Cassane dimer derivatives, process for their preparation and use thereof

Abstract

This invention discloses the cassane dimer derivatives represented by Formula I or II, their preparation methods, and applications, belonging to the field of pharmaceutical technology. It utilizes a GNPS molecular network strategy for targeted separation of petroleum ether extracts of *Cypripedium spp.* to rapidly identify novel cassane dimers. This method avoids the repetitive separation required by traditional methods, accurately locates the dimer enrichment sites, reduces time and reagent costs, and minimizes resource consumption. The cassane dimer can inhibit the expression of renal fibrosis markers in a TGF-β1-induced NRK-52E cell renal fibrosis model and can be used to prepare drugs for the treatment of chronic kidney disease.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to cassane dimer derivatives, their preparation methods, and their application in the preparation of anti-renal fibrosis drugs. Background Technology

[0002] Chronic kidney disease (CKD) is a global public health problem affecting 10% of the world's population, characterized by poor prognosis, limited interventions, and high mortality. Renal fibrosis is the leading cause of rapid deterioration of kidney function and ultimately the progression of CKD to end-stage renal disease. Extensive evidence suggests that inhibiting renal fibrosis is a crucial pathway to slowing CKD progression. Due to the limited therapeutic effects of RAS inhibitors and the side effects of steroids, there is a lack of safe and effective treatments for renal fibrosis, thus creating an urgent need for novel treatments with fewer side effects. Natural products are an important source for innovative drug development; therefore, finding highly effective and low-toxicity drugs from natural plants is crucial.

[0003] *Ticanto sinensis* (Hemsl.) R. Clark & ​​Gagnon is a vine belonging to the genus *Ticanto* in the family Leguminosae, mainly distributed in Yunnan, Sichuan, Guangxi, and Guangdong provinces. Previous research by our group revealed that cassane-type diterpenes obtained from the ethanol extract of *Ticanto sinensis* seed kernels can inhibit the expression of renal fibrosis markers, demonstrating good inhibitory activity against renal fibrosis. Cassane dimers are a relatively rare structural type among cassane compounds, with fewer than 10 isolated to date. These compounds are low in polarity, have large molecular weights, and are present in low concentrations in plants, making them difficult to isolate effectively using conventional methods, thus posing a significant challenge to our isolation efforts. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, this invention provides a method for preparing cassane dimer derivatives and their applications. This method overcomes the drawbacks of traditional extraction and separation methods, such as complexity, time consumption, and high solvent loss, and yields a very rare class of cassane dimer compounds. Furthermore, experiments have confirmed that these compounds possess excellent anti-renal fibrosis inhibitory activity.

[0005] This invention provides cassane dimer derivatives of Formula I-III or pharmaceutically acceptable salts thereof:

[0006]

[0007] In Formula I, rings A, B, F, and G are all cyclohexane, ring D is a benzene ring, ring C is fused with ring D to form a naphthone structure, ring E is an α,β-unsaturated cyclohexenone, and R is an aldehyde group or a ketal structure with two methoxy groups attached to a carbon atom.

[0008] In Formula ⅠI, rings A, B, F, and G are all cyclohexane, rings C and E are both α,β-unsaturated cyclohexenone, ring D is cyclohexadiene, and the two alkenes in ring D are conjugated, with the carbon atom of the alkene connected to an aldehyde group.

[0009] Furthermore, in Formula I, rings A, B, F, and G are all six-membered cyclohexane rings, ring D is an aromatic ring, ring C and ring D are fused to form a naphthone structure, ring E is an α,β-unsaturated six-membered cyclohexenone ring, acetoxy groups are attached to the C6 position of ring B and the C6′ position of ring F, and R is -CHO or -CH(OCH3)2.

[0010] Furthermore, in Formula II, rings A, B, F, and G are all six-membered cyclohexane rings, ring D is a cyclohexadiene containing a conjugated olefin, a double bond is formed between the C13 and C14 positions and a conjugated system is formed between the double bond between the C15 and C16′ positions, rings C and E are both α,β-unsaturated cyclohexenone six-membered rings, an acetoxy group is attached to the C6 position of ring B and the C6′ position of ring F, and an aldehyde group is attached to the C15 position of ring D.

[0011] The present invention provides three preferred biscaesalmin derivatives, the structures and names of which are as follows: biscaesalmin L1 (1), biscaesalmin L2 (2), and biscaesalmin L3 (3).

[0012]

[0013] The present invention also provides a method for preparing the aforementioned cassane dimer derivative, comprising the following steps:

[0014] (1) After drying, the kernels of the chicken beak seed are crushed, extracted with ethanol, and the extract is concentrated to obtain an alcohol extract.

[0015] (2) The alcohol extract was suspended in water, and the suspension was extracted with petroleum ether, dichloromethane or chloroform to obtain petroleum ether extract and dichloromethane or chloroform extract.

[0016] (3) The petroleum ether extract from step (2) was separated by silica gel column chromatography and gradient elution was performed using a petroleum ether-acetone system to obtain 6 eluents Fr.1-Fr.6;

[0017] (4) Perform LC-MS / MS analysis on fractions Fr.1-Fr.6 in step (3), collect secondary mass spectrometry data of the sample, convert the original data file into a data file in mzXML format and import it into the GNPS online platform to establish a visualized molecular network, analyze the molecular network diagram with the help of the GNPS data platform, select the quasi-molecular ion peak with a mass-to-charge ratio (m / z) of about 600-750, and then compare the similarities and differences of the mass spectrometry data between the relevant nodes according to the correlation between nodes in the molecular network diagram, find the new fraction enriched by the cassane dimer, which is found to be Fr.1, and perform guided separation on it.

[0018] (5) The fraction Fr.1 was eluted using a petroleum ether-acetone system, and the subfractions were collected. The subfraction Fr.1.6 was subjected to gradient elution using a petroleum ether-acetone system to obtain fractions Fr.1.6.1-Fr.1.6.13. Fr.1.6.8 was separated and purified by reversed-phase semi-preparative HPLC with a 78:22 acetonitrile-water system as the mobile phase, a flow rate of 3 mL / min, and UV detector wavelengths of 210 nm and 254 nm to obtain compound 3. Fr.1.6.10 was separated and purified by reversed-phase semi-preparative HPLC with a 75:25 acetonitrile-water system as the mobile phase, a flow rate of 3 mL / min, and UV detector wavelengths of 210 nm and 254 nm to obtain compounds 1-2.

[0019] In step (1), the kernels of the chicken beak seed are dried and then crushed. The extraction is carried out by heating and reflux extraction with 60%-95% ethanol (preferably 70%-80% ethanol) and the ethanol solvent is recovered under reduced pressure to obtain the alcohol extract.

[0020] In step (2), the alcohol extract is suspended in water, and the suspension is extracted with petroleum ether, dichloromethane or chloroform. The extraction is performed 1-5 times, and the volume ratio of the extraction solvent to the suspension is 1:1-2:1. The solvent is recovered to obtain the petroleum ether layer extract and the dichloromethane or chloroform layer extract.

[0021] In step (3), the petroleum ether extract from step (2) is separated by silica gel column chromatography, and gradient elution is performed using a petroleum ether-acetone system with a volume ratio of 100:0-0:100.

[0022] In step (5), a petroleum ether-acetone system with a volume ratio of 1:1-5:1 is used to elute fraction Fr.1 and collect subfraction Fr.1.6.

[0023] This invention provides a pharmaceutical composition comprising one or more of the aforementioned catazane dimer derivatives or pharmaceutically acceptable salts thereof and a pharmaceutically acceptable carrier. The pharmaceutical composition can be administered in various ways, specifically in dosage forms such as oral, injectable, topical, inhalant, suppository, or liposomal formulations.

[0024] The petroleum ether extract of *Gnaphalium affine* kernels described in this invention contains a cassane dimer derivative or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the derivative, which has anti-renal fibrosis activity and can be used to prepare a medicament for the treatment of chronic kidney disease.

[0025] The beneficial effects of this invention are as follows: Guided separation of petroleum ether extract of *Caulis Caulis Persicae* using a GNPS molecular network strategy rapidly identifies novel cassane dimers. This method avoids the repetitive separation required by traditional methods, accurately locates the dimer enrichment sites, reduces time and reagent costs, and minimizes resource consumption. Compounds 1-3 obtained in this invention are relatively rare cassane dimers. The cassane dimer derivatives prepared by this method can inhibit the expression of fibronectin, a marker of renal fibrosis, in a TGF-β1-induced NRK-52E cell renal fibrosis model, showing great promise for the preparation of drugs for the treatment of chronic kidney disease. Attached Figure Description

[0026] Figure 1 The effect of cassane dimer derivative (50 μM) on NRK-52E cell viability was investigated. Compared with the control group... *** P<0.001.

[0027] Figure 2 This study investigated the effect of a cassane dimer derivative (PFD, pirfenidone) on the protein expression of fibronectin, a marker of renal fibrosis, in a TGF-β1-induced NRK-52E cell renal fibrosis model. Compared with the control group... ### P < 0.001; *** P<0.001. Detailed Implementation

[0028] The following embodiments will further illustrate the present invention in detail, but are not intended to limit the present invention.

[0029] Example 1

[0030] A method for preparing a cassane dimer derivative or a pharmaceutically acceptable salt thereof, comprising the following specific steps:

[0031] 13.5 kg of seeds of *Gnaphalium affine* were harvested, yielding 8.0 kg of kernels after dehulling. The kernels were dried, pulverized, and extracted three times with 10 times the volume (8 L) of 75% industrial ethanol under reflux for 2 h each time. The ethanol extracts were combined and concentrated under reduced pressure until no alcohol odor remained, yielding an ethanol extract. The ethanol extract was dispersed in distilled water and extracted three times sequentially with petroleum ether and chloroform, with a solvent-to-suspension volume ratio of 1:1–2:1. The solvent was recovered, yielding 153.5 g of petroleum ether extract. The petroleum ether extract was separated using open-cell silica gel column chromatography with a gradient elution system of petroleum ether-acetone (100:0–0:100, v / v). The resulting fractions were analyzed by silica gel thin-layer chromatography. Six eluates, Fr.1–Fr.6, were obtained by combining identical fractions. LC-MS analysis of Fr.1–Fr.6 revealed that Fr.1–Fr.2 were fractions with concentrated dimers. Data from Fr.1-Fr.2 were uploaded to the GNPS website. Molecular network visualization analysis revealed an abundance of quasi-molecular ion peaks with molecular weights around 600-750 in the Fr.1 fraction, suggesting that Fr.1 contained cassane diterpene dimers. Therefore, silica gel column chromatography was used for further separation of Fr.1, with petroleum ether-acetone (5:1, v / v) elution, and subfraction Fr.1.6 was collected. Fr.1.6 was then separated by silica gel column chromatography using a gradient elution of petroleum ether-acetone (100:0-0:100, v / v) to obtain 13 eluates Fr.1.6.1-Fr.1.6.13. Fraction Fr. 1.6.8 was separated by reversed-phase semi-preparative HPLC with acetonitrile-water (78:22, v / v) as the mobile phase to obtain compound 3 (2.0 mg); Fraction Fr. 1.6.10 was separated by reversed-phase semi-preparative HPLC with acetonitrile-water (75:25, v / v) as the mobile phase to obtain compounds 1 (36.3 mg) and 2 (10.4 mg).

[0032]

[0033] The spectroscopic data of compounds 1-3 are as follows:

[0034] Compound 1 (biscaesalmin L1):

[0035] White powder (methanol) 1 H-NMR (600 MHz, CD3OD) δ H:10.35 (1H, s, H-16),7.35(1H, s, H-17), 7.20 (1H, s ,H-16'), 5.65 (1H, m, H-6), 5.62 (1H, m, H-6'),3.32 (1H, overlap, H-8), 2.86 (1H, td, J = 12.7, 3.0 Hz, H-8'), 2.79 (1H, dd,J = 15.4, 3.1 Hz, H a -11), 2.70 (1H, overlap, H b -11), 2.67 (1H, m, H a -7), 2.53(1H, dd, J = 15.2, 2.9 Hz, H a -11'), 2.45 (1H, t, J = 14.8 Hz, H b -11'), 2.40(1H, dt, J = 13.6, 3.2 Hz, H a -7'), 2.10 (3H, overlap, H3-6-OCOCH3), 2.10 (3H,overlap, H3-6'-OCOCH3), 1.83 (1H, m, Ha-1), 1.82 (1H, overlap, H-9), 1.77 (1H,overlap, H a -2'), 1.76 (1H, overlap, H a -2), 1.74 (1H, overlap, H-9'), 1.73 (1H,m, H a -1'), 1.73 (3H, s, H3-17'), 1.58 (1H, overlap, H b -7), 1.55 (1H, overlap,H b -2'), 1.54 (1H, overlap, H b -2), 1.53 (1H, overlap, H b -7'), 1.44 (1H,overlap, H a -3), 1.43 (1H, overlap, H a-3'), 1.36 (3H, s, H3-20), 1.31 (3H, s,H3-20'), 1.30 (1H, overlap, H-5'), 1.28 (1H, overlap, H b -3), 1.28 (1H,overlap, H-5), 1.28 (1H, overlap, H b -3'), 1.13 (1H, overlap, H b -1'), 1.09 (3H,s, H3-19), 1.08 (3H, s, H3-19'), 1.05 (1H, overlap, H b -1), 1.00 (3H, overlap,H3-18), 1.00 (3H, overlap, H3-18')。 13 1H-NMR spectrum (150 MHz, CD3OD) δ C : 202.1 (C-12), 200.8 (C-12'), 195.3 (C-16), 163.7 (C-14'), 149.4 (C-13), 143.7 (C-15), 140.0 (C-14), 137.9 (C-15'), 133.6 (C-17), 132.8 (C-13'), 128.7 (C-16'), 70.7 (C-6), 70.6 (C-6'), 56.2 (C-5), 56.1 (C-5'), 54.4 (C-9), 53.5 (C-9'), 45.0 (C-3), 44.9 (C-3'), 41.8 (C-1), 41.6 (C-1'), 40.1 (C-11), 38.8 (C-10), 38.5 (C-7), 38.5 (C-11'), 38.4 (C-10'), 38.3 (C-8'), 36.9 (C-7'), 34.9 (C-4), 34.8 (C-4'), 34.7 (C-8), 33.6 (C-18), 33.5 (C-18'), 23.9 (C-19), 23.8 (C-19'), 20.2 (C-17'), 19.9 (C-2), 19.8 (C-2'), 17.3 (C-20), 17.3 (C-20'), 172.3 / 21.8 (C-6-OCOCH3), 172.2 / 21.7 (C-6-OCOCH3)。

[0036] It should be noted that there seems to be a small error in the original text where "56.2 (C-5)" has a capital "5", which is likely a typo and is corrected to "5" in the translation.Compound 2 (biscaesalmin L2):

[0037] White powder (methanol), 1 1H-NMR (600 MHz, DMSO-d6) δ H : 9.51 (1H, s, H-16), 6.41(1H, d, J = 2.9 Hz, H-16'), 5.43 (1H, m, H-6), 5.42(1H, m, H-6'), 3.80 (1H,m, H-15'), 2.75 (1H, m, H-8), 2.58 (1H, m, H a -11'), 2.43 (1H, t, J = 14.2 Hz,H a -11), 2.36 (1H, m, H b -11), 2.35 (1H, m, H b -17), 2.32 (1H, m, H a -17), 2.27(1H, s, H-8'), 2.26 (1H, d, J= 9.2 Hz, H b -11'), 2.21 (1H, dt, J = 13.9, 3.4Hz, H a -7'), 2.15 (1H, dt, J = 13.5, 3.3 Hz, H a -7), 2.04 (3H, s, H3-6'-OCOCH3),2.01 (3H, s, H3-6-OCOCH3), 1.85 (3H, s, H3-17'), 1.67 (1H, m, H-9), 1.60 (1H,overlap, H a -2), 1.60 (1H, overlap, H a -1'), 1.59 (1H,overlap, H a -1), 1.59 (1H,overlap, H a -2'), 1.50 (1H, overlap, H b -7), 1.50 (1H, overlap, H-9'), 1.44 (1H,overlap, H b -2'), 1.43 (1H, overlap, H b -2), 1.37 (1H, overlap, H b-7'), 1.34(1H, overlap, H a -3), 1.33 (1H, overlap, H a -3'), 1.18 (3H, s, H3-20), 1.17 (1H,overlap, H b -3), 1.17 (1H, overlap, H b -3'),1.15 (1H, overlap, H-5), 1.15 (1H,overlap, H-5'), 1.14 (3H, s, H3-20'), 0.97 (3H, s, H3-19'), 0.96 (3H, s, H3-19), 0.93 (1H, overlap, H b -1), 0.93 (1H, overlap, H b -1'), 0.90 (3H, overlap,H3-18), 0.90 (3H, overlap, H3-18'). 13 13C-NMR spectrum (150 MHz, DMSO-d6) δ C: 197.8 (C-12'), 197.4 (C-12), 190.5 (C-16), 160.7 (C-14), 160.0 (C-14'), 143.1 (C-16'), 135.4 (C-13'), 133.9 (C-15), 127.9 (C-13), 68.5 (C-6), 68.4 (C-6'), 54.0 (C-5), 53.7 (C-5'), 52.7 (C-9), 51.1 (C-9'), 43.3 (C-3), 43.3 (C-3'), 39.7 (C-1), 39.7 (C-1'), 37.8 (C-11), 37.1 (C-8'), 37.1 (C-11'), 37.0 (C-10'), 36.6(C-10), 36.2 (C-7), 35.3 (C-7'), 34.4 (C-8), 33.4 (C-4), 33.4 (C-4'), 33.4(C-4'), 32.7 (C-18), 32.7 (C-18'), 32.2 (C-15'), 30.4 (C-17), 23.1 (C-19), 23.1 (C-19'), 18.8 (C-17'), 18.4 (C-2), 18.4 (C-2'), 16.4 (C-20), 16.4 (C-20'), 169.9 / 21.5 (C-6-OCOCH3), 169.8 / 21.5 (C-6'-OCOCH3).

[0038] Compound 3 (biscaesalmin L3):

[0039] White powder (methanol) 1 H-NMR (600 MHz, CD3OD) δ H : 7.29 (1H, s, H-16'), 7.11 (1H, s, H-17), 6.22 (1H, s, H-16), 5.62 (1H, m, H-6), 5.62 (1H, m, H-6'), 3.41 (3H, s, H3-16-OCH3), 3.34 (3H, s, H3-16-OCH3), 3.25 (1H, td, J = 12.0, 3.8 Hz, H-8), 2.84 (1H, m, H-8'), 2.65 (1H, dd, J = 14.5, 3.0 Hz, H a -11), 2.60 (H, m, Ha -7), 2.57 (1H, overlap, H b -11), 2.53 (1H, dd, J = 15.3, 3.4 Hz,H a -11'), 2.45 (1H, m, H b -11'), 2.40 (1H, dt, J = 13.9, 3.4 Hz, H a -7'), 2.10(3H, s, H3-6'-OCOCH3), 2.09 (3H, s, H3-6-OCOCH3), 1.79 (1H, m, H a -1), 1.74 (1H,overlap, H-9), 1.74 (1H, overlap, H-9'), 1.73 (1H, overlap, H a -2), 1.73 (1H,overlap, H a -2'), 1.73 (3H, s, H3-17'), 1.71 (1H, overlap, H a -1'), 1.55 (1H,overlap, H b -7), 1.54 (1H, m, H b -2'), 1.52 (1H, m, H b -2), 1.52 (1H, overlap,H b -7'), 1.44 (1H, m, H a -3'), 1.42 (1H, m, H a -3), 1.32 (3H, s, H3-20), 1.31(3H, s, H3-20'), 1.28 (1H, m, H b -3), 1.28 (1H, overlap, H-5), 1.28 (1H,overlap, H-5'), 1.28 (1H, m, H b -3'), 1.09 (1H, overlap, H b -1), 1.08 (3H,overlap, H-19'), 1.07 (3H, overlap, H3-19), 1.07 (1H, overlap, H b -1'), 1.00(3H, s, H3-18), 0.99 (3H, s, H3-18')。13 C-NMR spectrum (150 MHz, CD3OD) δ C : 203.0 (C-12), 201 (C-12'), 163.1 (C-14'), 149.3 (C-13), 142.8 (C-15), 140.1 (C-14), 138.6 (C-15'), 130.6 (C-13'), 130.3 (C-17), 128.1 (C-16'), 103.0 (C-16), 70.8(C-6), 70.8 (C-6'), 56.2 (C-5), 56.2 (C-5'), 55.6 (C-16-OCH3), 55.3 (C-16-OCH3), 54.3 (C-9), 53.6 (C-9'), 45.0 (C-3), 44.9 (C-3'), 41.8 (C-11), 41.7(C-1), 41.6 (C-1'), 39.1 (C-7), 38.7 (C-11'), 38.6 (C-10), 38.4 (C-10'), 38.3(C-8'), 37.0 (C-7'), 35.4 (C-8), 34.9 (C-4), 34.8 (C-4'), 33.5 (C-18), 33.5(C-18'), 23.9 (C-19), 23.9 (C-19'), 20.1 (C-17'), 19.9 (C-2), 19.8 (C-2'),17.3 (C-20), 17.2 (C-20'), 172.4 / 21.8 (C-6-OCOCH3), 172.3 / 21.7 (C-6'-OCOCH3).

[0040] Example 2

[0041] Screening of in vitro anti-renal fibrosis activity of cassane dimer derivatives:

[0042] (1) Cell culture:

[0043] NRK-52E cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2.

[0044] (2) Activity test:

[0045] MTT cytotoxic activity assay:

[0046] NRK-52E cells were collected after centrifugation and seeded into 96-well plates. After culturing in an incubator for 24 h, a final concentration of 50 μM of the test monomer compound was added to the 96-well plates, and the cells were cultured for another 24 h. Finally, cell viability was determined according to the MTT reagent instructions.

[0047] Screening for anti-renal fibrosis activity: NRK-52E cells were incubated in serum-free medium for 12 h. The control group was then cultured in medium containing 0.5% DMSO for 24 h. The TGF-β1 group was cultured in medium containing TGF-β1 for 24 h. The test compound group was cultured in medium containing TGF-β1 and the test compound (final concentration 30 μM) for 24 h. The PFD (pirfenidone) group was cultured in medium containing TGF-β1 and pirfenidone (final concentration 5 μM) for 24 h. Finally, ELISA was performed according to the reagent instructions.

[0048] The results of the MTT cell experiment are attached. Figure 1 The results showed that compound 2 had significant cytotoxicity against NRK-52E cells at a concentration of 50 μM, while compounds 1 and 3 had no effect on NRK-52E cell viability at a concentration of 50 μM.

[0049] Results of the compound's anti-renal fibrosis activity are as follows Figure 2 As shown, compounds 1 and 3 significantly reduced the protein expression of fibronectin, a fibrosis marker, in a TGF-β1-induced renal fibrosis model at a concentration of 30 μM.

Claims

1. A cassane dimer derivative or a pharmaceutically acceptable salt thereof, characterized in that, The cassane dimer derivative is selected from the following structural formulas: In Formula I: R is -CHO or -CH(OCH3)2.

2. The cassane dimer derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The aforementioned cassane dimer derivative is any one of the following compounds: 。 3. A method for preparing the cassane dimer derivative of claim 1 or 2 or a pharmaceutically acceptable salt thereof, characterized in that, The steps include the following: (1) After drying, the kernels of the chicken beak seed are crushed and extracted with ethanol. The extract is then concentrated to obtain an alcohol extract. (2) The alcohol extract was suspended in water and extracted with petroleum ether, dichloromethane or chloroform to obtain petroleum ether extract and dichloromethane or chloroform extract. (3) The petroleum ether extract was separated by silica gel column chromatography and eluted with a petroleum ether-acetone system to obtain 6 eluents Fr.1-Fr.6; (4) LC-MS / MS secondary mass spectrometry analysis was performed on fractions Fr.1-Fr.

6. The data were imported into the GNPS platform to construct a molecular network. By analyzing the network diagram, ion peaks with a mass-to-charge ratio of 600-750 were screened. The secondary mass spectrometry characteristics were determined based on the node correlation ratio. Finally, fraction Fr.1, which is rich in cassane dimers, was identified for subsequent guided separation. (5) Fr.1 was eluted using a petroleum ether-acetone system, and the sub-fractions were collected. The sub-fraction Fr.1.6 was eluted in a petroleum ether-acetone system to obtain fractions Fr.1.6.1-Fr.1.6.

13. Fr.1.6.8 or Fr.1.6.10 was separated and purified by reversed-phase semi-preparative HPLC to obtain the cassane dimer derivative.

4. The method for preparing the cassane dimer derivative or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that, In step (1), the kernels of the chicken beak seed are dried and then pulverized, and extracted by heating and reflux with ethanol of 60%-95% by volume.

5. The method for preparing the cassane dimer derivative or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that, In step (3), the petroleum ether extract is separated by silica gel column chromatography, and gradient elution is performed using a petroleum ether-acetone system with a volume ratio of 100:0-0:

100.

6. The method for preparing the cassane dimer derivative or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that, In step (5), Fr.1 is eluted using a petroleum ether-acetone system with a volume ratio of 1:1-5:1, and the sub-fraction is collected.

7. The method for preparing the cassane dimer derivative or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that, In step (5), Fr.1.6.8 was separated and purified by reversed-phase semi-preparative HPLC with a 78:22 acetonitrile-water system as the mobile phase, a flow rate of 3 mL / min, and a UV detector with wavelengths of 210 nm and 254 nm to obtain compound 3; Fr.1.6.10 was separated and purified by reversed-phase semi-preparative HPLC with a 75:25 acetonitrile-water system as the mobile phase, a flow rate of 3 mL / min, and a UV detector with wavelengths of 210 nm and 254 nm to obtain compounds 1-2.

8. A pharmaceutical composition, characterized in that, It comprises one or more of the cassane dimer derivatives of claim 1 or 2 or their pharmaceutically acceptable salts and a pharmaceutically acceptable carrier.

9. The use of the cassane dimer derivative of claim 1 or 2 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 8 in the preparation of a medicament for the treatment of chronic kidney disease.

10. The application of a *Gnaphalium affine* seed extract in the preparation of a drug for treating chronic kidney disease, characterized in that... The extract of *Gnaphalium affine* kernels comprises the cassane dimer derivative of claim 1 or 2 or a pharmaceutically acceptable salt thereof.

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