阅读说明：本技术 一类色原酮氮芥衍生物与抗肿瘤应用 (Chromone nitrogen mustard derivative and anti-tumor application ) 是由 曹昊 李达翃 华会明 孙迦南 穆家辉 高祥 于 2021-10-21 设计创作，主要内容包括：本发明公开了一类色原酮氮芥衍生物与抗肿瘤应用,属于天然药物及药物化学领域。具体涉及一系列具有抗肿瘤活性的色原酮氮芥衍生物的制备方法和在抗肿瘤药物方面新用途。本发明所述的色原酮氮芥衍生物及其药学上可接受的盐如通式I所示。其中,R和X如权利要求书和说明书中所述。(The invention discloses a chromone nitrogen mustard derivative and anti-tumor application, belonging to the fields of natural medicines and medicinal chemistry. In particular to a preparation method of a series of chromone nitrogen mustard derivatives with anti-tumor activity and new application in anti-tumor drugs. The chromone nitrogen mustard derivative and the pharmaceutically acceptable salt thereof are shown as a general formula I. Wherein R and X are as described in the claims and the description.)

1. A chromone mustard derivative of the general formula I:

wherein R is an alkyl group having 1 to 8 carbon atoms or an alkoxy group having 1 to 8 carbon atoms; the heteroatom X is N, O, S or Se.

2. The chromone mustard derivative of formula I and its pharmaceutically acceptable salts of claim 1, wherein:

in the general formula I, R is alkyl containing 1-6 carbon atoms or alkoxy containing 1-6 carbon atoms; the heteroatom X is N, O or S.

3. The chromone mustard derivative of formula I and its pharmaceutically acceptable salts of claim 1, wherein:

in the general formula I, R is alkyl containing 1-4 carbon atoms or alkoxy containing 1-4 carbon atoms; the heteroatom X is N or O.

4. The chromone mustard derivative of formula I, or a pharmaceutically acceptable salt thereof, according to any one of claims 1-3, wherein: the structural formula of the chromone nitrogen mustard derivative and the pharmaceutically acceptable salt thereof is shown as a-d:

5. a pharmaceutical composition characterized by: the pharmaceutical composition comprises a therapeutically effective amount of the chromone mustard derivative of formula I of any one of claims 1-4, and pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier.

6. Use of the chromone mustard derivative of the general formula I and its pharmaceutically acceptable salts in any of claims 1-4 for the preparation of a medicament for the treatment of a neoplastic disease.

7. Use of the pharmaceutical composition of claim 5 for the preparation of a medicament for the treatment of a neoplastic disease.

8. Use according to claim 6 or 7, characterized in that: the tumor is breast cancer tumor or liver cancer tumor.

Technical Field

The invention belongs to the field of natural medicines and medicinal chemistry, relates to a chromone nitrogen mustard derivative and anti-tumor application, and particularly relates to a preparation method of a series of chromone nitrogen mustard derivatives with anti-tumor activity and application of the chromone nitrogen mustard derivatives in the aspect of anti-tumor.

Background

The chromone backbone is an important component of a large number of biologically active molecules, and is also a core moiety of many natural products, lead compounds, and clinical drugs. The chromone can be synthesized into derivatives with various structures, and can be used as a lead compound for structural modification to synthesize various derivatives with different pharmacological activities. At present, chromone and derivatives thereof become one of the most important synthetic compounds with anticancer activity, and the synthesis and structure modification of chromone are actively researched at home and abroad.

Nitrogen mustards are a class of DNA alkylating agents that have now been developed into a variety of chemotherapeutic agents with a broad spectrum of anti-tumor activity. However, because of its non-specificity to cancer cells, it is often necessary to use a large dose to achieve the effect of killing cancer cells. The use of large doses of nitrogen mustard causes serious side effects and drug resistance, and limits the further clinical application of the nitrogen mustard. Therefore, many researchers have structurally modified nitrogen mustards in order to obtain more active, less toxic and more selective antitumor candidate compounds.

Disclosure of Invention

The invention aims to solve the technical problem of searching the chromone nitrogen mustard derivative with good anti-tumor activity and the pharmaceutically acceptable salt thereof and further providing a pharmaceutical composition.

In order to solve the technical problems, the invention provides the following technical scheme:

a chromone mustard derivative and pharmaceutically acceptable salts thereof have the following structural general formula I:

wherein R is an alkyl group having 1 to 8 carbon atoms or an alkoxy group having 1 to 8 carbon atoms; the heteroatom X is N, O, S or Se.

Preferably, R is an alkyl group having 1 to 6 carbon atoms or an alkoxy group having 1 to 6 carbon atoms; the heteroatom X is N, O or S.

More preferably, R is an alkyl group containing 1 to 4 carbon atoms or an alkoxy group containing 1 to 4 carbon atoms; the heteroatom X is N or O.

Further, the following derivatives and pharmaceutically acceptable salts thereof are preferably selected from the following structural formulas a to d:

the derivative of the invention can be prepared by the following method:

(1) reacting the compound 1a-b with phosphorus oxychloride in N, N-dimethylformamide at room temperature to obtain an intermediate 2 a-b; then the intermediate 2a-b is dissolved in isopropanol and alkaline Al is added₂O₃Carrying out reflux reaction to obtain a compound 3 a-b; dissolving the compounds 3a-b in dichloromethane, dropwise adding phosphorus tribromide under an ice bath condition, transferring to room temperature for reaction, dissolving the treated solid in N, N-dimethylformamide, adding ammonia water, and reacting at room temperature to obtain compounds 4 a-b;

(2) reacting the compound 5 with ethylene oxide in an acetic acid aqueous solution to obtain an intermediate 6; the intermediate 6 is firstly reacted with phosphorus oxychloride, and then the compound 7 is obtained under the condition of 10% hydrochloric acid aqueous solution.

(3) Dissolving the compound 3a-b or 4a-b in anhydrous dichloromethane, sequentially adding EDCI, DMAP or HOBt, and reacting with the compound 7 at room temperature to obtain a target compound 8 a-d.

A pharmaceutical composition, which comprises a therapeutically effective amount of the chromone nitrogen mustard derivative shown in the general formula I and pharmaceutically acceptable salts thereof and a pharmaceutically acceptable carrier.

The use of the chromone nitrogen mustard derivative shown in the general formula I and the pharmaceutically acceptable salt thereof in preparing medicaments for treating tumor diseases.

Furthermore, the tumor is breast cancer tumor or liver cancer tumor.

The application of the pharmaceutical composition in preparing medicines for treating tumor diseases.

Furthermore, the tumor is breast cancer tumor or liver cancer tumor.

The invention takes chromone as a lead compound, designs and synthesizes a series of chromone nitrogen mustard derivatives, and tests the biological activity of the synthesized derivatives in the aspect of anti-tumor.

Pharmacological tests prove that the chromone nitrogen mustard derivative has good anti-tumor cell proliferation effect and can be used for further preparing anti-tumor drugs.

Detailed Description

The following non-limiting examples will allow one of ordinary skill in the art to more fully understand the present invention, but are not intended to limit the invention in any way.

The synthesis route of the derivatives of the embodiment of the invention is as follows:

example 1

(1) 500mg of compound 1a (3.33mmol) was dissolved in 10mL of DMF, and 630. mu.L of phosphorus oxychloride (6.72mmol) was added dropwise and reacted at room temperature for 12 hours. TLC monitoring, reaction is almost complete, 15mL water is added, crystal is separated out, suction filtration and drying are carried out, and the intermediate 2a 402.4mg is obtained. 50mg of intermediate 2a (0.27mmol) are dissolved in 10mL of isopropanol and 1g of basic Al are added₂O₃(9.80mmol) and then the reaction was refluxed at 75 ℃ for 5 h. TLC monitoring, complete reaction, suction filtration,concentrating the filtrate to give crude product, and separating with silica gel column chromatography (DCM: MeOH) to give compound 3a (R ═ CH)₃)。

(2) 5mL of ethylene oxide (0.10mol) was added to a suspension of 1.53g of ethyl 4-aminobenzoate 5(9.26mmol) dissolved in 12mL of a 65% aqueous acetic acid solution, and stirred at room temperature for 24 hours. After completion of the reaction, the reaction mixture was extracted 3 times with ethyl acetate, washed 1 time with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to obtain 61.64 g of an intermediate. Slowly dropwise adding 2mL of POCl under ice bath condition₃In 1.5g of intermediate 6(6.00mmol), the reaction was carried out in an oil bath at 50 ℃ for 0.5 h. Then adding 3mL of 10% hydrochloric acid aqueous solution to react for 12h at room temperature, filtering, washing filter residue, and concentrating in vacuum to obtain a compound 7.

(3) 50mg of compound 3a (0.26mmol) was dissolved in 5mL of anhydrous dichloromethane, 151.6mg of EDCI (0.80mmol), 16.1mg of DMAP (0.13mmol), 68.9mg of compound 7(0.21mmol) were added, and the reaction was carried out at room temperature for 12 hours. After the reaction was completed, the reaction solution was poured into water, extracted with dichloromethane 3 times, washed with saturated brine 1 time, and anhydrous Na₂SO₄Drying and concentrating to obtain a crude product. Silica gel column chromatography (DCM: MeOH) afforded title compound 8 a. White oil, 66.5% yield.¹H NMR(CDCl₃,400MHz),δ:8.12(1H,s,2-H),8.02(1H,d,J＝2.0Hz,5-H),7.94(2H,d,J＝9.1Hz,Ar-H),7.47(1H,dd,J＝8.6,2.0Hz,7-H),7.34(1H,d,J＝8.6Hz,8-H),6.63(2H,d,J＝9.1Hz,Ar-H),5.27(2H,s,-CH₂),3.78(4H,t,J＝7.0Hz,-CH₂),3.64(4H,t,J＝7.0Hz,-CH₂),2.45(3H,s,-CH₃)；¹³C NMR(CDCl₃,100MHz),δ:176.76,166.40,155.42,154.75,149.83,135.36,135.07,131.98(2),125.20,123.75,119.77,118.59,117.95,110.86(2),58.12,53.27(2),40.20(2),20.95；HRMS(ESI)m/z calcd for C₂₂H₂₀Cl₂NO₄[M-H]^-432.0769,found 432.0748。

Example 2

Example 1 step (1) the step for the synthesis of 3a was replaced by: 553mg of Compound 1b (3.33mmol) were dissolved in 10mL of DMF, and then 630. mu.L of phosphorus oxychloride (6.72mmol) was added dropwise and reacted at room temperature for 12 hours. TLC monitoring, reaction is almost complete, 15mL water is added, crystal is separated out, suction filtration and drying are carried out, and the intermediate 2b 410mg is obtained. 55mg of intermediate 2b (0.27mmol) were dissolved in 10mL of isopropanol and 1g of basic Al was added₂O₃(9.80mmol) and then the reaction was refluxed at 75 ℃ for 5 h. TLC monitoring, reaction completed, suction filtration, filtrate concentration to get crude product, silica gel column chromatography (DCM: MeOH) separation to get compound 3b (R ═ OCH)₃)。

The remaining steps were performed according to the synthetic method of example 1 to obtain compound 8b as a yellow oil in 43.8% yield.¹H NMR(CDCl₃,400MHz),δ:8.14(1H,s,2-H),7.95(2H,d,J＝9.1Hz,Ar-H),7.61(1H,d,J＝3.1Hz,5-H),7.40(1H,d,J＝9.1Hz,8-H),7.26(1H,dd,J＝9.1,3.1Hz,7-H),6.64(2H,d,J＝9.1Hz,Ar-H),5.27(2H,s,-CH₂),3.90(3H,s,-OCH₃),3.79(4H,t,J＝6.8Hz,-CH₂),3.64(4H,t,J＝6.8Hz,-CH₂)；¹³C NMR(CDCl₃,100MHz),δ:176.56,166.41,157.08,155.32,151.34,149.83,132.00(2),124.70,123.95,119.62,119.18,118.62,110.86(2),104.95,58.13,55.95,53.29(2),40.10(2)；HRMS(ESI)m/z calcd for C₂₂H₂₀Cl₂NO₅[M-H]^-448.0719,found 448.0714。

Example 3

Example 1 the procedure for the synthesis of 3a in step (1) was replaced by: 500mg of compound 1a (3.33mmol) was dissolved in 10mL of DMF, and 630. mu.L of phosphorus oxychloride (6.72mmol) was added dropwise and reacted at room temperature for 12 hours. TLC monitoring, reaction is almost complete, 15mL water is added, crystal is separated out, suction filtration and drying are carried out, and the intermediate 2a 402.4mg is obtained. 50mg of intermediate 2a (0.27mmol) are dissolved in 10mL of isopropanol and 1g of basic Al are added₂O₃(9.80mmol) and then the reaction was refluxed at 75 ℃ for 5 h. TLC monitoring, reaction completion, suction filtration, concentration of filtrate to give crude product, silica gel column chromatography (D)CM: MeOH) to yield compound 3a 15 mg. 500mg of Compound 3a (2.62mmol) was dissolved in 10mL of DCM, 750. mu.L of phosphorus tribromide (7.90mmol) was added dropwise in an ice bath, reacted for 10min, and transferred to room temperature for reaction for 15 h. TLC monitoring, reaction completion, dichloromethane extraction 3 times, saturated salt water washing 1 time, anhydrous Na₂SO₄Dried, filtered and concentrated to give 652mg of a yellow solid. The resulting solid was dissolved in 10mL of DMF, and 10mL of aqueous ammonia was added to the solution to react at room temperature overnight. TLC monitoring, complete reaction, ethyl acetate extraction 3 times, saturated salt water washing 1 time, anhydrous Na₂SO₄Drying, filtering and concentrating to obtain a crude product. Separating with silica gel column chromatography (DCM: MeOH) to obtain compound 4a (R ═ CH)₃)。

Example 1 the procedure for the synthesis of 8a in step (3) was replaced by: 26.4mg of compound 4a (0.14mmol) was dissolved in 4mL of anhydrous dichloromethane, and 39.9mg of EDCI (0.21mmol), 22.5mg of HOBt (0.17mmol), 36.4mg of compound 7(0.14mmol) were added and reacted at room temperature for 6 h. After the reaction was completed, the reaction solution was poured into water, extracted with dichloromethane 3 times, washed with saturated brine 1 time, and anhydrous Na₂SO₄Drying and concentrating to obtain a crude product. Silica gel column chromatography (DCM: MeOH) afforded title compound 8 c.

The remaining steps were performed according to example 1 to obtain compound 8c as a white solid with a yield of 36.8%.¹H NMR(CDCl₃,400MHz),δ:8.17(1H,s,2-H),7.98(1H,d,J＝2.0Hz,5-H),7.70(2H,d,J＝8.9Hz,Ar-H),7.48(1H,dd,J＝8.6,2.0Hz,7-H),7.36(1H,d,J＝8.6Hz,8-H),7.14(1H,s,-NH),6.64(2H,d,J＝8.9Hz,Ar-H),4.45(2H,d,J＝5.6Hz,-CH₂),3.76(4H,m,-CH₂),3.62(4H,m,-CH₂),2.45(3H,s,-CH₃)；¹³C NMR(CDCl₃,100MHz),δ:178.5,167.0,154.9,154.5,148.7,135.3,135.2,129.1(2),124.8(2),123.6,121.0,118.1,111.1(2),53.3(2),40.2(2),36.3,20.9；HRMS(ESI)m/z calcd for C₂₂H₂₁Cl₂N₂O₃[M-H]^-431.0929,found 431.0921。

Example 4

Example 1 the procedure for the synthesis of 3a in step (1) was replaced by: 553mg of Compound 1b (3.33mmol) were dissolved in 10mL of DMF, and then 630. mu.L of phosphorus oxychloride (6.72mmol) was added dropwise and reacted at room temperature for 12 hours. TLC monitoring, reaction is almost complete, 15mL water is added, crystal is separated out, suction filtration and drying are carried out, and the intermediate 2b 410mg is obtained. 55mg of intermediate 2b (0.27mmol) were dissolved in 10mL of isopropanol and 1g of basic Al was added₂O₃(9.80mmol) and then the reaction was refluxed at 75 ℃ for 5 h. TLC detection, complete reaction, suction filtration, concentration of filtrate to give crude product, silica gel column chromatography (DCM: MeOH) separation to give compound 3b 22 mg. 540mg of compound 3b (2.62mmol) was dissolved in 10mL of DCM, 750. mu.L of phosphorus tribromide (7.90mmol) was added dropwise in an ice bath, reacted for 10min, and transferred to room temperature for reaction for 15 h. TLC monitoring, reaction completion, dichloromethane extraction 3 times, saturated salt water washing 1 time, anhydrous Na₂SO₄Dried, filtered and concentrated to afford 681mg of a yellow solid. The resulting solid was dissolved in 10mL of DMF, and 10mL of aqueous ammonia was added to the solution to react at room temperature overnight. TLC monitoring, complete reaction, ethyl acetate extraction 3 times, saturated salt water washing 1 time, anhydrous Na₂SO₄Drying, filtering and concentrating to obtain a crude product. Separating with silica gel column chromatography (DCM: MeOH) to obtain compound 4b (R ═ OCH)₃)。

Example 1 the procedure for the synthesis of 8a in step (3) was replaced by: 28.7mg of compound 4b (0.14mmol) were dissolved in 4mL of anhydrous dichloromethane, and 39.9mg of EDCI (0.21mmol), 22.5mg of HOBt (0.17mmol), 36.4mg of compound 7(0.14mmol) were added and reacted at room temperature for 6 h. After the reaction was completed, the reaction solution was poured into water, extracted with dichloromethane 3 times, washed with saturated brine 1 time, and anhydrous Na₂SO₄Drying and concentrating to obtain a crude product. Silica gel column chromatography (DCM: MeOH) afforded title compound 8 d.

The remaining steps were performed according to example 1 to obtain compound 8d as a white solid with a yield of 43.5%.¹H NMR(CDCl₃,400MHz),δ:8.17(1H,s,2-H),7.70(2H,d,J＝8.8Hz,Ar-H),7.55(1H,d,J＝3.1Hz,5-H),7.40(1H,d,J＝9.1Hz,8-H),7.27(1H,dd,J＝9.1,3.1Hz,7-H),7.11(1H,s,-NH),6.64(2H,d,J＝8.8Hz,Ar-H),4.46(2H,d,J＝5.7Hz,-CH₂),3.89(3H,d,-OCH₃),3.76(4H,m,-CH₂),3.62(4H,m,-CH₂)；¹³C NMR(CDCl₃,100MHz),δ:178.3,167.0,157.0,154.4,151.6,148.7,129.1(2),124.6,124.1,120.4,119.8,111.1(2),104.5,55.9,53.3(2),40.1(2),36.3,29.7；HRMS(ESI)m/z calcd for C₂₂H₂₁Cl₂N₂O₄[M-H]^-447.0878,found 447.0878。

Example 5

The following are the results of pharmacological experiments with some of the compounds of the invention:

experimental equipment and reagent

Instrument clean bench (Sujing group Antai company)

Constant temperature incubator (Thermo electronic Corporation)

Enzyme-linked immunosorbent assay (BIO-RAD company)

Inverted biological microscope (Chongqing optical instrument factory)

Reagent cell culture medium RPMI-1640, DMEM (high-sugar) (GIBCO Co., Ltd.) fetal bovine serum (Hangzhou Sijiqing Co., Ltd.)

CCK-8(Biosharp company product)

DMSO (Sigma Co.)

Cell lines of human breast cancer cell MCF-7, human breast cancer cell MDA-MB-231, human liver cancer cell HepG2 and Bel-7402

Experimental methods

Cell inhibitory activity test method

Cells were incubated at 37 ℃ with 5% CO₂Culturing in an incubator with saturated humidity. The culture medium is high-glucose DMEM cell culture medium containing 10% heat-inactivated fetal calf serum, penicillin 100U/mL and streptomycin 100U/mL. The culture medium was changed for 48h, and after the cells were attached to the wall, they were digested with 0.25% trypsin for passage. The cells for experiment are all in logarithmic growth phase, and the CCK-8 method shows the cell activity>95％。

Taking a bottle of cells in a logarithmic phase, adding a digestive juice (0.125% trypsin and 0.01% EDTA) for digestion, and counting by 2-4 × 10⁴cell/mL, preparing cell suspension, inoculating on 96-well plate, 100 μ L/well, and placing in constant temperature CO₂The culture was carried out in an incubator for 24 hours.The solution was changed, the test drug was added at 100. mu.L/well, and cultured for 72 hours. CCK-8 was added to 96-well plates at 50. mu.L/well and incubated in an incubator for 4 hours. The supernatant was aspirated, DMSO was added at 200. mu.L/well and shaken on a shaker for 10 min. The test substances were examined at 6 concentrations of 0.001 to 100. mu.M in ten-fold increments, and the cell inhibition rate at each concentration was calculated by measuring the absorbance of each well at a wavelength of 450nm using an enzyme-linked immunosorbent assay.

The inhibition rate calculation method comprises the following steps:

relative OD value of drug sensitive well (absolute OD value of drug sensitive well) — absolute OD value of blank control well

Results of the experiment

TABLE 1 examples IC for antiproliferative activity against 2 human breast cancer and 1 human hepatoma cell lines₅₀Value (μ M)

Pharmacological tests prove that the target derivative has better anti-breast cancer and anti-liver cancer cell proliferation activity, and can be used for further preparing anti-tumor drugs.