阅读说明：本技术 香豆素-二硫代氨基甲酸酯衍生物在制备抗肿瘤药物中的应用 (Application of coumarin-dithiocarbamate derivative in preparation of antitumor drugs ) 是由 潘远江 朱和平 金洪传 应士龙 梁霄 于 2020-12-01 设计创作，主要内容包括：本发明公开了一类香豆素-二硫代氨基甲酸酯衍生物在制备抗肿瘤药物中的应用,结构如式(I)所示,其中R可以为被不同的直链烷基、异丙基、叔丁基、环烷基、羟基、烷基醇基、炔基、氰基、哌嗪基、N-烷基哌嗪基、芳香基或取代芳香基取代的二级胺、乙撑亚胺、三甲烯亚胺、四氢吡咯基、哌啶基、环己亚胺、哌嗪基、吗啉基、咪唑基。本发明针对一类以香豆素-二硫代氨基甲酸酯衍生物技术领域进行大规模的设计、合成和抗肿瘤活性筛选,得出一类香豆素-二硫代氨基甲酸酯衍生物,并且本发明的香豆素-二硫代氨基甲酸酯衍生物具有潜在的抗肿瘤活性,尤其是抗结肠癌活性,在癌症及癌症相关疾病治疗方面有潜在的应用前景。(The invention discloses application of coumarin-dithiocarbamate derivatives in preparing antitumor drugsThe structure is shown as formula (I), wherein R can be a secondary amine, ethyleneimine, trimethyleneimine, tetrahydropyrrole, piperidyl, cycloheximide, piperazinyl, morpholinyl and imidazolyl which are substituted by different straight-chain alkyl groups, isopropyl groups, tert-butyl groups, cycloalkyl groups, hydroxyl groups, alkyl alcohol groups, alkynyl groups, cyano groups, piperazinyl groups, N-alkyl piperazinyl groups, aryl groups or substituted aryl groups. The coumarin-dithiocarbamate derivatives are obtained by large-scale design, synthesis and antitumor activity screening in the technical field of coumarin-dithiocarbamate derivatives, have potential antitumor activity, especially colon cancer resisting activity, and have potential application prospects in the treatment of cancers and cancer-related diseases.)

1. The application of the coumarin-dithiocarbamate derivative and the pharmaceutically acceptable salt thereof in preparing the antitumor drugs is characterized in that the coumarin-dithiocarbamate derivative is a compound with a structure shown in a formula (I):

wherein: r represents one of linear alkyl, isopropyl, tert-butyl, cycloalkyl, hydroxyl, alkyl alcohol group, alkynyl, cyano, piperazinyl, N-alkyl piperazinyl, secondary amine substituted by aryl or substituted aryl, ethylene imine, trimethyleneimine, tetrahydropyrrole, piperidyl, cycloheximide, piperazinyl, morpholinyl and imidazolyl which are different.

2. The use according to claim 1, wherein said coumarin-dithiocarbamate derivative is a compound of formula (I):

wherein: r represents

One of (1);

wherein the wavy line indicates the position of the connection.

3. The use of claim 1, wherein the tumor is at least one of gastric cancer, colon cancer, breast cancer, liver cancer, leukemia, lymphoma, ovarian cancer, prostate cancer, endometrial cancer, skin cancer, esophageal cancer, and central nervous system cancer.

4. The use according to claim 1, wherein the antitumor drug comprises an active ingredient and a pharmaceutical excipient, and the active ingredient is a coumarin-dithiocarbamate derivative.

5. The use as claimed in claim 1, wherein the antineoplastic agent is in the form of tablet, injection, aerosol, suppository, ointment, capsule, membrane, dripping pill, controlled-release or sustained-release preparation or nanometer preparation.

6. The application of the coumarin-dithiocarbamate derivative and pharmaceutically acceptable salt thereof in preparing anti-colon cancer drugs is characterized in that the coumarin-dithiocarbamate derivative is a compound with a structure shown in a formula (I):

wherein: r represents one of linear alkyl, isopropyl, tert-butyl, cycloalkyl, hydroxyl, alkyl alcohol group, alkynyl, cyano, piperazinyl, N-alkyl piperazinyl, secondary amine substituted by aryl or substituted aryl, ethylene imine, trimethyleneimine, tetrahydropyrrole, piperidyl, cycloheximide, piperazinyl, morpholinyl and imidazolyl which are different.

Technical Field

The invention belongs to the field of pharmaceutical chemistry, and particularly relates to application of coumarin-dithiocarbamate derivatives in preparation of antitumor drugs.

Background

Cancer has currently become an important killer that endangers human life and health. As predicted by the World Health Organization (WHO), by 2030, there will be 2200 million new cancer patients and 1300 million cancer-related death cases worldwide per year. Despite the current abundance of cancer treatments, a high incidence of cancer and mortality still occurs. Therefore, the active search for new antitumor drugs is a constant concern for scientists.

Coumarin, also known as benzopyrone, is a structural parent nucleus of a large class of coumarin compounds existing in the plant kingdom. Previous researches find that the coumarin compound has wide pharmacological activities including antitumor, anti-inflammatory, antibacterial, anticoagulant, antioxidant and other effects. It is worth mentioning that some compounds containing coumarin structures have been approved and widely used in the market, such as warfarin (anticoagulant), hydrocinnaman (anticoagulant), neomycin sodium (antibacterial), trimethanalin (psoriasis treatment), methoxsalen (psoriasis treatment), dicumyl (antithrombotic), carbopol (ischemic heart disease treatment), dicumarol (deep vein thrombosis treatment), aceroloumarin (thromboembolism treatment), clocrolimun (thromboembolism treatment), coumarone (bile secretion and biliary spasm treatment), and 5-methoxypsoralen (skin tumor treatment, aids, psoriasis and vitiligo treatment), and the like, and the wide use of these drugs accounts for the advantages of the coumarin skeleton. We have also noted that in the field of antitumor therapy, there are few drugs on the market that use coumarin backbones, and therefore there is a great room for development of novel antitumor molecules that use coumarin as a parent nucleus.

In order to find a small molecular compound with potential antitumor activity, a dithiocarbamate which is one of fragments with better antitumor activity is reported, a dithiocarbamate fragment is introduced during design, a novel coumarin-dithiocarbamate derivative is designed, and the antitumor activity of the coumarin-dithiocarbamate derivative is screened in a large amount, so that the compound is finally found to have potential antitumor activity, particularly the antitumor activity against colon cancer.

Disclosure of Invention

The invention provides application of coumarin-dithiocarbamate derivatives in preparation of antitumor drugs, and researches show that the coumarin-dithiocarbamate derivatives have good antitumor activity, especially colon cancer resisting activity.

The coumarin-dithiocarbamate derivatives are compounds shown in a formula (I) or medicinal salts thereof;

in formula (I): r represents a secondary amine, an ethyleneimine, a trimethyleneimine, a tetrahydropyrrolyl, a piperidyl, a cycloheximide, a piperazinyl, a morpholinyl or an imidazolyl group, which are substituted by different straight-chain alkyl groups, isopropyl groups, tert-butyl groups, cycloalkyl groups, hydroxyl groups, alkyl alcohol groups, alkynyl groups, cyano groups, piperazinyl groups, N-alkyl piperazinyl groups, aryl groups or substituted aryl groups.

Further, wherein R represents

One of (1);

wherein the wavy line indicates the position of the connection.

Preferably, the coumarin-dithiocarbamate derivative is one or more of the following compounds or pharmaceutically acceptable salts thereof:

preferably, the coumarin-dithiocarbamate derivative is one or a combination of the following compounds or pharmaceutically acceptable salts thereof:

preferably, the anti-tumor medicament is used for treating tumors or tumor-related diseases. These 20 compounds have a broad spectrum of anti-tumor activity and examples of cancers that can be treated include, but are not limited to, gastric cancer, colon cancer, breast cancer, liver cancer, leukemia, lymphoma, ovarian cancer, prostate cancer, endometrial cancer, skin cancer, esophageal cancer, central nervous system cancer. The coumarin-dithiocarbamate derivative has potential anti-tumor activity, especially anti-colon cancer activity, and has potential application prospect in the treatment of cancer and cancer-related diseases.

Preferably, the anti-tumor medicine comprises an active ingredient with a therapeutically effective amount and pharmaceutic adjuvants, wherein the active ingredient is a coumarin-dithiocarbamate derivative. The pharmaceutic adjuvant refers to a commonly used medicine carrier in the field of pharmacy, such as: fillers, binders, diluents, excipients, disintegrants, surfactants, lubricants, and the like. Other adjuvants such as sweetener, flavoring agent, etc. can also be added into the composition.

Preferably, the anti-tumor drug of the anti-tumor drug is a combination of coumarin-dithiocarbamate derivatives and other anti-tumor drugs, and the anti-tumor drug comprises anti-tumor antibiotics, various biological alkylating agents, anti-metabolism drugs, molecular targeting drugs, immune checkpoint inhibitors and the like.

Various dosage forms of the antitumor drug can be prepared according to the conventional production method in the pharmaceutical field. For example, the active ingredient may be combined with one or more carriers and then formulated into the desired dosage form. The preparation forms of the medicine comprise tablets, injections, aerosols, suppositories, ointments, capsules, films, dropping pills, controlled release or sustained release agents or nano preparations. The present invention may be administered in the form of a composition to a patient in need of such treatment by oral, nasal inhalation, rectal or parenteral administration. For oral administration, it can be made into conventional solid preparations such as tablet, powder, capsule, granule, etc., liquid preparations such as aqueous or oil suspension, or other liquid preparations such as syrup, elixir, etc.; for parenteral administration, it can be formulated into solution for injection, aqueous or oily suspension, etc.

Compared with the prior art, the invention has the beneficial effects that:

the coumarin-dithiocarbamate derivative has a novel structure and good anti-tumor activity, and can be used for preparing medicines for tumors and tumor-related diseases.

Drawings

FIG. 1 is a graph showing the inhibition of cell proliferation by the target compounds ID1-ID20 of the present invention against three different colon cancer cell lines. (A) Compound ID1-ID20 pairsInhibition of RKO cells (10. mu.M); (B) inhibition of SW620 cells by Compound ID1-ID20 (10. mu.M); (C) inhibition of SW480 cells by compound ID1-ID20 (10. mu.M); (D) IC of Compound ID11 on RKO cells₅₀A value; (E) IC of Compound ID11 on SW620 cells₅₀A value; (F) IC of Compound ID11 on SW480 cells₅₀A value;

FIG. 2 is a graph showing the inhibition of cell colony formation by the objective compound ID11 of the present invention on two different colon cancer cell lines. (A) SW620 cells; (B) SW480 cells;

FIG. 3 is a graph showing cell cycle arrest of the target compound ID11 of the present invention against two different colon cancer cell lines. (A) SW620 cell and SW480 cell cycle arrest results; (B) statistical plots of SW620 and SW480 cell cycle arrest results (G2/M phase); (C) immunoblotting results for SW620 cells and SW480 cell cycle-associated proteins. P < 0.01, P < 0.001;

FIG. 4 is a graph showing the induction of apoptosis by the target compound ID11 of the present invention on two different colon cancer cell lines. (A) SW620 and SW480 apoptosis results; (B) statistical plots of apoptosis results for SW620 and SW480 cells (total apoptotic cells); (C) immunoblotting results for SW620 and SW480 apoptosis-related proteins. P <0.05, P < 0.001.

Detailed Description

The present invention will be described in detail with reference to specific examples. It should be noted that technical features or combinations of technical features described in the following embodiments should not be considered as being isolated, and they may be combined with each other to achieve better technical effects.

Example 1

The structures of compounds ID1-ID20 are shown in Table 1.

TABLE 1

The method for preparing the coumarin-dithiocarbamate derivative comprises the following steps:

the following description of the synthetic method is made in conjunction with specific compounds, wherein step a is a unified step:

step a: synthesis of 6- (bromomethyl) -2H-benzopyran-2-one (3): 6-methylcoumarin (1) (1g, 6.24mmol), N-bromosuccinimide (2) (1.11g, 6.24mmol) and azobisisobutyronitrile (102.5mg, 0.624mmol) were weighed and dissolved in carbon tetrachloride, and the reaction system was placed in an oil bath and stirred at 90 ℃ for reflux reaction for 10 hours. The progress of the reaction was monitored by Thin Layer Chromatography (TLC). After the reaction is finished, the carbon tetrachloride is removed by reduced pressure distillation, and the residue is directly used for the next reaction without further treatment.

Example 2 Synthesis of Compound ID1

Step b: (2-oxo-2H-benzopyran-6-yl) methylthiodimethylcarbamate (ID 1): 6- (bromomethyl) -2H-benzopyran-2-one (3) (100mg, 0.418mmol), carbon disulfide (34. mu.l, 0.558mmol), dimethylamine (28. mu.l, 0.558mmol) and potassium hydroxide (31mg, 0.558mmol) were weighed out and dissolved in absolute ethanol, and the reaction flask was left to stir at room temperature for 24 hours. TLC monitored the progress of the reaction. After completion of the reaction, ethanol was distilled off under reduced pressure, the residue was extracted three times with ethyl acetate (100ml) and water (100ml), the organic layers were combined, dried over anhydrous magnesium sulfate, and ethyl acetate was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate ═ 2: 1) to give the final product dimethyl (2-oxo-2H-benzopyran-6-yl) methylthiodimethylcarbamate (ID1) (83mg, yield 71%, white solid). The physicochemical data for compound ID1 are as follows: melting point 151.6-152.5 ℃.¹H NMR(400MHz,CDCl₃)δ(ppm):7.67(1H,d,J＝9.2Hz,H-4),7.54(1H,d,J＝7.2Hz,H-7),7.53(1H,s,H-5),7.26(1H,d,J＝6.4Hz,H-8),6.41(1H,d,J＝9.2Hz,H-3),4.61(2H,s,-SCH ₂-),3.55(3H,s,-N(CH ₃)₂),3.36(3H,s,-N(CH ₃)₂).¹³C NMR(100MHz,CDCl₃)δ(ppm):195.9,160.7,153.2,143.3,133.3,132.8,128.3,118.7,117.0,116.8,45.6,41.5,41.0.HRMS(ESI,m/z)calcd.for C₁₃H₁₃NO₂S₂(M+H)⁺280.0460,found 208.0461.

EXAMPLE 3 Synthesis of Compound ID2

Synthesis of diethyl (2-oxo-2H-benzopyran-6-yl) methylthiodiethylaminocarbamate (ID2) the procedure was analogous to that of Synthesis b of Compound ID1 in example 2. It is noted that in the synthesis of ID2, the catalyst was potassium carbonate and the reaction solvent was dioxane/water 1/4 (v/v). The physicochemical data for compound ID2 are as follows: a white solid. The yield is 50%. The melting point is 100.1-101.0 ℃.¹H NMR(400MHz,DMSO-d₆)δ(ppm):8.04(1H,d,J＝9.6Hz,H-4),7.73(1H,d,J＝2.4Hz,H-5),7.62(1H,dd,J₁＝8.8Hz,J₂＝2.4Hz,H-7),7.35(1H,d,J＝8.4Hz,H-8),6.48(1H,d,J＝9.6Hz,H-3),4.58(2H,s,-SCH ₂-),3.96(2H,q,J＝6.8Hz,-N(CH ₂CH₃)₂),3.72(2H,q,J＝6.8Hz,-N(CH ₂CH₃)₂),1.21-1.15(6H,m,-N(CH₂CH ₃)₂).¹³C NMR(100MHz,DMSO-d₆)δ(ppm):193.0,159.8,152.6,144.0,133.0,132.7,128.6,118.5,116.4×2,49.1,46.5,39.6,12.3,11.3.HRMS(ESI,m/z)calcd.for C₁₅H₁₇NO₂S₂(M+Na)⁺330.0593,found 330.0599.

EXAMPLE 4 Synthesis of Compound ID3

Synthesis of dipropyl (2-oxo-2H-benzopyran-6-yl) methyldithiocarbamate (ID3) the procedure was analogous to Synthesis procedure b of Compound ID2 in example 3. The physicochemical data for compound ID3 are as follows: a pale yellow solid. The yield is 60%. Melting point of 52.3-53.2 ℃.¹H NMR(400MHz,CDCl₃)δ(ppm):7.65(1H,d,J＝9.6Hz,H-4),7.51-7.48(2H,m,H-5,H-7),7.20(1H,d,J＝8.4Hz,H-8),6.35(1H,d,J＝9.6Hz,H-3),4.55(2H,s,-SCH ₂-),3.86(2H,t,-N(CH ₂CH₂CH₃)₂),3.56(2H,t,-N(CH ₂CH₂CH₃)₂),1.76-1.63(4H,m,-N(CH₂CH ₂CH₃)₂),0.88(6H,t,-N(CH₂CH₂CH ₃)₂).¹³C NMR(100MHz,CDCl₃)δ(ppm):194.8,160.6,153.1,143.3,133.3,132.8,128.4,118.6,116.9,116.7,57.0,54.4,40.7,20.7,19.6,11.1×2.HRMS(ESI,m/z)calcd.for C₁₇H₂₁NO₂S₂(M+Na)⁺358.0906,found 358.0918.

EXAMPLE 5 Synthesis of Compound ID4

Synthesis of dibutyl (2-oxo-2H-benzopyran-6-yl) methyldithiocarbamate (ID4) the procedure was analogous to Synthesis procedure b of Compound ID2 in example 3. The physicochemical data for compound ID4 are as follows: a light yellow liquid. The yield is 70%.¹H NMR(400MHz,CDCl₃)δ(ppm):7.69(1H,d,J＝9.6Hz,H-4),7.55-7.53(2H,m,H-5,H-7),7.24(1H,d,J＝8.4Hz,H-8),6.40(1H,d,J＝9.6Hz,H-3),4.60(2H,s,-SCH ₂-),3.95(2H,t,J＝8.0Hz,-N(CH ₂CH₂CH₂CH₃)₂),3.64(2H,t,J＝8.0Hz,-N(CH ₂CH₂CH₂CH₃)₂),1.74-1.67(4H,m,-N(CH₂CH ₂CH₂CH₃)₂),1.38-1.31(4H,m,-N(CH₂CH₂CH ₂CH₃)₂),0.96-0.91(6H,m,-N(CH₂CH₂CH₂CH ₃)₂).¹³C NMR(100MHz,CDCl₃)δ(ppm):194.6,160.6,153.2,143.3,133.3,132.9,128.4,118.7,116.9,116.7,55.2,52.5,40.7,29.3,28.3,20.0×2,13.8,13.7.HRMS(ESI,m/z)calcd.for C₁₉H₂₅NO₂S₂(M+Na)⁺386.1219,found 386.1222.

EXAMPLE 6 Synthesis of Compound ID5

Synthesis of (2-oxo-2H-benzopyran-6-yl) methylazetidin-1-carbodiimide sulphate (ID5) the procedure was analogous to Synthesis procedure b of Compound ID2 in example 3. The physicochemical data for compound ID5 are as follows: a white solid. The yield was 56%. The melting point is 144.0-145.0 ℃.¹H NMR(400MHz,CDCl₃)δ(ppm):7.66(1H,d,J＝9.6Hz,H-4),7.55-7.53(2H,m,H-5,H-7),7.25(1H,d,J＝9.2Hz,H-8),6.40(1H,d,J＝9.6Hz,H-3),4.59(2H,s,-SCH ₂-),4.30(2H,t,J＝7.6Hz,H-a),4.17(2H,t,J＝7.6Hz,H-c),2.43-2.35(2H,m,H-b).¹³C NMR(100MHz,CDCl₃)δ(ppm):192.8,160.7,153.2,143.2,133.9,132.7,128.2,118.7,117.0,116.8,54.8,53.1,38.9,15.5.HRMS(ESI,m/z)calcd.for C₁₄H₁₃NO₂S₂(M+Na)⁺314.0280,found 314.0281.

EXAMPLE 7 Synthesis of Compound ID6

The procedure for the synthesis of (2-oxo-2H-benzopyran-6-yl) methyl 3-hydroxyazetidin-1-carbodiimide sulphate (ID6) is analogous to that of Synthesis b of Compound ID1 in example 2. The physicochemical data for compound ID6 are as follows: a white solid. The yield was 55%. The melting point is 137.8-138.7 ℃.¹H NMR(400MHz,DMSO-d₆)δ(ppm):8.04(1H,d,J＝9.6Hz,H-4),7.70(1H,d,J＝1.2Hz,H-5),7.61(1H,dd,J₁＝8.4Hz,J₂＝1.6Hz,H-7),7.35(1H,d,J＝8.4Hz,H-8),6.48(1H,d,J＝9.6Hz,H-3),5.94(1H,d,J＝6.4Hz,-N(CH₂)₂CHOH),4.58-4.52(3H,m,-SCH ₂-,-N(CH₂)₂CHOH),4.39(2H,m,-N(CH ₂)₂CHOH),3.90-3.84(2H,m,-N(CH ₂)₂CHOH).¹³C NMR(100MHz,DMSO-d₆)δ(ppm):191.7,159.8,152.6,144.0,133.8,132.6,128.4,118.5,116.4×2,64.4,62.5,59.8,38.2.HRMS(ESI,m/z)calcd.for(M+Na)⁺330.0229,found 330.0232.

EXAMPLE 8 Synthesis of Compound ID7

Synthesis of 2-oxo-2H-benzopyran-6-yl) methylpyrrolidine-1-carbothionate (ID7) the procedure was analogous to Synthesis procedure b of Compound ID1 in example 2. The physicochemical data for compound ID7 are as follows: a white solid. The yield is 50%. Melting point of 110.2-111.1 deg.c.¹H NMR(400MHz,CDCl₃)δ(ppm):7.67(1H,d,J＝9.6Hz,H-4),7.56-7.54(2H,m,H-5,H-7),7.25(1H,d,J＝9.2Hz,H-8),6.40(1H,d,J＝9.6Hz,H-3),4.63(2H,s,-SCH ₂-),3.93(2H,t,J＝6.8Hz,-NCH ₂CH₂CH₂CH₂N-(pyrrolidine)),3.63(2H,t,J＝6.8Hz,-NCH₂CH₂CH₂CH ₂N-(pyrrolidine)),2.10-2.03(2H,m,-NCH₂CH ₂CH₂CH₂N-(pyrrolidine)),2.01-1.94(2H,m,-NCH₂CH₂CH ₂CH₂N-(pyrrolidine)).¹³C NMR(100MHz,CDCl₃)δ(ppm):191.5,160.7,153.2,143.3,133.7,132.8,128.3,118.7,117.0,116.8,55.3,50.6,39.8,26.0,24.2.HRMS(ESI,m/z)calcd.for C₁₅H₁₅NO₂S₂(M+Na)⁺328.0436,found 328.0444.

EXAMPLE 9 Synthesis of Compound ID8

Synthesis of 2-oxo-2H-benzopyran-6-yl) methylpiperidine-1-carbothionate (ID8) the procedure was analogous to Synthesis procedure b of Compound ID2 in example 3. The physicochemical data for compound ID8 are as follows: a pale yellow solid. The yield is 70%. Melting point 130.1-131.0 deg.C.¹H NMR(400MHz,CDCl₃)δ(ppm):7.67(1H,d,J＝9.6Hz,H-4),7.56-7.53(2H,m,H-5,H-7),7.26(1H,d,J＝8.0Hz,H-8),6.40(1H,d,J＝9.6Hz,H-3),4.62(2H,s,-SCH ₂-),4.28(2H,br,H-a),3.86(2H,br,H-e),1.69(6H,br,H-b,H-c,H-d).¹³C NMR(100MHz,CDCl₃)δ(ppm):194.3,160.7,153.2,143.3,133.3,132.9,128.4,118.7,117.0,116.8,53.2,51.4,40.7,26.0,25.4,24.2.HRMS(ESI,m/z)calcd.for C₁₆H₁₇NO₂S₂(M+Na)⁺342.0593,found 342.0585.

EXAMPLE 10 Synthesis of Compound ID9

Synthesis of 2-oxo-2H-benzopyran-6-yl) methylazepan-1-carbothionate (ID9) the procedure was analogous to Synthesis procedure b of Compound ID2 in example 3. The physicochemical data for compound ID9 are as follows: a white solid. The yield is 66%. The melting point is 72.6-73.5 ℃.¹H NMR(400MHz,CDCl₃)δ(ppm):7.65(1H,d,J＝9.6Hz,H-4),7.53-7.50(2H,m,H-5,H-7),7.22(1H,d,J＝8.0Hz,H-8),6.37(1H,d,J＝9.6Hz,H-3),4.58(2H,s,-SCH ₂-),4.15(2H,t,J＝6.0Hz,H-a),3.83(2H,t,J＝6.0Hz,H-f),1.84-1.79(4H,m,H-b,H-e),1.23-1.20(2H,m,H-c),0.85-0.78(2H,m,H-d).¹³C NMR(100MHz,CDCl₃)δ(ppm):195.0,160.6,153.2,143.3,133.4,132.8,128.4,118.7,116.9,116.7,55.8,52.8,40.6,27.3,26.6,26.5,26.1.HRMS(ESI,m/z)calcd.for C₁₇H₁₉NO₂S₂(M+Na)⁺356.0749,found 356.0751.

EXAMPLE 11 Synthesis of Compound ID10

Synthesis of 2-oxo-2H-benzopyran-6-yl) methylmorpholine-4-carbothionate (ID10) the procedure was analogous to Synthesis b of Compound ID1 in example 2. The physicochemical data for compound ID10 are as follows: a white solid. The yield was 57%. The melting point is 135.4-136.3 ℃.¹H NMR(600MHz,DMSO-d₆)δ(ppm):8.04(1H,d,J＝9.6Hz,H-4),7.73(1H,s,H-5),7.63(1H,dd,J₁＝8.4Hz,J₂＝1.8Hz,H-7),7.35(1H,d,J＝9.0Hz,H-8),6.48(1H,d,J＝9.0Hz,H-3),4.63(2H,s,-SCH ₂-),4.22(2H,br,-N(CH ₂CH₂)₂O-(morpholine)),3.90(2H,br,-N(CH ₂CH₂)₂O-(morpholine)),3.65(4H,br,-N(CH₂CH ₂)₂O-(morpholine)).¹³C NMR(125MHz,DMSO-d₆)δ(ppm):197.8,162.9,155.8,147.1,136.0,135.9,131.8,121.7,119.5,119.5,68.6×2,54.4,53.3,43.1.HRMS(ESI,m/z)calcd.for C₁₅H₁₅NO₃S₂(M+Na)⁺344.0386,found 344.0391.

EXAMPLE 12 Synthesis of Compound ID11

Synthesis of 2-oxo-2H-benzopyran-6-yl) methyl 1H-imidazole-1-carbodiimide sulphate (ID11) the procedure was analogous to Synthesis b of Compound ID1 in example 2. It should be noted that, in the synthesis of ID11, the catalyst was potassium phosphate, and the reaction solvent was acetone. The physicochemical data for compound ID11 are as follows: yellow solid. The yield was 68%. The melting point is 139.1-140.0 ℃.¹H NMR(400MHz,DMSO-d₆)δ(ppm):8.64(1H,s,H-b),8.05(1H,d,J＝9.6Hz,H-4),8.01(1H,s,H-5),7.82(1H,s,H-e),7.71(1H,d,J＝8.4Hz,H-7),7.40(1H,d,J＝8.4Hz,H-8),7.17(1H,s,H-d),6.50(1H,d,J＝9.6Hz,H-3),4.78(2H,s,-SCH ₂-).¹³C NMR(100MHz,DMSO-d₆)δ(ppm):197.8,159.7,153.0,143.8,135.9,133.0,131.7,130.6,129.0,118.7,118.3,116.6×2,39.6.HRMS(ESI,m/z)calcd.for C₁₄H₁₀N₂O₂S₂(M+Na)⁺325.0076,found 325.0070.

EXAMPLE 13 Synthesis of Compound ID12

Synthesis of 2-oxo-2H-benzopyran-6-yl) methyl 4-methylpiperazine-1-carbodiimide sulphate (ID12) the procedure was analogous to Synthesis b of Compound ID1 in example 2. The physicochemical data for compound ID12 are as follows: a pale yellow solid. The yield was 53%. Melting point of 124.3-125.2 ℃.¹H NMR(400MHz,DMSO-d₆)δ(ppm):8.03(1H,d,J＝9.6Hz,H-4),7.73(1H,d,J＝2.0Hz,H-5),7.62(1H,dd,J₁＝8.8Hz,J₂＝2.0Hz,H-7),7.35(1H,d,J＝8.4Hz,H-8),6.48(1H,d,J＝9.6Hz,H-3),4.61(2H,s,-SCH ₂-),4.23(2H,br,-N(CH₂CH ₂)₂N-(piperazine)),3.87(2H,br,-N(CH₂CH ₂)₂N-(piperazine)),2.38(4H,br,-N(CH ₂CH₂)₂N-(piperazine)),2.19(3H,s,-NCH ₃).¹³C NMR(100MHz,DMSO-d₆)δ(ppm):194.1,159.8,152.6,143.9,132.9,132.8,128.7,118.5,116.4×2,53.9×2,50.9,49.5,45.0×2.HRMS(ESI,m/z)calcd.for C₁₆H₁₈N₂O₂S₂(M+Na)⁺357.0702,found 357.0709.

EXAMPLE 14 Synthesis of Compound ID13

Synthesis of 2-oxo-2H-benzopyran-6-yl) methyl 4-isopropylpiperazine-1-carbo-disulfate (ID13) the procedure was analogous to Synthesis procedure b of Compound ID2 in example 3. The physicochemical data for compound ID13 are as follows: a pale yellow solid. The yield was 58%. Melting point 73.6-74.5 deg.C.¹H NMR(400MHz,DMSO-d₆)δ(ppm):8.04(1H,d,J＝9.6Hz,H-4),7.73(1H,d,J＝2.0Hz,H-5),7.63(1H,dd,J₁＝8.8Hz,J₂＝2.4Hz,H-7),7.35(1H,d,J＝8.4Hz,H-8),6.48(1H,d,J＝9.6Hz,H-3),4.61(2H,s,-SCH ₂-),4.21(2H,br,-N(CH ₂CH₂)₂N-CH(CH₃)₂),3.86(2H,br,-N(CH ₂CH₂)₂N-CH(CH₃)₂),2.71-2.65(1H,m,-N(CH₂CH₂)₂N-CH(CH₃)₂),2.50-2.49(4H,m,-N(CH₂CH ₂)₂N-CH(CH₃)₂),0.96(3H,s,-N(CH₂CH₂)₂N-CH(CH ₃)₂),0.95(3H,s,-N(CH₂CH₂)₂N-CH(CH ₃)₂).¹³C NMR(100MHz,DMSO-d₆)δ(ppm):193.7,159.8,152.6,143.9,132.9,132.8,128.7,118.5,116.4×2,53.4,51.5,50.1,47.7,47.6×2,18.0×2.HRMS(ESI,m/z)calcd.for C₁₈H₂₂N₂O₂S₂(M+H)⁺363.1195,found 363.1201.

EXAMPLE 15 Synthesis of Compound ID14

The procedure for the synthesis of 2-oxo-2H-benzopyran-6-yl) methyl 4- (tert-butyl) piperazine-1-carbo-disulfate (ID14) is analogous to that of Synthesis b of Compound ID2 in example 3. The physicochemical data for compound ID14 are as follows: a pale yellow solid. The yield was 62%. Melting point 159.4-160.3 ℃.¹H NMR(400MHz,DMSO-d₆)δ(ppm):8.04(1H,d,J＝9.6Hz,H-4),7.73(1H,d,J＝2.0Hz,H-5),7.62(1H,dd,J₁＝8.8Hz,J₂＝2.4Hz,H-7),7.35(1H,d,J＝8.4Hz,H-8),6.48(1H,d,J＝9.6Hz,H-3),4.60(2H,s,-SCH ₂-),4.18(2H,br,-N(CH ₂CH₂)₂N-C(CH₃)₃),3.84(2H,br,-N(CH ₂CH₂)₂N-C(CH₃)₃),2.55(4H,br,-N(CH₂CH ₂)₂N-C(CH₃)₃),1.00(9H,s,-N(CH₂CH₂)₂N-C(CH ₃)₃).¹³C NMR(100MHz,DMSO-d₆)δ(ppm):193.5,159.8,152.6,143.9,133.0,132.8,128.7,118.5,116.4×2,53.3,51.7,50.4,45.4,45.3,45.2,25.5×3.HRMS(ESI,m/z)calcd.for C₁₉H₂₄N₂O₂S₂(M+H)⁺377.1352,found 377.1349.

EXAMPLE 16 Synthesis of Compound ID15

The procedure for the synthesis of 2-oxo-2H-benzopyran-6-yl) methyl 4- (2-hydroxyethyl) piperazine-1-carbo-disulfate (ID15) is analogous to that of Synthesis b of Compound ID1 in example 2. The physicochemical data for compound ID15 are as follows: a pale yellow solid. The yield was 43%. The melting point is 120.9-121.8 ℃.¹H NMR(400MHz,DMSO-d₆)δ(ppm):8.04(1H,d,J＝9.6Hz,H-4),7.73(1H,d,J＝2.0Hz,H-5),7.62(1H,dd,J₁＝8.4Hz,J₂＝2.0Hz,H-7),7.35(1H,d,J＝8.4Hz,H-8),6.48(1H,d,J＝9.6Hz,H-3),4.61(2H,s,-SCH ₂-),4.47(1H,t,J＝4.8Hz,-NCH₂CH₂OH),4.22(2H,br,-N(CH₂CH ₂)₂N-(piperazine)),3.87(2H,br,-N(CH₂CH ₂)₂N-(piperazine)),3.50(2H,q,J＝5.6Hz,-NCH₂CH ₂OH),2.50(4H,br,-N(CH ₂CH₂)₂N-(piperazine)),2.42(2H,t,J＝6.0Hz,-NCH ₂CH₂OH).¹³C NMR(100MHz,DMSO-d₆)δ(ppm):193.9,159.8,152.6,143.9,132.9,132.8,128.7,118.5,116.4×2,59.4,58.4,52.5,52.4,51.0,49.6,39.5.HRMS(ESI,m/z)calcd.for C₁₇H₂₀N₂O₃S₂(M+Na)⁺387.0808,found 387.0808.

EXAMPLE 17 Synthesis of Compound ID16

Synthesis procedure for 2-oxo-2H-benzopyran-6-yl) methyl 4-cyclohexylpiperazine-1-carbodiimide sulphate (ID16) analogously to Synthesis procedure b of Compound ID2 in example 3. The physicochemical data for compound ID16 are as follows: a pale yellow solid. The yield was 68%. Melting point 106.9-107.8 deg.c.¹H NMR(400MHz,DMSO-d₆)δ(ppm):8.04(1H,d,J＝9.6Hz,H-4),7.73(1H,d,J＝2.0Hz,H-5),7.62(1H,dd,J₁＝8.4Hz,J₂＝2.0Hz,H-7),7.35(1H,d,J＝8.8Hz,H-8),6.48(1H,d,J＝9.6Hz,H-3),4.60(2H,s,-SCH ₂-),4.19(2H,br,H-a),3.84(2H,br,H-d),2.56-2.54(5H,m,H-b,H-c,H-e),1.73-1.70(4H,m,H-f,H-j),1.56-1.53(1H,m,H-h),1.22-1.00(5H,m,H-g,H-h,H-i).¹³C NMR(100MHz,DMSO-d₆)δ(ppm):193.7,179.3,159.8,152.6,143.9,132.9,132.8,128.7,118.5,116.4,62.2,51.5,50.2,48.2,48.1,48.0,29.4,28.2,25.7,25.1×2.HRMS(ESI,m/z)calcd.for C₂₁H₂₆N₂O₂S₂(M+H)⁺403.1508,found 403.1509.

EXAMPLE 18 Synthesis of Compound ID17

Synthesis procedure for 2-oxo-2H-benzopyran-6-yl) methyl 4- (4-hydroxyphenyl) piperazine-1-carbo-disulfate (ID17) was analogous to Synthesis procedure b of Compound ID2 in example 3. The physicochemical data for compound ID17 are as follows: a pale yellow solid. The yield was 55%. Melting point 195.5-196.4 deg.C.¹H NMR(400MHz,DMSO-d₆)δ(ppm):8.91(1H,s,-OH),8.04(1H,d,J＝9.6Hz,H-4),7.74(1H,d,J＝2.0Hz,H-5),7.64(1H,dd,J₁＝8.4Hz,J₂＝2.0Hz,H-7),7.36(1H,d,J＝8.4Hz,H-8),6.80(2H,d,J＝8.8Hz,H-f,H-g),6.66(2H,d,J＝8.8Hz,H-e,H-h),6.48(1H,d,J＝9.6Hz,H-3),4.63(2H,s,-SCH ₂-),4.36(2H,br,H-a),4.05-3.99(2H,m,H-d),3.05(4H,br,H-b,H-c).¹³C NMR(100MHz,DMSO-d₆)δ(ppm):194.2,159.8,152.6,151.4,143.9,143.1,132.9,132.8,128.7,118.5,118.3×2,116.4×2,115.4×2,51.0,50.9,49.7×2,39.5.HRMS(ESI,m/z)calcd.for C₂₁H₂₀N₂O₃S₂(M-H)^-411.0843,found 411.0860.

EXAMPLE 19 Synthesis of Compound ID18

Synthesis procedure for 2-oxo-2H-benzopyran-6-yl) methyl 4- (4-cyanophenyl) piperazine-1-carbo disulfate (ID18) was analogous to Synthesis procedure b of Compound ID2 in example 3. The physicochemical data for compound ID18 are as follows: a pale yellow solid. The yield was 55%. The melting point is 181.5-182.4 ℃.¹H NMR(400MHz,DMSO-d₆)δ(ppm):8.04(1H,d,J＝9.6Hz,H-4),7.74(1H,d,J＝1.6Hz,H-5),7.64(1H,dd,J₁＝8.4Hz,J₂＝1.6Hz,H-7),7.60(2H,d,J＝8.8Hz,H-f,H-g),7.36(1H,d,J＝8.4Hz,H-8),6.94(2H,d,J＝8.8Hz,H-e,H-h),6.48(1H,d,J＝9.6Hz,H-3),4.65(2H,s,-SCH ₂-),4.35(2H,br,H-a),4.06(2H,br,H-d),3.55(4H,t,J＝5.2Hz,H-b,H-c).¹³C NMR(100MHz,DMSO-d₆)δ(ppm):194.4,159.8,152.6,151.9,143.9,133.3×2,132.9,132.8,128.7,119.9,118.5,116.4,116.4,113.3×2,98.1,50.2,50.1,48.6,48.5,44.9.HRMS(ESI,m/z)calcd.for C₂₂H₁₉N₃O₂S₂(M+Na)⁺444.0811,found 444.0827.

EXAMPLE 20 Synthesis of Compound ID19

Synthesis of 2-oxo-2H-benzopyran-6-yl) methyl 4-phenylpiperidin-1-carbodisulfate (ID19) the procedure was analogous to Synthesis b of Compound ID2 in example 3. The physicochemical data for compound ID19 are as follows: a white solid. The yield is 66%. The melting point is 115.6-116.5 ℃.¹H NMR(400MHz,CDCl₃)δ(ppm):7.68(1H,d,J＝9.6Hz,H-4),7.58-7.56(2H,m,H-5,H-7),7.33-7.27(3H,m,H-g,H-i,H-8),7.24-7.18(3H,m,H-f,H-h,H-j),6.42(1H,d,J＝9.2Hz,H-3),5.75(1H,br,H-a),4.72-4.64(3H,m,-SCH ₂-,H-a),3.26-3.16(2H,m,H-e),2.92-2.84(1H,m,H-c),2.00-1.96(2H,m,H-b),1.80(2H,br,H-d).¹³C NMR(100MHz,CDCl₃)δ(ppm):194.9,160.6,153.3,144.2,143.2,133.2,132.9,128.6×2,128.4,126.8,126.7×2,118.7,117.0,116.9,52.6,50.8,42.5,40.8,33.2,32.6.HRMS(ESI,m/z)calcd.for C₂₂H₂₁NO₂S₂(M+Na)⁺418.0906,found 418.0910.

EXAMPLE 21 Synthesis of Compound ID20

Synthesis of 2-oxo-2H-benzopyran-6-yl) methyl 4- (4-methylpiperazin-1-yl) piperidine-1-carbodisulfate (ID20) the procedure was analogous to Synthesis procedure b of Compound ID1 in example 2. The physicochemical data for compound ID20 are as follows: an orange solid. The yield is 66%. The melting point is 95.0-96.0 ℃.¹H NMR(400MHz,DMSO-d₆)δ(ppm):8.04(1H,d,J＝9.6Hz,H-4),7.73(1H,d,J＝2.0Hz,H-5),7.62(1H,dd,J₁＝8.4Hz,J₂＝2.4Hz,H-7),7.35(1H,d,J＝8.8Hz,H-8),6.48(1H,d,J＝9.6Hz,H-3),4.59(2H,d,J＝4.0Hz,-SCH ₂-),3.36-3.23(2H,m,-CH(CH₂CH ₂)N-(piperidine)),2.58-2.53(1H,m,-CH(CH₂CH₂)N-(piperidine)),2.52-2.49(2H,m,-CH(CH₂CH ₂)N-(piperidine)),2.45(4H,br,-N(CH ₂CH₂)₂N-CH₃-(piperazine)),2.30(4H,br,-N(CH₂CH ₂)₂N-CH₃-(piperazine)),2.14(3H,s,-N(CH₂CH₂)₂N-CH ₃),1.867-1.839(2H,m,-CH(CH ₂CH₂)N-(piperidine)),1.38-1.34(2H,m,-CH(CH₂CH ₂)N-(piperidine)).¹³C NMR(100MHz,DMSO-d₆)δ(ppm):193.2,159.8,152.6,144.0,133.0,132.8,128.7,118.5,116.4×2,59.7,54.9×2,48.3×3,45.5×2,39.8,28.0,27.5.HRMS(ESI,m/z)calcd.for C₂₁H₂₇N₃O₂S₂(M+H)⁺418.1617,found 418.1630.

EXAMPLE 22 in vitro antitumor Activity assay of Compounds

Cell lines and reagents: colon cancer cell lines SW620, SW480 and RKO cells were purchased from the cell bank of the typical culture collection committee of the chinese academy of sciences (shanghai, china); fetal Bovine Serum (FBS) was purchased from HyClone (utah, usa); RPMI-1640 medium and Phosphate Buffered Saline (PBS) were purchased from Zhejiang Senri Biotech, Inc. (Hu, China); primary antibodies include anti-Cdc2, anti-CyclinB1, anti-cleared PARP, anti-Bcl-2, anti-actin and secondary antibodies from Santa Cruz Biotechnology, Calif., USA. DMSO was purchased from Sigma-Aldrich (Mo.). MTS powder was purchased from Promega (Promega, usa).

Cell culture and administration: 5% CO at 37 ℃₂In the environment of an incubator, cells are cultured in RPMI-1640 medium containing 10% fetal bovine serum. The compound is prepared into a solution with the mother liquor concentration of 20mM by DMSO, the solution is placed in a refrigerator at the temperature of-30 ℃, and the solution is diluted into a corresponding concentration according to the experiment requirement.

EXAMPLE 23 Compound ID1-ID20 anti-colon cancer cell proliferation assay

SW620, SW480 and RKO cells were cultured in 96-well plates (6000 cells/well) in RPMI-1640 medium containing 10% FBS, and after overnight adherence different concentrations of the compounds containing the corresponding medium were given for 72 hours of action. The medium was discarded and fresh medium containing 10% MTS was added to each well and incubated at 37 ℃ for 30 minutes. And finally, detecting the absorbance value of the 96-well plate in a microplate reader under the set wavelength of 490 nm. Wherein, it is toThe compound concentration was set at 10 μ M for inhibition assay. For IC₅₀Values were determined with compound concentration gradients set at 0.625. mu.M, 1.25. mu.M, 2.5. mu.M, 5. mu.M and 10. mu.M. IC of the Compound₅₀Values can be calculated using Graphpad Prism 7.0 software. The results obtained by performing the assay with different compounds are shown in FIG. 1 (A-F).

Example 24 cell colony formation assay

SW620 and SW480 cells were cultured in 6-well plates (1000 cells/well) in RMPI-1640 medium containing 10% FBS and administered DMSO overnight adherent with different concentrations (4. mu.M, 8. mu.M, 16. mu.M and 32. mu.M) of compound ID11 containing the corresponding medium for 24 hours. Each well was then replaced with fresh RPMI-1640 medium and culture was continued until distinct colonies of cells (greater than 50 cells per colony) grew out. After completion of the culture, the medium was discarded, and 1ml of methanol was added to each well and fixed for 15 minutes. Methanol was discarded, and 1ml of 0.1% crystal violet solution was added to each well and stained for more than 30 minutes. Washing off the dye solution with tap water, drying, taking a picture, and finally analyzing the result. The obtained results are shown in FIG. 2(A, B).

Example 25 cell cycle assay

SW620 and SW480 cells were cultured in 6-well plates (5X 10) in RMPI-1640 medium containing 10% FBS⁵Individual cells/well) were administered overnight after adherence with different concentrations (8 μ M and 16 μ M) of compound ID11 containing the corresponding medium for 24 hours. Cells were then harvested and fixed by adding 75% glacial ethanol. After washing the cells with PBS, they were stained with Propidium Iodide (PI) for 30 minutes in the dark, after which the flow cytometry tubes were placed in a FACSCalibur flow cytometer (BDBiosciences, CA) to detect cell cycle distribution. The obtained results are shown in FIG. 3(A, B).

Example 26 apoptosis assay

SW620 and SW480 cells were cultured in 6-well plates (5X 10) in RMPI-1640 medium containing 10% FBS⁵Individual cells/well) were administered overnight after adherence with different concentrations (8 μ M and 16 μ M) of compound ID11 containing the corresponding medium for 24 hours. After cell collection, the cells were washed twice with ice-cold PBS. After that, FITC conjugated Annexin V and Propidium Iodide (PI) were added and protected from light for 30 minutesDouble staining was performed, after which the flow cytometry tubes were placed in a FACSCalibur flow cytometer to detect apoptosis. The obtained results are shown in FIG. 4(A, B).

Example 27 Western immunoblot assay

After compound treatment, cells were harvested, lysed with proteolytic lysis buffer, and then centrifuged at 4 ℃ to remove impurities. The protein concentration of the samples was determined using the Bradford protein assay kit (Bio-Rad, Hercules, Calif.). After adding the loading buffer to the protein sample, the protein sample was subjected to electrophoresis, and then transferred to a polyvinylidene fluoride membrane (PVDF membrane). After the transfer, blocking with freshly prepared 5% skim milk in 1 × TBST was performed, followed by incubation with the specific primary antibody overnight at 4 ℃ in 1 × TBST. Following 3 washes with 1 × TBST, incubation with horseradish peroxidase (HRP) -conjugated secondary antibodies for 1 hour at room temperature and detection of the immunofluorescence in an exposure apparatus by ECL kit (Bio-Rad, Hercules, Calif.). The results obtained by performing the assay with different compounds are shown in FIG. 3(C) and FIG. 4 (C).

Statistical analysis

All experiments were independently repeated three times and the results are presented as mean ± standard error (SD). The statistical significance of the experimental results was assessed using one-way ANOVA. P <0.05 was considered statistically significant.

While some embodiments of the present invention have been presented herein, it will be appreciated by those skilled in the art that changes may be made to the embodiments herein without departing from the spirit of the invention. The above examples are merely illustrative and should not be taken as limiting the scope of the invention.