Evodiamine derivative, rutaecarpin derivative and preparation method and application thereof

Abstract

The invention belongs to the technical field of chemical pharmacy, particularly relates to an anti-tumor drug, and particularly relates to an evodiamine derivative, a rutaecarpin derivative and a preparation method and application of the evodiamine derivative and the rutaecarpin derivative. The invention provides an evodiamine derivative and a rutaecarpin derivative with higher in-vivo and in-vitro anti-tumor activity. A new drug fragment is introduced to the N-13 site of a B ring of evodiamine or rutaecarpin for structural modification, and an acylaniline group is adopted to replace a hydrogen atom on an imino group of the B ring of evodiamine / rutaecarpin. A series of evodiamine derivative small molecules and rutaecarpin derivative small molecules which have novel structures and are used for targeted inhibition of DNA topoisomerase are obtained. In-vitro anti-tumor activity research shows that the molecules disclosed by the invention have very strong inhibitory activity on tumor cell strains and can promote apoptosis of tumor cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical pharmacy, and in particular relates to an anti-tumor drug, and specifically relates to an evodiamine derivative, an evodiamine derivative, and a preparation method and application thereof. Background Art

[0002] Evodiamine and Rutecarpine are pentacyclic alkaloids belonging to the tryptamine indole class. They are derived from a nearly mature Rutaceae plant, Evodiamine, and have multiple functions, including enhancing cardiac function, regulating hormone levels, and anti-inflammatory and analgesic effects. In addition, Evodiamine and Rutecarpine exhibit excellent anti-cancer properties. They inhibit the proliferation and invasion of tumor cells, induce cell apoptosis, and other ways, showing good inhibitory effects on some common tumors, such as glioblastoma cells, gastric cancer cells, breast cancer cells, and lung cancer cells.

[0003] Studies on the anticancer pharmacological mechanism have shown that evodiamine and rutaecarpine 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, which can induce DNA damage, but irinotecan and others will inhibit it in the S phase. In the process of exploring its anti-prostate cancer and colon cancer effects, it was found that cells treated with evodiamine / rutaecarpine can block the cell cycle in the G2 / M phase, which is different from the effect of previous Topo-I inhibitors. Therefore, further studies have found that evodiamine / rutaecarpine can affect the balance between microtubule depolymerization and separation in human cancer cells and inhibit the proliferation of human cancer cells.

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

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

[0006] The molecular docking of the B-ring substitution structure-activity relationship showed that in the docking conformation of the evodiamine / evodiamine complex with TopⅠ-DNA, the A, B and C rings of evodiamine / evodiamine have a unique L-shaped spatial conformation embedded in the DNA base pair, so that N-13 on the B ring interacts with Arg 364 of TopⅠ through hydrogen bonds and π-π interactions. The study found that only part of the evodiamine / evodiamine was inserted into the DNA base pair, and the N-13 position of the B ring pointed to the large groove of the DNA, indicating that there is still room for structural optimization. At present, the structural modification of the skeleton B ring of evodiamine / evodiamine mainly focuses on the introduction of alkyl, benzoyl, benzyl, ester, long-chain alkoxy, mesyl and other groups on the indole nitrogen atom.

[0007] Summary of the invention

[0008] The purpose of the present invention is to provide an evodiamine derivative and an evodiamine derivative with higher anti-tumor activity in vitro and in vivo. A series of novel evodiamine derivative small molecules and evodiamine derivative small molecules that target and inhibit DNA topoisomerase are obtained by introducing a new drug fragment at the N-13 position of the B ring of evodiamine or evodiamine for structural modification.

[0009] The present invention finds through rational drug structure-activity design analysis that based on the binding mode of evodiamine / evodiamine in the DNA topoisomerase binding pocket, the use of acylanilide groups to replace the hydrogen atoms on the imino group of the B ring of evodiamine / evodiamine can further enhance the targeting of evodiamine / evodiamine to TopⅠ and significantly improve its anti-tumor activity. In vitro anti-tumor activity studies have shown that this type of molecule has a strong inhibitory activity on tumor cell lines and can promote the apoptosis of tumor cells.

[0010] The specific technical solutions are as follows:

[0011] An evodiamine derivative, the general structural formula of which is shown in Formula S1:

[0012]

[0013] In the formula, R 1 , R 2 , R 3 , R 4 , R 5 Each independently selected from H, CH 3 、OCH 3 CF 3 , F, Cl or Br.

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

[0015] A rutaecarpine derivative, the general structural formula of which is shown in Formula S2:

[0016]

[0017] In the formula, R 1 , R 2 , R 3 , R 4 , R 5 Each independently selected from H, CH 3 、OCH 3 CF 3 , F, Cl or Br.

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

[0019] The preparation method of the evodiamine derivative or the evodiamine derivative comprises the following steps:

[0020] (1) Preparation of bromoacetylaniline compounds: Aniline compounds and bromoacetyl bromide are used as raw materials, an acid binding agent is added, and the reaction is stirred at room temperature (here room temperature refers to the indoor temperature regulated in accordance with the pharmaceutical industry standards, generally controlled at 20-25° C.) for at least 12 hours. The entire reaction process is monitored using a thin layer chromatography silica gel plate.

[0021] The specific operation of monitoring is: use a capillary to draw a trace amount of reaction system solution and spot it on a thin layer chromatography silica gel plate, and then use the chromatography fluid (V 乙酸乙酯 :V 石油醚 =1:1) unfold the sample, and judge the reaction conversion of the reactant by the fluorescence intensity at 365nm under the ultraviolet analyzer, so as to monitor the whole reaction process.

[0022] Among them, the general structural formula of aniline compounds is: Where R 1 , R 2 , R 3 , R 4 , R 5 Each independently selected from H, CH 3 、OCH 3 CF 3 , F, Cl or Br;

[0023] After the reaction is completed, water is added for quenching, and an extractant is added for extraction. The lower organic phase is removed from the water, filtered and spin-dried to obtain a crude bromoacetylaniline compound.

[0024] The structural formula of bromoacetylanilide compounds is: Where R 1 , R 2 , R 3 , R 4 , R 5 Each independently selected from H, CH 3 、OCH 3 CF 3 , F, Cl or Br.

[0025] The organic phase can be dehydrated by anhydrous sodium sulfate. The extractant in this step can be selected from any one of dichloromethane (DCM), ethyl acetate, ether or chloroform.

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

[0027]

[0028] (2) Preparation of evodiamine derivatives or evodiamine derivatives: First, evodiamine or evodiamine is added to the crude bromoacetylaniline compound obtained in step (1). When preparing evodiamine derivatives, evodiamine is added to the crude bromoacetylaniline compound obtained in step (1); when preparing evodiamine derivatives, evodiamine is added to the crude bromoacetylaniline compound obtained in step (1).

[0029] Then, a solvent and a catalyst sodium hydride (NaH) are added, and the mixture is refluxed at 80°C-90°C for at least 12 hours under nitrogen conditions, and the entire reaction process is monitored by thin layer chromatography silica gel plates. The solvent may be N,N-dimethylformamide (DMF).

[0030] After the reaction is completed, the reaction solution is cooled to room temperature, the reaction solution is extracted, the water is removed, the solution is filtered, and the product is separated and purified by column chromatography after being spin-dried to obtain the evodiamine derivative or the evodiamine derivative.

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

[0032] The reaction equation for preparing the evodiamine derivative in step (2) is:

[0033]

[0034] The reaction equation for preparing the Evodia rutaecarpa derivative in step (2) is:

[0035]

[0036] In the present invention, the preparation method, in step (1), the molar ratio of aniline compound to bromoacetyl bromide is 1:1.5-2; the molar ratio of acid-binding agent to aniline compound is 2-2.5:1; the acid-binding agent is triethylamine (Et 3 N).

[0037] In the preparation method of the present invention, in step (2), the molar ratio of the crude bromoacetylanilide compound to evodiamine is 1:0.8-1.2; the molar ratio of the crude bromoacetylanilide compound to evodiamine is 1:0.8-1.2; and the molar ratio of sodium hydride to the crude bromoacetylanilide compound is 2-2.5:1.

[0038] The use of the above-mentioned evodiamine derivatives or the above-mentioned evodiamine derivatives in inhibiting the activity of DNA topoisomerase. The above-mentioned evodiamine derivatives or the above-mentioned evodiamine derivatives can inhibit the ability of DNA topoisomerase to form a covalent complex with DNA, and have a topoisomerase inhibitory effect.

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

[0040] Application of the above-mentioned evodiamine derivative or the above-mentioned evodiamine derivative in the preparation of anti-tumor drugs.

[0041] Application of the evodiamine derivative or evodiamine 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 derivatives or the rutaecarpine derivatives; or contains at least one of the evodiamine derivatives or the rutaecarpine derivatives prepared by the above preparation method.

[0043] The beneficial effects of the present invention are as follows: the evodiamine derivatives or evodiamine derivatives described in the present invention not only enhance the anti-tumor activity to a certain extent, but also enhance the inhibitory effect on the viability of tumor cells. For example, the inhibitory effect of the evodiamine derivatives 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 derivatives or evodiamine derivatives provided by the present invention have higher selectivity and will not inhibit normal cells in the human body, thus having low toxicity and higher safety. In addition, the activity of the evodiamine derivatives provided by the present invention is nearly 10 times higher than that of its parent compound, evodiamine.

[0044] This shows that the structural modification strategy adopted by the present invention is effective. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The in vitro antiproliferative activity of evodiamine / evodiamine derivatives against human lung cancer cell lines A549 and H460.

[0046] Figure 2 The in vitro antiproliferative activity of evodiamine / evodiamine derivatives against human melanoma cell lines A375 and A827.

[0047] Figure 3 The in vitro antiproliferative activity of evodiamine / evodiamine derivatives against human colorectal cancer cell lines HCT-8 and HT-29.

[0048] Figure 4 The in vitro antiproliferative activity of evodiamine / evodiamine derivatives on human renal epithelial cell line (293T).

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

[0050] Figure 6 The effect of evodiamine / evodiamine derivatives on the cell cycle of A549 cells. The data are the mean ± SEM of three independent experiments (*P<0.05, **P<0.01).

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

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

[0053] Fig. 9 It is the NMR spectrum of Example 1.

[0054] Fig.10 It is the NMR spectrum of Example 2.

[0055] Fig.11 It is the NMR spectrum of Example 3.

[0056] Fig.12 It is the NMR spectrum of Example 4.

[0057] Fig.13 It is the NMR spectrum of Example 5.

[0058] Fig.14 It is the NMR spectrum of Example 6.

[0059] Fig.15 This is the NMR spectrum of Example 7.

[0060] Fig.16 This is the NMR spectrum of Example 8.

[0061] Fig.17 This is the NMR spectrum of Example 9.

[0062] Fig.18 This is the NMR spectrum of Example 10.

[0063] Fig.19 This is the NMR spectrum of Example 11.

[0064] Fig. 20 This is the NMR spectrum of Example 12.

[0065] Fig.21 This is the NMR spectrum of Example 13.

[0066] Fig. 22 This is the NMR spectrum of Example 14. DETAILED DESCRIPTION

[0067] The technical solution of the present invention is described in detail below in conjunction with the accompanying drawings.

[0068] Example 1

[0069] This embodiment is an evodiamine derivative as shown in the following formula:

[0070]

[0071] pass Fig. 9 From the spectrum analysis, we can see that 1 H NMR (600 MHz, CDCl 3 )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 in S1-1 comprises the following specific steps:

[0073] (1) In a 150 mL round-bottom flask, 1 mmol of aniline, 1.5 mmol of bromoacetyl bromide, 2 mmol of acid-binding agent triethylamine and 30 mL of purified dichloromethane were added in sequence, and the mixture was stirred at room temperature for 12 h. The reaction was monitored by thin layer chromatography on silica gel plate until completion.

[0074] After the reaction is complete, 18 mL of deionized water is added to the reaction solution for quenching, and then 18 mL of dichloromethane is added for extraction. The extraction is repeated three times, and the obtained lower organic phase liquid is combined.

[0075] The combined organic phase liquid was dehydrated by passing through anhydrous sodium sulfate, and the sodium sulfate was removed by suction filtration. The liquid was spin-dried to obtain a crude bromoacetylaniline.

[0076] (2) Add 0.9 mmol of evodiamine and 2 mmol of NaH to the crude bromoacetylaniline obtained in step (1), and add 20 mL of solvent N,N-dimethylformamide to dissolve it. Under nitrogen conditions, condense and reflux at 80° C. for 12 h, and monitor the reaction progress using 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, and 100 mg of anhydrous sodium sulfite is added to remove moisture, and then filtered, the solvent is dried using a rotary evaporator, and finally the product is separated and purified by column chromatography to obtain the S1-1 evodiamine derivative in the form of a white powder. The yield is 70%.

[0078] Example 2

[0079] This embodiment is an evodiamine derivative as shown in the following formula:

[0080]

[0081] pass Fig.10 From the spectrum analysis, we can see that 1 H NMR (600 MHz, CDCl 3)δ8.12(s,1H),7.67(d,J=7.7Hz,1H),7.42(m,5),7.18-7.09(m,5H),6.91(d,J=7.1Hz,1H),5.97(s,1H) ,5.29(s,2H),4.96(d,J=17.0Hz,2H),3.24(td,J=12.4,3.9Hz,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-methylaniline. The yield is 76%.

[0083] The rest is the same as in Example 1.

[0084] Example 3

[0085] This embodiment is an evodiamine derivative as shown in the following formula:

[0086]

[0087] pass Fig.11 From the spectrum analysis, we can see that 1 H NMR (600 MHz, CDCl 3 )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, the aniline compound added in step (1) is p-methoxyaniline. The yield is 66%.

[0089] The rest is the same as in Example 1.

[0090] Example 4

[0091] This embodiment is an evodiamine derivative as shown in the following formula:

[0092]

[0093] pass Fig.12 Spectral analysis shows 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 m-trifluoromethylaniline. The yield is 64%.

[0095] The rest is the same as in Example 1.

[0096] Example 5

[0097] This embodiment is an evodiamine derivative as shown in the following formula:

[0098]

[0099] pass Fig.13 From the spectrum analysis, we can see that 1 H NMR (600 MHz, CDCl 3 )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),7.21-7.0 6(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 p-fluoroaniline. The yield is 53%.

[0101] The rest is the same as in Example 1.

[0102] Example 6

[0103] This embodiment is an evodiamine derivative as shown in the following formula:

[0104]

[0105] pass Fig.14 From the spectrum analysis, we can see that 1 H NMR (400MHz, DMSO) δ10.57 (s, 1H), 7.92 (d, J = 7.2Hz, 1H), 7.82 (s, 1H), 7.61 (d,J=7.7Hz,1H),7.54(d,J=8.2Hz,1H),7.46(d,J=5.0Hz,2H),7.37(t,J=8 .0Hz,1H),7.23(m,2H),7.14(d,J=8.0Hz,2H),7.02(d,J=7.8Hz,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 m-chloroaniline. The yield is 63%.

[0107] The rest is the same as in Example 1.

[0108] Example 7

[0109] This embodiment is an evodiamine derivative as shown in the following formula:

[0110]

[0111] pass Fig.15 From the spectrum analysis, we can see that 1H NMR (600 MHz, CDCl 3 )δ8.23(d,J=7.8Hz,1H),7.61(t,J=7.4Hz,1H),7.57-7.52(m,2H),7.49(d ,J=8.3Hz,2H),7.41-7.38(m,1H),7.33(t,J=7.2Hz,1H),7.28(d,J=7.6Hz ,1H),7.25-7.20(m,2H),7.18(s,2H),7.09(t,J=7.4Hz,1H),4.50(t,J=6. 8Hz,2H),3.37-3.32(m,3H),3.14(t,J=6.8Hz,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, the aniline compound added in step (1) is p-chloroaniline. The yield is 72%.

[0113] The rest is the same as in Example 1.

[0114] Example 8

[0115] This embodiment is a rutaecarpine derivative as shown in the following formula:

[0116]

[0117] pass Fig.16 From the spectrum analysis, we can see that 1 H NMR (600 MHz, CDCl 3 )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 evodia rutaecarpine derivative shown in S2-1 comprises the following specific steps:

[0119] (1) In a 150 mL round-bottom flask, 1 mmol of aniline, 1.5 mmol of bromoacetyl bromide, 2 mmol of acid-binding agent triethylamine and 30 mL of purified dichloromethane were added in sequence, and the mixture was stirred at room temperature for 12 h. The reaction was monitored by thin layer chromatography on silica gel plate until completion.

[0120] After the reaction is complete, 18 mL of deionized water is added to the reaction solution for quenching, and then 18 mL of dichloromethane is added for extraction. The extraction is repeated three times, and the obtained lower organic phase liquid is combined.

[0121] The combined organic phase liquid was dehydrated by passing through anhydrous sodium sulfate, and the sodium sulfate was removed by suction filtration. The liquid was spin-dried to obtain a crude bromoacetylaniline.

[0122] (2) Add 0.9 mmol of evodia rutaecarpine and 2 mmol of NaH to the crude bromoacetylaniline obtained in step (1), and add 20 mL of solvent N,N-dimethylformamide to dissolve it. Under nitrogen conditions, condense and reflux 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, 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, and 100 mg of anhydrous sodium sulfite is added to remove moisture, and then filtered, the solvent is dried using a rotary evaporator, and finally the product is separated and purified by column chromatography to obtain the S2-1 rutaecarpine derivative in the form of a white powder. The yield is 52%.

[0124] Example 9

[0125] This embodiment is a rutaecarpine derivative as shown in the following formula:

[0126]

[0127] pass Fig.17 From the spectrum analysis, we can see that 1 H NMR(600MHz, CDCl3)δ8.06(dd,J=68.9,7.6Hz,3H),7.68-7.59(m,2H),7.58-7.53(m,1H),7.47(dd,J=14.0,7.3Hz,3H),7.3 8(d,J=7.9Hz,1H),7.30-7.26(m,2H),5.29(s,2H),4.58(dd,J=12.8,3.8Hz,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 evodia rutaecarpine derivative shown in S2-2, the aniline compound added in step (1) is m-toluidine. The yield is 64%.

[0129] The rest is the same as Example 8.

[0130] Example 10

[0131] This embodiment is a rutaecarpine derivative as shown in the following formula:

[0132]

[0133] pass Fig.18 From the spectrum analysis, we can see that 1 H NMR (600 MHz, CDCl 3 )δ7.98(d,J=7.9Hz,1H),7.70-7.60(m,2H),7.46-7.36(m,3H),7.29(d,J=7.8Hz,1H),7.12(d,J=7.2Hz,1H),6 .98-6.92(m,2H),6.86-6.74(m,2H),6.60(d,J=9.5Hz,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 evodia rutaecarpine derivative shown in S2-3, the aniline compound added in step (1) is p-methoxyaniline. The yield is 73%.

[0135] The rest is the same as Example 8.

[0136] Embodiment 11

[0137] This embodiment is a rutaecarpine derivative as shown in the following formula:

[0138]

[0139] pass Fig.19 From the spectrum analysis, we can see that 1 H NMR (600 MHz, CDCl 3)δ10.93(s,1H),8.39(d,J=7.9Hz,1H),7.84(s,1H),7.76(dd,J=19.2,8.4Hz,2H),7.67(d,J=8.0Hz,2H),7.63(d,J=8.0Hz,1H),7.52(t,J =7.5Hz,1H),7.48(t,J=7.5Hz,1H),7.34(t,J=7.9Hz,1H),7.31-7.26(m,2H),5.38(s,2H),4.59(s,2H),3.24(t,J=6.7Hz,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 evodia rutaecarpine derivative shown in S2-4, the aniline compound added in step (1) is m-trifluoromethylaniline. The yield is 58%.

[0141] The rest is the same as Example 8.

[0142] Example 12

[0143] This embodiment is a rutaecarpine derivative as shown in the following formula:

[0144]

[0145] pass Fig. 20 From the spectrum analysis, we can see that 1 H NMR (600 MHz, CDCl 3 )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 evodia rutaecarpine derivative shown in S2-5, the aniline compound added in step (1) is p-fluoroaniline. The yield is 66%.

[0147] The rest is the same as Example 8.

[0148] Example 13

[0149] This embodiment is a rutaecarpine derivative as shown in the following formula:

[0150]

[0151] pass Fig.21 From the spectrum analysis, we can see that 1 H NMR (600 MHz, CDCl 3 )δ10.72(s,1H),8.35(dd,J=46.1,7.9Hz,1H),7.81-7.61(m,4H),7.55-7.40(m,3H),7.37-7.22(m,3H),7 .16(dt,J=33.6,7.9Hz,1H),5.30(s,2H),4.59(t,J=6.9Hz,2H),3.24(dd,J=13.9,7.0Hz,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 evodia rutaecarpine derivative shown in S2-6, the aniline compound added in step (1) is m-chloroaniline. The yield is 75%.

[0153] The rest is the same as Example 8.

[0154] Embodiment 14

[0155] This embodiment is a rutaecarpine derivative as shown in the following formula:

[0156]

[0157] pass Fig. 22 From the spectrum analysis, we can see that 1H NMR (400MHz, DMSO) δ10.71(s,1H),8.18(d,J=7.9Hz,1H),7.82-7.70(m,5H),7.61(d,J=8.0Hz,1H),7.49(t,J=7.9Hz,1 H),7.42(d,J=8.7Hz,3H),7.25(t,J=7.4Hz,1H),5.71(s,2H),4.50(t,J=6.7Hz,2H),3.27(t,J=6.7Hz,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 evodia rutaecarpine derivative shown in S2-7, the aniline compound added in step (1) is p-chloroaniline. The yield is 61%.

[0159] The rest is the same as Example 8.

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

[0161] Application Example 1

[0162] In this application example, a CCK-8 detection kit was used to conduct a cell proliferation experiment, and evodiamine, evodiamine, etoposide, and teniposide were used as positive controls to detect the effects of the evodiamine derivatives described in Examples 1-7 and the evodiamine derivatives described in Examples 8-14 on the in vitro proliferation activity 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 lines (293T).

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

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

[0165] Among them, S1-2 (IC 50 =2.54 μM) had significant antiproliferative activity against A549 cells, which was 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 evodia rutaecarpine derivatives, and their antiproliferative activities are significantly better than evodia rutaecarpine.

[0167] From the perspective of structure and activity, it can be found that the benzene ring has an electron-donating group (CH 3 、OCH 3 CF 3 ) are significantly more active than compounds with electron-withdrawing groups (F, Cl) on the benzene ring. In addition, evodiamine / evodiamine derivatives have no obvious toxicity to human renal epithelial cells.

[0168] Table 1 In vitro inhibition of tumor cell proliferation activity of evodiamine and rutaecarpine derivatives

[0169]

[0170]

[0171] From the data analysis in Table 1, it can be seen that the in vitro antiproliferation experiment proved that the evodiamine derivatives / evodiamine derivatives all had significant antiproliferative activity against human non-small cell lung cancer A549 cells, and the median lethal concentration (IC 50The IC value of compound S2-2 on A549 cells was less than 10 μM. 50 The value was 2.54 μM, which was significantly lower than that of its parent compound evodiamine (IC 50 =7.29 μM), the anti-tumor activity was increased by more than 3 times.

[0172] Application Example 2

[0173] In this application example, the Annexin V-FITC-PI apoptosis kit was used to detect the apoptosis-inducing effects of evodiamine derivatives S1-2 and evodiamine derivatives S2-2 on A549 cells.

[0174] The specific operation is as follows: cells were seeded in 6-well plates (1×10 5 The cells were incubated with 100 μg / well for 12 hours and then treated with S1-2, S2-2, evodiamine, evodiamine and teniposide for 24 hours. The drug-treated cells were collected and washed twice with PBS, after which the cells were suspended in 1× binding buffer and stained with Annexin V-FITC and PI for 30 minutes in the dark and at room temperature. Apoptotic cells were quantified using a FACScan cell fluorescence instrument (BD, USA), and the data were analyzed using Flowjo7.6 software.

[0175] The experimental results are as attached Figure 5 As shown. Both evodiamine derivatives S1-2 and evodiamine derivatives 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 in human non-small cell lung cancer cells.

[0176] Application Example 3

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

[0178] The specific operation is as follows: cells were seeded in 6-well plates (1×10 5 / well) for 12 hours, and then treated with S1-2, S2-2, evodiamine, evodiamine and teniposide for 24 hours. 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 and at room temperature. Quantitative analysis was performed using a FACScan cell fluorescence instrument (BD, USA), and the data were analyzed using Flowjo7.6 software.

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

[0180] Application Example 4

[0181] This application example uses the evodia rutaecarpine derivative S2-2 to perform a cell scratch test to verify its ability to inhibit A549 cell migration. The healing of the cell scratches in each group was detected at 0, 24, and 48 hours after the drug treatment of the cells.

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

[0183] The results are as follows Figure 7 As shown, compared with the control group, the cell scratch healing rate in the group treated with compound S2-2 was the slowest, indicating that it can significantly and effectively inhibit the migration of A549 cells, while its parent compound evodipine and the positive control drug teniposide had no significant ability to inhibit the migration of A549 cells.

[0184] Application Example 5

[0185] This application example uses the Surflex docking in Sybyl-x2.1 to evaluate the possible binding modes of compounds evodiamine derivatives S1-2 and evodiamine S2-2 with DNA topoisomerase.

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

[0187] The results showed that the docking score of S1-2 and DNA topoisomerase was 9.143 (-log (K d)), represents the theoretical binding constant K between small molecules of toluidine and evodiamine and DNA topoisomerase d In 10 -9.143 , which may have nanomolar activity. The small molecule S1-2 forms hydrogen bonds with the amino acid residue A351 and the base DT10 in the active center of DNA topoisomerase and stably binds. The docking result of the small molecule S2-2 and DNA topoisomerase is scored as 11.1716 (-log (K d )), significantly better than S1-2. The amino acids and bases that the two small molecules interact with include A351, N352, R364, DT10, DC112, and DA113. The specific binding modes are as follows Figure 8 shown.

[0188] pass Figure 8 The docking mode shown shows that, taking human alveolar adenocarcinoma basal epithelial cells A549 as an example, S1-2 and the rutaecarpine derivative S2-2 can target and inhibit the DNA topoisomerase of A549. Therefore, the binding energy of small molecules S1-2 and S2-2 with DNA topoisomerase is low, indicating that both small molecules can effectively target DNA topoisomerase.

[0189] According to the above results, compared with its parent evodiamine / sub-basic, evodiamine / sub-basic derivatives have good anti-A549 proliferation activity and migration ability, which is mainly attributed to the group modification on the evodiamine / sub-basic nucleus. These groups may increase the targeting of the parent structure to DNA topoisomerase. Topoisomerase is a class of enzymes that play a key role in DNA repair and recombination. They can cut and reconnect the chains of DNA molecules, thereby changing the topological structure of DNA. Inhibition of DNA topoisomerase activity can lead to DNA replication disorders. This enzyme is an effective target for the design of anti-tumor drugs. Therefore, our research is expected to make a mechanistic contribution to the development of new evodiamine / sub-basic anticancer drugs.

Claims

1. An evodiamine derivative, characterized in that: The general structural formula is shown in Formula S1: In the formula, R1, R2, R3, R4, and R5 are each independently selected from H, CH3, OCH3, CF3, F, Cl or Br.

2. The evodiamine derivative according to claim 1, characterized in that The evodiamine derivative is any one of the derivatives shown in structural formulas S1-1 to S1-7:

3. A rutaecarpine derivative, characterized in that: The general structural formula is shown in Formula S2: In the formula, R1, R2, R3, R4, and R5 are each independently selected from H, CH3, OCH3, CF3, F, Cl or Br.

4. The evodiamine derivative according to claim 3, characterized in that The evodia rutaecarpine derivative is any one of the derivatives shown in structural formulas S2-1 to S2-7:

5. A method for preparing the evodiamine derivative according to claim 1 or the evodiamine derivative according to claim 3, characterized in that: The following steps are involved: (1) Preparation of bromoacetylaniline compounds: using aniline compounds and bromoacetyl bromide as raw materials, adding an acid binding agent, and stirring the reaction at room temperature for at least 12 hours; Among them, the general structural formula of aniline compounds is: R1, R2, R3, R4, R5 are each independently selected from H, CH3, OCH3, CF3, F, Cl or Br; After the reaction is completed, water is added to quench the reaction, and an extractant is added to extract the reaction mixture. The lower organic phase is removed from the reaction mixture, filtered, and dried to obtain a crude bromoacetylaniline compound. (2) preparing evodiamine derivatives or evodiamine derivatives: firstly, adding evodiamine or evodiamine to the crude bromoacetylanilide compound obtained in step (1), then adding a solvent and a catalyst sodium hydride, and reacting under condensation reflux at 80° C.-90° C. for at least 12 hours under nitrogen conditions; After the reaction is completed, the reaction solution is cooled to room temperature, the reaction solution is extracted, the water is removed, the solution is filtered, and the product is separated and purified by column chromatography after being spin-dried to obtain the evodiamine derivative or the evodiamine derivative.

6. The preparation method according to claim 5, characterized in that: In the step (1), the molar ratio of aniline compound to bromoacetyl bromide is 1:1.5-2; The molar ratio of acid binding agent: aniline compound is 2-2.5:1; The acid binding agent is triethylamine.

7. The preparation method according to claim 5, characterized in that: In the step (2), the molar ratio of the crude bromoacetylaniline compound to evodiamine is 1:0.8-1.2; the molar ratio of the crude bromoacetylaniline compound to evodiamine is 1:0.8-1.2; and the molar ratio of sodium hydride to the crude bromoacetylaniline compound is 2-2.5:

1.

8. Use of the evodiamine derivative according to any one of claims 1 to 2 or the evodiamine derivative according to any one of claims 3 to 4 in inhibiting the activity of DNA topoisomerase; Or the use of the evodiamine derivative or the evodiamine derivative obtained by the preparation method as described in any one of claims 5 to 7 in inhibiting the activity of DNA topoisomerase.

9. Use of the evodiamine derivative according to any one of claims 1 to 2 or the evodiamine derivative according to any one of claims 3 to 4 in the preparation of antitumor drugs; Or the use of the evodiamine derivative or the evodiamine derivative obtained by the preparation method according to any one of claims 5 to 7 in the preparation of anti-tumor drugs.

10. A DNA topoisomerase inhibitor, characterized in that The active ingredient of the DNA topoisomerase inhibitor contains at least one of the evodiamine derivatives according to any one of claims 1 to 2 or the evodiamine derivatives according to any one of claims 3 to 4; Or it contains at least one of the evodiamine derivatives or evodiamine derivatives prepared by the preparation method as described in any one of claims 5 to 7.

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