CN113185512B

CN113185512B - 5-aminopyrimido [4,5-b ] quinoline compound and preparation method and application thereof

Info

Publication number: CN113185512B
Application number: CN202110536683.2A
Authority: CN
Inventors: 黄婉云; 郑彬; 李芳耀; 周异欢; 徐燕艺; 梁云婷; 李瞻豪
Original assignee: Guilin Medical University
Current assignee: Guilin Medical University
Priority date: 2021-05-17
Filing date: 2021-05-17
Publication date: 2023-05-05
Anticipated expiration: 2041-05-17
Also published as: CN113185512A

Abstract

The invention provides a 5-aminopyrimido [4,5-b ]]Quinoline compound, preparation method and application thereof, and 5-aminopyrimido [4,5-b ]]Quinoline compounds have a general formula of

The invention recombines two structural advantage units of pyrimidine and quinoline, develops a series of 5-aminopyrimido [4,5-b ] by taking benzamidine hydrochloride, 2-cyano-3-ethoxyacrylic acid ethyl ester and the like as initial raw materials and performing four-step reactions such as halogenation, substitution reaction and the like]Quinoline compounds, and early research shows that the quinoline compounds have good anti-tumor activity in vitro, and 4g of the quinoline compounds have obvious topoisomerase I inhibition activity.

Description

5-aminopyrimido [4,5-b ] quinoline compound and preparation method and application thereof

Technical Field

The invention relates to the technical field of pharmaceutical chemistry, in particular to a 5-aminopyrimido [4,5-b ] quinoline derivative, a preparation method thereof and application of the compound in preparation of antitumor drugs.

Background

Cancer is reported to be the leading cause of death in countries around the world and the most important obstacle to increasing life expectancy. Currently, methods for treating cancer are various, but the use of chemotherapeutic drugs is still one of the main means for dealing with cancer treatment. The DNA topoisomerase I (Topo I) is used as an important anti-tumor target, and is used for designing, synthesizing and screening an anti-tumor new chemical entity with good Topo I inhibition activity, so that the application of the important target in the research and development of anti-cancer new drugs is further excavated, and the important target has important research significance.

The dominant structure theory is an important theory for guiding the development of new drugs and the design of bioactive compounds. Pyrimidine and quinoline are very important dominant structural units, and their derivatives have very important biological and pharmacological activities, and research reports on synthesizing heterocyclic compounds containing pyrimidine and quinoline structures are frequent. There are three main ways of fusing the heterocyclic compounds of the pyrimidoquinoline class, as shown in Figure 1:

depending on the manner of fusion, these can be classified into pyrimido [4,5-b ] quinolines, pyrimido [4,5-c ] quinolines, and pyrimido [5,4-c ] quinolines. Pyrimido [4,5-b ] quinolines are of particular interest to pharmaceutical chemists because of their structural similarity to natural flavins.

Referring to the literature, the synthesis and biological activity reports of pyrimido [4,5-b ] quinoline skeleton compounds are less. Development of pyrimido [4,5-b ] quinoline skeleton compounds has important significance in development of antitumor drugs.

Disclosure of Invention

The invention provides a 5-aminopyrimido [4,5-b ] quinoline compound, a preparation method and application thereof, wherein two structural advantage units of pyrimidine and quinoline are recombined, a series of 5-aminopyrimido [4,5-b ] quinoline compounds are synthesized by taking benzamidine hydrochloride, 2-cyano-3-ethoxyacrylic acid ethyl ester and the like as initial raw materials through four-step reactions such as halogenation, substitution and the like, and early researches show that the compounds have good anti-tumor activity in vitro.

In order to achieve the above purpose, the technical scheme of the invention is as follows:

the present invention provides 5-aminopyrimido [4,5-b ] quinolines of the general formula (I):

wherein: r is R ₁ Selected from methyl, aryl, methoxy or isopropyl.

Preferably, ar is selected from phenyl or 4-chlorophenyl.

Preferably, the compound is specifically:

the invention also provides a preparation method of the compound, which comprises the following steps:

s1, will

And 2-cyano-3-ethoxyacrylic acid ethyl ester under alkaline condition to synthesize compound 1

S2, halogenating the compound 1 to obtain a compound 2

/>

S3, adding the compound 2 into a reaction container,

And toluene, to obtain compound 3

S4, adding the compound 3, dichloroethane and trifluoromethanesulfonic acid into a reaction container, and reacting to obtain the 5-aminopyrimido [4,5-b ] quinoline compound shown in the general formula (I).

Preferably, the step S1 specifically includes: adding into a reaction vessel

The 2-cyano-3-ethoxyacrylic acid ethyl ester, naOH and EtOH react at 100-110 ℃ to prepare the compound 1.

Preferably, the step S2 specifically includes: the compound 1, N-dimethylaniline and POCl are added into a reaction vessel ₃ And (3) reacting at 105-120 ℃ to obtain the compound 2.

Preferably, in the step S3, the reaction temperature is 105 to 120 ℃.

Preferably, in the step S4, the reaction temperature is 55 to 70 ℃.

The preparation route of the invention is shown in Scheme 1:

the invention also provides application of the compound or the compound prepared by the preparation method in preparation of antitumor drugs or topoisomerase I activity inhibitors.

Further, the tumor is a tumor caused by nasopharyngeal carcinoma, liver cancer, gastric cancer, bladder cancer or lung cancer.

The invention synthesizes 5-aminopyrimido [4,5-b ] quinoline compounds with the general formula (I), and provides a preparation method thereof, and the 5-aminopyrimido [4,5-b ] quinoline compounds with the general formula (I) have good inhibition effect on tumor cells through in-vitro inhibition tests on human nasopharyngeal carcinoma (CNE-2), human liver cancer (HepG-2), human gastric cancer (MGC-803), human bladder cancer (T-24) or human lung cancer (NCI-H460).

The 5-aminopyrimido [4,5-b ] quinoline compound with the general formula (I) can be directly used, or can be combined with pharmaceutically acceptable auxiliary materials to form a pharmaceutical composition, wherein the pharmaceutical composition can be granules, capsules, spray, drops, injection, medicinal granules and the like, and can also have good inhibition effect on human nasopharyngeal carcinoma (CNE-2), human liver cancer (HepG-2), human gastric cancer (MGC-803), human bladder cancer (T-24) or human lung cancer (NCI-H460), thereby providing more choices for development of antitumor medicaments.

Meanwhile, the experiment proves that 4g of the compound has obvious inhibition activity to topoisomerase I, and a wider thought is provided for the research and development of new antitumor drugs.

Drawings

FIG. 1 is a gel electrophoresis chart of the inhibition of Topo I by compound 4g.

Detailed Description

The present invention is further illustrated with reference to the following examples, but the scope of the present invention is not limited to the following examples.

The compounds involved in this example are shown below:

example 1

Under electromagnetic stirring, the compound benzamidine hydrochloride (0.60 g,5 mmol), ethyl 2-cyano-3-ethoxyacrylate (0.845 g,5 mmol), naOH (0.2 g,5 mmol) and EtOH (60 mL) were added sequentially to a 100mL round bottom flask and reacted at 100deg.C for 4h (monitored by TLC, developer: V _EA :V _PE =1:2), and after the reaction was completed, the filtrate was filtered with a buchner funnel to give 6.1g of compound 1a as a white solid after being added to concentrated hydrochloric acid to neutralize most of NaOH

Yield: 61.9%. />

Compound 1a (2.0 g,10 mmol), N-dimethylaniline (1.5 mL) and POCl were stirred electromagnetically ₃ (15 mL) was added sequentially to a 100mL round-bottomed flask and reacted at 110℃for 12 hours (the reaction was monitored by TLC, developer: V) _EA :V _PE =1:1). Distilling off most of POCl ₃ Then adding ice water into the reaction bottle, stirring for 3-5 min to separate out most solid compounds, and suction filtering to obtain 1.8g of gray solid compound 2a

Yield: 81%.

In the electric fieldUnder magnetic stirring, compound 2a (0.2 g,1 mmol) and p-toluidine (0.373 g,4 mmol) were added sequentially to a 100mL round bottom flask, and after stirring, the mixture was reacted at 110℃for 1.5h (monitoring the reaction by TLC, developer: V) _EA :V _PE =1:2). After the reaction, the solvent is removed by rotary evaporation under reduced pressure, and the crude product is purified by silica gel column chromatography (eluent: V) _DCM ) And adding dichloromethane and petroleum ether of two times to recrystallize and separate out white solid. Then suction filtration gave 0.198g of compound 3a as a white solid

Yield: 69.2%.

Compound 3a (0.198 g,1 mmol), DCE (8 mL), trifluoromethanesulfonic acid (1.5 mL) were added sequentially to a 100mL round bottom flask with electromagnetic stirring and reacted at 60℃for 3h (monitored by TLC, developer: V) _DCM :V _EA :V _MeOH =9:3:1). After the reaction is finished, the solvent is removed under reduced pressure, then a proper amount of saturated sodium bicarbonate solution is added into a reaction bottle, the mixture is stirred for 3 to 5 minutes to separate out most of solid compounds, and the solid compounds are filtered by suction to obtain 0.128g of yellow solid compound 4a, and the yield is: 86.7%.

7-methyl-2-phenylpyrimido[4,5-b]quinolin-5-amine(4a)Yellow solid,yield 86.7％,m.p.281.0～283.0℃；IR(KBr),v/cm ^-1 :3466,3385,3304,3215,3064,2985,1669,1623,1590,1571,1505,869,824,782,727,707,665,638. ¹ H NMR(400MHz,DMSO)δ9.96(s,1H),8.55–8.52(m,1H),8.51(d,J＝2.4Hz,1H),8.22(s,1H),7.71(s,2H),7.58(d,J＝7.1Hz,3H),2.45(s,3H). ¹³ C NMR(100MHz,DMSO)δ164.5,159.8,156.3,154.3,145.6,137.3,137.1,133.8,132.2,129.3,129.1,124.6,123.5,114.7,104.5,21.5.HRMS(ESI ⁺ )m/z calcd for C ₁₈ H ₁₅ N ₄ [M+H] ⁺ 287.1297,found 287.1279.

Example 2

By using

The synthesis method is the same as that of example 1 except that p-toluidine is replaced, so that the compound 4b is obtained.

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7,9-dimethyl-2-phenylpyrimido[4,5-b]quinolin-5-amine(4b)Yellow solid,yield 76.1％,m.p.274.0～277.0℃；IR(KBr),v/cm ^-1 :3347,3190,1687,1620,1595,1576,1519,860,821,781,763,732,708,691,636. ¹ H NMR(400MHz,DMSO)δ9.97(s,1H),8.56(dd,J＝7.8,1.5Hz,2H),8.07(s,1H),7.60(m,J＝13.9,6.2Hz,4H),2.64(s,3H),2.41(s,3H). ¹³ C NMR(100MHz,DMSO)δ165.0,159.8,157.1,153.6,143.0,138.2,137.0,133.8,132.5,131.7,129.3,129.5,121.4,114.4,104.1,21.4,18.9.HRMS(ESI ⁺ )m/z calcd for C ₁₉ H ₁₇ N ₄ [M+H] ⁺ 301.1454,found 301.1437.

Example 3

By using

The synthesis method is the same as that of example 1 except that p-toluidine is replaced, so that the compound 4c is obtained.

10-phenylbenzo[h]pyrimido[4,5-b]quinolin-7-amine(4c).Yellow solid,yield 69.6％,m.p.>300℃；IR(KBr),v/cm ^-1 :3345,3189,1686,1631,1614,1599,1553,1520,818,792,772,738,693,639. ¹ H NMR(400MHz,DMSO)δ10.15(s,1H),9.34(d,J＝8.3Hz,1H),8.65(d,J＝6.6Hz,2H),8.38(d,J＝9.2Hz,1H),8.16(d,J＝7.9Hz,1H),8.06(d,J＝9.1Hz,1H),7.92(dt,J＝14.8,7.1Hz,2H),7.75–7.59(m,3H). ¹³ C NMR(100MHz,DMSO)δ166.3,159.7,158.4,152.9,141.2,136.3,136.3,133.3,132.1,129.6,129.6,129.5,128.6,126.6,125.3,123.2,119.9,111.3,105.7.HRMS(ESI ⁺ )m/z calcd for C ₂₁ H ₁₅ N ₄ [M+H] ⁺ 323.1298,found 323.1281.

Example 4

The compound 4d is obtained by substituting o-toluidine for p-toluidine and synthesizing the same method as in example 1.

9-Methyl-2-phenylbenzo[g]quinazolin-5-amine(4d).Yellow solid,yield 72.2％,m.p.>300℃；IR(KBr),v/cm ^-1 :3345,3181,1667,1594,1577,1519,825,767,706,677,623. ¹ HNMR(400MHz,DMSO-d ₆ )δ10.08(s,1H),8.62(dd,J＝6.6,2.9Hz,2H),8.31(d,J＝8.5Hz,1H),7.69(d,J＝6.7Hz,1H),7.62–7.54(m,3H),7.33(dd,J＝8.3,7.1Hz,1H),2.73(s,3H). ¹³ C NMR(100MHz,DMSO-d ₆ )δ163.3,160.0,160.0,154.8,152.2,138.2,136.7,133.0,131.5,129.1,129.0,122.9,122.0,114.7,104.3,19.4.HRMS(ESI ⁺ )m/z calcd for C ₁₈ H ₁₅ N ₄ [M+H] ⁺ 287.1298,found 287.1282.

Example 5

By using

The synthetic method is the same as that of example 1 to obtain the compound 4e.

7,8-dimethoxy-2-phenylpyrimido[4,5-b]quinolin-5-amine(4e).Yellow solid,yield 71.4％,m.p.305～307℃；IR(KBr),v/cm ^-1 :3374,3150,2835,1681,1628,1593,1575,850,816,783,749,701,661,614. ¹ H NMR(400MHz,DMSO)δ9.99(s,1H),8.61–8.54(m,2H),7.73(s,1H),7.55(dd,J＝5.3,1.8Hz,3H),7.23(s,1H),3.96(s,3H),3.93(s,3H). ¹³ C NMR(100MHz,DMSO)δ162.3,159.1,155.9,155.6,152.0,151.2,148.1,138.5,131.2,129.0,128.7,109.3,107.6,104.3,102.3,56.5,56.2.HRMS(ESI ⁺ )m/z calcd for C ₁₉ H ₁₇ O ₂ N ₄ [M+H] ⁺ 333.1353,found 333.1333.

Example 6

The compound 4f was obtained by substituting m-isopropylaniline for p-toluidine and by the same method as in example 1.

8-isopropyl-2-phenylpyrimido[4,5-b]quinolin-5-amine(4f).Yellow solid,yield 81.1％,m.p.>300℃；IR(KBr),v/cm ^-1 :3336,3169,2975,1686,1630,1596,1560,1514,882,819,780,704,689,667,635. ¹ H NMR(400MHz,DMSO)δ10.07(s,1H),8.58–8.51(m,3H),7.76–7.57(m,5H),3.15(dt,J＝13.7,6.8Hz,1H),1.34(s,3H),1.32(s,3H). ¹³ C NMR(100MHz,DMSO)δ166.6,160.0,159.4,159.2,152.6,141.4,136.1,133.3,129.6,129.4,125.9,125.2,116.3,112.5,104.6,34.4,23.5.HRMS(ESI ⁺ )m/z calcd for C ₂₀ H ₁₉ N ₄ [M+H] ⁺ 315.1611,found 315.1593.

Example 7

By using

The synthesis method was the same as that of example 1 except that p-toluidine was replaced, to obtain 4g of a compound. />

7-methoxy-8-methyl-2-phenylpyrimido[4,5-b]quinolin-5-amine(4g).Yellow solid,yield69.2％,m.p.288.0～290.0℃；IR(KBr),v/cm ^-1 :3377,3211,1684,1624,1594,1577,874,848,819,777,730,708,693,639. ¹ H NMR(400MHz,DMSO)δ9.97(s,1H),8.59–8.49(m,2H),7.74–7.52(m,5H),3.93(s,3H),2.35(s,3H). ¹³ C NMR(100MHz,DMSO)δ163.7,159.5,155.3,154.5,153.7,138.1,137.5,132.0,129.2,129.0,122.83,113.6,104.0,101.4,100.0,56.6,17.6.HRMS(ESI ⁺ )m/z calcd for C ₁₉ H ₁₇ ON ₄ [M+H] ⁺ 317.1404,found 317.1387.

Example 8

The benzamidine hydrochloride is replaced by p-chlorobenzamidine hydrochloride, the p-toluidine is replaced by o-methoxyaniline, and the compound 4h is obtained by the synthesis method same as that of the example 1.

2-(4-chlorophenyl)-9-methoxypyrimido[4,5-b]quinolin-5-amine(4h).Yellow solid,yield 76.2％,m.p.274.0～277.0℃；IR(KBr),v/cm ^-1 :3371,3148,1689,1624,1587,1558,1516,852,793,759,732,711,680,656,627. ¹ H NMR(400MHz,DMSO)δ10.11(s,1H),8.56(d,J＝8.6Hz,2H),8.15(d,J＝7.8Hz,1H),7.73–7.56(m,4H),4.08(s,3H). ¹³ C NMR(100MHz,DMSO)δ165.4,160.3,159.7,152.3,149.2,138.3,135.0,132.1,131.2,129.6,125.8,116.4,116.3,115.0,104.8,57.1.HRMS(ESI ⁺ )m/z calcd for C ₁₈ H ₁₄ ClON ₄ [M+H] ⁺ 337.0857,found 337.0840.

Example 9

By using

The o-methoxyaniline is replaced, and the synthesis method is the same as that of the example 8, so that the compound 4i is obtained.

2-(4-chlorophenyl)-7,9-dimethylpyrimido[4,5-b]quinolin-5-amine(4i).Yellow solid,yield 79.1％,m.p.269.0～270.0℃；IR(KBr),v/cm ^-1 :3343,3175,1666,1589,1516,848,810,781,733,636. ¹ H NMR(400MHz,DMSO)δ9.99(s,1H),8.56(d,J＝8.6Hz,2H),8.04(s,1H),7.61(d,J＝8.6Hz,2H),7.52(s,1H),2.66(s,3H),2.44(s,3H). ¹³ C NMR(100MHz,DMSO)δ162.2,160.0,154.9,154.4,149.4,136.9,136.4,135.8,135.7,132.4,130.6,129.2,120.6,114.6,104.3,21.7,19.2.HRMS(ESI ⁺ )m/z calcd for C ₁₉ H ₁₆ ClN ₄ [M+H] ⁺ 335.1064,found 335.1046.

Example 10

The compound 4j is obtained by replacing o-methoxyaniline with m-isopropylaniline and synthesizing method as same as example 8.

2-(4-chlorophenyl)-8-isopropylpyrimido[4,5-b]quinolin-5-amine(4j).Yellow solid,yield 72.8％,m.p.298.0～300.0℃；IR(KBr),v/cm ^-1 :3312,3230,2960,1632,1589,1500,884,854,798,718,680,652. ¹ HNMR(400MHz,DMSO)δ9.98(s,1H),8.56(d,J＝8.6Hz,2H),8.38(d,J＝8.8Hz,1H),7.62(d,J＝8.5Hz,3H),7.36(dd,J＝8.7,1.2Hz,1H),3.07(dt,J＝13.7,6.8Hz,1H),1.32(s,3H),1.31(s,3H). ¹³ C NMR(100MHz,DMSO)δ162.3,159.8,156.6,154.8,154.1,137.1,136.3,130.5,129.2,124.7,124.4,123.3,113.8,104.5,100.2,34.1,23.8.HRMS(ESI ⁺ )m/z calcd for C ₂₀ H ₁₈ ClN ₄ [M+H] ⁺ 349.1221,found 349.1204.

Example 11

The o-methylaniline is used for replacing o-methoxyaniline, and the compound 4k is obtained by the synthesis method in the same way as in the example 8.

2-(4-chlorophenyl)-9-methylpyrimido[4,5-b]quinolin-5-amine(4k).Yellow solid,yield 77.5％,m.p.287.0～290.0℃；IR(KBr),v/cm ^-1 :3375,3226,2986,1682,1627,1589,1553,1518,855,812,793,755,731,637. ¹ H NMR(400MHz,DMSO)δ10.03(s,1H),8.55(d,J＝8.6Hz,2H),8.39(d,J＝8.4Hz,1H),7.87(d,J＝7.0Hz,1H),7.66(d,J＝8.6Hz,2H),7.52(t,J＝7.8Hz,1H),2.69(s,3H). ¹³ C NMR(100MHz,DMSO)δ165.0,160.1,159.2,153.3,137.9,137.7,135.3,131.1,130.2,129.5,125.0,123.2,122.8,114.6,104.5,18.9.HRMS(ESI ⁺ )m/z calcd for C ₁₈ H ₁₄ ClN ₄ [M+H] ⁺ 321.0908,found 321.0893.

Example 12

By using

The synthesis method is the same as that of example 8 except that o-methoxyaniline is replaced, so that the compound 4l is obtained. />

10-(4-chlorophenyl)benzo[h]pyrimido[4,5-b]quinolin-7-amine(4l).Yellow solid,yield 72.4％,m.p.>300℃；IR(KBr),v/cm ^-1 :3444,3327,3236,1649,1617,1587,1570,1521,852,834,798,760,701,672,634. ¹ H NMR(400MHz,DMSO)δ10.12(s,1H),9.33(dd,J＝7.7,1.1Hz,1H),8.64(d,J＝8.7Hz,2H),8.28(d,J＝9.2Hz,1H),8.01–7.96(m,1H),7.83–7.72(m,3H),7.69–7.64(m,2H). ¹³ C NMR(100MHz,DMSO)δ162.4,159.5,156.6,153.4,152.4,137.1,136.4,135.3,131.3,130.7,130.1,129.3,128.2,127.2,126.4,124.2,120.8,111.1,106.1.HRMS(ESI ⁺ )m/z calcd for C ₂₁ H ₁₄ ClN ₄ [M+H] ⁺ 357.0908,found 357.0887.

Effect verification example 1: the MTT method screens compounds for antitumor activity:

(1) The ultra-clean bench and the main appliances are irradiated by an ultraviolet lamp for at least 30min before being used, the ultra-clean bench and the main appliances are wiped by alcohol cotton balls once before and after being used, 75% alcohol is used for disinfection before all articles are taken into the ultra-clean bench, and the articles are sterilized by fire before and after being used, so that a sterile environment is ensured.

(2) Culturing and detecting human gastric cancer cell (MGC-803), human bladder cancer cell (T24), human nasopharyngeal carcinoma cell (CNE-2), human lung cancer cell (NCI-H460), human liver cancer cell (HepG-2), human normal liver cell (HL-7702) to logarithmic phase, digesting with trypsin, blowing with fresh culture solution to obtain single cell suspension, adding appropriate culture mediumThe whole cell suspension concentration, taking a proper amount of cell solution, adding a certain amount of culture medium for dilution (10 mu L of cell solution is taken, 90 mu L of culture medium is added, namely 10 times dilution is carried out), counting (5000 cells are inoculated in each hole), then inoculating the diluted and uniformly mixed cell solution into a 96-well plate by a row gun, 90 mu L of each hole volume, adding sterile PBS (100 mu L) at the edge of the 96-well plate in a row to reduce the evaporation of medicines and the culture medium so as to reduce errors, and placing the 96-well plate inoculated with the cells at 37 ℃ and 5% CO ₂ Culturing in an incubator.

(3) Weighing a certain mass of compound, calculating the volume of DMSO (preferably special for dispensing to avoid bacterial contamination) required by preparing 20mM, adding the compound to be tested into 96-well plates planted in different tumor cells when cells are grown to 70-80% by adhering to the wall, enabling the final concentration of each well to be 20 mu M, arranging 5 compound wells in each group, performing primary screening, adding 10 mu L of DMSO culture medium with the content of 0.1% into each well of a control group, and continuously culturing for 24 hours. mu.L of MTT (5 mg/mL) solution was added to each well and the culture was continued for 4 hours.

(4) The culture was terminated, the culture medium in the wells was carefully discarded, 100. Mu.L of DMSO was added to each well, and the mixture was subjected to micro shaking by a shaker at a low speed for 5min to sufficiently dissolve the crystals. Absorbance values for each well were measured using an enzyme-linked immunosorbent assay, OD values at a wavelength of 490 nm. Compound with better effect is selected to carry out rescreening with different concentration gradients after preliminary screening, and IC is calculated ₅₀ 。IC ₅₀ Refers to a 50% inhibition concentration, i.e., the concentration at which the ratio of apoptotic cells to total cell number is equal to 50%. IC (integrated circuit) ₅₀ The value can be used to measure the ability of a drug to induce apoptosis, i.e., the stronger the induction, the more IC ₅₀ The lower the value. The lower the number, the more resistant a cell can of course be to the drug.

(5) Inhibition rate and IC ₅₀ Calculation of the values% inhibition = (1-average OD of sample group/average OD of control group) ×100%, and then calculation of IC of each compound on different tumor cells using SPSS22 software ₅₀ And (5) value, and carrying out statistical analysis on the data. The results of the experiments were obtained from 3 independent replicates and the data are expressed as mean ± standard deviation.

The test results are shown in table 1:

table 1 cytotoxic Activity of Compounds 4a-4l

HCPT 10-hydroxycamptothecin.

Effect verification example 2: inhibition activity assay of compounds on DNA Topo I

Take compound 4g as an example:

(1) The experimental steps are as follows:

a certain amount of the drug was weighed, 4g of the compound and camptothecin were dissolved in 1mL of DMSO to give solutions of 10mM initial concentration, and the solutions of initial concentration were diluted with PBS to the desired concentrations, respectively. The initial concentration of 20U/. Mu.L DNA Topo I was diluted with secondary water to a final concentration of 1U/. Mu.L. The 50 xTAE running buffer was diluted with secondary water to 1 xTAE running buffer and prepared for use.

Then preparing agarose gel with concentration of 1%, namely weighing about 0.7g agarose, placing the agarose gel into a proper conical flask, adding the agarose gel into about 70mL of prepared 1 xTAE electrophoresis buffer solution, sealing the solution by using tinfoil paper, and heating the solution for 3min by using a microwave oven until the agarose gel is completely dissolved, thus obtaining transparent clear solution. The organic glass inner groove in the electrophoresis tank is washed clean in advance, dried in the air, placed in a glue making glass plate, poured into a glue film inserted with a comb when the agarose solution which is uniformly dissolved is hot until the glue is completely solidified, the comb is vertically pulled out, the gel and the inner groove are placed in the electrophoresis tank, and the 1 xTAE electrophoresis buffer in the electrophoresis tank is about 1cm beyond the glue.

TABLE 2 preparation of gradient concentration Compound 4g and CPT and DNA, topo I reaction solution

Table 2 Preparation of compounds 4g and CPT in gradient concentration with DNA and Topo I reaction solution

Centrifuging, mixing, placing in a 37 ℃ constant temperature water bath, reacting for 30min, taking out, adding 2 mu L of 6 Xloading buffer, and stopping the reaction. Mixing uniformly, sucking 10 mu L of sample, adding the sample into each comb hole of the adhesive film by a liquid-transfering gun, and replacing the gun head to prevent the sample from pollution after one sample is added. After electrophoresis for 90min (current 110A, voltage 85V), taking out, putting into GelRed dye for dyeing for 30min, taking out, imaging in a gel imager, and obtaining the result shown in FIG. 1.

Wherein, the substances in each lane are respectively:

lane 1: DNA; lane 2 DNA+Topo I; lane 3: compound 4g (20 μm) +topoi+ DNA; lane 4: compound 4g (40 μm) +topoi+ DNA; lane 5: compound 4g (80 μm) +topoi+ DNA; lane 8: CPT (10. Mu.M) +TopoI+ DNA; lane 9: CPT (40. Mu.M) +TopoI+ DNA; lane 10: CPT (80. Mu.M) +TopoI+ DNA.

As can be seen from FIG. 1, the first lane is the negative control pBR322DNA, and the DNA is in a supercoiled state; the second lane is where DNA coexists with Topo I, which unwinds the DNA, where the DNA is in a relaxed state; the third to fifth lanes are the cases where 4g of the compound was allowed to coexist with DNA and Topo I at different concentrations, and no inhibition was shown at a concentration of 20. Mu.M, and weak Topo I-inhibiting activity was already started at a concentration of 40. Mu.M, and significant Topo I-inhibiting activity was already shown at a concentration of 80. Mu.M. The positive control camptothecins in lanes eight to tenth gradually increased in inhibiting Topo I with increasing concentration. Thus, 4g of the compound can achieve the effect of inhibiting topoisomerase I.

Claims

1. 5-aminopyrimido [4,5-b ] s of the general formula (I)]Quinoline compounds:

wherein: r is R ₁ Selected from methyl, methoxy or isopropyl;

ar is selected from phenyl or 4-chlorophenyl.

2. The following compounds, characterized in that:

the compound is specifically as follows:

3. a process for the preparation of a compound according to claim 1 or 2, characterized by comprising the steps of:

s1, will

S2, halogenating the compound 1 to obtain a compound 2

S3, adding the compound 2 into a reaction container,

And toluene, to obtain compound 3

4. A method of preparation according to claim 3, characterized in that:

the step S1 specifically comprises the following steps: adding into a reaction vessel

5. A method of preparation according to claim 3, characterized in that:

the step S2 specifically comprises the following steps: the compound 1, N-dimethylaniline and POCl are added into a reaction vessel ₃ And (3) reacting at 105-120 ℃ to obtain the compound 2.

6. A method of preparation according to claim 3, characterized in that:

in the step S3, the reaction temperature is 105-120 ℃.

7. A method of preparation according to claim 3, characterized in that:

in the step S4, the reaction temperature is 55-70 ℃.

8. Use of a compound according to claim 1 or 2 or a compound prepared by a method according to any one of claims 3 to 7 for the preparation of an anti-tumour agent or an inhibitor of topoisomerase I activity.

9. The use according to claim 8, characterized in that: the tumor is a tumor caused by nasopharyngeal carcinoma, liver cancer, gastric cancer, bladder cancer or lung cancer.