Cucurbitacin compound 11-hydrocucurbitacin B, pharmaceutical composition thereof and application

Abstract

Provide white sandalwood [ Aquilaria sinensis [Lour.)Spreng.] Compound 11-hydrocucurbitacin B (PGC34A) from stem bark, its preparation method, pharmaceutical composition, and applications. PGC34A exhibits significant inhibitory activity against human gastric cancer AGS, human gastric cancer HGC-27, human colon cancer SW480, human lung cancer A549, and the paclitaxel-resistant human lung cancer strain A549 / Taxol, with activity superior to cisplatin. Furthermore, PGC34A shows low toxicity (IC50) against the normal human lung epithelial cell line BEAS-2B. 50 >40μM). Cucurbitacin compound PGC34A and its pharmaceutical compositions represent a new option for cancer treatment.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to cucurbitacin compounds in the bark of Aquilaria sinensis stems, their pharmaceutical compositions, their preparation methods, and their application in the preparation of drugs for treating cancer. Background Technology

[0002] Aquilaria sinensis (Lour.) Spreng. is a plant belonging to the genus Aquilaria in the family Thymelaeaceae. Its resinous heartwood is the traditional Chinese medicine agarwood. The bark of Aquilaria sinensis is a Yi medicine, known as Liwazi in Yi ethnicity, and is used to treat abdominal pain, bloating, chronic gastritis, and other diseases [Edited by the Drug Inspection Institute of the Health Bureau of Chuxiong Yi Autonomous Prefecture, Yunnan Province. Records of Yi Medicine. Sichuan Nationalities Publishing House. Chengdu: 1983, 173-174]. Previous reports indicate that the main chemical components of the stem bark of Aquilaria sinensis are flavonoids, 2-(2-phenylethyl)chromene, steroids, and phenylpropanoids, etc. [Chen CT, Yeh YT, Chao D, Chen CY. Chemical constituents from the bark of Aquilaria sinensis. Chemistry of Natural Compounds, 2013, 48: 1074-1075; Wang SL, Hwang TL, Chung MI, Sung PJ, Shu CW, Cheng MJ, Chen JJ. Newflavones, a 2-(2-phenylethyl)-4H-chromen-4-one derivative, and anti-inflammatory constituents from the stem barks of Aquilaria sinensis. Molecules, 2015, 20: 20912-20925; Tsai YC, Wang SL, Wu MY, Liao CH, LinC-H, Chen JJ, Fu SL. Pilloin, a flavonoid isolated from Aquilaria sinensis, exhibits anti-inflammatory activity in vitro and in vivo. Molecules, 2018, 23:3177]. Summary of the Invention

[0003] To address the aforementioned problems in the prior art, this invention provides 11-hydrocucurbitacin B from the stem bark of Aquilaria sinensis, its pharmaceutical composition, a method for its preparation, and its application in the preparation of drugs for treating cancer.

[0004] To achieve the above-mentioned objectives of the present invention, the present invention provides the following technical solution:

[0005] The compound 11-hydrocucurbitacin B (PGC34A) is shown in the following structural formula.

[0006]

[0007] The method for preparing the compound 11-hydrocucurbitacin B (PGC34A) includes the following steps:

[0008] The dried bark of Aquilaria sinensis was pulverized and extracted three times with 90% ethanol at 60℃. The first extraction used 80L of solvent and was carried out for 2 hours; the second extraction used 80L of solvent and was carried out for 1 hour; the third extraction used 60L of solvent and was carried out for 1 hour. The extracts were combined and evaporated to dryness by reflux at 60℃ using a rotary evaporator to remove the solvent, yielding a crude extract. The crude extract was separated by column chromatography using 80-100 mesh silica gel, eluted with dichloromethane, ethyl acetate, acetone, and methanol, respectively. After removing the solvent from the eluent, the dichloromethane eluent, ethyl acetate eluent, acetone eluent, and methanol eluent were obtained.

[0009] The ethyl acetate eluent was separated by column chromatography using 80-100 mesh silica gel. A gradient elution of petroleum ether-ethyl acetate (10:1 → 0:1) was performed to obtain seven fractions B1–B7. The eluent from the petroleum ether-ethyl acetate (1:1) elution was analyzed by thin-layer chromatography and was combined into two fractions, B-5.1 and B-5.2.

[0010] B-5.2 was recrystallized from methanol to obtain cucurbitacin Q1. The mother liquor after B-5.2 crystallization was analyzed using ODS-C. 18 Ten fractions, B-5.2.1 to B-5.2.10, were obtained by reverse-phase column chromatography with methanol:water, 10% → 100%. Fraction B-5.2.5 was further purified by Sephadex LH-20 gel column chromatography with methanol elution to yield another ten fractions, B-5.2.5.1 to B-5.2.5.10. Fraction B-5.2.5.3 was purified by high-performance liquid chromatography with acetonitrile:water = 36:64 to obtain two mixtures, P1 and P2. P2 was purified by HPLC with acetonitrile:water = 34:66 to obtain 11-hydrocucurbitacin B.

[0011] The application of the compound 11-hydrocucurbitacin B (PGC34A) in the preparation of anticancer drugs.

[0012] The application of the compound 11-hydrocucurbitacin B (PGC34A) in the preparation of drugs for treating gastric cancer, leukemia, breast cancer, colon cancer, and lung cancer.

[0013] A pharmaceutical composition comprising the compound 11-hydrocucurbitacin B (PGC34A) and a pharmaceutically acceptable carrier.

[0014] The method for preparing the pharmaceutical composition involves first preparing 11-hydrocucurbitacin B, and then adding a pharmaceutically acceptable carrier.

[0015] The application of the pharmaceutical composition in the preparation of drugs for treating gastric cancer, leukemia, breast cancer, colon cancer, and lung cancer.

[0016] When the cucurbitacin compound 11-hydrocucurbitacin B (PGC34A) is used to prepare an anti-liver cancer drug, the present invention preferably uses the compound directly or in the form of a pharmaceutical composition.

[0017] The pharmaceutical composition provided by this invention comprises the aforementioned compound 11-hydrocucurbitacin B (PGC34A) and a pharmaceutically acceptable carrier. In this invention, the pharmaceutically acceptable carrier is preferably a solid, semi-solid, or liquid diluent, filler, or pharmaceutical excipient. This invention does not impose any particular limitation on the pharmaceutically acceptable carrier; any pharmaceutically acceptable carrier well-known in the art that is non-toxic and inert to humans and animals may be selected.

[0018] The present invention does not impose any particular limitation on the preparation method of the pharmaceutical composition. At least one of the compounds can be directly mixed with a pharmaceutically acceptable carrier. The present invention does not impose any particular limitation on the mixing process. Any process well known in the art that can obtain the pharmaceutical composition can be selected.

[0019] This invention provides the application of the pharmaceutical composition described in the above technical solution in the preparation of anticancer drugs. There are no special limitations on the method of application, and any method well known in the art can be used.

[0020] In this invention, when the pharmaceutical composition is used to prepare an anticancer drug, the content of the composition in the drug is preferably 0.1% to 99%; in the pharmaceutical composition, the content of compound 11-hydrocucurbitacin B (PGC34A) in the pharmaceutical composition is preferably 0.5% to 90%. The pharmaceutical composition of this invention is preferably used in the form of a dose per unit body weight. In this invention, the prepared drug is preferably administered by both injection (intravenous injection, intramuscular injection) and oral administration.

[0021] Compared with the prior art, the present invention has the following advantages.

[0022] 1. This invention provides a novel compound, 11-hydrocucurbitacin (BPGC34A), extracted from the bark of Aquilaria sinensis stems, and a pharmaceutical composition thereof. It also provides the application of compound 11-hydrocucurbitacin B (PGC34A) in the preparation of medicaments for treating cancer.

[0023] 2. The novel compound 11-hydrocucurbitacin (BPGC34A) exhibits superior inhibitory activity against human gastric cancer AGS, human gastric cancer HGC-27, human colon cancer SW480, human lung cancer A549, and human lung cancer paclitaxel-resistant strain A549 / Taxol compared to cisplatin.

[0024] 3. The novel compound 11-hydrocucurbitacin B (PGC34A) exhibits low toxicity (IC50) to normal human lung epithelial BEAS-2B cell line. 50 >40μM).

[0025] 4. The raw materials for preparing the compound 11-hydrocucurbitacin B (PGC34A) of the present invention are widely available and inexpensive. Attached Figure Description

[0026] Figure 1 Chemical structural formulas of compounds 11-hydrocucurbitacin B (PGC34A), PGC28B, PGC7, PGC27, PGC28C and PGC29 in the stem bark of Aquilaria sinensis;

[0027] Figure 2 Key two-dimensional nuclear magnetic resonance correlations of compound 11-hydrocucurbitacin B (PGC34A);

[0028] Figure 3 Experimental and computational electronic circular dichroism spectroscopy of compound 11-hydrocucurbitacin B (PGC34A);

[0029] Figure 4 A schematic diagram of the chemical structure of compound 11-hydrocucurbitacin B (PGC34A). Detailed Implementation

[0030] The following description, in conjunction with the accompanying drawings, uses embodiments of the present invention to further illustrate the substantive content of the invention, but does not limit the invention thereto.

[0031] Example 1

[0032] Cucurbitacin compounds in the bark of Aquilaria sinensis stem (structural formula shown) Figure 1 Preparation of )

[0033] 24 kg of dried Aquilaria sinensis (Lour.) Spreng. stem bark was pulverized and extracted three times with 90% ethanol at 60 °C. The first extraction used 80 L of solvent for 2 hours; the second used 80 L of solvent for 1 hour; and the third used 60 L of solvent for 1 hour. The extracts were combined and refluxed at 60 °C to remove the solvent, yielding 2.6 kg of crude extract. The crude extract was separated by column chromatography on 80–100 mesh silica gel, eluted with dichloromethane, ethyl acetate, acetone, and methanol, respectively. After removing the solvent from the eluent, the following fractions were obtained: dichloromethane eluent (A, 168.9 g), ethyl acetate eluent (B, 256.1 g), acetone eluent (C, 243.8 g), and methanol eluent (D, 1450.0 g).

[0034] The ethyl acetate eluent (B) was separated by column chromatography using 80-100 mesh silica gel, followed by gradient elution with petroleum ether-ethyl acetate (10:1→0:1) to obtain seven fractions (B1–B7). The eluent obtained from the petroleum ether-ethyl acetate (1:1) elution was analyzed by thin-layer chromatography (TLC) and combined into two fractions: B-5.1 (16.3 g) and B-5.2 (18.8 g).

[0035] B-5.2 was recrystallized from methanol to obtain cucurbitacin Q1 (PGC7, 435.8 mg). The mother liquor after crystallization of B-5.2 was analyzed using ODS-C. 18 Ten fractions, B-5.2.1 to B-5.2.10, were obtained by reversed-phase column chromatography (methanol:water, 10%→100%). Fractions B-5.2.5 (eluted with methanol:water = 50%:50% and methanol:water = 60%:40%) were obtained by Sephadex LH-20 gel column chromatography, eluted with methanol, yielding ten fractions, B-5.2.5.1 to B-5.2.5.10. Fraction B-5.2.5.3 (178.5 mg) was purified by high-performance liquid chromatography (HPLC; Agilent ZORBAX RX-C8 column, 5.0 μm, φ9.4×250 mm; v = 2 mL / min, acetonitrile:water = 36:64) to obtain cucurbitacin D (PGC27, 3.2 mg; t...). R =18.866min) and gentian violetin (PGC29, 8.5mg; t R =32.925min), and two mixtures P1 (3.8mg, t R =22.614min) and P2 (2.8mg, t R=40.871 min). P1 was purified by HPLC (Chiral CD-Ph column, 5.0 μm, φ4.6×250 mm; v = 1 mL / min, methanol:water = 58:42) to obtain 3-epicocucurbitacin Q1 (PGC28B, 1.1 mg; t R =14.017min) and isocucurbitacin D (PGC28C, 1.2mg; t R =16.142 min). P2 was purified by HPLC (Chiral CD-Ph column, 5.0 μm, φ4.6×250 mm; v = 1 mL / min, acetonitrile:water = 34:66) to obtain 11-hydrocucurbitacin B (PGC34A, 1.4 mg; t = 16.142 min). R =24.100min).

[0036] Example 2

[0037] Spectroscopic data and structural analysis of the new compound 11-hydrocucurbitacin B (PGC34A):

[0038] Based on the high-resolution mass spectrometry data of compound PGC34A, namely HRESIMS m / z 595.3041 [M+Cl]-(calcd.for C 32 H 48 ClO8,595.3043), and combined with 13 Based on the C10 NMR data (Table 1), the molecular formula of PGC34A is deduced to be C10. 32 H 48 O8 has 9 degrees of unsaturation. Tested in deuterated pyridine. 1 1H NMR spectrum (Table 1) shows that the compound contains 9 methyl [δ] groups. H 1.89(3H,s), 1.81(3H,s), 1.78(3H,s), 1.54(3H,s), 1.50(3H,s), 1.47(3H,s), 1.43(3H,s), 1.26(3H,s), 1.20(3H,s)], a trisubstituted double bond [δ H 5.67(1H,m)], and a disubstituted double bond with an unknown configuration [δ H 7.39 (2H, br s)]. By examining the NMR spectral data tested in deuterated methanol (Table 1), it was found that the configuration of this disubstituted double bond is trans [δ]. H 6.98 (1H,d,J = 16.0 Hz) and 6.84 (1H,d,J = 16.0 Hz)]. Tested in pyridine. 13The 12C NMR spectrum (Table 1) shows that the compound has 32 carbon signals, including signals from 3 carbonyl groups (δ¹⁺). C 213.7, 204.6, 169.6), signals of two double bonds (δ) C 149.3, 142.7, 122.6, 120.5), 9 methyl signals (δ C (28.9, 26.4, 26.0, 25.2, 22.4, 21.7, 21.5, 21.1, 18.5), 4 methylene signals, 6 sp signals 3 Hybridized methine signal (with 3 oxygen atoms: δ C 72.7, 71.5, and 71.1), and 6 quaternary carbon signals (2 of which are oxygen-related: δ C (80.2 and 79.7). The above signals indicate that this compound may be a cucurbitane-type triterpenoid. Careful comparison of the NMR data of this compound with that of cucurbitacin B [Ryu SY, Lee SH, Choi SU, Lee CO, No Z, Ahn JW. Antitumor activity of Trichosanthes kirilowii. Archives of Pharmacal Research, 1994, 17:348–353] revealed that PGC34A has one more oxymethylene signal and one less carbonyl signal than cucurbitacin B.

[0039] according to 1 H– 1 H COSY related ( Figure 2 From this, four connection segments can be deduced: H-10 / H2-1 / H-2, H-6 / H2-7 / H-8, H-11 / H2-12, and H2-15 / H-16 / H-17. (In HMBC related...) Figure 2In the diagram, we can see that H2-1, H3-28, and H3-29 are associated with C-3 and C-5, H-6 with C-4, H3-19 with C-8, C-9, C-10, and C-11, H3-30 with C-8, C-13, C-14, and C-15, and H3-18 with C-12, C-14, and C-17. Based on these associations, the tetracyclic skeleton (A–D rings) of cucurbitane triterpenes can be established. In the HMBC correlation, it can also be seen that H-16 is associated with C-20, H3-21 with C-17, C-20, and C-22, H-23 with C-25, H-24 with C-22, and H3-26 and H3-27 with C-24 and C-25. Based on these correlations, the side chain of the cucurbitane triterpene can be determined, and it is speculated that it is attached to C-17. According to the literature, if C-25 has an acetoxy group, its chemical shift is approximately 79 ppm; if C-25 is attached to a hydroxyl group, its chemical shift is approximately 71 ppm [Yang J, HuDB, Xia MY, Luo JF, Li XY, Wang YH. Bioassay-guided isolation of cytotoxic constituents from the flowers of Aquilaria sinensis. Natural Products and Bioprospecting, 2022, 12:11]. PGC34A 13 In the C10 NMR, the chemical shift at C-25 is 79.7 ppm, therefore it is speculated that the remaining acetoxy fragment should be attached to C-25. Thus, the planar structure of PGC34A is determined to be 25-acetoxy-3,11,16,20-tetrahydroxy-9-methyl-19-norlanster-5,23-diene-3,22-dione.

[0040] The relative configuration of PGC34A is determined by ROESY correlation ( Figure 2The configuration of PGC34A was determined by electron circular dichroism (ECD). The ROESY correlations of hydrogens H-1β / H3-19, H-8 / H3-19, H-8 / H3-18, and H-16 / H3-18 indicate that these hydrogens are β-oriented; while the correlations of hydrogens H-2 / H-10, H-10 / H-11, H-10 / H3-30, and H-17 / H3-30 indicate that these hydrogens are α-oriented. In other words, 2-OH and 11-OH are β-oriented, while 16-OH is α-oriented. Due to the rotational effect of the C-C single bond, the ROESY correlation cannot determine the configuration of C-20. Finally, the absolute configuration of PGC34A was determined using electron circular dichroism (ECD) calculations. The ECD spectra of the (2S,8S,9R,10R,11S,13R,14S,16R,17R,20R)- isomers and (2S,8S,9R,10R,11S,13R,14S,16R,17R,20S)- isomers were calculated. Figure 3 It was found that the ECD spectrum calculated for the (2S,8S,9R,10R,11S,13R,14S,16R,17R,20R)-isomer was similar to the experimental ECD spectrum because the absolute configuration of PGC34A was determined to be (3β,11β,16α,20R,23E)-25-acetoxy-3,11,16,20-tetrahydroxy-9-methyl-19-norlanster-5,23-diene-3,22-dione, i.e., 11-hydrocucurbitacin B.

[0041] Spectral data of 11-hydrocucurbitacin B:

[0042] 11-Hydrocucurbitacin B (PGC34A): White powder; UV(MeOH)λ max (logε)277(2.30),219(3.07),203(3.29)nm; ECD(c,0.086,MeOH)λ max (Δε)294(-0.31)nm; 1 H and 13 C10 NMR data are shown in Table 1; ESIMS m / z 583 [M+Na] + ;HRESIMS m / z595.3041[M+Cl]-(calcd for C 32 H 48 ClO8, 595.3043).

[0043] Table 1. Compound PGC34A 1 H(800MHz) and 13C NMR (201MHz) data (δin ppm, J in Hz)

[0044]

[0045] a Detected by HMBC.

[0046] Example 3:

[0047] Spectral data of compounds PGC28B, PGC7, PGC27, PGC28C and PGC29.

[0048] Table 2 PGC28B and PGC7 1 H and 13 C NMR data (δin ppm, J in Hz).

[0049]

[0050]

[0051] a Detected by HMBC.

[0052] 3-Epicucurbitacin Q1 (PGC28B): White powder, molecular formula C 32 H 48 O8, CAS No. 343567-22-6; ECD(c,0.072,MeOH)λ max (Δε)297(+1.80),223(-0.31),202(+7.34)nm; 1 H and 13 C10 NMR data, see Table 2; ESIMS m / z 583 [M+Na] + ;HRESIMS m / z605.3330[M+HCOO]-(calcd for C 33 H 49 O 103-Epicocucurbitacin Q1 is mentioned in the literature, but no NMR data are available [Dinan L, Bourne PC, Meng Y, Sarker SD, Tolentino RB, Whiting P. Assessment of natural products in the Drosophila melanogaster BII cellbioassay for ecdysteroid agonist and antagonist activities. Cellular and Molecular Life Sciences, 2001, 58, 321-342]. Two-dimensional NMR correlation confirmed that the structure of PGC28B is indeed 3-epicocucurbitacin Q1, and further analysis was conducted on... 1 H and 13 The C NMR data were assigned (Table 2).

[0053] Cucurbitacin Q1 (PGC7): Colorless needle crystals (methanol), mp. 232–233℃, molecular formula C 32 H 48 O8, CAS No. 99530-82-2; 1 H and 13 C10 NMR data are shown in Table 2; ESIMS m / z 583 [M+Na] + The NMR data for cucurbitacins Q1 are incomplete in the literature [Afifi MS, Ross SA, Elsohly MA, Naeem ZE, Halaweish FT. Cucurbitacins of Cucumis prophetarum and Cucumis prophetarum. Journal of Chemical Ecology, 1999, 25: 847-859]. Through two-dimensional NMR correlation, the structure of PGC7 was identified as cucurbitacins Q1, and its NMR data were fully assigned (Table 2).

[0054] Cucurbitacin D (PGC27): White powder, molecular formula C 30 H 44 O7, CAS No. 3877-86-9; 1 H NMR (800MHz, CDCl3): δ H7.12(1H,d,J=15.2Hz,H-24),6.67(1H,d,J=15.1Hz,H-23),5.79(1H,m,H-6),4.44(1H, dd,J=12.8,5.8Hz,H-2),3.27(1H,d,J=14.6Hz,H-12α),2.70(1H,d,J=14.6Hz,H-12β),2 .56(1H,d,J=6.9Hz,H-17),1.42(3H,s,H3-21),1.38(6H,s,H3-26,H3-27),1.35(3H,s,H 3-29),1.34(3H,s,H3-30),1.29(3H,s,H3-28),1.08(3H,s,H3-19),0.99(3H,s,H3-18); 13 C NMR (201MHz, CDCl3): δ C 213.1(C-11,C),212.2(C-3,C),202.9(C-22,C),155.6(C-24,CH),140.5(C-5,C),120.3(C-6,CH),119.0(C-23,CH),78.1(C-2 0,C),71.6(C-2,CH),71.4(C-16,CH),71.3(C-25,C),57.5(C-17,CH),50.8(C-14,C),50.3(C-4,C),48.6(C-12,CH2),48.4(C- 9,C),48.2(C-13,C),45.4(C-15,CH2),42.4(C-8,CH),36.0(C-1,CH2),33.8(C-10,CH),29.5(C-27,CH3),29.3(C-29,CH3),29 .2(C-26,CH3),24.5(C-7,CH2),23.9(C-21,CH3),21.2(C-28,CH3),20.1(C-30,CH3),19.9(C-18,CH3),19.1(C-19,CH3); ESIMS m / z 539[M+Na] + The above NMR data are basically consistent with the data in the literature [Ryu SY, Lee SH, Choi SU, Lee CO, No Z, Ahn JW. Antitumor activity of Trichosanthes kirilowii. Archives of Pharmacal Research, 1994, 17: 348-353]. Therefore, PGC27 is identified as cucurbitacin D.

[0055] Isocucurbitacin D (PGC28C): White powder, molecular formula C 30 H 44 O7, CAS No. 68422-20-8; 1 H NMR (800MHz, CDCl3): δ H 7.10(1H,d,J=15.2Hz,H-24),6.65(1H,d,J=15.2,Hz,H-23),5.94(1H,m ,H-6),3.12(1H,d,J=14.6Hz,H-12α),2.65(1H,d,J=14.6Hz,H-12β),1.4 0(3H,s,H3-21),1.38(6H,s,H3-26,H3-27),1.33(3H,s,H3-30),1.25(3H ,s,H3-29),1.18(3H,s,H3-28),0.97(3H,s,H3-19),0.81(3H,s,H3-18); 13 C NMR (201MHz, CDCl3): δ C 211.9(C-11,C),210.6(C-2,C),203.0(C-22,C),155.6(C-24,CH),138.2(C-5,C),121.8(C-6,CH),119.0(C-23,CH),80.2(C-3 ,CH),78.1(C-20,C),71.3(C-16,CH),71.2(C-25,C),57.5(C-17,CH),50.8(C-14,C),48.5(C-12,CH2),48.3(C-9,C),48.1(C- 13,C),46.7(C-4,C),45.3(C-15,CH2),42.7(C-8,CH),38.8(C-1,CH2),36.3(C-10,CH),29.5(C-27,CH3),29.4(C-26,CH3),24 .1(C-29,CH3),23.9(C-7,CH2),23.8(C-21,CH3),20.9(C-28,CH3),20.1(C-18,CH3),19.9(C-30,CH3),18.9(C-19,CH3); ESIMS m / z 539[M+Na] +The above NMR data are basically consistent with the data in the literature [Ryu SY, Lee SH, Choi SU, Lee CO, No Z, Ahn JW. Antitumor activity of Trichosanthes kirilowii. Archives of Pharmacal Research, 1994, 17:348-353]. Therefore, PGC28C is identified as isocucurbitacin D.

[0056] Hemslecin A (PGC29): Colorless needle crystals (methanol), mp. 229–230℃, molecular formula C 32 H 50 O8, CAS No. 58546-34-2; 1 H NMR (500MHz, CDCl3): δ H 5.73(1H,m,H-6),1.96(3H,s,CH3CO),1.46(3H,s.H3-26),1.43(3H,s,H3-27),1.40(3H,s,H3-21),1 .28(3H,s,H3-30),1.20(3H,s,H3-29),1.10(3H,s,H3-19),0.96(3H,s,H3-28),0.95(3H,s,H3-18); 13 CNMR (126MHz, CDCl3): δ C214.0(C-22,C),212.8(C-11,C),170.4(CH3CO),140.7(C-5,C),119.3(C-6,CH),81.3(C-25,C),81.0(C-3,CH),78.9(C-20,C),71. 10(C-16,CH),71.07(C-2,CH),57.8(C-17,CH),50.7(C-14,C),48.7(C-12,CH2),48.3(C-9,C),45.5(C-15,CH2),42.6(C-8,CH),41. 9(C-4,C),34.8(C-24,CH2),33.9(C-10,CH),33.3(C-1,CH2),30.7(C-23,CH2),26.2(C-26,CH3),25.9(C-27,CH3),24.7(C-21,CH3 ),24.5(C-29,CH3),23.8(C-7,CH2),22.4(CH3CO),21.5(C-28,CH3),20.3(C-18,CH3),19.8(C-30,CH3),18.9(C-19,CH3).ESIMSm / z 585[M+Na] + The above NMR data are basically consistent with the data in the literature [Reguero MT, Mata R, Bye R, Linares E, Delgado G. Chemical studies on Mexican plants used in traditional medicine, II: cucurbitacins from Hintonia latiflora. Journal of Natural Products, 1987, 50: 315-316; Li Zuqiang, Luo Lei, Ling Min. Anticancer active ingredients of bitter melon. Chinese Herbal Medicines, 1999, 30: 409-411]. Therefore, PGC29 was identified as cucurbitacins.

[0057] Example 4:

[0058] Inhibitory effects of PGC34A, PGC28B, PGC7, PGC27, PGC28C and PGC29 on cancer cell growth (Table 3).

[0059] Cytotoxic activity was tested using the MTS method [Yang J, Su Y, Luo JF, Gu W, Niu HM, Li Y, Wang YH, Long CL. New amide alkaloids from Piper longum fruits. Natural Products and Bioprospecting, 2013, 3, 277-281], with cisplatin and paclitaxel as positive controls. The results of the cytotoxic activity test are shown in Table 3. The results indicated that PGC34A, PGC28B, PGC7, PGC27, PGC28C, and PGC29 exhibited varying degrees of inhibitory effects on human gastric cancer AGS, human gastric cancer HGC-27, human leukemia HL-60, human breast cancer MDA-MB-231, human colon cancer SW480, human lung cancer A549, and the paclitaxel-resistant human lung cancer strain A549 / Taxol. Among them, the new compound PGC34A showed superior inhibitory activity against human gastric cancer AGS, human gastric cancer HGC-27, human colon cancer SW480, human lung cancer A549, and the paclitaxel-resistant human lung cancer strain A549 / Taxol compared to cisplatin, and exhibited lower toxicity (IC50) against the normal human lung epithelial cell line BEAS-2B. 50 >40μM).

[0060]

[0061] Formulation Examples

[0062] In the following formulation examples, conventional reagents were selected and the formulations were prepared according to existing conventional methods. This application example only demonstrates that at least one of the compounds PGC34A, PGC28B, PGC7, PGC27, PGC28C, and PGC29 described in this invention can be prepared into different formulations, and no specific limitations are made on the specific reagents and operations:

[0063] 1. Dissolve at least one of the compounds PGC34A, PGC28B, PGC7, PGC27, PGC28C and PGC29 of the present invention in a small amount of DMSO, add water for injection according to conventional methods, filter, fill and sterilize to prepare an injection solution, wherein the concentration of the injection solution is 0.5-5 mg / mL.

[0064] 2. Dissolve at least one of the compounds PGC34A, PGC28B, PGC7, PGC27, PGC28C and PGC29 of the present invention in a small amount of DMSO, then dissolve it in sterile water for injection, stir to dissolve, filter with a sterile suction funnel, then filter aseptically, dispense into ampoules, freeze-dry at low temperature and then seal aseptically to obtain a powder for injection.

[0065] 3. At least one of the compounds PGC34A, PGC28B, PGC7, PGC27, PGC28C and PGC29 of the present invention is added to an excipient at a weight ratio of 9:1 to prepare a powder.

[0066] 4. At least one of the compounds PGC34A, PGC28B, PGC7, PGC27, PGC28C and PGC29 of the present invention is added to the excipient at a weight ratio of 5:1, and then granulated and tableted.

[0067] 5. Prepare an oral liquid from at least one of the compounds PGC34A, PGC28B, PGC7, PGC27, PGC28C and PGC29 of the present invention using conventional oral liquid preparation methods.

[0068] 6. At least one of the compounds PGC34A, PGC28B, PGC7, PGC27, PGC28C and PGC29 of the present invention is added to an excipient at a weight ratio of 5:1 to the excipient to form a capsule.

[0069] 7. At least one of the compounds PGC34A, PGC28B, PGC7, PGC27, PGC28C and PGC29 of the present invention is added to an excipient at a weight ratio of 5:1 to the excipient to prepare granules.

[0070] As can be seen from the above embodiments, the present invention provides a class of PGC34A, PGC28B, PGC7, PGC27, PGC28C, and PGC29, their preparation methods and applications, as well as pharmaceutical compositions and their applications. The compounds provided by the present invention exhibit varying degrees of inhibitory effects on cancer cells, can be combined with pharmaceutically acceptable carriers to form pharmaceutical compositions, and can be used to prepare anticancer drugs.

[0071] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The compound 11-hydrocucurbitacin B, as shown in the following structural formula, 。 2. The method for preparing compound 11-hydrocucurbitacin B according to claim 1, characterized in that... The method includes the following steps: The dried bark of Aquilaria sinensis was pulverized and extracted three times with 90% ethanol at 60 °C. The first extraction used 80 L of solvent and was carried out for 2 hours; the second extraction used 80 L of solvent and was carried out for 1 hour; the third extraction used 60 L of solvent and was carried out for 1 hour. The extracts were combined and evaporated to dryness under reflux at 60 °C using a rotary evaporator to remove the solvent, yielding a crude extract. The crude extract was separated by column chromatography using 80-100 mesh silica gel, eluted with dichloromethane, ethyl acetate, acetone, and methanol, respectively. After removing the solvent from the eluent, the dichloromethane eluent, ethyl acetate eluent, acetone eluent, and methanol eluent were obtained. The ethyl acetate eluent was separated by column chromatography using 80-100 mesh silica gel. A gradient elution of petroleum ether-ethyl acetate (10:1 → 0:1) was performed to obtain seven fractions B1–B7. The eluent from the petroleum ether-ethyl acetate (1:1) elution was analyzed by thin-layer chromatography and was combined into two fractions, B-5.1 and B-5.

2. B-5.2 was recrystallized from methanol to obtain cucurbitacin Q1. The mother liquor after B-5.2 crystallization was analyzed using ODS-C. 18 Ten fractions, B-5.2.1 to B-5.2.10, were obtained by reverse-phase column chromatography with methanol:water, 10%→100%. Fraction B-5.2.5 was purified by Sephadex LH-20 gel column chromatography with methanol elution to obtain ten fractions, B-5.2.5.1 to B-5.2.5.

10. Fraction B-5.2.5.3 was purified by high-performance liquid chromatography with acetonitrile:water = 36:64 to obtain two mixtures, P1 and P2. P2 was purified by HPLC with acetonitrile:water = 34:66 to obtain 11-hydrocucurbitacin B.

3. The use of compound 11-hydrocucurbitacin B according to claim 1 in the preparation of drugs for treating gastric cancer, leukemia, colon cancer, and lung cancer.

4. A pharmaceutical composition comprising compound 11-hydrocucurbitacin B as described in claim 1 and a pharmaceutically acceptable carrier.

5. The use of the pharmaceutical composition according to claim 4 in the preparation of a medicament for treating gastric cancer, leukemia, colon cancer, and lung cancer.

6. The method for preparing the pharmaceutical composition according to claim 4, characterized in that... The method includes the following steps: The dried bark of Aquilaria sinensis was pulverized and extracted three times with 90% ethanol at 60 °C. The first extraction used 80 L of solvent and was carried out for 2 hours; the second extraction used 80 L of solvent and was carried out for 1 hour; the third extraction used 60 L of solvent and was carried out for 1 hour. The extracts were combined and evaporated to dryness under reflux at 60 °C using a rotary evaporator to remove the solvent, yielding a crude extract. The crude extract was separated by column chromatography using 80-100 mesh silica gel, eluted with dichloromethane, ethyl acetate, acetone, and methanol, respectively. After removing the solvent from the eluent, the dichloromethane eluent, ethyl acetate eluent, acetone eluent, and methanol eluent were obtained. The ethyl acetate eluent was separated by column chromatography using 80-100 mesh silica gel. A gradient elution of petroleum ether-ethyl acetate (10:1 → 0:1) was performed to obtain seven fractions B1–B7. The eluent from the petroleum ether-ethyl acetate (1:1) elution was analyzed by thin-layer chromatography and was combined into two fractions, B-5.1 and B-5.

2. B-5.2 was recrystallized from methanol to obtain cucurbitacin Q1. The mother liquor after B-5.2 crystallization was analyzed using ODS-C. 18 Ten fractions, B-5.2.1 to B-5.2.10, were obtained by reverse-phase column chromatography with methanol:water, 10%→100%. Fraction B-5.2.5 was further purified by Sephadex LH-20 gel column chromatography with methanol elution to yield another ten fractions, B-5.2.5.1 to B-5.2.5.

10. Fraction B-5.2.5.3 was purified by high-performance liquid chromatography with acetonitrile:water = 36:64 to obtain two mixtures, P1 and P2. P2 was purified by HPLC with acetonitrile:water = 34:66 to obtain 11-hydrocucurbitacin B, which was then added to a pharmaceutically acceptable carrier.

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