1, 3, 4-oxadiazino tetrahydroisoquinoline compound as well as preparation method and application thereof

Abstract

The invention provides a 1, 3, 4-oxadiazino tetrahydroisoquinoline compound as well as a preparation method and application thereof, and belongs to the technical field of heterocyclic compounds for medicines. The method comprises the following steps: by taking an azomethine imine substrate and a styrene oxide substrate as raw materials and cobalt perchlorate hexahydrate as a catalyst, adding the raw materials and the catalyst into a reaction solvent, reacting overnight at normal temperature, and performing column separation to obtain the 1, 3, 4-oxadiazino tetrahydroisoquinoline compound. According to the application, the reaction condition is mild, the selectivity is high, excellent cytotoxicity is shown on HeLa cervical cancer cells and A549 lung cancer cells, the half death inhibition ratio (IC50) value on tumor cells reaches the nanomole level, and the application has a good anti-cancer drug application prospect.

Description

Technical Field

[0001] The present application relates to a 1,3,4-oxadiazine tetrahydroisoquinoline compound, a preparation method and an application thereof, and belongs to the technical field of pharmaceutical heterocyclic compounds. Background Art

[0002] Cancer is a common and frequently occurring disease that threatens human health and life, becoming the second leading cause of death. Lung cancer, in particular, has the highest mortality rate of all cancers. Currently, traditional surgical or drug treatments, respectively, suffer from significant side effects and cell resistance, failing to meet the clinical needs of patients. Therefore, the development of innovative drugs for lung cancer is of paramount importance.

[0003] Oxadiazines are six-membered heterocyclic compounds consisting of one oxygen atom and come in many types. Among all types of oxadiazines, 1,3,4-oxadiazines exhibit the broadest range of biological activities. For example, 4H-1,3,4-oxadiazine-5-one compounds have a good inhibitory effect on monoamine oxidase and can be used clinically to treat depression. The antifungal drug ofloxacin has enhanced antifungal activity and a broader antibacterial spectrum after the introduction of a 1,3,4-oxadiazine ring. In addition, all 5,6-dihydro-4H-1,3,4-oxadiazine derivatives antagonize the ovicidal, acaricidal, and insecticidal activities of the insecticide indoxacarb. Therefore, the synthesis of 1,3,4-oxadiazines has always attracted much attention.

[0004]

[0005] Traditional synthesis methods for 1,3,4-oxadiazines mainly include [4+2] cycloaddition reactions of hydrazides with (chain) alkenes or dibromoethane and [3+3] cyclization reactions of hydrazones with styrene oxides or phenols (X. Guo, X. Chen, Y. Cheng, X. Chang, X. Li, P. Li, Organocatalytic enantioselective [2+4]-annulation of γ-substituted allenoates with N-acyldiazenes for the synthesis of optically active 1,3,4-oxadiazines, Org. Biomol. Chem. 2021, 19, 1727; H. Zuo, J. Qin, W. Zhang, M. A. Bashir, Q. Yu, W. Zhao, G. Wu, F. Zhong, Hemin-Catalyzed Oxidative Phenol-Hydrazone [3+3] Cycloaddition Enables Rapid Construction of1,3,4-Oxadiazines,Org.Lett.2020,22,6911; JACortés-Vázquez,J.Davis,VNNesterov,H.Wang,W.Luo,Sc(OTf)3-Catalyzed Formal[3+3]Cycloaddition Reaction of Diaziridines andQuinones for the Synthesis of Benzo[e][1,3,4]oxadiazines,Org.Lett.2021,23,3136; M.Mishra,PKMaharana,P.Karjee,T.Punniyamurthy,Expedient cobalt-catalyzed stereospecific cascade C-Nand CO bond formation of styrene oxideswith hydrazones,Chem.Commun.2022,58,7090;SHKang,BJPark,S.-G.Kim,Base-Promoted[3+3]-Cycloaddition ofγ-Hydroxy-α,β-unsaturated Carbonyls with N,N′-Cyclic Azomethine Imines for Synthesizing Bicyclic Oxadiazines,Eur.J.Org.Chem.2022,2022(37),1;SHKang,BJPark,S.-G.Kim,EnantioselectiveSynthesis of Diazobicyclic Oxadiazines via Organocatalytic[3+3]-Cycloadditionofγ-Hydroxy-α,β-Unsaturated Carbonyls with N,N′-Cyclic Azomethine Imines, Synthesis. 2023, 55, 1410; X. Qiao, Y. Han, S. Huang, Y. Sun, C. Wang, Z. Wang, F. Li, L. Wang, Visible-light-induced [3+3] cycloaddition reaction of phenol and hydrazone to access 1,3,4-oxadiazines scaffolds, J. Mol. Catal. 2024, 561, 114-156). These methods involve unstable substrate structures or low product selectivity (such as alkenes), complex reaction conditions (such as the need to add additional acid-binding agents or heat the reaction), or the production of toxic and harmful substances such as hydrocyanic acid, which can easily cause environmental pollution. In addition, these methods do not further clarify the biological activity value of 1,3,4-oxadiazines.

[0006] Furthermore, research on the biological activity of 1,3,4-oxadiazines has primarily focused on pesticides, antimicrobials, and herbicides. Studies on the anti-tumor activity of these compounds are limited, particularly regarding their toxicity to lung cancer cells. Summary of the Invention

[0007] In view of this, the present application provides a 1,3,4-oxadiazine tetrahydroisoquinoline compound and its selective preparation and application.

[0008] Specifically, this application is implemented through the following solutions:

[0009] The first object of the applicant is to provide a 1,3,4-oxadiazine tetrahydroisoquinoline compound, which has the following general structural formula:

[0010] R has no substituent or the substituent is any one of benzenesulfonyl, p-toluenesulfonyl, methanesulfonyl, p-methoxybenzenesulfonyl and p-nitrobenzenesulfonyl; R 1 is any one of hydrogen, methyl, tert-butyl, fluorine, phenyl, chlorine, bromine, acetoxy, and trifluoromethyl; R 2 It is any one of hydrogen, phenyl, bromine, fluorine and methyl.

[0011] When R 1 When the R group on the benzene ring is an electron-donating or weak electron-withdrawing group (such as hydrogen, methyl, tert-butyl, fluorine or phenyl, etc.), the reaction generates a 1,3,4-oxadiazine tetrahydroisoquinoline compound containing an R protecting group; when the R group on the benzene ring is 1 When the substituents are electron-withdrawing (e.g., chlorine, bromine, acetoxy, or trifluoromethyl), and the catalyst dosage is increased, the reaction primarily produces compounds with deprotected groups. The electronegativity of the substituents on the azomethine imine has little effect on the reaction selectivity, primarily producing 1,3,4-oxadiazinone tetrahydroisoquinoline compounds containing R-protecting groups.

[0012] Furthermore, as a preference:

[0013] The structural formula of the 1,3,4-oxadiazine tetrahydroisoquinoline compound is

[0014] Any of .

[0015] The second purpose of the applicant is to provide a method for preparing the above-mentioned 1,3,4-oxadiazine tetrahydroisoquinoline compounds, which is characterized in that: an azomethine imine substrate and a styrene oxide substrate are used as raw materials, cobalt perchlorate hexahydrate is used as a catalyst, the raw materials and the catalyst are added to a reaction solvent, and the reaction is carried out at room temperature overnight, and the 1,3,4-oxadiazine tetrahydroisoquinoline compounds are obtained by column separation.

[0016] Preferred:

[0017] The amount of catalyst added is 5-20% of the total molar amount of the raw materials. 1 When the substituent is an electron-withdrawing group, increasing the amount of catalyst is conducive to the formation of products with deprotected group structures, and when the catalyst amount is increased to 20 mol%, the main product is deprotected 1,3,4-oxadiazine tetrahydroisoquinoline compounds.

[0018] The molar ratio of the azomethine imine substrate to the styrene oxide substrate is 1:1.2-1.5.

[0019] The reaction solvent can be selected from any one of dichloromethane, dichloroethane, chloroform, ethyl acetate, tetrahydrofuran, and acetonitrile. In particular, when the reaction solvent is dichloromethane, the reaction effect in dichloromethane is the best, and the target product can be obtained with a yield of 80-96%. When diethyl ether or toluene is used as the reaction solvent, the reaction raw materials have poor solubility, resulting in low yield.

[0020] The reaction time is 12 to 18 hours.

[0021] The column chromatography eluent in the column separation is a mixture of petroleum ether and dichloromethane, and the volume ratio of petroleum ether to dichloromethane is 0.5-5:1.

[0022] The above-mentioned method for preparing a 1,3,4-oxadiazine tetrahydroisoquinoline compound is characterized in that the reaction formula of the preparation process is as follows:

[0023]

[0024] 1,3,4-oxadiazine tetrahydroisoquinoline compounds exhibit excellent cytotoxicity against HeLa and A549 tumor cells. Therefore, the third objective of the applicant is to provide the use of the above-mentioned 1,3,4-oxadiazine tetrahydroisoquinoline compounds in anti-tumor preparations:

[0025] When R 2 =H, R = Ts and R 1 =4-Cl, 4-Br or H, the structural formula of 1,3,4-oxadiazine tetrahydroisoquinoline compounds is Among them, LY1 and LY5 have an inhibitory effect on tumor cell IC 50 The values ​​can reach nanomolar level, and the IC 50 =0.9821μM, IC for A549 cells 50 =0.9290 μM, IC of LY5 for HeLa cells 50 =0.493 μM, IC for A549 cells 50 =0.09289μM, and can be used for the preparation of anti-tumor preparations such as cervical cancer and lung cancer.

[0026] When there is no substituent at the R position (i.e., no R), R 2 =H, R 1 =3-Br, the structural formula of 1,3,4-oxadiazine tetrahydroisoquinoline compounds is Its IC for A549 tumor cells 50The value reached nanomolar level, and the IC 50 =0.1671μM, which can be used for the preparation of anti-A549 lung cancer preparations.

[0027] The beneficial effects of this application are summarized as follows:

[0028] (1) The present application achieves a one-step reaction to prepare 1,3,4-oxadiazine tetrahydroisoquinoline compounds by controlling the electronegativity of the substituents on the benzene ring with the help of the catalytic effect of cobalt perchlorate. This provides a new method for the selective synthesis of tetrahydroisoquinoline and oxadiazine derivatives. The method has fewer reaction steps, simple operation, mild conditions, and reduces the synthesis cost.

[0029] (2) This application has determined that 1,3,4-oxadiazine tetrahydroisoquinoline compounds have excellent killing ability against HeLa and A549 tumor cells through tumor cell toxicity experiment screening. Its IC 50 The value reaches the nanomolar level and has potential application value in the development of anti-cancer drugs.

[0030] (3) The 1,3,4-oxadiazine tetrahydroisoquinoline compounds prepared by the above method provide lead compounds for the subsequent development of innovative drugs for anti-cervical cancer and anti-lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments or descriptions of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0032] Figure 1 is the inhibition curve of compound LY5;

[0033] Figure 2 This is the inhibition curve of compound LY7. DETAILED DESCRIPTION

[0034] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application more clearly understood, the technical solutions in the embodiments of this application will be further described in detail below in conjunction with the drawings in the embodiments of this application. It should be understood that the specific embodiments described herein are only used to explain this application and are not used to limit the technical solutions of this application. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of this application.

[0035] The analytical instruments and equipment used in this example are: nuclear magnetic resonance spectrometer, AVANCE DMXⅡ 400M and 500M (TMS internal standard, Bruker); microplate reader: Varioskan LUX (Thermo Fisher).

[0036] Example 1

[0037] This example is about the preparation of azomethine imine substrates.

[0038] Step 1: To a 1000 mL round-bottom flask, a stirring bar and dichloromethane (200 mL), 0.16 mol of isochroman (20 mL), and 8 mL of methanol were added sequentially. The mixture was stirred in an ice bath, followed by the slow addition of 0.18 mol of 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ). The mixture was stirred at room temperature for 48 hours. After completion of the reaction, the mixture was filtered, and the filtrate was extracted with 200 mL of saturated sodium bicarbonate solution. The aqueous phase was further extracted twice with dichloromethane (40 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to obtain 2-methanol isochroman with an 88% yield, which was then directly processed into the next step.

[0039] Step 2: To a 1000 ml round-bottom flask, 330 ml of toluene, 2-methanol obtained in step 1, 0.15 mol of tetrabutylammonium bromide, and 0.15 mol of trimethylsilyl bromide were added in sequence. The mixture was refluxed at 80° C. for 6 hours and then cooled to room temperature. 400 ml of saturated sodium bicarbonate solution was then poured into the mixture to quench the mixture. The aqueous phase was extracted twice with ethyl acetate (40 ml). The organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography with a 50:1 ratio of petroleum ether to ethyl acetate as the eluent to obtain 2-bromoethylbenzaldehyde in a 64% yield.

[0040] Step 3: To a 200 ml round-bottom flask, a stirring magnet and 45 mmol of 2-bromoethylbenzaldehyde and 90 ml of methanol were added in sequence, followed by the addition of 30 mmol of p-toluenesulfonylhydrazide under stirring. The mixture was heated under reflux at 70 ° C for 1 hour, then cooled to room temperature, 45 mmol of triethylamine was added to the reaction solution, and stirring was continued at room temperature for 10 minutes, after which the mixture was poured into 200 ml of water. The mixture was extracted three times with dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography with an eluent of dichloromethane:methanol = 70:1 to obtain azomethine imine compounds with a yield of 22%.

[0041] Example 2

[0042] This example is about the preparation of styrene oxide substrate.

[0043] To a 200 ml round-bottom flask, a stirring magnet, 0.1 mol of a styrene raw material, 70 ml of dichloromethane, and 0.15 mol of m-chloroperbenzoic acid were added in sequence and stirred at room temperature overnight. After the reaction was completed, 100 ml of a saturated sodium bicarbonate solution was poured into the mixture and separated. The aqueous phase was further extracted once with 20 ml of dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by silica gel column chromatography with a ratio of petroleum ether to ethyl acetate of 20:1 as the eluent. The product, styrene oxide substrate, was obtained by concentration.

[0044] Examples 3 to 5 are all R 1 This is the case with electron-donating or weakly electron-withdrawing groups such as hydrogen, methyl, tert-butyl, fluorine, or phenyl.

[0045] Example 3

[0046] In this example, 3-phenyl-4-p-toluenesulfonyl-3,4,7,11b-tetrahydro-2H,6H-[1,3,4]oxadiazine[2,3-a]isoquinoline (LY1) was synthesized.

[0047] To a 10 ml reaction flask, a magnetic stirrer, 1 mmol of azomethine imine (commercially available or prepared as described in Example 1), 1.2 mmol of styrene oxide (commercially available), 2 ml of dichloromethane, and 0.05 mmol of cobalt perchlorate hexahydrate were added in sequence. The mixture was stirred at room temperature for approximately 12 hours. After completion of the reaction, the product was purified by silica gel column chromatography using a 10:1 ratio of petroleum ether to ethyl acetate as the eluent to obtain LY1 as a white solid in a 91% yield.

[0048] The structural characterization data of the white solid LY1 are shown below:

[0049] 1 H NMR (400MHz, CDCl3) δ7.87 (d, J = 8.3Hz, 2H), 7.72 (d, J = 7.7Hz, 2H), 7.34 (t, J = 7. 4Hz,2H),7.31-7.27(m,3H),7.04-6.90(m,1H),5.27(s,1H),5.24(d,J=3.8Hz,1H ),4.81(d,J=12.3Hz,1H),4.34(dd,J=12.2,4.0Hz,1H),2.89(td,J=12.5,12.1, 3.3Hz, 1H), 2.59 (ddd, J=18.1, 12.7, 5.7Hz, 1H), 2.43 (s, 3H), 2.35-2.23 (m, 2H).

[0050] 13C NMR (126MHz, CDCl3) δ143.80,140.26,136.03,134.56,133.10,129.44,128.99,128.65,12 8.49,128.44,128.41,128.20,127.50,126.30,85.73,67.02,49.90,47.21,30.05,21.65.

[0051] According to the above data, the structural formula of the obtained product LY1 is as follows:

[0052] Example 4

[0053] In this example, 3-(p-tert-butylphenyl)-4-p-toluenesulfonyl-3,4,7,11b-tetrahydro-2H,6H-[1,3,4]oxadiazine[2,3-a]isoquinoline (LY2) was synthesized.

[0054] To a 10 ml reaction flask, a stirring magnet, 1 mmol of azomethine imine (commercially available or prepared according to the method in Example 1), 1.2 mmol of 4-tert-butylstyrene oxide (commercially available or prepared according to the method in Example 2, specifically 4-tert-butylstyrene), 2 ml of dichloromethane, and 0.05 mmol of cobalt perchlorate hexahydrate were added in sequence. The mixture was stirred at room temperature for 12 hours. After completion of the reaction, the product was purified by silica gel column chromatography using a 15:1 ratio of petroleum ether to ethyl acetate as the eluent to obtain LY2 as a white solid in a 94% yield.

[0055] The structural characterization data of the product are shown below:

[0056] 1 H NMR(400MHz,Chloroform-d)δ7.91(d,J=8.2Hz,2H),7.66(d,J=8.3Hz,2H),7.38(d,J=8.4Hz,2H), 7.33(d,J=8.1Hz,2H),7.28-7.21(m,3H),7.06-6.97(m,1H),5.32(s,1H),5.25(d,J=3.8Hz,1H),4 .83(d,J=12.1Hz,1H),4.36(dd,J=12.2,4.0Hz,1H),2.95(td,J=12.1,11.2,3.3Hz,1H),2.62(ddd ,J=17.5,12.9,5.7Hz,1H),2.47(s,3H),2.38(d,J=3.0Hz,1H),2.35(t,J=5.2Hz,1H),1.34(s,9H).

[0057] 13 C NMR (101MHz, CDCl3) δ150.45,143.68,137.08,136.14,134.58,133.20,129.38,128.98,128.57,1 28.44,128.14,128.00,126.25,125.33,85.72,67.20,49.73,47.21,34.48,31.33,30.06,21.62.

[0058] According to the above data, the structural formula of the obtained product LY2 is as follows:

[0059] Example 5

[0060] This example synthesizes 3-(p-tolyl)-4-p-toluenesulfonyl-3,4,7,11b-tetrahydro-2H,6H-[1,3,4]oxadiazine[2,3-a]isoquinoline (LY3):

[0061] To a 10 ml reaction flask, a magnetic stirrer, 1 mmol of azomethine imine (commercially available or prepared according to the method in Example 1), 1.2 mmol of 4-methylstyrene oxide (commercially available or prepared according to the method in Example 2, using p-methylstyrene as the corresponding styrene raw material), 2 ml of dichloromethane, and 0.05 mmol of cobalt perchlorate hexahydrate were added in sequence. The mixture was stirred at room temperature for 12 hours. After completion of the reaction, the product was purified by silica gel column chromatography using a 20:1 ratio of petroleum ether to ethyl acetate as the eluent to obtain LY3 as a white solid in a 96% yield.

[0062] The structural characterization data of white solid LY3 are shown below:

[0063] 1 H NMR(400MHz,Chloroform-d)δ7.86(d,J=8.3Hz,2H),7.59(d,J=8.0Hz,2H),7.28(d,J=8.0H z,2H),7.24-7.17(m,3H),7.14(d,J=8.0Hz,2H),7.01-6.95(m,1H),5.26(s,1H),5.20(d,J= 3.7Hz,1H),4.78(d,J=12.2Hz,1H),4.31(dd,J=12.2,4.0Hz,1H),2.93(td,J=12.0,11.0,3. 3Hz,1H),2.58(ddd,J=17.5,12.8,5.7Hz,1H),2.42(s,3H),2.33(s,3H),2.34-2.24(m,2H).

[0064] 13 C NMR (101MHz, CDCl3) δ143.73,137.24,137.18,136.11,134.58,133.14,129.40,129.15,128.9 7,128.61,128.40,128.29,128.18,126.27,85.72,67.17,49.75,47.24,30.03,21.63,21.10.

[0065] According to the above data, the structural formula of the obtained product LY3 is as follows: Comparative Example 1

[0066] The configuration of this comparative example is the same as that of Example 5, except that the added amounts of cobalt perchlorate hexahydrate are 0 (ie, no catalyst is added), 0.1 mmol, and 0.2 mmol, respectively.

[0067] The results showed that no reaction occurred without the addition of cobalt perchlorate hexahydrate. However, as the catalyst dosage increased, compared to the catalyst-to-azomethine imine molar ratio of 1:20 in Example 4, when the cobalt perchlorate hexahydrate dosage was increased to 0.1 mmol, the reaction primarily produced products with oxadiazinane structures, such as structures similar to LY1-LY5. Furthermore, when the cobalt perchlorate hexahydrate dosage was increased to 0.2 mmol, the reaction products were still primarily oxadiazinane structures.

[0068] Comparative Example 2

[0069] The configuration of this comparative example is the same as that of Example 5, except that the reaction time is 18 hours.

[0070] Comparative Example 3

[0071] The configuration of this comparative example is the same as that of Example 5, except that the amount of cobalt perchlorate hexahydrate added is 0.2 mmol and the reaction time is 18 hours.

[0072] It can be seen from the above examples 3 to 5 that: R 1 is an electron-donating or weak electron-withdrawing group (R 1 is hydrogen, and in Example 4 R 1 is tert-butyl, in Example 5 R 1 When the methyl group, fluorine group, and phenyl group are not specifically described), the reaction generates a 1,3,4-oxadiazinetetrahydroisoquinoline compound containing an R protecting group.

[0073] Combining Example 5 with Comparative Examples 1 to 3, it can be seen that: R 1 When it is an electron-donating or weak electron-withdrawing group, increasing the amount of catalyst and extending the reaction time still makes it difficult to obtain the deprotected product, and the reaction product is still mainly a product containing a protecting group.

[0074] Examples 6 to 9 are all R 1 This is an example of electron-withdrawing substitution (such as chlorine, bromine, acetoxy or trifluoromethyl).

[0075] Example 6

[0076] This example synthesizes 3-(p-chlorophenyl)-4-p-toluenesulfonyl-3,4,7,11b-tetrahydro-2H,6H-[1,3,4]oxadiazine[2,3-a]isoquinoline (LY4):

[0077] To a 10 ml reaction flask, a magnetic stirrer, 1 mmol of azomethine imine (commercially available or prepared according to the method in Example 1), 1.2 mmol of 4-chlorostyrene oxide (commercially available or prepared according to the method in Example 2, with the corresponding styrene raw material being 4-chlorostyrene), 2 ml of dichloromethane, and 0.05 mmol of cobalt perchlorate hexahydrate were added in sequence. The reaction was stirred at room temperature for 12 hours. After completion of the reaction, the product was purified by silica gel column chromatography using a 15:1 ratio of petroleum ether to ethyl acetate as the eluent to obtain LY4 as a white solid in a 76% yield.

[0078] The structural characterization data of the product are shown below:

[0079] 1 H NMR(400MHz,Chloroform-d)δ7.99-7.81(m,2H),7.75-7.64(m,2H),7.39-7.30(m, 4H),7.26(dd,J=4.5,2.3Hz,3H),7.10-6.90(m,1H),5.27(s,1H),5.23(d,J=3.9Hz ,1H),4.78(d,J=12.3Hz,1H),4.35(dd,J=12.4,4.0Hz,1H),2.91(ddd,J=12.2,10. 8,3.3Hz,1H),2.63(td,J=14.6,12.8,5.6Hz,1H),2.47(s,3H),2.42-2.24(m,2H).

[0080] 13 C NMR (101MHz, CDCl3) δ143.94,138.89,135.88,134.47,133.49,132.90,129.88,129.48,12 8.94,128.70,128.66,128.37,128.21,126.32,85.75,66.89,49.42,47.38,30.00,21.61.

[0081] Based on the above data, the structural formula of the obtained product LY4 is inferred to be: Example 6-1

[0082] This example synthesizes 3-(4-chlorophenyl)-7,11b-tetrahydro-2H,6H-[1,3,4]oxadiazine[2,3-a]isoquinoline (LY4-1):

[0083] To a 10 ml reaction flask, a stirring magnet, 1 mmol of azomethine imine (commercially available or prepared according to the method in Example 1), 1.2 mmol of p-chlorostyrene oxide (commercially available or prepared according to the method in Example 2, specifically p-chlorostyrene), 2 ml of dichloromethane, and 0.2 mmol of cobalt perchlorate hexahydrate were added in sequence. The reaction was stirred at room temperature for 18 hours. After completion of the reaction, the product was purified by silica gel column chromatography using a 2:1 ratio of petroleum ether to dichloromethane as the eluent to obtain LY4-1 as a white solid in a 67% yield.

[0084] The structural characterization data of the product are shown below:

[0085] 1 H NMR(500MHz,Chloroform-d)δ7.59-7.54(m,2H),7.51(dd,J=5.4,3.7Hz,1H),7.40-7.32(m,2H),7.29(dq,J=7.3,4.0Hz,2H),7.17(dd,J=5.2 ,3.6Hz,1H),5.12(s,1H),5.03(d,J=16.6Hz,1H),4.81(d,J=16.6Hz,1H),3.85-3.77(m,1H),3.44-3.27(m,2H),2.89(dd,J=15.6,3.4Hz,1H).

[0086] 13 C NMR (126MHz, CDCl3) δ144.40,135.09,134.80,133.34,132.74,128.73,128.43,128.41,126.57,126.46,125.48,84.14,64.21,50.24,29.16.

[0087] Based on the above data, it can be inferred that when the amount of cobalt perchlorate hexahydrate added is increased from 0.05 mmol to 0.2 mmol, the product LY4-1 obtained by removing the R protecting group has the structural formula:

[0088] Example 7

[0089] This example synthesizes 3-(p-bromophenyl)-4-p-toluenesulfonyl-3,4,7,11b-tetrahydro-2H,6H-[1,3,4]oxadiazine[2,3-a]isoquinoline (LY5):

[0090] To a 10 ml reaction flask, a stirring magnet, 1 mmol of azomethine imine (commercially available or prepared according to the method in Example 1), 1.2 mmol of 4-bromostyrene oxide (commercially available or prepared according to the method in Example 2, the corresponding styrene raw material is specifically p-bromostyrene), 2 ml of dichloromethane, and 0.05 mmol of cobalt perchlorate hexahydrate were added in sequence. The reaction was stirred at room temperature for 12 hours. After completion of the reaction, the product was purified by silica gel column chromatography using a 20:1 ratio of petroleum ether to ethyl acetate as the eluent to obtain LY5 as a white solid in an 86% yield.

[0091] The structural characterization data of the product are shown below:

[0092] 1 H NMR(400MHz,Chloroform-d)δ7.88(d,J=8.3Hz,2H),7.66-7.59(m,2H),7.50(d,J=8.6Hz ,2H),7.36-7.31(m,2H),7.25(d,J=3.7Hz,3H),7.08-6.98(m,1H),5.27(s,1H),5.21(d,J =3.8Hz,1H),4.77(dd,J=12.3,0.8Hz,1H),4.35(dd,J=12.4,4.0Hz,1H),2.91(ddd,J=12. 2,10.8,3.4Hz,1H),2.63(ddd,J=17.2,12.4,5.7Hz,1H),2.47(s,3H),2.40-2.26(m,2H).

[0093] 13 C NMR (101MHz, CDCl3) δ143.95,139.42,135.85,134.46,132.89,131.63,130.22,129.49,12 8.93,128.71,128.37,128.20,126.31,121.67,85.75,66.83,49.46,47.39,29.99,21.61.

[0094] Based on the above data, the structural formula of the obtained product LY5 is inferred to be:

[0095] Example 7-1

[0096] This example synthesizes 3-(p-bromophenyl)-7,11b-tetrahydro-2H,6H-[1,3,4]oxadiazine[2,3-a]isoquinoline (LY5-1):

[0097] To a 10 ml reaction flask, add a stirring magnet, 1 mmol of azomethine imine (commercially available or prepared according to the method in Example 1), 1.2 mmol of 4-bromostyrene oxide (commercially available or prepared according to the method in Example 2, the corresponding styrene raw material is specifically p-bromostyrene), 2 ml of dichloromethane, and 0.2 mmol of cobalt perchlorate hexahydrate. Stir and react at room temperature for 18 hours. After completion of the reaction, the product was purified by silica gel column chromatography using a 2:1 ratio of petroleum ether to dichloromethane as the eluent to obtain LY5-1 as a white solid in a 78% yield.

[0098] The structural characterization data of the product are shown below:

[0099] 1 H NMR(400MHz,Chloroform-d)δ7.70(d,J=1.8Hz,1H),7.67-7.62(m,2H),7.46-7.36(m,4H),7.07(d,J=8.2Hz,1H),5.08(s,1H),5 .06(d,J=16.8Hz,1H),4.87(d,J=16.8Hz,1H),3.94-3.79(m,1H),3.41-3.31(m,1H),3.31-3.20(m,1H),2.86(d,J=15.9Hz,1H).

[0100] 13 C NMR (101MHz, CDCl3) δ146.08,135.00,134.66,134.14,131.45,130.12,129.50,129.15,128.60,124.32,120.23,83.56,64.29,50.06,28.64.

[0101] Based on the above data, it can be inferred that when the amount of cobalt perchlorate hexahydrate added is increased from 0.05 mmol to 0.2 mmol, the product LY5-1 obtained by removing the R protecting group has the structural formula: Comparative Example 4

[0102] The configuration of this comparative example is the same as that of Example 7, except that the added amounts of cobalt perchlorate hexahydrate are 0 (ie, no catalyst is added) and 0.1 mmol, respectively.

[0103] The results showed that no reaction occurred without the addition of cobalt perchlorate hexahydrate. However, as the catalyst dosage increased, the reaction produced the protected group-containing product LY5 when the cobalt perchlorate hexahydrate dosage increased to 0.05-0.1 mmol, compared to the catalyst-to-azomethine imine molar ratio of 1:20 in Example 7. Furthermore, when the cobalt perchlorate hexahydrate dosage increased to 0.1 mmol or more, such as 0.2 mmol in Example 7-1, the deprotected product LY5-1 was produced.

[0104] Example 8

[0105] This example synthesizes 3-(2-chlorophenyl)-7,11b-tetrahydro-2H,6H-[1,3,4]oxadiazine[2,3-a]isoquinoline (LY6):

[0106] To a 10 ml reaction flask, a stirring magnet, 1 mmol of azomethine imine (commercially available or prepared according to the method in Example 1), 1.2 mmol of p-chlorostyrene oxide (commercially available or prepared according to the method in Example 2, with the corresponding styrene raw material being 2-chlorostyrene), 2 ml of dichloromethane, and 0.2 mmol of cobalt perchlorate hexahydrate were added in sequence. The mixture was stirred at room temperature for 18 hours. After completion of the reaction, the product was purified by silica gel column chromatography using a 2:1 ratio of petroleum ether to dichloromethane as the eluent to obtain LY6 as a white solid in a 75% yield.

[0107] The structural characterization data of the product are shown below:

[0108] 1 H NMR(500MHz,Chloroform-d)δ7.59-7.54(m,2H),7.51(dd,J=5.4,3.7Hz,1H),7.40-7.32(m,2H),7.29(dq,J=7.3,4.0Hz,2H),7.17(dd,J=5.2 ,3.6Hz,1H),5.12(s,1H),5.03(d,J=16.6Hz,1H),4.81(d,J=16.6Hz,1H),3.85-3.77(m,1H),3.44-3.27(m,2H),2.89(dd,J=15.6,3.4Hz,1H).

[0109] 13 C NMR (126MHz, CDCl3) δ144.40,135.09,134.80,133.34,132.74,128.73,128.43,128.41,126.57,126.46,125.48,84.14,64.21,50.24,29.16.

[0110] Based on the above data, the structural formula of the obtained product LY6 is inferred to be:

[0111] Example 9

[0112] This example synthesizes 3-(3-bromophenyl)-7,11b-tetrahydro-2H,6H-[1,3,4]oxadiazine[2,3-a]isoquinoline (LY7):

[0113] To a 10 ml reaction flask, add a stirring magnet, 1 mmol of azomethine imine (commercially available or prepared according to the method in Example 1), 1.2 mmol of 3-bromostyrene oxide (commercially available or prepared according to the method in Example 2, specifically 3-bromostyrene), 2 ml of dichloromethane, and 0.2 mmol of cobalt perchlorate hexahydrate. Stir and react at room temperature for 18 hours. After completion, the reaction was purified by silica gel column chromatography using a 2:1 ratio of petroleum ether to dichloromethane as the eluent to obtain LY7 as a white solid in a 64% yield.

[0114] The structural characterization data of the product are shown below:

[0115] 1 H NMR (400MHz, CDCl3) δ7.83 (s, 1H), 7.57-7.44 (m, 3H), 7.34-7.29 (m, 2H), 7.26 (d, J = 8.2Hz, 1H), 7.21-7.14 (m, 1H), 5.14 (s, 1H) ),5.02(d,J=16.7Hz,1H),4.81(d,J=16.7Hz,1H),3.95-3.79(m,1H),3.47-3.37(m,1H),3.37-3.26(m,1H),2.96-2.81(m,1H).

[0116] 13 C NMR (126MHz, CDCl3) δ148.97,135.04,134.84,132.84,132.62,130.59,130.16 ,129.99,128.38,128.36,127.20,126.55,126.46,84.40,65.96,50.16,29.09.

[0117] Based on the above data, the structural formula of the obtained product LY7 is inferred to be:

[0118] It can be seen from the above examples 6 to 9 that: R 1 is an electron-withdrawing group (R 1 is chlorine, and R 1When the amount of the catalyst is about 5% (such as 0.05 mmol in Example 6 and Example 7), a product containing a protecting group can be generated; and when it is increased to 20% (such as 0.2 mmol in Example 6-1, Example 7-1, Example 8, and Example 9), the reaction time is extended to 18 hours, and a deprotected product can be generated.

[0119] The above examples 3 to 9 control the electronegativity of the substituents on the benzene ring and use the catalytic effect of cobalt perchlorate to achieve a one-step reaction to prepare 1,3,4-oxadiazine tetrahydroisoquinoline compounds. 1 When the R-position is Ts in Example 3, Example 4, and Example 5, it is not easy to obtain the deprotected group product, and the product containing the protective group is still the main product; when R is on the benzene ring, the R-position is Ts. 1 The substituent is an electron-withdrawing substituent (such as chlorine in Examples 6 and 8, bromine, acetoxy or trifluoromethyl in Examples 7 and 9), and the reaction will generate 1,3,4-oxadiazine tetrahydroisoquinoline compounds containing a protecting group (such as Examples 6 and 7). However, as the amount of catalyst increases, compounds with deprotected groups will be generated (such as Examples 6-1 and 7-1).

[0120] Regarding the eluent: The composition of the eluent varies with the catalyst dosage. When the catalyst dosage is high, a suitable eluent is a mixture of petroleum ether and dichloromethane. For example, when the molar ratio of catalyst to azomethine imine is 1:5 (i.e., in the example, 0.2 mmol of cobalt perchlorate hexahydrate is added for every 1 mmol of azomethine), an eluent composition of petroleum ether:dichloromethane = 2:1 is suitable. When the catalyst dosage is low, a suitable eluent is a mixture of petroleum ether and ethyl acetate. For example, when the molar ratio of catalyst to azomethine imine is 1:20 (i.e., in the example, 0.05 mmol of cobalt perchlorate hexahydrate is added for every 1 mmol of azomethine), an eluent composition of petroleum ether:ethyl acetate = 10-20:1 is suitable.

[0121] Application Examples

[0122] Tumor cell toxicity analysis was performed on the synthesized compounds LY1, LY2, LY3, LY4, LY5, LY6, and LY7:

[0123] Tumor cell suspension was added to a 96-well plate, 100 μL per well, approximately 1×10 3 ~5×10 3cells / well. Culture medium was added to the edge wells, and a DMSO blank control group was set up. The cells were cultured in a carbon dioxide incubator at 37°C with saturated humidity and 5% CO2 for 24 hours. The synthesized compounds were prepared in four sample concentration gradients of 0.1, 1, 10, and 100 μM, and 100 μL of sample culture medium was added to each well. Cisplatin was set as a positive control group and cultured for another 48 hours. Subsequently, 10 μL of CCK-8 reagent was added to each well, mixed by oscillation, and incubated at 37°C in the dark for 4 hours. The absorbance of each well was measured on a microplate reader at a wavelength of 450 nm.

[0124] The formula for calculating cell survival rate is: survival rate (%) = (A 样品 -A 空白 ) / (A 对照 -A 空白 )×100%.

[0125] The cell survival rate was plotted as the vertical axis and the logarithm of the cell concentration as the horizontal axis. GraphPadPrism was used to draw a standard curve and calculate the half-maximal lethal inhibition concentration of the compound, i.e., IC 50 value.

[0126] Table 1: IC values ​​of the compounds prepared in this application 50 value

[0127]

[0128]

[0129] This case evaluated the cytotoxicity of each compound against four tumor cell lines: HeLa (cervical cancer cells), A549 (lung cancer cells), MDAMB231 (breast cancer cells), and DU145 (prostate cancer cells). As shown in Table 1, the compounds given in this application showed cytotoxicity against all four tumor cell lines. Compared with the common anti-tumor drug cisplatin, the 1,3,4-oxadiazine tetrahydroisoquinoline compounds synthesized in this case have resistance to most tumor cells, especially HeLa cells and A549 cells. The corresponding IC 50 The value is much lower than the IC of cisplatin 50 Value level.

[0130] Among all the compounds, LY1, LY5, and LY7 performed best overall: LY1 and LY5 showed excellent cytotoxicity against HeLa cells and A549 cells, and LY1 had an IC 50 The values ​​were 0.9821μM and 0.9290μM respectively; while the IC values ​​of compound LY5 for these two types of cells were 50The values ​​reached the nanomolar level, which were 0.4930 μM and 0.09289 μM respectively (see Figure 1 The compound LY7 after deprotection has certain cell killing ability against HeLa cells, IC 50 The value is 1.226μM; it has a good cell killing effect on A549, and its IC 50 The value reached the nanomolar level, which was 0.1671 μM (see Figure 2 ). The cell killing activity of LY4 on A549 cells was slightly lower than that of LY7, and the corresponding IC 50 The value is 0.3648 μM, and its killing ability on HeLa cells is far inferior to LY1, LY5, and LY7.

[0131] From the activity data analysis, compared with compound LY4, the toxicity of the deprotected product LY4-1 to HeLa cells was increased, and the toxicity to A549 cells was decreased; compared with compound LY5, the toxicity of the deprotected product LY5-1 to both HeLa cells and A549 cells was decreased.

[0132] The above-described embodiments merely represent several feasible implementation methods of the present invention. The description thereof is relatively specific and detailed, but it should not be understood as limiting the scope of the invention. The embodiments are not intended to limit the scope of protection in the claims of the present invention. For those skilled in the art, various modifications and improvements can be made without departing from the concept of the present invention. Any equivalent implementation or modification that does not depart from the scope of the present invention should be included in the technology of the present invention.

Claims

1. A 1,3,4-oxadiazine tetrahydroisoquinoline compound, characterized in that: It has the following general structural formula: R has no substituent or the substituent is any one of benzenesulfonyl, p-toluenesulfonyl, methanesulfonyl, p-methoxybenzenesulfonyl and p-nitrobenzenesulfonyl; R 1 is any one of hydrogen, methyl, tert-butyl, fluorine, phenyl, chlorine, bromine, acetoxy, and trifluoromethyl; R 2 It is any one of hydrogen, phenyl, bromine, fluorine and methyl.

2. A 1,3,4-oxadiazinone tetrahydroisoquinoline compound according to claim 1, characterized in that: The structural formula of the 1,3,4-oxadiazine tetrahydroisoquinoline compound is Any of .

3. A method for preparing the 1,3,4-oxadiazinone tetrahydroisoquinoline compound according to claim 1, characterized in that: Azomethine imine substrates and styrene oxide substrates are used as raw materials, cobalt perchlorate hexahydrate is used as a catalyst, the raw materials and the catalyst are added to a reaction solvent, and the reaction is carried out at room temperature overnight. After column separation, 1,3,4-oxadiazine tetrahydroisoquinoline compounds are obtained.

4. The method for preparing a 1,3,4-oxadiazinone tetrahydroisoquinoline compound according to claim 3, wherein: The amount of the catalyst added is 5-20% of the total molar amount of the raw materials.

5. The method for preparing 1,3,4-oxadiazinone tetrahydroisoquinoline compounds according to claim 3, wherein: The molar ratio of the azomethine imine substrate to the styrene oxide substrate is 1:1.2-1.

5.

6. The method for preparing 1,3,4-oxadiazinone tetrahydroisoquinoline compounds according to claim 3, wherein: The reaction solvent is selected from any one of dichloromethane, dichloroethane, chloroform, ethyl acetate, tetrahydrofuran, and acetonitrile.

7. The method for preparing 1,3,4-oxadiazinone tetrahydroisoquinoline compounds according to any one of claims 3 to 6, characterized in that: The reaction formula of the preparation process is as follows:

8. Use of the 1,3,4-oxadiazinotetrahydroisoquinoline compound according to claim 1, characterized in that: 1,3,4-Oxadiazinotetrahydroisoquinoline compounds are used for the preparation of anti-tumor preparations.

9. The use of 1,3,4-oxadiazinone tetrahydroisoquinoline compounds according to claim 8, characterized in that: The structural formula of 1,3,4-oxadiazine tetrahydroisoquinoline compounds is It showed cytotoxicity against HeLa and A549 tumor cells.

10. The use of 1,3,4-oxadiazinone tetrahydroisoquinoline compounds according to claim 8, characterized in that: The structural formula of 1,3,4-oxadiazine tetrahydroisoquinoline compounds is It showed cytotoxicity against A549 tumor cells.

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