14-Chloro-β-elemene nitric oxide donor derivatives, their preparation and use

Abstract

Provided are 14-chloro-β-elemene nitric oxide donor-type derivatives, their preparation and uses. The present invention relates to the field of medicinal chemistry, and the present invention discloses 14-chloro-β-elemene nitric oxide donor type derivatives, their preparation and use in the preparation of antitumor drugs. The 14-chloro-β-elemene nitric oxide donor type derivatives are represented by the formula (I) (wherein R 1 is a linear or cyclic alcoholamine structure containing nitrogen and oxygen atoms, and R 2 , R 3 are each independently 1-10 Alkyl group, C 3-12 Cycloalkyl groups, C 6-12 Aryl groups, 5-10 membered heteroaryl groups, C 2-10 Alkenyl group, C 2-10 Alkynyl group or C 2-10 The design strategy of the derivative according to the present invention is superior to conventional β-elemene nitric oxide donor derivatives, and an alcoholamine structure capable of improving antitumor activity in vivo is introduced as a linker, improving in vivo stability, and providing good therapeutic effects against malignant gliomas for which clinically effective therapeutic agents are lacking. [ka]

Description

[Technical field]

[0001] This application claims priority from a Chinese patent application filed with the China Patent Office on October 11, 2021, bearing application number CN202111180424.7 and entitled "14-Chloro-β-elemene nitric oxide donor-type derivatives, their preparation and use", the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to the field of medicinal chemistry, specifically to 14-chloro-β-elemene nitric oxide donor type derivatives, their preparation and uses. [Background technology]

[0003] Elemene is a sesquiterpene natural product isolated from Zedoary, and has a broad spectrum of antitumor activity. In 1994, oral emulsion and injection of Elemene were approved by the China Food and Drug Administration (CFDA) of the People's Republic of China and are used as a broad-spectrum anticancer drug for the treatment of lung cancer, liver cancer, esophageal cancer, nasopharyngeal cancer and brain cancer.

[0004] β-elemene is the most abundant component in elemene extract mixtures and is also the most important antitumor active component. However, since β-elemene structure only contains two elements, carbon and hydrogen, its lipid solubility is too high, water solubility is low, bioavailability is low, and it is difficult to be absorbed by the human body, which limits its clinical application. Take β-elemene injection as an example, in the treatment process, patients often need to be injected with large amounts, and the drug solution is highly irritating to the patient's blood vessels, which is easy to cause phlebitis. Therefore, it is necessary to carry out structural modification and modification on β-elemene in order to improve its physicochemical properties on the one hand and its antitumor activity on the other hand.

[0005] Nitric oxide (NO) is involved in various physiological and pathological processes, and high levels of NO can inhibit tumor cell proliferation through various signaling pathways, such as ERK and Akt. However, as a gaseous small molecule, NO is difficult to quantify and transport, so the preparation of portable and stable NO donors has become a research focus.

[0006] Research has shown that phenylsulfonylfurazan is a classic nitric oxide donor that can generate high levels of nitric oxide in vitro and in vivo. Therefore, it is expected that the introduction of nitric oxide donor into the structure of β-elemene can significantly improve the antitumor activity of β-elemene, improve the drug-likeness of β-elemene, and obtain an antitumor drug with better therapeutic effect.

[0007] Patent Document 1 discloses the synthesis of nitric oxide donor type β-elemene derivatives, and the specific synthesis route and general structural formula are shown in Figure 1. The compound reported in the patent uses 13-ol of β-elemene as an intermediate, which is linked with a furazan type nitric oxide donor through an esterification reaction to prepare an ester type nitric oxide β-elemene derivative. The series of compounds showed excellent in vitro antitumor activity and in vivo tumor inhibitory activity, but as shown in Figure 1, the ester bond at the 13th position of β-elemene in the series of compounds is very easily hydrolyzed and metabolized by esterase in vivo to produce 13-β-elemenol. Then, 13-β-elemenol is rapidly oxidized and metabolized to 3-β-elemenal. 13-β-elemenal has very strong cytotoxicity, and long-term administration of medium and high doses can directly cause the death of the administered animal, so long-term administration has a high safety risk and is not suitable for use in humans. In addition, the compounds disclosed in the patent are easily metabolized and decomposed, making the compounds less stable and less able to penetrate the blood-brain barrier, and therefore unable to effectively treat tumors in the brain, such as malignant gliomas. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] China Patent Application Publication No. 106866572 Summary of the Invention [Problem to be solved by the invention]

[0009] The 14-chloro-β-elemene nitric oxide donor derivative provided by the present invention uses an alcohol amine structure as a linker, first prepares a 13-β-elemene amine intermediate, then links it with a furazan nitric oxide donor, and finally prepares a β-elemene nitric oxide donor derivative with a new linker, which has excellent activity and does not have the toxicity problems that may occur in the above-mentioned Patent 201710066664.1. [Means for solving the problem]

[0010] In the present invention, the synthesis route of 13,14-dichloro-β-elemene is shown in FIG. 2, and the reaction conditions and reagents are N-chlorosuccinimide (NCS), ytterbium trifluoromethanesulfonate (Yb(OTf)3), trimethylchlorosilane (TMSCl), dichloromethane (CH2Cl2):tetrahydrofuran (THF) (volume ratio is 4:1), 0°C.

[0011] The present invention uses 13,14-dichloro-β-elemene as an antitumor active scaffold, and has the following important advantages: 1) In the chlorination reaction of β-elemene, 13-chloro-β-elemene (intermediate 2, main product), 14-chloro-β-elemene (intermediate 3, by-product) and 13,14-dichloro-β-elemene (intermediate 4) are generated simultaneously, but the polarity of the two monosubstituted β-elemene chlorinated derivatives is very similar, and even if separated by HPLC, it is difficult to isolate both, and the mixture of both can only be used as raw material for the subsequent reaction, resulting in a certain amount of 14-position derivatives in the final product, which is difficult to purify. Therefore, the prepared compound is difficult to purify, is not suitable for large-scale industrial production, and is not useful for the development of medicines that can be prepared on a large scale, making it difficult to carry out pharmaceutical development research. 2) The preparation of 13,14-dichloro-β-elemene is relatively easy, the polarity difference between the dichlorinated product and the monochlorinated β-elemene is large, and the dichlorinated β-elemene intermediate with high purity and the subsequent derivatized final product can be easily isolated and obtained, which is suitable for scale-up production. 3) The antitumor activity of 13,14-dichloro-β-elemene is comparable to that of 13-chloro-β-elemene, 14-chloro-β-elemene and β-elemene, and the polarity of the compound is slightly increased after dichlorination, showing better drug-likeness.

[0012] Therefore, the β-elemene nitric oxide donor derivative disclosed in the present invention is easy to prepare and isolate, has higher structural and metabolic stability, is safer for long-term administration, and can penetrate the blood-brain barrier to efficiently inhibit malignant glioma, and has excellent innovation and novelty.

[0013] The present invention provides 14-chloro-β-elemene nitric oxide donor-type derivatives, pharma- ceutically acceptable salts, solvates, enantiomers, or diastereomers thereof.

[0014] In the present invention, the 14-chloro-β-elemene nitric oxide donor derivative has a general structural formula represented by formula (I). [ka] (In formula (I), R 1 is a linear or cyclic alcoholamine structure containing a nitrogen atom and an oxygen atom, R 2 , R 3 are each independently 1-10 Alkyl group, C 3-12 Cycloalkyl groups, C 6-12 Aryl groups, 5-10 membered heteroaryl groups, C 2-10 Alkenyl group, C 2-10 Alkynyl group or C 2-10 is a group selected from an alkoxy group.

[0015] Preferably, in formula (I), R 1 is C 2-5 A linear alcohol amine structure containing nitrogen and oxygen atoms of C 5-6 is a cyclic alcohol amine structure represented by the formula: R 2 , R 3 are each independently 2-6 Alkyl groups of C 2-6 Alkenyl group, C 2-6 The alkynyl group is selected from the group consisting of alkynyl groups represented by the formula:

[0016] Preferably, in formula (I), R 1 teeth, [ka] is one of the following: R 2 , R 3 are each independently [ka] It is one of the following:

[0017] Preferably, the 14-chloro-β-elemene nitric oxide donor derivative is any one selected from formulas I-1 to I-6, [ka] In formulas I-1 to I-6, R 2 , R 3 are each independently 2-6 Alkyl groups of C 2-6 Alkenyl group, C 2-6 is one of the alkynyl groups selected from [ka] Contains one of the following:

[0018] Preferably, the 14-chloro-β-elemene nitric oxide donor derivative is any one selected from the compounds having the structures represented by formulas 1 to 24. [ka] [ka]

[0019] The present invention further relates to a 14-chloro-β-elemene nitric oxide donor type derivative as described in the above technical solution, its pharma- ceutically acceptable salt, solvate, enantiomer, or diastereomer. Using Antitumor drugs of preparation How to to provide.

[0020] Preferably, the tumor comprises lung cancer, colon cancer, malignant glioma. Effect of the Invention

[0021] Compared with the prior art, the main advantages of the technical solutions provided by the present invention include: In the present invention, an alcoholamine structure capable of improving antitumor activity in vivo is introduced as a linker, which improves in vivo stability and has very good therapeutic activity against malignant glioma, for which clinically effective therapeutic drugs are lacking. [Brief description of the drawings]

[0022] [Figure 1] The advantages of the compounds of the present application compared with the compounds of patent CN201710066664.1 are: [Diagram 2] A synthetic route to 13,14-dichloro-β-elemene. [Diagram 3] This is a synthetic route for intermediates 5 to 10. [Figure 4] Synthetic pathway for intermediates 12, 13, 14a, 14b, 15a, and 15b. [Diagram 5] Synthetic pathways for the final products Ia-d, IIa-b, IIIa-d, IVa-d, Va-b, and VIa-b of β-elemene nitric oxide donor derivatives. [Figure 6] Antitumor activity of β-elemene and Example 4 against malignant glioma (n=5). FIG. 6A shows body weight, FIG. 6B shows brain weight, FIG. 6C shows fluorescent signal intensity of brain tumor, and FIG. 6D shows inhibition rate of β-elemene and Example 4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The present invention will be further described below with reference to the drawings and specific examples.It should be understood that these examples are only for illustrating the present invention and do not limit the scope of the present invention.In the following examples, the operation methods without specific conditions are usually according to conventional conditions or according to the conditions recommended by the manufacturer.

[0024] (1) Preparation of intermediate 4

[0025] In the synthesis route diagram shown in Figure 2, under the condition of 0 ° C, NCS (1.34 g, 20 mmol), ytterbium trifluoromethanesulfonate (310 mg, 0.5 mmol), and trimethylchlorosilane (54 mg, 0.5 mmol) were added in sequence to a mixed solution of dichloromethane (8 mL) and tetrahydrofuran (2 mL) containing β-elemene (1.02 g, 5 mmol), and reacted for 8 h under the condition of 0 ° C. After the reaction was completed, the solvent was distilled off under reduced pressure, water (15 mL) was added to dilute, and the mixture was extracted with ethyl acetate (4 mL x 3), and the organic phases were combined, washed with water (20 mL x 2) and saturated saline (20 mL x 2) in sequence, dried with anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, and the residue was isolated by silica gel column chromatography (pure petroleum ether) to obtain intermediate 4 as a colorless liquid compound with a yield of 45%. The spectrum data is as follows.

[0026] Intermediate 4 1 H NMR

[0027] 1 H NMR(400MHz,CDCl3)δ5.85-5.72(m,1H),5.28(s,1H),5.18(s,1H),5.04(s,1H),4.98-4.89(m,3H ),4.15-4.05(m,3H),3.97(d,J=11.7Hz,1H),2.35-2.21(m,2H),1.77-1.42(m,6H),0.99(s,3H). 13 C NMR(100MHz, CDCl3)δ149.6,149.0,147.5,116.4,113.5,111.6,51.1,47.8,47.5,41.0,39.8,39.7,33.8,27.0,15.8.

[0028] (2) Preparation of intermediates 5-10

[0029] In the synthesis route diagram shown in Figure 3, the corresponding alcohol amine reactant (1.3 mmol) and N,N-diisopropylethylamine (DIPEA, 172 mg, 1.3 mmol) were added sequentially to N,N-dimethylformamide (DMF, 3 mL) containing intermediate 4 (304 mg, 1.1 mmol), and stirred at 60 ° C for 12. After the reaction was completed, the mixed solution was diluted with water (10 mL), extracted with ethyl acetate (10 mL), the organic liquid was combined, washed with water (10 mL) and saturated saline (10 mL), and dried over anhydrous sodium sulfate. The residue was isolated by silica gel column chromatography (volume ratio of dichloromethane and methanol is 150:1) to obtain intermediates 5 to 10 as pale yellow liquids. The spectrum data is as follows:

[0030] Intermediates 5-10 1 H NMR

[0031] 2-((2-((1R,3R,4S)-3-(3-chloroprop-1-en-2-yl)-4-methyl-4-vinylcyclohexyl))(methyl)amino)ethan-1-ol (5)

[0032] Pale yellow liquid, 44% yield. 1 H NMR(400MHz,CDCl3)δ5.78(dd,J=18.0,10.3Hz,1H),5.27(s,1H),5.01-4.88(m,5H),4.14-3.91(m,2H),3.61(t,J=5.4Hz,2H),3. 01(s,2H),2.56-2.51(m,2H),2.28(dd,J=12.5,3.4Hz,1H),2.20(s,3H),2.11(t,J=11.8Hz,1H),1.72-1.44(m,6H),0.98(s,3H). 13 C NMR(100MHz,CDCl3)δ151.0,149.3,147.8,116.2,111.4,111.3,63.1,59.0,58.6,51.0,41.9,41.7,40.0,39.9,34.1,27.3,16.0.HRMS(ESI)calcd for C 18 H 31 ClNO 312.2089[M+H]+ ,found 312.2080.

[0033] 2-((2-((1R,3R,4S)-3-(3-chloroprop-1-en-2-yl)-4-methyl-4-vinylcyclohexyl)allyl)(isopropyl)amino)ethan-1-ol (6)

[0034] Pale yellow liquid, 60% yield. 1 H NMR(400MHz,CDCl3)δ5.77(dd,J=17.1,11.2Hz,1H),5.26(s,1H),5.02-4.79(m,5H),4.02(dd,J=47.3,11.7Hz,2H),3.51(s,2H) ,3.02(d,J=18.7Hz,3H),2.54(s,2H),2.27(dd,J=12.6,3.1Hz,1H),2.14-2.02(m,1H),1.71-1.41(m,6H),0.98(d,J=7.2Hz,9H). 13 C NMR(100MHz,CDCl3)δ149.2,147.7,116.3,111.4,58.5,54.9,51.2,49.9,49.1,47.9,41.7,39.9,40.0,34.3,27.3,17.8,17.7,15.9.HRMS(ESI)calcd for C 20 H 35 ClNO 340.2402[M+H] + ,found 340.2394.

[0035] (S)-1-(2-((1R,3R,4S)-3-(3-chloroprop-1-en-2-yl)-4-methyl-4-vinylcyclohexyl)allyl)pyrrolidin-3-ol (7)

[0036] Pale yellow liquid, 48% yield. 1H NMR(400MHz,CDCl3)δ5.77(dd,J=17.1,11.1Hz,1H),5.26(s,1H),5.05-4.80(m, 5H),4.30(s,1H),4.09(d,J=11.6Hz,1H),3.96(d,J=11.7Hz,1H),3.05(q,J=13. 4Hz,2H),2.85(td,J=8.1,7.2,4.1Hz,1H),2.66(d,J=10.2Hz,1H),2.55(s,1H), 2.43(dd,J=10.2,5.1Hz,1H),2.31-2.07(m,4H),1.69-1.41(m,6H),0.96(s,3H). 13 C NMR(100MHz,CDCl3)δ151.4,149.3,147.7,116.3,111.3,110.2,71.5,63.1,60.9 ,52.6,51.1,47.7,42.0,39.9,39.8,35.0,34.0,27.1,15.8.HRMS(ESI)calcdfor C 19 H 31 ClNO 324.2089[M+H] + ,found 324.2083.

[0037] (1-(2-((1R,3R,4S)-3-(3-chloroprop-1-en-2-yl)-4-methyl-4-vinylcyclohexyl)allyl)piperidin-4-yl)methanol (8)

[0038] Pale yellow liquid, 60% yield. 1 H NMR(400MHz,CDCl3)δ5.79(dd,J=17.9,10.4Hz,1H),5.26(s,1H),4.98-4.86(m,5H),4.09(d,J=11.7Hz,1H),3.97(d,J=11.7Hz,1H),3.50(d,J =6.4Hz,2H),2.90(d,J=14.5Hz,4H),2.32-2.23(m,1H),2.13(d,J=7.9H z,1H),1.88(s,2H),1.75-1.57(m,7H),1.53-1.43(m,4H),0.98(s,3H). 13C NMR(100MHz,CDCl3)δ149.1,147.6,115.9,110.9,67.8,63.5,53.5,53.3,50.8,47.6,42.0,39.7,39.6,38.5,33.8,28.6,26.8,15.6.HRMS(ESI)calcd for C 21 H 35 ClNO 352.2402[M+H] + ,found352.2384.

[0039] (1-(2-((1R,3R,4S)-3-(3-chloroprop-1-en-2-yl)-4-methyl-4-vinylcyclohexyl)allyl)piperidin-3-yl)methanol (9)

[0040] Pale yellow liquid, 51% yield. 1 H NMR(400MHz,CDCl3)δ5.83-5.73(m,1H),5.26(s,1H),4.91(dd,J=10.5,6.4Hz,5 H),4.14-4.02(m,1H),3.97(d,J=11.7Hz,1H),3.69-3.54(m,2H),2.89(d,J=3.9 Hz,2H),2.68(d,J=9.7Hz,1H),2.47(s,2H),2.27(dd,J=11.0,5.0Hz,1H),2.09( ddt,J=12.2,8.2,4.3Hz,2H),1.82-1.74(m,2H),1.70-1.41(m,9H),0.97(s,3H). 13 C NMR(100MHz,CDCl3)δ150.7,149.4,147.8,147.8,116.2,111.3,111.0,67.5,64.1,57 .8,54.4,51.0,47.8,42.1,39.9,39.9,34.0,27.8,27.0,24.6,15.8.HRMS(ESI)calcd for C 21 H 35 ClNO 352.2402[M+H] + ,found 352.2412.

[0041] 2-((4-(2-(4-(2-((1R,3R,4S)-3-(3-chloroprop-1-en-2-yl)-4-methyl-4-vinylcyclohexyl)allyl)piperazin-1-yl)ethan-1-ol (10)

[0042] Pale yellow liquid, 44% yield. 1 H NMR(400MHz,CDCl3)δ5.78(dd,J=18.0,10.2Hz,1H),5.26(s,1H),4.97-4.85(m,5H),4.09(d,J=11.5Hz,1H),3.96(d,J=11.6Hz,1H),3 .61(t,J=5.4Hz,2H),2.91(d,J=4.2Hz,2H),2.62-2.38(m,10H),2.30-2.25(m,1H),2.15-2.08(m,1H),1.64-1.43(m,6H),0.97(s,3H). 13 C NMR(100MHz,CDCl3)δ150.7,149.4,147.8,116.3,111.3,111.0,63.5,59.4,57 .8,53.3,53.1,51.2,47.7,42.2,39.9,39.9,34.0,27.1,15.8.HRMS(ESI)calcd for C 21 H 36 ClNO 367.2511[M+H] + ,found367.2503.

[0043] (3) Preparation of intermediates 12 and 13

[0044] Under the condition of 0°C, 25% NaOH aqueous solution (0.2mL) was slowly added dropwise to a tetrahydrofuran solution containing 3,4-bis(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (183mg, 0.5mmol), and after the completion of the dropwise addition, stirring was continued for 10min, and the reactant ethylene glycol (124mg, 2mmol) was added and reacted at room temperature for 6h. The solvent was distilled off under reduced pressure, water (15mL) was added to dilute, and the mixture was extracted with dichloromethane (20mL x 3), and the organic phases were combined, washed with water (20mL x 2) and saturated saline (20mL x 2) in sequence, dried over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, and the residue was subjected to silica gel chromatography (volume ratio of dichloromethane to methanol was 400:1) to obtain intermediate 12 (72mg, 48%).

[0045] mp128~130℃. 1 H NMR(400MHz,CDCl3)δ8.07(d,J=8.2Hz,2H),7.77(t,J=7.5Hz,1H),7.63(t,J=7.8Hz,2H),4.59-4.49(m,2H),4.08-4.02(m,2H). 13 C NMR(100MHz,CDCl3)δ159.1,137.8,135.9,129.9,128.7,110.7,73.0,60.5.HRMS(ESI)calcd for C 10 H 10 N2NaO6S 309.0152[M+Na] + ,found 309.0142.

[0046] Following the same method as shown in FIG. 4 and described above, but substituting reactants (substituting 1,4-butynediol for ethylene glycol), intermediate 13 was prepared.

[0047] Intermediate 13 1 H NMR data

[0048] 4-((4-hydroxybutyl-2-yn-1-yl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (13)

[0049] White solid, yield 53%, mp 116~118℃. 1 H NMR(400MHz,CDCl3)δ8.07(d,J=7.7Hz,2H),7.76(t,J=7.4Hz,1H),7.63(t,J=7.7Hz,2H),5.10(s,2H),4.34(s,2H). 13 C NMR(100MHz,CDCl3)δ158.2,138.0,135.9,129.8,128.8,110.8,88.3,77.7,59.0,51.1.HRMS(ESI)calcd for C 12 H 10 N2NaO6S 333.0512[M+Na] + ,found 333.0512.

[0050] (4) Preparation of intermediates 14a, 14b, 15a, and 15b

[0051] As shown in FIG. 4, the reactants succinic anhydride (76 mg, 0.76 mmol) and 4-dimethylaminopyridine DMAP (38 mg, 0.32 mmol) were added sequentially to a dichloromethane (5 mL) solution containing intermediate 12 (182 mg, 0.63 mmol), and the mixture was stirred at room temperature for 6 h. Water (5 mL) was added to the mixture solution at room temperature to quench the mixture, and the mixture was stirred for 10 min. The mixture was extracted with dichloromethane (5 mL x 3), and the organic phases were combined and washed sequentially with water (20 mL x 2) and saturated saline (20 mL x 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was isolated by silica gel column chromatography (volume ratio of dichloromethane and methanol was 150:1) to obtain intermediate 14a (167 mg, 67%) as a white solid.

[0052] mp118~120℃. 1 H NMR(400MHz,CDCl3)δ8.07(d,J=7.3Hz,2H),7.76(t,J=7.5Hz,1H),7.63(t,J=7.9Hz,2H),4.72-4.59(m,2H),4.57-4.44(m,2H),2.92-2.51(m,4H). 13C NMR(100MHz,CDCl3)δ176.9,171.9,158.8,138.2,135.8,129.8,128.8,110.6,69.0,61.6,29.0,28.9.HRMS(ESI)calcd for C 14 H 14 N2NaO9S 409.0312[M+Na] + ,found 409.0295.

[0053] Following the same method as in Figure 4 and described above, with substitutions of reactants, intermediates 14b, 15a and 15b were prepared.

[0054] Intermediates 14b, 15a and 15b 1 H NMR

[0055] 4-(2-((4-carboxybutyryl)oxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (14b)

[0056] White solid, yield 53%, mp 91-93℃. 1 H NMR(400MHz,CDCl3)δ8.06(d,J=8.1Hz,2H),7.76(t,J=7.5Hz,1H),7.62(t,J=7.8Hz,2 H),4.66-4.60(m,2H),4.55-4.48(m,2H),2.47(q,J=7.0Hz,4H),1.98(p,J=7.2Hz,2H). 13 C NMR(100MHz,CDCl3)δ178.6,172.7,158.8,138.0,135.8,129.8,128.8,110.6,69.0,61.3,33.0,32.9,19.7.HRMS(ESI)calcd for C 15 H 16 N2NaO9S 423.0469[M+Na] + ,found 423.0465.

[0057] 4-((4-((3-carboxypropionyl)oxy)but-2-yn-1-yl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (15a)

[0058] White solid, yield 54%, mp 106~108℃. 1 H NMR(400MHz,CDCl3)δ8.07(d,J=7.4Hz,2H),7.77(t,J=7.5Hz,1H),7.63(t,J=7.9Hz,2H),5.10(s,2H),4.77(s,2H),2.70(dt,J=8.1,4.7Hz,4H). 13 C NMR(100MHz,CDCl3)δ177.2,171.4,158.1,138.1,135.9,129.9,128.8,110.8,83.9,78.8,58.8,52.3,28.8,28.8.HRMS(ESI)calcd for C 16 H 14 N2NaO9S 433.0312[M+Na] + ,found 433.0317.

[0059] 4-(4-((4-carboxybutyryl)oxy)but-2-yn-1-yloxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (15b)

[0060] White solid, yield 54%, mp 94-96℃. 1 H NMR(400MHz,CDCl3)δ8.07(d,J=7.6Hz,2H),7.77(t,J=7.5Hz,1H),7.63(t,J=7.8Hz ,2H),5.10(s,2H),4.74(s,2H),2.46(td,J=7.3,4.9Hz,4H),1.97(p,J=7.3Hz,2H). 13 C NMR(100MHz,CDCl3)δ179.0,172.1,158.0,137.7,135.8,129.8,128.7,110.6,83.9,78.6,58.6,52.0,32.8,19.6.HRMS(ESI)calcd for C 16 H 14N2NaO9S 447.0469[M+Na] + ,found 447.0486.

[0061] (5) Preparation of the final products of β-elemene nitric oxide donor derivatives Ia-Id, IIa-IIb, IIIa-IIId, IVa-IVd, Va-Vb and VIa-VIb

[0062] As shown in Figure 5, intermediates 5-10 (0.08 mmol), NO donor intermediates 14a, 14b and 15a, 15b (0.10 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 0.12 mmol) and DMAP (0.01 mmol) were added to anhydrous dichloromethane (2 mL) and stirred at room temperature for 8 h. The reaction was detected by thin layer chromatography. The reaction mixture was diluted with dichloromethane (5 mL). The organic layer was washed successively with water and brine, dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the residue was purified by column chromatography (dichloromethane / methanol) to obtain the product as a pale yellow liquid.

[0063] Each of the intermediates used can be prepared according to the above (1) to (4), and will not be described in detail here.

[0064] The technical solutions provided by the present invention are described in detail below with reference to examples, which should not be construed as limiting the protection scope of the present invention.

[0065] Example 1

[0066] [ka]

[0067] Preparation of 4-(2-((4-(2-((2-((1R,3R,4S)-3-(3-chloroprop-1-en-2-yl)-4-methyl-4-vinylcyclohexyl))(methyl)amino)ethoxy)-4-oxobutanoyl)oxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide.

[0068] To a dichloromethane (1.5 mL) solution containing intermediate 5 (26 mg, 0.084 mmol), intermediate 14a (39 mg, 0.101 mmol), DMAP (1 mg, 0.008 mmol), and EDCI (24 mg, 0.126 mmol) were added in that order, and the mixture was stirred at room temperature for 8 h. The mixture was diluted with dichloromethane (5 mL), washed with water (10 mL x 2) and saturated saline (10 mL x 2) in that order, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was isolated by silica gel column chromatography (volume ratio of dichloromethane to methanol was 400:1) to obtain a pale yellow liquid in 69% yield.

[0069] 1 H NMR(400MHz,CDCl3)δ8.07(d,J=7.8Hz,2H),7.75(d,J=7.5Hz,1H),7.63(t,J=7.8Hz,2H ),5.78(dd,J=17.8,10.4Hz,1H),5.26(s,1H),5.02-4.87(m,5H),4.65-4.59(m,2H),4. 57-4.48(m,2H),4.21(t,J=5.8Hz,2H),4.09(d,J=11.7Hz,1H),3.96(d,J=11.8Hz,1H), 3.01(s,2H),2.63(d,J=5.5Hz,6H),2.32-2.22(m,4H),1.66-1.42(m,6H),0.97(s,3H). 13 C NMR(100MHz,CDCl3)δ172.0,172.0,158.7,149.2,147.7,138.1,135.6,129.7,128.6,116.1,111.2,110.4 ,77.0,68.9,62.5,61.4,55.3,50.8,47.9,42.6,41.5,39.8,33.8,29.0,28.9,27.1,15.8.HRMS(ESI)calcd for C32 H 43 ClN3O9S 680.2403[M+H] + ,found 680.2441.

[0070] Example 2

[0071] [ka]

[0072] The preparation method of 4-(2-((5-(2-((2-((1R,3R,4S)-3-(3-chloroprop-1-en-2-yl)-4-methyl-4-vinylcyclohexyl)allyl)(methyl)amino)ethoxy)-5-oxopentanoyl)oxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide is basically the same as that in Example 1, except that succinic anhydride in (3) is replaced with glutaric anhydride.

[0073] In this example, a yellow waxy liquid was prepared in 71% yield.

[0074] 1 H NMR(400MHz,CDCl3)δ8.06(d,J=8.5Hz,2H),7.76(t,J=7.5Hz,1H),7.63(t,J=7.9Hz,2H),5.87-5.71(m, 1H),5.26(s,1H),5.01-4.86(m,5H),4.67-4.60(m,2H),4.53-4.46(m,2H),4.17(t,J=5.7Hz,2H),4.09( d,J=11.6Hz,1H),3.96(d,J=11.7Hz,1H),2.95(s,2H),2.57(t,J=5.1Hz,2H),2.43(dt,J=17.0,7.3Hz,4 H),2.28(dd,J=11.9,4.0Hz,1H),2.16-2.07(m,1H),2.02-1.93(m,2H),1.65-1.35(m,6H),0.96(s,3H). 13C NMR(100MHz,CDCl3)δ173.0,172.8,158.8,151.4,149.4,147.8,138.1,135.8,129.8,128.7,116.3,110.9,110.5,6 9.0,63.3,62.5,61.2,55.4,51.1,47.7,42.9,41.5,39.9,39.9,33.9,33.2,33.1,27.1,20.0,15.8.HRMS(ESI)calcd for C 33 H 45 ClN3O9S 694.2560[M+H] + ,found 694.2562.

[0075] Example 3

[0076] [ka]

[0077] The preparation method of 4-((4-((4-(2-((2-((1R,3R,4S)-3-(3-chloroprop-1-en-2-yl)-4-methyl-4-vinylcyclohexyl)allyl)(methyl)amino)ethoxy)-4-oxobutanoyl)oxy)but-2-yn-1-yl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide is basically the same as in Example 1, except that ethylene glycol in (3) is replaced with 1,4-butynediol.

[0078] In this example, a yellow waxy liquid was prepared in 74% yield.

[0079] 1H NMR(400MHz,CDCl3)δ8.07(d,J=7.4Hz,2H),7.77(t,J=7.5Hz,1H),7.64(t,J=7.9 Hz,2H),5.78(dd,J=16.9,11.3Hz,1H),5.26(s,1H),5.10(s,2H),5.05-4.85(m,5H ),4.76(s,2H),4.20(t,J=5.6Hz,2H),4.12-4.06(m,1H),3.96(d,J=11.7Hz,1H),2 .97(s,2H),2.70-2.56(m,6H),2.31-2.09(m,5H),1.70-1.41(m,6H),0.97(s,3H). 13 C NMR(100MHz,CDCl3)δ172.2,171.6,158.0,151.3,149.4,149.4,147.7,137.9,135.9,129.9,128.8,116.3,111.3,110.7,8 3.9,78.7,63.2,62.8,58.7,55.3,52.3,51.1,47.7,42.8,41.4,39.9,39.9,33.9,29.0,28.9,27.1,15.8.HRMS(ESI)calcd for C 34 H 43 ClN3O9S 704.2403[M+H] + ,found 704.2408.

[0080] Example 4

[0081] [ka]

[0082] The preparation method of 4-((4-((5-(2-((2-((1R,3R,4S)-3-(3-chloroprop-1-en-2-yl)-4-methyl-4-vinylcyclohexyl)allyl)(methyl)amino)ethoxy)-5-oxopentyloxy)but-2-yn-1-yloxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide is basically the same as that in Example 3, except that succinic anhydride in (3) is replaced with glutaric anhydride.

[0083] In this example, a yellow ワックス-like liquid was prepared with a yield of 73%.

[0084] 1 H NMR (400MHz, CDCl3) δ8.07(d,J=7.4Hz,2H),7.77(t,J=7.5Hz,1H),7.63(t,J=7.8Hz,2H),5.77(dd,J =17.1,11.2Hz,1H),5.25(s,1H),5.09(s,2H),4.98-4.83(m,5H),4.73(s,2H),4.17(t,J=5.8Hz,2H) ,4.09(d,J=11.7Hz,1H),3.96(d,J=11.7Hz,1H),2.96(s,2H),2.57(t,J=5.5Hz,2H),2.41(dt,J=17. 1,7.3Hz,4H),2.31-2.18(m,4H),2.12(s,1H),1.96(p,J=7.1Hz,2H),1.68-1.41(m,6H),0.96(s,3H). 13 C NMR (100MHz, CDCl3) δ172.9,172.2,158.0,151.3,149.3,147.7,137.8,135.9,129.8,128.8,116.3,111.3,110.9,11 0.7,84.0,78.6,63.2,62.4,58.7,55.4,52.0,51.1,47.7,39.9,33.8,33.2,33.0,27.1,20.0,15.8.HRMS(ESI)calcd for C 35 H 45 ClN3O9S 718.2560[M+H] + ,found 718.2553.

[0085] Example 5

[0086]

change

[0087] The preparation method of 4-(2-((4-(2-((2-((1R,3R,4S)-3-(3-chloroprop-1-en-2-yl)-4-methyl-4-vinylcyclohexyl)allyl)(isopropyl)amino)ethoxy)-4-oxobutanoyl)oxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide is basically the same as in Example 1, except that N-methyl-2-hydroxyethylamine in (1) is replaced with 2-(isopropylamino)ethanol.

[0088] In this example, a yellow waxy liquid was prepared in 58% yield.

[0089] 1 H NMR(400MHz,CDCl3)δ8.07(d,J=7.5Hz,2H),7.76(t,J=7.5Hz,1H),7.63(t,J =7.8Hz,2H),5.77(dd,J=17.6,10.6Hz,1H),5.25(s,1H),5.03-4.84(m,5H), 4.66-4.58(m,2H),4.56-4.47(m,2H),4.11-3.93(m,4H),3.06-2.82(m,3H), 2.72-2.56(m,6H),2.30-2.09(m,2H),1.65-1.40(m,6H),1.02-0.90(m,9H). 13 C NMR(100MHz, CDCl3)δ172.2,172.1,158.7,149.8,149.3,147.7,138.0,135.7,129.7,128.7,116.2,111.2,110.4,110. 0,68.9,64.0,61.4,55.6,51.2,50.2,47.7,41.3,39.9,39.9,34.0,28.9,28.9,27.1,18.0,17.7,15.7.HRMS(ESI)calcd for C 34 H 47 ClN3O9S 708.2716[M+H] + ,found 708.2703.

[0090] Example 6

[0091] [ka]

[0092] The preparation method of 4-(2-((5-(2-((2-((1R,3R,4S)-3-(3-chloroprop-1-en-2-yl)-4-methyl-4-vinylcyclohexyl)allyl)(isopropyl)amino)ethoxy)-5-oxopentyloxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide is basically the same as in Example 2, except that N-methyl-2-hydroxyethylamine in (1) is replaced with 2-(isopropylamino)ethanol.

[0093] In this example, a yellow waxy liquid was prepared in 68% yield.

[0094] 1 H NMR(400MHz,CDCl3)δ8.06(d,J=7.4Hz,2H),7.76(t,J=7.5Hz,1H),7.62(t,J=7.8Hz,2H),5.77(dd, J=16.9,11.3Hz,1H),5.25(s,1H),5.03-4.82(m,5H),4.65-4.56(m,2H),4.54-4.43(m,2H),4.12-3 .91(m,4H),3.08-2.79(m,3H),2.58(t,J=6.6Hz,2H),2.42(dt,J=26.0,7.3Hz,4H),2.26(dd,J=12. 7,3.4Hz,1H),2.13(t,J=11.3Hz,1H),1.97(p,J=7.3Hz,2H),1.66-1.41(m,6H),1.01-0.87(m,9H). 13 C NMR(100MHz,CDCl3)δ172.9,172.8,158.8,149.8,149.3,147.8,138.0,135.8,129.8,128.7,116.3,111.3,110.5,110.1,6 9.0,63.8,61.2,55.6,51.2,50.2,47.8,41.4,40.0,39.9,34.1,33.2,33.1,27.1,20.0,18.1,17.8,15.8.HRMS(ESI)calcd for C 35H 48 ClN3O9S 722.2873[M+H] + ,found 722.2873.

[0095] Example 7

[0096] [ka]

[0097] The preparation method of 4-((4-((4-(2-((2-((1R,3R,4S)-3-(3-chloroprop-1-en-2-yl)-4-methyl-4-vinylcyclohexyl)allyl)(isopropyl)amino)ethoxy)-4-oxobutanoyl)oxy)but-2-yn-1-yl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide is basically the same as in Example 3, except that N-methyl-2-hydroxyethylamine in (1) is replaced with 2-(isopropylamino)ethanol.

[0098] In this example, a yellow waxy liquid was prepared in 47% yield.

[0099] 1 H NMR(400MHz,CDCl3)δ8.08(d,J=7.7Hz,2H),7.77(t,J=7.4Hz,1H),7.64(t,J=7.8Hz,2H),5.78(dd,J=17.1,11.1Hz,1H),5.25(s,1H),5.10(s, 2H),4.76(s,2H),4.18-3.91(m,4H),2.98(d,J=45.6Hz,3H),2.65(dt, J=10.6,5.1Hz,6H),2.31-2.11(m,2H),1.64-1.41(m,6H),0.97(s,9H). 13C NMR(100MHz,CDCl3)δ172.1,171.6,158.1,149.4,147.9,138.1,135.8,129.9,128.8,116.2,111.3,110.7,84.0,78 .8,58.7,55.7,52.2,51.1,48.0,47.9,41.5,40.0,34.2,29.1,29.0,27.3,22.8,18.1,17.8,15.9.HRMS(ESI)calcd for C 36 H 47 ClN3O9S 732.2716[M+H] + ,found 732.2711.

[0100] Example 8

[0101] [ka]

[0102] The preparation method of 4-((4-((5-(2-((2-((1R,3R,4S)-3-(3-chloroprop-1-en-2-yl)-4-methyl-4-vinylcyclohexyl)allyl)(isopropylamino)ethoxy)-5-oxopentanoyl)oxy)but-2-yn-1-yl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide is basically the same as in Example 4, except that N-methyl-2-hydroxyethylamine in step 1 of (1) is replaced with 2-(isopropylamino)ethanol.

[0103] In this example, a yellow waxy liquid was prepared in 53% yield.

[0104] 11H NMR (400 MHz, CDCl3) δ 8.07 (d, J = 7.4 Hz, 2H), 7.77 (t, J = 7.5 Hz, 1H), 7.63 (t, J = 7.8 Hz, 2H), 5.78 (dd, J = 17.0, 11.2 Hz, 1H), 5.25 (s, 1H), 5.09 (s, 2H), 5.04 - 4.81 (m, 5H), 4.74 (s, 2H), 4.13 - 3.91 (m, 4H), 3.09 - 2.79 (m, 3H), 2.59 (t, J = 6.2 Hz, 2H), 2.40 (dt, J = 26.3, 7.4 Hz, 5H), 2.26 (dd, J = 12.2, 3.5 Hz, 1H), 2.14 (s, 1H), 1.96 (p, J = 7.3 Hz, 2H), 1.66 - 1.41 (m, 6H), 1.06 - 0.89 (m, 9H). 13 13C NMR (100 MHz, CDCl3) δ 172.7, 172.0, 157.9, 149.3, 147.7, 137.9, 135.7, 129.7, 128.7, 116.1, 111.1, 110.6, 110.0, 84.0, 78.5, 63.7, 58.6, 55.6, 51.8, 51.0, 50.3, 47.9, 47.9, 41.4, 39.9, 39.9, 34.1, 33.2, 33.0, 27.1, 20.0, 18.0, 17.7, 15.8. HRMS (ESI) calcd for C 37 H 49 ClN3O9S 746.2873 [M + H] + , found 746.2871.

[0105] Example 9

[0106]

Chemical formula

[0107] The preparation method of 4-(2-((4-(((S)-1-(2-((1R,3R,4S)-3-(3-chloroprop-1-en-2-yl)-4-methyl-4-vinylcyclohexyl)allyl)pyrrolidin-3-yl)oxy)-4-oxobutanoyl)oxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide is basically the same as in Example 1, except that N-methyl-2-hydroxyethylamine in (1) is replaced with (R)-3-pyrrolidinol.

[0108] In this example, a yellow waxy liquid was prepared in 68% yield.

[0109] 1 H NMR(400MHz,CDCl3)δ8.06(d,J=7.5Hz,2H),7.76(t,J=7.5Hz,1H),7.63(t,J=7.8Hz,2H),5.77(dd,J =17.1,11.2Hz,1H),5.26(s,1H),5.17(s,1H),5.00-4.83(m,5H),4.66-4.57(m,2H),4.56-4.48(m,2 H),4.09(d,J=11.6Hz,1H),3.96(d,J=11.7Hz,1H),3.12(d,J=13.4Hz,1H),2.99(d,J=13.4Hz,1H),2 .76-2.60(m,7H),2.40(s,1H),2.31-2.19(m,2H),1.87-1.79(m,1H),1.67-1.40(m,6H),0.96(s,3H). 13 C NMR(100MHz,CDCl3)δ172.2,172.2,158.8,149.3,147.7,138.0,135.8,129.8,128.7,116.2,111.3,110.5,74.7 ,68.9,61.5,60.8,60.0,53.0,51.1,47.6,42.0,39.9,39.8,34.0,31.9,29.2,29.0,27.1,15.8.HRMS(ESI)calcd for C 33 H 43 ClN3O9S 692.2403[M+H] + ,found 692.2394.

[0110] Example 10

[0111] [ka]

[0112] The preparation method of 4-(2-((5-(((S)-1-(2-((1R,3R,4S)-3-(3-chloroprop-1-en-2-yl)-4-methyl-4-vinylcyclohexyl)allyl)pyrrolidin-3-yl)oxy)-5-oxopentanoyl)oxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide is essentially the same as in Example 2, except that N-methyl-2-hydroxyethylamine in (1) is replaced with (R)-3-pyrrolidinol.

[0113] In this example, a yellow waxy liquid was prepared in 62% yield.

[0114] 1 H NMR(400MHz,CDCl3)δ8.05(d,J=7.5Hz,2H),7.76(t,J=7.5Hz,1H),7.63(t,J=7.8Hz,2H),5.77(dd,J=17.0,11.2 Hz,1H),5.25(s,1H),5.16(s,1H),5.03-4.85(m,5H),4.66-4.59(m,2H),4.53-4.46(m,2H),4.09(d,J=11.6Hz,1 H),3.96(d,J=11.7Hz,1H),3.17-2.91(m,2H),2.71(d,J=6.6Hz,2H),2.59(d,J=9.8Hz,1H),2.42(dt,J=23.1,7. 3Hz,5H),2.31-2.19(m,2H),2.11(s,1H),2.00-1.92(m,2H),1.85-1.76(m,1H),1.67-1.40(m,6H),0.96(s,3H). 13C NMR(100MHz,CDCl3)δ172.9,172.8,158.8,149.3,147.7,138.0,135.8,129.8,128.7,116.2,111.3,110.5,74.4,69 .0,61.2,60.9,60.0,53.0,51.1,47.6,42.0,39.9,39.8,34.0,33.4,33.1,32.0,27.1,20.0,15.8.HRMS(ESI)calcd for C 34 H 45 ClN3O9S 706.2560[M+H] + ,found 706.2555.

[0115] Example 11

[0116] [ka]

[0117] The preparation method of 4-((4-((4-(((S)-1-(2-((1R,3R,4S)-3-(3-chloroprop-1-en-2-yl)-4-methyl-4-vinylcyclohexyl)allyl)pyrrolidin-3-yl)oxy)-4-oxobutanoyl)oxy)but-2-yn-1-yl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide is basically the same as in Example 3, except that N-methyl-2-hydroxyethylamine in (1) is replaced with (R)-3-pyrrolidinol.

[0118] In this example, a yellow waxy liquid was prepared in 53% yield.

[0119] 11H NMR (400 MHz, CDCl3) δ 8.07 (d, J = 7.4 Hz, 2H), 7.77 (t, J = 7.58 Hz, 1H), 7.63 (t, J = 7.9 Hz, 2H), 5.78 (dd, J = 17.9, 10.3 Hz, 1H), 5.26 (s, 1H), 5.19 (s, 1H), 5.09 (s, 2H), 5.02 - 4.84 (m, 5H), 4.76 (s, 2H), 4.09 (d, J = 11.4 Hz, 1H), 3.97 (d, J = 11.8 Hz, 1H), 3.16 - 2.95 (m, 2H), 2.76 - 2.60 (m, 7H), 2.41 (s, 1H), 2.31 - 2.08 (m, 3H), 1.88 - 1.79 (m, 1H), 1.68 - 1.43 (m, 6H), 0.97 (s, 3H). 13 13C NMR (100 MHz, CDCl3) δ 172.0, 171.6, 158.1, 149.4, 147.8, 147.8, 138.0, 135.8, 129.8, 128.8, 116.2, 111.3, 110.7, 84.0, 78.8, 74.8, 60.9, 60.0, 58.7, 53.0, 52.2, 51.0, 47.9, 42.2, 39.9, 39.9, 34.1, 32.0, 29.2, 29.0, 27.2, 15.9. HRMS (ESI) calcd for C 35 H 43 ClN3O9S 716.2403 [M + H] + , found 716.2400.

[0120] Example 12

[0121]

Chemical Structure

[0122] The preparation method of 4-((4-((5-(((S)-1-(2-((1R,3R,4S)-3-(3-chloroprop-1-en-2-yl)-4-methyl-4-vinylcyclohexyl)allyl)pyrrolidin-3-yl)oxy)-5-oxopentanoyl)oxy)but-2-yn-1-yl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide is basically the same as in Example 4, except that N-methyl-2-hydroxyethylamine in (1) is replaced with (R)-3-pyrrolidinol.

[0123] In this example, a yellow waxy liquid was prepared in 63% yield.

[0124] 1 H NMR(400MHz,CDCl3)δ8.07(d,J=7.4Hz,2H),7.76(t,J=7.5Hz,1H),7.63(t,J=7.8Hz,2H),5.78(dd,J=17.9,10.3Hz,1H),5.26(s, 1H),5.17(td,J=6.2,3.2Hz,1H),5.09(s,2H),5.01-4.82(m,5H),4.74(s,2H),4.09(d,J=11.5Hz,1H),3.96(d,J=11.8Hz,1H),3.1 1(d,J=13.4Hz,1H),2.99(d,J=13.4Hz,1H),2.78-2.66(m,2H),2.60(d,J=10.9Hz,1H),2.40(dt,J=23.9,7.3Hz,5H),2.31-2.17( m,2H),2.10(dq,J=7.6,5.3,3.5Hz,1H),1.95(p,J=7.3Hz,2H),1.81(dq,J=13.7,8.7,6.9Hz,1H),1.70-1.39(m,6H),0.97(s,3H). 13C NMR(100MHz,CDCl3)δ172.8,172.2,158.0,151.1,149.3,147.7,137.8,135.9,129.8,128.7,116.2,111.3,110.7,110.4,84.0,7 8.6,74.4,60.8,60.0,58.7,53.0,52.0,51.0,47.6,42.0,39.9,39.8,34.0,33.3,33.0,31.9,27.1,20.0,15.8.HRMS(ESI)calcd for C 36 H 45 ClN3O9S 730.2560[M+H] + ,found 730.255.

[0125] Example 13

[0126] [ka]

[0127] The preparation method of 4-(2-((4-((1-(2-((1R,3R,4S)-3-(3-chloroprop-1-en-2-yl)-4-methyl-4-vinylcyclohexyl)allyl)piperidin-4-yl)methoxy)-4-oxobutanoyl)oxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide is basically the same as in Example 1, except that N-methyl-2-hydroxyethylamine in (1) is replaced with 4-hydroxymethylpiperidine.

[0128] In this example, a yellow waxy liquid was prepared in 67% yield.

[0129] 1H NMR(400MHz,CDCl3)δ8.06(d,J=7.7Hz,2H),7.76(t,J=7.5Hz,1H),7.63(t,J=7.8 Hz,2H),5.78(dd,J=17.1,11.1Hz,1H),5.25(s,1H),4.97-4.84(m,5H),4.66-4.5 6(m,2H),4.57-4.47(m,2H),4.13-3.88(m,3H),2.88(d,J=17.1Hz,4H),2.68(s,4 H),2.34-2.20(m,1H),2.12(s,1H),1.85(s,2H),1.70-1.38(m,10H),0.96(s,3H). 13 C NMR(100MHz,CDCl3)δ172.25,172.15,158.69,149.29,147.72,137.94,135.74,129.73,128.66,116.14,111.18,110.43,69.29,6 8.86,63.64,61.42,53.42,53.28,51.08,47.62,42.10,39.85,39.74,35.34,33.91,28.94,28.92,26.95,15.69.HRMS(ESI)calcd for C 35 H 47 ClN3O9S 720.2716[M+H] + ,found 720.2709.

[0130] Example 14

[0131] [ka]

[0132] The preparation method of 4-(2-((5-((1-(2-((1R,3R,4S)-3-(3-chloroprop-1-en-2-yl)-4-methyl-4-vinylcyclohexyl)allyl)piperidin-4-yl)methoxy)-5-oxopentanoyl)oxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide is basically the same as in Example 2, except that N-methyl-2-hydroxyethylamine in (1) is replaced with 4-hydroxymethylpiperidine.

[0133] In this example, a yellow ワックス-like liquid was prepared with a yield of 70%.

[0134] 1 H NMR(400MHz, CDCl3)δ8.06(d,J=7.4Hz,2H),7.76(t,J=7.5Hz,1H),7.62(t,J=7.9Hz,2H),5.7 8(dd,J=18.0,10.3Hz,1H),5.26(s,1H),5.01-4.85(m,5H),4.72-4.59(m,2H),4.54-4.46(m, 2H),4.08(d,J=11.7Hz,1H),3.98-3.90(m,3H),3.03-2.80(m,4H),2.42(dt,J=18.4,7.3Hz,4 H),2.31-2.23(m,1H),2.15-2.07(m,1H),2.03-1.86(m,4H),1.68-1.42(m,10H),0.97(s,3H). 13 C NMR (100MHz, CDCl3) δ173.0,172.8,158.8,149.4,147.8,138.1,135.8,129.8,128.7, 116.2,111.3,110.5,69.0,69.0,63.6,61.2,53.4,53.3,51.1,47.8,42.2,39.9,39.8, 35.4,34.0,33.3,33.2,29.8,28.8,27.1,20.1,15.8.HRMS(ESI)calcd for C 36 H 49 ClN3O9S 734.2873[M+H] + ,found 734.889.

[0135] Example 15

[0136]

change

[0137] The preparation method of 4-((4-((4-((1-(2-((1R,3R,4S)-3-(3-chloroprop-1-en-2-yl)-4-methyl-4-vinylcyclohexyl)allyl)piperidin-4-yl)methoxy)-4-oxobutanoyl)oxy)but-2-yn-1-yl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide is basically the same as in Example 3, except that N-methyl-2-hydroxyethylamine in (1) is replaced with 4-hydroxymethylpiperidine.

[0138] In this example, a yellow waxy liquid was prepared in 65% yield.

[0139] 1 H NMR(400MHz,CDCl3)δ8.07(d,J=7.4Hz,2H),7.76(t,J=7.5Hz,1H),7.63(t,J=7.8Hz,2H) ,5.78(dd,J=17.9,10.4Hz,1H),5.26(s,1H),5.09(s,2H),5.02-4.85(m,5H),4.76(s,2H) ),4.08(d,J=11.4Hz,1H),3.99-3.94(m,3H),2.98(s,4H),2.66(q,J=3.7Hz,4H),2.30-2 .25(m,1H),2.17-2.11(m,1H),1.97(d,J=17.5Hz,2H),1.71-1.43(m,10H),0.97(s,3H). 13 C NMR(100MHz,CDCl3)δ172.1,171.6,158.1,149.3,147.9,138.1,135.8,129.8,128.8,116.1,111.3,110.7,84.0,78.8,69.1 ,65.7,63.5,58.7,53.4,53.3,52.2,50.9,48.0,42.3,39.9,39.9,35.3,34.1,29.8,29.1,29.0,27.2,15.9.HRMS(ESI)calcd for C 37 H 47 ClN3O9S 744.2716[M+H] + ,found 744.2700.

[0140] Example 16

[0141] [ka]

[0142] The preparation method of 4-((4-((5-((1-(2-((1R,3R,4S)-3-(3-chloroprop-1-en-2-yl)-4-methyl-4-vinylcyclohexyl)allyl)piperidin-4-yl)methoxy)-5-oxopentanoyl)oxy)but-2-yn-1-yl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide is basically the same as in Example 4, except that N-methyl-2-hydroxyethylamine in (1) is replaced with 4-hydroxymethylpiperidine.

[0143] In this example, a yellow waxy liquid was prepared in 60% yield.

[0144] 1 H NMR(400MHz,CDCl3)δ8.07(d,J=7.5Hz,2H),7.77(t,J=7.5Hz,1H),7.63(t,J=7.8Hz,2H),5 .78(dd,J=17.5,10.8Hz,1H),5.25(s,1H),5.09(s,2H),5.00-4.85(m,5H),4.74(s,2H),4.0 9(d,J=11.6Hz,1H),4.01-3.87(m,3H),2.89(d,J=17.0Hz,4H),2.41(dt,J=18.4,7.3Hz,5H) ,2.30-2.23(m,1H),2.16-2.06(m,1H),2.04-1.77(m,5H),1.73-1.39(m,10H),0.96(s,3H). 13C NMR(100MHz,CDCl3)δ173.0,172.2,158.0,149.4,147.8,137.8,135.9,129.8,128.8,116.2,111.3,110.7,84.0,78.6,76.8,69 .1,63.7,58.7,53.5,53.4,52.0,51.1,47.7,42.2,39.9,39.8,35.4,34.0,33.2,33.0,29.0,27.0,20.1,15.8.HRMS(ESI)calcd for C 38 H 49 ClN3O9S 758.2873[M+H] + ,found 758.2866.

[0145] Example 17

[0146] [ka]

[0147] The preparation method of 4-(2-((4-((1-(2-((1R,3R,4S)-3-(3-chloroprop-1-en-2-yl)-4-methyl-4-vinylcyclohexyl)allyl)piperidin-3-yl)methoxy)-4-oxobutanoyl)oxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide is basically the same as in Example 1, except that N-methyl-2-hydroxyethylamine in (1) is replaced with 3-hydroxymethylpiperidine.

[0148] In this example, a yellow waxy liquid was prepared with a yield of 70%.

[0149] 1H NMR(400MHz,CDCl3)δ8.07(d,J=7.4Hz,2H),7.77(t,J=7.5Hz,1H),7.63(t,J=7.9 Hz,2H),5.78(dd,J=17.9,10.3Hz,1H),5.26(s,1H),4.98-4.82(m,5H),4.65-4.6 0(m,2H),4.54-4.50(m,2H),4.14-3.87(m,4H),2.88(s,2H),2.67(t,J=3.8Hz,6H ),2.32-2.23(m,1H),2.11(s,1H),1.91(s,2H),1.78-1.41(m,12H),0.97(s,3H). 13 C NMR(100MHz,CDCl3)δ172.3,172.2,158.8,151.1,149.4,147.8,138.1,135.8,129.8,128.8,116.2,111.3,111.3,110.5,69. 0,67.7,64.0,61.5,57.0,54.2,51.1,47.8,42.2,39.9,39.9,35.7,34.1,34.0,29.0,27.3,27.1,24.6,15.8.HRMS(ESI)calcd for C 35 H 47 ClN3O9S 720.2716[M+H]+,found 720.2721.

[0150] Example 18

[0151] [ka]

[0152] The preparation method of 4-(2-((5-((1-(2-((1R,3R,4S)-3-(3-chloroprop-1-en-2-yl)-4-methyl-4-vinylcyclohexyl)allyl)piperidin-3-yl)methoxy)-5-oxopentanoyl)oxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide is basically the same as in Example 2, except that N-methyl-2-hydroxyethylamine in (1) is replaced with 3-hydroxymethylpiperidine.

[0153] In this example, a yellow ワックス-like liquid was prepared with a yield of 74%.

[0154] 1 H NMR (400MHz, CDCl3) δ8.06(d,J=7.5Hz,2H),7.76(t,J=7.5Hz,1H),7.63(t,J=7.8Hz,2H),5.77( dd,J=17.3,10.9Hz,1H),5.25(s,1H),4.98-4.80(m,5H),4.68-4.57(m,2H),4.55-4.45(m,2H), 4.09(d,J=11.6Hz,1H),3.94(dd,J=21.4,11.3Hz,3H),2.99-2.54(m,4H),2.42(dt,J=20.6,7.3 Hz,4H),2.30-2.23(m,1H),2.11(s,1H),1.97(p,J=7.3Hz,4H),1.76-1.36(m,10H),0.97(s,3H). 13 C NMR (100MHz, CDCl3) δ173.0,172.8,158.8,149.4,147.8,138.0,135.8,129.8,128.7,116.2,111.3,110.5,69.0,67.3,63.9 ,61.2,57.0,54.2,51.1,47.8,42.2,39.9,39.8,35.7,34.0,34.0,33.2,33.1,27.3,27.0,24.6,20.0,15.8.HRMS(ESI)calcd for C 36 H 49 ClN3O9S 734.2873[M+H] + ,found 734.2871.

[0155] Example 19

[0156]

change

[0157] The preparation method of 4-((4-((4-((1-(2-((1R,3R,4S)-3-(3-chloroprop-1-en-2-yl)-4-methyl-4-vinylcyclohexyl)allyl)piperidin-3-yl)methoxy)-4-oxobutanoyl)oxy)but-2-yn-1-yl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide is basically the same as in Example 3, except that N-methyl-2-hydroxyethylamine in (1) is replaced with 3-hydroxymethylpiperidine.

[0158] In this example, a yellow waxy liquid was prepared with a yield of 70%.

[0159] 1 H NMR(400MHz,CDCl3)δ8.07(d,J=7.5Hz,2H),7.77(t,J=7.5Hz,1H),7.63(t,J=7. 8Hz,2H),5.78(dd,J=17.8,10.4Hz,1H),5.25(s,1H),5.10(s,2H),4.91(d,J=14. 0Hz,5H),4.76(s,2H),4.11-3.91(m,4H),2.88(s,2H),2.80-2.51(m,J=3.7Hz,7H ),2.31-2.21(m,1H),2.12(s,1H),1.92(s,3H),1.71-1.39(m,10H),0.97(s,3H). 13 C NMR(100MHz,CDCl3)δ172.2,171.6,158.0,149.4,147.8,137.9,135.9,129.8,128.8,116.2,111.3,110.7,83.9,78.7,67.7 ,63.9,58.7,57.0,54.2,52.2,51.1,47.8,42.2,39.9,39.8,35.7,34.0,29.0,28.9,27.3,27.0,24.6,15.8.HRMS(ESI)calcd for C 37 H 47 ClN3O9S 744.2716[M+H] + ,found 744.2709.

[0160] Example 20

[0161] [ka]

[0162] The preparation method of 4-((4-((5-((1-(2-((1R,3R,4S)-3-(3-chloroprop-1-en-2-yl)-4-methyl-4-vinylcyclohexyl)allyl)piperidin-3-yl)methoxy)-5-oxopentanoyl)oxy)but-2-yn-1-yl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide is basically the same as in Example 4, except that N-methyl-2-hydroxyethylamine in (1) is replaced with 3-hydroxymethylpiperidine.

[0163] In this example, a yellow waxy liquid was prepared in 73% yield.

[0164] 1 H NMR(400MHz,CDCl3)δ8.07(d,J=7.7Hz,2H),7.77(t,J=7.5Hz,1H),7.63(t,J=7.8Hz,2H),5.7 8(dd,J=17.9,10.4Hz,1H),5.26(s,1H),5.10(s,2H),4.92(d,J=14.0Hz,5H),4.74(s,2H),4.0 9(d,J=11.7Hz,1H),3.94(dd,J=18.4,11.3Hz,3H),3.02-2.57(m,5H),2.40(dt,J=20.6,7.3Hz ,5H),2.31-2.23(m,1H),2.11(s,1H),1.96(p,J=7.3Hz,4H),1.70-1.42(m,10H),0.97(s,3H). 13C NMR(100MHz,CDCl3)δ172.9,172.2,158.0,151.1,149.4,147.8,138.0,135.9,129.8,128.8,116.2,111.3,110.7,84.1,78.7,67.4 ,64.0,58.7,57.0,54.2,52.0,51.1,47.9,42.3,39.9,39.9,35.8,34.1,33.2,33.1,27.3,27.1,24.7,20.1,15.9.HRMS(ESI)calcd for C 38 H 49 ClN3O9S 758.2873[M+H] + ,found 758.2897.

[0165] Example 21

[0166] [ka]

[0167] The preparation method of 4-(2-((4-(2-(4-(2-((1R,3R,4S)-3-(3-chloroprop-1-en-2-yl)-4-methyl-4-vinylcyclohexyl)allyl)piperazin-1-yl)ethoxy)-4-oxobutanoyl)oxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide is basically the same as in Example 1, except that N-methyl-2-hydroxyethylamine in (1) is replaced with N-hydroxyethylpiperazine.

[0168] In this example, a yellow waxy liquid was prepared in 53% yield.

[0169] 11H NMR (400 MHz, CDCl3) δ 8.06 (d, J = 7.3 Hz, 2H), 7.75 (t, J = 7.5 Hz, 1H), 7.62 (t, J = 7.8 Hz, 2H), 5.78 (dd, J = 17.8, 10.4 Hz, 1H), 5.26 (s, 1H), 4.98 - 4.83 (m, 5H), 4.62 (dd, J = 5.6, 3.5 Hz, 2H), 4.51 (dd, J = 5.4, 3.6 Hz, 2H), 4.21 (t, J = 6.0 Hz, 2H), 4.08 (d, J = 11.6 Hz, 1H), 3.96 (d, J = 11.7 Hz, 1H), 2.97 - 2.84 (m, 2H), 2.67 (s, 4H), 2.63 (t, J = 6.0 Hz, 2H), 2.46 (d, J = 40.2 Hz, 8H), 2.29 - 2.24 (m, 1H), 2.15 - 2.08 (m, 1H), 1.64 - 1.43 (m, 6H), 0.97 (s, 3H). 13 13C NMR (100 MHz, CDCl3) δ 172.2, 172.1, 158.8, 150.7, 149.4, 147.9, 138.3, 135.7, 129.8, 128.8, 116.2, 111.2, 111.0, 110.5, 69.0, 63.5, 62.4, 61.5, 56.7, 53.7, 53.2, 51.0, 48.0, 42.4, 39.9, 34.1, 29.1, 29.1, 27.1, 15.9. HRMS (ESI) calcd for C 35 H 48 ClN4O9S 735.2825 [M + H] + , found 735.2831.

[0170] Example 22

[0171]

Chemical Structure

[0172] The preparation method of 4-(2-((4-(2-(4-(2-((1R,3R,4S)-3-(3-chloroprop-1-en-2-yl)-4-methyl-4-vinylcyclohexyl)allyl)piperazin-1-yl)ethoxy)-4-oxobutanoyl)oxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide is basically the same as in Example 2, except that N-methyl-2-hydroxyethylamine in (1) is replaced by N-hydroxyethylpiperazine.

[0173] In this example, a yellow waxy liquid was prepared in 56% yield.

[0174] 1 H NMR(400MHz,CDCl3)δ8.06(d,J=7.3Hz,2H),7.76(t,J=7.5Hz,1H),7.62(t,J=7.9Hz,2H),5.78(dd,J=17. 9,10.4Hz,1H),5.26(s,1H),4.99-4.86(m,5H),4.65-4.61(m,2H),4.53-4.47(m,2H),4.21(t,J=5.9Hz,2 H),4.09(d,J=11.1Hz,1H),3.97(d,J=11.7Hz,1H),2.92(s,2H),2.62(t,J=5.9Hz,2H),2.59-2.36(m,12H) ),2.30-2.24(m,1H),2.16-2.09(m,1H),2.01-1.95(m,2H),1.81(s,2H),1.64-1.41(m,6H),0.98(s,3H). 13 C NMR(100MHz,CDCl3)δ172.9,172.7,158.8,150.7,149.4,147.9,138.3,135.8,129.8,128.8,116.2,111.3,111.0,110.6,6 9.1,63.5,62.0,61.3,56.8,53.6,53.2,51.1,48.0,42.4,40.0,34.1,33.3,33.2,29.8,27.2,20.1,15.9.HRMS(ESI)calcd for C 36 H 50 ClN4O9S 749.2982[M+H] +,found 749.2993.

[0175] Example 23

[0176] [ka]

[0177] The preparation method of 4-((4-((4-(2-(4-(2-((1R,3R,4S)-3-(3-chloroprop-1-en-2-yl)-4-methyl-4-vinylcyclohexyl)allyl)piperazin-1-yl)ethoxy)-4-oxobutanoyl)oxy)but-2-yn-1-yl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide is basically the same as in Example 3, except that N-methyl-2-hydroxyethylamine in (1) is replaced with N-hydroxyethylpiperazine.

[0178] In this example, a yellow waxy liquid was prepared in 60% yield.

[0179] 1 H NMR(400MHz,CDCl3)δ8.07(d,J=7.5Hz,2H),7.77(t,J=7.5Hz,1H),7.63(t,J=7.8Hz,2H),5.78(d d,J=17.8,10.4Hz,1H),5.26(s,1H),5.10(s,2H),4.98-4.86(m,5H),4.76(s,2H),4.23(t,J=5.9H z,2H),4.09(d,J=11.6Hz,1H),3.97(d,J=11.7Hz,1H),2.92(s,2H),2.69-2.62(m,6H),2.48(d,J =40.6Hz,7H),2.30-2.24(m,1H),2.16-2.08(m,1H),1.89(s,1H),1.64-1.43(m,6H),0.98(s,3H). 13C NMR(100MHz,CDCl3)δ172.1,171.6,158.1,150.7,149.4,147.9,138.1,135.8,129.8,128.8,116.2,111.3,111.1,110.7,8 4.0,78.8,63.5,62.4,58.7,56.7,53.6,53.2,52.2,51.1,48.0,42.4,40.0,34.1,29.1,29.0,27.2,15.9.HRMS(ESI)calcd for C 37 H 48 ClN4O9S 759.2825[M+H] + ,found 759.2811.

[0180] Example 24

[0181] [ka]

[0182] The preparation method of 4-((4-((5-(2-(4-(2-((1R,3R,4S)-3-(3-chloroprop-1-en-2-yl)-4-methyl-4-vinylcyclohexyl)allyl)piperazin-1-yl)ethoxy)-5-oxyglutaryl)oxy)but-2-yn-1-yl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide is basically the same as in Example 4, except that N-methyl-2-hydroxyethylamine in (1) is replaced with N-hydroxyethylpiperazine.

[0183] In this example, a yellow waxy liquid was prepared in 58% yield.

[0184] 1H NMR(400MHz,CDCl3)δ8.06(d,J=7.5Hz,2H),7.76(t,J=7.5Hz,1H),7.63(t,J=7.9Hz,2H),5.77(dd,J= 17.5,10.7Hz,1H),5.25(s,1H),5.09(s,2H),4.96-4.86(m,5H),4.73(s,2H),4.20(t,J=5.9Hz,2H),4 .08(d,J=11.5Hz,1H),3.96(d,J=11.7Hz,1H),2.91(s,2H),2.62(t,J=5.9Hz,2H),2.42(dt,J=20.1,7 .3Hz,12H),2.29-2.24(m,1H),2.13-2.07(m,1H),1.98-1.91(m,2H),1.63-1.41(m,6H),0.96(s,3H). 13 C NMR(100MHz,CDCl3)δ172.8,172.1,157.9,150.5,149.3,147.7,137.8,135.8,129.8,128.7,116.2,111.2,111.0,110.6,84.0,7 8.6,63.3,61.8,58.6,56.7,53.5,53.0,51.9,51.1,47.6,42.1,39.8,39.7,33.9,33.1,32.9,27.0,19.9,15.7.HRMS(ESI)calcd for C 38 H 50 ClN4O9S 773.2982[M+H] + ,found 773.2974.

[0185] Pharmacodynamics Experiments

[0186] 1. In vitro nitric oxide release test

[0187] 1.1 Experimental equipment and reagents

[0188] Equipment

[0189] Multi-function microplate reader (MD Spectramac M3, USA) Clean bench (Suzhou Purification)

[0190] reagent

[0191] Potassium dihydrogen phosphate (Alata Chemical Reagents Co., Ltd.) Dipotassium hydrogen phosphate (Alata Chemical Reagents Co., Ltd.) L-cysteine ​​(Alading Chemical Reagent Co., Ltd.)

[0192] 1.2 Experimental Method

[0193] (1) Sodium nitrite solutions of different concentrations were prepared and a calibration curve was created.

[0194] (2) The samples were treated to prepare 100 μM solutions, and 2.5 mL of the prepared compound solution and 2.5 mL of the cysteine ​​solution were taken and incubated at 37° C. for 120 min, and then sampled at different time points.

[0195] (3) Blank wells and standard wells were set up, and 160 μL of sample was collected and mixed uniformly with 0.08 mL of color developer, and allowed to stand at 37° C. for 15 min. The absorbance (OD value) at λ=550 nm was measured with a microplate reader, and the values ​​were substituted into the standard curve to obtain the amount of NO released.

[0196] 1.3 Test results

[0197] Table 1. In vitro NO release results of β-elemene derivatives prepared in Examples 1 to 24 [Table 1] JPEG0007645007000033.jpg189170

[0198] As shown by the data in Table 1, all of the compounds in the examples were able to effectively release nitric oxide, and in most examples, the levels of nitric oxide release increased over time.

[0199] 2. In vitro antitumor activity evaluation test

[0200] 2.1 Experimental equipment and reagents

[0201] Equipment:

[0202] Clean bench (Suzhou Purification Equipment Co., Ltd.) CO2 incubator (Japan SANYO) Inverted biological microscope (OLYMPUS Japan) Microplate reader (BioTek, USA)

[0203] reagent:

[0204] Penicillin-streptomycin mixed solution (Jiangsu Kaiji Bio-Technology Co., Ltd.) Trypsin Digestive Solution (Jiangsu Kaiji Biotechnology Co., Ltd.) PBS (Jiangsu Kaiji Biotechnology Co., Ltd.) Fetal Bovine Serum (GIBCO) RPMI-1640 (Jiangsu Kaiji Biotechnology Co., Ltd.) DMEM (Jiangsu Kaiji Biotechnology Co., Ltd.) L-15 (Jiangsu Kaiji Biotechnology Co., Ltd.) CCK8 (Jiangsu Kaiki Biotechnology Co., Ltd.) DMSO (SIGMA)

[0205] 2.2 Experimental Method

[0206] (1) Digest, count, and prepare cell suspensions (H520, SW620 5.0 × 10 4 pieces / mL, U87MG3.5×10 4 cells / mL), and 100 μL of the cell suspension was added to each well of a 96-well cell culture plate.

[0207] (2) The 96-well cell culture plate was cultured in a 37°C, 5% CO2 incubator for 24 hours.

[0208] (3) The drugs were diluted with culture medium to the desired working solution concentration, and 100 μL of the corresponding drug-containing culture medium was added to each well, and a negative control group and a positive control group were set up at the same time.

[0209] (4) The 96-well cell culture plate was cultured in a 37°C, 5% CO2 incubator for 72 hours.

[0210] (5) The 96-well plate was stained with CCK-8, and the OD value at λ = 450 nm was measured.

[0211] 1) 10 μL of CCK-8 was added to each well and the plates were incubated in an incubator for 2 to 3 hours.

[0212] To remove air bubbles in the 96-well plate, the plates were shaken on a shaker for 10 minutes to mix uniformly.

[0213] 3) The OD value of each well at λ=450 nm was read using a microplate reader, and the inhibition rate was calculated.

[0214] (6) The tumor cell proliferation inhibition rate was calculated using the following formula (1). The results were expressed as IC 50 Substituting into the calculation software SPSS17.0, IC 50 Calculate the value, IC 50 The results are shown in Table 1.

[0215] Formula (1)

number

[0216] 2.3 Experimental results

[0217] Table 2. Antiproliferative activity of Examples 1 to 24 (1 μM) against three human cancer cell lines, as a percentage of inhibition. [Table 2] JPEG0007645007000036.jpg154170

[0218] As shown in Table 2, the 24 compounds showed different degrees of inhibitory effects against three types of human tumor cells, SW620, U87MG and NCI-H520, at a concentration of 1 μM, and the antiproliferative activities were all significantly stronger than those of β-elemene and 13,14-dichloro-β-elemene, indicating that the introduction of NO donors successfully improved the antitumor effect of β-elemene.

[0219] Table 3. Antiproliferative activity of preferred example compounds against three human cancer cell lines, IC 50 (μM) [Table 3] JPEG0007645007000038.jpg227170

[0220] Table 3 shows that the antiproliferative activities of most of the compounds were significantly stronger than those of β-elemene and 13,14-dichloro-β-elemene. For the SW620 cell line, the activities of Examples 4 and 14 were more than 110-fold stronger than β-elemene 110. For the U87MG cell line, the antiproliferative activities of Examples 2, 4, 5, 6, 9, 10 and 22 were more than 250-fold stronger than β-elemene. For the NCI-H520 cell line, the activities of Examples 2, 4, 10, 14 and 22 were more than 100-fold stronger than β-elemene.

[0221] 3. In vivo antitumor activity against malignant glioma

[0222] 3.1 Test animals and experimental equipment

[0223] Test animals:

[0224] Source, species, and lineage: BABLc / nude mice, provided by Shanghai Silica Laboratory Animal Co., Ltd.

[0225] Experimental Animal Production Permit: SCXK(Hu)2017-0005

[0226] Certificate number: 20170005040021

[0227] Experimental Animal Use Permit: SYXK(Su)2017-0040

[0228] Age: 4-5 weeks

[0229] Gender: Female

[0230] Number of animals: 5 per group, 15 in total.

[0231] Experimental equipment:

[0232] 3D imaging system for small animals, Spectrum, PerkinElmer

[0233] 3.2 Model creation

[0234] Cultured human glioma U87MG-LUC cell suspension at a concentration of 5 x 10 7 The cells were collected at 100 / mL and seeded orthotopically in the brain at 20 μL per mouse. Mice were anesthetized by intraperitoneal injection of 10% chloral hydrate, and the mouse head was fixed in a stereotaxic apparatus. The head skin was disinfected with an alcohol swab, and a slight incision was made to the right along the midline of the brain. The right hemisphere was removed, and the anterior fontanel was used as the reference point, and 2 mm to the right and 1 mm in front of the midline were used as the needle entry point, and the skull was ground with a microdrill. After the cells were thoroughly resuspended with a pipette tip, the cell suspension was extracted with a syringe, and the needle was inserted vertically 3.5 mm in a stereotaxic apparatus, withdrawn 0.5 mm, stopped for 1 min, and fixed to slowly inject the cells. After the injection was completed, the needle was stopped for 1 min and slowly withdrawn. The skin was disinfected with an alcohol swab, and the skin was sutured with a needle thread, and the mouse was returned to the breeding cage and allowed to wake up naturally.

[0235] 3.3 Grouping and Administration

[0236] 14 days after cell seeding, the animals were randomly divided into groups of 5. At the same time, the nude mice in each group were administered, and the administration schedule was based on the grouping and administration design. After the experiment was completed, the nude mice were immediately sacrificed, and the brain tissue was surgically removed, photographed, and weighed.

[0237] 3.4 Bioimaging Detection

[0238] Two animals were selected from each group, and 100 μL of Luc fluorescent substrate was intraperitoneally injected per animal before administration, and 1, 2, and 3 weeks after administration. After 10 min, the mice were anesthetized with isoflurane and placed on the darkroom stage of the instrument for bioimaging detection to observe the fluorescent images of the in situ tumors in the mouse brain.

[0239] 3.5 Experimental results

[0240] As shown in FIG. 6, after 3 weeks of administration of β-elemene and Example 4, the growth of malignant glioma in mice was effectively inhibited. Although the tumor volume of the model group continued to increase (FIG. 6), after treatment with β-elemene and Example 4, the tumor volume continued to be inhibited and was significantly smaller than that of the model group. In terms of brain weight, the body weight of the β-elemene and Example 4 group was significantly lighter than that of the model group, and the life and physical condition of the mice were also significantly better than that of the model group. Meanwhile, the fluorescence intensity of the glioma in the model group continued to increase rapidly, while the fluorescence intensity of the β-elemene and Example 4 group rapidly decreased. In the first week, the inhibitory activity of Example 4 was over 80%, significantly higher than that of β-elemene (>20%). In the second week, both β-elemene and Example 4 reached inhibition of over 80%. In the last week of administration, Example 4 inhibited tumor growth by over 90%, showing strong malignant glioma inhibitory activity. In any case, both β-elemene and Example 4 exhibited effective therapeutic activity, but Example 4 exhibited a stronger inhibitory effect than β-elemene.

[0241] These results suggest that the introduction of NO donors effectively enhanced the antitumor effect of β-elemene in vivo and is a viable strategy for the development of β-elemene-based antitumor drugs.

[0242] Furthermore, after reading the above description of the invention, one skilled in the art should understand that various changes or modifications can be made to the invention, the equivalents of which also fall within the scope defined by the claims appended to this application.

Claims

1. Formula (I) 【Chemistry 1】 (In the formula, R １ is a linear or cyclic alcohol amine structure containing a nitrogen atom and an oxygen atom, R ２ , R ３ are each independently １－１０ Alkyl group, C ３－１２ Cycloalkyl group, C ６－１２ Aryl group, 5-10 membered heteroaryl group, C ２－１０ Alkenyl group, C ２－１０ Alkynyl group or C ２－１０ is a group selected from alkoxy groups. The structural formula is represented by In formula (I), R １ teeth, 【Chemistry 2】 is one selected from R ２ , R ３ are each independently 【Chemistry 3】 14-chloro-β-elemene nitric oxide donor derivative, its pharma- ceutically acceptable salt, solvate, enantiomer, or diastereomer, characterized in that it is any one of the following:

2. The 14-chloro-β-elemene nitric oxide donor derivative is any one selected from formulas I-1 to I-6, 【Chemistry 4】 In formulas I-1 to I-6, R ２ , R ３ are each independently ２－６ Alkyl groups of C ２－６ an alkenyl group of C ２－６ The 14-chloro-β-elemene nitric oxide donor derivative according to claim 1, characterized in that the alkynyl group is any one selected from the group consisting of:

3. The 14-chloro-β-elemene nitric oxide donor derivative according to claim 1, characterized in that the 14-chloro-β-elemene nitric oxide donor derivative is any one selected from compounds having structures represented by formulas 1 to 24, or a pharma- ceutically acceptable salt, solvate, enantiomer, or diastereomer thereof. 【Chemistry 5】 【Chemistry 6】

4. A method for preparing an antitumor drug using the 14-chloro-β-elemene nitric oxide donor type derivative according to any one of claims 1 to 3, or a pharma- ceutically acceptable salt, solvate, enantiomer, or diastereomer thereof.

5. 5. The method of claim 4, wherein the tumor comprises lung cancer, colon cancer, or malignant glioma.

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