Sarpagine type alkaloid cooperating with platinum drug to resist ovarian cancer activity and preparation method and application thereof

Abstract

The invention discloses a class of sarpagine alkaloids cooperating with platinum drugs to resist ovarian cancer activity as well as a preparation method and application thereof, and belongs to the technical field of natural product chemistry. According to the invention, a chloroform part with a pH value of 9 of a medicinal plant rauvolfia vomitoria is separated and purified to obtain 14 arpagine-type alkaloids (compound 114) which are found for the first time, and the arpagine-type alkaloids (compound 114) are named as Rauvoines AN. By testing the synergistic cytotoxic effect of the combined use of the 14 sarpagine type compounds and 10 analogues (known structures) and cis-platinum on two ovarian cancer cell lines OVCAR3 and SKOV3, it is found for the first time that the sarpagine type compounds can significantly enhance the cytotoxicity of cis-platinum on ovarian cancer cells and show an obvious cis-platinum sensitization effect. It is fully indicated that the sarpagine type compound separated from rauvolfia devilpepper can be used for preparing the platinum drug sensitizer for ovarian cancer treatment, a new strategy is provided for ovarian cancer treatment, important clinical application value is achieved, and great technical progress is achieved.

Description

Technical Field

[0001] This invention belongs to the field of natural product chemistry technology, and relates to the medicinal plant Rauvolfia elegans (…). Rauvolfia vomitoria) The chloroform fraction was isolated and purified, the compound structure was confirmed, and its sensitizing effect on cisplatin in the fight against ovarian cancer was evaluated. This involved sarpagine-type compounds with synergistic anti-ovarian cancer activity of cisplatin, their isolation and preparation methods, and their applications. Specifically, it involved a class of sarpagine-type alkaloids with synergistic anti-ovarian cancer activity of platinum-based drugs, their preparation methods, and their applications. Background Technology

[0002] Ovarian cancer is one of the most common malignant tumors of the reproductive system and has the highest mortality rate among all gynecological cancers. Although standard treatment includes platinum-based chemotherapy after cytoreductive surgery... Despite paclitaxel chemotherapy, over 70% of patients relapse, often with platinum-based resistance, resulting in a global 5-year survival rate of only about 30%. 40%. Cisplatin, one of the most effective chemotherapy drugs for treating ovarian cancer, faces significant treatment challenges due to frequent drug resistance. Given its significant impact on patient survival and quality of life, improving the efficacy of cisplatin treatment remains a key issue in ovarian cancer treatment. Platinum sensitizers delay the development of drug resistance by reducing the dosage of platinum drugs, while simultaneously mitigating the side effects of high-dose treatment, such as nephrotoxicity, neurotoxicity, and myelosuppression. This dual advantage improves both treatment efficacy and patient quality of life. Therefore, the development of novel platinum sensitizers has become an urgent priority.

[0003] Emetic Rauvolfia ( Rauvolfia vomitoria Afzel. (Apocynaceae family) is a medicinal plant rich in monoterpenoid indole alkaloids (MIAs), and its alkaloids have been reported to possess acetylcholinesterase inhibitory activity and anti-inflammatory activity. However, research on sarpagine-type alkaloids is limited, and a large number of derivatives remain to be discovered. Furthermore, there are no reports of sarpagine-type alkaloids synergistically acting with platinum-based drugs in the treatment of ovarian cancer. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the present invention aims to provide a class of sarpagine-type alkaloids with synergistic anti-ovarian cancer activity against platinum-based drugs, their preparation method, and applications. To achieve the above objectives, the present invention employs the following technical solution: This invention discloses a class of sarpagine-type alkaloids or pharmaceutically acceptable salts thereof that exhibit synergistic anti-ovarian cancer activity with platinum-based drugs, the structure of which is shown in the following formula: ; R1 is selected from hydrogen or hydroxyl; R2 is selected from hydrogen or hydroxyl; R3 is selected from hydrogen, hydroxyl, or carboxyl groups; R4 is selected from hydrogen or hydroxyl groups; R5 is selected from hydrogen or carbonyl; R6 is selected from hydrogen, hydroxymethyl, and methyl ester. R7 is selected from hydrogen, chlorine, hydroxyl, hydroxymethyl, carboxyl, hydroxy(methoxy)methyl, glucose-1-hydroxymethyl, or azirmonoethyl ethylamine; R8 is selected from methyl, carbonyl, or none.

[0005] Preferably, the sarpagine-type alkaloids that synergize with platinum-based drugs against ovarian cancer specifically include compounds with structures shown in Formulas 1-14 or pharmaceutically acceptable salts thereof:

[0006]

[0007]

[0008]

[0009] .

[0011] This invention also discloses a method for extracting sarpagine-type alkaloids with the above-mentioned synergistic anti-ovarian cancer activity against platinum-based drugs, comprising the following steps: 1) Take the dried rhizome of Rauvolfia pulveratum, crush it, and extract it with alcohol to prepare the total extract; 2) After suspending the total extract in water, extract it several times with chloroform at a pH of 2-3. Then adjust the pH to 8-10 and extract it several times with chloroform again. Combine the dichloromethane extract fractions at pH 8-10 to obtain the total alkaloids. 3) The total alkaloids were separated by normal-phase silica gel chromatography column chromatography, and gradient elution was performed using a mixture of dichloromethane and methanol as the eluent. Sarpagine-type alkaloids were concentrated in the eluent. 4) The eluent containing sarpagine-type alkaloids is concentrated and then separated by reversed-phase column chromatography gradient elution. The eluents of each gradient are then separated by silica gel column chromatography, gel column chromatography and HPLC to obtain a series of sarpagine-type alkaloids.

[0012] Preferably, in step 1), 80% methanol is used for extraction at 40°C, and the ratio of dried rhizome of Rauvolfia pulveratum to 80% methanol is 1 kg: (2-4) L; the extraction is performed 4 times, each time for 2 days, and the solvent is removed by rotary evaporation of the combined alcohol extracts to obtain the total extract.

[0013] Preferably, in step 2), the total extract is suspended in warm water, the pH is adjusted to 2 with 2% hydrochloric acid, and then extracted with chloroform 5 times. Subsequently, the pH is adjusted to 9 with ammonia water, and then extracted with chloroform 5 times again. The chloroform extract fraction with a combined pH of 9 is the total alkaloid.

[0014] Preferably, in step 3), when performing separation using a normal-phase silica gel column, the elution ratio of dichloromethane and methanol in the eluent changes sequentially from 30:1, 20:1, 15:1, 10:1, 5:1 to the final 2:1, so that sarpagine alkaloids are concentrated in the 10:1 eluent.

[0015] Preferably, in step 4), the chromatographic conditions for reversed-phase column chromatography separation are as follows: The chromatographic packing material was C18, and the eluent was a mixture of methanol and water. The initial elution ratio was 10% methanol, and the final ratio was 100% methanol. The methanol ratio was increased by 10% for each intermediate gradient, and elution was performed at 12 L for each gradient.

[0016] This invention also discloses the application of the above-mentioned sarpagine-type alkaloids or their pharmaceutically acceptable salts that synergize with platinum-based drugs against ovarian cancer in the preparation of platinum-based drug sensitizers; And its application in the preparation of drugs for treating ovarian cancer; The sarpagine-type alkaloids mentioned above include compounds with the following structures:

[0017]

[0018]

[0019]

[0020]

[0021]

[0022]

[0023] .

[0025] Preferably, the platinum-based drug is cisplatin.

[0026] Compared with the prior art, the present invention has the following beneficial effects: This invention involves the isolation and purification of the chloroform fraction of the medicinal plant Rauvolfia vomitoria (pH 9), yielding 14 newly discovered sarpagine-type alkaloids (new compound 1). 14), named Rauvoines A N. By testing the synergistic cytotoxic effects of 14 sarpagine compounds and 10 of their analogues (with known structures) in combination with cisplatin on two ovarian cancer cell lines, OVCAR3 and SKOV3, it was discovered for the first time that sarpagine-type compounds can significantly enhance the cytotoxicity of cisplatin against ovarian cancer cells, exhibiting a significant cisplatin sensitization effect. This fully demonstrates that the sarpagine-type compounds isolated from Rauvolfia pulveratula in this invention can be used to prepare platinum-based drug sensitizers in the treatment of ovarian cancer, providing a new strategy for the treatment of ovarian cancer, possessing significant clinical application value, and representing a major technological advancement.

[0027] Furthermore, experimental verification showed that compounds 2 and 7 exhibited the strongest synergistic effect, with a combination index ranging from 0.24 to 0.52. The combined use of compounds 2 and 7 with cisplatin may promote cell cycle S-phase arrest by reducing the expression of CDK1 and Chk1, and induce a shift in the Bcl-2 / BAX ratio towards apoptosis.

[0028] Meanwhile, this invention proposes an extraction and separation method for the above-mentioned compounds with synergistic anti-ovarian cancer activity of platinum-based drugs. The process design is reasonable, highly operable, and the compounds with synergistic anti-ovarian cancer activity obtained are of high purity. Attached Figure Description

[0029] Figure 1 Compound 1 obtained by the present invention Flowchart of the extraction and separation process of 24. Figure 2 The crystal structure of compound 3 obtained by the present invention.

[0030] Figure 3 The crystal structure of compound 7 obtained by the present invention.

[0031] Figure 4 Compounds 2 and 7 in 10 m The M concentration significantly enhanced the effect of cisplatin, increasing its half-maximal inhibitory concentration (HMC) in the OVCAR3 cell line from 6.68 to 10.5%. m M decreased to 2.34 m M and 1.88 m M, in the SKOV3 cell line, was 26.11. m M drops to 17.60 m M and 15.95 m M indicates that they have a significant synergistic effect with cisplatin.

[0032] Figure 5 The number of cells in S phase significantly increased after incubation with cisplatin in combination with compounds 2 or 7, indicating that the combination of compounds 2 and 7 with cisplatin can arrest the cell cycle in S phase. The combination of cisplatin with compounds 2 and 7 significantly increased the apoptosis rate of SKOV3 cells compared to cisplatin or the compounds alone, indicating that compounds 2 or 7 can enhance the apoptosis-induced effect of cisplatin in ovarian cancer cells.

[0033] Figure 6 Western blot analysis revealed significant changes in BAX, Bcl-2, CDK1, and Chk1 proteins in the co-treated SKOV3 cells: the expression of the anti-apoptotic protein Bcl-2 was downregulated, the level of the pro-apoptotic protein BAX was increased, and the expression levels of the cell cycle proteins CDK1 and Chk1 were significantly reduced. This suggests that the combination of two sarpagine alkaloids with cisplatin may promote S-phase arrest of the cell cycle by inhibiting the expression of key cell cycle regulators CDK1 and Chk1. Simultaneously, it shifts the Bcl-2 / BAX ratio towards apoptosis. Detailed Implementation

[0034] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0035] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0036] The present invention will now be described in further detail with reference to the accompanying drawings: Example 1 I. Compound 1 isolated in this invention Preparation of 14 1. Plant Information The rhizome of this plant was collected in August 2017 from Xishuangbanna, Yunnan Province, People's Republic of China, and identified by Associate Professor Zhou Tao of Xi'an Jiaotong University as *Rauvolfia vomitingii*. Rauvolfia vomitoria) The plant specimens are stored in the herbarium of the Department of Natural Product Chemistry, School of Pharmacy, Xi'an Jiaotong University, specimen number 20170817.

[0037] 2. Extraction and separation (see...) Figure 1 ) 50 kg of dried rhizome of Rauvolfia purpurea was pulverized and extracted four times with 120 L of 80% methanol at 40°C for two days each time, yielding a total extract of 3.18 kg. This extract was suspended in warm water (10 L) and extracted with chloroform (5 × 4 L) at pH 2, resulting in an acidic aqueous layer and a chloroform fraction at pH 2. The acidic aqueous layer was adjusted to pH 9 with ammonia and extracted with chloroform (5 × 4 L) to obtain a chloroform layer (154 g) at pH 9. The chloroform fraction at pH 9 was subjected to silica gel column chromatography with dichloromethane:methanol (30:1). 2:1) Gradient elution was performed, and similar fractions were detected by TLC to obtain four fractions (I... (IV). Component IV was subjected to reversed-phase column chromatography, and similar fractions were detected by TLC, yielding 9 fractions: III1 III9.

[0038] Component III1 was subjected to gel column chromatography (MeOH) to yield two components: III1a III1b; fraction III1a was subjected to reversed-phase high-performance liquid chromatography (MeOH : H2O, 20 : 80) to yield compounds 12 and 24; fraction III1b was subjected to gel column chromatography (MeOH) and subsequent reversed-phase high-performance liquid chromatography (MeOH : H2O, 85 : 15) to yield compound 19.

[0039] Fraction III2 was subjected to silica gel column chromatography (CH2Cl2: MeOH, 8:1) to obtain three fractions: III2a After being subjected to gel column chromatography (MeOH), III2c and III2a were followed by reversed-phase high-performance liquid chromatography (MeCN: H2O, 11: 89) to obtain compound 11.

[0040] Fraction III3 was subjected to silica gel column chromatography (CH2Cl2: MeOH, 6:1 to 1:1) to obtain three fractions: III3a III3c; fraction III3a was subjected to silica gel column chromatography (CH2Cl2: MeOH, 5:1) to yield compounds 6 and 14; fraction III3c was subjected to gel column chromatography (MeOH) followed by reversed-phase high-performance liquid chromatography (MeOH: H2O, 80:20) to yield compounds 4, 13 and 20.

[0041] Component III4 was subjected to reverse-phase C 18 Column chromatography (MeOH: H2O, 20:80) 100 : 0), yielding 6 components: III4a Compound III4f and compound III4b were subjected to gel column chromatography (MeOH) followed by silica gel column chromatography (CH2Cl2:MeOH, 15:1) to obtain compound 22; compound III4f was subjected to reversed-phase high-performance liquid chromatography (MeOH:H2O, 50:50) to obtain compound 8.

[0042] Reversed-phase column chromatography of component III5 yielded three components: III5a III5c; component III5a was subjected to reversed-phase high-performance liquid chromatography (MeCN: H2O, 15: 85) to yield compounds 3 and 5; III5b was subjected to reversed-phase high-performance liquid chromatography (MeOH: H2O, 75: 25) to yield compound 1; component III5c was subjected to reversed-phase high-performance liquid chromatography (MeOH: H2O, 50: 50) to yield compound 18.

[0043] Reversed-phase column chromatography was performed on fraction III8 to obtain five fractions: III8a Component III8e; Component III8c was subjected to silica gel column chromatography to obtain four components: III8c1 Compound III8c4; fraction III8c1 was separated by reversed-phase high-performance liquid chromatography (MeOH: H2O, 45:55) after gel column chromatography (MeOH); compound III8c2 was separated by reversed-phase high-performance liquid chromatography (MeOH: H2O, 40:60) to obtain compounds 2 and 16; fraction III8c3 was separated by reversed-phase high-performance liquid chromatography (MeOH: H2O, 40:60) to obtain compounds 17 and 21; fraction III8c4 was separated by reversed-phase high-performance liquid chromatography (MeOH: H2O, 50:50) after gel column chromatography (MeOH); compound III8c4 was separated by reversed-phase high-performance liquid chromatography (MeOH: H2O, 50:50) to obtain compound 10; fraction III8c4 was separated by silica gel column chromatography (CH2Cl2: MeOH, 50:1) and then by reversed-phase high-performance liquid chromatography (MeOH: H2O, 65:1). 35) Compound 9 was obtained; after gel column chromatography (MeOH), component III8e was separated by reversed-phase high-performance liquid chromatography (MeOH : H2O, 85 : 15) to obtain compound 7.

[0044] Component III9 was subjected to silica gel column chromatography (CH2Cl2: MeOH, 50:1), followed by gel column chromatography (MeOH), and then separated by reversed-phase high-performance liquid chromatography (MeOH: H2O, 60:40) to obtain compound 23.

[0045] II. Compound 1 14 Structural Identification Through the analysis of new compound 1 A comprehensive analysis of data from nuclear magnetic resonance (NMR), high-resolution mass spectrometry (HMR), ultraviolet spectroscopy, infrared spectroscopy, optical rotation, circular dichroism spectroscopy, and X-ray single-crystal diffraction (XRD) was conducted to determine compound 1. The structure of 14.

[0046] Compound 1: colorless oil ; 70 ( c 0.1, MeOH); UV (MeOH) λ max (log e )225 (4.41), 280 (3.74), 289 (3.64) nm; ECD (MeOH) 218 ​​(Δ e , 5.89), 239 (Δ e , 3.32), 251 (Δ e , 0.24), 270 (Δ e , 7.77), 306 (Δ e , 0.41 nm; IR (KBr) n max 3393, 2928, 2854, 1701, 1594, 1385, 1351, 1088, 1032, 881, 738 cm -1 HRESIMS m / z 299.13091 [M + H] + (calcd for C 18 H 20 The nuclear magnetic resonance (NMR) data of compound 1 (N2Cl, 299.13150) are shown in Table 1.

[0047] Compound 2: white powder ; 19 ( c 0.1, MeOH); UV (MeOH) λ max (log e ) 223(4.39), 280 (3.79), 290 (3.61) nm; ECD (MeOH) 217 ​​(Δ e , 20.66), 239 (Δ e , 1.19), 249 (Δ e , 0.55), 269 (Δ e , 9.16), 303 (Δ e , 2.82), 333 (Δ e , 1.52) nm; IR (KBr) n max 3257, 2922, 2858, 1452, 1301, 1055, 1033, 1010, 977,736 cm -1 HRESIMS m / z 281.16332 [M + H] + (calcd for C 18 H 20 N2O, 281.16539). The nuclear magnetic resonance (NMR) data of compound 2 are shown in Table 1.

[0048] Compound 3: transparent crystal ; 48 ( c 0.1, MeOH); UV (MeOH) λmax (log e ) 213 (4.31), 253 (4.11), 279 (3.90), 301 (3.81) nm; ECD (MeOH) 219 (Δ e ,13.33), 237 (Δ e , 0.29), 249 (Δ e , 4.51), 269 (Δ e , 6.17), 310 (Δ e (0.48) nm; IR (KBr) n max 3387, 2921, 2860, 1645, 1585, 1486, 1452, 1385, 1203,1087, 1032, 902, 859 cm -1 HRESIMS m / z 325.15578 [M + H] + (calcd for C 19 H 21 N2O3, 325.15522). The nuclear magnetic resonance (NMR) data of compound 3 are shown in Tables 2 and 5. The crystal structure is as follows: Figure 1 As shown.

[0049] Compound 4: colorless oil ; 131 ( c 0.1, MeOH); UV (MeOH) λ max (log e )214 (4.36), 254 (4.10), 275 (3.96), 302 (3.88) nm; ECD (MeOH) 219 (Δ e ,16.00), 238 (Δ e , 0.39), 249 (Δ e , 5.42), 269 (Δ e , 7.40), 311 (Δ e (0.59) nm; IR (KBr) n max 3248, 2927, 2860, 1645, 1585, 1478, 1434, 1368, 1269,1102, 1035, 809 cm -1 HRESIMS m / z 341.15011 [M + H] + (calcd for C 19 H 21 N2O4, 341.15013). The nuclear magnetic resonance (NMR) data of compound 4 are shown in Tables 2 and 5.

[0050] Compound 5: colorless oil ; twenty one ( c 0.1, MeOH); UV (MeOH) λ max (log e )213 (4.31), 253 (4.12), 278 (3.92), 301 (3.87) nm; ECD (MeOH) 220 (Δ e ,12.70), 238 (Δ e , 0.49), 249 (Δ e , 2.48), 271 (Δ e , 8.75), 318 (Δ e ,1.23) nm; IR (KBr) n max 3381, 2930, 2855, 1643, 1640, 1486, 1324, 1090, 876,818 cm -1 HRESIMS m / z 341.15246 [M + H] + (calcd for C 19 H 21 N2O4, 341.15013). The nuclear magnetic resonance (NMR) data of compound 5 are shown in Tables 2 and 5.

[0051] Compound 6: colorless oil ; 74 ( c 0.1, MeOH); UV (MeOH) λ max (log e )226 (4.40), 276 (3.94), 312 (3.84) nm; ECD (MeOH) 216 (Δ e , 15.62), 238 (Δ e , 0.56), 248 (Δ e , 0.91), 269 (Δ e , 9.95), 295 (Δ e , 6.13) nm; IR (KBr) n max 3291, 2930, 2863, 1593, 1437, 1353, 1238, 1159, 1081, 1029, 803 cm -1 HRESIMS m / z 327.17085 [M + H] + (calcd for C 19 H 23 N2O3 (327.17087). The nuclear magnetic resonance (NMR) data of compound 6 are shown in Tables 2 and 5.

[0052] Compound 7: transparent crystal ; 144 ( c 0.1, MeOH); UV (MeOH) λ max (log e ) 223 (4.31), 280 (3.78), 290 (3.61) nm; ECD (MeOH) 219 (Δ e , 12.12),237 (Δ e , 0.27), 249 (Δ e , 4.11), 269 (Δ e , 5.61), 309 (Δ e (0.42) nm; IR(KBr) n max 2929, 2851, 1450, 1172, 1068, 1024, 968, 804, 735, 719 cm -1 HRESIMS m / z 350.25847 [M + H] + (calcd for C 23 H 32 N3, 350.25962). The nuclear magnetic resonance (NMR) data of compound 7 are shown in Tables 2 and 5. The crystal structure is as follows: Figure 2 As shown.

[0053] Compound 8: colorless oil ; –343 ( c 0.1, MeOH); UV (MeOH) λmax (log e )225 (4.35), 280 (3.74), 290 (3.57) nm; ECD (MeOH) 217 ​​(Δ e , 14.27), 237 (Δ e , 4.34), 249 (Δ e , 0.85), 270 (Δ e , 12.63), 309 (Δ e , 0.67) nm; IR (KBr) n max 3311, 2915, 2859, 1673, 1600, 1453, 1380, 1171, 1081, 1025, 746 cm -1 HRESIMS m / z 457.23363 [M + H] + (calcd for C 25 H 33 N2O6, 457.23386). The nuclear magnetic resonance (NMR) data of compound 8 are shown in Tables 3 and 5.

[0054] Compound 9: colorless oil ; 31 ( c 0.1, MeOH); UV (MeOH) λ max (log e )225 (4.38), 280 (3.78), 290 (3.48) nm; ECD (MeOH) 217 ​​(Δ e , 11.30), 239 (Δ e , 2.94), 250 (Δ e , 0.89), 270 (Δ e , 8.62), 306 (Δ e , 0.51 nm; IR (KBr) n max 3404, 2927, 2858, 1654, 1591, 1455, 1379, 1316, 1084, 1032, 740 cm -1HRESIMS m / z 325.19150 [M + H] + (calcd for C 20 H 25 N2O2 (325.19160). The nuclear magnetic resonance (NMR) data of compound 9 are shown in Table 4.

[0055] Compound 10: white powder ; 128 ( c 0.1, MeOH); UV (MeOH) λ max (log e )223 (4.35), 280 (3.86), 290 (3.58) nm; ECD (MeOH) 217 ​​(Δ e , 13.56), 239 (Δ e , 3.53), 250 (Δ e , 1.07), 271 (Δ e , 10.32), 306 (Δ e , 0.60 nm; IR (KBr) n max 3172, 2925, 1446, 1126, 1039, 1028, 814, 733, 632, 561,480, 453 cm -1 HRESIMS m / z 341.18544 [M + H] + (calcd for C 20 H 25 N2O3, 341.18597). The nuclear magnetic resonance (NMR) data of compound 10 are shown in Table 4.

[0056] Compound 11: colorless oil ; 32 ( c 0.1, MeOH); UV (MeOH) λ max (log e )225 (4.36), 276 (3.95), 306 (3.59) nm; ECD (MeOH) 217 ​​(Δ e , 14.69), 239 (Δ e , 2.07), 249 (Δ e , 0.01), 269 (Δ e , 8.46), 306 (Δ e , 1.01) nm; IR (KBr) n max 3271, 2930, 2860, 1672, 1596, 1457, 1379, 1316, 1171, 1087, 1040, 899, 822,812 cm -1 HRESIMS m / z 327.17085 [M + H] + (calcd for C 19 H 23 N2O3, 327.17087). The nuclear magnetic resonance (NMR) data of compound 11 are shown in Tables 3 and 5.

[0057] Compound 12: colorless oil ; 70 ( c 0.1, MeOH); UV (MeOH) λ max (log e )220 (4.42), 274 (3.72), 290 (3.52) nm; ECD (MeOH) 222 (Δ e , 7.21), 242 (Δ e , 0.83), 270 (Δ e , 3.12) nm; IR (KBr) n max 3417, 2956, 1649, 1457, 1325, 1261,1044, 856, 754, 659 cm -1 HRESIMS m / z 325.19075 [M] + (calcd for C 20 H 25 N2O2 + (325.19105). The nuclear magnetic resonance (NMR) data of compound 12 are shown in Tables 3 and 5.

[0058] Compound 13: colorless oil ; 68 ( c0.1, MeOH); UV (MeOH) λ max (log e )230 (4.36), 270 (3.98), 306 (3.63), 323 (3.90) nm; ECD (MeOH) 222 (Δ e , 6.81), 241 (Δ e , 1.33), 270 (Δ e , 2.91) nm; IR (KBr) n max 3370, 3257, 2924,2860, 1614, 1510, 1429, 1400, 1287, 1139, 1050, 830 cm -1 HRESIMS m / z 369.18075 [M] + (calcd for C 21 H 25 N2O4 + (369.18088). The nuclear magnetic resonance (NMR) data of compound 13 are shown in Tables 3 and 5.

[0059] Compound 14: colorless oil ; 30 ( c 0.1, MeOH); UV (MeOH) λ max (log e )228 (4.54), 280 (3.94), 295 (3.67) nm; ECD (MeOH) 222 (Δ e , 7.01), 241 (Δ e , 1.98), 270 (Δ e , 3.01) nm; IR (KBr) n max 3384, 3262, 2933, 2858, 1631, 1593,1457, 1368, 1240, 1203, 1145, 1032, 847, 798 cm -1 HRESIMS m / z 325.19007 [M] + (calcd for C 20 H 25 N2O2 +(325.19105). The nuclear magnetic resonance (NMR) data of compound 14 are shown in Tables 3 and 5.

[0060] Table 1. 1H and 1C NMR spectra of compounds 1 and 2 (δ in ppm, J in Hz)

[0061] Table 2 Compound 3 7.1H and 1C NMR spectra (δ in ppm, J in Hz).

[0062] Table 3. Compounds 8 and 11 14 1H and 1C NMR data ( d in ppm, J (in Hz)

[0063] Table 4. 1H and 1C NMR spectra of compounds 9a, 9b, 10a, and 10b ( d in ppm, J (in Hz)

[0064] Table 5 Compound 3 8 and 11 14 1H and 1C NMR data ( d in ppm, J (in Hz)

[0065] Example 2 Compound 1 Evaluation of the synergistic effect of 24-hydroxychloroquine with cisplatin in the fight against ovarian

[0066] I. Compound 1 Evaluation of the synergistic effect of 24 in combination with cisplatin SKOV3 and OVCAR3 cells were cultured in Dulbecco modified Eagle medium (DMEM) containing 20% ​​fetal bovine serum (FBS) at 37°C and 5% CO2. To assess antiproliferative activity, the test compound (10...) was used... m M) or cisplatin (OVCAR3 is 5) m M, SKOV3 is 20 m Cells were treated with M) (10,000 cells / well) for 48 hours. Cell viability was determined by the MTT assay. To assess the sensitizing effect of the compound on cisplatin, cells were simultaneously treated with cisplatin (5 or 20 mg / well). m M) and compound (10 m M), and their inhibition rates are shown in Tables 6 and 7.

[0067] The combination index (CI) is calculated using the following formula:

[0068] The combination index (CI) is used as a quantitative index to classify drug interactions: strong synergy (CI < 0.8), weak synergy (0.8 < CI < 1.0), additive effect (CI = 1.0), and antagonistic effect (CI > 1.0). The results showed that 20 compounds (2 20, 24) showed significant synergistic effects with cisplatin in SKOV3 cells, and 2 compounds (22 23) showed weak synergistic effects with cisplatin in SKOV3 cells; while 11 compounds (2 4, 6 8, 13, 16 19, 21) showed strong synergistic effects in OVCAR3 cells. Among them, the synergistic effects of compounds 2 and 7 were the most significant, with combination indices (CI) of 0.45 and 0.44 respectively in SKOV3 cells, and 0.52 and 0.24 respectively in OVCAR3 cells. As shown in Table 8.

[0069] Table 6 Survival rates (%) of two ovarian cancer cell lines when compound 2 24 and cisplatin were used alone

[0070] Table 7 Inhibitory rates (%) of compound 2 24 combined with cisplatin on the proliferation of two ovarian cancer cell lines

[0071] Table 8 Combination indices of compound 2 24 a and

[0072] II. Evaluation of the cisplatin-sensitizing effect of the optimal compound pair 2 and 7 To further evaluate the cisplatin-sensitizing effects of compounds 2 and 7 in the SKOV3 / OVCAR3 cell lines, we measured the IC 50 values ( Figure 4 ) when each compound was combined with cisplatin. The results showed that compounds 2 and 7 significantly enhanced the effect of cisplatin at a concentration of 10 m M, making its IC 50The values ​​ranged from 6.68. m M decreased to 2.34 m M and 1.88 m M, in the SKOV3 cell line, was 26.11. m M drops to 17.60 m M and 15.95 m M. We used the reversal fold (RF, i.e., the half-maximum inhibitory concentration of cisplatin divided by the compound / cisplatin combination value) as a parameter to evaluate the reversal ability of these compounds. Compounds 2 and 7 (10 m The RF values ​​of M in the OVCAR3 cell line were 2.85 and 3.55, respectively, and in the SKOV3 cell line were 1.48 and 1.64, respectively, indicating that they have a significant synergistic effect with cisplatin.

[0073] After incubation with cisplatin in combination with compounds 2 or 7, the number of cells in S phase increased significantly. Figure 5 This indicates that the combination of compounds 2 and 7 with cisplatin can arrest the cell cycle in the S phase. Simultaneously, the combination of cisplatin and compound 2 significantly increased the apoptosis rate of SKOV3 cells compared to cisplatin or compound 2 alone. Figure 5 This indicates that compounds 2 or 7 can enhance the apoptosis-induced effect of cisplatin in ovarian cancer cells.

[0074] Western blot analysis revealed significant changes in BAX, Bcl-2, CDK1, and Chk1 proteins in the co-treated SKOV3 cells: the expression of the anti-apoptotic protein Bcl-2 was downregulated, the level of the pro-apoptotic protein BAX was increased, and the expression levels of the cell cycle proteins CDK1 and Chk1 were significantly decreased. Figure 6 ) III. Experimental Conclusions Sarpagine alkaloids exhibit broad synergistic effects with cisplatin in the treatment of ovarian cancer. The combination of sarpagine alkaloids (particularly compounds 2 and 7) with cisplatin may promote S-phase cell cycle arrest by inhibiting the expression of key cell cycle regulators CDK1 and Chk1. Simultaneously, the combination therapy shifts the Bcl-2 / BAX ratio towards apoptosis. This synergistic regulation of cell cycle progression and apoptosis signaling pathways highlights the potential of compounds 2 and 7 as cisplatin sensitizers.

[0075] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A class of sarpagine-type alkaloids or pharmaceutically acceptable salts thereof that exhibit synergistic anti-ovarian cancer activity with platinum-based drugs, characterized in that, The structure of the sarpagine-type alkaloid is shown in the following formula: ； R1 is selected from hydrogen or hydroxyl; R2 is selected from hydrogen or hydroxyl; R3 is selected from hydrogen, hydroxyl, or carboxyl groups; R4 is selected from hydrogen or hydroxyl groups; R5 is selected from hydrogen or carbonyl; R6 is selected from hydrogen, hydroxymethyl, and methyl ester. R7 is selected from hydrogen, chlorine, hydroxyl, hydroxymethyl, carboxyl, hydroxy(methoxy)methyl, glucose-1-hydroxymethyl, or azirmonoethyl ethylamine; R8 is selected from methyl, carbonyl, or none.

2. The sarpagine-type alkaloid or its pharmaceutically acceptable salt, as described in claim 1, exhibiting synergistic anti-ovarian cancer activity against platinum-based drugs, characterized in that... Specifically, this includes compounds with structures shown in Formulas 1-14 below, or pharmaceutically acceptable salts thereof: 。 3. The extraction method of sarpagine-type alkaloids with synergistic anti-ovarian cancer activity against platinum-based drugs as described in claim 1 or 2, characterized in that, Includes the following steps: 1) Take the dried rhizome of Rauvolfia pulveratum, crush it, and extract it with alcohol to prepare the total extract; 2) After suspending the total extract in water, extract it several times with chloroform at a pH of 2-3. Then adjust the pH to 8-10 and extract it several times with chloroform again. Combine the dichloromethane extract fractions at pH 8-10 to obtain the total alkaloids. 3) The total alkaloids were separated by normal-phase silica gel chromatography column chromatography, and gradient elution was performed using a mixture of dichloromethane and methanol as the eluent. Sarpagine-type alkaloids were concentrated in the eluent. 4) The eluent containing sarpagine-type alkaloids is concentrated and then separated by reversed-phase column chromatography gradient elution. The eluents of each gradient are then separated by silica gel column chromatography, gel column chromatography and HPLC to obtain a series of sarpagine-type alkaloids.

4. The extraction method of sarpagine-type alkaloids with synergistic anti-ovarian cancer activity against platinum-based drugs according to claim 3, characterized in that, In step 1), 80% methanol was used for extraction at 40℃, and the ratio of dried rhizomes of Rauvolfia purpurea to 80% methanol was 1 kg: (2-4) L; the extraction was performed 4 times, each time for 2 days, and the solvent was removed by rotary evaporation of the combined alcohol extracts to obtain the total extract.

5. The extraction method of sarpagine-type alkaloids with synergistic anti-ovarian cancer activity against platinum-based drugs according to claim 3, characterized in that, In step 2), the total extract is suspended in warm water, the pH is adjusted to 2 with 2% hydrochloric acid, and then extracted with chloroform 5 times. Subsequently, the pH is adjusted to 9 with ammonia water, and then extracted with chloroform 5 times again. The chloroform extract fraction with a combined pH of 9 is the total alkaloid.

6. The extraction method of sarpagine-type alkaloids with synergistic anti-ovarian cancer activity against platinum-based drugs according to claim 3, characterized in that, In step 3), during normal-phase silica gel chromatography, the elution ratio of dichloromethane and methanol in the eluent changes from 30:1, 20:1, 15:1, 10:1, 5:1 to the final 2:1, and sarpagine alkaloids are concentrated in the 10:1 eluent.

7. The extraction method of sarpagine-type alkaloids with synergistic anti-ovarian cancer activity against platinum-based drugs according to claim 3, characterized in that, In step 4), the chromatographic conditions for reversed-phase column chromatography separation are as follows: The chromatographic packing material was C18, and the eluent was a mixture of methanol and water. The initial elution ratio was 10% methanol, and the final ratio was 100% methanol. The methanol ratio was increased by 10% for each intermediate gradient, and elution was performed at 12 L for each gradient.

8. The use of a class of sarpagine-type alkaloids or pharmaceutically acceptable salts thereof that synergize with platinum-based drugs against ovarian cancer in the preparation of platinum-based drug sensitizers, characterized in that, The sarpagine-type alkaloids mentioned above include compounds with the following structures: 。 9. The application as described in claim 8, characterized in that, The platinum-based drug in question is cisplatin.

10. The use of a sarpagine-type alkaloid or a pharmaceutically acceptable salt thereof, which exhibits synergistic anti-ovarian cancer activity with platinum-based drugs, in the preparation of a medicament for treating ovarian cancer, characterized in that... The sarpagine-type alkaloids mentioned above include compounds with the following structures: 。

Images