Guaiacolane-eucalyptane sesquiterpene dimer and preparation method and application thereof

Abstract

The present invention provides a new guaiacane-eudesane sesquiterpene dimer 1-7 shown in a structural formula, and a pharmaceutical composition thereof, a preparation method thereof and an application thereof, and belongs to the field of pharmaceutical technology. The preparation method of the present invention comprises preparing guaiacane-eudesane sesquiterpene dimer 1 (compound 1) by Diels-Alder reaction / deprotection with guaiacane diene and eudesane-type sesquiterpene isoinulin lactone, and further derivatizing to obtain 6 guaiacane-eudesane sesquiterpene dimers 2-7 (compound 2-7). The guaiacane-eudesane sesquiterpene dimer of the present invention has inhibitory activity on human liver cancer cell lines HepG2, Huh7 and SK-Hep-1, and compound 1 can significantly inhibit the growth of nude mouse transplanted tumors, can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition, and can be used to prepare anti-liver cancer drugs.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and in particular, relates to a novel guaiacane-eudesane sesquiterpene dimer, a preparation method thereof, a pharmaceutical composition for treating liver cancer with the compound as an active ingredient, and the use of the compound and the pharmaceutical composition thereof in the preparation of anti-liver cancer drugs. Background Art

[0002] Liver cancer is one of the common malignant tumors, including primary liver cancer and secondary liver cancer, of which hepatocellular carcinoma (HCC) accounts for more than 90%, which can be caused by liver fibrosis, cirrhosis, hepatitis infection, alcoholism, aflatoxin infection and other factors. Globally, the incidence of HCC ranks sixth among malignant tumors. Currently, there are more than 800,000 new cases of liver cancer worldwide each year, and more than 460,000 cases in my country each year. Liver cancer has the characteristics of hidden onset, long latency, strong invasiveness, easy metastasis, and high malignancy. For patients with unresectable or advanced disease, the 5-year survival rate is only 13%. More than 422,000 people die from liver cancer in my country each year. At present, the main drugs for the treatment of liver cancer in clinical practice are 4 small molecule synthetic drugs acting on tyrosine kinase inhibitors: sorafenib, regoratinib, lenvatinib; and 3 antibody drugs: nivolumab, pembrolizumab and ramucirumab. These drugs have played a significant role in the treatment of liver cancer in clinical practice and have prolonged the survival of patients to a certain extent. However, they still have disadvantages such as low objective remission rate (<20%), easy drug resistance, intolerance of some liver cancer patients, obvious toxicity and side effects, and high price, and their effects are not satisfactory.

[0003] Sesquiterpene lactones are active ingredients of various medicinal plants used in traditional medicine to treat inflammatory diseases. In recent years, sesquiterpene compounds have attracted great interest from researchers due to their anticancer activity, and a lot of work has been done to understand the molecular mechanism and potential anti-tumor mechanism of sesquiterpene. Guaiarene-eudesane sesquiterpene dimers are a class of complex sesquiterpene dimers with a skeleton of at least 30 carbon atoms, which are formed by connecting a guaiarene sesquiterpene unit with a eudesane sesquiterpene unit. A large number of proteins responsible for cell functions in living organisms exist in the form of dimers or need to be activated by dimerization before mediating certain signaling pathways. Targeting both monomers of dimeric proteins at the same time is an important strategy for drug research. Since sesquiterpene dimers have the potential to act on both monomers of dimeric proteins at the same time, it is of great significance to synthesize dimers and derivatives based on sesquiterpene with anti-liver cancer activity.

[0004] There is no report on the synthesis of the relevant guaiacane-eudes lactone sesquiterpene dimer in the prior art, nor on the pharmaceutical composition with the guaiacane-eudes lactone dimer as an active ingredient, nor on the application of the pharmaceutical composition in the preparation or treatment of liver cancer drugs. Summary of the invention

[0005] The object of the present invention is to provide new guaiacane-eudesane sesquiterpene dimers 1-7 and pharmaceutical compositions thereof and preparation methods and applications thereof. The preparation method of the present invention uses aglabin and isoinulin lactone as raw materials to prepare new guaiacane-eudesane sesquiterpene dimers. The raw materials of the method are easily available, easy to operate, and suitable for industrial production. The method has good substrate universality and can be used to prepare guaiacane-eudesane sesquiterpene dimers substituted with various functional groups.

[0006] In order to achieve the above-mentioned object of the present invention, the present invention provides the following technical solutions:

[0007] The present invention provides novel guaiacane-eudesane sesquiterpene dimers 1-7 shown in the following structural formula:

[0008]

[0009] The present invention also provides a method for preparing guaiacane-eudesane sesquiterpene dimer 1-7, which comprises the steps of preparing guaiacane-eudesane sesquiterpene dimer 1 by Diels-Alder reaction / demethylation of dimethylamine with guaiacane diene and eudesane-type sesquiterpene isoinulin lactone,

[0010]

[0011] The synthesis method of guaiacane-eudesane sesquiterpene dimer 1-7 mainly includes the following key reaction steps:

[0012] Preparation of guaiacane-eudesane sesquiterpene dimer 1: Guaiacane-type diene and eudesane-type sesquiterpene isoinulin lactone are reacted under appropriate conditions by Diels–Alder reaction / demethylamination to obtain guaiacane-eudesane sesquiterpene dimer 1. The appropriate conditions for the Diels–Alder reaction are that the diene and the dienophile are heated to react under solvent-free conditions;

[0013] Preparation of guaiacane-eudesane sesquiterpene dimer 2: Using 1,4-dioxane and water as solvents, compound 1 reacts with 3,5-dinitrosalicylic acid at room temperature to prepare guaiacane-eudesane sesquiterpene dimer 2;

[0014] Preparation of guaiacane-eudesane sesquiterpene dimers 3-4: guaiacane-eudesane sesquiterpene dimer 1 is reacted with selenium dioxide and tert-butyl hydroperoxide at room temperature to prepare guaiacane-eudesane sesquiterpene dimers 3 and 4;

[0015] Preparation of guaiacane-eudesane sesquiterpene dimer 5: guaiacane-eudesane sesquiterpene dimer 1 is reacted with m-chloroperbenzoic acid to prepare guaiacane-eudesane sesquiterpene dimer 5;

[0016] Preparation of guaiacane-eudesane sesquiterpene dimers 6 and 7: Using dichloromethane as solvent and 4-dimethylaminopyridine as base, guaiacane-eudesane sesquiterpene dimer 3 was reacted with acetic anhydride to prepare guaiacane-eudesane sesquiterpene dimer 6, and with benzoic anhydride to prepare guaiacane-eudesane sesquiterpene dimer 7.

[0017] The present invention also provides the use of the novel guaiacane-eudesane sesquiterpene dimer 1-7 in the preparation of anti-liver cancer drugs.

[0018] The present invention also provides a pharmaceutical composition, which comprises at least one of the above-mentioned guaiacane-eudesane sesquiterpene dimers 1-7 (compounds 1-7) and a pharmaceutically acceptable carrier.

[0019] The pharmaceutical composition provided by the present invention comprises at least one of the above compounds 1-7 and a pharmaceutically acceptable carrier. In the present invention, the pharmaceutically acceptable carrier is preferably a solid, semisolid or liquid diluent, a filler and a pharmaceutical product adjuvant. The present invention has no particular limitation on the pharmaceutically acceptable carrier, and any pharmaceutically acceptable carrier well known in the art, non-toxic and inert to humans and animals can be selected.

[0020] The present invention also provides a method for preparing a pharmaceutical composition, firstly obtaining guaiacane-eudesane sesquiterpene dimers 1-7 according to the above steps for preparing guaiacane-eudesane sesquiterpene dimers 1-7, and then taking any one or any several of them and adding a pharmaceutically acceptable carrier.

[0021] The present invention has no particular limitation on the method for preparing the pharmaceutical composition. At least one of compounds 1-7 can be directly mixed with a pharmaceutically acceptable carrier. The present invention has no particular limitation on the mixing process. A pharmaceutical composition can be obtained by selecting a process well known in the art.

[0022] The present invention provides the use of the pharmaceutical composition of the above technical solution in the preparation of anti-liver cancer drugs. The present invention has no special limitation on the method of the application, and any method well known in the art can be used.

[0023] In the present invention, when the pharmaceutical composition is used to prepare an anti-liver cancer drug, the content of the composition in the drug is preferably 0.1-99%; in the pharmaceutical composition, the content of at least one of the compounds 1-7 in the pharmaceutical composition is preferably 0.5-90%. The pharmaceutical composition of the present invention is preferably used in the form of a dosage per unit body weight. In the present invention, the prepared drug is preferably administered in two forms: injection (intravenous injection, intramuscular injection) and oral administration.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] 1. The present invention provides a series of new guaiacane-eudesane sesquiterpene dimers 1-7 (compounds 1-7).

[0026] 2. The present invention provides a new method for preparing new compounds 1-7, which has readily available raw materials, a simple process and is easy to operate.

[0027] 3. The present invention provides a pharmaceutical composition with new compounds 1-7 as active ingredients, providing a new anti-liver cancer drug with better medicinal effects.

[0028] 4. Compounds 1-7 of the present invention have strong cytotoxic activity against three liver cancer cell lines (HepG2, SK-Hep-1 and Huh7), IC 50 The values ​​ranged from 4.6 to 40.4 μM. In particular, compound 1 showed significant cytotoxicity, with IC 50 The values ​​were 5.6 (HepG2), 4.6 (Huh7) and 4.8 μM (SK-Hep-1), respectively, which were stronger than the positive drug sorafenib. Compound 1 inhibited tumor growth in nude mouse xenograft tumor models without causing obvious side effects. After intervention with compound 1 (60 mg / kg), the tumor inhibition rate was as high as 78.1%, which was comparable to sorafenib (73.1%, 60 mg / kg).

[0029] 5. The synthesized compound sesquiterpene dimer 1-7 can be used as a drug to treat diseases related to liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the structural formula of guaiacane-eudesane sesquiterpene dimer 1-7 (Compound 1-7) of the present invention.

[0031] Figure 2 The guaiacane-eudesane sesquiterpene dimer 1 (compound 1) (KGA-6006) of the present invention inhibits tumor growth in vivo. DETAILED DESCRIPTION

[0032] In order to better understand the present invention, the substantial content of the present invention is further described below with reference to the accompanying drawings using embodiments of the present invention, but the present invention is not limited thereto.

[0033] Embodiment 1:

[0034] Preparation of guaiacane-eudesane sesquiterpene dimer 1 (Compound 1).

[0035]

[0036] At room temperature, iso-inulin lactone (1.02 g, 4.4 mmol, 1.1 equiv) and diene (1.17 g, 4 mmol, 1 equiv) were dissolved in dichloromethane (10 mL), concentrated under reduced pressure, heated to 50 ° C in an oil bath, and reacted for 24 h. The crude product was separated by column chromatography and used for the next reaction. The sample was dissolved in methanol (20 mL) at room temperature, and iodomethane (1.24 mL, 20 mmol, 5 equiv) was added under stirring, and the reaction was continued for 4 h. The solvent was removed by concentration under reduced pressure, and 50 mL of saturated sodium bicarbonate solution and 50 mL of ethyl acetate were added. The mixture was transferred to a separatory funnel and mixed until the solid was completely dissolved. The organic phase was separated, the aqueous phase was extracted with dichloromethane, the organic phases were combined, and the solvent was removed by concentration under reduced pressure. The crude product was separated by column chromatography (ethyl acetate-petroleum ether, 20:80) to obtain compound 1 (1.20 g).

[0037] Compound 1

[0038] Yield: 63%

[0039] Molecular formula: C 30 H 38 O5

[0040] Molecular weight: 478.6290

[0041] Optical rotation: [α] D 25 +55.8 (c 0.066, MeOH)

[0042] HRMS (ESI, m / z): [M+Na] + [C 30 H 38 O5Na] + Calculated value 501.2617, measured value 517.2616.

[0043] IR max :3471,1757,1644,1445,1263,1157

[0044] 1H NMR (400MHz, CDCl3) δ6.21(d,J=3.6Hz,1H,H-13a),5.51(d,J=3.6Hz,1H,H-13b),5.13(d,J=9.6Hz,1H,H-6),4. 93-4.91(m,1H,H-8′),4.74(s,1H,H-15′a),4.43(s,1H,H-15′b),3.00-2.99(m,1H,H-2),2.85-2.81(m,1H,H-7 ),2.19-1.71(m,10H,H-1′a,H-1′b,H-3′a,H-6′a,H-9′a,H-13′a,H-8a,H-8b,H-9a,H-9b),1.59-1.48(m,11H,H -2′a,H-2′b,H-9′b,H-6′b,H-3′a,15-Me,14-Me),1.27-1.09(m,4H,H-3′b,H-13′b,H-3b),0.78(s,3H,14′-Me); 13 C NMR (100MHz, CDCl3) δ154.3(C-1),42.3(C-2),53.4(C-3),63.7(C-4),139.2(C-5),82.6(C-6),46.9(C-7 ),23.9(C-8),39.1(C-9),72.2(C-10),140.7(C-11),169.9(C-12),119.9(C-13),29.0(C-14),17.6(C-1 5),41.9(C-1′),22.8(C-2′),36.7(C-3′),149.6(C-4′),46.6(C-5′),25.6(C-6′),43.3(C-7′),76.9(C- 8′), 42.1(C-9′), 34.1(C-10′), 61.1(C-11′), 181.2(C-12′), 36.9(C-13′), 18.4(C-14′), 106.4(C-15′).

[0045] Embodiment 2:

[0046] Preparation of guaiacane-eudesane sesquiterpene dimer 2 (Compound 2).

[0047]

[0048] In a 10 mL round-bottom flask, compound 1 (100 mg, 0.2 mmol, 1 equiv) was dissolved in a mixed solvent of 1 mL 1,4-dioxane and 1 mL water, and 3,5-dinitrosalicylic acid (18.2 mg, 0.08 mmol, 2 equiv) was added and reacted at room temperature for 2 h. Saturated sodium bicarbonate solution was added to quench, and dichloromethane was extracted. The organic phases were combined, washed with saturated brine, and concentrated under reduced pressure to remove the solvent. The crude product was separated by column chromatography (ethyl acetate-petroleum ether, 20:80) to obtain compound 2 (8.6 mg) and unreacted compound 1.

[0049] Compound 2

[0050] Yield: 45%

[0051] Molecular formula: C 30 H 38 O5

[0052] Molecular weight: 478.6290

[0053] Optical rotation: [α] D 25 +90.5(c 0.063,MeOH)

[0054] HRMS (ESI, m / z): [M+Na] + [C 30 H 38 O5Na] + Calculated value 501.2617, measured value 501.2619

[0055] IR max :3472,1761,1644,1444,1256,1157,1008

[0056] 1H NMR (400MHz, CDCl3) δ6.23 (d, J=3.2Hz, 1H, H-13a), 5.52 (d, J=3.2Hz, 1H, H-13b), 4.95-4.93 (m, 1H, H-8′), 4. 81(d,J=10.0Hz,1H,H-6),4.76(s,1H,H-15′a),4.41(s,1H,H-15′b),3.21-3.20(m,1H,H-2),2.91-2.86(m,1H ,H-7),2.38-2.16(m,4H,H-1′a,H-5′,H-7′,H-13′a),2.07-1.13(m,22H,H-1′b,H-2′a,H-2′b,H-3′a,H-3′b, H-6′a,H-6′b,H-9′a,H-9′b,H-13′b,H-3a,H-3b,H-8a,H-8b,H-9a,H-9b,15-Me,14-Me),0.78(s,3H,14′-Me); 13 CNMR(100MHz, CDCl3)δ154.7(C-1),41.9(C-2),53.8(C-3),64.3(C-4),138.6(C-5),83.6(C-6),47.8(C- 7),25.0(C-8),40.5(C-9),73.5(C-10),139.0(C-11),169.6(C-12),120.0(C-13),27.0(C-14),17.6(C-1 5),41.9(C-1′),22.8(C-2′),36.7(C-3′),149.6(C-4′),46.6(C-5′),25.8(C-6′),43.4(C-7′),76.8(C- 8′), 42.1(C-9′), 34.2(C-10′), 61.0(C-11′), 180.9(C-12′), 36.9(C-13′), 18.4(C-14′), 106.3(C-15′).

[0057] Embodiment 3:

[0058] Preparation of guaiacane-eudesane sesquiterpene dimers 3 and 4 (compounds 3 and 4).

[0059]

[0060] At room temperature, compound 1 (144 mg, 0.3 mmol, 1.0 equiv) was dissolved in dichloromethane (15 mL) and stirred. Tert-butyl hydroperoxide (5 M decane solution, 0.15 mL, 0.75 mmol, 2.5 equiv) and SeO2 (6.6 mg, 0.06 mmol, 0.2 equiv) were added under stirring. The reaction was allowed to react for 2 h. The reaction was quenched with 10% sodium thiosulfate solution and extracted with ethyl acetate. The organic phases were combined and concentrated under reduced pressure to remove the solvent. The crude product was separated by column chromatography (ethyl acetate-petroleum ether, 10:90 and 20:80) to give compounds 3 (98 mg) and 4 (23 mg).

[0061] Compound 3

[0062] Yield: 66%

[0063] Molecular formula: C 30 H 38 O6

[0064] Molecular weight: 494.6280

[0065] Optical rotation: [α] D 25 +11.4 (c 0.168, MeOH)

[0066] HRMS (ESI, m / z): [M+Na] + [C 30 H 38 O6Na] + Calculated value 517.2566, measured value 517.2567

[0067] IR max :3480,1757,1647,1449,1154

[0068] 1H NMR(400MHz, CDCl3) δ6.23(d,J=3.6Hz,1H,H-13a),5.51(d,J=3.6Hz,1H,H-13b),5.09(d,J=9.6Hz,1H,H-6),4 .98(s,1H,H-15′a),4.95-4.93(m,1H,H-8′),4.59(s,1H,H-15′b),4.32(brs,1H,H-3′),2.99-2.98(m,1H,H-2) ,2.85-2.81(m,1H,H-7),2.36-2.04(m,6H,H-1a′,H-5′,H-7′,H-9a′,H-13a′,H-9a),1.89-1.05(m,18H,H-1′b, H-2′a,H-2′b,H-6′a,H-6′b,H-9′b,H-13′b,H-3a,H-3b,H-8a,H-8b,H-9b,15-Me,14-Me),0.78(s,3H,14′-Me); 13 C NMR (100MHz, CDCl3) δ154.0(C-1),40.7(C-2),53.4(C-3),63.7(C-4),139.1(C-5),82.5(C-6),46.9(C-7 ),23.9(C-8),39.2(C-9),72.5(C-10),141.0(C-11),169.7(C-12),119.9(C-13),29.1(C-14),18.4(C-1 5),35.6(C-1′),29.4(C-2′),73.4(C-3′),150.4(C-4′),42.3(C-5′),25.2(C-6′),43.3(C-7′),76.8(C- 8′),41.6(C-9′),34.0(C-10′),61.1(C-11′),181.0(C-12′),36.8(C-13′),16.9(C-14′),109.8(C-15′)

[0069] Compound 4

[0070] Yield: 16%

[0071] Molecular formula: C 30 H 36 O5

[0072] Molecular weight: 476.6130

[0073] Optical rotation: [α] D 25 +28.2 (c 0.076, MeOH)

[0074] HRMS (ESI, m / z): [M+Na] + [C 30 H 36 O5Na] + Calculated value 499.2460, measured value 499.2462

[0075] IR max :3437,1761,1629,1462,1261,1156

[0076] 1 H NMR (400MHz, CDCl3) δ6.27(d,J=3.6Hz,1H,H-13a),5.67-5.65(m,1H,H-9),5.54(d,J=3.6Hz,1H,H-13b),4.99- 4.97(m,2H,H-7′,H-15′a),5.13(d,J=8.8Hz,1H,H-6),4.58(s,1H,HH-15′b),4.32-4.30(m,1H,H-3′),3.19-3. 13(m,1H,H-7),3.02-3.01(m,1H,H-2),2.37-2.08(m,6H,H-1′a,H-5′,H-7′,H-9′a,H-13′a,H-8a),1.93(s,3H, 15-Me),1.87-1.38(m,12H,H-1′b,H-2′a,H-2′b,H-6′a,H-6′b,H-9′b,H-13′b,H-3a,H-3b,15-Me),0.78(s,3H); 13 C NMR (100MHz, CDCl3) δ140.6(C-1),42.6(C-2),52.7(C-3),63.5(C-4),145.0(C-5),83.5(C-6),46.3(C-7 ),32.1(C-8),127.2(C-9),129.9(C-10),139.0(C-11),169.9(C-12),120.8(C-13),24.9(C-14),18.0(C -15),35.6(C-1′),29.4(C-2′),73.3(C-3′),150.6(C-4′),40.7(C-5′),25.1(C-6′),43.5(C-7′),76.8( C-8′),41.5(C-9′),34.1(C-10′),60.7(C-11′),180.9(C-12′),36.3(C-13′),16.9(C-14′)109.6(C-15′)

[0077] Embodiment 4:

[0078] Preparation of guaiacane-eudesane sesquiterpene dimer 5 (Compound 5).

[0079]

[0080] At room temperature, compound 1 (19.1 mg, 0.04 mmol, 1.0 equiv) was dissolved in dichloromethane (1 mL), cooled to 0 ° C in an ice bath, and then m-chloroperbenzoic acid (75%, 11 mg, 0.048 mmol, 1.2 equiv) was added. After naturally warming to room temperature, the reaction was allowed to react for 4 hours, and the reaction was quenched with 10% sodium thiosulfate solution. The mixture was extracted with dichloromethane, the organic phases were combined, and the solvent was removed by concentration under reduced pressure. The crude product was separated by column chromatography (ethyl acetate-petroleum ether, 20:80) to obtain compound 5 (14.4 mg).

[0081] Compound 5

[0082] Yield: 73%

[0083] Molecular formula: C 30 H 38 O6

[0084] Molecular weight: 494.6280

[0085] Optical rotation: [α] D 25.1 +7.1(c 0.068,MeOH)

[0086] IR max :3454,1759,1630,1457,1263,1147,817

[0087] HRMS(ESI,m / z):[M+H] + [C 30 H 38 O6Na] + Calculated value 517.2566, measured value 517.2566

[0088] 1H NMR (400MHz, CDCl3) δ6.21(d,J=3.6Hz,1H,H-13a),5.51(d,J=3.6Hz,1H,H-13b),5.13(d,J=9.6Hz,1H,H-6),4.92-4.9 1(m,1H,H-8′),2.97-2.96(m,1H,H-2),2.84-2.80(m,1H,H-7),2.65(d,J=3.6Hz,1H,H-15′a),2.51(d,J=3.6Hz,1H,H-1 5′a),2.27-2.04(m,4H,H-7′a,H-9′a,H-13′a,H-9a),1.87-1.38(m,19H,H-1′a,H-2′a,H-2′b,H-3′a,H-3′b,H-5′,H-6 ′a,H-9′b,H-13′b,H-3a,H-8a,H-8b,H-9b,15-Me,14-Me),1.32-1.13(m,3H,H-1′b,H-6′b,H-3b),0.93(s,3H,14′-Me); 13 CNMR(100MHz, CDCl3)δ154.4(C-1),42.2(C-5),53.5(C-3),63.6(C-4),139.2(C-5),82.5(C-6),46.9(C- 7),24.0(C-8),39.1(C-9),72.0(C-10),140.6(C-11),169.8(C-12),119.8(C-13),29.0(C-14),18.5(C- 15),42.4(C-1′),20.3(C-2′),35.4(C-3′),58.9(C-4′),44.5(C-5′),20.9(C-6′),43.3(C-7′),76.6(C- 8′), 41.3(C-9′), 34.1(C-10′), 61.0(C-11′), 181.0(C-12′), 36.8(C-13′), 18.4(C-14′), 50.5(C-15′).

[0089] Embodiment 5:

[0090] Preparation of guaiacane-eudesane sesquiterpene dimers 6 and 7 (Compounds 6 and 7).

[0091]

[0092] At room temperature, compound 3 (30 mg, 0.06 mmol, 1.0 equiv) was dissolved in dichloromethane (2 mL), and 4-dimethylaminopyridine (3.7 mg, 0.03 mmol, 0.5 equiv) and acetic anhydride (6.8 μL, 0.072 mmol, 1.2 equiv) or benzoic anhydride (13.7 μL, 0.07 mmol, 1.2 equiv) were added thereto in sequence, and the mixture was reacted at room temperature for 3 h. 5 mL of water was added to quench the reaction, and the mixture was extracted with dichloromethane. The organic phases were combined, concentrated under reduced pressure to remove the solvent, and the crude product was separated by column chromatography (ethyl acetate-petroleum ether, 20:80) to obtain compound 6 (29 mg) or 7 (28 mg).

[0093] Compound 6

[0094] Yield: 89%

[0095] Molecular formula: C 32 H 40 O7

[0096] Molecular weight: 536.6656

[0097] Optical rotation: [α] D 25.0 +77.9(c 0.039,MeOH)

[0098] HRMS(ESI,m / z):[M+H] + [C 32 H 40 O7Na] + Calculated value 559.2672, measured value 559.2672

[0099] IR max :3457,1762,1648,1627,1452,1372,1258,1154

[0100] 1H NMR (400MHz, CDCl3) δ6.22 (d, J=3.6Hz, 1H, H-13a), 5.51 (d, J=3.6Hz, 1H, H-13b), 5.33 (brs, 1H, H-3′), 5.15 (d, J=9. 2Hz,1H,H-6),5.13(s,1H,H-15′a),4.96-4.94(m,1H,H-8′),4.71(s,1H,H-15′b),2.99-2.98(m,1H,H-2),2.85-2.81 (m,1H,H-7),2.37-2.33(m,1H,H-5′),2.22-2.06(m,8H,H-1a′,H-7′,H-9a′,H-13a′,H-9a,OAc),1.89-1.05(m,18H,H -1′b,H-2′a,H-2′b,H-6′a,H-6′b,H-9′b,H-13′b,H-3a,H-3b,H-8a,H-8b,H-9b,15-Me,14-Me),0.78(s,3H,14′-Me); 13 CNMR(100MHz, CDCl3)δ153.9(C-1),42.3(C-2),53.5(C-3),63.8(C-4),139.2(C-5),82.5(C-6),46.9(C-7),24 .2(C-8),39.1(C-9),72.4(C-10),141.0(C-11),169.7(C-12),119.9(C-13),29.3(C-14),18.3(C-15),36.2(C- 1′),27.1(C-2′),75.5(C-3′),146.1(C-4′),41.9(C-5′),25.2(C-6′),43.3(C-7′),76.6(C-8′),41.5(C-9′),3 3.7(C-10′), 61.1(C-11′), 180.9(C-12′), 36.9(C-13′), 17.0(C-14′), 112.4(C-15′), 21.6(OAc), 170.3(OAc).

[0101] Compound 7

[0102] Yield: 78%

[0103] Molecular formula: C 37 H 42 O7

[0104] Molecular weight: 598.7360

[0105] Optical rotation: [α] D 25.0-8.7(c 0.076,MeOH)

[0106] HRMS(ESI,m / z):[M+Na] + [C 37 H 42 O7Na] + Calculated value: 621.2828, Found value: 621.2826

[0107] IR v max : 3439, 1761, 1715, 1635, 1452, 1271, 1023

[0108] 1 H NMR(400MHz,CDCl3)δ8.10(d,J=7.2Hz,H-5″),7.59-7.45(m,4H,H-2″,H-3″,H-5″,H-6″),6.20(d,J=3.6Hz,1H,H-13a),5.58(brs,1H,H-3′),5.48(d,J=3.6Hz,1H,H-13b),5.26(s,1H,H-15′a),5.15(d,J=10.0Hz,1H,H-6),4.96-4.94(m,1H,H-8′),4.78(s,1H,H-15′b),2.99-2.98(m,1H,H-2),2.85-2.81(m,1H,H-7),2.37-2.33(m,1H,H-5′),2.26-2.06(m,5H,H-1a′,H-7′,H-9a′,H-13a′,H-9a),1.94-1.10(m,18H,H-1′b,H-2′a,H-2′b,H-6′a,H-6′b,H-9′b,H-13′b,H-3a,H-3b,H-8a,H-8b,H-9b,15-Me,14-Me),0.85(s,3H,14′-Me); 13C NMR (100MHz, CDCl3) δ154.2(C-1),42.3(C-2),53.4(C-3),63.8(C-4),139.2(C-5),82.3(C-6),46.9(C-7),23.9(C-8),39.1( C-9),72.4(C-10),140.8(C-11),169.8(C-12),119.9(C-13),29.1(C-14),18.3(C-15),36.6(C-1′),27.4(C-2′),75.9(C-3′) ,145.8(C-4′),42.1(C-5′),25.2(C-6′),43.1(C-7′),76.6(C-8′),41.6(C-9′),33.8(C-10′),61.0(C-11′),181.0(C-12′),3 7.0(C-13′),17.0(C-14′),113.0(C-15′),165.6(C-1″),130.9(C-2″),129.6(C-3″,C-7″),128.4(C-4″,C-6″),132.9(C-5″).

[0109] Embodiment 6:

[0110] The cytotoxic activity of guaiacane-eudesane sesquiterpene dimer 1-7 (compound 1-7) against liver cancer cell lines was evaluated.

[0111] 1. Materials and Methods

[0112] 1.1 Materials

[0113] Hepatocellular carcinoma cell lines (HepG2, Huh7 and SK-Hep-1) were purchased from Shanghai Jining Biotechnology Co., Ltd.; culture medium (Dulbecco's Modified Eagle Medium, DMEM) was purchased from Thermo Fisher Scientific (Suzhou, China); serum (fetal bovine serum, FBS) was purchased from Life Technologies (NY, USA); RPMI-1640 was purchased from ThermoFisher Biochemical Products (Beijing, China).

[0114] 1.2 Instruments

[0115] Flex Station 3 multifunctional microplate reader (Bio-RAD 680, USA); analytical balance (AG135, Metler Toledo, China); constant temperature box (DHP-9082, Shanghai).

[0116] 1.3 Experimental process

[0117] 1). Take the hepatoma cells in logarithmic phase, discard the old culture medium, wash twice with PBS, and discard the PBS;

[0118] 2) Digest the cells with 0.25% trypsin. When the cell outline becomes darker and tends to become round under the microscope, quickly remove the trypsin.

[0119] 3) Use DMEM complete medium containing 10% FBS to terminate digestion and resuspend the cells. Take 10 μL of the cell suspension, count it with a cell counter, and adjust the cell concentration to 1×10 4 / mL, inoculated on a 96-well plate, 100 μL of cell suspension was added to each well, and incubated in a 37°C, 5% CO2 incubator for 24 h to allow the cells to adhere;

[0120] 4). Aspirate the culture medium and add the diluted sample to the plate, add 100 μL to each well, set up 3 replicate wells for each concentration, and continue incubating in the incubator for 48 hours;

[0121] 5). Aspirate the culture medium, add the prepared MTT solution (1 mg / mL), add 100 μL to each well, and incubate in the incubator for 4 hours;

[0122] 6). Aspirate the MTT solution, add DMSO, add 100 μL to each well, and incubate in the incubator for 10 minutes;

[0123] 7). Use an ELISA reader to measure the absorbance at 490 nm, and calculate the cell inhibition rate using the formula: inhibition rate = (negative - experimental group) / (negative - blank group) × 100%, and use the statistical software GraphPad prism 5 to calculate IC 50 , the experiment was repeated 3 times.

[0124] 2. Results

[0125] Compound 1 showed significant cytostatic activity, IC 50 The values ​​were 5.6 (HepG2), 4.8 (Huh7) and 4.6 μM (SK-Hep-1) (Table 3.1), which were 2.6, 4.1 and 5.3 times stronger than its parent monomer, iso-inulin lactone, respectively, and had stronger anti-liver cancer cell inhibitory activity than sorafenib. Compounds 2, 5, 6 and 7 had good inhibitory activity against Huh7 cell line, with IC50 values ​​of 5.6, 9.2, 7.8 and 6.7 μM

[0126] Table 1. Inhibitory activity of compounds 1-7 against three liver cancer cells

[0127]

[0128] 3. Conclusion

[0129] The above results indicate that the new guaiacane-eudesane sesquiterpene dimers 1-7 are cytotoxic to three liver cancer cell lines (HepG2, Huh7 and SK-Hep-1) and can be used as drugs for liver cancer-related diseases.

[0130] Embodiment 7:

[0131] Inhibitory effect of KGA-6006 on the growth of xenograft tumors in nude mice.

[0132] (1) Experimental method: SK-Hep1 liver cancer cells were routinely cultured, and the cells were counted after trypsin digestion. The cell concentration was adjusted to 10 6 / mL. BALB / C male nude mice aged 5-6 weeks were selected and the treated tumor cells were inoculated subcutaneously in the left groin. The mice were raised for about two weeks until the tumor volume grew to 100 mm. 3 Around 2:00 pm, the drug was administered. 25 tumor-bearing nude mice were randomly divided into five groups, each with 5 mice, including the positive control group (sorafenib 60 mg / kg), the model control group (drug solvent group), and the high (60 mg / kg), medium (40 mg / kg), and low (20 mg / kg) dose groups of compound 1 (KGA-6006). Intratumoral injection was administered once every 5 days, and the tumor size was measured with a vernier caliper and the weight changes of tumor-bearing nude mice were recorded. The drug was administered continuously for 50 days. After the experiment, the mice were killed by cervical dislocation, dissected, and the tumors were removed and weighed. The antitumor activity was evaluated by the tumor diameter method and tumor weight method.

[0133] Experimental results ( Figure 2 ):

[0134] Different doses of compound 1 (20, 40, 60 mg / kg) were well tolerated in nude mice, and no significant changes in body weight were observed during compound treatment. Compared with the control group, the tumor size and weight of the compound 1 intervention group decreased dose-dependently, indicating that compound 1 delayed tumor progression. After administration of 20, 40, and 60 mg / kg of compound 1, the inhibition rates of tumor weight were as high as 36.7%, 46.3%, and 78.1%, respectively, compared with the control group. Sorafenib treatment (60 mg / kg) reached 73.1%, which is comparable to compound 1.

[0135] Preparation Example:

[0136] In the following formulation examples, conventional reagents are selected and the formulations are prepared according to conventional methods. This application example only reflects that at least one of the compounds 1-7 described in the present invention can be prepared into different formulations, and the specific reagents and operations are not specifically limited:

[0137] 1. Dissolve at least one of the compounds 1-7 in DMSO, add water for injection according to conventional methods, filter, and sterilize by filling to prepare an injection solution, wherein the concentration of the injection solution is 0.5-5 mg / mL.

[0138] 2. Dissolve at least one of the compounds 1-7 in DMSO, dissolve it in sterile water for injection, stir to dissolve it, filter it with a sterile suction funnel, filter it with sterile fine filtration, pack it into ampoules, freeze-dry it at low temperature, and seal it with sterile sealing to obtain a powder injection.

[0139] 3. Add at least one of compounds 1-7 to an excipient at a mass ratio of 9:1 to the excipient to prepare a powder.

[0140] 4. Add at least one of compounds 1-7 to the excipient at a mass ratio of 5:1, and granulate and tablet.

[0141] 5. Prepare at least one of compounds 1-7 into an oral liquid according to a conventional oral liquid preparation method.

[0142] 6. Add at least one of compounds 1-7 to the excipient at a mass ratio of 5:1 to the excipient to prepare capsules.

[0143] 7. Add at least one of compounds 1-7 to an excipient at a mass ratio of 5:1 to the excipient to prepare granules.

[0144] From the above examples, it can be seen that the present invention provides a guaiacane-eudesane sesquiterpene dimer and its preparation method and application, a pharmaceutical composition and its application. The new guaiacane-eudesane sesquiterpene dimers 1-7 provided by the present invention have different degrees of cytotoxic activity against liver cancer cells, can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition, and can be used to prepare anti-liver cancer drugs.

[0145] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Guaiarene-eudesane sesquiterpene dimer 1-7 as shown in the following structural formula, 2. The method for preparing the guaiacane-eudesane sesquiterpene dimer 1-7 represented by the structural formula according to claim 1, characterized in that: The synthetic route of this method is as follows: The key reaction steps include: Preparation of guaiacane-eudesane sesquiterpene dimer 1: Guaiacane-type diene and eudesane-type sesquiterpene isoinulin lactone are reacted under appropriate conditions by Diels–Alder reaction / demethylamination to obtain guaiacane-eudesane sesquiterpene dimer 1. The appropriate conditions for the Diels–Alder reaction are that the diene and the dienophile are heated to react under solvent-free conditions; Preparation of guaiacane-eudesane sesquiterpene dimer 2: Using 1,4-dioxane and water as solvents, compound 1 reacts with 3,5-dinitrosalicylic acid at room temperature to prepare guaiacane-eudesane sesquiterpene dimer 2; Preparation of guaiacane-eudesane sesquiterpene dimers 3-4: guaiacane-eudesane sesquiterpene dimer 1 is reacted with selenium dioxide and tert-butyl hydroperoxide at room temperature to prepare guaiacane-eudesane sesquiterpene dimers 3 and 4; Preparation of guaiacane-eudesane sesquiterpene dimer 5: guaiacane-eudesane sesquiterpene dimer 1 is reacted with m-chloroperbenzoic acid to prepare guaiacane-eudesane sesquiterpene dimer 5; Preparation of guaiacane-eudesane sesquiterpene dimers 6 and 7: Using dichloromethane as solvent and 4-dimethylaminopyridine as base, guaiacane-eudesane sesquiterpene dimer 3 was reacted with acetic anhydride to prepare guaiacane-eudesane sesquiterpene dimer 6, and with benzoic anhydride to prepare guaiacane-eudesane sesquiterpene dimer 7.

3. Use of the guaiacane-eudesane sesquiterpene dimers 1-7 represented by the structural formula according to claim 1 in the preparation of anti-liver cancer drugs.

4. A pharmaceutical composition comprising at least one of the guaiacane-eudesane sesquiterpene dimers 1-7 represented by the structural formula of claim 1 and a pharmaceutically acceptable carrier.

5. Use of the pharmaceutical composition according to claim 4 in the preparation of anti-liver cancer drugs.

6. The method for preparing the pharmaceutical composition according to claim 4, characterized in that: The synthetic route of this method is as follows: The key reaction steps include: Preparation of guaiacane-eudesane sesquiterpene dimer 1: Guaiacane-type diene and eudesane-type sesquiterpene isoinulin lactone are reacted under appropriate conditions by Diels–Alder reaction / demethylamination to obtain guaiacane-eudesane sesquiterpene dimer 1. The appropriate conditions for the Diels–Alder reaction are that the diene and the dienophile are heated to react under solvent-free conditions; Preparation of guaiacane-eudesane sesquiterpene dimer 2: Using 1,4-dioxane and water as solvents, compound 1 reacts with 3,5-dinitrosalicylic acid at room temperature to prepare guaiacane-eudesane sesquiterpene dimer 2; Preparation of guaiacane-eudesane sesquiterpene dimers 3-4: guaiacane-eudesane sesquiterpene dimer 1 is reacted with selenium dioxide and tert-butyl hydroperoxide at room temperature to prepare guaiacane-eudesane sesquiterpene dimers 3 and 4; Preparation of guaiacane-eudesane sesquiterpene dimer 5: guaiacane-eudesane sesquiterpene dimer 1 is reacted with m-chloroperbenzoic acid to prepare guaiacane-eudesane sesquiterpene dimer 5; Preparation of guaiacane-eudesane sesquiterpene dimers 6 and 7: Using dichloromethane as solvent and 4-dimethylaminopyridine as base, guaiacane-eudesane sesquiterpene dimer 3 reacts with acetic anhydride to prepare guaiacane-eudesane sesquiterpene dimer 6, and reacts with benzoic anhydride to prepare guaiacane-eudesane sesquiterpene dimer 7. Then, any one or any several of the guaiacane-eudesane sesquiterpene dimers 1-7 prepared in the above steps are added with a pharmaceutically acceptable carrier.

Images