Evodiamine derivative, rutaecarpine derivative, preparation method and application thereof

Abstract

The present invention belongs to the technical field of chemical pharmaceuticals, and particularly relates to anti-tumor drugs. Specifically, it relates to an evodiamine derivative, a rutaecarpine derivative, and their preparation methods and applications. The present invention provides an evodiamine derivative and a rutaecarpine derivative with higher anti-tumor activity in vitro and in vivo. By introducing a new drug fragment at the N-13 position of the B ring of evodiamine or rutaecarpine for structural modification, and using an acyl aniline group to replace the hydrogen atom on the imino group of the B ring of evodiamine / rutaecarpine, a series of novel small molecule evodiamine derivatives and small molecule rutaecarpine derivatives that target and inhibit DNA topoisomerase are obtained. The study of anti-tumor activity in vitro shows that this type of molecule described in the present invention has strong inhibitory activity against tumor cell lines and can promote the apoptosis of tumor cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical pharmaceuticals, and particularly relates to anti-tumor drugs, specifically to an evodiamine derivative, a rutaecarpine derivative, and their preparation methods and applications. Background Art

[0002] Evodiamine and rutecarpine belong to pentacyclic alkaloids of the tryptamine indole type, which are derived from a nearly mature Rutaceae plant, Evodia rutaecarpa, and have various effects, including enhancing heart function, regulating hormone levels, and anti-inflammatory and analgesic effects. In addition, evodiamine and rutecarpine exhibit excellent anti-cancer properties. They show good inhibitory effects on some common tumors, such as glioblastoma cells, gastric cancer cells, breast cancer cells, and lung cancer cells, by inhibiting the proliferation and invasion of tumor cells and inducing apoptosis in various ways.

[0003] Studies on the anti-cancer pharmacological mechanism show that evodiamine and rutecarpine have the ability to inhibit the formation of covalent complexes between DNA topoisomerase and DNA. The mechanism of action is similar to that of the Topo-I inhibitor irinotecan, and it can induce DNA damage, but irinotecan and others inhibit in the S phase. During the exploration of their anti-prostate cancer and colon cancer effects, it was found that after treating cells with evodiamine / rutecarpine, the cell cycle can be arrested in the G2 / M phase, which is different from the effects of previous Topo-I inhibitors. Therefore, further research found that evodiamine / rutecarpine can affect the balance between microtubule depolymerization and disassembly in human cancer cells and inhibit the proliferation of human cancer cells.

[0004] Although evodiamine / rutecarpine can inhibit the proliferation of human cancer cells, studies have found that the unmodified evodiamine / rutecarpine currently used has defects such as weak anti-tumor activity, poor water solubility, and poor pharmacokinetic properties, which affect its anti-tumor activity in vivo and in vitro, and thus limit its clinical development and application.

[0005] In order to improve the anti-tumor activity of evodiamine / rutecarpine in vivo and in vitro, in previous studies, evodiamine / rutecarpine was modified to form many new skeletons with better anti-tumor activity in vivo and in vitro.

[0006] Molecular docking was carried out to study the structure-activity relationship of B-ring substitution. The docking conformation of evodiamine / rutaecarpine with the TopⅠ-DNA complex showed that the A, B, and C rings of evodiamine / rutaecarpine had a unique L-shaped spatial conformation embedded in the DNA base pairs. Therefore, N-13 on the B ring interacted with Arg 364 of TopⅠ through hydrogen bonding and π-π interactions. It was found that only part of evodiamine / rutaecarpine was inserted into the DNA base pairs, and the N-13 position of the B ring pointed to the major groove of DNA, indicating that there was still room for structural optimization. At present, the structural modification of the B ring of the skeletons of evodiamine / rutaecarpine mainly focused on introducing groups such as alkyl, benzoyl, benzyl, ester, long-chain alkoxy, and methanesulfonyl groups on the indole nitrogen atom.

[0007] Summary of the Invention

[0008] The purpose of the present invention is to provide evodiamine derivatives and rutaecarpine derivatives with higher anti-tumor activities in vitro and in vivo. By introducing new drug fragments at the N-13 position of the B ring of evodiamine or rutaecarpine respectively for structural modification, a series of novel small molecule evodiamine derivatives and rutaecarpine derivatives targeting and inhibiting DNA topoisomerase were obtained.

[0009] Through reasonable drug structure-activity design and analysis, the present invention found that based on the binding mode of evodiamine / rutaecarpine in the binding pocket of DNA topoisomerase, replacing the hydrogen atom on the imino group of the B ring of evodiamine / rutaecarpine with an acyl aniline group could further enhance the targeting of evodiamine / rutaecarpine to TopⅠ and significantly improve its anti-tumor activity. The study of anti-tumor activity in vitro showed that these molecules had strong inhibitory activity against tumor cell lines and could promote the apoptosis of tumor cells.

[0010] The specific technical solution is as follows:

[0011] An evodiamine derivative, whose structural general formula is shown as formula S1:

[0012]

[0013] In the formula, R1, R2, R3, R4, and R5 are each independently selected from H, CH3, OCH3, CF3, F, Cl, or Br.

[0014] In the present invention, the evodiamine derivative is any one of the derivatives shown as structural formulas S1-1 to S1-7:

[0015] A rutaecarpine derivative, whose structural general formula is shown as formula S2:

[0016]

[0017] In the formula, R1, R2, R3, R4, and R5 are each independently selected from H, CH3, OCH3, CF3, F, Cl, or Br.

[0018] In the present invention, the rutaecarpine derivative is any one of the derivatives represented by structural formulas S2-1 to S2-7:

[0019] The preparation method of the above evodiamine derivative or the above rutaecarpine derivative includes the following steps:

[0020] (1) Prepare bromoacetyl aniline compounds: Using aniline compounds and bromoacetyl bromide as raw materials, adding an acid-binding agent, and stirring and reacting at room temperature (here, room temperature refers to the indoor temperature regulated according to the pharmaceutical industry guidelines, generally controlled at 20-25 °C) for at least 12 h. Monitor the entire reaction process using a thin-layer chromatography silica gel plate.

[0021] The specific operation of the monitoring is as follows: Use a capillary to absorb a small amount of the reaction system solution for spotting on the thin-layer chromatography silica gel plate, and develop the sample with a chromatography solution (V 乙酸乙酯 : V 石油醚 = 1:1), and judge the reaction conversion of the reactant to be reacted by the fluorescence intensity at 365 nm under an ultraviolet analyzer, so as to monitor the entire reaction process.

[0022] Among them, the general structural formula of the aniline compound is: In the formula, R1, R2, R3, R4, and R5 are each independently selected from H, CH3, OCH3, CF3, F, Cl, or Br;

[0023] After the reaction is completed, add water for quenching, and add an extractant for extraction. Take the lower organic phase, and after dehydration, suction filtration, and rotary evaporation, obtain the crude product of bromoacetyl aniline compounds.

[0024] The structural formula of the bromoacetyl aniline compound is: In the formula, R1, R2, R3, R4, and R5 are each independently selected from H, CH3, OCH3, CF3, F, Cl, or Br.

[0025] Anhydrous sodium sulfate can be used for dehydrating the organic phase. The extractant in this step can be any one of dichloromethane (DCM), ethyl acetate, diethyl ether, or chloroform.

[0026] The reaction equation of this step (1) is as follows:

[0027]

[0028] (2) Preparation of evodiamine derivatives or rutaecarpine derivatives: First, add evodiamine or rutaecarpine to the crude product of bromoacetyl aniline compounds obtained in step (1). When preparing evodiamine derivatives, add evodiamine to the crude product of bromoacetyl aniline compounds obtained in step (1); when preparing rutaecarpine derivatives, add rutaecarpine to the crude product of bromoacetyl aniline compounds obtained in step (1).

[0029] Then add a solvent and a catalyst sodium hydride (NaH), and under nitrogen conditions, carry out a condensation reflux reaction at 80 °C - 90 °C for at least 12 h. Also, monitor the entire reaction process using a thin-layer chromatography silica gel plate. The solvent can be selected as N,N-dimethylformamide (DMF).

[0030] After the reaction is completed, wait for the reaction solution to cool to room temperature, extract the reaction solution, remove the moisture, filter by suction, and after rotary evaporation, separate and purify the product by column chromatography to obtain the evodiamine derivative or the rutaecarpine derivative described above.

[0031] The extractant in this step can be ethyl acetate. Anhydrous sodium sulfite can be used to remove the moisture. After suction filtration, the solvent is rotary evaporated using a rotary evaporator, and finally the product is purified by column chromatography (V 石油醚 : V 乙酸乙酯 = 3:1).

[0032] The reaction equation for preparing evodiamine derivatives in step (2) is as follows:

[0033]

[0034] The reaction equation for preparing rutaecarpine derivatives in step (2) is as follows:

[0035]

[0036] In the present invention, in the described preparation method, the molar ratio of aniline compounds to bromoacetyl bromide in step (1) is 1:1.5 - 2; the molar ratio of the acid-binding agent to aniline compounds is 2 - 2.5:1; the acid-binding agent is triethylamine (Et3N).

[0037] In the present invention, in the described preparation method, the molar ratio of the crude product of bromoacetyl aniline compounds to evodiamine in step (2) is 1:0.8 - 1.2; the molar ratio of the crude product of bromoacetyl aniline compounds to rutaecarpine is 1:0.8 - 1.2; the molar ratio of sodium hydride to the crude product of bromoacetyl aniline compounds is 2 - 2.5:1.

[0038] Use of the evodiamine derivative or the rutaecarpine derivative as described above in inhibiting the activity of DNA topoisomerase. Both the evodiamine derivative and the rutaecarpine derivative as described above can inhibit the ability of DNA topoisomerase to form covalent complexes with DNA and have topoisomerase inhibitory effects.

[0039] Use of the evodiamine derivative or the rutaecarpine derivative prepared by the above preparation method in inhibiting the activity of DNA topoisomerase.

[0040] Use of the evodiamine derivative or the rutaecarpine derivative as described above in the preparation of anti-tumor drugs.

[0041] Use of the evodiamine derivative or the rutaecarpine derivative prepared by the above preparation method in the preparation of anti-tumor drugs.

[0042] A DNA topoisomerase inhibitor, the active ingredient of which contains at least one of the evodiamine derivative or the rutaecarpine derivative as described above; or contains at least one of the evodiamine derivative or the rutaecarpine derivative prepared by the above preparation method.

[0043] The beneficial effects of the present invention are as follows: The evodiamine derivative or the rutaecarpine derivative described in the present invention not only enhances the anti-tumor activity to a certain extent and strengthens the inhibitory effect on the viability of tumor cells. For example, the inhibitory effect of the evodiamine derivative on the viability of human lung cancer cell line A549 cells is at least 3.8 times that of the existing marketed positive drug etoposide. More importantly, the evodiamine derivative or the rutaecarpine derivative provided by the present invention has higher selectivity and will not inhibit normal human cells, thus having low toxicity and higher safety. In addition, the activity of the rutaecarpine derivative provided by the present invention is nearly 10 times higher than that of its parent compound rutaecarpine.

[0044] Thus, it can be seen that the structural modification strategy adopted by the present invention is effective. Description of the Drawings

[0045] Figure 1 In vitro anti-proliferative activities of evodiamine / rutaecarpine derivatives against human lung cancer cell lines A549 and H460.

[0046] Figure 2 In vitro anti-proliferative activities of evodiamine / rutaecarpine derivatives against human melanoma cell lines A375 and A827.

[0047] Figure 3 In vitro anti-proliferative activities of evodiamine / rutaecarpine derivatives against human colorectal cancer cell lines HCT-8 and HT-29.

[0048] Figure 4 Anti - proliferative activity of evodiamine / rutaecarpine derivatives against human renal epithelial cell line (293T) in vitro.

[0049] Figure 5 Flow cytometry analysis of apoptosis of A549 cells induced by evodiamine / rutaecarpine derivatives. Data are presented as the mean ± SEM of three independent experiments (*P < 0.05, **P < 0.01).

[0050] Figure 6 Effect of evodiamine / rutaecarpine derivatives on cell cycle progression of A549 cells. Data are presented as the mean ± SEM of three independent experiments (*P < 0.05, **P < 0.01).

[0051] Figure 7 Effect of rutaecarpine derivative S2 - 2 on migration ability of A549 cells. Data are presented as the mean ± SEM of three independent experiments (*P < 0.05, **P < 0.01).

[0052] Figure 8 Molecular docking simulation of evodiamine / rutaecarpine derivatives with the target protein DNA topoisomerase I. In the figure, (A) is evodiamine derivative S1 - 2; (B) is rutaecarpine derivative S2 - 2.

[0053] Figure 9 NMR spectrum of Example 1.

[0054] Figure 10 NMR spectrum of Example 2.

[0055] Figure 11 NMR spectrum of Example 3.

[0056] Figure 12 NMR spectrum of Example 4.

[0057] Figure 13 NMR spectrum of Example 5.

[0058] Figure 14 NMR spectrum of Example 6.

[0059] Figure 15 NMR spectrum of Example 7.

[0060] Figure 16 NMR spectrum of Example 8.

[0061] Figure 17 NMR spectrum of Example 9.

[0062] Figure 18 NMR spectrum of Example 10.

[0063] Figure 19 It is the NMR spectrum of Example 11.

[0064] Figure 20 It is the NMR spectrum of Example 12.

[0065] Figure 21 It is the NMR spectrum of Example 13.

[0066] Figure 22 It is the NMR spectrum of Example 14. Detailed implementation manners

[0067] The technical solutions of the present invention will be described in detail below with reference to the accompanying drawings.

[0068] Example 1

[0069] This example is evodiamine derivative shown by the following formula:

[0070]

[0071] Through Figure 9 spectrum analysis, it can be known that 1 H NMR(600MHz,CDCl3)δ9.60(s,1H),8.32(d,J=7.7Hz,1H),7.74 - 7.66(m,3H),7.63(d,J=8.0Hz,2H),7.43(t,J=7.9Hz,2H),7.39(d,J=8.3Hz,2H),7.32(t,J=7.6Hz,2H),7.18(t,J=7.5Hz,2H),4.59(t,J=6.9Hz,4H),3.24(t,J=6.9Hz,4H),1.28(s,1H).HR-MS(ESI + )m / z:437.1899[M + H] + .Found:437.1911[M + H] + .

[0072] The preparation method of the evodiamine derivative shown by S1-1 is as follows:

[0073] (1) Add 1 mmol of aniline, 1.5 mmol of bromoacetyl bromide, 2 mmol of acid-binding agent triethylamine and 30 mL of refined dichloromethane into a 150 mL round-bottom flask in sequence, stir and react at room temperature for 12 h, and monitor the completion of the reaction by using a thin-layer chromatography silica gel plate.

[0074] After the reaction is complete, add 18 mL of deionized water to the reaction solution for quenching, then add 18 mL of extraction agent dichloromethane for extraction, and extract repeatedly three times, and combine the obtained lower-layer organic phase liquid.

[0075] The combined organic phase liquid is dehydrated with anhydrous sodium sulfate, the sodium sulfate is removed by suction filtration, and after the liquid is rotary evaporated to dryness, the crude product of bromoacetyl aniline is obtained.

[0076] (2) 0.9 mmol of evodiamine and 2 mmol of NaH are added to the crude product of bromoacetyl aniline obtained in step (1), and 20 mL of the solvent N,N-dimethylformamide is added for dissolution. Under nitrogen conditions, the reaction is carried out under reflux condensation at 80 °C for 12 h, and the progress of the reaction is monitored by a thin-layer chromatography silica gel plate.

[0077] After the reaction is completed, the reaction solution is cooled to room temperature, extracted with 20 mL of ethyl acetate. The residue in the solution is dissolved in the aqueous phase, the organic phases are combined, 100 mg of anhydrous sodium sulfite is added to remove water, and after suction filtration, the solvent is rotary evaporated to dryness using a rotary evaporator. Finally, the product is separated and purified by column chromatography to obtain the white powdery S1-1 evodiamine derivative. The yield is 70%.

[0078] Example 2

[0079] The evodiamine derivative in this example is as shown in the following formula:

[0080]

[0081] By Figure 10 spectral analysis, it can be known that 1 H NMR (600 MHz, CDCl3) δ 8.12 (s, 1H), 7.67 (d, J = 7.7 Hz, 1H), 7.42 (m, 5), 7.18 - 7.09 (m, 5H), 6.91 (d, J = 7.1 Hz, 1H), 5.97 (s, 1H), 5.29 (s, 2H), 4.96 (d, J = 17.0 Hz, 2H), 3.24 (td, J = 12.4, 3.9 Hz, 2H), 2.27 (s, 3H), 1.26 (s, 3H). HR-MS (ESI + ) m / z: 451.2056 [M + H] + . Found: 451.2085 [M + H] + .

[0082] The difference from the preparation method described in Example 1 is that in the preparation method of the evodiamine derivative shown in S1-2, the aniline compound added in step (1) is m-toluidine. The yield is 76%.

[0083] Other steps are the same as those in Example 1.

[0084] Example 3

[0085] The evodiamine derivative in this example is as shown in the following formula:

[0086]

[0087] By Figure 11 spectral analysis, it can be known that 1 H NMR(600MHz,CDCl3)δ8.14 - 7.98(m,2H),7.69 - 7.53(m,4H),7.47(dd,J=13.5,7.4Hz,2H),7.28(d,J=7.0Hz,2H),6.99 - 6.92(m,2H),6.69(q,J=14.3Hz,1H),5.42(s,1H),5.30(s,2H),4.58(d,J=16.6Hz,2H),2.94 - 2.69(m,2H),2.39(s,3H),2.33(s,3H).HR-MS(ESI + )m / z:467.2005[M + H] + .Found:467.2001[M + H] + 。

[0088] The difference from the preparation method described in Example 1 is that in the preparation method of the evodiamine derivative shown in S1 - 3, in step (1), the aniline compound added is p - methoxyaniline. The yield is 66%.

[0089] Others are the same as in Example 1.

[0090] Example 4

[0091] This example is an evodiamine derivative shown by the following formula:

[0092]

[0093] By Figure 12 spectral analysis, it can be known that 1H NMR(600MHz,CDCl3)1H NMR(600MHz,CDCl3)δ8.63(s,2H),7.77(s,2H),7.67(d,J=8.0Hz,2H),7.34(d,J=8.0Hz,3H),7.29(d,J=8.1Hz,3H),7.18(s,1H),5.89(s,1H),4.94 - 4.78(m,1H),4.15(s,6H),3.01 - 2.82(m,2H).HR-MS(ESI+)m / z:505.1773[M + H]+.Found:505.1762[M + H] + 。

[0094] The difference from the preparation method described in Example 1 is that in the preparation method of the evodiamine derivative shown in S1-4, the aniline compound added in step (1) is 3-(trifluoromethyl)aniline. The yield is 64%.

[0095] Others are the same as in Example 1.

[0096] Example 5

[0097] This example is an evodiamine derivative shown by the following formula:

[0098]

[0099] Through Figure 13 spectral analysis, it can be known that 1 H NMR(600MHz,CDCl3)δ10.53(s,1H),8.42-8.29(m,1H),7.71(tdd,J=28.6,20.1,10.3Hz,4H),7.55-7.41(m,4H),7.33(dd,J=21.9,9.5Hz,1H),7.21-7.06(m,2H),6.92(t,J=8.7Hz,1H),5.33(d,J=40.0Hz,2H),4.58(t,J=6.9Hz,2H),3.23(dd,J=13.1,6.6Hz,2H),1.26(s,3H).HR-MS(ESI + )m / z:455.1805[M+H] + .Found:455.1821[M+H] + .

[0100] The difference from the preparation method described in Example 1 is that in the preparation method of the evodiamine derivative shown in S1-5, the aniline compound added in step (1) is 4-fluoroaniline. The yield is 53%.

[0101] Others are the same as in Example 1.

[0102] Example 6

[0103] This example is an evodiamine derivative shown by the following formula:

[0104]

[0105] Through Figure 14 spectral analysis, it can be known that 11H NMR (400 MHz, DMSO) δ 10.57 (s, 1H), 7.92 (d, J = 7.2 Hz, 1H), 7.82 (s, 1H), 7.61 (d, J = 7.7 Hz, 1H), 7.54 (d, J = 8.2 Hz, 1H), 7.46 (d, J = 5.0 Hz, 2H), 7.37 (t, J = 8.0 Hz, 1H), 7.23 (m, 2H), 7.14 (d, J = 8.0 Hz, 2H), 7.02 (d, J = 7.8 Hz, 1H), 6.08 (s, 1H), 5.15 (m, 2H), 3.40 (s, 3H), 3.16 (m, 2H), 2.86 - 2.76 (m, 2H). HR-MS (ESI + ) m / z: 471.1510 [M + H] + . Found: 471.1537 [M + H] + 。

[0106] The difference from the preparation method described in Example 1 is that in the preparation method of the evodiamine derivative shown in S1-6, the aniline compound added in step (1) is 3-chloroaniline. The yield is 63%.

[0107] Others are the same as in Example 1.

[0108] Example 7

[0109] The evodiamine derivative in this example is as shown in the following formula:

[0110]

[0111] By Figure 15 spectral analysis, it can be known that 1 1H NMR (600 MHz, CDCl3) δ 8.23 (d, J = 7.8 Hz, 1H), 7.61 (t, J = 7.4 Hz, 1H), 7.57 - 7.52 (m, 2H), 7.49 (d, J = 8.3 Hz, 2H), 7.41 - 7.38 (m, 1H), 7.33 (t, J = 7.2 Hz, 1H), 7.28 (d, J = 7.6 Hz, 1H), 7.25 - 7.20 (m, 2H), 7.18 (s, 2H), 7.09 (t, J = 7.4 Hz, 1H), 4.50 (t, J = 6.8 Hz, 2H), 3.37 - 3.32 (m, 3H), 3.14 (t, J = 6.8 Hz, 2H), 2.48 (s, 2H). HR-MS (ESI + ) m / z: 471.1510 [M + H] + . Found: 471.1536 [M + H] + 。

[0112] The difference from the preparation method described in Example 1 is that in the preparation method of the evodiamine derivative shown in S1-7, p-chloroaniline, an aniline compound, is added in step (1). The yield is 72%.

[0113] Others are the same as in Example 1.

[0114] Example 8

[0115] This example is the rutaecarpine derivative shown by the following formula:

[0116]

[0117] By Figure 16 spectral analysis, it can be known that 1 H NMR(600MHz,CDCl3)δ8.37(dd,J=18.1,8.0Hz,1H),8.25(s,1H),7.84-7.64(m,3H),7.57-7.43(m,4H),7.33-7.29(m,2H),7.12-7.07(m,2H),4.33(s,2H),3.06(s,2H),2.05(s,2H).HR-MS(ESI + )m / z:421.1586[M+H] + .Found:421.1585[M+H] + .

[0118] The preparation method of the rutaecarpine derivative shown in S2-1 is specifically as follows:

[0119] (1) Add 1 mmol of aniline, 1.5 mmol of bromoacetyl bromide, 2 mmol of the acid-binding agent triethylamine and 30 mL of refined dichloromethane into a 150 mL round-bottom flask in sequence, stir and react at room temperature for 12 h, and monitor the completion of the reaction using a thin-layer chromatography silica gel plate.

[0120] After the reaction is complete, add 18 mL of deionized water to the reaction solution for quenching, then add 18 mL of the extractant dichloromethane for extraction, and extract three times repeatedly. Combine the obtained lower-layer organic phase liquid.

[0121] The combined organic phase liquid is dehydrated with anhydrous sodium sulfate, the sodium sulfate is removed by suction filtration, and after the liquid is rotary evaporated, a crude product of bromoacetyl aniline is obtained.

[0122] (2) Add 0.9 mmol of rutaecarpine and 2 mmol of NaH to the crude bromoacetyl aniline obtained in step (1), and add 20 mL of the solvent N,N-dimethylformamide for dissolution. Under nitrogen conditions, reflux the reaction at 80 °C for 12 h, and monitor the reaction progress using a thin-layer chromatography silica gel plate.

[0123] After the reaction is completed, wait for the reaction solution to cool to room temperature, extract with 20 mL of ethyl acetate. The residue in the solution dissolves in the aqueous phase. Combine the organic phases, add 100 mg of anhydrous sodium sulfite to remove water, then after suction filtration, use a rotary evaporator to spin-dry the solvent, and finally obtain the white powdery S2-1 rutaecarpine derivative after separating and purifying the product by column chromatography. The yield is 52%.

[0124] Example 9

[0125] This example is a rutaecarpine derivative shown by the following formula:

[0126]

[0127] Through Figure 17 spectral analysis, it can be known that 1 H NMR (600 MHz, CDCl3) δ 8.06 (dd, J = 68.9, 7.6 Hz, 3H), 7.68 - 7.59 (m, 2H), 7.58 - 7.53 (m, 1H), 7.47 (dd, J = 14.0, 7.3 Hz, 3H), 7.38 (d, J = 7.9 Hz, 1H), 7.30 - 7.26 (m, 2H), 5.29 (s, 2H), 4.58 (dd, J = 12.8, 3.8 Hz, 2H), 2.94 - 2.88 (m, 2H), 2.39 (s, 3H). HR-MS (ESI + ) m / z: 435.1743 [M + H] + . Found: 435.1766 [M + H] + .

[0128] The difference from the preparation method described in Example 8 is that in the preparation method of the rutaecarpine derivative shown by S2-2, the aniline compound added in step (1) is m-toluidine. The yield is 64%.

[0129] Other steps are the same as in Example 8.

[0130] Example 10

[0131] This example is a rutaecarpine derivative shown by the following formula:

[0132]

[0133] By Figure 18 spectral analysis, it can be seen that 1 H NMR (600 MHz, CDCl3) δ 7.98 (d, J = 7.9 Hz, 1H), 7.70 - 7.60 (m, 2H), 7.46 - 7.36 (m, 3H), 7.29 (d, J = 7.8 Hz, 1H), 7.12 (d, J = 7.2 Hz, 1H), 6.98 - 6.92 (m, 2H), 6.86 - 6.74 (m, 2H), 6.60 (d, J = 9.5 Hz, 1H), 5.42 (s, 2H), 4.45 (s, 2H), 3.72 (s, 3H). HR-MS (ESI + ) m / z: 451.1692 [M + H] + . Found: 451.1682 [M + H] + .

[0134] The difference from the preparation method described in Example 8 is that in the preparation method of the rutaecarpine derivative shown in S2-3, the aniline compound added in step (1) is p-methoxyaniline. The yield is 73%.

[0135] Others are the same as in Example 8.

[0136] Example 11

[0137] This example is a rutaecarpine derivative shown by the following formula:

[0138]

[0139] By Figure 19 spectral analysis, it can be seen that 1 H NMR (600 MHz, CDCl3) δ 10.93 (s, 1H), 8.39 (d, J = 7.9 Hz, 1H), 7.84 (s, 1H), 7.76 (dd, J = 19.2, 8.4 Hz, 2H), 7.67 (d, J = 8.0 Hz, 2H), 7.63 (d, J = 8.0 Hz, 1H), 7.52 (t, J = 7.5 Hz, 1H), 7.48 (t, J = 7.5 Hz, 1H), 7.34 (t, J = 7.9 Hz, 1H), 7.31 - 7.26 (m, 2H), 5.38 (s, 2H), 4.59 (s, 2H), 3.24 (t, J = 6.7 Hz, 2H). HR-MS (ESI + ) m / z: 489.1460 [M + H] + . Found: 489.1482 [M + H] + .

[0140] The difference from the preparation method described in Example 8 is that in the preparation method of the rutaecarpine derivative shown in S2-4, the aniline compound added in step (1) is 3-trifluoromethylaniline. The yield is 58%.

[0141] Others are the same as in Example 8.

[0142] Example 12

[0143] This example is a rutaecarpine derivative represented by the following formula:

[0144]

[0145] By Figure 20 spectral analysis, it can be known that 1 H NMR(600MHz,CDCl3)δ8.13(d,J=7.6Hz,2H),7.60(d,J=7.9Hz,2H),7.50(t,J=7.5Hz,2H),7.42(d,J=8.2Hz,2H),7.28-7.21(m,2H),7.18(t,J=7.5Hz,2H),5.96(d,J=22.1Hz,2H),4.92(dd,J=49.1,14.5Hz,2H),3.33-3.24(m,2H).HR-MS(ESI + )m / z:439.1492[M+H] + .Found:439.1471[M+H] + .

[0146] The difference from the preparation method described in Example 8 is that in the preparation method of the rutaecarpine derivative shown in S2-5, the aniline compound added in step (1) is 4-fluoroaniline. The yield is 66%.

[0147] Others are the same as in Example 8.

[0148] Example 13

[0149] This example is a rutaecarpine derivative represented by the following formula:

[0150]

[0151] By Figure 21 spectral analysis, it can be known that 11H NMR (600 MHz, CDCl3) δ 10.72 (s, 1H), 8.35 (dd, J = 46.1, 7.9 Hz, 1H), 7.81 - 7.61 (m, 4H), 7.55 - 7.40 (m, 3H), 7.37 - 7.22 (m, 3H), 7.16 (dt, J = 33.6, 7.9 Hz, 1H), 5.30 (s, 2H), 4.59 (t, J = 6.9 Hz, 2H), 3.24 (dd, J = 13.9, 7.0 Hz, 2H). HR-MS (ESI + ) m / z: 455.1197 [M + H] + . Found: 455.1211 [M + H] + 。

[0152] The difference from the preparation method described in Example 8 is that in the preparation method of the rutaecarpine derivative shown in S2-6, the aniline compound added in step (1) is 3-chloroaniline. The yield is 75%.

[0153] Others are the same as in Example 8.

[0154] Example 14

[0155] This example is a rutaecarpine derivative shown by the following formula:

[0156]

[0157] Through Figure 22 spectrum analysis, it can be known that 1 1H NMR (400 MHz, DMSO) δ 10.71 (s, 1H), 8.18 (d, J = 7.9 Hz, 1H), 7.82 - 7.70 (m, 5H), 7.61 (d, J = 8.0 Hz, 1H), 7.49 (t, J = 7.9 Hz, 1H), 7.42 (d, J = 8.7 Hz, 3H), 7.25 (t, J = 7.4 Hz, 1H), 5.71 (s, 2H), 4.50 (t, J = 6.7 Hz, 2H), 3.27 (t, J = 6.7 Hz, 2H). HR-MS (ESI + ) m / z: 455.1197 [M + H] + . Found: 455.1164 [M + H] + 。

[0158] The difference from the preparation method described in Example 8 is that in the preparation method of the rutaecarpine derivative shown in S2-7, the aniline compound added in step (1) is 4-chloroaniline. The yield is 61%.

[0159] Others are the same as in Example 8.

[0160] Next, the drug action mechanisms of the evodiamine derivatives described in Examples 1-7 and the rutaecarpine derivatives described in Examples 8-14 were explored and verified by CCK-8 assay, cell scratch assay, and molecular docking.

[0161] Application Example 1

[0162] In this application example, a CCK-8 detection kit was used to perform cell proliferation experiments, and evodiamine, rutaecarpine, etoposide, and teniposide were used as positive controls to detect the effects of the evodiamine derivatives described in Examples 1-7 and the rutaecarpine derivatives described in Examples 8-14 on the in vitro proliferation activities of human lung cancer cell lines (A549, H460), human colorectal cancer cell lines (HCT-8, HT-29), melanoma cell lines (A375, B16F10), and human renal epithelial cell line (293T).

[0163] Specific experimental steps: The cell lines were grown to the logarithmic growth phase in complete medium and diluted to 2×10 4 cells / mL. Then, the cell suspension was inoculated into a 96-well flat-bottom plate, 100 μL per well, and incubated at 37 °C and 5% CO2 for 12 hours. The test compounds were diluted to preset concentrations (0.1, 1, 10, 100, 1000 μM) and added to the 96-well flat-bottom plate. After 48 hours of drug treatment, the supernatant in each well was aspirated, and 100 μL of fresh medium containing 10% CCK-8 solution was added again, and incubation was continued for about 2 hours. The absorbance was measured and recorded at a test wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader (ELx800, BioTek, USA). Each sample had three replicate wells, and each experiment was repeated three times. Finally, Origin 7.5 software was used to calculate the drug concentration required for 50% cell survival, that is, the IC 50 value.

[0164] The results are shown in Table 1 and Figure 1 , 2 , 3, and 4. Compounds S1-1 to S2-7 could all inhibit the viability of the above-mentioned tumor cells, but had no inhibitory effect on the in vitro proliferation activity of the human renal epithelial cell line (293T) of the human body itself, with low toxicity and higher safety.

[0165] Among them, S1-2 (IC 50 = 2.54 μM) had significant anti-proliferative activity against A549 cells, superior to evodiamine (IC 50 = 7.29 μM), etoposide (IC 50 = 38.46 μM), and teniposide (IC 50 = 4.23 μM).

[0166] Compounds S2-1 to S2-7 are rutaecarpine derivatives, and their anti-proliferative activities are significantly better than that of rutaecarpine.

[0167] From the perspective of structure and activity analysis, it can be found that the compounds with electron-donating groups (CH3, OCH3, CF3) on the benzene ring have significantly better activities than those with electron-withdrawing groups (F, Cl) on the benzene ring. In addition, evodiamine / rutaecarpine derivatives have no obvious toxicity to human renal epithelial cells.

[0168] Table 1 Anti-tumor cell proliferation activities of evodiamine and rutaecarpine derivatives in vitro

[0169]

[0170]

[0171] It can be known from the data analysis in Table 1 that through in vitro anti-proliferation experiments, it is proved that evodiamine derivatives / rutaecarpine derivatives all have significant anti-proliferation activities against human non-small cell lung cancer cell line A549. The half-maximal inhibitory concentration (IC 50 value) of some compounds is less than 10 μM. Especially for compound S2-2, the IC 50 value for A549 cells is 2.54 μM. Compared with its parent compound evodiamine (IC 50 = 7.29 μM), the anti-tumor activity is increased by more than 3 times.

[0172] Application Example 2

[0173] This application example uses the Annexin V-FITC-PI apoptosis kit to detect the induction of apoptosis of evodiamine derivative S1-2 and rutaecarpine derivative S2-2 on A549 cells.

[0174] The specific operation is as follows: Cells are seeded in 6-well plates (1×10 5 cells / well) and incubated for 12 hours, and then treated with S1-2, S2-2, evodiamine, rutaecarpine and teniposide for 24 hours respectively. The drug-treated cells are collected and washed twice with PBS. Then, the cells are suspended in 1× binding buffer and stained with Annexin V-FITC and PI for 30 minutes in the dark at room temperature. Apoptotic cells are quantified using a FACScan cell fluorescence analyzer (BD, USA), and the data are analyzed using Flowjo7.6 software.

[0175] The experimental results are as shown in the appendix Figure 5 It shows that both evodiamine derivative S1-2 and rutaecarpine derivative S2-2 can significantly increase the proportion of early and late apoptotic cells in A549 cells in a dose-dependent manner, indicating that they can effectively induce apoptosis of human non-small cell lung cancer cells.

[0176] Application Example 3

[0177] In this application example, evodiamine derivative S1-2 and rutaecarpine S2-2 were used to treat A549 cells respectively, with teniposide as the positive control, and the cell cycle distribution was detected.

[0178] The specific operation is as follows: The cells were seeded in 6-well plates (1×10 5 cells / well) and incubated for 12 hours, and then treated with S1-2, S2-2, evodiamine, rutaecarpine and teniposide for 24 hours respectively. The drug-treated cells were collected and washed twice with PBS, and stored in 500 μL of 70% ice ethanol for more than 24 hours. The cells were taken out and returned to room temperature, the ethanol solution was poured off after centrifugation, the cells were washed twice with PBS, and then the cells were suspended in 1× binding buffer and stained with PI solution for 30 minutes in the dark at room temperature. Quantitative analysis was performed using a FACScan cell fluorescence analyzer (BD, USA), and the data was analyzed using Flowjo 7.6 software.

[0179] As Figure 6 shown, both evodiamine derivative S1-2 and rutaecarpine S2-2 could arrest the cell cycle at the G2 / M phase, and the effect was better than that of the corresponding parent compounds evodiamine and rutaecarpine. It can be seen that evodiamine derivative S1-2 and rutaecarpine derivative S2-2 can effectively arrest the cell cycle of A549 cells.

[0180] Application Example 4

[0181] In this application example, the cell scratch assay was performed using rutaecarpine derivative S2-2 to verify its ability to inhibit the migration of A549 cells. The healing of cell scratches in each group was detected at 0, 24, and 48 hours after drug treatment of the cells.

[0182] The specific operation is as follows: The cells were seeded in 6-well plates (2×10 5 cells / cm 2 ) and incubated for more than 12 hours. When the cell density reached more than 90%, a scratch was made on the monolayer cells using a micropipette tip, and the same cell-free area was observed through an inverted microscope (NIKON, Japan), and the scratch healing was photographed at 0 and 48 hours of culture. Finally, the NIH Image J image analysis software was used to outline the scratch area and analyze the reduced cell-free area.

[0183] The results are as Figure 7As shown, compared with the control group, the cell scratch healing rate of the compound S2-2 treatment group was the slowest, indicating that it could significantly and effectively inhibit the migration of A549 cells, while the inhibitory ability of its parent compound rutaecarpine and the positive control drug teniposide on the migration of A549 cells was not significant.

[0184] Application Example 5

[0185] In this application example, Surflex docking in Sybyl-x2.1 was used to evaluate the possible binding modes of the compounds evodiamine derivative S1-2 and rutaecarpine S2-2 with DNA topoisomerase.

[0186] The specific operations are as follows: After retrieving the crystal structure of the TopⅠ protein complex from the RCSB Protein Data Bank, the compound molecules of S1-2 and S2-2 were hydrogenated and charged through the graphical user interface of Autodock Tool, and the small molecules S1-2 and S2-2 were docked with TopⅠ using AutoDock Vina 1.23. After molecular docking, the interaction types between the ligand-based pharmacophore model and the docked protein were analyzed.

[0187] The results showed that the docking score of S1-2 with DNA topoisomerase was 9.143 (-log(K d ))), indicating that the theoretical binding constant K d of the small molecule m-toluidine evodiamine with DNA topoisomerase was 10 -9.143 , that is, it might have nanomolar-level activity. The small molecule S1-2 stably bound by forming hydrogen bonds with the amino acid residue A351 at the active center of DNA topoisomerase and the base DT10 respectively. The docking score of the small molecule S2-2 with DNA topoisomerase was 11.1716 (-log(K d ))), which was significantly better than that of S1-2. The amino acids and bases involved in the co-action of the two small molecules included A351, N352, R364, DT10, DC112, DA113, and the specific binding mode was as shown in Figure 8 .

[0188] Through Figure 8 the docking mode shown, taking the human alveolar adenocarcinoma basal epithelial cell A549 as an example, S1-2 and the evodiamine derivative S2-2 could target and inhibit the DNA topoisomerase of A549. Therefore, the binding energy of the small molecules S1-2 and S2-2 with DNA topoisomerase was relatively low, indicating that both small molecules could effectively target DNA topoisomerase.

[0189] According to the above results, evodiamine / rutaecarpine derivatives have good anti - A549 proliferation activity and migration ability compared with their parent evodiamine / rutaecarpine. This is mainly attributed to the group modification on the evodiamine / rutaecarpine parent nucleus. These groups may increase the targeting of the parent structure to DNA topoisomerase. Topoisomerases are a class of enzymes that play a key role in DNA repair and recombination. They can cut and religate the strands of DNA molecules, thereby changing the topological structure of DNA. Inhibiting the activity of DNA topoisomerase can lead to DNA replication obstacles, and this enzyme is an effective target for anti - tumor drug design. Therefore, our research is expected to make a theoretical contribution to the development of new evodiamine / rutaecarpine anti - cancer drugs in terms of mechanism.

Claims

1. An evodiamine derivative, characterized in that, Its structural formula is:

2. A method for preparing the evodiamine derivative according to claim 1, characterized in that, It includes the following steps: (1) Prepare bromoacetyl aniline compounds: Using aniline compounds and bromoacetyl bromide as raw materials, add an acid-binding agent, and stir and react at room temperature for at least 12 h; Among them, the general structural formula of the aniline compound is: R2 is CF3, and R1, R3, R4, and R5 are all H; After the reaction is completed, add water for quenching, and add an extractant for extraction. Take the lower organic phase, dehydrate, filter by suction, and rotary evaporate to obtain the crude product of bromoacetyl aniline compounds; (2) Prepare evodiamine derivatives: First, add evodiamine to the crude product of bromoacetyl aniline compounds obtained in step (1), then add a solvent and a catalyst sodium hydride, and under nitrogen conditions, reflux and react at 80 °C - 90 °C for at least 12 h; After the reaction is completed, wait for the reaction solution to cool to room temperature, extract the reaction solution, remove water, filter by suction, rotary evaporate, and then separate and purify the product by column chromatography to obtain the evodiamine derivative.

3. The preparation method according to claim 2, characterized in that, In the step (1), the molar ratio of aniline compounds to bromoacetyl bromide is 1:1.5 - 2; The molar ratio of the acid-binding agent to aniline compounds is 2 - 2.5:1; The acid-binding agent is triethylamine.

4. The preparation method according to claim 2, wherein In the step (2), the molar ratio of the crude product of bromoacetyl aniline compounds to evodiamine is 1:0.8 - 1.2; sodium hydride: The molar ratio of sodium hydride to the crude product of bromoacetyl aniline compounds is 2 - 2.5:

1.

5. The application of the evodiamine derivative as described in claim 1 in the preparation of anti-lung cancer drugs, anti-melanoma drugs or anti-colorectal cancer drugs; or the application of the evodiamine derivative prepared by the preparation method as described in any one of claims 2 - 4 in the preparation of anti-lung cancer drugs, anti-melanoma drugs or anti-colorectal cancer drugs.

6. A DNA topoisomerase inhibitor, characterized in that, The active ingredient of this DNA topoisomerase inhibitor contains the evodiamine derivative as described in claim 1; or contains the evodiamine derivative prepared by the preparation method as described in any one of claims 2 - 4.

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