Benzo [b] [1, 6] naphthyridine compound as well as preparation method and application thereof

Abstract

The invention discloses a benzo [b] [1, 6] naphthyridine compound as well as a preparation method and application thereof, and belongs to the technical field of synthesis of organic compounds, acetyl o-toluidine is taken as an initiator, an aromatic ring is generated through cyclization reaction, a carboxylic ester compound is generated through oxidation and nucleophilic substitution, and the benzo [b] [1, 6] naphthyridine compound is obtained through cyclization, elimination reaction, amidation reaction and acylation reaction. And the benzo [b] [1, 6] naphthyridine compound is prepared. A benzo [b] [1, 6] naphthyridine skeleton is subjected to structural modification and transformation to prepare the novel compound with specific cancer cell inhibition biological activity, a candidate substance is provided for research and development of novel anti-tumor drugs, and the compound is suitable for preparing the anti-tumor drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of organic compound synthesis, and relates to a benzo[b][1,6]naphthyridine compound and a preparation method and application thereof. BACKGROUND

[0002] In the process of cell proliferation, the replication of DNA depends on the participation of topoisomerase (Topo). Studies have shown that the expression level and activity of Topo in cancer cells are usually significantly higher than those in normal cells, which makes Topo one of the important targets for the development of antitumor drugs. By inhibiting the activity of Topo, the process of DNA replication and transcription of tumor cells can be effectively interfered, and then the malignant proliferation thereof is inhibited.

[0003] At present, a variety of Topo I and Topo II inhibitors have been developed and applied to clinical treatment, in which quinoline compounds such as camptothecin and its derivatives are typical Topo I inhibitors. However, many existing topoisomerase inhibitors still face problems such as poor stability, short in-vivo drug efficacy maintenance time, and large systemic toxic side effects, which seriously limit their clinical application potential. Therefore, the development of new Topo inhibitors with high activity and good safety has become an urgent need for the research of antitumor drugs.

[0004] Among the numerous compounds with Topo inhibition potential, quinoline compounds are of great concern due to their wide pharmacological activity. As an important nitrogen-containing heterocyclic aromatic structure, quinoline and its derivatives exhibit unique value in the research and development of antitumor drugs. Among them, benzo[b][1,6]naphthyridine compounds, as structural derivatives of quinoline, not only have a planar aromatic backbone, but also can be optimized through side chain modification. Such compounds can be embedded into the DNA-Topo cleavage complex with high affinity, forming a stable ternary complex, thereby persistently inhibiting the religation function of Topo. SN28049, as a benzo[b][1,6]naphthyridine compound, has a dual inhibition mechanism of topoisomerase I / II, and can effectively interfere with the process of DNA replication and transcription, showing good anticancer potential. However, the inhibitory activity of this compound on specific cancer cells still needs to be further improved, and its pharmacodynamic performance has not yet reached the optimum, and breakthroughs still need to be achieved through means such as structural modification or dosage form optimization. SUMMARY

[0005] In view of the above deficiencies of the prior art, the purpose of the present application is to provide a benzo[b][1,6]naphthyridine compound and a preparation method and application thereof. By modifying and reforming the benzo[b][1,6]naphthyridine skeleton, a class of benzo[b][1,6]naphthyridine compounds with high tumor cell inhibition rate are prepared, achieving the purpose of being used for preparing antitumor drug intermediates.

[0006] To achieve the above object, the technical scheme adopted by the present application is:

[0007] A benz[b][1,6]naphthyridine compound has the following structural formula:

[0008]

[0009] In the formula, R is a substituted group with a nitrogen-containing heterocyclic structure, and the general formula is C n H 2n+1 N, n is an integer greater than or equal to 6. When the substitution is performed, the substituted group loses one H to form a chemical bond connected to -NH in the structural formula of the benz[b][1,6]naphthyridine compound.

[0010] As a limitation of the present application, the nitrogen-containing heterocyclic structure is a nitrogen-containing five-membered ring or a nitrogen-containing six-membered ring.

[0011] As another limitation of the present application, R is a substituted group made of one of

[0012] The present application also provides a preparation method of the benz[b][1,6]naphthyridine compound, which is prepared by acylating reaction of 2,6-dimethyl-1-oxo-1,2-dihydrobenz[b][1,6]naphthyridine-4-formyl chloride and an amine compound.

[0013] As a further limitation of the present application, the amine compound is

[0014] As a further limitation of the present application, the ratio of the use amount of 2,6-dimethyl-1-oxo-1,2-dihydrobenz[b][1,6]naphthyridine-4-formyl chloride to the amine compound is 70-90 mL:0.1 mol.

[0015] As a further limitation of the present application, the preparation method of 2,6-dimethyl-1-oxo-1,2-dihydrobenz[b][1,6]naphthyridine-4-formyl chloride comprises the following steps performed in sequence:

[0016] S1. Acetyl-o-toluidine and (chloromethylidene)dimethylammonium chloride are subjected to a cyclization reaction to obtain an intermediate 1, which has the following structural formula:

[0017] In the formula, (chloromethylidene)dimethylammonium chloride is a Vilsmeier reagent formed by reaction of DMF and POCl3;

[0018] S2. The intermediate 1, sodium dihydrogen phosphate monohydrate and sodium chlorite are subjected to a Pinnick oxidation reaction to obtain an intermediate 2, which has the following structural formula: ​

[0019] S3. Take intermediate 2 and ethyl acetoacetate, through nucleophilic substitution reaction, intermediate 3 is obtained, the structural formula is

[0020] S4. Take intermediate 3 and (chloromethylene) dimethyl ammonium chloride, through cyclization and elimination reaction, intermediate 4 is obtained, the structural formula is

[0021] S5. Take intermediate 4 and methylamine, through amidation reaction, intermediate 5 is obtained, the structural formula is

[0022] S6. Take intermediate 5 and chlorosulfoxide to occur acylation reaction at reflux temperature, 2,6-dimethyl-1-oxo-1,2-dihydrobenzo[b][1,6]naphthyridine-4-carbonyl chloride, i.e. intermediate 6, is obtained, the structural formula is

[0023] As a further limitation of the application, in step S1, the reaction temperature of the cyclization reaction is 70-100℃, preferably 80℃, and the reaction time is 4-5, preferably 5 days;

[0024] In step S2, the molar ratio of intermediate 1 to sodium dihydrogen phosphate monohydrate and sodium chlorite is 1:6-7:9-10, preferably 1:7.25:9.58; the reaction temperature of the Pinnick oxidation reaction is room temperature, and the reaction time is 2h;

[0025] Wherein, room temperature refers to indoor temperature, which is 20-25℃.

[0026] In step S3, the molar ratio of intermediate 2 to ethyl acetoacetate is 1:1.5; the reaction temperature of the nucleophilic substitution reaction is 80-95℃, preferably 85℃, and the reaction time is 2h.

[0027] As a further limitation of the application, in step S4, the reaction temperature of the cyclization and elimination reaction is 0-10℃, preferably 2℃, and the reaction time is 1.5h.

[0028] In step S5, the molar ratio of intermediate 4 to methylamine is 1:5; the reaction temperature of the amidation reaction is room temperature, and the reaction time is 5h;

[0029] In step S6, the reaction time of the acylation reaction is 7h.

[0030] The application also provides an application of a benzene [b][1,6] naphthyridine compound, which is specifically used for preparing a topoisomerase inhibitor or an antitumor drug.

[0031] The main principle of the present application is that the benz[b][1,6]naphthyridine compound as a structural derivative of quinoline has the effect of topoisomerase inhibitor or antitumor, and the present application unexpectedly finds that the amide bond has cis and trans isomerization due to the partial double bond property, if the substituent R and the carbonyl oxygen are in trans in the present application, the steric hindrance is small, which is the dominant conformation of most amides; if the substituent R and the carbonyl oxygen are on the same side, the steric hindrance is large, the energy is high, and it is usually not dominant. If the volume of the R group is very large, or has a rigid ring system, the steric hindrance of the trans conformation will also increase, the flexibility of the whole molecule will decrease, and the fitting degree with the protein binding pocket will decrease, and the activity will decrease, that is, the compound containing N-methylpiperidin-3-amine or 1-(3-aminopropyl)piperidine in the amide group has higher biological activity of inhibiting tumor cells, and the benz[b][1,6]naphthyridine compound with a specific structure prepared on this basis has higher tumor cell inhibition rate.

[0032] Compared with the prior art, the present application has the following technical progress:

[0033] The present application significantly improves the inhibition activity of the benz[b][1,6]naphthyridine compound on the growth of human colon cancer cells by optimizing the structure of the benz[b][1,6]naphthyridine compound, and the compound of the present application has higher inhibition activity on cancer cells compared with SN28049 with similar structure, which is manifested as the IC 50 value of the benz[b][1,6]naphthyridine compound prepared by the present application can reach 3.43nM, while the IC 50 value of SN28049 is 12.96nM.

[0034] The benz[b][1,6]naphthyridine compound of the present application has more prominent inhibition activity on cancer cells compared with other similar compounds due to the special R substituent, the planar aromatic skeleton is embedded into the DNA-topoisomerase cleavage complex with high affinity to form a stable ternary complex, thereby effectively inhibiting the activity of topoisomerase I and II, and interfering with the DNA replication and transcription process, and exhibiting a synergistic antitumor effect.

[0035] The preparation method of the benz[b][1,6]naphthyridine compound of the present application has mild process conditions, high operation safety, simple post-treatment process, and is more suitable for industrialized production, and is suitable for preparing topoisomerase inhibitors or antitumor drugs. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 The hydrogen nuclear magnetic spectrum of intermediate 1 prepared in example 1 of the present application is shown in the figure;

[0037] Figure 2 The hydrogen nuclear magnetic spectrum of intermediate 2 prepared in example 1 of the present application is shown in the figure;

[0038] Figure 3 The hydrogen nuclear magnetic spectrum of intermediate 3 prepared in Example 1 of the present application;

[0039] Figure 4 The hydrogen nuclear magnetic spectrum of intermediate 4 prepared in Example 1 of the present application;

[0040] Figure 5 The hydrogen nuclear magnetic spectrum of intermediate 5 prepared in Example 1 of the present application;

[0041] Figure 6 The hydrogen nuclear magnetic spectrum of compound A prepared in Example 1 of the present application;

[0042] Figure 7 The carbon nuclear magnetic spectrum of compound A prepared in Example 1 of the present application;

[0043] Figure 8 The hydrogen nuclear magnetic spectrum of compound B prepared in Example 2 of the present application;

[0044] Figure 9 The carbon nuclear magnetic spectrum of compound B prepared in Example 2 of the present application;

[0045] Figure 10 The hydrogen nuclear magnetic spectrum of compound C prepared in Example 3 of the present application;

[0046] Figure 11 The carbon nuclear magnetic spectrum of compound C prepared in Example 3 of the present application;

[0047] Figure 12 The hydrogen nuclear magnetic spectrum of compound D prepared in Comparative Example 1 of the present application;

[0048] Figure 13 The carbon nuclear magnetic spectrum of compound D prepared in Comparative Example 1 of the present application;

[0049] Figure 14 The reaction process schematic diagram of step S7 for preparing compound A, compound B, compound C and compound D in the present application;

[0050] Figure 15 The inhibition rate log fitting curve diagram of compound A obtained in Example 1 in the effect verification example of the present application;

[0051] Figure 16 The inhibition rate log fitting curve diagram of compound B obtained in Example 2 in the effect verification example of the present application;

[0052] Figure 17 The inhibition rate log fitting curve diagram of compound C obtained in Example 3 in the effect verification example of the present application;

[0053] Figure 18The log fitting curve of the inhibition rate of the compound D obtained in the comparative example 1 for verifying the effect of the present application is shown in the following figure. DETAILED DESCRIPTION

[0054] The present application will be further described in detail by specific examples. It should be understood that the described examples are only for explaining the present application and do not limit the present application.

[0055] The experimental methods used in the following examples are conventional methods in the art unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0056] Example 1

[0057] The present embodiment is a kind of benzene [b] [1,6] naphthyridine compound A and its preparation method, wherein the structural formula of compound A is as formula (I), wherein the general formula of the nitrogen-containing six-membered ring structure connected with N in the peptide bond is C6H 12 N;

[0058]

[0059] The preparation method of compound A specifically includes the following steps performed in sequence:

[0060] S1. Preparation of intermediate 1:

[0061] Under nitrogen protection, heavy distilled DMF (13.65 g, 0.19 mol) was added to a 250 mL four-necked flask, which was stirred under ice water bath, and heavy distilled phosphorus oxychloride (80.55 g, 0.53 mol) was added dropwise by constant pressure dropping funnel. The reaction was violent and released heat, and the reaction solution changed from colorless and clear liquid to light yellow and clear liquid. The reaction solution was moved to an oil bath, and the oil bath temperature was set to 80℃. Acetyl-o-toluidine (11.25 g, 0.08 mol) was added, and heating and stirring were carried out for 5 days to perform cyclization reaction. TLC was used to monitor the reaction (a small amount of reaction solution was added with water, and extracted with ethyl acetate). After the reaction was completed, the reaction solution was cooled by standing and then poured into 300 mL ice water. Stirring was started, a small amount of white smoke was generated, the reaction solution became yellow turbid liquid and released a large amount of heat. The mixture was stirred at room temperature for 1 h, and then filtered under suction to obtain yellow filtrate and yellow filter cake. The filter cake was dried at 50℃ under vacuum for 2 h to obtain 2-chloro-8-methylquinoline-3-carboxaldehyde, i.e. intermediate 1, as yellow solid (11.46 g, 74%). The melting point was detected to be 128-129℃, and the structural formula was

[0062] The reaction formula of the above reaction process is:

[0063]

[0064] The hydrogen nuclear magnetic spectrum of the prepared intermediate 1 was detected, as shown in the following figure.Figure 1 Its hydrogen nuclear magnetic characterization data are as follows: 1 HNMR (500 MHz, CDC13) δ 10.57 (s, 1H), 8.71 (s, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.72 (d, J = 7.0 Hz, 1H), 7.54-7.51 (m, 1H), 2.79 (s, 3H).

[0065] S2. Preparation of intermediate 2:

[0066] According to the molar ratio of intermediate 1 to sodium dihydrogen phosphate monohydrate and sodium chlorite of 1:7.25:9.58, 2-chloro-8-methylquinoline-3-carboxaldehyde (1.00 g, 4.85 mmol) was dissolved in 89 mL of hot tert-butanol and added to a 250 mL three-necked flask, which was stirred at room temperature. 4.85 g of sodium dihydrogen phosphate monohydrate and 4.20 g of sodium chlorite were weighed and dissolved in 39 mL of deionized water to form a solution. The solution was a yellow transparent liquid. The solution was poured into a constant pressure dropping funnel and added dropwise to the reaction solution. The reaction was stirred at room temperature for 2 h to perform Pinnick oxidation. TLC monitoring showed that the reaction was complete. The reaction solution was rotary evaporated to remove the solvent to obtain a yellow liquid with a small amount of white solid attached to the bottle wall. 68 mL of water was added to the residual liquid and stirred. The mixture was acidified with concentrated hydrochloric acid to pH = 4. Continue stirring and a large amount of white solid appears. 50 mL of ethyl acetate was added for extraction. TLC monitoring of the organic phase and aqueous phase showed that the product was in the organic phase. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was rotary evaporated to obtain 2-chloro-8-methylquinoline-3-carboxylic acid, i.e., intermediate 2, as a white solid (1.03 g, 96%), with a melting point of 215-217°C and a structural formula of

[0067] The reaction formula of the above reaction process is as follows:

[0068]

[0069] The hydrogen nuclear magnetic spectrum of the prepared intermediate 2 is as follows: Figure 2 Its hydrogen nuclear magnetic characterization data are as follows: 1 HNMR (500 MHz, DMSO-d6) δ 13.76 (s, 1H), 8.91 (s, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.79 (d, J = 7.0 Hz, 1H), 7.61-7.63 (m, 1H), 2.68 (s, 3H).

[0070] S3. Preparation of intermediate 3:

[0071] 2-Chloro-8-methylquinoline-3-carboxylic acid (5.00 g, 22.6 mmol) was placed in a 500 mL four-necked flask under nitrogen protection, with an intermediate-to-ethyl acetoacetate molar ratio of 1:1.5. Ethyl acetoacetate (4.41 g, 33.9 mmol), sodium ethoxide (4.00 g, 58.80 mmol), and copper acetate (0.18 g, 0.84 mmol) were added sequentially, followed by anhydrous ethanol (150 mL) to form a suspension. Heating and stirring were initiated, and the oil bath temperature was set to 85 °C. The nucleophilic substitution reaction was carried out for 2 h, and TLC was used to monitor the reaction until completion. After cooling to room temperature, the reaction solution was transferred to a 1 L beaker, and 130 mL of glacial acetic acid was added to quench the alkaline system. The reaction solution was a yellow liquid. After stirring for 30 min, the ethanol and acetic acid were removed by rotary evaporation, yielding a yellow viscous liquid. 130 mL of deionized water was added, and a pink solid precipitated. After stirring for 2 h, the mixture was filtered through a sintered glass funnel, yielding a pink solid and a yellow filtrate. A small amount of the solid product was dissolved in DMF and monitored by TLC. The product was relatively pure, and no product was present in the filtrate. The solid was dried in a vacuum drying oven at 50 °C for 30 min to obtain ethyl 3-carboxy-8-methyl-2-quinoline, i.e., intermediate 3, a pink solid (6.02 g, 97%) with a melting point of 120-121 °C and the structural formula is as follows.

[0072] The reaction equation for the above reaction process is:

[0073]

[0074] The proton NMR spectrum of the prepared intermediate 3 was detected, as shown in the figure. Figure 3 Its hydrogen NMR characterization data are as follows: 1 HNMR (500MHz, CDCl3) δ8.95 (s, 1H), 7.77 (d, J = 8.0Hz, 1H), 7.68 (d, J = 6.5Hz, 1H), 7.49 (dd, J=15Hz,7.5Hz,1H),4.50(s,2H),4.23(q,J=7.5Hz,2H),2.80(s,3H),1.30(t,J=7.0Hz,3H).

[0075] S4. Preparation of intermediate 4:

[0076] Under ice-water bath conditions, 40 mL of DMF was added to a 100 mL four-necked flask. The reaction solution temperature was lowered to 2 °C. 13 mL of phosphorus oxychloride was added dropwise over 0.5 h using a disposable dropper. After the addition was complete, the reaction solution was a clear, light pink color. Then, 10.00 g (36.63 mol) of ethyl 3-carboxy-8-methyl-2-quinoline was added. The reaction solution turned orange and a large amount of solid appeared. The mixture was stirred to allow for cyclization and elimination reactions for 1.5 h. The reaction was monitored by TLC until completion. The reaction solution was then filtered to obtain an orange solid and a red filtrate. The filter cake was further washed with acetone to obtain a dark red filtrate. TLC monitoring of the filter cake and filtrate showed that acetone washed away most of the impurities, resulting in a relatively pure product. The filter cake was then dried in a vacuum drying oven at 50°C for 1 hour to obtain 4-((dimethylamino)methylene)-6-methyl-1H-pyran[4,3-b]quinoline-1,3(4H)-dione, intermediate 4, which was an orange-yellow solid (10.60 g, yield 88%) with a melting point of 217°C and the following structural formula:

[0077] The reaction equation for the above reaction process is:

[0078]

[0079] The proton NMR spectrum of the prepared intermediate 4 was detected, as follows: Figure 4 Its hydrogen NMR characterization data are as follows: 1 HNMR (500MHz, DMSO-d6) δ8.94(s,1H),8.81(s,1H),7.90(d,J=8.0Hz,1H),7.69(d,J=7.0Hz,1H),7.37(m,1H),3.60(s,3H),3.31(s,3H),2.70(s,3H).

[0080] S5. Preparation of intermediate 5:

[0081] According to the molar ratio of intermediate 4 to methylamine of 1:5, 4-((dimethylamino)methylene)-6-methyl-1H-pyran[4,3-b]quinoline-1,3(4H)-dione (0.80g, 2.84mmol) and DMF (20mL) were added to a 250mL four-necked flask. The mixture was stirred until homogeneous, but not completely dissolved. A 30-33wt% methanol solution of methylamine (1.45g, 14.2mmol) was added to the reaction solution. The mixture was stirred at room temperature, and the solid gradually dissolved. The amidation reaction was carried out at room temperature for 5h. The reaction solution changed from a red turbid liquid to a light yellow turbid liquid, and a solid product was generated. The reaction process was monitored by TLC: After taking an appropriate amount of reaction solution with a dropper, it was placed in a centrifuge tube. After centrifugation, a solid precipitate and supernatant were obtained. The supernatant was discarded, and the solid particles were washed several times with an appropriate amount of methanol. The solid particle product (DMSO slightly soluble) was mixed with the raw material (DMF dissolved) and the supernatant together and the reaction was monitored by TLC until the reaction was completed.

[0082] The reaction solution was filtered to obtain a yellow solid and a light yellow filtrate. The solid was washed with cold acetone, and the filter cake was dried under vacuum at 50°C for 0.5 h to obtain 2,6-dimethyl-1-oxo-1,2-dihydrobenzo[b][1,6]naphthyl-4-carboxylic acid, i.e., intermediate 5, which was a yellow solid (0.50 g, yield 66%) with a melting point of 328–330°C. Intermediate 5 was purified by thermal recrystallization with dimethyl sulfoxide (DMSO). The structural formula of intermediate 5 is as follows:

[0083] The reaction equation for the above reaction process is:

[0084]

[0085] The proton NMR spectrum of the prepared intermediate 5 was detected, as shown in the figure. Figure 5 Its hydrogen NMR characterization data are as follows: 1 HNMR(500MHz,DMSO-d6)δ16.03(s,1H),9.52(s,1H),8.83(s,1H),8.25(d,J=8.0 Hz,1H),7.96(d,J=6.5Hz,1H),7.68(t,J=7.0Hz,1H),3.68(s,3H),2.75(s,3H).

[0086] S6. Preparation of intermediate 6:

[0087] Add 1.37 g (5.11 mmol) of 2,6-dimethyl-1-oxo-1,2-dihydrobenzo[b][1,6]naphthyl-4-carboxylic acid and 40 mL of thionyl chloride to a 100 mL single-necked flask. Heat under reflux for acylation for 7 h. Concentrate the reaction solution under reduced pressure to remove thionyl chloride. During rotary evaporation, a small amount of toluene can be added to remove the thionyl chloride, yielding a yellow solid, 2,6-dimethyl-1-oxo-1,2-dihydrobenzo[b][1,6]naphthyl-4-carboxylic chloride, i.e., intermediate 6, with the structural formula […].

[0088] The reaction equation for the above reaction process is:

[0089]

[0090] S7. Preparation of compound A:

[0091] Add 80 mL of dichloromethane to intermediate 6 to dissolve 2,6-dimethyl-1-oxo-1,2-dihydrobenzo[b][1,6]naphthyl-4-formyl chloride, and then add 0.10 mol of an amine compound. (That is, the ratio of intermediate 6 to amine compound is 80 mL: 0.1 mol). The mixture was stirred at room temperature for 8 h. The reaction was monitored by TLC until the reactants were completely reacted. A portion of the solvent was removed by rotary evaporation. The remaining reactants were directly purified by neutral alumina column chromatography with ethyl acetate-methanol gradient elution to obtain benzo[b][1,6]naphthidine-4-carboxamide derivative, denoted as compound A.

[0092] The proton NMR spectrum of compound A was obtained by detection, as follows: Figure 6 The proton NMR characterization data of compound A are as follows: 1 HNMR (400MHz, CDCl3) δ10.80(s,1H),9.35-9.18(m,1H),8.65(s,1H),7.90(d,J=8.4Hz,1H),7.76(d,J=6.8Hz,1H),7.55-7 .42(m,1H),4.40(s,1H),3.78-3.57(m,3H),3.03(s,2H),2.90(s,3H),2.68(m,2H),2.33(m,3H),2.16(s,2H),2.02(s,2H).

[0093] The carbon NMR spectrum of the prepared compound A was detected, as follows: Figure 7 The carbon NMR characterization data of compound A are as follows: 13CNMR (100MHz, CDCl3) δ163.85,162.94,148.99,148.41,143.93,140.04,135.96,133.01,127.5 0,126.67,126.06,124.23,121.51,119.41,109.66,60.91,55.67,46.38,37.21,30.19,19.23.

[0094] Example 2

[0095] The only difference between this embodiment and Example 1 is step S7, in which the amine compound is replaced with the same amount of substance. The other preparation methods were the same as in Example 1, yielding a benzo[b][1,6]naphthidine compound, denoted as Compound B, with the structural formula […].

[0096] The proton NMR spectrum of the prepared compound B was detected, as follows: Figure 8 The proton NMR characterization data of compound B are as follows: 1 HNMR (400MHz, CDCl3) δ11.12(s,1H),9.35-9.23(m,1H),8.62(s,1H),7.91(d,J=8.4Hz,1H),7.77(d,J=6.8Hz,1H),7.52(dd, J=15.2,8.0Hz,1H),3.72(s,3H),3.67-3.55(m,2H),2.84(s,3H),2.46(d,J=36Hz,6H),1.98(s,2H),1.51(d,J=66.8Hz,6H).

[0097] The carbon NMR spectrum of the prepared compound B was detected, as follows: Figure 9 The carbon NMR characterization data of compound B are as follows: 13 CNMR (100MHz, CDCl3) δ164.54,162.89,148.87,148.17,143.67,139.95,135.66,133.07,127. 48,126.74,126.00,119.42,109.47,56.99,54.57,44.15,38.05,37.26,25.77,24.30,18.82.

[0098] Example 3

[0099] The only difference between this embodiment and Example 1 is step S7, in which the amine compound is replaced with the same amount of substance. The other preparation methods were the same as in Example 1, yielding a benzo[b][1,6]naphthidine compound, denoted as compound C, with the structural formula […].

[0100] The proton NMR spectrum of the prepared compound C was obtained, as shown below. Figure 10 The proton NMR characterization data of compound C are as follows: 1 HNMR (400MHz, CDCl3) δ11.03(s,1H),9.35-9.12(m,1H),8.61-8.50(m,1H),7.89(d,J=8.0Hz,1H),7.75(d,J=6.8Hz,1H),7.52(dd,J=8.0, 2.8Hz,1H),3.72-3.71(m,3H),3.65-3.54(m,2H),3.09(t,J=7.2Hz,1H),2.83(s,3H),2.36(s,3H),2.25-1.97(m,4H),1.93-1.41(m,4H).

[0101] The carbon NMR spectrum of the prepared compound C was obtained, as shown in the figure. Figure 11 The carbon NMR characterization data of compound C are as follows: 13 CNMR (100MHz, CDCl3) δ164.40,162.83,148.83,148.11,143.69,139.89,135.57,133.03,127.49, 126.69,125.96,119.38,109.38,99.99,64.50,57.19,40.43,37.41,34.14,30.93,21.89,18.85.

[0102] Comparative Example 1

[0103] The difference between this comparative example and Example 1 is in step S7, where the amine compound is replaced with the same amount of substance. The other preparation methods were the same as in Example 1, yielding a benzo[b][1,6]naphthidine compound, denoted as Compound D, with the structural formula […].

[0104] The proton NMR spectrum of the prepared compound D was obtained, as shown below. Figure 12The proton NMR characterization data of compound D are as follows: ¹H NMR (400 MHz, CDCl₃) δ 11.13 (d, J = 6.4 Hz, ¹H), 9.30 (s, ¹H), 8.61 (s, ¹H), 7.89 (d, J = 8.0 Hz, ¹H), 7.76 (d, J = 6.8 Hz, ¹H), 7.55–7.48 (m, ¹H), 3.72 (s, ³H), 3.70–3.65 (m, ¹H), 2.90 (s, ³H), 2.87–2.76 (m, 4H), 2.53–2.46 (m, 2H), 2.43 (s, ³H).

[0105] The carbon NMR spectrum of the prepared compound D was detected, as follows: Figure 13 The carbon NMR characterization data of compound D are as follows: 13CNMR (100MHz, CDCl3) δ 163.91, 162.90, 148.81, 148.31, 143.71, 139.98, 136.00, 133.04, 127.45, 126.70, 126.03, 109.48, 62.86, 55.22, 49.44, 42.02, 37.25, 33.19, 19.01.

[0106] The schematic diagram of step S7 of the present invention for preparing compounds A, B, C and D is shown below. Figure 14 It can be seen that the side chain structures of benzo[b][1,6]naphthidine-4-carboxamide derivatives prepared by different amine compounds are different.

[0107] Effect verification example

[0108] The cell inhibitory activity of benzo[b][1,6]naphthidine compound SN28049, known to inhibit topoisomerases I and II and possess anticancer effects, as well as compounds A, B, C, and D prepared in Examples 1-3 and Comparative Example 1, on human colon cancer cells (HCT116) was detected. Cancer cell activity was assessed using the MTT assay, and absorbance was measured at 492 nm and 530 nm using an enzyme-linked immunosorbent assay (ELISA) scanner. The number of viable cells was indirectly quantitatively assessed, and the results were analyzed using IC50. 50 The value represents the half-inhibition concentration (the concentration at which the test compound achieves a 50% inhibition effect on HCT116).

[0109] The results of the anticancer activity detection of each compound are shown in Table 1, and the inhibition rate log fitting curves are shown in [Table 1]. Figures 15 to 17 .

[0110] Table 1. Results of the inhibitory effect of the compounds on cancer cells.

[0111] Compound No. Preparation method IC 50 values (nM) SN28049 / 12.96 Compound A Example 1 3.43 Compound B Example 2 6.24 Compound C Example 3 11.06 Compound D Comparative Example 1 20375.17

[0112] The results showed that the IC50 of SN28049, as measured using HCT116 cells, was... 50 The value was 12.96 nM. The anticancer activities of compounds A, B, and C were all higher than those of SN28049, with an IC50 value of 12.96 nM. 50 The values ​​are 3.43 nM, 6.24 nM, and 11.06 nM, respectively.

[0113] Figures 15 to 17 The log-fit curves of inhibition rates for compounds A, B, and C are shown in order. The results indicate that compounds A, B, and C all possess good anti-cancer cell growth activity. This demonstrates that the side chain structure linked by the amide bond directly affects the compound activity: higher activity is observed when the side chain is N-methylpiperidin-3-amine or 1-(3-aminopropyl)piperidine, while significantly reduced activity occurs when it is N-methyl-2-(2-aminoethyl)-pyrrolidine. The mechanism lies in the cis-trans isomerism of the amide bond; the volume and rigidity of the side chain affect the conformational flexibility and target fit through steric hindrance, ultimately determining the activity level. Therefore, compound A has the optimal conformation and highest activity, while compound C exhibits the lowest activity.

[0114] Figure 18 The figure shows the log-fit curve of the inhibition rate of compound D. Compared with Example 2, the results of Comparative Example 1 show that the compound containing 1-(3-aminopropyl)piperidine amide group is 3000 times more active than the compound containing 1-(3-aminopropyl)pyrrolidine.

[0115] The above results indicate that the benzo[b][1,6]naphthidine compounds of the present invention, compared with the similar compound SN28049, exhibit more prominent inhibitory activity against cancer cells due to the presence of a special R substituent. They effectively inhibit the activity of topoisomerases I and II and can interfere with DNA replication and transcription processes, demonstrating a synergistic anti-tumor effect. Therefore, they are suitable for the preparation of topoisomerase inhibitors or anti-tumor drugs.

[0116] In other embodiments, acetyl-o-toluidine and (chloromethylene)dimethylammonium chloride were subjected to a cyclization reaction at a reaction temperature of 70°C or 100°C for a reaction time of 4 or 5 days to obtain intermediate 1. The molar ratio of intermediate 1 to sodium dihydrogen phosphate monohydrate and sodium chlorite was 1:6:10 or 1:7:9; the molar ratio of 2,6-dimethyl-1-oxo-1,2-dihydrobenzo[b][1,6]naphthyl-4-carboxyl chloride to amine compounds was 70 mL:0.1 mol or 90 mL:0.1 mol. In both cases, benzo[b][1,6]naphthyl chloride compounds were successfully prepared, and the substituent R of the obtained benzo[b][1,6]naphthyl chloride compounds had the general formula C. n H 2n+1N and n are integers not less than 6, such as 6, 7, 8, or 9, and the nitrogen-containing heterocyclic structure in the substituent R is a nitrogen-containing five-membered ring or a six-membered ring.

[0117] In other embodiments, in step S3, the reaction temperature of the nucleophilic substitution reaction is 80°C, 90°C, or 95°C, and in step S4, the reaction temperature of the cyclization and elimination reactions is 0°C, 6°C, or 10°C. The resulting benzo[b][1,6]naphthidine compounds, after testing, also exhibit antitumor activity similar to that of SN28049 in Examples 1-3, and can be used as pharmaceutical intermediates or active ingredients to prepare topoisomerase inhibitors or antitumor drugs.

[0118] It should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still modify the technical solutions described in the above embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A benzo[b][1,6]naphthidine compound, characterized in that, It has the following structural formula: In the formula, R is a substituent group with a nitrogen-containing heterocyclic structure, with the general formula C1. n H 2n+1 N, n≥6 and are integers.

2. A benzo[b][1,6]naphthidine compound according to claim 1, characterized in that, The nitrogen-containing heterocyclic structure is a nitrogen-containing five-membered ring or a six-membered ring.

3. A benzo[b][1,6]naphthidine compound according to claim 1 or 2, characterized in that, R is One of them.

4. A method for preparing a benzo[b][1,6]naphthidine compound according to any one of claims 1 to 3, characterized in that, It is prepared by acylation reaction of 2,6-dimethyl-1-oxo-1,2-dihydrobenzo[b][1,6]naphthyl-4-carboxyl chloride with amine compounds.

5. The method for preparing a benzo[b][1,6]naphthidine compound according to claim 4, characterized in that, The amine compound is 6. The method for preparing a benzo[b][1,6]naphthidine compound according to claim 5, characterized in that, The ratio of 2,6-dimethyl-1-oxo-1,2-dihydrobenzo[b][1,6]naphthidine-4-carboxyl chloride to amine compounds is 70-90 mL: 0.1 mol.

7. A method for preparing a benzo[b][1,6]naphthidine compound according to claim 5 or 6, characterized in that, The method for preparing the 2,6-dimethyl-1-oxo-1,2-dihydrobenzo[b][1,6]naphthyl-4-formyl chloride includes the following steps performed sequentially: S1. Acetyl o-toluidine and (chloromethylene)dimethylammonium chloride were subjected to a cyclization reaction to obtain intermediate 1; S2. Take intermediate 1, sodium dihydrogen phosphate monohydrate and sodium chlorite, and perform Pinnick oxidation reaction to obtain intermediate 2; S3. Take intermediate 2 and ethyl acetoacetate, and carry out a nucleophilic substitution reaction to obtain intermediate 3; S4. Intermediate 3 and (chloromethylene)dimethylammonium chloride are subjected to cyclization and elimination reactions to obtain intermediate 4; S5. Take intermediate 4 and methylamine, and react them via amidation to obtain intermediate 5; S6. Intermediate 5 is reacted with thionyl chloride at reflux temperature to obtain the 2,6-dimethyl-1-oxo-1,2-dihydrobenzo[b][1,6]naphthidine-4-formyl chloride.

8. The method for preparing a benzo[b][1,6]naphthidine compound according to claim 7, characterized in that, In step S1, the cyclization reaction is carried out at a temperature of 70–100°C for 4–5 days. In step S2, the molar ratio of intermediate 1 to sodium dihydrogen phosphate monohydrate and sodium chlorite is 1:6-7:9-10; the reaction temperature of the Pinnick oxidation reaction is room temperature, and the reaction time is 2 hours. In step S3, the molar ratio of intermediate 2 to ethyl acetoacetate is 1:1.5; the reaction temperature of the nucleophilic substitution reaction is 80-95℃, and the reaction time is 2h.

9. The method for preparing a benzo[b][1,6]naphthidine compound according to claim 7, characterized in that, In step S4, the reaction temperature for the cyclization and elimination reactions is 0–10 °C, and the reaction time is 1.5 h. In step S5, the molar ratio of intermediate 4 to methylamine is 1:5; the amidation reaction is carried out at room temperature for 5 hours. In step S6, the reaction time for the acylation reaction is 7 hours.

10. The use of any one of the benzo[b][1,6]naphthidine compounds according to claims 1 to 3, characterized in that, Used to prepare topoisomerase inhibitors or antitumor drugs.

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