1. A diterpene derivative is characterized by having a structure shown as a formula I:

the R is₁Is oxygen, hydroxy or esterA group; the R is₂Is oxygen, hydroxyl or ester group; the R is₃Is ethyl or vinyl.

2. A diterpene derivative according to claim 1, which has the structure of formula ii, formula iii, formula iv, formula v or formula vi:

wherein R is₄As in one of compounds 3-26:

R₅as in one of compounds 27-38:

R₆or R₇Such as one of compounds 40-43:

R₈such as one of compounds 44-45:

R₉as in one of compounds 46-47:

3. the preparation method of the diterpene derivative is characterized in that the preparation route is route one, route two, route three, route four or route five;

route one:

and a second route:

and a third route:

and a fourth route:

and a fifth route:

4. use of a diterpene derivative according to claim 1 or 2 for the preparation of a medicament or food for treating cancer, preventing cancer and/or ameliorating symptoms of cancer.

5. The use according to claim 4, wherein the cancer comprises gynaecological cancer, endocrine cancer, bone cancer, lung cancer, brain and CNS tumors, gastrointestinal cancer, genitourinary cancer, head and neck tumors, blood cancer, blood disorders of bone marrow cancer, lymphatic cancer, eye cancer, skin cancer, soft tissue sarcoma.

6. Use according to claim 5, wherein said cancer comprises breast cancer, colorectal cancer, lung cancer.

7. A pharmaceutical composition characterized in that the active ingredient thereof is the diterpene derivative according to claim 1 or 2.

8. The pharmaceutical composition of claim 7, further comprising a solvent or a pharmaceutically acceptable carrier.

Background

Diterpenoid compounds are a kind of natural products with complex and various structures and important biological activity, are widely distributed in nature, plants, animals and marine organisms, and are a compound group with a molecular skeleton consisting of 4 isoprene units and 20 carbon atoms. The structure shows diversity, and the main types are kaurane, cromethane, abietane, aconitane and the like. Many oxygen-containing derivatives of diterpenes have various biological activities, and some of the oxygen-containing derivatives are important medicaments, such as paclitaxel, andrographolide, tanshinone, ginkgolide, triptolide, lilac daphne ester, stevia rebaudiana and the like, which have strong biological activities.

At present, no report on the research of the antitumor activity of diterpene compound derivatives exists, for example, pleuromutilin has insufficient killing capacity on most cancer cells and IC (integrated Circuit) in an in vitro tumor cell proliferation test₅₀The values are all greater than 100. mu.M.

Disclosure of Invention

To solve the foregoing problems, the present invention provides a diterpene derivative, a pharmaceutical composition for treating cancer comprising the diterpene derivative as an active ingredient, and a method for preparing a novel diterpene derivative.

In order to achieve the above object, the present invention provides the following technical solutions:

a diterpene derivative has a structure shown in a formula I:

the R is₁Is oxygen, hydroxyl or ester group; the R is₂Is oxygen, hydroxyl or ester group; the R is₃Is ethyl or vinyl.

Preferably, the diterpene derivative has a specific structure shown in formula II, formula III, formula IV, formula V or formula VI:

wherein R is₄As in one of compounds 3-26:

；R₅as in one of compounds 27-38:

；R₆or R₇Such as one of compounds 40-43:

；R₈such as one of compounds 44-45:

；R₉as in one of compounds 46-47:

the invention also provides a preparation method of the diterpene derivative, and the preparation route is route one, route two, route three, route four or route five;

route one:

and a second route:

and a third route:

and a fourth route:

and a fifth route:

the research of the inventor finds that the diterpene derivative can be applied to the preparation of medicines or foods for treating cancer, preventing cancer and/or improving cancer symptoms. Can be used as an active ingredient of a pharmaceutical composition for treating cancer, the composition further comprises a solvent or a pharmaceutically acceptable carrier.

The above cancers include: gynaecological cancers, for example: ovarian cancer, cervical cancer, vaginal cancer, pudendal cancer, uterine/endometrial cancer, gestational trophoblastic tumors, fallopian tube cancer, uterine sarcoma; endocrine cancers, for example: adrenocortical carcinoma, pituitary cancer, pancreatic cancer, thyroid cancer, parathyroid cancer, thymus gland cancer, multiple endocrine tumors; bone cancers, for example: osteosarcoma, Ewing's sarcoma, chondrosarcoma, etc.; lung cancers, for example: small cell lung cancer, non-small cell lung cancer; brain and CNS tumors, for example: neuroblastoma, acoustic neuroma, glioma and other brain tumors, spinal cord tumors, breast cancer, colorectal cancer, advanced colorectal adenocarcinoma; gastrointestinal cancers, for example: liver cancer, extrahepatic bile duct cancer, gastrointestinal carcinoid tumor, gallbladder cancer, gastric cancer, esophageal cancer, small intestine cancer; urogenital cancers, for example: penile cancer, emerald cancer, prostate cancer; head and neck tumors, for example: nasal cancer, sinus cancer, nasopharyngeal cancer, oral cancer, lip cancer, salivary gland cancer, laryngeal cancer, hypopharyngeal cancer, and eupharyngeal cancer; blood cancers, for example: acute myeloid leukemia, acute lymphoid leukemia, childhood leukemia, chronic lymphoid leukemia, chronic myeloid leukemia, hairy cell leukemia, acute promyelocytic leukemia, plasma cell leukemia; hematological disorders of bone marrow cancer, such as: myelodysplastic syndrome, myeloproliferative disorders, fanconi anemia, aplastic anemia, idiopathic macroglobulinemia; lymphoid cancers, for example: hodgkin's disease, non-hodgkin's lymphoma, peripheral T-cell lymphoma, cutaneous T-cell lymphoma, AIDS-related lymphoma; eye cancers, including: retinoblastoma, uveal melanoma; skin cancers, for example: melanoma, non-melanoma skin cancer, merkel cell carcinoma; soft tissue sarcomas, for example: kaposi's sarcoma, children's soft tissue sarcoma, adult's soft tissue sarcoma, urinary system cancers, for example: kidney cancer, wilms' tumor, cancer of the wing skin, cancer of the urethra, and metastatic cell cancer. Preferably used for treating breast cancer, colorectal cancer and lung cancer.

The invention has the beneficial effects that: the diterpene derivative provided by the invention shows strong activity for inhibiting proliferation of various tumor cells through pharmacological experiments, has obvious in-vitro and in-vivo anticancer activity, generally does not have antibiotic activity, and is suitable for development of antitumor drugs.

Drawings

FIG. 1 is a graph showing the results of a mouse model, in which FIG. 1(A) is a B16F10 xenograft tumor volume curve, FIG. 1(B) is a graph showing the quantitative analysis of tumor weight, FIG. 1(C) is a graph showing the quantitative analysis of mouse weight, and FIG. 1(D) is a graph showing the tumor removed from the mouse model.

Detailed Description

Unless defined otherwise, technical terms used in the following examples have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.

The invention will be described in detail with reference to the following examples.

Example 1: preparation of diterpene derivatives

(1) Preparation of compound 3:

the structure of compound 3 is as follows:

the preparation process comprises the following steps:

to a solution of pleuromutilin 1(7.00g, 22.1BmmoL) in ethanol (50mL) and water (32mL) was added 50% aqueous sodium hydroxide (3.70mL) at room temperature. After stirring at 65 ℃ for 1 hour, the reaction mixture was cooled to room temperature and filtered, and the filtrate was acidified to pH 2 with 1N hydrochloric acid and then diluted with ethyl acetate (200 mL); the organic phase was separated and the aqueous phase was extracted with ethyl acetate (3X 200 mL); the combined organic phases were washed with brine, dried over anhydrous sodium sulfate, concentrated in vacuo and used directly in the next step.

To a solution of the diol compound 2(5.50 g, 17.2 mmol) in N, N-dimethylformamide (60 ml) at room temperature were added tetramethylmethanediamine (60 ml) and acetic anhydride (60 ml), respectively, and after stirring overnight at 95 ℃, the reaction mixture was quenched with a saturated sodium bicarbonate solution (200ml) and diluted with ethyl acetate (200 ml); the organic phase was separated and the aqueous phase was extracted with ethyl acetate (3 × 200 ml); the combined organic phases were washed with brine, dried over anhydrous sodium sulfate and concentrated in vacuo to give the crude product; flash column chromatography on silica gel (petroleum ether: ethyl acetate 6: 1) gave compound 3(3.92 g, 68%, white solid).

Detecting Compound 3, the detection thereofThe measurement results are as follows: melting point 142-]_D ²¹＝-62.6°(c 1.45,CHCl₃).IR(KBr)ν_max 3563,3542,3500,2981,2929,2359,1744,1716,1455,1296,1029,996,921cm^-1.¹H NMR(400MHz,CDCl₃)δ6.16(dd,J＝17.8,11.1Hz,1H),6.07–6.02(m,1H),5.41–5.34(m,1H),5.30(d,J＝11.7Hz,2H),4.41–4.34(m,1H),3.41(t,J＝5.7Hz,1H),2.32(dt,J＝16.5,3.0Hz,1H),2.23–2.15(m,3H),1.94(dd,J＝15.9,7.7Hz,1H),1.75(dq,J＝14.4,2.8Hz,1H),1.68–1.56(m,3H),1.49(dd,J＝13.3,3.0Hz,1H),1.44(s,3H),1.36(ddd,J＝21.6,8.9,4.6Hz,2H),1.15(s,3H),1.06(dd,J＝14.0,4.4Hz,1H),0.98(d,J＝7.0Hz,3H),0.94(d,J＝7.1Hz,3H).¹³C NMR(100MHz,CDCl₃)δ205.5,143.4,139.4,118.3,115.9,75.1,66.7,59.6,45.3,45.1,43.1,42.8,37.0,36.1,34.8,31.5,28.6,27.1,18.2,13.6,11.5.HRMS(ESI):m/zcalcd for C₂₁H₃₁O₃ ^-[M-H]^-:331.2279；found 331.2276。

(2) Preparation of compound 4:

the structure of compound 4 is as follows:

the preparation process comprises the following steps:

to a solution of compound 3(200 mg, 0.602 mmol) in dichloromethane (2 ml) was added triethylamine (0.125 ml, 0.903 mmol) and DMAP (7.33 mg, 0.0602 mmol) at 0 ℃. After stirring for 10 minutes, acetic anhydride (68.0. mu.l, 0.722 mmol) was added. After stirring for 1 hour, the resulting mixture was quenched with water (3 ml) and diluted with dichloromethane (3 ml). The organic phase was separated and the aqueous phase was extracted with dichloromethane (3 × 5 ml). The combined organic phases were dried over anhydrous sodium sulfate and concentrated in vacuo to give the crude product which was purified by flash column chromatography on silica gel (PE: EA ═ 12:1) to give compound 4(126 mg, 56%, white solid).

Compound 4 was tested as follows: melting point 152-]_D ²¹＝-49.8°(c 1.24,CH Cl₃).IR(KBr)ν_max 3588,2991,2878,1712,1664,1358,1330,1240,1025,965c m^-1.¹H NMR(400MHz,CDCl₃)δ6.03(dd,J＝17.8,11.1Hz,1H),5.91(s,1H),5.25–5.08(m,3H),4.46(d,J＝8.8Hz,1H),3.08(d,J＝18.2Hz,1H),2.24–2.07(m,3H),2.02(s,3H),1.93(d,J＝13.5Hz,1H),1.77(dt,J＝13.3,2.8Hz,1H),1.73–1.65(m,2H),1.39(d,J＝3.9Hz,1H),1.35(s,3H),1.29(d,J＝12.1Hz,1H),1.15–0.99(m,2H),0.95(d,J＝6.5Hz,3H),0.86(s,3H),0.76(d,J＝7.1Hz,3H).¹³C NM R(100MHz,CDCl₃)δ203.5,170.6,146.8,141.9,114.3,113.5,79.2,49.5,47.7,46.5,44.3,39.4,37.6,37.1,34.9,33.9,27.9,27.1,27.0,20.7,19.4,12.8,12.3.HR MS(ESI):m/zcalcd for C₂₃H₃₄NaO₄ ⁺[M+Na]⁺:397.2349；found 397.2350。

(3) Preparation of compound 5:

the structure of compound 5 is as follows:

the preparation process comprises the following steps:

to a solution of compound 3(200 mg, 0.602 mmol) in dichloromethane (2 ml) was added N, N-diisopropylethylamine (0.124 ml, 0.722 mmol) at 0 ℃. After stirring for 10 min, propionyl chloride (58.0 μ l, 0.662 mmol) was added. After stirring for 1 hour, the resulting mixture was quenched with water (10 ml) and diluted with dichloromethane (10 ml). The organic phase was separated and the aqueous phase was extracted with dichloromethane (3 × 10 ml). The combined organic phases were dried over anhydrous sodium sulfate and concentrated in vacuo to give the crude product which was purified by flash column chromatography on silica gel (PE/EA ═ 12:1) to give compound 5(96.1 mg, 41%, white solid).

The compound 5 was detected as follows: melting point 156-]_D ²¹＝-46.6°(c 1.28,CHCl₃).IR(KBr)ν_max 3582,2987,2877,1718,1701,1640,1293,1223,1106,934cm^-1.¹H NMR(400MHz,CDCl₃)δ6.10(dd,J＝17.9,11.2Hz,1H),6.04–5.98(m,1H),5.43(d,J＝17.9Hz,1H),5.27(d,J＝11.2Hz,2H),4.82(d,J＝6.6Hz,1H),4.32(d,J＝7.2Hz,1H),2.68(d,J＝16.6Hz,1H),2.39(qd,J＝7.5,1.5Hz,2H),2.32–2.20(m,2H),2.07(d,J＝16.6Hz,1H),1.96(dd,J＝15.9,7.4Hz,1H),1.76–1.61(m,3H),1.45(d,J＝1.5Hz,4H),1.41–1.30(m,2H),1.27–1.21(m,1H),1.17(td,J＝7.6,1.6Hz,3H),1.07(td,J＝14.0,4.2Hz,1H),0.97(dd,J＝4.8,1.8H z,6H),0.78(d,J＝7.0Hz,3H).¹³C NMR(100MHz,CDCl3)δ205.4,173.8,143.5,140.0,118.2,115.6,76.6,66.9,59.5,44.6,44.2,42.9,42.7,36.9,36.4,34.9,31.5,29.0,27.6,27.2,18.2,13.6,12.0,9.3.HRMS(ESI):m/zcalcd for C₂₄H₃₆NaO₄[M+Na]⁺:411.2506；found 411.2508。

(4) Preparation of compound 6:

the structure of compound 6 is as follows:

the preparation process comprises the following steps:

to a solution of cyclobutanecarboxylic acid (120 mg, 1.20 mmol), N' -diisopropylcarbodiimide (0.152 ml, 0.963 mmol) and 4-dimethylaminopyridine (22.4 mg, 0.181 mmol) in N, N-dimethylformamide (2 ml) was added compound 3(200 mg, 0.602 mmol). After stirring at 40 ℃ overnight, the resulting mixture was quenched with water (3 ml) and diluted with ethyl acetate (3 ml). The organic phase was separated and the aqueous phase was extracted with ethyl acetate (3 × 5 ml). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate and concentrated in vacuo to give the crude product which was purified by flash column chromatography on silica gel (PE: EA ═ 12:1) to give compound 6(152 mg, 61%, white solid).

The compound 6 was detected as follows: melting point 190-]_D ²²＝-42.9°(c 1.53,CHCl₃).IR(KBr)ν_max 3554,2991,2953,2864,1718,1699,1402,1383,1357,1341,1027,923,892cm^-1.¹H NMR(400MHz,CDCl₃)δ6.11(ddd,J＝18.0,11.2,1.1Hz,1H),6.05–6.01(m,1H),5.44(dd,J＝17.9,1.3Hz,1H),5.29–5.25(m,2H),4.82(d,J＝6.7Hz,1H),4.33(t,J＝6.4Hz,1H),3.20(pd,J＝8.6,1.1Hz,1H),2.71(dt,J＝16.6,3.3Hz,1H),2.36–2.21(m,6H),2.08(dt,J＝16.5,1.7Hz,1H),2.04–1.95(m,2H),1.93(dd,J＝5.1,4.0Hz,1H),1.73–1.63(m,3H),1.58(s,1H),1.46(s,3H),1.34(dd,J＝20.9,4.5Hz,2H),1.12–1.04(m,1H),0.97(d,J＝7.0Hz,6H),0.78(d,J＝7.1Hz,3H).¹³C NMR(100MHz,CDCl₃)δ205.5,174.8,143.7,140.2,118.3,115.7,76.6,67.1,59.7,44.8,44.4,43.1,42.9,38.5,37.0,36.6,35.1,31.6,29.1,27.4,25.6,25.5,18.8,18.3,13.7,12.1.HR MS(ESI):m/zcalcd for C₂₆H₃₈NaO₄ ⁺[M+Na]⁺:437.2662；found 437.2669。

(5) Preparation of compound 7:

the structure of compound 7 is as follows:

the preparation process comprises the following steps:

the synthesis procedure described for compound 6 was followed using cyclopentanecarboxylic acid (137 mg, 1.20 mmol) to afford target compound 7. Flash column chromatography (petroleum ether: ethyl acetate: 12:1) afforded compound 7(100 mg, 39%, white solid).

(6) Preparation of compound 8:

the structure of compound 8 is as follows:

the preparation process comprises the following steps:

the synthesis procedure described for compound 6 was followed using 6-bromohexanoic acid (234 mg, 1.20 mmol) to afford target compound 8. Flash column chromatography (petroleum ether: ethyl acetate: 12:1) afforded compound 8(73.3 mg, 24%, white solid).

(7) Preparation of compound 9:

the structure of compound 9 is as follows:

the preparation process comprises the following steps:

the synthesis procedure described for compound 6 was followed using 8-bromooctanoic acid (268 mg, 1.20 mmol) to afford the title compound 9. Flash column chromatography (petroleum ether: ethyl acetate: 12:1) afforded compound 9(103 mg, 32%, white solid).

(8) Preparation of compound 10:

the structure of compound 10 is as follows:

the preparation process comprises the following steps:

di-tert-butyl dicarbonate (157 mg, 1.20 mmol) was used to obtain the title compound 10 following the synthetic procedure described for compound 4. Flash column chromatography (petroleum ether: ethyl acetate 12:1) afforded compound 10(150 mg, 58%, white solid).

(9) Preparation of compound 11:

the structure of compound 11 is as follows:

the preparation process comprises the following steps:

propyl carbonate (81.1 mg, 0.662 mmol) was used to obtain the title compound 11 following the procedure described for compound 5. Flash column chromatography (petroleum ether: ethyl acetate 12:1) afforded compound 11(146 mg, 58%, white solid).

(10) Preparation of compound 12:

the structure of compound 12 is as follows:

the preparation process comprises the following steps:

using isobutyl carbonate (90.0 mg, 0.662 mmol), the procedure described for Compound 5 was followed to afford Compound 12, title compound. Flash column chromatography (petroleum ether: ethyl acetate: 12:1) afforded compound 12(125 mg, 48%, white solid).

(11) Preparation of compound 13:

the structure of compound 13 is as follows:

the preparation process comprises the following steps:

the title compound 13 was obtained following the procedure described for compound 6, using benzoic acid (146 mg, 1.20 mmol). Flash column chromatography (petroleum ether: ethyl acetate: 12:1) afforded compound 13(68.3 mg, 26%, white solid).

(12) Preparation of compound 14:

the structure of compound 14 is as follows:

the preparation process comprises the following steps:

compound 14 was obtained following the synthetic procedure described for compound 6 using 4-fluorobenzoic acid (168 mg, 1.20 mmol). Flash column chromatography (polyethylene: ethyl acetate 12:1) afforded 14(109 mg, 40%, white solid).

(13) Preparation of compound 15:

the structure of compound 15 is as follows:

the preparation process comprises the following steps:

the synthesis procedure described for compound 6 was followed using 3-fluorobenzoic acid (168 mg, 1.20 mmol) to afford target compound 15. Flash column chromatography (petroleum ether: ethyl acetate: 12:1) afforded compound 15(161 mg, 59%, white solid).

The compound 15 was detected as follows: melting point 233-. [ alpha ] to]_D ²¹＝-38.6°(c 1.45,CHCl₃).IR(KBr)ν_max 3558,2981,2922,1716,1655,1538,1464,1129,974,758cm^-1.¹H NMR(400MHz,CDCl₃)δ7.86(d,J＝7.8Hz,1H),7.73(ddd,J＝9.3,2.7,1.5Hz,1H),7.46(td,J＝8.0,5.6Hz,1H),7.30(td,J＝8.3,2.0Hz,1H),6.26(dd,J＝17.9,11.2Hz,1H),6.07(s,1H),5.52(dd,J＝17.9,1.1Hz,1H),5.37(d,J＝11.3Hz,1H),5.30(s,1H),5.09(d,J＝6.8Hz,1H),4.40(t,J＝6.4Hz,1H),2.77(dt,J＝16.5,3.0Hz,1H),2.42(p,J＝7.0Hz,1H),2.35(d,J＝2.4Hz,1H),2.12(d,J＝16.5Hz,1H),2.05(dd,J＝16.0,7.5Hz,1H),1.75(d,J＝16.1Hz,1H),1.72–1.55(m,3H),1.49(s,3H),1.38(dd,J＝13.8,3.4Hz,2H),1.11(td,J＝14.0,4.3Hz,1H),1.02(s,3H),1.00(d,J＝7.1Hz,3H),0.84(d,J＝7.1Hz,3H).¹³C NMR(100MHz,CDCl₃)δ205.4,163.2(d,J_F-C＝335.6Hz),143.5,139.9,132.2(d,J_F-C＝7.5Hz),130.4(d,J_F-C＝7.6Hz),125.6,125.6,120.5(d,J_F-C＝21.7Hz),118.5,116.7(d,J_F-C＝22.9Hz),116.2,78.2,67.1,59.8,45.0,44.5,43.2,42.9,37.0,36.8,35.1,31.6,29.1,27.3,18.3,13.7,12.4.¹⁹F NMR(376MHz,CDCl₃)δ-62.7.HRMS(ESI):m/zcalcd for C₂₈H₃₅FNaO₄[M+Na]⁺:477.2412；found 477.2418。

(14) Preparation of compound 16:

the structure of compound 16 is as follows:

the preparation process comprises the following steps:

using 2-fluorobenzoic acid (168 mg, 1.20 mmol), the title compound 16 was obtained according to the procedure described in 6. Flash column chromatography (petroleum ether: ethyl acetate 12:1) afforded compound 16(139 mg, 51%, white solid).

(15) Preparation of compound 17:

the structure of compound 17 is as follows:

the preparation process comprises the following steps:

using 4- (trifluoromethyl) benzoic acid (228 mg, 1.20 mmol), the title compound 17 was obtained according to the procedure described in 6. Flash column chromatography (petroleum ether: ethyl acetate: 12:1) afforded compound 17(154 mg, 51%, white solid).

The compound 17 was detected as follows: melting point 175-. [ alpha ] to]_D ²¹＝-37.6°(c 1.19,CHCl₃).IR(KBr)ν_max 2985,2935,2863,1721,1645,1326,1311,1280,1170,1132,1101,862,773,704cm^-1.¹H NMR(400MHz,CDCl₃)δ8.17(d,J＝7.8H z,2H),7.73(d,J＝7.8Hz,2H),6.26(dd,J＝17.6,11.3Hz,1H),6.06(s,1H),5.52(d,J＝17.8Hz,1H),5.37(d,J＝11.2Hz,1H),5.29(s,1H),5.10(d,J＝6.2Hz,1H),4.39(d,J＝5.9Hz,1H),2.75(d,J＝16.1Hz,1H),2.49–2.38(m,1H),2.34(s,1H),2.11(d,J＝16.8Hz,1H),2.09–1.98(m,1H),1.81–1.62(m,3H),1.54–1.44(m,5H),1.37(d,J＝13.7Hz,1H),1.10(td,J＝16.7,15.5,7.6Hz,1H),1.01(s,3H),0.98(d,J＝6.7Hz,3H),0.83(d,J＝6.4Hz,3H).¹³C NMR(100MHz,CDCl₃)δ205.2,164.7,143.3,139.6,134.7(q,J_F-C＝32.5Hz),133.0,130.1,125.7(d,J_F-C＝3.6Hz),123.6(d,J_F-C＝274.1Hz),118.4,116.2,78.3,66.9,59.6,44.8,44.4,43.0,42.8,36.9,36.6,35.0,31.5,28.9,27.2,18.2,13.6,12.3.¹⁹F NMR(376MHz,CDCl₃)δ-63.1.HRMS(ESI):m/zcalcd for C₂₉H₃₅F₃NaO₄[M+Na]⁺:527.2380；found 527.2384。

(16) Preparation of compound 18:

the structure of compound 18 is as follows:

the preparation process comprises the following steps:

the synthesis procedure described for compound 6 was followed using 3- (trifluoromethyl) benzoic acid (228 mg, 1.20 mmol) to afford target compound 18. Flash column chromatography (petroleum ether: ethyl acetate: 12:1) afforded compound 18(124 mg, 41%, white solid).

(17) Preparation of compound 19:

the structure of compound 19 is as follows:

the preparation process comprises the following steps:

using anisic acid (186 mg, 1.20 mmol), the procedure described for the synthesis of compound 6 was followed to afford the title compound 19. Flash column chromatography (petroleum ether: ethyl acetate: 12:1) afforded compound 19(84.4 mg, 30%, white solid).

(18) Preparation of compound 20:

the structure of compound 20 is as follows:

the preparation process comprises the following steps:

target compound 20 was obtained according to the synthetic procedure described for compound 6 using 3-phenoxybenzoic acid (257 mg, 1.20 mmol). Flash column chromatography (petroleum ether: ethyl acetate: 12:1) afforded compound 20(152 mg, 48%, white solid).

(19) Preparation of compound 21:

the structure of compound 21 is as follows:

the preparation process comprises the following steps:

the title compound 21 was obtained following the synthetic procedure described for compound 6 using p-nitrobenzoic acid (200 mg, 1.20 mmol). Flash column chromatography (petroleum ether: ethyl acetate: 12:1) afforded compound 21(162 mg, 78%, white solid).

(20) Preparation of compound 22:

the structure of compound 22 is as follows:

the preparation process comprises the following steps:

the title compound 22 was obtained following the synthetic procedure described for compound 6 using 4-bromobenzoic acid (241 mg, 1.20 mmol). Flash column chromatography (petroleum ether: ethyl acetate: 12:1) afforded compound 22(178 mg, 56%, white solid).

The compound 22 was tested as follows: melting point 143-]_D ²²＝-44.6°(c 1.28,CHCl₃).IR(KBr)ν_max 3582,2980,2901,1720,1663,1431,1330,1301,1185,979cm^-1.¹H NMR(400MHz,CDCl₃)δ7.82(d,J＝8.3Hz,1H),7.48–7.39(m,2H),6.21(dd,J＝17.9,11.2Hz,1H),6.09–6.03(m,1H),5.48(d,J＝17.8Hz,1H),5.35–5.28(m,2H),5.09(d,J＝6.7Hz,1H),4.38(t,J＝6.1Hz,1H),2.79(d,J＝3.3Hz,1H),2.60(s,3H),2.43–2.34(m,2H),2.11(dt,J＝16.6,1.9Hz,1H),2.07–2.00(m,1H),1.72(s,3H),1.56–1.49(m,1H),1.47(s,3H),1.43–1.33(m,2H),1.12(dd,J＝14.2,4.6Hz,1H),1.01(s,3H),0.99(d,J＝7.0Hz,3H),0.85(d,J＝7.0Hz,3H).¹³C NMR(100MHz,CDCl₃)δ205.4,165.9,143.5,143.1,140.0,134.9,131.9,129.3,128.0,127.1,118.4,116.1,77.5,67.0,59.7,44.9,44.5,43.1,42.8,37.0,36.7,35.1,31.6,29.1,27.3,22.0,18.3,13.7,12.6.H RMS(ESI):m/zcalcd for C₂₇H₄₁ ⁷⁹BrNaO₄ ⁺[M+Na]⁺:531.2080；found 531.2085。

(21) Preparation of compound 23:

the structure of compound 23 is as follows:

the preparation process comprises the following steps:

using 3-bromo-2-methylbenzoic acid (258 mg, 1.20 mmol), the title compound 23 was obtained according to the procedure described in 6. Flash column chromatography (petroleum ether: ethyl acetate: 12:1) afforded compound 23(200 mg, 63%, white solid).

(22) Preparation of compound 24:

the structure of compound 24 is as follows:

the preparation process comprises the following steps:

using 5-trifluoromethylpyridine-2-carboxylic acid (229 mg, 1.20 mmol), the title compound 24 was obtained according to the procedure described in 6. Flash column chromatography (petroleum ether: ethyl acetate: 12:1) afforded compound 24(200 mg, 63%, white solid).

(23) Preparation of compound 25:

the structure of compound 25 is as follows:

the preparation process comprises the following steps:

the title compound 25 was obtained following the synthetic procedure described for compound 6 using 4-fluorophenylacetic acid (185 mg, 1.20 mmol). Flash column chromatography (petroleum ether: ethyl acetate: 12:1) afforded compound 25(152 mg, 54%, white solid).

(24) Preparation of compound 26:

the structure of compound 26 is as follows:

the preparation process comprises the following steps:

the title compound 26 was obtained following the synthetic procedure described for compound 4 using trifluoroacetic anhydride (151 mg, 0.722 mmol). Flash column chromatography (petroleum ether: ethyl acetate 12:1) afforded compound 26(137 mg, 53%, white solid).

(25) Preparation of compound 27:

the structure of compound 27 is as follows:

the preparation process comprises the following steps:

to a solution of α -methylenecarbonyl 3(200 mg, 0.602 mmol) in dichloromethane (2 ml) was added triethylamine (0.248 ml, 1.81 mmol) and 4-dimethylaminopyridine (14.6 mg, 0.120 mmol) at 0 ℃. After stirring for 10 min, acetic anhydride (0.142 ml, 1.44 mmol) was added. After stirring for 1 hour, the resulting mixture was quenched with water (3 ml) and diluted with dichloromethane (3 ml). The organic phase was separated and the aqueous phase was extracted with dichloromethane (3X 5 mL). The combined organic phases were dried over anhydrous sodium sulfate and concentrated in vacuo to give the crude product. Purification by flash column chromatography on silica gel (petroleum ether: ethyl acetate 18: 1) gave compound 27(223 mg, 89%, white solid).

(26) Preparation of compound 28:

the structure of compound 28 is as follows:

the preparation process comprises the following steps:

to a solution of cyclobutanecarboxylic acid (240 mg, 2.40 mmol), triethylamine (0.335 ml, 1.93 mmol) and 4-dimethylaminopyridine (44.2 mg, 0.362 mmol) in N, N-dimethylformamide (2 ml) was added α -methylenecarbonyl compound 3(200 mg, 0.602 mmol). After stirring at 40 ℃ overnight, the resulting mixture was quenched with water (3 ml) and diluted with ethyl acetate (3 ml). The organic phase was separated and the aqueous phase was extracted with ethyl acetate (3 × 5 ml). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate and concentrated in vacuo to give the crude product. Purification by flash column chromatography on silica gel (12: 1 petroleum ether/ethyl acetate) gave 28(225 mg, 76%, white solid).

(27) Preparation of compound 29:

the structure of compound 29 is as follows:

the preparation process comprises the following steps:

the title compound 29 was obtained following the synthetic procedure described for compound 28 using 6-bromohexanoic acid (468 mg, 2.40 mmol). Flash column chromatography (petroleum ether: ethyl acetate 18: 1) afforded compound 29(201 mg, 50%, white solid).

(28) Preparation of compound 30:

the structure of compound 30 is as follows:

the preparation process comprises the following steps:

title compound 30 was obtained following the procedure described for compound 28 using 8-bromooctanoic acid (535 mg, 2.40 mmol). Flash column chromatography (petroleum ether: ethyl acetate 18: 1) afforded 30(187 mg, 42%, white solid).

(29) Preparation of compound 31:

the structure of compound 31 is as follows:

the preparation process comprises the following steps:

using cyclopropanecarboxylic acid (207 mg, 2.40 mmol), the title compound 31 was obtained following the procedure described for compound 28. Flash column chromatography (petroleum ether: ethyl acetate 18: 1) afforded title compound 31(149 mg, 53%, white solid).

(30) Preparation of compound 32:

the structure of compound 32 is as follows:

the preparation process comprises the following steps:

the title compound 32 was obtained following the synthetic procedure described for compound 28 using cyclobutanecarboxylic acid (240 mg, 2.40 mmol). Flash column chromatography (petroleum ether: ethyl acetate 18: 1) afforded compound 32(125 mg, 42%, white solid).

(31) Preparation of compound 33:

the structure of compound 33 is as follows:

the preparation process comprises the following steps:

using cyclopentanecarboxylic acid (274 mg, 2.40 mmol), the title compound 33 was obtained according to the procedure described for 28. Flash column chromatography (petroleum ether: ethyl acetate 18: 1) afforded compound 33(139 mg, 44%, white solid).

(32) Preparation of compound 34:

compound 34 has the structure:

the preparation process comprises the following steps:

target compound 34 was obtained according to the procedure described for compound 28 using 4- (trifluoromethyl) benzoic acid (456 mg, 2.40 mmol). Flash column chromatography (petroleum ether: ethyl acetate 18: 1) afforded compound 34(240 mg, 59%, white solid).

(33) Preparation of compound 35:

compound 35 has the structure:

the preparation process comprises the following steps:

the title compound 35 was obtained according to the procedure described for compound 28, using 3-fluorobenzoic acid (336 mg, 2.40 mmol). Flash column chromatography (petroleum ether: ethyl acetate 18: 1) afforded compound 35(173 mg, 50%, white solid).

(34) Preparation of compound 36:

the structure of compound 36 is as follows:

the preparation process comprises the following steps:

target compound 36 was obtained as described for 28 using 3-phenoxybenzoic acid (514 mg, 2.40 mmol). Flash column chromatography (petroleum ether: ethyl acetate 18: 1) afforded compound 36(222 mg, 51%, white solid).

(35) Preparation of compound 37:

the structure of compound 37 is as follows:

the preparation process comprises the following steps:

the title compound 37 was obtained according to the synthetic procedure described for compound 28 using 2-chloro-6-methylisonicotinic acid (412 mg, 2.40 mmol). Flash column chromatography (petroleum ether: ethyl acetate 18: 1) afforded compound 37(165 mg, 43% white solid).

(36) Preparation of compound 38:

the structure of compound 38 is as follows:

the preparation process comprises the following steps:

using 5- (trifluoromethyl) picolinic acid (459 mg, 2.40 mmol), title compound 38 was obtained according to the procedure described for 28. Flash column chromatography (petroleum ether: ethyl acetate 18: 1) afforded compound 38(248 mg, 61%, white solid).

The compound 38 was tested as follows: melting point 204-. [ alpha ] to]_D ²¹＝-18.8°(c 1.45,CHCl₃).IR(KBr)ν_max 2989,2964,1722,1331,1310,1170,1140,1077,1016,941,853,706cm^-1.¹H NMR(400MHz,CDCl₃d)δ9.04(d,J＝8.1Hz,2H),8.19(d,J＝8.0Hz,2H),8.16–8.07(m,2H),6.59(dd,J＝17.5,11.2Hz,1H),6.08(s,2H),5.46–5.32(m,2H),5.31(s,1H),5.25(d,J＝6.7Hz,1H),2.85(d,J＝16.6Hz,1H),2.76(p,J＝6.9Hz,1H),2.54–2.49(m,1H),2.38(dd,J＝16.1,8.3Hz,1H),2.15(d,J＝16.6Hz,1H),1.84–1.57(m,7H),1.39(d,J＝12.5Hz,1H),1.20–1.03(m,4H),0.90(d,J＝6.9Hz,3H),0.69(d,J＝6.4Hz,3H).¹³C NMR(100MHz,CDCl₃)δ204.5,163.2,162.6,151.4,150.6,147.3(d,J_F-C＝4.6Hz),147.1(d,J_F-C＝4.0H z),143.2,139.3,136.1(d,J_F-C＝120.2Hz),134.8(dd,J_F-C＝6.7,3.5Hz),130.0(d,J_F-C＝37.2Hz),129.6(q,J_F-C＝32.9Hz),129.4(q,J_F-C＝33.4Hz),124.7,124.7,123.0(d,J_F-C＝273.0Hz),119.0,117.1,79.2,70.7,58.9,45.3,43.4,43.3,42.9,36.9,36.1,35.0,31.5,27.4,27.0,16.7,15.3,12.3.¹⁹F NMR(376MHz,CDCl₃)δ-62.7,-62.7.HRMS(ESI):m/zcalcd for C₃₅H₃₆F₆N₂NaO₅ ⁺[M+Na]⁺:701.2421；found 701.2425。

(37) Preparation of compound 40:

the structure of compound 40 is as follows:

the preparation process comprises the following steps:

compound 39(2.00 g, 6.32 mmol) was used to obtain the title compound 40 following the synthetic procedure described for compound 3. Flash column chromatography (petroleum ether: ethyl acetate 6: 1) afforded compound 40(1.14 g, 56%, white solid).

(38) Preparation of compound 41:

the structure of compound 41 is as follows:

the preparation process comprises the following steps:

the target compound 41 was obtained according to the procedure described for compound 4 using acetic anhydride (68.0 μ l, 0.722 mmol). Flash column chromatography (petroleum ether: ethyl acetate: 12:1) afforded compound 41(133 mg, 59%, white solid).

(39) Preparation of compound 42:

the structure of compound 42 is as follows:

the preparation process comprises the following steps:

the target compound 42 was obtained according to the procedure described for compound 27 using acetic anhydride (0.136 ml, 1.44 mmol). Flash column chromatography (petroleum ether: ethyl acetate 18: 1) afforded 42(173 mg, 69%, white solid).

(40) Preparation of compound 43:

the structure of compound 43 is as follows:

the preparation process comprises the following steps:

the title compound 43 was obtained according to the synthesis procedure described for compound 28 using 5- (trifluoromethyl) picolinic acid (459 mg, 2.40 mmol). Flash column chromatography (polyethylene: ethyl acetate 18: 1) afforded 43(236 mg, 58%, white solid).

(41) Preparation of compound 44:

the structure of compound 44 is as follows:

the preparation process comprises the following steps:

to a solution of compound 3(200 mg, 0.602 mmol) in dichloromethane (5 ml) was added dess-martin oxidizer (305 mg, 0.662 mmol) at room temperature. After stirring for 1 hour, the reaction was diluted with 30 ml of saturated sodium thiosulfate solution and 30 ml of saturated sodium bicarbonate solution, and the resulting mixture was diluted with dichloromethane (20 ml). The organic phase was separated and the aqueous phase was extracted with dichloromethane (3 × 5 ml). The combined organic phases were dried over anhydrous sodium sulfate and concentrated in vacuo to give the crude product which was purified by flash column chromatography on silica gel (PE: EA ═ 12:1) to give compound 44(169 mg, 85%, white solid).

(42) Preparation of compound 45:

compound 45 has the following structure:

the preparation process comprises the following steps:

to a solution of diol compound 3(200 mg, 0.602 mmol) in dichloromethane (5 ml) was added dess-martin oxidizer (560 mg, 1.32 mmol) at room temperature. After stirring for 1 hour, the reaction was diluted with 30 ml of saturated sodium thiosulfate solution and 30 ml of saturated sodium bicarbonate solution, and the resulting mixture was diluted with dichloromethane (20 ml). The organic phase was separated and the aqueous phase was extracted with dichloromethane (3 × 5 ml). The combined organic phases were dried over anhydrous sodium sulfate and concentrated in vacuo to give the crude product which was purified by flash column chromatography on silica gel (PE: EA ═ 12:1) to give compound 45(158 mg, 80%, white solid).

(43) Preparation of compound 46:

the structure of compound 46 is as follows:

the preparation process comprises the following steps:

target compound 46 was obtained following the synthetic procedure described for compound 44 using 40(200 mg, 0.602 mmol). Flash column chromatography (petroleum ether: ethyl acetate 12:1) afforded compound 46(136 mg, 68%, white solid).

(44) Preparation of compound 47:

compound 47 has the structure:

the preparation process comprises the following steps:

target compound 47 was obtained following the synthetic procedure described for compound 45 using 40(200 mg, 0.602 mmol). Flash column chromatography (petroleum ether: ethyl acetate 18: 1) afforded compound 47(163 mg, 82%, white solid).

Effect verification example:

in vitro cancer cell inhibition effect of monoterpene and diterpene derivatives

The inhibition effect of the pentacyclic triterpene derivative on cancer cells is detected by adopting a tetramethyltetrazole blue (MTT) colorimetric method.

MTT colorimetric assay procedure: cancer cells in logarithmic growth phase were measured at 5X 10 per ml⁴The density of cell numbers was seeded into 96-well cell culture plates and the wells were zeroed to normal medium without cells. Changing culture medium containing different concentration gradient iso-pentacyclic triterpene derivatives after 12 hours, changing normal culture in zero setting holeThe concentration gradient is provided with 5 compound holes which are placed at 37 ℃ and 5 percent CO₂Culturing in an incubator. After 24 hours, the state and growth of the cells were observed under a microscope. After 48 hours, a solution of tetramethylazodicarbonamide (Kaiybe, 5mg/mL) was added to each well and the reaction was continued at 37 ℃ with 5% CO₂Culturing in incubator, sucking out culture medium after 4 hr, adding DMSO 100 μ L into each well, measuring absorbance value at 570 nm wavelength with microplate reader, performing data statistical analysis with Graphpad software, and calculating half effective concentration (IC)₅₀The unit: μ M). The inhibitory effect is shown in table 1 below.

TABLE 1 Effect of diterpene derivatives on the proliferation of different cancer cell lines

Wherein 4T1, A375, B16F10, HCT-116, CT-26 and Kasumi-1 respectively represent mouse mammary gland tumor cells, human malignant melanoma cells, mouse melanoma cells, human colorectal adenocarcinoma cells, mouse colon carcinoma cells and human acute myelogenous leukemia cells.

Taken together, these synthetic analogs have a significantly enhanced (at least 50-fold stronger) antiproliferative effect compared to the native pleuromutilins. Malignant melanoma cells B16F10 were most sensitive to the therapeutic effects of the synthetic compounds. Compounds 15, 17, 22 and 38 have better anticancer activity and are therefore useful for subsequent studies.

Detection of antibacterial activity of diterpene derivatives

To verify whether the above-synthesized diterpene derivatives still have antibacterial activity, the test determined the Minimum Inhibitory Concentrations (MIC) of the selected compounds against staphylococcus aureus, escherichia coli, and bacillus subtilis, with pleuromutilin as a positive control (table 2).

TABLE 2 antibacterial Activity

As expected, pleuromutilin is a potent broad spectrum antibiotic, however the chosen analogue has no antibacterial activity (MIC >1000 μ g/mL). Based on the above results, structural modification of pleuromutilin can significantly improve its anticancer activity and reduce its antibacterial activity.

Third, in vivo anti-tumor effect

Test subjects: the selected animals for the test are C57BL/6J mice, female, 18-22 g, SPF grade.

Test inoculation content: B16F10 cells loaded tumors subcutaneously.

The method is a mouse subcutaneous tumor-bearing method, and cultured tumor cells are inoculated to the axilla of the back of a mouse one by one. And regrouping the tumor body after the obvious tumor body is observed at the tumor-bearing part to ensure that the size and the weight of the tumor body are uniformly distributed in each group, wherein the mice in the model control group are administered with 0.1mL of physiological saline every day, and the administration dose of the compound 38 is 20 mg/kg. Is treated by intraperitoneal injection. Mouse body weight and tumor size were recorded once daily (V ═ ab2/2, a for the tumor major diameter, b for the tumor minor diameter), mice were sacrificed two weeks after dosing and tumors were harvested. The results are shown in FIG. 1, in which FIG. 1(A) is a B16F10 xenograft tumor volume curve, FIG. 1(B) is a graph showing the quantitative analysis of tumor weight, FIG. 1(C) is a graph showing the quantitative analysis of mouse weight, and FIG. 1(D) is a tumor image removed from a mouse model.

The results show that compound 38 can significantly inhibit tumor growth and has little effect on mouse body weight.