acetone (200:1-1:1, v / v) on a silica gel column to obtain five subcomponents (C 2.1 -C 2.5 ), C 2.4 Five subfractions (C 2.4.1 -C 2.4.5 ). C 2.4.3Elution with a Sephadex LH-20 column gave a halimane-type diterpenoid compound (15 mg) of compound formula (II), which was recorded as compound JGX-58; a halimane-type diterpenoid compound (500 mg) of formula (V), which was recorded as compound JGX-39; and a halimane-type diterpenoid compound (16 g) of formula (IX), which was recorded as compound JGX-24; C2.4.5 was separated by semi-preparative HPLC (methanol / water, 70%, 1.0 mL / min) to give a halimane-type diterpenoid compound (555 mg) of formula (XII), which was recorded as compound JGX-16; and C3 fraction (11 g) was separated by gradient elution with petroleum ether / ethyl acetate (100:1-1:1, v / v) on a silica gel column to give five subfractions (C 3.1 -C 3.5 ), C 3.2 After elution with a Sephadex LH-20 column, the product was separated by semi-preparative HPLC (acetonitrile / water, 75%, 1.0 mL / min) to obtain a halimane-type diterpenoid compound (570 mg) of formula (III), designated as compound JGX-31; a halimane-type diterpenoid compound (200 mg) of formula (VIII), designated as compound JGX-35; a halimane-type diterpenoid compound (570 mg) of formula (X), designated as compound JGX-37; a halimane-type diterpenoid compound (35 mg) of formula (XI), designated as compound JGX-11; and C 3.4 The fraction was eluted with dichloromethane / ethyl acetate (100:1-2:1, v / v) through a silica gel column to obtain three subfractions (C 3.4.1 -C 3.4.3 ), C 3.4.1 Semi-preparative HPLC (methanol / water, 80%, 1.0 mL / min) was used to separate and obtain a halimane-type diterpenoid compound (50 mg) of formula (IV), designated as compound JGX-36; a halimane-type diterpenoid compound (750 mg) of formula (VI), designated as compound JGX-4; and a halimane-type diterpenoid compound (750 mg) of formula (VII), designated as compound JGX-54. The compound structures are shown in FIG. Figure 1 shown.

[0045] The 12 monomer compounds JGX-11, JGX-58, JGX-31, JGX-36, JGX-39, JGX-4, JGX-54, JGX-35, JGX-24, JGX-37, JGX-40, and JGX-16, their physical and chemical properties and structural formulas are as follows:

[0046] JGX-11, yellow oil, C 21 H 24 O4, 1 H NMR (400 MHz, CDCl3) δ H 8.55(s,H-16),7.42(t,J=

[0047] 1.7Hz,H-15),7.00(d,J=1.9Hz,H-14),3.56(s,H-18-OMe),2.81(d,J=15.7Hz,H-3a),2.72(d,J=17.0Hz,H-11a),2.66(d,J=17.0Hz,H-11b), 2.45(d,J=15.7Hz,H-3b),2.30(m,H-6),1.67(m,H-7a),1.57(m,H-7b) ,1.57(m,H-8),1.37(s,H-19),0.98(s,H-20),0.96(d,J=6.1Hz,H-17); 13 C NMR (400 MHz, CDCl3) δ C 195.2(s,C-2),174.8(s,C-18),150.5(s,C-10),146.8(d,C-16),142.8(d,C-15),13 9.8(s,C-12),128.1(s,C-1),125.2(s,C-5),122.0(s,C-13),111.1(d,C-14),52.4(q , C-18-OMe), 52.3(t, C-3), 50.5(t, C-11), 48.6(s, C-4), 42.6(s, C-9), 37.2(d, C-8), 27.2(t, C-7), 23.9(t, C-6), 22.4(q, C-19), 20.5(q, C-20), 16.6(q, C-17); shown in structural formula (I).

[0048]

[0049] JGX-58, yellow crystal, C 19 H 20 O3, 1 HNMR (400 MHz, CDCl3) δ H 8.26(s,H-16),7.37(t,J=1.7

[0050] Hz, H-15), 6.60 (m, H-14), 6.50 (brs, H-11), 5.95 (brs, H-3), 2.32 (dt, J = 13.5, 3.0 Hz, H-6a), 2.05 (s, H-18), 1.95 (m, H-7a), 1.51 (m, H-7b), 1.23 (s, H-20), 1.19 (m, H-8), 1.18 (m, H-6b), 1.15 (d, J = 6.2 Hz, H-17); 13 C NMR (100 MHz, CDCl3) δ C 183.5 (s, C-2), 172.5 (s, C-10), 160.7 (s, C-4), 143.0 (d, C-16), 142.4 (d, C-15), 142.1 (d, C-11), 132.9 (s, C-1), 130.9 (s, C-12), 127.7 (d, C-3), 119.7 (s, C-13), 110.2 (d, C-14), 71.2 (s, C-5), 57.3 (s, C-9), 43.6 (d, C-8), 40.1 (t, C-6), 26.9 (t, C-7), 18.0 (q, C-17), 16.8 (q, C-18), 13.2 (q, C-20); as shown in Structural Formula (II).

[0051]

[0052] JGX-31, white crystal, C 19 H 20 O5, 1 H NMR (400 MHz, CDCl3) δ H 7.42 (s, H-16), 7.42 (s, H-15), 6.35 (s, H-14), 5.36 (t, J = 8.0 Hz, H-12), 5.00 (m, H-6), 3.27 (m, H-10), 2.59 (dd, J = 13.9, 8.3 Hz, H-11), 2.32 (m, H-3), 2.32 (m, H-7), 2.16 (m, H-3), 2.16 (m, H-8), 2.02 (m, H-1), 1.98 (m, H-2), 1.62 (m, H-2), 1.47 (m, H-7), 1.40 (m, H-1), 1.34 (d, J = 7.3, H-17); 13 C NMR (100 MHz, CDCl3) δ C177.7(s,C-20),172.7(s,C-18),162.3(s,C-5),144.5(d,C-15),139.6(d, C-16),127.8(s,C-4),125.3(s,C-13),108.0(d,C-14),76.1(d,C-6),72.0( d,C-12),52.1(s,C-9),39.1(t,C-11),38.8(d,C-8),35.9(d,C-10),35.8(t ,C-7),23.4(t,C-1),21.5(t,C-2),20.1(t,C-3),14.4(q,C-17); shown in structural formula (III).

[0053]

[0054] JGX-36, yellow oil, C 24 H 28 O9, 1 H NMR (500 MHz, CDCl3) δ H 7.44(s,H-16),7.43(s,H-15),6.38(s,H-14),5.46(t,J=8.0,H-6),5.4 6(t,J=8.0Hz,H-12),3.72(s,18,19-OMe),2.22(m,H-2),2.17(m,H-1), 2.16(m,H-3),2.05(m,H-7),2.02(m,H-2),1.93(s,6-OAc),1.88(m,H-8 ),1.73(m,H-11),1.61(m,H-11),1.60(m,H-7),1.06(d,J=6.8Hz,H-17); 13 CNMR (125MHz, CDCl3)δ C176.6 (s, C-20), 171.7 (s, C-19), 171.5 (s, 18,19-OMe), 171.0 (s, C-18), 170.4 (s, 6-OAc), 144.2 (d, C-15), 139.5 (d, C-16), 136.4 (s, C-10), 130.4 (s, C-5), 125.3 (s, C-13), 108.1 (d, C-14), 72.4 (d, C-12), 70.1 (d, C-6), 57.1 (s, C-4), 54.0 (s, C-9), 52.8 (q, 18,19-OMe), 40.8 (t, C-11), 35.8 (d, C-8), 32.7 (t, C-7), 32.2 (t, C-3), 26.6 (t, C-1), 21.1 (q, 6-OAc), 19.0 (t, C-2), 16.7 (q, C-17); as shown in Structural Formula (IV).

[0055]

[0056] JGX-39, yellow oil, C 21 H 26 O6, 1 1H NMR (400 MHz, CDCl3) δ H 7.44 (s, H-16), 7.43 (s, H-15), 6.37 (s, H-14), 5.45 (t, J = 8.2 Hz, H-12), 4.00 (d, J = 12.0 Hz, H-19), 3.69 (s, 18-OMe), 3.55 (d, J = 12.0 Hz, H-19), 2.82 (dd, J = 13.6, 8.4 Hz, H-11), 2.20 (m, H-6), 2.18 (m, H-11), 2.17 (m, H-3), 2.06 (m, H-1), 1.84 (m, H-1), 1.84 (m, H-3), 1.70 (m, H-2), 1.70 (m, H-8), 1.52 (m, H-6), 1.00 (d, J = 7.0 Hz, H-17); 13 13C NMR (100 MHz, CDCl3) δ C178.1(s,C-20),177.1(s,C-18),144.2(d,C-15),139.4(d,C-16),131.9(s,C-10), 131.2(s,C-5),125.7(s,C-13),108.3(d,C-14),72.4(d,C-12),64.7(t,C-19),53.6 (s, C-9), 53.2 (q, 18-OMe), 52.2 (s, C-4), 41.0 (t, C-11), 37.8 (d, C-8), 28.0 (t, C-3), 26.6 (t, C-6), 26.4 (t, C-7), 25.4 (t, C-1), 18.4 (t, C-2), 16.5 (q, C-17); shown in structural formula (V).

[0057]

[0058] JGX-4, yellow oil, C 21 H 24 O5, 1 H NMR (500 MHz, CDCl3) δ H 7.47(s,H-16),7.33(t,J=1.7

[0059] Hz,H-15),6.40(m,H-14),5.90(s,H-1),4.80(s,H-11),3.54(s,18-OAc),2.70(d,J=16.4Hz,H-3),2.38(d,J=16.4Hz,H-3),2 .23(m,H-6),2.18(m,H-7),1.95(m,H-8),1.92(m,H-6),1.42(s,H-19),1.26(m,H-7),1.17(s,H-20),0.88(d,J=7.0Hz,H-17); 13 C NMR (125 MHz, CDCl3) δ C196.5(s,C-2),174.0(s,C-18),157.9(s,C-10),146.4(s,C-12),143.3(d,C-15),1 9.7(d,C-16),122.0(d,C-1),121.4(s,C-13),107.3(d,C-14),103.3(d,C-11),79.6( s,C-5), 52.3(q,18-OAc), 51.6(s,C-4), 45.7(t,C-3), 42.7(d,C-8), 41.5(s,C-9),3 1.9(t,C-6),26.7(t,C-7),22.4(q,C-20),20.3(q,C-19),14.5(q,C-17)

[0060]

[0061] JGX-54,Empty Railway,C 20 H 24 O4, 1 H NMR(500MHz,CDCl3)δ H 7.92(s,H-16),7.39(s,H-15),6.73(s,H-14),4.74(ddd,JK5.7、2.8、2.8Hz,H-2),2.84(d ,JC15.5Hz,H-11),2.72(d,JC15.5Hz,H-11),2.38(dddd,JC17.9、2.8、2.8、2.5Hz,H-1),2. 31(dd,J)17.9,2.7Hz,H-1),2.15(m,H-6),2.13(m,H-3),2.04(m,H-8),1.93(d,J610.9Hz, H-3), 1.75(m,H-7), 1.42(m,H-7), 1.31(s,H-19),1.05(s,H-20),0.84(d,J6.9Hz,H-17) 13 C NMR(125MHz,CDCl3)δ C193.7 (s, C-12), 178.4 (s, C-18), 147.1 (d, C-16), 144.2 (d, C-15), 132.6 (s, C-10), 132.1 (s, C-5), 129.3 (s, C-13), 108.7 (d, C-14), 74.1 (d, C-2), 47.8 (t, C-11), 43.7 (s, C-4), 41.2 (t, C-3), 40.4 (s, C-9), 33.3 (d, C-8), 31.7 (t, C-1), 25.6 (t, C-7), 22.3 (t, C-6), 22.0 (q, C-20), 16.6 (q, C-19), 15.3 (q, C-17); as shown in Structural Formula (VII).

[0062]

[0063] JGX-35, yellow crystal, C 25 H 30 O 10 , 1 H NMR (400 MHz, CDCl3) δ H 8.04 (s, H-16), 7.43 (s, H-15), 6.73 (s, H-14), 5.59 (m, H-6), 3.74 (s, 18-OMe), 3.73 (s, 19-OMe), 3.73 (20-OMe), 3.29 (d, J = 5.0 Hz, H-11), 2.37 (m, H-8), 2.18 (m, H-3), 2.13 (m, H-3), 2.01 (m, H-1), 1.94 (s, 6-OAc), 1.90 (m, H-7), 1.87 (m, H-7), 1.66 (m, H-1), 1.57 (m, H-2), 1.51 (m, H-2), 0.88 (d, J = 7.0 Hz, H-17); 13 C NMR (100 MHz, CDCl3) δ C190.9 (s, C-12), 173.8 (s, C-20), 172.2 (s, C-18), 170.6 (s, C-19), 170.3 (6-OAc), 146.7 (d, C-16), 144.1 (d, C-15), 135.0 (s, C-10), 129.6 (s, C-5), 128.6 (s, C-13), 108.5 (d, C-14), 71.2 (d, C-6), 56.6 (s, C-4), 54.7 (s, C-9), 52.4 (q, 20-OMe), 52.0 (q, 18-OMe), 51.9 (q, 19-OMe), 41.1 (t, C-11), 33.6 (t, C-7), 32.3 (t, C-3), 32.2 (d, C-8), 27.2 (t, C-1), 20.9 (q, 6-OAc), 18.9 (t, C-2), 16.9 (q, C-17); as shown in Structural Formula (VIII).

[0064]

[0065] JGX-24, colorless crystal, C 21 H 26 O6, 1 1H NMR (400 MHz, CDCl3) δ H 7.98 (s, H-16), 7.38 (t, J = 1.7

[0066] Hz, H-15), 6.62 (m, H-14), 5.79 (s, H-1), 3.68 (s, 18-OMe), 3.23 (d, J = 19.0 Hz, H-11), 3.11 (d, J = 19.0 Hz, H-11), 2.68 (d, J = 17.1 Hz, H-3), 2.50 (d, J = 17.1 Hz, H-3), 2.34 (m, H-8), 2.17 (m, H-6), 1.98 (m, H-6), 1.73 (m, H-7), 1.47 (m, H-7), 1.35 (s, H-19), 1.16 (s, H-20), 0.86 (d, J = 6.9 Hz, H-17); 13 13C NMR (100 MHz, CDCl3) δ C198.3 (s, C-12), 191.1 (s, C-2), 174.8 (s, C-18), 168.0 (s, C-10), 146.5 (d, C-16), 144.2 (d, C-15), 128.0 (s, C-13), 125.6 (d, C-1), 108.4 (d, C-14), 72.7 (s, C-5), 53.1 (t, C-3), 52.5 (q, 18-OMe), 47.9 (t, C-11), 43.3 (s, C-4), 41.3 (s, C-9), 35.3 (d, C-8), 31.8 (t, C-6), 26.1 (q, C-19), 25.2 (t, C-7), 19.6 (q, C-20), 16.7 (q, C-17); as shown in Structural Formula (IX).

[0067]

[0068] JGX-37, white powder, C 21 H 24 O5, 1 1H NMR (500 MHz, CDCl3) δ H 7.94 (s, H-16), 7.39 (t, J = 1.7

[0069] Hz, H-15), 6.68 (m, H-14), 6.04 (s, H-6), 5.87 (s, H-1), 3.59 (s, 18-OMe), 2.96 (d, J = 1 3.3 Hz, H-11), 2.94 (d, J = 13.2 Hz, H-3), 2.68 (m, H-11), 2.66 (m, H-7), 2.26 (m, H-8), 2.25 (m, H-3), 2.05 (m, H-7), 1.45 (s, H-19), 1.20 (s, H-20), 0.79 (d, J = 7.0 Hz, H-17); 13 13C NMR (125 MHz, CDCl3) δ C197.2(s,C-2),192.9(s,C-12),175.1(s,C-18),159.9(s,C-10),147.2(d,C-16),14 4.4(d,C-15),133.5(s,C-5),129.1(s,C-13),128.9(d,C-6),123.5(d,C-1),108.7(d ,C-14),52.7(q,18-OMe),49.2(s,C-4),47.7(t,C-3),47.0(t,C-11),41.6(s,C-9),34.3(d,C-8),31.8(t,C-7),23.1(q,C-19),21.8(q,C-20),15.9(q,C-17); shown in the structural formula (X).

[0070]

[0071] JGX-40, yellow powder, C 20 H 28 O3, 1 H NMR (400 MHz, CDCl3) δ H 7.34(s,H-15),7.22(s,H-12),7.20(s,H-16),6.27(s,H-14),2.10(m,H-1),1.90(m,H-1),1.90(m,H-3),1.90(m,H-6),1.77(m,H-2 ),1.75(m,H-8),1.67(m,H-3),1.65(m,H-11),1.55(m,H-7),1.38(m,H-6),1.30(s,H-19),0.88(d,J=6.9Hz,H-17),0.86(s,H-20); 13 C NMR (100 MHz, CDCl3) δ C 184.6(s,C-18),142.7(d,C-15),138.6(d,C-16),136.0(s,C-10),131.1(s,C-5 ),126.0(s,C-13),111.2(d,C-14),47.6(s,C-4),41.0(s,C-9),36.6(t,C-11), 35.5(t,C-3),33.4(d,C-8),26.9(t,C-7),26.1(t,C-6),23.0(q,C-19),21.0(q,C-20),20.1(t,C-1),19.6(t,C-2),19,6(t,C-12),16.2(q,C-17); shown in structural formula (XI).

[0072]

[0073] JGX-16, white crystal, C 20 H 28 O4, 1 H NMR (400 MHz, CDCl3) δ H 5.82(s,H-14),4.76(s,H-16),2.27(m,H-12),2.06(m,H-1),2.01(m,H-3),2.01(m,H-6),1.99(m,H-12),1.83(d,J=16.2Hz,H-6),1.63( m,H-1),1.60(m,H-8),1,60(m,H-11),1.58(m,H-2),1.43(m,H-7),1.42(m,H-3),1.26(s,H-19),0.91(s,H-20),0.86(d,J=6.8Hz,H-17); 13 C NMR (100 MHz, CDCl3) δ C 183.8(s,C-18),174.3(s,C-15),171.2(s,C-13),135.2(s,C-10),132.6(s,C-5 ),115.1(t,C-14),73.2(d,C-16),47.6(s,C-4),41.2(s,C-9),36.6(t,C-3),33 .8(d,C-8),33.4(t,C-11),27.8(t,C-6),26.8(t,C-7),25.3(t,C-1),24.4(q,C-19),23.7(t,C-12),20.8(q,C-20),20.1(t,C-2),16.2(q,C-17); shown in structural formula (XII).

[0074]

[0075] Experimental Example 1

[0076] Determination of the ferroptosis inhibitory activity of the compounds.

[0077] 1. Cell Culture

[0078] The cells used in this experiment were HT22 cells (mouse hippocampal neuronal cells).

[0079] HT22 cell culture and passaging: HT22 cells were maintained for laboratory use. After thawing, cells were cultured in DMEM high-glucose medium supplemented with 10 wt% FBS and 1 wt% double-antibody. When cells reached a density of approximately 80%, they were passaged. The original medium was aspirated, and the cells were gently washed with medium. The cells were passaged at a 1:5 ratio. All cells were incubated at 37°C in a 5% CO2 incubator.

[0080] 2. Cell viability test (MTT method)

[0081] (1) HT22 cells in good growth condition and in the logarithmic growth phase were seeded into 96-well plates at a density of 3000 cells / well, with 85 μL per well.

[0082] (2) After the cells were cultured in an incubator until adherent, ferroptosis inducers (1 μM erastin or 1 μM RSL3) and 10 μM of the test compounds JGX-11, JGX-58, JGX-31, JGX-36, JGX-39, JGX-4, JGX-54, JGX-35, JGX-24, JGX-37, JGX-40, and JGX-16 prepared in Example 1 were added and incubated for 24 h. 50 The test compound JGX-11 prepared in Example 1 was added at concentrations of 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM.

[0083] (3) Add 20 μL MTT solution (prepared in PBS, concentration of 5 mg / mL) to each well and incubate in a cell culture incubator at 37°C for 3 h.

[0084] (4) Discard the liquid and add 100 μL of analytical grade DMSO to each well. Shake rapidly on a shaker for 10 minutes. Measure the absorbance (OD value) at 492 nm using a multifunctional microplate reader. Calculate cell viability based on the absorbance of each well as follows:

[0085]

[0086] 3. Statistical Methods

[0087] GraphPad Prism 8.0 software was used to process and analyze the experimental data, and the differences between the groups were compared by one-way ANOVA. When P < 0.05, the differences between the groups were considered statistically significant.

[0088] Experimental Example 2 Detection of lipid peroxidation levels

[0089] 1. Laser confocal microscopy imaging detection:

[0090] (1) Cell seeding: HT22 cells were seeded at a density of 3000 cells / well in a 96-well glass-bottom cell culture plate.

[0091] (2) Compound treatment: After overnight culture, 1 μM RSL3 and 10 μM JGX-11 or 1 μM Fer-1 were added and incubated in a cell culture incubator until the cell morphology was clearly different (approximately 4 h).

[0092] (3) Probe incubation: Discard the culture medium and rinse the cells with PBS. Prepare the probe (5 μM BODIPY-C11) in complete culture medium, add it to the wells, and incubate in a cell culture incubator for 30 min. Discard the probe and add 1 μg / mL Hoechst 33342 to stain the cell nuclei for 10 min.

[0093] (4) Imaging analysis: Discard the Hoechst 33342 dye, rinse with PBS, add serum-free culture medium, and scan and record using an FV3000 laser confocal microscope.

[0094] 2. Flow cytometry detection:

[0095] (1) Cell seeding: 3×10 4 HT22 cells were seeded in 6-well plates at a density of 1 cell / well.

[0096] (2) Compound treatment: After the cells were cultured overnight in an incubator, 1 μM RSL3 and JGX-11 (2.5 μM, 5 μM, or 10 μM) or 1 μM Fer-1 were added and incubated in a cell culture incubator for 4 h.

[0097] (3) Probe incubation: Discard the culture medium, rinse the cells with PBS, and then prepare the probe (5 μM BODIPY-C11) with complete culture medium, add it to the wells, and incubate in a cell culture incubator at 37°C for 30 min.

[0098] (4) Detection: Discard the probe, rinse once with PBS, add trypsin to digest and collect cells, resuspend in 1 mL of serum-free culture medium after centrifugation, and detect using flow cytometry.

[0099] (5) Data processing: FlowJo 10 software was used to analyze the data and draw the result graphs.

[0100] Experimental Example 3 Detection of intracellular ROS levels

[0101] The steps were the same as those in Experimental Example 3, except that the probe was 10 μM carboxy-H2DCFDA prepared in serum-free medium.

[0102] Experimental Example 4 Other cell death induction experiments

[0103] (1) Cell seeding: HT22 cells were seeded in a 96-well plate at a density of 3000 cells / well.

[0104] (2) Compound treatment: After the cells were cultured in an incubator for 24 h to adhere to the wall, 10 μM JGX-11 and apoptosis inducer STS (0.3 μM) or necrosis inducer H2O2 (1 mM) were added.

[0105] (3) Detection: After 24 h of compound treatment, the cell viability was detected using the MTT assay.

[0106] Experimental Example 5: Antioxidant activity detection using DPPH method

[0107] (1) Solution preparation: Prepare a 200 μM DPPH solution in methanol, and dilute a 10 mM stock solution of the test compound dissolved in DMSO with methanol to twice the test concentration.

[0108] (2) Reaction: 100 μL of the above-prepared DPPH solution and compound solution were added to a 96-well plate, mixed, and incubated at room temperature in the dark for 30 min.

[0109] (3) Detection: Detect the absorbance at 517 nm using a multifunctional microplate reader.

[0110] Experimental Example 6: Ultraviolet Detection of Iron Chelation

[0111] (1) Solution preparation: Prepare buffer (20 mM HEPES pH 7.4, 150 mM NaCl) and 10 mM FeSO4 solution using ultrapure water. Dilute the 10 mM stock solution of the compound in DMSO to 50 μM using the buffer. All solutions should be prepared immediately before use.

[0112] (2) Data acquisition: 500 μL of compound solution was transferred to a micro-quartz cuvette and the UV absorbance of the solution was measured at 200–600 nm using a UV-visible spectrophotometer. Then, 2.5 μL of FeSO₄ solution was added to a final concentration of 50 μM. The solution was gently pipetted and mixed, and data was collected again.

[0113] Experimental Example 8 Ferrozine-iron chelation experiment

[0114] (1) Preparation of HEPES buffer: Prepare the buffer (20 mM HEPES pH 7.4, 150 mM NaCl) using ultrapure water.

[0115] (2) Preparation of FeSO4 solution: Weigh an appropriate amount of solid FeSO4, dissolve it in HEPES buffer and prepare it to 80μM. Prepare it immediately and use it immediately. The final working concentration is 20μM.

[0116] (3) Preparation of the test compound: 10 mM stock solution of the compound dissolved in DMSO was diluted to 400 μM with HEPES buffer, and the final working concentration was 100 μM.

[0117] (4) Preparation of Ferrozine solution: Weigh an appropriate amount of Ferrozine solid, dissolve it in HEPES buffer and prepare it to 1 mM, with a final working concentration of 500 μM. Prepare it for use immediately.

[0118] (5) Detection: 50 μL of FeSO4 and 50 μL of diluted compound were mixed in a 96-well plate and incubated at room temperature for 10 minutes. 100 μL of Ferrozine solution was then added to bring the final volume to 200 μL. After incubation at room temperature for 5 minutes, the absorbance at 562 nm was measured using a multifunctional microplate reader.

[0119] The MTT assay was used to test the inhibitory activity of 12 compounds (JGX-11, JGX-58, JGX-31, JGX-36, JGX-39, JGX-4, JGX-54, JGX-35, JGX-24, JGX-37, JGX-40, and JGX-16) against cell ferroptosis at 10 μM. Figure 1 As shown. Compounds JGX-11 and JGX-58 showed significant inhibitory activity against erastin-induced ferroptosis in HT22 cells at 10 μM. In particular, compound JGX-1 showed significant inhibitory activity against ferroptosis inducers erastin or RSL3-induced ferroptosis in HT22 cells, EC 50 4.3μM and 6.8μM respectively (as Figure 2 Further experiments have shown that JGX-11 can significantly inhibit the levels of lipid peroxidation and ROS in cells (such as Figure 3 、 Figure 4 ). JGX-11 has no inhibitory activity on cell apoptosis and necrosis (such as Figure 5 ), indicating that JGX-11 is a specific ferroptosis inhibitor. DPPH antioxidant activity assay ( Figure 6 ) and iron ion chelation experiments ( Figure 7 ) showed that JGX-11 had neither free radical scavenging activity nor the ability to chelate iron.

[0120] Example 2

[0121] Take any one of the compounds JGX-11, JGX-58, JGX-31, JGX-36, JGX-39, JGX-4, JGX-54, JGX-35, JGX-24, JGX-37, JGX-40, and JGX-16 prepared in Example 1, add injection water and Tween 80 as conventionally prepared, finely filter, and sterilize by filling to prepare an injection solution.

[0122] Example 3

[0123] Any one of the compounds JGX-11, JGX-58, JGX-31, JGX-36, JGX-39, JGX-4, JGX-54, JGX-35, JGX-24, JGX-37, JGX-40, or JGX-16 prepared in Example 1 was dissolved in sterile water for injection, stirred until dissolved, and filtered through a sterile filtration funnel. The mixture was sterile finely filtered and dispensed into ampoules. After freeze-drying at low temperature, the mixture was aseptically sealed to obtain a powder for injection.

[0124] Example 4

[0125] Any one of the compounds JGX-11, JGX-58, JGX-31, JGX-36, JGX-39, JGX-4, JGX-54, JGX-35, JGX-24, JGX-37, JGX-40, or JGX-16 prepared in Example 1 was added with an excipient (e.g., starch slurry) at a weight ratio of 5:1, and granulated and tableted.

[0126] Example 5

[0127] Any one of the compounds JGX-11, JGX-58, JGX-31, JGX-36, JGX-39, JGX-4, JGX-54, JGX-35, JGX-24, JGX-37, JGX-40, or JGX-16 prepared in Example 1 was added with an excipient (e.g., polyethylene glycol 400) at a weight ratio of 5:1 to prepare capsules.

[0128] Example 6

[0129] Any one of the compounds JGX-11, JGX-58, JGX-31, JGX-36, JGX-39, JGX-4, JGX-54, JGX-35, JGX-24, JGX-37, JGX-40, or JGX-16 prepared in Example 1 was added with an excipient (e.g., Tween 80) at a weight ratio of 3:1 to prepare capsules.

Claims

1. A halimane-type diterpenoid compound, characterized in that: The halimane-type diterpenoid compound is extracted and separated from the root of Cyperus rotundus and has a structural formula as shown in any one of formulas (I) to (XII):

2. The method for preparing the halimane-type diterpenoid compound according to claim 1, wherein The steps include: S1: drying, crushing, extracting and concentrating the roots of the Chinese herb, and obtaining a crude extract; S2: suspending the crude extract with water, extracting, concentrating the extract under reduced pressure, eluting through a normal phase chromatography column and a reverse phase chromatography column, and then separating through chromatography and high performance liquid chromatography to obtain the halimane-type diterpenoid compound.

3. The method for preparing a halimane-type diterpenoid compound according to claim 2, wherein: In S1, 85% to 95% ethanol solution is used for leaching; the number of leaching is 3 to 4 times, and the time of a single leaching is 2 to 5 days.

4. The method for preparing a halimane-type diterpenoid compound according to claim 3, wherein: The material-liquid ratio of the chicken bone incense and the ethanol solution in S1 is 1:

8.

5. The method for preparing a halimane-type diterpenoid compound according to claim 2, wherein: The normal phase chromatographic column is a silica gel column; the reverse phase chromatographic column is a C18 reverse phase column or a dextran gel column.

6. The method for preparing a halimane-type diterpenoid compound according to claim 2, wherein: S2 was extracted with ethyl acetate.

7. The method for preparing a halimane-type diterpenoid compound according to claim 2, wherein: The eluent for elution in S2 is a mixed solution of petroleum ether / ethyl acetate; the elution gradient is: the volume ratio of petroleum ether / ethyl acetate is 0→40min: 1:0→100:1; 40→80min: 100:1→50:1; 80→120min: 50:1→20:1; 120→160min: 20:1→9:1; 160→200min: 9:1→8:2; 200→240min: 8:2→7:3; 240→280min: 7:3→6:4; 280→320min: 6:4→1:

1.

8. Use of the halimane-type diterpenoid compound according to claim 1 in the preparation of a medicament for treating ferroptosis.

9. A drug for treating ferroptosis, characterized in that: The medicine contains the halimane-type diterpenoid compound according to claim 1 and one or more pharmaceutically acceptable carriers or excipients.

10. The drug for treating ferroptosis according to claim 9, characterized in that The mass fraction of the halimane-type diterpenoid compound and the pharmaceutically acceptable carrier or excipient in the medicine is 0.1% to 99%; the dosage form of the medicine is injection, tablet or capsule.

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