Eucalyptane type sesquiterpene lactone TBA and TBB nitrogen mustard derivative and application thereof

Abstract

The invention discloses an eucalyptol type sesquiterpene lactone TBA and TBB nitrogen mustard derivative as well as a preparation method and application thereof. The structural formulas of the eucalyptol type sesquiterpene lactone TBA and TBB nitrogen mustard derivative are respectively shown as a formula TBA-DJ-1-3 and a formula TBB-DJ-1-3. Cytotoxicity tests show that the nitrogen mustard derivatives can inhibit the proliferation activity of liver cancer cells, particularly, the proliferation inhibition effect of the compound TBA-DJ-2 on HepG2 cancer cells is remarkably better than that of a parent body, and the nitrogen mustard derivatives can be used for clinically preparing anti-liver cancer drugs.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, specifically to a eucalyptane-type sesquiterpene lactone derivative, TBA and TBB nitrogen mustard, and their applications. Background Technology

[0002] Cancer remains a major and persistent global health challenge, causing millions of deaths annually. Hepatocellular carcinoma (HCC), the leading histological type of primary liver cancer, accounts for approximately 90% of non-metastatic liver cancer cases, imposing a heavy clinical, economic, and psychological burden on patients. Therefore, developing more effective treatments for HCC remains a critical and urgent issue. Sesquiterpene lactones (SLs) are a class of highly bioactive natural products widely found in various plants, and their outstanding anticancer activity has attracted considerable attention from researchers.

[0003] The applicant has isolated a pair of eucalyptane-type sesquiterpene lactone epimers from *Wedelia trichotomum* (application number: CN202010069334.X), wherein compound 1 is (1S,4S,5S,6S,7S,8S,9R,10S)-1,9-diacetoxy-4-hydroxy-6-isobutyryloxyhalothrin lactone (hereinafter referred to as "compound TBA"), and compound 2 is (1S,4S,5R,6S,7S,8S,9R,10S)-1,9-diacetoxy-4-hydroxy-6-isobutyryloxyhalothrin lactone (hereinafter referred to as "compound TBB"). Both compounds TBA and TBB have shown certain inhibitory effects on the proliferation of breast cancer and liver cancer cells. However, as lead compounds for anti-liver cancer, the inhibitory efficacy of compounds TBA and TBB against liver cancer cells is still insufficient, especially against Huh7 liver cancer cells (TBA: IC50). 50 =19.9μM, TBB:IC 50 =15.2μM). Therefore, there is an urgent need to develop derivatives based on compounds TBA and TBB that can inhibit the proliferation of liver cancer cells. These derivatives can also lay an important material foundation for the subsequent development of novel anti-tumor drugs. Summary of the Invention

[0004] The purpose of this invention is to provide a eucalyptane-type sesquiterpene lactone derivative, TBA and TBB nitrogen mustard derivative, which can inhibit the proliferative activity of liver cancer cells and has the potential to be developed into an anti-liver cancer drug.

[0005] The above-mentioned objective of this invention is achieved through the following technical solution:

[0006] A eucalyptane-type sesquiterpene lactone derivative of TBA and TBB nitrogen mustard, wherein the eucalyptane-type sesquiterpene lactone derivatives of TBA and TBB nitrogen mustard are compounds TBA-DJ-1 to TBA-DJ-3 and compounds TBB-DJ-1 to TBB-DJ-3, respectively, and their chemical structures are as follows:

[0007]

[0008] Another objective of this invention is to provide a method for preparing eucalyptane-type sesquiterpene lactones TBA and TBB nitrogen mustard derivatives, comprising the following steps: first, mixing and reacting dicyclohexylcarbodiimide, 4-dimethylaminopyridine, p-nitrophenylbutyrate nitrogen mustard and dichloromethane; then adding eucalyptane-type sesquiterpene lactone compounds and copper(II) trifluoromethanesulfonate and heating the reaction; and finally separating and purifying to obtain a eucalyptane-type sesquiterpene lactone nitrogen mustard derivative.

[0009] Furthermore, the eucalyptane-type sesquiterpene lactones are selected from (1S,4S,5S,6S,7S,8S,9R,10S,11R)-4,6-dihydroxy-1,9-diacetoxyhalothia lactone, (1S,4S,5S,6S,7S,8S,9R,10S,11R)-1,4,9-trihydroxy-13-methoxy-6-isobutyryloxyhalothia lactone, (1S,4S,5R,6S,7S,8S,9R,10S,11R)-1,4,9-trihydroxy-6-isobutyryloxyhalothia lactone, ( One of (1S,4S,5R,6S,7S,8S,9R,10S,11R)-1,4,9-hydroxy-6-isobutyryloxy-13-methoxyhalothrin and (1S,4S,5R,6S,7S,8S,9R,10S,11R)-1,9-diacetoxy-4,6-hydroxy-13-methoxyhalothrin; the eucalyptane-type sesquiterpene lactone nitrogen mustard derivative is selected from at least one of compounds TBA-DJ-1 to TBA-DJ-3 or compounds TBB-DJ-1 to TBB-DJ-3.

[0010] Further, dicyclohexylcarbodiimide, 4-dimethylaminopyridine, p-nitrophenylbutyric acid mustard, and dichloromethane were mixed and reacted at room temperature. Then, (1S,4S,5S,6S,7S,8S,9R,10S,11R)-4,6-dihydroxy-1,9-diacetoxyhalothrin lactone and copper(II) trifluoromethanesulfonate were added and heated. The mixture was extracted with ethyl acetate, concentrated under reduced pressure, and then prepared by thin-layer preparative chromatography to obtain compound TBA-DJ-1.

[0011] Further, dicyclohexylcarbodiimide, 4-dimethylaminopyridine, p-nitrophenylbutyric acid mustard, and dichloromethane were mixed and reacted at room temperature. Then, (1S,4S,5S,6S,7S,8S,9R,10S,11R)-1,4,9-trihydroxy-6-isobutyryloxyhalothrin and copper(II) trifluoromethanesulfonate were added and heated. The mixture was extracted with ethyl acetate, concentrated under reduced pressure, and then prepared by high performance liquid chromatography to obtain compounds TBA-DJ-2 and TBA-DJ-3.

[0012] Further, dicyclohexylcarbodiimide, 4-dimethylaminopyridine, p-nitrophenylbutyric acid mustard, and dichloromethane were mixed and reacted at room temperature. Then, (1S,4S,5R,6S,7S,8S,9R,10S,11R)-1,4,9-trihydroxy-6-isobutyryloxyhalothrin and copper(II) trifluoromethanesulfonate were added and heated. The mixture was extracted with ethyl acetate, concentrated under reduced pressure, and then prepared by high performance liquid chromatography to obtain compound TBB-DJ-1.

[0013] Further, dicyclohexylcarbodiimide, 4-dimethylaminopyridine, p-nitrophenylbutyric acid mustard, and dichloromethane were mixed and reacted at room temperature. Copper(II) trifluoromethanesulfonate was added as a catalyst, followed by the addition of (1S,4S,5R,6S,7S,8S,9R,10S,11R)-1,4,9-hydroxy-6-isobutyryloxy-13-methoxyhalothrin, and the reaction was heated. The mixture was extracted with ethyl acetate, concentrated under reduced pressure, and then prepared by high performance liquid chromatography to obtain compound TBB-DJ-2.

[0014] Further, dicyclohexylcarbodiimide, 4-dimethylaminopyridine, p-nitrophenylbutyric acid mustard, and dichloromethane were mixed and reacted at room temperature. Copper(II) trifluoromethanesulfonate was added as a catalyst, followed by the addition of (1S,4S,5R,6S,7S,8S,9R,10S,11R)-1,9-diacetoxy-4,6-hydroxy-13-methoxyhalothrin, and the reaction was heated. The mixture was extracted with ethyl acetate, concentrated under reduced pressure, and then prepared by high performance liquid chromatography to obtain compound TBB-DJ-3.

[0015] Another object of the present invention is to provide the application of eucalyptane-type sesquiterpene lactones TBA and TBB nitrogen mustard derivatives in the preparation of anticancer drugs, especially in the preparation of liver cancer drugs.

[0016] Compared with the prior art, the beneficial effects of the present invention are:

[0017] This invention modifies the structures of eucalyptane-type sesquiterpene lactone epimers (compounds TBA and TBB) to obtain novel nitrogen mustard derivatives, namely compounds TBA-DJ-1 to TBA-DJ-3 and compounds TBB-DJ-1 to TBB-DJ-3. Experimental results show that these nitrogen mustard derivatives all inhibit the proliferation activity of hepatocellular carcinoma cells to a certain extent, and have potential applications in the preparation of anti-hepatocellular carcinoma drugs. Among them, compounds TBA-DJ-2 and TBB-DJ-2 significantly enhance the inhibitory efficacy against the proliferation activity of HepG2 hepatocellular carcinoma cells. These nitrogen mustard derivatives lay an important material foundation for the subsequent development of novel antitumor drugs. Detailed Implementation

[0018] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional experimental conditions.

[0019] The eucalyptane-type sesquiterpene lactones TBA and TBB nitrogen mustard derivatives provided by this invention are compounds TBA-DJ-1 to TBA-DJ-3 and compounds TBB-DJ-1 to TBB-DJ-3, respectively, and their synthetic routes are as follows:

[0020]

[0021] Synthetic routes of compounds TBA-DJ-1 to TBA-DJ-3

[0022]

[0023] Synthetic routes of compounds TBB-DJ-1 to TBB-DJ-3

[0024] The present invention will be further described below with reference to specific analysis.

[0025] Example 1

[0026] Preparation of compound TBA-DJ-1: 297 mg (0.657 mmol) of compound TBA, 50 mL of acetonitrile, and 88 mg of sodium borohydride were sequentially added to a 250 mL round-bottom flask and stirred at 60 °C for 26 h. After the reaction was complete as detected by TLC (chloroform:acetone = 6:1), the pH of the mixture was adjusted to 6-7 with 10% dilute hydrochloric acid. The mixture was extracted three times with ethyl acetate, dried with anhydrous sodium sulfate, concentrated under reduced pressure, and then 86 mg of compound 1 was obtained by semi-preparative high-performance liquid chromatography. Compound 1 is (1S,4S,5S,6S,7S,8S,9R,10S,11R)-4-hydroxy-1,9-diacetoxy-6-isobutyryloxyhalothrin lactone, with a yield of 28.77%.

[0027] 23.3 mg (0.051 mmol) of compound 1, 5 mL of methanol, and 22.6 mg of potassium hydroxide were sequentially added to a 50 mL round-bottom flask. The mixture was stirred at room temperature for 6 h. After the reaction was complete as detected by TLC (chloroform:acetone = 3:1), the pH of the mixture was adjusted to 6-7 with 10% dilute hydrochloric acid. The mixture was extracted three times with ethyl acetate, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was purified by column chromatography (chloroform:acetone = 6:1) to give 3.5 mg of compound 2, which is (1S,4S,5S,6S,7S,8S,9R,10S,11R)-4,6-dihydroxy-1,9-diacetoxyhalothrin lactone, with a yield of 17.65%.

[0028] In a 50 mL round-bottom flask, 110 mg of dicyclohexylcarbodiimide, 16 mg of 4-dimethylaminopyridine, 63 mg of p-nitrophenylbutyrate mustard, and 5 mL of dry dichloromethane were added. The mixture was stirred at room temperature for 3 h, and then 11.3 mg (0.065 mmol) of compound 2 and copper(II) trifluoromethanesulfonate were added. The mixture was heated to reflux at 95 °C for 4 h. After the reaction was complete as detected by TLC (trichloromethane:ethyl acetate = 15:1), a green liquid was obtained. The liquid was extracted three times with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by preparative thin-layer chromatography (trichloromethane:ethyl acetate = 10:1) to obtain 6 mg of compound TBA-DJ-1, with a yield of 11.85%.

[0029] Compound TBA-DJ-1 is (3R,3aS,4S,4aS,5S,8S,8aS,9R,9aS)-4-((4-(4-(bis(2-chloroethyl)amino)phenyl)butanoyl)oxy)-5-hydroxy-3,5,8a-trimethyl-2-oxododecahydronaphtho[2,3-b]furan-8,9-diyl diacetate. Spectroscopic data for compound TBA-DJ-1:

[0030] 1 H NMR (400MHz, CDCl3) δ: 7.06 (2H, d, J = 8.4Hz, H-7″, and H-9′′), 6.62 (2H, d, J = 8.4Hz, H-6″, and H-10″),5.79(1H,dd,J=11.1,8.5Hz,H-6),5.46(1H,t,J=8.3Hz,H-1),5.27(1 H,d,J=2.8Hz,H-9),4.80(1H,dd,J=10.3,2.9Hz,H-8),3.70(4H,m,H-11″,and H-13″),3.62(4H,m,H-12″,and H-14″),2.76(1H,m,H-11),2.58(2H,m,H-2″),2.42(1H,m,H-7),2.36(2H,m,H-4″),2.23,1.44(2H,m,H-2),2.03(3H,s,H-2″′),1.96(3H ,s,H-2′),1.91(2H,m,H-3″),1.87,1.70(2H,m,H-3),1.22(1H,d,J=11.2Hz,H-5),1.31(H-14),1.27(H-15),1.22(3H,d,J=7.3Hz,H-13). 13C NMR (100MHz, CDCl3) δ: 178.6 (C-12), 173.4 (C-1″), 170.7 (C-1′), 169.2 (C-1″′), 144.6 (C-8″), 130.3 (C-5″), 129.8 (2×C, C-7″, and C-9″),112.4(2×C,C-6″,and C-10″),73.7(C-8),73.1(C-1),72.8(C-9),70.5(C-4),68.5(C-6),53.7(2×C,C-11″,and C-13″),53.4(C-5),47.8(C-7),40.6(2×C,C-12″,and C-14″),40.6(C-10),39.7(C-11),35.2(C-3),34.2(C-4″),34.1(C-2″),32.5(C-14), 26.4(C-3″),22.5(C-15),22.2(C-2),21.4(C-2″′),21.0(C-2′),14.3(C-13).HRESIMS m / z:692.2351[M+H] + (calcd for C 33 H 45 Cl2NO9Na, 692.2364).

[0031] Example 2

[0032] Preparation of compounds TBA-DJ-2 and TBA-DJ-3: 64.4 mg (0.142 mmol) of TBA, 31.7 mg of potassium hydroxide, and 10 mL of methanol were added to a 50 mL round-bottom flask. The mixture was stirred at room temperature for 3.5 h. After the reaction was complete (trichloromethane:acetone = 3:1) as detected by TLC, the mixture was transferred to a 50 mL beaker. The pH of the mixture was adjusted to 6-7 with 10% dilute hydrochloric acid. The sample was transferred to a 60 mL separatory funnel, extracted three times with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The crude product was separated by column chromatography (chloroform:acetone = 3:1) to give 3.9 mg of compound 3, which is (1S,4S,5S,6S,7S,8S,9R,10S,11R)-1,4,9-trihydroxy-13-methoxy-6-isobutyryloxyhalothrin lactone, with a yield of 7.04%.

[0033] In a 50 mL round-bottom flask, 111 mg (0.533 mmol) of dicyclohexylcarbodiimide, 18 mg (0.149 mmol) of 4-dimethylaminopyridine, 93.5 mg (0.307 mmol) of p-nitrophenylbutyrate mustard, and 5 mL of dry dichloromethane were added. After stirring at room temperature for 3 h, 24 mg (0.06 mmol) of compound 3 and 5.1 mg (0.014 mmol) of copper(II) trifluoromethanesulfonate were added. The mixture was heated under reflux at 85 °C for 2 h. After the reaction was complete as detected by TLC (petroleum ether:acetone = 2:1), a green liquid was obtained. The liquid was extracted three times with ethyl acetate, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was prepared by high performance liquid chromatography to yield 6.2 mg of compound TBA-DJ-2 and 5.9 mg of compound TBA-DJ-3, with yields of 15% and 14.33%, respectively.

[0034] Compound TBA-DJ-2 is (3R,3aS,4S,4aS,5S,8S,8aR,9R,9aS)-5,9-dihydroxy-4-(isobutyryloxy)-3-(methoxymethyl)-5,8a-dimethyl-2-oxododecahydronaphtho[2,3-b]furan-8-yl4-(4-(bis(2-chloroethyl)amino)phenyl)butanoate. Spectral data for compound TBA-DJ-2:

[0035] 1H NMR(400MHz,CDCl3)δ:7.06(2H,d,J=8.4Hz,H-7′,and H-9′),6.63(2H,d,J=8.4Hz,H-6′,and H-10′),5.63(1H,dd,J=2.4Hz,H-6),4.23(1H,d,J=3.8Hz,H-1),4.8(1H,dd,J=9.2,3.6Hz,H-8),3.86(1H,d,J=3.6Hz,H-9),3.77-3.66(2H,m,H-13),3.69(4H,m,H-11′,and H-13′),3.63(4H,m,H-12′,and H-14′),3.34(3H,s,H-OCH3),3.28(1H,m,H-11),2.88(1H,m,H-7),2.56(2H,m,H-2′),2.52(1H,m,H-2″),2.35(2H,m,H-4′),2.28-1.59(2H,m,H-2),1.9(2H,m,H-3′),1.78(1H,d,J=2.4Hz,H-5),1.74-1.3(2H,m,H-3),1.31(3H,s,H-14),1.18(3H,d,J=3.6Hz,H-3″),1.16(3H,d,J=3.6Hz,H-4″),1.07(3H,s,H-15). 13 C NMR(100MHz,CDCl3)δ:177.7(C-12),175.7(C-1″),172.9(C-1′),144.6(C-8′),130.5(C-5′),129.8(2×C,C-7′,and C-9′),112.3(2×C,C-6′,and C-10′),75.1(C-1),74.7(C-8),71.7(C-9),70.3(C-6),69.1(C-13),69.0(C-4),59.2(C-OC H3),53.7(2×C,C-11′,and C-13′),51.6(C-5),44.2(C-11),41.1(C-10),40.7(2×C,C-12′,and C-14′),40.1(C-7),34.7(C-3),34.4(C-2″),34.3(C-4′),34.1(C-2′),29.7(C-14),27(C-3′),24.8(C-2),22.3(C-15),19.0(C-3″),18.8(C-4″).HRESIMS m / z:686.2852[M+H] + (calcd forC 34 H50 Cl2NO9,686.2857).

[0036] Compound TBA-DJ-3 is (3R,3aS,4S,4aS,5S,8S,8aS,9R,9aS)-5,8-dihydroxy-4-(isobutyryloxy)-3-(methoxymethyl)-5,8a-dimethyl-2-oxododecahydronaphtho[2,3-b]furan-9-yl 4-(4-(bis(2-chloroethyl)amino)phenyl)butanoate. The spectral data of compound TBA-DJ-3 are as follows:

[0037] 1 H NMR (400 MHz, CDCl3) δ: 7.07 (2H, d, J = 8.8 Hz, H-7″, and H-9″), 6.62 (2H, d, J = 8.4 Hz, H-6″, and H-10″), 5.74 (1H, dd, J = 11.2, 9.2 Hz, H-6), 5.23 (1H, d, J = 3.2 Hz, H-9), 4.89 (1H, dd, J = 10.2, 3.2 Hz, H-8), 4.23 (1H, H-1), 3.75 (1H, dd, J = 9.2, 2.8 Hz, H-13), 3.69 (4H, m, H-11″, and H-13″), 3.63 (4H, m, H-12″, and H-14″), 3.38 (1H, dd, J = 9.2, 3.2 Hz, H-13), 3.34 (3H, s, H-OCH3), 3.01 (1H, m, H-7), 2.81 (1H, m, H-11), 2.57 (1H, m, H-2′), 2.56 (2H, m, H-2″), 2.37 (2H, m, H-4″), 2.06 - 1.52 (2H, m, H-2), 1.93 (2H, m, H-3″), 1.79 - 1.71 (2H, m, H-3), 1.67 (1H, d, J = 10.8 Hz, H-5), 1.25 (3H, s, H-14), 1.23 (3H, d, J = 6.8 Hz, H-3′), 1.2 (3H, s, H-15), 1.19 (3H, d, J = 6.8 Hz, H-4′). 13C NMR (100MHz, CDCl3) δ: 177.7 (C-1′), 176.6 (C-12), 174.5 (C-1″), 144.5 (C-8″), 130.3 (C-5″), 129.9 (2×C, C-7″, and C-9″), 112.4 (2×C, C-6″, and C-10″),74.9(C-9),74.3(C-8),73.4(C-6),71.4(C-4),70.9(C-13),64.9(C-1),59.4(C-OCH3),54(C-5),53.8(2×C,C-11″,and C-13″),46.8(C-11),42.4(C-10),41.4(C-7),40.7(2×C,C-12″,and HRESIMS m / z:686.3458[M+H] + (calcd for C 34 H 50 Cl2NO9, 686.2857).

[0038] Example 3

[0039] Preparation method of compound TBB-DJ-1: Compound TBB, 50 mL of acetonitrile, and 67 mg of sodium borohydride were sequentially added to a 250 mL round-bottom flask and stirred at 60 °C for 26 h. After the reaction was complete as detected by TLC (chloroform:acetone = 6:1), the pH of the mixture was adjusted to 6-7 with 10% dilute hydrochloric acid. The mixture was extracted three times with ethyl acetate, dried with anhydrous sodium sulfate, concentrated under reduced pressure, and then 3 mg of compound 4 was prepared by semi-preparative high-performance liquid chromatography. Compound 4 is (1S,4S,5R,6S,7S,8S,9R,10S,11R)-1,4,9-trihydroxy-6-isobutyryloxyhalothrin lactone, with a yield of 1.18%.

[0040] In a 50 mL round-bottom flask, 90 mg of dicyclohexylcarbodiimide, 13 mg of 4-dimethylaminopyridine, 84 mg of p-nitrophenylbutyrate mustard, and 5 mL of dry dichloromethane were added. The mixture was stirred at room temperature for 3 h, and then 15 mg (0.04 mmol) of compound 4 and copper(II) trifluoromethanesulfonate were added. The mixture was heated to reflux at 85 °C for 6 h. After the reaction was complete as detected by TLC (trichloromethane:ethyl acetate = 1:1), a green liquid was obtained. The liquid was extracted three times with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (trichloromethane:ethyl acetate = 1:1.5) to give 3 mg of compound TBB-DJ-1, with a yield of 11.5%.

[0041] Compound TBB-DJ-1 is (3R,3aS,4S,4aR,5S,8S,8aR,9R,9aS)-5,9-dihydroxy-4-(isobutyryloxy)-3,5,8a-trimethyl-2-oxododecahydronaphtho[2,3-b]furan-8-yl4-(4-(bis(2-chloroethyl)amino)phenyl)butanoat). Spectral data for compound TBB-DJ-1:

[0042] 1 H NMR (400MHz, CDCl3) δ: 7.07 (2H, d, J = 8.6Hz, H-7′, and H-9′), 6.65 (2H, d, J = 8.7Hz, H-6′, and H-10′),5.63(1H,dd,J=3.2,1.2Hz,H-6),5.12(1H,dd,J=6.4,5.2Hz,H-1),4.59(1H,dd,J=8,4.4Hz,H-8),3.7(4H,m,H-11′,and H-13′),3.63(4H,m,H-12′,andH-14′),3.48(1H,d,J=3.6Hz,H-9),2.72 (1H,m,H-11),2.56(2H,m,H-2′),2.55(1H,m,H-2″),2.34(2H,m,H-4′), 2.21(1H,m,H-7),2.1(1H,d,J=3.2Hz,H-5),1.9(2H,m,H-3′),1.8-1.61 (4H,m,H-2,3),1.34(3H,s,H-14),1.27(3H,d,H-13),1.2(6H,H-3″,and H-4″),1.19(3H,s,H-15). 13C NMR (100MHz, CDCl3) δ: 179.7 (C-12), 176.3 (C-1″), 174.8 (C-1′), 144.7 (C-8′), 130.3 (C-5′), 129.8 (2×C, C-7′, and C-9′), 112.4 (2×C, C-6′, and C-10′),74.1(C-1),72.9(C-8),71.6(C-9),71.3(C-4),68.5(C-6),53.8(2×C,C-11′,and C-13′),49.4(C-7),43.3(C-10),42.1(C-5),42(C-3),40.7(2×C,C-12′,and C-14′),37(C-11),34.7(C-2″),34.1(C-2′),33.9(C-4′),26.9(C-3′),26.4( C-14),25(C-2),19.2(C-3″),18.5(C-4″),14.8(C-13),14.2(C-15).HRESIMS m / z:656.2709[M+H] + (calcd for C 33 H 48 Cl2NO8, 656.2751).

[0043] Example 4

[0044] Preparation of compounds TBB-DJ-2 and TBB-DJ-3: TBB, 29.7 mg potassium hydroxide, and 10 mL methanol were added to a 50 mL round-bottom flask and stirred at room temperature for 3.5 h. After the reaction was complete (chloroform:acetone = 3:1) as detected by TLC, the mixture was transferred to a 50 mL beaker. The pH of the mixture was adjusted to 6-7 with 10% dilute hydrochloric acid. The adjusted mixture was then transferred to a 60 mL separatory funnel and extracted three times with ethyl acetate. After drying with anhydrous sodium sulfate, the mixture was filtered and evaporated to dryness under reduced pressure. The crude product was subjected to column chromatography. Separation (chloroform:acetone = 3:2) yielded 4.1 mg of compound 5 and 10.4 mg of compound 6. Compound 5 was (1S,4S,5R,6S,7S,8S,9R,10S,11R)-1,4,9-hydroxy-6-isobutyryloxy-13-methoxyhalothrin, and compound 6 was (1S,4S,5R,6S,7S,8S,9R,10S,11R)-1,9-diacetoxy-4,6-hydroxy-13-methoxyhalothrin, with yields of 15.15% and 37.88%, respectively.

[0045] In a 50 mL round-bottom flask, 98 mg of dicyclohexylcarbodiimide, 15 mg of 4-dimethylaminopyridine, 87 mg of p-nitrophenylbutyrate mustard, and 5 mL of dry dichloromethane were added. The mixture was stirred at room temperature for 3 h, and then 30.2 mg (0.075 mmol) of compound 5 and copper(II) trifluoromethanesulfonate were added. The mixture was heated to reflux at 85 °C for 6 h. After the reaction was complete as detected by TLC (trichloromethane:ethyl acetate = 4:1), a green liquid was obtained. The liquid was extracted three times with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (trichloromethane:ethyl acetate = 10:1) to give 9.9 mg of compound TBB-DJ-2, with a yield of 18.67%.

[0046] In a 50 mL round-bottom flask, 90 mg of dicyclohexylcarbodiimide, 16 mg of 4-dimethylaminopyridine, 75 mg of p-nitrophenylbutyrate mustard, and 5 mL of dry dichloromethane were added. The mixture was stirred at room temperature for 3 h, and then 14 mg (0.034 mmol) of compound 6 and copper(II) trifluoromethanesulfonate were added. The mixture was heated to reflux at 85 °C for 6 h. After the reaction was complete as detected by TLC (trichloromethane:ethyl acetate = 2:1), a green liquid was obtained. The liquid was extracted three times with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (trichloromethane:ethyl acetate = 3:1) to give 4 mg of compound TBB-DJ-3, with a yield of 16.76%.

[0047] The compound TBB-DJ-2 is (3R,3aS,4S,4aR,5S,8S,8aR,9R,9aS)-5,9-dihydroxy-4-(isobutyryloxy)-3-(methoxymethyl)-5,8a-dimethyl-2-oxododecahydronaphtho[2,3-b]furan-8-yl4-(4-(bis(2-chloroethyl)amino)phenyl)butanoat). Spectral data for compound TBB-DJ-2:

[0048] 1H NMR(400MHz,CDCl3)δ:7.06(2H,d,J=8.7Hz,H-7′,and H-9′),6.63(2H,d,J=8.7Hz,H-6′,and H-10′),5.7(1H,dd,J=1.2,3.2Hz,H-6),5.11(1H,dd,J=10,5.2Hz,H-1),4.62(1H,dd,J=4,7.6Hz,H-8),3.48(1H,d,J=4Hz,H-9),3.73-3.6(2H,m,H-13),3.7(4H,m,H-11′,and H-13′),3.63(4H,m,H-12′,and H-14′),3.36(3H,s,H-OCH3),2.84(1H,m,H-11),2.73(1H,m,H-7),2.55(1H,m,H-2″),2.53(2H,m,H-2′),2.34(2H,m,H-4′),2.11(1H,d,J=3.2Hz,H-5),1.87(2H,m,H-3′),1.79-1.61(2H,m,H-3),1.75-1.73(2H,m,H-3),1.32(3H,s,H-14),1.21(3H,s,H-15),1.2(3H,H-3″),1.17(3H,H-4″). 13 C NMR(100MHz,CDCl3)δ:177.0(C-12),176.2(C-1″),174.8(C-1′),144.6(C-8′),130.3(C-5′),129.8(2×C,C-7′,and C-9′),112.4(2×C,C-6′,and C-10′),74.2(C-1),73(C-8),71.7(C-9),71.3(C-4),69.1(C-13),68.5(C-6),59.4(C-OCH3),53.8(2×C,C-11′,and C-13′),43.5(C-11),43.3(C-10),43.2(C-7),42.1(C-5),41.9(C-3),40.7(2×C,C-12′,and C-14′),34.7(C-2″),34.1(C-4′),33.9(C-2′),26.9(C-3′),26.3(C-14),25.0(C-2),19.2(C-3″),18.6(C-4″),14.1(C-15).HRESIMS m / z:686.2806[M+H] + (calcd for C 34 H 50Cl2NO9,686.2857).

[0049] Compound TBB-DJ-3 is (3R,3aS,4S,4aR,5S,8S,8aS,9R,9aS)-4-((4-(4-(bis(2-chloroethyl)amino)phenyl)butanoyl)oxy)-5-hydroxy-3-(methoxymethyl)-5,8a-dimethyl-2-oxododecahydronaphtho[2,3-b]furan-8,9-diyl diacetate. The spectral data of compound TBB-DJ-3 are as follows:

[0050] 1 H NMR(400MHz,CDCl3)δ: 7.06(2H,d,J = 8.7Hz,H-7″,and C-9′′), 6.63(2H,d,J = 8.7Hz,H-6″,and H-10″), 5.75(1H,dd,J = 1.6,3.2Hz,H-6), 5.27(1H,d,J = 4.4Hz,H-9), 4.74(1H,dd,J = 8,4.8Hz,H-8), 4.63(1H,dd,J = 4.4,11.6Hz,H-1), 3.73(2H,m,H-13), 3.7(4H,m,H-11″,and H-13″), 3.63(4H,m,H-12″,and H-14″), 3.36(3H,s,H-OCH3), 2.83(1H,m,H-7), 2.65(1H,m,H-11), 2.58(2H,m,H-2″), 2.36(2H,m,H-4″), 2.0(3H,s,H-2″′), 2.1(3H,s,H-2″), 1.95(2H,m,H-3″), 1.91(1H,d,J = 3.2Hz,H-5), 1.84 - 1.63(4H,m,H-2,3), 1.34(6H,s,H-14,and H-15). 13C NMR (100MHz, CDCl3) δ: 175.7 (C-1″), 172.7 (C-12), 170.8 (C-1″′), 169.2 (C-1′), 144.7 (C-8″), 130.2 (C-5″), 129.8 (2×C, C-7″, and C-9″),112.4(2×C,C-6″,and C-10″),73.2(C-1),71.8(C-4),71.4(C-8),71.1(C-9),68.9(C-13),68.(C- 6),59.5(C-OCH3),53.7(2×C,C-1″),43.4(C-5),43.2(C-11),42.8(C-7),41 .9(C-10),41.5(C-3),40.7(2×C,C-2″),34.5(C-4″),34.1(C-2″),26.4(C-3 ″),26.5(C-14),24.2(C-2),21.2(C-2″′),21.2(C-2′),14.6(C-15).HRESIMS m / z: 700.2608 [M+H] + (calcd forC 34 H 48 Cl2NO 10 ,700.2650).

[0051] Example 5

[0052] The toxicity experiments of eucalyptane-type sesquiterpene lactones TBA and TBB nitrogen mustard derivatives (compounds TBA-DJ-1~TBA-DJ-3, compounds TBB-DJ-1~TBB-DJ-3) against two hepatocellular carcinoma (HCC) cell lines, Huh7 and HepG2, are as follows:

[0053] Cell lines and cell culture: Huh7 and HepG2 cell lines were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Both cell lines were cultured in MEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and maintained in a constant temperature and humidity incubator at 37°C and 5% CO2.

[0054] Cytotoxicity assay: The cytotoxic effects of the compounds on Huh7 and HepG2 cells were evaluated using the CCK-8 assay. Cells were cultured in MEM medium containing 10% fetal bovine serum at 37°C. Cells were seeded at a density of 8000 cells per well in 96-well plates and cultured for 24 h to allow adherence. Subsequently, compounds TBA-DJ-1 to TBA-DJ-3 and compounds TBB-DJ-1 to TBB-DJ-3 were added, and the cells were incubated at 37°C for 24 h. After incubation, CCK-8 reagent was added, and the cells were incubated in the dark for 0.5–2 h. Cell viability was determined by measuring absorbance at 570 nm using a microplate reader. As shown in Table 1, compounds TBA-DJ-1 to TBA-DJ-3 and TBB-DJ-1 to TBB-DJ-3 all showed certain inhibitory effects on the proliferation of liver cancer cells. Among them, compounds TBA-DJ-2 and TBB-DJ-2 showed significantly better inhibitory effects on the proliferation of HepG2 liver cancer cells than their parent compound (compound TBA), significantly enhancing their inhibitory efficacy against HepG2. This indicates that these derivatives have potential clinical application value and can serve as candidate compounds for the development of anti-liver cancer drugs.

[0055] Table 1. Results of the toxic effects of compounds TBA-DJ-1~TBA-DJ-3 and TBB-DJ-1~TBB-DJ-3 on human hepatocellular carcinoma cells Huh7 and HepG2.

[0056]

[0057] 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 technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A eucalyptane-type sesquiterpene lactone derivative of TBA and TBB nitrogen mustard, characterized in that, The eucalyptane-type sesquiterpene lactones TBA and TBB nitrogen mustard derivatives are compounds TBA-DJ-1 to TBA-DJ-3 and compounds TBB-DJ-1 to TBB-DJ-3, respectively, and their chemical structures are as follows:

2. The method for preparing the eucalyptane-type sesquiterpene lactones TBA and TBB nitrogen mustard derivatives as described in claim 1, characterized in that, Includes the following steps: First, dicyclohexylcarbodiimide, 4-dimethylaminopyridine, p-nitrophenylbutyric acid nitrogen mustard and dichloromethane were mixed and reacted. Then, eucalyptane-type sesquiterpene lactone compounds and copper(II) trifluoromethanesulfonate were added and heated to react. After separation and purification, a eucalyptane-type sesquiterpene lactone nitrogen mustard derivative was obtained.

3. The method for preparing the eucalyptane-type sesquiterpene lactones TBA and TBB nitrogen mustard derivatives as described in claim 2, characterized in that, The eucalyptane-type sesquiterpene lactones are selected from (1S,4S,5S,6S,7S,8S,9R,10S,11R)-4,6-dihydroxy-1,9-diacetoxyhalothia lactone, (1S,4S,5S,6S,7S,8S,9R,10S,11R)-1,4,9-trihydroxy-13-methoxy-6-isobutyryloxyhalothia lactone, (1S,4S,5S,6S,7S,8S,9R,10S,11R)-1,4,9-trihydroxy-6-isobutyryloxyhalothia lactone, (1 (S,4S,5R,6S,7S,8S,9R,10S,11R)-1,4,9-hydroxy-6-isobutyryloxy-13-methoxyhalothrin and (1S,4S,5R,6S,7S,8S,9R,10S,11R)-1,9-diacetoxy-4,6-hydroxy-13-methoxyhalothrin; eucalyptane-type sesquiterpene lactone nitrogen mustard derivatives are selected from at least one of compounds TBA-DJ-1 to TBA-DJ-3 or compounds TBB-DJ-1 to TBB-DJ-3.

4. The method for preparing the eucalyptane-type sesquiterpene lactone TBA and TBB nitrogen mustard derivatives as described in claim 3, characterized in that, The process includes the following steps: dicyclohexylcarbodiimide, 4-dimethylaminopyridine, p-nitrophenylbutyric acid mustard, and dichloromethane are mixed and reacted at room temperature. Then, (1S,4S,5S,6S,7S,8S,9R,10S,11R)-4,6-dihydroxy-1,9-diacetoxyhalothrin lactone and copper(II) trifluoromethanesulfonate are added and heated. After extraction with ethyl acetate and concentration under reduced pressure, compound TBA-DJ-1 is prepared by thin-layer preparative chromatography.

5. The method for preparing the eucalyptane-type sesquiterpene lactones TBA and TBB nitrogen mustard derivatives as described in claim 3, characterized in that, The process includes the following steps: Dicyclohexylcarbodiimide, 4-dimethylaminopyridine, p-nitrophenylbutyric acid mustard, and dichloromethane are mixed and reacted at room temperature. Then, (1S,4S,5S,6S,7S,8S,9R,10S,11R)-1,4,9-trihydroxy-6-isobutyryloxyhalothrin lactone and copper(II) trifluoromethanesulfonate are added and heated. After extraction with ethyl acetate and concentration under reduced pressure, compounds TBA-DJ-2 and TBA-DJ-3 are prepared by high performance liquid chromatography.

6. The method for preparing the eucalyptane-type sesquiterpene lactone TBA and TBB nitrogen mustard derivatives as described in claim 3, characterized in that, The process includes the following steps: dicyclohexylcarbodiimide, 4-dimethylaminopyridine, p-nitrophenylbutyric acid mustard, and dichloromethane are mixed and reacted at room temperature. Then, (1S,4S,5R,6S,7S,8S,9R,10S,11R)-1,4,9-trihydroxy-6-isobutyryloxyhalothrin lactone and copper(II) trifluoromethanesulfonate are added and heated. After extraction with ethyl acetate and concentration under reduced pressure, compound TBB-DJ-1 is prepared by high performance liquid chromatography.

7. The method for preparing the eucalyptane-type sesquiterpene lactones TBA and TBB nitrogen mustard derivatives as described in claim 3, characterized in that, The process includes the following steps: dicyclohexylcarbodiimide, 4-dimethylaminopyridine, p-nitrophenylbutyric acid mustard, and dichloromethane are mixed and reacted at room temperature. Copper(II) trifluoromethanesulfonate is added as a catalyst. Subsequently, (1S,4S,5R,6S,7S,8S,9R,10S,11R)-1,4,9-hydroxy-6-isobutyryloxy-13-methoxyhalothrin is added and reacted under heat. After extraction with ethyl acetate and concentration under reduced pressure, compound TBB-DJ-2 is prepared by high performance liquid chromatography.

8. The method for preparing the eucalyptane-type sesquiterpene lactone TBA and TBB nitrogen mustard derivatives as described in claim 3, characterized in that, The process includes the following steps: a mixture of dicyclohexylcarbodiimide, 4-dimethylaminopyridine, p-nitrophenylbutyric acid mustard, and dichloromethane is reacted, copper(II) trifluoromethanesulfonate is added as a catalyst, and then (1S,4S,5R,6S,7S,8S,9R,10S,11R)-1,9-diacetoxy-4,6-hydroxy-13-methoxyhalothrin is added and heated to react. After extraction with ethyl acetate and concentration under reduced pressure, the compound TBB-DJ-3 is prepared by high performance liquid chromatography.

9. The use of the eucalyptane-type sesquiterpene lactones TBA and TBB nitrogen mustard derivatives as described in claim 1 in the preparation of anticancer drugs.

10. The use of the eucalyptane-type sesquiterpene lactones TBA and TBB nitrogen mustard derivatives as described in claim 9 in the preparation of anti-hepatocellular carcinoma drugs.

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