Ent-6, 7-closed-loop kaurene type oridonin hydroxyl modified derivative as well as preparation method and application thereof

Abstract

The invention discloses an ent-6, 7-closed-loop kaurene type oridonin hydroxyl modified derivative as well as a preparation method and application thereof. The derivative is an A-ring and 6, 7 and 14-site hydroxyl modified oridonin derivative with a structure as shown in a general formula I, and an optical active body or racemate, a diastereoisomer mixture or a medicinal salt of the oridonin derivative; the derivative has stronger anti-tumor activity, can obviously inhibit proliferation of various malignant tumor cells such as breast cancer and liver cancer, also has good pharmacokinetic stability and solubility, is low in toxic and side effects, and has great clinical application prospects. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a hydroxyl-modified oridonin derivative, in particular to an ent-6,7-closed-ring kaurene-type hydroxyl-modified oridonin derivative, and also to a preparation method and application of the hydroxyl-modified oridonin derivative. Background Art

[0002] Oridonin is a tetracyclic diterpene of the enantio-kaurane type and one of the main active ingredients of the plant Rabdosia of the Lamiaceae family. It has multiple physiological activities such as antibacterial, anti-inflammatory and anti-tumor. Oridonin has been used to treat a variety of cancers and has good inhibitory activity against pancreatic cancer, lung cancer, colorectal cancer and other tumor cells.

[0003] Studies have found that oridonin A exerts its anticancer activity mainly by inducing cell cycle arrest, apoptosis, autophagy and their cross-effects. Its unique α-methylenecyclopentanone structure has been confirmed as the core mechanism for exerting antitumor and antibacterial activities. Its tumor inhibition has been verified in in vivo and in vitro experiments and has shown excellent biological activity. In addition, its unique α-methylenecyclopentanone structure has been confirmed as the core for exerting antitumor and antibacterial activities. Its tumor inhibition has been verified in in vivo and in vitro experiments and has shown excellent biological activity. In vitro experimental results show that oridonin A has a good inhibitory effect on human esophageal squamous cell carcinoma cells, sl80, Ehrlich ascites carcinoma, human liver cancer BEL-7402, reticulum cell sarcoma solid type, lung cancer SPCA-1, breast cancer and leukemia. However, due to the poor water solubility, unknown target and poor selectivity of oridonin A, its clinical application as an antitumor drug is severely limited, and new drug compounds with stronger antitumor activity and lower toxicity are urgently needed. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide an ent-6,7-closed-ring kaurene-type oridonin A hydroxy-modified derivative having more excellent inhibitory activity against a variety of tumor cells, and also to provide a method for preparing the above-mentioned derivative and its application in anti-tumor drugs.

[0005] Technical solution: The present invention discloses an ent-6,7-closed-ring kaurene-type Rubescensine A hydroxyl-modified derivative, which is a Rubescensine A derivative with modified A-ring and 6, 7, and 14 hydroxyl groups, and has a structure as shown in Formula I:

[0006]

[0007] Wherein: R is selected from hydrogen atom, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, C 6-10Aryl, C 3-8 Cycloalkyl, oxirane, aziridine, thiirane, oxetane, azetidine, thietane, tetrahydrofuran, tetrahydropyrrole, tetrahydrothiophene, pyridine, pyrimidine, pyrazine, piperazine, piperidine, morpholine, pyrrole, furan, thiophene, imidazole, pyrazole, oxazole, thiazole, 5-6 membered aromatic ring, 5-6 membered aromatic ring, 6-6 membered aromatic ring, quinoline, isoquinoline, indole, benzofuran or benzothiophene;

[0008] or R is substituted by 1-4 identical or different R1: said R1 is selected from hydrogen atom, halogen, hydroxyl, cyano, nitro, carboxyl, -NH2, sulfonyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino; said C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino is substituted by 1, 2 or 3 substituents, said substituents are selected from halogen, hydroxyl, cyano, C1-C6 alkyl, C3-C7 cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl or phenyl;

[0009] n is 1, 2 or 3.

[0010] Wherein, the derivative is preferably: R is selected from C1-C6 alkyl, C1-C6 alkoxy, C 6-10 Aryl, C 3-8 Cycloalkyl, aziridine, thiirane, oxetane, tetrahydrofuran, tetrahydropyrrole, pyridine, pyrimidine, piperidine, morpholine, pyrrole, furan, imidazole, pyrazole, quinoline, indole, benzofuran; or R is substituted by 1-4 identical or different R1: R1 is selected from hydrogen atom, halogen, nitro, -NH2, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino.

[0011] Among them, the derivative is further preferably: R is selected from C1-C6 alkyl, C1-C6 alkoxy, C 6-10 Aryl, C 3-8 Cycloalkyl, aziridine, thiirane, oxetane, pyridine, pyrimidine, furan, imidazole, pyrazole, quinoline, indole, benzofuran; or R is substituted by 1-4 identical or different R1: R1 is selected from halogen, nitro, -NH2, C1-C6 alkoxy, C1-C6 alkylamino; n is 1 or 2.

[0012] Wherein, the derivative is any of the following structures:

[0013]

[0014]

[0015] The derivatives also include optically active forms or racemates, diastereoisomer mixtures, or pharmaceutically acceptable salts thereof.

[0016] The preparation method of the above derivative comprises the following steps:

[0017] (1) dissolving Rubescensine A in anhydrous acetone, adding 2,2-dimethoxypropane and p-toluenesulfonic acid, and reflux reaction. After the reaction is completed, cooling to room temperature, extracting, washing, drying, and concentrating to obtain compound II;

[0018] (2) dissolving compound II in dichloromethane, adding triethylamine, and dropping methanesulfonyl chloride to react. After the reaction is completed, cooling to room temperature, extracting, washing, drying, and concentrating to obtain compound III;

[0019] (3) Compound III was dissolved in DMF, and lithium carbonate and lithium bromide were added to react. After the reaction was completed, the mixture was cooled to room temperature, filtered, washed, concentrated, and dried by spin drying. Compound IV was quickly passed through a column with dichloromethane;

[0020] (4) Compound IV was dissolved in tetrahydrofuran, and hydrochloric acid solution was added to react. After the reaction was completed, the compound was dried by spin drying, washed with water, concentrated, and subjected to column chromatography to obtain compound V;

[0021] (5) Compound V was dissolved in dichloromethane and reacted at room temperature. After the reaction was completed, a small amount of water was added to quench the reaction. The compound was extracted, washed, dried, filtered, vacuum concentrated, and subjected to column chromatography to obtain compound VI.

[0022] (6) Compound VI and formaldehyde are dissolved in a mixed solution of toluene and ethanol, p-toluenesulfonic acid is added, and the mixture is heated under reflux for reaction; or Compound VI is dissolved in ethylene glycol dimethyl ether, and NaOH and methyl chloroacetate are added, and the mixture is heated under reflux for reaction; after the reaction is completed, vacuum concentration is performed, and column chromatography is performed to obtain an ent-6,7-closed-ring kaurene-type oridonin A hydroxy-modified derivative, and the general reaction formula is as follows:

[0023]

[0024] The present invention also discloses a pharmaceutical composition, comprising the above derivative, and its optically active form or racemic form, diastereoisomer mixture, or its pharmaceutically acceptable salt or mixture and pharmaceutically acceptable auxiliary materials or carriers.

[0025] The ent-6,7-closed-ring kaurene-type Rubescensine A hydroxy-modified derivative or pharmaceutical composition can also be used in the preparation of anti-tumor drugs.

[0026] Wherein, the tumor includes triple-negative breast cancer, leukemia, colon cancer or liver cancer.

[0027] The application is that the ent-6,7-closed-ring kaurene-type Rubescensine A hydroxy-modified derivative exerts anti-cancer activity by inducing cell cycle arrest, cell apoptosis, cell autophagy and their cross-effects.

[0028] Definitions of terms used in the present invention: Unless otherwise specified, the initial definitions provided for the groups or terms in this document are used for the groups or terms throughout the specification; for terms that are not specifically defined in this document, the meaning that can be given by a person skilled in the art should be given based on the disclosure and context. The term "substituted" refers to the replacement of a hydrogen atom or molecule in a molecule, including one or more substituents.

[0029] The term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), iodine (I) or astatine (At) ions.

[0030] The term "alkyl" means a straight or branched chain saturated hydrocarbon group having the stated number of carbon atoms. 16 "Aliphatic chain" refers to a straight or branched saturated hydrocarbon group having 1 to 6 carbon atoms. C1-C6 alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, 2,2-dimethylbutyl and 2,3-dimethylbutyl, etc.

[0031] The term "alkenyl" refers to a non-aromatic hydrocarbon group that is linear, branched or cyclic, and has a main chain containing 2 to 6 carbon atoms and at least one carbon-carbon double bond. Thus, "C2-C6 alkenyl" refers to an alkenyl group having a main chain of 2 to 6 carbon atoms. Alkenyl groups include ethenyl, propenyl, butenyl, 2-methylbutenyl and cyclohexenyl, etc. The linear, branched or cyclic portion of the alkenyl group may contain double bonds and may be substituted if a substituted alkenyl group is indicated.

[0032] The term "alkynyl" refers to a non-aromatic hydrocarbon group that is linear, branched or cyclic, and has 2 to 6 carbon atoms in the main chain and at least one carbon-carbon triple bond. Thus, "C2-C6 alkynyl" refers to an alkynyl group having 2 to 6 carbon atoms. Alkynyl groups include ethynyl, propynyl, butynyl, 3-methylbutynyl, and the like. The linear, branched or cyclic portion of the alkynyl group may contain a triple bond and may be substituted if a substituted alkynyl group is indicated.

[0033] The term "heterocycle" refers to a saturated cyclic group of 3 to 6 ring atoms, one or two of which are selected from N, O or S(O). m (wherein m is an integer from 0 to 2), the remaining ring atoms are C, one or two of which may be optionally replaced by a carbonyl group. The heterocyclic ring may be optionally substituted independently by one, two or three substituents.

[0034] The term "aromatic ring", "aryl" refers to an all-carbon monocyclic or fused polycyclic group of 6 to 10 carbon atoms, with a completely conjugated π electron system. Non-limiting examples of aromatic rings or aryl groups are phenyl, naphthyl and anthracenyl.

[0035] The term "heteroaromatic ring", "heteroaryl" means a monocyclic or fused ring group of 5 to 10 ring atoms, containing one, two, three or four ring heteroatoms selected from N, O or S, the remaining ring atoms being C, and having a completely conjugated π electron system. Non-limiting examples of unsubstituted heteroaromatic groups are pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline, purine, tetrazole, triazine and carbazole. The heteroaromatic ring or heteroaryl group may be substituted or unsubstituted. When substituted, the substituents are preferably one or more, more preferably one, two or three, and even more preferably one or two.

[0036] Principle of the invention: The ent-6,7-closed-ring kaurene-type Rubescensine A hydroxyl-modified derivative of the present invention is a Rubescensine A derivative with modified A-ring and 6, 7, and 14 hydroxyl groups. The 14 hydroxyl group is sulfonylated, and the two hydrogen bond receptors in the sulfonyl structure increase the binding force between the compound and the receptor; the 6 and 7 hydroxyl groups are protected by cyclization technology to improve the lipid solubility of the compound, thereby enhancing the anti-tumor activity and reducing the toxicity to normal cells; the present invention improves and optimizes the structure of Rubescensine A, designs and synthesizes a series of ent-6,7-closed-ring kaurene-type Rubescensine A hydroxyl-modified derivatives with more excellent inhibitory activity against a variety of tumor cells.

[0037] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The ent-6,7-closed-ring kaurene-type oridonin hydroxy-modified derivatives of the present invention have stronger anti-tumor activity and can significantly inhibit the proliferation of various malignant tumor cells such as breast cancer and liver cancer; (2) The derivatives of the present invention also have good pharmacokinetic stability and solubility, low toxic and side effects, and have great clinical application prospects. DETAILED DESCRIPTION

[0038] The technical scheme of the present invention is further described below in conjunction with the examples. The test materials used in the examples can all be purchased through conventional channels.

[0039] Example 1

[0040] The compound 1 of the present invention is shown below:

[0041]

[0042] The compound 1 of the present invention can be synthesized according to the following reaction route:

[0043]

[0044] (1) Preparation of compound (II): Rubescensine A (3 g, 8.23 ​​mmol) was dissolved in 40 mL of anhydrous acetone, and 3 mL of 2,2-dimethoxypropane and a catalytic amount of p-toluenesulfonic acid (150 mg) were added. The mixture was refluxed for 10 min under drying tube protection until the solution turned from white turbidity to clarity. 3 mL of 2,2-dimethoxypropane was added and the reaction was monitored by TLC until the starting material disappeared. The mixture was cooled to room temperature and saturated NaHCO3 solution was added. The mixture was extracted with dichloromethane three times, 30 mL each time, washed twice with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain 3.17 g (yield 95%) of a white solid, which was directly used in the next step. 1 H NMR(500MHz, CDCl3) δ5.69(d,J=3.3Hz,2H),4.12(d,J=11.0Hz,1H),4.02(d,J=11.2Hz,1H),3.84(dd,J=6 .8,5.1Hz,1H),3.42-3.36(m,2H),3.31-3.23(m,1H),3.03(d,J=5.1Hz,1H),2.47-2.37(m,1H),2.32(dddd ,J=8.1,4.7,3.3,2.3Hz,1H),1.77-1.71(m,1H),1.74-1.66(m,1H),1.70-1.63(m,1H),1.66-1.57(m,2H) ,1.60-1.49(m,1H),1.51-1.42(m,1H),1.40-1.30(m,1H),1.00(s,5H),0.94(ddd,J=6.7,3.8,2.6Hz,1H); 13 C NMR (125MHz, CDCl3) δ206.53,153.58,118.42,100.94,98.20,74.35,73.46,72.30,65.6 5,61.19,60.68,50.88,46.64,41.35,34.75,34.15,29.80,29.04,28.00,25.83; ESI-MS m / z:405.2272calcd for C 23 H 33 O6[M+H] + 405.2278.

[0045] (2) Preparation of compound (III): The product of step 1 was dissolved in 30 mL of pre-dried dichloromethane, and 2 mL of triethylamine was added. The mixture was stirred in an ice bath, and 2 mL of methanesulfonyl chloride was added dropwise over 15 min. The mixture was stirred for 1 h. After TLC detection, the starting material disappeared. 80 mL of dichloromethane was added, and the mixture was washed twice with water and once with saturated brine (20 mL each time). The mixture was dried over anhydrous sodium sulfate and concentrated to obtain 3 g of a light yellow solid (yield 79.4%). 1 H NMR (500MHz, CDCl3) δ5.69 (d, J = 3.3 Hz, 2H), 4.54 (dt, J = 6.4, 3.8 Hz, 1H), 4.12 (d, J = 11.0 Hz, 1H), 4.02 (d, J = 11. 2Hz,1H),3.84(dd,J=7.0,5.1Hz,1H),3.39(dd,J=4.6,3.7Hz,1H),3.03(d,J=5.1Hz,1H),2.98(s,2H),2.47-2.3 7(m,1H),2.32(dddd,J=8.1,4.7,3.3,2.3Hz,1H),1.93-1.83(m,1H),1.77-1.71(m,1H),1.74-1.63(m,1H),1.5 5(s,5H),1.66-1.42(m,3H),1.35(ddd,J=12.6,9.3,7.3Hz,1H),1.00(s,4H),0.94(ddd,J=7.0,3.7,2.4Hz,1H); 13 C NMR (125MHz, CDCl3) δ206.53,153.58,118.42,100.94,98.20,81.40,74.35,72.30,65.71,60.77,6 0.68,50.22,46.64,40.60,38.84,36.70,34.15,29.80,29.04,26.60,25.83,25.54,19.95; ESI-MS m / z:483.2047calcd forC 24 H 35 O8S[M+H] + 483.2042.

[0046] (3) Preparation of compound (IV): Compound (III) (3 g, 6.22 mmol) was dissolved in 25 mL of pre-dried DMF, and lithium carbonate (4.59 g, 62.16 mmol) and lithium bromide (5.4 g, 62.16 mmol) were added. The mixture was reacted at 110°C for 1 h, cooled to room temperature, and lithium carbonate and lithium bromide were removed by filtration using a sand core funnel. The solid was washed three times with dichloromethane. The organic layers were combined and dichloromethane was added to 120 mL. The mixture was washed twice with water and once with saturated brine to remove DMF, 30 mL each time. The mixture was concentrated and the residual DMF was spin-dried with an oil pump. The dichloromethane was quickly passed through a column to obtain 2.08 g of a light yellow solid (yield 86.7%). 1 H NMR (500MHz, CDCl3) δ5.88(dtt,J=8.2,1.8,0.9Hz,1H),5.69(d,J=3.3Hz,2H),5.36(dt,J=8.1,4.9Hz,1H),3.84(dd, J=7.0,4.8Hz,1H),3.42–3.35(m,2H),3.03(d,J=4.8Hz,1H),2.76(dd,J=10.2,0.9Hz,1H),2.42(dddd,J=12.8,8.4,5. 9,2.2Hz,1H),2.32(dddd,J=8.1,4.7,3.3,2.3Hz,1H),1.84(dddd,J=15.2,5.1,0.9Hz,1H),1.68–1.57(m,2H),1.60–1. 49(m,1H),1.39(dddt,J=7.7,6.0,4.0,1.8Hz,1H),1.38–1.28(m,1H),1.07(ddd,J=7.0,2.6,1.8Hz,1H),1.02(s,5H); 13 CNMR(125MHz, CDCl3)δ206.53,153.58,131.28,129.05,118.42,100.94,98.20,74.35,71.57,68 .94,60.75,59.13,49.37,46.64,41.44,40.11,34.58,31.73,29.80,25.54,23.17,19.77; ESI-MS m / z:387.2166calcd for C 23 H 31 O5[M+H] + 387.2173.

[0047] (4) Preparation of compound (V): Compound (IV) (2.08 g, 5.38 mmol) was dissolved in 35 mL of tetrahydrofuran, 10 mL of 10% hydrochloric acid solution was added, and the mixture was stirred at room temperature for 1.5 h. The tetrahydrofuran was dried by spin drying, and dichloromethane was added to 100 mL. The mixture was washed three times with water and once with saturated brine, each time with 25 mL. The mixture was concentrated and subjected to column chromatography with dichloromethane:methanol = 100:1 to obtain 1.657 g of a white solid (yield 84.4%). 1 H NMR (500MHz, CDCl3) δ6.39(d,J=4.6Hz,1H),5.88(dtt,J=8.2,1.8,0.9Hz,1H),5.69(d,J=1.8Hz,2H),5.36(dt,J=8.1,4.9Hz,1H ),4.92(ddd,J=5.7,4.6,3.7Hz,1H),4.31(s,1H),3.64(dd,J=8.2,4.9Hz,1H),3.38(dd,J=10.2,0.9Hz,1H),3.03(d,J=5.1Hz,1 H),3.02–2.96(m,1H),2.76(dd,J=10.2,0.9Hz,1H),2.42(dddd,J=13.0,8.3,5.8,2.5Hz,1H),1.84(ddd,J=15.2,5.1,0.9Hz,1H ),1.68–1.49(m,2H),1.39(dddd,J=6.4,5.5,2.7,1.8Hz,1H),1.38–1.28(m,1H),1.07(dddd,J=8.2,2.6,1.8Hz,1H),1.02(s,4H); 13 C NMR (125MHz, CDCl3) δ208.70,153.06,131.28,129.05,119.64,100.58,73.25,72.94,66. 78,61.56,60.13,51.80,44.95,41.44,40.11,34.58,31.73,30.27,23.17,18.02; ESI-MS m / z:347.1853calcd for C 20 H 37 O5[M+H] + 347.1846.

[0048] (5) Preparation of compound (VI): Compound (V) (150 mg, 0.43 mmol) was dissolved in 10 mL of dichloromethane and reacted at room temperature for 30 min. The reaction was completed when the starting material disappeared as detected by TLC. A small amount of water was added to quench the reaction, and the mixture was extracted three times with dichloromethane. The mixture was washed with saturated brine, dried, filtered, and concentrated in vacuo. 84.4 mg of a light yellow solid was obtained by column chromatography (yield 40%). 1 H NMR (500MHz, CDCl3) δ8.91(t,J=1.6Hz,1H),8.84(ddd,J=3.8,2.1,1.4Hz,1H),8.34(dt,J=8.2,2.0Hz,1H),7.59(dd,J=8.3,4.0Hz,1H),5.88(dtt, J=8.2,1.8,0.9Hz,1H),5.69(d,J=3.3Hz,2H),5.36(dt,J=8.1,4.9Hz,1H) ,4.89(dd,J=6.7,3.8Hz,1H),4.31(s,1H),3.64(dd,J=8.2,4.9Hz,1H),3. 38(dd,J=10.2,0.9Hz,1H),3.03(d,J=5.1Hz,1H),2.83-2.73(m,2H),2.42 (dddd,J=12.8,8.4,5.9,2.6Hz,1H),1.84(dddd,J=15.2,5.1,0.9Hz,1H),1 .68-1.49(m,3H),1.39(dddd,J=5.7,4.1,3.4,2.3Hz,1H),1.33(dddd,J=1 3.4,8.9,5.9,3.0Hz,1H),1.07(ddd,J=8.2,2.6,1.8Hz,1H),1.02(s,5H); 13 C NMR (125MHz, CDCl3) δ208.75,153.77,149.71,149.58,142.75,134.46,131.28,129.05,124.91,118.42,100. 54,83.61,72.94,66.78,61.83,60.13,51.46,46.02,41.44,40.11,34.58,31.73,30.27,23.17,18.02; ESI-MS m / z:488.1743calcd for C 25 H 30 NO7S[M+H] + 488.1750.

[0049] (6) Preparation of compound 1: Compound (VI) (30 mg, 0.06 mmol) and formaldehyde (2.5 mL, 0.07 mmol) were dissolved in a mixed solution of toluene and ethanol, and a catalytic amount of p-toluenesulfonic acid (5 mg) was added. The mixture was heated to reflux at 90°C for 1.5 h, concentrated in vacuo, and subjected to column chromatography to obtain 20 mg of a yellow solid (yield 65%). 1 H NMR (500MHz, CDCl3) δ8.93-8.89(m,1H),8.84(ddd,J=3.8,2.1,1.4Hz,1H),8.34(dt,J=8.2,2.0Hz,1H),7.59(dd,J=8.3,4 .0Hz,1H),5.88(dtt,J=8.2,1.8,0.9Hz,1H),5.69(d,J=3.3Hz,2H),5.36(dt,J=8.1,4.9Hz,1H),5.24(dd,J=3.0,1.7Hz,1 H),5.15(d,J=2.9Hz,1H),4.89(dd,J=6.9,3.8Hz,1H),3.54(dd,J=7.3,1.8Hz,1H),3.38(dd,J=10.2,0.9Hz,1H),2.83-2. 73(m,2H),2.42(dddd,J=12.8,8.4,5.9,2.6Hz,1H),1.88-1.80(m,1H),1.68-1.49(m,3H),1.43-1.24(m,3H),1.02(s,4H); 13 C NMR (125MHz, CDCl3) δ207.39,153.58,149.71,149.58,142.75,134.46,131.28,129.05,124.91,118.42,108.75, 92.92,84.20,76.93,68.94,60.34,53.38,48.51,47.08,42.12,40.11,34.53,31.73,29.80,23.17,19.77; ESI-MS m / z:500.1737calcd forC 27 H 32 N6O2 + [M+H] + 500.1744.

[0050] Example 2

[0051] The compound 2 of the present invention and its preparation method are as follows:

[0052]

[0053] Refer to the synthesis method of compound 1 in Example 1.1 H NMR (500MHz, CDCl3) δ7.50 (s, 1H), 5.88 (dtt, J = 8.2, 1.8, 0.9Hz, 1H), 5.69 (d, J = 3.3Hz, 2H), 5.36 (dt, J = 8 .1,4.9Hz,1H),5.24(dd,J=3.0,1.7Hz,1H),5.15(d,J=2.9Hz,1H),4.89(dd,J=6.9,3.8Hz,1H),4.03(s,3H ),3.54(dd,J=7.3,1.8Hz,1H),3.38(dd,J=10.2,0.9Hz,1H),2.83-2.73(m,2H),2.42(dddd,J=12.8,8.4, 5.9,2.6Hz,1H),1.88-1.80(m,1H),1.68-1.55(m,2H),1.58-1.49(m,1H),1.43-1.24(m,3H),1.02(s,5H); 13 CNMR(125MHz, CDCl3)δ207.39,153.58,134.79,131.28,129.05,124.30,120.31,118.42,108.75,92.92,85.5 5,76.93,68.94,60.34,53.38,48.51,47.08,42.12,40.11,38.86,34.53,31.73,29.80,23.17,19.77;; ESI-MS m / z:503.1846calcd for C 25 H 31 N2O7S + [M+H] + 503.1839.

[0054] Example 3

[0055] The compound 3 of the present invention and its preparation method are as follows:

[0056]

[0057] Refer to the synthesis method of compound 1 in Example 1. 1H NMR(500MHz, CDCl3)δ5.88(dtt,J=8.2,1.8,0.9Hz,1H),5.69(d,J=3.3Hz,2H),5.36(dt,J=8.1,4.9Hz,1H), 5.24(d,J=3.1Hz,1H),5.15(d,J=2.9Hz,1H),4.89(dd,J=7.0,3.8Hz,1H),3.54(d,J=6.2Hz,1H),3.38(dd,J= 10.2,0.9Hz,1H),3.01(t,J=8.5Hz,2H),2.83-2.73(m,2H),2.42(dddd,J=12.8,8.4,5.9,2.6Hz,1H),1.94(d ,J=8.6Hz,1H),1.93-1.80(m,2H),1.68-1.49(m,3H),1.43-1.24(m,3H),1.10(t,J=8.6Hz,3H),1.02(s,5H); 13 C NMR (125MHz, CDCl3) δ206.53,153.77,130.65,127.51,118.42,108.75,92.92,89.81,79.49,67.86, 60.34,54.35,49.64,48.51,46.02,42.12,40.81,32.91,30.27,27.78,18.54,17.85,13.09; ESI-MS m / z:465.1498calcd for C 27 H 32 N6O2[M+H] + 465.1505.

[0058] Example 4

[0059] The compound 4 of the present invention and its preparation method are as follows:

[0060]

[0061] Refer to the synthesis method of compound 1 in Example 1. 1H NMR (500MHz, CDCl3) δ7.42-7.34(m,3H),5.88(dtt,J=8.2,1.8,0.9Hz,1H),5.69(d,J=3.3Hz,2H),5.36(dt,J=8.1,4 .9Hz,1H),4.89(dd,J=6.9,3.8Hz,1H),4.33-4.27(m,3H),3.64(dd,J=8.2,4.9Hz,1H),3.38(dd,J=10.3,0.9Hz,1H) ,3.03(d,J=5.1Hz,1H),2.83-2.73(m,2H),2.42(dddd,J=12.8,8.4,5.9,2.6Hz,1H),1.88-1.80(m,1H),1.68-1.49( m,3H),1.42-1.36(m,1H),1.33(dddd,J=13.4,8.8,5.9,3.0Hz,1H),1.07(dddd,J=8.2,2.6,1.8Hz,1H),1.02(s,5H); 13 C NMR (125MHz, CDCl3) δ208.75,153.77,131.54,131.28,131.09,129.41,129.05,128.19,118.42,100.54,86.6 8,72.94,66.78,61.83,60.13,58.00,51.46,46.02,41.44,40.11,34.58,31.73,30.27,23.17,18.02; ESI-MS m / z:501.1942calcd for C 27 H 33 7S + [M+H] + 501.1939.

[0062] Example 5

[0063] The compound 5 of the present invention and its preparation method are as follows:

[0064]

[0065] Refer to the synthesis method of compound 1 in Example 1. 1H NMR (500MHz, CDCl3) δ8.79 (dd, J=4.1, 1.7Hz, 1H), 8.43 (d, J=2.0Hz, 1H), 8.10 (dd,J=7.6,1.8Hz,1H),8.06-7.96(m,2H),7.48(dd,J=7.6,4.1Hz,1H),5.74- 5.65(m,2H),5.49-5.40(m,2H),5.02(dd,J=6.9,3.8Hz,1H),4.84(d,J=2.9Hz ,1H),4.77(dd,J=2.9,1.8Hz,1H),4.64(dd,J=7.4,1.7Hz,1H),4.12(dd,J=10 .4,0.9Hz,1H),3.80(dd,J=10.2,0.9Hz,1H),3.32(dddd,J=6.8,5.8,4.5,2.9 Hz,1H),2.51-2.44(m,1H),2.05(dddd,J=13.4,8.8,5.8,3.1Hz,1H),2.01-1. 94(m,1H),1.96-1.91(m,1H),1.92(d,J=0.9Hz,1H),1.91(d,J=0.7Hz,1H),1. 86(ddt,J=13.4,8.6,5.9Hz,1H),1.78-1.68(m,1H),1.08(s,2H),1.02(s,2H); 13 C NMR (125MHz, CDCl3) δ202.18,150.62,149.62,146.94,139.15,136.44,136.09,131.75,127.94,126.88,125.89,125.11,122.68, 118.30,103.63,96.10,83.22,81.22,66.32,57.62,51.66,43.92,43.29,41.30,40.81,32.93,31.24,30.38,28.55,19.21; ESI-MS m / z:550.1894calcd forC 30 H 32 NO7S + [M+H] + 550.1901.

[0066] Example 6

[0067] The compound 6 of the present invention and its preparation method are as follows:

[0068]

[0069] Refer to the synthesis method of compound 1 in Example 1. 1 H NMR (500MHz, CDCl3) δ7.86-7.80(m,2H),7.71-7.64(m,2H),5.74-5.65(m,2H),5.49-5.40(m,2H),5.02(dd,J=6.9,3.8Hz,1H),4.84(d,J =2.9Hz,1H),4.77(dd,J=2.9,1.8Hz,1H),4.64(dd,J=7.4,1.7Hz,1H),4.12(dd,J=10.4,0.9Hz,1H),3.80(dd,J=10.3,0.9Hz,1H),3.32(d dddd,J=6.8,5.8,4.6,2.9Hz,1H),2.47(tdd,J=5.7,2.8,1.7Hz,1H),2.05(dddd,J=13.4,8.8,5.8,3.1Hz,1H),2.01-1.94(m,1H),1.96-1. 91(m,1H),1.92(d,J=0.9Hz,1H),1.91(d,J=0.7Hz,1H),1.86(ddt,J=13.4,8.6,5.9Hz,1H),1.78-1.68(m,1H),1.08(s,2H),1.02(s,2H); 13 C NMR (125MHz, CDCl3) δ202.18,149.62,139.15,135.88,133.33,130.00,129.71,125.89,118.30,103.63,96.1 0,83.21,81.22,66.32,57.62,51.66,43.92,43.29,41.30,40.81,32.93,31.24,30.38,28.55,19.21; ESI-MS m / z:577.0890calcd for C 27 H 30 B7 + [M+H] + 577.0894.

[0070] Example 7

[0071] The compound 7 of the present invention and its preparation method are as follows:

[0072]

[0073] Refer to the synthesis method of compound 1 in Example 1. 1H NMR (500MHz, CDCl3) δ7.68-7.61(m,3H),7.63-7.54(m,2H),5.74-5.65(m,2H),5.49-5.40(m,2H),5.02(dd,J=6.9,3.8Hz,1H),4.84(d,J= 2.9Hz,1H),4.77(dd,J=2.9,1.8Hz,1H),4.64(dd,J=7.4,1.7Hz,1H),4.12(dd,J=10.4,0.9Hz,1H),3.80(dd,J=10.2,0.9Hz,1H),3.32(ddd d,J=6.8,5.8,4.5,2.9Hz,1H),2.47(tdd,J=5.7,2.8,1.7Hz,1H),2.05(dddd,J=13.4,8.8,5.8,3.1Hz,1H),2.01-1.94(m,1H),1.96-1.91 13C NMR (125MHz, CDCl3) δ202.18,149.62,139.15,136.75,134.13,129.29,128.66,125.89,118.30,103.63,96.1 0,83.21,81.22,66.32,57.62,51.66,43.92,43.29,41.30,40.81,32.93,31.24,30.38,28.55,19.21; ESI-MS m / z:499.1785calcd for C 27 H 31 7S + [M+H] + 499.1791.

[0074] Example 8

[0075] The compound 8 of the present invention and its preparation method are as follows:

[0076]

[0077] Refer to the synthesis method of compound 1 in Example 1. 1H NMR (500MHz, CDCl3) δ5.74-5.65(m,2H),5.49-5.40(m,2H),4.97(dd,J=6.8,3.8Hz,1H),4.84(d,J=2 .9Hz,1H),4.77(dd,J=2.9,1.8Hz,1H),4.64(dd,J=7.4,1.7Hz,1H),4.12(dd,J=10.4,0.9Hz,1H),3.8 0(dd,J=10.2,0.9Hz,1H),3.34-3.26(m,1H),3.30-3.24(m,1H),2.51-2.44(m,1H),2.10-1.81(m,8H) ,1.80-1.63(m,5H),1.56(ddq,J=12.3,8.9,6.3Hz,2H),1.51-1.42(m,2H),1.08(s,2H),1.02(s,2H); 13 C NMR (125MHz, CDCl3) δ202.08,149.53,139.15,125.89,118.30,104.19,96.10,81.27,80.27,66.32,63.25, 57.33,51.66,43.89,43.43,41.30,40.81,32.93,31.30,30.38,28.55,27.42,26.52,25.57,19.21; ESI-MS m / z:505.2255calcd forC 27 H 37 7S + [M+H] + 505.2258.

[0078] Example 9

[0079] The compound 9 of the present invention and its preparation method are as follows:

[0080]

[0081] Refer to the synthesis method of compound 1 in Example 1. 1H NMR (500MHz, CDCl3) δ8.05-7.98(m,2H),7.93-7.87(m,2H),5.74-5.65(m,2H),5.49-5.40(m,2H),5.02(dd,J=6.9,3.8Hz,1H),4.84(d,J=2. 9Hz,1H),4.77(dd,J=2.9,1.8Hz,1H),4.64(dd,J=7.4,1.7Hz,1H),4.12(dd,J=10.4,0.9Hz,1H),3.91(s,2H),3.80(dd,J=10.2,0.9Hz,1H),3 .32(dddd,J=6.8,5.8,4.5,2.9Hz,1H),2.47(tdd,J=5.7,2.8,1.7Hz,1H),2.05(dddd,J=13.4,8.8,5.8,3.1Hz,1H),2.01-1.94(m,1H),1.96 -1.91(m,1H),1.92(d,J=0.9Hz,1H),1.91(d,J=0.7Hz,1H),1.86(ddt,J=13.4,8.6,5.9Hz,1H),1.78-1.68(m,1H),1.08(s,2H),1.02(s,2H); 13 C NMR (125MHz, CDCl3) δ202.18,166.23,149.62,140.79,139.15,135.00,130.55,128.62,125.89,118.30,103.63,96. 10,83.21,81.22,66.32,57.62,52.05,51.66,43.92,43.29,41.30,40.81,32.93,31.24,30.38,28.55,19.21; ESI-MS m / z:557.1840calcd forC 29 H 33 9S + [M+H] + 557.1844.

[0082] Example 10

[0083] The compound 10 of the present invention and its preparation method are as follows:

[0084]

[0085] Refer to the synthesis method of compound 1 in Example 1. 1H NMR (500MHz, CDCl3) δ5.74-5.65(m,2H),5.49-5.40(m,2H),4.97(dd,J=6.8,3.8Hz,1H),4.84(d,J=2.9Hz,1H),4.77(dd,J= 2.9,1.8Hz,1H),4.64(dd,J=7.4,1.7Hz,1H),4.12(dd,J=10.4,0.9Hz,1H),3.80(dd,J=10.3,0.9Hz,1H),3.39-3.24(m,2H), 2.47(tdd,J=5.7,2.8,1.7Hz,1H),2.05(dddd,J=13.4,8.8,5.7,3.1Hz,1H),2.00-1.93(m,1H),1.96-1.91(m,2H),1.91(d,J =0.7Hz,1H),1.86(ddt,J=13.4,8.6,5.9Hz,1H),1.78-1.67(m,2H),1.46(dd,J=11.2,7.9Hz,2H),1.08(s,2H),1.02(s,2H); 13 C NMR (125MHz, CDCl3) δ202.08,149.53,139.15,125.89,118.30,104.19,96.10,81.27,80.24,66.32, 57.33,51.66,43.89,43.43,41.30,40.81,37.35,32.93,31.30,30.38,28.55,19.21,10.29; ESI-MS m / z:463.1785calcd for C 24 H 31 7S + [M+H] + 463.1789.

[0086] Embodiment 11

[0087] The compound 11 of the present invention and its preparation method are as follows:

[0088]

[0089] Refer to the synthesis method of compound 1 in Example 1. 1H NMR (500MHz, CDCl3) δ7.86-7.80(m,2H),7.45-7.38(m,2H),5.74-5.65(m,2H),5.49-5.40(m,2H),5.02(dd,J=6.9,3.8Hz,1H),4.84(d,J=2.9 Hz,1H),4.77(dd,J=2.9,1.8Hz,1H),4.64(dd,J=7.4,1.7Hz,1H),4.12(dd,J=10.4,0.9Hz,1H),3.80(dd,J=10.3,0.9Hz,1H),3.65-3.59(m,4H ),3.32(dddd,J=6.8,5.8,4.6,2.9Hz,1H),2.47(tdd,J=5.7,2.8,1.7Hz,1H),2.05(dddd,J=13.4,8.8,5.8,3.1Hz,1H),2.01-1.94(m,1H),1.9 6-1.91(m,1H),1.92(d,J=0.9Hz,1H),1.91(d,J=0.7Hz,1H),1.86(ddt,J=13.4,8.6,5.9Hz,1H),1.78-1.68(m,1H),1.08(s,2H),1.02(s,2H); 13 C NMR (125MHz, CDCl3) δ202.18,172.64,149.62,139.15,138.79,135.12,130.52,127.90,125.89,118.30,103.63,96.10, 83.21,81.22,66.32,57.62,51.90,51.66,43.92,43.29,41.30,40.81,40.62,32.93,31.24,30.38,28.55,19.21; ESI-MS m / z:571.1996calcd for C 30 H 35 9S + [M+H] + 571.1998.

[0090] Example 12

[0091] The compound 12 of the present invention and its preparation method are as follows:

[0092]

[0093] Refer to the synthesis method of compound 1 in Example 1. 1H NMR (500MHz, CDCl3) δ7.44(d,J=3.1Hz,0H),6.99(dd,J=5.5,1.6Hz,1H),6.82(dd,J=5.5,1.3Hz,1H),5.74-5.65(m,2H),5.49-5.40(m,2H),5.11(d d,J=6.9,3.8Hz,1H),4.84(d,J=2.9Hz,1H),4.77(dd,J=2.9,1.8Hz,1H),4.64(dd,J=7.4,1.7Hz,1H),4.12(dd,J=10.4,0.9Hz,1H),3.80(dd,J=10.3 ,0.9Hz,1H),3.32(dddd,J=6.8,5.8,4.5,2.9Hz,1H),2.47(tdd,J=5.7,2.8,1.7Hz,1H),2.05(dddd,J=13.4,8.8,5.8,3.1Hz,1H),2.01-1.94(m,1H ),1.96-1.91(m,1H),1.92(d,J=0.9Hz,1H),1.91(d,J=0.7Hz,1H),1.86(d dt,J=13.4,8.6,5.9Hz,1H),1.78-1.68(m,1H),1.08(s,2H),1.02(s,2H); 13 C NMR (125MHz, CDCl3) δ201.93,149.65,146.94,144.73,139.15,125.89,118.30,115.25,113.56,104.19,96.1 0,82.19,81.22,66.32,58.12,51.66,43.93,43.31,41.30,40.81,32.93,31.27,30.38,28.55,19.21; ESI-MS m / z:489.1578calcd for C 25 H 29 8S + [M+H] + 489.1580.

[0094] Example 13

[0095] The compound 13 of the present invention and its preparation method are as follows:

[0096]

[0097] Refer to the synthesis method of compound 1 in Example 1. 1H NMR (500MHz, CDCl3) δ7.31-7.23(m,3H),7.18(ddddt,J=4.9,4.0,3.1,1.9,1.0Hz,2H),5.74-5.65(m,2H),5.49-5.40(m,2H),5.04(dd,J=7.0,3 .8Hz,1H),4.84(d,J=2.9Hz,1H),4.77(dd,J=2.9,1.8Hz,1H),4.64(dd,J=7.4,1.7Hz,1H),4.12(dd,J=10.4,0.9Hz,1H),3.80(dd,J=10.3,1.0Hz ,1H),3.42-3.24(m,3H),3.01(ddt,J=11.1,10.2,0.9Hz,2H),2.51-2.44(m,1H),2.05(dddd,J=13.4,8.8,5.7,3.1Hz,1H),2.00-1.94(m,1H),1 .96-1.91(m,1H),1.92(d,J=0.9Hz,1H),1.91(d,J=0.8Hz,1H),1.86(dd t,J=13.4,8.6,5.9Hz,1H),1.78-1.68(m,1H),1.08(s,2H),1.02(s,2H); 13 C NMR (125MHz, CDCl3) δ202.08,149.53,139.15,135.37,129.63,128.70,127.16,125.89,118.30,104.21,96.10,81.6 1,81.27,66.32,57.25,54.08,51.66,43.89,43.46,41.30,40.81,32.93,31.26,31.00,30.38,28.55,19.21; ESI-MS m / z:527.2098calcd forC 29 H 35 7S + [M+H] + 527.2091.

[0098] Embodiment 14

[0099] The compound 14 of the present invention and its preparation method are as follows:

[0100]

[0101] Refer to the synthesis method of compound 1 in Example 1. 1H NMR(500MHz, CDCl3)δ9.58(s,1H),7.84-7.78(m,2H),7.69-7.63(m,2H),5.74-5.65(m,2H),5.49-5.40(m,2H),5.02(dd,J=6.9,3.8Hz,1H),4.84 (d,J=2.9Hz,1H),4.77(dd,J=2.9,1.8Hz,1H),4.64(dd,J=7.4,1.7Hz,1H),4.12(dd,J=10.4,0.9Hz,1H),3.80(dd,J=10.2,0.9Hz,1H),3.32(ddd d,J=6.8,5.8,4.6,2.9Hz,1H),2.47(tdd,J=5.7,2.8,1.7Hz,1H),2.05(dddd,J=13.4,8.8,5.8,3.1Hz,1H),2.01-1.94(m,1H),1.96-1.91(m,1H) ,1.92(d,J=0.9Hz,1H),1.91(d,J=0.7Hz,1H),1.86(ddt,J=13.4,8.6,5.9Hz,1H),1.73(ddt,J=12.9,8.6,5.5Hz,1H),1.08(s,2H),1.02(s,2H); 13 C NMR (125MHz, CDCl3) δ202.18,169.62,149.62,143.62,139.15,130.38,129.43,125.89,120.89,118.30,103.63,96. 10,83.21,81.22,66.32,57.62,51.66,43.92,43.29,41.30,40.81,32.93,31.24,30.38,28.55,24.01,19.21; ESI-MS m / z:556.2000calcd forC 29 H 34 NO8S + [M+H] + 556.2004.

[0102] Embodiment 15

[0103] The compound 15 and preparation method of the present invention are as follows:

[0104]

[0105] Refer to the synthesis method of compound 1 in Example 1. 1H NMR (500MHz, CDCl3) δ8.77(d,J=6.8Hz,1H),8.19(t,J=2.1Hz,1H),7.74(dd,J=7.8,2.1Hz,1H),7.61(d,J=7.8Hz,1H),7.22-7.17(m,1H),6.64(dd,J=3. 2,2.2Hz,1H),5.74-5.65(m,2H),5.49-5.40(m,2H),5.02(dd,J=6.9,3.8Hz, 1H),4.84(d,J=2.9Hz,1H),4.77(dd,J=2.9,1.8Hz,1H),4.64(dd,J=7.4,1.7 Hz,1H),4.12(dd,J=10.4,0.9Hz,1H),3.80(dd,J=10.3,0.9Hz,1H),3.32(d ddd,J=6.8,5.8,4.5,2.9Hz,1H),2.47(tdd,J=5.7,2.8,1.7Hz,1H),2.05(dd dd,J=13.4,8.8,5.8,3.1Hz,1H),2.01-1.93(m,1H),1.96-1.90(m,3H),1.86 (ddt,J=13.4,8.6,5.9Hz,1H),1.78-1.68(m,1H),1.08(s,2H),1.02(s,2H); 13 C NMR (125MHz, CDCl3) δ202.18,149.62,139.15,138.62,130.84,129.59,125.89,125.75,123.58,121.32,118.30,111.86,103. 63,103.13,96.10,83.22,81.22,66.32,57.62,51.66,43.92,43.29,41.30,40.81,32.93,31.24,30.38,28.55,19.21; ESI-MS m / z:538.1894calcd for C 29 H 32 NO7S + [M+H] + 538.1891.

[0106] Example 16

[0107] The compound 16 of the present invention and its preparation method are as follows:

[0108]

[0109] Compound (VI) (30 mg, 0.06 mmol) was dissolved in 5 mL of ethylene glycol dimethyl ether, and NaOH (7 mg, 0.2 mmol) and methyl chloroacetate (0.08 mg, 0.07 mmol) were added. The mixture was heated under reflux at 70°C for 4 h. The reaction was completed when the starting material disappeared as detected by TLC. A small amount of water was added to quench the reaction, and the mixture was extracted three times with dichloromethane. The mixture was washed with saturated brine, dried, filtered, concentrated in vacuo, and subjected to column chromatography to obtain 15 mg of a white solid (yield 47%). 1 H NMR (500MHz, CDCl3) δ8.91(t,J=1.6Hz,1H),8.84(ddd,J=3.8,2.1,1.4Hz,1H),8.34(dt,J=8.2,2.0Hz,1H),7.59(dd,J =8.3,4.0Hz,1H),5.88(dtt,J=8.2,1.8,0.9Hz,1H),5.69(d,J=3.3Hz,2H),5.36(dt,J=8.1,4.9Hz,1H),4.89(dd,J=6. 9,3.8Hz,1H),3.80-3.73(m,2H),3.70-3.62(m,2H),3.54(d,J=7.7Hz,1H),3.38(dd,J=10.2,0.9Hz,1H),2.83-2.73(m ,2H),2.42(dddd,J=12.8,8.4,5.9,2.6Hz,1H),1.88-1.80(m,1H),1.68-1.49(m,3H),1.43-1.24(m,3H),1.02(s,4H); 13 CNMR(125MHz, CDCl3)δ207.74,153.58,149.71,149.58,142.75,134.46,131.28,128.17,124.91,118.42,101.75,84. 20,68.94,65.11,63.61,61.78,59.10,51.79,48.51,47.08,42.12,40.99,32.99,29.80,29.75,24.01,19.77; ESI-MS m / z:514.1894calcd for C 27 H 32 N6O2[M+H] + 514.1903.

[0110] Embodiment 17

[0111] The compound 17 of the present invention and its preparation method are as follows:

[0112]

[0113] Refer to the synthesis method of compound 16 in Example 16. 1 H NMR (500MHz, CDCl3) δ7.50 (s, 1H), 5.88 (dtt, J = 8.2, 1.8, 0.9Hz, 1H), 5.69 (d, J = 3.3Hz, 2H), 5.3 6(dt,J=8.1,4.9Hz,1H),4.89(dd,J=6.9,3.8Hz,1H),4.03(s,3H),3.80-3.73(m,2H),3.70-3.62 (m,2H),3.54(d,J=7.7Hz,1H),3.38(dd,J=10.3,0.9Hz,1H),2.83-2.73(m,2H),2.42(dddd,J=1 2.8,8.4,5.9,2.6Hz,1H),1.88-1.80(m,1H),1.68-1.49(m,3H),1.43-1.24(m,3H),1.02(s,5H); 13 CNMR(125MHz, CDCl3)δ207.74,153.58,134.79,131.28,128.17,124.30,120.31,118.42,101.75,85.55,68.94,6 5.11,63.61,61.78,59.10,51.79,48.51,47.08,42.12,40.99,38.86,32.99,29.80,29.75,24.01,19.77; ESI-MS m / z:517.2003calcd for C 26 H 37 N2O7S + [M+H] + 517.1997.

[0114] Embodiment 18

[0115] The compound 18 of the present invention and its preparation method are as follows:

[0116]

[0117] Refer to the synthesis method of compound 16 in Example 16. 1H NMR (500MHz, CDCl3) δ5.88(dtt,J=8.2,1.8,0.9Hz,1H),5.69(d,J=3.3Hz,2H),5.36(dt,J=8.1,4.9Hz,1H) ,4.89(dd,J=7.0,3.8Hz,1H),3.80-3.73(m,2H),3.69-3.62(m,2H),3.54(d,J=7.7Hz,1H),3.38(dd,J=10. 2,0.9Hz,1H),3.01(t,J=8.5Hz,2H),2.83-2.73(m,2H),2.42(dddd,J=12.8,8.4,5.9,2.6Hz,1H),1.94(d, J=8.6Hz,1H),1.93-1.80(m,2H),1.68-1.49(m,3H),1.43-1.24(m,3H),1.10(t,J=8.6Hz,3H),1.02(s,5H); 13 C NMR (125MHz, CDCl3) δ207.74,153.58,131.28,128.17,118.42,101.75,87.40,68.94,65.11,63.61,61.78, 59.10,54.35,51.79,48.51,47.08,42.12,40.99,32.99,29.80,29.75,24.01,19.77,17.85,13.09; ESI-MS m / z:479.2098calcd for C 25 H 35 7S + [M+H] + 479.2090.

[0118] Embodiment 19

[0119] The compound 19 of the present invention and its preparation method are as follows:

[0120]

[0121] Refer to the synthesis method of compound 16 in Example 16. 1H NMR (500MHz, CDCl3) δ7.42-7.34(m,3H),5.88(dtt,J=8.3,1.8,0.9Hz,1H),5.69(d,J=3.3Hz,2H),5.36( dt,J=8.1,4.9Hz,1H),4.89(dd,J=7.0,3.8Hz,1H),4.29(t,J=0.7Hz,2H),3.80-3.73(m,2H),3.69-3.62 (m,2H),3.54(d,J=7.7Hz,1H),3.38(dd,J=10.2,0.9Hz,1H),2.83-2.73(m,2H),2.42(dddd,J=12.8,8.4 ,5.9,2.6Hz,1H),1.84(ddd,J=15.2,5.1,0.9Hz,1H),1.68-1.49(m,3H),1.43-1.24(m,3H),1.02(s,5H); 13 C NMR (125MHz, CDCl3) δ207.74,153.58,131.54,131.28,131.09,129.41,128.19,128.17,118.42,101.75,87.40,68.9 4,65.11,63.61,61.78,59.10,58.00,51.79,48.51,47.08,42.12,40.99,32.99,29.80,29.75,24.01,19.77; ESI-MS m / z:527.2098calcd for C 29 H 35 7S + [M+H] + 527.2104.

[0122] Embodiment 20

[0123] The compound 20 of the present invention and its preparation method are as follows:

[0124]

[0125] Refer to the synthesis method of compound 16 in Example 16. 1H NMR (500MHz, CDCl3) δ9.06(dd,J=4.1,1.7Hz,1H),8.59(d,J=2.0Hz,1H),8.26(dd,J=7.5,1.6Hz,1H),8.11-8.02(m,2H),7. 48-7.43(m,1H),5.88(dtt,J=8.2,1.8,0.9Hz,1H),5.69(d,J=3.3Hz,2H),5.36(dt,J=8.1,4.9Hz,1H),4.89(dd,J=6.9,3.8H z,1H),3.80-3.73(m,2H),3.70-3.62(m,2H),3.54(d,J=7.7Hz,1H),3.38(dd,J=10.2,0.9Hz,1H),2.83-2.73(m,2H),2.42(d ddd,J=12.8,8.4,5.9,2.6Hz,1H),1.88-1.80(m,1H),1.68-1.55(m,2H),1.58-1.49(m,1H),1.43-1.24(m,3H),1.02(s,5H); 13 CNMR(125MHz, CDCl3)δ207.74,153.58,150.81,147.12,137.42,135.83,132.01,131.70,131.28,128.17,127.22,125.08,121.20,118 .42,101.75,84.20,68.94,65.11,63.61,61.78,59.10,51.79,48.51,47.08,42.12,40.99,32.99,29.80,29.75,24.01,19.77; ESI-MS m / z:564.2050calcd for C 31 H 34 NO7S + [M+H] + 564.2093.

[0126] Embodiment 21

[0127] The compound 21 of the present invention and its preparation method are as follows:

[0128]

[0129] Refer to the synthesis method of compound 16 in Example 16. 1H NMR (500MHz, CDCl3) δ7.84-7.78(m,2H),7.73-7.67(m,2H),5.88(dtt,J=8.2,1.8,0.9Hz,1H),5.69 (d,J=3.3Hz,2H),5.36(dt,J=8.1,4.9Hz,1H),4.89(dd,J=6.9,3.8Hz,1H),3.80-3.73(m,2H),3.69 -3.62(m,2H),3.54(d,J=7.7Hz,1H),3.38(dd,J=10.2,0.9Hz,1H),2.83-2.73(m,2H),2.42(dddd,J =12.8,8.4,5.9,2.6Hz,1H),1.88-1.80(m,1H),1.68-1.49(m,3H),1.43-1.24(m,3H),1.02(s,5H); 13 C NMR (125MHz, CDCl3) δ207.74,153.58,140.08,132.92,131.28,129.86,129.84,128.17,118.42,101.75,84.20,6 8.94,65.11,63.61,61.78,59.10,51.79,48.51,47.08,42.12,40.99,32.99,29.80,29.75,24.01,19.77; ESI-MS m / z:591.1047calcd forC 28 H 32 B7 + [M+H] + 591.1045.

[0130] Embodiment 22

[0131] The compound 22 of the present invention and its preparation method are as follows:

[0132]

[0133] Refer to the synthesis method of compound 16 in Example 16. 1H NMR (500MHz, CDCl3) δ7.99-7.93(m,2H),7.84-7.77(m,1H),7.72-7.64(m,2H),5.88(dtt,J=8.2,1.8,0. 9Hz,1H),5.69(d,J=3.3Hz,2H),5.36(dt,J=8.1,4.9Hz,1H),4.89(dd,J=6.9,3.8Hz,1H),3.80-3.73(m, 2H),3.70-3.62(m,2H),3.54(d,J=7.7Hz,1H),3.38(dd,J=10.2,0.9Hz,1H),2.83-2.73(m,2H),2.42(dd dd,J=12.8,8.4,5.9,2.6Hz,1H),1.88-1.80(m,1H),1.68-1.49(m,3H),1.43-1.24(m,3H),1.02(s,5H); 13 CNMR(125MHz, CDCl3)δ207.74,153.58,138.83,133.46,131.28,129.36,128.17,127.65,118.42,101.75,84.20, 68.94,65.11,63.61,61.78,59.10,51.79,48.51,47.08,42.12,40.99,32.99,29.80,29.75,24.01,19.77; ESI-MS m / z:513.1942calcd for C 28 H 33 7S + [M+H] + 513.1947.

[0134] Embodiment 23

[0135] The compound 23 of the present invention and its preparation method are as follows:

[0136]

[0137] Refer to the synthesis method of compound 16 in Example 16. 1H NMR (500MHz, CDCl3) δ5.88(dtt,J=8.2,1.8,0.9Hz,1H),5.69(d,J=3.3Hz,2H),5.36(dt,J=8.1,4.9 Hz,1H),4.89(dd,J=6.9,3.8Hz,1H),3.80-3.73(m,2H),3.69-3.62(m,2H),3.54(d,J=7.7Hz,1H),3. 38(dd,J=10.2,0.9Hz,1H),2.94(p,J=7.9Hz,1H),2.83-2.73(m,2H),2.42(dddd,J=12.8,8.4,5.9, 2.6Hz,1H),2.03-1.91(m,2H),1.84(ddd,J=15.2,5.1,0.9Hz,1H),1.78-1.24(m,11H),1.02(s,4H); 13 C NMR (125MHz, CDCl3) δ207.74,153.58,131.28,128.17,118.42,101.75,87.74,68.94,65.11,63.61,61.78,60. 93,59.10,51.79,48.51,47.08,42.12,40.99,32.99,29.80,29.75,26.02,24.42,24.01,23.52,19.77; ESI-MS m / z:519.2411calcd forC 28 H 39 7S + [M+H] + 519.2418.

[0138] Embodiment 24

[0139] The compound 24 of the present invention and its preparation method are as follows:

[0140]

[0141] Refer to the synthesis method of compound 16 in Example 16. 1H NMR (500MHz, CDCl3) δ8.06-8.00(m,2H),7.95-7.89(m,2H),5.88(dtt,J=8.2,1.8,0.9Hz,1H),5.69(d ,J=3.3Hz,2H),5.36(dt,J=8.1,4.9Hz,1H),4.89(dd,J=6.9,3.8Hz,1H),3.85(s,2H),3.80-3.73(m,2H ),3.69-3.62(m,2H),3.54(d,J=7.7Hz,1H),3.38(dd,J=10.2,0.9Hz,1H),2.83-2.73(m,2H),2.42(ddd d,J=12.8,8.4,5.9,2.6Hz,1H),1.88-1.80(m,1H),1.68-1.49(m,3H),1.43-1.24(m,3H),1.02(s,4H); 13 C NMR (125MHz, CDCl3) δ207.74,167.06,153.58,143.39,131.28,131.26,130.99,130.74,128.17,118.42,101.75,84.20, 68.94,65.11,63.61,61.78,59.10,52.18,51.79,48.51,47.08,42.12,40.99,32.99,29.80,29.75,24.01,19.77; ESI-MS m / z:571.1996calcd for C 30 H 35 9S + [M+H] + 571.2005.

[0142] Embodiment 25

[0143] The compound 25 of the present invention and its preparation method are as follows:

[0144]

[0145] Refer to the synthesis method of compound 16 in Example 16. 1H NMR (500MHz, CDCl3) δ5.88(dtt,J=8.2,1.8,0.9Hz,1H),5.69(d,J=3.3Hz,2H),5.36(dt,J=8.1,4.9Hz,1 H),4.89(dd,J=6.9,3.8Hz,1H),3.80-3.73(m,2H),3.70-3.62(m,2H),3.54(d,J=7.7Hz,1H),3.38(dd,J= 10.2,0.9Hz,1H),2.83-2.73(m,2H),2.42(dddd,J=12.8,8.4,5.9,2.6Hz,1H),1.88-1.80(m,1H),1.72(p ,J=7.8Hz,1H),1.67-1.49(m,3H),1.43-1.24(m,3H),1.02(s,5H),0.78-0.70(m,2H),0.53-0.44(m,2H); 13 CNMR (125MHz, CDCl3) δ207.74,153.58,131.28,128.17,118.42,101.75,87.74,68.94,65.11,63.61,61 .78,59.10,51.79,48.51,47.08,42.12,41.79,40.99,32.99,29.80,29.75,24.01,19.77,9.76; ESI-MS m / z:477.1942calcd forC 25 H 33 7S + [M+H] + 477.1951.

[0146] Embodiment 26

[0147] The compound 26 of the present invention and its preparation method are as follows:

[0148]

[0149] Refer to the synthesis method of compound 16 in Example 16. 1H NMR (500MHz, CDCl3) δ7.86-7.80(m,2H),7.45-7.38(m,2H),5.74-5.65(m,2H),5.49-5.40(m,2H),5.04(dd,J=6.8,3.8Hz, 1H),4.28(d,J=7.7Hz,1H),4.12(dd,J=10.3,0.9Hz,1H),4.04(ddd,J=11.3,4.9,2.4Hz,1H),3.97(ddd,J=11.7,5.0,2.3Hz ,1H),3.93-3.82(m,2H),3.85-3.77(m,1H),3.65-3.59(m,4H),3.32(dddd,J=6.8,5.8,4.5,2.9Hz,1H),2.50(tdd,J=5.7, 2.5,1.7Hz,1H),2.05(dddd,J=13.5,8.8,5.8,3.2Hz,1H),2.01-1.81(m,5H),1.78-1.68(m,1H),1.08(s,2H),1.02(s,2H); 13 C NMR (125MHz, CDCl3) δ201.44,172.64,149.76,139.03,138.79,135.12,130.52,127.90,125.89,118.23,100.80,82.92,75. 88,66.39,65.16,63.06,58.48,51.90,50.81,43.86,43.83,41.23,40.93,40.62,32.98,31.21,30.19,28.54,19.32; ESI-MS m / z:585.2153calcd for C 31 H 37 9S + [M+H] + 585.2151.

[0150] Embodiment 27

[0151] The compound 27 of the present invention and its preparation method are as follows:

[0152]

[0153] Refer to the synthesis method of compound 16 in Example 16. 1H NMR (500MHz, CDCl3) δ7.44(d,J=3.1Hz,0H),6.99(dd,J=5.5,1.6Hz,1H),6.82(dd,J=5.5,1.3Hz,1H),5.74-5.65(m,2H),5.49-5.40(m,2 H),5.11(dd,J=7.0,3.8Hz,1H),4.28(d,J=7.7Hz,1H),4.12(dd,J=10.3,0.9Hz,1H),4.04(ddd,J=11.3,4.9,2.4Hz,1H),3.97(ddd,J=11. 7,5.0,2.3Hz,1H),3.88(dddd,J=21.1,11.7,5.0,2.4Hz,2H),3.80(dd,J=10.3,0.8Hz,1H),3.32(dddd,J=8.4,6.6,3.2,2.4Hz,1H),2.5 0(tdd,J=5.7,2.5,1.7Hz,1H),2.05(dddd,J=13.6,8.8,5.8,3.2Hz,1H),2.01-1.81(m,5H),1.78-1.68(m,1H),1.08(s,2H),1.02(s,2H); 13 C NMR (125MHz, CDCl3) δ201.96,149.87,146.94,144.73,139.03,125.89,118.23,115.25,113.56,100.96,81.91,7 6.02,66.39,65.16,63.06,59.16,50.81,43.98,43.79,41.23,40.93,32.98,31.24,30.19,28.54,19.32; ESI-MS m / z:503.1734calcd for C 26 H 31 8S + [M+H]+503.1736.

[0154] Embodiment 28

[0155] The compound 28 of the present invention and its preparation method are as follows:

[0156]

[0157] Refer to the synthesis method of compound 16 in Example 16. 1H NMR (500MHz, CDCl3) δ7.31-7.23(m,3H),7.18(ddddt,J=5.0,4.0,3.1,2.0,1.0Hz,2H),5.74-5.65(m,2H),5.49-5.40(m,2H) ,5.09(dd,J=7.1,3.8Hz,1H),4.28(d,J=7.7Hz,1H),4.12(dd,J=10.2,0.9Hz,1H),4.04(ddd,J=11.3,4.9,2.4Hz,1H),3.97( ddd,J=11.7,5.0,2.3Hz,1H),3.93-3.82(m,2H),3.85-3.77(m,1H),3.42-3.23(m,3H),3.01(ddt,J=11.1,10.2,0.9Hz,2H), 2.52-2.45(m,1H),2.05(dddd,J=13.5,8.8,5.8,3.2Hz,1H),2.01-1.81(m,5H),1.78-1.68(m,1H),1.08(s,2H),1.02(s,2H); 13 C NMR (125MHz, CDCl3) δ202.02,149.76,139.02,135.37,129.63,128.70,127.16,125.89,118.23,101.27,80.46,76.05,6 6.42,65.16,63.06,58.34,54.08,50.81,43.72,43.59,41.22,40.93,32.98,31.22,31.00,30.19,28.54,19.35; ESI-MS m / z:541.2255calcd for C 30 H 37 7S + [M+H] + 541.2259.

[0158] Embodiment 29

[0159] The compound 29 of the present invention and its preparation method are as follows:

[0160]

[0161] Refer to the synthesis method of compound 16 in Example 16. 1H NMR(500MHz, CDCl3)δ9.58(s,1H),7.84-7.78(m,2H),7.69-7.63(m,2H),5.74-5.65(m,2H),5.49-5.40(m,2H),5.04(dd,J=6.8,3.8Hz ,1H),4.28(d,J=7.7Hz,1H),4.12(dd,J=10.2,0.9Hz,1H),4.04(ddd,J=11.3,4.9,2.4Hz,1H),3.97(ddd,J=11.7,5.0,2.3Hz,1H),3.8 8(dddd,J=21.1,11.7,5.0,2.4Hz,2H),3.80(dd,J=10.3,0.8Hz,1H),3.32(dddd,J=6.8,5.8,4.5,2.9Hz,1H),2.50(tdd,J=5.7,2.5,1 .7Hz,1H),2.05(dddd,J=13.6,8.8,5.8,3.2Hz,1H),2.01-1.81(m,5H),1.73(ddt,J=12.9,8.6,5.5Hz,1H),1.08(s,2H),1.02(s,2H); 13 C NMR (125MHz, CDCl3) δ201.44,169.62,149.76,143.62,139.03,130.38,129.43,125.89,120.89,118.23,100.80,82.95, 75.88,66.39,65.16,63.06,58.48,50.81,43.86,43.83,41.23,40.93,32.98,31.21,30.19,28.54,24.01,19.32; ESI-MS m / z:570.2156calcd for C 30 H 36 NO8S + [M+H] + 570.2158.

[0162] Embodiment 30

[0163] The compound 30 of the present invention and its preparation method are as follows:

[0164]

[0165] Refer to the synthesis method of compound 16 in Example 16. 1H NMR (500MHz, CDCl3) δ8.77(d,J=6.8Hz,1H),8.19(t,J=2.1Hz,1H),7.74(dd,J=7.8,2.1Hz,1H),7.61(d,J=7.8Hz,1H),7.22-7.17(m,1H),6 .64(dd,J=3.2,2.2Hz,1H),5.74-5.65(m,2H),5.49-5.40(m,2H),5.04(dd,J=6.8,3.8Hz,1H),4.28(d,J=7.7Hz,1H),4.12(dd,J=10.3,0.9H z,1H),4.04(ddd,J=11.3,4.9,2.4Hz,1H),3.97(ddd,J=11.7,5.0,2.3Hz,1H),3.93-3.77(m,3H),3.32(dddd,J=6.8,5.8,4.5,2.9Hz,1H),2 .50(tdd,J=5.7,2.5,1.7Hz,1H),2.05(dddd,J=13.5,8.8,5.8,3.2Hz,1H),2.01-1.81(m,5H),1.78-1.68(m,1H),1.08(s,2H),1.02(s,2H); 13 C NMR (125MHz, CDCl3) δ201.44,149.76,139.03,138.62,130.85,129.59,125.89,125.75,123.58,121.32,118.23,111.86,103.13, 100.80,82.95,75.88,66.39,65.16,63.06,58.48,50.81,43.86,43.83,41.23,40.93,32.98,31.21,30.19,28.54,19.32; ESI-MS m / z:552.2051calcd for C 30 H 34 NO7S + [M+H] + 552.2050.

[0166] Embodiment 31

[0167] Compound 14 obtained in Example 14 of the present invention was used to prepare tablets according to the formula in Table 1:

[0168] Table 1. Tablet formula

[0169]

[0170]

[0171] The tablets containing the ent-6,7-closed-ring kaurene-type Rubescensine A hydroxyl-modified derivative of the present invention are obtained.

[0172] Embodiment 32

[0173] Pharmacological experiments were performed to verify the ent-6,7-closed-ring kaurene-type Rubescensine A hydroxy-modified derivatives of the present invention:

[0174] Experimental equipment and reagents:

[0175] Instrument clean bench (Suzhou Aikelin Purification Equipment Co., Ltd.)

[0176] Constant temperature CO2 incubator (SANYO, Japan)

[0177] Enzyme-linked immunosorbent assay (BIO-RAD, USA)

[0178] Inverted biological microscope (Japan OLYMPUS)

[0179] Reagent penicillin and streptomycin mixed solution (Nanjing KeyGen Biotechnology Development Co., Ltd.)

[0180] Trypsin digestion solution (Nanjing KeyGen Biotechnology Development Co., Ltd.)

[0181] PBS (Nanjing KeyGen Biotechnology Development Co., Ltd.)

[0182] MTT(BIOSHARP)

[0183] DMSO(SIGMA)

[0184] Cell line: human breast cancer cell line MCF-7

[0185] Human chronic myeloid leukemia K562 cells

[0186] Human colon cancer cell line SW480

[0187] Human hepatocellular carcinoma cell line SMMC-7721

[0188] Human breast cell line MCF-10A

[0189] 1) Cell digestion, counting, and concentration of 3×10 4 100uL of cell suspension (5×10 cells / mL) was added to each well of a 96-well plate. 3 cells);

[0190] (2) The 96-well plate was placed in a 37°C, 5% CO2 incubator for 24 hours;

[0191] (3) Dilute the drug to the required concentration with complete culture medium, add 100 μL of the corresponding drug-containing culture medium to each well, and set up a negative control group, a solvent control group, and a positive control group;

[0192] (4) The 96-well plate was placed in a 37° C., 5% CO 2 incubator for 72 h; the 96-well plate was stained with MTT at λ=490 nm, and the OD value was measured.

[0193] (5) Add 20 μL of MTT (5 mg / mL) to each well and continue culturing in the incubator for 4 hours;

[0194] (6) Discard the culture medium, add 150 μL DMSO to each well and mix gently on a shaker for 10 minutes; read the OD value of each well with an ELISA reader at λ = 490 nm, and calculate the inhibition rate:

[0195] Cell inhibition rate % = 100% × (OD value of negative control group - OD value of compound group) / OD value of negative control group The experimental results of the effects of compounds on the growth of human cancer cells and normal cells are shown in Table 2:

[0196] Table 2. Effects of some compounds on the growth of five human cell lines in vitro (IC 50 :Unit: μmol / ml)

[0197]

[0198] As shown in Table 2, the derivatives of Rubescensine A have inhibitory effects on four types of cancer cells. Most of the derivatives show stronger inhibitory activity than Rubescensine A, and show good activity against MCF-7 cells. The degree of inhibition is the greatest compared with Rubescensine A, and the best one is more than 14 times the activity of Rubescensine A. In addition, the cyclization activity of the six-membered ring at positions 6 and 7 is better than that of the five-membered ring; the introduction of oxygen and nitrogen heterocycles also improves its activity and water solubility; at the same time, it shows good safety for normal breast cells MCF-10A.

[0199] Verification of the effect of the ent-6,7-closed-ring kaurene-type Rubescensine A hydroxy-modified derivatives of the present invention on tumor growth in vivo:

[0200] Female ICR mice weighing 18-22g and successfully inoculated with breast cancer MCF-7 cells (from Shanghai Esprit Experimental Animal Center) were selected and randomly divided into groups of 8 mice each. One group was a blank control, injected with the same volume of saline solution; the other group was a positive drug control group, given 25 mg / kg of oridonin (purchased from Merck, Germany, product number 496919); the remaining groups were given a certain dose of the compound. The dissolving solvent for oridonin and the above-mentioned compounds is DMF: Tween 80: 5% saline = 10:2:88 (V:V:V), and the administration method is once a day for 21 consecutive days. After the experiment, the mice were killed and the tumors were surgically removed and weighed. The data obtained were statistically analyzed using SPSS17.0:

[0201]

[0202] The results of the compounds inhibiting tumor growth in vivo are shown in Table 3:

[0203] Table 3. Results of compounds inhibiting tumor growth in vivo

[0204] Group drug dose Tumor inhibition rate 1 Normal saline - - 2 Compound 1 25 mg / kg 56.4％ 3 Compound 2 25 mg / kg 72.2％ 4 Compound 3 25 mg / kg 53.2％ 5 Compound 10 25 mg / kg 64.1％ 6 Compound 16 25 mg / kg 65.7％ 7 Compound 17 25 mg / kg 78.4％ 8 Compound 18 25 mg / kg 64.3％ 9 Compound 25 25 mg / kg 67.4％ 10 Rubescensine A 25 mg / kg 51.2％

[0205] As shown in Table 3, the oridonin derivatives have better in vivo antitumor activity than oridonin, and are consistent with the in vitro activity data.

[0206] Pharmacological experiments have shown that the ent-6,7-closed-ring kaurene-type oridonin hydroxy-modified derivatives of the present invention have an anti-cancer cell growth effect and can be used to prepare anti-tumor disease drugs. Preferred tumor diseases for treatment are breast cancer, leukemia, colon cancer and liver cancer; the corresponding tumor cell lines used in pharmacological experiments are MCF-7, K562, SW480, and SMC-7721 cell lines, which significantly inhibit the proliferation of various malignant tumor cells such as breast cancer and liver cancer.

[0207] Embodiment 33

[0208] Water solubility test of the ent-6,7-closed-ring kaurene-type Rubescensine A hydroxyl-modified derivative of the present invention:

[0209] 1. Experimental methods

[0210] (1) Experimental instruments and conditions

[0211] HPLC model: Agilent 1100; chromatographic column: Lichrospher C18 (5 μm, 4.6×250 mm); column temperature: 37°C; flow rate: 1.0 ml / min; injection volume: 10 μL; detection wavelength: 210 nm.

[0212] (2) Experimental procedures

[0213] Each sample was accurately weighed and prepared into a standard solution with a concentration of 0.01, 0.05, 0.1, 0.2, 0.4, and 1.0 mg / mL with methanol. The main peak area was obtained by HPLC injection, and the standard curve was calculated. Then, an appropriate excess of the compound to be tested (>10 mg) was dissolved in PBS and incubated in a constant temperature shaker at 37°C for 24 hours. After centrifugation, 10 μL of the supernatant was injected into HPLC to determine the main peak area of ​​each compound, and then the corresponding solubility was calculated through the standard curve. The experimental results are shown in Table 4.

[0214] 2. Experimental results

[0215] Table 4. Water solubility results of some compounds of the present invention

[0216]

[0217] As can be seen from Table 4, compared with Rubescensine A, the compound designed in the present invention has good water solubility and is convenient for drug development.

[0218] Embodiment 34

[0219] The liver microsomal stability experiment of the ent-6,7-closed-ring kaurene-type Rubescensine A hydroxy-modified derivatives of the present invention:

[0220] The compounds were co-incubated with human or mouse liver microsomes, and the coenzyme NADPH was added to start the reaction. At the incubation time of 0min, 5min, 15min, 30min, 60min, and 120min, 20μL of incubation solution was taken out and transferred to 200μL of acetonitrile containing internal standard to terminate the reaction. After protein precipitation, centrifuge at 3700rpm for 10min and take the supernatant. The supernatant was diluted 1:1 with water and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS). The test results were processed using Graphpadprism 8.3.0 to calculate the phase I metabolic stability parameter t of the compound in liver microsomes of different species. 1 / 2 (min).

[0221] Table 5. Liver microsomal stability of some compounds

[0222]

[0223] Therefore, the ent-6,7-closed-ring kaurene-type Rubescensine A hydroxy-modified derivative of the present invention can significantly inhibit the proliferation of various malignant tumor cells such as breast cancer and liver cancer, and has extremely strong anti-tumor activity; it also has good pharmacokinetic stability and solubility, is conducive to drug formulation, and has low toxic and side effects, and is very promising in clinical application.

Claims

1. An ent-6,7-closed-ring kaurene-type Rubescensine A hydroxyl-modified derivative, characterized in that: The derivative is a Rubescensine A derivative with modified A-ring and 6, 7, and 14 hydroxyl groups, having a structure of Formula I, and also includes a pharmaceutically acceptable salt thereof: in: R is selected from a hydrogen atom, a C1-C6 alkyl group, a C1-C6 alkoxy group, a C1-C6 alkylamino group, 6-10 Aryl, C 3-8 Cycloalkyl, oxirane, aziridine, thiirane, oxetane, azetidine, thietane, tetrahydrofuran, tetrahydropyrrole, tetrahydrothiophene, pyridine, pyrimidine, pyrazine, piperazine, piperidine, morpholine, pyrrole, furan, thiophene, imidazole, pyrazole, oxazole, thiazole, 5-6 membered aromatic ring, 5-6 membered aromatic ring, 6-6 membered aromatic ring, quinoline, isoquinoline, indole, benzofuran, benzothiophene; or R is substituted by 1-4 identical or different R1: the R1 is selected from hydrogen atom, halogen, hydroxyl, cyano, nitro, carboxyl, -NH2, sulfonyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino; the C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino is substituted by 1, 2 or 3 substituents selected from halogen, hydroxyl, cyano, C1-C6 alkyl, C3-C7 cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, phenyl; n is selected from 1, 2, and 3.

2. The ent-6,7-closed-ring kaurene-type Rubescensine A hydroxy-modified derivative according to claim 1, characterized in that: In the structure: R is selected from C1-C6 alkyl, C1-C6 alkoxy, 6-10 Aryl, C 3-8 Cycloalkyl, aziridine, thiirane, oxetane, tetrahydrofuran, tetrahydropyrrole, pyridine, pyrimidine, piperidine, morpholine, pyrrole, furan, imidazole, pyrazole, quinoline, indole, benzofuran; Or R is substituted by 1-4 identical or different R1: R1 is selected from hydrogen atom, halogen, nitro, -NH2, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino.

3. The ent-6,7-closed-ring kaurene-type Rubescensine A hydroxy-modified derivative according to claim 1, characterized in that: In the structure: R is selected from C1-C6 alkyl, C1-C6 alkoxy, 6-10 Aryl, C 3-8 Cycloalkyl, aziridine, thiirane, oxetane, pyridine, pyrimidine, furan, imidazole, pyrazole, quinoline, indole, benzofuran; or R is substituted by 1-4 identical or different R1: R1 is selected from halogen, nitro, -NH2, C1-C6 alkoxy, C1-C6 alkylamino; n is 1 and 2.

4. The ent-6,7-closed-ring kaurene-type Rubescensine A hydroxy-modified derivative according to claim 1, characterized in that: Select from any of the following structures:

5. The ent-6,7-closed-ring kaurene-type Rubescensine A hydroxy-modified derivative according to claim 1, characterized in that: The derivatives also include optically active forms, racemates, diastereomers or mixtures thereof.

6. A method for preparing the derivative according to claim 1, characterized in that: The following steps are involved: (1) dissolving Rubescensine A in anhydrous acetone, adding 2,2-dimethoxypropane and p-toluenesulfonic acid, and reflux reaction. After the reaction is completed, cooling to room temperature, extracting, washing, drying, and concentrating to obtain compound II; (2) Compound II is dissolved in dichloromethane, triethylamine is added, and methanesulfonyl chloride is added dropwise to react. After the reaction is completed, the mixture is cooled to room temperature, extracted, washed, dried, and concentrated to obtain Compound III; (3) Compound III was dissolved in DMF, and lithium carbonate and lithium bromide were added to react. After the reaction was completed, the mixture was cooled to room temperature, filtered, washed, concentrated, and dried by spin drying. Compound IV was quickly passed through a column with dichloromethane; (4) Compound IV was dissolved in tetrahydrofuran, and hydrochloric acid solution was added to react. After the reaction was completed, the compound was dried by spin drying, washed with water, concentrated, and subjected to column chromatography to obtain compound V; (5) Compound V was dissolved in dichloromethane and reacted at room temperature. After the reaction was completed, a small amount of water was added to quench the reaction. The compound was extracted, washed, dried, filtered, vacuum concentrated, and subjected to column chromatography to obtain compound VI. (6) Compound VI and formaldehyde are dissolved in a mixed solution of toluene and ethanol, p-toluenesulfonic acid is added, and the mixture is heated under reflux for reaction; or Compound VI is dissolved in ethylene glycol dimethyl ether, and NaOH and methyl chloroacetate are added, and the mixture is heated under reflux for reaction; after the reaction is completed, the mixture is concentrated under vacuum, and column chromatography is performed to obtain ent-6,7-closed-ring kaurene-type Rubescensine A hydroxy-modified derivative I, and the general reaction formula is as follows:

7. A pharmaceutical composition, characterized in that The invention comprises the derivative according to claim 1, and its optically active form or racemate, diastereoisomer mixture, or its pharmaceutically acceptable salt or mixture and pharmaceutically acceptable auxiliary materials or carriers.

8. Use of the ent-6,7-closed-ring kaurene-type Rubescensine A hydroxy-modified derivative according to claim 1 or the pharmaceutical composition according to claim 7 in the preparation of anti-tumor drugs.

9. The use according to claim 8, characterized in that: The tumors include triple-negative breast cancer, leukemia, colon cancer or liver cancer.

10. The use according to claim 8, characterized in that: The application is that the ent-6,7-closed-ring kaurene-type Rubescensine A hydroxy-modified derivative exerts anti-cancer activity by inducing cell cycle arrest, cell apoptosis, cell autophagy and their cross-effects.

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